Nell-1, a key Functional Mediator of Runx2, Partially Rescues Calvarial Defects in Runx2+/− Mice

PubMed Central

Zhang, Xinli; Ting, Kang; Bessette, Catherine M; Culiat, Cymbeline T; Sung, Sang Jin; Lee, Haofu; Chen, Feng; Shen, Jia; Wang, James J; Kuroda, Shun'ichi; Soo, Chia

2011-01-01

Mesenchymal stem cell commitment to an osteoprogenitor lineage requires the activity of Runx2, a molecule implicated in the etiopathology of multiple congenital craniofacial anomalies. Through promoter analyses, we have recently identified a new direct transcriptional target of Runx2, Nell-1, a craniosynostosis (CS)–associated molecule with potent osteogenic properties. This study investigated the mechanistic and functional relationship between Nell-1 and Runx2 in regulating osteoblast differentiation. The results showed that spatiotemporal distribution and expression levels of Nell-1 correlated closely with those of endogenous Runx2 during craniofacial development. Phenotypically, cross-mating Nell-1 overexpression transgenic (CMV-Nell-1) mice with Runx2 haploinsufficient (Runx2+/−) mice partially rescued the calvarial defects in the cleidocranial dysplasia (CCD)–like phenotype of Runx2+/− mice, whereas Nell-1 protein induced mineralization and bone formation in Runx2+/− but not Runx2−/− calvarial explants. Runx2-mediated osteoblastic gene expression and/or mineralization was severely reduced by Nell-1 siRNA oligos transfection into Runx2+/+ newborn mouse calvarial cells (NMCCs) or in N-ethyl-N-nitrosourea (ENU)–induced Nell-1−/− NMCCs. Meanwhile, Nell-1 overexpression partially rescued osteoblastic gene expression but not mineralization in Runx2 null (Runx2−/−) NMCCs. Mechanistically, irrespective of Runx2 genotype, Nell-1 signaling activates ERK1/2 and JNK1 mitogen-activated protein kinase (MAPK) pathways in NMCCs and enhances Runx2 phosphorylation and activity when Runx2 is present. Collectively, these data demonstrate that Nell-1 is a critical downstream Runx2 functional mediator insofar as Runx2-regulated Nell-1 promotes osteoblastic differentiation through, in part, activation of MAPK and enhanced phosphorylation of Runx2, and Runx2 activity is significantly reduced when Nell-1 is blocked or absent. © 2011 American Society for Bone and Mineral Research. PMID:20939017

Claudin-1 has tumor suppressive activity and is a direct target of RUNX3 in gastric epithelial cells.

PubMed

Chang, Ti Ling; Ito, Kosei; Ko, Tun Kiat; Liu, Qiang; Salto-Tellez, Manuel; Yeoh, Khay Guan; Fukamachi, Hiroshi; Ito, Yoshiaki

2010-01-01

The transcription factor RUNX3 is a gastric tumor suppressor. Tumorigenic Runx3(-/-) gastric epithelial cells attach weakly to each other, compared with nontumorigenic Runx3(+/+) cells. We aimed to identify RUNX3 target genes that promote cell-cell contact to improve our understanding of RUNX3's role in suppressing gastric carcinogenesis. We compared gene expression profiles of Runx3(+/+) and Runx3(-/-) cells and observed down-regulation of genes associated with cell-cell adhesion in Runx3(-/-) cells. Reporter, mobility shift, and chromatin immunoprecipitation assays were used to examine the regulation of these genes by RUNX3. Tumorigenesis assays and immunohistological analyses of human gastric tumors were performed to confirm the role of the candidate genes in gastric tumor development. Mobility shift and chromatin immunoprecipitation assays revealed that the promoter activity of the gene that encodes the tight junction protein claudin-1 was up-regulated via the binding of RUNX3 to the RUNX consensus sites. The tumorigenicity of gastric epithelial cells from Runx3(-/-) mice was significantly reduced by restoration of claudin-1 expression, whereas knockdown of claudin-1 increased the tumorigenicity of human gastric cancer cells. Concomitant expression of RUNX3 and claudin-1 was observed in human normal gastric epithelium and cancers. The tight junction protein claudin-1 has gastric tumor suppressive activity and is a direct transcriptional target of RUNX3. Claudin-1 is down-regulated during the epithelial-mesenchymal transition; RUNX3 might therefore act as a tumor suppressor to antagonize the epithelial-mesenchymal transition. Copyright 2010 AGA Institute. Published by Elsevier Inc. All rights reserved.

COACTIVATOR ACTIVATOR (CoAA) PREVENTS THE TRANSCRIPTIONAL ACTIVITY OF RUNT DOMAIN TRANSCRIPTION FACTORS

PubMed Central

Li, Xiaodong; Hoeppner, Luke H.; Jensen, Eric D.; Gopalakrishnan, Rajaram; Westendorf, Jennifer J.

2013-01-01

Runx proteins are essential for a number of developmental processes and are aberrantly expressed in many human cancers. Runx factors bind DNA and co-factors to activate or repress genes crucial for bone formation, hematopoiesis, and neuronal development. Co-activator activator (CoAA) is a nuclear protein that regulates gene expression, RNA splicing and is overexpressed in many human tumors. In this study, we identified CoAA as a Runx2 binding protein. CoAA repressed Runx factor-dependent activation of reporter genes in a histone deacetylase-independent manner. CoAA also blocked Runx2-mediated repression of the Axin2 promoter, a novel Runx target gene. The carboxy-terminus of CoAA is essential for binding the Runt domains of Runx1 and Runx2. In electophoretic mobility shift assays, CoAA inhibited Runx2 interactions with DNA. These data indicate that CoAA is an inhibitor of Runx factors and can negate Runx factor regulation of gene expression. CoAA is expressed at high levels in human fetal osteoblasts and osteosarcoma cell lines. Suppression of CoAA expression by RNA interference reduced osteosarcoma cell viability in vitro, suggesting that it contributes to the proliferation and/or survival of osteoblast lineage cells. PMID:19585539

The RUNX1 +24 enhancer and P1 promoter identify a unique subpopulation of hematopoietic progenitor cells derived from human pluripotent stem cells

PubMed Central

Ferrell, Patrick I; Xi, Jiafei; Ma, Chao; Adlakha, Mitali; Kaufman, Dan S.

2016-01-01

Derivation of hematopoietic stem cells from human pluripotent stem cells remains a key goal for the fields of developmental biology and regenerative medicine. Here, we use a novel genetic reporter system to prospectively identify and isolate early hematopoietic cells derived from human embryonic stem cells (hESCs) and human induced pluripotent cells (iPSCs). Cloning the human RUNX1c P1 promoter and +24 enhancer to drive expression of tdTomato (tdTom) in hESCs and iPSCs, we demonstrate that tdTom expression faithfully enriches for RUNX1c-expressing hematopoietic progenitor cells. Time-lapse microscopy demonstrated the tdTom+ hematopoietic cells to emerge from adherent cells. Furthermore, inhibition of primitive hematopoiesis by blocking Activin/Nodal signaling promoted the expansion and/or survival of tdTom+ population. Notably, RUNX1c/tdTom+ cells represent only a limited subpopuation of CD34+CD45+ and CD34+CD43+ cells with a unique genetic signature. Using gene array analysis, we find significantly lower expression of Let-7 and mir181a microRNAs in the RUNX1c/tdTom+ cell population. These phenotypic and genetic analyses comparing the RUNX1c/tdTom+ population to CD34+CD45+ umbilical cord blood and fetal liver demonstrate several key differences that likely impact the development of HSCs capable of long-term multilineage engraftment from hESCs and iPSCs. PMID:25546363

Wnt3a induces the expression of acetylcholinesterase during osteoblast differentiation via the Runx2 transcription factor.

PubMed

Xu, Miranda L; Bi, Cathy W C; Liu, Etta Y L; Dong, Tina T X; Tsim, Karl W K

2017-07-28

Acetylcholinesterase (AChE) hydrolyzes acetylcholine to terminate cholinergic transmission in neurons. Apart from this AChE activity, emerging evidence suggests that AChE could also function in other, non-neuronal cells. For instance, in bone, AChE exists as a proline-rich membrane anchor (PRiMA)-linked globular form in osteoblasts, in which it is proposed to play a noncholinergic role in differentiation. However, this hypothesis is untested. Here, we found that in cultured rat osteoblasts, AChE expression was increased in parallel with osteoblastic differentiation. Because several lines of evidence indicate that AChE activity in osteoblast could be triggered by Wnt/β-catenin signaling, we added recombinant human Wnt3a to cultured osteoblasts and found that this addition induced expression of the ACHE gene and protein product. This Wnt3a-induced AChE expression was blocked by the Wnt-signaling inhibitor Dickkopf protein-1 (DKK-1). We hypothesized that the Runt-related transcription factor 2 (Runx2), a downstream transcription factor in Wnt/β-catenin signaling, is involved in AChE regulation in osteoblasts, confirmed by the identification of a Runx2-binding site in the ACHE gene promoter, further corroborated by ChIP. Of note, Runx2 overexpression in osteoblasts induced AChE expression and activity of the ACHE promoter tagged with the luciferase gene. Moreover, deletion of the Runx2-binding site in the ACHE promoter reduced its activity during osteoblastic differentiation, and addition of 5-azacytidine and trichostatin A to differentiating osteoblasts affected AChE expression, suggesting epigenetic regulation of the ACHE gene. We conclude that AChE plays a role in osteoblastic differentiation and is regulated by both Wnt3a and Runx2. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

miR-152 regulated glioma cell proliferation and apoptosis via Runx2 mediated by DNMT1.

PubMed

Zhang, Peng; Sun, Hongwei; Yang, Bo; Luo, Wenzheng; Liu, Zengjin; Wang, Junkuan; Zuo, Yuchao

2017-08-01

Aberrant DNA methylation is associated with tumor onset and progression. Study has verified that the DNA methylation of miR-152 was mediated in many tumors, but whether it involved in glioblastomas was still unclear. This study enrolled 20 patients with glioma to analyze the expression pattern of miR-152. Real-time PCR and western blot were used to detect the mRNA or protein expression level, respectively. The relationship between miR-152 and runx2 was detected by Luciferase reporter assay. The methylation level of miR-152 was determined by methylation-specific PCR. Cell proliferation and apoptosis were detected by MTT and Annexin-FITC/PI assay. The expression of miR-152 was down-regulated while the expression of DNMT1 was up-regulated in both glioma tissue and cell lines. MiR-152 was hypermethylated and its expression was negatively correlated with DNMT in glioma cell lines. DNMT1 knockdown promoted the expression of miR-152, however, DNMT1 overexpression suppressed the expression of miR-152. MiR-152 overexpression promoted glioma cell apoptosis while miR-152 knockdown promoted cell proliferation. MiR-152 targets Runx2 to regulate its expression, Runx2 overexpression abolished the effects of miR-152 overexpression. MiR-152 regulated cell proliferation and apoptosis of glioma mediated by Runx2, while the mechanism of down regulated miR-152 in glioma tissues and cells was its hypermethylation. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

The Cancer-Related Transcription Factor Runx2 Modulates Cell Proliferation in Human Osteosarcoma Cell Lines

PubMed Central

Lucero, Claudia M.J.; Vega, Oscar A.; Osorio, Mariana M.; Tapia, Julio C.; Antonelli, Marcelo; Stein, Gary S.; Van Wijnen, Andre J.; Galindo, Mario A.

2013-01-01

Runx2 regulates osteogenic differentiation and bone formation, but also suppresses pre-osteoblast proliferation by affecting cell cycle progression in the G1 phase. The growth suppressive potential of Runx2 is normally inactivated in part by protein destabilization, which permits cell cycle progression beyond the G1/S phase transition, and Runx2 is again up-regulated after mitosis. Runx2 expression also correlates with metastasis and poor chemotherapy response in osteosarcoma. Here we show that six human osteosarcoma cell lines (SaOS, MG63, U2OS, HOS, G292, and 143B) have different growth rates, which is consistent with differences in the lengths of the cell cycle. Runx2 protein levels are cell cycle-regulated with respect to the G1/S phase transition in U2OS, HOS, G292, and 143B cells. In contrast, Runx2 protein levels are constitutively expressed during the cell cycle in SaOS and MG63 cells. Forced expression of Runx2 suppresses growth in all cell lines indicating that accumulation of Runx2 in excess of its pre-established levels in a given cell type triggers one or more anti-proliferative pathways in osteosarcoma cells. Thus, regulatory mechanisms controlling Runx2 expression in osteosarcoma cells must balance Runx2 protein levels to promote its putative oncogenic functions, while avoiding suppression of bone tumor growth. PMID:22949168

Histone Deacetylase Inhibitors Repress Tumoral Expression of the Proinvasive Factor RUNX2.

PubMed

Sancisi, Valentina; Gandolfi, Greta; Ambrosetti, Davide Carlo; Ciarrocchi, Alessia

2015-05-01

Aberrant reactivation of embryonic pathways occurs commonly in cancer. The transcription factor RUNX2 plays a fundamental role during embryogenesis and is aberrantly reactivated during progression and metastasization of different types of human tumors. In this study, we attempted to dissect the molecular mechanisms governing RUNX2 expression and its aberrant reactivation. We identified a new regulatory enhancer element, located within the RUNX2 gene, which is responsible for the activation of the RUNX2 promoter and for the regulation of its expression in cancer cells. Furthermore, we have shown that treatment with the anticancer compounds histone deacetylase inhibitor (HDACi) results in a profound inhibition of RUNX2 expression, which is determined by the disruption of the transcription-activating complex on the identified enhancer. These data envisage a possible targeting strategy to counteract the oncongenic function of RUNX2 in cancer cells and provide evidence that the cytotoxic activity of HDACi in cancer is not only dependent on the reactivation of silenced oncosuppressors but also on the repression of oncogenic factors that are necessary for survival and progression. ©2015 American Association for Cancer Research.

RUNX1 promote invasiveness in pancreatic ductal adenocarcinoma through regulating miR-93

PubMed Central

Cheng, Yin; Yang, Haiyan; Sun, Yang; Zhang, Hongkai; Yu, Shuangni; Lu, Zhaohui; Chen, Jie

2017-01-01

Runt-related transcription factor 1(RUNX1), a key factor in hematopoiesis that mediates specification and homeostasis of hematopoietic stem and progenitor cells (HSPCs), is also overexpressed in several solid human cancers, and correlated with tumor progression. However, the expression and function of RUNX1 in pancreatic ductal adenocarcinoma were still unclear. Here, we show that RUNX1 is highly expressed in pancreatic adenocarcinoma tissues and knocking down of RUNX1 attenuated aggressiveness in pancreatic cell lines. Moreover, we found that RUNX1 could negatively regulate the expression of miR-93. Bioinformatics method showed that there are two binding sites in the the promotor region of miR-93 precursor and through ChIP-qPCR and firefly luciferase reporter assay, we vertified that these two binding sites each have transcriptive activity in one pancreatic cell lines. This result supported our presumption that RUNX1 regulate miR-93 through binding to the promotor region of miR-93. Besides, the expression and function of miR-93 is quite the opposite, miR-93 overexpression suppresses migration and invasiveness in pancreatic cell lines supporting that RUNX1 negatively regulated miR-93. Our findings provided evidence regarding the role of RUNX1 as an oncogene through the inhibition of miR-93. Targeting RUNX1 can be a potential therapeutic strategy in pancreatic cancer. PMID:29245924

Human immunodeficiency virus type 1 enhancer-binding protein 3 is essential for the expression of asparagine-linked glycosylation 2 in the regulation of osteoblast and chondrocyte differentiation.

PubMed

Imamura, Katsuyuki; Maeda, Shingo; Kawamura, Ichiro; Matsuyama, Kanehiro; Shinohara, Naohiro; Yahiro, Yuhei; Nagano, Satoshi; Setoguchi, Takao; Yokouchi, Masahiro; Ishidou, Yasuhiro; Komiya, Setsuro

2014-04-04

Human immunodeficiency virus type 1 enhancer-binding protein 3 (Hivep3) suppresses osteoblast differentiation by inducing proteasomal degradation of the osteogenesis master regulator Runx2. In this study, we tested the possibility of cooperation of Hivep1, Hivep2, and Hivep3 in osteoblast and/or chondrocyte differentiation. Microarray analyses with ST-2 bone stroma cells demonstrated that expression of any known osteochondrogenesis-related genes was not commonly affected by the three Hivep siRNAs. Only Hivep3 siRNA promoted osteoblast differentiation in ST-2 cells, whereas all three siRNAs cooperatively suppressed differentiation in ATDC5 chondrocytes. We further used microarray analysis to identify genes commonly down-regulated in both MC3T3-E1 osteoblasts and ST-2 cells upon knockdown of Hivep3 and identified asparagine-linked glycosylation 2 (Alg2), which encodes a mannosyltransferase residing on the endoplasmic reticulum. The Hivep3 siRNA-mediated promotion of osteoblast differentiation was negated by forced Alg2 expression. Alg2 suppressed osteoblast differentiation and bone formation in cultured calvarial bone. Alg2 was immunoprecipitated with Runx2, whereas the combined transfection of Runx2 and Alg2 interfered with Runx2 nuclear localization, which resulted in suppression of Runx2 activity. Chondrocyte differentiation was promoted by Hivep3 overexpression, in concert with increased expression of Creb3l2, whose gene product is the endoplasmic reticulum stress transducer crucial for chondrogenesis. Alg2 silencing suppressed Creb3l2 expression and chondrogenesis of ATDC5 cells, whereas infection of Alg2-expressing virus promoted chondrocyte maturation in cultured cartilage rudiments. Thus, Alg2, as a downstream mediator of Hivep3, suppresses osteogenesis, whereas it promotes chondrogenesis. To our knowledge, this study is the first to link a mannosyltransferase gene to osteochondrogenesis.

RUNX3 promoter hypermethylation is frequent in leukaemia cell lines and associated with acute myeloid leukaemia inv(16) subtype.

PubMed

Estécio, Marcos R H; Maddipoti, Sirisha; Bueso-Ramos, Carlos; DiNardo, Courtney D; Yang, Hui; Wei, Yue; Kondo, Kimie; Fang, Zhihong; Stevenson, William; Chang, Kun-Sang; Pierce, Sherry A; Bohannan, Zachary; Borthakur, Gautam; Kantarjian, Hagop; Garcia-Manero, Guillermo

2015-05-01

Correlative and functional studies support the involvement of the RUNX gene family in haematological malignancies. To elucidate the role of epigenetics in RUNX inactivation, we evaluated promoter DNA methylation of RUNX1, 2, and 3 in 23 leukaemia cell lines and samples from acute myeloid leukaemia (AML), acute lymphocytic leukaemia (ALL) and myelodysplatic syndromes (MDS) patients. RUNX1 and RUNX2 gene promoters were mostly unmethylated in cell lines and clinical samples. Hypermethylation of RUNX3 was frequent among cell lines (74%) and highly variable among patient samples, with clear association to cytogenetic status. High frequency of RUNX3 hypermethylation (85% of the 20 studied cases) was found in AML patients with inv(16)(p13.1q22) compared to other AML subtypes (31% of the other 49 cases). RUNX3 hypermethylation was also frequent in ALL (100% of the six cases) but low in MDS (21%). In support of a functional role, hypermethylation of RUNX3 was correlated with low levels of protein, and treatment of cell lines with the DNA demethylating agent, decitabine, resulted in mRNA re-expression. Furthermore, relapse-free survival of non-inv(16)(p13.1q22) AML patients without RUNX3 methylation was significantly better (P = 0·016) than that of methylated cases. These results suggest that RUNX3 silencing is an important event in inv(16)(p13.1q22) leukaemias. © 2015 John Wiley & Sons Ltd.

RUNX2 correlates with subtype-specific breast cancer in a human tissue microarray, and ectopic expression of Runx2 perturbs differentiation in the mouse mammary gland

PubMed Central

McDonald, Laura; Ferrari, Nicola; Terry, Anne; Bell, Margaret; Mohammed, Zahra M.; Orange, Clare; Jenkins, Alma; Muller, William J.; Gusterson, Barry A.; Neil, James C.; Edwards, Joanne; Morris, Joanna S.; Cameron, Ewan R.; Blyth, Karen

2014-01-01

RUNX2, a master regulator of osteogenesis, is oncogenic in the lymphoid lineage; however, little is known about its role in epithelial cancers. Upregulation of RUNX2 in cell lines correlates with increased invasiveness and the capacity to form osteolytic disease in models of breast and prostate cancer. However, most studies have analysed the effects of this gene in a limited number of cell lines and its role in primary breast cancer has not been resolved. Using a human tumour tissue microarray, we show that high RUNX2 expression is significantly associated with oestrogen receptor (ER)/progesterone receptor (PR)/HER2-negative breast cancers and that patients with high RUNX2 expression have a poorer survival rate than those with negative or low expression. We confirm RUNX2 as a gene that has a potentially important functional role in triple-negative breast cancer. To investigate the role of this gene in breast cancer, we made a transgenic model in which Runx2 is specifically expressed in murine mammary epithelium under the control of the mouse mammary tumour virus (MMTV) promoter. We show that ectopic Runx2 perturbs normal development in pubertal and lactating animals, delaying ductal elongation and inhibiting lobular alveolar differentiation. We also show that the Runx2 transgene elicits age-related, pre-neoplastic changes in the mammary epithelium of older transgenic animals, suggesting that elevated RUNX2 expression renders such tissue more susceptible to oncogenic changes and providing further evidence that this gene might have an important, context-dependent role in breast cancer. PMID:24626992

PTEN loss promotes intratumoral androgen synthesis and tumor microenvironment remodeling via aberrant activation of RUNX2 in castration-resistant prostate cancer

PubMed Central

Yang, Yinhui; Bai, Yang; He, Yundong; Zhao, Yu; Chen, Jiaxiang; Ma, Linlin; Pan, Yunqian; Hinten, Michael; Zhang, Jun; Karnes, R. Jeffrey; Kohli, Manish; Westendorf, Jennifer J.; Li, Benyi; Zhu, Runzhi; Huang, Haojie; Xu, Wanhai

2018-01-01

Purpose Intratumoral androgen synthesis (IAS) is a key mechanism promoting androgen receptor (AR)reactivation and anti-androgen resistance in castration-resistant prostate cancer (CRPC). However, signaling pathways driving aberrant IAS remain poorly understood. Experimental Design The effect of components of the AKT-RUNX2-osteocalcin (OCN)-GPRC6A-CREB signaling axis on expression of steroidogenesis genes CYP11A1 and CYP17A1 and testosterone level were examined in PTEN-null human PCa cell lines. Pten knockout mice were employed to examine the effect of Runx2 heterozygous deletion or abiraterone acetate (ABA), a prodrug of the CYP17A1 inhibitor abiraterone on Cyp11a1 and Cyp17a1 expression, testosterone level and tumor microenvironment (TME) remodeling in vivo. Results We uncovered that activation of the AKT-RUNX2-OCN-GPRC6A-CREB signaling axis induced expression of CYP11A1 and CYP17A1 and testosterone production in PTEN-null PCa cell lines in culture. Deletion of Runx2 in Pten homozygous knockout prostate tumors decreased Cyp11a1 and Cyp17a1 expression, testosterone level and tumor growth in castrated mice. ABA treatment also inhibited testosterone synthesis and alleviated Pten loss-induced tumorigenesis in vivo. Pten deletion induced TME remodeling, but Runx2 heterozygous deletion or ABA treatment reversed the effect of Pten loss by decreasing expression of the collagenase Mmp9. Conclusions Abnormal RUNX2 activation plays a pivotal role in PTEN loss-induced IAS and TME remodeling, suggesting that the identified signaling cascade represents a viable target for effective treatment of PTEN-null PCa including CRPC. PMID:29167276

RUNX2 Mediates Plasmacytoid Dendritic Cell Egress from the Bone Marrow and Controls Viral Immunity.

PubMed

Chopin, Michaël; Preston, Simon P; Lun, Aaron T L; Tellier, Julie; Smyth, Gordon K; Pellegrini, Marc; Belz, Gabrielle T; Corcoran, Lynn M; Visvader, Jane E; Wu, Li; Nutt, Stephen L

2016-04-26

Plasmacytoid dendritic cells (pDCs) represent a unique immune cell type that responds to viral nucleic acids through the rapid production of type I interferons. Within the hematopoietic system, the transcription factor RUNX2 is exclusively expressed in pDCs and is required for their peripheral homeostasis. Here, we show that RUNX2 plays an essential role in promoting pDC localization and function. RUNX2 is required for the appropriate expression of the integrin-mediated adhesion machinery, as well as for the down-modulation of the chemokine receptor CXCR4, which allows pDC egress into the circulation. RUNX2 also facilitates the robust response to viral infection through the control of IRF7, the major regulator of type I interferon production. Mice lacking one copy of Runx2 have reduced numbers of peripheral pDCs and IFN-α expression, which might contribute to the reported difficulties of individuals with cleidocranial dysplasia, who are haploinsufficient for RUNX2, to clear viral infections. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Loss of RUNX3 expression inhibits bone invasion of oral squamous cell carcinoma

PubMed Central

Park, Junhee; Kim, Hyun-Jeong; Kim, Ki Rim; Lee, Sun Kyoung; Kim, Hyungkeun; Park, Kwang-Kyun; Chung, Won-Yoon

2017-01-01

High recurrence and lower survival rates in patients with oral squamous cell carcinoma (OSCC) are associated with its bone invasion. We identified the oncogenic role of RUNX3 during bone invasion by OSCC. Tumor growth and the generation of osteolytic lesions were significantly inhibited in mice that were subcutaneously inoculated with RUNX3-knockdown human OSCC cells. RUNX3 knockdown enhanced TGF-β-induced growth arrest and inhibited OSCC cell migration and invasion in the absence or presence of transforming growth factor-β (TGF-β), a major growth factor abundant in the bone microenvironment. RUNX3 knockdown induced cell cycle arrest at the G1 and G2 phases and promoted G2 arrest by TGF-β in Ca9.22 OSCC cells. RUNX3 knockdown also inhibited both the basal and TGF-β-induced epithelial-to-mesenchymal transition by increasing E-cadherin expression and suppressing the nuclear translocation of β-catenin. In addition, the expression and TGF-β-mediated induction of parathyroid hormone-related protein (PTHrP), one of key osteolytic factors, was blocked in RUNX3-knockdown OSCC cells. Furthermore, treating human osteoblastic cells with conditioned medium derived from RUNX3-knockdown OSCC cells reduced the receptor activator of nuclear factor-kappaB ligand (RANKL)/osteoprotegerin ratio compared with treatment with conditioned medium from RUNX3-expressing cells. These findings indicate that RUNX3 expression in OSCC cells contributes to their bone invasion and the resulting osteolysis by inducing their malignant behaviors and production of osteolytic factors. RUNX3 alone or in combination with TGF-β and PTHrP may be a useful predictive biomarker and therapeutic target for bone invasion by oral cancer. PMID:28030842

Loss of RUNX3 expression inhibits bone invasion of oral squamous cell carcinoma.

PubMed

Park, Junhee; Kim, Hyun-Jeong; Kim, Ki Rim; Lee, Sun Kyoung; Kim, Hyungkeun; Park, Kwang-Kyun; Chung, Won-Yoon

2017-02-07

High recurrence and lower survival rates in patients with oral squamous cell carcinoma (OSCC) are associated with its bone invasion. We identified the oncogenic role of RUNX3 during bone invasion by OSCC. Tumor growth and the generation of osteolytic lesions were significantly inhibited in mice that were subcutaneously inoculated with RUNX3-knockdown human OSCC cells. RUNX3 knockdown enhanced TGF-β-induced growth arrest and inhibited OSCC cell migration and invasion in the absence or presence of transforming growth factor-β (TGF-β), a major growth factor abundant in the bone microenvironment. RUNX3 knockdown induced cell cycle arrest at the G1 and G2 phases and promoted G2 arrest by TGF-β in Ca9.22 OSCC cells. RUNX3 knockdown also inhibited both the basal and TGF-β-induced epithelial-to-mesenchymal transition by increasing E-cadherin expression and suppressing the nuclear translocation of β-catenin. In addition, the expression and TGF-β-mediated induction of parathyroid hormone-related protein (PTHrP), one of key osteolytic factors, was blocked in RUNX3-knockdown OSCC cells. Furthermore, treating human osteoblastic cells with conditioned medium derived from RUNX3-knockdown OSCC cells reduced the receptor activator of nuclear factor-kappaB ligand (RANKL)/osteoprotegerin ratio compared with treatment with conditioned medium from RUNX3-expressing cells. These findings indicate that RUNX3 expression in OSCC cells contributes to their bone invasion and the resulting osteolysis by inducing their malignant behaviors and production of osteolytic factors. RUNX3 alone or in combination with TGF-β and PTHrP may be a useful predictive biomarker and therapeutic target for bone invasion by oral cancer.

The Bone-specific Expression of Runx2 Oscillates during the Cell Cycle to Support a G1-related Antiproliferative Function in Osteoblasts*

PubMed Central

Galindo, Mario; Pratap, Jitesh; Young, Daniel W.; Hovhannisyan, Hayk; Im, Hee-Jeong; Choi, Je-Yong; Lian, Jane B.; Stein, Janet L.; Stein, Gary S.; van Wijnen, Andre J.

2010-01-01

The Runx2 (CBFA1/AML3/PEBP2αA) transcription factor promotes skeletal cell differentiation, but it also has a novel cell growth regulatory activity in osteoblasts. We addressed here whether Runx2 activity is functionally linked to cell cycle-related mechanisms that control normal osteoblast proliferation and differentiation. We found that the levels of Runx2 gene transcription, mRNA and protein, are each up-regulated with cessation of cell growth (i.e. G0/G1 transition) in preconfluent MC3T3 osteoblastic cells that do not yet express mature bone phenotypic gene expression. Cell growth regulation of Runx2 is also observed in primary calvarial osteoblasts and other osteoblastic cells with relatively normal cell growth characteristics, but not in osteosarcoma cells (e.g. SAOS-2 and ROS17/2.8). Runx2 levels are cell cycle-regulated in MC3T3 cells with respect to the G1/S and M/G1 transitions: expression oscillates from maximal levels during early G1 to minimal levels during early S phase and mitosis. However, in normal or immortalized (e.g. ATDC5) chondrocytic cells, Runx2 expression is suppressed during quiescence, and Runx2 levels are not regulated during G1 and S phase in ATDC5 cells. Antisense or small interfering RNA-mediated reduction of the low physiological levels of Runx2 in proliferating MC3T3 cells does not accelerate cell cycle progression. However, forced expression of Runx2 suppresses proliferation of MC3T3 preosteoblasts or C2C12 mesenchymal cells which have osteogenic potential. Forced elevation of Runx2 in synchronized MC3T3 cells causes a delay in G1. We propose that Runx2 levels and function are biologically linked to a cell growth-related G1 transition in osteoblastic cells. PMID:15781466

Heparin-binding EGF-like growth factor and miR-1192 exert opposite effect on Runx2-induced osteogenic differentiation.

PubMed

Yu, S; Geng, Q; Ma, J; Sun, F; Yu, Y; Pan, Q; Hong, A

2013-10-17

Osteoblast differentiation is a pivotal event in bone formation. Runt-related transcription factor-2 (Runx2) is an essential factor required for osteoblast differentiation and bone formation. However, the underlying mechanism of Runx2-regulated osteogenic differentiation is still unclear. Here, we explored the corresponding mechanism using the C2C12/Runx2(Dox) subline, which expresses Runx2 in response to doxycycline (Dox). We found that Runx2-induced osteogenic differentiation of C2C12 cells results in a sustained decrease in the expression of heparin-binding EGF-like growth factor (HB-EGF), a member of the epidermal growth factor (EGF) family. Forced expression of HB-EGF or treatment with HB-EGF is capable of reducing the expression of alkaline phosphatase (ALP), a defined marker of early osteoblast differentiation. HB-EGF-mediated inhibition of ALP depends upon activation of the EGFR and the downstream extracellular signal-regulated kinase, c-Jun N-terminal kinase mitogen-activated protein kinase pathways as well as phosphatidylinositol 3-kinase/Akt pathway. Runx2 specifically binds to the Hbegf promoter, suggesting that Hbegf transcription is directly inhibited by Runx2. Runx2 can upregulate miR-1192, which enhances Runx2-induced osteogenic differentiation. Moreover, miR-1192 directly targets Hbegf through translational inhibition, suggesting enhancement of Runx2-induced osteogenic differentiation by miR-1192 through the downregulation of HB-EGF. Taken together, our results suggest that Runx2 induces osteogenic differentiation of C2C12 cells by inactivating HB-EGF-EGFR signaling through the downregulation of HB-EGF via both transcriptional and post-transcriptional mechanisms.

Heparin-binding EGF-like growth factor and miR-1192 exert opposite effect on Runx2-induced osteogenic differentiation

PubMed Central

Yu, S; Geng, Q; Ma, J; Sun, F; Yu, Y; Pan, Q; Hong, A

2013-01-01

Osteoblast differentiation is a pivotal event in bone formation. Runt-related transcription factor-2 (Runx2) is an essential factor required for osteoblast differentiation and bone formation. However, the underlying mechanism of Runx2-regulated osteogenic differentiation is still unclear. Here, we explored the corresponding mechanism using the C2C12/Runx2Dox subline, which expresses Runx2 in response to doxycycline (Dox). We found that Runx2-induced osteogenic differentiation of C2C12 cells results in a sustained decrease in the expression of heparin-binding EGF-like growth factor (HB-EGF), a member of the epidermal growth factor (EGF) family. Forced expression of HB-EGF or treatment with HB-EGF is capable of reducing the expression of alkaline phosphatase (ALP), a defined marker of early osteoblast differentiation. HB-EGF-mediated inhibition of ALP depends upon activation of the EGFR and the downstream extracellular signal-regulated kinase, c-Jun N-terminal kinase mitogen-activated protein kinase pathways as well as phosphatidylinositol 3-kinase/Akt pathway. Runx2 specifically binds to the Hbegf promoter, suggesting that Hbegf transcription is directly inhibited by Runx2. Runx2 can upregulate miR-1192, which enhances Runx2-induced osteogenic differentiation. Moreover, miR-1192 directly targets Hbegf through translational inhibition, suggesting enhancement of Runx2-induced osteogenic differentiation by miR-1192 through the downregulation of HB-EGF. Taken together, our results suggest that Runx2 induces osteogenic differentiation of C2C12 cells by inactivating HB-EGF-EGFR signaling through the downregulation of HB-EGF via both transcriptional and post-transcriptional mechanisms. PMID:24136232

[Effects of sika pilose antler type collagen on ROS1728 cell and its molecular mechanism].

PubMed

Wang, Yan-Shuang; Luo, Su; Zhang, Da-Fang; Qu, Xiao-Bo; Li, Feng

2016-09-01

In this paper, effect and molecular mechanism of sika pilose antler type I collagen(SPC-I) of ROS1728 cell were explored. For the SPC-I provides the theory basis for the treatment of osteoporosis. The adherent method was used to cultivate rat osteosarcoma osteogenesis sample cell line ROS1728. The effect of SPC-I on ROS1728 cells proliferation was tested by CCK-8 method. Runx2, osernix, ALP, Coll-I, OC osteogenesis related genes expression was tested by RT-PCR, and Runx2 protein expression was tested by Western-bolt. Results showed that 5 g•L ⁻¹ SPC-I could inhibit ROS1728 cell proliferation, and significantly promote the expression of ROS1728 cell specific transcription factor Runx2 and osterix mRNA, Runx2 protein and marker gene ALP, Coll-I, OC mRNA expression(P<0.01). 2.5 g•L ⁻¹ and 10 g•L ⁻¹ SPC-I could significantly inhibit the ROS1728 cell proliferation(P<0.01), and inhibit the expression of related genes. In conclusion, 5 g•L ⁻¹ SPC-I could inhibit ROS1728 cell proliferation, obviously enhance ROS1728 cell function, promote ROS1728 cell differentiation, maturation. Copyright© by the Chinese Pharmaceutical Association.

MiR-18a increased the permeability of BTB via RUNX1 mediated down-regulation of ZO-1, occludin and claudin-5.

PubMed

Miao, Yin-Sha; Zhao, Ying-Yu; Zhao, Li-Ni; Wang, Ping; Liu, Yun-Hui; Ma, Jun; Xue, Yi-Xue

2015-01-01

The purposes of this study were to investigate the possible molecular mechanisms of miR-18a regulating the permeability of blood-tumor barrier (BTB) via down-regulated expression and distribution of runt-related transcription factor 1 (RUNX1). An in vitro BTB model was established with hCMEC/D3 cells and U87MG cells to obtain glioma vascular endothelial cells (GECs). The endogenous expressions of miR-18a and RUNX1 were converse in GECs. The overexpression of miR-18a significantly impaired the integrity and increased the permeability of BTB, which respectively were detected by TEER and HRP flux assays, accompanied by down-regulated mRNA and protein expressions and distributions of ZO-1, occludin and claudin-5 in GECs. Dual-luciferase reporter assay was carried out and revealed RUNX1 is a target gene of miR-18a. Meanwhile, mRNA and protein expressions and distribution of RUNX1 were downregulated by miR-18a. Most important, miR-18a and RUNX1 could reversely regulate the permeability of BTB as well as the expressions and distributions of ZO-1, occludin and claudin-5. Finally, chromatin immunoprecipitation verified that RUNX1 interacted with "TGGGGT" DNA sequence in promoter region of ZO-1, occludin and claudin-5 respectively. Taken together, our present study indicated that miR-18a increased the permeability of BTB via RUNX1 mediated down-regulation of tight junction related proteins ZO-1, occludin and claudin-5, which would attract more attention to miR-18a and RUNX1 as potential targets of drug delivery across BTB and provide novel strategies for glioma treatment. Copyright © 2014 Elsevier Inc. All rights reserved.

MicroRNA-194 promotes osteoblast differentiation via downregulating STAT1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Jun; He, Xijing; Wei, Wenzhi

Osteoblast differentiation is a vital process in maintaining bone homeostasis in which various transcriptional factors, signaling molecules, and microRNAs (miRNAs) are involved. Recently, signal transducer and activator of transcription 1 (STAT1) has been found to play an important role in regulating osteoblast differentiation. Here, we identified that STAT1 expression was regulated by miR-194. Using mouse bone mesenchymal stem cells (BMSCs), we found that miR-194 expression was significantly increased following osteoblast differentiation induction. Overexpression of miR-194 by lentivirus-mediated gene transfer markedly increased osteoblast differentiation, whereas inhibition of miR-194 significantly suppressed osteoblast differentiation of BMSCs. Using a dual-luciferase reporter assay, a directmore » interaction between miR-194 and the 3′-untranslated region (UTR) of STAT1 was confirmed. Additionally, miR-194 regulated mRNA and protein expression of STAT1 in BMSCs. Further analysis showed that miR-194 overexpression promoted the nuclear translocation of runt-related transcription factor 2 (Runx2), which is critical for osteoblast differentiation. In contrast, inhibition of miR-194 blocked the nuclear translocation of Runx2. Moreover, overexpression of STAT1 significantly blocked Runx2 nuclear translocation and osteoblast differentiation mediated by miR-194 overexpression. Taken together, our data suggest that miR-194 regulates osteoblast differentiation through modulating STAT1-mediated Runx2 nuclear translocation. - Highlights: • Overexpression of miR-194 significantly increased osteoblast differentiation. • miR-194 directly targeted the 3′- UTR of STAT1. • miR-194 regulated the expression of STAT1. • Overexpression of miR-194 promoted the nuclear translocation of Runx2.« less

The flavonoid fisetin promotes osteoblasts differentiation through Runx2 transcriptional activity.

PubMed

Léotoing, Laurent; Davicco, Marie-Jeanne; Lebecque, Patrice; Wittrant, Yohann; Coxam, Véronique

2014-06-01

Flavonoids represent a group of polyphenolic compounds commonly found in daily nutrition with proven health benefits. Among this group, the flavonol fisetin has been previously shown to protect bone by repressing osteoclast differentiation. In the present study, we investigated the role of fisetin in regulating osteoblasts physiology. In vivo mice treated with LPSs exhibited osteoporosis features associated with a dramatic repression of osteoblast marker expression. In this model, inhibition of osteocalcin and type I collagen alpha 1 transcription was partially countered by a daily consumption of fisetin. Interestingly, in vitro, fisetin promoted both osteoblast alkaline phosphatase activity and mineralization process. To decipher how fisetin may exert its positive effect on osteoblastogenesis, we analyzed its ability to control the runt-related transcription factor 2 (Runx2), a key organizer in developing and maturing osteoblasts. While fisetin did not impact Runx2 mRNA and protein levels, it upregulated its transcriptional activity. Actually, fisetin stimulated the luciferase activity of a reporter plasmid driven by the osteocalcin gene promoter that contains Runx2 binding sites and promoted the mRNA expression of osteocalcin and type I collagen alpha 1 targets. Bone sparing properties of fisetin also rely on its positive influence on osteoblast differentiation and activity. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A role for Runx transcription factor signaling in dorsal root ganglion sensory neuron diversification.

PubMed

Kramer, Ina; Sigrist, Markus; de Nooij, Joriene C; Taniuchi, Ichiro; Jessell, Thomas M; Arber, Silvia

2006-02-02

Subpopulations of sensory neurons in the dorsal root ganglion (DRG) can be characterized on the basis of sensory modalities that convey distinct peripheral stimuli, but the molecular mechanisms that underlie sensory neuronal diversification remain unclear. Here, we have used genetic manipulations in the mouse embryo to examine how Runx transcription factor signaling controls the acquisition of distinct DRG neuronal subtype identities. Runx3 acts to diversify an Ngn1-independent neuronal cohort by promoting the differentiation of proprioceptive sensory neurons through erosion of TrkB expression in prospective TrkC+ sensory neurons. In contrast, Runx1 controls neuronal diversification within Ngn1-dependent TrkA+ neurons by repression of neuropeptide CGRP expression and controlling the fine pattern of laminar termination in the dorsal spinal cord. Together, our findings suggest that Runx transcription factor signaling plays a key role in sensory neuron diversification.

Epigenetic changes in localized gastric cancer: the role of RUNX3 in tumor progression and the immune microenvironment

PubMed Central

Ibarrola-Villava, Maider; Peña-Chilet, María; Mongort, Cristina; Martinez-Ciarpaglini, Carolina; Navarro, Lara; Gambardella, Valentina; Castillo, Josefa; Roselló, Susana; Navarro, Samuel; Ribas, Gloria; Cervantes, Andrés

2016-01-01

Gastric cancer (GC) pathogenesis involves genetic, epigenetic and environmental factors. Epigenetic alterations, such as DNA methylation are considered pivotal in the inactivation of tumor-related genes. We assessed a methylation panel of 5 genes to study their association to GC progression and microsatellite instability (MSI), and studied the role of RUNX3 in GC pathogenesis and the tumor immune microenvironment. The methylation status of 47 promoter-CpG islands was studied through MALDI-TOF mass spectrometry analysis in 35 Microsatellite stable (MSS) GC, 26 MSI, and 18 cancer-free samples (CFS), and 6 MSS GC and 4 MSI GC cell lines. We also studied RUNX3 expression by immunohistochemistry (IHC) in 40 samples, and validated differences in methylation levels between tumor, normal, and immune tissue in 14 additional samples. Unsupervised hierarchical clustering of methylation levels revealed no distinct subgroups between MSI and MSS samples or cell lines. CFSs clustered together showing higher levels of RUNX3 methylation compared to GC samples. RUNX3 showed protein silencing in cancer and normal mucosa, compared to inflammatory peritumoural infiltrate in almost all cases, showing a non-lymphocytic predominant pattern and being correlated with epigenetic silencing. Our results show aberrant promoter's methylation in APC, CDH1, CDKN2A, MLH1 and RUNX3 associated with GC, as well as a non-lymphocytic predominant infiltrate with high expression of RUNX3. Deep study of RUNX3 inflammation signaling could help in understanding inflammation and immune activation in the tumor microenvironment. PMID:27566570

Alteration of runt-related transcription factor 3 gene expression and biologic behavior of esophageal carcinoma TE-1 cells after 5-azacytidine intervention.

PubMed

Wang, Shuai; Liu, Hong; Akhtar, Javed; Chen, Hua-Xia; Wang, Zhou

2013-01-01

5-Azacytidine (5-azaC) was originally identified as an anticancer drug (NSC102876) which can cause hypomethylation of tumor suppressor genes. To assess its effects on runt-related transcription factor 3 (RUNX3), expression levels and the promoter methylation status of the RUNX3 gene were assessed. We also investigated alteration of biologic behavior of esophageal carcinoma TE-1 cells. MTT assays showed 5-azaC inhibited the proliferation of TE-1 cells in a time and dose-dependent way. Although other genes could be demethylated after 5-azaC intervention, we focused on RUNX3 gene in this study. The expression level of RUNX3 mRNA increased significantly in TE-1 cells after treatment with 5-azaC at hypotoxic levels. RT-PCR showed 5-azaC at 50 μM had the highest RUNX3-induction activity. Methylation-specific PCR indicated that 5-azaC induced RUNX3 expression through demethylation. Migration and invasion of TE-1 cells were inhibited by 5-azaC, along with growth of Eca109 xenografts in nude mice. In conclusion, we demonstrate that the RUNX3 gene can be reactivated by the demethylation reagent 5-azaC, which inhibits the proliferation, migration and invasion of esophageal carcinoma TE-1 cells.

Characterization of RUNX1T1, an Adipogenesis Regulator in Ovine Preadipocyte Differentiation.

PubMed

Deng, Kaiping; Ren, Caifang; Liu, Zifei; Gao, Xiaoxiao; Fan, Yixuan; Zhang, Guomin; Zhang, Yanli; Ma, Ei-Samahy; Wang, Feng; You, Peihua

2018-04-26

Runt-related transcription factor 1 translocation partner 1 (RUNX1T1), a potential novel regulator of adipogenesis, exists in two splice variants: a long (RUNX1T1-L) and a short (RUNX1T1-S) isoform. However, there is no data showing the existence of RUNX1T1 in ovine subcutaneous fat at different stages of developmental and its role on ovine adipogenesis. Therefore, the objectives of this study were to evaluate the presence of RUNX1T1 in subcutaneous fat of five-day-old to 24-month-old sheep and to investigate the role of RUNX1T1 in ovine adipogenesis. In this study, we detected a 1829 bp cDNA fragment of RUNX1T1 which contains a 1815 bp coding sequence that encodes 602-amino acid and 14 bp of 5' untranslated region, respectively. The amino acid sequence of RUNX1T1 has 31.18⁻94.21% homology with other species' protein sequences. During fat development, the RUNX1T1 protein expression was higher in subcutaneous fat of 24-month-old Hu sheep. In addition, the expression of RUNX1T1-L mRNA decreased first, then subsequently increased during ovine preadipocyte differentiation. Knockdown of RUNX1T1-L in ovine preadipocytes promoted preadipocyte differentiation and lipid accumulation. Taken together, our data suggests that RUNX1T1 is an important functional molecule in adipogenesis. Moreover, it showed for the first time that RUNX1T1-L was negatively correlated with the ovine preadipocyte differentiation.

HCV core protein promotes hepatocyte proliferation and chemoresistance by inhibiting NR4A1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tan, Yongsheng, E-mail: yongshengtanwhu@126.com; Li, Yan, E-mail: liyansd2@163.com

This study investigated the effect of HCV core protein on the proliferation of hepatocytes and hepatocellular carcinoma cells (HCC), the influence of HCV core protein on HCC apoptosis induced by the chemotherapeutic agent cisplatin, and the mechanism through which HCV core protein acts as a potential oncoprotein in HCV-related HCC by measuring the levels of NR4A1 and Runt-related transcription factor 3 (RUNX3), which are associated with tumor suppression and chemotherapy resistance. In the present study, PcDNA3.1-core and RUNX3 siRNA were transfected into LO2 and HepG2 cells using Lipofectamine 2000. LO2-core, HepG2-core, LO2-RUNX3 {sup low} and control cells were treated withmore » different concentrations of cisplatin for 72 h, and cell proliferation and apoptosis were assayed using the CellTiter 96{sup ®}Aqueous Non-Radioactive Cell Proliferation Assay Kit. Western blot and real time PCR analyses were used to detect NR4A1, RUNX3, smad7, Cyclin D1 and BAX. Confocal microscopy was used to determine the levels of NR4A1 in HepG2 and HepG2-core cells. The growth rate of HepG2-core cells was considerably greater than that of HepG2 cells. HCV core protein increased the expression of cyclin D1 and decreased the expressions of NR4A1 and RUNX3. In LO2 – RUNX3 {sup low}, the rate of cell proliferation and the level of cisplatin resistance were the same as in the LO2 -core. These results suggest that HCV core protein decreases the sensitivity of hepatocytes to cisplatin by inhibiting the expression of NR4A1 and promoting the expression of smad7, which negatively regulates the TGF-β pathway. This effect results in down regulation of RUNX3, a target of the TGF-β pathway. Taken together, these findings indicate that in hepatocytes, HCV core protein increases drug resistance and inhibits cell apoptosis by inhibiting the expressions of NR4A1 and RUNX3. - Highlights: • HCV core protein inhibits HepG2 cell sensitivity to cisplatin. • Core expression in HepG2 decreases expression of NR4A1. • Core protein increases the expression of smad7 in hepatocytes. • Core protein inhibits HepG2 cells apoptosis induced by cisplatin.« less

Smurf1 plays a role in EGF inhibition of BMP2-induced osteogenic differentiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Hye-Lim; Park, Hyun-Jung; Kwon, Arang

2014-05-01

It has been demonstrated that epidermal growth factor (EGF) plays a role in supporting the proliferation of bone marrow stromal cells in bone but inhibits their osteogenic differentiation. However, the mechanism underlying EGF inhibition of osteoblast differentiation remains unclear. Smurf1 is an E3 ubiquitin ligase that targets Smad1/5 and Runx2, which are critical transcription factors for bone morphogenetic protein 2 (BMP2)-induced osteoblast differentiation. In this study, we investigated the effect of EGF on the expression of Smurf1, and the role of Smurf1 in EGF inhibition of osteogenic differentiation using C2C12 cells, a murine myoblast cell line. EGF increased Smurf1 expression,more » which was blocked by inhibiting the activity of either JNK or ERK. Chromatin immunoprecipitation and Smurf1 promoter assays demonstrated that c-Jun and Runx2 play roles in the EGF induction of Smurf1 transcription. EGF suppressed BMP2-induced expression of osteogenic marker genes, which were rescued by Smurf1 knockdown. EGF downregulated the protein levels of Runx2 and Smad1 in a proteasome-dependent manner. EGF decreased the transcriptional activity of Runx2 and Smurf1, which was partially rescued by Smurf1 silencing. Taken together, these results suggest that EGF increases Smurf1 expression via the activation of JNK and ERK and the subsequent binding of c-Jun and Runx2 to the Smurf1 promoter and that Smurf1 mediates the inhibitory effect of EGF on BMP2-induced osteoblast differentiation. - Highlights: • EGF increases the expression level of Smurf1 in mesenchymal precursor cells. • EGF reduces the protein levels and transcriptional activity of Runx2 and Smad1. • EGF suppresses BMP2-induced osteogenic differentiation, which is rescued by Smurf1 knockdown.« less

RUNX1 positively regulates the ErbB2/HER2 signaling pathway through modulating SOS1 expression in gastric cancer cells.

PubMed

Mitsuda, Yoshihide; Morita, Ken; Kashiwazaki, Gengo; Taniguchi, Junichi; Bando, Toshikazu; Obara, Moeka; Hirata, Masahiro; Kataoka, Tatsuki R; Muto, Manabu; Kaneda, Yasufumi; Nakahata, Tatsutoshi; Liu, Pu Paul; Adachi, Souichi; Sugiyama, Hiroshi; Kamikubo, Yasuhiko

2018-04-23

The dual function of runt-related transcriptional factor 1 (RUNX1) as an oncogene or oncosuppressor has been extensively studied in various malignancies, yet its role in gastric cancer remains elusive. Up-regulation of the ErbB2/HER2 signaling pathway is frequently-encountered in gastric cancer and contributes to the maintenance of these cancer cells. This signaling cascade is partly mediated by son of sevenless homolog (SOS) family, which function as adaptor proteins in the RTK cascades. Herein we report that RUNX1 regulates the ErbB2/HER2 signaling pathway in gastric cancer cells through transactivating SOS1 expression, rendering itself an ideal target in anti-tumor strategy toward this cancer. Mechanistically, RUNX1 interacts with the RUNX1 binding DNA sequence located in SOS1 promoter and positively regulates it. Knockdown of RUNX1 led to the decreased expression of SOS1 as well as dephosphorylation of ErbB2/HER2, subsequently suppressed the proliferation of gastric cancer cells. We also found that our novel RUNX inhibitor (Chb-M') consistently led to the deactivation of the ErbB2/HER2 signaling pathway and was effective against several gastric cancer cell lines. Taken together, our work identified a novel interaction of RUNX1 and the ErbB2/HER2 signaling pathway in gastric cancer, which can potentially be exploited in the management of this malignancy.

C/EBPβ regulates homeostatic and oncogenic gastric cell proliferation.

PubMed

Regalo, Goncalo; Förster, Susann; Resende, Carlos; Bauer, Bianca; Fleige, Barbara; Kemmner, Wolfgang; Schlag, Peter M; Meyer, Thomas F; Machado, José C; Leutz, Achim

2016-12-01

Cancer of the stomach is among the leading causes of death from cancer worldwide. The transcription factor C/EBPβ is frequently overexpressed in gastric cancer and associated with the suppression of the differentiation marker TFF1. We show that the murine C/EBPβ knockout stomach displays unbalanced homeostasis and reduced cell proliferation and that tumorigenesis of human gastric cancer xenograft is inhibited by knockdown of C/EBPβ. Cross-species comparison of gene expression profiles between C/EBPβ-deficient murine stomach and human gastric cancer revealed a subset of tumors with a C/EBPβ signature. Within this signature, the RUNX1t1 tumor suppressor transcript was down-regulated in 38 % of gastric tumor samples. The RUNX1t1 promoter was frequently hypermethylated and ectopic expression of RUNX1t1 in gastric cancer cells inhibited proliferation and enhanced TFF1 expression. These data suggest that the tumor suppressor activity of both RUNX1t1 and TFF1 are mechanistically connected to C/EBPβ and that cross-regulation between C/EBPβ-RUNX1t1-TFF1 plays an important role in gastric carcinogenesis. C/EBPβ controls proliferation and differentiation balance in the stomach. Homeostatic differentiation/proliferation balance is altered in gastric cancer. RUNX1t1 is a C/EBPβ-associated tumor suppressor. RUNX1t1 negatively regulates C/EBPβ pro-oncogenic functions.

Autonomous feedback loop of RUNX1-p53-CBFB in acute myeloid leukemia cells.

PubMed

Morita, Ken; Noura, Mina; Tokushige, Chieko; Maeda, Shintaro; Kiyose, Hiroki; Kashiwazaki, Gengo; Taniguchi, Junichi; Bando, Toshikazu; Yoshida, Kenichi; Ozaki, Toshifumi; Matsuo, Hidemasa; Ogawa, Seishi; Liu, Pu Paul; Nakahata, Tatsutoshi; Sugiyama, Hiroshi; Adachi, Souichi; Kamikubo, Yasuhiko

2017-11-30

Although runt-related transcription factor 1 (RUNX1) and its associating core binding factor-β (CBFB) play pivotal roles in leukemogenesis, and inhibition of RUNX1 has now been widely recognized as a novel strategy for anti-leukemic therapies, it has been elusive how leukemic cells could acquire the serious resistance against RUNX1-inhibition therapies and also whether CBFB could participate in this process. Here, we show evidence that p53 (TP53) and CBFB are sequentially up-regulated in response to RUNX1 depletion, and their mutual interaction causes the physiological resistance against chemotherapy for acute myeloid leukemia (AML) cells. Mechanistically, p53 induced by RUNX1 gene silencing directly binds to CBFB promoter and stimulates its transcription as well as its translation, which in turn acts as a platform for the stabilization of RUNX1, thereby creating a compensative RUNX1-p53-CBFB feedback loop. Indeed, AML cells derived from relapsed cases exhibited higher CBFB expression levels compared to those from primary AML cells at diagnosis, and these CBFB expressions were positively correlated to those of p53. Our present results underscore the importance of RUNX1-p53-CBFB regulatory loop in the development and/or maintenance of AML cells, which could be targeted at any sides of this triangle in strategizing anti-leukemia therapies.

Restoration of miR-1305 relieves the inhibitory effect of nicotine on periodontal ligament-derived stem cell proliferation, migration, and osteogenic differentiation.

PubMed

Chen, Zhuo; Liu, Hui-Li

2017-04-01

Nicotine hinders the regenerative potentials of human periodontal ligament-derived stem cells (PDLSCs) and delays the healing process of periodontal diseases, but the underlying mechanism remains unclear. miR-1305 upregulation and its potential target RUNX2 downregulation exist in the PDLSCs exposed to nicotine. In this study, we aimed to investigate whether nicotine inhibits PDLSC proliferation, migration, and osteogenic differentiation by increasing miR-1305 level and decreasing RUNX2 level. Quantitative real-time PCR (qRT-PCR) and Western blot assays were performed to detect the expression levels of miR-1305 and RUNX2 in the PDLSCs exposed to nicotine, respectively. PDLSCs with miR-1305 overexpression, low expression, or RUNX2 overexpression were constructed by lipofectin transfection. MTT, migration, and Western blot assays were applied to assess the effect of miR-1305 on PDLSC proliferation, migration, and osteogenic differentiation, respectively. Target prediction and luciferase reporter assays were performed to investigate the targets of miR-1305. Nicotine promoted miR-1305 expression and inhibited RUNX2 expression in PDLSCs. Cell proliferation, migration, and differentiation detection showed that nicotine suppressed proliferation, migration, and osteogenic differentiation of PDLSCs, and restoration of miR-1305 relieved the inhibitory effect of nicotine on PDLSCs. Moreover, we identified and validated that RUNX2 was a direct target of miR-1305, and upregulation of RUNX2 had similar effects with the downregulation of miR-1305 on relieving the inhibitory effect of nicotine on PDLSCs. Nicotine suppresses proliferation, migration, and osteogenic differentiation of PDLSCs, and restoration of miR-1305 relieves the inhibitory effect of nicotine on PDLSCs depending on its target RUNX2. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Role of Ox-PAPCs in the Differentiation of Mesenchymal Stem Cells (MSCs) and Runx2 and PPARγ2 Expression in MSCs-Like of Osteoporotic Patients

PubMed Central

Valenti, Maria Teresa; Garbin, Ulisse; Pasini, Andrea; Zanatta, Mirko; Stranieri, Chiara; Manfro, Stefania; Zucal, Chiara; Dalle Carbonare, Luca

2011-01-01

Background Mesenchymal stem cells (MSCs) can differentiate into osteoblasts and adipocytes and conditions causing bone loss may induce a switch from the osteoblast to adipocyte lineage. In addition, the expression of Runx2 and the PPARγ2 transcription factor genes is essential for cellular commitment to an osteogenic and adipogenic differentiation, respectively. Modified lipoproteins derived from the oxidation of arachidonate-containing phospholipids (ox-PAPCs: POVPC, PGPC and PEIPC) are considered important factors in atherogenesis. Methodology We investigated the effect of ox-PAPCs on osteogenesis and adipogenesis in human mesenchymal stem cells (hMSCs). In particular, we analyzed the transcription factor Runx2 and the PPARγ2 gene expression during osteogenic and adipogenic differentiation in absence and in presence of ox-PAPCs. We also analyzed gene expression level in a panel of osteoblastic and adipogenic differentiation markers. In addition, as circulating blood cells can be used as a “sentinel” that responds to changes in the macro- or micro-environment, we analyzed the Runx2 and the PPARγ2 gene expression in MSCs-like and ox-PAPC levels in serum of osteoporotic patients (OPs). Finally, we examined the effects of sera obtained from OPs in hMSCs comparing the results with age-matched normal donors (NDs). Principal findings Quantitative RT-PCR demonstrated that ox-PAPCs enhanced PPARγ2 and adipogenic gene expression and reduced Runx2 and osteoblast differentiation marker gene expression in differentiating hMSCs. In OPs, ox-PAPC levels and PPARγ2 expression were higher than in NDs, whereas Runx2 was lower than in ND circulant MSCs-like. Conclusions Ox-PAPCs affect the osteogenic differentiation by promoting adipogenic differentiation and this effect may appear involved in bone loss in OPs. PMID:21674037

FGF2 Stimulation of the Pyrophosphate-Generating Enzyme, PC-1, in Pre-Osteoblast Cells Is Mediated by RUNX2

PubMed Central

Hatch, Nan E; Li, Yan; Franceschi, Renny T

2009-01-01

Pyrophosphate is an established inhibitor of hydroxyapatite deposition and crystal growth, yet when hydrolyzed into phosphate, it becomes a substrate for hydroxyapatite deposition. Pyrophosphate-generating enzyme (PC-1), Ank, and tissue nonspecific alkaline phosphatase (Tnap) are three factors that regulate extracellular pyrophosphate levels through its generation, transport, and hydrolysis. We previously showed that fibroblast growth factor 2 (FGF2) induces PC-1 and Ank while inhibiting Tnap expression and mineralization in MC3T3E1(C4) calvarial pre-osteoblast cells. In this study, we showed similar FGF2 regulation of these genes in primary pre-osteoblast cultures. In contrast to Ank and Tnap that are regulated by FGF2 in multiple cell types, we found regulation of PC-1 to be selective to pre-osteoblastic cells and to require the osteoblast-related transcription factor, Runx2. Specifically, FGF2 was unable to induce PC-1 expression in Runx2-negative nonbone cells or in calvarial cells from Runx2-deficient mice. Transfection of these cells with a Runx2 expression vector restored FGF2 responsiveness. FGF2 was also shown to stimulate recruitment of Runx2 to the endogenous PC-1 promoter in MC3T3E1(C4) cells, as measured by chromatin immunoprecipitation. Taken together, our results establish that FGF2 is a specific inducer of PC-1 in pre-osteoblast cells and that FGF2 induces PC-1 expression through a mechanism involving Runx2. PMID:19049325

Stromal-Cell-Derived Factor (SDF) 1-Alpha Overexpression Promotes Bone Regeneration by Osteogenesis and Angiogenesis in Osteonecrosis of the Femoral Head.

PubMed

Yang, Fan; Xue, Feng; Guan, Junjie; Zhang, Zeng; Yin, Jimin; Kang, Qingling

2018-05-07

Osteonecrosis of the femoral head (ONFH) is a devastating orthopedic disease. Previous studies suggested that stromal-cell-derived factor (SDF)-1 was involved in osteogenesis and angiogenesis. However, whether SDF-1 potentiates the angiogenesis and osteogenesis of bone marrow-derived stromal stem cells (BMSCs) in ONFH is not clear. BMSCs were transfected with green fluorescent protein (GFP) or the fusion gene encoding GFP and SDF-1α, and transgenic efficacy was monitored by immunofluorescence. The expression of SDF-1α, runt-related transcription factor 2 (Runx2, osteocalcin (OCN), and alkaline phosphatase (ALP) at the mRNA level was measured by real-time polymerase chain reactions (RT-PCR). The expression of SDF-1α, Runx2, OCN, and p-Smad1/5 were measured at the protein level by Western blot. Transwell migration assay and tube formation assay were utilized to detect the angiogenesis in vitro, whereas the in vivo angiogenesis was monitored by angiography. Immunohistological staining and micro-CT scanning were conducted to assess the histological changes in morphology. In vitro, SDF-1α overexpression in BMSCs promoted osteogenic differentiation and upregulated the expression of osteogenic-related proteins, such as ALP, Runx2, OCN, and p-Smadl/5. In the methylprednisolone induced ONFH rat model used in our investigation, the overexpression of SDF-1α in BMSCs promoted significantly more bone regeneration and the expression of OCN and Runx2 as compared with the effect of vehicle overexpression. Moreover, the morphology of ONFH was ameliorated after the transplantation of BMSCs with SDF-1α overexpression. Furthermore, SDF-1α overexpression in BMSCs significantly increased osteoblastic angiogenesis as indicated by the increased tube formation ability, CD31 expression, and vessel volume. SDF-1α overexpression in BMSCs promotes bone generation as indicated by osteogenesis and angiogenesis, suggesting SDF-1α may serve as a therapeutic drug target for ONFH treatment. © 2018 The Author(s). Published by S. Karger AG, Basel.

Collagen-derived dipeptide prolyl-hydroxyproline promotes differentiation of MC3T3-E1 osteoblastic cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kimira, Yoshifumi, E-mail: kimira@josai.ac.jp; Ogura, Kana; Taniuchi, Yuri

Highlights: • Pro-Hyp did not affect MC3T3-E1 cell proliferation and matrix mineralization. • Pro-Hyp significantly increased alkaline phosphatase activity. • Pro-Hyp significantly upregulated gene expression of Runx2, Osterix, and Col1α1. - Abstract: Prolyl-hydroxyproline (Pro-Hyp) is one of the major constituents of collagen-derived dipeptides. The objective of this study was to investigate the effects of Pro-Hyp on the proliferation and differentiation of MC3T3-E1 osteoblastic cells. Addition of Pro-Hyp did not affect MC3T3-E1 cell proliferation and matrix mineralization but alkaline phosphatase activity was significantly increased. Furthermore, cells treated with Pro-Hyp significantly upregulated gene expression of Runx2, Osterix, and Col1α1. These results indicatemore » that Pro-Hyp promotes osteoblast differentiation. This study demonstrates for the first time that Pro-Hyp has a positive effect on osteoblast differentiation with upregulation of Runx2, Osterix, and Collα1 gene expression.« less

A novel role for bone-derived cells in ankylosing spondylitis: Focus on IL-23.

PubMed

Jo, Sungsin; Koo, Bon San; Lee, Bitnara; Kwon, Eunji; Lee, Young Lim; Chung, Heekyoung; Sung, Il-Hoon; Park, Ye-Soo; Kim, Tae-Hwan

2017-09-23

The main aim of this study are to explore the role of bone-derived cells (BdCs) in ankylosing spondylitis (AS) and determine the underlying molecular mechanisms of IL-23 production. Primary BdCs were isolated from diced bone of facet joints obtained during surgery from seven AS patients and seven disease control (Ct) patients. Osteoblastic activity of BdCs was assessed by measuring their alkaline phosphatase activity and by alizarin red staining. Osteoblast and endoplasmic reticulum (ER) stress-related genes were assessed by quantitative PCR, immunoblotting, immunofluorescence, and immunohistochemistry. In addition, expression of IL-23 in response to BIX (selective BIP inducer X)-induced ER stress was evaluated by qPCR and ELISA. Protein interaction and binding to IL-23 promoter were confirmed by Immunoprecipitation and Chromatin immunoprecipitation, respectively. Transcript levels of genes involved in osteoblast function, as well as of the ER stress marker were higher in the AS group than the Ct group, and elevated RUNX2, BiP and IL-23 expression were observed in the BdCs, serum, and bone biopsies from the AS group. BIX-induced ER stress stimulated osteoblastic activity and IL-23 secretion by upregulating RUNX2 expression. Furthermore, in AS BdCs, RUNX2 interacted with C/EBPβ to bind to IL-23 promoter and RUNX2 knockdown suppressed IL-23 secretion. These finding may provide a molecular mechanism involved in sustained ER stress in AS BdCs stimulates the activation of RUNX2 and C/EBPβ genes, leading to IL-23 production. Copyright © 2017 Elsevier Inc. All rights reserved.

RUNX1 and RUNX3 protect against YAP-mediated EMT, stem-ness and shorter survival outcomes in breast cancer

PubMed Central

Kulkarni, Madhura; Tan, Tuan Zea; Syed Sulaiman, Nurfarhanah Bte; Lamar, John M.; Bansal, Prashali; Cui, Jianzhou; Qiao, Yiting; Ito, Yoshiaki

2018-01-01

Hippo pathway target, YAP has emerged as an important player in solid tumor progression. Here, we identify RUNX1 and RUNX3 as novel negative regulators of oncogenic function of YAP in the context of breast cancer. RUNX proteins are one of the first transcription factors identified to interact with YAP. RUNX1 or RUNX3 expression abrogates YAP-mediated pro-tumorigenic properties of mammary epithelial cell lines in an interaction dependent manner. RUNX1 and RUNX3 inhibit YAP-mediated migration and stem-ness properties of mammary epithelial cell lines by co-regulating YAP-mediated gene expression. Analysis of whole genome expression profiles of breast cancer samples revealed significant co-relation between YAP–RUNX1/RUNX3 expression levels and survival outcomes of breast cancer patients. High RUNX1/RUNX3 expression proved protective towards YAP-dependent patient survival outcomes. High YAP in breast cancer patients’ expression profiles co-related with EMT and stem-ness gene signature enrichment. High RUNX1/RUNX3 expression along with high YAP reflected lower enrichment of EMT and stem-ness signatures. This antagonistic activity of RUNX1 and RUNX3 towards oncogenic function of YAP identified in mammary epithelial cells as well as in breast cancer expression profiles gives a novel mechanistic insight into oncogene–tumor suppressor interplay in the context of breast cancer progression. The novel interplay between YAP, RUNX1 and RUNX3 and its significance in breast cancer progression can serve as a prognostic tool to predict cancer recurrence. PMID:29581836

MIR146A inhibits JMJD3 expression and osteogenic differentiation in human mesenchymal stem cells

PubMed Central

Huszar, Jessica M.; Payne, Christopher J.

2014-01-01

Chromatin remodeling is important for cell differentiation. Histone methyltransferase EZH2 and histone demethylase JMJD3 (KDM6B) modulate levels of histone H3 lysine 27 trimethylation (H3K27me3). Interplay between the two modulators influence lineage specification in stem cells. Here, we identified microRNA MIR146A to be a negative regulator of JMJD3. In the osteogenic differentiation of human mesenchymal stem cells (hMSCs), we observed an upregulation of JMJD3 and a downregulation of MIR146A. Blocking JMJD3 activity in differentiating hMSCs reduced transcript levels of osteogenic gene RUNX2. H3K27me3 levels decreased at the RUNX2 promoter during cell differentiation. Modulation of MIR146A levels in hMSCs altered JMJD3 and RUNX2 expression and affected osteogenic differentiation. We conclude that JMJD3 promotes osteogenesis in differentiating hMSCs, with MIR146A regulating JMJD3. PMID:24726732

Mangiferin positively regulates osteoblast differentiation and suppresses osteoclast differentiation

PubMed Central

Sekiguchi, Yuusuke; Mano, Hiroshi; Nakatani, Sachie; Shimizu, Jun; Kataoka, Aya; Ogura, Kana; Kimira, Yoshifumi; Ebata, Midori; Wada, Masahiro

2017-01-01

Mangiferin is a polyphenolic compound present in Salacia reticulata. It has been reported to reduce bone destruction and inhibit osteoclastic differentiation. This study aimed to determine whether mangiferin directly affects osteoblast and osteoclast proliferation and differentiation, and gene expression in MC3T3-E1 osteoblastic cells and osteoclast-like cells derived from primary mouse bone marrow macrophage cells. Mangiferin induced significantly greater WST-1 activity, indicating increased cell proliferation. Mangiferin induced significantly increased alkaline phosphatase staining, indicating greater cell differentiation. Reverse transcription-polymerase chain reaction (RT-PCR) demonstrated that mangiferin significantly increased the mRNA level of runt-related transcription factor 2 (RunX2), but did not affect RunX1 mRNA expression. Mangiferin significantly reduced the formation of tartrate-resistant acid phosphatase-positive multinuclear cells. RT-PCR demonstrated that mangiferin significantly increased the mRNA level of estrogen receptor β (ERβ), but did not affect the expression of other osteoclast-associated genes. Mangiferin may inhibit osteoclastic bone resorption by suppressing differentiation of osteoclasts and promoting expression of ERβ mRNA in mouse bone marrow macrophage cells. It also has potential to promote osteoblastic bone formation by promoting cell proliferation and inducing cell differentiation in preosteoblast MC3T3-E1 cells via RunX2. Mangiferin may therefore be useful in improving bone disease outcomes. PMID:28627701

Mangiferin positively regulates osteoblast differentiation and suppresses osteoclast differentiation.

PubMed

Sekiguchi, Yuusuke; Mano, Hiroshi; Nakatani, Sachie; Shimizu, Jun; Kataoka, Aya; Ogura, Kana; Kimira, Yoshifumi; Ebata, Midori; Wada, Masahiro

2017-08-01

Mangiferin is a polyphenolic compound present in Salacia reticulata. It has been reported to reduce bone destruction and inhibit osteoclastic differentiation. This study aimed to determine whether mangiferin directly affects osteoblast and osteoclast proliferation and differentiation, and gene expression in MC3T3‑E1 osteoblastic cells and osteoclast‑like cells derived from primary mouse bone marrow macrophage cells. Mangiferin induced significantly greater WST‑1 activity, indicating increased cell proliferation. Mangiferin induced significantly increased alkaline phosphatase staining, indicating greater cell differentiation. Reverse transcription‑polymerase chain reaction (RT‑PCR) demonstrated that mangiferin significantly increased the mRNA level of runt‑related transcription factor 2 (RunX2), but did not affect RunX1 mRNA expression. Mangiferin significantly reduced the formation of tartrate‑resistant acid phosphatase‑positive multinuclear cells. RT‑PCR demonstrated that mangiferin significantly increased the mRNA level of estrogen receptor β (ERβ), but did not affect the expression of other osteoclast‑associated genes. Mangiferin may inhibit osteoclastic bone resorption by suppressing differentiation of osteoclasts and promoting expression of ERβ mRNA in mouse bone marrow macrophage cells. It also has potential to promote osteoblastic bone formation by promoting cell proliferation and inducing cell differentiation in preosteoblast MC3T3‑E1 cells via RunX2. Mangiferin may therefore be useful in improving bone disease outcomes.

Pulsed electromagnetic fields promote osteogenesis and osseointegration of porous titanium implants in bone defect repair through a Wnt/β-catenin signaling-associated mechanism

PubMed Central

Jing, Da; Zhai, Mingming; Tong, Shichao; Xu, Fei; Cai, Jing; Shen, Guanghao; Wu, Yan; Li, Xiaokang; Xie, Kangning; Liu, Juan; Xu, Qiaoling; Luo, Erping

2016-01-01

Treatment of osseous defects remains a formidable clinical challenge. Porous titanium alloys (pTi) have been emerging as ideal endosseous implants due to the excellent biocompatibility and structural properties, whereas inadequate osseointegration poses risks for unreliable long-term implant stability. Substantial evidence indicates that pulsed electromagnetic fields (PEMF), as a safe noninvasive method, inhibit osteopenia/osteoporosis experimentally and clinically. We herein investigated the efficiency and potential mechanisms of PEMF on osteogenesis and osseointegration of pTi in vitro and in vivo. We demonstrate that PEMF enhanced cellular attachment and proliferation, and induced well-organized cytoskeleton for in vitro osteoblasts seeded in pTi. PEMF promoted gene expressions in Runx2, OSX, COL-1 and Wnt/β-catenin signaling. PEMF-stimulated group exhibited higher Runx2, Wnt1, Lrp6 and β-catenin protein expressions. In vivo results via μCT and histomorphometry show that 6-week and 12-week PEMF promoted osteogenesis, bone ingrowth and bone formation rate of pTi in rabbit femoral bone defect. PEMF promoted femoral gene expressions of Runx2, BMP2, OCN and Wnt/β-catenin signaling. Together, we demonstrate that PEMF improve osteogenesis and osseointegration of pTi by promoting skeletal anabolic activities through a Wnt/β-catenin signaling-associated mechanism. PEMF might become a promising biophysical modality for enhancing the repair efficiency and quality of pTi in bone defect. PMID:27555216

Thrombopoietin/MPL participates in initiating and maintaining RUNX1-ETO acute myeloid leukemia via PI3K/AKT signaling

PubMed Central

Pulikkan, John Anto; Madera, Dmitri; Xue, Liting; Bradley, Paul; Landrette, Sean Francis; Kuo, Ya-Huei; Abbas, Saman; Zhu, Lihua Julie; Valk, Peter

2012-01-01

Oncogenic mutations in components of cytokine signaling pathways elicit ligand-independent activation of downstream signaling, enhancing proliferation and survival in acute myeloid leukemia (AML). The myeloproliferative leukemia virus oncogene, MPL, a homodimeric receptor activated by thrombopoietin (THPO), is mutated in myeloproliferative disorders but rarely in AML. Here we show that wild-type MPL expression is increased in a fraction of human AML samples expressing RUNX1-ETO, a fusion protein created by chromosome translocation t(8;21), and that up-regulation of Mpl expression in mice induces AML when coexpressed with RUNX1-ETO. The leukemic cells are sensitive to THPO, activating survival and proliferative responses. Mpl expression is not regulated by RUNX1-ETO in mouse hematopoietic progenitors or leukemic cells. Moreover, we find that activation of PI3K/AKT but not ERK/MEK pathway is a critical mediator of the MPL-directed antiapoptotic function in leukemic cells. Hence, this study provides evidence that up-regulation of wild-type MPL levels promotes leukemia development and maintenance through activation of the PI3K/AKT axis, and suggests that inhibitors of this axis could be effective for treatment of MPL-positive AML. PMID:22613795

Thrombopoietin/MPL participates in initiating and maintaining RUNX1-ETO acute myeloid leukemia via PI3K/AKT signaling.

PubMed

Pulikkan, John Anto; Madera, Dmitri; Xue, Liting; Bradley, Paul; Landrette, Sean Francis; Kuo, Ya-Huei; Abbas, Saman; Zhu, Lihua Julie; Valk, Peter; Castilla, Lucio Hernán

2012-07-26

Oncogenic mutations in components of cytokine signaling pathways elicit ligand-independent activation of downstream signaling, enhancing proliferation and survival in acute myeloid leukemia (AML). The myeloproliferative leukemia virus oncogene, MPL, a homodimeric receptor activated by thrombopoietin (THPO), is mutated in myeloproliferative disorders but rarely in AML. Here we show that wild-type MPL expression is increased in a fraction of human AML samples expressing RUNX1-ETO, a fusion protein created by chromosome translocation t(8;21), and that up-regulation of Mpl expression in mice induces AML when coexpressed with RUNX1-ETO. The leukemic cells are sensitive to THPO, activating survival and proliferative responses. Mpl expression is not regulated by RUNX1-ETO in mouse hematopoietic progenitors or leukemic cells. Moreover, we find that activation of PI3K/AKT but not ERK/MEK pathway is a critical mediator of the MPL-directed antiapoptotic function in leukemic cells. Hence, this study provides evidence that up-regulation of wild-type MPL levels promotes leukemia development and maintenance through activation of the PI3K/AKT axis, and suggests that inhibitors of this axis could be effective for treatment of MPL-positive AML.

CCAAT/enhancer-binding protein beta promotes osteoblast differentiation by enhancing Runx2 activity with ATF4.

PubMed

Tominaga, Hiroyuki; Maeda, Shingo; Hayashi, Makoto; Takeda, Shu; Akira, Shizuo; Komiya, Setsuro; Nakamura, Takashi; Akiyama, Haruhiko; Imamura, Takeshi

2008-12-01

Although CCAAT/enhancer-binding protein beta (C/EBPbeta) is involved in osteocalcin gene expression in osteoblast in vitro, the physiological importance of and molecular mechanisms governing C/EBPbeta in bone formation remain to be elucidated. In particular, it remains unclear whether C/EBPbeta acts as a homodimer or a heterodimer with other proteins during osteoblast differentiation. Here, deletion of the C/EBPbeta gene from mice resulted in delayed bone formation with concurrent suppression of chondrocyte maturation and osteoblast differentiation. The expression of type X collagen as well as chondrocyte hypertrophy were suppressed in mutant bone, providing new insight into the possible roles of C/EBPbeta in chondrocyte maturation. In osteoblasts, luciferase reporter, gel shift, DNAP, and ChIP assays demonstrated that C/EBPbeta heterodimerized with activating transcription factor 4 (ATF4), another basic leucine zipper transcription factor crucial for osteoblast maturation. This complex interacted and transactivated osteocalcin-specific element 1 (OSE1) of the osteocalcin promoter. C/EBPbeta also enhanced the synergistic effect of ATF4 and Runx2 on osteocalcin promoter transactivation by enhancing their interaction. Thus, our results provide evidence that C/EBPbeta is a crucial cofactor in the promotion of osteoblast maturation by Runx2 and ATF4.

GASDERMIN, suppressed frequently in gastric cancer, is a target of LMO1 in TGF-beta-dependent apoptotic signalling.

PubMed

Saeki, N; Kim, D H; Usui, T; Aoyagi, K; Tatsuta, T; Aoki, K; Yanagihara, K; Tamura, M; Mizushima, H; Sakamoto, H; Ogawa, K; Ohki, M; Shiroishi, T; Yoshida, T; Sasaki, H

2007-10-04

Defining apoptosis-regulatory cascades of the epithelium is important for understanding carcinogenesis, since cancer cells are considered to arise as a result of the collapse of the cascades. We previously reported that a novel gene GASDERMIN (GSDM) is expressed in the stomach but suppressed in gastric cancer cell lines. Furthermore, in this study, we demonstrated that GSDM is expressed in the mucus-secreting pit cells of the gastric epithelium and frequently silenced in primary gastric cancers. We found that GSDM has a highly apoptotic activity and its expression is regulated by a transcription factor LIM domain only 1 (LMO1) through a sequence to which Runt-related transcription factor 3 (RUNX3) binds, in a GSDM promoter region. We observed coexpression of GSDM with LMO1, RUNX3 and type II transforming growth factor-beta receptor (TGF-betaRII) in the pit cells, and found that TGF-beta upregulates the LMO1- and GSDM-expression in the gastric epithelial cell line and induces apoptosis, which was confirmed by the finding that the apoptosis induction is inhibited by suppression of each LMO1-, RUNX3- and GSDM expression, respectively. The present data suggest that TGF-beta, LMO1, possibly RUNX3, and GSDM form a regulatory pathway for directing the pit cells to apoptosis.

TCF-1 participates in the occurrence of dedifferentiated chondrosarcoma.

PubMed

Xu, Xiaolong; Tang, Xiaodong; Guo, Wei; Yang, Kang; Ren, Tingting

2016-10-01

The present study demonstrated that T cell factor 1 (TCF-1) protein, a component of the canonical Wnt/β-catenin signaling pathway, can regulate the expression of runt-related transcription factor 2 (runx2) gene and Sry-related HMG box 9 (sox9) gene, which may participate in the differentiation of chondrosarcoma. Dedifferentiated chondrosarcoma (DDCS) is a special variant of conventional chondrosarcoma (CCS), associated with poor survival and high metastasis rate. However, little is known about the mechanism of its occurrence; thus, no effective treatment is available except surgery. Earlier, high expression of runx2 and low expression of sox9 were found in DDCS compared with CCS. Using Western blot to detect clinical tissue samples (including 8 CCS samples and 8 DDCS samples) and immunohistochemistry to detect 85 different-grade chondrosarcoma specimens, a high expression of TCF-1 in DDCS tissues was found compared with CCS tissues. This difference in expression was related to patients' prognosis. Results of luciferase, chromatin immunoprecipitation, and gel electrophoresis mobility shift assays demonstrated that TCF-1 protein could bind to the promoter of runx2 gene directly and sox9 gene indirectly. Hence, it could regulate expression of runx2 gene positively and sox9 gene negatively. Furthermore, in vitro and in vivo experiments showed that TCF-1 protein was closely related to the phenotype and aggressiveness of chondrosarcoma. In conclusion, this study proved that TCF-1 participates in the dedifferentiation of DDCS, which may be mediated by runx2 gene and sox9 gene. Also, TCF-1 can be of important prognostic value and a promising therapeutic target for DDCS patients.

Alk5/Runx1 signaling mediated by extracellular vesicles promotes vascular repair in acute respiratory distress syndrome.

PubMed

Shah, Trushil; Qin, Shanshan; Vashi, Mona; Predescu, Dan N; Jeganathan, Niranjan; Bardita, Cristina; Ganesh, Balaji; diBartolo, Salvatore; Fogg, Louis F; Balk, Robert A; Predescu, Sanda A

2018-06-22

Pulmonary endothelial cells' (ECs) injury and apoptotic death are necessary and sufficient for the pathogenesis of the acute respiratory distress syndrome (ARDS), regardless of epithelial damage. Interaction of dysfunctional ECs with circulatory extracellular vesicles (EVs) holds therapeutic promise in ARDS. However, the presence in the blood of long-term ARDS survivors of EVs with a distinct phenotype compared to the EVs of non-surviving patients is not reported. With a multidisciplinary translational approach, we studied EVs from the blood of 33 patients with moderate-to-severe ARDS. The EVs were isolated from the blood of ARDS and control subjects. Immunoblotting and magnetic beads immunoisolation complemented by standardized flow cytometry and nanoparticles tracking analyses identified in the ARDS patients a subset of EVs with mesenchymal stem cell (MSC) origin (CD73 + CD105 + Cd34 - CD45 - ). These EVs have 4.7-fold greater counts compared to controls and comprise the transforming growth factor-beta receptor I (TβRI)/Alk5 and the Runx1 transcription factor. Time course analyses showed that the expression pattern of two Runx1 isoforms is critical for ARDS outcome: the p52 isoform shows a continuous expression, while the p66 is short-lived. A high ratio Runx1p66/p52 provided a survival advantage, regardless of age, sex, disease severity or length of stay in the intensive care unit. Moreover, the Runx1p66 isoform is transiently expressed by cultured human bone marrow-derived MSCs, it is released in the EVs recoverable from the conditioned media and stimulates the proliferation of lipopolysaccharide (LPS)-treated ECs. The findings are consistent with a causal effect of Runx1p66 expression on EC proliferation. Furthermore, morphological and functional assays showed that the EVs bearing the Runx1p66 enhanced junctional integrity of LPS-injured ECs and decreased lung histological severity in the LPS-treated mice. The expression pattern of Runx1 isoforms might be a reliable circulatory biomarker of ARDS activity and a novel determinant of the molecular mechanism for lung vascular/tissue repair and recovery after severe injury.

Transcription Factors Runx1 to 3 Are Expressed in the Lacrimal Gland Epithelium and Are Involved in Regulation of Gland Morphogenesis and Regeneration

PubMed Central

Voronov, Dmitry; Gromova, Anastasia; Liu, Daren; Zoukhri, Driss; Medvinsky, Alexander; Meech, Robyn; Makarenkova, Helen P.

2013-01-01

Purpose. Lacrimal gland (LG) morphogenesis and repair are regulated by a complex interplay of intrinsic factors (e.g., transcription factors) and extrinsic signals (e.g., soluble growth/signaling factors). Many of these interconnections remain poorly characterized. Runt-related (Runx) factors belong to a small family of heterodimeric transcription factors known to regulate lineage-specific proliferation and differentiation of stem cells. The purpose of this study was to define the expression pattern and the role of Runx proteins in LG development and regeneration. Methods. Expression of epithelial-restricted transcription factors in murine LG was examined using immunostaining, qRT-PCR, and RT2Profiler PCR microarrays. The role of Runx transcription factors in LG morphogenesis was studied using siRNA and ex vivo LG cultures. Expression of Runx transcription factors during LG regeneration was assessed using in vivo model of LG regeneration. Results. We found that Runx factors are expressed in the epithelial compartment of the LG; in particular, Runx1 was restricted to the epithelium with highest level of expression in ductal and centroacinar cells. Downregulation of Runx1 to 3 expression using Runx-specific siRNAs abolished LG growth and branching and our data suggest that Runx1, 2, and 3 are partially redundant in LG development. In siRNA-treated LG, reduction of branching correlated with reduction of epithelial proliferation, as well as expression of cyclin D1 and the putative epithelial progenitor cell marker cytokeratin-5. Runx1, Runx3, and cytokeratin-5 expression increased significantly in regenerating LG and there was modest increase in Runx2 expression during LG differentiation. Conclusions. Runx1 and 2 are new markers of the LG epithelial lineage and Runx factors are important for normal LG morphogenesis and regeneration. PMID:23532528

The Relationship between RUNX3 Expression, Nursing Strategies and Nutritional Status in Elderly Patients with Advanced Gastric Cancer.

PubMed

Song, Wen; Teng, Wenhui; Shi, Xinyan; Liu, Xiaozhen; Cui, Zheng; Tian, Zibin

2017-06-01

The aim of this study was to explore the relationship between nutritional status and expression of RUNX3 in gastric cancer cells and to investigate the effects of nursing strategies on the nutritional status of elderly patients with advanced gastric cancer. Forty-eight elderly patients admitted at Affiliated Hospital of Qingdao University with advanced gastric cancer and 30 healthy controls were selected as subjects from 2014-15. The correlation between RNX3 gene expression and nutritional status of the gastric cancer patients was investigated. The patients with advanced gastric cancer who had low expression of RUNX3 gene were treated with holistic nursing while routine nursing was taken for those patients who had normal or high expression of RUNX3 gene. The nutritional statuses of these patients were evaluated after 3 months of nursing. After a follow-up of 1 year, the influence of different nursing methods on the survival time was evaluated. Compared with normal gastric tissue, the expression of RUNX3 gene and protein in tissues of advanced gastric cancer were significantly decreased ( P <0.01). Compared with patients with normal or high expressions of RUNX3, the nutritional statuses of advanced gastric cancer patients with low expressions of RUNX3 were lower ( P <0.01). The nutritional statuses of patients with low expressions of RUNX3 were notably improved after holistic nursing, becoming equivalent to those with normal or high expression of RUNX3 who received routine nursing ( P >0.05). The survival time of patients with low expression of RUNX3 who received holistic nursing were similar to patients with normal or high expression of RUNX3 who received routine nursing ( P >0.05). RUNX3 is correlated with the occurrence and development of advanced gastric cancer. The low nutritional status of elderly advanced gastric cancer patients with low expressions of RUNX3 can be significantly enhanced by holistic nursing, thereby prolonging survival time.

Histone H3 Lysine 36 Methyltransferase Whsc1 Promotes the Association of Runx2 and p300 in the Activation of Bone-Related Genes

PubMed Central

Lee, Yu Fei; Nimura, Keisuke; Lo, Wan Ning; Saga, Kotaro; Kaneda, Yasufumi

2014-01-01

The orchestration of histone modifiers is required to establish the epigenomic status that regulates gene expression during development. Whsc1 (Wolf-Hirschhorn Syndrome candidate 1), a histone H3 lysine 36 (H3K36) trimethyltransferase, is one of the major genes associated with Wolf-Hirshhorn syndrome, which is characterized by skeletal abnormalities. However, the role of Whsc1 in skeletal development remains unclear. Here, we show that Whsc1 regulates gene expression through Runt-related transcription factor (Runx) 2, a transcription factor central to bone development, and p300, a histone acetyltransferase, to promote bone differentiation. Whsc1 −/− embryos exhibited defects in ossification in the occipital bone and sternum. Whsc1 knockdown in pre-osteoblast cells perturbed histone modification patterns in bone-related genes and led to defects in bone differentiation. Whsc1 increased the association of p300 with Runx2, activating the bone-related genes Osteopontin (Opn) and Collagen type Ia (Col1a1), and Whsc1 suppressed the overactivation of these genes via H3K36 trimethylation. Our results suggest that Whsc1 fine-tunes the expression of bone-related genes by acting as a modulator in balancing H3K36 trimethylation and histone acetylation. Our results provide novel insight into the mechanisms by which this histone methyltransferase regulates gene expression. PMID:25188294

The long non-coding RNA H19-derived miR-675 modulates human gastric cancer cell proliferation by targeting tumor suppressor RUNX1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhuang, Ming; Department of Oncology, The First People’s Hospital of Lianyungang, Lianyungang, Jiangsu; Gao, Wen

Graphical abstract: - Highlights: • H19 regulates gastric cancer cell proliferation phenotype via miR-675. • MiR-675 modulates cell proliferation of gastric cancer cells by targeting tumor suppressor RUNX1. • The H19/miR-675/RUNX1 axis plays an important role in the tumorigenesis of gastric cancer. - Abstract: The lncRNA H19 has been recently shown to be upregulated and play important roles in gastric cancer tumorigenesis. However, the precise molecular mechanism of H19 and its mature product miR-675 in the carcinogenesis of gastric cancer remains unclear. In this study, we found that miR-675 was positively expressed with H19 and was a pivotal mediator inmore » H19-induced gastric cancer cell growth promotion. Subsequently, the tumor suppressor Runt Domain Transcription Factor1 (RUNX1) was confirmed to be a direct target of miR-675 using a luciferase reporter assay and Western blotting analyses. A series of rescue assays indicated that RUNX1 mediated H19/miR-67-induced gastric cancer cell phenotypic changes. Moreover, the inverse relationship between the expression of RUNX1 and H19/miR-675 was also revealed in gastric cancer tissues and gastric cancer cell lines. Taken together, our study demonstrated that the novel pathway H19/miR-675/RUNX1 regulates gastric cancer development and may serve as a potential target for gastric cancer therapy.« less

Smad4 deficiency impairs chondrocyte hypertrophy via the Runx2 transcription factor in mouse skeletal development.

PubMed

Yan, Jianyun; Li, Jun; Hu, Jun; Zhang, Lu; Wei, Chengguo; Sultana, Nishat; Cai, Xiaoqiang; Zhang, Weijia; Cai, Chen-Leng

2018-06-15

Chondrocyte hypertrophy is the terminal step in chondrocyte differentiation and is crucial for endochondral bone formation. How signaling pathways regulate chondrocyte hypertrophic differentiation remains incompletely understood. In this study, using a Tbx18:Cre ( Tbx18 Cre /+ ) gene-deletion approach, we selectively deleted the gene for the signaling protein SMAD family member 4 ( Smad4 f/f ) in the limbs of mice. We found that the Smad4 -deficient mice develop a prominent shortened limb, with decreased expression of chondrocyte differentiation markers, including Col2a1 and Acan , in the humerus at mid-to-late gestation. The most striking defects in these mice were the absence of stylopod elements and failure of chondrocyte hypertrophy in the humerus. Moreover, expression levels of the chondrocyte hypertrophy-related markers Col10a1 and Panx3 were significantly decreased. Of note, we also observed that the expression of runt-related transcription factor 2 ( Runx2 ), a critical mediator of chondrocyte hypertrophy, was also down-regulated in Smad4 -deficient limbs. To determine how the skeletal defects arose in the mouse mutants, we performed RNA-Seq with ChIP-Seq analyses and found that Smad4 directly binds to regulatory elements in the Runx2 promoter. Our results suggest a new mechanism whereby Smad4 controls chondrocyte hypertrophy by up-regulating Runx2 expression during skeletal development. The regulatory mechanism involving Smad4-mediated Runx2 activation uncovered here provides critical insights into bone development and pathogenesis of chondrodysplasia. © 2018 Yan et al.

Microtubule actin crosslinking factor 1 promotes osteoblast differentiation by promoting β-catenin/TCF1/Runx2 signaling axis.

PubMed

Hu, Lifang; Su, Peihong; Yin, Chong; Zhang, Yan; Li, Runzhi; Yan, Kun; Chen, Zhihao; Li, Dijie; Zhang, Ge; Wang, Liping; Miao, Zhiping; Qian, Airong; Xian, Cory J

2018-02-01

Osteoblast differentiation is a multistep process delicately regulated by many factors, including cytoskeletal dynamics and signaling pathways. Microtubule actin crosslinking factor 1 (MACF1), a key cytoskeletal linker, has been shown to play key roles in signal transduction and in diverse cellular processes; however, its role in regulating osteoblast differentiation is still needed to be elucidated. To further uncover the functions and mechanisms of action of MACF1 in osteoblast differentiation, we examined effects of MACF1 knockdown (MACF1-KD) in MC3T3-E1 osteoblastic cells on their osteoblast differentiation and associated molecular mechanisms. The results showed that knockdown of MACF1 significantly suppressed mineralization of MC3T3-E1 cells, down-regulated the expression of key osteogenic genes alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2) and type I collagen α1 (Col Iα1). Knockdown of MACF1 dramatically reduced the nuclear translocation of β-catenin, decreased the transcriptional activation of T cell factor 1 (TCF1), and down-regulated the expression of TCF1, lymphoid enhancer-binding factor 1 (LEF1), and Runx2, a target gene of β-catenin/TCF1. In addition, MACF1-KD increased the active level of glycogen synthase kinase-3β (GSK-3β), which is a key regulator for β-catenin signal transduction. Moreover, the reduction of nuclear β-catenin amount and decreased expression of TCF1 and Runx2 were significantly reversed in MACF1-KD cells when treated with lithium chloride, an agonist for β-catenin by inhibiting GSK-3β activity. Taken together, these findings suggest that knockdown of MACF1 in osteoblastic cells inhibits osteoblast differentiation through suppressing the β-catenin/TCF1-Runx2 axis. Thus, a novel role of MACF1 in and a new mechanistic insight of osteoblast differentiation are uncovered. © 2017 Wiley Periodicals, Inc.

Runx2 Expression in Smooth Muscle Cells Is Required for Arterial Medial Calcification in Mice

PubMed Central

Lin, Mu-En; Chen, Theodore; Leaf, Elizabeth M.; Speer, Mei Y.; Giachelli, Cecilia M.

2016-01-01

Arterial medial calcification (AMC) is a hallmark of aging, diabetes, and chronic kidney disease. Smooth muscle cell (SMC) transition to an osteogenic phenotype is a common feature of AMC, and is preceded by expression of runt-related transcription factor 2 (Runx2), a master regulator of bone development. Whether SMC-specific Runx2 expression is required for osteogenic phenotype change and AMC remains unknown. We therefore created an improved targeting construct to generate mice with floxed Runx2 alleles (Runx2f/f) that do not produce truncated Runx2 proteins after Cre recombination, thereby preventing potential off-target effects. SMC-specific deletion using SM22–recombinase transgenic allele mice (Runx2ΔSM) led to viable mice with normal bone and arterial morphology. After vitamin D overload, arterial SMCs in Runx2f/f mice expressed Runx2, underwent osteogenic phenotype change, and developed severe AMC. In contrast, vitamin D–treated Runx2ΔSM mice had no Runx2 in blood vessels, maintained SMC phenotype, and did not develop AMC. Runx2 deletion did not affect serum calcium, phosphate, fibroblast growth factor-23, or alkaline phosphatase levels. In vitro, Runx2f/f SMCs calcified to a much greater extent than those derived from Runx2ΔSM mice. These data indicate a critical role of Runx2 in SMC osteogenic phenotype change and mineral deposition in a mouse model of AMC, suggesting that Runx2 and downstream osteogenic pathways in SMCs may be useful therapeutic targets for treating or preventing AMC in high-risk patients. PMID:25987250

Gene expression of runx2, Osterix, c-fos, DLX-3, DLX-5, and MSX-2 in dental follicle cells during osteogenic differentiation in vitro.

PubMed

Morsczeck, C

2006-02-01

Recently, osteogenic precursor cells were isolated from human dental follicles, which differentiate into cementoblast- or osteoblast- like cells under in vitro conditions. However, mechanisms for osteogenic differentiation are not known in detail. Dental follicle cell long-term cultures supplemented with dexamethasone or with insulin resulted in mineralized nodules, whereas no mineralization or alkaline phosphatase activity was detected in the control culture without an osteogenic stimulus. A real-time reverse-transcriptase polymerase chain reaction (PCR) analysis was developed to investigate gene expression during osteogenic differentiation in vitro. Expression of the alkaline phosphatase (ALP) gene was detected during differentiation in the control culture and was similar to that in cultures with dexamethasone and insulin. DLX-3, DLX-5, runx2, and MSX-2 are differentially expressed during osteogenic differentiation in bone marrow mesenchymal stem cells. In dental follicle cells, gene expression of runx2, DLX-5, and MSX-2 was unaffected during osteogenic differentiation in vitro. Osteogenic differentiation appeared to be independent of MSX-2 expression; the same was true of runx2 and DLX-5, which were protagonists of osteogenic differentiation and osteocalcin promoter activity in bone marrow mesenchymal stem cells. Like in bone marrow-derived stem cells, DLX-3 gene expression was increased in dental follicle cells during osteogenic differentiation but similar to control cultures. However, gene expression of osterix was not detected in dental follicle cells during osteogenic differentiation; this gene is expressed during osteogenic differentiation in bone marrow stem cells. These real-time PCR results display molecular mechanisms in dental follicle precursor cells during osteogenic differentiation that are different from those in bone marrow-derived mesenchymal stem cells.

The association between runt-related transcription factor 3 gene promoter methylation and gastric cancer: A meta-analysis.

PubMed

Liu, Xu; Wang, Lina; Guo, Yongtie

2016-10-01

To systematically evaluate the relationship of the methylation of the human-runt-related transcription factor 3 (RUNX3) promoter region and gastric cancer risk through meta-analysis. The studies published in PubMed, EMBASE, Ovid, and CNKI were retrieved. The association between RUNX3 gene promoter methylation and gastric cancer was analyzed using Stata 11.0 (http://www.stata.com; Stata Corporation, College Station, TX, USA) and Review Man 5.0 software (http://ims.cochrane.org/revman/download). Seventeen studies are included in the analysis. Meta-analysis reveals that the odds ratio of the methylation of the RUNX3 promoter region in gastric was 7.32 (95% confidence interval: 5.12-10.47), which was significant higher than the normal gastric tissues (P < 0.05). The RUNX3 gene promoter methylation rate was much higher in tumor tissue than that in normal gastric tissue in patient with gastric cancer, which indicates a close association between gastric cancer and RUNX3 gene promoter methylation.

MiR-133a modulates osteogenic differentiation of vascular smooth muscle cells.

PubMed

Liao, Xiao-Bo; Zhang, Zhi-Yuan; Yuan, Ke; Liu, Yuan; Feng, Xiang; Cui, Rong-Rong; Hu, Ye-Rong; Yuan, Zhao-Shun; Gu, Lu; Li, Shi-Jun; Mao, Ding-An; Lu, Qiong; Zhou, Xin-Ming; de Jesus Perez, Vinicio A; Yuan, Ling-Qing

2013-09-01

Arterial calcification is a key pathologic component of vascular diseases such as atherosclerosis, coronary artery disease, and peripheral vascular disease. A hallmark of this pathological process is the phenotypic transition of vascular smooth muscle cells (VSMCs) to osteoblast-like cells. Several studies have demonstrated that microRNAs (miRNAs) regulate osteoblast differentiation, but it is unclear whether miRNAs also regulate VSMC-mediated arterial calcification. In the present study, we sought to characterize the role of miR-133a in regulating VSMC-mediated arterial calcification. Northern blotting analysis of VSMCs treated with β-glycerophosphate demonstrated that miR-133a was significantly decreased during osteogenic differentiation. Overexpression of miR-133a inhibited VSMC transdifferentiation into osteoblast-like cells as evidenced by a decrease in alkaline phosphatase activity, osteocalcin secretion, Runx2 expression, and mineralized nodule formation. Conversely, the knockdown of miR-133a using an miR-133a inhibitor promoted osteogenic differentiation of VSMCs by increasing alkaline phosphatase activity, osteocalcin secretion, and Runx2 expression. Runx2 was identified as a direct target of miR-133a by a cotransfection experiment in VSMCs with luciferase reporter plasmids containing wild-type or mutant 3'-untranslated region sequences of Runx2. Furthermore, the pro-osteogenic effects of miR-133a inhibitor were abrogated in Runx2-knockdown cells, and the inhibition of osteogenic differentiation by pre-miR-133a was reversed by overexpression of Runx2, providing functional evidence that the effects of miR-133a in osteogenic differentiation were mediated by targeting Runx2. These results demonstrate that miR-133a is a key negative regulator of the osteogenic differentiation of VSMCs.

RUNX3 is oncogenic in natural killer/T-cell lymphoma and is transcriptionally regulated by MYC

PubMed Central

Selvarajan, V; Osato, M; Nah, G S S; Yan, J; Chung, T-H; Voon, D C-C; Ito, Y; Ham, M F; Salto-Tellez, M; Shimizu, N; Choo, S-N; Fan, S; Chng, W-J; Ng, S-B

2017-01-01

RUNX3, runt-domain transcription factor, is a master regulator of gene expression in major developmental pathways. It acts as a tumor suppressor in many cancers but is oncogenic in certain tumors. We observed upregulation of RUNX3 mRNA and protein expression in nasal-type extranodal natural killer (NK)/T-cell lymphoma (NKTL) patient samples and NKTL cell lines compared to normal NK cells. RUNX3 silenced NKTL cells showed increased apoptosis and reduced cell proliferation. Potential binding sites for MYC were identified in the RUNX3 enhancer region. Chromatin immunoprecipitation–quantitative PCR revealed binding activity between MYC and RUNX3. Co-transfection of the MYC expression vector with RUNX3 enhancer reporter plasmid resulted in activation of RUNX3 enhancer indicating that MYC positively regulates RUNX3 transcription in NKTL cell lines. Treatment with a small-molecule MYC inhibitor (JQ1) caused significant downregulation of MYC and RUNX3, leading to apoptosis in NKTL cells. The growth inhibition resulting from depletion of MYC by JQ1 was rescued by ectopic MYC expression. In summary, our study identified RUNX3 overexpression in NKTL with functional oncogenic properties. We further delineate that MYC may be an important upstream driver of RUNX3 upregulation and since MYC is upregulated in NKTL, further study on the employment of MYC inhibition as a therapeutic strategy is warranted. PMID:28119527

A Genome-Wide RNAi Screen Identifies FOXO4 as a Metastasis-Suppressor through Counteracting PI3K/AKT Signal Pathway in Prostate Cancer

PubMed Central

Su, Bing; Gao, Lingqiu; Baranowski, Catherine; Gillard, Bryan; Wang, Jianmin; Ransom, Ryan; Ko, Hyun-Kyung; Gelman, Irwin H.

2014-01-01

Activation of the PI3K/AKT signal pathway is a known driving force for the progression to castration-recurrent prostate cancer (CR-CaP), which constitutes the major lethal phenotype of CaP. Here, we identify using a genomic shRNA screen the PI3K/AKT-inactivating downstream target, FOXO4, as a potential CaP metastasis suppressor. FOXO4 protein levels inversely correlate with the invasive potential of a panel of human CaP cell lines, with decreased mRNA levels correlating with increased incidence of clinical metastasis. Knockdown (KD) of FOXO4 in human LNCaP cells causes increased invasion in vitro and lymph node (LN) metastasis in vivo without affecting indices of proliferation or apoptosis. Increased Matrigel invasiveness was found by KD of FOXO1 but not FOXO3. Comparison of differentially expressed genes affected by FOXO4-KD in LNCaP cells in culture, in primary tumors and in LN metastases identified a panel of upregulated genes, including PIP, CAMK2N1, PLA2G16 and PGC, which, if knocked down by siRNA, could decrease the increased invasiveness associated with FOXO4 deficiency. Although only some of these genes encode FOXO promoter binding sites, they are all RUNX2-inducible, and RUNX2 binding to the PIP promoter is increased in FOXO4-KD cells. Indeed, the forced expression of FOXO4 reversed the increased invasiveness of LNCaP/shFOXO4 cells; the forced expression of FOXO4 did not alter RUNX2 protein levels, yet it decreased RUNX2 binding to the PIP promoter, resulting in PIP downregulation. Finally, there was a correlation between FOXO4, but not FOXO1 or FOXO3, downregulation and decreased metastasis-free survival in human CaP patients. Our data strongly suggest that increased PI3K/AKT-mediated metastatic invasiveness in CaP is associated with FOXO4 loss, and that mechanisms to induce FOXO4 re-expression might suppress CaP metastatic aggressiveness. PMID:24983969

RUNX family members are covalently modified and regulated by PIAS1-mediated sumoylation

PubMed Central

Kim, J-H; Jang, J-W; Lee, Y-S; Lee, J-W; Chi, X-Z; Li, Y-H; Kim, M-K; Kim, D-M; Choi, B-S; Kim, J; Kim, H-M; van Wijnen, A; Park, IlY; Bae, S-C

2014-01-01

Transcription factors of the RUNX family (RUNXs), which play pivotal roles in normal development and neoplasia, are regulated by various post-translational modifications. To understand the molecular mechanisms underlying the regulation of RUNXs, we performed a large-scale functional genetic screen of a fly mutant library. The screen identified dPias (the fly ortholog of mammalian PIASs), an E3 ligase for the SUMO (small ubiquitin-like modifier) modification, as a novel genetic modifier of lz (the fly ortholog of mammalian RUNX3). Molecular biological analysis revealed that lz/RUNXs are sumoylated by dPias/PIAS1 at an evolutionarily conserved lysine residue (K372 of lz, K144 of RUNX1, K181 of RUNX2 and K148 of RUNX3). PIAS1-mediated sumoylation inhibited RUNX3 transactivation activity, and this modification was promoted by the AKT1 kinase. Importantly, PIAS1 failed to sumoylate some RUNX1 mutants associated with breast cancer. In nude mice, tumorigenicity was promoted by RUNX3 bearing a mutation in the sumoylation site, but suppressed by wild-type RUNX3. Our results suggest that RUNXs are sumoylated by PIAS1, and that this modification could play a critical role in the regulation of the tumor-suppressive activity of these proteins. PMID:24777122

Integrative analysis of RUNX1 downstream pathways and target genes

PubMed Central

Michaud, Joëlle; Simpson, Ken M; Escher, Robert; Buchet-Poyau, Karine; Beissbarth, Tim; Carmichael, Catherine; Ritchie, Matthew E; Schütz, Frédéric; Cannon, Ping; Liu, Marjorie; Shen, Xiaofeng; Ito, Yoshiaki; Raskind, Wendy H; Horwitz, Marshall S; Osato, Motomi; Turner, David R; Speed, Terence P; Kavallaris, Maria; Smyth, Gordon K; Scott, Hamish S

2008-01-01

Background The RUNX1 transcription factor gene is frequently mutated in sporadic myeloid and lymphoid leukemia through translocation, point mutation or amplification. It is also responsible for a familial platelet disorder with predisposition to acute myeloid leukemia (FPD-AML). The disruption of the largely unknown biological pathways controlled by RUNX1 is likely to be responsible for the development of leukemia. We have used multiple microarray platforms and bioinformatic techniques to help identify these biological pathways to aid in the understanding of why RUNX1 mutations lead to leukemia. Results Here we report genes regulated either directly or indirectly by RUNX1 based on the study of gene expression profiles generated from 3 different human and mouse platforms. The platforms used were global gene expression profiling of: 1) cell lines with RUNX1 mutations from FPD-AML patients, 2) over-expression of RUNX1 and CBFβ, and 3) Runx1 knockout mouse embryos using either cDNA or Affymetrix microarrays. We observe that our datasets (lists of differentially expressed genes) significantly correlate with published microarray data from sporadic AML patients with mutations in either RUNX1 or its cofactor, CBFβ. A number of biological processes were identified among the differentially expressed genes and functional assays suggest that heterozygous RUNX1 point mutations in patients with FPD-AML impair cell proliferation, microtubule dynamics and possibly genetic stability. In addition, analysis of the regulatory regions of the differentially expressed genes has for the first time systematically identified numerous potential novel RUNX1 target genes. Conclusion This work is the first large-scale study attempting to identify the genetic networks regulated by RUNX1, a master regulator in the development of the hematopoietic system and leukemia. The biological pathways and target genes controlled by RUNX1 will have considerable importance in disease progression in both familial and sporadic leukemia as well as therapeutic implications. PMID:18671852

The role of Runx2 in facilitating autophagy in metastatic breast cancer cells.

PubMed

Tandon, Manish; Othman, Ahmad H; Ashok, Vivek; Stein, Gary S; Pratap, Jitesh

2018-01-01

Breast cancer metastases cause significant patient mortality. During metastases, cancer cells use autophagy, a catabolic process to recycle nutrients via lysosomal degradation, to overcome nutritional stress for their survival. The Runt-related transcription factor, Runx2, promotes cell survival under metabolic stress, and regulates breast cancer progression and bone metastases. Here, we identify that Runx2 enhances autophagy in metastatic breast cancer cells. We defined Runx2 function in cellular autophagy by monitoring microtubule-associated protein light chain (LC3B-II) levels, an autophagy-specific marker. The electron and confocal microscopic analyses were utilized to identify alterations in autophagic vesicles. The Runx2 knockdown cells accumulate LC3B-II protein and autophagic vesicles due to reduced turnover. Interestingly, Runx2 promotes autophagy by enhancing trafficking of LC3B vesicles. Our mechanistic studies revealed that Runx2 promotes autophagy by increasing acetylation of α-tubulin sub-units of microtubules. Inhibiting autophagy decreased cell adhesion and survival of Runx2 knockdown cells. Furthermore, analysis of LC3B protein in clinical breast cancer specimens and tumor xenografts revealed significant association between high Runx2 and low LC3B protein levels. Our studies reveal a novel regulatory mechanism of autophagy via Runx2 and provide molecular insights into the role of autophagy in metastatic cancer cells. © 2017 Wiley Periodicals, Inc.

Bone morphogenetic protein 2 activates Smad6 gene transcription through bone-specific transcription factor Runx2.

PubMed

Wang, Qing; Wei, Xiaochao; Zhu, Tianhui; Zhang, Ming; Shen, Run; Xing, Lianping; O'Keefe, Regis J; Chen, Di

2007-04-06

BMP-2 plays an essential role in osteoblast and chondrocyte differentiation, but its signaling mechanism has not been fully defined. In the present studies, we investigated the mechanism through which BMP-2 activates the Smad6 gene. A -2006/+45 Smad6 promoter-luciferase construct was generated along with deletions and Runx2 binding site mutations to examine the role of Smad1 and Runx2 signaling following BMP-2 stimulation in osteoblasts. Transfection of Runx2 or treatment with BMP-2-stimulated promoter activity of the -2006/+45 and -1191/+45 reporters but not the -829/+45 and -374/+45 reporters. No Smad1/5 binding site is present in the -1191/-829 region of the Smad6 promoter. Mutation of the OSE2-a site (-1036/-1031) completely abolished the stimulatory effect of Runx2 as well as BMP-2 on the -2006/+45 and -1191/+45 Smad6 reporters. Gel shift and chromatin immunoprecipitation (ChIP) assays showed that Runx2 binds the OSE2-a element. ChIP assays demonstrated that Smad1 also interacts with the OSE2-a site at the Smad6 promoter through Runx2. The protein degradation of Runx2 is mediated by the E3 ubiquitin ligase Smurf1. In the present studies, we found that Smurf1 binds the OSE2-a site through Runx2 and inhibits Smad6 gene transcription. Treatment with BMP-2 and transfection of Smad1 abolished Smurf1 binding to the OSE2 site. These results show that Smad1 binding excludes Smurf1 interaction with the OSE2 site and promotes Smad6 gene transcription.

RUNX1 regulates phosphoinositide 3-kinase/AKT pathway: role in chemotherapy sensitivity in acute megakaryocytic leukemia.

PubMed

Edwards, Holly; Xie, Chengzhi; LaFiura, Katherine M; Dombkowski, Alan A; Buck, Steven A; Boerner, Julie L; Taub, Jeffrey W; Matherly, Larry H; Ge, Yubin

2009-09-24

RUNX1 (AML1) encodes the core binding factor alpha subunit of a heterodimeric transcription factor complex which plays critical roles in normal hematopoiesis. Translocations or down-regulation of RUNX1 have been linked to favorable clinical outcomes in acute leukemias, suggesting that RUNX1 may also play critical roles in chemotherapy responses in acute leukemias; however, the molecular mechanisms remain unclear. The median level of RUNX1b transcripts in Down syndrome (DS) children with acute megakaryocytic leukemia (AMkL) were 4.4-fold (P < .001) lower than that in non-DS AMkL cases. Short hairpin RNA knockdown of RUNX1 in a non-DS AMkL cell line, Meg-01, resulted in significantly increased sensitivity to cytosine arabinoside, accompanied by significantly decreased expression of PIK3CD, which encodes the delta catalytic subunit of the survival kinase, phosphoinositide 3 (PI3)-kinase. Transcriptional regulation of PIK3CD by RUNX1 was further confirmed by chromatin immunoprecipitation and promoter reporter gene assays. Further, a PI3-kinase inhibitor, LY294002, and cytosine arabinoside synergized in antileukemia effects on Meg-01 and primary pediatric AMkL cells. Our results suggest that RUNX1 may play a critical role in chemotherapy response in AMkL by regulating the PI3-kinase/Akt pathway. Thus, the treatment of AMkL may be improved by integrating PI3-kinase or Akt inhibitors into the chemotherapy of this disease.

HIERARCHICAL ORGANIZATION OF OSTEOBLASTS REVEALS THE SIGNIFICANT ROLE OF CD166 IN HEMATOPOIETIC STEM CELL MAINTANANCE AND FUNCTION

PubMed Central

Chitteti, Brahmananda R.; Cheng, Ying-Hua; Kacena, Melissa A.; Srour, Edward F.

2013-01-01

The role of osteoblasts (OB) in maintaining hematopoietic stem cells (HSC) in their niche is well elucidated, but the exact definition, both phenotypically and hierarchically of OB responsible for these functions is not clearly known. We previously demonstrated that OB maturational status influences HSC function whereby immature OB with high Runx2 expression promote hematopoietic expansion. Here, we show that Activated Leukocyte Cell Adhesion Molecule (ALCAM) or CD166 expression on OB is directly correlated with Runx2 expression and high hematopoiesis enhancing activity (HEA). Fractionation of OB with lineage markers: Sca1, osteopontin (OPN), CD166, CD44, and CD90 revealed that Lin-Sca1-OPN+CD166+ cells (CD166+) and their subpopulations fractionated with CD44 and CD90 expressed high levels of Runx2 and low levels of osteocalcin (OC) demonstrating the relatively immature status of these cells. Conversely, the majority of the Lin-Sca1-OPN+CD166− cells (CD166−) expressed high OC levels suggesting that CD166− OB are more mature. In vitro hematopoietic potential of LSK cells co-cultured for 7 days with fresh OB or OB pre-cultured for 1, 2, or 3 weeks declined precipitously with increasing culture duration concomitant with loss of CD166 expression. Importantly, LSK cells co-cultured with CD166+CD44+CD90+ OB maintained their in vivo repopulating potential through primary and secondary transplantation, suggesting that robust HEA activity is best mediated by immature CD166+ OB with high Runx2 and low OC expression. These studies begin to define the hierarchical organization of osteoblastic cells and provide a more refined definition of OB that can mediate HEA. PMID:23369988

Combination immunohistochemistry for SMAD4 and Runt-related transcription factor 3 may identify a favorable prognostic subgroup of pancreatic ductal adenocarcinomas.

PubMed

Lee, Yangkyu; Lee, Hyejung; Park, Hyunjin; Kim, Jin-Won; Hwang, Jin-Hyeok; Kim, Jaihwan; Yoon, Yoo-Seok; Han, Ho-Seong; Kim, Haeryoung

2017-09-29

SMAD4/DPC4 mutations have been associated with aggressive behavior in pancreatic ductal adenocarcinomas (PDAC), and it has recently been suggested that RUNX3 expression combined with SMAD4 status may predict the metastatic potential of PDACs. We evaluated the prognostic utility of SMAD4/RUNX3 status in human PDACs by immunohistochemistry. Immunohistochemical stains were performed for SMAD4 and RUNX3 on 210 surgically resected PDACs, and the results were correlated with the clinicopathological features. Loss of SMAD4 expression was associated with poor overall survival (OS) ( p = 0.015) and progression-free survival (PFS) ( p = 0.044). Nuclear RUNX3 expression was associated with decreased OS ( p = 0.010) and PFS ( p = 0.009), and more frequent in poorly differentiated PDACs ( p = 0.037). On combining RUNX3/SMAD4 status, RUNX3-/SMAD4+ PDACs demonstrated longer OS ( p = 0.008, median time; RUNX3-/SMAD4+ 34 months, others 17 months) and PFS ( p = 0.009, median time; RUNX3-/SMAD4+ 29 months, others 8 months) compared to RUNX3+/SMAD4+ and SMAD4- groups; RUNX3-/SMAD4+ was a significant independent predictive factor for both OS [ p = 0.025, HR 1.842 (95% CI 1.079-3.143)] and PFS [ p = 0.020, HR 1.850 (95% CI 1.100-3.113)]. SMAD4-positivity with RUNX3-negativity was a significant independent predictive factor for favorable OS and PFS in PDAC. This is the first and large clinicopathological study of RUNX3/SMAD4 expression status in human PDAC. Combination immunohistochemistry for SMAD4 and RUNX3 may help identify a favorable prognostic subgroup of PDAC.

Combination immunohistochemistry for SMAD4 and Runt-related transcription factor 3 may identify a favorable prognostic subgroup of pancreatic ductal adenocarcinomas

PubMed Central

Lee, Yangkyu; Lee, Hyejung; Park, Hyunjin; Kim, Jin-Won; Hwang, Jin-Hyeok; Kim, Jaihwan; Yoon, Yoo-Seok; Han, Ho-Seong; Kim, Haeryoung

2017-01-01

Purposes SMAD4/DPC4 mutations have been associated with aggressive behavior in pancreatic ductal adenocarcinomas (PDAC), and it has recently been suggested that RUNX3 expression combined with SMAD4 status may predict the metastatic potential of PDACs. We evaluated the prognostic utility of SMAD4/RUNX3 status in human PDACs by immunohistochemistry. Materials and Methods Immunohistochemical stains were performed for SMAD4 and RUNX3 on 210 surgically resected PDACs, and the results were correlated with the clinicopathological features. Results Loss of SMAD4 expression was associated with poor overall survival (OS) (p = 0.015) and progression-free survival (PFS) (p = 0.044). Nuclear RUNX3 expression was associated with decreased OS (p = 0.010) and PFS (p = 0.009), and more frequent in poorly differentiated PDACs (p = 0.037). On combining RUNX3/SMAD4 status, RUNX3-/SMAD4+ PDACs demonstrated longer OS (p = 0.008, median time; RUNX3-/SMAD4+ 34 months, others 17 months) and PFS (p = 0.009, median time; RUNX3-/SMAD4+ 29 months, others 8 months) compared to RUNX3+/SMAD4+ and SMAD4- groups; RUNX3-/SMAD4+ was a significant independent predictive factor for both OS [p = 0.025, HR 1.842 (95% CI 1.079-3.143)] and PFS [p = 0.020, HR 1.850 (95% CI 1.100-3.113)]. Conclusions SMAD4-positivity with RUNX3-negativity was a significant independent predictive factor for favorable OS and PFS in PDAC. This is the first and large clinicopathological study of RUNX3/SMAD4 expression status in human PDAC. Combination immunohistochemistry for SMAD4 and RUNX3 may help identify a favorable prognostic subgroup of PDAC. PMID:29100342

Depletion of pro-oncogenic RUNX2 enhances gemcitabine (GEM) sensitivity of p53-mutated pancreatic cancer Panc-1 cells through the induction of pro-apoptotic TAp63.

PubMed

Ozaki, Toshinori; Nakamura, Mizuyo; Ogata, Takehiro; Sang, Meijie; Yoda, Hiroyuki; Hiraoka, Kiriko; Sang, Meixiang; Shimozato, Osamu

2016-11-01

Recently, we have described that siRNA-mediated silencing of runt-related transcription factor 2 (RUNX2) improves anti-cancer drug gemcitabine (GEM) sensitivity of p53-deficient human pancreatic cancer AsPC-1 cells through the augmentation of p53 family TAp63-dependent cell death pathway. In this manuscript, we have extended our study to p53-mutated human pancreatic cancer Panc-1 cells. According to our present results, knockdown of mutant p53 alone had a marginal effect on GEM-mediated cell death of Panc-1 cells. We then sought to deplete RUNX2 using siRNA in Panc-1 cells and examined its effect on GEM sensitivity. Under our experimental conditions, RUNX2 knockdown caused a significant enhancement of GEM sensitivity of Panc-1 cells. Notably, GEM-mediated induction of TAp63 but not of TAp73 was further stimulated in RUNX2-depleted Panc-1 cells, indicating that, like AsPC-1 cells, TAp63 might play a pivotal role in the regulation of GEM sensitivity of Panc-1 cells. Consistent with this notion, forced expression of TAp63α in Panc-1 cells promoted cell cycle arrest and/or cell death, and massively increased luciferase activities driven by TAp63-target gene promoters such as p21WAF1 and NOXA. In addition, immunoprecipitation experiments indicated that RUNX2 forms a complex with TAp63 in Panc-1 cells. Taken together, our current observations strongly suggest that depletion of RUNX2 enhances the cytotoxic effect of GEM on p53-mutated Panc-1 cells through the stimulation of TAp63-dependent cell death pathway even in the presence of a large amount of pro-oncogenic mutant p53, and might provide an attractive strategy to treat pancreatic cancer patients with p53 mutations.

Depletion of pro-oncogenic RUNX2 enhances gemcitabine (GEM) sensitivity of p53-mutated pancreatic cancer Panc-1 cells through the induction of pro-apoptotic TAp63

PubMed Central

Ozaki, Toshinori; Nakamura, Mizuyo; Ogata, Takehiro; Sang, Meijie; Yoda, Hiroyuki; Hiraoka, Kiriko; Sang, Meixiang; Shimozato, Osamu

2016-01-01

Recently, we have described that siRNA-mediated silencing of runt-related transcription factor 2 (RUNX2) improves anti-cancer drug gemcitabine (GEM) sensitivity of p53-deficient human pancreatic cancer AsPC-1 cells through the augmentation of p53 family TAp63-dependent cell death pathway. In this manuscript, we have extended our study to p53-mutated human pancreatic cancer Panc-1 cells. According to our present results, knockdown of mutant p53 alone had a marginal effect on GEM-mediated cell death of Panc-1 cells. We then sought to deplete RUNX2 using siRNA in Panc-1 cells and examined its effect on GEM sensitivity. Under our experimental conditions, RUNX2 knockdown caused a significant enhancement of GEM sensitivity of Panc-1 cells. Notably, GEM-mediated induction of TAp63 but not of TAp73 was further stimulated in RUNX2-depleted Panc-1 cells, indicating that, like AsPC-1 cells, TAp63 might play a pivotal role in the regulation of GEM sensitivity of Panc-1 cells. Consistent with this notion, forced expression of TAp63α in Panc-1 cells promoted cell cycle arrest and/or cell death, and massively increased luciferase activities driven by TAp63-target gene promoters such as p21WAF1 and NOXA. In addition, immunoprecipitation experiments indicated that RUNX2 forms a complex with TAp63 in Panc-1 cells. Taken together, our current observations strongly suggest that depletion of RUNX2 enhances the cytotoxic effect of GEM on p53-mutated Panc-1 cells through the stimulation of TAp63-dependent cell death pathway even in the presence of a large amount of pro-oncogenic mutant p53, and might provide an attractive strategy to treat pancreatic cancer patients with p53 mutations. PMID:27713122

Distinct Contributions of Conserved Modules to Runt Transcription Factor Activity

PubMed Central

Walrad, Pegine B.; Hang, Saiyu; Joseph, Genevieve S.; Salas, Julia

2010-01-01

Runx proteins play vital roles in regulating transcription in numerous developmental pathways throughout the animal kingdom. Two Runx protein hallmarks are the DNA-binding Runt domain and a C-terminal VWRPY motif that mediates interaction with TLE/Gro corepressor proteins. A phylogenetic analysis of Runt, the founding Runx family member, identifies four distinct regions C-terminal to the Runt domain that are conserved in Drosophila and other insects. We used a series of previously described ectopic expression assays to investigate the functions of these different conserved regions in regulating gene expression during embryogenesis and in controlling axonal projections in the developing eye. The results indicate each conserved region is required for a different subset of activities and identify distinct regions that participate in the transcriptional activation and repression of the segmentation gene sloppy-paired-1 (slp1). Interestingly, the C-terminal VWRPY-containing region is not required for repression but instead plays a role in slp1 activation. Genetic experiments indicating that Groucho (Gro) does not participate in slp1 regulation further suggest that Runt's conserved C-terminus interacts with other factors to promote transcriptional activation. These results provide a foundation for further studies on the molecular interactions that contribute to the context-dependent properties of Runx proteins as developmental regulators. PMID:20462957

Frequent Attenuation of the WWOX Tumor Suppressor in Osteosarcoma is Associated with Increased Tumorigenicity and Aberrant RUNX2 Expression

PubMed Central

Kurek, Kyle; Del Mare, Sara; Salah, Zaidoun; Abdeen, Suhaib; Sadiq, Hussain; Lee, Sukhee; Gaudio, Eugenio; Zanesi, Nicola; Jones, Kevin B.; DeYoung, Barry; Amir, Gail; Gebhardt, Mark; Warman, Matthew; Stein, Gary S.; Stein, Janet L.; Lian, Jane B.; Aqeilan, Rami I.

2011-01-01

The WW domain-containing oxidoreductase (WWOX) is a tumor suppressor that is deleted or attenuated in most human tumors. Wwox-deficient mice develop osteosarcoma (OS), an aggressive bone tumor with poor prognosis that often metastasizes to lung. On the basis of these observations, we examined the status of WWOX in human OS specimens and cell lines. In human OS clinical samples, WWOX expression was absent or reduced in 58% of tumors examined (P< 0.0001). Compared to the primary tumors, WWOX levels frequently increased in tumors resected following chemotherapy. In contrast, tumor metastases to lung often exhibited reduced WWOX levels, relative to the primary tumor. In human OS cell lines having reduced WWOX expression, ectopic expression of WWOX inhibited proliferation and attenuated invasion in vitro, and suppressed tumorgenicity in nude mice. Expression of WWOX was associated with reduced RUNX2 expression in OS cell lines, whereas Runx2 levels were elevated in femurs of Wwox-deficient mice. Furthermore, WWOX reconstitution in HOS cells was associated with downregulation of RUNX2 levels and RUNX2 target genes, consistent with the ability of WWOX to suppress RUNX2 transactivation activity. In clinical samples, RUNX2 was expressed in the majority of primary tumors and undetectable in most tumors resected following chemotherapy, whereas most metastases were RUNX2 positive. Our results deepen the evidence of a tumor suppressor role for WWOX in OS, furthering its prognostic and therapeutic significance in this disease. PMID:20530675

RUNX3 methylation in normal surrounding urothelium of patients with non-muscle-invasive bladder cancer: potential role in the prediction of tumor progression.

PubMed

Jeong, P; Min, B D; Ha, Y S; Song, P H; Kim, I Y; Ryu, K H; Kim, J H; Yun, S J; Kim, W J

2012-11-01

Previously, we reported a causal relationship between RUNX3 methylation and bladder tumor development. Thus, in order to clarify its role in tumorigenesis, this study aims to identify the function of RUNX3 methylation in normal adjacent urothelium of patients with non-muscle invasive bladder cancer (NMIBC). Tumor tissue and donor-matched normal adjacent tissue from 55 patients who underwent transurethral resection (TUR) were selected for the study, and RUNX3 promoter methylation was assessed using methylation-specific polymerase chain reaction (MS-PCR). RUNX3 promoter methylation occurred more frequently in tumor samples than in histologically normal urothelium in patients with NMIBC (P = 0.02). The methylation rates for the RUNX3 promoter in normal adjacent urothelium and tumor tissue were 47% and 69%, respectively. Interestingly, RUNX3 methylation in normal adjacent urothelium was associated with tumor number (P = 0.022) and progression (P = 0.035). Kaplan-Meier estimates revealed that RUNX3 methylation in normal urothelium showed a significant association with time to progression (P = 0.017) in NMIBC patients. Stratifying the patients into 'both methylation', 'one methylation' and 'no methylation' groups for tumors and normal urothelium revealed that no progression occurred in the 'no methylation' group during follow-up. Multivariate Cox regression analysis demonstrated that RUNX3 methylation in normal urothelium [hazards ratio (HR): 5.692, P = 0.042] was an independent predictor of progression. RUNX3 methylation was associated with transition from normal urothelium to bladder tumor. More importantly, RUNX3 methylation in normal adjacent urothelium may predict progression in NMIBC patients who have undergone TUR. Copyright © 2012 Elsevier Ltd. All rights reserved.

The transcription factor cyclic adenosine 3',5'-monophosphate response element-binding protein enhances the odonto/osteogenic differentiation of stem cells from the apical papilla.

PubMed

Su, S; Zhu, Y; Li, S; Liang, Y; Zhang, J

2017-09-01

To investigate the role of cAMP response element-binding protein (CREB) in the regulation of odonto/osteogenic differentiation of stem cells from the apical papilla (SCAPs). Stem cells from the apical papilla were obtained from human impacted third molars (n = 15). Isolated SCAPs were transfected with CREB overexpressing/silenced lentivirus. Transfected cells were stained with alizarin red to investigate mineralized nodule formation. The expression of the mineralization-related genes, alkaline phosphatase (ALP), collagen type I (Col I), runt-related transcription factor 2 (RUNX2), osterix (OSX) and osteocalcin (OCN), was determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Protein expression of the odontogenic-related marker dentine sialoprotein (DSP) and the osteogenic-related marker RUNX2 was measured by Western blotting analysis. One-way analysis of variance (anova) and Student's t-test were used for statistical analysis (a = 0.05). The overexpression of CREB enhanced mineralized nodule formation and up-regulated (P < 0.05) the mRNA levels of odonto/osteogenic-related markers, including ALP, Col I, RUNX2, OSX and OCN, and also increased (P < 0.05) the protein expression of DSP and RUNX2. In contrast, the silencing of CREB inhibited (P < 0.05) the mineralization capacity of the SCAPs and decreased (P < 0.05) the expression of odonto/osteogenic-related markers. Up-regulation of CREB expression promoted odonto/osteogenic differentiation of SCAPs and provided a potential method for the regeneration of the dentine-pulp complex. © 2016 International Endodontic Journal. Published by John Wiley & Sons Ltd.

Gene array analysis reveals a common Runx transcriptional program controlling cell adhesion and survival

PubMed Central

Wotton, Sandy; Terry, Anne; Kilbey, Anna; Jenkins, Alma; Herzyk, Pawel; Cameron, Ewan; Neil, James C.

2008-01-01

The Runx genes play divergent roles in development and cancer, where they can act either as oncogenes or tumour suppressors. We compared the effects of ectopic Runx expression in established fibroblasts, where all three genes produce an indistinguishable phenotype entailing epithelioid morphology and increased cell survival under stress conditions. Gene array analysis revealed a strongly overlapping transcriptional signature, with no examples of opposing regulation of the same target gene. A common set of 50 highly regulated genes was identified after further filtering on regulation by inducible RUNX1-ER. This set revealed a strong bias towards genes with annotated roles in cancer and development, and a preponderance of targets encoding extracellular or surface proteins, reflecting the marked effects of Runx on cell adhesion. Furthermore, in silico prediction of resistance to glucocorticoid growth inhibition was confirmed in fibroblasts and lymphoid cells expressing ectopic Runx. The effects of fibroblast expression of common RUNX1 fusion oncoproteins (RUNX1-ETO, TEL-RUNX1, CBFB-MYH11) were also tested. While two direct Runx activation target genes were repressed (Ncam1, Rgc32), the fusion proteins appeared to disrupt regulation of down-regulated targets (Cebpd, Id2, Rgs2) rather than impose constitutive repression. These results elucidate the oncogenic potential of the Runx family and reveal novel targets for therapeutic inhibition. PMID:18560354

DNA–PKcs–SIN1 complexation mediates low-dose X-ray irradiation (LDI)-induced Akt activation and osteoblast differentiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Yong; Fang, Shi-ji; Zhu, Li-juan

Highlights: • LDI increases ALP activity, promotes type I collagen (Col I)/Runx2 mRNA expression. • LDI induces DNA–PKcs activation, which is required for osteoblast differentiation. • Akt activation mediates LDI-induced ALP activity and Col I/Runx2 mRNA increase. • DNA–PKcs–SIN1 complexation mediates LDI-induced Akt Ser-473 phosphorylation. • DNA–PKcs–SIN1 complexation is important for osteoblast differentiation. - Abstract: Low-dose irradiation (LDI) induces osteoblast differentiation, however the underlying mechanisms are not fully understood. In this study, we explored the potential role of DNA-dependent protein kinase catalytic subunit (DNA–PKcs)–Akt signaling in LDI-induced osteoblast differentiation. We confirmed that LDI promoted mouse calvarial osteoblast differentiation, which wasmore » detected by increased alkaline phosphatase (ALP) activity as well as mRNA expression of type I collagen (Col I) and runt-related transcription factor 2 (Runx2). In mouse osteoblasts, LDI (1 Gy) induced phosphorylation of DNA–PKcs and Akt (mainly at Ser-473). The kinase inhibitors against DNA–PKcs (NU-7026 and NU-7441) or Akt (LY294002, perifosine and MK-2206), as well as partial depletion of DNA–PKcs or Akt1 by targeted-shRNA, dramatically inhibited LDI-induced Akt activation and mouse osteoblast differentiation. Further, siRNA-knockdown of SIN1, a key component of mTOR complex 2 (mTORC2), also inhibited LDI-induced Akt Ser-473 phosphorylation as well as ALP activity increase and Col I/Runx2 expression in mouse osteoblasts. Co-immunoprecipitation (Co-IP) assay results demonstrated that LDI-induced DNA–PKcs–SIN1 complexation, which was inhibited by NU-7441 or SIN1 siRNA-knockdown in mouse osteoblasts. In summary, our data suggest that DNA–PKcs–SIN1 complexation-mediated Akt activation (Ser-473 phosphorylation) is required for mouse osteoblast differentiation.« less

Runx2 contributes to the regenerative potential of the mammary epithelium.

PubMed

Ferrari, Nicola; Riggio, Alessandra I; Mason, Susan; McDonald, Laura; King, Ayala; Higgins, Theresa; Rosewell, Ian; Neil, James C; Smalley, Matthew J; Sansom, Owen J; Morris, Joanna; Cameron, Ewan R; Blyth, Karen

2015-10-22

Although best known for its role in bone development and associated structures the transcription factor RUNX2 is expressed in a wide range of lineages, including those of the mammary gland. Previous studies have indicated that Runx2 can regulate aspects of mammary cell function and influence the properties of cancer cells. In this study we investigate the role of Runx2 in the mammary stem/progenitor population and its relationship with WNT signalling. Results show that RUNX2 protein is differentially expressed throughout embryonic and adult development of the murine mammary gland with high levels of expression in mammary stem-cell enriched cultures. Importantly, functional analysis reveals a role for Runx2 in mammary stem/progenitor cell function in in vitro and in vivo regenerative assays. Furthermore, RUNX2 appears to be associated with WNT signalling in the mammary epithelium and is specifically upregulated in mouse models of WNT-driven breast cancer. Overall our studies reveal a novel function for Runx2 in regulating mammary epithelial cell regenerative potential, possibly acting as a downstream target of WNT signalling.

Runx2 contributes to the regenerative potential of the mammary epithelium

PubMed Central

Ferrari, Nicola; Riggio, Alessandra I.; Mason, Susan; McDonald, Laura; King, Ayala; Higgins, Theresa; Rosewell, Ian; Neil, James C.; Smalley, Matthew J.; Sansom, Owen J.; Morris, Joanna; Cameron, Ewan R.; Blyth, Karen

2015-01-01

Although best known for its role in bone development and associated structures the transcription factor RUNX2 is expressed in a wide range of lineages, including those of the mammary gland. Previous studies have indicated that Runx2 can regulate aspects of mammary cell function and influence the properties of cancer cells. In this study we investigate the role of Runx2 in the mammary stem/progenitor population and its relationship with WNT signalling. Results show that RUNX2 protein is differentially expressed throughout embryonic and adult development of the murine mammary gland with high levels of expression in mammary stem-cell enriched cultures. Importantly, functional analysis reveals a role for Runx2 in mammary stem/progenitor cell function in in vitro and in vivo regenerative assays. Furthermore, RUNX2 appears to be associated with WNT signalling in the mammary epithelium and is specifically upregulated in mouse models of WNT-driven breast cancer. Overall our studies reveal a novel function for Runx2 in regulating mammary epithelial cell regenerative potential, possibly acting as a downstream target of WNT signalling. PMID:26489514

RUNX3 Facilitates Growth of Ewing Sarcoma Cells

PubMed Central

Bledsoe, Krista L.; McGee-Lawrence, Meghan E.; Camilleri, Emily T.; Wang, Xiaoke; van Wijnen, Andre J.; Oliveira, Andre M.; Westendorf, Jennifer J.

2014-01-01

Ewing sarcoma is an aggressive pediatric small round cell tumor that predominantly occurs in bone. Approximately 85% of Ewing sarcomas harbor the EWS/FLI fusion protein, which arises from a chromosomal translocation, t(11:22)(q24:q12). EWS/FLI interacts with numerous lineage-essential transcription factors to maintain mesenchymal progenitors in an undifferentiated state. We previously showed that EWS/FLI binds the osteogenic transcription factor RUNX2 and prevents osteoblast differentiation. In this study, we investigated the role of another Runt-domain protein, RUNX3, in Ewing sarcoma. RUNX3 participates in mesenchymal-derived bone formation and is a context dependent tumor suppressor and oncogene. RUNX3 was detected in all Ewing sarcoma cells examined, whereas RUNX2 was detected in only 73% of specimens. Like RUNX2, RUNX3 binds to EWS/FLI via its Runt domain. EWS/FLI prevented RUNX3 from activating the transcription of a RUNX-responsive reporter, p6OSE2. Stable suppression of RUNX3 expression in the Ewing sarcoma cell line A673 delayed colony growth in anchorage independent soft agar assays and reversed expression of EWS/FLI-responsive genes. These results demonstrate an important role for RUNX3 in Ewing sarcoma. PMID:24812032

Silibinin promotes osteoblast differentiation of human bone marrow stromal cells via bone morphogenetic protein signaling.

PubMed

Ying, Xiaozhou; Sun, Liaojun; Chen, Xiaowei; Xu, Huazi; Guo, Xiaoshan; Chen, Hua; Hong, Jianjun; Cheng, Shaowen; Peng, Lei

2013-12-05

Silibinin is the major active constituent of the natural compound silymarin; several studies suggest that silibinin possesses antihepatotoxic properties and anticancer effects against carcinoma cells. However, no study has yet investigated the effect of silibinin on osteogenic differentiation of human bone marrow stem cells (hBMSCs). The aim of this study was to evaluate the effect of silibinin on osteogenic differentiation of hBMSCs. In this study, the hBMSCs were cultured in an osteogenic medium with 0, 1, 10 or 20 μmol/l silibinin respectively. hBMSCs viability was analyzed by cell number quantification assay and cells osteogenic differentiation was evaluated by alkaline phosphatas (ALP) activity assay, Von Kossa staining and real time-polymerase chain reaction (RT-PCR). We found that silibinin promoted ALP activity in hBMSCs without affecting their proliferation. The mineralization of hBMSCs was enhanced by treatment with silibinin. Silibinin also increased the mRNA expressions of Collagen type I (COL-I), ALP, Osteocalcin (OCN), Osterix, bone morphogenetic protein-2 (BMP-2) and Runt-related transcription factor 2 (RUNX2). The BMP antagonist noggin and its receptor kinase inhibitors dorsomorphin and LDN-193189 attenuated silibinin-promoted ALP activity. Furthermore, BMP-responsive and Runx2-responsive reporters were activated by silibinin treatment. These results indicate that silibinin enhances osteoblast differentiation probably by inducing the expressions of BMPs and activating BMP and RUNX2 pathways. Thus, silibinin may play an important therapeutic role in osteoporosis patients by improving osteogenic differentiation of BMSCs. © 2013 Elsevier B.V. All rights reserved.

RUNX3 is involved in caspase-3-dependent apoptosis induced by a combination of 5-aza-CdR and TSA in leukaemia cell lines.

PubMed

Zhai, Feng-Xian; Liu, Xiang-Fu; Fan, Rui-Fang; Long, Zi-Jie; Fang, Zhi-Gang; Lu, Ying; Zheng, Yong-Jiang; Lin, Dong-Jun

2012-03-01

Epigenetic therapy has had a significant impact on the management of haematologic malignancies. The aim of this study was to assess whether 5-aza-CdR and TSA inhibit the growth of leukaemia cells and induce caspase-3-dependent apoptosis by upregulating RUNX3 expression. K562 and Reh cells were treated with 5-aza-CdR, TSA or both compounds. RT-PCR and Western blot analyses were used to examine the expression of RUNX3 at the mRNA and protein levels, respectively. Immunofluorescence microscopy was used to detect the cellular location of RUNX3. Additionally, after K562 cells were transfected with RUNX3, apoptosis and proliferation were studied using Annexin V staining and MTT assays. The expression of RUNX3 in leukaemia cell lines was markedly less than that in the controls. Demethylating drug 5-aza-CdR could induce RUNX3 expression, but the combination of TSA and 5-aza-CdR had a greater effect than did treatment with a single compound. The combination of 5-aza-CdR and TSA induced the translocation of RUNX3 from the cytoplasm into the nucleus. TSA enhanced apoptosis induced by 5-aza-CdR, and Annexin V and Hoechst 33258 staining showed that the combination induced apoptosis but not necrosis. Furthermore, apoptosis was dependent on the caspase-3 pathway. RUNX3 overexpression in K562 cells led to growth inhibition and apoptosis and potentiated the effects of 5-aza-CdR induction. RUNX3 plays an important role in leukaemia cellular functions, and the induction of RUNX3-mediated effects may contribute to the therapeutic value of combination TSA and 5-aza-CdR treatment.

Inhibition of FOXO1/3 promotes vascular calcification.

PubMed

Deng, Liang; Huang, Lu; Sun, Yong; Heath, Jack M; Wu, Hui; Chen, Yabing

2015-01-01

Vascular calcification is a characteristic feature of atherosclerosis, diabetes mellitus, and end-stage renal disease. We have demonstrated that activation of protein kinase B (AKT) upregulates runt-related transcription factor 2 (Runx2), a key osteogenic transcription factor that is crucial for calcification of vascular smooth muscle cells (VSMC). Using mice with SMC-specific deletion of phosphatase and tensin homolog (PTEN), a major negative regulator of AKT, the present studies uncovered a novel molecular mechanism underlying PTEN/AKT/FOXO (forkhead box O)-mediated Runx2 upregulation and VSMC calcification. SMC-specific PTEN deletion mice were generated by crossing PTEN floxed mice with SM22α-Cre transgenic mice. The PTEN deletion resulted in sustained activation of AKT that upregulated Runx2 and promoted VSMC calcification in vitro and arterial calcification ex vivo. Runx2 knockdown did not affect proliferation but blocked calcification of the PTEN-deficient VSMC, suggesting that PTEN deletion promotes Runx2-depedent VSMC calcification that is independent of proliferation. At the molecular level, PTEN deficiency increased the amount of Runx2 post-transcriptionally by inhibiting Runx2 ubiquitination. AKT activation increased phosphorylation of FOXO1/3 that led to nuclear exclusion of FOXO1/3. FOXO1/3 knockdown in VSMC phenocopied the PTEN deficiency, demonstrating a novel function of FOXO1/3, as a downstream signaling of PTEN/AKT, in regulating Runx2 ubiquitination and VSMC calcification. Using heterozygous SMC-specific PTEN-deficient mice and atherogenic ApoE(-/-) mice, we further demonstrated AKT activation, FOXO phosphorylation, and Runx2 ubiquitination in vascular calcification in vivo. Our studies have determined a new causative effect of SMC-specific PTEN deficiency on vascular calcification and demonstrated that FOXO1/3 plays a crucial role in PTEN/AKT-modulated Runx2 ubiquitination and VSMC calcification. © 2014 American Heart Association, Inc.

Accelerated leukemogenesis by truncated CBFβ-SMMHC defective in high-affinity binding with RUNX1

PubMed Central

Kamikubo, Yasuhiko; Zhao, Ling; Wunderlich, Mark; Corpora, Takeshi; Hyde, R. Katherine; Paul, Thomas A.; Kundu, Mondira; Garrett, Lisa; Compton, Sheila; Huang, Gang; Wolff, Linda; Ito, Yoshiaki; Bushweller, John; Mulloy, James C.; Liu, P. Paul

2010-01-01

SUMMARY Dominant RUNX1 inhibition has been proposed as a common pathway for CBF-leukemia. CBFβ-SMMHC, a fusion protein in human acute myeloid leukemia (AML), dominantly inhibits RUNX1 largely through its RUNX1 high-affinity binding domain (HABD). However, the type I CBFβ-SMMHC fusion in AML patients lacks HABD. Here we report that the type I CBFβ-SMMHC protein binds RUNX1 inefficiently. Knock-in mice expressing CBFβ-SMMHC with a HABD deletion developed leukemia quickly, even though hematopoietic defects associated with Runx1-inhibition were partially rescued. A larger pool of leukemia initiating cells, increased MN1 expression, and retention of RUNX1 phosphorylation are potential mechanisms for accelerated leukemia development in these mice. Our data suggest that RUNX1 dominant inhibition may not be a critical step for leukemogenesis by CBFβ-SMMHC. PMID:20478528

Macrolactin F inhibits RANKL-mediated osteoclastogenesis by suppressing Akt, MAPK and NFATc1 pathways and promotes osteoblastogenesis through a BMP-2/smad/Akt/Runx2 signaling pathway.

PubMed

Li, Liang; Sapkota, Mahesh; Gao, Ming; Choi, Hyukjae; Soh, Yunjo

2017-11-15

The balance between bone formation and bone resorption is maintained by osteoblasts and osteoclasts. In the current study, macrolactin F (MF) was investigated for novel biological activity on the receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-induced osteoclastogenesis in primary bone marrow-derived macrophages (BMMs). We found that RANKL-induced osteoclast formation and differentiation from BMMs was significantly inhibited by MF in a dose-dependent manner without cytotoxicity. RANKL-induced F-actin ring formation and bone resorption activity in BMMs which was attenuated by MF. In addition, MF suppressed the expression of osteoclast-related genes, including c-myc, RANK, tartrate-resistant acid phosphatase (TRAP), nuclear factor of activated T cells c1 (NFATc1), cathepsin K and matrix metalloproteinase 9 (MMP9). Furthermore, the protein expression NFATc1, c-Fos, MMP9, cathepsin K and phosphorylation of Jun N-terminal kinase (JNK), p38 and Akt were also down-regulated by MF treatment. Interestingly, MF promoted pre-osteoblast cell differentiation on Alizarin Red-mineralization activity, alkaline phosphatase (ALP) activity, and the expression of osteoblastogenic markers including Runx2, Osterix, Smad4, ALP, type I collagen alpha 1 (Col1α), osteopontin (OPN), and osteocalcin (OCN) via activation of the BMP-2/smad/Akt/Runx2 pathway on MC3T3-E1. Taken together, these results indicate that MF may be useful as a therapeutic agent to enhance bone health and treat osteoporosis. Copyright © 2017 Elsevier B.V. All rights reserved.

runt Homology Domain Transcription Factors (Runx, Cbfa, and AML) Mediate Repression of the Bone Sialoprotein Promoter: Evidence for Promoter Context-Dependent Activity of Cbfa Proteins

PubMed Central

Javed, Amjad; Barnes, George L.; Jasanya, B. O.; Stein, Janet L.; Gerstenfeld, Louis; Lian, Jane B.; Stein, Gary S.

2001-01-01

Expression of the bone sialoprotein (BSP) gene, a marker of bone formation, is largely restricted to cells in mineralized tissues. Recent studies have shown that the Cbfa1 (also known as Runx2, AML-3, and PEBP2αA) transcription factor supports commitment and differentiation of progenitor cells to hypertrophic chondrocytes and osteoblasts. This study addresses the functional involvement of Cbfa sites in expression of the Gallus BSP gene. Gel mobility shift analyses with nuclear extracts from ROS 17/2.8 osteoblastic cells revealed that multiple Cbfa consensus sequences are functional Cbfa DNA binding sites. Responsiveness of the 1.2-kb Gallus BSP promoter to Cbfa factors Cbfa1, Cbfa2, and Cbfa3 was assayed in osseous and nonosseous cells. Each of the Cbfa factors mediated repression of the wild-type BSP promoter, in contrast to their well known activation of various hematopoietic and skeletal phenotypic genes. Suppression of BSP by Cbfa factors was not observed in BSP promoters in which Cbfa sites were deleted or mutated. Expression of the endogenous BSP gene in Gallus osteoblasts was similarly downregulated by forced expression of Cbfa factors. Our data indicate that Cbfa repression of the BSP promoter does not involve the transducin-like enhancer (TLE) proteins. Neither coexpression of TLE1 or TLE2 nor the absence of the TLE interaction motif of Cbfa1 (amino acids 501 to 513) influenced repressor activity. However, removal of the C terminus of Cbfa1 (amino acids 362 to 513) relieved suppression of the BSP promoter. Our results, together with the evolutionary conservation of the seven Cbfa sites in the Gallus and human BSP promoters, suggest that suppressor activity by Cbfa is of significant physiologic consequence and may contribute to spatiotemporal expression of BSP during bone development. PMID:11283267

Root bark of Sambucus Williamsii Hance promotes rat femoral fracture healing by the BMP-2/Runx2 signaling pathway.

PubMed

Yang, Bingyou; Lin, Xiaoying; Tan, Jinyan; She, Xian; Liu, Yan; Kuang, Haixue

2016-09-15

Sambucus Williamsii Hance (SWH) is a plant from a family of Caprifoliaceae, which has a long medical history of use as an effective folk treatment for fracture bruises. To evaluate the effects of 50% ethanol extracts of root-bark of Sambucus Williamsii Hance(EE-rbSWH) on fracture healing of rats and explore its mechanism of actions related to the BMP-2 signaling pathway. EE-rbSWH was orally administered at the doses of 340 and 680mg/kg to adult Sprague-Dawley rats with operation of open femur fracture completely for 2, 4 and 8 weeks. And the rats of sham operation and Model groups were administered Vehicle (distilled water 0.8mL/200g/day). Firstly, the bone X-ray morphology and bone mineral density(BMD) of the fracture site were observed and measured after anesthesia the rats at weeks 2, 4, and 8 after surgery, then the serum levels of alkaline phosphatase(ALP) and osteocalcin (BGP) were measured; Secondly, the tissue morphology of the fracture site was observed after sacrificed the rats; Thirdly, the formation of mineralized nodules in bone marrow stromal cells(BMSC) were evaluated at week 2; Lastly, the genes levels of BMP-2 and Runx2 in the femur were detected at week 2 and 4, and the proteins expression of BMP-2 signaling pathway (BMP-2, BMPRIB, BMPRII and Runx2) in the femur also were detected at week 2. EE-rbSWH remarkably accelerated fracture healing by promoting bone formation at all the time points of fracture healing. Mainly by increasing the BMD level at the fracture site, the levels of serum ALP and BGP, and also the numbers increasing of calcified nodules in BMSC. The mechanism studies, EE-rbSWH can promote fracture healing by enhancing the expressions of BMP-2 and Runx2 mRNA, and also the proteins of BMP-2, BMPRIB, BMPRII and Runx2 at the fracture site of rats. Our results suggested that 50% ethanol extracts of root-bark of Sambucus Williamsii Hance can accelerate fracture healing by recruitment of osteoblasts at the fracture site and through up-regulation of the BMP-2 signaling pathway. Copyright © 2016. Published by Elsevier Ireland Ltd.

Effects of PTHrP on chondrocytes of sika deer antler.

PubMed

Guo, Bin; Wang, Shou-Tang; Duan, Cui-Cui; Li, Dang-Dang; Tian, Xue-Chao; Wang, Qu-Yuan; Yue, Zhan-Peng

2013-11-01

Parathyroid-hormone-related peptide (PTHrP) is an important regulator of chondrocyte differentiation in growth plates but little is known about its role in deer antler cartilage. The aim of the present study was to use the deer antler as a model to determine the possible role of PTHrP in regulating chondrocyte differentiation of deer antler. PTHrP and its receptor PTH1R mRNA were highly expressed in the perichondrium and cartilage of sika deer antler, as shown by in situ hybridization. Chondrocytes of deer antler were identified by toluidine blue staining of glycosaminoglycan and immunocytochemical staining of type II collagen (Col II). Treatment with PTHrP (1-34) reduced the expression of prehypertrophic chondrocyte marker Col IX and hypertrophic chondrocyte marker Col X. In order to confirm the mechanism of action of PTHrP, we initially examined the expression of cyclin D1, Bcl-2 and runt-related transcription factor 2 (Runx2) in sika deer antler by in situ hybridization and found that cyclin D1, Runx2 and Bcl-2 mRNA were also expressed in antler chondrocytes. Exogenous PTHrP induced the expression of cyclin D1 and Bcl-2 mRNA by various signalling pathways, whereas it inhibited Runx2 expression through PKA, p38MAPK, MEK and PI3K signalling pathways. Thus, PTHrP might promote the proliferation of antler chondrocytes and prevent their differentiation; it might furthermore influence the growth and development of sika deer antler.

Klf10 regulates odontoblast differentiation and mineralization via promoting expression of dentin matrix protein 1 and dentin sialophosphoprotein genes

PubMed Central

Chen, Zhuo; Li, Wentong; Wang, Han; Wan, Chunyan; Luo, Daoshu; Deng, Shuli

2016-01-01

Klf10, a member of the Krüppel-like family of transcription factors, is critical for osteoblast differentiation, bone formation and mineralization. However, whether Klf10 is involved in odontoblastic differentiation and tooth development has not been determined. In this study, we investigate the expression patterns of Klf10 during murine tooth development in vivo and its role in odontoblastic differentiation in vitro. Klf10 protein was expressed in the enamel organ and the underlying mesenchyme, ameloblasts and odontoblasts at early and later stages of murine molar formation. Furthermore, the expression of Klf10, Dmp1, Dspp and Runx2 was significantly elevated during the process of mouse dental papilla mesenchymal differentiation and mineralization. The overexpression of Klf10 induced dental papilla mesenchymal cell differentiation and mineralization as detected by alkaline phosphatase staining and alizarin red S assay. Klf10 additionally up-regulated the expression of odontoblastic differentiation marker genes Dmp1, Dspp and Runx2 in mouse dental papilla mesenchymal cells. The molecular mechanism of Klf10 in controlling Dmp1 and Dspp expression is thus to activate their regulatory regions in a dosage-dependent manner. Our results suggest that Klf10 is involved in tooth development and promotes odontoblastic differentiation via the up-regulation of Dmp1 and Dspp transcription. PMID:26310138

Epigenetic heterogeneity affects the risk of relapse in children with t(8;21)RUNX1-RUNX1T1-rearranged AML.

PubMed

Zampini, Matteo; Tregnago, Claudia; Bisio, Valeria; Simula, Luca; Borella, Giulia; Manara, Elena; Zanon, Carlo; Zonta, Francesca; Serafin, Valentina; Accordi, Benedetta; Campello, Silvia; Buldini, Barbara; Pession, Andrea; Locatelli, Franco; Basso, Giuseppe; Pigazzi, Martina

2018-05-01

The somatic translocation t(8;21)(q22;q22)/RUNX1-RUNX1T1 is one of the most frequent rearrangements found in children with standard-risk acute myeloid leukemia (AML). Despite the favorable prognostic role of this aberration, we recently observed a higher than expected frequency of relapse. Here, we employed an integrated high-throughput approach aimed at identifying new biological features predicting relapse among 34 t(8;21)-rearranged patients. We found that the DNA methylation status of patients who suffered from relapse was peculiarly different from that of children maintaining complete remission. The epigenetic signature, made up of 337 differentially methylated regions, was then integrated with gene and protein expression profiles, leading to a network, where cell-to-cell adhesion and cell-motility pathways were found to be aberrantly activated in relapsed patients. We identified most of these factors as RUNX1-RUNX1T1 targets, with Ras Homolog Family Member (RHOB) overexpression being the core of this network. We documented how RHOB re-organized the actin cytoskeleton through its downstream ROCK-LIMK-COFILIN axis: this increases blast adhesion by stress fiber formation, and reduces mitochondrial apoptotic cell death after chemotherapy treatment. Altogether, our data show an epigenetic heterogeneity within t(8;21)-rearranged AML patients at diagnosis able to influence the program of the chimeric transcript, promoting blast re-emergence and progression to relapse.

miR-764-5p promotes osteoblast differentiation through inhibition of CHIP/STUB1 expression.

PubMed

Guo, Junwei; Ren, Fangli; Wang, Yinyin; Li, Shan; Gao, Zhengrong; Wang, Xiaoyan; Ning, Hongxiu; Wu, Jianguo; Li, Yi; Wang, Zhao; Chim, Shek Man; Xu, Jiake; Chang, Zhijie

2012-07-01

Differentiation of committed precursor cells into the osteoblast lineage is tightly regulated by several factors, including Runx2 and BMP2. We previously reported that C terminus of Hsc70-interacting protein/STIP1 homology and U-Box containing protein 1 (CHIP/STUB1) negatively regulated osteoblast differentiation through promoting Runx2 protein degradation. However, how CHIP is regulated during osteoblast differentiation remains unknown. In this study, we found that miR-764-5p is up-expressed during the osteoblast differentiation in calvarial and osteoblast progenitor cells, coupled with down-expression of CHIP protein. We observed that forced expression or inhibition of miR-764-5p decreased or increased the CHIP protein level through affecting its translation by targeting the 3'-UTR region. Perturbation of miR-764-5p resulted in altered differentiation fate of osteoblast progenitor cells and the role of miR-764-5p was reversed by overexpression of CHIP, whereas depletion of CHIP impaired the effect of miR-764-5p. Our data showed that miR-764-5p positively regulates osteoblast differentiation from osteoblast progenitor cells by repressing the translation of CHIP protein. Copyright © 2012 American Society for Bone and Mineral Research.

Epigenetic Control of Prostate Cancer Metastasis: Role of Runx2 Phosphorylation

DTIC Science & Technology

2014-04-01

prostate cancer cells. In the third budget year, we achieved the following: a. Generation of retrovirus and lentivirus vectors expressing WT RUNX2 and S301A... retrovirus vectors will be developed that express β-galactosidase (negative control), wild type Runx2, S301A/S319A (non-phosphorylated) or S301E/S310E...constitutively active) Runx2 mutants. As described last year, retrovirus and lentivirus vectors were constructed to stably introduce wild type and mutant

Core binding factor beta (Cbfβ) controls the balance of chondrocyte proliferation and differentiation by upregulating Indian hedgehog (Ihh) expression and inhibiting parathyroid hormone-related protein receptor (PPR) expression in postnatal cartilage and bone formation.

PubMed

Tian, Fei; Wu, Mengrui; Deng, Lianfu; Zhu, Guochun; Ma, Junqing; Gao, Bo; Wang, Lin; Li, Yi-Ping; Chen, Wei

2014-07-01

Core binding factor beta (Cbfβ) is essential for embryonic bone morphogenesis. Yet the mechanisms by which Cbfβ regulates chondrocyte proliferation and differentiation as well as postnatal cartilage and bone formation remain unclear. Hence, using paired-related homeobox transcription factor 1-Cre (Prx1-Cre) mice, mesenchymal stem cell-specific Cbfβ-deficient (Cbfβ(f/f) Prx1-Cre) mice were generated to study the role of Cbfβ in postnatal cartilage and bone development. These mutant mice survived to adulthood but exhibited severe sternum and limb malformations. Sternum ossification was largely delayed in the Cbfβ(f/f) Prx1-Cre mice and the xiphoid process was noncalcified and enlarged. In newborn and 7-day-old Cbfβ(f/f) Prx1-Cre mice, the resting zone was dramatically elongated, the proliferation zone and hypertrophic zone of the growth plates were drastically shortened and disorganized, and trabecular bone formation was reduced. Moreover, in 1-month-old Cbfβ(f/f) Prx1-Cre mice, the growth plates were severely deformed and trabecular bone was almost absent. In addition, Cbfβ deficiency impaired intramembranous bone formation both in vivo and in vitro. Interestingly, although the expression of Indian hedgehog (Ihh) was largely reduced, the expression of parathyroid hormone-related protein (PTHrP) receptor (PPR) was dramatically increased in the Cbfβ(f/f) Prx1-Cre growth plate, indicating that that Cbfβ deficiency disrupted the Ihh-PTHrP negative regulatory loop. Chromatin immunoprecipitation (ChIP) analysis and promoter luciferase assay demonstrated that the Runx/Cbfβ complex binds putative Runx-binding sites of the Ihh promoter regions, and also the Runx/Cbfβ complex directly upregulates Ihh expression at the transcriptional level. Consistently, the expressions of Ihh target genes, including CyclinD1, Ptc, and Pthlh, were downregulated in Cbfβ-deficient chondrocytes. Taken together, our study reveals not only that Cbfβ is essential for chondrocyte proliferation and differentiation for the growth and maintenance of the skeleton in postnatal mice, but also that it functions in upregulating Ihh expression to promoter chondrocyte proliferation and osteoblast differentiation, and inhibiting PPR expression to enhance chondrocyte differentiation. © 2014 American Society for Bone and Mineral Research.

Tumor Necrosis Factor Receptor-associated Factor 6 Is an Intranuclear Transcriptional Coactivator in Osteoclasts*

PubMed Central

Bai, Shuting; Zha, Jikun; Zhao, Haibo; Ross, F. Patrick; Teitelbaum, Steven L.

2008-01-01

Tumor necrosis factor receptor-associated factor 6 (TRAF6) associates with the cytoplasmic domain of receptor activator of NF-κB (RANK) and is an essential component of the signaling complex mediating osteoclastogenesis. However, the osteoclastic activity of TRAF6 is blunted by its association with four and half LIM domain 2 (FHL2), which functions as an adaptor protein in the cytoplasm and transcriptional regulator in the nucleus. We find that TRAF6 also localizes in the nuclei of osteoclasts but not their bone marrow macrophage precursors and that osteoclast intranuclear abundance is specifically increased by RANK ligand (RANKL). TRAF6 nuclear localization requires FHL2 and is diminished in fhl2-/- osteoclasts. Suggesting transcriptional activity, TRAF6 interacts with the transcription factor RUNX1 in the osteoclast nucleus. FHL2 also associates with RUNX1 but does so only in the presence of TRAF6. Importantly, TRAF6 recognizes FHL2 and RUNX1 in osteoclast nuclei, and the three molecules form a DNA-binding complex that recognizes and transactivates the RUNX1 response element in the fhl2 promoter. Finally, TRAF6 and its proximal activator, RANKL, polyubiquitinate FHL2, prompting its proteasomal degradation. These observations suggest a feedback mechanism whereby TRAF6 negatively regulates osteoclast formation by intracytoplasmic sequestration of FHL2 to blunt RANK activation and as a component of a transcription complex promoting FHL2 expression. PMID:18768464

MicroRNA-196b Inhibits Cell Growth and Metastasis of Lung Cancer Cells by Targeting Runx2.

PubMed

Bai, Xiaoxue; Meng, Lin; Sun, Huijie; Li, Zhuo; Zhang, Xiufang; Hua, Shucheng

2017-01-01

Lung cancer is one of the most common causes of cancer related deaths worldwide. The role of several microRNAs (miRNAs) including miR-196b in different cancers has already been established. The study was aimed to explore the role of miR-196b in lung cancer and its possible underlying mechanism. Human lung cancer cell line A549 was transfected with miR-196b mimic, miR-196b inhibitor and corresponding controls. Then cell viability, migration, invasion, and apoptosis of A549 lung cancer cells either with overexpression or with suppression of miR-196b were estimated sequentially. Next, dual luciferase activity assay was performed to clarify whether Runx2 was a direct target of miR-196b. Finally, the expressions of main factors associated with epithelial mesenchymal transition (EMT), PI3K/AKT/GSK3β, Smad, and JNK pathways were detected by western blot. MiR-196b expression was significantly decreased in A549, H1650 and H1299 cell lines compared with in WI-38 and HEL-1 cell lines. Overexpression of miR-196b suppressed cell viability, migration, invasion, and induced apoptosis as well as inhibited TGF-β induced EMT process in A549 cells. In addition, Runx2 was a putative target of miR-196b, and Runx2 silence remarkably increased cell apoptosis and abolished the promotive effects of miR-196b suppression on cell viability, migration and invasion. Finally, miR-196b also mediated its action by inactivation of PI3K/AKT/GSK3β, Smad, and JNK pathways by down-regulation of Runx2. MiR-196b functions as a tumor suppressor that inhibited cell growth and metastasis of lung cancer cells by targeting Runx2. These findings provided further evidences for treatment of lung cancer. The Author(s). Published by S. Karger AG, Basel.

Baghdadite ceramics modulate the cross talk between human adipose stem cells and osteoblasts for bone regeneration.

PubMed

Lu, Zufu; Wang, Guocheng; Roohani-Esfahani, Iman; Dunstan, Colin R; Zreiqat, Hala

2014-03-01

Understanding interactions among the three elements (cells, scaffolds, and bioactive factors) is critical for successful tissue engineering. This study was aimed to investigate how scaffolds would affect osteogenic gene expression in human adipose tissue-derived stem cells (ASCs) or human primary osteoblasts (HOBs), and their cross talk. Either ASCs or HOBs were seeded on Baghdadite (Ca3ZrSi2O9) and hydroxyapatite/tricalcium phosphate (HA/TCP) scaffolds, and osteogenic gene expression was assessed. To further evaluate how substrate affected HOB and ASC cross talk, an indirect co-culture system with semipermeable inserts placed on the culture plate was set up to co-culture ASCs or HOBs, which were grown in monolayer or seeded on Baghdadite or HA/TCP scaffolds, and osteogenic differentiation of the cells was assessed. We found that Baghdadite scaffolds induced a significantly greater increase in RUNX2, osteopontin, bone sialoprotein, and osteocalcin gene expression in HOBs in comparison to HA/TCP scaffolds; Baghdadite scaffolds also significantly induced RUNX2 and osteopontin, but not bone sialoprotein and osteocalcin gene expression in ASCs. In the co-culture system, the HOBs on Baghdadite scaffolds more markedly promoted osteogenic gene expression in ASCs compared to HOBs in monolayer or the HOBs on HA/TCP scaffolds. In addition, the ASCs seeded on Baghdadite scaffolds more markedly promoted osteogenic gene expression in HOBs than did the ASCs on HA/TCP scaffolds. BMP-2 expression in ASCs or HOBs was increased when they were seeded on Baghdadite scaffolds, and adding Noggin into the co-culture medium largely abrogated Baghdadite scaffold-modulated ASC-HOB cross talk. In summary, Baghdadite scaffolds not only promote the osteogenic differentiation of HOBs or ASCs but also modulate the cross talk between ASCs and HOBs, in part via increasing BMP2 expression, thereby promoting their osteogenic differentiation.

Wnt signaling inhibits cementoblast differentiation and promotes proliferation.

PubMed

Nemoto, Eiji; Koshikawa, Yohei; Kanaya, Sousuke; Tsuchiya, Masahiro; Tamura, Masato; Somerman, Martha J; Shimauchi, Hidetoshi

2009-05-01

Cementoblasts, tooth root lining cells, are responsible for laying down cementum on the root surface, a process that is indispensable for establishing a functional periodontal ligament. Cementoblasts share phenotypical features with osteoblasts. Wnt signaling has been implicated in increased bone formation by controlling mesenchymal stem cell or osteoblastic cell functions; however the role of Wnt signaling on cementogenesis has not been examined. In this study, we have identified a consistent expression profile of Wnt signaling molecules in cementoblasts, in vitro by RT-PCR. Exposure of cells to LiCl, which promotes canonical Wnt signaling by inhibiting GSK-3beta, increased beta-catenin nuclear translocation and up-regulated the transcriptional activity of a canonical Wnt-responsive promoters, suggesting that an endogenous canonical Wnt pathway functions in cementoblasts. Activation of endogenous canonical Wnt signaling with LiCl suppressed alkaline phosphatase (ALP) activity and expression of genes associated with cementum function; ALP, bone sialoprotein (BSP), and osteocalcin (OCN). Exposure to Wnt3a, as a representative canonical Wnt member, also inhibited the expression of ALP, BSP, and OCN gene. This effect was accompanied by decreased gene expression of Runx2 and Osterix and by increased gene expression of lymphoid enhancer factor-1. Pretreatment with Dickkopf (Dkk)-1, a potent canonical Wnt antagonist, which binds to a low-density lipoprotein-receptor-related protein (LRP)-5/6 co-receptor, attenuated the suppressive effects of Wnt3a on mRNA expression of Runx2 and OCN on cementoblasts. These findings suggest that canonical Wnt signaling inhibits cementoblast differentiation via regulation of expression of selective transcription factors. Wnt3a also increased the expression of cyclin D1, known as a cell cycle regulator, as well as cell proliferation. In conclusion, these observations suggest that Wnt signaling inhibits cementoblast differentiation and promotes cell proliferation. Elucidating the role of Wnt in controlling cementoblast function will provide new tools needed to improve on existing periodontal regeneration therapies.

Osteogenic Differentiation of Three-Dimensional Bioprinted Constructs Consisting of Human Adipose-Derived Stem Cells In Vitro and In Vivo.

PubMed

Wang, Xiao-Fei; Song, Yang; Liu, Yun-Song; Sun, Yu-Chun; Wang, Yu-Guang; Wang, Yong; Lyu, Pei-Jun

2016-01-01

Here, we aimed to investigate osteogenic differentiation of human adipose-derived stem cells (hASCs) in three-dimensional (3D) bioprinted tissue constructs in vitro and in vivo. A 3D Bio-plotter dispensing system was used for building 3D constructs. Cell viability was determined using live/dead cell staining. After 7 and 14 days of culture, real-time quantitative polymerase chain reaction (PCR) was performed to analyze the expression of osteogenesis-related genes (RUNX2, OSX, and OCN). Western blotting for RUNX2 and immunofluorescent staining for OCN and RUNX2 were also performed. At 8 weeks after surgery, osteoids secreted by osteogenically differentiated cells were assessed by hematoxylin-eosin (H&E) staining, Masson trichrome staining, and OCN immunohistochemical staining. Results from live/dead cell staining showed that most of the cells remained alive, with a cell viability of 89%, on day 1 after printing. In vitro osteogenic induction of the 3D construct showed that the expression levels of RUNX2, OSX, and OCN were significantly increased on days 7 and 14 after printing in cells cultured in osteogenic medium (OM) compared with that in normal proliferation medium (PM). Fluorescence microscopy and western blotting showed that the expression of osteogenesis-related proteins was significantly higher in cells cultured in OM than in cells cultured in PM. In vivo studies demonstrated obvious bone matrix formation in the 3D bioprinted constructs. These results indicated that 3D bioprinted constructs consisting of hASCs had the ability to promote mineralized matrix formation and that hASCs could be used in 3D bioprinted constructs for the repair of large bone tissue defects.

Osteogenic Differentiation of Three-Dimensional Bioprinted Constructs Consisting of Human Adipose-Derived Stem Cells In Vitro and In Vivo

PubMed Central

Liu, Yun-Song; Sun, Yu-chun; Wang, Yu-guang; Wang, Yong; Lyu, Pei-Jun

2016-01-01

Here, we aimed to investigate osteogenic differentiation of human adipose-derived stem cells (hASCs) in three-dimensional (3D) bioprinted tissue constructs in vitro and in vivo. A 3D Bio-plotter dispensing system was used for building 3D constructs. Cell viability was determined using live/dead cell staining. After 7 and 14 days of culture, real-time quantitative polymerase chain reaction (PCR) was performed to analyze the expression of osteogenesis-related genes (RUNX2, OSX, and OCN). Western blotting for RUNX2 and immunofluorescent staining for OCN and RUNX2 were also performed. At 8 weeks after surgery, osteoids secreted by osteogenically differentiated cells were assessed by hematoxylin-eosin (H&E) staining, Masson trichrome staining, and OCN immunohistochemical staining. Results from live/dead cell staining showed that most of the cells remained alive, with a cell viability of 89%, on day 1 after printing. In vitro osteogenic induction of the 3D construct showed that the expression levels of RUNX2, OSX, and OCN were significantly increased on days 7 and 14 after printing in cells cultured in osteogenic medium (OM) compared with that in normal proliferation medium (PM). Fluorescence microscopy and western blotting showed that the expression of osteogenesis-related proteins was significantly higher in cells cultured in OM than in cells cultured in PM. In vivo studies demonstrated obvious bone matrix formation in the 3D bioprinted constructs. These results indicated that 3D bioprinted constructs consisting of hASCs had the ability to promote mineralized matrix formation and that hASCs could be used in 3D bioprinted constructs for the repair of large bone tissue defects. PMID:27332814

Ibandronate promotes osteogenic differentiation of periodontal ligament stem cells by regulating the expression of microRNAs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhou, Qiang; Zhao, Zhi-Ning; Cheng, Jing-Tao

2011-01-07

Research highlights: {yields} Ibandronate significantly promote the proliferation of PDLSC cells. {yields} Ibandronate enhanced the expression of ALP, COL-1, OPG, OCN, Runx2. {yields} The expression of a class of miRNAs, e.g., miR-18a, miR-133a, miR-141 and miR-19a, was significantly modified in PDLSC cells cultured with ibandronate. {yields} Ibandronate regulates the expression of diverse bone formation-related genes via miRNAs in PDLSCs. {yields} Ibandronate can suppress the activity of osteoclast while promoting the proliferation of osteoblast by regulating the expression of microRNAs. -- Abstract: Bisphosphonates (BPs) have a profound effect on bone resorption and are widely used to treat osteoclast-mediated bone diseases. Theymore » suppress bone resorption by inhibiting the activity of mature osteoclasts and/or the formation of new osteoclasts. Osteoblasts may be an alternative target for BPs. Periodontal ligament stem cells (PDLSCs) exhibit osteoblast-like features and are capable of differentiating into osteoblasts or cementoblasts. This study aimed to determine the effects of ibandronate, a nitrogen-containing BP, on the proliferation and the differentiation of PDLSCs and to identify the microRNAs (miRNAs) that mediate these effects. The PDLSCs were treated with ibandronate, and cell proliferation was measured using the MTT (3-dimethylthiazol-2,5-diphenyltetrazolium bromide) assay. The expression of genes and miRNAs involved in osteoblastic differentiation was assayed using quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR). Ibandronate promoted the proliferation of PDLSCs and enhanced the expression of alkaline phosphatase (ALP), type I collagen (COL-1), osteoprotegerin (OPG), osteocalcin (OCN), and Runx2. The expression of miRNAs, including miR-18a, miR-133a, miR-141 and miR-19a, was significantly altered in the PDLSCs cultured with ibandronate. In PDLSCs, ibandronate regulates the expression of diverse bone formation-related genes via miRNAs. The exact mechanism underlying the role of ibandronate in osteoblasts has not been completely understood. Ibandronate may suppress the activity of osteoclasts while promoting the proliferation of osteoblasts by regulating the expression of miRNAs.« less

The temporal expression of estrogen receptor alpha-36 and runx2 in human bone marrow derived stromal cells during osteogenesis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Francis, W.R., E-mail: w.francis@swansea.ac.uk; Owens, S.E.; Wilde, C.

2014-10-24

Highlights: • ERα36 is the predominant ERα isoform involved in bone regulation in human BMSC. • ERα36 mRNA is significantly upregulated during the process of osteogenesis. • The pattern of ERα36 and runx2 mRNA expression is similar during osteogenesis. • ERα36 appears to be co-localised with runx2 during osteogenesis. - Abstract: During bone maintenance in vivo, estrogen signals through estrogen receptor (ER)-α. The objectives of this study were to investigate the temporal expression of ERα36 and ascertain its functional relevance during osteogenesis in human bone marrow derived stromal cells (BMSC). This was assessed in relation to runt-related transcription factor-2 (runx2),more » a main modulatory protein involved in bone formation. ERα36 and runx2 subcellular localisation was assessed using immunocytochemistry, and their mRNA expression levels by real time PCR throughout the process of osteogenesis. The osteogenically induced BMSCs demonstrated a rise in ERα36 mRNA during proliferation followed by a decline in expression at day 10, which represents a change in dynamics within the culture between the proliferative stage and the differentiative stage. The mRNA expression profile of runx2 mirrored that of ERα36 and showed a degree subcellular co-localisation with ERα36. This study suggests that ERα36 is involved in the process of osteogenesis in BMSCs, which has implications in estrogen deficient environments.« less

Intersection of FOXO- and RUNX1-mediated gene expression programs in single breast epithelial cells during morphogenesis and tumor progression.

PubMed

Wang, Lixin; Brugge, Joan S; Janes, Kevin A

2011-10-04

Gene expression networks are complicated by the assortment of regulatory factors that bind DNA and modulate transcription combinatorially. Single-cell measurements can reveal biological mechanisms hidden by population averages, but their value has not been fully explored in the context of mRNA regulation. Here, we adapted a single-cell expression profiling technique to examine the gene expression program downstream of Forkhead box O (FOXO) transcription factors during 3D breast epithelial acinar morphogenesis. By analyzing patterns of mRNA fluctuations among individual matrix-attached epithelial cells, we found that a subset of FOXO target genes was jointly regulated by the transcription factor Runt-related transcription factor 1 (RUNX1). Knockdown of RUNX1 causes hyperproliferation and abnormal morphogenesis, both of which require normal FOXO function. Down-regulating RUNX1 and FOXOs simultaneously causes widespread oxidative stress, which arrests proliferation and restores normal acinar morphology. In hormone-negative breast cancers lacking human epidermal growth factor receptor 2 (HER2) amplification, we find that RUNX1 down-regulation is strongly associated with up-regulation of FOXO1, which may be required to support growth of RUNX1-negative tumors. The coordinate function of these two tumor suppressors may provide a failsafe mechanism that inhibits cancer progression.

RUNX1 promotes cell growth in human T-cell acute lymphoblastic leukemia by transcriptional regulation of key target genes.

PubMed

Jenkins, Catherine E; Gusscott, Samuel; Wong, Rachel J; Shevchuk, Olena O; Rana, Gurneet; Giambra, Vincenzo; Tyshchenko, Kateryna; Islam, Rashedul; Hirst, Martin; Weng, Andrew P

2018-05-04

RUNX1 is frequently mutated in T-cell acute lymphoblastic leukemia (T-ALL). The spectrum of RUNX1 mutations has led to the notion that it acts as a tumor suppressor in this context; however, other studies have placed RUNX1 along with transcription factors TAL1 and NOTCH1 as core drivers of an oncogenic transcriptional program. To reconcile these divergent roles, we knocked down RUNX1 in human T-ALL cell lines and deleted Runx1 or Cbfb in primary mouse T-cell leukemias. RUNX1 depletion consistently resulted in reduced cell proliferation and increased apoptosis. RUNX1 upregulated variable sets of target genes in each cell line, but consistently included a core set of oncogenic effectors including IGF1R and NRAS. Our results support the conclusion that RUNX1 has a net positive effect on cell growth in the context of established T-ALL. Copyright © 2018. Published by Elsevier Inc.

RUNX1 reshapes the epigenetic landscape at the onset of haematopoiesis.

PubMed

Lichtinger, Monika; Ingram, Richard; Hannah, Rebecca; Müller, Dorothee; Clarke, Deborah; Assi, Salam A; Lie-A-Ling, Michael; Noailles, Laura; Vijayabaskar, M S; Wu, Mengchu; Tenen, Daniel G; Westhead, David R; Kouskoff, Valerie; Lacaud, Georges; Göttgens, Berthold; Bonifer, Constanze

2012-11-14

Cell fate decisions during haematopoiesis are governed by lineage-specific transcription factors, such as RUNX1, SCL/TAL1, FLI1 and C/EBP family members. To gain insight into how these transcription factors regulate the activation of haematopoietic genes during embryonic development, we measured the genome-wide dynamics of transcription factor assembly on their target genes during the RUNX1-dependent transition from haemogenic endothelium (HE) to haematopoietic progenitors. Using a Runx1-/- embryonic stem cell differentiation model expressing an inducible Runx1 gene, we show that in the absence of RUNX1, haematopoietic genes bind SCL/TAL1, FLI1 and C/EBPβ and that this early priming is required for correct temporal expression of the myeloid master regulator PU.1 and its downstream targets. After induction, RUNX1 binds to numerous de novo sites, initiating a local increase in histone acetylation and rapid global alterations in the binding patterns of SCL/TAL1 and FLI1. The acquisition of haematopoietic fate controlled by Runx1 therefore does not represent the establishment of a new regulatory layer on top of a pre-existing HE program but instead entails global reorganization of lineage-specific transcription factor assemblies.

RUNX1 reshapes the epigenetic landscape at the onset of haematopoiesis

PubMed Central

Lichtinger, Monika; Ingram, Richard; Hannah, Rebecca; Müller, Dorothee; Clarke, Deborah; Assi, Salam A; Lie-A-Ling, Michael; Noailles, Laura; Vijayabaskar, M S; Wu, Mengchu; Tenen, Daniel G; Westhead, David R; Kouskoff, Valerie; Lacaud, Georges; Göttgens, Berthold; Bonifer, Constanze

2012-01-01

Cell fate decisions during haematopoiesis are governed by lineage-specific transcription factors, such as RUNX1, SCL/TAL1, FLI1 and C/EBP family members. To gain insight into how these transcription factors regulate the activation of haematopoietic genes during embryonic development, we measured the genome-wide dynamics of transcription factor assembly on their target genes during the RUNX1-dependent transition from haemogenic endothelium (HE) to haematopoietic progenitors. Using a Runx1−/− embryonic stem cell differentiation model expressing an inducible Runx1 gene, we show that in the absence of RUNX1, haematopoietic genes bind SCL/TAL1, FLI1 and C/EBPβ and that this early priming is required for correct temporal expression of the myeloid master regulator PU.1 and its downstream targets. After induction, RUNX1 binds to numerous de novo sites, initiating a local increase in histone acetylation and rapid global alterations in the binding patterns of SCL/TAL1 and FLI1. The acquisition of haematopoietic fate controlled by Runx1 therefore does not represent the establishment of a new regulatory layer on top of a pre-existing HE program but instead entails global reorganization of lineage-specific transcription factor assemblies. PMID:23064151

ET-1 Promotes Differentiation of Periodontal Ligament Stem Cells into Osteoblasts through ETR, MAPK, and Wnt/β-Catenin Signaling Pathways under Inflammatory Microenvironment

PubMed Central

Liang, Li; Zhou, Wei; Yang, Nan; Yu, Jifeng; Liu, Hongchen

2016-01-01

Periodontitis is a kind of chronic inflammatory disease that affects the tooth-supporting tissues. ET-1 is related to periodontitis and involved in the regulation of cytokines, but the mechanisms remain unclear. The aim of this study is to investigate how ET-1 affects proinflammatory cytokine expression and differentiation in human periodontal ligament stem cells (PDLSCs). PDLSCs were isolated from the periodontal ligament tissues of periodontitis patients and then treated with ET-1 (1, 10, or 100 nM) for 12 h, 24 h, or 72 h. The osteogenic potential of PDLSCs was tested using ALP staining. TNF-α, IL-1β, and IL-6 levels were evaluated by ELISA and western blot. Runx2, OCN, and COL1 mRNA and western levels were detected by RT-PCR and western blot, respectively. To examine the signaling pathways and molecular mechanisms involved in ET-1-mediated cytokine expression and osteogenic differentiation, ETR pathway, MAPKs pathway, Wnt/β-catenin pathway, and Wnt/Ca2+ pathway were detected by RT-PCR and western blot, respectively. ET-1 promoted differentiation of PDLSCs into osteoblasts by increasing secretion of TNF-α, IL-1β, and IL-6 in a dose- and time-dependent manner. ET-1 also increased expression of Runx2, OCN, and COL1. ET-1 promotes differentiation of PDLSCs into osteoblasts through ETR, MAPK, and Wnt/β-catenin signaling pathways under inflammatory microenvironment. PMID:26884650

Participation of GATA-3 in regulation of bone healing through transcriptional upregulation of bcl-xL expression

PubMed Central

Liao, Mei-Hsiu; Lin, Pei-I; Ho, Wei-Pin; Chan, Wing P; Chen, Ta-Liang; Chen, Ruei-Ming

2017-01-01

We have previously demonstrated the expression of GATA-DNA-binding protein (GATA)-3, a transcription factor, in osteoblasts and have verified its function in transducing cell survival signaling. This translational study was further designed to evaluate the roles of GATA-3 in regulating bone healing and to explore its possible mechanisms. A metaphyseal bone defect was created in the left femurs of male ICR mice. Analysis by micro-computed topography showed that the bone volume, trabecular bone number and trabecular thickness were augmented and that the trabecular pattern factor decreased. Interestingly, immunohistological analyses showed specific expression of GATA-3 in the defect area. In addition, colocalized expression of GATA-3 and alkaline phosphatase was observed at the wound site. As the fracture healed, the amounts of phosphorylated and non-phosphorylated GATA-3 concurrently increased. Separately, GATA-3 mRNA was induced during bone healing, and, levels of Runx2 mRNA and protein were also increased. The results of confocal microscopy and co-immunoprecipitation showed an association between nuclear GATA-3 and Runx2 in the area of insult. In parallel with fracture healing, Bcl-XL mRNA was significantly triggered. A bioinformatic search revealed the existence of a GATA-3-specific DNA-binding element in the promoter region of the bcl-xL gene. Analysis by chromatin immunoprecipitation assays further demonstrated transactivation activity by which GATA-3 regulated bcl-xL gene expression. Therefore, this study shows that GATA-3 participates in the healing of bone fractures via regulating bcl-xL gene expression, owing to its association with Runx2. In the clinic, GATA-3 may be used as a biomarker for diagnoses/prognoses or as a therapeutic target for bone diseases, such as bone fractures. PMID:29170477

Codonolactone, a sesquiterpene lactone isolated from Chloranthus henryi Hemsl, inhibits breast cancer cell invasion, migration and metastasis by downregulating the transcriptional activity of Runx2.

PubMed

Wang, Wei; Chen, Bin; Zou, Ruolan; Tu, Xiuying; Tan, Songlin; Lu, Hong; Liu, Zhaojie; Fu, Jianjiang

2014-11-01

Metastasis is the most insidious aspect of breast cancer, but effective strategies to control this malignant process are still lacking. In previous studies, we screened over 200 extracts from plants of genus Chloranthaceae by bioactivity-guided fractionation, and found that Codonolactone (CLT) exhibited potential antimetastatic properties in breast cancer cells. This sesquiterpene lactone was isolated from Chloranthus henryi Hemsl, and is also found in other medical herbs, such as Codonopsis pilosula, Atractylodes macrocephala Koidz and others. Here, we report that CLT inhibited the ability of invasion and migration in metastatic breast cancer cells. Furthermore, CLT exhibited significant suppression on formation of lung metastatic foci of breast cancer in vivo. We next investigated the mechanism of CLT-induced metastasis inhibitory effects in breast cancer cells. A significant inhibition on activity and expression of MMP-9 and MMP-13 was observed. Moreover, data from western blotting, Runx2 transcription factor assay and chromatin immunoprecipitation assay showed that binding ability of Runx2 to sequences of the mmp-13 promoter was inhibited by CLT. Collectively, these findings suggested that the antimetastatic properties of CLT in breast cancer were due to the inhibition of MMPs, which might be associated with a downregulation of Runx2 transcriptional activity.

Nemo-like kinase (NLK) expression in osteoblastic cells and suppression of osteoblastic differentiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nifuji, Akira, E-mail: nifuji-a@tsurumi-u.ac.jp; Department of Pharmacology, Tsurumi University School of Dental Medicine, Yokohama; Ideno, Hisashi

2010-04-15

Mitogen-activated protein kinases (MAPKs) regulate proliferation and differentiation in osteoblasts. The vertebral homologue of nemo, nemo-like kinase (NLK), is an atypical MAPK that targets several signaling components, including the T-cell factor/lymphoid enhancer factor (TCF/Lef1) transcription factor. Recent studies have shown that NLK forms a complex with the histone H3-K9 methyltransferase SETDB1 and suppresses peroxisome proliferator-activated receptor (PPAR)-gamma:: action in the mesenchymal cell line ST2. Here we investigated whether NLK regulates osteoblastic differentiation. We showed that NLK mRNA is expressed in vivo in osteoblasts at embryonic day 18.5 (E18.5) mouse calvariae. By using retrovirus vectors, we performed forced expression of NLKmore » in primary calvarial osteoblasts (pOB cells) and the mesenchymal cell line ST2. Wild-type NLK (NLK-WT) suppressed alkaline phosphatase activity and expression of bone marker genes such as alkaline phosphatase, type I procollagen, runx2, osterix, steopontin and osteocalcin in these cells. NLK-WT also decreased type I collagen protein expression in pOB and ST2 cells. Furthermore, mineralized nodule formation was reduced in pOB cells overexpressing NLK-WT. In contrast, kinase-negative form of NLK (NLK-KN) did not suppress or partially suppress ALP activity and bone marker gene expression in pOB and ST2 cells. NLK-KN did not suppress nodule formation in pOB cells. In addition to forced expression, suppression of endogenous NLK expression by siRNA increased bone marker gene expression in pOB and ST2 cells. Finally, transcriptional activity analysis of gene promoters revealed that NLK-WT suppressed Wnt1 activation of TOP flash promoter and Runx2 activation of the osteocalcin promoter. Taken together, these results suggest that NLK negatively regulates osteoblastic differentiation.« less

Immunohistochemical expression of hard tissue related factors in the mouse dental pulp after immediate teeth separation

PubMed Central

2011-01-01

We examined change of Runx2 and ALP expression in mouse tooth pulp which exposed to teeth separation experiment by immunohistochemistry as a model for conservative dentistry treatment. 8-week-old 36 male ddY mice were used and wedge was inserted between upper 1st and 2nd molars. The wedge was removed 30 minutes as well as 3 hours after the insertion and the samples were prepared extending up to 1 week of time period for regular histopathological and immunohistochemical examinations for ALP and Runx2 expression. The opposite sides without wedge insertion were taken as controls. In the control group pulp, weak expressions of Runx2 and ALP in the vessel endothelial cells as well as the pulp cells were revealed, suggesting the appearance of these genes upon mechanical stress induced by mastication and tongue pressure etc. On the other hand in the experiment group, Runx2 expression increased both in 30-minute and 3-hour teeth separation group. The expression became maximum at 24 hours. Then it gradually decreased and became similar level with the control group at 1-week after the wedge insertion. Similarly ALP expression increased after the wedge insertion and was maximum at 24 hours and then gradually decreased to the levels similar with the control group. These results suggest that when immunohistochemical expression of Runx2 as well as ALP was used as an index, no severe damage occur upon clinical application of wedge insertion. PMID:22027645

Biomechanical forces promote embryonic haematopoiesis

PubMed Central

Adamo, Luigi; Naveiras, Olaia; Wenzel, Pamela L.; McKinney-Freeman, Shannon; Mack, Peter J.; Gracia-Sancho, Jorge; Suchy-Dicey, Astrid; Yoshimoto, Momoko; Lensch, M. William; Yoder, Mervin C.; García-Cardeña, Guillermo; Daley, George Q.

2009-01-01

Biomechanical forces are emerging as critical regulators of embryogenesis, particularly in the developing cardiovascular system1,2. After initiation of the heartbeat in vertebrates, cells lining the ventral aspect of the dorsal aorta, the placental vessels, and the umbilical and vitelline arteries initiate expression of the transcription factor Runx1 (refs 3–5), a master regulator of haematopoiesis, and give rise to haematopoietic cells4. It remains unknown whether the biomechanical forces imposed on the vascular wall at this developmental stage act as a determinant of haematopoietic potential6. Here, using mouse embryonic stem cells differentiated in vitro, we show that fluid shear stress increases the expression of Runx1 in CD41+c-Kit+ haematopoietic progenitor cells7,concomitantly augmenting their haematopoietic colony-forming potential. Moreover, we find that shear stress increases haematopoietic colony-forming potential and expression of haematopoietic markers in the paraaortic splanchnopleura/aorta–gonads–mesonephros of mouse embryos and that abrogation of nitric oxide, a mediator of shear-stress-induced signalling8, compromises haematopoietic potential in vitro and in vivo. Collectively, these data reveal a critical role for biomechanical forces in haematopoietic development. PMID:19440194

Notch signaling pathways in human thoracic ossification of the ligamentum flavum.

PubMed

Qu, Xiaochen; Chen, Zhongqiang; Fan, Dongwei; Sun, Chuiguo; Zeng, Yan; Hou, Xiaofei; Ning, Shanglong

2016-08-01

This study investigated the pathological process of Notch signaling in the osteogenesis of ligamentum flavum tissues and cells, and the associated regulatory mechanisms. Notch receptors, ligands, and target genes were identified by quantitative polymerase chain reaction (qPCR) in ligamentum flavum cells and immunohistochemistry in ligamentum flavum sections from ossification of the ligamentum flavum (OLF) patients and controls. The temporospatial expression patterns of JAG1/Notch2/HES1 in human ligamentum flavum cells during osteogenic differentiation were determined by qPCR. Lentiviral vectors for Notch2 overexpression and knockdown were constructed and transfected into ligamentum flavum cells before osteogenic differentiation to examine the function of Notch signaling pathways in the osteogenic differentiation of ligamentum flavum cells. Alkaline phosphatase, Runx2, Osterix, osteocalcin, and osteopontin mRNA levels, alkaline phosphatase activity, and Alizarin Red staining were used as indicators of osteogenic differentiation. JAG1/Notch2/HES1 mRNA levels were up-regulated in ligamentum flavum cells from OLF patients, which increased during osteogenic differentiation. Immunohistochemical analysis suggested positive Notch2 expression at the ossification front. Down-regulation of Notch2 expression decelerated osteogenic differentiation of ligamentum flavum cells, and Notch2 overexpression promoted osteogenic differentiation of ligamentum flavum cells. Expression of Runx2 and Osterix increased in a manner similar to that of Notch2 during osteogenic differentiation of ligamentum flavum cells, and Notch2 knockdown and overexpression influenced their expression levels. Notch signaling plays an important role in OLF, and Notch may affect the osteogenic differentiation of ligamentum flavum cells via interactions with Runx2 and Osterix.© 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1481-1491, 2016. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Cell-Autonomous Function of Runx1 Transcriptionally Regulates Mouse Megakaryocytic Maturation

PubMed Central

Pencovich, Niv; Jaschek, Ram; Dicken, Joseph; Amit, Ayelet; Lotem, Joseph; Tanay, Amos; Groner, Yoram

2013-01-01

RUNX1 transcription factor (TF) is a key regulator of megakaryocytic development and when mutated is associated with familial platelet disorder and predisposition to acute myeloid leukemia (FPD-AML). We used mice lacking Runx1 specifically in megakaryocytes (MK) to characterized Runx1-mediated transcriptional program during advanced stages of MK differentiation. Gene expression and chromatin-immunoprecipitation-sequencing (ChIP-seq) of Runx1 and p300 identified functional Runx1 bound MK enhancers. Runx1/p300 co-bound regions showed significant enrichment in genes important for MK and platelet homeostasis. Runx1 occupied genomic regions were highly enriched in RUNX and ETS motifs and to a lesser extent in GATA motif. Megakaryocytic specificity of Runx1/P300 bound enhancers was validated by transfection mutagenesis and Runx1/P300 co-bound regions of two key megakaryocytic genes Nfe2 and Selp were tested by in vivo transgenesis. The data provides the first example of genome wide Runx1/p300 occupancy in maturating primary FL-MK, unravel the Runx1-regulated program controlling MK maturation in vivo and identify a subset of its bona fide regulated genes. It advances our understanding of the molecular events that upon RUNX1mutations in human lead to the predisposition to familial platelet disorders and FPD-AML. PMID:23717578

Loss of Runx2 sensitises osteosarcoma to chemotherapy-induced apoptosis

PubMed Central

Roos, Alison; Satterfield, Laura; Zhao, Shuying; Fuja, Daniel; Shuck, Ryan; Hicks, M John; Donehower, Lawrence A; Yustein, Jason T

2015-01-01

Background: Osteosarcoma (OS) is the most common bone malignancy in the paediatric population, principally affecting adolescents and young adults. Minimal advancements in patient prognosis have been made over the past two decades because of the poor understanding of disease biology. Runx2, a critical transcription factor in bone development, is frequently amplified and overexpressed in OS. However, the molecular and biological consequences of Runx2 overexpression remain unclear. Methods: si/shRNA and overexpression technology to alter Runx2 levels in OS cells. In vitro assessment of doxorubicin (doxo)-induced apoptosis and in vivo chemosensitivity studies. Small-molecule inhibitor of c-Myc transcriptional activity was used to assess its role. Results: Loss of Runx2 sensitises cells to doxo-induced apoptosis both in vitro and in vivo. Furthermore, in conjunction with chemotherapy, decreasing Runx2 protein levels activates both the intrinsic and extrinsic apoptotic pathways. Transplanted tumour studies demonstrated that loss of endogenous Runx2 protein expression enhances caspase-3 cleavage and tumour necrosis in response to chemotherapy. Finally, upon doxo-treated Runx2 knockdown OS cells there was evidence of enhanced c-Myc expression and transcriptional activity. Inhibition of c-Myc under these conditions resulted in decreased activation of apoptosis, therefore insinuating a role for c-Myc in dox-induced activation of apoptotic pathways. Conclusions: Therefore, we have established a novel molecular mechanism by which Runx2 provides a chemoprotective role in OS, indicating that in conjunction to standard chemotherapy, targeting Runx2 may be a new therapeutic strategy for patients with OS. PMID:26528706

Loss of Osteoblast Runx3 Produces Severe Congenital Osteopenia

PubMed Central

Bauer, Omri; Sharir, Amnon; Kimura, Ayako; Hantisteanu, Shay; Takeda, Shu

2015-01-01

Congenital osteopenia is a bone demineralization condition that is associated with elevated fracture risk in human infants. Here we show that Runx3, like Runx2, is expressed in precommitted embryonic osteoblasts and that Runx3-deficient mice develop severe congenital osteopenia. Runx3-deficient osteoblast-specific (Runx3fl/fl/Col1α1-cre), but not chondrocyte-specific (Runx3fl/fl/Col1α2-cre), mice are osteopenic. This demonstrates that an osteoblastic cell-autonomous function of Runx3 is required for proper osteogenesis. Bone histomorphometry revealed that decreased osteoblast numbers and reduced mineral deposition capacity in Runx3-deficient mice cause this bone formation deficiency. Neonatal bone and cultured primary osteoblast analyses revealed a Runx3-deficiency-associated decrease in the number of active osteoblasts resulting from diminished proliferation and not from enhanced osteoblast apoptosis. These findings are supported by Runx3-null culture transcriptome analyses showing significant decreases in the levels of osteoblastic markers and increases in the levels of Notch signaling components. Thus, while Runx2 is mandatory for the osteoblastic lineage commitment, Runx3 is nonredundantly required for the proliferation of these precommitted cells, to generate adequate numbers of active osteoblasts. Human RUNX3 resides on chromosome 1p36, a region that is associated with osteoporosis. Therefore, RUNX3 might also be involved in human bone mineralization. PMID:25605327

Mediator MED23 cooperates with RUNX2 to drive osteoblast differentiation and bone development.

PubMed

Liu, Zhen; Yao, Xiao; Yan, Guang; Xu, YiChi; Yan, Jun; Zou, Weiguo; Wang, Gang

2016-04-01

How lineage specifiers are regulated during development is an outstanding question, and the molecular regulation of osteogenic factor RUNX2 remains to be fully understood. Here we report that the Mediator subunit MED23 cooperates with RUNX2 to regulate osteoblast differentiation and bone development. Med23 deletion in mesenchymal stem cells or osteoblast precursors results in multiple bone defects similar to those observed in Runx2(+/-) mice. In vitro, Med23-deficient progenitor cells are refractory to osteoblast differentiation, and Med23 deficiency reduces Runx2-target gene activity without changing Runx2 expression. Mechanistically, MED23 binds to RUNX2 and modulates its transcriptional activity. Moreover, Med23 deficiency in osteoprogenitor cells exacerbates the skeletal abnormalities observed in Runx2(+/-) mice. Collectively, our results establish a genetic and physical interaction between RUNX2 and MED23, suggesting that MED23 constitutes a molecular node in the regulatory network of anabolic bone formation and related diseases.

Ethanol extract of Lithospermum erythrorhizon Sieb. et Zucc. promotes osteoblastogenesis through the regulation of Runx2 and Osterix.

PubMed

Choi, You Hee; Kim, Geum Soog; Choi, Jae Ho; Jin, Sun Woo; Kim, Hyung Gyun; Han, Younho; Lee, Dae Young; Choi, Soo Im; Kim, Seung Yu; Ahn, Young Sup; Lee, Kwang Youl; Jeong, Hye Gwang

2016-08-01

Bone remodeling and homeostasis are largely the result of the coordinated action of osteoblasts and osteoclasts. Osteoblasts are responsible for bone formation. The differentiation of osteoblasts is regulated by the transcription factors, Runx2 and Osterix. Natural products of plant origin are still a major part of traditional medicinal systems in Korea. The root of Lithospermum erythrorhizon Sieb. et Zucc. (LR), the purple gromwell, is an herbal medicine used for inflammatory and infectious diseases. LR is an anti-inflammatory and exerts anticancer effects by inducing the apoptosis of cancer cells. However, the precise molecular signaling mechanisms of osteoblastogenesis as regards LR and osteoblast transcription are not yet known. In this study, we investigated the effects of ethanol (EtOH) extract of LR (LES) on the osteoblast differentiation of C2C12 myoblasts induced by bone morphogenetic protein 4 (BMP4) and the potential involvement of Runx2 and Osterix in these effects. We found that the LES exhibited an ability to induce osteoblast differentiation. LES increased the expression of the osteoblast marker, alkaline phosphatase (ALP), as well as its activity, as shown by ALP staining and ALP activity assay. LES also increased mineralization, as shown by Alizarin Red S staining. Treatment with LES increased the protein levels (as shown by immunoblotting), as well as the transcriptional activity of Runx2 and Osterix and enhanced osteogenic activity. These results suggest that LES modulates osteoblast differentiation at least in part through Runx2 and Osterix.

Lyophilized platelet-rich fibrin (PRF) promotes craniofacial bone regeneration through Runx2.

PubMed

Li, Qi; Reed, David A; Min, Liu; Gopinathan, Gokul; Li, Steve; Dangaria, Smit J; Li, Leo; Geng, Yajun; Galang, Maria-Therese; Gajendrareddy, Praveen; Zhou, Yanmin; Luan, Xianghong; Diekwisch, Thomas G H

2014-05-14

Freeze-drying is an effective means to control scaffold pore size and preserve its composition. The purpose of the present study was to determine the applicability of lyophilized Platelet-rich fibrin (LPRF) as a scaffold for craniofacial tissue regeneration and to compare its biological effects with commonly used fresh Platelet-rich fibrin (PRF). LPRF caused a 4.8-fold±0.4-fold elevation in Runt-related transcription factor 2 (Runx2) expression in alveolar bone cells, compared to a 3.6-fold±0.2-fold increase when using fresh PRF, and a more than 10-fold rise of alkaline phosphatase levels and mineralization markers. LPRF-induced Runx2 expression only occurred in alveolar bone and not in periodontal or dental follicle cells. LPRF also caused a 1.6-fold increase in osteoblast proliferation (p<0.001) when compared to fresh PRF. When applied in a rat craniofacial defect model for six weeks, LPRF resulted in 97% bony coverage of the defect, compared to 84% for fresh PRF, 64% for fibrin, and 16% without scaffold. Moreover, LPRF thickened the trabecular diameter by 25% when compared to fresh PRF and fibrin, and only LPRF and fresh PRF resulted in the formation of interconnected trabeculae across the defect. Together, these studies support the application of lyophilized PRF as a biomimetic scaffold for craniofacial bone regeneration and mineralized tissue engineering.

The nucleolar protein NIFK promotes cancer progression via CK1α/β-catenin in metastasis and Ki-67-dependent cell proliferation

PubMed Central

Lin, Tsung-Chieh; Su, Chia-Yi; Wu, Pei-Yu; Lai, Tsung-Ching; Pan, Wen-An; Jan, Yi-Hua; Chang, Yu-Chang; Yeh, Chi-Tai; Chen, Chi-Long; Ger, Luo-Ping; Chang, Hong-Tai; Yang, Chih-Jen; Huang, Ming-Shyan; Liu, Yu-Peng; Lin, Yuan-Feng; Shyy, John Y-J; Tsai, Ming-Daw; Hsiao, Michael

2016-01-01

Nucleolar protein interacting with the FHA domain of pKi-67 (NIFK) is a Ki-67-interacting protein. However, its precise function in cancer remains largely uninvestigated. Here we show the clinical significance and metastatic mechanism of NIFK in lung cancer. NIFK expression is clinically associated with poor prognosis and metastasis. Furthermore, NIFK enhances Ki-67-dependent proliferation, and promotes migration, invasion in vitro and metastasis in vivo via downregulation of casein kinase 1α (CK1α), a suppressor of pro-metastatic TCF4/β-catenin signaling. Inversely, CK1α is upregulated upon NIFK knockdown. The silencing of CK1α expression in NIFK-silenced cells restores TCF4/β-catenin transcriptional activity, cell migration, and metastasis. Furthermore, RUNX1 is identified as a transcription factor of CSNK1A1 (CK1α) that is negatively regulated by NIFK. Our results demonstrate the prognostic value of NIFK, and suggest that NIFK is required for lung cancer progression via the RUNX1-dependent CK1α repression, which activates TCF4/β-catenin signaling in metastasis and the Ki-67-dependent regulation in cell proliferation. DOI: http://dx.doi.org/10.7554/eLife.11288.001 PMID:26984280

Prostacyclin Suppresses Twist Expression in the Presence of Indomethacin in Bone Marrow-Derived Mesenchymal Stromal Cells

PubMed Central

Kemper, Oliver; Herten, Monika; Fischer, Johannes; Haversath, Marcel; Beck, Sascha; Classen, Tim; Warwas, Sebastian; Tassemeier, Tjark; Landgraeber, Stefan; Lensing-Höhn, Sabine; Krauspe, Rüdiger; Jäger, Marcus

2014-01-01

Background Iloprost, a stable prostacyclin I2 analogue, seems to have an osteoblast-protective potential, whereas indomethacin suppresses new bone formation. The aim of this study was to investigate human bone marrow stromal cell (BMSC) proliferation and differentiation towards the osteoblastic lineage by administration of indomethacin and/or iloprost. Material/Methods Human bone marrow cells were obtained from 3 different donors (A=26 yrs/m; B=25 yrs/f, C=35 yrs/m) via vacuum aspiration of the iliac crest followed by density gradient centrifugation and flow cytometry with defined antigens (CD105+/73+/45−/14−). The cells were seeded and incubated as follows: without additives (Group 0; donor A/B/C), with 10−7 M iloprost only (Group 0+ilo; A/B), with indomethacin only in concentrations of 10−6 M (Group 1, A), 10−5 M (Group 2, B), 10−4 M (Group 3, A/B), and together with 10−7 M iloprost (Groups 4–6, A/B/C). On Day 10 and 28, UV/Vis spectrometric and immunocytochemical assays (4 samples per group and donor) were performed to investigate cell proliferation (cell count measurement) and differentiation towards the osteoblastic lineage (CD34−, CD45−, CD105+, type 1 collagen (Col1), osteocalcin (OC), alkaline phosphatase (ALP), Runx2, Twist, specific ALP-activity). Results Indomethacin alone suppressed BMSC differentiation towards the osteoblastic lineage by downregulation of Runx2, Col1, and ALP. In combination with indomethacin, iloprost increased cell proliferation and differentiation and it completely suppressed Twist expression at Day 10 and 28. Iloprost alone did not promote cell proliferation, but moderately enhanced Runx2 and Twist expression. However, the proliferative effects and the specific ALP-activity varied donor-dependently. Conclusions Iloprost partially antagonized the suppressing effects of indomethacin on BMSC differentiation towards the osteoblast lineage. It enhanced the expression of Runx2 and, only in the presence of indomethacin, it completely suppressed Twist. Thus, in the treatment of avascular osteonecrosis or painful bone marrow edema, the undesirable effects of indomethacin might be counterbalanced by iloprost. PMID:25382306

Graphene oxide enrichment of collagen membranes improves DPSCs differentiation and controls inflammation occurrence.

PubMed

Radunovic, Milena; De Colli, Marianna; De Marco, Patrizia; Di Nisio, Chiara; Fontana, Antonella; Piattelli, Adriano; Cataldi, Amelia; Zara, Susi

2017-08-01

Collagen membranes are used in oral surgery for bone defects treatment acting as a barrier that does not allow the invasion of soft tissue into the growing bone. To improve biocompatibility collagen membranes were coated with graphene oxide (GO), a graphene derivative. The aim of this study was to investigate the biocompatibility of GO coated collagen membranes on human dental pulp stem cells (DPSCs) focusing on biomaterial cytotoxicity, ability to promote DPSCs differentiation process and to control inflammation event induction. DPSCs were cultured on uncoated membranes and on both 2 and 10 μg mL -1 GO coated membranes up to 28 days. Alamar blue and LDH cytotocicity assay, PGE2 ELISA assay, real time RT-PCR for RUNX2, BMP2, SP7, TNFα and COX2 genes expression were performed. Proliferation is higher on GO coated membranes at days 14 and 28. LDH assay evidences no cytotoxicity. BMP2 and RUNX2 expression is higher on coated membranes, BMP2 at early and RUNX2 and SP7 at late experimental times. PGE2 levels are lower on GO coated membranes at days 14 and 28, both TNFα and COX2 expression is significantly decreased when GO is applied. GO coated membranes are not toxic for DPSCs, induce a faster DPSCs differentiation into odontoblasts/osteoblasts and may represent good alternative to conventional membranes thus ensuring more efficient bone formation and improving the clinical performance. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2312-2320, 2017. © 2017 Wiley Periodicals, Inc.

[Effects of different mechanical stretch conditions on differentiation of rat tendon stem cells].

PubMed

Li, Pao; Gao, Shang; Zhou, Mei; Tang, Hong; Mu, Miduo; Zhang, Jiqiang; Tang, Kanglai

2017-04-01

To investigate the effects of different mechanical stretch conditions on the differentiation of rat tendon stem cells (TSCs), to find the best uniaxial cyclic stretching for TSCs tenogenic differentiation, osteogenic differentiation, and adipogenic differentiation. TSCs were isolated from the Achilles tendons of 8-week-old male Sprague Dawley rats by enzymatic digestion method and cultured. The TSCs at passage 3 were randomly divided into 5 groups: group A (stretch strength of 4% and frequency of 1 Hz), group B (stretch strength of 4% and frequency of 2 Hz), group C (stretch strength of 8% and frequency of 1 Hz), group D (stretch strength of 8% and frequency of 2 Hz), and group E (static culture). At 12, 24, and 48 hours after mechanical stretch, the mRNA expressions of the tenogenic differentiation related genes [Scleraxis (SCX) and Tenascin C (TNC)], the osteogenic differentiation related genes [runt related transcription factor 2 (RUNX2) and distal-less homeobox 5 (DLX5)], and the adipogenic differentiation related genes [CCAAT-enhancer-binding protein-α (CEBPα) and lipoprteinlipase (LPL)] were detected by real-time fluorescent quantitative PCR and the protein expressions of TNC, CEBPα, and RUNX2 were detected by Western blot. The mRNA expressions of SCX and TNC in group B were significantly higher than those in groups A, C, D, and E at 24 hours after mechanical stretch ( P <0.05). The mRNA expressions of CEBPα and LPL in group D were significantly higher than those in groups A, B, C, and E at 48 hours after mechanical stretch ( P <0.05). The mRNA expressions of RUNX2 and DLX5 in group C were significantly higher than those in groups A, B, D, and E at 24 hours after mechanical stretch ( P <0.05). Western blot detection showed that higher protein expression of TNC in group B than group E at each time point after mechanical stretch ( P <0.05), and the protein expression of CEBPα was significantly inhibited when compared with group E at 24 hours after mechanical stretch ( P <0.05). At 24 hours after mechanical stretch, the protein expression of RUNX2 in group C was significantly higher than that in group E ( P <0.05); and the protein expression of TNC was significantly lower than that in group E at 24 and 48 hours after mechanical stretch ( P <0.05). At 48 hours after mechanical stretch, the protein expression of CEBPα was significantly increased and the protein expression of TNC was significantly decreased in group D when compared with group E ( P <0.05), but no significant difference was found in the protein expression of RUNX2 between groups D and E ( P >0.05). The mechanical strain could promote differentiation of TSCs, and different parameter of stretch will lead to different differentiation. The best stretch condition for tenogenic differentiation is 4% strength and 2 Hz frequency for 24 hours; the best stretch condition for osteogenic differentiation is 8% strength and 1 Hz frequency for 24 hours; and the best stretch condition for adipogenic differentiation is 8% strength and 2 Hz frequency for 48 hours.

[Investigation of Wnt/β-catenin signaling pathway on regulation of Runx2 in cementoblasts under mechanical stress in vitro].

PubMed

Shuqin, Li; Shan, Yang; Aishu, Ren; Hongwei, Dai

2015-02-01

Periodontal tissue remodeling includes remodeling of alveolar bone, periodontal ligament, and cementum. Cementoblast plays a main role in repairing root resorption. Canonical Wnt/β-catenin signaling can promote the odontogenic differentiation in osteoblast. However, the mechanism on how the orthodontic force influences the function of cementoblast and the relationship between the canonical Wnt/β-catenin signaling and Runx2 of cementoblast are not yet known. The aim of this study is focus on this relationship. OCCM30 cementoblasts were subjected to mechanical strain by four-point bending system with tension stress for 0, 3, 6, and 12 h. They were pretreated with different concentrations of Dikkopf-1 (DKK1) for 48 h. Western blot analysis was performed to detect the β-catenin levels in the nucleus. Runx2 mRNA was observed by real-time quantitative polymerase chain reaction (RT-PCR). OCCM30 cementoblasts were then pretreated with 150 ng · mL(-1) DKK1 for 48 h and subjected to mechanical strain by FX4000T system with tension stress for 12 h. Western blot analysis was conducted to detect the β-catenin levels in the nucleus, and Runx2 mRNA was observed by RT-PCR. OCCM30 cementoblasts had significantly higher Runx2 mRNA and β-catenin levels after being loaded with mechanical stress. The amount of Runx2 mRNA in OCCM30 cementoblasts was significantly decreased by DKK1. When OCCM30 cemento-blasts were pretreated with DKK1 without stress, their β-catenin level was significantly decreased by DKK1 and Wnt signaling was blocked. When they were not pretreated with stress, the β-catenin level with DKK1 was lower than that without DKK1. Without DKK1, the β-catenin level in OCCM30 cemento- blasts increased afterbeing loaded with mechanical stress. With DKK1, the β-catenin level in OCCM30 cementoblasts, which were loaded with mechanical stress, was higher than that without mechanical stress. Cementoblasts had higher Runx2 mRNA expression under mechanical stress because of the Wnt/β-catenin signaling pathway effect.

Bromocriptine modulates the expression of PTHrP receptor, Indian hedgehog, and Runx2 proteins in the growth plate of lactating rats.

PubMed

Wongdee, Kannikar; Thonapan, Natchayaporn; Saengamnart, Wasana; Krishnamra, Nateetip; Charoenphandhu, Narattaphol

2013-09-01

In lactating rats, the endochondral bone growth is markedly enhanced, leading to the lengthening of long bone. This lactation-induced bone elongation could be abolished by a dopaminergic D2 receptor agonist bromocriptine, but how bromocriptine altered the expression of major chondroregulatory proteins in the growth plate cartilage was elusive. Here, we performed a quantitative immunohistochemical analysis to determine the expression of various peptides and transcription factors known to control the growth plate chondrocyte proliferation and differentiation [i.e., parathyroid hormone-related protein (PTHrP), PTHrP receptor, Indian hedgehog (Ihh), and runt-related transcription factor 2 (Runx2)], in bromocriptine-treated lactating rats. The results showed that bromocriptine markedly increased Ihh expression in hypertrophic chondrocytes during early and mid-lactation, while the expression of PTHrP receptor, but not its ligand PTHrP, was upregulated in the proliferative and hypertrophic zones during mid and late lactation. In contrast, the expression of Runx2, an important transcription factor for chondrocyte differentiation, was suppressed in the hypertrophic chondrocytes of bromocriptine-treated rats. In conclusion, bromocriptine increased Ihh and PTHrP receptor expressions and decreased Runx2 expression, which might, in turn, enhance chondrocyte proliferation and delay chondrocyte hypertrophy, thereby slowing down endochondral bone growth. This finding could explain how bromocriptine compromised the lactation-induced bone elongation.

Diethylstilbestrol induces vaginal adenosis by disrupting SMAD/RUNX1-mediated cell fate decision in the Müllerian duct epithelium.

PubMed

Laronda, Monica M; Unno, Kenji; Ishi, Kazutomo; Serna, Vanida A; Butler, Lindsey M; Mills, Alea A; Orvis, Grant D; Behringer, Richard R; Deng, Chuxia; Sinha, Satrajit; Kurita, Takeshi

2013-09-01

Women exposed to diethylstilbestrol (DES) in utero frequently develop vaginal adenosis, from which clear cell adenocarcinoma can arise. Despite decades of extensive investigation, the molecular pathogenesis of DES-associated vaginal adenosis remains elusive. Here we report that DES induces vaginal adenosis by inhibiting the BMP4/Activin A-regulated vaginal cell fate decision through a downregulation of RUNX1. BMP4 and Activin A produced by vaginal mesenchyme synergistically activated the expression of ΔNp63, thus deciding vaginal epithelial cell fate in the Müllerian duct epithelial cells (MDECs) via direct binding of SMADs on the highly conserved 5' sequence of ΔNp63. Therefore, mice in which Smad4 was deleted in MDECs failed to express ΔNp63 in vaginal epithelium and developed adenosis. This SMAD-dependent ΔNp63 activation required RUNX1, a binding partner of SMADs. Conditional deletion of Runx1 in the MDECs induced adenosis in the cranial portion of vagina, which mimicked the effect of developmental DES-exposure. Furthermore, neonatal DES exposure downregulated RUNX1 in the fornix of the vagina, where DES-associated adenosis is frequently found. This observation strongly suggests that the downregulation of RUNX1 is the cause of vaginal adenosis. However, once cell fate was determined, the BMP/Activin-SMAD/RUNX1 signaling pathway became dispensable for the maintenance of ΔNp63 expression in vaginal epithelium. Instead, the activity of the ΔNp63 locus in vaginal epithelium was maintained by a ΔNp63-dependent mechanism. This is the first demonstration of a molecular mechanism through which developmental chemical exposure causes precancerous lesions by altering cell fate. Copyright © 2013 Elsevier Inc. All rights reserved.

Diethylstilbestrol induces vaginal adenosis by disrupting SMAD/RUNX1-mediated cell fate decision in the Müllerian duct epithelium

PubMed Central

Laronda, Monica M.; Unno, Kenji; Ishi, Kazutomo; Serna, Vanida A.; Butler, Lindsey M.; Mills, Alea A.; Orvis, Grant D.; Behringer, Richard R.; Deng, Chuxia; Sinha, Satrajit; Kurita, Takeshi

2013-01-01

Women exposed to diethylstilbestrol (DES) in utero frequently develop vaginal adenosis, from which clear cell adenocarcinoma can arise. Despite decades of extensive investigation, the molecular pathogenesis of DES-associated vaginal adenosis remains elusive. Here we report that DES induces vaginal adenosis by inhibiting the BMP4/Activin A-regulated vaginal cell fate decision through a downregulation of RUNX1. BMP4 and Activin A produced by vaginal mesenchyme synergistically activated the expression of ΔNp63, thus deciding vaginal epithelial cell fate in the Müllerian duct epithelial cells (MDECs) via direct binding of SMADs on the highly conserved 5′sequence of ΔNp63. Therefore, mice in which Smad4 was deleted in MDECs failed to express ΔNp63 in vaginal epithelium and developed adenosis. This SMAD-dependent ΔNp63 activation required RUNX1, a binding partner of SMADs. Conditional deletion of Runx1 in the MDECs induced adenosis in the cranial portion of vagina, which mimicked the effect of developmental DES-exposure. Furthermore, neonatal DES exposure downregulated RUNX1 in the fornix of the vagina, where DES-associated adenosis is frequently found. This observation strongly suggests that the downregulation of RUNX1 is the cause of vaginal adenosis. However, once cell fate was determined, the BMP/Activin-SMAD/RUNX1 signaling pathway became dispensable for the maintenance of ΔNp63 expression in vaginal epithelium. Instead, the activity of the ΔNp63 locus in vaginal epithelium was maintained by a ΔNp63-dependent mechanism. This is the first demonstration of a molecular mechanism through which developmental chemical exposure causes precancerous lesions by altering cell fate. PMID:23830984

17β-estradiol regulates the differentiation of cementoblasts via Notch signaling cascade

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liao, Jing; Zhou, Zeyuan; Huang, Li

Estrogen has been well recognized as a key factor in the homeostasis of bone and periodontal tissue, but the way it regulates the activities of cementoblasts, the cell population maintaining cementum has not been fully understood. In this study, we examined the expression of estrogen receptor in OCCM-30 cells and the effect of 17β-estradiol (E2) on the proliferation and differentiation of OCCM-30 cells. We found that both estrogen receptor α and β were expressed in OCCM-30 cells. E2 exerted no significant influence on the proliferation of OCCM-30 cells, but inhibited the transcription and translation of BSP and Runx2 in the early phase of osteogenicmore » induction except the BSP mRNA. Afterwards in the late phase of osteogenic induction, E2 enhanced the transcription and translation of BSP and Runx2 and promoted the calcium deposition. In addition, the expression level of Notch1, NICD and Hey1 mRNAs responded to exogenous E2 in a pattern similar to that of the osteoblastic markers. DAPT could attenuate the effect of E2 on the expression of osteoblastic markers. These findings indicated that E2 might regulate the differentiation of cementoblasts via Notch signaling. - Highlights: • 17β-estradiol showed no significant effect on the proliferation of cementoblasts. • 17β-estradiol promoted the osteoblastic differentiation of cementoblasts despite of an early transient inhibition. • Notch signaling was regulated by 17β-estradiol and was responsible for mediating the effect of E2 on cementoblasts. • Hey1 might display an opposite expression pattern to Notch signaling in certain circumstances.« less

Connexin43 Potentiates Osteoblast Responsiveness to Fibroblast Growth Factor 2 via a Protein Kinase C-Delta/Runx2–dependent Mechanism

PubMed Central

Lima, Florence; Niger, Corinne; Hebert, Carla

2009-01-01

In this study, we examine the role of the gap junction protein, connexin43 (Cx43), in the transcriptional response of osteocalcin to fibroblast growth factor 2 (FGF2) in MC3T3 osteoblasts. By luciferase reporter assays, we identify that the osteocalcin transcriptional response to FGF2 is markedly increased by overexpression of Cx43, an effect that is mediated by Runx2 via its OSE2 cognate element, but not by a previously identified connexin-responsive Sp1/Sp3-binding element. Furthermore, disruption of Cx43 function with Cx43 siRNAs or overexpression of connexin45 markedly attenuates the response to FGF2. Inhibition of protein kinase C delta (PKCδ) with rottlerin or siRNA-mediated knockdown abrogates the osteocalcin response to FGF2. Additionally, we show that upon treatment with FGF2, PKCδ translocates to the nucleus, PKCδ and Runx2 are phosphorylated and these events are enhanced by Cx43 overexpression, suggesting that the degree of activation is enhanced by increased Cx43 levels. Indeed, chromatin immunoprecipitations of the osteocalcin proximal promoter with antibodies against Runx2 demonstrate that the recruitment of Runx2 to the osteocalcin promoter in response to FGF2 treatment is dramatically enhanced by Cx43 overexpression. Thus, Cx43 plays a critical role in regulating the ability of osteoblasts to respond to FGF2 by impacting PKCδ and Runx2 function. PMID:19339281

The RUNX complex: reaching beyond haematopoiesis into immunity.

PubMed

Voon, Dominic Chih-Cheng; Hor, Yit Teng; Ito, Yoshiaki

2015-12-01

Among their diverse roles as transcriptional regulators during development and cell fate specification, the RUNX transcription factors are best known for the parts they play in haematopoiesis. RUNX proteins are expressed throughout all haematopoietic lineages, being necessary for the emergence of the first haematopoietic stem cells to their terminal differentiation. Although much progress has been made since their discoveries almost two decades ago, current appreciation of RUNX in haematopoiesis is largely grounded in their lineage-specifying roles. In contrast, the importance of RUNX to immunity has been mostly obscured for historic, technical and conceptual reasons. However, this paradigm is likely to shift over time, as a primary purpose of haematopoiesis is to resource the immune system. Furthermore, recent evidence suggests a role for RUNX in the innate immunity of non-haematopoietic cells. This review takes a haematopoiesis-centric approach to collate what is known of RUNX's contribution to the overall mammalian immune system and discuss their growing prominence in areas such as autoimmunity, inflammatory diseases and mucosal immunity. © 2015 John Wiley & Sons Ltd.

Application of Green Tea Catechin for Inducing the Osteogenic Differentiation of Human Dedifferentiated Fat Cells in Vitro

PubMed Central

Kaida, Koji; Honda, Yoshitomo; Hashimoto, Yoshiya; Tanaka, Masahiro; Baba, Shunsuke

2015-01-01

Despite advances in stem cell biology, there are few effective techniques to promote the osteogenic differentiation of human primary dedifferentiated fat (DFAT) cells. We attempted to investigate whether epigallocatechin-3-gallate (EGCG), the main component of green tea catechin, facilitates early osteogenic differentiation and mineralization on DFAT cells in vitro. DFAT cells were treated with EGCG (1.25–10 μM) in osteogenic medium (OM) with or without 100 nM dexamethasone (Dex) for 12 days (hereafter two osteogenic media were designated as OM(Dex) and OM). Supplementation of 1.25 μM EGCG to both the media effectively increased the mRNA expression of collagen 1 (COL1A1) and runt-related transcription factor 2 (RUNX2) and also increased proliferation and mineralization. Compared to OM(Dex) with EGCG, OM with EGCG induced earlier expression for COL1A1 and RUNX2 at day 1 and higher mineralization level at day 12. OM(Dex) with 10 μM EGCG remarkably hampered the proliferation of the DFAT cells. These results suggest that OM(without Dex) with EGCG might be a preferable medium to promote proliferation and to induce osteoblast differentiation of DFAT cells. Our findings provide an insight for the combinatory use of EGCG and DFAT cells for bone regeneration and stem cell-based therapy. PMID:26602917

Clinicopathological and prognostic significance of the RUNX3 expression in gastric cancer: a systematic review and meta-analysis.

PubMed

Liu, Baiying; Han, Yao; Jiang, Lu; Jiang, Dongdong; Li, Wenbin; Zhang, Taotao; Zu, Guo; Zhang, Xiangwen

2018-05-01

The relationship between expression of runt related transcription factor 3 (RUNX3) and clinicopathological parameters of the patients with gastric cancer (GC) is controversial. The studies were retrieved from those already published essay in PubMed, EMBASE, Wan Fang, CNKI (China National Knowledge Infrastructure), the Cochrane Library and Google Scholar. All statistical tests in this meta-analysis were performed using Stata 10.0 software (Stata Corp, College Station, TX). A P value less than 0.05 was considered statistically significant. A total of nine studies involving 796 patients were included in final meta-analysis. The pooled data showed that expression of RUNX3 was significant correlated with tumor's differentiation (OR = 0.387; 95%CI: 0.237-0.633; P = 0.000), depth of invasion (OR = 0.443; 95%CI: 0.273-0.717; P = 0.001), lymph node metastasis (OR = 0.394; 95%CI: 0.259-0.598; P = 0.000), distant metastasis (OR = 0.403; 95%CI: 0.213-0.764; P = 0.005) and TNM stage (OR = 0.461; 95%CI, 0.322-0.659; P = 0.000) in GC. Expression of RUNX3 was significant correlated with good overall survival (OS) [1-year OS (OR = 2.735; 95%CI: 1.966-3.806; P = 0.000), 3-year OS (OR = 4.782; 95%CI: 3.634-6.292; P = 0.000), 5-year OS (OR = 5.191; 95%CI: 3.775-7.138; P = 0.000]. However, RUNX3 was not correlated with gender (OR = 1.409; 95%CI: 0.986-2.014; P = 0.060). RUNX3 expression correlates with tumor's differentiation, depth of invasion, lymph node metastasis, distant metastasis, TNM stage and OS of GC patients. Copyright © 2018 IJS Publishing Group Ltd. Published by Elsevier Ltd. All rights reserved.

Nfatc1 Is a Functional Transcriptional Factor Mediating Nell-1-Induced Runx3 Upregulation in Chondrocytes.

PubMed

Li, Chenshuang; Zheng, Zhong; Zhang, Xinli; Asatrian, Greg; Chen, Eric; Song, Richard; Culiat, Cymbeline; Ting, Kang; Soo, Chia

2018-01-06

Neural EGFL like 1 (Nell-1) is essential for chondrogenic differentiation, maturation, and regeneration. Our previous studies have demonstrated that Nell-1's pro-chondrogenic activities are predominantly reliant upon runt-related transcription factor 3 (Runx3)-mediated Indian hedgehog (Ihh) signaling. Here, we identify the nuclear factor of activated T-cells 1 (Nfatc1) as the key transcriptional factor mediating the Nell-1 → Runx3 signal transduction in chondrocytes. Using chromatin immunoprecipitation assay, we were able to determine that Nfatc1 binds to the -833--810 region of the Runx3 -promoter in response to Nell-1 treatment. By revealing the Nell-1 → Nfatc1 → Runx3 → Ihh cascade, we demonstrate the involvement of Nfatc1, a nuclear factor of activated T-cells, in chondrogenesis, while providing innovative insights into developing a novel therapeutic strategy for cartilage regeneration and other chondrogenesis-related conditions.

Transient changes in oxygen tension inhibit osteogenic differentiation and Runx2 expression in osteoblasts.

PubMed

Salim, Ali; Nacamuli, Randall P; Morgan, Elise F; Giaccia, Amato J; Longaker, Michael T

2004-09-17

Vascular disruption following bony injury results in a hypoxic gradient within the wound microenvironment. Nevertheless, the effects of low oxygen tension on osteogenic precursors remain to be fully elucidated. In the present study, we investigated in vitro osteoblast and mesenchymal stem cell differentiation following exposure to 21% O(2) (ambient oxygen), 2% O(2) (hypoxia), and <0.02% O(2) (anoxia). Hypoxia had little effect on osteogenic differentiation. In contrast, short-term anoxic treatment of primary osteoblasts and mesenchymal precursors inhibited in vitro bone nodule formation and extracellular calcium deposition. Cell viability assays revealed that this effect was not caused by immediate or delayed cell death. Microarray profiling implicated down-regulation of the key osteogenic transcription factor Runx2 as a potential mechanism for the anoxic inhibition of differentiation. Subsequent analysis revealed not only a short-term differential regulation of Runx2 and its targets by anoxia and hypoxia, but a long-term inhibition of Runx2 transcriptional and protein levels after only 12-24 h of anoxic insult. Furthermore, we present evidence that Runx2 inhibition may, at least in part, be because of anoxic repression of BMP2, and that restoring Runx2 levels during anoxia by pretreatment with recombinant BMP2 rescued the anoxic inhibition of differentiation. Taken together, our findings indicate that brief exposure to anoxia (but not 2% hypoxia) down-regulated BMP2 and Runx2 expression, thus inhibiting critical steps in the osteogenic differentiation of pluripotent mesenchymal precursors and committed osteoblasts.

Inducible overexpression of RUNX1b/c in human embryonic stem cells blocks early hematopoiesis from mesoderm.

PubMed

Chen, B; Teng, Jiawen; Liu, Hongwei; Pan, X; Zhou, Y; Huang, Shu; Lai, Mowen; Bian, Guohui; Mao, Bin; Sun, Wencui; Zhou, Qiongxiu; Yang, Shengyong; Nakahata, Tatsutoshi; Ma, Feng

2017-08-01

RUNX1 is absolutely required for definitive hematopoiesis, but the function of RUNX1b/c, two isoforms of human RUNX1, is unclear. We established inducible RUNX1b/c-overexpressing human embryonic stem cell (hESC) lines, in which RUNX1b/c overexpression prevented the emergence of CD34+ cells from early stage, thereby drastically reducing the production of hematopoietic stem/progenitor cells. Simultaneously, the expression of hematopoiesis-related factors was downregulated. However, such blockage effect disappeared from day 6 in hESC/AGM-S3 cell co-cultures, proving that the blockage occurred before the generation of hemogenic endothelial cells. This blockage was partially rescued by RepSox, an inhibitor of the transforming growth factor (TGF)-β signaling pathway, indicating a close relationship between RUNX1b/c and TGF-β pathway. Our results suggest a unique inhibitory function of RUNX1b/c in the development of early hematopoiesis and may aid further understanding of its biological function in normal and diseased models. © The Author (2017). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved.

[Mechanism of losartan suppressing vascular calcification in rat aortic artery].

PubMed

Shao, Juan; Wu, Panfeng; Wu, Jiliang; Li, Mincai

2016-08-01

Objective To investigate the effect of the angiotensin II receptor 1 (AT1R) blocker losartan on vascular calcification in rat aortic artery and explore the underlying mechanisms. Methods SD rats were divided randomly into control group, vascular calcification model group and treatment group. Vascular calcification models were made by subcutaneous injection of warfarin plus vitamin K1 for two weeks. Rats in the treatment group were subcutaneously injected with losartan (10 mg/kg) at the end of the first week and consecutively for one week. We observed the morphological changes by HE staining and the calcium deposition by Alizarin red staining in the artery vascular wall. The mRNA expressions of bone morphogenetic protein 2 (BMP2) and Runt-related transcription factor 2 (RUNX2) were analyzed by reverse transcription PCR. The BMP2 and RUNX2 protein expressions were determined by Western blotting. The apoptosis of smooth muscle cells (SMCs) were detected by TUNEL. The AT1R expression was tested by fluorescent immunohistochemistry. Results The aortic vascular calcification was induced by warfarin and vitamin K1. Compared with the vascular calcification model group, the mRNA and protein expressions of BMP2 and RUNX2 were significantly downregulated in the aorta in the losartan treatment group. Furthermore, the apoptosis of SMCs and the AT1R expression obviously decreased. Conclusion AT1R blocker losartan inhibits the apoptosis of SMCs and reduces AT1R expression; it downregulates the BMP2 and RUNX2 expressions in the vascular calcification process.

Genome-wide DNase hypersensitivity, and occupancy of RUNX2 and CTCF reveal a highly dynamic gene regulome during MC3T3 pre-osteoblast differentiation.

PubMed

Tai, Phillip W L; Wu, Hai; van Wijnen, André J; Stein, Gary S; Stein, Janet L; Lian, Jane B

2017-01-01

The ability to discover regulatory sequences that control bone-related genes during development has been greatly improved by massively parallel sequencing methodologies. To expand our understanding of cis-regulatory regions critical to the control of gene expression during osteoblastogenesis, we probed the presence of open chromatin states across the osteoblast genome using global DNase hypersensitivity (DHS) mapping. Our profiling of MC3T3 mouse pre-osteoblasts during differentiation has identified more than 224,000 unique DHS sites. Approximately 65% of these sites are dynamic during temporal stages of osteoblastogenesis, and a majority of them are located within non-promoter (intergenic and intronic) regions. Nearly half of all DHS sites (both constitutive and dynamic) overlap binding events of the bone-essential RUNX2 and/or the chromatin-related CTCF transcription factors. This finding reinforces the role of these regulatory proteins as essential components of the bone gene regulome. We observe a reduction in chromatin accessibility throughout the genome between pre-osteoblast and early osteoblasts. Our analysis also defined a class of differentially expressed genes that harbor DHS peaks centered within 1 kb downstream of transcriptional end sites (TES). These DHSs at the 3'-flanks of genes exhibit dynamic changes during differentiation that may impact regulation of the osteoblast genome. Taken together, the distribution of DHS regions within non-promoter locations harboring osteoblast and chromatin related transcription factor binding motifs, reflect novel cis-regulatory requirements to support temporal gene expression in differentiating osteoblasts.

Oxidized low-density lipoprotein acts synergistically with beta-glycerophosphate to induce osteoblast differentiation in primary cultures of vascular smooth muscle cells.

PubMed

Bear, Mackenzie; Butcher, Martin; Shaughnessy, Stephen G

2008-09-01

Previous studies have localized osteoblast specific markers to sites of calcified atherosclerotic lesions. We therefore decided to use an established in vitro model of vascular calcification in order to confirm earlier reports of oxidized low-density lipoprotein (oxLDL) promoting the osteogenic differentiation of vascular smooth muscle cells. Treatment of primary bovine aortic smooth muscle cells (BASMCs) with beta-glycerophosphate was found to induce a time-dependent increase in osteoblast differentiation. In contrast, no effect was seen when BASMCs were cultured in the presence of oxLDL alone. However, when the BASMCs were cultured in the presence of both beta-glycerophosphate and oxLDL, beta-glycerophosphate's ability to induce osteoblast differentiation was significantly enhanced. In an attempt to resolve the mechanism by which this effect was occurring, we examined the effect of beta-glycerophosphate and oxLDL on several pathways known to be critical to the differentiation of osteoblasts. Surprisingly, beta-glycerophosphate alone was found to enhance Osterix (Osx) expression by inducing both Smad 1/5/8 activation and Runx2 expression. In contrast, oxLDL did not affect either Smad 1/5/8 activation or Runx2 activation but rather, it enhanced both beta-glycerophosphate-induced Osx expression and osteoblast differentiation in an extracellular signal-regulated kinase 1 and 2 (Erk 1 and 2) -dependent manner. When taken together, these findings suggest a plausible mechanism by which oxLDL may promote osteogenic differentiation and vascular calcification in vivo. J. Cell. Biochem. 105: 185-193, 2008. (c) 2008 Wiley-Liss, Inc. (c) 2008 Wiley-Liss, Inc.

CBFβ enhances de novo protein biosynthesis of its binding partners HIV-1 Vif and RUNX1 and potentiates the Vif-induced degradation of APOBEC3G.

PubMed

Miyagi, Eri; Kao, Sandra; Yedavalli, Venkat; Strebel, Klaus

2014-05-01

Vif is a lentiviral accessory protein that regulates viral infectivity in part by inducing proteasomal degradation of APOBEC3G (A3G). Recently, CBFβ was found to facilitate Vif-dependent degradation of A3G. However, the exact role of CBFβ remains unclear. Several studies noted reduced Vif expression in CBFβ knockdown cells while others saw no significant impact of CBFβ on Vif stability. Here, we confirmed that CBFβ increases Vif steady-state levels. CBFβ affected expression of neither viral Gag nor Vpu protein, indicating that CBFβ regulates Vif expression posttranscriptionally. Kinetic studies revealed effects of CBFβ on both metabolic stability and the rate of Vif biosynthesis. These effects were dependent on the ability of CBFβ to interact with Vif. Importantly, at comparable Vif levels, CBFβ further enhanced A3G degradation, suggesting that CBFβ facilitates A3G degradation by increasing the levels of Vif and by independently augmenting the ability of Vif to target A3G for degradation. CBFβ also increased expression of RUNX1 by enhancing RUNX1 biosynthesis. Unlike Vif, however, CBFβ had no detectable effect on RUNX1 metabolic stability. We propose that CBFβ acts as a chaperone to stabilize Vif during and after synthesis and to facilitate interaction of Vif with cellular cofactors required for the efficient degradation of A3G. In this study, we show that CBFβ has a profound effect on the expression of the HIV-1 infectivity factor Vif and the cellular transcription factor RUNX1, two proteins that physically interact with CBFβ. Kinetic studies revealed that CBFβ increases the rate of Vif and RUNX1 biosynthesis at the level of translation. Mutants of Vif unable to physically interact with CBFβ were nonresponsive to CBFβ. Our data suggest that CBFβ exerts a chaperone-like activity (i) to minimize the production of defective ribosomal products (DRiPs) by binding to nascent protein to prevent premature termination and (ii) to stabilize mature protein conformation to ensure proper function of Vif and RUNX1. Thus, we identified a novel mechanism of protein regulation that affects both viral and cellular factors and thus has broad implications beyond the immediate HIV field.

NF-κB RelB Negatively Regulates Osteoblast Differentiation and Bone Formation

PubMed Central

Yao, Zhenqiang; Li, Yanyun; Yin, Xiaoxiang; Dong, Yufeng; Xing, Lianping; Boyce, Brendan F.

2013-01-01

RelA-mediated NF-κB canonical signaling promotes mesenchymal progenitor cell (MPC) proliferation, but inhibits differentiation of mature osteoblasts (OBs) and thus negatively regulates bone formation. Previous studies suggest that NF-κB RelB may also negatively regulate bone formation through non-canonical signaling, but they involved a complex knockout mouse model and the molecular mechanisms involved were not investigated. Here, we report that RelB−/− mice develop age-related increased trabecular bone mass associated with increased bone formation. RelB−/− bone marrow stromal cells expanded faster in vitro and have enhanced OB differentiation associated with increased expression of the osteoblastogenic transcription factor, Runx2. In addition, RelB directly targeted the Runx2 promoter to inhibit its activation. Importantly, RelB−/− bone-derived MPCs formed bone more rapidly than wild-type cells after they were injected into a murine tibial bone defect model. Our findings indicate that RelB negatively regulates bone mass as mice age and limits bone formation in healing bone defects, suggesting that inhibition of RelB could reduce age-related bone loss and enhance bone repair. PMID:24115294

Novel in Vitro Modification of Bone for an Allograft with Improved Toughness Osteoconductivity

DTIC Science & Technology

2015-06-01

osteocalcin, Runx2, and col1a1 by RT-PCR. Spectrophotometry and fluorescence microscopy were used to quantify AGEs. 2. KEYWORDS Fracture toughness, R...markers (alkaline phosphatase, osteocalcin, RUNX2 and COL1A1 ) Completed Task 10 Data analysis, publications, reports Completed Task 1. Retrieval...FEMALE 25 Task 9. Measure expression of molecular markers of mineralization, osteocalcin, RUNX2 and COL1A1 using quantitative RT-PCR with specific

Combined cistrome and transcriptome analysis of SKI in AML cells identifies SKI as a co-repressor for RUNX1

PubMed Central

Feld, Christine; Sahu, Peeyush; Frech, Miriam; Finkernagel, Florian; Nist, Andrea; Stiewe, Thorsten; Bauer, Uta-Maria; Neubauer, Andreas

2018-01-01

Abstract SKI is a transcriptional co-regulator and overexpressed in various human tumors, for example in acute myeloid leukemia (AML). SKI contributes to the origin and maintenance of the leukemic phenotype. Here, we use ChIP-seq and RNA-seq analysis to identify the epigenetic alterations induced by SKI overexpression in AML cells. We show that approximately two thirds of differentially expressed genes are up-regulated upon SKI deletion, of which >40% harbor SKI binding sites in their proximity, primarily in enhancer regions. Gene ontology analysis reveals that many of the differentially expressed genes are annotated to hematopoietic cell differentiation and inflammatory response, corroborating our finding that SKI contributes to a myeloid differentiation block in HL60 cells. We find that SKI peaks are enriched for RUNX1 consensus motifs, particularly in up-regulated SKI targets upon SKI deletion. RUNX1 ChIP-seq displays that nearly 70% of RUNX1 binding sites overlap with SKI peaks, mainly at enhancer regions. SKI and RUNX1 occupy the same genomic sites and cooperate in gene silencing. Our work demonstrates for the first time the predominant co-repressive function of SKI in AML cells on a genome-wide scale and uncovers the transcription factor RUNX1 as an important mediator of SKI-dependent transcriptional repression. PMID:29471413

Effects of strontium on proliferation and differentiation of rat bone marrow mesenchymal stem cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Yunfeng; Li, Jihua; Zhu, Songsong

Highlights: Black-Right-Pointing-Pointer Strontium ranelate (SrR) inhibits proliferation of BMMSCs. Black-Right-Pointing-Pointer SrR increases osteoblastic but decreases adipocytic differentiation of BMMSCs. Black-Right-Pointing-Pointer SrR increases expression of Runx2, BSP and OCN by BMMSCs in osteogenic medium. Black-Right-Pointing-Pointer SrR decreases expression of PPAR{gamma}, aP2/ALBP and LPL by BMMSCs in adipogenic medium. -- Abstract: Strontium ranelate (SrR) was an effective anti-osteoporotic drug to increase bone formation and decrease bone resorption. However, reports about the effect of SR on osteoblastic and adipocytic differentiation from bone marrow mesenchymal stem cells (BMMSCs) are limited. The purpose of this study is to evaluate whether SrR affects the ability ofmore » BMMSCs to differentiate into osteoblasts or adipocytes. Rat BMMSCs were identified by flow cytometry and exposed to SR (0.1 and 1.0 mM Sr{sup 2+}) under osteogenic or adipogenic medium for 1 and 2 weeks. The proliferation and differentiation of BMMSCs were analyzed by MTT, alkaline phosphatase (ALP), Oil red O staining, quantitative real-time RT-PCR and Western blot assays. SrR significantly inhibited the proliferation, increased osteoblastic but decreased adipocytic differentiation of rat BMMSCs dose-dependently. In osteogenic medium, SrR increased the expression of ALP, the mRNA levels of Cbfa1/Runx2, bone sialoprotein, and osteocalcin by RT-PCR, and the protein levels of Cbfa1/Runx2 by Western blot. In adipogenic medium, SrR decreased the mRNA levels of PPAR{gamma}2, adipocyte lipid-binding protein 2 (aP2/ALBP), and lipoprotein lipase (LPL) by RT-PCR, and the protein expression of PPAR{gamma} in Western blot analysis. These results indicated that the effects of SrR to promote osteoblastic but inhibit adipocytic differentiation of BMMSCs might contribute to its effect on osteoporosis treatment.« less

Long non-coding RNA RUNXOR accelerates MDSC-mediated immunosuppression in lung cancer.

PubMed

Tian, Xinyu; Ma, Jie; Wang, Ting; Tian, Jie; Zheng, Yu; Peng, Rongrong; Wang, Yungang; Zhang, Yue; Mao, Lingxiang; Xu, Huaxi; Wang, Shengjun

2018-06-18

RUNX1 overlapping RNA (RUNXOR) is a long non-coding RNA that has been indicated as a key regulator in the development of myeloid cells by targeting runt-related transcription factor 1 (RUNX1). Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of cells consisting of immature granulocytes and monocytes with immunosuppression. However, the impact of lncRNA RUNXOR on the development of MDSCs remains unknown. Both the expressions of RUNXOR and RUNX1 in the peripheral blood were measured by qRT-PCR. Human MDSCs used in this study were isolated from tumor tissue of patients with lung cancer by FCM or induced from PBMCs of healthy donors with IL-1β + GM-CSF. Specific siRNA was used to knockdown the expression of RUNXOR in MDSCs. In this study, we found that the lncRNA RUNXOR was upregulated in the peripheral blood of lung cancer patients. In addition, as a target gene of RUNXOR, the expression of RUNX1 was downregulated in lung cancer patients. Finally, the expression of RUNXOR was higher in MDSCs isolated from the tumor tissues of lung cancer patients compared with cells from adjacent tissue. In addition, RUNXOR knockdown decreased Arg1 expression in MDSCs. Based on our findings, it is illustrated that RUNXOR is significantly associated with the immunosuppression induced by MDSCs in lung cancer patients and may be a target of anti-tumor therapy.

Loss of RUNX1/AML1 arginine-methylation impairs in peripheral T cell homeostasis

PubMed Central

Mizutani, Shinsuke; Yoshida, Tatsushi; Zhao, Xinyang; Nimer, Stephen D.; Taniwaki, Masafumi; Okuda, Tsukasa

2016-01-01

Summary RUNX1 (previously termed AML1) is a frequent target of human leukaemia-associated gene aberrations, and it encodes the DNA-binding subunit of the Core-Binding Factor transcription factor complex. RUNX1 expression is essential for the initiation of definitive haematopoiesis, for steady-state thrombopoiesis, and for normal lymphocytes development. Recent studies revealed that protein arginine methyltransferase 1 (PRMT1), which accounts for the majority of the type I PRMT activity in cells, methylates two arginine residues in RUNX1 (R206 and R210), and these modifications inhibit corepressor-binding to RUNX1 thereby enhancing its transcriptional activity. In order to elucidate the biological significance of these methylations, we established novel knock-in mouse lines with non-methylable, double arginine-to-lysine (RTAMR-to-KTAMK) mutations in RUNX1. Homozygous Runx1KTAMK/KTAMK mice are born alive and appear normal during adulthood. However, Runx1KTAMK/KTAMK mice showed a reduction in CD3+ T lymphoid cells and a decrease in CD4+ T cells in peripheral lymphoid organs, in comparison to their wild-type littermates, leading to a reduction in the CD4+ to CD8+ T-cell ratio. These findings suggest that arginine-methylation of RUNX1 in the RTAMR-motif is dispensable for the development of definitive haematopoiesis and for steady-state platelet production, however this modification affects the role of RUNX1 in the maintenance of the peripheral CD4+ T-cell population. PMID:26010396

Differential requirements for Runx proteins in CD4 repression and epigenetic silencing during T lymphocyte development.

PubMed

Taniuchi, Ichiro; Osato, Motomi; Egawa, Takeshi; Sunshine, Mary Jean; Bae, Suk Chul; Komori, Toshihisa; Ito, Yoshiaki; Littman, Dan R

2002-11-27

T lymphocytes differentiate in discrete stages within the thymus. Immature thymocytes lacking CD4 and CD8 coreceptors differentiate into double-positive cells (CD4(+)CD8(+)), which are selected to become either CD4(+)CD8(-)helper cells or CD4(-)CD8(+) cytotoxic cells. A stage-specific transcriptional silencer regulates expression of CD4 in both immature and CD4(-)CD8(+) thymocytes. We show here that binding sites for Runt domain transcription factors are essential for CD4 silencer function at both stages, and that different Runx family members are required to fulfill unique functions at each stage. Runx1 is required for active repression in CD4(-)CD8(-) thymocytes whereas Runx3 is required for establishing epigenetic silencing in cytotoxic lineage thymocytes. Runx3-deficient cytotoxic T cells, but not helper cells, have defective responses to antigen, suggesting that Runx proteins have critical functions in lineage specification and homeostasis of CD8-lineage T lymphocytes.

Space microgravity drives transdifferentiation of human bone marrow-derived mesenchymal stem cells from osteogenesis to adipogenesis.

PubMed

Zhang, Cui; Li, Liang; Jiang, Yuanda; Wang, Cuicui; Geng, Baoming; Wang, Yanqiu; Chen, Jianling; Liu, Fei; Qiu, Peng; Zhai, Guangjie; Chen, Ping; Quan, Renfu; Wang, Jinfu

2018-03-13

Bone formation is linked with osteogenic differentiation of mesenchymal stem cells (MSCs) in the bone marrow. Microgravity in spaceflight is known to reduce bone formation. In this study, we used a real microgravity environment of the SJ-10 Recoverable Scientific Satellite to examine the effects of space microgravity on the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hMSCs). hMSCs were induced toward osteogenic differentiation for 2 and 7 d in a cell culture device mounted on the SJ-10 Satellite. The satellite returned to Earth after going through space experiments in orbit for 12 d, and cell samples were harvested and analyzed for differentiation potentials. The results showed that space microgravity inhibited osteogenic differentiation and resulted in adipogenic differentiation, even under osteogenic induction conditions. Under space microgravity, the expression of 10 genes specific for osteogenesis decreased, including collagen family members, alkaline phosphatase ( ALP), and runt-related transcription factor 2 ( RUNX2), whereas the expression of 4 genes specific for adipogenesis increased, including adipsin ( CFD), leptin ( LEP), CCAAT/enhancer binding protein β ( CEBPB), and peroxisome proliferator-activated receptor-γ ( PPARG). In the analysis of signaling pathways specific for osteogenesis, we found that the expression and activity of RUNX2 was inhibited, expression of bone morphogenetic protein-2 ( BMP2) and activity of SMAD1/5/9 were decreased, and activity of focal adhesion kinase (FAK) and ERK-1/2 declined significantly under space microgravity. These data indicate that space microgravity plays a dual role by decreasing RUNX2 expression and activity through the BMP2/SMAD and integrin/FAK/ERK pathways. In addition, we found that space microgravity increased p38 MAPK and protein kinase B (AKT) activities, which are important for the promotion of adipogenic differentiation of hMSCs. Space microgravity significantly decreased the expression of Tribbles homolog 3 ( TRIB3), a repressor of adipogenic differentiation. Y15, a specific inhibitor of FAK activity, was used to inhibit the activity of FAK under normal gravity; Y15 decreased protein expression of TRIB3. Therefore, it appears that space microgravity decreased FAK activity and thereby reduced TRIB3 expression and derepressed AKT activity. Under space microgravity, the increase in p38 MAPK activity and the derepression of AKT activity seem to synchronously lead to the activation of the signaling pathway specifically promoting adipogenesis.-Zhang, C., Li, L., Jiang, Y., Wang, C., Geng, B., Wang, Y., Chen, J., Liu, F., Qiu, P., Zhai, G., Chen, P., Quan, R., Wang, J. Space microgravity drives transdifferentiation of human bone marrow-derived mesenchymal stem cells from osteogenesis to adipogenesis.

Effect of low-level mechanical vibration on osteogenesis and osseointegration of porous titanium implants in the repair of long bone defects

NASA Astrophysics Data System (ADS)

Jing, Da; Tong, Shichao; Zhai, Mingming; Li, Xiaokang; Cai, Jing; Wu, Yan; Shen, Guanghao; Zhang, Xuhui; Xu, Qiaoling; Guo, Zheng; Luo, Erping

2015-11-01

Emerging evidence substantiates the potential of porous titanium alloy (pTi) as an ideal bone-graft substitute because of its excellent biocompatibility and structural properties. However, it remains a major clinical concern for promoting high-efficiency and high-quality osseointegration of pTi, which is beneficial for securing long-term implant stability. Accumulating evidence demonstrates the capacity of low-amplitude whole-body vibration (WBV) in preventing osteopenia, whereas the effects and mechanisms of WBV on osteogenesis and osseointegration of pTi remain unclear. Our present study shows that WBV enhanced cellular attachment and proliferation, and induced well-organized cytoskeleton of primary osteoblasts in pTi. WBV upregulated osteogenesis-associated gene and protein expression in primary osteoblasts, including OCN, Runx2, Wnt3a, Lrp6 and β-catenin. In vivo findings demonstrate that 6-week and 12-week WBV stimulated osseointegration, bone ingrowth and bone formation rate of pTi in rabbit femoral bone defects via μCT, histological and histomorphometric analyses. WBV induced higher ALP, OCN, Runx2, BMP2, Wnt3a, Lrp6 and β-catenin, and lower Sost and RANKL/OPG gene expression in rabbit femora. Our findings demonstrate that WBV promotes osteogenesis and osseointegration of pTi via its anabolic effect and potential anti-catabolic activity, and imply the promising potential of WBV for enhancing the repair efficiency and quality of pTi in osseous defects.

Effects of the 1, 4-dihydropyridine L-type calcium channel blocker benidipine on bone marrow stromal cells.

PubMed

Ma, Zhong-ping; Liao, Jia-cheng; Zhao, Chang; Cai, Dao-zhang

2015-08-01

Osteoporosis (OP) often increases the risk of bone fracture and other complications and is a major clinical problem. Previous studies have found that high blood pressure is associated with bone formation abnormalities, resulting in increased calcium loss. We have investigated the effect of the antihypertensive drug benidipine on bone marrow stromal cell (BMSC) differentiation into osteoblasts and bone formation under osteoporotic conditions. We used a combination of in vitro and in vivo approaches to test the hypothesis that benidipine promotes murine BMSC differentiation into osteoblasts. Alkaline phosphatase (ALP), osteocalcin (OCN), runt-related transcription factor 2 (RUNX2), β-catenin, and low-density lipoprotein receptor-related protein 5 (LRP5) protein expression was evaluated in primary femoral BMSCs from C57/BL6 mice cultured under osteogenic conditions for 2 weeks to examine the effects of benidipine. An ovariectomized (OVX) mouse model was used to investigate the effect of benidipine treatment for 3 months in vivo. We found that ALP, OCN, and RUNX2 expression was up-regulated and WNT/β-catenin signaling was enhanced in vitro and in vivo. In OVX mice that were intragastrically administered benidipine, bone parameters (trabecular thickness, bone mineral density, and trabecular number) in the distal femoral metaphysis were significantly increased compared with control OVX mice. Consistently, benidipine promoted BMSC differentiation into osteoblasts and protected against bone loss in OVX mice. Therefore, benidipine might be a suitable candidate for the treatment of patients with postmenopausal osteoporosis and hypertension.

Effect of low-level mechanical vibration on osteogenesis and osseointegration of porous titanium implants in the repair of long bone defects

PubMed Central

Jing, Da; Tong, Shichao; Zhai, Mingming; Li, Xiaokang; Cai, Jing; Wu, Yan; Shen, Guanghao; Zhang, Xuhui; Xu, Qiaoling; Guo, Zheng; Luo, Erping

2015-01-01

Emerging evidence substantiates the potential of porous titanium alloy (pTi) as an ideal bone-graft substitute because of its excellent biocompatibility and structural properties. However, it remains a major clinical concern for promoting high-efficiency and high-quality osseointegration of pTi, which is beneficial for securing long-term implant stability. Accumulating evidence demonstrates the capacity of low-amplitude whole-body vibration (WBV) in preventing osteopenia, whereas the effects and mechanisms of WBV on osteogenesis and osseointegration of pTi remain unclear. Our present study shows that WBV enhanced cellular attachment and proliferation, and induced well-organized cytoskeleton of primary osteoblasts in pTi. WBV upregulated osteogenesis-associated gene and protein expression in primary osteoblasts, including OCN, Runx2, Wnt3a, Lrp6 and β-catenin. In vivo findings demonstrate that 6-week and 12-week WBV stimulated osseointegration, bone ingrowth and bone formation rate of pTi in rabbit femoral bone defects via μCT, histological and histomorphometric analyses. WBV induced higher ALP, OCN, Runx2, BMP2, Wnt3a, Lrp6 and β-catenin, and lower Sost and RANKL/OPG gene expression in rabbit femora. Our findings demonstrate that WBV promotes osteogenesis and osseointegration of pTi via its anabolic effect and potential anti-catabolic activity, and imply the promising potential of WBV for enhancing the repair efficiency and quality of pTi in osseous defects. PMID:26601709

Estrogenic gper signaling regulates mir144 expression in cancer cells and cancer-associated fibroblasts (cafs).

PubMed

Vivacqua, Adele; De Marco, Paola; Santolla, Maria Francesca; Cirillo, Francesca; Pellegrino, Michele; Panno, Maria Luisa; Abonante, Sergio; Maggiolini, Marcello

2015-06-30

MicroRNAs (miRNAs) are small non coding RNA molecules that play a crucial role in several pathophysiological conditions, including cancer. The stimulation of hormone-sensitive tumors by estrogens are mediated by estrogen receptor (ER)α and G protein estrogen receptor (GPER). Previous studies have reported that ERα regulates miRNA expression, while this ability of GPER remains to be elucidated. Here, we demonstrate that in SkBr3 breast cancer and HepG2 hepatocarcinoma cells, 17β-estradiol (E2) and the selective GPER ligand G-1 induce miR144 expression through GPER and the involvement of the PI3K/ERK1/2/Elk1 transduction pathway. Moreover, we show that E2 and G-1 down-regulate through miR144 the onco-suppressor Runx1 and increase cell cycle progression. The capability of E2 and G-1 in triggering the induction of miR144 and the down-regulation of Runx1 was also confirmed in cancer-associated fibroblasts (CAFs) that are main components of the tumor microenvironment driving cancer progression. Further confirming these results, Runx1 protein levels were found decreased in tumor xenografts upon G-1 treatment. On the basis of our findings miR144 and Runx1 may be included among the oncotargets of GPER action. Moreover, the present data provide new insights regarding the ability of estrogens to trigger the GPER/miR144/Runx1 transduction pathway toward the stimulation of cancer progression.

Runx2 is required for early stages of endochondral bone formation but delays final stages of bone repair in Axin2-deficient mice

PubMed Central

McGee-Lawrence, Meghan E.; Carpio, Lomeli R.; Bradley, Elizabeth W.; Dudakovic, Amel; Lian, Jane B.; van Wijnen, Andre J.; Kakar, Sanjeev; Hsu, Wei; Westendorf, Jennifer J.

2014-01-01

Runx2 and Axin2 regulate skeletal development. We recently determined that Axin2 and Runx2 molecularly interact in differentiating osteoblasts to regulate intramembranous bone formation, but the relationship between these factors in endochondral bone formation was unresolved. To address this, we examined the effects of Axin2 deficiency on the cleidocranial dysplasia (CCD) phenotype of Runx2+/− mice, focusing on skeletal defects attributed to improper endochondral bone formation. Axin2 deficiency unexpectedly exacerbated calvarial components of the CCD phenotype in the Runx2+/− mice; the endocranial layer of the frontal suture, which develops by endochondral bone formation, failed to mineralize in the Axin2−/−:Runx2+/− mice, resulting in a cartilaginous, fibrotic and larger fontanel than observed in Runx2+/− mice. Transcripts associated with cartilage development (e.g., Acan, miR140) were expressed at higher levels, whereas blood vessel morphogenesis transcripts (e.g., Slit2) were suppressed in Axin2−/−:Runx2+/− calvaria. Cartilage maturation was impaired, as primary chondrocytes from double mutant mice demonstrated delayed differentiation and produced less calcified matrix in vitro. The genetic dominance of Runx2 was also reflected during endochondral fracture repair, as both Runx2+/− and double mutant Axin2−/−:Runx2+/− mice had enlarged fracture calluses at early stages of healing. However, by the end stages of fracture healing, double mutant animals diverged from the Runx2+/− mice, showing smaller calluses and increased torsional strength indicative of more rapid end stage bone formation as seen in the Axin2−/− mice. Taken together, our data demonstrate a dominant role for Runx2 in chondrocyte maturation, but implicate Axin2 as an important modulator of the terminal stages of endochondral bone formation. PMID:24973690

Periostin promotes migration and osteogenic differentiation of human periodontal ligament mesenchymal stem cells via the Jun amino-terminal kinases (JNK) pathway under inflammatory conditions.

PubMed

Tang, Yi; Liu, Lin; Wang, Pei; Chen, Donglei; Wu, Ziqiang; Tang, Chunbo

2017-12-01

Mesenchymal stem cell (MSC)-mediated periodontal tissue regeneration is considered to be a promising method for periodontitis treatment. The molecular mechanism of functional regulation by MSCs remains unclear, thus limiting their application. Our previous study discovered that Periostin (POSTN) promoted the migration and osteogenic differentiation of periodontal ligament mesenchymal stem cells (PDLSCs), but it is still unclear whether POSTN is able to restore the regenerative potential of PDLSCs under inflammatory conditions. In this study, we investigated the effect of POSTN on PDLSCs under inflammatory conditions and its mechanism. PDLSCs were isolated from periodontal ligament tissue. TNF-α was used at 10 ng/mL to mimic inflammatory conditions. Lentivirus POSTN shRNA was used to knock down POSTN. Recombinant human POSTN (rhPOSTN) was used to stimulate PDLSCs. A scratch assay was used to analyse cell migration. Alkaline phosphatase (ALP) activity, Alizarin Red staining and expression of osteogenesis-related genes were used to investigate the osteogenic differentiation potential. Western blot analysis was used to detect the mitogen-activated protein kinases (MAPK) and AKT signalling pathways. After a 10 ng/mL TNF-α treatment, knockdown of POSTN impeded scratch closure, inhibited ALP activity and mineralization in vitro, and decreased expression of RUNX2, OSX, OPN and OCN in PDLSCs, while 75 ng/mL rhPOSTN significantly accelerated scratch closure, enhanced ALP activity and mineralization in vitro, and increased expression of RUNX2, OSX, OPN and OCN. In addition, knockdown of POSTN inhibited expression of phosphorylated c-Jun N-terminal kinase (p-JNK), while 75 ng/mL rhPOSTN increased expression of p-JNK in PDLSCs with TNF-α treatment. Furthermore, inhibition of JNK by its inhibitor SP600125 dramatically blocked POSTN-enhanced scratch closure, ALP activity and mineralization in PDLSCs. Our results revealed that POSTN might promote the migration and osteogenic differentiation potential of PDLSCs via the JNK pathway, providing insight into the mechanism underlying MSC biology under inflammatory conditions and identifying a potential target for improving periodontal tissue regeneration. © 2017 John Wiley & Sons Ltd.

Mutant Runx2 regulates amelogenesis and osteogenesis through a miR-185-5p-Dlx2 axis.

PubMed

Chang, Huaiguang; Wang, Yue; Liu, Haochen; Nan, Xu; Wong, Singwai; Peng, Saihui; Gu, Yajuan; Zhao, Hongshan; Feng, Hailan

2017-12-14

Regulation of microRNAs (miRNA) has been extensively investigated in diseases; however, little is known about the roles of miRNAs in cleidocranial dysplasia (CCD). The aim of the present study was to investigate the potential involvement of miRNAs in CCD. In vitro site-directed mutagenesis was performed to construct three mutant Runx2 expression vectors, which were then transfected into LS8 cells and MC3T3-E1 cells, to determine the impact on amelogenesis and osteogenesis, respectively. miRCURY LNA miRNA microarray identify miR-185-5p as a miRNA target commonly induced by all three Runx2 mutants. Real-time quantitative PCR was applied to determine the expression of miR-185-5p and Dlx2 in samples. Dual-luciferase reporter assays were conducted to confirm Dlx2 as a legitimate target of miR-185-5p. The suppressive effect of miR-185-5p on amelogenesis and osteogenesis of miR-185-5p was evaluated by RT-PCR and western blot examination of Amelx, Enam, Klk4, and Mmp20 gene and protein expression, and by Alizarin Red stain. We found that mutant Runx2 suppressed amelogenesis and osteogenesis. miR-185-5p, induced by Runx2, suppressed amelogenesis and osteogenesis. Furthermore, we identified Dlx2 as direct target of miR-185-5p. Consistently, Dlx2 expression was inversely correlated with miR-185-5p levels. This study highlights the molecular etiology and significance of miR-185-5p in CCD, and suggests that targeting miR-185-5p may represent a new therapeutic strategy in prevention or intervention of CCD.

Quantitative analysis of tumor-derived methylated RUNX3 sequences in the serum of gastric cancer patients.

PubMed

Sakakura, Chouhei; Hamada, Takuo; Miyagawa, Koji; Nishio, Minoru; Miyashita, Atushi; Nagata, Hiroyuki; Ida, Hiroshi; Yazumi, Shujiro; Otsuji, Eigo; Chiba, Tsutomu; Ito, Kosei; Ito, Yoshiaki

2009-07-01

Using real-time quantitative methylation-specific PCR (RTQ-MSP), methylated RUNX3 sequences were quantified and the fractional concentrations of circulating tumor DNA in serum were determined, along with peripheral blood cells collected preoperatively, intraoperatively and postoperatively from 65 patients with gastric cancer. RTQ-MSP was sufficiently sensitive to detect RUNX3 methylation. Quantitative MSP data were expressed in terms of the methylation index, which was defined as the relative amount of methylated RUNX3 sequences divided by the concentration of methylated actin. High levels of methylated RUNX3 sequences were detected in the peripheral circulation of 29% (19 of 65) of the gastric cancer patients. The RUNX3 methylation index was concordant with cancer stage, histology, lymphatic and vascular invasion, and was more sensitive than carcinoembryonic antigen (CEA) as a biomarker. Twenty-nine percent (19 out of 65) of preoperative serum samples had methylated RUNX3 sequences, ranging from 5.2 to 1625955 (median quantity=43 m-index, sensitivity 95.5%, specificity 62.5%, AUC 0.8651). After surgical resection, the median RUNX3 methylation index in serum significantly decreased. These results demonstrate the clinical usefulness and effectiveness of peripheral blood RTQ-MSP for detecting and monitoring gastric cancer after treatment. Furthermore, 5 out of the 30 preoperative control samples of benign disease (cases of panperitonitis due to acute appendicitis or cholecystitis) showed transient RUNX3 methylation which decreased after the operation in accordance with recovery. Quantification of epigenetic changes in serum RUNX3 methylation using RTQ-MSP is useful for the detection and monitoring of gastric cancer.

Losartan Inhibits Vascular Calcification by Suppressing the BMP2 and Runx2 Expression in Rats In Vivo.

PubMed

Li, Mincai; Wu, Panfeng; Shao, Juan; Ke, Zhiqiang; Li, Dan; Wu, Jiliang

2016-04-01

The blockade of renin-angiotensin II system has been shown to reduce morbidity and mortality in hypertension, atherosclerosis, diabetes and chronic kidney disease. Since vascular calcification (VC) is commonly found in these diseases, the aim of this study was to examine whether or not losartan, a widely used angiotensin II receptor blockers, inhibits VC in rats in vivo. A rat model of VC was generated by treating rats with a combination of warfarin and vitamin K1. Two weeks after the treatments, the rats were treated with vehicle or without losartan (100 ng/kg/day) for 2 weeks. At the end of the experiments, aortic arteries were isolated for the examination of calcification morphology, mRNA and protein expression of BMP2 and Runx2, and osteoblast differentiation. Warfarin and vitamin K instigated vascular remodeling with calcified plaques in the aortic arteries in rats. Losartan significantly attenuated warfarin- and vitamin K-induced vascular injury and calcification. Consistently, losartan suppressed the levels of mRNA and protein expression of BMP2 and Runx2, two key factors for VC. Further, vascular calcified lesion areas expressed angiotensin II 1 receptor (AT1R). Finally, losartan treatment significantly inhibited apoptosis in vascular smooth muscle cell (VSMC) in rat arteries. We conclude that losartan suppresses VC by lowering the expression of AT1R, Runx2 and BMP2, and by inhibiting the apoptosis of VSMC in rat aortic arteries.

Nano-copper-bearing stainless steel promotes fracture healing by accelerating the callus evolution process.

PubMed

Wang, Lei; Li, Guoyuan; Ren, Ling; Kong, Xiangdong; Wang, Yugang; Han, Xiuguo; Jiang, Wenbo; Dai, Kerong; Yang, Ke; Hao, Yongqiang

2017-01-01

Treatment for fractures requires internal fixation devices, which are mainly produced from stainless steel or titanium alloy without biological functions. Therefore, we developed a novel nano-copper-bearing stainless steel with nano-sized copper-precipitation (317L-Cu SS). Based on previous studies, this work explores the effect of 317L-Cu SS on fracture healing; that is, proliferation, osteogenic differentiation, osteogenesis-related gene expression, and lysyl oxidase activity of human bone mesenchymal stem cells were detected in vitro. Sprague-Dawley rats were used to build an animal fracture model, and fracture healing and callus evolution were investigated by radiology (X-ray and micro-CT), histology (H&E, Masson, and safranin O/fast green staining), and histomorphometry. Further, the Cu 2+ content and Runx2 level in the callus were determined, and local mechanical test of the fracture was performed to assess the healing quality. Our results revealed that 317L-Cu SS did not affect the proliferation of human bone mesenchymal stem cells, but promoted osteogenic differentiation and the expression of osteogenesis-related genes. In addition, 317L-Cu SS upregulated the lysyl oxidase activity. The X-ray and micro-CT results showed that the callus evolution efficiency and fracture healing speed were superior for 317L-Cu SS. Histological staining displayed large amounts of fibrous tissues at 3 weeks, and cartilage and new bone at 6 weeks. Further, histomorphometric analysis indicated that the callus possessed higher osteogenic efficiency at 6 weeks, and a high Cu 2+ content and increased Runx2 expression were observed in the callus for 317L-Cu SS. Besides, the mechanical strength of the fracture site was much better than that of the control group. Overall, we conclude that 317L-Cu SS possesses the ability to increase Cu 2+ content and promote osteogenesis in the callus, which could accelerate the callus evolution process and bone formation to provide faster and better fracture healing.

Nano-copper-bearing stainless steel promotes fracture healing by accelerating the callus evolution process

PubMed Central

Kong, Xiangdong; Wang, Yugang; Han, Xiuguo; Jiang, Wenbo; Dai, Kerong; Yang, Ke; Hao, Yongqiang

2017-01-01

Treatment for fractures requires internal fixation devices, which are mainly produced from stainless steel or titanium alloy without biological functions. Therefore, we developed a novel nano-copper-bearing stainless steel with nano-sized copper-precipitation (317L-Cu SS). Based on previous studies, this work explores the effect of 317L-Cu SS on fracture healing; that is, proliferation, osteogenic differentiation, osteogenesis-related gene expression, and lysyl oxidase activity of human bone mesenchymal stem cells were detected in vitro. Sprague–Dawley rats were used to build an animal fracture model, and fracture healing and callus evolution were investigated by radiology (X-ray and micro-CT), histology (H&E, Masson, and safranin O/fast green staining), and histomorphometry. Further, the Cu2+ content and Runx2 level in the callus were determined, and local mechanical test of the fracture was performed to assess the healing quality. Our results revealed that 317L-Cu SS did not affect the proliferation of human bone mesenchymal stem cells, but promoted osteogenic differentiation and the expression of osteogenesis-related genes. In addition, 317L-Cu SS upregulated the lysyl oxidase activity. The X-ray and micro-CT results showed that the callus evolution efficiency and fracture healing speed were superior for 317L-Cu SS. Histological staining displayed large amounts of fibrous tissues at 3 weeks, and cartilage and new bone at 6 weeks. Further, histomorphometric analysis indicated that the callus possessed higher osteogenic efficiency at 6 weeks, and a high Cu2+ content and increased Runx2 expression were observed in the callus for 317L-Cu SS. Besides, the mechanical strength of the fracture site was much better than that of the control group. Overall, we conclude that 317L-Cu SS possesses the ability to increase Cu2+ content and promote osteogenesis in the callus, which could accelerate the callus evolution process and bone formation to provide faster and better fracture healing. PMID:29225463

Does the calcification of adamantinomatous craniopharyngioma resemble the calcium deposition of osteogenesis/odontogenesis?

PubMed

Song-Tao, Qi; Xiao-Rong, Yan; Jun, Pan; Yong-Jian, Deng; Jin, Liang; Guang-Long, Huang; Yun-Tao, Lu; Jian, Ruan; Xiang-Zhao, Li; Jia-Ming, Xu

2014-02-01

Calcification in adamantinomatous craniopharyngioma (ACP) is troublesome for surgical intervention. The aim of this study was to examine the osteogenic proteins that play important roles in the calcium deposition of the odontogenic/osteogenic tissues in craniopharyngioma. Craniopharyngiomas (n = 89) were investigated for the presence and expression pattern of the osteoinductive/odontoinductive factor bone morphogenetic protein-2 (Bmp2) and two osteoblastic differentiation makers, Runt-related transcription factor-2 (Runx2) and Osterix, using immunohistochemistry and Western blotting. Our results showed that Bmp2, Runx2 and Osterix levels increased in cases with high calcification and correlated positively with the degree of calcification in ACP, whereas they showed little or no expression in squamous papillary craniopharyngioma. In ACP, Bmp2 was expressed primarily in the stellate reticulum and whorl-like array cells; Runx2 and Osterix tended to be expressed in calcification-related epithelia, including whorl-like array cells and epithelia in/around wet keratin and calcification lesions. Our study indicated, for the first time, that osteogenic factor Bmp2 may play an important role in the calcification of ACP via autocrine or paracrine mechanisms. Given the presence of osteogenic markers (Runx2 and Osterix), craniopharyngioma cells could differentiate into an osteoblast-like lineage, and the process of craniopharyngioma calcification resembles that which occurs in osteogenesis/odontogenesis. © 2014 John Wiley & Sons Ltd.

MiR-133 is Involved in Estrogen Deficiency-Induced Osteoporosis through Modulating Osteogenic Differentiation of Mesenchymal Stem Cells.

PubMed

Lv, Hao; Sun, Yujie; Zhang, Yuchen

2015-05-27

MiR-133 expression is dysregulated in postmenopausal osteoporosis. However, its role in postmenopausal osteoporosis is still not well understood. In the current study, we explore how estrogen deficiency affects miR-133 expression and how miR-133 is involved in osteogenic differentiation of mesenchymal stem cells (MSCs). qRT-PCR analysis was performed to assess miR-133 expression in MSCs isolated from bone marrow of an ovariectomized (OVX) animal model and postmenopausal osteoporosis patients (PMOP) and their corresponding controls. The binding between miR-133 and predicted target SLC39A1 was verified using dual luciferase assay and Western blot analysis. The effect of miR-133 and SLC39A1 on osteogenic differentiation of MSCs was assessed through measuring alkaline phosphatase (ALP), mineralization nodules, and osteoblast-specific genes Runx2 and Osterix expression. miR-133 expression is significantly enhanced as a result of estrogen deficiency. Its overexpression is negatively correlated to osteogenic differentiation of hMSCs. SLC39A1 showed an inverse expression trend to miR-133 during the differentiation. miR-133 can directly target 3'UTR of SLC39A1 and thereby modulate its expression in hMSCs. The miR-133-SLC39A1 axis might play an important role in osteogenic differentiation of hMSCs. SLC39A1 can promote ALP activity and formation of mineralization nodules. In addition, SLC39A1 expression level is also positively correlated with RUNX2 and Osterix. Estrogen deficiency is associated with miR-133 overexpression. MiR-133 can induce postmenopausal osteoporosis by weakening osteogenic differentiation of hMSCs, at least partly through repressing SLC39A1 expression.

MiR-133 is Involved in Estrogen Deficiency-Induced Osteoporosis through Modulating Osteogenic Differentiation of Mesenchymal Stem Cells

PubMed Central

Lv, Hao; Sun, Yujie; Zhang, Yuchen

2015-01-01

Background MiR-133 expression is dysregulated in postmenopausal osteoporosis. However, its role in postmenopausal osteoporosis is still not well understood. In the current study, we explore how estrogen deficiency affects miR-133 expression and how miR-133 is involved in osteogenic differentiation of mesenchymal stem cells (MSCs). Material/Methods qRT-PCR analysis was performed to assess miR-133 expression in MSCs isolated from bone marrow of an ovariectomized (OVX) animal model and postmenopausal osteoporosis patients (PMOP) and their corresponding controls. The binding between miR-133 and predicted target SLC39A1 was verified using dual luciferase assay and Western blot analysis. The effect of miR-133 and SLC39A1 on osteogenic differentiation of MSCs was assessed through measuring alkaline phosphatase (ALP), mineralization nodules, and osteoblast-specific genes Runx2 and Osterix expression. Results miR-133 expression is significantly enhanced as a result of estrogen deficiency. Its overexpression is negatively correlated to osteogenic differentiation of hMSCs. SLC39A1 showed an inverse expression trend to miR-133 during the differentiation. miR-133 can directly target 3′UTR of SLC39A1 and thereby modulate its expression in hMSCs. The miR-133-SLC39A1 axis might play an important role in osteogenic differentiation of hMSCs. SLC39A1 can promote ALP activity and formation of mineralization nodules. In addition, SLC39A1 expression level is also positively correlated with RUNX2 and Osterix. Conclusions Estrogen deficiency is associated with miR-133 overexpression. MiR-133 can induce postmenopausal osteoporosis by weakening osteogenic differentiation of hMSCs, at least partly through repressing SLC39A1 expression. PMID:26013661

Core-binding factor beta interacts with Runx2 and is required for skeletal development.

PubMed

Yoshida, Carolina A; Furuichi, Tatsuya; Fujita, Takashi; Fukuyama, Ryo; Kanatani, Naoko; Kobayashi, Shinji; Satake, Masanobu; Takada, Kenji; Komori, Toshihisa

2002-12-01

Core-binding factor beta (CBFbeta, also called polyomavirus enhancer binding protein 2beta (PEBP2B)) is associated with an inversion of chromosome 16 and is associated with acute myeloid leukemia in humans. CBFbeta forms a heterodimer with RUNX1 (runt-related transcription factor 1), which has a DNA binding domain homologous to the pair-rule protein runt in Drosophila melanogaster. Both RUNX1 and CBFbeta are essential for hematopoiesis. Haploinsufficiency of another runt-related protein, RUNX2 (also called CBFA1), causes cleidocranial dysplasia in humans and is essential in skeletal development by regulating osteoblast differentiation and chondrocyte maturation. Mice deficient in Cbfb (Cbfb(-/-)) die at midgestation, so the function of Cbfbeta in skeletal development has yet to be ascertained. To investigate this issue, we rescued hematopoiesis of Cbfb(-/-) mice by introducing Cbfb using the Gata1 promoter. The rescued Cbfb(-/-) mice recapitulated fetal liver hematopoiesis in erythroid and megakaryocytic lineages and survived until birth, but showed severely delayed bone formation. Although mesenchymal cells differentiated into immature osteoblasts, intramembranous bones were poorly formed. The maturation of chondrocytes into hypertrophic cells was markedly delayed, and no endochondral bones were formed. Electrophoretic mobility shift assays and reporter assays showed that Cbfbeta was necessary for the efficient DNA binding of Runx2 and for Runx2-dependent transcriptional activation. These findings indicate that Cbfbeta is required for the function of Runx2 in skeletal development.

Epigenetically Modified Bone Marrow Stromal Cells in Silk Scaffolds Promote Craniofacial Bone Repair and Wound Healing.

PubMed

Han, Qianqian; Yang, Pishan; Wu, Yuwei; Meng, Shu; Sui, Lei; Zhang, Lan; Yu, Liming; Tang, Yin; Jiang, Hua; Xuan, Dongying; Kaplan, David L; Kim, Sung Hoon; Tu, Qisheng; Chen, Jake

2015-08-01

Epigenetic regulation of gene expression is a central mechanism that governs cell stemness, determination, commitment, and differentiation. It has been recently found that PHF8, a major H4K20/H3K9 demethylase, plays a critical role in craniofacial and bone development. In this study, we hypothesize that PHF8 promotes osteoblastogenesis by epigenetically regulating the expression of a nuclear matrix protein, special AT-rich sequence-binding protein 2 (SATB2) that plays pivotal roles in skeletal patterning and osteoblast differentiation. Our results showed that expression levels of PHF8 and SATB2 in preosteoblasts and bone marrow stromal cells (BMSCs) increased simultaneously during osteogenic induction. Overexpressing PHF8 in these cells upregulated the expression of SATB2, Runx2, osterix, and bone matrix proteins. Conversely, knockdown of PHF8 reduced the expression of these genes. Furthermore, ChIP assays confirmed that PHF8 specifically bound to the transcription start site (TSS) of the SATB2 promoter, and the expression of H3K9me1 at the TSS region of SATB2 decreased in PHF8 overexpressed group. Implantation of the BMSCs overexpressing PHF8 with silk protein scaffolds promoted bone regeneration in critical-sized defects in mouse calvaria. Taken together, our results demonstrated that PHF8 epigenetically modulates SATB2 activity, triggering BMSCs osteogenic differentiation and facilitating bone formation and regeneration in biodegradable silk scaffolds.

Combined experience of six independent laboratories attempting to create an Ewing sarcoma mouse model

PubMed Central

Han, Jenny; Han, Zhi-Yan; Sax, Barbara; Kream, Barbara E.; Hong, Sung-Hyeok; Çelik, Haydar; Tirode, Franck; Tuckermann, Jan; Toretsky, Jeffrey A.; Kenner, Lukas; Kovar, Heinrich; Lee, Sean; Sweet-Cordero, E. Alejandro; Nakamura, Takuro; Moriggl, Richard; Delattre, Olivier; Üren, Aykut

2017-01-01

Ewing sarcoma (ES) involves a tumor-specific chromosomal translocation that produces the EWS-FLI1 protein, which is required for the growth of ES cells both in vitro and in vivo. However, an EWS-FLI1-driven transgenic mouse model is not currently available. Here, we present data from six independent laboratories seeking an alternative approach to express EWS-FLI1 in different murine tissues. We used the Runx2, Col1a2.3, Col1a3.6, Prx1, CAG, Nse, NEFL, Dermo1, P0, Sox9 and Osterix promoters to target EWS-FLI1 or Cre expression. Additional approaches included the induction of an endogenous chromosomal translocation, in utero knock-in, and the injection of Cre-expressing adenovirus to induce EWS-FLI1 expression locally in multiple lineages. Most models resulted in embryonic lethality or developmental defects. EWS-FLI1-induced apoptosis, promoter leakiness, the lack of potential cofactors, and the difficulty of expressing EWS-FLI1 in specific sites were considered the primary reasons for the failed attempts to create a transgenic mouse model of ES. PMID:27191748

Combined experience of six independent laboratories attempting to create an Ewing sarcoma mouse model.

PubMed

Minas, Tsion Zewdu; Surdez, Didier; Javaheri, Tahereh; Tanaka, Miwa; Howarth, Michelle; Kang, Hong-Jun; Han, Jenny; Han, Zhi-Yan; Sax, Barbara; Kream, Barbara E; Hong, Sung-Hyeok; Çelik, Haydar; Tirode, Franck; Tuckermann, Jan; Toretsky, Jeffrey A; Kenner, Lukas; Kovar, Heinrich; Lee, Sean; Sweet-Cordero, E Alejandro; Nakamura, Takuro; Moriggl, Richard; Delattre, Olivier; Üren, Aykut

2017-05-23

Ewing sarcoma (ES) involves a tumor-specific chromosomal translocation that produces the EWS-FLI1 protein, which is required for the growth of ES cells both in vitro and in vivo. However, an EWS-FLI1-driven transgenic mouse model is not currently available. Here, we present data from six independent laboratories seeking an alternative approach to express EWS-FLI1 in different murine tissues. We used the Runx2, Col1a2.3, Col1a3.6, Prx1, CAG, Nse, NEFL, Dermo1, P0, Sox9 and Osterix promoters to target EWS-FLI1 or Cre expression. Additional approaches included the induction of an endogenous chromosomal translocation, in utero knock-in, and the injection of Cre-expressing adenovirus to induce EWS-FLI1 expression locally in multiple lineages. Most models resulted in embryonic lethality or developmental defects. EWS-FLI1-induced apoptosis, promoter leakiness, the lack of potential cofactors, and the difficulty of expressing EWS-FLI1 in specific sites were considered the primary reasons for the failed attempts to create a transgenic mouse model of ES.

Differentiation of Human Dental Stem Cells Reveal a Role for microRNA-218

PubMed Central

Gay, Isabel; Cavender, Adriana; Peto, David; Sun, Zhao; Speer, Aline; Cao, Huojun; Amendt, Brad A.

2013-01-01

Background Regeneration of the lost periodontium is the ultimate goal of periodontal therapy. Advances in tissue engineering have demonstrated the multilineage potential and plasticity of adult stem cells located in the periodontal apparatus. However, it remains unclear how epigenetic mechanisms controlling signals determine tissue specification and cell lineage decisions. To date, no data is available on micro-RNAs (miRNAs) activity behind human-derived dental stem cells. Methods In this study, we isolated periodontal ligament stem cells (PDLSCs), dental pulp stem cells (DPSCs), and gingival stem cells (GSCs) from extracted third molars; human bone marrow stem cells (BMSCs) were used as a positive control. The expression of OCT4A and NANOG was confirmed in these undifferentiated cells. All cells were cultured under osteogenic inductive conditions and RUNX2 expression was analyzed as a marker of mineralized tissue differentiation. A miRNA expression profile was obtained at baseline and after osteogenic induction in all cell types. Results RUNX2 expression demonstrated the successful osteogenic induction of all cell types, which was confirmed by alizarin red stain. The analysis of 765 miRNAs demonstrated a shift in miRNA expression occurred in all four stem cell types, including a decrease in hsa-mir-218 across all differentiated cell populations. Hsa-mir-218 targets RUNX2 and decreases RUNX2 expression in undifferentiated human dental stem cells (DSCs). DSC mineralized tissue type differentiation is associated with a decrease in hsa-mir-218 expression. Conclusions These data reveal a miRNA regulated pathway for the differentiation of human DSCs and a select network of human microRNAs that control DSC osteogenic differentiation. PMID:23662917

Effect of hydrogel elasticity and ephrinB2-immobilized manner on Runx2 expression of human mesenchymal stem cells.

PubMed

Toda, Hiroyuki; Yamamoto, Masaya; Uyama, Hiroshi; Tabata, Yasuhiko

2017-08-01

The objective of this study is to design the manner of ephrinB2 immobilized onto polyacrylamide (PAAm) hydrogels with varied elasticity and evaluate the effect of hydrogels elasticity and the immobilized manner of ephrinB2 on the Runx2 expression of human mesenchymal stem cells (hMSC). The PAAm hydrogels were prepared by the radical polymerization of acrylamide (AAm), and N,N'-methylenebisacrylamide (BIS). By changing the BIS concentration, the elasticity of PAAm hydrogels changed from 1 to 70kPa. For the bio-specific immobilization of ephrinB2, a chimeric protein of ephrinB2 and Fc domain was immobilized onto protein A-conjugated PAAm hydrogels by making use of the bio-specific interaction between the Fc domain and protein A. When hMSC were cultured on the ephrinB2-immobilized PAAm hydrogels with varied elasticity, the morphology of hMSC was of cuboidal shape on the PAAm hydrogels immobilized with ephrinB2 compared with non-conjugated ones, irrespective of the hydrogels elasticity. The bio-specific immobilization of ephrinB2 enhanced the level of Runx2 expression. The expression level was significantly high for the hydrogels of 3.6 and 5.9kPa elasticity with bio-specific immobilization of ephrinB2 compared with other hydrogels with the same elasticity. The hydrogels showed a significantly down-regulated RhoA activity. It is concluded that the Runx2 expression of hMSC is synergistically influenced by the hydrogels elasticity and their immobilized manner of ephrinB2 immobilized. Differentiation fate of mesenchymal stem cells (MSC) is modified by biochemical and biophysical factors, such as elasticity and signal proteins. However, there are few experiments about combinations of them. In this study, to evaluate the synergistic effect of them on cell properties of MSC, we established to design the manner of Eph signal ligand, ephrinB2, immobilized onto polyacrylamide hydrogels with varied elasticity. The gene expression level of an osteogenic maker, Runx2, was enhanced by the immobilized manner, and significantly enhanced for the hydrogels of around 4kPa elasticity with bio-specific immobilization of ephrinB2. This is the novel report describing to demonstrate that the Runx2 expression of MSC is synergistically influenced by the hydrogels elasticity and their manner of ephrinB2 immobilized. Copyright © 2017 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Synthetic octacalcium phosphate-enhanced reparative dentine formation via induction of odontoblast differentiation.

PubMed

Wang, Xiaogu; Suzawa, Tetsuo; Miyauchi, Tomohiko; Zhao, Baohong; Yasuhara, Rika; Anada, Takahisa; Nakamura, Masanori; Suzuki, Osamu; Kamijo, Ryutaro

2015-11-01

Synthetic octacalcium phosphate (OCP) has been suggested to be a useful biomaterial for the regeneration of hard tissues, including bone. However, it remains unknown whether OCP induces dentine formation by dental pulp. We investigated biomineralization of dental pulp exposed to synthetic OCP in vitro and in vivo. When dental pulp was exposed directly to OCP, rapid formation of reparative dentine (RD) was induced and expression of dentine sialoprotein synthesis was observed in dental pulp adjacent to newly synthesized RD. OCP inhibited the proliferation of rat pulp cells and also promoted their odontoblastic differentiation in vitro, as alkaline phosphatase activity, mineralization of pulp cells and the expression level of dentine sialophosphoprotein were enhanced. Direct contact between OCP and pulp cells is required for OCP to exhibit its effects in vitro. The expression level of Runx2, a transcription factor whose downregulation is closely related to odontoblast differentiation, was downregulated in pulp cells cultured with OCP. Structural changes of OCP during culture were determined by Fourier transform infrared spectroscopy. OCP tended to be converted to carbonate hydroxyapatite after incubation with or without pulp cells, which may be analogous to biological apatite crystals. Taken together, our data suggest that synthetic OCP supports RD formation by dental pulp and downregulation of Runx2 may be involved in that stimulatory activity. Furthermore, OCP-apatite conversion is involved in this stimulatory capacity of OCP. Copyright © 2013 John Wiley & Sons, Ltd.

Platelet-Rich Fibrin Promotes Periodontal Regeneration and Enhances Alveolar Bone Augmentation

PubMed Central

Li, Qi; Pan, Shuang; Dangaria, Smit J.; Gopinathan, Gokul; Kolokythas, Antonia; Chu, Shunli; Geng, Yajun; Zhou, Yanmin; Luan, Xianghong

2013-01-01

In the present study we have determined the suitability of platelet-rich fibrin (PRF) as a complex scaffold for periodontal tissue regeneration. Replacing PRF with its major component fibrin increased mineralization in alveolar bone progenitors when compared to periodontal progenitors, suggesting that fibrin played a substantial role in PRF-induced osteogenic lineage differentiation. Moreover, there was a 3.6-fold increase in the early osteoblast transcription factor RUNX2 and a 3.1-fold reduction of the mineralization inhibitor MGP as a result of PRF application in alveolar bone progenitors, a trend not observed in periodontal progenitors. Subcutaneous implantation studies revealed that PRF readily integrated with surrounding tissues and was partially replaced with collagen fibers 2 weeks after implantation. Finally, clinical pilot studies in human patients documented an approximately 5 mm elevation of alveolar bone height in tandem with oral mucosal wound healing. Together, these studies suggest that PRF enhances osteogenic lineage differentiation of alveolar bone progenitors more than of periodontal progenitors by augmenting osteoblast differentiation, RUNX2 expression, and mineralized nodule formation via its principal component fibrin. They also document that PRF functions as a complex regenerative scaffold promoting both tissue-specific alveolar bone augmentation and surrounding periodontal soft tissue regeneration via progenitor-specific mechanisms. PMID:23586051

Platelet-rich fibrin promotes periodontal regeneration and enhances alveolar bone augmentation.

PubMed

Li, Qi; Pan, Shuang; Dangaria, Smit J; Gopinathan, Gokul; Kolokythas, Antonia; Chu, Shunli; Geng, Yajun; Zhou, Yanmin; Luan, Xianghong

2013-01-01

In the present study we have determined the suitability of platelet-rich fibrin (PRF) as a complex scaffold for periodontal tissue regeneration. Replacing PRF with its major component fibrin increased mineralization in alveolar bone progenitors when compared to periodontal progenitors, suggesting that fibrin played a substantial role in PRF-induced osteogenic lineage differentiation. Moreover, there was a 3.6-fold increase in the early osteoblast transcription factor RUNX2 and a 3.1-fold reduction of the mineralization inhibitor MGP as a result of PRF application in alveolar bone progenitors, a trend not observed in periodontal progenitors. Subcutaneous implantation studies revealed that PRF readily integrated with surrounding tissues and was partially replaced with collagen fibers 2 weeks after implantation. Finally, clinical pilot studies in human patients documented an approximately 5 mm elevation of alveolar bone height in tandem with oral mucosal wound healing. Together, these studies suggest that PRF enhances osteogenic lineage differentiation of alveolar bone progenitors more than of periodontal progenitors by augmenting osteoblast differentiation, RUNX2 expression, and mineralized nodule formation via its principal component fibrin. They also document that PRF functions as a complex regenerative scaffold promoting both tissue-specific alveolar bone augmentation and surrounding periodontal soft tissue regeneration via progenitor-specific mechanisms.

Mitotic inheritance of mRNA facilitates translational activation of the osteogenic-lineage commitment factor Runx2 in progeny of osteoblastic cells

PubMed Central

Varela, Nelson; Aranguiz, Alejandra; Lizama, Carlos; Sepulveda, Hugo; Antonelli, Marcelo; Thaler, Roman; Moreno, Ricardo D.; Montecino, Martin; Stein, Gary S.; van Wijnen, Andre J.; Galindo, Mario

2017-01-01

Epigenetic mechanisms mediate the acquisition of specialized cellular phenotypes during tissue development, maintenance and repair. When phenotype-committed cells transit through mitosis, chromosomal condensation counteracts epigenetic activation of gene expression. Subsequent post-mitotic re-activation of transcription depends on epigenetic DNA and histone modifications, as well as other architecturally bound proteins that ‘bookmark’ the genome. Osteogenic lineage commitment, differentiation and progenitor proliferation require the bone-related runt-related transcription factor Runx2. Here, we characterized a non-genomic mRNA mediated mechanism by which osteoblast precursors retain their phenotype during self-renewal. We show that osteoblasts produce maximal levels of Runx2 mRNA, but not protein, prior to mitotic cell division. Runx2 mRNA partitions symmetrically between daughter cells in a non-chromosomal tubulin-containing compartment. Subsequently, transcription-independent de novo synthesis of Runx2 protein in early G1 phase results in increased functional interactions of Runx2 with a representative osteoblast-specific target gene (osteocalcin/BGLAP2) in chromatin. Somatic transmission of Runx2 mRNAs in osteoblasts and osteosarcoma cells represents a versatile mechanism for translational rather than transcriptional induction of this principal gene regulator to maintain osteoblast phenotype identity after mitosis. PMID:26381402

Mutations in RAD21 Disrupt Regulation of APOB in Patients with Chronic Intestinal Pseudo-obstruction

PubMed Central

Bonora, Elena; Bianco, Francesca; Cordeddu, Lina; Bamshad, Michael; Francescatto, Ludmila; Dowless, Dustin; Stanghellini, Vincenzo; Cogliandro, Rosanna F.; Lindberg, Greger; Mungan, Zeynel; Cefle, Kivanc; Ozcelik, Tayfun; Palanduz, Sukru; Ozturk, Sukru; Gedikbasi, Asuman; Gori, Alessandra; Pippucci, Tommaso; Graziano, Claudio; Volta, Umberto; Caio, Giacomo; Barbara, Giovanni; D'Amato, Mauro; Seri, Marco; Katsanis, Nicholas; Romeo, Giovanni; De Giorgio, Roberto

2015-01-01

Background & Aims Chronic intestinal pseudo-obstruction (CIPO) is characterized by severe intestinal dysmotility that mimicks a mechanical sub-occlusion with no evidence of gut obstruction. We searched for genetic variants associated with CIPO to increase our understanding of its pathogenesis and indentify potential biomarkers. Methods We performed whole-exome sequencing of genomic DNA from patients with familial CIPO syndrome. Blood and lymphoblastoid cells were collected from patients and controls (individuals without CIPO); levels of mRNA and proteins were analyzed by quantitative reverse transcription PCR, immunoblot, and mobility shift assays. cDNAs were transfected into HEK293 cells. Expression of rad21 was suppressed in zebrafish embryos using a splice-blocking morpholino (rad21a MO). Gut tissues were collected and analyzed. Results We identified a homozygous mutation (p.622, encodes Ala>Thr) in RAD21 in patients from a consanguineous family with CIPO. Expression of RUNX1, a target of RAD21, was reduced in cells from patients with CIPO compared with controls. In zebrafish, suppression of rad21a reduced expression of runx1; this phenotype was corrected by injection of human RAD21 mRNA, but not with the mRNA from the mutated p.622 allele. rad21a MO zebrafish had delayed intestinal transit and greatly reduced numbers of enteric neurons, similar to patients with CIPO. This defect was greater in zebrafish with suppressed expression of ret and rad21, indicating their interaction in regulation of gut neurogenesis. The promoter region of APOB bound RAD21 but not RAD21 p.622 Ala>Thr; expression of wild-type RAD21 in HEK293 cells repressed expression of APOB, compared with control vector. The gut-specific isoform of APOB (APOB48) is overexpressed in sera from patients with CIPO who carry the RAD21 mutation. APOB48 is also overexpressed in sporadic CIPO in sera and gut biopsies. Conclusions Some patients with CIPO carry mutations in RAD21 that disrupt the ability of its product to regulate genes such as RUNX1 and APOB. Reduced expression of rad21 in zebrafish, and dysregulation of these target genes, disrupts intestinal transit and development of enteric neurons. PMID:25575569

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhu Lingling; Zaidi, Samir; Peng Yuanzhen

Strontium ranelate, a new agent for the treatment of osteoporosis, has been shown stimulate bone formation in various experimental models. This study examines the effect of strontium ranelate on gene expression in osteoblasts, as well as the formation of mineralized (von Kossa-positive) colony-forming unit-osteoblasts (CFU-obs). Bone marrow-derived stromal cells cultured for 21 days under differentiating conditions, when exposed to strontium ranelate, displayed a significant time- and concentration-dependent increase in the expression of the master gene, Runx2, as well as bone sialoprotein (BSP), but interestingly without effects on osteocalcin. This was associated with a significant increase in the formation of CFU-obsmore » at day 21 of culture. In U-33 pre-osteoblastic cells, strontium ranelate significantly enhanced the expression of Runx2 and osteocalcin, but not BSP. Late, more mature osteoblastic OB-6 cells showed significant elevations in BSP and osteocalcin, but with only minimal effects on Runx2. In conclusion, strontium ranelate stimulates osteoblast differentiation, but the induction of the program of gene expression appears to be cell type-specific. The increased osteoblastic differentiation is the likely basis underlying the therapeutic bone-forming actions of strontium ranelate.« less

RUNX1-induced silencing of non-muscle myosin heavy chain IIB contributes to megakaryocyte polyploidization.

PubMed

Lordier, Larissa; Bluteau, Dominique; Jalil, Abdelali; Legrand, Céline; Pan, Jiajia; Rameau, Philippe; Jouni, Dima; Bluteau, Olivier; Mercher, Thomas; Leon, Catherine; Gachet, Christian; Debili, Najet; Vainchenker, William; Raslova, Hana; Chang, Yunhua

2012-03-06

Megakaryocytes are unique mammalian cells that undergo polyploidization (endomitosis) during differentiation, leading to an increase in cell size and protein production that precedes platelet production. Recent evidence demonstrates that endomitosis is a consequence of a late failure in cytokinesis associated with a contractile ring defect. Here we show that the non-muscle myosin IIB heavy chain (MYH10) is expressed in immature megakaryocytes and specifically localizes in the contractile ring. MYH10 downmodulation by short hairpin RNA increases polyploidization by inhibiting the return of 4N cells to 2N, but other regulators, such as of the G1/S transition, might regulate further polyploidization of the 4N cells. Conversely, re-expression of MYH10 in the megakaryocytes prevents polyploidization and the transition of 2N to 4N cells. During polyploidization, MYH10 expression is repressed by the major megakaryocyte transcription factor RUNX1. Thus, RUNX1-mediated silencing of MYH10 is required for the switch from mitosis to endomitosis, linking polyploidization with megakaryocyte differentiation.

BAG3 promotes chondrosarcoma progression by upregulating the expression of β-catenin

PubMed Central

Shi, Huijuan; Chen, Wenfang; Dong, Yu; Lu, Xiaofang; Zhang, Wenhui; Wang, Liantang

2018-01-01

To investigate the roles of B-cell lymphoma-2 associated athanogene 3 (BAG3) in human chondrosarcoma and the potential mechanisms, the expression levels of BAG3 were detected in the present study, and the associations between BAG3 and clinical pathological parameters, clinical stage as well as the survival of patients were analyzed. The present study detected BAG3 mRNA and protein expression in the normal cartilage cell line HC-a and in SW1353 chondrosarcoma cells by reverse transcription-quantitative polymerase chain reaction and western blot analysis. The BAG3 protein expression in 59 cases of chondrosarcoma, 30 patients with endogenous chondroma and 8 cases of normal cartilage was semi-quantitatively analyzed using the immunohistochemical method. In addition, the BAG3 protein expression level, the clinical pathological parameters, clinical stage and the survival time of patients with chondrosarcoma were analyzed. The plasmid transfection method was employed to upregulate the expression BAG3 and small RNA interference to downregulate the expression of BAG3 in SW1353 cells. The expression levels of BAG3 protein and mRNA were significantly increased in the chondrosarcoma cell line when compared with the normal cartilage cell line. The immunohistochemistry results indicated that BAG3 protein was overexpressed in the tissue of human chondrosarcoma. Statistical analysis showed that the expression level of BAG3 was significantly increased in the different Enneking staging of patients with chondrosarcoma and Tumor staging, and there were no statistical differences in age, gender, histological classification and tumor size. In the in vitro experiments, the data revealed that BAG3 significantly promoted chondrosarcoma cell proliferation, colony-formation, migration and invasion; however, it inhibited chondrosarcoma cell apoptosis. It was observed that BAG3 upregulated β-catenin expression at the mRNA and protein levels. In addition, BAG3 induced the expression of runt-related transcription factor 2 (RUNX2) in chondrosarcoma cells by upregulating β-catenin. These clinical analyses revealed a positive association between β-catenin and BAG3 in chondrosarcoma tumors. BAG3 was significantly increased in chondrosarcoma cells and tissues compared with the normal cartilage cells, tissue and cartilage benign tumors. Thus, BAG3 may serve as an oncogene in the development of chondrosarcoma via the induction of RUNX2 expression. The results of the present study contribute to further research on the biological development of chondrosarcoma. PMID:29484408

RUNX1 and NF-E2 upregulation is not specific for MPNs, but is seen in polycythemic disorders with augmented HIF signaling

PubMed Central

Kapralova, Katarina; Lanikova, Lucie; Lorenzo, Felipe; Song, Jihyun; Horvathova, Monika; Divoky, Vladimir

2014-01-01

Overexpression of transcription factors runt-related transcription factor 1 (RUNX1) and nuclear factor, erythroid-derived 2 (NF-E2) was reported in granulocytes of patients with polycythemia vera and other myeloproliferative neoplasms (MPNs). Further, a transgenic mouse overexpressing the NF-E2 transgene was reported to be a model of MPN. We hypothesized that increased transcripts of RUNX1 and NF-E2 might characterize other polycythemic states with primary polycythemic features, that is, those with exaggerated erythropoiesis due to augmented erythropoietin (EPO) sensitivity. We tested the expression of RUNX1 and NF-E2 in polycythemic patients of diverse phenotypes and molecular causes. We report that RUNX1 and NF-E2 overexpression is not specific for MPN; these transcripts were also significantly elevated in polycythemias with augmented hypoxia-inducible factor activity whose erythroid progenitors were hypersensitive to EPO. RUNX1 and NF-E2 overexpression was not detected in patients with EPO receptor (EPOR) gain-of-function, suggesting distinct mechanisms by which erythroid progenitors in polycythemias with defects of hypoxia sensing and EPOR mutations exert their EPO hypersensitivity. PMID:24297870

Low-magnitude, high-frequency vibration promotes the adhesion and the osteogenic differentiation of bone marrow-derived mesenchymal stem cells cultured on a hydroxyapatite-coated surface: The direct role of Wnt/β-catenin signaling pathway activation.

PubMed

Chen, Bailing; Lin, Tao; Yang, Xiaoxi; Li, Yiqiang; Xie, Denghui; Zheng, Wenhui; Cui, Haowen; Deng, Weimin; Tan, Xin

2016-11-01

The positive effect of low-magnitude, high‑frequency (LMHF) vibration on implant osseointegration has been demonstrated; however, the underlying cellular and molecular mechanisms remain unknown. The aim of this study was to explore the effect of LMHF vibration on the adhesion and the osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) cultured on hydroxyapatite (HA)-coated surfaces in an in vitro model as well as to elucidate the molecular mechanism responsible for the effects of LMHF vibration on osteogenesis. LMHF vibration resulted in the increased expression of fibronectin, which was measured by immunostaining and RT-qPCR. Stimulation of BMSCs by LMHF vibration resulted in the rearrangement of the actin cytoskeleton with more prominent F-actin. Moreover, the expression of β1 integrin, vinculin and paxillin was notably increased following LMHF stimulation. Scanning electron microscope observations revealed that there were higher cell numbers and more extracellular matrix attached to the HA-coated surface in the LMHF group. Alkaline phosphatase activity as well as the expression of osteogenic-specific genes, namely Runx2, osterix, collagen I and osteocalcin, were significantly elevated in the LMHF group. In addition, the protein expression of Wnt10B, β-catenin, Runx2 and osterix was increased following exposure to LMHF vibration. Taken together, the findings of this study indicate that LMHF vibration promotes the adhesion and the osteogenic differentiation of BMSCs on HA-coated surfaces in vitro, and LMHF vibration may directly induce osteogenesis by activating the Wnt/β‑catenin signaling pathway. These data suggest that LMHF vibration enhances the osseointegration of bone to a HA-coated implant, and provide a scientific foundation for improving bone-implant osseointegration through the application of LMHF vibration.

Deletion of Core-binding factor β (Cbfβ) in mesenchymal progenitor cells provides new insights into Cbfβ/Runxs complex function in cartilage and bone development

PubMed Central

Wu, Mengrui; Li, Chenguan; Zhu, Guochun; Wang, Yiping; Jules, Joel; Lu, Yun; McConnell, Matthew; Wang, Yong-Jun; Shao, Jian-Zhong; Li, Yi-Ping; Chen, Wei

2015-01-01

Core-binding factor β (Cbfβ) is a subunit of the Cbf family of heterodimeric transcription factors which plays a critical role in skeletal development through its interaction with the Cbfα subunits, also known as Runt-related transcription factors (Runxs). However, the mechanism by which Cbfβ regulates cartilage and bone development remains unclear. Existing Cbfβ-deficient mouse models cannot specify the role of Cbfβ in skeletal cell lineage. Herein, we sought to specifically address the role of Cbfβ in cartilage and bone development by using a conditional knockout (CKO) approach. A mesenchymal-specific Cbfβ CKO mouse model was generated by using the Dermo1-Cre mouse line to specifically delete Cbfβ in mesenchymal stem cells, which give rise to osteoblasts and chondrocytes. Surprisingly, the mutant mice had under-developed larynx and tracheal cartilage causing alveolus defects which led to death shortly after birth from suffocation. Also, the mutant mice exhibited severe skeletal deformities from defective intramembranous and endochondral ossification, owing to delayed chondrocyte maturation and impaired osteoblast differentiation. Almost all bones of the mutant mice, including the calvariae, vertebrae, tibiae, femurs, ribs, limbs and sternums were defective. Importantly, we showed that Cbfβ was expressed throughout the skeleton during both embryonic and postnatal development, which explains the multiple-skeletal defects observed in the mutant mice. Consistently, Cbfβ deficiency impaired both chondrocyte proliferation and hypertrophy zone hypertrophy during growth-plate development in the long bones of mutant mice. Notably, Cbfβ, Runx1 and Runx2 displayed different expression patterns in the growth plates of the wildtype mice indicating that Cbfβ/Runx1 complex and Cbfβ/Runx2 complex may regulate chondrocyte proliferation and hypertrophy, respectively, in a spatial and temporal manner. Cbfβ deletion in the mesenchymal progenitors impacted bone development by dramatically down-regulating Collagen X (Col X) and Osterix (Osx), but had a dispensable effect on osteoclast development. Collectively, the results demonstrate that Cbfβ mediates cartilage and bone development by interacting with Runx1 and Runx2 to regulate the expressions of Col X and Osx for chondrocyte and osteoblast development. These findings not only reveal a critical role for Cbfβ in cartilage and bone development, but also facilitate the design of novel therapeutic approaches for skeletal diseases. PMID:24798493

Deletion of core-binding factor β (Cbfβ) in mesenchymal progenitor cells provides new insights into Cbfβ/Runxs complex function in cartilage and bone development.

PubMed

Wu, Mengrui; Li, Chenguan; Zhu, Guochun; Wang, Yiping; Jules, Joel; Lu, Yun; McConnell, Matthew; Wang, Yong-Jun; Shao, Jian-Zhong; Li, Yi-Ping; Chen, Wei

2014-08-01

Core-binding factor β (Cbfβ) is a subunit of the Cbf family of heterodimeric transcription factors, which plays a critical role in skeletal development through its interaction with the Cbfα subunits, also known as Runt-related transcription factors (Runxs). However, the mechanism by which Cbfβ regulates cartilage and bone development remains unclear. Existing Cbfβ-deficient mouse models cannot specify the role of Cbfβ in skeletal cell lineage. Herein, we sought to specifically address the role of Cbfβ in cartilage and bone development by using a conditional knockout (CKO) approach. A mesenchymal-specific Cbfβ CKO mouse model was generated by using the Dermo1-Cre mouse line to specifically delete Cbfβ in mesenchymal stem cells, which give rise to osteoblasts and chondrocytes. Surprisingly, the mutant mice had under-developed larynx and tracheal cartilage, causing alveolus defects that led to death shortly after birth from suffocation. Also, the mutant mice exhibited severe skeletal deformities from defective intramembranous and endochondral ossification, owing to delayed chondrocyte maturation and impaired osteoblast differentiation. Almost all bones of the mutant mice, including the calvariae, vertebrae, tibiae, femurs, ribs, limbs and sternums were defective. Importantly, we showed that Cbfβ was expressed throughout the skeleton during both embryonic and postnatal development, which explains the multiple-skeletal defects observed in the mutant mice. Consistently, Cbfβ deficiency impaired both chondrocyte proliferation and hypertrophy zone hypertrophy during growth-plate development in the long bones of mutant mice. Notably, Cbfβ, Runx1 and Runx2 displayed different expression patterns in the growth plates of the wild-type mice, indicating that Cbfβ/Runx1 complex and Cbfβ/Runx2 complex may regulate chondrocyte proliferation and hypertrophy, respectively, in a spatial and temporal manner. Cbfβ deletion in the mesenchymal progenitors affected bone development by dramatically down-regulating Collagen X (Col X) and Osterix (Osx) but had a dispensable effect on osteoclast development. Collectively, the results demonstrate that Cbfβ mediates cartilage and bone development by interacting with Runx1 and Runx2 to regulate the expressions of Col X and Osx for chondrocyte and osteoblast development. These findings not only reveal a critical role for Cbfβ in cartilage and bone development but also facilitate the design of novel therapeutic approaches for skeletal diseases. Copyright © 2014. Published by Elsevier Inc.

RUNX3 confers sensitivity to pheophorbide a-photodynamic therapy in human oral squamous cell carcinoma cell lines.

PubMed

Moon, Sook; Bae, Jung Yoon; Son, Hwa-Kyung; Lee, Doo Young; Park, Gyeongju; You, Hyun; Ko, Hyojin; Kim, Yong-Chul; Kim, Jin

2015-02-01

Photodynamic therapy (PDT) with photosensitizer is one of the promising modalities for cancer treatment. For clinical use of PDT, screening process should be preceded to enhance sensitivity to PDT. Thus, we investigated a molecular biomarker to determine the sensitivity to pheophorbide a (Pa)-PDT in immortalized human oral keratinocytes (IHOK) and oral squamous cell carcinoma (OSCC) cell lines. Two IHOK and several OSCC cell lines were used. After Pa-PDT, cell viability was reduced by more than 50%, and reactive oxygen species were generated in IHOK and OSCC cell lines. Additionally, apoptosis occurred in PDT-treated cells. IHOK(S) and IHOK(P), the two IHOK cell lines derived from the same source, showed a difference in cytotoxicity after Pa-PDT. To explain this difference in cytotoxicity, we looked at the expression of Wnt signaling-related genes in these two cell lines, for the morphology of IHOK(S) which was spindle like and elongated and distinct from IHOK(P) and the parent cell. Among the relevant genes, runt-related transcription factor 3 (RUNX3), an apoptosis-related gene, was selected as a potential marker that confers sensitivity to PDT. We found that the cytotoxicity by Pa-PDT was proportional to RUNX3 expression in OSCC cell lines. Additionally, knockdown of RUNX3 expression reduced cytotoxicity by Pa-PDT, suggesting that RUNX3 might be a biomarker to determine sensitivity to Pa-PDT. This was the first study to find a new target molecule that enhances Pa-PDT effects in IHOK and OSCC cell lines. Hence, the development of a PDT-dependent biomarker could provide a novel approach to improve the effects of PDT on oral precancerous and cancerous lesions.

MicroRNA-29a mitigates glucocorticoid induction of bone loss and fatty marrow by rescuing Runx2 acetylation.

PubMed

Ko, Jih-Yang; Chuang, Pei-Chin; Ke, Huei-Jin; Chen, Yu-Shan; Sun, Yi-Chih; Wang, Feng-Sheng

2015-12-01

Glucocorticoid treatment reportedly increases the morbidity of osteoporotic or osteonecrotic disorders. Exacerbated bone acquisition and escalated marrow adipogenesis are prominent pathological features of glucocorticoid-mediated skeletal disorders. MicroRNAs reportedly modulate tissue metabolism and remodeling. This study was undertaken to investigate the biological roles of microRNA-29a (miR-29a) in skeletal and fat metabolism in the pathogenesis of glucocorticoid-induced osteoporosis. Transgenic mice overexpressing miR-29a precursor or wild-type mice were given methylprednisolone. Bone mass, microarchitecture and histology were assessed by dual energy X-ray absorptiometry, μCT and histomorphometry. Differential gene expression and signaling components were delineated by quantitative RT-PCR and immunoblotting. Glucocorticoid treatment accelerated bone loss and marrow fat accumulation in association with decreased miR-29a expression. The miR-29a transgenic mice had high bone mineral density, trabecular microarchitecture and cortical thickness. miR-29a overexpression mitigated the glucocorticoid-induced impediment of bone mass, skeletal microstructure integrity and mineralization reaction and attenuated fatty marrow histopathology. Ex vivo, miR-29a increased osteogenic differentiation capacity and alleviated the glucocorticoid-induced promotion of adipocyte formation in primary bone-marrow mesenchymal progenitor cell cultures. Through inhibition of histone deacetylase 4 (HDAC4) expression, miR-29a restored acetylated Runx2 and β-catenin abundances and reduced RANKL, leptin and glucocorticoid receptor expression in glucocorticoid-mediated osteoporosis bone tissues. Taken together, glucocorticoid suppression of miR-29a signaling disturbed the balances between osteogenic and adipogenic activities, and thereby interrupted bone formation and skeletal homeostasis. miR-29a inhibition of HDAC4 stabilized the acetylation state of Runx2 and β-catenin that ameliorated the detrimental effects of glucocorticoid on mineralization and lipogenesis reactions in bone tissue microenvironments. This study highlighted emerging skeletal-anabolic actions of miR-29a signaling in the progression of glucocorticoid-induced bone tissue destruction. Sustaining miR-29a actions is beneficial in protecting against glucocorticoid-mediated osteoporosis. Copyright © 2015 Elsevier Inc. All rights reserved.

IGF-1/IGF-1R/hsa-let-7c axis regulates the committed differentiation of stem cells from apical papilla

PubMed Central

Ma, Shu; Liu, Genxia; Jin, Lin; Pang, Xiyao; Wang, Yanqiu; Wang, Zilu; Yu, Yan; Yu, Jinhua

2016-01-01

Insulin-like growth factor-1 (IGF-1) and its receptor IGF-1R play a paramount role in tooth/bone formation while hsa-let-7c actively participates in the osteogenic differentiation of mesenchymal stem cells. However, the interaction between IGF-1/IGF-1R and hsa-let-7c on the committed differentiation of stem cells from apical papilla (SCAPs) remains unclear. In this study, human SCAPs were isolated and treated with IGF-1 and hsa-let-7c over/low-expression viruses. The odonto/osteogenic differentiation of these stem cells and the involvement of mitogen-activated protein kinase (MAPK) pathway were subsequently investigated. Alizarin red staining showed that hsa-let-7c low-expression can significantly promote the mineralization of IGF-1 treated SCAPs, while hsa-let-7c over-expression can decrease the calcium deposition of IGF-1 treated SCAPs. Western blot assay and real-time reverse transcription polymerase chain reaction further demonstrated that the expression of odonto/osteogenic markers (ALP, RUNX2/RUNX2, OSX/OSX, OCN/OCN, COL-I/COL-I, DSPP/DSP, and DMP-1/DMP-1) in IGF-1 treated SCAPs were significantly upregulated in Let-7c-low group. On the contrary, hsa-let-7c over-expression could downregulate the expression of these odonto/osteogenic markers. Moreover, western blot assay showed that the JNK and p38 MAPK signaling pathways were activated in Let-7c-low SCAPs but inhibited in Let-7c-over SCAPs. Together, the IGF-1/IGF-1R/hsa-let-7c axis can control the odonto/osteogenic differentiation of IGF-1-treated SCAPs via the regulation of JNK and p38 MAPK signaling pathways. PMID:27833148

Epigenetically Modified Bone Marrow Stromal Cells in Silk Scaffolds Promote Craniofacial Bone Repair and Wound Healing

PubMed Central

Han, Qianqian; Yang, Pishan; Wu, Yuwei; Meng, Shu; Sui, Lei; Zhang, Lan; Yu, Liming; Tang, Yin; Jiang, Hua; Xuan, Dongying; Kaplan, David L.; Kim, Sung Hoon

2015-01-01

Epigenetic regulation of gene expression is a central mechanism that governs cell stemness, determination, commitment, and differentiation. It has been recently found that PHF8, a major H4K20/H3K9 demethylase, plays a critical role in craniofacial and bone development. In this study, we hypothesize that PHF8 promotes osteoblastogenesis by epigenetically regulating the expression of a nuclear matrix protein, special AT-rich sequence-binding protein 2 (SATB2) that plays pivotal roles in skeletal patterning and osteoblast differentiation. Our results showed that expression levels of PHF8 and SATB2 in preosteoblasts and bone marrow stromal cells (BMSCs) increased simultaneously during osteogenic induction. Overexpressing PHF8 in these cells upregulated the expression of SATB2, Runx2, osterix, and bone matrix proteins. Conversely, knockdown of PHF8 reduced the expression of these genes. Furthermore, ChIP assays confirmed that PHF8 specifically bound to the transcription start site (TSS) of the SATB2 promoter, and the expression of H3K9me1 at the TSS region of SATB2 decreased in PHF8 overexpressed group. Implantation of the BMSCs overexpressing PHF8 with silk protein scaffolds promoted bone regeneration in critical-sized defects in mouse calvaria. Taken together, our results demonstrated that PHF8 epigenetically modulates SATB2 activity, triggering BMSCs osteogenic differentiation and facilitating bone formation and regeneration in biodegradable silk scaffolds. PMID:25923143

Fibrinogen Induces RUNX2 Activity and Osteogenic Development from Human Pluripotent Stem Cells

PubMed Central

Kidwai, Fahad; Edwards, Jessica; Zou, Li; Kaufman, Dan S.

2016-01-01

Pluripotent stem cells, both human embryonic stem cells (hESC) and induced pluripotent stem cells (iPSC), provide an important resource to produce specialized cells such as osteogenic cells for therapeutic applications such as repair or replacement of injured, diseased or damaged bone. hESCs and iPSCs can also be used to better define basic cellular and genetic mechanisms that regulate the earliest stages of human bone development. However, current strategies to mediate osteogenic differentiation of hESC and iPSC are typically limited by the use of xenogeneic components such as fetal bovine serum (FBS) that make defining specific agents that mediate human osteogenesis difficult. Runt-related transcription factor 2 (RUNX2) is a key regulator required for osteogenic differentiation. Here, we used a RUNX2-YFP reporter system to characterize the novel ability of fibrinogen to mediate human osteogenic development from hESC and iPSC in defined (serum-free) conditions. These studies demonstrate that fibrinogen mediates significant osteo-induction potential. Specifically, fibrinogen binds to the surface integrin (α9β1) to mediate RUNX2 gene expression through the SMAD1/5/8 signaling pathway. Additional studies characterize the fibrinogen-induced hESC/iPSC-derived osteogenic cells to demonstrate these osteogenic cells retain the capacity to express typical mature osteoblastic markers. Together, these studies define a novel fibrinogen-α9β1-SMAD1/5/8-RUNX2 signaling axis can efficiently induce osteogenic differentiation from hESCs and iPSCs. PMID:27331788

Short-term application of dexamethasone on stem cells derived from human gingiva reduces the expression of RUNX2 and β-catenin.

PubMed

Kim, Bo-Bae; Kim, Minji; Park, Yun-Hee; Ko, Youngkyung; Park, Jun-Beom

2017-06-01

Objective Next-generation sequencing was performed to evaluate the effects of short-term application of dexamethasone on human gingiva-derived mesenchymal stem cells. Methods Human gingiva-derived stem cells were treated with a final concentration of 10 -7  M dexamethasone and the same concentration of vehicle control. This was followed by mRNA sequencing and data analysis, gene ontology and pathway analysis, quantitative real-time polymerase chain reaction of mRNA, and western blot analysis of RUNX2 and β-catenin. Results In total, 26,364 mRNAs were differentially expressed. Comparison of the results of dexamethasone versus control at 2 hours revealed that 7 mRNAs were upregulated and 25 mRNAs were downregulated. The application of dexamethasone reduced the expression of RUNX2 and β-catenin in human gingiva-derived mesenchymal stem cells. Conclusion The effects of dexamethasone on stem cells were evaluated with mRNA sequencing, and validation of the expression was performed with qualitative real-time polymerase chain reaction and western blot analysis. The results of this study can provide new insights into the role of mRNA sequencing in maxillofacial areas.

Effect of micro-nano-hybrid structured hydroxyapatite bioceramics on osteogenic and cementogenic differentiation of human periodontal ligament stem cell via Wnt signaling pathway.

PubMed

Mao, Lixia; Liu, Jiaqiang; Zhao, Jinglei; Chang, Jiang; Xia, Lunguo; Jiang, Lingyong; Wang, Xiuhui; Lin, Kaili; Fang, Bing

2015-01-01

The surface structure of bioceramic scaffolds is crucial for its bioactivity and osteoinductive ability, and in recent years, human periodontal ligament stem cells have been certified to possess high osteogenic and cementogenic differential ability. In the present study, hydroxyapatite (HA) bioceramics with micro-nano-hybrid surface (mnHA [the hybrid of nanorods and microrods]) were fabricated via hydrothermal reaction of the α-tricalcium phosphate granules as precursors in aqueous solution, and the effects of mnHA on the attachment, proliferation, osteogenic and cementogenic differentiations of human periodontal ligament stem cells as well as the related mechanisms were systematically investigated. The results showed that mnHA bioceramics could promote cell adhesion, proliferation, alkaline phosphatase (ALP) activity, and expression of osteogenic/cementogenic-related markers including runt-related transcription factor 2 (Runx2), ALP, osteocalcin (OCN), cementum attachment protein (CAP), and cementum protein (CEMP) as compared to the HA bioceramics with flat and dense surface. Moreover, mnHA bioceramics stimulated gene expression of low-density lipoprotein receptor-related protein 5 (LRP5) and β-catenin, which are the key genes of canonical Wnt signaling. Moreover, the stimulatory effect on ALP activity and osteogenic and cementogenic gene expression, including that of ALP, OCN, CAP, CEMP, and Runx2 of mnHA bioceramics could be repressed by canonical Wnt signaling inhibitor dickkopf1 (Dkk1). The results suggested that the HA bioceramics with mnHA could act as promising grafts for periodontal tissue regeneration.

Calcium Signaling Pathway Genes RUNX2 and CACNA1C Are Associated With Calcific Aortic Valve Disease

PubMed Central

Guauque-Olarte, Sandra; Messika-Zeitoun, David; Droit, Arnaud; Lamontagne, Maxime; Tremblay-Marchand, Joël; Lavoie-Charland, Emilie; Gaudreault, Nathalie; Arsenault, Benoit J.; Dubé, Marie-Pierre; Tardif, Jean-Claude; Body, Simon C.; Seidman, Jonathan G.; Boileau, Catherine; Mathieu, Patrick; Pibarot, Philippe; Bossé, Yohan

2016-01-01

Background Calcific aortic valve stenosis (AS) is a life-threatening disease with no medical therapy. The genetic architecture of AS remains elusive. This study combines genome-wide association studies, gene expression, and expression quantitative trait loci mapping in human valve tissues to identify susceptibility genes of AS. Methods and Results A meta-analysis was performed combining the results of 2 genome-wide association studies in 474 and 486 cases from Quebec City (Canada) and Paris (France), respectively. Corresponding controls consisted of 2988 and 1864 individuals with European ancestry from the database of genotypes and phenotypes. mRNA expression levels were evaluated in 9 calcified and 8 normal aortic valves by RNA sequencing. The results were integrated with valve expression quantitative trait loci data obtained from 22 AS patients. Twenty-five single-nucleotide polymorphisms had P<5×10−6 in the genome-wide association studies meta-analysis. The calcium signaling pathway was the top gene set enriched for genes mapped to moderately AS-associated single-nucleotide polymorphisms. Genes in this pathway were found differentially expressed in valves with and without AS. Two single-nucleotide polymorphisms located in RUNX2 (runt-related transcription factor 2), encoding an osteogenic transcription factor, demonstrated some association with AS (genome-wide association studies P=5.33×10−5). The mRNA expression levels of RUNX2 were upregulated in calcified valves and associated with eQTL-SNPs. CACNA1C encoding a subunit of a voltage-dependent calcium channel was upregulated in calcified valves. The eQTL-SNP with the most significant association with AS located in CACNA1C was associated with higher expression of the gene. Conclusions This integrative genomic study confirmed the role of RUNX2 as a potential driver of AS and identified a new AS susceptibility gene, CACNA1C, belonging to the calcium signaling pathway. PMID:26553695

Posttranslationally modified progesterone receptors direct ligand-specific expression of breast cancer stem cell-associated gene programs.

PubMed

Knutson, Todd P; Truong, Thu H; Ma, Shihong; Brady, Nicholas J; Sullivan, Megan E; Raj, Ganesh; Schwertfeger, Kathryn L; Lange, Carol A

2017-04-17

Estrogen and progesterone are potent breast mitogens. In addition to steroid hormones, multiple signaling pathways input to estrogen receptor (ER) and progesterone receptor (PR) actions via posttranslational events. Protein kinases commonly activated in breast cancers phosphorylate steroid hormone receptors (SRs) and profoundly impact their activities. To better understand the role of modified PRs in breast cancer, we measured total and phospho-Ser294 PRs in 209 human breast tumors represented on 2754 individual tissue spots within a tissue microarray and assayed the regulation of this site in human tumor explants cultured ex vivo. To complement this analysis, we assayed PR target gene regulation in T47D luminal breast cancer models following treatment with progestin (promegestone; R5020) and antiprogestins (mifepristone, onapristone, or aglepristone) in conditions under which the receptor is regulated by Lys388 SUMOylation (K388 intact) or is SUMO-deficient (via K388R mutation to mimic persistent Ser294 phosphorylation). Selected phospho-PR-driven target genes were validated by qRT-PCR and following RUNX2 shRNA knockdown in breast cancer cell lines. Primary and secondary mammosphere assays were performed to implicate phospho-Ser294 PRs, epidermal growth factor signaling, and RUNX2 in breast cancer stem cell biology. Phospho-Ser294 PR species were abundant in a majority (54%) of luminal breast tumors, and PR promoter selectivity was exquisitely sensitive to posttranslational modifications. Phospho-PR expression and target gene programs were significantly associated with invasive lobular carcinoma (ILC). Consistent with our finding that activated phospho-PRs undergo rapid ligand-dependent turnover, unique phospho-PR gene signatures were most prevalent in breast tumors clinically designated as PR-low to PR-null (luminal B) and included gene sets associated with cancer stem cell biology (HER2, PAX2, AHR, AR, RUNX). Validation studies demonstrated a requirement for RUNX2 in the regulation of selected phospho-PR target genes (SLC37A2). In vitro mammosphere formation assays support a role for phospho-Ser294-PRs via growth factor (EGF) signaling as well as RUNX2 as potent drivers of breast cancer stem cell fate. We conclude that PR Ser294 phosphorylation is a common event in breast cancer progression that is required to maintain breast cancer stem cell fate, in part via cooperation with growth factor-initiated signaling pathways and key phospho-PR target genes including SLC37A2 and RUNX2. Clinical measurement of phosphorylated PRs should be considered a useful marker of breast tumor stem cell potential. Alternatively, unique phospho-PR target gene sets may provide useful tools with which to identify patients likely to respond to selective PR modulators that block PR Ser294 phosphorylation as part of rational combination (i.e., with antiestrogens) endocrine therapies designed to durably block breast cancer recurrence.

RAG-mediated recombination is the predominant driver of oncogenic rearrangement in ETV6-RUNX1 acute lymphoblastic leukemia

PubMed Central

Papaemmanuil, Elli; Rapado, Inmaculada; Li, Yilong; Potter, Nicola E; Wedge, David C; Tubio, Jose; Alexandrov, Ludmil B; Van Loo, Peter; Cooke, Susanna L; Marshall, John; Martincorena, Inigo; Hinton, Jonathan; Gundem, Gunes; van Delft, Frederik W; Nik-Zainal, Serena; Jones, David R; Ramakrishna, Manasa; Titley, Ian; Stebbings, Lucy; Leroy, Catherine; Menzies, Andrew; Gamble, John; Robinson, Ben; Mudie, Laura; Raine, Keiran; O’Meara, Sarah; Teague, Jon W; Butler, Adam P; Cazzaniga, Giovanni; Biondi, Andrea; Zuna, Jan; Kempski, Helena; Muschen, Markus; Ford, Anthony M; Stratton, Michael R; Greaves, Mel; Campbell, Peter J

2014-01-01

The ETV6-RUNX1 fusion gene, found in 25% of childhood acute lymphoblastic leukemia (ALL), is acquired in utero but requires additional somatic mutations for overt leukemia. We used exome and low-coverage whole-genome sequencing to characterize secondary events associated with leukemic transformation. RAG-mediated deletions emerge as the dominant mutational process, characterized by recombination signal sequence motifs near the breakpoints; incorporation of non-templated sequence at the junction; ~30-fold enrichment at promoters and enhancers of genes actively transcribed in B-cell development and an unexpectedly high ratio of recurrent to non-recurrent structural variants. Single cell tracking shows that this mechanism is active throughout leukemic evolution with evidence of localized clustering and re-iterated deletions. Integration of point mutation and rearrangement data identifies ATF7IP and MGA as two new tumor suppressor genes in ALL. Thus, a remarkably parsimonious mutational process transforms ETV6-RUNX1 lymphoblasts, targeting the promoters, enhancers and first exons of genes that normally regulate B-cell differentiation. PMID:24413735

Use of RUNX2 Expression to Identify Osteogenic Progenitor Cells Derived from Human Embryonic Stem Cells

PubMed Central

Zou, Li; Kidwai, Fahad K.; Kopher, Ross A.; Motl, Jason; Kellum, Cory A.; Westendorf, Jennifer J.; Kaufman, Dan S.

2015-01-01

Summary We generated a RUNX2-yellow fluorescent protein (YFP) reporter system to study osteogenic development from human embryonic stem cells (hESCs). Our studies demonstrate the fidelity of YFP expression with expression of RUNX2 and other osteogenic genes in hESC-derived osteoprogenitor cells, as well as the osteogenic specificity of YFP signal. In vitro studies confirm that the hESC-derived YFP+ cells have similar osteogenic phenotypes to osteoprogenitor cells generated from bone-marrow mesenchymal stem cells. In vivo studies demonstrate the hESC-derived YFP+ cells can repair a calvarial defect in immunodeficient mice. Using the engineered hESCs, we monitored the osteogenic development and explored the roles of osteogenic supplements BMP2 and FGF9 in osteogenic differentiation of these hESCs in vitro. Taken together, this reporter system provides a novel system to monitor the osteogenic differentiation of hESCs and becomes useful to identify soluble agents and cell signaling pathways that mediate early stages of human bone development. PMID:25680477

Tcf7 Is an Important Regulator of the Switch of Self-Renewal and Differentiation in a Multipotential Hematopoietic Cell Line

PubMed Central

Schulz, Vincent P.; Hariharan, Manoj; Tuck, David; Lian, Jin; Du, Jiang; Shi, Minyi; Ye, Zhijia; Gerstein, Mark; Snyder, Michael P.; Weissman, Sherman

2012-01-01

A critical problem in biology is understanding how cells choose between self-renewal and differentiation. To generate a comprehensive view of the mechanisms controlling early hematopoietic precursor self-renewal and differentiation, we used systems-based approaches and murine EML multipotential hematopoietic precursor cells as a primary model. EML cells give rise to a mixture of self-renewing Lin-SCA+CD34+ cells and partially differentiated non-renewing Lin-SCA-CD34− cells in a cell autonomous fashion. We identified and validated the HMG box protein TCF7 as a regulator in this self-renewal/differentiation switch that operates in the absence of autocrine Wnt signaling. We found that Tcf7 is the most down-regulated transcription factor when CD34+ cells switch into CD34− cells, using RNA–Seq. We subsequently identified the target genes bound by TCF7, using ChIP–Seq. We show that TCF7 and RUNX1 (AML1) bind to each other's promoter regions and that TCF7 is necessary for the production of the short isoforms, but not the long isoforms of RUNX1, suggesting that TCF7 and the short isoforms of RUNX1 function coordinately in regulation. Tcf7 knock-down experiments and Gene Set Enrichment Analyses suggest that TCF7 plays a dual role in promoting the expression of genes characteristic of self-renewing CD34+ cells while repressing genes activated in partially differentiated CD34− state. Finally a network of up-regulated transcription factors of CD34+ cells was constructed. Factors that control hematopoietic stem cell (HSC) establishment and development, cell growth, and multipotency were identified. These studies in EML cells demonstrate fundamental cell-intrinsic properties of the switch between self-renewal and differentiation, and yield valuable insights for manipulating HSCs and other differentiating systems. PMID:22412390

Wnt/β-catenin pathway regulates Bmp2-mediated differentiation of dental follicle cells

PubMed Central

Silvério, Karina G.; Davidson, Kathryn C.; James, Richard G.; Adams, Allison M.; Foster, Brian L.; Nociti, Francisco H.; Somermam, Martha J.; Moon, Randall T.

2013-01-01

Background and Objectives Bmp2-induced osteogenic differentiation has been shown to occur through the canonical Wnt/β-catenin pathway, whereas factors promoting canonical Wnt signaling in cementoblasts inhibited cell differentiation and promoted cell proliferation in vitro. The aim of this study was to investigate whether putative precursor cells of cementoblasts, dental follicle cells (murine SVF4 cells), when stimulated with Bmp2, would exhibit changes in genes/proteins associated with the Wnt/β-catenin pathway. Materials and Methods SVF4 cells were stimulated with Bmp2, and the following assays were carried out: 1) Wnt/β-catenin pathway activation assessed by western blot, β-catenin/TCF reporter assay, and gene expression of lymphoid enhancer-binding factor-1 (Lef1), transcription factor 7 (Tcf7), Wnt inhibitor factor 1 (Wif1) and Axin2, and 2) cementoblast/osteoblast differentiation assessed by mineralization in vitro, and mRNA levels of runt-related transcription factor 2 (Runx2), osterix (Osx), alkaline phosphatase (Alp), osteocalcin (Ocn) and bone sialoprotein (Bsp) by qPCR after Wnt3a treatment and knockdown of β-catenin. Results Wnt3a induced β-catenin nuclear translocation and upregulated the transcriptional activity of a canonical Wnt-responsive reporter, suggesting the Wnt/β-catenin pathway functions in SVF4 cells. Activation of Wnt signaling with Wnt3a suppressed Bmp2-mediated induction of cementoblast/osteoblast maturation of SVF4 cells. However, β-catenin knockdown showed that Bmp2-induced expression of cementoblast/osteoblast differentiation markers requires endogenous β-catenin. Wnt3a down-regulated transcripts for Runx2, Alp and Ocn in SVF4 cells compared to untreated cells. In contrast, Bmp2 induction of Bsp transcripts occurred independent of Wnt/β-catenin signaling. Conclusions These data suggest that stabilization of β-catenin by Wnt-3a treatment inhibits Bmp2-mediated induction of cementoblast/osteoblast differentiation in SVF4 cells, although Bmp2 requires endogenous Wnt/β-catenin signaling to promote cell maturation. PMID:22150562

Aberrant gene methylation in non-neoplastic mucosa as a predictive marker of ulcerative colitis-associated CRC.

PubMed

Scarpa, Marco; Scarpa, Melania; Castagliuolo, Ignazio; Erroi, Francesca; Kotsafti, Andromachi; Basato, Silvia; Brun, Paola; D'Incà, Renata; Rugge, Massimo; Angriman, Imerio; Castoro, Carlo

2016-03-01

BACKGROUND PROMOTER: hypermethylation plays a major role in cancer through transcriptional silencing of critical genes. The aim of our study is to evaluate the methylation status of these genes in the colonic mucosa without dysplasia or adenocarcinoma at the different steps of sporadic and UC-related carcinogenesis and to investigate the possible role of genomic methylation as a marker of CRC. The expression of Dnmts 1 and 3A was significantly increased in UC-related carcinogenesis compared to non inflammatory colorectal carcinogenesis. In non-neoplastic colonic mucosa, the number of methylated genes resulted significantly higher in patients with CRC and in those with UC-related CRC compared to the HC and UC patients and patients with dysplastic lesion of the colon. The number of methylated genes in non-neoplastic colonic mucosa predicted the presence of CRC with good accuracy either in non inflammatory and inflammatory related CRC. Colonic mucosal samples were collected from healthy subjects (HC) (n = 30) and from patients with ulcerative colitis (UC) (n = 29), UC and dysplasia (n = 14), UC and cancer (n = 10), dysplastic adenoma (n = 14), and colon adenocarcinoma (n = 10). DNA methyltransferases-1, -3a, -3b, mRNA expression were quantified by real time qRT-PCR. The methylation status of CDH13, APC, MLH1, MGMT1 and RUNX3 gene promoters was assessed by methylation-specific PCR. Methylation status of APC, CDH13, MGMT, MLH1 and RUNX3 in the non-neoplastic mucosa may be used as a marker of CRC: these preliminary results could allow for the adjustment of a patient's surveillance interval and to select UC patients who should undergo intensive surveillance.

17beta-estradiol promotes the odonto/osteogenic differentiation of stem cells from apical papilla via mitogen-activated protein kinase pathway.

PubMed

Li, Yao; Yan, Ming; Wang, Zilu; Zheng, Yangyu; Li, Junjun; Ma, Shu; Liu, Genxia; Yu, Jinhua

2014-11-17

Estrogen plays an important role in the osteogenic differentiation of mesenchymal stem cells, while stem cells from apical papilla (SCAP) can contribute to the formation of dentin/bone-like tissues. To date, the effects of estrogen on the differentiation of SCAP remain unclear. SCAP was isolated and treated with 10⁻⁷ M 17beta-estradiol (E2). The odonto/osteogenic potency and the involvement of mitogen-activated protein kinase (MAPK) signaling pathway were subsequently investigated by using methyl-thiazolyl-tetrazolium (MTT) assay, and other methods. MTT and flow cytometry results demonstrated that E2 treatment had no effect on the proliferation of SCAP in vitro, while alkaline phosphatase (ALP) assay and alizarin red staining showed that E2 can significantly promote ALP activity and mineralization ability in SCAP. Real-time reverse transcription polymerase chain reaction (RT-PCR) and western blot assay revealed that the odonto/osteogenic markers (ALP, DMP1/DMP1, DSPP/DSP, RUNX2/RUNX2, OSX/OSX and OCN/OCN) were significantly upregulated in E2-treated SCAP. In addition, the expression of phosphor-p38 and phosphor-JNK in these stem cells was enhanced by E2 treatment, as was the expression of the nuclear downstream transcription factors including phosphor-Sp1, phosphor-Elk-1, phosphor-c-Jun and phosphor-c-Fos, indicating the activation of MAPK signaling pathway during the odonto/osteogenic differentiation of E2-treated SCAP. Conversely, the differentiation of E2-treated SCAP was inhibited in the presence of MAPK specific inhibitors. The ondonto/osteogenic differentiation of SCAP is enhanced by 10⁻⁷ M 17beta-estradiol via the activation of MAPK signaling pathway.

Antiaging Gene Klotho Deficiency Promoted High-Fat Diet-Induced Arterial Stiffening via Inactivation of AMP-Activated Protein Kinase.

PubMed

Lin, Yi; Chen, Jianglei; Sun, Zhongjie

2016-03-01

Klotho was originally discovered as an aging-suppressor gene. The objective of this study is to investigate whether klotho gene deficiency affects high-fat diet (HFD)-induced arterial stiffening. Heterozygous Klotho-deficient (KL(+/-)) mice and WT littermates were fed on HFD or normal diet. HFD increased pulse wave velocity within 5 weeks in KL(+/-) mice but not in wild-type mice, indicating that klotho deficiency accelerates and exacerbates HFD-induced arterial stiffening. A greater increase in blood pressure was found in KL(+/-) mice fed on HFD. Protein expressions of phosphorylated AMP-activated protein kinase-α (AMPKα), phosphorylated endothelial nitric oxide synthase (eNOS), and manganese-dependent superoxide dismutase (Mn-SOD) were decreased, whereas protein expressions of collagen I, transforming growth factor-β1, and Runx2 were increased in aortas of KL(+/-) mice fed on HFD. Interestingly, daily injections of an AMPKα activator, 5-aminoimidazole-4-carboxamide-3-ribonucleoside, abolished the increases in pulse wave velocity, blood pressure, and blood glucose in KL(+/-) mice fed on HFD. Treatment with 5-aminoimidazole-4-carboxamide-3-ribonucleoside for 2 weeks not only abolished the downregulation of phosphorylated AMPKα, phosphorylated eNOS, and Mn-SOD levels but also attenuated the increased levels of collagen I, transforming growth factor-β1, Runx2, superoxide, elastic lamellae breaks, and calcification in aortas of KL(+/-) mice fed on HFD. In cultured mouse aortic smooth muscle cells, cholesterol plus KL-deficient serum decreased phosphorylation levels of AMPKα and LKB1 (an important upstream regulator of AMPKα activity) but increased collagen I synthesis, which can be eliminated by activation of AMPKα by 5-aminoimidazole-4-carboxamide-3-ribonucleoside. In conclusions, Klotho deficiency promoted HFD-induced arterial stiffening and hypertension via downregulation of AMPKα activity. © 2016 American Heart Association, Inc.

Role of Klotho in Osteoporosis and Renal Osteodystrophy

DTIC Science & Technology

2015-10-01

uremia induced increases in FGF23 transcription (Figure 6). VEGFa Runx2 Osx Col1a1 ALP OC 0 2 4 6 8 KL fl/fl Prx1-Cre; KL fl/fl m R N A ex pr es...week old mice revealed that Prx1cre;Klothofl/fl mice have significantly higher expression of osteoblastic and osteocytic markers such as Col1a1 , Runx2

The posterior HOXD locus: Its contribution to phenotype and malignancy of Ewing sarcoma

PubMed Central

von Heyking, Kristina; Schmidt, Oxana; Calzada-Wack, Julia; Neff, Frauke; Lawlor, Elizabeth R.; Burdach, Stefan; Richter, Günther H.S.

2016-01-01

Microarray analysis revealed genes of the posterior HOXD locus normally involved in bone formation to be over-expressed in primary Ewing sarcoma (ES). The expression of posterior HOXD genes was not influenced via ES pathognomonic EWS/ETS translocations. However, knock down of the dickkopf WNT signaling pathway inhibitor 2 (DKK2) resulted in a significant suppression of HOXD10, HOXD11 and HOXD13 while over-expression of DKK2 and stimulation with factors of the WNT signaling pathway such as WNT3a, WNT5a or WNT11 increased their expression. RNA interference demonstrated that individual HOXD genes promoted chondrogenic differentiation potential, and enhanced expression of the bone-associated gene RUNX2. Furthermore, HOXD genes increased the level of the osteoblast- and osteoclast-specific genes, osteocalcin (BGLAP) and platelet-derived growth factor beta polypeptide (PDGFB), and may further regulate endochondral bone development via induction of parathyroid hormone-like hormone (PTHLH). Additionally, HOXD11 and HOXD13 promoted contact independent growth of ES, while in vitro invasiveness of ES lines was enhanced by all 3 HOXD genes investigated and seemed mediated via matrix metallopeptidase 1 (MMP1). Consequently, knock down of HOXD11 or HOXD13 significantly suppressed lung metastasis in a xeno-transplant model in immune deficient mice, providing overall evidence that posterior HOXD genes promote clonogenicity and metastatic potential of ES. PMID:27363011

Epigenetic remodeling and modification to preserve skeletogenesis in vivo.

PubMed

Godfrey, Tanner C; Wildman, Benjamin J; Javed, Amjad; Lengner, Christopher J; Hassan, Mohammad Quamarul

2018-12-01

Current studies offer little insight on how epigenetic remodeling of bone-specific chromatin maintains bone mass in vivo. Understanding this gap and precise mechanism is pivotal for future therapeutic innovation to prevent bone loss. Recently, we found that low bone mass is associated with decreased H3K27 acetylation (activating histone modification) of bone specific gene promoters. Here, we aim to elucidate the epigenetic mechanisms by which a miRNA cluster controls bone synthesis and homeostasis by regulating chromatin accessibility and H3K27 acetylation. In order to decipher the epigenetic axis that regulates osteogenesis, we studied a drug inducible anti-miR-23a cluster (miR-23a Cl ZIP ) knockdown mouse model. MiR-23a cluster knockdown (heterozygous) mice developed high bone mass. These mice displayed increased expression of Runx2 and Baf45a, essential factors for skeletogenesis; and decreased expression of Ezh2, a chromatin repressor indispensable for skeletogenesis. ChIP assays using miR-23a Cl knockdown calvarial cells revealed a BAF45A-EZH2 epigenetic antagonistic mechanism that maintains bone formation. Together, our findings support that the miR-23a Cl connection with tissue-specific RUNX2-BAF45A-EZH2 function is a novel molecular epigenetic axis through which a miRNA cluster orchestrates chromatin modification to elicit major effects on osteogenesis in vivo.

The effect of noncoherent red light irradiation on proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells.

PubMed

Peng, Fei; Wu, Hua; Zheng, Yadong; Xu, Xiqiang; Yu, Jizhe

2012-05-01

Mesenchymal stem cells (MSCs) are promising for use in regenerative medicine. Low-level light irradiation (LLLI) has been shown to modulate various processes in different biological systems. The aim of our study was to investigate the effect of red light emitted from a light-emitting diode (LED) on bone marrow MSCs with or without osteogenic supplements. MSCs both with and without osteogenic supplements were divided into four groups, and each group was irradiated at doses of 0, 1, 2 and 4 J/cm(2). Cellular proliferation was evaluated using WST-8 and 5-ethynyl-2'-deoxyuridine (EdU) fluorescence staining. The alkaline phosphatase activity, mineralization, and expression of osteoblast master genes (Col1α1, Alpl, Bglap and Runx2) were monitored as indicators of MSC differentiation towards osteoblasts. In groups without osteogenic supplements, red light at all doses significantly stimulated cellular proliferation, whereas the osteogenic phenotype of the MSCs was not enhanced. In groups with osteogenic supplements, red light increased alkaline phosphatase activity and mineralized nodule formation, and stimulated the expression of Bglap and Runx2, but decreased cellular proliferation. In conclusion, nonconherent red light can promote proliferation but cannot induce osteogenic differentiation of MSCs in normal media, while it enhances osteogenic differentiation and decreases proliferation of MSCs in media with osteogenic supplements.

O-GlcNAc Modification of the runt-Related Transcription Factor 2 (Runx2) Links Osteogenesis and Nutrient Metabolism in Bone Marrow Mesenchymal Stem Cells*

PubMed Central

Nagel, Alexis K.; Ball, Lauren E.

2014-01-01

Runx2 is the master switch controlling osteoblast differentiation and formation of the mineralized skeleton. The post-translational modification of Runx2 by phosphorylation, ubiquitinylation, and acetylation modulates its activity, stability, and interactions with transcriptional co-regulators and chromatin remodeling proteins downstream of osteogenic signals. Characterization of Runx2 by electron transfer dissociation tandem mass spectrometry revealed sites of O-linked N-acetylglucosamine (O-GlcNAc) modification, a nutrient-responsive post-translational modification that modulates the action of numerous transcriptional effectors. O-GlcNAc modification occurs in close proximity to phosphorylated residues and novel sites of arginine methylation within regions known to regulate Runx2 transactivation. An interaction between Runx2 and the O-GlcNAcylated, O-GlcNAc transferase enzyme was also detected. Pharmacological inhibition of O-GlcNAcase (OGA), the enzyme responsible for the removal of O-GlcNAc from Ser/Thr residues, enhanced basal (39.9%) and BMP2/7-induced (43.3%) Runx2 transcriptional activity in MC3T3-E1 pre-osteoblasts. In bone marrow-derived mesenchymal stem cells differentiated for 6 days in osteogenic media, inhibition of OGA resulted in elevated expression (24.3%) and activity (65.8%) of alkaline phosphatase (ALP) an early marker of bone formation and a transcriptional target of Runx2. Osteogenic differentiation of bone marrow-derived mesenchymal stem cells in the presence of BMP2/7 for 8 days culminated in decreased OGA activity (39.0%) and an increase in the abundance of O-GlcNAcylated Runx2, as compared with unstimulated cells. Furthermore, BMP2/7-induced ALP activity was enhanced by 35.6% in bone marrow-derived mesenchymal stem cells differentiated in the presence of the OGA inhibitor, demonstrating that direct or BMP2/7-induced inhibition of OGA is associated with increased ALP activity. Altogether, these findings link O-GlcNAc cycling to the Runx2-dependent regulation of the early ALP marker under osteoblast differentiation conditions. PMID:25187572

Decoding of exon splicing patterns in the human RUNX1-RUNX1T1 fusion gene.

PubMed

Grinev, Vasily V; Migas, Alexandr A; Kirsanava, Aksana D; Mishkova, Olga A; Siomava, Natalia; Ramanouskaya, Tatiana V; Vaitsiankova, Alina V; Ilyushonak, Ilia M; Nazarov, Petr V; Vallar, Laurent; Aleinikova, Olga V

2015-11-01

The t(8;21) translocation is the most widespread genetic defect found in human acute myeloid leukemia. This translocation results in the RUNX1-RUNX1T1 fusion gene that produces a wide variety of alternative transcripts and influences the course of the disease. The rules of combinatorics and splicing of exons in the RUNX1-RUNX1T1 transcripts are not known. To address this issue, we developed an exon graph model of the fusion gene organization and evaluated its local exon combinatorics by the exon combinatorial index (ECI). Here we show that the local exon combinatorics of the RUNX1-RUNX1T1 gene follows a power-law behavior and (i) the vast majority of exons has a low ECI, (ii) only a small part is represented by "exons-hubs" of splicing with very high ECI values, and (iii) it is scale-free and very sensitive to targeted skipping of "exons-hubs". Stochasticity of the splicing machinery and preferred usage of exons in alternative splicing can explain such behavior of the system. Stochasticity may explain up to 12% of the ECI variance and results in a number of non-coding and unproductive transcripts that can be considered as a noise. Half-life of these transcripts is increased due to the deregulation of some key genes of the nonsense-mediated decay system in leukemia cells. On the other hand, preferred usage of exons may explain up to 75% of the ECI variability. Our analysis revealed a set of splicing-related cis-regulatory motifs that can explain "attractiveness" of exons in alternative splicing but only when they are considered together. Cis-regulatory motifs are guides for splicing trans-factors and we observed a leukemia-specific profile of expression of the splicing genes in t(8;21)-positive blasts. Altogether, our results show that alternative splicing of the RUNX1-RUNX1T1 transcripts follows strict rules and that the power-law component of the fusion gene organization confers a high flexibility to this process. Copyright © 2015 Elsevier Ltd. All rights reserved.

Conversion of adult endothelium to immunocompetent haematopoietic stem cells.

PubMed

Lis, Raphael; Karrasch, Charles C; Poulos, Michael G; Kunar, Balvir; Redmond, David; Duran, Jose G Barcia; Badwe, Chaitanya R; Schachterle, William; Ginsberg, Michael; Xiang, Jenny; Tabrizi, Arash Rafii; Shido, Koji; Rosenwaks, Zev; Elemento, Olivier; Speck, Nancy A; Butler, Jason M; Scandura, Joseph M; Rafii, Shahin

2017-05-25

Developmental pathways that orchestrate the fleeting transition of endothelial cells into haematopoietic stem cells remain undefined. Here we demonstrate a tractable approach for fully reprogramming adult mouse endothelial cells to haematopoietic stem cells (rEC-HSCs) through transient expression of the transcription-factor-encoding genes Fosb, Gfi1, Runx1, and Spi1 (collectively denoted hereafter as FGRS) and vascular-niche-derived angiocrine factors. The induction phase (days 0-8) of conversion is initiated by expression of FGRS in mature endothelial cells, which results in endogenous Runx1 expression. During the specification phase (days 8-20), RUNX1 + FGRS-transduced endothelial cells commit to a haematopoietic fate, yielding rEC-HSCs that no longer require FGRS expression. The vascular niche drives a robust self-renewal and expansion phase of rEC-HSCs (days 20-28). rEC-HSCs have a transcriptome and long-term self-renewal capacity similar to those of adult haematopoietic stem cells, and can be used for clonal engraftment and serial primary and secondary multi-lineage reconstitution, including antigen-dependent adaptive immune function. Inhibition of TGFβ and CXCR7 or activation of BMP and CXCR4 signalling enhanced generation of rEC-HSCs. Pluripotency-independent conversion of endothelial cells into autologous authentic engraftable haematopoietic stem cells could aid treatment of haematological disorders.

Conversion of adult endothelium to immunocompetent haematopoietic stem cells

PubMed Central

Lis, Raphael; Karrasch, Charles C.; Poulos, Michael G.; Kunar, Balvir; Redmond, David; Barcia Duran, Jose G.; Badwe, Chaitanya R.; Schachterle, Will; Ginsberg, Michael; Xiang, Jenny; Tabrizi, Arash Rafii; Shido, Koji; Rosenwaks, Zev; Elemento, Olivier; Speck, Nancy; Butler, Jason M.; Scandura, Joseph M.; Rafii, Shahin

2018-01-01

Developmental pathways that orchestrate the fleeting transition of endothelial cells into haematopoietic stem cells remain undefined. Here we demonstrate a tractable approach for fully converting adult mouse endothelial cells to haematopoietic stem cells (rEC-HSCs) through transient expression of genes encoding the transcription factors Fosb, Gfi1, Runx1, and Spi1 (also known as Fgrs) and vascular-niche-derived angiocrine factors. The induction phase (day 0–8) of conversion is initiated by expression of Fgrs in mature endothelial cells, which results in endogenous Runx1 expression. During the specification phase (day 8–20), Runx1+ Fgrs-transduced endothelial cells commit to a haematopoietic fate yielding rEC-HSCs that no longer require Fgrs expression. The vascular niche drives a robust self-renewal and expansion phase of rEC-HSCs (at day 20–28). rEC-HSCs have a transcriptome and long-term self-renewal capacity similar to those of adult haematopoietic stem cells, are competent for clonal engraftment and serial primary and secondary multi-lineage reconstituting potential, including antigen-dependent adaptive immune function. Inhibition of TGF-β and CXCR7 or activation of BMP and CXCR4 signalling enhanced generation of rEC-HSCs. Conversion of endothelial cells into autologous authentic engraftable haematopoietic stem cells could aid treatment of haematological disorders. PMID:28514438

BAG3 promotes chondrosarcoma progression by upregulating the expression of β-catenin.

PubMed

Shi, Huijuan; Chen, Wenfang; Dong, Yu; Lu, Xiaofang; Zhang, Wenhui; Wang, Liantang

2018-04-01

To investigate the roles of B‑cell lymphoma‑2 associated athanogene 3 (BAG3) in human chondrosarcoma and the potential mechanisms, the expression levels of BAG3 were detected in the present study, and the associations between BAG3 and clinical pathological parameters, clinical stage as well as the survival of patients were analyzed. The present study detected BAG3 mRNA and protein expression in the normal cartilage cell line HC‑a and in SW1353 chondrosarcoma cells by reverse transcription‑quantitative polymerase chain reaction and western blot analysis. The BAG3 protein expression in 59 cases of chondrosarcoma, 30 patients with endogenous chondroma and 8 cases of normal cartilage was semi-quantitatively analyzed using the immunohistochemical method. In addition, the BAG3 protein expression level, the clinical pathological parameters, clinical stage and the survival time of patients with chondrosarcoma were analyzed. The plasmid transfection method was employed to upregulate the expression BAG3 and small RNA interference to downregulate the expression of BAG3 in SW1353 cells. The expression levels of BAG3 protein and mRNA were significantly increased in the chondrosarcoma cell line when compared with the normal cartilage cell line. The immunohistochemistry results indicated that BAG3 protein was overexpressed in the tissue of human chondrosarcoma. Statistical analysis showed that the expression level of BAG3 was significantly increased in the different Enneking staging of patients with chondrosarcoma and Tumor staging, and there were no statistical differences in age, gender, histological classification and tumor size. In the in vitro experiments, the data revealed that BAG3 significantly promoted chondrosarcoma cell proliferation, colony‑formation, migration and invasion; however, it inhibited chondrosarcoma cell apoptosis. It was observed that BAG3 upregulated β‑catenin expression at the mRNA and protein levels. In addition, BAG3 induced the expression of runt‑related transcription factor 2 (RUNX2) in chondrosarcoma cells by upregulating β‑catenin. These clinical analyses revealed a positive association between β‑catenin and BAG3 in chondrosarcoma tumors. BAG3 was significantly increased in chondrosarcoma cells and tissues compared with the normal cartilage cells, tissue and cartilage benign tumors. Thus, BAG3 may serve as an oncogene in the development of chondrosarcoma via the induction of RUNX2 expression. The results of the present study contribute to further research on the biological development of chondrosarcoma.

Transfection of gene regulation nanoparticles complexed with pDNA and shRNA controls multilineage differentiation of hMSCs.

PubMed

Kim, Hye Jin; Yi, Se Won; Oh, Hyun Jyung; Lee, Jung Sun; Park, Ji Sun; Park, Keun-Hong

2018-05-29

Overexpression and knockdown of specific proteins can control stem cell differentiation for therapeutic purposes. In this study, we fabricated RUNX2, SOX9, and C/EBPα plasmid DNAs (pDNAs) and ATF4-targeting shRNA (shATF4) to induce osteogenesis, chondrogenesis, and adipogenesis of human mesenchymal stem cells (hMSCs). The pDNAs and shATF4 were complexed with TRITC-gene regulation nanoparticles (GRN). Osteogenesis-related gene expression was reduced at early (12 h) and late (36 h) time points after co-delivery of shATF4 and SOX9 or C/EBPα pDNA, respectively, and osteogenesis was inhibited in these hMSCs. By contrast, osteogenesis-related genes were highly expressed upon co-delivery of RUNX2 and ATF4 pDNAs. DEX in GRN enhanced chondrogenic differentiation. Expression of osteogenesis-, chondrogenesis-, and adipogenesis-related genes was higher in hMSCs transfected with NPs complexed with RUNX2 and ATF4 pDNAs, shATF4 and SOX9 pDNA, and shATF4 and C/EBPα pDNA for 72 h than in control hMSCs, respectively. Moreover, delivery of these NPs also increased expression of osteogenesis-, chondrogenesis-, and adipogenesis-related proteins. These alterations in expression led to morphological changes, indicating that hMSCs differentiated into osteoblasts, chondrocytes, and adipose cells. Copyright © 2018 Elsevier Ltd. All rights reserved.

Prolonged use of alendronate alters the biology of cranial repair in estrogen-deficient rats' associated simultaneous immunohistochemical expression of TGF-β1+, α-ER+, and BMPR1B.

PubMed

Giovanini, Allan Fernando; de Sousa Passoni, Giuliene Nunes; Göhringer, Isabella; Deliberador, Tatiana Miranda; Zielak, João Cesar; Storrer, Carmem Lucia Muller; Costa-Casagrande, Thais Andrade; Scariot, Rafaela

2018-06-01

TGF-β1 is a cytokine that may induce both osteoneogenesis through Runx-2 or fibrosis via the transcription of α-smooth muscle actin (α-SMA). Because it has been previously known that alendronate increases the level of TGF-β1 and that under the usual condition of bone metabolism the estrogen may prevent the fibrotic effect of TGF-β1, the aim of this study was to evaluate if alendronate alters the cellular differentiation process post calvarial surgery in estrogen-deficient specimens. A transosseous defect that was 5 mm in diameter was created on the calvarium of each of 32 female rats with previous ovarian-salpingo-oophorectomy. All defects were treated with autografts, and 16 rats received the administration of 1 mg/kg of alendronate three times a week until euthanasia on the 15th and 60th day post surgery. Histomorphometric and immunohistochemical analyses of the expression of TGF-β1, estrogen receptor alpha nuclear (α-ER), α-SMA, BMPR1B, and Runx-2 were performed, and ELISA was used to measure the level of estrogen. All animals demonstrated low levels of estrogen post ovarian-salpingo-oophorectomy. The histological results demonstrated larger bone matrix deposition in specimens treated with alendronate on the 15th day post surgery. The result was associated with a higher co-expression of TGF-β1, BMPR1B, and Runx-2 when compared with the control group. In addition, on the 60th day post surgery, the increase of bone matrix deposition from 15th to 60th day was discrete in specimens treated with alendronate compared with the control group. This result coincided with the intense simultaneous expression of TGF-β1, α-ER, and α-SMA, whereas the expression of BMPR1B and Runx-2 decreased. The prolonged administration of alendronate altered the cranial repair in ovarian-salpingo-oophorectomized specimens due to the simultaneous occurrence of low estrogen and the presence of TGF-β1+/α-ER+ inducing the presence of α-SMA + , whereas BMPR1B and Runx-2 were suppressed. The prolonged administration of alendronate alters osteoneogenesis and induces an unusual microenvironment in the bone that seems to imitate the physiological tissue damage that culminates in the loss of the functional layer of endometrium.

MicroRNA-433 Dampens Glucocorticoid Receptor Signaling, Impacting Circadian Rhythm and Osteoblastic Gene Expression.

PubMed

Smith, Spenser S; Dole, Neha S; Franceschetti, Tiziana; Hrdlicka, Henry C; Delany, Anne M

2016-10-07

Serum glucocorticoids play a critical role in synchronizing circadian rhythm in peripheral tissues, and multiple mechanisms regulate tissue sensitivity to glucocorticoids. In the skeleton, circadian rhythm helps coordinate bone formation and resorption. Circadian rhythm is regulated through transcriptional and post-transcriptional feedback loops that include microRNAs. How microRNAs regulate circadian rhythm in bone is unexplored. We show that in mouse calvaria, miR-433 displays robust circadian rhythm, peaking just after dark. In C3H/10T1/2 cells synchronized with a pulse of dexamethasone, inhibition of miR-433 using a tough decoy altered the period and amplitude of Per2 gene expression, suggesting that miR-433 regulates rhythm. Although miR-433 does not directly target the Per2 3'-UTR, it does target two rhythmically expressed genes in calvaria, Igf1 and Hif1α. miR-433 can target the glucocorticoid receptor; however, glucocorticoid receptor protein abundance was unaffected in miR-433 decoy cells. Rather, miR-433 inhibition dramatically enhanced glucocorticoid signaling due to increased nuclear receptor translocation, activating glucocorticoid receptor transcriptional targets. Last, in calvaria of transgenic mice expressing a miR-433 decoy in osteoblastic cells (Col3.6 promoter), the amplitude of Per2 and Bmal1 mRNA rhythm was increased, confirming that miR-433 regulates circadian rhythm. miR-433 was previously shown to target Runx2, and mRNA for Runx2 and its downstream target, osteocalcin, were also increased in miR-433 decoy mouse calvaria. We hypothesize that miR-433 helps maintain circadian rhythm in osteoblasts by regulating sensitivity to glucocorticoid receptor signaling. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

MicroRNA-433 Dampens Glucocorticoid Receptor Signaling, Impacting Circadian Rhythm and Osteoblastic Gene Expression*

PubMed Central

Smith, Spenser S.; Dole, Neha S.; Franceschetti, Tiziana; Hrdlicka, Henry C.; Delany, Anne M.

2016-01-01

Serum glucocorticoids play a critical role in synchronizing circadian rhythm in peripheral tissues, and multiple mechanisms regulate tissue sensitivity to glucocorticoids. In the skeleton, circadian rhythm helps coordinate bone formation and resorption. Circadian rhythm is regulated through transcriptional and post-transcriptional feedback loops that include microRNAs. How microRNAs regulate circadian rhythm in bone is unexplored. We show that in mouse calvaria, miR-433 displays robust circadian rhythm, peaking just after dark. In C3H/10T1/2 cells synchronized with a pulse of dexamethasone, inhibition of miR-433 using a tough decoy altered the period and amplitude of Per2 gene expression, suggesting that miR-433 regulates rhythm. Although miR-433 does not directly target the Per2 3′-UTR, it does target two rhythmically expressed genes in calvaria, Igf1 and Hif1α. miR-433 can target the glucocorticoid receptor; however, glucocorticoid receptor protein abundance was unaffected in miR-433 decoy cells. Rather, miR-433 inhibition dramatically enhanced glucocorticoid signaling due to increased nuclear receptor translocation, activating glucocorticoid receptor transcriptional targets. Last, in calvaria of transgenic mice expressing a miR-433 decoy in osteoblastic cells (Col3.6 promoter), the amplitude of Per2 and Bmal1 mRNA rhythm was increased, confirming that miR-433 regulates circadian rhythm. miR-433 was previously shown to target Runx2, and mRNA for Runx2 and its downstream target, osteocalcin, were also increased in miR-433 decoy mouse calvaria. We hypothesize that miR-433 helps maintain circadian rhythm in osteoblasts by regulating sensitivity to glucocorticoid receptor signaling. PMID:27551048

miR-30 Family Members Negatively Regulate Osteoblast Differentiation*

PubMed Central

Wu, Tingting; Zhou, Haibo; Hong, Yongfeng; Li, Jing; Jiang, Xinquan; Huang, Hui

2012-01-01

miRNAs are endogenously expressed 18- to 25-nucleotide RNAs that regulate gene expression through translational repression by binding to a target mRNA. Recently, it has been indicated that miRNAs are closely related to osteogenesis. Our previous data suggested that miR-30 family members might be important regulators during the biomineralization process. However, whether and how they modulate osteogenic differentiation have not been explored. In this study, we demonstrated that miR-30 family members negatively regulate BMP-2-induced osteoblast differentiation by targeting Smad1 and Runx2. Evidentially, overexpression of miR-30 family members led to a decrease of alkaline phosphatase activity, whereas knockdown of them increased the activity. Then bioinformatic analysis identified potential target sites of the miR-30 family located in the 3′ untranslated regions of Smad1 and Runx2. Western blot analysis and quantitative RT-PCR assays demonstrated that miR-30 family members inhibit Smad1 gene expression on the basis of repressing its translation. Furthermore, dual-luciferase reporter assays confirmed that Smad1 is a direct target of miR-30 family members. Rescue experiments that overexpress Smad1 and Runx2 significantly eliminated the inhibitory effect of miR-30 on osteogenic differentiation and provided strong evidence that miR-30 mediates the inhibition of osteogenesis by targeting Smad1 and Runx2. Also, the inhibitory effects of the miR-30 family were validated in mouse bone marrow mesenchymal stem cells. Therefore, our study uncovered that miR-30 family members are key negative regulators of BMP-2-mediated osteogenic differentiation. PMID:22253433

FGFR2IIIb-MAPK Activity Is Required for Epithelial Cell Fate Decision in the Lower Müllerian Duct

PubMed Central

Terakawa, Jumpei; Rocchi, Altea; Serna, Vanida A.; Bottinger, Erwin P.; Graff, Jonathan M.

2016-01-01

Cell fate of lower Müllerian duct epithelium (MDE), to become uterine or vaginal epithelium, is determined by the absence or presence of ΔNp63 expression, respectively. Previously, we showed that SMAD4 and runt-related transcription factor 1 (RUNX1) were independently required for MDE to express ΔNp63. Here, we report that vaginal mesenchyme directs vaginal epithelial cell fate in MDE through paracrine activation of fibroblast growth factor (FGF) receptor-MAPK pathway. In the developing reproductive tract, FGF7 and FGF10 were enriched in vaginal mesenchyme, whereas FGF receptor 2IIIb was expressed in epithelia of both the uterus and vagina. When Fgfr2 was inactivated, vaginal MDE underwent uterine cell fate, and this differentiation defect was corrected by activation of MEK-ERK pathway. In vitro, FGF10 in combination with bone morphogenetic protein 4 and activin A (ActA) was sufficient to induce ΔNp63 in MDE, and ActA was essential for induction of RUNX1 through SMAD-independent pathways. Accordingly, inhibition of type 1 receptors for activin in neonatal mice induced uterine differentiation in vaginal epithelium by down-regulating RUNX1, whereas conditional deletion of Smad2 and Smad3 had no effect on vaginal epithelial differentiation. In conclusion, vaginal epithelial cell fate in MDE is induced by FGF7/10-MAPK, bone morphogenetic protein 4-SMAD, and ActA-RUNX1 pathway activities, and the disruption in any one of these pathways results in conversion from vaginal to uterine epithelial cell fate. PMID:27164167

Effect of micro-nano-hybrid structured hydroxyapatite bioceramics on osteogenic and cementogenic differentiation of human periodontal ligament stem cell via Wnt signaling pathway

PubMed Central

Mao, Lixia; Liu, Jiaqiang; Zhao, Jinglei; Chang, Jiang; Xia, Lunguo; Jiang, Lingyong; Wang, Xiuhui; Lin, Kaili; Fang, Bing

2015-01-01

The surface structure of bioceramic scaffolds is crucial for its bioactivity and osteoinductive ability, and in recent years, human periodontal ligament stem cells have been certified to possess high osteogenic and cementogenic differential ability. In the present study, hydroxyapatite (HA) bioceramics with micro-nano-hybrid surface (mnHA [the hybrid of nanorods and microrods]) were fabricated via hydrothermal reaction of the α-tricalcium phosphate granules as precursors in aqueous solution, and the effects of mnHA on the attachment, proliferation, osteogenic and cementogenic differentiations of human periodontal ligament stem cells as well as the related mechanisms were systematically investigated. The results showed that mnHA bioceramics could promote cell adhesion, proliferation, alkaline phosphatase (ALP) activity, and expression of osteogenic/cementogenic-related markers including runt-related transcription factor 2 (Runx2), ALP, osteocalcin (OCN), cementum attachment protein (CAP), and cementum protein (CEMP) as compared to the HA bioceramics with flat and dense surface. Moreover, mnHA bioceramics stimulated gene expression of low-density lipoprotein receptor-related protein 5 (LRP5) and β-catenin, which are the key genes of canonical Wnt signaling. Moreover, the stimulatory effect on ALP activity and osteogenic and cementogenic gene expression, including that of ALP, OCN, CAP, CEMP, and Runx2 of mnHA bioceramics could be repressed by canonical Wnt signaling inhibitor dickkopf1 (Dkk1). The results suggested that the HA bioceramics with mnHA could act as promising grafts for periodontal tissue regeneration. PMID:26648716

Fabrication of hydrogels with elasticity changed by alkaline phosphatase for stem cell culture.

PubMed

Toda, Hiroyuki; Yamamoto, Masaya; Uyama, Hiroshi; Tabata, Yasuhiko

2016-01-01

The objective of this study is to design hydrogels whose elasticity can be changed by alkaline phosphatase (ALP) in cell culture and evaluate the effect of hydrogel elasticity on an osteogenic gene expression of cells. Hydrogels were prepared by the radical polymerization of acrylamide (AAm), N,N'-methylenebisacrylamide (BIS), and Phosmer™M containing phosphate groups (PE-PAAm hydrogels). The storage modulus of PE-PAAm hydrogels prepared was changed by the preparation conditions. When human mesenchymal stem cells (hMSC) were cultured on the ALP-responsive PE-PAAm hydrogels in the presence or absence of ALP, the morphology of hMSC was observed and one of the osteogenic differentiation markers, Runx2, was evaluated. By ALP addition into the culture medium, the morphology of hMSC was changed into an elongated shape without cell damage. ALP addition modified the level of Runx2 gene expression, which was influenced by the modulus of PE-PAAm hydrogels. It is concluded that the elasticity change of hydrogel substrates in cell culture had an influence on the Runx2 gene expression of hMSC. Stem cells sense the surface elasticity of culture substrates, and their differentiation fate is biologically modified by substrate properties. Most of experiments have been performed in static conditions during cell culture, while the in vivo microenvironment is dynamically changed. In this study, we established to design an enzyme-responsive hydrogel whose elasticity can be changed by alkaline phosphatase (ALP) in cell culture to mimic in vivo conditions. As a result, the cells were deformed and the gene expression level of an osteogenic maker, Runx2, was modified by ALP treatment. This is the novel report describing to demonstrate that the dynamic alteration of hydrogel substrate elasticity could modulate the osteoblastic gene expression of human MSC in vitro. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

Assessment of the Role of Noni (Morinda citrifolia) Juice for Inducing Osteoblast Differentiation in Isolated Rat Bone Marrow Derived Mesenchymal Stem Cells

PubMed Central

Hussain, Sharmila; Tamizhselvi, Ramasamy; George, Leema; Manickam, Venkatraman

2016-01-01

Background and Objectives Morinda citrifolia (Noni), an important traditional medicinal plant still used in patients with bone fractures or dislocation to promote connective tissue repair and to reduce inflammation. However, the effects of Noni on bone metabolism and whether it influences the osteogenic differentiation is yet to be clarified. In this study, we investigated the effect of Morinda citrifolia (Noni) juice on the proliferation rate of rat bone marrow derived mesenchymal stem cells (BMSC) and the osteoblastic differentiation as shown by alkaline phosphatase (ALP), Runt-related transcription factor 2 (Runx2) and osteocalcin (OCN) mRNA expression in vitro. Methods and Results Treatment with 200 μg/ml Noni juice enhanced the proliferation rate of the BMSC and also upregulated the osteogenic differentiation marker genes ALP and OCN, and Runx2 measured by RTPCR. Consistent with these results collagen scaffolds implanted in vivo, which were loaded with BMSC pre-exposed to Noni, showed increased bone density measured by computed tomography and histological analysis revealed neo-angiogenesis for bone formation. Conclusions These results suggest that Noni stimulates osteoblastogenesis and can be used as adjuvant natural medicine for bone diseases such as osteoporosis. PMID:27572713

Assessment of the Role of Noni (Morinda citrifolia) Juice for Inducing Osteoblast Differentiation in Isolated Rat Bone Marrow Derived Mesenchymal Stem Cells.

PubMed

Hussain, Sharmila; Tamizhselvi, Ramasamy; George, Leema; Manickam, Venkatraman

2016-11-30

Morinda citrifolia (Noni), an important traditional medicinal plant still used in patients with bone fractures or dislocation to promote connective tissue repair and to reduce inflammation. However, the effects of Noni on bone metabolism and whether it influences the osteogenic differentiation is yet to be clarified. In this study, we investigated the effect of Morinda citrifolia (Noni) juice on the proliferation rate of rat bone marrow derived mesenchymal stem cells (BMSC) and the osteoblastic differentiation as shown by alkaline phosphatase (ALP), Runt-related transcription factor 2 (Runx2) and osteocalcin (OCN) mRNA expression in vitro . Treatment with 200 μg/ml Noni juice enhanced the proliferation rate of the BMSC and also upregulated the osteogenic differentiation marker genes ALP and OCN, and Runx2 measured by RTPCR. Consistent with these results collagen scaffolds implanted in vivo , which were loaded with BMSC pre-exposed to Noni, showed increased bone density measured by computed tomography and histological analysis revealed neo-angiogenesis for bone formation. These results suggest that Noni stimulates osteoblastogenesis and can be used as adjuvant natural medicine for bone diseases such as osteoporosis.

AML1 is overexpressed in patients with myeloproliferative neoplasms and mediates JAK2V617F-independent overexpression of NF-E2

PubMed Central

Wang, Wei; Schwemmers, Sven; Hexner, Elizabeth O.

2010-01-01

The transcription factor NF-E2 is overexpressed in the majority of patients with polycythemia vera (PV). Concomitantly, 95% of these patients carry the JAK2V617F mutation. Although NF-E2 levels correlate with JAK2V671F allele burden in some PV cohorts, the molecular mechanism causing aberrant NF-E2 expression has not been described. Here we show that NF-E2 expression is also increased in patients with essential thrombocythemia and primary myelofibrosis independent of the presence of the JAK2V617F mutation. Characterization of the NF-E2 promoter revealed multiple functional binding sites for AML1/RUNX-1. Chromatin immunoprecipitation demonstrated AML1 binding to the NF-E2 promoter in vivo. Moreover, AML1 binding to the NF-E2 promoter was significantly increased in granulocytes from PV patients compared with healthy controls. AML1 mRNA expression was elevated in patients with PV, essential thrombocythemia, and primary myelofibrosis both in the presence and absence of JAK2V617F. In addition, AML1 and NF-E2 expression were highly correlated. RNAi-mediated suppression of either AML1 or of its binding partner CBF-β significantly decreased NF-E2 expression. Moreover, expression of the leukemic fusion protein AML/ETO drastically decreased NF-E2 protein levels. Our data identify NF-E2 as a novel AML1 target gene and delineate a role for aberrant AML1 expression in mediating elevated NF-E2 expression in MPN patients. PMID:20339092

Vitamin D Impacts the Expression of Runx2 Target Genes and Modulates Inflammation, Oxidative Stress and Membrane Vesicle Biogenesis Gene Networks in 143B Osteosarcoma Cells

PubMed Central

Garimella, Rama; Tadikonda, Priyanka; Tawfik, Ossama; Gunewardena, Sumedha; Rowe, Peter; Van Veldhuizen, Peter

2017-01-01

Osteosarcoma (OS) is an aggressive malignancy of bone affecting children, adolescents and young adults. Understanding vitamin D metabolism and vitamin D regulated genes in OS is an important aspect of vitamin D/cancer paradigm, and in evaluating vitamin D as adjuvant therapy for human OS. Vitamin D treatment of 143B OS cells induced significant and novel changes in the expression of genes that regulate: (a) inflammation and immunity; (b) formation of reactive oxygen species, metabolism of cyclic nucleotides, sterols, vitamins and mineral (calcium), quantity of gap junctions and skeletogenesis; (c) bone mineral density; and (d) cell viability of skeletal cells, aggregation of bone cancer cells and exocytosis of secretory vesicles. Ingenuity pathway analysis revealed significant reduction in Runx2 target genes such as fibroblast growth factor -1, -12 (FGF1 and FGF12), bone morphogenetic factor-1 (BMP1), SWI/SNF related, matrix associated actin dependent regulator of chromatin subfamily a, member 4 (SMARCA4), Matrix extracellular phosphoglycoprotein (MEPE), Integrin, β4 (ITGBP4), Matrix Metalloproteinase -1, -28 (MMP1 and MMP28), and signal transducer and activator of transcription-4 (STAT4) in vitamin D treated 143B OS cells. These genes interact with the inflammation, oxidative stress and membrane vesicle biogenesis gene networks. Vitamin D not only inhibited the expression of Runx2 target genes MMP1, MMP28 and kallikrein related peptidase-7 (KLK7), but also migration and invasion of 143B OS cells. Vitamin D regulated Runx2 target genes or their products represent potential therapeutic targets and laboratory biomarkers for applications in translational oncology. PMID:28300755

Domain retention in transcription factor fusion genes and its biological and clinical implications: a pan-cancer study

PubMed Central

Kim, Pora; Ballester, Leomar Y.; Zhao, Zhongming

2017-01-01

Genomic rearrangements involving transcription factors (TFs) can form fusion proteins resulting in either enhanced, weakened, or even loss of TF activity. Functional domain (FD) retention is a critical factor in the activity of transcription factor fusion genes (TFFGs). A systematic investigation of FD retention in TFFGs and their outcome (e.g. expression changes) in a pan-cancer study has not yet been completed. Here, we examined the FD retention status in 386 TFFGs across 13 major cancer types and identified 83 TFFGs involving 67 TFs that retained FDs. To measure the potential biological relevance of TFs in TFFGs, we introduced a Major Active Isofusion Index (MAII) and built a prioritized TFFG network using MAII scores and the observed frequency of fusion positive samples. Interestingly, the four TFFGs (PML-RARA, RUNX1-RUNX1T1, TMPRSS2-ERG, and SFPQ-TFE3) with the highest MAII scores showed 50 differentially expressed target genes (DETGs) in fusion-positive versus fusion-negative cancer samples. DETG analysis revealed that they were involved in tumorigenesis-related processes in each cancer type. PLAU, which encodes plasminogen activator urokinase and serves as a biomarker for tumor invasion, was found to be consistently activated in the samples with the highest MAII scores. Among the 50 DETGs, 21 were drug targetable genes. Fourteen of these 21 DETGs were expressed in acute myeloid leukemia (AML) samples. Accordingly, we constructed an AML-specific TFFG network, which included 38 DETGs in RUNX1-RUNX1T1 or PML-RARA positive samples. In summary, this study revealed several TFFGs and their potential target genes, and provided insights into the clinical implications of TFFGs. PMID:29299133

Vitamin D Impacts the Expression of Runx2 Target Genes and Modulates Inflammation, Oxidative Stress and Membrane Vesicle Biogenesis Gene Networks in 143B Osteosarcoma Cells.

PubMed

Garimella, Rama; Tadikonda, Priyanka; Tawfik, Ossama; Gunewardena, Sumedha; Rowe, Peter; Van Veldhuizen, Peter

2017-03-16

Osteosarcoma (OS) is an aggressive malignancy of bone affecting children, adolescents and young adults. Understanding vitamin D metabolism and vitamin D regulated genes in OS is an important aspect of vitamin D/cancer paradigm, and in evaluating vitamin D as adjuvant therapy for human OS. Vitamin D treatment of 143B OS cells induced significant and novel changes in the expression of genes that regulate: (a) inflammation and immunity; (b) formation of reactive oxygen species, metabolism of cyclic nucleotides, sterols, vitamins and mineral (calcium), quantity of gap junctions and skeletogenesis; (c) bone mineral density; and (d) cell viability of skeletal cells, aggregation of bone cancer cells and exocytosis of secretory vesicles. Ingenuity pathway analysis revealed significant reduction in Runx2 target genes such as fibroblast growth factor -1, -12 ( FGF1 and FGF12 ), bone morphogenetic factor-1 ( BMP1 ), SWI/SNF related, matrix associated actin dependent regulator of chromatin subfamily a, member 4 ( SMARCA4 ), Matrix extracellular phosphoglycoprotein ( MEPE ), Integrin, β4 ( ITGBP4 ), Matrix Metalloproteinase -1, -28 ( MMP1 and MMP28 ), and signal transducer and activator of transcription-4 ( STAT4 ) in vitamin D treated 143B OS cells. These genes interact with the inflammation, oxidative stress and membrane vesicle biogenesis gene networks. Vitamin D not only inhibited the expression of Runx2 target genes MMP1 , MMP28 and kallikrein related peptidase-7 ( KLK7 ), but also migration and invasion of 143B OS cells. Vitamin D regulated Runx2 target genes or their products represent potential therapeutic targets and laboratory biomarkers for applications in translational oncology.

Prospective signs of cleidocranial dysplasia in Cebpb deficiency

PubMed Central

2014-01-01

Background Although runt-related transcription factor 2 (RUNX2) has been considered a determinant of cleidocranial dysplasia (CCD), some CCD patients were free of RUNX2 mutations. CCAAT/enhancer-binding protein beta (Cebpb) is a key factor of Runx2 expression and our previous study has reported two CCD signs including hyperdontia and elongated coronoid process of the mandible in Cebpb deficient mice. Following that, this work aimed to conduct a case-control study of thoracic, zygomatic and masticatory muscular morphology to propose an association between musculoskeletal phenotypes and deficiency of Cebpb, using a sample of Cebpb-/-, Cebpb+/- and Cebpb+/+ adult mice. Somatic skeletons and skulls of mice were inspected with soft x-rays and micro-computed tomography (μCT), respectively. Zygomatic inclination was assessed using methods of coordinate geometry and trigonometric function on anatomic landmarks identified with μCT. Masseter and temporal muscles were collected and weighed. Expression of Cebpb was examined with a reverse transcriptase polymerase chain reaction (RT-PCR) technique. Results Cebpb-/- mice displayed hypoplastic clavicles, a narrow thoracic cage, and a downward tilted zygomatic arch (p < 0.001). Although Cebpb+/- mice did not show the phenotypes above (p = 0.357), a larger mass percentage of temporal muscles over masseter muscles was seen in Cebpb+/- littermates (p = 0.012). The mRNA expression of Cebpb was detected in the clavicle, the zygoma, the temporal muscle and the masseter muscle, respectively. Conclusions Prospective signs of CCD were identified in mice with Cebpb deficiency. These could provide an additional aetiological factor of CCD. Succeeding investigation into interactions among Cebpb, Runx2 and musculoskeletal development is indicated. PMID:24885110

Effect of TAK1 on osteogenic differentiation of mesenchymal stem cells by regulating BMP-2 via Wnt/β-catenin and MAPK pathway.

PubMed

Yang, Hongpeng; Guo, Yue; Wang, Dawei; Yang, Xiaofei; Ha, Chengzhi

2018-01-02

Mesenchymal stem cells (MSCs) have the ability to differentiate into osteoblasts and chondrocytes. In vitro osteogenic differentiation is critical but the molecular mechanism has yet to be further clarified. The role of TGF-β activated kinase 1 (TAK1) in MSCs osteogenesis differentiation has not been reported. By adding si-TAK1 and rhTAK1, the osteogenic differentiation of MSCs was measured. Expression levels of the osteoblastic marker genes during osteogenic differentiation of MSCs were checked. As well as molecules involved in BMP and Wnt/β-catenin signaling pathways. The phosphorylation of p38 and JNK was also checked. TAK1 is essential for mineralization of MSCs at low concentration, but excessive rhTAK1 inhibits mineralization of MSCs. It up regulates the expression levels of bone sialoprotein (BSP), osteocalcin (OSC), Alkaline phosphatase (ALP), and RUNX2 during osteogenic differentiation of MSCs. It can also promote TGF-β/BMP-2 gene expression and β-catenin expression, and down regulate GSK-3β expression. Meanwhile, TAK1 promotes the phosphorylation of p38 and JNK. Additionally, TAK1 up regulates the expression of BMP-2 at all concentration under the inhibition of p38 and JNK. Our results suggested that TAK1 is essential in MSCs osteogenesis differentiation, and functions as a double-edged sword, probably through regulation of β-catenin and p38/JNK.

Wnt signaling regulates pulp volume and dentin thickness

PubMed Central

Lim, Won Hee; Liu, Bo; Cheng, Du; Hunter, Daniel J; Zhong, Zhendong; Ramos, Daniel M; Williams, Bart O; Sharpe, Paul T; Bardet, Claire; Mah, Su-jung; Helms, Jill A

2015-01-01

Odontoblasts, cementoblasts, ameloblasts and osteoblasts all form mineralized tissues in the craniofacial complex, and all these cell types exhibit active Wnt signaling during postnatal life. We set out to understand the functions of this Wnt signaling, by evaluating the phenotypes of mice in which the essential Wnt chaperone protein, Wingless was eliminated. The deletion of Wls was restricted to cells expressing Osteocalcin, which in addition to osteoblasts includes odontoblasts, cementoblasts, and ameloblasts. Dentin, cementum, enamel, and bone all formed in OCN-Cre;Wlsfl/fl mice but their homeostasis was dramatically affected. The most notable feature was a significant increase in dentin volume and density. We attribute this gain in dentin volume to a Wnt-mediated mis-regulation of Runx2. Normally, Wnt signaling stimulates Runx2, which in turn inhibits DSP; this inhibition must be relieved for odontoblasts to differentiate. In OCN-Cre;Wlsfl/fl mice, Wnt pathway activation is reduced and Runx2 levels decline. The Runx2-mediated repression of DSP is relieved and odontoblast differentiation is accordingly enhanced. This study demonstrates the importance of Wnt signaling in the homeostasis of mineralized tissues of the craniofacial complex. PMID:23996396

A small-molecule inhibitor of the aberrant transcription factor CBFβ-SMMHC delays leukemia in mice

PubMed Central

Illendula, Anuradha; Pulikkan, John A.; Zong, Hongliang; Grembecka, Jolanta; Xue, Liting; Sen, Siddhartha; Zhou, Yunpeng; Boulton, Adam; Kuntimaddi, Aravinda; Gao, Yan; Rajewski, Roger A.; Guzman, Monica L.; Castilla, Lucio H.; Bushweller, John H.

2015-01-01

Acute myeloid leukemia (AML) is the most common form of adult leukemia. The transcription factor fusion CBFβ-SMMHC (core binding factor β and the smooth-muscle myosin heavy chain), expressed in AML with the chromosome inversion inv(16)(p13q22), outcompetes wild-type CBFβ for binding to the transcription factor RUNX1, deregulates RUNX1 activity in hematopoiesis, and induces AML. Current inv(16) AML treatment with nonselective cytotoxic chemotherapy results in a good initial response but limited long-term survival. Here, we report the development of a protein-protein interaction inhibitor, AI-10-49, that selectively binds to CBFβ-SMMHC and disrupts its binding to RUNX1. AI-10-49 restores RUNX1 transcriptional activity, displays favorable pharmacokinetics, and delays leukemia progression in mice. Treatment of primary inv(16) AML patient blasts with AI-10-49 triggers selective cell death. These data suggest that direct inhibition of the oncogenic CBFβ-SMMHC fusion protein may be an effective therapeutic approach for inv(16) AML, and they provide support for transcription factor targeted therapy in other cancers. PMID:25678665

The creatine kinase pathway is a metabolic vulnerability in EVI1-positive acute myeloid leukemia

PubMed Central

Fenouille, Nina; Bassil, Christopher F.; Ben-Sahra, Issam; Benajiba, Lina; Alexe, Gabriela; Ramos, Azucena; Pikman, Yana; Conway, Amy S.; Burgess, Michael R.; Li, Qing; Luciano, Frédéric; Auberger, Patrick; Galinsky, Ilene; DeAngelo, Daniel J.; Stone, Richard M.; Zhang, Yi; Perkins, Archibald S.; Shannon, Kevin; Hemann, Michael T.; Puissant, Alexandre; Stegmaier, Kimberly

2017-01-01

Expression of the EVI1 proto-oncogene is deregulated by chromosomal translocations in some cases of acute myeloid leukemia (AML) and is associated with poor clinical outcome. Here, through transcriptomic and metabolomic profiling of hematopoietic cells, we reveal that EVI1 overexpression alters cellular metabolism. A pooled shRNA screen identified the ATP-buffering, mitochondrial creatine kinase CKMT1 as a metabolic dependency in EVI1-positive AML. EVI1 promotes CKMT1 expression by repressing the myeloid differentiation regulator RUNX1. Suppression of arginine-creatine metabolism by CKMT1-directed shRNAs or by the small molecule cyclocreatine selectively decreased the viability, promoted cell cycle arrest and apoptosis of human EVI1-positive AML cells, and prolonged survival in human orthotopic and mouse primary AML models. CKMT1 inhibition alters mitochondrial respiration and ATP production, an effect that is abrogated by phospho-creatine-mediated reactivation of the arginine-creatine pathway. Targeting CKMT1 is a promising therapeutic strategy for this EVI1-driven AML subtype that is highly resistant to current treatment regimens. PMID:28191887

Consecutive epigenetically-active agent combinations act in ID1-RUNX3-TET2 and HOXA pathways for Flt3ITD+ve AML.

PubMed

Sayar, Hamid; Liu, Yan; Gao, Rui; Zaid, Mohammad Abu; Cripe, Larry D; Weisenbach, Jill; Sargent, Katie J; Nassiri, Mehdi; Li, Lang; Konig, Heiko; Suvannasankha, Attaya; Pan, Feng; Shanmugam, Rajasubramaniam; Goswami, Chirayu; Kapur, Reuben; Xu, Mingjiang; Boswell, H Scott

2018-01-19

Co-occurrence of Flt3ITD and TET2 mutations provoke an animal model of AML by epigenetic repression of Wnt pathway antagonists, including RUNX3, and by hyperexpression of ID1, encoding Wnt agonist. These affect HOXA over-expression and treatment resistance. A comparable epigenetic phenotype was identified among adult AML patients needing novel intervention. We chose combinations of targeted agents acting on distinct effectors, at the levels of both signal transduction and chromatin remodeling, in relapsed/refractory AML's, including Flt3ITD+ve, described with a signature of repressed tumor suppressor genes, involving Wnt antagonist RUNX3 , occurring along with ID1 and HOXA over-expressions. We tracked patient response to combination of Flt3/Raf inhibitor, Sorafenib, and Vorinostat, pan-histone deacetylase inhibitor, without or with added Bortezomib, in consecutive phase I trials. A striking association of rapid objective remissions (near-complete, complete responses) was noted to accompany induced early pharmacodynamic changes within patient blasts in situ, involving these effectors, significantly linking RUNX3 /Wnt antagonist de-repression (80%) and ID1 downregulation (85%), to a response, also preceded by profound HOXA9 repression. Response occurred in context of concurrent TET2 mutation/hypomorphy and Flt3ITD+ve mutation (83% of complete responses). Addition of Bortezomib to the combination was vital to attainment of complete response in Flt3ITD+ve cases exhibiting such Wnt pathway dysregulation.

PRELP (proline/arginine-rich end leucine-rich repeat protein) promotes osteoblastic differentiation of preosteoblastic MC3T3-E1 cells by regulating the β-catenin pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Haiying; Cui, Yazhou; Luan, Jing

Proline/arginine-rich end leucine-rich repeat protein (PRELP) is a collagen-binding proteoglycan highly expressed in the developing bones. Recent studies indicated that PRELP could inhibit osteoclastogenesis as a NF-κB inhibitor. However, its role during osteoblast differentiation is still unclear. In this study, we confirmed that the expression of PRELP increased with the osteogenesis induction of preosteoblastic MC3T3-E1 cells. Down-regulation of PRELP expression by shRNA reduced ALP activity, mineralization and expression of osteogenic marker gene Runx2. Our microarray analysis data suggested that β-catenin may act as a hub gene in the PRELP-mediated gene network. We validated furtherly that PRELP knockdown could inhibit themore » level of connexin43, a key regulator of osteoblast differentiation by affecting β-catenin protein expression, and its nuclear translocation in MC3T3-E1 preosteoblasts. Therefore, this study established a new role of PRELP in modulating β-catenin/connexin43 pathway and osteoblast differentiation.« less

Activation of the mitogen-activated protein kinase pathway by bone sialoprotein regulates osteoblast differentiation.

PubMed

Gordon, Jonathan A R; Hunter, Graeme K; Goldberg, Harvey A

2009-01-01

Bone sialoprotein (BSP) is an abundant protein in the extracellular matrix of bone that has been suggested to have several different physiological functions, including the nucleation of hydroxyapatite (HA), promotion of cell attachment and binding of collagen. Studies in our lab have demonstrated that increased expression of BSP in osteoblast cells can increase expression of the osteoblast-related genes Runx2 and Osx as well as alkaline phosphatase and osteocalcin and increase matrix mineralization. To determine the molecular mechanisms responsible for the BSP-mediated increase in osteoblastic differentiation, several functional domain mutants of BSP were expressed in primary rat bone osteoblastic cells, including the contiguous glutamic acid sequences (polyGlu) and the arginine-glycine-aspartic acid (RGD) motif. Markers of osteoblast differentiation, including matrix mineralization and alkaline phosphatase staining, were increased in cells expressing BSP mutants of the polyGlu sequences but not in cells expressing RGD-mutated BSP. We also determined the dependence on integrin-associated pathways in promoting BSP-mediated differentiation responses in osteoblasts by demonstrating the activation of focal adhesion kinase, MAP kinase-associated proteins ERK1/2, ribosomal s6 kinase 2 and the AP-1 protein cFos. Thus, the mechanism regulating osteoblast differentiation by BSP was determined to be dependent on integrin-mediated intracellular signaling pathways. Copyright 2008 S. Karger AG, Basel.

Osteoblast differentiation and skeletal development are regulated by Mdm2–p53 signaling

PubMed Central

Lengner, Christopher J.; Steinman, Heather A.; Gagnon, James; Smith, Thomas W.; Henderson, Janet E.; Kream, Barbara E.; Stein, Gary S.; Lian, Jane B.; Jones, Stephen N.

2006-01-01

Mdm2 is required to negatively regulate p53 activity at the peri-implantation stage of early mouse development. However, the absolute requirement for Mdm2 throughout embryogenesis and in organogenesis is unknown. To explore Mdm2–p53 signaling in osteogenesis, Mdm2-conditional mice were bred with Col3.6-Cre–transgenic mice that express Cre recombinase in osteoblast lineage cells. Mdm2-conditional Col3.6-Cre mice die at birth and display multiple skeletal defects. Osteoblast progenitor cells deleted for Mdm2 have elevated p53 activity, reduced proliferation, reduced levels of the master osteoblast transcriptional regulator Runx2, and reduced differentiation. In contrast, p53-null osteoprogenitor cells have increased proliferation, increased expression of Runx2, increased osteoblast maturation, and increased tumorigenic potential, as mice specifically deleted for p53 in osteoblasts develop osteosarcomas. These results demonstrate that p53 plays a critical role in bone organogenesis and homeostasis by negatively regulating bone development and growth and by suppressing bone neoplasia and that Mdm2-mediated inhibition of p53 function is a prerequisite for Runx2 activation, osteoblast differentiation, and proper skeletal formation. PMID:16533949

Glucocorticoid-mediated BIM induction and apoptosis are regulated by Runx2 and c-Jun in leukemia cells

PubMed Central

Heidari, N; Miller, A V; Hicks, M A; Marking, C B; Harada, H

2012-01-01

Glucocorticoids (GCs) are common components of many chemotherapeutic regimens for lymphoid malignancies. GC-induced apoptosis involves an intrinsic mitochondria-dependent pathway. BIM (BCL-2-interacting mediator of cell death), a BCL-2 homology 3-only pro-apoptotic protein, is upregulated by dexamethasone (Dex) treatment in acute lymphoblastic leukemia cells and has an essential role in Dex-induced apoptosis. It has been indicated that Dex-induced BIM is regulated mainly by transcription, however, the molecular mechanisms including responsible transcription factors are unclear. In this study, we found that Dex treatment induced transcription factor Runx2 and c-Jun in parallel with BIM induction. Dex-induced BIM and apoptosis were decreased in cells harboring dominant-negative c-Jun and were increased in cells with c-Jun overexpression. Cells harboring short hairpin RNA for Runx2 also decreased BIM induction and apoptosis. On the Bim promoter, c-Jun bound to and activated the AP-1-binding site at about −2.7 kb from the transcription start site. Treatment with RU486, a GC receptor antagonist, blocked Dex-induced Runx2, c-Jun and BIM induction, as well as apoptosis. Furthermore, pretreatment with SB203580, a p38-mitogen-activated protein kinase (MAPK) inhibitor, decreased Dex-induced Runx2, c-Jun and BIM, suggesting that p38-MAPK activation is upstream of the induction of these molecules. In conclusion, we identified the critical signaling pathway for GC-induced apoptosis, and targeting these molecules may be an alternative approach to overcome GC-resistance in leukemia treatment. PMID:22825467

Conditions Inducing Excessive O-GlcNAcylation Inhibit BMP2-Induced Osteogenic Differentiation of C2C12 Cells.

PubMed

Gu, Hanna; Song, Mina; Boonanantanasarn, Kanitsak; Baek, Kyunghwa; Woo, Kyung Mi; Ryoo, Hyun-Mo; Baek, Jeong-Hwa

2018-01-09

Hyperglycemic conditions in diabetic patients can affect various cellular functions, including the modulation of osteogenic differentiation. However, the molecular mechanisms by which hyperglycemia affects osteogenic differentiation are yet to be clarified. This study aimed to investigate whether the aberrant increase in protein O -linked-β- N -acetylglucosamine glycosylation ( O -GlcNAcylation) contributes to the suppression of osteogenic differentiation due to hyperglycemia. To induce osteogenic differentiation, C2C12 cells were cultured in the presence of recombinant human bone morphogenetic protein 2 (BMP2). Excessive protein O -GlcNAcylation was induced by treating C2C12 cells with high glucose, glucosamine, or N -acetylglucosamine concentrations or by O -GlcNAc transferase (OGT) overexpression. The effect of O -GlcNAcylation on osteoblast differentiation was then confirmed by examining the expression levels of osteogenic marker gene mRNAs, activity of alkaline phosphatase, and transcriptional activity of Runx2, a critical transcription factor for osteoblast differentiation and bone formation. Cell treatment with high glucose, glucosamine or N -acetylglucosamine increased O -GlcNAcylation of Runx2 and the total levels of O -GlcNAcylated proteins, which led to a decrease in the transcriptional activity of Runx2, expression levels of osteogenic marker genes (Runx2, osterix, alkaline phosphatase, and type I collagen), and activity of alkaline phosphatase. These inhibitory effects were rescued by lowering protein O -GlcNAcylation levels by adding STO45849, an OGT inhibitor, or by overexpressing β- N -acetylglucosaminidase. Our findings suggest that excessive protein O -GlcNAcylation contributes to high glucose-suppressed osteogenic differentiation.

Increased baseline RUNX2, caspase 3 and p21 gene expressions in the peripheral blood of disease-modifying anti-rheumatic drug-naïve rheumatoid arthritis patients are associated with improved clinical response to methotrexate therapy.

PubMed

Tchetina, Elena V; Demidova, Natalia V; Markova, Galina A; Taskina, Elena A; Glukhova, Svetlana I; Karateev, Dmitry E

2017-10-01

To investigate the potential of the baseline gene expression in the whole blood of disease-modifying anti-rheumatic drug-naïve rheumatoid arthritis (RA) patients for predicting the response to methotrexate (MTX) treatment. Twenty-six control subjects and 40 RA patients were examined. Clinical, immunological and radiographic parameters were assessed before and after 24 months of follow-up. The gene expressions in the whole blood were measured using real-time reverse transcription polymerase chain reaction. The protein concentrations in peripheral blood mononuclear cells were quantified using enzyme-linked immunosorbent assay. Receiver operating characteristic curve analyses were used to suggest thresholds that were associated with the prediction of the response. Decreases in the disease activity at the end of the study were accompanied by significant increases in joint space narrowing score (JSN). Positive correlations between the expressions of the Unc-51-like kinase 1 (ULK1) and matrix metalloproteinase 9 (MMP-9) genes with the level of C-reactive protein and MMP-9 expression with Disease Activity Score of 28 joints (DAS28) and swollen joint count were noted at baseline. The baseline tumor necrosis factor (TNF)α gene expression was positively correlated with JSN at the end of the follow-up, whereas p21, caspase 3, and runt-related transcription factor (RUNX)2 were correlated with the ΔDAS28 values. Our results suggest that the expressions of MMP-9 and ULK1 might be associated with disease activity. Increased baseline gene expressions of RUNX2, p21 and caspase 3 in the peripheral blood might predict better responses to MTX therapy. © 2017 Asia Pacific League of Associations for Rheumatology and John Wiley & Sons Australia, Ltd.

Continuous hydrostatic pressure induces differentiation phenomena in chondrocytes mediated by changes in polycystins, SOX9, and RUNX2.

PubMed

Karamesinis, Konstantinos; Spyropoulou, Anastasia; Dalagiorgou, Georgia; Katsianou, Maria A; Nokhbehsaim, Marjan; Memmert, Svenja; Deschner, James; Vastardis, Heleni; Piperi, Christina

2017-01-01

The present study aimed to investigate the long-term effects of hydrostatic pressure on chondrocyte differentiation, as indicated by protein levels of transcription factors SOX9 and RUNX2, on transcriptional activity of SOX9, as determined by pSOX9 levels, and on the expression of polycystin-encoding genes Pkd1 and Pkd2. ATDC5 cells were cultured in insulin-supplemented differentiation medium (ITS) and/or exposed to 14.7 kPa of hydrostatic pressure for 12, 24, 48, and 96 h. Cell extracts were assessed for SOX9, pSOX9, and RUNX2 using western immunoblotting. The Pkd1 and Pkd2 mRNA levels were detected by real-time PCR. Hydrostatic pressure resulted in an early drop in SOX9 and pSOX9 protein levels at 12 h followed by an increase from 24 h onwards. A reverse pattern was followed by RUNX2, which reached peak levels at 24 h of hydrostatic pressure-treated chondrocytes in ITS culture. Pkd1 and Pkd2 mRNA levels increased at 24 h of combined hydrostatic pressure and ITS treatment, with the latter remaining elevated up to 96 h. Our data indicate that long periods of continuous hydrostatic pressure stimulate chondrocyte differentiation through a series of molecular events involving SOX9, RUNX2, and polycystins-1, 2, providing a theoretical background for functional orthopedic mechanotherapies.

Low-magnitude mechanical vibration regulates expression of osteogenic proteins in ovariectomized rats.

PubMed

Li, Ming; Wu, Wei; Tan, Lei; Mu, Degong; Zhu, Dong; Wang, Jian; Zhao, Bin

2015-09-25

The present study aimed to investigate the impact of low-magnitude and high-frequency mechanical vibration with various lengths of resting period incorporated between loading cycles on the expression of osteogenesis-related proteins in a rat model of osteoporosis. The rats in the mechanical loading groups received low-magnitude and high-frequency vibration (35 Hz and acceleration of 0.25 g, 15 min/day) for 8 weeks. Bilateral humeral heads and femoral heads were then isolated, and protein levels of bone morphogenetic protein 2 (BMP-2), extracellular signal-regulated kinase 1/2 (ERK1/2), phosphorylated ERK1/2 (p-ERK1/2), runt-related transcription factor 2 (Runx2) and osteocalcin (OCN) were determined by Western blotting. Increased levels of BMP-2, Runx2 and OCN were observed in rats receiving mechanical vibration. Total ERK1/2 protein remained unchanged, whereas the level of activated ERK1/2 (p-ERK1/2) increased after mechanical vibration. Vibration with incorporated resting period, regardless of length, was more effective in inducing expression of these osteogenic proteins, and the vibration with 7-day resting period had the most profound impact. Signals from low-magnitude and high-frequency mechanical vibration upregulated the expression of BMP-2 and Runx2, activated the ERK1/2 signaling pathway, and consequently led to increased expression of OCN. The anabolic effect of mechanical stimulation was enhanced with incorporation of resting period between loadings, and the one with 7-day resting period exhibited the strongest effect among all. Our results could provide a reference for development of mechanical stimulation as a non-pharmacological intervention for osteoporosis. Copyright © 2015 Elsevier Inc. All rights reserved.

Effects of a metabolic syndrome induced by a fructose-rich diet on bone metabolism in rats.

PubMed

Felice, Juan Ignacio; Gangoiti, María Virginia; Molinuevo, María Silvina; McCarthy, Antonio Desmond; Cortizo, Ana María

2014-02-01

The aims of this study were: first, to evaluate the possible effects of a fructose rich diet (FRD)-induced metabolic syndrome (MS) on different aspects of long bone histomorphometry in young male rats; second, to investigate the effects of this diet on bone tissue regeneration; and third, to correlate these morphometric alterations with changes in the osteogenic/adipogenic potential and expression of specific transcription factors, of marrow stromal cells (MSC) isolated from rats with fructose-induced MS. MS was induced in rats by treatment with a FRD for 28 days. Halfway through treatment, a parietal wound was made and bone healing was evaluated 14 days later. After treatments, histomorphometric analysis was performed in dissected femoral and parietal bones. MSC were isolated from the femora of control or fructose-treated rats and differentiated either to osteoblasts (evaluated by type 1 collagen, Alkaline phosphatase and extracellular nodule mineralization) or to adipocytes (evaluated by intracellular triglyceride accumulation). Expression of Runx2 and PPARγ was assessed by Western blot. Fructose-induced MS induced deleterious effects on femoral metaphysis microarchitecture and impaired bone regeneration. Fructose treatment decreased the osteogenic potential of MSC and Runx2 expression. In addition, it increased the adipogenic commitment of MSC and PPARγ expression. Fructose-induced MS is associated with deleterious effects on bone microarchitecture and with a decrease in bone repair. These alterations could be due to a deviation in the adipogenic/osteogenic commitment of MSC, probably by modulation of the Runx2/PPARγ ratio. Copyright © 2014 Elsevier Inc. All rights reserved.

[The crosstalk between canonical and noncanonical Wnt signaling pathway in osteoblast differentiation of periodontal ligament stem cells in inflammatory microenvironments].

PubMed

Liu, N; Shi, H G; Zhang, W; Gu, B

2016-11-09

Objective: To investigate the crosstalk between canonical Wnt/β-catenin and noncanonical Wnt/Ca 2+ pathway in osteoblast differentiation process of periodontal ligament stem cell (PDLSC) in inflammatory microenvironments. Methods: PDLSCs were obtained from human healthy individuals(H-PDLSC) and patients with periodontitis(P-PDLSC). The H/P-PDLSCs were transfected with β-catenin siRNA. Cell morphology was observed under fluorescent microscope and transfection efficiency was easured by Western blotting after transfection of PDLSC. The mRNA expressions of Runt-related transcription factor 2(Runx2), β-catenin and nemo like kinase(NLK) were detected by real time PCR, the protein expressions of calcium/calmodulin-dependent protein kinase Ⅱ (CaMK Ⅱ) and NLK were examined by Western blotting and the CaMK Ⅱ was observed by immunofluorescence staining, respectively. Results: The β-catenin expressions in H/P-PDLSCs were inhibited specifically and efficiently by treatment of β-catenin-siRNA for 24 h. After a 3-day-osteogenic process, results of real-time quantitative PCR showed that the Runx2 mRNA expression in P-PDLSC siRNA β-catenin transfected group(4.553 ± 0.659) was significantly higher than that in P-PDLSC empty plasmid control group(1.918 ± 0.315) ( P= 0.000). A similar trend was observed in the NLK mRNA expression tests(7.341 ± 1.331 vs. 5.664 ± 0.792) ( P= 0.030). Accordingly, the protein expression levels of CaMK Ⅱ, NLK were higher in P-PDLSC siRNA β-catenin transfected group than that in P-PDLSC empty plasmid control group in osteogenic differentiation condition for 3 days. CaMKⅡ was more strongly induced in P-PDLSC siRNA β-catenin group than that in P-PDLSC empty plasmid control group after PDLSC cultured in osteogenic medium for 3 days. Conclusions: Both canonical Wnt/β-catenin and noncanonical Wnt/Ca 2+ pathway could regulate the osteogenic differentiation potential of P-PDLSC. Suppression of β-catenin by siRNA promoted osteogenic differentiation via increasing noncanonical Wnt signaling pathway of PDLSC in inflammatory microenvironments.

Effects of quercetin and quercetin 3-glucuronide on the expression of bone sialoprotein gene.

PubMed

Kim, Dong-Soon; Takai, Hideki; Arai, Masato; Araki, Shouta; Mezawa, Masaru; Kawai, Yoshichika; Murota, Kaeko; Terao, Junji; Ogata, Yorimasa

2007-06-01

Quercetin is a typical flavonol-type flavonoid and is present in a variety of vegetables, and their antioxidant effect implies their possible role in the prevention of oxidative stress related chronic diseases. Bone sialoprotein (BSP) is a noncollagenous protein of the extracellular matrix in the mineralized connective tissues that has been implicated in the nucleation of hydroxyapatite crystals. Previously, we reported that isoflavone (genistein) activated BSP gene transcription is mediated through an inverted CCAAT box in the proximal BSP gene promoter. The present study investigates the regulation of BSP transcription in a rat osteoblast-like cell line, ROS 17/2.8 cells, by quercetin and its conjugated metabolite quercetin 3-glucuronide. Quercetin and quercetin 3-glucuronide (5 microM) increased the BSP mRNA levels at 12 h and quercetin upregulated the Cbfa1/Runx2 mRNA expression at 12 h. From transient transfection assays using various sized BSP promoter-luciferase constructs, quercetin increased the luciferase activity of the construct (pLUC3), including the promoter sequence nucleotides -116 to -43. Transcriptional stimulations by quercetin were almost completely abrogated in the constructs that included 2 bp mutations in the inverted CCAAT and FRE elements whereas the CCAAT-protein complex did not change after stimulation by quercetin according to gel shift assays. Quercetin increased the nuclear protein binding to the FRE and 3'-FRE. These data suggest that quercetin and quercetin 3-glucuronide increased the BSP mRNA expression, and that the inverted CCAAT and FRE elements in the promoter of the BSP gene are required for quercetin induced BSP transcription.

RUNX in Invertebrates.

PubMed

Hughes, S; Woollard, A

2017-01-01

Runx genes have been identified in all metazoans and considerable conservation of function observed across a wide range of phyla. Thus, insight gained from studying simple model organisms is invaluable in understanding RUNX biology in higher animals. Consequently, this chapter will focus on the Runx genes in the diploblasts, which includes sea anemones and sponges, as well as the lower triploblasts, including the sea urchin, nematode, planaria and insect. Due to the high degree of functional redundancy amongst vertebrate Runx genes, simpler model organisms with a solo Runx gene, like C. elegans, are invaluable systems in which to probe the molecular basis of RUNX function within a whole organism. Additionally, comparative analyses of Runx sequence and function allows for the development of novel evolutionary insights. Strikingly, recent data has emerged that reveals the presence of a Runx gene in a protist, demonstrating even more widespread occurrence of Runx genes than was previously thought. This review will summarize recent progress in using invertebrate organisms to investigate RUNX function during development and regeneration, highlighting emerging unifying themes.

ETV6-RUNX1 + Acute Lymphoblastic Leukaemia in Identical Twins.

PubMed

Ford, Anthony M; Greaves, Mel

2017-01-01

Acute leukaemia is the major subtype of paediatric cancer with a cumulative risk of 1 in 2000 for children up to the age of 15 years. Childhood acute lymphoblastic leukaemia (ALL) is a biologically and clinically diverse disease with distinctive subtypes; multiple chromosomal translocations exist within the subtypes and each carries its own prognostic relevance. The most common chromosome translocation observed is the t(12;21) that results in an in-frame fusion between the first five exons of ETV6 (TEL) and almost the entire coding region of RUNX1 (AML1).The natural history of childhood ALL is almost entirely clinically silent and is well advanced at the point of diagnosis. It has, however, been possible to backtrack this process through molecular analysis of appropriate clinical samples: (i) leukaemic clones in monozygotic twins that are either concordant or discordant for ALL; (ii) archived neonatal blood spots or Guthrie cards from individuals who later developed leukaemia; and (iii) stored, viable cord blood cells.Here, we outline our studies on the aetiology and pathology of childhood ALL that provide molecular evidence for a monoclonal, prenatal origin of ETV6-RUNX1+ leukaemia in monozygotic identical twins. We provide mechanistic support for the concept that altered patterns of infection during early childhood can deliver the necessary promotional drive for the progression of ETV6-RUNX1+ pre-leukaemic cells into a postnatal overt leukaemia.

Foxp1/2/4 regulate endochondral ossification as a suppresser complex

PubMed Central

Zhao, Haixia; Zhou, Wenrong; Yao, Zhengju; Wan, Yong; Cao, Jingjing; Zhang, Lingling; Zhao, Jianzhi; Li, Hanjun; Zhou, Rujiang; Li, Baojie; Wei, Gang; Zhang, Zhenlin; French, Catherine A.; Dekker, Joseph D.; Yang, Yingzi; Fisher, Simon E.; lucker, Haley O.; Guo, Xizhi

2015-01-01

Osteoblast induction and differentiation in developing long bones is dynamically controlled by the opposing action of transcriptional activators and repressors. In contrast to the long list of activators that have been discovered over past decades, the network of repressors is not well-defined. Here we identify the expression of Foxp1/2/4 proteins, comprised of Forkhead-box (Fox) transcription factors of the Foxp subfamily, in both perichondrial skeletal progenitors and proliferating chondrocytes during endochondral ossification. Mice carrying loss-of-function and gain-of-function Foxp mutations had gross defects in appendicular skeleton formation. At the cellular level, over-expression of Foxp1/2/4 in chondroctyes abrogated osteoblast formation and chondrocyte hypertrophy. Conversely, single or compound deficiency of Foxp1/2/4 in skeletal progenitors or chondrocytes resulted in premature osteoblast differentiation in the perichondrium, coupled with impaired proliferation, survival, and hypertrophy of chondrocytes in the growth plate. Foxp1/2/4 and Runx2 proteins interacted in vitro and in vivo, and Foxp1/2/4 repressed Runx2 transactivation function in heterologous cells. This study establishes Foxp1/2/4 proteins as coordinators of osteogenesis and chondrocyte hypertrophy in developing long bones and suggests that a novel transcriptional repressor network involving Foxp1/2/4 may regulate Runx2 during endochondral ossification. PMID:25527076

[Experimental study on the transforming growth factor β3 combined with dental pulp stem cells in early bone integration of implant].

PubMed

Guzalinuer, Ababaikeli; Muhetaer, Huojia; Wu, H; Paerhati, Abudureheman

2018-04-09

Objective: To establish the experimental model of rabbit mandibular anterior implant repair and evaluate the effects of transforming growth factor (TGF)-β3 and dental pulp stem cells (DPSC) in promoting the bone integration of implant. Methods: The New Zealand rabbits were randomly divided into experimental group, control group and blank group (6 rabbits for each group) . In the experimental group, the implant area was filled with the mixture of TGF-β3, DPSC and Bio-oss powder. In the control group, the implant area was filled with the mixture of DPSC and Bio-oss powder. In the blank group, the implant area was filled with the mixture of phosphate buffer solution and Bio-oss powder. Eighteen New Zealand rabbits were sacrificed in 2 weeks after procedure. The treated alveolar bone tissue was observed. The bone tissue around the implant were estimated by HE staining, immunocytochemical staining and real-time quantitative PCR. Results: The implants were no shedding nor loose. HE staining shows the blank group had a sparse trabecular bone and a small amount of blood vessel around the implant and no obvious new bone formation. The control group showed that the bone trabecula around the implant was sparse and slender, the osteoblasts were arranged linearly around the trabecular bone, a small amount of new bone formation was found around the implant. In the experimental group, there were more thick and dense trabecular bone around the implant, the surrounding osteoblasts were arranged in clusters. The osteoblasts were active and many new bone formed. Typical bone lacunae, bone cells and a large number of new blood vessels can be observed. Immunohistochemistry showed that the proportion of average positive area in the experimental group, control group, blank group were (24.6±5.3) %, (11.3±2.8) % and (7.6±3.8) % respectively. The expression of bone sialoprotein in experimental group were significantly higher than the other 2 groups( P= 0.000). Real-time quantitative PCR results showed that the expression level of Runt-related transcription factor 2 (RUNX2), type Ⅰcollagen (COL-Ⅰ), alkaline phosphatase in the experimental group was higher than in the blank group. The expression level of RUNX2 and COL-Ⅰ in the experimental group was higher than that of the control group ( P= 0.023). Conclusions: TGF-β3 has potential to promote the transformation of DPSC into osteoblasts, which can promote the integration of bone around the implant.

Regulation of MHC class I expression by Foxp3 and its effect on Treg cell function

PubMed Central

Mu, Jie; Tai, Xuguang; Iyer, Shankar S.; Weissman, Jocelyn D.; Singer, Alfred; Singer, Dinah S.

2014-01-01

Expression of MHC class I molecules, which provide immune surveillance against intracellular pathogens, is higher on lymphoid cells than on any other cell types. In T cells, this is a result of activation of class I transcription by the T cell enhanceosome consisting of Runx1, CBFβ and LEF1. We now report that MHC class I transcription in T cells also is enhanced by Foxp3, resulting in higher levels of class I in CD4+CD25+ T regulatory cells than in conventional CD4+CD25− T cells. Interestingly, the effect of Foxp3 regulation of MHC class I transcription is cell-type specific: Foxp3 increases MHC class I expression in T cells but represses it in epithelial tumor cells. In both cell types, Foxp3 targets the upstream IRE and downstream core promoter of the class I gene. Importantly, expression of MHC class I contributes to the function of CD4+CD25+ T regulatory cells by enhancing immune suppression, both in in vitro and in vivo. These findings identify MHC class I genes as direct targets of Foxp3 whose expression augments regulatory T cell function. PMID:24523508

Accumulation of type VI collagen in the primary osteon of the rat femur during postnatal development

PubMed Central

Kohara, Yukihiro; Soeta, Satoshi; Izu, Yayoi; Amasaki, Hajime

2015-01-01

In rodents, the long bone diaphysis is expanded by forming primary osteons at the periosteal surface of the cortical bone. This ossification process is thought to be regulated by the microenvironment in the periosteum. Type VI collagen (Col VI), a component of the extracellular matrix (ECM) in the periosteum, is involved in osteoblast differentiation at early stages. In several cell types, Col VI interacts with NG2 on the cytoplasmic membrane to promote cell proliferation, spreading and motility. However, the detailed functions of Col VI and NG2 in the ossification process in the periosteum are still under investigation. In this study, to clarify the relationship between localization of Col VI and formation of the primary osteon, we examined the distribution of Col VI and osteoblast lineages expressing NG2 in the periosteum of rat femoral diaphysis during postnatal growing periods by immunohistochemistry. Primary osteons enclosing the osteonal cavity were clearly identified in the cortical bone from 2 weeks old. The size of the osteonal cavities decreased from the outer to the inner region of the cortical bone. In addition, the osteonal cavities of newly formed primary osteons at the outermost region started to decrease in size after rats reached the age of 4 weeks. Immunohistochemistry revealed concentrated localization of Col VI in the ECM in the osteonal cavity. Col VI-immunoreactive areas were reduced and they disappeared as the osteonal cavities became smaller from the outer to the inner region. In the osteonal cavities of the outer cortical regions, Runx2-immunoreactive spindle-shaped cells and mature osteoblasts were detected in Col VI-immunoreactive areas. The numbers of Runx2-immunoreactive cells were significantly higher in the osteonal cavities than in the osteogenic layers from 2 to 4 weeks. Most of these Runx2-immunoreactive cells showed NG2-immunoreactivity. Furthermore, PCNA-immunoreactivity was detected in the Runx2-immunoreactive spindle cells in the osteonal cavities. These results indicate that Col VI provides a characteristic microenvironment in the osteonal cavity of the primary osteon, and that differentiation and proliferation of the osteoblast lineage occur in the Col VI-immunoreactive area. Interaction of Col VI and NG2 may be involved in the structural organization of the primary osteon by regulating osteoblast lineages. PMID:25943007

Accumulation of type VI collagen in the primary osteon of the rat femur during postnatal development.

PubMed

Kohara, Yukihiro; Soeta, Satoshi; Izu, Yayoi; Amasaki, Hajime

2015-05-01

In rodents, the long bone diaphysis is expanded by forming primary osteons at the periosteal surface of the cortical bone. This ossification process is thought to be regulated by the microenvironment in the periosteum. Type VI collagen (Col VI), a component of the extracellular matrix (ECM) in the periosteum, is involved in osteoblast differentiation at early stages. In several cell types, Col VI interacts with NG2 on the cytoplasmic membrane to promote cell proliferation, spreading and motility. However, the detailed functions of Col VI and NG2 in the ossification process in the periosteum are still under investigation. In this study, to clarify the relationship between localization of Col VI and formation of the primary osteon, we examined the distribution of Col VI and osteoblast lineages expressing NG2 in the periosteum of rat femoral diaphysis during postnatal growing periods by immunohistochemistry. Primary osteons enclosing the osteonal cavity were clearly identified in the cortical bone from 2 weeks old. The size of the osteonal cavities decreased from the outer to the inner region of the cortical bone. In addition, the osteonal cavities of newly formed primary osteons at the outermost region started to decrease in size after rats reached the age of 4 weeks. Immunohistochemistry revealed concentrated localization of Col VI in the ECM in the osteonal cavity. Col VI-immunoreactive areas were reduced and they disappeared as the osteonal cavities became smaller from the outer to the inner region. In the osteonal cavities of the outer cortical regions, Runx2-immunoreactive spindle-shaped cells and mature osteoblasts were detected in Col VI-immunoreactive areas. The numbers of Runx2-immunoreactive cells were significantly higher in the osteonal cavities than in the osteogenic layers from 2 to 4 weeks. Most of these Runx2-immunoreactive cells showed NG2-immunoreactivity. Furthermore, PCNA-immunoreactivity was detected in the Runx2-immunoreactive spindle cells in the osteonal cavities. These results indicate that Col VI provides a characteristic microenvironment in the osteonal cavity of the primary osteon, and that differentiation and proliferation of the osteoblast lineage occur in the Col VI-immunoreactive area. Interaction of Col VI and NG2 may be involved in the structural organization of the primary osteon by regulating osteoblast lineages. © 2015 Anatomical Society.

Evaluation of markers for CpG island methylator phenotype (CIMP) in colorectal cancer by a large population-based sample.

PubMed

Ogino, Shuji; Kawasaki, Takako; Kirkner, Gregory J; Kraft, Peter; Loda, Massimo; Fuchs, Charles S

2007-07-01

The CpG island methylator phenotype (CIMP or CIMP-high) with extensive promoter methylation is a distinct phenotype in colorectal cancer. However, a choice of markers for CIMP has been controversial. A recent extensive investigation has selected five methylation markers (CACNA1G, IGF2, NEUROG1, RUNX3, and SOCS1) as surrogate markers for epigenomic aberrations in tumor. The use of these markers as a CIMP-specific panel needs to be validated by an independent, large dataset. Using MethyLight assays on 920 colorectal cancers from two large prospective cohort studies, we quantified DNA methylation in eight CIMP-specific markers [the above five plus CDKN2A (p16), CRABP1, and MLH1]. A CIMP-high cutoff was set at > or = 6/8 or > or = 5/8 methylated promoters, based on tumor distribution and BRAF/KRAS mutation frequencies. All but two very specific markers [MLH1 (98% specific) and SOCS1 (93% specific)] demonstrated > or = 85% sensitivity and > or = 80% specificity, indicating overall good concordance in methylation patterns and good performance of these markers. Based on sensitivity, specificity, and false positives and negatives, the eight markers were ranked in order as: RUNX3, CACNA1G, IGF2, MLH1, NEUROG1, CRABP1, SOCS1, and CDKN2A. In conclusion, a panel of markers including at least RUNX3, CACNA1G, IGF2, and MLH1 can serve as a sensitive and specific marker panel for CIMP-high.

Overexpression of Galnt3 in chondrocytes resulted in dwarfism due to the increase of mucin-type O-glycans and reduction of glycosaminoglycans.

PubMed

Yoshida, Carolina Andrea; Kawane, Tetsuya; Moriishi, Takeshi; Purushothaman, Anurag; Miyazaki, Toshihiro; Komori, Hisato; Mori, Masako; Qin, Xin; Hashimoto, Ayako; Sugahara, Kazuyuki; Yamana, Kei; Takada, Kenji; Komori, Toshihisa

2014-09-19

Galnt3, UDP-N-acetyl-α-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 3, transfers N-acetyl-D-galactosamine to serine and threonine residues, initiating mucin type O-glycosylation of proteins. We searched the target genes of Runx2, which is an essential transcription factor for chondrocyte maturation, in chondrocytes and found that Galnt3 expression was up-regulated by Runx2 and severely reduced in Runx2(-/-) cartilaginous skeletons. To investigate the function of Galnt3 in chondrocytes, we generated Galnt3(-/-) mice and chondrocyte-specific Galnt3 transgenic mice under the control of the Col2a1 promoter-enhancer. Galnt3(-/-) mice showed a delay in endochondral ossification and shortened limbs at embryonic day 16.5, suggesting that Galnt3 is involved in chondrocyte maturation. Galnt3 transgenic mice presented dwarfism, the chondrocyte maturation was retarded, the cell cycle in chondrocytes was accelerated, premature chondrocyte apoptosis occurred, and the growth plates were disorganized. The binding of Vicia villosa agglutinin, which recognizes the Tn antigen (GalNAc-O-Ser/Thr), was drastically increased in chondrocytes, and aggrecan (Acan) was highly enriched with Tn antigen. However, safranin O staining, which recognizes glycosaminoglycans (GAGs), and Acan were severely reduced. Chondroitin sulfate was reduced in amount, but the elongation of chondroitin sulfate chains had not been severely disturbed in the isolated GAGs. These findings indicate that overexpression of Galnt3 in chondrocytes caused dwarfism due to the increase of mucin-type O-glycans and the reduction of GAGs, probably through competition with xylosyltransferases, which initiate GAG chains by attaching O-linked xylose to serine residues, suggesting a negative effect of Galnt family proteins on Acan deposition in addition to the positive effect of Galnt3 on chondrocyte maturation. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Overexpression of Galnt3 in Chondrocytes Resulted in Dwarfism Due to the Increase of Mucin-type O-Glycans and Reduction of Glycosaminoglycans*

PubMed Central

Yoshida, Carolina Andrea; Kawane, Tetsuya; Moriishi, Takeshi; Purushothaman, Anurag; Miyazaki, Toshihiro; Komori, Hisato; Mori, Masako; Qin, Xin; Hashimoto, Ayako; Sugahara, Kazuyuki; Yamana, Kei; Takada, Kenji; Komori, Toshihisa

2014-01-01

Galnt3, UDP-N-acetyl-α-d-galactosamine:polypeptide N-acetylgalactosaminyltransferase 3, transfers N-acetyl-d-galactosamine to serine and threonine residues, initiating mucin type O-glycosylation of proteins. We searched the target genes of Runx2, which is an essential transcription factor for chondrocyte maturation, in chondrocytes and found that Galnt3 expression was up-regulated by Runx2 and severely reduced in Runx2−/− cartilaginous skeletons. To investigate the function of Galnt3 in chondrocytes, we generated Galnt3−/− mice and chondrocyte-specific Galnt3 transgenic mice under the control of the Col2a1 promoter-enhancer. Galnt3−/− mice showed a delay in endochondral ossification and shortened limbs at embryonic day 16.5, suggesting that Galnt3 is involved in chondrocyte maturation. Galnt3 transgenic mice presented dwarfism, the chondrocyte maturation was retarded, the cell cycle in chondrocytes was accelerated, premature chondrocyte apoptosis occurred, and the growth plates were disorganized. The binding of Vicia villosa agglutinin, which recognizes the Tn antigen (GalNAc-O-Ser/Thr), was drastically increased in chondrocytes, and aggrecan (Acan) was highly enriched with Tn antigen. However, safranin O staining, which recognizes glycosaminoglycans (GAGs), and Acan were severely reduced. Chondroitin sulfate was reduced in amount, but the elongation of chondroitin sulfate chains had not been severely disturbed in the isolated GAGs. These findings indicate that overexpression of Galnt3 in chondrocytes caused dwarfism due to the increase of mucin-type O-glycans and the reduction of GAGs, probably through competition with xylosyltransferases, which initiate GAG chains by attaching O-linked xylose to serine residues, suggesting a negative effect of Galnt family proteins on Acan deposition in addition to the positive effect of Galnt3 on chondrocyte maturation. PMID:25107907

Adrenaline inhibits osteogenesis via repressing miR-21 expression.

PubMed

Chen, Danying; Wang, Zuolin

2017-01-01

Sympathetic signaling is involved in bone homeostasis; however, the cellular and molecular mechanisms remain unknown. In this study, we found that the psychological stress mediator adrenaline inhibited osteogenic differentiation of human bone marrow-derived stem cells (hMSC) by reducing microRNA-21 (miR-21) expression. Briefly, adrenaline significantly inhibited the osteogenic differentiation of hMSCs, as observed with both Alizarin red staining and maker gene expression (RUNX2, OSX, OCN, and OPN). During this process, miR-21 was suppressed by adrenaline via inhibition of histone acetylation, as verified by H3K9Ac chromatin immunoprecipitation (ChIP) assay. MiR-21 was confirmed to promote hMSC osteogenic differentiation, and overexpression of miR-21 reversed the impeditive effect of adrenaline on hMSC osteogenic differentiation. Our results demonstrate that down-regulation of miR-21 is responsible for the adrenaline-mediated inhibition of hMSC osteogenic differentiation. These findings indicate a regulation of bone metabolism by psychological stress and also provide a molecular basis for psychological stress-associated bone diseases. © 2016 International Federation for Cell Biology.

Prognostic significance of CDKN2A (p16) promoter methylation and loss of expression in 902 colorectal cancers: Cohort study and literature review.

PubMed

Shima, Kaori; Nosho, Katsuhiko; Baba, Yoshifumi; Cantor, Mami; Meyerhardt, Jeffrey A; Giovannucci, Edward L; Fuchs, Charles S; Ogino, Shuji

2011-03-01

A cyclin-dependent kinase inhibitor CDKN2A (p16/Ink4a) is a tumor suppressor and upregulated in cellular senescence. CDKN2A promoter methylation and gene silencing are associated with the CpG island methylator phenotype (CIMP) in colon cancer. However, prognostic significance of CDKN2A methylation or loss of CDKN2A (p16) expression independent of CIMP status remains uncertain. Using a database of 902 colorectal cancers in 2 independent cohort studies (the Nurses' Health Study and the Health Professionals Follow-up Study), we quantified CDKN2A promoter methylation and detected hypermethylation in 269 tumors (30%). By immunohistochemistry, we detected loss of CDKN2A (p16) expression in 25% (200/804) of tumors. We analyzed for LINE-1 hypomethylation and hypermethylation at 7 CIMP-specific CpG islands (CACNA1G, CRABP1, IGF2, MLH1, NEUROG1, RUNX3 and SOCS1); microsatellite instability (MSI); KRAS, BRAF and PIK3CA mutations; and expression of TP53 (p53), CTNNB1 (β-catenin), CDKN1A (p21), CDKN1B (p27), CCND1 (cyclin D1), FASN (fatty acid synthase) and PTGS2 (cyclooxygenase-2). CDKN2A promoter methylation and loss of CDKN2A (p16) were associated with shorter overall survival in univariate Cox regression analysis [hazard ratio (HR): 1.36, 95% CI: 1.10-1.66, p = 0.0036 for CDKN2A methylation; HR: 1.30, 95% CI: 1.03-1.63, p = 0.026 for CDKN2A (p16) loss] but not in multivariate analysis that adjusted for clinical and tumor variables, including CIMP, MSI and LINE-1 methylation. Neither CDKN2A promoter methylation nor loss of CDKN2A (p16) was associated with colorectal cancer-specific mortality in uni- or multivariate analysis. Despite its well-established role in carcinogenesis, CDKN2A (p16) promoter methylation or loss of expression in colorectal cancer is not independently associated with patient prognosis. Copyright © 2010 UICC.

Combined VEGF and LMP-1 delivery enhances osteoprogenitor cell differentiation and ectopic bone formation.

PubMed

Wang, Xiuli; Cui, Fuai; Madhu, Vedavathi; Dighe, Abhijit S; Balian, Gary; Cui, Quanjun

2011-02-01

A novel strategy to enhance bone repair is to combine angiogenic factors and osteogenic factors. We combined vascular endothelial growth factor (VEGF) and LIM mineralization protein-1 (LMP-1) by using an internal ribosome entry site to link the genes within a single plasmid. We then evaluated the effects on osteoblastic differentiation in vitro and ectopic bone formation in vivo with a subcutaneously placed PLAGA scaffold loaded with a cloned mouse osteoprogenitor cell line, D1, transfected with plasmids containing VEGF and LMP-1 genes. The cells expressing both genes elevated mRNA expression of RunX2 and β-catenin and alkaline phosphatase activity compared to cells from other groups. In vivo, X-ray and micro-CT analysis of the retrieved implants revealed more ectopic bone formation at 2 and 3 weeks but not at 4 weeks compared to other groups. The results indicate that the combination of the therapeutic growth factors potentiates cell differentiation and may promote osteogenesis.

Gelatin-Derived Graphene–Silicate Hybrid Materials Are Biocompatible and Synergistically Promote BMP9-Induced Osteogenic Differentiation of Mesenchymal Stem Cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zou, Yulong; Qazvini, Nader Taheri; Zane, Kylie

Graphene-based materials are used in many fields but have found only limited applications in biomedicine, including bone tissue engineering. Here, we demonstrate that novel hybrid materials consisting of gelatin-derived graphene and silicate nanosheets of Laponite (GL) are biocompatible and promote osteogenic differentiation of mesenchymal stem cells (MSCs). Homogeneous cell attachment, long-term proliferation, and osteogenic differentiation of MSCs on a GL-scaffold were confirmed using optical microscopy and scanning electron microscopy. GL-powders made by pulverizing the GL-scaffold were shown to promote bone morphogenetic protein (BMP9)-induced osteogenic differentiation. GL-powders increased the alkaline phosphatase (ALP) activity in immortalized mouse embryonic fibroblasts but decreased themore » ALP activity in more-differentiated immortalized mouse adipose-derived cells. Note, however, that GL-powders promoted BMP9-induced calcium mineral deposits in both MSC lines, as assessed using qualitative and quantitative alizarin red assays. Furthermore, the expression of chondro-osteogenic regulator markers such as Runx2, Sox9, osteopontin, and osteocalcin was upregulated by the GL-powder, independent of BMP9 stimulation; although the powder synergistically upregulated the BMP9-induced Osterix expression, the adipogenic marker PPAR gamma was unaffected. Furthermore, in vivo stem cell implantation experiments demonstrated that GL-powder could significantly enhance the BMP9-induced ectopic bone formation from MSCs. Collectively, our results strongly suggest that the GL hybrid materials promote BMP9-induced osteogenic differentiation of MSCs and hold promise for the development of bone tissue engineering platforms.« less

Variable transcriptional responsiveness of the P2X3 receptor gene during CFA-induced inflammatory hyperalgesia.

PubMed

Nuñez-Badinez, Paulina; Sepúlveda, Hugo; Diaz, Emilio; Greffrath, Wolfgang; Treede, Rolf-Detlef; Stehberg, Jimmy; Montecino, Martin; van Zundert, Brigitte

2018-05-01

The purinergic receptor P2X3 (P2X3-R) plays important roles in molecular pathways of pain, and reduction of its activity or expression effectively reduces chronic inflammatory and neuropathic pain sensation. Inflammation, nerve injury, and cancer-induced pain can increase P2X3-R mRNA and/or protein levels in dorsal root ganglia (DRG). However, P2X3-R expression is unaltered or even reduced in other pain studies. The reasons for these discrepancies are unknown and might depend on the applied traumatic intervention or on intrinsic factors such as age, gender, genetic background, and/or epigenetics. In this study, we sought to get insights into the molecular mechanisms responsible for inflammatory hyperalgesia by determining P2X3-R expression in DRG neurons of juvenile male rats that received a Complete Freund's Adjuvant (CFA) bilateral paw injection. We demonstrate that all CFA-treated rats showed inflammatory hyperalgesia, however, only a fraction (14-20%) displayed increased P2X3-R mRNA levels, reproducible across both sides. Immunostaining assays did not reveal significant increases in the percentage of P2X3-positive neurons, indicating that increased P2X3-R at DRG somas is not critical for inducing inflammatory hyperalgesia in CFA-treated rats. Chromatin immunoprecipitation (ChIP) assays showed a correlated (R 2  = 0.671) enrichment of the transcription factor Runx1 and the epigenetic active mark histone H3 acetylation (H3Ac) at the P2X3-R gene promoter in a fraction of the CFA-treated rats. These results suggest that animal-specific increases in P2X3-R mRNA levels are likely associated with the genetic/epigenetic context of the P2X3-R locus that controls P2X3-R gene transcription by recruiting Runx1 and epigenetic co-regulators that mediate histone acetylation. © 2017 Wiley Periodicals, Inc.

Differences in the calcification of preosteoblast cultured on sputter-deposited titanium, zirconium, and gold.

PubMed

Chen, Peng; Nagai, Akiko; Tsutsumi, Yusuke; Ashida, Maki; Doi, Hisashi; Hanawa, Takao

2016-03-01

In this study, osteogenic differentiation and calcification of preosteoblast (MC3T3-E1) cultured on sputter-deposited titanium (Ti), zirconium (Zr), and gold (Au) on cover glasses were evaluated to understand the differences in bone formation ability among these three metals; these metals show the same high corrosion resistance, but Ti and Zr are covered by surface passive oxide film while Au is not covered by the oxide film. Ti and Zr promoted cellular proliferation without osteogenic differentiation. Cells cultured on Ti and Zr expressed higher levels of Runx2, Col1α1, and Akp2 at an earlier stage, which indicated faster promotion of osteogenic differentiation, as compared to those cultured on Au. Moreover, after 21 days of culture, the Bglap1 and Ifitm5 expression peaks in cells cultured on Ti and Zr were higher than those in cells cultured on Au, which indicated faster promotion of calcification. Cells cultured on Ti showed an advantage in osteogenic differentiation at an early stage, while cells on Zr showed better calcification promotion with a long-term culture. The amount of extracellular calcified deposits was in good agreement with the gene expression results. On the other hand, the intracellular calcium content of cells on Au specimens was higher than that of cells on Ti and Zr specimens. The results indicate that preosteoblasts on Ti and Zr showed faster osteogenic differentiation and calcification than those on Au, whereas Au improved the intracellular calcium content. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 639-651, 2016. © 2015 Wiley Periodicals, Inc.

Osteosarcoma cells promote the production of pro-tumor cytokines in mesenchymal stem cells by inhibiting their osteogenic differentiation through the TGF-β/Smad2/3 pathway.

PubMed

Tu, Bing; Peng, Zhao-Xiang; Fan, Qi-Ming; Du, Lin; Yan, Wei; Tang, Ting-Ting

2014-01-01

Mesenchymal stem cells (MSCs) are among the most important components of the osteosarcoma microenvironment and are reported to promote tumor progression. However, the means by which osteosarcoma cells modulate MSC behavior remains unclear. The aim of this study was to determine the effects of osteosarcoma cells on both the production of pro-tumor cytokines by mesenchymal stem cells (MSCs) and the osteogenic differentiation of MSCs. High level of transforming growth factor-β (TGF-β) was detected in three osteosarcoma cell lines. Conditioned media (CM) from the osteosarcoma cell lines Saos-2 and U2-OS were used to stimulate the cultured MSCs. We found that osteosarcoma cells promoted the production of IL-6 and VEGF in MSCs by inhibiting their osteogenic differentiation. Furthermore, TGF-β in tumor CM was proved to be an important factor. The TGF-β neutralizing antibody antagonized the effects induced by osteosarcoma CM. The inhibition of Smad2/3 by siRNA significantly decreased the production of IL-6 and VEGF in MSCs and induced their osteogenic differentiation. We also found that Smad2/3 enhanced the expression of β-catenin in MSCs by decreasing the level of Dickkopf-1 (DKK1). Although the inhibition of β-catenin did not affect the production of IL-6 or VEGF, or the gene expression of the early osteogenic markers Runx2 and ALP, it did enhance the gene expression of osteocalcin. Taken together, our data indicate that osteosarcoma cells secrete TGF-β to maintain the stemness of MSCs and promote the production of pro-tumor cytokines by these cells. © 2013 Published by Elsevier Inc.

Low-level overexpression of p53 promotes warfarin-induced calcification of porcine aortic valve interstitial cells by activating Slug gene transcription.

PubMed

Gao, Li; Ji, Yue; Lu, Yan; Qiu, Ming; Shen, Yejiao; Wang, Yaqing; Kong, Xiangqing; Shao, Yongfeng; Sheng, Yanhui; Sun, Wei

2018-03-09

The most frequently used oral anti-coagulant warfarin has been implicated in inducing calcification of aortic valve interstitial cells (AVICs), whereas the mechanism is not fully understood. The low-level activation of p53 is found to be involved in osteogenic transdifferentiation and calcification of AVICs. Whether p53 participates in warfarin-induced AVIC calcification remains unknown. In this study, we investigated the role of low-level p53 overexpression in warfarin-induced porcine AVIC (pAVIC) calcification. Immunostaining, quantitative PCR, and Western blotting revealed that p53 was expressed in human and pAVICs and that p53 expression was slightly increased in calcific human aortic valves compared with non-calcific valves. Terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling staining indicated that apoptosis slightly increased in calcific aortic valves than in non-calcific valves. Warfarin treatment led to a low-level increase of p53 mRNA and protein in both pAVICs and mouse aortic valves. Low-level overexpression of p53 in pAVICs via an adenovirus vector did not affect pAVIC apoptosis but promoted warfarin-induced calcium deposition and expression of osteogenic markers. shRNA-mediated p53 knockdown attenuated the pAVIC calcium deposition and osteogenic marker expression. Moreover, ChIP and luciferase assays showed that p53 was recruited to the slug promoter and activated slug expression in calcific pAVICs. Of note, overexpression of Slug increased osteogenic marker Runx2 expression, but not pAVIC calcium deposition, and Slug knockdown attenuated pAVIC calcification and p53-mediated pAVIC calcium deposition and expression of osteogenic markers. In conclusion, we found that p53 plays an important role in warfarin induced pAVIC calcification, and increased slug transcription by p53 is required for p53-mediated pAVIC calcification. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Porous titanium and Ti-35Nb alloy: effects on gene expression of osteoblastic cells derived from human alveolar bone.

PubMed

do Prado, Renata Falchete; Rabêlo, Sylvia Bicalho; de Andrade, Dennia Perez; Nascimento, Rodrigo Dias; Henriques, Vinicius André Rodrigues; Carvalho, Yasmin Rodarte; Cairo, Carlos Alberto Alves; de Vasconcellos, Luana Marotta Reis

2015-11-01

Tests on titanium alloys that possess low elastic modulus, corrosion resistance and minimal potential toxicity are ongoing. This study aimed to evaluate the behavior of human osteoblastic cells cultured on dense and porous Titanium (Ti) samples comparing to dense and porous Ti-35 Niobium (Ti-35Nb) samples, using gene expression analysis. Scanning electronic microscopy confirmed surface porosity and pore interconnectivity and X-ray diffraction showed titanium beta-phase stabilization in Ti-35Nb alloy. There were no differences in expression of transforming growth factor-β, integrin-β1, alkaline phosphatase, osteopontin, macrophage colony stimulating factor, prostaglandin E synthase, and apolipoprotein E regarding the type of alloy, porosity and experimental period. The experimental period was a significant factor for the markers: bone sialoprotein II and interleukin 6, with expression increasing over time. Porosity diminished Runt-related transcription factor-2 (Runx-2) expression. Cells adhering to the Ti-35Nb alloy showed statistically similar expression to those adhering to commercially pure Ti grade II, for all the markers tested. In conclusion, the molecular mechanisms of interaction between human osteoblasts and the Ti-35Nb alloy follow the principal routes of osseointegration of commercially pure Ti grade II. Porosity impaired the route of transcription factor Runx-2.

Transcriptional network systems in cartilage development and disease.

PubMed

Nishimura, Riko; Hata, Kenji; Nakamura, Eriko; Murakami, Tomohiko; Takahata, Yoshifumi

2018-04-01

Transcription factors play important roles in the regulation of cartilage development by controlling the expression of chondrogenic genes. Genetic studies have revealed that Sox9/Sox5/Sox6, Runx2/Runx3 and Osterix in particular are essential for the sequential steps of cartilage development. Importantly, these transcription factors form network systems that are also required for appropriate cartilage development. Molecular cloning approaches have largely contributed to the identification of several transcriptional partners for Sox9 and Runx2 during cartilage development. Although the importance of a negative-feedback loop between Indian hedgehog (Ihh) and parathyroid hormone-related protein (PTHrP) in chondrocyte hypertrophy has been well established, recent studies indicate that several transcription factors interact with the Ihh-PTHrP loop and demonstrated that Ihh has multiple functions in the regulation of cartilage development. The most common cartilage disorder, osteoarthritis, has been reported to result from the pathological action of several transcription factors, including Runx2, C/EBPβ and HIF-2α. On the other hand, NFAT family members appear to play roles in the protection of cartilage from osteoarthritis. It is also becoming important to understand the homeostasis and regulation of articular chondrocytes, because they have different cellular and molecular features from chondrocytes of the growth plate. This review summarizes the regulation and roles of transcriptional network systems in cartilage development and their pathological roles in osteoarthritis.

Long noncoding RNA ANCR suppresses bone formation of periodontal ligament stem cells via sponging miRNA-758.

PubMed

Peng, Wei; Deng, Wei; Zhang, Jing; Pei, Gengwang; Rong, Qiong; Zhu, Shuangxi

2018-06-22

Long noncoding RNAs (lncRNAs) were proposed to be important regulators influencing various differentiation processes. Yet, the molecular mechanisms of lncRNAs governing osteogenic differentiation of Periodontal Ligament Stem Cells (PDLSCs) remain unclear. Here, PDLSCs were isolated from normal periodontal ligament of human (PDL) whereas P-PDLSCs were isolated from periodontitis affected PDL. Quantitative real-time PCR (qRT-PCR) was performed to examine the relative expression level of lncRNA-ANCR and of Osterix (OSX), Alkaline Phosphatase (ALP) as well as Runt-related transcription factor 2 (RUNX2) in PDLSCs. Gain- and loss-of- function experiments was performed to study the role of lncRNA-ANCR. Alizarin Red staining was used to evaluate the function of lncRNA-ANCR and miRNA-758 on osteogenic differentiation. In addition, via dual luciferase reporter assay and RNA immunoprecipitation the microRNA sponge potential of lncRNA-ANCR was assessed. A luciferase reporter assay identified the correlation between miR-758 and Notch2. Our results showed that the expression of ALP, RUNX2 and OSX were increased whereas lncRNA-ANCR was decreased during the process of differentiation in PDLSCs. Overexpression of lncRNA-ANCR decreased the expression of ALP, RUNX2 and OSX as confirmed by Alizarin red staining. Overexpression of lncRNA-ANCR resulted in reduction of the miR-758 expression level. Furthermore, RNA immunoprecipitation proved that lncRNA-ANCR targets miR-758 directly. The results of dual luciferase reporter assay also demonstrated that miR-758 regulated Notch2 expression by targeting 3'-UTR of Notch2. In conclusion, the novel pathway lncRNA-ANCR/miR-758/Notch2 plays an important role in the process of regulating osteogenic differentiation of PDLSCs. Copyright © 2018 Elsevier Inc. All rights reserved.

Correlation of beta-catenin localization with cyclooxygenase-2 expression and CpG island methylator phenotype (CIMP) in colorectal cancer.

PubMed

Kawasaki, Takako; Nosho, Katsuhiko; Ohnishi, Mutsuko; Suemoto, Yuko; Kirkner, Gregory J; Dehari, Reiko; Meyerhardt, Jeffrey A; Fuchs, Charles S; Ogino, Shuji

2007-07-01

The WNT/beta-catenin (CTNNB1) pathway is commonly activated in the carcinogenic process. Cross-talks between the WNT and cyclooxygenase-2 (COX-2 or PTGS2)/prostaglandin pathways have been suggested. The relationship between beta-catenin activation and microsatellite instability (MSI) in colorectal cancer has been controversial. The CpG island methylator phenotype (CIMP or CIMP-high) with widespread promoter methylation is a distinct epigenetic phenotype in colorectal cancer, which is associated with MSI-high. However, no study has examined the relationship between beta-catenin activation and CIMP status. Using 832 population-based colorectal cancer specimens, we assessed beta-catenin localization by immunohistochemistry. We quantified DNA methylation in eight CIMP-specific promoters [CACNA1G, CDKN2A(p16), CRABP1, IGF2, MLH1, NEUROG1, RUNX3, and SOCS1] by real-time polymerase chain reaction (MethyLight). MSI-high, CIMP-high, and BRAF mutation were associated inversely with cytoplasmic and nuclear beta-catenin expressions (i.e., beta-catenin activation) and associated positively with membrane expression. The inverse relation between beta-catenin activation and CIMP was independent of MSI. COX-2 overexpression correlated with cytoplasmic beta-catenin expression (even after tumors were stratified by CIMP status), but did not correlate significantly with nuclear or membrane expression. In conclusion, beta-catenin activation is inversely associated with CIMP-high independent of MSI status. Cytoplasmic beta-catenin is associated with COX-2 overexpression, supporting the role of cytoplasmic beta-catenin in stabilizing PTGS2 (COX-2) mRNA.

[Effect of endoplasmic reticulum stress on the expression and osteogenic differentiation of periodontal ligament stem cells].

PubMed

Xue, Peng; Li, Bei; Tan, Jun; An, Ying; Jin, Yan; Wang, Qintao

2015-09-01

To determine the activity of endoplasmic reticulum stress (ERS) and its effect on osteogenic differentiation of periodontal ligament stem cells (PDLSC) in inflammatory microenvironment. PDLSC were obtained from the primary culture of the human tooth and cloned with limited diluted method. Real-time reverse transcription (RT)-PCR was used to examine the different expression of thapsigargin (TG) treated PDLSC and lipopolysaccharide (LPS) treated PDLSC. Real-time RT-PCR, alizarin red staining and cetyl pyridine chloride quantitative analyze were used to examine the osteogenic differentiation of PDLSC, TG + PDLSC, LPS + PDLSC and LPS + PDLSC + 4-PBA. Protein kinase receptor like endoplasmic reticulum kinase (PERK), glucose regulated protein 78 (GRP78), transcription activation factor 4(ATF4), CCAAT/enhancer-binding protein-homologous protein (CHOP) mRNA expression in group PDLSC + TG in 6 h were respectively 1.49 ± 0.24, 2.77 ± 0.60, 1.75 ± 0.16, 2.16 ± 0.32, which were all greater than that in group PDLSC (P < 0.05). PERK, CHOP mRNA expression reached the peak at 6 h (1.76 ± 0.08, 2.31 ± 0.17) and were greater than group PDLSC (P < 0.05). ERS could suppress osteogenic differentiation of TG + PDLSC and LPS + PDLSC. The runt-related transcription factor-2 (RUNX2), alkaline phosphatase (ALP), osteocalcin (OCN) mRNA expression of group TG + PDLSC was respectively 0.73 ± 0.06, 0.01 ± 0.00, 0.20 ± 0.06 (P < 0.05). The RUNX2, ALP, OCN mRNA expression of group LPS + PDLSC was respectively 0.80 ± 0.06, 0.48 ± 0.05, 0.29 ± 0.04 (P < 0.05). The RUNX2, ALP, OCN mRNA expression of group PDLSC + TG + 4-PBA was respectively 1.10 ± 0.09, 0.74 ± 0.05, 0.67 ± 0.13, which were greater higher than that of group LPS + PDLSC (P < 0.05). ERS was activated in PDLSC and suppressed osteogenic differentiation of PDLSC, which can simulate inflammatory microenvironment in vitro. This effect can be recovered by using ERS inhibitor 4-PBA.

Insulin-like growth factor 1 can promote proliferation and osteogenic differentiation of human dental pulp stem cells via mTOR pathway.

PubMed

Feng, Xingmei; Huang, Dan; Lu, Xiaohui; Feng, Guijuan; Xing, Jing; Lu, Jun; Xu, Ke; Xia, Weiwei; Meng, Yan; Tao, Tao; Li, Liren; Gu, Zhifeng

2014-12-01

Insulin-like growth factor 1 (IGF-1) is a multifunctional peptide that can enhance osteogenic differentiation of bone marrow mesenchymal stem cells (BMMSCs). However, it remains unclear whether IGF-1 can promote osteogenic differentiation of human dental pulp stem cells (DPSCs). In our study, DPSCs were isolated from the impacted third molars, and treated with IGF-1. Osteogenic differentiation abilities were investigated. We found that IGF-1 activated the mTOR signaling pathway during osteogenic differentiation of DPSCs. IGF-1 also increased the expression of runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), osterix (OSX) and collagen type I (COL I) during this process. Rapamycin, an mTOR inhibitor, blocked osteogenic differentiation induced by IGF-1. Meanwhile, CCK-8 assay and flow cytometry results demonstrated that 10-200 ng/mL IGF-1 could enhance proliferation ability of DPSCs and 100 ng/mL was the optimal concentration. In summary, IGF-1 could promote proliferation and osteogenic differentiation of DPSCs via mTOR pathways, which might have clinical implications for osteoporosis. © 2014 The Authors Development, Growth & Differentiation © 2014 Japanese Society of Developmental Biologists.

Differentiation of human embryonic stem cells to HOXA+ hemogenic vasculature that resembles the aorta-gonad-mesonephros.

PubMed

Ng, Elizabeth S; Azzola, Lisa; Bruveris, Freya F; Calvanese, Vincenzo; Phipson, Belinda; Vlahos, Katerina; Hirst, Claire; Jokubaitis, Vanta J; Yu, Qing C; Maksimovic, Jovana; Liebscher, Simone; Januar, Vania; Zhang, Zhen; Williams, Brenda; Conscience, Aude; Durnall, Jennifer; Jackson, Steven; Costa, Magdaline; Elliott, David; Haylock, David N; Nilsson, Susan K; Saffery, Richard; Schenke-Layland, Katja; Oshlack, Alicia; Mikkola, Hanna K A; Stanley, Edouard G; Elefanty, Andrew G

2016-11-01

The ability to generate hematopoietic stem cells from human pluripotent cells would enable many biomedical applications. We find that hematopoietic CD34 + cells in spin embryoid bodies derived from human embryonic stem cells (hESCs) lack HOXA expression compared with repopulation-competent human cord blood CD34 + cells, indicating incorrect mesoderm patterning. Using reporter hESC lines to track the endothelial (SOX17) to hematopoietic (RUNX1C) transition that occurs in development, we show that simultaneous modulation of WNT and ACTIVIN signaling yields CD34 + hematopoietic cells with HOXA expression that more closely resembles that of cord blood. The cultures generate a network of aorta-like SOX17 + vessels from which RUNX1C + blood cells emerge, similar to hematopoiesis in the aorta-gonad-mesonephros (AGM). Nascent CD34 + hematopoietic cells and corresponding cells sorted from human AGM show similar expression of cell surface receptors, signaling molecules and transcription factors. Our findings provide an approach to mimic in vitro a key early stage in human hematopoiesis for the generation of AGM-derived hematopoietic lineages from hESCs.

Thymidine phosphorylase exerts complex effects on bone resorption and formation in myeloma

PubMed Central

Liu, Huan; Liu, Zhiqiang; Du, Juan; He, Jin; Lin, Pei; Amini, Behrang; Starbuck, Michael W.; Novane, Nora; Shah, Jatin J.; Davis, Richard E.; Hou, Jian; Gagel, Robert F.; Yang, Jing

2016-01-01

Myelomatous bone disease is characterized by the development of lytic bone lesions and a concomitant reduction in bone formation, leading to chronic bone pain and fractures. To understand the underlying mechanism, we investigated the contribution of myeloma-expressed thymidine phosphorylase (TP) to bone lesions. In osteoblast progenitors, TP upregulated the methylation of RUNX2 and osterix, leading to decreased bone formation. In osteoclast progenitors, TP upregulated the methylation of IRF8, thereby enhanced expression of NFATc1, leading to increased bone resorption. TP reversibly catalyzes thymidine into thymine and 2DDR. Myeloma-secreted 2DDR bound to integrin αVβ3/α5β1 in the progenitors, activated PI3K/Akt signaling, and increased DNMT3A expression, resulting in hypermethylation of RUNX2, osterix, and IRF8. This study elucidates an important mechanism for myeloma-induced bone lesions, suggesting that targeting TP may be a viable approach to healing resorbed bone in patients. As TP overexpression is common in bone-metastatic tumors, our findings could have additional mechanistic implications. PMID:27559096

MicroRNA 665 Regulates Dentinogenesis through MicroRNA-Mediated Silencing and Epigenetic Mechanisms.

PubMed

Heair, Hannah M; Kemper, Austin G; Roy, Bhaskar; Lopes, Helena B; Rashid, Harunur; Clarke, John C; Afreen, Lubana K; Ferraz, Emanuela P; Kim, Eddy; Javed, Amjad; Beloti, Marcio M; MacDougall, Mary; Hassan, Mohammad Q

2015-09-01

Studies of proteins involved in microRNA (miRNA) processing, maturation, and silencing have indicated the importance of miRNAs in skeletogenesis, but the specific miRNAs involved in this process are incompletely defined. Here, we identified miRNA 665 (miR-665) as a potential repressor of odontoblast maturation. Studies with cultured cell lines and primary embryonic cells showed that miR-665 represses the expression of early and late odontoblast marker genes and stage-specific proteases involved in dentin maturation. Notably, miR-665 directly targeted Dlx3 mRNA and decreased Dlx3 expression. Furthermore, RNA-induced silencing complex (RISC) immunoprecipitation and biotin-labeled miR-665 pulldown studies identified Kat6a as another potential target of miR-665. KAT6A interacted physically and functionally with RUNX2, activating tissue-specific promoter activity and prompting odontoblast differentiation. Overexpression of miR-665 reduced the recruitment of KAT6A to Dspp and Dmp1 promoters and prevented KAT6A-induced chromatin remodeling, repressing gene transcription. Taken together, our results provide novel molecular evidence that miR-665 functions in an miRNA-epigenetic regulatory network to control dentinogenesis. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

UnPAKing RUNX3 functions-Both sides of the coin.

PubMed

Kumar, Arun; Sundaram, Sandhya; Rayala, Suresh K; Venkatraman, Ganesh

2017-06-19

Post translational modifications of RUNX3 have been shown to play an important role in directing RUNX3 functions. In this review we highlight the phosphorylation dependent functions of RUNX3 as regulated by PAK1 and its implications on tumorigenesis.

10(-7)  m 17β-oestradiol enhances odonto/osteogenic potency of human dental pulp stem cells by activation of the NF-κB pathway.

PubMed

Wang, Y; Zheng, Y; Wang, Z; Li, J; Wang, Z; Zhang, G; Yu, J

2013-12-01

Oestrogen has been proven to significantly enhance osteogenic potency, while oestrogen deficiency usually leads to impaired osteogenic differentiation of mesenchymal stem cells. However, little is known concerning direct effects of oestrogen on differentiation of human dental pulp stem cells (DPSCs). In this study, human DPSCs were isolated and treated with 10(-7)  m 17β-oestradiol (E2). Alkaline phosphatase (ALP) assay and alizarin red staining were performed. Alkaline phosphatase and alizarin red showed that E2 treatment significantly enhanced ALP activity and mineralization ability of DPSCs, but had no effect on cell proliferation. Real-time RT-PCR and western blot assay demonstrated that odonto/osteogenic markers (ALP, RUNX2/RUNX2, OSX/OSX, OCN/OCN and DSPP/DSP) were significantly upregulated in the cells after E2 treatment. Moreover, phosphorylation of cytoplasmic IκBα/P65 and expression of nuclear P65 were enhanced in a time-dependent manner following E2 treatment, suggesting activation of NF-κB signaling. Conversely, inhibition of the NF-κB pathway suppressed E2-mediated upregulation of odonto/osteogenic markers, indicating that the NF-κB pathway was pivotal for E2-mediated differentiation. These findings provide evidence that 10(-7)  m 17β-oestradiol promoted odonto/osteogenic differentiation of human DPSCs via activation of the NF-κB signaling pathway. © 2013 The Authors. Cell Proliferation published by John Wiley & Sons Ltd.

10−7 m 17β-oestradiol enhances odonto/osteogenic potency of human dental pulp stem cells by activation of the NF-κB pathway

PubMed Central

Wang, Y; Zheng, Y; Wang, Z; Li, J; Wang, Z; Zhang, G; Yu, J

2013-01-01

Objectives Oestrogen has been proven to significantly enhance osteogenic potency, while oestrogen deficiency usually leads to impaired osteogenic differentiation of mesenchymal stem cells. However, little is known concerning direct effects of oestrogen on differentiation of human dental pulp stem cells (DPSCs). Materials and methods In this study, human DPSCs were isolated and treated with 10−7 m 17β-oestradiol (E2). Alkaline phosphatase (ALP) assay and alizarin red staining were performed. Results Alkaline phosphatase and alizarin red showed that E2 treatment significantly enhanced ALP activity and mineralization ability of DPSCs, but had no effect on cell proliferation. Real-time RT-PCR and western blot assay demonstrated that odonto/osteogenic markers (ALP, RUNX2/RUNX2, OSX/OSX, OCN/OCN and DSPP/DSP) were significantly upregulated in the cells after E2 treatment. Moreover, phosphorylation of cytoplasmic IκBα/P65 and expression of nuclear P65 were enhanced in a time-dependent manner following E2 treatment, suggesting activation of NF-κB signaling. Conversely, inhibition of the NF-κB pathway suppressed E2-mediated upregulation of odonto/osteogenic markers, indicating that the NF-κB pathway was pivotal for E2-mediated differentiation. Conclusion These findings provide evidence that 10−7 m 17β-oestradiol promoted odonto/osteogenic differentiation of human DPSCs via activation of the NF-κB signaling pathway. PMID:24152244

Akermanite bioceramics promote osteogenesis, angiogenesis and suppress osteoclastogenesis for osteoporotic bone regeneration

PubMed Central

Xia, Lunguo; Yin, Zhilan; Mao, Lixia; Wang, Xiuhui; Liu, Jiaqiang; Jiang, Xinquan; Zhang, Zhiyuan; Lin, Kaili; Chang, Jiang; Fang, Bing

2016-01-01

It is a big challenge for bone healing under osteoporotic pathological condition with impaired angiogenesis, osteogenesis and remodeling. In the present study, the effect of Ca, Mg, Si containing akermanite bioceramics (Ca2MgSi2O7) extract on cell proliferation, osteogenic differentiation and angiogenic factor expression of BMSCs derived from ovariectomized rats (BMSCs-OVX) as well as the expression of osteoclastogenic factors was evaluated. The results showed that akermanite could enhance cell proliferation, ALP activity, expression of Runx2, BMP-2, BSP, OPN, OCN, OPG and angiogenic factors including VEGF and ANG-1. Meanwhile, akermanite could repress expression of osteoclastogenic factors including RANKL and TNF-α. Moreover, akermanite could activate ERK, P38, AKT and STAT3 signaling pathways, while crosstalk among these signaling pathways was evident. More importantly, the effect of akermanite extract on RANKL-induced osteoclastogenesis was evaluated by TRAP staining and real-time PCR assay. The results showed that akermanite could suppress osteoclast formation and expression of TRAP, cathepsin K and NFATc1. The in vivo experiments revealed that akermanite bioceramics dramatically stimulated osteogenesis and angiogenesis in an OVX rat critical-sized calvarial defect model. All these results suggest that akermanite bioceramics with the effects of Mg and Si ions on osteogenesis, angiogenesis and osteoclastogenesis are promising biomaterials for osteoporotic bone regeneration. PMID:26911441

Tridax procumbens flavonoids promote osteoblast differentiation and bone formation.

PubMed

Al Mamun, Md Abdullah; Hosen, Mohammad Jakir; Islam, Kamrul; Khatun, Amina; Alam, M Masihul; Al-Bari, Md Abdul Alim

2015-11-18

Tridax procumbens flavonoids (TPFs) are well known for their medicinal properties among local natives. Besides traditionally used for dropsy, anemia, arthritis, gout, asthma, ulcer, piles, and urinary problems, it is also used in treating gastric problems, body pain, and rheumatic pains of joints. TPFs have been reported to increase osteogenic functioning in mesenchymal stem cells. Our previous study showed that TPFs were significantly suppressed the RANKL-induced differentiation of osteoclasts and bone resorption. However, the effects of TPFs to promote osteoblasts differentiation and bone formation remain unclear. TPFs were isolated from Tridax procumbens and investigated for their effects on osteoblasts differentiation and bone formation by using primary mouse calvarial osteoblasts. TPFs promoted osteoblast differentiation in a dose-dependent manner demonstrated by up-regulation of alkaline phosphatase and osteocalcin. TPFs also upregulated osteoblast differentiation related genes, including osteocalcin, osterix, and Runx2 in primary osteoblasts. TPFs treated primary osteoblast cells showed significant upregulation of bone morphogenetic proteins (BMPs) including Bmp-2, Bmp-4, and Bmp-7. Addition of noggin, a BMP specific-antagonist, inhibited TPFs induced upregulation of the osteocalcin, osterix, and Runx2. Our findings point towards the induction of osteoblast differentiation by TPFs and suggested that TPFs could be a potential anabolic agent to treat patients with bone loss-associated diseases such as osteoporosis.

Correlation between Gene Expression and Osteoarthritis Progression in Human.

PubMed

Zhong, Leilei; Huang, Xiaobin; Karperien, Marcel; Post, Janine N

2016-07-14

Osteoarthritis (OA) is a multifactorial disease characterized by gradual degradation of joint cartilage. This study aimed to quantify major pathogenetic factors during OA progression in human cartilage. Cartilage specimens were isolated from OA patients and scored 0-5 according to the Osteoarthritis Research Society International (OARSI) guidelines. Protein and gene expressions were measured by immunohistochemistry and qPCR, respectively. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays were used to detect apoptotic cells. Cartilage degeneration in OA is a gradual progress accompanied with gradual loss of collagen type II and a gradual decrease in mRNA expression of SOX9, ACAN and COL2A1. Expression of WNT antagonists DKK1 and FRZB was lost, while hypertrophic markers (RUNX2, COL10A1 and IHH) increased during OA progression. Moreover, DKK1 and FRZB negatively correlated with OA grading, while RUNX2 and IHH showed a significantly positive correlation with OA grading. The number of apoptotic cells was increased with the severity of OA. Taken together, our results suggested that genetic profiling of the gene expression could be used as markers for staging OA at the molecular level. This helps to understand the molecular pathology of OA and may lead to the development of therapies based on OA stage.

Second generation sequencing of microRNA in Human Bone Cells treated with Parathyroid Hormone or Dexamethasone.

PubMed

Laxman, Navya; Rubin, Carl-Johan; Mallmin, Hans; Nilsson, Olle; Tellgren-Roth, Christian; Kindmark, Andreas

2016-03-01

We investigated the impact of treatment with parathyroid hormone (PTH) and dexamethasone (DEX) for 2 and 24h by RNA sequencing of miRNAs in primary human bone (HOB) cells. A total of 207 million reads were obtained, and normalized absolute expression retrieved for 373 most abundant miRNAs. In naïve control cells, 7 miRNAs were differentially expressed (FDR<0.05) between the two time points. Ten miRNAs exhibited differential expression (FDR <0.05) across two time points and treatments after adjusting for expression in controls and were selected for downstream analyses. Results show significant effects on miRNA expression when comparing PTH with DEX at 2h with even more pronounced effects at 24h. Interestingly, several miRNAs exhibiting differences in expression are predicted to target genes involved in bone metabolism e.g. miR-30c2, miR-203 and miR-205 targeting RUNX2, and miR-320 targeting β-catenin (CTNNB1) mRNA expression. CTNNB1and RUNX2 levels were decreased after DEX treatment and increased after PTH treatment. Our analysis also identified 2 putative novel miRNAs in PTH and DEX treated cells at 24h. RNA sequencing showed that PTH and DEX treatment affect miRNA expression in HOB cells and that regulated miRNAs in turn are correlated with expression levels of key genes involved in bone metabolism. Copyright © 2016 Elsevier Inc. All rights reserved.

Stem cell exhaustion due to Runx1 deficiency is prevented by Evi5 activation in leukemogenesis

PubMed Central

Jacob, Bindya; Yamashita, Namiko; Wang, Chelsia Qiuxia; Taniuchi, Ichiro; Littman, Dan R.; Asou, Norio

2010-01-01

The RUNX1/AML1 gene is the most frequently mutated gene in human leukemia. Conditional deletion of Runx1 in adult mice results in an increase of hematopoietic stem cells (HSCs), which serve as target cells for leukemia; however, Runx1−/− mice do not develop spontaneous leukemia. Here we show that maintenance of Runx1−/− HSCs is compromised, progressively resulting in HSC exhaustion. In leukemia development, the stem cell exhaustion was rescued by additional genetic changes. Retroviral insertional mutagenesis revealed Evi5 activation as a cooperating genetic alteration and EVI5 overexpression indeed prevented Runx1−/− HSC exhaustion in mice. Moreover, EVI5 was frequently overexpressed in human RUNX1-related leukemias. These results provide insights into the mechanism for maintenance of pre-leukemic stem cells and may provide a novel direction for therapeutic applications. PMID:20008790

TCDD disrupts posterior palatogenesis and causes cleft palate.

PubMed

Yamada, Tomohiro; Hirata, Azumi; Sasabe, Eri; Yoshimura, Tomohide; Ohno, Seiji; Kitamura, Naoya; Yamamoto, Tetsuya

2014-01-01

Dioxins (e.g. 2,3,7,8-tetrachlorodibenzo-p-dioxin; TCDD) cause cleft palate at a high rate. A post-fusional split may contribute to the pathogenesis, and tissue fragility may be a concern. The objective of this study was to investigate the effects of TCDD on the palatal epithelium, bone and muscle, which contribute to tissue integrity. ICR mice (10-12 weeks old) were used. TCDD was administered on E12.5 at 40 mg/kg. Immunohistochemical staining for AhR, ER-α, laminin, collagen IV, osteopontin, Runx2, MyoD, and desmin were performed. Furthermore, western blot analysis for osteopontin, Runx2, MyoD, and desmin were performed to evaluate protein expression in the palatal tissue. Immunohistologically, there was little difference in the collagen IV and laminin localization in the palatal epithelium between control versus TCDD-treated mice. Runx2 and osteopontin immunoreactivity decreased in the TCDD-treated palatal bone, and MyoD and desmin decreased in the TCDD-treated palatal muscle. AhR and ER-α immunoreactivity were localized to the normal palatal bone, but ER-α was diminished in the TCDD-treated palate. On western blot analysis, Runx2, MyoD, and desmin were all downregulated in the TCDD-treated palate. TCDD may suppress palatal osteogenesis and myogenesis via AhR, and cause cleft palates via a post-fusional split mechanism, in addition to a failure of palatal fusion. Copyright © 2013 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.

Tet2 Regulates Osteoclast Differentiation by Interacting with Runx1 and Maintaining Genomic 5-Hydroxymethylcytosine (5hmC).

PubMed

Chu, Yajing; Zhao, Zhigang; Wayne Sant, David; Zhu, Ganqian; Greenblatt, Sarah M; Liu, Lin; Wang, Jinhuan; Cao, Zeng; Cheng Tho, Jeanette; Chen, Shi; Liu, Xiaochen; Zhang, Peng; Maciejewski, Jaroslaw P; Nimer, Stephen; Wang, Gaofeng; Yuan, Weiping; Yang, Feng-Chun; Xu, Mingjiang

2018-06-13

As a dioxygenase, Ten-eleven translocation 2 (TET2) catalyzes subsequent steps of 5-methylcytosine (5mC) oxidation. Tet2 plays a critical role in the self-renewal, proliferation, and differentiation of hematopoietic stem cells, but its impact on mature hematopoietic cells is not well-characterized. Here we show that Tet2 plays an essential role in osteoclastogenesis. Deletion of Tet2 impairs the differentiation of osteoclast precursor cells (macrophages) and their maturation into bone-resorbing osteoclasts in vitro. Furthermore, Tet2 -/- mice exhibit mild osteopetrosis, accompanied by decreased number of osteoclasts in vivo. Tet2 loss in macrophages results in the altered expression of a set of genes implicated in osteoclast differentiation, such as Cebpa, Mafb, and Nfkbiz. Tet2 deletion also leads to a genome-wide alteration in the level of 5-hydroxymethylcytosine (5hmC) and altered expression of a specific subset of macrophage genes associated with osteoclast differentiation. Furthermore, Tet2 interacts with Runx1 and negatively modulates its transcriptional activity. Our studies demonstrate a novel molecular mechanism controlling osteoclast differentiation and function by Tet2, that is, through interactions with Runx1 and the maintenance of genomic 5hmC. Targeting Tet2 and its pathway could be a potential therapeutic strategy for the prevention and treatment of abnormal bone mass caused by the deregulation of osteoclast activities. Copyright © 2018. Production and hosting by Elsevier B.V.

Mathematical model of a gene regulatory network reconciles effects of genetic perturbations on hematopoietic stem cell emergence.

PubMed

Narula, Jatin; Williams, C J; Tiwari, Abhinav; Marks-Bluth, Jonathon; Pimanda, John E; Igoshin, Oleg A

2013-07-15

Interlinked gene regulatory networks (GRNs) are vital for the spatial and temporal control of gene expression during development. The hematopoietic transcription factors (TFs) Scl, Gata2 and Fli1 form one such densely connected GRN which acts as a master regulator of embryonic hematopoiesis. This triad has been shown to direct the specification of the hemogenic endothelium and emergence of hematopoietic stem cells (HSCs) in response to Notch1 and Bmp4-Smad signaling. Here we employ previously published data to construct a mathematical model of this GRN network and use this model to systematically investigate the network dynamical properties. Our model uses a statistical-thermodynamic framework to describe the combinatorial regulation of gene expression and reconciles, mechanistically, several previously published but unexplained results from different genetic perturbation experiments. In particular, our results demonstrate how the interactions of Runx1, an essential hematopoietic TF, with components of the Bmp4 signaling pathway allow it to affect triad activation and acts as a key regulator of HSC emergence. We also explain why heterozygous deletion of this essential TF, Runx1, speeds up the network dynamics leading to accelerated HSC emergence. Taken together our results demonstrate that the triad, a master-level controller of definitive hematopoiesis, is an irreversible bistable switch whose dynamical properties are modulated by Runx1 and components of the Bmp4 signaling pathway. Copyright © 2013 Elsevier Inc. All rights reserved.

RUNX1 mutations in acute myeloid leukemia are associated with distinct clinico-pathologic and genetic features.

PubMed

Gaidzik, V I; Teleanu, V; Papaemmanuil, E; Weber, D; Paschka, P; Hahn, J; Wallrabenstein, T; Kolbinger, B; Köhne, C H; Horst, H A; Brossart, P; Held, G; Kündgen, A; Ringhoffer, M; Götze, K; Rummel, M; Gerstung, M; Campbell, P; Kraus, J M; Kestler, H A; Thol, F; Heuser, M; Schlegelberger, B; Ganser, A; Bullinger, L; Schlenk, R F; Döhner, K; Döhner, H

2016-11-01

We evaluated the frequency, genetic architecture, clinico-pathologic features and prognostic impact of RUNX1 mutations in 2439 adult patients with newly-diagnosed acute myeloid leukemia (AML). RUNX1 mutations were found in 245 of 2439 (10%) patients; were almost mutually exclusive of AML with recurrent genetic abnormalities; and they co-occurred with a complex pattern of gene mutations, frequently involving mutations in epigenetic modifiers (ASXL1, IDH2, KMT2A, EZH2), components of the spliceosome complex (SRSF2, SF3B1) and STAG2, PHF6, BCOR. RUNX1 mutations were associated with older age (16-59 years: 8.5%; ⩾60 years: 15.1%), male gender, more immature morphology and secondary AML evolving from myelodysplastic syndrome. In univariable analyses, RUNX1 mutations were associated with inferior event-free (EFS, P<0.0001), relapse-free (RFS, P=0.0007) and overall survival (OS, P<0.0001) in all patients, remaining significant when age was considered. In multivariable analysis, RUNX1 mutations predicted for inferior EFS (P=0.01). The effect of co-mutation varied by partner gene, where patients with the secondary genotypes RUNX1 mut /ASXL1 mut (OS, P=0.004), RUNX1 mut /SRSF2 mut (OS, P=0.007) and RUNX1 mut /PHF6 mut (OS, P=0.03) did significantly worse, whereas patients with the genotype RUNX1 mut /IDH2 mut (OS, P=0.04) had a better outcome. In conclusion, RUNX1-mutated AML is associated with a complex mutation cluster and is correlated with distinct clinico-pathologic features and inferior prognosis.

Evaluation of the Effects of Photobiomodulation on Partial Osteotomy in Streptozotocin-Induced Diabetes in Rats.

PubMed

Mostafavinia, Ataroalsadat; Masteri Farahani, Reza; Abdollahifar, Mohammad-Amin; Ghatrehsamani, Mahdi; Ghoreishi, Seyed Kamran; Hajihossainlou, Behnam; Chien, Sufan; Dadras, Sara; Rezaei, Fatemehalsadat; Bayat, Mohammad

2018-05-31

We examined the effects of photobiomodulation (PBM) on stereological parameters, and gene expression of Runt-related transcription factor 2 (RUNX2), osteocalcin, and receptor activator of nuclear factor kappa-B ligand (RANKL) in repairing tissue of tibial bone defect in streptozotocin (STZ)-induced type 1 diabetes mellitus (TIDM) in rats during catabolic response of fracture healing. There were conflicting results regarding the efficacy of PBM on bone healing process in healthy and diabetic animals. Forty-eight rats have been distributed into four groups: group 1 (healthy control, no TIDM and no PBM), group 2 (healthy test, no TIDM and PBM), group 3 (diabetic control, TIDM and no PBM), and group 4 (diabetic test, no TIDM and PBM). TIDM was induced in the groups 3 and 4. A partial bone defect in tibia was made in all groups. The bone defects of groups second and fourth were irradiated by a laser (890 nm, 80 Hz, 1.5 J/cm 2 ). Thirty days after the surgery, all bone defects were extracted and were submitted to stereological examination and real-time polymerase chain reaction (RT-PCR). PBM significantly increased volumes of total callus, total bone, bone marrow, trabecular bone, and cortical bone, and the numbers of osteocytes and osteoblasts of callus in TIDM rats compared to those of callus in diabetic control. In addition, TIDM increased RUNX2, and osteocalcin in callus of tibial bone defect compared to healthy group. PBM significantly decreased osteocalcin gene expression in TIDM rats. PBM significantly increased many stereological parameters of bone repair in an STZ-induced TIDM during catabolic response of fracture healing. Further RT-PCR test demonstrated that bone repair was modulated in diabetic rats during catabolic response of fracture healing by significant increase in mRNA expression of RUNX2, and osteocalcin compared to healthy control rats. PBM also decreased osteocalcin mRNA expression in TIDM rats.

The Role of Epigenetic Regulation in Epstein-Barr Virus-Associated Gastric Cancer

PubMed Central

Nishikawa, Jun; Iizasa, Hisashi; Nakamura, Munetaka; Saito, Mari; Sasaki, Sho; Shimokuri, Kanami; Yanagihara, Masashi; Sakai, Kouhei; Suehiro, Yutaka; Yamasaki, Takahiro; Sakaida, Isao

2017-01-01

The Epstein–Barr virus (EBV) is detected in about 10% of gastric carcinoma cases throughout the world. In EBV-associated gastric carcinoma (EBVaGC), all tumor cells harbor the clonal EBV genome. The expression of latent EBV genes is strictly regulated through the methylation of EBV DNA. The methylation of viral DNA regulates the type of EBV latency, and methylation of the tumor suppressor genes is a key abnormality in EBVaGC. The methylation frequencies of several tumor suppressor genes and cell adhesion molecules are significantly higher in EBVaGC than in control cases. EBV-derived microRNAs repress translation from viral and host mRNAs. EBV regulates the expression of non-coding RNA in gastric carcinoma. With regard to the clinical application of demethylating agents against EBVaGC, we investigated the effects of decitabine against the EBVaGC cell lines. Decitabine inhibited the cell growth of EBVaGC cells. The promoter regions of p73 and Runt-related transcription factor 3(RUNX3) were demethylated, and their expression was upregulated by the treatment. We review the role of epigenetic regulation in the development and maintenance of EBVaGC and discuss the therapeutic application of DNA demethylating agents for EBVaGC. PMID:28757548

The Role of Epigenetic Regulation in Epstein-Barr Virus-Associated Gastric Cancer.

PubMed

Nishikawa, Jun; Iizasa, Hisashi; Yoshiyama, Hironori; Nakamura, Munetaka; Saito, Mari; Sasaki, Sho; Shimokuri, Kanami; Yanagihara, Masashi; Sakai, Kouhei; Suehiro, Yutaka; Yamasaki, Takahiro; Sakaida, Isao

2017-07-25

The Epstein-Barr virus (EBV) is detected in about 10% of gastric carcinoma cases throughout the world. In EBV-associated gastric carcinoma (EBVaGC), all tumor cells harbor the clonal EBV genome. The expression of latent EBV genes is strictly regulated through the methylation of EBV DNA. The methylation of viral DNA regulates the type of EBV latency, and methylation of the tumor suppressor genes is a key abnormality in EBVaGC. The methylation frequencies of several tumor suppressor genes and cell adhesion molecules are significantly higher in EBVaGC than in control cases. EBV-derived microRNAs repress translation from viral and host mRNAs. EBV regulates the expression of non-coding RNA in gastric carcinoma. With regard to the clinical application of demethylating agents against EBVaGC, we investigated the effects of decitabine against the EBVaGC cell lines. Decitabine inhibited the cell growth of EBVaGC cells. The promoter regions of p73 and Runt-related transcription factor 3(RUNX3) were demethylated, and their expression was upregulated by the treatment. We review the role of epigenetic regulation in the development and maintenance of EBVaGC and discuss the therapeutic application of DNA demethylating agents for EBVaGC.

Histone deacetylase 3 is required for maintenance of bone mass during aging

PubMed Central

McGee-Lawrence, Meghan E.; Bradley, Elizabeth W.; Dudakovic, Amel; Carlson, Samuel W.; Ryan, Zachary C.; Kumar, Rajiv; Dadsetan, Mahrokh; Yaszemski, Michael J.; Chen, Qingshan; An, Kai-Nan; Westendorf, Jennifer J.

2012-01-01

Histone deacetylase 3 (Hdac3) is a nuclear enzyme that removes acetyl groups from lysine residues in histones and other proteins to epigenetically regulate gene expression. Hdac3 interacts with bone-related transcription factors and co-factors such as Runx2 and Zfp521, and thus is poised to play a key role in the skeletal system. To understand the role of Hdac3 in osteoblasts and osteocytes, Hdac3 conditional knockout (CKO) mice were created with the Osteocalcin (OCN) promoter driving Cre expression. Hdac3 CKOOCN mice were of normal size and weight, but progressively lost trabecular and cortical bone mass with age. The Hdac3 CKOOCN mice exhibited reduced cortical bone mineralization and material properties and suffered frequent fractures. Bone resorption was lower, not higher, in the Hdac3 CKOOCN mice, suggesting that primary defects in osteoblasts caused the reduced bone mass. Indeed, reductions in bone formation were observed. Osteoblasts and osteocytes from Hdac3 CKOOCN mice showed increased DNA damage and reduced functional activity in vivo and in vitro. Thus, Hdac3 expression in osteoblasts and osteocytes is essential for bone maintenance during aging. PMID:23085085

Extracellular Purines Promote the Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells to the Osteogenic and Adipogenic Lineages

PubMed Central

Zini, Roberta; Rossi, Lara; Salvestrini, Valentina; Ferrari, Davide; Manfredini, Rossella; Lemoli, Roberto M.

2013-01-01

Extracellular nucleotides are potent signaling molecules mediating cell-specific biological functions, mostly within the processes of tissue damage and repair and flogosis. We previously demonstrated that adenosine 5′-triphosphate (ATP) inhibits the proliferation of human bone marrow-derived mesenchymal stem cells (BM-hMSCs), while stimulating, in vitro and in vivo, their migration. Here, we investigated the effects of ATP on BM-hMSC differentiation capacity. Molecular analysis showed that ATP treatment modulated the expression of several genes governing adipogenic and osteoblastic (ie, WNT-pathway-related genes) differentiation of MSCs. Functional studies demonstrated that ATP, under specific culture conditions, stimulated adipogenesis by significantly increasing the lipid accumulation and the expression levels of the adipogenic master gene PPARγ (peroxisome proliferator-activated receptor-gamma). In addition, ATP stimulated osteogenic differentiation by promoting mineralization and expression of the osteoblast-related gene RUNX2 (runt-related transcription factor 2). Furthermore, we demonstrated that ATP stimulated adipogenesis via its triphosphate form, while osteogenic differentiation was induced by the nucleoside adenosine, resulting from ATP degradation induced by CD39 and CD73 ectonucleotidases expressed on the MSC membrane. The pharmacological profile of P2 purinergic receptors (P2Rs) suggests that adipogenic differentiation is mainly mediated by the engagement of P2Y1 and P2Y4 receptors, while stimulation of the P1R adenosine-specific subtype A2B is involved in adenosine-induced osteogenic differentiation. Thus, we provide new insights into molecular regulation of MSC differentiation. PMID:23259837

Epstein-Barr virus exploits intrinsic B-lymphocyte transcription programs to achieve immortal cell growth.

PubMed

Zhao, Bo; Zou, James; Wang, Hongfang; Johannsen, Eric; Peng, Chih-wen; Quackenbush, John; Mar, Jessica C; Morton, Cynthia Casson; Freedman, Matthew L; Blacklow, Stephen C; Aster, Jon C; Bernstein, Bradley E; Kieff, Elliott

2011-09-06

Epstein-Barr virus nuclear antigen 2 (EBNA2) regulation of transcription through the cell transcription factor RBPJ is essential for resting B-lymphocyte (RBL) conversion to immortal lymphoblast cell lines (LCLs). ChIP-seq of EBNA2 and RBPJ sites in LCL DNA found EBNA2 at 5,151 and RBPJ at 10,529 sites. EBNA2 sites were enriched for RBPJ (78%), early B-cell factor (EBF, 39%), RUNX (43%), ETS (39%), NFκB (22%), and PU.1 (22%) motifs. These motif associations were confirmed by LCL RBPJ ChIP-seq finding 72% RBPJ occupancy and Encyclopedia Of DNA Elements LCL ChIP-seq finding EBF, NFκB RELA, and PU.1 at 54%, 31%, and 17% of EBNA2 sites. EBNA2 and RBPJ were predominantly at intergene and intron sites and only 14% at promoter sites. K-means clustering of EBNA2 site transcription factors identified RELA-ETS, EBF-RUNX, EBF, ETS, RBPJ, and repressive RUNX clusters, which ranked from highest to lowest in H3K4me1 signals and nucleosome depletion, indicative of active chromatin. Surprisingly, although quantitatively less, the same genome sites in RBLs exhibited similar high-level H3K4me1 signals and nucleosome depletion. The EBV genome also had an LMP1 promoter EBF site, which proved critical for EBNA2 activation. LCL HiC data mapped intergenic EBNA2 sites to EBNA2 up-regulated genes. FISH and chromatin conformation capture linked EBNA2/RBPJ enhancers 428 kb 5' of MYC to MYC. These data indicate that EBNA2 evolved to target RBL H3K4me1 modified, nucleosome-depleted, nonpromoter sites to drive B-lymphocyte proliferation in primary human infection. The primed RBL program likely supports antigen-induced proliferation.

Fabrication and evaluation of osteoblastic differentiation of human mesenchymal stem cells on novel CaO-SiO2-P2O5-B2O3 glass-ceramics.

PubMed

Lee, Jae Hyup; Seo, Jun-Hyuk; Lee, Kyung Mee; Ryu, Hyun-Seung; Baek, Hae-Ri

2013-07-01

Apatite-wollastonite glass-ceramics have high mechanical strength, and CaO-SiO2 -B2 O3 glass-ceramics showed excellent bioactivity and high biodegradability. A new type of CaO-SiO2 -P2 O5 -B2 O3 system of bioactive glass-ceramics (BGS-7) was fabricated, and the effect and usefulness was evaluated via bioactivity using simulated body fluid and human mesenchymal stem cells (hMSCs). The purpose of this study was to compare BGS-7 and hydroxyapatite (HA) using hMSCs in order to evaluate the bioactivity of BGS-7 and its possibility as a bone graft extender. Alkaline phosphatase (ALP) staining, ALP activity, cell proliferation 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay, Alizarin Red-S (AR-S) staining, calcium levels, the mRNA expression of ALP, osteocalcin, osteopontin, and runt-related transcription factor 2 (runx-2) using reverse-transcription polymerase chain reaction (RT-PCR) and the protein expression of osteocalcin and runx-2 using Western blot were measured by transplanting hMSC onto a tissue culture plate, HA, and BGS-7. The ALP staining and AR-S staining of BGS-7 was greater than that of HA and control. The ALP value of BGS-7 was significantly higher than that of HA and control. The MTS results showed that BGS-7 had a higher value than the groups transplanted onto HA and control on day 15. The calcium level was higher than the control in both HA and BGS-7, and was especially high in BGS-7. There were more mineral products on BGS-7 than on the HA when analyzed by scanning electron microscopy. The mRNA expression of ALP, osteopontin, osteocalcin, and runx-2 were higher on BGS-7 than on HA and the control when analyzed by RT-PCR. The relative gene expression of osteopontin and runx-2 were found to be higher on BGS-7 than on HA and the control by Western blot. Accordingly, it is predicted that BGS-7 would have high biocompatibility and good osteoconductivity, and presents a possibility as a new bone graft extender. © 2013, Copyright the Authors. Artificial Organs © 2013, International Center for Artificial Organs and Transplantation and Wiley Periodicals, Inc.

[Regulation effects of liuwei dihuang pill, jingui shenqi pill, jiangu erxian pill containing serums on adipogenic and osteogenic differentiation-related genes expressions in the differentiation process of preadipocytes to osteoblasts].

PubMed

Cheng, Zhi-An; Han, Ling; Wei, Jian-An; Sun, Jing; Duan, Xiao-Dong

2013-02-01

To study the effects of Chinese medical recipes for invigorating Shen on rat bone marrow mesenchymal stem cells (BMSCs)-derived preadipocytes' differentiation to osteoblasts. The BMSCs were cultured using whole bone marrow adherence wall method. The BMSCs were induced to preadipocytes by classic chemical method. The osteogenic differentiation process of preadipocytes was intervened by Liuwei Dihuang Pill (LDP), Jingui Shenqi Pill (JSP), or Jiangu Erxian Pill (JEP)-containing serums (with the concentRation of 10%, on behalf of tonifying Shen yin, tonifying Shen yang, and tonifying Shen essence). Reverse transcription-real time fluorescent quantitative-PCR (RT real time qPCR) was used to detect RUNX2, ALP, BGP, BMP2, BMP4, SPP1, and IGF1 mRNA expressions of osteogenic differentiation-related genes, mRNA expressions of LPL, FABP4, and PPARgamma of adipogenic differentiation-related genes on the 6th, the 12th, and the 18th day. As for the osteogenic differentiation-related gene, when compared with the control group, there was no statistical difference in the gene expression level in the experimental groups on the 6th day (2.0 > Ratio > 0.5). On the 12th day, the mRNA expressions of IGF1 and Runx2 increased more significantly in the JSP group, with their relative quantification (Ratio) being 2.97 and 1.81 respectively. On the 18th day the IGF1 mRNA expression significantly increased, being the Ratio value of 3.74, 12.60, and 8.35, respectively, in the LDP group, the JSP group, and the JEP group. The SPP1 mRNA expression also significantly increased, with the Ratio value of 2.94, 3.18, and 2.62, respectively, in the LDP group, the JSP group, and the JEP group. As for adipogenic differentiation-related genes, on the 6th day, when compared with the control group, FABP4 mRNA expression significantly decreased in the LDP group and the JSP group (with the Ratio value of 0.47 and 0.40 respectively). The expression levels of other genes were all down-regulated, but not significantly. On the 12th day and 18th day, there was no statistical change in the adipogenic differentiation-related genes expressions (2.0 > Ratio > 0.5). Up-regulation of osteogenic differentiation-related genes expression occurred in later time, while down-regulation of adipogenic differentiation-related genes expression occurred in earlier time after treatment by Chinese medical recipes for invigorating Shen. In general, above data indicated that tonifying Shen yang was more effective in promoting osteogenic differentiation and inhibiting adipogenic differentiation of BMSCs.

Direct comparison of regulators of calcification between bone and vessels in humans.

PubMed

Schweighofer, N; Aigelsreiter, A; Trummer, O; Graf-Rechberger, M; Hacker, N; Kniepeiss, D; Wagner, D; Stiegler, P; Trummer, C; Pieber, T; Obermayer-Pietsch, B; Müller, H

2016-07-01

Calcification is not only physiologically present in bone but is a main pathophysiological process in vasculature, favouring cardiovascular diseases. Our aim was to investigate changes in the expression of calcification regulators during vascular calcification in bone and vasculature. Levels of gene expression of osteoprotegerin (OPG), receptor activator of NF-κB ligand (RANKL), osteopontin (OPN), matrix gla protein (MGP), bone sialoprotein (BSP), SMAD6, and runt-related transcription factor 2 (RUNX2) were determined in bone, aorta, and external iliac artery tissue samples of transplant donors. Histological stages of atherosclerosis (AS) in vessels are defined as "no changes", "intima thickening", or "intima calcification". Patients' bone samples were subgrouped accordingly. We demonstrate that in vessels BSP and OPN expression significantly increased during intima thickening and decreased during intima calcification, whereas the expression of regulators of calcification did not significantly change in bone during intima thickening and intima calcification. At the stage of intima thickening, MGP, OPG, and SMAD6 expression and at stage of intima calcification only MGP expression was lower in bone than in vessel. The expression of BSP and RANKL was regulated in opposite ways in bone and vessels, whereas the expression of MGP, OC, RUNX2, and OPN was regulated in a tissue-specific manner. Our study is the first direct comparison of gene expression changes during AS progression in bone and vessels. Our results indicate that changes in the expression of regulators of calcification in the vessel wall as well as in bone occur early in the calcification process, even prior to deposition of calcium/phosphate precipitation. Copyright © 2016. Published by Elsevier Inc.

TGF-beta1 modulates focal adhesion kinase expression in rat intestinal epithelial IEC-6 cells via stimulatory and inhibitory Smad binding elements.

PubMed

Walsh, Mary F; Ampasala, Dinakar R; Rishi, Arun K; Basson, Marc D

2009-02-01

TGF-beta and FAK modulate cell migration, differentiation, proliferation and apoptosis, and TGF-beta promotes FAK transcription in intestinal epithelial cells via Smad-dependent and independent pathways. We utilized a 1320 bp FAK promoter-luciferase construct to characterize basal and TGF-beta-mediated FAK gene transcription in IEC-6 cells. Inhibiting JNK or Akt negated TGF-beta-stimulated promoter activity; ERK inhibition did not block the TGF-beta effect but increased basal activity. Co-transfection with Co-Smad4 enhanced the TGF-beta response while the inhibitory Smad7 abolished it. Serial deletions sequentially removing the four Smad binding elements (SBE) in the 5' untranslated region of the promoter revealed that the two most distal SBE's are positive regulators while SBE3 exerts a negative influence. Mutational deletion of two upstream p53 sites enhanced basal but did not affect TGF-beta-stimulated increases in promoter activity. TGF-beta increased DNA binding of Smad4, phospho-Smad2/3 and Runx1/AML1a to the most distal 435 bp containing 3 SBE and 2 AML1a sites by ChIP assay. However, although point mutation of SBE1 ablated the TGF-beta-mediated rise in SV40-promoter activity, mutation of AML1a sites did not. TGF-beta regulation of FAK transcription reflects a complex interplay between positive and negative non-Smad signals and SBE's, the last independent of p53 or AML1a.

Phosphatase of regenerating liver-3 is expressed in acute lymphoblastic leukemia and mediates leukemic cell adhesion, migration and drug resistance

PubMed Central

Hjort, Magnus A.; Abdollahi, Pegah; Vandsemb, Esten N.; Fenstad, Mona H.; Lund, Bendik; Slørdahl, Tobias S.; Børset, Magne; Rø, Torstein B.

2018-01-01

Phosphatase of regenerating liver-3 (PRL-3/PTP4A3) is upregulated in multiple cancers, including BCR-ABL1- and ETV6-RUNX-positive acute lymphoblastic leukemia (ALL). With this study, we aim to characterize the biological role of PRL-3 in B cell ALL (B-ALL). Here, we demonstrate that PRL-3 expression at mRNA and protein level was higher in B-ALL cells than in normal cells, as measured by qRT-PCR or flow cytometry. Further, we demonstrate that inhibition of PRL-3 using shRNA or a small molecular inhibitor reduced cell migration towards an SDF-1α gradient in the preB-ALL cell lines Reh and MHH-CALL-4. Knockdown of PRL-3 also reduced cell adhesion towards fibronectin in Reh cells. Mechanistically, PRL-3 mediated SDF-1α stimulated calcium release, and activated focal adhesion kinase (FAK) and Src, important effectors of migration and adhesion. Finally, PRL-3 expression made Reh cells more resistance to cytarabine treatment. In conclusion, the expression level of PRL-3 was higher in B-ALL cells than in normal cells. PRL-3 promoted adhesion, migration and resistance to cytarabine. PRL-3 may represent a novel target in the treatment of B-ALL. PMID:29423065

RUNX1 Amplification Increases the Risk for Thrombosis in Children With B-cell Acute Lymphoblastic Leukemia.

PubMed

Boucher, Maria O; Smitherman, Andrew B; Pahl, Kristy S; Rao, Kathleen W; Deal, Allison M; Blatt, Julie

2016-04-01

RUNX1 (AML1) amplification in patients with B-cell acute lymphoblastic leukemia (B-ALL) has been associated with poor survival for unclear reasons. Our anecdotal experience suggests that children with B-ALL and RUNX1 amplification might be predisposed to thrombosis. We performed a retrospective cohort study of children with B-ALL treated from 2008 to 2014 at the North Carolina Children's Hospital. Patient demographics, cytogenetics, and diagnosis of thrombosis were extracted by blinded chart review. Analysis was performed examining the relationship between RUNX1 amplification and thrombosis. We identified 119 patients with B-ALL and a median age of 4.9 years (interquartile range, 2.9 to 8.6 y) at diagnosis. Four patients (3%) had RUNX1 amplification. The average number of RUNX1 copies among those with amplification was 5 (SD 0.81 [range, 4 to 6]). Eighteen thromboses were diagnosed within 6 months of starting treatment. These events were more likely among patients with RUNX1 amplification than in patients without amplification (75% vs. 13%; RR 5.75, 95% confidence interval, 2.75-12.01). RUNX1 amplification may predispose to early thrombotic events in children with B-ALL which could, in part, contribute to their poorer outcomes. Treatment implications, including possible prophylactic anticoagulation of patients with of RUNX1 amplification, justify larger studies to confirm these findings.

Effects of titanium surface anodization with CaP incorporation on human osteoblastic response

PubMed Central

OLIVEIRA, Natássia Cristina Martins; MOURA, Camilla Christian Gomes; ZANETTA-BARBOSA, Darceny; MENDONÇA, Daniela Baccelli Silveira; MENDONÇA, Gustavo; DECHICHI, Paula

2015-01-01

In this study we investigated whether anodization with calcium phosphate (CaP) incorporation (Vulcano®) enhances growth factors secretion, osteoblast-specific gene expression, and cell viability, when compared to acid etched surfaces (Porous®) and machined surfaces (Screw®) after 3 and 7 days. Results showed significant cell viability for Porous and Vulcano at day 7, when compared with Screw (p=0.005). At the same time point, significant differences regarding runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP) and bone sialoprotein (BSP) expression were found for all surfaces (p<0.05), but with greater fold induction for Porous and Vulcano. The secretion of transforming growth factor β1 (TGF-β1) and bone morphogenetic protein 2 (BMP-2) was not significantly affected by surface treatment in any experimental time (p>0.05). Although no significant correlation was found for growth factors secretion and Runx2 expression, a significant positive correlation between this gene and ALP/BSP expression showed that their strong association is independent on the type of surface. The incorporation of CaP affected the biological parameters evaluated similar to surfaces just acid etched. The results presented here support the observations that roughness also may play an important role in determining cell response. PMID:23498218

A simple PCR-based strategy for estimating species-specific contributions in chimeras and xenografts

PubMed Central

Ealba, Erin L.; Schneider, Richard A.

2013-01-01

Many tissue-engineering approaches for repair and regeneration involve transplants between species. Yet a challenge is distinguishing donor versus host effects on gene expression. This study provides a simple molecular strategy to quantify species-specific contributions in chimeras and xenografts. Species-specific primers for reverse transcription quantitative real-time PCR (RT-qPCR) were designed by identifying silent mutations in quail, duck, chicken, mouse and human ribosomal protein L19 (RPL19). cDNA from different pairs of species was mixed in a dilution series and species-specific RPL19 primers were used to generate standard curves. Then quail cells were transplanted into transgenic-GFP chick and resulting chimeras were analyzed with species-specific primers. Fluorescence-activated cell sorting (FACS) confirmed that donor- and host-specific levels of RPL19 expression represent actual proportions of cells. To apply the RPL19 strategy, we measured Runx2 expression in quail-duck chimeras. Elevated Runx2 levels correlated with higher percentages of donor cells. Finally, RPL19 primers also discriminated mouse from human and chick. Thus, this strategy enables chimeras and/or xenografts to be screened rapidly at the molecular level. PMID:23785056

Effects of {gamma}-secretase inhibition on the proliferation and vitamin D{sub 3} induced osteogenesis in adipose derived stem cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jing, Wei; Xiong, Zhonghua; Cai, Xiaoxiao

2010-02-12

As a {gamma}-secretase inhibitor, DAPT has been widely used to evaluate the biological behaviors and Notch signaling pathway in various cells. This study was aimed to examine the effects of DAPT on the growth and vitamin D{sub 3} induced osteogenesis in adipose derived stem cells (ASCs). The cells were treated with or without DAPT and induced to osteoblastic lineage in the presence of vitamin D{sub 3}. Alizarin red staining and real-time PCR results indicated that the addition of DAPT to vitamin D{sub 3} treatments enhanced osteogenesis in ASCs. According to the fold increase and colony-forming unit assay results, the cellsmore » cultured in DAPT exhibited lower proliferation rate than those cultured in control medium. Hey1, expressed in the nucleus of ASCs to act as a transcriptional repressor, was downregulated when Notch signaling was inhibited by DAPT. Whereas the expression of Runx2 increased in the nucleus of osteogenic induced ASCs after DAPT treatment. This study demonstrated that DAPT reduced the proliferation and enhanced the osteogenesis in ASCs via regulation of Notch and Runx2 expression.« less

Osteogenic differentiation of human mesenchymal bone marrow cells in silk scaffolds is regulated by nitric oxide.

PubMed

Damoulis, Petros D; Drakos, Dimitrios E; Gagari, Eleni; Kaplan, David L

2007-11-01

Bone marrow-derived mesenchymal stem cells (BMSC) are a powerful tool for tissue engineering and can be used in the regeneration of bone and other tissues. Nitric oxide (NO) produced by the endothelial NO synthase (eNOS) plays an important role in bone development and healing. We hypothesized that NO plays a role in osteogenic differentiation of BMSC cultured in three-dimensional silk scaffolds. eNOS protein was measured by Western Analysis and its activity was assessed by measuring nitrite in culture supernatants. Mineralization was evaluated through calcium deposition and the expression of genes associated with osteogenic differentiation (collagen I, RUNX2, and osteocalcin) was quantified using real-time RT-PCR. eNOS was consistently expressed with minor fluctuations, but NO production significantly increased at later time points (weeks 4 and 5). Addition of a competitive NOS inhibitor (L-NAME) resulted in a modest decrease in calcium deposition, which became statistically significant in week 5. This was preceded by a dramatic decrease in RUNX2 and osteocalcin expression in week 4. These results support our hypothesis and implicate NO as an important player in bone tissue engineering.

Runx1 and Cbfβ regulate the development of Flt3+ dendritic cell progenitors and restrict myeloproliferative disorder

PubMed Central

Satpathy, Ansuman T.; Briseño, Carlos G.; Cai, Xiongwei; Michael, Drew G.; Chou, Chun; Hsiung, Sunnie; Bhattacharya, Deepta; Speck, Nancy A.

2014-01-01

Runx1 and Cbfβ are critical for the establishment of definitive hematopoiesis and are implicated in leukemic transformation. Despite the absolute requirements for these factors in the development of hematopoietic stem cells and lymphocytes, their roles in the development of bone marrow progenitor subsets have not been defined. Here, we demonstrate that Cbfβ is essential for the development of Flt3+ macrophage-dendritic cell (DC) progenitors in the bone marrow and all DC subsets in the periphery. Besides the loss of DC progenitors, pan-hematopoietic Cbfb-deficient mice also lack CD105+ erythroid progenitors, leading to severe anemia at 3 to 4 months of age. Instead, Cbfb deficiency results in aberrant progenitor differentiation toward granulocyte-macrophage progenitors (GMPs), resulting in a myeloproliferative phenotype with accumulation of GMPs in the periphery and cellular infiltration of the liver. Expression of the transcription factor Irf8 is severely reduced in Cbfb-deficient progenitors, and overexpression of Irf8 restors DC differentiation. These results demonstrate that Runx proteins and Cbfβ restrict granulocyte lineage commitment to facilitate multilineage hematopoietic differentiation and thus identify their novel tumor suppressor function in myeloid leukemia. PMID:24677539

Asporin and transforming growth factor-beta gene expression in osteoblasts from subchondral bone and osteophytes in osteoarthritis.

PubMed

Sakao, Kei; Takahashi, Kenji A; Arai, Yuji; Saito, Masazumi; Honjyo, Kuniaki; Hiraoka, Nobuyuki; Kishida, Tsunao; Mazda, Osam; Imanishi, Jiro; Kubo, Toshikazu

2009-11-01

To clarify the significance of subchondral bone and osteophytes in the pathology of osteoarthritis (OA), we investigated the expression of asporin (ASPN), transforming growth factor-beta1 (TGF-beta1), TGF-beta2, TGF-beta3, and runt-related transcription factor-2 (Runx2) genes involved in bone metabolism. Osteoblasts were isolated from 19 patients diagnosed with knee OA and from 4 patients diagnosed with femoral neck fracture. Osteoblast expression of mRNA encoding ASPN, TGF-beta1, TGF-beta2, TGF-beta3, and Runx2 was analyzed using real-time RT-PCR. Expression of ASPN, TGF-beta1, and TGF-beta3 mRNA in the subchondral bone and osteophytes of OA patients increased compared with that of non-OA patients. The ratio of ASPN to TGF-beta1 mRNA in patients with severe cartilage damage was higher than that in patients with mild cartilage damage. The increased ratio of ASPN mRNA to TGF-beta1 mRNA in patients with severe relative to mild cartilage damage indicates that increased ASPN mRNA expression was significantly associated with the severity of cartilage degeneration. This finding suggests that ASPN may regulate TGF-beta1-mediated factors in the development of OA, which may provide clues as to the underlying pathology of OA.

SIRT6 deficiency culminates in low-turnover osteopenia.

PubMed

Sugatani, Toshifumi; Agapova, Olga; Malluche, Hartmut H; Hruska, Keith A

2015-12-01

Deficiency of Sirtuin 6 (SIRT6), a chromatin-related deacetylase, in mice reveals severe premature aging phenotypes including osteopenia. However, the underlying molecular mechanisms of SIRT6 in bone metabolism are unknown. Here we show that SIRT6 deficiency in mice produces low-turnover osteopenia caused by impaired bone formation and bone resorption, which are mechanisms similar to those of age-related bone loss. Mechanistically, SIRT6 interacts with runt-related transcription factor 2 (Runx2) and osterix (Osx), which are the two key transcriptional regulators of osteoblastogenesis, and deacetylates histone H3 at Lysine 9 (H3K9) at their promoters. Hence, excessively elevated Runx2 and Osx in SIRT6(-/-) osteoblasts lead to impaired osteoblastogenesis. In addition, SIRT6 deficiency produces hyperacetylation of H3K9 in the promoter of dickkopf-related protein 1 (Dkk1), a potent negative regulator of osteoblastogenesis, and osteoprotegerin, an inhibitor of osteoclastogenesis. Therefore, the resulting up-regulation of Dkk1 and osteoprotegerin levels contribute to impaired bone remodeling, leading to osteopenia with a low bone turnover in SIRT6-deficient mice. These results establish a new link between SIRT6 and bone remodeling that positively regulates osteoblastogenesis and osteoclastogenesis. Copyright © 2015 Elsevier Inc. All rights reserved.

Evaluation of bone substitutes for treatment of peri-implant bone defects: biomechanical, histological, and immunohistochemical analyses in the rabbit tibia

PubMed Central

2016-01-01

Purpose We sought to evaluate the effectiveness of bone substitutes in circumferential peri-implant defects created in the rabbit tibia. Methods Thirty rabbits received 45 implants in their left and right tibia. A circumferential bone defect (6.1 mm in diameter/4 mm depth) was created in each rabbit tibia using a trephine bur. A dental implant (4.1 mm × 8.5 mm) was installed after the creation of the defect, providing a 2-mm gap. The bone defect gaps between the implant and the bone were randomly filled according to the following groups: blood clot (CO), particulate Bio-Oss® (BI), and Bio-Oss® Collagen (BC). Ten animals were euthanized after periods of 15, 30, and 60 days. Biomechanical analysis by means of the removal torque of the implants, as well as histologic and immunohistochemical analyses for protein expression of osteocalcin (OC), Runx2, OPG, RANKL, and TRAP were evaluated. Results For biomechanics, BC showed a better biological response (61.00±15.28 Ncm) than CO (31.60±14.38 Ncm) at 30 days. Immunohistochemical analysis showed significantly different OC expression in CO and BC at 15 days, and also between the CO and BI groups, and between the CO and BC groups at 60 days. After 15 days, Runx2 expression was significantly different in the BI group compared to the CO and BC groups. RANKL expression was significantly different in the BI and CO groups and between the BI and BC groups at 15 days, and also between the BI and CO groups at 60 days. OPG expression was significantly higher at 60 days postoperatively in the BI group than the CO group. Conclusions Collectively, our data indicate that, compared to CO and BI, BC offered better bone healing, which was characterized by greater RUNX2, OC, and OPG immunolabeling, and required greater reversal torque for implant removal. Indeed, along with BI, BC presents promising biomechanical and biological properties supporting its possible use in osteoconductive grafts for filling peri-implant gaps. PMID:27382506

Focal Adhesion Kinase Signaling Mediated the Enhancement of Osteogenesis of Human Mesenchymal Stem Cells Induced by Extracorporeal Shockwave

NASA Astrophysics Data System (ADS)

Hu, Jun; Liao, Haojie; Ma, Zebin; Chen, Hongjiang; Huang, Zhonglian; Zhang, Yuantao; Yu, Menglei; Chen, Youbin; Xu, Jiankun

2016-02-01

Extracorporeal shockwave (ESW) has been shown of great potential in promoting the osteogenesis of bone marrow mesenchymal stem cells (BMSCs), but it is unknown whether this osteogenic promotion effect can also be achieved in other MSCs (i.e., tendon-derived stem cells (TDSCs) and adipose-derived stem cells (ADSCs)). In the current study, we aimed not only to compare the osteogenic effects of BMSCs induced by ESW to those of TDSCs and ADSCs; but also to investigate the underlying mechanisms. We show here that ESW (0.16 mj/mm2) significantly promoted the osteogenic differentiation in all the tested types of MSCs, accompanied with the downregulation of miR-138, but the activation of FAK, ERK1/2, and RUNX2. The enhancement of osteogenesis in these MSCs was consistently abolished when the cells were pretreated with one of the following conditions: overexpression of miR-138, FAK knockdown using specific siRNA, and U0126, implying that all of these elements are indispensable for mediating the effect of ESW. Moreover, our study provides converging genetic and molecular evidence that the miR-138-FAK-ERK1/2-RUNX2 machinery can be generally activated in ESW-preconditioned MSCs, suggesting that ESW may be a promising therapeutic strategy for the enhancement of osteogenesis of MSCs, regardless of their origins.

A Microfluidic-Based Multi-Shear Device for Investigating the Effects of Low Fluid-Induced Stresses on Osteoblasts

PubMed Central

Yu, Weiliang; Qu, Hong; Hu, Guoqing; Zhang, Qian; Song, Kui; Guan, Haijie; Liu, Tingjiao; Qin, Jianhua

2014-01-01

Interstitial fluid flow (IFF) within the extracellular matrix (ECM) produces low magnitude shear stresses on cells. Fluid flow-induced stress (FSS) plays an important role during tissue morphogenesis. To investigate the effect of low FSS generated by IFF on cells, we developed a microfluidic-based cell culture device that can generate multiple low shear stresses. By changing the length and width of the flow-in channels, different continuous low level shear stresses could be generated in individual cell culture chambers. Numerical calculations demonstrate uniform shear stress distributions of the major cell culture area of each chamber. This calculation is further confirmed by the wall shear stress curves. The effects of low FSS on MC3T3-E1 proliferation and differentiation were studied using this device. It was found that FSS ranging from 1.5 to 52.6 µPa promoted MC3T3-E1 proliferation and differentiation, but FSS over 412 µPa inhibited the proliferation and differentiation of MC3T3-E1 cells. FSS ranging from 1.5 to 52.6 µPa also increased the expression of Runx2, a key transcription factor regulating osteoblast differentiation. It is suggested that Runx2 might be an important regulator in low FSS-induced MC3T3-E1 differentiation. This device allows for detailed study of the effect of low FSS on the behaviors of cells; thus, it would be a useful tool for analysis of the effects of IFF-induced shear stresses on cells. PMID:24587156

Hypoxia Promotes Osteogenesis but Suppresses Adipogenesis of Human Mesenchymal Stromal Cells in a Hypoxia-Inducible Factor-1 Dependent Manner

PubMed Central

Lohanatha, Ferenz L.; Hahne, Martin; Strehl, Cindy; Fangradt, Monique; Tran, Cam Loan; Schönbeck, Kerstin; Hoff, Paula; Ode, Andrea; Perka, Carsten; Duda, Georg N.; Buttgereit, Frank

2012-01-01

Background Bone fracture initiates a series of cellular and molecular events including the expression of hypoxia-inducible factor (HIF)-1. HIF-1 is known to facilitate recruitment and differentiation of multipotent human mesenchymal stromal cells (hMSC). Therefore, we analyzed the impact of hypoxia and HIF-1 on the competitive differentiation potential of hMSCs towards adipogenic and osteogenic lineages. Methodology/Principal Findings Bone marrow derived primary hMSCs cultured for 2 weeks either under normoxic (app. 18% O2) or hypoxic (less than 2% O2) conditions were analyzed for the expression of MSC surface markers and for expression of the genes HIF1A, VEGFA, LDHA, PGK1, and GLUT1. Using conditioned medium, adipogenic or osteogenic differentiation as verified by Oil-Red-O or von-Kossa staining was induced in hMSCs under either normoxic or hypoxic conditions. The expression of HIF1A and VEGFA was measured by qPCR. A knockdown of HIF-1α by lentiviral transduction was performed, and the ability of the transduced hMSCs to differentiate into adipogenic and osteogenic lineages was analyzed. Hypoxia induced HIF-1α and HIF-1 target gene expression, but did not alter MSC phenotype or surface marker expression. Hypoxia (i) suppressed adipogenesis and associated HIF1A and PPARG gene expression in hMSCs and (ii) enhanced osteogenesis and associated HIF1A and RUNX2 gene expression. shRNA-mediated knockdown of HIF-1α enhanced adipogenesis under both normoxia and hypoxia, and suppressed hypoxia-induced osteogenesis. Conclusions/Significance Hypoxia promotes osteogenesis but suppresses adipogenesis of human MSCs in a competitive and HIF-1-dependent manner. We therefore conclude that the effects of hypoxia are crucial for effective bone healing, which may potentially lead to the development of novel therapeutic approaches. PMID:23029528

The Effects of Targeted Deliveries of Lovastatin and Tocotrienol on Ossification-Related Gene Expressions in Fracture Healing in an Osteoporosis Rat Model

PubMed Central

Ibrahim, Nurul ‘Izzah; Mohamed, Norazlina; Soelaiman, Ima Nirwana; Shuid, Ahmad Nazrun

2015-01-01

Osteoporotic drugs are used to prevent fragility fractures, but their role in fracture healing still remains unknown. Thus, alternative agents with suitable mode of delivery are needed to promote fracture healing. This study was performed to investigate the effects of direct deliveries of lovastatin and tocotrienol to fracture sites on ossification-related gene expression in fracture healing in a postmenopausal osteoporosis model. Forty-eight Sprague Dawley female rats were divided into six groups. Group I comprised the sham-operated rats, while Groups II–VI were ovariectomized rats. After 8 weeks, the right tibiae of all rats were fractured and stabilized. Group I and Group II were given two single injections of lovastatin and tocotrienol carriers. Group III was given an estrogen preparation at 64.5 µg/kg daily via oral gavages. Group IV was injected with lovastatin particles (750 µg/kg), while Group V was injected with tocotrienol particles (60 mg/kg). Group VI received two single injections of 750 µg/kg lovastatin particles and 60 mg/kg tocotrienol particles. After 4 weeks, the gene expressions were measured. Group VI showed significantly higher gene expressions of osteocalcin, BMP-2, VEGF-α, and RUNX-2 compared to Group II. In conclusion, combined treatment of lovastatin and tocotrienol upregulated the expression of genes related to fracture healing. PMID:26501302

The Effects of Targeted Deliveries of Lovastatin and Tocotrienol on Ossification-Related Gene Expressions in Fracture Healing in an Osteoporosis Rat Model.

PubMed

Ibrahim, Nurul 'Izzah; Mohamed, Norazlina; Soelaiman, Ima Nirwana; Shuid, Ahmad Nazrun

2015-10-16

Osteoporotic drugs are used to prevent fragility fractures, but their role in fracture healing still remains unknown. Thus, alternative agents with suitable mode of delivery are needed to promote fracture healing. This study was performed to investigate the effects of direct deliveries of lovastatin and tocotrienol to fracture sites on ossification-related gene expression in fracture healing in a postmenopausal osteoporosis model. Forty-eight Sprague Dawley female rats were divided into six groups. Group I comprised the sham-operated rats, while Groups II-VI were ovariectomized rats. After 8 weeks, the right tibiae of all rats were fractured and stabilized. Group I and Group II were given two single injections of lovastatin and tocotrienol carriers. Group III was given an estrogen preparation at 64.5 µg/kg daily via oral gavages. Group IV was injected with lovastatin particles (750 µg/kg), while Group V was injected with tocotrienol particles (60 mg/kg). Group VI received two single injections of 750 µg/kg lovastatin particles and 60 mg/kg tocotrienol particles. After 4 weeks, the gene expressions were measured. Group VI showed significantly higher gene expressions of osteocalcin, BMP-2, VEGF-α, and RUNX-2 compared to Group II. In conclusion, combined treatment of lovastatin and tocotrienol upregulated the expression of genes related to fracture healing.

Prognostic Value of RUNX1 Mutations in AML: A Meta-Analysis

PubMed

Jalili, Mahdi; Yaghmaie, Marjan; Ahmadvand, Mohammad; Alimoghaddam, Kamran; Mousavi, Seyed Asadollah; Vaezi, Mohammad; Ghavamzadeh, Ardeshir

2018-02-26

The RUNX1 (AML1) gene is a relatively infrequent mutational target in cases of acute myeloid leukemia (AML). Previous work indicated that RUNX1 mutations can have pathological and prognostic implications. To evaluate prognostic value, we conducted a meta-analysis of 4 previous published works with data for survival according to RUNX1 mutation status. Pooled hazard ratios for overall survival and disease-free survival were 1.55 (95% confidence interval (CI) = 1.11–2.15; p-value = 0.01) and 1.76 (95% CI = 1.24–2.52; p-value = 0.002), respectively, for cases positive for RUNX1 mutations. This evidence supports clinical implications of RUNX1 mutations in the development and progression of AML cases and points to the possibility of a distinct category within the newer WHO classification. Though it must be kept in mind that the present work was based on data extracted from observational studies, the findings suggest that the RUNX1 status can contribute to risk-stratification and decision-making in management of AML. Creative Commons Attribution License

The influence of stereolithographic scaffold architecture and composition on osteogenic signal expression with rat bone marrow stromal cells

PubMed Central

Kim, Kyobum; Dean, David; Wallace, Jonathan; Breithaupt, Rob; Mikos, Antonios G.; Fisher, John P.

2011-01-01

Scaffold design parameters, especially physical construction factors such as mechanical stiffness of substrate materials, pore size of 3D porous scaffolds, and channel geometry, are known to influence the osteogenic signal expression and subsequent differentiation of a transplanted cell population. In this study of photocrosslinked poly(propylene fumarate) (PPF) and diethyl fumarate (DEF) scaffolds, the effect of DEF incorporation ratio and pore size on the osteogenic signal expression of rat bone marrow stromal cells (BMSCs) was investigated. Results demonstrated that DEF concentrations and pore sizes that led to increased scaffold mechanical stiffness also upregulated osteogenic signal expression, including bone morphogenic protein-2 (BMP-2), fibroblast growth factors-2 (FGF-2), transforming growth factor-β1 (TGF-β1), vascular endothelial growth factor (VEGF), and Runx2 transcriptional factor. Similar scaffold fabrication parameters supported rapid BMSC osteoblastic differentiation, as demonstrated by increased alkaline phosphatase (ALP) and osteocalcin expression. When scaffolds with random architecture, fabricated by porogen leaching, were compared to those with controlled architecture, fabricated by stereolithography (SLA), results showed that SLA scaffolds with the highly permeable and porous channels also have significantly higher expression of FGF-2, TGF-β1, and VEGF. Subsequent ALP expression and osteopontin secretion were also significantly increased in SLA scaffolds. Based upon these results, we conclude that scaffold properties provided by additive manufacturing techniques such as SLA fabrication, particularly increased mechanical stiffness and high permeability, may stimulate dramatic BMSC responses that promote rapid bone tissue regeneration. PMID:21396709

The use of chitosan/PLA nano-fibers by emulsion eletrospinning for periodontal tissue engineering.

PubMed

Shen, Renze; Xu, Weihong; Xue, Yanxiang; Chen, Luyuan; Ye, Haicheng; Zhong, Enyi; Ye, Zhanchao; Gao, Jie; Yan, Yurong

2018-04-16

In this study, nanofibrous scaffolds base on pure polylactic acid (PLA) and chitosan/PLA blends were fabricated by emulsion eletrospinning. By modulating their mechanical and biological properties, cell-compatible and biodegradable scaffolds were developed for periodontal bone regeneration. Pure PLA and different weight ratios of chitosan nano-particle/PLA nano-fibers were fabricated by emulsion eletrospinning. Scanning electron microscope (SEM) was performed to observe the morphology of nano-fibers. Mechanical properties of nano-fibers were tested by single fiber strength tester. Hydrophilic/hydrophobic nature of the nano-fibers was observed by stereomicroscope. In vitro degradation was also tested. Cells were seeded on nano-fibers scaffolds. Changes in cell adhesion, proliferation and osteogenic differentiation were tested by MTT assay and Alizarin Red S staining. Reverse transcription-polymerase chain reaction (RT-PCR) assay was used to evaluate the expression of (Toll-like receptor 4) TLR4, IL-6, IL-8, IL-1β, OPG, RUNX2 mRNA. It is shown that the mean diameter of nano-fibers is about 200 nm. The mean diameter of chitosan nano-particles is about 50 nm. The combination of chitosan nano-particles enhanced the mechanical properties of pure PLA nano-fibers. By adding a certain amount of chitosan nano-particles, it promoted cell adhesion. It also promoted the osteogenic differentiation of bone marrow stem cells (BMSCs) by elevating the expression of osteogenic marker genes such as BSP, Ocn, collagen I, and OPN and enhanced ECM mineralization. Nonetheless, it caused higher expression of inflammatory mediators and TLR4 of human periodontal ligament cells (hPDLCs). The combination of chitosan nano-particles enhanced the mechanical properties of pure PLA nano-fibers and increased its hydrophilicity. Pure PLA nano-fibers scaffold facilitated BMSCs proliferation. Adding an appropriate amount of chitosan nano-particles may promote its properties of cell proliferation and osteogenic differentiation. The higher expression of inflammatory mediators caused by nano-fibers may be regulated via TLR4 pathway.

Insulin-like growth factor 1 promotes the proliferation and committed differentiation of human dental pulp stem cells through MAPK pathways.

PubMed

Lv, Taohong; Wu, Yongzheng; Mu, Chao; Liu, Genxia; Yan, Ming; Xu, Xiangqin; Wu, Huayin; Du, Jinyin; Yu, Jinhua; Mu, Jinquan

2016-12-01

Insulin-like growth factor 1 (IGF-1) is a broad-spectrum growth-promoting factor that plays a key role in natural tooth development. Human dental pulp stem cells (hDPSCs) are multipotent and can influence the reparative regeneration of dental pulp and dentin. This study was designed to evaluate the effects of IGF-1 on the proliferation and differentiation of human dental pulp stem cells. HDPSCs were isolated and purified from human dental pulps. The proliferation and osteo/odontogenic differentiation of hDPSCs treated with 100ng/ml exogenous IGF-1 were subsequently investigated. MTT assays revealed that IGF-1 enhanced the proliferation of hDPSCs. ALP activity in IGF-1-treated group was obviously enhanced compared to the control group from days 3 to 9. Alizarin red staining revealed that the IGF-1-treated cells contained a greater number of mineralization nodules and had higher calcium concentrations. Moreover, western blot and qRT-PCR analyses demonstrated that the expression levels of several osteogenic genes (e.g., RUNX2, OSX, and OCN) and an odontoblast-specific marker (DSPP) were significantly up-regulated in IGF-1-treated hDPSCs as compared with untreated cells (P<0.01). Interestingly, the expression of phospho-ERK and phospho-p38 were also up-regulated, indicating that the MAPK signaling pathway is activated during the differentiation of hDPSCs. IGF-1 can promote the proliferation and osteo/odontogenic differentiation of hDPSCs by activating MAPK pathways. Copyright © 2016 Elsevier Ltd. All rights reserved.

A novel inherited mutation of the transcription factor RUNX1 causes thrombocytopenia and may predispose to acute myeloid leukaemia.

PubMed

Walker, Logan C; Stevens, Jane; Campbell, Hamish; Corbett, Rob; Spearing, Ruth; Heaton, David; Macdonald, Donald H; Morris, Christine M; Ganly, Peter

2002-06-01

The RUNX1 (AML1, CBFA2) gene is a member of the runt transcription factor family, responsible for DNA binding and heterodimerization of other non-DNA binding transcription factors. RUNX1 plays an important part in regulating haematopoiesis and it is frequently disrupted by illegitimate somatic recombination in both acute myeloid and lymphoblastic leukaemia. Germline mutations of RUNX1 have also recently been described and are dominantly associated with inherited leukaemic conditions. We have identified a unique point mutation of the RUNX1 gene (A107P) in members of a family with autosomal dominant inheritance of thrombocytopenia. One member has developed acute myeloid leukaemia (AML).

Epigenetic inactivation of TWIST2 in acute lymphoblastic leukemia modulates proliferation, cell survival and chemosensitivity

PubMed Central

Thathia, Shabnam H.; Ferguson, Stuart; Gautrey, Hannah E.; van Otterdijk, Sanne D.; Hili, Michela; Rand, Vikki; Moorman, Anthony V.; Meyer, Stefan; Brown, Robert; Strathdee, Gordon

2012-01-01

Background Altered regulation of many transcription factors has been shown to be important in the development of leukemia. TWIST2 modulates the activity of a number of important transcription factors and is known to be a regulator of hematopoietic differentiation. Here, we investigated the significance of epigenetic regulation of TWIST2 in the control of cell growth and survival and in response to cytotoxic agents in acute lymphoblastic leukemia. Design and Methods TWIST2 promoter methylation status was assessed quantitatively, by combined bisulfite and restriction analysis (COBRA) and pyrosequencing assays, in multiple types of leukemia and TWIST2 expression was determined by quantitative reverse transcriptase polymerase chain reaction analysis. The functional role of TWIST2 in cell proliferation, survival and response to chemotherapy was assessed in transient and stable expression systems. Results We found that TWIST2 was inactivated in more than 50% of cases of childhood and adult acute lymphoblastic leukemia through promoter hypermethylation and that this epigenetic regulation was especially prevalent in RUNX1-ETV6-driven cases. Re-expression of TWIST2 in cell lines resulted in a dramatic reduction in cell growth and induction of apoptosis in the Reh cell line. Furthermore, re-expression of TWIST2 resulted in increased sensitivity to the chemotherapeutic agents etoposide, daunorubicin and dexamethasone and TWIST2 hypermethylation was almost invariably found in relapsed adult acute lymphoblastic leukemia (91% of samples hypermethylated). Conclusions This study suggests a dual role for epigenetic inactivation of TWIST2 in acute lymphoblastic leukemia, initially through altering cell growth and survival properties and subsequently by increasing resistance to chemotherapy. PMID:22058208

The Effect of Cyclooxygenase Inhibition on Tendon-Bone Healing in an In Vitro Coculture Model

PubMed Central

Schwarting, Tim; Pretzsch, Sebastian; Debus, Florian; Ruchholtz, Steffen; Lechler, Philipp

2015-01-01

The effects of cyclooxygenase (COX) inhibition following the reconstruction of the anterior cruciate ligament remain unclear. We examined the effects of selective COX-2 and nonselective COX inhibition on bone-tendon integration in an in vitro model. We measured the dose-dependent effects of ibuprofen and parecoxib on the viability of lipopolysaccharide- (LPS-) stimulated and unstimulated mouse MC3T3-E1 and 3T3 cells, the influence on gene expression at the osteoblast, interface, and fibroblast regions measured by quantitative PCR, and cellular outgrowth assessed on histological sections. Ibuprofen led to a dose-dependent suppression of MC3T3 cell viability, while parecoxib reduced the viability of 3T3 cultures. Exposure to ibuprofen significantly suppressed expression of Alpl (P < 0.01), Bglap (P < 0.001), and Runx2 (P < 0.01), and although parecoxib reduced expression of Alpl (P < 0.001), Fmod (P < 0.001), and Runx2 (P < 0.01), the expression of Bglap was increased (P < 0.01). Microscopic analysis showed a reduction in cellular outgrowth in LPS-stimulated cultures following exposure to ibuprofen and parecoxib. Nonselective COX inhibition and the specific inhibition of COX-2 led to region-specific reductions in markers of calcification and cell viability. We suggest further in vitro and in vivo studies examining the biologic and biomechanical effects of selective and nonselective COX inhibition. PMID:26063979

Low-intensity pulsed ultrasound produced an increase of osteogenic genes expression during the process of bone healing in rats.

PubMed

Fávaro-Pípi, Elaine; Bossini, Paulo; de Oliveira, Poliani; Ribeiro, Juliana Uema; Tim, Carla; Parizotto, Nivaldo A; Alves, Jose Marcos; Ribeiro, Daniel Araki; Selistre de Araújo, Heloísa Sobreiro; Renno, Ana Claudia Muniz

2010-12-01

The aim of this study was to measure the temporal expression of osteogenic genes during the process of bone healing in low-intensity pulsed ultrasound (LIPUS) treated bone defects by means of histopathologic and real-time polymerase chain reaction (PCR) analysis. Animals were randomly distributed into two groups (n = 30): control group (bone defect without treatment) and LIPUS treated (bone defect treated with LIPUS). On days 7, 13 and 25 postinjury, 10 rats per group were sacrificed. Rats were treated with a 30 mW/cm(2) LIPUS. The results pointed out intense new bone formation surrounded by highly vascularized connective tissue presenting a slight osteogenic activity, with primary bone deposition was observed in the group exposed to LIPUS in the intermediary (13 days) and late stages of repair (25 days) in the treated animals. In addition, quantitative real-time polymerase chain reaction (RT-qPCR) showed an upregulation of bone morphogenetic protein 4 (BMP4), osteocalcin and Runx2 genes 7 days after the surgery. In the intermediary period, there was no increase in the expression. The expression of alkaline phosphatase, BMP4 and Runx2 was significantly increased at the last period. Our results indicate that LIPUS therapy improves bone repair in rats and upregulated osteogenic genes, mainly at the late stages of recovery. Copyright © 2010. Published by Elsevier Inc.

Hydrostatic pressure enhances chondrogenic differentiation of human bone marrow stromal cells in osteochondrogenic medium.

PubMed

Wagner, Diane R; Lindsey, Derek P; Li, Kelvin W; Tummala, Padmaja; Chandran, Sheena E; Smith, R Lane; Longaker, Michael T; Carter, Dennis R; Beaupre, Gary S

2008-05-01

This study demonstrated the chondrogenic effect of hydrostatic pressure on human bone marrow stromal cells (MSCs) cultured in a mixed medium containing osteogenic and chondrogenic factors. MSCs seeded in type I collagen sponges were exposed to 1 MPa of intermittent hydrostatic pressure at a frequency of 1 Hz for 4 h per day for 10 days, or remained in identical culture conditions but without exposure to pressure. Afterwards, we compared the proteoglycan content of loaded and control cell/scaffold constructs with Alcian blue staining. We also used real-time PCR to evaluate the change in mRNA expression of selected genes associated with chondrogenic and osteogenic differentiation (aggrecan, type I collagen, type II collagen, Runx2 (Cbfa-1), Sox9, and TGF-beta1). With the hydrostatic pressure loading regime, proteoglycan staining increased markedly. Correspondingly, the mRNA expression of chondrogenic genes such as aggrecan, type II collagen, and Sox9 increased significantly. We also saw a significant increase in the mRNA expression of type I collagen, but no change in the expression of Runx2 or TGF-beta1 mRNA. This study demonstrated that hydrostatic pressure enhanced differentiation of MSCs in the presence of multipotent differentiation factors in vitro, and suggests the critical role that this loading regime may play during cartilage development and regeneration in vivo.

Setd7 and its contribution to Boron-induced bone regeneration in B-MBG scaffolds.

PubMed

Yin, Chengcheng; Jia, Xiaoshi; Miron, Richard J; Long, Qiaoyun; Xu, Hudi; Wei, Yan; Wu, Min; Zhang, Yufeng; Li, Zubing

2018-04-20

Boron (B), a trace element found in the human body, plays an important role for health of bone by promoting the proliferation and differentiation of osteoblasts. Our research group previously fabricated B-mesoporous bioactive glass (MBG) scaffolds, which successfully promoted osteogenic differentiation of osteoblasts when compared to pure MBG scaffolds without boron. However, the mechanisms of the positive effect of B-MBG scaffolds on osteogenesis remain unknown. Therefore, we performed in-vivo experiments in OVX rat models with pure MBG scaffolds and compared them to B-MBG scaffold. As a result, we found that B-MBG scaffold induced more new bone regeneration compared to pure MBG scaffold and examined genes related to bone regeneration induced by B-MBG scaffold through RNA-seq to obtain target genes and epigenetic mechanisms. The results demonstrated an increased expression and affiliation of Setd7 in the B-MBG group when compared to the MBG group. Immunofluorescent staining from our in vivo samples further demonstrated a higher localization of Setd7 and H3K4me3 in Runx2-positive cells in defects treated with B-MBG scaffolds. KEGG results suggested that specifically Wnt/β-catenin signaling pathway was highly activated in new bone area associated with B-MBG scaffolds. Thereafter, in vitro studies with human bone marrow stem cells (hBMSCs) stimulated by extracted liquid of B-MBG scaffolds was associated with significantly elevated levels of Setd7, as well as H3K4me3 when compared to MBG scaffolds alone. To verify the role of Setd7 in new bone formation in the presence of Boron, Setd7 was knocked down in hBMSCs with stimulation of the extracted liquids of B-MBG or MBG scaffolds. The result showed that osteoblast differentiation of hBMSCs was inhibited when Setd7 was knocked down, which could not be rescued by the extracted liquids of B-MBG scaffolds confirming its role in osteoblast differentiation and bone regeneration. As a histone methylase, Setd7 may be expected to be a potential epigenetic target for new treatment schemes of osteoporosis. Boron-containing MBG scaffold has already been proved to promote bone regeneration in femoral defects of OVX rats by our research group, however, the epigenetic mechanism of Boron's positive effects on bone generation remains ill-informed. In our present study, we found an increased expression and affiliation of Setd7 and H3K4me3 in Runx2-positive osteoblasts in vivo. And in vitro, the higher expression of Setd7 enhanced osteogenic differentiation of human BMSCs stimulated by extracted liquids of B-MBG scaffold compared to MBG scaffold, which was associated with the activation of Wnt/β-catenin signaling pathway. Above all, it suggests that Setd7 plays an positive role in osteogenic differentiation and it may become a potential epigenetic target for new schemes for osteoporosis. Copyright © 2018. Published by Elsevier Ltd.

Sumoylation of CCAAT/enhancer-binding protein α is implicated in hematopoietic stem/progenitor cell development through regulating runx1 in zebrafish.

PubMed

Yuan, Hao; Zhang, Tao; Liu, Xiaohui; Deng, Min; Zhang, Wenqing; Wen, Zilong; Chen, Saijuan; Chen, Zhu; de The, Hugues; Zhou, Jun; Zhu, Jun

2015-03-11

The small ubiquitin-related modifier (SUMO) participates in various cellular processes, including maintenance of genome integrity, nuclear transport, transcription and signal transduction. However, the biological function of sumoylation in hematopoiesis has not been fully explored. We show here that definitive hematopoietic stem/progenitor cells (HSPCs) are depleted in SUMO-deficient zebrafish embryos. Impairment of sumoylation attenuates HSPC generation and proliferation. The hyposumoylation triggered HSPC defects are CCAAT/enhancer-binding protein α (C/ebpα) dependent. Critically, a SUMO-C/ebpα fusion rescues the defective hematopoiesis in SUMO-deficient embryos, at least in part through restored runx1 expression. While C/ebpα-dependent transcription is involved in myeloid differentiation, our studies here reveal that C/ebpα sumoylation is essential for HSPC development during definitive hematopoiesis.

Sumoylation of CCAAT/enhancer-binding protein α is implicated in hematopoietic stem/progenitor cell development through regulating runx1 in zebrafish

PubMed Central

Yuan, Hao; Zhang, Tao; Liu, Xiaohui; Deng, Min; Zhang, Wenqing; Wen, Zilong; Chen, Saijuan; Chen, Zhu; de The, Hugues; Zhou, Jun; Zhu, Jun

2015-01-01

The small ubiquitin-related modifier (SUMO) participates in various cellular processes, including maintenance of genome integrity, nuclear transport, transcription and signal transduction. However, the biological function of sumoylation in hematopoiesis has not been fully explored. We show here that definitive hematopoietic stem/progenitor cells (HSPCs) are depleted in SUMO-deficient zebrafish embryos. Impairment of sumoylation attenuates HSPC generation and proliferation. The hyposumoylation triggered HSPC defects are CCAAT/enhancer-binding protein α (C/ebpα) dependent. Critically, a SUMO-C/ebpα fusion rescues the defective hematopoiesis in SUMO-deficient embryos, at least in part through restored runx1 expression. While C/ebpα-dependent transcription is involved in myeloid differentiation, our studies here reveal that C/ebpα sumoylation is essential for HSPC development during definitive hematopoiesis. PMID:25757417

A novel bioactive osteogenesis scaffold delivers ascorbic acid, β-glycerophosphate, and dexamethasone in vivo to promote bone regeneration.

PubMed

Wang, Chao; Cao, Xuecheng; Zhang, Yongxian

2017-05-09

Ascorbic acid, β-glycerophosphate, and dexamethasone have been used in osteogenesis differentiation medium for in vitro cell culture, nothing is known for delivering these three bioactive compounds in vivo. In this study, we synthesized a novel bioactive scaffold by combining these three compounds with a lysine diisocyanate-based polyurethane. These bioactive compounds were released from the scaffold during the degradation process. The cell culture showed that the sponge-like structure in the scaffold was critical in providing a large surface area to support cell growth and all degradation products of the polymer were non-toxic. This bioactive scaffold enhanced the bone regeneration as evidenced by increasing the expression of three bone-related genes including collagen type I, Runx-2 and osteocalcin in rabbit bone marrow stem cells (BMSCs) in vitro and in vivo. The osteogenesis differentiation of BMSCs cultured in this bioactive scaffold was similar to that in osteogenesis differentiation medium and more extensive in this bioactive scaffold compared to the scaffold without these three bioactive compounds. These results indicated that the scaffold containing three bioactive compounds was a good osteogenesis differentiation promoter to enhance the osteogenesis differentiation and new bone formation in vivo.

A novel bioactive osteogenesis scaffold delivers ascorbic acid, β-glycerophosphate, and dexamethasone in vivo to promote bone regeneration

PubMed Central

Wang, Chao; Cao, Xuecheng; Zhang, Yongxian

2017-01-01

Ascorbic acid, β-glycerophosphate, and dexamethasone have been used in osteogenesis differentiation medium for in vitro cell culture, nothing is known for delivering these three bioactive compounds in vivo. In this study, we synthesized a novel bioactive scaffold by combining these three compounds with a lysine diisocyanate-based polyurethane. These bioactive compounds were released from the scaffold during the degradation process. The cell culture showed that the sponge-like structure in the scaffold was critical in providing a large surface area to support cell growth and all degradation products of the polymer were non-toxic. This bioactive scaffold enhanced the bone regeneration as evidenced by increasing the expression of three bone-related genes including collagen type I, Runx-2 and osteocalcin in rabbit bone marrow stem cells (BMSCs) in vitro and in vivo. The osteogenesis differentiation of BMSCs cultured in this bioactive scaffold was similar to that in osteogenesis differentiation medium and more extensive in this bioactive scaffold compared to the scaffold without these three bioactive compounds. These results indicated that the scaffold containing three bioactive compounds was a good osteogenesis differentiation promoter to enhance the osteogenesis differentiation and new bone formation in vivo. PMID:28404942

Structure and Biophysics of CBFβ/RUNX and Its Translocation Products.

PubMed

Tahirov, Tahir H; Bushweller, John

2017-01-01

The core binding factor (CBF) transcription factor is somewhat unique in that it is composed of a DNA binding RUNX subunit (RUNX1, 2, or 3) and a non-DNA binding CBFβ subunit, which modulates RUNX protein activity by modulating the auto-inhibition of the RUNX subunits. Since the discovery of this fascinating transcription factor more than 20 years ago, there has been a robust effort to characterize the structure as well as the biochemical properties of CBF. More recently, these efforts have also extended to the fusion proteins that arise from the subunits of CBF in leukemia. This chapter highlights the work of numerous labs which has provided a detailed understanding of the structure and function of this transcription factor and its fusion proteins.

Palmitate Attenuates Osteoblast Differentiation of Fetal Rat Calvarial Cells

PubMed Central

Yeh, Lee-Chuan C.; Ford, Jeffery J.; Lee, John C.; Adamo, Martin L.

2014-01-01

Aging is associated with the accumulation of ectopic lipid resulting in the inhibition of normal organ function, a phenomenon known as lipotoxicity. Within the bone marrow microenvironment, elevation in fatty acid levels may produce an increase in osteoclast activity and a decrease in osteoblast number and function, thus contributing to age-related osteoporosis. However, little is known about lipotoxic mechanisms in intramembraneous bone. Previously we reported that the long chain saturated fatty acid palmitate inhibited the expression of the osteogenic markers RUNX2 and osteocalcin in fetal rat calvarial cell (FRC) cultures. Moreover, the acetyl Co-A carboxylase inhibitor TOFA blocked the inhibitory effect of palmitate on expression of these two markers. In the current study we have extended these observations to show that palmitate inhibits spontaneous mineralized bone formation in FRC cultures in association with reduced mRNA expression of RUNX2, alkaline phosphatase, osteocalcin, and bone sialoprotein and reduced alkaline phosphatase activity. The effects of palmitate on osteogenic marker expression were inhibited by TOFA. Palmitate also inhibited the mRNA expression of fatty acid synthase and PPAR gamma in FRC cultures, and as with osteogenic markers, this effect was inhibited by TOFA. Palmitate had no effect on FRC cell proliferation or apoptosis, but inhibited BMP-7-induced alkaline phosphatase activity. We conclude that palmitate accumulation may lead to lipotoxic effects on osteoblast differentiation and mineralization and that increases in fatty acid oxidation may help to prevent these lipotoxic effects. PMID:24955854

Palmitate attenuates osteoblast differentiation of fetal rat calvarial cells.

PubMed

Yeh, Lee-Chuan C; Ford, Jeffery J; Lee, John C; Adamo, Martin L

2014-07-18

Aging is associated with the accumulation of ectopic lipid resulting in the inhibition of normal organ function, a phenomenon known as lipotoxicity. Within the bone marrow microenvironment, elevation in fatty acid levels may produce an increase in osteoclast activity and a decrease in osteoblast number and function, thus contributing to age-related osteoporosis. However, little is known about lipotoxic mechanisms in intramembraneous bone. Previously we reported that the long chain saturated fatty acid palmitate inhibited the expression of the osteogenic markers RUNX2 and osteocalcin in fetal rat calvarial cell (FRC) cultures. Moreover, the acetyl CoA carboxylase inhibitor TOFA blocked the inhibitory effect of palmitate on expression of these two markers. In the current study we have extended these observations to show that palmitate inhibits spontaneous mineralized bone formation in FRC cultures in association with reduced mRNA expression of RUNX2, alkaline phosphatase, osteocalcin, and bone sialoprotein and reduced alkaline phosphatase activity. The effects of palmitate on osteogenic marker expression were inhibited by TOFA. Palmitate also inhibited the mRNA expression of fatty acid synthase and PPARγ in FRC cultures, and as with osteogenic markers, this effect was inhibited by TOFA. Palmitate had no effect on FRC cell proliferation or apoptosis, but inhibited BMP-7-induced alkaline phosphatase activity. We conclude that palmitate accumulation may lead to lipotoxic effects on osteoblast differentiation and mineralization and that increases in fatty acid oxidation may help to prevent these lipotoxic effects. Copyright © 2014 Elsevier Inc. All rights reserved.

Osteogenic Differentiation of Human and Ovine Bone Marrow Stromal Cells in response to β-Glycerophosphate and Monosodium Phosphate.

PubMed

Bottagisio, Marta; Lovati, Arianna B; Lopa, Silvia; Moretti, Matteo

2015-08-01

Bone defects are severe burdens in clinics, and thus cell therapy offers an alternative strategy exploiting the features of bone marrow stromal cells (BMSCs). Sheep are a suitable orthopedic preclinical model for similarities with humans. This study compares the influence of two phosphate sources combined with bone morphogenetic protein-2 (BMP-2) on the osteogenic potential of human and ovine BMSCs. β-Glycerophosphate (β-GlyP) and monosodium phosphate (NaH2PO4) were used as organic and inorganic phosphate sources. Osteogenic differentiation of the BMSCs was assessed by calcified matrix, alkaline phosphatase (ALP) activity, and gene expression analysis. A higher calcified matrix deposition was detected in BMSCs cultured with NaH2PO4. Although no significant differences were detected among media for human BMSCs, β-GlyP with or without BMP-2 determined a positive trend in ALP levels compared to NaH2PO4. In contrast, NaH2PO4 had a positive effect on ALP levels in ovine BMSCs. β-GlyP better supported the expression of COL1A1 in human BMSCs, whereas all media enhanced RUNX2 and SPARC expression. Ovine BMSCs responded poorly to any media for RUNX2, COL1A1, and SPARC expression. NaH2PO4 improved calcified matrix deposition without upregulating the transcriptional expression of osteogenic markers. A further optimization of differentiation protocols needs to be performed to translate the procedures from preclinical to clinical models.

Conditioned medium from bone marrow-derived mesenchymal stem cells inhibits vascular calcification through blockade of the BMP2-Smad1/5/8 signaling pathway.

PubMed

Wang, Shuangshuang; Hu, Siwang; Wang, Jian; Liu, Yahui; Zhao, Ruochi; Tong, Maoqing; Cui, Hanbin; Wu, Nan; Chen, Xiaomin

2018-06-13

Arterial calcification is associated with cardiovascular disease as a complication of advanced atherosclerosis and is a significant contributor to cardiovascular morbidity and mortality. Osteoblastic differentiation of vascular smooth muscle cells (VSMCs) plays an important role in arterial calcification and is characterized by cellular necrosis, inflammation, and lipoprotein and phospholipid complexes, especially in atherosclerotic calcification. The conditioned medium from bone marrow-derived mesenchymal stem cells (MSC-CM) is well known as a rich source of autologous cytokines and is universally used for tissue regeneration in current clinical medicine. Here, we demonstrate that MSC-CM inhibits beta-glycerophosphate (β-GP)-induced vascular calcification through blockade of the bone morphogenetic protein-2 (BMP2)-Smad1/5/8 signaling pathway. VSMC calcification was induced by β-GP followed by treatment with MSC-CM. Mineral deposition was assessed by Alizarin Red S staining. Intracellular calcium content was determined colorimetrically by the o-cresolphthalein complexone method and alkaline phosphatase (ALP) activity was measured by the para-nitrophenyl phosphate method. Expression of BMP2, BMPR1A, BMPR1B, BMPR2, msh homeobox 2 (Msx2), Runt-related transcription factor 2 (Runx2), and osteocalcin (OC), representative osteoblastic markers, was assessed using real-time polymerase chain reaction analysis while the protein expression of BMP2, Runx2, and phosphorylated Smad1/5/8 was detected by western blot analysis. Our data demonstrated that MSC-CM inhibits osteoblastic differentiation and mineralization of VSMCs as evidenced by decreased calcium content, ALP activity, and decreased expression of BMP-2, Runx2, Msx2, and OC. MSC-CM suppressed the expression of phosphorylated Smad1/5/8 and the β-GP-induced translocation from the cytoplasm to the nucleus. Further study demonstrated that human recombinant BMP-2 overcame the suppression of VSMC calcification by MSC-CM. MSC-CM may act as a novel therapy for VSMC calcification by mediating the BMP2-Smad1/5/8 signaling pathway.

Thymidine phosphorylase exerts complex effects on bone resorption and formation in myeloma.

PubMed

Liu, Huan; Liu, Zhiqiang; Du, Juan; He, Jin; Lin, Pei; Amini, Behrang; Starbuck, Michael W; Novane, Nora; Shah, Jatin J; Davis, Richard E; Hou, Jian; Gagel, Robert F; Yang, Jing

2016-08-24

Myelomatous bone disease is characterized by the development of lytic bone lesions and a concomitant reduction in bone formation, leading to chronic bone pain and fractures. To understand the underlying mechanism, we investigated the contribution of myeloma-expressed thymidine phosphorylase (TP) to bone lesions. In osteoblast progenitors, TP up-regulated the methylation of RUNX2 and osterix, leading to decreased bone formation. In osteoclast progenitors, TP up-regulated the methylation of IRF8 and thereby enhanced expression of NFATc1 (nuclear factor of activated T cells, cytoplasmic 1 protein), leading to increased bone resorption. TP reversibly catalyzes thymidine into thymine and 2-deoxy-d-ribose (2DDR). Myeloma-secreted 2DDR bound to integrin αVβ3/α5β1 in the progenitors, activated PI3K (phosphoinositide 3-kinase)/Akt signaling, and increased DNMT3A (DNA methyltransferase 3A) expression, resulting in hypermethylation of RUNX2, osterix, and IRF8 This study elucidates an important mechanism for myeloma-induced bone lesions, suggesting that targeting TP may be a viable approach to healing resorbed bone in patients. Because TP overexpression is common in bone-metastatic tumors, our findings could have additional mechanistic implications. Copyright © 2016, American Association for the Advancement of Science.

Tumor Suppressor WWOX inhibits osteosarcoma metastasis by modulating RUNX2 function.

PubMed

Del Mare, Sara; Aqeilan, Rami I

2015-08-10

Osteosarcoma (OS) is among the most frequently occurring primary bone tumors, primarily affecting adolescents and young adults. This malignant osteoid forming tumor is characterized by its metastatic potential, mainly to lungs. We recently demonstrated that WW domain-containing oxidoreductase (WWOX) is frequently inactivated in human OS and that WWOX restoration in WWOX-negative OS cells suppresses tumorigenicity. Of note, WWOX levels are reduced in paired OS samples of post-treatment metastastectomies as compared to pre-treatment biopsies suggesting that decreased WWOX levels are associated with a more aggressive phenotype at the metastatic site. Nevertheless, little is known about WWOX function in OS metastasis. Here, we investigated the role of tumor suppressor WWOX in suppressing pulmonary OS metastasis both in vitro and in vivo. We demonstrated that ectopic expression of WWOX in OS cells, HOS and LM-7, inhibits OS invasion and cell migration in vitro. Furthermore, WWOX expression reduced tumor burden in vivo and inhibited metastases' seeding and colonization. Mechanistically, WWOX function is associated with reduced levels of RUNX2 metastatic target genes implicated in adhesion and motility. Our results suggest that WWOX plays a critical role in determining the aggressive phenotype of OS, and its expression could be an attractive therapeutic target to combat this devastating adolescent disease.

Tumor Suppressor WWOX inhibits osteosarcoma metastasis by modulating RUNX2 function

PubMed Central

Del Mare, Sara; Aqeilan, Rami I.

2015-01-01

Osteosarcoma (OS) is among the most frequently occurring primary bone tumors, primarily affecting adolescents and young adults. This malignant osteoid forming tumor is characterized by its metastatic potential, mainly to lungs. We recently demonstrated that WW domain-containing oxidoreductase (WWOX) is frequently inactivated in human OS and that WWOX restoration in WWOX-negative OS cells suppresses tumorigenicity. Of note, WWOX levels are reduced in paired OS samples of post-treatment metastastectomies as compared to pre-treatment biopsies suggesting that decreased WWOX levels are associated with a more aggressive phenotype at the metastatic site. Nevertheless, little is known about WWOX function in OS metastasis. Here, we investigated the role of tumor suppressor WWOX in suppressing pulmonary OS metastasis both in vitro and in vivo. We demonstrated that ectopic expression of WWOX in OS cells, HOS and LM-7, inhibits OS invasion and cell migration in vitro. Furthermore, WWOX expression reduced tumor burden in vivo and inhibited metastases’ seeding and colonization. Mechanistically, WWOX function is associated with reduced levels of RUNX2 metastatic target genes implicated in adhesion and motility. Our results suggest that WWOX plays a critical role in determining the aggressive phenotype of OS, and its expression could be an attractive therapeutic target to combat this devastating adolescent disease. PMID:26256646

X-ray induced alterations in the differentiation and mineralization potential of murine preosteoblastic cells

NASA Astrophysics Data System (ADS)

Hu, Yueyuan; Lau, Patrick; Baumstark-Khan, Christa; Hellweg, Christine E.; Reitz, Günther

2012-05-01

To evaluate the effects of ionizing radiation (IR) on murine preosteoblastic cell differentiation, we directed OCT-1 cells to the osteoblastic lineage by treatment with a combination of β-glycerophosphate (β-GP), ascorbic acid (AA), and dexamethasone (Dex). In vitro mineralization was evaluated based on histochemical staining and quantification of the hydroxyapatite content of the extracellular bone matrix. Expression of mRNA encoding Runx2, transforming growth factor β1 (TGF-β1), osteocalcin (OCN), and p21CDKN1A was analyzed. Exposure to IR reduced the growth rate and diminished cell survival of OCT-1 cells under standard conditions. Notably, calcium content analysis revealed that deposition of mineralized matrix increased significantly under osteogenic conditions after X-ray exposure in a time-dependent manner. In this study, higher radiation doses exert significant overall effects on TGF-β1, OCN, and p21CDKN1A gene expression, suggesting that gene expression following X-ray treatment is affected in a dose-dependent manner. Additionally, we verified that Runx2 was suppressed within 24 h after irradiation at 2 and 4 Gy. Although further studies are required to verify the molecular mechanism, our observations strongly suggest that treatment with IR markedly alters the differentiation and mineralization process of preosteoblastic cells.

Development of a cell-based high throughput luciferase enzyme fragment complementation assay to identify nuclear-factor-e2-related transcription factor 2 activators.

PubMed

Xie, Wensheng; Pao, Christina; Graham, Taylor; Dul, Ed; Lu, Quinn; Sweitzer, Thomas D; Ames, Robert S; Li, Hu

2012-12-01

Nuclear-factor-E2-related transcription factor 2 (Nrf2) regulates a large panel of Phase II genes and plays an important role in cell survival. Nrf2 activation has been shown as preventing cigarette smoke-induced alveolar enlargement in mice. Therefore, activation of the Nrf2 protein by small-molecule activators represents an attractive therapeutic strategy that is used for chronic obstructive pulmonary disease. In this article, we describe a cell-based luciferase enzyme fragment complementation assay that identifies Nrf2 activators. This assay is based on the interaction of Nrf2 with its nuclear partner MafK or runt-related transcription factor 2 (RunX2) and is dependent on the reconstitution of a "split" luciferase. Firefly luciferase is split into two fragments, which are genetically fused to Nrf2 and MafK or RunX2, respectively. BacMam technology was used to deliver the fusion constructs into cells for expression of the tagged proteins. When the BacMam-transduced cells were treated with Nrf2 activators, the Nrf2 protein was stabilized and translocated into the nucleus where it interacted with MafK or RunX2. The interaction of Nrf2 and MafK or RunX2 brought together the two luciferase fragments that form an active luciferase. The assay was developed in a 384-well format and was optimized by titrating the BacMam concentration, transduction time, cell density, and fetal bovine serum concentration. It was further validated with known Nrf2 activators. Our data show that this assay is robust, sensitive, and amenable to high throughput screening of a large compound collection for the identification of novel Nrf2 activators.

Myeloid leukemia factor is a conserved regulator of RUNX transcription factor activity involved in hematopoiesis.

PubMed

Bras, Stéphanie; Martin-Lannerée, Séverine; Gobert, Vanessa; Augé, Benoît; Breig, Osman; Sanial, Matthieu; Yamaguchi, Masamitsu; Haenlin, Marc; Plessis, Anne; Waltzer, Lucas

2012-03-27

Defining the function of the genes that, like RUNX1, are deregulated in blood cell malignancies represents an important challenge. Myeloid leukemia factors (MLFs) constitute a poorly characterized family of conserved proteins whose founding member, MLF1, has been associated with acute myeloid leukemia in humans. To gain insight into the functions of this family, we investigated the role of the Drosophila MLF homolog during blood cell development. Here we report that mlf controls the homeostasis of the Drosophila hematopoietic system. Notably, mlf participates in a positive feedback loop to fine tune the activity of the RUNX transcription factor Lozenge (LZ) during development of the crystal cells, one of the two main blood cell lineages in Drosophila. At the molecular level, our data in cell cultures and in vivo strongly suggest that MLF controls the number of crystal cells by protecting LZ from degradation. Remarkably, it appears that the human MLF1 protein can substitute for MLF in the crystal cell lineage. In addition, MLF stabilizes the human oncogenic fusion protein RUNX1-ETO and is required for RUNX1-ETO-induced blood cell disorders in a Drosophila model of leukemia. Finally, using the human leukemic blood cell line Kasumi-1, we show that MLF1 depletion impairs RUNX1-ETO accumulation and reduces RUNX1-ETO-dependent proliferation. Thus, we propose that the regulation of RUNX protein levels is a conserved feature of MLF family members that could be critical for normal and pathological blood cell development.

Fibroblast growth factor 2 regulates bone sialoprotein gene transcription in human breast cancer cells.

PubMed

Li, Zhengyang; Wang, Zhitao; Yang, Li; Li, Xinyue; Sasaki, Yoko; Wang, Shuang; Araki, Shouta; Mezawa, Masaru; Takai, Hideki; Nakayama, Youhei; Ogata, Yorimasa

2010-03-01

Bone sialoprotein (BSP) is a major non-collagenous, extracellular matrix glycoprotein associated with mineralized tissues. Fibroblast growth factor 2 (FGF2) is recognized as a potent mitogen for a variety of mesenchymal cells. FGF2 produced by osteoblasts accumulates in the bone matrix and acts as an autocrine/paracrine regulator of osteoblasts. We previously reported that FGF2 regulates BSP gene transcription through the FGF2 response element (FRE) and activator protein 1 (AP1) binding site overlapping with the glucocorticoid response element in the rat BSP gene promoter. In the present study, FGF2 (10 ng/ml) increased BSP and Runx2 mRNA levels at 6 h in MCF7 human breast cancer cells. Transient transfection analyses were performed using chimeric constructs of the human BSP gene promoter linked to a luciferase reporter gene. Treatment of MCF7 cells with FGF2 (10 ng/ml) increased the luciferase activity of the constructs between -84LUC and -927LUC. Gel mobility shift analyses showed that FGF2 increased the binding of AP1 and CRE2. The CRE2- and AP1-protein complexes were disrupted by antibodies against CREB1, c-Fos, c-Jun, Fra2, p300 and Runx2. These studies demonstrate that FGF2 stimulates BSP transcription in MCF7 human breast cancer cells by targeting the AP1 and CRE2 elements in the human BSP gene promoter.

Comparison of Genome-Wide Binding of MyoD in Normal Human Myogenic Cells and Rhabdomyosarcomas Identifies Regional and Local Suppression of Promyogenic Transcription Factors

PubMed Central

MacQuarrie, Kyle L.; Yao, Zizhen; Fong, Abraham P.; Diede, Scott J.; Rudzinski, Erin R.; Hawkins, Douglas S.

2013-01-01

Rhabdomyosarcoma is a pediatric tumor of skeletal muscle that expresses the myogenic basic helix-loop-helix protein MyoD but fails to undergo terminal differentiation. Prior work has determined that DNA binding by MyoD occurs in the tumor cells, but myogenic targets fail to activate. Using MyoD chromatin immunoprecipitation coupled to high-throughput sequencing and gene expression analysis in both primary human muscle cells and RD rhabdomyosarcoma cells, we demonstrate that MyoD binds in a similar genome-wide pattern in both tumor and normal cells but binds poorly at a subset of myogenic genes that fail to activate in the tumor cells. Binding differences are found both across genomic regions and locally at specific sites that are associated with binding motifs for RUNX1, MEF2C, JDP2, and NFIC. These factors are expressed at lower levels in RD cells than muscle cells and rescue myogenesis when expressed in RD cells. MEF2C is located in a genomic region that exhibits poor MyoD binding in RD cells, whereas JDP2 exhibits local DNA hypermethylation in its promoter in both RD cells and primary tumor samples. These results demonstrate that regional and local silencing of differentiation factors contributes to the differentiation defect in rhabdomyosarcomas. PMID:23230269

MicroRNA-34b/c inhibits aldosterone-induced vascular smooth muscle cell calcification via a SATB2/Runx2 pathway.

PubMed

Hao, Jianbing; Zhang, Lei; Cong, Guangting; Ren, Liansheng; Hao, Lirong

2016-12-01

Increasing evidence shows that aldosterone and specific microRNAs (miRs) contribute to vascular smooth muscle cell (VSMC) calcification. In this study, we aim to explore the mechanistic links between miR-34b/c and aldosterone in VSMC calcification. VSMC calcification models were established both in vitro and in vivo. First, the levels of aldosterone, miR-34b/c and special AT-rich sequence-binding protein 2 (SATB2) were measured. Then, miR-34b/c mimics or inhibitors were transfected into VSMCs to evaluate the function of miR-34b/c. Luciferase reporter assays were used to demonstrate whether SATB2 was a direct target of miR-34b/c. Aldosterone and SATB2 were found to be markedly upregulated during VSMC calcification, whereas miR-34b/c expression was downregulated. Treatment with the mineralocorticoid receptor (MR) antagonist eplerenone inhibited VSMC calcification. In aldosterone-induced VSMC calcification, miR-34b/c levels were downregulated and SATB2 protein was upregulated. Furthermore, miR-34b/c overexpression alleviated aldosterone-induced VSMC calcification as well as inhibited the expression of SATB2 protein, whereas miR-34b/c inhibition markedly enhanced VSMC calcification and upregulated SATB2 protein. In addition, luciferase reporter assays showed that SATB2 is a direct target of miR-34b/c in VSMCs. Overexpression of SATB2 induced Runx2 overproduction and VSMC calcification. Therefore, miR-34b/c participates in aldosterone-induced VSMC calcification via a SATB2/Runx2 pathway. As miR-34b/c appears to be a negative regulator, it has potential as a therapeutic target of VSMC calcification.

Biphasic influence of dexamethasone exposure on embryonic vertebrate skeleton development

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cheng, Xin; Chen, Jian-long; Ma, Zheng-lai

Dexamethasone (Dex) has anti-inflammatory and immunomodulatory properties against many conditions. There is a potential teratogenic risk, however, for pregnant women receiving Dex treatment. It has been claimed that Dex exposure during pregnancy could affect osteogenesis in the developing embryo, which still remains highly controversial. In this study, we employed chick embryos to investigate the effects of Dex exposure on skeletal development using combined in vivo and in vitro approach. First, we demonstrated that Dex (10{sup −8}–10{sup −6} μmol/egg) exposure resulted in a shortening of the developing long bones of chick embryos, and it accelerated the deposition of calcium salts. Secondly,more » histological analysis of chick embryo phalanxes exhibited Dex exposure inhibited the proliferation of chondrocytes, increased apoptosis of chondrocytes and osteocytes, and led to atypical arranged hypertrophic chondrocytes. The expression of genes related to skeletogenesis was also analyzed by semi-quantitative RT-PCR. The expression of ALP, Col1a2 and Col2a1 was decreased in the Dex treated phalanxes. A detectable increase was observed in Runx-2 and Mmp-13 expression. We next examined how Dex affected the different stages of skeletogenesis in vitro. Utilizing limb bud mesenchyme micromass cultures, we determined that Dex exposure exerted no effect on apoptosis but impaired chondrogenic cell proliferation. Interestingly, low dose of Dex moderately prompted nodule formation as revealed by alcian blue staining, but higher doses of Dex significantly inhibited similar chondrogenic differentiation. Dex exposure did not induce apoptosis when the chondrogenic precursors were still at the mesenchymal stage, however, cell viability was suppressed when the mesenchyme differentiated into chondrocytes. Alizarin red staining revealed that the capacity to form mineralized bone nodules was correspondingly enhanced as Dex concentrations increased. The mRNA level of Sox-9 was slightly increased in mesenchymal cell mass treated by low concentration of Dex. Mmp-13 expression was obviously up-regulated by Dex in both mesenchymal cells and primary chondrocyte cultures. And Col10a1 expression was also increased by Dex exposure in chondrocyte. In summary, we have revealed that different concentrations of Dex exposure during early gestation could exert a biphasic effect on vertebrate skeletal development. - Highlights: • Chick embryos occurred shortening of the long bone following Dex exposure. • Dex suppressed chondrocytes proliferation and promoted apoptosis. • Dex exposure decreased ALP production and up-regulated Runx-2 and Mmp-13. • Dex exhibited biphasic effects on chondrogenic proliferation and nodule formation. • The hypertrophy and ossification were accelerated by Dex both in vivo and in vitro.« less

Maf promotes osteoblast differentiation in mice by mediating the age-related switch in mesenchymal cell differentiation

PubMed Central

Nishikawa, Keizo; Nakashima, Tomoki; Takeda, Shu; Isogai, Masashi; Hamada, Michito; Kimura, Ayako; Kodama, Tatsuhiko; Yamaguchi, Akira; Owen, Michael J.; Takahashi, Satoru; Takayanagi, Hiroshi

2010-01-01

Aging leads to the disruption of the homeostatic balance of multiple biological systems. In bone marrow multipotent mesenchymal cells undergo differentiation into various anchorage-dependent cell types, including osteoblasts and adipocytes. With age as well as with treatment of antidiabetic drugs such as thiazolidinediones, mesenchymal cells favor differentiation into adipocytes, resulting in an increased number of adipocytes and a decreased number of osteoblasts, causing osteoporosis. The mechanism behind this differentiation switch is unknown. Here we show an age-related decrease in the expression of Maf in mouse mesenchymal cells, which regulated mesenchymal cell bifurcation into osteoblasts and adipocytes by cooperating with the osteogenic transcription factor Runx2 and inhibiting the expression of the adipogenic transcription factor Pparg. The crucial role of Maf in both osteogenesis and adipogenesis was underscored by in vivo observations of delayed bone formation in perinatal Maf–/– mice and an accelerated formation of fatty marrow associated with bone loss in aged Maf+/– mice. This study identifies a transcriptional mechanism for an age-related switch in cell fate determination and may provide a molecular basis for novel therapeutic strategies against age-related bone diseases. PMID:20877012

Myeloid Leukemia Factor Acts in a Chaperone Complex to Regulate Transcription Factor Stability and Gene Expression.

PubMed

Dyer, Jamie O; Dutta, Arnob; Gogol, Madelaine; Weake, Vikki M; Dialynas, George; Wu, Xilan; Seidel, Christopher; Zhang, Ying; Florens, Laurence; Washburn, Michael P; Abmayr, Susan M; Workman, Jerry L

2017-06-30

Mutations that affect myelodysplasia/myeloid leukemia factor (MLF) proteins are associated with leukemia and several other cancers. However, with no strong homology to other proteins of known function, the role of MLF proteins in the cell has remained elusive. Here, we describe a proteomics approach that identifies MLF as a member of a nuclear chaperone complex containing a DnaJ protein, BCL2-associated anthanogene 2, and Hsc70. This complex associates with chromatin and regulates the expression of target genes. The MLF complex is bound to sites of nucleosome depletion and sites containing active chromatin marks (e.g., H3K4me3 and H3K4me1). Hence, MLF binding is enriched at promoters and enhancers. Additionally, the MLF-chaperone complex functions to regulate transcription factor stability, including the RUNX transcription factor involved in hematopoiesis. Although Hsc70 and other co-chaperones have been shown to play a role in nuclear translocation of a variety of proteins including transcription factors, our findings suggest that MLF and the associated co-chaperones play a direct role in modulating gene transcription. Copyright © 2016 Elsevier Ltd. All rights reserved.

DLX3 regulates bone mass by targeting genes supporting osteoblast differentiation and mineral homeostasis in vivo

PubMed Central

Isaac, J; Erthal, J; Gordon, J; Duverger, O; Sun, H-W; Lichtler, A C; Stein, G S; Lian, J B; Morasso, M I

2014-01-01

Human mutations and in vitro studies indicate that DLX3 has a crucial function in bone development, however, the in vivo role of DLX3 in endochondral ossification has not been established. Here, we identify DLX3 as a central attenuator of adult bone mass in the appendicular skeleton. Dynamic bone formation, histologic and micro-computed tomography analyses demonstrate that in vivo DLX3 conditional loss of function in mesenchymal cells (Prx1-Cre) and osteoblasts (OCN-Cre) results in increased bone mass accrual observed as early as 2 weeks that remains elevated throughout the lifespan owing to increased osteoblast activity and increased expression of bone matrix genes. Dlx3OCN-conditional knockout mice have more trabeculae that extend deeper in the medullary cavity and thicker cortical bone with an increased mineral apposition rate, decreased bone mineral density and increased cortical porosity. Trabecular TRAP staining and site-specific Q-PCR demonstrated that osteoclastic resorption remained normal on trabecular bone, whereas cortical bone exhibited altered osteoclast patterning on the periosteal surface associated with high Opg/Rankl ratios. Using RNA sequencing and chromatin immunoprecipitation-Seq analyses, we demonstrate that DLX3 regulates transcription factors crucial for bone formation such as Dlx5, Dlx6, Runx2 and Sp7 as well as genes important to mineral deposition (Ibsp, Enpp1, Mepe) and bone turnover (Opg). Furthermore, with the removal of DLX3, we observe increased occupancy of DLX5, as well as increased and earlier occupancy of RUNX2 on the bone-specific osteocalcin promoter. Together, these findings provide novel insight into mechanisms by which DLX3 attenuates bone mass accrual to support bone homeostasis by osteogenic gene pathway regulation. PMID:24948010

Redundant miR-3077-5p and miR-705 mediate the shift of mesenchymal stem cell lineage commitment to adipocyte in osteoporosis bone marrow.

PubMed

Liao, L; Yang, X; Su, X; Hu, C; Zhu, X; Yang, N; Chen, X; Shi, S; Shi, S; Jin, Y

2013-04-18

During the process of aging, especially for postmenopausal females, the cell lineage commitment of mesenchymal stem cells (MSCs) shift to adipocyte in bone marrow, resulting in osteoporosis. However, the cell-intrinsic mechanism of this cell lineage commitment switch is poorly understood. As the post-transcription regulation by microRNAs (miRNAs) has a critical role in MSCs differentiation and bone homeostasis, we performed comprehensive miRNAs profiling and found miR-705 and miR-3077-5p were significantly enhanced in MSCs from osteoporosis bone marrow. Both miR-705 and miR-3077-5p acted as inhibitors of MSCs osteoblast differentiation and promoters of adipocyte differentiation, by targeting on the 3'untranslated region (3'UTR) of HOXA10 and RUNX2 mRNA separately. Combined inhibition of miR-705 and miR-3077-5p rescued the cell lineage commitment disorder of MSCs through restoring HOXA10 and RUNX2 protein level. Furthermore, we found excessive TNFα and reactive oxygen species caused by estrogen deficiency led to the upregulation of both miRNAs through NF-κB pathway. In conclusion, our findings showed that redundant miR-705 and miR-3077-5p synergistically mediated the shift of MSCs cell lineage commitment to adipocyte in osteoporosis bone marrow, providing new insight into the etiology of osteoporosis at the post-transcriptional level. Moreover, the rescue of MSCs lineage commitment disorder by regulating miRNAs expression suggested a novel potential therapeutic target for osteoporosis as well as stem cell-mediated regenerative medicine.

Myeloid leukemia factor is a conserved regulator of RUNX transcription factor activity involved in hematopoiesis

PubMed Central

Bras, Stéphanie; Martin-Lannerée, Séverine; Gobert, Vanessa; Augé, Benoît; Breig, Osman; Sanial, Matthieu; Yamaguchi, Masamitsu; Haenlin, Marc; Plessis, Anne; Waltzer, Lucas

2012-01-01

Defining the function of the genes that, like RUNX1, are deregulated in blood cell malignancies represents an important challenge. Myeloid leukemia factors (MLFs) constitute a poorly characterized family of conserved proteins whose founding member, MLF1, has been associated with acute myeloid leukemia in humans. To gain insight into the functions of this family, we investigated the role of the Drosophila MLF homolog during blood cell development. Here we report that mlf controls the homeostasis of the Drosophila hematopoietic system. Notably, mlf participates in a positive feedback loop to fine tune the activity of the RUNX transcription factor Lozenge (LZ) during development of the crystal cells, one of the two main blood cell lineages in Drosophila. At the molecular level, our data in cell cultures and in vivo strongly suggest that MLF controls the number of crystal cells by protecting LZ from degradation. Remarkably, it appears that the human MLF1 protein can substitute for MLF in the crystal cell lineage. In addition, MLF stabilizes the human oncogenic fusion protein RUNX1-ETO and is required for RUNX1-ETO–induced blood cell disorders in a Drosophila model of leukemia. Finally, using the human leukemic blood cell line Kasumi-1, we show that MLF1 depletion impairs RUNX1-ETO accumulation and reduces RUNX1-ETO–dependent proliferation. Thus, we propose that the regulation of RUNX protein levels is a conserved feature of MLF family members that could be critical for normal and pathological blood cell development. PMID:22411814

Integrated analysis of gene expression and methylation profiles of 48 candidate genes in breast cancer patients.

PubMed

Li, Zibo; Heng, Jianfu; Yan, Jinhua; Guo, Xinwu; Tang, Lili; Chen, Ming; Peng, Limin; Wu, Yepeng; Wang, Shouman; Xiao, Zhi; Deng, Zhongping; Dai, Lizhong; Wang, Jun

2016-11-01

Gene-specific methylation and expression have shown biological and clinical importance for breast cancer diagnosis and prognosis. Integrated analysis of gene methylation and gene expression may identify genes associated with biology mechanism and clinical outcome of breast cancer and aid in clinical management. Using high-throughput microfluidic quantitative PCR, we analyzed the expression profiles of 48 candidate genes in 96 Chinese breast cancer patients and investigated their correlation with gene methylation and associations with breast cancer clinical parameters. Breast cancer-specific gene expression alternation was found in 25 genes with significant expression difference between paired tumor and normal tissues. A total of 9 genes (CCND2, EGFR, GSTP1, PGR, PTGS2, RECK, SOX17, TNFRSF10D, and WIF1) showed significant negative correlation between methylation and gene expression, which were validated in the TCGA database. Total 23 genes (ACADL, APC, BRCA2, CADM1, CAV1, CCND2, CST6, EGFR, ESR2, GSTP1, ICAM5, NPY, PGR, PTGS2, RECK, RUNX3, SFRP1, SOX17, SYK, TGFBR2, TNFRSF10D, WIF1, and WRN) annotated with potential TFBSs in the promoter regions showed negative correlation between methylation and expression. In logistics regression analysis, 31 of the 48 genes showed improved performance in disease prediction with combination of methylation and expression coefficient. Our results demonstrated the complex correlation and the possible regulatory mechanisms between DNA methylation and gene expression. Integration analysis of methylation and expression of candidate genes could improve performance in breast cancer prediction. These findings would contribute to molecular characterization and identification of biomarkers for potential clinical applications.

Identification of ETV6-RUNX1-like and DUX4-rearranged subtypes in paediatric B-cell precursor acute lymphoblastic leukaemia.

PubMed

Lilljebjörn, Henrik; Henningsson, Rasmus; Hyrenius-Wittsten, Axel; Olsson, Linda; Orsmark-Pietras, Christina; von Palffy, Sofia; Askmyr, Maria; Rissler, Marianne; Schrappe, Martin; Cario, Gunnar; Castor, Anders; Pronk, Cornelis J H; Behrendtz, Mikael; Mitelman, Felix; Johansson, Bertil; Paulsson, Kajsa; Andersson, Anna K; Fontes, Magnus; Fioretos, Thoas

2016-06-06

Fusion genes are potent driver mutations in cancer. In this study, we delineate the fusion gene landscape in a consecutive series of 195 paediatric B-cell precursor acute lymphoblastic leukaemia (BCP ALL). Using RNA sequencing, we find in-frame fusion genes in 127 (65%) cases, including 27 novel fusions. We describe a subtype characterized by recurrent IGH-DUX4 or ERG-DUX4 fusions, representing 4% of cases, leading to overexpression of DUX4 and frequently co-occurring with intragenic ERG deletions. Furthermore, we identify a subtype characterized by an ETV6-RUNX1-like gene-expression profile and coexisting ETV6 and IKZF1 alterations. Thus, this study provides a detailed overview of fusion genes in paediatric BCP ALL and adds new pathogenetic insights, which may improve risk stratification and provide therapeutic options for this disease.

Osteo-maturation of adipose-derived stem cells required the combined action of vitamin D3, beta-glycerophosphate, and ascorbic acid.

PubMed

Gupta, Anurag; Leong, David Tai; Bai, Hui Fen; Singh, Shiv Brat; Lim, Thiam-Chye; Hutmacher, Dietmar Werner

2007-10-12

This study investigated the effects of various components [vitamin D3 (VD3), beta-glycerophosphate (BGP), and ascorbic acid (AA)] on the potential of human adipose-derived progenitor cells (ADPCs) to transdifferentiate into osteoblast-like cells. ADPCs were induced under four different supplement groups: (1) VD3+BGP+AA, (2) VD3 alone, (3) BGP+AA, and (4) no VD3, BGP or AA. Mineralization studies and presence of bone matrix-related proteins by immunostaining showed that the Group 1 ADPCs showed their ability to undergo osteoblastic differentiation. Further evaluation was made by estimation of levels of RUNX-2 and TAZ genes. Group 1 ADPCs showed the consistent expression of RUNX-2 and TAZ levels over the study period of 28days. The study showed good correlation among various parameters evaluated to conclude that ADPCs could be an alternative source for generating osteoblast-like cells.

Effects of dexamethasone, ascorbic acid and β-glycerophosphate on the osteogenic differentiation of stem cells in vitro.

PubMed

Langenbach, Fabian; Handschel, Jörg

2013-01-01

The standard procedure for the osteogenic differentiation of multipotent stem cells is treatment of a confluent monolayer with a cocktail of dexamethasone (Dex), ascorbic acid (Asc) and β-glycerophosphate (β-Gly). This review describes the effects of these substances on intracellular signaling cascades that lead to osteogenic differentiation of bone marrow stroma-derived stem cells. We conclude that Dex induces Runx2 expression by FHL2/β-catenin-mediated transcriptional activation and that Dex enhances Runx2 activity by upregulation of TAZ and MKP1. Asc leads to the increased secretion of collagen type I (Col1), which in turn leads to increased Col1/α2β1 integrin-mediated intracellular signaling. The phosphate from β-Gly serves as a source for the phosphate in hydroxylapatite and in addition influences intracellular signaling molecules. In this context we give special attention to the differences between dystrophic and bone-specific mineralization.

Anabolic and Antiresorptive Modulation of Bone Homeostasis by the Epigenetic Modulator Sulforaphane, a Naturally Occurring Isothiocyanate*

PubMed Central

Thaler, Roman; Maurizi, Antonio; Roschger, Paul; Sturmlechner, Ines; Khani, Farzaneh; Spitzer, Silvia; Rumpler, Monika; Zwerina, Jochen; Karlic, Heidrun; Dudakovic, Amel; Klaushofer, Klaus; Teti, Anna; Rucci, Nadia; Varga, Franz; van Wijnen, Andre J.

2016-01-01

Bone degenerative pathologies like osteoporosis may be initiated by age-related shifts in anabolic and catabolic responses that control bone homeostasis. Here we show that sulforaphane (SFN), a naturally occurring isothiocyanate, promotes osteoblast differentiation by epigenetic mechanisms. SFN enhances active DNA demethylation via Tet1 and Tet2 and promotes preosteoblast differentiation by enhancing extracellular matrix mineralization and the expression of osteoblastic markers (Runx2, Col1a1, Bglap2, Sp7, Atf4, and Alpl). SFN decreases the expression of the osteoclast activator receptor activator of nuclear factor-κB ligand (RANKL) in osteocytes and mouse calvarial explants and preferentially induces apoptosis in preosteoclastic cells via up-regulation of the Tet1/Fas/Caspase 8 and Caspase 3/7 pathway. These mechanistic effects correlate with higher bone volume (∼20%) in both normal and ovariectomized mice treated with SFN for 5 weeks compared with untreated mice as determined by microcomputed tomography. This effect is due to a higher trabecular number in these mice. Importantly, no shifts in mineral density distribution are observed upon SFN treatment as measured by quantitative backscattered electron imaging. Our data indicate that the food-derived compound SFN epigenetically stimulates osteoblast activity and diminishes osteoclast bone resorption, shifting the balance of bone homeostasis and favoring bone acquisition and/or mitigation of bone resorption in vivo. Thus, SFN is a member of a new class of epigenetic compounds that could be considered for novel strategies to counteract osteoporosis. PMID:26757819

AP1 binding site is another target of FGF2 regulation of bone sialoprotein gene transcription.

PubMed

Takai, Hideki; Araki, Shouta; Mezawa, Masaru; Kim, Dong-Soon; Li, Xinyue; Yang, Li; Li, Zhengyang; Wang, Zhitao; Nakayama, Youhei; Ogata, Yorimasa

2008-02-29

Bone sialoprotein (BSP) is an early marker of osteoblast differentiation. We previously reported that fibroblast growth factor 2 (FGF2) regulates BSP gene transcription via FGF2 response element (FRE) in the proximal promoter of rat BSP gene. We here report that activator protein 1 (AP1) binding site overlapping with glucocorticoid response element (GRE) AP1/GRE in the rat BSP gene promoter is another target of FGF2. Using the osteoblastic cell line ROS17/2.8, we determined that BSP mRNA levels increased by 10 ng/ml FGF2 at 6 and 12 h. Runx2 protein levels increased by FGF2 (10 ng/ml) at 3 h. Treatment of ROS17/2.8 cells with FGF2 (10 ng/ml, 12 h) increased luciferase activities of constructs including -116 to +60 and -938 to +60 of the rat BSP gene promoter. Effects of FGF2 abrogated in constructs included 2 bp mutations in the FRE and AP1/GRE elements. Luciferase activities induced by FGF2 were blocked by tyrosine kinase inhibitor herbimycin A, src-tyrosine kinase inhibitor PP1 and MAP kinase kinase inhibitor U0126. Gel shift analyses showed that FGF2 increased binding of FRE and AP1/GRE elements. Notably, the AP1/GRE-protein complexes were supershifted by Smad1 and c-Fos antibodies, c-Jun and Dlx5 antibodies disrupted the complexes formation, on the other hand AP1/GRE-protein complexes did not change by Runx2 antibody. These studies demonstrate that FGF2 stimulates BSP gene transcription by targeting the FRE and AP1/GRE elements in the rat BSP gene promoter.

Gravity affects the responsiveness of Runx2 to 1, 25-dihydroxyvitamin D3 (VD3)

NASA Astrophysics Data System (ADS)

Guo, Feima; Dai, Zhongquan; Wu, Feng; Liu, Zhaoxia; Tan, Yingjun; Wan, Yumin; Shang, Peng; Li, Yinghui

2013-03-01

Bone loss resulting from spaceflight is mainly caused by decreased bone formation, and decreased osteoblast proliferation and differentiation. Transcription factor Runx2 plays an important role in osteoblast differentiation and function by responding to microenvironment changes including cytokine and mechanical factors. The effects of 1, 25-dihydroxyvitamin D3 (VD3) on Runx2 in terms of mechanical competence is far less clear. This study describes how gravity affects the response of Runx2 to VD3. A MC3T3-6OSE2-Luc osteoblast model was constructed in which the activity of Runx2 was reflected by reporter luciferase activity identifed by bone-related cytokines. The results showed that luciferase activity in MC3T3-6OSE2-Luc cells transfected with Runx2 was twice that of the vacant vector. Alkaline phosphatase (ALP) activity was increased in MC3T3-6OSE2-Luc cells by different concentrations of IGF-I and BMP2. MC3T3-6OSE2-Luc cells were cultured under simulated microgravity or centrifuge with or without VD3. In simulated microgravity, luciferase activity was decreased after 48 h of clinorotation culture, but increased in the centrifuge culture. Luciferase activity was increased after VD3 treatment in normal conditions and simulated microgravity, the increase in luciferase activity in simulated microgravity was lower than that in the 1 g condition when simultaneously treated with VD3 and higher than that in the centrifuge condition. Co-immunoprecipitation showed that the interaction between the VD3 receptor (VDR) and Runx2 was decreased by simulated microgravity, but increased by centrifugation. From these results, we conclude that gravity affects the response of Runx2 to VD3 which results from an alteration in the interaction between VDR and Runx2 under different gravity conditions.

Vitamin C treatment promotes mesenchymal stem cell sheet formation and tissue regeneration by elevating telomerase activity.

PubMed

Wei, Fulan; Qu, Cunye; Song, Tieli; Ding, Gang; Fan, Zhipeng; Liu, Dayong; Liu, Yi; Zhang, Chunmei; Shi, Songtao; Wang, Songlin

2012-09-01

Cell sheet engineering has been developed as an alternative approach to improve mesenchymal stem cell-mediated tissue regeneration. In this study, we found that vitamin C (Vc) was capable of inducing telomerase activity in periodontal ligament stem cells (PDLSCs), leading to the up-regulated expression of extracellular matrix type I collagen, fibronectin, and integrin β1, stem cell markers Oct4, Sox2, and Nanog as well as osteogenic markers RUNX2, ALP, OCN. Under Vc treatment, PDLSCs can form cell sheet structures because of increased cell matrix production. Interestingly, PDLSC sheets demonstrated a significant improvement in tissue regeneration compared with untreated control dissociated PDLSCs and offered an effective treatment for periodontal defects in a swine model. In addition, bone marrow mesenchymal stem cell sheets and umbilical cord mesenchymal stem cell sheets were also well constructed using this method. The development of Vc-mediated mesenchymal stem cell sheets may provide an easy and practical approach for cell-based tissue regeneration. Copyright © 2011 Wiley Periodicals, Inc.

Haematopoietic stem and progenitor cells from human pluripotent stem cells

PubMed Central

Sugimura, Ryohichi; Jha, Deepak Kumar; Han, Areum; Soria-Valles, Clara; da Rocha, Edroaldo Lummertz; Lu, Yi-Fen; Goettel, Jeremy A.; Serrao, Erik; Rowe, R. Grant; Malleshaiah, Mohan; Wong, Irene; Sousa, Patricia; Zhu, Ted N.; Ditadi, Andrea; Keller, Gordon; Engelman, Alan N.; Snapper, Scott B.; Doulatov, Sergei; Daley, George Q.

2018-01-01

A variety of tissue lineages can be differentiated from pluripotent stem cells by mimicking embryonic development through stepwise exposure to morphogens, or by conversion of one differentiated cell type into another by enforced expression of master transcription factors. Here, to yield functional human haematopoietic stem cells, we perform morphogen-directed differentiation of human pluripotent stem cells into haemogenic endothelium followed by screening of 26 candidate haematopoietic stem-cell-specifying transcription factors for their capacity to promote multi-lineage haematopoietic engraftment in mouse hosts. We recover seven transcription factors (ERG, HOXA5, HOXA9, HOXA10, LCOR, RUNX1 and SPI1) that are sufficient to convert haemogenic endothelium into haematopoietic stem and progenitor cells that engraft myeloid, B and T cells in primary and secondary mouse recipients. Our combined approach of morphogen-driven differentiation and transcription-factor-mediated cell fate conversion produces haematopoietic stem and progenitor cells from pluripotent stem cells and holds promise for modelling haematopoietic disease in humanized mice and for therapeutic strategies in genetic blood disorders. PMID:28514439

Adenosine signaling promotes hematopoietic stem and progenitor cell emergence

PubMed Central

Jing, Lili; Tamplin, Owen J.; Chen, Michael J.; Deng, Qing; Patterson, Shenia; Kim, Peter G.; Durand, Ellen M.; McNeil, Ashley; Green, Julie M.; Matsuura, Shinobu; Ablain, Julien; Brandt, Margot K.; Schlaeger, Thorsten M.; Huttenlocher, Anna; Daley, George Q.; Ravid, Katya

2015-01-01

Hematopoietic stem cells (HSCs) emerge from aortic endothelium via the endothelial-to-hematopoietic transition (EHT). The molecular mechanisms that initiate and regulate EHT remain poorly understood. Here, we show that adenosine signaling regulates hematopoietic stem and progenitor cell (HSPC) development in zebrafish embryos. The adenosine receptor A2b is expressed in the vascular endothelium before HSPC emergence. Elevated adenosine levels increased runx1+/cmyb+ HSPCs in the dorsal aorta, whereas blocking the adenosine pathway decreased HSPCs. Knockdown of A2b adenosine receptor disrupted scl+ hemogenic vascular endothelium and the subsequent EHT process. A2b adenosine receptor activation induced CXCL8 via cAMP–protein kinase A (PKA) and mediated hematopoiesis. We further show that adenosine increased multipotent progenitors in a mouse embryonic stem cell colony-forming assay and in embryonic day 10.5 aorta-gonad-mesonephros explants. Our results demonstrate that adenosine signaling plays an evolutionary conserved role in the first steps of HSPC formation in vertebrates. PMID:25870200

Transforming growth factor-beta1 promotes articular cartilage repair through canonical Smad and Hippo pathways in bone mesenchymal stem cells.

PubMed

Ying, Jun; Wang, Pinger; Zhang, Shanxing; Xu, Taotao; Zhang, Lei; Dong, Rui; Xu, Shibing; Tong, Peijian; Wu, Chengliang; Jin, Hongting

2018-01-01

Transforming growth factor-β1 (TGF-β1) is a chondrogenic factor and has been reported to be able to enhance chondrocyte differentiation from bone marrow mesenchymal stem cells (BMSCs). Here we investigate the molecular mechanism through which TGF-β1 chronically promotes the repair of cartilage defect and inhibit chondrocyte hypertrophy. Animal models of full thickness cartilage defects were divided into three groups: model group, BMSCs group (treated with BMSCs/calcium alginate gel) and BMSCs+TGF-β1 group (treated with Lentivirus-TGF-β1-EGFP transduced BMSCs/calcium alginate gel). 4 and 8weeks after treatment, macroscopic observation, histopathological study and quantitative reverse transcription-polymerase chain reaction (qRT-PCR) were done to analyze phenotypes of the animals. BMSCs were transduced with Lentivirus-TGF-β1-EGFP in vitro and Western blot analysis was performed. We found that TGF-β1-expressiing BMSCs improved the repair of the cartilage defect. The impaired cartilage contained higher amount of GAG and type II collagen and was integrated to the surrounding normal cartilage and higher content of GAG and type II collagen. The major events include increased expression of type II collagen following Smad2/3 phosphorylation, and inhibition of cartilage hypertrophy by increasing Yes-associated protein-1 (YAP-1) and inhibiting Runx2 and Col10 after the completion of chondrogenic differentiation. We conclude that TGF-β1 is beneficial to chondrogenic differentiation of BMSCs via canonical Smad pathway to promote early-repairing of cartilage defect. Furthermore, TGF-β1 inhibits chondrocyte hypertrophy by decreasing hypertrophy marker gene expression via Hippo signaling. Long-term rational use of TGF-β1 may be an alternative approach in clinic for cartilage repair and regeneration. Copyright © 2017. Published by Elsevier Inc.

The effect of globin scaffold on osteoblast adhesion and phenotype expression in vitro.

PubMed

Hamdan, Ahmad A; Loty, Sabine; Isaac, Juliane; Tayot, Jean-Louis; Bouchard, Philippe; Khraisat, Ameen; Bedral, Ariane; Sautier, Jean-Michel

2012-01-01

Different synthetic and natural biomaterials have been used in bone tissue regeneration. However, several limitations are associated with the use of synthetic as well as allogenous or xenogenous natural materials. This study evaluated, in an in vitro model, the behavior of rat osteoblastic cells cultured on a human globin scaffold. Rat osteoblastic cells were isolated from the calvaria of 21-day-old fetal Sprague-Dawley rats. They were then grown in the presence of globin. Real-time polymerase chain reaction (RT-PCR) was performed to study the expression of cyclin D1, integrin Β1, Msx2, Dlx5, Runx2, and osteocalcin on days 1, 5, and 9. Moreover, alkaline phosphatase activity was measured on days 1, 3, 5, and 7. Alizarin red staining was performed on day 9 to observe calcium deposition. Cells were able to adhere, proliferate, and differentiate on globin scaffolds. Moreover, RT-PCR showed that globin may stimulate some key genes of osteoblastic differentiation (Runx2, osteocalcin, Dlx5). Globin had an inhibitory effect on alkaline phosphatase activity. Calcium deposits were seen after 9 days of culture. These results indicate that purified human globin might be a suitable scaffold for bone tissue regeneration.

Characterization and comparison of osteoblasts derived from mouse embryonic stem cells and induced pluripotent stem cells.

PubMed

Ma, Ming-San; Kannan, Vishnu; de Vries, Anneriek E; Czepiel, Marcin; Wesseling, Evelyn M; Balasubramaniyan, Veerakumar; Kuijer, Roel; Vissink, Arjan; Copray, Sjef C V M; Raghoebar, Gerry M

2017-01-01

New developments in stem cell biology offer alternatives for the reconstruction of critical-sized bone defects. One of these developments is the use of induced pluripotent stem (iPS) cells. These stem cells are similar to embryonic stem (ES) cells, but can be generated from adult somatic cells and therefore do not raise ethical concerns. Proper characterization of iPS-derived osteoblasts is important for future development of safe clinical applications of these cells. For this reason, we differentiated mouse ES and iPS cells toward osteoblasts using osteogenic medium and compared their functionality. Immunocytochemical analysis showed significant expression of bone markers (osteocalcin and collagen type I) in osteoblasts differentiated from ES and iPS cells on days 7 and 30. An in vitro mineralization assay confirmed the functionality of osteogenically differentiated ES and iPS cells. Gene expression arrays focusing on osteogenic differentiation were performed in order to compare the gene expression pattern in both differentiated and undifferentiated ES cells and iPS cells. We observed a significant upregulation of osteogenesis-related genes such as Runx2, osteopontin, collagen type I, Tnfsf11, Csf1, and alkaline phosphatase upon osteogenic differentiation of the ES and iPS cells. We further validated the expression of key osteogenic genes Runx2, osteopontin, osteocalcin, collagen type I, and osterix in both differentiated and undifferentiated ES and iPS cells by means of quantified real-time polymerase chain reaction. We conclude that ES and iPS cells are similar in their osteogenic differentiation capacities, as well as in their gene expression patterns.

Biochemical changes of salivary gland adenoid cystic carcinoma cells induced by SGI-1776

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hou, Xiuxiu, E-mail: show-1989@163.com; The First Affiliated Hospital of Wenzhou Medical University, Wenzhou 325000; Yu, Yunfang, E-mail: yyf_8247425@163.com

Provirus integration site for Moloney murine leukemia virus 1 (Pim-1) has proved to be an oncogene and it is known that to depress Pim-1 activity may be a novel oncological treatment strategy. SGI-1776, a small molecule, is the first clinically tested inhibitor of the Pim kinase family. Here, we aimed to explore the effect of SGI-1776 on salivary adenoid cystic carcinoma (SACC). Expression of Pim-1 was confirmed in SACC and control tissues by qRT-PCR. After SGI-1776 treatment, the Pim-1 expressions and Pim-1 kinase activity in both SACC-83 and SACC-LM cell lines were measured. Cell proliferation, cell invasion, cell cycle, apoptosismore » and mitochondrial membrane potential were analyzed. Also, the expression of FOXO3a, p-FOXO3a, RUNX3, Bcl-2, BAD, p-BAD, Bim and p-Bim were detected by Western blot. The results showed that Pim-1 was significantly overexpressed in SACC tissues. SGI-1776 down-regulated the Pim-1 expression, inhibited Pim-1 kinase activity, reduced cell proliferation, decreased invasive ability, increased caspase-3 activity and induced apoptosis, cell cycle arrest and mitochondrial depolarization. Reduced expression was also seen in p-FOXO3a, RUNX3, Bcl-2, p-BAD and p-Bim, whereas no significant changes were observed from FOXO3a, BAD and Bim. These results confirm the pivotal role of Pim-1 in SACC and suggest that targeting Pim-1 kinase signal pathway by SGI-1776 might be a promising therapeutic modality for SACC.« less

Biochemical changes of salivary gland adenoid cystic carcinoma cells induced by SGI-1776.

PubMed

Hou, Xiuxiu; Yu, Yunfang; Feng, Jianguo; Wang, Jiafeng; Zheng, Chuanming; Ling, Zhiqiang; Ge, Minghua; Zhu, Xin

2017-03-15

Provirus integration site for Moloney murine leukemia virus 1 (Pim-1) has proved to be an oncogene and it is known that to depress Pim-1 activity may be a novel oncological treatment strategy. SGI-1776, a small molecule, is the first clinically tested inhibitor of the Pim kinase family. Here, we aimed to explore the effect of SGI-1776 on salivary adenoid cystic carcinoma (SACC). Expression of Pim-1 was confirmed in SACC and control tissues by qRT-PCR. After SGI-1776 treatment, the Pim-1 expressions and Pim-1 kinase activity in both SACC-83 and SACC-LM cell lines were measured. Cell proliferation, cell invasion, cell cycle, apoptosis and mitochondrial membrane potential were analyzed. Also, the expression of FOXO3a, p-FOXO3a, RUNX3, Bcl-2, BAD, p-BAD, Bim and p-Bim were detected by Western blot. The results showed that Pim-1 was significantly overexpressed in SACC tissues. SGI-1776 down-regulated the Pim-1 expression, inhibited Pim-1 kinase activity, reduced cell proliferation, decreased invasive ability, increased caspase-3 activity and induced apoptosis, cell cycle arrest and mitochondrial depolarization. Reduced expression was also seen in p-FOXO3a, RUNX3, Bcl-2, p-BAD and p-Bim, whereas no significant changes were observed from FOXO3a, BAD and Bim. These results confirm the pivotal role of Pim-1 in SACC and suggest that targeting Pim-1 kinase signal pathway by SGI-1776 might be a promising therapeutic modality for SACC. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Targeted reversion of induced pluripotent stem cells from patients with human cleidocranial dysplasia improves bone regeneration in a rat calvarial bone defect model.

PubMed

Saito, Akiko; Ooki, Akio; Nakamura, Takashi; Onodera, Shoko; Hayashi, Kamichika; Hasegawa, Daigo; Okudaira, Takahito; Watanabe, Katsuhito; Kato, Hiroshi; Onda, Takeshi; Watanabe, Akira; Kosaki, Kenjiro; Nishimura, Ken; Ohtaka, Manami; Nakanishi, Mahito; Sakamoto, Teruo; Yamaguchi, Akira; Sueishi, Kenji; Azuma, Toshifumi

2018-01-22

Runt-related transcription factor 2 (RUNX2) haploinsufficiency causes cleidocranial dysplasia (CCD) which is characterized by supernumerary teeth, short stature, clavicular dysplasia, and osteoporosis. At present, as a therapeutic strategy for osteoporosis, mesenchymal stem cell (MSC) transplantation therapy is performed in addition to drug therapy. However, MSC-based therapy for osteoporosis in CCD patients is difficult due to a reduction in the ability of MSCs to differentiate into osteoblasts resulting from impaired RUNX2 function. Here, we investigated whether induced pluripotent stem cells (iPSCs) properly differentiate into osteoblasts after repairing the RUNX2 mutation in iPSCs derived from CCD patients to establish normal iPSCs, and whether engraftment of osteoblasts derived from properly reverted iPSCs results in better regeneration in immunodeficient rat calvarial bone defect models. Two cases of CCD patient-derived induced pluripotent stem cells (CCD-iPSCs) were generated using retroviral vectors (OCT3/4, SOX2, KLF4, and c-MYC) or a Sendai virus SeVdp vector (KOSM302L). Reverted iPSCs were established using programmable nucleases, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas-derived RNA-guided endonucleases, to correct mutations in CCD-iPSCs. The mRNA expressions of osteoblast-specific markers were analyzed using quantitative reverse-transcriptase polymerase chain reaction. iPSCs-derived osteoblasts were transplanted into rat calvarial bone defects, and bone regeneration was evaluated using microcomputed tomography analysis and histological analysis. Mutation analysis showed that both contained nonsense mutations: one at the very beginning of exon 1 and the other at the initial position of the nuclear matrix-targeting signal. The osteoblasts derived from CCD-iPSCs (CCD-OBs) expressed low levels of several osteoblast differentiation markers, and transplantation of these osteoblasts into calvarial bone defects created in rats with severe combined immunodeficiency showed poor regeneration. However, reverted iPSCs improved the abnormal osteoblast differentiation which resulted in much better engraftment into the rat calvarial bone defect. Taken together, these results demonstrate that patient-specific iPSC technology can not only provide a useful disease model to elucidate the role of RUNX2 in osteoblastic differentiation but also raises the tantalizing prospect that reverted iPSCs might provide a practical medical treatment for CCD.

Effects of intermittent versus continuous parathyroid hormone administration on condylar chondrocyte proliferation and differentiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Qi; Wan, Qilong; Yang, Rongtao

Highlights: Black-Right-Pointing-Pointer Different PTH administration exerts different effects on condylar chondrocyte. Black-Right-Pointing-Pointer Intermittent PTH administration suppresses condylar chondrocyte proliferation. Black-Right-Pointing-Pointer Continuous PTH administration maintains condylar chondrocyte proliferating. Black-Right-Pointing-Pointer Intermittent PTH administration enhances condylar chondrocyte differentiation. -- Abstract: Endochondral ossification is a complex process involving chondrogenesis and osteogenesis regulated by many hormones and growth factors. Parathyroid hormone (PTH), one of the key hormones regulating bone metabolism, promotes osteoblast differentiation and osteogenesis by intermittent administration, whereas continuous PTH administration inhibits bone formation. However, the effects of PTH on chondrocyte proliferation and differentiation are still unclear. In this study, intermittent PTH administration presentedmore » enhanced effects on condylar chondrocyte differentiation and bone formation, as demonstrated by increased mineral nodule formation and alkaline phosphatase (ALP) activity, up-regulated runt-related transcription factor 2 (RUNX2), ALP, collagen type X (COL10a1), collagen type I (COL1a1), osteocalcin (OCN), bone sialoprotein (BSP), bone morphogenetic protein 2 (BMP2) and osterix (OSX) mRNA and/or protein expression. On the contrary, continuous PTH administration promoted condylar chondrocyte proliferation and suppressed its differentiation, as demonstrated by up-regulated collagen type II (COL2a1) mRNA expression, reduced mineral nodule formation and down-regulated expression of the mRNAs and/or proteins mentioned above. Our data suggest that PTH can regulate condylar chondrocyte proliferation and differentiation, depending on the type of PTH administration. These results provide new insight into the effects of PTH on condylar chondrocytes and new evidence for using local PTH administration to cure mandibular asymmetry.« less

Incorporation of cerium oxide into hydroxyapatite coating regulates osteogenic activity of mesenchymal stem cell and macrophage polarization.

PubMed

Li, Kai; Shen, Qingyi; Xie, Youtao; You, Mingyu; Huang, Liping; Zheng, Xuebin

2017-02-01

Biomedical coatings for orthopedic implants should facilitate osseointegration and mitigate implant-induced inflammatory reactions. Cerium oxide (CeO 2 ) ceramics possess anti-oxidative properties and can be used to decrease mediators of inflammation, which makes them attractive for biomedical applications. In our work, two kinds of CeO 2 incorporated hydroxyapatite coatings (HA-10Ce and HA-30Ce) were prepared via plasma spraying technique and the effects of CeO 2 addition on the responses of bone mesenchymal stem cells (BMSCs) and RAW264.7 macrophages were investigated. An increase in CeO 2 content in the HA coatings resulted in better osteogenic behaviors of BMSCs in terms of cell proliferation, alkaline phosphatase (ALP) activity and mineralized nodule formation. RT-PCR and western blot analysis suggested that the incorporation of CeO 2 may promote the osteogenic differentiation of BMSCs through the Smad-dependent BMP signaling pathway, which activated Runx2 expression and subsequently enhanced the expression of ALP and OCN. The expression profiles of macrophages cultured on the CeO 2 modified coating revealed a tendency toward a M2 phenotype, because of an upregulation of M2 surface markers (CD163 and CD206), anti-inflammatory cytokines (TNF-α and IL-6) and osteoblastogenesis-related genes (BMP2 and TGF-β1) as well as a downregulation of M1 surface markers (CCR7 and CD11c), proinflammatory cytokines (IL-10 and IL-1ra) and reactive oxygen species production. The results suggested the regulation of BMSCs behaviors and macrophage-mediated responses at the coating's surface were associated with CeO 2 incorporation. The incorporation of CeO 2 in HA coatings can be a valuable strategy to promote osteogenic responses and reduce inflammatory reactions.

Effect of molecular weight and concentration of hyaluronan on cell proliferation and osteogenic differentiation in vitro

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhao, Ningbo, E-mail: curl-zhao@163.com; Wang, Xin, E-mail: 394041230@qq.com; Qin, Lei, E-mail: qinlei30@126.com

Hyaluronan (HA), the simplest glycosaminoglycan and a major component of the extracellular matrix, exists in various tissues. It is involved in some critical biological procedures, including cellular signaling, cell adhesion and proliferation, and cell differentiation. The effect of molecular weight (MW) and concentration of HA on cell proliferation and differentiation was controversial. In this study, we investigated the effect of MW and concentration of HA on the proliferation and osteogenic differentiation of rabbit bone marrow-derived stem cells in vitro. Results showed that high MW HA decreased the cell adhesion rate in a concentration-dependant manner. The cell adhesion rate was decreased bymore » increasing MW of HA. Cell proliferation was significantly enhanced by low MW HA (P < 0.05). The factorial analysis indicated that MW and concentration had an interactive effect on the cell adhesion rate and cell proliferation (P < 0.05). High MW HA increased the mRNA expressions of ALP, RUNX-2 and OCN. The higher the MW was, the higher the mRNA expressions were. The factorial analysis indicated that MW and concentration had an interactive effect on ALP mRNA expression (P < 0.05). HA of higher MW and higher concentration promoted bone formation. These findings provide some useful information in understanding the mechanism underlying the effect of MW and concentration of HA on cell proliferation and differentiation. - Highlights: • Effect of hyaluronan on cell proliferation and differentiation is evaluated in vitro. • Hyaluronan of low molecular weight increases cell proliferation. • Hyaluronan of high molecular weight promotes cell osteogenic differentiation. • Molecular weight and concentration of hyaluronan show interactive effect.« less

A new platelet cryoprecipitate glue promoting bone formation after ectopic mesenchymal stromal cell-loaded biomaterial implantation in nude mice.

PubMed

Trouillas, Marina; Prat, Marie; Doucet, Christelle; Ernou, Isabelle; Laplace-Builhé, Corinne; Blancard, Patrick Saint; Holy, Xavier; Lataillade, Jean-Jacques

2013-01-04

This study investigated the promising effect of a new Platelet Glue obtained from Cryoprecipitation of Apheresis Platelet products (PGCAP) used in combination with Mesenchymal Stromal Cells (MSC) loaded on ceramic biomaterials to provide novel strategies enhancing bone repair. PGCAP growth factor content was analyzed by ELISA and compared to other platelet and plasma-derived products. MSC loaded on biomaterials (65% hydroxyapatite/35% beta-TCP or 100% beta-TCP) were embedded in PGCAP and grown in presence or not of osteogenic induction medium for 21 days. Biomaterials were then implanted subcutaneously in immunodeficient mice for 28 days. Effect of PGCAP on MSC was evaluated in vitro by proliferation and osteoblastic gene expression analysis and in vivo by histology and immunohistochemistry. We showed that PGCAP, compared to other platelet-derived products, allowed concentrating large amount of growth factors and cytokines which promoted MSC and osteoprogenitor proliferation. Next, we found that PGCAP improves the proliferation of MSC and osteogenic-induced MSC. Furthermore, we demonstrated that PGCAP up-regulates the mRNA expression of osteogenic markers (Collagen type I, Osteonectin, Osteopontin and Runx2). In vivo, type I collagen expressed in ectopic bone-like tissue was highly enhanced in biomaterials embedded in PGCAP in the absence of osteogenic pre-induction. Better results were obtained with 65% hydroxyapatite/35% beta-TCP biomaterials as compared to 100% beta-TCP. We have demonstrated that PGCAP is able to enhance in vitro MSC proliferation, osteoblastic differentiation and in vivo bone formation in the absence of osteogenic pre-induction. This clinically adaptable platelet glue could be of interest for improving bone repair.

A new platelet cryoprecipitate glue promoting bone formation after ectopic mesenchymal stromal cell-loaded biomaterial implantation in nude mice

PubMed Central

2013-01-01

Introduction This study investigated the promising effect of a new Platelet Glue obtained from Cryoprecipitation of Apheresis Platelet products (PGCAP) used in combination with Mesenchymal Stromal Cells (MSC) loaded on ceramic biomaterials to provide novel strategies enhancing bone repair. Methods PGCAP growth factor content was analyzed by ELISA and compared to other platelet and plasma-derived products. MSC loaded on biomaterials (65% hydroxyapatite/35% beta-TCP or 100% beta-TCP) were embedded in PGCAP and grown in presence or not of osteogenic induction medium for 21 days. Biomaterials were then implanted subcutaneously in immunodeficient mice for 28 days. Effect of PGCAP on MSC was evaluated in vitro by proliferation and osteoblastic gene expression analysis and in vivo by histology and immunohistochemistry. Results We showed that PGCAP, compared to other platelet-derived products, allowed concentrating large amount of growth factors and cytokines which promoted MSC and osteoprogenitor proliferation. Next, we found that PGCAP improves the proliferation of MSC and osteogenic-induced MSC. Furthermore, we demonstrated that PGCAP up-regulates the mRNA expression of osteogenic markers (Collagen type I, Osteonectin, Osteopontin and Runx2). In vivo, type I collagen expressed in ectopic bone-like tissue was highly enhanced in biomaterials embedded in PGCAP in the absence of osteogenic pre-induction. Better results were obtained with 65% hydroxyapatite/35% beta-TCP biomaterials as compared to 100% beta-TCP. Conclusions We have demonstrated that PGCAP is able to enhance in vitro MSC proliferation, osteoblastic differentiation and in vivo bone formation in the absence of osteogenic pre-induction. This clinically adaptable platelet glue could be of interest for improving bone repair. PMID:23290259

Epigenetic Control of Prostate Cancer Metastasis: Role of Runx2 Phosphorylation

DTIC Science & Technology

2015-05-01

primary tumors.5•8•10 Lastly, transgenic overexpression of Runx2 predisposes mice to T -cell lymphomas , suggesting an oncogene function.11•12...leukemias while Runx3 may function as a tumor suppressor in gastric cancers (for reviews, see Blyth et a/.12’ 13). MAP kinase (MAPK), P13K/AKT and non

The chimeric transcript RUNX1-GLRX5: a biomarker for good postoperative prognosis in Stage IA non-small-cell lung cancer.

PubMed

Ishikawa, Rie; Amano, Yosuke; Kawakami, Masanori; Sunohara, Mitsuhiro; Watanabe, Kousuke; Kage, Hidenori; Ohishi, Nobuya; Yatomi, Yutaka; Nakajima, Jun; Fukayama, Masashi; Nagase, Takahide; Takai, Daiya

2016-02-01

Stage IA non-small-cell lung cancer cases have been recognized as having a low risk of relapse; however, occasionally, relapse may occur. To predict clinical outcome in Stage IA non-small-cell lung cancer patients, we searched for chimeric transcripts that can be used as biomarkers and identified a novel chimeric transcript, RUNX1-GLRX5, comprising RUNX1, a transcription factor, and GLRX5. This chimera was detected in approximately half of the investigated Stage IA non-small-cell lung cancer patients (44/104 cases, 42.3%). Although there was no significant difference in the overall survival rate between RUNX1-GLRX5-positive and -negative cases (P = 0.088), a significantly lower relapse rate was observed in the RUNX1-GLRX5-positive cases (P = 0.039), indicating that this chimera can be used as a biomarker for good prognosis in Stage IA patients. Detection of the RUNX1-GLRX5 chimeric transcript may therefore be useful for the determination of a postoperative treatment plan for Stage IA non-small-cell lung cancer patients. © The Author 2015. Published by Oxford University Press.

Breast cancer cells obtain an osteomimetic feature via epithelial-mesenchymal transition that have undergone BMP2/RUNX2 signaling pathway induction.

PubMed

Tan, Cong-Cong; Li, Gui-Xi; Tan, Li-Duan; Du, Xin; Li, Xiao-Qing; He, Rui; Wang, Qing-Shan; Feng, Yu-Mei

2016-11-29

Bone is one of the most common organs of breast cancer metastasis. Cancer cells that mimic osteoblasts by expressing bone matrix proteins and factors have a higher likelihood of metastasizing to bone. However, the molecular mechanisms of osteomimicry formation of cancer cells remain undefined. Herein, we identified a set of bone-related genes (BRGs) that are ectopically co-expressed in primary breast cancer tissues and determined that osteomimetic feature is obtained due to the osteoblast-like transformation of epithelial breast cancer cells that have undergone epithelial-mesenchymal transition (EMT) followed by bone morphogenetic protein-2 (BMP2) stimulation. Furthermore, we demonstrated that breast cancer cells that transformed into osteoblast-like cells with high expression of BRGs showed enhanced chemotaxis, adhesion, proliferation and multidrug resistance in an osteoblast-mimic bone microenvironment in vitro. During these processes, runt-related transcription factor 2 (RUNX2) functioned as a master mediator by suppressing or activating the transcription of BRGs that underlie the dynamic antagonism between the TGF-β/SMAD and BMP/SMAD signaling pathways in breast cancer cells. Our findings suggest a novel mechanism of osteomimicry formation that arises in primary breast tumors, which may explain the propensity of breast cancer to metastasize to the skeleton and contribute to potential strategies for predicting and targeting breast cancer bone metastasis and multidrug resistance.

Epigenetically induced ectopic expression of UNCX impairs the proliferation and differentiation of myeloid cells

PubMed Central

Daniele, Giulia; Simonetti, Giorgia; Fusilli, Caterina; Iacobucci, Ilaria; Lonoce, Angelo; Palazzo, Antonio; Lomiento, Mariana; Mammoli, Fabiana; Marsano, Renè Massimiliano; Marasco, Elena; Mantovani, Vilma; Quentmeier, Hilmar; Drexler, Hans G; Ding, Jie; Palumbo, Orazio; Carella, Massimo; Nadarajah, Niroshan; Perricone, Margherita; Ottaviani, Emanuela; Baldazzi, Carmen; Testoni, Nicoletta; Papayannidis, Cristina; Ferrari, Sergio; Mazza, Tommaso; Martinelli, Giovanni; Storlazzi, Clelia Tiziana

2017-01-01

We here describe a leukemogenic role of the homeobox gene UNCX, activated by epigenetic modifications in acute myeloid leukemia (AML). We found the ectopic activation of UNCX in a leukemia patient harboring a t(7;10)(p22;p14) translocation, in 22 of 61 of additional cases [a total of 23 positive patients out of 62 (37.1%)], and in 6 of 75 (8%) of AML cell lines. UNCX is embedded within a low-methylation region (canyon) and encodes for a transcription factor involved in somitogenesis and neurogenesis, with specific expression in the eye, brain, and kidney. UNCX expression turned out to be associated, and significantly correlated, with DNA methylation increase at its canyon borders based on data in our patients and in archived data of patients from The Cancer Genome Atlas. UNCX-positive and -negative patients displayed significant differences in their gene expression profiles. An enrichment of genes involved in cell proliferation and differentiation, such as MAP2K1 and CCNA1, was revealed. Similar results were obtained in UNCX-transduced CD34+ cells, associated with low proliferation and differentiation arrest. Accordingly, we showed that UNCX expression characterizes leukemia cells at their early stage of differentiation, mainly M2 and M3 subtypes carrying wild-type NPM1. We also observed that UNCX expression significantly associates with an increased frequency of acute promyelocytic leukemia with PML-RARA and AML with t(8;21)(q22;q22.1); RUNX1-RUNX1T1 classes, according to the World Health Organization disease classification. In summary, our findings suggest a novel leukemogenic role of UNCX, associated with epigenetic modifications and with impaired cell proliferation and differentiation in AML. PMID:28411256

Epigenetically induced ectopic expression of UNCX impairs the proliferation and differentiation of myeloid cells.

PubMed

Daniele, Giulia; Simonetti, Giorgia; Fusilli, Caterina; Iacobucci, Ilaria; Lonoce, Angelo; Palazzo, Antonio; Lomiento, Mariana; Mammoli, Fabiana; Marsano, Renè Massimiliano; Marasco, Elena; Mantovani, Vilma; Quentmeier, Hilmar; Drexler, Hans G; Ding, Jie; Palumbo, Orazio; Carella, Massimo; Nadarajah, Niroshan; Perricone, Margherita; Ottaviani, Emanuela; Baldazzi, Carmen; Testoni, Nicoletta; Papayannidis, Cristina; Ferrari, Sergio; Mazza, Tommaso; Martinelli, Giovanni; Storlazzi, Clelia Tiziana

2017-07-01

We here describe a leukemogenic role of the homeobox gene UNCX , activated by epigenetic modifications in acute myeloid leukemia (AML). We found the ectopic activation of UNCX in a leukemia patient harboring a t(7;10)(p22;p14) translocation, in 22 of 61 of additional cases [a total of 23 positive patients out of 62 (37.1%)], and in 6 of 75 (8%) of AML cell lines. UNCX is embedded within a low-methylation region (canyon) and encodes for a transcription factor involved in somitogenesis and neurogenesis, with specific expression in the eye, brain, and kidney. UNCX expression turned out to be associated, and significantly correlated, with DNA methylation increase at its canyon borders based on data in our patients and in archived data of patients from The Cancer Genome Atlas. UNCX -positive and -negative patients displayed significant differences in their gene expression profiles. An enrichment of genes involved in cell proliferation and differentiation, such as MAP2K1 and CCNA1 , was revealed. Similar results were obtained in UNCX -transduced CD34 + cells, associated with low proliferation and differentiation arrest. Accordingly, we showed that UNCX expression characterizes leukemia cells at their early stage of differentiation, mainly M2 and M3 subtypes carrying wild-type NPM1 We also observed that UNCX expression significantly associates with an increased frequency of acute promyelocytic leukemia with PML-RARA and AML with t(8;21)(q22;q22.1); RUNX1-RUNX1T1 classes, according to the World Health Organization disease classification. In summary, our findings suggest a novel leukemogenic role of UNCX , associated with epigenetic modifications and with impaired cell proliferation and differentiation in AML. Copyright© 2017 Ferrata Storti Foundation.

Brn3a and Islet1 act epistatically to regulate the gene expression program of sensory differentiation

PubMed Central

Dykes, Iain M.; Tempest, Lynne; Lee, Su-In; Turner, Eric E.

2011-01-01

The combinatorial expression of transcription factors frequently marks cellular identity in the nervous system, yet how these factors interact to determine specific neuronal phenotypes is not well understood. Sensory neurons of the trigeminal (TG) and dorsal root ganglia (DRG) co-express the homeodomain transcription factors Brn3a and Islet1, and past work has revealed partially overlapping programs of gene expression downstream of these factors. Here we examine sensory development in Brn3a/Islet1 double knockout mice (DKO mice). Sensory neurogenesis and the formation of the TG and DRG occur in DKO embryos, but the DRG are dorsally displaced, and the peripheral projections of the ganglia are markedly disturbed. Sensory neurons in DKO embryos show a profound loss of all early markers of sensory subtypes, including the Ntrk neurotrophin receptors, and the runt-family transcription factors Runx1 and Runx3. Examination of global gene expression in the E12.5 DRG of single and double mutant embryos shows that Brn3a and Islet1 are together required for nearly all aspects of sensory-specific gene expression, including several newly identified sensory markers. On a majority of targets Brn3a and Islet1 exhibit negative epistasis, in which the effects of the individual knockout alleles are less than additive in the DKO. Smaller subsets of targets exhibit positive epistasis, or are regulated exclusively by one factor. Brn3a/Islet1 double mutants also fail to developmentally repress neurogenic bHLH genes, and in vivo chromatin immunoprecipitation shows that Islet1 binds to a known Brn3a -regulated enhancer in the neurod4 gene, suggesting a mechanism of interaction between these genes. PMID:21734270

Effects of Titanium Surface Microtopography and Simvastatin on Growth and Osteogenic Differentiation of Human Mesenchymal Stem Cells in Estrogen-Deprived Cell Culture.

PubMed

Arpornmaeklong, Premjit; Pripatnanont, Prisana; Chookiatsiri, Chonticha; Tangtrakulwanich, Boonsin

This study aimed to investigate the effects of titanium surface topography and simvastatin on growth and osteogenic differentiation of human bone marrow stromal cells (hBMSCs) in estrogen-deprived (ED) cell culture. Human BMSCs were seeded on cell culture plates, smooth-surface titanium (Ti) disks, and sandblasted with large grits and acid etched (SLA)-surface Ti disks; and subsequently cultured in regular (fetal bovine serum [FBS]), ED, and ED-with 100 nM simvastatin (ED-SIM) culture media for 14 to 21 days. Live/dead cell staining, scanning electron microscope examination, and cell viability assay were performed to determine cell attachment, morphology, and growth. Expression levels of osteoblast-associated genes, Runx2 and bone sialoprotein and levels of alkaline phosphatase (ALP) activity, calcium content, and osteocalcin in culture media were measured to determine osteoblastic differentiation. Expression levels of bone morphogenetic protein-2 (BMP-2) were investigated to examine stimulating effects of simvastatin (n = 4 to 5, mean ± SD). In vitro mineralization was verified by calcein staining. Human BMSCs exhibited different attachment and shapes on smooth and SLA titanium surfaces. Estrogen-deprived cell culture decreased cell attachment and growth, particularly on the SLA titanium surface, but cells were able to grow to reach confluence on day 21 in the ED-osteogenic (OS) culture medium. Promoting effects of the SLA titanium surface in ED-OS were significantly decreased. Simvastatin significantly increased osteogenic differentiation of human BMSCs on the SLA titanium surface in the ED-OS medium, and the promoting effects of simvastatin corresponded with the increasing of BMP-2 gene expression on the SLA titanium surface in ED-OS-SIM culture medium. The ED cell culture model provided a well-defined platform for investigating the effects of hormones and growth factors on cells and titanium surface interaction. Titanium, the SLA surface, and simvastatin synergistically promoted osteoblastic differentiation of hBMSCs in ED condition and might be useful to promote osteointegration in osteoporotic bone.

Tet1 and Tet2 maintain mesenchymal stem cell homeostasis via demethylation of the P2rX7 promoter.

PubMed

Yang, Ruili; Yu, Tingting; Kou, Xiaoxing; Gao, Xiang; Chen, Chider; Liu, Dawei; Zhou, Yanheng; Shi, Songtao

2018-06-01

Ten-eleven translocation (Tet) family-mediated DNA oxidation represents an epigenetic modification capable of converting 5-methylcytosine (5-mC) to 5-hydroxymethylcytosine (5-hmC), which regulates various biological processes. However, it is unknown whether Tet family affects mesenchymal stem cells (MSCs) or the skeletal system. Here we show that depletion of Tet1 and Tet2 results in impaired self-renewal and differentiation of bone marrow MSCs (BMMSCs) and a significant osteopenia phenotype. Tet1 and Tet2 deficiency reduces demethylation of the P2rX7 promoter and downregulates exosome release, leading to intracellular accumulation of miR-297a-5p, miR-297b-5p, and miR-297c-5p. These miRNAs inhibit Runx2 signaling to impair BMMSC function. We show that overexpression of P2rX7 rescues the impaired BMMSCs and osteoporotic phenotype in Tet1 and Tet2 double knockout mice. These results indicate that Tet1 and Tet2 play a critical role in maintaining BMMSC and bone homeostasis through demethylation of P2rX7 to control exosome and miRNA release. This Tet/P2rX7/Runx2 cascade may serve as a target for the development of novel therapies for osteopenia disorders.

The effect of collagen coating on titanium with nanotopography on in vitro osteogenesis.

PubMed

Costa, Daniel G; Ferraz, Emanuela P; Abuna, Rodrigo P F; de Oliveira, Paulo T; Morra, Marco; Beloti, Marcio M; Rosa, Adalberto L

2017-10-01

Several studies have shown the positive effects of Ti either with nanotopography or coated with collagen on osteoblast differentiation. Thus, we hypothesized that the association of nanotopography with collagen may increase the in vitro osteogenesis on Ti surface. Ti discs with nanotopography with or without collagen coating were characterized by scanning electron microscopy and atomic force microscopy. Rat calvaria-derived osteoblastic cells were cultured on both Ti surfaces for up to 14 days and the following parameters were evaluated: cell proliferation, alkaline phosphatase (ALP) activity, extracellular matrix mineralization, protein expression of bone sialoprotein (BSP) and osteopontin (OPN), and gene expression of collagen type 1a (Coll1a), runt-related transcription factor 2 (Runx2), osterix (OSX), osteocalcin (OC), Ki67, Survivin, and Bcl2-associated X protein (BAX). Surface characterization evidenced that collagen coating did not alter the nanotopography. Collagen coating increased cell proliferation, ALP activity, extracellular matrix mineralization, and Coll1a, OSX, OC, and BAX gene expression. Also, OPN and BSP proteins were strongly detected in cultures grown on both Ti surfaces. In conclusion, our results showed that the combination of nanotopography with collagen coating stimulates the early, intermediate, and final events of the in vitro osteogenesis and may be considered a potential approach to promote osseointegration of Ti implants. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 2783-2788, 2017. © 2017 Wiley Periodicals, Inc.

Identification of a novel long noncoding RNA that promotes osteoblast differentiation.

PubMed

Nardocci, Gino; Carrasco, Margarita E; Acevedo, Elvis; Hodar, Christian; Meneses, Claudio; Montecino, Martín

2018-05-28

Long noncoding RNAs (lncRNAs) are a heterogeneous class of transcripts, longer than 200 nucleotides, 5'-capped, polyadenylated, and poorly conserved among mammalian species. Several studies have shown the contribution of lncRNAs to different cellular processes, including regulation of the chromatin structure, control of messenger RNA translation, regulation of gene transcription, regulation of embryonic pluripotency, and differentiation. Although limited numbers of functional lncRNAs have been identified so far, the immense regulatory potential of these RNAs is already evident, indicating that a functional characterization of lncRNAs is needed. In this study, mouse preosteoblastic cells were induced to differentiate into osteoblasts. At 3 sequential differentiation stages, total RNA was isolated and libraries were constructed for Illumina sequencing. The resulting sequences were aligned and transcript abundances were determined. New lncRNA candidates that displayed differential expression patterns during osteoblast differentiation were identified by combining bioinformatics and reverse transcription polymerase chain reaction analyses. Among these, lncRNA-1 that exhibited increased expression during osteogenesis and was downregulated during myogenesis. Importantly, knockdown of lncRNA-1 expression in primary mouse preosteoblasts was found to inhibit osteogenic differentiation, reflected by a reduced transcription of the Runx2/p57 and Sp7 bone master genes. Together, our results indicate that lncRNA-1 represents a new regulatory RNA that plays a relevant role during the early stages of osteogenesis. © 2018 Wiley Periodicals, Inc.

Long Noncoding RNA uc002yug.2 Activates HIV-1 Latency through Regulation of mRNA Levels of Various RUNX1 Isoforms and Increased Tat Expression.

PubMed

Huan, Chen; Li, Zhaolong; Ning, Shanshan; Wang, Hong; Yu, Xiao-Fang; Zhang, Wenyan

2018-05-01

The HIV-1 reservoir is a major obstacle to complete eradication of the virus. Although many proteins and RNAs have been characterized as regulators in HIV-1/AIDS pathogenesis and latency, only a few long noncoding RNAs (lncRNAs) have been shown to be closely associated with HIV-1 replication and latency. In this study, we demonstrated that lncRNA uc002yug.2 plays a key role in HIV-1 replication and latency. uc002yug.2 potentially enhances HIV-1 replication, long terminal repeat (LTR) activity, and the activation of latent HIV-1 in both cell lines and CD4 + T cells from patients. Further investigation revealed that uc002yug.2 activates latent HIV-1 through downregulating RUNX1b and -1c and upregulating Tat protein expression. The accumulated evidence supports our model that the Tat protein has the key role in the uc002yug.2-mediated regulatory effect on HIV-1 reactivation. Moreover, uc002yug.2 showed an ability to activate HIV-1 similar to that of suberoylanilide hydroxamic acid or phorbol 12-myristate 13-acetate using latently infected cell models. These findings improve our understanding of lncRNA regulation of HIV-1 replication and latency, providing new insights into potential targeted therapeutic interventions. IMPORTANCE The latent viral reservoir is the primary obstacle to curing HIV-1 disease. To date, only a few lncRNAs, which play major roles in various biological processes, including viral infection, have been identified as regulators in HIV-1 latency. In this study, we demonstrated that lncRNA uc002yug.2 is important for both HIV-1 replication and activation of latent viruses. Moreover, uc002yug.2 was shown to activate latent HIV-1 through regulating alternative splicing of RUNX1 and increasing the expression of Tat protein. These findings highlight the potential merit of targeting lncRNA uc002yug.2 as an activating agent for latent HIV-1. Copyright © 2018 American Society for Microbiology.

Chitosan-Graphene Oxide 3D scaffolds as Promising Tools for Bone Regeneration in Critical-Size Mouse Calvarial Defects.

PubMed

Hermenean, Anca; Codreanu, Ada; Herman, Hildegard; Balta, Cornel; Rosu, Marcel; Mihali, Ciprian Valentin; Ivan, Alexandra; Dinescu, Sorina; Ionita, Mariana; Costache, Marieta

2017-11-30

Limited self-regenerating capacity of human skeleton makes the reconstruction of critical size bone defect a significant challenge for clinical practice. Aimed for regenerating bone tissues, this study was designed to investigate osteogenic differentiation, along with bone repair capacity of 3D chitosan (CHT) scaffolds enriched with graphene oxide (GO) in critical-sized mouse calvarial defect. Histopathological/histomorphometry and scanning electron microscopy(SEM) analysis of the implants revealed larger amount of new bone in the CHT/GO-filled defects compared with CHT alone (p < 0.001). When combined with GO, CHT scaffolds synergistically promoted the increase of alkaline phosphatase activity both in vitro and in vivo experiments. This enhanced osteogenesis was corroborated with increased expression of bone morphogenetic protein (BMP) and Runx-2 up to week 4 post-implantation, which showed that GO facilitates the differentiation of osteoprogenitor cells. Meanwhile, osteogenesis was promoted by GO at the late stage as well, as indicated by the up-regulation of osteopontin and osteocalcin at week 8 and overexpressed at week 18, for both markers. Our data suggest that CHT/GO biomaterial could represent a promising tool for the reconstruction of large bone defects, without using exogenous living cells or growth factors.

Development of t(8;21) and RUNX1-RUNX1T1 in the Philadelphia-positive clone of a patient with chronic myelogenous leukemia: additional evidence for multiple steps involved in disease progression.

PubMed

Najfeld, Vesna; Wisch, Nathaniel; Mascarenhas, John; Issa, Leonard; Tripodi, Joseph; Sidhu, Manpreet; Hoffman, Ronald

2011-03-01

A 65-year-old patient with a high hemoglobin and hematocrit was treated for 14 months with therapeutic phlebotomy when cytogenetics of bone marrow revealed 100% cells with the Ph chromosome and 45% of the Ph+ cells contained trisomy 8. Treatment with tyrosine kinase inhibitors did not reduce the BCR-ABL1 fusion positive clone. Instead, the Ph positive cells acquired further the t(8;21)/RUNX1-RUNX1T1, del(4q) and trisomy 15 chromosomal abnormalities which were resistant to further treatment. Literature review revealed eight other patients who either had t(9;22) and t(8;21) simultaneously or developed t(8;21) in the Ph positive clone. We conclude that there are rare patients with CML who either present in blast crisis with coexistence of t(9;22) and t(8;21) with or without +8, or progress to blast crisis with acquiring RUNX1-RUNX1T1 in the BCR-ABL1 clone which may or may not be therapy related and represent a later event in a multistep pathogenesis. Copyright © 2011 Elsevier Inc. All rights reserved.

Control of RUNX-induced repression of Notch signaling by MLF and its partner DnaJ-1 during Drosophila hematopoiesis

PubMed Central

Gobert, Vanessa; Augé, Benoit; Burlet-Schiltz, Odile; Haenlin, Marc

2017-01-01

A tight regulation of transcription factor activity is critical for proper development. For instance, modifications of RUNX transcription factors dosage are associated with several diseases, including hematopoietic malignancies. In Drosophila, Myeloid Leukemia Factor (MLF) has been shown to control blood cell development by stabilizing the RUNX transcription factor Lozenge (Lz). However, the mechanism of action of this conserved family of proteins involved in leukemia remains largely unknown. Here we further characterized MLF’s mode of action in Drosophila blood cells using proteomic, transcriptomic and genetic approaches. Our results show that MLF and the Hsp40 co-chaperone family member DnaJ-1 interact through conserved domains and we demonstrate that both proteins bind and stabilize Lz in cell culture, suggesting that MLF and DnaJ-1 form a chaperone complex that directly regulates Lz activity. Importantly, dnaj-1 loss causes an increase in Lz+ blood cell number and size similarly as in mlf mutant larvae. Moreover we find that dnaj-1 genetically interacts with mlf to control Lz level and Lz+ blood cell development in vivo. In addition, we show that mlf and dnaj-1 loss alters Lz+ cell differentiation and that the increase in Lz+ blood cell number and size observed in these mutants is caused by an overactivation of the Notch signaling pathway. Finally, using different conditions to manipulate Lz activity, we show that high levels of Lz are required to repress Notch transcription and signaling. All together, our data indicate that the MLF/DnaJ-1-dependent increase in Lz level allows the repression of Notch expression and signaling to prevent aberrant blood cell development. Thus our findings establish a functional link between MLF and the co-chaperone DnaJ-1 to control RUNX transcription factor activity and Notch signaling during blood cell development in vivo. PMID:28742844

Preleukemic and second-hit mutational events in an acute myeloid leukemia patient with a novel germline RUNX1 mutation.

PubMed

Ng, Isaac Ks; Lee, Joanne; Ng, Christopher; Kosmo, Bustamin; Chiu, Lily; Seah, Elaine; Mok, Michelle Meng Huang; Tan, Karen; Osato, Motomi; Chng, Wee-Joo; Yan, Benedict; Tan, Lip Kun

2018-01-01

Germline mutations in the RUNX1 transcription factor give rise to a rare autosomal dominant genetic condition classified under the entity: Familial Platelet Disorders with predisposition to Acute Myeloid Leukaemia (FPD/AML). While several studies have identified a myriad of germline RUNX1 mutations implicated in this disorder, second-hit mutational events are necessary for patients with hereditary thrombocytopenia to develop full-blown AML. The molecular picture behind this process remains unclear. We describe a patient of Malay descent with an unreported 7-bp germline RUNX1 frameshift deletion, who developed second-hit mutations that could have brought about the leukaemic transformation from a pre-leukaemic state. These mutations were charted through the course of the treatment and stem cell transplant, showing a clear correlation between her clinical presentation and the mutations present. The patient was a 27-year-old Malay woman who presented with AML on the background of hereditary thrombocytopenia affecting her father and 3 brothers. Initial molecular testing revealed the same novel RUNX1 mutation in all 5 individuals. The patient received standard induction, consolidation chemotherapy, and a haploidentical stem cell transplant from her mother with normal RUNX1 profile. Comprehensive genomic analyses were performed at diagnosis, post-chemotherapy and post-transplant. A total of 8 mutations ( RUNX1 , GATA2 , DNMT3A , BCORL1 , BCOR , 2 PHF6 and CDKN2A ) were identified in the pre-induction sample, of which 5 remained ( RUNX1 , DNMT3A , BCORL1 , BCOR and 1 out of 2 PHF6 ) in the post-treatment sample and none were present post-transplant. In brief, the 3 mutations which were lost along with the leukemic cells at complete morphological remission were most likely acquired leukemic driver mutations that were responsible for the AML transformation from a pre-leukemic germline RUNX1 -mutated state. On the contrary, the 5 mutations that persisted post-treatment, including the germline RUNX1 mutation, were likely to be part of the preleukemic clone. Further studies are necessary to assess the prevalence of these preleukemic and secondary mutations in the larger FPD/AML patient cohort and establish their prognostic significance. Given the molecular heterogeneity of FPD/AML and other AML subtypes, a better understanding of mutational classes and their involvement in AML pathogenesis can improve risk stratification of patients for more effective and targeted therapy.

Myelodysplastic Syndrome with concomitant t(5;21)(q15;q22) and del(5)(q13q33): case report and review of literature

PubMed Central

Weckbaugh, Brandon; Sirridge, Christopher; Woodroof, Janet; Persons, Diane

2016-01-01

Chromosomal abnormalities lead to the development of hematologic malignancies such as Myelodysplastic Syndrome (MDS). Known chromosomal changes causing MDS include deletion of the long arm of chromosome 5, runt-related transcription factor 1 (RUNX1) also known as acute myeloid leukemia 1 protein (AML1), and very rarely fusion genes involving RUNX1 at t(5;21)(q15;q22). We present a case of a 71-year-old female with MDS, refractory anemia with excess blasts, type 1, with a combination of two cytogenetic abnormalities, specifically a concomitant translocation between chromosomes 5q15 and 21q22 and deletion of chromosome 5q13q33. Fluorescence in-situ hybridization (FISH) using a probe for RUNX1 (AML1), localized to 21q22, showed three FISH signals for RUNX1, consistent with rearrangement of RUNX1. Therapy was started with Lenalidomide leading to normal blood counts. Most significantly, repeat cytogenetics revealed normal karyotype and resolution of deletion on the long arm of chromosome 5 and a t(5;21). FISH negative for deletion 5q. The results altogether meet criteria for a complete cytogenetic remission (CR). We report a new case of t(5;21)(q15;q22) involving the RUNX1 gene and del(5)(q13q33) in a MDS patient, a combination of chromosomal abnormalities heretofore not reported in the literature. RUNX1 rearrangement is usually associated with an adverse prognosis in AML and MDS. Deletions of 5q are typically associated with poor prognosis in AML, however it is usually associated with a favorable prognosis in MDS. Our patient responded very well to Lenalidomide therapy with achievement of CR. Lenalidomide is approved for treatment of anemia in low and intermediate risk MDS with del (5q), however based on a search of literature it seems that RUNX1 mutations are also more prominent in patients who have responded to Lenalidomide therapy. MDS is a genomically unstable disease. Hence, it is conceivable that our patient started with a 5q minus syndrome and then acquired the second hit RUNX1 translocation leading to an accelerated phase of myeloid neoplasm or refractory anemia with excess blasts, type 1. Hence, the temporal relationship between acquisition of del 5q and RUNX1 rearrangement may have influenced the clinical outcome and possibly response to therapy. PMID:27358895

Mineral trioxide aggregate upregulates odonto/osteogenic capacity of bone marrow stromal cells from craniofacial bones via JNK and ERK MAPK signalling pathways.

PubMed

Wang, Y; Li, J; Song, W; Yu, J

2014-06-01

The aim of this study was to investigate effects of mineral trioxide aggregate (MTA) on odonto/osteogenic differentiation of bone marrow stromal cells (BMSCs) from craniofacial bones. Craniofacial BMSCs were isolated from rat mandible and effects of MTA on their proliferation, differentiation and MAPK pathway involvement were subsequently investigated, in vitro. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2,5-tetrazoliumbromide) assay was performed to evaluate proliferation of the MTA-treated cells. Alkaline phosphatase (ALP) activity, alizarin red staining, real-time reverse transcription polymerase chain reaction and western blot assays were used to assess differentiation capacity as well as MAPK pathway involvement. 0.02 mg/ml MTA-treated BMSCs had significantly higher ALP activity and formed more mineralized nodules than the untreated group. Odonto/osteoblastic marker genes/proteins (Alp, Runx2/RUNX2, Osx/OSX, Ocn/OCN and Dspp/DSP respectively) in MTA-treated cells were remarkably upregulated compared to untreated ones. Mechanistically, phosphorylated Jun N-terminal kinase (P-JNK) and phosphorylated extracellular regulated protein kinases (P-ERK) in MTA-treated BMSCs increased significantly in a time-dependent manner, while inhibition of JNK and ERK MAPK pathways dramatically blocked MTA-induced odonto/osteoblastic differentiation, as indicated by reduced ALP levels, weakened mineralization capacity and downregulated levels of odonto/osteoblastic marker genes (Alp, Runx2, Osx, Ocn and Dspp). Mineral trioxide aggregate promoted odonto/osteogenic capacity of craniofacial BMSCs via JNK and ERK MAPK signalling pathways. © 2014 John Wiley & Sons Ltd.

Adenoviral Mediated Expression of BMP2 by Bone Marrow Stromal Cells Cultured in 3D Copolymer Scaffolds Enhances Bone Formation.

PubMed

Sharma, Sunita; Sapkota, Dipak; Xue, Ying; Sun, Yang; Finne-Wistrand, Anna; Bruland, Ove; Mustafa, Kamal

2016-01-01

Selection of appropriate osteoinductive growth factors, suitable delivery method and proper supportive scaffold are critical for a successful outcome in bone tissue engineering using bone marrow stromal cells (BMSC). This study examined the molecular and functional effect of a combination of adenoviral mediated expression of bone morphogenetic protein-2 (BMP2) in BMSC and recently developed and characterized, biodegradable Poly(L-lactide-co-є-caprolactone){poly(LLA-co-CL)}scaffolds in osteogenic molecular changes and ectopic bone formation by using in vitro and in vivo approaches. Pathway-focused custom PCR array, validation using TaqMan based quantitative RT-PCR (qRT-PCR) and ALP staining showed significant up-regulation of several osteogenic and angiogenic molecules, including ALPL and RUNX2 in ad-BMP2 BMSC group grown in poly(LLA-co-CL) scaffolds both at 3 and 14 days. Micro CT and histological analyses of the subcutaneously implanted scaffolds in NOD/SCID mice revealed significantly increased radiopaque areas, percentage bone volume and formation of vital bone in ad-BMP2 scaffolds as compared to the control groups both at 2 and 8 weeks. The increased bone formation in the ad-BMP2 group in vivo was paralleled at the molecular level with concomitant over-expression of a number of osteogenic and angiogenic genes including ALPL, RUNX2, SPP1, ANGPT1. The increased bone formation in ad-BMP2 explants was not found to be associated with enhanced endochondral activity as evidenced by qRT-PCR (SOX9 and FGF2) and Safranin O staining. Taken together, combination of adenoviral mediated BMP-2 expression in BMSC grown in the newly developed poly(LLA-co-CL) scaffolds induced expression of osteogenic markers and enhanced bone formation in vivo.

Palmitate attenuates osteoblast differentiation of fetal rat calvarial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yeh, Lee-Chuan C.; Ford, Jeffery J.; Lee, John C.

Highlights: • Palmitate inhibits osteoblast differentiation. • Fatty acid synthase. • PPARγ. • Acetyl Co-A carboxylase inhibitor TOFA. • Fetal rat calvarial cell culture. - Abstract: Aging is associated with the accumulation of ectopic lipid resulting in the inhibition of normal organ function, a phenomenon known as lipotoxicity. Within the bone marrow microenvironment, elevation in fatty acid levels may produce an increase in osteoclast activity and a decrease in osteoblast number and function, thus contributing to age-related osteoporosis. However, little is known about lipotoxic mechanisms in intramembraneous bone. Previously we reported that the long chain saturated fatty acid palmitate inhibitedmore » the expression of the osteogenic markers RUNX2 and osteocalcin in fetal rat calvarial cell (FRC) cultures. Moreover, the acetyl CoA carboxylase inhibitor TOFA blocked the inhibitory effect of palmitate on expression of these two markers. In the current study we have extended these observations to show that palmitate inhibits spontaneous mineralized bone formation in FRC cultures in association with reduced mRNA expression of RUNX2, alkaline phosphatase, osteocalcin, and bone sialoprotein and reduced alkaline phosphatase activity. The effects of palmitate on osteogenic marker expression were inhibited by TOFA. Palmitate also inhibited the mRNA expression of fatty acid synthase and PPARγ in FRC cultures, and as with osteogenic markers, this effect was inhibited by TOFA. Palmitate had no effect on FRC cell proliferation or apoptosis, but inhibited BMP-7-induced alkaline phosphatase activity. We conclude that palmitate accumulation may lead to lipotoxic effects on osteoblast differentiation and mineralization and that increases in fatty acid oxidation may help to prevent these lipotoxic effects.« less

EDTA conditioning of dentine promotes adhesion, migration and differentiation of dental pulp stem cells.

PubMed

Galler, K M; Widbiller, M; Buchalla, W; Eidt, A; Hiller, K-A; Hoffer, P C; Schmalz, G

2016-06-01

To evaluate the effect of dentine conditioning on migration, adhesion and differentiation of dental pulp stem cells. Dentine discs prepared from extracted human molars were pre-treated with EDTA (10%), NaOCl (5.25%) or H2 O. Migration of dental pulp stem cells towards pre-treated dentine after 24 and 48 h was assessed in a modified Boyden chamber assay. Cell adhesion was evaluated indirectly by measuring cell viability. Expression of mineralization-associated genes (COL1A1, ALP, BSP, DSPP, RUNX2) in cells cultured on pre-treated dentine for 7 days was determined by RT-qPCR. Nonparametric statistical analysis was performed for cell migration and cell viability data to compare different groups and time-points (Mann-Whitney U-test, α = 0.05). Treatment of dentine with H2 O or EDTA allowed for cell attachment, which was prohibited by NaOCl with statistical significance (P = 0.000). Furthermore, EDTA conditioning induced cell migration towards dentine. The expression of mineralization-associated genes was increased in dental pulp cells cultured on dentine after EDTA conditioning compared to H2 O-pre-treated dentine discs. EDTA conditioning of dentine promoted the adhesion, migration and differentiation of dental pulp stem cells towards or onto dentine. A pre-treatment with EDTA as the final step of an irrigation protocol for regenerative endodontic procedures has the potential to act favourably on new tissue formation within the root canal. © 2015 International Endodontic Journal. Published by John Wiley & Sons Ltd.

Vascular niche promotes hematopoietic multipotent progenitor formation from pluripotent stem cells

PubMed Central

Gori, Jennifer L.; Butler, Jason M.; Chan, Yan-Yi; Chandrasekaran, Devikha; Poulos, Michael G.; Ginsberg, Michael; Nolan, Daniel J.; Elemento, Olivier; Wood, Brent L.; Adair, Jennifer E.; Rafii, Shahin; Kiem, Hans-Peter

2015-01-01

Pluripotent stem cells (PSCs) represent an alternative hematopoietic stem cell (HSC) source for treating hematopoietic disease. The limited engraftment of human PSC–derived (hPSC-derived) multipotent progenitor cells (MPP) has hampered the clinical application of these cells and suggests that MPP require additional cues for definitive hematopoiesis. We hypothesized that the presence of a vascular niche that produces Notch ligands jagged-1 (JAG1) and delta-like ligand-4 (DLL4) drives definitive hematopoiesis. We differentiated hes2 human embryonic stem cells (hESC) and Macaca nemestrina–induced PSC (iPSC) line-7 with cytokines in the presence or absence of endothelial cells (ECs) that express JAG1 and DLL4. Cells cocultured with ECs generated substantially more CD34+CD45+ hematopoietic progenitors compared with cells cocultured without ECs or with ECs lacking JAG1 or DLL4. EC-induced cells exhibited Notch activation and expressed HSC-specific Notch targets RUNX1 and GATA2. EC-induced PSC-MPP engrafted at a markedly higher level in NOD/SCID/IL-2 receptor γ chain–null (NSG) mice compared with cytokine-induced cells, and low-dose chemotherapy-based selection further increased engraftment. Long-term engraftment and the myeloid-to-lymphoid ratio achieved with vascular niche induction were similar to levels achieved for cord blood–derived MPP and up to 20-fold higher than those achieved with hPSC-derived MPP engraftment. Our findings indicate that endothelial Notch ligands promote PSC-definitive hematopoiesis and production of long-term engrafting CD34+ cells, suggesting these ligands are critical for HSC emergence. PMID:25664855

Activation of the PI3K/Akt pathway by oxidative stress mediates high glucose-induced increase of adipogenic differentiation in primary rat osteoblasts.

PubMed

Zhang, Yu; Yang, Jian-Hong

2013-11-01

Diabetes mellitus is associated with increased risk of osteopenia and bone fracture that may be related to hyperglycemia. However, the mechanisms accounting for diabetic bone disorder are unclear. Here, we showed that high glucose significantly promoted the production of reactive oxygen species (ROS) in rat primary osteoblasts. Most importantly, we reported for the first time that ROS induced by high glucose increased alkaline phosphatase activity, inhibited type I collagen (collagen I) protein level and cell mineralization, as well as gene expression of osteogenic markers including runt-related transcription factor 2 (Runx2), collagen I, and osteocalcin, but promoted lipid droplet formation and gene expression of adipogenic markers including peroxisome proliferator-activated receptor gamma, adipocyte fatty acid binding protein (aP2), and adipsin, which were restored by pretreatment with N-acetyl-L-cysteine (NAC), a ROS scavenger. Moreover, high glucose-induced oxidative stress activated PI3K/Akt pathway to inhibited osteogenic differentiation but stimulated adipogenic differentiation. In contrast, NAC and a PI3K inhibitor, LY-294002, reversed the down-regulation of osteogenic markers and the up-regulation of adipogenic markers as well as the activation of Akt under high glucose. These results indicated that oxidative stress played a key role in high glucose-induced increase of adipogenic differentiation, which contributed to the inhibition of osteogenic differentiation. This process was mediated by PI3K/Akt pathway in rat primary osteoblasts. Hence, suppression of oxidative stress could be a potential therapeutic approach for diabetic osteopenia. © 2013 Wiley Periodicals, Inc.

Short term sodium alendronate administration improves the peri-implant bone quality in osteoporotic animals

PubMed Central

de OLIVEIRA, Danila; HASSUMI, Jaqueline Suemi; GOMES-FERREIRA, Pedro Henrique da Silva; POLO, Tárik Ocon Braga; FERREIRA, Gabriel Ramalho; FAVERANI, Leonardo Perez; OKAMOTO, Roberta

2017-01-01

Abstract Sodium alendronate is a bisphosphonate drug that exerts antiresorptive action and is used to treat osteoporosis. Objective The aim of this study was to evaluate the bone repair process at the bone/implant interface of osteoporotic rats treated with sodium alendronate through the analysis of microtomography, real time polymerase chain reactions and immunohistochemistry (RUNX2 protein, bone sialoprotein (BSP), alkaline phosphatase, osteopontin and osteocalcin). Material and Methods A total of 42 rats were used and divided in to the following experimental groups: CTL: control group (rats submitted to fictitious surgery and fed with a balanced diet), OST: osteoporosis group (rats submitted to a bilateral ovariectomy and fed with a low calcium diet) and ALE: alendronate group (rats submitted to a bilateral ovariectomy, fed with a low calcium diet and treated with sodium alendronate). A surface treated implant was installed in both tibial metaphyses of each rat. Euthanasia of the animals was conducted at 14 (immunhostochemistry) and 42 days (immunohistochemistry, micro CT and PCR). Data were subjected to statistical analysis with a 5% significance level. Results Bone volume (BV) and total pore volume were higher for ALE group (P<0.05). Molecular data for RUNX2 and BSP proteins were significantly expressed in the ALE group (P<0.05), in comparison with the other groups. ALP expression was higher in the CTL group (P<0.05). The immunostaining for RUNX2 and osteopontin was positive in the osteoblastic lineage cells of neoformed bone for the CTL and ALE groups in both periods (14 and 42 days). Alkaline phosphatase presented a lower staining area in the OST group compared to the CTL in both periods and the ALE at 42 days. Conclusion There was a decrease of osteocalcin precipitation at 42 days for the ALE and OST groups. Therefore, treatment with short-term sodium alendronate improved bone repair around the implants installed in the tibia of osteoporotic rats. PMID:28198975

ERK1/2 signaling mediated naringin-induced osteogenic differentiation of immortalized human periodontal ligament stem cells.

PubMed

Wei, Kai; Xie, Yuansheng; Chen, Tianyu; Fu, Bo; Cui, Shaoyuan; Wang, Yan; Cai, Guangyan; Chen, Xiangmei

2017-07-29

Periodontal ligament stem cells (PDLSCs) are promising tools for the investigations of cell differentiation and bone regeneration. However, the limited life span significantly restricts their usefulness. In this study, we established an immortalized PDLSC cell line by the introduction of Bmi1 (PDLSC-Bmi1). Several genes related to cell cycle, cell replication and stemness were found to be changed with the overexpression of Bmi1. Compared with primary PDLSCs, the immortalized cells had a slower aging rate, maintained in a proliferative state without crisis for more than 30 passages, and retained the molecular markers and biological functions of primary ones. Using the PDLSC-Bmi1, we confirmed the promotive effect of naringin on osteogenesis. Naringin promoted the osteogenic differentiation of PDLSC-Bmi1 manifested as the increased activity of alkaline phosphatase (ALP), expression of the runt-related transcription factor 2 (Runx2) and osteocalcin (OCN), and formation of mineralized nodules. In addition, the extracellular regulated protein kinases (ERK) 1/2 was found to be activated by naringin, and the ERK1/2 specific inhibitor significantly inhibited naringin-induced osteogenic differentiation in PDLSC-Bmi1. Our results indicated that the overexpression of Bmi1 extended the life span of PDLSCs without perturbing their biological functions, and that naringin promoted the osteogenesis of PDLSC-Bmi1 at least partially through the ERK1/2 signaling pathway. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

Calvarial cleidocraniodysplasia-like defects with ENU-induced Nell-1 deficiency.

PubMed

Zhang, Xinli; Ting, Kang; Pathmanathan, Dharmini; Ko, Theodore; Chen, Weiwei; Chen, Feng; Lee, Haofu; James, Aaron W; Siu, Ronald K; Shen, Jia; Culiat, Cymbeline T; Soo, Chia

2012-01-01

Nell-1, first identified by its overexpression in synostotic cranial sutures, is a novel osteoinductive growth and differentiation factor. To further define Nell-1's role in craniofacial patterning, we characterized defects of the ENU-induced Nell-1-deficient (END) mice, focusing on both intramembranous and endochondral cranial bones. Results showed that calvarial bones of neonatal END mice were reduced in thickness and density, with a phenotype resembling calvarial cleidocraniodysplasia. In addition, a global reduction in osteoblast markers was observed, including reductions in Runx2, alkaline phosphatase, and osteocalcin. Remarkably, detailed analysis of endochondral bones showed dysplasia as well. The chondrocranium in the END mouse showed enrichment for early, proliferating Sox9⁺ chondrocytes, whereas in contrast markers of chondrocytes maturation were reduced. These data suggest that Nell-1 is an important growth factor for regulation of osteochondral differentiation, by regulating both Runx2 and Sox9 expression within the calvarium. In summary, Nell-1 is required for normal craniofacial membranous and endochondral skeletal development.

Next generation bone tissue engineering: non-viral miR-133a inhibition using collagen-nanohydroxyapatite scaffolds rapidly enhances osteogenesis

NASA Astrophysics Data System (ADS)

Mencía Castaño, Irene; Curtin, Caroline M.; Duffy, Garry P.; O'Brien, Fergal J.

2016-06-01

Bone grafts are the second most transplanted materials worldwide at a global cost to healthcare systems valued over $30 billion every year. The influence of microRNAs in the regenerative capacity of stem cells offers vast therapeutic potential towards bone grafting; however their efficient delivery to the target site remains a major challenge. This study describes how the functionalisation of porous collagen-nanohydroxyapatite (nHA) scaffolds with miR-133a inhibiting complexes, delivered using non-viral nHA particles, enhanced human mesenchymal stem cell-mediated osteogenesis through the novel focus on a key activator of osteogenesis, Runx2. This study showed enhanced Runx2 and osteocalcin expression, as well as increased alkaline phosphatase activity and calcium deposition, thus demonstrating a further enhanced therapeutic potential of a biomaterial previously optimised for bone repair applications. The promising features of this platform offer potential for a myriad of applications beyond bone repair and tissue engineering, thus presenting a new paradigm for microRNA-based therapeutics.

Effects of dexamethasone, ascorbic acid and β-glycerophosphate on the osteogenic differentiation of stem cells in vitro

PubMed Central

2013-01-01

The standard procedure for the osteogenic differentiation of multipotent stem cells is treatment of a confluent monolayer with a cocktail of dexamethasone (Dex), ascorbic acid (Asc) and β-glycerophosphate (β-Gly). This review describes the effects of these substances on intracellular signaling cascades that lead to osteogenic differentiation of bone marrow stroma-derived stem cells. We conclude that Dex induces Runx2 expression by FHL2/β-catenin-mediated transcriptional activation and that Dex enhances Runx2 activity by upregulation of TAZ and MKP1. Asc leads to the increased secretion of collagen type I (Col1), which in turn leads to increased Col1/α2β1 integrin-mediated intracellular signaling. The phosphate from β-Gly serves as a source for the phosphate in hydroxylapatite and in addition influences intracellular signaling molecules. In this context we give special attention to the differences between dystrophic and bone-specific mineralization. PMID:24073831

Highly efficient in vitro and in vivo delivery of functional RNAs using new versatile MS2-chimeric retrovirus-like particles

PubMed Central

Prel, Anne; Caval, Vincent; Gayon, Régis; Ravassard, Philippe; Duthoit, Christine; Payen, Emmanuel; Maouche-Chretien, Leila; Creneguy, Alison; Nguyen, Tuan Huy; Martin, Nicolas; Piver, Eric; Sevrain, Raphaël; Lamouroux, Lucille; Leboulch, Philippe; Deschaseaux, Frédéric; Bouillé, Pascale; Sensébé, Luc; Pagès, Jean-Christophe

2015-01-01

RNA delivery is an attractive strategy to achieve transient gene expression in research projects and in cell- or gene-based therapies. Despite significant efforts investigating vector-directed RNA transfer, there is still a requirement for better efficiency of delivery to primary cells and in vivo. Retroviral platforms drive RNA delivery, yet retrovirus RNA-packaging constraints limit gene transfer to two genome-molecules per viral particle. To improve retroviral transfer, we designed a dimerization-independent MS2-driven RNA packaging system using MS2-Coat-retrovirus chimeras. The engineered chimeric particles promoted effective packaging of several types of RNAs and enabled efficient transfer of biologically active RNAs in various cell types, including human CD34+ and iPS cells. Systemic injection of high-titer particles led to gene expression in mouse liver and transferring Cre-recombinase mRNA in muscle permitted widespread editing at the ROSA26 locus. We could further show that the VLPs were able to activate an osteoblast differentiation pathway by delivering RUNX2- or DLX5-mRNA into primary human bone-marrow mesenchymal-stem cells. Thus, the novel chimeric MS2-lentiviral particles are a versatile tool for a wide range of applications including cellular-programming or genome-editing. PMID:26528487

The Role of KV7.3 in Regulating Osteoblast Maturation and Mineralization

PubMed Central

Yang, Ji Eun; Song, Min Seok; Shen, Yiming; Ryu, Pan Dong; Lee, So Yeong

2016-01-01

KCNQ (KV7) channels are voltage-gated potassium (KV) channels, and the function of KV7 channels in muscles, neurons, and sensory cells is well established. We confirmed that overall blockade of KV channels with tetraethylammonium augmented the mineralization of bone-marrow-derived human mesenchymal stem cells during osteogenic differentiation, and we determined that KV7.3 was expressed in MG-63 and Saos-2 cells at the mRNA and protein levels. In addition, functional KV7 currents were detected in MG-63 cells. Inhibition of KV7.3 by linopirdine or XE991 increased the matrix mineralization during osteoblast differentiation. This was confirmed by alkaline phosphatase, osteocalcin, and osterix in MG-63 cells, whereas the expression of Runx2 showed no significant change. The extracellular glutamate secreted by osteoblasts was also measured to investigate its effect on MG-63 osteoblast differentiation. Blockade of KV7.3 promoted the release of glutamate via the phosphorylation of extracellular signal-regulated kinase 1/2-mediated upregulation of synapsin, and induced the deposition of type 1 collagen. However, activation of KV7.3 by flupirtine did not produce notable changes in matrix mineralization during osteoblast differentiation. These results suggest that KV7.3 could be a novel regulator in osteoblast differentiation. PMID:26999128

The Role of KV7.3 in Regulating Osteoblast Maturation and Mineralization.

PubMed

Yang, Ji Eun; Song, Min Seok; Shen, Yiming; Ryu, Pan Dong; Lee, So Yeong

2016-03-18

KCNQ (KV7) channels are voltage-gated potassium (KV) channels, and the function of KV7 channels in muscles, neurons, and sensory cells is well established. We confirmed that overall blockade of KV channels with tetraethylammonium augmented the mineralization of bone-marrow-derived human mesenchymal stem cells during osteogenic differentiation, and we determined that KV7.3 was expressed in MG-63 and Saos-2 cells at the mRNA and protein levels. In addition, functional KV7 currents were detected in MG-63 cells. Inhibition of KV7.3 by linopirdine or XE991 increased the matrix mineralization during osteoblast differentiation. This was confirmed by alkaline phosphatase, osteocalcin, and osterix in MG-63 cells, whereas the expression of Runx2 showed no significant change. The extracellular glutamate secreted by osteoblasts was also measured to investigate its effect on MG-63 osteoblast differentiation. Blockade of KV7.3 promoted the release of glutamate via the phosphorylation of extracellular signal-regulated kinase 1/2-mediated upregulation of synapsin, and induced the deposition of type 1 collagen. However, activation of KV7.3 by flupirtine did not produce notable changes in matrix mineralization during osteoblast differentiation. These results suggest that KV7.3 could be a novel regulator in osteoblast differentiation.

Secreted Phosphoprotein 1 Is a Determinant of Lung Function Development in Mice

PubMed Central

Martin, Timothy M.; Concel, Vincent J.; Upadhyay, Swapna; Bein, Kiflai; Brant, Kelly A.; George, Leema; Mitra, Ankita; Thimraj, Tania A.; Fabisiak, James P.; Vuga, Louis J.; Fattman, Cheryl; Kaminski, Naftali; Schulz, Holger; Leikauf, George D.

2014-01-01

Secreted phosphoprotein 1 (Spp1) is located within quantitative trait loci associated with lung function that was previously identified by contrasting C3H/HeJ and JF1/Msf mouse strains that have extremely divergent lung function. JF1/Msf mice with diminished lung function had reduced lung SPP1 transcript and protein during the peak stage of alveologenesis (postnatal day [P]14–P28) as compared with C3H/HeJ mice. In addition to a previously identified genetic variant that altered runt-related transcription factor 2 (RUNX2) binding in the Spp1 promoter, we identified another promoter variant in a putative RUNX2 binding site that increased the DNA protein binding. SPP1 induced dose-dependent mouse lung epithelial-15 cell proliferation. Spp1(−/−) mice have decreased specific total lung capacity/body weight, higher specific compliance, and increased mean airspace chord length (Lm) compared with Spp1(+/+) mice. Microarray analysis revealed enriched gene ontogeny categories, with numerous genes associated with lung development and/or respiratory disease. Insulin-like growth factor 1, Hedgehog-interacting protein, wingless-related mouse mammary tumor virus integration site 5A, and NOTCH1 transcripts decreased in the lung of P14 Spp1(−/−) mice as determined by quantitative RT-PCR analysis. SPP1 promotes pneumocyte growth, and mice lacking SPP1 have smaller, more compliant lungs with enlarged airspace (i.e., increased Lm). Microarray analysis suggests a dysregulation of key lung developmental transcripts in gene-targeted Spp1(−/−) mice, particularly during the peak phase of alveologenesis. In addition to its known roles in lung disease, this study supports SPP1 as a determinant of lung development in mice. PMID:24816281

IGF-1 Release Kinetics from Chitosan Microparticles Fabricated Using Environmentally Benign Conditions

PubMed Central

Mantripragada, Venkata P.; Jayasuriya, Ambalangodage C.

2014-01-01

The main objective of this study is to maximize growth factor encapsulation efficiency into microparticles. The novelty of this study is to maximize the encapsulated growth factors into microparticles by minimizing the use of organic solvents and using relatively low temperatures. The microparticles were fabricated using chitosan biopolymer as a base polymer and cross-linked with tripolyphosphate (TPP). Insulin like-growth factor-1 (IGF-1) was encapsulated into microparticles to study release kinetics and bioactivity. In order to authenticate the harms of using organic solvents like hexane and acetone during microparticle preparation, IGF-1 encapsulated microparticles prepared by the emulsification and coacervation methods were compared. The microparticles fabricated by emulsification method have shown a significant decrease (p<0.05) in IGF-1 encapsulation efficiency, and cumulative release during the two-week period. The biocompatibility of chitosan microparticles and the bioactivity of the released IGF-1 were determined in vitro by live/dead viability assay. The mineralization data observed with Von Kossa assay, was supported by mRNA expression levels of osterix and runx2, which are transcription factors necessary for osteoblasts differentiation. Real time RT-PCR data showed an increased expression of runx 2 and a decreased expression of osterix over time, indicating differentiating osteoblasts. Chitosan microparticles prepared in optimum environmental conditions are a promising controlled delivery system for cells to attach, proliferate, differentiate and mineralize, thereby acting as a suitable bone repairing material. PMID:25063148

Reverse engineering of TLX oncogenic transcriptional networks identifies RUNX1 as tumor suppressor in T-ALL.

PubMed

Della Gatta, Giusy; Palomero, Teresa; Perez-Garcia, Arianne; Ambesi-Impiombato, Alberto; Bansal, Mukesh; Carpenter, Zachary W; De Keersmaecker, Kim; Sole, Xavier; Xu, Luyao; Paietta, Elisabeth; Racevskis, Janis; Wiernik, Peter H; Rowe, Jacob M; Meijerink, Jules P; Califano, Andrea; Ferrando, Adolfo A

2012-02-26

The TLX1 and TLX3 transcription factor oncogenes have a key role in the pathogenesis of T cell acute lymphoblastic leukemia (T-ALL). Here we used reverse engineering of global transcriptional networks to decipher the oncogenic regulatory circuit controlled by TLX1 and TLX3. This systems biology analysis defined T cell leukemia homeobox 1 (TLX1) and TLX3 as master regulators of an oncogenic transcriptional circuit governing T-ALL. Notably, a network structure analysis of this hierarchical network identified RUNX1 as a key mediator of the T-ALL induced by TLX1 and TLX3 and predicted a tumor-suppressor role for RUNX1 in T cell transformation. Consistent with these results, we identified recurrent somatic loss-of-function mutations in RUNX1 in human T-ALL. Overall, these results place TLX1 and TLX3 at the top of an oncogenic transcriptional network controlling leukemia development, show the power of network analyses to identify key elements in the regulatory circuits governing human cancer and identify RUNX1 as a tumor-suppressor gene in T-ALL.

Divalent Metal Ions Induced Osteogenic Differentiation of MC3T3E1

NASA Astrophysics Data System (ADS)

Wang, Guoshou; Su, Wenta; Chen, Pohung; Huang, Teyang

2017-12-01

Biomaterial scaffolds blended with biochemical signal molecules with adequate osteoinductive and osteoconductive properties have attracted significant interest in bone tissue engineering regeneration. The divalent metal ions can gradually release from the scaffold into the culture medium and then induced osteoblastic differentiation of MC3T3E1. These MC3T3E1 cells expressed high activity of alkaline phosphatase, bone-related gene expression of collagen type I, Runx2, osteopontin, osteocalcin, and significantly enhanced deposited minerals on scaffold after 21 days of culture. This experiment provided a useful inducer for osteogenic differentiation in bone repair.

Alendronate promotes osteoblast differentiation and bone formation in ovariectomy-induced osteoporosis through interferon-β/signal transducer and activator of transcription 1 pathway

PubMed Central

Ma, Xiaoqing; Xu, Zhongyang; Ding, Shaofeng; Yi, Guangkun; Wang, Qian

2018-01-01

Alendronate is commonly used for the treatment of postmenopausal osteoporosis; however, the underlying pathological molecular mechanisms of its action remain unclear. In the present study, the alendronate-treated signaling pathway in bone metabolism in rats with ovariectomy induced by osteoporosis was investigated. Rats with osteoporosis were orally administered alendronate or phosphate-buffered saline (control). In addition, the interferon-β (IFN-β)/signal transducer and activator of transcription 1 (STAT1) signaling pathway was investigated in osteoblasts following treatment with alendronate in vitro and in vivo. During the differentiation period, IFN-β (100 ng/ml) was used to treat the osteoblast cells, and the activity, viability and bone metabolism-associated gene expression levels (STAT1, p-STAT1, Fra1, TRAF6 and SOCS1) were analyzed in osteoblast cells. Histopathological changes were used to evaluate osteoblasts, osteoclasts, inflammatory phase of bone healing and osteonecrotic areas. The results demonstrated that alendronate significantly inhibited the activity of osteoporotic osteoclasts by stimulating expression of IFN-β, as well as markedly improved the viability and activity of osteoblasts compared with the control group. In addition, alendronate increased the expression and phosphorylation levels of STAT1 in osteoclasts, enhanced osteoblast differentiation, upregulated the expression levels of alkaline phosphatase and osteocalcin, and increased the expression of osteoblast differentiation-associated genes (osteocalcin, osterix and Runx2). Inhibition of IFN-β expression canceled the benefits of alendronate-mediated osteoblast differentiation. Notably, alendronate enhanced bone formation in rats with osteoporosis induced by ovariectomy. In conclusion, these findings suggest that alendronate can regulate osteoblast differentiation and bone formation in rats with osteoporosis induced by ovariectomy through upregulation of IFN-β/STAT1 signaling pathway. PMID:29375681

A Genome-wide Regulatory Network Identifies Key Transcription Factors for Memory CD8+ T Cell Development

PubMed Central

Hu, Guangan; Chen, Jianzhu

2014-01-01

Memory CD8+ T cell development is defined by the expression of a specific set of memory signature genes (MSGs). Despite recent progress, many components of the transcriptional control of memory CD8+ T cell development are still unknown. To identify transcription factors (TFs) and their interactions in memory CD8+ T cell development, we construct a genome-wide regulatory network and apply it to identify key TFs that regulate MSGs. Most of the known TFs in memory CD8+ T cell development are rediscovered and about a dozen new TFs are also identified. Sox4, Bhlhe40, Bach2 and Runx2 are experimentally verified and Bach2 is further shown to promote both development and recall proliferation of memory CD8+ T cells through Prdm1 and Id3. Gene perturbation study identifies the mode of interactions among the TFs with Sox4 as a hub. The identified TFs and insights into their interactions should facilitate further dissection of molecular mechanisms underlying memory CD8+ T cell development. PMID:24335726

[Effect of trichostatin A on the osteogenic differentiation potential of periodontal ligament stem cells in inflammatory microenvironment induced by tumor necrosis factor-α stimulation].

PubMed

Wang, H; Chen, Q; Liu, W J; Yang, Z H; Li, D; Jin, F

2016-04-09

To compare the expression of histone deacetylase(HDAC)1-11 of human periodontal ligament stem cells(PDLSC)in normal and inflammatory microenvironments, and to investigate the effect of histone deacetylase inhibitor trichostatin A(TSA)on the osteogenic differentiation potential of PDLSC in inflammatory microenvironment induced by tumor necrosis factor-α(TNF-α)stimulation. PDLSC were isolated from periodontal ligament tissues obtained from the surgically extracted human teeth and cultured by single-colony selection. The expression of HDAC1-11 in cells with or without TNF-α(10 μg/L)stimulation was evaluated by quantitative real time-PCR(RT-PCR). The effect of TSA on cell proliferation was investigated by methyl thiazolyl tetrazolium(MTT)assay. The influence of TSA on osteogenic differentiation of PDLSC in inflammatory microenvironment with TNF-α stimulation was assessed by alizarin red staining, quantitative RT-PCR and Western blotting, respectively. The expression of HDAC in PDLSC with TNF-α stimulation was significantly higher than that in normal PDLSC(P<0.05)(except HDAC7, P=0.243). TSA had no significant effect on PDLSC proliferation at the concentration of 50 nmol/L(P=0.232). The alizarin red staining showed that PDLSC in TNF-α group generated less mineralized nodule than the control group, while the cell matrix mineralization in TSA group was improved obviously. TNF-α had an inhibitory effect on the expression of osteogenesis related genes, runt-related transcription factor-2(RUNX2)and alkaline phosphatase(ALP), with relative gene expression ratio(experimental/control)decreased to 0.17 ± 0.02 and 0.32 ± 0.03, while TSA could significantly increase the genes' expression to 0.67±0.03 and 0.89±0.02(P<0.01). Western blotting test showed that in TNF-α group the expression of osteogenesis related proteins was obviously reduced, and compared with the TNF-α group, TSA could significantly promote the expression of proteinsin inflammatory microenvironment. PDLSC in inflammatory microenvironment by TNF-α stimulation had a higher expression of HDAC than that in normal conditions. TSA, as a histone deacetylase inhibitor, could significantly promote the osteogenic differentiation potential of PDLSC in inflammatory microenvironment by suppressing HDAC.

Effects of ß-TCP scaffolds on neurogenic and osteogenic differentiation of human embryonic stem cells.

PubMed

Arpornmaeklong, Premjit; Pressler, Michael J

2018-01-01

Extracellular matrix (ECM) and adhesion molecules play crucial roles in regulating growth and differentiation of stem cells. The current study aimed to investigate the effects of beta-tricalcium phosphate (ß-TCP) scaffolds on differentiation and expression of ECM and adhesion molecules of human embryonic stem cells (hESCs). Undifferentiated hESCs were seeded on ß-TCP scaffolds and cell culture plates and cultured in growth and osteogenic medium for 21 days. Scanning electron microscopy (SEM) displayed adhesion and growth of hESCs on the porous ß-TCP scaffolds. Histological analysis, immunohistochemical staining and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) demonstrated that the scaffolds supported growth and differentiation of hESCs. Expression levels of neural crest related genes (AP2a, FoxD3, HNK1, P75, Sox1, Sox10) and osteoblast-related genes (Runx2, SPP1 and BGLA) on the scaffolds in osteogenic medium were significantly higher than on the scaffolds in growth and cell culture plates in osteogenic medium, respectively (p<0.05). Polymerase chain reaction array experiments demonstrated increased expression of ECM and adhesion molecule-related genes on the scaffolds. In conclusion, osteoconductive scaffolds such as ß-TCP scaffolds promoted differentiation of hESCs, particularly expression of genes related to neural crest stem cell and osteoblastic differentiations. Beta-TCP scaffolds could be an alternative cell culture substrate for neural crest and osteogenic differentiation of hESCs. Optimization of culture medium may be necessary to enhance lineage restriction of hESCs on the ß-TCP scaffolds. Copyright © 2017 Elsevier GmbH. All rights reserved.

Kaempferol stimulates bone sialoprotein gene transcription and new bone formation.

PubMed

Yang, Li; Takai, Hideki; Utsunomiya, Tadahiko; Li, Xinyue; Li, Zhengyang; Wang, Zhitao; Wang, Shuang; Sasaki, Yoko; Yamamoto, Hirotsugu; Ogata, Yorimasa

2010-08-15

Kaempferol is a typical flavonol-type flavonoid that is present in a variety of vegetables and fruits, and has a protective effect on postmenopausal bone loss. Bone sialoprotein (BSP) is thought to function in the initial mineralization of bone and could be crucial for osteoblast differentiation, bone matrix mineralization and tumor metastasis. In the present study we investigated the regulation of BSP transcription by kaempferol in rat osteoblast-like UMR106 cells, and the effect of kaempferol on new bone formation. Kaempferol (5 microM) increased BSP and Osterix mRNA levels at 12 h and up-regulated Runx2 mRNA expression at 6 h. Kaempferol increased luciferase activity of the construct pLUC3, which including the promoter sequence between nucleotides -116 to +60. Transcriptional stimulation by kaempferol abrogated in constructs included 2 bp mutations in the inverted CCAAT, CRE, and FRE elements. Gel shift analyses showed that kaempferol increased nuclear protein binding to CRE and FRE elements, whereas the CCAAT-protein complex did not change after kaempferol stimulation. Twelve daily injections of 5 microM kaempferol directly into the periosteum of parietal bones of newborn rats increased new bone formation. These data suggest that kaempferol increased BSP gene transcription mediated through inverted CCAAT, CRE, and FRE elements in the rat BSP gene promoter, and could induce osteoblast activities in the early stage of bone formation. (c) 2010 Wiley-Liss, Inc.

Gli1 protein participates in Hedgehog-mediated specification of osteoblast lineage during endochondral ossification.

PubMed

Hojo, Hironori; Ohba, Shinsuke; Yano, Fumiko; Saito, Taku; Ikeda, Toshiyuki; Nakajima, Keiji; Komiyama, Yuske; Nakagata, Naomi; Suzuki, Kentaro; Takato, Tsuyoshi; Kawaguchi, Hiroshi; Chung, Ung-il

2012-05-18

With regard to Hedgehog signaling in mammalian development, the majority of research has focused on Gli2 and Gli3 rather than Gli1. This is because Gli1(-/-) mice do not show any gross abnormalities in adulthood, and no detailed analyses of fetal Gli1(-/-) mice are available. In this study, we investigated the physiological role of Gli1 in osteogenesis. Histological analyses revealed that bone formation was impaired in Gli1(-/-) fetuses compared with WT fetuses. Gli1(-/-) perichondrial cells expressed neither runt-related transcription factor 2 (Runx2) nor osterix, master regulators of osteogenesis, in contrast to WT cells. In vitro analyses showed that overexpression of Gli1 up-regulated early osteogenesis-related genes in both WT and Runx2(-/-) perichondrial cells, and Gli1 activated transcription of those genes via its association with their 5'-regulatory regions, underlying the function of Gli1 in the perichondrium. Moreover, Gli1(-/-);Gli2(-/-) mice showed more severe phenotypes of impaired bone formation than either Gli1(-/-) or Gli2(-/-) mice, and osteoblast differentiation was impaired in Gli1(-/-);Gli3(-/-) perichondrial cells compared with Gli3(-/-) cells in vitro. These data suggest that Gli1 itself can induce early osteoblast differentiation, at least to some extent, in a Runx2-independent manner. It also plays a redundant role with Gli2 and is involved in the repressor function of Gli3 in osteogenesis. On the basis of these findings, we propose that upon Hedgehog input, Gli1 functions collectively with Gli2 and Gli3 in osteogenesis.

Histomorphometric and immunohistochemical analysis of human maxillary sinus-floor augmentation using porous β-tricalcium phosphate for dental implant treatment.

PubMed

Miyamoto, Shinji; Shinmyouzu, Kouhei; Miyamoto, Ikuya; Takeshita, Kenji; Terada, Toshihisa; Takahashi, Tetsu

2013-08-01

This study utilized the constitution and expression of Runx2/Cbfa1 to conduct 6-month-post-operation histomorphometrical and histochemical analysis of osteocalcin in bone regeneration following sinus-floor augmentation procedures using β-tricalcium phosphate (β-TCP) and autogenous cortical bone. Thirteen sinuses of nine patients were treated with sinus-floor augmentation using 50% β-TCP and 50% autogenous cancellous bone harvested from the ramus of the mandible. Biopsies of augmented sinuses were taken at 6 months for histomorphometric and immunohistochemical measurements. Runx2/Cbfa1- and osteocalcin-positive cells were found around TCP particles and on the bone surface. Approximately 60% of cells found around TCP particles stained positive for Runx2/Cbfa1. Fewer cells stained positive for osteocalcin. These positive cells decreased apically with increasing vertical distance from the maxillary bone surface. Histomorphometric analysis showed that the augmented site close to residual bone and periosteum contained approximately 42% bony tissue and 42% soft connective tissue, and the remaining 16% consisted of TCP particles. On the other hand, the augmented bone far from residual bone and periosteum contained 35% bony tissue and 50% soft connective tissue. Our data suggest that TCP particles attract osteoprogenitor cells that migrate into the interconnecting micropores of the bone-substitute material by 6 months. The augmented site close to residual bone contained a higher proportion of bony tissue and a lower proportion of soft connective tissue than did the augmented site far from residual bone. © 2012 John Wiley & Sons A/S.

Lipopolysaccharide can modify differentiation and immunomodulatory potential of periodontal ligament stem cells via ERK1,2 signaling.

PubMed

Kukolj, Tamara; Trivanović, Drenka; Djordjević, Ivana Okić; Mojsilović, Slavko; Krstić, Jelena; Obradović, Hristina; Janković, Srdja; Santibanez, Juan Francisco; Jauković, Aleksandra; Bugarski, Diana

2018-01-01

Lipopolysaccharide (LPS) is a pertinent deleterious factor in oral microenvironment for cells which are carriers of regenerative processes. The aim of this study was to investigate the emerging in vitro effects of LPS (Escherichia coli) on human periodontal ligament stem cell (PDLSC) functions and associated signaling pathways. We demonstrated that LPS did not affect immunophenotype, proliferation, viability, and cell cycle of PDLSCs. However, LPS modified lineage commitment of PDLSCs inhibiting osteogenesis by downregulating Runx2, ALP, and Ocn mRNA expression, while stimulating chondrogenesis and adipogenesis by upregulating Sox9 and PPARγ mRNA expression. LPS promoted myofibroblast-like phenotype of PDLSCs, since it significantly enhanced PDLSC contractility, as well as protein and/or gene expression of TGF-β, fibronectin (FN), α-SMA, and NG2. LPS also increased protein and gene expression levels of anti-inflammatory COX-2 and pro-inflammatory IL-6 molecules in PDLSCs. Inhibition of peripheral blood mononuclear cells (MNCs) transendothelial migration in presence of LPS-treated PDLSCs was accompanied by the reduction of CD29 expression within MNCs. However, LPS treatment did not change the inhibitory effect of PDLSCs on mitogen-stimulated proliferation of CD4 + and the ratio of CD4 + CD25 high /CD4 + CD25 low lymphocytes. LPS-treated PDLSCs did not change the frequency of CD34 + and CD45 + cells, but decreased the frequency of CD33 + and CD14 + myeloid cells within MNCs. Moreover, LPS treatment attenuated the stimulatory effect of PDLSCs on CFC activity of MNCs, predominantly the CFU-GM number. The results indicated that LPS-activated ERK1,2 was at least partly involved in the observed effects on PDLSC differentiation capacity, acquisition of myofibroblastic attributes, and changes of their immunomodulatory features. © 2017 Wiley Periodicals, Inc.

Effects of the micro-nano surface topography of titanium alloy on the biological responses of osteoblast.

PubMed

Yin, Chengcheng; Zhang, Yanjing; Cai, Qing; Li, Baosheng; Yang, Hua; Wang, Heling; Qi, Hua; Zhou, Yanmin; Meng, Weiyan

2017-03-01

In clinical applications, osseointegration is essential for the long-term stability of dental implants. Inspired by the hierarchical structure of natural bone, we applied the electrochemical etching (EC) technique to form a micro-nano structure on a titanium alloy (Ti6Al4V) substrate, called EC surface. Sand blasting and acid etching (SLA) and machined (M) methods were employed to generate micro and smooth textures, respectively, as the control groups. The surface topographies of the three substrates were characterized using scanning electron microscopy (SEM). Then, human osteoblast-like cells (MG63) were cultured on substrates, and adhesion, proliferation, morphology, alkaline phosphatase activity (ALP), and gene expression levels of Runt-related transcription factor 2 (RUNX2), osteocalcin (OCN), osteopontin (OPN), and type I collagen (COLIA 1) were analyzed. MG63 cells cultured on the EC Ti alloy substrates displayed better cell adhesion, significant proliferation, and a higher production level of ALP, gene expressions of RUNX2, OCN, OPN and COLIA 1 (p < 0.01 or p < 0.05) compared with those of SLA and M substrates. These results indicate that the micro-nano structure fabricated by electrochemical etching method is beneficial for the biological functions of MG63 cells and may be a promising candidate in dental implants. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 757-769, 2017. © 2016 Wiley Periodicals, Inc.

Silk Fibroin-Alginate-Hydroxyapatite Composite Particles in Bone Tissue Engineering Applications In Vivo

PubMed Central

Jo, You-Young; Kim, Seong-Gon; Kwon, Kwang-Jun; Kweon, HaeYong; Chae, Weon-Sik; Yang, Won-Geun; Lee, Eun-Young; Seok, Hyun

2017-01-01

The aim of this study was to evaluate the in vivo bone regeneration capability of alginate (AL), AL/hydroxyapatite (HA), and AL/HA/silk fibroin (SF) composites. Forty Sprague Dawley rats were used for the animal experiments. Central calvarial bone (diameter: 8.0 mm) defects were grafted with AL, AL/HA, or AL/HA/SF. New bone formation was evaluated by histomorphometric analysis. To demonstrate the immunocompatibility of each group, the level of tumor necrosis factor (TNF)-α expression was studied by immunohistochemistry (IHC) and quantitative reverse transcription polymerase chain reaction (qRT-PCR) at eight weeks post implantation. Additionally, osteogenic markers, such as fibroblast growth factor (FGF)-23, osteoprotegerin (OPG), and Runt-related transcription factor (Runx2) were evaluated by qPCR or IHC at eight weeks post implantation. The AL/HA/SF group showed significantly higher new bone formation than did the control group (p = 0.044) and the AL group (p = 0.035) at four weeks post implantation. Additionally, the AL/HA/SF group showed lower relative TNF-α mRNA levels and higher FGF-23 mRNA levels than the other groups did at eight weeks post implantation. IHC results demonstrated that the AL/HA/SF group had lower TNF-α expression and higher OPG and Runx2 expression at eight weeks post implantation. Additionally, no evidence of the inflammatory reaction or giant cell formation was observed around the residual graft material. We concluded that the AL/HA/SF composite could be effective as a scaffold for bone tissue engineering. PMID:28420224

Dental Cell Sheet Biomimetic Tooth Bud Model

PubMed Central

Monteiro, Nelson; Smith, Elizabeth E.; Angstadt, Shantel; Zhang, Weibo; Khademhosseini, Ali

2016-01-01

Tissue engineering and regenerative medicine technologies offer promising therapies for both medicine and dentistry. Our long-term goal is to create functional biomimetic tooth buds for eventual tooth replacement in humans. Here, our objective was to create a biomimetic 3D tooth bud model consisting of dental epithelial (DE) – dental mesenchymal (DM) cell sheets (CSs) combined with biomimetic enamel organ and pulp organ layers created using GelMA hydrogels. Pig DE or DM cells seeded on temperature-responsive plates at various cell densities (0.02, 0.114 and 0.228 cells 106/cm2) and cultured for 7, 14 and 21 days were used to generate DE and DM cell sheets, respectively. Dental CSs were combined with GelMA encapsulated DE and DM cell layers to form bioengineered 3D tooth buds. Biomimetic 3D tooth bud constructs were cultured in vitro, or implanted in vivo for 3 weeks. Analyses were performed using micro-CT, H&E staining, polarized light (Pol) microscopy, immunofluorescent (IF) and immunohistochemical (IHC) analyses. H&E, IHC and IF analyses showed that in vitro cultured multilayered DE-DM CSs expressed appropriate tooth marker expression patterns including SHH, BMP2, RUNX2, tenascin and syndecan, which normally direct DE-DM interactions, DM cell condensation, and dental cell differentiation. In vivo implanted 3D tooth bud constructs exhibited mineralized tissue formation of specified size and shape, and SHH, BMP2 and RUNX2and dental cell differentiation marker expression. We propose our biomimetic 3D tooth buds as models to study optimized DE-DM cell interactions leading to functional biomimetic replacement tooth formation. PMID:27565550

Silk Fibroin-Alginate-Hydroxyapatite Composite Particles in Bone Tissue Engineering Applications In Vivo.

PubMed

Jo, You-Young; Kim, Seong-Gon; Kwon, Kwang-Jun; Kweon, HaeYong; Chae, Weon-Sik; Yang, Won-Geun; Lee, Eun-Young; Seok, Hyun

2017-04-18

The aim of this study was to evaluate the in vivo bone regeneration capability of alginate (AL), AL/hydroxyapatite (HA), and AL/HA/silk fibroin (SF) composites. Forty Sprague Dawley rats were used for the animal experiments. Central calvarial bone (diameter: 8.0 mm) defects were grafted with AL, AL/HA, or AL/HA/SF. New bone formation was evaluated by histomorphometric analysis. To demonstrate the immunocompatibility of each group, the level of tumor necrosis factor (TNF)-α expression was studied by immunohistochemistry (IHC) and quantitative reverse transcription polymerase chain reaction (qRT-PCR) at eight weeks post implantation. Additionally, osteogenic markers, such as fibroblast growth factor (FGF)-23, osteoprotegerin (OPG), and Runt-related transcription factor (Runx2) were evaluated by qPCR or IHC at eight weeks post implantation. The AL/HA/SF group showed significantly higher new bone formation than did the control group ( p = 0.044) and the AL group ( p = 0.035) at four weeks post implantation. Additionally, the AL/HA/SF group showed lower relative TNF-α mRNA levels and higher FGF-23 mRNA levels than the other groups did at eight weeks post implantation. IHC results demonstrated that the AL/HA/SF group had lower TNF-α expression and higher OPG and Runx2 expression at eight weeks post implantation. Additionally, no evidence of the inflammatory reaction or giant cell formation was observed around the residual graft material. We concluded that the AL/HA/SF composite could be effective as a scaffold for bone tissue engineering.

DNA methylation and childhood asthma in the inner city.

PubMed

Yang, Ivana V; Pedersen, Brent S; Liu, Andrew; O'Connor, George T; Teach, Stephen J; Kattan, Meyer; Misiak, Rana Tawil; Gruchalla, Rebecca; Steinbach, Suzanne F; Szefler, Stanley J; Gill, Michelle A; Calatroni, Agustin; David, Gloria; Hennessy, Corinne E; Davidson, Elizabeth J; Zhang, Weiming; Gergen, Peter; Togias, Alkis; Busse, William W; Schwartz, David A

2015-07-01

Epigenetic marks are heritable, influenced by the environment, direct the maturation of T lymphocytes, and in mice enhance the development of allergic airway disease. Thus it is important to define epigenetic alterations in asthmatic populations. We hypothesize that epigenetic alterations in circulating PBMCs are associated with allergic asthma. We compared DNA methylation patterns and gene expression in inner-city children with persistent atopic asthma versus healthy control subjects by using DNA and RNA from PBMCs. Results were validated in an independent population of asthmatic patients. Comparing asthmatic patients (n = 97) with control subjects (n = 97), we identified 81 regions that were differentially methylated. Several immune genes were hypomethylated in asthma, including IL13, RUNX3, and specific genes relevant to T lymphocytes (TIGIT). Among asthmatic patients, 11 differentially methylated regions were associated with higher serum IgE concentrations, and 16 were associated with percent predicted FEV1. Hypomethylated and hypermethylated regions were associated with increased and decreased gene expression, respectively (P < 6 × 10(-12) for asthma and P < .01 for IgE). We further explored the relationship between DNA methylation and gene expression using an integrative analysis and identified additional candidates relevant to asthma (IL4 and ST2). Methylation marks involved in T-cell maturation (RUNX3), TH2 immunity (IL4), and oxidative stress (catalase) were validated in an independent asthmatic cohort of children living in the inner city. Our results demonstrate that DNA methylation marks in specific gene loci are associated with asthma and suggest that epigenetic changes might play a role in establishing the immune phenotype associated with asthma. Published by Elsevier Inc.

Myostatin inhibits osteoblastic differentiation by suppressing osteocyte-derived exosomal microRNA-218: A novel mechanism in muscle-bone communication.

PubMed

Qin, Yiwen; Peng, Yuanzhen; Zhao, Wei; Pan, Jianping; Ksiezak-Reding, Hanna; Cardozo, Christopher; Wu, Yingjie; Divieti Pajevic, Paola; Bonewald, Lynda F; Bauman, William A; Qin, Weiping

2017-06-30

Muscle and bone are closely associated in both anatomy and function, but the mechanisms that coordinate their synergistic action remain poorly defined. Myostatin, a myokine secreted by muscles, has been shown to inhibit muscle growth, and the disruption of the myostatin gene has been reported to cause muscle hypertrophy and increase bone mass. Extracellular vesicle-exosomes that carry microRNA (miRNA), mRNA, and proteins are known to perform an important role in cell-cell communication. We hypothesized that myostatin may play a crucial role in muscle-bone interactions and may promote direct effects on osteocytes and on osteocyte-derived exosomal miRNAs, thereby indirectly influencing the function of other bone cells. We report herein that myostatin promotes expression of several bone regulators such as sclerostin (SOST), DKK1, and RANKL in cultured osteocytic (Ocy454) cells, concomitant with the suppression of miR-218 in both parent Ocy454 cells and derived exosomes. Exosomes produced by Ocy454 cells that had been pretreated with myostatin could be taken up by osteoblastic MC3T3 cells, resulting in a marked reduction of Runx2, a key regulator of osteoblastic differentiation, and in decreased osteoblastic differentiation via the down-regulation of the Wnt signaling pathway. Importantly, the inhibitory effect of myostatin-modified osteocytic exosomes on osteoblast differentiation is completely reversed by expression of exogenous miR-218, through a mechanism involving miR-218-mediated inhibition of SOST. Together, our findings indicate that myostatin directly influences osteocyte function and thereby inhibits osteoblastic differentiation, at least in part, through the suppression of osteocyte-derived exosomal miR-218, suggesting a novel mechanism in muscle-bone communication. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Heterodimeric BMP-2/7 Antagonizes the Inhibition of All-Trans Retinoic Acid and Promotes the Osteoblastogenesis

PubMed Central

Bi, Wenjuan; Gu, Zhiyuan; Zheng, Yuanna; Zhang, Xiao; Guo, Jing; Wu, Gang

2013-01-01

Objectives Hypervitaminosis A and alcoholism can result in a low mineral density and compromised regenerative capacity of bone, thus delaying implant osteointegration. The inhibitory effect of all-trans retinoic acid on osteoblastogenesis is considered to be one of the mechanisms. We hypothesized that heterodimeric bone morphogenetic protein-2/7 could antagonize all-trans retinoic acid and enhance osteoblastogenesis, with an aim to accelerate and enhance bone regeneration and implant osteointegration. Materials and Methods We applied 5 ng/ml or 50 ng/ml bone morphogenetic protein-2/7 to restore the osteoblastogenesis of pre-osteoblasts (MC3T3-E1 cell line) that was inhibited by 1 µM all-trans retinoic acid. We evaluated the efficacy by assessing cell numbers (proliferation), alkaline phosphatase activity (a marker for early differentiation), osteocalcin (a marker for late differentiation), calcium deposition (a marker for final mineralization) and the expression of osteoblastogenic genes (such as Runx2, Collagen Ia, alkaline phosphatase and osteocalcin) at different time points. Results All-trans retinoic acid significantly inhibited the expression of all the tested osteoblastogenic genes and proteins except alkaline phosphatase activity. In the presence of ATRA, 50 ng/ml bone morphogenetic protein-2/7 not only completely restored but also significantly enhanced all the osteoblastogenic genes and proteins. On the 28th day, mineralization was completely inhibited by all-trans retinoic acid. In contrast, 50 ng/ml BMP-2/7 could antagonize ATRA and significantly enhance the mineralization about 2.5 folds in comparison with the control treatment (no ATRA, no BMP2/7). Conclusions Heterodimeric bone morphogenetic protein-2/7 bears a promising application potential to significantly promote bone regeneration and implant osteointegration for the patients with hypervitaminosis A and alcoholism. PMID:24205156

Smad7 mediates inhibition of Saos2 osteosarcoma cell differentiation by NF{kappa}B

DOE Office of Scientific and Technical Information (OSTI.GOV)

Eliseev, Roman A.; Schwarz, Edward M.; Zuscik, Michael J.

2006-01-01

The transcription factor NF{kappa}B is constitutively activated in various tumor cells where it promotes proliferation and represses apoptosis. The bone morphogenetic proteins (BMPs) delay cell proliferation and promote differentiation and apoptosis of bone cells through activation of Smad downstream effectors and via Smad-independent mechanisms. Thus, NF{kappa}B and BMP pathways play opposing roles in regulating osteoblastic cell fate. Here, we show that in osteosarcoma Saos2 osteoblasts, NF{kappa}B regulates the activity of the BMP/Smad signaling. Inhibition of NF{kappa}B by overexpression of mI{kappa}B leads to the induction of osteoblast differentiation. Saos2 cells overexpressing mI{kappa}B (Saos2-mI{kappa}B) exhibit higher expression of osteoblast phenotypic genes suchmore » as alkaline phosphatase, Runx2 and osteocalcin and are more responsive to BMP2 in comparison to wild-type cells (Saos2-wt) or empty vector infected controls (Saos2-EV). Furthermore, BMP-2 signaling and Smad phosphorylation are significantly increased in Saos2-mI{kappa}B cells in comparison to Saos2-EV cells. Inhibition of NF{kappa}B signaling in Saos2-mI{kappa}B cells is associated with decreased expression of the BMP signaling inhibitor Smad7. While gain of Smad7 function in Saos2-mI{kappa}B cells results in inhibition of BMP signaling, anti-sense knockdown of Smad7 in Saos2-EV cells leads to upregulation of BMP signaling. We therefore conclude that in osteosarcoma Saos2 cells, NF{kappa}B represses BMP/Smad signaling and BMP2-induced differentiation through Smad7.« less

Salmon DNA Accelerates Bone Regeneration by Inducing Osteoblast Migration

PubMed Central

Sato, Ayako; Kajiya, Hiroshi; Mori, Nana; Sato, Hironobu; Fukushima, Tadao; Kido, Hirofumi

2017-01-01

The initial step of bone regeneration requires the migration of osteogenic cells to defective sites. Our previous studies suggest that a salmon DNA-based scaffold can promote the bone regeneration of calvarial defects in rats. We speculate that the salmon DNA may possess osteoinductive properties, including the homing of migrating osteogenic cells. In the present study, we investigated the influence of the salmon DNA on osteoblastic differentiation and induction of osteoblast migration using MG63 cells (human preosteoblasts) in vitro. Moreover, we analyzed the bone regeneration of a critical-sized in vivo calvarial bone defect (CSD) model in rats. The salmon DNA enhanced both mRNA and protein expression of the osteogenesis-related factors, runt-related transcription factor 2 (Runx2), alkaline phosphatase, and osterix (OSX) in the MG63 cells, compared with the cultivation using osteogenic induction medium alone. From the histochemical and immunohistochemical assays using frozen sections of the bone defects from animals that were implanted with DNA disks, many cells were found to express aldehyde dehydrogenase 1, one of the markers for mesenchymal stem cells. In addition, OSX was observed in the replaced connective tissue of the bone defects. These findings indicate that the DNA induced the migration and accumulation of osteogenic cells to the regenerative tissue. Furthermore, an in vitro transwell migration assay showed that the addition of DNA enhanced an induction of osteoblast migration, compared with the medium alone. The implantation of the DNA disks promoted bone regeneration in the CSD of rats, compared with that of collagen disks. These results indicate that the salmon DNA enhanced osteoblastic differentiation and induction of migration, resulting in the facilitation of bone regeneration. PMID:28060874

Diamagnetic levitation promotes osteoclast differentiation from RAW264.7 cells.

PubMed

Sun, Yu-Long; Chen, Zhi-Hao; Chen, Xiao-Hu; Yin, Chong; Li, Di-Jie; Ma, Xiao-Li; Zhao, Fan; Zhang, Ge; Shang, Peng; Qian, Ai-Rong

2015-03-01

The superconducting magnet with a high magnetic force field can levitate diamagnetic materials. In this study, a specially designed superconducting magnet with large gradient high magnetic field (LGHMF), which provides three apparent gravity levels (μg, 1 g, and 2 g), was used to study its influence on receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation from preosteoclast cell line RAW264.7. The effects of LGHMF on the viability, nitric oxide (NO) production, morphology in RAW264.7 cells were detected by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method, the Griess method, and the immunofluorescence staining, respectively. The changes induced by LGHMF in osteoclast formation, mRNA expression, and bone resorption were determined by tartrate-resistant acid phosphatase staining, semiquantity PCR, and bone resorption test, respectively. The results showed that: 1) LGHMF had no lethal effect on osteoclast precursors but attenuated NO release in RAW264.7 cells. 2) Diamagnetic levitation (μg) enhanced both the formation and bone resorption capacity of osteoclast. Moreover, diamagnetic levitation up-regulated mRNA expression of RANK, Cathepsin K, MMP-9, and NFATc1, while down-regulated RunX2 in comparison with controls. Furthermore, diamagnetic levitation induced obvious morphological alterations in osteoclast, including active cytoplasmic peripheral pseudopodial expansion, formation of pedosome belt, and aggregation of actin ring. 3) Magnetic field produced by LGHMF attenuated osteoclast resorption activity. Collectively, LGHMF with combined effects has multiple effects on osteoclast, which attenuated osteoclast resorption with magnetic field, whereas promoted osteoclast differentiation with diamagnetic levitation. Therefore, these findings indicate that diamagnetic levitation could be used as a novel ground-based microgravity simulator, which facilitates bone cell research of weightlessness condition.

Epigenetic Marks Define the Lineage and Differentiation Potential of Two Distinct Neural Crest-Derived Intermediate Odontogenic Progenitor Populations

PubMed Central

Gopinathan, Gokul; Kolokythas, Antonia

2013-01-01

Epigenetic mechanisms, such as histone modifications, play an active role in the differentiation and lineage commitment of mesenchymal stem cells. In the present study, epigenetic states and differentiation profiles of two odontogenic neural crest-derived intermediate progenitor populations were compared: dental pulp (DP) and dental follicle (DF). ChIP on chip assays revealed substantial H3K27me3-mediated repression of odontoblast lineage genes DSPP and dentin matrix protein 1 (DMP1) in DF cells, but not in DP cells. Mineralization inductive conditions caused steep increases of mineralization and patterning gene expression levels in DP cells when compared to DF cells. In contrast, mineralization induction resulted in a highly dynamic histone modification response in DF cells, while there was only a subdued effect in DP cells. Both DF and DP progenitors featured H3K4me3-active marks on the promoters of early mineralization genes RUNX2, MSX2, and DLX5, while OSX, IBSP, and BGLAP promoters were enriched for H3K9me3 or H3K27me3. Compared to DF cells, DP cells expressed higher levels of three pluripotency-associated genes, OCT4, NANOG, and SOX2. Finally, gene ontology comparison of bivalent marks unique for DP and DF cells highlighted cell–cell attachment genes in DP cells and neurogenesis genes in DF cells. In conclusion, the present study indicates that the DF intermediate odontogenic neural crest lineage is distinguished from its DP counterpart by epigenetic repression of DSPP and DMP1 genes and through dynamic histone enrichment responses to mineralization induction. Findings presented here highlight the crucial role of epigenetic regulatory mechanisms in the terminal differentiation of odontogenic neural crest lineages. PMID:23379639

Coexistence of iAMP21 and ETV6-RUNX1 fusion in an adolescent with B cell acute lymphoblastic leukemia: literature review of six additional cases.

PubMed

Gu, Jun; Reynolds, Alexandra; Fang, Lianghua; DeGraffenreid, Corrie; Sterns, Kenneth; Patel, Keyur P; Medeiros, L Jeffrey; Lin, Pei; Lu, Xinyan

2016-01-01

Intrachromosomal amplification of chromosome 21 (iAMP21) results from breakage-fusion-bridge cycles and chromothripsis is a distinct marker of a subgroup of B cell acute lymphoblastic leukemia (B-ALL) cases associated with a poor prognosis. iAMP21 accounts for 2% of pediatric B-ALL and occurs predominantly in older children or adolescents. ETV6-RUNX1 fusion, resulting from t(12;21)(p13;q22), is associated with an excellent outcome in younger children with B-ALL. Coexistence of iAMP21 with ETV6-RUNX1 fusion is extremely rare with limited clinical information available. We report the case of an 18-year old Caucasian man diagnosed with ETV6-RUNX1 fusion positive B-ALL. He was treated with intensive chemotherapy and achieved remission for 6 months before relapse, 15 months after the initial diagnosis. G-band karyotyping and Fluorescence in situ hybridization (FISH) analyses performed on bone marrow revealed complex abnormalities: 41,X,-Y,der(3)t(3;20)(p11.2;q11.2),-4,t(5;22)(q32;q11.2),del(9)(p13),dic(9;17)(p13;p11.2),t(12;21)(p13;q22),der(14)t(14;17)(p11.2;q11.2),der(17;22)(q11.2;q11.2),-20,add(21)(q22),-22[4]/46,XY[15] with an iAMP21 and an ETV6-RUNX1 . Additional molecular studies confirmed ETV6-RUNX1 fusion and with a TP53 mutation. High-resolution single nucleotide polymorphism microarray (SNP array) revealed the iAMP21 to be chromothripsis of 21q and subsequent metaphase FISH further delineated complex genomic aberrations. Although the patient received intensive chemotherapy with allogenic stem cell transplant, he died 26 months after initial diagnosis. We searched the literature and identified six cases showing coexisting iAMP21 and ETV6-RUNX1 . The median age for these six patients was 10 years (range, 2-18) and males predominated. The median overall survival (OS) was 28 months. Patients with B-ALL associated with both iAMP21 and ETV6-RUNX1 tend to be older children or adolescents and have a poor prognosis.

Sika pilose antler type I collagen promotes BMSC differentiation via the ERK1/2 and p38-MAPK signal pathways.

PubMed

Wang, Yanshuang; Luo, Su; Zhang, Dafang; Qu, Xiaobo; Tan, Yinfeng

2017-12-01

Sika pilose antler type I collagen (SPC-I) have been reported to promote bone marrow mesenchymal stem cell (BMSC) proliferation and differentiation. However, the underlying mechanism is still unclear. This study investigates the molecular mechanisms of SPC-I on the BMSC proliferation and differentiation of osteoblast (OB) in vitro. The primary rat BMSC was cultured and exposed to SPC-I at different concentrations (2.5, 5.0 and 10.0 mg/mL) for 20 days. The effect of SPC-I on the differentiation of BMSCs was evaluated through detecting the activity of alkaline phosphatase (ALP), ALP staining, collagen I (Col-I) content, and calcified nodules. The markers of osteoblastic differentiation were evaluated using RT-PCR and Western-blot analysis. SPC-I treatment (2.5 mg/mL) significantly increased the proliferation of BMSCs (p < 0.01), whereas, SPC-I (5.0 and 10.0 mg/mL) significantly inhibited the proliferation of BMSCs (p < 0.01). SPC-I (2.5 mg/mL) significantly increased ALP activity and Col-I content (p < 0.01), and increased positive cells in ALP staining and the formation of calcified nodules. Additionally, the gene expression of ALP, Col-I, Osteocalcin (OC), Runx2, Osterix (Osx), ERK1/2, BMP2 and p38-MAPK, along with the protein expression of ERK1/2, p-ERK1/2, p-p38 MAPK were markedly increased in the SPC-I (5.0 mg/mL) treatment group (p < 0.01) compared to the control group. SPC-I can induce BMSC differentiation into OBs and enhance the function of osteogenesis through ERK1/2 and p38-MAPK signal transduction pathways and regulating the gene expression of osteogenesis-specific transcription factors.

[Effects of intermediate conductance calcium-activated potassium channel blocker TARAM-34 on β-glycerophosphate induced vascular smooth muscle cells calcification].

PubMed

Zhang, S L; Xu, J S; Yang, S; Bai, Y L; Zhang, J X; Cui, L W; Yu, Q Y

2016-06-24

To observe the role of TRAM-34 (1-((2-chlorophenyl)diphenylmethyl)-1H-pyrazole), the blocker of intermediate conductance calcium-activated potassium channel (KCa3.1), on β-glycerophosphate induced vascular calcification in vitro. Vascular smooth muscle cells(VSMCs) were obtained from rat thoracic aorta, and VSMCs after the fourth passage and aortic rings were divided into control group (cultured in DMEM with 10% fetal bovine serum), high phosphorus group (cultured in DMEM with 10% fetal bovine serum and 10% β-glycerophosphate) and TRAM-34 group(20 nmol/L TRAM-34 was added into high phosphorus DMEM). Calcium deposition of VSMCs and aortic rings were measured by o-cresolphthalein complexone method.Calcium influx of VSMCs was measured by immunofluorescence probe Fluo-3 AM.The expression of runt-related transcription factor 2(Runx2)was detected by RT-PCR and Western blot for cells and immunohistochemistry for aortic rings.ALP activity was measured by alkaline phosphatase activity detection kit. (1) Compared with control group, calcification was significantly increased in high phosphorus group ((121.67±6.17) mg/g vs. (84.38±8.17) mg/g, P<0.05) and this effect could be attenuated by TRAM-34 ((93.31±11.36) mg/g, P<0.05 vs. high phosphorus group) after 12 days culture. Similar results were found in aortic rings cultured for 12 days-high phosphorus group: (7.17±0.57) mg/g vs. (1.18±0.13) mg/g (P<0.05) and TRAM-34: (4.71±0.42) mg/g, P<0.05 vs. high phosphorus group.(2) Compared with control group, the calcium influx was higher in high phosphorus group (349.22±40.47 vs. 151.67±16.94, P<0.05) and reduced in TRAM-34 group (194.67±22.21, P<0.05 vs. high phosphorus group) in VSMCs simulated for 4 days. (3) Both mRNA and protein expressions of Runx2 in high phosphorus groups were higher than in control group (0.630±0.033 vs.0.340±0.058 and 0.865±0.031 vs.0.414±0.011, both P<0.05) and lower in TRAM-34 group (0.399±0.023 and 0.575±0.014, both P<0.05 vs. high phosphorus group) in VSMCs simulated for 4 days.Besides, compared with high phosphorus group, the expression of Runx2 was decreased in control group(0.113±0.010 vs.0.067±0.008, P<0.05) and TRAM-34 group (0.069±0.006, P<0.05) after aortic rings were cultured for 4 days. (4) Compared with control group, the activity of ALP was significantly increased in high phosphorus group (96.56±9.84 vs.46.92±4.60, P<0.05) and decreased in TRAM-34 group(70.20±8.41, P<0.05 vs. high phosphorus group) in VSMCs simulated for 12 days. KCa3.1 blocker TRAM-34 can inhibit β-glycerophosphate induced VSMCs and aortic ring calcification through inhibiting calcium influx, downregulating Runx2 expression and attenuating osteogenic differentiation.

Osteogenic potential of in situ TiO2 nanowire surfaces formed by thermal oxidation of titanium alloy substrate

NASA Astrophysics Data System (ADS)

Tan, A. W.; Ismail, R.; Chua, K. H.; Ahmad, R.; Akbar, S. A.; Pingguan-Murphy, B.

2014-11-01

Titanium dioxide (TiO2) nanowire surface structures were fabricated in situ by a thermal oxidation process, and their ability to enhance the osteogenic potential of primary osteoblasts was investigated. Human osteoblasts were isolated from nasal bone and cultured on a TiO2 nanowires coated substrate to assess its in vitro cellular interaction. Bare featureless Ti-6Al-4V substrate was used as a control surface. Initial cell adhesion, cell proliferation, cell differentiation, cell mineralization, and osteogenic related gene expression were examined on the TiO2 nanowire surfaces as compared to the control surfaces after 2 weeks of culturing. Cell adhesion and cell proliferation were assayed by field emission scanning electron microscope (FESEM) and Alamar Blue reduction assay, respectively. The nanowire surfaces promoted better cell adhesion and spreading than the control surface, as well as leading to higher cell proliferation. Our results showed that osteoblasts grown onto the TiO2 nanowire surfaces displayed significantly higher production levels of alkaline phosphatase (ALP), extracellular (ECM) mineralization and genes expression of runt-related transcription factor (Runx2), bone sialoprotein (BSP), ostoepontin (OPN) and osteocalcin (OCN) compared to the control surfaces. This suggests the potential use of such surface modification on Ti-6Al-4V substrates as a promising means to improve the osteointegration of titanium based implants.

Influence of the intensity and loading time of direct current electric field on the directional migration of rat bone marrow mesenchymal stem cells.

PubMed

Wang, Xiaoyu; Gao, Yuxuan; Shi, Haigang; Liu, Na; Zhang, Wei; Li, Hongbo

2016-09-01

Exogenic electric fields can effectively accelerate bone healing and remodeling through the enhanced migration of bone marrow mesenchymal stem cells (BMSCs) toward the injured area. This study aimed to determine the following: (1) the direction of rat BMSC (rBMSC) migration upon exposure to a direct current electric field (DCEF), (2) the optimal DCEF intensity and duration, and (3) the possible regulatory role of SDF-1/CXCR4 axis in rBMSC migration as induced by DCEF. Results showed that rBMSCs migrated to the positive electrode of the DCEF, and that the DCEF of 200 mV/mm for 4 h was found to be optimal in enhancing rBMSC migration. This DCEF strength and duration also upregulated the expression of osteoblastic genes, including ALP and OCN, and upregulated the expression of ALP and Runx2 proteins. Moreover, when CXCR4 was inhibited, rBMSC migration due to DCEF was partially blocked. These findings indicated that DCEF can effectively induce rBMSC migration. A DCEF of 200 mV/mm for 4 h was recommended because of its ability to promote rBMSC migration, proliferation, and osteogenic differentiation. The SDF-1/CXCR4 signaling pathway may play an important role in regulating the DCEF-induced migration of rBMSCs.

Effect of Different Titanium Surfaces on Maturation of Murine Bone Marrow-Derived Dendritic Cells

NASA Astrophysics Data System (ADS)

Zheng, Xiaofei; Zhou, Fengjuan; Gu, Yifei; Duan, Xiaobo; Mo, Anchun

2017-02-01

Dendritic cells (DCs) play a pivotal role in the host response to implanted biomaterials. Osseointegration of titanium (Ti) implant is an immunological and inflammatory-driven process. However, the role of DCs in this complex process is largely unknown. This study aimed to investigate the effect of different Ti surfaces on DC maturation, and evaluate its subsequent potential on osteogenic differentiation of preosteoblasts. Murine bone marrow-derived DCs were seeded on Ti disks with different surface treatments, including pretreatment (PT), sandblasted/acid-etched (SLA) and modified SLA (modSLA) surface. Compared with DCs cultured on PT and SLA surfaces, the cells seeded on modSLA surface demonstrated a more round morphology with lower expression of CD86 and MHC-II, the DC maturation markers. Those cells also secreted high levels of anti-inflammatory cytokine IL-10 and TGF-β. Notably, addition of conditioned medium (CM) from modSLA-induced DCs significantly increased the mRNA expression of Runx2 and ALP as well as ALP activity by murine preosteoblast MC3T3-E1 cells. Our data demonstrated that Ti disks with different surfaces lead to differential DCs responses. PT and SLA surfaces induce DCs mature, while DCs seeded on modSLA-Ti surface maintain an immature phenotype and exhibit a potential of promoting osteogenic differentiation of MC3T3-E1 cells.

A novel osteogenic oxysterol compound for therapeutic development to promote bone growth: activation of hedgehog signaling and osteogenesis through smoothened binding.

PubMed

Montgomery, Scott R; Nargizyan, Taya; Meliton, Vicente; Nachtergaele, Sigrid; Rohatgi, Rajat; Stappenbeck, Frank; Jung, Michael E; Johnson, Jared S; Aghdasi, Bayan; Tian, Haijun; Weintraub, Gil; Inoue, Hirokazu; Atti, Elisa; Tetradis, Sotirios; Pereira, Renata C; Hokugo, Akishige; Alobaidaan, Raed; Tan, Yanlin; Hahn, Theodor J; Wang, Jeffrey C; Parhami, Farhad

2014-08-01

Osteogenic factors are often used in orthopedics to promote bone growth, improve fracture healing, and induce spine fusion. Osteogenic oxysterols are naturally occurring molecules that were shown to induce osteogenic differentiation in vitro and promote spine fusion in vivo. The purpose of this study was to identify an osteogenic oxysterol more suitable for clinical development than those previously reported, and evaluate its ability to promote osteogenesis in vitro and spine fusion in rats in vivo. Among more than 100 oxysterol analogues synthesized, Oxy133 induced significant expression of osteogenic markers Runx2, osterix (OSX), alkaline phosphatase (ALP), bone sialoprotein (BSP), and osteocalcin (OCN) in C3H10T1/2 mouse embryonic fibroblasts and in M2-10B4 mouse marrow stromal cells. Oxy133-induced activation of an 8X-Gli luciferase reporter, its direct binding to Smoothened, and the inhibition of Oxy133-induced osteogenic effects by the Hedgehog (Hh) pathway inhibitor, cyclopamine, demonstrated the role of Hh pathway in mediating osteogenic responses to Oxy133. Oxy133 did not stimulate osteogenesis via BMP or Wnt signaling. Oxy133 induced the expression of OSX, BSP, and OCN, and stimulated robust mineralization in primary human mesenchymal stem cells. In vivo, bilateral spine fusion occurred through endochondral ossification and was observed in animals treated with Oxy133 at the fusion site on X-ray after 4 weeks and confirmed with manual assessment, micro-CT (µCT), and histology after 8 weeks, with equal efficiency to recombinant human bone morphogenetic protein-2 (rhBMP-2). Unlike rhBMP-2, Oxy133 did not induce adipogenesis in the fusion mass and resulted in denser bone evidenced by greater bone volume/tissue volume (BV/TV) ratio and smaller trabecular separation. Findings here suggest that Oxy133 has significant potential as an osteogenic molecule with greater ease of synthesis and improved time to fusion compared to previously studied oxysterols. Small molecule osteogenic oxysterols may serve as the next generation of bone anabolic agents for therapeutic development. © 2014 American Society for Bone and Mineral Research.

A Novel Osteogenic Oxysterol Compound for Therapeutic Development to Promote Bone Growth: Activation of Hedgehog Signaling and Osteogenesis through Smoothened Binding

PubMed Central

Montgomery, Scott R.; Nargizyan, Taya; Meliton, Vicente; Nachtergaele, Sigrid; Rohatgi, Rajat; Stappenbeck, Frank; Jung, Michael E.; Johnson, Jared S.; Aghdasi, Bayan; Tian, Haijun; Weintraub, Gil; Inoue, Hirokazu; Atti, Elisa; Tetradis, Sotirios; Pereira, Renata C; Hokugo, Akishige; Alobaidaan, Raed; Tan, Yanlin; Hahn, Theodor J.; Wang, Jeffrey C; Parhami, Farhad

2015-01-01

Osteogenic factors are often used in orthopedics to promote bone growth, improve fracture healing, and induce spine fusion. Osteogenic oxysterols are naturally occurring molecules that were shown to induce osteogenic differentiation in vitro and promote spine fusion in vivo. The purpose of this study was to identify an osteogenic oxysterol more suitable for clinical development than those previously reported, and evaluate its ability to promote osteogenesis in vitro and spine fusion in rats in vivo. Among more than 100 oxysterol analogues synthesized, Oxy133 induced significant expression of osteogenic markers Runx2, osterix (OSX), alkaline phosphatase (ALP), bone sialoprotein (BSP), and osteocalcin (OCN) in C3H10T1/2 mouse embryonic fibroblasts and in M2-10B4 mouse marrow stromal cells. Oxy133-induced activation of an 8×-Gli luciferase reporter, its direct binding to Smoothened, and the inhibition of Oxy133-induced osteogenic effects by the Hedgehog (Hh) pathway inhibitor, cyclopamine, demonstrated the role of Hh pathway in mediating osteogenic responses to Oxy133. Oxy133 did not stimulate osteogenesis via BMP or Wnt signaling. Oxy133 induced the expression of OSX, BSP, and OCN and stimulated robust mineralization in primary human mesenchymal stem cells. In vivo, bilateral spine fusion occurred through endochondral ossification and was observed in animals treated with Oxy133 at the fusion site on xray after 4 weeks and confirmed with manual assessment, micro CT (μCT), and histology after 8 weeks, with equal efficiency to recombinant human bone morphogenetic protein-2 (rhBMP-2). Unlike rhBMP-2, Oxy133 did not induce adipogenesis in the fusion mass and resulted in denser bone evidenced by greater BV/TV ratio and smaller trabecular separation. Findings here suggest that Oxy133 has significant potential as an osteogenic molecule with greater ease of synthesis and improved time to fusion compared to previously studied oxysterols. Small molecule osteogenic oxysterols may serve as the next generation of bone anabolic agents for therapeutic development. PMID:24591126

In Vitro Osteoblast Model for Bone Wound Infections and Antimicrobial Therapy

DTIC Science & Technology

2012-10-01

such as  osteocalcin, osteopontin, and bone  sialoprotein . In addition, osteoblast‐produced  of circulating cytokines and growth factors also influence...genes such osteocalcin, bone  sialoprotein , osteopontin, and collagen type I .  When infected with the AB, KP or PA, RUNX2 expression was not significant

Chemical and genetic blockade of HDACs enhances osteogenic differentiation of human adipose tissue-derived stem cells by oppositely affecting osteogenic and adipogenic transcription factors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Maroni, Paola; Brini, Anna Teresa; Dipartimento di Scienze Biomediche, Chirurgiche ed Odontoiatriche, Universita degli Studi di Milano, Milano

2012-11-16

Highlights: Black-Right-Pointing-Pointer Acetylation affected hASCs osteodifferentiation through Runx2-PPAR{gamma}. Black-Right-Pointing-Pointer HDACs knocking-down favoured the commitment effect of osteogenic medium. Black-Right-Pointing-Pointer HDACs silencing early activated Runx2 and ALP. Black-Right-Pointing-Pointer PPAR{gamma} reduction and calcium/collagen deposition occurred later. Black-Right-Pointing-Pointer Runx2/PPAR{gamma} target genes were modulated in line with HDACs role in osteo-commitment. -- Abstract: The human adipose-tissue derived stem/stromal cells (hASCs) are an interesting source for bone-tissue engineering applications. Our aim was to clarify in hASCs the role of acetylation in the control of Runt-related transcription factor 2 (Runx2) and Peroxisome proliferator activated receptor (PPAR) {gamma}. These key osteogenic and adipogenic transcription factors are oppositelymore » involved in osteo-differentiation. The hASCs, committed or not towards bone lineage with osteoinductive medium, were exposed to HDACs chemical blockade with Trichostatin A (TSA) or were genetically silenced for HDACs. Alkaline phosphatase (ALP) and collagen/calcium deposition, considered as early and late osteogenic markers, were evaluated concomitantly as index of osteo-differentiation. TSA pretreatment, useful experimental protocol to analyse pan-HDAC-chemical inhibition, and switch to osteogenic medium induced early-osteoblast maturation gene Runx2, while transiently decreased PPAR{gamma} and scarcely affected late-differentiation markers. Time-dependent effects were observed after knocking-down of HDAC1 and 3: Runx2 and ALP underwent early activation, followed by late-osteogenic markers increase and by PPAR{gamma}/ALP activity diminutions mostly after HDAC3 silencing. HDAC1 and 3 genetic blockade increased and decreased Runx2 and PPAR{gamma} target genes, respectively. Noteworthy, HDACs knocking-down favoured the commitment effect of osteogenic medium. Our results reveal a role for HDACs in orchestrating osteo-differentiation of hASCs at transcriptional level, and might provide new insights into the modulation of hASCs-based regenerative therapy.« less

Molecular Analysis of Neutrophil Differentiation from Human Induced Pluripotent Stem Cells Delineates the Kinetics of Key Regulators of Hematopoiesis.

PubMed

Sweeney, Colin L; Teng, Ruifeng; Wang, Hongmei; Merling, Randall K; Lee, Janet; Choi, Uimook; Koontz, Sherry; Wright, Daniel G; Malech, Harry L

2016-06-01

In vitro generation of mature neutrophils from human induced pluripotent stem cells (iPSCs) requires hematopoietic progenitor development followed by myeloid differentiation. The purpose of our studies was to extensively characterize this process, focusing on the critical window of development between hemogenic endothelium, hematopoietic stem/progenitor cells (HSPCs), and myeloid commitment, to identify associated regulators and markers that might enable the stem cell field to improve the efficiency and efficacy of iPSC hematopoiesis. We utilized a four-stage differentiation protocol involving: embryoid body (EB) formation (stage-1); EB culture with hematopoietic cytokines (stage-2); HSPC expansion (stage-3); and neutrophil maturation (stage-4). CD34(+) CD45(-) putative hemogenic endothelial cells were observed in stage-3 cultures, and expressed VEGFR-2/Flk-1/KDR and VE-cadherin endothelial markers, GATA-2, AML1/RUNX1, and SCL/TAL1 transcription factors, and endothelial/HSPC-associated microRNAs miR-24, miR-125a-3p, miR-126/126*, and miR-155. Upon further culture, CD34(+) CD45(-) cells generated CD34(+) CD45(+) HSPCs that produced hematopoietic CFUs. Mid-stage-3 CD34(+) CD45(+) HSPCs exhibited increased expression of GATA-2, AML1/RUNX1, SCL/TAL1, C/EBPα, and PU.1 transcription factors, but exhibited decreased expression of HSPC-associated microRNAs, and failed to engraft in immune-deficient mice. Mid-stage-3 CD34(-) CD45(+) cells maintained PU.1 expression and exhibited increased expression of hematopoiesis-associated miR-142-3p/5p and a trend towards increased miR-223 expression, indicating myeloid commitment. By late Stage-4, increased CD15, CD16b, and C/EBPɛ expression were observed, with 25%-65% of cells exhibiting morphology and functions of mature neutrophils. These studies demonstrate that hematopoiesis and neutrophil differentiation from human iPSCs recapitulates many features of embryonic hematopoiesis and neutrophil production in marrow, but reveals unexpected molecular signatures that may serve as a guide for enhancing iPSC hematopoiesis. Stem Cells 2016;34:1513-1526. © 2016 AlphaMed Press.

FLT3 mutation and expression did not adversely affect clinical outcome of childhood acute leukaemia: a study of 531 Southeast Asian children by the Ma-Spore study group.

PubMed

Leow, Shuangjie; Kham, Shirley Kow-Yin; Ariffin, Hany; Quah, Thuan Chong; Yeoh, Allen Eng-Juh

2011-12-01

FMS-like tyrosine kinase 3 (FLT3) is critical for normal haematopoiesis and have been reported to be expressed in the majority of acute myeloid and lymphoid malignancies. We correlated the impact of FLT3 mutations and its expression with age, WHO 2008 classification and treatment outcome in 531 childhood acute leukaemias. Of 150 acute myeloid leukaemia (AMLs), 18 (12%) harboured FLT3-ITD while nine (6%) had FLT3-TKD. FLT3-ITD and -TKD were rare in acute megakaryoblastic leukaemia (AMKL; FLT3-ITD 0/26, FLT3-TKD 1/26) and children below 3 years (n = 2/48). Acute promyelocytic leukaemia (APL) with t(15;17);PML-RARα (n = 7/18; 39%) harboured the highest frequency of FLT3 mutations, followed by myelomonocytic (n = 4/18; 22%) and AML with t(8;21);RUNX1-RUNX1T1 (n = 2/21; 9%). FLT3 expression levels were also lowest in AMKL, both in Down's and non-Down's (p = 0.002) followed by patients <3 years (p = 0.001). The rarity of FLT3 mutations and expression levels in AMKL were independent of age. Conversely, only 2% of childhood acute lymphoblastic leukaemia (ALL) harboured FLT3 mutations (ITD = 1/381; TKD = 6/381). FLT3 was highly expressed in hyperdiploid ALL (p < 0.001). Of the 121 AMLs with clinical history, there were no significant differences in 4-year event-free survival (EFS) (46% vs. 38%; p = 0.46) and overall-survival (OS) (55% vs. 43%; p = 0.30) between FLT3-wildtype and ITD+ patients. Similarly, FLT3 expression levels did not influence survival in AML in both the good risk and non-good risk subgroups. FLT3 does not appear to be involved in the pathogenesis of AMKL, both in Down's and non-Down's. Therapeutic targets using FLT3 inhibitors may not be useful in AMKL and in young children with AML. Copyright © 2011 John Wiley & Sons, Ltd.

Molecular analysis of neutrophil differentiation from human iPSCs delineates the kinetics of key regulators of hematopoiesis

PubMed Central

Sweeney, Colin L.; Teng, Ruifeng; Wang, Hongmei; Merling, Randall K.; Lee, Janet; Choi, Uimook; Koontz, Sherry; Wright, Daniel G.; Malech, Harry L.

2016-01-01

In vitro generation of mature neutrophils from human induced pluripotent stem cells (iPSCs) requires hematopoietic progenitor development followed by myeloid differentiation. The purpose of our studies was to extensively characterize this process, focusing on the critical window of development between hemogenic endothelium, hematopoietic stem/progenitor cells (HSPCs), and myeloid commitment, to identify associated regulators and markers that might enable the stem cell field to improve the efficiency and efficacy of iPSC hematopoiesis. We utilized a 4-stage differentiation protocol involving: embryoid body (EB) formation (Stage-1); EB culture with hematopoietic cytokines (Stage-2); HSPC expansion (Stage-3); and neutrophil maturation (Stage-4). CD34+CD45− putative hemogenic endothelial cells were observed in Stage-3 cultures, and expressed VEGFR-2/Flk-1/KDR and VE-cadherin endothelial markers, GATA-2, AML1/RUNX1, and SCL/TAL1 transcription factors, and endothelial/HSPC-associated microRNAs miR-24, miR-125a-3p, miR-126/126*, and miR-155. Upon further culture, CD34+CD45− cells generated CD34+CD45+ HSPCs that produced hematopoietic CFUs. Mid-Stage-3 CD34+CD45+ HSPCs exhibited increased expression of GATA-2, AML1/RUNX1, SCL/TAL1, C/EBPα, and PU.1 transcription factors, but exhibited decreased expression of HSPC-associated microRNAs, and failed to engraft in immune-deficient mice. Mid-stage-3 CD34−CD45+ cells maintained PU.1 expression and exhibited increased expression of hematopoiesis-associated miR-142-3p/5p and a trend towards increased miR-223 expression, indicating myeloid commitment. By late Stage-4, increased CD15, CD16b, and C/EBPε expression were observed, with 25–65% of cells exhibiting morphology and functions of mature neutrophils. These studies demonstrate that hematopoiesis and neutrophil differentiation from human iPSCs recapitulates many features of embryonic hematopoiesis and neutrophil production in marrow, but reveals unexpected molecular signatures that may serve as a guide for enhancing iPSC hematopoiesis. PMID:26866427

Influence of different intensities of vibration on proliferation and differentiation of human periodontal ligament stem cells.

PubMed

Zhang, Chunxiang; Lu, Yanqin; Zhang, Linkun; Liu, Yang; Zhou, Yi; Chen, Yangxi; Yu, Haiyang

2015-06-19

To understand the effects of low-magnitude, high-frequency (LMHF) mechanical vibration at different intensities on human periodontal ligament stem cell (hPDLSC) proliferation and osteogenic differentiation. The effect of vibration on hPDLSC proliferation, osteogenic differentiation, tenogenic differentiation and cytoskeleton was assessed at the cellular, genetic and protein level. The PDLSC proliferation was decreased after different magnitudes of mechanical vibration; however, there were no obvious senescent cells in the experimental and the static control group. Expression of osteogenesis markers was increased. The expression of alkaline phosphatase (ALP) and osteocalcin (OCN) mRNA was up-regulated at 0.1 g, 0.3 g, 0.6 g and 0.9 g magnitude, with the peak at 0.3 g. The type I collagen (Col-I) level was increased after vibration exposure at 0.1 g, 0.3 g, and 0.6 g, peaking at 0.3 g. The expression levels of both mRNA and protein of Runx2 and osterix (OSX) significantly increased at a magnitude of 0.1 g to 0.9 g, reached a peak at 0.3 g and then decreased slowly. The scleraxis, tenogenic markers, and mRNA expression decreased at 0.05 g, 0.1 g, and 0.3 g, and significantly increased at 0.6 g and 0.9 g. Compared with the static group, the F-actin stress fibers of hPDLSCs became thicker and clearer following vibration. The LMHF mechanical vibration promotes PDLSC osteogenic differentiation and implies the existence of a magnitude-dependent effect of vibration on determining PDLSC commitment to the osteoblast lineage. Changes in the cytoskeleton of hPDLSCs after vibration may be one of the mechanisms of the biological effects.

Influence of different intensities of vibration on proliferation and differentiation of human periodontal ligament stem cells

PubMed Central

Zhang, Chunxiang; Lu, Yanqin; Zhang, Linkun; Liu, Yang; Zhou, Yi; Chen, Yangxi

2015-01-01

Introduction To understand the effects of low-magnitude, high-frequency (LMHF) mechanical vibration at different intensities on human periodontal ligament stem cell (hPDLSC) proliferation and osteogenic differentiation. Material and methods The effect of vibration on hPDLSC proliferation, osteogenic differentiation, tenogenic differentiation and cytoskeleton was assessed at the cellular, genetic and protein level. Results The PDLSC proliferation was decreased after different magnitudes of mechanical vibration; however, there were no obvious senescent cells in the experimental and the static control group. Expression of osteogenesis markers was increased. The expression of alkaline phosphatase (ALP) and osteocalcin (OCN) mRNA was up-regulated at 0.1 g, 0.3 g, 0.6 g and 0.9 g magnitude, with the peak at 0.3 g. The type I collagen (Col-I) level was increased after vibration exposure at 0.1 g, 0.3 g, and 0.6 g, peaking at 0.3 g. The expression levels of both mRNA and protein of Runx2 and osterix (OSX) significantly increased at a magnitude of 0.1 g to 0.9 g, reached a peak at 0.3 g and then decreased slowly. The scleraxis, tenogenic markers, and mRNA expression decreased at 0.05 g, 0.1 g, and 0.3 g, and significantly increased at 0.6 g and 0.9 g. Compared with the static group, the F-actin stress fibers of hPDLSCs became thicker and clearer following vibration. Conclusions The LMHF mechanical vibration promotes PDLSC osteogenic differentiation and implies the existence of a magnitude-dependent effect of vibration on determining PDLSC commitment to the osteoblast lineage. Changes in the cytoskeleton of hPDLSCs after vibration may be one of the mechanisms of the biological effects. PMID:26170859

The effects of strength training and raloxifene on bone health in aging ovariectomized rats.

PubMed

Stringhetta-Garcia, Camila Tami; Singulani, Monique Patrício; Santos, Leandro Figueiredo; Louzada, Mário Jefferson Quirino; Nakamune, Ana Cláudia Stevanato; Chaves-Neto, Antonio Hernandes; Rossi, Ana Cláudia; Ervolino, Edilson; Dornelles, Rita Cássia Menegati

2016-04-01

The aim of this study was to investigate the effects of strength training (ST) and raloxifene (Ral), alone or in combination, on the prevention of bone loss in an aging estrogen-deficient rat model. Aging Wistar female rats were ovariectomized at 14months and allocated to four groups: (1) non-trained and treated with vehicle, NT-Veh; (2) strength training and treated with vehicle, ST-Veh; (3) non-trained and treated with raloxifene, NT-Ral; and (4) strength training and treated with raloxifene, ST-Ral. ST was performed on a ladder three times per week and Ral was administered daily by gavage (1mg/kg/day), both for 120days. Areal bone mineral density (aBMD), strength, microarchitecture, and biomarkers (osteocalcin, OCN; osteoprotegerin, OPG; and tartrate-resistant acid phosphatase, TRAP) were assessed. Immunohistochemistry was performed for runt-related transcription factor 2 (RUNX2), osterix (OSX), OCN, OPG, TRAP, and receptor activator of nuclear factor kappa-B ligand (RANKL). The rats that performed ST (ST-Veh) or were treated with Ral (NT-Ral) showed significant improvements in aBMD (p=0.001 and 0.004), bone strength (p=0.001), and bone microarchitecture, such as BV/TV (%) (p=0.001), BS/TV (mm(2)/mm(3)) (p=0.023 and 0.002), Conn.Dn (1/mm(3)) (p=0.001), Tb.N (1/mm) (p=0.012 and 0.011), Tb.Th (1/mm) (p=0.001), SMI (p=0.001 and 0.002), Tb.Sp (p=0.001), and DA (p=0.002 and 0.007); there was also a significant decrease in plasma levels of OCN (p=0.001 and 0.002) and OPG (p=0.003 and 0.014), compared with animals in the NT-Veh group. Ral, with or without ST, promoted an increased immunolabeling pattern for RUNX2 (p=0.0105 and p=0.0006) and OSX (p=0.0105), but a reduced immunolabeling pattern for TRAP (p=0.0056) and RANKL (p=0.033 and 0.004). ST increased the immunolabeling pattern for RUNX2 (p=0.0105), and association with Ral resulted in an increased immunolabeling pattern for OPG (p=0.0034) and OCN (p=0.0024). In summary, ST and Ral administration in aged, estrogen-deficient Wistar female rats is associated with a decrease in bone turnover marker plasma levels, increased activity of cells that promote osteoblastogenesis, and decreased activity of cells that promote osteoclastogenesis; these are correlated with higher aBMD, bone strength, and bone microarchitecture at the femoral neck. The results indicate that strength training and Ral are potential tools to reduce the risk of fractures at clinically relevant sites. Copyright © 2016 Elsevier Inc. All rights reserved.

The PAX2-null immunophenotype defines multiple lineages with common expression signatures in benign and neoplastic oviductal epithelium

PubMed Central

Ning, Gang; Bijron, Jonathan G.; Yamamoto, Yusuke; Wang, Xia; Howitt, Brooke E.; Herfs, Michael; Yang, Eric; Hong, Yue; Cornille, Maxence; Wu, Lingyan; Hanamornroongruang, Suchanan; McKeon, Frank D.; Crum, Christopher P.; Xian, Wa

2014-01-01

The oviducts contain high grade serous cancer (HGSC) precursors (serous tubal intraepithelial neoplasia or STINs), which are γ-H2AXp- and TP53 mutation-positive. Although they express wild type p53, secretory cell outgrowths (SCOUTs) are associated with older age and serous cancer; moreover both STINs and SCOUTs share a loss of PAX2 expression (PAX2n). We evaluated PAX2 expression in proliferating adult and embryonic oviductal cells, normal mucosa, SCOUTs, Walthard cell nests (WCNs), STINs and HGSCs, and the expression of genes chosen empirically or from SCOUT expression arrays. Clones generated in vitro from embryonic gynecologic tract and adult fallopian tube were Krt7p/PAX2n/EZH2p and underwent ciliated (PAX2n/EZH2n/FOXJ1p) and basal (Krt7n/EZH2n/Krt5p) differentiation. Similarly non-ciliated cells in normal mucosa were PAX2p but became PAX2n in multilayered epithelium undergoing ciliated or basal (Walthard cell nests or WCN) cell differentiation. PAX2n SCOUTs fell into two groups; Type I were secretory or secretory/ciliated with a “tubal” phenotype and were ALDH1n and β-cateninmem (membraneous only). Type II displayed a columnar to pseudostratified (endometrioid) phenotype, with an EZH2p, ALDH1p, β-cateninnc (nuclear and cytoplasmic), stathminp, LEF1p, RCN1p and RUNX2p expression signature. STINs and HGSCs shared the Type I immunophenotype of PAX2n, ALDH1n, β-cateninmem, but highly expressed EZH2p, LEF1p, RCN1p, and stathminp. This study, for the first time, links PAX2n with proliferating fetal and adult oviductal cells undergoing basal and ciliated differentiation and shows that this expression state is maintained in SCOUTs, STINs and HGSCs. All three entities can demonstrate a consistent perturbation of genes involved in potential tumor suppressor gene silencing (EZH2), transcriptional regulation (LEF1), regulation of differentiation (RUNX2), calcium binding (RCN1) and oncogenesis (stathmin). This shared expression signature between benign and neoplastic entities links normal progenitor cell expansion to abnormal and neoplastic outgrowth in the oviduct and exposes a common pathway that could be a target for early prevention. PMID:25130537

Magnesium Attenuates Phosphate-Induced Deregulation of a MicroRNA Signature and Prevents Modulation of Smad1 and Osterix during the Course of Vascular Calcification.

PubMed

Louvet, Loïc; Metzinger, Laurent; Büchel, Janine; Steppan, Sonja; Massy, Ziad A

2016-01-01

Vascular calcification (VC) is prevalent in patients suffering from chronic kidney disease (CKD). High phosphate levels promote VC by inducing abnormalities in mineral and bone metabolism. Previously, we demonstrated that magnesium (Mg(2+)) prevents inorganic phosphate- (Pi-) induced VC in human aortic vascular smooth muscle cells (HAVSMC). As microRNAs (miR) modulate gene expression, we investigated the role of miR-29b, -30b, -125b, -133a, -143, and -204 in the protective effect of Mg(2+) on VC. HAVSMC were cultured in the presence of 3 mM Pi with or without 2 mM Mg(2+) chloride. Total RNA was extracted after 4 h, 24 h, day 3, day 7, and day 10. miR-30b, -133a, and -143 were downregulated during the time course of Pi-induced VC, whereas the addition of Mg(2+) restored (miR-30b) or improved (miR-133a, miR-143) their expression. The expression of specific targets Smad1 and Osterix was significantly increased in the presence of Pi and restored by coincubation with Mg(2+). As miR-30b, miR-133a, and miR-143 are negatively regulated by Pi and restored by Mg(2+) with a congruent modulation of their known targets Runx2, Smad1, and Osterix, our results provide a potential mechanistic explanation of the observed upregulation of these master switches of osteogenesis during the course of VC.

Magnesium Attenuates Phosphate-Induced Deregulation of a MicroRNA Signature and Prevents Modulation of Smad1 and Osterix during the Course of Vascular Calcification

PubMed Central

Louvet, Loïc; Metzinger, Laurent; Büchel, Janine; Steppan, Sonja; Massy, Ziad A.

2016-01-01

Vascular calcification (VC) is prevalent in patients suffering from chronic kidney disease (CKD). High phosphate levels promote VC by inducing abnormalities in mineral and bone metabolism. Previously, we demonstrated that magnesium (Mg2+) prevents inorganic phosphate- (Pi-) induced VC in human aortic vascular smooth muscle cells (HAVSMC). As microRNAs (miR) modulate gene expression, we investigated the role of miR-29b, -30b, -125b, -133a, -143, and -204 in the protective effect of Mg2+ on VC. HAVSMC were cultured in the presence of 3 mM Pi with or without 2 mM Mg2+ chloride. Total RNA was extracted after 4 h, 24 h, day 3, day 7, and day 10. miR-30b, -133a, and -143 were downregulated during the time course of Pi-induced VC, whereas the addition of Mg2+ restored (miR-30b) or improved (miR-133a, miR-143) their expression. The expression of specific targets Smad1 and Osterix was significantly increased in the presence of Pi and restored by coincubation with Mg2+. As miR-30b, miR-133a, and miR-143 are negatively regulated by Pi and restored by Mg2+ with a congruent modulation of their known targets Runx2, Smad1, and Osterix, our results provide a potential mechanistic explanation of the observed upregulation of these master switches of osteogenesis during the course of VC. PMID:27419135

Interaction between bone marrow stromal cells and neuroblastoma cells leads to a VEGFA-mediated osteoblastogenesis

PubMed Central

HaDuong, Josephine H.; Blavier, Laurence; Baniwal, Sanjeev K.; Frenkel, Baruch; Malvar, Jemily; Punj, Vasu; Sposto, Richard; DeClerck, Yves A.

2017-01-01

The potential role of osteoblasts in bone and bone marrow (BM) metastases in neuroblastoma (NBL) remains unclear. In this study, we examined the effect of NBL cells on the osteoblastic differentiation of bone marrow-derived mesenchymal stromal cells (BMMSC). We show that the presence of NBL cells enhanced the osteoblastic differentiation of BMMSC driven by bone morphogenetic protein (BMP)-4, in the absence of any effect on NBL cell proliferation. Expression profiles of BMMSC driven towards osteoblastic differentiation revealed an increase in vascular endothelial growth factor A (Vegfa) expression in the presence of NBL cells. We demonstrated that NBL cells increased BMMSC-derived VEGFA mRNA and protein and that this was enhanced by BMP-4. However, in similar conditions, neither the addition of an mVEGFA blocking antibody nor exogenous recombinant (r) mVEGFA affected osteoblastic differentiation. In contrast, siRNA-mediated knock-down of VEGFA in BMMSC prevented osteoblastic differentiation in BMP-4-treated co-cultures, an effect that was not reversed in the presence of rmVEGFA. An analysis of murine bones injected with hNBL cells revealed an increase of mVEGFA producing cells near tumor cells concomitantly with an increase in Vegfa and Runx2 mRNA. This coincided with an increase in osteoclasts, in Rankl/Opg mRNA ratio and with the formation of osteolytic lesions. Thus NBL cells promote osteoblastogenesis in the BM by increasing VEGFA expression in BMMSC. Our study provides a new insight into the role of VEGFA in NBL metastases by pointing to the role of stroma-derived intracrine VEGFA in osteoblastogenesis. PMID:25648303

Interaction between bone marrow stromal cells and neuroblastoma cells leads to a VEGFA-mediated osteoblastogenesis.

PubMed

HaDuong, Josephine H; Blavier, Laurence; Baniwal, Sanjeev K; Frenkel, Baruch; Malvar, Jemily; Punj, Vasu; Sposto, Richard; DeClerck, Yves A

2015-08-15

The potential role of osteoblasts in bone and bone marrow (BM) metastases in neuroblastoma (NBL) remains unclear. In this study, we examined the effect of NBL cells on the osteoblastic differentiation of BM-derived mesenchymal stromal cells (BMMSC). We show that the presence of NBL cells enhanced the osteoblastic differentiation of BMMSC driven by bone morphogenetic protein (BMP)-4, in the absence of any effect on NBL cell proliferation. Expression profiles of BMMSC driven toward osteoblastic differentiation revealed an increase in vascular endothelial growth factor A (Vegfa) expression in the presence of NBL cells. We demonstrated that NBL cells increased BMMSC-derived VEGFA mRNA and protein and that this was enhanced by BMP-4. However, in similar conditions, neither the addition of an mVEGFA blocking antibody nor exogenous recombinant (r) mVEGFA affected osteoblastic differentiation. In contrast, siRNA- mediated knock-down of VEGFA in BMMSC prevented osteoblastic differentiation in BMP-4-treated cocultures, an effect that was not reversed in the presence of rmVEGFA. An analysis of murine bones injected with hNBL cells revealed an increase of mVEGFA producing cells near tumor cells concomitantly with an increase in Vegfa and Runx2 mRNA. This coincided with an increase in osteoclasts, in Rankl/Opg mRNA ratio and with the formation of osteolytic lesions. Thus NBL cells promote osteoblastogenesis in the BM by increasing VEGFA expression in BMMSC. Our study provides a new insight into the role of VEGFA in NBL metastases by pointing to the role of stroma-derived intracrine VEGFA in osteoblastogenesis. © 2015 UICC.

Effects of naringin on the proliferation and osteogenic differentiation of human amniotic fluid-derived stem cells.

PubMed

Liu, Meimei; Li, Yan; Yang, Shang-Tian

2017-01-01

Human amniotic fluid-derived stem cells (hAFSCs) are a novel cell source for generating osteogenic cells to treat bone diseases. Effective induction of osteogenic differentiation from hAFSCs is critical to fulfil their therapeutic potential. In this study, naringin, the main active compound of Rhizoma drynariae (a Chinese herbal medicine), was used to stimulate the proliferation and osteogenic differentiation of hAFSCs. The results showed that naringin enhanced the proliferation and alkaline phosphatase activity (ALP) of hAFSCs in a dose-dependent manner in the range 1-100 µg/ml, while an inhibition effect was observed at 200 µg/ml. Consistently, the calcium content also increased with naringin concentration up to 100 µg/ml. The enhanced osteogenic differentiation of hAFSCs by naringin was further confirmed by the dose-dependent upregulation of marker genes, including osteopontin (OPN) and Collagen I from RT-PCR analysis. The increased osteoprotegerin (OPG) expression and minimal expression of receptor activator of nuclear factor-κB ligand (RANKL) suggested that naringin also inhibited osteoclastogenesis of hAFSCs. In addition, the gene expressions of bone morphogenetic protein 4 (BMP4), runt-related transcription factor 2 (RUNX2), β-catenin and Cyclin D1 also increased significantly, indicating that naringin promotes the osteogenesis of hAFSCs via the BMP and Wnt-β-catenin signalling pathways. These results suggested that naringin can be used to upregulate the osteogenic differentiation of hAFSCs, which could provide an attractive and promising treatment for bone disorders. Copyright © 2014 John Wiley & Sons, Ltd. Copyright © 2014 John Wiley & Sons, Ltd.

Epigallocatechin-3-gallate (EGCG) as a pro-osteogenic agent to enhance osteogenic differentiation of mesenchymal stem cells from human bone marrow: an in vitro study.

PubMed

Jin, Pan; Wu, Huayu; Xu, Guojie; Zheng, Li; Zhao, Jinmin

2014-05-01

The proliferation and osteogenic capacity of mesenchymal stem cells (MSCs) needs to be improved for their use in cell-based therapy for osteoporosis. (-)-Epigallocatechin-3-gallate (EGCG), one of the green tea catechins, has been widely investigated in studies of osteoblasts and osteoclasts. However, no consensus on its role as an osteogenic inducer has been reached, possibly because of the various types of cell lines examined and the range of concentrations of EGCG used. In this study, the osteogenic effects of EGCG are studied in primary human bone-marrow-derived MSCs (hBMSCs) by detecting cell proliferation, alkaline phosphatase (ALP) activity and the expression of relevant osteogenic markers. Our results show that EGCG has a strong stimulatory effect on hBMSCs developing towards the osteogenic lineage, especially at a concentration of 5 μM, as evidenced by an increased ALP activity, the up-regulated expression of osteogenic genes and the formation of bone-like nodules. Further exploration has indicated that EGCG directes osteogenic differentiation via the continuous up-regulation of Runx2. The underlying mechanism might involve EGCG affects on osteogenic differentiation through the modulation of bone morphogenetic protein-2 expression. EGCG has also been found to promote the proliferation of hBMSCs in a dose-dependent manner. This might be associated with its antioxidative effect leading to favorable amounts of reactive oxygen species in the cellular environment. Our study thus indicates that EGCG can be used as a pro-osteogenic agent for the stem-cell-based therapy of osteoporosis.

Differential analysis of genome-wide methylation and gene expression in mesenchymal stem cells of patients with fractures and osteoarthritis

PubMed Central

del Real, Alvaro; Pérez-Campo, Flor M.; Fernández, Agustín F.; Sañudo, Carolina; Ibarbia, Carmen G.; Pérez-Núñez, María I.; Criekinge, Wim Van; Braspenning, Maarten; Alonso, María A.; Fraga, Mario F.

2017-01-01

ABSTRACT Insufficient activity of the bone-forming osteoblasts leads to low bone mass and predisposes to fragility fractures. The functional capacity of human mesenchymal stem cells (hMSCs), the precursors of osteoblasts, may be compromised in elderly individuals, in relation with the epigenetic changes associated with aging. However, the role of hMSCs in the pathogenesis of osteoporosis is still unclear. Therefore, we aimed to characterize the genome-wide methylation and gene expression signatures and the differentiation capacity of hMSCs from patients with hip fractures. We obtained hMSCs from the femoral heads of women undergoing hip replacement due to hip fractures and controls with hip osteoarthritis. DNA methylation was explored with the Infinium 450K bead array. Transcriptome analysis was done by RNA sequencing. The genomic analyses revealed that most differentially methylated loci were situated in genomic regions with enhancer activity, distant from gene bodies and promoters. These regions were associated with differentially expressed genes enriched in pathways related to hMSC growth and osteoblast differentiation. hMSCs from patients with fractures showed enhanced proliferation and upregulation of the osteogenic drivers RUNX2/OSX. Also, they showed some signs of accelerated methylation aging. When cultured in osteogenic medium, hMSCs from patients with fractures showed an impaired differentiation capacity, with reduced alkaline phosphatase activity and poor accumulation of a mineralized matrix. Our results point to 2 areas of potential interest for discovering new therapeutic targets for low bone mass disorders and bone regeneration: the mechanisms stimulating MSCs proliferation after fracture and those impairing their terminal differentiation. PMID:27982725

Synergistic interaction of platelet derived growth factor (PDGF) with the surface of PLLA/Col/HA and PLLA/HA scaffolds produces rapid osteogenic differentiation.

PubMed

Raghavendran, Hanumantha Rao Balaji; Mohan, Saktiswaren; Genasan, Krishnamurithy; Murali, Malliga Raman; Naveen, Sangeetha Vasudevaraj; Talebian, Sepehr; McKean, Robert; Kamarul, Tunku

2016-03-01

Scaffolds with structural features similar to the extracellular matrix stimulate rapid osteogenic differentiation in favorable microenvironment and with growth factor supplementation. In this study, the osteogenic potential of electrospun poly-l-lactide/hydroxyapatite/collagen (PLLA/Col/HA, PLLA/HA and PLLA/Col) scaffolds were tested in vitro with the supplementation of platelet derived growth factor-BB (PDGF-BB). Cell attachment and topography, mineralization, extracellular matrix protein localization, and gene expression of the human mesenchymal stromal cells were compared between the fibrous scaffolds PLLA/Col/HA, PLLA/Col, and PLLA/HA. The levels of osteocalcin, calcium, and mineralization were significantly greater in the PLLA/Col/HA and PLLA/HA compared with PLLA/Col. High expression of fibronectin, intracellular adhesion molecule, cadherin, and collagen 1 (Col1) suggests that PLLA/Col/HA and PLLA/HA scaffolds had superior osteoinductivity than PLLA/Col. Additionally, osteopontin, osteocalcin, osterix, Runt-related transcription factor 2 (Runx2), and bone morphogenic protein (BMP2) expression were higher in PLLA/Col/HA and PLLA/HA compared with PLLA/Col. In comparison with PLLA/Col, the PLLA/Col/HA and PLLA/HA scaffolds presented a significant upregulation of the genes Runx2, Col 1, Integrin, osteonectin (ON), bone gamma-carboxyglutamic acid-containing protein (BGALP), osteopontin (OPN), and BMP2. The upregulation of these genes was further increased with PDGF-BB supplementation. These results show that PDGF-BB acts synergistically with PLLA/Col/HA and PLLA/HA to enhance the osteogenic differentiation potential. Therefore, this combination can be used for the rapid expansion of bone marrow stromal cells into bone-forming cells for tissue engineering. Copyright © 2015 Elsevier B.V. All rights reserved.

Naringin protects human adipose-derived mesenchymal stem cells against hydrogen peroxide-induced inhibition of osteogenic differentiation.

PubMed

Wang, Lei; Zhang, Yu-Ge; Wang, Xiu-Mei; Ma, Long-Fei; Zhang, Yuan-Min

2015-12-05

Extensive evidence indicates that oxidative stress plays a pivotal role in the development of osteoporosis. We show that naringin, a natural antioxidant and anti-inflammatory compound, effectively protects human adipose-derived mesenchymal stem cells (hADMSCs) against hydrogen peroxide (H2O2)-induced inhibition of osteogenic differentiation. Naringin increased viability of hAMDSCs and attenuated H2O2-induced cytotoxicity. Naringin also reversed H2O2-induced oxidative stress. Oxidative stress induced by H2O2 inhibits osteogenic differentiation by decreasing alkaline phosphatase (ALP) activity, calcium content and mRNA expression levels of osteogenesis marker genes RUNX2 and OSX in hADMSCs. However, addition of naringin leads to a significant recovery, suggesting the protective effects of naringin against H2O2-induced inhibition of osteogenic differentiation. Furthermore, the H2O2-induced decrease of protein expressions of β-catenin and clyclin D1, two important transcriptional regulators of Wnt-signaling, was successfully rescued by naringin treatment. Also, in the presence of Wnt inhibitor DKK-1, naringin is no longer effective in stimulating ALP activity, increasing calcium content and mRNA expression levels of RUNX2 and OSX in H2O2-exposed hADMSCs. These data clearly demonstrates that naringin protects hADMSCs against oxidative stress-induced inhibition of osteogenic differentiation, which may involve Wnt signaling pathway. Our work suggests that naringin may be a useful addition to the treatment armamentarium for osteoporosis and activation of Wnt signaling may represent attractive therapeutic strategy for the treatment of degenerative disease of bone tissue. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Pioglitazone-induced bone loss in diabetic rats and its amelioration by berberine: A portrait of molecular crosstalk.

PubMed

Adil, Mohammad; Mansoori, Mohd Nizam; Singh, Divya; Kandhare, Amit Dattatraya; Sharma, Manju

2017-10-01

Diabetes mellitus and osteoporosis both are high prevalence disorders, especially in the elderly population. Pioglitazone, a PPAR-γ agonist associated with bone loss and risk of fracture in type 2 diabetes mellitus patients. In this study, ameliorative effect of berberine against pioglitazone-induced bone loss in diabetic rats and possible mechanisms has been explored. Diabetes was induced in male Wistar albino rats by streptozotocin (65 mg/kg, i.v.) after 15min of nicotinamide (230mg/kg, i.p.) administration. Diabetic rats were treated orally with pioglitazone (10mg/kg) and berberine (100mg/kg) alone and in combination of both for 12 weeks. Femur of each rat was isolated and evaluated for the bone micro-architecture, BMD, histology and mRNA expression of PPAR-γ, AMPK, and bone turnover markers (RANKL, OPG, Runx2, and osteocalcin). Urinary calcium and serum TRAP was also measured. Treatment of pioglitazone and berberine alone and in combination significantly ameliorate abnormal blood glucose, serum insulin, and HbA1c levels in streptozotocin-induced diabetic rats. Pioglitazone treatment significantly increased urinary calcium, serum TRAP, mRNA expression of RANKL, PPAR-γ as well as significantly decreased Runx2, OPG, osteocalcin and AMPK levels in diabetic rats. Pioglitazone administration also shows detrimental effect on femur epiphysis micro-architecture, BMD and histology. Whereas, berberine treatment alone and in combination with pioglitazone remarkably ameliorates the abnormal urinary calcium, mRNA expression of AMPK, bone turnover markers, femur epiphysis micro-architecture, histology and also increases BMD in diabetic rats. In conclusion, berberine shows protective effect against pioglitazone-induced bone loss in diabetic rats possibly through AMPK activation pathway. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Pulsed electromagnetic fields promote the proliferation and differentiation of osteoblasts by reinforcing intracellular calcium transients.

PubMed

Tong, Jie; Sun, Lijun; Zhu, Bin; Fan, Yun; Ma, Xingfeng; Yu, Liyin; Zhang, Jianbao

2017-10-01

Pulsed electromagnetic fields (PEMF) can be used to treat bone-related diseases, but the underlying mechanism remains unclear, especially the process by which PEMFs initiate biological effects. In this study, we demonstrated the effects of PEMF on proliferation and differentiation of osteoblasts using the model of calcium transients induced by high extracellular calcium. Our results showed that PEMF can increase both the percentage of responding cells and amplitude of intracellular calcium transients induced by high extracellular calcium stimulation. Compared with corresponding extracellular calcium levels, PEMF stimulation increased proliferation and differentiation of osteoblasts and related gene expressions, such as insulin-like growth factor 1 (IGF-1), alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), and osteocalcin (OCN), which can be completely abolished by BAPTA-AM. Moreover, PEMF did not affect proliferation and differentiation of osteoblasts if no intracellular calcium transient was present in osteoblasts during PEMF exposure. Our results revealed that PEMF affects osteoblast proliferation and differentiation through enhanced intracellular calcium transients, which provided a cue to treat bone-related diseases with PEMF. Bioelectromagnetics. 38:541-549, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

Bradykinin regulates osteoblast differentiation by Akt/ERK/NFκB signaling axis.

PubMed

Srivastava, Swati; Sharma, Kirti; Kumar, Narender; Roy, Partha

2014-12-01

Bradykinin (BK), a well known mediator of pain and inflammation, is also known to be involved in the process of bone resorption. The present study therefore evaluated the role of BK in osteoblast lineage commitment. Our data showed that BK inhibits the migration of bone marrow mesenchymal stem cells, but does not affect their viability. Moreover, BK also inhibits osteoblastic differentiation by significantly downregulating the levels of mRNAs for osteopontin, runX2, col24, osterix, osteocalcin genes and bone mineralization (P < 0.05). Further, BK was found to elicit the BK receptors (BDKR1 and BDKR2) mediated activation of ERK1/2 and Akt pathways, which finally led to the activation of NFκB. BK also promoted the osteoclast differentiation of bone marrow derived preosteoclast cells by upregulating the expression of c-fos, NFATC1, TRAP, clcn7, cathK, and OSCAR genes and increasing TRAP activity through NFκB pathway. In conclusion, our data suggest that BK decreases the differentiation of osteoblasts with concomitant increase in osteoclast formation and thus provides new insight into the mechanism of action of BK in modulating bone resorption. © 2014 Wiley Periodicals, Inc.

Moderate treadmill running exercise prior to tendon injury enhances wound healing in aging rats

PubMed Central

Zhang, Jianying; Yuan, Ting; Wang, James H-C.

2016-01-01

The effect of exercise on wound healing in aging tendon was tested using a rat moderate treadmill running (MTR) model. The rats were divided into an MTR group that ran on a treadmill for 4 weeks and a control group that remained in cages. After MTR, a window defect was created in the patellar tendons of all rats and wound healing was analyzed. We found that MTR accelerated wound healing by promoting quicker closure of wounds, improving the organization of collagen fibers, and decreasing senescent cells in the wounded tendons when compared to the cage control. MTR also lowered vascularization, increased the numbers of tendon stem/progenitor cells (TSCs) and TSC proliferation than the control. Besides, MTR significantly increased the expression of stem cell markers, OCT-4 and Nanog, and tenocyte genes, Collagen I, Collagen III and tenomodulin, and down-regulated PPAR-γ, Collagen II and Runx-2 (non-tenocyte genes). These findings indicated that moderate exercise enhances healing of injuries in aging tendons through TSC based mechanisms, through which exercise regulates beneficial effects in tendons. This study reveals that appropriate exercise may be used in clinics to enhance tendon healing in aging patients. PMID:26885754

Moderate treadmill running exercise prior to tendon injury enhances wound healing in aging rats.

PubMed

Zhang, Jianying; Yuan, Ting; Wang, James H-C

2016-02-23

The effect of exercise on wound healing in aging tendon was tested using a rat moderate treadmill running (MTR) model. The rats were divided into an MTR group that ran on a treadmill for 4 weeks and a control group that remained in cages. After MTR, a window defect was created in the patellar tendons of all rats and wound healing was analyzed. We found that MTR accelerated wound healing by promoting quicker closure of wounds, improving the organization of collagen fibers, and decreasing senescent cells in the wounded tendons when compared to the cage control. MTR also lowered vascularization, increased the numbers of tendon stem/progenitor cells (TSCs) and TSC proliferation than the control. Besides, MTR significantly increased the expression of stem cell markers, OCT-4 and Nanog, and tenocyte genes, Collagen I, Collagen III and tenomodulin, and down-regulated PPAR-γ, Collagen II and Runx-2 (non-tenocyte genes). These findings indicated that moderate exercise enhances healing of injuries in aging tendons through TSC based mechanisms, through which exercise regulates beneficial effects in tendons. This study reveals that appropriate exercise may be used in clinics to enhance tendon healing in aging patients.

Nanoparticle-antagomiR based targeting of miR-31 to induce osterix and osteocalcin expression in mesenchymal stem cells.

PubMed

McCully, Mark; Conde, João; V Baptista, Pedro; Mullin, Margaret; Dalby, Matthew J; Berry, Catherine C

2018-01-01

Mesenchymal stem cells are multipotent adult stem cells capable of generating bone, cartilage and fat, and are thus currently being exploited for regenerative medicine. When considering osteogenesis, developments have been made with regards to chemical induction (e.g. differentiation media) and physical induction (e.g. material stiffness, nanotopography), targeting established early transcription factors or regulators such as runx2 or bone morphogenic proteins and promoting increased numbers of cells committing to osteo-specific differentiation. Recent research highlighted the involvement of microRNAs in lineage commitment and terminal differentiation. Herein, gold nanoparticles that confer stability to short single stranded RNAs were used to deliver MiR-31 antagomiRs to both pre-osteoblastic cells and primary human MSCs in vitro. Results showed that blocking miR-31 led to an increase in osterix protein in both cell types at day 7, with an increase in osteocalcin at day 21, suggesting MSC osteogenesis. In addition, it was noted that antagomiR sequence direction was important, with the 5 prime reading direction proving more effective than the 3 prime. This study highlights the potential that miRNA antagomiR-tagged nanoparticles offer as novel therapeutics in regenerative medicine.

Wedelolactone enhances osteoblastogenesis by regulating Wnt/β-catenin signaling pathway but suppresses osteoclastogenesis by NF-κB/c-fos/NFATc1 pathway.

PubMed

Liu, Yan-Qiu; Hong, Zhi-Lai; Zhan, Li-Bin; Chu, Hui-Ying; Zhang, Xiao-Zhe; Li, Guo-Hui

2016-08-25

Bone homeostasis is maintained by formation and destruction of bone, which are two processes tightly coupled and controlled. Targeting both stimulation on bone formation and suppression on bone resorption becomes a promising strategy for treating osteoporosis. In this study, we examined the effect of wedelolactone, a natural product from Ecliptae herba, on osteoblastogenesis as well as osteoclastogenesis. In mouse bone marrow mesenchymal stem cells (BMSC), wedelolactone stimulated osteoblast differentiation and bone mineralization. At the molecular level, wedelolactone directly inhibited GSK3β activity and enhanced the phosphorylation of GSK3β, thereafter stimulated the nuclear translocation of β-catenin and runx2. The expression of osteoblastogenesis-related marker gene including osteorix, osteocalcin and runx2 increased. At the same concentration range, wedelolactone inhibited RANKL-induced preosteoclastic RAW264.7 actin-ring formation and bone resorption pits. Further, wedelolactone blocked NF-kB/p65 phosphorylation and abrogated the NFATc1 nuclear translocation. As a result, osteoclastogenesis-related marker gene expression decreased, including c-src, c-fos, and cathepsin K. In ovariectomized mice, administration of wedelolactone prevented ovariectomy-induced bone loss by enhancing osteoblast activity and inhibiting osteoclast activity. Together, these data demonstrated that wedelolactone facilitated osteoblastogenesis through Wnt/GSK3β/β-catenin signaling pathway and suppressed RANKL-induced osteoclastogenesis through NF-κB/c-fos/NFATc1 pathway. These results suggested that wedelolacone could be a novel dual functional therapeutic agent for osteoporosis.

Regulation of osteogenesis of human amniotic mesenchymal stem cells by sodium butyrate.

PubMed

Fan, Xiaoting; Li, Lei; Ye, Zhaoyang; Zhou, Yan; Tan, Wen-Song

2018-04-01

Human amniotic membrane-derived mesenchymal stem cells (hAMSCs) draw great interests for regenerative medicine due to convenient availability and low immunogenicity. However, suboptimal culture conditions limit their application. In recent years, small molecules have proven powerful in regulating stem cell fates and can be applied to stimulate their function. In the present study, the impacts of sodium butyrate (NaBu), a histone deacetylase inhibitor (HDACi), on hAMSCs were investigated. It was shown that NaBu at a low concentration inhibited cell proliferation by arresting cell cycle at G0/G1 rather than inducing apoptosis. When NaBu was supplemented at a concentration of <1.0 mM for 3 days during osteogenic induction, significantly more mineralized nodules were generated and the expression of osteogenesis-related genes (ALP, Runx2, Opn, and Ocn) and proteins (Col1a1, OPN, OCN, Runx2, and TAZ) were both significantly enhanced. However, a higher concentration (1.0 mM) and longer exposure time (14 days) of NaBu showed no such effects, which may be partially attributed to both the increased expression of histone deacetylase 8 (HDAC8) and reduced level of H3K9-Ace, thus leading to the transcriptional inhibition during osteogenesis. Further, it was indicated that ERK might be involved in the stimulatory effects of NaBu. These findings may be helpful to develop an efficient culture process for hAMSCs towards bone regeneration. © 2018 International Federation for Cell Biology.

Zein nanoparticle as a novel BMP6 derived peptide carrier for enhanced osteogenic differentiation of C2C12 cells.

PubMed

Hadavi, Mahvash; Hasannia, Sadegh; Faghihi, Shahab; Mashayekhi, Farhad; Homazadeh, Homayoun; Mostofi, Seyed Behrooz

2018-01-26

Zein nanoparticles as a carrier system for BMP6-derived peptide were prepared by liquid-liquid phase separation procedure and characterized with SEM, DLS, FTIR and thermogravimetric methods. After peptide encapsulation, nanoparticle size increased from 236.3 ± 92.2 nm to 379.4 ± 116.8 nm. The encapsulation efficiency of peptide was 72.6% and the release of peptide from Zein nanoparticles was partly sustained in trypsin containing phosphate buffered saline (pH 7.4) for up to 14 days. Peptide-loaded nanoparticles showed similar cell viability compared with blank ones. ALP activity of C2C12 cells treated with peptide-loaded nanoparticles (500 µg/mL) was evaluated 7, 14, 21 and 28 days after culture. In peptide-loaded nanoparticles, ALP activity was significantly higher (p < .05) compared with other groups at day 14. Alizarin Red S staining showed, C2C12 cells behind peptide-loaded nanoparticles had significantly (p < .05) higher calcium deposition at day 21. The results of RT-qPCR show that the BMP-6 peptide activated expression of RUNX2 as a transcription factor. In turn, RUNX2 regulates SPP1 and BGLAP gene expression, as osteogenic marker genes. The results confirm that the peptide-loaded Zein nanoparticles, as osteoinductive material, may be used to repair small area of bone defects, with low load bearing.

Endothelin-1 regulates rat bone sialoprotein gene transcription.

PubMed

Li, Xinyue; Wang, Zhitao; Yang, Li; Li, Zhengyang; Ogata, Yorimasa

2010-06-01

Endothelin-1 (ET-1) was originally discovered as a vasoconstrictor protein excreted by vascular endothelial cells. Recently, tumor-produced ET-1 has been considered to stimulate osteoblasts to form new bone, and to be an important mediator of osteoblastic bone metastasis. ET-1 has high affinity for two different membrane receptors, ET(A)R and ET(B)R, which are expressed by many types of cells including osteoblasts. Bone sialoprotein (BSP) is a phosphorylated and sulfated glycoprotein associated with mineralized connective tissues. To investigate the effects of ET-1 on BSP transcription, we used rat osteoblast-like ROS17/2.8 cells. Levels of BSP and osteopontin mRNA were increased at 12 h after treatment with ET-1 (10 ng/ml), and ET-1 at the same concentration induced luciferase activity of a -116 to +60 BSP promoter construct at 6 h. Transcriptional activity of -84BSPLUC, which contains the cAMP response element (CRE), was increased by ET-1. Furthermore, at 6 h, ET-1 (10 ng/ml) increased the binding of nuclear protein to CRE, the FGF2 response element (FRE) and the homeodomain protein-binding site (HOX). Antibodies against CREB1, JunD and Fra2 disrupted the formation of CRE-protein complexes, while antibodies against Runx2 and Dlx5 reduced the formation of FRE- and HOX-protein complexes. These findings indicate that ET-1 increases BSP transcription via the CRE, FRE and HOX sites in the rat BSP gene promoter.

Mg-Al and Zn-Al Layered Double Hydroxides Promote Dynamic Expression of Marker Genes in Osteogenic Differentiation by Modulating Mitogen-Activated Protein Kinases.

PubMed

Kang, Ha Ram; da Costa Fernandes, Célio Junior; da Silva, Rodrigo Augusto; Constantino, Vera Regina Leopoldo; Koh, Ivan Hong Jun; Zambuzzi, Willian F

2018-02-01

The effect of LDH samples comprised of chloride anions intercalated between positive layers of magnesium/aluminum (Mg-Al LDH) or zinc/aluminum (Zn-Al LDH) chemical composition on pre-osteoblast performance is investigated. Non-cytotoxic concentrations of both LDHs modulated pre-osteoblast adhesion by triggering cytoskeleton rearrangement dependent on recruiting of Cofilin, which is modulated by the inhibition of the Protein Phosphatase 2A (PP2A), culminating in osteoblast differentiation with a significant increase of osteogenic marker genes. The alkaline phosphatase (ALP) and bone sialoprotein (BSP) are significantly up-modulated by both LDHs; however, Mg-Al LDH nanomaterial promoted even more significance than both experimental controls, while the phosphorylations of mitogen-activated protein kinase (MAPKs)- extracellular signal-regulated kinases (ERK) and c-Jun N-terminal kinase (JNK) significantly increased. MAPK signaling is necessary to activate Runt-related transcription factor 2 (RUNX2) gene. Concomitantly, it is also investigated whether challenged osteoblasts are able to modulate osteoclastogenesis by investigating both osteoprotegerin (OPG) and Receptor activator of nuclear factor kappa-ligand (RANKL) in this model; a dynamic reprogramming of both these genes is found, suggesting LDHs in modulating osteoclastogenesis. These results suggest that LDHs interfere in bone remodeling, and they can be considered as nanomaterials in graft-based bone healing or drug-delivery materials for bone disorders. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Impact of ionizing radiation exposure on in vitro differentiation of preosteoblastic cell lines

NASA Astrophysics Data System (ADS)

Hu, Yueyuan; Lau, Patrick; Hellweg, Christine; Baumstark-Khan, Christa; Reitz, Guenther

Bone demineralization of astronauts during residence in microgravity is a well known phe-nomenon during space travel. Besides altered gravity conditions, radiation risk is considered to be one of the major health hazards for astronauts in both orbital and interplanetary space. Un-til know, little is known about the effects of space radiation on the skeletal system especially on the bone forming osteoblasts. Accelerator facilities are used to simulate parts of the radiation environment in space. We examined the effects of heavy ion exposure on osteoblastic differ-entiation of murine preosteoblastic cell lines to gain insight into potential cellular mechanisms involved in bone cellular response after exposure to heavy ions. Therefore, we examined gene expression modulation of bone specific transcription factors, osteoblast specific marker genes as well as genes function as coupling factors that link bone resorption to bone formation. mRNA levels were determined using quantitative real time reverse transcriptase PCR (qRT-PCR). Expression of a target gene was standardized to unregulated reference genes. We investigated the transcriptional regulation of Osteocalcin (OCN) as well as TGF-β1, p21(CDKN1A) and the bone specific transcription factor Runx2 (cbfa1). We investigated gene expression modula-tions after exposure to energetic carbon ions (35 MeV/u, 73 keV/µm), iron ions (1000 MeV/u, 150 keV/µm) and lead ions (29 MeV/u, 9600 keV/µm) versus low LET X-rays. X-irradiation dose-dependently increased the mRNA levels of p21(CDKN1A) and Runx2 (cbfa1) whereas expression of OCN and TGF-β1 were elevated at later time points. Exposure to heavy ions provoked a more pronounced effect on osteoblastic specific gene expression within the dif-ferentiation process. Collectively, our results indicate that heavy ions facilitate osteoblastic differentiation more effectively than X-ray. Using the proposed in vitro model we confirmed that exposure to ionizing radiation significantly modulates gene expression levels of marker genes involved in the differentiation of osteoblasts. The data presented allow us to suggest that exposure to ionizing radiation interferes with bone formation at the level of cell differentiation.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tu Qisheng; Valverde, Paloma; Chen, Jake

Osterix (Osx) is a zinc-finger-containing transcription factor that is expressed in osteoblasts of all endochondral and membranous bones. In Osx null mice osteoblast differentiation is impaired and bone formation is absent. In this study, we hypothesized that overexpression of Osx in murine bone marrow stromal cells (BMSC) would be able to enhance their osteoblastic differentiation and mineralization in vitro. Retroviral transduction of Osx in BMSC cultured in non-differentiating medium did not affect expression of Runx2/Cbfa1, another key transcription factor of osteoblast differentiation, but induced an increase in the expression of other markers associated with the osteoblastic lineage including alkaline phosphatase,more » bone sialoprotein, osteocalcin, and osteopontin. Retroviral transduction of Osx in BMSC also increased their proliferation, alkaline phosphatase activity, and ability to form bone nodules. These events occurred without significant changes in the expression of {alpha}1(II) procollagen or lipoprotein lipase, which are markers of chondrogenic and adipogenic differentiation, respectively.« less

Osteogenic potential of mesenchymal cells embedded in the provisional matrix after a 6-week healing period in augmented and non-augmented extraction sockets: an immunohistochemical prospective pilot study in humans.

PubMed

Heberer, Susanne; Wustlich, Alexander; Lage, Hermann; Nelson, John J; Nelson, Katja

2012-01-01

The aim of the present clinical study was the evaluation of the osteogenic potential of mesenchymal cells embedded in the provisional matrix of non-augmented and with Bio-Oss collagen-augmented human extraction sockets after 6 weeks of healing time. Twenty-five patients with 47 extraction sites participated in the present study. After tooth removal, the extraction sockets were augmented with Bio-Oss collagen or not augmented. At implant placement, bone biopsies of the extraction sockets were obtained. The immunohistochemical analysis of the osteogenic potential of the mesenchymal cells in the provisional matrix was performed using three monoclonal antibodies: core-binding factor α1 (Cbfa1)/runt-related protein (Runx)2, osteonectin (OSN/secreted protein acidic and rich in cyst [SPARC]) and osteocalcin (OC). The statistical analysis was performed using two-factorial analysis for repeated measures, Mann-Whitney U-test and Spearman's rank-order correlation coefficient. Of 47 extraction sockets examined, 17 sockets demonstrated an almost complete ossification. Hence, the provisional matrix of the 30 remaining extraction sockets (21 non-augmented, 9 augmented) was immunohistochemically investigated. No evidence of acute or chronic inflammation was noted in any of the specimens. In the provisional matrix of the non-grafted socket, the median amount of Cbfa1/Runx2-positive cells was 72.3%, of OSN (SPARC) 66.9% and of OC 23.4%, whereas in the grafted sockets the median rate of immunopositive cells staining with Cbfa1/Runx2 was 73.3%, of OSN (SPARC) 61.4% and of OC 20.1%. There was no significant difference in the proportion of positive cells expressed by Cbfa1/Runx2, OSN/SPARC and OC between the grafted and non-grafted socket. Furthermore, the cell density did not correlate to the quantity of stained cells independent of the used proteins. After a 6-week healing period, the provisional matrix was demonstrated to have a high proportion of cells displaying a maturation of mature osteoprogenitor cells to osteoblasts. The grafting procedure did not influence the quantity of osteogenic cells in the extraction socket. © 2011 John Wiley & Sons A/S.

Stepwise differentiation of pluripotent stem cells into osteoblasts using four small molecules under serum-free and feeder-free conditions.

PubMed

Kanke, Kosuke; Masaki, Hideki; Saito, Taku; Komiyama, Yuske; Hojo, Hironori; Nakauchi, Hiromitsu; Lichtler, Alexander C; Takato, Tsuyoshi; Chung, Ung-Il; Ohba, Shinsuke

2014-06-03

Pluripotent stem cells are a promising tool for mechanistic studies of tissue development, drug screening, and cell-based therapies. Here, we report an effective and mass-producing strategy for the stepwise differentiation of mouse embryonic stem cells (mESCs) and mouse and human induced pluripotent stem cells (miPSCs and hiPSCs, respectively) into osteoblasts using four small molecules (CHIR99021 [CHIR], cyclopamine [Cyc], smoothened agonist [SAG], and a helioxanthin-derivative 4-(4-methoxyphenyl)pyrido[4',3':4,5]thieno[2,3-b]pyridine-2-carboxamide [TH]) under serum-free and feeder-free conditions. The strategy, which consists of mesoderm induction, osteoblast induction, and osteoblast maturation phases, significantly induced expressions of osteoblast-related genes and proteins in mESCs, miPSCs, and hiPSCs. In addition, when mESCs defective in runt-related transcription factor 2 (Runx2), a master regulator of osteogenesis, were cultured by the strategy, they molecularly recapitulated osteoblast phenotypes of Runx2 null mice. The present strategy will be a platform for biological and pathological studies of osteoblast development, screening of bone-augmentation drugs, and skeletal regeneration.

N-AC-l-Leu-PEI-mediated miR-34a delivery improves osteogenic differentiation under orthodontic force.

PubMed

Yu, Wenwen; Zheng, Yi; Yang, Zhujun; Fei, Hongbo; Wang, Yang; Hou, Xu; Sun, Xinhua; Shen, Yuqin

2017-12-15

Rare therapeutic genes or agents are reported to control orthodontic bone remodeling. MicroRNAs have recently been associated with bone metabolism. Here, we report the in vitro and in vivo effects of miR-34a on osteogenic differentiation under orthodontic force using an N -acetyl-L-leucine-modified polyethylenimine ( N -Ac-l-Leu-PEI) carrier. N -Ac-l-Leu-PEI exhibited low cytotoxicity and high miR-34a transfection efficiency in rat bone mineral stem cells and local alveolar bone tissue. After transfection, miR-34a enhanced the osteogenic differentiation of Runx2 and ColI , Runx2 and ColI protein levels, and early osteogenesis function under orthodontic strain in vitro . MiR-34a also enhanced alveolar bone remodeling under orthodontic force in vivo , as evidenced by elevated gene and protein expression, upregulated indices of alveolar bone anabolism, and diminished tooth movement. We determined that the mechanism miR-34a in osteogenesis under orthodontic force may be associated with GSK-3β. These results suggested that miR-34a delivered by N -Ac-l-Leu-PEI could be a potential therapeutic target for orthodontic treatment.

Saxagliptin affects long-bone microarchitecture and decreases the osteogenic potential of bone marrow stromal cells.

PubMed

Sbaraglini, María Laura; Molinuevo, María Silvina; Sedlinsky, Claudia; Schurman, León; McCarthy, Antonio Desmond

2014-03-15

Diabetes mellitus is associated with a decrease in bone quality and an increase in fracture incidence. Additionally, treatment with anti-diabetic drugs can either adversely or positively affect bone metabolism. In this study we evaluated: the effect of a 3-week oral treatment with saxagliptin on femoral microarchitecture in young male non-type-2-diabetic Sprague Dawley rats; and the in vitro effect of saxagliptin and/or fetal bovine serum (FBS), insulin or insulin-like growth factor-1 (IGF1), on the proliferation, differentiation (Runx2 and PPAR-gamma expression, type-1 collagen production, osteocalcin expression, mineralization) and extracellular-regulated kinase (ERK) activation, in bone marrow stromal cells (MSC) obtained from control (untreated) rats and in MC3T3E1 osteoblast-like cells. In vivo, oral saxagliptin treatment induced a significant decrease in the femoral osteocytic and osteoblastic density of metaphyseal trabecular bone and in the average height of the proximal cartilage growth plate; and an increase in osteoclastic tartrate-resistant acid phosphatase (TRAP) activity of the primary spongiosa. In vitro, saxagliptin inhibited FBS-, insulin- and IGF1-induced ERK phosphorylation and cell proliferation, in both MSC and MC3T3E1 preosteoblasts. In the absence of growth factors, saxagliptin had no effect on ERK activation or cell proliferation. In both MSC and MC3T3E1 cells, saxagliptin in the presence of FBS inhibited Runx2 and osteocalcin expression, type-1 collagen production and mineralization, while increasing PPAR-gamma expression. In conclusion, orally administered saxagliptin induced alterations in long-bone microarchitecture that could be related to its in vitro down-regulation of the ERK signaling pathway for insulin and IGF1 in MSC, thus decreasing the osteogenic potential of these cells. Copyright © 2014 Elsevier B.V. All rights reserved.

Altered thermogenesis and impaired bone remodeling in Misty mice

PubMed Central

Motyl, Katherine J; Bishop, Kathleen A; DeMambro, Victoria E; Bornstein, Sheila A; Le, Phuong; Kawai, Masanobu; Lotinun, Sutada; Horowitz, Mark C; Baron, Roland; Bouxsein, Mary L; Rosen, Clifford J

2013-01-01

Fat mass may be modulated by the number of brown-like adipocytes in white adipose tissue (WAT) in humans and rodents. Bone remodeling is dependent on systemic energy metabolism and, with age, bone remodeling becomes uncoupled and brown adipose tissue (BAT) function declines. To test the interaction between BAT and bone, we employed Misty (m/m) mice, which were reported be deficient in BAT. We found that Misty mice have accelerated age-related trabecular bone loss and impaired brown fat function (including reduced temperature, lower expression of Pgc1a and less sympathetic innervation compared to wildtype (+/+)). Despite reduced BAT function, Misty mice had normal core body temperature, suggesting heat is produced from other sources. Indeed, upon acute cold exposure (4°C for 6 hr), inguinal WAT from Misty mice compensated for BAT dysfunction by increasing expression of Acadl, Pgc1a, Dio2 and other thermogenic genes. Interestingly, acute cold exposure also decreased Runx2 and increased Rankl expression in Misty bone, but only Runx2 was decreased in wildtype. Browning of WAT is under the control of the sympathetic nervous system (SNS) and, if present at room temperature, could impact bone metabolism. To test whether SNS activity could be responsible for accelerated trabecular bone loss, we treated wildtype and Misty mice with the β-blocker, propranolol. As predicted, propranolol slowed trabecular BV/TV loss in the distal femur of Misty mice without affecting wildtype. Finally, the Misty mutation (a truncation of DOCK7) also has a significant cell-autonomous role. We found DOCK7 expression in whole bone and osteoblasts. Primary osteoblast differentiation from Misty calvaria was impaired, demonstrating a novel role for DOCK7 in bone remodeling. Despite the multifaceted effects of the Misty mutation, we have shown that impaired brown fat function leads to altered SNS activity and bone loss, and for the first time that cold exposure negatively affects bone remodeling. PMID:23553822

Influence of high glucose and advanced glycation end-products (ages) levels in human osteoblast-like cells gene expression.

PubMed

Miranda, Cristina; Giner, Mercè; Montoya, M José; Vázquez, M Angeles; Miranda, M José; Pérez-Cano, Ramón

2016-08-31

Type 2 diabetes mellitus (T2DM) is associated with an increased risk of osteoporotic fracture. Several factors have been identified as being potentially responsible for this risk, such as alterations in bone remodelling that may have been induced by changes in circulating glucose or/and by the presence of non-oxidative end products of glycosylation (AGEs). The aim of this study is to assess whether such variations generate a change in the gene expression related to the differentiation and osteoblast activity (OPG, RANKL, RUNX2, OSTERIX, and AGE receptor) in primary cultures of human osteoblast-like cells (hOB). We recruited 32 patients; 10 patients had osteoporotic hip fractures (OP group), 12 patients had osteoporotic hip fractures with T2DM (T2DM group), and 10 patients had hip osteoarthritis (OA group) with no osteoporotic fractures and no T2DM. The gene expression was analyzed in hOB cultures treated with physiological glucose concentration (4.5 mM) as control, high glucose (25 mM), and high glucose plus AGEs (2 μg/ml) for 24 h. The hOB cultures from patients with hip fractures presented slower proliferation. Additionally, the hOB cultures from the T2DM group were the most negatively affected with respect to RUNX2 and OSX gene expression when treated solely with high glucose or with high glucose plus AGEs. Moreover, high levels of glucose induced a major decrease in the RANKL/OPG ratio when comparing the OP and the T2DM groups to the OA group. Our data indicates an altered bone remodelling rate in the T2DM group, which may, at least partially, explain the reduced bone strength and increased incidence of non-traumatic fractures in diabetic patients.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Carmona-Rodriguez, Bruno; Alvarez-Perez, Marco Antonio; Narayanan, A. Sampath

We recently presented evidence showing that a human cementoblastoma-derived protein, named Cementum Protein 1 (CEMP1) may play a role as a local regulator of cementoblast differentiation and cementum-matrix mineralization. This protein was shown to be expressed by cementoblasts and progenitor cells localized in the periodontal ligament. In this study we demonstrate that transfection of CEMP1 into human gingival fibroblasts (HGF) induces mineralization and expression of bone and cementum-matrix proteins. The transfected HGF cells had higher alkaline phosphatase activity and proliferation rate and they expressed genes for alkaline phosphatase, bone sialoprotein, osteocalcin, osteopontin, the transcription factor Runx2/Cbfa1, and cementum attachment proteinmore » (CAP). They also produced biological-type hydroxyapatite. These findings indicate that the CEMP1 might participate in differentiation and mineralization of nonosteogenic cells, and that it might have a potential function in cementum and bone formation.« less

Enhancement of osteogenic differentiation of human adipose derived stem cells by the controlled release of platelet lysates from hybrid scaffolds produced by supercritical fluid foaming.

PubMed

Santo, Vítor E; Duarte, Ana Rita C; Popa, Elena G; Gomes, Manuela E; Mano, João F; Reis, Rui L

2012-08-20

A new generation of scaffolds capable of acting not only as support for cells but also as a source of biological cues to promote tissue regeneration is currently a hot topic of in bone Tissue Engineering (TE) research. The inclusion of growth factor (GF) controlled release functionalities in the scaffolds is a possible strategy to achieve such goal. Platelet Lysate (PL) is an autologous source of GFs, providing several bioactive agents known to act on bone regeneration. In this study, chitosan-chondroitin sulfate nanoparticles loaded with PL were included in a poly(D,L-lactic acid) foam produced by supercritical fluid foaming. The tridimensional (3D) structures were then seeded with human adipose-derived stem cells (hASCs) and cultured in vitro under osteogenic stimulus. The osteogenic differentiation of the seeded hASCs was observed earlier for the PL-loaded constructs, as shown by the earlier alkaline phosphatase peak and calcium detection and stronger Runx2 expression at day 7 of culture, in comparison with the control scaffolds. Osteocalcin gene expression was upregulated in presence of PL during all culture period, which indicates an enhanced osteogenic induction. These results suggest the synergistic effect of PL and hASCs in combinatory TE strategies and support the potential of PL to increase the multifunctionality of the 3D hybrid construct for bone TE applications. Copyright © 2012 Elsevier B.V. All rights reserved.

Overexpression of Hif-1α in Mesenchymal Stem Cells Affects Cell-Autonomous Angiogenic and Osteogenic Parameters.

PubMed

Lampert, F M; Kütscher, C; Stark, G B; Finkenzeller, G

2016-03-01

Reconstruction of large bone defects still represents a major medical challenge. In recent years tissue engineering has developed techniques based on adult mesenchymal stem cells (MSCs) that could represent an attractive therapeutical option to treat large bone defects in the future. It has been demonstrated in various animal models that ex vivo expanded MSCs are capable of promoting the regeneration of skeletal defects after implantation. However, for the efficient regeneration of bone in tissue engineering applications, a rapid vascularization of implanted grafts is essential to ensure the survival of cells in the early post-implantational phase. A promising strategy to enhance vascularization of MSC-containing implants could consist of overexpression of the angiogenic master transcription factor Hypoxia-inducible factor 1 (Hif-1) in the MSCs in order to induce angiogenesis and support osteogenesis. In the present study, we overexpressed Hif-1α in MSCs by using recombinant adenoviruses and investigated cell-autonomous effects. Overexpression of Hif-1α enhanced proliferation, migration, cell survival and expression of pro-angiogenic genes. Other parameters such as expression of the osteogenic markers BMP-2 and RunX2 were decreased. Hif-1α overexpression had no effect on invasion, senescence and osteogenic differentiation of MSCs. Our experiments revealed multifarious effects of Hif-1α overexpression on cell-autonomous parameters. Therefore, Hif-1α overexpression may represent a therapeutic option to improve cellular functions of MSCs to treat critical sized bone defects. © 2015 Wiley Periodicals, Inc.

Enhanced osteogenic potential of human mesenchymal stem cells on electrospun nanofibrous scaffolds prepared from eri-tasar silk fibroin.

PubMed

Panda, Niladri Nath; Biswas, Amit; Pramanik, Krishna; Jonnalagadda, Sriramakamal

2015-07-01

This study evaluated the mechanical properties and osteogenic potential of a silk fibroin scaffold prepared from a 70:30 blend of Eri (Philosamia ricini) and Tasar (Antheraea mylitta) silk, respectively (ET scaffolds). An electrospinning process was used to prepare uniformly blended, fibrous scaffolds of nanoscale dimensions, as confirmed by scanning and transmission electron microscopy (fiber diameter < 300 nm). Similarly prepared scaffolds derived from gelatin and Bombyx mori (BM) silk fibroin were used as controls. Mechanical testing and atomic force microscopy showed that the ET scaffolds had significantly higher tensile strength (1.83 ± 0.13 MPa) and surface roughness (0.44 μm) compared with BM (1.47 ± 0.10 MPa; 0.37 μm) and gelatin scaffolds (0.6 ± 0.07 MPa; 0.28 μm). All scaffolds were exposed to mesenchymal stem cells isolated to human chord blood (hMSCs) for up to 28 days in vitro. Alamar blue and alkaline phosphatase assay showed greater attachment and proliferation for both ET and BM scaffolds compared with gelatin. The ET scaffolds also promoted greater differentiation of the attached hMSCs as evidenced by higher expression of RunX2, osteocalcin, and CD29/CD44 expression. ET scaffolds also showed significantly higher mineralization, as evidenced by glycosaminoglycan assay, alizarin red staining, and elemental analysis of crystalline composites isolated from the scaffolds. © 2014 Wiley Periodicals, Inc.

Topographical cues of direct metal laser sintering titanium surfaces facilitate osteogenic differentiation of bone marrow mesenchymal stem cells through epigenetic regulation.

PubMed

Zheng, Guoying; Guan, Binbin; Hu, Penghui; Qi, Xingying; Wang, Pingting; Kong, Yu; Liu, Zihao; Gao, Ping; Li, Rui; Zhang, Xu; Wu, Xudong; Sui, Lei

2018-04-27

To investigate the role of hierarchical micro/nanoscale topography of direct metal laser sintering (DMLS) titanium surfaces in osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), as well as the possible underlying epigenetic mechanism. Three groups of titanium specimens were prepared, including DMLS group, sandblasted, large-grit, acid-etched (SLA) group and smooth titanium (Ti) group. BMSCs were cultured on discs followed by surface characterization. Cell adhesion and proliferation were examined by SEM and CCK-8 assay, while osteogenic-related gene expression was detected by real-time RT-PCR. Immunofluorescence, western blotting and in vivo study were also performed to evaluate the potential for osteogenic induction of materials. In addition, to investigate the underlying epigenetic mechanisms, immunofluorescence and western blotting were performed to evaluate the global level of H3K4me3 during osteogenesis. The H3K4me3 and H3K27me3 levels at the promoter area of the osteogenic gene Runx2 were detected by ChIP assay. The DMLS surface exhibits greater protein adsorption ability and shows better cell adhesion performance than SLA and Ti surfaces. Moreover, both in vitro and in vivo studies demonstrated that the DMLS surface is more favourable for the osteogenic differentiation of BMSCs than SLA and Ti surfaces. Accordingly, osteogenesis-associated gene expression in BMSCs is efficiently induced by a rapid H3K27 demethylation and increase in H3K4me3 levels at gene promoters upon osteogenic differentiation on DMLS titanium surface. Topographical cues of DMLS surfaces have greater potential for the induction of osteogenic differentiation of BMSCs than SLA and Ti surfaces both in vitro and in vivo. A potential epigenetic mechanism is that the appropriate topography allows rapid H3K27 demethylation and an increased H3K4me3 level at the promoter region of osteogenesis-associated genes during the osteogenic differentiation of BMSCs. © 2018 John Wiley & Sons Ltd.

[A preliminary study for the effect of nano hydroxyapatite on human adipose-derived mesenchymal stem cells mixture 3D bio-printing].

PubMed

Song, Y; Wang, X F; Wang, Y G; Dong, F; Lv, P J

2016-10-18

To study the effect of nano hydroxyapatite on human adipose-derived mesenchymal stem cells(hASCs) mixture 3D bio-printing for cells' proliferation and osteogenesis. P5 hASCs were used as seed cells, 10 g/L nano hydroxyapatite was added into the cell-sodium alginate-gelatin mixture (concentration: 20 g/L sodium alginate, 80 g/L gelatin; cell density: 1×10 6 /mL), then the mixture was printed by 3D bio-printer as the experimental group. And the cell-sodium alginate-gelatin mixture without nano hydroxyapatite was printed as the control group. Respectively, both the experimental and control groups were detected by microscope, CCK-8, Western blot and PCR at certain time pointsafter being printed, whose cells' proliferation and osteogenic differentiation were analyzed. The microscopic observation and CCK-8 results showed that the cells of the experimental group and the control group both had a good proliferation 24 h and 7 d after being printed. The Western blot results showed that 14 d after printing, the expression of Runt-related transcription factor 2 (RUNX2) had no statistical difference between the experimental group and control group. The PCR results showed that 14 d after printing, the expression of osteogenesis-related genes (RUNX2, osterix, and osteocalcin) was significantly higher in the experimental group than in the control group. Nano hydroxyapatite can increase osteogenic differentiation of the hASCs mixture after bio-printing, in which the cells still have a good proliferation.

The Morphogenesis of Cranial Sutures in Zebrafish

PubMed Central

Topczewska, Jolanta M.; Shoela, Ramy A.; Tomaszewski, Joanna P.; Mirmira, Rupa B.; Gosain, Arun K.

2016-01-01

Using morphological, histological, and TEM analyses of the cranium, we provide a detailed description of bone and suture growth in zebrafish. Based on expression patterns and localization, we identified osteoblasts at different degrees of maturation. Our data confirm that, unlike in humans, zebrafish cranial sutures maintain lifelong patency to sustain skull growth. The cranial vault develops in a coordinated manner resulting in a structure that protects the brain. The zebrafish cranial roof parallels that of higher vertebrates and contains five major bones: one pair of frontal bones, one pair of parietal bones, and the supraoccipital bone. Parietal and frontal bones are formed by intramembranous ossification within a layer of mesenchyme positioned between the dermal mesenchyme and meninges surrounding the brain. The supraoccipital bone has an endochondral origin. Cranial bones are separated by connective tissue with a distinctive architecture of osteogenic cells and collagen fibrils. Here we show RNA in situ hybridization for col1a1a, col2a1a, col10a1, bglap/osteocalcin, fgfr1a, fgfr1b, fgfr2, fgfr3, foxq1, twist2, twist3, runx2a, runx2b, sp7/osterix, and spp1/ osteopontin, indicating that the expression of genes involved in suture development in mammals is preserved in zebrafish. We also present methods for examining the cranium and its sutures, which permit the study of the mechanisms involved in suture patency as well as their pathological obliteration. The model we develop has implications for the study of human disorders, including craniosynostosis, which affects 1 in 2,500 live births. PMID:27829009

[The effect of Foxc2 overexpression on the osteogenic properties of C3H10T1/2 cells].

PubMed

Wang, Min-Jiao; Si, Jia-Wen; Li, Hong-Liang; Ouyang, Ning-Juan; Shen, Guo-Fang

2016-08-01

To investigate the effect of Foxc2 overexpression on osteogenic and adipogenic differentiation of C3H10T1/2 cells. C3H10T1/2 cells were transfected with plenti-Foxc2 and selected with puromycin for stable clones. The expression of Foxc2 was determined by real-time PCR and Western blot. Cell proliferation was detected by CCK-8 kit. Cell cycle and apoptosis were detected by flow cytometry. The level of osteogenic biomarkers Runx2, OPN, OCN and adipogenic biomarker PPARγ were quantified by real-time PCR and Western blot. Alkaline phosphatase (ALP) staining and oil red staining were conducted to evaluate the effect of Foxc2 overexpression on osteogenic and adipogenic differentiation. Statistical analysis was performed using SPSS 17.0 software package. C3H10T1/2-Foxc2 cell line was successfully constructed and verified by direct sequencing and Foxc2 overexpression in vitro. Cell proliferation was reduced and cell cycle was blocked in G1/G0 phase. Enhanced ALP staining and reduced oil red staining were observed in C3H10T1/2-Foxc2 cells as compared with the control. Foxc2 overexpression up-regulated Runx2, OPN, OCN during osteogenic differentiation and down-regulated PPARγduring adipogenic differentiation. C3H10T1/2 cell line stably expressing Foxc2 gene was successfully established, cell proliferation was reduced, osteogenesis biomarkers were up-regulated during the osteogenesis by overexpression Foxc2, PPARγwas down-regulated during adipogenesis.

The effects of biodegradable poly(lactic-co-glycolic acid)-based microspheres loaded with quercetin on stemness, viability and osteogenic differentiation potential of stem cell spheroids.

PubMed

Lee, H; Nguyen, T T; Kim, M; Jeong, J-H; Park, J-B

2018-05-31

Quercetin has been reported to exert many beneficial effects on the protection against various diseases, such as diabetes, cancer, and inflammation. The aim of this study is to evaluate the potential osteogenic differentiation ability of mesenchymal stem cells in the presence of quercetin. Quercetin-loaded poly(lactic-co-glycolic acid) microspheres were prepared using an electrospraying technique. Characterization of the microspheres was evaluated with a scanning electron microscope and release profile. Three-dimensional cell spheroids were fabricated using silicon elastomer-based concave microwells. Qualitative results of cellular viability were seen under a confocal microscope, and quantitative cellular viability was evaluated using the Cell Counting Kit-8 assay. The alkaline phosphatase activity and Alizarin Red S staining were performed. A quantitative real-time polymerase chain reaction and a western blot analysis were performed. Spheroids were well formed irrespective of quercetin concentration. Most of the cells in spheroids emitted green fluorescence, and the morphology was round without significant changes. The application of quercetin-loaded microspheres produced a significant increase in the alkaline phosphatase activity. The real-time polymerase chain reaction results showed a significant increase in Runx2, and western blot results showed higher expression of Runx2 protein expression. Biodegradable microspheres loaded with quercetin produced prolonged release profiles with increased mineralization. Microspheres loaded with quercetin can be used for the enhancement of osteoblastic differentiation in cell therapy. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

CCN3 Protein Participates in Bone Regeneration as an Inhibitory Factor*

PubMed Central

Matsushita, Yuki; Sakamoto, Kei; Tamamura, Yoshihiro; Shibata, Yasuaki; Minamizato, Tokutaro; Kihara, Tasuku; Ito, Masako; Katsube, Ken-ichi; Hiraoka, Shuichi; Koseki, Haruhiko; Harada, Kiyoshi; Yamaguchi, Akira

2013-01-01

CCN3, a member of the CCN protein family, inhibits osteoblast differentiation in vitro. However, the role of CCN3 in bone regeneration has not been well elucidated. In this study, we investigated the role of CCN3 in bone regeneration. We identified the Ccn3 gene by microarray analysis as a highly expressed gene at the early phase of bone regeneration in a mouse bone regeneration model. We confirmed the up-regulation of Ccn3 at the early phase of bone regeneration by RT-PCR, Western blot, and immunofluorescence analyses. Ccn3 transgenic mice, in which Ccn3 expression was driven by 2.3-kb Col1a1 promoter, showed osteopenia compared with wild-type mice, but Ccn3 knock-out mice showed no skeletal changes compared with wild-type mice. We analyzed the bone regeneration process in Ccn3 transgenic mice and Ccn3 knock-out mice by microcomputed tomography and histological analyses. Bone regeneration in Ccn3 knock-out mice was accelerated compared with that in wild-type mice. The mRNA expression levels of osteoblast-related genes (Runx2, Sp7, Col1a1, Alpl, and Bglap) in Ccn3 knock-out mice were up-regulated earlier than those in wild-type mice, as demonstrated by RT-PCR. Bone regeneration in Ccn3 transgenic mice showed no significant changes compared with that in wild-type mice. Phosphorylation of Smad1/5 was highly up-regulated at bone regeneration sites in Ccn3 KO mice compared with wild-type mice. These results indicate that CCN3 is up-regulated in the early phase of bone regeneration and acts as a negative regulator for bone regeneration. This study may contribute to the development of new strategies for bone regeneration therapy. PMID:23653360

Role of diabetes- and obesity-related protein in the regulation of osteoblast differentiation

PubMed Central

Linares, Gabriel R.; Xing, Weirong; Burghardt, Hans; Baumgartner, Bernhard; Chen, Shin-Tai; Ricart, Wifredo; Fernández-Real, José Manuel; Zorzano, Antonio

2011-01-01

Although thyroid hormone (TH) is known to exert important effects on the skeleton, the nuclear factors constituting the TH receptor coactivator complex and the molecular pathways by which TH mediates its effects on target gene expression in osteoblasts remain poorly understood. A recent study demonstrated that the actions of TH on myoblast differentiation are dependent on diabetes- and obesity-related protein (DOR). However, the role of DOR in osteoblast differentiation is unknown. We found DOR expression increased during in vitro differentiation of bone marrow stromal cells into osteoblasts and also in MC3T3-E1 cells treated with TH. However, DOR expression decreased during cellular proliferation. To determine whether DOR acts as a modulator of TH action during osteoblast differentiation, we examined whether overexpression or knockdown of DOR in MC3T3-E1 cells affects the ability of TH to induce osteoblast differentiation by evaluating alkaline phosphatase (ALP) activity. ALP activity was markedly increased in DOR-overexpressing cells treated with TH. In contrast, loss of DOR dramatically reduced TH stimulation of ALP activity in MC3T3-E1 cells and primary calvaria osteoblasts transduced with lentiviral DOR shRNA. Consistent with reduced ALP activity, mRNA levels of osteocalcin, ALP, and Runx2 were decreased significantly in DOR shRNA cells. In addition, a common single nucleotide polymorphism (SNP), DOR1 found on the promoter of human DOR gene, was associated with circulating osteocalcin levels in nondiabetic subjects. Based on these data, we conclude that DOR plays an important role in TH-mediated osteoblast differentiation, and a DOR SNP associates with plasma osteocalcin in men. PMID:21467300

Combined use of leptin and mechanical stress has osteogenic effects on ossification of the posterior longitudinal ligament.

PubMed

Chen, Shuai; Zhu, Haifeng; Wang, Gangliang; Xie, Ziang; Wang, Jiying; Chen, Jian

2018-06-16

To evaluate the effects of leptin/leptin receptor (LepR) combined with mechanical stress on the development of ossification of the posterior longitudinal ligament (OPLL), which is a disease characterized by ectopic bone formation of the posterior longitudinal ligament (PLL) and can lead to radiculopathy and myelopathy. Six human samples of the PLL were analyzed for the expression of leptin and LepR by RT-PCR and western blotting. PLL cells were stimulated with leptin and mechanical stress delivered via a Flexcell tension system, and osteogenic differentiation was evaluated by RT-PCR and western blotting analysis of osteogenic marker expression as well as by alkaline phosphatase (ALP) staining and alizarin red S staining. Activation of mitogen-activated protein kinase (MAPK), Janus kinase (JAK) 2-signal transducer, activator of transcription (STAT) 3 and phosphatidylinositol 3-kinase (PI3K)-Akt was evaluated by western blotting. Samples from the OPLL group had higher LepR mRNA and protein levels and lower leptin levels than those from healthy controls. Exposure to leptin and Flexcell increased the number of ALP-positive cells and calcium nodules in a dose-dependent manner; this effect was accompanied by upregulation of the osteogenic markers osteocalcin, runt-related transcription factor 2 (RUNX2) and osteopontin. Extracellular signal-regulated kinase, P38 MAPK, JAK2, STAT3, PI3K and Akt signaling, was also activated by the combined effects of leptin and mechanical stress. Leptin and LepR are differentially expressed in OPLL tissues, and the combined use of leptin/LepR and mechanical stress promotes osteogenic differentiation of PLL cells via MAPK, JAK2-STAT3 and PI3K/Akt signaling. These slides can be retrieved under Electronic Supplementary Material.

DNA methylation patterns in ulcerative colitis-associated cancer: a systematic review.

PubMed

Emmett, Ruth A; Davidson, Katherine L; Gould, Nicholas J; Arasaradnam, Ramesh P

2017-07-01

Evidence points to the role of DNA methylation in ulcerative colitis (UC)-associated cancer (UCC), the most serious complication of ulcerative colitis. A better understanding of the etiology of UCC may facilitate the development of new therapeutic targets and help to identify biomarkers of the disease risk. A search was performed in three databases following PRISMA protocol. DNA methylation in UCC was compared with sporadic colorectal cancer (SCRC), and individual genes differently methylated in UCC identified. While there were some similarities in the methylation patterns of UCC compared with SCRC, generally lower levels of hypermethylation in promoter regions of individual genes was evident in UCC. Certain individual genes are, however, highly methylated in colitis-associated cancer: RUNX3, MINT1, MYOD and p16 exon1 and the promoter regions of EYA4 and ESR. Patterns of DNA methylation differ between UCC and SCRC. Seven genes appear to be promising putative biomarkers.

Methylation analysis of p16, SLIT2, SCARA5, and Runx3 genes in hepatocellular carcinoma

PubMed Central

Sun, Gaofeng; Zhang, Chen; Feng, Min; Liu, Wensheng; Xie, Huifang; Qin, Qin; Zhao, E.; Wan, Li

2017-01-01

Abstract This study is to investigate the methylation status of multiple tumor suppressor 1 (p16), secreted glycoprotein 2 (SLIT2), scavenger receptor class A, member 5 putative (SCARA5), and human runt-related transcription factor 3 (Runx3) genes in the peripheral blood of hepatocellular carcinoma (HCC). This is a case–control study. The peripheral blood samples were collected from 25 HCC patients, 25 patients with high risk of HCC (defined as “internal control group”), and 25 healthy individuals (defined as “external control group”), respectively. Then the methylation status of p16, SLIT2, SCARA5, and Runx3 genes in the blood samples were analyzed by pyrosequencing. The relationship between the methylation and the clinical features of HCC patients were evaluated. The methylation levels in the 7 CpG loci of p16 gene in HCC patients were low and without statistically significant difference (P > .05) compared to the control groups. Although the methylation levels of CpG3 and CpG4 in SLIT2 gene loci were higher than those of the control groups, there was no statistically significant difference (P > .05). However, the methylation rate of CpG2 locus in SCARA5 gene in HCC patients was significantly higher (P < .05). And the methylation rates of CpG1, CpG2, CpG3, CpG4, CpG5, and CpG8 in Runx3 gene in HCC patients were significantly different to that of control groups (P < .05). We also have analyzed the correlations between the CpG islands methylation of Runx3 or SCARA5 genes and the age, gender, hepatitis B, liver cirrhosis, alpha fetal protein, or hepatitis B surface antigen (HBsAg) of the HCC patients, which all showed no significant correlations (P > .05). The methylation status of SCARA5 and Runx3 genes are abnormal in HCC patients, which may further be used as molecular markers for early auxiliary diagnosis of liver cancer. PMID:29019900

Study of the role of functional variants of SLC22A4, RUNX1 and SUMO4 in systemic lupus erythematosus

PubMed Central

Orozco, G; Sánchez, E; Gómez, L M; González‐Gay, M A; López‐Nevot, M A; Torres, B; Ortego‐Centeno, N; Jiménez‐Alonso, J; de Ramón, E; Román, J Sánchez; Anaya, J M; Sturfelt, G; Gunnarsson, I; Svennungsson, E; Alarcón‐Riquelme, M; González‐Escribano, M F; Martín, J

2006-01-01

Background Functional polymorphisms of the solute carrier family 22, member 4 (SLC22A4), runt related transcription factor 1 (RUNX1) and small ubiquitin‐like modifier 4 (SUMO4) genes have been shown to be associated with several autoimmune diseases. Objective To test the possible role of these variants in susceptibility to or severity of systemic lupus erythematosus (SLE), on the basis that common genetic bases are shared by autoimmune disorders. Methods 597 SLE patients and 987 healthy controls of white Spanish origin were studied. Two additional cohorts of 228 SLE patients from Sweden and 122 SLE patients from Colombia were included. A case–control association study was carried out with six single nucleotide polymorphisms (SNP) spanning the SLC22A4 gene, one SNP in RUNX1 gene, and one additional SNP in SUM04 gene. Results No significant differences were observed between SLE patients and healthy controls when comparing the distribution of the genotypes or alleles of any of the SLC22A4, RUNX1, or SUMO4 polymorphisms tested. Significant differences were found in the distribution of the SUMO4 genotypes and alleles among SLE patients with and without nephritis, but after multiple testing correction, the significance of the association was lost. The association of SUMO4 with nephritis could not be verified in two independent SLE cohorts from Sweden and Colombia. Conclusions These results suggest that the SLC22A4, RUNX1, and SUMO4 polymorphisms analysed do not play a role in the susceptibility to or severity of SLE. PMID:16249223

Facing the facts: The Runx2 gene is associated with variation in facial morphology in primates.

PubMed

Ritzman, Terrence B; Banovich, Nicholas; Buss, Kaitlin P; Guida, Jennifer; Rubel, Meagan A; Pinney, Jennifer; Khang, Bao; Ravosa, Matthew J; Stone, Anne C

2017-10-01

The phylogenetic and adaptive factors that cause variation in primate facial form-including differences among the major primate clades and variation related to feeding and/or social behavior-are relatively well understood. However, comparatively little is known about the genetic mechanisms that underlie diversity in facial form in primates. Because it is essential for osteoblastic differentiation and skeletal development, the runt-related transcription factor 2 (Runx2) is one gene that may play a role in these genetic mechanisms. Specifically, polymorphisms in the QA ratio (determined by the ratio of the number of polyglutamines to polyalanines in one functional domain of Runx2) have been shown to be correlated with variation in facial length and orientation in other mammal groups. However, to date, the relationship between variation in this gene and variation in facial form in primates has not been explicitly tested. To test the hypothesis that the QA ratio is correlated with facial form in primates, the current study quantified the QA ratio, facial length, and facial angle in a sample of 33 primate species and tested for correlation using phylogenetic generalized least squares. The results indicate that the QA ratio of the Runx2 gene is positively correlated with variation in relative facial length in anthropoid primates. However, no correlation was found in strepsirrhines, and there was no correlation between facial angle and the QA ratio in any groups. These results suggest that, in primates, the QA ratio of the Runx2 gene may play a role in modulating facial size, but not facial orientation. This study therefore provides important clues about the genetic and developmental mechanisms that may underlie variation in facial form in primates. Copyright © 2017 Elsevier Ltd. All rights reserved.

FOXO1-suppressed miR-424 regulates the proliferation and osteogenic differentiation of MSCs by targeting FGF2 under oxidative stress

NASA Astrophysics Data System (ADS)

Li, Liangping; Qi, Qihua; Luo, Jiaquan; Huang, Sheng; Ling, Zemin; Gao, Manman; Zhou, Zhiyu; Stiehler, Maik; Zou, Xuenong

2017-02-01

Recently, microRNAs (miRNAs) have been identified as key regulators of the proliferation and differentiation of mesenchymal stem cells (MSCs). Our previous in vivo study and other in vitro studies using miRNA microarrays suggest that miR-424 is involved in the regulation of bone formation. However, the role and mechanism of miR-424 in bone formation still remain unknown. Here, we identified that the downregulation of miR-424 mediates bone formation under oxidative stress, and we explored its underlying mechanism. Our results showed that miR-424 was significantly downregulated in an anterior lumbar interbody fusion model of pigs and in a cell model of oxidative stress induced by H2O2. The overexpression of miR-424 inhibited proliferation and osteogenic differentiation shown by a decrease in alkaline phosphatase (ALP) activity, mineralization and osteogenic markers, including RUNX2 and ALP, whereas the knockdown of miR-424 led to the opposite results. Moreover, miR-424 exerts its effects by targeting FGF2. Furthermore, we found that FOXO1 suppressed miR-424 expression and bound to its promoter region. FOXO1 enhanced proliferation and osteogenic differentiation in part through the miR-424/FGF2 pathway. These results indicated that FOXO1-suppressed miR-424 regulates both the proliferation and osteogenic differentiation of MSCs via targeting FGF2, suggesting that miR-424 might be a potential novel therapeutic strategy for promoting bone formation.

Naringin enhances osteogenic differentiation through the activation of ERK signaling in human bone marrow mesenchymal stem cells.

PubMed

Wang, Huichao; Li, Chunbo; Li, Jianming; Zhu, Yingjie; Jia, Yudong; Zhang, Ying; Zhang, Xiaodong; Li, Wenlong; Cui, Lei; Li, Wuyin; Liu, Youwen

2017-04-01

Naringin has been reported to regulate bone metabolism. However, its effect on osteogenesis remains unclear. The aim was to investigate the effect of naringin on osteogenic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) through the activation of the ERK signaling pathway in osteogenic differentiation. Annexin V-FITC assay and MTT assay were used to measure the effect of naringin on cytotoxicity and proliferation of hBMSCs, respectively. Alkaline phosphatase activity analysis, Alizarin Red S staining, Western blotting, and real-time PCR assay were used to evaluate both the potential effect of naringin on osteogenic differentiation and the role of ERK signaling pathway in osteogenic differentiation. Our results showed that naringin had no obvious toxicity on hBMSCs, and could significantly promote the proliferation of hBMSCs. Naringin also enhanced the osteogenic differentiation of hBMSCs and increased the protein and mRNA expression levels of osteogenic markers such as Runx-2, OXS, OCN, and Col1 in a dose-dependent manner. In addition, we found that the enhancing effect of naringin on osteogenic differentiation was related to the activation of phosphor-ERK, with an increase in duration of activity from 30 min to 120 min. More importantly, both the enhancing effect of naringin on osteogenic differentiation and the activity effect of naringin on ERK signaling pathway were reversed by U0126 addition. Our findings demonstrated that naringin promoted proliferation and osteogenesis of hBMSCs by activating the ERK signaling pathway and it might be a potential therapeutic agent for treating or preventing osteoporosis.

Chitosan nanofiber scaffold improves bone healing via stimulating trabecular bone production due to upregulation of the Runx2/osteocalcin/alkaline phosphatase signaling pathway

PubMed Central

Ho, Ming-Hua; Yao, Chih-Jung; Liao, Mei-Hsiu; Lin, Pei-I; Liu, Shing-Hwa; Chen, Ruei-Ming

2015-01-01

Osteoblasts play critical roles in bone formation. Our previous study showed that chitosan nanofibers can stimulate osteoblast proliferation and maturation. This translational study used an animal model of bone defects to evaluate the effects of chitosan nanofiber scaffolds on bone healing and the possible mechanisms. In this study, we produced uniform chitosan nanofibers with fiber diameters of approximately 200 nm. A bone defect was surgically created in the proximal femurs of male C57LB/6 mice, and then the left femur was implanted with chitosan nanofiber scaffolds for 21 days and compared with the right femur, which served as a control. Histological analyses revealed that implantation of chitosan nanofiber scaffolds did not lead to hepatotoxicity or nephrotoxicity. Instead, imaging analyses by X-ray transmission and microcomputed tomography showed that implantation of chitosan nanofiber scaffolds improved bone healing compared with the control group. In parallel, microcomputed tomography and bone histomorphometric assays further demonstrated augmentation of the production of new trabecular bone in the chitosan nanofiber-treated group. Furthermore, implantation of chitosan nanofiber scaffolds led to a significant increase in the trabecular bone thickness but a reduction in the trabecular parameter factor. As to the mechanisms, analysis by confocal microscopy showed that implantation of chitosan nanofiber scaffolds increased levels of Runt-related transcription factor 2 (Runx2), a key transcription factor that regulates osteogenesis, in the bone defect sites. Successively, amounts of alkaline phosphatase and osteocalcin, two typical biomarkers that can simulate bone maturation, were augmented following implantation of chitosan nanofiber scaffolds. Taken together, this translational study showed a beneficial effect of chitosan nanofiber scaffolds on bone healing through stimulating trabecular bone production due to upregulation of Runx2-mediated alkaline phosphatase and osteocalcin gene expressions. Our results suggest the potential of chitosan nanofiber scaffolds for therapy of bone diseases, including bone defects and bone fractures. PMID:26451104

Caffeic acid phenethyl ester protects against glucocorticoid-induced osteoporosis in vivo: Impact on oxidative stress and RANKL/OPG signals

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tolba, Mai F.

Glucocorticoid-induced osteoporosis (GIO) is one of the most common causes of secondary osteoporosis. Given that glucocorticoids are considered as a main component of the treatment protocols for a variety of inflammation and immune-mediated diseases besides its use as adjuvant to several chemotherapeutic agents, it is crucial to find ways to overcome this critical adverse effect. Caffeic acid phenethyl ester (CAPE), which is a natural compound derived from honeybee propolis displayed promising antiosteoporotic effects against mechanical bone injury in various studies. The current work aimed at investigating the potential protective effect of CAPE against GIO in vivo with emphasis on themore » modulation of oxidative status and receptor activator of NF-kB ligand (RANKL)/osteoprotegrin (OPG) signaling. The results showed that CAPE opposed dexamethasone (DEX)-mediated alterations in bone histology and tartarate-resistant acid phosphatase (TRAP) activity. In addition, CAPE restored oxidative balance, Runt-related transcription factor 2 (RunX2) expression and reduced caspase-3 activity in femur tissues. Co-administration of CAPE with DEX normalized RANKL/OPG ratio and Akt activation indicating a reduction in DEX-osteoclastogenesis. In conclusion, concurrent treatment of CAPE with DEX exhibited promising effects in the protection against DEX-induced osteoporosis through opposing osteoclastogenesis and protecting osteoblasts. The potent antioxidant activity of CAPE is, at least in part, involved in its anti-apoptotic effects and modulation of RunX2 and RANKL/OPG signals. The use of CAPE-enriched propolis formulas is strongly recommended for patients on chronic glucocorticoid therapy to help in the attenuation of GIO. - Highlights: • Caffeic acid phenethyl ester (CAPE) counteracts DEX-induced osteoporosis. • CAPE hinders DEX-induced alterations in oxidation parameters as GSH, SOD and MDA. • CAPE opposes osteoclastogenesis via suppressing RANL/OPG ratio and Akt signals. • CAPE supports the osteoblasts via modulating caspase-3 and RUNX2 signals.« less

[Vascular Calcification - Pathological Mechanism and Clinical Application - . Role of vascular smooth muscle cells in vascular calcification].

PubMed

Kurabayashi, Masahiko

2015-05-01

Vascular calcification is commonly seen with aging, chronic kidney disese (CKD), diabetes, and atherosclerosis, and is closely associated with cardiovascular morbidity and mortality. Vascular calcification has long been regarded as the final stage of degeneration and necrosis of arterial wall and a passive, unregulated process. However, it is now known to be an active and tightly regulated process involved with phenotypic transition of vascular smooth muscle cells (VSMC) that resembles bone mineralization. Briefly, calcium deposits of atherosclerotic plaque consist of hydroxyapatite and may appear identical to fully formed lamellar bone. By using a genetic fate mapping strategy, VSMC of the vascular media give rise to the majority of the osteochondrogenic precursor- and chondrocyte-like cells observed in the calcified arterial media of MGP (- / -) mice. Osteogenic differentiation of VSMC is characterized by the expression of bone-related molecules including bone morphogenetic protein (BMP) -2, Msx2 and osteopontin, which are produced by osteoblasts and chondrocytes. Our recent findings are that (i) Runx2 and Notch1 induce osteogenic differentiation, and (ii) advanced glycation end-product (AGE) /receptor for AGE (RAGE) and palmitic acid promote osteogenic differentiation of VSMC. To understand of the molecular mechanisms of vascular calcification is now under intensive research area.

Osteogenic Response to BMP-2 of hMSCs Grown on Apatite-Coated Scaffolds

PubMed Central

Davis, Hillary E.; Case, Erin M.; Miller, Stephanie L.; Genetos, Damian C.; Leach, J. Kent

2011-01-01

Osteoconductive materials play a critical role in promoting integration with surrounding bone tissue and resultant bone repair in vivo. However, the impact of 3D osteoconductive substrates coupled with soluble signals on progenitor cell differentiation is not clear. In this study, we investigated the influence of bone morphogenetic protein-2 (BMP-2) concentration on the osteogenic differentiation of human mesenchymal stem cells (hMSCs) when seeded in carbonated apatite-coated polymer scaffolds. Mineralized scaffolds were more hydrophilic and adsorbed more BMP-2 compared to nonmineralized scaffolds. Changes in alkaline phosphatase (ALP) activity within stimulated hMSCs were dependent on the dose of BMP-2 and the scaffold composition. We detected more cell-secreted calcium on mineralized scaffolds at all time points, and higher BMP-2 concentrations resulted in increased ALP and calcium levels. RUNX2 and IBSP gene expression within hMSCs was affected by both substrate and soluble signals, SP7 by soluble factors, and SPARC by substrate-mediated cues. The present data indicate that a combination of apatite and BMP-2 do not simply enhance the osteogenic response of hMSCs, but act through multiple pathways that may be both substrate- and growth factor-mediated. Thus, multiple signaling strategies will likely be necessary to achieve optimal bone regeneration. PMID:21656707

Effect of lactoferrin on osteogenic differentiation of human adipose stem cells.

PubMed

Ying, Xiaozhou; Cheng, Shaowen; Wang, Wei; Lin, Zhongqin; Chen, Qingyu; Zhang, Wei; Kou, Dongquan; Shen, Yue; Cheng, Xiaojie; Peng, Lei; Zi Xu, Hua; Zhu Lu, Chuan

2012-03-01

Many in vitro studies of the analysis of the lactoferrin (LF) effect on cells have been reported. However, no study has yet investigated the effect of LF on osteogenic differentiation of human adipose-derived stem cells (hADSCs). The aim of this study was to evaluate the effect of LF on osteogenic differentiation of human adipose stem cells. The hADSCs were cultured in an osteogenic medium with 0, 10, 50 and 100 μg/ml LF, respectively. hADSC proliferation was analysed by Cell Counting Kit-8 (CCK-8) assay, and cell osteogenic differentiation was evaluated by alkaline phosphatase (ALP) activity assay, von Kossa staining and real-time polymerase chain reaction (RT-PCR). Cell proliferation was significantly increased by LF in a dose-dependent manner from days 4 to 14. Cells cultured with 100 μg/ml LF presented a higher activity compared with the control. The deposition of calcium was increased after the addition of LF. The mRNA expression of type I collagen (COL-I), ALP, osteocalcin (OCN) and RUNX2 increased markedly as a result of LF treatment. We have shown for the first time that LF could promote the proliferation and osteogenic differentiation of hADSCs, which could be a promising approach for enhancing osteogenic capacity of cell-based construction in bone tissue engineering.

Phelligridin D-loaded oral nanotube titanium implant enhances osseointegration and prevents osteolysis in rat mandible.

PubMed

Kim, Ji-Eun; Takanche, Jyoti Shrestha; Kim, Jeong-Seok; Lee, Min-Ho; Jeon, Jae-Gyu; Park, Il-Song; Yi, Ho-Keun

2018-04-12

Poor bone quality and osteolysis are the major causes of implant failure in dentistry. Here, this study tested the effect of phelligridin D-loaded nanotubes titanium (Ti) for bone formation around the dental implants. The purpose of this study was to enhance osseointegration of phelligridin D-loaded implant into the bone for bone formation and prevention of osteolysis. Cell viability, crystal violet staining, Western blot, alizarin red S staining, alkaline phosphatase activity, tartrate-resistant acid phosphatase staining, micro-computed tromography (μ-CT), hematoxylin and eosin (H&E) and immunohistochemical staining were used in vitro and in vivo to test the biocompatibility of phelligridin D. Phelligridin D enhanced osteoblast differentiation and mineralization by increasing bone morphogenic protein-2/7 (BMP-2/7), Osterix, Runx-2, osteoprotegerin (OPG), alkaline phosphatase and inhibited osteoclast differentiation by decreasing receptor activator of nuclear factor kappa-B ligand (RANKL) in MC-3T3 E1 cells. Further, phelligridin D promoted bone regeneration around nanotube Ti implant surface by increasing the levels of BMP-2/7 and OPG in a rat model. Phelligridin D also inhibited osteolysis by suppressing the expression of RANKL. These findings strongly suggest that phelligridin D is a new compound representing a potential therapeutic candidate for implant failure caused by osteolysis and poor bone quality of teeth.

Exclusion of MYF5, GSC, RUNX2, and TCOF1 mutation in a case of cerebro-costo-mandibular syndrome.

PubMed

Su, Pen-Hua; Chen, Jia-Yuh; Chiang, Chin-Lung; Ng, Yan-Yan; Chen, Suh-Jen

2010-04-01

Cerebro-costo-mandibular syndrome (CCMS) is an uncommon multiple congenital anomaly syndrome characterized by severe micrognathia, posterior rib-gap defects, and developmental delay. The cause of CCMS is unknown. Genes hypothesized to have a causal role in CCMS, include myogenic factor 5 (MYF5), goosecoid homeobox (GSC) and runt-related transcription factor 2 (RUNX2) [formerly known as core-binding factor (CBFA1)]. We report an infant with typical features of CCMS who, on prenatal ultrasound, was found to have severe micrognathia. We present the first image by three-dimensional computed tomography of posterior rib-defect, and we exclude mutations of the MYF5, GSC, RUNX2, and TCOF1 genes in our patient. Further molecular studies are needed to evaluate the cause of CCMS.

Molecular Genetic Studies of Bone Mechanical Strain and of Pedigrees with Very High Bone Density

DTIC Science & Technology

2007-11-01

regulating the expression of BSP. 17 0-25 Map3k4 Clcn7 Thbs2 Traf7 Tnf Notch3 Vegfa Runx2 Involved in normal skeletal patterning. Critical for...Chrs cM Genes 8 38–69 Ptger1 Junb Mt1, 2 Cdh11 Hsd11b2 Cdh1 Cbfb Hsd17b2 Il17c 16 30–46 Col8a1 EphA3 Pit1 17 0–25 Map3k4 Clcn7 Thbs2 Traf7 Tnf Notch3

Mechanisms of Reactive Stroma-Induced Tumorigenesis in Prostate Cancer

DTIC Science & Technology

2016-11-01

PREPARED FOR: U.S. Army Medical Research and Materiel Command Fort Detrick, Maryland 21702-5012 DISTRIBUTION STATEMENT: Approved for Public... Research and Materiel Command Fort Detrick, Maryland 21702-5012 11. SPONSOR/MONITOR’S REPORT NUMBER(S) 12. DISTRIBUTION / AVAILABILITY STATEMENT...and co-culture models. We have found that RUNX1 is critical mediator of TGF-beta action in mesenchymal stem /progenitor cells. RUNX1 is critical for

Zoledronate, ibandronate and clodronate enhance osteoblast differentiation in a dose dependent manner--a quantitative in vitro gene expression analysis of Dlx5, Runx2, OCN, MSX1 and MSX2.

PubMed

Koch, Felix Peter; Merkel, Christina; Al-Nawas, Bilal; Smeets, Ralf; Ziebart, Thomas; Walter, Christian; Wagner, Wilfried

2011-12-01

Bisphosphonates are widely used in the clinical treatment of bone diseases with increased bone resorption. In terms of side effects, they are known to be associated with osteonecrosis of the jaw (BONJ). There are two groups of bisphosphonates: the nitrogen-containing bisphosphonates, e.g. zoledronate and ibandronate, and the non-nitrogen-containing bisphosphonates, e.g. clodronate. Their impact on bone metabolism seems to differ. The objective of this study was to compare the osteogenic differentiation potency of these two pharmacologic groups. Human osteoblasts were stimulated with zoledronate and ibandronate at concentrations of 5×10(-5) M, 5×10(-6) M and 5×10(-7) M over the experimental periods of 1, 2, 5, 10 and 14 days. Clodronate was applied with concentrations of 5×10(-3), 5×10(-5) M and 5×10(-6) M. At each time point, the cells were dissolved, the mRNA extracted, and the gene expression level of the osteoblast specific differentiation markers of the homeobox transcription factors MSX1 and MSX2, the distal-less homeobox 5 (Dlx5), the Runt-related transcription factor 2 (Runx2/CBF1a) and osteocalcin (OCN) were quantified by Real-Time PCR. The gene expression was compared to an unstimulated osteoblast cell culture as control. The results showed a significant difference between the nitrogen-containing and the non-nitrogen-containing bisphosphonates. Zoledronate and ibandronate at concentrations of 5×10(-5) M enhanced the gene expression of all differentiation markers by several hundred folds compared to unstimulated control after 10 days, whereas clodronate had less influence on gene expression, even at higher concentrations of 5×10(-3) M. Lower concentrations of zoledronate and ibandronate, however, led to a decreased gene expression. These data confirm the results of other studies which have shown the osteogenic stimulus on osteoblasts in a dose dependent manner. The nitrogen-containing bisphosphonates appear to enhance bone density by stimulation of osteoblast differentiation. Non-nitrogen-containing bisphosphonates seem to have less influence on osteoblast differentiation. Copyright © 2010 European Association for Cranio-Maxillo-Facial Surgery. Published by Elsevier Ltd. All rights reserved.

Influence of Bone and Muscle Injuries on the Osteogenic Potential of Muscle Progenitors: Contribution of Tissue Environment to Heterotopic Ossification

PubMed Central

Molligan, Jeremy; Mitchell, Reed; Schon, Lew; Achilefu, Samuel; Zahoor, Talal; Cho, Young; Loube, Jeffery

2016-01-01

By using surgical mouse models, this study investigated how the tissue environment influences the osteogenic potential of muscle progenitors (m-progenitors) and potentially contributes to heterotopic ossification (HO). Injury was induced by clamping the gluteus maximus and medius (group M) or osteotomy of greater trochanter (group O) on the right hip, as well as combined muscle injury and osteotomy of greater trochanter (group M+O). The gluteus maximus and medius of the operated hips were harvested at days 1, 3, 5, and 10 for isolation of m-progenitors. The cells were cultured in an osteogenic medium for 3 weeks, and osteogenesis was evaluated by matrix mineralization and the expression of osteogenesis-related genes. The expression of type I collagen, RUNX2 (runt-related transcription factor 2), and osteocalcin by the m-progenitors of group M+O was significantly increased, compared with groups M and O. Osteogenic m-progenitors in group O increased the expression of bone morphogenetic protein 2 and also bone morphogenetic protein antagonist differential screening-selected gene aberrative in neuroblastoma. On histology, there was calcium deposition mostly in the muscles of group M+O harvested at day 10. CD56, representing myogenic progenitors, was highly expressed in the m-progenitors isolated from group M (day 10), but m-progenitors of group M+O (day 10) exhibited the highest expression of platelet-derived growth factor receptor α (PDGFR-α), a marker of muscle-derived mesenchymal stem cells (M-MSCs). The expressions of PDGFR-α and RUNX2 were colocalized in osteogenic m-progenitors. The data indicate that the tissue environment simulated in the M+O model is a favorable condition for HO formation. Most likely, M-MSCs, rather than myogenic progenitors, in the m-progenitors participate in HO formation. Significance The prevalence of traumatic heterotopic ossification (HO) is high in war injury. The pathogenesis of HO is still unknown. This study clarified the contribution of a tissue environment created by bone or muscle injury to the formation of HO. The study also found that muscle-derived mesenchymal stem cells, but not myogenic progenitors, are involved in the formation of HO. The findings of this study could be used to strategize the prevention and treatment of HO. PMID:27112178