Rps14 haploinsufficiency causes a block in erythroid differentiation mediated by S100A8/S100A9

PubMed Central

Schneider, Rebekka K.; Schenone, Monica; Ferreira, Monica Ventura; Kramann, Rafael; Joyce, Cailin E.; Hartigan, Christina; Beier, Fabian; Brümmendorf, Tim H.; Gehrming, Ulrich; Platzbecker, Uwe; Büsche, Guntram; Knüchel, Ruth; Chen, Michelle C.; Waters, Christopher S.; Chen, Edwin; Chu, Lisa P.; Novina, Carl D.; Lindsley, R. Coleman; Carr, Steven A.; Ebert, Benjamin L.

2016-01-01

Heterozygous deletion of RPS14 occurs in del(5q) MDS and has been linked to impaired erythropoiesis, characteristic of this disease subtype. We generated a murine model with conditional inactivation of Rps14 and demonstrated a p53-dependent erythroid differentiation defect with apoptosis at the transition from polychromatic to orthochromatic erythroblasts resulting in age-dependent progressive anemia, megakaryocyte dysplasia, and loss of hematopoietic stem cell (HSC) quiescence. Using quantitative proteomics, we identified significantly increased expression of proteins involved in innate immune signaling, particularly the heterodimeric S100a8/S100a9 proteins in purified erythroblasts. S100a8 expression was significantly increased in erythroblasts, monocytes and macrophages and recombinant S100a8 was sufficient to induce an erythroid differentiation defect in wild-type cells. We rescued the erythroid differentiation defect in Rps14 haploinsufficient HSCs by genetic inactivation of S100a8 expression. Our data link Rps14 haploinsufficiency to activation of the innate immune system via induction of S100A8/A9 and the p53-dependant erythroid differentiation defect in del(5q) MDS. PMID:26878232

The cAMP Response Element Binding protein (CREB) is activated by Insulin-like Growth Factor-1 (IGF-1) and regulates myostatin gene expression in skeletal myoblast

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

Zuloaga, R.; Fuentes, E.N.; Molina, A.

2013-10-18

Highlights: •IGF-1 induces the activation of CREB via IGF-1R/PI3K/PLC signaling pathway. •Calcium dependent signaling pathways regulate myostatin gene expression. •IGF-1 regulates myostatin gene expression via CREB transcription in skeletal myoblast. -- Abstract: Myostatin, a member of the Transforming Growth Factor beta (TGF-β) superfamily, plays an important role as a negative regulator of skeletal muscle growth and differentiation. We have previously reported that IGF-1 induces a transient myostatin mRNA expression, through the activation of the Nuclear Factor of Activated T cells (NFAT) in an IP{sub 3}/calcium-dependent manner. Here we examined the activation of CREB transcription factor as downstream targets of IGF-1more » during myoblast differentiation and its role as a regulator of myostatin gene expression. In cultured skeletal myoblast, IGF-1 induced the phosphorylation and transcriptional activation of CREB via IGF-1 Receptor/Phosphatidylinositol 3-Kinase (PI3K)/Phospholipase C gamma (PLC γ), signaling pathways. Also, IGF-1 induced calcium-dependent molecules such as Calmodulin Kinase II (CaMK II), Extracellular signal-regulated Kinases (ERK), Protein Kinase C (PKC). Additionally, we examined myostatin mRNA levels and myostatin promoter activity in differentiated myoblasts stimulated with IGF-1. We found a significant increase in mRNA contents of myostatin and its reporter activity after treatment with IGF-1. The expression of myostatin in differentiated myoblast was downregulated by the transfection of siRNA–CREB and by pharmacological inhibitors of the signaling pathways involved in CREB activation. By using pharmacological and genetic approaches together these data demonstrate that IGF-1 regulates the myostatin gene expression via CREB transcription factor during muscle cell differentiation.« less

Expression of POEM, a positive regulator of osteoblast differentiation, is suppressed by TNF-{alpha}

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

Tsukasaki, Masayuki; Yamada, Atsushi, E-mail: yamadaa@dent.showa-u.ac.jp; Suzuki, Dai

2011-07-15

Highlights: {yields} TNF-{alpha} inhibits POEM gene expression. {yields} Inhibition of POEM gene expression is caused by NF-{kappa}B activation by TNF-{alpha}. {yields} Over-expression of POEM recovers inhibition of osteoblast differentiation by TNF-{alpha}. -- Abstract: POEM, also known as nephronectin, is an extracellular matrix protein considered to be a positive regulator of osteoblast differentiation. In the present study, we found that tumor necrosis factor-{alpha} (TNF-{alpha}), a key regulator of bone matrix properties and composition that also inhibits terminal osteoblast differentiation, strongly inhibited POEM expression in the mouse osteoblastic cell line MC3T3-E1. TNF-{alpha}-induced down-regulation of POEM gene expression occurred in both time- andmore » dose-dependent manners through the nuclear factor kappa B (NF-{kappa}B) pathway. In addition, expressions of marker genes in differentiated osteoblasts were down-regulated by TNF-{alpha} in a manner consistent with our findings for POEM, while over-expression of POEM recovered TNF-{alpha}-induced inhibition of osteoblast differentiation. These results suggest that TNF-{alpha} inhibits POEM expression through the NF-{kappa}B signaling pathway and down-regulation of POEM influences the inhibition of osteoblast differentiation by TNF-{alpha}.« less

Tumor necrosis factor-alpha inhibits differentiation of myogenic cells in human urethral rhabdosphincter.

PubMed

Shinohara, Mayuka; Sumino, Yasuhiro; Sato, Fuminori; Kiyono, Tohru; Hashimoto, Naohiro; Mimata, Hiromitsu

2017-06-01

To examine the inhibitory effects of tumor necrosis factor-α on myogenic differentiation of human urethral rhabdosphincter cells. A rhabdosphincter sample was obtained from a patient who underwent total cystectomy. To expand the lifespan of the primary cultured cells, rhabdosphincter myogenic cells were immortalized with mutated cyclin-dependent kinase 4, cyclin D1 and telomerase. The differential potential of the cells was investigated. The transfected human rhabdosphincter cells were induced for myogenic differentiation with recombinant human tumor necrosis factor-α and/or the tumor necrosis factor-α antagonist etanercept at different concentrations, and activation of signaling pathways was monitored. Human rhabdosphincter cells were selectively cultured for at least 40 passages. Molecular analysis confirmed the expression of myosin heavy chain, which is a specific marker of differentiated muscle cells, significantly increased after differentiation induction. Although tumor necrosis factor-α treatment reduced the myosin heavy chain expression in a concentration-dependent manner, etanercept inhibited this suppression. Tumor necrosis factor-α suppressed phosphorylation of protein kinase B and p38, whereas etanercept pretreatment promoted phosphorylation and myosin heavy chain expression in a concentration-dependent manner. Tumor necrosis factor-α inhibits differentiation of urethral rhabdosphincter cells in part through the p38 mitogen-activated protein kinase and phosphoinositide 3-kinase pathways. Inhibition of tumor necrosis factor-α might be a useful strategy to treat stress urinary incontinence. © 2017 The Japanese Urological Association.

Comparative toxicogenomic analysis of oral Cr(VI) exposure effects in rat and mouse small intestinal epithelia

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

Kopec, Anna K.; Thompson, Chad M.; Kim, Suntae

2012-07-15

Continuous exposure to high concentrations of hexavalent chromium [Cr(VI)] in drinking water results in intestinal tumors in mice but not rats. Concentration-dependent gene expression effects were evaluated in female F344 rat duodenal and jejunal epithelia following 7 and 90 days of exposure to 0.3–520 mg/L (as sodium dichromate dihydrate, SDD) in drinking water. Whole-genome microarrays identified 3269 and 1815 duodenal, and 4557 and 1534 jejunal differentially expressed genes at 8 and 91 days, respectively, with significant overlaps between the intestinal segments. Functional annotation identified gene expression changes associated with oxidative stress, cell cycle, cell death, and immune response that weremore » consistent with reported changes in redox status and histopathology. Comparative analysis with B6C3F1 mouse data from a similarly designed study identified 2790 differentially expressed rat orthologs in the duodenum compared to 5013 mouse orthologs at day 8, and only 1504 rat and 3484 mouse orthologs at day 91. Automated dose–response modeling resulted in similar median EC{sub 50}s in the rodent duodenal and jejunal mucosae. Comparative examination of differentially expressed genes also identified divergently regulated orthologs. Comparable numbers of differentially expressed genes were observed at equivalent Cr concentrations (μg Cr/g duodenum). However, mice accumulated higher Cr levels than rats at ≥ 170 mg/L SDD, resulting in a ∼ 2-fold increase in the number of differentially expressed genes. These qualitative and quantitative differences in differential gene expression, which correlate with differences in tissue dose, likely contribute to the disparate intestinal tumor outcomes. -- Highlights: ► Cr(VI) elicits dose-dependent changes in gene expression in rat intestine. ► Cr(VI) elicits less differential gene expression in rats compared to mice. ► Cr(VI) gene expression can be phenotypically anchored to intestinal changes. ► Species-specific and divergent changes are consistent with species-specific tumors.« less

Age-dependent epigenetic control of differentiation inhibitors is critical for remyelination efficiency

PubMed Central

Shen, Siming; Sandoval, Juan; Swiss, Victoria A; Li, Jiadong; Dupree, Jeff; Franklin, Robin J M; Casaccia-Bonnefil, Patrizia

2009-01-01

The efficiency of remyelination decreases with age, but the molecular mechanisms responsible for this decline remain only partially understood. In this study, we show that remyelination is regulated by age-dependent epigenetic control of gene expression. In demyelinated young brains, new myelin synthesis is preceded by downregulation of oligodendrocyte differentiation inhibitors and neural stem cell markers, and this is associated with recruitment of histone deacetylases (HDACs) to promoter regions. In demyelinated old brains, HDAC recruitment is inefficient, and this allows the accumulation of transcriptional inhibitors and prevents the subsequent surge in myelin gene expression. Defective remyelination can be recapitulated in vivo in mice receiving systemic administration of pharmacological HDAC inhibitors during cuprizone treatment and is consistent with in vitro results showing defective differentiation of oligodendrocyte progenitors after silencing specific HDAC isoforms. Thus, we suggest that inefficient epigenetic modulation of the oligodendrocyte differentiation program contributes to the age-dependent decline in remyelination efficiency. PMID:19160500

Gene expression analysis of a porcine hepatocyte/bile duct in vitro differentiaion model

USDA-ARS?s Scientific Manuscript database

A serum-free, feeder-cell-dependent, inductive differentiation culture system of porcine hepatocytes and bile ductules was analyzed for differential gene expression on a porcine genome microarray. Primary cultures of baby pig hepatocytes (BPH) were matured in culture as a monolayer of hepatocytes w...

Reversing SKI-SMAD4-mediated suppression is essential for TH17 cell differentiation.

PubMed

Zhang, Song; Takaku, Motoki; Zou, Liyun; Gu, Ai-di; Chou, Wei-Chun; Zhang, Ge; Wu, Bing; Kong, Qing; Thomas, Seddon Y; Serody, Jonathan S; Chen, Xian; Xu, Xiaojiang; Wade, Paul A; Cook, Donald N; Ting, Jenny P Y; Wan, Yisong Y

2017-11-02

T helper 17 (T H 17) cells are critically involved in host defence, inflammation, and autoimmunity. Transforming growth factor β (TGFβ) is instrumental in T H 17 cell differentiation by cooperating with interleukin-6 (refs 6, 7). Yet, the mechanism by which TGFβ enables T H 17 cell differentiation remains elusive. Here we reveal that TGFβ enables T H 17 cell differentiation by reversing SKI-SMAD4-mediated suppression of the expression of the retinoic acid receptor (RAR)-related orphan receptor γt (RORγt). We found that, unlike wild-type T cells, SMAD4-deficient T cells differentiate into T H 17 cells in the absence of TGFβ signalling in a RORγt-dependent manner. Ectopic SMAD4 expression suppresses RORγt expression and T H 17 cell differentiation of SMAD4-deficient T cells. However, TGFβ neutralizes SMAD4-mediated suppression without affecting SMAD4 binding to the Rorc locus. Proteomic analysis revealed that SMAD4 interacts with SKI, a transcriptional repressor that is degraded upon TGFβ stimulation. SKI controls histone acetylation and deacetylation of the Rorc locus and T H 17 cell differentiation via SMAD4: ectopic SKI expression inhibits H3K9 acetylation of the Rorc locus, Rorc expression, and T H 17 cell differentiation in a SMAD4-dependent manner. Therefore, TGFβ-induced disruption of SKI reverses SKI-SMAD4-mediated suppression of RORγt to enable T H 17 cell differentiation. This study reveals a critical mechanism by which TGFβ controls T H 17 cell differentiation and uncovers the SKI-SMAD4 axis as a potential therapeutic target for treating T H 17-related diseases.

Lamin A/C Is Required for ChAT-Dependent Neuroblastoma Differentiation.

PubMed

Guglielmi, Loredana; Nardella, Marta; Musa, Carla; Iannetti, Ilaria; Arisi, Ivan; D'Onofrio, Mara; Storti, Andrea; Valentini, Alessandra; Cacci, Emanuele; Biagioni, Stefano; Augusti-Tocco, Gabriella; D'Agnano, Igea; Felsani, Armando

2017-07-01

The mouse neuroblastoma N18TG2 clone is unable to differentiate and is defective for the enzymes of the biosynthesis of neurotransmitters. The forced expression of choline acetyltransferase (ChAT) in these cells results in the synthesis and release of acetylcholine (Ach) and hence in the expression of neurospecific features and markers. To understand how the expression of ChAT triggered neuronal differentiation, we studied the differences in genome-wide transcription profiles between the N18TG2 parental cells and its ChAT-expressing 2/4 derived clone. The engagement of the 2/4 cells in the neuronal developmental program was confirmed by the increase of the expression level of several differentiation-related genes and by the reduction of the amount of transcripts of cell cycle genes. At the same time, we observed a massive reorganization of cytoskeletal proteins in terms of gene expression, with the accumulation of the nucleoskeletal lamina component Lamin A/C in differentiating cells. The increase of the Lmna transcripts induced by ChAT expression in 2/4 cells was mimicked treating the parental N18TG2 cells with the acetylcholine receptor agonist carbachol, thus demonstrating the direct role played by this receptor in neuron nuclei maturation. Conversely, a treatment of 2/4 cells with the muscarinic receptor antagonist atropine resulted in the reduction of the amount of Lmna RNA. Finally, the hypothesis that Lmna gene product might play a crucial role in the ChAT-dependent molecular differentiation cascade was strongly supported by Lmna knockdown in 2/4 cells leading to the downregulation of genes involved in differentiation and cytoskeleton formation and to the upregulation of genes known to regulate self-renewal and stemness.

In vitro differentiation of HT-29 M6 mucus-secreting colon cancer cells involves a trychostatin A and p27(KIP1)-inducible transcriptional program of gene expression.

PubMed

Mayo, Clara; Lloreta, Josep; Real, Francisco X; Mayol, Xavier

2007-07-01

Tumor cell dedifferentiation-such as the loss of cell-to-cell adhesion in epithelial tumors-is associated with tumor progression. To better understand the mechanisms that maintain carcinoma cells in a differentiated state, we have dissected in vitro differentiation pathways in the mucus-secretor HT-29 M6 colon cancer cell line, which spontaneously differentiates in postconfluent cultures. By lowering the extracellular calcium concentration to levels that prevent intercellular adhesion and epithelial polarization, our results reveal that differentiation is calcium-dependent and involves: (i) a process of cell cycle exit to G(0) and (ii) the induction of a transcriptional program of differentiation gene expression (i.e., mucins MUC1 and MUC5AC, and the apical membrane peptidase DPPIV). In calcium-deprived, non-differentiated postconfluent cultures, differentiation gene promoters are repressed by a trichostatin A (TSA)-sensitive mechanism, indicating that loss of gene expression by dedifferentiation is driven by histone deacetylases (HDAC). Since TSA treatment or extracellular calcium restoration allow gene promoter activation to similar levels, we suggest that induction of differentiation is one mechanism of HDAC inhibitor antitumor action. Moreover, transcriptional de-repression can also be induced in non-differentiating culture conditions by overexpressing the cyclin-dependent kinase inhibitor p27(KIP1), which is normally induced during spontaneous differentiation. Since p27(KIP1) downregulation in colon cancer is associated with poor prognosis independently of tumor cell division rates, we propose that p27 (KIP1) may prevent tumor progression by, at least in part, enhancing the expression of some differentiation genes. Therefore, the HT-29 M6 model allows the identification of some basic mechanisms of cancer cell differentiation control, so far revealing HDAC and p27(KIP1) as key regulatory factors of differentiation gene expression.

Activation of MRTF-A–dependent gene expression with a small molecule promotes myofibroblast differentiation and wound healing

PubMed Central

Velasquez, Lissette S.; Sutherland, Lillian B.; Liu, Zhenan; Grinnell, Frederick; Kamm, Kristine E.; Schneider, Jay W.; Olson, Eric N.; Small, Eric M.

2013-01-01

Myocardin-related transcription factors (MRTFs) regulate cellular contractility and motility by associating with serum response factor (SRF) and activating genes involved in cytoskeletal dynamics. We reported previously that MRTF-A contributes to pathological cardiac remodeling by promoting differentiation of fibroblasts to myofibroblasts following myocardial infarction. Here, we show that forced expression of MRTF-A in dermal fibroblasts stimulates contraction of a collagen matrix, whereas contractility of MRTF-A null fibroblasts is impaired under basal conditions and in response to TGF–β1 stimulation. We also identify an isoxazole ring-containing small molecule, previously shown to induce smooth muscle α-actin gene expression in cardiac progenitor cells, as an agonist of myofibroblast differentiation. Isoxazole stimulates myofibroblast differentiation via induction of MRTF-A–dependent gene expression. The MRTF-SRF signaling axis is activated in response to skin injury, and treatment of dermal wounds with isoxazole accelerates wound closure and suppresses the inflammatory response. These results reveal an important role for MRTF-SRF signaling in dermal myofibroblast differentiation and wound healing and suggest that targeting MRTFs pharmacologically may prove useful in treating diseases associated with inappropriate myofibroblast activity. PMID:24082095

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

He, Yonghan; Aquatic and Crop Resource Development, Life Sciences Branch, National Research Council Canada, Charlottetown, PE, Canada C1A 4P3; State Key Laboratory of Genetic Resources and Evolution, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming 650223

Highlights: •Radicicol suppressed intracellular fat accumulation in 3T3-L1 adipocytes. •Radicicol inhibited the expression of FAS and FABP4. •Radicicol blocked cell cycle at the G1-S phase during cell differentiation. •Radicicol inhibited the PDK1/Akt pathway in adipocyte differentiation. -- Abstract: Heat shock protein 90 (Hsp90) is involved in various cellular processes, such as cell proliferation, differentiation and apoptosis. As adipocyte differentiation plays a critical role in obesity development, the present study investigated the effect of an Hsp90 inhibitor radicicol on the differentiation of 3T3-L1 preadipocytes and potential mechanisms. The cells were treated with different concentrations of radicicol during the first 8 daysmore » of cell differentiation. Adipogenesis, the expression of adipogenic transcriptional factors, differentiation makers and cell cycle were determined. It was found that radicicol dose-dependently decreased intracellular fat accumulation through down-regulating the expression of peroxisome proliferator-activated receptor γ (PPAR{sub γ}) and CCAAT element binding protein α (C/EBP{sub α}), fatty acid synthase (FAS) and fatty acid-binding protein 4 (FABP4). Flow cytometry analysis revealed that radicicol blocked cell cycle at G1-S phase. Radicicol redcued the phosphorylation of Akt while showing no effect on β-catenin expression. Radicicol decreased the phosphorylation of phosphoinositide-dependent kinase 1 (PDK1). The results suggest that radicicol inhibited 3T3-L1 preadipocyte differentiation through affecting the PDK1/Akt pathway and subsequent inhibition of mitotic clonal expansion and the expression/activity of adipogenic transcriptional factors and their downstream adipogenic proteins.« less

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

Zhang, Long; Shi, Songting; Zhang, Juan

Highlights: Black-Right-Pointing-Pointer Expression of Id3 but not Id1 is induced by Wnt3a stimulation in C2C12 cells. Black-Right-Pointing-Pointer Wnt3a induces Id3 expression via canonical Wnt/{beta}-catenin pathway. Black-Right-Pointing-Pointer Wnt3a-induced Id3 expression does not depend on BMP signaling activation. Black-Right-Pointing-Pointer Induction of Id3 expression is critical determinant in Wnt3a-induced cell proliferation and differentiation. -- Abstract: Canonical Wnt signaling plays important roles in regulating cell proliferation and differentiation. In this study, we report that inhibitor of differentiation (Id)3 is a Wnt-inducible gene in mouse C2C12 myoblasts. Wnt3a induced Id3 expression in a {beta}-catenin-dependent manner. Bone morphogenetic protein (BMP) also potently induced Id3 expression. However,more » Wnt-induced Id3 expression occurred independent of the BMP/Smad pathway. Functional studies showed that Id3 depletion in C2C12 cells impaired Wnt3a-induced cell proliferation and alkaline phosphatase activity, an early marker of osteoblast cells. Id3 depletion elevated myogenin induction during myogenic differentiation and partially impaired Wnt3a suppressed myogenin expression in C2C12 cells. These results suggest that Id3 is an important Wnt/{beta}-catenin induced gene in myoblast cell fate determination.« less

The expression of VE-cadherin in breast cancer cells modulates cell dynamics as a function of tumor differentiation and promotes tumor-endothelial cell interactions.

PubMed

Rezaei, Maryam; Cao, Jiahui; Friedrich, Katrin; Kemper, Björn; Brendel, Oliver; Grosser, Marianne; Adrian, Manuela; Baretton, Gustavo; Breier, Georg; Schnittler, Hans-Joachim

2018-01-01

The cadherin switch has profound consequences on cancer invasion and metastasis. The endothelial-specific vascular endothelial cadherin (VE-cadherin) has been demonstrated in diverse cancer types including breast cancer and is supposed to modulate tumor progression and metastasis, but underlying mechanisms need to be better understood. First, we evaluated VE-cadherin expression by tissue microarray in 392 cases of breast cancer tumors and found a diverse expression and distribution of VE-cadherin. Experimental expression of fluorescence-tagged VE-cadherin (VE-EGFP) in undifferentiated, fibroblastoid and E-cadherin-negative MDA-231 (MDA-VE-EGFP) as well as in differentiated E-cadherin-positive MCF-7 human breast cancer cell lines (MCF-VE-EGFP), respectively, displayed differentiation-dependent functional differences. VE-EGFP expression reversed the fibroblastoid MDA-231 cells to an epithelial-like phenotype accompanied by increased β-catenin expression, actin and vimentin remodeling, increased cell spreading and barrier function and a reduced migration ability due to formation of VE-cadherin-mediated cell junctions. The effects were largely absent in both MDA-VE-EGFP and in control MCF-EGFP cell lines. However, MCF-7 cells displayed a VE-cadherin-independent planar cell polarity and directed cell migration that both developed in MDA-231 only after VE-EGFP expression. Furthermore, VE-cadherin expression had no effect on tumor cell proliferation in monocultures while co-culturing with endothelial cells enhanced tumor cell proliferation due to integration of the tumor cells into monolayer where they form VE-cadherin-mediated cell contacts with the endothelium. We propose an interactive VE-cadherin-based crosstalk that might activate proliferation-promoting signals. Together, our study shows a VE-cadherin-mediated cell dynamics and an endothelial-dependent proliferation in a differentiation-dependent manner.

Regulation of collagenase-3 and osteocalcin gene expression by collagen and osteopontin in differentiating MC3T3-E1 cells

NASA Technical Reports Server (NTRS)

D'Alonzo, Richard C.; Kowalski, Aaron J.; Denhardt, David T.; Nickols, G. Allen; Partridge, Nicola C.

2002-01-01

Both collagenase-3 and osteocalcin mRNAs are expressed maximally during the later stages of osteoblast differentiation. Here, we demonstrate that collagenase-3 mRNA expression in differentiating MC3T3-E1 cells is dependent upon the presence of ascorbic acid, is inhibited in the presence of the collagen synthesis inhibitor, 3,4-dehydroproline, and is stimulated by growth on collagen in the absence of ascorbic acid. Transient transfection studies show that collagenase-3 promoter activity increases during cell differentiation and requires the presence of ascorbic acid. Additionally, we show that, in differentiating MC3T3-E1 cells, collagenase-3 gene expression increases in the presence of an anti-osteopontin monoclonal antibody that binds near the RGD motif of this protein, whereas osteocalcin expression is inhibited. Furthermore, an RGD peptidomimetic compound, designed to block interaction of ligands to the alpha(v) integrin subunit, increases osteocalcin expression and inhibits collagenase-3 expression, suggesting that the RGD peptidomimetic initiates certain alpha(v) integrin signaling in osteoblastic cells. Overall, these studies demonstrate that stimulation of collagenase-3 expression during osteoblast differentiation requires synthesis of a collagenous matrix and that osteopontin and alpha(v) integrins exert divergent regulation of collagenase-3 and osteocalcin expression during osteoblast differentiation.

Functional studies of TcRjl, a novel GTPase of Trypanosoma cruzi, reveals phenotypes related with MAPK activation during parasite differentiation and after heterologous expression in Drosophila model system

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

Reis Monteiro dos-Santos, Guilherme Rodrigo; Fontenele, Marcio Ribeiro; Dias, Felipe de Almeida

The life cycle of the protozoan parasite Trypanosoma cruzi comprises rounds of proliferative cycles and differentiation in distinct host environments. Ras GTPases are molecular switches that play pivotal regulatory functions in cell fate. Rjl is a novel GTPase with unknown function. Herein we show that TcRjl blocks in vivo cell differentiation. The forced expression of TcRjl leads to changes in the overall tyrosine protein phosphorylation profile of parasites. TcRjl expressing parasites sustained DNA synthesis regardless the external stimuli for differentiation. Heterologous expression in the Drosophila melanogaster genetic system strongly suggests a role from TcRjl protein in RTK-dependent pathways and MAPK activation.

Purification and differentiation of human adipose-derived stem cells by membrane filtration and membrane migration methods

PubMed Central

Lin, Hong Reng; Heish, Chao-Wen; Liu, Cheng-Hui; Muduli, Saradaprasan; Li, Hsing-Fen; Higuchi, Akon; Kumar, S. Suresh; Alarfaj, Abdullah A.; Munusamy, Murugan A.; Hsu, Shih-Tien; Chen, Da-Chung; Benelli, Giovanni; Murugan, Kadarkarai; Cheng, Nai-Chen; Wang, Han-Chow; Wu, Gwo-Jang

2017-01-01

Human adipose-derived stem cells (hADSCs) are easily isolated from fat tissue without ethical concerns, but differ in purity, pluripotency, differentiation ability, and stem cell marker expression, depending on the isolation method. We isolated hADSCs from a primary fat tissue solution using: (1) conventional culture, (2) a membrane filtration method, (3) a membrane migration method where the primary cell solution was permeated through membranes, adhered hADSCs were cultured, and hADSCs migrated out from the membranes. Expression of mesenchymal stem cell markers and pluripotency genes, and osteogenic differentiation were compared for hADSCs isolated by different methods using nylon mesh filter membranes with pore sizes ranging from 11 to 80 μm. hADSCs isolated by the membrane migration method had the highest MSC surface marker expression and efficient differentiation into osteoblasts. Osteogenic differentiation ability of hADSCs and MSC surface marker expression were correlated, but osteogenic differentiation ability and pluripotent gene expression were not. PMID:28071738

Different profiles of neuroendocrine cell differentiation evolve in the PC-310 human prostate cancer model during long-term androgen deprivation.

PubMed

Jongsma, Johan; Oomen, Monique H; Noordzij, Marinus A; Van Weerden, Wytske M; Martens, Gerard J M; van der Kwast, Theodorus H; Schröder, Fritz H; van Steenbrugge, Gert J

2002-03-01

Neuroendocrine (NE) cells are androgen-independent cells and secrete growth-modulating peptide hormones via a regulated secretory pathway (RSP). We studied NE differentiation after long-term androgen withdrawal in the androgen-dependent human prostate cancer xenograft PC-310. Tumor-bearing nude mice were killed at 0, 2, 5, 7, 14, 21, 47, 84, and 154 days after castration. The half-life of the PC-310 tumor was 10 days, with a stable residual tumor volume of 30--40% after 21 days and longer periods of androgen deprivation. Proliferative activity and prostate-specific antigen serum levels decreased to zero after castration, whereas cell-cycle arrest was manifested by increased p27(kip1) expression. A temporary downregulation of androgen receptor (AR) expression was noted after androgen deprivation. The expression of chromogranin A, secretogranin III, and secretogranin V (7B2) increased 5 days after castration and later. Subsequently, pro-hormone convertase 1 and peptidyl alpha--amidating monooxygenase as well as vascular endothelial growth factor were expressed from 7 days after castration on. Finally, such growth factors as gastrin-releasing peptide and serotonin were expressed in a small part of the NE cells 21 days after castration, but strong expression was induced late during androgen deprivation, that is, 84 and 154 days after castration, respectively. Androgen deprivation of the NE-differentiated PC-310 model induced the formation of NE-differentiated AR(minus sign) and non-NE AR(+) tumor residues. The NE-differentiated cells actively produced growth factors via an RSP that may lead to hormone-refractory disease. The dormant non-NE AR(+) tumor cells were shown to remain androgen sensitive even after long-term androgen deprivation. In the PC-310 xenograft, time-dependent NE differentiation and subsequent maturation were induced after androgen depletion. The androgen-dependent PC-310 xenograft model constitutes an excellent model for studying the role of NE cells in the progression of clinical prostate cancer. Copyright 2002 Wiley-Liss, Inc.

MERP1: a mammalian ependymin-related protein gene differentially expressed in hematopoietic cells.

PubMed

Gregorio-King, Claudia C; McLeod, Janet L; Collier, Fiona McL; Collier, Gregory R; Bolton, Karyn A; Van Der Meer, Gavin J; Apostolopoulos, Jim; Kirkland, Mark A

2002-03-20

We have utilized differential display polymerase chain reaction to investigate the gene expression of hematopoietic progenitor cells from adult bone marrow and umbilical cord blood. A differentially expressed gene was identified in CD34+ hematopoietic progenitor cells, with low expression in CD34- cells. We have obtained the full coding sequence of this gene which we designated human mammalian ependymin-related protein 1 (MERP1). Expression of MERP1 was found in a variety of normal human tissues, and is 4- and 10-fold higher in adult bone marrow and umbilical cord blood CD34+ cells, respectively, compared to CD34- cells. Additionally, MERP1 expression in a hematopoietic stem cell enriched population was down-regulated with proliferation and differentiation. Conceptual translation of the MERP1 open reading frame reveals significant homology to two families of glycoprotein calcium-dependant cell adhesion molecules: ependymins and protocadherins.

MiR-132 regulates osteogenic differentiation via downregulating Sirtuin1 in a peroxisome proliferator-activated receptor β/δ–dependent manner

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

Gong, Kai; Qu, Bo; Liao, Dongfa

MicroRNAs (miRNAs) play significant roles in multiple diseases by regulating the expression of their target genes. Type 2 diabetes mellitus (T2DM) is a chronic endocrine and metabolic disease with complex mechanisms. T2DM can result in diabetic osteoporosis (DO), which is characterized by bone loss, decreased bone mineral density and increased bone fractures. The promotion of osteogenic differentiation of osteoblasts is an effective way to treat osteoporosis. In the present study, high glucose (HG) and free fatty acids (FFA) were employed to mimic T2DM in MC3T3-E1 cells. To induce osteogenic differentiation, MC3T3-E1 cells were cultured in osteogenic medium. The results showedmore » that osteogenic differentiation was significantly suppressed by HG and FFA. We found that miR-132 expression was significantly upregulated and much higher in HG-FFA–induced cells than other selected miRNAs, indicating that miR-132 might play an important role in DO. Furthermore, overexpression of miR-132 markedly inhibited the expression of key markers of osteogenic differentiation and alkaline phosphatase (ALP) activity. Reciprocally, inhibition of miR-132 restored osteogenic differentiation, even under treatment with HG-FFA. We also showed that Sirtuin 1 (Sirt1) was one of the target genes of miR-132, whose expression was controlled by miR-132. Ectopic expression of Sirt1 reversed the decrease in osteogenic differentiation caused by miR-132 and HG-FFA. These results demonstrated the direct role of miR-132 in suppressing osteogenic differentiation through downregulating Sirt1. Moreover, we demonstrated that peroxisome proliferator-activated receptor β/δ (PPARβ/δ) was a downstream molecule of Sirt1, and its knockout by PPARβ/δ siRNA significantly abolished the promotive effects of Sirt1 on osteogenic differentiation, indicating that Sirt1 functioned in a PPARβ/δ–dependent manner. Taken together, we provide crucial evidence that miR-132 plays a key role in regulating osteogenic differentiation through Sirt1 in a PPARβ/δ–dependent manner, indicating that miR-132 and Sirt1-PPARβ/δ may act as potential therapeutic targets for T2DM–induced osteoporosis. - Highlights: • MiR-132 participates in regulating osteogenic differentiation of MC3T3-E1 cells. • Sirt1 is a target gene of miR-132. • Sirt1 is the effector of miR-132 in regulating osteogenic differentiation. • MiR-132-Sirt1 regulates osteogenic differentiation in a PPARβ/δ–dependent manner.« less

Identification of transcription coactivator OCA-B-dependent genes involved in antigen-dependent B cell differentiation by cDNA array analyses.

PubMed

Kim, Unkyu; Siegel, Rachael; Ren, Xiaodi; Gunther, Cary S; Gaasterland, Terry; Roeder, Robert G

2003-07-22

The tissue-specific transcriptional coactivator OCA-B is required for antigen-dependent B cell differentiation events, including germinal center formation. However, the identity of OCA-B target genes involved in this process is unknown. This study has used large-scale cDNA arrays to monitor changes in gene expression patterns that accompany mature B cell differentiation. B cell receptor ligation alone induces many genes involved in B cell expansion, whereas B cell receptor and helper T cell costimulation induce genes associated with B cell effector function. OCA-B expression is induced by both B cell receptor ligation alone and helper T cell costimulation, suggesting that OCA-B is involved in B cell expansion as well as B cell function. Accordingly, several genes involved in cell proliferation and signaling, such as Lck, Kcnn4, Cdc37, cyclin D3, B4galt1, and Ms4a11, have been identified as OCA-B-dependent genes. Further studies on the roles played by these genes in B cells will contribute to an understanding of B cell differentiation.

Cell Aggregation-induced FGF8 Elevation Is Essential for P19 Cell Neural Differentiation

PubMed Central

Wang, Chen; Xia, Caihong; Bian, Wei; Liu, Li; Lin, Wei; Chen, Ye-Guang; Ang, Siew-Lan

2006-01-01

FGF8, a member of the fibroblast growth factor (FGF) family, has been shown to play important roles in different developing systems. Mouse embryonic carcinoma P19 cells could be induced by retinoic acid (RA) to differentiate into neuroectodermal cell lineages, and this process is cell aggregation dependent. In this report, we show that FGF8 expression is transiently up-regulated upon P19 cell aggregation, and the aggregation-dependent FGF8 elevation is pluripotent stem cell related. Overexpressing FGF8 promotes RA-induced monolayer P19 cell neural differentiation. Inhibition of FGF8 expression by RNA interference or blocking FGF signaling by the FGF receptor inhibitor, SU5402, attenuates neural differentiation of the P19 cell. Blocking the bone morphogenetic protein (BMP) pathway by overexpressing Smad6 in P19 cells, we also show that FGF signaling plays a BMP inhibition–independent role in P19 cell neural differentiation. PMID:16641368

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

Shim, Ki Shuk; Department of Neonatology, Medical University of Vienna, Waehringer Guertel 18-20, A-1090 Vienna; Rosner, Margit

Bach1 and Bach2 are evolutionarily related members of the BTB-basic region leucine zipper transcription factor family. We found that Bach2 downregulates cell proliferation of N1E-115 cells and negatively affects their potential to differentiate. Nuclear localization of the cyclin-dependent kinase inhibitor p21 is known to arrest cell cycle progression, and cytoplasmic p21 has been shown to promote neuronal differentiation of N1E-115 cells. We found that ectopic Bach2 causes upregulation of p21 expression in the nucleus and in the cytoplasm in undifferentiated N1E-115 cells. In differentiated cells, Bach2 specifically triggers upregulation of cytoplasmic p21. Our data suggest that Bach2 expression could representmore » a switch during the process of neuronal differentiation. Bach2 is not expressed in neuronal precursor cells. It would have negative effects on proliferation and differentiation of these cells. In differentiated neuronal cells Bach2 expression is upregulated, which could allow Bach2 to function as a gatekeeper of the differentiated status.« 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.

Scoparone attenuates RANKL-induced osteoclastic differentiation through controlling reactive oxygen species production and scavenging

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

Lee, Sang-Hyun; Jang, Hae-Dong, E-mail: haedong@hnu.kr

Scoparone, one of the bioactive components of Artemisia capillaris Thunb, has various biological properties including immunosuppressive, hepatoprotective, anti-allergic, anti-inflammatory, and antioxidant effects. This study aims at evaluating the anti-osteoporotic effect of scoparone and its underlying mechanism in vitro. Scoparone demonstrated potent cellular antioxidant capacity. It was also found that scoparone inhibited the receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast differentiation and suppressed cathepsin K and tartrate-resistant acid phosphatase (TRAP) expression via c-jun N-terminal kinase (JNK)/extracellular signal-regulated kinase (ERK)/p38-mediated c-Fos–nuclear factor of activated T cells, cytoplasmic 1 (NFATc1) signaling pathway. During osteoclast differentiation, the production of general reactive oxygen speciesmore » (ROS) and superoxide anions was dose-dependently attenuated by scoparone. In addition, scoparone diminished NADPH (nicotinamide adenine dinucleotide phosphate) oxidase 1 (Nox1) expression and activation via the tumor necrosis factor receptor-associated factor 6 (TRAF6)–cSrc–phosphatidylinositol 3-kinase (PI3k) signaling pathway and prevented the disruption of mitochondrial electron transport chain system. Furthermore, scoparone augmented the expression of superoxide dismutase 1 (SOD1) and catalase (CAT). The overall results indicate that the inhibitory effect of scoparone on RANKL-induced osteoclast differentiation is attributed to the suppressive effect on ROS and superoxide anion production by inhibiting Nox1 expression and activation and protecting the mitochondrial electron transport chain system and the scavenging effect of ROS resulting from elevated SOD1 and CAT expression. - Highlights: • Scoparone dose-dependently inhibited RANKL-induced osteoclast differentiation. • Scoparone diminished general ROS and superoxide anions in a dose-dependent manner. • Scoparone inhibited Nox1 expression and activation. • Scoparone prevented the disruption of mitochondrial electron transport chain system. • Scoparone augmented superoxide dismutase and catalase expression.« less

Differential Gene Expression in Liver, Gill, and Olfactory Rosettes of Coho Salmon (Oncorhynchus kisutch) After Acclimation to Salinity.

PubMed

Maryoung, Lindley A; Lavado, Ramon; Bammler, Theo K; Gallagher, Evan P; Stapleton, Patricia L; Beyer, Richard P; Farin, Federico M; Hardiman, Gary; Schlenk, Daniel

2015-12-01

Most Pacific salmonids undergo smoltification and transition from freshwater to saltwater, making various adjustments in metabolism, catabolism, osmotic, and ion regulation. The molecular mechanisms underlying this transition are largely unknown. In the present study, we acclimated coho salmon (Oncorhynchus kisutch) to four different salinities and assessed gene expression through microarray analysis of gills, liver, and olfactory rosettes. Gills are involved in osmotic regulation, liver plays a role in energetics, and olfactory rosettes are involved in behavior. Between all salinity treatments, liver had the highest number of differentially expressed genes at 1616, gills had 1074, and olfactory rosettes had 924, using a 1.5-fold cutoff and a false discovery rate of 0.5. Higher responsiveness of liver to metabolic changes after salinity acclimation to provide energy for other osmoregulatory tissues such as the gills may explain the differences in number of differentially expressed genes. Differentially expressed genes were tissue- and salinity-dependent. There were no known genes differentially expressed that were common to all salinity treatments and all tissues. Gene ontology term analysis revealed biological processes, molecular functions, and cellular components that were significantly affected by salinity, a majority of which were tissue-dependent. For liver, oxygen binding and transport terms were highlighted. For gills, muscle, and cytoskeleton-related terms predominated and for olfactory rosettes, immune response-related genes were accentuated. Interaction networks were examined in combination with GO terms and determined similarities between tissues for potential osmosensors, signal transduction cascades, and transcription factors.

The zinc sensing receptor, ZnR/GPR39, controls proliferation and differentiation of colonocytes and thereby tight junction formation in the colon

PubMed Central

Cohen, L; Sekler, I; Hershfinkel, M

2014-01-01

The intestinal epithelium is a renewable tissue that requires precise balance between proliferation and differentiation, an essential process for the formation of a tightly sealed barrier. Zinc deficiency impairs the integrity of the intestinal epithelial barrier and is associated with ulcerative and diarrheal pathologies, but the mechanisms underlying the role of Zn2+ are not well understood. Here, we determined a role of the colonocytic Zn2+ sensing receptor, ZnR/GPR39, in mediating Zn2+-dependent signaling and regulating the proliferation and differentiation of colonocytes. Silencing of ZnR/GPR39 expression attenuated Zn2+-dependent activation of ERK1/2 and AKT as well as downstream activation of mTOR/p70S6K, pathways that are linked with proliferation. Consistently, ZnR/GPR39 silencing inhibited HT29 and Caco-2 colonocyte proliferation, while not inducing caspase-3 cleavage. Remarkably, in differentiating HT29 colonocytes, silencing of ZnR/GPR39 expression inhibited alkaline phosphatase activity, a marker of differentiation. Furthermore, Caco-2 colonocytes showed elevated expression of ZnR/GPR39 during differentiation, whereas silencing of ZnR/GPR39 decreased monolayer transepithelial electrical resistance, suggesting compromised barrier formation. Indeed, silencing of ZnR/GPR39 or chelation of Zn2+ by the cell impermeable chelator CaEDTA was followed by impaired expression of the junctional proteins, that is, occludin, zonula-1 (ZO-1) and E-cadherin. Importantly, colon tissues of GPR39 knockout mice also showed a decrease in expression levels of ZO-1 and occludin compared with wildtype mice. Altogether, our results indicate that ZnR/GPR39 has a dual role in promoting proliferation of colonocytes and in controlling their differentiation. The latter is followed by ZnR/GPR39-dependent expression of tight junctional proteins, thereby leading to formation of a sealed intestinal epithelial barrier. Thus, ZnR/GPR39 may be a therapeutic target for promoting epithelial function and tight junction barrier integrity during ulcerative colon diseases. PMID:24967969

The zinc sensing receptor, ZnR/GPR39, controls proliferation and differentiation of colonocytes and thereby tight junction formation in the colon.

PubMed

Cohen, L; Sekler, I; Hershfinkel, M

2014-06-26

The intestinal epithelium is a renewable tissue that requires precise balance between proliferation and differentiation, an essential process for the formation of a tightly sealed barrier. Zinc deficiency impairs the integrity of the intestinal epithelial barrier and is associated with ulcerative and diarrheal pathologies, but the mechanisms underlying the role of Zn(2+) are not well understood. Here, we determined a role of the colonocytic Zn(2+) sensing receptor, ZnR/GPR39, in mediating Zn(2+)-dependent signaling and regulating the proliferation and differentiation of colonocytes. Silencing of ZnR/GPR39 expression attenuated Zn(2+)-dependent activation of ERK1/2 and AKT as well as downstream activation of mTOR/p70S6K, pathways that are linked with proliferation. Consistently, ZnR/GPR39 silencing inhibited HT29 and Caco-2 colonocyte proliferation, while not inducing caspase-3 cleavage. Remarkably, in differentiating HT29 colonocytes, silencing of ZnR/GPR39 expression inhibited alkaline phosphatase activity, a marker of differentiation. Furthermore, Caco-2 colonocytes showed elevated expression of ZnR/GPR39 during differentiation, whereas silencing of ZnR/GPR39 decreased monolayer transepithelial electrical resistance, suggesting compromised barrier formation. Indeed, silencing of ZnR/GPR39 or chelation of Zn(2+) by the cell impermeable chelator CaEDTA was followed by impaired expression of the junctional proteins, that is, occludin, zonula-1 (ZO-1) and E-cadherin. Importantly, colon tissues of GPR39 knockout mice also showed a decrease in expression levels of ZO-1 and occludin compared with wildtype mice. Altogether, our results indicate that ZnR/GPR39 has a dual role in promoting proliferation of colonocytes and in controlling their differentiation. The latter is followed by ZnR/GPR39-dependent expression of tight junctional proteins, thereby leading to formation of a sealed intestinal epithelial barrier. Thus, ZnR/GPR39 may be a therapeutic target for promoting epithelial function and tight junction barrier integrity during ulcerative colon diseases.

Serum from patients with SLE instructs monocytes to promote IgG and IgA plasmablast differentiation

PubMed Central

Joo, HyeMee; Coquery, Christine; Xue, Yaming; Gayet, Ingrid; Dillon, Stacey R.; Punaro, Marilynn; Zurawski, Gerard; Banchereau, Jacques; Pascual, Virginia

2012-01-01

The development of autoantibodies is a hallmark of systemic lupus erythematosus (SLE). SLE serum can induce monocyte differentiation into dendritic cells (DCs) in a type I IFN–dependent manner. Such SLE-DCs activate T cells, but whether they promote B cell responses is not known. In this study, we demonstrate that SLE-DCs can efficiently stimulate naive and memory B cells to differentiate into IgG- and IgA-plasmablasts (PBs) resembling those found in the blood of SLE patients. SLE-DC–mediated IgG-PB differentiation is dependent on B cell–activating factor (BAFF) and IL-10, whereas IgA-PB differentiation is dependent on a proliferation-inducing ligand (APRIL). Importantly, SLE-DCs express CD138 and trans-present CD138-bound APRIL to B cells, leading to the induction of IgA switching and PB differentiation in an IFN-α–independent manner. We further found that this mechanism of providing B cell help is relevant in vivo, as CD138-bound APRIL is expressed on blood monocytes from active SLE patients. Collectively, our study suggests that a direct myeloid DC–B cell interplay might contribute to the pathogenesis of SLE. PMID:22689824

Identification of embryonic precursor cells that differentiate into thymic epithelial cells expressing autoimmune regulator

PubMed Central

Takizawa, Nobukazu; Miyauchi, Maki; Yanai, Hiromi; Tateishi, Ryosuke; Shinzawa, Miho; Yoshinaga, Riko; Kurihara, Masaaki; Yasuda, Hisataka; Sakamoto, Reiko; Yoshida, Nobuaki

2016-01-01

Medullary thymic epithelial cells (mTECs) expressing autoimmune regulator (Aire) are critical for preventing the onset of autoimmunity. However, the differentiation program of Aire-expressing mTECs (Aire+ mTECs) is unclear. Here, we describe novel embryonic precursors of Aire+ mTECs. We found the candidate precursors of Aire+ mTECs (pMECs) by monitoring the expression of receptor activator of nuclear factor-κB (RANK), which is required for Aire+ mTEC differentiation. pMECs unexpectedly expressed cortical TEC molecules in addition to the mTEC markers UEA-1 ligand and RANK and differentiated into mTECs in reaggregation thymic organ culture. Introduction of pMECs in the embryonic thymus permitted long-term maintenance of Aire+ mTECs and efficiently suppressed the onset of autoimmunity induced by Aire+ mTEC deficiency. Mechanistically, pMECs differentiated into Aire+ mTECs by tumor necrosis factor receptor-associated factor 6-dependent RANK signaling. Moreover, nonclassical nuclear factor-κB activation triggered by RANK and lymphotoxin-β receptor signaling promoted pMEC induction from progenitors exhibiting lower RANK expression and higher CD24 expression. Thus, our findings identified two novel stages in the differentiation program of Aire+ mTECs. PMID:27401343

A subset of replication-dependent histone mRNAs are expressed as polyadenylated RNAs in terminally differentiated tissues.

PubMed

Lyons, Shawn M; Cunningham, Clark H; Welch, Joshua D; Groh, Beezly; Guo, Andrew Y; Wei, Bruce; Whitfield, Michael L; Xiong, Yue; Marzluff, William F

2016-11-02

Histone proteins are synthesized in large amounts during S-phase to package the newly replicated DNA, and are among the most stable proteins in the cell. The replication-dependent (RD)-histone mRNAs expressed during S-phase end in a conserved stem-loop rather than a polyA tail. In addition, there are replication-independent (RI)-histone genes that encode histone variants as polyadenylated mRNAs. Most variants have specific functions in chromatin, but H3.3 also serves as a replacement histone for damaged histones in long-lived terminally differentiated cells. There are no reported replacement histone genes for histones H2A, H2B or H4. We report that a subset of RD-histone genes are expressed in terminally differentiated tissues as polyadenylated mRNAs, likely serving as replacement histone genes in long-lived non-dividing cells. Expression of two genes, HIST2H2AA3 and HIST1H2BC, is conserved in mammals. They are expressed as polyadenylated mRNAs in fibroblasts differentiated in vitro, but not in serum starved fibroblasts, suggesting that their expression is part of the terminal differentiation program. There are two histone H4 genes and an H3 gene that encode mRNAs that are polyadenylated and expressed at 5- to 10-fold lower levels than the mRNAs from H2A and H2B genes, which may be replacement genes for the H3.1 and H4 proteins. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Human cytomegalovirus tegument protein pp150 acts as a cyclin A2-CDK-dependent sensor of the host cell cycle and differentiation state.

PubMed

Bogdanow, Boris; Weisbach, Henry; von Einem, Jens; Straschewski, Sarah; Voigt, Sebastian; Winkler, Michael; Hagemeier, Christian; Wiebusch, Lüder

2013-10-22

Upon cell entry, herpesviruses deliver a multitude of premade virion proteins to their hosts. The interplay between these incoming proteins and cell-specific regulatory factors dictates the outcome of infections at the cellular level. Here, we report a unique type of virion-host cell interaction that is essential for the cell cycle and differentiation state-dependent onset of human cytomegalovirus (HCMV) lytic gene expression. The major tegument 150-kDa phosphoprotein (pp150) of HCMV binds to cyclin A2 via a functional RXL/Cy motif resulting in its cyclin A2-dependent phosphorylation. Alanine substitution of the RXL/Cy motif prevents this interaction and allows the virus to fully escape the cyclin-dependent kinase (CDK)-mediated block of immediate early (IE) gene expression in S/G2 phase that normally restricts the onset of the HCMV replication cycle to G0/G1. Furthermore, the cyclin A2-CDK-pp150 axis is also involved in the establishment of HCMV quiescence in NTera2 cells, showing the importance of this molecular switch for differentiation state-dependent regulation of IE gene expression. Consistent with the known nucleocapsid-binding function of pp150, its RXL/Cy-dependent phosphorylation affects gene expression of the parental virion only, suggesting a cis-acting, virus particle-associated mechanism of control. The pp150 homologs of other primate and mammalian CMVs lack an RXL/Cy motif and accordingly even the nearest relative of HCMV, chimpanzee CMV, starts its lytic cycle in a cell cycle-independent manner. Thus, HCMV has evolved a molecular sensor for cyclin A2-CDK activity to restrict its IE gene expression program as a unique level of self-limitation and adaptation to its human host.

A novel DLX3-PKC integrated signaling network drives keratinocyte differentiation.

PubMed

Palazzo, Elisabetta; Kellett, Meghan D; Cataisson, Christophe; Bible, Paul W; Bhattacharya, Shreya; Sun, Hong-Wei; Gormley, Anna C; Yuspa, Stuart H; Morasso, Maria I

2017-04-01

Epidermal homeostasis relies on a well-defined transcriptional control of keratinocyte proliferation and differentiation, which is critical to prevent skin diseases such as atopic dermatitis, psoriasis or cancer. We have recently shown that the homeobox transcription factor DLX3 and the tumor suppressor p53 co-regulate cell cycle-related signaling and that this mechanism is functionally involved in cutaneous squamous cell carcinoma development. Here we show that DLX3 expression and its downstream signaling depend on protein kinase C α (PKCα) activity in skin. We found that following 12-O-tetradecanoyl-phorbol-13-acetate (TPA) topical treatment, DLX3 expression is significantly upregulated in the epidermis and keratinocytes from mice overexpressing PKCα by transgenic targeting (K5-PKCα), resulting in cell cycle block and terminal differentiation. Epidermis lacking DLX3 (DLX3cKO), which is linked to the development of a DLX3-dependent epidermal hyperplasia with hyperkeratosis and dermal leukocyte recruitment, displays enhanced PKCα activation, suggesting a feedback regulation of DLX3 and PKCα. Of particular significance, transcriptional activation of epidermal barrier, antimicrobial peptide and cytokine genes is significantly increased in DLX3cKO skin and further increased by TPA-dependent PKC activation. Furthermore, when inhibiting PKC activity, we show that epidermal thickness, keratinocyte proliferation and inflammatory cell infiltration are reduced and the PKC-DLX3-dependent gene expression signature is normalized. Independently of PKC, DLX3 expression specifically modulates regulatory networks such as Wnt signaling, phosphatase activity and cell adhesion. Chromatin immunoprecipitation sequencing analysis of primary suprabasal keratinocytes showed binding of DLX3 to the proximal promoter regions of genes associated with cell cycle regulation, and of structural proteins and transcription factors involved in epidermal differentiation. These results indicate that Dlx3 potentially regulates a set of crucial genes necessary during the epidermal differentiation process. Altogether, we demonstrate the existence of a robust DLX3-PKCα signaling pathway in keratinocytes that is crucial to epidermal differentiation control and cutaneous homeostasis.

A novel DLX3–PKC integrated signaling network drives keratinocyte differentiation

PubMed Central

Palazzo, Elisabetta; Kellett, Meghan D; Cataisson, Christophe; Bible, Paul W; Bhattacharya, Shreya; Sun, Hong-wei; Gormley, Anna C; Yuspa, Stuart H; Morasso, Maria I

2017-01-01

Epidermal homeostasis relies on a well-defined transcriptional control of keratinocyte proliferation and differentiation, which is critical to prevent skin diseases such as atopic dermatitis, psoriasis or cancer. We have recently shown that the homeobox transcription factor DLX3 and the tumor suppressor p53 co-regulate cell cycle-related signaling and that this mechanism is functionally involved in cutaneous squamous cell carcinoma development. Here we show that DLX3 expression and its downstream signaling depend on protein kinase C α (PKCα) activity in skin. We found that following 12-O-tetradecanoyl-phorbol-13-acetate (TPA) topical treatment, DLX3 expression is significantly upregulated in the epidermis and keratinocytes from mice overexpressing PKCα by transgenic targeting (K5-PKCα), resulting in cell cycle block and terminal differentiation. Epidermis lacking DLX3 (DLX3cKO), which is linked to the development of a DLX3-dependent epidermal hyperplasia with hyperkeratosis and dermal leukocyte recruitment, displays enhanced PKCα activation, suggesting a feedback regulation of DLX3 and PKCα. Of particular significance, transcriptional activation of epidermal barrier, antimicrobial peptide and cytokine genes is significantly increased in DLX3cKO skin and further increased by TPA-dependent PKC activation. Furthermore, when inhibiting PKC activity, we show that epidermal thickness, keratinocyte proliferation and inflammatory cell infiltration are reduced and the PKC-DLX3-dependent gene expression signature is normalized. Independently of PKC, DLX3 expression specifically modulates regulatory networks such as Wnt signaling, phosphatase activity and cell adhesion. Chromatin immunoprecipitation sequencing analysis of primary suprabasal keratinocytes showed binding of DLX3 to the proximal promoter regions of genes associated with cell cycle regulation, and of structural proteins and transcription factors involved in epidermal differentiation. These results indicate that Dlx3 potentially regulates a set of crucial genes necessary during the epidermal differentiation process. Altogether, we demonstrate the existence of a robust DLX3–PKCα signaling pathway in keratinocytes that is crucial to epidermal differentiation control and cutaneous homeostasis. PMID:28186503

The effect of syndecan-4 and glypican-1 knockdown on the proliferation and differentiation of turkey satellite cells differing in age and growth rates.

PubMed

Velleman, Sandra G; Clark, Daniel L; Tonniges, Jeffrey R

2018-09-01

Posthatch skeletal muscle growth requires myogenic satellite cells and the dynamic expression of cell membrane-associated proteins. The membrane associated heparan sulfate proteoglycans, syndecan-4 and glypican-1, link the satellite cell niche to the intracellular environment. Sydnecan-4 and glypican-1 are differentially expressed with age in turkey satellite cells and their over-expression impacts both satellite cell proliferation and differentiation, but their effect on satellite cells from lines with different growth potentials is not known. The objective of the current study was to determine if syndecan-4 and glypican-1 regulation of satellite cell proliferation and differentiation is affected by age and growth selection. Pectoralis major satellite cells isolated at 1 d, 7 and 16-wk of age from a Randombred Control 2 (RBC2) line and a 16-wk body weight (F) line selected from the RBC2 line turkeys were studied. Syndecan-4 and glypican-1 expression was knocked down in both lines. The F-line cells proliferated faster than RBC2 line cells regardless of age, while differentiation tended to be greater in RBC2 line cells than F-line cells at each age. Syndecan-4 knockdown decreased proliferation at 7- and 16-wk but not 1 d cells, and increased differentiation at 1 d and 7 wk but not 16 wk cells. Glypican-1 knockdown differentially affected proliferation depending on cell age, whereas differentiation was decreased for 7- and 16-wk but not 1 d cells. These data suggest syndecan-4 and glypican-1 differentially affected satellite cell function in an age-dependent manner, but had little impact on differences in proliferation and differentiation due to growth selection. Copyright © 2018. Published by Elsevier Inc.

Genome-wide gene expression effects in B6C3F1 mouse intestinal epithelia following 7 and 90 days of exposure to hexavalent chromium in drinking water

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

Kopec, Anna K.; Kim, Suntae; Forgacs, Agnes L.

2012-02-15

Chronic administration of high doses of hexavalent chromium [Cr(VI)] as sodium dichromate dihydrate (SDD) elicits alimentary cancers in mice. To further elucidate key events underlying tumor formation, a 90-day drinking water study was conducted in B6C3F1 mice. Differential gene expression was examined in duodenal and jejunal epithelial samples following 7 or 90 days of exposure to 0, 0.3, 4, 14, 60, 170 or 520 mg/L SDD in drinking water. Genome-wide microarray analyses identified 6562 duodenal and 4448 jejunal unique differentially expressed genes at day 8, and 4630 and 4845 unique changes, respectively, in the duodenum and jejunum at day 91.more » Comparative analysis identified significant overlap in duodenal and jejunal differential gene expression. Automated dose–response modeling identified > 80% of the differentially expressed genes exhibited sigmoidal dose–response curves with EC{sub 50} values ranging from 10 to 100 mg/L SDD. Only 16 genes satisfying the dose-dependent differential expression criteria had EC{sub 50} values < 10 mg/L SDD, 3 of which were regulated by Nrf2, suggesting oxidative stress in response to SDD at low concentrations. Analyses of differentially expressed genes identified over-represented functions associated with oxidative stress, cell cycle, lipid metabolism, and immune responses consistent with the reported effects on redox status and histopathology at corresponding SDD drinking water concentrations. Collectively, these data are consistent with a mode of action involving oxidative stress and cytotoxicity as early key events. This suggests that the tumorigenic effects of chronic Cr(VI) oral exposure likely require chronic tissue damage and compensatory epithelial cell proliferation. Highlights: ► Mouse small intestine gene expression is highly responsive to hexavalent chromium [Cr(VI)]. ► Cr(VI) elicits more differential gene expression after 7 days of exposure than 90 days of exposure. ► Oral exposure to Cr(VI) leads to oxidative stress, cell cycle, lipid and immune dysregulation. ► Cr(VI) elicits dose-dependent changes in gene expression with an overall median EC{sub 50} of 47 mg/L SDD.« less

Down-regulated RPS3a/nbl expression during retinoid-induced differentiation of HL-60 cells: a close association with diminished susceptibility to actinomycin D-stimulated apoptosis.

PubMed

Russell, L; Naora, H; Naora, H

2000-04-01

The efficacy of anticancer agents significantly depends on the differential susceptibility of undifferentiated cancer cells and differentiated normal cells to undergo apoptosis. We previously found that enhanced expression of RPS3a/nbl, which apparently encodes a ribosomal protein, seems to prime cells for apoptosis, while suppressing such enhanced expression triggers cell death. The present study found that HL-60 cells induced to differentiate by all-trans retinoic acid did not undergo apoptosis following treatment with actinomycin D whereas undifferentiated HL-60 cells were highly apoptosis-susceptible, confirming earlier suggestions that differentiated cells have diminished apoptosis-susceptibility. Undifferentiated HL-60 cells highly expressed RPS3a/nbl whereas all-trans retinoic acid -induced differentiated cells exhibited markedly reduced levels, suggesting that apoptosis-resistance of differentiated cells could be due to low RPS3a/nbl expression. Down-regulation of enhanced RPS3a/nbl expression was also observed in cells induced to differentiate with the retinoid 4-[(E)-2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-napthalenyl)-1- propenyl]benzoic acid without any significant induction of cell death. While down-regulation of RPS3a/nbl expression during differentiation did not apparently induce apoptosis, RPS3a/nbl antisense oligomers triggered death of undifferentiated HL-60 cells, but not of retinoid-induced differentiated cells. It therefore seems that while down-regulation of enhanced RPS3a/nbl expression can induce apoptosis in undifferentiated cells, down-regulation of enhanced RPS3a/nbl expression during differentiation occurs independently of apoptosis, and could be regarded as reverting the primed condition to the unprimed (low RPS3a/nbl) state.

Expression of toll-like receptors 2 and 4 and CD14 during differentiation of HL-60 cells induced by phorbol 12-myristate 13-acetate and 1 alpha, 25-dihydroxy-vitamin D(3).

PubMed

Li, Changlin; Wang, Yibing; Gao, Li; Zhang, Jingsong; Shao, Jie; Wang, Shengnian; Feng, Weiguo; Wang, Xingyu; Li, Minglie; Chang, Zongliang

2002-01-01

Macrophages form a crucial bridge between the innate and adaptive immune response. One of their most important functions is to recognize infectious microorganisms. Toll-like receptors (TLRs) are key elements in pathogen recognition, and among them, TLR2 and TLR4 are most discussed. However, expression patterns of TLRs during myeloid cell differentiation to macrophage are unknown. In this study, we examined differentiation in the model human myeloid cell line, HL-60, treated with phorbol 12-myristate 13-acetate (PMA) or VitD(3). Expression of TLR2, TLR4, and CD14 were measured by reverse transcription-PCR, RNase protection assay, and fluorescence-activated cell sorter assays. After treatment by PMA (1, 10, and 100 nM) for 12, 24, and 48 h, expression of TLR2 and CD14 mRNA was increased in a time- and dose-dependent manner. However, VitD(3) only induced expression of CD14 but not TLR2 in HL-60 cells. TLR4 was expressed constitutively before differentiation and increased slightly after that. Thus, PMA-mediated differentiation of HL-60 cells to macrophages is associated largely with TLR2 expression and, to a much lesser extent, with TLR4. Furthermore, up-regulation of TLR2 and CD14 mRNA expression by PMA was abrogated by a protein kinase C inhibitor, Calphostine C, suggesting the up-regulation of TLR2 and CD14 mRNA is dependent on the activation of protein kinase C. Coexpression of CD14/TLR2 and/or CD14/TLR4 may be essential but not sufficient for the production of tumor necrosis factor-alpha in response to lipopolysaccharide in our system.

Bach2 is involved in neuronal differentiation of N1E-115 neuroblastoma cells.

PubMed

Shim, Ki Shuk; Rosner, Margit; Freilinger, Angelika; Lubec, Gert; Hengstschläger, Markus

2006-07-15

Bach1 and Bach2 are evolutionarily related members of the BTB-basic region leucine zipper transcription factor family. We found that Bach2 downregulates cell proliferation of N1E-115 cells and negatively affects their potential to differentiate. Nuclear localization of the cyclin-dependent kinase inhibitor p21 is known to arrest cell cycle progression, and cytoplasmic p21 has been shown to promote neuronal differentiation of N1E-115 cells. We found that ectopic Bach2 causes upregulation of p21 expression in the nucleus and in the cytoplasm in undifferentiated N1E-115 cells. In differentiated cells, Bach2 specifically triggers upregulation of cytoplasmic p21. Our data suggest that Bach2 expression could represent a switch during the process of neuronal differentiation. Bach2 is not expressed in neuronal precursor cells. It would have negative effects on proliferation and differentiation of these cells. In differentiated neuronal cells Bach2 expression is upregulated, which could allow Bach2 to function as a gatekeeper of the differentiated status.

A Study of the Differential Effects of Eicosapentaenoic Acid (EPA) and Docosahexaenoic Acid (DHA) on Gene Expression Profiles of Stimulated Thp-1 Macrophages.

PubMed

Allam-Ndoul, Bénédicte; Guénard, Frédéric; Barbier, Olivier; Vohl, Marie-Claude

2017-04-25

Background: An appropriate intake of omega-3 ( n -3) fatty acids (FAs) such as eicosapentaenoic and docosahexaenoic acid (EPA/DHA) from marine sources is known to have anti-inflammatory effects. However, molecular mechanisms underlying their beneficial effects on health are not fully understood. The aim of the present study was to characterize gene expression profiles of THP-1 macrophages, incubated in either EPA or DHA and stimulated with lipopolysaccharide (LPS), a pro-inflammatory agent. Methods: THP-1 macrophages were incubated into 10, 50 and 75 µM of EPA or DHA for 24 h, and 100 nM of LPS was added to the culture media for 18 h. Total mRNA was extracted and gene expression examined by microarray analysis using Illumina Human HT-12 expression beadchips (Illumina). Results: Pathway analysis revealed that EPA and DHA regulate genes involved in cell cycle regulation, apoptosis, immune response and inflammation, oxidative stress and cancer pathways in a differential and dose-dependent manner. Conclusions: EPA and DHA appear to exert differential effects on gene expression in THP-1 macrophages. Specific effects of n -3 FAs on gene expression levels are also dose-dependent.

Enforced expression of KDR receptor promotes proliferation, survival and megakaryocytic differentiation of TF1 progenitor cell line.

PubMed

Coppola, S; Narciso, L; Feccia, T; Bonci, D; Calabrò, L; Morsilli, O; Gabbianelli, M; De Maria, R; Testa, U; Peschle, C

2006-01-01

Vascular endothelial growth factor (VEGF) receptor-2/kinase insert domain-containing receptor (KDR) is expressed in primitive hematopoietic cells, in megakaryocytes and platelets. In primitive hematopoiesis KDR mediates cell survival via autocrine VEGF, while its effect on cell growth and differentiation has not been elucidated. We induced enforced KDR expression in the granulocyte macrophage-colony-stimulating factor (GM-CSF)-dependent TF1 progenitor cell line (TF1-KDR), treated the cells with VEGF and analyzed their response. In GM-CSF-deprived cells, VEGF induces cell proliferation and protection against apoptosis, followed by enhanced expression of megakaryocytic (MK) markers. Combined with GM-CSF, VEGF induces a mild proliferative stimulus, followed by cell adherence, accumulation in G0/G1, massive MK differentiation and Fas-mediated apoptosis. Accordingly, we observed that MK-differentiating cells, derived from hematopoietic progenitors, produce VEGF, express KDR, inhibition of which reduces MK differentiation, indicating a key role of KDR in megakaryopoiesis. In conclusion, TF1-KDR cells provide a reliable model to investigate the biochemical and molecular mechanisms underlying hematopoietic progenitor proliferation, survival and MK differentiation.

Differential Gene Expression (DEX) and Alternative Splicing Events (ASE) for Temporal Dynamic Processes Using HMMs and Hierarchical Bayesian Modeling Approaches.

PubMed

Oh, Sunghee; Song, Seongho

2017-01-01

In gene expression profile, data analysis pipeline is categorized into four levels, major downstream tasks, i.e., (1) identification of differential expression; (2) clustering co-expression patterns; (3) classification of subtypes of samples; and (4) detection of genetic regulatory networks, are performed posterior to preprocessing procedure such as normalization techniques. To be more specific, temporal dynamic gene expression data has its inherent feature, namely, two neighboring time points (previous and current state) are highly correlated with each other, compared to static expression data which samples are assumed as independent individuals. In this chapter, we demonstrate how HMMs and hierarchical Bayesian modeling methods capture the horizontal time dependency structures in time series expression profiles by focusing on the identification of differential expression. In addition, those differential expression genes and transcript variant isoforms over time detected in core prerequisite steps can be generally further applied in detection of genetic regulatory networks to comprehensively uncover dynamic repertoires in the aspects of system biology as the coupled framework.

A Common Position-Dependent Mechanism Controls Cell-Type Patterning and GLABRA2 Regulation in the Root and Hypocotyl Epidermis of Arabidopsis1

PubMed Central

Hung, Chen-Yi; Lin, Yan; Zhang, Meng; Pollock, Susan; David Marks, M.; Schiefelbein, John

1998-01-01

A position-dependent pattern of epidermal cell types is produced during root development in Arabidopsis thaliana. This pattern is reflected in the expression pattern of GLABRA2 (GL2), a homeobox gene that regulates cell differentiation in the root epidermis. GL2 promoter::GUS fusions were used to show that the TTG gene, a regulator of root epidermis development, is necessary for maximal GL2 activity but is not required for the pattern of GL2 expression. Furthermore, GL2-promoter activity is influenced by expression of the myc-like maize R gene (35S::R) in Arabidopsis but is not affected by gl2 mutations. A position-dependent pattern of cell differentiation and GL2-promoter activity was also discovered in the hypocotyl epidermis that was analogous to the pattern in the root. Non-GL2-expressing cell files in the hypocotyl epidermis located outside anticlinal cortical cell walls exhibit reduced cell length and form stomata. Like the root, the hypocotyl GL2 activity was shown to be influenced by ttg and 35S::R but not by gl2. The parallel pattern of cell differentiation in the root and hypocotyl indicates that TTG and GL2 participate in a common position-dependent mechanism to control cell-type patterning throughout the apical-basal axis of the Arabidopsis seedling. PMID:9576776

Reversible Block of Mouse Neural Stem Cell Differentiation in the Absence of Dicer and MicroRNAs

PubMed Central

Sansom, Stephen N.; Alsiö, Jessica M.; Kaneda, Masahiro; Smith, James; O'Carroll, Donal; Tarakhovsky, Alexander; Livesey, Frederick J.

2010-01-01

Background To investigate the functions of Dicer and microRNAs in neural stem (NS) cell self-renewal and neurogenesis, we established neural stem cell lines from the embryonic mouse Dicer-null cerebral cortex, producing neural stem cell lines that lacked all microRNAs. Principal Findings Dicer-null NS cells underwent normal self-renewal and could be maintained in vitro indefinitely, but had subtly altered cell cycle kinetics and abnormal heterochromatin organisation. In the absence of all microRNAs, Dicer-null NS cells were incapable of generating either glial or neuronal progeny and exhibited a marked dependency on exogenous EGF for survival. Dicer-null NS cells assumed complex differences in mRNA and protein expression under self-renewing conditions, upregulating transcripts indicative of self-renewing NS cells and expressing genes characteristic of differentiating neurons and glia. Underlining the growth-factor dependency of Dicer-null NS cells, many regulators of apoptosis were enriched in expression in these cells. Dicer-null NS cells initiate some of the same gene expression changes as wild-type cells under astrocyte differentiating conditions, but also show aberrant expression of large sets of genes and ultimately fail to complete the differentiation programme. Acute replacement of Dicer restored their ability to differentiate to both neurons and glia. Conclusions The block in differentiation due to loss of Dicer and microRNAs is reversible and the significantly altered phenotype of Dicer-null NS cells does not constitute a permanent transformation. We conclude that Dicer and microRNAs function in this system to maintain the neural stem cell phenotype and to facilitate the completion of differentiation. PMID:20976144

N-Myc Differentially Regulates Expression of MXI1 Isoforms in Neuroblastoma1

PubMed Central

Armstrong, Michael B; Mody, Rajen J; Ellis, D Christian; Hill, Adam B; Erichsen, David A; Wechsler, Daniel S

2013-01-01

Amplification of the MYCN proto-oncogene is associated with a poor prognosis in patients with metastatic neuroblastoma (NB). MYCN encodes the N-Myc protein, a transcriptional regulator that dimerizes with the Max transcription factor, binds to E-box DNA sequences, and regulates genes involved in cell growth and apoptosis. Overexpression of N-Myc leads to transcriptional activation and an increase in NB cell proliferation. Mxi1, a member of the Myc family of transcriptional regulators, also binds to Max. However, Mxi1 is a transcriptional repressor and inhibits proliferation of NB cells, suggesting that Mxi1 functions as an N-Myc antagonist. Our laboratory previously identified Mxi1-0, an alternatively transcribed Mxi1 isoform. Mxi1-0 has properties distinct from those of Mxi1; in contrast to Mxi1, Mxi1-0 is unable to suppress c-Myc-dependent transcription. We now show that Mxi1-0 expression increases in response to MYCN overexpression in NB cells, with a positive correlation between MYCN and MXI1-0 RNA levels. We also show that N-Myc expression differentially regulates the MXI1 and MXI1-0 promoters: Increased MYCN expression suppresses MXI1 promoter activity while enhancing transcription through the MXI1-0 promoter. Finally, induction of Mxi1-0 leads to increased proliferation, whereas expression of Mxi1 inhibits cell growth, indicating differential roles for these two proteins. These data suggest that N-Myc differentially regulates the expression of MXI1 and MXI1-0 and can alter the balance between the two transcription factors. Furthermore, MXI1-0 appears to be a downstream target of MYCN-dependent signaling pathways and may contribute to N-Myc-dependent cell growth and proliferation. PMID:24403858

Activity-Dependent Bidirectional Regulation of GAD Expression in a Homeostatic Fashion Is Mediated by BDNF-Dependent and Independent Pathways

PubMed Central

Hanno-Iijima, Yoko; Tanaka, Masami; Iijima, Takatoshi

2015-01-01

Homeostatic synaptic plasticity, or synaptic scaling, is a mechanism that tunes neuronal transmission to compensate for prolonged, excessive changes in neuronal activity. Both excitatory and inhibitory neurons undergo homeostatic changes based on synaptic transmission strength, which could effectively contribute to a fine-tuning of circuit activity. However, gene regulation that underlies homeostatic synaptic plasticity in GABAergic (GABA, gamma aminobutyric) neurons is still poorly understood. The present study demonstrated activity-dependent dynamic scaling in which NMDA-R (N-methyl-D-aspartic acid receptor) activity regulated the expression of GABA synthetic enzymes: glutamic acid decarboxylase 65 and 67 (GAD65 and GAD67). Results revealed that activity-regulated BDNF (brain-derived neurotrophic factor) release is necessary, but not sufficient, for activity-dependent up-scaling of these GAD isoforms. Bidirectional forms of activity-dependent GAD expression require both BDNF-dependent and BDNF-independent pathways, both triggered by NMDA-R activity. Additional results indicated that these two GAD genes differ in their responsiveness to chronic changes in neuronal activity, which could be partially caused by differential dependence on BDNF. In parallel to activity-dependent bidirectional scaling in GAD expression, the present study further observed that a chronic change in neuronal activity leads to an alteration in neurotransmitter release from GABAergic neurons in a homeostatic, bidirectional fashion. Therefore, the differential expression of GAD65 and 67 during prolonged changes in neuronal activity may be implicated in some aspects of bidirectional homeostatic plasticity within mature GABAergic presynapses. PMID:26241953

Activity-Dependent Bidirectional Regulation of GAD Expression in a Homeostatic Fashion Is Mediated by BDNF-Dependent and Independent Pathways.

PubMed

Hanno-Iijima, Yoko; Tanaka, Masami; Iijima, Takatoshi

2015-01-01

Homeostatic synaptic plasticity, or synaptic scaling, is a mechanism that tunes neuronal transmission to compensate for prolonged, excessive changes in neuronal activity. Both excitatory and inhibitory neurons undergo homeostatic changes based on synaptic transmission strength, which could effectively contribute to a fine-tuning of circuit activity. However, gene regulation that underlies homeostatic synaptic plasticity in GABAergic (GABA, gamma aminobutyric) neurons is still poorly understood. The present study demonstrated activity-dependent dynamic scaling in which NMDA-R (N-methyl-D-aspartic acid receptor) activity regulated the expression of GABA synthetic enzymes: glutamic acid decarboxylase 65 and 67 (GAD65 and GAD67). Results revealed that activity-regulated BDNF (brain-derived neurotrophic factor) release is necessary, but not sufficient, for activity-dependent up-scaling of these GAD isoforms. Bidirectional forms of activity-dependent GAD expression require both BDNF-dependent and BDNF-independent pathways, both triggered by NMDA-R activity. Additional results indicated that these two GAD genes differ in their responsiveness to chronic changes in neuronal activity, which could be partially caused by differential dependence on BDNF. In parallel to activity-dependent bidirectional scaling in GAD expression, the present study further observed that a chronic change in neuronal activity leads to an alteration in neurotransmitter release from GABAergic neurons in a homeostatic, bidirectional fashion. Therefore, the differential expression of GAD65 and 67 during prolonged changes in neuronal activity may be implicated in some aspects of bidirectional homeostatic plasticity within mature GABAergic presynapses.

DIDO as a Switchboard that Regulates Self-Renewal and Differentiation in Embryonic Stem Cells.

PubMed

Fütterer, Agnes; de Celis, Jésus; Navajas, Rosana; Almonacid, Luis; Gutiérrez, Julio; Talavera-Gutiérrez, Amaia; Pacios-Bras, Cristina; Bernascone, Ilenia; Martin-Belmonte, Fernando; Martinéz-A, Carlos

2017-04-11

Transition from symmetric to asymmetric cell division requires precise coordination of differential gene expression. We show that embryonic stem cells (ESCs) mainly express DIDO3 and that their differentiation after leukemia inhibitory factor withdrawal requires DIDO1 expression. C-terminal truncation of DIDO3 (Dido3ΔCT) impedes ESC differentiation while retaining self-renewal; small hairpin RNA-Dido1 ESCs have the same phenotype. Dido3ΔCT ESC differentiation is rescued by ectopic expression of DIDO3, which binds the Dido locus via H3K4me3 and RNA POL II and induces DIDO1 expression. DIDO1, which is exported to cytoplasm, associates with, and is N-terminally phosphorylated by PKCiota. It binds the E3 ubiquitin ligase WWP2, which contributes to cell fate by OCT4 degradation, to allow expression of primitive endoderm (PE) markers. PE formation also depends on phosphorylated DIDO3 localization to centrosomes, which ensures their correct positioning for PE cell polarization. We propose that DIDO isoforms act as a switchboard that regulates genetic programs for ESC transition from pluripotency maintenance to promotion of differentiation. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Time-dependent regulation of morphological changes and cartilage differentiation markers in the mouse pubic symphysis during pregnancy and postpartum recovery.

PubMed

Castelucci, Bianca Gazieri; Consonni, Sílvio Roberto; Rosa, Viviane Souza; Sensiate, Lucimara Aparecida; Delatti, Paula Cristina Rugno; Alvares, Lúcia Elvira; Joazeiro, Paulo Pinto

2018-01-01

Animal models commonly serve as a bridge between in vitro experiments and clinical applications; however, few physiological processes in adult animals are sufficient to serve as proof-of-concept models for cartilage regeneration. Intriguingly, some rodents, such as young adult mice, undergo physiological connective tissue modifications to birth canal elements such as the pubic symphysis during pregnancy; therefore, we investigated whether the differential expression of cartilage differentiation markers is associated with cartilaginous tissue morphological modifications during these changes. Our results showed that osteochondral progenitor cells expressing Runx2, Sox9, Col2a1 and Dcx at the non-pregnant pubic symphysis proliferated and differentiated throughout pregnancy, giving rise to a complex osteoligamentous junction that attached the interpubic ligament to the pubic bones until labour occurred. After delivery, the recovery of pubic symphysis cartilaginous tissues was improved by the time-dependent expression of these chondrocytic lineage markers at the osteoligamentous junction. This process potentially recapitulates embryologic chondrocytic differentiation to successfully recover hyaline cartilaginous pads at 10 days postpartum. Therefore, we propose that this physiological phenomenon represents a proof-of-concept model for investigating the mechanisms involved in cartilage restoration in adult animals.

p39, the Primary Activator for Cyclin-dependent Kinase 5 (Cdk5) in Oligodendroglia, Is Essential for Oligodendroglia Differentiation and Myelin Repair*

PubMed Central

Bankston, Andrew N.; Li, Wenqi; Zhang, Hui; Ku, Li; Liu, Guanglu; Papa, Filomena; Zhao, Lixia; Bibb, James A.; Cambi, Franca; Tiwari-Woodruff, Seema K.; Feng, Yue

2013-01-01

Cyclin-dependent kinase 5 (Cdk5) plays key roles in normal brain development and function. Dysregulation of Cdk5 may cause neurodegeneration and cognitive impairment. Besides the well demonstrated role of Cdk5 in neurons, emerging evidence suggests the functional requirement of Cdk5 in oligodendroglia (OL) and CNS myelin development. However, whether neurons and OLs employ similar or distinct mechanisms to regulate Cdk5 activity remains elusive. We report here that in contrast to neurons that harbor high levels of two Cdk5 activators, p35 and p39, OLs express abundant p39 but negligible p35. In addition, p39 is selectively up-regulated in OLs during differentiation along with elevated Cdk5 activity, whereas p35 expression remains unaltered. Specific knockdown of p39 by siRNA significantly attenuates Cdk5 activity and OL differentiation without affecting p35. Finally, expression of p39, but not p35, is increased during myelin repair, and remyelination is impaired in p39−/− mice. Together, these results reveal that neurons and OLs harbor distinct preference of Cdk5 activators and demonstrate important functions of p39-dependent Cdk5 activation in OL differentiation during de novo myelin development and myelin repair. PMID:23645679

Effector Regulatory T Cell Differentiation and Immune Homeostasis Depend on the Transcription Factor Myb.

PubMed

Dias, Sheila; D'Amico, Angela; Cretney, Erika; Liao, Yang; Tellier, Julie; Bruggeman, Christine; Almeida, Francisca F; Leahy, Jamie; Belz, Gabrielle T; Smyth, Gordon K; Shi, Wei; Nutt, Stephen L

2017-01-17

FoxP3-expressing regulatory T (Treg) cells are essential for maintaining immune homeostasis. Activated Treg cells undergo further differentiation into an effector state that highly expresses genes critical for Treg cell function, although how this process is coordinated on a transcriptional level is poorly understood. Here, we demonstrate that mice lacking the transcription factor Myb in Treg cells succumbed to a multi-organ inflammatory disease. Myb was specifically expressed in, and required for the differentiation of, thymus-derived effector Treg cells. The combination of transcriptome and genomic footprint analyses revealed that Myb directly regulated a large proportion of the gene expression specific to effector Treg cells, identifying Myb as a critical component of the gene regulatory network controlling effector Treg cell differentiation and function. Copyright © 2017 Elsevier Inc. All rights reserved.

Differential Gene Expression in Liver, Gill, and Olfactory Rosettes of Coho Salmon (Oncorhynchus kisutch) After Acclimation to Salinity

PubMed Central

Lavado, Ramon; Bammler, Theo K.; Gallagher, Evan P.; Stapleton, Patricia L.; Beyer, Richard P.; Farin, Federico M.; Hardiman, Gary; Schlenk, Daniel

2015-01-01

Most Pacific salmonids undergo smoltification and transition from freshwater to saltwater, making various adjustments in metabolism, catabolism, osmotic, and ion regulation. The molecular mechanisms underlying this transition are largely unknown. In the present study, we acclimated coho salmon (Oncorhynchus kisutch) to four different salinities and assessed gene expression through microarray analysis of gills, liver, and olfactory rosettes. Gills are involved in osmotic regulation, liver plays a role in energetics, and olfactory rosettes are involved in behavior. Between all salinity treatments, liver had the highest number of differentially expressed genes at 1616, gills had 1074, and olfactory rosettes had 924, using a 1.5-fold cutoff and a false discovery rate of 0.5. Higher responsiveness of liver to metabolic changes after salinity acclimation to provide energy for other osmoregulatory tissues such as the gills may explain the differences in number of differentially expressed genes. Differentially expressed genes were tissue- and salinity-dependent. There were no known genes differentially expressed that were common to all salinity treatments and all tissues. Gene ontology term analysis revealed biological processes, molecular functions, and cellular components that were significantly affected by salinity, a majority of which were tissue-dependent. For liver, oxygen binding and transport terms were highlighted. For gills, muscle, and cytoskeleton-related terms predominated and for olfactory rosettes, immune response-related genes were accentuated. Interaction networks were examined in combination with GO terms and determined similarities between tissues for potential osmosensors, signal transduction cascades, and transcription factors. PMID:26260986

Mechanical stimuli differentially control stem cell behavior: morphology, proliferation, and differentiation

PubMed Central

Maul, Timothy M.; Chew, Douglas W.; Nieponice, Alejandro

2011-01-01

Mesenchymal stem cell (MSC) therapy has demonstrated applications in vascular regenerative medicine. Although blood vessels exist in a mechanically dynamic environment, there has been no rigorous, systematic analysis of mechanical stimulation on stem cell differentiation. We hypothesize that mechanical stimuli, relevant to the vasculature, can differentiate MSCs toward smooth muscle (SMCs) and endothelial cells (ECs). This was tested using a unique experimental platform to differentially apply various mechanical stimuli in parallel. Three forces, cyclic stretch, cyclic pressure, and laminar shear stress, were applied independently to mimic several vascular physiologic conditions. Experiments were conducted using subconfluent MSCs for 5 days and demonstrated significant effects on morphology and proliferation depending upon the type, magnitude, frequency, and duration of applied stimulation. We have defined thresholds of cyclic stretch that potentiate SMC protein expression, but did not find EC protein expression under any condition tested. However, a second set of experiments performed at confluence and aimed to elicit the temporal gene expression response of a select magnitude of each stimulus revealed that EC gene expression can be increased with cyclic pressure and shear stress in a cell-contact-dependent manner. Further, these MSCs also appear to express genes from multiple lineages simultaneously which may warrant further investigation into post-transcriptional mechanisms for controlling protein expression. To our knowledge, this is the first systematic examination of the effects of mechanical stimulation on MSCs and has implications for the understanding of stem cell biology, as well as potential bioreactor designs for tissue engineering and cell therapy applications. PMID:21253809

Coordinating cell proliferation and differentiation: Antagonism between cell cycle regulators and cell type-specific gene expression

PubMed Central

Ruijtenberg, Suzan; van den Heuvel, Sander

2016-01-01

ABSTRACT Cell proliferation and differentiation show a remarkable inverse relationship. Precursor cells continue division before acquiring a fully differentiated state, while terminal differentiation usually coincides with proliferation arrest and permanent exit from the division cycle. Mechanistic insight in the temporal coordination between cell cycle exit and differentiation has come from studies of cells in culture and genetic animal models. As initially described for skeletal muscle differentiation, temporal coordination involves mutual antagonism between cyclin-dependent kinases that promote cell cycle entry and transcription factors that induce tissue-specific gene expression. Recent insights highlight the contribution of chromatin-regulating complexes that act in conjunction with the transcription factors and determine their activity. In particular SWI/SNF chromatin remodelers contribute to dual regulation of cell cycle and tissue-specific gene expression during terminal differentiation. We review the concerted regulation of the cell cycle and cell type-specific transcription, and discuss common mutations in human cancer that emphasize the clinical importance of proliferation versus differentiation control. PMID:26825227

Dual functions of silver nanoparticles in F9 teratocarcinoma stem cells, a suitable model for evaluating cytotoxicity- and differentiation-mediated cancer therapy.

PubMed

Han, Jae Woong; Gurunathan, Sangiliyandi; Choi, Yun-Jung; Kim, Jin-Hoi

2017-01-01

Silver nanoparticles (AgNPs) exhibit strong antibacterial and anticancer activity owing to their large surface-to-volume ratios and crystallographic surface structure. Owing to their various applications, understanding the mechanisms of action, biological interactions, potential toxicity, and beneficial effects of AgNPs is important. Here, we investigated the toxicity and differentiation-inducing effects of AgNPs in teratocarcinoma stem cells. AgNPs were synthesized and characterized using various analytical techniques such as UV-visible spectroscopy, X-ray diffraction, energy-dispersive X-ray spectroscopy, and transmission electron microscopy. The cellular responses of AgNPs were analyzed by a series of cellular and biochemical assays. Gene and protein expressions were analyzed by reverse transcription-quantitative polymerase chain reaction and western blotting, respectively. The AgNPs showed typical crystalline structures and spherical shapes (average size =20 nm). High concentration of AgNPs induced cytotoxicity in a dose-dependent manner by increasing lactate dehydrogenase leakage and reactive oxygen species. Furthermore, AgNPs caused mitochondrial dysfunction, DNA fragmentation, increased expression of apoptotic genes, and decreased expression of antiapoptotic genes. Lower concentrations of AgNPs induced neuronal differentiation by increasing the expression of differentiation markers and decreasing the expression of stem cell markers. Cisplatin reduced the viability of F9 cells that underwent AgNPs-induced differentiation. The results showed that AgNPs caused differentially regulated cytotoxicity and induced neuronal differentiation of F9 cells in a concentration-dependent manner. Therefore, AgNPs can be used for differentiation therapy, along with chemotherapeutic agents, for improving cancer treatment by targeting specific chemotherapy-resistant cells within a tumor. Furthermore, understanding the molecular mechanisms of apoptosis and differentiation in stem cells could also help in developing new strategies for cancer stem cell (CSC) therapies. The findings of this study could significantly contribute to the nanomedicine because this study is the first of its kind, and our results will lead to new strategies for cancer and CSC therapies.

Local Adaptation of Sun-Exposure-Dependent Gene Expression Regulation in Human Skin.

PubMed

Kita, Ryosuke; Fraser, Hunter B

2016-10-01

Sun-exposure is a key environmental variable in the study of human evolution. Several skin-pigmentation genes serve as classical examples of positive selection, suggesting that sun-exposure has significantly shaped worldwide genomic variation. Here we investigate the interaction between genetic variation and sun-exposure, and how this impacts gene expression regulation. Using RNA-Seq data from 607 human skin samples, we identified thousands of transcripts that are differentially expressed between sun-exposed skin and non-sun-exposed skin. We then tested whether genetic variants may influence each individual's gene expression response to sun-exposure. Our analysis revealed 10 sun-exposure-dependent gene expression quantitative trait loci (se-eQTLs), including genes involved in skin pigmentation (SLC45A2) and epidermal differentiation (RASSF9). The allele frequencies of the RASSF9 se-eQTL across diverse populations correlate with the magnitude of solar radiation experienced by these populations, suggesting local adaptation to varying levels of sunlight. These results provide the first examples of sun-exposure-dependent regulatory variation and suggest that this variation has contributed to recent human adaptation.

Chimeric calcium/calmodulin-dependent protein kinase in tobacco: differential regulation by calmodulin isoforms

NASA Technical Reports Server (NTRS)

Liu, Z.; Xia, M.; Poovaiah, B. W.

1998-01-01

cDNA clones of chimeric Ca2+/calmodulin-dependent protein kinase (CCaMK) from tobacco (TCCaMK-1 and TCCaMK-2) were isolated and characterized. The polypeptides encoded by TCCaMK-1 and TCCaMK-2 have 15 different amino acid substitutions, yet they both contain a total of 517 amino acids. Northern analysis revealed that CCaMK is expressed in a stage-specific manner during anther development. Messenger RNA was detected when tobacco bud sizes were between 0.5 cm and 1.0 cm. The appearance of mRNA coincided with meiosis and became undetectable at later stages of anther development. The reverse polymerase chain reaction (RT-PCR) amplification assay using isoform-specific primers showed that both of the CCaMK mRNAs were expressed in anther with similar expression patterns. The CCaMK protein expressed in Escherichia coli showed Ca2+-dependent autophosphorylation and Ca2+/calmodulin-dependent substrate phosphorylation. Calmodulin isoforms (PCM1 and PCM6) had differential effects on the regulation of autophosphorylation and substrate phosphorylation of tobacco CCaMK, but not lily CCaMK. The evolutionary tree of plant serine/threonine protein kinases revealed that calmodulin-dependent kinases form one subgroup that is distinctly different from Ca2+-dependent protein kinases (CDPKs) and other serine/threonine kinases in plants.

Fibroblast growth factor receptors in in vitro and in vivo chondrogenesis: relating tissue engineering using adult mesenchymal stem cells to embryonic development.

PubMed

Hellingman, Catharine A; Koevoet, Wendy; Kops, Nicole; Farrell, Eric; Jahr, Holger; Liu, Wei; Baatenburg de Jong, Robert J; Frenz, Dorothy A; van Osch, Gerjo J V M

2010-02-01

Adult mesenchymal stem cells (MSCs) are considered promising candidate cells for therapeutic cartilage and bone regeneration. Because tissue regeneration and embryonic development may involve similar pathways, understanding common pathways may lead to advances in regenerative medicine. In embryonic limb development, fibroblast growth factor receptors (FGFRs) play a role in chondrogenic differentiation. The aim of this study was to investigate and compare FGFR expression in in vivo embryonic limb development and in vitro chondrogenesis of MSCs. Our study showed that in in vitro chondrogenesis of MSCs three sequential stages can be found, as in embryonic limb development. A mesenchymal condensation (indicated by N-cadherin) is followed by chondrogenic differentiation (indicated by collagen II), and hypertrophy (indicated by collagen X). FGFR1-3 are expressed in a stage-dependent pattern during in vitro differentiation and in vivo embryonic limb development. In both models FGFR2 is clearly expressed by cells in the condensation phase. No FGFR expression was observed in differentiating and mature hyaline chondrocytes, whereas hypertrophic chondrocytes stained strongly for all FGFRs. To evaluate whether stage-specific modulation of chondrogenic differentiation in MSCs is possible with different subtypes of FGF, FGF2 and FGF9 were added to the chondrogenic medium during different stages in the culture process (early or late). FGF2 and FGF9 differentially affected the amount of cartilage formed by MSCs depending on the stage in which they were added. These results will help us understand the role of FGF signaling in chondrogenesis and find new tools to monitor and control chondrogenic differentiation.

Chronic Ethanol Exposure Produces Time- and Brain Region-Dependent Changes in Gene Coexpression Networks

PubMed Central

Osterndorff-Kahanek, Elizabeth A.; Becker, Howard C.; Lopez, Marcelo F.; Farris, Sean P.; Tiwari, Gayatri R.; Nunez, Yury O.; Harris, R. Adron; Mayfield, R. Dayne

2015-01-01

Repeated ethanol exposure and withdrawal in mice increases voluntary drinking and represents an animal model of physical dependence. We examined time- and brain region-dependent changes in gene coexpression networks in amygdala (AMY), nucleus accumbens (NAC), prefrontal cortex (PFC), and liver after four weekly cycles of chronic intermittent ethanol (CIE) vapor exposure in C57BL/6J mice. Microarrays were used to compare gene expression profiles at 0-, 8-, and 120-hours following the last ethanol exposure. Each brain region exhibited a large number of differentially expressed genes (2,000-3,000) at the 0- and 8-hour time points, but fewer changes were detected at the 120-hour time point (400-600). Within each region, there was little gene overlap across time (~20%). All brain regions were significantly enriched with differentially expressed immune-related genes at the 8-hour time point. Weighted gene correlation network analysis identified modules that were highly enriched with differentially expressed genes at the 0- and 8-hour time points with virtually no enrichment at 120 hours. Modules enriched for both ethanol-responsive and cell-specific genes were identified in each brain region. These results indicate that chronic alcohol exposure causes global ‘rewiring‘ of coexpression systems involving glial and immune signaling as well as neuronal genes. PMID:25803291

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

Different coding strategies for the perception of stable and changeable facial attributes.

PubMed

Taubert, Jessica; Alais, David; Burr, David

2016-09-01

Perceptual systems face competing requirements: improving signal-to-noise ratios of noisy images, by integration; and maximising sensitivity to change, by differentiation. Both processes occur in human vision, under different circumstances: they have been termed priming, or serial dependencies, leading to positive sequential effects; and adaptation or habituation, which leads to negative sequential effects. We reasoned that for stable attributes, such as the identity and gender of faces, the system should integrate: while for changeable attributes like facial expression, it should also engage contrast mechanisms to maximise sensitivity to change. Subjects viewed a sequence of images varying simultaneously in gender and expression, and scored each as male or female, and happy or sad. We found strong and consistent positive serial dependencies for gender, and negative dependency for expression, showing that both processes can operate at the same time, on the same stimuli, depending on the attribute being judged. The results point to highly sophisticated mechanisms for optimizing use of past information, either by integration or differentiation, depending on the permanence of that attribute.

Coordinated activation of AMP-activated protein kinase, extracellular signal-regulated kinase, and autophagy regulates phorbol myristate acetate-induced differentiation of SH-SY5Y neuroblastoma cells.

PubMed

Zogovic, Nevena; Tovilovic-Kovacevic, Gordana; Misirkic-Marjanovic, Maja; Vucicevic, Ljubica; Janjetovic, Kristina; Harhaji-Trajkovic, Ljubica; Trajkovic, Vladimir

2015-04-01

We explored the interplay between the intracellular energy sensor AMP-activated protein kinase (AMPK), extracellular signal-regulated kinase (ERK), and autophagy in phorbol myristate acetate (PMA)-induced neuronal differentiation of SH-SY5Y human neuroblastoma cells. PMA-triggered expression of neuronal markers (dopamine transporter, microtubule-associated protein 2, β-tubulin) was associated with an autophagic response, measured by the conversion of microtubule-associated protein light chain 3 (LC3)-I to autophagosome-bound LC3-II, increase in autophagic flux, and expression of autophagy-related (Atg) proteins Atg7 and beclin-1. This coincided with the transient activation of AMPK and sustained activation of ERK. Pharmacological inhibition or RNA interference-mediated silencing of AMPK suppressed PMA-induced expression of neuronal markers, as well as ERK activation and autophagy. A selective pharmacological blockade of ERK prevented PMA-induced neuronal differentiation and autophagy induction without affecting AMPK phosphorylation. Conversely, the inhibition of autophagy downstream of AMPK/ERK, either by pharmacological agents or LC3 knockdown, promoted the expression of neuronal markers, thus indicating a role of autophagy in the suppression of PMA-induced differentiation of SH-SY5Y cells. Therefore, PMA-induced neuronal differentiation of SH-SY5Y cells depends on a complex interplay between AMPK, ERK, and autophagy, in which the stimulatory effects of AMPK/ERK signaling are counteracted by the coinciding autophagic response. Phorbol myristate acetate (PMA) induces the expression of dopamine transporter, microtubule-associated protein 2, and β-tubulin, and subsequent neuronal differentiation of SH-SY5Y neuroblastoma cells through AMP-activated protein kinase (AMPK)-dependent activation of extracellular signal-regulated kinase (ERK). The activation of AMPK/ERK axis also induces the expression of beclin-1 and Atg7, and increases LC3 conversion, thereby triggering the autophagic response that counteracts differentiation process. © 2014 International Society for Neurochemistry.

Differential DNases are selectively used in neuronal apoptosis depending on the differentiation state.

PubMed

Shiokawa, D; Tanuma, S

2004-10-01

In this study, we investigate the roles of two apoptotic endonucleases, CAD and DNase gamma, in neuronal apoptosis. High expression of CAD, but not DNase gamma, is detected in proliferating N1E-115 neuroblastoma cells, and apoptotic DNA fragmentation induced by staurosporine under proliferating conditions is abolished by the expression of a caspase-resistant form of ICAD. After the induction of neuronal differentiation, CAD disappearance and the induction of DNase gamma occur simultaneously in N1E-115 cells. Apoptotic DNA fragmentation that occurs under differentiating conditions is suppressed by the downregulation of DNase gamma caused by its antisense RNA. The induction of DNase gamma is also observed during neuronal differentiation of PC12 cells, and apoptotic DNA fragmentation induced by NGF deprivation is inhibited by the antisense-mediated downregulation of DNase gamma. These observations suggest that DNA fragmentation in neuronal apoptosis is catalyzed by either CAD or DNase gamma depending on the differentiation state. Furthermore, DNase gamma is suggested to be involved in naturally occurring apoptosis in developing nervous systems.

Cyp15F1: a novel cytochrome P450 gene linked to juvenile hormone-dependent caste differention in the termite Reticulitermes flavipes.

PubMed

Tarver, Matthew R; Coy, Monique R; Scharf, Michael E

2012-07-01

Termites are eusocial insects that jointly utilize juvenile hormone (JH), pheromones, and other semiochemicals to regulate caste differentiation and achieve caste homeostasis. Prior EST sequencing from the symbiont-free gut transcriptome of Reticulitermes flavipes unexpectedly revealed a number of unique cytochrome P450 (Cyp) transcripts, including fragments of a Cyp15 family gene (Cyp15F1) with homology to other insect Cyp15s that participate in JH biosynthesis. The present study investigated the role of Cyp15F1 in termite caste polyphenism and specifically tested the hypothesis that it plays a role in JH-dependent caste differentiation. After assembling the full-length Cyp15F1 cDNA sequence, we (i) determined its mRNA tissue expression profile, (ii) investigated mRNA expression changes in response to JH and the caste-regulatory primer pheromones γ-cadinene (CAD) and γ-cadinenal (ALD), and (iii) used RNA interference (RNAi) in combination with caste differentiation bioassays to investigate gene function at the phenotype level. Cyp15F1 has ubiquitous whole-body expression (including gut tissue); is rapidly and sustainably induced from 3 h to 48 h by JH, CAD, and ALD; and functions at least in part by facilitating JH-dependent soldier caste differentiation. These findings provide the second example of a termite caste regulatory gene identified through the use of RNAi, and significantly build upon our understanding of termite caste homeostatic mechanisms. These results also reinforce the concept of environmental caste determination in termites by revealing how primer pheromones, as socioenvironmental factors, can directly influence Cyp15 expression and caste differentiation. © 2012 Wiley Periodicals, Inc.

Coordinated Proliferation and Differentiation of Human-Induced Pluripotent Stem Cell-Derived Cardiac Progenitor Cells Depend on Bone Morphogenetic Protein Signaling Regulation by GREMLIN 2

PubMed Central

Bylund, Jeffery B.; Trinh, Linh T.; Awgulewitsch, Cassandra P.; Paik, David T.; Jetter, Christopher; Jha, Rajneesh; Zhang, Jianhua; Nolan, Kristof; Xu, Chunhui; Thompson, Thomas B.; Kamp, Timothy J.

2017-01-01

Heart development depends on coordinated proliferation and differentiation of cardiac progenitor cells (CPCs), but how the two processes are synchronized is not well understood. Here, we show that the secreted Bone Morphogenetic Protein (BMP) antagonist GREMLIN 2 (GREM2) is induced in CPCs shortly after cardiac mesoderm specification during differentiation of human pluripotent stem cells. GREM2 expression follows cardiac lineage differentiation independently of the differentiation method used, or the origin of the pluripotent stem cells, suggesting that GREM2 is linked to cardiogenesis. Addition of GREM2 protein strongly increases cardiomyocyte output compared to established procardiogenic differentiation methods. Our data show that inhibition of canonical BMP signaling by GREM2 is necessary to promote proliferation of CPCs. However, canonical BMP signaling inhibition alone is not sufficient to induce cardiac differentiation, which depends on subsequent JNK pathway activation specifically by GREM2. These findings may have broader implications in the design of approaches to orchestrate growth and differentiation of pluripotent stem cell-derived lineages that depend on precise regulation of BMP signaling. PMID:28125926

Coordinated Proliferation and Differentiation of Human-Induced Pluripotent Stem Cell-Derived Cardiac Progenitor Cells Depend on Bone Morphogenetic Protein Signaling Regulation by GREMLIN 2.

PubMed

Bylund, Jeffery B; Trinh, Linh T; Awgulewitsch, Cassandra P; Paik, David T; Jetter, Christopher; Jha, Rajneesh; Zhang, Jianhua; Nolan, Kristof; Xu, Chunhui; Thompson, Thomas B; Kamp, Timothy J; Hatzopoulos, Antonis K

2017-05-01

Heart development depends on coordinated proliferation and differentiation of cardiac progenitor cells (CPCs), but how the two processes are synchronized is not well understood. Here, we show that the secreted Bone Morphogenetic Protein (BMP) antagonist GREMLIN 2 (GREM2) is induced in CPCs shortly after cardiac mesoderm specification during differentiation of human pluripotent stem cells. GREM2 expression follows cardiac lineage differentiation independently of the differentiation method used, or the origin of the pluripotent stem cells, suggesting that GREM2 is linked to cardiogenesis. Addition of GREM2 protein strongly increases cardiomyocyte output compared to established procardiogenic differentiation methods. Our data show that inhibition of canonical BMP signaling by GREM2 is necessary to promote proliferation of CPCs. However, canonical BMP signaling inhibition alone is not sufficient to induce cardiac differentiation, which depends on subsequent JNK pathway activation specifically by GREM2. These findings may have broader implications in the design of approaches to orchestrate growth and differentiation of pluripotent stem cell-derived lineages that depend on precise regulation of BMP signaling.

Spectral characterization of differential group delay in uniform fiber Bragg gratings.

PubMed

Bette, S; Caucheteur, C; Wuilpart, M; Mégret, P; Garcia-Olcina, R; Sales, S; Capmany, J

2005-12-12

In this paper, we completely study the wavelength dependency of differential group delay (DGD) in uniform fiber Bragg gratings (FBG) exhibiting birefringence. An analytical expression of DGD is established. We analyze the impact of grating parameters (physical length, index modulation and apodization profile) on the wavelength dependency of DGD. Experimental results complete the paper. A very good agreement between theory and experience is reported.

Threshold-dependent cooperativity of Pdx1 and Oc1 in pancreatic progenitors establishes competency for endocrine differentiation and β-cell function

PubMed Central

Wright, Christopher V.E.; Won, Kyoung-Jae

2016-01-01

Summary Pdx1 and Oc1 are co-expressed in multipotent pancreatic progenitors and regulate the pro-endocrine gene Neurog3. Their expression diverges in later organogenesis, with Oc1 absent from hormone+ cells and Pdx1 maintained in mature β cells. In a classical genetic test for cooperative functional interactions, we derived mice with combined Pdx1 and Oc1 heterozygosity. Endocrine development in double-heterozygous pancreata was normal at embryonic day (e)13.5, but defects in specification and differentiation were apparent at e15.5, the height of the second wave of differentiation. Pancreata from double heterozygotes showed alterations in the expression of genes crucial for β-cell development and function, decreased numbers and altered allocation of Neurog3-expressing endocrine progenitors, and defective endocrine differentiation. Defects in islet gene expression and β-cell function persisted in double heterozygous neonates. These results suggest that Oc1 and Pdx1 cooperate prior to their divergence, in pancreatic progenitors, to allow for proper differentiation and functional maturation of β cells. PMID:27292642

NPH4, a Conditional Modulator of Auxin-Dependent Differential Growth Responses in Arabidopsis1

PubMed Central

Stowe-Evans, Emily L.; Harper, Reneé M.; Motchoulski, Andrei V.; Liscum, Emmanuel

1998-01-01

Although sessile in nature, plants are able to use a number of mechanisms to modify their morphology in response to changing environmental conditions. Differential growth is one such mechanism. Despite its importance in plant development, little is known about the molecular events regulating the establishment of differential growth. Here we report analyses of the nph4 (nonphototropic hypocotyl) mutants of Arabidopsis that suggest that the NPH4 protein plays a central role in the modulation of auxin-dependent differential growth. Results from physiological studies demonstrate that NPH4 activity is conditionally required for a number of differential growth responses, including phototropism, gravitropism, phytochrome-dependent hypocotyl curvature, apical hook maintenance, and abaxial/adaxial leaf-blade expansion. The nph4 mutants exhibited auxin resistance and severely impaired auxin-dependent gene expression, indicating that the defects associated with differential growth likely arise because of altered auxin responsiveness. Moreover, the auxin signaling events mediating phototropism are genetically correlated with the abundance of the NPH4 protein. PMID:9847100

Involvement of WNT Signaling in the Regulation of Gestational Age-Dependent Umbilical Cord-Derived Mesenchymal Stem Cell Proliferation

PubMed Central

Shono, Akemi; Yoshida, Makiko; Yamana, Keiji; Thwin, Khin Kyae Mon; Kuroda, Jumpei; Kurokawa, Daisuke; Koda, Tsubasa; Nishida, Kosuke; Ikuta, Toshihiko; Mizobuchi, Masami; Taniguchi-Ikeda, Mariko

2017-01-01

Mesenchymal stem cells (MSCs) are a heterogeneous cell population that is isolated initially from the bone marrow (BM) and subsequently almost all tissues including umbilical cord (UC). UC-derived MSCs (UC-MSCs) have attracted an increasing attention as a source for cell therapy against various degenerative diseases due to their vigorous proliferation and differentiation. Although the cell proliferation and differentiation of BM-derived MSCs is known to decline with age, the functional difference between preterm and term UC-MSCs is poorly characterized. In the present study, we isolated UC-MSCs from 23 infants delivered at 22–40 weeks of gestation and analyzed their gene expression and cell proliferation. Microarray analysis revealed that global gene expression in preterm UC-MSCs was distinct from term UC-MSCs. WNT signaling impacts on a variety of tissue stem cell proliferation and differentiation, and its pathway genes were enriched in differentially expressed genes between preterm and term UC-MSCs. Cell proliferation of preterm UC-MSCs was significantly enhanced compared to term UC-MSCs and counteracted by WNT signaling inhibitor XAV939. Furthermore, WNT2B expression in UC-MSCs showed a significant negative correlation with gestational age (GA). These results suggest that WNT signaling is involved in the regulation of GA-dependent UC-MSC proliferation. PMID:29138639

FGF/EGF signaling regulates the renewal of early nephron progenitors during embryonic development.

PubMed

Brown, Aaron C; Adams, Derek; de Caestecker, Mark; Yang, Xuehui; Friesel, Robert; Oxburgh, Leif

2011-12-01

Recent studies indicate that nephron progenitor cells of the embryonic kidney are arranged in a series of compartments of an increasing state of differentiation. The earliest progenitor compartment, distinguished by expression of CITED1, possesses greater capacity for renewal and differentiation than later compartments. Signaling events governing progression of nephron progenitor cells through stages of increasing differentiation are poorly understood, and their elucidation will provide key insights into normal and dysregulated nephrogenesis, as well as into regenerative processes that follow kidney injury. In this study, we found that the mouse CITED1(+) progenitor compartment is maintained in response to receptor tyrosine kinase (RTK) ligands that activate both FGF and EGF receptors. This RTK signaling function is dependent on RAS and PI3K signaling but not ERK. In vivo, RAS inactivation by expression of sprouty 1 (Spry1) in CITED1(+) nephron progenitors results in loss of characteristic molecular marker expression and in increased death of progenitor cells. Lineage tracing shows that surviving Spry1-expressing progenitor cells are impaired in their subsequent epithelial differentiation, infrequently contributing to epithelial structures. These findings demonstrate that the survival and developmental potential of cells in the earliest embryonic nephron progenitor cell compartment are dependent on FGF/EGF signaling through RAS.

A Study of the Differential Effects of Eicosapentaenoic Acid (EPA) and Docosahexaenoic Acid (DHA) on Gene Expression Profiles of Stimulated Thp-1 Macrophages

PubMed Central

Allam-Ndoul, Bénédicte; Guénard, Frédéric; Barbier, Olivier; Vohl, Marie-Claude

2017-01-01

Background: An appropriate intake of omega-3 (n-3) fatty acids (FAs) such as eicosapentaenoic and docosahexaenoic acid (EPA/DHA) from marine sources is known to have anti-inflammatory effects. However, molecular mechanisms underlying their beneficial effects on health are not fully understood. The aim of the present study was to characterize gene expression profiles of THP-1 macrophages, incubated in either EPA or DHA and stimulated with lipopolysaccharide (LPS), a pro-inflammatory agent. Methods: THP-1 macrophages were incubated into 10, 50 and 75 µM of EPA or DHA for 24 h, and 100 nM of LPS was added to the culture media for 18 h. Total mRNA was extracted and gene expression examined by microarray analysis using Illumina Human HT-12 expression beadchips (Illumina). Results: Pathway analysis revealed that EPA and DHA regulate genes involved in cell cycle regulation, apoptosis, immune response and inflammation, oxidative stress and cancer pathways in a differential and dose-dependent manner. Conclusions: EPA and DHA appear to exert differential effects on gene expression in THP-1 macrophages. Specific effects of n-3 FAs on gene expression levels are also dose-dependent. PMID:28441337

Orphan nuclear receptor chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) protein negatively regulates bone morphogenetic protein 2-induced osteoblast differentiation through suppressing runt-related gene 2 (Runx2) activity.

PubMed

Lee, Kkot-Nim; Jang, Won-Gu; Kim, Eun-Jung; Oh, Sin-Hye; Son, Hye-Ju; Kim, Sun-Hun; Franceschi, Renny; Zhang, Xiao-Kun; Lee, Shee-Eun; Koh, Jeong-Tae

2012-06-01

Chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) is an orphan nuclear receptor of the steroid-thyroid hormone receptor superfamily. COUP-TFII is widely expressed in multiple tissues and organs throughout embryonic development and has been shown to regulate cellular growth, differentiation, and organ development. However, the role of COUP-TFII in osteoblast differentiation has not been systematically evaluated. In the present study, COUP-TFII was strongly expressed in multipotential mesenchymal cells, and the endogenous expression level decreased during osteoblast differentiation. Overexpression of COUP-TFII inhibited bone morphogenetic protein 2 (BMP2)-induced osteoblastic gene expression. The results of alkaline phosphatase, Alizarin Red staining, and osteocalcin production assay showed that COUP-TFII overexpression blocks BMP2-induced osteoblast differentiation. In contrast, the down-regulation of COUP-TFII synergistically induced the expression of BMP2-induced osteoblastic genes and osteoblast differentiation. Furthermore, the immunoprecipitation assay showed that COUP-TFII and Runx2 physically interacted and COUP-TFII significantly impaired the Runx2-dependent activation of the osteocalcin promoter. From the ChIP assay, we found that COUP-TFII repressed DNA binding of Runx2 to the osteocalcin gene, whereas Runx2 inhibited COUP-TFII expression via direct binding to the COUP-TFII promoter. Taken together, these findings demonstrate that COUP-TFII negatively regulates osteoblast differentiation via interaction with Runx2, and during the differentiation state, BMP2-induced Runx2 represses COUP-TFII expression and promotes osteoblast differentiation.

The preferentially expressed antigen in melanoma (PRAME) inhibits myeloid differentiation in normal hematopoietic and leukemic progenitor cells

PubMed Central

Guthrie, Katherine A.; Cummings, Carrie L.; Sabo, Kathleen; Wood, Brent L.; Gooley, Ted; Yang, Taimei; Epping, Mirjam T.; Shou, Yaping; Pogosova-Agadjanyan, Era; Ladne, Paula; Stirewalt, Derek L.; Abkowitz, Janis L.; Radich, Jerald P.

2009-01-01

The preferentially expressed antigen in melanoma (PRAME) is expressed in several hematologic malignancies, but either is not expressed or is expressed at only low levels in normal hematopoietic cells, making it a target for cancer therapy. PRAME is a tumor-associated antigen and has been described as a corepressor of retinoic acid signaling in solid tumor cells, but its function in hematopoietic cells is unknown. PRAME mRNA expression increased with chronic myeloid leukemia (CML) disease progression and its detection in late chronic-phase CML patients before tyrosine kinase inhibitor therapy was associated with poorer therapeutic responses and ABL tyrosine kinase domain point mutations. In leukemia cell lines, PRAME protein expression inhibited granulocytic differentiation only in cell lines that differentiate along this lineage after all-trans retinoic acid (ATRA) exposure. Forced PRAME expression in normal hematopoietic progenitors, however, inhibited myeloid differentiation both in the presence and absence of ATRA, and this phenotype was reversed when PRAME was silenced in primary CML progenitors. These observations suggest that PRAME inhibits myeloid differentiation in certain myeloid leukemias, and that its function in these cells is lineage and phenotype dependent. Lastly, these observations suggest that PRAME is a target for both prognostic and therapeutic applications. PMID:19625708

Voltage-gated sodium channel expression and action potential generation in differentiated NG108-15 cells.

PubMed

Liu, Jinxu; Tu, Huiyin; Zhang, Dongze; Zheng, Hong; Li, Yu-Long

2012-10-25

The generation of action potential is required for stimulus-evoked neurotransmitter release in most neurons. Although various voltage-gated ion channels are involved in action potential production, the initiation of the action potential is mainly mediated by voltage-gated Na+ channels. In the present study, differentiation-induced changes of mRNA and protein expression of Na+ channels, Na+ currents, and cell membrane excitability were investigated in NG108-15 cells. Whole-cell patch-clamp results showed that differentiation (9 days) didn't change cell membrane excitability, compared to undifferentiated state. But differentiation (21 days) induced the action potential generation in 45.5% of NG108-15 cells (25/55 cells). In 9-day-differentiated cells, Na+ currents were mildly increased, which was also found in 21-day differentiated cells without action potential. In 21-day differentiated cells with action potential, Na+ currents were significantly enhanced. Western blot data showed that the expression of Na+ channels was increased with differentiated-time dependent manner. Single-cell real-time PCR data demonstrated that the expression of Na+ channel mRNA was increased by 21 days of differentiation in NG108-15 cells. More importantly, the mRNA level of Na+ channels in cells with action potential was higher than that in cells without action potential. Differentiation induces expression of voltage-gated Na+ channels and action potential generation in NG108-15 cells. A high level of the Na+ channel density is required for differentiation-triggered action potential generation.

Efficient experimental design and analysis strategies for the detection of differential expression using RNA-Sequencing

PubMed Central

2012-01-01

Background RNA sequencing (RNA-Seq) has emerged as a powerful approach for the detection of differential gene expression with both high-throughput and high resolution capabilities possible depending upon the experimental design chosen. Multiplex experimental designs are now readily available, these can be utilised to increase the numbers of samples or replicates profiled at the cost of decreased sequencing depth generated per sample. These strategies impact on the power of the approach to accurately identify differential expression. This study presents a detailed analysis of the power to detect differential expression in a range of scenarios including simulated null and differential expression distributions with varying numbers of biological or technical replicates, sequencing depths and analysis methods. Results Differential and non-differential expression datasets were simulated using a combination of negative binomial and exponential distributions derived from real RNA-Seq data. These datasets were used to evaluate the performance of three commonly used differential expression analysis algorithms and to quantify the changes in power with respect to true and false positive rates when simulating variations in sequencing depth, biological replication and multiplex experimental design choices. Conclusions This work quantitatively explores comparisons between contemporary analysis tools and experimental design choices for the detection of differential expression using RNA-Seq. We found that the DESeq algorithm performs more conservatively than edgeR and NBPSeq. With regard to testing of various experimental designs, this work strongly suggests that greater power is gained through the use of biological replicates relative to library (technical) replicates and sequencing depth. Strikingly, sequencing depth could be reduced as low as 15% without substantial impacts on false positive or true positive rates. PMID:22985019

Efficient experimental design and analysis strategies for the detection of differential expression using RNA-Sequencing.

PubMed

Robles, José A; Qureshi, Sumaira E; Stephen, Stuart J; Wilson, Susan R; Burden, Conrad J; Taylor, Jennifer M

2012-09-17

RNA sequencing (RNA-Seq) has emerged as a powerful approach for the detection of differential gene expression with both high-throughput and high resolution capabilities possible depending upon the experimental design chosen. Multiplex experimental designs are now readily available, these can be utilised to increase the numbers of samples or replicates profiled at the cost of decreased sequencing depth generated per sample. These strategies impact on the power of the approach to accurately identify differential expression. This study presents a detailed analysis of the power to detect differential expression in a range of scenarios including simulated null and differential expression distributions with varying numbers of biological or technical replicates, sequencing depths and analysis methods. Differential and non-differential expression datasets were simulated using a combination of negative binomial and exponential distributions derived from real RNA-Seq data. These datasets were used to evaluate the performance of three commonly used differential expression analysis algorithms and to quantify the changes in power with respect to true and false positive rates when simulating variations in sequencing depth, biological replication and multiplex experimental design choices. This work quantitatively explores comparisons between contemporary analysis tools and experimental design choices for the detection of differential expression using RNA-Seq. We found that the DESeq algorithm performs more conservatively than edgeR and NBPSeq. With regard to testing of various experimental designs, this work strongly suggests that greater power is gained through the use of biological replicates relative to library (technical) replicates and sequencing depth. Strikingly, sequencing depth could be reduced as low as 15% without substantial impacts on false positive or true positive rates.

Activation of the kinase activity of ATM by retinoic acid is required for CREB-dependent differentiation of neuroblastoma cells.

PubMed

Fernandes, Norvin D; Sun, Yingli; Price, Brendan D

2007-06-01

The ATM protein kinase is mutated in ataxia telangiectasia, a genetic disease characterized by defective DNA repair, neurodegeneration, and growth factor signaling defects. The activity of ATM kinase is activated by DNA damage, and this activation is required for cells to survive genotoxic events. In addition to this well characterized role in DNA repair, we now demonstrate a novel role for ATM in the retinoic acid (RA)-induced differentiation of SH-SY5Y neuroblastoma cells into post-mitotic, neuronal-like cells. RA rapidly activates the activity of ATM kinase, leading to the ATM-dependent phosphorylation of the CREB protein, extrusion of neuritic processes, and differentiation of SH-SY5Y cells into neuronal-like cells. When ATM protein expression was suppressed by short hairpin RNA, the ATM-dependent phosphorylation of CREB was blocked. Furthermore, ATM-negative cells failed to differentiate into neuronal-like cells when exposed to retinoic acid; instead, they underwent cell death. Expression of a constitutively active CREBVP16 construct, or exposure to forskolin to induce CREB phosphorylation, rescued ATM negative cells and restored differentiation. Furthermore, when dominant negative CREB proteins with mutations in either the CREB phosphorylation site (CREBS133A) or the DNA binding domain (KCREB) were introduced into SH-SY5Y cells, retinoic acid-induced differentiation was blocked and the cells underwent cell death. The results demonstrate that ATM is required for the retinoic acid-induced differentiation of SH-SY5Y cells through the ATM dependent-phosphorylation of serine 133 of CREB. These results therefore define a novel mechanism for activation of the activity of ATM kinase by RA, and implicate ATM in the regulation of CREB function during RA-induced differentiation.

Neurobiological Signatures of Alcohol Dependence Revealed by Protein Profiling

PubMed Central

Gorini, Giorgio; Roberts, Amanda J.; Mayfield, R. Dayne

2013-01-01

Alcohol abuse causes dramatic neuroadaptations in the brain, which contribute to tolerance, dependence, and behavioral modifications. Previous proteomic studies in human alcoholics and animal models have identified candidate alcoholism-related proteins. However, recent evidences suggest that alcohol dependence is caused by changes in co-regulation that are invisible to single protein-based analysis. Here, we analyze global proteomics data to integrate differential expression, co-expression networks, and gene annotations to unveil key neurobiological rearrangements associated with the transition to alcohol dependence modeled by a Chronic Intermittent Ethanol (CIE), two-bottle choice (2BC) paradigm. We analyzed cerebral cortices (CTX) and midbrains (MB) from male C57BL/6J mice subjected to a CIE, 2BC paradigm, which induces heavy drinking and represents one of the best available animal models for alcohol dependence and relapse drinking. CIE induced significant changes in protein levels in dependent mice compared with their non-dependent controls. Multiple protein isoforms showed region-specific differential regulation as a result of post-translational modifications. Our integrative analysis identified modules of co-expressed proteins that were highly correlated with CIE treatment. We found that modules most related to the effects of CIE treatment coordinate molecular imbalances in endocytic- and energy-related pathways, with specific proteins involved, such as dynamin-1. The qRT-PCR experiments validated both differential and co-expression analyses, and the correspondence among our data and previous genomic and proteomic studies in humans and rodents substantiates our findings. The changes identified above may play a key role in the escalation of ethanol consumption associated with dependence. Our approach to alcohol addiction will advance knowledge of brain remodeling mechanisms and adaptive changes in response to drug abuse, contribute to understanding of organizational principles of CTX and MB proteomes, and define potential new molecular targets for treating alcohol addiction. The integrative analysis employed here highlight the advantages of systems approaches in studying the neurobiology of alcohol addiction. PMID:24358215

Andrographolide inhibits adipogenesis of 3T3-L1 cells by suppressing C/EBPβ expression and activation

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

Chen, Ching-Chu

Andrographolide, a diterpenoid, is the most abundant terpenoid in Andrographis paniculata, a popular Chinese herbal medicine. Andrographolide displays diverse biological activities including hypoglycemia, hypolipidemia, anti-inflammation, and anti-tumorigenesis. Recent evidence indicates that andrographolide displays anti-obesity property by inhibiting lipogenic gene expression, however, the underlying mechanisms remain to be elucidated. In this study, the effects of andrographolide on transcription factor cascade and mitotic clonal expansion in 3T3-L1 preadipocyte differentiation into adipocyte were determined. Andrographolide dose-dependently (0–15 μM) inhibited CCAAT/enhancer-binding protein α (C/EBPα) and C/EBPβ mRNA and protein expression as well as peroxisome proliferator-activated receptor γ (PPARγ) protein level during the adipogenesis ofmore » 3T3-L1 cells. Concomitantly, fatty acid synthase and stearoyl-CoA desaturase expression and lipid accumulation were attenuated by andrographolide. Oil-red O staining further showed that the first 48 h after the initiation of differentiation was critical for andrographolide inhibition of adipocyte formation. Andrographolide inhibited the phosphorylation of PKA and the activation of cAMP response element-binding protein (CREB) in response to a differentiation cocktail, which led to attenuated C/EBPβ expression. In addition, ERK and GSK3β-dependent C/EBPβ phosphorylation was attenuated by andrographolide. Moreover, andrographolide suppressed cyclin A, cyclin E, and CDK2 expression and impaired the progression of mitotic clonal expansion (MCE) by arresting the cell cycle at the Go/G1 phase. Taken together, these results indicate that andrographolide has a potent anti-obesity action by inhibiting PKA-CREB-mediated C/EBPβ expression as well as C/EBPβ transcriptional activity, which halts MCE progression and attenuates C/EBPα and PPARγ expression. - Highlights: • Andrographolide is a diterpenoid phytochemical. • Andrographolide inhibits adipogenesis of 3 T3-L1 adipocytes. • Andrographolide suppresses differentiation cocktail-induced C/EBPβ expression. • Andrographolide attenuates ERK and GSK3β-dependent C/EBPβ activation. • Andrographolide arrests 3 T3-L1 adipocytes at G0/G1 phase.« less

Modulation of the differentiation of dental pulp stem cells by different concentrations of β-glycerophosphate.

PubMed

Liu, Mingyue; Sun, Yao; Liu, Yang; Yuan, Mengtong; Zhang, Zhihui; Hu, Weiping

2012-01-31

Dentinogenesis is a necessary prerequisite for dental tissue engineering. One of the steps for dentinogenesis is to obtain large quantities of highly purified odontoblasts. Therefore, we have undertaken an experiment applying different concentrations of β-glycerophosphate (β-GP) to induce the differentiation of dental pulp stem cells (DPSCs) in a long-term 28-day culture. In the meanwhile, we have studied the time- and maturation-dependent expression of matrix extracellular phosphoglycoprotein (MEPE) and that of the odontoblast-like marker-dentin sialoprotein (DSP), in order to investigate an optimized mineralized condition. Western blot results revealed that the expression of DSP became lower when accompanied by the increase of the β-GP concentration, and there was also an influence on MEPE expression when different concentrations of β-GP were applied. Meanwhile, the mineralized groups had an inhibitory function on the expression of MEPE as compared with the control group. Above all, all experimental groups successfully generated mineralized nodules by Alizarin Red S and the 5 mM β-GP group formed more mineralized nodules quantitated using the CPC extraction method. In conclusion, there is a significant modulation of the β-GP during the differentiation of the DPSCs. The degree of odontoblast differentiation is β-glycerophosphate concentration dependent. A low concentration of β-GP (5 mM) has been shown to be the optimal concentration for stimulating the maturation of the DPSCs. Moreover, MEPE accompanied with DSP clearly demonstrates the degree of the differentiation.

Glycogen synthase kinase-3 (GSK-3) regulates TGF-β1-induced differentiation of pulmonary fibroblasts

PubMed Central

Baarsma, Hoeke A; Engelbertink, Lilian HJM; van Hees, Lonneke J; Menzen, Mark H; Meurs, Herman; Timens, Wim; Postma, Dirkje S; Kerstjens, Huib AM; Gosens, Reinoud

2013-01-01

Background Chronic lung diseases such as asthma, COPD and pulmonary fibrosis are characterized by abnormal extracellular matrix (ECM) turnover. TGF-β is a key mediator stimulating ECM production by recruiting and activating lung fibroblasts and initiating their differentiation process into more active myofibroblasts. Glycogen synthase kinase-3 (GSK-3) regulates various intracellular signalling pathways; its role in TGF-β1-induced myofibroblast differentiation is currently largely unknown. Purpose To determine the contribution of GSK-3 signalling in TGF-β1-induced myofibroblast differentiation. Experimental Approach We used MRC5 human lung fibroblasts and primary pulmonary fibroblasts of individuals with and without COPD. Protein and mRNA expression were determined by immunoblotting and RT-PCR analysis respectively. Results Stimulation of MRC5 and primary human lung fibroblasts with TGF-β1 resulted in time- and dose-dependent increases of α-sm-actin and fibronectin expression, indicative of myofibroblast differentiation. Pharmacological inhibition of GSK-3 by SB216763 dose-dependently attenuated TGF-β1-induced expression of these myofibroblasts markers. Moreover, silencing of GSK-3 by siRNA or pharmacological inhibition by CT/CHIR99021 fully inhibited the TGF-β1-induced expression of α-sm-actin and fibronectin. The effect of GSK-3 inhibition on α-sm-actin expression was similar in fibroblasts from individuals with and without COPD. Neither smad, NF-κB nor ERK1/2 were involved in the inhibitory actions of GSK-3 inhibition by SB126763 on myofibroblast differentiation. Rather, SB216763 increased the phosphorylation of CREB, which in its phosphorylated form acts as a functional antagonist of TGF-β/smad signalling. Conclusion and Implication We demonstrate that GSK-3 signalling regulates TGF-β1-induced myofibroblast differentiation by regulating CREB phosphorylation. GSK-3 may constitute a useful target for treatment of chronic lung diseases. PMID:23297769

The role of ROS in hydroquinone-induced inhibition of K562 cell erythroid differentiation.

PubMed

Yu, Chun Hong; Suriguga; Li, Yang; Li, Yi Ran; Tang, Ke Ya; Jiang, Liang; Yi, Zong Chun

2014-03-01

The role of ROS in hydroquinone-induced inhibition of K562 cell erythroid differentiation was investigated. After K562 cells were treated with hydroquinone for 24 h, and hemin was later added to induce erythroid differentiation for 48 h, hydroquinone inhibited hemin-induced hemoglobin synthesis and mRNA expression of γ-globin in K562 cells in a concentration-dependent manner. The 24-h exposure to hydroquinone also caused a concentration-dependent increase at an intracellular ROS level, while the presence of N- acetyl-L-cysteine prevented hydroquinone- induced ROS production in K562 cells. The presence of N-acetyl-L-cysteine also prevented hydroquinone inhibiting hemin-induced hemoglobin synthesis and mRNA expression of γ-globin in K562 cells. These evidences indicated that ROS production played a role in hydroquinone-induced inhibition of erythroid differentiation. Copyright © 2014 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.

Chromosomal position effects in chicken lysozyme gene transgenic mice are correlated with suppression of DNase I hypersensitive site formation.

PubMed Central

Huber, M C; Bosch, F X; Sippel, A E; Bonifer, C

1994-01-01

The complete chicken lysozyme gene locus is expressed copy number dependently and at a high level in macrophages of transgenic mice. Gene expression independent of genomic position can only be achieved by the concerted action of all cis regulatory elements located on the lysozyme gene domain. Position independency of expression is lost if one essential cis regulatory region is deleted. Here we compared the DNase I hypersensitive site (DHS) pattern formed on the chromatin of position independently and position dependently expressed transgenes in order to assess the influence of deletions within the gene domain on active chromatin formation. We demonstrate, that in position independently expressed transgene all DHSs are formed with the authentic relative frequency on all genes. This is not the case for position dependently expressed transgenes. Our results show that the formation of a DHS during cellular differentiation does not occur autonomously. In case essential regulatory elements of the chicken lysozyme gene domain are lacking, the efficiency of DHS formation on remaining cis regulatory elements during myeloid differentiation is reduced and influenced by the chromosomal position. Hence, no individual regulatory element on the lysozyme domain is capable of organizing the chromatin structure of the whole locus in a dominant fashion. Images PMID:7937145

Sexual dimorphism in parental imprint ontogeny and contribution to embryonic development.

PubMed

Bourc'his, Déborah; Proudhon, Charlotte

2008-01-30

Genomic imprinting refers to the functional non-equivalence of parental genomes in mammals that results from the parent-of-origin allelic expression of a subset of genes. Parent-specific expression is dependent on the germ line acquisition of DNA methylation marks at imprinting control regions (ICRs), coordinated by the DNA-methyltransferase homolog DNMT3L. We discuss here how the gender-specific stages of DNMT3L expression may have influenced the various sexually dimorphic aspects of genomic imprinting: (1) the differential developmental timing of methylation establishment at paternally and maternally imprinted genes in each parental germ line, (2) the differential dependence on DNMT3L of parental methylation imprint establishment, (3) the unequal duration of paternal versus maternal methylation imprints during germ cell development, (4) the biased distribution of methylation-dependent ICRs towards the maternal genome, (5) the different genomic organization of paternal versus maternal ICRs, and finally (6) the overwhelming contribution of maternal germ line imprints to development compared to their paternal counterparts.

The Nuclear Receptor AhR Controls Bone Homeostasis by Regulating Osteoclast Differentiation via the RANK/c-Fos Signaling Axis

PubMed Central

Izawa, Takashi; Arakaki, Rieko; Mori, Hiroki; Tsunematsu, Takaaki; Kudo, Yasusei; Tanaka, Eiji

2016-01-01

The aryl hydrocarbon receptor (AhR) pathway plays a key role in receptor activator of NF-κB ligand (RANKL)–mediated osteoclastogenesis. However, the mechanism underlying the regulation of AhR expression in osteoclasts and the signaling pathway through which AhR controls osteoclastogenesis remain unclear. We found that the expression of AhR in bone marrow–derived osteoclasts was upregulated by RANKL at an earlier stage than was the expression of signature osteoclast genes such as those encoding cathepsin K and NFAT, cytoplasmic, calcineurin-dependent 1. In response to RANKL, bone marrow macrophages isolated from AhR−/− mice exhibited impaired phosphorylation of Akt and MAPK as well as NF-κB, whereas their response to M-CSF remained unchanged. Osteoclast differentiation mediated by the AhR signaling pathway was also regulated in an RANKL/c-Fos–dependent manner. Furthermore, ligand activation of AhR by the smoke toxin benzo[a]pyrene accelerated osteoclast differentiation in a receptor-dependent manner, and AhR-dependent regulation of mitochondrial biogenesis in osteoclasts was observed. Moreover, AhR−/− mice exhibited impaired bone healing with delayed endochondral ossification. Taken together, the present results suggest that the RANKL/AhR/c-Fos signaling axis plays a critical role in osteoclastogenesis, thereby identifying the potential of AhR in treating pathological, inflammatory, or metabolic disorders of the bone. PMID:27849171

Control of germline stem cell self-renewal and differentiation in the Drosophila ovary: concerted actions of niche signals and intrinsic factors.

PubMed

Xie, Ting

2013-01-01

In the Drosophila ovary, germline stem cells (GSCs) physically interact with their niche composed of terminal filament cells, cap cells, and possibly GSC-contacting escort cells (ECs). A GSC divides to generate a self-renewing stem cell that remains in the niche and a differentiating daughter that moves away from the niche. The GSC niche provides a bone morphogenetic protein (BMP) signal that maintains GSC self-renewal by preventing stem cell differentiation via repression of the differentiation-promoting gene bag of marbles (bam). In addition, it expresses E-cadherin, which mediates cell adhesion for anchoring GSCs in the niche, enabling continuous self-renewal. GSCs themselves also express different classes of intrinsic factors, including signal transducers, transcription factors, chromatin remodeling factors, translation regulators, and miRNAs, which control self-renewal by strengthening interactions with the niche and repressing various differentiation pathways. Differentiated GSC daughters, known as cystoblasts (CBs), also express distinct classes of intrinsic factors to inhibit self-renewal and promote germ cell differentiation. Surprisingly, GSC progeny are also dependent on their surrounding ECs for proper differentiation at least partly by preventing BMP from diffusing to the differentiated germ cell zone and by repressing ectopic BMP expression. Therefore, both GSC self-renewal and CB differentiation are controlled by collaborative actions of extrinsic signals and intrinsic factors. Copyright © 2012 Wiley Periodicals, Inc.

Dual functions of silver nanoparticles in F9 teratocarcinoma stem cells, a suitable model for evaluating cytotoxicity- and differentiation-mediated cancer therapy

PubMed Central

Han, Jae Woong; Gurunathan, Sangiliyandi; Choi, Yun-Jung; Kim, Jin-Hoi

2017-01-01

Background Silver nanoparticles (AgNPs) exhibit strong antibacterial and anticancer activity owing to their large surface-to-volume ratios and crystallographic surface structure. Owing to their various applications, understanding the mechanisms of action, biological interactions, potential toxicity, and beneficial effects of AgNPs is important. Here, we investigated the toxicity and differentiation-inducing effects of AgNPs in teratocarcinoma stem cells. Materials and methods AgNPs were synthesized and characterized using various analytical techniques such as UV–visible spectroscopy, X-ray diffraction, energy-dispersive X-ray spectroscopy, and transmission electron microscopy. The cellular responses of AgNPs were analyzed by a series of cellular and biochemical assays. Gene and protein expressions were analyzed by reverse transcription-quantitative polymerase chain reaction and western blotting, respectively. Results The AgNPs showed typical crystalline structures and spherical shapes (average size =20 nm). High concentration of AgNPs induced cytotoxicity in a dose-dependent manner by increasing lactate dehydrogenase leakage and reactive oxygen species. Furthermore, AgNPs caused mitochondrial dysfunction, DNA fragmentation, increased expression of apoptotic genes, and decreased expression of antiapoptotic genes. Lower concentrations of AgNPs induced neuronal differentiation by increasing the expression of differentiation markers and decreasing the expression of stem cell markers. Cisplatin reduced the viability of F9 cells that underwent AgNPs-induced differentiation. Conclusion The results showed that AgNPs caused differentially regulated cytotoxicity and induced neuronal differentiation of F9 cells in a concentration-dependent manner. Therefore, AgNPs can be used for differentiation therapy, along with chemotherapeutic agents, for improving cancer treatment by targeting specific chemotherapy-resistant cells within a tumor. Furthermore, understanding the molecular mechanisms of apoptosis and differentiation in stem cells could also help in developing new strategies for cancer stem cell (CSC) therapies. The findings of this study could significantly contribute to the nanomedicine because this study is the first of its kind, and our results will lead to new strategies for cancer and CSC therapies. PMID:29066898

Lung Metabolic Activation as an Early Biomarker of the Acute Respiratory Distress Syndrome and Local Gene Expression Heterogeneity

PubMed Central

Wellman, Tyler J.; de Prost, Nicolas; Tucci, Mauro; Winkler, Tilo; Baron, Rebecca M.; Filipczak, Piotr; Raby, Benjamin; Chu, Jen-hwa; Harris, R. Scott; Musch, Guido; dos Reis Falcao, Luiz F.; Capelozzi, Vera; Venegas, Jose; Melo, Marcos F. Vidal

2016-01-01

Background The acute respiratory distress syndrome (ARDS) is an inflammatory condition comprising diffuse lung edema and alveolar damage. ARDS frequently results from regional injury mechanisms. However, it is unknown whether detectable inflammation precedes lung edema and opacification, and whether topographically differential gene expression consistent with heterogeneous injury occurs in early ARDS. We aimed to determine the temporal relationship between pulmonary metabolic activation and density in a large animal model of early ARDS, and to assess gene expression in differentially activated regions. Methods We produced ARDS in sheep with intravenous LPS (10ng/kg/h) and mechanical ventilation for 20h. Using positron emission tomography, we assessed regional cellular metabolic activation with 2-deoxy-2-[(18)F]fluoro-D-glucose, perfusion and ventilation with 13NN-saline, and aeration using transmission scans. Species-specific micro-array technology was used to assess regional gene expression. Results Metabolic activation preceded detectable increases in lung density (as required for clinical diagnosis) and correlated with subsequent histological injury, suggesting its predictive value for severity of disease progression. Local time-courses of metabolic activation varied, with highly perfused and less aerated dependent lung regions activated earlier than non-dependent regions. These regions of distinct metabolic trajectories demonstrated differential gene expression for known and potential novel candidates for ARDS pathogenesis. Conclusions Heterogeneous lung metabolic activation precedes increases in lung density in the development of ARDS due to endotoxemia and mechanical ventilation. Local differential gene expression occurs in these early stages and reveals molecular pathways relevant to ARDS biology and of potential use as treatment targets. PMID:27611185

Phosphate and calcium are required for TGFbeta-mediated stimulation of ANK expression and function during chondrogenesis.

PubMed

Oca, Paulina; Zaka, Raihana; Dion, Arnold S; Freeman, Theresa A; Williams, Charlene J

2010-08-01

The expression of ANK, a key player in biomineralization, is stimulated by treatment with TGFbeta. The purpose of this study was to determine whether TGFbeta stimulation of ANK expression during chondrogenesis was dependent upon the influx of calcium and phosphate into cells. Treatment of ATDC5 cells with TGFbeta increased ANK expression during all phases of chondrogenic differentiation, particularly at day 14 (proliferation) and day 32 (mineralizing hypertrophy) of culture. Phosphate uptake studies in the presence and absence of phosphonoformic acid (PFA), a competitive inhibitor of the type III Na(+)/Pi channels Pit-1 and Pit-2, indicated that the stimulation of ANK expression by TGFbeta required the influx of phosphate, specifically by the Pit-1 transporter, at all phases of differentiation. At hypertrophy, when alkaline phosphatase is highly expressed, inhibition of its activity with levamisole also abrogated the stimulatory effect of TGFbeta on ANK expression, further illustrating that Pi availability and uptake by the cells is necessary for stimulation of ANK expression in response to TGFbeta. Since previous studies of endochondral ossification in the growth plate have shown that L-type calcium channels are essential for chondrogenesis, we investigated their role in the TGFbeta-stimulated ANK response in ATDC5 cells. Treatment with nifedipine to inhibit calcium influx via the L-type channel Cav1.2 (alpha(1C)) inhibited the TGFbeta stimulated increase in ANK expression at all phases of chondrogenesis. Our findings indicate that TGFbeta stimulation of ANK expression is dependent upon the influx of phosphate and calcium into ATDC5 cells at all stages of differentiation.

Detergents with different chemical properties induce variable degree of cytotoxicity and mRNA expression of lipid-metabolizing enzymes and differentiation markers in cultured keratinocytes.

PubMed

Wei, Tianling; Geijer, Sophia; Lindberg, Magnus; Berne, Berit; Törmä, Hans

2006-12-01

The knowledge how detergents with different chemical properties influence epidermal keratinocytes is sparse. In the present study, the effects of five detergents were examined with respect to cell-toxicity and mRNA expression of key-enzymes in barrier lipid production and keratinocyte differentiation markers. First, the LD(50) for each detergent were determined. Secondly, keratinocytes were exposed to sub-toxic concentrations and the mRNA expression was analysed by real-time PCR after 24 h exposure to the detergents. SLS and CAPB induced a concentration-dependent increase in the expression of enzymes producing cholesterol and ceramides, while transcripts of enzymes producing fatty acids were unaffected. SLES and cocoglucoside increased the expression of certain enzymes involved in cholesterol and fatty acid synthesis while sodium cocoamphoacetate (SCAA) stimulated expression of transcripts involved in fatty acid synthesis. The expression of differentiation markers were increased by SLS, SLES and CAPB, while SCAA and cocoglucoside exhibited no effect. The present findings show that detergents have variable effects on lipid synthesis and keratinocyte differentiation, which could partly explain their barrier destruction potential in vivo.

Critical role of histone demethylase Jmjd3 in the regulation of CD4+ T cell differentiation

PubMed Central

Ding, Xilai; Chepelev, Iouri; Zhou, Xikun; Zhao, Wei; Wei, Gang; Cui, Jun; Zhao, Keji; Wang, Helen Y.; Wang, Rong-Fu

2014-01-01

Epigenetic factors have been implicated in the regulation of CD4+ T cell differentiation. Jmjd3 plays a role in many biological processes, but its in vivo function in T cell differentiation remains unknown. Here, we report that Jmjd3 ablation promotes CD4+ T cell differentiation into Th2 and Th17 cells in the small intestine and colon, and inhibits T cell differentiation into Th1 cells under different cytokine-polarizing conditions and in a Th1-dependent colitis model. Jmjd3 deficiency also restrains the plasticity of the conversion of Th2, Th17 or Treg cells to Th1 cells. The skewing of T cell differentiation is concomitant with changes in the expression of key transcription factors and cytokines. H3K27me3 and H3K4me3 levels in Jmjd3-deficient cells are correlated with altered gene expression through interactions with specific transcription factors. Our results identify Jmjd3 as an epigenetic factor in T cell differentiation via changes in histone methylation and target gene expression. PMID:25531312

Differential expression of pancreatic protein and chemosensing receptor mRNAs in NKCC1-null intestine.

PubMed

Bradford, Emily M; Vairamani, Kanimozhi; Shull, Gary E

2016-02-15

To investigate the intestinal functions of the NKCC1 Na(+)-K(+)-2Cl cotransporter (SLC12a2 gene), differential mRNA expression changes in NKCC1-null intestine were analyzed. Microarray analysis of mRNA from intestines of adult wild-type mice and gene-targeted NKCC1-null mice (n = 6 of each genotype) was performed to identify patterns of differential gene expression changes. Differential expression patterns were further examined by Gene Ontology analysis using the online Gorilla program, and expression changes of selected genes were verified using northern blot analysis and quantitative real time-polymerase chain reaction. Histological staining and immunofluorescence were performed to identify cell types in which upregulated pancreatic digestive enzymes were expressed. Genes typically associated with pancreatic function were upregulated. These included lipase, amylase, elastase, and serine proteases indicative of pancreatic exocrine function, as well as insulin and regenerating islet genes, representative of endocrine function. Northern blot analysis and immunohistochemistry showed that differential expression of exocrine pancreas mRNAs was specific to the duodenum and localized to a subset of goblet cells. In addition, a major pattern of changes involving differential expression of olfactory receptors that function in chemical sensing, as well as other chemosensing G-protein coupled receptors, was observed. These changes in chemosensory receptor expression may be related to the failure of intestinal function and dependency on parenteral nutrition observed in humans with SLC12a2 mutations. The results suggest that loss of NKCC1 affects not only secretion, but also goblet cell function and chemosensing of intestinal contents via G-protein coupled chemosensory receptors.

The expression of hematopoietic progenitor cell antigen CD34 is regulated by DNA methylation in a site-dependent manner in gastrointestinal stromal tumours.

PubMed

Bure, Irina; Braun, Alexander; Kayser, Claudia; Geddert, Helene; Schaefer, Inga-Marie; Cameron, Silke; Ghadimi, Michael B; Ströbel, Philipp; Werner, Martin; Hartmann, Arndt; Wiemann, Stefan; Agaimy, Abbas; Haller, Florian; Moskalev, Evgeny A

2017-12-01

The anatomic site-dependent expression of hematopoietic progenitor cell antigen CD34 is a feature of gastrointestinal stromal tumours (GISTs). The basis for the differential CD34 expression is only incompletely understood. This study aimed at understanding the regulation of CD34 in GISTs and clarification of its site-dependent expression. Two sample sets of primary GISTs were interrogated including 52 fresh-frozen and 134 paraffin-embedded and formalin-fixed specimens. DNA methylation analysis was performed by HumanMethylation450 BeadChip array in three cell lines derived from gastric and intestinal GISTs, and differentially methylated CpG sites were established upstream of CD34. The methylation degree was further quantified by pyrosequencing, and inverse correlation with CD34 mRNA and protein abundance was revealed. The gene's expression could be activated upon induction of DNA hypomethylation with 5-aza-2'-deoxycytidine in GIST-T1 cells. In patient samples, a strong inverse correlation of DNA methylation degree with immunohistochemically evaluated CD34 expression was documented. Both CD34 expression and DNA methylation levels were specific to the tumours' anatomic location and mutation status. A constant decrease in methylation levels was observed ranging from almost 100% hypermethylation in intestinal GISTs from duodenum to hypomethylation in rectum. CD34 was heavily methylated in gastric PDGFRA-mutant GISTs in comparison to hypomethylated KIT-mutant counterparts. Next to CD34 hypermethylation, miR-665 was predicted and experimentally confirmed to target CD34 mRNA in GIST-T1 cells. Our results suggest that CD34 expression in GISTs may undergo a complex control by DNA methylation and miR-665. Differential methylation and expression of CD34 in GISTs along the gastrointestinal tract axis and in tumours that harbour different gain-of-function mutations suggest the origin from different cell populations in the gastrointestinal tract. © 2017 UICC.

Differential regulation of cyclin-dependent kinase inhibitors in neuroblastoma cells

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

Qiao, Lan; Department of Pharmaceutical Sciences, Jilin University, Changchun 130021; Paul, Pritha

2013-05-31

Highlights: •GRP-R signaling differentially regulated the expression of p21 and p27. •Silencing GRP/GRP-R downregulated p21, while p27 expression was upregulated. •Inhibition of GRP/GRP-R signaling enhanced PTEN expression, correlative to the increased expression of p27. •PTEN and p27 co-localized in cytoplasm and silencing PTEN decreased p27 expression. -- Abstract: Gastrin-releasing peptide (GRP) and its receptor (GRP-R) are highly expressed in undifferentiated neuroblastoma, and they play critical roles in oncogenesis. We previously reported that GRP activates the PI3K/AKT signaling pathway to promote DNA synthesis and cell cycle progression in neuroblastoma cells. Conversely, GRP-R silencing induces cell cycle arrest. Here, we speculated thatmore » GRP/GRP-R signaling induces neuroblastoma cell proliferation via regulation of cyclin-dependent kinase (CDK) inhibitors. Surprisingly, we found that GRP/GRP-R differentially induced expressions of p21 and p27. Silencing GRP/GRP-R decreased p21, but it increased p27 expressions in neuroblastoma cells. Furthermore, we found that the intracellular localization of p21 and p27 in the nuclear and cytoplasmic compartments, respectively. In addition, we found that GRP/GRP-R silencing increased the expression and accumulation of PTEN in the cytoplasm of neuroblastoma cells where it co-localized with p27, thus suggesting that p27 promotes the function of PTEN as a tumor suppressor by stabilizing PTEN in the cytoplasm. GRP/GRP-R regulation of CDK inhibitors and tumor suppressor PTEN may be critical for tumoriogenesis of neuroblastoma.« less

Increased numbers of preexisting memory CD8 T cells and decreased T-bet expression can restrain terminal differentiation of secondary effector and memory CD8 T cells.

PubMed

Joshi, Nikhil S; Cui, Weiguo; Dominguez, Claudia X; Chen, Jonathan H; Hand, Timothy W; Kaech, Susan M

2011-10-15

Memory CD8 T cells acquire effector memory cell properties after reinfection and may reach terminally differentiated, senescent states ("Hayflick limit") after multiple infections. The signals controlling this process are not well understood, but we found that the degree of secondary effector and memory CD8 T cell differentiation was intimately linked to the amount of T-bet expressed upon reactivation and preexisting memory CD8 T cell number (i.e., primary memory CD8 T cell precursor frequency) present during secondary infection. Compared with naive cells, memory CD8 T cells were predisposed toward terminal effector (TE) cell differentiation because they could immediately respond to IL-12 and induce T-bet, even in the absence of Ag. TE cell formation after secondary (2°) or tertiary infections was dependent on increased T-bet expression because T-bet(+/-) cells were resistant to these phenotypic changes. Larger numbers of preexisting memory CD8 T cells limited the duration of 2° infection and the amount of IL-12 produced, and consequently, this reduced T-bet expression and the proportion of 2° TE CD8 T cells that formed. Together, these data show that over repeated infections, memory CD8 T cell quality and proliferative fitness is not strictly determined by the number of serial encounters with Ag or cell divisions, but is a function of the CD8 T cell differentiation state, which is genetically controlled in a T-bet-dependent manner. This differentiation state can be modulated by preexisting memory CD8 T cell number and the intensity of inflammation during reinfection. These results have important implications for vaccinations involving prime-boost strategies.

Cardiac differentiation of cardiosphere-derived cells in scaffolds mimicking morphology of the cardiac extracellular matrix.

PubMed

Xu, Yanyi; Patnaik, Sourav; Guo, Xiaolei; Li, Zhenqing; Lo, Wilson; Butler, Ryan; Claude, Andrew; Liu, Zhenguo; Zhang, Ge; Liao, Jun; Anderson, Peter M; Guan, Jianjun

2014-08-01

Stem cell therapy has the potential to regenerate heart tissue after myocardial infarction (MI). The regeneration is dependent upon cardiac differentiation of the delivered stem cells. We hypothesized that timing of the stem cell delivery determines the extent of cardiac differentiation as cell differentiation is dependent on matrix properties such as biomechanics, structure and morphology, and these properties in cardiac extracellular matrix (ECM) continuously vary with time after MI. In order to elucidate the relationship between ECM properties and cardiac differentiation, we created an in vitro model based on ECM-mimicking fibers and a type of cardiac progenitor cell, cardiosphere-derived cells (CDCs). A simultaneous fiber electrospinning and cell electrospraying technique was utilized to fabricate constructs. By blending a highly soft hydrogel with a relatively stiff polyurethane and modulating fabrication parameters, tissue constructs with similar cell adhesion property but different global modulus, single fiber modulus, fiber density and fiber alignment were achieved. The CDCs remained alive within the constructs during a 1week culture period. CDC cardiac differentiation was dependent on the scaffold modulus, fiber volume fraction and fiber alignment. Two constructs with relatively low scaffold modulus, ∼50-60kPa, most significantly directed the CDC differentiation into mature cardiomyocytes as evidenced by gene expressions of cardiac troponin T (cTnT), calcium channel (CACNA1c) and cardiac myosin heavy chain (MYH6), and protein expressions of cardiac troponin I (cTnI) and connexin 43 (CX43). Of these two low-modulus constructs, the extent of differentiation was greater for lower fiber alignment and higher fiber volume fraction. These results suggest that cardiac ECM properties may have an effect on cardiac differentiation of delivered stem cells. Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

MicroRNA network changes in the brain stem underlie the development of hypertension.

PubMed

DeCicco, Danielle; Zhu, Haisun; Brureau, Anthony; Schwaber, James S; Vadigepalli, Rajanikanth

2015-09-01

Hypertension is a major chronic disease whose molecular mechanisms remain poorly understood. We compared neuroanatomical patterns of microRNAs in the brain stem of the spontaneous hypertensive rat (SHR) to the Wistar Kyoto rat (WKY, control). We quantified 419 well-annotated microRNAs in the nucleus of the solitary tract (NTS) and rostral ventrolateral medulla (RVLM), from SHR and WKY rats, during three main stages of hypertension development. Changes in microRNA expression were stage- and region-dependent, with a majority of SHR vs. WKY differential expression occurring at the hypertension onset stage in NTS versus at the prehypertension stage in RVLM. Our analysis identified 24 microRNAs showing time-dependent differential expression in SHR compared with WKY in at least one brain region. We predicted potential gene regulatory targets corresponding to catecholaminergic processes, neuroinflammation, and neuromodulation using the miRWALK and RNA22 databases, and we tested those bioinformatics predictions using high-throughput quantitative PCR to evaluate correlations of differential expression between the microRNAs and their predicted gene targets. We found a novel regulatory network motif consisting of microRNAs likely downregulating a negative regulator of prohypertensive processes such as angiotensin II signaling and leukotriene-based inflammation. Our results provide new evidence on the dynamics of microRNA expression in the development of hypertension and predictions of microRNA-mediated regulatory networks playing a region-dependent role in potentially altering brain-stem cardiovascular control circuit function leading to the development of hypertension. Copyright © 2015 the American Physiological Society.

The hedgehog system machinery controls transforming growth factor-β-dependent myofibroblastic differentiation in humans: involvement in idiopathic pulmonary fibrosis.

PubMed

Cigna, Natacha; Farrokhi Moshai, Elika; Brayer, Stéphanie; Marchal-Somme, Joëlle; Wémeau-Stervinou, Lidwine; Fabre, Aurélie; Mal, Hervé; Lesèche, Guy; Dehoux, Monique; Soler, Paul; Crestani, Bruno; Mailleux, Arnaud A

2012-12-01

Idiopathic pulmonary fibrosis (IPF) is a devastating disease of unknown cause. Key signaling developmental pathways are aberrantly expressed in IPF. The hedgehog pathway plays a key role during fetal lung development and may be involved in lung fibrogenesis. We determined the expression pattern of several Sonic hedgehog (SHH) pathway members in normal and IPF human lung biopsies and primary fibroblasts. The effect of hedgehog pathway inhibition was assayed by lung fibroblast proliferation and differentiation with and without transforming growth factor (TGF)-β1. We showed that the hedgehog pathway was reactivated in the IPF lung. Importantly, we deciphered the cross talk between the hedgehog and TGF-β pathway in human lung fibroblasts. TGF-β1 modulated the expression of key components of the hedgehog pathway independent of Smoothened, the obligatory signal transducer of the pathway. Smoothened was required for TGF-β1-induced myofibroblastic differentiation of control fibroblasts, but differentiation of IPF fibroblasts was partially resistant to Smoothened inhibition. Furthermore, functional hedgehog pathway machinery from the primary cilium, as well as GLI-dependent transcription in the nucleus, was required for the TGF-β1 effects on normal and IPF fibroblasts during myofibroblastic differentiation. These data identify the GLI transcription factors as potential therapeutic targets in lung fibrosis. Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Cell recognition molecule L1 promotes embryonic stem cell differentiation through the regulation of cell surface glycosylation

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

Li, Ying; Department of Clinical Laboratory, Second Affiliated Hospital of Dalian Medical University, Dalian 116023; Huang, Xiaohua

2013-10-25

Highlights: •Down-regulating FUT9 and ST3Gal4 expression blocks L1-induced neuronal differentiation of ESCs. •Up-regulating FUT9 and ST3Gal4 expression in L1-ESCs depends on the activation of PLCγ. •L1 promotes ESCs to differentiate into neuron through regulating cell surface glycosylation. -- Abstract: Cell recognition molecule L1 (CD171) plays an important role in neuronal survival, migration, differentiation, neurite outgrowth, myelination, synaptic plasticity and regeneration after injury. Our previous study has demonstrated that overexpressing L1 enhances cell survival and proliferation of mouse embryonic stem cells (ESCs) through promoting the expression of FUT9 and ST3Gal4, which upregulates cell surface sialylation and fucosylation. In the present study,more » we examined whether sialylation and fucosylation are involved in ESC differentiation through L1 signaling. RNA interference analysis showed that L1 enhanced differentiation of ESCs into neurons through the upregulation of FUT9 and ST3Gal4. Furthermore, blocking the phospholipase Cγ (PLCγ) signaling pathway with either a specific PLCγ inhibitor or knockdown PLCγ reduced the expression levels of both FUT9 and ST3Gal4 mRNAs and inhibited L1-mediated neuronal differentiation. These results demonstrate that L1 promotes neuronal differentiation from ESCs through the L1-mediated enhancement of FUT9 and ST3Gal4 expression.« less

The Aux/IAA gene rum1 involved in seminal and lateral root formation controls vascular patterning in maize (Zea mays L.) primary roots.

PubMed

Zhang, Yanxiang; Paschold, Anja; Marcon, Caroline; Liu, Sanzhen; Tai, Huanhuan; Nestler, Josefine; Yeh, Cheng-Ting; Opitz, Nina; Lanz, Christa; Schnable, Patrick S; Hochholdinger, Frank

2014-09-01

The maize (Zea mays L.) Aux/IAA protein RUM1 (ROOTLESS WITH UNDETECTABLE MERISTEMS 1) controls seminal and lateral root initiation. To identify RUM1-dependent gene expression patterns, RNA-Seq of the differentiation zone of primary roots of rum1 mutants and the wild type was performed in four biological replicates. In total, 2 801 high-confidence maize genes displayed differential gene expression with Fc ≥2 and FDR ≤1%. The auxin signalling-related genes rum1, like-auxin1 (lax1), lax2, (nam ataf cuc 1 nac1), the plethora genes plt1 (plethora 1), bbm1 (baby boom 1), and hscf1 (heat shock complementing factor 1) and the auxin response factors arf8 and arf37 were down-regulated in the mutant rum1. All of these genes except nac1 were auxin-inducible. The maize arf8 and arf37 genes are orthologues of Arabidopsis MP/ARF5 (MONOPTEROS/ARF5), which controls the differentiation of vascular cells. Histological analyses of mutant rum1 roots revealed defects in xylem organization and the differentiation of pith cells around the xylem. Moreover, histochemical staining of enlarged pith cells surrounding late metaxylem elements demonstrated that their thickened cell walls displayed excessive lignin deposition. In line with this phenotype, rum1-dependent mis-expression of several lignin biosynthesis genes was observed. In summary, RNA-Seq of RUM1-dependent gene expression in maize primary roots, in combination with histological and histochemical analyses, revealed the specific regulation of auxin signal transduction components by RUM1 and novel functions of RUM1 in vascular development. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Compound-specific effects of diverse neurodevelopmental toxicants on global gene expression in the neural embryonic stem cell test (ESTn)

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

Theunissen, P.T., E-mail: Peter.Theunissen@rivm.nl; Department of Toxicogenomics, Maastricht University, Maastricht; Robinson, J.F.

Alternative assays for developmental toxicity testing are needed to reduce animal use in regulatory toxicology. The in vitro murine neural embryonic stem cell test (ESTn) was designed as an alternative for neurodevelopmental toxicity testing. The integration of toxicogenomic-based approaches may further increase predictivity as well as provide insight into underlying mechanisms of developmental toxicity. In the present study, we investigated concentration-dependent effects of six mechanistically diverse compounds, acetaldehyde (ACE), carbamazepine (CBZ), flusilazole (FLU), monoethylhexyl phthalate (MEHP), penicillin G (PENG) and phenytoin (PHE), on the transcriptome and neural differentiation in the ESTn. All compounds with the exception of PENG altered ESTnmore » morphology (cytotoxicity and neural differentiation) in a concentration-dependent manner. Compound induced gene expression changes and corresponding enriched gene ontology biological processes (GO–BP) were identified after 24 h exposure at equipotent differentiation-inhibiting concentrations of the compounds. Both compound-specific and common gene expression changes were observed between subsets of tested compounds, in terms of significance, magnitude of regulation and functionality. For example, ACE, CBZ and FLU induced robust changes in number of significantly altered genes (≥ 687 genes) as well as a variety of GO–BP, as compared to MEHP, PHE and PENG (≤ 55 genes with no significant changes in GO–BP observed). Genes associated with developmentally related processes (embryonic morphogenesis, neuron differentiation, and Wnt signaling) showed diverse regulation after exposure to ACE, CBZ and FLU. In addition, gene expression and GO–BP enrichment showed concentration dependence, allowing discrimination of non-toxic versus toxic concentrations on the basis of transcriptomics. This information may be used to define adaptive versus toxic responses at the transcriptome level.« less

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.

Differential expression of the phthalate syndrome in male Sprague-Dawley and Wistar rats after in utero DEHP exposure

EPA Science Inventory

Exposure to phthalate esters during sexual differentiation disrupts testosterone and insulin-like three hormones resulting in malformations of androgen- and insulin-like three-dependent tissues. The current study was designed to test the hypothesis that gubernacular lesions would...

Regulation of ITAM adaptor molecules and their receptors by inhibition of calcineurin-NFAT signalling during late stage osteoclast differentiation

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

Zawawi, M.S.F.; Discipline of Anatomy and Pathology, School of Medical Sciences, University of Adelaide, Adelaide, SA 5005; Dharmapatni, A.A.S.S.K.

2012-10-19

Highlights: Black-Right-Pointing-Pointer Calcineurin/NFAT inhibitors FK506 and VIVIT treated human PBMC derived osteoclasts in vitro. Black-Right-Pointing-Pointer Differential regulation of ITAM receptors and adaptor molecules by calcineurin/NFAT inhibitors. Black-Right-Pointing-Pointer FK506 and VIVIT suppress ITAM factors during late phase osteoclast differentiation. -- Abstract: Osteoclasts are specialised bone resorptive cells responsible for both physiological and pathological bone loss. Osteoclast differentiation and activity is dependent upon receptor activator NF-kappa-B ligand (RANKL) interacting with its receptor RANK to induce the transcription factor, nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 (NFATc1). The immunoreceptor tyrosine-based activation motif (ITAM)-dependent pathway has been identified as a co-stimulatory pathway inmore » osteoclasts. Osteoclast-associated receptor (OSCAR) and triggering receptor expressed in myeloid cells (TREM2) are essential receptors that pair with adaptor molecules Fc receptor common gamma chain (FcR{gamma}) and DNAX-activating protein 12 kDa (DAP12) respectively to induce calcium signalling. Treatment with calcineurin-NFAT inhibitors, Tacrolimus (FK506) and the 11R-VIVIT (VIVIT) peptide, reduces NFATc1 expression consistent with a reduction in osteoclast differentiation and activity. This study aimed to investigate the effects of inhibiting calcineurin-NFAT signalling on the expression of ITAM factors and late stage osteoclast genes including cathepsin K (CathK), Beta 3 integrin ({beta}3) and Annexin VIII (AnnVIII). Human peripheral blood mononuclear cells (PBMCs) were differentiated with RANKL and macrophage-colony stimulating factor (M-CSF) over 10 days in the presence or absence of FK506 or VIVIT. Osteoclast formation (as assessed by tartrate resistant acid phosphatase (TRAP)) and activity (assessed by dentine pit resorption) were significantly reduced with treatment. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis demonstrated that FK506 treatment significantly (p < 0.05) reduced the expression of NFATc1, CathK, OSCAR, FcR{gamma}, TREM2 and DAP12 during the terminal stage of osteoclast formation. VIVIT treatment significantly (p < 0.05) decreased CathK, OSCAR, FcR{gamma}, and AnnVIII, gene expression. This data suggest FK506 and VIVIT act differently in targeting the calcineurin-NFAT signalling cascade to suppress key mediators of the ITAM pathway during late stage osteoclast differentiation and this is associated with a reduction in both osteoclast differentiation and activity.« less

Heparan sulfates and the decrease of N-glycans promote early adipogenic differentiation rather than myogenesis of murine myogenic progenitor cells.

PubMed

Grassot, Vincent; Bouchatal, Amel; Da Silva, Anne; Chantepie, Sandrine; Papy-Garcia, Dulce; Maftah, Abderrahman; Gallet, Paul-François; Petit, Jean-Michel

In vitro, extracted muscle satellite cells, called myogenic progenitor cells, can differentiate either in myotubes or preadipocytes, depending on environmental factors and the medium. Transcriptomic analyses on glycosylation genes during satellite cells differentiation into myotubes showed that 31 genes present a significant variation of expression at the early stages of murine myogenic progenitor cells (MPC) differentiation. In the present study, we analyzed the expression of 383 glycosylation related genes during murine MPC differentiation into preadipocytes and compared the data to those previously obtained during their differentiation into myotubes. Fifty-six glycosylation related genes are specifically modified in their expression during early adipogenesis. The variations correspond mainly to: a decrease of N-glycans, and of alpha (2,3) and (2,6) linked sialic acids, and to a high level of heparan sulfates. A high amount of TGF-β1 in extracellular media during early adipogenesis was also observed. It seems that the increases of heparan sulfates and TGF-β1 favor pre-adipogenic differentition of MPC and possibly prevent their myogenic differentiation. Copyright © 2016 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

Control of regulatory T cell lineage commitment and maintenance.

PubMed

Josefowicz, Steven Z; Rudensky, Alexander

2009-05-01

Foxp3-expressing regulatory T (Treg) cells suppress pathology mediated by immune responses against self and foreign antigens and commensal microorganisms. Sustained expression of the transcription factor Foxp3, a key distinguishing feature of Treg cells, is required for their differentiation and suppressor function. In addition, Foxp3 expression prevents deviation of Treg cells into effector T cell lineages and confers dependence of Treg cell survival and expansion on growth factors, foremost interleukin-2, provided by activated effector T cells. In this review we discuss Treg cell differentiation and maintenance with a particular emphasis on molecular regulation of Foxp3 expression, arguably a key to mechanistic understanding of biology of regulatory T cells.

Differential regulation of cyclo-oxygenase-2 and 5-lipoxygenase-activating protein (FLAP) expression by glucocorticoids in monocytic cells.

PubMed

Goppelt-Struebe, M; Schaefer, D; Habenicht, A J

1997-10-01

1. The objective of the present study was to determine the effects of dexamethasone on key constituents of prostaglandin and leukotriene biosynthesis, cyclo-oxygenase-2 (COX-2) and 5-lipoxygenase activating protein (FLAP). The human monocytic cell line THP-1 was used as a model system. mRNA and protein levels of COX-2 and FLAP were determined by Northern and Western blot analyses, respectively. 2. Low levels of COX-2 and FLAP mRNA were expressed in undifferentiated THP-1 cells, but were induced upon differentiation of the cells along the monocytic pathway by treatment with phorbol ester (TPA, 5 nM). Maximal expression was observed after two days. 3. Coincubation of the undifferentiated cells with dexamethasone (10(-9) - 10(-6) M) and phorbol ester prevented induction of COX-2 mRNA, but did not affect the induction of FLAP mRNA. 4. Dexamethasone downregulated COX-2 mRNA and protein in differentiated, monocyte-like THP-1 cells. In contrast, FLAP mRNA and protein were upregulated by dexamethasone in differentiated THP-1 cells. After 24 h, FLAP mRNA levels were increased more than 2 fold. Dexamethasone did not change 5-lipoxygenase mRNA expression. 5. Release of prostaglandin E2 (PGE2) and peptidoleukotrienes was determined in cell culture supernatants of differentiated THP-1 cells by ELISA. Calcium ionophore-dependent PGE2 synthesis was associated with COX-2 expression, whereas COX-1 and COX-2 seemed to participate in arachidonic acid-dependent PGE2 synthesis. Very low levels of peptidoleukotrienes were released from differentiated THP-1 cells upon incubation with ionophore. Treatment with dexamethasone did not significantly affect leukotriene release. 6. These data provide evidence that prostaglandin synthesis is consistently downregulated by glucocorticoids. However, the glucocorticoid-mediated induction of FLAP may provide a mechanism to maintain leukotriene biosynthesis through more efficient transfer of arachidonic acid to the 5-lipoxygenase reaction, in spite of inhibitory effects on other enzymes of the biosynthetic pathway.

Mechanisms controlling neurite outgrowth in a pheochromocytoma cell line: The role of TRPC channels

PubMed Central

Kumar, Sanjay; Chakraborty, Saikat; Barbosa, Cindy; Brustovetsky, Tatiana; Brustovetsky, Nickolay; Obukhov, Alexander G.

2014-01-01

Transient Receptor Potential Canonical (TRPC) channels are implicated in modulating neurite outgrowth. The expression pattern of TRPC changes significantly during brain development, suggesting that fine-tuning TRPC expression may be important for orchestrating neuritogenesis. To study how alterations in the TRPC expression pattern affect neurite outgrowth, we used nerve growth factor (NGF)-differentiated rat pheochromocytoma 12 (PC12) cells, a model system for neuritogenesis. In PC12 cells, NGF markedly up-regulated TRPC1 and TRPC6 expression, but down-regulated TRPC5 expression while promoting neurite outgrowth. Overexpression of TRPC1 augmented, whereas TRPC5 overexpression decelerated NGF-induced neurite outgrowth. Conversely, shRNA-mediated knockdown of TRPC1 decreased, whereas shRNA-mediated knockdown of TRPC5 increased NGF-induced neurite extension. Endogenous TRPC1 attenuated the anti-neuritogenic effect of overexpressed TRPC5 in part by forming the heteromeric TRPC1–TRPC5 channels. Previous reports suggested that TRPC6 may facilitate neurite outgrowth. However, we found that TRPC6 overexpression slowed down neuritogenesis, whereas dominant negative TRPC6 (DN-TRPC6) facilitated neurite outgrowth in NGF-differentiated PC12 cells. Consistent with these findings, hyperforin, a neurite outgrowth promoting factor, decreased TRPC6 expression in NGF-differentiated PC12 cells. Using pharmacological and molecular biological approaches, we determined that NGF up-regulated TRPC1 and TRPC6 expression via a p75NTR-IKK2-dependent pathway that did not involve TrkA receptor signaling in PC12 cells. Similarly, NGF up-regulated TRPC1 and TRPC6 via an IKK2 dependent pathway in primary cultured hippocampal neurons. Thus, our data suggest that a balance of TRPC1, TRPC5, and TRPC6 expression determines neurite extension rate in neural cells, with TRPC6 emerging as an NGF-dependent “molecular damper” maintaining a submaximal velocity of neurite extension. PMID:21618530

Similarities in temperature-dependent gene expression plasticity across timescales in threespine stickleback (Gasterosteus aculeatus).

PubMed

Metzger, David C H; Schulte, Patricia M

2018-04-14

Phenotypic plasticity occurs at a variety of timescales, but little is known about the degree to which plastic responses at different timescales are associated with similar underlying molecular processes, which is critical for assessing the effects of plasticity on evolutionary trajectories. To address this issue, we identified differential gene expression in response to developmental temperature in the muscle transcriptome of adult threespine stickleback (Gasterosteus aculeatus) exposed to 12, 18 and 24°C until hatch and then held at 18°C for 9 months and compared these results to differential gene expression in response to adult thermal acclimation in stickleback developed at 18°C and then acclimated to 5 and 25°C as adults. Adult thermal acclimation affected the expression of 7,940 and 7,015 genes in response to cold and warm acclimation, respectively, and 4,851 of these genes responded in both treatments. In contrast, the expression of only 33 and 29 genes was affected by cold and warm development, respectively. The majority of the genes affected by developmental temperature were also affected by adult acclimation temperature. Many genes that were differentially expressed as a result of adult acclimation were associated with previously identified temperature-dependent effects on DNA methylation patterns, suggesting a role of epigenetic mechanisms in regulating gene expression plasticity during acclimation. Taken together, these results demonstrate similarities between the persistent effects of developmental plasticity on gene expression and the effects of adult thermal acclimation, emphasizing the potential for mechanistic links between plasticity acting at these different life stages. © 2018 John Wiley & Sons Ltd.

[Cytokine-mediated regulation of expression of Gfi1 and U2afll4 genes activated by T-cells with different differentiation status in vitro].

PubMed

Yurova, K A; Sokhonevich, N A; Khaziakhmatova, O G; Litvinova, L S

2016-01-01

The dose-dependent effects of cytokines (IL-2, IL-7, and IL-15), which have a common g-chain, on mRNA expression of U2afll4 and GFi1 genes involved in regulation of alternative splicing of the Ptprc gene, have been investigated in vitro using T-lymphocyte cultures with different degrees of differentiation. IL-2, IL-7, and IL-15 caused a similar unidirectional inhibitory effect of various severity on restimulated CD45RO+ T-cells exposed to an antigen-independent activation; they caused a dose-dependent decrease of the U2af1l4 gene expression, and an increase of Gfi1 gene expression. This may suggest formation of active forms of the CD45 receptor, and also limitation of the formation of low-molecular short splice variants of the CD45RO receptor. Under conditions of antigen-independent stimulation of naive CD45RA+-cells rIL-7 and IL-15 exhibited opposite effects on U2af1l4 and Gfi1 gene expression. The increase of IL-7 concentrations in the incubation medium of naive cells was accompanied by a decrease in expression of both genes. IL-15 IL-7 exhibited opposite effects. Cytokines possessing a common g-chain (IL-2, IL-7, and IL-15) prevented antigen-independent differentiation of naive T-cells, by preventing the formation of polyclonal "surrogate" cells. In general, the study of the molecular mechanisms of genetic control determining homeostatic processes of T-cells in response to exposure to antigenic or non-antigenic treatments may be important for construction of a general model of self-maintenance and differentiation of immune cells.

Temporal regulation of Stat5 activity in determination of cell differentiation program

PubMed Central

Hoshino, Akemi; Fujii, Hodaka

2007-01-01

Although Stat5 is activated by various cytokines, only ethrytopoietin (Epo) and a small number of cytokines induce Stat5-dependent erythroid differentiation. Here, by using a reporter gene system to monitor transcriptional activity of Stat5, we showed that Epo but not interleukin (IL)-3 supports sustained activation of Stat5, which induces globin gene expression. IL-3 or IL-2 stimulation inhibits Epo-induced globin gene expression. The acidic region of the IL-2 receptor β chain was essential for this inhibition. These results underscore the importance of temporal regulation of Stat activity for regulation of cytokine-specific cell differentiation. PMID:17511959

AhR-mediated gene expression in the developing mouse telencephalon.

PubMed

Gohlke, Julia M; Stockton, Pat S; Sieber, Stella; Foley, Julie; Portier, Christopher J

2009-11-01

We hypothesize that TCDD-induced developmental neurotoxicity is modulated through an AhR-dependent interaction with key regulatory neuronal differentiation pathways during telencephalon development. To test this hypothesis we examined global gene expression in both dorsal and ventral telencephalon tissues in E13.5 AhR-/- and wildtype mice exposed to TCDD or vehicle. Consistent with previous biochemical, pathological and behavioral studies, our results suggest TCDD initiated changes in gene expression in the developing telencephalon are primarily AhR-dependent, as no statistically significant gene expression changes are evident after TCDD exposure in AhR-/- mice. Based on a gene regulatory network for neuronal specification in the developing telencephalon, the present analysis suggests differentiation of GABAergic neurons in the ventral telencephalon is compromised in TCDD exposed and AhR-/- mice. In addition, our analysis suggests Sox11 may be directly regulated by AhR based on gene expression and comparative genomics analyses. In conclusion, this analysis supports the hypothesis that AhR has a specific role in the normal development of the telencephalon and provides a mechanistic framework for neurodevelopmental toxicity of chemicals that perturb AhR signaling.

Development and regeneration of the electric organ.

PubMed

Zakon, H H; Unguez, G A

1999-05-01

The electric organ has evolved independently from muscle in at least six lineages of fish. How does a differentiated muscle cell change its fate to become an electrocyte? Is the process by which this occurs similar in different lineages? We have begun to answer these questions by studying the formation and maintenance of electrocytes in the genus Sternopygus, a weakly electric teleost. Electrocytes arise from the fusion of fully differentiated muscle fibers, mainly those expressing fast isoforms of myosin. Electrocytes briefly co-express sarcomeric proteins, such as myosin and tropomyosin, and keratin, a protein not found in mature muscle. The sarcomeric proteins are subsequently down-regulated, but keratin expression persists. We investigated whether the maintenance of the electrocyte phenotype depends on innervation. We found that, after spinal cord transection, which silences the electromotor neurons that innervate the electrocytes, or destruction of the spinal cord, which denervates the electrocytes, mature electrocytes re-express sarcomeric myosin and tropomyosin, although keratin expression persists. Ultrastructural examination of denervated electrocytes revealed nascent sarcomeres. Thus, the maintenance of the electrocyte phenotype depends on neural activity.

DREAM mediates cAMP-dependent, Ca2+-induced stimulation of GFAP gene expression and regulates cortical astrogliogenesis.

PubMed

Cebolla, Beatriz; Fernández-Pérez, Antonio; Perea, Gertrudis; Araque, Alfonso; Vallejo, Mario

2008-06-25

In the developing mouse brain, once the generation of neurons is mostly completed during the prenatal period, precisely coordinated signals act on competent neural precursors to direct their differentiation into astrocytes, which occurs mostly after birth. Among these signals, those provided by neurotrophic cytokines and bone morphogenetic proteins appear to have a key role in triggering the neurogenic to gliogenic switch and in regulating astrocyte numbers. In addition, we have reported previously that the neurotrophic peptide pituitary adenylate cyclase-activating polypeptide (PACAP) is able to promote astrocyte differentiation of cortical precursors via activation of a cAMP-dependent pathway. Signals acting on progenitor cells of the developing cortex to generate astrocytes activate glial fibrillary acidic protein (GFAP) gene expression, but the transcriptional mechanisms that regulate this activation are unclear. Here, we identify the previously known transcriptional repressor downstream regulatory element antagonist modulator (DREAM) as an activator of GFAP gene expression. We found that DREAM occupies specific sites on the GFAP promoter before and after differentiation is initiated by exposure of cortical progenitor cells to PACAP. PACAP raises intracellular calcium concentration via a mechanism that requires cAMP, and DREAM-mediated transactivation of the GFAP gene requires the integrity of calcium-binding domains. Cortical progenitor cells from dream(-/-) mice fail to express GFAP in response to PACAP. Moreover, the neonatal cortex of dream(-/-) mice exhibits a reduced number of astrocytes and increased number of neurons. These results identify the PACAP-cAMP-Ca(2+)-DREAM cascade as a new pathway to activate GFAP gene expression during astrocyte differentiation.

Differential expression of pancreatic protein and chemosensing receptor mRNAs in NKCC1-null intestine

PubMed Central

Bradford, Emily M; Vairamani, Kanimozhi; Shull, Gary E

2016-01-01

AIM: To investigate the intestinal functions of the NKCC1 Na+-K+-2Cl cotransporter (SLC12a2 gene), differential mRNA expression changes in NKCC1-null intestine were analyzed. METHODS: Microarray analysis of mRNA from intestines of adult wild-type mice and gene-targeted NKCC1-null mice (n = 6 of each genotype) was performed to identify patterns of differential gene expression changes. Differential expression patterns were further examined by Gene Ontology analysis using the online Gorilla program, and expression changes of selected genes were verified using northern blot analysis and quantitative real time-polymerase chain reaction. Histological staining and immunofluorescence were performed to identify cell types in which upregulated pancreatic digestive enzymes were expressed. RESULTS: Genes typically associated with pancreatic function were upregulated. These included lipase, amylase, elastase, and serine proteases indicative of pancreatic exocrine function, as well as insulin and regenerating islet genes, representative of endocrine function. Northern blot analysis and immunohistochemistry showed that differential expression of exocrine pancreas mRNAs was specific to the duodenum and localized to a subset of goblet cells. In addition, a major pattern of changes involving differential expression of olfactory receptors that function in chemical sensing, as well as other chemosensing G-protein coupled receptors, was observed. These changes in chemosensory receptor expression may be related to the failure of intestinal function and dependency on parenteral nutrition observed in humans with SLC12a2 mutations. CONCLUSION: The results suggest that loss of NKCC1 affects not only secretion, but also goblet cell function and chemosensing of intestinal contents via G-protein coupled chemosensory receptors. PMID:26909237

RNA-binding Protein Quaking Stabilizes Sirt2 mRNA during Oligodendroglial Differentiation*

PubMed Central

Thangaraj, Merlin P.; Furber, Kendra L.; Gan, Jotham K.; Ji, Shaoping; Sobchishin, Larhonda; Doucette, J. Ronald; Nazarali, Adil J.

2017-01-01

Myelination is controlled by timely expression of genes involved in the differentiation of oligodendrocyte precursor cells (OPCs) into myelinating oligodendrocytes (OLs). Sirtuin 2 (SIRT2), a NAD+-dependent deacetylase, plays a critical role in OL differentiation by promoting both arborization and downstream expression of myelin-specific genes. However, the mechanisms involved in regulating SIRT2 expression during OL development are largely unknown. The RNA-binding protein quaking (QKI) plays an important role in myelination by post-transcriptionally regulating the expression of several myelin specific genes. In quaking viable (qkv/qkv) mutant mice, SIRT2 protein is severely reduced; however, it is not known whether these genes interact to regulate OL differentiation. Here, we report for the first time that QKI directly binds to Sirt2 mRNA via a common quaking response element (QRE) located in the 3′ untranslated region (UTR) to control SIRT2 expression in OL lineage cells. This interaction is associated with increased stability and longer half-lives of Sirt2.1 and Sirt2.2 transcripts leading to increased accumulation of Sirt2 transcripts. Consistent with this, overexpression of qkI promoted the expression of Sirt2 mRNA and protein. However, overexpression of the nuclear isoform qkI-5 promoted the expression of Sirt2 mRNA, but not SIRT2 protein, and delayed OL differentiation. These results suggest that the balance in the subcellular distribution and temporal expression of QKI isoforms control the availability of Sirt2 mRNA for translation. Collectively, our study demonstrates that QKI directly plays a crucial role in the post-transcriptional regulation and expression of Sirt2 to facilitate OL differentiation. PMID:28188285

The Nuclear Receptor AhR Controls Bone Homeostasis by Regulating Osteoclast Differentiation via the RANK/c-Fos Signaling Axis.

PubMed

Izawa, Takashi; Arakaki, Rieko; Mori, Hiroki; Tsunematsu, Takaaki; Kudo, Yasusei; Tanaka, Eiji; Ishimaru, Naozumi

2016-12-15

The aryl hydrocarbon receptor (AhR) pathway plays a key role in receptor activator of NF-κB ligand (RANKL)-mediated osteoclastogenesis. However, the mechanism underlying the regulation of AhR expression in osteoclasts and the signaling pathway through which AhR controls osteoclastogenesis remain unclear. We found that the expression of AhR in bone marrow-derived osteoclasts was upregulated by RANKL at an earlier stage than was the expression of signature osteoclast genes such as those encoding cathepsin K and NFAT, cytoplasmic, calcineurin-dependent 1. In response to RANKL, bone marrow macrophages isolated from AhR -/- mice exhibited impaired phosphorylation of Akt and MAPK as well as NF-κB, whereas their response to M-CSF remained unchanged. Osteoclast differentiation mediated by the AhR signaling pathway was also regulated in an RANKL/c-Fos-dependent manner. Furthermore, ligand activation of AhR by the smoke toxin benzo[a]pyrene accelerated osteoclast differentiation in a receptor-dependent manner, and AhR-dependent regulation of mitochondrial biogenesis in osteoclasts was observed. Moreover, AhR -/- mice exhibited impaired bone healing with delayed endochondral ossification. Taken together, the present results suggest that the RANKL/AhR/c-Fos signaling axis plays a critical role in osteoclastogenesis, thereby identifying the potential of AhR in treating pathological, inflammatory, or metabolic disorders of the bone. Copyright © 2016 by The American Association of Immunologists, Inc.

Dendritic cell MST1 inhibits Th17 differentiation

PubMed Central

Li, Chunxiao; Bi, Yujing; Li, Yan; Yang, Hui; Yu, Qing; Wang, Jian; Wang, Yu; Su, Huilin; Jia, Anna; Hu, Ying; Han, Linian; Zhang, Jiangyuan; Li, Simin; Tao, Wufan; Liu, Guangwei

2017-01-01

Although the differentiation of CD4+T cells is widely studied, the mechanisms of antigen-presenting cell-dependent T-cell modulation are unclear. Here, we investigate the role of dendritic cell (DC)-dependent T-cell differentiation in autoimmune and antifungal inflammation and find that mammalian sterile 20-like kinase 1 (MST1) signalling from DCs negatively regulates IL-17 producing-CD4+T helper cell (Th17) differentiation. MST1 deficiency in DCs increases IL-17 production by CD4+T cells, whereas ectopic MST1 expression in DCs inhibits it. Notably, MST1-mediated DC-dependent Th17 differentiation regulates experimental autoimmune encephalomyelitis and antifungal immunity. Mechanistically, MST1-deficient DCs promote IL-6 secretion and regulate the activation of IL-6 receptor α/β and STAT3 in CD4+T cells in the course of inducing Th17 differentiation. Activation of the p38 MAPK signal is responsible for IL-6 production in MST1-deficient DCs. Thus, our results define the DC MST1–p38MAPK signalling pathway in directing Th17 differentiation. PMID:28145433

Dimorphic DNA methylation during temperature-dependent sex determination in the sea turtle Lepidochelys olivacea.

PubMed

Venegas, Daniela; Marmolejo-Valencia, Alejandro; Valdes-Quezada, Christian; Govenzensky, Tzipe; Recillas-Targa, Félix; Merchant-Larios, Horacio

2016-09-15

Sex determination in vertebrates depends on the expression of a conserved network of genes. Sea turtles such as Lepidochelys olivacea have temperature-dependent sex determination. The present work analyses some of the epigenetic processes involved in this. We describe sexual dimorphism in global DNA methylation patterns between ovaries and testes of L. olivacea and show that the differences may arise from a combination of DNA methylation and demethylation events that occur during sex determination. Irrespective of incubation temperature, 5-hydroxymethylcytosine was abundant in the bipotential gonad; however, following sex determination, this modification was no longer found in pre-Sertoli cells in the testes. These changes correlate with the establishment of the sexually dimorphic DNA methylation patterns, down regulation of Sox9 gene expression in ovaries and irreversible gonadal commitment towards a male or female differentiation pathway. Thus, DNA methylation changes may be necessary for the stabilization of the gene expression networks that drive the differentiation of the bipotential gonad to form either an ovary or a testis in L. olivacea and probably among other species that manifest temperature-dependent sex determination. Copyright © 2016 Elsevier Inc. All rights reserved.

Derivation, expansion and differentiation of induced pluripotent stem cells in continuous suspension cultures

PubMed Central

Fluri, David A.; Tonge, Peter D.; Song, Hannah; Baptista, Ricardo P.; Shakiba, Nika; Shukla, Shreya; Clarke, Geoffrey; Nagy, Andras; Zandstra, Peter W.

2016-01-01

We demonstrate derivation of induced pluripotent stem cells (iPSCs) from terminally differentiated mouse cells in serum- and feeder-free stirred suspension cultures. Temporal analysis of global gene expression revealed high correlations between cells reprogrammed in suspension and cells reprogrammed in adhesion-dependent conditions. Suspension (S) reprogrammed iPSCs (SiPSCs) could be differentiated into all three germ layers in vitro and contributed to chimeric embryos in vivo. SiPSC generation allowed for efficient selection of reprogramming factor expressing cells based on their differential survival and proliferation in suspension. Seamless integration of SiPSC reprogramming and directed differentiation enabled the scalable production of functionally and phenotypically defined cardiac cells in a continuous single cell- and small aggregate-based process. This method is an important step towards the development of a robust PSC generation, expansion and differentiation technology. PMID:22447133

The absence of p27Kip1, an inhibitor of G1 cyclin-dependent kinases, uncouples differentiation and growth arrest during the granulosa->luteal transition.

PubMed

Tong, W; Kiyokawa, H; Soos, T J; Park, M S; Soares, V C; Manova, K; Pollard, J W; Koff, A

1998-09-01

The involvement of cyclin-dependent kinase inhibitors in differentiation remains unclear: are the roles of cyclin-dependent kinase inhibitors restricted to cell cycle arrest; or also required for completion of the differentiation program; or both? Here, we report that differentiation of luteal cells can be uncoupled from growth arrest in p27-deficient mice. In these mice, female-specific infertility correlates with a failure of embryos to implant at embryonic day 4.5. We show by ovarian transplant and hormone reconstitution experiments that failure to regulate luteal cell estradiol is one physiological mechanism for infertility in these mice. This failure is not due to a failure of p27-deficient granulosa cells to differentiate after hormonal stimulation; P450scc, a marker for luteal progesterone biosynthesis, is expressed and granulosa cell-specific cyclin D2 expression is reduced. However, unlike their wild-type counterparts, p27-deficient luteal cells continue to proliferate for up to 3.5 days after hormonal stimulation. By day 5.5, however, these cells withdraw from the cell cycle, suggesting that p27 plays a role in the early events regulating withdrawal of cells from the cell cycle. We have further shown that in the absence of this timely withdrawal, estradiol regulation is perturbed, explaining in part how fertility is compromised at the level of implantation. These data support the interpretation of our previous observations on oligodendrocyte differentiation about a role for p27 in establishing the nonproliferative state, which in some cases (oligodendrocytes) is required for differentiation, whereas in other cases it is required for the proper functioning of a differentiated cell (luteal cell).

Pharmacologic inhibition of lactate production prevents myofibroblast differentiation.

PubMed

Kottmann, Robert Matthew; Trawick, Emma; Judge, Jennifer L; Wahl, Lindsay A; Epa, Amali P; Owens, Kristina M; Thatcher, Thomas H; Phipps, Richard P; Sime, Patricia J

2015-12-01

Myofibroblasts are one of the primary cell types responsible for the accumulation of extracellular matrix in fibrosing diseases, and targeting myofibroblast differentiation is an important therapeutic strategy for the treatment of pulmonary fibrosis. Transforming growth factor-β (TGF-β) has been shown to be an important inducer of myofibroblast differentiation. We previously demonstrated that lactate dehydrogenase and its metabolic product lactic acid are important mediators of myofibroblast differentiation, via acid-induced activation of latent TGF-β. Here we explore whether pharmacologic inhibition of LDH activity can prevent TGF-β-induced myofibroblast differentiation. Primary human lung fibroblasts from healthy patients and those with pulmonary fibrosis were treated with TGF-β and or gossypol, an LDH inhibitor. Protein and RNA were analyzed for markers of myofibroblast differentiation and extracellular matrix generation. Gossypol inhibited TGF-β-induced expression of the myofibroblast marker α-smooth muscle actin (α-SMA) in a dose-dependent manner in both healthy and fibrotic human lung fibroblasts. Gossypol also inhibited expression of collagen 1, collagen 3, and fibronectin. Gossypol inhibited LDH activity, the generation of extracellular lactic acid, and the rate of extracellular acidification in a dose-dependent manner. Furthermore, gossypol inhibited TGF-β bioactivity in a dose-dependent manner. Concurrent treatment with an LDH siRNA increased the ability of gossypol to inhibit TGF-β-induced myofibroblast differentiation. Gossypol inhibits TGF-β-induced myofibroblast differentiation through inhibition of LDH, inhibition of extracellular accumulation of lactic acid, and inhibition of TGF-β bioactivity. These data support the hypothesis that pharmacologic inhibition of LDH may play an important role in the treatment of pulmonary fibrosis. Copyright © 2015 the American Physiological Society.

Modulation of lens cell adhesion molecules by particle beams

NASA Technical Reports Server (NTRS)

McNamara, M. P.; Bjornstad, K. A.; Chang, P. Y.; Chou, W.; Lockett, S. J.; Blakely, E. A.

2001-01-01

Cell adhesion molecules (CAMs) are proteins which anchor cells to each other and to the extracellular matrix (ECM), but whose functions also include signal transduction, differentiation, and apoptosis. We are testing a hypothesis that particle radiations modulate CAM expression and this contributes to radiation-induced lens opacification. We observed dose-dependent changes in the expression of beta 1-integrin and ICAM-1 in exponentially-growing and confluent cells of a differentiating human lens epithelial cell model after exposure to particle beams. Human lens epithelial (HLE) cells, less than 10 passages after their initial culture from fetal tissue, were grown on bovine corneal endothelial cell-derived ECM in medium containing 15% fetal bovine serum and supplemented with 5 ng/ml basic fibroblast growth factor (FGF-2). Multiple cell populations at three different stages of differentiation were prepared for experiment: cells in exponential growth, and cells at 5 and 10 days post-confluence. The differentiation status of cells was characterized morphologically by digital image analysis, and biochemically by Western blotting using lens epithelial and fiber cell-specific markers. Cultures were irradiated with single doses (4, 8 or 12 Gy) of 55 MeV protons and, along with unirradiated control samples, were fixed using -20 degrees C methanol at 6 hours after exposure. Replicate experiments and similar experiments with helium ions are in progress. The intracellular localization of beta 1-integrin and ICAM-1 was detected by immunofluorescence using monoclonal antibodies specific for each CAM. Cells known to express each CAM were also processed as positive controls. Both exponentially-growing and confluent, differentiating cells demonstrated a dramatic proton-dose-dependent modulation (upregulation for exponential cells, downregulation for confluent cells) and a change in the intracellular distribution of the beta 1-integrin, compared to unirradiated controls. In contrast, there was a dose-dependent increase in ICAM-1 immunofluorescence in confluent, but not exponentially-growing cells. These results suggest that proton irradiation downregulates beta 1-integrin and upregulates ICAM-1, potentially contributing to cell death or to aberrant differentiation via modulation of anchorage and/or signal transduction functions. Quantification of the expression levels of the CAMs by Western analysis is in progress.

Activity-Dependent Human Brain Coding/Noncoding Gene Regulatory Networks

PubMed Central

Lipovich, Leonard; Dachet, Fabien; Cai, Juan; Bagla, Shruti; Balan, Karina; Jia, Hui; Loeb, Jeffrey A.

2012-01-01

While most gene transcription yields RNA transcripts that code for proteins, a sizable proportion of the genome generates RNA transcripts that do not code for proteins, but may have important regulatory functions. The brain-derived neurotrophic factor (BDNF) gene, a key regulator of neuronal activity, is overlapped by a primate-specific, antisense long noncoding RNA (lncRNA) called BDNFOS. We demonstrate reciprocal patterns of BDNF and BDNFOS transcription in highly active regions of human neocortex removed as a treatment for intractable seizures. A genome-wide analysis of activity-dependent coding and noncoding human transcription using a custom lncRNA microarray identified 1288 differentially expressed lncRNAs, of which 26 had expression profiles that matched activity-dependent coding genes and an additional 8 were adjacent to or overlapping with differentially expressed protein-coding genes. The functions of most of these protein-coding partner genes, such as ARC, include long-term potentiation, synaptic activity, and memory. The nuclear lncRNAs NEAT1, MALAT1, and RPPH1, composing an RNAse P-dependent lncRNA-maturation pathway, were also upregulated. As a means to replicate human neuronal activity, repeated depolarization of SY5Y cells resulted in sustained CREB activation and produced an inverse pattern of BDNF-BDNFOS co-expression that was not achieved with a single depolarization. RNAi-mediated knockdown of BDNFOS in human SY5Y cells increased BDNF expression, suggesting that BDNFOS directly downregulates BDNF. Temporal expression patterns of other lncRNA-messenger RNA pairs validated the effect of chronic neuronal activity on the transcriptome and implied various lncRNA regulatory mechanisms. lncRNAs, some of which are unique to primates, thus appear to have potentially important regulatory roles in activity-dependent human brain plasticity. PMID:22960213

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

Zeadin, Melec G.; Butcher, Martin K.; Shaughnessy, Stephen G.

Highlights: Black-Right-Pointing-Pointer Leptin promotes osteoblast differentiation of primary smooth muscle cells. Black-Right-Pointing-Pointer Leptin regulates the expression of genes involved in osteoblast differentiation. Black-Right-Pointing-Pointer Constitutively active GSK-3{beta} attenuates leptin-induced osteoblast differentiation. Black-Right-Pointing-Pointer This suggests that leptin signals through GSK-3{beta} to promote osteoblast differentiation. -- Abstract: In this study, we begin to investigate the underlying mechanism of leptin-induced vascular calcification. We found that treatment of cultured bovine aortic smooth muscle cells (BASMCs) with leptin (0.5-4 {mu}g/ml) induced osteoblast differentiation in a dose-dependent manner. Furthermore, we found that leptin significantly increased the mRNA expression of osteopontin and bone sialoprotein, while down-regulating matrix glamore » protein (MGP) expression in BASMCs. Key factors implicated in osteoblast differentiation, including members of the Wnt signaling pathway, were examined. Exposure to leptin enhanced phosphorylation of GSK-3{beta} on serine-9 thereby inhibiting activity and promoting the nuclear accumulation of {beta}-catenin. Transfection of BASMCs with an adenovirus that expressed constitutively active GSK-3{beta} (Ad-GSK-3{beta} S9A) resulted in a >2-fold increase in GSK-3{beta} activity and a significant decrease in leptin-induced alkaline phosphatase (ALP) activity. In addition, qRT-PCR analysis showed that GSK-3{beta} activation resulted in a significant decrease in the expression of osteopontin and bone sialoprotein, but a marked increase in MGP mRNA expression. When taken together, our results suggest a mechanism by which leptin promotes osteoblast differentiation and vascular calcification in vivo.« less

Drosophila E-Cadherin Functions in Hematopoietic Progenitors to Maintain Multipotency and Block Differentiation

PubMed Central

Gao, Hongjuan; Wu, Xiaorong; Fossett, Nancy

2013-01-01

A fundamental question in stem cell biology concerns the regulatory strategies that control the choice between multipotency and differentiation. Drosophila blood progenitors or prohemocytes exhibit key stem cell characteristics, including multipotency, quiescence, and niche dependence. As a result, studies of Drosophila hematopoiesis have provided important insights into the molecular mechanisms that control these processes. Here, we show that E-cadherin is an important regulator of prohemocyte fate choice, maintaining prohemocyte multipotency and blocking differentiation. These functions are reminiscent of the role of E-cadherin in mammalian embryonic stem cells. We also show that mis-expression of E-cadherin in differentiating hemocytes disrupts the boundary between these cells and undifferentiated prohemocytes. Additionally, upregulation of E-cadherin in differentiating hemocytes increases the number of intermediate cell types expressing the prohemocyte marker, Patched. Furthermore, our studies indicate that the Drosophila GATA transcriptional co-factor, U-shaped, is required for E-cadherin expression. Consequently, E-cadherin is a downstream target of U-shaped in the maintenance of prohemocyte multipotency. In contrast, we showed that forced expression of the U-shaped GATA-binding partner, Serpent, repressed E-cadherin expression and promoted lamellocyte differentiation. Thus, U-shaped may maintain E-cadherin expression by blocking the inhibitory activity of Serpent. Collectively, these observations suggest that GATA:FOG complex formation regulates E-cadherin levels and, thereby, the choice between multipotency and differentiation. The work presented in this report further defines the molecular basis of prohemocyte cell fate choice, which will provide important insights into the mechanisms that govern stem cell biology. PMID:24040319

Glucose availability controls adipogenesis in mouse 3T3-L1 adipocytes via up-regulation of nicotinamide metabolism.

PubMed

Jackson, Robert M; Griesel, Beth A; Gurley, Jami M; Szweda, Luke I; Olson, Ann Louise

2017-11-10

Expansion of adipose tissue in response to a positive energy balance underlies obesity and occurs through both hypertrophy of existing cells and increased differentiation of adipocyte precursors (hyperplasia). To better understand the nutrient signals that promote adipocyte differentiation, we investigated the role of glucose availability in regulating adipocyte differentiation and maturation. 3T3-L1 preadipocytes were grown and differentiated in medium containing a standard differentiation hormone mixture and either 4 or 25 mm glucose. Adipocyte maturation at day 9 post-differentiation was determined by key adipocyte markers, including glucose transporter 4 (GLUT4) and adiponectin expression and Oil Red O staining of neutral lipids. We found that adipocyte differentiation and maturation required a pulse of 25 mm glucose only during the first 3 days of differentiation. Importantly, fatty acids were unable to substitute for the 25 mm glucose pulse during this period. The 25 mm glucose pulse increased adiponectin and GLUT4 expression and accumulation of neutral lipids via distinct mechanisms. Adiponectin expression and other early markers of differentiation required an increase in the intracellular pool of total NAD/P. In contrast, GLUT4 protein expression was only partially restored by increased NAD/P levels. Furthermore, GLUT4 mRNA expression was mediated by glucose-dependent activation of GLUT4 gene transcription through the cis-acting GLUT4-liver X receptor element (LXRE) promoter element. In summary, this study supports the conclusion that high glucose promotes adipocyte differentiation via distinct metabolic pathways and independently of fatty acids. This may partly explain the mechanism underlying adipocyte hyperplasia that occurs much later than adipocyte hypertrophy in the development of obesity. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Survival and differentiation defects contribute to neutropenia in glucose-6-phosphatase-β (G6PC3) deficiency in a model of mouse neutrophil granulocyte differentiation.

PubMed

Gautam, S; Kirschnek, S; Gentle, I E; Kopiniok, C; Henneke, P; Häcker, H; Malleret, L; Belaaouaj, A; Häcker, G

2013-08-01

Differentiation of neutrophil granulocytes (neutrophils) occurs through several steps in the bone marrow and requires a coordinate regulation of factors determining survival and lineage-specific development. A number of genes are known whose deficiency disrupts neutrophil generation in humans and in mice. One of the proteins encoded by these genes, glucose-6-phosphatase-β (G6PC3), is involved in glucose metabolism. G6PC3 deficiency causes neutropenia in humans and in mice, linked to enhanced apoptosis and ER stress. We used a model of conditional Hoxb8 expression to test molecular and functional differentiation as well as survival defects in neutrophils from G6PC3(-/-) mice. Progenitor lines were established and differentiated into neutrophils when Hoxb8 was turned off. G6PC3(-/-) progenitor cells underwent substantial apoptosis when differentiation was started. Transgenic expression of Bcl-XL rescued survival; however, Bcl-XL-protected differentiated cells showed reduced proliferation, immaturity and functional deficiency such as altered MAP kinase signaling and reduced cytokine secretion. Impaired glucose utilization was found and was associated with ER stress and apoptosis, associated with the upregulation of Bim and Bax; downregulation of Bim protected against apoptosis during differentiation. ER-stress further caused a profound loss of expression and secretion of the main neutrophil product neutrophil elastase during differentiation. Transplantation of wild-type Hoxb8-progenitor cells into irradiated mice allowed differentiation into neutrophils in the bone marrow in vivo. Transplantation of G6PC3(-/-) cells yielded few mature neutrophils in bone marrow and peripheral blood. Transgenic Bcl-XL permitted differentiation of G6PC3(-/-) cells in vivo. However, functional deficiencies and differentiation abnormalities remained. Differentiation of macrophages from Hoxb8-dependent progenitors was only slightly disturbed. A combination of defects in differentiation and survival thus underlies neutropenia in G6PC3(-/-) deficiency, both originating from a reduced ability to utilize glucose. Hoxb8-dependent cells are a model to study differentiation and survival of the neutrophil lineage.

Diverse effects of lead nitrate on the proliferation, differentiation, and gene expression of stem cells isolated from a dental origin.

PubMed

Abdullah, Mariam; Rahman, Fazliny Abd; Gnanasegaran, Nareshwaran; Govindasamy, Vijayendran; Abu Kasim, Noor Hayaty; Musa, Sabri

2014-01-01

Lead (Pb(2+)) exposure continues to be a significant public health problem. Therefore, it is vital to have a continuous epidemiological dataset for a better understanding of Pb(2+) toxicity. In the present study, we have exposed stem cells isolated from deciduous and permanent teeth, periodontal ligament, and bone marrow to five different types of Pb(2+) concentrations (160, 80, 40, 20, and 10 µM) for 24 hours to identify the adverse effects of Pb(2+) on the proliferation, differentiation, and gene expression on these cell lines. We found that Pb(2+) treatment altered the morphology and adhesion of the cells in a dose-dependent manner. There were no significant changes in terms of cell surface phenotypes. Cells exposed to Pb(2+) continued to differentiate into chondrogenesis and adipogenesis, and a severe downregulation was observed in osteogenesis. Gene expression studies revealed a constant expression of key markers associated with stemness (Oct 4, Rex 1) and DNA repair enzyme markers, but downregulation occurred with some ectoderm and endoderm markers, demonstrating an irregular and untimely differentiation trail. Our study revealed for the first time that Pb(2+) exposure not only affects the phenotypic characteristics but also induces significant alteration in the differentiation and gene expression in the cells.

STAR and AKR1B10 are down-regulated in high-grade endometrial cancer.

PubMed

Sinreih, Maša; Štupar, Saša; Čemažar, Luka; Verdenik, Ivan; Frković Grazio, Snježana; Smrkolj, Špela; Rižner, Tea Lanišnik

2017-07-01

Endometrial cancer is the most frequent gynecological malignancy in the developed world. The majority of cases are estrogen dependent, and are associated with diminished protective effects of progesterone. Endometrial cancer is also related to enhanced inflammation and decreased differentiation. In our previous studies, we examined the expression of genes involved in estrogen and progesterone actions in inflammation and tumor differentiation, in tissue samples from endometrial cancer and adjacent control endometrium. The aims of the current study were to examine correlations between gene expression and several demographic characteristics, and to evaluate changes in gene expression with regard to histopathological and clinical characteristics of 51 patients. We studied correlations and differences in expression of 38 genes involved in five pathophysiological processes: (i) estrogen-stimulated proliferation; (ii) estrogen-dependent carcinogenesis; (iii) diminished biosynthesis of progesterone: (iv) enhanced formation of progesterone metabolites; and (v) increased inflammation and decreased differentiation. Spearman correlation coefficient analysis shows that expression of PAQR7 correlates with age, expression of SRD5A1, AKR1B1 and AKR1B10 correlate with body mass, while expression of SRD5A1 and AKR1B10 correlate with body mass index. When patients with endometrial cancer were stratified based on menopausal status, histological grade, myometrial invasion, lymphovascular invasion, and FIGO stage, Mann-Whitney U tests revealed significantly decreased expression of STAR (4.4-fold; adjusted p=0.009) and AKR1B10 (9-fold; adjusted p=0.003) in high grade versus low grade tumors. Lower levels of STAR might lead to decreased de-novo steroid hormone synthesis and tumor differentiation, and lower levels of AKR1B10 to diminished elimination of toxic electrophilic carbonyl compounds in high-grade endometrial cancer. These data thus reveal the potential of STAR and AKR1B10 as prognostic biomarkers, which calls for further validation at the protein level. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

Uremic Conditions Drive Human Monocytes to Pro-Atherogenic Differentiation via an Angiotensin-Dependent Mechanism

PubMed Central

Trojanowicz, Bogusz; Ulrich, Christof; Seibert, Eric; Fiedler, Roman; Girndt, Matthias

2014-01-01

Aims Elevated expression levels of monocytic-ACE have been found in haemodialysis patients. They are not only epidemiologically linked with increased mortality and cardiovascular disease, but may also directly participate in the initial steps of atherosclerosis. To further address this question we tested the role of monocytic-ACE in promotion of atherosclerotic events in vitro under conditions mimicking those of chronic renal failure. Methods and Results Treatment of human primary monocytes or THP-1 cells with uremic serum as well as PMA-induced differentiation led to significantly up-regulated expression of ACE, further increased by additional treatment with LPS. Functionally, these monocytes revealed significantly increased adhesion and transmigration through endothelial monolayers. Overexpression of ACE in transfected monocytes or THP-1 cells led to development of more differentiated, macrophage-like phenotype with up-regulated expression of Arg1, MCSF, MCP-1 and CCR2. Expression of pro-inflammatory cytokines TNFa and IL-6 were also noticeably up-regulated. ACE overexpression resulted in significantly increased adhesion and transmigration properties. Transcriptional screening of ACE-overexpressing monocytes revealed noticeably increased expression of Angiotensin II receptors and adhesion- as well as atherosclerosis-related ICAM-1 and VCAM1. Inhibition of monocyte ACE or AngII-receptor signalling led to decreased adhesion potential of ACE-overexpressing cells. Conclusions Taken together, these data demonstrate that uremia induced expression of monocytic-ACE mediates the development of highly pro-atherogenic cells via an AngII-dependent mechanism. PMID:25003524

Transcription factor-dependent chromatin remodeling of Il18r1 during Th1 and Th2 differentiation 1

PubMed Central

Yu, Qing; Chang, Hua-Chen; Ahyi, Ayele-Nati N.; Kaplan, Mark H.

2008-01-01

The IL-18Rα chain is expressed on Th1 but not Th2 cells. We have recently shown that Stat4 is an important component of programming the Il18r1 locus (encoding IL-18Rα) for maximal expression in Th1 cells. Il18r1 is reciprocally repressed during Th2 development. In this report we demonstrate that the establishment of DNase hypersensitivity patterns that are distinct among undifferentiated CD4 T cells, Th1 and Th2 cells. Stat6 is required for the repression of Il18r1 expression and in Stat6-deficient Th2 cultures, mRNA levels, histone acetylation and H3K4 methylation levels are intermediate between levels observed in Th1 and Th2 cells. Despite the repressive effects of IL-4 during Th2 differentiation, we observed only modest binding of Stat6 to the Il18r1 locus. In contrast, we observed robust GATA-3 binding to a central region of the locus where DNase hypersensitivity sites overlapped with conserved non-coding sequences in Il18r1 introns. Ectopic expression of GATA-3 in differentiated Th1 cells repressed Il18r1 mRNA and surface expression of IL-18Rα. These data provide further mechanistic insight into transcription factor dependent establishment of Th subset-specific patterns of gene expression. PMID:18714006

Differential pathway dependency discovery associated with drug response across cancer cell lines* | Office of Cancer Genomics

Cancer.gov

The effort to personalize treatment plans for cancer patients involves the identification of drug treatments that can effectively target the disease while minimizing the likelihood of adverse reactions. In this study, the gene-expression profile of 810 cancer cell lines and their response data to 368 small molecules from the Cancer Therapeutics Research Portal (CTRP) are analyzed to identify pathways with significant rewiring between genes, or differential gene dependency, between sensitive and non-sensitive cell lines.

CKIP-1 suppresses odontoblastic differentiation of dental pulp stem cells via BMP2 pathway and can interact with NRP1.

PubMed

Song, Yihua; Wang, Chenfei; Gu, Zhifeng; Cao, Peipei; Huang, Dan; Feng, Guijuan; Lian, Min; Zhang, Ye; Feng, Xingmei; Gao, Zhenran

2018-05-31

Casein kinase 2 interacting protein-1 (CKIP-1) is a recently discovered intracellular regulator of bone formation, muscle cell differentiation and tumor cell proliferation. Our study aims to identify the inhibition of BMP2-Smad1/5 signaling by CKIP-1 in odontoblastic differentiation of human dental pulp stem cells (DPSCs). DPSCs infected CKIP-1 siRNA or transfected CKIP-1 full-length plasmid were cultured in odontoblastic differentiation medium or added noggin (200 ng/mL) for 21 days. We examined the effects of CKIP-1 on odontoblastic differentiation, mineralized nodules formation and interaction by western blot, real-time polymerase chain reaction (RT-PCR), alkaline phosphatase (ALP) staining, alizarin red S staining and immunoprecipitation. Firstly, we have demonstrated that CKIP-1 expression markedly decreased time-dependently along with cell odontoblastic differentiation. Indeed, the silence of CKIP-1 up-regulated odontoblastic differentiation via BMP2-Smad1/5 signaling, while CKIP-1 over-expression had a negative effect on odontoblastic differentiation of DPSCs. Furthermore, CKIP-1 could interact with Neuropilin-1 (NRP1). This work provides data that it advocates a novel perception on odontoblastic differentiation of DPSCs. Therefore, inhibiting the expression of CKIP-1 may be of great significance to the development of dental caries.

DNA methyltransferase inhibitor CDA-II inhibits myogenic differentiation

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

Chen, Zirong; Department of Molecular Genetics and Microbiology, Shands Cancer Center, University of Florida, Gainesville, FL 32610; Jin, Guorong

2012-06-08

Highlights: Black-Right-Pointing-Pointer CDA-II inhibits myogenic differentiation in a dose-dependent manner. Black-Right-Pointing-Pointer CDA-II repressed expression of muscle transcription factors and structural proteins. Black-Right-Pointing-Pointer CDA-II inhibited proliferation and migration of C2C12 myoblasts. -- Abstract: CDA-II (cell differentiation agent II), isolated from healthy human urine, is a DNA methyltransferase inhibitor. Previous studies indicated that CDA-II played important roles in the regulation of cell growth and certain differentiation processes. However, it has not been determined whether CDA-II affects skeletal myogenesis. In this study, we investigated effects of CDA-II treatment on skeletal muscle progenitor cell differentiation, migration and proliferation. We found that CDA-II blocked differentiationmore » of murine myoblasts C2C12 in a dose-dependent manner. CDA-II repressed expression of muscle transcription factors, such as Myogenin and Mef2c, and structural proteins, such as myosin heavy chain (Myh3), light chain (Mylpf) and MCK. Moreover, CDA-II inhibited C1C12 cell migration and proliferation. Thus, our data provide the first evidence that CDA-II inhibits growth and differentiation of muscle progenitor cells, suggesting that the use of CDA-II might affect skeletal muscle functions.« less

MUC4, a novel immunohistochemical marker identified by gene expression profiling, differentiates pleural sarcomatoid mesothelioma from lung sarcomatoid carcinoma.

PubMed

Amatya, Vishwa Jeet; Kushitani, Kei; Mawas, Amany Sayed; Miyata, Yoshihiro; Okada, Morihito; Kishimoto, Takumi; Inai, Kouki; Takeshima, Yukio

2017-05-01

Sarcomatoid mesothelioma, a histological subtype of malignant pleural mesothelioma, is a very aggressive tumor with a poor prognosis. Histological diagnosis of sarcomatoid mesothelioma largely depends on the histomorphological feature of spindled tumor cells with immunohistochemical reactivity to cytokeratins. Diagnosis also requires clinico-radiological and/or macroscopic evidence of an extrapulmonary location to differentiate it from lung sarcomatoid carcinoma. Although there are promising immunohistochemical antibody panels to differentiate mesothelioma from lung carcinoma, a consensus on the immunohistochemical markers that distinguish sarcomatoid mesothelioma from lung sarcomatoid carcinoma has not been reached and requires further study. We performed whole gene expression analysis of formalin-fixed paraffin-embedded tissue from sarcomatoid mesothelioma and lung sarcomatoid carcinoma and observed significant differences in the expression of MUC4 and other genes between sarcomatoid mesothelioma and lung sarcomatoid carcinoma. Immunohistochemistry demonstrated that MUC4 was expressed in the spindled tumor cells of lung sarcomatoid carcinoma (21/29, 72%) but was not expressed in any sarcomatoid mesothelioma (0/31, 0%). To differentiate sarcomatoid mesothelioma from lung sarcomatoid carcinoma, negative MUC4 expression showed 100% sensitivity and 72% specificity and accuracy rate of 87%, which is higher than immunohistochemical markers such as calretinin, D2-40 and Claudin-4. Therefore, we recommend to include MUC4 as a novel and useful negative immunohistochemical marker for differentiating sarcomatoid mesothelioma from lung sarcomatoid carcinoma.

The effects of dexamethasone, ascorbic acid, and β-glycerophosphate on osteoblastic differentiation by regulating estrogen receptor and osteopontin expression.

PubMed

Park, Jun-Beom

2012-03-01

Ascorbic acid (AA), β-glycerophosphate (GP), and dexamethasone (DEX) are the compounds known to favor the expression of the osteoblastic phenotype in several bone cell systems. In this report, the combination effects of differentiation agents on osteoprecursor cells were evaluated. The effect on cell proliferation was determined by a cell viability test with morphologic analysis. Differentiation and mineralization were evaluated using an alkaline phosphatase activity test and alizarin red-S staining. Protein expressions related to bone formation, such as transforming growth factor-beta (TGF-β), estrogen receptor-alpha (ER-α), and osteopontin (OPN) were evaluated by using a Western blot analysis. AA and GP provided an inductive effect for differentiation of osteoprecusor cells, while short-term application of DEX seemed to lead to a dose-dependent increase of cellular differentiation. Long-term use of DEX seemed to reduce mineralization. These effects may seem to be regulated by the expression of ER-α, OPN, and TGF-β. Further studies related to this mechanism within the in vivo model may be necessary to ascertain greater detail. Copyright © 2012 Elsevier Inc. All rights reserved.

Platelet-Released Growth Factors Induce Differentiation of Primary Keratinocytes

PubMed Central

Tohidnezhad, Mersedeh; Lammel, Justus; Lippross, Sebastian; Behrendt, Peter; Klüter, Tim; Pufe, Thomas; Jahr, Holger; Cremer, Jochen; Rademacher, Franziska; Gläser, Regine; Harder, Jürgen

2017-01-01

Autologous thrombocyte concentrate lysates, for example, platelet-released growth factors, (PRGFs) or their clinically related formulations (e.g., Vivostat PRF®) came recently into the physicians' focus as they revealed promising effects in regenerative and reparative medicine such as the support of healing of chronic wounds. To elucidate the underlying mechanisms, we analyzed the influence of PRGF and Vivostat PRF on human keratinocyte differentiation in vitro and on epidermal differentiation status of skin wounds in vivo. Therefore, we investigated the expression of early (keratin 1 and keratin 10) and late (transglutaminase-1 and involucrin) differentiation markers. PRGF treatment of primary human keratinocytes decreased keratin 1 and keratin 10 gene expression but induced involucrin and transglutaminase-1 gene expression in an epidermal growth factor receptor- (EGFR-) dependent manner. In concordance with these results, microscopic analyses revealed that PRGF-treated human keratinocytes displayed morphological features typical of keratinocytes undergoing terminal differentiation. In vivo treatment of artificial human wounds with Vivostat PRF revealed a significant induction of involucrin and transglutaminase-1 gene expression. Together, our results indicate that PRGF and Vivostat PRF induce terminal differentiation of primary human keratinocytes. This potential mechanism may contribute to the observed beneficial effects in the treatment of hard-to-heal wounds with autologous thrombocyte concentrate lysates in vivo. PMID:28808357

Nitric oxide enhances Oct-4 expression in bone marrow stem cells and promotes endothelial differentiation.

PubMed

Chu, Ling; Jiang, Yuehua; Hao, Hong; Xia, Yong; Xu, Jian; Liu, Zehao; Verfaillie, Catherine M; Zweier, Jay L; Liu, Zhenguo

2008-09-04

This study was designed to investigate the role of nitric oxide (NO) in bone marrow stem cells and their differentiation into endothelial cells in vitro. Adult mouse bone marrow multipotent progenitor cells (MAPCs) were used as the source of stem cells. Oct-4 expression (both mRNA and protein) was significantly increased by up to 68.0% in MAPCs when incubated with NO donors DETA-NONOate or sodium nitroprusside (SNP) in a concentration-dependant manner (n=3, P<0.05). However, the cell proliferation was dramatically decreased by over 3-folds when treated with DETA-NONOate or SNP for 48 h (n=3, P<0.05). When MAPCs were exposed to DETA-NONOate (100 microM) for the first 48 h during differentiation, the expression (both mRNA and protein) of vWF was significantly increased at day 14 in the differentiating cells. The effects of DETA-NONOate or SNP on cell proliferation, Oct-4 expression and endothelial differentiation of MAPCs were not affected by the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one or cGMP analog 8-Br-cGMP. These data indicate that NO may regulate both the pluripotency and differentiation of MAPCs via a cGMP-independent mechanism.

Contexts for dopamine specification by calcium spike activity in the central nervous system

PubMed Central

Velázquez-Ulloa, Norma A.; Spitzer, Nicholas C.; Dulcis, Davide

2011-01-01

Calcium-dependent electrical activity plays a significant role in neurotransmitter specification at early stages of development. To test the hypothesis that activity-dependent differentiation depends on molecular context we investigated the development of dopaminergic neurons in the central nervous system of larval Xenopus laevis. We find that different dopaminergic nuclei respond to manipulation of this early electrical activity by ion channel misexpression with different increases and decreases in numbers of dopaminergic neurons. Focusing on the ventral suprachiasmatic nucleus and the spinal cord in order to gain insight into these differences, we identify distinct subpopulations of neurons that express characteristic combinations of GABA and NPY as co-transmitters and Lim1,2 and Nurr1 transcription factors. We demonstrate that the developmental state of neurons identified by their spatial location and expression of these molecular markers is correlated with characteristic spontaneous calcium spike activity. Different subpopulations of dopaminergic neurons respond differently to manipulation of this early electrical activity. Moreover, retinohypothalamic circuit activation of the ventral suprachiasmatic nucleus recruits expression of dopamine selectively in reserve pool neurons that already express GABA and neuropeptide Y. The results are consistent with the hypothesis that spontaneously active neurons expressing GABA are most susceptible to activity-dependent expression of dopamine both in the spinal cord and in the brain. Because loss of dopaminergic neurons plays a role in neurological disorders such as Parkinson’s disease, understanding how subpopulations of neurons become dopaminergic may lead to protocols for differentiation of neurons in vitro to replace those that have been lost in vivo. PMID:21209192

Glutathione S-transferase expression and isoenzyme composition during cell differentiation of Caco-2 cells.

PubMed

Scharmach, E; Hessel, S; Niemann, B; Lampen, A

2009-11-30

The human colon adenocarcinoma cell line Caco-2 is frequently used to study human intestinal metabolism and transport of xenobiotica. Previous studies have shown that both Caco-2 cells and human colon cells constitutively express the multigene family of detoxifying enzymes glutathione S-transferases (GSTs), particularly GST alpha and GST pi. GSTs may play a fundamental role in the molecular interplay between phase I, II enzymes and ABC-transporters. The gut fermentation product, butyrate, can modulate the potential for detoxification. The aim of this study was to investigate the basal expression of further cytosolic GSTs in Caco-2 cells during cell differentiation. In addition, a comparison was made with expression levels in MCF-7 and HepG2, two other cell types with barrier functions. Finally, the butyrate-mediated modulation of gene and protein expression was determined by real time PCR and western blot analysis. In Caco-2, gene and protein expression levels of GST alpha increased during cell differentiation. High levels of GSTO1 and GSTP1 were constantly expressed. No expression of GSTM5 and GSTT1 was detected. HepG2 expressed GSTO1 and MCF-7 GSTZ1 most intensively. No expression of GSTA5, GSTM5, or GSTP1 was detected in either cell. Incubation of Caco-2 cells with butyrate (5 mM) significantly induced GSTA1 and GSTM2 in proliferating Caco-2 cells. In differentiated cells, butyrate tended to increase GSTO1 and GSTP1. The results of this study show that a differentiation-dependent expression of GSTs in Caco-2 cells may reflect the in vivo situation and indicate the potential of butyrate to modify intestinal metabolism. GSTA1-A4 have been identified as good markers for cell differentiation. The Caco-2 cell line is a useful model for assessing the potential of food-related substances to modulate the GST expression pattern.

Lactoferrin promote primary rat osteoblast proliferation and differentiation via up-regulation of insulin-like growth factor-1 expression.

PubMed

Hou, Jian-ming; Wu, Man; Lin, Qing-ming; Lin, Fan; Xue, Ying; Lan, Xu-hua; Chen, En-yu; Wang, Mei-li; Yang, Hai-yan; Wang, Feng-xiong

2014-08-01

The aim of this study was to explore the effect of lactoferrin (LF) in primary fetal rat osteoblasts proliferation and differentiation and investigate the underlying molecular mechanisms. Primary rat osteoblasts were obtained from the calvarias of neonatal rats. Osteoblasts were treated with LF (0.1-1000 μg/mL), or OSI-906 [a selective inhibitor of insulin-like growth factor 1 (IGF-1) receptor and insulin receptor]. The IGF-1 was then knocked down by small hairpin RNA (shRNA) technology and then was treated with recombinant human IGF-1 or LF. Cell proliferation and differentiation were measured by MTT assay and alkaline phosphatase (ALP) assay, respectively. The expression of IGF-1 and IGF binding protein 2 (IGFBP2) mRNA were analyzed using real-time PCR. LF promotes the proliferation and differentiation of osteoblasts in a certain range (1-100 μg/mL) in time- and dose-dependent manner. The mRNA level of IGF-1 was significantly increased, while the expression of IGFBP2 was suppressed by LF treatment. Knockdown of IGF-1 by shRNA in primary rat osteoblast dramatically decreased the abilities of proliferation and differentiation of osteoblasts and blocked the proliferation and differentiation effect of LF in osteoblasts. OSI906 (5 μM) blocked the mitogenic and differentiation of LF in osteoblasts. Proliferation and differentiation of primary rat osteoblasts in response to LF are mediated in part by stimulating of IGF-1 gene expression and alterations in the gene expression of IGFBP2.

Successful pod infections by Moniliophthora roreri result in differential Theobroma cacao gene expression depending on the clone's level of tolerance.

PubMed

Ali, Shahin S; Melnick, Rachel L; Crozier, Jayne; Phillips-Mora, Wilberth; Strem, Mary D; Shao, Jonathan; Zhang, Dapeng; Sicher, Richard; Meinhardt, Lyndel; Bailey, Bryan A

2014-09-01

An understanding of the tolerance mechanisms of Theobroma cacao used against Moniliophthora roreri, the causal agent of frosty pod rot, is important for the generation of stable disease-tolerant clones. A comparative view was obtained of transcript populations of infected pods from two susceptible and two tolerant clones using RNA sequence (RNA-Seq) analysis. A total of 3009 transcripts showed differential expression among clones. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis of differentially expressed genes indicated shifts in 152 different metabolic pathways between the tolerant and susceptible clones. Real-time quantitative reverse transcription polymerase chain reaction (real-time qRT-PCR) analyses of 36 genes verified the differential expression. Regression analysis validated a uniform progression in gene expression in association with infection levels and fungal loads in the susceptible clones. Expression patterns observed in the susceptible clones diverged in tolerant clones, with many genes showing higher expression at a low level of infection and fungal load. Principal coordinate analyses of real-time qRT-PCR data separated the gene expression patterns between susceptible and tolerant clones for pods showing malformation. Although some genes were constitutively differentially expressed between clones, most results suggested that defence responses were induced at low fungal load in the tolerant clones. Several elicitor-responsive genes were highly expressed in tolerant clones, suggesting rapid recognition of the pathogen and induction of defence genes. Expression patterns suggested that the jasmonic acid-ethylene- and/or salicylic acid-mediated defence pathways were activated in the tolerant clones, being enhanced by reduced brassinosteroid (BR) biosynthesis and catabolic inactivation of both BR and abscisic acids. Finally, several genes associated with hypersensitive response-like cell death were also induced in tolerant clones. © 2014 BSPP AND JOHN WILEY & SONS LTD.

ErbB2 and EGFR are downmodulated during the differentiation of 3T3-L1 preadipocytes.

PubMed

Pagano, Eleonora; Calvo, Juan Carlos

2003-10-15

The expression of receptors belonging to the epidermal growth factor receptor subfamily has been largely studied these last years in epithelial cells mainly as involved in cell proliferation and malignant progression. Although much work has focused on the role of these growth factor receptors in the differentiation of a variety of tissues, there is little information in regards to normal stromal cells. We investigated erbB2 expression in the murine fibroblast cell line Swiss 3T3L1, which naturally or hormonally induced undergoes adipocyte differentiation. We found that the Swiss 3T3-L1 fibroblasts express erbB2, in addition to EGFR, and in a quantity comparable to or even greater than the breast cancer cell line T47D. Proliferating cells increased erbB2 and EGFR levels when reaching confluence up to 4- and 10-fold, respectively. This expression showed a significant decrease when growth-arrested cells were stimulated to differentiate with dexamethasone and isobutyl-methylxanthine. Differentiated cells presented a decreased expression of both erbB2 and EGFR regardless of whether the cells were hormonally or spontaneously differentiated. EGF stimulation of serum-starved cells increased erbB2 tyrosine phosphorylation and retarded erbB2 migration in SDS-PAGE, suggesting receptor association and activation. Heregulin-alpha1 and -beta1, two EGF related factors, had no effect on erbB2 or EGFR phosphorylation. Although 3T3-L1 cells expressed heregulin, its specific receptors, erbB3 and erbB4, were not found. This is the first time in which erbB2 is reported to be expressed in an adipocytic cell line which does not depend on non EGF family growth factors (thyroid hormone, growth hormone, etc.) to accomplish adipose differentiation. Since erbB2 and EGFR expression were downmodulated as differentiation progressed it is conceivable that a mechanism of switching from a mitogenic to a differentiating signaling pathway may be involved, through regulation of the expression of these growth factor receptors. Copyright 2003 Wiley-Liss, Inc.

Identification of Gene Expression Signatures in the Chicken Intestinal Intraepithelial Lymphocytes in Response to Herb Additive Supplementations

PubMed Central

Won, Kyeong-Hye; Song, Ki-Duk; Park, Jong-Eun; Kim, Duk-Kyung; Na, Chong-Sam

2016-01-01

Anethole and garlic have an immune modulatory effects on avian coccidiosis, and these effects are correlated with gene expression changes in intestinal epithelial lymphocytes (IELs). In this study, we integrated gene expression datasets from two independent experiments and investigated gene expression profile changes by anethole and garlic respectively, and identified gene expression signatures, which are common targets of these herbs as they might be used for the evaluation of the effect of plant herbs on immunity toward avian coccidiosis. We identified 4,382 and 371 genes, which were differentially expressed in IELs of chickens supplemented with garlic and anethole respectively. The gene ontology (GO) term of differentially expressed genes (DEGs) from garlic treatment resulted in the biological processes (BPs) related to proteolysis, e.g., “modification-dependent protein catabolic process”, “proteolysis involved in cellular protein catabolic process”, “cellular protein catabolic process”, “protein catabolic process”, and “ubiquitin-dependent protein catabolic process”. In GO analysis, one BP term, “Proteolysis”, was obtained. Among DEGs, 300 genes were differentially regulated in response to both garlic and anethole, and 234 and 59 genes were either up- or down-regulated in supplementation with both herbs. Pathway analysis resulted in enrichment of the pathways related to digestion such as “Starch and sucrose metabolism” and “Insulin signaling pathway”. Taken together, the results obtained in the present study could contribute to the effective development of evaluation system of plant herbs based on molecular signatures related with their immunological functions in chicken IELs. PMID:26954117

Differential muscle regulatory factor gene expression between larval and adult myogenesis in the frog Xenopus laevis: adult myogenic cell-specific myf5 upregulation and its relation to the notochord suppression of adult muscle differentiation.

PubMed

Yamane, Hitomi; Nishikawa, Akio

2013-08-01

During Xenopus laevis metamorphosis, larval-to-adult muscle conversion depends on the differential responses of adult and larval myogenic cells to thyroid hormone. Essential differences in cell growth, differentiation, and hormone-dependent life-or-death fate have been reported between cultured larval (tail) and adult (hindlimb) myogenic cells. A previous study revealed that tail notochord cells suppress terminal differentiation in adult (but not larval) myogenic cells. However, little is known about the differences in expression patterns of myogenic regulatory factors (MRF) and the satellite cell marker Pax7 between adult and larval myogenic cells. In the present study, we compared mRNA expression of these factors between the two types. At first, reverse transcription polymerase chain reaction analysis of hindlimb buds showed sequential upregulation of myf5, myogenin, myod, and mrf4 during stages 50-54, when limb buds elongate and muscles begin to form. By contrast, in the tail, there was no such increase during the same period. Secondary, these results were duplicated in vitro: adult myogenic cells upregulated myf5, myod, and pax7 in the early culture period, followed by myogenin upregulation and myotube differentiation, while larval myogenic cells did not upregulate these genes and precociously started myotube differentiation. Thirdly, myf5 upregulation and early-phase proliferation in adult myogenic cells were potently inhibited by the presence of notochord cells, suggesting that notochord cells suppress adult myogenesis through inhibiting the transition from Myf5(-) stem cells to Myf5(+) committed myoblasts. All of the data presented here suggest that myf5 upregulation can be a good criterion for the activation of adult myogenesis during X. laevis metamorphosis.

Organotins Are Potent Activators of PPARγ and Adipocyte Differentiation in Bone Marrow Multipotent Mesenchymal Stromal Cells

PubMed Central

Yanik, Susan C.; Baker, Amelia H.; Mann, Koren K.; Schlezinger, Jennifer J.

2011-01-01

Adipocyte differentiation in bone marrow is potentially deleterious to both bone integrity and lymphopoiesis. Here, we examine the hypothesis that organotins, common environmental contaminants that are dual ligands for peroxisome proliferator–activated receptor (PPAR) γ and its heterodimerization partner retinoid X receptor (RXR), are potent activators of bone marrow adipogenesis. A C57Bl/6-derived bone marrow multipotent mesenchymal stromal cell (MSC) line, BMS2, was treated with rosiglitazone, a PPARγ agonist, bexarotene, an RXR agonist, or a series of organotins. Rosiglitazone and bexarotene potently activated adipocyte differentiation; however, bexarotene had a maximal efficacy of only 20% of that induced by rosiglitazone. Organotins (tributyltin [TBT], triphenyltin, and dibutyltin) also stimulated adipocyte differentiation (EC50 of 10–20nM) but with submaximal, structure-dependent efficacy. In coexposures, both bexarotene and TBT enhanced rosiglitazone-induced adipogenesis. To investigate the contribution of PPARγ to TBT-induced adipogenesis, we examined expression of PPARγ2, as well as its transcriptional target FABP4. TBT-induced PPARγ2 and FABP4 protein expression with an efficacy intermediate between rosiglitazone and bexarotene, similar to lipid accumulation. A PPARγ antagonist and PPARγ-specific small hairpin RNA suppressed TBT-induced differentiation, although to a lesser extent than rosiglitazone-induced differentiation, suggesting that TBT may engage alternate pathways. TBT and bexarotene, but not rosiglitazone, also induced the expression of TGM2 (an RXR target) and ABCA1 (a liver X receptor target). The results show that an environmental contaminant, acting with the same potency as a therapeutic drug, induces PPARγ-dependent adipocyte differentiation in bone marrow MSCs. Activation of multiple nuclear receptor pathways by organotins may have significant implications for bone physiology. PMID:21622945

Male-specific expression of Sox9 during gonad development of crocodile and mouse is mediated by alternative splicing of its proline-glutamine-alanine rich domain.

PubMed

Agrawal, Raman; Wessely, Oliver; Anand, Amit; Singh, Lalji; Aggarwal, Ramesh K

2009-08-01

The initial trigger for sexual differentiation is regulated by multiple ways during embryonic development. In vertebrates, chromosome-based mechanisms generally known as genetic sex determination are prevalent; however, some species, such as many reptilians, display temperature-dependent sex determination. The Sry-related transcription factor, Sox9, which is expressed by an evolutionary conserved gene, has been shown to be a key player in the process of sex determination. In the present study, we report the identification and expression of crocodile homolog of Sox9 (cpSox9) from the Indian Mugger, Crocodylus palustris. We show that cpSox9 undergoes extensive alternative splicing around the proline-glutamine-alanine rich transactivation domain that results in cpSox9 variants with presumably impaired or reduced transactivation potential. The multiple isoforms were also detected in various embryonic tissues, with some of them displaying a differential expression profile. With respect to sex differentiation, a putative unspliced full-length cpSox9 could be detected only in the genital ridge-adrenal-mesonephros complex of male, but not female embryos during the temperature-sensitive period. Importantly, we further show that this phenomenon was not restricted to the temperature-dependent sex determination species C. palustris, but was also observed in the mouse, a species exhibiting genetic sex determination. Thus, the present study describes, for the first time, a complete coding locus of Sox9 homolog from a temperature-dependent sex determination species. More importantly, we demonstrate an evolutionarily conserved role of alternative splicing resulting in transcriptional diversity and male-sex specific expression of Sox9 during testis development in vertebrates (i.e. irrespective of their underlying sex-determination mechanisms).

Protein arginine methyltransferase 1 modulates innate immune responses through regulation of peroxisome proliferator-activated receptor γ-dependent macrophage differentiation.

PubMed

Tikhanovich, Irina; Zhao, Jie; Olson, Jody; Adams, Abby; Taylor, Ryan; Bridges, Brian; Marshall, Laurie; Roberts, Benjamin; Weinman, Steven A

2017-04-28

Arginine methylation is a common posttranslational modification that has been shown to regulate both gene expression and extranuclear signaling events. We recently reported defects in protein arginine methyltransferase 1 (PRMT1) activity and arginine methylation in the livers of cirrhosis patients with a history of recurrent infections. To examine the role of PRMT1 in innate immune responses in vivo , we created a cell type-specific knock-out mouse model. We showed that myeloid-specific PRMT1 knock-out mice demonstrate higher proinflammatory cytokine production and a lower survival rate after cecal ligation and puncture. We found that this defect is because of defective peroxisome proliferator-activated receptor γ (PPARγ)-dependent M2 macrophage differentiation. PPARγ is one of the key transcription factors regulating macrophage polarization toward a more anti-inflammatory and pro-resolving phenotype. We found that PRMT1 knock-out macrophages failed to up-regulate PPARγ expression in response to IL4 treatment resulting in 4-fold lower PPARγ expression in knock-out cells than in wild-type cells. Detailed study of the mechanism revealed that PRMT1 regulates PPARγ gene expression through histone H4R3me2a methylation at the PPARγ promoter. Supplementing with PPARγ agonists rosiglitazone and GW1929 was sufficient to restore M2 differentiation in vivo and in vitro and abrogated the difference in survival between wild-type and PRMT1 knock-out mice. Taken together these data suggest that PRMT1-dependent regulation of macrophage PPARγ expression contributes to the infection susceptibility in PRMT1 knock-out mice. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Differentially expressed genes of Coptotermes formosanus (Isoptera: Rhinotermitidae) challenged by chemical insecticides.

PubMed

Zhang, Yi; Zhao, Yuanyuan; Qiu, Xuehong; Han, Richou

2013-08-01

Coptotermes formosanus Shiraki (Isoptera: Rhinotermitidae) termites are harmful social insects to wood constructions. The current control methods heavily depend on the chemical insecticides with increasing resistance. Analysis of the differentially expressed genes mediated by chemical insecticides will contribute to the understanding of the termite resistance to chemicals and to the establishment of alternative control measures. In the present article, a full-length cDNA library was constructed from the termites induced by a mixture of commonly used insecticides (0.01% sulfluramid and 0.01% triflumuron) for 24 h, by using the RNA ligase-mediated Rapid Amplification cDNA End method. Fifty-eight differentially expressed clones were obtained by polymerase chain reaction and confirmed by dot-blot hybridization. Forty-six known sequences were obtained, which clustered into 33 unique sequences grouped in 6 contigs and 27 singlets. Sixty-seven percent (22) of the sequences had counterpart genes from other organisms, whereas 33% (11) were undescribed. A Gene Ontology analysis classified 33 unique sequences into different functional categories. In general, most of the differential expression genes were involved in binding and catalytic activity.

Emerin and histone deacetylase 3 (HDAC3) cooperatively regulate expression and nuclear positions of MyoD, Myf5, and Pax7 genes during myogenesis

PubMed Central

Demmerle, Justin; Koch, Adam J.; Holaska, James M.

2016-01-01

The spatial organization of chromatin is critical in establishing cell-type dependent gene expression programs. The inner nuclear membrane protein emerin has been implicated in regulating global chromatin architecture. We show emerin associates with genomic loci of muscle differentiation promoting factors in murine myogenic progenitors, including Myf5 and MyoD. Prior to their transcriptional activation Myf5 and MyoD loci localized to the nuclear lamina in proliferating progenitors and moved to the nucleoplasm upon transcriptional activation during differentiation. The Pax7 locus, which is transcribed in proliferating progenitors, localized to the nucleoplasm and Pax7 moved to the nuclear lamina upon repression during differentiation. Localization of Myf5, MyoD, and Pax7 to the nuclear lamina and proper temporal expression of these genes required emerin and HDAC3. Interestingly, activation of HDAC3 catalytic activity rescued both Myf5 localization to the nuclear lamina and its expression. Collectively, these data support a model whereby emerin facilitates repressive chromatin formation at the nuclear lamina by activating the catalytic activity of HDAC3 to regulate the coordinated spatiotemporal expression of myogenic differentiation genes. PMID:24062260

Estimating differential expression from multiple indicators

PubMed Central

Ilmjärv, Sten; Hundahl, Christian Ansgar; Reimets, Riin; Niitsoo, Margus; Kolde, Raivo; Vilo, Jaak; Vasar, Eero; Luuk, Hendrik

2014-01-01

Regardless of the advent of high-throughput sequencing, microarrays remain central in current biomedical research. Conventional microarray analysis pipelines apply data reduction before the estimation of differential expression, which is likely to render the estimates susceptible to noise from signal summarization and reduce statistical power. We present a probe-level framework, which capitalizes on the high number of concurrent measurements to provide more robust differential expression estimates. The framework naturally extends to various experimental designs and target categories (e.g. transcripts, genes, genomic regions) as well as small sample sizes. Benchmarking in relation to popular microarray and RNA-sequencing data-analysis pipelines indicated high and stable performance on the Microarray Quality Control dataset and in a cell-culture model of hypoxia. Experimental-data-exhibiting long-range epigenetic silencing of gene expression was used to demonstrate the efficacy of detecting differential expression of genomic regions, a level of analysis not embraced by conventional workflows. Finally, we designed and conducted an experiment to identify hypothermia-responsive genes in terms of monotonic time-response. As a novel insight, hypothermia-dependent up-regulation of multiple genes of two major antioxidant pathways was identified and verified by quantitative real-time PCR. PMID:24586062

The differential expression of alternatively polyadenylated transcripts is a common stress-induced response mechanism that modulates mammalian mRNA expression in a quantitative and qualitative fashion.

PubMed

Hollerer, Ina; Curk, Tomaz; Haase, Bettina; Benes, Vladimir; Hauer, Christian; Neu-Yilik, Gabriele; Bhuvanagiri, Madhuri; Hentze, Matthias W; Kulozik, Andreas E

2016-09-01

Stress adaptation plays a pivotal role in biological processes and requires tight regulation of gene expression. In this study, we explored the effect of cellular stress on mRNA polyadenylation and investigated the implications of regulated polyadenylation site usage on mammalian gene expression. High-confidence polyadenylation site mapping combined with global pre-mRNA and mRNA expression profiling revealed that stress induces an accumulation of genes with differentially expressed polyadenylated mRNA isoforms in human cells. Specifically, stress provokes a global trend in polyadenylation site usage toward decreased utilization of promoter-proximal poly(A) sites in introns or ORFs and increased utilization of promoter-distal polyadenylation sites in intergenic regions. This extensively affects gene expression beyond regulating mRNA abundance by changing mRNA length and by altering the configuration of open reading frames. Our study highlights the impact of post-transcriptional mechanisms on stress-dependent gene regulation and reveals the differential expression of alternatively polyadenylated transcripts as a common stress-induced mechanism in mammalian cells. © 2016 Hollerer et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

A combined gene expression and functional study reveals the crosstalk between N-Myc and differentiation-inducing microRNAs in neuroblastoma cells

PubMed Central

Zhao, Zhenze; Ma, Xiuye; Shelton, Spencer D.; Sung, Derek C.; Li, Monica; Hernandez, Daniel; Zhang, Maggie; Losiewicz, Michael D.; Chen, Yidong; Pertsemlidis, Alexander; Yu, Xiaojie; Liu, Yuanhang; Du, Liqin

2016-01-01

MYCN amplification is the most common genetic alteration in neuroblastoma and plays a critical role in neuroblastoma tumorigenesis. MYCN regulates neuroblastoma cell differentiation, which is one of the mechanisms underlying its oncogenic function. We recently identified a group of differentiation-inducing microRNAs. Given the demonstrated inter-regulation between MYCN and microRNAs, we speculated that MYCN and the differentiation-inducing microRNAs might form an interaction network to control the differentiation of neuroblastoma cells. In this study, we found that eight of the thirteen differentiation-inducing microRNAs, miR-506-3p, miR-124-3p, miR-449a, miR-34a-5p, miR-449b-5p, miR-103a-3p, miR-2110 and miR-34b-5p, inhibit N-Myc expression by either directly targeting the MYCN 3′UTR or through indirect regulations. Further investigation showed that both MYCN-dependent and MYCN-independent pathways play roles in mediating the differentiation-inducing function of miR-506-3p and miR-449a, two microRNAs that dramatically down-regulate MYCN expression. On the other hand, we found that N-Myc inhibits the expression of multiple differentiation-inducing microRNAs, suggesting that these miRNAs play a role in mediating the function of MYCN. In examining the published dataset collected from clinical neuroblastoma specimens, we found that expressions of two miRNAs, miR-137 and miR-2110, were significantly anti-correlated with MYCN mRNA levels, suggesting their interactions with MYCN play a clinically-relevant role in maintaining the MYCN and miRNA expression levels in neuroblastoma. Our findings altogether suggest that MYCN and differentiation-inducing miRNAs form an interaction network that play an important role in neuroblastoma tumorigenesis through regulating cell differentiation. PMID:27764804

A combined gene expression and functional study reveals the crosstalk between N-Myc and differentiation-inducing microRNAs in neuroblastoma cells.

PubMed

Zhao, Zhenze; Ma, Xiuye; Shelton, Spencer D; Sung, Derek C; Li, Monica; Hernandez, Daniel; Zhang, Maggie; Losiewicz, Michael D; Chen, Yidong; Pertsemlidis, Alexander; Yu, Xiaojie; Liu, Yuanhang; Du, Liqin

2016-11-29

MYCN amplification is the most common genetic alteration in neuroblastoma and plays a critical role in neuroblastoma tumorigenesis. MYCN regulates neuroblastoma cell differentiation, which is one of the mechanisms underlying its oncogenic function. We recently identified a group of differentiation-inducing microRNAs. Given the demonstrated inter-regulation between MYCN and microRNAs, we speculated that MYCN and the differentiation-inducing microRNAs might form an interaction network to control the differentiation of neuroblastoma cells. In this study, we found that eight of the thirteen differentiation-inducing microRNAs, miR-506-3p, miR-124-3p, miR-449a, miR-34a-5p, miR-449b-5p, miR-103a-3p, miR-2110 and miR-34b-5p, inhibit N-Myc expression by either directly targeting the MYCN 3'UTR or through indirect regulations. Further investigation showed that both MYCN-dependent and MYCN-independent pathways play roles in mediating the differentiation-inducing function of miR-506-3p and miR-449a, two microRNAs that dramatically down-regulate MYCN expression. On the other hand, we found that N-Myc inhibits the expression of multiple differentiation-inducing microRNAs, suggesting that these miRNAs play a role in mediating the function of MYCN. In examining the published dataset collected from clinical neuroblastoma specimens, we found that expressions of two miRNAs, miR-137 and miR-2110, were significantly anti-correlated with MYCN mRNA levels, suggesting their interactions with MYCN play a clinically-relevant role in maintaining the MYCN and miRNA expression levels in neuroblastoma. Our findings altogether suggest that MYCN and differentiation-inducing miRNAs form an interaction network that play an important role in neuroblastoma tumorigenesis through regulating cell differentiation.

Role of alpha- and beta-adrenergic receptors in cardiomyocyte differentiation from murine-induced pluripotent stem cells.

PubMed

Li, Xiao-Li; Zeng, Di; Chen, Yan; Ding, Lu; Li, Wen-Ju; Wei, Ting; Ou, Dong-Bo; Yan, Song; Wang, Bin; Zheng, Qiang-Sun

2017-02-01

Induced pluripotent stem cell (iPSC)-derived cardiomyocytes are a promising source of cells for regenerative heart disease therapies, but progress towards their use has been limited by their low differentiation efficiency and high cellular heterogeneity. Previous studies have demonstrated expression of adrenergic receptors (ARs) in stem cells after differentiation; however, roles of ARs in fate specification of stem cells, particularly in cardiomyocyte differentiation and development, have not been characterized. Murine-induced pluripotent stem cells (miPSCs) were cultured in hanging drops to form embryoid bodies, cells of which were then differentiated into cardiomyocytes. To determine whether ARs regulated miPSC differentiation into cardiac lineages, effects of the AR agonist, epinephrine (EPI), on miPSC differentiation and underlying signalling mechanisms, were evaluated. Treatment with EPI, robustly enhanced miPSC cardiac differentiation, as indicated by increased expression levels of cardiac-specific markers, GATA4, Nkx2.5 and Tnnt2. Although β-AR signalling is the foremost signalling pathway in cardiomyocytes, EPI-enhanced cardiac differentiation depended more on α-AR signalling than β-AR signalling. In addition, selective activation of α 1 -AR signalling with specific agonists induced vigorous cardiomyocyte differentiation, whereas selective activation of α 2 - or β-AR signalling induced no or less differentiation, respectively. EPI- and α 1 -AR-dependent cardiomyocyte differentiation from miPSCs occurred through specific promotion of CPC proliferation via the MEK-ERK1/2 pathway and regulation of miPS cell-cycle progression. These results demonstrate that activation of ARs, particularly of α 1 -ARs, promoted miPSC differentiation into cardiac lineages via MEK-ERK1/2 signalling. © 2016 John Wiley & Sons Ltd.

Ca2+-dependent localization of integrin-linked kinase to cell junctions in differentiating keratinocytes.

PubMed

Vespa, Alisa; Darmon, Alison J; Turner, Christopher E; D'Souza, Sudhir J A; Dagnino, Lina

2003-03-28

Integrin complexes are necessary for proper proliferation and differentiation of epidermal keratinocytes. Differentiation of these cells is accompanied by down-regulation of integrins and focal adhesions as well as formation of intercellular adherens junctions through E-cadherin homodimerization. A central component of integrin adhesion complexes is integrin-linked kinase (ILK), which can induce loss of E-cadherin expression and epithelial-mesenchymal transformation when ectopically expressed in intestinal and mammary epithelia. In cultured primary mouse keratinocytes, we find that ILK protein levels are independent of integrin expression and signaling, since they remain constant during Ca(2+)-induced differentiation. In contrast, keratinocyte differentiation is accompanied by marked reduction in kinase activity in ILK immunoprecipitates and altered ILK subcellular distribution. Specifically, ILK distributes in close apposition to actin fibers along intercellular junctions in differentiated but not in undifferentiated keratinocytes. ILK localization to cell-cell borders occurs independently of integrin signaling and requires Ca(2+) as well as an intact actin cytoskeleton. Further, and in contrast to what is observed in other epithelial cells, ILK overexpression in differentiated keratinocytes does not promote E-cadherin down-regulation and epithelial-mesenchymal transition. Thus, novel tissue-specific mechanisms control the formation of ILK complexes associated with cell-cell junctions in differentiating murine epidermal keratinocytes.

Piezo type mechanosensitive ion channel component 1 functions as a regulator of the cell fate determination of mesenchymal stem cells.

PubMed

Sugimoto, Asuna; Miyazaki, Aya; Kawarabayashi, Keita; Shono, Masayuki; Akazawa, Yuki; Hasegawa, Tomokazu; Ueda-Yamaguchi, Kimiko; Kitamura, Takamasa; Yoshizaki, Keigo; Fukumoto, Satoshi; Iwamoto, Tsutomu

2017-12-18

The extracellular environment regulates the dynamic behaviors of cells. However, the effects of hydrostatic pressure (HP) on cell fate determination of mesenchymal stem cells (MSCs) are not clearly understood. Here, we established a cell culture chamber to control HP. Using this system, we found that the promotion of osteogenic differentiation by HP is depend on bone morphogenetic protein 2 (BMP2) expression regulated by Piezo type mechanosensitive ion channel component 1 (PIEZO1) in MSCs. The PIEZO1 was expressed and induced after HP loading in primary MSCs and MSC lines, UE7T-13 and SDP11. HP and Yoda1, an activator of PIEZO1, promoted BMP2 expression and osteoblast differentiation, whereas inhibits adipocyte differentiation. Conversely, PIEZO1 inhibition reduced osteoblast differentiation and BMP2 expression. Furthermore, Blocking of BMP2 function by noggin inhibits HP induced osteogenic maker genes expression. In addition, in an in vivo model of medaka with HP loading, HP promoted caudal fin ray development whereas inhibition of piezo1 using GsMTx4 suppressed its development. Thus, our results suggested that PIEZO1 is responsible for HP and could functions as a factor for cell fate determination of MSCs by regulating BMP2 expression.

Adiabatic reduction of a model of stochastic gene expression with jump Markov process.

PubMed

Yvinec, Romain; Zhuge, Changjing; Lei, Jinzhi; Mackey, Michael C

2014-04-01

This paper considers adiabatic reduction in a model of stochastic gene expression with bursting transcription considered as a jump Markov process. In this model, the process of gene expression with auto-regulation is described by fast/slow dynamics. The production of mRNA is assumed to follow a compound Poisson process occurring at a rate depending on protein levels (the phenomena called bursting in molecular biology) and the production of protein is a linear function of mRNA numbers. When the dynamics of mRNA is assumed to be a fast process (due to faster mRNA degradation than that of protein) we prove that, with appropriate scalings in the burst rate, jump size or translational rate, the bursting phenomena can be transmitted to the slow variable. We show that, depending on the scaling, the reduced equation is either a stochastic differential equation with a jump Poisson process or a deterministic ordinary differential equation. These results are significant because adiabatic reduction techniques seem to have not been rigorously justified for a stochastic differential system containing a jump Markov process. We expect that the results can be generalized to adiabatic methods in more general stochastic hybrid systems.

The inhibitory effect of vitamin K on RANKL-induced osteoclast differentiation and bone resorption.

PubMed

Wu, Wei-Jie; Kim, Min Seuk; Ahn, Byung-Yong

2015-10-01

To further understand the correlation between vitamin K and bone metabolism, the effects of vitamins K1, menaquinone-4 (MK-4), and menaquinone-7 (MK-7) on RANKL-induced osteoclast differentiation and bone resorption were comparatively investigated. Vitamin K2 groups (MK-4 and MK-7) were found to significantly inhibit RANKL-medicated osteoclast cell formation of bone marrow macrophages (BMMs) in a dose-dependent manner, without any evidence of cytotoxicity. The mRNA expression of specific osteoclast differentiation markers, such as c-Fos, NFATc1, OSCAR, and TRAP, as well as NFATc1 protein expression and TRAP activity in RANKL-treated BMMs were inhibited by vitamin K2, although MK-4 exhibited a significantly greater efficiency compared to MK-7. In contrast, the same dose of vitamin K1 had no inhibitory effect on RANKL-induced osteoclast cell formation, but increased the expression of major osteoclastogenic genes. Interestingly, vitamins K1, MK-4 and MK-7 all strongly inhibited osteoclastic bone resorption (p < 0.01) in a dose dependent manner. These results suggest that vitamins K1, MK-4 and MK-7 have anti-osteoporotic properties, while their regulation effects on osteoclastogenesis are somewhat different.

Retinoic acid-induced CHD5 upregulation and neuronal differentiation of neuroblastoma.

PubMed

Higashi, Mayumi; Kolla, Venkatadri; Iyer, Radhika; Naraparaju, Koumudi; Zhuang, Tiangang; Kolla, Sriharsha; Brodeur, Garrett M

2015-08-07

Chromodomain-helicase DNA binding protein 5 (CHD5) is an important tumor suppressor gene deleted from 1p36.31 in neuroblastomas (NBs). High CHD5 expression is associated with a favorable prognosis, but deletion or low expression is frequent in high-risk tumors. We explored the role of CHD5 expression in the neuronal differentiation of NB cell lines. NB cell lines SH-SY5Y (SY5Y), NGP, SK-N-DZ, IMR5, LAN5, SK-N-FI, NB69 and SH-EP were treated with 1-10 μM 13-cis-retinoic acid (13cRA) for 3-12 days. qRT-PCR and Western blot analyses were performed to measure mRNA and protein expression levels, respectively. Morphological differences were examined by both phase contrast and immunofluorescence studies. Treatment of SY5Y cells with 13cRA caused upregulation of CHD5 expression in a time- and dose-dependent manner (1, 5, or 10 μM for 7 or 12 days) and also induced neuronal differentiation. Furthermore, both NGP and SK-N-DZ cells showed CHD5 upregulation and neuronal differentiation after 13cRA treatment. In contrast, 13cRA treatment of IMR5, LAN5, or SK-N-FI induced neither CHD5 expression nor neuronal differentiation. NB69 cells showed two different morphologies (neuronal and substrate adherent) after 12 days treatment with 10 μM of 13cRA. CHD5 expression was high in the neuronal cells, but low/absent in the flat, substrate adherent cells. Finally, NGF treatment caused upregulation of CHD5 expression and neuronal differentiation in SY5Y cells transfected to express TrkA (SY5Y-TrkA) but not in TrkA-null parental SY5Y cells, and both changes were blocked by a pan-TRK inhibitor. Treatment with 13cRA induces neuronal differentiation only in NB cells that upregulate CHD5. In addition, NGF induced CHD5 upregulation and neuronal differentiation only in TrkA expressing cells. Together, these results suggest that CHD5 is downstream of TrkA, and CHD5 expression may be crucial for neuronal differentiation induced by either 13cRA or TrkA/NGF signaling.

Differential Utilization and Localization of ErbB Receptor Tyrosine Kinases in Skin Compared to Normal and Malignant Keratinocytes1

PubMed Central

Stoll, Stefan W; Kansra, Sanjay; Peshick, Scott; Fry, David W; Leopold, Wilbur R; Wiesen, Jane F; Sibilia, Maria; Zhang, Tong; Werb, Zena; Derynck, Rik; Wagner, Erwin F; Elder, James T

2001-01-01

Abstract Induction of heparin-binding epidermal growth factor-like growth factor (HB-EGF) mRNA in mouse skin organ culture was blocked by two pan-ErbB receptor tyrosine kinase (RTK) inhibitors but not by genetic ablation of ErbB1, suggesting involvement of multiple ErbB species in skin physiology. Human skin, cultured normal keratinocytes, and A431 skin carcinoma cells expressed ErbB1, ErbB2, and ErbB3, but not ErbB4. Skin and A431 cells expressed more ErbB3 than did keratinocytes. Despite strong expression of ErbB2 and ErbB3, heregulin was inactive in stimulating tyrosine phosphorylation in A431 cells. In contrast, it was highly active in MDA-MB-453 breast carcinoma cells. ErbB2 displayed punctate cytoplasmic staining in A431 and keratinocytes, compared to strong cell surface staining in MDA-MB-453. In skin, ErbB2 was cytoplasmic in basal keratinocytes, assuming a cell surface pattern in the upper suprabasal layers. In contrast, ErbB1 retained a cell surface distribution in all epidermal layers. Keratinocyte proliferation in culture was found to be ErbB1-RTK-dependent, using a selective inhibitor. These results suggest that in skin keratinocytes, ErbB2 transduces ligand-dependent differentiation signals, whereas ErbB1 transduces ligand-dependent proliferation/survival signals. Intracellular sequestration of ErbB2 may contribute to the malignant phenotype of A431 cells, by allowing them to respond to ErbB1-dependent growth/survival signals, while evading ErbB2-dependent differentiation signals. PMID:11571634

Prenatal stress-induced programming of genome-wide promoter DNA methylation in 5-HTT-deficient mice.

PubMed

Schraut, K G; Jakob, S B; Weidner, M T; Schmitt, A G; Scholz, C J; Strekalova, T; El Hajj, N; Eijssen, L M T; Domschke, K; Reif, A; Haaf, T; Ortega, G; Steinbusch, H W M; Lesch, K P; Van den Hove, D L

2014-10-21

The serotonin transporter gene (5-HTT/SLC6A4)-linked polymorphic region has been suggested to have a modulatory role in mediating effects of early-life stress exposure on psychopathology rendering carriers of the low-expression short (s)-variant more vulnerable to environmental adversity in later life. The underlying molecular mechanisms of this gene-by-environment interaction are not well understood, but epigenetic regulation including differential DNA methylation has been postulated to have a critical role. Recently, we used a maternal restraint stress paradigm of prenatal stress (PS) in 5-HTT-deficient mice and showed that the effects on behavior and gene expression were particularly marked in the hippocampus of female 5-Htt+/- offspring. Here, we examined to which extent these effects are mediated by differential methylation of DNA. For this purpose, we performed a genome-wide hippocampal DNA methylation screening using methylated-DNA immunoprecipitation (MeDIP) on Affymetrix GeneChip Mouse Promoter 1.0 R arrays. Using hippocampal DNA from the same mice as assessed before enabled us to correlate gene-specific DNA methylation, mRNA expression and behavior. We found that 5-Htt genotype, PS and their interaction differentially affected the DNA methylation signature of numerous genes, a subset of which showed overlap with the expression profiles of the corresponding transcripts. For example, a differentially methylated region in the gene encoding myelin basic protein (Mbp) was associated with its expression in a 5-Htt-, PS- and 5-Htt × PS-dependent manner. Subsequent fine-mapping of this Mbp locus linked the methylation status of two specific CpG sites to Mbp expression and anxiety-related behavior. In conclusion, hippocampal DNA methylation patterns and expression profiles of female prenatally stressed 5-Htt+/- mice suggest that distinct molecular mechanisms, some of which are promoter methylation-dependent, contribute to the behavioral effects of the 5-Htt genotype, PS exposure and their interaction.

Stabilin-2 modulates the efficiency of myoblast fusion during myogenic differentiation and muscle regeneration

PubMed Central

Park, Seung-Yoon; Yun, Youngeun; Lim, Jung-Suk; Kim, Mi-Jin; Kim, Sang-Yeob; Kim, Jung-Eun; Kim, In-San

2016-01-01

Myoblast fusion is essential for the formation of skeletal muscle myofibres. Studies have shown that phosphatidylserine is necessary for myoblast fusion, but the underlying mechanism is not known. Here we show that the phosphatidylserine receptor stabilin-2 acts as a membrane protein for myoblast fusion during myogenic differentiation and muscle regeneration. Stabilin-2 expression is induced during myogenic differentiation, and is regulated by calcineurin/NFAT signalling in myoblasts. Forced expression of stabilin-2 in myoblasts is associated with increased myotube formation, whereas deficiency of stabilin-2 results in the formation of small, thin myotubes. Stab2-deficient mice have myofibres with small cross-sectional area and few myonuclei and impaired muscle regeneration after injury. Importantly, myoblasts lacking stabilin-2 have reduced phosphatidylserine-dependent fusion. Collectively, our results show that stabilin-2 contributes to phosphatidylserine-dependent myoblast fusion and provide new insights into the molecular mechanism by which phosphatidylserine mediates myoblast fusion during muscle growth and regeneration. PMID:26972991

Peroxisome Proliferator-Activated Receptor γ Target Gene Encoding a Novel Angiopoietin-Related Protein Associated with Adipose Differentiation

PubMed Central

Yoon, J. Cliff; Chickering, Troy W.; Rosen, Evan D.; Dussault, Barry; Qin, Yubin; Soukas, Alexander; Friedman, Jeffrey M.; Holmes, William E.; Spiegelman, Bruce M.

2000-01-01

The nuclear receptor peroxisome proliferator-activated receptor γ regulates adipose differentiation and systemic insulin signaling via ligand-dependent transcriptional activation of target genes. However, the identities of the biologically relevant target genes are largely unknown. Here we describe the isolation and characterization of a novel target gene induced by PPARγ ligands, termed PGAR (for PPARγ angiopoietin related), which encodes a novel member of the angiopoietin family of secreted proteins. The transcriptional induction of PGAR follows a rapid time course typical of immediate-early genes and occurs in the absence of protein synthesis. The expression of PGAR is predominantly localized to adipose tissues and placenta and is consistently elevated in genetic models of obesity. Hormone-dependent adipocyte differentiation coincides with a dramatic early induction of the PGAR transcript. Alterations in nutrition and leptin administration are found to modulate the PGAR expression in vivo. Taken together, these data suggest a possible role for PGAR in the regulation of systemic lipid metabolism or glucose homeostasis. PMID:10866690

Changes in Gene Expression of Arabidopsis Thaliana Cell Cultures Upon Exposure to Real and Simulated Partial- g Forces

NASA Astrophysics Data System (ADS)

Fengler, Svenja; Spirer, Ina; Neef, Maren; Ecke, Margret; Hauslage, Jens; Hampp, Rüdiger

2016-06-01

Cell cultures of the plant model organism Arabidopsis thaliana were exposed to partial- g forces during parabolic flight and clinostat experiments (0.16 g, 0.38 g and 0.5 g were tested). In order to investigate gravity-dependent alterations in gene expression, samples were metabolically quenched by the fixative RNA later Ⓡ to stabilize nucleic acids and used for whole-genome microarray analysis. An attempt to identify the potential threshold acceleration for the gravity-dependent response showed that the smaller the experienced g-force, the greater was the susceptibility of the cell cultures. Compared to short-term μ g during a parabolic flight, the number of differentially expressed genes under partial- g was lower. In addition, the effect on the alteration of amounts of transcripts decreased during partial- g parabolic flight due to the sequence of the different parabolas (0.38 g, 0.16 g and μ g). A time-dependent analysis under simulated 0.5 g indicates that adaptation occurs within minutes. Differentially expressed genes (at least 2-fold up- or down-regulated in expression) under real flight conditions were to some extent identical with those affected by clinorotation. The highest number of homologuous genes was detected within seconds of exposure to 0.38 g (both flight and clinorotation). To a considerable part, these genes deal with cell wall properties. Additionally, responses specific for clinorotation were observed.

Socio-environmental and endocrine influences on developmental and caste-regulatory gene expression in the eusocial termite Reticulitermes flavipes

PubMed Central

2010-01-01

Background Strict regulation of caste differentiation, at the molecular level, is thought to be important to maintain social structure in insect societies. Previously, a number of extrinsic and intrinsic factors have been shown to influence caste composition in termite colonies. One important factor is the influence of nestmates; in particular, soldier termites are known to inhibit hormone-dependent worker-to-soldier differentiation. However, soldier influences on nestmates at the molecular level are virtually unknown. Here, to test the hypothesis that soldiers can influence nestmate gene expression, we investigated the impact of four treatments on whole-body gene expression in totipotent Reticulitermes flavipes workers: (i) juvenile hormone III (JHIII; a morphogenetic hormone), (ii) soldier head extracts (SHE), (iii) JHIII+SHE, and (iv) live soldiers. Results Using quantitative-real-time PCR we determined the expression patterns of 49 previously identified candidate genes in response to the four treatments at assay days 1, 5, and 10. Thirty-eight total genes from three categories (chemical production/degradation, hemolymph protein, and developmental) showed significant differential expression among treatments. Most importantly, SHE and live soldier treatments had a significant impact on a number of genes from families known to play roles in insect development, supporting previous findings and hypotheses that soldiers regulate nestmate caste differentiation via terpene primer pheromones contained in their heads. Conclusions This research provides new insights into the impacts that socio-environmental factors (JH, soldiers, primer pheromones) can have on termite gene expression and caste differentiation, and reveals a number of socially-relevant genes for investigation in subsequent caste differentiation research. PMID:20416061

Socio-environmental and endocrine influences on developmental and caste-regulatory gene expression in the eusocial termite Reticulitermes flavipes.

PubMed

Tarver, Matthew R; Zhou, Xuguo; Scharf, Michael E

2010-04-23

Strict regulation of caste differentiation, at the molecular level, is thought to be important to maintain social structure in insect societies. Previously, a number of extrinsic and intrinsic factors have been shown to influence caste composition in termite colonies. One important factor is the influence of nestmates; in particular, soldier termites are known to inhibit hormone-dependent worker-to-soldier differentiation. However, soldier influences on nestmates at the molecular level are virtually unknown. Here, to test the hypothesis that soldiers can influence nestmate gene expression, we investigated the impact of four treatments on whole-body gene expression in totipotent Reticulitermes flavipes workers: (i) juvenile hormone III (JHIII; a morphogenetic hormone), (ii) soldier head extracts (SHE), (iii) JHIII+SHE, and (iv) live soldiers. Using quantitative-real-time PCR we determined the expression patterns of 49 previously identified candidate genes in response to the four treatments at assay days 1, 5, and 10. Thirty-eight total genes from three categories (chemical production/degradation, hemolymph protein, and developmental) showed significant differential expression among treatments. Most importantly, SHE and live soldier treatments had a significant impact on a number of genes from families known to play roles in insect development, supporting previous findings and hypotheses that soldiers regulate nestmate caste differentiation via terpene primer pheromones contained in their heads. This research provides new insights into the impacts that socio-environmental factors (JH, soldiers, primer pheromones) can have on termite gene expression and caste differentiation, and reveals a number of socially-relevant genes for investigation in subsequent caste differentiation research.

The role of catechol-O-methyltransferase in catechol-enhanced erythroid differentiation of K562 cells.

PubMed

Suriguga; Li, Xiao-Fei; Li, Yang; Yu, Chun-Hong; Li, Yi-Ran; Yi, Zong-Chun

2013-12-15

Catechol is widely used in pharmaceutical and chemical industries. Catechol is also one of phenolic metabolites of benzene in vivo. Our previous study showed that catechol improved erythroid differentiation potency of K562 cells, which was associated with decreased DNA methylation in erythroid specific genes. Catechol is a substrate for the catechol-O-methyltransferase (COMT)-mediated methylation. In the present study, the role of COMT in catechol-enhanced erythroid differentiation of K562 cells was investigated. Benzidine staining showed that exposure to catechol enhanced hemin-induced hemoglobin accumulation and induced mRNA expression of erythroid specific genes in K562 cells. Treatment with catechol caused a time- and concentration-dependent increase in guaiacol concentration in the medium of cultured K562 cells. When COMT expression was knocked down by COMT shRNA expression in K562 cells, the production of guaiacol significantly reduced, and the sensitivity of K562 cells to cytotoxicity of catechol significantly increased. Knockdown of COMT expression by COMT shRNA expression also eliminated catechol-enhanced erythroid differentiation of K562 cells. In addition, the pre-treatment with methyl donor S-adenosyl-L-methionine or its demethylated product S-adenosyl-L-homocysteine induced a significant increase in hemin-induced Hb synthesis in K562 cells and the mRNA expression of erythroid specific genes. These findings indicated that O-methylation catalyzed by COMT acted as detoxication of catechol and involved in catechol-enhanced erythroid differentiation of K562 cells, and the production of S-adenosyl-L-homocysteine partly explained catechol-enhanced erythroid differentiation. © 2013.

Necdin interacts with the Msx2 homeodomain protein via MAGE-D1 to promote myogenic differentiation of C2C12 cells.

PubMed

Kuwajima, Takaaki; Taniura, Hideo; Nishimura, Isao; Yoshikawa, Kazuaki

2004-09-24

Necdin is a potent growth suppressor that is expressed predominantly in postmitotic cells such as neurons and skeletal muscle cells. Necdin shows a significant homology to MAGE (melanoma antigen) family proteins, all of which contain a large homology domain. MAGE-D1 (NRAGE, Dlxin-1) interacts with the Dlx/Msx family homeodomain proteins via an interspersed hexapeptide repeat domain distinct from the homology domain. Here we report that necdin associates with the Msx homeodomain proteins via MAGE-D1 to modulate their function. In vitro binding and co-immunoprecipitation analyses revealed that MAGE-D1 directly interacted with necdin via the homology domain and Msx1 (or Msx2) via the repeat domain. A ternary complex of necdin, MAGE-D1, and Msx2 was formed in vitro, and an endogenous complex containing these three proteins was detected in differentiating embryonal carcinoma cells. Co-expression of necdin and MAGE-D1 released Msx-dependent transcriptional repression. C2C12 myoblast cells that were stably transfected with Msx2 cDNA showed a marked reduction in myogenic differentiation, and co-expression of necdin and MAGE-D1 canceled the Msx2-dependent repression. These results suggest that necdin and MAGE-D1 cooperate to modulate the function of Dlx/Msx homeodomain proteins in cellular differentiation. Copyright 2004 American Society for Biochemistry and Molecular Biology, Inc.

β-Catenin Mediates Anti-adipogenic and Anticancer Effects of Arctigenin in Preadipocytes and Breast Cancer Cells.

PubMed

Lee, Jihye; Imm, Jee-Young; Lee, Seong-Ho

2017-03-29

Arctigenin is a lignan abundant in Asteraceae plants and has anti-inflammatory, antiobesity, and anticancer activities. Obesity is one of the leading causes of several types of cancers including breast cancer. The current study was performed to investigate if arctigenin suppresses differentiation of preadipocytes and proliferation of breast cancer cells and to explore potential molecular mechanisms. Treatment of arctigenin reduced lipid accumulation in differentiated 3T3-L1 adipocytes in a dose- and time-dependent manner without toxicity. Arctigenin suppressed the expression of peroxisome proliferator-activated receptor-gamma (PPARγ), CCAAT/enhancer-binding protein-alpha (C/EBPα), perilipin, and fatty acid binding protein 4 (FABP4) in a dose-dependent manner in differentiated 3T3-L1 cells. Both total and unphosphorylated (active) β-catenin were increased in whole cell lysates and the nuclear fraction of differentiated 3T3-L1 cells treated with 25 μM arctigenin. On the other hand, arctigenin decreased proliferation of two human breast cancer cells (MCF-7 and MDA-MB-231). Arctigenin induced apoptosis and decreased expression of total and unphosphorylated (active) β-catenin and cyclin D1 in MCF-7, but not in MDA-MB-231. These data indicate that arctigenin suppressed adipogenesis in preadipocytes and activated apoptosis in estrogen receptor (ER) positive breast cancer cells through modulating expression of β-catenin.

RhoA, Rac1, and Cdc42 differentially regulate αSMA and collagen I expression in mesenchymal stem cells.

PubMed

Ge, Jianfeng; Burnier, Laurent; Adamopoulou, Maria; Kwa, Mei Qi; Schaks, Matthias; Rottner, Klemens; Brakebusch, Cord

2018-06-15

Mesenchymal stem cells (MSC) are suggested to be important progenitors of myofibroblasts in fibrosis. To understand the role of Rho GTPase signaling in TGFβ-induced myofibroblast differentiation of MSC, we generated a novel MSC line and its descendants lacking functional Rho GTPases and Rho GTPase signaling components. Unexpectedly, our data revealed that Rho GTPase signaling is required for TGFβ-induced expression of α-smooth muscle actin (αSMA) but not of collagen I α1 ( col1a1 ). Whereas loss of RhoA and Cdc42 reduced αSMA expression, ablation of the Rac1 gene had the opposite effect. Although actin polymerization and MRTFa were crucial for TGFβ-induced αSMA expression, neither Arp2/3-dependent actin polymerization nor cofilin-dependent severing and depolymerization of F-actin were required. Instead, F-actin levels were dependent on cell contraction, and TGFβ-induced actin polymerization correlated with increased cell contraction mediated by RhoA and Cdc42. Finally, we observed impaired collagen I secretion in MSC lacking RhoA or Cdc42. These data give novel molecular insights into the role of Rho GTPases in TGFβ signaling and have implications for our understanding of MSC function in fibrosis. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Vacuolar H+-ATPase Is Expressed in Response to Gibberellin during Tomato Seed Germination1

PubMed Central

Cooley, Michael B.; Yang, Hong; Dahal, Peetambar; Mella, R. Alejandra; Downie, A. Bruce; Haigh, Anthony M.; Bradford, Kent J.

1999-01-01

Completion of germination (radicle emergence) by gibberellin (GA)-deficient (gib-1) mutant tomato (Lycopersicon esculentum Mill.) seeds is dependent upon exogenous GA, because weakening of the endosperm tissue enclosing the radicle tip requires GA. To investigate genes that may be involved in endosperm weakening or embryo growth, differential cDNA display was used to identify mRNAs differentially expressed in gib-1 seeds imbibed in the presence or absence of GA4+7. Among these was a GA-responsive mRNA encoding the 16-kD hydrophobic subunit c of the V0 membrane sector of vacuolar H+-translocating ATPases (V-ATPase), which we termed LVA-P1. LVA-P1 mRNA expression in gib-1 seeds was dependent on GA and was particularly abundant in the micropylar region prior to radicle emergence. Both GA dependence and tissue localization of LVA-P1 mRNA expression were confirmed directly in individual gib-1 seeds using tissue printing. LVA-P1 mRNA was also expressed in wild-type seeds during development and germination, independent of exogenous GA. Specific antisera detected protein subunits A and B of the cytoplasmic V1 sector of the V-ATPase holoenzyme complex in gib-1 seeds only in the presence of GA, and expression was localized to the micropylar region. The results suggest that V-ATPase plays a role in GA-regulated germination of tomato seeds. PMID:10594121

Genome-wide gene expression effects in B6C3F1 mouse intestinal epithelia following 7 and 90days of exposure to hexavalent chromium in drinking water.

PubMed

Kopec, Anna K; Kim, Suntae; Forgacs, Agnes L; Zacharewski, Timothy R; Proctor, Deborah M; Harris, Mark A; Haws, Laurie C; Thompson, Chad M

2012-02-15

Chronic administration of high doses of hexavalent chromium [Cr(VI)] as sodium dichromate dihydrate (SDD) elicits alimentary cancers in mice. To further elucidate key events underlying tumor formation, a 90-day drinking water study was conducted in B6C3F1 mice. Differential gene expression was examined in duodenal and jejunal epithelial samples following 7 or 90days of exposure to 0, 0.3, 4, 14, 60, 170 or 520mg/L SDD in drinking water. Genome-wide microarray analyses identified 6562 duodenal and 4448 jejunal unique differentially expressed genes at day 8, and 4630 and 4845 unique changes, respectively, in the duodenum and jejunum at day 91. Comparative analysis identified significant overlap in duodenal and jejunal differential gene expression. Automated dose-response modeling identified >80% of the differentially expressed genes exhibited sigmoidal dose-response curves with EC(50) values ranging from 10 to 100mg/L SDD. Only 16 genes satisfying the dose-dependent differential expression criteria had EC(50) values <10mg/L SDD, 3 of which were regulated by Nrf2, suggesting oxidative stress in response to SDD at low concentrations. Analyses of differentially expressed genes identified over-represented functions associated with oxidative stress, cell cycle, lipid metabolism, and immune responses consistent with the reported effects on redox status and histopathology at corresponding SDD drinking water concentrations. Collectively, these data are consistent with a mode of action involving oxidative stress and cytotoxicity as early key events. This suggests that the tumorigenic effects of chronic Cr(VI) oral exposure likely require chronic tissue damage and compensatory epithelial cell proliferation. Copyright © 2011 Elsevier Inc. All rights reserved.

Altered MENIN expression disrupts the MAFA differentiation pathway in insulinoma.

PubMed

Hamze, Z; Vercherat, C; Bernigaud-Lacheretz, A; Bazzi, W; Bonnavion, R; Lu, J; Calender, A; Pouponnot, C; Bertolino, P; Roche, C; Stein, R; Scoazec, J Y; Zhang, C X; Cordier-Bussat, M

2013-12-01

The protein MENIN is the product of the multiple endocrine neoplasia type I (MEN1) gene. Altered MENIN expression is one of the few events that are clearly associated with foregut neuroendocrine tumours (NETs), classical oncogenes or tumour suppressors being not involved. One of the current challenges is to understand how alteration of MENIN expression contributes to the development of these tumours. We hypothesised that MENIN might regulate factors maintaining endocrine-differentiated functions. We chose the insulinoma model, a paradigmatic example of well-differentiated pancreatic NETs, to study whether MENIN interferes with the expression of v-MAF musculoaponeurotic fibrosarcoma oncogene homologue A (MAFA), a master glucose-dependent transcription factor in differentiated β-cells. Immunohistochemical analysis of a series of human insulinomas revealed a correlated decrease in both MENIN and MAFA. Decreased MAFA expression resulting from targeted Men1 ablation was also consistently observed in mouse insulinomas. In vitro analyses using insulinoma cell lines showed that MENIN regulated MAFA protein and mRNA levels, and bound to Mafa promoter sequences. MENIN knockdown concomitantly decreased mRNA expression of both Mafa and β-cell differentiation markers (Ins1/2, Gck, Slc2a2 and Pdx1) and, in parallel, increased the proliferation rate of tumours as measured by bromodeoxyuridine incorporation. Interestingly, MAFA knockdown alone also increased proliferation rate but did not affect the expression of candidate proliferation genes regulated by MENIN. Finally, MENIN variants with missense mutations detected in patients with MEN1 lost the WT MENIN properties to regulate MAFA. Together, our findings unveil a previously unsuspected MENIN/MAFA connection regarding control of the β-cell differentiation/proliferation balance, which could contribute to tumorigenesis.

Miz1, a Novel Target of ING4, Can Drive Prostate Luminal Epithelial Cell Differentiation.

PubMed

Berger, Penny L; Winn, Mary E; Miranti, Cindy K

2017-01-01

How prostate epithelial cells differentiate and how dysregulation of this process contributes to prostate tumorigenesis remain unclear. We recently identified a Myc target and chromatin reader protein, ING4, as a necessary component of human prostate luminal epithelial cell differentiation, which is often lost in primary prostate tumors. Furthermore, loss of ING4 in the context of oncogenic mutations is required for prostate tumorigenesis. Identifying the gene targets of ING4 can provide insight into how its loss disrupts differentiation and leads to prostate cancer. Using a combination of RNA-Seq, a best candidate approach, and chromatin immunoprecipitation (ChIP), we identified Miz1 as a new ING4 target. ING4 or Miz1 overexpression, shRNA knock-down, and a Myc-binding mutant were used in a human in vitro differentiation assay to assess the role of Miz1 in luminal cell differentiation. ING4 directly binds the Miz1 promoter and is required to induce Miz1 mRNA and protein expression during luminal cell differentiation. Miz1 mRNA was not induced in shING4 expressing cells or tumorigenic cells in which ING4 is not expressed. Miz1 dependency on ING4 was unique to differentiating luminal cells; Miz1 mRNA expression was not induced in basal cells. Although Miz1 is a direct target of ING4, and its overexpression can drive luminal cell differentiation, Miz1 was not required for differentiation. Miz1 is a newly identified ING4-induced target gene which can drive prostate luminal epithelial cell differentiation although it is not absolutely required. Prostate 77:49-59, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Role of C/EBPβ-LAP and C/EBPβ-LIP in early adipogenic differentiation of human white adipose-derived progenitors and at later stages in immature adipocytes.

PubMed

Lechner, Stefan; Mitterberger, Maria C; Mattesich, Monika; Zwerschke, Werner

2013-01-01

We investigated the role of the major isoforms of CCAAT enhancer binding protein β (C/EBPβ), C/EBPβ-LAP and C/EBPβ-LIP, in adipogenesis of human white adipose-derived stromal/progenitor cells (ASC). C/EBPβ gene expression was transiently induced early in adipogenesis. At later stages, in immature adipocytes, the C/EBPβ mRNA and protein levels declined. The C/EBPβ-LIP protein steady-state level decreased considerably stronger than the C/EBPβ-LAP level and the C/EBPβ-LIP half-life was significantly shorter than the C/EBPβ-LAP half-life. The turn-over of both C/EBPβ-isoforms was regulated by ubiquitin/proteasome-dependent degradation. These data suggest that the protein stability of the C/EBPβ-isoforms is differentially regulated in the course of adipogenesis and in immature adipocytes. Constitutive overexpression of C/EBPβ-LIP had antiadipogenic activity in human ASC. C/EBPβ-LAP, which promotes adipogenesis in mouse 3T3-L1 preadipocytes by directly activating expression of the adipogenic keyregulator PPARγ2, induced the expression of PPARγ2 and of the adipocyte differentiation gene product FABP4 in confluent ASC in the absence of adipogenic hormones. At later stages after hormone cocktail-induced adipogenesis, in immature adipocytes, constitutive overexpression of C/EBPβ-LAP led to reduced expression of PPARγ2 and FABP4, C/EBPα expression was downregulated and the expression of the adipocyte differentiation gene products adiponectin and leptin was impaired. These findings suggest that constitutive overexpression of C/EBPβ-LAP induces adipogenesis in human ASC and negatively regulates the expression of adipogenic regulators and certain adipocyte differentiation gene products in immature adipocytes. We conclude the regulation of both C/EBPβ gene expression and C/EBPβ-LIP and C/EBPβ-LAP protein turn-over plays an important role for the expression of adipogenic regulators and/or adipocyte differentiation genes in early adipogenic differentiation of human ASC and at later stages in human immature adipocytes. Copyright © 2012 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

Prostaglandin E2 inhibits Tr1 cell differentiation through suppression of c-Maf

PubMed Central

Hooper, Kirsten Mary; Kong, Weimin

2017-01-01

Prostaglandin E2 (PGE2), a major lipid mediator abundant at inflammatory sites, acts as a proinflammatory agent in models of inflammatory/autoimmune diseases by promoting CD4 Th1/Th17 differentiation. Regulatory T cells, including the IL-10 producing Tr1 cells counterbalance the proinflammatory activity of effector Th1/Th17 cells. Tr1 cell differentiation and function are induced by IL-27, and depend primarily on sustained expression of c-Maf in addition to AhR and Blimp-1. In agreement with the in vivo proinflammatory role of PGE2, here we report for the first time that PGE2 inhibits IL-27-induced differentiation and IL-10 production of murine CD4+CD49b+LAG-3+Foxp3- Tr1 cells. The inhibitory effect of PGE2 was mediated through EP4 receptors and induction of cAMP, leading to a significant reduction in c-Maf expression. Although PGE2 reduced IL-21 production in differentiating Tr1 cells, its inhibitory effect on Tr1 differentiation and c-Maf expression also occurred independent of IL-21 signaling. PGE2 did not affect STAT1/3 activation, AhR expression and only marginally reduced Egr-2/Blimp-1 expression. The effect of PGE2 on CD4+CD49b+LAG-3+ Tr1 differentiation was not associated with either induction of Foxp3 or IL-17 production, suggesting a lack of transdifferentiation into Foxp3+ Treg or effector Th17 cells. We recently reported that PGE2 inhibits the expression and production of IL-27 from activated conventional dendritic cells (cDC) in vivo and in vitro. The present study indicates that PGE2 also reduces murine Tr1 differentiation and function directly by acting on IL-27-differentiating Tr1 cells. Together, the ability of PGE2 to inhibit IL-27 production by cDC, and the direct inhibitory effect on Tr1 differentiation mediated through reduction in c-Maf expression, represent a new mechanistic perspective for the proinflammatory activity of PGE2. PMID:28604806

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

Aoyagi, Yasuyuki; Department of Genome Research and Clinical Application, Graduate School of Medicine, Chiba University, Chiba; Kuroda, Masayuki, E-mail: kurodam@faculty.chiba-u.jp

Adipose tissue is expected to provide a source of cells for protein replacement therapies via auto-transplantation. However, the conditioning of the environment surrounding the transplanted adipocytes for their long-term survival and protein secretion properties has not been established. We have recently developed a preparation procedure for preadipocytes, ceiling culture-derived proliferative adipocytes (ccdPAs), as a therapeutic gene vehicle suitable for stable gene product secretion. We herein report the results of our evaluation of using fibrin glue as a scaffold for the transplanted ccdPAs for the expression of a transduced gene in a three-dimensional culture system. The ccdPAs secreted the functional proteinmore » translated from an exogenously transduced gene, as well as physiological adipocyte proteins, and the long viability of ccdPAs (up to 84 days) was dependent on the fibrinogen concentrations. The ccdPAs spontaneously accumulated lipid droplets, and their expression levels of the transduced exogenous gene with its product were maintained for at least 56 days. The fibrinogen concentration modified the adipogenic differentiation of ccdPAs and their exogenous gene expression levels, and the levels of exogenously transduced gene expression at the different fibrinogen concentrations were dependent on the extent of adipogenic differentiation in the gel. These results indicate that fibrin glue helps to maintain the high adipogenic potential of cultured adipocytes after passaging in a 3D culture system, and suggests that once they are successfully implanted at the transplantation site, the cells exhibit increased expression of the transduced gene with adipogenic differentiation.« less

Increased numbers of pre-existing memory CD8 T cells and decreased T-bet expression can restrain terminal differentiation of secondary effector and memory CD8 T cells1

PubMed Central

Joshi, Nikhil S.; Cui, Weiguo; Dominguez, Claudia; Chen, Jonathan H.; Hand, Timothy W.; Kaech, Susan M.

2011-01-01

Memory CD8 T cells acquire TEM properties following reinfection, and may reach terminally differentiated, senescent states (“Hayflick limit”) after multiple infections. The signals controlling this process are not well understood, but we found that the degree of 2o effector and memory CD8 T cell differentiation was intimately linked to the amount of T-bet expressed upon reactivation and pre-existing memory CD8 T cell number (i.e., 1o memory CD8 T cell precursor frequency) present during secondary infection. Compared to naïve cells, memory CD8 T cells were predisposed towards terminal effector (TE) cell differentiation because they could immediately respond to IL-12 and induce T-bet, even in the absence of antigen. TE cell formation following 2o or 3o infections was dependent on increased T-bet expression because T-bet+/− cells were resistant to these phenotypic changes. Larger numbers of pre-existing memory CD8 T cells limited the duration of 2o infection and the amount of IL-12 produced, and consequently, this reduced T-bet expression and the proportion of 2o TE CD8 T cells that formed. Together, these data show that, over repeated infections, memory CD8 T cell quality and proliferative fitness is not strictly determined by the number of serial encounters with antigen or cell divisions, but is a function of the CD8 T cell differentiation state, which is genetically controlled in a T-bet-dependent manner. This differentiation state can be modulated by pre-existing memory CD8 T cell number and the intensity of inflammation during reinfection. These results have important implications for vaccinations involving prime-boost strategies. PMID:21930973

Secreted Clusterin protein inhibits osteoblast differentiation of bone marrow mesenchymal stem cells by suppressing ERK1/2 signaling pathway.

PubMed

Abdallah, Basem M; Alzahrani, Abdullah M; Kassem, Moustapha

2018-05-01

Secreted Clusterin (sCLU, also known as Apolipoprotein J) is an anti-apoptotic glycoprotein involved in the regulation of cell proliferation, lipid transport, extracellular tissue remodeling and apoptosis. sCLU is expressed and secreted by mouse bone marrow-derived skeletal (stromal or mesenchymal) stem cells (mBMSCs), but its functional role in MSC biology is not known. In this study, we demonstrated that Clusterin mRNA expression and protein secretion in conditioned medium increased during adipocyte differentiation and decreased during osteoblast differentiation of mBMSCs. Treatment of mBMSC cultures with recombinant sCLU protein increased cell proliferation and exerted an inhibitory effect on the osteoblast differentiation while stimulated adipocyte differentiation in a dose-dependent manner. siRNA-mediated silencing of Clu expression in mBMSCs reduced adipocyte differentiation and stimulated osteoblast differentiation of mBMSCs. Furthermore, the inhibitory effect of sCLU on the osteoblast differentiation of mBMSCs was mediated by the suppression of extracellular signal-regulated kinase (ERK1/2) phosphorylation. In conclusion, we identified sCLU as a regulator of mBMSCs lineage commitment to osteoblasts versus adipocytes through a mechanism mediated by ERK1/2 signaling. Inhibiting sCLU is a possible therapeutic approach for enhancing osteoblast differentiation and consequently bone formation. Copyright © 2018 Elsevier Inc. All rights reserved.

Differential Expression of Osteo-Modulatory Molecules in Periodontal Ligament Stem Cells in Response to Modified Titanium Surfaces

PubMed Central

Kim, So Yeon; Yoo, Ji-Yeon; Ohe, Joo-Young; Lee, Jung-Woo; Moon, Ji-Hoi; Kwon, Yong-Dae; Heo, Jung Sun

2014-01-01

This study assessed differential gene expression of signaling molecules involved in osteogenic differentiation of periodontal ligament stem cells (PDLSCs) subjected to different titanium (Ti) surface types. PDLSCs were cultured on tissue culture polystyrene (TCPS), and four types of Ti discs (PT, SLA, hydrophilic PT (pmodPT), and hydrophilic SLA (modSLA)) with no osteoinductive factor and then osteogenic activity, including alkaline phosphatase (ALP) activity, mRNA expression of runt-related gene 2, osterix, FOSB, FRA1, and protein levels of osteopontin and collagen type IA, were examined. The highest osteogenic activity appeared in PDLSCs cultured on SLA, compared with the TCPS and other Ti surfaces. The role of surface properties in affecting signaling molecules to modulate PDLSC behavior was determined by examining the regulation of Wnt pathways. mRNA expression of the canonical Wnt signaling molecules, Wnt3a and β-catenin, was higher on SLA and modSLA than on smooth surfaces, but gene expression of the calcium-dependent Wnt signaling molecules Wnt5a, calmodulin, and NFATc1 was increased significantly on PT and pmodPT. Moreover, integrin α2/β1, sonic hedgehog, and Notch signaling molecules were affected differently by each surface modification. In conclusion, surface roughness and hydrophilicity can affect differential Wnt pathways and signaling molecules, targeting the osteogenic differentiation of PDLSCs. PMID:25057487

Differential expression of osteo-modulatory molecules in periodontal ligament stem cells in response to modified titanium surfaces.

PubMed

Kim, So Yeon; Yoo, Ji-Yeon; Ohe, Joo-Young; Lee, Jung-Woo; Moon, Ji-Hoi; Kwon, Yong-Dae; Heo, Jung Sun

2014-01-01

This study assessed differential gene expression of signaling molecules involved in osteogenic differentiation of periodontal ligament stem cells (PDLSCs) subjected to different titanium (Ti) surface types. PDLSCs were cultured on tissue culture polystyrene (TCPS), and four types of Ti discs (PT, SLA, hydrophilic PT (pmodPT), and hydrophilic SLA (modSLA)) with no osteoinductive factor and then osteogenic activity, including alkaline phosphatase (ALP) activity, mRNA expression of runt-related gene 2, osterix, FOSB, FRA1, and protein levels of osteopontin and collagen type IA, were examined. The highest osteogenic activity appeared in PDLSCs cultured on SLA, compared with the TCPS and other Ti surfaces. The role of surface properties in affecting signaling molecules to modulate PDLSC behavior was determined by examining the regulation of Wnt pathways. mRNA expression of the canonical Wnt signaling molecules, Wnt3a and β-catenin, was higher on SLA and modSLA than on smooth surfaces, but gene expression of the calcium-dependent Wnt signaling molecules Wnt5a, calmodulin, and NFATc1 was increased significantly on PT and pmodPT. Moreover, integrin α2/β1, sonic hedgehog, and Notch signaling molecules were affected differently by each surface modification. In conclusion, surface roughness and hydrophilicity can affect differential Wnt pathways and signaling molecules, targeting the osteogenic differentiation of PDLSCs.

Taurine suppresses osteoblastic differentiation of aortic valve interstitial cells induced by beta-glycerophosphate disodium, dexamethasone and ascorbic acid via the ERK pathway.

PubMed

Feng, Xiang; Li, Jian-ming; Liao, Xiao-bo; Hu, Ye-rong; Shang, Bao-peng; Zhang, Zhi-yuan; Yuan, Ling-qing; Xie, Hui; Sheng, Zhi-feng; Tang, Hao; Zhang, Wei; Gu, Lu; Zhou, Xin-min

2012-10-01

Aortic valve calcification (AVC) is an active process characterized by osteoblastic differentiation of the aortic valve interstitial cells (AVICs). Taurine is a free β-amino acid and plays important physiological roles including protective effect of cardiovascular events. To evaluate the possible role of taurine in AVC, we isolated human AVICs from patients with type A dissection without leaflet disease. We demonstrated that the cultured AVICs express SM α-actin, vimentin and taurine transporter (TAUT), but not CD31, SM-myosin or desmin. We also established the osteoblastic differentiation model of the AVICs induced by pro-calcific medium (PCM) containing β-glycerophosphate disodium, dexamethasone and ascorbic acid in vitro. The results showed that taurine attenuated the PCM-induced osteoblastic differentiation of AVICs by decreasing the alkaline phosphate (ALP) activity/expression and the expression of the core binding factor α1 (Cbfα1) in a dose-dependent manner (reaching the maximum protective effect at 10 mM), and taurine (10 mM) inhibited the mineralization level of AVICs in the form of calcium content significantly. Furthermore, taurine activated the extracellular signal-regulated protein kinase (ERK) pathway via TAUT, and the inhibitor of ERK (PD98059) abolished the effect of taurine on both ALP activity/expression and Cbfα1 expression. These results suggested that taurine could inhibit osteoblastic differentiation of AVIC via the ERK pathway.

Human pancreatic islet-derived extracellular vesicles modulate insulin expression in 3D-differentiating iPSC clusters

PubMed Central

Andersson, Eva-Marie; Heath, Nikki; Persson-kry, Anette; Collins, Richard; Hicks, Ryan; Dekker, Niek; Forslöw, Anna

2017-01-01

It has been suggested that extracellular vesicles (EVs) can mediate crosstalk between hormones and metabolites within pancreatic tissue. However, the possible effect of pancreatic EVs on stem cell differentiation into pancreatic lineages remains unknown. Herein, human islet-derived EVs (h-Islet-EVs) were isolated, characterized and subsequently added to human induced pluripotent stem cell (iPSC) clusters during pancreatic differentiation. The h-islet-EVs had a mean size of 117±7 nm and showed positive expression of CD63 and CD81 EV markers as measured by ELISA. The presence of key pancreatic transcription factor mRNA, such as NGN3, MAFA and PDX1, and pancreatic hormone proteins such as C-peptide and glucagon, were confirmed in h-Islet-EVs. iPSC clusters were differentiated in suspension and at the end stages of the differentiation protocol, the mRNA expression of the main pancreatic transcription factors and pancreatic hormones was increased. H-Islet-EVs were supplemented to the iPSC clusters in the later stages of differentiation. It was observed that h-Islet-EVs were able to up-regulate the intracellular levels of C-peptide in iPSC clusters in a concentration-dependent manner. The effect of h-Islet-EVs on the differentiation of iPSC clusters cultured in 3D-collagen hydrogels was also assessed. Although increased mRNA expression for pancreatic markers was observed when culturing the iPSC clusters in 3D-collagen hydrogels, delivery of EVs did not affect the insulin or C-peptide intracellular content. Our results provide new information on the role of h-Islet-EVs in the regulation of insulin expression in differentiating iPSC clusters, and are highly relevant for pancreatic tissue engineering applications. PMID:29117231

Novel murine clonal cell lines either express slow or mixed (fast and slow) muscle markers following differentiation in vitro.

PubMed

Peltzer, J; Colman, L; Cebrian, J; Musa, H; Peckham, M; Keller, A

2008-05-01

We have investigated whether the phenotype of myogenic clones derived from satellite cells of different muscles from the transgenic immortomouse depended on muscle type origin. Clones derived from neonatal, or 6- to 12-week-old fast and slow muscles, were analyzed for myosin and enolase isoforms as phenotypic markers. All clones derived from slow-oxidative muscles differentiated into myotubes with a preferentially slow contractile phenotype, whereas some clones derived from rapid-glycolytic or neonatal muscles expressed both fast and slow myosin isoforms. Thus, muscle origin appears to bias myosin isoform expression in myotubes. The neonatal clone (WTt) was cultivated in various medium and substrate conditions, allowing us to determine optimized conditions for their differentiation. Matrigel allowed expressions of adult myosin isoforms, and an isozymic switch from embryonic alpha- toward muscle-specific beta-enolase, never previously observed in vitro. These cells will be a useful model for in vitro studies of muscle fiber maturation and plasticity.

Barx2 is Expressed in Satellite Cells and is Required for Normal Muscle Growth and Regeneration

PubMed Central

Meech, Robyn; Gonzalez, Katie N.; Barro, Marietta; Gromova, Anastasia; Zhuang, Lizhe; Hulin, Julie-Ann; Makarenkova, Helen P.

2015-01-01

Muscle growth and regeneration are regulated through a series of spatiotemporally dependent signaling and transcriptional cascades. Although the transcriptional program controlling myogenesis has been extensively investigated, the full repertoire of transcriptional regulators involved in this process is far from defined. Various homeodomain transcription factors have been shown to play important roles in both muscle development and muscle satellite cell-dependent repair. Here, we show that the homeodomain factor Barx2 is a new marker for embryonic and adult myoblasts and is required for normal postnatal muscle growth and repair. Barx2 is coexpressed with Pax7, which is the canonical marker of satellite cells, and is upregulated in satellite cells after muscle injury. Mice lacking the Barx2 gene show reduced postnatal muscle growth, muscle atrophy, and defective muscle repair. Moreover, loss of Barx2 delays the expression of genes that control proliferation and differentiation in regenerating muscle. Consistent with the in vivo observations, satellite cell-derived myoblasts cultured from Barx2−/− mice show decreased proliferation and ability to differentiate relative to those from wild-type or Barx2+/− mice. Barx2−/− myoblasts show reduced expression of the differentiation-associated factor myogenin as well as cell adhesion and matrix molecules. Finally, we find that mice lacking both Barx2 and dystrophin gene expression have severe early onset myopathy. Together, these data indicate that Barx2 is an important regulator of muscle growth and repair that acts via the control of satellite cell proliferation and differentiation. PMID:22076929

Microtubule-regulating proteins and cAMP-dependent signaling in neuroblastoma differentiation.

PubMed

Muñoz-Llancao, Pablo; de Gregorio, Cristian; Las Heras, Macarena; Meinohl, Christopher; Noorman, Kevin; Boddeke, Erik; Cheng, Xiaodong; Lezoualc'h, Frank; Schmidt, Martina; Gonzalez-Billault, Christian

2017-03-01

Neurons are highly differentiated cells responsible for the conduction and transmission of information in the nervous system. The proper function of a neuron relies on the compartmentalization of their intracellular domains. Differentiated neuroblastoma cells have been extensively used to study and understand the physiology and cell biology of neuronal cells. Here, we show that differentiation of N1E-115 neuroblastoma cells is more pronounced upon exposure of a chemical analog of cyclic AMP (cAMP), db-cAMP. We next analysed the expression of key microtubule-regulating proteins in differentiated cells and the expression and activation of key cAMP players such as EPAC, PKA and AKAP79/150. Most of the microtubule-promoting factors were up regulated during differentiation of N1E-115 cells, while microtubule-destabilizing proteins were down regulated. We observed an increase in tubulin post-translational modifications related to microtubule stability. As expected, db-cAMP increased PKA- and EPAC-dependent signalling. Consistently, pharmacological modulation of EPAC activity instructed cell differentiation, number of neurites, and neurite length in N1E-115 cells. Moreover, disruption of the PKA-AKAP interaction reduced these morphometric parameters. Interestingly, PKA and EPAC act synergistically to induce neuronal differentiation in N1E-115. Altogether these results show that the changes observed in the differentiation of N1E-115 cells proceed by regulating several microtubule-stabilizing factors, and the acquisition of a neuronal phenotype is a process involving concerted although independent functions of EPAC and PKA. © 2017 Wiley Periodicals, Inc.

Interleukin-1β modulates smooth muscle cell phenotype to a distinct inflammatory state relative to PDGF-DD via NF-κB-dependent mechanisms.

PubMed

Alexander, Matthew R; Murgai, Meera; Moehle, Christopher W; Owens, Gary K

2012-04-02

Smooth muscle cell (SMC) phenotypic modulation in atherosclerosis and in response to PDGF in vitro involves repression of differentiation marker genes and increases in SMC proliferation, migration, and matrix synthesis. However, SMCs within atherosclerotic plaques can also express a number of proinflammatory genes, and in cultured SMCs the inflammatory cytokine IL-1β represses SMC marker gene expression and induces inflammatory gene expression. Studies herein tested the hypothesis that IL-1β modulates SMC phenotype to a distinct inflammatory state relative to PDGF-DD. Genome-wide gene expression analysis of IL-1β- or PDGF-DD-treated SMCs revealed that although both stimuli repressed SMC differentiation marker gene expression, IL-1β distinctly induced expression of proinflammatory genes, while PDGF-DD primarily induced genes involved in cell proliferation. Promoters of inflammatory genes distinctly induced by IL-1β exhibited over-representation of NF-κB binding sites, and NF-κB inhibition in SMCs reduced IL-1β-induced upregulation of proinflammatory genes as well as repression of SMC differentiation marker genes. Interestingly, PDGF-DD-induced SMC marker gene repression was not NF-κB dependent. Finally, immunofluorescent staining of mouse atherosclerotic lesions revealed the presence of cells positive for the marker of an IL-1β-stimulated inflammatory SMC, chemokine (C-C motif) ligand 20 (CCL20), but not the PDGF-DD-induced gene, regulator of G protein signaling 17 (RGS17). Results demonstrate that IL-1β- but not PDGF-DD-induced phenotypic modulation of SMC is characterized by NF-κB-dependent activation of proinflammatory genes, suggesting the existence of a distinct inflammatory SMC phenotype. In addition, studies provide evidence for the possible utility of CCL20 and RGS17 as markers of inflammatory and proliferative state SMCs within atherosclerotic plaques in vivo.

TBR2 antagonizes retinoic acid dependent neuronal differentiation by repressing Zfp423 during corticogenesis.

PubMed

Massimino, Luca; Flores-Garcia, Lisbeth; Di Stefano, Bruno; Colasante, Gaia; Icoresi-Mazzeo, Cecilia; Zaghi, Mattia; Hamilton, Bruce A; Sessa, Alessandro

2018-02-15

During cerebral cortex development, neural progenitors are required to elaborate a variety of cell differentiation signals to which they are continuously exposed. RA acid is a potent inducer of neuronal differentiation as it was found to influence cortical development. We report herein that TBR2, a transcription factor specific to Intermediate (Basal) Neural Progenitors (INPs), represses activation of the RA responsive element and expression of RA target genes in cell lines. This repressive action on RA signaling was functionally confirmed by the decrease of RA-mediated neuronal differentiation in neural stem cells stably overexpressing TBR2. In vivo mapping of RA activity in the developing cortex indicated that RA activity is detected in radial glial cells and subsequently downregulated in INPs, revealing a fine cell-type specific regulation of its signaling. Thus, TBR2 might be a molecular player in opposing RA signaling in INPs. Interestingly, this negative regulation is achieved at least in part by directly repressing the critical nuclear RA co-factor ZFP423. Indeed, we found ZFP423 to be expressed in the developing cortex and promote RA-dependent neuronal differentiation. These data indicate that TBR2 contributes to suppressing RA signaling in INPs, thereby enabling them to re-enter the cell cycle and delay neuronal differentiation. Copyright © 2018 Elsevier Inc. All rights reserved.

CORNAS: coverage-dependent RNA-Seq analysis of gene expression data without biological replicates.

PubMed

Low, Joel Z B; Khang, Tsung Fei; Tammi, Martti T

2017-12-28

In current statistical methods for calling differentially expressed genes in RNA-Seq experiments, the assumption is that an adjusted observed gene count represents an unknown true gene count. This adjustment usually consists of a normalization step to account for heterogeneous sample library sizes, and then the resulting normalized gene counts are used as input for parametric or non-parametric differential gene expression tests. A distribution of true gene counts, each with a different probability, can result in the same observed gene count. Importantly, sequencing coverage information is currently not explicitly incorporated into any of the statistical models used for RNA-Seq analysis. We developed a fast Bayesian method which uses the sequencing coverage information determined from the concentration of an RNA sample to estimate the posterior distribution of a true gene count. Our method has better or comparable performance compared to NOISeq and GFOLD, according to the results from simulations and experiments with real unreplicated data. We incorporated a previously unused sequencing coverage parameter into a procedure for differential gene expression analysis with RNA-Seq data. Our results suggest that our method can be used to overcome analytical bottlenecks in experiments with limited number of replicates and low sequencing coverage. The method is implemented in CORNAS (Coverage-dependent RNA-Seq), and is available at https://github.com/joel-lzb/CORNAS .

Comparison of Long Noncoding RNA and mRNA Expression Profiles in Mesenchymal Stem Cells Derived from Human Periodontal Ligament and Bone Marrow

PubMed Central

Dong, Rui; Du, Juan; Wang, Liping; Wang, Jinsong; Ding, Gang; Wang, Songlin; Fan, Zhipeng

2014-01-01

Mesenchymal stem cells (MSCs) in different anatomic locations possess diverse biological activities. Maintaining the pluripotent state and differentiation depend on the expression and regulation of thousands of genes, but it remains unclear which molecular mechanisms underlie MSC diversity. Thus, potential MSC applications are restricted. Long noncoding RNAs (lncRNAs) are implicated in the complex molecular circuitry of cellular processes. We investigated differences in lncRNA and mRNA expression profiles between bone marrow stem cells (BMSCs) and periodontal ligament stem cells (PDLSCs) with lncRNA microarray assays and bioinformatics analysis. In PDLSCs, numerous lncRNAs were significantly upregulated (n = 457) or downregulated (n = 513) compared to BMSCs. Furthermore, 1,578 mRNAs were differentially expressed. These genes implicated cellular pathways that may be associated with MSC characteristics, including apoptosis, MAPK, cell cycle, and Wnt signaling pathway. Signal-net analysis indicated that phospholipase C beta 4, filamin B beta, calcium/calmodulin-dependent protein kinase II gamma, and the ionotropic glutamate receptor, AMPA 1, had the highest betweenness centrality among significant genes in the differential gene profile network. A comparison between the coding-noncoding gene coexpression networks of PDLSCs and BMSCs identified chemokine (C-X-C motif) ligand 12 as a core regulatory factor in MSC biology. These results provided insight into the mechanisms underlying MSC biology. PMID:24790996

Role of the POZ zinc finger transcription factor FBI-1 in human and murine adipogenesis.

PubMed

Laudes, Matthias; Christodoulides, Constantinos; Sewter, Ciaran; Rochford, Justin J; Considine, Robert V; Sethi, Jaswinder K; Vidal-Puig, Antonio; O'Rahilly, Stephen

2004-03-19

Poxvirus zinc finger (POZ) zinc finger domain transcription factors have been shown to play a role in the control of growth arrest and differentiation in several types of mesenchymal cells but not, as yet, adipocytes. We found that a POZ domain protein, factor that binds to inducer of short transcripts-1 (FBI-1), was induced during both murine and human preadipocyte differentiation with maximal expression levels seen at days 2-4. FBI-1 mRNA was expressed in human adipose tissue with the highest levels found in samples from morbidly obese subjects. Murine cell lines constitutively expressing FBI-1 showed evidence for accelerated adipogenesis with earlier induction of markers of differentiation and enhanced lipid accumulation, suggesting that FBI-1 may be an active participant in the differentiation process. Consistent with the properties of this family of proteins in other cell systems, 3T3L1 cells stably overexpressing FBI-1 showed reduced DNA synthesis and reduced expression of cyclin A, cyclin-dependent kinase 2, and p107, proteins known to be involved in the regulation of mitotic clonal expansion. In addition, FBI-1 reduced the transcriptional activity of the cyclin A promoter. Thus, FBI-1, a POZ zinc finger transcription factor, is induced during the early phases of human and murine preadipocyte differentiation where it may contribute to adipogenesis through influencing the switch from cellular proliferation to terminal differentiation.

Adenosine A₂A receptors inhibit delayed rectifier potassium currents and cell differentiation in primary purified oligodendrocyte cultures.

PubMed

Coppi, Elisabetta; Cellai, Lucrezia; Maraula, Giovanna; Pugliese, Anna Maria; Pedata, Felicita

2013-10-01

Oligodendrocyte progenitor cells (OPCs) are a population of cycling cells which persist in the adult central nervous system (CNS) where, under opportune stimuli, they differentiate into mature myelinating oligodendrocytes. Adenosine A(2A) receptors are Gs-coupled P1 purinergic receptors which are widely distributed throughout the CNS. It has been demonstrated that OPCs express A(2A) receptors, but their functional role in these cells remains elusive. Oligodendrocytes express distinct voltage-gated ion channels depending on their maturation. Here, by electrophysiological recordings coupled with immunocytochemical labeling, we studied the effects of adenosine A(2A) receptors on membrane currents and differentiation of purified primary OPCs isolated from the rat cortex. We found that the selective A(2A) agonist, CGS21680, inhibits sustained, delayed rectifier, K(+) currents (I(K)) without modifying transient (I(A)) conductances. The effect was observed in all cells tested, independently from time in culture. CGS21680 inhibition of I(K) current was concentration-dependent (10-200 nM) and blocked in the presence of the selective A(2A) antagonist SCH58261 (100 nM). It is known that I(K) currents play an important role during OPC development since their block decreases cell proliferation and differentiation. In light of these data, our further aim was to investigate whether A(2A) receptors modulate these processes. CGS21680, applied at 100 nM in the culture medium of oligodendrocyte cultures, inhibits OPC differentiation (an effect prevented by SCH58261) without affecting cell proliferation. Data demonstrate that cultured OPCs express functional A(2A) receptors whose activation negatively modulate I(K) currents. We propose that, by this mechanism, A(2A) adenosine receptors inhibit OPC differentiation. Copyright © 2013 Elsevier Ltd. All rights reserved.

All-Trans Retinoic Acid-Induced Deficiency of the Wnt/β-Catenin Pathway Enhances Hepatic Carcinoma Stem Cell Differentiation

PubMed Central

Zhang, Xia; Bai, Jianhua; Chen, Gang; Li, Li; Li, Meizhang

2015-01-01

Retinoic acid (RA) is an important biological signal that directly differentiates cells during embryonic development and tumorigenesis. However, the molecular mechanism of RA-mediated differentiation in hepatic cancer stem cells (hCSCs) is not well understood. In this study, we found that mRNA expressions of RA-biosynthesis-related dehydrogenases were highly expressed in hepatocellular carcinoma. All-trans retinoic acid (ATRA) differentiated hCSCs through inhibiting the function of β-catenin in vitro. ATRA also inhibited the function of PI3K-AKT and enhanced GSK-3β-dependent degradation of phosphorylated β-catenin. Furthermore, ATRA and β-catenin silencing both increased hCSC sensitivity to docetaxel treatment. Our results suggest that targeting β-catenin will provide extra benefits for ATRA-mediated treatment of hepatic cancer patients. PMID:26571119

A GATA-2/estrogen receptor chimera functions as a ligand-dependent negative regulator of self-renewal

PubMed Central

Heyworth, Clare; Gale, Karin; Dexter, Michael; May, Gillian; Enver, Tariq

1999-01-01

The transcription factor GATA-2 is expressed in hematopoietic stem and progenitor cells and is functionally implicated in their survival and proliferation. We have used estrogen and tamoxifen-inducible forms of GATA-2 to modulate the levels of GATA-2 in the IL-3-dependent multipotential hematopoietic progenitor cell model FDCP mix. Ligand-dependent induction of exogenous GATA-2 activity did not rescue cells deprived of IL-3 from apoptosis. However, induction of GATA-2 activity in cells cultured in IL-3 blocked factor-dependent self-renewal but not factor-dependent survival: Cells undergo cell cycle arrest and cease proliferating but do not apoptose. This was accompanied by differentiation down the monocytic and granulocytic pathways. Differentiation occurred in the presence of IL-3 and did not require addition of exogenous differentiation growth factors such as G-CSF or GM-CSF normally required to induce granulomonocytic differentiation of FDCP-mix cells. Conversely, EPO-dependent erythroid differentiation was inhibited by GATA-2 activation. These biological effects were obtained with levels of exogenous GATA-2 representing less than twofold increases over endogenous GATA-2 levels and were not observed in cells overexpressing GATA-1/ER. Similar effects on proliferation and differentiation were also observed in primary progenitor cells, freshly isolated from murine bone marrow and transduced with a GATA-2/ER-containing retrovirus. Taken together, these data suggest that threshold activities of GATA-2 in hematopoietic progenitor cells are a critical determinant in influencing self-renewal versus differentiation outcomes. PMID:10421636

Transcription factors SOHLH1 and SOHLH2 coordinate oocyte differentiation without affecting meiosis I.

PubMed

Shin, Yong-Hyun; Ren, Yu; Suzuki, Hitomi; Golnoski, Kayla J; Ahn, Hyo Won; Mico, Vasil; Rajkovic, Aleksandar

2017-06-01

Following migration of primordial germ cells to the genital ridge, oogonia undergo several rounds of mitotic division and enter meiosis at approximately E13.5. Most oocytes arrest in the dictyate (diplotene) stage of meiosis circa E18.5. The genes necessary to drive oocyte differentiation in parallel with meiosis are unknown. Here, we have investigated whether expression of spermatogenesis and oogenesis bHLH transcription factor 1 (Sohlh1) and Sohlh2 coordinates oocyte differentiation within the embryonic ovary. We found that SOHLH2 protein was expressed in the mouse germline as early as E12.5 and preceded SOHLH1 protein expression, which occurred circa E15.5. SOHLH1 protein appearance at E15.5 correlated with SOHLH2 translocation from the cytoplasm into the nucleus and was dependent on SOHLH1 expression. NOBOX oogenesis homeobox (NOBOX) and LIM homeobox protein 8 (LHX8), two important regulators of postnatal oogenesis, were coexpressed with SOHLH1. Single deficiency of Sohlh1 or Sohlh2 disrupted the expression of LHX8 and NOBOX in the embryonic gonad without affecting meiosis. Sohlh1-KO infertility was rescued by conditional expression of the Sohlh1 transgene after the onset of meiosis. However, Sohlh1 or Sohlh2 transgene expression could not rescue Sohlh2-KO infertility due to a lack of Sohlh1 or Sohlh2 expression in rescued mice. Our results indicate that Sohlh1 and Sohlh2 are essential regulators of oocyte differentiation but do not affect meiosis I.

Glutamine Synthetase Is a Genetic Determinant of Cell Type–Specific Glutamine Independence in Breast Epithelia

PubMed Central

Kung, Hsiu-Ni; Marks, Jeffrey R.; Chi, Jen-Tsan

2011-01-01

Although significant variations in the metabolic profiles exist among different cells, little is understood in terms of genetic regulations of such cell type–specific metabolic phenotypes and nutrient requirements. While many cancer cells depend on exogenous glutamine for survival to justify the therapeutic targeting of glutamine metabolism, the mechanisms of glutamine dependence and likely response and resistance of such glutamine-targeting strategies among cancers are largely unknown. In this study, we have found a systematic variation in the glutamine dependence among breast tumor subtypes associated with mammary differentiation: basal- but not luminal-type breast cells are more glutamine-dependent and may be susceptible to glutamine-targeting therapeutics. Glutamine independence of luminal-type cells is associated mechanistically with lineage-specific expression of glutamine synthetase (GS). Luminal cells can also rescue basal cells in co-culture without glutamine, indicating a potential for glutamine symbiosis within breast ducts. The luminal-specific expression of GS is directly induced by GATA3 and represses glutaminase expression. Such distinct glutamine dependency and metabolic symbiosis is coupled with the acquisition of the GS and glutamine independence during the mammary differentiation program. Understanding the genetic circuitry governing distinct metabolic patterns is relevant to many symbiotic relationships among different cells and organisms. In addition, the ability of GS to predict patterns of glutamine metabolism and dependency among tumors is also crucial in the rational design and application of glutamine and other metabolic pathway targeted therapies. PMID:21852960

Insulin and Wnt1 Pathways Cooperate to Induce Reserve Cell Activation in Differentiation and Myotube Hypertrophy

PubMed Central

Rochat, Anne; Fernandez, Anne; Vandromme, Marie; Molès, Jeàn-Pierre; Bouschet, Triston; Carnac, Gilles; Lamb, Ned J. C.

2004-01-01

During ex vivo myoblast differentiation, a pool of quiescent mononucleated myoblasts, reserve cells, arise alongside myotubes. Insulin/insulin-like growth factor (IGF) and PKB/Akt-dependent phosphorylation activates skeletal muscle differentiation and hypertrophy. We have investigated the role of glycogen synthase kinase 3 (GSK-3) inhibition by protein kinase B (PKB)/Akt and Wnt/β-catenin pathways in reserve cell activation during myoblast differentiation and myotube hypertrophy. Inhibition of GSK-3 by LiCl or SB216763, restored insulin-dependent differentiation of C2ind myoblasts in low serum, and cooperated with insulin in serum-free medium to induce MyoD and myogenin expression in C2ind myoblasts, quiescent C2 or primary human reserve cells. We show that LiCl treatment induced nuclear accumulation of β-catenin in C2 myoblasts, thus mimicking activation of canonical Wnt signaling. Similarly to the effect of GSK-3 inhibitors with insulin, coculturing C2 reserve cells with Wnt1-expressing fibroblasts enhanced insulin-stimulated induction of MyoD and myogenin in reserve cells. A similar cooperative effect of LiCl or Wnt1 with insulin was observed during late ex vivo differentiation and promoted increased size and fusion of myotubes. We show that this synergistic effect on myotube hypertrophy involved an increased fusion of reserve cells into preexisting myotubes. These data reveal insulin and Wnt/β-catenin pathways cooperate in muscle cell differentiation through activation and recruitment of satellite cell-like reserve myoblasts. PMID:15282335

Differential anti-tumor activity of coriolus versicolor (Yunzhi) extract through p53- and/or Bcl-2-dependent apoptotic pathway in human breast cancer cells.

PubMed

Ho, Cheong-Yip; Kim, Chi-Fai; Leung, Kwok-Nam; Fung, Kwok-Pui; Tse, Tak-Fu; Chan, Helen; Lau, Clara Bik-San

2005-06-01

Coriolus versicolor (CV), also called Yunzhi, has been demonstrated to exert anti-tumor effects on various types of cancer cells, but the underlying mechanism has not been fully elucidated. The present study aimed to evaluate the in vitro anti-tumor activity of a standardized aqueous ethanol extract prepared from CV on four breast cancer cell lines using MTT assay, and test whether the mechanism involves apoptosis induction and modulation of p53 and Bcl-2 protein expressions using cell death detection ELISA, p53 and Bcl-2 ELISAs respectively. Our results demonstrated that the CV extract dose-dependently suppressed the proliferation of three breast tumor cell lines, with ascending order of IC50 values: T-47D, MCF-7, MDA-MB-231, while BT-20 cells were not significantly affected. Tumoricidal activity of the CV extract was found to be comparable to a chemotherapeutic anti-cancer drug, mitomycin C. Nucleosome productions in apoptotic MDA-MB-231, MCF-7 and T-47D cells were significantly augmented in a time-dependent manner and paralleled the anti-proliferative activity of CV extract. Expression of p53 protein was significantly upregulated only in T-47D cells treated with the CV extract in a dose- and time-dependent fashion, but not in MCF-7 (except at 400 mug/ml after 16 h) and MDA-MB-231 cells. The CV extract significantly induced a dose-dependent downregulation of Bcl-2 protein expression in MCF-7 and T-47D cells, but not in MDA-MB-231 cells. These results suggested that apoptosis induction, differentially dependent of p53 and Bcl-2 expressions, might be the possible mechanism of CV extract-mediated cytotoxicity in human breast cancer cells in vitro.

T cell exit from quiescence and differentiation into Th2 cells depend on Raptor-mTORC1-mediated metabolic programming

PubMed Central

Yang, Kai; Shrestha, Sharad; Zeng, Hu; Karmaus, Peer W.F.; Neale, Geoffrey; Vogel, Peter; Guertin, David A.; Lamb, Richard F.; Chi, Hongbo

2014-01-01

SUMMARY Naïve T cells respond to antigen stimulation by exiting from quiescence and initiating clonal expansion and functional differentiation, but the control mechanism is elusive. Here we describe that Raptor-mTORC1-dependent metabolic programming is a central determinant of this transitional process. Loss of Raptor abrogated T cell priming and Th2 cell differentiation, although Raptor function is less important for continuous proliferation of actively cycling cells. mTORC1 coordinated multiple metabolic programs in T cells including glycolysis, lipid synthesis and oxidative phosphorylation to mediate antigen-triggered exit from quiescence. mTORC1 further linked glucose metabolism to the initiation of Th2 cell differentiation by orchestrating cytokine receptor expression and cytokine responsiveness. Activation of Raptor-mTORC1 integrated T cell receptor and CD28 co-stimulatory signals in antigen-stimulated T cells. Our studies identify a Raptor-mTORC1-dependent pathway linking signal-dependent metabolic reprogramming to quiescence exit, and this in turn coordinates lymphocyte activation and fate decisions in adaptive immunity. PMID:24315998

Gene position in a long operon governs motility development in Bacillus subtilis

PubMed Central

Cozy, Loralyn M.; Kearns, Daniel B.

2010-01-01

Growing cultures of Bacillus subtilis bifurcate into subpopulations of motile individuals and non-motile chains of cells that are differentiated at the level of gene expression. The motile cells are ON and the chaining cells are OFF for transcription that depends on RNA polymerase and the alternative sigma factor σD. Here we show that chaining cells were OFF for σD-dependent gene expression because σD levels fell below a threshold, and σD activity was inhibited by the anti-sigma factor FlgM. The probability that σD exceeded the threshold was governed by the position of the sigD genes. The proportion of ON cells increased when sigD was artificially moved forward in the 27kb fla/che operon. In addition, we identified a new σD-dependent promoter that increases sigD expression and may provide positive feedback to stabilize the ON state. Finally, we demonstrate that ON/OFF motility states in B. subtilis are a form of development because mosaics of stable and differentiated epigenotypes were evident when the normally dispersed bacteria were forced to grow in one dimension. PMID:20233303

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.

Redox-sensitive MAPK and Notch3 regulate fibroblast differentiation and activation: a dual role of ERK1/2.

PubMed

Lai, Jun-Mei; Zhang, Xiong; Liu, Fang-Fang; Yang, Rui; Li, Shen-Yu; Zhu, Lan-Bing; Zou, Ming; Cheng, Wen-Hsing; Zhu, Jian-Hong

2016-07-12

Myofibroblastic transformation, characterized by upregulation of α-smooth muscle actin in response to profibrotic agents such as TGF-β1, is considered as a major event leading to fibrosis. The mechanistic basis linking myofibroblast differentiation to idiopathic pulmonary fibrosis and the disease treatment remain elusive. In this study, we studied roles of MAPK, Notch, and reactive oxygen species (ROS) during the differentiation of IMR-90 lung fibroblasts at basal level and induced by TGF-β1. Our results demonstrated that ROS-dependent activation of p38, JNK1/2 and Notch3 promoted basal and TGF-β1-induced differentiation and expression of extracellular matrix proteins. In stark contrast, ERK1/2 was suppressed by ROS and exhibited an inhibitory effect on the differentiation but showed a weak promotion on the expression of extracellular matrix proteins. TGF-β1-induced Notch3 expression depended on p38 and JNK1/2. Interestingly, Notch3 was also downstream of ERK1/2, suggesting a complex role of ERK1/2 in lung function. Our results suggest a novel ROS-mediated shift of dominance from the inhibitory ERK1/2 to the stimulatory p38, JNK1/2 and Notch3 during the pathological progression of IPF. Thus, targeting ERK1/2 signaling for activation and p38, JNK1/2 and Notch3 for inhibition may be of clinical potential against lung fibrosis.

Analysis of host response to bacterial infection using error model based gene expression microarray experiments

PubMed Central

Stekel, Dov J.; Sarti, Donatella; Trevino, Victor; Zhang, Lihong; Salmon, Mike; Buckley, Chris D.; Stevens, Mark; Pallen, Mark J.; Penn, Charles; Falciani, Francesco

2005-01-01

A key step in the analysis of microarray data is the selection of genes that are differentially expressed. Ideally, such experiments should be properly replicated in order to infer both technical and biological variability, and the data should be subjected to rigorous hypothesis tests to identify the differentially expressed genes. However, in microarray experiments involving the analysis of very large numbers of biological samples, replication is not always practical. Therefore, there is a need for a method to select differentially expressed genes in a rational way from insufficiently replicated data. In this paper, we describe a simple method that uses bootstrapping to generate an error model from a replicated pilot study that can be used to identify differentially expressed genes in subsequent large-scale studies on the same platform, but in which there may be no replicated arrays. The method builds a stratified error model that includes array-to-array variability, feature-to-feature variability and the dependence of error on signal intensity. We apply this model to the characterization of the host response in a model of bacterial infection of human intestinal epithelial cells. We demonstrate the effectiveness of error model based microarray experiments and propose this as a general strategy for a microarray-based screening of large collections of biological samples. PMID:15800204

ERα inhibited myocardin-induced differentiation in uterine fibroids

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

Liao, Xing-Hua, E-mail: xinghualiao@hotmail.com; Key Laboratory of Industrial Fermentation Microbiology, Ministry of Education and Tianjin, College of Biotechnology, Tianjin University of Science and Technology, Tianjin 300457; Li, Jun-Yan

Uterine fibroids, also known as uterine leiomyomas, are a benign tumor of the human uterus and the commonest estrogen-dependent benign tumor found in women. Myocardin is an important transcriptional regulator in smooth and cardiac muscle development. The role of myocardin and its relationship with ERα in uterine fibroids have barely been addressed. We noticed that the expression of myocardin was markedly reduced in human uterine fibroid tissue compared with corresponding normal or adjacent myometrium tissue. Here we reported that myocardin induced the transcription and expression of differentiation markers SM22α and alpha smooth muscle actin (α-SMA) in rat primary uterine smoothmore » muscle cells (USMCs) and this effect was inhibited by ERα. Notably, we showed that, ERα induced expression of proliferation markers PCNA and ki-67 in rat primary USMCs. We also found ERα interacted with myocardin and formed complex to bind to CArG box and inhibit the SM22α promoter activity. Furthermore, ERα inhibited the transcription and expression of myocardin, and reduced the levels of transcription and expression of downstream target SM22α, a SMC differentiation marker. Our data thus provided important and novel insights into how ERα and myocardin interact to control the cell differentiation and proliferation of USMCs. Thus, it may provide potential therapeutic target for uterine fibroids.« less

A balance of FGF and BMP signals regulates cell cycle exit and Equarin expression in lens cells

PubMed Central

Jarrin, Miguel; Pandit, Tanushree; Gunhaga, Lena

2012-01-01

In embryonic and adult lenses, a balance of cell proliferation, cell cycle exit, and differentiation is necessary to maintain physical function. The molecular mechanisms regulating the transition of proliferating lens epithelial cells to differentiated primary lens fiber cells are poorly characterized. To investigate this question, we used gain- and loss-of-function analyses to modulate fibroblast growth factor (FGF) and/or bone morphogenetic protein (BMP) signals in chick lens/retina explants. Here we show that FGF activity plays a key role for proliferation independent of BMP signals. Moreover, a balance of FGF and BMP signals regulates cell cycle exit and the expression of Ccdc80 (also called Equarin), which is expressed at sites where differentiation of lens fiber cells occurs. BMP activity promotes cell cycle exit and induces Equarin expression in an FGF-dependent manner. In contrast, FGF activity is required but not sufficient to induce cell cycle exit or Equarin expression. Furthermore, our results show that in the absence of BMP activity, lens cells have increased cell cycle length or are arrested in the cell cycle, which leads to decreased cell cycle exit. Taken together, these findings suggest that proliferation, cell cycle exit, and early differentiation of primary lens fiber cells are regulated by counterbalancing BMP and FGF signals. PMID:22718906

Toxicogenomic outcomes predictive of forestomach carcinogenesis following exposure to benzo(a)pyrene: Relevance to human cancer risk

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

Labib, Sarah, E-mail: Sarah.Labib@hc-sc.gc.ca; Guo, Charles H., E-mail: Charles.Guo@hc-sc.gc.ca; Williams, Andrew, E-mail: Andrew.Williams@hc-sc.gc.ca

2013-12-01

Forestomach tumors are observed in mice exposed to environmental carcinogens. However, the relevance of this data to humans is controversial because humans lack a forestomach. We hypothesize that an understanding of early molecular changes after exposure to a carcinogen in the forestomach will provide mode-of-action information to evaluate the applicability of forestomach cancers to human cancer risk assessment. In the present study we exposed mice to benzo(a)pyrene (BaP), an environmental carcinogen commonly associated with tumors of the rodent forestomach. Toxicogenomic tools were used to profile gene expression response in the forestomach. Adult Muta™Mouse males were orally exposed to 25, 50,more » and 75 mg BaP/kg-body-weight/day for 28 consecutive days. The forestomach was collected three days post-exposure. DNA microarrays, real-time RT-qPCR arrays, and protein analyses were employed to characterize responses in the forestomach. Microarray results showed altered expression of 414 genes across all treatment groups (± 1.5 fold; false discovery rate adjusted P ≤ 0.05). Significant downregulation of genes associated with phase II xenobiotic metabolism and increased expression of genes implicated in antigen processing and presentation, immune response, chemotaxis, and keratinocyte differentiation were observed in treated groups in a dose-dependent manner. A systematic comparison of the differentially expressed genes in the forestomach from the present study to differentially expressed genes identified in human diseases including human gastrointestinal tract cancers using the NextBio Human Disease Atlas showed significant commonalities between the two models. Our results provide molecular evidence supporting the use of the mouse forestomach model to evaluate chemically-induced gastrointestinal carcinogenesis in humans. - Highlights: • Benzo(a)pyrene-mediated transcriptomic response in the forestomach was examined. • The immunoproteosome subunits and MHC class I pathway were the most affected. • Keratinocyte differentiation associated gene expression changes were dose-dependent. • Molecular similarities exist between cancers of the forestomach and human stomach.« less

Differential display RT PCR of total RNA from human foreskin fibroblasts for investigation of androgen-dependent gene expression

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

Nitsche, E.M.; Moquin, A.; Adams, P.S.

1996-05-03

Male sexual differentiation is a process that involves androgen action via the androgen receptor. Defects in the androgen receptor, many resulting from point mutations in the androgen receptor gene, lead to varying degrees of impaired masculinization in chromosomally male individuals. To date no specific androgen regulated morphogens involved in this process have been identified and no marker genes are known that would help to predict further virilization in infants with partial androgen insensitivity. In the present study we first show data on androgen regulated gene expression investigated by differential display reverse transcription PCR (dd RT PCR) on total RNA frommore » human neonatal genital skin fibroblasts cultured in the presence or absence of 100 nM testosterone. Using three different primer combinations, 54 cDNAs appeared to be regulated by androgens. Most of these sequences show the characteristics of expressed mRNAs but showed no homology to sequences in the database. However 15 clones with significant homology to previously cloned sequences were identified. Seven cDNAs appear to be induced by androgen withdrawal. Of these, five are similar to ETS (expression tagged sequences) from unknown genes; the other two show significant homology to the cDNAs of ubiquitin and human guanylate binding protein 2 (GBP-2). In addition, we have identified 8 cDNA clones which show homologies to other sequences in the database and appear to be upregulated in the presence of testosterone. Three differential expressed sequences show significant homology to the cDNAs of L-plastin and one to the cDNA of testican. This latter gene codes for a proteoglycan involved in cell social behavior and therefore of special interest in this context. The results of this study are of interest in further investigation of normal and disturbed androgen-dependent gene expression. 49 refs., 2 figs., 5 tabs.« less

Induction of differentiation in human promyelocytic HL-60 leukemia cells activates p21, WAF1/CIP1, expression in the absence of p53.

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

Jiang, H.; Lin, J.; Su, Z.-Z.

The melanoma differentiation associated gene, mda-6, which is identical to the P53-inducible gene WAF1/CIP1, encodes an M(r) 21,000 protein (p21) that can directly inhibit cell growth by repressing cyclin dependent kinases. mda-6 was identified using subtraction hybridization by virtue of its enhanced expression in human melanoma cells induced to terminally differentiate by treatment with human fibroblast interferon and the anti-leukemic compound mezerein (Jiang and Fisher, 1993). In the present study, we demonstrate that mda-6 (WAF1/CIP1) is an immediate early response gene induced during differentiation of the promyelocytic HL-60 leukemia cell line along the granulocytic or macrophage/monocyte pathway. mda-6 gene expressionmore » in HL-60 cells is induced within 1 to 3 h during differentiation along the macrophage/monocyte pathway evoked by 12-0-tetradecanoyl phorbol-13-acetate (TPA) or 1,25-dihydroxyvitamin D3 (Vit D3) or the granulocytic pathway produced by retinoic acid (RA) or dimethylsulfoxide (DMSO). Immunoprecipitation analyses using an anti-p21 antibody indicate a temporal induction of p21 protein following treatment with TPA, DMSO or RA. A relationship between rapid induction of mda-6 gene expression and differentiation is indicated by a delay in this expression in an HL-60 cell variant resistant to TPA-induced growth arrest and differentiation. A similar delay in mda-6 gene expression is not observed in Vit D3 treated TPA-resistant variant cells that are also sensitive to induction of monocytic differentiation. Since HL-60 cells have a null-p53 phenotype, these results demonstrate that p21 induction occurs during initiation of terminal differentiation in a p53-independent manner. In this context, p21 may play a more global role in growth control and differentiation than originally envisioned.« less

Spi-C has opposing effects to PU.1 on gene expression in progenitor B cells.

PubMed

Schweitzer, Brock L; Huang, Kelly J; Kamath, Meghana B; Emelyanov, Alexander V; Birshtein, Barbara K; DeKoter, Rodney P

2006-08-15

The Ets transcription factor Spi-C, expressed in B cells and macrophages, is closely related to PU.1 and has the ability to recognize the same DNA consensus sequence. However, the function of Spi-C has yet to be determined. The purpose of this study is to further examine Spi-C activity in B cell development. First, using retroviral vectors to infect PU.1(-/-) fetal liver progenitors, Spi-C was found to be inefficient at inducing cytokine-dependent proliferation and differentiation of progenitor B (pro-B) cells or macrophages relative to PU.1 or Spi-B. Next, Spi-C was ectopically expressed in fetal liver-derived, IL-7-dependent pro-B cell lines. Wild-type (WT) pro-B cells ectopically expressing Spi-C (WT-Spi-C) have several phenotypic characteristics of pre-B cells such as increased CD25 and decreased c-Kit surface expression. In addition, WT-Spi-C pro-B cells express increased levels of IgH sterile transcripts and reduced levels of expression and transcription of the FcgammaRIIb gene. Gel-shift analysis suggests that Spi-C, ectopically expressed in pro-B cells, can bind PU.1 consensus sites in the IgH intronic enhancer and FcgammaRIIb promoter. Transient transfection analysis demonstrated that PU.1 functions to repress the IgH intronic enhancer and activate the FcgammaRIIb promoter, while Spi-C opposes these activities. WT-Spi-C pro-B cells have reduced levels of dimethylation on lysine 9 of histone H3 within the IgH 3' regulatory region, indicating that Spi-C can contribute to removal of repressive features in the IgH locus. Overall, these studies suggest that Spi-C may promote B cell differentiation by modulating the activity of PU.1-dependent genes.

Insulin-like growth factor-1 suppresses the Myostatin signaling pathway during myogenic differentiation

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

Retamales, A.; Zuloaga, R.; Valenzuela, C.A.

Myogenic differentiation is a complex and well-coordinated process for generating mature skeletal muscle fibers. This event is autocrine/paracrine regulated by growth factors, principally Myostatin (MSTN) and Insulin-like Growth Factor-1 (IGF-1). Myostatin, a member of the transforming growth factor-β superfamily, is a negative regulator of skeletal muscle growth in vertebrates that exerts its inhibitory function by activating Smad transcription factors. In contrast, IGF-1 promotes the differentiation of skeletal myoblasts by activating the PI3K/Akt signaling pathway. This study reports on a novel functional crosstalk between the IGF-1 and MSTN signaling pathways, as mediated through interaction between PI3K/Akt and Smad3. Stimulation of skeletalmore » myoblasts with MSTN resulted in a transient increase in the pSmad3:Smad3 ratio and Smad-dependent transcription. Moreover, MSTN inhibited myod gene expression and myoblast fusion in an Activin receptor-like kinase/Smad3-dependent manner. Preincubation of skeletal myoblasts with IGF-1 blocked MSTN-induced Smad3 activation, promoting myod expression and myoblast differentiation. This inhibitory effect of IGF-1 on the MSTN signaling pathway was dependent on IGF-1 receptor, PI3K, and Akt activities. Finally, immunoprecipitation assay analysis determined that IGF-1 pretreatment increased Akt and Smad3 interaction. These results demonstrate that the IGF-1/PI3K/Akt pathway may inhibit MSTN signaling during myoblast differentiation, providing new insight to existing knowledge on the complex crosstalk between both growth factors. - Highlights: • IGF-1 inhibits Myostatin canonical signaling pathway through IGF-1R/PI3K/Akt pathway. • IGF-1 promotes myoblast differentiation through a direct blocking of Myostatin signaling pathway. • IGF-1 induces the interaction of Akt with Smad3 in skeletal myoblast.« less

FOXO1 opposition of CD8+ T cell effector programming confers early memory properties and phenotypic diversity.

PubMed

Delpoux, Arnaud; Lai, Chen-Yen; Hedrick, Stephen M; Doedens, Andrew L

2017-10-17

The factors and steps controlling postinfection CD8 + T cell terminal effector versus memory differentiation are incompletely understood. Whereas we found that naive TCF7 (alias "Tcf-1") expression is FOXO1 independent, early postinfection we report bimodal, FOXO1-dependent expression of the memory-essential transcription factor TCF7 in pathogen-specific CD8 + T cells. We determined the early postinfection TCF7 high population is marked by low TIM3 expression and bears memory signature hallmarks before the appearance of established memory precursor marker CD127 (IL-7R). These cells exhibit diminished TBET, GZMB, mTOR signaling, and cell cycle progression. Day 5 postinfection, TCF7 high cells express higher memory-associated BCL2 and EOMES, as well as increased accumulation potential and capacity to differentiate into memory phenotype cells. TCF7 retroviral transduction opposes GZMB expression and the formation of KLRG1 pos phenotype cells, demonstrating an active role for TCF7 in extinguishing the effector program and forestalling terminal differentiation. Past the peak of the cellular immune response, we report a gradient of FOXO1 and TCF7 expression, which functions to oppose TBET and orchestrate a continuum of effector-to-memory phenotypes.

TRACING CO-REGULATORY NETWORK DYNAMICS IN NOISY, SINGLE-CELL TRANSCRIPTOME TRAJECTORIES.

PubMed

Cordero, Pablo; Stuart, Joshua M

2017-01-01

The availability of gene expression data at the single cell level makes it possible to probe the molecular underpinnings of complex biological processes such as differentiation and oncogenesis. Promising new methods have emerged for reconstructing a progression 'trajectory' from static single-cell transcriptome measurements. However, it remains unclear how to adequately model the appreciable level of noise in these data to elucidate gene regulatory network rewiring. Here, we present a framework called Single Cell Inference of MorphIng Trajectories and their Associated Regulation (SCIMITAR) that infers progressions from static single-cell transcriptomes by employing a continuous parametrization of Gaussian mixtures in high-dimensional curves. SCIMITAR yields rich models from the data that highlight genes with expression and co-expression patterns that are associated with the inferred progression. Further, SCIMITAR extracts regulatory states from the implicated trajectory-evolvingco-expression networks. We benchmark the method on simulated data to show that it yields accurate cell ordering and gene network inferences. Applied to the interpretation of a single-cell human fetal neuron dataset, SCIMITAR finds progression-associated genes in cornerstone neural differentiation pathways missed by standard differential expression tests. Finally, by leveraging the rewiring of gene-gene co-expression relations across the progression, the method reveals the rise and fall of co-regulatory states and trajectory-dependent gene modules. These analyses implicate new transcription factors in neural differentiation including putative co-factors for the multi-functional NFAT pathway.

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.

Postnatal ablation of osteoblast Smad4 enhances proliferative responses to canonical Wnt signaling through interactions with β-catenin

PubMed Central

Salazar, Valerie S.; Zarkadis, Nicholas; Huang, Lisa; Watkins, Marcus; Kading, Jacqueline; Bonar, Sheri; Norris, Jin; Mbalaviele, Gabriel; Civitelli, Roberto

2013-01-01

Summary Canonical Wnt (cWnt) signaling through β-catenin regulates osteoblast proliferation and differentiation to enhance bone formation. We previously reported that osteogenic action of β-catenin is dependent on BMP signaling. Here, we further examined interactions between cWnt and BMP in bone. In osteoprogenitors stimulated with BMP2, β-catenin localizes to the nucleus, physically interacts with Smad4, and is recruited to DNA-binding transcription complexes containing Smad4, R-Smad1/5 and TCF4. Furthermore, Tcf/Lef-dependent transcription, Ccnd1 expression and proliferation all increase when Smad4, 1 or 5 levels are low, whereas TCF/Lef activities decrease when Smad4 expression is high. The ability of Smad4 to antagonize transcription of Ccnd1 is dependent on DNA-binding activity but Smad4-dependent transcription is not required. In mice, conditional deletion of Smad4 in osterix+ cells increases mitosis of cells on trabecular bone surfaces as well as in primary osteoblast cultures from adult bone marrow and neonatal calvaria. By contrast, ablation of Smad4 delays differentiation and matrix mineralization by primary osteoblasts in response to Wnt3a, indicating that loss of Smad4 perturbs the balance between proliferation and differentiation in osteoprogenitors. We propose that Smad4 and Tcf/Lef transcription complexes compete for β-catenin, thus restraining cWnt-dependent proliferative signals while favoring the matrix synthesizing activity of osteoblasts. PMID:24101723

Identification and Characterization of FGF2-Dependent mRNA: microRNA Networks During Lens Fiber Cell Differentiation

PubMed Central

Wolf, Louise; Gao, Chun S.; Gueta, Karen; Xie, Qing; Chevallier, Tiphaine; Podduturi, Nikhil R.; Sun, Jian; Conte, Ivan; Zelenka, Peggy S.; Ashery-Padan, Ruth; Zavadil, Jiri; Cvekl, Ales

2013-01-01

MicroRNAs (miRNAs) and fibroblast growth factor (FGF) signaling regulate a wide range of cellular functions, including cell specification, proliferation, migration, differentiation, and survival. In lens, both these systems control lens fiber cell differentiation; however, a possible link between these processes remains to be examined. Herein, the functional requirement for miRNAs in differentiating lens fiber cells was demonstrated via conditional inactivation of Dicer1 in mouse (Mus musculus) lens. To dissect the miRNA-dependent pathways during lens differentiation, we used a rat (Rattus norvegicus) lens epithelial explant system, induced by FGF2 to differentiate, followed by mRNA and miRNA expression profiling. Transcriptome and miRNome analysis identified extensive FGF2-regulated cellular responses that were both independent and dependent on miRNAs. We identified 131 FGF2-regulated miRNAs. Seventy-six of these miRNAs had at least two in silico predicted and inversely regulated target mRNAs. Genes modulated by the greatest number of FGF-regulated miRNAs include DNA-binding transcription factors Nfib, Nfat5/OREBP, c-Maf, Ets1, and N-Myc. Activated FGF signaling influenced bone morphogenetic factor/transforming growth factor-β, Notch, and Wnt signaling cascades implicated earlier in lens differentiation. Specific miRNA:mRNA interaction networks were predicted for c-Maf, N-Myc, and Nfib (DNA-binding transcription factors); Cnot6, Cpsf6, Dicer1, and Tnrc6b (RNA to miRNA processing); and Ash1l, Med1/PBP, and Kdm5b/Jarid1b/Plu1 (chromatin remodeling). Three miRNAs, including miR-143, miR-155, and miR-301a, down-regulated expression of c-Maf in the 3′-UTR luciferase reporter assays. These present studies demonstrate for the first time global impact of activated FGF signaling in lens cell culture system and predicted novel gene regulatory networks connected by multiple miRNAs that regulate lens differentiation. PMID:24142921

Differential levels of Neurod establish zebrafish endocrine pancreas cell fates

PubMed Central

Dalgin, Gökhan; Prince, Victoria E.

2015-01-01

During development a network of transcription factors functions to differentiate foregut cells into pancreatic endocrine cells. Differentiation of appropriate numbers of each hormone-expressing endocrine cell type is essential for the normal development of the pancreas and ultimately for effective maintenance of blood glucose levels. A fuller understanding of the details of endocrine cell differentiation may contribute to development of cell replacement therapies to treat diabetes. In this study, by using morpholino and gRNA/Cas9 mediated knockdown we establish that differential levels of the basic-helix loop helix (bHLH) transcription factor Neurod are required for the differentiation of distinct endocrine cell types in developing zebrafish. While Neurod plays a role in the differentiation of all endocrine cells, we find that differentiation of glucagon-expressing alpha cells is disrupted by a minor reduction in Neurod levels, whereas differentiation of insulin-expressing beta cells is less sensitive to Neurod depletion. The endocrine cells that arise during embryonic stages to produce the primary islet, and those that arise subsequently during larval stages from the intra-pancreatic duct (IPD) to ultimately contribute to the secondary islets, show similar dependence on differential Neurod levels. Intriguingly, Neurod-deficiency triggers premature formation of endocrine precursors from the IPD during early larval stages. However, the Neurod-deficient endocrine precursors fail to differentiate appropriately, and the larvae are unable to maintain normal glucose levels. In summary, differential levels of Neurod are required to generate endocrine pancreas subtypes from precursors during both embryonic and larval stages, and Neurod function is in turn critical to endocrine function. PMID:25797153

Tumor Necrosis Factor Alpha and Insulin-Like Growth Factor 1 Induced Modifications of the Gene Expression Kinetics of Differentiating Skeletal Muscle Cells

PubMed Central

Meyer, Swanhild U.; Krebs, Stefan; Thirion, Christian; Blum, Helmut; Krause, Sabine; Pfaffl, Michael W.

2015-01-01

Introduction TNF-α levels are increased during muscle wasting and chronic muscle degeneration and regeneration processes, which are characteristic for primary muscle disorders. Pathologically increased TNF-α levels have a negative effect on muscle cell differentiation efficiency, while IGF1 can have a positive effect; therefore, we intended to elucidate the impact of TNF-α and IGF1 on gene expression during the early stages of skeletal muscle cell differentiation. Methodology/Principal Findings This study presents gene expression data of the murine skeletal muscle cells PMI28 during myogenic differentiation or differentiation with TNF-α or IGF1 exposure at 0 h, 4 h, 12 h, 24 h, and 72 h after induction. Our study detected significant coregulation of gene sets involved in myoblast differentiation or in the response to TNF-α. Gene expression data revealed a time- and treatment-dependent regulation of signaling pathways, which are prominent in myogenic differentiation. We identified enrichment of pathways, which have not been specifically linked to myoblast differentiation such as doublecortin-like kinase pathway associations as well as enrichment of specific semaphorin isoforms. Moreover to the best of our knowledge, this is the first description of a specific inverse regulation of the following genes in myoblast differentiation and response to TNF-α: Aknad1, Cmbl, Sepp1, Ndst4, Tecrl, Unc13c, Spats2l, Lix1, Csdc2, Cpa1, Parm1, Serpinb2, Aspn, Fibin, Slc40a1, Nrk, and Mybpc1. We identified a gene subset (Nfkbia, Nfkb2, Mmp9, Mef2c, Gpx, and Pgam2), which is robustly regulated by TNF-α across independent myogenic differentiation studies. Conclusions This is the largest dataset revealing the impact of TNF-α or IGF1 treatment on gene expression kinetics of early in vitro skeletal myoblast differentiation. We identified novel mRNAs, which have not yet been associated with skeletal muscle differentiation or response to TNF-α. Results of this study may facilitate the understanding of transcriptomic networks underlying inhibited muscle differentiation in inflammatory diseases. PMID:26447881

Naringin promotes differentiation of bone marrow stem cells into osteoblasts by upregulating the expression levels of microRNA-20a and downregulating the expression levels of PPARγ.

PubMed

Fan, Jifeng; Li, Jie; Fan, Qinbo

2015-09-01

Naringin is a dihydrotestosterone flavonoid compound that significantly inhibits bone loss, improves bone density, and enhances biomechanical anti‑compression performance. Previous studies have demonstrated that naringin improves the activity levels of osteocalcin (OC) and alkaline phosphatase (ALP) in MC3T3‑E1 osteoblast precursor cells. The present study investigated the effects of naringin on osteoblastic differentiation and inhibition of adipocyte formation in bone marrow stem cells (BMSCs). The levels of osteogenesis were modulated via upregulation of the expression levels of microRNA (miR)‑20a, and downregulation of the expression levels of peroxisome proliferator‑activated receptor γ (PPARγ). The results indicated that naringin significantly enhanced BMSC proliferation in a dose‑dependent manner. In addition, naringin significantly increased the mRNA expression levels of OC, ALP, and collagen type I. Furthermore, naringin decreased the protein expression levels of PPARγ, and increased the expression levels of miR‑20a in the BMSCs. These results suggested that miR‑20a may regulate the expression of PPARγ in BMSCs. To our knowledge, this is the first study to report naringin‑induced osteogenesis via upregulation of the expression levels of miR‑20a, and downregulation of the expression levels of PPARγ. These results indicated the important role of naringin in BMSC differentiation.

Biased gene expression in early honeybee larval development

PubMed Central

2013-01-01

Background Female larvae of the honeybee (Apis mellifera) develop into either queens or workers depending on nutrition. This nutritional stimulus triggers different developmental trajectories, resulting in adults that differ from each other in physiology, behaviour and life span. Results To understand how these trajectories are established we have generated a comprehensive atlas of gene expression throughout larval development. We found substantial differences in gene expression between worker and queen-destined larvae at 6 hours after hatching. Some of these early changes in gene expression are maintained throughout larval development, indicating that caste-specific developmental trajectories are established much earlier than previously thought. Within our gene expression data we identified processes that potentially underlie caste differentiation. Queen-destined larvae have higher expression of genes involved in transcription, translation and protein folding early in development with a later switch to genes involved in energy generation. Using RNA interference, we were able to demonstrate that one of these genes, hexamerin 70b, has a role in caste differentiation. Both queen and worker developmental trajectories are associated with the expression of genes that have alternative splice variants, although only a single variant of a gene tends to be differentially expressed in a given caste. Conclusions Our data, based on the biases in gene expression early in development together with published data, supports the idea that caste development in the honeybee consists of two phases; an initial biased phase of development, where larvae can still switch to the other caste by differential feeding, followed by commitment to a particular developmental trajectory. PMID:24350621

Transcriptional and post-transcriptional down-regulation of cyclin D1 contributes to C6 glioma cell differentiation induced by forskolin.

PubMed

He, Songmin; Zhu, Wenbo; Zhou, Yuxi; Huang, Yijun; Ou, Yanqiu; Li, Yan; Yan, Guangmei

2011-09-01

Malignant gliomas are the most common and lethal intracranial tumors, and differentiation therapy shows great potential to be a promising candidate for their treatment. Here, we have elaborated that a PKA activator, forskolin, represses cell growth via cell cycle arrest in the G0/G1 phase and induces cell differentiation characteristic with elongated processes and restoration of GFAP expression. In mechanisms, we verified that forskolin significantly diminishes the mRNA and protein level of a key cell cycle regulator cyclin D1, and maintenance of low cyclin D1 expression level was required for forskolin-induced proliferation inhibition and differentiation by gain and loss of function approaches. In addition, that forskolin down-regulated the cyclin D1 by proteolytic (post-transcriptional) mechanisms was dependent on GSK-3β activation at Ser9. The pro-differentiation activity of forskolin and related molecular mechanisms imply that forskolin can be developed into a candidate for the future in differentiation therapy of glioma, and cyclin D1 is a promising target for pro-differentiation strategy. Copyright © 2011 Wiley-Liss, Inc.

Immunoregulatory cytokines in mouse placental extracts inhibit in vitro osteoclast differentiation of murine macrophages.

PubMed

Canellada, A; Custidiano, A; Abraham, F; Rey, E; Gentile, T

2013-03-01

Previous studies showed that placental extracts (PE) alleviates arthritic symptoms in animal models of arthritis. To evaluate whether murine PEs obtained at embryonic days 7.5 (PE7) and 17.5 (PE18) regulate RANKL-induced osteoclast differentiation, RAW 264.7 cells were cultured with RANKL and MCSF in presence or not of PEs. Tartrate-resistant acid phosphatase (TRAP) was stained and multinucleated TRAP positive cells were visualized under a light microscope. Cathepsin K and metalloprotease expression was assessed by RT-PCR and gelatin zymography respectively. NFATc1 expression was determined by immunoblot. To analyze NFAT-dependent transcription, macrophages were transfected with a luciferase reporter plasmid. Cytokines were determined in PEs by ELISA and immunoblot. Transforming growth factor (TGF)- beta and Interleukin (IL)-10 receptor were inhibited in cell cultures with specific antibodies. PE7 and PE18 inhibited RANKL-induced multinucleated TRAP positive cells, Cathepsin K expression and metalloprotease activity, as well as NFATc1 expression and activity, thereby inhibiting osteoclast differentiation of RAW cells. Inflammatory/Regulatory cytokine ratio was higher in PE7 than in PE18. Blocking TGF-beta abolished the effect of both, PE7 and PE18, on multinucleated TRAP positive cells and metalloprotease expression, whereas blocking IL-10 receptor reverted the effect of PE18 but not of PE7. Inhibition of osteoclast differentiation by PEs was not unexpected, since cytokines detected in extracts were previously found to regulate osteoclast differentiation. PEs inhibited osteoclast differentiation of macrophages in vitro. Downregulation of NFATc1 might be involved in this effect. Regulatory/Th2 cytokines play a role in the effect of PEs on osteoclast differentiation. Copyright © 2013 Elsevier Ltd. All rights reserved.

A Cbfa1-dependent genetic pathway controls bone formation beyond embryonic development

PubMed Central

Ducy, Patricia; Starbuck, Michael; Priemel, Matthias; Shen, Jianhe; Pinero, Gerald; Geoffroy, Valerie; Amling, Michael; Karsenty, Gerard

1999-01-01

The molecular mechanisms controlling bone extracellular matrix (ECM) deposition by differentiated osteoblasts in postnatal life, called hereafter bone formation, are unknown. This contrasts with the growing knowledge about the genetic control of osteoblast differentiation during embryonic development. Cbfa1, a transcriptional activator of osteoblast differentiation during embryonic development, is also expressed in differentiated osteoblasts postnatally. The perinatal lethality occurring in Cbfa1-deficient mice has prevented so far the study of its function after birth. To determine if Cbfa1 plays a role during bone formation we generated transgenic mice overexpressing Cbfa1 DNA-binding domain (ΔCbfa1) in differentiated osteoblasts only postnatally. ΔCbfa1 has a higher affinity for DNA than Cbfa1 itself, has no transcriptional activity on its own, and can act in a dominant-negative manner in DNA cotransfection assays. ΔCbfa1-expressing mice have a normal skeleton at birth but develop an osteopenic phenotype thereafter. Dynamic histomorphometric studies show that this phenotype is caused by a major decrease in the bone formation rate in the face of a normal number of osteoblasts thus indicating that once osteoblasts are differentiated Cbfa1 regulates their function. Molecular analyses reveal that the expression of the genes expressed in osteoblasts and encoding bone ECM proteins is nearly abolished in transgenic mice, and ex vivo assays demonstrated that ΔCbfa1-expressing osteoblasts were less active than wild-type osteoblasts. We also show that Cbfa1 regulates positively the activity of its own promoter, which has the highest affinity Cbfa1-binding sites characterized. This study demonstrates that beyond its differentiation function Cbfa1 is the first transcriptional activator of bone formation identified to date and illustrates that developmentally important genes control physiological processes postnatally. PMID:10215629

Enhancement of ATRA-induced differentiation of neuroblastoma cells with LOX/COX inhibitors: an expression profiling study.

PubMed

Chlapek, Petr; Redova, Martina; Zitterbart, Karel; Hermanova, Marketa; Sterba, Jaroslav; Veselska, Renata

2010-05-11

We performed expression profiling of two neuroblastoma cell lines, SK-N-BE(2) and SH-SY5Y, after combined treatment with all-trans retinoic acid (ATRA) and inhibitors of lipoxygenases (LOX) and cyclooxygenases (COX). This study is a continuation of our previous work confirming the possibility of enhancing ATRA-induced cell differentiation in these cell lines by the application of LOX/COX inhibitors and brings more detailed information concerning the mechanisms of the enhancement of ATRA-induced differentiation of neuroblastoma cells. Caffeic acid, as an inhibitor of 5-lipoxygenase, and celecoxib, as an inhibitor on cyclooxygenase-2, were used in this study. Expression profiling was performed using Human Cancer Oligo GEArray membranes that cover 440 cancer-related genes. Cluster analyses of the changes in gene expression showed the concentration-dependent increase in genes known to be involved in the process of retinoid-induced neuronal differentiation, especially in cytoskeleton remodeling. These changes were detected in both cell lines, and they were independent of the type of specific inhibitors, suggesting a common mechanism of ATRA-induced differentiation enhancement. Furthermore, we also found overexpression of some genes in the same cell line (SK-N-BE(2) or SH-SY5Y) after combined treatment with both ATRA and CA, or ATRA and CX. Finally, we also detected that gene expression was changed after treatment with the same inhibitor (CA or CX) in combination with ATRA in both cell lines. Obtained results confirmed our initial hypothesis of the common mechanism of enhancement in ATRA-induced cell differentiation via inhibition of arachidonic acid metabolic pathway.

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

Liraglutide attenuates the osteoblastic differentiation of MC3T3-E1 cells by modulating AMPK/mTOR signaling

PubMed Central

Hu, Xiong-Ke; Yin, Xin-Hua; Zhang, Hong-Qi; Guo, Chao-Feng; Tang, Ming-Xing

2016-01-01

Liraglutide, a synthetic analogue of glucagon-like peptide-1, is utilized in the treatment of type 2 diabetes and obesity. Liraglutide has been previously demonstrated to prevent osteoblastic differentiation of human vascular smooth muscle cells, resulting in the slowing of arterial calcification, however, its effect on bone formation remains unclear. The present study investigated the effect of liraglutide on osteoblastic differentiation using Alizarin Red S staining, and examined the molecular mechanisms underlying the regulatory effect by western blot analysis. The present study demonstrated that protein expression levels of phosphorylated adenosine monophosphate-activated protein kinase (p-AMPK) were downregulated in MC3T3-E1 cells during osteoblastic differentiation in commercial osteogenic differentiation medium, whereas protein expression levels of transforming growth factor-β (TGF-β) and phosphorylated mammalian target of rapamycin (p-mTOR) increased. Liraglutide was subsequently demonstrated to dose-dependently attenuate the osteoblastic differentiation of MC3T3-E1 cells, to upregulate p-AMPK, and downregulate p-mTOR and TGF-β protein expression levels. Treatment with an AMPK-specific inhibitor, Compound C, eradicated the effect of liraglutide on osteoblastic differentiation, and p-mTOR and TGF-β downregulation. An mTOR activator, MHY1485, also abolished the inhibitory effect of liraglutide on osteoblastic differentiation, and resulted in p-mTOR and TGF-β downregulation, but did not attenuate the liraglutide-induced increase in p-AMPK protein expression levels. The results of the present study demonstrate that liraglutide attenuates osteoblastic differentiation of MC3T3-E1 cells via modulation of AMPK/mTOR signaling. The present study revealed a novel function of liraglutide, which contributes to the understanding of its pharmacological and physiological effects in clinical settings. PMID:27600753

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.

Peroxisome proliferator-activated receptor-alpha regulates fatty acid utilization in primary human skeletal muscle cells.

PubMed

Muoio, Deborah M; Way, James M; Tanner, Charles J; Winegar, Deborah A; Kliewer, Steven A; Houmard, Joseph A; Kraus, William E; Dohm, G Lynis

2002-04-01

In humans, skeletal muscle is a major site of peroxisome proliferator-activated receptor-alpha (PPAR-alpha) expression, but its function in this tissue is unclear. We investigated the role of hPPAR-alpha in regulating muscle lipid utilization by studying the effects of a highly selective PPAR-alpha agonist, GW7647, on [(14)C]oleate metabolism and gene expression in primary human skeletal muscle cells. Robust induction of PPAR-alpha protein expression occurred during muscle cell differentiation and corresponded with differentiation-dependent increases in oleate oxidation. In mature myotubes, 48-h treatment with 10-1,000 nmol/l GW7647 increased oleate oxidation dose-dependently, up to threefold. Additionally, GW7647 decreased oleate esterification into myotube triacylglycerol (TAG), up to 45%. This effect was not abolished by etomoxir, a potent inhibitor of beta-oxidation, indicating that PPAR-alpha-mediated TAG depletion does not depend on reciprocal changes in fatty acid catabolism. Consistent with its metabolic actions, GW7647 induced mRNA expression of mitochondrial enzymes that promote fatty acid catabolism; carnitine palmityltransferase 1 and malonyl-CoA decarboxylase increased approximately 2-fold, whereas pyruvate dehydrogenase kinase 4 increased 45-fold. Expression of several genes that regulate glycerolipid synthesis was not changed by GW7647 treatment, implicating involvement of other targets to explain the TAG-depleting effect of the compound. These results demonstrate a role for hPPAR-alpha in regulating muscle lipid homeostasis.

Ectopic Expression of Nolz-1 in Neural Progenitors Promotes Cell Cycle Exit/Premature Neuronal Differentiation Accompanying with Abnormal Apoptosis in the Developing Mouse Telencephalon

PubMed Central

Chang, Sunny Li-Yun; Chen, Shih-Yun; Huang, Huai-Huei; Ko, Hsin-An; Liu, Pei-Tsen; Liu, Ya-Chi; Chen, Ping-Hau; Liu, Fu-Chin

2013-01-01

Nolz-1, as a murine member of the NET zinc-finger protein family, is expressed in post-mitotic differentiating neurons of striatum during development. To explore the function of Nolz-1 in regulating the neurogenesis of forebrain, we studied the effects of ectopic expression of Nolz-1 in neural progenitors. We generated the Cre-loxP dependent conditional transgenic mice in which Nolz-1 was ectopically expressed in proliferative neural progenitors. Ectopic expression of Nolz-1 in neural progenitors by intercrossing the Nolz-1 conditional transgenic mice with the nestin-Cre mice resulted in hypoplasia of telencephalon in double transgenic mice. Decreased proliferation of neural progenitor cells were found in the telencephalon, as evidenced by the reduction of BrdU−, Ki67− and phospho-histone 3-positive cells in E11.5–12.5 germinal zone of telencephalon. Transgenic Nolz-1 also promoted cell cycle exit and as a consequence might facilitate premature differentiation of progenitors, because TuJ1-positive neurons were ectopically found in the ventricular zone and there was a general increase of TuJ1 immunoreactivity in the telencephalon. Moreover, clusters of strong TuJ1-expressing neurons were present in E12.5 germinal zone. Some of these strong TuJ1-positive clusters, however, contained apoptotic condensed DNA, suggesting that inappropriate premature differentiation may lead to abnormal apoptosis in some progenitor cells. Consistent with the transgenic mouse analysis in vivo, similar effects of Nozl-1 over-expression in induction of apoptosis, inhibition of cell proliferation and promotion of neuronal differentiation were also observed in three different N18, ST14A and N2A neural cell lines in vitro. Taken together, our study indicates that ectopic expression of Nolz-1 in neural progenitors promotes cell cycle exit/premature neuronal differentiation and induces abnormal apoptosis in the developing telencephalon. PMID:24073229

Ectopic expression of nolz-1 in neural progenitors promotes cell cycle exit/premature neuronal differentiation accompanying with abnormal apoptosis in the developing mouse telencephalon.

PubMed

Chang, Sunny Li-Yun; Chen, Shih-Yun; Huang, Huai-Huei; Ko, Hsin-An; Liu, Pei-Tsen; Liu, Ya-Chi; Chen, Ping-Hau; Liu, Fu-Chin

2013-01-01

Nolz-1, as a murine member of the NET zinc-finger protein family, is expressed in post-mitotic differentiating neurons of striatum during development. To explore the function of Nolz-1 in regulating the neurogenesis of forebrain, we studied the effects of ectopic expression of Nolz-1 in neural progenitors. We generated the Cre-loxP dependent conditional transgenic mice in which Nolz-1 was ectopically expressed in proliferative neural progenitors. Ectopic expression of Nolz-1 in neural progenitors by intercrossing the Nolz-1 conditional transgenic mice with the nestin-Cre mice resulted in hypoplasia of telencephalon in double transgenic mice. Decreased proliferation of neural progenitor cells were found in the telencephalon, as evidenced by the reduction of BrdU-, Ki67- and phospho-histone 3-positive cells in E11.5-12.5 germinal zone of telencephalon. Transgenic Nolz-1 also promoted cell cycle exit and as a consequence might facilitate premature differentiation of progenitors, because TuJ1-positive neurons were ectopically found in the ventricular zone and there was a general increase of TuJ1 immunoreactivity in the telencephalon. Moreover, clusters of strong TuJ1-expressing neurons were present in E12.5 germinal zone. Some of these strong TuJ1-positive clusters, however, contained apoptotic condensed DNA, suggesting that inappropriate premature differentiation may lead to abnormal apoptosis in some progenitor cells. Consistent with the transgenic mouse analysis in vivo, similar effects of Nozl-1 over-expression in induction of apoptosis, inhibition of cell proliferation and promotion of neuronal differentiation were also observed in three different N18, ST14A and N2A neural cell lines in vitro. Taken together, our study indicates that ectopic expression of Nolz-1 in neural progenitors promotes cell cycle exit/premature neuronal differentiation and induces abnormal apoptosis in the developing telencephalon.

Peroxisome proliferator-activated receptor-β/δ inhibits human neuroblastoma cell tumorigenesis by inducing p53- and SOX2-mediated cell differentiation.

PubMed

Yao, Pei-Li; Chen, Liping; Dobrzański, Tomasz P; Zhu, Bokai; Kang, Boo-Hyon; Müller, Rolf; Gonzalez, Frank J; Peters, Jeffrey M

2017-05-01

Neuroblastoma is a common childhood cancer typically treated by inducing differentiation with retinoic acid (RA). Peroxisome proliferator-activated receptor-β/δ, (PPARβ/δ) is known to promote terminal differentiation of many cell types. In the present study, PPARβ/δ was over-expressed in three human neuroblastoma cell lines, NGP, SK-N-BE(2), and IMR-32, that exhibit high, medium, and low sensitivity, respectively, to retinoic acid-induced differentiation to determine if PPARβ/δ and retinoic acid receptors (RARs) could be jointly targeted to increase the efficacy of treatment. All-trans-RA (atRA) decreased expression of SRY (sex determining region Y)-box 2 (SOX2), a stem cell regulator and marker of de-differentiation, in NGP and SK-N-BE(2) cells with inactive or mutant tumor suppressor p53, respectively. However, atRA did not suppress SOX2 expression in IMR-32 cells carrying wild-type p53. Over-expression and/or ligand activation of PPARβ/δ reduced the average volume and weight of ectopic tumor xenografts from NGP, SK-N-BE(2), or IMR-32 cells compared to controls. Compared with that found with atRA, PPARβ/δ suppressed SOX2 expression in NGP and SK-N-BE(2) cells and ectopic xenografts, and was also effective in suppressing SOX2 expression in IMR-32 cells that exhibit higher p53 expression compared to the former cell lines. Combined, these observations demonstrate that activating or over-expressing PPARβ/δ induces cell differentiation through p53- and SOX2-dependent signaling pathways in neuroblastoma cells and tumors. This suggests that combinatorial activation of both RARα and PPARβ/δ may be suitable as an alternative therapeutic approach for RA-resistant neuroblastoma patients. Published [2016]. This article is a U.S. Government work and is in the public domain in the USA.

Flow cytometric analysis of immunoglobulin heavy chain expression in B-cell lymphoma and reactive lymphoid hyperplasia

PubMed Central

Grier, David D; Al-Quran, Samer Z; Cardona, Diana M; Li, Ying; Braylan, Raul C

2012-01-01

The diagnosis of B-cell lymphoma (BCL) is often dependent on the detection of clonal immunoglobulin (Ig) light chain expression. In some BCLs, the determination of clonality based on Ig light chain restriction may be difficult. The aim of our study was to assess the utility of flow cytometric analysis of surface Ig heavy chain (HC) expression in lymphoid tissues in distinguishing lymphoid hyperplasias from BCLs, and also differentiating various BCL subtypes. HC expression on B-cells varied among different types of hyperplasias. In follicular hyperplasia, IgM and IgD expression was high in mantle cells while germinal center cells showed poor HC expression. In other hyperplasias, B cell compartments were blurred but generally showed high IgD and IgM expression. Compared to hyperplasias, BCLs varied in IgM expression. Small lymphocytic lymphomas had lower IgM expression than mantle cell lymphomas. Of importance, IgD expression was significantly lower in BCLs than in hyperplasias, a finding that can be useful in differentiating lymphoma from reactive processes. PMID:22400070

Role of the EGF-Related Growth Factor Cripto in Murine Mammary Tumorigenesis

DTIC Science & Technology

1999-10-01

release or disclosure of technical data (other than detailed manufacturing or process data) to, or use of such data by, a foreign government that is in...function, we have made constructs that express secreted processed NODAL protein in transfected mammalian cells. For this purpose, we have used a wild-type...that processing of unmodified NODAL protein is dependent upon the cell line used for expression, which presumably reflects differential expression of

Solvent effects in time-dependent self-consistent field methods. II. Variational formulations and analytical gradients

DOE PAGES

Bjorgaard, J. A.; Velizhanin, K. A.; Tretiak, S.

2015-08-06

This study describes variational energy expressions and analytical excited state energy gradients for time-dependent self-consistent field methods with polarizable solvent effects. Linear response, vertical excitation, and state-specific solventmodels are examined. Enforcing a variational ground stateenergy expression in the state-specific model is found to reduce it to the vertical excitation model. Variational excited state energy expressions are then provided for the linear response and vertical excitation models and analytical gradients are formulated. Using semiempiricalmodel chemistry, the variational expressions are verified by numerical and analytical differentiation with respect to a static external electric field. Lastly, analytical gradients are further tested by performingmore » microcanonical excited state molecular dynamics with p-nitroaniline.« less

Lactic Acid Is Elevated in Idiopathic Pulmonary Fibrosis and Induces Myofibroblast Differentiation via pH-Dependent Activation of Transforming Growth Factor-β

PubMed Central

Kottmann, Robert Matthew; Kulkarni, Ajit A.; Smolnycki, Katie A.; Lyda, Elizabeth; Dahanayake, Thinesh; Salibi, Rami; Honnons, Sylvie; Jones, Carolyn; Isern, Nancy G.; Hu, Jian Z.; Nathan, Steven D.; Grant, Geraldine; Phipps, Richard P.

2012-01-01

Rationale: Idiopathic pulmonary fibrosis (IPF) is a complex disease for which the pathogenesis is poorly understood. In this study, we identified lactic acid as a metabolite that is elevated in the lung tissue of patients with IPF. Objectives: This study examines the effect of lactic acid on myofibroblast differentiation and pulmonary fibrosis. Methods: We used metabolomic analysis to examine cellular metabolism in lung tissue from patients with IPF and determined the effects of lactic acid and lactate dehydrogenase-5 (LDH5) overexpression on myofibroblast differentiation and transforming growth factor (TGF)-β activation in vitro. Measurements and Main Results: Lactic acid concentrations from healthy and IPF lung tissue were determined by nuclear magnetic resonance spectroscopy; α-smooth muscle actin, calponin, and LDH5 expression were assessed by Western blot of cell culture lysates. Lactic acid and LDH5 were significantly elevated in IPF lung tissue compared with controls. Physiologic concentrations of lactic acid induced myofibroblast differentiation via activation of TGF-β. TGF-β induced expression of LDH5 via hypoxia-inducible factor 1α (HIF1α). Importantly, overexpression of both HIF1α and LDH5 in human lung fibroblasts induced myofibroblast differentiation and synergized with low-dose TGF-β to induce differentiation. Furthermore, inhibition of both HIF1α and LDH5 inhibited TGF-β–induced myofibroblast differentiation. Conclusions: We have identified the metabolite lactic acid as an important mediator of myofibroblast differentiation via a pH-dependent activation of TGF-β. We propose that the metabolic milieu of the lung, and potentially other tissues, is an important driving force behind myofibroblast differentiation and potentially the initiation and progression of fibrotic disorders. PMID:22923663

Formation of Cartilage and Synovial Tissue by Human Gingival Stem Cells

PubMed Central

Larjava, Hannu; Loison-Robert, Ludwig-Stanislas; Berbar, Tsouria; Owen, Gethin R.; Berdal, Ariane; Chérifi, Hafida; Gogly, Bruno; Häkkinen, Lari; Fournier, Benjamin P.J.

2014-01-01

Human gingival stem cells (HGSCs) can be easily isolated and manipulated in culture to investigate their multipotency. Osteogenic differentiation of bone-marrow-derived mesenchymal stem/stromal cells has been well documented. HGSCs derive from neural crests, however, and their differentiation capacity has not been fully established. The aim of the present report was to investigate whether HGSCs can be induced to differentiate to osteoblasts and chondrocytes. HGSCs were cultured either in a classical monolayer culture or in three-dimensional floating micromass pellet cultures in specific differentiation media. HGSC differentiation to osteogenic and chondrogenic lineages was determined by protein and gene expression analyses, and also by specific staining of cells and tissue pellets. HGSCs cultured in osteogenic differentiation medium showed induction of Runx2, alkaline phosphatase (ALPL), and osterix expression, and subsequently formed mineralized nodules consistent with osteogenic differentiation. Interestingly, HGSC micromass cultures maintained in chondrogenic differentiation medium showed SOX9-dependent differentiation to both chondrocyte and synoviocyte lineages. Chondrocytes at different stages of differentiation were identified by gene expression profiles and by histochemical and immunohistochemical staining. In 3-week-old cultures, peripheral cells in the micromass cultures organized in layers of cuboidal cells with villous structures facing the medium. These cells were strongly positive for cadherin-11, a marker of synoviocytes. In summary, the findings indicate that HGSCs have the capacity to differentiate to osteogenic, chondrogenic, and synoviocyte lineages. Therefore, HGSCs could serve as an alternative source for stem cell therapies in regenerative medicine for patients with cartilage and joint destructions, such as observed in rheumatoid arthritis. PMID:25003637

Differentiation and upregulation of heat shock protein 70 induced by a subset of histone deacetylase inhibitors in mouse and human embryonic stem cells.

PubMed

Park, Jeong-A; Kim, Young-Eun; Seok, Hyun-Jeong; Park, Woo-Youn; Kwon, Hyung-Joo; Lee, Younghee

2011-03-01

Inhibiting histone deacetylase (HDAC) activity modulates the epigenetic status of cells, resulting in an alteration of gene expression and cellular function. Here, we investigated the effects of HDAC inhibitors on mouse embryonic stem (ES) cells. The HDAC inhibitors trichostatin A, suberoylanilide hydroxamic acid, sodium butyrate, and valproic acid induced early differentiation of mouse ES cells and triggered induction of heatshock protein (HSP)70. In contrast, class III HDAC inhibitors failed to induce differentiation or HSP70 expression. Transcriptional upregulation of HSP70 was confirmed by mRNA expression analysis, an inhibitor study, and chromatin immunoprecipitation. HSP70 induction was dependent on the SAPK/ JNK, p38, and PI3K/Akt pathways. Differentiation and induction of HSP70 by a subset of HDAC inhibitors was also examined in human ES cells, which suggests that the phenomenon generally occurs in ES cells. A better understanding of the effects of HDAC inhibitors may give more insight into their application in stem cell biology.

Nobiletin enhances differentiation and lipolysis of 3T3-L1 adipocytes

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

Saito, Takeshi; Abe, Daigo; Sekiya, Keizo

2007-06-01

Nobiletin is a polymethoxylated flavone found in certain citrus fruits. Here we demonstrate that nobiletin enhance differentiation of 3T3-L1 preadipocytes. Nobiletin dose-dependently increased accumulation of lipid droplets in adipocytes. Quantitative RT-PCR analyses indicated that nobiletin increased the expression of genes critical for acquisition of the adipocyte phenotype. Some of them were known peroxisome proliferator activated receptor {gamma} (PPAR{gamma}) targets and PPAR{gamma} itself, however, nobiletin did not exhibit PPAR{gamma} ligand activity. We observed the expression of CCAAT/enhancer binding protein {beta} (C/EBP{beta}), a transcription factor for PPAR{gamma}, was increased by nobiletin. The activation of cAMP-responsive element binding protein (CREB) and extracellular signal-regulatedmore » kinase (ERK), which play important roles in C/EBP{beta} expression were also potentiated by nobiletin. Furthermore, nobiletin stimulated lipolysis in differentiated adipocytes, which is known to be stimulated by cAMP pathway. These results suggested that nobiletin enhanced both differentiation and lipolysis of adipocyte through activation of signaling cascades mediated by cAMP/CREB.« less

Prohibitin regulates the FSH signaling pathway in rat granulosa cell differentiation.

PubMed

Chowdhury, Indrajit; Thomas, Kelwyn; Zeleznik, Anthony; Thompson, Winston E

2016-05-01

Published results from our laboratory identified prohibitin (PHB), a gene product expressed in granulosa cells (GCs) that progressively increases during follicle maturation. Our current in vitro studies demonstrate that follicle-stimulating hormone (FSH) stimulates Phb expression in rat primary GCs. The FSH-dependent expression of PHB was primarily localized within mitochondria, and positively correlates with the morphological changes in GCs organelles, and synthesis and secretions of estradiol (E2) and progesterone (P4). In order to confirm that PHB plays a regulatory role in rat GC differentiation, endogenous PHB-knockdown studies were carried out in undifferentiated GCs using adenoviral (Ad)-mediated RNA interference methodology. Knockdown of PHB in GCs resulted in the suppression of the key steroidogenic enzymes including steroidogenic acute regulatory protein (StAR), p450 cholesterol side-chain cleavage enzyme (p450scc), 3β-hydroxysteroid dehydrogenase (3β-HSD), and aromatase (Cyp19a1); and decreased E2 and P4 synthesis and secretions in the presence of FSH stimulation. Furthermore, these experimental studies also provided direct evidence that PHB within the mitochondrial fraction in GCs is phosphorylated at residues Y249, T258, and Y259 in response to FSH stimulation. The observed levels of phosphorylation of PHB at Y249, T258, and Y259 were significantly low in GCs in the absence of FSH stimulation. In addition, during GC differentiation FSH-induced expression of phospho-PHB (pPHB) requires the activation of MEK1-ERK1/2 signaling pathway. Taken together, these studies provide new evidence supporting FSH-dependent PHB/pPHB upregulation in GCs is required to sustain the differentiated state of GCs. © 2016 The authors.

Wnt/β-catenin signaling enhances osteoblastogenic differentiation from human periodontal ligament fibroblasts.

PubMed

Heo, Jung Sun; Lee, Seung-Youp; Lee, Jeong-Chae

2010-11-01

Wnt/β-catenin signaling has been known to influence bone formation and homeostasis. In this study, we investigated the canonical Wnt signaling regulation of osteogenic differentiation from periodontal ligament (PDL) fibroblasts. Stimulating PDL fibroblasts with lithium chloride (LiCl), a canonical Wnt activator, significantly increased mineralized nodule and alkaline phosphatase (ALP) activity in a time- and dose-dependent manner. LiCl up-regulated protein expression of osteogenic transcription factors, including the runt-related gene 2, Msx2, and Osterix 2, in the PDL fibroblasts. Treatment of these cells with LiCl also increased the mRNA levels of ALP, FosB, and Fra1 in a dose-dependent manner. Blockage of canonical Wnt signaling by treating the cells with DKK1 inhibited Wnt1-stimulated mRNA expression of these osteogenic factors. Furthermore, pretreatment with DKK1 reduced the ALP activity and matrix mineralization stimulated by Wnt1. Collectively, these results suggest that canonical Wnt signaling leads to the differentiation of PDL fibroblasts into osteogenic lineage with the attendant stimulation of osteogenic transcription factors.

Co-Expression of α9β1 Integrin and VEGF-D Confers Lymphatic Metastatic Ability to a Human Breast Cancer Cell Line MDA-MB-468LN

PubMed Central

Majumder, Mousumi; Rodriguez-Torres, Mauricio; Torres-Garcia, Jose; Wiebe, Ryan; Timoshenko, Alexander V.; Bhattacharjee, Rabindra N.; Chambers, Ann F.; Lala, Peeyush K.

2012-01-01

Introduction and Objectives Lymphatic metastasis is a common occurrence in human breast cancer, mechanisms remaining poorly understood. MDA-MB-468LN (468LN), a variant of the MDA-MB-468GFP (468GFP) human breast cancer cell line, produces extensive lymphatic metastasis in nude mice. 468LN cells differentially express α9β1 integrin, a receptor for lymphangiogenic factors VEGF-C/-D. We explored whether (1) differential production of VEGF-C/-D by 468LN cells provides an autocrine stimulus for cellular motility by interacting with α9β1 and a paracrine stimulus for lymphangiogenesis in vitro as measured with capillary-like tube formation by human lymphatic endothelial cells (HMVEC-dLy); (2) differential expression of α9 also promotes cellular motility/invasiveness by interacting with macrophage derived factors; (3) stable knock-down of VEGF-D or α9 in 468LN cells abrogates lymphangiogenesis and lymphatic metastasis in vivo in nude mice. Results A comparison of expression of cyclo-oxygenase (COX)-2 (a VEGF-C/-D inducer), VEGF-C/-D and their receptors revealed little COX-2 expression by either cells. However, 468LN cells showed differential VEGF-D and α9β1 expression, VEGF-D secretion, proliferative, migratory/invasive capacities, latter functions being stimulated further with VEGF-D. The requirement of α9β1 for native and VEGF-D-stimulated proliferation, migration and Erk activation was demonstrated by treating with α9β1 blocking antibody or knock-down of α9. An autocrine role of VEGF-D in migration was shown by its impairment by silencing VEGF-D and restoration with VEGF-D. 468LN cells and their soluble products stimulated tube formation, migration/invasiveness of HMVEC-dLy cell in a VEGF-D dependent manner as indicated by the loss of stimulation by silencing VEGF-D in 468LN cells. Furthermore, 468LN cells showed α9-dependent stimulation of migration/invasiveness by macrophage products. Finally, capacity for intra-tumoral lymphangiogenesis and lymphatic metastasis in nude mice was completely abrogated by stable knock-down of either VEGF-D or α9 in 468LN cells. Conclusion Differential capacity for VEGF-D production and α9β1 integrin expression by 468LN cells jointly contributed to their lymphatic metastatic phenotype. PMID:22545097

Global gene expression analyses of hematopoietic stem cell-like cell lines with inducible Lhx2 expression

PubMed Central

Richter, Karin; Wirta, Valtteri; Dahl, Lina; Bruce, Sara; Lundeberg, Joakim; Carlsson, Leif; Williams, Cecilia

2006-01-01

Background Expression of the LIM-homeobox gene Lhx2 in murine hematopoietic cells allows for the generation of hematopoietic stem cell (HSC)-like cell lines. To address the molecular basis of Lhx2 function, we generated HSC-like cell lines where Lhx2 expression is regulated by a tet-on system and hence dependent on the presence of doxycyclin (dox). These cell lines efficiently down-regulate Lhx2 expression upon dox withdrawal leading to a rapid differentiation into various myeloid cell types. Results Global gene expression of these cell lines cultured in dox was compared to different time points after dox withdrawal using microarray technology. We identified 267 differentially expressed genes. The majority of the genes overlapping with HSC-specific databases were those down-regulated after turning off Lhx2 expression and a majority of the genes overlapping with those defined as late progenitor-specific genes were the up-regulated genes, suggesting that these cell lines represent a relevant model system for normal HSCs also at the level of global gene expression. Moreover, in situ hybridisations of several genes down-regulated after dox withdrawal showed overlapping expression patterns with Lhx2 in various tissues during embryonic development. Conclusion Global gene expression analysis of HSC-like cell lines with inducible Lhx2 expression has identified genes putatively linked to self-renewal / differentiation of HSCs, and function of Lhx2 in organ development and stem / progenitor cells of non-hematopoietic origin. PMID:16600034

Sex-based differences in gene expression in hippocampus following postnatal lead exposure

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

Schneider, J.S., E-mail: jay.schneider@jefferson.edu; Anderson, D.W.; Sonnenahalli, H.

The influence of sex as an effect modifier of childhood lead poisoning has received little systematic attention. Considering the paucity of information available concerning the interactive effects of lead and sex on the brain, the current study examined the interactive effects of lead and sex on gene expression patterns in the hippocampus, a structure involved in learning and memory. Male or female rats were fed either 1500 ppm lead-containing chow or control chow for 30 days beginning at weaning.Blood lead levels were 26.7 {+-} 2.1 {mu}g/dl and 27.1 {+-} 1.7 {mu}g/dl for females and males, respectively. The expression of 175more » unique genes was differentially regulated between control male and female rats. A total of 167 unique genes were differentially expressed in response to lead in either males or females. Lead exposure had a significant effect without a significant difference between male and female responses in 77 of these genes. In another set of 71 genes, there were significant differences in male vs. female response. A third set of 30 genes was differentially expressed in opposite directions in males vs. females, with the majority of genes expressed at a lower level in females than in males. Highly differentially expressed genes in males and females following lead exposure were associated with diverse biological pathways and functions. These results show that a brief exposure to lead produced significant changes in expression of a variety of genes in the hippocampus and that the response of the brain to a given lead exposure may vary depending on sex. - Highlights: > Postnatal lead exposure has a significant effect on hippocampal gene expression patterns. > At least one set of genes was affected in opposite directions in males and females. > Differentially expressed genes were associated with diverse biological pathways.« less

A protein phosphatase network controls the temporal and spatial dynamics of differentiation commitment in human epidermis

PubMed Central

Walko, Gernot; Viswanathan, Priyalakshmi; Tihy, Matthieu; Nijjher, Jagdeesh; Dunn, Sara-Jane; Lamond, Angus I

2017-01-01

Epidermal homeostasis depends on a balance between stem cell renewal and terminal differentiation. The transition between the two cell states, termed commitment, is poorly understood. Here, we characterise commitment by integrating transcriptomic and proteomic data from disaggregated primary human keratinocytes held in suspension to induce differentiation. Cell detachment induces several protein phosphatases, five of which - DUSP6, PPTC7, PTPN1, PTPN13 and PPP3CA – promote differentiation by negatively regulating ERK MAPK and positively regulating AP1 transcription factors. Conversely, DUSP10 expression antagonises commitment. The phosphatases form a dynamic network of transient positive and negative interactions that change over time, with DUSP6 predominating at commitment. Boolean network modelling identifies a mandatory switch between two stable states (stem and differentiated) via an unstable (committed) state. Phosphatase expression is also spatially regulated in vivo and in vitro. We conclude that an auto-regulatory phosphatase network maintains epidermal homeostasis by controlling the onset and duration of commitment. PMID:29043977

Bone Morphogenetic Protein-Induced Msx1 and Msx2 Inhibit Myocardin-Dependent Smooth Muscle Gene Transcription▿

PubMed Central

Hayashi, Ken'ichiro; Nakamura, Seiji; Nishida, Wataru; Sobue, Kenji

2006-01-01

During the onset and progression of atherosclerosis, the vascular smooth muscle cell (VSMC) phenotype changes from differentiated to dedifferentiated, and in some cases, this change is accompanied by osteogenic transition, resulting in vascular calcification. One characteristic of dedifferentiated VSMCs is the down-regulation of smooth muscle cell (SMC) marker gene expression. Bone morphogenetic proteins (BMPs), which are involved in the induction of osteogenic gene expression, are detected in calcified vasculature. In this study, we found that the BMP2-, BMP4-, and BMP6-induced expression of Msx transcription factors (Msx1 and Msx2) preceded the down-regulation of SMC marker expression in cultured differentiated VSMCs. Either Msx1 or Msx2 markedly reduced the myocardin-dependent promoter activities of SMC marker genes (SM22α and caldesmon). We further investigated interactions between Msx1 and myocardin/serum response factor (SRF)/CArG-box motif (cis element for SRF) using coimmunoprecipitation, gel-shift, and chromatin immunoprecipitation assays. Our results showed that Msx1 or Msx2 formed a ternary complex with SRF and myocardin and inhibited the binding of SRF or SRF/myocardin to the CArG-box motif, resulting in inhibition of their transcription. PMID:17030628

Bone morphogenetic protein-induced MSX1 and MSX2 inhibit myocardin-dependent smooth muscle gene transcription.

PubMed

Hayashi, Ken'ichiro; Nakamura, Seiji; Nishida, Wataru; Sobue, Kenji

2006-12-01

During the onset and progression of atherosclerosis, the vascular smooth muscle cell (VSMC) phenotype changes from differentiated to dedifferentiated, and in some cases, this change is accompanied by osteogenic transition, resulting in vascular calcification. One characteristic of dedifferentiated VSMCs is the down-regulation of smooth muscle cell (SMC) marker gene expression. Bone morphogenetic proteins (BMPs), which are involved in the induction of osteogenic gene expression, are detected in calcified vasculature. In this study, we found that the BMP2-, BMP4-, and BMP6-induced expression of Msx transcription factors (Msx1 and Msx2) preceded the down-regulation of SMC marker expression in cultured differentiated VSMCs. Either Msx1 or Msx2 markedly reduced the myocardin-dependent promoter activities of SMC marker genes (SM22alpha and caldesmon). We further investigated interactions between Msx1 and myocardin/serum response factor (SRF)/CArG-box motif (cis element for SRF) using coimmunoprecipitation, gel-shift, and chromatin immunoprecipitation assays. Our results showed that Msx1 or Msx2 formed a ternary complex with SRF and myocardin and inhibited the binding of SRF or SRF/myocardin to the CArG-box motif, resulting in inhibition of their transcription.

Role of the POZ Zinc Finger Transcription Factor FBI-1 in Human and Murine Adipogenesis

PubMed Central

Laudes, Matthias; Christodoulides, Constantinos; Sewter, Ciaran; Rochford, Justin J.; Considine, Robert V.; Sethi, Jaswinder K.; Vidal-Puig, Antonio; O’Rahilly, Stephen

2015-01-01

Poxvirus zinc finger (POZ) zinc finger domain transcription factors have been shown to play a role in the control of growth arrest and differentiation in several types of mesenchymal cells but not, as yet, adipocytes. We found that a POZ domain protein, factor that binds to inducer of short transcripts-1 (FBI-1), was induced during both murine and human preadipocyte differentiation with maximal expression levels seen at days 2–4. FBI-1 mRNA was expressed in human adipose tissue with the highest levels found in samples from morbidly obese subjects. Murine cell lines constitutively expressing FBI-1 showed evidence for accelerated adipogenesis with earlier induction of markers of differentiation and enhanced lipid accumulation, suggesting that FBI-1 may be an active participant in the differentiation process. Consistent with the properties of this family of proteins in other cell systems, 3T3L1 cells stably overexpressing FBI-1 showed reduced DNA synthesis and reduced expression of cyclin A, cyclin-dependent kinase 2, and p107, proteins known to be involved in the regulation of mitotic clonal expansion. In addition, FBI-1 reduced the transcriptional activity of the cyclin A promoter. Thus, FBI-1, a POZ zinc finger transcription factor, is induced during the early phases of human and murine preadipocyte differentiation where it may contribute to adipogenesis through influencing the switch from cellular proliferation to terminal differentiation. PMID:14701838

Differentiation Induces Dramatic Changes in miRNA Profile, Where Loss of Dicer Diverts Differentiating SH-SY5Y Cells Toward Senescence.

PubMed

Jauhari, Abhishek; Singh, Tanisha; Pandey, Ankita; Singh, Parul; Singh, Nishant; Srivastava, Ankur Kumar; Pant, Aditya Bhushan; Parmar, Devendra; Yadav, Sanjay

2017-09-01

MicroRNAs (miRNAs) are generated by endonuclease activity of Dicer, which also helps in loading of miRNAs to their target sequences. SH-SY5Y, a human neuroblastoma and a cellular model of neurodevelopment, consistently expresses genes related to neurodegenerative disorders at different biological levels (DNA, RNA, and proteins). Using SH-SY5Y cells, we have studied the role of Dicer and miRNAs in neuronal differentiation and explored involvement of P53, a master regulator of gene expression in differentiation-induced induction of miRNAs. Knocking down Dicer gene induced senescence in differentiating SH-SY5Y cells, which indicate the essential role of Dicer in brain development. Differentiation of SH-SY5Y cells by retinoic acid (RA) or RA + brain-derived neurotrophic factor (BDNF) induced dramatic changes in global miRNA expression. Fully differentiated SH-SY5Y cells (5-day RA followed by 3-day BDNF) significantly (p < 0.05 and atleast >3-fold change) upregulated and downregulated the expression of 77 and 17 miRNAs, respectively. Maximum increase was observed in the expression of miR-193-5p, miR-199a-5p, miR-192, miR-145, miR-28-5p, miR-29b, and miR-222 after RA exposure and miR-193-5p, miR-146a, miR-21, miR-199a-5p, miR-153, miR-29b, and miR-222 after RA + BDNF exposure in SH-SY5Y cells. Exploring the role of P53 in differentiating SH-SY5Y cells, we have observed that induction of miR-222, miR-192, and miR-145 is P53 dependent and expression of miR-193a-5p, miR-199a-5p, miR-146a, miR-21, miR-153, and miR-29b is P53 independent. In conclusion, decreased Dicer level enforces differentiating cells to senescence, and differentiating SH-SY5Y cells needs increased expression of P53 to cope up with changes in protein levels of mature neurons.

Dose-dependent Effect of Boric Acid on Myogenic Differentiation of Human Adipose-derived Stem Cells (hADSCs).

PubMed

Apdik, Hüseyin; Doğan, Ayşegül; Demirci, Selami; Aydın, Safa; Şahin, Fikrettin

2015-06-01

Boron, a vital micronutrient for plant metabolism, is not fully elucidated for embryonic and adult body development, and tissue regeneration. Although optimized amount of boron supplement has been shown to be essential for normal gestational development in zebrafish and frog and beneficial for bone regeneration in higher animals, effects of boron on myogenesis and myo-regeneration remains to be solved. In the current study, we investigated dose-dependent activity of boric acid on myogenic differentiation of human adipose-derived stem cells (hADSCs) using immunocytochemical, gene, and protein expression analysis. The results revealed that while low- (81.9 μM) and high-dose (819.6 μM) boron treatment increased myogenic gene expression levels such as myosin heavy chain (MYH), MyoD, myogenin, and desmin at day 4 of differentiation, high-dose treatment decreased myogenic-related gene and protein levels at day 21 of differentiation, confirmed by immunocytochemical analysis. The findings of the study present not only an understanding of boron's effect on myogenic differentiation but also an opportunity for the development of scaffolds to be used in skeletal tissue engineering and supplements for embryonic muscle growth. However, fine dose tuning and treatment period arranging are highly warranted as boron treatment over required concentrations and time might result in detrimental outcomes to myogenesis and myo-regeneration.

SAA drives proinflammatory heterotypic macrophage differentiation in the lung via CSF-1R-dependent signaling

PubMed Central

Anthony, Desiree; McQualter, Jonathan L.; Bishara, Maria; Lim, Ee X.; Yatmaz, Selcuk; Seow, Huei Jiunn; Hansen, Michelle; Thompson, Michelle; Hamilton, John A.; Irving, Louis B.; Levy, Bruce D.; Vlahos, Ross; Anderson, Gary P.; Bozinovski, Steven

2014-01-01

Serum amyloid A (SAA) is expressed locally in chronic inflammatory conditions such as chronic obstructive pulmonary disease (COPD), where macrophages that do not accord with the classic M1/M2 paradigm also accumulate. In this study, the role of SAA in regulating macrophage differentiation was investigated in vitro using human blood monocytes from healthy subjects and patients with COPD and in vivo using an airway SAA challenge model in BALB/c mice. Differentiation of human monocytes with SAA stimulated the proinflammatory monokines IL-6 and IL-1β concurrently with the M2 markers CD163 and IL-10. Furthermore, SAA-differentiated macrophages stimulated with lipopolysaccharide (LPS) expressed markedly higher levels of IL-6 and IL-1β. The ALX/FPR2 antagonist WRW4 reduced IL-6 and IL-1β expression but did not significantly inhibit phagocytic and efferocytic activity. In vivo, SAA administration induced the development of a CD11chighCD11bhigh macrophage population that generated higher levels of IL-6, IL-1β, and G-CSF following ex vivo LPS challenge. Blocking CSF-1R signaling effectively reduced the number of CD11chighCD11bhigh macrophages by 71% and also markedly inhibited neutrophilic inflammation by 80%. In conclusion, our findings suggest that SAA can promote a distinct CD11chighCD11bhigh macrophage phenotype, and targeting this population may provide a novel approach to treating chronic inflammatory conditions associated with persistent SAA expression.—Anthony, D., McQualter, J. L., Bishara, M., Lim, E. X., Yatmaz, S., Seow, H. J., Hansen, M., Thompson, M., Hamilton, J. A., Irving, L. B., Levy, B. D., Vlahos, R., Anderson, G. P., Bozinovski, S. SAA drives proinflammatory heterotypic macrophage differentiation in the lung via CSF-1R-dependent signaling. PMID:24846388

Differential expression profiling of the hepatic proteome in a rat model of dioxin resistance: correlation with genomic and transcriptomic analyses.

PubMed

Pastorelli, Roberta; Carpi, Donatella; Campagna, Roberta; Airoldi, Luisa; Pohjanvirta, Raimo; Viluksela, Matti; Hakansson, Helen; Boutros, Paul C; Moffat, Ivy D; Okey, Allan B; Fanelli, Roberto

2006-05-01

One characteristic feature of acute 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) toxicity is dramatic interspecies and interstrain variability in sensitivity. This complicates dioxin risk assessment for humans. However, this variability also provides a means of characterizing mechanisms of dioxin toxicity. Long-Evans (Turku/AB) rats are orders of magnitude more susceptible to TCDD lethality than Han/Wistar (Kuopio) rats, and this difference constitutes a very useful model for identifying mechanisms of dioxin toxicity. We adopted a proteomic approach to identify the differential effects of TCDD exposure on liver protein expression in Han/Wistar rats as compared with Long-Evans rats. This allows determination of which, if any, protein markers are indicative of differences in dioxin susceptibility and/or responsible for conferring resistance. Differential protein expression in total liver protein was assessed using two-dimensional gel electrophoresis, computerized gel image analysis, in-gel digestion, and mass spectrometry. We observed significant changes in the abundance of several proteins, which fall into three general classes: (i) TCDD-independent and exclusively strain-specific (e.g. isoforms of the protein-disulfide isomerase A3, regucalcin, and agmatine ureohydrolase); (ii) strain-independent and only dependent on TCDD exposure (e.g. aldehyde dehydrogenase 3A1 and rat selenium-binding protein 2); (iii) dependent on both TCDD exposure and strain (e.g. oxidative stress-related proteins, apoptosis-inducing factor, and MAWD-binding protein). By integrating transcriptomic (microarray) data and genomic data (computational search of regulatory elements), we found that protein expression levels were mainly controlled at the level of transcription. These results reveal, for the first time, a subset of hepatic proteins that are differentially regulated in response to TCDD in a strain-specific manner. Some of these differential responses may play a role in establishing the major differences in TCDD response between these two strains of rats. As such, our work is expected to lead to new insights into the mechanism of TCDD toxicity and resistance.

Inducible and Conditional Deletion of Extracellular Signal-regulated Kinase 5 Disrupts Adult Hippocampal Neurogenesis*

PubMed Central

Pan, Yung-Wei; Zou, Junhui; Wang, Wenbin; Sakagami, Hiroyuki; Garelick, Michael G.; Abel, Glen; Kuo, Chay T.; Storm, Daniel R.; Xia, Zhengui

2012-01-01

Recent studies have led to the exciting idea that adult-born neurons in the dentate gyrus of the hippocampus may play a role in hippocampus-dependent memory formation. However, signaling mechanisms that regulate adult hippocampal neurogenesis are not well defined. Here we report that extracellular signal-regulated kinase 5 (ERK5), a member of the mitogen-activated protein kinase family, is selectively expressed in the neurogenic regions of the adult mouse brain. We present evidence that shRNA suppression of ERK5 in adult hippocampal neural stem/progenitor cells (aNPCs) reduces the number of neurons while increasing the number of cells expressing markers for stem/progenitor cells or proliferation. Furthermore, shERK5 attenuates both transcription and neuronal differentiation mediated by Neurogenin 2, a transcription factor expressed in adult hippocampal neural progenitor cells. By contrast, ectopic activation of endogenous ERK5 signaling via expression of constitutive active MEK5, an upstream activating kinase for ERK5, promotes neurogenesis in cultured aNPCs and in the dentate gyrus of the mouse brain. Moreover, neurotrophins including NT3 activate ERK5 and stimulate neuronal differentiation in aNPCs in an ERK5-dependent manner. Finally, inducible and conditional deletion of ERK5 specifically in the neurogenic regions of the adult mouse brain delays the normal progression of neuronal differentiation and attenuates adult neurogenesis in vivo. These data suggest ERK5 signaling as a critical regulator of adult hippocampal neurogenesis. PMID:22645146

Myostatin inhibits porcine intramuscular preadipocyte differentiation in vitro.

PubMed

Sun, W X; Dodson, M V; Jiang, Z H; Yu, S G; Chu, W W; Chen, J

2016-04-01

This study assessed the effect of myostatin on adipogenesis by porcine intramuscular preadipocytes. Intramuscular preadipocytes were isolated from the longissimus dorsi muscle of newborn pigs. Myostatin inhibited intramuscular preadipocyte differentiation in a dose-dependent manner. Myostatin treatment during preadipocyte differentiation significantly (P < 0.05) inhibited the expression of the adipogenic marker genes CCAAT/enhancer-binding protein β, CCAAT/enhancer-binding protein α, peroxisome proliferator-activated receptor γ, sterol regulatory element-binding protein-1c, fatty acid-binding protein, and adiponectin. Myostatin also significantly (P < 0.05) reduced the release of glycerol and decreased both adipose triglyceride lipase and hormone-sensitive lipase expression in intramuscular adipocytes. Our study suggests that myostatin acts as an extrinsic regulatory factor in regulating intramuscular adipogenesis. Copyright © 2016 Elsevier Inc. All rights reserved.

A central role for Notch in effector CD8+ T cell differentiation

PubMed Central

Backer, Ronald A.; Helbig, Christina; Gentek, Rebecca; Kent, Andrew; Laidlaw, Brian J.; Dominguez, Claudia X.; de Souza, Yevan S.; van Trierum, Stella E.; van Beek, Ruud; Rimmelzwaan, Guus F.; ten Brinke, Anja; Willemsen, A. Marcel; van Kampen, Antoine H. C.; Kaech, Susan M.; Blander, J. Magarian; van Gisbergen, Klaas; Amsen, Derk

2014-01-01

Activated CD8+ T cells choose between terminal effector cell (TEC) or memory precursor cell (MPC) fates. We show that Notch controls this choice. Notch promoted differentiation of immediately protective TECs and was correspondingly required for clearance of an acute influenza virus infection. Notch activated a major portion of the TEC-specific gene expression program and suppressed the MPC-specific program. Expression of Notch receptors was induced on naïve CD8+ T cells by inflammatory mediators and interleukin 2 (IL-2) via mTOR and T-bet dependent pathways. These pathways were subsequently amplified downstream of Notch, creating a positive feedback loop. Notch thus functions as a central hub where information from different sources converges to match effector T cell differentiation to the demands of the infection. PMID:25344724

Dectin-1 diversifies Aspergillus fumigatus–specific T cell responses by inhibiting T helper type 1 CD4 T cell differentiation

PubMed Central

Hohl, Tobias M.; Collins, Nichole; Leiner, Ingrid; Gallegos, Alena; Saijo, Shinobu; Coward, Jesse W.; Iwakura, Yoichiro

2011-01-01

Pulmonary infection of mice with Aspergillus fumigatus induces concurrent T helper type 1 (Th1) and Th17 responses that depend on Toll-like receptor/MyD88 and Dectin-1, respectively. However, the mechanisms balancing Th1 and Th17 CD4 T cell populations during infection remain incompletely defined. In this study, we show that Dectin-1 deficiency disproportionally increases Th1 responses and decreases Th17 differentiation after A. fumigatus infection. Dectin-1 signaling in A. fumigatus–infected wild-type mice reduces IFN-γ and IL-12p40 expression in the lung, thereby decreasing T-bet expression in responding CD4 T cells and enhancing Th17 responses. Absence of IFN-γ or IL-12p35 in infected mice or T-bet in responding CD4 T cells enhances Th17 differentiation, independent of Dectin-1 expression, in A. fumigatus–infected mice. Transient deletion of monocyte-derived dendritic cells also reduces Th1 and boosts Th17 differentiation of A. fumigatus–specific CD4 T cells. Our findings indicate that Dectin-1–mediated signals alter CD4 T cell responses to fungal infection by decreasing the production of IL-12 and IFN-γ in innate cells, thereby decreasing T-bet expression in A. fumigatus–specific CD4 T cells and enabling Th17 differentiation. PMID:21242294

Communication-dependent mineralization of osteoblasts via gap junctions.

PubMed

Hashida, Yukihiko; Nakahama, Ken-ichi; Shimizu, Kaori; Akiyama, Masako; Harada, Kiyoshi; Morita, Ikuo

2014-04-01

Connexin43 (Cx43) is a major gap junction (GJ) protein in bone and plays a critical role in osteoblast differentiation. Several studies show that osteoblast differentiation is delayed by Cx43 ablation. However, the precise mechanism underlying the role of Cx43 in osteoblast differentiation is not fully understood. Firstly, we analyzed the phenotype of a conditional knockout mouse, which was generated by mating of an osterix promoter-driven Cre expressing mouse with a Cx43-floxed mouse. As expected, delayed ossification was observed. Secondly, we demonstrated that the cell communication via gap junctions played an important role in osteoblast differentiation using a tamoxifen-inducible knockout system in vitro. Genetic ablation of Cx43 resulted in both the disruption of cell-communications and the attenuation of osteoblast mineralization induced by BMP-2, but not by ascorbic acid. Moreover, restoring full-length Cx43 (382aa) expression rescued the impairment of osteoblast cell-communication and osteoblast mineralization; however, the expression of the Cx43 N-terminal mutant (382aaG2V) did not rescue either of them. Comparing the gene expression profiles, the genes directly regulated by BMP-2 were attenuated by Cx43 gene ablation. These results suggested that the cell-communication mediated by gap junctions was indispensable for normal differentiation of osteoblast induced by BMP-2. Copyright © 2013 Elsevier Inc. All rights reserved.

Effect of anomalous tbW vertex on decay-lepton distributions in e+ e-® tt(bar) and CP-violating asymmetries

NASA Astrophysics Data System (ADS)

Rindani, Saurabh D.

2000-06-01

We obtain analytic expressions for the energy and polar-angle double differential distributions of a secondary lepton l+(l-) arising from the decay of t (tbar) in with an anomalous tbW decay vertex. We also obtain analytic expressions for the various differential cross-sections with the lepton energy integrated over. In this case, we find that the angular distributions of the secondary lepton do not depend on the anomalous coupling in the decay, regardless of possible anomalous couplings occurring in the production amplitude for . Our study includes the effect of longitudinal e- and e+ beam polarization. We also study the lepton energy and beam polarization dependence of certain CP-violating lepton angular asymmetries arising from an anomalous tbW decay vertex and compare them with the asymmetries arising due to CP-violation in the production process due to the top electric or weak dipole moment.

RIP2: A novel player in the regulation of keratinocyte proliferation and cutaneous wound repair?

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

Adams, Stephanie; Valchanova, Ralitsa S.; Munz, Barbara, E-mail: barbara.munz@charite.de

2010-03-10

We could recently demonstrate an important role of receptor interacting protein 4 (RIP4) in the regulation of keratinocyte differentiation. Now, we analyzed a potential role of the RIP4 homolog RIP2 in keratinocytes. Specifically, we demonstrate here that rip2 expression is induced by scratch-wounding and after the induction of differentiation in these cells. Furthermore, serum growth factors and cytokines can induce rip2, with TNF-{alpha}-dependent induction being dependent on p38 MAPK. In addition, we demonstrate that scratch-induced upregulation of rip2 expression is completely blocked by the steroid dexamethasone. Since we also show that RIP2 is an important player in the regulation ofmore » keratinocyte proliferation, these data suggest that inhibition of rip2 upregulation after wounding might contribute to the reduced and delayed wound re-epithelialization phenotype seen in glucocorticoid-treated patients.« less

Heterocellular interaction enhances recruitment of {alpha} and {beta}-catenins and ZO-2 into functional gap-junction complexes and induces gap junction-dependant differentiation of mammary epithelial cells

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

Talhouk, Rabih S.; Mroue, Rana; Mokalled, Mayssa

2008-11-01

Gap junctions (GJ) are required for mammary epithelial differentiation. Using epithelial (SCp2) and myoepithelial-like (SCg6) mouse-derived mammary cells, the role of heterocellular interaction in assembly of GJ complexes and functional differentiation ({beta}-casein expression) was evaluated. Heterocellular interaction is critical for {beta}-casein expression, independent of exogenous basement membrane or cell anchoring substrata. Functional differentiation of SCp2, co-cultured with SCg6, is more sensitive to GJ inhibition relative to homocellular SCp2 cultures differentiated by exogenous basement membrane. Connexin (Cx)32 and Cx43 levels were not regulated across culture conditions; however, GJ functionality was enhanced under differentiation-permissive conditions. Immunoprecipitation studies demonstrated association of junctional complexmore » components ({alpha}-catenin, {beta}-catenin and ZO-2) with Cx32 and Cx43, in differentiation conditions, and additionally with Cx30 in heterocellular cultures. Although {beta}-catenin did not shuttle between cadherin and GJ complexes, increased association between connexins and {beta}-catenin in heterocellular cultures was observed. This was concomitant with reduced nuclear {beta}-catenin, suggesting that differentiation in heterocellular cultures involves sequestration of {beta}-catenin in GJ complexes.« less

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

Genome-wide analysis of miRNAs in the ovaries of Jining Grey and Laiwu Black goats to explore the regulation of fecundity.

PubMed

Miao, Xiangyang; Luo, Qingmiao; Zhao, Huijing; Qin, Xiaoyu

2016-11-29

Goat fecundity is important for agriculture and varies depending on the genetic background of the goat. Two excellent domestic breeds in China, the Jining Grey and Laiwu Black goats, have different fecundity and prolificacies. To explore the potential miRNAs that regulate the expression of the genes involved in these prolific differences and to potentially discover new miRNAs, we performed a genome-wide analysis of the miRNAs in the ovaries from these two goats using RNA-Seq technology. Thirty miRNAs were differentially expressed between the Jining Grey and Laiwu Black goats. Gene Ontology and KEGG pathway analyses revealed that the target genes of the differentially expressed miRNAs were significantly enriched in several biological processes and pathways. A protein-protein interaction analysis indicated that the miRNAs and their target genes were related to the reproduction complex regulation network. The differential miRNA expression profiles found in the ovaries between the two distinctive breeds of goats studied here provide a unique resource for addressing fecundity differences in goats.

Interleukins 1alpha and 1beta secreted by some melanoma cell lines strongly reduce expression of MITF-M and melanocyte differentiation antigens.

PubMed

Kholmanskikh, Olga; van Baren, Nicolas; Brasseur, Francis; Ottaviani, Sabrina; Vanacker, Julie; Arts, Nathalie; van der Bruggen, Pierre; Coulie, Pierre; De Plaen, Etienne

2010-10-01

We report that melanoma cell lines expressing the interleukin-1 receptor exhibit 4- to 10-fold lower levels of mRNA of microphthalmia-associated transcription factor (MITF-M) when treated with interleukin-1beta. This effect is NF-kappaB and JNK-dependent. MITF-M regulates the expression of melanocyte differentiation genes such as MLANA, tyrosinase and gp100, which encode antigens recognized on melanoma cells by autologous cytolytic T lymphocytes. Accordingly, treating some melanoma cells with IL-1beta reduced by 40-100% their ability to activate such antimelanoma cytolytic T lymphocytes. Finally, we observed large amounts of biologically active IL-1alpha or IL-1beta secreted by two melanoma cell lines that did not express MITF-M, suggesting an autocrine MITF-M downregulation. We estimate that approximately 13% of melanoma cell lines are MITF-M-negative and secrete IL-1 cytokines. These results indicate that the repression of melanocyte-differentiation genes by IL-1 produced by stromal cells or by tumor cells themselves may represent an additional mechanism of melanoma immune escape.

Host- and stage-dependent secretome of the arbuscular mycorrhizal fungus Rhizophagus irregularis.

PubMed

Zeng, Tian; Holmer, Rens; Hontelez, Jan; Te Lintel-Hekkert, Bas; Marufu, Lucky; de Zeeuw, Thijs; Wu, Fangyuan; Schijlen, Elio; Bisseling, Ton; Limpens, Erik

2018-05-01

Arbuscular mycorrhizal fungi form the most wide-spread endosymbiosis with plants. There is very little host specificity in this interaction, however host preferences as well as varying symbiotic efficiencies have been observed. We hypothesize that secreted proteins (SPs) may act as fungal effectors to control symbiotic efficiency in a host-dependent manner. Therefore, we studied whether arbuscular mycorrhizal (AM) fungi adjust their secretome in a host- and stage-dependent manner to contribute to their extremely wide host range. We investigated the expression of SP-encoding genes of Rhizophagus irregularis in three evolutionary distantly related plant species, Medicago truncatula, Nicotiana benthamiana and Allium schoenoprasum. In addition we used laser microdissection in combination with RNA-seq to study SP expression at different stages of the interaction in Medicago. Our data indicate that most expressed SPs show roughly equal expression levels in the interaction with all three host plants. In addition, a subset shows significant differential expression depending on the host plant. Furthermore, SP expression is controlled locally in the hyphal network in response to host-dependent cues. Overall, this study presents a comprehensive analysis of the R. irregularis secretome, which now offers a solid basis to direct functional studies on the role of fungal SPs in AM symbiosis. © 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.

Differential role of the estrogen receptors ESR1 and ESR2 on the regulation of proteins involved with proliferation and differentiation of Sertoli cells from 15-day-old rats.

PubMed

Lucas, Thaís F G; Lazari, Maria Fatima M; Porto, Catarina S

2014-01-25

The aim of the present study was to investigate the role of each estrogen receptors on the regulation of proteins involved with proliferation and differentiation of Sertoli cells from 15-day-old rats. Activation of ESR1 by 17β-estradiol (E2) and ESR1-selective agonist PPT increased CCND1 expression, and this effect was dependent on NF-kB activation. E2 and the ESR2-selective agonist DPN, but not PPT, increased, in a PI3K and CREB-dependent manner, the expression of CDKN1B and the transcription factors GATA-1 and DMRT1. Analyzing the expression of ESR1 and ESR2 in different stages of development of Sertoli cells, we observed that the ESR1/ESR2 ratio decreased with age, and this ratio seems to be important to determine the end of cell proliferation and the start of cell differentiation. In Sertoli cells from 15-day-old rats, the ESR1/ESR2 ratio favors the effect of ESR1 and the activation of this receptor increased [Methyl-(3)H]thymidine incorporation. We propose that in Sertoli cells from 15-day-old rats E2 modulates Sertoli cell proliferation through ESR1/NF-kB-mediated increase of CCND1, and cell cycle exit and differentiation through ESR2/CREB-mediated increase of CDKN1B, GATA-1 and DMRT1. The present study reinforces the important role of estrogen for normal testis development. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

The role of DNA methylation in catechol-enhanced erythroid differentiation of K562 cells

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

Li, Xiao-Fei; Wu, Xiao-Rong; Xue, Ming

2012-11-15

Catechol is one of phenolic metabolites of benzene in vivo. Catechol is also widely used in pharmaceutical and chemical industries. In addition, fruits, vegetables and cigarette smoke also contain catechol. Our precious study showed that several benzene metabolites (phenol, hydroquinone, and 1,2,4-benzenetriol) inhibited erythroid differentiation of K562 cells. In present study, the effect of catechol on erythroid differentiation of K562 cells was investigated. Moreover, to address the role of DNA methylation in catechol-induced effect on erythroid differentiation in K562 cells, methylation levels of erythroid-specific genes were analyzed by Quantitative MassARRAY methylation analysis platform. Benzidine staining showed that exposure to catecholmore » enhanced hemin-induced hemoglobin accumulation in K562 cells in concentration- and time-dependent manners. The mRNA expression of erythroid specific genes, including α-globin, β-globin, γ-globin, erythroid 5-aminolevulinate synthase, erythroid porphobilinogen deaminase, and transcription factor GATA-1 genes, showed a significant concentration-dependent increase in catechol-treated K562 cells. The exposure to catechol caused a decrease in DNA methylation levels at a few CpG sites in some erythroid specific genes including α-globin, β-globin and erythroid porphobilinogen deaminase genes. These results indicated that catechol improved erythroid differentiation potency of K562 cells at least partly via up-regulating transcription of some erythroid related genes, and suggested that inhibition of DNA methylation might be involved in up-regulated expression of some erythroid related genes. -- Highlights: ► Catechol enhanced hemin-induced hemoglobin accumulation. ► Exposure to catechol resulted in up-regulated expression of erythroid genes. ► Catechol reduced methylation levels at some CpG sites in erythroid genes.« less

In Vivo Exposure to Inorganic Arsenic Alters Differentiation-Specific Gene Expression of Adipose-Derived Mesenchymal Stem/Stromal Cells in C57BL/6J Mouse Model

PubMed Central

Shearer, Joseph J.; Figueiredo Neto, Manoel; Umbaugh, C. Samuel; Figueiredo, Marxa L.

2017-01-01

Abstract The number of mesenchymal stem cell (MSC) therapeutic modalities has grown in recent years. Adipose-derived mesenchymal stem/stromal cells (ASCs) can be isolated and expanded relatively easily as compared with their bone-marrow counterparts, making them a particularly promising source of MSCs. And although the biological mechanisms surrounding ASCs are actively being investigated, little is known about the effects that in vivo environmental exposures might have on their ability to properly differentiate. Therefore, we hypothesized that ASCs isolated from mice exposed to inorganic arsenic (iAs) would have an altered response towards adipogenic, osteogenic, and/or chondrogenic differentiation. To test this hypothesis, C57BL/6J male mice were provided drinking water containing 0, 300, or 1000 ppb iAs. ASCs were then isolated and differentiated, which was assessed by immunocytochemistry and real-time quantitative PCR (RT-qPCR). Our results showed that total urinary arsenic equilibrated within 1 week of exposure to iAs and was maintained throughout the study. ASCs isolated from each exposure group maintained differentiation capabilities for each lineage. The magnitude of differentiation-specific gene expression, however, appeared to be concentration dependent. For osteogenesis and chondrogenesis, differentiation-specific gene expression decreased, whereas adipogenesis showed a biphasic response with an initial decrease followed by an increase in adipogenic-related gene expression following iAs exposure. These results suggest that the level in which differentiation-specific genes are induced within these stromal cells might be sensitive to environmental contaminants. These findings highlight the need to take into account potential environmental exposures prior to selecting stromal cell donors, so ASCs can achieve optimal efficiency in regenerative therapy applications. PMID:28206643

ERK1/2 mediates glucose-regulated POMC gene expression in hypothalamic neurons.

PubMed

Zhang, Juan; Zhou, Yunting; Chen, Cheng; Yu, Feiyuan; Wang, Yun; Gu, Jiang; Ma, Lian; Ho, Guyu

2015-04-01

Hypothalamic glucose-sensing neurons regulate the expression of genes encoding feeding-related neuropetides POMC, AgRP, and NPY - the key components governing metabolic homeostasis. AMP-activated protein kinase (AMPK) is postulated to be the molecular mediator relaying glucose signals to regulate the expression of these neuropeptides. Whether other signaling mediator(s) plays a role is not clear. In this study, we investigated the role of ERK1/2 using primary hypothalamic neurons as the model system. The primary neurons were differentiated from hypothalamic progenitor cells. The differentiated neurons possessed the characteristic neuronal cell morphology and expressed neuronal post-mitotic markers as well as leptin-regulated orexigenic POMC and anorexigenic AgRP/NPY genes. Treatment of cells with glucose dose-dependently increased POMC and decreased AgRP/NPY expression with a concurrent suppression of AMPK phosphorylation. In addition, glucose treatment dose-dependently increased the ERK1/2 phosphorylation. Blockade of ERK1/2 activity with its specific inhibitor PD98059 partially (approximately 50%) abolished glucose-induced POMC expression, but had little effect on AgRP/NPY expression. Conversely, blockade of AMPK activity with its specific inhibitor produced a partial (approximately 50%) reversion of low-glucose-suppressed POMC expression, but almost completely blunted the low-glucose-induced AgRP/NPY expression. The results indicate that ERK1/2 mediated POMC but not AgRP/NPY expression. Confirming the in vitro findings, i.c.v. administration of PD98059 in rats similarly attenuated glucose-induced POMC expression in the hypothalamus, but again had little effect on AgRP/NPY expression. The results are indicative of a novel role of ERK1/2 in glucose-regulated POMC expression and offer new mechanistic insights into hypothalamic glucose sensing. © 2015 Society for Endocrinology.

TPA induces a block of differentiation and increases the susceptibility to neoplastic transformation of a rat thyroid epithelial cell line.

PubMed

Portella, G; Vitagliano, D; Li, Z; Sferratore, F; Santoro, M; Vecchio, G; Fusco, A

1998-01-01

The PC Cl 3 cell line is a well-characterized epithelial cell line of rat thyroid origin. This cell line retains in vitro the typical markers of thyroid differentiation: thyroglobulin (TG) synthesis and secretion, iodide uptake, thyroperoxidase (TPO) expression, and dependency on TSH for growth. Although the differentiated phenotype of thyroid cells has been relatively well described, the molecular mechanisms that regulate both differentiation and neoplastic transformation of thyroid cells still need to be investigated in detail. Protein kinase C (PKC), the target of tetradecanoylphorbol acetate (TPA), regulates growth and differentiation of several cell types. Here we show that treatment of PC Cl 3 cells with TPA induces an acute block of thyroid differentiation. TPA-treated PC Cl 3 cells are unable to trap iodide and the expression levels of thyroglobulin, TSH receptor, and TPO genes are drastically reduced by TPA treatment. This differentiation block is not caused by a reduced expression of one of the master genes of thyroid differentiation, the thyroid transcription factor 1 (TTF-1). TPA-treated PC Cl 3 cells display an increased growth rate indicating that, in addition to the differentiation block, TPA also significantly affects the growth regulation of thyroid cells. Finally, TPA treatment dramatically increases the number of transformation foci induced in PC Cl 3 cells by retroviruses carrying v-Ki-ras, v-Ha-ras, and v-mos oncogenes. These findings support the notion that the PKC pathway can influence proliferation, differentiation, and neoplastic transformation of thyroid cells in culture.

Biphasic effects of FGF2 on odontoblast differentiation involve changes in the BMP and Wnt signaling pathways.

PubMed

Sagomonyants, Karen; Mina, Mina

2014-08-01

Odontoblast differentiation during physiological and reparative dentinogenesis is dependent upon multiple signaling molecules, including fibroblast growth factors (FGFs), bone morphogenetic proteins (BMPs) and Wingless/Integrated (Wnt) ligands. Recent studies in our laboratory showed that continuous exposure of primary dental pulp cultures to FGF2 exerted biphasic effects on the expression of markers of dentinogenesis. In the present study, we examined the possible involvement of the BMP and Wnt signaling pathways in mediating the effects of FGF2 on dental pulp cells. Our results showed that stimulatory effects of FGF2 on dentinogenesis during the proliferation phase of growth were associated with increased expression of the components of the BMP (Bmp2, Dlx5, Msx2, Osx) and Wnt (Wnt10a, Wisp2) pathways, and decreased expression of an inhibitor of the Wnt signaling, Nkd2. Further addition of FGF2 during the differentiation/mineralization phase of growth resulted in decreased expression of components of the BMP signaling (Bmp2, Runx2, Osx) and increased expression of inhibitors of the Wnt signaling (Nkd2, Dkk3). This suggests that both BMP and Wnt pathways may be involved in mediating the effects of FGF2 on dental pulp cells.

Retinoids Modulate Expression of the Endocytic Partners Megalin, Cubilin, and Disabled-2 and Uptake of Vitamin D-Binding Protein in Human Mammary Cells12

PubMed Central

Chlon, Timothy M.; Taffany, David A.; Welsh, JoEllen; Rowling, Matthew J.

2008-01-01

The major circulating form of vitamin D, 25-hydroxycholecalciferol (25D3), circulates bound to vitamin D-binding protein (DBP). Prior to activation to 1,25-dihydroxycholecalciferol in the kidney, the 25D3-DBP complex is internalized via receptor-mediated endocytosis, which is absolutely dependent on the membrane receptors megalin and cubilin and the adaptor protein disabled-2 (Dab2). We recently reported that mammary epithelial cells (T-47D) expressing megalin, cubilin, and Dab2 rapidly internalize DBP via endocytosis, whereas cells that do not express all 3 proteins (MCF-7) do not. The objectives of this study were to characterize megalin, cubilin, and Dab2 expression and transport of DBP in human mammary epithelial cells. Using immunoblotting and real-time PCR, we found that megalin, cubilin, and Dab2 were expressed and dose dependently induced by all-trans-retinoic acid (RA) in T-47D human breast cancer cells and that RA-treated T-47D cells exhibited enhanced DBP internalization. These are the first studies to our knowledge to demonstrate that mammary epithelial cells express megalin, cubilin, and Dab2, which are enhanced during differentiation and may explain, at least in part, our finding that receptor-mediated endocytosis of DBP is upregulated in differentiated mammary epithelial cells. PMID:18567755

Dual Role of Cyanidin-3-glucoside on the Differentiation of Bone Cells.

PubMed

Park, K H; Gu, D R; So, H S; Kim, K J; Lee, S H

2015-12-01

Cyanidin-3-glucoside (C3G) is one of the major components of anthocyanin, a water-soluble phytochemical. Recent studies demonstrated the chemopreventive and chemotherapeutic activities of C3G in various conditions, including cancer, although the precise effects of C3G on osteoclast and osteoblast differentiation remain unclear. Here, we investigated the role of C3G in the differentiation of bone-associated cells and its underlying mechanism. C3G inhibited receptor activator of nuclear factor kappa-B ligand (RANKL)-mediated osteoclast differentiation and formation in a dose-dependent manner and downregulated the expression of osteoclast differentiation marker genes. Pretreatment with C3G considerably reduced the induction of extracellular signal-regulated kinase, c-Jun N-terminal kinase, and p38 mitogen-activated kinases activation by RANKL in osteoclast precursor cells. Furthermore, C3G dramatically inhibited the expression of c-Fos and nuclear factor of activated T-cells, cytoplasmic 1, which are important transcription factors for osteoclast differentiation and activation. The formation of osteoclasts in coculture of bone marrow cells and calvaria-derived osteoblasts was also inhibited by C3G treatment, although the expression of macrophage colony-stimulating factor and RANKL (master factors for osteoclast differentiation and formation) and osteoprotegerin (a decoy receptor for RANKL) on osteoblasts was unaffected. The inhibitory effect of C3G on osteoclastogenesis is therefore targeted specifically to osteoclasts but not osteoblasts. Moreover, analysis of the expression levels of osteoblast differentiation marker genes and alizarin red staining showed that osteoblast differentiation and matrix formation increased after C3G treatment. Taken together, these results strongly suggest that C3G has a dual role in bone metabolism, as an effective inhibitor of osteoclast differentiation but also as an activator of osteoblast differentiation. Therefore, C3G may be used as a potent preventive or therapeutic agent for bone-related diseases, such as osteoporosis, rheumatoid arthritis, and periodontitis. © International & American Associations for Dental Research 2015.

PPARγ ligand production is tightly linked to clonal expansion during initiation of adipocyte differentiation[S

PubMed Central

Hallenborg, Philip; Petersen, Rasmus Koefoed; Feddersen, Søren; Sundekilde, Ulrik; Hansen, Jacob B.; Blagoev, Blagoy; Madsen, Lise; Kristiansen, Karsten

2014-01-01

Adipocyte differentiation is orchestrated by the ligand-activated nuclear receptor PPARγ. Endogenous ligands comprise oxidized derivatives of arachidonic acid and structurally similar PUFAs. Although expression of PPARγ peaks in mature adipocytes, ligands are produced primarily at the onset of differentiation. Concomitant with agonist production, murine fibroblasts undergo two rounds of mitosis referred to as mitotic clonal expansion. Here we show that mouse embryonic fibroblasts deficient in either of two cell cycle inhibitors, the transcription factor p53 or its target gene encoding the cyclin-dependent kinase inhibitor p21, exhibit increased adipogenic potential. The antiadipogenic effect of p53 relied on its transcriptional activity and p21 expression but was circumvented by administration of an exogenous PPARγ agonist suggesting a linkage between cell cycling and PPARγ ligand production. Indeed, cell cycle inhibitory compounds decreased PPARγ ligand production in differentiating 3T3-L1 preadipocytes. Furthermore, these inhibitors abolished the release of arachidonic acid induced by the hormonal cocktail initiating adipogenesis. Collectively, our results suggest that murine fibroblasts require clonal expansion for PPARγ ligand production at the onset of adipocyte differentiation. PMID:25312885

Genome-wide Analysis of the H3K4 Histone Demethylase RBP2 Reveals a Transcriptional Program Controlling Differentiation

PubMed Central

Lopez-Bigas, Nuria; Kisiel, Tomasz A.; DeWaal, Dannielle C.; Holmes, Katie B.; Volkert, Tom L.; Gupta, Sumeet; Love, Jennifer; Murray, Heather L.; Young, Richard A.; Benevolenskaya, Elizaveta V.

2010-01-01

SUMMARY Retinoblastoma protein (pRB) mediates cell-cycle withdrawal and differentiation by interacting with a variety of proteins. RB-Binding Protein 2 (RBP2) has been shown to be a key effector. We sought to determine transcriptional regulation by RBP2 genome-wide by using location analysis and gene expression profiling experiments. We describe that RBP2 shows high correlation with the presence of H3K4me3 and its target genes are separated into two functionally distinct classes: differentiation-independent and differentiation-dependent genes. The former class is enriched by genes that encode mitochondrial proteins, while the latter is represented by cell-cycle genes. We demonstrate the role of RBP2 in mitochondrial biogenesis, which involves regulation of H3K4me3-modified nucleosomes. Analysis of expression changes upon RBP2 depletion depicted genes with a signature of differentiation control, analogous to the changes seen upon reintroduction of pRB. We conclude that, during differentiation, RBP2 exerts inhibitory effects on multiple genes through direct interaction with their promoters. PMID:18722178

Vitamin E isomer δ-tocopherol enhances the efficiency of neural stem cell differentiation via L-type calcium channel.

PubMed

Deng, Sihao; Hou, Guoqiang; Xue, Zhiqin; Zhang, Longmei; Zhou, Yuye; Liu, Chao; Liu, Yanqing; Li, Zhiyuan

2015-01-12

The effects of the vitamin E isomer δ-tocopherol on neural stem cell (NSC) differentiation have not been investigated until now. Here we investigated the effects of δ-tocopherol on NSC neural differentiation, maturation and its possible mechanisms. Neonatal rat NSCs were grown in suspended neurosphere cultures, and were identified by their expression of nestin protein and their capacity for self-renewal. Treatment with a low concentration of δ-tocopherol induced a significant increase in the percentage of β-III-tubulin-positive cells. δ-Tocopherol also stimulated morphological maturation of neurons in culture. We further observed that δ-tocopherol stimulation increased the expression of voltage-dependent Ca(2+) channels. Moreover, a L-type specific Ca(2+) channel blocker verapamil reduced the percentage of differentiated neurons after δ-tocopherol treatment, and blocked the effects of δ-tocopherol on NSC differentiation into neurons. Together, our study demonstrates that δ-tocopherol may act through elevation of L-type calcium channel activity to increase neuronal differentiation. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Effects of nutrient deprivation and differentiation on the expression of growth-arrest genes (gas and gadd) in F9 embryonal carcinoma cells.

PubMed Central

Fleming, J V; Hay, S M; Harries, D N; Rees, W D

1998-01-01

The growth-arrest genes (gas and gadd) are widely expressed during mammalian embryogenesis and may be useful as markers of nutritional stress in the embryo. F9 embryonal carcinoma cells have been used to characterize the effect of serum or amino acid deficiency on growth-arrest gene expression in a differentiating embryonic cell. The differentiation markers, homeobox B2 (HoxB2), collagen type IV and laminin B2, were not induced by growth arrest. Treatment with all-trans retinoic acid (RA) produced a dose-dependent increase in alkaline phosphatase activity, which was unchanged in lysine-deficient medium and reduced in low-serum medium. Low-serum medium also reduced HoxB2 expression. There was a transient 2-6-fold increase in mRNAs for C/EBP-beta, gadd153/CHOP-10 and gas5 genes 24 h after transfer to amino-acid-deficient media. The mRNAs for the gas2 and gas6 genes began to rise slowly by 5-10-fold after a delay of approx. 24 h. The transient increases did not occur in low-serum medium where there was a much smaller and slower increase. Differentiation caused 1-2-fold increases in gas2, gas3 and gas6 mRNA levels. The transient overexpression of gas5, gadd153/CHOP-10 and CCAAT-enhancer-binding protein-beta, and the later expression of gas6 mRNAs in response to amino acid deficiency, were not affected by differentiation. RA treatment increased the expression of gas3 and caused gas2 to be transiently overexpressed in amino-acid-deficient medium. Differentiation in serum-deficient medium did not significantly alter the levels of the growth-arrest gene mRNAs. These results show that in F9 cells the growth-arrest genes are expressed sequentially as a result of nutrient stress. PMID:9461558

Effects of nutrient deprivation and differentiation on the expression of growth-arrest genes (gas and gadd) in F9 embryonal carcinoma cells.

PubMed

Fleming, J V; Hay, S M; Harries, D N; Rees, W D

1998-02-15

The growth-arrest genes (gas and gadd) are widely expressed during mammalian embryogenesis and may be useful as markers of nutritional stress in the embryo. F9 embryonal carcinoma cells have been used to characterize the effect of serum or amino acid deficiency on growth-arrest gene expression in a differentiating embryonic cell. The differentiation markers, homeobox B2 (HoxB2), collagen type IV and laminin B2, were not induced by growth arrest. Treatment with all-trans retinoic acid (RA) produced a dose-dependent increase in alkaline phosphatase activity, which was unchanged in lysine-deficient medium and reduced in low-serum medium. Low-serum medium also reduced HoxB2 expression. There was a transient 2-6-fold increase in mRNAs for C/EBP-beta, gadd153/CHOP-10 and gas5 genes 24 h after transfer to amino-acid-deficient media. The mRNAs for the gas2 and gas6 genes began to rise slowly by 5-10-fold after a delay of approx. 24 h. The transient increases did not occur in low-serum medium where there was a much smaller and slower increase. Differentiation caused 1-2-fold increases in gas2, gas3 and gas6 mRNA levels. The transient overexpression of gas5, gadd153/CHOP-10 and CCAAT-enhancer-binding protein-beta, and the later expression of gas6 mRNAs in response to amino acid deficiency, were not affected by differentiation. RA treatment increased the expression of gas3 and caused gas2 to be transiently overexpressed in amino-acid-deficient medium. Differentiation in serum-deficient medium did not significantly alter the levels of the growth-arrest gene mRNAs. These results show that in F9 cells the growth-arrest genes are expressed sequentially as a result of nutrient stress.

Normal neutrophil differentiation and secondary granule gene expression in the EML and MPRO cell lines.

PubMed

Lawson, N D; Krause, D S; Berliner, N

1998-11-01

The EML and MPRO cell lines express a dominant negative retinoic acid receptor alpha that causes a block at specific stages of myelopoiesis. The EML cell line is multipotent and gives rise to erythroid, lymphoid, and myeloid lineages depending on the presence of appropriate cytokines. The MPRO cell line is promyelocytic and undergoes neutrophilic differentiation when induced with all-trans retinoic acid in the presence of granulocyte/macrophage colony-stimulating factor. Previous studies have shown that both of these cell lines undergo morphological differentiation into neutrophils. In this study, we show that unlike other models of neutrophil differentiation such as NB4 and HL60, both EML and MPRO cell lines undergo complete, normal granulocytic differentiation programs. Similar to HL60, MPRO and EML induce expression of CD11b/CD18 and also exhibit downregulation of CD34 on differentiation. In contrast to HL60 and NB4, EML and MPRO cell lines coordinately upregulate secondary granule transcripts for lactoferrin and neutrophil gelatinase. Furthermore, we have confirmed previous observations that serum can induce a low level of differentiation in MPRO cells and that it is possible to grow these cells in serum-free medium, thereby eliminating this effect. Based on these studies, it appears that these lines can serve as a model for normal retinoic acid-induced neutrophil differentiation and provide insight into the role of the retinoic acid-responsive pathway in normal and leukemic myelopoiesis.

Interleukin-9 enhances interleukin-5 receptor expression, differentiation, and survival of human eosinophils.

PubMed

Gounni, A S; Gregory, B; Nutku, E; Aris, F; Latifa, K; Minshall, E; North, J; Tavernier, J; Levit, R; Nicolaides, N; Robinson, D; Hamid, Q

2000-09-15

Interleukin-9 (IL-9) has been implicated in the pathogenesis of allergic disorders. To examine the interaction between IL-9 and eosinophils, we evaluated mature peripheral blood eosinophils for their expression of the specific alpha-subunit of the IL-9 receptor (IL-9R-alpha). The expression of IL-9R-alpha by human eosinophils was detected at the messenger RNA (mRNA) and protein levels by reverse transcriptase-polymerase chain reaction (RT-PCR), flow cytometry, and immunocytochemical analysis, respectively. Functional analyses demonstrated that recombinant human (rh)IL-9 inhibited in vitro peripheral blood human eosinophil apoptosis in a concentration-dependent manner. We then examined the role of IL-9 in eosinophil differentiation using the human cord blood CD34(+) cells and human promyelocytic leukemia cells (HL-60). The addition of IL-9 to CD34(+) cells cultured in IL-3 and IL-5 enhanced eosinophil development, and IL-9 alone induced the expression of IL-5R-alpha. IL-9 also up-regulated the IL-5R-alpha chain cell surface expression during terminal eosinophil differentiation of the HL-60 cell line. Our findings suggest that IL-9 may potentiate in vivo eosinophil function by increasing their survival and IL-5-mediated differentiation and maturation. Taken together, these results suggest a mechanism by which IL-9 potentiates airway and tissue eosinophilia.

ERα inhibited myocardin-induced differentiation in uterine fibroids.

PubMed

Liao, Xing-Hua; Li, Jun-Yan; Dong, Xiu-Mei; Wang, Xiuhong; Xiang, Yuan; Li, Hui; Yu, Cheng-Xi; Li, Jia-Peng; Yuan, Bai-Yin; Zhou, Jun; Zhang, Tong-Cun

2017-01-01

Uterine fibroids, also known as uterine leiomyomas, are a benign tumor of the human uterus and the commonest estrogen-dependent benign tumor found in women. Myocardin is an important transcriptional regulator in smooth and cardiac muscle development. The role of myocardin and its relationship with ERα in uterine fibroids have barely been addressed. We noticed that the expression of myocardin was markedly reduced in human uterine fibroid tissue compared with corresponding normal or adjacent myometrium tissue. Here we reported that myocardin induced the transcription and expression of differentiation markers SM22α and alpha smooth muscle actin (α-SMA) in rat primary uterine smooth muscle cells (USMCs) and this effect was inhibited by ERα. Notably, we showed that, ERα induced expression of proliferation markers PCNA and ki-67 in rat primary USMCs. We also found ERα interacted with myocardin and formed complex to bind to CArG box and inhibit the SM22α promoter activity. Furthermore, ERα inhibited the transcription and expression of myocardin, and reduced the levels of transcription and expression of downstream target SM22α, a SMC differentiation marker. Our data thus provided important and novel insights into how ERα and myocardin interact to control the cell differentiation and proliferation of USMCs. Thus, it may provide potential therapeutic target for uterine fibroids. Copyright © 2016 Elsevier Inc. All rights reserved.

Protein Kinase Inhibitor γ reciprocally regulates osteoblast and adipocyte differentiation by downregulating Leukemia Inhibitory Factor

PubMed Central

Chen, Xin; Hausman, Bryan S.; Luo, Guangbin; Zhou, Guang; Murakami, Shunichi; Rubin, Janet; Greenfield, Edward M.

2013-01-01

The Protein Kinase Inhibitor (Pki) gene family inactivates nuclear PKA and terminates PKA-induced gene expression. We previously showed that Pkig is the primary family member expressed in osteoblasts and that Pkig knockdown increases the effects of parathyroid hormone and isoproterenol on PKA activation, gene expression, and inhibition of apoptosis. Here, we determined whether endogenous levels of Pkig regulate osteoblast differentiation. Pkig is the primary family member in MEFs, murine marrow-derived mesenchymal stem cells, and human mesenchymal stem cells. Pkig deletion increased forskolin-dependent nuclear PKA activation and gene expression and Pkig deletion or knockdown increased osteoblast differentiation. PKA signaling is known to stimulate adipogenesis; however, adipogenesis and osteogenesis are often reciprocally regulated. We found that the reciprocal regulation predominates over the direct effects of PKA since adipogenesis was decreased by Pkig deletion or knockdown. Pkig deletion or knockdown simultaneously increased osteogenesis and decreased adipogenesis in mixed osteogenic/adipogenic medium. Pkig deletion increased PKA-induced expression of Leukemia Inhibitory Factor (Lif) mRNA and LIF protein. LIF neutralizing antibodies inhibited the effects on osteogenesis and adipogenesis of either Pkig deletion in MEFs or PKIγ knockdown in both murine and human mesenchymal stem cells. Collectively, our results show that endogenous levels of Pkig reciprocally regulate osteoblast and adipocyte differentiation and that this reciprocal regulation is mediated in part by LIF. PMID:23963683

The aryl hydrocarbon receptor suppresses osteoblast proliferation and differentiation through the activation of the ERK signaling pathway

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

Yu, Haitao; Du, Yuxuan; Zhang, Xulong

Ahr activation is known to be associated with synovitis and exacerbated rheumatoid arthritis (RA), but its contributions to bone loss have not been completely elucidated. Osteoblast proliferation and differentiation are abnormal at the erosion site in RA. Here, we reported that the expression of Ahr was increased in the hind paws' bone upon collagen-induced arthritis (CIA) in mice, and the levels of Ahr were negatively correlated with bone mineral density (BMD). In addition, immunofluorescent staining showed that the high expression of Ahr was mainly localized in osteoblasts from the CIA mice compared to normal controls. Moreover, the luciferase intensity ofmore » Ahr in the nucleus increased by 12.5% in CIA osteoblasts compared to that in normal controls. In addition, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) activation of the Ahr inhibited pre-osteoblast MC3T3-E1 cellular proliferation and differentiation in a dose-dependent manner. Interestingly, the levels of alkaline phosphatase (ALP) mRNA expression in the osteoblasts of CIA mice were reduced compared to normal controls. In contrast, decreased ALP expression by activated Ahr was completely reversed after pretreatment with an Ahr inhibitor (CH-223191) in MC3T3-E1 cell lines and primary osteoblasts on day 5. Our data further showed that activation of Ahr promoted the phosphorylation of ERK after 5 days. Moreover, Ahr-dependent activation of the ERK signaling pathway decreased the levels of proliferation cells and inhibited ALP activity in MC3T3-E1 cells. These results demonstrated that the high expression of Ahr may suppress osteoblast proliferation and differentiation through activation of the ERK signaling pathway, further enabling bone erosion in CIA mice. - Highlights: • The upregulation of Ahr was localized in osteoblasts of CIA mice. • The overexpression of Ahr suppressed osteoblast development. • The Ahr activated ERK signaling pathway to exacerbate bone erosion.« less

Gene expression underlying enhanced, steroid-dependent auditory sensitivity of hair cell epithelium in a vocal fish.

PubMed

Fergus, Daniel J; Feng, Ni Y; Bass, Andrew H

2015-10-14

Successful animal communication depends on a receiver's ability to detect a sender's signal. Exemplars of adaptive sender-receiver coupling include acoustic communication, often important in the context of seasonal reproduction. During the reproductive summer season, both male and female midshipman fish (Porichthys notatus) exhibit similar increases in the steroid-dependent frequency sensitivity of the saccule, the main auditory division of the inner ear. This form of auditory plasticity enhances detection of the higher frequency components of the multi-harmonic, long-duration advertisement calls produced repetitively by males during summer nights of peak vocal and spawning activity. The molecular basis of this seasonal auditory plasticity has not been fully resolved. Here, we utilize an unbiased transcriptomic RNA sequencing approach to identify differentially expressed transcripts within the saccule's hair cell epithelium of reproductive summer and non-reproductive winter fish. We assembled 74,027 unique transcripts from our saccular epithelial sequence reads. Of these, 6.4 % and 3.0 % were upregulated in the reproductive and non-reproductive saccular epithelium, respectively. Gene ontology (GO) term enrichment analyses of the differentially expressed transcripts showed that the reproductive saccular epithelium was transcriptionally, translationally, and metabolically more active than the non-reproductive epithelium. Furthermore, the expression of a specific suite of candidate genes, including ion channels and components of steroid-signaling pathways, was upregulated in the reproductive compared to the non-reproductive saccular epithelium. We found reported auditory functions for 14 candidate genes upregulated in the reproductive midshipman saccular epithelium, 8 of which are enriched in mouse hair cells, validating their hair cell-specific functions across vertebrates. We identified a suite of differentially expressed genes belonging to neurotransmission and steroid-signaling pathways, consistent with previous work showing the importance of these characters in regulating hair cell auditory sensitivity in midshipman fish and, more broadly, vertebrates. The results were also consistent with auditory hair cells being generally more physiologically active when animals are in a reproductive state, a time of enhanced sensory-motor coupling between the auditory periphery and the upper harmonics of vocalizations. Together with several new candidate genes, our results identify discrete patterns of gene expression linked to frequency- and steroid-dependent plasticity of hair cell auditory sensitivity.

Human Keratinocytes That Express hTERT and Also Bypass a p16INK4a-Enforced Mechanism That Limits Life Span Become Immortal yet Retain Normal Growth and Differentiation Characteristics

PubMed Central

Dickson, Mark A.; Hahn, William C.; Ino, Yasushi; Ronfard, Vincent; Wu, Jenny Y.; Weinberg, Robert A.; Louis, David N.; Li, Frederick P.; Rheinwald, James G.

2000-01-01

Normal human cells exhibit a limited replicative life span in culture, eventually arresting growth by a process termed senescence. Progressive telomere shortening appears to trigger senescence in normal human fibroblasts and retinal pigment epithelial cells, as ectopic expression of the telomerase catalytic subunit, hTERT, immortalizes these cell types directly. Telomerase expression alone is insufficient to enable certain other cell types to evade senescence, however. Such cells, including keratinocytes and mammary epithelial cells, appear to require loss of the pRB/p16INK4a cell cycle control mechanism in addition to hTERT expression to achieve immortality. To investigate the relationships among telomerase activity, cell cycle control, senescence, and differentiation, we expressed hTERT in two epithelial cell types, keratinocytes and mesothelial cells, and determined the effect on proliferation potential and on the function of cell-type-specific growth control and differentiation systems. Ectopic hTERT expression immortalized normal mesothelial cells and a premalignant, p16INK4a-negative keratinocyte line. In contrast, when four keratinocyte strains cultured from normal tissue were transduced to express hTERT, they were incompletely rescued from senescence. After reaching the population doubling limit of their parent cell strains, hTERT+ keratinocytes entered a slow growth phase of indefinite length, from which rare, rapidly dividing immortal cells emerged. These immortal cell lines frequently had sustained deletions of the CDK2NA/INK4A locus or otherwise were deficient in p16INK4a expression. They nevertheless typically retained other keratinocyte growth controls and differentiated normally in culture and in xenografts. Thus, keratinocyte replicative potential is limited by a p16INK4a-dependent mechanism, the activation of which can occur independent of telomere length. Abrogation of this mechanism together with telomerase expression immortalizes keratinocytes without affecting other major growth control or differentiation systems. PMID:10648628

Reversible upregulation of tropomyosin-related kinase receptor B by geranylgeranoic acid in human neuroblastoma SH-SY5Y cells.

PubMed

Sakane, Chiharu; Shidoji, Yoshihiro

2011-09-01

All-trans retinoic acid (ATRA) plays crucial roles in cell survival and differentiation of neuroblastoma cells. In the present study, we investigated the effects of geranylgeranoic acid (GGA), an acyclic retinoid, on differentiation and tropomyosin-related kinase receptor B (TrkB) gene expression in SH-SY5Y human neuroblastoma cells in comparison with ATRA. GGA induced growth suppression and neural differentiation to the same extent as ATRA. Two variants (145 and 95 kD) of the TrkB protein were dramatically increased by GGA treatment, comparable to the effect of ATRA. Following 6- to 8-day GGA treatment, the effect of GGA on TrkB was reversed after 2-4 days of its removal, whereas the effect of ATRA was irreversible under the same conditions. Both GGA and ATRA upregulated the cellular levels of three major TrkB messenger RNA splice variants in a time-dependent manner. Time-dependent induction of cell cycle-related genes, such as cyclin D1 and retinoblastoma protein, and amplification of the neural progenitor cell marker, brain lipid binding protein, were suppressed by GGA treatment and were completely abolished by ATRA. ATRA and GGA induced retinoic acid receptor β (RARβ) expression, whereas the time-dependent expression of both RARα and RARγ was abolished by ATRA, but not by GGA. Our results suggest that GGA may be able to restore neuronal properties of SH-SY5Y human neuroblastoma cells in a similar but not identical way to ATRA.

cDNA cloning and functional characterization of the mouse Ca2+-gated K+ channel, mIK1. Roles in regulatory volume decrease and erythroid differentiation.

PubMed

Vandorpe, D H; Shmukler, B E; Jiang, L; Lim, B; Maylie, J; Adelman, J P; de Franceschi, L; Cappellini, M D; Brugnara, C; Alper, S L

1998-08-21

We have cloned from murine erythroleukemia (MEL) cells, thymus, and stomach the cDNA encoding the Ca2+-gated K+ (KCa) channel, mIK1, the mouse homolog of hIK1 (Ishii, T. M., Silvia, C., Hirschberg, B., Bond, C. T., Adelman, J. P., and Maylie, J. (1997) Proc. Natl. Acad. Sci.(U. S. A. 94, 11651-11656). mIK1 mRNA was detected at varied levels in many tissue types. mIK1 KCa channel activity expressed in Xenopus oocytes closely resembled the Kca of red cells (Gardos channel) and MEL cells in its single channel conductance, lack of voltage-sensitivity of activation, inward rectification, and Ca2+ concentration dependence. mIK1 also resembled the erythroid channel in its pharmacological properties, mediating whole cell and unitary currents sensitive to low nM concentrations of both clotrimazole (CLT) and its des-imidazolyl metabolite, 2-chlorophenyl-bisphenyl-methanol, and to low nM concentrations of iodocharybdotoxin. Whereas control oocytes subjected to hypotonic swelling remained swollen, mIK1 expression conferred on oocytes a novel, Ca2+-dependent, CLT-sensitive regulatory volume decrease response. Hypotonic swelling of voltage-clamped mIK1-expressing oocytes increased outward currents that were Ca2+-dependent, CLT-sensitive, and reversed near the K+ equilibrium potential. mIK1 mRNA levels in ES cells increased steadily during erythroid differentiation in culture, in contrast to other KCa mRNAs examined. Low nanomolar concentrations of CLT inhibited proliferation and erythroid differentiation of peripheral blood stem cells in liquid culture.

Oxidative phosphorylation in Debaryomyces hansenii: physiological uncoupling at different growth phases.

PubMed

Cabrera-Orefice, Alfredo; Guerrero-Castillo, Sergio; Díaz-Ruíz, Rodrigo; Uribe-Carvajal, Salvador

2014-07-01

Physiological uncoupling of mitochondrial oxidative phosphorylation (OxPhos) was studied in Debaryomyces hansenii. In other species, such as Yarrowia lipolytica and Saccharomyces cerevisiae, OxPhos can be uncoupled through differential expression of branched respiratory chain enzymes or by opening of a mitochondrial unspecific channel (ScMUC), respectively. However D. hansenii mitochondria, which contain both a branched respiratory chain and a mitochondrial unspecific channel (DhMUC), selectively uncouple complex I-dependent rate of oxygen consumption in the stationary growth phase. The uncoupled complex I-dependent respiration was only 20% of the original activity. Inhibition was not due to inactivation of complex I, lack of protein expression or to differential expression of alternative oxidoreductases. Furthermore, all other respiratory chain activities were normal. Decrease of complex I-dependent respiration was due to NAD(+) loss from the matrix, probably through an open of DhMUC. When NAD(+) was added back, coupled complex I-activity was recovered. NAD(+) re-uptake was independent of DhMUC opening and seemed to be catalyzed by a NAD(+)-specific transporter, which was sensitive to bathophenanthroline, bromocresol purple or pyridoxal-5'-phosphate as described for S. cerevisiae mitochondrial NAD(+) transporters. Loss of NAD(+) from the matrix through an open MUC is proposed as an additional mechanism to uncouple OxPhos. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Zebrafish hhex, nk2.1a, and pax2.1 regulate thyroid growth and differentiation downstream of Nodal-dependent transcription factors.

PubMed

Elsalini, Osama A; von Gartzen, Julia; Cramer, Matthias; Rohr, Klaus B

2003-11-01

During zebrafish development, the thyroid primordium initiates expression of molecular markers such as hhex and nk2.1a in the endoderm prior to pharynx formation. As expected for an endodermally derived organ, initiation of thyroid development depends on Nodal signalling. We find that it also depends on three downstream effectors of Nodal activity, casanova (cas), bonnie and clyde (bon), and faust (fau)/gata5. Despite their early Nodal-dependent expression in the endoderm, both hhex and nk2.1a are only required relatively late during thyroid development. In hhex and nk2.1a loss-of-function phenotypes, thyroid development is initiated and arrests only after the primordium has evaginated from the pharyngeal epithelium. Thus, like pax2.1, both hhex and nk2.1a have similarly late roles in differentiation or growth of thyroid follicular cells, and here, we show that all three genes act in parallel rather than in a single pathway. Our functional analysis suggests that these genes have similar roles as in mammalian thyroid development, albeit in a different temporal mode of organogenesis.

MicroRNA-205 targets tight junction-related proteins during urothelial cellular differentiation.

PubMed

Chung, Pei-Jung Katy; Chi, Lang-Ming; Chen, Chien-Lun; Liang, Chih-Lung; Lin, Chung-Tzu; Chang, Yu-Xun; Chen, Chun-Hsien; Chang, Yu-Sun

2014-09-01

The mammalian bladder urothelium classified as basal, intermediate, and terminally differentiated umbrella cells offers one of the most effective permeability barrier functions known to exist in nature because of the formation of apical uroplakin plaques and tight junctions. To improve our understanding of urothelial differentiation, we analyzed the microRNA (miRNA) expression profiles of mouse urinary tissues and by TaqMan miRNA analysis of microdissected urothelial layers and in situ miRNA-specific hybridization to determine the dependence of these miRNAs on the differentiation stage. Our in situ hybridization studies revealed that miR-205 was enriched in the undifferentiated basal and intermediate cell layers. We then used a quantitative proteomics approach to identify miR-205 target genes in primary cultured urothelial cells subjected to antagomir-mediated knockdown of specific miRNAs. Twenty-four genes were reproducibly regulated by miR-205; eleven of them were annotated as cell junction- and tight junction-related molecules. Western blot analysis demonstrated that antagomir-induced silencing of miR-205 in primary cultured urothelial cells elevated the expression levels of Tjp1, Cgnl1, and Cdc42. Ectopic expression of miR-205 in MDCK cells inhibited the expression of tight junction proteins and the formation of tight junctions. miR-205- knockdown urothelial cells showed alterations in keratin synthesis and increases of uroplakin Ia and Ib, which are the urothelial differentiation products. These results suggest that miR-205 may contribute a role in regulation of urothelial differentiation by modulating the expression of tight junction-related molecules. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

STATs shape the active enhancer landscape of T cell populations.

PubMed

Vahedi, Golnaz; Takahashi, Hayato; Nakayamada, Shingo; Sun, Hong-Wei; Sartorelli, Vittorio; Kanno, Yuka; O'Shea, John J

2012-11-21

Signaling pathways are intimately involved in cellular differentiation, allowing cells to respond to their environment by regulating gene expression. Although enhancers are recognized as key elements that regulate selective gene expression, the interplay between signaling pathways and actively used enhancer elements is not clear. Here, we use CD4(+) T cells as a model of differentiation, mapping the activity of cell-type-specific enhancer elements in T helper 1 (Th1) and Th2 cells. Our data establish that STAT proteins have a major impact on the activation of lineage-specific enhancers and the suppression of enhancers associated with alternative cell fates. Transcriptome analysis further supports a functional role for enhancers regulated by STATs. Importantly, expression of lineage-defining master regulators in STAT-deficient cells fails to fully recover the chromatin signature of STAT-dependent enhancers. Thus, these findings point to a critical role of STATs as environmental sensors in dynamically molding the specialized enhancer architecture of differentiating cells. Copyright © 2012 Elsevier Inc. All rights reserved.

STATs Shape the Active Enhancer Landscape of T Cell Populations

PubMed Central

Vahedi, Golnaz; Takahashi, Hayato; Nakayamada, Shingo; Sun, Hong-wei; Sartorelli, Vittorio; Kanno, Yuka; O’Shea, John J.

2012-01-01

SUMMARY Signaling pathways are intimately involved in cellular differentiation, allowing cells to respond to their environment by regulating gene expression. While enhancers are recognized as key elements that regulate selective gene expression, the interplay between signaling pathways and actively used enhancer elements is not clear. Here, we use CD4+ T cells as a model of differentiation, mapping the acquisition of cell-type-specific enhancer elements in T-helper 1 (Th1) and Th2 cells. Our data establish that STAT proteins have a major impact on the acquisition of lineage-specific enhancers and the suppression of enhancers associated with alternative cell fates. Transcriptome analysis further supports a functional role for enhancers regulated by STATs. Importantly, expression of lineage-defining master regulators in STAT-deficient cells fails to fully recover the chromatin signature of STAT-dependent enhancers. Thus, these findings point to a critical role of STATs as environmental sensors in dynamically molding the specialized enhancer architecture of differentiating cells. PMID:23178119

[Differentiation of human umbilical cord derived mesenchymal stem cells into low immunogenic and functional hepatocyte-like cells in vitro].

PubMed

Ren, Hong-ying; Zhao, Qin-jun; Xing, Wen; Yang, Shao-guang; Lu, Shi-hong; Ren, Qian; Zhang, Lei; Han, Zhong-chao

2010-04-01

To investigate the biological function of hepatocyte-like cells derived from mesenchymal stem cells that isolated from human umbilical cord UC-MSCs in vitro, and to detect the changes in the immunogenicity of the differentiated hepatocyte-like cells (DHC). Transdifferentiation of UC-MSCs into hepatic lineage in vitro was induced in modified two-step induction medium. The expressions of hepatic specific markers were detected by RT-PCR analysis and immunofluorescence staining at different time points after induction. The levels of albumin and urea in the supernatants of cultures were measured by enzyme-linked immunosorbent assay. Furthermore, the immunosuppressive property of DHC was detected by one-way mixed lymphocyte culture. The mRNA and proteins of alpha fetoprotein (AFP), albumin (ALB),and cytokeratin-19 (CK-19) were expressed in naive UC-MSCs at low levels. DHC highly expressed hepatic markers AFP, ALB, CK-19, and tryptophan 2, 3-dioxygenase 14 and 28 days after hepatic differentiation and were accompanied by an increased production of ALB and urea in supernatant in a time-dependent manner. DHC did not express human leukocyte antigen DR antigen and significantly decreased the lymphocyte proliferation. UC-MSCs are able to differentiate into functional hepatocyte-like cells in vitro, while the immunogenicity of DHC remains low.

The expression of native and cultured human retinal pigment epithelial cells grown in different culture conditions.

PubMed

Tian, J; Ishibashi, K; Honda, S; Boylan, S A; Hjelmeland, L M; Handa, J T

2005-11-01

To determine the transcriptional proximity of retinal pigment epithelium (RPE) cells grown under different culture conditions and native RPE. ARPE-19 cells were grown under five conditions in 10% CO(2): "subconfluent" in DMEM/F12+10% FBS, "confluent" in serum and serum withdrawn, and "differentiated" for 2.5 months in serum and serum withdrawn medium. Native RPE was laser microdissected. Total RNA was extracted, reverse transcribed, and radiolabelled probes were hybridised to an array containing 5,353 genes. Arrays were evaluated by hierarchical cluster analysis and significance analysis of microarrays. 78% of genes were expressed by native RPE while 45.3--47.7% were expressed by ARPE-19 cells, depending on culture condition. While the most abundant genes were expressed by native and cultured cells, significant differences in low abundance genes were seen. Hierarchical cluster analysis showed that confluent and differentiated, serum withdrawn cultures clustered closest to native RPE, and that serum segregated cultured cells from native RPE. The number of differentially expressed genes and their function, and profile of expressed and unexpressed genes, demonstrate differences between native and cultured cells. While ARPE-19 cells have significant value for studying RPE behaviour, investigators must be aware of how culture conditions can influence the mRNA phenotype of the cell.

Differentially Expressed Genes Associated with Low-Dose Gamma Radiation

NASA Astrophysics Data System (ADS)

Hegyesi, Hargita; Sándor, Nikolett; Schilling, Boglárka; Kis, Enikő; Lumniczky, Katalin; Sáfrány, Géza

We have studied low dose radiation induced gene expression alterations in a primary human fibroblast cell line using Agilent's whole human genome microarray. Cells were irradiated with 60Co γ-rays (0; 0.1; 0.5 Gy) and 2 hours later total cellular RNA was isolated. We observed differential regulation of approximately 300-500 genes represented on the microarray. Of these, 126 were differentially expressed at both doses, among them significant elevation of GDF-15 and KITLG was confirmed by qRT-PCR. Based on the transcriptional studies we selected GDF-15 to assess its role in radiation response, since GDF-15 is one of the p53 gene targets and is believed to participate in mediating p53 activities. First we confirmed gamma-radiation induced dose-dependent changes in GDF-15 expression by qRT-PCR. Next we determined the effect of GDF-15 silencing on radiosensitivity. Four GDF-15 targeting shRNA expressing lentiviral vectors were transfected into immortalized human fibroblast cells. We obtained efficient GDF-15 silencing in one of the four constructs. RNA interference inhibited GDF-15 gene expression and enhanced the radiosensitivity of the cells. Our studies proved that GDF-15 plays an essential role in radiation response and may serve as a promising target in radiation therapy.

A switch in the mode of Wnt signaling orchestrates the formation of germline stem cell differentiation niche in Drosophila

PubMed Central

Upadhyay, Maitreyi; Kuna, Michael; Tudor, Sara; Martino Cortez, Yesenia

2018-01-01

Germline stem cell (GSC) self-renewal and differentiation into gametes is regulated by both intrinsic factors in the germ line as well as extrinsic factors from the surrounding somatic niche. dWnt4, in the escort cells of the adult somatic niche promotes GSC differentiation using the canonical β-catenin-dependent transcriptional pathway to regulate escort cell survival, adhesion to the germ line and downregulation of self-renewal signaling. Here, we show that in addition to the β-catenin-dependent canonical pathway, dWnt4 also uses downstream components of the Wnt non-canonical pathway to promote escort cell function earlier in development. We find that the downstream non-canonical components, RhoA, Rac1 and cdc42, are expressed at high levels and are active in escort cell precursors of the female larval gonad compared to the adult somatic niche. Consistent with this expression pattern, we find that the non-canonical pathway components function in the larval stages but not in adults to regulate GSC differentiation. In the larval gonad, dWnt4, RhoA, Rac1 and cdc42 are required to promote intermingling of escort cell precursors, a function that then promotes proper escort cell function in the adults. We find that dWnt4 acts by modulating the activity of RhoA, Rac1 and cdc42, but not their protein levels. Together, our results indicate that at different points of development, dWnt4 switches from using the non-canonical pathway components to using a β-catenin-dependent canonical pathway in the escort cells to facilitate the proper differentiation of GSCs. PMID:29370168

Differential Regulation of the Ascorbic Acid Transporter SVCT2 during Development and in Response to Ascorbic Acid Depletion

PubMed Central

Meredith, M. Elizabeth; Harrison, Fiona E.; May, James M.

2011-01-01

The sodium-dependent vitamin C transporter-2 (SVCT2) is the only ascorbic acid (ASC) transporter significantly expressed in brain. It is required for life and critical during brain development to supply adequate levels of ASC. To assess SVCT2 function in the developing brain, we studied time-dependent SVCT2 mRNA and protein expression in mouse brain, using liver as a comparison tissue because it is the site of ASC synthesis. We found that SVCT2 expression followed an inverse relationship with ASC levels in the developing brain. In cortex and cerebellum, ASC levels were high throughout late embryonic stages and early post-natal stages and decreased with age, whereas SVCT2 mRNA and protein levels were low in embryos and increased with age. A different response was observed for liver, in which ASC levels and SVCT2 expression were both low throughout embryogenesis and increased post-natally. To determine whether low intracellular ASC might be capable of driving SVCT2 expression, we depleted ASC by diet in adult mice unable to synthesize ASC. We observed that SVCT2 mRNA and protein were not affected by ASC depletion in brain cortex, but SVCT2 protein expression was increased by ASC depletion in the cerebellum and liver. The results suggest that expression of the SVCT2 is differentially regulated during embryonic development and in adulthood. PMID:22001929

State-dependent differential Riccati equation to track control of time-varying systems with state and control nonlinearities.

PubMed

Korayem, M H; Nekoo, S R

2015-07-01

This work studies an optimal control problem using the state-dependent Riccati equation (SDRE) in differential form to track for time-varying systems with state and control nonlinearities. The trajectory tracking structure provides two nonlinear differential equations: the state-dependent differential Riccati equation (SDDRE) and the feed-forward differential equation. The independence of the governing equations and stability of the controller are proven along the trajectory using the Lyapunov approach. Backward integration (BI) is capable of solving the equations as a numerical solution; however, the forward solution methods require the closed-form solution to fulfill the task. A closed-form solution is introduced for SDDRE, but the feed-forward differential equation has not yet been obtained. Different ways of solving the problem are expressed and analyzed. These include BI, closed-form solution with corrective assumption, approximate solution, and forward integration. Application of the tracking problem is investigated to control robotic manipulators possessing rigid or flexible joints. The intention is to release a general program for automatic implementation of an SDDRE controller for any manipulator that obeys the Denavit-Hartenberg (D-H) principle when only D-H parameters are received as input data. Copyright © 2015 ISA. Published by Elsevier Ltd. All rights reserved.

Differential gene expression by Moniliophthora roreri while overcoming cacao tolerance in the field.

PubMed

Bailey, Bryan A; Melnick, Rachel L; Strem, Mary D; Crozier, Jayne; Shao, Jonathan; Sicher, Richard; Phillips-Mora, Wilberth; Ali, Shahin S; Zhang, Dapeng; Meinhardt, Lyndel

2014-09-01

Frosty pod rot (FPR) of Theobroma cacao (cacao) is caused by the hemibiotrophic fungus Moniliophthora roreri. Cacao clones tolerant to FPR are being planted throughout Central America. To determine whether M. roreri shows a differential molecular response during successful infections of tolerant clones, we collected field-infected pods at all stages of symptomatology for two highly susceptible clones (Pound-7 and CATIE-1000) and three tolerant clones (UF-273, CATIE-R7 and CATIE-R4). Metabolite analysis was carried out on clones Pound-7, CATIE-1000, CATIE-R7 and CATIE-R4. As FPR progressed, the concentrations of sugars in pods dropped, whereas the levels of trehalose and mannitol increased. Associations between symptoms and fungal loads and some organic and amino acid concentrations varied depending on the clone. RNA-Seq analysis identified 873 M. roreri genes that were differentially expressed between clones, with the primary difference being whether the clone was susceptible or tolerant. Genes encoding transcription factors, heat shock proteins, transporters, enzymes modifying membranes or cell walls and metabolic enzymes, such as malate synthase and alternative oxidase, were differentially expressed. The differential expression between clones of 43 M. roreri genes was validated by real-time quantitative reverse transcription polymerase chain reaction. The expression profiles of some genes were similar in susceptible and tolerant clones (other than CATIE-R4) and varied with the biotrophic/necrotropic shift. Moniliophthora roreri genes associated with stress metabolism and responses to heat shock and anoxia were induced early in tolerant clones, their expression profiles resembling that of the necrotrophic phase. Moniliophthora roreri stress response genes, induced during the infection of tolerant clones, may benefit the fungus in overcoming cacao defense mechanisms. © 2014 BSPP AND JOHN WILEY & SONS LTD.

Differential Changes in Expression of Stress- and Metabolic-Related Neuropeptides in the Rat Hypothalamus during Morphine Dependence and Withdrawal

PubMed Central

Núnez, Cristina; Zelei, Edina; Polyák, Ágnes; Milanés, M. Victoria

2013-01-01

Chronic morphine treatment and naloxone precipitated morphine withdrawal activates stress-related brain circuit and results in significant changes in food intake, body weight gain and energy metabolism. The present study aimed to reveal hypothalamic mechanisms underlying these effects. Adult male rats were made dependent on morphine by subcutaneous implantation of constant release drug pellets. Pair feeding revealed significantly smaller weight loss of morphine treated rats compared to placebo implanted animals whose food consumption was limited to that eaten by morphine implanted pairs. These results suggest reduced energy expenditure of morphine-treated animals. Chronic morphine exposure or pair feeding did not significantly affect hypothalamic expression of selected stress- and metabolic related neuropeptides - corticotropin-releasing hormone (CRH), urocortin 2 (UCN2) and proopiomelanocortin (POMC) compared to placebo implanted and pair fed animals. Naloxone precipitated morphine withdrawal resulted in a dramatic weight loss starting as early as 15–30 min after naloxone injection and increased adrenocorticotrophic hormone, prolactin and corticosterone plasma levels in morphine dependent rats. Using real-time quantitative PCR to monitor the time course of relative expression of neuropeptide mRNAs in the hypothalamus we found elevated CRH and UCN2 mRNA and dramatically reduced POMC expression. Neuropeptide Y (NPY) and arginine vasopressin (AVP) mRNA levels were transiently increased during opiate withdrawal. These data highlight that morphine withdrawal differentially affects expression of stress- and metabolic-related neuropeptides in the rat hypothalamus, while relative mRNA levels of these neuropeptides remain unchanged either in rats chronically treated with morphine or in their pair-fed controls. PMID:23805290

Global gene expression under nitrogen starvation in Xylella fastidiosa: contribution of the σ54 regulon

PubMed Central

2010-01-01

Background Xylella fastidiosa, a Gram-negative fastidious bacterium, grows in the xylem of several plants causing diseases such as citrus variegated chlorosis. As the xylem sap contains low concentrations of amino acids and other compounds, X. fastidiosa needs to cope with nitrogen limitation in its natural habitat. Results In this work, we performed a whole-genome microarray analysis of the X. fastidiosa nitrogen starvation response. A time course experiment (2, 8 and 12 hours) of cultures grown in defined medium under nitrogen starvation revealed many differentially expressed genes, such as those related to transport, nitrogen assimilation, amino acid biosynthesis, transcriptional regulation, and many genes encoding hypothetical proteins. In addition, a decrease in the expression levels of many genes involved in carbon metabolism and energy generation pathways was also observed. Comparison of gene expression profiles between the wild type strain and the rpoN null mutant allowed the identification of genes directly or indirectly induced by nitrogen starvation in a σ54-dependent manner. A more complete picture of the σ54 regulon was achieved by combining the transcriptome data with an in silico search for potential σ54-dependent promoters, using a position weight matrix approach. One of these σ54-predicted binding sites, located upstream of the glnA gene (encoding glutamine synthetase), was validated by primer extension assays, confirming that this gene has a σ54-dependent promoter. Conclusions Together, these results show that nitrogen starvation causes intense changes in the X. fastidiosa transcriptome and some of these differentially expressed genes belong to the σ54 regulon. PMID:20799976

Distinct transcriptome profiles differentiate NSAID-dependent from NSAID-independent food anaphylaxis

PubMed Central

Muñoz-Cano, Rosa; Pascal, Mariona; Bartra, Joan; Picado, Cesar; Valero, Antonio; Kim, Do-Kyun; Brooks, Stephen; Ombrello, Michael; Metcalfe, Dean D.; Rivera, Juan; Olivera, Ana

2015-01-01

Background Lipid transfer protein (LTP), an abundant protein in fruits, vegetables and nuts, is a common food allergen in Mediterranean areas causing diverse allergic reactions. Approximately 40% of food anaphylaxis induced by LTP require non-steroidal anti-inflammatory drugs (NSAIDs) as a triggering cofactor. Objective To better understand the determinants of NSAID-dependent (NSAID-LTP-A) and NSAID-independent LTP-anaphylaxis (LTP-A) Methods Selection of patients was based on a proven clinical history of NSAID-dependent or -independent anaphylaxis to LTP, positive skin prick test to LTP and serum LTP-IgE. Whole transcriptome (RNA-Seq) analysis of blood cells from 14 individuals with NSAID-LTP-A, 7 with LTP-A and 13 healthy controls was performed to identify distinct gene expression signatures. Results Expression of genes regulating gastrointestinal epithelium renewal was altered in both patient sets, particularly in LTP-A, who also presented gene expression profiles characteristic of an inflammatory syndrome. These included altered B cell pathways, increased neutrophil activation markers and elevated levels of reactive oxygen species. Increased expression of the IgG receptor (CD64) in LTP-A patients was mirrored by the presence of LTP-specific IgG1 and 3. Conversely, NSAID-LTP-A patients were characterized by reduced expression of IFN-γ-regulated genes and IFN-γ levels as well as up-regulated adenosine receptor 3 (ADORA3) expression and genes related to adenosine metabolism. Conclusions Gene ontology analysis suggests disturbances in gut epithelium homeostasis in both LTP-related anaphylaxis groups with potential integrity breaches in LTP-A that may explain their distinct inflammatory signature. Differential regulation in LTP-A and NSAID-LTP-A of the IFN-γ pathway, IgG receptors and ADORA3 may provide the pathogenic basis of their distinct responses. PMID:26194548

Stressor and Glucocorticoid-Dependent Induction of the Immediate Early Gene Krüppel-Like Factor 9: Implications for Neural Development and Plasticity

PubMed Central

Bonett, Ronald M.; Hu, Fang; Bagamasbad, Pia; Denver, Robert J.

2009-01-01

Krüppel-like factor 9 (KLF9) is a thyroid hormone-induced, immediate early gene implicated in neural development in vertebrates. We analyzed stressor and glucocorticoid (GC)-dependent regulation of KLF9 expression in the brain of the frog Xenopus laevis, and investigated a possible role for KLF9 in neuronal differentiation. Exposure to shaking/confinement stressor increased plasma corticosterone (CORT) concentration, and KLF9 immunoreactivity in several brain regions, which included the medial amygdala and bed nucleus of the stria terminalis, anterior preoptic area (homologous to the mammalian paraventricular nucleus), and optic tectum (homologous to the mammalian superior colliculus). The stressor-induced KLF9 mRNA expression in the brain was blocked by pretreatment with the GC receptor antagonist RU486, or mimicked by injection of CORT. Treatment with CORT also caused a rapid and dose-dependent increase in KLF9 mRNA in X. laevis XTC-2 cells that was resistant to inhibition of protein synthesis. The action of CORT on KLF9 expression in XTC-2 cells was blocked by RU486, but not by the mineralocorticoid receptor antagonist spironolactone. To test for functional consequences of up-regulation of KLF9, we introduced a KLF9 expression plasmid into living tadpole brain by electroporation-mediated gene transfer. Forced expression of KLF9 in tadpole brain caused an increase in Golgi-stained cells, reflective of neuronal differentiation/maturation. Our results support that KLF9 is a direct, GC receptor target gene that is induced by stress, and functions as an intermediary in the actions of GCs on brain gene expression and neuronal structure. PMID:19036875

Chronic ethanol exposure changes dopamine D2 receptor splicing during retinoic acid-induced differentiation of human SH-SY5Y cells.

PubMed

Wernicke, Catrin; Hellmann, Julian; Finckh, Ulrich; Rommelspacher, Hans

2010-01-01

There is evidence for ethanol-induced impairment of the dopaminergic system in the brain during development. The dopamine D2 receptor (DRD2) and the dopamine transporter (DAT) are decisively involved in dopaminergic signaling. Two splice variants of DRD2 are known, with the short one (DRD2s) representing the autoreceptor and the long one (DRD2l) the postsynaptic receptor. We searched for a model to investigate the impact of chronic ethanol exposure and withdrawal on the expression of these proteins during neuronal differentiation. RA-induced differentiation of human neuroblastoma SH-SY5Y cells seems to represent such a model. Our real-time RT-PCR, Western blot, and immunocytochemistry analyses of undifferentiated and RA-differentiated cells have demonstrated the enhanced expression of both splice variants of DRD2, with the short one being stronger enhanced than the long one under RA-treatment, and the DRD2 distribution on cell bodies and neurites under both conditions. In contrast, DAT was down-regulated by RA. The DAT is functional both in undifferentiated and RA-differentiated cells as demonstrated by [(3)H]dopamine uptake. Chronic ethanol exposure during differentiation for up to 4 weeks resulted in a delayed up-regulation of DRD2s. Ethanol withdrawal caused an increased expression of DRD2l and a normalization of DRD2s. Thus the DRD2s/DRD2l ratio was still disturbed. The dopamine level was increased by RA-differentiation compared to controls and was diminished under RA/ethanol treatment and ethanol withdrawal compared to RA-only treated cells. In conclusion, chronic ethanol exposure impairs differentiation-dependent adaptation of dopaminergic proteins, specifically of DRD2s. RA-differentiating SH-SY5Y cells are suited to study the impact of chronic ethanol exposure and withdrawal on expression of dopaminergic proteins during neuronal differentiation.

Brain and muscle Arnt-like protein-1 (BMAL1), a component of the molecular clock, regulates adipogenesis

PubMed Central

Shimba, Shigeki; Ishii, Norimasa; Ohta, Yuki; Ohno, Toshiharu; Watabe, Yuichi; Hayashi, Mitsuaki; Wada, Taira; Aoyagi, Toshinori; Tezuka, Masakatsu

2005-01-01

Brain and muscle Arnt-like protein-1 (BMAL1; also known as MOP3 or Arnt3) is a transcription factor known to regulate circadian rhythm. Here, we established its involvement in the control of adipogenesis and lipid metabolism activity in mature adipocytes. During adipose differentiation in 3T3-L1 cells, the level of BMAL1 mRNA began to increase 4 days after induction and was highly expressed in differentiated cells. In white adipose tissues isolated from C57BL/6J mice, BMAL1 was predominantly expressed in a fraction containing adipocytes, as compared with the stromal-vascular fraction. BMAL1 knockout mice embryonic fibroblast cells failed to be differentiated into adipocytes. Importantly, adding BMAL1 back by adenovirus gene transfer restored the ability of BMAL1 knockout mice embryonic fibroblast cells to differentiate. Knock-down of BMAL1 expression in 3T3-L1 cells by an RNA interference technique allowed the cells to accumulate only minimum amounts of lipid droplets in the cells. Adenovirus-mediated expression of BMAL1 in 3T3-L1 adipocytes resulted in induction of several factors involved in lipogenesis. The promoter activity of these genes was stimulated in a BMAL1-dependent manner. Interestingly, expression of these factors showed clear circadian rhythm in mice adipose tissue. Furthermore, overexpression of BMAL1 in adipocytes increased lipid synthesis activity. These results indicate that BMAL1, a master regulator of circadian rhythm, also plays important roles in the regulation of adipose differentiation and lipogenesis in mature adipocytes. PMID:16093318

Identification of choriogenin cis-regulatory elements and production of estrogen-inducible, liver-specific transgenic Medaka.

PubMed

Ueno, Tetsuro; Yasumasu, Shigeki; Hayashi, Shinji; Iuchi, Ichiro

2004-07-01

Choriogenins (chg-H, chg-L) are precursor proteins of egg envelope of medaka and synthesized in the spawning female liver in response to estrogen. We linked a gene construct chg-L1.5 kb/GFP (a 1.5 kb 5'-upstream region of the chg-L gene fused with a green fluorescence protein (GFP) gene) to another construct emgb/RFP (a cis-regulatory region of embryonic globin gene fused with an RFP gene), injected the double fusion gene construct into 1- or 2-cell-stage embryos, and selected embryos expressing the RFP in erythroid cells. From the embryos, we established two lines of chg-L1.5 kb/GFP-emgb/RFP-transgenic medaka. The 3-month-old spawning females and estradiol-17beta (E2)-exposed males displayed the liver-specific GFP expression. The E2-dependent GFP expression was detected in the differentiating liver of the stage 37-38 embryos. In addition, RT-PCR and whole-mount in situ hybridization showed that the E2-dependent chg expression was found in the liver of the stage 34 embryos of wild medaka, suggesting that such E2-dependency is achieved shortly after differentiation of the liver. Analysis using serial deletion mutants fused with GFP showed that the region -426 to -284 of the chg-L gene or the region -364 to -265 of the chg-H gene had the ability to promote the E2-dependent liver-specific GFP expression of its downstream gene. Further analyses suggested that an estrogen response element (ERE) at -309, an ERE half-site at -330 and a binding site for C/EBP at -363 of the chg-L gene played important roles in its downstream chg-L gene expression. In addition, this transgenic medaka may be useful as one of the test animals for detecting environmental estrogenic steroids.

The Expression Pattern of the Cell Cycle Inhibitor p19INK4d by Progenitor Cells of the Rat Embryonic Telencephalon and Neonatal Anterior Subventricular Zone

PubMed Central

Coskun, Volkan; Luskin, Marla B.

2014-01-01

In this study we investigated whether the pattern of expression of the cyclin-dependent kinase inhibitor p19INK4d by the unique progenitor cells of the neonatal anterior subventricular zone (SVZa) can account for their ability to divide even though they express phenotypic characteristics of differentiated neurons. p19INK4d was chosen for analysis because it usually acts to block permanently the cell cycle at the G1 phase. p19INK4d immunoreactivity and the incorporation of bromodeoxyuridine (BrdU) by SVZa cells were compared with that of the more typical progenitor cells of the prenatal telencephalic ventricular zone. In the developing telencephalon, p19INK4d is expressed by postmitotic cells and has a characteristic perinuclear distribution depending on the laminar position and state of differentiation of a cell. Moreover, the laminar-specific staining of the developing cerebral cortex revealed that the ventricular zone (VZ) is divided into p19INK4d(+) and p19INK4d(−) sublaminae, indicating that the VZ has a previously unrecognized level of functional organization. Furthermore, the rostral migratory stream, traversed by the SVZa-derived cells, exhibits an anteriorhigh–posteriorlow gradient of p19INK4d expression. On the basis of the p19INK4d immunoreactivity and BrdU incorporation, SVZa-derived cells appear to exit and reenter the cell cycle successively. Thus, in contrast to telencephalic VZ cells, SVZa cells continue to undergo multiple rounds of division and differentiation before becoming postmitotic. PMID:11312294

Roles of Estrogen Receptor-α and the Coactivator MED1 During Human Endometrial Decidualization

PubMed Central

Kaya Okur, Hatice S.; Das, Amrita; Taylor, Robert N.; Bagchi, Indrani C.

2016-01-01

The steroid hormones 17β-estradiol and progesterone are critical regulators of endometrial stromal cell differentiation, known as decidualization, which is a prerequisite for successful establishment of pregnancy. The present study using primary human endometrial stromal cells (HESCs) addressed the role of estrogen receptor-α (ESR1) in decidualization. Knockdown of ESR1 transcripts by RNA interference led to a marked reduction in decidualization of HESCs. Gene expression profiling at an early stage of decidualization indicated that ESR1 negatively regulates several cell cycle regulatory factors, thereby suppressing the proliferation of HESCs as these cells enter the differentiation program. ESR1 also controls the expression of WNT4, FOXO1, and progesterone receptor (PGR), well-known mediators of decidualization. Whereas ESR1 knockdown strongly inhibited the expression of FOXO1 and WNT4 transcripts within 24 hours of the initiation of decidualization, PGR expression remained unaffected at this early time point. Our study also revealed a major role of cAMP signaling in influencing the function of ESR1 during decidualization. Using a proteomic approach, we discovered that the cAMP-dependent protein kinase A (PKA) phosphorylates Mediator 1 (MED1), a subunit of the mediator coactivator complex, during HESC differentiation. Using immunoprecipitation, we demonstrated that PKA-phosphorylated MED1 interacts with ESR1. The PKA-dependent phosphorylation of MED1 was also correlated with its enhanced recruitment to estrogen-responsive elements in the WNT4 gene. Knockdown of MED1 transcripts impaired the expression of ESR1-induced WNT4 and FOXO1 transcripts and blocked decidualization. Based on these findings, we conclude that modulation of ESR1-MED1 interactions by cAMP signaling plays a critical role in human decidualization. PMID:26849466

Isoniazid suppresses antioxidant response element activities and impairs adipogenesis in mouse and human preadipocytes

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

Chen, Yanyan; The First Affiliated Hospital, China Medical University, Shenyang 110001; Xue, Peng

2013-12-15

Transcriptional signaling through the antioxidant response element (ARE), orchestrated by the Nuclear factor E2-related factor 2 (Nrf2), is a major cellular defense mechanism against oxidative or electrophilic stress. Here, we reported that isoniazid (INH), a widely used antitubercular drug, displays a substantial inhibitory property against ARE activities in diverse mouse and human cells. In 3T3-L1 preadipocytes, INH concentration-dependently suppressed the ARE-luciferase reporter activity and mRNA expression of various ARE-dependent antioxidant genes under basal and oxidative stressed conditions. In keeping with our previous findings that Nrf2-ARE plays a critical role in adipogenesis by regulating expression of CCAAT/enhancer-binding protein β (C/EBPβ) andmore » peroxisome proliferator-activated receptor γ (PPARγ), suppression of ARE signaling by INH hampered adipogenic differentiation of 3T3-L1 cells and human adipose-derived stem cells (ADSCs). Following adipogenesis induced by hormonal cocktails, INH-treated 3T3-L1 cells and ADSCs displayed significantly reduced levels of lipid accumulation and attenuated expression of C/EBPα and PPARγ. Time-course studies in 3T3-L1 cells revealed that inhibition of adipogenesis by INH occurred in the early stage of terminal adipogenic differentiation, where reduced expression of C/EBPβ and C/EBPδ was observed. To our knowledge, the present study is the first to demonstrate that INH suppresses ARE signaling and interrupts with the transcriptional network of adipogenesis, leading to impaired adipogenic differentiation. The inhibition of ARE signaling may be a potential underlying mechanism by which INH attenuates cellular antioxidant response contributing to various complications. - Highlights: • Isoniazid suppresses ARE-mediated transcriptional activity. • Isoniazid inhibits adipogenesis in preadipocytes. • Isoniazid suppresses adipogenic gene expression during adipogenesis.« less

Stretch and interleukin 1 beta: pro-labour factors with similar mitogen-activated protein kinase effects but differential patterns of transcription factor activation and gene expression.

PubMed

Sooranna, S R; Engineer, N; Liang, Z; Bennett, P R; Johnson, M R

2007-07-01

IL-1beta and stretch increase uterine smooth muscle cell (USMC) prostaglandin H synthase 2 (PGHS-2) and interleukin (IL)-8 mRNA expression in a mitogen-activated protein kinase (MAPK) dependent mechanism. We have tested our hypothesis that stretch and IL-1beta activate different components of the MAPK cascade in USMC and investigated the effects of specific MAPK inhibitors on these components. Further, we have used a Jun N-terminal kinase (JNK) and p38 activator, anisomycin, to compare the effect of differential MAPK activation on the expression of PGHS-2, IL-8 and oxytocin receptor (OTR) mRNA with that seen in response to stretch and IL-1beta. Stretch, IL-1beta and anisomycin activated similar components of the MAPK cascade and specific inhibitors of MAPK altered phosphorylation of MAPK and downstream cascade components as expected. Expression of OTR mRNA was increased by stretch and anisomycin in a MAPK-independent manner. All three stimuli increased PGHS-2 and IL-8 mRNA expression in a MAPK-dependent manner, but while the MAPK inhibitors reduced the IL-1beta-induced activation of activating transcription factor (ATF)-2, liver activating protein (LAP) and c-jun, the stretch-induced increase in LAP was unaffected by MAPK-inhibition and only JNK inhibition appeared to reduce c-jun activation. These observations show that stretch, IL-1beta and anisomycin activate the same components of the MAPK cascade, but differentially activate LAP and liver inhibitory protein (LIP) perhaps accounting for the increase in OTR by stretch and anisomycin but not IL-1beta observed in this study.

ARTD1 regulates osteoclastogenesis and bone homeostasis by dampening NF-κB-dependent transcription of IL-1β

PubMed Central

Robaszkiewicz, Agnieszka; Qu, Chao; Wisnik, Ewelina; Ploszaj, Tomasz; Mirsaidi, Ali; Kunze, Friedrich A.; Richards, Peter J.; Cinelli, Paolo; Mbalaviele, Gabriel; Hottiger, Michael O.

2016-01-01

While ADP-ribosyltransferase diphtheria toxin-like 1 (ARTD1, formerly PARP1) and its enzymatic activity have been shown to be important for reprogramming and differentiation of cells, such as during adipogenesis, their role and mechanism in regulating osteoclastogenesis and bone homeostasis are largely unknown. Here, in cell culture-based RANKL-induced osteoclastogenesis models, we show that silencing of ARTD1 or inhibition of its enzymatic activity enhances osteoclast differentiation and function. As a consequence of ARTD1 silencing or inhibition, the recruitment of p65/RelA to the IL-1β promoter, which is associated with transcriptionally active histone marks, IL-1β expression and inflammasome-dependent secretion of IL-1β are enhanced. This subsequently promotes sustained induction of the transcription factor Nfatc1/A and osteoclastogenesis in an autocrine manner via the IL-1 receptor. In vivo, Artd1-deficient mice display significantly decreased bone mass as a consequence of increased osteoclast differentiation. Accordingly, the expression of osteoclast markers is enhanced in mutant compared to wild-type mice. Together, these results indicate that ARTD1 controls osteoclast development and bone remodelling via its enzymatic activity by modulating the epigenetic marks surrounding the IL-1β promoter and expression of IL-1β and subsequently also Nfatc1/A. PMID:26883084

Characterisation of FAP-1 expression and CD95 mediated apoptosis in the A818-6 pancreatic adenocarcinoma differentiation system.

PubMed

Winterhoff, Boris J N; Arlt, Alexander; Duttmann, Angelika; Ungefroren, Hendrik; Schäfer, Heiner; Kalthoff, Holger; Kruse, Marie-Luise

2012-03-01

The present study investigated the expression and localisation of FAP-1 (Fas associated phosphatase-1) and CD95 in a 3D differentiation model in comparison to 2D monolayers of the pancreatic adenocarcinoma cell line A818-6. Under non-adherent growth conditions, A818-6 cells differentiate into 3D highly organised polarised epithelial hollow spheres, resembling duct-like structures. A818-6 cells showed a differentiation-dependent FAP-1 localisation. Cells grown as 2D monolayers revealed FAP-1 staining in a juxtanuclear cisternal position, as well as localisation in the nucleus. After differentiation into hollow spheres, FAP-1 was relocated towards the actin cytoskeleton beneath the outer plasma membrane of polarised cells and no further nuclear localisation was observed. CD95 surface staining was found only in a subset of A818-6 monolayer cells, while differentiated hollow spheres appeared to express CD95 in all cells of a given sphere. We rarely observed co-localisation of CD95 and FAP-1 in A818-6 monolayer cells, but strong co-localisation beneath the outer plasma membrane in polarised cells. Analysis of surface expression by flow cytometry revealed that only a subset (36%) of monolayer cells showed CD95 surface expression, and after induction of hollow spheres, CD95 presentation at the outer plasma membrane was reduced to 13% of hollow spheres. Induction of apoptosis by stimulation with agonistic anti-CD95 antibodies, resulted in increased caspase activity in both, monolayer cells and hollow spheres. Knock down of FAP-1 mRNA in A818-6 monolayer cells did not alter resposiveness to CD95 agonistic antibodies. These data suggested that CD95 signal transduction was not affected by FAP-1 expression in A818-6 monolayer cells. In differentiated 3D hollow spheres, we found a polarisation-induced co-localisation of CD95 and FAP-1. A tight control of receptor surface representation and signalling induced apoptosis ensures controlled removal of individual cells instead of a "snowball effect" of apoptotic events. Copyright © 2011 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

Dissecting the Calcium-Induced Differentiation of Human Primary Keratinocytes Stem Cells by Integrative and Structural Network Analyses

PubMed Central

Toufighi, Kiana; Yang, Jae-Seong; Luis, Nuno Miguel; Aznar Benitah, Salvador; Lehner, Ben; Serrano, Luis; Kiel, Christina

2015-01-01

The molecular details underlying the time-dependent assembly of protein complexes in cellular networks, such as those that occur during differentiation, are largely unexplored. Focusing on the calcium-induced differentiation of primary human keratinocytes as a model system for a major cellular reorganization process, we look at the expression of genes whose products are involved in manually-annotated protein complexes. Clustering analyses revealed only moderate co-expression of functionally related proteins during differentiation. However, when we looked at protein complexes, we found that the majority (55%) are composed of non-dynamic and dynamic gene products (‘di-chromatic’), 19% are non-dynamic, and 26% only dynamic. Considering three-dimensional protein structures to predict steric interactions, we found that proteins encoded by dynamic genes frequently interact with a common non-dynamic protein in a mutually exclusive fashion. This suggests that during differentiation, complex assemblies may also change through variation in the abundance of proteins that compete for binding to common proteins as found in some cases for paralogous proteins. Considering the example of the TNF-α/NFκB signaling complex, we suggest that the same core complex can guide signals into diverse context-specific outputs by addition of time specific expressed subunits, while keeping other cellular functions constant. Thus, our analysis provides evidence that complex assembly with stable core components and competition could contribute to cell differentiation. PMID:25946651

Development of an in vitro culture method for stepwise differentiation of mouse embryonic stem cells and induced pluripotent stem cells into mature osteoclasts.

PubMed

Nishikawa, Keizo; Iwamoto, Yoriko; Ishii, Masaru

2014-05-01

The development of methods for differentiation of embryonic stem cells (ESCs) and induced pluripotent stem cell (iPSCs) into functional cells have helped to analyze the mechanism regulating cellular processes and to explore cell-based assays for drug discovery. Although several reports have demonstrated methods for differentiation of mouse ESCs into osteoclast-like cells, it remains unclear whether these methods are applicable for differentiation of iPSCs to osteoclasts. In this study, we developed a simple method for stepwise differentiation of mouse ESCs and iPSCs into bone-resorbing osteoclasts based upon a monoculture approach consisting of three steps. First, based on conventional hanging-drop methods, embryoid bodies (EBs) were produced from mouse ESCs or iPSCs. Second, EBs were cultured in medium supplemented with macrophage colony-stimulating factor (M-CSF), and differentiated to osteoclast precursors, which expressed CD11b. Finally, ESC- or iPSC-derived osteoclast precursors stimulated with receptor activator of nuclear factor-B ligand (RANKL) and M-CSF formed large multinucleated osteoclast-like cells that expressed tartrate-resistant acid phosphatase and were capable of bone resorption. Molecular analysis showed that the expression of osteoclast marker genes such as Nfatc1, Ctsk, and Acp5 are increased in a RANKL-dependent manner. Thus, our procedure is simple and easy and would be helpful for stem cell-based bone research.

Epidermal Notch signalling: differentiation, cancer and adhesion.

PubMed

Watt, Fiona M; Estrach, Soline; Ambler, Carrie A

2008-04-01

The Notch pathway plays an important role in regulating epidermal differentiation. Notch ligands, receptors and effectors are expressed in a complex and dynamic pattern in embryonic and adult skin. Genetic ablation or activation of the pathway reveals that Notch signalling promotes differentiation of the hair follicle, sebaceous gland and interfollicular epidermal lineages and that Notch acts as an epidermal tumour suppressor. Notch signalling interacts with a range of other pathways to fulfil these functions and acts via RBP-Jkappa dependent and independent mechanisms. The effects on differentiation can be cell autonomous and non-autonomous, and Notch contributes to stem cell clustering via modulation of cell adhesion.

11-Hydroxy-β-steroid dehydrogenase gene expression in canine adipose tissue and adipocytes: stimulation by lipopolysaccharide and tumor necrosis factor α.

PubMed

Ryan, V H; Trayhurn, P; Hunter, L; Morris, P J; German, A J

2011-10-01

The enzyme 11β-hydroxysteroid dehydrogenase 1 (11β-HSD-1) is expressed in a number of tissues in rodents and humans and is responsible for the reactivation of inert cortisone into cortisol. Its gene expression and activity are increased in white adipose tissue (WAT) from obese humans and may contribute to the adverse metabolic consequences of obesity and the metabolic syndrome. The extent to which 11β-HSD-1 contributes to adipose tissue function in dogs is unknown; the aim of the present study was to examine 11β-HSD-1 gene expression and its regulation by proinflammatory and anti-inflammatory agents in canine adipocytes. Real-time PCR was used to examine the expression of 11β-HSD-1 in canine adipose tissue and canine adipocytes differentiated in culture. The mRNA encoding 11β-HSD-1 was identified in all the major WAT depots in dogs and also in liver, kidney, and spleen. Quantification by real-time PCR showed that 11β-HSD-1 mRNA was least in perirenal and falciform depots and greatest in subcutaneous, omental, and gonadal depots. Greater expression was seen in the omental depot in female than in male dogs (P=0.05). Gene expression for 11β-HSD-1 was also seen in adipocytes, from both subcutaneous and visceral depots, differentiated in culture; expression was evident throughout differentiation but was generally greatest in preadipocytes and during early differentiation, declining as cells progressed to maturity. The inflammatory mediators lipopolysaccharide and tumor necrosis factor α had a main stimulatory effect on 11β-HSD-1 gene expression in canine subcutaneous adipocytes, but IL-6 had no significant effect. Treatment with dexamethasone resulted in a significant time- and dose-dependent increase in 11β-HSD-1 gene expression, with greatest effects seen at 24 h (2 nM: approximately 4-fold; 20 nM: approximately 14-fold; P=0.010 for both). When subcutaneous adipocytes were treated with the peroxisome proliferator activated receptor γ agonist rosiglitazone, similar dose- and time-dependent effects were noted. However, no effects were seen when adipocytes from the gonadal WAT depot were treated with rosiglitazone. The induction of 11β-HSD-1 expression, by the pro-inflammatory cytokine tumor necrosis factor α and by lipopolysaccharide may have implications for the pathogenesis of obesity and its associated diseases in the dog. Copyright © 2011 Elsevier Inc. All rights reserved.

Pod-1/Capsulin shows a sex- and stage-dependent expression pattern in the mouse gonad development and represses expression of Ad4BP/SF-1.

PubMed

Tamura, M; Kanno, Y; Chuma, S; Saito, T; Nakatsuji, N

2001-04-01

Mammalian sex-determination and differentiation are controlled by several genes, such as Sry, Sox-9, Dax-1 and Mullerian inhibiting substance (MIS), but their upstream and downstream genes are largely unknown. Ad4BP/SF-1, encoding a zinc finger transcription factor, plays important roles in gonadogenesis. Disruption of this gene caused disappearance of the urogenital system including the gonad. Ad4BP/SF-1, however, is also involved in the sex differentiation of the gonad at later stages, such as the regulation of steroid hormones and MIS. Pod-1/Capsulin, a member of basic helix-loop-helix transcription factors, is expressed in a pattern closely related but mostly complimentary to that of the Ad4BP/SF-1 expression in the developing gonad. In the co-transfection experiment using cultured cells, overexpression of Pod-1/Capsulin repressed expression of a reporter gene that carried the upstream regulatory region of the Ad4BP/SF-1 gene. Furthermore, forced expression of Pod-1/Capsulin repressed expression of Ad4BP/SF-1 in the Leydig cell-derived I-10 cells. These results suggest that Pod-1/Capsulin may play important roles in the development and sex differentiation of the mammalian gonad via transcriptional regulation of Ad4BP/SF-1.

The Third Intron of the Interferon Regulatory Factor-8 Is an Initiator of Repressed Chromatin Restricting Its Expression in Non-Immune Cells

PubMed Central

Barnea-Yizhar, Ofer; Ram, Sigal; Kovalev, Ekaterina; Azriel, Aviva; Rand, Ulfert; Nakayama, Manabu; Hauser, Hansjörg; Gepstein, Lior; Levi, Ben-Zion

2016-01-01

Interferon Regulatory Factor-8 (IRF-8) serves as a key factor in the hierarchical differentiation towards monocyte/dendritic cell lineages. While much insight has been accumulated into the mechanisms essential for its hematopoietic specific expression, the mode of restricting IRF-8 expression in non-hematopoietic cells is still unknown. Here we show that the repression of IRF-8 expression in restrictive cells is mediated by its 3rd intron. Removal of this intron alleviates the repression of Bacterial Artificial Chromosome (BAC) IRF-8 reporter gene in these cells. Fine deletion analysis points to conserved regions within this intron mediating its restricted expression. Further, the intron alone selectively initiates gene silencing only in expression-restrictive cells. Characterization of this intron’s properties points to its role as an initiator of sustainable gene silencing inducing chromatin condensation with suppressive histone modifications. This intronic element cannot silence episomal transgene expression underlining a strict chromatin-dependent silencing mechanism. We validated this chromatin-state specificity of IRF-8 intron upon in-vitro differentiation of induced pluripotent stem cells (iPSCs) into cardiomyocytes. Taken together, the IRF-8 3rd intron is sufficient and necessary to initiate gene silencing in non-hematopoietic cells, highlighting its role as a nucleation core for repressed chromatin during differentiation. PMID:27257682

N-CADHERIN MEDIATES NITRIC OXIDE-INDUCED NEUROGENESIS IN YOUNG AND RETIRED BREEDER NEUROSPHERES

PubMed Central

CHEN, J.; ZACHAREK, A.; LI, Y.; LI, A.; WANG, L.; KATAKOWSKI, M.; ROBERTS, C.; LU, M.; CHOPP, M.

2009-01-01

Neurogenesis may contribute to functional recovery after neural injury. Nitric oxide donors such as DETA-NONOate promote functional recovery after stroke. However, the mechanisms underlying functional improvement have not been ascertained. We therefore investigated the effects of DETA-NONOate on neural progenitor/stem cell neurospheres derived from the subventricular zone from young and retired breeder rat brain. Subventricular zone cells were dissociated from normal young adult male Wistar rats (2–3 months old) and retired breeder rats (14 months old), treated with or without DETA-NONOate. Subventricular zone neurosphere formation, proliferation, telomerase activity, and Neurogenin 1 mRNA expression were significantly decreased and glial fibrillary acidic protein expression was significantly increased in subventricular zone neurospheres from retired breeder rats compared with young rats. Treatment of neurospheres with DETA-NONOate significantly decreased neurosphere formation and telomerase activity, and promoted neuronal differentiation and neurite outgrowth concomitantly with increased N-cadherin and β-catenin mRNA expression in both young and old neurospheres. DETA-NONOate selectively increased Neurogenin 1 and decreased glial fibrillary acidic protein mRNA expression in retired breeder neurospheres. N-cadherin significantly increased Neurogenin 1 mRNA expression in young and old neurospheres. Anti-N-cadherin reversed DETA-NONOate-induced neurosphere adhesion, neuronal differentiation, neurite outgrowth, and β-catenin mRNA expression. Our data indicate that age has a potent effect on the characteristics of subventricular zone neurospheres; neurospheres from young rats show significantly higher formation, proliferation and telomerase activity than older neurospheres. In contrast, older neurospheres exhibit significantly increased glial differentiation than young neurospheres. DETA-NONOate promotes neuronal differentiation and neurite outgrowth in both young and older neurospheres. The molecular mechanisms associated with the DETA-NONOate modulation of neurospheres from young and older animals as well age dependent effects of neurospheres appear to be controlled by N-cadherin and β-catenin gene expression, which subsequently regulates the neuronal differentiating factor Neurogenin expression in both young and old neural progenitor cells. PMID:16580782

Transcriptome Analysis of CD4+ T Cells in Coeliac Disease Reveals Imprint of BACH2 and IFNγ Regulation

PubMed Central

Molloy, Ben; Dominguez Castro, Patricia; Cormican, Paul; Trimble, Valerie; Mahmud, Nasir; McManus, Ross

2015-01-01

Genetic studies have to date identified 43 genome wide significant coeliac disease susceptibility (CD) loci comprising over 70 candidate genes. However, how altered regulation of such disease associated genes contributes to CD pathogenesis remains to be elucidated. Recently there has been considerable emphasis on characterising cell type specific and stimulus dependent genetic variants. Therefore in this study we used RNA sequencing to profile over 70 transcriptomes of CD4+ T cells, a cell type crucial for CD pathogenesis, in both stimulated and resting samples from individuals with CD and unaffected controls. We identified extensive transcriptional changes across all conditions, with the previously established CD gene IFNy the most strongly up-regulated gene (log2 fold change 4.6; Padjusted = 2.40x10-11) in CD4+ T cells from CD patients compared to controls. We show a significant correlation of differentially expressed genes with genetic studies of the disease to date (Padjusted = 0.002), and 21 CD candidate susceptibility genes are differentially expressed under one or more of the conditions used in this study. Pathway analysis revealed significant enrichment of immune related processes. Co-expression network analysis identified several modules of coordinately expressed CD genes. Two modules were particularly highly enriched for differentially expressed genes (P<2.2x10-16) and highlighted IFNy and the genetically associated transcription factor BACH2 which showed significantly reduced expression in coeliac samples (log2FC -1.75; Padjusted = 3.6x10-3) as key regulatory genes in CD. Genes regulated by BACH2 were very significantly over-represented among our differentially expressed genes (P<2.2x10-16) indicating that reduced expression of this master regulator of T cell differentiation promotes a pro-inflammatory response and strongly corroborates genetic evidence that BACH2 plays an important role in CD pathogenesis. PMID:26444573

Improved differentiation of umbilical cord blood-derived mesenchymal stem cells into insulin-producing cells by PDX-1 mRNA transfection.

PubMed

Van Pham, Phuc; Thi-My Nguyen, Phuoc; Thai-Quynh Nguyen, Anh; Minh Pham, Vuong; Nguyen-Tu Bui, Anh; Thi-Tung Dang, Loan; Gia Nguyen, Khue; Kim Phan, Ngoc

2014-06-01

Numerous studies have sought to identify diabetes mellitus treatment strategies with fewer side effects. Mesenchymal stem cell (MSC) therapy was previously considered as a promising therapy; however, it requires the cells to be trans-differentiated into cells of the pancreatic-endocrine lineage before transplantation. Previous studies have shown that PDX-1 expression can facilitate MSC differentiation into insulin-producing cells (IPCs), but the methods employed to date use viral or DNA-based tools to express PDX-1, with the associated risks of insertional mutation and immunogenicity. Thus, this study aimed to establish a new method to induce PDX-1 expression in MSCs by mRNA transfection. MSCs were isolated from human umbilical cord blood and expanded in vitro, with stemness confirmed by surface markers and multipotentiality. MSCs were transfected with PDX-1 mRNA by nucleofection and chemically induced to differentiate into IPCs (combinatorial group). This IPC differentiation was then compared with that of untransfected chemically induced cells (inducer group) and uninduced cells (control group). We found that PDX-1 mRNA transfection significantly improved the differentiation of MSCs into IPCs, with 8.3±2.5% IPCs in the combinatorial group, 3.21±2.11% in the inducer group and 0% in the control. Cells in the combinatorial group also strongly expressed several genes related to beta cells (Pdx-1, Ngn3, Nkx6.1 and insulin) and could produce C-peptide in the cytoplasm and insulin in the supernatant, which was dependent on the extracellular glucose concentration. These results indicate that PDX-1 mRNA may offer a promising approach to produce safe IPCs for clinical diabetes mellitus treatment. Copyright © 2014 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

NDM29, a RNA polymerase III-dependent non coding RNA, promotes amyloidogenic processing of APP and amyloid β secretion.

PubMed

Massone, Sara; Ciarlo, Eleonora; Vella, Serena; Nizzari, Mario; Florio, Tullio; Russo, Claudio; Cancedda, Ranieri; Pagano, Aldo

2012-07-01

Neuroblastoma Differentiation Marker 29 (NDM29) is a RNA polymerase (pol) III-transcribed non-coding (nc) RNA whose synthesis drives neuroblastoma (NB) cell differentiation to a nonmalignant neuron-like phenotype. Since in this process a complex pattern of molecular changes is associated to plasma membrane protein repertoire we hypothesized that the expression of NDM29 might influence also key players of neurodegenerative pathways. In this work we show that the NDM29-dependent cell maturation induces amyloid precursor protein (APP) synthesis, leading to the increase of amyloid β peptide (Aβ) secretion and the concomitant increment of Aβ x-42/Aβ x-40 ratio. We also demonstrate that the expression of NDM29 RNA, and the consequent increase of Aβ formation, can be promoted by inflammatory stimuli (and repressed by anti-inflammatory drugs). Moreover, NDM29 expression was detected in normal human brains although an abnormal increased synthesis of this ncRNA is induced in patients affected by neurodegenerative diseases. Therefore, the complex of events triggered by NDM29 expression induces a condition that favors the formation of Aβ peptides in the extracellular space, as it may occur in Alzheimer's Disease (AD). In addition, these data unexpectedly show that a pol III-dependent small RNA can act as key regulator of brain physiology and/or pathology suggesting that a better knowledge of this portion of the human transcriptome might provide hints for neurodegeneration studies. Copyright © 2012 Elsevier B.V. All rights reserved.

Mesenchymal stem cell-derived exosomes have altered microRNA profiles and induce osteogenic differentiation depending on the stage of differentiation

PubMed Central

Wang, Xiaoqin; Omar, Omar; Vazirisani, Forugh; Thomsen, Peter

2018-01-01

Human mesenchymal stem cell (hMSC)-derived exosomes have shown regenerative effects, but their role in osteogenesis and the underlying mechanism are yet to be determined. In this study, we examined the time-course secretion of exosomes by hMSCs during the entire process of osteogenic differentiation. Exosomes derived from hMSCs in various stages of osteogenic differentiation committed homotypic cells to differentiate towards osteogenic lineage, but only exosomes from late stages of osteogenic differentiation induced extracellular matrix mineralisation. Exosomes from expansion and early and late stages of osteogenic differentiation were internalised by a subpopulation of hMSCs. MicroRNA profiling revealed a set of differentially expressed exosomal microRNAs from the late stage of osteogenic differentiation, which were osteogenesis related. Target prediction demonstrated that these microRNAs enriched pathways involved in regulation of osteogenic differentiation and general mechanisms how exosomes exert their functions, such as “Wnt signalling pathway” and “endocytosis”. Taken together, the results show that MSCs secrete exosomes with different biological properties depending on differentiation stage of their parent cells. The exosomal cargo transferred from MSCs in the late stage of differentiation induces osteogenic differentiation and mineralisation. Moreover, it is suggested that the regulatory effect on osteogenesis by exosomes is at least partly exerted by exosomal microRNA. PMID:29447276

An in vitro ES cell imprinting model shows that imprinted expression of the Igf2r gene arises from an allele-specific expression bias

PubMed Central

Latos, Paulina A.; Stricker, Stefan H.; Steenpass, Laura; Pauler, Florian M.; Huang, Ru; Senergin, Basak H.; Regha, Kakkad; Koerner, Martha V.; Warczok, Katarzyna E.; Unger, Christine; Barlow, Denise P.

2010-01-01

Genomic imprinting is an epigenetic process that results in parental-specific gene expression. Advances in understanding the mechanism that regulates imprinted gene expression in mammals have largely depended on generating targeted manipulations in embryonic stem (ES) cells that are analysed in vivo in mice. However, genomic imprinting consists of distinct developmental steps, some of which occur in post-implantation embryos, indicating that they could be studied in vitro in ES cells. The mouse Igf2r gene shows imprinted expression only in post-implantation stages, when repression of the paternal allele has been shown to require cis-expression of the Airn non-coding (nc) RNA and to correlate with gain of DNA methylation and repressive histone modifications. Here we follow the gain of imprinted expression of Igf2r during in vitro ES cell differentiation and show that it coincides with the onset of paternal-specific expression of the Airn ncRNA. Notably, although Airn ncRNA expression leads, as predicted, to gain of repressive epigenetic marks on the paternal Igf2r promoter, we unexpectedly find that the paternal Igf2r promoter is expressed at similar low levels throughout ES cell differentiation. Our results further show that the maternal and paternal Igf2r promoters are expressed equally in undifferentiated ES cells, but during differentiation expression of the maternal Igf2r promoter increases up to 10-fold, while expression from the paternal Igf2r promoter remains constant. This indicates, contrary to expectation, that the Airn ncRNA induces imprinted Igf2r expression not by silencing the paternal Igf2r promoter, but by generating an expression bias between the two parental alleles. PMID:19141673

Activation of peroxisome proliferator-activated receptor-{alpha} enhances fatty acid oxidation in human adipocytes

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

Lee, Joo-Young; Hashizaki, Hikari; Goto, Tsuyoshi

2011-04-22

Highlights: {yields} PPAR{alpha} activation increased mRNA expression levels of adipocyte differentiation marker genes and GPDH activity in human adipocytes. {yields} PPAR{alpha} activation also increased insulin-dependent glucose uptake in human adipocytes. {yields} PPAR{alpha} activation did not affect lipid accumulation in human adipocytes. {yields} PPAR{alpha} activation increased fatty acid oxidation through induction of fatty acid oxidation-related genes in human adipocytes. -- Abstract: Peroxisome proliferator-activated receptor-{alpha} (PPAR{alpha}) is a key regulator for maintaining whole-body energy balance. However, the physiological functions of PPAR{alpha} in adipocytes have been unclarified. We examined the functions of PPAR{alpha} using human multipotent adipose tissue-derived stem cells as a humanmore » adipocyte model. Activation of PPAR{alpha} by GW7647, a potent PPAR{alpha} agonist, increased the mRNA expression levels of adipocyte differentiation marker genes such as PPAR{gamma}, adipocyte-specific fatty acid-binding protein, and lipoprotein lipase and increased both GPDH activity and insulin-dependent glucose uptake level. The findings indicate that PPAR{alpha} activation stimulates adipocyte differentiation. However, lipid accumulation was not changed, which is usually observed when PPAR{gamma} is activated. On the other hand, PPAR{alpha} activation by GW7647 treatment induced the mRNA expression of fatty acid oxidation-related genes such as CPT-1B and AOX in a PPAR{alpha}-dependent manner. Moreover, PPAR{alpha} activation increased the production of CO{sub 2} and acid soluble metabolites, which are products of fatty acid oxidation, and increased oxygen consumption rate in human adipocytes. The data indicate that activation of PPAR{alpha} stimulates both adipocyte differentiation and fatty acid oxidation in human adipocytes, suggesting that PPAR{alpha} agonists could improve insulin resistance without lipid accumulation in adipocytes. The expected effects of PPAR{alpha} activation are very valuable for managing diabetic conditions accompanied by obesity, because PPAR{gamma} agonists, usually used as antidiabetic drugs, induce excessive lipid accumulation in adipocytes in addition to improvement of insulin resistance.« less

Embryotoxic and pharmacologic potency ranking of six azoles in the rat whole embryo culture by morphological and transcriptomic analysis

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

Dimopoulou, Myrto, E-mail: myrto.dimopoulou@wur.nl

Differential gene expression analysis in the rat whole embryo culture (WEC) assay provides mechanistic insight into the embryotoxicity of test compounds. In our study, we hypothesized that comparative analysis of the transcriptomes of rat embryos exposed to six azoles (flusilazole, triadimefon, ketoconazole, miconazole, difenoconazole and prothioconazole) could lead to a better mechanism-based understanding of their embryotoxicity and pharmacological action. For evaluating embryotoxicity, we applied the total morphological scoring system (TMS) in embryos exposed for 48 h. The compounds tested showed embryotoxicity in a dose-response fashion. Functional analysis of differential gene expression after 4 h exposure at the ID{sub 10} (effectivemore » dose for 10% decreased TMS), revealed the sterol biosynthesis pathway and embryonic development genes, dominated by genes in the retinoic acid (RA) pathway, albeit in a differential way. Flusilazole, ketoconazole and triadimefon were the most potent compounds affecting the RA pathway, while in terms of regulation of sterol function, difenoconazole and ketoconazole showed the most pronounced effects. Dose-dependent analysis of the effects of flusilazole revealed that the RA pathway related genes were already differentially expressed at low dose levels while the sterol pathway showed strong regulation at higher embryotoxic doses, suggesting that this pathway is less predictive for the observed embryotoxicity. A similar analysis at the 24-hour time point indicated an additional time-dependent difference in the aforementioned pathways regulated by flusilazole. In summary, the rat WEC assay in combination with transcriptomics could add a mechanistic insight into the embryotoxic potency ranking and pharmacological mode of action of the tested compounds. - Highlights: • Embryonic exposure to azoles revealed concentration-dependent malformations. • Transcriptomics could enhance the mechanistic knowledge of embryotoxicants. • Retinoic acid gene set identifies early embryotoxic responses to azoles. • Toxic versus pharmacologic potency determines functional efficacy.« less

Fibronectin Matrix Polymerization Regulates Smooth Muscle Cell Phenotype through a Rac1 Dependent Mechanism

PubMed Central

Shi, Feng; Long, Xiaochun; Hendershot, Allison; Miano, Joseph M.; Sottile, Jane

2014-01-01

Smooth muscle cells are maintained in a differentiated state in the vessel wall, but can be modulated to a synthetic phenotype following injury. Smooth muscle phenotypic modulation is thought to play an important role in the pathology of vascular occlusive diseases. Phenotypically modulated smooth muscle cells exhibit increased proliferative and migratory properties that accompany the downregulation of smooth muscle cell marker proteins. Extracellular matrix proteins, including fibronectin, can regulate the smooth muscle phenotype when used as adhesive substrates. However, cells produce and organize a 3-dimensional fibrillar extracellular matrix, which can affect cell behavior in distinct ways from the protomeric 2-dimensional matrix proteins that are used as adhesive substrates. We previously showed that the deposition/polymerization of fibronectin into the extracellular matrix can regulate the deposition and organization of other extracellular matrix molecules in vitro. Further, our published data show that the presence of a fibronectin polymerization inhibitor results in increased expression of smooth muscle cell differentiation proteins and inhibits vascular remodeling in vivo. In this manuscript, we used an in vitro cell culture system to determine the mechanism by which fibronectin polymerization affects smooth muscle phenotypic modulation. Our data show that fibronectin polymerization decreases the mRNA levels of multiple smooth muscle differentiation genes, and downregulates the levels of smooth muscle α-actin and calponin proteins by a Rac1-dependent mechanism. The expression of smooth muscle genes is transcriptionally regulated by fibronectin polymerization, as evidenced by the increased activity of luciferase reporter constructs in the presence of a fibronectin polymerization inhibitor. Fibronectin polymerization also promotes smooth muscle cell growth, and decreases the levels of actin stress fibers. These data define a Rac1-dependent pathway wherein fibronectin polymerization promotes the SMC synthetic phenotype by modulating the expression of smooth muscle cell differentiation proteins. PMID:24752318

MicroRNAs-Dependent Regulation of PPARs in Metabolic Diseases and Cancers

PubMed Central

Portius, Dorothea; Sobolewski, Cyril

2017-01-01

Peroxisome proliferator-activated receptors (PPARs) are a family of ligand-dependent nuclear receptors, which control the transcription of genes involved in energy homeostasis and inflammation and cell proliferation/differentiation. Alterations of PPARs' expression and/or activity are commonly associated with metabolic disorders occurring with obesity, type 2 diabetes, and fatty liver disease, as well as with inflammation and cancer. Emerging evidence now indicates that microRNAs (miRNAs), a family of small noncoding RNAs, which fine-tune gene expression, play a significant role in the pathophysiological mechanisms regulating the expression and activity of PPARs. Herein, the regulation of PPARs by miRNAs is reviewed in the context of metabolic disorders, inflammation, and cancer. The reciprocal control of miRNAs expression by PPARs, as well as the therapeutic potential of modulating PPAR expression/activity by pharmacological compounds targeting miRNA, is also discussed. PMID:28167956

o-p′-DDT-mediated uterotrophy and gene expression in immature C57BL/6 mice and Sprague–Dawley rats

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

Kwekel, Joshua C.; Forgacs, Agnes L.; Center for Integrative Toxicology, Michigan State University, East Lansing, MI

1,1,1-Trichloro-2,2-bis(2-chlorophenyl-4-chlorophenyl)ethane (o,p′-DDT) is an organochlorine pesticide and endocrine disruptor known to activate the estrogen receptor. Comprehensive ligand- and species-comparative dose- and time-dependent studies were conducted to systematically assess the uterine physiological, morphological and gene expression responses elicited by o,p′-DDT and ethynyl estradiol (EE) in immature ovariectomized C57BL/6 mice and Sprague–Dawley rats. Custom cDNA microarrays were used to identify conserved and divergent differential gene expression responses. A total of 1256 genes were differentially expressed by both ligands in both species, 559 of which exhibited similar temporal expression profiles suggesting that o,p′-DDT elicits estrogenic effects at high doses when compared to EE.more » However, 51 genes exhibited species-specific uterine expression elicited by o,p′-DDT. For example, carbonic anhydrase 2 exhibited species- and ligand-divergent expression as confirmed by quantitative real-time PCR. The identification of comparable temporal phenotypic responses linked to gene expression demonstrates that systematic comparative gene expression assessments are valuable for elucidating conserved and divergent estrogen signaling mechanisms in rodent uterotrophy. - Highlights: • o,p′-DDT and enthynyl estradiol (EE) both elicit uterotrophy in mice and rats. • o,p′-DDT and EE have different kinetics in uterine wet weight induction. • o,p′-DDT elicited stromal hypertrophy in rats but myometrial hypertrophy in mice. • 1256 genes were differentially expressed by both ligands in both species. • Only 51 genes had species-specific uterine expression.« less

Mesenchymal Stem Cells Modulate Differentiation of Myeloid Progenitor Cells During Inflammation.

PubMed

Amouzegar, Afsaneh; Mittal, Sharad K; Sahu, Anuradha; Sahu, Srikant K; Chauhan, Sunil K

2017-06-01

Mesenchymal stem cells (MSCs) possess distinct immunomodulatory properties and have tremendous potential for use in therapeutic applications in various inflammatory diseases. MSCs have been shown to regulate pathogenic functions of mature myeloid inflammatory cells, such as macrophages and neutrophils. Intriguingly, the capacity of MSCs to modulate differentiation of myeloid progenitors (MPs) to mature inflammatory cells remains unknown to date. Here, we report the novel finding that MSCs inhibit the expression of differentiation markers on MPs under inflammatory conditions. We demonstrate that the inhibitory effect of MSCs is dependent on direct cell-cell contact and that this intercellular contact is mediated through interaction of CD200 expressed by MSCs and CD200R1 expressed by MPs. Furthermore, using an injury model of sterile inflammation, we show that MSCs promote MP frequencies and suppress infiltration of inflammatory cells in the inflamed tissue. We also find that downregulation of CD200 in MSCs correlates with abrogation of their immunoregulatory function. Collectively, our study provides unequivocal evidence that MSCs inhibit differentiation of MPs in the inflammatory environment via CD200-CD200R1 interaction. Stem Cells 2017;35:1532-1541. © 2017 AlphaMed Press.

CD1d-dependent expansion of NKT follicular helper cells in vivo and in vitro is a product of cellular proliferation and differentiation.

PubMed

Rampuria, Pragya; Lang, Mark L

2015-05-01

NKT follicular helper cells (NKTfh cells) are a recently discovered functional subset of CD1d-restricted NKT cells. Given the potential for NKTfh cells to promote specific antibody responses and germinal center reactions, there is much interest in determining the conditions under which NKTfh cells proliferate and/or differentiate in vivo and in vitro. We confirm that NKTfh cells expressing the canonical semi-invariant Vα14 TCR were CXCR5(+)/ICOS(+)/PD-1(+)/Bcl6(+) and increased in number following administration of the CD1d-binding glycolipid α-galactosylceramide (α-GC) to C57Bl/6 mice. We show that the α-GC-stimulated increase in NKTfh cells was CD1d-dependent since the effect was diminished by reduced CD1d expression. In vivo and in vitro treatment with α-GC, singly or in combination with IL-2, showed that NKTfh cells increased in number to a greater extent than total NKT cells, but proliferation was near-identical in both populations. Acquisition of the NKTfh phenotype from an adoptively transferred PD-1-depleted cell population was also evident, showing that peripheral NKT cells differentiated into NKTfh cells. Therefore, the α-GC-stimulated, CD1d-dependent increase in peripheral NKTfh cells is a result of cellular proliferation and differentiation. These findings advance our understanding of the immune response following immunization with CD1d-binding glycolipids. © The Japanese Society for Immunology. 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Interruptin B induces brown adipocyte differentiation and glucose consumption in adipose-derived stem cells

PubMed Central

KAEWSUWAN, SIREEWAN; PLUBRUKARN, ANUCHIT; UTSINTONG, MALEERUK; KIM, SEOK-HO; JEONG, JIN-HYUN; CHO, JIN GU; PARK, SANG GYU; SUNG, JONG-HYUK

2016-01-01

Interruptin B has been isolated from Cyclosorus terminans, however, its pharamcological effect has not been fully identified. In the present study, the effects of interruptin B, from C. terminans, on brown adipocyte differentiation and glucose uptake in adipose-derived stem cells (ASCs) were investigated. The results revealed that interruptin B dose-dependently enhanced the adipogenic differentiation of ASCs, with an induction in the mRNA expression levels of peroxisome proliferator-activated receptor (PPAR)-α and PPAR-γ. In addition, interruptin B efficiently increased the number and the membrane potential of mitochondria and upregulated the mRNA expression levels of uncoupling protein (UCP)-1 and cyclooxygenase (COX)-2, which are all predominantly expressed in brown adipocytes. Interruptin B increased glucose consumption in differentiated ASCs, accompanied by the upregulation in the mRNA expression levels of glucose transporter (GLUT)-1 and GLUT-4. The computational analysis of molecular docking, a luciferase reporter assay and surface plasmon resonance confirmed the marked binding affinity of interruptin B to PPAR-α and PPAR-γ (KD values of 5.32 and 0.10 µM, respectively). To the best of our knowledge, the present study is the first report to show the stimulatory effects of interruptin B on brown adipocyte differentiation and glucose uptake in ASCs, through its role as a dual PPAR-α and PPAR-γ ligand. Therefore, interruptin B could be further developed as a therapeutic agent for the treatment of diabetes. PMID:26781331

Muscle-specific gene expression is underscored by differential stressor responses and coexpression changes.

PubMed

Moreno-Sánchez, Natalia; Rueda, Julia; Reverter, Antonio; Carabaño, María Jesús; Díaz, Clara

2012-03-01

Variations on the transcriptome from one skeletal muscle type to another still remain unknown. The reliable identification of stable gene coexpression networks is essential to unravel gene functions and define biological processes. The differential expression of two distinct muscles, M. flexor digitorum (FD) and M. psoas major (PM), was studied using microarrays in cattle to illustrate muscle-specific transcription patterns and to quantify changes in connectivity regarding the expected gene coexpression pattern. A total of 206 genes were differentially expressed (DE), 94 upregulated in PM and 112 in FD. The distribution of DE genes in pathways and biological functions was explored in the context of system biology. Global interactomes for genes of interest were predicted. Fast/slow twitch genes, genes coding for extracellular matrix, ribosomal and heat shock proteins, and fatty acid uptake centred the specific gene expression patterns per muscle. Genes involved in repairing mechanisms, such as ribosomal and heat shock proteins, suggested a differential ability of muscles to react to similar stressing factors, acting preferentially in slow twitch muscles. Muscle attributes do not seem to be completely explained by the muscle fibre composition. Changes in connectivity accounted for 24% of significant correlations between DE genes. Genes changing their connectivity mostly seem to contribute to the main differential attributes that characterize each specific muscle type. These results underscore the unique flexibility of skeletal muscle where a substantial set of genes are able to change their behavior depending on the circumstances.

PACAP signaling to DREAM: a cAMP-dependent pathway that regulates cortical astrogliogenesis.

PubMed

Vallejo, Mario

2009-04-01

Astrocytes constitute a very abundant cell type in the mammalian central nervous system and play critical roles in brain function. During development, astrocytes are generated from neural progenitor cells only after these cells have generated neurons. This so called gliogenic switch is tightly regulated by intrinsic factors that inhibit the generation of astrocytes during the neurogenic period. Once neural progenitors acquire gliogenic competence, they differentiate into astrocytes in response to specific extracellular signals. Some of these signals are delivered by neurotrophic cytokines via activation of the gp130-JAK-signal transducer and activator of transcription system, whereas others depend on the activity of pituitary adenylate cyclase-activating polypeptide (PACAP) on specific PAC1 receptors that stimulate the production of cAMP. This results in the activation of the small GTPases Rap1 and Ras, and in the cAMP-dependent entry of extracellular calcium into the cell. Calcium, in turn, stimulates the transcription factor downstream regulatory element antagonist modulator (DREAM), which is bound to specific sites of the promoter of the glial fibrillary acidic protein gene, stimulating its expression during astrocyte differentiation. Lack of DREAM in vivo results in alterations in the number of neurons and astrocytes generated during development. Thus, the PACAP-cAMP-Ca(2+)-DREAM signaling cascade constitutes an important pathway to activate glial-specific gene expression during astrocyte differentiation.

Phosphate Uptake-Independent Signaling Functions of the Type III Sodium-Dependent Phosphate Transporter, PiT-1, in Vascular Smooth Muscle Cells

PubMed Central

Chavkin, Nicholas W.; Jun Chia, Jia; Crouthamel, Matthew H.; Giachelli, Cecilia M.

2015-01-01

Vascular calcification (VC) is prevalent in chronic kidney disease and elevated serum inorganic phosphate (Pi) is a recognized risk factor. The type III sodium-dependent phosphate transporter, PiT-1, is required for elevated Pi-induced osteochondrogenic differentiation and matrix mineralization in vascular smooth muscle cells (VSMCs). However, the molecular mechanism(s) by which PiT-1 promotes these processes is unclear. In the present study, we confirmed that the Pi concentration required to induce osteochondrogenic differentiation and matrix mineralization of mouse VSMCs was well above that required for maximal Pi uptake, suggesting a signaling function of PiT-1 that was independent of Pi transport. Elevated Pi-induced signaling via ERK1/2 phosphorylation was abrogated in PiT-1 deficient VSMCs, but could be rescued by wild-type (WT) and a Pi transport-deficient PiT-1 mutant. Furthermore, both WT and transport-deficient PiT-1 mutants promoted osteochondrogenic differentiation as measured by decreased SM22α and increased osteopontin mRNA expression. Finally, compared to vector alone, expression of transport-deficient PiT-1 mutants promoted VSMC matrix mineralization, but not to the extent observed with PiT-1 WT. These data suggest that both Pi uptake-dependent and -independent functions of PiT-1 are important for VSMC processes mediating vascular calcification. PMID:25684711

Dual Role of Act1 in Keratinocyte Differentiation and Host Defense: TRAF3IP2 Silencing Alters Keratinocyte Differentiation and Inhibits IL-17 Responses.

PubMed

Lambert, Sylviane; Swindell, William R; Tsoi, Lam C; Stoll, Stefan W; Elder, James T

2017-07-01

TRAF3IP2 is a candidate psoriasis susceptibility gene encoding Act1, an adaptor protein with ubiquitin ligase activity that couples the IL-17 receptor to downstream signaling pathways. We investigated the role of Act1 in keratinocyte responses to IL-17 using a tetracycline inducible short hairpin RNA targeting TRAF3IP2. Tetracycline exposure for 7 days effectively silenced TRAF3IP2 mRNA and Act1 protein, resulting in 761 genes with significant changes in expression (495 down, 266 up; >1.5-fold, P < 0.05). Gene ontology analysis showed that genes affected by TRAF3IP2 silencing are involved in epidermal differentiation, with early differentiation genes (KRT1, KRT10, DSC1, DSG1) being down-regulated and late differentiation genes (SPRR2, SPRR3, LCE3) being up-regulated. AP1 binding sites were enriched upstream of genes up-regulated by TRAF3IP2 silencing. Correspondingly, nuclear expression of FosB and Fra1 was increased in TRAF3IP2-silenced cells. Many genes involved in host defense were induced by IL-17 in a TRAF3IP2-dependent fashion. Inflammatory differentiation conditions (serum addition for 4 days postconfluence) markedly amplified these IL-17 responses and increased basal levels and TRAF3IP2 silencing-dependent up-regulation of multiple late differentiation genes. These findings suggest that TRAF3IP2 may alter both epidermal homeostasis and keratinocyte defense responses to influence psoriasis risk. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

Indole-3-Carbinol Promotes Goblet-Cell Differentiation Regulating Wnt and Notch Signaling Pathways AhR-Dependently.

PubMed

Park, Joo-Hung; Lee, Jeong-Min; Lee, Eun-Jin; Hwang, Won-Bhin; Kim, Da-Jeong

2018-04-30

Using an in vitro model of intestinal organoids derived from intestinal crypts, we examined effects of indole-3-carbinol (I3C), a phytochemical that has anticancer and aryl hydrocarbon receptor (AhR)-activating abilities and thus is sold as a dietary supplement, on the development of intestinal organoids and investigated the underlying mechanisms. I3C inhibited the in vitro development of mouse intestinal organoids. Addition of α-naphthoflavone, an AhR antagonist or AhR siRNA transfection, suppressed I3C function, suggesting that I3C-mediated interference with organoid development is AhR-dependent. I3C increased the expression of Muc2 and lysozyme, lineage-specific genes for goblet cells and Paneth cells, respectively, but inhibits the expression of IAP, a marker gene for enterocytes. In the intestines of mice treated with I3C, the number of goblet cells was reduced, but the number of Paneth cells and the depth and length of crypts and villi were not changed. I3C increased the level of active nonphosphorylated β-catenin, but suppressed the Notch signal. As a result, expression of Hes1, a Notch target gene and a transcriptional repressor that plays a key role in enterocyte differentiation, was reduced, whereas expression of Math1, involved in the differentiation of secretory lineages, was increased. These results provide direct evidence for the role of AhR in the regulation of the development of intestinal stem cells and indicate that such regulation is likely mediated by regulation of Wnt and Notch signals.

Aging Reduces an ERRalpha-Directed Mitochondrial Glutaminase Expression Suppressing Glutamine Anaplerosis and Osteogenic Differentiation of Mesenchymal Stem Cells.

PubMed

Huang, Tongling; Liu, Renzhong; Fu, Xuekun; Yao, Dongsheng; Yang, Meng; Liu, Qingli; Lu, William W; Wu, Chuanyue; Guan, Min

2017-02-01

Aging deteriorates osteogenic capacity of mesenchymal stem/stromal cells (MSCs), contributing to imbalanced bone remodeling and osteoporosis. Glutaminase (Gls) catabolizes glutamine into glutamate at the first step of mitochondrial glutamine (Gln)-dependent anaplerosis which is essential for MSCs upon osteogenic differentiation. Estrogen-related receptor α (ERRα) regulates genes required for mitochondrial function. Here, we found that ERRα and Gls are upregulated by osteogenic induction in human MSCs (hMSCs). In contrast, osteogenic differentiation capacity and glutamine consumption of MSCs, as well as ERRα, Gls and osteogenic marker genes are significantly reduced with age. We demonstrated that ERRα binds to response elements on Gls promoter and affects glutamine anaplerosis through transcriptional induction of Gls. Conversely, mTOR inhibitor rapamycin, ERRα inverse agonist compound 29 or Gls inhibitor BPTES leads to reduced Gln anaplerosis and deteriorated osteogenic differentiation of hMSCs. Importantly, overexpression of ERRα or Gls restored impairment by these inhibitors. Finally, we proved that compensated ERRα or Gls expression indeed potentiated Gln anaplerosis and osteogenic capability of elderly mice MSCs in vitro. Together, we establish that Gls is a novel ERRα target gene and ERRα/Gls signaling pathway plays an important role in osteogenic differentiation of MSCs, providing new sights into novel regenerative therapeutics development. Our findings suggest that restoring age-related mitochondrial Gln-dependent anaplerosis may be beneficial for degenerative bone disorders such as osteoporosis. Stem Cells 2017;35:411-424. © 2016 AlphaMed Press.

Gene selection for the reconstruction of stem cell differentiation trees: a linear programming approach.

PubMed

Ghadie, Mohamed A; Japkowicz, Nathalie; Perkins, Theodore J

2015-08-15

Stem cell differentiation is largely guided by master transcriptional regulators, but it also depends on the expression of other types of genes, such as cell cycle genes, signaling genes, metabolic genes, trafficking genes, etc. Traditional approaches to understanding gene expression patterns across multiple conditions, such as principal components analysis or K-means clustering, can group cell types based on gene expression, but they do so without knowledge of the differentiation hierarchy. Hierarchical clustering can organize cell types into a tree, but in general this tree is different from the differentiation hierarchy itself. Given the differentiation hierarchy and gene expression data at each node, we construct a weighted Euclidean distance metric such that the minimum spanning tree with respect to that metric is precisely the given differentiation hierarchy. We provide a set of linear constraints that are provably sufficient for the desired construction and a linear programming approach to identify sparse sets of weights, effectively identifying genes that are most relevant for discriminating different parts of the tree. We apply our method to microarray gene expression data describing 38 cell types in the hematopoiesis hierarchy, constructing a weighted Euclidean metric that uses just 175 genes. However, we find that there are many alternative sets of weights that satisfy the linear constraints. Thus, in the style of random-forest training, we also construct metrics based on random subsets of the genes and compare them to the metric of 175 genes. We then report on the selected genes and their biological functions. Our approach offers a new way to identify genes that may have important roles in stem cell differentiation. tperkins@ohri.ca Supplementary data are available at Bioinformatics online. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

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

Generation of high-yield insulin producing cells from human bone marrow mesenchymal stem cells.

PubMed

Jafarian, Arefeh; Taghikhani, Mohammad; Abroun, Saeid; Pourpak, Zahra; Allahverdi, Amir; Soleimani, Masoud

2014-07-01

Allogenic islet transplantation is a most efficient approach for treatment of diabetes mellitus. However, the scarcity of islets and long term need for an immunosuppressant limits its application. Recently, cell replacement therapies that generate of unlimited sources of β cells have been developed to overcome these limitations. In this study we have described a stage specific differentiation protocol for the generation of insulin producing islet-like clusters from human bone marrow mesenchymal stem cells (hBM-MSCs). This specific stepwise protocol induced differentiation of hMSCs into definitive endoderm, pancreatic endoderm and pancreatic endocrine cells that expressed of sox17, foxa2, pdx1, ngn3, nkx2.2, insulin, glucagon, somatostatin, pancreatic polypeptide, and glut2 transcripts respectively. In addition, immunocytochemical analysis confirmed protein expression of the above mentioned genes. Western blot analysis discriminated insulin from proinsulin in the final differentiated cells. In derived insulin producing cells (IPCs), secreted insulin and C-peptide was in a glucose dependent manner. We have developed a protocol that generates effective high-yield human IPCs from hBM-MSCs in vitro. These finding suggest that functional IPCs generated by this procedure can be used as a cell-based approach for insulin dependent diabetes mellitus.

Differentiation-associated microRNAs antagonize the Rb–E2F pathway to restrict proliferation

PubMed Central

Marzi, Matteo J.; Puggioni, Eleonora M. R.; Dall'Olio, Valentina; Bucci, Gabriele; Bernard, Loris; Bianchi, Fabrizio; Crescenzi, Marco

2012-01-01

The cancer-associated loss of microRNA (miRNA) expression leads to a proliferative advantage and aggressive behavior through largely unknown mechanisms. Here, we exploit a model system that recapitulates physiological terminal differentiation and its reversal upon oncogene expression to analyze coordinated mRNA/miRNA responses. The cell cycle reentry of myotubes, forced by the E1A oncogene, was associated with a pattern of mRNA/miRNA modulation that was largely reciprocal to that induced during the differentiation of myoblasts into myotubes. The E1A-induced mRNA response was preponderantly Retinoblastoma protein (Rb)-dependent. Conversely, the miRNA response was mostly Rb-independent and exerted through tissue-specific factors and Myc. A subset of these miRNAs (miR-1, miR-34, miR-22, miR-365, miR-29, miR-145, and Let-7) was shown to coordinately target Rb-dependent cell cycle and DNA replication mRNAs. Thus, a dual level of regulation—transcriptional regulation via Rb–E2F and posttranscriptional regulation via miRNAs—confers robustness to cell cycle control and provides a molecular basis to understand the role of miRNA subversion in cancer. PMID:23027903

The Scaffold Attachment Factor SAFB1: A New Player in G2/M Checkpoint Control

DTIC Science & Technology

2007-04-01

RNA was obtained from locally advanced breast tumors in 24 patients before they underwent four cycles of neoadjuvant docetaxel treatment . Gene...expression analysis was performed and correlated to the treatment response to determine genes that are differentially expressed in docetaxel-sensitive...decreased sensitivity to drugs, depending on the chemotherapeutic agent used 2) Association of SAFB1 loss with resistance to docetaxel treatment , both

Trefoil factor 3 is required for differentiation of thyroid follicular cells and acts as a context-dependent tumor suppressor.

PubMed

Abols, A; Ducena, K; Andrejeva, D; Sadovska, L; Zandberga, E; Vilmanis, J; Narbuts, Z; Tars, J; Eglitis, J; Pirags, V; Line, A

2015-01-01

Trefoil factor 3 (TFF3) is overexpressed in a variety of solid epithelial cancers, where it has been shown to promote migration, invasion, proliferation, survival and angiogenesis. On the contrary, in the majority of thyroid tumors, it is downregulated, yet its role in the development of thyroid cancer remains unknown. Here we show that TFF3 exhibits strong cytoplasmic staining of normal thyroid follicular cells and colloid and the staining is increased in hyperfunctioning thyroid nodules, while it is decreased in all thyroid cancers of follicular cell origin. By meta-analysis of gene expression datasets, we found that in the thyroid cancer, conversely to the breast cancer, the expression of TFF3 mRNA was downregulated by estrogen signaling and confirmed this by treating thyroid cancer cells with estradiol. Forced expression of TFF3 in anaplastic thyroid cancer cells resulted in decreased cell proliferation, clonal spheroid formation and entry into the S phase. Furthermore, it induced acquisition of epithelial-like cell morphology and expression of the differentiation markers of thyroid follicular cells and transcription factors implicated in the thyroid morphogenesis and function. Taken together, this study provides the first evidence that TFF3 may act as a tumor suppressor or an oncogene depending on the cellular context.

Independent AMP and NAD signaling regulates C2C12 differentiation and metabolic adaptation.

PubMed

Hsu, Chia George; Burkholder, Thomas J

2016-12-01

The balance of ATP production and consumption is reflected in adenosine monophosphate (AMP) and nicotinamide adenine dinucleotide (NAD) content and has been associated with phenotypic plasticity in striated muscle. Some studies have suggested that AMPK-dependent plasticity may be an indirect consequence of increased NAD synthesis and SIRT1 activity. The primary goal of this study was to assess the interaction of AMP- and NAD-dependent signaling in adaptation of C2C12 myotubes. Changes in myotube developmental and metabolic gene expression were compared following incubation with 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) and nicotinamide mononucleotide (NMN) to activate AMPK- and NAD-related signaling. AICAR showed no effect on NAD pool or nampt expression but significantly reduced histone H3 acetylation and GLUT1, cytochrome C oxidase subunit 2 (COX2), and MYH3 expression. In contrast, NMN supplementation for 24 h increased NAD pool by 45 % but did not reduce histone H3 acetylation nor promote mitochondrial gene expression. The combination of AMP and NAD signaling did not induce further metabolic adaptation, but NMN ameliorated AICAR-induced myotube reduction. We interpret these results as indication that AMP and NAD contribute to C2C12 differentiation and metabolic adaptation independently.

Naringenin Inhibits Adipogenesis and Reduces Insulin Sensitivity and Adiponectin Expression in Adipocytes

PubMed Central

Richard, Allison J.; Ribnicky, David M.; Stephens, Jacqueline M.

2013-01-01

Adipose tissue development and function are widely studied to examine the relationship between obesity and the metabolic syndrome. It is well documented that the inability of adipose tissue to properly increase its lipid storage capacity during the obese state can lead to metabolic dysfunction. In a blind screen of 425 botanicals, we identified naringenin as an inhibitor of adipocyte differentiation. Naringenin is one of the most abundant citrus flavonoids, and recent studies have demonstrated antihyperlipidemic capabilities. These studies have largely focused on the effects of naringenin on the liver. Our biochemical studies clearly demonstrate that naringenin inhibits adipogenesis and impairs mature fat cell function. Naringenin specifically inhibited adipogenesis in a dose-dependent fashion as judged by examining lipid accumulation and induction of adipocyte marker protein expression. In mature 3T3-L1 adipocytes, naringenin reduced the ability of insulin to induce IRS-1 tyrosine phosphorylation and substantially inhibited insulin-stimulated glucose uptake in a dose-dependent manner and over a time frame of 1.5 to 24 hours. Exposure to naringenin also inhibited adiponectin protein expression in mature murine and human adipocytes. Our studies have revealed that naringenin may have a negative impact on adipocyte-related diseases by limiting differentiation of preadipocytes, by significantly inducing insulin resistance, and by decreasing adiponectin expression in mature fat cells. PMID:23983791

Identification of differentially expressed proteins during human urinary bladder cancer progression.

PubMed

Memon, Ashfaque A; Chang, Jong W; Oh, Bong R; Yoo, Yung J

2005-01-01

Comparative proteome analysis was performed between RT4 (grade-1) and T24 (grade-3) bladder cancer cell lines, in an attempt to identify differentially expressed proteins during bladder cancer progression. Among those relatively abundant proteins, seven spots changed more than two-fold reproducibly and identified by peptide mass fingerprinting using mass spectrometry and database search. We found most extensive and reproducible down-regulation of NADP dependent isocitrate dehydrogenase cytoplasmic (IDPc) and peroxiredoxin-II (Prx-II), in poorly differentiated T24 compared to well-differentiated RT4 bladder cancer cell line. Subsequent Western blotting analysis of human biopsy samples from bladder cancer patient revealed significant loss of IDPc and Prx-II in more advance tumor samples, in agreement with data on cell lines. These results suggest that loss of IDPc and Prx-II during tumor development may involve in tumor progression and metastasis. However, additional investigations are needed on large number of human samples to further verify these findings.

Neuronal differentiation is associated with a redox-regulated increase of copper flow to the secretory pathway

PubMed Central

Hatori, Yuta; Yan, Ye; Schmidt, Katharina; Furukawa, Eri; Hasan, Nesrin M.; Yang, Nan; Liu, Chin-Nung; Sockanathan, Shanthini; Lutsenko, Svetlana

2016-01-01

Brain development requires a fine-tuned copper homoeostasis. Copper deficiency or excess results in severe neuro-pathologies. We demonstrate that upon neuronal differentiation, cellular demand for copper increases, especially within the secretory pathway. Copper flow to this compartment is facilitated through transcriptional and metabolic regulation. Quantitative real-time imaging revealed a gradual change in the oxidation state of cytosolic glutathione upon neuronal differentiation. Transition from a broad range of redox states to a uniformly reducing cytosol facilitates reduction of the copper chaperone Atox1, liberating its metal-binding site. Concomitantly, expression of Atox1 and its partner, a copper transporter ATP7A, is upregulated. These events produce a higher flux of copper through the secretory pathway that balances copper in the cytosol and increases supply of the cofactor to copper-dependent enzymes, expression of which is elevated in differentiated neurons. Direct link between glutathione oxidation and copper compartmentalization allows for rapid metabolic adjustments essential for normal neuronal function. PMID:26879543

Neuronal differentiation is associated with a redox-regulated increase of copper flow to the secretory pathway.

PubMed

Hatori, Yuta; Yan, Ye; Schmidt, Katharina; Furukawa, Eri; Hasan, Nesrin M; Yang, Nan; Liu, Chin-Nung; Sockanathan, Shanthini; Lutsenko, Svetlana

2016-02-16

Brain development requires a fine-tuned copper homoeostasis. Copper deficiency or excess results in severe neuro-pathologies. We demonstrate that upon neuronal differentiation, cellular demand for copper increases, especially within the secretory pathway. Copper flow to this compartment is facilitated through transcriptional and metabolic regulation. Quantitative real-time imaging revealed a gradual change in the oxidation state of cytosolic glutathione upon neuronal differentiation. Transition from a broad range of redox states to a uniformly reducing cytosol facilitates reduction of the copper chaperone Atox1, liberating its metal-binding site. Concomitantly, expression of Atox1 and its partner, a copper transporter ATP7A, is upregulated. These events produce a higher flux of copper through the secretory pathway that balances copper in the cytosol and increases supply of the cofactor to copper-dependent enzymes, expression of which is elevated in differentiated neurons. Direct link between glutathione oxidation and copper compartmentalization allows for rapid metabolic adjustments essential for normal neuronal function.

CBX7 gene expression plays a negative role in adipocyte cell growth and differentiation

PubMed Central

Forzati, Floriana; Federico, Antonella; Pallante, Pierlorenzo; Colamaio, Marianna; Esposito, Francesco; Sepe, Romina; Gargiulo, Sara; Luciano, Antonio; Arra, Claudio; Palma, Giuseppe; Bon, Giulia; Bucher, Stefania; Falcioni, Rita; Brunetti, Arturo; Battista, Sabrina; Fedele, Monica; Fusco, Alfredo

2014-01-01

ABSTRACT We have recently generated knockout mice for the Cbx7 gene, coding for a polycomb group protein that is downregulated in human malignant neoplasias. These mice develop liver and lung adenomas and carcinomas, which confirms a tumour suppressor role for CBX7. The CBX7 ability to downregulate CCNE1 expression likely accounts for the phenotype of the Cbx7-null mice. Unexpectedly, Cbx7-knockout mice had a higher fat tissue mass than wild-type, suggesting a role of CBX7 in adipogenesis. Consistently, we demonstrate that Cbx7-null mouse embryonic fibroblasts go towards adipocyte differentiation more efficiently than their wild-type counterparts, and this effect is Cbx7 dose-dependent. Similar results were obtained when Cbx7-null embryonic stem cells were induced to differentiate into adipocytes. Conversely, mouse embryonic fibroblasts and human adipose-derived stem cells overexpressing CBX7 show an opposite behaviour. These findings support a negative role of CBX7 in the control of adipocyte cell growth and differentiation. PMID:25190058

Efficient and Reproducible Myogenic Differentiation from Human iPS Cells: Prospects for Modeling Miyoshi Myopathy In Vitro

PubMed Central

Tanaka, Akihito; Woltjen, Knut; Miyake, Katsuya; Hotta, Akitsu; Ikeya, Makoto; Yamamoto, Takuya; Nishino, Tokiko; Shoji, Emi; Sehara-Fujisawa, Atsuko; Manabe, Yasuko; Fujii, Nobuharu; Hanaoka, Kazunori; Era, Takumi; Yamashita, Satoshi; Isobe, Ken-ichi; Kimura, En; Sakurai, Hidetoshi

2013-01-01

The establishment of human induced pluripotent stem cells (hiPSCs) has enabled the production of in vitro, patient-specific cell models of human disease. In vitro recreation of disease pathology from patient-derived hiPSCs depends on efficient differentiation protocols producing relevant adult cell types. However, myogenic differentiation of hiPSCs has faced obstacles, namely, low efficiency and/or poor reproducibility. Here, we report the rapid, efficient, and reproducible differentiation of hiPSCs into mature myocytes. We demonstrated that inducible expression of myogenic differentiation1 (MYOD1) in immature hiPSCs for at least 5 days drives cells along the myogenic lineage, with efficiencies reaching 70–90%. Myogenic differentiation driven by MYOD1 occurred even in immature, almost completely undifferentiated hiPSCs, without mesodermal transition. Myocytes induced in this manner reach maturity within 2 weeks of differentiation as assessed by marker gene expression and functional properties, including in vitro and in vivo cell fusion and twitching in response to electrical stimulation. Miyoshi Myopathy (MM) is a congenital distal myopathy caused by defective muscle membrane repair due to mutations in DYSFERLIN. Using our induced differentiation technique, we successfully recreated the pathological condition of MM in vitro, demonstrating defective membrane repair in hiPSC-derived myotubes from an MM patient and phenotypic rescue by expression of full-length DYSFERLIN (DYSF). These findings not only facilitate the pathological investigation of MM, but could potentially be applied in modeling of other human muscular diseases by using patient-derived hiPSCs. PMID:23626698

CHD1 regulates cell fate determination by activation of differentiation-induced genes

PubMed Central

Baumgart, Simon J.; Najafova, Zeynab; Hossan, Tareq; Xie, Wanhua; Nagarajan, Sankari; Kari, Vijayalakshmi; Ditzel, Nicholas; Kassem, Moustapha

2017-01-01

Abstract The coordinated temporal and spatial activation of gene expression is essential for proper stem cell differentiation. The Chromodomain Helicase DNA-binding protein 1 (CHD1) is a chromatin remodeler closely associated with transcription and nucleosome turnover downstream of the transcriptional start site (TSS). In this study, we show that CHD1 is required for the induction of osteoblast-specific gene expression, extracellular-matrix mineralization and ectopic bone formation in vivo. Genome-wide occupancy analyses revealed increased CHD1 occupancy around the TSS of differentiation-activated genes. Furthermore, we observed that CHD1-dependent genes are mainly induced during osteoblast differentiation and are characterized by higher levels of CHD1 occupancy around the TSS. Interestingly, CHD1 depletion resulted in increased pausing of RNA Polymerase II (RNAPII) and decreased H2A.Z occupancy close to the TSS, but not at enhancer regions. These findings reveal a novel role for CHD1 during osteoblast differentiation and provide further insights into the intricacies of epigenetic regulatory mechanisms controlling cell fate determination. PMID:28475736

CHD1 regulates cell fate determination by activation of differentiation-induced genes.

PubMed

Baumgart, Simon J; Najafova, Zeynab; Hossan, Tareq; Xie, Wanhua; Nagarajan, Sankari; Kari, Vijayalakshmi; Ditzel, Nicholas; Kassem, Moustapha; Johnsen, Steven A

2017-07-27

The coordinated temporal and spatial activation of gene expression is essential for proper stem cell differentiation. The Chromodomain Helicase DNA-binding protein 1 (CHD1) is a chromatin remodeler closely associated with transcription and nucleosome turnover downstream of the transcriptional start site (TSS). In this study, we show that CHD1 is required for the induction of osteoblast-specific gene expression, extracellular-matrix mineralization and ectopic bone formation in vivo. Genome-wide occupancy analyses revealed increased CHD1 occupancy around the TSS of differentiation-activated genes. Furthermore, we observed that CHD1-dependent genes are mainly induced during osteoblast differentiation and are characterized by higher levels of CHD1 occupancy around the TSS. Interestingly, CHD1 depletion resulted in increased pausing of RNA Polymerase II (RNAPII) and decreased H2A.Z occupancy close to the TSS, but not at enhancer regions. These findings reveal a novel role for CHD1 during osteoblast differentiation and provide further insights into the intricacies of epigenetic regulatory mechanisms controlling cell fate determination. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

STIM1- and Orai1-dependent store-operated calcium entry regulates human myoblast differentiation.

PubMed

Darbellay, Basile; Arnaudeau, Serge; König, Stéphane; Jousset, Hélène; Bader, Charles; Demaurex, Nicolas; Bernheim, Laurent

2009-02-20

Our previous work on human myoblasts suggested that a hyperpolarization followed by a rise in [Ca(2+)](in) involving store-operated Ca(2+) entry (SOCE) channels induced myoblast differentiation. Advances in the understanding of the SOCE pathway led us to examine more precisely its role in post-natal human myoblast differentiation. We found that SOCE orchestrated by STIM1, the endoplasmic reticulum Ca(2+) sensor activating Orai Ca(2+) channels, is crucial. Silencing STIM1, Orai1, or Orai3 reduced SOCE amplitude and myoblast differentiation, whereas Orai2 knockdown had no effect. Conversely, overexpression of STIM1 with Orai1 increased SOCE and accelerated myoblast differentiation. STIM1 or Orai1 silencing decreased resting [Ca(2+)](in) and intracellular Ca(2+) store content, but correction of these parameters did not rescue myoblast differentiation. Remarkably, SOCE amplitude correlated linearly with the expression of two early markers of myoblast differentiation, MEF2 and myogenin, regardless of the STIM or Orai isoform that was silenced. Unexpectedly, we found that the hyperpolarization also depends on SOCE, placing SOCE upstream of K(+) channel activation in the signaling cascade that controls myoblast differentiation. These findings indicate that STIM1 and Orai1 are key molecules for the induction of human myoblast differentiation.

Activation of TRPV2 negatively regulates the differentiation of mouse brown adipocytes.

PubMed

Sun, Wuping; Uchida, Kunitoshi; Takahashi, Nobuyuki; Iwata, Yuko; Wakabayashi, Shigeo; Goto, Tsuyoshi; Kawada, Teruo; Tominaga, Makoto

2016-09-01

Transient receptor potential vanilloid 2 (TRPV2) acts as a Ca(2+)-permeable non-selective cation channel that has been reported to be sensitive to temperature, mechanical force, and some chemicals. We recently showed that TRPV2 is critical for maintenance of the thermogenic function of brown adipose tissue in mice. However, the involvement of TRPV2 in the differentiation of brown adipocytes remains unexplored. We found that the expression of TRPV2 was dramatically increased during the differentiation of brown adipocytes. Non-selective TRPV2 agonists (2-aminoethoxydiphenyl borate and lysophosphatidylcholine) inhibited the differentiation of brown adipocytes in a dose-dependent manner during the early stage of differentiation of brown adipocytes. The inhibition was rescued by a TRPV2-selective antagonist, SKF96365 (SKF). Mechanical force, which activates TRPV2, also inhibited the differentiation of brown adipocytes in a strength-dependent manner, and the effect was reversed by SKF. In addition, the inhibition of adipocyte differentiation by either TRPV2 ligand or mechanical stimulation was significantly smaller in the cells from TRPV2KO mice. Moreover, calcineurin inhibitors, cyclosporine A and FK506, partially reversed TRPV2 activation-induced inhibition of brown adipocyte differentiation. Thus, we conclude that TRPV2 might be involved in the modulation of brown adipocyte differentiation partially via a calcineurin pathway.

TULA-2, a novel histidine phosphatase regulates bone remodeling by modulating osteoclast function

PubMed Central

Back, Steven H.; Adapala, Naga Suresh; Barbe, Mary F.; Carpino, Nick C.; Tsygankov, Alexander Y.; Sanjay, Archana

2013-01-01

Bone is a dynamic tissue that depends on the intricate relationship between protein tyrosine kinases (PTK) and protein tyrosine phosphatases (PTP) for maintaining homeostasis. PTKs and PTPs act like molecular on and off switches and help modulate differentiation and the attachment of osteoclasts to bone matrix regulating bone resorption. The novel protein T-cell Ubiquitin Ligand-2 (TULA-2), which is abundantly expressed in osteoclasts, is a novel histidine phosphatase. Our results show that of the two family members only TULA-2 is expressed in osteoclasts and that its expression is sustained throughout the course of osteoclast differentiation suggesting that TULA-2 may play a role during early as well late stages of osteoclast differentiation. Skeletal analysis of mice that do not express TULA or TULA-2 proteins (DKO Mice) revealed that there was a decrease in bone volume due to increased osteoclast numbers and function. Furthermore, in vitro experiments indicated that bone marrow precursor cells from DKO mice have an increased potential to form osteoclasts. At the molecular level, the absence of TULA-2 in osteoclasts results in increased Syk phosphorylation at the Y352 and Y525/526 residues and activation of phospholipase C gamma 2 (PLCγ2) upon engagement of Immune-receptor-Tyrosine-based-Activation-Motif (ITAM)–mediated signaling. Furthermore, expression of a phosphatase-dead TULA-2 leads to increased osteoclast function. Taken together, these results suggest that TULA-2 negatively regulates osteoclast differentiation and function. PMID:23149425

TULA-2, a novel histidine phosphatase, regulates bone remodeling by modulating osteoclast function.

PubMed

Back, Steven H; Adapala, Naga Suresh; Barbe, Mary F; Carpino, Nick C; Tsygankov, Alexander Y; Sanjay, Archana

2013-04-01

Bone is a dynamic tissue that depends on the intricate relationship between protein tyrosine kinases (PTK) and protein tyrosine phosphatases (PTP) for maintaining homeostasis. PTKs and PTPs act like molecular on and off switches and help modulate differentiation and the attachment of osteoclasts to bone matrix regulating bone resorption. The protein T cell ubiquitin ligand-2 (TULA-2), which is abundantly expressed in osteoclasts, is a novel histidine phosphatase. Our results show that of the two family members, only TULA-2 is expressed in osteoclasts and that its expression is sustained throughout the course of osteoclast differentiation, suggesting that TULA-2 may play a role during early as well late stages of osteoclast differentiation. Skeletal analysis of mice that do not express TULA or TULA-2 proteins (DKO mice) revealed that there was a decrease in bone volume due to increased osteoclast numbers and function. Furthermore, in vitro experiments indicated that bone marrow precursor cells from DKO mice have an increased potential to form osteoclasts. At the molecular level, the absence of TULA-2 in osteoclasts results in increased Syk phosphorylation at the Y352 and Y525/526 residues and activation of phospholipase C gamma 2 (PLCγ2) upon engagement of immune-receptor-tyrosine-based-activation-motif (ITAM)-mediated signaling. Furthermore, expression of a phosphatase-dead TULA-2 leads to increased osteoclast function. Taken together, these results suggest that TULA-2 negatively regulates osteoclast differentiation and function.

The transcriptional repressor DREAM is involved in thyroid gene expression

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

D'Andrea, Barbara; Di Palma, Tina; Mascia, Anna

2005-04-15

Downstream regulatory element antagonistic modulator (DREAM) was originally identified in neuroendocrine cells as a calcium-binding protein that specifically binds to downstream regulatory elements (DRE) on DNA, and represses transcription of its target genes. To explore the possibility that DREAM may regulate the endocrine activity of the thyroid gland, we analyzed its mRNA expression in undifferentiated and differentiated thyroid cells. We demonstrated that DREAM is expressed in the normal thyroid tissue as well as in differentiated thyroid cells in culture while it is absent in FRT poorly differentiated cells. In the present work, we also show that DREAM specifically binds tomore » DRE sites identified in the 5' untranslated region (UTR) of the thyroid-specific transcription factors Pax8 and TTF-2/FoxE1 in a calcium-dependent manner. By gel retardation assays we demonstrated that thapsigargin treatment increases the binding of DREAM to the DRE sequences present in Pax8 and TTF-2/Foxe1 5' UTRs, and this correlates with a significant reduction of the expression of these genes. Interestingly, in poorly differentiated thyroid cells overexpression of exogenous DREAM strongly inhibits Pax8 expression. Moreover, we provide evidence that a mutated form of DREAM unable to bind Ca{sup 2+} interferes with thyroid cell proliferation. Therefore, we propose that in thyroid cells DREAM is a mediator of the calcium-signaling pathway and it is involved in the regulation of thyroid cell function.« less

Inhibition of 5α-Reductase in Rat Prostate Reveals Differential Regulation of Androgen-Response Gene Expression by Testosterone and Dihydrotestosterone

PubMed Central

Dadras, Soheil S.; Cai, Xiaoyan; Abasolo, Ibane; Wang, Zhou

2001-01-01

The growth and development of some of the male sex accessory organs such as the prostate requires the conversion of testosterone to dihydrotestosterone (DHT) by 5α-reductase. To provide insights into the role of testosterone versus DHT in the prostate, we studied the impact of finasteride, a potent and specific inhibitor of 5α-reductase, on the expression of prostatic androgen-response genes in testis-intact rats and in 7-day castrated rats. Finasteride inhibition of the conversion of testosterone to DHT was confirmed by measuring serum and intraprostatic androgens. As expected, finasteride treatment caused a reduction in the wet weight of the prostate in the testis-intact rats and inhibited the testosterone-stimulated prostatic regrowth in the 7-day castrated rats. Although finasteride treatment had little or no effect on the expression of the surveyed androgen-response genes in testis-intact rats, its administration enhanced the expression of many androgen-response genes during the testosterone-stimulated regrowth of the regressed prostate in castrated rats. These observations suggest that testosterone is more potent than DHT in stimulating the expression of many androgen-response genes in the regressed prostate. The expression of androgen-response genes is mainly prostate specific and thus is likely to be associated with androgen-dependent prostatic differentiation. Therefore, testosterone is more potent than DHT in inducing differentiation and weaker in stimulating proliferation during prostate regrowth. The fact that testosterone is a strong inducer of prostatic differentiation has potential clinical implications. PMID:11444528

Binary Cell Fate Decisions and Fate Transformation in the Drosophila Larval Eye

PubMed Central

Rister, Jens; Ng, June; Celik, Arzu; Sprecher, Simon G.

2013-01-01

The functionality of sensory neurons is defined by the expression of specific sensory receptor genes. During the development of the Drosophila larval eye, photoreceptor neurons (PRs) make a binary choice to express either the blue-sensitive Rhodopsin 5 (Rh5) or the green-sensitive Rhodopsin 6 (Rh6). Later during metamorphosis, ecdysone signaling induces a cell fate and sensory receptor switch: Rh5-PRs are re-programmed to express Rh6 and become the eyelet, a small group of extraretinal PRs involved in circadian entrainment. However, the genetic and molecular mechanisms of how the binary cell fate decisions are made and switched remain poorly understood. We show that interplay of two transcription factors Senseless (Sens) and Hazy control cell fate decisions, terminal differentiation of the larval eye and its transformation into eyelet. During initial differentiation, a pulse of Sens expression in primary precursors regulates their differentiation into Rh5-PRs and repression of an alternative Rh6-cell fate. Later, during the transformation of the larval eye into the adult eyelet, Sens serves as an anti-apoptotic factor in Rh5-PRs, which helps in promoting survival of Rh5-PRs during metamorphosis and is subsequently required for Rh6 expression. Comparably, during PR differentiation Hazy functions in initiation and maintenance of rhodopsin expression. Hazy represses Sens specifically in the Rh6-PRs, allowing them to die during metamorphosis. Our findings show that the same transcription factors regulate diverse aspects of larval and adult PR development at different stages and in a context-dependent manner. PMID:24385925

Binary cell fate decisions and fate transformation in the Drosophila larval eye.

PubMed

Mishra, Abhishek Kumar; Tsachaki, Maria; Rister, Jens; Ng, June; Celik, Arzu; Sprecher, Simon G

2013-01-01

The functionality of sensory neurons is defined by the expression of specific sensory receptor genes. During the development of the Drosophila larval eye, photoreceptor neurons (PRs) make a binary choice to express either the blue-sensitive Rhodopsin 5 (Rh5) or the green-sensitive Rhodopsin 6 (Rh6). Later during metamorphosis, ecdysone signaling induces a cell fate and sensory receptor switch: Rh5-PRs are re-programmed to express Rh6 and become the eyelet, a small group of extraretinal PRs involved in circadian entrainment. However, the genetic and molecular mechanisms of how the binary cell fate decisions are made and switched remain poorly understood. We show that interplay of two transcription factors Senseless (Sens) and Hazy control cell fate decisions, terminal differentiation of the larval eye and its transformation into eyelet. During initial differentiation, a pulse of Sens expression in primary precursors regulates their differentiation into Rh5-PRs and repression of an alternative Rh6-cell fate. Later, during the transformation of the larval eye into the adult eyelet, Sens serves as an anti-apoptotic factor in Rh5-PRs, which helps in promoting survival of Rh5-PRs during metamorphosis and is subsequently required for Rh6 expression. Comparably, during PR differentiation Hazy functions in initiation and maintenance of rhodopsin expression. Hazy represses Sens specifically in the Rh6-PRs, allowing them to die during metamorphosis. Our findings show that the same transcription factors regulate diverse aspects of larval and adult PR development at different stages and in a context-dependent manner.

Differential replication dynamics for large and small Vibrio chromosomes affect gene dosage, expression and location

PubMed Central

Dryselius, Rikard; Izutsu, Kaori; Honda, Takeshi; Iida, Tetsuya

2008-01-01

Background Replication of bacterial chromosomes increases copy numbers of genes located near origins of replication relative to genes located near termini. Such differential gene dosage depends on replication rate, doubling time and chromosome size. Although little explored, differential gene dosage may influence both gene expression and location. For vibrios, a diverse family of fast growing gammaproteobacteria, gene dosage may be particularly important as they harbor two chromosomes of different size. Results Here we examined replication dynamics and gene dosage effects for the separate chromosomes of three Vibrio species. We also investigated locations for specific gene types within the genome. The results showed consistently larger gene dosage differences for the large chromosome which also initiated replication long before the small. Accordingly, large chromosome gene expression levels were generally higher and showed an influence from gene dosage. This was reflected by a higher abundance of growth essential and growth contributing genes of which many locate near the origin of replication. In contrast, small chromosome gene expression levels were low and appeared independent of gene dosage. Also, species specific genes are highly abundant and an over-representation of genes involved in transcription could explain its gene dosage independent expression. Conclusion Here we establish a link between replication dynamics and differential gene dosage on one hand and gene expression levels and the location of specific gene types on the other. For vibrios, this relationship appears connected to a polarisation of genetic content between its chromosomes, which may both contribute to and be enhanced by an improved adaptive capacity. PMID:19032792

Perceived facial expressions of emotion as motivational incentives: evidence from a differential implicit learning paradigm.

PubMed

Schultheiss, Oliver C; Pang, Joyce S; Torges, Cynthia M; Wirth, Michelle M; Treynor, Wendy; Derryberry, Douglas

2005-03-01

Participants (N = 216) were administered a differential implicit learning task during which they were trained and tested on 3 maximally distinct 2nd-order visuomotor sequences, with sequence color serving as discriminative stimulus. During training, 1 sequence each was followed by an emotional face, a neutral face, and no face, using backward masking. Emotion (joy, surprise, anger), face gender, and exposure duration (12 ms, 209 ms) were varied between participants; implicit motives were assessed with a picture-story exercise. For power-motivated individuals, low-dominance facial expressions enhanced and high-dominance expressions impaired learning. For affiliation-motivated individuals, learning was impaired in the context of hostile faces. These findings did not depend on explicit learning of fixed sequences or on awareness of sequence-face contingencies. Copyright 2005 APA, all rights reserved.

Expression profiles of amhy and major sex-related genes during gonadal sex differentiation and their relation with genotypic and temperature-dependent sex determination in pejerrey Odontesthes bonariensis.

PubMed

Zhang, Yan; Hattori, Ricardo S; Sarida, Munti; García, Estefany L; Strüssmann, Carlos Augusto; Yamamoto, Yoji

2018-03-15

To shed light on the mechanisms of and interactions of GSD and TSD in pejerrey, we investigated how the transcriptional profiles of amhy and amha are affected by feminizing (17 °C) and masculinizing (29 °C) temperatures during the critical period of sex determination/differentiation and their relation with the expression profiles of AMH receptor type II (amhrII), gonadal aromatase (cyp19a1a), and 11 beta-hydroxysteroid dehydrogenase 2 (hsd11b2). Careful consideration of the results of this study and all information currently available for this species, including similar analyzes for an intermediate, mixed-sex promoting temperature (25 °C), suggests a model for genotypic/temperature-dependent sex determination and gonadal sex differentiation that involves a) cyp19a1a-dependent, developmentally-programmed ovarian development as the default state that becomes self-sustaining in the absence of a potent and timely masculinizing stimulus, b) early, developmentally-programmed amhy expression and high temperature as masculinization signals that antagonize the putative female pathway by suppressing cyp19a1a expression, c) increasing stress response, cortisol, and the synthesis of the masculinizing androgen 11-keto-testosterone via hsd11b2 with increasing temperature that is important for masculinization in both genotypes but particularly so in XX individuals, and d) an endocrine network with positive/negative feedback mechanisms that ensure fidelity of the male/female pathway once started. The proposed model, albeit tentative and non-all inclusive, accounts for the continuum of responses, from all-females at low temperatures to all-males at high temperatures and for the balanced-, genotype-linked sex ratios obtained at intermediate temperatures, and therefore supports the coexistence of TSD and GSD in pejerrey across the range of viable temperatures for this species. Copyright © 2018 Elsevier Inc. All rights reserved.

Age-Dependent Decline in Mouse Lung Regeneration with Loss of Lung Fibroblast Clonogenicity and Increased Myofibroblastic Differentiation

PubMed Central

Paxson, Julia A.; Gruntman, Alisha; Parkin, Christopher D.; Mazan, Melissa R.; Davis, Airiel; Ingenito, Edward P.; Hoffman, Andrew M.

2011-01-01

While aging leads to a reduction in the capacity for regeneration after pneumonectomy (PNX) in most mammals, this biological phenomenon has not been characterized over the lifetime of mice. We measured the age-specific (3, 9, 24 month) effects of PNX on physiology, morphometry, cell proliferation and apoptosis, global gene expression, and lung fibroblast phenotype and clonogenicity in female C57BL6 mice. The data show that only 3 month old mice were fully capable of restoring lung volumes by day 7 and total alveolar surface area by 21 days. By 9 months, the rate of regeneration was slower (with incomplete regeneration by 21 days), and by 24 months there was no regrowth 21 days post-PNX. The early decline in regeneration rate was not associated with changes in alveolar epithelial cell type II (AECII) proliferation or apoptosis rate. However, significant apoptosis and lack of cell proliferation was evident after PNX in both total cells and AECII cells in 24 mo mice. Analysis of gene expression at several time points (1, 3 and 7 days) post-PNX in 9 versus 3 month mice was consistent with a myofibroblast signature (increased Tnc, Lox1, Col3A1, Eln and Tnfrsf12a) and more alpha smooth muscle actin (αSMA) positive myofibroblasts were present after PNX in 9 month than 3 month mice. Isolated lung fibroblasts showed a significant age-dependent loss of clonogenicity. Moreover, lung fibroblasts isolated from 9 and 17 month mice exhibited higher αSMA, Col3A1, Fn1 and S100A expression, and lower expression of the survival gene Mdk consistent with terminal differentiation. These data show that concomitant loss of clonogenicity and progressive myofibroblastic differentiation contributes to the age-dependent decline in the rate of lung regeneration. PMID:21912590

Global analysis of epigenetic regulation of gene expression in response to drought stress in Sorghum.

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

Reddy, Anireddy; Ben-Hur, Asa

Abiotic stresses including drought are major limiting factors of crop yields and cause significant crop losses. Acquisition of stress tolerance to abiotic stresses requires coordinated regulation of a multitude of biochemical and physiological changes, and most of these changes depend on alterations in gene expression. The goal of this work is to perform global analysis of differential regulation of gene expression and alternative splicing, and their relationship with chromatin landscape in drought sensitive and tolerant cultivars. our Iso-Seq study revealed transcriptome-wide full-length isoforms at an unprecedented scale with over 11000 novel splice isoforms. Additionally, we uncovered alternative polyadenylation sites ofmore » ~11000 expressed genes and many novel genes. Overall, Iso-Seq results greatly enhanced sorghum gene annotations that are not only useful in analyzing all our RNA-seq, ChIP-seq and ATAC-seq data but also serve as a great resource to the plant biology community. Our studies identified differentially expressed genes and splicing events that are correlated with the drought-resistant phenotype. An association between alternative splicing and chromatin accessibility was also revealed. Several computational tools developed here (TAPIS and iDiffIR) have been made freely available to the research community in analyzing alternative splicing and differential alternative splicing.« less

Does the liposuction method influence the phenotypic characteristic of human adipose-derived stem cells?

PubMed

Bajek, Anna; Gurtowska, Natalia; Gackowska, Lidia; Kubiszewska, Izabela; Bodnar, Magdalena; Marszałek, Andrzej; Januszewski, Rafał; Michalkiewicz, Jacek; Drewa, Tomasz

2015-05-14

Adipose-derived stem cells (ASCs) possess a high differentiation and proliferation potential. However, the phenotypic characterization of ASCs is still difficult. Until now, there is no extensive analysis of ASCs markers depending on different liposuction methods. Therefore, the aim of the present study was to analyse 242 surface markers and determine the differences in the phenotypic pattern between ASCs obtained during mechanical and ultrasound-assisted liposuction. ASCs were isolated from healthy donors, due to mechanical and ultrasound-assisted liposuction and cultured in standard medium to the second passage. Differentiation potential and markers expression was evaluated to confirm the mesenchymal nature of cells. Then, the BD LyoplateTM Human Cell Surface Marker Screening Panel was used. Results shown that both population of ASCs are characterized by high expression of markers specific for ASCs: cluster of differentiation (CD)9, CD10, CD34, CD44, CD49d, CD54, CD55, CD59, CD71 and low expression of CD11a, CD11c and CD144. Moreover, we have noticed significant differences in antigen expression in 58 markers from the 242 studied. Presented study shows for the first time that different liposuction methods are not a significant factor which can influence the expression of human ASCs surface markers. © 2015 The Authors.

The NO signaling pathway differentially regulates KCC3a and KCC3b mRNA expression.

PubMed

Di Fulvio, Mauricio; Lauf, Peter K; Adragna, Norma C

2003-11-01

Nitric oxide (NO) donors and protein kinase G (PKG) acutely up-regulate K-Cl cotransporter-1 and -3 (KCC1 and KCC3) mRNA expression in vascular smooth muscle cells (VSMCs). Here, we report the presence, relative abundance, and regulation by sodium nitroprusside (SNP) of the novel KCC3a and KCC3b mRNAs, in primary cultures of rat VSMCs. KCC3a and KCC3b mRNAs were expressed in an approximate 3:1 ratio, as determined by semiquantitative RT-PCR analysis. SNP as well as YC-1 and 8-Br-cGMP, a NO-independent stimulator of soluble guanylyl cyclase (sGC) and PKG, respectively, increased KCC3a and KCC3b mRNA expression by 2.5-fold and 8.1-fold in a time-dependent manner, following a differential kinetics. Stimulation of the NO/sGC/PKG signaling pathway with either SNP, YC-1, or 8-Br-cGMP decreased the KCC3a/KCC3b ratio from 3.0+/-0.4 to 0.9+/-0.1. This is the first report on a differential regulation by the NO/sGC/PKG signaling pathway of a cotransporter and of KCC3a and KCC3b mRNA expression.

Monocyte-lymphocyte fusion induced by the HIV-1 envelope generates functional heterokaryons with an activated monocyte-like phenotype.

PubMed

Martínez-Méndez, David; Rivera-Toledo, Evelyn; Ortega, Enrique; Licona-Limón, Ileana; Huerta, Leonor

2017-03-01

Enveloped viruses induce cell-cell fusion when infected cells expressing viral envelope proteins interact with target cells, or through the contact of cell-free viral particles with adjoining target cells. CD4 + T lymphocytes and cells from the monocyte-macrophage lineage express receptors for HIV envelope protein. We have previously reported that lymphoid Jurkat T cells expressing the HIV-1 envelope protein (Env) can fuse with THP-1 monocytic cells, forming heterokaryons with a predominantly myeloid phenotype. This study shows that the expression of monocytic markers in heterokaryons is stable, whereas the expression of lymphoid markers is mostly lost. Like THP-1 cells, heterokaryons exhibited FcγR-dependent phagocytic activity and showed an enhanced expression of the activation marker ICAM-1 upon stimulation with PMA. In addition, heterokaryons showed morphological changes compatible with maturation, and high expression of the differentiation marker CD11b in the absence of differentiation-inducing agents. No morphological change nor increase in CD11b expression were observed when an HIV-fusion inhibitor blocked fusion, or when THP-1 cells were cocultured with Jurkat cells expressing a non-fusogenic Env protein, showing that differentiation was not induced merely by cell-cell interaction but required cell-cell fusion. Inhibition of TLR2/TLR4 signaling by a TIRAP inhibitor greatly reduced the expression of CD11b in heterokaryons. Thus, lymphocyte-monocyte heterokaryons induced by HIV-1 Env are stable and functional, and fusion prompts a phenotype characteristic of activated monocytes via intracellular TLR2/TLR4 signaling. Copyright © 2017 Elsevier Inc. All rights reserved.

Transcriptomic analysis reveals differential gene expression in response to aluminium in common bean (Phaseolus vulgaris) genotypes

PubMed Central

Eticha, Dejene; Zahn, Marc; Bremer, Melanie; Yang, Zhongbao; Rangel, Andrés F.; Rao, Idupulapati M.; Horst, Walter J.

2010-01-01

Background and Aims Aluminium (Al) resistance in common bean is known to be due to exudation of citrate from the root after a lag phase, indicating the induction of gene transcription and protein synthesis. The aims of this study were to identify Al-induced differentially expressed genes and to analyse the expression of candidate genes conferring Al resistance in bean. Methods The suppression subtractive hybridization (SSH) method was used to identify differentially expressed genes in an Al-resistant bean genotype (‘Quimbaya’) during the induction period. Using quantitative real-time PCR the expression patterns of selected genes were compared between an Al-resistant and an Al-sensitive genotype (‘VAX 1’) treated with Al for up to 24 h. Key Results Short-term Al treatment resulted in up-regulation of stress-induced genes and down-regulation of genes involved in metabolism. However, the expressions of genes encoding enzymes involved in citrate metabolism were not significantly affected by Al. Al treatment dramatically increased the expression of common bean expressed sequence tags belonging to the citrate transporter gene family MATE (multidrug and toxin extrusion family protein) in both the Al-resistant and -sensitive genotype in close agreement with Al-induced citrate exudation. Conclusions The expression of a citrate transporter MATE gene is crucial for citrate exudation in common bean. However, although the expression of the citrate transporter is a prerequisite for citrate exudation, genotypic Al resistance in common bean particularly depends on the capacity to sustain the synthesis of citrate for maintaining the cytosolic citrate pool that enables exudation. PMID:20237115

Functional link between DNA damage responses and transcriptional regulation by ATM in response to a histone deacetylase inhibitor TSA.

PubMed

Lee, Jong-Soo

2007-09-01

Mutations in the ATM (ataxia-telangiectasia mutated) gene, which encodes a 370 kd protein with a kinase catalytic domain, predisposes people to cancers, and these mutations are also linked to ataxia-telangiectasia (A-T). The histone acetylaion/deacetylation- dependent chromatin remodeling can activate the ATM kinase-mediated DNA damage signal pathway (in an accompanying work, Lee, 2007). This has led us to study whether this modification can impinge on the ATM-mediated DNA damage response via transcriptional modulation in order to understand the function of ATM in the regulation of gene transcription. To identify the genes whose expression is regulated by ATM in response to histone deaceylase (HDAC) inhibition, we performed an analysis of oligonucleotide microarrays with using the appropriate cell lines, isogenic A-T (ATM(-)) and control (ATM(+)) cells, following treatment with a HDAC inhibitor TSA. Treatment with TSA reprograms the differential gene expression profile in response to HDAC inhibition in ATM(-) cells and ATM(+) cells. We analyzed the genes that are regulated by TSA in the ATM-dependent manner, and we classified these genes into different functional categories, including those involved in cell cycle/DNA replication, DNA repair, apoptosis, growth/differentiation, cell- cell adhesion, signal transduction, metabolism and transcription. We found that while some genes are regulated by TSA without regard to ATM, the patterns of gene regulation are differentially regulated in an ATM-dependent manner. Taken together, these finding indicate that ATM can regulate the transcription of genes that play critical roles in the molecular response to DNA damage, and this response is modulated through an altered HDAC inhibition-mediated gene expression.

LIF inhibits osteoblast differentiation at least in part by regulation of HAS2 and its product hyaluronan.

PubMed

Falconi, Dominic; Aubin, Jane E

2007-08-01

LIF arrests osteogenesis in fetal rat calvaria cells in a differentiation stage-specific manner. Differential display identified HAS2 as a LIF-induced gene and its product, HA, modulated osteoblast differentiation similarly to LIF. Our data suggest that LIF arrests osteoblast differentiation by altering HA content of the extracellular matrix. Leukemia inhibitory factor (LIF) elicits both anabolic and catabolic effects on bone. We previously showed in the fetal rat calvaria (RC) cell system that LIF inhibits osteoblast differentiation at the late osteoprogenitor/early osteoblast stage. To uncover potential molecular mediators of this inhibitory activity, we used a positive-negative genome-wide differential display screen to identify LIF-induced changes in the developing osteoblast transcriptome. Although LIF signaling is active throughout the RC cell proliferation-differentiation sequence, only a relatively small number of genes, in several different functional clusters, are modulated by LIF specifically during the LIF-sensitive inhibitory time window. Based on their known and predicted functions, most of the LIF-regulated genes identified are plausible candidates to be involved in the LIF-induced arrest of osteoprogenitor differentiation. To test this hypothesis, we further analyzed the function of one of the genes identified, hyaluronan synthase 2 (HAS2), in the LIF-induced inhibition. Synthesis of hyaluronan (HA), the product of HAS enzymatic activity, was stimulated by LIF and mimicked the HAS2 expression profile, with highest expression in early/proliferative and late/maturing cultures and lowest levels in intermediate/late osteoprogenitor-early osteoblast cultures. Exogenously added high molecular weight HA, the product of HAS2, dose-dependently inhibited osteoblast differentiation, with pulse-treatment effective in the same differentiation stage-specific inhibitory window as seen with LIF. In addition, however, pulse treatment with HA in early cultures slightly increased bone nodule formation. Treatment with hyaluronidase, on the other hand, stimulated bone nodule formation in early cultures but caused a small dose-dependent inhibition of osteoblast differentiation in the LIF- and HA-sensitive late time window. Together the data suggest that osteoblast differentiation is acutely sensitive to HA levels and that LIF inhibits osteoblast development at least in part by stimulating high molecular weight HA synthesis through HAS2.

The calcineurin pathway links hyperpolarization (Kir2.1)-induced Ca2+ signals to human myoblast differentiation and fusion.

PubMed

Konig, Stéphane; Béguet, Anne; Bader, Charles R; Bernheim, Laurent

2006-08-01

In human myoblasts triggered to differentiate, a hyperpolarization, resulting from K+ channel (Kir2.1) activation, allows the generation of an intracellular Ca2+ signal. This signal induces an increase in expression/activity of two key transcription factors of the differentiation process, myogenin and MEF2. Blocking hyperpolarization inhibits myoblast differentiation. The link between hyperpolarization-induced Ca2+ signals and the four main regulatory pathways involved in myoblast differentiation was the object of this study. Of the calcineurin, p38-MAPK, PI3K and CaMK pathways, only the calcineurin pathway was inhibited when Kir2.1-linked hyperpolarization was blocked. The CaMK pathway, although Ca2+ dependent, is unaffected by changes in membrane potential or block of Kir2.1 channels. Concerning the p38-MAPK and PI3K pathways, their activity is present already in proliferating myoblasts and they are unaffected by hyperpolarization or Kir2.1 channel block. We conclude that the Kir2.1-induced hyperpolarization triggers human myoblast differentiation via the activation of the calcineurin pathway, which, in turn, induces expression/activity of myogenin and MEF2.

LIN28A enhances the therapeutic potential of cultured neural stem cells in a Parkinson's disease model.

PubMed

Rhee, Yong-Hee; Kim, Tae-Ho; Jo, A-Young; Chang, Mi-Yoon; Park, Chang-Hwan; Kim, Sang-Mi; Song, Jae-Jin; Oh, Sang-Min; Yi, Sang-Hoon; Kim, Hyeon Ho; You, Bo-Hyun; Nam, Jin-Wu; Lee, Sang-Hun

2016-10-01

The original properties of tissue-specific stem cells, regardless of their tissue origins, are inevitably altered during in vitro culturing, lessening the clinical and research utility of stem cell cultures. Specifically, neural stem cells derived from the ventral midbrain lose their dopamine neurogenic potential, ventral midbrain-specific phenotypes, and repair capacity during in vitro cell expansion, all of which are critical concerns in using the cultured neural stem cells in therapeutic approaches for Parkinson's disease. In this study, we observed that the culture-dependent changes of neural stem cells derived from the ventral midbrain coincided with loss of RNA-binding protein LIN28A expression. When LIN28A expression was forced and sustained during neural stem cell expansion using an inducible expression-vector system, loss of dopamine neurogenic potential and midbrain phenotypes after long-term culturing was blocked. Furthermore, dopamine neurons that differentiated from neural stem cells exhibited remarkable survival and resistance against toxic insults. The observed effects were not due to a direct action of LIN28A on the differentiated dopamine neurons, but rather its action on precursor neural stem cells as exogene expression was switched off in the differentiating/differentiated cultures. Remarkable and reproducible behavioural recovery was shown in all Parkinson's disease rats grafted with neural stem cells expanded with LIN28A expression, along with extensive engraftment of dopamine neurons expressing mature neuronal and midbrain-specific markers. These findings suggest that LIN28A expression during stem cell expansion could be used to prepare therapeutically competent donor cells. © The Author (2016). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Levetiracetam attenuates hippocampal expression of synaptic plasticity-related immediate early and late response genes in amygdala-kindled rats

PubMed Central

2010-01-01

Background The amygdala-kindled rat is a model for human temporal lobe epilepsy and activity-dependent synaptic plasticity. Hippocampal RNA isolated from amygdala-kindled rats at different kindling stages was analyzed to identify kindling-induced genes. Furthermore, effects of the anti-epileptic drug levetiracetam on kindling-induced gene expression were examined. Results Cyclooxygenase-2 (Cox-2), Protocadherin-8 (Pcdh8) and TGF-beta-inducible early response gene-1 (TIEG1) were identified and verified as differentially expressed transcripts in the hippocampus of kindled rats by in situ hybridization and quantitative RT-PCR. In addition, we identified a panel of 16 additional transcripts which included Arc, Egr3/Pilot, Homer1a, Ania-3, MMP9, Narp, c-fos, NGF, BDNF, NT-3, Synaptopodin, Pim1 kinase, TNF-α, RGS2, Egr2/krox-20 and β-A activin that were differentially expressed in the hippocampus of amygdala-kindled rats. The list consists of many synaptic plasticity-related immediate early genes (IEGs) as well as some late response genes encoding transcription factors, neurotrophic factors and proteins that are known to regulate synaptic remodelling. In the hippocampus, induction of IEG expression was dependent on the afterdischarge (AD) duration. Levetiracetam, 40 mg/kg, suppressed the development of kindling measured as severity of seizures and AD duration. In addition, single animal profiling also showed that levetiracetam attenuated the observed kindling-induced IEG expression; an effect that paralleled the anti-epileptic effect of the drug on AD duration. Conclusions The present study provides mRNA expression data that suggest that levetiracetam attenuates expression of genes known to regulate synaptic remodelling. In the kindled rat, levetiracetam does so by shortening the AD duration thereby reducing the seizure-induced changes in mRNA expression in the hippocampus. PMID:20105316

Human serum amyloid A genes are expressed in monocyte/macrophage cell lines.

PubMed

Urieli-Shoval, S; Meek, R L; Hanson, R H; Eriksen, N; Benditt, E P

1994-09-01

Serum amyloid A (apoSAA) is a family of proteins found, mainly associated with high density lipoproteins, in the blood plasma of mammals and at least one avian species, the Pekin duck. These proteins are present in small amounts under normal circumstances, but their concentration is capable of rising 100- to 1,000-fold in situations involving tissue injury or infection. Like classic acute phase proteins they are produced in the liver; however, expression of one of the apoSAA genes is known to occur in activated macrophages of mice. We examined three human macrophage precursor cell lines (THP-1, U-937, and HL-60), before and after differentiation with phorbol 12-myristate 13-acetate or 1 alpha,25-dihydroxy-vitamin D3, for apoSAA messenger (m)-RNA expression and found that: 1) induction of steady-state apoSAA mRNA by lipopolysaccharide, interleukin-1, or interleukin-6 required the presence of the synthetic glucocorticoid dexamethasone; 2) the three known active genes, apoSAA1, apoSAA2, and apoSAA4, were induced in THP-1 cells, whereas the pseudogene apoSAA3 was not; 3) differentiated and undifferentiated THP-1 cells expressed apoSAA mRNA, but U-937 cells expressed apoSAA mRNA (low levels) only after phorbol 12-myristate 13-acetate differentiation and HL-60 cells did not express apoSAA mRNA whether differentiated or not; 4) apoSAA protein was detectable immunologically at a low level in lyophilized medium from induced THP-1 cells. Our findings are compatible with the hypotheses that 1) apoSAA gene expression in human monocytes/macrophages in vivo is differentiation dependent; 2) activated macrophages provide a local source of apoSAA at sites of tissue injury or inflammation; 3) apoSAA is induced in tissue macrophages by local stimuli, under conditions that may not evoke the systemic acute phase response.

Sertoli cell androgen receptor expression regulates temporal fetal and adult Leydig cell differentiation, function, and population size.

PubMed

Hazra, Rasmani; Jimenez, Mark; Desai, Reena; Handelsman, David J; Allan, Charles M

2013-09-01

We recently created a mouse model displaying precocious Sertoli cell (SC) and spermatogenic development induced by SC-specific transgenic androgen receptor expression (TgSCAR). Here we reveal that TgSCAR regulates the development, function, and absolute number of Leydig cells (LCs). Total fetal and adult type LC numbers were reduced in postnatal and adult TgSCAR vs control testes, despite normal circulating LH levels. Normal LC to SC ratios found in TgSCAR testes indicate that SC androgen receptor (SCAR)-mediated activity confers a quorum-dependent relationship between total SC and LC numbers. TgSCAR enhanced LC differentiation, shown by elevated ratios of advanced to immature LC types, and reduced LC proliferation in postnatal TgSCAR vs control testes. Postnatal TgSCAR testes displayed up-regulated expression of coupled ligand-receptor transcripts (Amh-Amhr2, Dhh-Ptch1, Pdgfa-Pdgfra) for potential SCAR-stimulated paracrine pathways, which may coordinate LC differentiation. Neonatal TgSCAR testes displayed normal T and dihydrotestosterone levels despite differential changes to steroidogenic gene expression, with down-regulated Star, Cyp11a1, and Cyp17a1 expression contrasting with up-regulated Hsd3b1, Hsd17b3, and Srd5a1 expression. TgSCAR males also displayed elevated postnatal and normal adult serum testosterone levels, despite reduced LC numbers. Enhanced adult-type LC steroidogenic output was revealed by increased pubertal testicular T, dihydrotestosterone, 3α-diol and 3β-diol levels per LC and up-regulated steroidogenic gene (Nr5a1, Lhr, Cyp11a1, Cyp17a1, Hsd3b6, Srd5a1) expression in pubertal or adult TgSCAR vs control males, suggesting regulatory mechanisms maintain androgen levels independently of absolute LC numbers. Our unique gain-of-function TgSCAR model has revealed that SCAR activity controls temporal LC differentiation, steroidogenic function, and population size.

[Molecular mechanisms of thymocyte differentiation].

PubMed

Kuklina, E M

2003-01-01

A review of the main molecular events occurring during differentiation of T-lymphocytes in the thymus: T-cell specialization of early intrathymic precursors, formation and expression of antigen receptor, formation of antigen recognizing cell repertoire, and alpha beta/gamma beta- and CD4/CD8-commitment. The mechanisms of glucocorticoid-induced apoptosis of thymocytes and its blockade during antigen-dependent activation are considered. A special attention is paid to the analysis of intracellular signals underlying the clonal selection of thymocytes.

Sensitivity of hiPSC-derived neural stem cells (NSC) to Pyrroloquinoline quinone depends on their developmental stage.

PubMed

Augustyniak, J; Lenart, J; Zychowicz, M; Lipka, G; Gaj, P; Kolanowska, M; Stepien, P P; Buzanska, L

2017-12-01

Pyrroloquinoline quinone (PQQ) is a factor influencing on the mitochondrial biogenesis. In this study the PQQ effect on viability, total cell number, antioxidant capacity, mitochondrial biogenesis and differentiation potential was investigated in human induced Pluripotent Stem Cells (iPSC) - derived: neural stem cells (NSC), early neural progenitors (eNP) and neural progenitors (NP). Here we demonstrated that sensitivity to PQQ is dependent upon its dose and neural stage of development. Induction of the mitochondrial biogenesis by PQQ at three stages of neural differentiation was evaluated at mtDNA, mRNA and protein level. Changes in NRF1, TFAM and PPARGC1A gene expression were observed at all developmental stages, but only at eNP were correlated with the statistically significant increase in the mtDNA copy numbers and enhancement of SDHA, COX-1 protein level. Thus, the "developmental window" of eNP for PQQ-evoked mitochondrial biogenesis is proposed. This effect was independent of high antioxidant capacity of PQQ, which was confirmed in all tested cell populations, regardless of the stage of hiPSC neural differentiation. Furthermore, a strong induction of GFAP, with down regulation of MAP2 gene expression upon PQQ treatment was observed. This indicates a possibility of shifting the balance of cell differentiation in the favor of astroglia, but more research is needed at this point. Copyright © 2017 Elsevier Ltd. All rights reserved.

Cell cycle activation in p21 dependent pathway: An alternative mechanism of organophosphate induced dopaminergic neurodegeneration.

PubMed

Wani, Willayat Yousuf; Kandimalla, Ramesh J L; Sharma, Deep Raj; Kaushal, Alka; Ruban, Anand; Sunkaria, Aditya; Vallamkondu, Jayalakshmi; Chiarugi, Alberto; Reddy, P Hemachandra; Gill, Kiran Dip

2017-07-01

In the previous study, we demonstrated that dichlorvos induces oxidative stress in dopaminergic neuronal cells and subsequent caspase activation mediates apoptosis. In the present study, we evaluated the effect and mechanism of dichlorvos induced oxidative stress on cell cycle activation in NGF-differentiated PC12 cells. Dichlorvos exposure resulted in oxidative DNA damage along with activation of cell cycle machinery in differentiated PC12 cells. Dichlorvos exposed cells exhibited an increased expression of p53, cyclin-D1, pRb and decreased expression of p21suggesting a re-entry of differentiated cells into the cell cycle. Cell cycle analysis of dichlorvos exposed cells revealed a reduction of cells in the G 0 /G 1 phase of the cell cycle (25%), and a concomitant increase of cells in S phase (30%) and G2/M phase (43.3%) compared to control PC12 cells. Further, immunoblotting of cytochrome c, Bax, Bcl-2 and cleaved caspase-3 revealed that dichlorvos induces a caspase-dependent cell death in PC12 cells. These results suggest that Dichlorvos exposure has the potential to generate oxidative stress which evokes activation of cell cycle machinery leading to apoptotic cell death via cytochrome c release from mitochondria and subsequent caspase-3 activation in differentiated PC12 cells. Copyright © 2016 Elsevier B.V. All rights reserved.

Electrical Stimulation Promotes Cardiac Differentiation of Human Induced Pluripotent Stem Cells

PubMed Central

Hernández, Damián; Millard, Rodney; Sivakumaran, Priyadharshini; Wong, Raymond C. B.; Crombie, Duncan E.; Hewitt, Alex W.; Liang, Helena; Hung, Sandy S. C.; Pébay, Alice; Shepherd, Robert K.; Dusting, Gregory J.; Lim, Shiang Y.

2016-01-01

Background. Human induced pluripotent stem cells (iPSCs) are an attractive source of cardiomyocytes for cardiac repair and regeneration. In this study, we aim to determine whether acute electrical stimulation of human iPSCs can promote their differentiation to cardiomyocytes. Methods. Human iPSCs were differentiated to cardiac cells by forming embryoid bodies (EBs) for 5 days. EBs were then subjected to brief electrical stimulation and plated down for 14 days. Results. In iPS(Foreskin)-2 cell line, brief electrical stimulation at 65 mV/mm or 200 mV/mm for 5 min significantly increased the percentage of beating EBs present by day 14 after plating. Acute electrical stimulation also significantly increased the cardiac gene expression of ACTC1, TNNT2, MYH7, and MYL7. However, the cardiogenic effect of electrical stimulation was not reproducible in another iPS cell line, CERA007c6. Beating EBs from control and electrically stimulated groups expressed various cardiac-specific transcription factors and contractile muscle markers. Beating EBs were also shown to cycle calcium and were responsive to the chronotropic agents, isoproterenol and carbamylcholine, in a concentration-dependent manner. Conclusions. Our results demonstrate that brief electrical stimulation can promote cardiac differentiation of human iPS cells. The cardiogenic effect of brief electrical stimulation is dependent on the cell line used. PMID:26788064

Oncogenic RAS Enables DNA Damage- and p53-Dependent Differentiation of Acute Myeloid Leukemia Cells in Response to Chemotherapy

PubMed Central

Meyer, Mona; Rübsamen, Daniela; Slany, Robert; Illmer, Thomas; Stabla, Kathleen; Roth, Petra; Stiewe, Thorsten

2009-01-01

Acute myeloid leukemia (AML) is a clonal disease originating from myeloid progenitor cells with a heterogeneous genetic background. High-dose cytarabine is used as the standard consolidation chemotherapy. Oncogenic RAS mutations are frequently observed in AML, and are associated with beneficial response to cytarabine. Why AML-patients with oncogenic RAS benefit most from high-dose cytarabine post-remission therapy is not well understood. Here we used bone marrow cells expressing a conditional MLL-ENL-ER oncogene to investigate the interaction of oncogenic RAS and chemotherapeutic agents. We show that oncogenic RAS synergizes with cytotoxic agents such as cytarabine in activation of DNA damage checkpoints, resulting in a p53-dependent genetic program that reduces clonogenicity and increases myeloid differentiation. Our data can explain the beneficial effects observed for AML patients with oncogenic RAS treated with higher dosages of cytarabine and suggest that induction of p53-dependent differentiation, e.g. by interfering with Mdm2-mediated degradation, may be a rational approach to increase cure rate in response to chemotherapy. The data also support the notion that the therapeutic success of cytotoxic drugs may depend on their ability to promote the differentiation of tumor-initiating cells. PMID:19890398

Sea urchin tube feet are photosensory organs that express a rhabdomeric-like opsin and PAX6

PubMed Central

Lesser, Michael P.; Carleton, Karen L.; Böttger, Stefanie A.; Barry, Thomas M.; Walker, Charles W.

2011-01-01

All echinoderms have unique hydraulic structures called tube feet, known for their roles in light sensitivity, respiration, chemoreception and locomotion. In the green sea urchin, the most distal portion of these tube feet contain five ossicles arranged as a light collector with its concave surface facing towards the ambient light. These ossicles are perforated and lined with pigment cells that express a PAX6 protein that is universally involved in the development of eyes and sensory organs in other bilaterians. Polymerase chain reaction (PCR)-based sequencing and real time quantitative PCR (qPCR) also demonstrate the presence and differential expression of a rhabdomeric-like opsin within these tube feet. Morphologically, nerves that could serve to transmit information to the test innervate the tube feet, and the differential expression of opsin transcripts in the tube feet is inversely, and significantly, related to the amount of light that tube feet are exposed to depending on their location on the test. The expression of these genes, the differential expression of opsin based on light exposure and the unique morphological features at the distal portion of the tube foot strongly support the hypothesis that in addition to previously identified functional roles of tube feet they are also photosensory organs that detect and respond to changes in the underwater light field. PMID:21450733

CaMKIIα-GluA1 activity underlies vulnerability to adolescent binge alcohol drinking

PubMed Central

Agoglia, Abigail E.; Holstein, Sarah E.; Reid, Grant; Hodge, Clyde W.

2015-01-01

Background Binge drinking during adolescence is associated with increased risk for developing alcohol use disorders (AUDs); however, the neural mechanisms underlying this liability are unclear. In this study, we sought to determine if binge-drinking alters expression or phosphorylation of two molecular mechanisms of neuroplasticity, calcium/calmodulin dependent kinase II alpha (CaMKIIα) and the GluA1 subunit of AMPA receptors (AMPAR) in addiction-associated brain regions. We also asked if activation of CaMKIIα-dependent AMPAR activity escalates binge-like drinking. Methods To address these questions, CaMKIIαT286 and GluA1S831 protein phosphorylation and expression were assessed in the amygdala and striatum of adolescent and adult male C57BL/6J mice immediately after voluntary binge-like alcohol drinking (blood alcohol > 80mg/dL). In separate mice, effects of the CaMKIIα-dependent pGluA1S831-enhancing drug tianeptine were tested on binge-like alcohol consumption in both age groups. Results Binge-like drinking decreased CaMKIIαT286 phosphorylation (pCaMKIIαT286) selectively in adolescent amygdala with no effect in adults. Alcohol also produced a trend for reduced pGluA1S831 expression in adolescent amygdala but differentially increased pGluA1S831 in adult amygdala. No effects were observed in the nucleus accumbens or dorsal striatum. Tianeptine increased binge-like alcohol consumption in adolescents but decreased alcohol consumption in adults. Sucrose consumption was similarly decreased by tianeptine pretreatment in both ages. Conclusions These data show that the adolescent and adult amygdalae are differentially sensitive to effects of binge-like alcohol drinking on plasticity-linked glutamate signaling molecules. Tianeptine-induced increases in binge-like drinking only in adolescents suggest that differential CaMKIIα-dependent AMPAR activation may underlie age-related escalation of binge drinking. PMID:26247621

Epidermal cornification is preceded by the expression of a keratinocyte-specific set of pyroptosis-related genes.

PubMed

Lachner, Julia; Mlitz, Veronika; Tschachler, Erwin; Eckhart, Leopold

2017-12-12

The homeostasis of the epidermis depends on keratinocyte differentiation and cornification, a mode of programmed cell death that does not elicit inflammation. Here, we report that cornification is associated with the expression of specific genes that control multiple steps of pyroptosis, another form of cell death that involves the processing and release of interleukin-1 family (IL1F) cytokines. Expression levels of pro-inflammatory IL1A and IL1B and of the pyroptotic pore-forming gasdermin (GSDM) D were downregulated during terminal differentiation of human keratinocytes in vitro. By contrast, negative regulators of IL-1 processing, including NLR family pyrin domain containing 10 (NLRP10) and pyrin domain-containing 1 (PYDC1), the anti-inflammatory IL1F members IL-37 (IL1F7) and IL-38 (IL1F10), and GSDMA, were strongly induced in differentiated keratinocytes. In human tissues, these keratinocyte differentiation-associated genes are expressed in the skin at higher levels than in any other organ, and mammalian species, that have lost the epidermal cornification program during evolution, i.e. whales and dolphins, lack homologs of these genes. Together, our results suggest that human epidermal cornification is accompanied by a tight control of pyroptosis and warrant further studies of potential defects in the balance between cornification and pyroptosis in skin pathologies.

[Effects of nicotine on bone marrow stromal cells proliferation and differentiation of chondrocyte in vitro].

PubMed

Ying, Xiao-zhou; Peng, Lei; Cheng, Shao-wen; Chen, Qing-yu; Zhang, Wei; Kou, Dong-quan; Shen, Yue

2011-11-01

To examine the effects of various concentration of nicotine on bone marrow stromal cells (BMSCs) proliferation and differentiation of cartilaginous in vitro. BMSCs was obtained from femoral bone and tibia of New-Zealand albino rabbit. The cells of the 3rd generation were used in study. Different concentration of nicotine (0, 1 x 10(-7), 1 x 10(-6), 1 x 10(-5) M) were added into BMSCs. BMSCs proliferation was analyzed by MTT assay at the 1, 4, 7, 14 days. The expression of collagen type II and aggrecan as the marker genes of cartilaginous differentiation from BMSCs were detected by reverse transcriptase-polymerase chain reaction (RT-PCR). Microscope showed that BMSCs transformed from round to fusiform shape. The concentration of nicotine in 1 x 10(-7), 1 x 10(-6) M had a significant positive effect on cell proliferation and the expression of type II collagen in a time-dependent manner when supplemented in commonly used induction media (P<0.05). Concentrations of nicotine in 1 x 10(-7) can promote the expression of aggrecan at the 7th day after induction,and in 1 x 10(-5) M may inhibit the expression of type II collagen and aggrecan. It was implied that local application of nicotine at an appropriate concentration may be a promising approach for enhancing cartilaginous differentiation capacity of BMSCs in cartilage tissue engineering.

Odour discrimination learning in the Indian greater short-nosed fruit bat (Cynopterus sphinx): differential expression of Egr-1, C-fos and PP-1 in the olfactory bulb, amygdala and hippocampus.

PubMed

Mukilan, Murugan; Bogdanowicz, Wieslaw; Marimuthu, Ganapathy; Rajan, Koilmani Emmanuvel

2018-06-15

Activity-dependent expression of immediate-early genes (IEGs) is induced by exposure to odour. The present study was designed to investigate whether there is differential expression of IEGs ( Egr-1 , C-fos ) in the brain region mediating olfactory memory in the Indian greater short-nosed fruit bat, Cynopterus sphinx We assumed that differential expression of IEGs in different brain regions may orchestrate a preference odour (PO) and aversive odour (AO) memory in C. sphinx We used preferred (0.8% w/w cinnamon powder) and aversive (0.4% w/v citral) odour substances, with freshly prepared chopped apple, to assess the behavioural response and induction of IEGs in the olfactory bulb, hippocampus and amygdala. After experiencing PO and AO, the bats initially responded to both, later only engaging in feeding bouts in response to the PO food. The expression pattern of EGR-1 and c-Fos in the olfactory bulb, hippocampus and amygdala was similar at different time points (15, 30 and 60 min) following the response to PO, but was different for AO. The response to AO elevated the level of c-Fos expression within 30 min and reduced it at 60 min in both the olfactory bulb and the hippocampus, as opposed to the continuous increase noted in the amygdala. In addition, we tested whether an epigenetic mechanism involving protein phosphatase-1 (PP-1) acts on IEG expression. The observed PP-1 expression and the level of unmethylated/methylated promoter revealed that C-fos expression is possibly controlled by odour-mediated regulation of PP-1. These results in turn imply that the differential expression of C-fos in the hippocampus and amygdala may contribute to olfactory learning and memory in C. sphinx . © 2018. Published by The Company of Biologists Ltd.

Essential Dosage-Dependent Functions of the Transcription Factor Yin Yang 1 in Late Embryonic Development and Cell Cycle Progression†

PubMed Central

Affar, El Bachir; Gay, Frédérique; Shi, Yujiang; Liu, Huifei; Huarte, Maite; Wu, Su; Collins, Tucker; Li, En; Shi, Yang

2006-01-01

Constitutive ablation of the Yin Yang 1 (YY1) transcription factor in mice results in peri-implantation lethality. In this study, we used homologous recombination to generate knockout mice carrying yy1 alleles expressing various amounts of YY1. Phenotypic analysis of yy1 mutant embryos expressing ∼75%, ∼50%, and ∼25% of the normal complement of YY1 identified a dosage-dependent requirement for YY1 during late embryogenesis. Indeed, reduction of YY1 levels impairs embryonic growth and viability in a dose-dependent manner. Analysis of the corresponding mouse embryonic fibroblast cells also revealed a tight correlation between YY1 dosage and cell proliferation, with a complete ablation of YY1 inducing cytokinesis failure and cell cycle arrest. Consistently, RNA interference-mediated inhibition of YY1 in HeLa cells prevents cytokinesis, causes proliferative arrest, and increases cellular sensitivity to various apoptotic agents. Genome-wide expression profiling identified a plethora of YY1 target genes that have been implicated in cell growth, proliferation, cytokinesis, apoptosis, development, and differentiation, suggesting that YY1 coordinates multiple essential biological processes through a complex transcriptional network. These data not only shed new light on the molecular basis for YY1 developmental roles and cellular functions, but also provide insight into the general mechanisms controlling eukaryotic cell proliferation, apoptosis, and differentiation. PMID:16611997

Differentiation Generates Paracrine Cell Pairs That Maintain Basaloid Mouse Mammary Tumors: Proof of Concept

PubMed Central

Kim, Soyoung; Goel, Shruti; Alexander, Caroline M.

2011-01-01

There is a paradox offered up by the cancer stem cell hypothesis. How are the mixed populations that are characteristic of heterogeneous solid tumors maintained at constant proportion, given their high, and different, mitotic indices? In this study, we evaluate a well-characterized mouse model of human basaloid tumors (induced by the oncogene Wnt1), which comprise mixed populations of mammary epithelial cells resembling their normal basal and luminal counterparts. We show that these cell types are substantially inter-dependent, since the MMTV LTR drives expression of Wnt1 ligand in luminal cells, whereas the functional Wnt1-responsive receptor (Lrp5) is expressed by basal cells, and both molecules are necessary for tumor growth. There is a robust tumor initiating activity (tumor stem cell) in the basal cell population, which is associated with the ability to differentiate into luminal and basal cells, to regenerate the oncogenic paracrine signaling cell pair. However, we found an additional tumor stem cell activity in the luminal cell population. Knowing that tumors depend upon Wnt1-Lrp5, we hypothesized that this stem cell must express Lrp5, and found that indeed, all the stem cell activity could be retrieved from the Lrp5-positive cell population. Interestingly, this reflects post-transcriptional acquisition of Lrp5 protein expression in luminal cells. Furthermore, this plasticity of molecular expression is reflected in plasticity of cell fate determination. Thus, in vitro, Wnt1-expressing luminal cells retro-differentiate to basal cell types, and in vivo, tumors initiated with pure luminal cells reconstitute a robust basal cell subpopulation that is indistinguishable from the populations initiated by pure basal cells. We propose this is an important proof of concept, demonstrating that bipotential tumor stem cells are essential in tumors where oncogenic ligand-receptor pairs are separated into different cell types, and suggesting that Wnt-induced molecular and fate plasticity can close paracrine loops that are usually separated into distinct cell types. PMID:21541292

Mesenchymal stem cells induce mature dendritic cells into a novel Jagged-2-dependent regulatory dendritic cell population.

PubMed

Zhang, Bin; Liu, Rui; Shi, Dan; Liu, Xingxia; Chen, Yuan; Dou, Xiaowei; Zhu, Xishan; Lu, Chunhua; Liang, Wei; Liao, Lianming; Zenke, Martin; Zhao, Robert C H

2009-01-01

Mesenchymal stem cells (MSCs), in addition to their multilineage differentiation, exert immunomodulatory effects on immune cells, even dendritic cells (DCs). However, whether they influence the destiny of full mature DCs (maDCs) remains controversial. Here we report that MSCs vigorously promote proliferation of maDCs, significantly reduce their expression of Ia, CD11c, CD80, CD86, and CD40 while increasing CD11b expression. Interestingly, though these phenotypes clearly suggest their skew to immature status, bacterial lipopolysaccharide (LPS) stimulation could not reverse this trend. Moreover, high endocytosic capacity, low immunogenicity, and strong immunoregulatory function of MSC-treated maDCs (MSC-DCs) were also observed. Furthermore we found that MSCs, partly via cell-cell contact, drive maDCs to differentiate into a novel Jagged-2-dependent regulatory DC population and escape their apoptotic fate. These results further support the role of MSCs in preventing rejection in organ transplantation and treatment of autoimmune disease.

Epigenetic control of skin differentiation genes by phytocannabinoids

PubMed Central

Pucci, Mariangela; Rapino, Cinzia; Di Francesco, Andrea; Dainese, Enrico; D'Addario, Claudio; Maccarrone, Mauro

2013-01-01

BACKGROUND AND PURPOSE Endocannabinoid signalling has been shown to have a role in the control of epidermal physiology, whereby anandamide is able to regulate the expression of skin differentiation genes through DNA methylation. Here, we investigated the possible epigenetic regulation of these genes by several phytocannabinoids, plant-derived cannabinoids that have the potential to be novel therapeutics for various human diseases. EXPERIMENTAL APPROACH The effects of cannabidiol, cannabigerol and cannabidivarin on the expression of skin differentiation genes keratins 1 and 10, involucrin and transglutaminase 5, as well as on DNA methylation of keratin 10 gene, were investigated in human keratinocytes (HaCaT cells). The effects of these phytocannabinoids on global DNA methylation and the activity and expression of four major DNA methyltransferases (DNMT1, 3a, 3b and 3L) were also examined. KEY RESULTS Cannabidiol and cannabigerol significantly reduced the expression of all the genes tested in differentiated HaCaT cells, by increasing DNA methylation of keratin 10 gene, but cannabidivarin was ineffective. Remarkably, cannabidiol reduced keratin 10 mRNA through a type-1 cannabinoid (CB1) receptor-dependent mechanism, whereas cannabigerol did not affect either CB1 or CB2 receptors of HaCaT cells. In addition, cannabidiol, but not cannabigerol, increased global DNA methylation levels by selectively enhancing DNMT1 expression, without affecting DNMT 3a, 3b or 3L. CONCLUSIONS AND IMPLICATIONS These findings show that the phytocannabinoids cannabidiol and cannabigerol are transcriptional repressors that can control cell proliferation and differentiation. This indicates that they (especially cannabidiol) have the potential to be lead compounds for the development of novel therapeutics for skin diseases. PMID:23869687

Csf2 null mutation alters placental gene expression and trophoblast glycogen cell and giant cell abundance in mice.

PubMed

Sferruzzi-Perri, Amanda N; Macpherson, Anne M; Roberts, Claire T; Robertson, Sarah A

2009-07-01

Genetic deficiency in granulocyte-macrophage colony-stimulating factor (CSF2, GM-CSF) results in altered placental structure in mice. To investigate the mechanism of action of CSF2 in placental morphogenesis, the placental gene expression and cell composition were examined in Csf2 null mutant and wild-type mice. Microarray and quantitative RT-PCR analyses on Embryonic Day (E) 13 placentae revealed that the Csf2 null mutation caused altered expression of 17 genes not previously known to be associated with placental development, including Mid1, Cd24a, Tnfrsf11b, and Wdfy1. Genes controlling trophoblast differentiation (Ascl2, Tcfeb, Itgav, and Socs3) were also differentially expressed. The CSF2 ligand and the CSF2 receptor alpha subunit were predominantly synthesized in the placental junctional zone. Altered placental structure in Csf2 null mice at E15 was characterized by an expanded junctional zone and by increased Cx31(+) glycogen cells and cyclin-dependent kinase inhibitor 1C (CDKN1C(+), P57(Kip2+)) giant cells, accompanied by elevated junctional zone transcription of genes controlling spongiotrophoblast and giant cell differentiation and secretory function (Ascl2, Hand1, Prl3d1, and Prl2c2). Granzyme genes implicated in tissue remodeling and potentially in trophoblast invasion (Gzmc, Gzme, and Gzmf) were downregulated in the junctional zone of Csf2 null mutant placentae. These data demonstrate aberrant placental gene expression in Csf2 null mutant mice that is associated with altered differentiation and/or functional maturation of junctional zone trophoblast lineages, glycogen cells, and giant cells. We conclude that CSF2 is a regulator of trophoblast differentiation and placental development, which potentially influences the functional capacity of the placenta to support optimal fetal growth in pregnancy.

Defective IL-17- and IL-22-dependent mucosal host response to Candida albicans determines susceptibility to oral candidiasis in mice expressing the HIV-1 transgene.

PubMed

Goupil, Mathieu; Cousineau-Côté, Vincent; Aumont, Francine; Sénéchal, Serge; Gaboury, Louis; Hanna, Zaher; Jolicoeur, Paul; de Repentigny, Louis

2014-10-26

The tissue-signaling cytokines IL-17 and IL-22 are critical to host defense against oral Candida albicans infection, by their induction of oral antimicrobial peptide expression and recruitment of neutrophils. Mucosal Th17 cells which produce these cytokines are preferentially depleted in HIV-infected patients. Here, we tested the hypothesis that defective IL-17- and IL-22-dependent host responses to C. albicans determine the phenotype of susceptibility to oropharyngeal candidiasis (OPC) in transgenic (Tg) mice expressing HIV-1. Naïve CD4+ T-cells and the differentiated Th1, Th2, Th17, Th1Th17 and Treg lineages were all profoundly depleted in cervical lymph nodes (CLNs) of these Tg mice. However, naive CD4+ cells from Tg mice maintained the capacity to differentiate into these lineages in response to polarizing cytokines in vitro. Expression of Il17, Il22, S100a8 and Ccl20 was enhanced in oral mucosal tissue of non-Tg, but not of Tg mice, after oral infection with C. albicans. Treatment of infected Tg mice with the combination of IL-17 and IL-22, but not IL-17 or Il-22 alone, significantly reduced oral burdens of C. albicans and abundance of Candida hyphae in the epithelium of tongues of infected Tg mice, and restored the ability of the Tg mice to up-regulate expression of S100a8 and Ccl20 in response to C. albicans infection. These findings demonstrate that defective IL-17- and IL-22-dependent induction of innate mucosal immunity to C. albicans is central to the phenotype of susceptibility to OPC in these HIV transgenic mice.

High glucose impaired estrogen receptor alpha signaling via β-catenin in osteoblastic MC3T3-E1.

PubMed

Wang, Rui; Gao, Dong; Zhou, Yin; Chen, Lu; Luo, Bin; Yu, Yanrong; Li, Hao; Hu, Jiawei; Huang, Qiren; He, Ming; Peng, Weijie; Luo, Dan

2017-11-01

Diabetic Mellitus is a risk factor for osteoporosis. It has been suggested that altered estrogen or estrogen receptor α/β (ERα/β) signaling may be involved in diabetic osteoporosis. The present study is to investigate the effects of high glucose on ERα/β signaling in osteoblastic MC3T3-E1 and how the altered signaling of ERα/β affect osteoblastic bone formation. ERα/β signaling was demonstrated as ERα/β protein expression (Western Blotting) and ER transcription activity (Luciferase Reporter assays). Proliferation (WSK-1 assaying), differentiation (ALP staining) and mineralization (Alizalard Red staining) of MC3T3-E1 were examined to evaluate bone formation function. It has been found that high glucose increased ERα/β expression dose-dependently and time-dependently, but high glucose (33mM) decreased ERα transcription activity. 17β-estradiol increased the ERα/β expression dose-dependently in normal medium, but decreased the ERα/β expression dose-dependently in medium with high glucose (33mM). High glucose decreased bone formation and also decreased the osteogenic effects of 17β-estradiol (10 -8 M). High glucose decreased β-catenin expression dose-dependently and time-dependently. LiCl, an inhibitor of β-catenin degradation, decreased ERα expression but increased ERα transcription activity. When compared with high glucose treatment, LiCl (5mM) increased ALP activity and calcified nodes. Besides, high glucose also decreased the protein expression PI-3K, pAKT/AKT, GSK-3β. In conclusion, the present study suggested that high glucose may impair ERα transcription activity by inhibiting β-catenin signaling in osteoblastic MC3T3-E1, leading decreased bone formation ligand-dependently or ligand-independently. Copyright © 2017 Elsevier Ltd. All rights reserved.

Myogenin Recruits the Histone Chaperone Facilitates Chromatin Transcription (FACT) to Promote Nucleosome Disassembly at Muscle-specific Genes*

PubMed Central

Lolis, Alexandra A.; Londhe, Priya; Beggs, Benjamin C.; Byrum, Stephanie D.; Tackett, Alan J.; Davie, Judith K.

2013-01-01

Facilitates chromatin transcription (FACT) functions to reorganize nucleosomes by acting as a histone chaperone that destabilizes and restores nucleosomal structure. The FACT complex is composed of two subunits: SSRP1 and SPT16. We have discovered that myogenin interacts with the FACT complex. Transfection of FACT subunits with myogenin is highly stimulatory for endogenous muscle gene expression in 10T1/2 cells. We have also found that FACT subunits do not associate with differentiation-specific genes while C2C12 cells are proliferating but are recruited to muscle-specific genes as differentiation initiates and then dissociate as differentiation proceeds. The recruitment is dependent on myogenin, as knockdowns of myogenin show no recruitment of the FACT complex. These data suggest that FACT is involved in the early steps of gene activation through its histone chaperone activities that serve to open the chromatin structure and facilitate transcription. Consistent with this hypothesis, we find that nucleosomes are depleted at muscle-specific promoters upon differentiation and that this activity is dependent on the presence of FACT. Our results show that the FACT complex promotes myogenin-dependent transcription and suggest that FACT plays an important role in the establishment of the appropriate transcription profile in a differentiated muscle cell. PMID:23364797

Somatostatin and its receptors contribute in a tissue-specific manner to the sex-dependent metabolic (fed/fasting) control of growth hormone axis in mice

PubMed Central

Córdoba-Chacón, José; Gahete, Manuel D.; Castaño, Justo P.; Kineman, Rhonda D.

2011-01-01

Somatostatin (SST) inhibits growth hormone (GH) secretion and regulates multiple processes by signaling through its receptors sst1–5. Differential expression of SST/ssts may contribute to sex-specific GH pattern and fasting-induced GH rise. To further delineate the tissue-specific roles of SST and sst1–5 in these processes, their expression patterns were evaluated in hypothalamus, pituitary, and stomach of male and female mice under fed/fasted conditions in the presence (wild type) or absence (SST-knockout) of endogenous SST. Under fed conditions, hypothalamic/stomach SST/ssts expression did not differ between sexes, whereas male pituitary expressed more SST and sst2A/2B/3/5A/5TMD2/5TMD1 and less sst1, and male pituitary cell cultures were more responsive to SST inhibitory actions on GH release compared with females. This suggests that local pituitary SST/ssts can contribute to the sexually dimorphic pattern of GH release. Fasting (48 h) reduced stomach sst2A/B and hypothalamic SST/sst2A expression in both sexes, whereas it caused a generalized downregulation of pituitary sst subtypes in male and of sst2A only in females. Thus, fasting can reduce SST sensitivity across tissues and SST input to the pituitary, thereby jointly contributing to enhance GH release. In SST-knockout mice, lack of SST differentially altered sst subtype expression levels in both sexes, supporting an important role for SST in sex-dependent control of GH axis. Evaluation of SST, IGF-I, and glucocorticoid effects on hypothalamic and pituitary cell cultures revealed that these hormones could directly account for alterations in sst2/5 expression in the physiological states examined. Taken together, these results indicate that changes in SST output and sensitivity can contribute critically to precisely define, in a tissue-dependent manner, the sex-specific metabolic regulation of the GH axis. PMID:20943754

Two-dimensional integrating matrices on rectangular grids. [solving differential equations associated with rotating structures

NASA Technical Reports Server (NTRS)

Lakin, W. D.

1981-01-01

The use of integrating matrices in solving differential equations associated with rotating beam configurations is examined. In vibration problems, by expressing the equations of motion of the beam in matrix notation, utilizing the integrating matrix as an operator, and applying the boundary conditions, the spatial dependence is removed from the governing partial differential equations and the resulting ordinary differential equations can be cast into standard eigenvalue form. Integrating matrices are derived based on two dimensional rectangular grids with arbitrary grid spacings allowed in one direction. The derivation of higher dimensional integrating matrices is the initial step in the generalization of the integrating matrix methodology to vibration and stability problems involving plates and shells.

Decoding the Regulatory Landscape of Ageing in Musculoskeletal Engineered Tissues Using Genome-Wide DNA Methylation and RNASeq

PubMed Central

Peffers, Mandy Jayne; Goljanek-Whysall, Katarzyna; Collins, John; Fang, Yongxiang; Rushton, Michael; Loughlin, John; Proctor, Carole; Clegg, Peter David

2016-01-01

Mesenchymal stem cells (MSC) are capable of multipotent differentiation into connective tissues and as such are an attractive source for autologous cell-based regenerative medicine and tissue engineering. Epigenetic mechanisms, like DNA methylation, contribute to the changes in gene expression in ageing. However there was a lack of sufficient knowledge of the role that differential methylation plays during chondrogenic, osteogenic and tenogenic differentiation from ageing MSCs. This study undertook genome level determination of the effects of DNA methylation on expression in engineered tissues from chronologically aged MSCs. We compiled unique DNA methylation signatures from chondrogenic, osteogenic, and tenogenic engineered tissues derived from young; n = 4 (21.8 years ± 2.4 SD) and old; n = 4 (65.5 years±8.3SD) human MSCs donors using the Illumina HumanMethylation 450 Beadchip arrays and compared these to gene expression by RNA sequencing. Unique and common signatures of global DNA methylation were identified. There were 201, 67 and 32 chondrogenic, osteogenic and tenogenic age-related DE protein-coding genes respectively. Findings inferred the nature of the transcript networks was predominantly for ‘cell death and survival’, ‘cell morphology’, and ‘cell growth and proliferation’. Further studies are required to validate if this gene expression effect translates to cell events. Alternative splicing (AS) was dysregulated in ageing with 119, 21 and 9 differential splicing events identified in chondrogenic, osteogenic and tenogenic respectively, and enrichment in genes associated principally with metabolic processes. Gene ontology analysis of differentially methylated loci indicated age-related enrichment for all engineered tissue types in ‘skeletal system morphogenesis’, ‘regulation of cell proliferation’ and ‘regulation of transcription’ suggesting that dynamic epigenetic modifications may occur in genes associated with shared and distinct pathways dependent upon engineered tissue type. An altered phenotype in engineered tissues was observed with ageing at numerous levels. These changes represent novel insights into the ageing process, with implications for stem cell therapies in older patients. In addition we have identified a number of tissue-dependant pathways, which warrant further studies. PMID:27533049

Organic Cation Transporter-Mediated Ergothioneine Uptake in Mouse Neural Progenitor Cells Suppresses Proliferation and Promotes Differentiation into Neurons

PubMed Central

Ishimoto, Takahiro; Nakamichi, Noritaka; Hosotani, Hiroshi; Masuo, Yusuke; Sugiura, Tomoko; Kato, Yukio

2014-01-01

The aim of the present study is to clarify the functional expression and physiological role in neural progenitor cells (NPCs) of carnitine/organic cation transporter OCTN1/SLC22A4, which accepts the naturally occurring food-derived antioxidant ergothioneine (ERGO) as a substrate in vivo. Real-time PCR analysis revealed that mRNA expression of OCTN1 was much higher than that of other organic cation transporters in mouse cultured cortical NPCs. Immunocytochemical analysis showed colocalization of OCTN1 with the NPC marker nestin in cultured NPCs and mouse embryonic carcinoma P19 cells differentiated into neural progenitor-like cells (P19-NPCs). These cells exhibited time-dependent [3H]ERGO uptake. These results demonstrate that OCTN1 is functionally expressed in murine NPCs. Cultured NPCs and P19-NPCs formed neurospheres from clusters of proliferating cells in a culture time-dependent manner. Exposure of cultured NPCs to ERGO or other antioxidants (edaravone and ascorbic acid) led to a significant decrease in the area of neurospheres with concomitant elimination of intracellular reactive oxygen species. Transfection of P19-NPCs with small interfering RNA for OCTN1 markedly promoted formation of neurospheres with a concomitant decrease of [3H]ERGO uptake. On the other hand, exposure of cultured NPCs to ERGO markedly increased the number of cells immunoreactive for the neuronal marker βIII-tubulin, but decreased the number immunoreactive for the astroglial marker glial fibrillary acidic protein (GFAP), with concomitant up-regulation of neuronal differentiation activator gene Math1. Interestingly, edaravone and ascorbic acid did not affect such differentiation of NPCs, in contrast to the case of proliferation. Knockdown of OCTN1 increased the number of cells immunoreactive for GFAP, but decreased the number immunoreactive for βIII-tubulin, with concomitant down-regulation of Math1 in P19-NPCs. Thus, OCTN1-mediated uptake of ERGO in NPCs inhibits cellular proliferation via regulation of oxidative stress, and also promotes cellular differentiation by modulating the expression of basic helix-loop-helix transcription factors via an unidentified mechanism different from antioxidant action. PMID:24586778

Differentiation expression during proliferative activity induced through different pathways: in situ hybridization study of thyroglobulin gene expression in thyroid epithelial cells

PubMed Central

1990-01-01

In canine thyrocytes in primary culture, our previous studies have identified three mitogenic agents and pathways: thyrotropin (TSH) acting through cyclic AMP (cAMP), EGF and its receptor tyrosine protein kinase, and the phorbol esters that stimulate protein kinase C. TSH enhances, while EGF and phorbol esters inhibit, the expression of differentiation. Given that growth and differentiation expression are often considered as mutually exclusive activities of the cells, it was conceivable that the differentiating action of TSH was restricted to noncycling (Go) cells, while the inhibition of the differentiation expression by EGF and phorbol esters only concerned proliferating cells. Therefore, the capacity to express the thyroglobulin (Tg) gene, the most prominent marker of differentiation in thyrocytes, was studied in proliferative cells (with insulin) and in quiescent cells (without insulin). Using cRNA in situ hybridization, we observed that TSH (and, to a lesser extent, insulin and insulin-like growth factor I) restored or maintained the expression of the Tg gene. Without these hormones, the Tg mRNA content became undetectable in most of the cells. EGF and 12-0-tetradecanoyl phorbol-13-acetate (TPA) inhibited the Tg mRNA accumulation induced by TSH (and/or insulin). Most of the cells (up to 90%) responded to both TSH and EGF. Nevertheless, the range of individual response was quite variable. The effects of TSH and EGF on differentiation expression were not dependent on insulin and can therefore be dissociated from their mitogenic effects. Cell cycling did not affect the induction of Tg gene. Indeed, the same cell distribution of Tg mRNA content was observed in quiescent cells stimulated by TSH alone, or in cells approximately 50% of which had performed one mitotic cycle in response to TSH + insulin. Moreover, after proliferation in "dedifferentiating" conditions (EGF + serum + insulin), thyrocytes had acquired a fusiform fibroblast-like morphology, and responded to TSH by regaining a characteristic epithelial shape and high Tg mRNA content. 32 h after the replacement of EGF by TSH, cells in mitosis presented the same distribution of the Tg mRNA content as the rest of the cell population. This implies that cell cycling (at least 27 h, as previously shown) did not affect the induction of the Tg gene which is clearly detectable after a time lag of at least 24 h. The data unequivocally show that the reexpression of differentiation and proliferative activity are separate but fully compatible processes when induced by cAMP in thyrocytes.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:2199463

Intracellular fragment of NLRR3 (NLRR3-ICD) stimulates ATRA-dependent neuroblastoma differentiation

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

Akter, Jesmin; Takatori, Atsushi, E-mail: atakatori@chiba-cc.jp; Islam, Md. Sazzadul

2014-10-10

Highlights: • NLRR3 is a membrane protein highly expressed in favorable neuroblastoma. • NLRR3-ICD was produced through proteolytic processing by secretases. • NLRR3-ICD was induced to be translocated into cell nucleus following ATRA exposure. • NLRR3-ICD plays a pivotal role in ATRA-mediated neuroblastoma differentiation. - Abstract: We have previously identified neuronal leucine-rich repeat protein-3 (NLRR3) gene which is preferentially expressed in favorable human neuroblastomas as compared with unfavorable ones. In this study, we have found for the first time that NLRR3 is proteolytically processed by secretases and its intracellular domain (NLRR3-ICD) is then released to translocate into cell nucleus duringmore » ATRA-mediated neuroblastoma differentiation. According to our present observations, NLRR3-ICD was induced to accumulate in cell nucleus of neuroblastoma SH-SY5Y cells following ATRA treatment. Since the proteolytic cleavage of NLRR3 was blocked by α- or γ-secretase inhibitor, it is likely that NLRR3-ICD is produced through the secretase-mediated processing of NLRR3. Intriguingly, forced expression of NLRR3-ICD in neuroblastoma SK-N-BE cells significantly suppressed their proliferation as examined by a live-cell imaging system and colony formation assay. Similar results were also obtained in neuroblastoma TGW cells. Furthermore, overexpression of NLRR3-ICD stimulated ATRA-dependent neurite elongation in SK-N-BE cells. Together, our present results strongly suggest that NLRR3-ICD produced by the secretase-mediated proteolytic processing of NLRR3 plays a crucial role in ATRA-mediated neuronal differentiation, and provide a clue to develop a novel therapeutic strategy against aggressive neuroblastomas.« less

Selective estrogen receptor modulators differentially alter the immune response of gilthead seabream juveniles.

PubMed

Rodenas, M C; Cabas, I; García-Alcázar, A; Meseguer, J; Mulero, V; García-Ayala, A

2016-05-01

17α-ethynylestradiol (EE2), a synthetic estrogen used in oral contraceptives and hormone replacement therapy, tamoxifen (Tmx), a selective estrogen-receptor modulator used in hormone replacement therapy, and G1, a G protein-coupled estrogen receptor (GPER) selective agonist, differentially increased the hepatic vitellogenin (vtg) gene expression and altered the immune response in adult gilthead seabream (Sparus aurata L.) males. However, no information exists on the effects of these compounds on the immune response of juveniles. This study aims, for the first time, to investigate the effects of the dietary intake of EE2, Tmx or G1 on the immune response of gilthead seabream juveniles and the capacity of the immune system of the specimens to recover its functionality after ceasing exposures (recovery period). The specimens were immunized with hemocyanin in the presence of aluminium adjuvant 1 (group A) or 120 (group B) days after the treatments ceased (dpt). The results indicate that EE2 and Tmx, but not G1, differentially promoted a transient alteration in hepatic vtg gene expression. Although all three compounds did not affect the production of reactive oxygen intermediates, they inhibited the induction of interleukin-1β (il1b) gene expression after priming. Interestingly, although Tmx increased the percentage of IgM-positive cells in both head kidney and spleen during the recovery period, the antibody response of vaccinated fish varied depending on the compound used and when the immunization was administered. Taken together, our results suggest that these compounds differentially alter the capacity of fish to respond to infection during ontogeny and, more interestingly, that the adaptive immune response remained altered to an extent that depends on the compound. Copyright © 2016 Elsevier Ltd. All rights reserved.

The ADAMTS5 Metzincin Regulates Zebrafish Somite Differentiation

PubMed Central

Dancevic, Carolyn M.; Gibert, Yann; Smith, Adam D.; Ward, Alister C.; McCulloch, Daniel R.

2018-01-01

The ADAMTS5 metzincin, a secreted zinc-dependent metalloproteinase, modulates the extracellular matrix (ECM) during limb morphogenesis and other developmental processes. Here, the role of ADAMTS5 was investigated by knockdown of zebrafish adamts5 during embryogenesis. This revealed impaired Sonic Hedgehog (Shh) signaling during somite patterning and early myogenesis. Notably, synergistic regulation of myod expression by ADAMTS5 and Shh during somite differentiation was observed. These roles were not dependent upon the catalytic activity of ADAMTS5. These data identify a non-enzymatic function for ADAMTS5 in regulating an important cell signaling pathway that impacts on muscle development, with implications for musculoskeletal diseases in which ADAMTS5 and Shh have been associated. PMID:29518972

Cyclin-dependent kinase 4 signaling acts as a molecular switch between syngenic differentiation and neural transdifferentiation in human mesenchymal stem cells

PubMed Central

Lee, Janet; Baek, Jeong-Hwa; Choi, Kyu-Sil; Kim, Hyun-Soo; Park, Hye-Young; Ha, Geun-Hyoung; Park, Ho; Lee, Kyo-Won; Lee, Chang Geun; Yang, Dong-Yun; Moon, Hyo Eun; Paek, Sun Ha; Lee, Chang-Woo

2013-01-01

Multipotent mesenchymal stem/stromal cells (MSCs) are capable of differentiating into a variety of cell types from different germ layers. However, the molecular and biochemical mechanisms underlying the transdifferentiation of MSCs into specific cell types still need to be elucidated. In this study, we unexpectedly found that treatment of human adipose- and bone marrow-derived MSCs with cyclin-dependent kinase (CDK) inhibitor, in particular CDK4 inhibitor, selectively led to transdifferentiation into neural cells with a high frequency. Specifically, targeted inhibition of CDK4 expression using recombinant adenovial shRNA induced the neural transdifferentiation of human MSCs. However, the inhibition of CDK4 activity attenuated the syngenic differentiation of human adipose-derived MSCs. Importantly, the forced regulation of CDK4 activity showed reciprocal reversibility between neural differentiation and dedifferentiation of human MSCs. Together, these results provide novel molecular evidence underlying the neural transdifferentiation of human MSCs; in addition, CDK4 signaling appears to act as a molecular switch from syngenic differentiation to neural transdifferentiation of human MSCs. PMID:23324348

A Novel Regulatory Function of Sweet Taste-Sensing Receptor in Adipogenic Differentiation of 3T3-L1 Cells

PubMed Central

Masubuchi, Yosuke; Nakagawa, Yuko; Ma, Jinhui; Sasaki, Tsutomu; Kitamura, Tadahiro; Yamamoto, Yoritsuna; Kurose, Hitoshi; Kojima, Itaru; Shibata, Hiroshi

2013-01-01

Background Sweet taste receptor is expressed not only in taste buds but also in nongustatory organs such as enteroendocrine cells and pancreatic beta-cells, and may play more extensive physiological roles in energy metabolism. Here we examined the expression and function of the sweet taste receptor in 3T3-L1 cells. Methodology/Principal Findings In undifferentiated preadipocytes, both T1R2 and T1R3 were expressed very weakly, whereas the expression of T1R3 but not T1R2 was markedly up-regulated upon induction of differentiation (by 83.0 and 3.8-fold, respectively at Day 6). The α subunits of Gs (Gαs) and G14 (Gα14) but not gustducin were expressed throughout the differentiation process. The addition of sucralose or saccharin during the first 48 hours of differentiation considerably reduced the expression of peroxisome proliferator activated receptor γ (PPARγ and CCAAT/enhancer-binding protein α (C/EBPα at Day 2, the expression of aP2 at Day 4 and triglyceride accumulation at Day 6. These anti-adipogenic effects were attenuated by short hairpin RNA-mediated gene-silencing of T1R3. In addition, overexpression of the dominant-negative mutant of Gαs but not YM-254890, an inhibitor of Gα14, impeded the effects of sweeteners, suggesting a possible coupling of Gs with the putative sweet taste-sensing receptor. In agreement, sucralose and saccharin increased the cyclic AMP concentration in differentiating 3T3-L1 cells and also in HEK293 cells heterologously expressing T1R3. Furthermore, the anti-adipogenic effects of sweeteners were mimicked by Gs activation with cholera toxin but not by adenylate cyclase activation with forskolin, whereas small interfering RNA-mediated knockdown of Gαs had the opposite effects. Conclusions 3T3-L1 cells express a functional sweet taste-sensing receptor presumably as a T1R3 homomer, which mediates the anti-adipogenic signal by a Gs-dependent but cAMP-independent mechanism. PMID:23336004

Cloning and expression of R-Spondin1 in different vertebrates suggests a conserved role in ovarian development.

PubMed

Smith, Craig A; Shoemaker, Christina M; Roeszler, Kelly N; Queen, Joanna; Crews, David; Sinclair, Andrew H

2008-07-24

R-Spondin1 (Rspo1) is a novel regulator of the Wnt/beta-catenin signalling pathway. Loss-of-function mutations in human RSPO1 cause testicular differentiation in 46, XX females, pointing to a role in ovarian development. Here we report the cloning and comparative expression analysis of R-SPONDIN1 orthologues in the mouse, chicken and red-eared slider turtle, three species with different sex-determining mechanisms. Evidence is presented that this gene is an ancient component of the vertebrate ovary-determining pathway. Gonadal RSPO1 gene expression is female up-regulated in the embryonic gonads in each species at the onset of sexual differentiation. In the mouse gonad, Rspo1 mRNA is expressed in the somatic cell lineage at the time of ovarian differentiation (E12.5-E15.5), with little expression in germ cells. However, the protein is localised in the cytoplasm and at the cell surface of both somatic (pre-follicular) and germ cells. In the chicken embryo, RSPO1 expression becomes elevated in females at the time of ovarian differentiation, coinciding with female-specific activation of the FOXL2 gene and estrogen synthesis. RSPO1 protein in chicken is localised in the outer cortical zone of the developing ovary, the site of primordial follicle formation and germ cell differentiation. Inhibition of estrogen synthesis with a specific aromatase inhibitor results in a decline in chicken RSPO1 expression, indicating that RSPO1 is influenced by estrogen. In the red-eared slider turtle, which exhibits temperature-dependent sex determination, up-regulation of RSPO1 occurs during the temperature-sensitive period, when gonadal development is responsive to temperature. Accordingly, RSPO1 expression is temperature-responsive, and is down-regulated in embryos shifted from female- to male-producing incubation temperatures. These results indicate that RSPO1 is up-regulated in the embryonic gonads of female vertebrates with different sex-determining mechanisms. In all instances, RSPO1 is expressed in the incipient ovary. These findings suggest that R-SPONDIN1 is an ancient, conserved part of the vertebrate ovary-determining pathway.

Cloning and expression of R-Spondin1 in different vertebrates suggests a conserved role in ovarian development

PubMed Central

Smith, Craig A; Shoemaker, Christina M; Roeszler, Kelly N; Queen, Joanna; Crews, David; Sinclair, Andrew H

2008-01-01

Background R-Spondin1 (Rspo1) is a novel regulator of the Wnt/β-catenin signalling pathway. Loss-of-function mutations in human RSPO1 cause testicular differentiation in 46, XX females, pointing to a role in ovarian development. Here we report the cloning and comparative expression analysis of R-SPONDIN1 orthologues in the mouse, chicken and red-eared slider turtle, three species with different sex-determining mechanisms. Evidence is presented that this gene is an ancient component of the vertebrate ovary-determining pathway. Results Gonadal RSPO1 gene expression is female up-regulated in the embryonic gonads in each species at the onset of sexual differentiation. In the mouse gonad, Rspo1 mRNA is expressed in the somatic cell lineage at the time of ovarian differentiation (E12.5–E15.5), with little expression in germ cells. However, the protein is localised in the cytoplasm and at the cell surface of both somatic (pre-follicular) and germ cells. In the chicken embryo, RSPO1 expression becomes elevated in females at the time of ovarian differentiation, coinciding with female-specific activation of the FOXL2 gene and estrogen synthesis. RSPO1 protein in chicken is localised in the outer cortical zone of the developing ovary, the site of primordial follicle formation and germ cell differentiation. Inhibition of estrogen synthesis with a specific aromatase inhibitor results in a decline in chicken RSPO1 expression, indicating that RSPO1 is influenced by estrogen. In the red-eared slider turtle, which exhibits temperature-dependent sex determination, up-regulation of RSPO1 occurs during the temperature-sensitive period, when gonadal development is responsive to temperature. Accordingly, RSPO1 expression is temperature-responsive, and is down-regulated in embryos shifted from female- to male-producing incubation temperatures. Conclusion These results indicate that RSPO1 is up-regulated in the embryonic gonads of female vertebrates with different sex-determining mechanisms. In all instances, RSPO1 is expressed in the incipient ovary. These findings suggest that R-SPONDIN1 is an ancient, conserved part of the vertebrate ovary-determining pathway. PMID:18651984

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.

Transcriptomic profile adaptations following exposure of equine satellite cells to nutriactive phytochemical gamma-oryzanol.

PubMed

Szcześniak, K A; Ciecierska, A; Ostaszewski, P; Sadkowski, T

2016-01-01

Adult skeletal muscle myogenesis depends on the activation of satellite cells that have the potential to differentiate into new fibers. Gamma-oryzanol (GO), a commercially available nutriactive phytochemical, has gained global interest on account of its muscle-building and regenerating effects. Here, we investigated GO for its potential influence on myogenesis, using equine satellite cell culture model, since the horse is a unique animal, bred and exercised for competitive sport. To our knowledge, this is the first report where the global gene expression in cultured equine satellite cells has been described. Equine satellite cells were isolated from semitendinosus muscle and cultured until the second day of differentiation. Differentiating cells were incubated with GO for the next 24 h. Subsequently, total RNA from GO-treated and control cells was isolated, amplified, labeled, and hybridized to two-color Horse Gene Expression Microarray slides. Quantitative PCR was used for the validation of microarray data. Our results revealed 58 genes with changed expression in GO-treated vs. control cells. Analysis of expression changes suggests that various processes are reinforced by GO in differentiating equine satellite cells, including inhibition of myoblast differentiation, increased proliferation and differentiation, stress response, and increased myogenic lineage commitment. The present study may confirm putative muscle-enhancing abilities of GO; however, the collective role of GO in skeletal myogenesis remains equivocal. The diversity of these changes is likely due to heterogenous growth rate of cells in primary culture. Genes identified in our study, modulated by the presence of GO, may become potential targets of future research investigating impact of this supplement in skeletal muscle on proteomic and biochemical level.

Combinatorial programming of human neuronal progenitors using magnetically-guided stoichiometric mRNA delivery.

PubMed

Azimi, Sayyed M; Sheridan, Steven D; Ghannad-Rezaie, Mostafa; Eimon, Peter M; Yanik, Mehmet Fatih

2018-05-01

Identification of optimal transcription-factor expression patterns to direct cellular differentiation along a desired pathway presents significant challenges. We demonstrate massively combinatorial screening of temporally-varying mRNA transcription factors to direct differentiation of neural progenitor cells using a dynamically-reconfigurable magnetically-guided spotting technology for localizing mRNA, enabling experiments on millimetre size spots. In addition, we present a time-interleaved delivery method that dramatically reduces fluctuations in the delivered transcription-factor copy-numbers per cell. We screened combinatorial and temporal delivery of a pool of midbrain-specific transcription factors to augment the generation of dopaminergic neurons. We show that the combinatorial delivery of LMX1A, FOXA2 and PITX3 is highly effective in generating dopaminergic neurons from midbrain progenitors. We show that LMX1A significantly increases TH -expression levels when delivered to neural progenitor cells either during proliferation or after induction of neural differentiation, while FOXA2 and PITX3 increase expression only when delivered prior to induction, demonstrating temporal dependence of factor addition. © 2018, Azimi et al.

Na+ influx via Orai1 inhibits intracellular ATP-induced mTORC2 signaling to disrupt CD4 T cell gene expression and differentiation.

PubMed

Miao, Yong; Bhushan, Jaya; Dani, Adish; Vig, Monika

2017-05-11

T cell effector functions require sustained calcium influx. However, the signaling and phenotypic consequences of non-specific sodium permeation via calcium channels remain unknown. α-SNAP is a crucial component of Orai1 channels, and its depletion disrupts the functional assembly of Orai1 multimers. Here we show that α-SNAP hypomorph, hydrocephalus with hopping gait, Napa hyh/hyh mice harbor significant defects in CD4 T cell gene expression and Foxp3 regulatory T cell (Treg) differentiation. Mechanistically, TCR stimulation induced rapid sodium influx in Napa hyh/hyh CD4 T cells, which reduced intracellular ATP, [ATP] i . Depletion of [ATP] i inhibited mTORC2 dependent NFκB activation in Napa hyh/hyh cells but ablation of Orai1 restored it. Remarkably, TCR stimulation in the presence of monensin phenocopied the defects in Napa hyh/hyh signaling and Treg differentiation, but not IL-2 expression. Thus, non-specific sodium influx via bonafide calcium channels disrupts unexpected signaling nodes and may provide mechanistic insights into some divergent phenotypes associated with Orai1 function.

Increased cyclooxygenase-2 and thromboxane synthase expression is implicated in diosgenin-induced megakaryocytic differentiation in human erythroleukemia cells.

PubMed

Cailleteau, C; Liagre, B; Battu, S; Jayat-Vignoles, C; Beneytout, J L

2008-09-01

Differentiation induction as a therapeutic strategy has, so far, the greatest impact in hematopoietic malignancies, most notably leukemia. Diosgenin is a very interesting natural product because, depending on the specific dose used, its biological effect is very different in HEL (human erythroleukemia) cells. For example, at 10 microM, diosgenin induced megakaryocytic differentiation, in contrast to 40 microM diosgenin, which induced apoptosis in HEL cells previously demonstrated using sedimentation field-flow fractionation (SdFFF). The goal of this work focused on the correlation between cyclooxygenase-2 (COX-2) and thromboxane synthase (TxS) and megakaryocytic differentiation induced by diosgenin in HEL cells. Furthermore, the technique of SdFFF, having been validated in our models, was used in this new study as an analytical tool that provided us with more or less enriched differentiated cell fractions that could then be used for further analyses of enzyme protein expression and activity for the first time. In our study, we showed the implication of COX-2 and TxS in diosgenin-induced megakaryocytic differentiation in HEL cells. Furthermore, we showed that the analytical technique of SdFFF may be used as a tool to confirm our results as a function of the degree of cell differentiation.

Clonal yeast biofilms can reap competitive advantages through cell differentiation without being obligatorily multicellular

PubMed Central

Hanghøj, Kristian Ebbesen; Andersen, Kaj Scherz; Boomsma, Jacobus J.

2016-01-01

How differentiation between cell types evolved is a fundamental question in biology, but few studies have explored single-gene phenotypes that mediate first steps towards division of labour with selective advantage for groups of cells. Here, we show that differential expression of the FLO11 gene produces stable fractions of Flo11+ and Flo11− cells in clonal Saccharomyces cerevisiae biofilm colonies on medium with intermediate viscosity. Differentiated Flo11+/− colonies, consisting of adhesive and non-adhesive cells, obtain a fourfold growth advantage over undifferentiated colonies by overgrowing glucose resources before depleting them, rather than depleting them while they grow as undifferentiated Flo11− colonies do. Flo11+/− colonies maintain their structure and differentiated state by switching non-adhesive cells to adhesive cells with predictable probability. Mixtures of Flo11+ and Flo11− cells from mutant strains that are unable to use this epigenetic switch mechanism produced neither integrated colonies nor growth advantages, so the condition-dependent selective advantages of differentiated FLO11 expression can only be reaped by clone-mate cells. Our results show that selection for cell differentiation in clonal eukaryotes can evolve before the establishment of obligate undifferentiated multicellularity, and without necessarily leading to more advanced organizational complexity. PMID:27807261

Generation of insulin-producing cells from human bone marrow-derived mesenchymal stem cells: comparison of three differentiation protocols.

PubMed

Gabr, Mahmoud M; Zakaria, Mahmoud M; Refaie, Ayman F; Khater, Sherry M; Ashamallah, Sylvia A; Ismail, Amani M; El-Badri, Nagwa; Ghoneim, Mohamed A

2014-01-01

Many protocols were utilized for directed differentiation of mesenchymal stem cells (MSCs) to form insulin-producing cells (IPCs). We compared the relative efficiency of three differentiation protocols. Human bone marrow-derived MSCs (HBM-MSCs) were obtained from three insulin-dependent type 2 diabetic patients. Differentiation into IPCs was carried out by three protocols: conophylline-based (one-step protocol), trichostatin-A-based (two-step protocol), and β -mercaptoethanol-based (three-step protocol). At the end of differentiation, cells were evaluated by immunolabeling for insulin production, expression of pancreatic endocrine genes, and release of insulin and c-peptide in response to increasing glucose concentrations. By immunolabeling, the proportion of generated IPCs was modest ( ≃ 3%) in all the three protocols. All relevant pancreatic endocrine genes, insulin, glucagon, and somatostatin, were expressed. There was a stepwise increase in insulin and c-peptide release in response to glucose challenge, but the released amounts were low when compared with those of pancreatic islets. The yield of functional IPCs following directed differentiation of HBM-MSCs was modest and was comparable among the three tested protocols. Protocols for directed differentiation of MSCs need further optimization in order to be clinically meaningful. To this end, addition of an extracellular matrix and/or a suitable template should be attempted.

Murine bone cell lines as models for spaceflight induced effects on differentiation and gene expression

NASA Astrophysics Data System (ADS)

Lau, P.; Hellweg, C. E.; Baumstark-Khan, C.; Reitz, G.

Critical health factors for space crews especially on long-term missions are radiation exposure and the absence of gravity DNA double strand breaks DSB are presumed to be the most deleterious DNA lesions after radiation as they disrupt both DNA strands in close proximity Besides radiation risk the absence of gravity influences the complex skeletal apparatus concerning muscle and especially bone remodelling which results from mechanical forces exerting on the body Bone is a dynamic tissue which is life-long remodelled by cells from the osteoblast and osteoclast lineage Any imbalance of this system leads to pathological conditions such as osteoporosis or osteopetrosis Osteoblastic cells play a crucial role in bone matrix synthesis and differentiate either into bone-lining cells or into osteocytes Premature terminal differentiation has been reported to be induced by a number of DNA damaging or cell stress inducing agents including ionising and ultraviolet radiation as well as treatment with mitomycin C In the present study we compare the effects of sequential differentiation by adding osteoinductive substances ss -glycerophosphate and ascorbic acid Radiation-induced premature differentiation was investigated regarding the biosynthesis of specific osteogenic marker molecules and the differentiation dependent expression of marker genes The bone cell model established in our laboratory consists of the osteocyte cell line MLO-Y4 the osteoblast cell line OCT-1 and the subclones 4 and 24 of the osteoblast cell line MC3T3-E1 expressing several

Galectin-3 is essential for proper bone cell differentiation and activity, bone remodeling and biomechanical competence in mice.

PubMed

Iacobini, Carla; Fantauzzi, Claudia Blasetti; Bedini, Rossella; Pecci, Raffaella; Bartolazzi, Armando; Amadio, Bruno; Pesce, Carlo; Pugliese, Giuseppe; Menini, Stefano

2018-02-09

Galectin-3 is constitutively expressed in bone cells and was recently shown to modulate osteogenic transdifferentiation of vascular smooth muscle cells and atherosclerotic calcification. However, the role of galectin-3 in bone physiology is largely undefined. To address this issue, we analyzed (1) the skeletal features of 1-, 3- and 6-month-old galectin-3 null (Lgals3 -/- ) and wild type (WT) mice and (2) the differentiation and function of osteoblasts and osteoclasts derived from these animals. Long bone phenotype, gene expression profile, and remodeling were investigated by micro-computed tomography, real time-PCR, static and dynamic histomorphometry, and assessment of biochemical markers of bone resorption and formation. Bone competence was also evaluated by biomechanical testing at 3 months. In vitro, the effects of galectin-3 deficiency on bone cell differentiation and function were investigated by assessing (a) gene expression of osteoblast markers, alkaline phosphatase activity, mineralization assay, and WNT/β-catenin signaling (of which galectin-3 is a known regulator) in osteoblasts; and (b) tartrate-resistant acid phosphatase activity and bone resorption activity in osteoclasts. Lgals3 -/- mice revealed a wide range of age-dependent alterations including lower bone formation and higher bone resorption, accelerated age-dependent trabecular bone loss (p < 0.01 vs. WT at 3 months) and reduced bone strength (p < 0.01 vs. WT at 3 months). These abnormalities were accompanied by a steady inflammatory state, as revealed by higher bone expression of the pro-inflammatory cytokines interleukin (IL)-1β and IL-6 (p < 0.001 vs. WT at 3 months), increased content of osteal macrophages (p < 0.01 vs. WT at 3 months), and reduced expression of markers of alternative (M2) macrophage activation. Lgals3 -/- osteoblasts and osteoclasts showed impaired terminal differentiation, reduced mineralization capacity (p < 0.01 vs. WT cells) and resorption activity (p < 0.01 vs. WT cells). Mechanistically, impaired differentiation and function of Lgals3 -/- osteoblasts was associated with altered WNT/β-catenin signaling (p < 0.01 vs. WT cells). These data provide evidence for a contribution of galectin-3 to bone cell maturation and function, bone remodeling, and biomechanical competence, thus identifying galectin-3 as a promising therapeutic target for age-related disorders of bone remodeling. Copyright © 2018. Published by Elsevier Inc.

Screening and analysis of genes expressed upon infection of broad bean with Clover yellow vein virus causing lethal necrosis.

PubMed

Nakahara, Kenji S; Kitazawa, Hiroaki; Atsumi, Go; Choi, Sun Hee; Suzuki, Yuji; Uyeda, Ichiro

2011-07-18

Clover yellow vein virus (ClYVV) causes lethal systemic necrosis in legumes, including broad bean (Vicia faba) and pea (Pisum sativum). To identify host genes involved in necrotic symptom expression after ClYVV infection, we screened cDNA fragments in which expression was changed in advance of necrotic symptom expression in broad bean (V. faba cv. Wase) using the differential display technique and secondarily with Northern blot analysis. Expression changes were confirmed in 20 genes, and the six that exhibited the most change were analyzed further. These six genes included a gene that encodes a putative nitrate-induced NOI protein (VfNOI), and another was homologous to an Arabidopsis gene that encodes a glycine- and proline-rich protein GPRP (VfGPRP). We recently reported that necrotic symptom development in ClYVV-infected pea is associated with expression of salicylic acid (SA)-dependent pathogenesis-related (PR) proteins and requires SA-dependent host responses. Interestingly, VfNOI and VfGPRP expression was correlated with that of the putative SA-dependent PR proteins in ClYVV-infected broad bean. However, broad bean infected with a recombinant ClYVV expressing the VfGPRP protein showed weaker symptoms and less viral multiplication than that infected with ClYVV expressing the GFP protein. These results imply that VfGPRP plays a role in defense against ClYVV rather than in necrotic symptom expression.

Magnolol Inhibits RANKL-induced osteoclast differentiation of raw 264.7 macrophages through heme oxygenase-1-dependent inhibition of NFATc1 expression.

PubMed

Lu, Sheng-Hua; Chen, Tso-Hsiao; Chou, Tz-Chong

2015-01-23

Magnolol (1) isolated from Magnolia officinalis exhibits many beneficial effects such as anti-inflammatory and antioxidant activity. The aim of this study was to evaluate the effects of magnolol (1) on RANKL-induced osteoclast differentiation and investigate the underlying molecular mechanisms. Treatment with magnolol (1) significantly inhibited osteoclast differentiation of RAW 264.7 macrophages and bone-resorbing activity of osteoclasts in the RANKL-induced system. Moreover, RANKL-activated JNK/ERK/AP-1 and NF-κB signaling, ROS formation, and NFATc1 activation were attenuated by magnolol (1). A novel finding of this study is that magnolol (1) can increase heme oxygenase-1 (HO-1) expression and Nrf2 activation in RANKL-stimulated cells. Blocking HO-1 activity with tin protoporphyrin IX markedly reversed magnolol (1)-mediated inhibition of osteoclast differentiation, NFATc1 nuclear translocation, and MMP-9 activity, suggesting that HO-1 contributes to the attenuation of NFATc1-mediated osteoclastogenesis by magnolol (1). Therefore, the inhibitory effect of magnolol (1) on osteoclast differentiation is due to inhibition of MAPK/c-fos/AP-1 and NF-κB signaling as well as ROS production and up-regulation of HO-1 expression, which ultimately suppresses NFATc1 induction. These findings indicate that magnolol (1) may have potential to treat bone diseases associated with excessive osteoclastogenesis.

microRNA regulation of T lymphocyte immunity: modulation of molecular networks responsible for T cell activation, differentiation and development

PubMed Central

Podshivalova, Katie; Salomon, Daniel R.

2014-01-01

MicroRNAs (miRNA) are a class of small non-coding RNAs that constitute an essential and evolutionarily conserved mechanism for post-transcriptional gene regulation. Multiple miRNAs have been described to play key roles in T lymphocyte development, differentiation and function. In this review we highlight the current literature regarding the differential expression of miRNAs in various models of mouse and human T cell biology and emphasize mechanistic understandings of miRNA regulation of thymocyte development, T cell activation, and differentiation into effector and memory subsets. We describe the participation of miRNAs in complex regulatory circuits shaping T cell proteomes in a context-dependent manner. It is striking that some miRNAs regulate multiple processes, while others only appear in limited functional contexts. It is also evident that the expression and function of specific miRNAs can differ between mouse and human systems. Ultimately, it is not always correct to simplify the complex events of T cell biology into a model driven by only one or two master regulator miRNAs. In reality, T cell activation and differentiation involves the expression of multiple miRNAs with many mRNA targets and thus, the true extent of miRNA regulation of T cell biology is likely far more vast than currently appreciated. PMID:24099302

RNA-Seq and Gene Network Analysis Uncover Activation of an ABA-Dependent Signalosome During the Cork Oak Root Response to Drought

PubMed Central

Magalhães, Alexandre P.; Verde, Nuno; Reis, Francisca; Martins, Inês; Costa, Daniela; Lino-Neto, Teresa; Castro, Pedro H.; Tavares, Rui M.; Azevedo, Herlânder

2016-01-01

Quercus suber (cork oak) is a West Mediterranean species of key economic interest, being extensively explored for its ability to generate cork. Like other Mediterranean plants, Q. suber is significantly threatened by climatic changes, imposing the need to quickly understand its physiological and molecular adaptability to drought stress imposition. In the present report, we uncovered the differential transcriptome of Q. suber roots exposed to long-term drought, using an RNA-Seq approach. 454-sequencing reads were used to de novo assemble a reference transcriptome, and mapping of reads allowed the identification of 546 differentially expressed unigenes. These were enriched in both effector genes (e.g., LEA, chaperones, transporters) as well as regulatory genes, including transcription factors (TFs) belonging to various different classes, and genes associated with protein turnover. To further extend functional characterization, we identified the orthologs of differentially expressed unigenes in the model species Arabidopsis thaliana, which then allowed us to perform in silico functional inference, including gene network analysis for protein function, protein subcellular localization and gene co-expression, and in silico enrichment analysis for TFs and cis-elements. Results indicated the existence of extensive transcriptional regulatory events, including activation of ABA-responsive genes and ABF-dependent signaling. We were then able to establish that a core ABA-signaling pathway involving PP2C-SnRK2-ABF components was induced in stressed Q. suber roots, identifying a key mechanism in this species’ response to drought. PMID:26793200

TDP-43 causes differential pathology in neuronal versus glial cells in the mouse brain

PubMed Central

Yan, Sen; Wang, Chuan-En; Wei, Wenjie; Gaertig, Marta A.; Lai, Liangxue; Li, Shihua; Li, Xiao-Jiang

2014-01-01

Mutations in TAR DNA-binding protein 43 (TDP-43) are associated with familial forms of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Although recent studies have revealed that mutant TDP-43 in neuronal and glial cells is toxic, how mutant TDP-43 causes primarily neuronal degeneration in an age-dependent manner remains unclear. Using adeno-associated virus (AAV) that expresses mutant TDP-43 (M337V) ubiquitously, we found that mutant TDP-43 accumulates preferentially in neuronal cells in the postnatal mouse brain. We then ubiquitously or selectively expressed mutant TDP-43 in neuronal and glial cells in the striatum of adult mouse brains via stereotaxic injection of AAV vectors and found that it also preferentially accumulates in neuronal cells. Expression of mutant TDP-43 in neurons in the striatum causes more severe degeneration, earlier death and more robust symptoms in mice than expression of mutant TDP-43 in glial cells; however, aging increases the expression of mutant TDP-43 in glial cells, and expression of mutant TDP-43 in older mice caused earlier onset of phenotypes and more severe neuropathology than that in younger mice. Although expression of mutant TDP-43 in glial cells via stereotaxic injection does not lead to robust neurological phenotypes, systemic inhibition of the proteasome activity via MG132 in postnatal mice could exacerbate glial TDP-43-mediated toxicity and cause mice to die earlier. Consistently, this inhibition increases the expression of mutant TDP-43 in glial cells in mouse brains. Thus, the differential accumulation of mutant TDP-43 in neuronal versus glial cells contributes to the preferential toxicity of mutant TDP-43 in neuronal cells and age-dependent pathology. PMID:24381309

TDP-43 causes differential pathology in neuronal versus glial cells in the mouse brain.

PubMed

Yan, Sen; Wang, Chuan-En; Wei, Wenjie; Gaertig, Marta A; Lai, Liangxue; Li, Shihua; Li, Xiao-Jiang

2014-05-15

Mutations in TAR DNA-binding protein 43 (TDP-43) are associated with familial forms of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Although recent studies have revealed that mutant TDP-43 in neuronal and glial cells is toxic, how mutant TDP-43 causes primarily neuronal degeneration in an age-dependent manner remains unclear. Using adeno-associated virus (AAV) that expresses mutant TDP-43 (M337V) ubiquitously, we found that mutant TDP-43 accumulates preferentially in neuronal cells in the postnatal mouse brain. We then ubiquitously or selectively expressed mutant TDP-43 in neuronal and glial cells in the striatum of adult mouse brains via stereotaxic injection of AAV vectors and found that it also preferentially accumulates in neuronal cells. Expression of mutant TDP-43 in neurons in the striatum causes more severe degeneration, earlier death and more robust symptoms in mice than expression of mutant TDP-43 in glial cells; however, aging increases the expression of mutant TDP-43 in glial cells, and expression of mutant TDP-43 in older mice caused earlier onset of phenotypes and more severe neuropathology than that in younger mice. Although expression of mutant TDP-43 in glial cells via stereotaxic injection does not lead to robust neurological phenotypes, systemic inhibition of the proteasome activity via MG132 in postnatal mice could exacerbate glial TDP-43-mediated toxicity and cause mice to die earlier. Consistently, this inhibition increases the expression of mutant TDP-43 in glial cells in mouse brains. Thus, the differential accumulation of mutant TDP-43 in neuronal versus glial cells contributes to the preferential toxicity of mutant TDP-43 in neuronal cells and age-dependent pathology.

Trophic Mode-Dependent Proteomic Analysis Reveals Functional Significance of Light-Independent Chlorophyll Synthesis in Synechocystis sp. PCC 6803.

PubMed

Fang, Longfa; Ge, Haitao; Huang, Xiahe; Liu, Ye; Lu, Min; Wang, Jinlong; Chen, Weiyang; Xu, Wu; Wang, Yingchun

2017-01-09

The photosynthetic model organism Synechocystis sp. PCC 6803 can grow in different trophic modes, depending on the availability of light and exogenous organic carbon source. However, how the protein profile changes to facilitate the cells differentially propagate in different modes has not been comprehensively investigated. Using isobaric labeling-based quantitative proteomics, we simultaneously identified and quantified 45% Synechocystis proteome across four different trophic modes, i.e., autotrophic, heterotrophic, photoheterotrophic, and mixotrophic modes. Among the 155 proteins that are differentially expressed across four trophic modes, proteins involved in nitrogen assimilation and light-independent chlorophyll synthesis are dramatically upregulated in the mixotrophic mode, concomitant with a dramatic increase of P II phosphorylation that senses carbon and nitrogen assimilation status. Moreover, functional study using a mutant defective in light-independent chlorophyll synthesis revealed that this pathway is important for chlorophyll accumulation under a cycled light/dark illumination regime, a condition mimicking day/night cycles in certain natural habitats. Collectively, these results provide the most comprehensive information on trophic mode-dependent protein expression in cyanobacterium, and reveal the functional significance of light-independent chlorophyll synthesis in trophic growth. Copyright © 2017 The Author. Published by Elsevier Inc. All rights reserved.

Delta-like 1/Fetal Antigen-1 (Dlk1/FA1) Is a Novel Regulator of Chondrogenic Cell Differentiation via Inhibition of the Akt Kinase-dependent Pathway*

PubMed Central

Chen, Li; Qanie, Diyako; Jafari, Abbas; Taipaleenmaki, Hanna; Jensen, Charlotte H.; Säämänen, Anna-Marja; Sanz, Maria Luisa Nueda; Laborda, Jorge; Abdallah, Basem M.; Kassem, Moustapha

2011-01-01

Delta-like 1 (Dlk1, also known as fetal antigen-1, FA1) is a member of Notch/Delta family that inhibits adipocyte and osteoblast differentiation; however, its role in chondrogenesis is still not clear. Thus, we overexpressed Dlk1/FA1 in mouse embryonic ATDC5 cells and tested its effects on chondrogenic differentiation. Dlk1/FA1 inhibited insulin-induced chondrogenic differentiation as evidenced by reduction of cartilage nodule formation and gene expression of aggrecan, collagen Type II and X. Similar effects were obtained either by using Dlk1/FA1-conditioned medium or by addition of a purified, secreted, form of Dlk1 (FA1) directly to the induction medium. The inhibitory effects of Dlk1/FA1 were dose-dependent and occurred irrespective of the chondrogenic differentiation stage: proliferation, differentiation, maturation, or hypertrophic conversion. Overexpression or addition of the Dlk1/FA1 protein to the medium strongly inhibited the activation of Akt, but not the ERK1/2, or p38 MAPK pathways, and the inhibition of Akt by Dlk1/FA1 was mediated through PI3K activation. Interestingly, inhibition of fibronectin expression by siRNA rescued the Dlk1/FA1-mediated inhibition of Akt, suggesting interaction of Dlk1/FA1 and fibronectin in chondrogenic cells. Our results identify Dlk1/FA1 as a novel regulator of chondrogenesis and suggest Dlk1/FA1 acts as an inhibitor of the PI3K/Akt pathways that leads to its inhibitory effects on chondrogenesis. PMID:21724852

The SKI proto-oncogene enhances the in vivo repopulation of hematopoietic stem cells and causes myeloproliferative disease.

PubMed

Singbrant, Sofie; Wall, Meaghan; Moody, Jennifer; Karlsson, Göran; Chalk, Alistair M; Liddicoat, Brian; Russell, Megan R; Walkley, Carl R; Karlsson, Stefan

2014-04-01

The proto-oncogene SKI is highly expressed in human myeloid leukemia and also in murine hematopoietic stem cells. However, its operative relevance in these cells remains elusive. We have over-expressed SKI to define its intrinsic role in hematopoiesis and myeloid neoplasms, which resulted in a robust competitive advantage upon transplantation, a complete dominance of the stem and progenitor compartments, and a marked enhancement of myeloid differentiation at the expense of other lineages. Accordingly, enforced expression of SKI induced a gene signature associated with hematopoietic stem cells and myeloid differentiation, as well as hepatocyte growth factor signaling. Here we demonstrate that, in contrast to what has generally been assumed, the significant impact of SKI on hematopoiesis is independent of its ability to inhibit TGF-beta signaling. Instead, myeloid progenitors expressing SKI are partially dependent on functional hepatocyte growth factor signaling. Collectively our results demonstrate that SKI is an important regulator of hematopoietic stem cell activity and its overexpression leads to myeloproliferative disease.

Induction of UO-44 gene expression by tamoxifen in the rat uterus and ovary.

PubMed

Huynh, H; Ng, C Y; Lim, K B; Ong, C K; Ong, C S; Tran, E; Tuyen Nguyen, T T; Chan, T W

2001-07-01

A complementary DNA, uterine-ovarian-specific gene 44 (UO-44), has been isolated from tamoxifen-induced rat uterine complementary DNA library using differential display techniques. UO-44 transcripts are found to be abundant in the uterus and ovary. UO-44 gene expression in the uterus is strictly regulated by estrogens, tamoxifen, and GH, whereas the pure antiestrogen ICI 182780 is inhibitory. Treatment of ovariectomized rats and hypophysectomized rats with tamoxifen and GH, respectively, resulted in up-regulation of UO-44 expression in a dose-dependent manner. In situ hybridization revealed that UO-44 gene expression was restricted to the luminal and glandular epithelial cells of the uterus and to granulosa cells of medium-size ovarian follicles. Transfection studies showed that UO-44 was a membrane-associated protein. Because estrogens, tamoxifen, and GH are stimulators of uterine luminal epithelial cell growth in vivo, UO-44 protein may serve as a mediator of the effect of these compounds in inducing epithelial proliferation and differentiation in these tissues.

The SKI proto-oncogene enhances the in vivo repopulation of hematopoietic stem cells and causes myeloproliferative disease

PubMed Central

Singbrant, Sofie; Wall, Meaghan; Moody, Jennifer; Karlsson, Göran; Chalk, Alistair M.; Liddicoat, Brian; Russell, Megan R.; Walkley, Carl R.; Karlsson, Stefan

2014-01-01

The proto-oncogene SKI is highly expressed in human myeloid leukemia and also in murine hematopoietic stem cells. However, its operative relevance in these cells remains elusive. We have over-expressed SKI to define its intrinsic role in hematopoiesis and myeloid neoplasms, which resulted in a robust competitive advantage upon transplantation, a complete dominance of the stem and progenitor compartments, and a marked enhancement of myeloid differentiation at the expense of other lineages. Accordingly, enforced expression of SKI induced a gene signature associated with hematopoietic stem cells and myeloid differentiation, as well as hepatocyte growth factor signaling. Here we demonstrate that, in contrast to what has generally been assumed, the significant impact of SKI on hematopoiesis is independent of its ability to inhibit TGF-beta signaling. Instead, myeloid progenitors expressing SKI are partially dependent on functional hepatocyte growth factor signaling. Collectively our results demonstrate that SKI is an important regulator of hematopoietic stem cell activity and its overexpression leads to myeloproliferative disease. PMID:24415629

The Quest for Targets Executing MYC-Dependent Cell Transformation.

PubMed

Hartl, Markus

2016-01-01

MYC represents a transcription factor with oncogenic potential converting multiple cellular signals into a broad transcriptional response, thereby controlling the expression of numerous protein-coding and non-coding RNAs important for cell proliferation, metabolism, differentiation, and apoptosis. Constitutive activation of MYC leads to neoplastic cell transformation, and deregulated MYC alleles are frequently observed in many human cancer cell types. Multiple approaches have been performed to isolate genes differentially expressed in cells containing aberrantly activated MYC proteins leading to the identification of thousands of putative targets. Functional analyses of genes differentially expressed in MYC-transformed cells had revealed that so far more than 40 upregulated or downregulated MYC targets are actively involved in cell transformation or tumorigenesis. However, further systematic and selective approaches are required for determination of the known or yet unidentified targets responsible for processing the oncogenic MYC program. The search for critical targets in MYC-dependent tumor cells is exacerbated by the fact that during tumor development, cancer cells progressively evolve in a multistep process, thereby acquiring their characteristic features in an additive manner. Functional expression cloning, combinatorial gene expression, and appropriate in vivo tests could represent adequate tools for dissecting the complex scenario of MYC-specified cell transformation. In this context, the central goal is to identify a minimal set of targets that suffices to phenocopy oncogenic MYC. Recently developed genomic editing tools could be employed to confirm the requirement of crucial transformation-associated targets. Knowledge about essential MYC-regulated genes is beneficial to expedite the development of specific inhibitors to interfere with growth and viability of human tumor cells in which MYC is aberrantly activated. Approaches based on the principle of synthetic lethality using MYC-overexpressing cancer cells and chemical or RNAi libraries have been employed to search for novel anticancer drugs, also leading to the identification of several druggable targets. Targeting oncogenic MYC effector genes instead of MYC may lead to compounds with higher specificities and less side effects. This class of drugs could also display a wider pharmaceutical window because physiological functions of MYC, which are important for normal cell growth, proliferation, and differentiation would be less impaired.

The Quest for Targets Executing MYC-Dependent Cell Transformation

PubMed Central

Hartl, Markus

2016-01-01

MYC represents a transcription factor with oncogenic potential converting multiple cellular signals into a broad transcriptional response, thereby controlling the expression of numerous protein-coding and non-coding RNAs important for cell proliferation, metabolism, differentiation, and apoptosis. Constitutive activation of MYC leads to neoplastic cell transformation, and deregulated MYC alleles are frequently observed in many human cancer cell types. Multiple approaches have been performed to isolate genes differentially expressed in cells containing aberrantly activated MYC proteins leading to the identification of thousands of putative targets. Functional analyses of genes differentially expressed in MYC-transformed cells had revealed that so far more than 40 upregulated or downregulated MYC targets are actively involved in cell transformation or tumorigenesis. However, further systematic and selective approaches are required for determination of the known or yet unidentified targets responsible for processing the oncogenic MYC program. The search for critical targets in MYC-dependent tumor cells is exacerbated by the fact that during tumor development, cancer cells progressively evolve in a multistep process, thereby acquiring their characteristic features in an additive manner. Functional expression cloning, combinatorial gene expression, and appropriate in vivo tests could represent adequate tools for dissecting the complex scenario of MYC-specified cell transformation. In this context, the central goal is to identify a minimal set of targets that suffices to phenocopy oncogenic MYC. Recently developed genomic editing tools could be employed to confirm the requirement of crucial transformation-associated targets. Knowledge about essential MYC-regulated genes is beneficial to expedite the development of specific inhibitors to interfere with growth and viability of human tumor cells in which MYC is aberrantly activated. Approaches based on the principle of synthetic lethality using MYC-overexpressing cancer cells and chemical or RNAi libraries have been employed to search for novel anticancer drugs, also leading to the identification of several druggable targets. Targeting oncogenic MYC effector genes instead of MYC may lead to compounds with higher specificities and less side effects. This class of drugs could also display a wider pharmaceutical window because physiological functions of MYC, which are important for normal cell growth, proliferation, and differentiation would be less impaired. PMID:27313991

Differential activation of catalase expression and activity by PPAR agonists: Implications for astrocyte protection in anti-glioma therapy☆

PubMed Central

Khoo, Nicholas K.H.; Hebbar, Sachin; Zhao, Weiling; Moore, Steven A.; Domann, Frederick E.; Robbins, Mike E.

2013-01-01

Glioma survival is dismal, in part, due to an imbalance in antioxidant expression and activity. Peroxisome proliferator-activated receptor (PPAR) agonists have antineoplastic properties which present new redox-dependent targets for glioma anticancer therapies. Herein, we demonstrate that treatment of primary cultures of normal rat astrocytes with PPAR agonists increased the expression of catalase mRNA protein, and enzymatic activity. In contrast, these same agonists had no effect on catalase expression and activity in malignant rat glioma cells. The increase in steady-state catalase mRNA observed in normal rat astrocytes was due, in part, to de novo mRNA synthesis as opposed to increased catalase mRNA stability. Moreover, pioglitazone-mediated induction of catalase activity in normal rat astrocytes was completely blocked by transfection with a PPARγ-dominant negative plasmid. These data suggest that defects in PPAR-mediated signaling and gene expression may represent a block to normal catalase expression and induction in malignant glioma. The ability of PPAR agonists to differentially increase catalase expression and activity in normal astrocytes but not glioma cells suggests that these compounds might represent novel adjuvant therapeutic agents for the treatment of gliomas. PMID:24024139

Cadmium inhibits neurite outgrowth in differentiating human SH-SY5Y neuroblastoma cells.

PubMed

Pak, Eun Joo; Son, Gi Dong; Yoo, Byung Sun

2014-01-01

Cadmium, a highly ubiquitous heavy metal, is well known to induce neurotoxicity. However, the underlying mechanism of cadmium-mediated neurotoxicity remains unclear. We have studied cadmium inhibition of neurite outgrowth using human SH-SY5Y neuroblastoma cells induced to differentiate by all-trans-retinoic acid (RA). Cadmium, at a concentration of 3 μmol/L, had no significant effect on the viability of differentiating SH-SY5Y cells. However, the neurite outgrowth of the differentiating SH-SY5Y cells 48 hours after cadmium treatment (1-3 μmol/L cadmium) was significantly inhibited in a dose-dependent manner. Treatment of RA-stimulated differentiating SH-SY5Y cells with 1 to 3 μmol/L cadmium resulted in decreased level of cross-reactivities with 43-kDa growth-associated protein (GAP-43) in a dose-dependent manner. The reactive oxygen species (ROS) scavenger, NAC (N-acetyl-l-cysteine), recovered the expression of GAP-43 in cadmium-treated cells. The results indicate that cadmium is able to inhibit neurite outgrowth of differentiating SH-SY5Y cells and that this effect might result from ROS generation by cadmium. © The Author(s) 2014.

Ski can negatively regulates macrophage differentiation through its interaction with PU.1

PubMed Central

Ueki, N; Zhang, L; Haymann, MJ

2010-01-01

In the hematopoietic cell system, the oncoprotein Ski dramatically affects growth and differentiation programs, in some cases leading to malignant leukemia. However, little is known about the interaction partners or signaling pathways involved in the Ski-mediated block of differentiation in hematopoietic cells. Here we show that Ski interacts with PU.1, a lineage-specific transcription factor essential for terminal myeloid differentiation, and thereby represses PU.1-dependent transcriptional activation. Consistent with this, Ski inhibits the biological function of PU.1 to promote myeloid cells to differentiate into macrophage colony-stimulating factor receptor (M-CSFR)-positive macrophages. Using a Ski mutant deficient in PU.1 binding, we demonstrate that Ski–PU.1 interaction is critical for Ski's ability to repress PU.1-dependent transcription and block macrophage differentiation. Furthermore, we provide evidence that Ski-mediated repression of PU.1 is due to Ski's ability to recruit histone deacetylase 3 to PU.1 bound to DNA. Since inactivation of PU.1 is closely related to the development of myeloid leukemia and Ski strongly inhibits PU.1 function, we propose that aberrant Ski expression in certain types of myeloid cell lineages might contribute to leukemogenesis. PMID:17621263

Sulforaphane inhibits mitotic clonal expansion during adipogenesis through cell cycle arrest.

PubMed

Choi, Kyeong-Mi; Lee, Youn-Sun; Sin, Dong-Mi; Lee, Seunghyun; Lee, Mi Kyeong; Lee, Yong-Moon; Hong, Jin-Tae; Yun, Yeo-Pyo; Yoo, Hwan-Soo

2012-07-01

Obesity is a risk factor for numerous metabolic disorders such as type 2 diabetes, hypertension, and coronary heart disease. Adipocyte differentiation is triggered by adipocyte hyperplasia, which leads to obesity. In this study, the inhibitory effect of sulforaphane, an isothiocyanate, on adipogenesis in 3T3-L1 cells was investigated. Sulforaphane decreased the accumulation of lipid droplets stained with Oil Red O and inhibited the elevation of triglycerides in the adipocytes (half-maximal inhibitory concentration = 7.3 µmol/l). The expression of peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein α (C/EBPα), major transcription factors for adipocyte differentiation, was significantly reduced by sulforaphane. The major effects of sulforaphane on the inhibition of adipocyte differentiation occurred during the early stage of adipogenesis. Thus, the expression of C/EBPβ, an early-stage biomarker of adipogenesis, decreased in a concentration-dependent manner when the adipocytes were exposed to sulforaphane (0, 5, 10, and 20 µmol/l). The proliferation of adipocytes treated with 20 µmol/l sulforaphane for 24 and 48 h was also suppressed. These results indicate that sulforaphane may specifically affect mitotic clonal expansion to inhibit adipocyte differentiation. Sulforaphane arrested the cell cycle at the G(0)/G(1) phase, increased p27 expression, and decreased retinoblastoma (Rb) phosphorylation. Additionally, sulforaphane modestly decreased the phosphorylation of ERK1/2 and Akt. Our results indicate that the inhibition of early-stage adipocyte differentiation by sulforaphane may be associated with cell cycle arrest at the G(0)/G(1) phase through upregulation of p27 expression.

Sex-lethal enables germline stem cell differentiation by down-regulating Nanos protein levels during Drosophila oogenesis

PubMed Central

Chau, Johnnie; Kulnane, Laura Shapiro; Salz, Helen K.

2012-01-01

Drosophila ovarian germ cells require Sex-lethal (Sxl) to exit from the stem cell state and to enter the differentiation pathway. Sxl encodes a female-specific RNA binding protein and in somatic cells serves as the developmental switch gene for somatic sex determination and X-chromosome dosage compensation. None of the known Sxl target genes are required for germline differentiation, leaving open the question of how Sxl promotes the transition from stem cell to committed daughter cell. We address the mechanism by which Sxl regulates this transition through the identification of nanos as one of its target genes. Previous studies have shown that Nanos protein is necessary for GSC self-renewal and is rapidly down-regulated in the daughter cells fated to differentiate in the adult ovary. We find that this dynamic expression pattern is limited to female germ cells and is under Sxl control. In the absence of Sxl, or in male germ cells, Nanos protein is continuously expressed. Furthermore, this female-specific expression pattern is dependent on the presence of canonical Sxl binding sites located in the nanos 3′ untranslated region. These results, combined with the observation that nanos RNA associates with the Sxl protein in ovarian extracts and loss and gain of function studies, suggest that Sxl enables the switch from germline stem cell to committed daughter cell by posttranscriptional down-regulation of nanos expression. These findings connect sexual identity to the stem cell self-renewal/differentiation decision and highlight the importance of posttranscriptional gene regulatory networks in controlling stem cell behavior. PMID:22645327

Sex-lethal enables germline stem cell differentiation by down-regulating Nanos protein levels during Drosophila oogenesis.

PubMed

Chau, Johnnie; Kulnane, Laura Shapiro; Salz, Helen K

2012-06-12

Drosophila ovarian germ cells require Sex-lethal (Sxl) to exit from the stem cell state and to enter the differentiation pathway. Sxl encodes a female-specific RNA binding protein and in somatic cells serves as the developmental switch gene for somatic sex determination and X-chromosome dosage compensation. None of the known Sxl target genes are required for germline differentiation, leaving open the question of how Sxl promotes the transition from stem cell to committed daughter cell. We address the mechanism by which Sxl regulates this transition through the identification of nanos as one of its target genes. Previous studies have shown that Nanos protein is necessary for GSC self-renewal and is rapidly down-regulated in the daughter cells fated to differentiate in the adult ovary. We find that this dynamic expression pattern is limited to female germ cells and is under Sxl control. In the absence of Sxl, or in male germ cells, Nanos protein is continuously expressed. Furthermore, this female-specific expression pattern is dependent on the presence of canonical Sxl binding sites located in the nanos 3' untranslated region. These results, combined with the observation that nanos RNA associates with the Sxl protein in ovarian extracts and loss and gain of function studies, suggest that Sxl enables the switch from germline stem cell to committed daughter cell by posttranscriptional down-regulation of nanos expression. These findings connect sexual identity to the stem cell self-renewal/differentiation decision and highlight the importance of posttranscriptional gene regulatory networks in controlling stem cell behavior.

Identification and validation of differentially expressed transcripts by RNA-sequencing of formalin-fixed, paraffin-embedded (FFPE) lung tissue from patients with Idiopathic Pulmonary Fibrosis.

PubMed

Vukmirovic, Milica; Herazo-Maya, Jose D; Blackmon, John; Skodric-Trifunovic, Vesna; Jovanovic, Dragana; Pavlovic, Sonja; Stojsic, Jelena; Zeljkovic, Vesna; Yan, Xiting; Homer, Robert; Stefanovic, Branko; Kaminski, Naftali

2017-01-12

Idiopathic Pulmonary Fibrosis (IPF) is a lethal lung disease of unknown etiology. A major limitation in transcriptomic profiling of lung tissue in IPF has been a dependence on snap-frozen fresh tissues (FF). In this project we sought to determine whether genome scale transcript profiling using RNA Sequencing (RNA-Seq) could be applied to archived Formalin-Fixed Paraffin-Embedded (FFPE) IPF tissues. We isolated total RNA from 7 IPF and 5 control FFPE lung tissues and performed 50 base pair paired-end sequencing on Illumina 2000 HiSeq. TopHat2 was used to map sequencing reads to the human genome. On average ~62 million reads (53.4% of ~116 million reads) were mapped per sample. 4,131 genes were differentially expressed between IPF and controls (1,920 increased and 2,211 decreased (FDR < 0.05). We compared our results to differentially expressed genes calculated from a previously published dataset generated from FF tissues analyzed on Agilent microarrays (GSE47460). The overlap of differentially expressed genes was very high (760 increased and 1,413 decreased, FDR < 0.05). Only 92 differentially expressed genes changed in opposite directions. Pathway enrichment analysis performed using MetaCore confirmed numerous IPF relevant genes and pathways including extracellular remodeling, TGF-beta, and WNT. Gene network analysis of MMP7, a highly differentially expressed gene in both datasets, revealed the same canonical pathways and gene network candidates in RNA-Seq and microarray data. For validation by NanoString nCounter® we selected 35 genes that had a fold change of 2 in at least one dataset (10 discordant, 10 significantly differentially expressed in one dataset only and 15 concordant genes). High concordance of fold change and FDR was observed for each type of the samples (FF vs FFPE) with both microarrays (r = 0.92) and RNA-Seq (r = 0.90) and the number of discordant genes was reduced to four. Our results demonstrate that RNA sequencing of RNA obtained from archived FFPE lung tissues is feasible. The results obtained from FFPE tissue are highly comparable to FF tissues. The ability to perform RNA-Seq on archived FFPE IPF tissues should greatly enhance the availability of tissue biopsies for research in IPF.

In vitro synthesis of oncogenic human papillomaviruses requires episomal genomes for differentiation-dependent late expression.

PubMed Central

Frattini, M G; Lim, H B; Laimins, L A

1996-01-01

Human papillomavirus (HPV) types 16, 18, 31, and 51 are the etiologic agents of many anogenital cancers including those of the cervix. These "high risk" HPVs specifically target genital squamous epithelia, and their lytic life cycle is closely linked to epithelial differentiation. We have developed a genetic assay for HPV functions during pathogenesis using recircularized cloned HPV 31 genomes that were transfected together with a drug resistance marker into monolayer cultures of normal human foreskin keratinocytes, the natural host cell. After drug selection, cell lines were isolated that stably maintained HPV 31 DNA as episomes and underwent terminal differentiation when grown in organotypic raft cultures. In differentiated rafts, the expression of late viral genes, amplification of viral DNA, and production of viral particles were detected in suprabasal cells. This demonstrated the ability to synthesize HPV 31 virions from transfected DNA templates and allowed an examination of HPV functions during the vegetative viral life cycle. We then used this system to investigate whether an episomal genome was required for the induction of late viral gene expression. When an HPV 31 genome (31E1*) containing a missense mutation in the E1 open reading frame was transfected into normal human keratinocytes, the mutant viral sequences were found to integrate into the host cell chromosomal DNA with both early and late regions intact. While high levels of early viral gene transcription were observed, no late gene expression was detected in rafts of cell lines containing the mutant viral genome despite evidence of terminal differentiation. Therefore, the induction of late viral gene expression required that the viral genomes be maintained as extrachromosomal elements, and terminal differentiation alone was not sufficient. These studies provide the basis for a detailed examination of HPV functions during viral pathogenesis. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 Fig. 6 Fig. 7 PMID:8610168

Parkin Knockout Inhibits Neuronal Development via Regulation of Proteasomal Degradation of p21

PubMed Central

Park, Mi Hee; Lee, Hwa-Jeong; Lee, Hye Lim; Son, Dong Ju; Ju, Jung Hoon; Hyun, Byung Kook; Jung, Sung Hee; Song, Ju-Kyoung; Lee, Dong Hun; Hwang, Chul Ju; Han, Sang Bae; Kim, Sanghyeon; Hong, Jin Tae

2017-01-01

PARK2 encodes for the E3 ubiquitin ligase parkin and is implicated in the development of Parkinson's disease (PD). Although the neuroprotective role of parkin is well known, the mechanism of PARK2's function in neural stem differentiation has not yet been thoroughly studied. Co-expressions network analysis showed that synaptosomal-associated protein 25 (SNAP-25) and brain-derived neurotrophic factor (BDNF) were positively correlated with parkin, but negatively correlated with p21 in human patient brain. We investigated a link between the ubiquitin E3 ligase parkin and proteasomal degradation of p21 for the control of neural stem cell differentiation. We found that the neurogenesis was lowered in PARK2 knockout (KO) mice compared with non-tg mice. Expression of the marker protein for neural cell differentiation such as class III beta tubulin (TUBBIII), glial fibrillary acidic protein (GFAP) and neurofilament, as well as SNAP25 and BDNF, was down regulated in PARK2 KO mice. Associated with the loss of differentiation function, p21 protein was highly accumulated in the neural stem cells of PARK2 KO mice. We discovered that p21 directly binds with parkin and is ubiquitinated by parkin which resulted in the loss of cell differentiation ability. Introduction of p21 shRNA in PARK2 KO mice significantly rescued the differentiation efficacy as well as SNAP25 and BDNF expression. c-Jun N-terminal kinase (JNK) pathway is implicated in neurogenesis and p21 degradation. We also defined the decreased p21 ubiquitination and differentiation ability were reversed after treatment with JNK inhibitor, SP600125 in PARK2 KO mice derived neural stem cells. Thus, the present study indicated that parkin knockout inhibits neural stem cell differentiation by JNK-dependent proteasomal degradation of p21. PMID:28656059

Parkin Knockout Inhibits Neuronal Development via Regulation of Proteasomal Degradation of p21.

PubMed

Park, Mi Hee; Lee, Hwa-Jeong; Lee, Hye Lim; Son, Dong Ju; Ju, Jung Hoon; Hyun, Byung Kook; Jung, Sung Hee; Song, Ju-Kyoung; Lee, Dong Hun; Hwang, Chul Ju; Han, Sang Bae; Kim, Sanghyeon; Hong, Jin Tae

2017-01-01

PARK2 encodes for the E3 ubiquitin ligase parkin and is implicated in the development of Parkinson's disease (PD). Although the neuroprotective role of parkin is well known, the mechanism of PARK2's function in neural stem differentiation has not yet been thoroughly studied. Co-expressions network analysis showed that synaptosomal-associated protein 25 (SNAP-25) and brain-derived neurotrophic factor (BDNF) were positively correlated with parkin, but negatively correlated with p21 in human patient brain. We investigated a link between the ubiquitin E3 ligase parkin and proteasomal degradation of p21 for the control of neural stem cell differentiation. We found that the neurogenesis was lowered in PARK2 knockout (KO) mice compared with non-tg mice. Expression of the marker protein for neural cell differentiation such as class III beta tubulin (TUBBIII), glial fibrillary acidic protein (GFAP) and neurofilament, as well as SNAP25 and BDNF, was down regulated in PARK2 KO mice. Associated with the loss of differentiation function, p21 protein was highly accumulated in the neural stem cells of PARK2 KO mice. We discovered that p21 directly binds with parkin and is ubiquitinated by parkin which resulted in the loss of cell differentiation ability. Introduction of p21 shRNA in PARK2 KO mice significantly rescued the differentiation efficacy as well as SNAP25 and BDNF expression. c-Jun N-terminal kinase (JNK) pathway is implicated in neurogenesis and p21 degradation. We also defined the decreased p21 ubiquitination and differentiation ability were reversed after treatment with JNK inhibitor, SP600125 in PARK2 KO mice derived neural stem cells. Thus, the present study indicated that parkin knockout inhibits neural stem cell differentiation by JNK-dependent proteasomal degradation of p21.

The Effects of Orbital Spaceflight on Human Osteoblastic Cell Physiology and Gene Expression

NASA Technical Reports Server (NTRS)

Turner, R. T.

1999-01-01

The purpose of the proposed study is to establish whether changes in gravitational loading have a direct effect on osteoblasts to regulate TGF-6 expression. The effects of spaceflight and reloading on TGF-B MRNA and peptide levels will be studied in a newly developed line of immortalized human fetal osteoblasts (HFOB) transfected with an SV-40 temperature dependent mutant to generate proliferating, undifferentiated hFOB cells at 33-34 C and a non-proliferating, differentiated HFOB cells at 37-39'C. Unlike previous cell culture models, HFOB cells have unlimited proliferative capacity yet can be precisely regulated to differentiate into mature cells which express mature osteoblast function. If isolated osteoblasts respond to changes in mechanical loading in a manner similar to their response in animals, the cell system could provide a powerful model to investigate the signal transduction pathway for gravitational loading.

PI3K/Akt-dependent functions of TFII-I transcription factors in mouse embryonic stem cells.

PubMed

Chimge, Nyam-Osor; Makeyev, Aleksandr V; Waigel, Sabine J; Enkhmandakh, Badam; Bayarsaihan, Dashzeveg

2012-04-01

Activation of PI3K/Akt signaling is sufficient to maintain the pluripotency of mouse embryonic stem cells (mESC) and results in down-regulation of Gtf2i and Gtf2ird1 encoding TFII-I family transcription factors. To investigate how these genes might be involved in the process of embryonic stem cell differentiation, we performed expression microarray profiling of mESC upon inhibition of PI3K by LY294002. This analysis revealed significant alterations in expression of genes for specific subsets of chromatin-modifying enzymes. Surprisingly, genome-wide promoter ChIP-chip mapping indicated that the majority of differently expressed genes could be direct targets of TFII-I regulation. The data support the hypothesis that upregulation of TFII-I factors leads to activation of a specific group of developmental genes during mESC differentiation. © 2011 Wiley Periodicals, Inc.

Nicotine induces cell proliferation in association with cyclin D1 up-regulation and inhibits cell differentiation in association with p53 regulation in a murine pre-osteoblastic cell line

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

Sato, Tsuyoshi; Abe, Takahiro; Nakamoto, Norimichi

Recent studies have suggested that nicotine critically affects bone metabolism. Many studies have examined the effects of nicotine on proliferation and differentiation, but the underlying molecular mechanisms remain unclear. We examined cell cycle regulators involved in the proliferation and differentiation of MC3T3-E1 cells. Nicotine induced cell proliferation in association with p53 down-regulation and cyclin D1 up-regulation. In differentiated cells, nicotine reduced alkaline phosphatase activity and mineralized nodule formation in dose-dependent manners. Furthermore, p53 expression was sustained in nicotine-treated cells during differentiation. These findings indicate that nicotine promotes the cell cycle and inhibits differentiation in association with p53 regulation in pre-osteoblasticmore » cells.« less

Transforming and differentiation-inducing potential of constitutively activated c-kit mutant genes in the IC-2 murine interleukin-3-dependent mast cell line.

PubMed Central

Hashimoto, K.; Tsujimura, T.; Moriyama, Y.; Yamatodani, A.; Kimura, M.; Tohya, K.; Morimoto, M.; Kitayama, H.; Kanakura, Y.; Kitamura, Y.

1996-01-01

Two mutations of c-kit receptor tyrosine kinase (KIT), valine-559 to glycine (G559) and aspartic acid-814 to valine (V814), resulted in its constitutive activation. To examine the transforming and differentiation-inducing potential of the mutant KIT, we used the murine interleukin-3-dependent IC-2 mast cell line as a transfectant. The IC-2 cells contained few basophilic granules and did not express KIT on the surface. The KITG559 or KITV814 gene was introduced into IC-2 cells using a retroviral vector. KITG559 and KITV814 expressed in IC-2 cells were constitutively phosphorylated on tyrosine and demonstrated kinase activity in the absence of stem cell factor, which is a ligand for KIT. IC-2 cells expressing either KITG559 or KITV814 (IC-2G559 or IC-2V814 cells) showed factor-independent growth in suspension culture and produced tumors in nude athymic mice. In addition, IC-2G559 and IC-2V814 cells showed a more mature phenotype compared with the phenotype of the original IC-2 cells, especially after transplantation into nude mice. The number of basophilic granules and the content of histamine increased remarkably. KITG559 and KITV814 also influenced the transcriptional phenotype of mouse mast cell proteases (MMCP) in IC-2 cells. The expression of MMCP-2, MMCP-4, and MMCP-6 was much greater in IC-2G559 and IC-2V814 cells than in the original IC-2 cells. The results indicated that constitutively activated KIT had not only oncogenic activity but also differentiation-inducing activity in mast cells. Images Figure 1 Figure 4 Figure 5 Figure 6 PMID:8546206

Adipose cell differentiation: evidence for a two-step process in the polyamine-dependent Ob1754 clonal line.

PubMed Central

Amri, E Z; Dani, C; Doglio, A; Etienne, J; Grimaldi, P; Ailhaud, G

1986-01-01

A subclone of preadipocyte Ob17 cells has been isolated (Ob1754 clonal line). Confluent Ob1754 cells treated with an inhibitor of spermidine and spermine synthesis, methylglyoxal bis(guanylhydrazone), were totally dependent upon putrescine addition for the expression of glycerol-3-phosphate dehydrogenase which behaved as a late marker of adipose conversion. Under these conditions, the early expression of lipoprotein lipase during growth arrest remained unchanged. Studies at the mRNA level showed that the expression of unidentified pOb24 and pGH3 mRNAs, which was parallel to that of lipoprotein lipase, is independent of polyamine addition whereas the late emergence of glycerol-3-phosphate dehydrogenase mRNA was putrescine-dependent and co-ordinated with the expression of pAL422 mRNA encoding for a myelin-P2 homologue [Bernlohr, Angus, Lane, Bolanowski & Kelly (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 5468-5472]. The appearance of lipoprotein lipase preceded DNA synthesis and post-confluent mitoses which were both putrescine-dependent and which took place before the appearance of glycerol-3-phosphate dehydrogenase. Thus the adipose conversion of Ob1754 cells involves the expression of at least two separate sets of markers which are differently regulated. Images Fig. 3. Fig. 6. PMID:3800927

Coordinated dysregulation of mRNAs and microRNAs in the rat medial prefrontal cortex following a history of alcohol dependence

PubMed Central

Tapocik, Jenica D.; Solomon, Matthew; Flanigan, Meghan; Meinhardt, Marcus; Barbier, Estelle; Schank, Jesse; Schwandt, Melanie; Sommer, Wolfgang H.; Heilig, Markus

2012-01-01

Long-term changes in brain gene expression have been identified in alcohol dependence, but underlying mechanisms remain unknown. Here, we examined the potential role of microRNAs for persistent gene expression changes in the rat medial prefrontal cortex after a history of alcohol dependence. Two-bottle free-choice alcohol consumption increased following 7-week exposure to intermittent alcohol intoxication. A bioinformatic approach using microarray analysis, qPCR, bioinformatic analysis, and microRNA-mRNA integrative analysis identified expression patterns indicative of a disruption in synaptic processes and neuroplasticity. 41 rat-microRNAs and 165 mRNAs in the medial prefrontal cortex were significantly altered after chronic alcohol exposure. A subset of the microRNAs and mRNAs was confirmed by qPCR. Gene ontology categories of differential expression pointed to functional processes commonly associated with neurotransmission, neuroadaptation, and synaptic plasticity. microRNA-mRNA expression pairing identified 33 microRNAs putatively targeting 89 mRNAs suggesting transcriptional networks involved in axonal guidance and neurotransmitter signaling. Our results demonstrate a significant shift in microRNA expression patterns in the medial prefrontal cortex following a history of dependence. Due to their global regulation of multiple downstream target transcripts, microRNAs may play a pivotal role in the reorganization of synaptic connections and long term neuroadaptations in alcohol dependence. microRNA-mediated alterations of transcriptional networks may be involved in disrupted prefrontal control over alcohol-drinking observed in alcoholic patients. PMID:22614244

Identification of an osteoclast transcription factor that binds to the human T cell leukemia virus type I-long terminal repeat enhancer element.

PubMed

Inoue, D; Santiago, P; Horne, W C; Baron, R

1997-10-03

Transgenic mice expressing human T cell leukemia virus type I (HTLV-I)-tax under the control of HTLV-I-long terminal repeat (LTR) promoter develop skeletal abnormalities with high bone turnover and myelofibrosis. In these animals, Tax is highly expressed in bone with a pattern of expression restricted to osteoclasts and spindle-shaped cells within the endosteal myelofibrosis. To test the hypothesis that lineage-specific transcription factors promote transgene expression from the HTLV-I-LTR in osteoclasts, we first examined tax expression in transgenic bone marrow cultures. Expression was dependent on 1alpha,25-dihydroxycholecalciferol and coincided with tartrate-resistant acid phosphatase (TRAP) expression, a marker of osteoclast differentiation. Furthermore, Tax was expressed in vitronectin receptor-positive mononuclear precursors as well as in mature osteoclast-like cells (OCLs). Consistent with our hypothesis, electrophoretic mobility shift assays revealed the presence of an OCL nuclear factor (NFOC-1) that binds to the LTR 21-base pair direct repeat, a region critical for the promoter activity. This binding is further enhanced by Tax. Since NFOC-1 is absent in macrophages and conserved in osteoclasts among species including human, such a factor may play a role in lineage determination and/or in expression of the differentiated osteoclast phenotype.

Transcription profiling of the chilling requirement for bud break in apples: a putative role for FLC-like genes.

PubMed

Porto, Diogo Denardi; Bruneau, Maryline; Perini, Pâmela; Anzanello, Rafael; Renou, Jean-Pierre; dos Santos, Henrique Pessoa; Fialho, Flávio Bello; Revers, Luís Fernando

2015-05-01

Apple production depends on the fulfilment of a chilling requirement for bud dormancy release. Insufficient winter chilling results in irregular and suboptimal bud break in the spring, with negative impacts on apple yield. Trees from apple cultivars with contrasting chilling requirements for bud break were used to investigate the expression of the entire set of apple genes in response to chilling accumulation in the field and controlled conditions. Total RNA was analysed on the AryANE v.1.0 oligonucleotide microarray chip representing 57,000 apple genes. The data were tested for functional enrichment, and differential expression was confirmed by real-time PCR. The largest number of differentially expressed genes was found in samples treated with cold temperatures. Cold exposure mostly repressed expression of transcripts related to photosynthesis, and long-term cold exposure repressed flavonoid biosynthesis genes. Among the differentially expressed selected candidates, we identified genes whose annotations were related to the circadian clock, hormonal signalling, regulation of growth, and flower development. Two genes, annotated as FLOWERING LOCUS C-like and MADS AFFECTING FLOWERING, showed strong differential expression in several comparisons. One of these two genes was upregulated in most comparisons involving dormancy release, and this gene's chromosomal position co-localized with the confidence interval of a major quantitative trait locus for the timing of bud break. These results indicate that photosynthesis and auxin transport are major regulatory nodes of apple dormancy and unveil strong candidates for the control of bud dormancy. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Hypophyseal corticosteroids stimulate somatotrope differentiation in the embryonic chicken pituitary gland.

PubMed

Zheng, Jun; Takagi, Hiroyasu; Tsutsui, Chihiro; Adachi, Akihito; Sakai, Takafumi

2008-03-01

Although it is known that glucocorticoids induce differentiation of growth hormone (GH)-producing cells in rodents and birds, the effect of mineralocorticoids on GH mRNA expression and the origin of corticosteroids affecting somatotrope differentiation have not been elucidated. In this study, we therefore carried out experiments to determine the effect of mineralocorticoids on GH mRNA expression in the chicken anterior pituitary gland in vitro and to determine whether corticosteroids are synthesized in the chicken embryonic pituitary gland. In a pituitary culture experiment with E11 embryos, both corticosterone and aldosterone stimulated GH mRNA expression and increased the number of GH cells in both lobes of the pituitary gland in a dose-dependent manner. These effects of the corticosteroids were significantly reversed by pretreatment with mifepristone, a glucocorticoid receptor (GR) antagonist, or spironolactone, a mineralocorticoid receptor (MR) antagonist. Interestingly, an in vitro serum-free culture experiment with an E11 pituitary gland showed that the GH mRNA level spontaneously increased during cultivation for 2 days without any extra stimulation, and this increase in GH mRNA level was completely suppressed by metyrapone, a corticosterone-producing enzyme P450C11 inhibitor. Moreover, progesterone, the corticosterone precursor, also stimulated GH mRNA expression in the cultured chicken pituitary gland, and this effect was blocked by pretreatment with metyrapone. We also detected mRNA expression of enzymes of cytochrome P450 cholesterol side chain cleavage (P450scc) and 3beta-hydroxysteroid dehydrogenase1 (3beta-HSD1) in the developmental chicken pituitary gland from E14 and E18, respectively. These results suggest that mineralocorticoids as well as glucocorticoids can stimulate GH mRNA expression and that corticosteroids generated in the embryonic pituitary gland by intrinsic steroidogenic enzymes stimulate somatotrope differentiation.

Epigenomics and bolting tolerance in sugar beet genotypes.

PubMed

Hébrard, Claire; Peterson, Daniel G; Willems, Glenda; Delaunay, Alain; Jesson, Béline; Lefèbvre, Marc; Barnes, Steve; Maury, Stéphane

2016-01-01

In sugar beet (Beta vulgaris altissima), bolting tolerance is an essential agronomic trait reflecting the bolting response of genotypes after vernalization. Genes involved in induction of sugar beet bolting have now been identified, and evidence suggests that epigenetic factors are involved in their control. Indeed, the time course and amplitude of DNA methylation variations in the shoot apical meristem have been shown to be critical in inducing sugar beet bolting, and a few functional targets of DNA methylation during vernalization have been identified. However, molecular mechanisms controlling bolting tolerance levels among genotypes are still poorly understood. Here, gene expression and DNA methylation profiles were compared in shoot apical meristems of three bolting-resistant and three bolting-sensitive genotypes after vernalization. Using Cot fractionation followed by 454 sequencing of the isolated low-copy DNA, 6231 contigs were obtained that were used along with public sugar beet DNA sequences to design custom Agilent microarrays for expression (56k) and methylation (244k) analyses. A total of 169 differentially expressed genes and 111 differentially methylated regions were identified between resistant and sensitive vernalized genotypes. Fourteen sequences were both differentially expressed and differentially methylated, with a negative correlation between their methylation and expression levels. Genes involved in cold perception, phytohormone signalling, and flowering induction were over-represented and collectively represent an integrative gene network from environmental perception to bolting induction. Altogether, the data suggest that the genotype-dependent control of DNA methylation and expression of an integrative gene network participate in bolting tolerance in sugar beet, opening up perspectives for crop improvement. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Differential regional expression patterns of α-synuclein, TNF-α, and IL-1β; and variable status of dopaminergic neurotoxicity in mouse brain after Paraquat treatment

PubMed Central

2011-01-01

Background Paraquat (1, 1-dimethyl-4, 4-bipyridium dichloride; PQ) causes neurotoxicity, especially dopaminergic neurotoxicity, and is a supposed risk factor for Parkinson's disease (PD). However, the cellular and molecular mechanisms of PQ-induced neurodegeneration are far from clear. Previous studies have shown that PQ induces neuroinflammation and dopaminergic cell loss, but the prime cause of those events is still in debate. Methods We examined the neuropathological effects of PQ not only in substantia nigra (SN) but also in frontal cortex (FC) and hippocampus of the progressive mouse (adult Swiss albino) model of PD-like neurodegeneration, using immunohistochemistry, western blots, and histological and biochemical analyses. Results PQ caused differential patterns of changes in cellular morphology and expression of proteins related to PD and neuroinflammation in the three regions examined (SN, FC and hippocampus). Coincident with behavioral impairment and brain-specific ROS generation, there was differential immunolocalization and decreased expression levels of tyrosine hydroxylase (TH) in the three regions, whereas α-synuclein immunopositivity increased in hippocampus, increased in FC and decreased in SN. PQ-induced neuroinflammation was characterized by area-specific changes in localization and appearances of microglial cells with or without activation and increment in expression patterns of tumor necrosis factor-α in the three regions of mouse brain. Expression of interleukin-1β was increased in FC and hippocampus but not significantly changed in SN. Conclusion The present study demonstrates that PQ induces ROS production and differential α-synuclein expression that promotes neuroinflammation in microglia-dependent or -independent manners, and produces different patterns of dopaminergic neurotoxicity in three different regions of mouse brain. PMID:22112368

Exercise reduces adipose tissue via cannabinoid receptor type 1 which is regulated by peroxisome proliferator-activated receptor-delta.

PubMed

Yan, Zhen Cheng; Liu, Dao Yan; Zhang, Li Li; Shen, Chen Yi; Ma, Qun Li; Cao, Ting Bing; Wang, Li Juan; Nie, Hai; Zidek, Walter; Tepel, Martin; Zhu, Zhi Ming

2007-03-09

Obesity is one major cardiovascular risk factor. We tested effects of endurance exercise on cannabinoid receptor type 1 (CB1) and peroxisome proliferator-activated receptor-delta (PPAR-delta)-dependent pathways in adipose tissue. Male Wistar rats were randomly assigned to standard laboratory chow or a high-fat diet without and with regular endurance exercise. Exercise in rats on high-fat diet significantly reduced visceral fat mass, blood pressure, and adipocyte size (each p<0.05). Adipocyte hypertrophy induced by high-fat diet was accompanied by increased CB1 expression in adipose tissue, whereas exercise significantly reduced CB1 expression (each p<0.05). CB1 receptor expression and adipocyte differentiation were directly regulated by PPAR-delta. Adipocyte hypertrophy induced by high-fat diet was accompanied by reduced PPAR-delta. Furthermore, selective silencing of PPAR-delta by RNA interference in 3T3-L1-preadipocyte cells significantly increased CB1 expression from 1.00+/-0.06 (n=3) to 1.91+/-0.06 (n=3; p<0.01) and increased adipocyte differentiation, whereas adenovirus-mediated overexpression of PPAR-delta significantly reduced CB1 expression to 0.39+/-0.03 (n=3; p<0.01) and reduced adipocyte differentiation. In the presence of the CB1 antagonist rimonabant adipocyte differentiation in stimulated 3T3 L1 preadipocyte cells was significantly reduced. The study indicates that high-fat diet-induced hypertrophy of adipocytes is associated with increased CB1 receptor expression which is directly regulated by PPAR-delta. Both CB1 and PPAR-delta are intimately involved in therapeutic interventions against a most important cardiovascular risk factor.

BIGEL analysis of gene expression in HL60 cells exposed to X rays or 60 Hz magnetic fields

NASA Technical Reports Server (NTRS)

Balcer-Kubiczek, E. K.; Zhang, X. F.; Han, L. H.; Harrison, G. H.; Davis, C. C.; Zhou, X. J.; Ioffe, V.; McCready, W. A.; Abraham, J. M.; Meltzer, S. J.

1998-01-01

We screened a panel of 1,920 randomly selected cDNAs to discover genes that are differentially expressed in HL60 cells exposed to 60 Hz magnetic fields (2 mT) or X rays (5 Gy) compared to unexposed cells. Identification of these clones was accomplished using our two-gel cDNA library screening method (BIGEL). Eighteen cDNAs differentially expressed in X-irradiated compared to control HL60 cells were recovered from a panel of 1,920 clones. Differential expression in experimental compared to control cells was confirmed independently by Northern blotting of paired total RNA samples hybridized to each of the 18 clone-specific cDNA probes. DNA sequencing revealed that 15 of the 18 cDNA clones produced matches with the database for genes related to cell growth, protein synthesis, energy metabolism, oxidative stress or apoptosis (including MYC, neuroleukin, copper zinc-dependent superoxide dismutase, TC4 RAS-like protein, peptide elongation factor 1alpha, BNIP3, GATA3, NF45, cytochrome c oxidase II and triosephosphate isomerase mRNAs). In contrast, BIGEL analysis of the same 1,920 cDNAs revealed no differences greater than 1.5-fold in expression levels in magnetic-field compared to sham-exposed cells. Magnetic-field-exposed and control samples were analyzed further for the presence of mRNA encoding X-ray-responsive genes by hybridization of the 18 specific cDNA probes to RNA from exposed and control HL60 cells. Our results suggest that differential gene expression is induced in approximately 1% of a random pool of cDNAs by ionizing radiation but not by 60 Hz magnetic fields under the present experimental conditions.

TGFβ1-induced down-regulation of microRNA-138 contributes to epithelial-mesenchymal transition in primary lung cancer cells.

PubMed

Zhang, Fang; Li, Tiepeng; Han, Lu; Qin, Peng; Wu, Zhao; Xu, Benling; Gao, Quanli; Song, Yongping

2018-02-19

The existence of cancer stem cells within the tumor could lead to cancer therapy resistance. TGFβ1 is considered as one of the most powerful players in the generation of CSCs through induction of epithelial-mesenchymal transition in different types of cancer including lung cancer, however, the detailed mechanisms by which TGFβ1 contribute to EMT induction and CSC maintenance remains unclear. Here, we showed primary lung cancer cells treated by TGFβ1 exhibit mesenchymal features, including morphology and expression of mesenchymal marker in a time-dependent manner. We also observed long-term TGFβ1 exposure leads to an enrichment of a sub-population of CD44 + CD90 + cells which represent CSCs in lung cancer cells. Moreover, the differential expression microRNAs between CSCs and non-CSCs were identified using next-generation sequencing to screen key miRNAs which might contribute to TGFβ1-induced EMT and CSCs generation. Among those differentially expressed miRNAs, the expression of microRNA-138 was time-dependently down-regulated by TGFβ1 treatment. We further demonstrated primary lung cancer cells, in which we knockdown the expression of miR-138, exhibit mesenchymal phenotypes and stem cell properties. Taken together, these findings indicate TGFβ1-induced down-regulation of microRNA-138 contributes to EMT in primary lung cancer cells, and suggest that miR-138 might serve as a potential therapeutic target. Copyright © 2018 Elsevier Inc. All rights reserved.

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

Svensson, Emelie; Eriksson, Helena; Gekas, Christos

The Wilms tumor gene 1 (WT1) encodes a zinc-finger-containing transcription factor highly expressed in immature hematopoietic progenitor cells. Overexpression and presence of somatic mutations in acute leukemia indicate a role for WT1 in the pathogenesis of leukemia. CD34{sup +} progenitor cells were transduced with one splice variant of human WT1 without the KTS insert in the zinc-finger domain, WT1(+/-), and with a deleted mutant of WT1 lacking the entire zinc-finger region, WT1(delZ), thus incapable of binding DNA. We show that inhibition of erythroid colony formation and differentiation is absolutely dependent on the DNA-binding zinc-finger domain of WT1. Unexpectedly, however, WT1(delZ)more » was equally effective as wild type protein in the reduction of myeloid clonogenic growth as well as in stimulation of myeloid differentiation, as judged by the expression of cell surface CD11b. Expression of neither WT1(+/-) nor WT1(delZ) upregulated mRNA for the cdk inhibitor p21{sup Waf1/Cip1} or p27{sup Kip1}. Our results demonstrate that WT1 affects proliferation and differentiation in erythroid and myeloid cells by different molecular mechanisms, and suggest that mutations affecting the zinc-finger domain of WT1 could interfere with normal differentiation in the pathogenesis of leukemia.« less

PPARγ agonists promote differentiation of cancer stem cells by restraining YAP transcriptional activity

PubMed Central

Rattanakorn, Kirk; Gadi, Abhilash; Verma, Narendra; Maurizi, Giulia; Gunaratne, Preethi H.; Coarfa, Cristian; Kennedy, Oran D.; Garabedian, Michael J.; Basilico, Claudio; Mansukhani, Alka

2016-01-01

Osteosarcoma (OS) is a highly aggressive pediatric bone cancer in which most tumor cells remain immature and fail to differentiate into bone-forming osteoblasts. However, OS cells readily respond to adipogenic stimuli suggesting they retain mesenchymal stem cell-like properties. Here we demonstrate that nuclear receptor PPARγ agonists such as the anti-diabetic, thiazolidinedione (TZD) drugs induce growth arrest and cause adipogenic differentiation in human, mouse and canine OS cells as well as in tumors in mice. Gene expression analysis reveals that TZDs induce lipid metabolism pathways while suppressing targets of the Hippo-YAP pathway, Wnt signaling and cancer-related proliferation pathways. Significantly, TZD action appears to be restricted to the high Sox2 expressing cancer stem cell population and is dependent on PPARγ expression. TZDs also affect growth and cell fate by causing the cytoplasmic sequestration of the transcription factors SOX2 and YAP that are required for tumorigenicity. Finally, we identify a TZD-regulated gene signature based on Wnt/Hippo target genes and PPARγ that predicts patient outcomes. Together, this work highlights a novel connection between PPARγ agonist in inducing adipogenesis and mimicking the tumor suppressive hippo pathway. It also illustrates the potential of drug repurposing for TZD-based differentiation therapy for osteosarcoma. PMID:27528232

Non-cytotoxic differentiation treatment of renal cell cancer

PubMed Central

Negrotto, Soledad; Hu, Zhenbo; Alcazar, Oscar; Ng, Kwok Peng; Triozzi, Pierre; Lindner, Daniel; Rini, Brian; Saunthararajah, Yogen

2013-01-01

Current drug therapy for metastatic renal cell cancer (RCC) results in temporary disease control but not cure, necessitating continued investigation into alternative mechanistic approaches. Drugs that inhibit chromatin-modifying enzymes involved in transcription repression (chromatin-relaxing drugs) could have a role, by inducing apoptosis, and/or through differentiation pathways. At low doses, the cytosine analogue decitabine can be used to deplete DNA methyl-transferase 1 (DNMT1), modify chromatin and alter differentiation without causing apoptosis (cytotoxicity). Non-cytotoxic regimens of decitabine were evaluated for in vitro and in vivo efficacy against RCC cell lines, including a p53 mutated RCC cell line developed from a patient with treatment refractory metastatic RCC. The cell-division permissive mechanism of action, absence of early apoptosis or DNA damage, increase in expression of HNF4α (a key driver associated with the mesenchymal to epithelial transition), decrease in mesenchymal marker expression, increase in epithelial marker expression, and late increase in cyclin dependent kinase inhibitor CDKN1B (p27) protein, was consistent with differentiation-mediated cell cycle exit. In vivo blood counts and animal weights were consistent with minimal toxicity of therapy. The distinctive mechanism of action of a dose and schedule of decitabine designed for non-cytotoxic depletion of DNMT1 suggests a potential role in treating RCC. PMID:21303982

Medicago truncatula Zinc-Iron Permease6 provides zinc to rhizobia-infected nodule cells.

PubMed

Abreu, Isidro; Saéz, Ángela; Castro-Rodríguez, Rosario; Escudero, Viviana; Rodríguez-Haas, Benjamín; Senovilla, Marta; Larue, Camille; Grolimund, Daniel; Tejada-Jiménez, Manuel; Imperial, Juan; González-Guerrero, Manuel

2017-11-01

Zinc is a micronutrient required for symbiotic nitrogen fixation. It has been proposed that in model legume Medicago truncatula, zinc is delivered by the root vasculature into the nodule and released in the infection/differentiation zone. There, transporters must introduce this element into rhizobia-infected cells to metallate the apoproteins that use zinc as a cofactor. MtZIP6 (Medtr4g083570) is an M. truncatula Zinc-Iron Permease (ZIP) that is expressed only in roots and nodules, with the highest expression levels in the infection/differentiation zone. Immunolocalization studies indicate that it is located in the plasma membrane of nodule rhizobia-infected cells. Down-regulating MtZIP6 expression levels with RNAi does not result in any strong phenotype when plants are fed mineral nitrogen. However, these plants displayed severe growth defects when they depended on nitrogen fixed by their nodules, losing of 80% of their nitrogenase activity. The reduction of this activity was likely an indirect effect of zinc being retained in the infection/differentiation zone and not reaching the cytosol of rhizobia-infected cells. These data are consistent with a model in which MtZIP6 would be responsible for zinc uptake by rhizobia-infected nodule cells in the infection/differentiation zone. © 2017 John Wiley & Sons Ltd.

Functional characterization of the Gentiana lutea zeaxanthin epoxidase (GlZEP) promoter in transgenic tomato plants.

PubMed

Yang, Qingjie; Yuan, Dawei; Shi, Lianxuan; Capell, Teresa; Bai, Chao; Wen, Nuan; Lu, Xiaodan; Sandmann, Gerhard; Christou, Paul; Zhu, Changfu

2012-10-01

The accumulation of carotenoids in plants depends critically on the spatiotemporal expression profiles of the genes encoding enzymes in the carotenogenic pathway. We cloned and characterized the Gentiana lutea zeaxanthin epoxidase (GlZEP) promoter to determine its role in the regulation of carotenogenesis, because the native gene is expressed at high levels in petals, which contain abundant chromoplasts. We transformed tomato (Solanum lycopersicum cv. Micro-Tom) plants with the gusA gene encoding the reporter enzyme β-glucuronidase (GUS) under the control of the GlZEP promoter, and investigated the reporter expression profile at the mRNA and protein levels. We detected high levels of gusA expression and GUS activity in chromoplast-containing flowers and fruits, but minimal levels in immature fruits containing green chloroplasts, in sepals, leaves, stems and roots. GlZEP-gusA expression was strictly associated with fruit development and chromoplast differentiation, suggesting an evolutionarily-conserved link between ZEP and the differentiation of organelles that store carotenoid pigments. The impact of our results on current models for the regulation of carotenogenesis in plants is discussed.

Multiclass cancer diagnosis using tumor gene expression signatures

DOE PAGES

Ramaswamy, S.; Tamayo, P.; Rifkin, R.; ...

2001-12-11

The optimal treatment of patients with cancer depends on establishing accurate diagnoses by using a complex combination of clinical and histopathological data. In some instances, this task is difficult or impossible because of atypical clinical presentation or histopathology. To determine whether the diagnosis of multiple common adult malignancies could be achieved purely by molecular classification, we subjected 218 tumor samples, spanning 14 common tumor types, and 90 normal tissue samples to oligonucleotide microarray gene expression analysis. The expression levels of 16,063 genes and expressed sequence tags were used to evaluate the accuracy of a multiclass classifier based on a supportmore » vector machine algorithm. Overall classification accuracy was 78%, far exceeding the accuracy of random classification (9%). Poorly differentiated cancers resulted in low-confidence predictions and could not be accurately classified according to their tissue of origin, indicating that they are molecularly distinct entities with dramatically different gene expression patterns compared with their well differentiated counterparts. Taken together, these results demonstrate the feasibility of accurate, multiclass molecular cancer classification and suggest a strategy for future clinical implementation of molecular cancer diagnostics.« less

Reading Faces: Differential Lateral Gaze Bias in Processing Canine and Human Facial Expressions in Dogs and 4-Year-Old Children

PubMed Central

Racca, Anaïs; Guo, Kun; Meints, Kerstin; Mills, Daniel S.

2012-01-01

Sensitivity to the emotions of others provides clear biological advantages. However, in the case of heterospecific relationships, such as that existing between dogs and humans, there are additional challenges since some elements of the expression of emotions are species-specific. Given that faces provide important visual cues for communicating emotional state in both humans and dogs, and that processing of emotions is subject to brain lateralisation, we investigated lateral gaze bias in adult dogs when presented with pictures of expressive human and dog faces. Our analysis revealed clear differences in laterality of eye movements in dogs towards conspecific faces according to the emotional valence of the expressions. Differences were also found towards human faces, but to a lesser extent. For comparative purpose, a similar experiment was also run with 4-year-old children and it was observed that they showed differential processing of facial expressions compared to dogs, suggesting a species-dependent engagement of the right or left hemisphere in processing emotions. PMID:22558335

Pleiotrophin (PTN) Expression and Function and in the Mouse Mammary Gland and Mammary Epithelial Cells

PubMed Central

Rosenfield, Sonia M.; Bowden, Emma T.; Cohen-Missner, Shani; Gibby, Krissa A.; Ory, Virginie; Henke, Ralf T.; Riegel, Anna T.; Wellstein, Anton

2012-01-01

Expression of the heparin-binding growth factor, pleiotrophin (PTN) in the mammary gland has been reported but its function during mammary gland development is not known. We examined the expression of PTN and its receptor ALK (Anaplastic Lymphoma Kinase) at various stages of mouse mammary gland development and found that their expression in epithelial cells is regulated in parallel during pregnancy. A 30-fold downregulation of PTN mRNA expression was observed during mid-pregnancy when the mammary gland undergoes lobular-alveolar differentiation. After weaning of pups, PTN expression was restored although baseline expression of PTN was reduced significantly in mammary glands of mice that had undergone multiple pregnancies. We found PTN expressed in epithelial cells of the mammary gland and thus used a monoclonal anti-PTN blocking antibody to elucidate its function in cultured mammary epithelial cells (MECs) as well as during gland development. Real-time impedance monitoring of MECs growth, migration and invasion during anti-PTN blocking antibody treatment showed that MECs motility and invasion but not proliferation depend on the activity of endogenous PTN. Increased number of mammospheres with laminin deposition after anti-PTN blocking antibody treatment of MECs in 3D culture and expression of progenitor markers suggest that the endogenously expressed PTN inhibits the expansion and differentiation of epithelial progenitor cells by disrupting cell-matrix adhesion. In vivo, PTN activity was found to inhibit ductal outgrowth and branching via the inhibition of phospho ERK1/2 signaling in the mammary epithelial cells. We conclude that PTN delays the maturation of the mammary gland by maintaining mammary epithelial cells in a progenitor phenotype and by inhibiting their differentiation during mammary gland development. PMID:23077670

Next-generation sequencing identifies deregulation of microRNAs involved in both innate and adaptive immune response in ALK+ ALCL.

PubMed

Steinhilber, Julia; Bonin, Michael; Walter, Michael; Fend, Falko; Bonzheim, Irina; Quintanilla-Martinez, Leticia

2015-01-01

Anaplastic large cell lymphoma (ALCL) is divided into two systemic diseases according to the expression of the anaplastic lymphoma kinase (ALK). We investigated the differential expression of miRNAs between ALK+ ALCL, ALK- ALCL cells and normal T-cells using next generation sequencing (NGS). In addition, a C/EBPβ-dependent miRNA profile was generated. The data were validated in primary ALCL cases. NGS identified 106 miRNAs significantly differentially expressed between ALK+ and ALK- ALCL and 228 between ALK+ ALCL and normal T-cells. We identified a signature of 56 miRNAs distinguishing ALK+ ALCL, ALK- ALCL and T-cells. The top candidates significant differentially expressed between ALK+ and ALK- ALCL included 5 upregulated miRNAs: miR-340, miR-203, miR-135b, miR-182, miR-183; and 7 downregulated: miR-196b, miR-155, miR-146a, miR-424, miR-503, miR-424*, miR-542-3p. The miR-17-92 cluster was also upregulated in ALK+ cells. Additionally, we identified a signature of 3 miRNAs significantly regulated by the transcription factor C/EBPβ, which is specifically overexpressed in ALK+ ALCL, including the miR-181 family. Of interest, miR-181a, which regulates T-cell differentiation and modulates TCR signalling strength, was significantly downregulated in ALK+ ALCL cases. In summary, our data reveal a miRNA signature linking ALK+ ALCL to a deregulated immune response and may reflect the abnormal TCR antigen expression known in ALK+ ALCL.

Filaggrin-dependent secretion of sphingomyelinase protects against staphylococcal α-toxin-induced keratinocyte death.

PubMed

Brauweiler, Anne M; Bin, Lianghua; Kim, Byung Eui; Oyoshi, Michiko K; Geha, Raif S; Goleva, Elena; Leung, Donald Y M

2013-02-01

The skin of patients with atopic dermatitis (AD) has defects in keratinocyte differentiation, particularly in expression of the epidermal barrier protein filaggrin. AD skin lesions are often exacerbated by Staphylococcus aureus-mediated secretion of the virulence factor α-toxin. It is unknown whether lack of keratinocyte differentiation predisposes to enhanced lethality from staphylococcal toxins. We investigated whether keratinocyte differentiation and filaggrin expression protect against cell death induced by staphylococcal α-toxin. Filaggrin-deficient primary keratinocytes were generated through small interfering RNA gene knockdown. RNA expression was determined by using real-time PCR. Cell death was determined by using the lactate dehydrogenase assay. Keratinocyte cell survival in filaggrin-deficient (ft/ft) mouse skin biopsies was determined based on Keratin 5 staining. α-Toxin heptamer formation and acid sphingomyelinase expression were determined by means of immunoblotting. We found that filaggrin expression, occurring as the result of keratinocyte differentiation, significantly inhibits staphylococcal α-toxin-mediated pathogenicity. Furthermore, filaggrin plays a crucial role in protecting cells by mediating the secretion of sphingomyelinase, an enzyme that reduces the number of α-toxin binding sites on the keratinocyte surface. Finally, we determined that sphingomyelinase enzymatic activity directly prevents α-toxin binding and protects keratinocytes against α-toxin-induced cytotoxicity. The current study introduces the novel concept that S aureus α-toxin preferentially targets and destroys filaggrin-deficient keratinocytes. It also provides a mechanism to explain the increased propensity for S aureus-mediated exacerbation of AD skin disease. Copyright © 2012 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

Estimating the age of Lucilia illustris during the intrapuparial period using two approaches: Morphological changes and differential gene expression.

PubMed

Wang, Yu; Gu, Zhi-Ya; Xia, Shui-Xiu; Wang, Jiang-Feng; Zhang, Ying-Na; Tao, Lu-Yang

2018-06-01

Lucilia illustris (Meigen, 1826) (Diptera: Calliphoridae) is a cosmopolitan species of fly that has forensic and medical significance. However, there is no relevant study regarding the determination of the age of this species during the intrapuparial period. In this study, we investigated the changes in both morphology and differential gene expression during intrapuparial development, with an aim to estimate the age of L. illustris during the intrapuparial stage. The overall intrapuparial morphological changes of L. illustris were divided into 12 substages. Structures such as the compound eyes, mouthparts, antennae, thorax, legs, wings, and abdomen, each capable of indicating age during the intrapuparial stage, were observed in detail, and the developmental progression of each of these structures was divided into six to eight stages. We recorded the time range over which each substage or structure appeared. The differential expression of the three genes 15_2, actin, and tbp previously identified for predicting the timing of intrapuparial development was measured during L. illustris metamorphosis. The expression of these genes was quantified by real-time PCR, and the results revealed that these genes can be used to estimate the age of L. illustris during the intrapuparial period, as they exhibit regular changes and temperature dependence. This study provides an important basis for estimating the minimum postmortem interval (PMI min ) in forensic entomology according to changes in intrapuparial development and differential gene expression. Furthermore, combination of the two approaches can generate a more precise PMI min than either approach alone. Copyright © 2018 Elsevier B.V. All rights reserved.

Aging-dependent DNA hypermethylation and gene expression of GSTM1 involved in T cell differentiation.

PubMed

Yeh, Shu-Hui; Liu, Cheng-Ling; Chang, Ren-Chieh; Wu, Chih-Chiang; Lin, Chia-Hsueh; Yang, Kuender D

2017-07-25

This study investigated whether aging was associated with epigenetic changes of DNA hypermethylation on immune gene expression and lymphocyte differentiation. We screened CG sites of methylation in blood leukocytes from different age populations, picked up genes with age-related increase of CG methylation content more than 15%, and validated immune related genes with CG hypermethylation involved in lymphocyte differentiation in the aged population. We found that 12 genes (EXHX1、 IL-10、 TSP50、 GSTM1、SLC5A5、SPI1、F2R、LMO2、PTPN6、FGFR2、MMP9、MET) were associated with promoter or exon one DNA hypermethylation in the aged group. Two immune related genes, GSTM1 and LMO2, were chosen to validate its aging-related CG hypermethylation in different leukocytes. We are the first to validate that GSTM1_P266 and LMO2_E128 CG methylation contents in T lymphocytes but not polymorphonuclear cells (PMNs) or mononuclear cells (MNCs) were significantly increased in the aged population. The GSTM1 mRNA expression in T lymphocytes but not PMNs or MNCs was inversely associated with the GSTM1 CG hypermethylation levels in the aged population studied. Further studies showed that lower GSTM1 CG methylation content led to the higher GSTM1 mRNA expression in T cells and knockdown of GSTM1 mRNA expression decreased type 1 T helper cell (Th1) differentiation in Jurkat T cells and normal adult CD4 T cells. The GSTM1_P266 hypermethylation in the aged population associated with lower GSTM1 mRNA expression was involved in Th1 differentiation, highlighting that modulation of aging-associated GSTM1 methylation may be able to enhance T helper cell immunity in the elders.

Directed Neural Differentiation of Mouse Embryonic Stem Cells Is a Sensitive System for the Identification of Novel Hox Gene Effectors

PubMed Central

Bami, Myrto; Episkopou, Vasso; Gavalas, Anthony; Gouti, Mina

2011-01-01

The evolutionarily conserved Hox family of homeodomain transcription factors plays fundamental roles in regulating cell specification along the anterior posterior axis during development of all bilaterian animals by controlling cell fate choices in a highly localized, extracellular signal and cell context dependent manner. Some studies have established downstream target genes in specific systems but their identification is insufficient to explain either the ability of Hox genes to direct homeotic transformations or the breadth of their patterning potential. To begin delineating Hox gene function in neural development we used a mouse ES cell based system that combines efficient neural differentiation with inducible Hoxb1 expression. Gene expression profiling suggested that Hoxb1 acted as both activator and repressor in the short term but predominantly as a repressor in the long run. Activated and repressed genes segregated in distinct processes suggesting that, in the context examined, Hoxb1 blocked differentiation while activating genes related to early developmental processes, wnt and cell surface receptor linked signal transduction and cell-to-cell communication. To further elucidate aspects of Hoxb1 function we used loss and gain of function approaches in the mouse and chick embryos. We show that Hoxb1 acts as an activator to establish the full expression domain of CRABPI and II in rhombomere 4 and as a repressor to restrict expression of Lhx5 and Lhx9. Thus the Hoxb1 patterning activity includes the regulation of the cellular response to retinoic acid and the delay of the expression of genes that commit cells to neural differentiation. The results of this study show that ES neural differentiation and inducible Hox gene expression can be used as a sensitive model system to systematically identify Hox novel target genes, delineate their interactions with signaling pathways in dictating cell fate and define the extent of functional overlap among different Hox genes. PMID:21637844

Interferon regulatory factor 4 (IRF4) controls myeloid-derived suppressor cell (MDSC) differentiation and function.

PubMed

Nam, Sorim; Kang, Kyeongah; Cha, Jae Seon; Kim, Jung Woo; Lee, Hee Gu; Kim, Yonghwan; Yang, Young; Lee, Myeong-Sok; Lim, Jong-Seok

2016-12-01

Myeloid-derived suppressor cells (MDSCs) are immature cells that do not differentiate into mature myeloid cells. Two major populations of PMN-MDSCs (Ly6G high Ly6C low Gr1 high CD11b + ) and MO-MDSCs (Ly6G - Ly6C high Gr-1 int CD11b + ) have an immune suppressive function. Interferon regulatory factor 4 (IRF4) has a role in the negative regulation of TLR signaling and is associated with lymphoid cell development. However, the roles of IRF4 in myeloid cell differentiation are unclear. In this study, we found that IRF4 expression was remarkably suppressed during the development of MDSCs in the tumor microenvironment. Both the mRNA and protein levels of IRF4 in MDSCs were gradually reduced, depending on the development of tumors in the 4T1 model. siRNA-mediated knockdown of IRF4 in bone marrow cells promoted the differentiation of PMN-MDSCs. Similarly, IRF4 inhibition in bone marrow cells using simvastatin, which has been known to inhibit IRF4 expression, increased PMN-MDSC numbers. In contrast, IRF4 overexpression in bone marrow cells inhibited the total numbers of MDSCs, especially PMN-MDSCs. Notably, treatment with IL-4, an upstream regulator of IRF4, induced IRF4 expression in the bone marrow cells, and consequently, IL-4-induced IRF4 expression resulted in a decrease in PMN-MDSC numbers. Finally, we confirmed that IRF4 expression in MDSCs can modulate their activity to inhibit T cell proliferation through IL-10 production and ROS generation, and myeloid-specific deletion of IRF4 leads to the increase of MDSC differentiation. Our present findings indicate that IRF4 reduction induced by tumor formation can increase the number of MDSCs, and increases in the IRF4 expression in MDSCs may infringe on the immune-suppressive function of MDSCs. © Society for Leukocyte Biology.

Gene set differential analysis of time course expression profiles via sparse estimation in functional logistic model with application to time-dependent biomarker detection.

PubMed

Kayano, Mitsunori; Matsui, Hidetoshi; Yamaguchi, Rui; Imoto, Seiya; Miyano, Satoru

2016-04-01

High-throughput time course expression profiles have been available in the last decade due to developments in measurement techniques and devices. Functional data analysis, which treats smoothed curves instead of originally observed discrete data, is effective for the time course expression profiles in terms of dimension reduction, robustness, and applicability to data measured at small and irregularly spaced time points. However, the statistical method of differential analysis for time course expression profiles has not been well established. We propose a functional logistic model based on elastic net regularization (F-Logistic) in order to identify the genes with dynamic alterations in case/control study. We employ a mixed model as a smoothing method to obtain functional data; then F-Logistic is applied to time course profiles measured at small and irregularly spaced time points. We evaluate the performance of F-Logistic in comparison with another functional data approach, i.e. functional ANOVA test (F-ANOVA), by applying the methods to real and synthetic time course data sets. The real data sets consist of the time course gene expression profiles for long-term effects of recombinant interferon β on disease progression in multiple sclerosis. F-Logistic distinguishes dynamic alterations, which cannot be found by competitive approaches such as F-ANOVA, in case/control study based on time course expression profiles. F-Logistic is effective for time-dependent biomarker detection, diagnosis, and therapy. © The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

The MHV68 M2 protein drives IL-10 dependent B cell proliferation and differentiation.

PubMed

Siegel, Andrea M; Herskowitz, Jeremy H; Speck, Samuel H

2008-04-04

Murine gammaherpesvirus 68 (MHV68) establishes long-term latency in memory B cells similar to the human gammaherpesvirus Epstein Barr Virus (EBV). EBV encodes an interleukin-10 (IL-10) homolog and modulates cellular IL-10 expression; however, the role of IL-10 in the establishment and/or maintenance of chronic EBV infection remains unclear. Notably, MHV68 does not encode an IL-10 homolog, but virus infection has been shown to result in elevated serum IL-10 levels in wild-type mice, and IL-10 deficiency results in decreased establishment of virus latency. Here we show that a unique MHV68 latency-associated gene product, the M2 protein, is required for the elevated serum IL-10 levels observed at 2 weeks post-infection. Furthermore, M2 protein expression in primary murine B cells drives high level IL-10 expression along with increased secretion of IL-2, IL-6, and MIP-1alpha. M2 expression was also shown to significantly augment LPS driven survival and proliferation of primary murine B cells. The latter was dependent on IL-10 expression as demonstrated by the failure of IL10-/- B cells to proliferate in response to M2 protein expression and rescue of M2-associated proliferation by addition of recombinant murine IL-10. M2 protein expression in primary B cells also led to upregulated surface expression of the high affinity IL-2 receptor (CD25) and the activation marker GL7, along with down-regulated surface expression of B220, MHC II, and sIgD. The cells retained CD19 and sIgG expression, suggesting differentiation to a pre-plasma memory B cell phenotype. These observations are consistent with previous analyses of M2-null MHV68 mutants that have suggested a role for the M2 protein in expansion and differentiation of MHV68 latently infected B cells-perhaps facilitating the establishment of virus latency in memory B cells. Thus, while the M2 protein is unique to MHV68, analysis of M2 function has revealed an important role for IL-10 in MHV68 pathogenesis-identifying a strategy that appears to be conserved between at least EBV and MHV68.

Alteration in gene expression in Nile tilapia (Oreochromis niloticus) juveniles submitted to fasting and refeeding.

USDA-ARS?s Scientific Manuscript database

One of the most important biological processes in living organisms that are affected by environmental fluctuations is growth, and the skeletal muscle growth in fish is dependent on proliferation and differentiation of myogenic precursor cells that are activated by Myogenic Regulatory Factors or inhi...

Statins impact primary embryonic mouse neural stem cell survival, cell death, and fate through distinct mechanisms.

PubMed

Carson, Ross A; Rudine, Anthony C; Tally, Serena J; Franks, Alexis L; Frahm, Krystle A; Waldman, Jacob K; Silswal, Neerupma; Burale, Suban; Phan, James V; Chandran, Uma R; Monaghan, A Paula; DeFranco, Donald B

2018-01-01

Statins inhibit HMG-CoA reductase, the rate-limiting enzyme in the cholesterol biosynthesis pathway (CBP), and are used for the prevention of cardiovascular disease. The anti-inflammatory effects of statins may also provide therapeutic benefits and have led to their use in clinical trials for preeclampsia, a pregnancy-associated inflammatory condition, despite their current classification as category X (i.e. contraindicated during pregnancy). In the developing neocortex, products of the CBP play essential roles in proliferation and differentiation of neural stem-progenitor cells (NSPCs). To understand how statins could impact the developing brain, we studied effects of pravastatin and simvastatin on primary embryonic NSPC survival, proliferation, global transcription, and cell fate in vitro. We found that statins dose dependently decrease NSPC expansion by promoting cell death and autophagy of NSPCs progressing through the G1 phase of the cell cycle. Genome-wide transcriptome analysis demonstrates an increase in expression of CBP genes following pravastatin treatment, through activation of the SREBP2 transcription factor. Co-treatment with farnesyl pyrophosphate (FPP), a CBP metabolite downstream of HMG-CoA reductase, reduces SREBP2 activation and pravastatin-induced PARP cleavage. Finally, pravastatin and simvastatin differentially alter NSPC cell fate and mRNA expression during differentiation, through a non-CBP dependent pathway.

Modulation of tyrosine hydroxylase expression by melatonin in human SH-SY5Y neuroblastoma cells.

PubMed

McMillan, Catherine R; Sharma, Rohita; Ottenhof, Tom; Niles, Lennard P

2007-06-04

We have previously reported in vivo preservation of tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis, following treatment with physiological doses of melatonin, in a 6-hydroxydopamine model of Parkinson's disease. Based on these findings, we postulated that melatonin would similarly modulate the expression of TH in vitro. Therefore, using human SH-SY5Y neuroblastoma cells which can differentiate into dopaminergic neurons following treatment with retinoic acid, we first examined whether these cells express melatonin receptors. Subsequently, the physiological dose-dependent effects of melatonin on TH expression were examined in both undifferentiated and differentiated cells. The novel detection of the G protein-coupled melatonin MT(1) receptor in SH-SY5Y cells by RT-PCR was confirmed by sequencing and Western blotting. In addition, following treatment of SH-SY5Y cells with melatonin (0.1-100 nM) for 24h, Western analysis revealed a significant increase in TH protein levels. A biphasic response, with significant increases in TH protein at 0.5 and 1 nM melatonin and a reversal at higher doses was seen in undifferentiated cells; whereas in differentiated cells, melatonin was effective at doses of 1 and 100 nM. These findings suggest a physiological role for melatonin in modulating TH expression, possibly via the MT(1) receptor.

Reactive oxygen species promote ovarian cancer progression via the HIF-1α/LOX/E-cadherin pathway.

PubMed

Wang, Yu; Ma, Jun; Shen, Haoran; Wang, Chengjie; Sun, Yueping; Howell, Stephen B; Lin, Xinjian

2014-11-01

Reactive oxygen species (ROS) can drive the de‑differentiation of tumor cells leading to the process of epithelial-to-mesenchymal transition (EMT) to enhance invasion and metastasis. The invasive and metastatic phenotype of malignant cells is often linked to loss of E-cadherin expression, a hallmark of EMT. Recent studies have demonstrated that hypoxic exposure causes HIF-1-dependent repression of E-cadherin. However, the mechanism by which ROS and/or HIF suppresses E-cadherin expression remains less clear. In the present study, we found that ROS accumulation in ovarian carcinoma cells upregulated HIF-1α expression and subsequent transcriptional induction of lysyl oxidase (LOX) which repressed E-cadherin. Loss of E-cadherin facilitated ovarian cancer (OC) cell migration in vitro and promoted tumor growth in vivo. E-cadherin immunoreactivity correlated with International Federation of Gynecology and Obstetrics (FIGO) stage, tumor differentiation and metastasis. Negative E-cadherin expression along with FIGO stage, tumor differentiation and metastasis significantly predicted for a lower 5-year survival rate. These findings suggest that ROS play an important role in the initiation of metastatic growth of OC cells and support a molecular pathway from ROS to aggressive transformation which involves upregulation of HIF-1α and its downstream target LOX to suppress E-cadherin expression leading to an increase in cell motility and invasiveness.

Differentially-Expressed Genes Associated with Faster Growth of the Pacific Abalone, Haliotis discus hannai

PubMed Central

Choi, Mi-Jin; Kim, Gun-Do; Kim, Jong-Myoung; Lim, Han Kyu

2015-01-01

The Pacific abalone Haliotis discus hannai is used for commercial aquaculture in Korea. We examined the transcriptome of Pacific abalone Haliotis discus hannai siblings using NGS technology to identify genes associated with high growth rates. Pacific abalones grown for 200 days post-fertilization were divided into small-, medium-, and large-size groups with mean weights of 0.26 ± 0.09 g, 1.43 ± 0.405 g, and 5.24 ± 1.09 g, respectively. RNA isolated from the soft tissues of each group was subjected to RNA sequencing. Approximately 1%–3% of the transcripts were differentially expressed in abalones, depending on the growth rate. RT-PCR was carried out on thirty four genes selected to confirm the relative differences in expression detected by RNA sequencing. Six differentially-expressed genes were identified as associated with faster growth of the Pacific abalone. These include five up-regulated genes (including one specific to females) encoding transcripts homologous to incilarin A, perlucin, transforming growth factor-beta-induced protein immunoglobulin-heavy chain 3 (ig-h3), vitelline envelope zona pellucida domain 4, and defensin, and one down-regulated gene encoding tomoregulin in large abalones. Most of the transcripts were expressed predominantly in the hepatopancreas. The genes identified in this study will lead to development of markers for identification of high-growth-rate abalones and female abalones. PMID:26593905

Differentially-Expressed Genes Associated with Faster Growth of the Pacific Abalone, Haliotis discus hannai.

PubMed

Choi, Mi-Jin; Kim, Gun-Do; Kim, Jong-Myoung; Lim, Han Kyu

2015-11-18

The Pacific abalone Haliotis discus hannai is used for commercial aquaculture in Korea. We examined the transcriptome of Pacific abalone Haliotis discus hannai siblings using NGS technology to identify genes associated with high growth rates. Pacific abalones grown for 200 days post-fertilization were divided into small-, medium-, and large-size groups with mean weights of 0.26 ± 0.09 g, 1.43 ± 0.405 g, and 5.24 ± 1.09 g, respectively. RNA isolated from the soft tissues of each group was subjected to RNA sequencing. Approximately 1%-3% of the transcripts were differentially expressed in abalones, depending on the growth rate. RT-PCR was carried out on thirty four genes selected to confirm the relative differences in expression detected by RNA sequencing. Six differentially-expressed genes were identified as associated with faster growth of the Pacific abalone. These include five up-regulated genes (including one specific to females) encoding transcripts homologous to incilarin A, perlucin, transforming growth factor-beta-induced protein immunoglobulin-heavy chain 3 (ig-h3), vitelline envelope zona pellucida domain 4, and defensin, and one down-regulated gene encoding tomoregulin in large abalones. Most of the transcripts were expressed predominantly in the hepatopancreas. The genes identified in this study will lead to development of markers for identification of high-growth-rate abalones and female abalones.

Indirect presentation in the thymus limits naive and regulatory T-cell differentiation by promoting deletion of self-reactive thymocytes.

PubMed

Yap, Jin Yan; Wirasinha, Rushika C; Chan, Anna; Howard, Debbie R; Goodnow, Christopher C; Daley, Stephen R

2018-02-07

Acquisition of T-cell central tolerance involves distinct pathways of self-antigen presentation to thymocytes. One pathway termed indirect presentation requires a self-antigen transfer step from thymic epithelial cells (TECs) to bone marrow-derived cells before the self-antigen is presented to thymocytes. The role of indirect presentation in central tolerance is context-dependent, potentially due to variation in self-antigen expression, processing and presentation in the thymus. Here, we report experiments in mice in which TECs expressed a membrane-bound transgenic self-antigen, hen egg lysozyme (HEL), from either the insulin (insHEL) or thyroglobulin (thyroHEL) promoter. Intrathymic HEL expression was less abundant and more confined to the medulla in insHEL mice compared with thyroHEL mice. When indirect presentation was impaired by generating mice lacking MHC class II expression in bone marrow-derived antigen-presenting cells, insHEL-mediated thymocyte deletion was abolished, whereas thyroHEL-mediated deletion occurred at a later stage of thymocyte development and Foxp3 + regulatory T-cell differentiation increased. Indirect presentation increased the strength of T-cell receptor signalling that both self-antigens induced in thymocytes, as assessed by Helios expression. Hence, indirect presentation limits the differentiation of naive and regulatory T cells by promoting deletion of self-reactive thymocytes. © 2018 John Wiley & Sons Ltd.

Increased expression of urokinase plasminogen activator in Quebec platelet disorder is linked to megakaryocyte differentiation

PubMed Central

Veljkovic, D. Kika; Rivard, Georges E.; Diamandis, Maria; Blavignac, Jessica; Cramer-Bordé, Elisabeth M.

2009-01-01

Quebec platelet disorder (QPD) is an inherited bleeding disorder associated with increased urokinase plasminogen activator (uPA) in platelets but not in plasma, intraplatelet plasmin generation, and α-granule protein degradation. These abnormalities led us to investigate uPA expression by QPD CD34+ progenitors, cultured megakaryocytes, and platelets, and whether uPA was stored in QPD α-granules. Although QPD CD34+ progenitors expressed normal amounts of uPA, their differentiation into megakaryocytes abnormally increased expression of the uPA gene but not the flanking genes for vinculin or calcium/calmodulin-dependent protein kinase IIγ on chromosome 10. The increased uPA production by cultured QPD megakaryocytes mirrored their production of α-granule proteins, which was normal. uPA was localized to QPD α-granules and it showed extensive colocalization with α-granule proteins in both cultured QPD megakaryocytes and platelets, and with plasminogen in QPD platelets. In QPD megakaryocytes, cultured without or with plasma as a source of plasminogen, α-granule proteins were stored undegraded and this was associated with much less uPA-plasminogen colocalization than in QPD platelets. Our studies indicate that the overexpression of uPA in QPD emerges with megakaryocyte differentiation, without altering the expression of flanking genes, and that uPA is costored with α-granule proteins prior to their proteolysis in QPD. PMID:19029443

Increased expression of urokinase plasminogen activator in Quebec platelet disorder is linked to megakaryocyte differentiation.

PubMed

Veljkovic, D Kika; Rivard, Georges E; Diamandis, Maria; Blavignac, Jessica; Cramer-Bordé, Elisabeth M; Hayward, Catherine P M

2009-02-12

Quebec platelet disorder (QPD) is an inherited bleeding disorder associated with increased urokinase plasminogen activator (uPA) in platelets but not in plasma, intraplatelet plasmin generation, and alpha-granule protein degradation. These abnormalities led us to investigate uPA expression by QPD CD34(+) progenitors, cultured megakaryocytes, and platelets, and whether uPA was stored in QPD alpha-granules. Although QPD CD34(+) progenitors expressed normal amounts of uPA, their differentiation into megakaryocytes abnormally increased expression of the uPA gene but not the flanking genes for vinculin or calcium/calmodulin-dependent protein kinase IIgamma on chromosome 10. The increased uPA production by cultured QPD megakaryocytes mirrored their production of alpha-granule proteins, which was normal. uPA was localized to QPD alpha-granules and it showed extensive colocalization with alpha-granule proteins in both cultured QPD megakaryocytes and platelets, and with plasminogen in QPD platelets. In QPD megakaryocytes, cultured without or with plasma as a source of plasminogen, alpha-granule proteins were stored undegraded and this was associated with much less uPA-plasminogen colocalization than in QPD platelets. Our studies indicate that the overexpression of uPA in QPD emerges with megakaryocyte differentiation, without altering the expression of flanking genes, and that uPA is costored with alpha-granule proteins prior to their proteolysis in QPD.