CSF-1R regulates non-small cell lung cancer cells dissemination through Wnt3a signaling.

PubMed

Yu, Yan Xia; Wu, Hai Jian; Tan, Bing Xu; Qiu, Chen; Liu, Hui Zhong

2017-01-01

Therapeutic antibodies targeting colony stimulating factor 1 receptor (CSF-1R) to block colony stimulating factor-1/colony stimulating factor 1 receptor (CSF-1/CSF-R) signaling axis have exhibit remarkable efficacy in the treatment of malignant tumor. Yet, little is known about the effects of intrinsic CSF-1R in human non-small-cell carcinoma (NSCLC). Here we demonstrated that NSCLC cell-intrinsic CSF-1R promoted cells growth and metastasis both in vitro and in vivo. CSF-1R knocked-down by transfecting with shRNA target CSF-1R suppressed NSCLC cells proliferation and tumor growth in nude mice. Conversely, ectopic expression of CSF-1R promoted cells proliferation and accelerated tumor growth. Mechanistically, the NSCLC CSF-1R modulated downstream effectors of phosphatidylinositol 3-kinase (PI3K) signaling. In addition, CSF-1R overexpression significantly enhanced NSCLC cells mobility, invasion and epithelial-mesenchymal transition (EMT) process, whereas silencing CSF-1R inhibits these phenotypes. Microarray analysis suggested that Wnt family member 3a (Wnt3a) function as a downstream factor of CSF-1R. On account of this, we future identified CSF-1R/Wnt3a a signaling pathway sustained NSCLC cells metastasis. Finally, in patients, CSF-1R and Wnt3a expression positively correlated with the of NSCLC patients. Our results identify NSCLC cell intrinsic functions of CSF-1R/Wnt3a axis in dissemination of NSCLC.

CSF-1R regulates non-small cell lung cancer cells dissemination through Wnt3a signaling

PubMed Central

Yu, Yan Xia; Wu, Hai Jian; Tan, Bing Xu; Qiu, Chen; Liu, Hui Zhong

2017-01-01

Therapeutic antibodies targeting colony stimulating factor 1 receptor (CSF-1R) to block colony stimulating factor-1/colony stimulating factor 1 receptor (CSF-1/CSF-R) signaling axis have exhibit remarkable efficacy in the treatment of malignant tumor. Yet, little is known about the effects of intrinsic CSF-1R in human non-small-cell carcinoma (NSCLC). Here we demonstrated that NSCLC cell-intrinsic CSF-1R promoted cells growth and metastasis both in vitro and in vivo. CSF-1R knocked-down by transfecting with shRNA target CSF-1R suppressed NSCLC cells proliferation and tumor growth in nude mice. Conversely, ectopic expression of CSF-1R promoted cells proliferation and accelerated tumor growth. Mechanistically, the NSCLC CSF-1R modulated downstream effectors of phosphatidylinositol 3-kinase (PI3K) signaling. In addition, CSF-1R overexpression significantly enhanced NSCLC cells mobility, invasion and epithelial-mesenchymal transition (EMT) process, whereas silencing CSF-1R inhibits these phenotypes. Microarray analysis suggested that Wnt family member 3a (Wnt3a) function as a downstream factor of CSF-1R. On account of this, we future identified CSF-1R/Wnt3a a signaling pathway sustained NSCLC cells metastasis. Finally, in patients, CSF-1R and Wnt3a expression positively correlated with the of NSCLC patients. Our results identify NSCLC cell intrinsic functions of CSF-1R/Wnt3a axis in dissemination of NSCLC. PMID:29218239

Exposure to febrile-range hyperthermia potentiates Wnt signalling and epithelial-mesenchymal transition gene expression in lung epithelium.

PubMed

Potla, Ratnakar; Tulapurkar, Mohan E; Luzina, Irina G; Atamas, Sergei P; Singh, Ishwar S; Hasday, Jeffrey D

2018-02-01

As environmental and body temperatures vary, lung epithelial cells experience temperatures significantly different from normal core temperature. Our previous studies in human lung epithelium showed that: (i) heat shock accelerates wound healing and activates profibrotic gene expression through heat shock factor-1 (HSF1); (ii) HSF1 is activated at febrile temperatures (38-41 °C) and (iii) hypothermia (32 °C) activates and hyperthermia (39.5 °C) reduces expression of a subset of miRNAs that target protein kinase-Cα (PKCα) and enhance proliferation. We analysed the effect of hypo- and hyperthermia exposure on Wnt signalling by exposing human small airway epithelial cells (SAECs) and HEK293T cells to 32, 37 or 39.5 °C for 24 h, then analysing Wnt-3a-induced epithelial-mesenchymal transition (EMT) gene expression by qRT-PCR and TOPFlash reporter plasmid activity. Effects of miRNA mimics and inhibitors and the HSF1 inhibitor, KNK437, were evaluated. Exposure to 39.5 °C for 24 h increased subsequent Wnt-3a-induced EMT gene expression in SAECs and Wnt-3a-induced TOPFlash activity in HEK293T cells. Increased Wnt responsiveness was associated with HSF1 activation and blocked by KNK437. Overexpressing temperature-responsive miRNA mimics reduced Wnt responsiveness in 39.5 °C-exposed HEK293T cells, but inhibitors of the same miRNAs failed to restore Wnt responsiveness in 32 °C-exposed HEK293T cells. Wnt responsiveness, including expression of genes associated with EMT, increases after exposure to febrile-range temperature through an HSF1-dependent mechanism that is independent of previously identified temperature-dependent miRNAs. This process may be relevant to febrile fibrosing lung diseases, including the fibroproliferative phase of acute respiratory distress syndrome (ARDS) and exacerbations of idiopathic pulmonary fibrosis (IPF).

Antiangiogenic and Antineuroinflammatory Effects of Kallistatin Through Interactions With the Canonical Wnt Pathway

PubMed Central

Liu, Xiaochen; Zhang, Bin; McBride, Jeffrey D.; Zhou, Kevin; Lee, Kyungwon; Zhou, Yueping; Liu, Zuguo; Ma, Jian-xing

2013-01-01

Kallistatin is a member of the serine proteinase inhibitor superfamily. Kallistatin levels have been shown to be decreased in the vitreous while increased in the circulation of patients with diabetic retinopathy (DR). Overactivation of the Wnt pathway is known to play pathogenic roles in DR. To investigate the role of kallistatin in DR and in Wnt pathway activation, we generated kallistatin transgenic (kallistatin-TG) mice overexpressing kallistatin in multiple tissues including the retina. In the oxygen-induced retinopathy (OIR) model, kallistatin overexpression attenuated ischemia-induced retinal neovascularization. In diabetic kallistatin-TG mice, kallistatin overexpression ameliorated retinal vascular leakage, leukostasis, and overexpression of vascular endothelial growth factor and intracellular adhesion molecule. Furthermore, kallistatin overexpression also suppressed Wnt pathway activation in the retinas of the OIR and diabetic models. In diabetic Wnt reporter (BAT-gal) mice, kallistatin overexpression suppressed retinal Wnt reporter activity. In cultured retinal cells, kallistatin blocked Wnt pathway activation induced by high glucose and by Wnt ligand. Coprecipitation and ligand-binding assays both showed that kallistatin binds to a Wnt coreceptor LRP6 with high affinity (Kd = 4.5 nmol/L). These observations suggest that kallistatin is an endogenous antagonist of LRP6 and inhibitor of Wnt signaling. The blockade of Wnt signaling may represent a mechanism for its antiangiogenic and antineuroinflammatory effects. PMID:23884893

The Wnt5A/Protein Kinase C Pathway Mediates Motility in Melanoma Cells via the Inhibition of Metastasis Suppressors and Initiation of an Epithelial to Mesenchymal Transition*S

PubMed Central

Dissanayake, Samudra K.; Wade, Michael; Johnson, Carrie E.; O’Connell, Michael P.; Leotlela, Poloko D.; French, Amanda D.; Shah, Kavita V.; Hewitt, Kyle J.; Rosenthal, Devin T.; Indig, Fred E.; Jiang, Yuan; Nickoloff, Brian J.; Taub, Dennis D.; Trent, Jeffrey M.; Moon, Randall T.; Bittner, Michael; Weeraratna, Ashani T.

2008-01-01

We have shown that Wnt5A increases the motility of melanoma cells. To explore cellular pathways involving Wnt5A, we compared gain-of-function (WNT5A stable transfectants) versus loss-of-function (siRNA knockdown) of WNT5A by microarray analysis. Increasing WNT5A suppressed the expression of several genes, which were re-expressed after small interference RNA-mediated knockdown of WNT5A. Genes affected by WNT5A include KISS-1, a metastasis suppressor, and CD44, involved in tumor cell homing during metastasis. This could be validated at the protein level using both small interference RNA and recombinant Wnt5A (rWnt5A). Among the genes up-regulated by WNT5A was the gene vimentin, associated with an epithelial to mesenchymal transition (EMT), which involves decreases in E-cadherin, due to up-regulation of the transcriptional repressor, Snail. rWnt5A treatment increases Snail and vimentin expression, and decreases E-cadherin, even in the presence of dominant-negativeTCF4, suggesting that this activation is independent of Wnt/β-catenin signaling. Because Wnt5A can signal via protein kinase C (PKC), the role of PKC in Wnt5A-mediated motility and EMT was also assessed using PKC inhibition and activation studies. Treating cells expressing low levels of Wnt5A with phorbol ester increased Snail expression inhibiting PKC in cells expressing high levels of Wnt5A decreased Snail. Furthermore, inhibition of PKC before Wnt5A treatment blocked Snail expression, implying that Wnt5A can potentiate melanoma metastasis via the induction of EMT in a PKC-dependent manner. PMID:17426020

Wnt4 coordinates directional cell migration and extension of the Müllerian duct essential for ontogenesis of the female reproductive tract

PubMed Central

Prunskaite-Hyyryläinen, Renata; Skovorodkin, Ilya; Xu, Qi; Miinalainen, Ilkka; Shan, Jingdong; Vainio, Seppo J.

2016-01-01

The Müllerian duct (MD) is the anlage of the oviduct, uterus and upper part of the vagina, the main parts of the female reproductive tract. Several wingless-type mouse mammary tumor virus (MMTV) integration site family member (Wnt) genes, including Wnt4, Wnt5a and Wnt7a, are involved in the development of MD and its derivatives, with Wnt4 particularly critical, since the MD fails to develop in its absence. We use, here, Wnt4EGFPCre-based fate mapping to demonstrate that the MD tip cells and the subsequent MD cells are derived from Wnt4+ lineage cells. Moreover, Wnt4 is required for the initiation of MD-forming cell migration. Application of anti-Wnt4 function-blocking antibodies after the initiation of MD elongation indicated that Wnt4 is necessary for the elongation as well, and consistent with this, cell culture wound-healing assays with NIH3T3 cells overexpressing Wnt4 promoted cell migration by comparison with controls. In contrast to the Wnt4 null embryos, some Wnt4monomeric cherry/monomeric cherry (Wnt4mCh/mCh) hypomorphic mice survived to adulthood and formed MD in ∼45% of cases. Nevertheless, the MD of the Wnt4mCh/mCh females had altered cell polarization and basement membrane deposition relative to the controls. Examination of the reproductive tract of the Wnt4mCh/mCh females indicated a poorly coiled oviduct, absence of the endometrial glands and an undifferentiated myometrium, and these mice were prone to develop a hydro-uterus. In conclusion, the results suggest that the Wnt4 gene encodes signals that are important for various aspects of female reproductive tract development. PMID:26721931

Ganoderma lucidum (Reishi) suppresses proliferation and migration of breast cancer cells via inhibiting Wnt/β-catenin signaling.

PubMed

Zhang, Yu

2017-07-08

The medical mushroom Ganoderma lucidum (Reishi), a traditional Chinese medicine, has exhibited a promising anti-cancer effect. However, the molecular mechanism of its action on cancer cells remains unclear. Aberrant activation of Wnt/β-catenin signaling pathway is the cause of many types of cancer, including breast cancer. Here we investigated the effect of Reishi on Wnt/β-catenin signaling pathway and elucidated the molecular mechanism of its function in inhibiting breast cancer cells. We found that Reishi blocked Wnt/β-catenin signaling through inhibiting the phosphorylation of Wnt co-receptor LRP6. In human (MDA-MB-231) and mouse (4T1) breast cancer cell lines, Reishi significantly decreased the phosphorylation of LRP6 and suppressed Wnt3a-activated Wnt target gene Axin2 expression. Administration of Reishi inhibited Wnt-induced hyper-proliferation of breast cancer cells and MDA-MB-231 cell migration. Our results provide evidence that Reishi suppresses breast cancer cell growth and migration through inhibiting Wnt/β-catenin signaling, indicating that Reishi may be a potential natural inhibitor for breast cancer. Copyright © 2017 Elsevier Inc. All rights reserved.

JNK signalling is necessary for a Wnt- and stem cell-dependent regeneration programme

PubMed Central

Tejada-Romero, Belen; Carter, Jean-Michel; Mihaylova, Yuliana; Neumann, Bjoern; Aboobaker, A. Aziz

2015-01-01

Regeneration involves the integration of new and old tissues in the context of an adult life history. It is clear that the core conserved signalling pathways that orchestrate development also play central roles in regeneration, and further study of conserved signalling pathways is required. Here we have studied the role of the conserved JNK signalling cascade during planarian regeneration. Abrogation of JNK signalling by RNAi or pharmacological inhibition blocks posterior regeneration and animals fail to express posterior markers. While the early injury-induced expression of polarity markers is unaffected, the later stem cell-dependent phase of posterior Wnt expression is not established. This defect can be rescued by overactivation of the Hh or Wnt signalling pathway to promote posterior Wnt activity. Together, our data suggest that JNK signalling is required to establish stem cell-dependent Wnt expression after posterior injury. Given that Jun is known to be required in vertebrates for the expression of Wnt and Wnt target genes, we propose that this interaction may be conserved and is an instructive part of planarian posterior regeneration. PMID:26062938

Tmem88a mediates GATA-dependent specification of cardiomyocyte progenitors by restricting WNT signaling

PubMed Central

Novikov, Natasha; Evans, Todd

2013-01-01

Biphasic control of WNT signaling is essential during cardiogenesis, but how the pathway switches from promoting cardiac mesoderm to restricting cardiomyocyte progenitor fate is unknown. We identified genes expressed in lateral mesoderm that are dysregulated in zebrafish when both gata5 and gata6 are depleted, causing a block to cardiomyocyte specification. This screen identified tmem88a, which is expressed in the early cardiac progenitor field and was previously implicated in WNT modulation by overexpression studies. Depletion of tmem88a results in a profound cardiomyopathy, secondary to impaired cardiomyocyte specification. In tmem88a morphants, activation of the WNT pathway exacerbates the cardiomyocyte deficiency, whereas WNT inhibition rescues progenitor cells and cardiogenesis. We conclude that specification of cardiac fate downstream of gata5/6 involves activation of the tmem88a gene to constrain WNT signaling and expand the number of cardiac progenitors. Tmem88a is a novel component of the regulatory mechanism controlling the second phase of biphasic WNT activity essential for embryonic cardiogenesis. PMID:23903195

The conserved transmembrane RING finger protein PLR-1 downregulates Wnt signaling by reducing Frizzled, Ror and Ryk cell-surface levels in C. elegans

PubMed Central

Moffat, Laura L.; Robinson, Ryan E.; Bakoulis, Anastasia; Clark, Scott G.

2014-01-01

Wnts control a wide range of essential developmental processes, including cell fate specification, axon guidance and anteroposterior neuronal polarization. We identified a conserved transmembrane RING finger protein, PLR-1, that governs the response to Wnts by lowering cell-surface levels of the Frizzled family of Wnt receptors in Caenorhabditis elegans. Loss of PLR-1 activity in the neuron AVG causes its anteroposterior polarity to be symmetric or reversed because signaling by the Wnts CWN-1 and CWN-2 are inappropriately activated, whereas ectopic PLR-1 expression blocks Wnt signaling and target gene expression. Frizzleds are enriched at the cell surface; however, when PLR-1 and Frizzled are co-expressed, Frizzled is not detected at the surface but instead is colocalized with PLR-1 in endosomes. The Frizzled cysteine-rich domain (CRD) and invariant second intracellular loop lysine are crucial for PLR-1 downregulation. The PLR-1 RING finger and protease-associated (PA) domain are essential for activity. In a Frizzled-dependent manner, PLR-1 reduces surface levels of the Wnt receptors CAM-1/Ror and LIN-18/Ryk. PLR-1 is a homolog of the mammalian transmembrane E3 ubiquitin ligases RNF43 and ZNRF3, which control Frizzled surface levels in an R-spondin-sensitive manner. We propose that PLR-1 downregulates Wnt receptor surface levels via lysine ubiquitylation of Frizzled to coordinate spatial and temporal responses to Wnts during neuronal development. PMID:24401370

Crosstalk between Wnt Signaling and RNA Processing in Colorectal Cancer.

PubMed

Bordonaro, Michael

2013-01-01

RNA processing involves a variety of processes affecting gene expression, including the removal of introns through RNA splicing, as well as 3' end processing (cleavage and polyadenylation). Alternative RNA processing is fundamentally important for gene regulation, and aberrant processing is associated with the initiation and progression of cancer. Deregulated Wnt signaling, which is the initiating event in the development of most cases of human colorectal cancer (CRC), has been linked to modified RNA processing, which may contribute to Wnt-mediated colonic carcinogenesis. Crosstalk between Wnt signaling and alternative RNA splicing with relevance to CRC includes effects on the expression of Rac1b, an alternatively spliced gene associated with tumorigenesis, which exhibits alternative RNA splicing that is influenced by Wnt activity. In addition, Tcf4, a crucial component of Wnt signaling, also exhibits alternative splicing, which is likely involved in colonic tumorigenesis. Modulation of 3' end formation, including of the Wnt target gene COX-2, also can influence the neoplastic process, with implications for CRC. While many human genes are dependent on introns and splicing for normal levels of gene expression, naturally intronless genes exist with a unique metabolism that allows for intron-independent gene expression. Effects of Wnt activity on the RNA metabolism of the intronless Wnt-target gene c-jun is a likely contributor to cancer development. Further, butyrate, a breakdown product of dietary fiber and a histone deacetylase inhibitor, upregulates Wnt activity in CRC cells, and also modulates RNA processing; therefore, the interplay between Wnt activity, the modulation of this activity by butyrate, and differential RNA metabolism in colonic cells can significantly influence tumorigenesis. Determining the role played by altered RNA processing in Wnt-mediated neoplasia may lead to novel interventions aimed at restoring normal RNA metabolism for therapeutic benefit. Therefore, this minireview presents a brief overview of several aspects of RNA processing of relevance to cancer, which potentially influence, or are influenced by, Wnt signaling activity.

Cellular demise and inflammatory microglial activation during beta-amyloid toxicity are governed by Wnt1 and canonical signaling pathways.

PubMed

Chong, Zhao Zhong; Li, Faqi; Maiese, Kenneth

2007-06-01

Initially described as a modulator of embryogenesis for a number of organ systems, Wnt1 has recently been linked to the development of several neurodegenerative disorders, none being of greater significance than Alzheimer's disease. We therefore examined the ability of Wnt1 to oversee vital pathways responsible for cell survival during beta-amyloid (Abeta1-42) exposure. Here we show that Wnt1 is critical for protection in the SH-SY5Y neuronal cell line against genomic DNA degradation, membrane phosphatidylserine (PS) exposure, and microglial activation, since these neuroprotective attributes of Wnt1 are lost during gene silencing of Wnt1 protein expression. Intimately tied to Wnt1 protection is the presence and activation of Akt1. Pharmacological inhibition of the PI 3-K pathway or gene silencing of Akt1 expression can abrogate the protective capacity of Wnt1. Closely aligned with Wnt1 and Akt1 are the integrated canonical pathways of synthase kinase-3beta (GSK-3beta) and beta-catenin. Through Akt1 dependent pathways, Wnt1 phosphorylates GSK-3beta and maintains beta-catenin integrity to insure its translocation from the cytoplasm to the nucleus to block apoptosis. Our work outlines a highly novel role for Wnt1 and its integration with Akt1, GSK-3beta, and beta-catenin to foster neuronal cell survival and repress inflammatory microglial activation that can identify new avenues of therapy against neurodegenerative disorders.

Inhibition of canonical WNT signaling attenuates human leiomyoma cell growth

PubMed Central

Ono, Masanori; Yin, Ping; Navarro, Antonia; Moravek, Molly B.; Coon, John S.; Druschitz, Stacy A.; Gottardi, Cara J.; Bulun, Serdar E.

2014-01-01

Objective Dysregulation of WNT signaling plays a central role in tumor cell growth and progression. Our goal was to assess the effect of three WNT/β-catenin pathway inhibitors, Inhibitor of β-Catenin And TCF4 (ICAT), niclosamide, and XAV939 on the proliferation of primary cultures of human uterine leiomyoma cells. Design Prospective study of human leiomyoma cells obtained from myomectomy or hysterectomy. Setting University research laboratory. Patient(s) Women (n=38) aged 27–53 years undergoing surgery. Intervention(s) Adenoviral ICAT overexpression or treatment with varying concentrations of niclosamide or XAV939. Main Outcome Measure(s) Cell proliferation, cell death, WNT/β-catenin target gene expression or reporter gene regulation, β-catenin levels and cellular localization. Result(s) ICAT, niclosamide, or XAV939 inhibit WNT/β-catenin pathway activation and exert anti-proliferative effects in primary cultures of human leiomyoma cells. Conclusion(s) Three WNT/β-catenin pathway inhibitors specifically block human leiomyoma growth and proliferation, suggesting that the canonical WNT pathway may be a potential therapeutic target for the treatment of uterine leiomyoma. Our findings provide rationale for further preclinical and clinical evaluation of ICAT, niclosamide, and XAV939 as candidate anti-tumor agents for uterine leiomyoma. PMID:24534281

Wnt-mediated reciprocal regulation between cartilage and bone development during endochondral ossification.

PubMed

Lu, Cheng; Wan, Yong; Cao, Jingjing; Zhu, Xuming; Yu, Jian; Zhou, Rujiang; Yao, Yiyun; Zhang, Lingling; Zhao, Haixia; Li, Hanjun; Zhao, Jianzhi; He, Lin; Ma, Gang; Yang, Xiao; Yao, Zhengju; Guo, Xizhi

2013-04-01

The role of Wnt signaling is extensively studied in skeletal development and postnatal bone remodeling, mostly based on the genetic approaches of β-catenin manipulation. However, given their independent function, a requirement for β-catenin is not the same as that for Wnt. Here, we investigated the effect of Wnt proteins in both tissues through generating cartilage- or bone-specific Wls null mice, respectively. Depletion of Wls by Col2-Cre, which would block Wnt secretion in the chondrocytes and perichondrium, delayed chondrocyte hypertrophy in the growth plate and impaired perichondrial osteogenesis. Loss of Wls in chondrocytes also disturbed the proliferating chondrocyte morphology and division orientation, which was similar to the defect observed in Wnt5a null mice. On the other hand, inactivation of Wls in osteoblasts by Col1-Cre resulted in a shorter hypertrophic zone and an increase of TRAP positive cell number in the chondro-osseous junction of growth plate, coupled with a decrease in bone mass. Taken together, our studies reveal that Wnt proteins not only modulate differentiation and cellular communication within populations of chondrocytes, but also mediate the cross regulation between the chondrocytes and osteoblasts in growth plate. Copyright © 2012 Elsevier Inc. All rights reserved.

Conditional Expression of Wnt4 during Chondrogenesis Leads to Dwarfism in Mice

PubMed Central

Lee, Hu-Hui; Behringer, Richard R.

2007-01-01

Wnts are expressed in the forming long bones, suggesting roles in skeletogenesis. To examine the action of Wnts in skeleton formation, we developed a genetic system to conditionally express Wnt4 in chondrogenic tissues of the mouse. A mouse Wnt4 cDNA was introduced into the ubiquitously expressed Rosa26 (R26) locus by gene targeting in embryonic stem (ES) cells. The expression of Wnt4 from the R26 locus was blocked by a neomycin selection cassette flanked by loxP sites (floxneo) that was positioned between the Rosa26 promoter and the Wnt4 cDNA, creating the allele designated R26floxneoWnt4. Wnt4 expression was activated during chondrogenesis using Col2a1-Cre transgenic mice that express Cre recombinase in differentiating chondrocytes. R26floxneoWnt4; Col2a1-Cre double heterozygous mice exhibited a growth deficiency, beginning approximately 7 to 10 days after birth, that resulted in dwarfism. In addition, they also had craniofacial abnormalities, and delayed ossification of the lumbar vertebrae and pelvic bones. Histological analysis revealed a disruption in the organization of the growth plates and a delay in the onset of the primary and secondary ossification centers. Molecular studies showed that Wnt4 overexpression caused decreased proliferation and altered maturation of chondrocytes. In addition, R26floxneoWnt4; Col2a1-Cre mice had decreased expression of vascular endothelial growth factor (VEGF). These studies demonstrate that Wnt4 overexpression leads to dwarfism in mice. The data indicate that Wnt4 levels must be regulated in chondrocytes for normal growth plate development and skeletogenesis. Decreased VEGF expression suggests that defects in vascularization may contribute to the dwarf phenotype. PMID:17505543

Wnt signaling during tooth replacement in zebrafish (Danio rerio): pitfalls and perspectives

PubMed Central

Huysseune, Ann; Soenens, Mieke; Elderweirdt, Fien

2014-01-01

The canonical (β-catenin dependent) Wnt signaling pathway has emerged as a likely candidate for regulating tooth replacement in continuously renewing dentitions. So far, the involvement of canonical Wnt signaling has been experimentally demonstrated predominantly in amniotes. These studies tend to show stimulation of tooth formation by activation of the Wnt pathway, and inhibition of tooth formation when blocking the pathway. Here, we report a strong and dynamic expression of the soluble Wnt inhibitor dickkopf1 (dkk1) in developing zebrafish (Danio rerio) tooth germs, suggesting an active repression of Wnt signaling during morphogenesis and cytodifferentiation of a tooth, and derepression of Wnt signaling during start of replacement tooth formation. To further analyse the role of Wnt signaling, we used different gain-of-function approaches. These yielded disjunct results, yet none of them indicating enhanced tooth replacement. Thus, masterblind (mbl) mutants, defective in axin1, mimic overexpression of Wnt, but display a normally patterned dentition in which teeth are replaced at the appropriate times and positions. Activating the pathway with LiCl had variable outcomes, either resulting in the absence, or the delayed formation, of first-generation teeth, or yielding a regular dentition with normal replacement, but no supernumerary teeth or accelerated tooth replacement. The failure so far to influence tooth replacement in the zebrafish by perturbing Wnt signaling is discussed in the light of (i) potential technical pitfalls related to dose- or time-dependency, (ii) the complexity of the canonical Wnt pathway, and (iii) species-specific differences in the nature and activity of pathway components. Finally, we emphasize the importance of in-depth knowledge of the wild-type pattern for reliable interpretations. It is hoped that our analysis can be inspiring to critically assess and elucidate the role of Wnt signaling in tooth development in polyphyodonts. PMID:25339911

Cirmtuzumab inhibits Wnt5a-induced Rac1 activation in chronic lymphocytic leukemia treated with ibrutinib

PubMed Central

Yu, J; Chen, L; Cui, B; Wu, Christina; Choi, M Y; Chen, Y; Zhang, L; Rassenti, L Z; Widhopf II, G F; Kipps, T J

2017-01-01

Signaling via the B cell receptor (BCR) plays an important role in the pathogenesis and progression of chronic lymphocytic leukemia (CLL). This is underscored by the clinical effectiveness of ibrutinib, an inhibitor of Bruton's tyrosine kinase (BTK) that can block BCR-signaling. However, ibrutinib cannot induce complete responses (CR) or durable remissions without continued therapy, suggesting alternative pathways also contribute to CLL growth/survival that are independent of BCR-signaling. ROR1 is a receptor for Wnt5a, which can promote activation of Rac1 to enhance CLL-cell proliferation and survival. In this study, we found that CLL cells of patients treated with ibrutinib had activated Rac1. Moreover, Wnt5a could induce Rac1 activation and enhance proliferation of CLL cells treated with ibrutinib at concentrations that were effective in completely inhibiting BTK and BCR-signaling. Wnt5a-induced Rac1 activation could be blocked by cirmtuzumab (UC-961), an anti-ROR1 mAb. We found that treatment with cirmtuzumab and ibrutinib was significantly more effective than treatment with either agent alone in clearing leukemia cells in vivo. This study indicates that cirmtuzumab may enhance the activity of ibrutinib in the treatment of patients with CLL or other ROR1+ B-cell malignancies. PMID:27904138

Cirmtuzumab inhibits Wnt5a-induced Rac1 activation in chronic lymphocytic leukemia treated with ibrutinib.

PubMed

Yu, J; Chen, L; Cui, B; Wu, Christina; Choi, M Y; Chen, Y; Zhang, L; Rassenti, L Z; Widhopf Ii, G F; Kipps, T J

2017-06-01

Signaling via the B cell receptor (BCR) plays an important role in the pathogenesis and progression of chronic lymphocytic leukemia (CLL). This is underscored by the clinical effectiveness of ibrutinib, an inhibitor of Bruton's tyrosine kinase (BTK) that can block BCR-signaling. However, ibrutinib cannot induce complete responses (CR) or durable remissions without continued therapy, suggesting alternative pathways also contribute to CLL growth/survival that are independent of BCR-signaling. ROR1 is a receptor for Wnt5a, which can promote activation of Rac1 to enhance CLL-cell proliferation and survival. In this study, we found that CLL cells of patients treated with ibrutinib had activated Rac1. Moreover, Wnt5a could induce Rac1 activation and enhance proliferation of CLL cells treated with ibrutinib at concentrations that were effective in completely inhibiting BTK and BCR-signaling. Wnt5a-induced Rac1 activation could be blocked by cirmtuzumab (UC-961), an anti-ROR1 mAb. We found that treatment with cirmtuzumab and ibrutinib was significantly more effective than treatment with either agent alone in clearing leukemia cells in vivo. This study indicates that cirmtuzumab may enhance the activity of ibrutinib in the treatment of patients with CLL or other ROR1 + B-cell malignancies.

Opposing activities of Notch and Wnt signaling regulate intestinal stem cells and gut homeostasis

PubMed Central

Tian, Hua; Biehs, Brian; Chiu, Cecilia; Siebel, Chris; Wu, Yan; Costa, Mike; de Sauvage, Frederic J.; Klein, Ophir D.

2015-01-01

Summary Proper organ homeostasis requires tight control of adult stem cells and differentiation through integration of multiple inputs. In the mouse small intestine, Notch and Wnt signaling are required both for stem cell maintenance and for a proper balance of differentiation between secretory and absorptive cell lineages. In the absence of Notch signaling, stem cells preferentially generate secretory cells at the expense of absorptive cells. Here, we use function-blocking antibodies against Notch receptors to demonstrate that Notch blockade perturbs intestinal stem cell function by causing a de-repression of the Wnt signaling pathway, leading to mis-expression of prosecretory genes. Importantly, attenuation of the Wnt pathway rescued the phenotype associated with Notch blockade. These studies bring to light a negative regulatory mechanism that maintains stem cell activity and balanced differentiation, and we propose that the interaction between Wnt and Notch signaling described here represents a common theme in adult stem cell biology. PMID:25818302

Wnt and the Wnt signaling pathway in bone development and disease

PubMed Central

Wang, Yiping; Li, Yi-Ping; Paulson, Christie; Shao, Jian-Zhong; Zhang, Xiaoling; Wu, Mengrui; Chen, Wei

2014-01-01

Wnt signaling affects both bone modeling, which occurs during development, and bone remodeling, which is a lifelong process involving tissue renewal. Wnt signals are especially known to affect the differentiation of osteoblasts. In this review, we summarize recent advances in understanding the mechanisms of Wnt signaling, which is divided into two major branches: the canonical pathway and the noncanonical pathway. The canonical pathway is also called the Wnt/β-catenin pathway. There are two major noncanonical pathways: the Wnt-planar cell polarity pathway (Wnt-PCP pathway) and the Wnt-calcium pathway (Wnt-Ca2+ pathway). This review also discusses how Wnt ligands, receptors, intracellular effectors, transcription factors, and antagonists affect both the bone modeling and bone remodeling processes. We also review the role of Wnt ligands, receptors, intracellular effectors, transcription factors, and antagonists in bone as demonstrated in mouse models. Disrupted Wnt signaling is linked to several bone diseases, including osteoporosis, van Buchem disease, and sclerosteosis. Studying the mechanism of Wnt signaling and its interactions with other signaling pathways in bone will provide potential therapeutic targets to treat these bone diseases. PMID:24389191

Illegitimate WNT signaling promotes proliferation of multiple myeloma cells

PubMed Central

Derksen, Patrick W. B.; Tjin, Esther; Meijer, Helen P.; Klok, Melanie D.; Mac Gillavry, Harold D.; van Oers, Marinus H. J.; Lokhorst, Henk M.; Bloem, Andries C.; Clevers, Hans; Nusse, Roel; van der Neut, Ronald; Spaargaren, Marcel; Pals, Steven T.

2004-01-01

The unrestrained growth of tumor cells is generally attributed to mutations in essential growth control genes, but tumor cells are also influenced by signals from the environment. In multiple myeloma (MM), the factors and signals coming from the bone marrow microenvironment are possibly even essential for the growth of the tumor cells. As targets for intervention, these signals may be equally important as mutated oncogenes. Given their oncogenic potential, WNT signals form a class of paracrine growth factors that could act to influence MM cell growth. In this paper, we report that MM cells have hallmarks of active WNT signaling, whereas the cells have not undergone detectable mutations in WNT signaling genes such as adenomatous polyposis coli and β-catenin (CTNNB1). We show that the malignant MM plasma cells overexpress β-catenin, including its N-terminally unphosphorylated form, suggesting active β-catenin/T cell factor-mediated transcription. Further accumulation and nuclear localization of β-catenin, and/or increased cell proliferation, was achieved by stimulation of WNT signaling with either Wnt3a, LiCl, or the constitutively active S33Y mutant of β-catenin. In contrast, by blocking WNT signaling by dominant-negative T cell factor, we can interfere with the growth of MM cells. We therefore suggest that MM cells are dependent on an active WNT signal, which may have important implications for the management of this incurable form of cancer. PMID:15067127

Wnt/β-catenin activation and macrophage induction during liver cancer development following steatosis.

PubMed

Debebe, A; Medina, V; Chen, C-Y; Mahajan, I M; Jia, C; Fu, D; He, L; Zeng, N; Stiles, B W; Chen, C-L; Wang, M; Aggarwal, K-R; Peng, Z; Huang, J; Chen, J; Li, M; Dong, T; Atkins, S; Borok, Z; Yuan, W; Machida, K; Ju, C; Kahn, M; Johnson, D; Stiles, B L

2017-10-26

Obesity confers an independent risk for carcinogenesis. In the liver, steatosis often proceeds cancer formation; however, the mechanisms by which steatosis promotes carcinogenesis is unknown. We hypothesize that steatosis alters the microenvironment to promote proliferation of tumor initiating cells (TICs) and carcinogenesis. We used several liver cancer models to address the mechanisms underlying the role of obesity in cancer and verified these findings in patient populations. Using bioinformatics analysis and verified by biochemical assays, we identified that hepatosteatosis resulting from either Pten deletion or transgenic expression of HCV core/NS5A proteins, promotes the activation of Wnt/β-catenin. We verified that high fat diet lipid accumulation is also capable of inducing Wnt/β-catenin. Caloric restriction inhibits hepatosteatosis, reduces Wnt/β-catenin activation and blocks the expansion of TICs leading to complete inhibition of tumorigenesis without affecting the phosphatase and tensin homologue deleted on chromosome 10 (PTEN) loss regulated protein kinase B (AKT) activation. Pharmacological inhibition or loss of the Wnt/β-catenin signal represses TIC growth in vitro, and decreases the accumulation of TICs in vivo. In human liver cancers, ontology analysis of gene set enrichment analysis (GSEA)-defined Wnt signature genes indicates that Wnt signaling is significantly induced in tumor samples compared with healthy livers. Indeed, Wnt signature genes predict 90% of tumors in a cohort of 558 patient samples. Selective depletion of macrophages leads to reduction of Wnt and suppresses tumor development, suggesting infiltrating macrophages as a key source for steatosis-induced Wnt expression. These data established Wnt/β-catenin as a novel signal produced by infiltrating macrophages induced by steatosis that promotes growth of tumor progenitor cells, underlying the increased risk of liver tumor development in obese individuals.

Secretion and extracellular space travel of Wnt proteins.

PubMed

Gross, Julia Christina; Boutros, Michael

2013-08-01

Wnt signaling pathways control many processes during development, stem cell maintenance and homeostasis, and their aberrant regulation has been linked to diseases in man including diabetes, neurodegeneration and cancer. Wnts are hydrophobic proteins, however, quite paradoxically, they can travel over distances to induce cell-type specific responses. While there has been an initial focus on elucidating the intracellular signaling cascade, discoveries in the past few years have shed light on a highly complex, and regulated secretory process that guides Wnt proteins through the exocytic pathway. Wnt proteins are at least in portion packaged onto extracellular carriers such as exosomes. Similar to dysregulation of components in the Wnt receiving cell, failure to regulate Wnt secretion has been linked to cancer. Here, we review recent discoveries on factors and processes implicated in Wnt secretion. Copyright © 2013 Elsevier Ltd. All rights reserved.

Canonical Wnt signaling differently modulates osteogenic differentiation of mesenchymal stem cells derived from bone marrow and from periodontal ligament under inflammatory conditions.

PubMed

Liu, Wenjia; Konermann, Anna; Guo, Tao; Jäger, Andreas; Zhang, Liqiang; Jin, Yan

2014-03-01

Cellular plasticity and complex functional requirements of the periodontal ligament (PDL) assume a local stem cell (SC) niche to maintain tissue homeostasis and repair. Here, pathological alterations caused by inflammatory insults might impact the regenerative capacities of these cells. As bone homeostasis is fundamentally controlled by Wnt-mediated signals, it was the aim of this study to characterize the SC-like capacities of cells derived from PDL and to investigate their involvement in bone pathophysiology especially regarding the canonical Wnt pathway. PDLSCs were investigated for their SC characteristics via analysis of cell surface marker expression, colony forming unit efficiency, proliferation, osteogenic differentiation and adipogenic differentiation, and compared to bone marrow derived mesenchymal SCs (BMMSCs). To determine the impact of both inflammation and the canonical Wnt pathway on osteogenic differentiation, cells were challenged with TNF-α, maintained with or without Wnt3a or DKK-1 under osteogenic induction conditions and investigated for p-IκBα, p-NF-κB, p-Akt, β-catenin, p-GSK-3β, ALP and Runx2. PDLSCs exhibit weaker adipogenic and osteogenic differentiation capacities compared to BMMSCs. TNF-α inhibited osteogenic differentiation of PDLSCs more than BMMSCs mainly through regulating canonical Wnt pathway. Blocking the canonical Wnt pathway by DKK-1 reconstituted osteogenic differentiation of PDLSCs under inflammatory conditions, whereas activation by Wnt3a increased osteogenic differentiation of BMMSCs. Our results suggest a diverse regulation of the inhibitory effect of TNF-α in BMMSCs and PDLSCs via canonical Wnt pathway modulation. These findings provide novel insights on PDLSC SC-like capacities and their involvement in bone pathophysiology under the impact of the canonical Wnt pathway. Copyright © 2013 Elsevier B.V. All rights reserved.

A Genome-Wide RNA Interference Screen Identifies a Role for Wnt/β-Catenin Signaling during Rift Valley Fever Virus Infection.

PubMed

Harmon, Brooke; Bird, Sara W; Schudel, Benjamin R; Hatch, Anson V; Rasley, Amy; Negrete, Oscar A

2016-08-15

Rift Valley fever virus (RVFV) is an arbovirus within the Bunyaviridae family capable of causing serious morbidity and mortality in humans and livestock. To identify host factors involved in bunyavirus replication, we employed genome-wide RNA interference (RNAi) screening and identified 381 genes whose knockdown reduced infection. The Wnt pathway was the most represented pathway when gene hits were functionally clustered. With further investigation, we found that RVFV infection activated Wnt signaling, was enhanced when Wnt signaling was preactivated, was reduced with knockdown of β-catenin, and was blocked using Wnt signaling inhibitors. Similar results were found using distantly related bunyaviruses La Crosse virus and California encephalitis virus, suggesting a conserved role for Wnt signaling in bunyaviral infection. We propose a model where bunyaviruses activate Wnt-responsive genes to regulate optimal cell cycle conditions needed to promote efficient viral replication. The findings in this study should aid in the design of efficacious host-directed antiviral therapeutics. RVFV is a mosquito-borne bunyavirus that is endemic to Africa but has demonstrated a capacity for emergence in new territories (e.g., the Arabian Peninsula). As a zoonotic pathogen that primarily affects livestock, RVFV can also cause lethal hemorrhagic fever and encephalitis in humans. Currently, there are no treatments or fully licensed vaccines for this virus. Using high-throughput RNAi screening, we identified canonical Wnt signaling as an important host pathway regulating RVFV infection. The beneficial role of Wnt signaling was observed for RVFV, along with other disparate bunyaviruses, indicating a conserved bunyaviral replication mechanism involving Wnt signaling. These studies supplement our knowledge of the fundamental mechanisms of bunyavirus infection and provide new avenues for countermeasure development against pathogenic bunyaviruses. Copyright © 2016 Harmon et al.

A genome-wide RNA interference screen identifies a role for Wnt/β-catenin signaling during Rift Valley Fever Virus infection

DOE PAGES

Harmon, Brooke; Bird, Sara W.; Schudel, Benjamin R.; ...

2016-05-25

Rift Valley fever virus (RVFV) is an arbovirus within the Bunyaviridae family capable of causing serious morbidity and mortality in humans and livestock. To identify host factors involved in bunyavirus replication, we employed genome-wide RNA interference (RNAi) screening and identified 381 genes whose knockdown reduced infection. The Wnt pathway was the most represented pathway when gene hits were functionally clustered. With further investigation, we found that RVFV infection activated Wnt signaling, was enhanced when Wnt signaling was preactivated, was reduced with knockdown of β-catenin, and was blocked using Wnt signaling inhibitors. Similar results were found using distantly related bunyaviruses Lamore » Crosse virus and California encephalitis virus, suggesting a conserved role for Wnt signaling in bunyaviral infection. We propose a model where bunyaviruses activate Wnt-responsive genes to regulate optimal cell cycle conditions needed to promote efficient viral replication. The findings in this study should aid in the design of efficacious host-directed antiviral therapeutics. IMPORTANCE RVFV is a mosquito-borne bunyavirus that is endemic to Africa but has demonstrated a capacity for emergence in new territories (e.g., the Arabian Peninsula). As a zoonotic pathogen that primarily affects livestock, RVFV can also cause lethal hemorrhagic fever and encephalitis in humans. Currently, there are no treatments or fully licensed vaccines for this virus. Using high-throughput RNAi screening, we identified canonical Wnt signaling as an important host pathway regulating RVFV infection. The beneficial role of Wnt signaling was observed for RVFV, along with other disparate bunyaviruses, indicating a conserved bunyaviral replication mechanism involving Wnt signaling. Lastly, these studies supplement our knowledge of the fundamental mechanisms of bunyavirus infection and provide new avenues for countermeasure development against pathogenic bunyaviruses.« less

A genome-wide RNA interference screen identifies a role for Wnt/β-catenin signaling during Rift Valley Fever Virus infection

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

Harmon, Brooke; Bird, Sara W.; Schudel, Benjamin R.

Rift Valley fever virus (RVFV) is an arbovirus within the Bunyaviridae family capable of causing serious morbidity and mortality in humans and livestock. To identify host factors involved in bunyavirus replication, we employed genome-wide RNA interference (RNAi) screening and identified 381 genes whose knockdown reduced infection. The Wnt pathway was the most represented pathway when gene hits were functionally clustered. With further investigation, we found that RVFV infection activated Wnt signaling, was enhanced when Wnt signaling was preactivated, was reduced with knockdown of β-catenin, and was blocked using Wnt signaling inhibitors. Similar results were found using distantly related bunyaviruses Lamore » Crosse virus and California encephalitis virus, suggesting a conserved role for Wnt signaling in bunyaviral infection. We propose a model where bunyaviruses activate Wnt-responsive genes to regulate optimal cell cycle conditions needed to promote efficient viral replication. The findings in this study should aid in the design of efficacious host-directed antiviral therapeutics. IMPORTANCE RVFV is a mosquito-borne bunyavirus that is endemic to Africa but has demonstrated a capacity for emergence in new territories (e.g., the Arabian Peninsula). As a zoonotic pathogen that primarily affects livestock, RVFV can also cause lethal hemorrhagic fever and encephalitis in humans. Currently, there are no treatments or fully licensed vaccines for this virus. Using high-throughput RNAi screening, we identified canonical Wnt signaling as an important host pathway regulating RVFV infection. The beneficial role of Wnt signaling was observed for RVFV, along with other disparate bunyaviruses, indicating a conserved bunyaviral replication mechanism involving Wnt signaling. Lastly, these studies supplement our knowledge of the fundamental mechanisms of bunyavirus infection and provide new avenues for countermeasure development against pathogenic bunyaviruses.« less

WNT signaling controls expression of pro-apoptotic BOK and BAX in intestinal cancer

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

Zeilstra, Jurrit; Joosten, Sander P.J.; Wensveen, Felix M.

Research highlights: {yields} Intestinal adenomas initiated by aberrant activation of the WNT pathway displayed an increased sensitivity to apoptosis. {yields} Expression profiling of apoptosis-related genes in Apc{sup Min/+} mice revealed the differential expression of pro-apoptotic Bok and Bax. {yields} APC-mutant adenomatous crypts in FAP patients showed strongly increased BAX immunoreactivity. {yields} Blocking of {beta}-catenin/TCF-4-mediated signaling in colon cancer cells reduced the expression of BOK and BAX. -- Abstract: In a majority of cases, colorectal cancer is initiated by aberrant activation of the WNT signaling pathway. Mutation of the genes encoding the WNT signaling components adenomatous polyposis coli or {beta}-catenin causesmore » constitutively active {beta}-catenin/TCF-mediated transcription, driving the transformation of intestinal crypts to cancer precursor lesions, called dysplastic aberrant crypt foci. Deregulated apoptosis is a hallmark of adenomatous colon tissue. However, the contribution of WNT signaling to this process is not fully understood. We addressed this role by analyzing the rate of epithelial apoptosis in aberrant crypts and adenomas of the Apc{sup Min/+} mouse model. In comparison with normal crypts and adenomas, aberrant crypts displayed a dramatically increased rate of apoptotic cell death. Expression profiling of apoptosis-related genes along the crypt-villus axis and in Apc mutant adenomas revealed increased expression of two pro-apoptotic Bcl-2 family members in intestinal adenomas, Bok and Bax. Analysis of the colon of familial adenomatous polyposis (FAP) patients along the crypt-to-surface axis, and of dysplastic crypts, corroborated this expression pattern. Disruption of {beta}-catenin/TCF-4-mediated signaling in the colorectal cancer cell line Ls174T significantly decreased BOK and BAX expression, confirming WNT-dependent regulation in intestinal epithelial cells. Our results suggest a feedback mechanism by which uncontrolled epithelial cell proliferation in the stem cell compartment can be counterbalanced by an increased propensity to undergo cell death.« less

Norrin mediates neuroprotective effects on retinal ganglion cells via activation of the Wnt/beta-catenin signaling pathway and the induction of neuroprotective growth factors in Muller cells.

PubMed

Seitz, Roswitha; Hackl, Simon; Seibuchner, Thomas; Tamm, Ernst R; Ohlmann, Andreas

2010-04-28

Norrin is a secreted protein that binds to frizzled 4 and controls development of capillaries in retina and inner ear. We provide evidence that Norrin has distinct neuroprotective properties that are independent from its effects on vascular development. The function of Norrin was investigated in a mouse model of excitotoxic retinal ganglion cell (RGC) damage after intravitreal injection of NMDA, and in cultured Müller glia or immortalized RGC-5 cells. Intravitreal injection of Norrin significantly increased the number of surviving RGC axons in the optic nerve and decreased apoptotic death of retinal neurons following NMDA-mediated damage. This effect could be blocked by adding dickkopf (DKK)-1, an inhibitor of the Wnt/beta-catenin signaling pathway. Treatment of eyes with combined Norrin/NMDA activated Wnt/beta-catenin signaling and increased the retinal expression of leukemia inhibitory factor and endothelin-2, as well as that of neurotrophic growth factors such as fibroblast growth factor-2, brain-derived neurotrophic factor, lens epithelium-derived growth factor, and ciliary neurotrophic factor. A similar activation of Wnt/beta-catenin signaling and an increased expression of neurotrophic factors was observed in cultured Müller cells after treatment with Norrin, effects that again could be blocked by adding DKK-1. In addition, conditioned cell culture medium of Norrin-treated Müller cells increased survival of differentiated RGC-5 cells. We conclude that Norrin has pronounced neuroprotective properties on retinal neurons with the distinct potential to decrease the damaging effects of NMDA-induced RGC loss. The effects of Norrin involve activation of Wnt/beta-catenin signaling and subsequent induction of neurotrophic growth factors in Müller cells.

Wnt affects symmetry and morphogenesis during post-embryonic development in colonial chordates.

PubMed

Di Maio, Alessandro; Setar, Leah; Tiozzo, Stefano; De Tomaso, Anthony W

2015-01-01

Wnt signaling is one of the earliest and most highly conserved regulatory pathways for the establishment of the body axes during regeneration and early development. In regeneration, body axes determination occurs independently of tissue rearrangement and early developmental cues. Modulation of the Wnt signaling in either process has shown to result in unusual body axis phenotypes. Botryllus schlosseri is a colonial ascidian that can regenerate its entire body through asexual budding. This processes leads to an adult body via a stereotypical developmental pathway (called blastogenesis), without proceeding through any embryonic developmental stages. In this study, we describe the role of the canonical Wnt pathway during the early stages of asexual development. We characterized expression of three Wnt ligands (Wnt2B, Wnt5A, and Wnt9A) by in situ hybridization and qRT-PCR. Chemical manipulation of the pathway resulted in atypical budding due to the duplication of the A/P axes, supernumerary budding, and loss of the overall cell apical-basal polarity. Our results suggest that Wnt signaling is used for equivalent developmental processes both during embryogenesis and asexual development in an adult organism, suggesting that patterning mechanisms driving morphogenesis are conserved, independent of embryonic, or regenerative development.

Androgens Up-regulate Transcription of the Notch Inhibitor Numb in C2C12 Myoblasts via Wnt/β-Catenin Signaling to T Cell Factor Elements in the Numb Promoter*

PubMed Central

Liu, Xin-Hua; Wu, Yong; Yao, Shen; Levine, Alice C.; Kirschenbaum, Alexander; Collier, Lauren; Bauman, William A.; Cardozo, Christopher P.

2013-01-01

Androgen signaling via the androgen receptor is a key pathway that contributes to development, cell fate decisions, and differentiation, including that of myogenic progenitors. Androgens and synthetic steroids have well established anabolic actions on skeletal muscle. Wnt and Notch signaling pathways are also essential to myogenic cell fate decisions during development and tissue repair. However, the interactions among these pathways are largely unknown. Androgenic regulation of Wnt signaling has been reported. Nandrolone, an anabolic steroid, has been shown to inhibit Notch signaling and up-regulate Numb, a Notch inhibitor. To elucidate the mechanisms of interaction between nandrolone and Wnt/Notch signaling, we investigated the effects of nandrolone on Numb expression and Wnt signaling and determined the roles of Wnt signaling in nandrolone-induced Numb expression in C2C12 myoblasts. Nandrolone increased Numb mRNA and protein levels and T cell factor (Tcf) transcriptional activity via inhibition of glycogen synthase kinase 3β. Up-regulation of Numb expression by nandrolone was blocked by the Wnt inhibitors, sFRP1 and DKK1, whereas Wnt3a increased Numb mRNA and protein expression. In addition, we observed that the proximal promoter of the Numb gene had functional Tcf binding elements to which β-catenin was recruited in a manner enhanced by both nandrolone and Wnt3a. Moreover, site-directed mutagenesis indicated that the Tcf binding sites in the Numb promoter are required for the nandrolone-induced Numb transcriptional activation in this cell line. These results reveal a novel molecular mechanism underlying up-regulation of Numb transcription with a critical role for increased canonical Wnt signaling. In addition, the data identify Numb as a novel target gene of the Wnt signaling pathway by which Wnts would be able to inhibit Notch signaling. PMID:23649620

Ectodermal Wnt signaling regulates abdominal myogenesis during ventral body wall development.

PubMed

Zhang, Lingling; Li, Hanjun; Yu, Jian; Cao, Jingjing; Chen, Huihui; Zhao, Haixia; Zhao, Jianzhi; Yao, Yiyun; Cheng, Huihui; Wang, Lifang; Zhou, Rujiang; Yao, Zhengju; Guo, Xizhi

2014-03-01

Defects of the ventral body wall are prevalent birth anomalies marked by deficiencies in body wall closure, hypoplasia of the abdominal musculature and multiple malformations across a gamut of organs. However, the mechanisms underlying ventral body wall defects remain elusive. Here, we investigated the role of Wnt signaling in ventral body wall development by inactivating Wls or β-catenin in murine abdominal ectoderm. The loss of Wls in the ventral epithelium, which blocks the secretion of Wnt proteins, resulted in dysgenesis of ventral musculature and genito-urinary tract during embryonic development. Molecular analyses revealed that the dermis and myogenic differentiation in the underlying mesenchymal progenitor cells was perturbed by the loss of ectodermal Wls. The activity of the Wnt-Pitx2 axis was impaired in the ventral mesenchyme of the mutant body wall, which partially accounted for the defects in ventral musculature formation. In contrast, epithelial depletion of β-catenin or Wnt5a did not resemble the body wall defects in the ectodermal Wls mutant. These findings indicate that ectodermal Wnt signaling instructs the underlying mesodermal specification and abdominal musculature formation during ventral body wall development, adding evidence to the theory that ectoderm-mesenchyme signaling is a potential unifying mechanism for the origin of ventral body wall defects. Copyright © 2013 Elsevier Inc. All rights reserved.

Sequence analysis and molecular characterization of Wnt4 gene in metacestodes of Taenia solium.

PubMed

Hou, Junling; Luo, Xuenong; Wang, Shuai; Yin, Cai; Zhang, Shaohua; Zhu, Xueliang; Dou, Yongxi; Cai, Xuepeng

2014-04-01

Wnt proteins are a family of secreted glycoproteins that are evolutionarily conserved and considered to be involved in extensive developmental processes in metazoan organisms. The characterization of wnt genes may improve understanding the parasite's development. In the present study, a wnt4 gene encoding 491amino acids was amplified from cDNA of metacestodes of Taenia solium using reverse transcription PCR (RT-PCR). Bioinformatics tools were used for sequence analysis. The conserved domain of the wnt gene family was predicted. The expression profile of Wnt4 was investigated using real-time PCR. Wnt4 expression was found to be dramatically increased in scolex evaginated cysticerci when compared to invaginated cysticerci. In situ hybridization showed that wnt4 gene was distributed in the posterior end of the worm along the primary body axis in evaginated cysticerci. These findings indicated that wnt4 may take part in the process of cysticerci evagination and play a role in scolex/bladder development of cysticerci of T. solium.

Effect of Wnt3a on Keratinocytes Utilizing in Vitro and Bioinformatics Analysis

PubMed Central

Nam, Ju-Suk; Chakraborty, Chiranjib; Sharma, Ashish Ranjan; Her, Young; Bae, Kee-Jeong; Sharma, Garima; Doss, George Priya; Lee, Sang-Soo; Hong, Myung-Sun; Song, Dong-Keun

2014-01-01

Wingless-type (Wnt) signaling proteins participate in various cell developmental processes. A suppressive role of Wnt5a on keratinocyte growth has already been observed. However, the role of other Wnt proteins in proliferation and differentiation of keratinocytes remains unknown. Here, we investigated the effects of the Wnt ligand, Wnt3a, on proliferation and differentiation of keratinocytes. Keratinocytes from normal human skin were cultured and treated with recombinant Wnt3a alone or in combination with the inflammatory cytokine, tumor necrosis factor α (TNFα). Furthermore, using bioinformatics, we analyzed the biochemical parameters, molecular evolution, and protein–protein interaction network for the Wnt family. Application of recombinant Wnt3a showed an anti-proliferative effect on keratinocytes in a dose-dependent manner. After treatment with TNFα, Wnt3a still demonstrated an anti-proliferative effect on human keratinocytes. Exogenous treatment of Wnt3a was unable to alter mRNA expression of differentiation markers of keratinocytes, whereas an altered expression was observed in TNFα-stimulated keratinocytes. In silico phylogenetic, biochemical, and protein–protein interaction analysis showed several close relationships among the family members of the Wnt family. Moreover, a close phylogenetic and biochemical similarity was observed between Wnt3a and Wnt5a. Finally, we proposed a hypothetical mechanism to illustrate how the Wnt3a protein may inhibit the process of proliferation in keratinocytes, which would be useful for future researchers. PMID:24686518

Secreted and Transmembrane Wnt Inhibitors and Activators

PubMed Central

Cruciat, Cristina-Maria; Niehrs, Christof

2013-01-01

Signaling by the Wnt family of secreted glycoproteins plays important roles in embryonic development and adult homeostasis. Wnt signaling is modulated by a number of evolutionarily conserved inhibitors and activators. Wnt inhibitors belong to small protein families, including sFRP, Dkk, WIF, Wise/SOST, Cerberus, IGFBP, Shisa, Waif1, APCDD1, and Tiki1. Their common feature is to antagonize Wnt signaling by preventing ligand–receptor interactions or Wnt receptor maturation. Conversely, the Wnt activators, R-spondin and Norrin, promote Wnt signaling by binding to Wnt receptors or releasing a Wnt-inhibitory step. With few exceptions, these antagonists and agonists are not pure Wnt modulators, but also affect additional signaling pathways, such as TGF-β and FGF signaling. Here we discuss their interactions with Wnt ligands and Wnt receptors, their role in developmental processes, as well as their implication in disease. PMID:23085770

Characterization of wise protein and its molecular mechanism to interact with both Wnt and BMP signals.

PubMed

Lintern, Katherine B; Guidato, Sonia; Rowe, Alison; Saldanha, José W; Itasaki, Nobue

2009-08-21

Cross-talk of BMP and Wnt signaling pathways has been implicated in many aspects of biological events during embryogenesis and in adulthood. A secreted protein Wise and its orthologs (Sostdc1, USAG-1, and Ectodin) have been shown to modulate Wnt signaling and also inhibit BMP signals. Modulation of Wnt signaling activity by Wise is brought about by an interaction with the Wnt co-receptor LRP6, whereas BMP inhibition is by binding to BMP ligands. Here we have investigated the mode of action of Wise on Wnt and BMP signals. It was found that Wise binds LRP6 through one of three loops formed by the cystine knot. The Wise deletion construct lacking the LRP6-interacting loop domain nevertheless binds BMP4 and inhibits BMP signals. Moreover, BMP4 does not interfere with Wise-LRP6 binding, suggesting separate domains for the physical interaction. Functional assays also show that the ability of Wise to block Wnt1 activity through LRP6 is not impeded by BMP4. In contrast, the ability of Wise to inhibit BMP4 is prevented by additional LRP6, implying a preference of Wise in binding LRP6 over BMP4. In addition to the interaction of Wise with BMP4 and LRP6, the molecular characteristics of Wise, such as glycosylation and association with heparan sulfate proteoglycans on the cell surface, are suggested. This study helps to understand the multiple functions of Wise at the molecular level and suggests a possible role for Wise in balancing Wnt and BMP signals.

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

Santiago, Francisco; Oguma, Junya; Brown, Anthony M.C.

Highlights: Black-Right-Pointing-Pointer First demonstration of direct role for noncanonical Wnt in osteoclast differentiation. Black-Right-Pointing-Pointer Demonstration of Ryk as a Wnt5a/b receptor in inhibition of canonical Wnt signaling. Black-Right-Pointing-Pointer Modulation of noncanonical Wnt signaling by a clinically important drug, ritonavir. Black-Right-Pointing-Pointer Establishes a mechanism for an important clinical problem: HIV-associated bone loss. -- Abstract: Wnt proteins that signal via the canonical Wnt/{beta}-catenin pathway directly regulate osteoblast differentiation. In contrast, most studies of Wnt-related effects on osteoclasts involve indirect changes. While investigating bone mineral density loss in the setting of human immunodeficiency virus (HIV) infection and its treatment with the protease inhibitormore » ritonavir (RTV), we observed that RTV decreased nuclear localization of {beta}-catenin, critical to canonical Wnt signaling, in primary human and murine osteoclast precursors. This occurred in parallel with upregulation of Wnt5a and Wnt5b transcripts. These Wnts typically stimulate noncanonical Wnt signaling, and this can antagonize the canonical Wnt pathway in many cell types, dependent upon Wnt receptor usage. We now document RTV-mediated upregulation of Wnt5a/b protein in osteoclast precursors. Recombinant Wnt5b and retrovirus-mediated expression of Wnt5a enhanced osteoclast differentiation from human and murine monocytic precursors, processes facilitated by RTV. In contrast, canonical Wnt signaling mediated by Wnt3a suppressed osteoclastogenesis. Both RTV and Wnt5b inhibited canonical, {beta}-catenin/T cell factor-based Wnt reporter activation in osteoclast precursors. RTV- and Wnt5-induced osteoclast differentiation were dependent upon the receptor-like tyrosine kinase Ryk, suggesting that Ryk may act as a Wnt5a/b receptor in this context. This is the first demonstration of a direct role for Wnt signaling pathways and Ryk in regulation of osteoclast differentiation, and its modulation by a clinically important drug, ritonavir. These studies also reveal a potential role for noncanonical Wnt5a/b signaling in acceleration of bone mineral density loss in HIV-infected individuals, and illuminate a potential means of influencing such processes in disease states that involve enhanced osteoclast activity.« less

DNA Demethylation Rescues the Impaired Osteogenic Differentiation Ability of Human Periodontal Ligament Stem Cells in High Glucose

PubMed Central

Liu, Zhi; Chen, Tian; Sun, Wenhua; Yuan, Zongyi; Yu, Mei; Chen, Guoqing; Guo, Weihua; Xiao, Jingang; Tian, Weidong

2016-01-01

Diabetes mellitus, characterized by abnormally high blood glucose levels, gives rise to impaired bone remodeling. In response to high glucose (HG), the attenuated osteogenic differentiation capacity of human periodontal ligament stem cells (hPDLSCs) is associated with the loss of alveolar bone. Recently, DNA methylation was reported to affect osteogenic differentiation of stem cells in pathological states. However, the intrinsic mechanism linking DNA methylation to osteogenic differentiation ability in the presence of HG is still unclear. In this study, we found that diabetic rats with increased DNA methylation levels in periodontal ligaments exhibited reduced bone mass and density. In vitro application of 5-aza-2′-deoxycytidine (5-aza-dC), a DNA methyltransferase inhibitor, to decrease DNA methylation levels in hPDLSCs, rescued the osteogenic differentiation capacity of hPDLSCs under HG conditions. Moreover, we demonstrated that the canonical Wnt signaling pathway was activated during this process and, under HG circumstances, the 5-aza-dC-rescued osteogenic differentiation capacity was blocked by Dickkopf-1, an effective antagonist of the canonical Wnt signaling pathway. Taken together, these results demonstrate for the first time that suppression of DNA methylation is able to facilitate the osteogenic differentiation capacity of hPDLSCs exposed to HG, through activation of the canonical Wnt signaling pathway. PMID:27273319

Stroma provides an intestinal stem cell niche in the absence of epithelial Wnts.

PubMed

Kabiri, Zahra; Greicius, Gediminas; Madan, Babita; Biechele, Steffen; Zhong, Zhendong; Zaribafzadeh, Hamed; Edison; Aliyev, Jamal; Wu, Yonghui; Bunte, Ralph; Williams, Bart O; Rossant, Janet; Virshup, David M

2014-06-01

Wnt/β-catenin signaling supports intestinal homeostasis by regulating proliferation in the crypt. Multiple Wnts are expressed in Paneth cells as well as other intestinal epithelial and stromal cells. Ex vivo, Wnts secreted by Paneth cells can support intestinal stem cells when Wnt signaling is enhanced with supplemental R-Spondin 1 (RSPO1). However, in vivo, the source of Wnts in the stem cell niche is less clear. Genetic ablation of Porcn, an endoplasmic reticulum resident O-acyltransferase that is essential for the secretion and activity of all vertebrate Wnts, confirmed the role of intestinal epithelial Wnts in ex vivo culture. Unexpectedly, mice lacking epithelial Wnt activity (Porcn(Del)/Villin-Cre mice) had normal intestinal proliferation and differentiation, as well as successful regeneration after radiation injury, indicating that epithelial Wnts are dispensable for these processes. Consistent with a key role for stroma in the crypt niche, intestinal stromal cells endogenously expressing Wnts and Rspo3 support the growth of Porcn(Del) organoids ex vivo without RSPO1 supplementation. Conversely, increasing pharmacologic PORCN inhibition, affecting both stroma and epithelium, reduced Lgr5 intestinal stem cells, inhibited recovery from radiation injury, and at the highest dose fully blocked intestinal proliferation. We conclude that epithelial Wnts are dispensable and that stromal production of Wnts can fully support normal murine intestinal homeostasis.

An RNAi-based chemical genetic screen identifies three small-molecule inhibitors of the Wnt/wingless signaling pathway

PubMed Central

Gonsalves, Foster C.; Klein, Keren; Carson, Brittany B.; Katz, Shauna; Ekas, Laura A.; Evans, Steve; Nagourney, Robert; Cardozo, Timothy; Brown, Anthony M. C.; DasGupta, Ramanuj

2011-01-01

Misregulated β-catenin responsive transcription (CRT) has been implicated in the genesis of various malignancies, including colorectal carcinomas, and it is a key therapeutic target in combating various cancers. Despite significant effort, successful clinical implementation of CRT inhibitory therapeutics remains a challenging goal. This is, in part, because of the challenge of identifying inhibitory compounds that specifically modulate the nuclear transcriptional activity of β-catenin while not affecting its cytoskeletal function in stabilizing adherens junctions at the cell membrane. Here, we report an RNAi-based modifier screening strategy for the identification of CRT inhibitors. Our data provide support for the specificity of these inhibitory compounds in antagonizing the transcriptional function of nuclear β-catenin. We show that these inhibitors efficiently block Wnt/β-catenin–induced target genes and phenotypes in various mammalian and cancer cell lines. Importantly, these Wnt inhibitors are specifically cytotoxic to human colon tumor biopsy cultures as well as colon cancer cell lines that exhibit deregulated Wnt signaling. PMID:21393571

Induced Wnt5a expression perturbs embryonic outgrowth and intestinal elongation, but is well-tolerated in adult mice.

PubMed

Bakker, Elvira R M; Raghoebir, Lalini; Franken, Patrick F; Helvensteijn, Werner; van Gurp, Léon; Meijlink, Frits; van der Valk, Martin A; Rottier, Robbert J; Kuipers, Ernst J; van Veelen, Wendy; Smits, Ron

2012-09-01

Wnt5a is essential during embryonic development, as indicated by mouse Wnt5a knockout embryos displaying outgrowth defects of multiple structures including the gut. The dynamics of Wnt5a involvement in these processes is unclear, and perinatal lethality of Wnt5a knockout embryos has hampered investigation of Wnt5a during postnatal stages in vivo. Although in vitro studies have suggested a relevant role for Wnt5a postnatally, solid evidence for a significant impact of Wnt5a within the complexity of an adult organism is lacking. We generated a tightly-regulated inducible Wnt5a transgenic mouse model and investigated the effects of Wnt5a induction during different time-frames of embryonic development and in adult mice, focusing on the gastrointestinal tract. When induced in embryos from 10.5 dpc onwards, Wnt5a expression led to severe outgrowth defects affecting the gastrointestinal tracts, limbs, facial structures and tails, closely resembling the defects observed in Wnt5a knockout mice. However, Wnt5a induction from 13.5 dpc onwards did not cause this phenotype, indicating that the most critical period for Wnt5a in embryonic development is prior to 13.5 dpc. In adult mice, induced Wnt5a expression did not reveal abnormalities, providing the first in vivo evidence that Wnt5a has no major impact on mouse intestinal homeostasis postnatally. Protein expression of Wnt5a receptor Ror2 was strongly reduced in adult intestine compared to embryonic stages. Moreover, we uncovered a regulatory process where induction of Wnt5a causes downregulation of its receptor Ror2. Taken together, our results indicate a role for Wnt5a during a restricted time-frame of embryonic development, but suggest no impact during homeostatic postnatal stages. Copyright © 2012 Elsevier Inc. All rights reserved.

AUTONOMOUS AND NONAUTONOMOUS REGULATION OF WNT-MEDIATED NEURONAL POLARITY BY THE C. ELEGANS ROR KINASE CAM-1

PubMed Central

Chien, Shih-Chieh Jason; Gurling, Mark; Kim, Changsung; Craft, Teresa; Forrester, Wayne; Garriga, Gian

2015-01-01

Wnts are a conserved family of secreted glycoproteins that regulate various developmental processes in metazoans. Three of the five C. elegans Wnts, CWN-1, CWN-2 and EGL-20, and the sole Wnt receptor of the Ror kinase family, CAM-1, are known to regulate the anterior polarization of the mechanosensory neuron ALM. Here we show that CAM-1 and the Frizzled receptor MOM-5 act in parallel pathways to control ALM polarity. We also show that CAM-1 has two functions in this process: an autonomous signaling function that promotes anterior polarization and a nonautonomous Wnt-antagonistic function that inhibits anterior polarization. These antagonistic activities can account for the weak ALM phenotypes displayed by cam-1 mutants. Our observations suggest that CAM-1 could function as a Wnt receptor in many developmental processes, but the analysis of cam-1 mutants may fail to reveal CAM-1’s role as a receptor in these processes because of its Wnt-antagonistic activity. In this model, loss of CAM-1 results in increased levels of Wnts that act through other Wnt receptors, masking CAM-1’s autonomous role as a Wnt receptor. PMID:25917219

Signal transduction by the Wnt family of ligands.

PubMed Central

Dale, T C

1998-01-01

The Wnt genes encode a large family of secreted polypeptides that mediate cell-cell communication in diverse developmental processes. The loss or inappropriate activation of Wnt expression has been shown to alter cell fate, morphogenesis and mitogenesis. Recent progress has identified Wnt receptors and components of an intracellular signalling pathway that mediate Wnt-dependent transcription. This review will highlight this 'core' Wnt signal-transduction pathway, but also aims to reveal the potential diversity of Wnt signalling targets. Particular attention will be paid to the overlap between developmental biology and oncogenesis, since recent progress shows Wnt signalling forms a paradigm for an interdisciplinary approach. PMID:9425102

Wnt5 is required for notochord cell intercalation in the ascidian Halocynthia roretzi.

PubMed

Niwano, Tomoko; Takatori, Naohito; Kumano, Gaku; Nishida, Hiroki

2009-08-25

In the embryos of various animals, the body elongates after gastrulation by morphogenetic movements involving convergent extension. The Wnt/PCP (planar cell polarity) pathway plays roles in this process, particularly mediolateral polarization and intercalation of the embryonic cells. In ascidians, several factors in this pathway, including Wnt5, have been identified and found to be involved in the intercalation process of notochord cells. In the present study, the role of the Wnt5 genes, Hr-Wnt5alpha (Halocynthia roretzi Wnt5alpha) and Hr-Wnt5beta, in convergent extension was investigated in the ascidian H. roretzi by injecting antisense oligonucleotides and mRNAs into single precursor blastomeres of various tissues, including notochord, at the 64-cell stage. Hr-Wnt5alpha is expressed in developing notochord and was essential for notochord morphogenesis. Precise quantitative control of its expression level was crucial for proper cell intercalation. Overexpression of Wnt5 proteins in notochord and other tissues that surround the notochord indicated that Wnt5alpha plays a role within the notochord, and is unlikely to be the source of polarizing cues arising outside the notochord. Detailed mosaic analysis of the behaviour of individual notochord cells overexpressing Wnt5alpha indicated that a Wnt5alpha-manipulated cell does not affect the behaviour of neighbouring notochord cells, suggesting that Wnt5alpha works in a cell-autonomous manner. This is further supported by comparison of the results of Wnt5alpha and Dsh (Dishevelled) knockdown experiments. In addition, our results suggest that the Wnt/PCP pathway is also involved in mediolateral intercalation of cells of the ventral row of the nerve cord (floor plate) and the endodermal strand. The present study highlights the role of the Wnt5alpha signal in notochord convergent extension movements in ascidian embryos. Our results raise the novel possibility that Wnt5alpha functions in a cell-autonomous manner in activation of the Wnt/PCP pathway to polarize the protrusive activity that drives convergent extension.

The sclerostin-neutralizing antibody AbD09097 recognizes an epitope adjacent to sclerostin's binding site for the Wnt co-receptor LRP6

PubMed Central

Boschert, V.; Frisch, C.; Back, J. W.; van Pee, K.; Weidauer, S. E.; Muth, E.-M.; Schmieder, P.; Beerbaum, M.; Knappik, A.; Timmerman, P.

2016-01-01

The glycoprotein sclerostin has been identified as a negative regulator of bone growth. It exerts its function by interacting with the Wnt co-receptor LRP5/6, blocks the binding of Wnt factors and thereby inhibits Wnt signalling. Neutralizing anti-sclerostin antibodies are able to restore Wnt activity and enhance bone growth thereby presenting a new osteoanabolic therapy approach for diseases such as osteoporosis. We have generated various Fab antibodies against human and murine sclerostin using a phage display set-up. Biochemical analyses have identified one Fab developed against murine sclerostin, AbD09097 that efficiently neutralizes sclerostin's Wnt inhibitory activity. In vitro interaction analysis using sclerostin variants revealed that this neutralizing Fab binds to sclerostin's flexible second loop, which has been shown to harbour the LRP5/6 binding motif. Affinity maturation was then applied to AbD09097, providing a set of improved neutralizing Fab antibodies which particularly bind human sclerostin with enhanced affinity. Determining the crystal structure of AbD09097 provides first insights into how this antibody might recognize and neutralize sclerostin. Together with the structure–function relationship derived from affinity maturation these new data will foster the rational design of new and highly efficient anti-sclerostin antibodies for the therapy of bone loss diseases such as osteoporosis. PMID:27558933

Calcitonin protects chondrocytes from lipopolysaccharide-induced apoptosis and inflammatory response through MAPK/Wnt/NF-κB pathways.

PubMed

Zhang, Lai-Bo; Man, Zhen-Tao; Li, Wei; Zhang, Wei; Wang, Xian-Quan; Sun, Shui

2017-07-01

Calcitonin (CT) is an anti-absorbent, which has long been used for treatment of osteoporosis. However, little information is available about the effects of CT on osteoarthritis (OA). This study was mainly aimed to explore the effects of CT on the treatment of OA, as well as the underlying mechanisms. Chondrocytes were isolated from immature mice and then were incubated with lipopolysaccharide (LPS), CT, small interfering (si) RNA against bone morphogenetic protein (BMP)-2, and/or the inhibitors of MAPK/Wnt/NF-κB pathway. Thereafter, cell viability, apoptosis, nitric oxide (NO) and inflammatory factors productions, and expression levels of cartilage synthesis protein key factors, cartilage-derived morphogenetic protein (CDMP) 1, SRY (sex-determining region Y)-box 9 protein (SOX9), and MAPK/Wnt/NF-κB pathways key factors were determined. CT significantly reversed LPS-induced cell viability decrease, apoptosis increase, the inflammatory factors and NO secretion, the abnormally expression of cartilage synthesis proteins and the activation of MAPK/Wnt/NF-κB pathways (P<0.05). In addition, we observed that administration of the inhibitors of MAPK/Wnt/NF-κB pathways statistically further increased the levels of CDMP1 and SOX9 (P<0.05). Suppression of BMP-2 decreased the levels of CDMP1 and SOX9 and activated MAPK/Wnt/NF-κB pathways, and could partially abolish CT-modulated the expression changes in CDMP1 and SOX9, and MAPK/Wnt/NF-κB pathways key factors (P<0.05). The results showed that CT protects chondrocytes from LPS-induced apoptosis and inflammatory response by regulating BMP-2 and thus blocking MAPK/Wnt/NF-κB pathways. Copyright © 2017 Elsevier Ltd. All rights reserved.

Smad, PI3K/Akt, and Wnt-dependent signaling pathways are involved in BMP-4-induced ESC self-renewal.

PubMed

Lee, Min Young; Lim, Hyun Woo; Lee, Sang Hun; Han, Ho Jae

2009-08-01

It is known that bone morphogenetic protein 4 (BMP-4) has a diverse effect on ESCs. However, its precise mechanism in mouse ESCs is not fully understood. We evaluated the effect of BMP-4 on ESC proliferation and its related signal cascades in this study. BMP-4 significantly increased the level of [(3)H]-thymidine incorporation in time- (> or =8 hours) and dose- (> or =10 ng/ml) dependent manners. Additionally, BMP-4 increased cyclin D1 and decreased p27(kip1) expression values in a time-dependent manner. The increases in BMP-4-induced [(3)H]-thymidine incorporation and cyclin D1 expression were inhibited by the BMP-4 receptor antagonist noggin. BMP-4 increased Wnt1 expression. Wnt1 expression was attenuated by Smad4 small interfering RNA (siRNA), and BMP-4-induced cyclin D1 expression was inhibited by Smad4 and Wnt1 siRNAs. BMP-4 also activated beta-catenin, which was blocked by Smad4 and Wnt1 siRNAs. In addition, BMP-4 induced Akt phosphorylation. BMP-4-induced beta-catenin activation and cyclin D1 expression were attenuated by phosphatidyl inositol 3-kinase (PI3K) siRNA and Akt inhibitor. Additionally, downregulation of Smad4, Wnt1, and PI3K expression by siRNA decreased the levels of pluripotency marker mRNAs of ESCs, including Oct4, Sox2, and FoxD3. Our results suggested that BMP-4-induced [(3)H]-thymidine incorporation was significantly attenuated by Smad4, Wnt1, and PI3K knockdown. In conclusion, BMP-4 contributed to the maintenance of cell proliferation and the pluripotent state by Smad, PI3K/Akt, and Wnt1/beta-catenin in mouse ESCs.

Autocrine stimulation of osteoblast activity by Wnt5a in response to TNF-α in human mesenchymal stem cells

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

Briolay, A.; Lencel, P.; Bessueille, L.

2013-01-18

Highlights: ► Ankylosing spondylitis (AS) leads to bone fusions and ankylosis. ► TNF-α stimulates osteoblasts through growth factors in AS. ► We compare the involvement of canonical vs non-canonical Wnt signaling. ► Canonical Wnt signaling is not involved in TNF-α effects in differentiating hMSCs. ► TNF-α stimulates osteoblasts through Wnt5a autocrine secretion in hMSCs. -- Abstract: Although anti-tumor necrosis factor (TNF)-α treatments efficiently block inflammation in ankylosing spondylitis (AS), they are inefficient to prevent excessive bone formation. In AS, ossification seems more prone to develop in sites where inflammation has resolved following anti-TNF therapy, suggesting that TNF-α indirectly stimulates ossification.more » In this context, our objectives were to determine and compare the involvement of Wnt proteins, which are potent growth factors of bone formation, in the effects of TNF-α on osteoblast function. In human mesenchymal stem cells (MSCs), TNF-α significantly increased the levels of Wnt10b and Wnt5a. Associated with this effect, TNF-α stimulated tissue-non specific alkaline phosphatase (TNAP) and mineralization. This effect was mimicked by activation of the canonical β-catenin pathway with either anti-Dkk1 antibodies, lithium chloride (LiCl) or SB216763. TNF-α reduced, and activation of β-catenin had little effect on expression of osteocalcin, a late marker of osteoblast differentiation. Surprisingly, TNF-α failed to stabilize β-catenin and Dkk1 did not inhibit TNF-α effects. In fact, Dkk1 expression was also enhanced in response to TNF-α, perhaps explaining why canonical signaling by Wnt10b was not activated by TNF-α. However, we found that Wnt5a also stimulated TNAP in MSCs cultured in osteogenic conditions, and increased the levels of inflammatory markers such as COX-2. Interestingly, treatment with anti-Wnt5a antibodies reduced endogenous TNAP expression and activity. Collectively, these data suggest that increased levels of Dkk1 may blunt the autocrine effects of Wnt10b, but not that of Wnt5a, acting through non-canonical signaling. Thus, Wnt5a may be potentially involved in the effects of inflammation on bone formation.« less

Interaction between the Wnt/β-catenin signaling pathway and the EMMPRIN/MMP-2, 9 route in periodontitis.

PubMed

Liu, X; Zhang, Z; Pan, S; Shang, S; Li, C

2018-06-14

In periodontitis, the Wnt/β-catenin signaling pathway is related to the metabolism of the alveolar bone; further, extracellular matrix metalloproteinase inducer (EMMPRIN) expression is correlated with matrix metalloproteinases (MMPs) expression and inflammation severity. The aim of this study was to perform a preliminary investigation of the interaction between the Wnt/β-catenin signaling pathway and the EMMPRIN/MMPs route in periodontitis. Chronic periodontitis and healthy gingival tissues were obtained to detect the expression of Wnt3a, β-catenin, EMMPRIN and MMP-2, 9 by using immunohistochemical analysis. The human immortalized oral epithelial cell/human gingival fibroblast direct co-culture model was treated with 10 μg/mL Porphyromonas gingivalis lipopolysaccharide (Pg. LPS). Anti-EMMPRIN antibody was used to block the effect of EMMPRIN. Dickkopf-1 (DKK-1) and Wnt3a were used as the inhibitor and activator of the Wnt/β-catenin signaling pathway, respectively. Immunofluorescence was performed to visualize the localization of β-catenin and EMMPRIN. Expression of the EMMPRIN, MMP-2, 9 and Wnt pathway's components was confirmed by western blotting and quantitative real-time polymerase chain reaction. Higher levels of Wnt3a, β-catenin, EMMPRIN and MMP-2, 9 were observed in chronic periodontitis gingival tissues compared with controls. Pg. LPS significantly enhanced β-catenin, p-GSK-3β, EMMPRIN and MMP-2, 9 inductions in the human immortalized oral epithelial cell/human gingival fibroblast co-culture model. Anti-EMMPRIN antibody markedly reduced the expression of MMP-2, 9 only in the presence of Pg. LPS. Co-expression of β-catenin and EMMPRIN was detected in the co-culture model. DKK-1 inhibited Wnt pathway, but upregulated the EMMPRIN/MMP-2, 9 routes. In contrast, activating Wnt pathway by Wnt3a repressed the EMMPRIN/MMP-2, 9 routes. The promotion effect of DKK-1 on MMP-2, 9 expressions was partially inhibited by the anti-EMMPRIN antibody. In addition, anti-EMMPRIN antibody led to a drastic decrease in β-catenin and p-GSK-3β. In periodontitis, EMMPRIN regulates MMP-2, 9 expressions, the activation of Wnt/β-catenin signaling pathway downregulates the EMMPRIN/MMP-2, 9 routes and the blockade of EMMPRIN attenuates Wnt/β-catenin signaling pathway. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

A rare human syndrome provides genetic evidence that WNT signaling is required for reprogramming of fibroblasts to induced pluripotent stem cells

PubMed Central

Ross, Jason; Busch, Julia; Mintz, Ellen; Ng, Damian; Stanley, Alexandra; Brafman, David; Sutton, V. Reid; Van den Veyver, Ignatia; Willert, Karl

2015-01-01

SUMMARY WNT signaling promotes the reprogramming of somatic cells to an induced pluripotent state. We provide genetic evidence that WNT signaling is a requisite step during the induction of pluripotency. Fibroblasts from individuals with Focal Dermal Hypoplasia (FDH), a rare genetic syndrome caused by mutations in the essential WNT processing enzyme PORCN, fail to reprogram using standard methods. This blockade in reprogramming is overcome by ectopic WNT signaling and by PORCN overexpression, thus demonstrating that WNT signaling is essential for reprogramming. The rescue of reprogramming is critically dependent on the level of WNT signaling: steady baseline activation of the WNT pathway yields karyotypically normal iPS cells, whereas daily stimulation with Wnt3a produces FDH-iPS cells with severely abnormal karyotypes. Therefore, although WNT signaling is required for cellular reprogramming, inappropriate activation of WNT signaling induces chromosomal instability, highlighting the precarious nature of ectopic WNT activation, and its tight relationship with oncogenic transformation. PMID:25464842

Wnt/β-catenin signaling pathway inhibits the proliferation and apoptosis of U87 glioma cells via different mechanisms

PubMed Central

Gao, Liyang; Chen, Bing; Li, Jinhong; Yang, Fan; Cen, Xuecheng; Liao, Zhuangbing; Long, Xiao’ao

2017-01-01

The Wnt signaling pathway is necessary for the development of the central nervous system and is associated with tumorigenesis in various cancers. However, the mechanism of the Wnt signaling pathway in glioma cells has yet to be elucidated. Small-molecule Wnt modulators such as ICG-001 and AZD2858 were used to inhibit and stimulate the Wnt/β-catenin signaling pathway. Techniques including cell proliferation assay, colony formation assay, Matrigel cell invasion assay, cell cycle assay and Genechip microarray were used. Gene Ontology Enrichment Analysis and Gene Set Enrichment Analysis have enriched many biological processes and signaling pathways. Both the inhibiting and stimulating Wnt/β-catenin signaling pathways could influence the cell cycle, moreover, reduce the proliferation and survival of U87 glioma cells. However, Affymetrix expression microarray indicated that biological processes and networks of signaling pathways between stimulating and inhibiting the Wnt/β-catenin signaling pathway largely differ. We propose that Wnt/β-catenin signaling pathway might prove to be a valuable therapeutic target for glioma. PMID:28837560

The R-spondin family of proteins: emerging regulators of WNT signaling

PubMed Central

Jin, Yong-Ri; Yoon, Jeong Kyo

2012-01-01

Recently, the R-spondin (RSPO) family of proteins has emerged as important regulators of WNT signaling. Considering the wide spectrum of WNT signaling functions in normal biological processes and disease conditions, there has been a significantly growing interest in understanding the functional roles of RSPOs in multiple biological processes and determining the molecular mechanisms by which RSPOs regulate the WNT signaling pathway. Recent advances in the RSPO research field revealed some of the in vivo functions of RSPOs and provided new information regarding the mechanistic roles of RSPO activity in regulation of WNT signaling. Herein, we review recent progress in RSPO research with an emphasis on signaling mechanisms and biological functions. PMID:22982762

A WNT/beta-catenin signaling activator, R-spondin, plays positive regulatory roles during skeletal myogenesis.

PubMed

Han, Xiang Hua; Jin, Yong-Ri; Seto, Marianne; Yoon, Jeong Kyo

2011-03-25

R-spondins (RSPOs) are a recently characterized family of secreted proteins that activate WNT/β-catenin signaling. In this study, we investigated the potential roles of the RSPO proteins during myogenic differentiation. Overexpression of the Rspo1 gene or administration of recombinant RSPO2 protein enhanced mRNA and protein expression of a basic helix-loop-helix (bHLH) class myogenic determination factor, MYF5, in both C2C12 myoblasts and primary satellite cells, whereas MYOD or PAX7 expression was not affected. RSPOs also promoted myogenic differentiation and induced hypertrophic myotube formation in C2C12 cells. In addition, Rspo2 and Rspo3 gene knockdown by RNA interference significantly compromised MYF5 expression, myogenic differentiation, and myotube formation. Furthermore, Myf5 expression was reduced in the developing limbs of mouse embryos lacking the Rspo2 gene. Finally, we demonstrated that blocking of WNT/β-catenin signaling by DKK1 or a dominant-negative form of TCF4 reversed MYF5 expression, myogenic differentiation, and hypertrophic myotube formation induced by RSPO2, indicating that RSPO2 exerts its activity through the WNT/β-catenin signaling pathway. Our results provide strong evidence that RSPOs are key positive regulators of skeletal myogenesis acting through the WNT/β-catenin signaling pathway.

A WNT/β-Catenin Signaling Activator, R-spondin, Plays Positive Regulatory Roles during Skeletal Myogenesis*

PubMed Central

Han, Xiang Hua; Jin, Yong-Ri; Seto, Marianne; Yoon, Jeong Kyo

2011-01-01

R-spondins (RSPOs) are a recently characterized family of secreted proteins that activate WNT/β-catenin signaling. In this study, we investigated the potential roles of the RSPO proteins during myogenic differentiation. Overexpression of the Rspo1 gene or administration of recombinant RSPO2 protein enhanced mRNA and protein expression of a basic helix-loop-helix (bHLH) class myogenic determination factor, MYF5, in both C2C12 myoblasts and primary satellite cells, whereas MYOD or PAX7 expression was not affected. RSPOs also promoted myogenic differentiation and induced hypertrophic myotube formation in C2C12 cells. In addition, Rspo2 and Rspo3 gene knockdown by RNA interference significantly compromised MYF5 expression, myogenic differentiation, and myotube formation. Furthermore, Myf5 expression was reduced in the developing limbs of mouse embryos lacking the Rspo2 gene. Finally, we demonstrated that blocking of WNT/β-catenin signaling by DKK1 or a dominant-negative form of TCF4 reversed MYF5 expression, myogenic differentiation, and hypertrophic myotube formation induced by RSPO2, indicating that RSPO2 exerts its activity through the WNT/β-catenin signaling pathway. Our results provide strong evidence that RSPOs are key positive regulators of skeletal myogenesis acting through the WNT/β-catenin signaling pathway. PMID:21252233

Characterization of the Interaction of Sclerostin with the Low Density Lipoprotein Receptor-related Protein (LRP) Family of Wnt Co-receptors*

PubMed Central

Holdsworth, Gill; Slocombe, Patrick; Doyle, Carl; Sweeney, Bernadette; Veverka, Vaclav; Le Riche, Kelly; Franklin, Richard J.; Compson, Joanne; Brookings, Daniel; Turner, James; Kennedy, Jeffery; Garlish, Rachael; Shi, Jiye; Newnham, Laura; McMillan, David; Muzylak, Mariusz; Carr, Mark D.; Henry, Alistair J.; Ceska, Thomas; Robinson, Martyn K.

2012-01-01

LRP5 and LRP6 are proteins predicted to contain four six-bladed β-propeller domains and both bind the bone-specific Wnt signaling antagonist sclerostin. Here, we report the crystal structure of the amino-terminal region of LRP6 and using NMR show that the ability of sclerostin to bind to this molecule is mediated by the central core of sclerostin and does not involve the amino- and carboxyl-terminal flexible arm regions. We show that this structured core region interacts with LRP5 and LRP6 via an NXI motif (found in the sequence PNAIG) within a flexible loop region (loop 2) within the central core region. This sequence is related closely to a previously identified motif in laminin that mediates its interaction with the β-propeller domain of nidogen. However, the NXI motif is not involved in the interaction of sclerostin with LRP4 (another β-propeller containing protein in the LRP family). A peptide derived from the loop 2 region of sclerostin blocked the interaction of sclerostin with LRP5/6 and also inhibited Wnt1 but not Wnt3A or Wnt9B signaling. This suggests that these Wnts interact with LRP6 in different ways. PMID:22696217

RAC1 GTP-ase signals Wnt-beta-catenin pathway mediated integrin-directed metastasis-associated tumor cell phenotypes in triple negative breast cancers.

PubMed

De, Pradip; Carlson, Jennifer H; Jepperson, Tyler; Willis, Scooter; Leyland-Jones, Brian; Dey, Nandini

2017-01-10

The acquisition of integrin-directed metastasis-associated (ID-MA) phenotypes by Triple-Negative Breast Cancer (TNBC) cells is caused by an upregulation of the Wnt-beta-catenin pathway (WP). We reported that WP is one of the salient genetic features of TNBC. RAC-GTPases, small G-proteins which transduce signals from cell surface proteins including integrins, have been implicated in tumorigenesis and metastasis by their role in essential cellular functions like motility. The collective percentage of alteration(s) in RAC1 in ER+ve BC was lower as compared to ER-ve BC (35% vs 57%) (brca/tcga/pub2015). High expression of RAC1 was associated with poor outcome for RFS with HR=1.48 [CI: 1.15-1.9] p=0.0019 in the Hungarian ER-veBC cohort. Here we examined how WP signals are transduced via RAC1 in the context of ID-MA phenotypes in TNBC. Using pharmacological agents (sulindac sulfide), genetic tools (beta-catenin siRNA), WP modulators (Wnt-C59, XAV939), RAC1 inhibitors (NSC23766, W56) and WP stimulations (LWnt3ACM, Wnt3A recombinant) in a panel of 6-7 TNBC cell lines, we studied fibronectin-directed (1) migration, (2) matrigel invasion, (3) RAC1 and Cdc42 activation, (4) actin dynamics (confocal microscopy) and (5) podia-parameters. An attenuation of WP, which (a) decreased cellular levels of beta-catenin, as well as its nuclear active-form, (b) decreased fibronectin-induced migration, (c) decreased invasion, (d) altered actin dynamics and (e) decreased podia-parameters was successful in blocking fibronectin-mediated RAC1/Cdc42 activity. Both Wnt-antagonists and RAC1 inhibitors blocked fibronectin-induced RAC1 activation and inhibited the fibronectin-induced ID-MA phenotypes following specific WP stimulation by LWnt3ACM as well as Wnt3A recombinant protein. To test a direct involvement of RAC1-activation in WP-mediated ID-MA phenotypes, we stimulated brain-metastasis specific MDA-MB231BR cells with LWnt3ACM. LWnt3ACM-stimulated fibronectin-directed migration was blocked by RAC1 inhibition in MDA-MB231BR cells. In the light of our previous report that WP upregulation causes ID-MA phenotypes in TNBC tumor cells, here we provide the first mechanism based evidence to demonstrate that WP upregulation signals ID-MA tumor cell phenotypes in a RAC1-GTPase dependent manner involving exchange-factors like TIAM1 and VAV2. Our study demonstrates for the first time that beta-catenin-RAC1 cascade signals integrin-directed metastasis-associated tumor cell phenotypes in TNBC.

Wnt transmembrane signaling and long-term spatial memory

PubMed Central

Tabatadze, Nino; Tomas, Caroline; McGonigal, Rhona; Lin, Brian; Schook, Andrew; Routtenberg, Aryeh

2011-01-01

Transmembrane signaling mechanisms are critical for regulating the plasticity of neuronal connections underlying the establishment of long-lasting memory (e.g., Linden and Routtenberg, 1989, Brain Res Rev. 14: 279–296; Sossin, 1996, Trends Neurosci 19: 215–218; Mayr and Montminy, 2001, Nat Rev Mol Cell Biol. 2: 599–609; Chen et al., 2011, Nature 469: 491–497). One signaling mechanism that has received surprisingly little attention in this regard is the well-known Wnt transmembrane signaling pathway even though this pathway in the adult plays a significant role, for example, in postsynaptic dendritic spine morphogenesis and presynaptic terminal neurotransmitter release (Inestrosa and Arenas, 2010, Nature Rev Neurosci 11: 77–86). The present report now provides the first evidence of Wnt signaling in spatial information storage processes. Importantly, this Wnt participation is specific and selective. Thus, spatial, but not cued, learning in a water maze selectively elevates the levels in hippocampus of Wnt 7 and Wnt 5a, but not the Wnt 3 isoform, indicating behavioral selectivity and isoform specificity. Wnt 7 elevation is subfield-specific: granule cells show an increase with no detectable change in CA3 neurons. Wnt 7 elevation is temporally specific: increased Wnt signaling is not observed during training, but is seen 7 days and, unexpectedly, 30 days later. If the Wnt elevation after learning is activity-dependent, then it may be possible to model this effect in primary hippocampal neurons in culture. Here we evaluate the consequence of potassium or glutamate depolarization on Wnt signaling. This represents, to our knowledge, the first demonstration of an activation-dependent elevation of Wnt levels. Additionally, the novel finding emerged of an increased number of Wnt-stained puncta in neuritis suggestive of trafficking from the cell body to neuronal processes, probably dendrites. It is proposed that Wnt signaling pathways, both canonical and non-canonical, regulate long-term information storage in a behavioral-, cellular- and isoform-specific manner. PMID:22180023

Wnt-1 Signaling in Mammary Carcinogenesis

DTIC Science & Technology

2000-04-01

and the notochord (4), Wnt-5A/LRP6 or LRP6 (higher doses) alone induced trunk axis duplication with muscle and neural tissues but lacking head or the... notochord (Fig. lb). It remains unclear whether this is due to quantitative or qualitative differences between Wnt-5a/LRP6 and Wnt-5a/hFz5 co...injections under these experimental conditions. We also analyzed LRP6 effect on neural crest formation, which is another Wnt- dependent developmental process

Wnt1 and wnt10b function redundantly at the zebrafish midbrain-hindbrain boundary.

PubMed

Lekven, Arne C; Buckles, Gerri R; Kostakis, Nicholas; Moon, Randall T

2003-02-15

Wnt signals have been shown to be involved in multiple steps of vertebrate neural patterning, yet the relative contributions of individual Wnts to the process of brain regionalization is poorly understood. Wnt1 has been shown in the mouse to be required for the formation of the midbrain and the anterior hindbrain, but this function of wnt1 has not been explored in other model systems. Further, wnt1 is part of a Wnt cluster conserved in all vertebrates comprising wnt1 and wnt10b, yet the function of wnt10b during embryogenesis has not been explored. Here, we report that in zebrafish wnt10b is expressed in a pattern overlapping extensively with that of wnt1. We have generated a deficiency allele for these closely linked loci and performed morpholino antisense oligo knockdown to show that wnt1 and wnt10b provide partially redundant functions in the formation of the midbrain-hindbrain boundary (MHB). When both loci are deleted, the expression of pax2.1, en2, and her5 is lost in the ventral portion of the MHB beginning at the 8-somite stage. However, wnt1 and wnt10b are not required for the maintenance of fgf8, en3, wnt8b, or wnt3a expression. Embryos homozygous for the wnt1-wnt10b deficiency display a mild MHB phenotype, but are sensitized to reductions in either Pax2.1 or Fgf8; that is, in combination with mutant alleles of either of these loci, the morphological MHB is lost. Thus, wnt1 and wnt10b are required to maintain threshold levels of Pax2.1 and Fgf8 at the MHB. Copyright 2003 Elsevier Science (USA)

Downregulation of Pygopus 2 inhibits vascular mimicry in glioma U251 cells by suppressing the canonical Wnt signaling pathway

PubMed Central

WANG, HAIDONG; FU, JIANHUA; XU, DIANSHUANG; XU, WEIWEI; WANG, SHIYONG; ZHANG, LIU; XIANG, YONGSHENG

2016-01-01

Gliomas are the most common type of malignant primary brain tumor, and the Wnt signaling pathway is associated with glioma malignancy. Pygopus protein plays an important role in developmental brain patterning, and has been identified to be a component of the Wnt signaling pathway. In the present study, the Pygopus 2 (Pygo2) protein was examined in 80 glioma tissue samples. Short hairpin (sh)RNA-Pygo2 was transfected into glioma U251 cells, and the cell proliferation, colony formation and bromodeoxyuridine (BrdU) incorporation were analyzed. Western blot analysis and reverse transcription-polymerase chain reaction were used to detect the expression of Pygo2. A vascular mimicry assay was performed to examine the vascular mimicry of U251 cells. A luciferase reporter assay was used to detect the β-catenin/Wnt system. The cyclin D1 protein was also detected using western blot analysis. The results demonstrated that inhibition of the expression of Pygo2 significantly triggered the decrease of cell proliferation, colony formation and BrdU incorporation compared with the cells treated with scramble control shRNA (shRNA-Scr). shRNA-Pygo2 transfection was found to inhibit vascular-mimicry and block the Wnt signaling pathway compared to the cells transfected with shRNA-Scr. The transfection of shRNA-Pygo2 also decreased the expression of the Wnt target gene cyclin D1. In conclusion, shRNA-Pygo2 suppressed glioma cell proliferation effectively and inhibited vascular mimicry by inhibiting the expression of cyclin D1 in the canonical Wnt/β-catenin pathway in brain glioma cells. PMID:26870266

Identification of two novel activities of the Wnt signaling regulator Dickkopf 3 and characterization of its expression in the mouse retina

PubMed Central

Nakamura, Rei EI; Hunter, Dale D; Yi, Hyun; Brunken, William J; Hackam, Abigail S

2007-01-01

Background The Wnt signaling pathway is a cellular communication pathway that plays critical roles in development and disease. A major class of Wnt signaling regulators is the Dickkopf (Dkk) family of secreted glycoproteins. Although the biological properties of Dickkopf 1 (Dkk1) and Dickkopf 2 (Dkk2) are well characterized, little is known about the function of the related Dickkopf 3 (Dkk3) protein in vivo or in cell lines. We recently demonstrated that Dkk3 transcripts are upregulated during photoreceptor death in a mouse model of retinal degeneration. In this study, we characterized the activity of Dkk3 in Wnt signaling and cell death. Results Dkk3 was localized to Müller glia and retinal ganglion cells in developing and adult mouse retina. Western blotting confirmed that Dkk3 is secreted from Müller glia cells in culture. We demonstrated that Dkk3 potentiated Wnt signaling in Müller glia and HEK293 cells but not in COS7 cells, indicating that it is a cell-type specific regulator of Wnt signaling. This unique Dkk3 activity was blocked by co-expression of Dkk1. Additionally, Dkk3 displayed pro-survival properties by decreasing caspase activation and increasing viability in HEK293 cells exposed to staurosporine and H2O2. In contrast, Dkk3 did not protect COS7 cells from apoptosis. Conclusion These data demonstrate that Dkk3 is a positive regulator of Wnt signaling, in contrast to its family member Dkk1. Furthermore, Dkk3 protects against apoptosis by reducing caspase activity, suggesting that Dkk3 may play a cytoprotective role in the retina. PMID:18093317

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

PubMed

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

2017-07-28

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

DKK1 mediated inhibition of Wnt signaling in postnatal mice leads to loss of TEC progenitors and thymic degeneration.

PubMed

Osada, Masako; Jardine, Logan; Misir, Ruth; Andl, Thomas; Millar, Sarah E; Pezzano, Mark

2010-02-08

Thymic epithelial cell (TEC) microenvironments are essential for the recruitment of T cell precursors from the bone marrow, as well as the subsequent expansion and selection of thymocytes resulting in a mature self-tolerant T cell repertoire. The molecular mechanisms, which control both the initial development and subsequent maintenance of these critical microenvironments, are poorly defined. Wnt signaling has been shown to be important to the development of several epithelial tissues and organs. Regulation of Wnt signaling has also been shown to impact both early thymocyte and thymic epithelial development. However, early blocks in thymic organogenesis or death of the mice have prevented analysis of a role of canonical Wnt signaling in the maintenance of TECs in the postnatal thymus. Here we demonstrate that tetracycline-regulated expression of the canonical Wnt inhibitor DKK1 in TECs localized in both the cortex and medulla of adult mice, results in rapid thymic degeneration characterized by a loss of DeltaNP63(+) Foxn1(+) and Aire(+) TECs, loss of K5K8DP TECs thought to represent or contain an immature TEC progenitor, decreased TEC proliferation and the development of cystic structures, similar to an aged thymus. Removal of DKK1 from DKK1-involuted mice results in full recovery, suggesting that canonical Wnt signaling is required for the differentiation or proliferation of TEC populations needed for maintenance of properly organized adult thymic epithelial microenvironments. Taken together, the results of this study demonstrate that canonical Wnt signaling within TECs is required for the maintenance of epithelial microenvironments in the postnatal thymus, possibly through effects on TEC progenitor/stem cell populations. Downstream targets of Wnt signaling, which are responsible for maintenance of these TEC progenitors may provide useful targets for therapies aimed at counteracting age associated thymic involution or the premature thymic degeneration associated with cancer therapy and bone marrow transplants.

Wnt5a attenuates Wnt3a-induced alkaline phosphatase expression in dental follicle cells

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

Sakisaka, Yukihiko; Tsuchiya, Masahiro; Tohoku Fukushi University, Sendai 989-3201

Wnt signaling regulates multiple cellular events such as cell proliferation, differentiation, and apoptosis through β-catenin-dependent canonical and β-catenin-independent noncanonical pathways. Canonical Wnt/β-catenin signaling can promote the differentiation of dental follicle cells, putative progenitor cells for cementoblasts, osteoblasts, and periodontal ligament cells, toward a cementoblast/osteoblast phenotype during root formation, but little is known about the biological significance of noncanonical Wnt signaling in this process. We identified the expression of Wnt5a, a representative noncanonical Wnt ligand, in tooth root lining cells (i.e. precementoblasts/cementoblasts) and dental follicle cells during mouse tooth root development, as assessed by immunohistochemistry. Silencing expression of the Wnt5a genemore » in a dental follicle cell line resulted in enhancement of the Wnt3a (a representative canonical Wnt ligand)-mediated increase in alkaline phosphatase (ALP) expression. Conversely, treatment with recombinant Wnt5a inhibited the increase in ALP expression, suggesting that Wnt5a signaling functions as a negative regulator of canonical Wnt-mediated ALP expression of dental follicle cells. Wnt5a did not affect the nuclear translocation of β-catenin as well as β-catenin-mediated transcriptional activation of T-cell factor (Tcf) triggered by Wnt3a, suggesting that Wnt5a inhibits the downstream part of the β-catenin-Tcf pathway. These findings suggest the existence of a feedback mechanism between canonical and noncanonical Wnt signaling during the differentiation of dental follicle cells. - Highlights: • Dental follicle cells express Wnt5a during tooth root development. • Silencing of Wnt5a enhances Wnt3a-mediated ALP expression of dental follicle cells. • Conversely, treatment with rWnt5a inhibited the increase in ALP expression. • Wnt5a functions as a negative regulator of Wnt3a-mediated ALP expression.« less

Cooperation between both Wnt/β-catenin and PTEN/PI3K/Akt signaling promotes primitive hematopoietic stem cell self-renewal and expansion

PubMed Central

Perry, John M.; He, Xi C.; Sugimura, Ryohichi; Grindley, Justin C.; Haug, Jeffrey S.; Ding, Sheng; Li, Linheng

2011-01-01

Although self-renewal is the central property of stem cells, the underlying mechanism remains inadequately defined. Using a hematopoietic stem and progenitor cell (HSPC)-specific conditional induction line, we generated a compound genetic model bearing both Pten deletion and β-catenin activation. These double mutant mice exhibit a novel phenotype, including expansion of phenotypic long-term hematopoietic stem cells (LT-HSCs) without extensive differentiation. Unexpectedly, constitutive activation of β-catenin alone results in apoptosis of HSCs. However, together, the Wnt/β-catenin and PTEN/PI3k/Akt pathways interact to drive phenotypic LT-HSC expansion by inducing proliferation while simultaneously inhibiting apoptosis and blocking differentiation, demonstrating the necessity of complementary cooperation between the two pathways in promoting self-renewal. Mechanistically, β-catenin activation reduces multiple differentiation-inducing transcription factors, blocking differentiation partially through up-regulation of Inhibitor of differentiation 2 (Id2). In double mutants, loss of Pten enhances the HSC anti-apoptotic factor Mcl-1. All of these contribute in a complementary way to HSC self-renewal and expansion. While permanent, genetic alteration of both pathways in double mutant mice leads to expansion of phenotypic HSCs, these HSCs cannot function due to blocked differentiation. We developed a pharmacological approach to expand normal, functional HSCs in culture using factors that reversibly activate both Wnt/β-catenin and PI3K/Akt signaling simultaneously. We show for the first time that activation of either single pathway is insufficient to expand primitive HSCs, but in combination, both pathways drive self-renewal and expansion of HSCs with long-term functional capacity. PMID:21890648

Chronic skin inflammation leads to bone loss by IL-17-mediated inhibition of Wnt signaling in osteoblasts.

PubMed

Uluçkan, Özge; Jimenez, Maria; Karbach, Susanne; Jeschke, Anke; Graña, Osvaldo; Keller, Johannes; Busse, Björn; Croxford, Andrew L; Finzel, Stephanie; Koenders, Marije; van den Berg, Wim; Schinke, Thorsten; Amling, Michael; Waisman, Ari; Schett, Georg; Wagner, Erwin F

2016-03-16

Inflammation has important roles in tissue regeneration, autoimmunity, and cancer. Different inflammatory stimuli can lead to bone loss by mechanisms that are not well understood. We show that skin inflammation induces bone loss in mice and humans. In psoriasis, one of the prototypic IL-17A-mediated inflammatory human skin diseases, low bone formation and bone loss correlated with increased serum IL-17A levels. Similarly, in two mouse models with chronic IL-17A-mediated skin inflammation,K14-IL17A(ind)andJunB(Δep), strong inhibition of bone formation was observed, different from classical inflammatory bone loss where osteoclast activation leads to bone degradation. We show that under inflammatory conditions, skin-resident cells such as keratinocytes, γδ T cells, and innate lymphoid cells were able to express IL-17A, which acted systemically to inhibit osteoblast and osteocyte function by a mechanism involving Wnt signaling. IL-17A led to decreased Wnt signaling in vitro, and importantly, pharmacological blockade of IL-17A rescued Wnt target gene expression and bone formation in vivo. These data provide a mechanism where IL-17A affects bone formation by regulating Wnt signaling in osteoblasts and osteocytes. This study suggests that using IL-17A blocking agents in psoriasis could be beneficial against bone loss in these patients. Copyright © 2016, American Association for the Advancement of Science.

Differential angiogenic properties of lithium chloride in vitro and in vivo.

PubMed

Zeilbeck, Ludwig F; Müller, Birgit; Knobloch, Verena; Tamm, Ernst R; Ohlmann, Andreas

2014-01-01

Wnt/β-catenin signaling induced by the Norrin/Frizzled-4 pathway has been shown to improve capillary repair following oxygen induced retinopathy (OIR) in the mouse, a model for retinopathy of prematurity. Here we investigated if treatment with the monovalent cation lithium that has been shown to augment Wnt/β-catenin signaling in vitro and in vivo has similar effects. In cultured human microvascular endothelial cells, LiCl as well as SB 216763, another small molecule that activates Wnt/β-catenin signaling, induced proliferation, survival and migration, which are all common parameters for angiogenic properties in vitro. Moreover, treatment with both agents caused an increase in the levels of β-catenin and their translocation to nuclei while quercetin, an inhibitor of Wnt/β-catenin signaling, completely blocked the effects of LiCl on proliferation. In mice with OIR, intraperitonal or intravitreal treatment with LiCl markedly increased the retinal levels of β-catenin, but did not improve capillary repair. In contrast, repair was significantly improved following intravitreal treatment with Norrin. The effects of LiCl on HDMEC in vitro have minor relevance for OIR in vivo, and the influence of the Norrin/Frizzled-4 pathway on capillary repair in OIR is not reproducible upon enhancing Wnt/β-catenin signaling by LiCl treatment strongly indicating the presence of additional and essential mechanisms.

The Wnt coreceptor Ryk regulates Wnt/planar cell polarity by modulating the degradation of the core planar cell polarity component Vangl2.

PubMed

Andre, Philipp; Wang, Qianyi; Wang, Na; Gao, Bo; Schilit, Arielle; Halford, Michael M; Stacker, Steven A; Zhang, Xuemin; Yang, Yingzi

2012-12-28

The Wnt signaling pathways control many critical developmental and adult physiological processes. In vertebrates, one fundamentally important function of Wnts is to provide directional information by regulating the evolutionarily conserved planar cell polarity (PCP) pathway during embryonic morphogenesis. However, despite the critical roles of Wnts and PCP in vertebrate development and disease, little is known about the molecular mechanisms underlying Wnt regulation of PCP. Here, we have found that the receptor-like tyrosine kinase (Ryk), a Wnt5a-binding protein required in axon guidance, regulates PCP signaling. We show that Ryk interacts with Vangl2 genetically and biochemically, and such interaction is potentiated by Wnt5a. Loss of Ryk in a Vangl2(+/-) background results in classic PCP defects, including open neural tube, misalignment of sensory hair cells in the inner ear, and shortened long bones in the limbs. Complete loss of both Ryk and Vangl2 results in more severe phenotypes that resemble the Wnt5a(-/-) mutant in many aspects such as shortened anterior-posterior body axis, limb, and frontonasal process. Our data identify the Wnt5a-binding protein Ryk as a general regulator of the mammalian Wnt/PCP signaling pathway. We show that Ryk transduces Wnt5a signaling by forming a complex with Vangl2 and that Ryk regulates PCP by at least in part promoting Vangl2 stability. As human mutations in WNT5A and VANGL2 are found to cause Robinow syndrome and neural tube defects, respectively, our results further suggest that human mutations in RYK may also be involved in these diseases.

Multi-level interactions between the nuclear receptor TRα1 and the WNT effectors β-catenin/Tcf4 in the intestinal epithelium.

PubMed

Sirakov, Maria; Skah, Seham; Lone, Imtiaz Nisar; Nadjar, Julien; Angelov, Dimitar; Plateroti, Michelina

2012-01-01

Intestinal homeostasis results from complex cross-regulation of signaling pathways; their alteration induces intestinal tumorigenesis. Previously, we found that the thyroid hormone nuclear receptor TRα1 activates and synergizes with the WNT pathway, inducing crypt cell proliferation and promoting tumorigenesis. Here, we investigated the mechanisms and implications of the cross-regulation between these two pathways in gut tumorigenesis in vivo and in vitro. We analyzed TRα1 and WNT target gene expression in healthy mucosae and tumors from mice overexpressing TRα1 in the intestinal epithelium in a WNT-activated genetic background (vil-TRα1/Apc mice). Interestingly, increased levels of β-catenin/Tcf4 complex in tumors from vil-TRα1/Apc mice blocked TRα1 transcriptional activity. This observation was confirmed in Caco2 cells, in which TRα1 functionality on a luciferase reporter-assay was reduced by the overexpression of β-catenin/Tcf4. Moreover, TRα1 physically interacted with β-catenin/Tcf4 in the nuclei of these cells. Using molecular approaches, we demonstrated that the binding of TRα1 to its DNA target sequences within the tumors was impaired, while it was newly recruited to WNT target genes. In conclusion, our observations strongly suggest that increased β-catenin/Tcf4 levels i) correlated with reduced TRα1 transcriptional activity on its target genes and, ii) were likely responsible for the shift of TRα1 binding on WNT targets. Together, these data suggest a novel mechanism for the tumor-promoting activity of the TRα1 nuclear receptor.

Beta-catenin-dependent Wnt signaling in mandibular bone regeneration.

PubMed

Leucht, Philipp; Kim, Jae-Beom; Helms, Jill A

2008-02-01

Osteoblasts are derived from two distinct embryonic lineages: cranial neural crest, and mesoderm. Both populations of cells are capable of forming bone and cartilage during fetal development and during adult bone repair, but whether they use equivalent molecular pathways to achieve osteoblast differentiation is unknown. We addressed this question in the context of cranial repair and focused on the role of Wnt signaling in mandibular skeletal healing. Transgenic Wnt reporter mice were used to pinpoint Wnt-responsive cells in the injury callus, and in situ hybridization was used to identify some of the Wnt ligands expressed by cells during the repair process. A gene transfer technique was employed to abrogate Wnt signaling during mandibular healing, and we found that reparative intramembranous ossification requires a functional Wnt pathway. Finally, we evaluated how constitutive activation of the Wnt pathway, caused by mutation of the LRP5 receptor, affected bone repair in the mandible. Taken together, these data underscore the functional requirement for Wnt signaling in cranial skeletal healing.

Boehmenan, a lignan from Hibiscus ficulneus, showed Wnt signal inhibitory activity.

PubMed

Shono, Takumi; Ishikawa, Naoki; Toume, Kazufumi; Arai, Midori A; Ahmed, Firoj; Sadhu, Samir K; Ishibashi, Masami

2015-07-15

The Wnt signal pathway modulates numerous biological processes, and its aberrant activation is related to various diseases. Therefore, inhibition of the Wnt signal may provide an effective (or efficient) strategy for these diseases. Cell-based luciferase assay targeting the Wnt signal (TOP assay) revealed that Hibiscus ficulneus extract inhibited the Wnt signal. The activity-guided isolation of the MeOH extract of H. ficulneus stems yielded four known (1-4) lignans along with myriceric acid (5). Compounds 1-4 potently inhibited the Wnt signal with TOPflash IC50 values of 1.0, 4.5, 6.3, and 1.9 μM, respectively. Compound 1 exhibited cytotoxicity against both Wnt-dependent (HCT116) and Wnt-independent (RKO) cells. Western blot analysis showed that 1 decreased the expression of full, cytosolic and nuclear β-catenin along with c-myc in STF/293 cells. Our results suggested that 1 may have inhibited the Wnt signal by decreasing β-catenin levels. Copyright © 2015 Elsevier Ltd. All rights reserved.

Ubiquitination of the Dishevelled DIX domain blocks its head-to-tail polymerization

PubMed Central

Madrzak, Julia; Fiedler, Marc; Johnson, Christopher M.; Ewan, Richard; Knebel, Axel; Bienz, Mariann; Chin, Jason W.

2015-01-01

Dishevelled relays Wnt signals from the plasma membrane to different cytoplasmic effectors. Its signalling activity depends on its DIX domain, which undergoes head-to-tail polymerization to assemble signalosomes. The DIX domain is ubiquitinated in vivo at multiple lysines, which can be antagonized by various deubiquitinases (DUBs) including the CYLD tumour suppressor that attenuates Wnt signalling. Here, we generate milligram quantities of pure human Dvl2 DIX domain mono-ubiquitinated at two lysines (K54 and K58) by genetically encoded orthogonal protection with activated ligation (GOPAL), to investigate their effect on DIX polymerization. We show that the ubiquitination of DIX at K54 blocks its polymerization in solution, whereas DIX58-Ub remains oligomerization-competent. DUB profiling identified 28 DUBs that cleave DIX-ubiquitin conjugates, half of which prefer, or are specific for, DIX54-Ub, including Cezanne and CYLD. These DUBs thus have the potential to promote Dvl polymerization and signalosome formation, rather than antagonize it as previously thought for CYLD. PMID:25907794

A Wnt5 Activity Asymmetry and Intercellular Signaling via PCP Proteins Polarize Node Cells for Left-Right Symmetry Breaking.

PubMed

Minegishi, Katsura; Hashimoto, Masakazu; Ajima, Rieko; Takaoka, Katsuyoshi; Shinohara, Kyosuke; Ikawa, Yayoi; Nishimura, Hiromi; McMahon, Andrew P; Willert, Karl; Okada, Yasushi; Sasaki, Hiroshi; Shi, Dongbo; Fujimori, Toshihiko; Ohtsuka, Toshihisa; Igarashi, Yasunobu; Yamaguchi, Terry P; Shimono, Akihiko; Shiratori, Hidetaka; Hamada, Hiroshi

2017-03-13

Polarization of node cells along the anterior-posterior axis of mouse embryos is responsible for left-right symmetry breaking. How node cells become polarized has remained unknown, however. Wnt5a and Wnt5b are expressed posteriorly relative to the node, whereas genes for Sfrp inhibitors of Wnt signaling are expressed anteriorly. Here we show that polarization of node cells is impaired in Wnt5a -/- Wnt5b -/- and Sfrp mutant embryos, and also in the presence of a uniform distribution of Wnt5a or Sfrp1, suggesting that Wnt5 and Sfrp proteins act as instructive signals in this process. The absence of planar cell polarity (PCP) core proteins Prickle1 and Prickle2 in individual cells or local forced expression of Wnt5a perturbed polarization of neighboring wild-type cells. Our results suggest that opposing gradients of Wnt5a and Wnt5b and of their Sfrp inhibitors, together with intercellular signaling via PCP proteins, polarize node cells along the anterior-posterior axis for breaking of left-right symmetry. Copyright © 2017 Elsevier Inc. All rights reserved.

Wnt signal transduction pathways: modules, development and evolution.

PubMed

Nayak, Losiana; Bhattacharyya, Nitai P; De, Rajat K

2016-08-01

Wnt signal transduction pathway (Wnt STP) is a crucial intracellular pathway mainly due to its participation in important biological processes, functions, and diseases, i.e., embryonic development, stem-cell management, and human cancers among others. This is why Wnt STP is one of the highest researched signal transduction pathways. Study and analysis of its origin, expansion and gradual development to the present state as found in humans is one aspect of Wnt research. The pattern of development and evolution of the Wnt STP among various species is not clear till date. A phylogenetic tree created from Wnt STPs of multiple species may address this issue. In this respect, we construct a phylogenetic tree from modules of Wnt STPs of diverse species. We term it as the 'Module Tree'. A module is nothing but a self-sufficient minimally-dependent subset of the original Wnt STP. Authenticity of the module tree is tested by comparing it with the two reference trees. The module tree performs better than an alternative phylogenetic tree constructed from pathway topology of Wnt STPs. Moreover, an evolutionary emergence pattern of the Wnt gene family is created and the module tree is tallied with it to showcase the significant resemblances.

β catenin in health: A review

PubMed Central

Prakash, Sharada; Swaminathan, Uma

2015-01-01

β catenin belongs to the armadillo family of proteins. It plays a crucial role in developmental and homeostatic processes. Wnts are a family of 19 secreted glycoproteins that transduce multiple signaling cascades, including the canonical Wnt/β catenin pathway, Wnt/Ca2+ pathway and the Wnt/polarity pathway. This is a review on β catenin, Wnt proteins and their secretion, the signaling pathway, the associated factors and the crucial role of β catenin in odontogenesis. PMID:26604501

Wnt5a Increases the Glycolytic Rate and the Activity of the Pentose Phosphate Pathway in Cortical Neurons

PubMed Central

Cisternas, Pedro; Salazar, Paulina; Silva-Álvarez, Carmen; Barros, L. Felipe

2016-01-01

In the last few years, several reports have proposed that Wnt signaling is a general metabolic regulator, suggesting a role for this pathway in the control of metabolic flux. Wnt signaling is critical for several neuronal functions, but little is known about the correlation between this pathway and energy metabolism. The brain has a high demand for glucose, which is mainly used for energy production. Neurons use energy for highly specific processes that require a high energy level, such as maintaining the electrical potential and synthesizing neurotransmitters. Moreover, an important metabolic impairment has been described in all neurodegenerative disorders. Despite the key role of glucose metabolism in the brain, little is known about the cellular pathways involved in regulating this process. We report here that Wnt5a induces an increase in glucose uptake and glycolytic rate and an increase in the activity of the pentose phosphate pathway; the effects of Wnt5a require the intracellular generation of nitric oxide. Our data suggest that Wnt signaling stimulates neuronal glucose metabolism, an effect that could be important for the reported neuroprotective role of Wnt signaling in neurodegenerative disorders. PMID:27688915

Wnt5a Increases the Glycolytic Rate and the Activity of the Pentose Phosphate Pathway in Cortical Neurons.

PubMed

Cisternas, Pedro; Salazar, Paulina; Silva-Álvarez, Carmen; Barros, L Felipe; Inestrosa, Nibaldo C

In the last few years, several reports have proposed that Wnt signaling is a general metabolic regulator, suggesting a role for this pathway in the control of metabolic flux. Wnt signaling is critical for several neuronal functions, but little is known about the correlation between this pathway and energy metabolism. The brain has a high demand for glucose, which is mainly used for energy production. Neurons use energy for highly specific processes that require a high energy level, such as maintaining the electrical potential and synthesizing neurotransmitters. Moreover, an important metabolic impairment has been described in all neurodegenerative disorders. Despite the key role of glucose metabolism in the brain, little is known about the cellular pathways involved in regulating this process. We report here that Wnt5a induces an increase in glucose uptake and glycolytic rate and an increase in the activity of the pentose phosphate pathway; the effects of Wnt5a require the intracellular generation of nitric oxide. Our data suggest that Wnt signaling stimulates neuronal glucose metabolism, an effect that could be important for the reported neuroprotective role of Wnt signaling in neurodegenerative disorders.

Butyrate and bioactive proteolytic form of Wnt-5a regulate colonic epithelial proliferation and spatial development

PubMed Central

Uchiyama, Kazuhiko; Sakiyama, Toshio; Hasebe, Takumu; Musch, Mark W.; Miyoshi, Hiroyuki; Nakagawa, Yasushi; He, Tong-Chuan; Lichtenstein, Lev; Naito, Yuji; Itoh, Yoshito; Yoshikawa, Toshikazu; Jabri, Bana; Stappenbeck, Thaddeus; Chang, Eugene B.

2016-01-01

Proliferation and spatial development of colonic epithelial cells are highly regulated along the crypt vertical axis, which, when perturbed, can result in aberrant growth and carcinogenesis. In this study, two key factors were identified that have important and counterbalancing roles regulating these processes: pericrypt myofibroblast-derived Wnt-5a and the microbial metabolite butyrate. Cultured YAMC cell proliferation and heat shock protein induction were analzyed after butryate, conditioned medium with Wnt5a activity, and FrzB containing conditioned medium. In vivo studies to modulate Hsp25 employed intra-colonic wall Hsp25 encoding lentivirus. To silence Wnt-5a in vivo, intra-colonic wall Wnt-5a silencing RNA was used. Wnt-5a, secreted by stromal myofibroblasts of the lower crypt, promotes proliferation through canonical β-catenin activation. Essential to this are two key requirements: (1) proteolytic conversion of the highly insoluble ~40 kD Wnt-5a protein to a soluble 36 mer amino acid peptide that activates epithelial β-catenin and cellular proliferation, and (2) the simultaneous inhibition of butyrate-induced Hsp25 by Wnt-5a which is necessary to arrest the proliferative process in the upper colonic crypt. The interplay and spatial gradients of these factors insures that crypt epithelial cell proliferation and development proceed in an orderly fashion, but with sufficient plasticity to adapt to physiological perturbations including inflammation. PMID:27561676

Ror receptor tyrosine kinases: orphans no more.

PubMed

Green, Jennifer L; Kuntz, Steven G; Sternberg, Paul W

2008-11-01

Receptor tyrosine kinase-like orphan receptor (Ror) proteins are a conserved family of tyrosine kinase receptors that function in developmental processes including skeletal and neuronal development, cell movement and cell polarity. Although Ror proteins were originally named because the associated ligand and signaling pathway were unknown, recent studies in multiple species have now established that Ror proteins are Wnt receptors. Depending on the cellular context, Ror proteins can either activate or repress transcription of Wnt target genes and can modulate Wnt signaling by sequestering Wnt ligands. New evidence implicates Ror proteins in planar cell polarity, an alternative Wnt pathway. Here, we review the progress made in understanding these mysterious proteins and, in particular, we focus on their function as Wnt receptors.

A context-dependent combination of Wnt receptors controls axis elongation and leg development in a short germ insect

PubMed Central

Beermann, Anke; Prühs, Romy; Lutz, Rebekka; Schröder, Reinhard

2011-01-01

Short germ embryos elongate their primary body axis by consecutively adding segments from a posteriorly located growth zone. Wnt signalling is required for axis elongation in short germ arthropods, including Tribolium castaneum, but the precise functions of the different Wnt receptors involved in this process are unclear. We analysed the individual and combinatorial functions of the three Wnt receptors, Frizzled-1 (Tc-Fz1), Frizzled-2 (Tc-Fz2) and Frizzled-4 (Tc-Fz4), and their co-receptor Arrow (Tc-Arr) in the beetle Tribolium. Knockdown of gene function and expression analyses revealed that Frizzled-dependent Wnt signalling occurs anteriorly in the growth zone in the presegmental region (PSR). We show that simultaneous functional knockdown of the Wnt receptors Tc-fz1 and Tc-fz2 via RNAi resulted in collapse of the growth zone and impairment of embryonic axis elongation. Although posterior cells of the growth zone were not completely abolished, Wnt signalling within the PSR controls axial elongation at the level of pair-rule patterning, Wnt5 signalling and FGF signalling. These results identify the PSR in Tribolium as an integral tissue required for the axial elongation process, reminiscent of the presomitic mesoderm in vertebrates. Knockdown of Tc-fz1 alone interfered with the formation of the proximo-distal and the dorso-ventral axes during leg development, whereas no effect was observed with single Tc-fz2 or Tc-fz4 RNAi knockdowns. We identify Tc-Arr as an obligatory Wnt co-receptor for axis elongation, leg distalisation and segmentation. We discuss how Wnt signalling is regulated at the receptor and co-receptor levels in a dose-dependent fashion. PMID:21652652

Use of an activated beta-catenin to identify Wnt pathway target genes in caenorhabditis elegans, including a subset of collagen genes expressed in late larval development.

PubMed

Jackson, Belinda M; Abete-Luzi, Patricia; Krause, Michael W; Eisenmann, David M

2014-04-16

The Wnt signaling pathway plays a fundamental role during metazoan development, where it regulates diverse processes, including cell fate specification, cell migration, and stem cell renewal. Activation of the beta-catenin-dependent/canonical Wnt pathway up-regulates expression of Wnt target genes to mediate a cellular response. In the nematode Caenorhabditis elegans, a canonical Wnt signaling pathway regulates several processes during larval development; however, few target genes of this pathway have been identified. To address this deficit, we used a novel approach of conditionally activated Wnt signaling during a defined stage of larval life by overexpressing an activated beta-catenin protein, then used microarray analysis to identify genes showing altered expression compared with control animals. We identified 166 differentially expressed genes, of which 104 were up-regulated. A subset of the up-regulated genes was shown to have altered expression in mutants with decreased or increased Wnt signaling; we consider these genes to be bona fide C. elegans Wnt pathway targets. Among these was a group of six genes, including the cuticular collagen genes, bli-1 col-38, col-49, and col-71. These genes show a peak of expression in the mid L4 stage during normal development, suggesting a role in adult cuticle formation. Consistent with this finding, reduction of function for several of the genes causes phenotypes suggestive of defects in cuticle function or integrity. Therefore, this work has identified a large number of putative Wnt pathway target genes during larval life, including a small subset of Wnt-regulated collagen genes that may function in synthesis of the adult cuticle.

The Canonical Wnt Pathway Drives Macropinocytosis in Cancer.

PubMed

Redelman-Sidi, Gil; Binyamin, Anna; Gaeta, Isabella; Palm, Wilhelm; Thompson, Craig B; Romesser, Paul B; Lowe, Scott W; Bagul, Mukta; Doench, John G; Root, David E; Glickman, Michael S

2018-06-05

Macropinocytosis has emerged as an important pathway of protein acquisition in cancer cells, particularly in tumors with activated Ras such as pancreatic and colon cancer. Macropinocytosis is also the route of entry of Bacillus Calmette-Guerin (BCG) and other microbial therapies of cancer. Despite this important role in tumor biology and therapy, the full mechanisms by which cancer cells can activate macropinocytosis remain incompletely defined. Using BCG uptake to assay macropinocytosis, we executed a genome-wide shRNA screen for macropinocytosis activators and identified Wnt pathway activation as a strong driver of macropinocytosis. Wnt-driven macropinocytosis was downstream of the beta catenin-dependent canonical Wnt pathway, was Pak1 dependent, and supported albumin-dependent growth in Ras-WT cells. In cells with activated Ras-dependent macropinocytosis, pharmacologic or genetic inhibition of Wnt signaling suppressed macropinocytosis. In a mouse model of Wnt-driven colonic hyperplasia via APC silencing, Wnt-activated macropinocytosis stimulated uptake of luminal microbiota, a process reversed by topical pharmacologic inhibition of macropinocytosis. Our findings indicate that Wnt pathway activation drives macropinocytosis in cancer, and its inhibition could provide a therapeutic vulnerability in Wnt-driven intestinal polyposis and cancers with Wnt activation. Copyright ©2018, American Association for Cancer Research.

Inhibition of Wnt signaling induces amyloidogenic processing of amyloid precursor protein and the production and aggregation of Amyloid-β (Aβ)42 peptides.

PubMed

Tapia-Rojas, Cheril; Burgos, Patricia V; Inestrosa, Nibaldo C

2016-12-01

Alzheimer's disease (AD) is the most common neurodegenerative disorder and the most frequent cause of dementia in the aged population. According to the amyloid hypothesis, the amyloid-β (Aβ) peptide plays a key role in the pathogenesis of AD. Aβ is generated from the amyloidogenic processing of amyloid precursor protein and can aggregate to form oligomers, which have been described as a major synaptotoxic agent in neurons. Dysfunction of Wnt signaling has been linked to increased Aβ formation; however, several other studies have argued against this possibility. Herein, we use multiple experimental approaches to confirm that the inhibition of Wnt signaling promoted the amyloidogenic proteolytic processing of amyloid precursor protein. We also demonstrate that inhibiting Wnt signaling increases the production of the Aβ 42 peptide, the Aβ 42 /Aβ 40 ratio, and the levels of Aβ oligomers such as trimers and tetramers. Moreover, we show that activating Wnt signaling reduces the levels of Aβ 42 and its aggregates, increases Aβ 40 levels, and reduces the Aβ 42 /Aβ 40 ratio. Finally, we show that the protective effects observed in response to activation of the Wnt pathway rely on β-catenin-dependent transcription, which is demonstrated experimentally via the expression of various 'mutant forms of β-catenin'. Together, our findings indicate that loss of the Wnt signaling pathway may contribute to the pathogenesis of AD. © 2016 International Society for Neurochemistry.

Distinct requirements for cranial ectoderm and mesenchyme-derived wnts in specification and differentiation of osteoblast and dermal progenitors.

PubMed

Goodnough, L Henry; Dinuoscio, Gregg J; Ferguson, James W; Williams, Trevor; Lang, Richard A; Atit, Radhika P

2014-02-01

The cranial bones and dermis differentiate from mesenchyme beneath the surface ectoderm. Fate selection in cranial mesenchyme requires the canonical Wnt effector molecule β-catenin, but the relative contribution of Wnt ligand sources in this process remains unknown. Here we show Wnt ligands are expressed in cranial surface ectoderm and underlying supraorbital mesenchyme during dermal and osteoblast fate selection. Using conditional genetics, we eliminate secretion of all Wnt ligands from cranial surface ectoderm or undifferentiated mesenchyme, to uncover distinct roles for ectoderm- and mesenchyme-derived Wnts. Ectoderm Wnt ligands induce osteoblast and dermal fibroblast progenitor specification while initiating expression of a subset of mesenchymal Wnts. Mesenchyme Wnt ligands are subsequently essential during differentiation of dermal and osteoblast progenitors. Finally, ectoderm-derived Wnt ligands provide an inductive cue to the cranial mesenchyme for the fate selection of dermal fibroblast and osteoblast lineages. Thus two sources of Wnt ligands perform distinct functions during osteoblast and dermal fibroblast formation.

Cell intrinsic modulation of Wnt signaling controls neuroblast migration in C. elegans.

PubMed

Mentink, Remco A; Middelkoop, Teije C; Rella, Lorenzo; Ji, Ni; Tang, Chung Yin; Betist, Marco C; van Oudenaarden, Alexander; Korswagen, Hendrik C

2014-10-27

Members of the Wnt family of secreted signaling proteins are key regulators of cell migration and axon guidance. In the nematode C. elegans, the migration of the QR neuroblast descendants requires multiple Wnt ligands and receptors. We found that the migration of the QR descendants is divided into three sequential phases that are each mediated by a distinct Wnt signaling mechanism. Importantly, the transition from the first to the second phase, which is the main determinant of the final position of the QR descendants along the anteroposterior body axis, is mediated through a cell-autonomous process in which the time-dependent expression of a Wnt receptor turns on the canonical Wnt/β-catenin signaling response that is required to terminate long-range anterior migration. Our results show that, in addition to direct guidance of cell migration by Wnt morphogenic gradients, cell migration can also be controlled indirectly through cell-intrinsic modulation of Wnt signaling responses.

Effect of JNK inhibitor SP600125 on hair cell regeneration in zebrafish (Danio rerio) larvae

PubMed Central

Sun, Shaoyang; Wang, Xu; Li, Wenyan; Li, Huawei

2016-01-01

The c-Jun amino-terminal kinase (JNK) proteins are a subgroup of the mitogen-activated protein kinase family. They play a complex role in cell proliferation, survival, and apoptosis. Here, we report a novel role of JNK signalling in hair cell regeneration. We eliminated hair cells of 5-day post-fertilization zebrafish larvae using neomycin followed by JNK inhibition with SP600125. JNK inhibition strongly decreased the number of regenerated hair cells in response to neomycin damage. These changes were associated with reduced proliferation. JNK inhibition also increased cleaved caspase-3 activity and induced apoptosis in regenerating neuromasts. Finally, JNK inhibition with SP600125 decreased the expression of genes related to Wnt. Over-activation of the Wnt signalling pathway partly rescued the hair cell regeneration defects induced by JNK inhibition. Together, our findings provide novel insights into the function of JNK and show that JNK inhibition blocks hair cell regeneration by controlling the Wnt signalling pathway. PMID:27438150

SIRT1 suppresses adipogenesis by activating Wnt/β-catenin signaling in vivo and in vitro

PubMed Central

Zhou, Yuanfei; Song, Tongxing; Peng, Jie; Zhou, Zheng; Wei, Hongkui; Zhou, Rui; Jiang, Siwen; Peng, Jian

2016-01-01

Sirtuin 1 (SIRT1) regulates adipocyte and osteoblast differentiation. However, the underlying mechanism should be investigated. This study revealed that SIRT1 acts as a crucial repressor of adipogenesis. RNA-interference-mediated SIRT1 knockdown or genetic ablation enhances adipogenic potential, whereas SIRT1 overexpression inhibits adipogenesis in mesenchymal stem cells (MSCs). SIRT1 also deacetylates the histones of sFRP1, sFRP2, and Dact1 promoters; inhibits the mRNA expression of sFRP1, sFRP2, and Dact1; activates Wnt signaling pathways; and suppresses adipogenesis. SIRT1 deacetylates β-catenin to promote its accumulation in the nucleus and thus induces the transcription of genes that block MSC adipogenesis. In mice, the partial absence of SIRT1 promotes the formation of white adipose tissues without affecting the development of the body of mice. Our study described the regulatory role of SIRT1 in Wnt signaling and proposed a regulatory mechanism of adipogenesis. PMID:27776347

How the Wnt signaling pathway protects from neurodegeneration: the mitochondrial scenario

PubMed Central

Arrázola, Macarena S.; Silva-Alvarez, Carmen; Inestrosa, Nibaldo C.

2015-01-01

Alzheimer’s disease (AD) is the most common neurodegenerative disorder and is characterized by progressive memory loss and cognitive decline. One of the hallmarks of AD is the overproduction of amyloid-beta aggregates that range from the toxic soluble oligomer (Aβo) form to extracellular accumulations in the brain. Growing evidence indicates that mitochondrial dysfunction is a common feature of neurodegenerative diseases and is observed at an early stage in the pathogenesis of AD. Reports indicate that mitochondrial structure and function are affected by Aβo and can trigger neuronal cell death. Mitochondria are highly dynamic organelles, and the balance between their fusion and fission processes is essential for neuronal function. Interestingly, in AD, the process known as “mitochondrial dynamics” is also impaired by Aβo. On the other hand, the activation of the Wnt signaling pathway has an essential role in synaptic maintenance and neuronal functions, and its deregulation has also been implicated in AD. We have demonstrated that canonical Wnt signaling, through the Wnt3a ligand, prevents the permeabilization of mitochondrial membranes through the inhibition of the mitochondrial permeability transition pore (mPTP), induced by Aβo. In addition, we showed that non-canonical Wnt signaling, through the Wnt5a ligand, protects mitochondria from fission-fusion alterations in AD. These results suggest new approaches by which different Wnt signaling pathways protect neurons in AD, and support the idea that mitochondria have become potential therapeutic targets for the treatment of neurodegenerative disorders. Here we discuss the neuroprotective role of the canonical and non-canonical Wnt signaling pathways in AD and their differential modulation of mitochondrial processes, associated with mitochondrial dysfunction and neurodegeneration. PMID:25999816

Cloning and characterization of wnt4a gene and evidence for positive selection in half-smooth tongue sole (Cynoglossus semilaevis).

PubMed

Hu, Qiaomu; Zhu, Ying; Liu, Yang; Wang, Na; Chen, Songlin

2014-11-24

Wnt4 gene plays a role in developmental processes in mammals. However, little is known regarding its function in teleosts. We cloned and characterized the full-length half-smooth tongue sole (Cynoglossus semilaevis) wnt4a gene (CS-wnt4a). CS-wnt4a cDNA was 1746 bp in length encoding 353aa. CS-wnt4a expression level was highest in the testis, and gradually increased in the developing gonads until 1 year of age. In situ hybridization revealed that CS-wnt4a expression level was highest in stage II oocytes and sperm in the adult ovary and testis, respectively. CS-wnt4a expression level was significantly up-regulated in the gonads after exposure to high temperature. The level of methylation of the CS-wnt4a first exon was negatively correlated with the expression of CS-wnt4a. The branch-site model suggested that vertebrate wnt4a differed significantly from that of wnt4b, and that the selective pressures differed between ancestral aquatic and terrestrial organisms. Two positively selected sites were found in the ancestral lineages of teleost fish, but none in the ancestral lineages of mammals. One positively selected site was located on the α-helices of the 3D structure, the other on the random coil. Our results are of value for further study of the function of wnt4 and the mechanism of selection.

Cloning and characterization of wnt4a gene and evidence for positive selection in half-smooth tongue sole (Cynoglossus semilaevis)

NASA Astrophysics Data System (ADS)

Hu, Qiaomu; Zhu, Ying; Liu, Yang; Wang, Na; Chen, Songlin

2014-11-01

Wnt4 gene plays a role in developmental processes in mammals. However, little is known regarding its function in teleosts. We cloned and characterized the full-length half-smooth tongue sole (Cynoglossus semilaevis) wnt4a gene (CS-wnt4a). CS-wnt4a cDNA was 1746 bp in length encoding 353aa. CS-wnt4a expression level was highest in the testis, and gradually increased in the developing gonads until 1 year of age. In situ hybridization revealed that CS-wnt4a expression level was highest in stage II oocytes and sperm in the adult ovary and testis, respectively. CS-wnt4a expression level was significantly up-regulated in the gonads after exposure to high temperature. The level of methylation of the CS-wnt4a first exon was negatively correlated with the expression of CS-wnt4a. The branch-site model suggested that vertebrate wnt4a differed significantly from that of wnt4b, and that the selective pressures differed between ancestral aquatic and terrestrial organisms. Two positively selected sites were found in the ancestral lineages of teleost fish, but none in the ancestral lineages of mammals. One positively selected site was located on the α-helices of the 3D structure, the other on the random coil. Our results are of value for further study of the function of wnt4 and the mechanism of selection.

Rspo3 binds syndecan 4 and induces Wnt/PCP signaling via clathrin-mediated endocytosis to promote morphogenesis.

PubMed

Ohkawara, Bisei; Glinka, Andrei; Niehrs, Christof

2011-03-15

The R-Spondin (Rspo) family of secreted Wnt modulators is involved in development and disease and holds therapeutic promise as stem cell growth factors. Despite growing biological importance, their mechanism of action is poorly understood. Here, we show that Rspo3 binds syndecan 4 (Sdc4) and that together they activate Wnt/PCP signaling. In Xenopus embryos, Sdc4 and Rspo3 are essential for two Wnt/PCP-driven processes-gastrulation movements and head cartilage morphogenesis. Rspo3/PCP signaling during gastrulation requires Wnt5a and is transduced via Fz7, Dvl, and JNK. Rspo3 functions by inducing Sdc4-dependent, clathrin-mediated endocytosis. We show that this internalization is essential for PCP signal transduction, suggesting that endocytosis of Wnt-receptor complexes is a key mechanism by which R-spondins promote Wnt signaling. Copyright © 2011 Elsevier Inc. All rights reserved.

Nano Let‑7b sensitization of eliminating esophageal cancer stem‑like cells is dependent on blockade of Wnt activation of symmetric division.

PubMed

Pang, Yamei; Liu, Jian; Li, Xiang; Zhang, Yiwen; Zhang, Boxiang; Zhang, Jing; Du, Ning; Xu, Chongwen; Liang, Rui; Ren, Hong; Tang, Shou-Ching; Sun, Xin

2017-10-01

The poor therapy response and poor prognosis of esophageal cancer has made it one of the most malignant carcinoma, and the complicated multidisciplinary treatment failed to achieve a long-term disease-free survival. To diagnose esophageal cancer at an earlier stage, and to improve the effect of anticancer therapy would improve the therapeutic efficacy. After retrospective analysis of the cancer samples of patients who received esophagectomy, we found the relevance between ratio of either ALDH1 or CD133-positive cancer stem cells and 2-year recurrence. Higher ratios of cancer stem cells indicated later clinical stages, and Wnt signaling activation was more frequent in later esophageal carcinoma. Further in bench studies, we explored the suppressive roles and the mechanisms involved in Let‑7 on self-renewal in ECA‑109 and ECA‑9706 esophageal cancer stem cells. Isolated cancer stem cells naturally divide symmetrically and are therapy resistant. Therapy of fluorouracil and docetaxel both enriched the stem cells, proving the resistant characteristics of cancer stem cells. Wnt activation stimulated more symmetric division of stem cells, resulting in self-renewal promotion, which could be blocked by Let‑7 overexpression. Furthermore, enforced Let‑7 sensitized the stem cells to chemotherapies in a Wnt pathway inhibition-dependent manner, contributing to Let‑7 sensitization of chemotherapeutic response. Wnt activation weakened the suppressive Let‑7b through the sponge functions of CCAT-1, forming the negative feedback loop of Let‑7b/Wnt/CCAT1. These results identified the crucial participation of stem cells in esophageal cancer occurrence and progression as the potent indicator, and also indicate the potential powerful agent of Let‑7 nano-particles in treatment of cancer.

Dynamic association with donor cell filopodia and lipid-modification are essential features of Wnt8a during patterning of the zebrafish neuroectoderm.

PubMed

Luz, Marta; Spannl-Müller, Stephanie; Özhan, Günes; Kagermeier-Schenk, Birgit; Rhinn, Muriel; Weidinger, Gilbert; Brand, Michael

2014-01-01

Wnt proteins are conserved signaling molecules that regulate pattern formation during animal development. Many Wnt proteins are post-translationally modified by addition of lipid adducts. Wnt8a provides a crucial signal for patterning the anteroposterior axis of the developing neural plate in vertebrates. However, it is not clear how this protein propagates from its source, the blastoderm margin, to the target cells in the prospective neural plate, and how lipid-modifications might influence Wnt8a propagation and activity. We have dynamically imaged biologically active, fluorescently tagged Wnt8a in living zebrafish embryos. We find that Wnt8a localizes to membrane-associated, punctate structures in live tissue. In Wnt8a expressing cells, these puncta are found on filopodial cellular processes, from where the protein can be released. In addition, Wnt8a is found colocalized with Frizzled receptor-containing clusters on signal receiving cells. Combining in vitro and in vivo assays, we compare the roles of conserved Wnt8a residues in cell and non-cell-autonomous signaling activity and secretion. Non-signaling Wnt8 variants show these residues can regulate Wnt8a distribution in producing cell membranes and filopodia as well as in the receiving tissue. Together, our results show that Wnt8a forms dynamic clusters found on filopodial donor cell and on signal receiving cell membranes. Moreover, they demonstrate a differential requirement of conserved residues in Wnt8a protein for distribution in producing cells and receiving tissue and signaling activity during neuroectoderm patterning.

Dynamic Association with Donor Cell Filopodia and Lipid-Modification Are Essential Features of Wnt8a during Patterning of the Zebrafish Neuroectoderm

PubMed Central

Luz, Marta; Spannl-Müller, Stephanie; Özhan, Günes; Kagermeier-Schenk, Birgit; Rhinn, Muriel; Weidinger, Gilbert; Brand, Michael

2014-01-01

Background Wnt proteins are conserved signaling molecules that regulate pattern formation during animal development. Many Wnt proteins are post-translationally modified by addition of lipid adducts. Wnt8a provides a crucial signal for patterning the anteroposterior axis of the developing neural plate in vertebrates. However, it is not clear how this protein propagates from its source, the blastoderm margin, to the target cells in the prospective neural plate, and how lipid-modifications might influence Wnt8a propagation and activity. Results We have dynamically imaged biologically active, fluorescently tagged Wnt8a in living zebrafish embryos. We find that Wnt8a localizes to membrane-associated, punctate structures in live tissue. In Wnt8a expressing cells, these puncta are found on filopodial cellular processes, from where the protein can be released. In addition, Wnt8a is found colocalized with Frizzled receptor-containing clusters on signal receiving cells. Combining in vitro and in vivo assays, we compare the roles of conserved Wnt8a residues in cell and non-cell-autonomous signaling activity and secretion. Non-signaling Wnt8 variants show these residues can regulate Wnt8a distribution in producing cell membranes and filopodia as well as in the receiving tissue. Conclusions Together, our results show that Wnt8a forms dynamic clusters found on filopodial donor cell and on signal receiving cell membranes. Moreover, they demonstrate a differential requirement of conserved residues in Wnt8a protein for distribution in producing cells and receiving tissue and signaling activity during neuroectoderm patterning. PMID:24427298

The Wnt signaling pathway in familial exudative vitreoretinopathy and Norrie disease.

PubMed

Warden, Scott M; Andreoli, Christopher M; Mukai, Shizuo

2007-01-01

The Wnt signaling pathway is highly conserved among species and has an important role in many cell biological processes throughout the body. This signaling cascade is involved in regulating ocular growth and development, and recent findings indicate that this is particularly true in the retina. Mutations involving different aspects of the Wnt signaling pathway are being linked to several diseases of retinal development. The aim of this article is to first review the Wnt signaling pathway. We will then describe two conditions, familial exudative vitreoretinopathy (FEVR) and Norrie disease (ND), which have been shown to be caused in part by defects in the Wnt signaling cascade.

Early planarian brain regeneration is independent of blastema polarity mediated by the Wnt/β-catenin pathway.

PubMed

Iglesias, Marta; Almuedo-Castillo, Maria; Aboobaker, A Aziz; Saló, Emili

2011-10-01

Analysis of anteroposterior (AP) axis specification in regenerating planarian flatworms has shown that Wnt/β-catenin signaling is required for posterior specification and that the FGF-like receptor molecule nou-darake (ndk) may be involved in restricting brain regeneration to anterior regions. The relationship between re-establishment of AP identity and correct morphogenesis of the brain is, however, still poorly understood. Here we report the characterization of two axin paralogs in the planarian Schmidtea mediterranea. Although Axins are well known negative regulators of Wnt/β-catenin signaling, no role in AP specification has previously been reported for axin genes in planarians. We show that silencing of Smed-axin genes by RNA interference (RNAi) results in two-tailed planarians, a phenotype previously reported after silencing of Smed-APC-1, another β-catenin inhibitor. More strikingly, we show for the first time that while early brain formation at anterior wounds remains unaffected, subsequent development of the brain is blocked in the two-tailed planarians generated after silencing of Smed-axin genes and Smed-APC-1. These findings suggest that the mechanisms underlying early brain formation can be uncoupled from the specification of AP identity by the Wnt/β-catenin pathway. Finally, the posterior expansion of the brain observed following Smed-ndk RNAi is enhanced by silencing Smed-APC-1, revealing an indirect relationship between the FGFR/Ndk and Wnt/β-catenin signaling systems in establishing the posterior limits of brain differentiation. Copyright © 2011 Elsevier Inc. All rights reserved.

An Epha4/Sipa1l3/Wnt pathway regulates eye development and lens maturation.

PubMed

Rothe, Melanie; Kanwal, Noreen; Dietmann, Petra; Seigfried, Franziska A; Hempel, Annemarie; Schütz, Desiree; Reim, Dominik; Engels, Rebecca; Linnemann, Alexander; Schmeisser, Michael J; Bockmann, Juergen; Kühl, Michael; Boeckers, Tobias M; Kühl, Susanne J

2017-01-15

The signal-induced proliferation-associated family of proteins comprises four members, SIPA1 and SIPA1L1-3. Mutations of the human SIPA1L3 gene result in congenital cataracts. In Xenopus, loss of Sipa1l3 function led to a severe eye phenotype that was distinguished by smaller eyes and lenses including lens fiber cell maturation defects. We found a direct interaction between Sipa1l3 and Epha4, building a functional platform for proper ocular development. Epha4 deficiency phenocopied loss of Sipa1l3 and rescue experiments demonstrated that Epha4 acts upstream of Sipa1l3 during eye development, with both Sipa1l3 and Epha4 required for early eye specification. The ocular phenotype, upon loss of either Epha4 or Sipa1l3, was partially mediated by rax We demonstrate that canonical Wnt signaling is inhibited downstream of Epha4 and Sipa1l3 during normal eye development. Depletion of either Sipa1l3 or Epha4 resulted in an upregulation of axin2 expression, a direct Wnt/β-catenin target gene. In line with this, Sipa1l3 or Epha4 depletion could be rescued by blocking Wnt/β-catenin or activating non-canonical Wnt signaling. We therefore conclude that this pathomechanism prevents proper eye development and maturation of lens fiber cells, resulting in congenital cataracts. © 2017. Published by The Company of Biologists Ltd.

LncRNA MEG3 repressed malignant melanoma progression via inactivating Wnt signaling pathway.

PubMed

Li, Peng; Gao, Ying; Li, Jun; Zhou, Yu; Yuan, Jing; Guan, Huiwen; Yao, Peng

2018-05-21

Accumulating evidence has indicated that MEG3 can serve as a tumor suppressive lncRNA in various tumors. It is aberrantly expressed in multiple cancers. However, the biological roles of MEG3 in melanoma are poorly understood. Therefore, in our study, we concentrated on the biological mechanism of MEG3 in melanoma progression. First, we observed that MEG3 was obviously decreased in melanoma cells including A375, SK-MEL-1, B16, and A2058 cells compared to human epidermal melanocytes HEMa-LP. MEG3 was restored by transfecting LV-MEG3 in to A375 and A2058 cells. Subsequently, we found that overexpression of MEG3 was able to inhibit cell proliferation and colony formation capacity. Meanwhile, melanoma cell apoptosis was induced by up-regulation of MEG3. Overexpression of MEG3 greatly repressed melanoma cell migration and invasion ability. In addition, Wnt signaling pathway has been identified in the progression of various cancers. Here, in our study, it was indicated that Wnt signaling was highly activated in melanoma cells with β-catenin expression significantly increased and GSK-3β decreased. Interestingly, MEG restoration strongly inactivated Wnt signaling pathway by reducing β-catenin and CyclinD1, elevating GSK-3β levels in vitro. Finally, in vivo experiments were carried out to confirm the inhibitory roles of MEG3 in vivo. Taken these together, we suggested that MEG3 can inhibit melanoma development through blocking Wnt signaling pathway. © 2018 Wiley Periodicals, Inc.

Enhanced Renal Afferent Arteriolar Reactive Oxygen Species and Contractility to Endothelin-1 Are Associated with Canonical Wnt Signaling in Diabetic Mice.

PubMed

Zhang, Suping; Huang, Qian; Wang, Qiaoling; Wang, Qin; Cao, Xiaoyun; Zhao, Liang; Xu, Nan; Zhuge, Zhengbing; Mao, Jianhua; Fu, Xiaodong; Liu, Ruisheng; Wilcox, Christopher S; Patzak, Andreas; Li, Lingli; Lai, En Yin

2018-05-30

Canonical Wnt signaling is involved in oxidative stress, vasculopathy and diabetes mellitus but its role in diabetic renal microvascular dysfunction is unclear. We tested the hypothesis that enhanced canonical Wnt signaling in renal afferent arterioles from diabetic mice increases reactive oxygen species (ROS) and contractions to endothelin-1 (ET-1). Streptozotocin-induced diabetes or control C57Bl/6 mice received vehicle or sulindac (40 mg·kg-1·day-1) to block Wnt signaling for 4 weeks. ET-1 contractions were measured by changes of afferent arteriolar diameter. Arteriolar H2O2, O2 -, protein expression and enzymatic activity were assessed using sensitive fluorescence probes, immunoblotting and colorimetric assay separately. Compared to control, diabetic mouse afferent arteriole had increased O2- (+ 84%) and H2O2 (+ 91%) and enhanced responses to ET-1 at 10-8 mol·l-1 (-72±4% of versus -43±4%, P< 0.05) accompanied by reduced protein expressions and activities for catalase and superoxide dismutase 2 (SOD2). Arteriolar O2 - was increased further by ET-1 and contractions to ET-1 reduced by PEG-SOD in both groups whereas H2O2 unchanged by ET-1 and contractions were reduced by PEG-catalase selectively in diabetic mice. The Wnt signaling protein β-catenin was upregulated (3.3-fold decrease in p-β-catenin/β-catenin) while the glycogen synthase kinase-3β (GSK-3β) was downregulated (2.6-fold increase in p-GSK-3β/ GSK-3β) in preglomerular vessels of diabetic mice. Sulindac normalized the Wnt signaling proteins, arteriolar O2 -, H2O2 and ET-1 contractions while doubling microvascular catalase and SOD2 expression in diabetic mice. Increased ROS, notably H2O2 contributes to enhanced afferent arteriolar responses to ET-1 in diabetes, which is closely associated with Wnt signaling. Antioxidant pharmacological strategies targeting Wnt signaling may improve vascular function in diabetic nephropathy. © 2018 The Author(s). Published by S. Karger AG, Basel.

Direct Interactions Between Gli3, Wnt8b, and Fgfs Underlie Patterning of the Dorsal Telencephalon.

PubMed

Hasenpusch-Theil, Kerstin; Watson, Julia A; Theil, Thomas

2017-02-01

A key step in the development of the cerebral cortex is a patterning process, which subdivides the telencephalon into several molecularly distinct domains and is critical for cortical arealization. This process is dependent on a complex network of interactions between signaling molecules of the Fgf and Wnt gene families and the Gli3 transcription factor gene, but a better knowledge of the molecular basis of the interplay between these factors is required to gain a deeper understanding of the genetic circuitry underlying telencephalic patterning. Using DNA-binding and reporter gene assays, we here investigate the possibility that Gli3 and these signaling molecules interact by directly regulating each other's expression. We show that Fgf signaling is required for Wnt8b enhancer activity in the cortical hem, whereas Wnt/β-catenin signaling represses Fgf17 forebrain enhancer activity. In contrast, Fgf and Wnt/β-catenin signaling cooperate to regulate Gli3 expression. Taken together, these findings indicate that mutual interactions between Gli3, Wnt8b, and Fgf17 are crucial elements of the balance between these factors thereby conferring robustness to the patterning process. Hence, our study provides a framework for understanding the genetic circuitry underlying telencephalic patterning and how defects in this process can affect the formation of cortical areas. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Apcdd1 is a novel Wnt inhibitor Mutated in Hereditary Hypotrichosis Simplex

PubMed Central

Shimomura, Yutaka; Agalliu, Dritan; Vonica, Alin; Luria, Victor; Wajid, Muhammad; Baumer, Alessandra; Belli, Serena; Petukhova, Lynn; Schinzel, Albert; Brivanlou, Ali H.; Barres, Ben A.; Christiano, Angela M.

2011-01-01

Hereditary hypotrichosis simplex (HHS) is a rare autosomal dominant form of hair loss characterized by hair follicle (HF) miniaturization1, 2. Using genetic linkage analysis, we mapped a novel locus for HHS to chromosome 18p11.22, and identified a mutation (L9R) in the APCDD1 gene in three families. We show that APCDD1 is a membrane-bound glycoprotein that is abundantly expressed in human HFs, and can interact in vitro with WNT3A and LRP5, two essential components of Wnt signaling. Functional studies revealed that APCDD1 inhibits Wnt signaling in a cell-autonomous manner and functions upstream of β-catenin. Moreover, APCDD1 represses activation of Wnt reporters and target genes, and inhibits the biological effects of Wnt signaling during both the generation of neurons from progenitors in the developing chick nervous system, and axis specification in Xenopus embryos. The mutation L9R is located in the signal peptide of APCDD1, and perturbs its translational processing from ER to the plasma membrane. L9R-APCDD1 likely functions in a dominant-negative manner to inhibit the stability and membrane localization of the wild-type protein. These findings describe a novel inhibitor of the Wnt signaling pathway with an essential role in human hair growth. Since APCDD1 is expressed in a broad repertoire of cell types3, our findings suggest that APCDD1 may regulate a diversity of biological processes controlled by Wnt signaling. PMID:20393562

Wnt5a inhibits K(+) currents in hippocampal synapses through nitric oxide production.

PubMed

Parodi, Jorge; Montecinos-Oliva, Carla; Varas, Rodrigo; Alfaro, Iván E; Serrano, Felipe G; Varas-Godoy, Manuel; Muñoz, Francisco J; Cerpa, Waldo; Godoy, Juan A; Inestrosa, Nibaldo C

2015-09-01

Hippocampal synapses play a key role in memory and learning processes by inducing long-term potentiation and depression. Wnt signaling is essential in the development and maintenance of synapses via several mechanisms. We have previously found that Wnt5a induces the production of nitric oxide (NO), which modulates NMDA receptor expression in the postsynaptic regions of hippocampal neurons. Here, we report that Wnt5a selectively inhibits a voltage-gated K(+) current (Kv current) and increases synaptic activity in hippocampal slices. Further supporting a specific role for Wnt5a, the soluble Frizzled receptor protein (sFRP-2; a functional Wnt antagonist) fully inhibits the effects of Wnt5a. We additionally show that these responses to Wnt5a are mediated by activation of a ROR2 receptor and increased NO production because they are suppressed by the shRNA-mediated knockdown of ROR2 and by 7-nitroindazole, a specific inhibitor of neuronal NOS. Together, our results show that Wnt5a increases NO production by acting on ROR2 receptors, which in turn inhibit Kv currents. These results reveal a novel mechanism by which Wnt5a may regulate the excitability of hippocampal neurons. Copyright © 2015 Elsevier Inc. All rights reserved.

Interrelationship of canonical and non-canonical Wnt signalling pathways in chronic metabolic diseases.

PubMed

Ackers, Ian; Malgor, Ramiro

2018-01-01

Chronic diseases account for approximately 45% of all deaths in developed countries and are particularly prevalent in countries with the most sophisticated and robust public health systems. Chronic metabolic diseases, specifically lifestyle-related diseases pertaining to diet and exercise, continue to be difficult to treat clinically. The most prevalent of these chronic metabolic diseases include obesity, diabetes, non-alcoholic fatty liver disease, chronic kidney disease and cardiovascular disease and will be the focus of this review. Wnt proteins are highly conserved glycoproteins best known for their role in development and homeostasis of tissues. Given the importance of Wnt signalling in homeostasis, aberrant Wnt signalling likely regulates metabolic processes and may contribute to the development of chronic metabolic diseases. Expression of Wnt proteins and dysfunctional Wnt signalling has been reported in multiple chronic diseases. It is interesting to speculate about an interrelationship between the Wnt signalling pathways as a potential pathological mechanism in chronic metabolic diseases. The aim of this review is to summarize reported findings on the contrasting roles of Wnt signalling in lifestyle-related chronic metabolic diseases; specifically, the contribution of Wnt signalling to lipid accumulation, fibrosis and chronic low-grade inflammation.

Compartmentalization of the somite and myogenesis in chick embryos are influenced by wnt expression.

PubMed

Wagner, J; Schmidt, C; Nikowits, W; Christ, B

2000-12-01

Muscles of the body and bones of the axial skeleton derive from specialized regions of somites. Somite development is influenced by adjacent structures. In particular, the dorsal neural tube and the overlying ectoderm have been shown to be necessary for the induction of myogenic precursor cells in the dermomyotome. Members of the Wnt family of signaling molecules, which are expressed in the dorsal neural tube and the ectoderm, are postulated to be responsible for this process. It is shown here that ectopically implanted Wnt-1-, -3a-, and -4-expressing cells alter the process of somite compartmentalization in vivo. An enlarged dorsal compartment results from the implantation of Wnt-expressing cells ventrally between the neural tube/notochord and epithelial somites, at the expense of the ventral compartment, the sclerotome. Thus, ectopic Wnt expression is able to override the influence of ventralizing signals arising from notochord and floor plate. This shift of the border between the two compartments was identified by an increase in the domain of Pax-3 expression and a complete loss of Pax-1 expression in somites close to the ectopic Wnt signal. The expanded expression of MyoD and desmin provides evidence that it is the myotome which increases as a result of Wnt signaling. Paraxis expression is also drastically amplified after implantation of Wnt-expressing cells indicating that Wnts are involved in the formation and maintenance of somite epithelium and suggesting that Paraxis is activated through Wnt signaling pathways. Taken together these results suggest that ectopic Wnts disturb the normal balance of signaling molecules within the somite, resulting in an enhanced recruitment of somitic cells into the myogenic lineage. Copyright 2000 Academic Press.

Wnt signaling-mediated redox regulation maintains the germ line stem cell differentiation niche

PubMed Central

Wang, Su; Gao, Yuan; Song, Xiaoqing; Ma, Xing; Zhu, Xiujuan; Mao, Ying; Yang, Zhihao; Ni, Jianquan; Li, Hua; Malanowski, Kathryn E; Anoja, Perera; Park, Jungeun; Haug, Jeff; Xie, Ting

2015-01-01

Adult stem cells continuously undergo self-renewal and generate differentiated cells. In the Drosophila ovary, two separate niches control germ line stem cell (GSC) self-renewal and differentiation processes. Compared to the self-renewing niche, relatively little is known about the maintenance and function of the differentiation niche. In this study, we show that the cellular redox state regulated by Wnt signaling is critical for the maintenance and function of the differentiation niche to promote GSC progeny differentiation. Defective Wnt signaling causes the loss of the differentiation niche and the upregulated BMP signaling in differentiated GSC progeny, thereby disrupting germ cell differentiation. Mechanistically, Wnt signaling controls the expression of multiple glutathione-S-transferase family genes and the cellular redox state. Finally, Wnt2 and Wnt4 function redundantly to maintain active Wnt signaling in the differentiation niche. Therefore, this study has revealed a novel strategy for Wnt signaling in regulating the cellular redox state and maintaining the differentiation niche. DOI: http://dx.doi.org/10.7554/eLife.08174.001 PMID:26452202

Wnt signaling inhibits cementoblast differentiation and promotes proliferation.

PubMed

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

2009-05-01

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

Smed-Evi/Wntless is required for beta-catenin-dependent and -independent processes during planarian regeneration.

PubMed

Adell, Teresa; Salò, Emili; Boutros, Michael; Bartscherer, Kerstin

2009-03-01

Planarians can regenerate a whole animal from only a small piece of their body, and have become an important model for stem cell biology. To identify regenerative processes dependent on Wnt growth factors in the planarian Schmidtea mediterranea (Smed), we analyzed RNAi phenotypes of Evi, a transmembrane protein specifically required for the secretion of Wnt ligands. We show that, during regeneration, Smed-evi loss-of-function prevents posterior identity, leading to two-headed planarians that resemble Smed-beta-catenin1 RNAi animals. In addition, we observe regeneration defects of the nervous system that are not found after Smed-beta-catenin1 RNAi. By systematic knockdown of all putative Smed Wnts in regenerating planarians, we identify Smed-WntP-1 and Smed-Wnt11-2 as the putative posterior organizers, and demonstrate that Smed-Wnt5 is a regulator of neuronal organization and growth. Thus, our study provides evidence that planarian Wnts are major regulators of regeneration, and that they signal through beta-catenin-dependent and -independent pathways.

WNT4 signaling in female gonadal development.

PubMed

Pellegrino, Miriam; Maiorino, Raffaella; Schonauer, Sergio

2010-06-01

WNT4 signaling pathways represent an important step in the multi-faceted process of mammalian gonadal differentiation and the development of internal genitalia. WNT4 protein controls the cytoplasmatic stability of specific transcriptional coactivator beta catenin during both embriogenesis and adult homeostasis. The biological significance of WNT4 consists in determining the final female reproductive system, inhibiting Wolff ducts' differentiation, male steroidogenesis and vascular cell migration. An overview of WNT4 cellular mechanisms is given in order to understand its critical role in the genesis of various human diseases such as congenital malformations and gynecological disorders like polycystic ovary syndrome (PCOS). The final discussion focusses on several possible therapeutic uses of Wnt4 both during pregnancy in order to correct the genetic loss of function of the protein and during adulthood in order to normalize fertility in PCOS-affected females planning pregnancy.

A linear model of population dynamics

NASA Astrophysics Data System (ADS)

Lushnikov, A. A.; Kagan, A. I.

2016-08-01

The Malthus process of population growth is reformulated in terms of the probability w(n,t) to find exactly n individuals at time t assuming that both the birth and the death rates are linear functions of the population size. The master equation for w(n,t) is solved exactly. It is shown that w(n,t) strongly deviates from the Poisson distribution and is expressed in terms either of Laguerre’s polynomials or a modified Bessel function. The latter expression allows for considerable simplifications of the asymptotic analysis of w(n,t).

Non-equivalence of Wnt and R-spondin ligands during Lgr5+ intestinal stem-cell self-renewal.

PubMed

Yan, Kelley S; Janda, Claudia Y; Chang, Junlei; Zheng, Grace X Y; Larkin, Kathryn A; Luca, Vincent C; Chia, Luis A; Mah, Amanda T; Han, Arnold; Terry, Jessica M; Ootani, Akifumi; Roelf, Kelly; Lee, Mark; Yuan, Jenny; Li, Xiao; Bolen, Christopher R; Wilhelmy, Julie; Davies, Paige S; Ueno, Hiroo; von Furstenberg, Richard J; Belgrader, Phillip; Ziraldo, Solongo B; Ordonez, Heather; Henning, Susan J; Wong, Melissa H; Snyder, Michael P; Weissman, Irving L; Hsueh, Aaron J; Mikkelsen, Tarjei S; Garcia, K Christopher; Kuo, Calvin J

2017-05-11

The canonical Wnt/β-catenin signalling pathway governs diverse developmental, homeostatic and pathological processes. Palmitoylated Wnt ligands engage cell-surface frizzled (FZD) receptors and LRP5 and LRP6 co-receptors, enabling β-catenin nuclear translocation and TCF/LEF-dependent gene transactivation. Mutations in Wnt downstream signalling components have revealed diverse functions thought to be carried out by Wnt ligands themselves. However, redundancy between the 19 mammalian Wnt proteins and 10 FZD receptors and Wnt hydrophobicity have made it difficult to attribute these functions directly to Wnt ligands. For example, individual mutations in Wnt ligands have not revealed homeostatic phenotypes in the intestinal epithelium-an archetypal canonical, Wnt pathway-dependent, rapidly self-renewing tissue, the regeneration of which is fueled by proliferative crypt Lgr5 + intestinal stem cells (ISCs). R-spondin ligands (RSPO1-RSPO4) engage distinct LGR4-LGR6, RNF43 and ZNRF3 receptor classes, markedly potentiate canonical Wnt/β-catenin signalling, and induce intestinal organoid growth in vitro and Lgr5 + ISCs in vivo. However, the interchangeability, functional cooperation and relative contributions of Wnt versus RSPO ligands to in vivo canonical Wnt signalling and ISC biology remain unknown. Here we identify the functional roles of Wnt and RSPO ligands in the intestinal crypt stem-cell niche. We show that the default fate of Lgr5 + ISCs is to differentiate, unless both RSPO and Wnt ligands are present. However, gain-of-function studies using RSPO ligands and a new non-lipidated Wnt analogue reveal that these ligands have qualitatively distinct, non-interchangeable roles in ISCs. Wnt proteins are unable to induce Lgr5 + ISC self-renewal, but instead confer a basal competency by maintaining RSPO receptor expression that enables RSPO ligands to actively drive and specify the extent of stem-cell expansion. This functionally non-equivalent yet cooperative interaction between Wnt and RSPO ligands establishes a molecular precedent for regulation of mammalian stem cells by distinct priming and self-renewal factors, with broad implications for precise control of tissue regeneration.

Molecular genetics and targeted therapy of WNT-related human diseases (Review)

PubMed Central

Katoh, Masuko; Katoh, Masaru

2017-01-01

Canonical WNT signaling through Frizzled and LRP5/6 receptors is transduced to the WNT/β-catenin and WNT/stabilization of proteins (STOP) signaling cascades to regulate cell fate and proliferation, whereas non-canonical WNT signaling through Frizzled or ROR receptors is transduced to the WNT/planar cell polarity (PCP), WNT/G protein-coupled receptor (GPCR) and WNT/receptor tyrosine kinase (RTK) signaling cascades to regulate cytoskeletal dynamics and directional cell movement. WNT/β-catenin signaling cascade crosstalks with RTK/SRK and GPCR-cAMP-PKA signaling cascades to regulate β-catenin phosphorylation and β-catenin-dependent transcription. Germline mutations in WNT signaling molecules cause hereditary colorectal cancer, bone diseases, exudative vitreoretinopathy, intellectual disability syndrome and PCP-related diseases. APC or CTNNB1 mutations in colorectal, endometrial and prostate cancers activate the WNT/β-catenin signaling cascade. RNF43, ZNRF3, RSPO2 or RSPO3 alterations in breast, colorectal, gastric, pancreatic and other cancers activate the WNT/β-catenin, WNT/STOP and other WNT signaling cascades. ROR1 upregulation in B-cell leukemia and solid tumors and ROR2 upregulation in melanoma induce invasion, metastasis and therapeutic resistance through Rho-ROCK, Rac-JNK, PI3K-AKT and YAP signaling activation. WNT signaling in cancer, stromal and immune cells dynamically orchestrate immune evasion and antitumor immunity in a cell context-dependent manner. Porcupine (PORCN), RSPO3, WNT2B, FZD5, FZD10, ROR1, tankyrase and β-catenin are targets of anti-WNT signaling therapy, and ETC-159, LGK974, OMP-18R5 (vantictumab), OMP-54F28 (ipafricept), OMP-131R10 (rosmantuzumab), PRI-724 and UC-961 (cirmtuzumab) are in clinical trials for cancer patients. Different classes of anti-WNT signaling therapeutics are necessary for the treatment of APC/CTNNB1-, RNF43/ZNRF3/RSPO2/RSPO3- and ROR1-types of human cancers. By contrast, Dickkopf-related protein 1 (DKK1), SOST and glycogen synthase kinase 3β (GSK3β) are targets of pro-WNT signaling therapy, and anti-DKK1 (BHQ880 and DKN-01) and anti-SOST (blosozumab, BPS804 and romosozumab) monoclonal antibodies are being tested in clinical trials for cancer patients and osteoporotic post-menopausal women. WNT-targeting therapeutics have also been applied as reagents for in vitro stem-cell processing in the field of regenerative medicine. PMID:28731148

Pax8 modulates the expression of Wnt4 that is necessary for the maintenance of the epithelial phenotype of thyroid cells

PubMed Central

2014-01-01

Background The transcription factor Pax8 is expressed during thyroid development and is involved in the morphogenesis of the thyroid gland and maintenance of the differentiated phenotype. In particular, Pax8 has been shown to regulate genes that are considered markers of thyroid differentiation. Recently, the analysis of the gene expression profile of FRTL-5 differentiated thyroid cells after the silencing of Pax8 identified Wnt4 as a novel target. Like the other members of the Wnt family, Wnt4 has been implicated in several developmental processes including regulation of cell fate and patterning during embryogenesis. To date, the only evidence on Wnt4 in thyroid concerns its down-regulation necessary for the progression of thyroid epithelial tumors. Results Here we demonstrate that Pax8 is involved in the transcriptional modulation of Wnt4 gene expression directly binding to its 5’-flanking region, and that Wnt4 expression in FRTL-5 cells is TSH-dependent. Interestingly, we also show that in thyroid cells a reduced expression of Wnt4 correlates with the alteration of the epithelial phenotype and that the overexpression of Wnt4 in thyroid cancer cells is able to inhibit cellular migration. Conclusions We have identified and characterized a functional Pax8 binding site in the 5’-flanking region of the Wnt4 gene and we show that Pax8 modulates the expression of Wnt4 in thyroid cells. Taken together, our results suggest that in thyroid cells Wnt4 expression correlates with the integrity of the epithelial phenotype and is reduced when this integrity is perturbed. In the end, we would like to suggest that the overexpression of Wnt4 in thyroid cancer cells is able to revert the mesenchymal phenotype. PMID:25270402

Effect of Wnt-1 inducible signaling pathway protein-2 (WISP-2/CCN5), a downstream protein of Wnt signaling, on adipocyte differentiation

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

Inadera, Hidekuni; Shimomura, Akiko; Tachibana, Shinjiro

2009-02-20

Wnt signaling negatively regulates adipocyte differentiation, and ectopic expression of Wnt-1 in 3T3-L1 cells induces several downstream molecules of Wnt signaling, including Wnt-1 inducible signaling pathway protein (WISP)-2. In this study, we examined the role of WISP-2 in the process of adipocyte differentiation using an in vitro cell culture system. In the differentiation of 3T3-L1 cells, WISP-2 expression was observed in growing cells and declined thereafter. In the mitotic clonal expansion phase of adipocyte differentiation, WISP-2 expression was transiently down-regulated concurrently with up-regulation of CCAAT/enhancer-binding protein {delta} expression. Treatment of 3T3-L1 cells in the differentiation medium with lithium, an activatormore » of Wnt signaling, inhibited the differentiation process with concomitant induction of WISP-2. Treatment of differentiated cells with lithium induced de-differentiation as evidenced by profound reduction of peroxisome proliferator-activator receptor {gamma} expression and concomitant induction of WISP-2. However, de-differentiation of differentiated cells induced by tumor necrosis factor-{alpha} did not induce WISP-2 expression. To directly examine the effect of WISP-2 on adipocyte differentiation, 3T3-L1 cells were infected with a retrovirus carrying WISP-2. Although forced expression of WISP-2 inhibited preadipocyte proliferation, it had no effect on adipocyte differentiation. Thus, although WISP-2 is a downstream protein of Wnt signaling, the role of WISP-2 on adipocyte differentiation may be marginal, at least in this in vitro culture model.« less

Role of Wnt Signaling in the Control of Adult Hippocampal Functioning in Health and Disease: Therapeutic Implications

PubMed Central

Ortiz-Matamoros, Abril; Salcedo-Tello, Pamela; Avila-Muñoz, Evangelina; Zepeda, Angélica; Arias, Clorinda

2013-01-01

It is well recognized the role of the Wnt pathway in many developmental processes such as neuronal maturation, migration, neuronal connectivity and synaptic formation. Growing evidence is also demonstrating its function in the mature brain where is associated with modulation of axonal remodeling, dendrite outgrowth, synaptic activity, neurogenesis and behavioral plasticity. Proteins involved in Wnt signaling have been found expressed in the adult hippocampus suggesting that Wnt pathway plays a role in the hippocampal function through life. Indeed, Wnt ligands act locally to regulate neurogenesis, neuronal cell shape and pre- and postsynaptic assembly, events that are thought to underlie changes in synaptic function associated with long-term potentiation and with cognitive tasks such as learning and memory. Recent data have demonstrated the increased expression of the Wnt antagonist Dickkopf-1 (DKK1) in brains of Alzheimer´s disease (AD) patients suggesting that dysfunction of Wnt signaling could also contribute to AD pathology. We review here evidence of Wnt-associated molecules expression linked to physiological and pathological hippocampal functioning in the adult brain. The basic aspects of Wnt related mechanisms underlying hippocampal plasticity as well as evidence of how hippocampal dysfunction may rely on Wnt dysregulation is analyzed. This information would provide some clues about the possible therapeutic targets for developing treatments for neurodegenerative diseases associated with aberrant brain plasticity. PMID:24403870

Role of wnt signaling in the control of adult hippocampal functioning in health and disease: therapeutic implications.

PubMed

Ortiz-Matamoros, Abril; Salcedo-Tello, Pamela; Avila-Muñoz, Evangelina; Zepeda, Angélica; Arias, Clorinda

2013-09-01

It is well recognized the role of the Wnt pathway in many developmental processes such as neuronal maturation, migration, neuronal connectivity and synaptic formation. Growing evidence is also demonstrating its function in the mature brain where is associated with modulation of axonal remodeling, dendrite outgrowth, synaptic activity, neurogenesis and behavioral plasticity. Proteins involved in Wnt signaling have been found expressed in the adult hippocampus suggesting that Wnt pathway plays a role in the hippocampal function through life. Indeed, Wnt ligands act locally to regulate neurogenesis, neuronal cell shape and pre- and postsynaptic assembly, events that are thought to underlie changes in synaptic function associated with long-term potentiation and with cognitive tasks such as learning and memory. Recent data have demonstrated the increased expression of the Wnt antagonist Dickkopf-1 (DKK1) in brains of Alzheimer´s disease (AD) patients suggesting that dysfunction of Wnt signaling could also contribute to AD pathology. We review here evidence of Wnt-associated molecules expression linked to physiological and pathological hippocampal functioning in the adult brain. The basic aspects of Wnt related mechanisms underlying hippocampal plasticity as well as evidence of how hippocampal dysfunction may rely on Wnt dysregulation is analyzed. This information would provide some clues about the possible therapeutic targets for developing treatments for neurodegenerative diseases associated with aberrant brain plasticity.

Notch-signalling is required for head regeneration and tentacle patterning in Hydra.

PubMed

Münder, Sandra; Tischer, Susanne; Grundhuber, Maresa; Büchels, Nathalie; Bruckmeier, Nadine; Eckert, Stefanie; Seefeldt, Carolin A; Prexl, Andrea; Käsbauer, Tina; Böttger, Angelika

2013-11-01

Local self-activation and long ranging inhibition provide a mechanism for setting up organising regions as signalling centres for the development of structures in the surrounding tissue. The adult hydra hypostome functions as head organiser. After hydra head removal it is newly formed and complete heads can be regenerated. The molecular components of this organising region involve Wnt-signalling and β-catenin. However, it is not known how correct patterning of hypostome and tentacles are achieved in the hydra head and whether other signals in addition to HyWnt3 are needed for re-establishing the new organiser after head removal. Here we show that Notch-signalling is required for re-establishing the organiser during regeneration and that this is due to its role in restricting tentacle activation. Blocking Notch-signalling leads to the formation of irregular head structures characterised by excess tentacle tissue and aberrant expression of genes that mark the tentacle boundaries. This indicates a role for Notch-signalling in defining the tentacle pattern in the hydra head. Moreover, lateral inhibition by HvNotch and its target HyHes are required for head regeneration and without this the formation of the β-catenin/Wnt dependent head organiser is impaired. Work on prebilaterian model organisms has shown that the Wnt-pathway is important for setting up signalling centres for axial patterning in early multicellular animals. Our data suggest that the integration of Wnt-signalling with Notch-Delta activity was also involved in the evolution of defined body plans in animals. © 2013 Elsevier Inc. All rights reserved.

Constitutive overexpression of Norrin activates Wnt/β-catenin and endothelin-2 signaling to protect photoreceptors from light damage.

PubMed

Braunger, Barbara M; Ohlmann, Andreas; Koch, Marcus; Tanimoto, Naoyuki; Volz, Cornelia; Yang, Ying; Bösl, Michael R; Cvekl, Ales; Jägle, Herbert; Seeliger, Mathias W; Tamm, Ernst R

2013-02-01

Norrin is a retinal signaling molecule which is expressed in Müller glia and binds to Frizzled-4 to activate canonical Wnt/β-catenin signaling. Norrin is part of an essential signaling system that controls the formation of retinal capillaries during development. To evaluate neuroprotective properties of Norrin independently from its function during retinal angiogenesis, we generated transgenic mice (Rpe65-Norrin) that constitutively express Norrin in the retinal pigmented epithelium. Substantial amounts of Norrin were secreted into the outer retina, which triggered retinal Wnt/β-catenin signaling in conjunction with an increase in the expression of endothelin-2 (EDN2), endothelin receptor B (EDNRB), and glial fibrillary acidic protein (GFAP). Photoreceptors of Norrin-overexpressing mice were significantly less vulnerable to light-induced damage compared to their wild-type littermates. Following light damage, we observed less apoptotic death of photoreceptors and a better retinal function than in controls. The protective effects were abolished if either Wnt/β-catenin or EDN2 signaling was blocked by intravitreal injection of Dickkopf-1 or BQ788, respectively. Light-damaged retinae from transgenic mice contained higher amounts of brain-derived neurotrophic factor (BDNF) and pAkt than those of wild-type littermates. We conclude that constitutive overexpression of Norrin protects photoreceptors from light damage, an effect that is mediated by Wnt/β-catenin and EDN2 signaling and involves neurotrophic activities of BDNF. The findings suggest that Norrin and its associated signaling pathways have strong potentials to attenuate photoreceptor death following injury. Copyright © 2012 Elsevier Inc. All rights reserved.

The canonical way to make a heart: β-catenin and plakoglobin in heart development and remodeling.

PubMed

Piven, Oksana O; Winata, Cecilia L

2017-12-01

The main mediator of the canonical Wnt pathway, β-catenin, is a major effector of embryonic development, postnatal tissue homeostasis, and adult tissue regeneration. The requirement for β-catenin in cardiogenesis and embryogenesis has been well established. However, many questions regarding the molecular mechanisms by which β-catenin and canonical Wnt signaling regulate these developmental processes remain unanswered. An interesting question that emerged from our studies concerns how β-catenin signaling is modulated through interaction with other factors. Recent experimental data implicate new players in canonical Wnt signaling, particularly those which modulate β-catenin function in many its biological processes, including cardiogenesis. One of the interesting candidates is plakoglobin, a little-studied member of the catenin family which shares several mechanistic and functional features with its close relative, β-catenin. Here we have focused on the function of β-catenin in cardiogenesis. We also summarize findings on plakoglobin signaling function and discuss possible interplays between β-catenin and plakoglobin in the regulation of embryonic heart development. Impact statement Heart development, function, and remodeling are complex processes orchestrated by multiple signaling networks. This review examines our current knowledge of the role of canonical Wnt signaling in cardiogenesis and heart remodeling, focusing primarily on the mechanistic action of its effector β-catenin. We summarize the generally accepted understanding of the field based on experimental in vitro and in vivo data, and address unresolved questions in the field, specifically relating to the role of canonical Wnt signaling in heart maturation and regeneration. What are the modulators of canonical Wnt, and particularly what are the potential roles of plakoglobin, a close relative of β-catenin, in regulating Wnt signaling?Answers to these questions will enhance our understanding of the mechanism by which the canonical Wnt signaling regulates development of the heart and its regeneration after damage.

Wnt pathway curation using automated natural language processing: combining statistical methods with partial and full parse for knowledge extraction.

PubMed

Santos, Carlos; Eggle, Daniela; States, David J

2005-04-15

Wnt signaling is a very active area of research with highly relevant publications appearing at a rate of more than one per day. Building and maintaining databases describing signal transduction networks is a time-consuming and demanding task that requires careful literature analysis and extensive domain-specific knowledge. For instance, more than 50 factors involved in Wnt signal transduction have been identified as of late 2003. In this work we describe a natural language processing (NLP) system that is able to identify references to biological interaction networks in free text and automatically assembles a protein association and interaction map. A 'gold standard' set of names and assertions was derived by manual scanning of the Wnt genes website (http://www.stanford.edu/~rnusse/wntwindow.html) including 53 interactions involved in Wnt signaling. This system was used to analyze a corpus of peer-reviewed articles related to Wnt signaling including 3369 Pubmed and 1230 full text papers. Names for key Wnt-pathway associated proteins and biological entities are identified using a chi-squared analysis of noun phrases over-represented in the Wnt literature as compared to the general signal transduction literature. Interestingly, we identified several instances where generic terms were used on the website when more specific terms occur in the literature, and one typographic error on the Wnt canonical pathway. Using the named entity list and performing an exhaustive assertion extraction of the corpus, 34 of the 53 interactions in the 'gold standard' Wnt signaling set were successfully identified (64% recall). In addition, the automated extraction found several interactions involving key Wnt-related molecules which were missing or different from those in the canonical diagram, and these were confirmed by manual review of the text. These results suggest that a combination of NLP techniques for information extraction can form a useful first-pass tool for assisting human annotation and maintenance of signal pathway databases. The pipeline software components are freely available on request to the authors. dstates@umich.edu http://stateslab.bioinformatics.med.umich.edu/software.html.

Demyelination in Multiple Sclerosis: Reprogramming Energy Metabolism and Potential PPARγ Agonist Treatment Approaches

PubMed Central

Lecarpentier, Yves; Guillevin, Rémy; Vallée, Jean-Noël

2018-01-01

Demyelination in multiple sclerosis (MS) cells is the site of several energy metabolic abnormalities driven by dysregulation between the opposed interplay of peroxisome proliferator-activated receptor γ (PPARγ) and WNT/β-catenin pathways. We focus our review on the opposing interactions observed in demyelinating processes in MS between the canonical WNT/β-catenin pathway and PPARγ and their reprogramming energy metabolism implications. Demyelination in MS is associated with chronic inflammation, which is itself associated with the release of cytokines by CD4+ Th17 cells, and downregulation of PPARγ expression leading to the upregulation of the WNT/β-catenin pathway. Upregulation of WNT/β-catenin signaling induces activation of glycolytic enzymes that modify their energy metabolic behavior. Then, in MS cells, a large portion of cytosolic pyruvate is converted into lactate. This phenomenon is called the Warburg effect, despite the availability of oxygen. The Warburg effect is the shift of an energy transfer production from mitochondrial oxidative phosphorylation to aerobic glycolysis. Lactate production is correlated with increased WNT/β-catenin signaling and demyelinating processes by inducing dysfunction of CD4+ T cells leading to axonal and neuronal damage. In MS, downregulation of PPARγ decreases insulin sensitivity and increases neuroinflammation. PPARγ agonists inhibit Th17 differentiation in CD4+ T cells and then diminish release of cytokines. In MS, abnormalities in the regulation of circadian rhythms stimulate the WNT pathway to initiate the demyelination process. Moreover, PPARγ contributes to the regulation of some key circadian genes. Thus, PPARγ agonists interfere with reprogramming energy metabolism by directly inhibiting the WNT/β-catenin pathway and circadian rhythms and could appear as promising treatments in MS due to these interactions. PMID:29659554

Demyelination in Multiple Sclerosis: Reprogramming Energy Metabolism and Potential PPARγ Agonist Treatment Approaches.

PubMed

Vallée, Alexandre; Lecarpentier, Yves; Guillevin, Rémy; Vallée, Jean-Noël

2018-04-16

Demyelination in multiple sclerosis (MS) cells is the site of several energy metabolic abnormalities driven by dysregulation between the opposed interplay of peroxisome proliferator-activated receptor γ (PPARγ) and WNT/β-catenin pathways. We focus our review on the opposing interactions observed in demyelinating processes in MS between the canonical WNT/β-catenin pathway and PPARγ and their reprogramming energy metabolism implications. Demyelination in MS is associated with chronic inflammation, which is itself associated with the release of cytokines by CD4⁺ Th17 cells, and downregulation of PPARγ expression leading to the upregulation of the WNT/β-catenin pathway. Upregulation of WNT/β-catenin signaling induces activation of glycolytic enzymes that modify their energy metabolic behavior. Then, in MS cells, a large portion of cytosolic pyruvate is converted into lactate. This phenomenon is called the Warburg effect, despite the availability of oxygen. The Warburg effect is the shift of an energy transfer production from mitochondrial oxidative phosphorylation to aerobic glycolysis. Lactate production is correlated with increased WNT/β-catenin signaling and demyelinating processes by inducing dysfunction of CD4⁺ T cells leading to axonal and neuronal damage. In MS, downregulation of PPARγ decreases insulin sensitivity and increases neuroinflammation. PPARγ agonists inhibit Th17 differentiation in CD4⁺ T cells and then diminish release of cytokines. In MS, abnormalities in the regulation of circadian rhythms stimulate the WNT pathway to initiate the demyelination process. Moreover, PPARγ contributes to the regulation of some key circadian genes. Thus, PPARγ agonists interfere with reprogramming energy metabolism by directly inhibiting the WNT/β-catenin pathway and circadian rhythms and could appear as promising treatments in MS due to these interactions.

Non-conventional Frizzled ligands and Wnt receptors.

PubMed

Hendrickx, Marijke; Leyns, Luc

2008-05-01

The Wnt family of secreted signaling factors plays numerous roles in embryonic development and in stem cell biology. In the adult, Wnt signaling is involved in tissue homeostasis and mutations that lead to the overexpression of Wnt can be linked to cancer. Wnt signaling is transduced intracellularly by the Frizzled (Fzd) family of receptors. In the canonical pathway, accumulation of beta-catenin and the subsequent formation of a complex with T cell factors (TCF) or lymphoid enhancing factors (Lef) lead to target gene activation. The identification of Ryk as an alternative Wnt receptor and the discovery of the novel Fzd ligands Norrie disease protein (NDP) and R-Spondin, changed the traditional view of Wnts binding to Fzd receptors. Mouse R-Spondin cooperates with Wnt signaling and Low density lipoprotein (LDL) receptor related protein (LRP) to activate beta-catenin dependent gene expression and is involved in processes such as limb and placental development in the mouse. NDP is the product of the Norrie disease gene and controls vascular development in the retina, inner ear and in the female reproductive system during pregnancy. In this review a functional overview of the interactions of the different Wnt and non-Wnt ligands with the Fzd receptors is given as well as a survey of Wnts binding to Ryk and we discuss the biological significance of these interactions.

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

Railo, Antti; Nagy, Irina I.; Kilpelaeinen, Pekka

The Wnt family of glycoprotein growth factors controls a number of central cellular processes such as proliferation, differentiation and ageing. All the Wnt proteins analyzed so far either activate or inhibit the canonical {beta}-catenin signaling pathway that regulates transcription of the target genes. In addition, some of them activate noncanonical signaling pathways that involve components such as the JNK, heterotrimeric G proteins, protein kinase C, and calmodulin-dependent protein kinase II, although the precise signaling mechanisms are only just beginning to be revealed. We demonstrate here that Wnt-11 signaling is sufficient to inhibit not only the canonical {beta}-catenin mediated Wnt signalingmore » but also JNK/AP-1 and NF-{kappa}B signaling in the CHO cells, thus serving as a noncanonical Wnt ligand in this system. Inhibition of the JNK/AP-1 pathway is mediated in part by the MAPK kinase MKK4 and Akt. Moreover, protein kinase C is involved in the regulation of JNK/AP-1 by Wnt-11, but not of the NF-{kappa}B pathway. Consistent with the central role of Akt, JNK and NF-{kappa}B in cell survival and stress responses, Wnt-11 signaling promotes cell viability. Hence Wnt-11 is involved in coordination of key signaling pathways.« less

Tip cell-specific requirement for an atypical Gpr124- and Reck-dependent Wnt/β-catenin pathway during brain angiogenesis

PubMed Central

Vanhollebeke, Benoit; Stone, Oliver A; Bostaille, Naguissa; Cho, Chris; Zhou, Yulian; Maquet, Emilie; Gauquier, Anne; Cabochette, Pauline; Fukuhara, Shigetomo; Mochizuki, Naoki; Nathans, Jeremy; Stainier, Didier YR

2015-01-01

Despite the critical role of endothelial Wnt/β-catenin signaling during central nervous system (CNS) vascularization, how endothelial cells sense and respond to specific Wnt ligands and what aspects of the multistep process of intra-cerebral blood vessel morphogenesis are controlled by these angiogenic signals remain poorly understood. We addressed these questions at single-cell resolution in zebrafish embryos. We identify the GPI-anchored MMP inhibitor Reck and the adhesion GPCR Gpr124 as integral components of a Wnt7a/Wnt7b-specific signaling complex required for brain angiogenesis and dorsal root ganglia neurogenesis. We further show that this atypical Wnt/β-catenin signaling pathway selectively controls endothelial tip cell function and hence, that mosaic restoration of single wild-type tip cells in Wnt/β-catenin-deficient perineural vessels is sufficient to initiate the formation of CNS vessels. Our results identify molecular determinants of ligand specificity of Wnt/β-catenin signaling and provide evidence for organ-specific control of vascular invasion through tight modulation of tip cell function. DOI: http://dx.doi.org/10.7554/eLife.06489.001 PMID:26051822

Activation of Wnt Signaling in Cortical Neurons Enhances Glucose Utilization through Glycolysis*

PubMed Central

Cisternas, Pedro; Salazar, Paulina; Silva-Álvarez, Carmen; Barros, L. Felipe; Inestrosa, Nibaldo C.

2016-01-01

The Wnt signaling pathway is critical for a number of functions in the central nervous system, including regulation of the synaptic cleft structure and neuroprotection against injury. Deregulation of Wnt signaling has been associated with several brain pathologies, including Alzheimer's disease. In recent years, it has been suggested that the Wnt pathway might act as a central integrator of metabolic signals from peripheral organs to the brain, which would represent a new role for Wnt signaling in cell metabolism. Energy metabolism is critical for normal neuronal function, which mainly depends on glucose utilization. Brain energy metabolism is important in almost all neurological disorders, to which a decrease in the capacity of the brain to utilize glucose has been linked. However, little is known about the relationship between Wnt signaling and neuronal glucose metabolism in the cellular context. In the present study, we found that acute treatment with the Wnt3a ligand induced a large increase in glucose uptake, without changes in the expression or localization of glucose transporter type 3. In addition, we observed that Wnt3a treatment increased the activation of the metabolic sensor Akt. Moreover, we observed an increase in the activity of hexokinase and in the glycolytic rate, and both processes were dependent on activation of the Akt pathway. Furthermore, we did not observe changes in the activity of glucose-6-phosphate dehydrogenase or in the pentose phosphate pathway. The effect of Wnt3a was independent of both the transcription of Wnt target genes and synaptic effects of Wnt3a. Together, our results suggest that Wnt signaling stimulates glucose utilization in cortical neurons through glycolysis to satisfy the high energy demand of these cells. PMID:27703002

Activation of Wnt Signaling in Cortical Neurons Enhances Glucose Utilization through Glycolysis.

PubMed

Cisternas, Pedro; Salazar, Paulina; Silva-Álvarez, Carmen; Barros, L Felipe; Inestrosa, Nibaldo C

2016-12-09

The Wnt signaling pathway is critical for a number of functions in the central nervous system, including regulation of the synaptic cleft structure and neuroprotection against injury. Deregulation of Wnt signaling has been associated with several brain pathologies, including Alzheimer's disease. In recent years, it has been suggested that the Wnt pathway might act as a central integrator of metabolic signals from peripheral organs to the brain, which would represent a new role for Wnt signaling in cell metabolism. Energy metabolism is critical for normal neuronal function, which mainly depends on glucose utilization. Brain energy metabolism is important in almost all neurological disorders, to which a decrease in the capacity of the brain to utilize glucose has been linked. However, little is known about the relationship between Wnt signaling and neuronal glucose metabolism in the cellular context. In the present study, we found that acute treatment with the Wnt3a ligand induced a large increase in glucose uptake, without changes in the expression or localization of glucose transporter type 3. In addition, we observed that Wnt3a treatment increased the activation of the metabolic sensor Akt. Moreover, we observed an increase in the activity of hexokinase and in the glycolytic rate, and both processes were dependent on activation of the Akt pathway. Furthermore, we did not observe changes in the activity of glucose-6-phosphate dehydrogenase or in the pentose phosphate pathway. The effect of Wnt3a was independent of both the transcription of Wnt target genes and synaptic effects of Wnt3a. Together, our results suggest that Wnt signaling stimulates glucose utilization in cortical neurons through glycolysis to satisfy the high energy demand of these cells. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

The Roles of the Wnt-Antagonists Axin and Lrp4 during Embryogenesis of the Red Flour Beetle Tribolium castaneum

PubMed Central

Prühs, Romy

2017-01-01

In both vertebrates and invertebrates, the Wnt-signaling pathway is essential for numerous processes in embryogenesis and during adult life. Wnt activity is fine-tuned at various levels by the interplay of a number of Wnt-agonists (Wnt ligands, Frizzled-receptors, Lrp5/6 coreceptors) and Wnt-antagonists (among them Axin, Secreted frizzled and Lrp4) to define anterior–posterior polarity of the early embryo and specify cell fate in organogenesis. So far, the functional analysis of Wnt-pathway components in insects has concentrated on the roles of Wnt-agonists and on the Wnt-antagonist Axin. We depict here additional features of the Wnt-antagonist Axin in the flour beetle Tribolium castaneum. We show that Tc-axin is dynamically expressed throughout embryogenesis and confirm its essential role in head development. In addition, we describe an as yet undetected, more extreme Tc-axin RNAi-phenotype, the ectopic formation of posterior abdominal segments in reverse polarity and a second hindgut at the anterior. For the first time, we describe here that an lrp4 ortholog is involved in axis formation in an insect. The Tribolium Lrp4 ortholog is ubiquitously expressed throughout embryogenesis. Its downregulation via maternal RNAi results in the reduction of head structures but not in axis polarity reversal. Furthermore, segmentation is impaired and larvae develop with a severe gap-phenotype. We conclude that, as in vertebrates, Tc-lrp4 functions as a Wnt-inhibitor in Tribolium during various stages of embryogenesis. We discuss the role of both components as negative modulators of Wnt signaling in respect to axis formation and segmentation in Tribolium. PMID:29615567

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

Perkins, Timothy N.; Dentener, Mieke A.

Growth and development of the mature lung is a complex process orchestrated by a number of intricate developmental signaling pathways. Wingless-type MMTV-integration site (WNT) signaling plays critical roles in controlling branching morphogenesis cell differentiation, and formation of the conducting and respiratory airways. In addition, WNT pathways are often re-activated in mature lungs during repair and regeneration. WNT- signaling has been elucidated as a crucial contributor to the development of idiopathic pulmonary fibrosis as well as other hyper-proliferative lung diseases. Silicosis, a detrimental occupational lung disease caused by excessive inhalation of crystalline silica dust, is hallmarked by repeated cycles of damagingmore » inflammation, epithelial hyperplasia, and formation of dense, hyalinized nodules of whorled collagen. However, mechanisms of epithelial cell hyperplasia and matrix deposition are not well understood, as most research efforts have focused on the pronounced inflammatory response. Microarray data from our previous studies has revealed a number of WNT-signaling and WNT-target genes altered by crystalline silica in human lung epithelial cells. In the present study, we utilize pathway analysis to designate connections between genes altered by silica in WNT-signaling networks. Furthermore, we confirm microarray findings by QRT-PCR and demonstrate both activation of canonical (β-catenin) and down-regulation of non-canonical (WNT5A) signaling in immortalized (BEAS-2B) and primary (PBEC) human bronchial epithelial cells. These findings suggest that WNT-signaling and cross-talk with other pathways (e.g. Notch), may contribute to proliferative, fibrogenic and inflammatory responses to silica in lung epithelial cells. - Highlights: • Pathway analysis reveals silica-induced WNT-signaling in lung epithelial cells. • Silica-induced canonical WNT-signaling is mediated by autocrine/paracrine signals. • Crystalline silica decreases non-canonical WNT5A signaling. • Microarray reveals WNT as a novel complex signaling network in silica-mediated injury.« less

Molecular genetics and targeted therapy of WNT-related human diseases (Review).

PubMed

Katoh, Masuko; Katoh, Masaru

2017-09-01

Canonical WNT signaling through Frizzled and LRP5/6 receptors is transduced to the WNT/β-catenin and WNT/stabilization of proteins (STOP) signaling cascades to regulate cell fate and proliferation, whereas non-canonical WNT signaling through Frizzled or ROR receptors is transduced to the WNT/planar cell polarity (PCP), WNT/G protein-coupled receptor (GPCR) and WNT/receptor tyrosine kinase (RTK) signaling cascades to regulate cytoskeletal dynamics and directional cell movement. WNT/β-catenin signaling cascade crosstalks with RTK/SRK and GPCR-cAMP-PKA signaling cascades to regulate β-catenin phosphorylation and β-catenin-dependent transcription. Germline mutations in WNT signaling molecules cause hereditary colorectal cancer, bone diseases, exudative vitreoretinopathy, intellectual disability syndrome and PCP-related diseases. APC or CTNNB1 mutations in colorectal, endometrial and prostate cancers activate the WNT/β-catenin signaling cascade. RNF43, ZNRF3, RSPO2 or RSPO3 alterations in breast, colorectal, gastric, pancreatic and other cancers activate the WNT/β-catenin, WNT/STOP and other WNT signaling cascades. ROR1 upregulation in B-cell leukemia and solid tumors and ROR2 upregulation in melanoma induce invasion, metastasis and therapeutic resistance through Rho-ROCK, Rac-JNK, PI3K-AKT and YAP signaling activation. WNT signaling in cancer, stromal and immune cells dynamically orchestrate immune evasion and antitumor immunity in a cell context-dependent manner. Porcupine (PORCN), RSPO3, WNT2B, FZD5, FZD10, ROR1, tankyrase and β-catenin are targets of anti-WNT signaling therapy, and ETC-159, LGK974, OMP-18R5 (vantictumab), OMP-54F28 (ipafricept), OMP-131R10 (rosmantuzumab), PRI-724 and UC-961 (cirmtuzumab) are in clinical trials for cancer patients. Different classes of anti-WNT signaling therapeutics are necessary for the treatment of APC/CTNNB1-, RNF43/ZNRF3/RSPO2/RSPO3- and ROR1-types of human cancers. By contrast, Dickkopf-related protein 1 (DKK1), SOST and glycogen synthase kinase 3β (GSK3β) are targets of pro-WNT signaling therapy, and anti-DKK1 (BHQ880 and DKN-01) and anti-SOST (blosozumab, BPS804 and romosozumab) monoclonal antibodies are being tested in clinical trials for cancer patients and osteoporotic post-menopausal women. WNT-targeting therapeutics have also been applied as reagents for in vitro stem-cell processing in the field of regenerative medicine.

Is resistant starch protective against colorectal cancer via modulation of the WNT signalling pathway?

PubMed

Malcomson, Fiona C; Willis, Naomi D; Mathers, John C

2015-08-01

Epidemiological and experimental evidence suggests that non-digestible carbohydrates (NDC) including resistant starch are protective against colorectal cancer. These anti-neoplastic effects are presumed to result from the production of the SCFA, butyrate, by colonic fermentation, which binds to the G-protein-coupled receptor GPR43 to regulate inflammation and other cancer-related processes. The WNT pathway is central to the maintenance of homeostasis within the large bowel through regulation of processes such as cell proliferation and migration and is frequently aberrantly hyperactivated in colorectal cancers. Abnormal WNT signalling can lead to irregular crypt cell proliferation that favours a hyperproliferative state. Butyrate has been shown to modulate the WNT pathway positively, affecting functional outcomes such as apoptosis and proliferation. Butyrate's ability to regulate gene expression results from epigenetic mechanisms, including its role as a histone deacetylase inhibitor and through modulating DNA methylation and the expression of microRNA. We conclude that genetic and epigenetic modulation of the WNT signalling pathway may be an important mechanism through which butyrate from fermentation of resistant starch and other NDC exert their chemoprotective effects.

rbm47, a novel RNA binding protein, regulates zebrafish head development.

PubMed

Guan, Rui; El-Rass, Suzan; Spillane, David; Lam, Simon; Wang, Yuodong; Wu, Jing; Chen, Zhuchu; Wang, Anan; Jia, Zhengping; Keating, Armand; Hu, Jim; Wen, Xiao-Yan

2013-12-01

Vertebrate trunk induction requires inhibition of bone morphogenetic protein (BMP) signaling, whereas vertebrate head induction requires concerted inhibition of both Wnt and BMP signaling. RNA binding proteins play diverse roles in embryonic development and their roles in vertebrate head development remain to be elucidated. We first characterized the human RBM47 as an RNA binding protein that specifically binds RNA but not single-stranded DNA. Next, we knocked down rbm47 gene function in zebrafish using morpholinos targeting the start codon and exon-1/intron-1 splice junction. Down-regulation of rbm47 resulted in headless and small head phenotypes, which can be rescued by a wnt8a blocking morpholino. To further reveal the mechanism of rbm47's role in head development, microarrays were performed to screen genes differentially expressed in normal and knockdown embryos. epcam and a2ml were identified as the most significantly up- and down-regulated genes, respectively. The microarrays also confirmed up-regulation of several genes involved in head development, including gsk3a, otx2, and chordin, which are important regulators of Wnt signaling. Altogether, our findings reveal that Rbm47 is a novel RNA-binding protein critical for head formation and embryonic patterning during zebrafish embryogenesis which may act through a Wnt8a signaling pathway. Copyright © 2013 Wiley Periodicals, Inc.

TGFβ regulates epithelial-mesenchymal interactions through WNT signaling activity to control muscle development in the soft palate.

PubMed

Iwata, Jun-ichi; Suzuki, Akiko; Yokota, Toshiaki; Ho, Thach-Vu; Pelikan, Richard; Urata, Mark; Sanchez-Lara, Pedro A; Chai, Yang

2014-02-01

Clefting of the soft palate occurs as a congenital defect in humans and adversely affects the physiological function of the palate. However, the molecular and cellular mechanism of clefting of the soft palate remains unclear because few animal models exhibit an isolated cleft in the soft palate. Using three-dimensional microCT images and histological reconstruction, we found that loss of TGFβ signaling in the palatal epithelium led to soft palate muscle defects in Tgfbr2(fl/fl);K14-Cre mice. Specifically, muscle mass was decreased in the soft palates of Tgfbr2 mutant mice, following defects in cell proliferation and differentiation. Gene expression of Dickkopf (Dkk1 and Dkk4), negative regulators of WNT-β-catenin signaling, is upregulated in the soft palate of Tgfbr2(fl/fl);K14-Cre mice, and WNT-β-catenin signaling is disrupted in the palatal mesenchyme. Importantly, blocking the function of DKK1 and DKK4 rescued the cell proliferation and differentiation defects in the soft palate of Tgfbr2(fl/fl);K14-Cre mice. Thus, our findings indicate that loss of TGFβ signaling in epithelial cells compromises activation of WNT signaling and proper muscle development in the soft palate through tissue-tissue interactions, resulting in a cleft soft palate. This information has important implications for prevention and non-surgical correction of cleft soft palate.

Interactions between TGF-β1, canonical WNT/β-catenin pathway and PPAR γ in radiation-induced fibrosis

PubMed Central

Vallée, Alexandre; Lecarpentier, Yves; Guillevin, Rémy; Vallée, Jean-Noël

2017-01-01

Radiation therapy induces DNA damage and inflammation leading to fibrosis. Fibrosis can occur 4 to 12 months after radiation therapy. This process worsens with time and years. Radiation-induced fibrosis is characterized by fibroblasts proliferation, myofibroblast differentiation, and synthesis of collagen, proteoglycans and extracellular matrix. Myofibroblasts are non-muscle cells that can contract and relax. Myofibroblasts evolve towards irreversible retraction during fibrosis process. In this review, we discussed the interplays between transforming growth factor-β1 (TGF-β1), canonical WNT/β-catenin pathway and peroxisome proliferator-activated receptor gamma (PPAR γ) in regulating the molecular mechanisms underlying the radiation-induced fibrosis, and the potential role of PPAR γ agonists. Overexpression of TGF-β and canonical WNT/β-catenin pathway stimulate fibroblasts accumulation and myofibroblast differentiation whereas PPAR γ expression decreases due to the opposite interplay of canonical WNT/β-catenin pathway. Both TGF-β1 and canonical WNT/β-catenin pathway stimulate each other through the Smad pathway and non-Smad pathways such as phosphatidylinositol 3-kinase/serine/threonine kinase (PI3K/Akt) signaling. WNT/β-catenin pathway and PPAR γ interact in an opposite manner. PPAR γ agonists decrease β-catenin levels through activation of inhibitors of the WNT pathway such as Smad7, glycogen synthase kinase-3 (GSK-3 β) and dickkopf-related protein 1 (DKK1). PPAR γ agonists also stimulate phosphatase and tensin homolog (PTEN) expression, which decreases both TGF-β1 and PI3K/Akt pathways. PPAR γ agonists by activating Smad7 decrease Smads pathway and then TGF-β signaling leading to decrease radiation-induced fibrosis. TGF-β1 and canonical WNT/β-catenin pathway promote radiation-induced fibrosis whereas PPAR γ agonists can prevent radiation-induced fibrosis. PMID:29163854

Spatio-temporal expression patterns of Wnt signaling pathway during the development of temporomandibular condylar cartilage.

PubMed

Chen, Kan; Quan, Huixin; Chen, Gang; Xiao, Di

2017-11-01

There is a growing body of evidence supporting the involvement of the Wnt signaling pathway in various aspects of skeletal and joint development; however, it is unclear whether it is involved in the process of temporomandibular joint development. In order to clarify this issue, we examined the spatio-temporal distribution of mRNAs and proteins of the Wnt family during the formation of the mandibular condylar cartilage at the prenatal and postnatal stages. An in situ hybridization test revealed no mRNAs of β-catenin and Axin2 during early mesenchymal condensation; the ligands surveyed in this study (including Wnt-4, 5a, and 9a) were clearly detected at various ranges of expression, mainly in the condylar blastema and later distinct cartilaginous layers. Apart from β-catenin and Axin2, the Wnt family members surveyed in this study, including Lef-1, were found to be immunopositive during early chondrogenesis in the condylar cartilage at E14.5. After distinct chondrocyte layers were identified within the cartilage at E16.5, the expression of the Wnt signaling members was different and mainly restricted to proliferating cells and mineralized hypertrophic chondrocytes. In the adult mandibular condylar cartilage, the Wnt-4 mRNA, as well as the Wnt-4 and Wnt-9a proteins, was not observed. Our findings demonstrated that the Wnt signaling pathway was associated with the development of mandibular condylar cartilage. Copyright © 2017 Elsevier B.V. All rights reserved.

Selective enhancement of wnt4 expression by cyclic AMP-associated cooperation between rat central astrocytes and microglia

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

Ohnishi, Masatoshi, E-mail: ohnishi@fupharm.fukuyama-u.ac.jp; Department of Pharmacotherapeutics, Faculty of Pharmacy and Pharmaceutical Sciences, Fukuyama University, 985-1 Sanzo, Higashimura-cho, Fukuyama, Hiroshima, 729-0292; Urasaki, Tomoka

2015-11-13

The wnt protein family has important members involved in cell differentiation, proliferation and plasticity expression; however, little is known about its biosynthesis processes. On the other hand, an increase in the intracerebral cyclic adenosine 3′, 5’-monophosphate (cAMP) level leads to synaptic plasticity via the de novo synthesis of any protein. Here, the effect of dibutyryl cAMP (dbcAMP), a membrane permeability cAMP analog, on the wnt family was investigated in rat primary-cultured glial cells containing astrocytes and microglia. Among wnt3a, 4, 5a, 7a and 11 mRNA, only wnt4 expression was increased by longer treatment (24 h), compared with short treatment (2 h), withmore » dbcAMP in a concentration-dependent manner, and its effect reached statistical significance at 1 mM. In cultures of isolated astrocytes or microglia, wnt4 expression was not affected by 1 mM dbcAMP for 24 h, and microglial wnt4 protein was undetectable even when cells were treated with the drug. Mixed glial cells treated for 24 h with 1 mM dbcAMP showed significantly increased wnt4 protein, as well as mRNA. Immunofluorescence manifested that cells that expressed wnt4 protein were astrocytes, but not microglia. Intraperitoneal injection of 1.25 mg/kg rolipram, a phosphodiesterase (PDE) IV inhibitor that can pass through the blood brain barrier and inhibits cAMP degradation specifically, showed a tendency to increase wnt4 expression in the adult rat brain after 24 h, and the increases in wnt4 mRNA and protein levels reached statistical significance in the hippocampus and striatum, respectively. This is the first finding to help elucidate the selective biosynthesis of central wnt4 through cAMP-stimulated microglia and astrocytes interaction. - Highlights: • Dibutyryl cAMP increased wnt4, but not wnt3a, 5a, 7a and 11, mRNA in mixed glia. • Wnt4 protein increased in astrocytes co-cultivated with microglia. • It took a long time to robustly increase wnt4 expression. • Rolipram increased wnt4 expression in the rat striatum and hippocampus.« less

Wnt5a signaling is a substantial constituent in bone morphogenetic protein-2-mediated osteoblastogenesis

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

Nemoto, Eiji, E-mail: e-nemoto@dent.tohoku.ac.jp; Ebe, Yukari; Kanaya, Sousuke

2012-06-15

Highlights: Black-Right-Pointing-Pointer Wnt5a is identified in osteoblasts in tibial growth plate and bone marrow. Black-Right-Pointing-Pointer Osteoblastic differentiation is associated with increased expression of Wnt5a/Ror2. Black-Right-Pointing-Pointer Wnt5a/Ror2 signaling is important for BMP-2-mediated osteoblastic differentiation. Black-Right-Pointing-Pointer Wnt5a/Ror2 operates independently of BMP-Smad pathway. -- Abstract: Wnts are secreted glycoproteins that mediate developmental and post-developmental physiology by regulating cellular processes including proliferation, differentiation, and apoptosis through {beta}-catenin-dependent canonical and {beta}-catenin-independent noncanonical pathway. It has been reported that Wnt5a activates noncanonical Wnt signaling through receptor tyrosine kinase-like orphan receptor 2 (Ror2). Although it appears that Wnt5a/Ror2 signaling supports normal bone physiology, the biological significance ofmore » noncanonical Wnts in osteogenesis is essentially unknown. In this study, we identified expression of Wnt5a in osteoblasts in the ossification zone of the tibial growth plate as well as bone marrow of the rat tibia as assessed by immunohistochemistry. In addition, we show that osteoblastic differentiation mediated by BMP-2 is associated with increased expression of Wnt5a and Ror2 using cultured pre-osteoblasts, MC3T3-E1 cells. Silencing gene expression of Wnt5a and Ror2 in MC3T3-E1 cells results in suppression of BMP-2-mediated osteoblastic differentiation, suggesting that Wnt5a and Ror2 signaling are of substantial importance for BMP-2-mediated osteoblastic differentiation. BMP-2 stimulation induced phosphorylation of Smad1/5/8 in a similar fashion in both siWnt5a-treated cells and control cells, suggesting that Wnt5a was dispensable for the phosphorylation of Smads by BMP-2. Taken together, our results suggest that Wnt5a/Ror2 signaling appears to be involved in BMP-2-mediated osteoblast differentiation in a Smad independent pathway.« less

The Identification and Cloning of the Wnt-1 Receptor

DTIC Science & Technology

1996-10-01

examination of embryos with duplicated axes revealed that Xwnt-5A and hFz5 induced a full array of dorsal tissues, including notochord , neural tube...tube, a notochord and somites in both axes. c). Xwnt-5A plus hfz5 induce ectopic goosecoid (gsc) expression in stage 11 embryos, as visualized by whole...Lai CJ, Olson DJ, Kelly GM: Dissecting Wnt signalling pathways and Wnt-sensitive developmental processes through transient misexpression analyses in

Spatio-temporal Model of Endogenous ROS and Raft-Dependent WNT/Beta-Catenin Signaling Driving Cell Fate Commitment in Human Neural Progenitor Cells

PubMed Central

Haack, Fiete; Lemcke, Heiko; Ewald, Roland; Rharass, Tareck; Uhrmacher, Adelinde M.

2015-01-01

Canonical WNT/β-catenin signaling is a central pathway in embryonic development, but it is also connected to a number of cancers and developmental disorders. Here we apply a combined in-vitro and in-silico approach to investigate the spatio-temporal regulation of WNT/β-catenin signaling during the early neural differentiation process of human neural progenitors cells (hNPCs), which form a new prospect for replacement therapies in the context of neurodegenerative diseases. Experimental measurements indicate a second signal mechanism, in addition to canonical WNT signaling, being involved in the regulation of nuclear β-catenin levels during the cell fate commitment phase of neural differentiation. We find that the biphasic activation of β-catenin signaling observed experimentally can only be explained through a model that combines Reactive Oxygen Species (ROS) and raft dependent WNT/β-catenin signaling. Accordingly after initiation of differentiation endogenous ROS activates DVL in a redox-dependent manner leading to a transient activation of down-stream β-catenin signaling, followed by continuous auto/paracrine WNT signaling, which crucially depends on lipid rafts. Our simulation studies further illustrate the elaborate spatio-temporal regulation of DVL, which, depending on its concentration and localization, may either act as direct inducer of the transient ROS/β-catenin signal or as amplifier during continuous auto-/parcrine WNT/β-catenin signaling. In addition we provide the first stochastic computational model of WNT/β-catenin signaling that combines membrane-related and intracellular processes, including lipid rafts/receptor dynamics as well as WNT- and ROS-dependent β-catenin activation. The model’s predictive ability is demonstrated under a wide range of varying conditions for in-vitro and in-silico reference data sets. Our in-silico approach is realized in a multi-level rule-based language, that facilitates the extension and modification of the model. Thus, our results provide both new insights and means to further our understanding of canonical WNT/β-catenin signaling and the role of ROS as intracellular signaling mediator. PMID:25793621

Skeletal metastasis: treatments, mouse models, and the Wnt signaling

PubMed Central

Valkenburg, Kenneth C.; Steensma, Matthew R.; Williams, Bart O.; Zhong, Zhendong

2013-01-01

Skeletal metastases result in significant morbidity and mortality. This is particularly true of cancers with a strong predilection for the bone, such as breast, prostate, and lung cancers. There is currently no reliable cure for skeletal metastasis, and palliative therapy options are limited. The Wnt signaling pathway has been found to play an integral role in the process of skeletal metastasis and may be an important clinical target. Several experimental models of skeletal metastasis have been used to find new biomarkers and test new treatments. In this review, we discuss pathologic process of bone metastasis, the roles of the Wnt signaling, and the available experimental models and treatments. PMID:23327798

Wnt Signaling in Cardiac Disease.

PubMed

Hermans, Kevin C M; Blankesteijn, W Matthijs

2015-07-01

Wnt signaling encompasses multiple and complex signaling cascades and is involved in many developmental processes such as tissue patterning, cell fate specification, and control of cell division. Consequently, accurate regulation of signaling activities is essential for proper embryonic development. Wnt signaling is mostly silent in the healthy adult organs but a reactivation of Wnt signaling is generally observed under pathological conditions. This has generated increasing interest in this pathway from a therapeutic point of view. In this review article, the involvement of Wnt signaling in cardiovascular development will be outlined, followed by its implication in myocardial infarct healing, cardiac hypertrophy, heart failure, arrhythmias, and atherosclerosis. The initial experiments not always offer consensus on the effects of activation or inactivation of the pathway, which may be attributed to (i) the type of cardiac disease, (ii) timing of the intervention, and (iii) type of cells that are targeted. Therefore, more research is needed to determine the exact implication of Wnt signaling in the conditions mentioned above to exploit it as a powerful therapeutic target. © 2015 American Physiological Society.

Role of the Wnt/β-catenin pathway in gastric cancer: An in-depth literature review

PubMed Central

Chiurillo, Miguel Angel

2015-01-01

Gastric cancer remains one of the most common cancers worldwide and one of the leading cause for cancer-related deaths. Gastric adenocarcinoma is a multifactorial disease that is genetically, cytologically and architecturally more heterogeneous than other gastrointestinal carcinomas. The aberrant activation of the Wnt/β-catenin signaling pathway is involved in the development and progression of a significant proportion of gastric cancer cases. This review focuses on the participation of the Wnt/β-catenin pathway in gastric cancer by offering an analysis of the relevant literature published in this field. Indeed, it is discussed the role of key factors in Wnt/β-catenin signaling and their downstream effectors regulating processes involved in tumor initiation, tumor growth, metastasis and resistance to therapy. Available data indicate that constitutive Wnt signalling resulting from Helicobacter pylori infection and inactivation of Wnt inhibitors (mainly by inactivating mutations and promoter hypermethylation) play an important role in gastric cancer. Moreover, a number of recent studies confirmed CTNNB1 and APC as driver genes in gastric cancer. The identification of specific membrane, intracellular, and extracellular components of the Wnt pathway has revealed potential targets for gastric cancer therapy. High-throughput “omics” approaches will help in the search for Wnt pathway antagonist in the near future. PMID:25992323

Wnt signaling induces vulva development in the nematode Pristionchus pacificus.

PubMed

Tian, Huiyu; Schlager, Benjamin; Xiao, Hua; Sommer, Ralf J

2008-01-22

The Caenorhabditis elegans vulva is induced by a member of the epidermal growth factor (EGF) family that is expressed in the gonadal anchor cell, representing a prime example of signaling processes in animal development. Comparative studies indicated that vulva induction has changed rapidly during evolution. However, nothing was known about the molecular mechanisms underlying these differences. By analyzing deletion mutants in five Wnt pathway genes, we show that Wnt signaling induces vulva formation in Pristionchus pacificus. A Ppa-bar-1/beta-catenin deletion is completely vulvaless. Several Wnt ligands and receptors act redundantly in vulva induction, and Ppa-egl-20/Wnt; Ppa-mom-2/Wnt; Ppa-lin-18/Ryk triple mutants are strongly vulvaless. Wnt ligands are differentially expressed in the somatic gonad, the anchor cell, and the posterior body region, respectively. In contrast, previous studies indicated that Ppa-lin-17, one of the Frizzled-type receptors, has a negative role in vulva formation. We found that mutations in Ppa-bar-1 and Ppa-egl-20 suppress the phenotype of Ppa-lin-17. Thus, an unexpected complexity of Wnt signaling is involved in vulva induction and vulva repression in P. pacificus. This study provides the first molecular identification of the inductive vulva signal in a nematode other than Caenorhabditis.

FGFR Inhibitor Ameliorates Hypophosphatemia and Impaired Engrailed-1/Wnt Signaling in FGF2 High Molecular Weight Isoform Transgenic Mice.

PubMed

Du, Erxia; Xiao, Liping; Hurley, Marja M

2016-09-01

High molecular weight FGF2 transgenic (HMWTg) mouse phenocopies the Hyp mouse, homolog of human X-linked hypophosphatemic rickets with hypophosphatemis, and abnormal FGF23, FGFR, Klotho signaling in kidney. Since abnormal Wnt signaling was reported in Hyp mice we assessed whether Wnt signaling was impaired in HMWTg kidneys and the effect of blocking FGF receptor (FGFR) signaling. Bone mineral density and bone mineral content in female HMWTg mice were significantly reduced. HMWTg mice were gavaged with FGFR inhibitor NVP-BGJ398, or vehicle and were euthanized 24 h post treatment. Serum phosphate was significantly reduced and urine phosphate was significantly increased in HMWTg and was rescued by NVP-BGJ398. Analysis of kidneys revealed a significant reduction in Npt2a mRNA in HMWTg that was significantly increased by NVP-BGJ398. Increased FGFR1, KLOTHO, P-ERK1/2, and decreased NPT2a protein in HMWTg were rescued by NVP-BGJ398. Wnt inhibitor Engrailed-1 mRNA and protein was increased in HMWTg and was decreased by BGJ398. Akt mRNA and protein was decreased in HMWTg and was increased by NVP-BGJ398. The active form of glycogen synthase 3 beta (pGSK3-β) and phosphor-β-catenin were increased in HMWTg and were both decreased by NVP-BGJ398 while decreased active-β-catenin in HMWTg was increased by NVP-BGJ398. We conclude that FGFR blockade rescued hypophosphatemia by regulating FGF and WNT signaling in HMWTg kidneys. J. Cell. Biochem. 117: 1991-2000, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Retinoic acid receptor signalling directly regulates osteoblast and adipocyte differentiation from mesenchymal progenitor cells

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

Green, A.C.; Department of Medicine at St. Vincent's Hospital, The University of Melbourne, Victoria 3065; Kocovski, P.

Low and high serum retinol levels are associated with increased fracture risk and poor bone health. We recently showed retinoic acid receptors (RARs) are negative regulators of osteoclastogenesis. Here we show RARs are also negative regulators of osteoblast and adipocyte differentiation. The pan-RAR agonist, all-trans retinoic acid (ATRA), directly inhibited differentiation and mineralisation of early osteoprogenitors and impaired the differentiation of more mature osteoblast populations. In contrast, the pan-RAR antagonist, IRX4310, accelerated differentiation of early osteoprogenitors. These effects predominantly occurred via RARγ and were further enhanced by an RARα agonist or antagonist, respectively. RAR agonists similarly impaired adipogenesis in osteogenicmore » cultures. RAR agonist treatment resulted in significant upregulation of the Wnt antagonist, Sfrp4. This accompanied reduced nuclear and cytosolic β-catenin protein and reduced expression of the Wnt target gene Axin2, suggesting impaired Wnt/β-catenin signalling. To determine the effect of RAR inhibition in post-natal mice, IRX4310 was administered to male mice for 10 days and bones were assessed by µCT. No change to trabecular bone volume was observed, however, radial bone growth was impaired. These studies show RARs directly influence osteoblast and adipocyte formation from mesenchymal cells, and inhibition of RAR signalling in vivo impairs radial bone growth in post-natal mice. - Graphical abstract: Schematic shows RAR ligand regulation of osteoblast differentiation in vitro. RARγ antagonists±RARα antagonists promote osteoblast differentiation. RARγ and RARα agonists alone or in combination block osteoblast differentiation, which correlates with upregulation of Sfrp4, and downregulation of nuclear and cytosolic β-catenin and reduced expression of the Wnt target gene Axin2. Red arrows indicate effects of RAR agonists on mediators of Wnt signalling.« less

Construction and Experimental Validation of a Petri Net Model of Wnt/β-Catenin Signaling.

PubMed

Jacobsen, Annika; Heijmans, Nika; Verkaar, Folkert; Smit, Martine J; Heringa, Jaap; van Amerongen, Renée; Feenstra, K Anton

2016-01-01

The Wnt/β-catenin signaling pathway is important for multiple developmental processes and tissue maintenance in adults. Consequently, deregulated signaling is involved in a range of human diseases including cancer and developmental defects. A better understanding of the intricate regulatory mechanism and effect of physiological (active) and pathophysiological (hyperactive) WNT signaling is important for predicting treatment response and developing novel therapies. The constitutively expressed CTNNB1 (commonly and hereafter referred to as β-catenin) is degraded by a destruction complex, composed of amongst others AXIN1 and GSK3. The destruction complex is inhibited during active WNT signaling, leading to β-catenin stabilization and induction of β-catenin/TCF target genes. In this study we investigated the mechanism and effect of β-catenin stabilization during active and hyperactive WNT signaling in a combined in silico and in vitro approach. We constructed a Petri net model of Wnt/β-catenin signaling including main players from the plasma membrane (WNT ligands and receptors), cytoplasmic effectors and the downstream negative feedback target gene AXIN2. We validated that our model can be used to simulate both active (WNT stimulation) and hyperactive (GSK3 inhibition) signaling by comparing our simulation and experimental data. We used this experimentally validated model to get further insights into the effect of the negative feedback regulator AXIN2 upon WNT stimulation and observed an attenuated β-catenin stabilization. We furthermore simulated the effect of APC inactivating mutations, yielding a stabilization of β-catenin levels comparable to the Wnt-pathway activities observed in colorectal and breast cancer. Our model can be used for further investigation and viable predictions of the role of Wnt/β-catenin signaling in oncogenesis and development.

Construction and Experimental Validation of a Petri Net Model of Wnt/β-Catenin Signaling

PubMed Central

Heijmans, Nika; Verkaar, Folkert; Smit, Martine J.; Heringa, Jaap

2016-01-01

The Wnt/β-catenin signaling pathway is important for multiple developmental processes and tissue maintenance in adults. Consequently, deregulated signaling is involved in a range of human diseases including cancer and developmental defects. A better understanding of the intricate regulatory mechanism and effect of physiological (active) and pathophysiological (hyperactive) WNT signaling is important for predicting treatment response and developing novel therapies. The constitutively expressed CTNNB1 (commonly and hereafter referred to as β-catenin) is degraded by a destruction complex, composed of amongst others AXIN1 and GSK3. The destruction complex is inhibited during active WNT signaling, leading to β-catenin stabilization and induction of β-catenin/TCF target genes. In this study we investigated the mechanism and effect of β-catenin stabilization during active and hyperactive WNT signaling in a combined in silico and in vitro approach. We constructed a Petri net model of Wnt/β-catenin signaling including main players from the plasma membrane (WNT ligands and receptors), cytoplasmic effectors and the downstream negative feedback target gene AXIN2. We validated that our model can be used to simulate both active (WNT stimulation) and hyperactive (GSK3 inhibition) signaling by comparing our simulation and experimental data. We used this experimentally validated model to get further insights into the effect of the negative feedback regulator AXIN2 upon WNT stimulation and observed an attenuated β-catenin stabilization. We furthermore simulated the effect of APC inactivating mutations, yielding a stabilization of β-catenin levels comparable to the Wnt-pathway activities observed in colorectal and breast cancer. Our model can be used for further investigation and viable predictions of the role of Wnt/β-catenin signaling in oncogenesis and development. PMID:27218469

Crosstalk Between Peroxisome Proliferator-Activated Receptor Gamma and the Canonical WNT/β-Catenin Pathway in Chronic Inflammation and Oxidative Stress During Carcinogenesis

PubMed Central

Vallée, Alexandre; Lecarpentier, Yves

2018-01-01

Inflammation and oxidative stress are common and co-substantial pathological processes accompanying, promoting, and even initiating numerous cancers. The canonical WNT/β-catenin pathway and peroxisome proliferator-activated receptor gamma (PPARγ) generally work in opposition. If one of them is upregulated, the other one is downregulated and vice versa. WNT/β-catenin signaling is upregulated in inflammatory processes and oxidative stress and in many cancers, although there are some exceptions for cancers. The opposite is observed with PPARγ, which is generally downregulated during inflammation and oxidative stress and in many cancers. This helps to explain in part the opposite and unidirectional profile of the canonical WNT/β-catenin signaling and PPARγ in these three frequent and morbid processes that potentiate each other and create a vicious circle. Many intracellular pathways commonly involved downstream will help maintain and amplify inflammation, oxidative stress, and cancer. Thus, many WNT/β-catenin target genes such as c-Myc, cyclin D1, and HIF-1α are involved in the development of cancers. Nuclear factor-kappaB (NFκB) can activate many inflammatory factors such as TNF-α, TGF-β, interleukin-6 (IL-6), IL-8, MMP, vascular endothelial growth factor, COX2, Bcl2, and inducible nitric oxide synthase. These factors are often associated with cancerous processes and may even promote them. Reactive oxygen species (ROS), generated by cellular alterations, stimulate the production of inflammatory factors such as NFκB, signal transducer and activator transcription, activator protein-1, and HIF-α. NFκB inhibits glycogen synthase kinase-3β (GSK-3β) and therefore activates the canonical WNT pathway. ROS activates the phosphatidylinositol 3 kinase/protein kinase B (PI3K/Akt) signaling in many cancers. PI3K/Akt also inhibits GSK-3β. Many gene mutations of the canonical WNT/β-catenin pathway giving rise to cancers have been reported (CTNNB1, AXIN, APC). Conversely, a significant reduction in the expression of PPARγ has been observed in many cancers. Moreover, PPARγ agonists promote cell cycle arrest, cell differentiation, and apoptosis and reduce inflammation, angiogenesis, oxidative stress, cell proliferation, invasion, and cell migration. All these complex and opposing interactions between the canonical WNT/β-catenin pathway and PPARγ appear to be fairly common in inflammation, oxidative stress, and cancers. PMID:29706964

Epigenetic mediated transcriptional activation of WNT5A participates in arsenical-associated malignant transformation

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

Jensen, Taylor J.; Wozniak, Ryan J.; Arizona Cancer Center, University of Arizona, Tucson, AZ 85724

2009-02-15

Arsenic is a human carcinogen with exposure associated with cancer of the lung, skin, and bladder. Many potential mechanisms have been implicated as playing a role in the process of arsenical-induced malignancy including the perturbation of signaling pathways and aberrant epigenetic regulation. We initiated studies to examine the role of a member of the non-canonical WNT signaling pathway, WNT5A, in UROtsa cells and arsenite [URO-ASSC] and monomethylarsonous acid [URO-MSC] malignantly transformed variants. We present data herein that suggest that WNT5A is transcriptionally activated during arsenical-induced malignant transformation. This WNT5A transcriptional activation is correlated with the enrichment of permissive histone modificationsmore » and the reduction of repressive modifications in the WNT5A promoter region. The epigenetic activation of WNT5A expression and acetylation of its promoter remain after the removal of the arsenical, consistent with the maintenance of an anchorage independent growth phenotype in these cells. Additionally, treatment with epigenetic modifying drugs supports a functional role for these epigenetic marks in controlling gene expression. Reduction of WNT5A using lentiviral shRNA greatly attenuated the ability of these cells to grow in an anchorage independent fashion. Extension of our model into human bladder cancer cell lines indicates that each of the cell lines examined also express WNT5A. Taken together, these data suggest that the epigenetic remodeling of the WNT5A promoter is correlated with its transcriptional activation and this upregulation likely participates in arsenical-induced malignant transformation.« less

The role of nutraceuticals in the regulation of Wnt and Hedgehog signaling in cancer

PubMed Central

Li, Yiwei; Wang, Zhiwei; Kong, Dejuan

2010-01-01

Multiple cellular signaling pathways have been involved in the processes of cancer cell invasion and metastasis. Among many signaling pathways, Wnt and Hedgehog (Hh) signaling pathways are critically involved in embryonic development, in the biology of cancer stem cells (CSCs) and in the acquisition of epithelial to mesenchymal transition (EMT), and thus this article will remain focused on Wnt and Hh signaling. Since CSCs and EMT are also known to be responsible for cancer cell invasion and metastasis, the Wnt and Hedgehog signaling pathways are also intimately associated with cancer invasion and metastasis. Emerging evidence suggests the beneficial role of chemopreventive agents commonly known as nutraceutical in cancer. Among many such agents, soy isoflavones, curcumin, green tea polyphenols, 3,3′-diindolylmethane, resveratrol, lycopene, vitamin D, etc. have been found to prevent, reverse, or delay the carcinogenic process. Interestingly, these agents have also shown to prevent or delay the progression of cancer, which could in part be due to their ability to attack CSCs or EMT-type cells by attenuating the Wnt and Hedgehog signaling pathways. In this review, we summarize the current state of our knowledge on the role of Wnt and Hedgehog signaling pathways, and their targeted inactivation by chemopreventive agents (nutraceuticals) for the prevention of tumor progression and/or treatment of human malignancies. PMID:20711635

Haplotypes and effects on growth traits of bovine Wnt7a gene in Chinese Qinchuan cattle.

PubMed

Xue, Jing; Sun, Yujia; Guo, Wenjiao; Yang, Ziqi; Tian, Huibin; Zhang, Chunlei; Lei, Chuzhao; Lan, Xianyong; Chen, Hong

2013-07-25

Wnt7a is a member of the WNT gene family, which encodes secreted signaling proteins and responds to many biological processes. Specifically Wnt7a influences satellite stem cells and regulates the regenerative potential of the muscle. However, similar researches about the bovine Wnt7a gene are lacking. Therefore, in this study, polymorphisms of the bovine Wnt7a gene were detected in 488 individuals from Chinese Qinchuan cattle by DNA pooling, forced PCR-RFLP, and DNA sequencing methods. 3 novel SNPs were identified, two SNPs (g.T4926C and g.A21943G) were in the intron and the last one (g.C63777T) was in the exon. Five haplotypes involved in these three variant sites in the Wnt7a gene were identified and their effects on growth traits were analyzed. The results revealed that haplotype 1 had the highest haplotype frequencies and was highly significantly associated with body height (P<0.01), body weight (P<0.05), chest width (P<0.05) and height at hip cross (P<0.01) respectively. Copyright © 2013 Elsevier B.V. All rights reserved.

Structure and functional properties of Norrin mimic Wnt for signalling with Frizzled4, Lrp5/6, and proteoglycan

PubMed Central

Chang, Tao-Hsin; Hsieh, Fu-Lien; Zebisch, Matthias; Harlos, Karl; Elegheert, Jonathan; Jones, E Yvonne

2015-01-01

Wnt signalling regulates multiple processes including angiogenesis, inflammation, and tumorigenesis. Norrin (Norrie Disease Protein) is a cystine-knot like growth factor. Although unrelated to Wnt, Norrin activates the Wnt/β-catenin pathway. Signal complex formation involves Frizzled4 (Fz4), low-density lipoprotein receptor related protein 5/6 (Lrp5/6), Tetraspanin-12 and glycosaminoglycans (GAGs). Here, we report crystallographic and small-angle X-ray scattering analyses of Norrin in complex with Fz4 cysteine-rich domain (Fz4CRD), of this complex bound with GAG analogues, and of unliganded Norrin and Fz4CRD. Our structural, biophysical and cellular data, map Fz4 and putative Lrp5/6 binding sites to distinct patches on Norrin, and reveal a GAG binding site spanning Norrin and Fz4CRD. These results explain numerous disease-associated mutations. Comparison with the Xenopus Wnt8–mouse Fz8CRD complex reveals Norrin mimics Wnt for Frizzled recognition. The production and characterization of wild-type and mutant Norrins reported here open new avenues for the development of therapeutics to combat abnormal Norrin/Wnt signalling. DOI: http://dx.doi.org/10.7554/eLife.06554.001 PMID:26158506

Expression of secreted Wnt pathway components reveals unexpected complexity of the planarian amputation response.

PubMed

Gurley, Kyle A; Elliott, Sarah A; Simakov, Oleg; Schmidt, Heiko A; Holstein, Thomas W; Sánchez Alvarado, Alejandro

2010-11-01

Regeneration is widespread throughout the animal kingdom, but our molecular understanding of this process in adult animals remains poorly understood. Wnt/β-catenin signaling plays crucial roles throughout animal life from early development to adulthood. In intact and regenerating planarians, the regulation of Wnt/β-catenin signaling functions to maintain and specify anterior/posterior (A/P) identity. Here, we explore the expression kinetics and RNAi phenotypes for secreted members of the Wnt signaling pathway in the planarian Schmidtea mediterranea. Smed-wnt and sFRP expression during regeneration is surprisingly dynamic and reveals fundamental aspects of planarian biology that have been previously unappreciated. We show that after amputation, a wounding response precedes rapid re-organization of the A/P axis. Furthermore, cells throughout the body plan can mount this response and reassess their new A/P location in the complete absence of stem cells. While initial stages of the amputation response are stem cell independent, tissue remodeling and the integration of a new A/P address with anatomy are stem cell dependent. We also show that WNT5 functions in a reciprocal manner with SLIT to pattern the planarian mediolateral axis, while WNT11-2 patterns the posterior midline. Moreover, we perform an extensive phylogenetic analysis on the Smed-wnt genes using a method that combines and integrates both sequence and structural alignments, enabling us to place all nine genes into Wnt subfamilies for the first time. Copyright © 2010 Elsevier Inc. All rights reserved.

Combined effects of dentin sialoprotein and bone morphogenetic protein-2 on differentiation in human cementoblasts.

PubMed

Lee, So-Youn; Auh, Q-Schick; Kang, Soo-Kyung; Kim, Hyung-Joon; Lee, Jung-Woo; Noh, Kwantae; Jang, Jun-Hyeog; Kim, Eun-Cheol

2014-07-01

The aim of this study is to determine the effects of the combination of recombinant human BMP-2 (rh-BMP-2) and dentin sialoprotein (rh-DSP) on growth and differentiation in human cementoblasts and determine the underlying signal transduction mechanism. Compared to treatment of cementoblasts with either rh-BMP-2 or rh-DSP alone, the combination of rh-BMP-2 and rh-DSP synergistically increased cell growth, ALP activity, nodule formation and expression of differentiation markers. The differentiation-promoting effect was also observed in periodontal ligament cells and an osteoblastic cell line. Likewise, combination of rh-DSP and rh-BMP-2 increased BMP-2 mRNA expression and Smad1/5/8 phosphorylation, which was blocked by the BMP antagonist noggin. The expression levels of α2β1 integrin and RhoA, as well as the phosphorylation status of FAK and Akt, were increased by the combination of rh-BMP-2 and rh-DSP in a time-dependent manner. In addition, rh-BMP-2 and rh-DSP enhanced expression of Wnt ligands, β-catenin activation and GSK-3β phosphorylation, all of which were inhibited by the Wnt receptor antagonist DKK1. Furthermore, treatment with rh-DSP plus rh-BMP-2 resulted in phosphorylation of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 and also induced the nuclear translocation of the NF-κB p65 subunit, which was blocked by noggin. This study demonstrates for the first time that rh-DSP and rh-BMP-2 act synergistically, enhancing each other's ability to stimulate cementoblastic cell growth and differentiation in vitro via autocrine BMP, integrin, Wnt/β-catenin, MAP kinase and NF-κB pathways. These results support the therapeutic potential of a combination strategy for aiding periodontal regeneration.

Novel inhibitors of the cellular renin-angiotensin system components, poricoic acids, target Smad3 phosphorylation and Wnt/β-catenin pathway against renal fibrosis.

PubMed

Wang, Ming; Chen, Dan-Qian; Chen, Lin; Cao, Gang; Zhao, Hui; Liu, Dan; Vaziri, Nosratola D; Guo, Yan; Zhao, Ying-Yong

2018-07-01

Tubulo-interstitial fibrosis is the final pathway in the progression of chronic kidney disease (CKD) to kidney failure. The renin-angiotensin system (RAS) plays a major role in CKD progression. Hence, we determined the efficacy of novel RAS inhibitors isolated from Poria cocos against renal fibrosis. Effects of three novel tetracyclic triterpenoid compounds, poricoic acid ZC (PZC), poricoic acid ZD (PZD) and poricoic acid ZE (PZE), were investigated on TGFβ1- and angiotensin II (AngII)-treated HK-2 cells and unilateral ureteral obstruction (UUO) in mice. Immunofluorescence staining, quantitative real-time PCR, siRNA, co-immunoprecipitation and Western blot analyses were used to evaluate expression of key molecules in RAS, Wnt/β-catenin and TGFβ/Smad pathways. Addition of the above compounds to culture media and their administration to UUO mice: (i) significantly attenuated epithelial-to-mesenchymal transition and extracellular matrix production in TGFβ1- and AngII-treated HK-2 cells and UUO mice by inhibiting Wnt/β-catenin pathway activation and Smad3 phosphorylation; (ii) selectively inhibited Smad3 phosphorylation by blocking the interaction of TGFBR1 with Smad3; and (iii) specifically inhibited Smad3 activation. PZC and PZD showed a strong inhibitory effect on all RAS components, and PZE showed a strong inhibitory effect on renin. Furthermore, the secolanostane tetracyclic triterpenoids, PZC and PZD, showed a stronger inhibitory effect than the lanostane tetracyclic triterpenoid PZE. Therefore, compounds with secolanostance skeleton showed stronger bioactivity than those with lanostance skeleton. The secolanostane tetracyclic triterpenoids effectively blocked RAS by simultaneously targeting multiple RAS components and lanostane tetracyclic triterpenoids inhibited renin and protected against tubulo-interstitial fibrosis. © 2018 The British Pharmacological Society.

Soy protein isolate inhibits hepatic tumor promotion in mice fed a high-fat liquid diet.

PubMed

Mercer, Kelly E; Pulliam, Casey F; Pedersen, Kim B; Hennings, Leah; Ronis, Martin Jj

2017-03-01

Alcoholic and nonalcoholic fatty liver diseases are risk factors for development of hepatocellular carcinoma, but the underlying mechanisms are poorly understood. On the other hand, ingestion of soy-containing diets may oppose the development of certain cancers. We previously reported that replacing casein with a soy protein isolate reduced tumor promotion in the livers of mice with alcoholic liver disease after feeding a high fat ethanol liquid diet following initiation with diethylnitrosamine. Feeding soy protein isolate inhibited processes that may contribute to tumor promotion including inflammation, sphingolipid signaling, and Wnt/β-catenin signaling. We have extended these studies to characterize liver tumor promotion in a model of nonalcoholic fatty liver disease produced by chronic feeding of high-fat liquid diets in the absence of ethanol. Mice treated with diethylnitrosamine on postnatal day 14 were fed a high-fat liquid diet made with casein or SPI as the sole protein source for 16 weeks in adulthood. Relative to mice fed normal chow, a high fat/casein diet led to increased tumor promotion, hepatocyte proliferation, steatosis, and inflammation. Replacing casein with soy protein isolate counteracted these effects. The high fat diets also resulted in a general increase in transcripts for Wnt/β-catenin pathway components, which may be an important mechanism, whereby hepatic tumorigenesis is promoted. However, soy protein isolate did not block Wnt signaling in this nonalcoholic fatty liver disease model. We conclude that replacing casein with soy protein isolate blocks development of steatosis, inflammation, and tumor promotion in diethylnitrosamine-treated mice fed high fat diets. Impact statement The impact of dietary components on cancer is a topic of great interest for both the general public and the scientific community. Liver cancer is currently the second leading form of cancer deaths worldwide. Our study has addressed the effect of the protein source on hepatic tumor promotion in a mouse model reflecting aspects of non-alcoholic fatty liver disease (NAFLD). A high-fat liquid diet with casein as the protein source promotes hepatic injury and tumor promotion in diethylnitrosamine-treated mice. Replacing casein with a soy protein isolate led to a pronounced diminishment of tumor promotion and associated hepatic injury and inflammation. The study thus demonstrates that a dietary protein source can have beneficial, preventative effects on hepatic tumor promotion.

Wnt ligands from the embryonic surface ectoderm regulate ‘bimetallic strip’ optic cup morphogenesis in mouse

PubMed Central

Carpenter, April C.; Smith, April N.; Wagner, Heidi; Cohen-Tayar, Yamit; Rao, Sujata; Wallace, Valerie; Ashery-Padan, Ruth; Lang, Richard A.

2015-01-01

The Wnt/β-catenin response pathway is central to many developmental processes. Here, we assessed the role of Wnt signaling in early eye development using the mouse as a model system. We showed that the surface ectoderm region that includes the lens placode expressed 12 out of 19 possible Wnt ligands. When these activities were suppressed by conditional deletion of wntless (Le-cre; Wlsfl/fl) there were dramatic consequences that included a saucer-shaped optic cup, ventral coloboma, and a deficiency of periocular mesenchyme. This phenotype shared features with that produced when the Wnt/β-catenin pathway co-receptor Lrp6 is mutated or when retinoic acid (RA) signaling in the eye is compromised. Consistent with this, microarray and cell fate marker analysis identified a series of expression changes in genes known to be regulated by RA or by the Wnt/β-catenin pathway. Using pathway reporters, we showed that Wnt ligands from the surface ectoderm directly or indirectly elicit a Wnt/β-catenin response in retinal pigment epithelium (RPE) progenitors near the optic cup rim. In Le-cre; Wlsfl/fl mice, the numbers of RPE cells are reduced and this can explain, using the principle of the bimetallic strip, the curvature of the optic cup. These data thus establish a novel hypothesis to explain how differential cell numbers in a bilayered epithelium can lead to shape change. PMID:25715397

An Inhibitory Role of Osthole in Rat MSCs Osteogenic Differentiation and Proliferation via Wnt/β-Catenin and Erk1/2-MAPK Pathways.

PubMed

Hu, Hongyang; Chen, Min; Dai, Guangzu; Du, Guoqing; Wang, Xuezong; He, Jie; Zhao, Yongfang; Han, Dapeng; Cao, Yuelong; Zheng, Yuxin; Ding, Daofang

2016-01-01

Bone marrow-derived mesenchymal stem cells (MSCs) are responsible for new bone formation during adulthood. Accumulating evidences showed that Osthole promotes the osteogenic differentiation in primary osteoblasts. The aim of this study was to investigate whether Osthole exhibits a potential to stimulate the osteogenic differentiation of MSCs and the underlying mechanism. MSCs were treated with a gradient concentration of Osthole (6.25 µM, 12.5 µM, and 25 µM). Cell proliferation was assessed by western blotting with the proliferating cell nuclear antigen (PCNA) and Cyclin D1 antibodies, fluorescence activated cell sorting (FACS), and cell counting kit 8 (CCK8). MSCs were cultured in osteogenesis-induced medium for one or two weeks. The osteogenic differentiation of MSCs was estimated by Alkaline Phosphatase (ALP) staining, Alizarin red staining, Calcium influx, and quantitative PCR (qPCR). The underlying mechanism of Osthole-induced osteogenesis was further evaluated by western blotting with antibodies in Wnt/β-catenin, PI3K/Akt, BMPs/smad1/5/8, and MAPK signaling pathways. Osthole inhibited proliferation of rat MSCs in a dose-dependent manner. Osthole suppressed osteogenic differentiation of rat MSCs by down-regulating the activities of Wnt/β-catenin and Erk1/2-MAPK signaling. Osthole inhibits the proliferation and osteogenic differentiation of rat MSCs, which might be mediated through blocking the Wnt/β-catenin and Erk1/2-MAPK signaling pathways. © 2016 The Author(s) Published by S. Karger AG, Basel.

Wnt Signaling Cross-Talks with JH Signaling by Suppressing Met and gce Expression

PubMed Central

Abdou, Mohamed; Peng, Cheng; Huang, Jianhua; Zyaan, Ola; Wang, Sheng; Li, Sheng; Wang, Jian

2011-01-01

Juvenile hormone (JH) plays key roles in controlling insect growth and metamorphosis. However, relatively little is known about the JH signaling pathways. Until recent years, increasing evidence has suggested that JH modulates the action of 20-hydroxyecdysone (20E) by regulating expression of broad (br), a 20E early response gene, through Met/Gce and Kr-h1. To identify other genes involved in JH signaling, we designed a novel Drosophila genetic screen to isolate mutations that derepress JH-mediated br suppression at early larval stages. We found that mutations in three Wnt signaling negative regulators in Drosophila, Axin (Axn), supernumerary limbs (slmb), and naked cuticle (nkd), caused precocious br expression, which could not be blocked by exogenous JHA. A similar phenotype was observed when armadillo (arm), the mediator of Wnt signaling, was overexpressed. qRT-PCR revealed that Met, gce and Kr-h1expression was suppressed in the Axn, slmb and nkd mutants as well as in arm gain-of-function larvae. Furthermore, ectopic expression of gce restored Kr-h1 expression but not Met expression in the arm gain-of-function larvae. Taken together, we conclude that Wnt signaling cross-talks with JH signaling by suppressing transcription of Met and gce, genes that encode for putative JH receptors. The reduced JH activity further induces down-regulation of Kr-h1expression and eventually derepresses br expression in the Drosophila early larval stages. PMID:22087234

Wnt/β-catenin signaling in dermal condensates is required for hair follicle formation

PubMed Central

Tsai, Su-Yi; Sennett, Rachel; Rezza, Amélie; Clavel, Carlos; Grisanti, Laura; Zemla, Roland; Najam, Sara; Rendl, Michael

2014-01-01

Broad dermal Wnt signaling is required for patterned induction of hair follicle placodes and subsequent Wnt signaling in placode stem cells is essential for induction of dermal condensates, cell clusters of precursors for the hair follicle dermal papilla (DP). Progression of hair follicle formation then requires coordinated signal exchange between dermal condensates and placode stem cells. However, it remains unknown whether continued Wnt signaling in DP precursor cells plays a role in this process, largely due to the long-standing inability to specifically target dermal condensates for gene ablation. Here we use the Tbx18Cre knockin mouse line to ablate the Wnt-responsive transcription factor β-catenin specifically in these cells at E14.5 during the first wave of guard hair follicle formation. In the absence of β-catenin, canonical Wnt signaling is effectively abolished in these cells. Sox2+ dermal condensates initiate normally, however by E16.5 guard hair follicle numbers are strongly reduced and by E18.5 most whiskers and guard hair follicles are absent, suggesting that active Wnt signaling in dermal condensates is important for hair follicle formation to proceed after induction. To explore the molecular mechanisms by which Wnt signaling in dermal condensates regulates hair follicle formation, we analyze genome-wide the gene expression changes in embryonic β-catenin null DP precursor cells. We find altered expression of several signaling pathway genes, including Fgfs and Activin, both previously implicated in hair follicle formation. In summary, these data reveal a functional role of Wnt signaling in DP precursors for embryonic hair follicle formation and identify Fgf and Activin signaling as potential effectors of Wnt signaling-regulated events. PMID:24309208

Wnt/β-Catenin Signaling Modulates Human Airway Sensitization Induced by β2-Adrenoceptor Stimulation

PubMed Central

Faisy, Christophe; Grassin-Delyle, Stanislas; Blouquit-Laye, Sabine; Brollo, Marion; Naline, Emmanuel; Chapelier, Alain; Devillier, Philippe

2014-01-01

Background Regular use of β2-agonists may enhance non-specific airway responsiveness. The wingless/integrated (Wnt) signaling pathways are responsible for several cellular processes, including airway inflammation and remodeling while cAMP–PKA cascade can activate the Wnt signaling. We aimed to investigate whether the Wnt signaling pathways are involved in the bronchial hyperresponsiveness induced by prolonged exposure to β2-adrenoceptor agonists in human isolated airways. Methods Bronchi were surgically removed from 44 thoracic surgery patients. After preparation, bronchial rings and primary cultures of bronchial epithelial cells were incubated with fenoterol (0.1 µM, 15 hours, 37°C), a β2-agonist with high intrinsic efficacy. The effects of inhibitors/blockers of Wnt signaling on the fenoterol-induced airway sensitization were examined and the impact of fenoterol exposure on the mRNA expression of genes interacting with Wnt signaling or cAMP–PKA cascade was assessed in complete bronchi and in cultured epithelial cells. Results Compared to paired controls, fenoterol-sensitization was abolished by inhibition/blockage of the Wnt/β-catenin signaling, especially the cell-surface LRP5/6 co-receptors or Fzd receptors (1 µM SFRP1 or 1 µM DKK1) and the nuclear recruitment of TCF/LEF transcriptions factors (0.3 µM FH535). Wnt proteins secretion did not seem to be involved in the fenoterol-induced sensitization since the mRNA expression of Wnt remained low after fenoterol exposure and the inactivator of Wnt secretion (1 µM IWP2) had no effect on the fenoterol-sensitization. Fenoterol exposure did not change the mRNA expression of genes regulating Wnt signaling or cAMP–PKA cascade. Conclusions Collectively, our pharmacological investigations indicate that fenoterol-sensitization is modulated by the inhibition/blockage of canonical Wnt/β-catenin pathway, suggesting a phenomenon of biased agonism in connection with the β2-adrenoceptor stimulation. Future experiments based on the results of the present study will be needed to determine the impact of prolonged fenoterol exposure on the extra- and intracellular Wnt signaling pathways at the protein expression level. PMID:25360795

Wnt/β-catenin signaling modulates human airway sensitization induced by β2-adrenoceptor stimulation.

PubMed

Faisy, Christophe; Grassin-Delyle, Stanislas; Blouquit-Laye, Sabine; Brollo, Marion; Naline, Emmanuel; Chapelier, Alain; Devillier, Philippe

2014-01-01

Regular use of β2-agonists may enhance non-specific airway responsiveness. The wingless/integrated (Wnt) signaling pathways are responsible for several cellular processes, including airway inflammation and remodeling while cAMP-PKA cascade can activate the Wnt signaling. We aimed to investigate whether the Wnt signaling pathways are involved in the bronchial hyperresponsiveness induced by prolonged exposure to β2-adrenoceptor agonists in human isolated airways. Bronchi were surgically removed from 44 thoracic surgery patients. After preparation, bronchial rings and primary cultures of bronchial epithelial cells were incubated with fenoterol (0.1 µM, 15 hours, 37 °C), a β2-agonist with high intrinsic efficacy. The effects of inhibitors/blockers of Wnt signaling on the fenoterol-induced airway sensitization were examined and the impact of fenoterol exposure on the mRNA expression of genes interacting with Wnt signaling or cAMP-PKA cascade was assessed in complete bronchi and in cultured epithelial cells. Compared to paired controls, fenoterol-sensitization was abolished by inhibition/blockage of the Wnt/β-catenin signaling, especially the cell-surface LRP5/6 co-receptors or Fzd receptors (1 µM SFRP1 or 1 µM DKK1) and the nuclear recruitment of TCF/LEF transcriptions factors (0.3 µM FH535). Wnt proteins secretion did not seem to be involved in the fenoterol-induced sensitization since the mRNA expression of Wnt remained low after fenoterol exposure and the inactivator of Wnt secretion (1 µM IWP2) had no effect on the fenoterol-sensitization. Fenoterol exposure did not change the mRNA expression of genes regulating Wnt signaling or cAMP-PKA cascade. Collectively, our pharmacological investigations indicate that fenoterol-sensitization is modulated by the inhibition/blockage of canonical Wnt/β-catenin pathway, suggesting a phenomenon of biased agonism in connection with the β2-adrenoceptor stimulation. Future experiments based on the results of the present study will be needed to determine the impact of prolonged fenoterol exposure on the extra- and intracellular Wnt signaling pathways at the protein expression level.

RSPOs facilitated HSC activation and promoted hepatic fibrogenesis

PubMed Central

Yin, Xinguang; Yi, Huixing; Wang, Linlin; Wu, Wanxin; Wu, Xiaojun; Yu, Linghua

2016-01-01

Roof plate-specific spondin (RSPO) proteins are potent Wnt pathway agonists and involve in a broad range of developmental and physiological processes. This study investigated the activities and mechanisms of RSPOs in liver fibrogenesis, especially in hepatic stellate cell (HSC) activation. HSC activation was assessed by fibrosis biomarker (α-smooth muscle actin and Collagen-I), phenotypic change (accumulation of lipid droplets), and increased proliferation. Similarly, Wnt pathway activity was evaluated by the expression of nuclear β-catenin and T cell-specific transcription factors (TCF) activity. We found RSPOs were overexpressed in human fibrotic liver tissue and the expressions were correlated with liver fibrosis stages. In vitro studies showed RSPOs level increased during HSC activation, and stimuli with RSPOs enhanced Wnt pathway activity and promoted HSC activation subsequently. Furthermore, in vivo experiments demonstrated that the knockdown of RSPOs suppressed both Wnt pathway activity and HSC activation. Interestingly, the inhibitor of the Wnt signaling pathway Dickkopf1 impairs RSPOs effects on HSCs. Taken together, our results revealed that RSPOs facilitated HSC activation and promote liver fibrogenesis by enhancing the Wnt pathway. PMID:27572318

RSPOs facilitated HSC activation and promoted hepatic fibrogenesis.

PubMed

Yin, Xinguang; Yi, Huixing; Wang, Linlin; Wu, Wanxin; Wu, Xiaojun; Yu, Linghua

2016-09-27

Roof plate-specific spondin (RSPO) proteins are potent Wnt pathway agonists and involve in a broad range of developmental and physiological processes. This study investigated the activities and mechanisms of RSPOs in liver fibrogenesis, especially in hepatic stellate cell (HSC) activation. HSC activation was assessed by fibrosis biomarker (α-smooth muscle actin and Collagen-I), phenotypic change (accumulation of lipid droplets), and increased proliferation. Similarly, Wnt pathway activity was evaluated by the expression of nuclear β-catenin and T cell-specific transcription factors (TCF) activity. We found RSPOs were overexpressed in human fibrotic liver tissue and the expressions were correlated with liver fibrosis stages. In vitro studies showed RSPOs level increased during HSC activation, and stimuli with RSPOs enhanced Wnt pathway activity and promoted HSC activation subsequently. Furthermore, in vivo experiments demonstrated that the knockdown of RSPOs suppressed both Wnt pathway activity and HSC activation. Interestingly, the inhibitor of the Wnt signaling pathway Dickkopf1 impairs RSPOs effects on HSCs. Taken together, our results revealed that RSPOs facilitated HSC activation and promote liver fibrogenesis by enhancing the Wnt pathway.

Recessive palmoplantar keratodermas: a tale of wings, hands, hair and cancer.

PubMed

Van Steensel, M A M

2010-12-01

The palmoplantar keratodermas (PPKs) are a heterogeneous group of disorders of cornification affecting the palms and soles. Of late, a number of rare, recessive PPKs such as odonto-onycho-dermal dysplasia have been elucidated. Surprisingly, these results indicate that correct palmoplantar keratinization depends on intact Wingless (WNT) signalling. WNT was originally discovered in the fruit fly where it is required for wing morphogenesis. This ancient signalling pathway is now emerging as a master regulator of differentiation in a variety of tissues, including the intestine and the hair follicle. It is also becoming increasingly clear that deregulation of WNT signalling is involved in neoplasia. Thus, a single pathway unites several seemingly disparate processes and disorders. The keratodermas are emerging as model systems in which to study WNT signalling. Moreover, as agents that are in the dermatological arsenal can modulate WNT signalling, some insight into its workings is of importance to the practicing dermatologist. In this review, I outline how WNT signalling is involved in epidermal differentiation and skin cancer and what these new insights mean for everyday dermatology.

Wnt7a interaction with Fzd5 and detection of signaling activation using a split eGFP

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

Carmon, Kendra S.; Loose, David S.

2008-04-04

Wnts are secreted glycoproteins that regulate important cellular processes including proliferation, differentiation, and cell fate. In the {beta}-catenin/canonical pathway, Wnt interacts with Fzd receptors to inhibit degradation of {beta}-catenin and promote its translocation into the nucleus where it regulates transcription of a number of genes. Dysregulation of this pathway has been attributed to a host of diseases including cancer. As a result, components of the {beta}-catenin/canonical pathway have been gaining recognition as promising targets for the discovery of novel therapeutic agents. Here, we show, using an ELISA-based protein-protein binding assay that purified Wnt7a binds to the extracellular cysteine-rich domain ofmore » Fzd5 in the nanomolar range. We have developed a novel split eGFP complementation assay to visually detect Wnt7a-Fzd5 interactions and subsequent pathway activation in cells. These biological tools could help lead to a better understanding of Wnt-Fzd interactions and the identification of new modulators of Wnt signaling.« less

Wnt/beta-catenin pathway activation and myogenic differentiation are induced by cholesterol depletion.

PubMed

Mermelstein, Cláudia S; Portilho, Débora M; Mendes, Fábio A; Costa, Manoel L; Abreu, José Garcia

2007-03-01

Myogenic differentiation is a multistep process that begins with the commitment of mononucleated precursors that withdraw from cell cycle. These myoblasts elongate while aligning to each other, guided by the recognition between their membranes. This step is followed by cell fusion and the formation of long and striated multinucleated myotubes. We have recently shown that cholesterol depletion by methyl-beta-cyclodextrin (MbetaCD) induces myogenic differentiation by enhancing myoblast recognition and fusion. Here, we further studied the signaling pathways responsible for early steps of myogenesis. As it is known that Wnt plays a role in muscle differentiation, we used the chemical MbetaCD to deplete membrane cholesterol and investigate the involvement of the Wnt/beta-catenin pathway during myogenesis. We show that cholesterol depletion promoted a significant increase in expression of beta-catenin, its nuclear translocation and activation of the Wnt pathway. Moreover, we show that the activation of the Wnt pathway after cholesterol depletion can be inhibited by the soluble protein Frzb-1. Our data suggest that membrane cholesterol is involved in Wnt/beta-catenin signaling in the early steps of myogenic differentiation.

FAK and BMP-9 synergistically trigger osteogenic differentiation and bone formation of adipose derived stem cells through enhancing Wnt-β-catenin signaling.

PubMed

Yuan, Cheng; Gou, Xiaoli; Deng, Jiang; Dong, Zhijun; Ye, Peng; Hu, Zhenming

2018-06-14

Adipose derived stem cells (ADSCs) could undergo osteogenesis via focal adhesion kinase (FAK) and bone morphogenetic protein (BMP) 9 signals, both of which could affect Wnt-β-catenin signal, a signal pathway closely related to ADSCs osteogenesis. It's still enigma whether FAK and BMP-9 contribute to osteogenesis. Here, we examined the effect of FAK on BMP9-inducedosteogenic differentiation, unveiled the possible molecular mechanism underling this process. In the present study, ADSCs were isolated and purified, and cells of passage 3 underwent virus mediated transfection to prepare ADSCs with stable FAK shRNA expression. Cell viability and migration were detected by MTT and transwell assay, respectively. Expression of osteogenic gene, phosphorylation of FAK and GSK were detected by western blot. Osteogenic potential was evaluated by activity of alkaline phosphatase (ALP) and calcium deposition by ALP staining and Alizarin Red S staining. BMP-9 administration promoted ADSCs osteogenesis. Knocking down FAK attenuated this process, inhibited osteogenic proteins expression through Wnt-β-catenin signal. BMP-9 also triggered ADSCs proliferation and migration, and shFAK antagonized such effects too. Although Wnt signal is affected by FAK shRNA, Smad signal remains intact in ADSCs with shFAK. FAK and BMP-9 could cross talk on Wnt signal pathway and promote ADSCs osteogenesis. FAK could participate in BMP-9 induced ADSCs osteogenesis via Wnt signal pathway other than Smads signals (see in graph). Copyright © 2018. Published by Elsevier Masson SAS.

Emerging Insights into Wnt/β-catenin Signaling in Head and Neck Cancer.

PubMed

Alamoud, K A; Kukuruzinska, M A

2018-06-01

Head and neck cancer presents primarily as head and neck squamous cell carcinoma (HNSCC), a debilitating malignancy fraught with high morbidity, poor survival rates, and limited treatment options. Mounting evidence indicates that the Wnt/β-catenin signaling pathway plays important roles in the pathobiology of HNSCC. Wnt/β-catenin signaling affects multiple cellular processes that endow cancer cells with the ability to maintain and expand immature stem-like phenotypes, proliferate, extend survival, and acquire aggressive characteristics by adopting mesenchymal traits. A central component of canonical Wnt signaling is β-catenin, which balances its role as a structural component of E-cadherin junctions with its function as a transcriptional coactivator of numerous target genes. Recent genomic characterization of head and neck cancer revealed that while β-catenin is not frequently mutated in HNSCC, its activity is unchecked by more common mutations in genes encoding upstream regulators of β-catenin, NOTCH1, FAT1, and AJUBA. Wnt/β-catenin signaling affects a wide range epigenetic and transcriptional activities, mediated by the interaction of β-catenin with different transcription factors and transcriptional coactivators and corepressors. Furthermore, Wnt/β-catenin functions in a network with many signaling and metabolic pathways that modulate its activity. In addition to its effects on tumor epithelia, β-catenin activity regulates the tumor microenvironment by regulating extracellular matrix remodeling, fibrotic processes, and immune response. These multifunctional oncogenic effects of β-catenin make it an attractive bona fide target for HNSCC therapy.

Wnt/Yes-Associated Protein Interactions During Neural Tissue Patterning of Human Induced Pluripotent Stem Cells.

PubMed

Bejoy, Julie; Song, Liqing; Zhou, Yi; Li, Yan

2018-04-01

Human induced pluripotent stem cells (hiPSCs) have special ability to self-assemble into neural spheroids or mini-brain-like structures. During the self-assembly process, Wnt signaling plays an important role in regional patterning and establishing positional identity of hiPSC-derived neural progenitors. Recently, the role of Wnt signaling in regulating Yes-associated protein (YAP) expression (nuclear or cytoplasmic), the pivotal regulator during organ growth and tissue generation, has attracted increasing interests. However, the interactions between Wnt and YAP expression for neural lineage commitment of hiPSCs remain poorly explored. The objective of this study is to investigate the effects of Wnt signaling and YAP expression on the cellular population in three-dimensional (3D) neural spheroids derived from hiPSCs. In this study, Wnt signaling was activated using CHIR99021 for 3D neural spheroids derived from human iPSK3 cells through embryoid body formation. Our results indicate that Wnt activation induces nuclear localization of YAP and upregulates the expression of HOXB4, the marker for hindbrain/spinal cord. By contrast, the cells exhibit more rostral forebrain neural identity (expression of TBR1) without Wnt activation. Cytochalasin D was then used to induce cytoplasmic YAP and the results showed the decreased HOXB4 expression. In addition, the incorporation of microparticles in the neural spheroids was investigated for the perturbation of neural patterning. This study may indicate the bidirectional interactions of Wnt signaling and YAP expression during neural tissue patterning, which have the significance in neurological disease modeling, drug screening, and neural tissue regeneration.

TCF3, a novel positive regulator of osteogenesis, plays a crucial role in miR-17 modulating the diverse effect of canonical Wnt signaling in different microenvironments

PubMed Central

Liu, W; Liu, Y; Guo, T; Hu, C; Luo, H; Zhang, L; Shi, S; Cai, T; Ding, Y; Jin, Y

2013-01-01

Wnt signaling pathways are a highly conserved pathway, which plays an important role from the embryonic development to bone formation. The effect of Wnt pathway on osteogenesis relies on their cellular environment and the expression of target genes. However, the molecular mechanism of that remains unclear. On the basis of the preliminary results, we observed the contrary effect of canonical Wnt signaling on osteogenic differentiation of periodontal ligament stem cells (PDLSCs) in the different culture environment. Furthermore, we found that the expression level of miR-17 was also varied with the change in the culture environment. Therefore, we hypothesized that miR-17 and canonical Wnt signaling may have potential interactions, particularly the inner regulation relationship in different microenvironments. In this paper, we observed that canonical Wnt signaling promoted osteogenesis of PDLSCs in the fully culture medium, while inhibited it in the osteogenic differentiation medium. Interestingly, alteration in the expression level of endogenous miR-17 could partially reverse the different effect of canonical Wnt signaling. Furthermore, the role of miR-17 was because of its target gene TCF3 (transcription factor 3), a key transcription factor of canonical Wnt pathway. Overexpression of TCF3 attenuated the effect of miR-17 on modulating canonical Wnt signaling. Finally, we elucidated that TCF3 enhanced osteogenesis both in vitro and in vivo. In brief, the different level of miR-17 was the main cause of the different effect of canonical Wnt signaling, and TCF3 was the crucial node of miR-17–canonial Wnt signaling regulation loop. This understanding of microRNAs regulating signaling pathways in different microenvironments may pave the way for fine-tuning the process of osteogenesis in bone-related disorders. PMID:23492770

TCF3, a novel positive regulator of osteogenesis, plays a crucial role in miR-17 modulating the diverse effect of canonical Wnt signaling in different microenvironments.

PubMed

Liu, W; Liu, Y; Guo, T; Hu, C; Luo, H; Zhang, L; Shi, S; Cai, T; Ding, Y; Jin, Y

2013-03-14

Wnt signaling pathways are a highly conserved pathway, which plays an important role from the embryonic development to bone formation. The effect of Wnt pathway on osteogenesis relies on their cellular environment and the expression of target genes. However, the molecular mechanism of that remains unclear. On the basis of the preliminary results, we observed the contrary effect of canonical Wnt signaling on osteogenic differentiation of periodontal ligament stem cells (PDLSCs) in the different culture environment. Furthermore, we found that the expression level of miR-17 was also varied with the change in the culture environment. Therefore, we hypothesized that miR-17 and canonical Wnt signaling may have potential interactions, particularly the inner regulation relationship in different microenvironments. In this paper, we observed that canonical Wnt signaling promoted osteogenesis of PDLSCs in the fully culture medium, while inhibited it in the osteogenic differentiation medium. Interestingly, alteration in the expression level of endogenous miR-17 could partially reverse the different effect of canonical Wnt signaling. Furthermore, the role of miR-17 was because of its target gene TCF3 (transcription factor 3), a key transcription factor of canonical Wnt pathway. Overexpression of TCF3 attenuated the effect of miR-17 on modulating canonical Wnt signaling. Finally, we elucidated that TCF3 enhanced osteogenesis both in vitro and in vivo. In brief, the different level of miR-17 was the main cause of the different effect of canonical Wnt signaling, and TCF3 was the crucial node of miR-17-canonial Wnt signaling regulation loop. This understanding of microRNAs regulating signaling pathways in different microenvironments may pave the way for fine-tuning the process of osteogenesis in bone-related disorders.

Wnt inhibition enhances browning of mouse primary white adipocytes.

PubMed

Lo, Kinyui Alice; Ng, Pei Yi; Kabiri, Zahra; Virshup, David; Sun, Lei

2016-01-01

The global epidemic in obesity and metabolic syndrome requires novel approaches to tackle. White adipose tissue, traditionally seen as a passive energy-storage organ, can be induced to take on certain characteristics of brown fat in a process called browning. The "browned" white adipose tissue, or beige fat, is a potential anti-obesity target. Various signaling pathways can enhance browning. Wnt is a key regulator of adipocyte biology, but its role in browning has not been explored. In this study, we found that in primary mouse adipocytes derived from the inguinal depot, Wnt inhibition by both chemical and genetic methods significantly enhanced browning. The effect of Wnt inhibition on browning most likely targets the beige precursor cells in selected adipose depots.

Chondroitin sulfatases differentially regulate Wnt signaling in prostate stem cells through effects on SHP2, phospho-ERK1/2, and Dickkopf Wnt signaling pathway inhibitor (DKK3)

PubMed Central

Bhattacharyya, Sumit; Feferman, Leo; Tobacman, Joanne K.

2017-01-01

The chondroitin sulfatases N-acetylgalactosamine-4-sulfatase (ARSB) and galactosamine-N-acetyl-6-sulfatase (GALNS) remove either the 4-sulfate group at the non-reducing end of chondroitin 4-sulfate (C4S) and dermatan sulfate, or the 6-sulfate group of chondroitin 6-sulfate, chondroitin 4,6-disulfate (chondroitin sulfate E), or keratan sulfate. In human prostate cancer tissues, the ARSB activity was reduced and the GALNS activity was increased, compared to normal prostate tissue. In human prostate stem cells, when ARSB was reduced by silencing or GALNS was increased by overexpression, activity of SHP2, the ubiquitous non-receptor tyrosine phosphatase, declined, attributable to increased binding of SHP2 with C4S. This led to increases in phospho-ERK1/2, Myc/Max nuclear DNA binding, DNA methyltransferase (DNMT) activity and expression, and methylation of the Dickkopf Wnt signaling pathway inhibitor (DKK)3 promoter and to reduced DKK3 expression. Since DKK3 negatively regulates Wnt/β-catenin signaling, silencing of ARSB or overexpression of GALNS disinhibited (increased) Wnt/β-catenin signaling. These findings indicate that the chondroitin sulfatases can exert profound effects on Wnt-mediated processes, due to epigenetic effects that modulate Wnt signaling. PMID:29245974

The tangled web of non-canonical Wnt signalling in neural migration.

PubMed

Clark, Charlotte E J; Nourse, C Cathrin; Cooper, Helen M

2012-01-01

In all multicellular animals, successful embryogenesis is dependent on the ability of cells to detect the status of the local environment and respond appropriately. The nature of the extracellular environment is communicated to the intracellular compartment by ligand/receptor interactions at the cell surface. The Wnt canonical and non-canonical signalling pathways are found in the most primitive metazoans, and they play an essential role in the most fundamental developmental processes in all multicellular organisms. Vertebrates have expanded the number of Wnts and Frizzled receptors and have additionally evolved novel Wnt receptor families (Ryk, Ror). The multiplicity of potential interactions between Wnts, their receptors and downstream effectors has exponentially increased the complexity of the signal transduction network. Signalling through each of the Wnt pathways, as well as crosstalk between them, plays a critical role in the establishment of the complex architecture of the vertebrate central nervous system. In this review, we explore the signalling networks triggered by non-canonical Wnt/receptor interactions, focussing on the emerging roles of the non-conventional Wnt receptors Ryk and Ror. We describe the role of these pathways in neural tube formation and axon guidance where Wnt signalling controls tissue polarity, coordinated cell migration and axon guidance via remodelling of the cytoskeleton. Copyright © 2012 S. Karger AG, Basel.

Mutation of KREMEN1, a modulator of Wnt signaling, is responsible for ectodermal dysplasia including oligodontia in Palestinian families.

PubMed

Issa, Yasmin A; Kamal, Lara; Rayyan, Amal Abu; Dweik, Dima; Pierce, Sarah; Lee, Ming K; King, Mary-Claire; Walsh, Tom; Kanaan, Moien

2016-10-01

Tooth development is controlled by the same processes that regulate formation of other ectodermal structures. Mutations in the genes underlying these processes may cause ectodermal dysplasia, including severe absence of primary or permanent teeth. Four consanguineous Palestinian families presented with oligodontia and hair and skin features of ectodermal dysplasia. Appearance of ectodermal dysplasia was consistent with autosomal recessive inheritance. Exome sequencing followed by genotyping of 56 informative relatives in the 4 families suggests that the phenotype is due to homozygosity for KREMEN1 p.F209S (c.626 T>C) on chromosome 22 at g.29,521,399 (hg19). The variant occurs in the highly conserved extracellular WSC domain of KREMEN1, which is known to be a high affinity receptor of Dickkopf-1, a component of the Dickkopf-Kremen-LRP6 complex, and a potent regulator of Wnt signaling. The Wnt signaling pathway is critical to development of ectodermal structures. Mutations in WNT10A, LRP6, EDA, and other genes in this pathway lead to tooth agenesis with or without other ectodermal anomalies. Our results implicate KREMEN1 for the first time in a human disorder and provide additional details on the role of the Wnt signaling in ectodermal and dental development.

The Tumor Suppressor Actions of the Vitamin D Receptor in Skin

DTIC Science & Technology

2014-10-01

induced tumor formation. In previous studies we determined that the hedgehog (HH) and wnt/β-catenin pathways were activated in the skin of VDR null...SUBJECT TERMS epidermal tumors, keratinocytes, vitamin D receptor, sonic hedgehog , β-catenin, UVB 16. SECURITY CLASSIFICATION OF: 17. LIMITATION...epidermal tumor formation by blocking the β-catenin and hedgehog pathways, key pathways in keratinocyte proliferation that if left unchecked lead to

Frizzled 2 and frizzled 7 function redundantly in convergent extension and closure of the ventricular septum and palate: evidence for a network of interacting genes

PubMed Central

Yu, Huimin; Ye, Xin; Guo, Nini; Nathans, Jeremy

2012-01-01

Frizzled (Fz) 2 and Fz7, together with Fz1, form a distinct subfamily within the Frizzled family of Wnt receptors. Using targeted gene deletion, we show that: Fz7−/− mice exhibit tail truncation and kinking with 100% penetrance and ventricular septal defects (VSDs) with ~15% penetrance; Fz2+/−;Fz7−/− mice exhibit VSDs with ~50% penetrance and cleft palate with less than 10% penetrance; and Fz2−/−;Fz7−/− mice exhibit convergent extension defects and mid-gestational lethality with 100% penetrance. When Fz2 and/or Fz7 mutations are combined with mutations in Vangl2, Dvl3, Wnt3a, Wnt5a or Wnt11, an increased frequency of VSDs is observed with Dvl3, Wnt3a and Wnt11; an increased frequency of palate closure defects is observed with Vangl2; and early lethality and enhanced tail shortening are observed with Wnt5a. To assess the signaling pathways that underlie these and other Frizzled-mediated genetic interactions, we used transfected mammalian cells to analyze (1) canonical Wnt signaling induced by all pairwise combinations of the ten mouse Frizzleds and the 19 mouse Wnts and (2) localization of each Frizzled at cell-cell junctional complexes formed by mouse Celsr1, a likely indicator of competence for planar cell polarity signaling. These in vitro experiments indicate that Fz2 and Fz7 are competent to signal via the canonical pathway. Taken together, the data suggest that genetic interactions between Fz2, Fz7 and Vangl2, Dvl3 and Wnt genes reflect interactions among different signaling pathways in developmental processes that are highly sensitive to perturbations in Frizzled signaling. PMID:23095888

Identification of Wnt Pathway Target Genes Regulating the Division and Differentiation of Larval Seam Cells and Vulval Precursor Cells in Caenorhabditis elegans

PubMed Central

Gorrepati, Lakshmi; Krause, Michael W.; Chen, Weiping; Brodigan, Thomas M.; Correa-Mendez, Margarita; Eisenmann, David M.

2015-01-01

The evolutionarily conserved Wnt/β-catenin signaling pathway plays a fundamental role during metazoan development, regulating numerous processes including cell fate specification, cell migration, and stem cell renewal. Wnt ligand binding leads to stabilization of the transcriptional effector β-catenin and upregulation of target gene expression to mediate a cellular response. During larval development of the nematode Caenorhabditis elegans, Wnt/β-catenin pathways act in fate specification of two hypodermal cell types, the ventral vulval precursor cells (VPCs) and the lateral seam cells. Because little is known about targets of the Wnt signaling pathways acting during larval VPC and seam cell differentiation, we sought to identify genes regulated by Wnt signaling in these two hypodermal cell types. We conditionally activated Wnt signaling in larval animals and performed cell type–specific "mRNA tagging" to enrich for VPC and seam cell–specific mRNAs, and then used microarray analysis to examine gene expression compared to control animals. Two hundred thirty-nine genes activated in response to Wnt signaling were identified, and we characterized 50 genes further. The majority of these genes are expressed in seam and/or vulval lineages during normal development, and reduction of function for nine genes caused defects in the proper division, fate specification, fate execution, or differentiation of seam cells and vulval cells. Therefore, the combination of these techniques was successful at identifying potential cell type–specific Wnt pathway target genes from a small number of cells and at increasing our knowledge of the specification and behavior of these C. elegans larval hypodermal cells. PMID:26048561

Identification of Wnt Pathway Target Genes Regulating the Division and Differentiation of Larval Seam Cells and Vulval Precursor Cells in Caenorhabditis elegans.

PubMed

Gorrepati, Lakshmi; Krause, Michael W; Chen, Weiping; Brodigan, Thomas M; Correa-Mendez, Margarita; Eisenmann, David M

2015-06-05

The evolutionarily conserved Wnt/β-catenin signaling pathway plays a fundamental role during metazoan development, regulating numerous processes including cell fate specification, cell migration, and stem cell renewal. Wnt ligand binding leads to stabilization of the transcriptional effector β-catenin and upregulation of target gene expression to mediate a cellular response. During larval development of the nematode Caenorhabditis elegans, Wnt/β-catenin pathways act in fate specification of two hypodermal cell types, the ventral vulval precursor cells (VPCs) and the lateral seam cells. Because little is known about targets of the Wnt signaling pathways acting during larval VPC and seam cell differentiation, we sought to identify genes regulated by Wnt signaling in these two hypodermal cell types. We conditionally activated Wnt signaling in larval animals and performed cell type-specific "mRNA tagging" to enrich for VPC and seam cell-specific mRNAs, and then used microarray analysis to examine gene expression compared to control animals. Two hundred thirty-nine genes activated in response to Wnt signaling were identified, and we characterized 50 genes further. The majority of these genes are expressed in seam and/or vulval lineages during normal development, and reduction of function for nine genes caused defects in the proper division, fate specification, fate execution, or differentiation of seam cells and vulval cells. Therefore, the combination of these techniques was successful at identifying potential cell type-specific Wnt pathway target genes from a small number of cells and at increasing our knowledge of the specification and behavior of these C. elegans larval hypodermal cells. Copyright © 2015 Gorrepati et al.

Psoriasis: in between the skin and the fat.

PubMed

Ståhle, Mona

2015-03-01

Substantial epidemiological evidence indicates that psoriasis associates with a predisposition to develop metabolic dysregulation leading to obesity and insulin resistance. However, the nature of this association and the potential underlying mechanisms remain unclear. In a recent report, Gerdes et al. explored the hypothesis that wingless-type MMTV integration site, Wnt5a, which has been linked to aberrant fat cell metabolism, may be driving this process. In this study, the authors compare circulating serum levels of Wnt5a in individuals with psoriasis and compare with healthy controls matched for age, gender and BMI. The bottom-line results show higher levels of Wnt5a in psoriasis patients irrespective of BMI compared to the matched non-psoriatic controls, indicating that psoriasis per se may result in increased secretion of Wnt5a into the circulation. In addition, there was a significant difference among patients with higher levels of Wnt5a in the obese psoriasis population. The study, even though being purely descriptive, may serve to inspire a more mechanistic approach exploring not only Wnt5a, but other inflammatory pathways in between the skin and the fat. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Preclinical validation and imaging of Wnt-induced repair in human 3D lung tissue cultures.

PubMed

Uhl, Franziska E; Vierkotten, Sarah; Wagner, Darcy E; Burgstaller, Gerald; Costa, Rita; Koch, Ina; Lindner, Michael; Meiners, Silke; Eickelberg, Oliver; Königshoff, Melanie

2015-10-01

Chronic obstructive pulmonary disease (COPD) is characterised by a progressive loss of lung tissue. Inducing repair processes within the adult diseased lung is of major interest and Wnt/β-catenin signalling represents a promising target for lung repair. However, the translation of novel therapeutic targets from model systems into clinical use remains a major challenge.We generated murine and patient-derived three-dimensional (3D) ex vivo lung tissue cultures (LTCs), which closely mimic the 3D lung microenvironment in vivo. Using two well-known glycogen synthase kinase-3β inhibitors, lithium chloride (LiCl) and CHIR 99021 (CT), we determined Wnt/β-catenin-driven lung repair processes in high spatiotemporal resolution using quantitative PCR, Western blotting, ELISA, (immuno)histological assessment, and four-dimensional confocal live tissue imaging.Viable 3D-LTCs exhibited preserved lung structure and function for up to 5 days. We demonstrate successful Wnt/β-catenin signal activation in murine and patient-derived 3D-LTCs from COPD patients. Wnt/β-catenin signalling led to increased alveolar epithelial cell marker expression, decreased matrix metalloproteinase-12 expression, as well as altered macrophage activity and elastin remodelling. Importantly, induction of surfactant protein C significantly correlated with disease stage (per cent predicted forced expiratory volume in 1 s) in patient-derived 3D-LTCs.Patient-derived 3D-LTCs represent a valuable tool to analyse potential targets and drugs for lung repair. Enhanced Wnt/β-catenin signalling attenuated pathological features of patient-derived COPD 3D-LTCs. Copyright ©ERS 2015.

Effect of perineoplasm perinephric adipose tissues on migration of clear cell renal cell carcinoma cells: a potential role of WNT signaling.

PubMed

Zi, Xiaolin; Lusch, Achim; Blair, Christopher A; Okhunov, Zhamshid; Yokoyama, Noriko N; Liu, Shuman; Baker, Molly; Huynh, Victor; Landman, Jaime

2016-08-16

To investigate the cellular and molecular interactions between clear-cell renal cell carcinoma (ccRCC) and perinephric adipose tissue (PAT), perineoplasm PAT, PAT away from the neoplasm, renal sinus and subcutaneous adipose tissues were collected at the time of renal surgery for renal masses and conditioned medium (CM) was generated from 62 patients. Perineoplasm PAT CMs from 44 out of 62 (about 71%) of patients with ccRCC or benign renal diseases (e.g. oncocytomas, angiomyolipomas, multicystic kidney, interstitial fibrosis, etc.) enhanced the migration of CaKi-2 cells. Perineoplasm PAT CMs from ccRCC significantly increased migration of ACHN and CaKi-2 cells by ~8.2 and ~2.4 folds, respectively, relative to those from benign renal diseases, whereas there is no significant difference in migration between ccRCC and benign renal diseases in CMs collected from culturing PAT away from neoplasm, renal sinus and subcutaneous adipose tissues. High Fuhrman Grade was associated with increased migration of Caki-2 cells by perineoplasm PAT CMs. Perineoplasm PATs from pT3 RCCs overexpressed multiple WNTs and their CMs exhibited higher WNT/ß-catenin activity and increased the migration of Caki-2 cells compared to CMs from benign neoplasms. Addition of secreted WNT inhibitory factor-1 recombinant protein into perineoplasm PAT CMs completely blocked the cell migration. These results indicate that WNT related factors from perineoplasm PAT may promote progression of local ccRCC to locally advanced (pT3) disease by increasing ccRCC cell mobility.

Nuclear factor I-C reciprocally regulates adipocyte and osteoblast differentiation via control of canonical Wnt signaling.

PubMed

Zhou, Jie; Wang, Shan; Qi, Qi; Yang, Xiaoyue; Zhu, Endong; Yuan, Hairui; Li, Xuemei; Liu, Ying; Li, Xiaoxia; Wang, Baoli

2017-05-01

Nuclear factor I-C (NFIC) has recently been identified as an important player in osteogenesis and bone homeostasis in vivo However, the molecular mechanisms involved have yet to be defined. In the current study, Nfic expression was altered in primary marrow stromal cells and established progenitor lines after adipogenic and osteogenic treatment. Overexpression of Nfic in stromal cells ST2, mesenchymal cells C3H10T1/2, and primary marrow stromal cells inhibited adipogenic differentiation, whereas it promoted osteogenic differentiation. Conversely, silencing of endogenous Nfic in the cell lines enhanced adipogenic differentiation, whereas it blocked osteogenic differentiation. Mechanism investigations revealed that Nfic overexpression promoted nuclear translocation of β-catenin and increased nuclear protein levels of β-catenin and transcription factor 7-like 2 (TCF7L2). Promoter studies and the chromatin immunoprecipitation (ChIP) assay revealed that NFIC directly binds to the promoter of low-density lipoprotein receptor-related protein 5 (Lrp5) and thereafter transactivates the promoter. Finally, inactivation of canonical Wnt signaling in ST2 attenuated the inhibition of adipogenic differentiation and stimulation of osteogenic differentiation by NFIC. Our study suggests that NFIC balances adipogenic and osteogenic differentiation from progenitor cells through controlling canonical Wnt signaling and highlights the potential of NFIC as a target for new therapies to control metabolic disorders like osteoporosis and obesity.-Zhou, J., Wang, S., Qi, Q., Yang, X., Zhu, E., Yuan, H., Li, X., Liu, Y., Li, X., Wang, B. Nuclear factor I-C reciprocally regulates adipocyte and osteoblast differentiation via control of canonical Wnt signaling. © FASEB.

The PTK7-Related Transmembrane Proteins Off-track and Off-track 2 Are Co-receptors for Drosophila Wnt2 Required for Male Fertility

PubMed Central

Honemann-Capito, Mona; Brechtel-Curth, Katja; Hedderich, Marie; Wodarz, Andreas

2014-01-01

Wnt proteins regulate many developmental processes and are required for tissue homeostasis in adult animals. The cellular responses to Wnts are manifold and are determined by the respective Wnt ligand and its specific receptor complex in the plasma membrane. Wnt receptor complexes contain a member of the Frizzled family of serpentine receptors and a co-receptor, which commonly is a single-pass transmembrane protein. Vertebrate protein tyrosine kinase 7 (PTK7) was identified as a Wnt co-receptor required for control of planar cell polarity (PCP) in frogs and mice. We found that flies homozygous for a complete knock-out of the Drosophila PTK7 homolog off track (otk) are viable and fertile and do not show PCP phenotypes. We discovered an otk paralog (otk2, CG8964), which is co-expressed with otk throughout embryonic and larval development. Otk and Otk2 bind to each other and form complexes with Frizzled, Frizzled2 and Wnt2, pointing to a function as Wnt co-receptors. Flies lacking both otk and otk2 are viable but male sterile due to defective morphogenesis of the ejaculatory duct. Overexpression of Otk causes female sterility due to malformation of the oviduct, indicating that Otk and Otk2 are specifically involved in the sexually dimorphic development of the genital tract. PMID:25010066

The PTK7-related transmembrane proteins off-track and off-track 2 are co-receptors for Drosophila Wnt2 required for male fertility.

PubMed

Linnemannstöns, Karen; Ripp, Caroline; Honemann-Capito, Mona; Brechtel-Curth, Katja; Hedderich, Marie; Wodarz, Andreas

2014-07-01

Wnt proteins regulate many developmental processes and are required for tissue homeostasis in adult animals. The cellular responses to Wnts are manifold and are determined by the respective Wnt ligand and its specific receptor complex in the plasma membrane. Wnt receptor complexes contain a member of the Frizzled family of serpentine receptors and a co-receptor, which commonly is a single-pass transmembrane protein. Vertebrate protein tyrosine kinase 7 (PTK7) was identified as a Wnt co-receptor required for control of planar cell polarity (PCP) in frogs and mice. We found that flies homozygous for a complete knock-out of the Drosophila PTK7 homolog off track (otk) are viable and fertile and do not show PCP phenotypes. We discovered an otk paralog (otk2, CG8964), which is co-expressed with otk throughout embryonic and larval development. Otk and Otk2 bind to each other and form complexes with Frizzled, Frizzled2 and Wnt2, pointing to a function as Wnt co-receptors. Flies lacking both otk and otk2 are viable but male sterile due to defective morphogenesis of the ejaculatory duct. Overexpression of Otk causes female sterility due to malformation of the oviduct, indicating that Otk and Otk2 are specifically involved in the sexually dimorphic development of the genital tract.

Feedback Activation of Basic Fibroblast Growth Factor Signaling via the Wnt/β-Catenin Pathway in Skin Fibroblasts

PubMed Central

Wang, Xu; Zhu, Yuting; Sun, Congcong; Wang, Tao; Shen, Yingjie; Cai, Wanhui; Sun, Jia; Chi, Lisha; Wang, Haijun; Song, Na; Niu, Chao; Shen, Jiayi; Cong, Weitao; Zhu, Zhongxin; Xuan, Yuanhu; Li, Xiaokun; Jin, Litai

2017-01-01

Skin wound healing is a complex process requiring the coordinated behavior of many cell types, especially in the proliferation and migration of fibroblasts. Basic fibroblast growth factor (bFGF) is a member of the FGF family that promotes fibroblast migration, but the underlying molecular mechanism remains elusive. The present RNA sequencing study showed that the expression levels of several canonical Wnt pathway genes, including Wnt2b, Wnt3, Wnt11, T-cell factor 7 (TCF7), and Frizzled 8 (FZD8) were modified by bFGF stimulation in fibroblasts. Enzyme-linked immunosorbent assay (ELISA) analysis also showed that Wnt pathway was activated under bFGF treatment. Furthermore, treatment of fibroblasts with lithium chloride or IWR-1, an inducer and inhibitor of the Wnt signaling pathway, respectively, promoted and inhibited cell migration. Also, levels of cytosolic glycogen synthase kinase 3 beta phosphorylated at serine9 (pGSK3β Ser9) and nuclear β-catenin were increased upon exposure to bFGF. Molecular and biochemical assays indicated that phosphoinositide 3-kinase (PI3K) signaling activated the GSK3β/β-catenin/Wnt signaling pathway via activation of c-Jun N-terminal kinase (JNK), suggesting that PI3K and JNK act at the upstream of β-catenin. In contrast, knock-down of β-catenin delayed fibroblast cell migration even under bFGF stimulation. RNA sequencing analysis of β-catenin knock-down fibroblasts demonstrated that β-catenin positively regulated the transcription of bFGF and FGF21. Moreover, FGF21 treatment activated AKT and JNK, and accelerated fibroblast migration to a similar extent as bFGF does. In addition, ELISA analysis demonstrated that both of bFGF and FGF21 were auto secretion factor and be regulated by Wnt pathway stimulators. Taken together, our analyses define a feedback regulatory loop between bFGF (FGF21) and Wnt signaling acting through β-catenin in skin fibroblasts. PMID:28217097

The impact of Wnt signalling and hypoxia on osteogenic and cementogenic differentiation in human periodontal ligament cells

PubMed Central

Li, Shuigen; Shao, Jin; Zhou, Yinghong; Friis, Thor; Yao, Jiangwu; Shi, Bin; Xiao, Yin

2016-01-01

Cementum is a periodontal support tissue that is directly connected to the periodontal ligament. It shares common traits with bone tissues, however, unlike bone, the cementum has a limited capacity for regeneration. As a result, following damage the cementum rarely, if ever, regenerates. Periodontal ligament cells (PDLCs) are able to differentiate into osteoblastic and cementogenic lineages according to specific local environmental conditions, including hypoxia, which is induced by inflammation or activation of the Wnt signalling pathway by local loading. The interactions between the Wnt signalling pathway and hypoxia during cementogenesis are of particular interest to improve the understanding of periodontal tissue regeneration. In the present study, osteogenic and cementogenic differentiation of PDLCs was investigated under hypoxic conditions in the presence and absence of Wnt pathway activation. Protein and gene expression of the osteogenic markers type 1 collagen (COL1) and runt-related transcription factor 2 (RUNX2), and cementum protein 1 (CEMP1) were used as markers for osteogenic and cementogenic differentiation, respectively. Wnt signalling activation inhibited cementogenesis, whereas hypoxia alone did not affect PDLC differentiation. However, hypoxia reversed the inhibition of cementogenesis that resulted from overexpression of Wnt signalling. Cross-talk between hypoxia and Wnt signalling pathways was, therefore, demonstrated to be involved in the differentiation of PDLCs to the osteogenic and cementogenic lineages. In summary, the present study suggests that the differentiation of PDLCs into osteogenic and cementogenic lineages is partially regulated by the Wnt signalling pathway and that hypoxia is also involved in this process. PMID:27840938

Wnt Signaling Specifies Anteroposterior Progenitor Zone Identity in the Drosophila Visual Center.

PubMed

Suzuki, Takumi; Trush, Olena; Yasugi, Tetsuo; Takayama, Rie; Sato, Makoto

2016-06-15

During brain development, various types of neuronal populations are produced from different progenitor pools to produce neuronal diversity that is sufficient to establish functional neuronal circuits. However, the molecular mechanisms that specify the identity of each progenitor pool remain obscure. Here, we show that Wnt signaling is essential for the specification of the identity of posterior progenitor pools in the Drosophila visual center. In the medulla, the largest component of the visual center, different types of neurons are produced from two progenitor pools: the outer proliferation center (OPC) and glial precursor cells (GPCs; also known as tips of the OPC). We found that OPC-type neurons are produced from the GPCs at the expense of GPC-type neurons when Wnt signaling is suppressed in the GPCs. In contrast, GPC-type neurons are ectopically induced when Wnt signaling is ectopically activated in the OPC. These results suggest that Wnt signaling is necessary and sufficient for the specification of the progenitor pool identity. We also found that Homothorax (Hth), which is temporally expressed in the OPC, is ectopically induced in the GPCs by suppression of Wnt signaling and that ectopic induction of Hth phenocopies the suppression of Wnt signaling in the GPCs. Thus, Wnt signaling is involved in regionalization of the fly visual center through the specification of the progenitor pool located posterior to the medulla by suppressing Hth expression. Brain consists of considerably diverse neurons of different origins. In mammalian brain, excitatory and inhibitory neurons derive from the dorsal and ventral telencephalon, respectively. Multiple progenitor pools also contribute to the neuronal diversity in fly brain. However, it has been unclear how differences between these progenitor pools are established. Here, we show that Wnt signaling, an evolutionarily conserved signaling, is involved in the process that establishes the differences between these progenitor pools. Because β-catenin signaling, which is under the control of Wnt ligands, specifies progenitor pool identity in the developing mammalian thalamus, Wnt signaling-mediated specification of progenitor pool identity may be conserved in insect and mammalian brains. Copyright © 2016 the authors 0270-6474/16/366503-11$15.00/0.

DIXDC1 activates the Wnt signaling pathway and promotes gastric cancer cell invasion and metastasis.

PubMed

Tan, Cong; Qiao, Fan; Wei, Ping; Chi, Yayun; Wang, Weige; Ni, Shujuan; Wang, Qifeng; Chen, Tongzhen; Sheng, Weiqi; Du, Xiang; Wang, Lei

2016-04-01

DIXDC1 (Dishevelled-Axin domain containing 1) is a DIX (Dishevelled-Axin) domain-possessing protein that promotes colon cancer cell proliferation and increases the invasion and migration ability of non-small-cell lung cancer via the PI3K pathway. As a positive regulator of the Wnt/β-catenin pathway, the biological role of DIXDC1 in human gastric cancer and the relationship between DIXDC1 and the Wnt pathway are unclear. In the current study, the upregulation of DIXDC1 was detected in gastric cancer and was associated with advanced TNM stage cancer, lymph node metastasis, and poor prognosis. We also found that the overexpression of DIXDC1 could promote the invasion and migration of gastric cancer cells. The upregulation of MMPs and the downregulation of E-cadherin were found to be involved in the process. DIXDC1 enhanced β-catenin nuclear accumulation, which activated the Wnt pathway. Additionally, the inhibition of β-catenin in DIXDC1-overexpressing cells reversed the metastasis promotion effects of DIXDC1. These results demonstrate that the expression of DIXDC1 is associated with poor prognosis of gastric cancer patients and that DIXDC1 promotes gastric cancer invasion and metastasis through the activation of the Wnt pathway; E-cadherin and MMPs are also involved in this process. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

Lineage-specific evolution of cnidarian Wnt ligands.

PubMed

Hensel, Katrin; Lotan, Tamar; Sanders, Steve M; Cartwright, Paulyn; Frank, Uri

2014-09-01

We have studied the evolution of Wnt genes in cnidarians and the expression pattern of all Wnt ligands in the hydrozoan Hydractinia echinata. Current views favor a scenario in which 12 Wnt sub-families were jointly inherited by cnidarians and bilaterians from their last common ancestor. Our phylogenetic analyses clustered all medusozoan genes in distinct, well-supported clades, but many orthologous relationships between medusozoan Wnts and anthozoan and bilaterian Wnt genes were poorly supported. Only seven anthozoan genes, Wnt2, Wnt4, Wnt5, Wnt6, Wnt 10, Wnt11, and Wnt16 were recovered with strong support with bilaterian genes and of those, only the Wnt2, Wnt5, Wnt11, and Wnt16 clades also included medusozoan genes. Although medusozoan Wnt8 genes clustered with anthozoan and bilaterian genes, this was not well supported. In situ hybridization studies revealed poor conservation of expression patterns of putative Wnt orthologs within Cnidaria. In polyps, only Wnt1, Wnt3, and Wnt7 were expressed at the same position in the studied cnidarian models Hydra, Hydractinia, and Nematostella. Different expression patterns are consistent with divergent functions. Our data do not fully support previous assertions regarding Wnt gene homology, and suggest a more complex history of Wnt family genes than previously suggested. This includes high rates of sequence divergence and lineage-specific duplications of Wnt genes within medusozoans, followed by functional divergence over evolutionary time scales. © 2014 Wiley Periodicals, Inc.

Lrp4 and Wise interplay controls the formation and patterning of mammary and other skin appendage placodes by modulating Wnt signaling.

PubMed

Ahn, Youngwook; Sims, Carrie; Logue, Jennifer M; Weatherbee, Scott D; Krumlauf, Robb

2013-02-01

The future site of skin appendage development is marked by a placode during embryogenesis. Although Wnt/β-catenin signaling is known to be essential for skin appendage development, it is unclear which cellular processes are controlled by the signaling and how the precise level of the signaling activity is achieved during placode formation. We have investigated roles for Lrp4 and its potential ligand Wise (Sostdc1) in mammary and other skin appendage placodes. Lrp4 mutant mice displayed a delay in placode initiation and changes in distribution and number of mammary precursor cells leading to abnormal morphology, number and position of mammary placodes. These Lrp4 mammary defects, as well as limb defects, were associated with elevated Wnt/β-catenin signaling and were rescued by reducing the dose of the Wnt co-receptor genes Lrp5 and Lrp6, or by inactivating the gene encoding β-catenin. Wise-null mice phenocopied a subset of the Lrp4 mammary defects and Wise overexpression reduced the number of mammary precursor cells. Genetic epistasis analyses suggest that Wise requires Lrp4 to exert its function and that, together, they have a role in limiting mammary fate, but Lrp4 has an early Wise-independent role in facilitating placode formation. Lrp4 and Wise mutants also share defects in vibrissa and hair follicle development, suggesting that the roles played by Lrp4 and Wise are common to skin appendages. Our study presents genetic evidence for interplay between Lrp4 and Wise in inhibiting Wnt/β-catenin signaling and provides an insight into how modulation of Wnt/β-catenin signaling controls cellular processes important for skin placode formation.

Wnt5a suppresses tumor formation and redirects tumor phenotype in MMTV-Wnt1 tumors.

PubMed

Easter, Stephanie L; Mitchell, Elizabeth H; Baxley, Sarah E; Desmond, Renee; Frost, Andra R; Serra, Rosa

2014-01-01

Wnt5a is a non-canonical signaling Wnt that has been implicated in tumor suppression. We previously showed that loss of Wnt5a in MMTV-PyVmT tumors resulted in a switch in tumor phenotype resulting in tumors with increased basal phenotype and high Wnt/β-catenin signaling. The object of this study was to test the hypothesis that Wnt5a can act to inhibit tumors formed by activation of Wnt/β-catenin signaling. To this end, we characterized tumor and non-tumor mammary tissue from MMTV-Wnt1 and double transgenic MMTV-Wnt1;MMTV-Wnt5a mice. Wnt5a containing mice demonstrated fewer tumors with increased latency when compared to MMTV-Wnt1 controls. Expression of markers for basal-like tumors was down-regulated in the tumors that formed in the presence of Wnt5a indicating a phenotypic switch. Reduced canonical Wnt signaling was detected in double transgenic tumors as a decrease in active β-catenin protein and a decrease in Axin2 mRNA transcript levels. In non-tumor tissues, over-expression of Wnt5a in MMTV-Wnt1 mammary glands resulted in attenuation of phenotypes normally observed in MMTV-Wnt1 glands including hyperbranching and increased progenitor and basal cell populations. Even though Wnt5a could antagonize Wnt/β-catenin signaling in primary mammary epithelial cells in culture, reduced Wnt/β-catenin signaling was not detected in non-tumor MMTV-Wnt1;Wnt5a tissue in vivo. The data demonstrate that Wnt5a suppresses tumor formation and promotes a phenotypic shift in MMTV-Wnt1 tumors.

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

Kim, Inae; Hur, Jung; Jeong, Sunjoo, E-mail: sjsj@dankook.ac.kr

Highlights: • Wnt signaling as well as β-catenin overexpression enhance HuR cytoplasmic export. • HuR overexpression promotes cytoplasmic localization of β-catenin from the perinuclear fraction. • Wnt/β-catenin-mediated transcriptional activity is repressesed by HuR. - Abstract: β-Catenin is the key transcriptional activator of canonical Wnt signaling in the nucleus; thus, nuclear accumulation of β-catenin is a critical step for expressing target genes. β-Catenin accumulates in the nucleus of cancer cells where it activates oncogenic target genes. Hu antigen R (HuR) is a RNA binding protein that regulates multiple post-transcriptional processes including RNA stability. Thus, cytoplasmic HuR protein may be involved inmore » tumorigenesis by stabilizing oncogenic transcripts, but the molecular mechanism remains unclear. Here, we observed that Wnt/β-catenin signaling induced export of the HuR protein, whereas HuR overexpression promoted accumulation of the β-catenin protein in the cytoplasm. Thus, Wnt/β-catenin-mediated transcriptional activity in the nucleus was reduced by overexpressing HuR. These results suggest novel and uncharacterized cytoplasmic β-catenin functions related to HuR-mediated RNA metabolism in cancer cells.« less

Embryonic attenuated Wnt/β-catenin signaling defines niche location and long-term stem cell fate in hair follicle

PubMed Central

Xu, Zijian; Wang, Wenjie; Jiang, Kaiju; Yu, Zhou; Huang, Huanwei; Wang, Fengchao; Zhou, Bin; Chen, Ting

2015-01-01

Long-term adult stem cells sustain tissue regeneration throughout the lifetime of an organism. They were hypothesized to originate from embryonic progenitor cells that acquire long-term self-renewal ability and multipotency at the end of organogenesis. The process through which this is achieved often remains unclear. Here, we discovered that long-term hair follicle stem cells arise from embryonic progenitor cells occupying a niche location that is defined by attenuated Wnt/β-catenin signaling. Hair follicle initiation is marked by placode formation, which depends on the activation of Wnt/β-catenin signaling. Soon afterwards, a region with attenuated Wnt/β-catenin signaling emerges in the upper follicle. Embryonic progenitor cells residing in this region gain expression of adult stem cell markers and become definitive long-term hair follicle stem cells at the end of organogenesis. Attenuation of Wnt/β-catenin signaling is a prerequisite for hair follicle stem cell specification because it suppresses Sox9, which is required for stem cell formation. DOI: http://dx.doi.org/10.7554/eLife.10567.001 PMID:26653852

Dkk2/Frzb in the dermal papillae regulates feather regeneration

PubMed Central

Chu, Qiqi; Cai, Linyan; Fu, Yu; Chen, Xi; Yan, Zhipeng; Lin, Xiang; Zhou, Guixuan; Han, Hao; Widelitz, Randall B.; Chuong, Cheng-ming; Wu, Wei; Yue, Zhicao

2015-01-01

Avian feathers have robust growth and regeneration capability. To evaluate the contribution of signaling molecules and pathways in these processes, we profiled gene expression in the feather follicle using an absolute quantification approach. We identified hundreds of genes that mark specific components of the feather follicle: the dermal papillae (DP) which controls feather regeneration and axis formation, the pulp mesenchyme (Pp) which is derived from DP cells and nourishes the feather follicle, and the ramogenic zone epithelium (Erz) where a feather starts to branch. The feather DP is enriched in BMP/TGF-β signaling molecules and inhibitors for Wnt signaling including Dkk2/Frzb. Wnt ligands are mainly expressed in the feather epithelium and pulp. We find that while Wnt signaling is required for the maintenance of DP marker gene expression and feather regeneration, excessive Wnt signaling delays regeneration and reduces pulp formation. Manipulating Dkk2/Frzb expression by lentiviral-mediated overexpression, shRNA-knockdown, or by antibody neutralization resulted in dual feather axes formation. Our results suggest that the Wnt signaling in the proximal feather follicle is fine-tuned to accommodate feather regeneration and axis formation. PMID:24463139

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

Adachi, Atsuo; Takahashi, Tomosaburo, E-mail: ttaka@koto.kpu-m.ac.jp; Ogata, Takehiro

Highlights: Black-Right-Pointing-Pointer NFAT5 protein expression is downregulated during cardiomyogenesis. Black-Right-Pointing-Pointer Inhibition of NFAT5 function suppresses canonical Wnt signaling. Black-Right-Pointing-Pointer Inhibition of NFAT5 function attenuates mesodermal induction. Black-Right-Pointing-Pointer NFAT5 function is required for cardiomyogenesis. -- Abstract: While nuclear factor of activated T cells 5 (NFAT5), a transcription factor implicated in osmotic stress response, is suggested to be involved in other processes such as migration and proliferation, its role in cardiomyogenesis is largely unknown. Here, we examined the role of NFAT5 in cardiac differentiation of P19CL6 cells, and observed that it was abundantly expressed in undifferentiated P19CL6 cells, and its protein expressionmore » was significantly downregulated by enhanced proteasomal degradation during DMSO-induced cardiomyogenesis. Expression of a dominant negative mutant of NFAT5 markedly attenuated cardiomyogenesis, which was associated with the inhibition of mesodermal differentiation. TOPflash reporter assay revealed that the transcriptional activity of canonical Wnt signaling was activated prior to mesodermal differentiation, and this activation was markedly attenuated by NFAT5 inhibition. Pharmacological activation of canonical Wnt signaling by [2 Prime Z, 3 Prime E]-6-bromoindirubin-3 Prime -oxime (BIO) restored Brachyury expression in NFAT5DN-expressing cells. Inhibition of NFAT5 markedly attenuated Wnt3 and Wnt3a induction. Expression of Dkk1 and Cerberus1, which are secreted Wnt antagonists, was also inhibited by NFAT5 inhibition. Thus, endogenous NFAT5 regulates the coordinated expression of Wnt ligands and antagonists, which are essential for cardiomyogenesis through the canonical Wnt pathway. These results demonstrated a novel role of NFAT5 in cardiac differentiation of stem cells.« less

The Response of wnt/ ß-Catenin Signaling Pathway in Osteocytes Under Simulated Microgravity

NASA Astrophysics Data System (ADS)

Yang, Xiao; Sun, Lian-Wen; Liang, Meng; Wang, Xiao-Nan; Fan, Yu-Bo

2015-11-01

Osteocytes were considered as potential sensors of mechanical loading and orchestrate the bone remodeling adapted to mechanical loading. On the other hand, osteocytes are also considered as the unloading sensors in vivo. Previous studies showed that the mechanosensation and mechanotransduction of osteocytes may play an essential role in mediating bone response to microgravity, and one of the most important molecular signaling pathway involved in the mechanotransduction is the Wnt/ ß-catenin signaling pathway. In order to investigate the effect of simulated microgravity on the Wnt/ ß-catenin signaling pathway in osteocytes, MLO-Y4 cells (an osteocyte-like cell line) were cultured under controlled rotation to simulate microgravity for 5 days. The cytoskeleton and ß-catenin nuclear translocation of MLO-Y4 cells were detected by laser scanning confocal microscope and the fluorescence intensity was quantified; the mRNA expressions of upstream and downstream key components in Wnt canonical signaling were detected with RT-PCR. Two regulators of the Wnt/ ß-catenin pathway, NMP4/CIZ and Smads, were also investigated by RT-PCR; finally the expression of Wnt target genes and Sost protein level were detected with the absence or presence of the Sclerostin antibody (Scl-AbI) under simulated microgravity. The results showed that under simulated microgravity, (1) F-actin filaments were disassembled and some short dendritic processes appeared at the cell periphery; (2) the gene expression of Wnt3a, Wnt5a, DKK1, CyclinD1, LEF-1 and CX43 in the simulated microgravity group were significantly lower whereas Wnt1 and Sost in the simulated microgravity group were significantly higher than the control group; (3) the gene and protein level of ß-catenin were reduced, and no ß-catenin nuclear translocation observed; (4) the gene expression of Smad1, Smad4 and Smad7 were significantly lower whereas NMP4/CIZ and Smad3 in the simulated microgravity were significantly higher than the control group; (5) Scl-AbI partially inhibited the down-regulation of simulated microgravity to Wnt target gene expression and Sclerostin protein expression. The results suggested that firstly the cytoskeleton was disturbed in MLO-Y4 by simulated microgravity; secondly the activity of Wnt/ ß-catenin signaling pathway was depressed, with the nuclear translocation of ß-catenin suppressed by simulated microgravity; thirdly the Wnt/ ß-catenin signaling pathway positive regulators (Smads) were decreased, while the negative regulator (NMP4/CIZ) was increased under simulated microgravity; finally Scl-AbI could partially restore the adverse effect of simulated microgravity to Wnt signaling. This study may help us to understand the mechanotransduction alteration of Wnt/ ß-catenin signaling pathway in osteocytes under simulated microgravity, and further may partly clarify the mechanism of microgravity-induced osteoporosis.

Planarian yorkie/YAP functions to integrate adult stem cell proliferation, organ homeostasis and maintenance of axial patterning.

PubMed

Lin, Alexander Y T; Pearson, Bret J

2014-03-01

During adult homeostasis and regeneration, the freshwater planarian must accomplish a constant balance between cell proliferation and cell death, while also maintaining proper tissue and organ size and patterning. How these ordered processes are precisely modulated remains relatively unknown. Here we show that planarians use the downstream effector of the Hippo signaling cascade, yorkie (yki; YAP in vertebrates) to control a diverse set of pleiotropic processes in organ homeostasis, stem cell regulation, regeneration and axial patterning. We show that yki functions to maintain the homeostasis of the planarian excretory (protonephridial) system and to limit stem cell proliferation, but does not affect the differentiation process or cell death. Finally, we show that Yki acts synergistically with WNT/β-catenin signaling to repress head determination by limiting the expression domains of posterior WNT genes and that of the WNT-inhibitor notum. Together, our data show that yki is a key gene in planarians that integrates stem cell proliferation control, organ homeostasis, and the spatial patterning of tissues.

The role of the Hes1 crosstalk hub in Notch-Wnt interactions of the intestinal crypt

PubMed Central

Harrington, Heather A.; Dale, Trevor; Gavaghan, David J.

2017-01-01

The Notch pathway plays a vital role in determining whether cells in the intestinal epithelium adopt a secretory or an absorptive phenotype. Cell fate specification is coordinated via Notch’s interaction with the canonical Wnt pathway. Here, we propose a new mathematical model of the Notch and Wnt pathways, in which the Hes1 promoter acts as a hub for pathway crosstalk. Computational simulations of the model can assist in understanding how healthy intestinal tissue is maintained, and predict the likely consequences of biochemical knockouts upon cell fate selection processes. Chemical reaction network theory (CRNT) is a powerful, generalised framework which assesses the capacity of our model for monostability or multistability, by analysing properties of the underlying network structure without recourse to specific parameter values or functional forms for reaction rates. CRNT highlights the role of β-catenin in stabilising the Notch pathway and damping oscillations, demonstrating that Wnt-mediated actions on the Hes1 promoter can induce dynamic transitions in the Notch system, from multistability to monostability. Time-dependent model simulations of cell pairs reveal the stabilising influence of Wnt upon the Notch pathway, in which β-catenin- and Dsh-mediated action on the Hes1 promoter are key in shaping the subcellular dynamics. Where Notch-mediated transcription of Hes1 dominates, there is Notch oscillation and maintenance of fate flexibility; Wnt-mediated transcription of Hes1 favours bistability akin to cell fate selection. Cells could therefore regulate the proportion of Wnt- and Notch-mediated control of the Hes1 promoter to coordinate the timing of cell fate selection as they migrate through the intestinal epithelium and are subject to reduced Wnt stimuli. Furthermore, mutant cells characterised by hyperstimulation of the Wnt pathway may, through coupling with Notch, invert cell fate in neighbouring healthy cells, enabling an aberrant cell to maintain its neighbours in mitotically active states. PMID:28245235

A Systematic Survey of Expression and Function of Zebrafish frizzled Genes

PubMed Central

Nikaido, Masataka; Law, Edward W. P.; Kelsh, Robert N.

2013-01-01

Wnt signaling is crucial for the regulation of numerous processes in development. Consistent with this, the gene families for both the ligands (Wnts) and receptors (Frizzleds) are very large. Surprisingly, while we have a reasonable understanding of the Wnt ligands likely to mediate specific Wnt-dependent processes, the corresponding receptors usually remain to be elucidated. Taking advantage of the zebrafish model's excellent genomic and genetic properties, we undertook a comprehensive analysis of the expression patterns of frizzled (fzd) genes in zebrafish. To explore their functions, we focused on testing their requirement in several developmental events known to be regulated by Wnt signaling, convergent extension movements of gastrulation, neural crest induction, and melanocyte specification. We found fourteen distinct fzd genes in the zebrafish genome. Systematic analysis of their expression patterns between 1-somite and 30 hours post-fertilization revealed complex, dynamic and overlapping expression patterns. This analysis demonstrated that only fzd3a, fzd9b, and fzd10 are expressed in the dorsal neural tube at stages corresponding to the timing of melanocyte specification. Surprisingly, however, morpholino knockdown of these, alone or in combination, gave no indication of reduction of melanocytes, suggesting the important involvement of untested fzds or another type of Wnt receptor in this process. Likewise, we found only fzd7b and fzd10 expressed at the border of the neural plate at stages appropriate for neural crest induction. However, neural crest markers were not reduced by knockdown of these receptors. Instead, these morpholino knockdown studies showed that fzd7a and fzd7b work co-operatively to regulate convergent extension movement during gastrulation. Furthermore, we show that the two fzd7 genes function together with fzd10 to regulate epiboly movements and mesoderm differentiation. PMID:23349976

Crossroads of Wnt and Hippo in epithelial tissues.

PubMed

Bernascone, Ilenia; Martin-Belmonte, Fernando

2013-08-01

Epithelial tissues undergo constant growth and differentiation during embryonic development and to replace damaged tissue in adult organs. These processes are governed by different signaling pathways that ultimately control the expression of genes associated with cell proliferation, patterning, and death. One essential pathway is Wnt, which controls tubulogenesis in several epithelial organs. Recently, Wnt has been closely linked to other signaling pathways, such as Hippo, that orchestrate proliferation and apoptosis to control organ size. There is evidence that epithelial cell junctions may sequester the transcription factors that act downstream of these signaling pathways, which would represent an important aspect of their functional regulation and their influence on cell behavior. Here, we review the transcriptional control exerted by the Wnt and Hippo signaling pathways during epithelial growth, patterning, and differentiation and recent advances in understanding of the regulation and crosstalk of these pathways in epithelial tissues. Copyright © 2013 Elsevier Ltd. All rights reserved.

Mechanical stress regulates osteogenic differentiation and RANKL/OPG ratio in periodontal ligament stem cells by the Wnt/β-catenin pathway.

PubMed

Zhang, Liqiang; Liu, Wenjia; Zhao, Jiangdong; Ma, Xiaojie; Shen, Lin; Zhang, Yongjie; Jin, Fang; Jin, Yan

2016-10-01

The balance between osteoblastic and osteoclastic activity is critical in orthodontic tooth movement (OTM). Mesenchymal stem cells (MSCs) play an important role in maintaining bone homeostasis, and periodontal ligament stem cells (PDLSCs) are tissue-specific MSCs in the periodontal ligament. However, whether PDLSCs are required for periodontal tissue remodeling during OTM is not fully understood. Here, we used PDGFRα and Nestin to trace PDLSCs during OTM in rats. We treat human PDLSCs with 100kpa static pressure for 1h or 12h in vitro, and examined the phenotypic changes and expression of RANKL and OPG in these cells. In vivo, we found that positive signals of PDGFRα and Nestin in the PDL gradually increased and then decreased on the pressure side to which pressure was applied. In vitro, the osteogenic differentiation of PDLSCs was significantly increased after force treatment for 1h relative to 12h. In contrast, the expression ratio of RANKL/OPG was reduced at 1h and significantly increased at 12h. Furthermore, we found that the Wnt/β-catenin pathway was dynamically activated in the PDL and in PDLSCs after mechanical stimulation. Importantly, the canonical Wnt pathway inhibitor DKK1 blocked the osteogenesis effect and rescued the ratio of RANKL/OPG in PDLSCs under force treatment for 1h. Our findings reveal that PDLSCs participate in OTM and that the Wnt/β-catenin pathway maintains bone homeostasis during tooth movement by regulating the balance between osteoblastic and osteoclastic activity. We describe a novel potential mechanism related to tooth movement. Copyright © 2016 Elsevier B.V. All rights reserved.

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

PDGFRα + pericryptal stromal cells are the critical source of Wnts and RSPO3 for murine intestinal stem cells in vivo.

PubMed

Greicius, Gediminas; Kabiri, Zahra; Sigmundsson, Kristmundur; Liang, Chao; Bunte, Ralph; Singh, Manvendra K; Virshup, David M

2018-04-03

Wnts and R-spondins (RSPOs) support intestinal homeostasis by regulating crypt cell proliferation and differentiation. Ex vivo, Wnts secreted by Paneth cells in organoids can regulate the proliferation and differentiation of Lgr5 -expressing intestinal stem cells. However, in vivo, Paneth cell and indeed all epithelial Wnt production is completely dispensable, and the cellular source of Wnts and RSPOs that maintain the intestinal stem-cell niche is not known. Here we investigated both the source and the functional role of stromal Wnts and RSPO3 in regulation of intestinal homeostasis. RSPO3 is highly expressed in pericryptal myofibroblasts in the lamina propria and is several orders of magnitude more potent than RSPO1 in stimulating both Wnt/β-catenin signaling and organoid growth. Stromal Rspo3 ablation ex vivo resulted in markedly decreased organoid growth that was rescued by exogenous RSPO3 protein. Pdgf receptor alpha ( PdgfRα ) is known to be expressed in pericryptal myofibroblasts. We therefore evaluated if PdgfRα identified the key stromal niche cells. In vivo, Porcn excision in PdgfRα + cells blocked intestinal crypt formation, demonstrating that Wnt production in the stroma is both necessary and sufficient to support the intestinal stem-cell niche. Mice with Rspo3 excision in the PdgfRα + cells had decreased intestinal crypt Wnt/β-catenin signaling and Paneth cell differentiation and were hypersensitive when stressed with dextran sodium sulfate. The data support a model of the intestinal stem-cell niche regulated by both Wnts and RSPO3 supplied predominantly by stromal pericryptal myofibroblasts marked by PdgfRα . Copyright © 2018 the Author(s). Published by PNAS.

PDGFRα+ pericryptal stromal cells are the critical source of Wnts and RSPO3 for murine intestinal stem cells in vivo

PubMed Central

Greicius, Gediminas; Kabiri, Zahra; Sigmundsson, Kristmundur; Liang, Chao; Bunte, Ralph; Singh, Manvendra K.

2018-01-01

Wnts and R-spondins (RSPOs) support intestinal homeostasis by regulating crypt cell proliferation and differentiation. Ex vivo, Wnts secreted by Paneth cells in organoids can regulate the proliferation and differentiation of Lgr5-expressing intestinal stem cells. However, in vivo, Paneth cell and indeed all epithelial Wnt production is completely dispensable, and the cellular source of Wnts and RSPOs that maintain the intestinal stem-cell niche is not known. Here we investigated both the source and the functional role of stromal Wnts and RSPO3 in regulation of intestinal homeostasis. RSPO3 is highly expressed in pericryptal myofibroblasts in the lamina propria and is several orders of magnitude more potent than RSPO1 in stimulating both Wnt/β-catenin signaling and organoid growth. Stromal Rspo3 ablation ex vivo resulted in markedly decreased organoid growth that was rescued by exogenous RSPO3 protein. Pdgf receptor alpha (PdgfRα) is known to be expressed in pericryptal myofibroblasts. We therefore evaluated if PdgfRα identified the key stromal niche cells. In vivo, Porcn excision in PdgfRα+ cells blocked intestinal crypt formation, demonstrating that Wnt production in the stroma is both necessary and sufficient to support the intestinal stem-cell niche. Mice with Rspo3 excision in the PdgfRα+ cells had decreased intestinal crypt Wnt/β-catenin signaling and Paneth cell differentiation and were hypersensitive when stressed with dextran sodium sulfate. The data support a model of the intestinal stem-cell niche regulated by both Wnts and RSPO3 supplied predominantly by stromal pericryptal myofibroblasts marked by PdgfRα. PMID:29559533

Discovery of small molecule inhibitors of the Wnt/β-catenin signaling pathway by targeting β-catenin/Tcf4 interactions.

PubMed

Yan, Maocai; Li, Guanqun; An, Jing

2017-06-01

The Wnt/β-catenin signaling pathway typically shows aberrant activation in various cancer cells, especially colorectal cancer cells. This signaling pathway regulates the expression of a variety of tumor-related proteins, including c-myc and cyclin D1, and plays essential roles in tumorigenesis and in the development of many cancers. Small molecules that block the interactions between β-catenin and Tcf4, a downstream stage of activation of the Wnt/β-catenin signaling pathway, could efficiently cut off this signal transduction and thereby act as a novel class of anticancer drugs. This paper reviews the currently reported inhibitors that target β-catenin/Tcf4 interactions, focusing on the discovery approaches taken in the design of these inhibitors and their bioactivities. A brief perspective is then shared on the future discovery and development of this class of inhibitors. Impact statement This mini-review summarized the current knowledge of inhibitors of interactions of beta-catenin/Tcf4 published to date according to their discovery approaches, and discussed their in vitro and in vivo activities, selectivities, and pharmacokinetic properties. Several reviews presently available now in this field describe modulators of the Wnt/beta-catenin pathway, but are generally focused on the bioactivities of these inhibitors. By contrast, this review focused on the drug discovery approaches taken in identifying these types of inhibitors and provided our perspective on further strategies for future drug discoveries. This review also integrated many recently published and important works on highly selective inhibitors as well as rational drug design. We believe that the findings and strategies summarized in this review have broad implications and will be of interest throughout the biochemical and pharmaceutical research community.

Cross-talk of WNT and FGF signaling pathways at GSK3beta to regulate beta-catenin and SNAIL signaling cascades.

PubMed

Katoh, Masuko; Katoh, Masaru

2006-09-01

WNT and FGF signaling pathways cross-talk during a variety of cellular processes, such as human colorectal carcinogenesis, mouse mammary tumor virus (MMTV)-induced carcinogenesis, E2A-Pbx-induced leukemogenesis, early embryogenesis, body-axis formation, limb-bud formation, and neurogenesis. Canonical WNT signals are transduced through Frizzled receptor and LRP5/6 coreceptor to downregulate GSK3beta (GSK3B) activity not depending on Ser 9 phosphorylation. FGF signals are transduced through FGF receptor to the FRS2-GRB2-GAB1-PI3K-AKT signaling cascade to downregulate GSK3beta activity depending on Ser 9 phosphorylation. Because GSK3beta-dependent phosphorylation of beta-catenin and SNAIL leads to FBXW1 (betaTRCP)-mediated ubiquitination and degradation, GSK3beta downregulation results in the stabilization and the nuclear accumulation of beta-catenin and SNAIL. Nuclear beta-catenin is complexed with TCF/LEF, Legless (BCL9 or BCL9L) and PYGO (PYGO1 or PYGO2) to activate transcription of CCND1, MYC, FGF18 and FGF20 genes for the cell-fate determination. Nuclear SNAIL represses transcription of CDH1 gene, encoding E-cadherin, to induce the epithelial-mesenchymal transition (EMT). Mammary carcinogenesis in MMTV-Wnt1 transgenic mice is accelerated by MMTV infection due to MMTV integration around Fgf3-Fgf4 or Fgf8 loci, and mammary carcinogenesis in MMTV-Fgf3 transgenic mice due to MMTV integration around Wnt1-Wnt10b locus. Coactivation of WNT and FGF signaling pathways in tumors leads to more malignant phenotypes. Single nucleotide polymorphism (SNP) and copy number polymorphism (CNP) of WNT and FGF signaling molecules could be utilized as screening method of cancer predisposition. cDNA-PCR, microarray or ELISA reflecting aberrant activation of WNT and FGF signaling pathways could be developed as novel cancer-related biomarkers for diagnosis, prognosis, and therapy. Cocktail therapy using WNT and FGF inhibitors, such as small-molecule compounds and human neutralizing antibodies, should be developed to increase the efficacy of chemotherapy through the inhibition of recurrence by destructing cancer stem cells.

R-spondin 2 facilitates differentiation of proliferating chondrocytes into hypertrophic chondrocytes by enhancing Wnt/β-catenin signaling in endochondral ossification

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

Takegami, Yasuhiko; Department of Orthopaedic Surgery, Nagoya University School of Medicine, Nagoya; Ohkawara, Bisei

Endochondral ossification is a crucial process for longitudinal growth of bones. Differentiating chondrocytes in growth cartilage form four sequential zones of proliferation, alignment into column, hypertrophy, and substitution of chondrocytes with osteoblasts. Wnt/β-catenin signaling is essential for differentiation of proliferating chondrocytes into hypertrophic chondrocytes in growth cartilage. R-spondin 2 (Rspo2), a member of R-spondin family, is an agonist for Wnt signaling, but its role in chondrocyte differentiation remains unknown. Here we report that growth cartilage of Rspo2-knockout mice shows a decreased amount of β-catenin and increased amounts collagen type II (CII) and Sox9 in the abnormally extended proliferating zone. Inmore » contrast, expression of collagen type X (CX) in the hypertrophic zone remains unchanged. Differentiating chondrogenic ATDC5 cells, mimicking proliferating chondrocytes, upregulate Rspo2 and its putative receptor, Lgr5, in parallel. Addition of recombinant human Rspo2 to differentiating ATDC5 cells decreases expressions of Col2a1, Sox9, and Acan, as well as production of proteoglycans. In contrast, lentivirus-mediated knockdown of Rspo2 has the opposite effect. The effect of Rspo2 on chondrogenic differentiation is mediated by Wnt/β-catenin signaling, and not by Wnt/PCP or Wnt/Ca{sup 2+} signaling. We propose that Rspo2 activates Wnt/β-catenin signaling to reduce Col2a1 and Sox9 and to facilitate differentiation of proliferating chondrocytes into hypertrophic chondrocytes in growth cartilage. - Highlights: • Rspo2 is a secreted activator of Wnt, and its knockout shows extended proliferating chondrocytes in endochondral ossification. • In proliferating chondrocytes of Rspo2-knockout mice, Sox9 and collagen type 2 are increased and β-catenin is decreased. • Rspo2 and its receptor Lgr5, as well as Sox9 and collagen type 2, are expressed in differentiating ATDC5 chondrogenic cells. • In ATDC5 cells, Rspo2 decreases expressions of Sox9, collagen type 2, and aggrecan through Wnt/β-catenin signaling. • We propose that Rspo2 activates Wnt/β-catenin to facilitate chondrocyte differentiation in endochondral ossification.« less

Hair follicle stem cell proliferation, Akt and Wnt signaling activation in TPA-induced hair regeneration.

PubMed

Qiu, Weiming; Lei, Mingxing; Zhou, Ling; Bai, Xiufeng; Lai, Xiangdong; Yu, Yu; Yang, Tian; Lian, Xiaohua

2017-06-01

Regeneration of hair follicles relies on activation of hair follicle stem cells during telogen to anagen transition process in hair cycle. This process is rigorously controlled by intrinsic and environmental factors. 12-o-tetradecanoylphorbol-13-acetate (TPA), a tumor promoter, accelerates reentry of hair follicles into anagen phase. However, it is unclear that how TPA promotes the hair regeneration. In the present study, we topically applied TPA onto the dorsal skin of 2-month-old C57BL/6 female mice to examine the activity of hair follicle stem cells and alteration of signaling pathways during hair regeneration. We found that refractory telogen hair follicles entered anagen prematurely after TPA treatment, with the enhanced proliferation of CD34-positive hair follicle stem cells. Meanwhile, we observed Akt signaling was activated in epidermis, hair infundibulum, bulge and hair bulb, and Wnt signaling was also activated after hair follicle stem cells proliferation. Importantly, after overexpression of DKK1, a specific Wnt signaling inhibitor, the accelerated reentry of hair follicles into anagen induced by TPA was abolished. Our data indicated that TPA-induced hair follicle regeneration is associated with activation of Akt and Wnt/β-catenin signaling.

Paracrine Activation of the Wnt/β-Catenin Pathway by Bone Marrow Stem Cell Attenuates Cisplatin-Induced Kidney Injury.

PubMed

Jiao, Xiaoyan; Cai, Jieru; Yu, Xiaofang; Ding, Xiaoqiang

2017-01-01

Cisplatin-induced acute kidney injury (AKI) involves damage to tubular cells via excess reactive oxygen species (ROS) generation. Stem cell-based therapies have shown great promise in AKI treatment. In this study, we aimed to assess the protective effect and mechanism of bone marrow mesenchymal stem cell (BMSC)-derived conditioned medium (CM) against cisplatin-induced AKI. In vitro, NRK-52E cells were incubated with cisplatin in the presence or absence of CM, followed by the assessment of cell viability, apoptosis and cell cycle distribution. Then, ICG-001 and IWR-1 were used to inhibit the wnt/β-catenin pathway. Furthermore, intracellular and mitochondrial ROS levels were evaluated using DCFH-DA and MitoSOX, respectively. In vivo, after cisplatin injection, rats were intravenously injected with CM or BMSCs. Sera and kidney tissues were collected on day 3 after cisplatin injection to evaluate changes in renal function and histology. Western blotting and qRT-PCR were employed to determine the expression of wnt/β-catenin pathway-related genes and proteins. Immunohistochemical staining was used to evaluate tubular β-catenin expression in kidney biopsy from AKI patients. CM protected NRK-52E cells from cisplatin-induced injury by restoring the wnt4/β-catenin pathway. In response to ICG-001 and IWR-1, the protective effect of CM was attenuated, characterized by a decrease in cell proliferation and an increase in cell apoptosis and intracellular and mitochondrial ROS levels. Knockdown of β-catenin using siRNAs also suppressed the mitochondrial biogenesis regulators PGC-1α, TFAM and NRF-1. In the rat model, CM significantly alleviated renal function and histology associated with tubular injury and upregulated wnt4 and β-catenin. However, the renoprotective effect of CM was blocked by ICG-001, characterized by exacerbated renal function, suppressed PGC-1α expression and increased mitochondrial ROS. Clinical data showed that the tubular β-catenin level was lower in AKI patients experiencing partial recovery than in patients experiencing complete recovery. The activation of the wnt/β-catenin pathway by CM protects against cisplatin-induced kidney injury, resulting in reduced apoptosis and intracellular ROS levels. © 2017 The Author(s). Published by S. Karger AG, Basel.

WNT16 antagonises excessive canonical WNT activation and protects cartilage in osteoarthritis.

PubMed

Nalesso, Giovanna; Thomas, Bethan Lynne; Sherwood, Joanna Claire; Yu, Jing; Addimanda, Olga; Eldridge, Suzanne Elizabeth; Thorup, Anne-Sophie; Dale, Leslie; Schett, Georg; Zwerina, Jochen; Eltawil, Noha; Pitzalis, Costantino; Dell'Accio, Francesco

2017-01-01

Both excessive and insufficient activation of WNT signalling results in cartilage breakdown and osteoarthritis. WNT16 is upregulated in the articular cartilage following injury and in osteoarthritis. Here, we investigate the function of WNT16 in osteoarthritis and the downstream molecular mechanisms. Osteoarthritis was induced by destabilisation of the medial meniscus in wild-type and WNT16-deficient mice. Molecular mechanisms and downstream effects were studied in vitro and in vivo in primary cartilage progenitor cells and primary chondrocytes. The pathway downstream of WNT16 was studied in primary chondrocytes and using the axis duplication assay in Xenopus. WNT16-deficient mice developed more severe osteoarthritis with reduced expression of lubricin and increased chondrocyte apoptosis. WNT16 supported the phenotype of cartilage superficial-zone progenitor cells and lubricin expression. Increased osteoarthritis in WNT16-deficient mice was associated with excessive activation of canonical WNT signalling. In vitro, high doses of WNT16 weakly activated canonical WNT signalling, but, in co-stimulation experiments, WNT16 reduced the capacity of WNT3a to activate the canonical WNT pathway. In vivo, WNT16 rescued the WNT8-induced primary axis duplication in Xenopus embryos. In osteoarthritis, WNT16 maintains a balanced canonical WNT signalling and prevents detrimental excessive activation, thereby supporting the homeostasis of progenitor cells. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/.

Klotho, stem cells, and aging.

PubMed

Bian, Ao; Neyra, Javier A; Zhan, Ming; Hu, Ming Chang

2015-01-01

Aging is an inevitable and progressive biological process involving dysfunction and eventually destruction of every tissue and organ. This process is driven by a tightly regulated and complex interplay between genetic and acquired factors. Klotho is an antiaging gene encoding a single-pass transmembrane protein, klotho, which serves as an aging suppressor through a wide variety of mechanisms, such as antioxidation, antisenescence, antiautophagy, and modulation of many signaling pathways, including insulin-like growth factor and Wnt. Klotho deficiency activates Wnt expression and activity contributing to senescence and depletion of stem cells, which consequently triggers tissue atrophy and fibrosis. In contrast, the klotho protein was shown to suppress Wnt-signaling transduction, and inhibit cell senescence and preserve stem cells. A better understanding of the potential effects of klotho on stem cells could offer novel insights into the cellular and molecular mechanisms of klotho deficiency-related aging and disease. The klotho protein may be a promising therapeutic agent for aging and aging-related disorders.

Methods to Manipulate and Monitor Wnt Signaling in Human Pluripotent Stem Cells.

PubMed

Huggins, Ian J; Brafman, David; Willert, Karl

2016-01-01

Human pluripotent stem cells (hPSCs) may revolutionize medical practice by providing: (a) a renewable source of cells for tissue replacement therapies, (b) a powerful system to model human diseases in a dish, and (c) a platform for examining efficacy and safety of novel drugs. Furthermore, these cells offer a unique opportunity to study early human development in vitro, in particular, the process by which a seemingly uniform cell population interacts to give rise to the three main embryonic lineages: ectoderm, endoderm. and mesoderm. This process of lineage allocation is regulated by a number of inductive signals that are mediated by growth factors, including FGF, TGFβ, and Wnt. In this book chapter, we introduce a set of tools, methods, and protocols to specifically manipulate the Wnt signaling pathway with the intention of altering the cell fate outcome of hPSCs.

Metyrapone Alleviates Deleterious Effects of Maternal Food Restriction on Lung Development and Growth of Rat Offspring

PubMed Central

Paek, David S.; Sakurai, Reiko; Saraswat, Aditi; Li, Yishi; Khorram, Omid; Torday, John S.

2015-01-01

Maternal food restriction (MFR) causes intrauterine growth restriction, a known risk factor for developing chronic lung disease. However, it is unknown whether this negative outcome is gender specific or preventable by blocking the MFR-induced hyperglucocorticoidism. Using a well-established rat model, we used metyrapone (MTP), an inhibitor of glucocorticoid synthesis, to study the MFR-induced lung changes on postnatal day (p) 21 in a gender-specific manner. From embryonic day 10 until delivery, pregnant dams were fed either an ad libitum diet or a 50% caloric restricted diet with or without MTP supplementation. Postnatally, the offspring were fed ad libitum from healthy dams until p21. Morphometric, Western blot, and immunohistochemical analysis of the lungs demonstrated that MTP mitigated the MFR-mediated decrease in alveolar count, decrease in adipogenic protein peroxisome proliferator-activated receptor γ, increase in myogenic proteins (fibronectin, α-smooth muscle actin, and calponin), increase in Wnt signaling intermediates (lymphoid enhancer-binding factor 1 and β-catenin), and increase in glucocorticoid receptor (GR) levels. The MFR-induced lung phenotype and the effects of MTP were similar in both genders. To elucidate the mechanism of MFR-induced shift of the adipogenic-to-myogenic phenotype, lung fibroblasts were used to independently study the effects of (1) nutrient restriction and (2) excess steroid exposure. Nutrient deprivation increased myogenic proteins, Wnt signaling intermediates, and GR, all changes blocked by protein supplementation. MTP also blocked, likely by normalizing nicotinamide adenine dinucleotide phosphate levels, the corticosterone-induced increase in myogenic proteins, but had no effect on GR levels. In summary, protein restriction and increased glucocorticoid levels appear to be the key players in MFR-induced lung disease, affecting both genders. PMID:24916330

Metyrapone alleviates deleterious effects of maternal food restriction on lung development and growth of rat offspring.

PubMed

Paek, David S; Sakurai, Reiko; Saraswat, Aditi; Li, Yishi; Khorram, Omid; Torday, John S; Rehan, Virender K

2015-02-01

Maternal food restriction (MFR) causes intrauterine growth restriction, a known risk factor for developing chronic lung disease. However, it is unknown whether this negative outcome is gender specific or preventable by blocking the MFR-induced hyperglucocorticoidism. Using a well-established rat model, we used metyrapone (MTP), an inhibitor of glucocorticoid synthesis, to study the MFR-induced lung changes on postnatal day (p) 21 in a gender-specific manner. From embryonic day 10 until delivery, pregnant dams were fed either an ad libitum diet or a 50% caloric restricted diet with or without MTP supplementation. Postnatally, the offspring were fed ad libitum from healthy dams until p21. Morphometric, Western blot, and immunohistochemical analysis of the lungs demonstrated that MTP mitigated the MFR-mediated decrease in alveolar count, decrease in adipogenic protein peroxisome proliferator-activated receptor γ, increase in myogenic proteins (fibronectin, α-smooth muscle actin, and calponin), increase in Wnt signaling intermediates (lymphoid enhancer-binding factor 1 and β-catenin), and increase in glucocorticoid receptor (GR) levels. The MFR-induced lung phenotype and the effects of MTP were similar in both genders. To elucidate the mechanism of MFR-induced shift of the adipogenic-to-myogenic phenotype, lung fibroblasts were used to independently study the effects of (1) nutrient restriction and (2) excess steroid exposure. Nutrient deprivation increased myogenic proteins, Wnt signaling intermediates, and GR, all changes blocked by protein supplementation. MTP also blocked, likely by normalizing nicotinamide adenine dinucleotide phosphate levels, the corticosterone-induced increase in myogenic proteins, but had no effect on GR levels. In summary, protein restriction and increased glucocorticoid levels appear to be the key players in MFR-induced lung disease, affecting both genders. © The Author(s) 2014.

Combinatorial Wnt control of zebrafish midbrain-hindbrain boundary formation.

PubMed

Buckles, Gerri R; Thorpe, Christopher J; Ramel, Marie-Christine; Lekven, Arne C

2004-05-01

Wnt signaling is known to be required for the normal development of the vertebrate midbrain and hindbrain, but genetic loss of function analyses in the mouse and zebrafish yield differing results regarding the relative importance of specific Wnt loci. In the zebrafish, Wnt1 and Wnt10b functionally overlap in their control of gene expression in the ventral midbrain-hindbrain boundary (MHB), but they are not required for the formation of the MHB constriction. Whether other wnt loci are involved in zebrafish MHB development is unclear, although the expression of at least two wnts, wnt3a and wnt8b, is maintained in wnt1/wnt10b mutants. In order to address the role of wnt3a in zebrafish, we have isolated a full length cDNA and examined its expression and function via knockdown by morpholino antisense oligonucleotide (MO)-mediated knockdown. The expression pattern of wnt3a appears to be evolutionarily conserved between zebrafish and mouse, and MO knockdown shows that Wnt3a, while not uniquely required for MHB development, is required in the absence of Wnt1 and Wnt10b for the formation of the MHB constriction. In zebrafish embryos lacking Wnt3a, Wnt1 and Wnt10b, the expression of engrailed orthologs, pax2a and fgf8 is not maintained after mid-somitogenesis. In contrast to acerebellar and no isthmus mutants, in which midbrain and hindbrain cells acquire new fates but cell number is not significantly affected until late in embryogenesis, zebrafish embryos lacking Wnt3a, Wnt1 and Wnt10b undergo extensive apoptosis in the midbrain and cerebellum anlagen beginning in mid-somitogenesis, which results in the absence of a significant portion of the midbrain and cerebellum. Thus, the requirement for Wnt signaling in forming the MHB constriction is evolutionarily conserved in vertebrates and it is possible in zebrafish to dissect the relative impact of multiple Wnt loci in midbrain and hindbrain development.

Ectopic Activation of Wnt/β-Catenin Signaling in Lens Fiber Cells Results in Cataract Formation and Aberrant Fiber Cell Differentiation

PubMed Central

Antosova, Barbora; Smolikova, Jana; Borkovcova, Romana; Strnad, Hynek; Lachova, Jitka; Machon, Ondrej; Kozmik, Zbynek

2013-01-01

The Wnt/β-catenin signaling pathway controls many processes during development, including cell proliferation, cell differentiation and tissue homeostasis, and its aberrant regulation has been linked to various pathologies. In this study we investigated the effect of ectopic activation of Wnt/β-catenin signaling during lens fiber cell differentiation. To activate Wnt/β-catenin signaling in lens fiber cells, the transgenic mouse referred to as αA-CLEF was generated, in which the transactivation domain of β-catenin was fused to the DNA-binding protein LEF1, and expression of the transgene was controlled by αA-crystallin promoter. Constitutive activation of Wnt/β-catenin signaling in lens fiber cells of αA-CLEF mice resulted in abnormal and delayed fiber cell differentiation. Moreover, adult αA-CLEF mice developed cataract, microphthalmia and manifested downregulated levels of γ-crystallins in lenses. We provide evidence of aberrant expression of cell cycle regulators in embryonic lenses of αA-CLEF transgenic mice resulting in the delay in cell cycle exit and in the shift of fiber cell differentiation to the central fiber cell compartment. Our results indicate that precise regulation of the Wnt/β-catenin signaling activity during later stages of lens development is essential for proper lens fiber cell differentiation and lens transparency. PMID:24205179

Embryonic ablation of osteoblast Smad4 interrupts matrix synthesis in response to canonical Wnt signaling and causes an osteogenesis-imperfecta-like phenotype

PubMed Central

Salazar, Valerie S.; Zarkadis, Nicholas; Huang, Lisa; Norris, Jin; Grimston, Susan K.; Mbalaviele, Gabriel; Civitelli, Roberto

2013-01-01

Summary To examine interactions between bone morphogenic protein (BMP) and canonical Wnt signaling during skeletal growth, we ablated Smad4, a key component of the TGF-β–BMP pathway, in Osx1+ cells in mice. We show that loss of Smad4 causes stunted growth, spontaneous fractures and a combination of features seen in osteogenesis imperfecta, cleidocranial dysplasia and Wnt-deficiency syndromes. Bones of Smad4 mutant mice exhibited markers of fully differentiated osteoblasts but lacked multiple collagen-processing enzymes, including lysyl oxidase (Lox), a BMP2-responsive gene regulated by Smad4 and Runx2. Accordingly, the collagen matrix in Smad4 mutants was disorganized, but also hypomineralized. Primary osteoblasts from these mutants did not mineralize in vitro in the presence of BMP2 or Wnt3a, and Smad4 mutant mice failed to accrue new bone following systemic inhibition of the Dickkopf homolog Dkk1. Consistent with impaired biological responses to canonical Wnt, ablation of Smad4 causes cleavage of β-catenin and depletion of the low density lipoprotein receptor Lrp5, subsequent to increased caspase-3 activity and apoptosis. In summary, Smad4 regulates maturation of skeletal collagen and osteoblast survival, and is required for matrix-forming responses to both BMP2 and canonical Wnt. PMID:24006258

Dishevelled is essential for neural connectivity and planar cell polarity in planarians.

PubMed

Almuedo-Castillo, Maria; Saló, Emili; Adell, Teresa

2011-02-15

The Wingless/Integrated (Wnt) signaling pathway controls multiple events during development and homeostasis. It comprises multiple branches, mainly classified according to their dependence on β-catenin activation. The Wnt/β-catenin branch is essential for the establishment of the embryonic anteroposterior (AP) body axis throughout the phylogenetic tree. It is also required for AP axis establishment during planarian regeneration. Wnt/β-catenin-independent signaling encompasses several different pathways, of which the most extensively studied is the planar cell polarity (PCP) pathway, which is responsible for planar polarization of cell structures within an epithelial sheet. Dishevelled (Dvl) is the hub of Wnt signaling because it regulates and channels the Wnt signal into every branch. Here, we analyze the role of Schmidtea mediterranea Dvl homologs (Smed-dvl-1 and Smed-dvl-2) using gene silencing. We demonstrate that in addition to a role in AP axis specification, planarian Dvls are involved in at least two different β-catenin-independent processes. First, they are essential for neural connectivity through Smed-wnt5 signaling. Second, Smed-dvl-2, together with the S. mediterranea homologs of Van-Gogh (Vang) and Diversin (Div), is required for apical positioning of the basal bodies of epithelial cells. These data represent evidence not only of the function of the PCP network in lophotrocozoans but of the involvement of the PCP core elements Vang and Div in apical positioning of the cilia.

Dishevelled is essential for neural connectivity and planar cell polarity in planarians

PubMed Central

Almuedo-Castillo, Maria; Saló, Emili; Adell, Teresa

2011-01-01

The Wingless/Integrated (Wnt) signaling pathway controls multiple events during development and homeostasis. It comprises multiple branches, mainly classified according to their dependence on β-catenin activation. The Wnt/β-catenin branch is essential for the establishment of the embryonic anteroposterior (AP) body axis throughout the phylogenetic tree. It is also required for AP axis establishment during planarian regeneration. Wnt/β-catenin–independent signaling encompasses several different pathways, of which the most extensively studied is the planar cell polarity (PCP) pathway, which is responsible for planar polarization of cell structures within an epithelial sheet. Dishevelled (Dvl) is the hub of Wnt signaling because it regulates and channels the Wnt signal into every branch. Here, we analyze the role of Schmidtea mediterranea Dvl homologs (Smed-dvl-1 and Smed-dvl-2) using gene silencing. We demonstrate that in addition to a role in AP axis specification, planarian Dvls are involved in at least two different β-catenin–independent processes. First, they are essential for neural connectivity through Smed-wnt5 signaling. Second, Smed-dvl-2, together with the S. mediterranea homologs of Van-Gogh (Vang) and Diversin (Div), is required for apical positioning of the basal bodies of epithelial cells. These data represent evidence not only of the function of the PCP network in lophotrocozoans but of the involvement of the PCP core elements Vang and Div in apical positioning of the cilia. PMID:21282632

Wnt/β-catenin signaling inhibitor ICG-001 enhances pigmentation of cultured melanoma cells.

PubMed

Kim, Kyung-Il; Jeong, Do-Sun; Jung, Eui Chang; Lee, Jeung-Hoon; Kim, Chang Deok; Yoon, Tae-Jin

2016-11-01

Wnt/β-catenin signaling is important in development and differentiation of melanocytes. The object of this study was to evaluate the effects of several Wnt/β-catenin signaling inhibitors on pigmentation using melanoma cells. Melanoma cells were treated with Wnt/β-catenin signaling inhibitors, and then melanin content and tyrosinase activity were checked. Although some inhibitors showed slight inhibition of pigmentation, we failed to observe potential inhibitory effect of those chemicals on pigmentation of HM3KO melanoma cells. Rather, one of powerful Wnt/β-catenin signaling inhibitors, ICG-001, increased the pigmentation of HM3KO melanoma cells. Pigmentation-enhancing effect of ICG-001 was reproducible in other melanoma cell line MNT-1. Consistent with these results. ICG-001 increased the expression of pigmentation-related genes, such as MITF, tyrosinase and TRP1. When ICG-001 was treated, the phosphorylation of CREB was significantly increased. In addition, ICG-001 treatment led to quick increase of intracellular cAMP level, suggesting that ICG-001 activated PKA signaling. The blockage of PKA signaling with pharmaceutical inhibitor H89 inhibited the ICG-001-induced pigmentation significantly. These results suggest that PKA signaling is pivotal in pigmentation process itself, while the importance of Wnt/β-catenin signaling should be emphasized in the context of development and differentiation. Copyright © 2016 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

Specification of hepatopancreas progenitors in zebrafish by hnf1ba and wnt2bb

PubMed Central

Lancman, Joseph J.; Zvenigorodsky, Natasha; Gates, Keith P.; Zhang, Danhua; Solomon, Keely; Humphrey, Rohan K.; Kuo, Taiyi; Setiawan, Linda; Verkade, Heather; Chi, Young-In; Jhala, Ulupi S.; Wright, Christopher V. E.; Stainier, Didier Y. R.; Dong, P. Duc Si

2013-01-01

Although the liver and ventral pancreas are thought to arise from a common multipotent progenitor pool, it is unclear whether these progenitors of the hepatopancreas system are specified by a common genetic mechanism. Efforts to determine the role of Hnf1b and Wnt signaling in this crucial process have been confounded by a combination of factors, including a narrow time frame for hepatopancreas specification, functional redundancy among Wnt ligands, and pleiotropic defects caused by either severe loss of Wnt signaling or Hnf1b function. Using a novel hypomorphic hnf1ba zebrafish mutant that exhibits pancreas hypoplasia, as observed in HNF1B monogenic diabetes, we show that hnf1ba plays essential roles in regulating β-cell number and pancreas specification, distinct from its function in regulating pancreas size and liver specification, respectively. By combining Hnf1ba partial loss of function with conditional loss of Wnt signaling, we uncover a crucial developmental window when these pathways synergize to specify the entire ventrally derived hepatopancreas progenitor population. Furthermore, our in vivo genetic studies demonstrate that hnf1ba generates a permissive domain for Wnt signaling activity in the foregut endoderm. Collectively, our findings provide a new model for HNF1B function, yield insight into pancreas and β-cell development, and suggest a new mechanism for hepatopancreatic specification. PMID:23720049

[Effect of Biejiajian Pills on Wnt signal pathway molecules β-catenin and GSK-3β and the target genes CD44v6 and VEGF in hepatocellular carcinoma cells].

PubMed

Sun, Haitao; He, Songqi; Wen, Bin; Jia, Wenyan; Fan, Eryan; Zheng, Yan

2014-10-01

To investigate the effect of Biejiajian Pills on the expressions of the signal molecules and target genes of Wnt signal pathway in HepG2 cells and explore the mechanisms by which Biejiajian pills suppress the invasiveness of hepatocellular carcinoma. HepG2 cells were cultured for 48 h in the presence of serum collected from rats fed with Biejiajian Pills. The expressions of β-catenin, GSK-3β and P-GSK-3β in the cultured cells were assessed by Western blotting and the expressions of CD44v6 and VEGF were detected using immunohistochemistry. HepG2 cells cultured with the serum of rats fed with Biejiajian Pills showed lowered expressions of β-catenin protein both in the cytoplasm and the nuclei with also inhibition of phosphorylation of GSK-3β and reduced expression of CD44v6 and VEGF. Biejiajian Pills can significantly reduce the expression of β-catenin by decreasing the phosphorylation of GSK-3β and blocking the Wnt/β-catenin signaling pathway to cause down-regulation of the target genes CD44v6 and VEGF, which may be one of the molecular mechanisms by which Biejiajian Pills suppress the proliferation and invasiveness of hepatocellular carcinoma.

[Effect of Biejiajian Pills on Wnt/β-catenin signal pathway and DKK-1 and FrpHe gene expressions in hepatocellular carcinoma cells].

PubMed

He, Songqi; Cheng, Yang; Zhu, Yun; Fan, Qin; Sun, Haitao; Jia, Wenyan

2013-01-01

To investigate the effect of Biejiajian Pills on Wnt signal pathway and its inhibitory gene (DKK-1 and FrpHe) expressions and explore the mechanism underlying the action of Biejiajian Pills to suppress the invasiveness of hepatocellular carcinoma. Twenty-four Wistar rats were randomized equally into 3 groups for gavage of normal saline and Biejiajian Pills at 20- and 10-fold clinical doses for 3 days. Blood samples were then collected from the rats, and the serum was separated and added in HepG2 cell cultures. After 48 h of culture, the cells were collected to determine the cellular content of β-catenin protein using flow cytometry and detect DKK-1 and FrpHe mRNA expressions using qRT-PCR. HepG2 cells cultured in the presence of sera from rats fed with Biejiajian Pills showed significantly lowered β-catenin protein expression and obvious down-regulation of DKK-1 mRNA expression, and the effect was correlated with the doses of the drug administered. The expression of FrpHe mRNA showed no significant differences between the 3 groups. Biejiajian Pills can effectively inhibit the invasiveness and migration of hepatocellular carcinoma cells, which is closely related to decreased expressions of β-catenin and DKK-1 to cause block of the Wnt/β-catenin signal pathway.

Computational biophysical, biochemical, and evolutionary signature of human R-spondin family proteins, the member of canonical Wnt/β-catenin signaling pathway.

PubMed

Sharma, Ashish Ranjan; Chakraborty, Chiranjib; Lee, Sang-Soo; Sharma, Garima; Yoon, Jeong Kyo; George Priya Doss, C; Song, Dong-Keun; Nam, Ju-Suk

2014-01-01

In human, Wnt/β-catenin signaling pathway plays a significant role in cell growth, cell development, and disease pathogenesis. Four human (Rspo)s are known to activate canonical Wnt/β-catenin signaling pathway. Presently, (Rspo)s serve as therapeutic target for several human diseases. Henceforth, basic understanding about the molecular properties of (Rspo)s is essential. We approached this issue by interpreting the biochemical and biophysical properties along with molecular evolution of (Rspo)s thorough computational algorithm methods. Our analysis shows that signal peptide length is roughly similar in (Rspo)s family along with similarity in aa distribution pattern. In Rspo3, four N-glycosylation sites were noted. All members are hydrophilic in nature and showed alike GRAVY values, approximately. Conversely, Rspo3 contains the maximum positively charged residues while Rspo4 includes the lowest. Four highly aligned blocks were recorded through Gblocks. Phylogenetic analysis shows Rspo4 is being rooted with Rspo2 and similarly Rspo3 and Rspo1 have the common point of origin. Through phylogenomics study, we developed a phylogenetic tree of sixty proteins (n = 60) with the orthologs and paralogs seed sequences. Protein-protein network was also illustrated. Results demonstrated in our study may help the future researchers to unfold significant physiological and therapeutic properties of (Rspo)s in various disease models.

Computational Biophysical, Biochemical, and Evolutionary Signature of Human R-Spondin Family Proteins, the Member of Canonical Wnt/β-Catenin Signaling Pathway

PubMed Central

Sharma, Ashish Ranjan; Lee, Sang-Soo; Yoon, Jeong Kyo; George Priya Doss, C.; Song, Dong-Keun

2014-01-01

In human, Wnt/β-catenin signaling pathway plays a significant role in cell growth, cell development, and disease pathogenesis. Four human (Rspo)s are known to activate canonical Wnt/β-catenin signaling pathway. Presently, (Rspo)s serve as therapeutic target for several human diseases. Henceforth, basic understanding about the molecular properties of (Rspo)s is essential. We approached this issue by interpreting the biochemical and biophysical properties along with molecular evolution of (Rspo)s thorough computational algorithm methods. Our analysis shows that signal peptide length is roughly similar in (Rspo)s family along with similarity in aa distribution pattern. In Rspo3, four N-glycosylation sites were noted. All members are hydrophilic in nature and showed alike GRAVY values, approximately. Conversely, Rspo3 contains the maximum positively charged residues while Rspo4 includes the lowest. Four highly aligned blocks were recorded through Gblocks. Phylogenetic analysis shows Rspo4 is being rooted with Rspo2 and similarly Rspo3 and Rspo1 have the common point of origin. Through phylogenomics study, we developed a phylogenetic tree of sixty proteins (n = 60) with the orthologs and paralogs seed sequences. Protein-protein network was also illustrated. Results demonstrated in our study may help the future researchers to unfold significant physiological and therapeutic properties of (Rspo)s in various disease models. PMID:25276837

The Dishevelled-binding protein CXXC5 negatively regulates cutaneous wound healing

PubMed Central

Lee, Soung-Hoon; Kim, Mi-Yeon; Kim, Hyun-Yi; Lee, Young-Mi; Kim, Heesu; Nam, Kyoung Ae; Roh, Mi Ryung; Min, Do Sik; Chung, Kee Yang

2015-01-01

Wnt/β-catenin signaling plays important roles in cutaneous wound healing and dermal fibrosis. However, its regulatory mechanism has not been fully elucidated, and a commercially available wound-healing agent targeting this pathway is desirable but currently unavailable. We found that CXXC-type zinc finger protein 5 (CXXC5) serves as a negative feedback regulator of the Wnt/β-catenin pathway by interacting with the Dishevelled (Dvl) protein. In humans, CXXC5 protein levels were reduced in epidermal keratinocytes and dermal fibroblasts of acute wounds. A differential regulation of β-catenin, α-smooth muscle actin (α-SMA), and collagen I by overexpression and silencing of CXXC5 in vitro indicated a critical role for this factor in myofibroblast differentiation and collagen production. In addition, CXXC5−/− mice exhibited accelerated cutaneous wound healing, as well as enhanced keratin 14 and collagen synthesis. Protein transduction domain (PTD)–Dvl-binding motif (DBM), a competitor peptide blocking CXXC5-Dvl interactions, disrupted this negative feedback loop and activated β-catenin and collagen production in vitro. Co-treatment of skin wounds with PTD-DBM and valproic acid (VPA), a glycogen synthase kinase 3β (GSK3β) inhibitor which activates the Wnt/β-catenin pathway, synergistically accelerated cutaneous wound healing in mice. Together, these data suggest that CXXC5 would represent a potential target for future therapies aimed at improving wound healing. PMID:26056233

Cholesterol depletion by methyl-β-cyclodextrin enhances cell proliferation and increases the number of desmin-positive cells in myoblast cultures.

PubMed

Portilho, Débora M; Soares, Carolina P; Morrot, Alexandre; Thiago, Leandro S; Butler-Browne, Gillian; Savino, Wilson; Costa, Manoel L; Mermelstein, Cláudia

2012-11-05

Skeletal myogenesis comprises myoblast replication and differentiation into striated multinucleated myotubes. Agents that interfere with myoblast replication are important tools for the understanding of myogenesis. Recently, we showed that cholesterol depletion by methyl-β-cyclodextrin (MCD) enhances the differentiation step in chick-cultured myogenic cells, involving the activation of the Wnt/β-catenin signaling pathway. However, the effects of cholesterol depletion on myoblast replication have not been carefully studied. Here we show that MCD treatment increases cell proliferation in primary chick myogenic cell cultures. Treatment of myogenic cells with the anti-mitotic reagent cytosine arabinoside, immediately following cholesterol depletion, blocks the MCD-induced effects on proliferation. Cholesterol depletion induced an increase in the number of desmin-positive mononucleated cells, and an increase in desmin expression. MCD induces an increase in the expression of the cell cycle regulator p53 and the master switch gene MyoD1. Treatment with BIO, a specific inhibitor of GSK3β, induced effects similar to MCD on cell proliferation; while treatment with Dkk1, a specific inhibitor of the Wnt/β-catenin pathway, neutralized the effects of MCD. These findings indicate that rapid changes in the cholesterol content in cell membranes of myoblasts can induce cell proliferation, possibly by the activation of the Wnt/β-catenin signaling pathway. Copyright © 2012 Elsevier B.V. All rights reserved.

Wnt5b-associated exosomes promote cancer cell migration and proliferation.

PubMed

Harada, Takeshi; Yamamoto, Hideki; Kishida, Shosei; Kishida, Michiko; Awada, Chihiro; Takao, Toshifumi; Kikuchi, Akira

2017-01-01

Wnt5b is a member of the same family of proteins as Wnt5a, the overexpression of which is associated with cancer aggressiveness. Wnt5b is also suggested to be involved in cancer progression, however, details remain unclarified. We analyzed the biochemical properties of purified Wnt5b and the mode of secretion of Wnt5b by cancer cells. Wnt5b was glycosylated at three asparagine residues and lipidated at one serine residue, and these post-translational modifications of Wnt5b were essential for secretion. Purified Wnt5b showed Dvl2 phosphorylation and Rac activation abilities to a similar extent as Wnt5a. In cultured-cell conditioned medium, Wnt5b was detected in supernatant or precipitation fractions that were separated by centrifugation at 100 000 g. In PANC-1 pancreatic cancer cells, 55% of secreted endogenous Wnt5b was associated with exosomes. Exosomes from wild-type PANC-1 cells, but not those from Wnt5b-knockout PANC-1 cells, activated Wnt5b signaling in CHO cells and stimulated migration and proliferation of A549 lung adenocarcinoma cells, suggesting that endogenous, Wnt5b-associated exosomes are active. The exosomes were taken up by CHO cells and immunoelectron microscopy revealed that Wnt5b is indeed associated with exosomes. In Caco-2 colon cancer cells, most Wnt5b was recovered in precipitation fractions when Wnt5b was ectopically expressed (Caco-2/Wnt5b cells). Knockdown of TSG101, an exosome marker, decreased the secretion of Wnt5b-associated exosomes from Caco-2/Wnt5b cells and inhibited Wnt5b-dependent cell proliferation. Exosomes secreted from Caco-2/Wnt5b cells stimulated migration and proliferation of A549 cells. These results suggest that Wnt5b-associated exosomes promote cancer cell migration and proliferation in a paracrine manner. © 2016 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

Wnt5a and Wnt11 regulate mammalian anterior-posterior axis elongation

PubMed Central

Andre, Philipp; Song, Hai; Kim, Wantae; Kispert, Andreas; Yang, Yingzi

2015-01-01

Mesoderm formation and subsequent anterior-posterior (A-P) axis elongation are fundamental aspects of gastrulation, which is initiated by formation of the primitive streak (PS). Convergent extension (CE) movements and epithelial-mesenchymal transition (EMT) are important for A-P axis elongation in vertebrate embryos. The evolutionarily conserved planar cell polarity (PCP) pathway regulates CE, and Wnts regulate many aspects of gastrulation including CE and EMT. However, the Wnt ligands that regulate A-P axis elongation in mammalian development remain unknown. Wnt11 and Wnt5a regulate axis elongation in lower vertebrates, but only Wnt5a, not Wnt11, regulates mammalian PCP signaling and A-P axis elongation in development. Here, by generating Wnt5a; Wnt11 compound mutants, we show that Wnt11 and Wnt5a play redundant roles during mouse A-P axis elongation. Both genes regulate trunk notochord extension through PCP-controlled CE of notochord cells, establishing a role for Wnt11 in mammalian PCP. We show that Wnt5a and Wnt11 are required for proper patterning of the neural tube and somites by regulating notochord formation, and provide evidence that both genes are required for the generation and migration of axial and paraxial mesodermal precursor cells by regulating EMT. Axial and paraxial mesodermal precursors ectopically accumulate in the PS at late gastrula stages in Wnt5a−/−; Wnt11−/− embryos and these cells ectopically express epithelial cell adhesion molecules. Our data suggest that Wnt5a and Wnt11 regulate EMT by inducing p38 (Mapk14) phosphorylation. Our findings provide new insights into the role of Wnt5a and Wnt11 in mouse early development and also in cancer metastasis, during which EMT plays a crucial role. PMID:25813538

Wedelolactone enhances osteoblastogenesis by regulating Wnt/β-catenin signaling pathway but suppresses osteoclastogenesis by NF-κB/c-fos/NFATc1 pathway.

PubMed

Liu, Yan-Qiu; Hong, Zhi-Lai; Zhan, Li-Bin; Chu, Hui-Ying; Zhang, Xiao-Zhe; Li, Guo-Hui

2016-08-25

Bone homeostasis is maintained by formation and destruction of bone, which are two processes tightly coupled and controlled. Targeting both stimulation on bone formation and suppression on bone resorption becomes a promising strategy for treating osteoporosis. In this study, we examined the effect of wedelolactone, a natural product from Ecliptae herba, on osteoblastogenesis as well as osteoclastogenesis. In mouse bone marrow mesenchymal stem cells (BMSC), wedelolactone stimulated osteoblast differentiation and bone mineralization. At the molecular level, wedelolactone directly inhibited GSK3β activity and enhanced the phosphorylation of GSK3β, thereafter stimulated the nuclear translocation of β-catenin and runx2. The expression of osteoblastogenesis-related marker gene including osteorix, osteocalcin and runx2 increased. At the same concentration range, wedelolactone inhibited RANKL-induced preosteoclastic RAW264.7 actin-ring formation and bone resorption pits. Further, wedelolactone blocked NF-kB/p65 phosphorylation and abrogated the NFATc1 nuclear translocation. As a result, osteoclastogenesis-related marker gene expression decreased, including c-src, c-fos, and cathepsin K. In ovariectomized mice, administration of wedelolactone prevented ovariectomy-induced bone loss by enhancing osteoblast activity and inhibiting osteoclast activity. Together, these data demonstrated that wedelolactone facilitated osteoblastogenesis through Wnt/GSK3β/β-catenin signaling pathway and suppressed RANKL-induced osteoclastogenesis through NF-κB/c-fos/NFATc1 pathway. These results suggested that wedelolacone could be a novel dual functional therapeutic agent for osteoporosis.

Discovery of Klotho peptide antagonists against Wnt3 and Wnt3a target proteins using combination of protein engineering, protein-protein docking, peptide docking and molecular dynamics simulations.

PubMed

Mirza, Shaher Bano; Ekhteiari Salmas, Ramin; Fatmi, M Qaiser; Durdagi, Serdar

2017-12-01

The Klotho is known as lifespan enhancing protein involved in antagonizing the effect of Wnt proteins. Wnt proteins are stem cell regulators, and uninterrupted exposure of Wnt proteins to the cell can cause stem and progenitor cell senescence, which may lead to aging. Keeping in mind the importance of Klotho in Wnt signaling, in silico approaches have been applied to study the important interactions between Klotho and Wnt3 and Wnt3a (wingless-type mouse mammary tumor virus (MMTV) integration site family members 3 and 3a). The main aim of the study is to identify important residues of the Klotho that help in designing peptides which can act as Wnt antagonists. For this aim, a protein engineering study is performed for Klotho, Wnt3 and Wnt3a. During the theoretical analysis of homology models, unexpected role of number of disulfide bonds and secondary structure elements has been witnessed in case of Wnt3 and Wnt3a proteins. Different in silico experiments were carried out to observe the effect of correct number of disulfide bonds on 3D protein models. For this aim, total of 10 molecular dynamics (MD) simulations were carried out for each system. Based on the protein-protein docking simulations of selected protein models of Klotho with Wnt3 and Wnt3a, different peptides derived from Klotho have been designed. Wnt3 and Wnt3a proteins have three important domains: Index finger, N-terminal domain and a patch of ∼10 residues on the solvent exposed surface of palm domain. Protein-peptide docking of designed peptides of Klotho against three important domains of palmitoylated Wnt3 and Wnt3a yields encouraging results and leads better understanding of the Wnt protein inhibition by proposed Klotho peptides. Further in vitro studies can be carried out to verify effects of novel designed peptides as Wnt antagonists.

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

PubMed

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

2015-02-01

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

MEK and TAK1 Regulate Apoptosis in Colon Cancer Cells with KRAS-Dependent Activation of Proinflammatory Signaling.

PubMed

McNew, Kelsey L; Whipple, William J; Mehta, Anita K; Grant, Trevor J; Ray, Leah; Kenny, Connor; Singh, Anurag

2016-12-01

MEK inhibitors have limited efficacy in treating RAS-RAF-MEK pathway-dependent cancers due to feedback pathway compensation and dose-limiting toxicities. Combining MEK inhibitors with other targeted agents may enhance efficacy. Here, codependencies of MEK, TAK1, and KRAS in colon cancer were investigated. Combined inhibition of MEK and TAK1 potentiates apoptosis in KRAS-dependent cells. Pharmacologic studies and cell-cycle analyses on a large panel of colon cancer cell lines demonstrate that MEK/TAK1 inhibition induces cell death, as assessed by sub-G 1 accumulation, in a distinct subset of cell lines. Furthermore, TAK1 inhibition causes G 2 -M cell-cycle blockade and polyploidy in many of the cell lines. MEK plus TAK1 inhibition causes reduced G 2 -M/polyploid cell numbers and additive cytotoxic effects in KRAS/TAK1-dependent cell lines as well as a subset of BRAF-mutant cells. Mechanistically, sensitivity to MEK/TAK1 inhibition can be conferred by KRAS and BMP receptor activation, which promote expression of NF-κB-dependent proinflammatory cytokines, driving tumor cell survival and proliferation. MEK/TAK1 inhibition causes reduced mTOR, Wnt, and NF-κB signaling in TAK1/MEK-dependent cell lines concomitant with apoptosis. A Wnt/NF-κB transcriptional signature was derived that stratifies primary tumors into three major subtypes: Wnt-high/NF-κB-low, Wnt-low/NF-κB-high and Wnt-high/NF-κB-high, designated W, N, and WN, respectively. These subtypes have distinct characteristics, including enrichment for BRAF mutations with serrated carcinoma histology in the N subtype. Both N and WN subtypes bear molecular hallmarks of MEK and TAK1 dependency seen in cell lines. Therefore, N and WN subtype signatures could be utilized to identify tumors that are most sensitive to anti-MEK/TAK1 therapeutics. This study describes a potential therapeutic strategy for a subset of colon cancers that are dependent on oncogenic KRAS signaling pathways, which are currently difficult to block with selective agents. Mol Cancer Res; 14(12); 1204-16. ©2016 AACR. ©2016 American Association for Cancer Research.

JS-K, a glutathione/glutathione S-transferase-activated nitric oxide releasing prodrug inhibits androgen receptor and WNT-signaling in prostate cancer cells.

PubMed

Laschak, Martin; Spindler, Klaus-Dieter; Schrader, Andres J; Hessenauer, Andrea; Streicher, Wolfgang; Schrader, Mark; Cronauer, Marcus V

2012-03-30

Nitric oxide (NO) and its oxidative reaction products have been repeatedly shown to block steroid receptor function via nitrosation of zinc finger structures in the DNA-binding domain (DBD). In consequence NO-donors could be of special interest for the treatment of deregulated androgen receptor(AR)-signaling in castration resistant prostate cancer (CRPC). Prostate cancer (PCa) cells were treated with JS-K, a diazeniumdiolate derivate capable of generating large amounts of intracellular NO following activation by glutathione S-transferase. Generation of NO was determined indirectly by the detection of nitrate in tissue culture medium or by immunodetection of nitrotyrosine in the cytoplasm. Effects of JS-K on intracellular AR-levels were determined by western blotting. AR-dimerization was analyzed by mammalian two hybrid assay, nuclear translocation of the AR was visualized in PCa cells transfected with a green fluorescent AR-Eos fusion protein using fluorescence microscopy. Modulation of AR- and WNT-signalling by JS-K was investigated using reporter gene assays. Tumor cell proliferation following JS-K treatment was measured by MTT-Assay. The NO-releasing compound JS-K was shown to inhibit AR-mediated reporter gene activity in 22Rv1 CRPC cells. Inhibition of AR signaling was neither due to an inhibition of nuclear import nor to a reduction in AR-dimerization. In contrast to previously tested NO-donors, JS-K was able to reduce the intracellular concentration of functional AR. This could be attributed to the generation of extremely high intracellular levels of the free radical NO as demonstrated indirectly by high levels of nitrotyrosine in JS-K treated cells. Moreover, JS-K diminished WNT-signaling in AR-positive 22Rv1 cells. In line with these observations, castration resistant 22Rv1 cells were found to be more susceptible to the growth inhibitory effects of JS-K than the androgen dependent LNCaP which do not exhibit an active WNT-signaling pathway. Our results suggest that small molecules able to inhibit WNT- and AR-signaling via NO-release represent a promising platform for the development of new compounds for the treatment of CRPC.

JS-K, a glutathione/glutathione S-transferase-activated nitric oxide releasing prodrug inhibits androgen receptor and WNT-signaling in prostate cancer cells

PubMed Central

2012-01-01

Background Nitric oxide (NO) and its oxidative reaction products have been repeatedly shown to block steroid receptor function via nitrosation of zinc finger structures in the DNA-binding domain (DBD). In consequence NO-donors could be of special interest for the treatment of deregulated androgen receptor(AR)-signaling in castration resistant prostate cancer (CRPC). Methods Prostate cancer (PCa) cells were treated with JS-K, a diazeniumdiolate derivate capable of generating large amounts of intracellular NO following activation by glutathione S-transferase. Generation of NO was determined indirectly by the detection of nitrate in tissue culture medium or by immunodetection of nitrotyrosine in the cytoplasm. Effects of JS-K on intracellular AR-levels were determined by western blotting. AR-dimerization was analyzed by mammalian two hybrid assay, nuclear translocation of the AR was visualized in PCa cells transfected with a green fluorescent AR-Eos fusion protein using fluorescence microscopy. Modulation of AR- and WNT-signalling by JS-K was investigated using reporter gene assays. Tumor cell proliferation following JS-K treatment was measured by MTT-Assay. Results The NO-releasing compound JS-K was shown to inhibit AR-mediated reporter gene activity in 22Rv1 CRPC cells. Inhibition of AR signaling was neither due to an inhibition of nuclear import nor to a reduction in AR-dimerization. In contrast to previously tested NO-donors, JS-K was able to reduce the intracellular concentration of functional AR. This could be attributed to the generation of extremely high intracellular levels of the free radical NO as demonstrated indirectly by high levels of nitrotyrosine in JS-K treated cells. Moreover, JS-K diminished WNT-signaling in AR-positive 22Rv1 cells. In line with these observations, castration resistant 22Rv1 cells were found to be more susceptible to the growth inhibitory effects of JS-K than the androgen dependent LNCaP which do not exhibit an active WNT-signaling pathway. Conclusions Our results suggest that small molecules able to inhibit WNT- and AR-signaling via NO-release represent a promising platform for the development of new compounds for the treatment of CRPC. PMID:22462810

WNT signaling in stem cell biology and regenerative medicine.

PubMed

Katoh, Masaru

2008-07-01

WNT family members are secreted-type glycoproteins to orchestrate embryogenesis, to maintain homeostasis, and to induce pathological conditions. FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, LRP5, LRP6, and ROR2 are transmembrane receptors transducing WNT signals based on ligand-dependent preferentiality for caveolin- or clathrin-mediated endocytosis. WNT signals are transduced to canonical pathway for cell fate determination, and to non-canonical pathways for regulation of planar cell polarity, cell adhesion, and motility. MYC, CCND1, AXIN2, FGF20, WISP1, JAG1, DKK1 and Glucagon are target genes of canonical WNT signaling cascade, while CD44, Vimentin and STX5 are target genes of non-canonical WNT signaling cascades. However, target genes of WNT signaling cascades are determined in a context-dependent manner due to expression profile of transcription factors and epigenetic status. WNT signaling cascades network with Notch, FGF, BMP and Hedgehog signaling cascades to regulate the balance of stem cells and progenitor cells. Here WNT signaling in embryonic stem cells, neural stem cells, mesenchymal stem cells, hematopoietic stem cells, and intestinal stem cells will be reviewed. WNT3, WNT5A and WNT10B are expressed in undifferentiated human embryonic stem cells, while WNT6, WNT8B and WNT10B in endoderm precursor cells. Wnt6 is expressed in intestinal crypt region for stem or progenitor cells. TNF/alpha-WNT10B signaling is a negative feedback loop to maintain homeostasis of adipose tissue and gastrointestinal mucosa with chronic inflammation. Recombinant WNT protein or WNT mimetic (circular peptide, small molecule compound, or RNA aptamer) in combination with Notch mimetic, FGF protein, and BMP protein opens a new window to tissue engineering for regenerative medicine.

Blocking epithelial-to-mesenchymal transition in glioblastoma with a sextet of repurposed drugs: the EIS regimen.

PubMed

Kast, Richard E; Skuli, Nicolas; Karpel-Massler, Georg; Frosina, Guido; Ryken, Timothy; Halatsch, Marc-Eric

2017-09-22

This paper outlines a treatment protocol to run alongside of standard current treatment of glioblastoma- resection, temozolomide and radiation. The epithelial to mesenchymal transition (EMT) inhibiting sextet, EIS Regimen, uses the ancillary attributes of six older medicines to impede EMT during glioblastoma. EMT is an actively motile, therapy-resisting, low proliferation, transient state that is an integral feature of cancers' lethality generally and of glioblastoma specifically. It is believed to be during the EMT state that glioblastoma's centrifugal migration occurs. EMT is also a feature of untreated glioblastoma but is enhanced by chemotherapy, by radiation and by surgical trauma. EIS Regimen uses the antifungal drug itraconazole to block Hedgehog signaling, the antidiabetes drug metformin to block AMP kinase (AMPK), the analgesic drug naproxen to block Rac1, the anti-fibrosis drug pirfenidone to block transforming growth factor-beta (TGF-beta), the psychiatric drug quetiapine to block receptor activator NFkB ligand (RANKL) and the antibiotic rifampin to block Wnt- all by their previously established ancillary attributes. All these systems have been identified as triggers of EMT and worthy targets to inhibit. The EIS Regimen drugs have a good safety profile when used individually. They are not expected to have any new side effects when combined. Further studies of the EIS Regimen are needed.

Second hit in cervical carcinogenesis process: involvement of wnt/beta catenin pathway

PubMed Central

Perez-Plasencia, Carlos; Duenas-Gonzalez, Alfonso; Alatorre-Tavera, Brenda

2008-01-01

The Human papillomavirus plays an important role in the initiation and progression of cervical cancer. However, it is a necessary but not sufficient cause to develop invasive carcinoma; hence, other factors are required in the pathogenesis of this malignancy. In this review we explore the hypothesis of the deregulation of wnt/β-catenin signaling pathway as a "second hit" required to develop cervical cancer. PMID:18606007

WNT2B2 mRNA, up-regulated in primary gastric cancer, is a positive regulator of the WNT- beta-catenin-TCF signaling pathway.

PubMed

Katoh, M; Kirikoshi, H; Terasaki, H; Shiokawa, K

2001-12-21

Genetic alterations of WNT signaling molecules lead to carcinogenesis through activation of the beta-catenin-TCF signaling pathway. We have previously cloned and characterized WNT2B/WNT13 gene on human chromosome 1p13, which is homologous to proto-oncogene WNT2 on human chromosome 7q31. WNT2B1 and WNT2B2 mRNAs, generated from the WNT2B gene due to alternative splicing of the alternative promoter type, encode almost identical polypeptides with divergence in the N-terminal region. WNT2B2 mRNA rather than WNT2B1 mRNA is preferentially expressed in NT2 cells with the potential of neuronal differentiation. Here, we describe our investigations of expression of WNT2B mRNAs in various types of human primary cancer. Matched tumor/normal expression array analysis revealed that WNT2B mRNAs were significantly up-regulated in 2 of 8 cases of primary gastric cancer. WNT2B2 mRNA rather than WNT2B1 mRNA was found to be preferentially up-regulated in a case of primary gastric cancer (signet ring cell carcinoma). Function of WNT2B1 mRNA and that of WNT2B2 mRNA were investigated by using Xenopus axis duplication assay. Injection of synthetic WNT2B1 mRNA into the ventral marginal zone of fertilized Xenopus eggs at the 4-cell stage did not induce axis duplication. In contrast, ventral injection of synthetic WNT2B2 mRNA induced axis duplication in 90% of embryos (complete axis duplication, 24%). These results strongly suggest that WNT2B2 up-regulation in some cases of gastric cancer might lead to carcinogenesis through activation of the beta-catenin-TCF signaling pathway.

Hepatocyte Growth Factor and MET Support Mouse Enteric Nervous System Development, the Peristaltic Response, and Intestinal Epithelial Proliferation in Response to Injury

PubMed Central

Avetisyan, Marina; Wang, Hongtao; Schill, Ellen Merrick; Bery, Saya; Grider, John R.; Hassell, John A.; Stappenbeck, Thaddeus

2015-01-01

Factors providing trophic support to diverse enteric neuron subtypes remain poorly understood. We tested the hypothesis that hepatocyte growth factor (HGF) and the HGF receptor MET might support some types of enteric neurons. HGF and MET are expressed in fetal and adult enteric nervous system. In vitro, HGF increased enteric neuron differentiation and neurite length, but only if vanishingly small amounts (1 pg/ml) of glial cell line-derived neurotrophic factor were included in culture media. HGF effects were blocked by phosphatidylinositol-3 kinase inhibitor and by MET-blocking antibody. Both of these inhibitors and MEK inhibition reduced neurite length. In adult mice, MET was restricted to a subset of calcitonin gene-related peptide-immunoreactive (IR) myenteric plexus neurons thought to be intrinsic primary afferent neurons (IPANs). Conditional MET kinase domain inactivation (Metfl/fl; Wnt1Cre+) caused a dramatic loss of myenteric plexus MET-IR neurites and 1–1′-dioctodecyl-3,3,3′,3′-tetramethylindocarbocyamine perchlorate (DiI) labeling suggested reduced MET-IR neurite length. In vitro, Metfl/fl; Wnt1Cre+ mouse bowel had markedly reduced peristalsis in response to mucosal deformation, but normal response to radial muscle stretch. However, whole-bowel transit, small-bowel transit, and colonic-bead expulsion were normal in Metfl/fl; Wnt1Cre+ mice. Finally, Metfl/fl; Wnt1Cre+ mice had more bowel injury and reduced epithelial cell proliferation compared with WT animals after dextran sodium sulfate treatment. These results suggest that HGF/MET signaling is important for development and function of a subset IPANs and that these cells regulate intestinal motility and epithelial cell proliferation in response to bowel injury. SIGNIFICANCE STATEMENT The enteric nervous system has many neuronal subtypes that coordinate and control intestinal activity. Trophic factors that support these neuron types and enhance neurite growth after fetal development are not well understood. We show that a subset of adult calcitonin gene-related peptide (CGRP)-expressing myenteric neurons produce MET, the receptor for hepatocyte growth factor, and that loss of MET activity affects peristalsis in response to mucosal stroking, reduces MET-immunoreactive neurites, and increases susceptibility to dextran sodium sulfate-induced bowel injury. These observations may be relevant for understanding and treating intestinal motility disorders and also suggest that enhancing the activity of MET-expressing CGRP neurons might be a useful strategy to reduce bowel inflammation. PMID:26290232

Activation of the Wnt/{beta}-catenin signaling pathway is associated with glial proliferation in the adult spinal cord of ALS transgenic mice

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

Chen, Yanchun; Department of Histology and Embryology, Shandong University School of Medicine, Jinan, Shandong; Guan, Yingjun, E-mail: guanyj@wfmc.edu.cn

Highlights: Black-Right-Pointing-Pointer Wnt3a and Cyclin D1 were upregulated in the spinal cord of the ALS mice. Black-Right-Pointing-Pointer {beta}-catenin translocated from the cell membrane to the nucleus in the ALS mice. Black-Right-Pointing-Pointer Wnt3a, {beta}-catenin and Cyclin D1 co-localized for astrocytes were all increased. Black-Right-Pointing-Pointer BrdU/Cyclin D1 double-positive cells were increased in the spinal cord of ALS mice. Black-Right-Pointing-Pointer BrdU/Cyclin D1/GFAP triple-positive cells were detected in the ALS mice. -- Abstract: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the progressive and fatal loss of motor neurons. In ALS, there is a significant cell proliferation in response to neurodegeneration; however,more » the exact molecular mechanisms of cell proliferation and differentiation are unclear. The Wnt signaling pathway has been shown to be involved in neurodegenerative processes. Wnt3a, {beta}-catenin, and Cyclin D1 are three key signaling molecules of the Wnt/{beta}-catenin signaling pathway. We determined the expression of Wnt3a, {beta}-catenin, and Cyclin D1 in the adult spinal cord of SOD1{sup G93A} ALS transgenic mice at different stages by RT-PCR, Western blot, and immunofluorescence labeling techniques. We found that the mRNA and protein of Wnt3a and Cyclin D1 in the spinal cord of the ALS mice were upregulated compared to those in wild-type mice. In addition, {beta}-catenin translocated from the cell membrane to the nucleus and subsequently activated transcription of the target gene, Cyclin D1. BrdU and Cyclin D1 double-positive cells were increased in the spinal cord of these mice. Moreover, Wnt3a, {beta}-catenin, and Cyclin D1 were also expressed in both neurons and astrocytes. The expression of Wnt3a, {beta}-catenin or Cyclin D1 in mature GFAP{sup +} astrocytes increased. Moreover, BrdU/Cyclin D1/GFAP triple-positive cells were detected in the ALS mice. Our findings suggest that neurodegeneration activates the Wnt/{beta}-catenin signaling pathway, which is associated with glial proliferation in the adult spinal cord of ALS transgenic mice. This mechanism may be significant in clinical gene therapy.« less

The Wnt/β-catenin signaling pathway tips the balance between apoptosis and reprograming of cell fusion hybrids.

PubMed

Lluis, Frederic; Pedone, Elisa; Pepe, Stefano; Cosma, Maria Pia

2010-11-01

Cell-cell fusion contributes to cell differentiation and developmental processes. We have previously showed that activation of Wnt/β-catenin enhances somatic cell reprograming after polyethylene glycol (PEG)-mediated fusion. Here, we show that neural stem cells and ESCs can fuse spontaneously in cocultures, although with very low efficiency (about 2%), as the hybrids undergo apoptosis. In contrast, when Wnt/β-catenin signaling is activated in ESCs and leads to accumulation of low amounts of β-catenin in the nucleus, activated ESCs can reprogram somatic cells with very high efficiency after spontaneous fusion. Furthermore, we also show that different levels of β-catenin accumulation in the ESC nuclei can modulate cell proliferation, although in our experimental setting, cell proliferation does not modulate the reprograming efficiency per se. Overall, the present study provides evidence that spontaneous fusion occurs, while the survival of the reprogramed clones is strictly dependent on induction of a Wnt-mediated reprograming pathway. Copyright © 2010 AlphaMed Press.

[3D structure of DKK1 indicates its involvement in both canonical and non-canonical Wnt pathways].

PubMed

Khalili, S; Rasaee, M J; Bamdad, T

2017-01-01

Dikkoppf-1 (DKK1) is an antagonist of the canonical Wnt signaling pathway. The importance of DKK1 as a diagnostic and therapeutic agent in a wide range of diseases along with its significance in a variety of biological processes accentuate the necessity to decipher its 3D structure that would pave the way towards the development of relevant selective inhibitors. A DKK1 structure model predicted by the Robetta server with structural refinements including a 10 ns molecular dynamics run was subjected to functional and docking analyses. We hypothesize that the N-terminal region of the DKK1 molecule could be functionally important for both canonical and noncanonical Wnt pathways. Moreover, it seems that DKK1 could be involved in interactions with the Frizzled receptors, leading to the activation of the Planar Cell Polarity (PCP) pathway (activation of Jun N-terminal kinase (JNK) Pathway) and Wnt/Ca^(2+) pathway (activation of CamKII).

Cited1 Deficiency Suppresses Intestinal Tumorigenesis

PubMed Central

Young, Madeleine; Poetz, Oliver; Parry, Lee; Jenkins, John R.; Williams, Geraint T.; Dunwoodie, Sally L.; Watson, Alastair; Clarke, Alan R.

2013-01-01

Conditional deletion of Apc in the murine intestine alters crypt-villus architecture and function. This process is accompanied by multiple changes in gene expression, including upregulation of Cited1, whose role in colorectal carcinogenesis is unknown. Here we explore the relevance of Cited1 to intestinal tumorigenesis. We crossed Cited1 null mice with ApcMin/+ and AhCre+Apcfl/fl mice and determined the impact of Cited1 deficiency on tumour growth/initiation including tumour multiplicity, cell proliferation, apoptosis and the transcriptome. We show that Cited1 is up-regulated in both human and murine tumours, and that constitutive deficiency of Cited1 increases survival in ApcMin/+ mice from 230.5 to 515 days. However, paradoxically, Cited1 deficiency accentuated nearly all aspects of the immediate phenotype 4 days after conditional deletion of Apc, including an increase in cell death and enhanced perturbation of differentiation, including of the stem cell compartment. Transcriptome analysis revealed multiple pathway changes, including p53, PI3K and Wnt. The activation of Wnt through Cited1 deficiency correlated with increased transcription of β-catenin and increased levels of dephosphorylated β-catenin. Hence, immediately following deletion of Apc, Cited1 normally restrains the Wnt pathway at the level of β-catenin. Thus deficiency of Cited1 leads to hyper-activation of Wnt signaling and an exaggerated Wnt phenotype including elevated cell death. Cited1 deficiency decreases intestinal tumourigenesis in ApcMin/+ mice and impacts upon a number of oncogenic signaling pathways, including Wnt. This restraint imposed by Cited1 is consistent with a requirement for Cited1 to constrain Wnt activity to a level commensurate with optimal adenoma formation and maintenance, and provides one mechanism for tumour repression in the absence of Cited1. PMID:23935526

Fyn/Yes and non-canonical Wnt signalling converge on RhoA in vertebrate gastrulation cell movements

PubMed Central

Jopling, Chris; den Hertog, Jeroen

2005-01-01

Convergent extension (CE) cell movements during gastrulation mediate extension of the anterior–posterior body axis of vertebrate embryos. Non-canonical Wnt5 and Wnt11 signalling is essential for normal CE movements in vertebrate gastrulation. Here, we show that morpholino (MO)-mediated double knock-down of the Fyn and Yes tyrosine kinases in zebrafish embryos impaired normal CE cell movements, resembling the silberblick and pipetail mutants, caused by mutations in wnt11 and wnt5, respectively. Co-injection of Fyn/Yes- and Wnt11- or Wnt5-MO was synergistic, but wnt11 or wnt5 RNA did not rescue the Fyn/Yes knockdown or vice versa. Remarkably, active RhoA rescued the Fyn/Yes knockdown as well as the Wnt11 knockdown, indicating that Fyn/Yes and Wnt11 signalling converged on RhoA. Our results show that Fyn and Yes act together with non-canonical Wnt signalling via RhoA in CE cell movements during gastrulation. PMID:15815683

Taurodontism, variations in tooth number, and misshapened crowns in Wnt10a null mice and human kindreds

PubMed Central

Yang, Jie; Wang, Shih-Kai; Choi, Murim; Reid, Bryan M; Hu, Yuanyuan; Lee, Yuan-Ling; Herzog, Curtis R; Kim-Berman, Hera; Lee, Moses; Benke, Paul J; Kent Lloyd, K C; Simmer, James P; Hu, Jan C-C

2015-01-01

WNT10A is a signaling molecule involved in tooth development, and WNT10A defects are associated with tooth agenesis. We characterized Wnt10a null mice generated by the knockout mouse project (KOMP) and six families with WNT10A mutations, including a novel p.Arg104Cys defect, in the absence of EDA,EDAR, or EDARADD variations. Wnt10a null mice exhibited supernumerary mandibular fourth molars, and smaller molars with abnormal cusp patterning and root taurodontism. Wnt10a−/− incisors showed distinctive apical–lingual wedge-shaped defects. These findings spurred us to closely examine the dental phenotypes of our WNT10A families. WNT10A heterozygotes exhibited molar root taurodontism and mild tooth agenesis (with incomplete penetrance) in their permanent dentitions. Individuals with two defective WNT10A alleles showed severe tooth agenesis and had fewer cusps on their molars. The misshapened molar crowns and roots were consistent with the Wnt10a null phenotype and were not previously associated with WNT10A defects. The missing teeth contrasted with the presence of supplemental teeth in the Wnt10a null mice and demonstrated mammalian species differences in the roles of Wnt signaling in early tooth development. We conclude that molar crown and root dysmorphologies are caused by WNT10A defects and that the severity of the tooth agenesis correlates with the number of defective WNT10A alleles. PMID:25629078

Plakophilin-1, a Novel Wnt Signaling Regulator, Is Critical for Tooth Development and Ameloblast Differentiation

PubMed Central

Arai, Chieko; Yamada, Aya; Saito, Kan; Ishikawa, Masaki; Xue, Han; Funada, Keita; Haruyama, Naoto; Yamada, Yoshihiko; Fukumoto, Satoshi; Takahashi, Ichiro

2016-01-01

Tooth morphogenesis is initiated by reciprocal interactions between the ectoderm and neural crest-derived mesenchyme, and the Wnt signaling pathway is involved in this process. We found that Plakophilin (PKP)1, which is associated with diseases such as ectodermal dysplasia/skin fragility syndrome, was highly expressed in teeth and skin, and was upregulated during tooth development. We hypothesized that PKP1 regulates Wnt signaling via its armadillo repeat domain in a manner similar to β-catenin. To determine its role in tooth development, we performed Pkp1 knockdown experiments using ex vivo organ cultures and cell cultures. Loss of Pkp1 reduced the size of tooth germs and inhibited dental epithelial cell proliferation, which was stimulated by Wnt3a. Furthermore, transfected PKP1-emerald green fluorescent protein was translocated from the plasma membrane to the nucleus upon stimulation with Wnt3a and LiCl, which required the PKP1 N terminus (amino acids 161 to 270). Localization of PKP1, which is known as an adhesion-related desmosome component, shifted to the plasma membrane during ameloblast differentiation. In addition, Pkp1 knockdown disrupted the localization of Zona occludens 1 in tight junctions and inhibited ameloblast differentiation; the two proteins were shown to directly interact by immunoprecipitation. These results implicate the participation of PKP1 in early tooth morphogenesis as an effector of canonical Wnt signaling that controls ameloblast differentiation via regulation of the cell adhesion complex. PMID:27015268

Frodo proteins: modulators of Wnt signaling in vertebrate development.

PubMed

Brott, Barbara K; Sokol, Sergei Y

2005-09-01

The Frodo/dapper (Frd) proteins are recently discovered signaling adaptors, which functionally and physically interact with Wnt and Nodal signaling pathways during vertebrate development. The Frd1 and Frd2 genes are expressed in dynamic patterns in early embryos, frequently in cells undergoing epithelial-mesenchymal transition. The Frd proteins function in multiple developmental processes, including mesoderm and neural tissue specification, early morphogenetic cell movements, and organogenesis. Loss-of-function studies using morpholino antisense oligonucleotides demonstrate that the Frd proteins regulate Wnt signal transduction in a context-dependent manner and may be involved in Nodal signaling. The identification of Frd-associated factors and cellular targets of the Frd proteins should shed light on the molecular mechanisms underlying Frd functions in embryonic development and in cancer.

Lens regeneration from the cornea requires suppression of Wnt/β-catenin signaling.

PubMed

Hamilton, Paul W; Sun, Yu; Henry, Jonathan J

2016-04-01

The frog, Xenopus laevis, possesses a high capacity to regenerate various larval tissues, including the lens, which is capable of complete regeneration from the cornea epithelium. However, the molecular signaling mechanisms of cornea-lens regeneration are not fully understood. Previous work has implicated the involvement of the Wnt signaling pathway, but molecular studies have been very limited. Iris-derived lens regeneration in the newt (Wolffian lens regeneration) has shown a necessity for active Wnt signaling in order to regenerate a new lens. Here we provide evidence that the Wnt signaling pathway plays a different role in the context of cornea-lens regeneration in Xenopus. We examined the expression of frizzled receptors and wnt ligands in the frog cornea epithelium. Numerous frizzled receptors (fzd1, fzd2, fzd3, fzd4, fzd6, fzd7, fzd8, and fzd10) and wnt ligands (wnt2b.a, wnt3a, wnt4, wnt5a, wnt5b, wnt6, wnt7b, wnt10a, wnt11, and wnt11b) are expressed in the cornea epithelium, demonstrating that this tissue is transcribing many of the ligands and receptors of the Wnt signaling pathway. When compared to flank epithelium, which is lens regeneration incompetent, only wnt11 and wnt11b are different (present only in the cornea epithelium), identifying them as potential regulators of cornea-lens regeneration. To detect changes in canonical Wnt/β-catenin signaling occurring within the cornea epithelium, axin2 expression was measured over the course of regeneration. axin2 is a well-established reporter of active Wnt/β-catenin signaling, and its expression shows a significant decrease at 24 h post-lentectomy. This decrease recovers to normal endogenous levels by 48 h. To test whether this signaling decrease was necessary for lens regeneration to occur, regenerating eyes were treated with either 6-bromoindirubin-3'-oxime (BIO) or 1-azakenpaullone - both activators of Wnt signaling - resulting in a significant reduction in the percentage of cases with successful regeneration. In contrast, inhibition of Wnt signaling using either the small molecule IWR-1, treatment with recombinant human Dickkopf-1 (rhDKK1) protein, or transgenic expression of Xenopus DKK1, did not significantly affect the percentage of successful regeneration. Together, these results suggest a model where Wnt/β-catenin signaling is active in the cornea epithelium and needs to be suppressed during early lens regeneration in order for these cornea cells to give rise to a new lentoid. While this finding differs from what has been described in the newt, it closely resembles the role of Wnt signaling during the initial formation of the lens placode from the surface ectoderm during early embryogenesis. Copyright © 2016 Elsevier Ltd. All rights reserved.

Renal hypodysplasia associates with a WNT4 variant that causes aberrant canonical WNT signaling.

PubMed

Vivante, Asaf; Mark-Danieli, Michal; Davidovits, Miriam; Harari-Steinberg, Orit; Omer, Dorit; Gnatek, Yehudit; Cleper, Roxana; Landau, Daniel; Kovalski, Yael; Weissman, Irit; Eisenstein, Israel; Soudack, Michalle; Wolf, Haike Reznik; Issler, Naomi; Lotan, Danny; Anikster, Yair; Dekel, Benjamin

2013-03-01

Abnormal differentiation of the renal stem/progenitor pool into kidney tissue can lead to renal hypodysplasia (RHD), but the underlying causes of RHD are not well understood. In this multicenter study, we identified 20 Israeli pedigrees with isolated familial, nonsyndromic RHD and screened for mutations in candidate genes involved in kidney development, including PAX2, HNF1B, EYA1, SIX1, SIX2, SALL1, GDNF, WNT4, and WT1. In addition to previously reported RHD-causing genes, we found that two affected brothers were heterozygous for a missense variant in the WNT4 gene. Functional analysis of this variant revealed both antagonistic and agonistic canonical WNT stimuli, dependent on cell type. In HEK293 cells, WNT4 inhibited WNT3A induced canonical activation, and the WNT4 variant significantly enhanced this inhibition of the canonical WNT pathway. In contrast, in primary cultures of human fetal kidney cells, which maintain WNT activation and more closely represent WNT signaling in renal progenitors during nephrogenesis, this mutation caused significant loss of function, resulting in diminished canonical WNT/β-catenin signaling. In conclusion, heterozygous WNT4 variants are likely to play a causative role in renal hypodysplasia.

Renal Hypodysplasia Associates with a Wnt4 Variant that Causes Aberrant Canonical Wnt Signaling

PubMed Central

Vivante, Asaf; Mark-Danieli, Michal; Davidovits, Miriam; Harari-Steinberg, Orit; Omer, Dorit; Gnatek, Yehudit; Cleper, Roxana; Landau, Daniel; Kovalski, Yael; Weissman, Irit; Eisenstein, Israel; Soudack, Michalle; Wolf, Haike Reznik; Issler, Naomi; Lotan, Danny; Anikster, Yair

2013-01-01

Abnormal differentiation of the renal stem/progenitor pool into kidney tissue can lead to renal hypodysplasia (RHD), but the underlying causes of RHD are not well understood. In this multicenter study, we identified 20 Israeli pedigrees with isolated familial, nonsyndromic RHD and screened for mutations in candidate genes involved in kidney development, including PAX2, HNF1B, EYA1, SIX1, SIX2, SALL1, GDNF, WNT4, and WT1. In addition to previously reported RHD-causing genes, we found that two affected brothers were heterozygous for a missense variant in the WNT4 gene. Functional analysis of this variant revealed both antagonistic and agonistic canonical WNT stimuli, dependent on cell type. In HEK293 cells, WNT4 inhibited WNT3A induced canonical activation, and the WNT4 variant significantly enhanced this inhibition of the canonical WNT pathway. In contrast, in primary cultures of human fetal kidney cells, which maintain WNT activation and more closely represent WNT signaling in renal progenitors during nephrogenesis, this mutation caused significant loss of function, resulting in diminished canonical WNT/β-catenin signaling. In conclusion, heterozygous WNT4 variants are likely to play a causative role in renal hypodysplasia. PMID:23520208

HnRNP C, YB-1 and hnRNP L coordinately enhance skipping of human MUSK exon 10 to generate a Wnt-insensitive MuSK isoform

PubMed Central

Nasrin, Farhana; Rahman, Mohammad Alinoor; Masuda, Akio; Ohe, Kenji; Takeda, Jun-ichi; Ohno, Kinji

2014-01-01

Muscle specific receptor tyrosine kinase (MuSK) is an essential postsynaptic transmembrane molecule that mediates clustering of acetylcholine receptors (AChR). MUSK exon 10 is alternatively skipped in human, but not in mouse. Skipping of this exon disrupts a cysteine-rich region (Fz-CRD), which is essential for Wnt-mediated AChR clustering. To investigate the underlying mechanisms of alternative splicing, we exploited block-scanning mutagenesis with human minigene and identified a 20-nucleotide block that contained exonic splicing silencers. Using RNA-affinity purification, mass spectrometry, and Western blotting, we identified that hnRNP C, YB-1 and hnRNP L are bound to MUSK exon 10. siRNA-mediated knockdown and cDNA overexpression confirmed the additive, as well as the independent, splicing suppressing effects of hnRNP C, YB-1 and hnRNP L. Antibody-mediated in vitro protein depletion and scanning mutagenesis additionally revealed that binding of hnRNP C to RNA subsequently promotes binding of YB-1 and hnRNP L to the immediate downstream sites and enhances exon skipping. Simultaneous tethering of two splicing trans-factors to the target confirmed the cooperative effect of YB-1 and hnRNP L on hnRNP C-mediated exon skipping. Search for a similar motif in the human genome revealed nine alternative exons that were individually or coordinately regulated by hnRNP C and YB-1. PMID:25354590

Adenovirus-mediated Wnt5a expression inhibits the telogen-to-anagen transition of hair follicles in mice.

PubMed

Xing, Yi-Zhan; Wang, Rui-Min; Yang, Ke; Guo, Hai-Ying; Deng, Fang; Li, Yu-Hong; Ye, Ji-Xing; He, Long; Lian, Xiao-Hua; Yang, Tian

2013-01-01

The canonical Wnt/β-catenin pathway plays an important role in hair cycle induction. Wnt5a is a non-canonical Wnt family member that generally antagonizes canonical Wnt signaling in other systems. In hair follicles, Wnt5a and canonical Wnt are both expressed in cells in the telogen stage. Wnt5a has been shown to be critical for controlling hair cell fate. However, the role that Wnt5a plays in the transition from the telogen to anagen stage is unknown. In this study, using whole-mount in situ hybridization, we show that Wnt5a is produced by several other cell types, excluding dermal papilla cells, throughout the hair cycle. For example, Wnt5a is expressed in bulge and secondary hair germ cells in the telogen stage. Our studies focused on the depilated 8-week-old mouse as a synchronized model of hair growth. Interestingly, overexpression of adenovirus Wnt5a in the dorsal skin of mice led to the elongation of the telogen stage and inhibition of the initiation of the anagen stage. However, following an extended period of time, four pelage hair types grew from hairless skin that was induced by Wnt5a, and the structure of these new hair shafts was normal. Using microarray analysis and quantitative arrays, we showed that the expression of β-catenin and some target genes of canonical Wnt signaling decreased after Wnt5a treatment. These data demonstrate that Wnt5a may inhibit the telogen stage to maintain a quiescent state of the hair follicle.

Myeloid Wnt ligands are required for normal development of dermal lymphatic vasculature.

PubMed

Muley, Ajit; Odaka, Yoshi; Lewkowich, Ian P; Vemaraju, Shruti; Yamaguchi, Terry P; Shawber, Carrie; Dickie, Belinda H; Lang, Richard A

2017-01-01

Resident tissue myeloid cells play a role in many aspects of physiology including development of the vascular systems. In the blood vasculature, myeloid cells use VEGFC to promote angiogenesis and can use Wnt ligands to control vascular branching and to promote vascular regression. Here we show that myeloid cells also regulate development of the dermal lymphatic vasculature using Wnt ligands. Using myeloid-specific deletion of the WNT transporter Wntless we show that myeloid Wnt ligands are active at two distinct stages of development of the dermal lymphatics. As lymphatic progenitors are emigrating from the cardinal vein and intersomitic vessels, myeloid Wnt ligands regulate both their numbers and migration distance. Later in lymphatic development, myeloid Wnt ligands regulate proliferation of lymphatic endothelial cells (LEC) and thus control lymphatic vessel caliber. Myeloid-specific deletion of WNT co-receptor Lrp5 or Wnt5a gain-of-function also produce elevated caliber in dermal lymphatic capillaries. These data thus suggest that myeloid cells produce Wnt ligands to regulate lymphatic development and use Wnt pathway co-receptors to regulate the balance of Wnt ligand activity during the macrophage-LEC interaction.

CD44 functions in Wnt signaling by regulating LRP6 localization and activation

PubMed Central

Schmitt, M; Metzger, M; Gradl, D; Davidson, G; Orian-Rousseau, V

2015-01-01

Wnt reception at the membrane is complex and not fully understood. CD44 is a major Wnt target gene in the intestine and is essential for Wnt-induced tumor progression in colorectal cancer. Here we show that CD44 acts as a positive regulator of the Wnt receptor complex. Downregulation of CD44 expression decreases, whereas CD44 overexpression increases Wnt activity in a concentration-dependent manner. Epistasis experiments place CD44 function at the level of the Wnt receptor LRP6. Mechanistically, CD44 physically associates with LRP6 upon Wnt treatment and modulates LRP6 membrane localization. Moreover, CD44 regulates Wnt signaling in the developing brain of Xenopus laevis embryos as shown by a decreased expression of Wnt targets tcf-4 and en-2 in CD44 morphants. PMID:25301071

Wise Regulates Bone Deposition through Genetic Interactions with Lrp5

PubMed Central

Ellies, Debra L.; Economou, Androulla; Viviano, Beth; Rey, Jean-Philippe; Paine-Saunders, Stephenie; Krumlauf, Robb; Saunders, Scott

2014-01-01

In this study using genetic approaches in mouse we demonstrate that the secreted protein Wise plays essential roles in regulating early bone formation through its ability to modulate Wnt signaling via interactions with the Lrp5 co-receptor. In Wise−/− mutant mice we find an increase in the rate of osteoblast proliferation and a transient increase in bone mineral density. This change in proliferation is dependent upon Lrp5, as Wise;Lrp5 double mutants have normal bone mass. This suggests that Wise serves as a negative modulator of Wnt signaling in active osteoblasts. Wise and the closely related protein Sclerostin (Sost) are expressed in osteoblast cells during temporally distinct early and late phases in a manner consistent with the temporal onset of their respective increased bone density phenotypes. These data suggest that Wise and Sost may have common roles in regulating bone development through their ability to control the balance of Wnt signaling. We find that Wise is also required to potentiate proliferation in chondrocytes, serving as a potential positive modulator of Wnt activity. Our analyses demonstrate that Wise plays a key role in processes that control the number of osteoblasts and chondrocytes during bone homeostasis and provide important insight into mechanisms regulating the Wnt pathway during skeletal development. PMID:24789067

Wise regulates bone deposition through genetic interactions with Lrp5.

PubMed

Ellies, Debra L; Economou, Androulla; Viviano, Beth; Rey, Jean-Philippe; Paine-Saunders, Stephenie; Krumlauf, Robb; Saunders, Scott

2014-01-01

In this study using genetic approaches in mouse we demonstrate that the secreted protein Wise plays essential roles in regulating early bone formation through its ability to modulate Wnt signaling via interactions with the Lrp5 co-receptor. In Wise-/- mutant mice we find an increase in the rate of osteoblast proliferation and a transient increase in bone mineral density. This change in proliferation is dependent upon Lrp5, as Wise;Lrp5 double mutants have normal bone mass. This suggests that Wise serves as a negative modulator of Wnt signaling in active osteoblasts. Wise and the closely related protein Sclerostin (Sost) are expressed in osteoblast cells during temporally distinct early and late phases in a manner consistent with the temporal onset of their respective increased bone density phenotypes. These data suggest that Wise and Sost may have common roles in regulating bone development through their ability to control the balance of Wnt signaling. We find that Wise is also required to potentiate proliferation in chondrocytes, serving as a potential positive modulator of Wnt activity. Our analyses demonstrate that Wise plays a key role in processes that control the number of osteoblasts and chondrocytes during bone homeostasis and provide important insight into mechanisms regulating the Wnt pathway during skeletal development.

Overview of the mutation spectrum in familial exudative vitreoretinopathy and Norrie disease with identification of 21 novel variants in FZD4, LRP5, and NDP.

PubMed

Nikopoulos, Konstantinos; Venselaar, Hanka; Collin, Rob W J; Riveiro-Alvarez, Rosa; Boonstra, F Nienke; Hooymans, Johanna M M; Mukhopadhyay, Arijit; Shears, Deborah; van Bers, Marleen; de Wijs, Ilse J; van Essen, Anthonie J; Sijmons, Rolf H; Tilanus, Mauk A D; van Nouhuys, C Erik; Ayuso, Carmen; Hoefsloot, Lies H; Cremers, Frans P M

2010-06-01

Wnt signaling is a crucial component of the cell machinery orchestrating a series of physiological processes such as cell survival, proliferation, and migration. Among the plethora of roles that Wnt signaling plays, its canonical branch regulates eye organogenesis and angiogenesis. Mutations in the genes encoding the low density lipoprotein receptor protein 5 (LRP5) and frizzled 4 (FZD4), acting as coreceptors for Wnt ligands, cause familial exudative vitreoretinopathy (FEVR). Moreover, mutations in the gene encoding NDP, a ligand for these Wnt receptors, cause Norrie disease and FEVR. Both FEVR and Norrie disease share similar phenotypic characteristics, including abnormal vascularization of the peripheral retina and formation of fibrovascular masses in the eye that can lead to blindness. In this mutation update, we report 21 novel variants for FZD4, LRP5, and NDP, and discuss the putative functional consequences of missense mutations. In addition, we provide a comprehensive overview of all previously published variants in the aforementioned genes and summarize the phenotypic characteristics in mouse models carrying mutations in the orthologous genes. The increasing molecular understanding of Wnt signaling, related to ocular development and blood supply, offers more tools for accurate disease diagnosis that may be important in the development of therapeutic interventions.

A C-terminally truncated form of β-catenin acts as a novel regulator of Wnt/β-catenin signaling in planarians

PubMed Central

Rabaneda-Lombarte, Neus; Gelabert, Maria; Xie, Jianlei; Wu, Wei

2017-01-01

β-Catenin, the core element of the Wnt/β-catenin pathway, is a multifunctional and evolutionarily conserved protein which performs essential roles in a variety of developmental and homeostatic processes. Despite its crucial roles, the mechanisms that control its context-specific functions in time and space remain largely unknown. The Wnt/β-catenin pathway has been extensively studied in planarians, flatworms with the ability to regenerate and remodel the whole body, providing a ‘whole animal’ developmental framework to approach this question. Here we identify a C-terminally truncated β-catenin (β-catenin4), generated by gene duplication, that is required for planarian photoreceptor cell specification. Our results indicate that the role of β-catenin4 is to modulate the activity of β-catenin1, the planarian β-catenin involved in Wnt signal transduction in the nucleus, mediated by the transcription factor TCF-2. This inhibitory form of β-catenin, expressed in specific cell types, would provide a novel mechanism to modulate nuclear β-catenin signaling levels. Genomic searches and in vitro analysis suggest that the existence of a C-terminally truncated form of β-catenin could be an evolutionarily conserved mechanism to achieve a fine-tuned regulation of Wnt/β-catenin signaling in specific cellular contexts. PMID:28976975

Wnt1a maintains characteristics of dermal papilla cells that induce mouse hair regeneration in a 3D preculture system.

PubMed

Dong, Liang; Hao, Haojie; Liu, Jiejie; Tong, Chuan; Ti, Dongdong; Chen, Deyun; Chen, Li; Li, Meirong; Liu, Huiling; Fu, Xiaobing; Han, Weidong

2017-05-01

Hair follicle morphogenesis and regeneration depend on intensive but well-orchestrated interactions between epithelial and mesenchymal components. Therefore, an alternative strategy to reproduce the process of epithelial-mesenchymal interaction in vitro could use a 3D system containing appropriate cell populations. The 3D air-liquid culture system for reproducibly generating hair follicles from dissociated epithelial and dermal papilla (DP) cells combined with a collagen-chitosan scaffold is described in this study. Wnt-CM was prepared from the supernatant of Wnt1a-expressing bone marrow mesenchymal stem cells (BM-MSCs) that maintain the hair-inducing gene expression of DP cells. The collagen-chitosan scaffold cells (CCS cells) were constructed using a two-step method by inoculating the Wnt-CM-treated DP cells and epidermal (EP) cells into the CCS. The cells in the air-liquid culture formed dermal condensates and a proliferative cell layer in vitro. The CCS cells were able to induce hair regeneration in nude mice. The results demonstrate that Wnt-CM can maintain the hair induction ability of DP cells in expansion cultures, and this approach can be used for large-scale preparation of CCS cells in vitro to treat hair loss. Copyright © 2015 John Wiley & Sons, Ltd. Copyright © 2015 John Wiley & Sons, Ltd.

A C-terminally truncated form of β-catenin acts as a novel regulator of Wnt/β-catenin signaling in planarians.

PubMed

Su, Hanxia; Sureda-Gomez, Miquel; Rabaneda-Lombarte, Neus; Gelabert, Maria; Xie, Jianlei; Wu, Wei; Adell, Teresa

2017-10-01

β-Catenin, the core element of the Wnt/β-catenin pathway, is a multifunctional and evolutionarily conserved protein which performs essential roles in a variety of developmental and homeostatic processes. Despite its crucial roles, the mechanisms that control its context-specific functions in time and space remain largely unknown. The Wnt/β-catenin pathway has been extensively studied in planarians, flatworms with the ability to regenerate and remodel the whole body, providing a 'whole animal' developmental framework to approach this question. Here we identify a C-terminally truncated β-catenin (β-catenin4), generated by gene duplication, that is required for planarian photoreceptor cell specification. Our results indicate that the role of β-catenin4 is to modulate the activity of β-catenin1, the planarian β-catenin involved in Wnt signal transduction in the nucleus, mediated by the transcription factor TCF-2. This inhibitory form of β-catenin, expressed in specific cell types, would provide a novel mechanism to modulate nuclear β-catenin signaling levels. Genomic searches and in vitro analysis suggest that the existence of a C-terminally truncated form of β-catenin could be an evolutionarily conserved mechanism to achieve a fine-tuned regulation of Wnt/β-catenin signaling in specific cellular contexts.

Integrative analyses of conserved WNT clusters and their co-operative behaviour in human breast cancer

PubMed Central

Qurrat-ul-Ain; Seemab, Umair; Nawaz, Sulaman; Rashid, Sajid

2011-01-01

In human, WNT gene clusters are highly conserved at specie level and associated with carcinogenesis. Among them, WNT-10A and WNT-6 genes clustered in chromosome 2q35 are homologous to WNT-10B and WNT-1 located in chromosome 12q13, respectively. In an attempt to study co-regulation, the coordinated expression of these genes was monitored in human breast cancer tissues. As compared to normal tissue, both WNT-10A and WNT-10B genes exhibited lower expression while WNT-6 and WNT-1 showed increased expression in breast cancer tissues. The co-expression pattern was elaborated by detailed phylogenetic and syntenic analyses. Moreover, the intergenic and intragenic regions for these gene clusters were analyzed for studying the transcriptional regulation. In this context, adequate conserved binding sites for SOX and TCF family of transcriptional factors were observed. We propose that SOX9 and TCF4 may compete for binding at the promoters of WNT family genes thus regulating the disease phenotype. PMID:22355234

The effects of letrozole and clomiphene citrate on ligands expression of Wnt3, Wnt7a, and Wnt8b in proliferative endometrium of women with Polycystic ovarian syndrome.

PubMed

Mehdinejadiani, Shayesteh; Amidi, Fardin; Mehdizadeh, Mehdi; Barati, Mahmood; Safdarian, Leili; Aflatoonian, Reza; Alyasin, Ashraf; Aghahosseini, Marzieh; Pazhohan, Azar; Hayat, Parisa; Mohammadzadeh Kazorgah, Farzaneh; Sobhani, Aligholi

2018-03-06

Polycystic ovarian syndrome (PCOS) is a common endocrinologic disorder in women of reproductive age characterized by polycystic ovaries, oligo/anovulation, and hyperandrogenism. Not only anovulation but also endometrial dysfunction can reduce fertility in PCOS patients. Wnt pathway is responsible for endometrial proliferation which be strongly regulated by estradiol. To determine the effects of clomiphene citrate (CC) and letrozole, we measured the expression of some main ligands of Wnt/β-catenin signaling including Wnt7a, Wnt3, and Wnt8b in the endometrial samples taken from PCOS women on day 12 of the menses who received 100 mg CC or 5 mg letrozole as well as from women without treatment. Significantly, the mean estrogen and progesterone concentration were lower and higher, respectively, in letrozole than CC. The mean endometrial thickness (ET) was significantly greater in letrozole compared to CC. Assessment of the mRNA and protein expression of Wnt7a, Wnt3, and Wnt8b showed significantly lower expression in CC than the letrozole and control groups. Collectively, letrozole provided a better molecular response in the endometrium of PCOS patients during the proliferative phase, similar to natural cycles, compared to CC. CC decreased the ligands expression of Wnt3, Wnt7a, and Wnt8b, resulting in endometrial dysfunction.

Synergistic effects of TGFβ2, WNT9a, and FGFR4 signals attenuate satellite cell differentiation during skeletal muscle development.

PubMed

Zhang, Weiya; Xu, Yueyuan; Zhang, Lu; Wang, Sheng; Yin, Binxu; Zhao, Shuhong; Li, Xinyun

2018-06-04

Satellite cells play a key role in the aging, generation, and damage repair of skeletal muscle. The molecular mechanism of satellite cells in these processes remains largely unknown. This study systematically investigated for the first time the characteristics of mouse satellite cells at ten different ages. Results indicated that the number and differentiation capacity of satellite cells decreased with age during skeletal muscle development. Transcriptome analysis revealed that 2,907 genes were differentially expressed at six time points at postnatal stage. WGCNA and GO analysis indicated that 1,739 of the 2,907 DEGs were mainly involved in skeletal muscle development processes. Moreover, the results of WGCNA and protein interaction analysis demonstrated that Tgfβ2, Wnt9a, and Fgfr4 were the key genes responsible for the differentiation of satellite cells. Functional analysis showed that TGFβ2 and WNT9a inhibited, whereas FGFR4 promoted the differentiation of satellite cells. Furthermore, each two of them had a regulatory relationship at the protein level. In vivo study also confirmed that TGFβ2 could regulate the regeneration of skeletal muscle, as well as the expression of WNT9a and FGFR4. Therefore, we concluded that the synergistic effects of TGFβ2, WNT9a, and FGFR4 were responsible for attenuating of the differentiation of aging satellite cells during skeletal muscle development. This study provided new insights into the molecular mechanism of satellite cell development. The target genes and signaling pathways investigated in this study would be useful for improving the muscle growth of livestock or treating muscle diseases in clinical settings. © 2018 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.

Induction of Canonical Wnt Signaling by the Alarmins S100A8/A9 in Murine Knee Joints: Implications for Osteoarthritis.

PubMed

van den Bosch, Martijn H; Blom, Arjen B; Schelbergen, Rik F P; Vogl, Thomas; Roth, Johannes P; Slöetjes, Annet W; van den Berg, Wim B; van der Kraan, Peter M; van Lent, Peter L E M

2016-01-01

Both alarmins S100A8/A9 and canonical Wnt signaling have been found to play active roles in the development of experimental osteoarthritis (OA). However, what activates canonical Wnt signaling remains unknown. This study was undertaken to investigate whether S100A8 induces canonical Wnt signaling and whether S100 proteins exert their effects via activation of Wnt signaling. Expression of the genes for S100A8/A9 and Wnt signaling pathway members was measured in an experimental OA model. Selected Wnt signaling pathway members were overexpressed, and levels of S100A8/A9 were measured. Activation of canonical Wnt signaling was determined after injection of S100A8 into naive joints and induction of collagenase-induced OA in S100A9-deficient mice. Expression of Wnt signaling pathway members was tested in macrophages and fibroblasts after S100A8 stimulation. Canonical Wnt signaling was inhibited in vivo to determine if the effects of S100A8 injections were dependent on Wnt signaling. The alarmins S100A8/A9 and members of the Wnt signaling pathway showed coinciding expression in synovial tissue in an experimental OA model. Synovial overexpression of selected Wnt signaling pathway members did not result in increased expression of S100 proteins. In contrast, intraarticular injection of S100A8 increased canonical Wnt signaling, whereas canonical Wnt signaling was decreased after induction of experimental OA in S100A9-deficient mice. S100A8 stimulation of macrophages, but not fibroblasts, resulted in increased expression of canonical Wnt signaling members. Overexpression of Dkk-1 to inhibit canonical Wnt signaling decreased the induction of matrix metalloproteinase 3, interleukin-6, and macrophage inflammatory protein 1α after injection of S100A8. Our findings indicate that the alarmin S100A8 induces canonical Wnt signaling in macrophages and murine knee joints. The effects of S100A8 are partially dependent on activation of canonical Wnt signaling. © 2016, American College of Rheumatology.

Upstream open reading frames regulate the expression of the nuclear Wnt13 isoforms

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

Tang Tao; Rector, Kyle; Barnett, Corey D.

2008-02-22

Wnt proteins control cell survival and cell fate during development. Although Wnt expression is tightly regulated in a spatio-temporal manner, the mechanisms involved both at the transcriptional and translational levels are poorly defined. We have identified a downstream translation initiation codon, AUG(+74), in Wnt13B and Wnt13C mRNAs responsible for the expression of Wnt13 nuclear forms. In this report, we demonstrate that the expression of the nuclear Wnt13C form is translationally regulated in response to stress and apoptosis. Though the 5'-leaders of both Wnt13C and Wnt13B mRNAs have an inhibitory effect on translation, they did not display an internal ribosome entrymore » site activity as demonstrated by dicistronic reporter assays. However, mutations or deletions of the upstream AUG(-99) and AUG(+1) initiation codons abrogate these translation inhibitory effects, demonstrating that Wnt13C expression is controlled by upstream open reading frames. Since long 5'-untranslated region with short upstream open reading frames characterize other Wnt transcripts, our present data on the translational control of Wnt13 expression open the way to further studies on the translation control of Wnt expression as a modulator of their subcellular localization and activity.« less

Chronic infusion of Wnt7a, Wnt5a and Dkk-1 in the adult hippocampus induces structural synaptic changes and modifies anxiety and memory performance.

PubMed

Ortiz-Matamoros, Abril; Arias, Clorinda

2018-05-01

Wnt signaling plays an important role in the adult brain function and its dysregulation has been implicated in some neurodegenerative pathways. Despite the functional role of the Wnt signaling in adult neural circuits, there is currently no evidence regarding the relationships between exogenously Wnt signaling activation or inhibition and hippocampal structural changes in vivo. Thus, we analyzed the effect of the chronic infusion of Wnt agonists, Wnt7a and Wnt5a, and antagonist, Dkk-1, on different markers of plasticity such as neuronal MAP-2, Tau, synapse number and morphology, and behavioral changes. We observed that Wnt7a and Wnt5a increased the number of perforated synapses and the content of pre-and postsynaptic proteins associated with synapse assembly compared to control and Dkk-1 infusion. These two Wnt agonists also reduced anxiety-like behavior. Conversely, the canonical antagonist, Dkk-1, increased anxiety and inhibited spatial memory recall. Therefore, the present study elucidates the potential participation of Wnt signaling in the remodeling of hippocampal circuits underlying plasticity events in vivo, and provides evidence of the potential benefits of Wnt agonist infusion for the treatment of some neurodegenerative conditions. Copyright © 2018 Elsevier Inc. All rights reserved.

Ror2-Src signaling in metastasis of mouse melanoma cells is inhibited by NRAGE.

PubMed

Lai, Shan-Shan; Xue, Bin; Yang, Yang; Zhao, Li; Chu, Chao-Shun; Hao, Jia-Yin; Wen, Chuan-Jun

2012-11-01

The receptor tyrosine kinase (RTK) Ror2 plays important roles in developmental morphogenesis and mediates the filopodia formation in Wnt5a-induced cell migration. However, the function of Ror2 in noncanonical Wnt signaling resulting in cancer metastasis is largely unknown. Here, we show that Ror2 expression is higher in the highly metastatic murine B16-BL6 melanoma cells than in the low metastatic variant B16 cells. Overexpression of Ror2 increases the metastasis ability of B16 cells, and knockdown of Ror2 reduces the migration ability of B16-BL6 cells. Furthermore, the inhibition of Src kinase activity is critical for the Ror2-mediated cell migration upon Wnt5a treatment. The C-terminus of Ror2, which is deleted in brachydactyly type B (BDB), is essential for the mutual interaction with the SH1 domain of Src. Intriguingly, the Neurotrophin receptor-interacting MAGE homologue (NRAGE), which, as we previously reported, can remodel the cellular skeleton and inhibit cell-cell adhesion and metastasis of melanoma and pancreatic cancer, sharply blocks the interaction between Src and Ror2 and inhibits Ror2-mediated B16 cell migration by decreasing the activity of Src and focal adhesion kinase (FAK). Our data show that Ror2 is a potential factor in the tumorigenesis and metastasis in a Src-dependent manner that is negatively regulated by NRAGE. Copyright © 2012. Published by Elsevier Inc.

Stabilization of beta-catenin induces pancreas tumor formation.

PubMed

Heiser, Patrick W; Cano, David A; Landsman, Limor; Kim, Grace E; Kench, James G; Klimstra, David S; Taketo, Maketo M; Biankin, Andrew V; Hebrok, Matthias

2008-10-01

beta-Catenin signaling within the canonical Wnt pathway is essential for pancreas development. However, the pathway is normally down-regulated in the adult organ. Increased cytoplasmic and nuclear localization of beta-catenin can be detected in nearly all human solid pseudopapillary neoplasms (SPN), a rare tumor with low malignant potential. Conversely, pancreatic ductal adenocarcinoma (PDA) accounts for the majority of pancreatic tumors and is among the leading causes of cancer death. Whereas activating mutations within beta-catenin and other members of the canonical Wnt pathway are rare, recent reports have implicated Wnt signaling in the development and progression of human PDA. Here, we sought to address the role of beta-catenin signaling in pancreas tumorigenesis. Using Cre/lox technology, we conditionally activated beta-catenin in a subset of murine pancreatic cells in vivo. Activation of beta-catenin results in the formation of large pancreatic tumors at a high frequency in adult mice. These tumors resemble human SPN based on morphologic and immunohistochemical comparisons. Interestingly, stabilization of beta-catenin blocks the formation of pancreatic intraepithelial neoplasia (PanIN) in the presence of an activating mutation in Kras that is known to predispose individuals to PDA. Instead, mice in which beta-catenin and Kras are concurrently activated develop distinct ductal neoplasms that do not resemble PanIN lesions. These results demonstrate that activation of beta-catenin is sufficient to induce pancreas tumorigenesis. Moreover, they indicate that the sequence in which oncogenic mutations are acquired has profound consequences on the phenotype of the resulting tumor.

Connective Tissue Growth Factor Domain 4 Amplifies Fibrotic Kidney Disease through Activation of LDL Receptor-Related Protein 6.

PubMed

Johnson, Bryce G; Ren, Shuyu; Karaca, Gamze; Gomez, Ivan G; Fligny, Cécile; Smith, Benjamin; Ergun, Ayla; Locke, George; Gao, Benbo; Hayes, Sebastian; MacDonnell, Scott; Duffield, Jeremy S

2017-06-01

Connective tissue growth factor (CTGF), a matrix-associated protein with four distinct cytokine binding domains, has roles in vasculogenesis, wound healing responses, and fibrogenesis and is upregulated in fibroblasts and myofibroblasts in disease. Here, we investigated the role of CTGF in fibrogenic cells. In mice, tissue-specific inducible overexpression of CTGF by kidney pericytes and fibroblasts had no bearing on nephrogenesis or kidney homeostasis but exacerbated inflammation and fibrosis after ureteral obstruction. These effects required the WNT receptor LDL receptor-related protein 6 (LRP6). Additionally, pericytes isolated from these mice became hypermigratory and hyperproliferative on overexpression of CTGF. CTGF is cleaved in vivo into distinct domains. Treatment with recombinant domain 1, 1+2 (N terminus), or 4 (C terminus) independently activated myofibroblast differentiation and wound healing responses in cultured pericytes, but domain 4 showed the broadest profibrotic activity. Domain 4 exhibited low-affinity binding to LRP6 in in vitro binding assays, and inhibition of LRP6 or critical signaling cascades downstream of LRP6, including JNK and WNT/ β -catenin, inhibited the biologic activity of domain 4. Administration of blocking antibodies specifically against CTGF domain 4 or recombinant Dickkopf-related protein-1, an endogenous inhibitor of LRP6, effectively inhibited inflammation and fibrosis associated with ureteral obstruction in vivo Therefore, domain 4 of CTGF and the WNT signaling pathway are important new targets in fibrosis. Copyright © 2017 by the American Society of Nephrology.

Structure–Function Dissection of the Frizzled Receptor in Drosophila melanogaster Suggests Different Mechanisms of Action in Planar Polarity and Canonical Wnt Signaling

PubMed Central

Strutt, David; Madder, Daisy; Artymiuk, Peter J.

2012-01-01

Members of the Frizzled family of sevenpass transmembrane receptors signal via the canonical Wnt pathway and also via noncanonical pathways of which the best characterized is the planar polarity pathway. Activation of both canonical and planar polarity signaling requires interaction between Frizzled receptors and cytoplasmic proteins of the Dishevelled family; however, there has been some dispute regarding whether the Frizzled–Dishevelled interactions are the same in both cases. Studies looking at mutated forms of Dishevelled suggested that stable recruitment of Dishevelled to membranes by Frizzled was required only for planar polarity activity, implying that qualitatively different Frizzled–Dishevelled interactions underlie canonical signaling. Conversely, studies looking at the sequence requirements of Frizzled receptors in the fruit fly Drosophila melanogaster for canonical and planar polarity signaling have concluded that there is most likely a common mechanism of action. To understand better Frizzled receptor function, we have carried out a large-scale mutagenesis in Drosophila to isolate novel mutations in frizzled that affect planar polarity activity and have identified a group of missense mutations in cytosolic-facing regions of the Frizzled receptor that block Dishevelled recruitment. Interestingly, although some of these affect both planar polarity and canonical activity, as previously reported for similar lesions, we find a subset that affect only planar polarity activity. These results support the view that qualitatively different Frizzled–Dishevelled interactions underlie planar polarity and canonical Wnt signaling. PMID:23023003

Tissue- and stage-specific Wnt target gene expression is controlled subsequent to β-catenin recruitment to cis-regulatory modules.

PubMed

Nakamura, Yukio; de Paiva Alves, Eduardo; Veenstra, Gert Jan C; Hoppler, Stefan

2016-06-01

Key signalling pathways, such as canonical Wnt/β-catenin signalling, operate repeatedly to regulate tissue- and stage-specific transcriptional responses during development. Although recruitment of nuclear β-catenin to target genomic loci serves as the hallmark of canonical Wnt signalling, mechanisms controlling stage- or tissue-specific transcriptional responses remain elusive. Here, a direct comparison of genome-wide occupancy of β-catenin with a stage-matched Wnt-regulated transcriptome reveals that only a subset of β-catenin-bound genomic loci are transcriptionally regulated by Wnt signalling. We demonstrate that Wnt signalling regulates β-catenin binding to Wnt target genes not only when they are transcriptionally regulated, but also in contexts in which their transcription remains unaffected. The transcriptional response to Wnt signalling depends on additional mechanisms, such as BMP or FGF signalling for the particular genes we investigated, which do not influence β-catenin recruitment. Our findings suggest a more general paradigm for Wnt-regulated transcriptional mechanisms, which is relevant for tissue-specific functions of Wnt/β-catenin signalling in embryonic development but also for stem cell-mediated homeostasis and cancer. Chromatin association of β-catenin, even to functional Wnt-response elements, can no longer be considered a proxy for identifying transcriptionally Wnt-regulated genes. Context-dependent mechanisms are crucial for transcriptional activation of Wnt/β-catenin target genes subsequent to β-catenin recruitment. Our conclusions therefore also imply that Wnt-regulated β-catenin binding in one context can mark Wnt-regulated transcriptional target genes for different contexts. © 2016. Published by The Company of Biologists Ltd.

Wise, a context-dependent activator and inhibitor of Wnt signalling.

PubMed

Itasaki, Nobue; Jones, C Michael; Mercurio, Sara; Rowe, Alison; Domingos, Pedro M; Smith, James C; Krumlauf, Robb

2003-09-01

We have isolated a novel secreted molecule, Wise, by a functional screen for activities that alter the anteroposterior character of neuralised Xenopus animal caps. Wise encodes a secreted protein capable of inducing posterior neural markers at a distance. Phenotypes arising from ectopic expression or depletion of Wise resemble those obtained when Wnt signalling is altered. In animal cap assays, posterior neural markers can be induced by Wnt family members, and induction of these markers by Wise requires components of the canonical Wnt pathway. This indicates that in this context Wise activates the Wnt signalling cascade by mimicking some of the effects of Wnt ligands. Activation of the pathway was further confirmed by nuclear accumulation of beta-catenin driven by Wise. By contrast, in an assay for secondary axis induction, extracellularly Wise antagonises the axis-inducing ability of Wnt8. Thus, Wise can activate or inhibit Wnt signalling in a context-dependent manner. The Wise protein physically interacts with the Wnt co-receptor, lipoprotein receptor-related protein 6 (LRP6), and is able to compete with Wnt8 for binding to LRP6. These activities of Wise provide a new mechanism for integrating inputs through the Wnt coreceptor complex to modulate the balance of Wnt signalling.

Atlas of Wnt and R-spondin gene expression in the developing male mouse lower urogenital tract.

PubMed

Mehta, Vatsal; Abler, Lisa L; Keil, Kimberly P; Schmitz, Christopher T; Joshi, Pinak S; Vezina, Chad M

2011-11-01

Prostate development is influenced by β-catenin signaling, but it is unclear which β-catenin activators are involved, where they are synthesized, and whether their mRNA abundance is influenced by androgens. We identified WNT/β-catenin-responsive β-galactosidase activity in the lower urogenital tract (LUT) of transgenic reporter mice, but β-galactosidase activity differed among the four mouse strains we examined. We used in situ hybridization to compare patterns of Wnts, r-spondins (Rspos, co-activators of β-catenin signaling), β-catenin-responsive mRNAs, and an androgen receptor-responsive mRNA in wild type fetal male, fetal female, and neonatal male LUT. Most Wnt and Rspo mRNAs were present in LUT during prostate development. Sexually dimorphic expression patterns were observed for WNT/β-catenin-responsive genes, and for Wnt2b, Wnt4, Wnt7a, Wnt9b, Wnt10b, Wnt11, Wnt16, and Rspo3 mRNAs. These results reveal sexual differences in WNT/β-catenin signaling in fetal LUT, supporting the idea that this pathway may be directly or indirectly responsive to androgens during prostate ductal development. Copyright © 2011 Wiley-Liss, Inc.

Distinct requirements of wls, wnt9a, wnt5b and gpc4 in regulating chondrocyte maturation and timing of endochondral ossification

PubMed Central

Ling, Irving TC; Rochard, Lucie; Liao, Eric C.

2017-01-01

Formation of the mandible requires progressive morphologic change, proliferation, differentiation and organization of chondrocytes preceding osteogenesis. The Wnt signaling pathway is involved in regulating bone development and maintenance. Chondrocytes that are fated to become bone require Wnt to polarize and orientate appropriately to initiate the endochondral ossification program. Although the canonical Wnt signaling has been well studied in the context of bone development, the effects of non-canonical Wnt signaling in regulating the timing of cartilage maturation and subsequent bone formation in shaping ventral craniofacial structure is not fully understood.. Here we examined the role of the non-canonical Wnt signaling pathway (wls, gpc4, wnt5b and wnt9a) in regulating zebrafish Meckel’s cartilage maturation to the onset of osteogenic differentiation. We found that disruption of wls resulted in a significant loss of craniofacial bone, whereas lack of gpc4, wnt5b and wnt9a resulted in severely delayed endochondral ossification. This study demonstrates the importance of the non-canonical Wnt pathway in regulating coordinated ventral cartilage morphogenesis and ossification. PMID:27908786

Fresh WNT into the regulation of mitosis.

PubMed

Stolz, Ailine; Bastians, Holger

2015-01-01

Canonical Wnt signaling triggering β-catenin-dependent gene expression contributes to cell cycle progression, in particular at the G1/S transition. Recently, however, it became clear that the cell cycle can also feed back on Wnt signaling at the G2/M transition. This is illustrated by the fact that mitosis-specific cyclin-dependent kinases can phosphorylate the Wnt co-receptor LRP6 to prime the pathway for incoming Wnt signals when cells enter mitosis. In addition, there is accumulating evidence that various Wnt pathway components might exert additional, Wnt-independent functions that are important for proper regulation of mitosis. The importance of Wnt pathways during mitosis was most recently enforced by the discovery of Wnt signaling contributing to the stabilization of proteins other than β-catenin, specifically at G2/M and during mitosis. This Wnt-mediated stabilization of proteins, now referred to as Wnt/STOP, might on one hand contribute to maintaining a critical cell size required for cell division and, on the other hand, for the faithful execution of mitosis itself. In fact, most recently we have shown that Wnt/STOP is required for ensuring proper microtubule dynamics within mitotic spindles, which is pivotal for accurate chromosome segregation and for the maintenance of euploidy.

Targeting the Wnt/β-catenin pathway in primary ovarian cancer with the porcupine inhibitor WNT974.

PubMed

Boone, Jonathan D; Arend, Rebecca C; Johnston, Bobbi E; Cooper, Sara J; Gilchrist, Scott A; Oelschlager, Denise K; Grizzle, William E; McGwin, Gerald; Gangrade, Abhishek; Straughn, J Michael; Buchsbaum, Donald J

2016-02-01

Preclinical studies in ovarian cancer have demonstrated upregulation of the Wnt/β-catenin pathway promoting tumor proliferation and chemoresistance. Our objective was to evaluate the effect of the Wnt/β-catenin pathway inhibitor, WNT974, in primary ovarian cancer ascites cells. Ascites cells from patients with papillary serous ovarian cancer were isolated and treated with 1 μM WNT974±100 μM carboplatin. Viability was evaluated with the ATPlite assay. The IC50 was calculated using a dose-response analysis. Immunohistochemistry (IHC) was performed on ascites cells and tumor. Expression of R-spondin 2 (RSPO2), RSPO3, PORCN, WLS, AXIN2, and three previously characterized RSPO fusion transcripts were assessed using Taqman assays. Sixty ascites samples were analyzed for response to WNT974. The ascites samples that showed a decrease in ATP concentration after treatment demonstrated no difference from the untreated cells in percent viability with trypan blue staining. Flow cytometry demonstrated fewer cells in the G2 phase and more in the G1 and S phases after treatment with WNT974. Combination therapy with WNT974 and carboplatin resulted in a higher percentage of samples that showed ≥30% reduction in ATP concentration than either single drug treatment. IHC analysis of Wnt pathway proteins suggests cell cycle arrest rather than cytotoxicity after WNT974 treatment. QPCR indicated that RSPO fusions are not prevalent in ovarian cancer tissues or ascites. However, higher PORCN expression correlated to sensitivity to WNT974 (P=0.0073). In conclusion, WNT974 produces cytostatic effects in patient ascites cells with primary ovarian cancer through inhibition of the Wnt/β-catenin pathway. The combination of WNT974 and carboplatin induces cytotoxicity plus cell cycle arrest in a higher percentage of ascites samples than with single drug treatment. RSPO fusions do not contribute to WNT974 sensitivity; however, higher PORCN expression indicates increased WNT974 sensitivity.

IL-1β-induced, matrix metalloproteinase-3-regulated proliferation of embryonic stem cell-derived odontoblastic cells is mediated by the Wnt5 signaling pathway

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

Ozeki, Nobuaki; Hase, Naoko; Hiyama, Taiki

2014-10-15

We previously established a method for differentiating induced pluripotent stem cells and embryonic stem (ES) cells into α2 integrin-positive odontoblast-like cells. We also reported that interleukin (IL)-1β induces matrix metalloproteinase (MMP)-3-regulated cell proliferation and suppresses apoptosis in these cells, suggesting that MMP-3 plays a potentially unique physiological role in the regeneration of odontoblast-like cells. Here, we examined whether up-regulation of MMP-3 activity by IL-1β was mediated by Wnt signaling and led to increased proliferation of odontoblast-like cells. IL-1β increased mRNA and protein levels of Wnt5a, Wnt5b and the Wnt receptor Lrp5. Exogenous Wnt5a and Wnt5b were found to increase MMP-3more » mRNA, protein and activity, and interestingly the rate of proliferation in these cells. Treatment with siRNAs against Wnt5a, Wnt5b and Lrp5 suppressed the IL-1β-induced increase in MMP-3 expression and suppressed cell proliferation, an effect rescued by application of exogenous Wnt5. These results demonstrate the sequential involvement of Wnt5, Lrp5 and MMP-3 in effecting IL-1β-induced proliferation of ES cell-derived odontoblast-like cells. - Highlights: • IL-1β induces Wnt5, Lrp5/Fzd9 and MMP-3 in ES cell-derived odontoblast-like cells. • IL-1β-induced Wnt5 expression results in increased cell proliferation. • Exogenous Wnt5 increases MMP-3 activity and cell proliferation. • Exogenous Wnt5 rescues IL-1β-driven proliferation with anti-Wnt5 siRNA suppression. • IL-1β-induced cell proliferation involves Wnt5, Lrp5, and MMP-3 sequentially.« less

Wnt7a activates canonical Wnt signaling, promotes bladder cancer cell invasion, and is suppressed by miR-370-3p.

PubMed

Huang, Xiaojing; Zhu, Hongwen; Gao, Zemin; Li, Junzun; Zhuang, Junlong; Dong, Yu; Shen, Bing; Li, Meiqian; Zhou, Hu; Guo, Hongqian; Huang, Ruimin; Yan, Jun

2018-05-04

Once urinary bladder cancer (UBC) develops into muscle-invasive bladder cancer, its mortality rate increases dramatically. However, the molecular mechanisms of UBC invasion and metastasis remain largely unknown. Herein, using 5637 UBC cells, we generated two sublines with low (5637 NMI) and high (5637 HMI) invasive capabilities. Mass spectrum analyses revealed that the Wnt family protein Wnt7a is more highly expressed in 5637 HMI cells than in 5637 NMI cells. We also found that increased Wnt7a expression is associated with UBC metastasis and predicted worse clinical outcome in UBC patients. Wnt7a depletion in 5637 HMI and T24 cells reduced UBC cell invasion and decreased levels of active β-catenin and its downstream target genes involved in the epithelial-to-mesenchymal transition (EMT) and extracellular matrix (ECM) degradation. Consistently, treating 5637 NMI and J82 cells with recombinant Wnt7a induced cell invasion, EMT, and expression of ECM degradation-associated genes. Moreover, TOP/FOPflash luciferase assays indicated that Wnt7a activated canonical β-catenin signaling in UBC cells, and increased Wnt7a expression was associated with nuclear β-catenin in UBC samples. Wnt7a ablation suppressed matrix metalloproteinase 10 (MMP10) expression, and Wnt7a overexpression increased MMP10 promoter activity through two TCF/LEF promoter sites, confirming that Wnt7a-mediated MMP10 activation is mediated by the canonical Wnt/β-catenin pathway. Of note, the microRNA miR-370-3p directly repressed Wnt7a expression and thereby suppressed UBC cell invasion, which was partially restored by Wnt7a overexpression. Our results have identified an miR-370-3p/Wnt7a axis that controls UBC invasion through canonical Wnt/β-catenin signaling, which may offer prognostic and therapeutic opportunities. © 2018 Huang et al.

Organized Emergence of Multiple-Generations of Teeth in Snakes Is Dysregulated by Activation of Wnt/Beta-Catenin Signalling

PubMed Central

Gaete, Marcia; Tucker, Abigail S.

2013-01-01

In contrast to mammals, most reptiles constantly regenerate their teeth. In the snake, the epithelial dental lamina ends in a successional lamina, which proliferates and elongates forming multiple tooth generations, all linked by a permanent dental lamina. To investigate the mechanisms used to control the initiation of new tooth germs in an ordered sequential pattern we utilized the polyphodont (multiple-generation) corn snake (Pantherophis guttatus). We observed that the dental lamina expressed the transcription factor Sox2, a multipotent stem cell marker, whereas the successional lamina cells expressed the transcription factor Lef1, a Wnt/β-catenin pathway target gene. Activation of the Wnt/β-catenin pathway in culture increased the number of developing tooth germs, in comparison to control untreated cultures. These additional tooth germs budded off from ectopic positions along the dental lamina, rather than in an ordered sequence from the successional lamina. Wnt/β-catenin activation enhanced cell proliferation, particularly in normally non-odontogenic regions of the dental lamina, which widely expressed Lef1, restricting the Sox2 domain. This suggests an expansion of the successional lamina at the expense of the dental lamina. Activation of the Wnt/β-catenin pathway in cultured snake dental organs, therefore, led to changes in proliferation and to the molecular pattern of the dental lamina, resulting in loss of the organised emergence of tooth germs. These results suggest that epithelial compartments are critical for the arrangement of organs that develop in sequence, and highlight the role of Wnt/β-catenin signalling in such processes. PMID:24019968

ECTODERMAL WNT/β-CATENIN SIGNALING SHAPES THE MOUSE FACE

PubMed Central

Reid, Bethany S.; Yang, Hui; Melvin, Vida Senkus; Taketo, Makoto M.; Williams, Trevor

2010-01-01

The canonical Wnt/β-catenin pathway is an essential component of multiple developmental processes. To investigate the role of this pathway in the ectoderm during facial morphogenesis, we generated conditional β-catenin mouse mutants using a novel ectoderm-specific Cre recombinase transgenic line. Our results demonstrate that ablating or stabilizing β-catenin in the embryonic ectoderm causes dramatic changes in facial morphology. There are accompanying alterations in the expression of Fgf8 and Shh, key molecules that establish a signaling center critical for facial patterning, the frontonasal ectodermal zone (FEZ). These data indicate that Wnt/β-catenin signaling within the ectoderm is critical for facial development and further suggest that this pathway is an important mechanism for generating the diverse facial shapes of vertebrates during evolution. PMID:21087601

Bone Marrow Mesenchymal Stem Cell-Derived CD63+ Exosomes Transport Wnt3a Exteriorly and Enhance Dermal Fibroblast Proliferation, Migration, and Angiogenesis In Vitro.

PubMed

McBride, Jeffrey D; Rodriguez-Menocal, Luis; Guzman, Wellington; Candanedo, Ambar; Garcia-Contreras, Marta; Badiavas, Evangelos V

2017-10-01

Wnts are secreted glycoproteins that regulate stem cell self-renewal, differentiation, and cell-to-cell communication during embryonic development and in adult tissues. Bone marrow mesenchymal stem cells (BM-MSCs) have been shown to stimulate dermis repair and regeneration; however, it is unclear how BM-MSCs may modulate downstream Wnt signaling. While recent reports implicate that Wnt ligands and Wnt messenger RNAs (such as Wnt4) exist within the interior compartment of exosomes, it has been debated whether or not Wnts exist on the exterior surface of exosomes to travel in the extracellular space. To help answer this question, we utilized flow cytometry of magnetic beads coated with anti-CD63 antibodies and found, for the first time, that Wnt3a protein is detectable exteriorly on CD63 + exosomes derived from BM-MSCs over-secreting Wnt3a into serum-free conditioned media (Wnt3a CM). Our data suggest that CD63 + exosomes significantly help transport exterior Wnt3a signal to recipient cells to promote fibroblast and endothelial functions. During purification of exosomes, we unexpectedly found that use of ultracentrifugation alone significantly decreased the ability to detect exteriorly bound Wnt3a on CD63 + exosomes, however, polyethylene glycol (PEG)-mediated exosome-enrichment before exosome-purification (with ultracentrifugation into a sucrose cushion) resulted in exosomes more likely to retain exterior Wnt3a detectability and downstream Wnt/beta-catenin activity. Our findings indicate the important role that purification methods may have on stem cell-derived Wnt-exosome activity in downstream assays. The ability for BM-MSC Wnt3a CM and exosomes to stimulate dermal fibroblast proliferation and migration, and endothelial angiogenesis in vitro, was significantly decreased after CD63 + -exosome depletion or knockdown of Wnt coreceptor LRP6 in recipient cells, suggesting both are required for optimal Wnt-exosome activity in our system. Thus, BM-MSC-derived CD63 + exosomes are a significant carrier of exterior Wnt3a within high Wnt environments, resulting in downstream fibroblast proliferation, migration, and angiogenesis in vitro.

β-Arrestin-1 deficiency ameliorates renal interstitial fibrosis by blocking Wnt1/β-catenin signaling in mice.

PubMed

Xu, Huiyan; Li, Quanxin; Liu, Jiang; Zhu, Jiaqing; Li, Liang; Wang, Ziying; Zhang, Yan; Sun, Yu; Sun, Jinpeng; Wang, Rong; Yi, Fan

2018-01-01

Despite substantial progress being made in understanding the mechanisms contributing to the pathogenesis of renal fibrosis, there are only a few therapies available to treat or prevent renal fibrosis in clinical use today. Therefore, identifying the key cellular and molecular mediators involved in the pathogenesis of renal fibrosis will provide new therapeutic strategy for treating patients with chronic kidney disease (CKD). β-Arrestin-1, a member of β-arrestin family, not only is a negative adaptor of G protein-coupled receptors (GPCRs), but also acts as a scaffold protein and regulates a diverse array of cellular functions independent of GPCR activation. In this study, we identified for the first time that β-arrestin-1 was upregulated in the kidney from mice with unilateral ureteral obstruction nephropathy as well as in the paraffin-embedded sections of human kidneys from the patients with diabetic nephropathy, polycystic kidney, or uronephrosis, which normally causes renal fibrosis. Deficiency of β-arrestin-1 in mice significantly alleviated renal fibrosis by the regulation of inflammatory responses, kidney fibroblast activation, and epithelial-mesenchymal transition (EMT) in both in vivo and in vitro studies. Furthermore, we found that among the major isoforms of Wnts, Wnt1 was regulated by β-arrestin-1 and gene silencing of Wnt1 inhibited the activation of β-catenin and suppressed β-arrestin-1-mediated renal fibrosis. Collectively, our results indicate that β-arrestin-1 is one of the critical components of signal transduction pathways in the development of renal fibrosis. Modulation of these pathways may be an innovative therapeutic strategy for treating patients with renal fibrosis. β-Arrestin-1 was upregulated in the kidney from mice with UUO nephropathy. β-Arrestin-1 regulated kidney fibroblast activation and epithelial-mesenchymal transition. β-Arrestin-1 exacerbated renal fibrosis via mediating Wnt1/β-catenin signaling.

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

Misu, Masayasu; Ouji, Yukiteru, E-mail: oujix@naramed-u.ac.jp; Kawai, Norikazu

In spite of the strong expression of Wnt-10b in melanomas, its role in melanoma cells has not been elucidated. In the present study, the biological effects of Wnt-10b on murine B16F10 (B16) melanoma cells were investigated using conditioned medium from Wnt-10b-producing COS cells (Wnt-CM). After 2 days of culture in the presence of Wnt-CM, proliferation of B16 melanoma cells was inhibited, whereas tyrosinase activity was increased. An in vitro wound healing assay demonstrated that migration of melanoma cells to the wound area was inhibited with the addition of Wnt-CM. Furthermore, evaluation of cellular senescence revealed prominent induction of SA-β-gal-positive senescent cellsmore » in cultures with Wnt-CM. Finally, the growth of B16 melanoma cell aggregates in collagen 3D-gel cultures was markedly suppressed in the presence of Wnt-CM. These results suggest that Wnt-10b represses tumor cell properties, such as proliferation and migration of B16 melanoma cells, driving them toward a more differentiated state along a melanocyte lineage. - Highlights: • Wnt-10b inhibited proliferation and migration of melanoma cells. • Wnt-10b induced tyrosinase activity and senescence of melanoma cells. • Wnt-10b suppressed growth of cell aggregates in collagen 3D-gel cultures. • Wnt-10b represses tumor cell properties, driving them toward a more differentiated state along a melanocyte lineage.« less

Genome-wide analysis of a Wnt1-regulated transcriptional network implicates neurodegenerative pathways.

PubMed

Wexler, Eric M; Rosen, Ezra; Lu, Daning; Osborn, Gregory E; Martin, Elizabeth; Raybould, Helen; Geschwind, Daniel H

2011-10-04

Wnt proteins are critical to mammalian brain development and function. The canonical Wnt signaling pathway involves the stabilization and nuclear translocation of β-catenin; however, Wnt also signals through alternative, noncanonical pathways. To gain a systems-level, genome-wide view of Wnt signaling, we analyzed Wnt1-stimulated changes in gene expression by transcriptional microarray analysis in cultured human neural progenitor (hNP) cells at multiple time points over a 72-hour time course. We observed a widespread oscillatory-like pattern of changes in gene expression, involving components of both the canonical and the noncanonical Wnt signaling pathways. A higher-order, systems-level analysis that combined independent component analysis, waveform analysis, and mutual information-based network construction revealed effects on pathways related to cell death and neurodegenerative disease. Wnt effectors were tightly clustered with presenilin1 (PSEN1) and granulin (GRN), which cause dominantly inherited forms of Alzheimer's disease and frontotemporal dementia (FTD), respectively. We further explored a potential link between Wnt1 and GRN and found that Wnt1 decreased GRN expression by hNPs. Conversely, GRN knockdown increased WNT1 expression, demonstrating that Wnt and GRN reciprocally regulate each other. Finally, we provided in vivo validation of the in vitro findings by analyzing gene expression data from individuals with FTD. These unbiased and genome-wide analyses provide evidence for a connection between Wnt signaling and the transcriptional regulation of neurodegenerative disease genes.

A Wnt1 regulated Frizzled-1/β-Catenin signaling pathway as a candidate regulatory circuit controlling mesencephalic dopaminergic neuron-astrocyte crosstalk: Therapeutical relevance for neuron survival and neuroprotection

PubMed Central

2011-01-01

Background Dopamine-synthesizing (dopaminergic, DA) neurons in the ventral midbrain (VM) constitute a pivotal neuronal population controlling motor behaviors, cognitive and affective brain functions, which generation critically relies on the activation of Wingless-type MMTV integration site (Wnt)/β-catenin pathway in their progenitors. In Parkinson's disease, DA cell bodies within the substantia nigra pars compacta (SNpc) progressively degenerate, with causes and mechanisms poorly understood. Emerging evidence suggests that Wnt signaling via Frizzled (Fzd) receptors may play a role in different degenerative states, but little is known about Wnt signaling in the adult midbrain. Using in vitro and in vivo model systems of DA degeneration, along with functional studies in both intact and SN lesioned mice, we herein highlight an intrinsic Wnt1/Fzd-1/β-catenin tone critically contributing to the survival and protection of adult midbrain DA neurons. Results In vitro experiments identifie Fzd-1 receptor expression at a mRNA and protein levels in dopamine transporter (DAT) expressing neurons, and demonstrate the ability of exogenous Wnt1 to exert robust neuroprotective effects against Caspase-3 activation, the loss of tyrosine hydroxylase-positive (TH+) neurons and [3H] dopamine uptake induced by different DA-specific insults, including serum and growth factor deprivation, 6-hydroxydopamine and MPTP/MPP+. Co-culture of DA neurons with midbrain astrocytes phenocopies Wnt1 neuroprotective effects, whereas RNA interference-mediated knockdown of Wnt1 in midbrain astrocytes markedly reduces astrocyte-induced TH+ neuroprotection. Likewise, silencing β-catenin mRNA or knocking down Fzd-1 receptor expression in mesencephalic neurons counteract astrocyte-induced TH+ neuroprotection. In vivo experiments document Fzd-1 co-localization with TH+ neurons within the intact SNpc and blockade of Fzd/β-catenin signaling by unilateral infusion of a Fzd/β-catenin antagonist within the SN induces reactive astrocytosis and acutely inhibits TH+ neuron survival in ipsilateral SNpc, an effect efficiently prevented by pharmacological activation of β-catenin signaling within the SNpc. Conclusion These results defining a novel Wnt1/Fzd-1/β-catenin astrocyte-DA autoprotective loop provide a new mechanistic inside into the regulation of pro-survival processes, with potentially relevant consequences for drug design or drug action in Parkinson's disease. PMID:21752258

Transdifferentiation from cornea to lens in Xenopus laevis depends on BMP signalling and involves upregulation of Wnt signalling

PubMed Central

2011-01-01

Background Surgical removal of the lens from larval Xenopus laevis results in a rapid transdifferention of central corneal cells to form a new lens. The trigger for this process is understood to be an induction event arising from the unprecedented exposure of the cornea to the vitreous humour that occurs following lens removal. The molecular identity of this trigger is unknown. Results Here, we have used a functional transgenic approach to show that BMP signalling is required for lens regeneration and a microarray approach to identify genes that are upregulated specifically during this process. Analysis of the array data strongly implicates Wnt signalling and the Pitx family of transcription factors in the process of cornea to lens transdifferentiation. Our analysis also captured several genes associated with congenital cataract in humans. Pluripotency genes, in contrast, were not upregulated, supporting the idea that corneal cells transdifferentiate without returning to a stem cell state. Several genes from the array were expressed in the forming lens during embryogenesis. One of these, Nipsnap1, is a known direct target of BMP signalling. Conclusions Our results strongly implicate the developmental Wnt and BMP signalling pathways in the process of cornea to lens transdifferentiation (CLT) in Xenopus, and suggest direct transdifferentiation between these two anterior eye tissues. PMID:21896182

Inhibition by Chondroitin Sulfate E Can Specify Functional Wnt/β-Catenin Signaling Thresholds in NIH3T3 Fibroblasts*

PubMed Central

Willis, Catherine M.; Klüppel, Michael

2012-01-01

Aberrant activation of the Wnt/β-catenin signaling pathway is frequently associated with human disease, including cancer, and thus represents a key therapeutic target. However, Wnt/β-catenin signaling also plays critical roles in many aspects of normal adult tissue homeostasis. The identification of mechanisms and strategies to selectively inhibit the disease-related functions of Wnt signaling, while preserving normal physiological functions, is in its infancy. Here, we report the identification of exogenous chondroitin sulfate-E (CS-E) as an inhibitor of specific molecular and biological outcomes of Wnt3a signaling in NIH3T3 fibroblasts. We demonstrate that CS-E can decrease Wnt3a signaling through the negative regulation of LRP6 receptor activation. However, this inhibitory effect of CS-E only affected Wnt3a-mediated induction, but not repression, of target gene expression. We went on to identify a critical Wnt3a signaling threshold that differentially affects target gene induction versus repression. This signaling threshold also controlled the effects of Wnt3a on proliferation and serum starvation-induced apoptosis. Limiting Wnt3a signaling to this critical threshold, either by CS-E treatment or by ligand dilution, interfered with Wnt3a-mediated stimulation of proliferation but did not impair Wnt3a-mediated reduction of serum starvation-induced apoptosis. Treatment with pharmacological inhibitors demonstrated that both induction and repression of Wnt3a target genes in NIH3T3 cells require the canonical Wnt/β-catenin signaling cascade. Our data establish the feasibility of selective inhibition of Wnt/β-catenin transcriptional programs and biological outcomes through the exploitation of intrinsic signaling thresholds. PMID:22915582

Ror2 Receptor Mediates Wnt11 Ligand Signaling and Affects Convergence and Extension Movements in Zebrafish*

PubMed Central

Bai, Yan; Tan, Xungang; Zhang, Haifeng; Liu, Chengdong; Zhao, Beibei; Li, Yun; Lu, Ling; Liu, Yunzhang; Zhou, Jianfeng

2014-01-01

The receptor-tyrosine kinase Ror2 acts as an alternative receptor or co-receptor for Wnt5a and mediates Wnt5a-induced convergent extension movements during embryogenesis in mice and Xenopus as well as the polarity and migration of several cell types during development. However, little is known about whether Ror2 function is conserved in other vertebrates or is involved in other non-canonical Wnt ligands in vivo. In this study we demonstrated that overexpression of dominant-negative ror2 (ror2-TM) mRNA in zebrafish embryos resulted in convergence and extension defects and incompletely separated eyes, which is consistent with observations from slb/wnt11 mutants or wnt11 knockdown morphants. Moreover, the co-injection of ror2-TM mRNA and a wnt11 morpholino or the coexpression of ror2 and wnt11 in zebrafish embryos synergetically induced more severe convergence and extension defects. Transplantation studies further demonstrated that the Ror2 receptor responded to the Wnt11 ligand and regulated cell migration and cell morphology during gastrulation. DnRor2 inhibited the action of Wnt11, which was revealed by a decreased percentage of Wnt11-induced convergence and extension defects. Ror2 physically interacts with Wnt11. The intracellular Tyr-647 and Ser-863 sites of Ror2 are essential for mediating the action of Wnt11. Dishevelled and RhoA act downstream of Wnt11-Ror2 to regulate convergence and extension movements. Overall, our data suggest an important role of Ror2 in mediating Wnt11 signaling and in regulating convergence and extension movements in zebrafish. PMID:24928507

Wnt5a Promotes Inflammatory Responses via Nuclear Factor κB (NF-κB) and Mitogen-activated Protein Kinase (MAPK) Pathways in Human Dental Pulp Cells*

PubMed Central

Zhao, Yuan; Wang, Chen-Lin; Li, Rui-Min; Hui, Tian-Qian; Su, Ying-Ying; Yuan, Quan; Zhou, Xue-Dong; Ye, Ling

2014-01-01

Wnt5a has been found recently to be involved in inflammation regulation through a mechanism that remains unclear. Immunohistochemical staining of infected human dental pulp and tissue from experimental dental pulpitis in rats showed that Wnt5a levels were increased. In vitro, Wnt5a was increased 8-fold in human dental pulp cells (HDPCs) after TNF-α stimulation compared with control cells. We then investigated the role of Wnt5a in HDPCs. In the presence of TNF-α, Wnt5a further increased the production of cytokines/chemokines, whereas Wnt5a knockdown markedly reduced cytokine/chemokine production induced by TNF-α. In addition, in HDPCs, Wnt5a efficiently induced cytokine/chemokine expression and, in particular, expression of IL-8 (14.5-fold) and CCL2 (25.5-fold), as assessed by a Luminex assay. The cytokine subsets regulated by Wnt5a overlap partially with those induced by TNF-α. However, no TNF-α and IL-1β was detected after Wnt5a treatment. We then found that Wnt5a alone and the supernatants of Wnt5a-treated HDPCs significantly increased macrophage migration, which supports a role for Wnt5a in macrophage recruitment and as an inflammatory mediator in human dental pulp inflammation. Finally, Wnt5a participates in dental pulp inflammation in a MAPK-dependent (p38-, JNK-, and ERK-dependent) and NF-κB-dependent manner. Our data suggest that Wnt5a, as an inflammatory mediator that drives the integration of cytokines and chemokines, acts downstream of TNF-α. PMID:24891513

Blocking epithelial-to-mesenchymal transition in glioblastoma with a sextet of repurposed drugs: the EIS regimen

PubMed Central

Kast, Richard E; Skuli, Nicolas; Karpel-Massler, Georg; Frosina, Guido; Ryken, Timothy; Halatsch, Marc-Eric

2017-01-01

This paper outlines a treatment protocol to run alongside of standard current treatment of glioblastoma- resection, temozolomide and radiation. The epithelial to mesenchymal transition (EMT) inhibiting sextet, EIS Regimen, uses the ancillary attributes of six older medicines to impede EMT during glioblastoma. EMT is an actively motile, therapy-resisting, low proliferation, transient state that is an integral feature of cancers’ lethality generally and of glioblastoma specifically. It is believed to be during the EMT state that glioblastoma’s centrifugal migration occurs. EMT is also a feature of untreated glioblastoma but is enhanced by chemotherapy, by radiation and by surgical trauma. EIS Regimen uses the antifungal drug itraconazole to block Hedgehog signaling, the antidiabetes drug metformin to block AMP kinase (AMPK), the analgesic drug naproxen to block Rac1, the anti-fibrosis drug pirfenidone to block transforming growth factor-beta (TGF-beta), the psychiatric drug quetiapine to block receptor activator NFkB ligand (RANKL) and the antibiotic rifampin to block Wnt- all by their previously established ancillary attributes. All these systems have been identified as triggers of EMT and worthy targets to inhibit. The EIS Regimen drugs have a good safety profile when used individually. They are not expected to have any new side effects when combined. Further studies of the EIS Regimen are needed. PMID:28977822

Wnt signaling in cancer

PubMed Central

Zhan, T; Rindtorff, N; Boutros, M

2017-01-01

Wnt signaling is one of the key cascades regulating development and stemness, and has also been tightly associated with cancer. The role of Wnt signaling in carcinogenesis has most prominently been described for colorectal cancer, but aberrant Wnt signaling is observed in many more cancer entities. Here, we review current insights into novel components of Wnt pathways and describe their impact on cancer development. Furthermore, we highlight expanding functions of Wnt signaling for both solid and liquid tumors. We also describe current findings how Wnt signaling affects maintenance of cancer stem cells, metastasis and immune control. Finally, we provide an overview of current strategies to antagonize Wnt signaling in cancer and challenges that are associated with such approaches. PMID:27617575

Blockade of LGR4 inhibits proliferation and odonto/osteogenic differentiation of stem cells from apical papillae.

PubMed

Zhou, Meng; Guo, Shuyu; Yuan, Lichan; Zhang, Yuxin; Zhang, Mengnan; Chen, Huimin; Lu, Mengting; Yang, Jianrong; Ma, Junqing

2017-12-01

During tooth root development, stem cells from apical papillae (SCAPs) are indispensable, and their abilities of proliferation, migration and odontoblast differentiation are linked to root formation. Leucine-rich repeat-containing GPCR 4 (LGR4) modulates the biological processes of proliferation and differentiation in multiple stem cells. In this study, we showed that LGR4 is expressed in all odontoblast cell lineage cells and Hertwig's epithelial root sheath (HERS) during the mouse root formation in vivo. In vitro we determined that LGR4 is involved in the Wnt/β-catenin signaling pathway regulating proliferation and odonto/osteogenic differentiation of SCAPs. Quantitative reverse-transcription PCR (qRT-PCR) confirmed that LGR4 is expressed during odontogenic differentiation of SCAPs. CCK8 assays and in vitro scratch tests, together with cell cycle flow cytometric analysis, demonstrated that downregulation of LGR4 inhibited SCAPs proliferation, delayed migration and arrested cell cycle progression at the S and G2/M phases. ALP staining revealed that blockade of LGR4 decreased ALP activity. QRT-PCR and Western blot analysis demonstrated that LGR4 silencing reduced the expression of odonto/osteogenic markers (RUNX2, OSX, OPN, OCN and DSPP). Further Western blot and immunofluorescence studies clarified that inhibition of LGR4 disrupted β-catenin stabilization. Taken together, downregulation of LGR4 gene expression inhibited SCAPs proliferation, migration and odonto/osteogenic differentiation by blocking the Wnt/β-catenin signaling pathway. These results indicate that LGR4 might play a vital role in SCAPs proliferation and odontoblastic differentiation.

Identifying novel members of the Wntless interactome through genetic and candidate gene approaches.

PubMed

Petko, Jessica; Tranchina, Trevor; Patel, Goral; Levenson, Robert; Justice-Bitner, Stephanie

2018-04-01

Wnt signaling is an important pathway that regulates several aspects of embryogenesis, stem cell maintenance, and neural connectivity. We have recently determined that opioids decrease Wnt secretion, presumably by inhibiting the recycling of the Wnt trafficking protein Wntless (Wls). This effect appears to be mediated by protein-protein interaction between Wls and the mu-opioid receptor (MOR), the primary cellular target of opioid drugs. The goal of this study was to identify novel protein interactors of Wls that are expressed in the brain and may also play a role in reward or addiction. Using genetic and candidate gene approaches, we show that among a variety of protein, Wls interacts with the dopamine transporter (target of cocaine), cannabinoid receptors (target of THC), Adenosine A2A receptor (target of caffeine), and SGIP1 (endocytic regulator of cannabinoid receptors). Our study shows that aside from opioid receptors, Wntless interacts with additional proteins involved in reward and/or addiction. Future studies will determine whether Wntless and WNT signaling play a more universal role in these processes. Copyright © 2017 Elsevier Inc. All rights reserved.

Neuronal Target Identification Requires AHA-1-Mediated Fine-Tuning of Wnt Signaling in C. elegans

PubMed Central

Zhang, Jingyan; Li, Xia; Jevince, Angela R.; Guan, Liying; Wang, Jiaming; Hall, David H.; Huang, Xun; Ding, Mei

2013-01-01

Electrical synaptic transmission through gap junctions is a vital mode of intercellular communication in the nervous system. The mechanism by which reciprocal target cells find each other during the formation of gap junctions, however, is poorly understood. Here we show that gap junctions are formed between BDU interneurons and PLM mechanoreceptors in C. elegans and the connectivity of BDU with PLM is influenced by Wnt signaling. We further identified two PAS-bHLH family transcription factors, AHA-1 and AHR-1, which function cell-autonomously within BDU and PLM to facilitate the target identification process. aha-1 and ahr-1 act genetically upstream of cam-1. CAM-1, a membrane-bound receptor tyrosine kinase, is present on both BDU and PLM cells and likely serves as a Wnt antagonist. By binding to a cis-regulatory element in the cam-1 promoter, AHA-1 enhances cam-1 transcription. Our study reveals a Wnt-dependent fine-tuning mechanism that is crucial for mutual target cell identification during the formation of gap junction connections. PMID:23825972

Delamination of neural crest cells requires transient and reversible Wnt inhibition mediated by Dact1/2.

PubMed

Rabadán, M Angeles; Herrera, Antonio; Fanlo, Lucia; Usieto, Susana; Carmona-Fontaine, Carlos; Barriga, Elias H; Mayor, Roberto; Pons, Sebastián; Martí, Elisa

2016-06-15

Delamination of neural crest (NC) cells is a bona fide physiological model of epithelial-to-mesenchymal transition (EMT), a process that is influenced by Wnt/β-catenin signalling. Using two in vivo models, we show that Wnt/β-catenin signalling is transiently inhibited at the time of NC delamination. In attempting to define the mechanism underlying this inhibition, we found that the scaffold proteins Dact1 and Dact2, which are expressed in pre-migratory NC cells, are required for NC delamination in Xenopus and chick embryos, whereas they do not affect the motile properties of migratory NC cells. Dact1/2 inhibit Wnt/β-catenin signalling upstream of the transcriptional activity of T cell factor (TCF), which is required for EMT to proceed. Dact1/2 regulate the subcellular distribution of β-catenin, preventing β-catenin from acting as a transcriptional co-activator to TCF, yet without affecting its stability. Together, these data identify a novel yet important regulatory element that inhibits β-catenin signalling, which then affects NC delamination. © 2016. Published by The Company of Biologists Ltd.

FGF signaling refines Wnt gradients to regulate the patterning of taste papillae.

PubMed

Prochazkova, Michaela; Häkkinen, Teemu J; Prochazka, Jan; Spoutil, Frantisek; Jheon, Andrew H; Ahn, Youngwook; Krumlauf, Robb; Jernvall, Jukka; Klein, Ophir D

2017-06-15

The patterning of repeated structures is a major theme in developmental biology, and the inter-relationship between spacing and size of such structures is an unresolved issue. Fungiform papillae are repeated epithelial structures that house taste buds on the anterior tongue. Here, we report that FGF signaling is a crucial regulator of fungiform papillae development. We found that mesenchymal FGF10 controls the size of the papillary area, while overall patterning remains unchanged. Our results show that FGF signaling negatively affects the extent of canonical Wnt signaling, which is the main activation pathway during fungiform papillae development; however, this effect does not occur at the level of gene transcription. Rather, our experimental data, together with computational modeling, indicate that FGF10 modulates the range of Wnt effects, likely via induction of Sostdc1 expression. We suggest that modification of the reach of Wnt signaling could be due to local changes in morphogen diffusion, representing a novel mechanism in this tissue context, and we propose that this phenomenon might be involved in a broader array of mammalian developmental processes. © 2017. Published by The Company of Biologists Ltd.

Regulation of the Wnt/β-Catenin Signaling Pathway by Human Papillomavirus E6 and E7 Oncoproteins

PubMed Central

Muñoz Bello, Jesus Omar; Olmedo Nieva, Leslie; Contreras Paredes, Adriana; Fuentes Gonzalez, Alma Mariana; Rocha Zavaleta, Leticia; Lizano, Marcela

2015-01-01

Cell signaling pathways are the mechanisms by which cells transduce external stimuli, which control the transcription of genes, to regulate diverse biological effects. In cancer, distinct signaling pathways, such as the Wnt/β-catenin pathway, have been implicated in the deregulation of critical molecular processes that affect cell proliferation and differentiation. For example, changes in β-catenin localization have been identified in Human Papillomavirus (HPV)-related cancers as the lesion progresses. Specifically, β-catenin relocates from the membrane/cytoplasm to the nucleus, suggesting that this transcription regulator participates in cervical carcinogenesis. The E6 and E7 oncoproteins are responsible for the transforming activity of HPV, and some studies have implicated these viral oncoproteins in the regulation of the Wnt/β-catenin pathway. Nevertheless, new interactions of HPV oncoproteins with cellular proteins are emerging, and the study of the biological effects of such interactions will help to understand HPV-related carcinogenesis. This review addresses the accumulated evidence of the involvement of the HPV E6 and E7 oncoproteins in the activation of the Wnt/β-catenin pathway. PMID:26295406

R-spondin 1 is required for specification of hematopoietic stem cells through Wnt16 and Vegfa signaling pathways

PubMed Central

2017-01-01

Hematopoietic stem cells (HSCs) are the therapeutic component of bone marrow transplants, but finding immune-compatible donors limits treatment availability and efficacy. Recapitulation of endogenous specification during development is a promising approach to directing HSC specification in vitro, but current protocols are not capable of generating authentic HSCs with high efficiency. Across phyla, HSCs arise from hemogenic endothelium in the ventral floor of the dorsal aorta concurrent with arteriovenous specification and intersegmental vessel (ISV) sprouting, processes regulated by Notch and Wnt. We hypothesized that coordination of HSC specification with vessel patterning might involve modulatory regulatory factors such as R-spondin 1 (Rspo1), an extracellular protein that enhances β-catenin-dependent Wnt signaling and has previously been shown to regulate ISV patterning. We find that Rspo1 is required for HSC specification through control of parallel signaling pathways controlling HSC specification: Wnt16/DeltaC/DeltaD and Vegfa/Tgfβ1. Our results define Rspo1 as a key upstream regulator of two crucial pathways necessary for HSC specification. PMID:28087636

Canonical wnt signaling regulates hematopoiesis in a dosage-dependent fashion.

PubMed

Luis, Tiago C; Naber, Brigitta A E; Roozen, Paul P C; Brugman, Martijn H; de Haas, Edwin F E; Ghazvini, Mehrnaz; Fibbe, Willem E; van Dongen, Jacques J M; Fodde, Riccardo; Staal, Frank J T

2011-10-04

Canonical Wnt signaling has been implicated in the regulation of hematopoiesis. By employing a Wnt-reporter mouse, we observed that Wnt signaling is differentially activated during hematopoiesis, suggesting an important regulatory role for specific Wnt signaling levels. To investigate whether canonical Wnt signaling regulates hematopoiesis in a dosage-dependent fashion, we analyzed the effect of different mutations in the Adenomatous polyposis coli gene (Apc), a negative modulator of the canonical Wnt pathway. By combining different targeted hypomorphic alleles and a conditional deletion allele of Apc, a gradient of five different Wnt signaling levels was obtained in vivo. We here show that different, lineage-specific Wnt dosages regulate hematopoietic stem cells (HSCs), myeloid precursors, and T lymphoid precursors during hematopoiesis. Differential, lineage-specific optimal Wnt dosages provide a unifying concept that explains the differences reported among inducible gain-of-function approaches, leading to either HSC expansion or depletion of the HSC pool. Copyright © 2011 Elsevier Inc. All rights reserved.

Wnt3a regulates proliferation and migration of HUVEC via canonical and non-canonical Wnt signaling pathways

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

Samarzija, Ivana; Sini, Patrizia; Schlange, Thomas

2009-08-28

Untangling the signaling pathways involved in endothelial cell biology is of central interest for the development of antiangiogenesis based therapies. Here we report that Wnt3a induces the proliferation and migration of HUVECs, but does not affect their survival. Wnt3a-induced proliferation was VEGFR signaling independent, but reduced upon CamKII inhibition. In a search for the downstream mediators of Wnt3a's effects on HUVEC biology, we found that Wnt3a treatment leads to phosphorylation of DVL3 and stabilization of {beta}-catenin. Moreover, under the same conditions we observed an upregulation in c-MYC, TIE-2 and GJA1 mRNA transcripts. Although treatment of HUVECs with Wnt5a induced DVL3more » phosphorylation, we did not observe any of the other effects seen upon Wnt3a stimulation. Taken together, our data indicate that Wnt3a induces canonical and non-canonical Wnt signaling in HUVECs, and stimulates their proliferation and migration.« less

Phosphorylation of PPP(S/T)P motif of the free LRP6 intracellular domain is not required to activate the Wnt/beta-catenin pathway and attenuate GSK3beta activity.

PubMed

Beagle, Brandon; Mi, Kaihong; Johnson, Gail V W

2009-11-01

The canonical Wnt/beta-catenin signaling pathway plays a critical role in numerous physiological and pathological processes. LRP6 is an essential co-receptor for Wnt/beta-catenin signaling; as transduction of the Wnt signal is strongly dependent upon GSK3beta-mediated phosphorylation of multiple PPP(S/T)P motifs within the membrane-anchored LRP6 intracellular domain. Previously, we showed that the free LRP6 intracellular domain (LRP6-ICD) can activate the Wnt/beta-catenin pathway in a beta-catenin and TCF/LEF-1 dependent manner, as well as interact with and attenuate GSK3beta activity. However, it is unknown if the ability of LRP6-ICD to attenuate GSK3beta activity and modulate activation of the Wnt/beta-catenin pathway requires phosphorylation of the LRP6-ICD PPP(S/T)P motifs, in a manner similar to the membrane-anchored LRP6 intracellular domain. Here we provide evidence that the LRP6-ICD does not have to be phosphorylated at its PPP(S/T)P motif by GSK3beta to stabilize endogenous cytosolic beta-catenin resulting in activation of TCF/LEF-1 and the Wnt/beta-catenin pathway. LRP6-ICD and a mutant in which all 5 PPP(S/T)P motifs were changed to PPP(A)P motifs equivalently interacted with and attenuated GSK3beta activity in vitro, and both constructs inhibited the in situ GSK3beta-mediated phosphorylation of beta-catenin and tau to the same extent. These data indicate that the LRP6-ICD attenuates GSK3beta activity similar to other GSK3beta binding proteins, and is not a result of it being a GSK3beta substrate. Our findings suggest the functional and regulatory mechanisms governing the free LRP6-ICD may be distinct from membrane-anchored LRP6, and that release of the LRP6-ICD may provide a complimentary signaling cascade capable of modulating Wnt-dependent gene expression. (c) 2009 Wiley-Liss, Inc.

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

PubMed Central

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

2013-01-01

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

Wnt5a Signaling Promotes Host Defense against Leishmania donovani Infection.

PubMed

Chakraborty, Arijit; Kurati, Sony Priya; Mahata, Sushil K; Sundar, Shyam; Roy, Syamal; Sen, Malini

2017-08-01

Leishmania donovani infects macrophages, disrupting immune homeostasis. The underlying mechanism that sustains infection remains unresolved. In view of the potential of Wnt5a signaling to support immune homeostasis, we evaluated the interrelationship of Wnt5a signaling and Leishmania donovani infection. Upon infecting macrophages separately with antimony drug-sensitive and -resistant L. donovani , we noted disruption in the steady-state level of Wnt5a. Moreover, inhibition of Wnt5a signaling by small interfering RNA transfection in vitro or by use of inhibitor of Wnt production in vivo led to an increase in cellular parasite load. In contrast, treatment of macrophages with recombinant Wnt5a caused a decrease in the load of antimony-sensitive and -resistant parasites, thus confirming that Wnt5a signaling antagonizes L. donovani infection. Using inhibitors of the Wnt5a signaling intermediates Rac1 and Rho kinase, we demonstrated that Wnt5a-mediated inhibition of parasite infection in macrophages is Rac1/Rho dependent. Furthermore, phalloidin staining and reactive oxygen species estimation of Wnt5a-treated macrophages suggested that a Wnt5a-Rac/Rho-mediated decrease in parasite load is associated with an increase in F- actin assembly and NADPH oxidase activity. Moreover, live microscopy of L. donovani -infected macrophages treated with Wnt5a demonstrated increased endosomal/lysosomal fusions with parasite-containing vacuoles (parasitophorous vacuoles [PV]). An increase in PV-endosomal/lysosomal fusion accompanied by augmented PV degradation in Wnt5a-treated macrophages was also apparent from transmission electron microscopy of infected cells. Our results suggest that, although L. donovani evades host immune response, at least in part through inhibition of Wnt5a signaling, revamping Wnt5a signaling can inhibit L. donovani infection, irrespective of drug sensitivity or resistance. Copyright © 2017 by The American Association of Immunologists, Inc.

Wnt5a suppresses inflammation-driven intervertebral disc degeneration via a TNF-α/NF-κB-Wnt5a negative-feedback loop.

PubMed

Li, Z; Zhang, K; Li, X; Pan, H; Li, S; Chen, F; Zhang, J; Zheng, Z; Wang, J; Liu, H

2018-04-12

This study was to investigate the molecular role of Wnt5a on inflammation-driven intervertebral disc degeneration (IVDD). The expression of Wnt5a was analyzed in human nucleus pulposus (NP) tissues with immunohistochemical staining. The effects of Wnt5a on matrix production were assessed by RT-qPCR and western blotting. Small interfering RNAs (siRNAs), promoter deletion assay, and promoter binding site mutant were used to reveal the molecular role of Wnt5a in TNF-α-induced matrix metalloproteinase (MMP) expression. The regulatory effects of TNF-α on Wnt5a were investigated with pharmachemical inhibitors and siRNA experiment. The expression of Wnt5a was elevated in moderately degenerated human NP tissue with similar expression pattern of TNF-α. In NP cells, Wnt5a significantly increased aggrecan and collagen II expression. Inhibition of JNK or interfering Sox9 gene expression significantly suppressed Wnt5a-induced matrix production. AP-1(JunB) binding sites were located in Sox9 promoter and mutation of these sites sabotaged Wnt5a-induced Sox9 up-regulation and subsequent matrix genes expression. Notably, Wnt5a, which was induced by TNF-α, on the other way round suppressed TNF-α-NF-κB (p65) signaling and subsequent MMPs expression. In vivo studies with MR imaging confirmed the protective role of Wnt5a in IVDD. Wnt5a, which can be induced by TNF-α, increased matrix production in a Sox9-dependent manner through the activation of JNK-AP1 (JunB) signaling, and antagonized TNF-α-induced up-regulation of MMPs through the inhibition of NF-κB signaling. It indicates that Wnt5a suppresses IVDD through a TNF-α/NF-κB-Wnt5a negative-feedback loop. Copyright © 2018 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

miRNA-431 Prevents Amyloid-β-Induced Synapse Loss in Neuronal Cell Culture Model of Alzheimer's Disease by Silencing Kremen1.

PubMed

Ross, Sean P; Baker, Kelly E; Fisher, Amanda; Hoff, Lee; Pak, Elena S; Murashov, Alexander K

2018-01-01

Synapse loss is well regarded as the underlying cause for the progressive decline of memory function over the course of Alzheimer's disease (AD) development. Recent observations suggest that the accumulation of the Wnt antagonist Dickkopf-1 (Dkk1) in the AD brain plays a critical role in triggering synaptic degeneration. Mechanistically, Dkk1 cooperates with Kremen1 (Krm1), its transmembrane receptor, to block the Wnt/β-catenin signaling pathway. Here, we show that silencing Krm1 with miR-431 prevents amyloid-β-mediated synapse loss in cortico-hippocampal cultures isolated from triple transgenic 3xTg-AD mice. Exposure to AβDDL (an amyloid-β derived diffusive ligand) or Dkk1 reduced the number of pre- and post-synaptic puncta in primary neuronal cultures, while treatment with miR-431 prevented synapse loss. In addition, treatment with miR-431 also prevented neurite degeneration. Our findings demonstrate that miR-431 protects synapses and neurites from Aβ-toxicity in an AD cell culture model and may be a promising therapeutic target.

Activation of Hedgehog signaling by loss of GNAS causes heterotopic ossification

PubMed Central

Regard, Jean B.; Malhotra, Deepti; Gvozdenovic-Jeremic, Jelena; Josey, Michelle; Chen, Min; Weinstein, Lee S.; Lu, Jianming; Shore, Eileen M.; Kaplan, Frederick S.; Yang, Yingzi

2014-01-01

Bone formation is exquisitely controlled in space and time. Heterotopic ossification (HO), the pathologic formation of extra-skeletal bone, occurs as a common complication of trauma or in genetic disorders and can be disabling and lethal. However, the underlying molecular mechanisms are largely unknown. Here we demonstrate that Gαs restricts bone formation to the skeleton by inhibiting Hedgehog (Hh) signaling in mesenchymal progenitor cells. In progressive osseous heteroplasia (POH), a human disease caused by null mutations in GNAS that encodes Gαs, HH signaling is upregulated in ectopic osteoblasts and progenitor cells. Ectopic Hh signaling is sufficient to induce HO, while Hh signaling inhibition blocks HO in animal models. As our previous work has shown that GNAS gain of function mutations upregulate WNT/β-Catenin signaling in fibrous dysplasia (FD), our findings identify Gαs as a critical regulator of osteoblast differentiation by maintaining a balance between two key signaling pathways: Wnt/β-catenin and Hh. HH signaling inhibitors developed for cancer therapy may be repurposed to treat HO and other diseases caused by GNAS inactivation. PMID:24076664

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

PubMed

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

2016-11-09

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

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

Shen, Longxiang; Glowacki, Julie; Zhou, Shuanhu, E-mail: szhou@rics.bwh.harvard.edu

The WNT signaling pathway plays important roles in the self-renewal and differentiation of mesenchymal stem cells (MSCs). Little is known about WNT signaling in adipocyte differentiation of human MSCs. In this study, we tested the hypothesis that canonical and non-canonical WNTs differentially regulate in vitro adipocytogenesis in human MSCs. The expression of adipocyte gene PPAR{gamma}2, lipoprotein lipase, and adipsin increased during adipocytogenesis of hMSCs. Simultaneously, the expression of canonical WNT2, 10B, 13, and 14 decreased, whereas non-canonical WNT4 and 11 increased, and WNT5A was unchanged. A small molecule WNT mimetic, SB-216763, increased accumulation of {beta}-catenin protein, inhibited induction of WNT4more » and 11 and inhibited adipocytogenesis. In contrast, knockdown of {beta}-catenin with siRNA resulted in spontaneous adipocytogenesis. These findings support the view that canonical WNT signaling inhibits and non-canonical WNT signaling promotes adipocytogenesis in adult human marrow-derived mesenchymal stem cells.« less

WNT5A inhibits human dental papilla cell proliferation and migration

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

Peng, L.; State Key Laboratory of Oral Diseases, Sichuan University, Chengdu, Sichuan; Ye, L.

WNT proteins are a large family of cysteine-rich secreted molecules that are linked to both canonical and non-canonical signal pathways, and have been implicated in oncogenesis and tissue development. Canonical WNT proteins have been proven to play critical roles in tooth development, while little is known about the role of non-canonical WNT proteins such as WNT5A. In this study, WNT5A was localized to human dental papilla tissue and human dental papilla cells (HDPCs) cultured in vitro, using immunochemistry and RT-PCR. Recombinant adenovirus encoding full-length Wnt5a cDNA was constructed to investigate the biological role of WNT5A on HDPCs. The BrdU incorporationmore » assay, the MTT assay and flow cytometric analysis showed that over-expression of Wnt5a strongly inhibited the proliferation of HDPCs in vitro. Wound healing and transwell migration assays indicated that over-expression of WNT5A reduced migration of HDPCs. In conclusion, our results showed that WNT5A negatively regulates both proliferation and migration of HDPCs, suggesting its important role in odontogenesis via controlling the HDPCs.« less

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

Ramos-Solano, Moisés, E-mail: mrsolano84@gmail.com; Programa de Doctorado en Ciencias Biomédica, Centro Universitario de Ciencias de la Salud; Meza-Canales, Ivan D., E-mail: imezacanales@ice.mpg.de

According to the multifactorial model of cervical cancer (CC) causation, it is now recognized that other modifications, in addition to Human papillomavirus (HPV) infection, are necessary for the development of this neoplasia. Among these, it has been proposed that a dysregulation of the WNT pathway might favor malignant progression of HPV-immortalized keratinocytes. The aim of this study was to identify components of the WNT pathway differentially expressed in CC vs. non-tumorigenic, but immortalized human keratinocytes. Interestingly, WNT7A expression was found strongly downregulated in cell lines and biopsies derived from CC. Restoration of WNT7A in CC-derived cell lines using a lentiviralmore » gene delivery system or after adding a recombinant human protein decreases cell proliferation. Likewise, WNT7A silencing in non-tumorigenic cells markedly accelerates proliferation. Decreased WNT7A expression was due to hypermethylation at particular CpG sites. To our knowledge, this is the first study reporting reduced WNT7A levels in CC-derived cells and that ectopic WNT7A restoration negatively affects cell proliferation and migration. - Highlights: • WNT7A is expressed in normal keratinocytes or cervical cells without lesion. • WNT7A is significantly reduced in cervical cancer-derived cells. • Restoration of WNT7A expression in HeLa decreases proliferation and cell migration. • Silencing of WNT7A in HaCaT induces an increased proliferation and migration rate. • Decreased WNT7A expression in this model is due to hypermethylation.« less

Expression of WNT5A in Idiopathic Pulmonary Fibrosis and Its Control by TGF-β and WNT7B in Human Lung Fibroblasts.

PubMed

Newman, Donna R; Sills, W Shane; Hanrahan, Katherine; Ziegler, Amanda; Tidd, Kathleen McGinnis; Cook, Elizabeth; Sannes, Philip L

2016-02-01

The wingless (Wnt) family of signaling ligands contributes significantly to lung development and is highly expressed in patients with usual interstitial pneumonia (UIP). We sought to define the cellular distribution of Wnt5A in the lung tissue of patients with idiopathic pulmonary fibrosis (IPF) and the signaling ligands that control its expression in human lung fibroblasts and IPF myofibroblasts. Tissue sections from 40 patients diagnosed with IPF or UIP were probed for the immunolocalization of Wnt5A. Further, isolated lung fibroblasts from normal or IPF human lungs, adenovirally transduced for the overexpression or silencing of Wnt7B or treated with TGF-β1 or its inhibitor, were analyzed for Wnt5A protein expression. Wnt5A was expressed in IPF lungs by airway and alveolar epithelium, smooth muscle cells, endothelium, and myofibroblasts of fibroblastic foci and throughout the interstitium. Forced overexpression of Wnt7B with or without TGF-β1 treatment significantly increased Wnt5A protein expression in normal human smooth muscle cells and fibroblasts but not in IPF myofibroblasts where Wnt5A was already highly expressed. The results demonstrate a wide distribution of Wnt5A expression in cells of the IPF lung and reveal that it is significantly increased by Wnt7B and TGF-β1, which, in combination, could represent key signaling pathways that modulate the pathogenesis of IPF. © 2016 The Histochemical Society.

A Novel Strategy to Increase the Proliferative Potential of Adult Human β-Cells While Maintaining Their Differentiated Phenotype

PubMed Central

Aly, Haytham; Rohatgi, Nidhi; Marshall, Connie A.; Grossenheider, Tiffani C.; Miyoshi, Hiroyuki; Stappenbeck, Thaddeus S.; Matkovich, Scot J.; McDaniel, Michael L.

2013-01-01

Our previous studies demonstrated that Wnt/GSK-3/β-catenin and mTOR signaling are necessary to stimulate proliferative processes in adult human β-cells. Direct inhibition of GSK-3, that engages Wnt signaling downstream of the Wnt receptor, increases β-catenin nuclear translocation and β-cell proliferation but results in lower insulin content. Our current goal was to engage canonical and non-canonical Wnt signaling at the receptor level to significantly increase human β-cell proliferation while maintaining a β-cell phenotype in intact islets. We adopted a system that utilized conditioned medium from L cells that expressed Wnt3a, R-spondin-3 and Noggin (L-WRN conditioned medium). In addition we used a ROCK inhibitor (Y-27632) and SB-431542 (that results in RhoA inhibition) in these cultures. Treatment of intact human islets with L-WRN conditioned medium plus inhibitors significantly increased DNA synthesis ∼6 fold in a rapamycin-sensitive manner. Moreover, this treatment strikingly increased human β-cell proliferation ∼20 fold above glucose alone. Only the combination of L-WRN conditioned medium with RhoA/ROCK inhibitors resulted in substantial proliferation. Transcriptome-wide gene expression profiling demonstrated that L-WRN medium provoked robust changes in several signaling families, including enhanced β-catenin-mediated and β-cell-specific gene expression. This treatment also increased expression of Nr4a2 and Irs2 and resulted in phosphorylation of Akt. Importantly, glucose-stimulated insulin secretion and content were not downregulated by L-WRN medium treatment. Our data demonstrate that engaging Wnt signaling at the receptor level by this method leads to necessary crosstalk between multiple signaling pathways including activation of Akt, mTOR, Wnt/β-catenin, PKA/CREB, and inhibition of RhoA/ROCK that substantially increase human β-cell proliferation while maintaining the β-cell phenotype. PMID:23776620

A novel strategy to increase the proliferative potential of adult human β-cells while maintaining their differentiated phenotype.

PubMed

Aly, Haytham; Rohatgi, Nidhi; Marshall, Connie A; Grossenheider, Tiffani C; Miyoshi, Hiroyuki; Stappenbeck, Thaddeus S; Matkovich, Scot J; McDaniel, Michael L

2013-01-01

Our previous studies demonstrated that Wnt/GSK-3/β-catenin and mTOR signaling are necessary to stimulate proliferative processes in adult human β-cells. Direct inhibition of GSK-3, that engages Wnt signaling downstream of the Wnt receptor, increases β-catenin nuclear translocation and β-cell proliferation but results in lower insulin content. Our current goal was to engage canonical and non-canonical Wnt signaling at the receptor level to significantly increase human β-cell proliferation while maintaining a β-cell phenotype in intact islets. We adopted a system that utilized conditioned medium from L cells that expressed Wnt3a, R-spondin-3 and Noggin (L-WRN conditioned medium). In addition we used a ROCK inhibitor (Y-27632) and SB-431542 (that results in RhoA inhibition) in these cultures. Treatment of intact human islets with L-WRN conditioned medium plus inhibitors significantly increased DNA synthesis ∼6 fold in a rapamycin-sensitive manner. Moreover, this treatment strikingly increased human β-cell proliferation ∼20 fold above glucose alone. Only the combination of L-WRN conditioned medium with RhoA/ROCK inhibitors resulted in substantial proliferation. Transcriptome-wide gene expression profiling demonstrated that L-WRN medium provoked robust changes in several signaling families, including enhanced β-catenin-mediated and β-cell-specific gene expression. This treatment also increased expression of Nr4a2 and Irs2 and resulted in phosphorylation of Akt. Importantly, glucose-stimulated insulin secretion and content were not downregulated by L-WRN medium treatment. Our data demonstrate that engaging Wnt signaling at the receptor level by this method leads to necessary crosstalk between multiple signaling pathways including activation of Akt, mTOR, Wnt/β-catenin, PKA/CREB, and inhibition of RhoA/ROCK that substantially increase human β-cell proliferation while maintaining the β-cell phenotype.

Anteroposterior Patterning of Gene Expression in the Human Infant Sclera: Chondrogenic Potential and Wnt Signaling.

PubMed

Seko, Yuko; Azuma, Noriyuki; Yokoi, Tadashi; Kami, Daisuke; Ishii, Ryuga; Nishina, Sachiko; Toyoda, Masashi; Shimokawa, Hitoyata; Umezawa, Akihiro

2017-01-01

Purpose/Aim: We sought to identify the anteroposterior spatial gene expression hierarchy in the human sclera to develop a hypothesis for axial elongation and deformity of the eyeball. We analyzed the global gene expression of human scleral cells derived from distinct parts of the human infant sclera obtained from surgically enucleated eyes with retinoblastoma, using Affymetrix GeneChip oligonucleotide arrays, and compared, in particular, gene expression levels between the anterior and posterior parts of the sclera. The ages of three donors were 10M, 4M, and 1Y9M. K-means clustering analysis of gene expression revealed that expression levels of cartilage-associated genes such as COLXIA and ACAN increased from the anterior to the posterior part of the sclera. Microarray analyses and RT-PCR data showed that the expression levels of MGP, COLXIA, BMP4, and RARB were significantly higher in the posterior than in the anterior sclera of two independent infant eyes. Conversely, expression levels of WNT2, DKK2, GREM1, and HOXB2 were significantly higher in the anterior sclera. Among several Wnt-family genes examined, WNT2B was found to be expressed at a significantly higher level in the posterior sclera, and the reverse order was observed for WNT2. The results of luciferase reporter assays suggested that a GSK-3β inhibitor stimulated Wnt/β-catenin signaling particularly strongly in the posterior sclera. The expression pattern of RARB, a myopia-related gene, was similar in three independent eyes. Chondrogenic potential was higher and Wnt/β-catenin signaling was more potently activated by a GSK-3β inhibitor in the posterior than in the anterior part of the human infant sclera. Although the differences in the gene expression profiles between the anterior and posterior sclera might be involved only in normal growth processes, this anteroposterior hierarchy in the sclera might contribute to disorders involving abnormal elongation and deformity of the eyeball, including myopia.

Genetic disruption of tubulin acetyltransferase, αTAT1, inhibits proliferation and invasion of colon cancer cells through decreases in Wnt1/β-catenin signaling

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

Oh, Somi; You, Eunae; Ko, Panseon

Microtubules are required for diverse cellular processes, and abnormal regulation of microtubule dynamics is closely associated with severe diseases including malignant tumors. In this study, we report that α-tubulin N-acetyltransferase (αTAT1), a regulator of α-tubulin acetylation, is required for colon cancer proliferation and invasion via regulation of Wnt1 and its downstream genes expression. Public transcriptome analysis showed that expression of ATAT1 is specifically upregulated in colon cancer tissue. A knockout (KO) of ATAT1 in the HCT116 colon cancer cell line, using the CRISPR/Cas9 system showed profound inhibition of proliferative and invasive activities of these cancer cells. Overexpression of αTAT1 ormore » the acetyl-mimic K40Q α-tubulin mutant in αTAT1 KO cells restored the invasiveness, indicating that microtubule acetylation induced by αTAT1 is critical for HCT116 cell invasion. Analysis of colon cancer-related gene expression in αTAT1 KO cells revealed that the loss of αTAT1 decreased the expression of WNT1. Mechanistically, abrogation of tubulin acetylation by αTAT1 knockout inhibited localization of β-catenin to the plasma membrane and nucleus, thereby resulting in the downregulation of Wnt1 and of its downstream genes including CCND1, MMP-2, and MMP-9. These results suggest that αTAT1-mediated Wnt1 expression via microtubule acetylation is important for colon cancer progression. - Highlights: • Ablation of αTAT1 inhibits HCT116 colon cancer cell invasion. • αTAT1/acetylated microtubules regulate expression of Wnt1/β-catenin target genes. • Acetylated microtubules regulate cellular localization of β-catenin. • Loss of αTAT1 prevents Wnt1 from inducing β-catenin-dependent and -independent pathways.« less

WNTLESS IS REQUIRED FOR PERIPHERAL LUNG DIFFERENTIATION AND PULMONARY VASCULAR DEVELOPMENT

PubMed Central

Cornett, Bridget; Snowball, John; Varisco, Brian M.; Lang, Richard; Whitsett, Jeffrey; Sinner, Debora

2013-01-01

Wntless (Wls), a gene highly conserved across the animal kingdom, encodes for a transmembrane protein that mediates Wnt ligand secretion. Wls is expressed in developing lung, wherein Wnt signaling is necessary for pulmonary morphogenesis. We hypothesize that Wls plays a critical role in modulating Wnt signaling during lung development and therefore affects processes critical for pulmonary morphogenesis. We generated conditional Wls mutant mice utilizing Shh-Cre and Dermo1-Cre mice to delete Wls in the embryonic respiratory epithelium and mesenchyme, respectively. Epithelial deletion of Wls disrupted lung branching morphogenesis, peripheral lung development and pulmonary endothelial differentiation. Epithelial Wls mutant mice died at birth due to respiratory failure caused by lung hypoplasia and pulmonary hemorrhage. In the lungs of these mice, VEGF and Tie2-angiopoietin signaling pathways, which mediate vascular development, were downregulated from early stages of development. In contrast, deletion of Wls in mesenchymal cells of the developing lung did not alter branching morphogenesis or early mesenchymal differentiation. In vitro assays support the concept that Wls acts in part via Wnt5a to regulate pulmonary vascular development. We conclude that epithelial Wls modulates Wnt ligand activities critical for pulmonary vascular differentiation and peripheral lung morphogenesis. These studies provide a new framework for understanding the molecular mechanisms underlying normal pulmonary vasculature formation and the dysmorphic pulmonary vasculature development associated with congenital lung disease. PMID:23523683

Wntless is required for peripheral lung differentiation and pulmonary vascular development.

PubMed

Cornett, Bridget; Snowball, John; Varisco, Brian M; Lang, Richard; Whitsett, Jeffrey; Sinner, Debora

2013-07-01

Wntless (Wls), a gene highly conserved across the animal kingdom, encodes for a transmembrane protein that mediates Wnt ligand secretion. Wls is expressed in developing lung, wherein Wnt signaling is necessary for pulmonary morphogenesis. We hypothesize that Wls plays a critical role in modulating Wnt signaling during lung development and therefore affects processes critical for pulmonary morphogenesis. We generated conditional Wls mutant mice utilizing Shh-Cre and Dermo1-Cre mice to delete Wls in the embryonic respiratory epithelium and mesenchyme, respectively. Epithelial deletion of Wls disrupted lung branching morphogenesis, peripheral lung development and pulmonary endothelial differentiation. Epithelial Wls mutant mice died at birth due to respiratory failure caused by lung hypoplasia and pulmonary hemorrhage. In the lungs of these mice, VEGF and Tie2-angiopoietin signaling pathways, which mediate vascular development, were downregulated from early stages of development. In contrast, deletion of Wls in mesenchymal cells of the developing lung did not alter branching morphogenesis or early mesenchymal differentiation. In vitro assays support the concept that Wls acts in part via Wnt5a to regulate pulmonary vascular development. We conclude that epithelial Wls modulates Wnt ligand activities critical for pulmonary vascular differentiation and peripheral lung morphogenesis. These studies provide a new framework for understanding the molecular mechanisms underlying normal pulmonary vasculature formation and the dysmorphic pulmonary vasculature development associated with congenital lung disease. Copyright © 2013 Elsevier Inc. All rights reserved.

Disruption of estrogen receptor signaling enhances intestinal neoplasia in Apc(Min/+) mice.

PubMed

Cleveland, Alicia G; Oikarinen, Seija I; Bynoté, Kimberly K; Marttinen, Maija; Rafter, Joseph J; Gustafsson, Jan-Ake; Roy, Shyamal K; Pitot, Henry C; Korach, Kenneth S; Lubahn, Dennis B; Mutanen, Marja; Gould, Karen A

2009-09-01

Estrogen receptors (ERs) [ERalpha (Esr1) and ERbeta (Esr2)] are expressed in the human colon, but during the multistep process of colorectal carcinogenesis, expression of both ERalpha and ERbeta is lost, suggesting that loss of ER function might promote colorectal carcinogenesis. Through crosses between an ERalpha knockout and Apc(Min) mouse strains, we demonstrate that ERalpha deficiency is associated with a significant increase in intestinal tumor multiplicity, size and burden in Apc(Min/+) mice. Within the normal intestinal epithelium of Apc(Min/+) mice, ERalpha deficiency is associated with an accumulation of nuclear beta-catenin, an indicator of activation of the Wnt-beta-catenin-signaling pathway, which is known to play a critical role in intestinal cancers. Consistent with the hypothesis that ERalpha deficiency is associated with activation of Wnt-beta-catenin signaling, ERalpha deficiency in the intestinal epithelium of Apc(Min/+) mice also correlated with increased expression of Wnt-beta-catenin target genes. Through crosses between an ERbeta knockout and Apc(Min) mouse strains, we observed some evidence that ERbeta deficiency is associated with an increased incidence of colon tumors in Apc(Min/+) mice. This effect of ERbeta deficiency does not involve modulation of Wnt-beta-catenin signaling. Our studies suggest that ERalpha and ERbeta signaling modulate colorectal carcinogenesis, and ERalpha does so, at least in part, by regulating the activity of the Wnt-beta-catenin pathway.

Alteration of Wnt5a expression and of the non-canonical Wnt/PCP and Wnt/PKC-Ca2+ pathways in human osteoarthritis osteoblasts

PubMed Central

Martineau, Xavier; Abed, Élie; Martel-Pelletier, Johanne; Pelletier, Jean-Pierre; Lajeunesse, Daniel

2017-01-01

Objective Clinical and in vitro studies suggest that subchondral bone sclerosis due to abnormal osteoblasts (Ob) is involved in the progression and/or onset of osteoarthritis (OA). Human Ob isolated from sclerotic subchondral OA bone tissue show an altered phenotype, a decreased canonical Wnt/β-catenin signaling pathway (cWnt), and a reduced mineralization in vitro. In addition to the cWnt pathway, at least two non-canonical signaling pathways, the Wnt/PKC and Wnt/PCP pathway have been described. However, there are no reports of either pathway in OA Ob. Here, we studied the two non-canonical pathways in OA Ob and if they influence their phenotype. Methods Human primary subchondral Ob were isolated from the subchondral bone plate of tibial plateaus of OA patients undergoing total knee arthroplasty, or of normal individuals at autopsy. The expression of genes involved in non-canonical Wnt signaling was evaluated by qRT-PCR and their protein production by Western blot analysis. Alkaline phosphatase activity and osteocalcin secretion (OC) were determined with substrate hydrolysis and EIA, respectively. Mineralization levels were evaluated with Alizarin Red Staining, Wnt/PKC and Wnt/PCP pathways by target gene expression and their respective activity using the NFAT and AP-1 luciferase reporter assays. Results OA Ob showed an altered phenotype as illustrated by an increased alkaline phosphatase activity and osteocalcin release compared to normal Ob. The expression of the non-canonical Wnt5a ligand was increased in OA Ob compared to normal. Whereas, the expression of LGR5 was significantly increased in OA Ob compared to normal Ob, the expression of LGR4 was similar. Wnt5a directly stimulated the expression and production of LGR5, contrasting, Wnt5a did not stimulate the expression of LGR4. Wnt5a also stimulated the phosphorylation of both JNK and PKC, as well as the activity of both NFAT and AP-1 transcription factors. The inhibition of Wnt5a expression partially corrects the abnormal mineralization, OC secretion and ALPase activity of OA Ob. Conclusion These data indicate that the alteration of Wnt5a, a non-canonical Wnt signaling activator, is implicated in the modified signalisation and phenotype observed in OA Ob. PMID:28777797

Diphtheria Toxin-Induced Cell Death Triggers Wnt-Dependent Hair Cell Regeneration in Neonatal Mice.

PubMed

Hu, Lingxiang; Lu, Jingrong; Chiang, Hao; Wu, Hao; Edge, Albert S B; Shi, Fuxin

2016-09-07

Cochlear hair cells (HCs), the sensory cells that respond to sound, do not regenerate after damage in adult mammals, and their loss is a major cause of deafness. Here we show that HC regeneration in newborn mouse ears occurred spontaneously when the original cells were ablated by treatment with diphtheria toxin (DT) in ears that had been engineered to overexpress the DT receptor, but was not detectable when HCs were ablated in vivo by the aminoglycoside antibiotic neomycin. A variety of Wnts (Wnt1, Wnt2, Wnt2b, Wnt4, Wnt5a, Wnt7b, Wnt9a, Wnt9b, and Wnt11) and Wnt pathway component Krm2 were upregulated after DT damage. Nuclear β-catenin was upregulated in HCs and supporting cells of the DT-damaged cochlea. Pharmacological inhibition of Wnt decreased spontaneous regeneration, confirming a role of Wnt signaling in HC regeneration. Inhibition of Notch signaling further potentiated supporting cell proliferation and HC differentiation that occurred spontaneously. The absence of new HCs in the neomycin ears was correlated to less robust Wnt pathway activation, but the ears subjected to neomycin treatment nonetheless showed increased cell division and HC differentiation after subsequent forced upregulation of β-catenin. These studies suggest, first, that Wnt signaling plays a key role in regeneration, and, second, that the outcome of a regenerative response to damage in the newborn cochlea is determined by reaching a threshold level of Wnt signaling rather than its complete absence or presence. Sensory HCs of the inner ear do not regenerate in the adult, and their loss is a major cause of deafness. We found that HCs regenerated spontaneously in the newborn mouse after diphtheria toxin (DT)-induced, but not neomycin-induced, HC death. Regeneration depended on activation of Wnt signaling, and regeneration in DT-treated ears correlated to a higher level of Wnt activation than occurred in nonregenerating neomycin-treated ears. This is significant because insufficient regeneration caused by a failure to reach a threshold level of signaling, if true in the adult, has the potential to be exploited for development of clinical approaches for the treatment of deafness caused by HC loss. Copyright © 2016 the authors 0270-6474/16/369479-11$15.00/0.

Diphtheria Toxin-Induced Cell Death Triggers Wnt-Dependent Hair Cell Regeneration in Neonatal Mice

PubMed Central

Hu, Lingxiang; Lu, Jingrong; Chiang, Hao; Shi, Fuxin

2016-01-01

Cochlear hair cells (HCs), the sensory cells that respond to sound, do not regenerate after damage in adult mammals, and their loss is a major cause of deafness. Here we show that HC regeneration in newborn mouse ears occurred spontaneously when the original cells were ablated by treatment with diphtheria toxin (DT) in ears that had been engineered to overexpress the DT receptor, but was not detectable when HCs were ablated in vivo by the aminoglycoside antibiotic neomycin. A variety of Wnts (Wnt1, Wnt2, Wnt2b, Wnt4, Wnt5a, Wnt7b, Wnt9a, Wnt9b, and Wnt11) and Wnt pathway component Krm2 were upregulated after DT damage. Nuclear β-catenin was upregulated in HCs and supporting cells of the DT-damaged cochlea. Pharmacological inhibition of Wnt decreased spontaneous regeneration, confirming a role of Wnt signaling in HC regeneration. Inhibition of Notch signaling further potentiated supporting cell proliferation and HC differentiation that occurred spontaneously. The absence of new HCs in the neomycin ears was correlated to less robust Wnt pathway activation, but the ears subjected to neomycin treatment nonetheless showed increased cell division and HC differentiation after subsequent forced upregulation of β-catenin. These studies suggest, first, that Wnt signaling plays a key role in regeneration, and, second, that the outcome of a regenerative response to damage in the newborn cochlea is determined by reaching a threshold level of Wnt signaling rather than its complete absence or presence. SIGNIFICANCE STATEMENT Sensory HCs of the inner ear do not regenerate in the adult, and their loss is a major cause of deafness. We found that HCs regenerated spontaneously in the newborn mouse after diphtheria toxin (DT)-induced, but not neomycin-induced, HC death. Regeneration depended on activation of Wnt signaling, and regeneration in DT-treated ears correlated to a higher level of Wnt activation than occurred in nonregenerating neomycin-treated ears. This is significant because insufficient regeneration caused by a failure to reach a threshold level of signaling, if true in the adult, has the potential to be exploited for development of clinical approaches for the treatment of deafness caused by HC loss. PMID:27605621

Noncanonical Wnt signaling promotes obesity-induced adipose tissue inflammation and metabolic dysfunction independent of adipose tissue expansion.

PubMed

Fuster, José J; Zuriaga, María A; Ngo, Doan Thi-Minh; Farb, Melissa G; Aprahamian, Tamar; Yamaguchi, Terry P; Gokce, Noyan; Walsh, Kenneth

2015-04-01

Adipose tissue dysfunction plays a pivotal role in the development of insulin resistance in obese individuals. Cell culture studies and gain-of-function mouse models suggest that canonical Wnt proteins modulate adipose tissue expansion. However, no genetic evidence supports a role for endogenous Wnt proteins in adipose tissue dysfunction, and the role of noncanonical Wnt signaling remains largely unexplored. Here we provide evidence from human, mouse, and cell culture studies showing that Wnt5a-mediated, noncanonical Wnt signaling contributes to obesity-associated metabolic dysfunction by increasing adipose tissue inflammation. Wnt5a expression is significantly upregulated in human visceral fat compared with subcutaneous fat in obese individuals. In obese mice, Wnt5a ablation ameliorates insulin resistance, in parallel with reductions in adipose tissue inflammation. Conversely, Wnt5a overexpression in myeloid cells augments adipose tissue inflammation and leads to greater impairments in glucose homeostasis. Wnt5a ablation or overexpression did not affect fat mass or adipocyte size. Mechanistically, Wnt5a promotes the expression of proinflammatory cytokines by macrophages in a Jun NH2-terminal kinase-dependent manner, leading to defective insulin signaling in adipocytes. Exogenous interleukin-6 administration restores insulin resistance in obese Wnt5a-deficient mice, suggesting a central role for this cytokine in Wnt5a-mediated metabolic dysfunction. Taken together, these results demonstrate that noncanonical Wnt signaling contributes to obesity-induced insulin resistance independent of adipose tissue expansion. © 2015 by the American Diabetes Association. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered.

Down-regulation of Wnt10a affects odontogenesis and proliferation in mesenchymal cells

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

Liu, Yang, E-mail: Ly10160624@163.com; Han, Dong, E-mail: Donghan@bjmu.edu.cn; Wang, Lei, E-mail: wanglei_dentist@163.com

Highlights: •Down-regulation of Wnt10a in dental mesenchymal cells impairs odontogenesis of reassociated tooth germs. •Dspp is down- and up-regulated after Wnt10a-knockdown and overexpression in dental mesenchymal cells. •Down-regulation of Wnt10a inhibits proliferation of dental mesenchymal cells. -- Abstract: The WNT10a mutation has been found in patients with abnormal odontogenesis. In mice, Wnt10a expression is found in the tooth germ, but its role has not yet been elucidated. We aimed to investigate the role of Wnt10a in odontogenesis. Mesenchymal cells of the first mandibular molar germ at the bell stage were isolated, transfected with Wnt10a SiRNA or plasmid, and reassociated withmore » epithelial part of the molar germ. Scrambled SiRNA or empty vector was used in the control group. The reassociated tooth germs were transplanted into mice subrenal capsules. After gene modification, dental mesenchymal cells cultured in vitro were checked for cell proliferation and the expression of Dspp was examined. All 12 reassociated tooth germs in the control group resumed odontogenesis, while only 5 of 12 in the Wnt10a knockdown group developed into teeth. After Wnt10a knockdown, the mesenchymal cells cultured in vitro presented repressed proliferation. Wnt10a knockdown and overexpression led to both down- and up-regulation of Dspp. We conclude that the down-regulation of Wnt10a impairs odontogensis and cell proliferation, and that Wnt10a regulates Dspp expression in mesenchymal cells. These findings help to elucidate the mechanism of abnormal tooth development in patients with the WNT10A mutation.« 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

Canonical Wnt signaling in megakaryocytes regulates proplatelet formation

PubMed Central

Macaulay, Iain C.; Thon, Jonathan N.; Tijssen, Marloes R.; Steele, Brian M.; MacDonald, Bryan T.; Meade, Gerardene; Burns, Philippa; Rendon, Augusto; Salunkhe, Vishal; Murphy, Ronan P.; Bennett, Cavan; Watkins, Nicholas A.; He, Xi; Fitzgerald, Desmond J.; Italiano, Joseph E.

2013-01-01

Wnt signaling is involved in numerous aspects of vertebrate development and homeostasis, including the formation and function of blood cells. Here, we show that canonical and noncanonical Wnt signaling pathways are present and functional in megakaryocytes (MKs), with several Wnt effectors displaying MK-restricted expression. Using the CHRF288-11 cell line as a model for human MKs, the canonical Wnt3a signal was found to induce a time and dose-dependent increase in β-catenin expression. β-catenin accumulation was inhibited by the canonical antagonist dickkopf-1 (DKK1) and by the noncanonical agonist Wnt5a. Whole genome expression analysis demonstrated that Wnt3a and Wnt5a regulated distinct patterns of gene expression in MKs, and revealed a further interplay between canonical and noncanonical Wnt pathways. Fetal liver cells derived from low-density-lipoprotein receptor-related protein 6-deficient mice (LRP6−/−), generated dramatically reduced numbers of MKs in culture of lower ploidy (2N and 4N) than wild-type controls, implicating LRP6-dependent Wnt signaling in MK proliferation and maturation. Finally, in wild-type mature murine fetal liver-derived MKs, Wnt3a potently induced proplatelet formation, an effect that could be completely abrogated by DKK1. These data identify novel extrinsic regulators of proplatelet formation, and reveal a profound role for Wnt signaling in platelet production. PMID:23160460

Activation of Wnt Planar Cell Polarity (PCP) signaling promotes growth plate column formation in vitro.

PubMed

Randall, Rachel M; Shao, Yvonne Y; Wang, Lai; Ballock, R Tracy

2012-12-01

Disrupting the Wnt Planar Cell Polarity (PCP) signaling pathway in vivo results in loss of columnar growth plate architecture, but it is unknown whether activation of this pathway in vitro is sufficient to promote column formation. We hypothesized that activation of the Wnt PCP pathway in growth plate chondrocyte cell pellets would promote columnar organization in these cells that are normally oriented randomly in culture. Rat growth plate chondrocytes were transfected with plasmids encoding the Fzd7 cell-surface Wnt receptor, a Fzd7 deletion mutant lacking the Wnt-binding domain, or Wnt receptor-associated proteins Ror2 or Vangl2, and then cultured as three-dimensional cell pellets in the presence of recombinant Wnt5a or Wnt5b for 21 days. Cellular morphology was evaluated using histomorphometric measurements. Activation of Wnt PCP signaling components promoted the initiation of columnar morphogenesis in the chondrocyte pellet culture model, as measured by histomorphometric analysis of the column index (ANOVA p = 0.01). Activation of noncanonical Wnt signaling through overexpression of both the cell-surface Wnt receptor Fzd7 and receptor-associated protein Ror2 with addition of recombinant Wnt5a promotes the initiation of columnar architecture of growth plate chondrocytes in vitro, representing an important step toward growth plate regeneration. Copyright © 2012 Orthopaedic Research Society.

Promotion of hair follicle development and trichogenesis by Wnt-10b in cultured embryonic skin and in reconstituted skin

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

Ouji, Yukiteru; Yoshikawa, Masahide; Shiroi, Akira

2006-06-30

We previously showed that Wnt-10b promoted the differentiation of primary skin epithelial cells (MPSEC) toward hair shaft and inner root sheath of the hair follicle (IRS) cells in vitro. In the present study, we found that Wnt-10b promotes the development of hair follicles using a culture of mouse embryonic skin tissue and trichogenesis using a reconstitution experiment with nude mice. Hair follicle development was observed in skin taken from mouse embryos on embryonic day 10.5 following a 2-day culture with recombinant Wnt-10b (rWnt-10b), however, not without rWnt-10b. Brown hair growth was observed at the site of reconstituted skin in Balb/cmore » nude mice where dermal fibroblasts and keratinocytes, derived from C3H/HeN new born mice, were transplanted with Wnt-10b-producing COS cells (Wnt-COS). Without the co-transplantation of Wnt-COS, no hair growth was observed. Our results suggest an important role of Wnt-10b in the initiation of hair follicle development and following trichogenesis.« less

Genetic Polymorphism in Extracellular Regulators of Wnt Signaling Pathway

PubMed Central

Sharma, Ashish Ranjan; Seo, Eun-Min; Nam, Ju-Suk

2015-01-01

The Wnt signaling pathway is mediated by a family of secreted glycoproteins through canonical and noncanonical mechanism. The signaling pathways are regulated by various modulators, which are classified into two classes on the basis of their interaction with either Wnt or its receptors. Secreted frizzled-related proteins (sFRPs) are the member of class that binds to Wnt protein and antagonizes Wnt signaling pathway. The other class consists of Dickkopf (DKK) proteins family that binds to Wnt receptor complex. The present review discusses the disease related association of various polymorphisms in Wnt signaling modulators. Furthermore, this review also highlights that some of the sFRPs and DKKs are unable to act as an antagonist for Wnt signaling pathway and thus their function needs to be explored more extensively. PMID:25945348

Wnt-10b, uniquely among Wnts, promotes epithelial differentiation and shaft growth

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

Ouji, Yukiteru; Yoshikawa, Masahide; Moriya, Kei

2008-03-07

Although Wnts are expressed in hair follicles throughout life from embryo to adult, and considered to be critical for their development and maturation, their roles remain largely unknown. In the present study, we investigated the effects of Wnts (Wnt-3a, Wnt-5a, Wnt-10b, and Wnt-11) on epithelial cell differentiation using adult mouse-derived primary skin epithelial cell (MPSEC) cultures and hair growth using hair follicle organ cultures. Only Wnt-10b showed evident promotion of epithelial cell differentiation and hair shaft growth, in contrast to Wnt-3a, 5a, and 11. Our results suggest that Wnt-10b is unique and plays an important role in differentiation of epithelialmore » cells in the hair follicle.« less

Direct visualization of the Wntless-induced redistribution of WNT1 in developing chick embryos.

PubMed

Galli, Lisa M; Santana, Frederick; Apollon, Chantilly; Szabo, Linda A; Ngo, Keri; Burrus, Laura W

2018-04-30

Paracrine Wnt signals are critical regulators of cell proliferation, specification, and differentiation during embryogenesis. Consistent with the discovery that Wnt ligands are post-translationally modified with palmitoleate (a 16 carbon mono-unsaturated fatty acid), our studies show that the vast majority of bioavailable chick WNT1 (cWNT1) produced in stably transfected L cells is cell-associated. Thus, it seems unlikely that the WNT1 signal is propagated by diffusion alone. Unfortunately, the production and transport of vertebrate Wnt proteins has been exceedingly difficult to study as few antibodies are able to detect endogenous Wnt proteins and fixation is known to disrupt the architecture of cells and tissues. Furthermore, vertebrate Wnts have been extraordinarily refractory to tagging. To help overcome these obstacles, we have generated a number of tools that permit the detection of WNT1 in palmitoylation assays and the visualization of chick and zebrafish WNT1 in live cells and tissues. Consistent with previous studies in fixed cells, live imaging of cells and tissues with overexpressed cWNT1-moxGFP shows predominant localization of the protein to a reticulated network that is likely to be the endoplasmic reticulum. As PORCN and WLS are important upstream regulators of Wnt gradient formation, we also undertook the generation of mCherry-tagged variants of both proteins. While co-expression of PORCN-mCherry had no discernible effect on the localization of WNT1-moxGFP, co-expression of WLS-mCherry caused a marked redistribution of WNT1-moxGFP to the cell surface and cellular projections in cultured cells as well as in neural crest and surface ectoderm cells in developing chick embryos. Our studies further establish that the levels of WLS, and not PORCN, are rate limiting with respect to WNT1 trafficking. Copyright © 2018. Published by Elsevier Inc.

Wnt6 regulates epithelial cell differentiation and is dysregulated in renal fibrosis.

PubMed

Beaton, Hayley; Andrews, Darrell; Parsons, Martin; Murphy, Mary; Gaffney, Andrew; Kavanagh, David; McKay, Gareth J; Maxwell, Alexander P; Taylor, Cormac T; Cummins, Eoin P; Godson, Catherine; Higgins, Debra F; Murphy, Paula; Crean, John

2016-07-01

Diabetic nephropathy is the most common microvascular complication of diabetes mellitus, manifesting as mesangial expansion, glomerular basement membrane thickening, glomerular sclerosis, and progressive tubulointerstitial fibrosis leading to end-stage renal disease. Here we describe the functional characterization of Wnt6, whose expression is progressively lost in diabetic nephropathy and animal models of acute tubular injury and renal fibrosis. We have shown prominent Wnt6 and frizzled 7 (FzD7) expression in the mesonephros of the developing mouse kidney, suggesting a role for Wnt6 in epithelialization. Importantly, TCF/Lef reporter activity is also prominent in the mesonephros. Analysis of Wnt family members in human renal biopsies identified differential expression of Wnt6, correlating with severity of the disease. In animal models of tubular injury and fibrosis, loss of Wnt6 was evident. Wnt6 signals through the canonical pathway in renal epithelial cells as evidenced by increased phosphorylation of GSK3β (Ser9), nuclear accumulation of β-catenin and increased TCF/Lef transcriptional activity. FzD7 was identified as a putative receptor of Wnt6. In vitro Wnt6 expression leads to de novo tubulogenesis in renal epithelial cells grown in three-dimensional culture. Importantly, Wnt6 rescued epithelial cell dedifferentiation in response to transforming growth factor-β (TGF-β); Wnt6 reversed TGF-β-mediated increases in vimentin and loss of epithelial phenotype. Wnt6 inhibited TGF-β-mediated p65-NF-κB nuclear translocation, highlighting cross talk between the two pathways. The critical role of NF-κB in the regulation of vimentin expression was confirmed in both p65(-/-) and IKKα/β(-/-) embryonic fibroblasts. We propose that Wnt6 is involved in epithelialization and loss of Wnt6 expression contributes to the pathogenesis of renal fibrosis. Copyright © 2016 the American Physiological Society.

Wnt-5a/Frizzled9 Receptor Signaling through the Gαo-Gβγ Complex Regulates Dendritic Spine Formation.

PubMed

Ramírez, Valerie T; Ramos-Fernández, Eva; Henríquez, Juan Pablo; Lorenzo, Alfredo; Inestrosa, Nibaldo C

2016-09-02

Wnt ligands play crucial roles in the development and regulation of synapse structure and function. Specifically, Wnt-5a acts as a secreted growth factor that regulates dendritic spine formation in rodent hippocampal neurons, resulting in postsynaptic development that promotes the clustering of the PSD-95 (postsynaptic density protein 95). Here, we focused on the early events occurring after the interaction between Wnt-5a and its Frizzled receptor at the neuronal cell surface. Additionally, we studied the role of heterotrimeric G proteins in Wnt-5a-dependent synaptic development. We report that FZD9 (Frizzled9), a Wnt receptor related to Williams syndrome, is localized in the postsynaptic region, where it interacts with Wnt-5a. Functionally, FZD9 is required for the Wnt-5a-mediated increase in dendritic spine density. FZD9 forms a precoupled complex with Gαo under basal conditions that dissociates after Wnt-5a stimulation. Accordingly, we found that G protein inhibition abrogates the Wnt-5a-dependent pathway in hippocampal neurons. In particular, the activation of Gαo appears to be a key factor controlling the Wnt-5a-induced dendritic spine density. In addition, we found that Gβγ is required for the Wnt-5a-mediated increase in cytosolic calcium levels and spinogenesis. Our findings reveal that FZD9 and heterotrimeric G proteins regulate Wnt-5a signaling and dendritic spines in cultured hippocampal neurons. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Wnt signaling is involved in human articular chondrocyte de-differentiation in vitro.

PubMed

Sassi, N; Laadhar, L; Allouche, M; Zandieh-Doulabi, B; Hamdoun, M; Klein-Nulend, J; Makni, S; Sellami, S

2014-01-01

Osteoarthritis is the most prevalent form of arthritis in the world. Certain signaling pathways, such as the wnt pathway, are involved in cartilage pathology. Osteoarthritic chondrocytes undergo morphological and biochemical changes that lead to chondrocyte de-differentiation. We investigated whether the Wnt pathway is involved in de-differentiation of human articular chondrocytes in vitro. Human articular chondrocytes were cultured for four passages in the presence or absence of IL-1 in monolayer or micromass culture. Changes in cell morphology were monitored by light microscopy. Protein and gene expression of chondrocyte markers and Wnt pathway components were determined by Western blotting and qPCR after culture. After culturing for four passages, chondrocytes exhibited a fibroblast-like morphology. Collagen type II and aggrecan protein and gene expression decreased, while collagen type I, matrix metalloproteinase 13, and nitric oxide synthase expressions increased. Wnt molecule expression profiles changed; Wnt5a protein expression, the Wnt target gene, c-jun, and in Wnt pathway regulator, sFRP4 increased. Treatment with IL-1 caused chondrocyte morphology to become more filament-like. This change in morphology was accompanied by extinction of col II expression and increased col I, MMP13 and eNOS expression. Changes in expression of the Wnt pathway components also were observed. Wnt7a decreased significantly, while Wnt5a, LRP5, β-catenin and c-jun expressions increased. Culture of human articular chondrocytes with or without IL-1 not only induced chondrocyte de-differentiation, but also changed the expression profiles of Wnt components, which suggests that the Wnt pathway is involved in chondrocyte de-differentiation in vitro.

Transduction of Wnt11 promotes mesenchymal stem cell transdifferentiation into cardiac phenotypes.

PubMed

He, Zhisong; Li, Hongxia; Zuo, Shi; Pasha, Zeeshan; Wang, Yigang; Yang, Yueting; Jiang, Wenping; Ashraf, Muhammad; Xu, Meifeng

2011-10-01

Transplantation of mesenchymal stem cells (MSCs) has emerged as a potential treatment for ischemic heart repair. Previous studies have suggested that Wnt11 plays a critical role in cardiac specification and morphogenesis. In this study, we examined whether transduction of Wnt11 directly increases MSC differentiation into cardiac phenotypes. MSCs harvested from rat bone marrow were transduced with both Wnt11 and green fluorescent protein (GFP) (MSC(Wnt11)) using the murine stem cell virus (pMSCV) retroviral expression system; control cells were only GFP-transfected (MSC(Null)). Compared with control cells, MSC(Wnt11) was shown to have higher expression of Wnt11 by immunofluorescence, real-time polymerase chain reaction, and western blotting. MSC(Wnt11) shows a higher expression of cardiac-specific genes, including GATA-4, brain natriuretic peptide (BNP), islet-1, and α-actinin, after being cultured with cardiomyocytes (CMs) isolated from ventricles of neonatal (1-3 day) SD rats. Some MSC(Wnt11) were positive for α-actinin when MSCs were cocultured with native CMs for 7 days. Electron microscopy further confirmed the appearance of sarcomeres in MSC(Wnt11). Connexin 43 was found between GFP-positive MSCs and neonatal rat CMs labeled with red fluorescent probe PKH26. The transdifferentiation rate was significantly higher in MSC(Wnt11) than in MSC(Null), as assessed by flow cytometry. Functional studies indicated that the differentiation of MSC(Wnt11) was diminished by knockdown of GATA-4 with GATA-4-siRNA. Transduction of Wnt11 into MSCs increases their differentiation into CMs by upregulating GATA-4.

1α,25-dihydroxyvitamin D3 modulates the hair-inductive capacity of dermal papilla cells: therapeutic potential for hair regeneration.

PubMed

Aoi, Noriyuki; Inoue, Keita; Chikanishi, Toshihiro; Fujiki, Ryoji; Yamamoto, Hanako; Kato, Harunosuke; Eto, Hitomi; Doi, Kentaro; Itami, Satoshi; Kato, Shigeaki; Yoshimura, Kotaro

2012-08-01

Dermal papilla cells (DPCs) have the potential to induce differentiation of epithelial stem cells into hair, and Wnt signaling is deeply involved in the initiation process. The functional limitation of expanded adult DPCs has been a difficult challenge for cell-based hair regrowth therapy. We previously reported that 1α,25-dihydroxyvitamin D(3) (VD(3)) upregulates expression of transforming growth factor (TGF)-β2 and alkaline phosphatase (ALP) activity, both features of hair-inducing human DPCs (hDPCs). In this study, we further examined the effects and signaling pathways associated with VD(3) actions on DPCs. VD(3) suppressed hDPC proliferation in a dose-dependent, noncytotoxic manner. Among the Wnt-related genes investigated, Wnt10b expression was significantly upregulated by VD(3) in hDPCs. Wnt10b upregulation, as well as upregulation of ALPL (ALP, liver/bone/kidney) and TGF-β2, by VD(3) was specific in hDPCs and not detected in human dermal fibroblasts. Screening of paracrine or endocrine factors in the skin indicated that all-trans retinoic acid (atRA) upregulated Wnt10b gene expression, although synergistic upregulation (combined atRA and VD(3)) was not seen. RNA interference with vitamin D receptor (VDR) revealed that VD(3) upregulation of Wnt10b, ALPL, and TGF-β2 was mediated through the genomic VDR pathway. In a rat model of de novo hair regeneration by murine DPC transplantation, pretreatment with VD(3) significantly enhanced hair folliculogenesis. Specifically, a greater number of outgrowing hair shafts and higher maturation of regenerated follicles were observed. Together, these data suggest that VD(3) may promote functional differentiation of DPCs and be useful in preserving the hair follicle-inductive capacity of cultured DPCs for hair regeneration therapies.

Subtypes of medulloblastoma have distinct developmental origins

PubMed Central

Gibson, Paul; Tong, Yiai; Robinson, Giles; Thompson, Margaret C.; Currle, D. Spencer; Eden, Christopher; Kranenburg, Tanya A.; Hogg, Twala; Poppleton, Helen; Martin, Julie; Finkelstein, David; Pounds, Stanley; Weiss, Aaron; Patay, Zoltan; Scoggins, Matthew; Ogg, Robert; Pei, Yanxin; Yang, Zeng-Jie; Brun, Sonja; Lee, Youngsoo; Zindy, Frederique; Lindsey, Janet C.; Taketo, Makoto M.; Boop, Frederick A.; Sanford, Robert A.; Gajjar, Amar; Clifford, Steven C.; Roussel, Martine F.; McKinnon, Peter J.; Gutmann, David H.; Ellison, David W.; Wechsler-Reya, Robert; Gilbertson, Richard J.

2010-01-01

Medulloblastoma encompasses a collection of clinically and molecularly diverse tumor subtypes that together comprise the most common malignant childhood brain tumor1–4. These tumors are thought to arise within the cerebellum, with approximately 25% originating from granule neuron precursor cells (GNPCs) following aberrant activation of the Sonic Hedgehog pathway (hereafter, SHH-subtype)3–8. The pathological processes that drive heterogeneity among the other medulloblastoma subtypes are not known, hindering the development of much needed new therapies. Here, we provide evidence that a discrete subtype of medulloblastoma that contains activating mutations in the WNT pathway effector CTNNB1 (hereafter, WNT-subtype)1,3,4, arises outside the cerebellum from cells of the dorsal brainstem. We found that genes marking human WNT-subtype medulloblastomas are more frequently expressed in the lower rhombic lip (LRL) and embryonic dorsal brainstem than in the upper rhombic lip (URL) and developing cerebellum. Magnetic resonance imaging (MRI) and intra-operative reports showed that human WNT-subtype tumors infiltrate the dorsal brainstem, while SHH-subtype tumors are located within the cerebellar hemispheres. Activating mutations in Ctnnb1 had little impact on progenitor cell populations in the cerebellum, but caused the abnormal accumulation of cells on the embryonic dorsal brainstem that included aberrantly proliferating Zic1+ precursor cells. These lesions persisted in all mutant adult mice and in 15% of cases in which Tp53 was concurrently deleted, progressed to form medulloblastomas that recapitulated the anatomy and gene expression profiles of human WNT-subtype medulloblastoma. We provide the first evidence that subtypes of medulloblastoma have distinct cellular origins. Our data provide an explanation for the marked molecular and clinical differences between SHH and WNT-subtype medulloblastomas and have profound implications for future research and treatment of this important childhood cancer. PMID:21150899

Priming 3D cultures of human mesenchymal stromal cells toward cartilage formation via developmental pathways.

PubMed

Centola, Matteo; Tonnarelli, Beatrice; Schären, Stefan; Glaser, Nicolas; Barbero, Andrea; Martin, Ivan

2013-11-01

The field of regenerative medicine has increasingly recognized the importance to be inspired by developmental processes to identify signaling pathways crucial for 3D organogenesis and tissue regeneration. Here, we aimed at recapitulating the first events occurring during limb development (ie, cell condensation and expansion of an undifferentiated mesenchymal cell population) to prime 3D cultures of human bone marrow-derived mesenchymal stromal/stem cells (hBM-MSC) toward the chondrogenic route. Based on embryonic development studies, we hypothesized that Wnt3a and fibroblast growth factor 2 (FGF2) induce hBM-MSC to proliferate in 3D culture as an undifferentiated pool of progenitors (defined by clonogenic capacity and expression of typical markers), retaining chondrogenic potential upon induction by suitable morphogens. hBM-MSC were responsive to Wnt signaling in 3D pellet culture, as assessed by significant upregulation of main target genes and increase of unphosphorylated β-catenin levels. Wnt3a was able to induce a five-fold increase in the number of proliferating hBM-MSC (6.4% vs. 1.3% in the vehicle condition), although total DNA content of the 3D construct was decreasing over time. Preconditioning with Wnt3a improved transforming growth factor-β1 mediated chondrogenesis (30% more glycosaminoglycans/cell in average). In contrast to developmental and 2D MSC culture models, FGF2 antagonized the Wnt-mediated effects. Interestingly, the CD146⁺ subpopulation was found to be more responsive to Wnt3a. The presented data indicate a possible strategy to prime 3D cultures of hBM-MSC by invoking a "developmental engineering" approach. The study also identifies some opportunities and challenges to cross-fertilize skeletal development models and 3D hBM-MSC culture systems.

Reck and Gpr124 Are Essential Receptor Cofactors for Wnt7a/Wnt7b-Specific Signaling in Mammalian CNS Angiogenesis and Blood-Brain Barrier Regulation.

PubMed

Cho, Chris; Smallwood, Philip M; Nathans, Jeremy

2017-08-30

Reck, a GPI-anchored membrane protein, and Gpr124, an orphan GPCR, have been implicated in Wnt7a/Wnt7b signaling in the CNS vasculature. We show here that vascular endothelial cell (EC)-specific reduction in Reck impairs CNS angiogenesis and that EC-specific postnatal loss of Reck, combined with loss of Norrin, impairs blood-brain barrier (BBB) maintenance. The most N-terminal domain of Reck binds to the leucine-rich repeat (LRR) and immunoglobulin (Ig) domains of Gpr124, and weakening this interaction by targeted mutagenesis reduces Reck/Gpr124 stimulation of Wnt7a signaling in cell culture and impairs CNS angiogenesis. Finally, a soluble Gpr124(LRR-Ig) probe binds to cells expressing Frizzled, Wnt7a or Wnt7b, and Reck, and a soluble Reck(CC1-5) probe binds to cells expressing Frizzled, Wnt7a or Wnt7b, and Gpr124. These experiments indicate that Reck and Gpr124 are part of the cell surface protein complex that transduces Wnt7a- and Wnt7b-specific signals in mammalian CNS ECs to promote angiogenesis and regulate the BBB. Copyright © 2017 Elsevier Inc. All rights reserved.

p38 MAP kinase is required for Wnt3a-mediated osterix expression independently of Wnt-LRP5/6-GSK3β signaling axis in dental follicle cells

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

Sakisaka, Yukihiko; Kanaya, Sousuke; Liason Center for Innovative Dentistry, Tohoku University Graduate School of Dentistry, Sendai 980-8575

Wnt3a is a secreted glycoprotein that activates the glycogen synthase kinase-3β (GSK3β)/β-catenin signaling pathway through low-density-lipoprotein receptor-related protein (LRP)5/6 co-receptors. Wnt3a has been implicated in periodontal development and homeostasis, as well as in cementum formation. Recently, we have reported that Wnt3a increases alkaline phosphatase expression through the induction of osterix (Osx) expression in dental follicle cells, a precursor of cementoblasts. However, the molecular mechanism by which Wnt3a induces Osx expression is still unknown. In this study, we show that Wnt3a-induced Osx expression was inhibited in the presence of p38 mitogen-activated protein kinase (MAPK) inhibitors (SB203580 and SB202190) at gene andmore » protein levels, as assessed by real-time PCR and immunocytohistochemistry, respectively. Pretreatment of cells with Dickkopf-1, a potent canonical Wnt antagonist binding to LRP5/6 co-receptors, did not influence Wnt3a-mediated p38 MAPK phosphorylation, suggesting that Wnt3a activates p38 MAPK through LRP5/6-independent signaling. On the other hand, pretreatment with p38 MAPK inhibitors had no effects on the phosphorylated status of GSK3β and β-catenin as well as β-catenin nuclear translocation, but inhibited Wnt3a-mediated β-catenin transcriptional activity. These findings suggest that p38 MAPK modulates canonical Wnt signaling at the β-catenin transcriptional level without any crosstalk with the Wnt3a-mediated LRP5/6-GSK3β signaling axis and subsequent β-catenin nuclear translocation. These findings expand our knowledge of the mechanisms controlling periodontal development and regeneration. - Highlights: • Wnt3a induces Osx expression via p38 MAPK signaling in dental follicle cells. • p38 MAPK has no crosstalk with Wnt3a-mediated LRP5/6 and GSK3β signaling. • p38 MAPK is required for Wnt signaling at the β-catenin transcriptional level.« less

SIRT-1 regulates TGF-β-induced dermal fibroblast migration via modulation of Cyr61 expression.

PubMed

Kwon, Eun-Jeong; Park, Eun-Jung; Yu, Hyeran; Huh, Jung-Sik; Kim, Jinseok; Cho, Moonjae

2018-05-01

SIRT1 is a NAD-dependent protein deacetylase that participates in cellular regulation. The increased migration of fibroblasts is an important phenotype in fibroblast activation. The role of SIRT1 in cell migration remains controversial as to whether SIRT1 acts as an activator or suppressor of cell migration. Therefore, we have established the role of SIRT1 in the migration of human dermal fibroblasts and explored targets of SIRT1 during dermal fibroblast migration. SIRT1 and Cyr61 were expressed in human dermal fibroblasts and the stimulation with TGF-β further induced their expression. Treatment with resveratrol (RSV), a SIRT1 agonist, or overexpression of SIRT1 also promoted the expression Cyr61 in human dermal fibroblasts, whereas the inhibition of SIRT1 activity by nicotinamide or knockdown of SIRT1 decreased the level of Cyr61, as well as TGF-β or RSV-induced Cyr61 expression. Blocking of ERK signaling by PD98509 reduced the expression of Cyr61 induced by TGF-β or RSV. TGF-β, RSV, or SIRT1 overexpression enhanced β-catenin as well as Cyr61 expression. This stimulation was reduced by the Wnt inhibitor XAV939. RSV increased migration and nicotinamide attenuated RSV-induced migration of human dermal fibroblasts. Furthermore, SIRT1 overexpression promoted cell migration, whereas blocking Cyr61 attenuated SIRT1-stimulated migration of human dermal fibroblasts. SIRT1 increased cell migration by stimulating Cyr61 expression and the ERK and Wnt/β-catenin signaling. SIRT1-induced Cyr61 activity is very important for human dermal fibroblasts migration.

The opposing roles of Wnt-5a in cancer

PubMed Central

McDonald, S L; Silver, A

2009-01-01

Wnt-5a is one of the most highly investigated non-canonical Wnts and has been implicated in almost all aspects of non-canonical Wnt signalling. In terms of cancer development, Wnt-5a has, until recently, lived in the shadow of its better-characterised relatives. This was largely because of its apparent inability to transform cells or signal through the canonical β-catenin pathway that is so important in cancer, particularly colorectal cancer. Recent work in a wide range of human tumours has pointed to a critical role for Wnt-5a in malignant progression, but there is conflicting evidence whether Wnt-5a has a tumour-promoting or -suppressing role. Emerging evidence suggests that the functions of Wnt-5a can be drastically altered depending on the availability of key receptors. Hence, the presence or absence of these receptors may go some way to explain the conflicting role of Wnt-5a in different cancers. This review summarises our current understanding of Wnt-5a and cancer. PMID:19603030

Mutations in WNT1 Cause Different Forms of Bone Fragility

PubMed Central

Keupp, Katharina; Beleggia, Filippo; Kayserili, Hülya; Barnes, Aileen M.; Steiner, Magdalena; Semler, Oliver; Fischer, Björn; Yigit, Gökhan; Janda, Claudia Y.; Becker, Jutta; Breer, Stefan; Altunoglu, Umut; Grünhagen, Johannes; Krawitz, Peter; Hecht, Jochen; Schinke, Thorsten; Makareeva, Elena; Lausch, Ekkehart; Cankaya, Tufan; Caparrós-Martín, José A.; Lapunzina, Pablo; Temtamy, Samia; Aglan, Mona; Zabel, Bernhard; Eysel, Peer; Koerber, Friederike; Leikin, Sergey; Garcia, K. Christopher; Netzer, Christian; Schönau, Eckhard; Ruiz-Perez, Victor L.; Mundlos, Stefan; Amling, Michael; Kornak, Uwe; Marini, Joan; Wollnik, Bernd

2013-01-01

We report that hypofunctional alleles of WNT1 cause autosomal-recessive osteogenesis imperfecta, a congenital disorder characterized by reduced bone mass and recurrent fractures. In consanguineous families, we identified five homozygous mutations in WNT1: one frameshift mutation, two missense mutations, one splice-site mutation, and one nonsense mutation. In addition, in a family affected by dominantly inherited early-onset osteoporosis, a heterozygous WNT1 missense mutation was identified in affected individuals. Initial functional analysis revealed that altered WNT1 proteins fail to activate canonical LRP5-mediated WNT-regulated β-catenin signaling. Furthermore, osteoblasts cultured in vitro showed enhanced Wnt1 expression with advancing differentiation, indicating a role of WNT1 in osteoblast function and bone development. Our finding that homozygous and heterozygous variants in WNT1 predispose to low-bone-mass phenotypes might advance the development of more effective therapeutic strategies for congenital forms of bone fragility, as well as for common forms of age-related osteoporosis. PMID:23499309

Overexpression of Wnt5a in mouse epidermis causes no psoriasis phenotype but an impairment of hair follicle anagen development.

PubMed

Zhu, Xuming; Wu, Yumei; Huang, Sixia; Chen, Yingwei; Tao, Yixin; Wang, Yushu; He, Shigang; Shen, Sanbing; Wu, Ji; Guo, Xizhi; Li, Baojie; He, Lin; Ma, Gang

2014-12-01

Increased Wnt5a expression has been observed in psoriatic plaques. However, whether Wnt5a overexpression directly causes psoriasis is unknown. In this study, we generated transgenic (TG) mice with epidermal Wnt5a overexpression under the control of the human K14 promoter. The skin of Wnt5a TG mice was not psoriatic, but characterized with normal proliferation and homeostasis of epidermis. Instead, these TG mice displayed impaired hair follicle transition from telogen to anagen, most likely due to impaired canonical Wnt signalling. These results suggest that increased Wnt5a expression alone is inadequate to induce psoriasis in the skin and possible involvement of Wnt5a in hair follicle cycling. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Isolation and characterization of recombinant murine Wnt3a.

PubMed

Witkowski, Andrzej; Krishnamoorthy, Aparna; Su, Betty; Beckstead, Jennifer A; Ryan, Robert O

2015-02-01

Wnt proteins are a family of morphogens that possess potent biological activity. Structure-function studies have been impeded by poor yield of biologically active recombinant Wnt as well as a propensity of isolated Wnt to self-associate in the absence of detergent. Using stably transfected Drosophila S2 cells, studies have been conducted to improve recovery of recombinant murine Wnt3a, establish conditions for a detergent-free Wnt preparation and examine the effects of limited proteolysis. S2 cell culture conditioned media was subjected to a 3-step protocol including dye-ligand chromatography, immobilized metal affinity chromatography and gel filtration chromatography. Through selective pooling of column fractions, homogeneous and purified Wnt3a preparations were obtained. Limited proteolysis of Wnt3a with thrombin resulted in site-specific cleavage within the N-terminal saposin-like motif. To generate detergent-free protein, Wnt3a was immobilized on Cu(2+)-charged, iminodiacetic acid-derivatized Sepharose beads, detergent-free buffer was applied and Wnt3a eluted from the beads with buffer containing imidazole plus 30mM methyl-ß-cyclodextrin (MßCD). Wnt3a recovered in MßCD-containing buffer was soluble and biologically active. Insofar as MßCD is a member of a family of non-toxic, low molecular weight compounds capable of binding and solubilizing small hydrophobic ligands, Wnt-cyclodextrin complexes may facilitate structure-activity studies in the absence of adverse detergent effects. Copyright © 2014 Elsevier Inc. All rights reserved.

Characteristic Markers of the WNT Signaling Pathways Are Differentially Expressed in Osteoarthritic Cartilage

PubMed Central

Dehne, T.; Lindahl, A.; Brittberg, M.; Pruss, A.; Ringe, J.; Sittinger, M.; Karlsson, C.

2012-01-01

Objective: It is well known that expression of markers for WNT signaling is dysregulated in osteoarthritic (OA) bone. However, it is still not fully known if the expression of these markers also is affected in OA cartilage. The aim of this study was therefore to examine this issue. Methods: Human cartilage biopsies from OA and control donors were subjected to genome-wide oligonucleotide microarrays. Genes involved in WNT signaling were selected using the BioRetis database, KEGG pathway analysis was searched using DAVID software tools, and cluster analysis was performed using Genesis software. Results from the microarray analysis were verified using quantitative real-time PCR and immunohistochemistry. In order to study the impact of cytokines for the dysregulated WNT signaling, OA and control chondrocytes were stimulated with interleukin-1 and analyzed with real-time PCR for their expression of WNT-related genes. Results: Several WNT markers displayed a significantly altered expression in OA compared to normal cartilage. Interestingly, inhibitors of the canonical and planar cell polarity WNT signaling pathways displayed significantly increased expression in OA cartilage, while the Ca2+/WNT signaling pathway was activated. Both real-time PCR and immunohistochemistry verified the microarray results. Real-time PCR analysis demonstrated that interleukin-1 upregulated expression of important WNT markers. Conclusions: WNT signaling is significantly affected in OA cartilage. The result suggests that both the canonical and planar cell polarity WNT signaling pathways were partly inhibited while the Ca2+/WNT pathway was activated in OA cartilage. PMID:26069618

Identification and expression characterization of WntA during intestinal regeneration in the sea cucumber Apostichopus japonicus.

PubMed

Li, Xiaoni; Sun, Lina; Yang, Hongsheng; Zhang, Libin; Miao, Ting; Xing, Lili; Huo, Da

2017-08-01

Wnt genes encode secreted glycoproteins that act as signaling molecules; these molecules direct cell proliferation, migration, differentiation and survival during animal development, maintenance of homeostasis and regeneration. At present, although the regeneration mechanism in Apostichopus japonicus has been studied, there is a little research on the Wnt signaling pathway in A. japonicus. To understand the potential role of the Wnt signaling pathway in A. japonicus, we cloned and sequenced the WntA gene in A. japonicus. Protein localization analysis showed that WntA protein was ubiquitously expressed in epidermal cells, the muscle and submucosa of the intestinal tissue. After stimulation and evisceration, the dynamic changes in expression of the WntA gene and protein showed that WntA was constitutively expressed during different stages of intestine regeneration in A. japonicus, with higher levels during the early wound healing stage and late lumen formation in the residual and nascent intestinal tissues, indicating its response to intestinal regeneration. Simultaneously, cell proliferation and apoptosis analysis showed that the patterns of cell proliferation were similar to the patterns of WntA protein expression during different intestinal regeneration stages in this organism. Taken together, these results suggested that WntA might participate in intestinal regeneration and may be connected with cell proliferation, apoptosis in different intestinal layers. This research could establish a basis for further examination of WntA functions in A. japonicus and Wnt genes in other echinoderms. Copyright © 2017 Elsevier Inc. All rights reserved.

Isolation and characterization of recombinant murine Wnt3a

PubMed Central

Witkowski, Andrzej; Krishnamoorthy, Aparna; Su, Betty; Beckstead, Jennifer A.; Ryan, Robert O.

2014-01-01

Wnt proteins are a family of morphogens that possess potent biological activity. Structure – function studies have been impeded by poor yield of biologically active recombinant Wnt as well as a propensity of isolated Wnt to self-associate in the absence of detergent. Using stably transfected Drosophila S2 cells, studies have been conducted to improve recovery of recombinant murine Wnt3a, establish conditions for a detergent-free Wnt preparation and examine the effects of limited proteolysis. S2 cell culture conditioned media was subjected to a 3-step protocol including dye-ligand chromatography, immobilized metal affinity chromatography and gel filtration chromatography. Through selective pooling of column fractions, homogeneous and purified Wnt3a preparations were obtained. Limited proteolysis of Wnt3a with thrombin resulted in site-specific cleavage within the N-terminal saposin-like motif. To generate detergent-free protein, Wnt3a was immobilized on Cu2+-charged, iminodiacetic acid-derivatized Sepharose beads, detergent-free buffer was applied and Wnt3a eluted from the beads with buffer containing imidazole plus 30 mM methyl-β-cyclodextrin (MβCD). Wnt3a recovered in MβCD-containing buffer was soluble and biologically active. Insofar as MβCD is a member of a family of non-toxic, low molecular weight compounds capable of binding and solubilizing small hydrophobic ligands, Wnt-cyclodextrin complexes may facilitate structure-activity studies in the absence of adverse detergent effects. PMID:25448592

The Wnt-1 (int-1) oncogene promoter and its mechanism of activation by insertion of proviral DNA of the mouse mammary tumor virus.

PubMed Central

Nusse, R; Theunissen, H; Wagenaar, E; Rijsewijk, F; Gennissen, A; Otte, A; Schuuring, E; van Ooyen, A

1990-01-01

Wnt-1 (int-1) is a cellular oncogene often activated by insertion of proviral DNA of the mouse mammary tumor virus. We have mapped the 5' end and the promoter area of the Wnt-1 gene by nuclease protection and primer extension assays. In differentiating P19 embryonal carcinoma cells, in which Wnt-1 is naturally expressed, two start sites of transcription were found, one preceded by two TATA boxes and one preceded by several GC boxes. In P19 cells, a 1-kilobase upstream sequence of Wnt-1 was able to confer differentiation-specific expression on a heterologous gene. We have investigated how Wnt-1 transcription was affected by mouse mammary tumor virus proviral integrations in various configurations near the promoters of the gene. One provirus has been inserted in the 5' nontranslated part of Wnt-1, in the same transcriptional orientation, and has functionally replaced the Wnt-1 promoters. Wnt-1 transcription in this tumor starts in the right long terminal repeat of the provirus, with considerable readthrough transcription from the left long terminal repeat. Another provirus has been inserted in the orientation opposite that of Wnt-1 into a GC box, disrupting the first Wnt-1 transcription start site but not the downstream start site. Most insertions have not structurally altered the Wnt-1 transcripts and have enhanced the activity of the normal two promoters. Images PMID:1695322

Winding through the WNT pathway during cellular development and demise.

PubMed

Li, F; Chong, Z Z; Maiese, K

2006-01-01

In slightly over a period of twenty years, our comprehension of the cellular and molecular mechanisms that govern the Wnt signaling pathway continue to unfold. The Wnt proteins were initially implicated in viral carcinogenesis experiments associated with mammary tumors, but since this period investigations focusing on the Wnt pathways and their transmembrane receptors termed Frizzled have been advanced to demonstrate the critical nature of Wnt for the development of a variety of cell populations as well as the potential of the Wnt pathway to avert apoptotic injury. In particular, Wnt signaling plays a significant role in both the cardiovascular and nervous systems during embryonic cell patterning, proliferation, differentiation, and orientation. Furthermore, modulation of Wnt signaling under specific cellular influences can either promote or prevent the early and late stages of apoptotic cellular injury in neurons, endothelial cells, vascular smooth muscle cells, and cardiomyocytes. A number of downstream signal transduction pathways can mediate the biological response of the Wnt proteins that include Dishevelled, beta-catenin, intracellular calcium, protein kinase C, Akt, and glycogen synthase kinase-3beta. Interestingly, these cellular cascades of the Wnt-Frizzled pathways can participate in several neurodegenerative, vascular, and cardiac disorders and may be closely integrated with the function of trophic factors. Identification of the critical elements that modulate the Wnt-Frizzled signaling pathway should continue to unlock the potential of Wnt pathway for the development of new therapeutic options against neurodegenerative and vascular diseases.

A proteomic analysis of LRRK2 binding partners reveals interactions with multiple signaling components of the WNT/PCP pathway.

PubMed

Salašová, Alena; Yokota, Chika; Potěšil, David; Zdráhal, Zbyněk; Bryja, Vítězslav; Arenas, Ernest

2017-07-11

Autosomal-dominant mutations in the Park8 gene encoding Leucine-rich repeat kinase 2 (LRRK2) have been identified to cause up to 40% of the genetic forms of Parkinson's disease. However, the function and molecular pathways regulated by LRRK2 are largely unknown. It has been shown that LRRK2 serves as a scaffold during activation of WNT/β-catenin signaling via its interaction with the β-catenin destruction complex, DVL1-3 and LRP6. In this study, we examine whether LRRK2 also interacts with signaling components of the WNT/Planar Cell Polarity (WNT/PCP) pathway, which controls the maturation of substantia nigra dopaminergic neurons, the main cell type lost in Parkinson's disease patients. Co-immunoprecipitation and tandem mass spectrometry was performed in a mouse substantia nigra cell line (SN4741) and human HEK293T cell line in order to identify novel LRRK2 binding partners. Inhibition of the WNT/β-catenin reporter, TOPFlash, was used as a read-out of WNT/PCP pathway activation. The capacity of LRRK2 to regulate WNT/PCP signaling in vivo was tested in Xenopus laevis' early development. Our proteomic analysis identified that LRRK2 interacts with proteins involved in WNT/PCP signaling such as the PDZ domain-containing protein GIPC1 and Integrin-linked kinase (ILK) in dopaminergic cells in vitro and in the mouse ventral midbrain in vivo. Moreover, co-immunoprecipitation analysis revealed that LRRK2 binds to two core components of the WNT/PCP signaling pathway, PRICKLE1 and CELSR1, as well as to FLOTILLIN-2 and CULLIN-3, which regulate WNT secretion and inhibit WNT/β-catenin signaling, respectively. We also found that PRICKLE1 and LRRK2 localize in signalosomes and act as dual regulators of WNT/PCP and β-catenin signaling. Accordingly, analysis of the function of LRRK2 in vivo, in X. laevis revelaed that LRKK2 not only inhibits WNT/β-catenin pathway, but induces a classical WNT/PCP phenotype in vivo. Our study shows for the first time that LRRK2 activates the WNT/PCP signaling pathway through its interaction to multiple WNT/PCP components. We suggest that LRRK2 regulates the balance between WNT/β-catenin and WNT/PCP signaling, depending on the binding partners. Since this balance is crucial for homeostasis of midbrain dopaminergic neurons, we hypothesize that its alteration may contribute to the pathophysiology of Parkinson's disease.

Expression of WNT genes in cervical cancer-derived cells: Implication of WNT7A in cell proliferation and migration.

PubMed

Ramos-Solano, Moisés; Meza-Canales, Ivan D; Torres-Reyes, Luis A; Alvarez-Zavala, Monserrat; Alvarado-Ruíz, Liliana; Rincon-Orozco, Bladimiro; Garcia-Chagollan, Mariel; Ochoa-Hernández, Alejandra B; Ortiz-Lazareno, Pablo C; Rösl, Frank; Gariglio, Patricio; Jave-Suárez, Luis F; Aguilar-Lemarroy, Adriana

2015-07-01

According to the multifactorial model of cervical cancer (CC) causation, it is now recognized that other modifications, in addition to Human papillomavirus (HPV) infection, are necessary for the development of this neoplasia. Among these, it has been proposed that a dysregulation of the WNT pathway might favor malignant progression of HPV-immortalized keratinocytes. The aim of this study was to identify components of the WNT pathway differentially expressed in CC vs. non-tumorigenic, but immortalized human keratinocytes. Interestingly, WNT7A expression was found strongly downregulated in cell lines and biopsies derived from CC. Restoration of WNT7A in CC-derived cell lines using a lentiviral gene delivery system or after adding a recombinant human protein decreases cell proliferation. Likewise, WNT7A silencing in non-tumorigenic cells markedly accelerates proliferation. Decreased WNT7A expression was due to hypermethylation at particular CpG sites. To our knowledge, this is the first study reporting reduced WNT7A levels in CC-derived cells and that ectopic WNT7A restoration negatively affects cell proliferation and migration. Copyright © 2015 Elsevier Inc. All rights reserved.

Wnt4 is essential to normal mammalian lung development.

PubMed

Caprioli, Arianna; Villasenor, Alethia; Wylie, Lyndsay A; Braitsch, Caitlin; Marty-Santos, Leilani; Barry, David; Karner, Courtney M; Fu, Stephen; Meadows, Stryder M; Carroll, Thomas J; Cleaver, Ondine

2015-10-15

Wnt signaling is essential to many events during organogenesis, including the development of the mammalian lung. The Wnt family member Wnt4 has been shown to be required for the development of kidney, gonads, thymus, mammary and pituitary glands. Here, we show that Wnt4 is critical for proper morphogenesis and growth of the respiratory system. Using in situ hybridization in mouse embryos, we identify a previously uncharacterized site of Wnt4 expression in the anterior trunk mesoderm. This expression domain initiates as early as E8.25 in the mesoderm abutting the tracheoesophageal endoderm, between the fusing dorsal aortae and the heart. Analysis of Wnt4(-/-) embryos reveals severe lung hypoplasia and tracheal abnormalities; however, aortic fusion and esophageal development are unaffected. We find decreased cell proliferation in Wnt4(-/-) lung buds, particularly in tip domains. In addition, we observe reduction of the important lung growth factors Fgf9, Fgf10, Sox9 and Wnt2 in the lung bud during early stages of organogenesis, as well as decreased tracheal expression of the progenitor factor Sox9. Together, these data reveal a previously unknown role for the secreted protein Wnt4 in respiratory system development. Copyright © 2015. Published by Elsevier Inc.

Mesothelial- and epithelial-derived FGF9 have distinct functions in the regulation of lung development

PubMed Central

Yin, Yongjun; Wang, Fen; Ornitz, David M.

2011-01-01

Fibroblast growth factor (FGF) 9 is a secreted signaling molecule that is expressed in lung mesothelium and epithelium and is required for lung development. Embryos lacking FGF9 show mesenchymal hypoplasia, decreased epithelial branching and, by the end of gestation, hypoplastic lungs that cannot support life. Mesenchymal FGF signaling interacts with β-catenin-mediated WNT signaling in a feed-forward loop that functions to sustain mesenchymal FGF responsiveness and mesenchymal WNT/β-catenin signaling. During pseudoglandular stages of lung development, Wnt2a and Wnt7b are the canonical WNT ligands that activate mesenchymal WNT/β-catenin signaling, whereas FGF9 is the only known ligand that signals to mesenchymal FGF receptors (FGFRs). Here, we demonstrate that mesothelial- and epithelial-derived FGF9, mesenchymal Wnt2a and epithelial Wnt7b have unique functions in lung development in mouse. Mesothelial FGF9 and mesenchymal WNT2A are principally responsible for maintaining mesenchymal FGF-WNT/β-catenin signaling, whereas epithelial FGF9 primarily affects epithelial branching. We show that FGF signaling is primarily responsible for regulating mesenchymal proliferation, whereas β-catenin signaling is a required permissive factor for mesenchymal FGF signaling. PMID:21750028

Context-dependent activation of Wnt signaling by tumor suppressor RUNX3 in gastric cancer cells

PubMed Central

Ju, Xiaoli; Ishikawa, Tomo-o; Naka, Kazuhito; Ito, Kosei; Ito, Yoshiaki; Oshima, Masanobu

2014-01-01

RUNX3 is a tumor suppressor for a variety of cancers. RUNX3 suppresses the canonical Wnt signaling pathway by binding to the TCF4/β-catenin complex, resulting in the inhibition of binding of the complex to the Wnt target gene promoter. Here, we confirmed that RUNX3 suppressed Wnt signaling activity in several gastric cancer cell lines; however, we found that RUNX3 increased the Wnt signaling activity in KatoIII and SNU668 gastric cancer cells. Notably, RUNX3 expression increased the ratio of the Wnt signaling-high population in the KatoIII cells. although the maximum Wnt activation level of individual cells was similar to that in the control. As found previously, RUNX3 also binds to TCF4 and β-catenin in KatoIII cells, suggesting that these molecules form a ternary complex. Moreover, the ChIP analyses revealed that TCF4, β-catenin and RUNX3 bind the promoter region of the Wnt target genes, Axin2 and c-Myc, and the occupancy of TCF4 and β-catenin in these promoter regions is increased by the RUNX3 expression. These results suggest that RUNX3 stabilizes the TCF4/β-catenin complex on the Wnt target gene promoter in KatoIII cells, leading to activation of Wnt signaling. Although RUNX3 increased the Wnt signaling activity, its expression resulted in suppression of tumorigenesis of KatoIII cells, indicating that RUNX3 plays a tumor-suppressing role in KatoIII cells through a Wnt-independent mechanism. These results indicate that RUNX3 can either suppress or activate the Wnt signaling pathway through its binding to the TCF4/β-catenin complex by cell context-dependent mechanisms. PMID:24447505

Bone Mass and Strength are Significantly Improved in Mice Overexpressing Human WNT16 in Osteocytes.

PubMed

Alam, Imranul; Reilly, Austin M; Alkhouli, Mohammed; Gerard-O'Riley, Rita L; Kasipathi, Charishma; Oakes, Dana K; Wright, Weston B; Acton, Dena; McQueen, Amie K; Patel, Bhavmik; Lim, Kyung-Eun; Robling, Alexander G; Econs, Michael J

2017-04-01

Recently, we demonstrated that osteoblast-specific overexpression of human WNT16 increased both cortical and trabecular bone mass and structure in mice. To further identify the cell-specific role of Wnt16 in bone homeostasis, we created transgenic (TG) mice overexpressing human WNT16 in osteocytes using Dmp1 promoter (Dmp1-hWNT16 TG) on C57BL/6 (B6) background. We analyzed bone phenotypes and serum bone biomarkers, performed gene expression analysis and measured dynamic bone histomorphometry in Dmp1-hWNT16 TG and wild-type (WT) mice. Compared to WT mice, Dmp1-hWNT16 TG mice exhibited significantly higher whole-body, spine and femoral aBMD, BMC and trabecular (BV/TV, Tb.N, and Tb.Th) and cortical (bone area and thickness) parameters in both male and female at 12 weeks of age. Femur stiffness and ultimate force were also significantly improved in the Dmp1-hWNT16 TG female mice, compared to sex-matched WT littermates. In addition, female Dmp1-hWNT16 TG mice displayed significantly higher MS/BS, MAR and BFR/BS compared to the WT mice. Gene expression analysis demonstrated significantly higher mRNA level of Alp in both male and female Dmp1-hWNT16 TG mice and significantly higher levels of Osteocalcin, Opg and Rankl in the male Dmp1-hWNT16 TG mice in bone tissue compared to sex-matched WT mice. These results indicate that WNT16 plays a critical role for acquisition of both cortical and trabecular bone mass and strength. Strategies designed to use WNT16 as a target for therapeutic interventions will be valuable to treat osteoporosis and other low bone mass conditions.

Bone Mass and Strength are Significantly Improved in Mice Overexpressing Human WNT16 in Osteocytes

PubMed Central

Alam, Imranul; Reilly, Austin M.; Alkhouli, Mohammed; Gerard-O’Riley, Rita L.; Kasipathi, Charishma; Oakes, Dana K.; Wright, Weston B.; Acton, Dena; McQueen, Amie K.; Patel, Bhavmik; Lim, Kyung-Eun; Robling, Alexander G.; Econs, Michael J.

2017-01-01

Recently, we demonstrated that osteoblast-specific overexpression of human WNT16 increased both cortical and trabecular bone mass and structure in mice. To further identify the cell-specific role of Wnt16 in bone homeostasis, we created transgenic (TG) mice over-expressing human WNT16 in osteocytes using Dmp1 promoter (Dmp1-hWNT16 TG) on C57BL/6 (B6) background. We analyzed bone phenotypes and serum bone biomarkers, performed gene expression analysis and measured dynamic bone histomorphometry in Dmp1-hWNT16 TG and wild-type (WT) mice. Compared to WT mice, Dmp1-hWNT16 TG mice exhibited significantly higher whole body, spine and femoral aBMD, BMC and trabecular (BV/TV, Tb.N, and Tb.Th) and cortical (bone area and thickness) parameters in both male and female at 12 weeks of age. Femur stiffness and ultimate force were also significantly improved in the Dmp1-hWNT16 TG female mice, compared to sex-matched WT littermates. In addition, female Dmp1-hWNT16 TG mice displayed significantly higher MS/BS, MAR and BFR/BS compared to the WT mice. Gene expression analysis demonstrated significantly higher mRNA level of Alp in both male and female Dmp1-hWNT16 TG mice and significantly higher levels of Osteocalcin, Opg and Rankl in the male Dmp1-hWNT16 TG mice in bone tissue compared to sex-matched WT mice. These results indicate that WNT16 plays a critical role for acquisition of both cortical and trabecular bone mass and strength. Strategies designed to use WNT16 as a target for therapeutic interventions will be valuable to treat osteoporosis and other low bone mass conditions. PMID:28013361

Concurrent Transient Activation of Wnt/{beta}-Catenin Pathway Prevents Radiation Damage to Salivary Glands

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

Hai Bo; Yang Zhenhua; Shangguan Lei

2012-05-01

Purpose: Many head and neck cancer survivors treated with radiotherapy suffer from permanent impairment of their salivary gland function, for which few effective prevention or treatment options are available. This study explored the potential of transient activation of Wnt/{beta}-catenin signaling in preventing radiation damage to salivary glands in a preclinical model. Methods and Materials: Wnt reporter transgenic mice were exposed to 15 Gy single-dose radiation in the head and neck area to evaluate the effects of radiation on Wnt activity in salivary glands. Transient Wnt1 overexpression in basal epithelia was induced in inducible Wnt1 transgenic mice before together with, after,more » or without local radiation, and then saliva flow rate, histology, apoptosis, proliferation, stem cell activity, and mRNA expression were evaluated. Results: Radiation damage did not significantly affect activity of Wnt/{beta}-catenin pathway as physical damage did. Transient expression of Wnt1 in basal epithelia significantly activated the Wnt/{beta}-catenin pathway in submandibular glands of male mice but not in those of females. Concurrent transient activation of the Wnt pathway prevented chronic salivary gland dysfunction following radiation by suppressing apoptosis and preserving functional salivary stem/progenitor cells. In contrast, Wnt activation 3 days before or after irradiation did not show significant beneficial effects, mainly due to failure to inhibit acute apoptosis after radiation. Excessive Wnt activation before radiation failed to inhibit apoptosis, likely due to extensive induction of mitosis and up-regulation of proapoptosis gene PUMA while that after radiation might miss the critical treatment window. Conclusion: These results suggest that concurrent transient activation of the Wnt/{beta}-catenin pathway could prevent radiation-induced salivary gland dysfunction.« less

Inhibition of Wnt/β-Catenin Signaling by a Soluble Collagen-Derived Frizzled Domain Interacting with Wnt3a and the Receptors Frizzled 1 and 8

PubMed Central

Hendaoui, Ismaïl; Lavergne, Elise; Lee, Heun-Sik; Hong, Seong Hyun; Kim, Hak-Zoo; Parent, Christelle; Heuzé-Vourc'h, Nathalie; Clément, Bruno; Musso, Orlando

2012-01-01

The Wnt/β-catenin pathway controls cell proliferation, death and differentiation. Several families of extracellular proteins can antagonize Wnt/β-catenin signaling, including the decoy receptors known as secreted frizzled related proteins (SFRPs), which have a cysteine-rich domain (CRD) structurally similar to the extracellular Wnt-binding domain of the frizzled receptors. SFRPs inhibit Wnt signaling by sequestering Wnts through the CRD or by forming inactive complexes with the frizzled receptors. Other endogenous molecules carrying frizzled CRDs inhibit Wnt signaling, such as V3Nter, which is proteolytically derived from the cell surface component collagen XVIII and contains a biologically active frizzled domain (FZC18) inhibiting in vivo cell proliferation and tumor growth in mice. We recently showed that FZC18 expressing cells deliver short-range signals to neighboring cells, decreasing their proliferation in vitro and in vivo through the Wnt/β-catenin signaling pathway. Here, using low concentrations of soluble FZC18 and Wnt3a, we show that they physically interact in a cell-free system. In addition, soluble FZC18 binds the frizzled 1 and 8 receptors' CRDs, reducing cell sensitivity to Wnt3a. Conversely, inhibition of Wnt/β-catenin signaling was partially rescued by the expression of full-length frizzled 1 and 8 receptors, but enhanced by the expression of a chimeric cell-membrane-tethered frizzled 8 CRD. Moreover, soluble, partially purified recombinant FZC18_CRD inhibited Wnt3a-induced β-catenin activation. Taken together, the data indicate that collagen XVIII-derived frizzled CRD shifts Wnt sensitivity of normal cells to a lower pitch and controls their growth. PMID:22303445

Using Ambystoma mexicanum (Mexican Axolotl) Embryos, Chemical Genetics, and Microarray Analysis to Identify Signaling Pathways Associated with Tissue Regeneration

PubMed Central

Ponomareva, Larissa V.; Athippozhy, Antony; Thorson, Jon S.; Voss, S. Randal

2015-01-01

Amphibian vertebrates are important models in regenerative biology because they present exceptional regenerative capabilities throughout life. However, it takes considerable effort to rear amphibians to juvenile and adult stages for regeneration studies and the relatively large sizes that frogs and salamanders achieve during development make them difficult to use in chemical screens. Here we introduce a new tail regeneration model using late stage Mexican axolotl embryos. We show that axolotl embryos completely regenerate amputated tails in 7 days before they exhaust their yolk supply and begin to feed. Further, we show that axolotl embryos can be efficiently reared in microtiter plates to achieve moderate throughput screening of soluble chemicals to investigate toxicity and identify molecules that alter regenerative outcome. As proof of principle, we identified integration 1 / wingless (Wnt), transforming growth factor beta (Tgf-β), and fibroblast growth factor (Fgf) pathway antagonists that completely block tail regeneration and additional chemicals that significantly affected tail outgrowth. Furthermore, we used microarray analysis to show that inhibition of Wnt signaling broadly affects transcription of genes associated with Wnt, Fgf, Tgf-β, epidermal growth factor (Egf), Notch, nerve growth factor (Ngf), homeotic gene (Hox), rat sarcoma/mitogen-activated protein kinase (Ras/Mapk), myelocytomatosis viral oncogene (Myc), tumor protein 53 (p53), and retinoic acid (RA) pathways. Punctuated changes in the expression of genes known to regulate vertebrate development were observed; this suggests the tail regeneration transcriptional program is hierarchically structured and temporally ordered. Our study establishes the axolotl as a chemical screening model to investigate signaling pathways associated with tissue regeneration. PMID:26092703

WNT/β-catenin signaling mediates human neural crest induction via a pre-neural border intermediate.

PubMed

Leung, Alan W; Murdoch, Barbara; Salem, Ahmed F; Prasad, Maneeshi S; Gomez, Gustavo A; García-Castro, Martín I

2016-02-01

Neural crest (NC) cells arise early in vertebrate development, migrate extensively and contribute to a diverse array of ectodermal and mesenchymal derivatives. Previous models of NC formation suggested derivation from neuralized ectoderm, via meso-ectodermal, or neural-non-neural ectoderm interactions. Recent studies using bird and amphibian embryos suggest an earlier origin of NC, independent of neural and mesodermal tissues. Here, we set out to generate a model in which to decipher signaling and tissue interactions involved in human NC induction. Our novel human embryonic stem cell (ESC)-based model yields high proportions of multipotent NC cells (expressing SOX10, PAX7 and TFAP2A) in 5 days. We demonstrate a crucial role for WNT/β-catenin signaling in launching NC development, while blocking placodal and surface ectoderm fates. We provide evidence of the delicate temporal effects of BMP and FGF signaling, and find that NC development is separable from neural and/or mesodermal contributions. We further substantiate the notion of a neural-independent origin of NC through PAX6 expression and knockdown studies. Finally, we identify a novel pre-neural border state characterized by early WNT/β-catenin signaling targets that displays distinct responses to BMP and FGF signaling from the traditional neural border genes. In summary, our work provides a fast and efficient protocol for human NC differentiation under signaling constraints similar to those identified in vivo in model organisms, and strengthens a framework for neural crest ontogeny that is separable from neural and mesodermal fates. © 2016. Published by The Company of Biologists Ltd.

Targeted repression of AXIN2 and MYC gene expression using designer TALEs

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

Rennoll, Sherri A.; Scott, Samantha A.; Yochum, Gregory S., E-mail: gsy3@psu.edu

Highlights: • We designed TALE–SID fusion proteins to target AXIN2 and MYC. • TALE–SIDs bound the chromosomal AXIN2 and MYC genes and repressed their expression. • TALE–SIDs repress β-catenin{sup S45F}-dependent AXIN2 and MYC transcription. - Abstract: Designer TALEs (dTALEs) are chimeric transcription factors that can be engineered to regulate gene expression in mammalian cells. Whether dTALEs can block gene transcription downstream of signal transduction cascades, however, has yet to be fully explored. Here we tested whether dTALEs can be used to target genes whose expression is controlled by Wnt/β-catenin signaling. TALE DNA binding domains were engineered to recognize sequences adjacentmore » to Wnt responsive enhancer elements (WREs) that control expression of axis inhibition protein 2 (AXIN2) and c-MYC (MYC). These custom DNA binding domains were linked to the mSin3A interaction domain (SID) to generate TALE–SID chimeric repressors. The TALE–SIDs repressed luciferase reporter activity, bound their genomic target sites, and repressed AXIN2 and MYC expression in HEK293 cells. We generated a novel HEK293 cell line to determine whether the TALE–SIDs could function downstream of oncogenic Wnt/β-catenin signaling. Treating these cells with doxycycline and tamoxifen stimulates nuclear accumulation of a stabilized form of β-catenin found in a subset of colorectal cancers. The TALE–SIDs repressed AXIN2 and MYC expression in these cells, which suggests that dTALEs could offer an effective therapeutic strategy for the treatment of colorectal cancer.« less

Breast cancer stem-like cells are sensitized to tamoxifen induction of self-renewal inhibition with enforced Let-7c dependent on Wnt blocking

PubMed Central

Meng, Jinying; Wang, Jichang; Tang, Shou-Ching; Qin, Sida; Du, Ning; Li, Gang

2018-01-01

Let-7 microRNAs have been reported to have tumor suppressive functions; however, the effect of Let-7 when used in combination with chemotherapies is uncertain, but may have potential for use in clinical practice. In this study, we used RT-qPCR, western blot analysis, cell proliferation assay, flow cytometry analysis, immunohistochemistry (IHC) staining, luciferase assays, cell sorting analysis and xenografted tumor model to explore the role of Let-7 in the chemotherapy sensitivity of breast cancer stem cells. The findings of the current study indicated that Let-7 enhances the effects of endocrine therapy potentially by regulating the self-renewal of cancer stem cells. Let-7c increased the anticancer functions of tamoxifen and reduced the ratio of cancer stem-like cells (CSCs), sensitizing cells to therapy-induced repression in an estrogen receptor (ER)-dependent manner. Notably, Let-7 decreased the tumor formation ability of estrogen-treated breast CSCs in vivo and suppressed Wnt signaling, which further consolidated the previously hypothesis that Let-7 decreases the self-renewal ability, contributing to reduced tumor formation ability of stem cells. The suppressive effects exerted by Let-7 on stem-like cells involved Let-7c/ER/Wnt signaling, and the functions of Let-7c exerted with tamoxifen were dependent on ER. Taken together, the findings identified a biochemical and functional link between Let-7 and endocrine therapy in breast CSCs, which may facilitate clinical treatment in the future using delivery of suppressive Let-7. PMID:29336465

Stabilization of β-catenin induces pancreas tumor formation

PubMed Central

Heiser, Patrick W.; Cano, David A.; Landsman, Limor; Kim, Grace E.; Kench, James G.; Klimstra, David S.; Taketo, Maketo M.; Biankin, Andrew V.; Hebrok, Matthias

2008-01-01

Background & Aims β-catenin signaling within the canonical Wnt pathway is essential for pancreas development. However, the pathway is normally down-regulated in the adult organ. Increased cytoplasmic and nuclear localization of β-catenin can be detected in nearly all human solid pseudopapillary neoplasms (SPN), a rare tumor with low malignant potential. Conversely, pancreatic ductal adenocarcinoma (PDA) accounts for the majority of pancreatic tumors and is one of the leading causes of cancer death. While activating mutations within β-catenin and other members of the canonical Wnt pathway are rare, recent reports have implicated Wnt signaling in the development and progression of human PDA. Here, we sought to address the role of β-catenin signaling in pancreas tumorigenesis. Methods Using Cre/lox technology, we conditionally activated β-catenin in a subset of murine pancreatic cells, in vivo. Results Activation of β-catenin results in the formation of large pancreatic tumors at a high frequency in adult mice. These tumors resemble human SPN based upon morphological and immunohistochemical comparisons. Interestingly, stabilization of β-catenin blocks the formation of pancreatic intraepithlelial neoplasia (PanIN) in the presence of an activating mutation in Kras that is known to predispose individuals to pancreatic ductal adenocarcinoma (PDA). Instead, mice in which β-catenin and Kras are concurrently activated develop distinct ductal neoplasms that do not resemble PanIN lesions. Conclusions These results demonstrate that activation of β-catenin is sufficient to induce pancreas tumorigenesis. Moreover, they indicate that the sequence in which oncogenic mutations are acquired has profound consequences on the phenotype of the resulting tumor. PMID:18725219

Therapeutic effect of a novel Wnt pathway inhibitor on cardiac regeneration after myocardial infarction.

PubMed

Yang, Dezhong; Fu, Wenbin; Li, Liangpeng; Xia, Xuewei; Liao, Qiao; Yue, Rongchuan; Chen, Hongmei; Chen, Xiongwen; An, Songzhu; Zeng, Chunyu; Wang, Wei Eric

2017-12-15

After myocardial infarction (MI), the heart is difficult to repair because of great loss of cardiomyoctyes and lack of cardiac regeneration. Novel drug candidates that aim at reducing pathological remodeling and stimulating cardiac regeneration are highly desirable. In the present study, we identified if and how a novel porcupine inhibitor CGX1321 influenced MI and cardiac regeneration. Permanent ligation of left anterior descending (LAD) coronary artery was performed in mice to induce MI injury. Cardiac function was measured by echocardiography, infarct size was examined by TTC staining. Fibrosis was evaluated with Masson's trichrome staining and vimentin staining. As a result, CGX1321 administration blocked the secretion of Wnt proteins, and inhibited both canonical and non-canonical Wnt signaling pathways. CGX1321 improved cardiac function, reduced myocardial infarct size, and fibrosis of post-MI hearts. CGX1321 significantly increased newly formed cardiomyocytes in infarct border zone of post-MI hearts, evidenced by the increased EdU + cardiomyocytes. Meanwhile, CGX1321 increased Ki67 + and phosphohistone H3 (PH3 + ) cardiomyocytes in culture, indicating enhanced cardiomyocyte proliferation. The mRNA microarray showed that CGX1321 up-regulated cell cycle regulating genes such as Ccnb1 and Ccne1 CGX1321 did not alter YAP protein phosphorylation and nuclear translocation in cardiomyocytes. In conclusion, porcupine inhibitor CGX1321 reduces MI injury by limiting fibrosis and promoting regeneration. It promotes cardiomyocyte proliferation by stimulating cell cycle regulating genes with a Hippo/YAP-independent pathway. © 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Wnt2 and WISP-1/CCN4 Induce Intimal Thickening via Promotion of Smooth Muscle Cell Migration.

PubMed

Williams, Helen; Mill, Carina A E; Monk, Bethan A; Hulin-Curtis, Sarah; Johnson, Jason L; George, Sarah J

2016-07-01

Increased vascular smooth muscle cell (VSMC) migration leads to intimal thickening which acts as a soil for atherosclersosis, as well as causing coronary artery restenosis after stenting and vein graft failure. Investigating factors involved in VSMC migration may enable us to reduce intimal thickening and improve patient outcomes. In this study, we determined whether Wnt proteins regulate VSMC migration and thereby intimal thickening. Wnt2 mRNA and protein expression were specifically increased in migrating mouse aortic VSMCs. Moreover, VSMC migration was induced by recombinant Wnt2 in vitro. Addition of recombinant Wnt2 protein increased Wnt1-inducible signaling pathway protein-1 (WISP-1) mRNA by ≈1.7-fold, via β-catenin/T-cell factor signaling, whereas silencing RNA knockdown of Wnt-2 reduced WISP-1 mRNA by ≈65%. Treatment with rWISP-1 significantly increased VSMC migration by ≈1.5-fold, whereas WISP-1 silencing RNA knockdown reduced migration by ≈40%. Wnt2 and WISP-1 effects were integrin-dependent and not additive, indicating that Wnt2 promoted VSMC migration via WISP-1. Additionally, Wnt2 and WISP-1 were significantly increased and colocated in human coronary arteries with intimal thickening. Reduced Wnt2 and WISP-1 levels in mouse carotid arteries from Wnt2(+/-) and WISP-1(-/-) mice, respectively, significantly suppressed intimal thickening in response to carotid artery ligation. In contrast, elevation of plasma WISP-1 via an adenovirus encoding WISP-1 significantly increased intimal thickening by ≈1.5-fold compared with mice receiving control virus. Upregulation of Wnt2 expression enhanced WISP-1 and promoted VSMC migration and thereby intimal thickening. As novel regulators of VSMC migration and intimal thickening, Wnt2 or WISP-1 may provide a potential therapy for restenosis and vein graft failure. © 2016 American Heart Association, Inc.

Wnt-11 overexpression promoting the invasion of cervical cancer cells.

PubMed

Wei, Heng; Wang, Ning; Zhang, Yao; Wang, Shizhuo; Pang, Xiaoao; Zhang, Shulan

2016-09-01

Wnt-11 is a positive regulator of the Wnt signaling pathway, which plays a crucial role in carcinogenesis. However, Wnt-11 expression in cervical cancer has not been well investigated. The aim of this study was to investigate the role of Wnt-11 in cervical tumor proliferation and invasion. This study examined 24 normal cervical squamous epithelia, 29 cervical intraepithelial neoplasia (CIN), and 78 cervical cancer samples. The expression of Wnt-11 was investigated by immunohistochemistry and quantitative reverse transcription-polymerase chain reaction analysis. The expression of the high-risk human papilloma virus (HR-HPV) E6 oncoprotein was also investigated by immunohistochemistry. In addition, the expression of Wnt-11, HR-HPV E6, JNK-1, phosphorylated JNK-1(P-JNK1), and β-catenin was examined by western blot analysis following Wnt-11 knockdown or overexpression in HeLa or SiHa cells, respectively. The promotion of cervical cancer cell proliferation and invasion was investigated using the cell counting kit-8 and Matrigel invasion assay, respectively. Wnt-11 and HR-HPV E6 expression increased in a manner that corresponded with the progression of cervical cancer and was significantly correlated with the International Federation of Gynecology and Obstetrics cancer stage, lymph node metastasis, tumor size, and HPV infection. Wnt-11 protein expression was positively associated with HR-HPV E6 protein expression in all 78 cervical cancer samples (P < 0.001). Furthermore, Wnt-11 was positively associated with P-JNK1 expression and promoted cervical cancer cell proliferation and invasion. These observations suggest that the increased Wnt-11 expression observed in cervical cancer cells may lead to the phosphorylation and activation of JNK-1 and significantly promote tumor cell proliferation and cell migration/invasion through activation of the Wnt/JNK pathway. Consequently, Wnt-11 may serve as a novel target for cervical cancer therapy.

Wnt signaling in bone formation and its therapeutic potential for bone diseases

PubMed Central

Kim, Jeong Hwan; Liu, Xing; Wang, Jinhua; Chen, Xiang; Zhang, Hongyu; Kim, Stephanie H.; Cui, Jing; Li, Ruidong; Zhang, Wenwen; Kong, Yuhan; Zhang, Jiye; Shui, Wei; Lamplot, Joseph; Rogers, Mary Rose; Zhao, Chen; Wang, Ning; Rajan, Prashant; Tomal, Justin; Statz, Joseph; Wu, Ningning; Luu, Hue H.; Haydon, Rex C.

2013-01-01

The Wnt signaling pathway plays an important role not only in embryonic development but also in the maintenance and differentiation of the stem cells in adulthood. In particular, Wnt signaling has been shown as an important regulatory pathway in the osteogenic differentiation of mesenchymal stem cells. Induction of the Wnt signaling pathway promotes bone formation while inactivation of the pathway leads to osteopenic states. Our current understanding of Wnt signaling in osteogenesis elucidates the molecular mechanisms of classic osteogenic pathologies. Activating and inactivating aberrations of the canonical Wnt signaling pathway in osteogenesis results in sclerosteosis and osteoporosis respectively. Recent studies have sought to target the Wnt signaling pathway to treat osteogenic disorders. Potential therapeutic approaches attempt to stimulate the Wnt signaling pathway by upregulating the intracellular mediators of the Wnt signaling cascade and inhibiting the endogenous antagonists of the pathway. Antibodies against endogenous antagonists, such as sclerostin and dickkopf-1, have demonstrated promising results in promoting bone formation and fracture healing. Lithium, an inhibitor of glycogen synthase kinase 3β, has also been reported to stimulate osteogenesis by stabilizing β catenin. Although manipulating the Wnt signaling pathway has abundant therapeutic potential, it requires cautious approach due to risks of tumorigenesis. The present review discusses the role of the Wnt signaling pathway in osteogenesis and examines its targeted therapeutic potential. PMID:23514963

Moving to the Rhythm with Clock (Circadian) Genes, Autophagy, mTOR, and SIRT1 in Degenerative Disease and Cancer.

PubMed

Maiese, Kenneth

2017-01-01

The mammalian circadian clock and its associated clock genes are increasingly been recognized as critical components for a number of physiological and disease processes that extend beyond hormone release, thermal regulation, and sleep-wake cycles. New evidence suggests that clinical behavior disruptions that involve prolonged shift work and even space travel may negatively impact circadian rhythm and lead to multi-system disease. In light of the significant role circadian rhythm can hold over the body's normal physiology as well as disease processes, we examined and discussed the impact circadian rhythm and clock genes hold over lifespan, neurodegenerative disorders, and tumorigenesis. In experimental models, lifespan is significantly reduced with the introduction of arrhythmic mutants and leads to an increase in oxidative stress exposure. Interestingly, patients with Alzheimer's disease and Parkinson's disease may suffer disease onset or progression as a result of alterations in the DNA methylation of clock genes as well as prolonged pharmacological treatment for these disorders that may lead to impairment of circadian rhythm function. Tumorigenesis also can occur with the loss of a maintained circadian rhythm and lead to an increased risk for nasopharyngeal carcinoma, breast cancer, and metastatic colorectal cancer. Interestingly, the circadian clock system relies upon the regulation of the critical pathways of autophagy, the mechanistic target of rapamycin (mTOR), AMP activated protein kinase (AMPK), and silent mating type information regulation 2 homolog 1 (Saccharomyces cerevisiae) (SIRT1) as well as proliferative mechanisms that involve the wingless pathway of Wnt/β-catenin pathway to foster cell survival during injury and block tumor cell growth. Future targeting of the pathways of autophagy, mTOR, SIRT1, and Wnt that control mammalian circadian rhythm may hold the key for the development of novel and effective therapies against aging- related disorders, neurodegenerative disease, and tumorigenesis. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Genome-wide network analysis of Wnt signaling in three pediatric cancers

NASA Astrophysics Data System (ADS)

Bao, Ju; Lee, Ho-Jin; Zheng, Jie J.

2013-10-01

Genomic structural alteration is common in pediatric cancers, and analysis of data generated by the Pediatric Cancer Genome Project reveals such tumor-related alterations in many Wnt signaling-associated genes. Most pediatric cancers are thought to arise within developing tissues that undergo substantial expansion during early organ formation, growth and maturation, and Wnt signaling plays an important role in this development. We examined three pediatric tumors--medullobastoma, early T-cell precursor acute lymphoblastic leukemia, and retinoblastoma--that show multiple genomic structural variations within Wnt signaling pathways. We mathematically modeled this pathway to investigate the effects of cancer-related structural variations on Wnt signaling. Surprisingly, we found that an outcome measure of canonical Wnt signaling was consistently similar in matched cancer cells and normal cells, even in the context of different cancers, different mutations, and different Wnt-related genes. Our results suggest that the cancer cells maintain a normal level of Wnt signaling by developing multiple mutations.

Targeting Wnts at the source--new mechanisms, new biomarkers, new drugs.

PubMed

Madan, Babita; Virshup, David M

2015-05-01

Wnt signaling is dysregulated in many cancers and is therefore an attractive therapeutic target. The focus of drug development has recently shifted away from downstream inhibitors of β-catenin. Active inhibitors of Wnt secretion and Wnt/receptor interactions have been developed that are now entering clinical trials. Such agents include inhibitors of Wnt secretion, as well as recombinant proteins that minimize Wnt-Frizzled interactions. These new therapies arrive together with the recent insight that cancer-specific upregulation of Wnt receptors at the cell surface regulates cellular sensitivity to Wnts. Loss-of-function mutations in RNF43 or ZNRF3 and gain-of-function chromosome translocations involving RSPO2 and RSPO3 are surprisingly common and markedly increase Wnt/β-catenin signaling in response to secreted Wnts. These mutations may be predictive biomarkers to select patients responsive to newly developed upstream Wnt inhibitors. ©2015 American Association for Cancer Research.

Parallel states of pathological Wnt signaling in neonatal brain injury and colon cancer

PubMed Central

Fancy, Stephen P.J.; Harrington, Emily P.; Baranzini, Sergio E.; Silbereis, John C.; Shiow, Lawrence R.; Yuen, Tracy J.; Huang, Eric J.; Lomvardas, Stavros; Rowitch, David H.

2014-01-01

In colon cancer, mutation of the Wnt repressor Adenomatous polyposis coli (APC) leads to a state of aberrant and unrestricted “high-activity” signaling. However, relevance of high Wnt tone in non-genetic human disease is unknown. Here we demonstrate that distinct Wnt activity functional states determine oligodendrocyte precursor (OPC) differentiation and myelination. Murine OPCs with genetic Wnt dysregulation (high tone) express multiple genes in common with colon cancer including Lef1, SP5, Ets2, Rnf43 and Dusp4. Surprisingly, we find that OPCs in lesions of hypoxic human neonatal white matter injury upregulate markers of high Wnt activity and lack expression of APC. Finally, we show lack of Wnt repressor tone promotes permanent white matter injury after mild hypoxic insult. These findings suggest a state of pathological high-activity Wnt signaling in human disease tissues that lack pre-disposing genetic mutation. PMID:24609463

In vivo activation of Wnt signaling pathway enhances cognitive function of adult mice and reverses cognitive deficits in an Alzheimer's disease model.

PubMed

Vargas, Jessica Y; Fuenzalida, Marco; Inestrosa, Nibaldo C

2014-02-05

The role of the Wnt signaling pathway during synaptic development has been well established. In the adult brain, different components of Wnt signaling are expressed, but little is known about its role in mature synapses. Emerging in vitro studies have implicated Wnt signaling in synaptic plasticity. Furthermore, activation of Wnt signaling has shown to protect against amyloid-β-induced synaptic impairment. The present study provides the first evidence that in vivo activation of Wnt signaling improves episodic memory, increases excitatory synaptic transmission, and enhances long-term potentiation in adult wild-type mice. Moreover, the activation of Wnt signaling also rescues memory loss and improves synaptic dysfunction in APP/PS1-transgenic mice that model the amyloid pathology of Alzheimer's diseases. These findings indicate that Wnt signaling modulates cognitive function in the adult brain and could be a novel promising target for Alzheimer's disease therapy.

Activation of the hypoxia-inducible factor 1α promotes myogenesis through the noncanonical Wnt pathway, leading to hypertrophic myotubes.

PubMed

Cirillo, Federica; Resmini, Giulia; Ghiroldi, Andrea; Piccoli, Marco; Bergante, Sonia; Tettamanti, Guido; Anastasia, Luigi

2017-05-01

Regeneration of skeletal muscle is a complex process that requires the activation of quiescent adult stem cells, called satellite cells, which are resident in hypoxic niches in the tissue. Hypoxia has been recognized as a key factor to maintain stem cells in an undifferentiated state. Herein we report that hypoxia plays a fundamental role also in activating myogenesis. In particular, we found that the activation of the hypoxia-inducible factor (HIF)-1α under hypoxia, in murine skeletal myoblasts, leads to activation of MyoD through the noncanonical Wnt/β-catenin pathway. Moreover, chemical inhibition of HIF-1α activity significantly reduces differentiation, thus confirming its crucial role in the process. Furthermore, hypoxia-preconditioned myoblasts, once induced to differentiate under normoxic conditions, tend to form hypertrophic myotubes. These results support the notion that hypoxia plays a pivotal role in activating the regeneration process by directly inducing myogenesis through HIF-1α. Although preliminary, these findings may suggest new perspective for novel therapeutic targets in the treatment of several muscle diseases.-Cirillo, F., Resmini, G., Ghiroldi, A., Piccoli, M., Bergante, S., Tettamanti, G., Anastasia, L. Activation of the hypoxia-inducible factor 1α promotes myogenesis through the noncanonical Wnt pathway, leading to hypertrophic myotubes. © FASEB.

Wnt modulates MCL1 to control cell survival in triple negative breast cancer

PubMed Central

2014-01-01

Background Triple negative breast cancer (TNBC) has higher rates of recurrence and distant metastasis, and poorer outcome as compared to non-TNBC. Aberrant activation of WNT signaling has been detected in TNBC, which might be important for triggering oncogenic conversion of breast epithelial cell. Therefore, we directed our focus on identifying the WNT ligand and its underlying mechanism in TNBC cells. Methods We performed large-scale analysis of public microarray data to screen the WNT ligands and the clinical significance of the responsible ligand in TNBC. WNT5B was identified and its overexpression in TNBC was confirmed by immunohistochemistry staining, Western blot and ELISA. ShRNA was used to knockdown WNT5B expression (shWNT5B). Cellular functional alteration with shWNT5B treatment was determined by using wound healing assay, mammosphere assay; while cell cycle and apoptosis were examined by flowcytometry. Mitochondrial morphology was photographed by electron microscope. Biological change of mitochondria was detected by RT-PCR and oxygen consumption assay. Activation of WNT pathway and its downstream targets were evaluated by liciferase assay, immunohistochemistry staining and immunoblot analysis. Statistical methods used in the experiments besides microarray analysis was two-tailed t-test. Results WNT5B was elevated both in the tumor and the patients’ serum. Suppression of WNT5B remarkably impaired cell growth, migration and mammosphere formation. Additionally, G0/G1 cell cycle arrest and caspase-independent apoptosis was observed. Study of the possible mechanism indicated that these effects occurred through suppression of mitochondrial biogenesis, as evidenced by reduced mitochondrial DNA (MtDNA) and compromised oxidative phosphorylation (OXPHOS). In Vivo and in vitro data uncovered that WNT5B modulated mitochondrial physiology was mediated by MCL1, which was regulated by WNT/β-catenin responsive gene, Myc. Clinic data analysis revealed that both WNT5B and MCL1 are associated with enhanced metastasis and decreased disease-free survival. Conclusions All our findings suggested that WNT5B/MCL1 cascade is critical for TNBC and understanding its regulatory apparatus provided valuable insight into the pathogenesis of the tumor development and the guidance for targeting therapeutics. PMID:24564888

The pan-PI3K inhibitor GDC-0941 activates canonical WNT signaling to confer resistance in TNBC cells: resistance reversal with WNT inhibitor.

PubMed

Tzeng, Huey-En; Yang, Lixin; Chen, Kemin; Wang, Yafan; Liu, Yun-Ru; Pan, Shiow-Lin; Gaur, Shikha; Hu, Shuya; Yen, Yun

2015-05-10

The pan-PI3K inhibitors are one treatment option for triple-negative breast cancer (TNBC). However, this treatment is ineffective for unknown reasons. Here, we report that aberrant expression of wingless-type MMTV integration site family (WNT) and activated WNT signals, which crosstalk with the PI3K-AKT-mTOR signaling pathway through GSK3β, plays the most critical role in resistance to pan-PI3K inhibitors in TNBC cells. GDC-0941 is a pan-PI3K inhibitor that activates the WNT/beta-catenin pathway in TNBC cells through stimulation of WNT secretion. GDC-0941-triggered WNT/beta-catenin pathway activation was observed in MDA-MB-231 and HCC1937 cells, which are TNBC cell lines showing aberrant WNT/beta-catenin activation, and not in SKBR3 and MCF7 cells. This observation is further investigated in vivo. GDC-0941 exhibited minimal tumor inhibition in MDA-MB-231 cells, but it significantly suppressed tumor growth in HER-positive SK-BR3 cells. In vivo mechanism study revealed the activation of WNT/beta-catenin pathway by GDC-0941. A synergistic effect was observed when combined treatment with GDC-0941 and the WNT inhibitor LGK974 at low concentrations in MDA-MB-231 cells. These findings indicated that WNT pathway activation conferred resistance in TNBC cells treated with GDC-0941. This resistance may be further circumvented through combined treatment with pan-PI3K and WNT inhibitors. Future clinical trials of these two inhibitors are warranted.

The pan-PI3K inhibitor GDC-0941 activates canonical WNT signaling to confer resistance in TNBC cells: resistance reversal with WNT inhibitor

PubMed Central

Tzeng, Huey-En; Yang, Lixin; Chen, Kemin; Wang, Yafan; Liu, Yun-Ru; Pan, Shiow-Lin; Gaur, Shikha; Hu, Shuya; Yen, Yun

2015-01-01

The pan-PI3K inhibitors are one treatment option for triple-negative breast cancer (TNBC). However, this treatment is ineffective for unknown reasons. Here, we report that aberrant expression of wingless-type MMTV integration site family (WNT) and activated WNT signals, which crosstalk with the PI3K-AKT-mTOR signaling pathway through GSK3β, plays the most critical role in resistance to pan-PI3K inhibitors in TNBC cells. GDC-0941 is a pan-PI3K inhibitor that activates the WNT/beta-catenin pathway in TNBC cells through stimulation of WNT secretion. GDC-0941-triggered WNT/beta-catenin pathway activation was observed in MDA-MB-231 and HCC1937 cells, which are TNBC cell lines showing aberrant WNT/beta-catenin activation, and not in SKBR3 and MCF7 cells. This observation is further investigated in vivo. GDC-0941 exhibited minimal tumor inhibition in MDA-MB-231 cells, but it significantly suppressed tumor growth in HER-positive SK-BR3 cells. In vivo mechanism study revealed the activation of WNT/beta-catenin pathway by GDC-0941. A synergistic effect was observed when combined treatment with GDC-0941 and the WNT inhibitor LGK974 at low concentrations in MDA-MB-231 cells. These findings indicated that WNT pathway activation conferred resistance in TNBC cells treated with GDC-0941. This resistance may be further circumvented through combined treatment with pan-PI3K and WNT inhibitors. Future clinical trials of these two inhibitors are warranted. PMID:25857298

Expression profile and function of Wnt signaling mechanisms in malignant mesothelioma cells

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

Fox, Simon A., E-mail: s.fox@curtin.edu.au; Richards, Alex K.; Kusumah, Ivonne

Highlights: •Expression profile of Wnt pathway related genes in mesothelioma cells. •Differential expression of key Wnt pathway molecules and regulators. •Wnt3a stimulated mesothelioma growth whereas sFRP4 was inhibitory. •Targeting β-Catenin can sensitise mesothelioma cells to cytotoxic drugs. -- Abstract: Malignant mesothelioma (MM) is an uncommon and particularly aggressive cancer associated with asbestos exposure, which currently presents an intractable clinical challenge. Wnt signaling has been reported to play a role in the neoplastic properties of mesothelioma cells but has not been investigated in detail in this cancer. We surveyed expression of Wnts, their receptors, and other key molecules in this pathwaymore » in well established in vitro mesothelioma models in comparison with primary mesothelial cultures. We also tested the biological response of MM cell lines to exogenous Wnt and secreted regulators, as well as targeting β-catenin. We detected frequent expression of Wnt3 and Wnt5a, as well as Fzd 2, 4 and 6. The mRNA of Wnt4, Fzd3, sFRP4, APC and axin2 were downregulated in MM relative to mesothelial cells while LEF1 was overexpressed in MM. Functionally, we observed that Wnt3a stimulated MM proliferation while sFRP4 was inhibitory. Furthermore, directly targeting β-catenin expression could sensitise MM cells to cytotoxic drugs. These results provide evidence for altered expression of a number of Wnt/Fzd signaling molecules in MM. Modulation of Wnt signaling in MM may prove a means of targeting proliferation and drug resistance in this cancer.« less

WNT10B Functional Dualism: β-Catenin/Tcf-dependent Growth Promotion or Independent Suppression with Deregulated Expression in Cancer

PubMed Central

Yoshikawa, Hirohide; Matsubara, Kenichi; Zhou, Xiaoling; Okamura, Shu; Kubo, Takahiko; Murase, Yaeko; Shikauchi, Yuko; Esteller, Manel; Herman, James G.; Wei Wang, Xin

2007-01-01

We found aberrant DNA methylation of the WNT10B promoter region in 46% of primary hepatocellular carcinoma (HCC) and 15% of colon cancer samples. Three of 10 HCC and one of two colon cancer cell lines demonstrated low or no expression, and 5-aza-2′deoxycytidine reactivated WNT10B expression with the induction of demethylation, indicating that WNT10B is silenced by DNA methylation in some cancers, whereas WNT10B expression is up-regulated in seven of the 10 HCC cell lines and a colon cancer cell line. These results indicate that WNT10B can be deregulated by either overexpression or silencing in cancer. We found that WNT10B up-regulated β-catenin/Tcf activity. However, WNT10B-overexpressing cells demonstrated a reduced growth rate and anchorage-independent growth that is independent of the β-catenin/Tcf activation, because mutant β-catenin–transduced cells did not suppress growth, and dominant-negative hTcf-4 failed to alleviate the growth suppression by WNT10B. Although WNT10B expression alone inhibits cell growth, it acts synergistically with the fibroblast growth factor (FGF) to stimulate cell growth. WNT10B is bifunctional, one function of which is involved in β-catenin/Tcf activation, and the other function is related to the down-regulation of cell growth through a different mechanism. We suggest that FGF switches WNT10B from a negative to a positive cell growth regulator. PMID:17761539

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

Ouji, Yukiteru, E-mail: oujix@naramed-u.ac.jp; Nakamura-Uchiyama, Fukumi; Yoshikawa, Masahide, E-mail: myoshika@naramed-u.ac.jp

Highlights: •First report on effects of various Wnts on DP cells. •Wnt-10b promoted trichogenesis, while Wnt-3a showed to a limited extent. •Canonical Wnts, specifically Wnt-10b, is important for DP cells maintenance. -- Abstract: Although Wnts are expressed in hair follicles (HFs) and considered to be crucial for maintaining dermal papilla (DP) cells, the functional differences among them remain largely unknown. In the present study, we investigated the effects of Wnts (Wnt-3a, 5a, 10b, 11) on the proliferation of mouse-derived primary DP cells in vitro as well as their trichogenesis-promoting ability using an in vivo skin reconstitution protocol. Wnt-10b promoted cellmore » proliferation and trichogenesis, while Wnt-3a showed those abilities to a limited extent, and Wnt-5a and 11 had no effects. Furthermore, we investigated the effects of these Wnts on cultured DP cells obtained from versican-GFP transgenic mice and found that Wnt-10b had a potent ability to sustain their GFP-positivity. These results suggest that canonical Wnts, specifically Wnt-10b, play important roles in the maintenance of DP cells and trichogenesis.« less

Syndecan-4 inhibits Wnt/β-catenin signaling through regulation of low-density-lipoprotein receptor-related protein (LRP6) and R-spondin 3.

PubMed

Astudillo, Pablo; Carrasco, Héctor; Larraín, Juan

2014-01-01

Regulation of Wnt signaling is crucial for embryonic development and adult homeostasis. Here we study the role of Syndecan-4 (SDC4), a cell-surface heparan sulphate proteoglycan, and Fibronectin (FN), in Wnt/β-catenin signaling. Gain- and loss-of-function experiments in mammalian cell lines and Xenopus embryos demonstrate that SDC4 and FN inhibit Wnt/β-catenin signaling. Epistatic and biochemical experiments show that this inhibition occurs at the cell membrane level through regulation of LRP6. R-spondin 3, a ligand that promotes canonical and non-canonical Wnt signaling, is more prone to potentiate Wnt/β-catenin signaling when SDC4 levels are reduced, suggesting a model whereby SDC4 tunes the ability of R-spondin to modulate the different Wnt signaling pathways. Since SDC4 has been previously related to non-canonical Wnt signaling, our results also suggest that this proteoglycan can be a key component in the regulation of Wnt signaling. Copyright © 2013 Elsevier Ltd. All rights reserved.

ATG5 mediates a positive feedback loop between Wnt signaling and autophagy in melanoma

PubMed Central

Ndoye, Abibatou; Budina-Kolomets, Anna; Kugel, Curtis H.; Webster, Marie; Kaur, Amanpreet; Behera, Reeti; Rebecca, Vito; Li, Ling; Brafford, Patricia; Liu, Qin; Gopal, Y.N. Vashisht; Davies, Michael A.; Mills, Gordon B.; Xu, Xiaowei; Wu, Hong; Herlyn, Meenhard; Nicastri, Michael; Winkler, Jeffrey; Soengas, Maria S.; Amaravadi, Ravi; Murphy, Maureen; Weeraratna, Ashani T.

2017-01-01

Autophagy mediates resistance to various anticancer agents. In melanoma, resistance to targeted therapy has been linked to expression of Wnt5A, an intrinsic inhibitor of β-catenin, which also promotes invasion. In this study, we assessed the interplay between Wnt5A and autophagy by combining expression studies in human clinical biopsies with functional analyses in cell lines and mouse models. Melanoma cells with high Wnt5A and low β-catenin displayed increased basal autophagy. Genetic blockade of autophagy revealed an unexpected feedback loop whereby knocking down the autophagy factor ATG5 in Wnt5Ahigh cells decreased Wnt5A and increased β-catenin. To define the physiological relevance of this loop, melanoma cells with different Wnt status were treated in vitro and in vivo with the potent lysosomotropic compound Lys05. Wnt5Ahigh cells were less sensitive to Lys05 and could be reverted by inducing β-catenin activity. Our results suggest the efficacy of autophagy inhibitors might be improved by taking the Wnt signature of melanoma cells into account. PMID:28887323

Derricin and Derricidin Inhibit Wnt/β-Catenin Signaling and Suppress Colon Cancer Cell Growth In Vitro

PubMed Central

Fonseca, Barbara F.; Predes, Danilo; Cerqueira, Debora M.; Reis, Alice H.; Amado, Nathalia G.; Cayres, Marina C. L.; Kuster, Ricardo M.; Oliveira, Felipe L.; Mendes, Fabio A.; Abreu, Jose G.

2015-01-01

Overactivation of the Wnt/β-catenin pathway in adult tissues has been implicated in many diseases, such as colorectal cancer. Finding chemical substances that can prevent this phenomenon is an emerging problem. Recently, several natural compounds have been described as Wnt/β-catenin inhibitors and might be promising agents for the control of carcinogenesis. Here, we describe two natural substances, derricin and derricidin, belonging to the chalcone subclass, that show potent transcriptional inhibition of the Wnt/β-catenin pathway. Both chalcones are able to affect the cell distribution of β-catenin, and inhibit Wnt-specific reporter activity in HCT116 cells and in Xenopus embryos. Derricin and derricidin also strongly inhibited canonical Wnt activity in vitro, and rescued the Wnt-induced double axis phenotype in Xenopus embryos. As a consequence of Wnt/β-catenin inhibition, derricin and derricidin treatments reduce cell viability and lead to cell cycle arrest in colorectal cancer cell lines. Taken together, our results strongly support these chalcones as novel negative modulators of the Wnt/β-catenin pathway and colon cancer cell growth in vitro. PMID:25775405

The Anti-Helminthic Niclosamide Inhibits Wnt/Frizzled1 Signaling†

PubMed Central

Chen, Minyong; Wang, Jiangbo; Lu, Jiuyi; Bond, Michael C.; Ren, Xiu-Rong; Lyerly, H. Kim; Barak, Larry S.; Chen, Wei

2009-01-01

Wnt proteins bind to seven-transmembrane Frizzled receptors to mediate the important developmental, morphogenetic, and tissue-regenerative effects of Wnt signaling. Dysregulated Wnt signaling is associated with many cancers. Currently there exist no drug candidates, or even tool compounds that modulate Wnt-mediated receptor trafficking, and subsequent Wnt signaling. We examined libraries of FDA-approved drugs for their utility as Frizzled internalization modulators, employing a primary imaged-based GFP-fluorescence assay that uses Frizzled1 endocytosis as the readout. We now report that the anti-helminthic niclosamide, a drug used for the treatment of tapeworm, promotes Frizzled1 endocytosis, down regulates Dishevelled-2 protein, and inhibits Wnt3A-stimulated β-catenin stabilization and LEF/TCF reporter activity. Additionally, following niclosamide mediated internalization, the Frizzled1 receptor co-localizes in vesicles containing Transferrin and agonist-activated β2-adrenergic receptor. Therefore, niclosamide may serve as a negative modulator of Wnt/Frizzled1 signaling by depleting up-stream signaling molecules (i.e. Frizzled and Dishevelled), and moreover may provide a valuable means to study the physiological consequences of Wnt signaling. PMID:19772353

Evolutionary divergence in the catalytic activity of the CAM-1, ROR1 and ROR2 kinase domains.

PubMed

Bainbridge, Travis W; DeAlmeida, Venita I; Izrael-Tomasevic, Anita; Chalouni, Cécile; Pan, Borlan; Goldsmith, Joshua; Schoen, Alia P; Quiñones, Gabriel A; Kelly, Ryan; Lill, Jennie R; Sandoval, Wendy; Costa, Mike; Polakis, Paul; Arnott, David; Rubinfeld, Bonnee; Ernst, James A

2014-01-01

Receptor tyrosine kinase-like orphan receptors (ROR) 1 and 2 are atypical members of the receptor tyrosine kinase (RTK) family and have been associated with several human diseases. The vertebrate RORs contain an ATP binding domain that deviates from the consensus amino acid sequence, although the impact of this deviation on catalytic activity is not known and the kinase function of these receptors remains controversial. Recently, ROR2 was shown to signal through a Wnt responsive, β-catenin independent pathway and suppress a canonical Wnt/β-catenin signal. In this work we demonstrate that both ROR1 and ROR2 kinase domains are catalytically deficient while CAM-1, the C. elegans homolog of ROR, has an active tyrosine kinase domain, suggesting a divergence in the signaling processes of the ROR family during evolution. In addition, we show that substitution of the non-consensus residues from ROR1 or ROR2 into CAM-1 and MuSK markedly reduce kinase activity, while restoration of the consensus residues in ROR does not restore robust kinase function. We further demonstrate that the membrane-bound extracellular domain alone of either ROR1 or ROR2 is sufficient for suppression of canonical Wnt3a signaling, and that this domain can also enhance Wnt5a suppression of Wnt3a signaling. Based on these data, we conclude that human ROR1 and ROR2 are RTK-like pseudokinases.

zic-1 Expression in Planarian Neoblasts after Injury Controls Anterior Pole Regeneration

PubMed Central

Vásquez-Doorman, Constanza; Petersen, Christian P.

2014-01-01

Mechanisms that enable injury responses to prompt regenerative outgrowth are not well understood. Planarians can regenerate essentially any tissue removed by wounding, even after decapitation, due to robust regulation of adult pluripotent stem cells of the neoblast population. Formation of pole signaling centers involving Wnt inhibitors or Wnt ligands promotes head or tail regeneration, respectively, and this process requires the use of neoblasts early after injury. We used expression profiling of purified neoblasts to identify factors needed for anterior pole formation. Using this approach, we identified zic-1, a Zic-family transcription factor, as transcriptionally activated in a subpopulation of neoblasts near wound sites early in head regeneration. As head regeneration proceeds, the Wnt inhibitor notum becomes expressed in the newly forming anterior pole in zic-1-expressing cells descended from neoblasts. Inhibition of zic-1 by RNAi resulted in a failure to express notum at the anterior pole and to regenerate a head, but did not affect tail regeneration. Both injury and canonical Wnt signaling inhibition are required for zic-1 expression, and double-RNAi experiments suggest zic-1 inhibits Wnt signaling to allow head regeneration. Analysis of neoblast fate determinants revealed that zic-1 controls specification of notum-expressing cells from foxD-expressing neoblasts to form the anterior pole, which organizes subsequent outgrowth. Specialized differentiation programs may in general underlie injury-dependent formation of tissue organizing centers used for regenerative outgrowth. PMID:24992682

Unsaturated fatty acyl recognition by Frizzled receptors mediates dimerization upon Wnt ligand binding

PubMed Central

Nile, Aaron H.; Mukund, Susmith; Stanger, Karen; Wang, Weiru; Hannoush, Rami N.

2017-01-01

Frizzled (FZD) receptors mediate Wnt signaling in diverse processes ranging from bone growth to stem cell activity. Moreover, high FZD receptor expression at the cell surface contributes to overactive Wnt signaling in subsets of pancreatic, ovarian, gastric, and colorectal tumors. Despite the progress in biochemical understanding of Wnt–FZD receptor interactions, the molecular basis for recognition of Wnt cis-unsaturated fatty acyl groups by the cysteine-rich domain (CRD) of FZD receptors remains elusive. Here, we determined a crystal structure of human FZD7 CRD unexpectedly bound to a 24-carbon fatty acid. We also report a crystal structure of human FZD5 CRD bound to C16:1 cis-Δ9 unsaturated fatty acid. Both structures reveal a dimeric arrangement of the CRD. The lipid-binding groove exhibits flexibility and spans both monomers, adopting a U-shaped geometry that accommodates the fatty acid. Re-evaluation of the published mouse FZD8 CRD structure reveals that it also shares the same architecture as FZD5 and FZD7 CRDs. Our results define a common molecular mechanism for recognition of the cis-unsaturated fatty acyl group, a necessary posttranslational modification of Wnts, by multiple FZD receptors. The fatty acid bridges two CRD monomers, implying that Wnt binding mediates FZD receptor dimerization. Our data uncover possibilities for the arrangement of Wnt–FZD CRD complexes and shed structural insights that could aide in the identification of pharmacological strategies to modulate FZD receptor function. PMID:28377511

Sonic Hedgehog switches on Wnt/planar cell polarity signaling in commissural axon growth cones by reducing levels of Shisa2

PubMed Central

Onishi, Keisuke

2017-01-01

Commissural axons switch on responsiveness to Wnt attraction during midline crossing and turn anteriorly only after exiting the floor plate. We report here that Sonic Hedgehog (Shh)-Smoothened signaling downregulates Shisa2, which inhibits the glycosylation and cell surface presentation of Frizzled3 in rodent commissural axon growth cones. Constitutive Shisa2 expression causes randomized turning of post-crossing commissural axons along the anterior–posterior (A–P) axis. Loss of Shisa2 led to precocious anterior turning of commissural axons before or during midline crossing. Post-crossing commissural axon turning is completely randomized along the A–P axis when Wntless, which is essential for Wnt secretion, is conditionally knocked out in the floor plate. This regulatory link between Shh and planar cell polarity (PCP) signaling may also occur in other developmental processes. PMID:28885142

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

Rawal, Nina; Corti, Olga; CNRS, UMR 7225, Paris

Parkinson's disease (PD) is caused by degeneration of the dopaminergic (DA) neurons of the substantia nigra but the molecular mechanisms underlying the degenerative process remain elusive. Several reports suggest that cell cycle deregulation in post-mitotic neurons could lead to neuronal cell death. We now show that Parkin, an E3 ubiquitin ligase linked to familial PD, regulates {beta}-catenin protein levels in vivo. Stabilization of {beta}-catenin in differentiated primary ventral midbrain neurons results in increased levels of cyclin E and proliferation, followed by increased levels of cleaved PARP and loss of DA neurons. Wnt3a signaling also causes death of post-mitotic DA neuronsmore » in parkin null animals, suggesting that both increased stabilization and decreased degradation of {beta}-catenin results in DA cell death. These findings demonstrate a novel regulation of Wnt signaling by Parkin and suggest that Parkin protects DA neurons against excessive Wnt signaling and {beta}-catenin-induced cell death.« less

Canonical Wnt Signaling as a Specific Mark of Normal and Tumorigenic Mammary Stem Cells

DTIC Science & Technology

2011-02-01

aggressive mammary tumors. 15. SUBJECT TERMS Breast cancer stem cells, Wnt signaling, canonical Wnt signaling, B-catenin, normal stem cells, adult stem...Wnt pathway is associated with abnormal mouse mammary development, tumorigenesis, and human breast cancer. In addition, increasing evidence suggests...activation occurs in human breast cancer and is required for proliferation of various other stem cell compartments, addressing how Wnt signaling promotes

Methylation of Wnt7a Is Modulated by DNMT1 and Cigarette Smoke Condensate in Non-Small Cell Lung Cancer

PubMed Central

Tennis, Meredith A.; VanScoyk, Michelle M.; Wilson, Lora A.; Kelley, Nicole; Winn, Robert A.

2012-01-01

Wnt7a is known to be a tumor suppressor that is lost in NSCLC, but no mechanism of loss has been established. Methylation of promoter regions has been established as a common mechanism of loss of tumor suppressor expression in NSCLC. We previously demonstrated that loss of Wnt7a in non-transformed lung epithelial cell lines led to increased cell growth, altered 3-D culture growth, and increased migration. The Wnt7a promoter has a higher percentage of methylation in NSCLC tumor tissue compared to matched normal lung tissue and methylation of the promoter region leads to decreased activity. We treated H157 and H1299 NSCLC cell lines with 5-Aza-2′-deoxycytidine and detected loss of Wnt7a promoter methylation, increased Wnt7a expression, and increased activity of the Wnt7a lung signaling pathway. When DNMT1 expression was knocked down by shRNA, expression of Wnt7a increased and methylation decreased. Together these data suggest that in NSCLC, Wnt7a is lost by methylation in a subset of tumors and that this methylation is maintained by DNMT1. Restoration of Wnt7a expression through demethylation could be an important therapeutic approach in the treatment of NSCLC. PMID:22403725

Wnt/β-catenin signaling enables developmental transitions during valvulogenesis

PubMed Central

Bosada, Fernanda M.; Devasthali, Vidusha; Jones, Kimberly A.; Stankunas, Kryn

2016-01-01

Heart valve development proceeds through coordinated steps by which endocardial cushions (ECs) form thin, elongated and stratified valves. Wnt signaling and its canonical effector β-catenin are proposed to contribute to endocardial-to-mesenchymal transformation (EMT) through postnatal steps of valvulogenesis. However, genetic redundancy and lethality have made it challenging to define specific roles of the canonical Wnt pathway at different stages of valve formation. We developed a transgenic mouse system that provides spatiotemporal inhibition of Wnt/β-catenin signaling by chemically inducible overexpression of Dkk1. Unexpectedly, this approach indicates canonical Wnt signaling is required for EMT in the proximal outflow tract (pOFT) but not atrioventricular canal (AVC) cushions. Furthermore, Wnt indirectly promotes pOFT EMT through its earlier activity in neighboring myocardial cells or their progenitors. Subsequently, Wnt/β-catenin signaling is activated in cushion mesenchymal cells where it supports FGF-driven expansion of ECs and then AVC valve extracellular matrix patterning. Mice lacking Axin2, a negative Wnt regulator, have larger valves, suggesting that accumulating Axin2 in maturing valves represents negative feedback that restrains tissue overgrowth rather than simply reporting Wnt activity. Disruption of these Wnt/β-catenin signaling roles that enable developmental transitions during valvulogenesis could account for common congenital valve defects. PMID:26893350

Wnt signaling regulates pancreatic β cell proliferation

PubMed Central

Rulifson, Ingrid C.; Karnik, Satyajit K.; Heiser, Patrick W.; ten Berge, Derk; Chen, Hainan; Gu, Xueying; Taketo, Makoto M.; Nusse, Roel; Hebrok, Matthias; Kim, Seung K.

2007-01-01

There is widespread interest in defining factors and mechanisms that stimulate proliferation of pancreatic islet cells. Wnt signaling is an important regulator of organ growth and cell fates, and genes encoding Wnt-signaling factors are expressed in the pancreas. However, it is unclear whether Wnt signaling regulates pancreatic islet proliferation and differentiation. Here we provide evidence that Wnt signaling stimulates islet β cell proliferation. The addition of purified Wnt3a protein to cultured β cells or islets promoted expression of Pitx2, a direct target of Wnt signaling, and Cyclin D2, an essential regulator of β cell cycle progression, and led to increased β cell proliferation in vitro. Conditional pancreatic β cell expression of activated β-catenin, a crucial Wnt signal transduction protein, produced similar phenotypes in vivo, leading to β cell expansion, increased insulin production and serum levels, and enhanced glucose handling. Conditional β cell expression of Axin, a potent negative regulator of Wnt signaling, led to reduced Pitx2 and Cyclin D2 expression by β cells, resulting in reduced neonatal β cell expansion and mass and impaired glucose tolerance. Thus, Wnt signaling is both necessary and sufficient for islet β cell proliferation, and our study provides previously unrecognized evidence of a mechanism governing endocrine pancreas growth and function. PMID:17404238

Atypical regulators of Wnt/β-catenin signaling as potential therapeutic targets in Hepatocellular Carcinoma.

PubMed

Chen, Jianxiang; Rajasekaran, Muthukumar; Hui, Kam M

2017-06-01

Hepatocellular carcinoma is one of the most common causes of cancer-related death worldwide. Hepatocellular carcinoma development depends on the inhibition and activation of multiple vital pathways, including the Wnt signaling pathway. The Wnt/β-catenin pathway lies at the center of various signaling pathways that regulate embryonic development, tissue homeostasis and cancers. Activation of the Wnt/β-catenin pathway has been observed frequently in hepatocellular carcinoma. However, activating mutations in β-catenin, Axin and Adenomatous Polyposis Coli only contribute to a portion of the Wnt signaling hyper-activation observed in hepatocellular carcinoma. Therefore, besides mutations in the canonical Wnt components, there must be additional atypical regulation or regulators during Wnt signaling activation that promote liver carcinogenesis. In this mini-review, we have tried to summarize some of these well-established factors and to highlight some recently identified novel factors in the Wnt/β-catenin signaling pathway in hepatocellular carcinoma. Impact statement Early recurrence of human hepatocellular carcinoma (HCC) is a frequent cause of poor survival after potentially curative liver resection. Among the deregulated signaling cascades in HCC, evidence indicates that alterations in the Wnt/β-catenin signaling pathway play key roles in hepatocarcinogenesis. In this review, we summarize the potential molecular mechanisms how the microtubule-associated Protein regulator of cytokinesis 1 (PRC1), a direct Wnt signaling target previously identified in our laboratory to be up-regulated in HCC, in promoting cancer proliferation, stemness, metastasis and tumorigenesis through a complex regulatory circuitry of Wnt3a activities.

Testis development requires the repression of Wnt4 by Fgf signaling

PubMed Central

Jameson, Samantha A.; Lin, Yi-Tzu; Capel, Blanche

2013-01-01

The bipotential gonad expresses genes associated with both the male and female pathways. Adoption of the male testicular fate is associated with the repression of many female genes including Wnt4. However, the importance of repression of Wnt4 to the establishment of male development was not previously determined. Deletion of either Fgf9 or Fgfr2 in an XY gonad resulted in up-regulation of Wnt4 and male-to-female sex reversal. We investigated whether the deletion if Wnt4 could rescue sex reversal in Fgf9 and Fgfr2 mutants. XY Fgf9/Wnt4 and Fgfr2/Wnt4 double mutants developed testes with male somatic and germ cells present, suggesting that the primary role of Fgf signaling is the repression of female-promoting genes. Thus, the decision to adopt the male fate is based not only on whether male genes, such as Sox9, are expressed, but also on the active repression of female genes, such as Wnt4. Because loss of Wnt4 results in the up-regulation of Fgf9, we also tested the possibility that derepression of Fgf9 was responsible for the aspects of male development observed in XX Wnt4 mutants. However, we found that the relationship between these two signaling factors is not symmetric: loss of Fgf9 in XX Wnt4−/− gonads does not rescue their partial female-to-male sex-reversal. PMID:22705479

Sequential changes in the expression of Wnt- and Notch-related genes in the vagina and uterus of ovariectomized mice after estrogen exposure.

PubMed

Nakamura, Takeshi; Miyagawa, Shinichi; Katsu, Yoshinao; Sato, Tomomi; Iguchi, Taisen; Ohta, Yasuhiko

2012-01-01

Estrogen regulates morphological changes in reproductive organs, such as the vagina and uterus, during the estrous cycles in mice. Estrogen depletion by ovariectomy in adults results in atrophy accompanied by apoptosis in vaginal and uterine cells, while estrogen treatment following ovariectomy elicits cell proliferation in both organs. Sequential changes in mRNA expression of wingless-related MMTV integration site (Wnt) and Notch signaling genes were analyzed in the vagina and uterus of ovariectomized adult mice after a single injection of 17β-estradiol to provide understanding over the molecular basis of differences in response to estrogen in these organs. We found estrogen-dependent up-regulation of Wnt4, Wnt5a and p21 and down-regulation of Wnt11, hairy/enhancer-of-split related with YRPW motif-1 (Hey1) and delta-like 4 (Dll4) in the vagina, and up-regulation of Wnt4, Wnt5a, Hey1, Heyl, Dll1, p21 and p53 and down-regulation of Wnt11, Hey2 and Dll4 in the uterus. The expression of Wnt4, Hey1, Hey2, Heyl, Dll1 and p53 showed different patterns after the estrogen injection. Expression patterns for Wnt5a, Wnt11, Dll4 and p21 in the vagina and uterus were similar, suggesting that these genes are involved in the proliferation of cells in both those organs in mice.

WNT4 is a key regulator of normal postnatal uterine development and progesterone signaling during embryo implantation and decidualization in the mouse

PubMed Central

Franco, Heather L.; Dai, Daisy; Lee, Kevin Y.; Rubel, Cory A.; Roop, Dennis; Boerboom, Derek; Jeong, Jae-Wook; Lydon, John P.; Bagchi, Indrani C.; Bagchi, Milan K.; DeMayo, Francesco J.

2011-01-01

WNT4, a member of the Wnt family of ligands, is critical for the development of the female reproductive tract. Analysis of Wnt4 expression in the adult uterus during pregnancy indicates that it may play a role in the regulation of endometrial stromal cell proliferation, survival, and differentiation, which is required to support the developing embryo. To investigate the role of Wnt4 in adult uterine physiology, conditional ablation of Wnt4 using the PRcre mouse model was accomplished. Ablation of Wnt4 rendered female mice subfertile due to a defect in embryo implantation and subsequent defects in endometrial stromal cell survival, differentiation, and responsiveness to progesterone signaling. In addition to altered stromal cell function, the uteri of PRcre/+Wnt4f/f (Wnt4d/d) mice displayed altered epithelial differentiation characterized by a reduction in the number of uterine glands and the emergence of a p63-positive basal cell layer beneath the columnar luminal epithelial cells. The altered epithelial cell phenotype was further escalated by chronic estrogen treatment, which caused squamous cell metaplasia of the uterine epithelium in the Wnt4d/d mice. Thus, WNT4 is a critical regulator not only of proper postnatal uterine development, but also embryo implantation and decidualization.—Franco, H. L., Dai, D., Lee, K. Y., Rubel, C. S., Roop, D., Boerboom, D., Jeong, J.-W., Lydon, J.-P., Bagchi, I. C., Bagchi, M. K., DeMayo, F. J. WNT4 is a key regulator of normal postnatal uterine development and progesterone signaling during embryo implantation and decidualization in the mouse. PMID:21163860

Wnt pathway in Dupuytren disease: connecting profibrotic signals.

PubMed

van Beuge, Marike M; Ten Dam, Evert-Jan P M; Werker, Paul M N; Bank, Ruud A

2015-12-01

A role of Wnt signaling in Dupuytren disease, a fibroproliferative disease of the hand and fingers, has not been fully elucidated. We examined a large set of Wnt pathway components and signaling targets and found significant dysregulation of 41 Wnt-related genes in tissue from the Dupuytren nodules compared with patient-matched control tissue. A large proportion of genes coding for Wnt proteins themselves was downregulated. However, both canonical Wnt targets and components of the noncanonical signaling pathway were upregulated. Immunohistochemical analysis revealed that protein expression of Wnt1-inducible secreted protein 1 (WISP1), a known Wnt target, was increased in nodules compared with control tissue, but knockdown of WISP1 using small interfering RNA (siRNA) in the Dupuytren myofibroblasts did not confirm a functional role. The protein expression of noncanonical pathway components Wnt5A and VANGL2 as well as noncanonical coreceptors Ror2 and Ryk was increased in nodules. On the contrary, the strongest downregulated genes in this study were 4 antagonists of Wnt signaling (DKK1, FRZB, SFRP1, and WIF1). Downregulation of these genes in the Dupuytren tissue was mimicked in vitro by treating normal fibroblasts with transforming growth factor β1 (TGF-β1), suggesting cross talk between different profibrotic pathways. Furthermore, siRNA-mediated knockdown of these antagonists in normal fibroblasts led to increased nuclear translocation of Wnt target β-catenin in response to TGF-β1 treatment. In conclusion, we have shown extensive dysregulation of Wnt signaling in affected tissue from Dupuytren disease patients. Components of both the canonical and the noncanonical pathways are upregulated, whereas endogenous antagonists are downregulated, possibly via interaction with other profibrotic pathways. Copyright © 2015 Elsevier Inc. All rights reserved.

WIF1 re-expression in glioblastoma inhibits migration through attenuation of non-canonical WNT signaling by downregulating the lncRNA MALAT1.

PubMed

Vassallo, I; Zinn, P; Lai, M; Rajakannu, P; Hamou, M-F; Hegi, M E

2016-01-07

Glioblastoma is the most aggressive primary brain tumor in adults and due to the invasive nature cannot be completely removed. The WNT inhibitory factor 1 (WIF1), a secreted inhibitor of WNTs, is systematically downregulated in glioblastoma and acts as strong tumor suppressor. The aim of this study was the dissection of WIF1-associated tumor-suppressing effects mediated by canonical and non-canonical WNT signaling. We found that WIF1 besides inhibiting the canonical WNT pathway selectively downregulates the WNT/calcium pathway associated with significant reduction of p38-MAPK (p38-mitogen-activated protein kinase) phosphorylation. Knockdown of WNT5A, the only WNT ligand overexpressed in glioblastoma, phenocopied this inhibitory effect. WIF1 expression inhibited cell migration in vitro and in an orthotopic brain tumor model, in accordance with the known regulatory function of the WNT/Ca(2+) pathway on migration and invasion. In search of a mediator for this function differential gene expression profiles of WIF1-expressing cells were performed. Metastasis-associated lung adenocarcinoma transcript 1 (MALAT1), a long non-coding RNA and key positive regulator of invasion, emerged as the top downregulated gene. Indeed, knockdown of MALAT1 reduced migration in glioblastoma cells, without effect on proliferation. Hence, loss of WIF1 enhances the migratory potential of glioblastoma through WNT5A that activates the WNT/Ca(2+) pathway and MALAT1. These data suggest the involvement of canonical and non-canonical WNT pathways in glioblastoma promoting key features associated with this deadly disease, proliferation on one hand and invasion on the other. Successful targeting will require a dual strategy affecting both canonical and non-canonical WNT pathways.

Roles of circulating WNT-signaling proteins and WNT-inhibitors in human adiposity, insulin resistance, insulin secretion, and inflammation.

PubMed

Almario, R U; Karakas, S E

2015-02-01

Wingless-type MMTV integration site family member (WNT) signaling and WNT-inhibitors have been implicated in regulation of adipogenesis, insulin resistance, pancreatic function, and inflammation. Our goal was to determine serum proteins involved in WNT signaling (WNT5 and WISP2) and WNT inhibition (SFRP4 and SFRP5) as they relate to obesity, serum adipokines, insulin resistance, insulin secretion, and inflammation in humans. Study population comprised 57 insulin resistant women with polycystic ovary syndrome (PCOS) and 27 reference women. In a cross-sectional study, blood samples were obtained at fasting, during oral, and frequently sampled intravenous glucose tolerance tests. Serum WNT5, WISP2, and SFRP4 concentrations did not differ between PCOS vs. reference women. Serum WNT5 correlated inversely with weight both in PCOS and reference women, and correlated directly with insulin response during oral glucose tolerance test in PCOS women. Serum WISP2 correlated directly with fatty acid binding protein 4. Serum SFRP5 did not differ between obese (n=32) vs. nonobese (n=25) PCOS women, but reference women had lower SFRP5 (p<5×10(-6) as compared to both PCOS groups). Serum SFRP5 correlated inversely with IL-1β, TNF-α, cholesterol, and apoprotein B. These findings demonstrated that WNT5 correlated inversely with adiposity and directly with insulin response, and the WNT-inhibitor SFRP5 may be anti-inflammatory. Better understanding of the role of WNT signaling in obesity, insulin resistance, insulin secretion, lipoprotein metabolism, and inflammation is important for prevention and treatment of metabolic syndrome, diabetes and cardiovascular disease. © Georg Thieme Verlag KG Stuttgart · New York.

Wnt10b and Dkk-1 gene therapy differentially influenced trabecular bone architecture, soft tissue integrity, and osteophytosis in a skeletally mature rat model of osteoarthritis.

PubMed

Mason, Jeffrey B; Gurda, Brittney L; Hankenson, Kurt D; Harper, Lindsey R; Carlson, Cathy S; Wilson, James M; Richardson, Dean W

2017-11-01

Our goals in the current experiments were to determine if (a) upregulation of Wnt signaling would induce osteoarthritis changes in stable stifle joints and (b) if downregulation of Wnt signaling in destabilized joints would influence the progression of OA. At 37 weeks of age, rats were injected in the stifle joint with a recombinant adeno-associated viral vector containing the Wnt-inhibitor Dkk-1 or a Wnt10b transgene. At 40 weeks of age, rats underwent surgical destabilization of the joint. At 50 weeks of age, stifle joints were submitted for micro-computed tomography and histopathological analysis. Injection of either Wnt10b or Dkk-1 transgenes in stable joints improved bone architectural parameters, but worsened soft tissue integrity. Osteophytosis was decreased by Dkk-1, but unchanged by Wnt10b. Destabilization negatively influenced bone architecture, increased osteophytosis, and decreased soft tissue integrity. Dkk-1 exacerbated the negative effects of destabilization, whereas Wnt10b had little effect on these parameters. Osteophytosis was improved, whereas soft tissue integrity was worsened by both transgenes in destabilized joints. The Wnt-inhibitor Dkk-1 does not appear to completely inhibit the effects of Wnt signaling on bone remodeling. In vivo upregulation of Wnt10b and its inhibitor, Dkk-1, can produce both parallel or contrasting phenotypic responses depending on the specific parameter measured and the fidelity of the examined joint. These observations elucidate different roles for Wnt signaling in stable versus destabilized joints and may help to explain the conflicting results previously reported for the role of Dkk-1 in joint disease.

Wnt5a Is Associated with Cigarette Smoke-Related Lung Carcinogenesis via Protein Kinase C

PubMed Central

Sung, Jae Sook; Ju, Hyun Jung; Kim, Hyun Kyung; Park, Kyong Hwa; Lee, Jong Won; Koh, In Song; Kim, Yeul Hong

2013-01-01

Wnt5a is overexpressed during the progression of human non-small cell lung cancer. However, the roles of Wnt5a during smoking-related lung carcinogenesis have not been clearly elucidated. We investigated the associations between Wnt5a and the early development of cigarette smoke related lung cancer using human bronchial epithelial (HBE) cells (NHBE, BEAS-2B, 1799, 1198 and 1170I) at different malignant stages established by exposure to cigarette smoke condensate (CSC). Abnormal up-regulation of Wnt5a mRNA and proteins was detected in CSC-exposed transformed 1198 and tumorigenic 1170I cells as compared with other non-CSC exposed HBE cells. Tumor tissues obtained from smokers showed higher Wnt5a expressions than matched normal tissues. In non-CSC exposed 1799 cells, treatment of recombinant Wnt5a caused the activations of PKC and Akt, and the blockage of Wnt5a and PKC significantly decreased the viabilities of CSC-transformed 1198 cells expressing high levels of Wnt5a. This reduced cell survival rate was associated with increased apoptosis via the down-regulation of Bcl2 and the induction of cleaved poly ADP-ribose polymerase. Moreover, CSC-treated 1799 cells showed induction of Wnt5a expression and enhanced colony-forming capacity. The CSC-induced colony forming efficiency was suppressed by the co-incubation with a PKC inhibitor. In conclusion, these results suggest that cigarette smoke induces Wnt5a-coupled PKC activity during lung carcinogenesis, which causes Akt activity and anti-apoptosis in lung cancer. Therefore, current study provides novel clues for the crucial role of Wnt5a in the smoking-related lung carcinogenesis. PMID:23349696

Wnt5a is associated with cigarette smoke-related lung carcinogenesis via protein kinase C.

PubMed

Whang, Young Mi; Jo, Ukhyun; Sung, Jae Sook; Ju, Hyun Jung; Kim, Hyun Kyung; Park, Kyong Hwa; Lee, Jong Won; Koh, In Song; Kim, Yeul Hong

2013-01-01

Wnt5a is overexpressed during the progression of human non-small cell lung cancer. However, the roles of Wnt5a during smoking-related lung carcinogenesis have not been clearly elucidated. We investigated the associations between Wnt5a and the early development of cigarette smoke related lung cancer using human bronchial epithelial (HBE) cells (NHBE, BEAS-2B, 1799, 1198 and 1170I) at different malignant stages established by exposure to cigarette smoke condensate (CSC). Abnormal up-regulation of Wnt5a mRNA and proteins was detected in CSC-exposed transformed 1198 and tumorigenic 1170I cells as compared with other non-CSC exposed HBE cells. Tumor tissues obtained from smokers showed higher Wnt5a expressions than matched normal tissues. In non-CSC exposed 1799 cells, treatment of recombinant Wnt5a caused the activations of PKC and Akt, and the blockage of Wnt5a and PKC significantly decreased the viabilities of CSC-transformed 1198 cells expressing high levels of Wnt5a. This reduced cell survival rate was associated with increased apoptosis via the down-regulation of Bcl2 and the induction of cleaved poly ADP-ribose polymerase. Moreover, CSC-treated 1799 cells showed induction of Wnt5a expression and enhanced colony-forming capacity. The CSC-induced colony forming efficiency was suppressed by the co-incubation with a PKC inhibitor. In conclusion, these results suggest that cigarette smoke induces Wnt5a-coupled PKC activity during lung carcinogenesis, which causes Akt activity and anti-apoptosis in lung cancer. Therefore, current study provides novel clues for the crucial role of Wnt5a in the smoking-related lung carcinogenesis.

WINGLESS (WNT) signaling is a progesterone target for rat uterine stromal cell proliferation

PubMed Central

Talbott, Alex; Bhusri, Anuradha; Krumsick, Zach; Foster, Sierra; Wormington, Joshua; Kimler, Bruce F

2016-01-01

Preparation of mammalian uterus for embryo implantation requires a precise sequence of cell proliferation. In rodent uterus, estradiol stimulates proliferation of epithelial cells. Progesterone operates as a molecular switch and redirects proliferation to the stroma by down-regulating glycogen synthase kinase-3β (GSK-3β) and stimulating β-catenin accumulation in the periluminal stromal cells. In this study, the WNT signal involved in the progesterone-dependent proliferative switch was investigated. Transcripts of four candidate Wnt genes were measured in the uteri from ovariectomized (OVX) rats, progesterone-pretreated (3 days of progesterone, 2mg/daily) rats, and progesterone-pretreated rats given a single dose (0.2µg) of estradiol. The spatial distribution of the WNT proteins was determined in the uteri after the same treatments. Wnt5a increased in response to progesterone and the protein emerged in the periluminal stromal cells of progesterone-pretreated rat uteri. To investigate whether WNT5A was required for proliferation, uterine stromal cell lines were stimulated with progesterone (1µM) and fibroblast growth factor (FGF, 50ng/mL). Proliferating stromal cells expressed a two-fold increase in WNT5A protein at 12h post stimulation. Stimulated stromal cells were cultured with actinomycin D (25µg/mL) to inhibit new RNA synthesis. Relative Wnt5a expression increased at 4 and 6 h of culture, suggesting that progesterone plus FGF preferentially increased Wnt5a mRNA stability. Knockdown of Wnt5a in uterine stromal cell lines inhibited stromal cell proliferation and decreased Wnt5a mRNA. The results indicate that progesterone initiates and synchronizes uterine stromal cell proliferation by increasing WNT5A expression and signaling. PMID:26975616

WNT-1 Signaling in Mammary Carcinogenesis

DTIC Science & Technology

2002-04-01

segment polarity gene whose mutant phenotype resembles that of the wingless (Drosophila Wnt-1) mutation (3). arrow encodes a transmembrane receptor...and function ofSpemann’s organizer. Annu. Rev. C Drv. of those caused by mutations in individual Wnt genes . Further- Biaol 13, 611-667 (1997). more, we... mutations of multiple Wnt genes [31]. In the 0.5 nM and thus is significantly higher than Wnt-Fz bind- Xenopus embryo, inhibition of LRP6 function

c-Kit mediates chemoresistance and tumor-initiating capacity of ovarian cancer cells through activation of Wnt/β-catenin-ATP-binding cassette G2 signaling.

PubMed

Chau, W K; Ip, C K; Mak, A S C; Lai, H-C; Wong, A S T

2013-05-30

Cisplatin and paclitaxel are standard chemotherapy for metastatic ovarian cancer, but with limited efficacy. Cancer stem/progenitor cells (or tumor-initiating cells, TICs) are hypothesized to be chemoresistant, and the existence of TICs in ovarian cancer has been previously demonstrated. However, the key signals and molecular events regulating the formation and expansion of ovarian tumor-initiating cells (OTICs) remain elusive. Here, we show that c-Kit is not just a marker of OTICs, but also a critical mediator of the phenotype that can be a viable target for the treatment of ovarian cancer. In contrast to non-OICs, c-Kit was overexpressed in OTICs. Moreover, the use of small interfering RNA to inhibit c-Kit expression markedly attenuated the number and size of OTIC subpopulations, inhibited the expression of stem cell markers and decreased the tumorigenic capabilities of OTICs. Imatinib (Gleevec), a clinical drug that blocks c-Kit kinase activity, also demonstrated its inhibition potency on OTICs. In addition, cisplatin/paclitaxel, which killed non-OTICs, with c-Kit knockdown or imatinib revealed that this was critically required for intervening ovarian cancer progression and recurrence in vitro and in xenograft tumors in vivo. Similar results were obtained with OTICs derived from ovarian carcinoma patients. Studies into the mechanisms suggest an important role for the activation of Wnt/β-catenin and ATP-binding cassette G2 downstream of c-Kit. The tumor-promoting microenvironment, such as hypoxia, could promote OTICs via upregulation of c-Kit expression. These results unravel an integral role for c-Kit in ovarian neoplastic processes and shed light on its mechanisms of action.

Association of Wnt1-inducible signaling pathway protein-1 with the proliferation, migration and invasion in gastric cancer cells.

PubMed

Jia, Shuqin; Qu, Tingting; Feng, Mengmeng; Ji, Ke; Li, Ziyu; Jiang, Wenguo; Ji, Jiafu

2017-06-01

Wnt1-inducible signaling pathway protein-1 is a cysteine-rich protein that belongs to the CCN family, which has been implicated in mediating the occurrence and progression through distinct molecular mechanisms in several tumor types. However, the association of Wnt1-inducible signaling pathway protein-1 with gastric cancer and the related molecular mechanisms remain to be elucidated. Therefore, this study aimed to clarify the biological role of Wnt1-inducible signaling pathway protein-1 in the proliferation, migration, and invasion in gastric cancer cells and further investigated the associated molecular mechanism on these biological functions. We first detected the expression level of Wnt1-inducible signaling pathway protein-1 in gastric cancer, and the reverse transcription polymerase chain reaction have shown that Wnt1-inducible signaling pathway protein-1 expression levels were upregulated in gastric cancer tissues. The expression of Wnt1-inducible signaling pathway protein-1 in gastric cancer cell lines was also detected by quantitative real-time polymerase chain reaction and Western blotting. Furthermore, two gastric cancer cell lines with high expression of Wnt1-inducible signaling pathway protein-1 were selected to explore the biological function of Wnt1-inducible signaling pathway protein-1 in gastric cancer. Function assays indicated that knockdown of Wnt1-inducible signaling pathway protein-1 suppressed cell proliferation, migration, and invasion in BGC-823 and AGS gastric cancer cells. Further investigation of mechanisms suggested that cyclinD1 was identified as one of Wnt1-inducible signaling pathway protein-1 related genes to accelerate proliferation in gastric cancer cells. In addition, one pathway of Wnt1-inducible signaling pathway protein-1 induced migration and invasion was mainly through the enhancement of epithelial-to-mesenchymal transition progression. Taken together, our findings presented the first evidence that Wnt1-inducible signaling pathway protein-1 was upregulated in gastric cancer and acted as an oncogene by promoting proliferation, migration, and invasion in gastric cancer cells.

Wnt signaling: Ig-norrin the dogma.

PubMed

Clevers, Hans

2004-06-08

Secreted Wnt proteins trigger the intracellular Wnt signaling cascade upon engagement of dedicated Frizzled-Lrp receptor complexes. Unexpectedly, a non-Wnt ligand for this receptor complex has now been discovered. This novel ligand, Norrin, is mutated in the hereditary ocular Norrie syndrome. Copyright 2004 Elsevier Ltd.

Notum is required for neural and head induction via Wnt deacylation, oxidation, and inactivation.

PubMed

Zhang, Xinjun; Cheong, Seong-Moon; Amado, Nathalia G; Reis, Alice H; MacDonald, Bryan T; Zebisch, Matthias; Jones, E Yvonne; Abreu, Jose Garcia; He, Xi

2015-03-23

Secreted Wnt morphogens are essential for embryogenesis and homeostasis and require a lipid/palmitoleoylate modification for receptor binding and activity. Notum is a secreted Wnt antagonist that belongs to the α/β hydrolase superfamily, but its mechanism of action and roles in vertebrate embryogenesis are not fully understood. Here, we report that Notum hydrolyzes the Wnt palmitoleoylate adduct extracellularly, resulting in inactivated Wnt proteins that form oxidized oligomers incapable of receptor binding. Thus, Notum is a Wnt deacylase, and palmitoleoylation is obligatory for the Wnt structure that maintains its active monomeric conformation. Notum is expressed in naive ectoderm and neural plate in Xenopus and is required for neural and head induction. These findings suggest that Notum is a prerequisite for the "default" neural fate and that distinct mechanisms of Wnt inactivation by the Tiki protease in the Organizer and the Notum deacylase in presumptive neuroectoderm orchestrate vertebrate brain development. Copyright © 2015 Elsevier Inc. All rights reserved.

Non-canonical Wnt4 prevents skeletal aging and inflammation by inhibiting NF-κB

PubMed Central

Yu, Bo; Chang, Jia; Liu, Yunsong; Li, Jiong; Kevork, Kareena; Al-Hezaimi, Khalid; Graves, Dana T; Park, No-Hee; Wang, Cun-Yu

2014-01-01

Aging-related bone loss and osteoporosis affect millions of patients worldwide. Chronic inflammation associated with aging and arthritis promotes bone resorption and impairs bone formation. Here we show that Wnt4 attenuated bone loss in osteoporosis and skeletal aging by inhibiting nuclear factor-kappa B (NF-κB) via non-canonical Wnt signaling. Transgenic mice expressing Wnt4 from osteoblasts were significantly protected from bone loss and chronic inflammation induced by ovariectomy, tumor necrosis factor or natural aging. In addition to promoting bone formation, Wnt4 could inhibit osteoclast formation and bone resorption. Mechanistically, Wnt4 inhibited transforming growth factor beta-activated kinase 1-mediated NF-κB activation in macrophages and osteoclast precursors independent of β-catenin. Moreover, recombinant Wnt4 proteins were able to alleviate osteoporotic bone loss and inflammation by inhibiting NF-κB in vivo. Taken together, our results suggest that Wnt4 might be used as a therapeutic agent for treating osteoporosis by attenuating NF-κB. PMID:25108526

RIPK4 phosphorylates Dishevelled proteins to regulate canonical Wnt signaling

PubMed Central

Huang, XiaoDong; McGann, James C.; Liu, Bob Y.; Hannoush, Rami N.; Lill, Jennie R.; Pham, Victoria; Newton, Kim; Kakunda, Michael; Liu, Jinfeng; Yu, Christine; Hymowitz, Sarah G.; Hongo, Jo-Anne; Wynshaw-Boris, Anthony; Polakis, Paul; Harland, Richard M.; Dixit, Vishva M.

2014-01-01

Receptor interacting protein kinase 4 (RIPK4) is required for epidermal differentiation (1–4) and is mutated in Bartsocas-Papas syndrome (5, 6). While RIPK4 binds protein kinase C (5, 6), RIPK4 signaling mechanisms are largely unknown. We show that ectopic RIPK4 induces cytosolic β-catenin accumulation and a transcriptional program similar to Wnt3a, whereas kinase-defective or Bartsocas-Papas syndrome RIPK4 mutants do not. Ectopic ripk4 synergized with Wnt family member xwnt8 in Xenopus, whereas ripk4 morpholinos or kinase-defective RIPK4 antagonized Wnt signaling. Mechanistically, RIKP4 interacted constitutively with the Wnt adaptor protein DVL2 and, after Wnt3a stimulation, with the co-receptor LRP6. Phosphorylation of DVL2 at Ser298 and Ser480 by RIPK4 favored canonical Wnt signaling. Growth of a Wnt-dependent N-Tera2 xenograft tumor model was suppressed by RIPK4 knockdown, suggesting that RIPK4 overexpression may contribute to the growth of certain tumor types. PMID:23371553

Aldolase positively regulates of the canonical Wnt signaling pathway

PubMed Central

2014-01-01

The Wnt signaling pathway is an evolutionary conserved system, having pivotal roles during animal development. When over-activated, this signaling pathway is involved in cancer initiation and progression. The canonical Wnt pathway regulates the stability of β-catenin primarily by a destruction complex containing a number of different proteins, including Glycogen synthase kinase 3β (GSK-3β) and Axin, that promote proteasomal degradation of β-catenin. As this signaling cascade is modified by various proteins, novel screens aimed at identifying new Wnt signaling regulators were conducted in our laboratory. One of the different genes that were identified as Wnt signaling activators was Aldolase C (ALDOC). Here we report that ALDOC, Aldolase A (ALDOA) and Aldolase B (ALDOB) activate Wnt signaling in a GSK-3β-dependent mechanism, by disrupting the GSK-3β-Axin interaction and targeting Axin to the dishevelled (Dvl)-induced signalosomes that positively regulate the Wnt pathway thus placing the Aldolase proteins as novel Wnt signaling regulators. PMID:24993527

Wnt inhibition promotes vascular specification of embryonic cardiac progenitors

PubMed Central

Reichman, David E.; Park, Laura; Man, Limor; Redmond, David; Chao, Kenny; Harvey, Richard P.; Taketo, Makoto M.; Rosenwaks, Zev

2018-01-01

ABSTRACT Several studies have demonstrated a multiphasic role for Wnt signaling during embryonic cardiogenesis and developed protocols that enrich for cardiac derivatives during in vitro differentiation of human pluripotent stem cells (hPSCs). However, few studies have investigated the role of Wnt signaling in the specification of cardiac progenitor cells (CPCs) toward downstream fates. Using transgenic mice and hPSCs, we tracked endothelial cells (ECs) that originated from CPCs expressing NKX2.5. Analysis of EC-fated CPCs at discrete phenotypic milestones during hPSC differentiation identified reduced Wnt activity as a hallmark of EC specification, and the enforced activation or inhibition of Wnt reduced or increased, respectively, the degree of vascular commitment within the CPC population during both hPSC differentiation and mouse embryogenesis. Wnt5a, which has been shown to exert an inhibitory influence on Wnt signaling during cardiac development, was dynamically expressed during vascular commitment of hPSC-derived CPCs, and ectopic Wnt5a promoted vascular specification of hPSC-derived and mouse embryonic CPCs. PMID:29217753

Adipose-derived stem cells cooperate with fractional carbon dioxide laser in antagonizing photoaging: a potential role of Wnt and β-catenin signaling.

PubMed

Xu, Xiao; Wang, Hong-Yi; Zhang, Yu; Liu, Yang; Li, Yan-Qi; Tao, Kai; Wu, Chu-Tse; Jin, Ji-de; Liu, Xiao-Yan

2014-01-01

It is well established that adipose-derived stem cells (ADSCs) produce and secrete cytokines/growth factors that antagonize UV-induced photoaging of skin. However, the exact molecular basis underlying the anti-photoaging effects exerted by ADSCs is not well understood, and whether ADSCs cooperate with fractional carbon dioxide (CO2) laser to facilitate photoaging skin healing process has not been explored. Here, we investigated the impacts of ADSCs on photoaging in a photoaging animal model, its associated mechanisms, and its functional cooperation with fractional CO2 laser in treatment of photoaging skin. We showed that ADSCs improved dermal thickness and activated the proliferation of dermal fibroblast. We further demonstrated that the combined treatment of ADSCs and fractional CO2 laser, the latter which is often used to resurface skin and treat wrinkles, had more beneficial effects on the photoaging skin compared with each individual treatment. In our prepared HDF photoaging model, flow cytometry showed that, after adipose derived stem cells conditioned medium (ADSC-CM) co-cultured HDF photoaging model, the cell proliferation rate is higher than UVB irradiation induced HDF modeling (p < 0.05). Additionally, the expressions of β-catenin and Wnt3a, which were up-regulated after the transplantation of ADSCs alone or in combination with fractional CO2 laser treatment. And the expression of wnt3a and β-catenin has the positive correlation with photoaging related protein TGF-β2 and COLI. We also verified these protein expressions in tissue level. In addition, after injected SFRP2 into ADSC-CM co-cultured HDF photoaging model, wnt3a inhibitor, compared with un-intervened group, wnt3a, β-catenin protein level significantly decreased. Both ADSCs and fractional CO2 laser improved photoaging skin at least partially via targeting dermal fibroblast activity which was increased in photoaging skin. The combinatorial use of ADSCs and fractional CO2 laser synergistically improved the healing process of photoaging skin. Thus, we provide a strong rationale for a combined use of ADSCs and fractional CO2 laser in treatment of photoaging skin in clinic in the future. Moreover, we provided evidence that the Wnt/β-catenin signaling pathway may contribute to the activation of dermal fibroblast by the transplantation of ADSCs in both vitro and vivo experiment.

Adipose-derived stem cells cooperate with fractional carbon dioxide laser in antagonizing photoaging: a potential role of Wnt and β-catenin signaling

PubMed Central

2014-01-01

Background It is well established that adipose-derived stem cells (ADSCs) produce and secrete cytokines/growth factors that antagonize UV-induced photoaging of skin. However, the exact molecular basis underlying the anti-photoaging effects exerted by ADSCs is not well understood, and whether ADSCs cooperate with fractional carbon dioxide (CO2) laser to facilitate photoaging skin healing process has not been explored. Here, we investigated the impacts of ADSCs on photoaging in a photoaging animal model, its associated mechanisms, and its functional cooperation with fractional CO2 laser in treatment of photoaging skin. Results We showed that ADSCs improved dermal thickness and activated the proliferation of dermal fibroblast. We further demonstrated that the combined treatment of ADSCs and fractional CO2 laser, the latter which is often used to resurface skin and treat wrinkles, had more beneficial effects on the photoaging skin compared with each individual treatment. In our prepared HDF photoaging model, flow cytometry showed that, after adipose derived stem cells conditioned medium (ADSC-CM) co-cultured HDF photoaging model, the cell proliferation rate is higher than UVB irradiation induced HDF modeling (p < 0.05). Additionally, the expressions of β-catenin and Wnt3a, which were up-regulated after the transplantation of ADSCs alone or in combination with fractional CO2 laser treatment. And the expression of wnt3a and β-catenin has the positive correlation with photoaging related protein TGF-β2 and COLI. We also verified these protein expressions in tissue level. In addition, after injected SFRP2 into ADSC-CM co-cultured HDF photoaging model, wnt3a inhibitor, compared with un-intervened group, wnt3a, β-catenin protein level significantly decreased. Conclusion Both ADSCs and fractional CO2 laser improved photoaging skin at least partially via targeting dermal fibroblast activity which was increased in photoaging skin. The combinatorial use of ADSCs and fractional CO2 laser synergistically improved the healing process of photoaging skin. Thus, we provide a strong rationale for a combined use of ADSCs and fractional CO2 laser in treatment of photoaging skin in clinic in the future. Moreover, we provided evidence that the Wnt/β-catenin signaling pathway may contribute to the activation of dermal fibroblast by the transplantation of ADSCs in both vitro and vivo experiment. PMID:24917925

Wnt signaling balances specification of the cardiac and pharyngeal muscle fields

PubMed Central

Mandal, Amrita; Holowiecki, Andrew; Song, Yuntao Charlie; Waxman, Joshua S.

2017-01-01

Canonical Wnt/β-catenin (Wnt) signaling plays multiple conserved roles during fate specification of cardiac progenitors in developing vertebrate embryos. Although lineage analysis in ascidians and mice has indicated there is a close relationship between the cardiac second heart field (SHF) and pharyngeal muscle (PM) progenitors, the signals underlying directional fate decisions of the cells within the cardio-pharyngeal muscle field in vertebrates are not yet understood. Here, we examined the temporal requirements of Wnt signaling in cardiac and PM development. In contrast to a previous report in chicken embryos that suggested Wnt inhibits PM development during somitogenesis, we find that in zebrafish embryos Wnt signaling is sufficient to repress PM development during anterior-posterior patterning. Importantly, the temporal sensitivity of dorso-anterior PMs to increased Wnt signaling largely overlaps with when Wnt signaling promotes specification of the adjacent cardiac progenitors. Furthermore, we find that excess early Wnt signaling can cell autonomously promote expansion of the first heart field (FHF) progenitors at the expense of PM and SHF within the anterior lateral plate mesoderm (ALPM). Our study provides insight into an antagonistic developmental mechanism that balances the sizes of the adjacent cardiac and PM progenitor fields in early vertebrate embryos. PMID:28087459

Binding of canonical Wnt ligands to their receptor complexes occurs in ordered plasma membrane environments.

PubMed

Sezgin, Erdinc; Azbazdar, Yagmur; Ng, Xue W; Teh, Cathleen; Simons, Kai; Weidinger, Gilbert; Wohland, Thorsten; Eggeling, Christian; Ozhan, Gunes

2017-08-01

While the cytosolic events of Wnt/β-catenin signaling (canonical Wnt signaling) pathway have been widely studied, only little is known about the molecular mechanisms involved in Wnt binding to its receptors at the plasma membrane. Here, we reveal the influence of the immediate plasma membrane environment on the canonical Wnt-receptor interaction. While the receptors are distributed both in ordered and disordered environments, Wnt binding to its receptors selectively occurs in more ordered membrane environments which appear to cointernalize with the Wnt-receptor complex. Moreover, Wnt/β-catenin signaling is significantly reduced when the membrane order is disturbed by specific inhibitors of certain lipids that prefer to localize at the ordered environments. Similarly, a reduction in Wnt signaling activity is observed in Niemann-Pick Type C disease cells where trafficking of ordered membrane lipid components to the plasma membrane is genetically impaired. We thus conclude that ordered plasma membrane environments are essential for binding of canonical Wnts to their receptor complexes and downstream signaling activity. © 2017 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.

The fast track to canonical Wnt signaling in MC3T3-E1 cells protected by substance P against serum deprivation-induced apoptosis.

PubMed

Yang, Jianguo; Nie, Jiping; Fu, Su; Liu, Song; Wu, Jianqun; Cui, Liang; Zhang, Yongtao; Yu, Bin

2017-01-01

The canonical Wnt pathway is vital to bone physiology by increasing bone mass through elevated osteoblast survival. Although investigated extensively in stem cells, its role in osteoblastic MC3T3-E1 cells has not been completely determined. To explore how this pathway is regulated by different conditions, we assessed the anti-apoptotic effects of substance P on the canonical Wnt pathway in MC3T3-E1 cells by treating cells with serum deprivation or serum starving with "substance P," a neuropeptide demonstrated to promote bone growth and stimulate Wnt signaling. The results showed that serum deprivation both induced apoptosis and activated Wnt signal transduction while substance P further stimulated the Wnt pathway via the NK-1 receptor but protected the cells from apoptotic death. Fast-tracking of Wnt signaling by substance P was also noted. These results indicate that nutritional deprivation and substance P synergistically activated the canonical Wnt pathway, a finding that helps to reveal the role of Wnt signaling in bone physiology affected by nutritional deprivation and neuropeptide substance P. © 2016 International Federation for Cell Biology.

Crosstalk between MLO-Y4 osteocytes and C2C12 muscle cells is mediated by the Wnt/β-catenin pathway.

PubMed

Huang, Jian; Romero-Suarez, Sandra; Lara, Nuria; Mo, Chenglin; Kaja, Simon; Brotto, Leticia; Dallas, Sarah L; Johnson, Mark L; Jähn, Katharina; Bonewald, Lynda F; Brotto, Marco

2017-10-01

We examined the effects of osteocyte secreted factors on myogenesis and muscle function. MLO-Y4 osteocyte-like cell conditioned media (CM) (10%) increased ex vivo soleus muscle contractile force by ~25%. MLO-Y4 and primary osteocyte CM (1-10%) stimulated myogenic differentiation of C2C12 myoblasts, but 10% osteoblast CMs did not enhance C2C12 cell differentiation. Since WNT3a and WNT1 are secreted by osteocytes, and the expression level of Wnt3a is increased in MLO-Y4 cells by fluid flow shear stress, both were compared, showing WNT3a more potent than WNT1 in inducing myogenesis. Treatment of C2C12 myoblasts with WNT3a at concentrations as low as 0.5ng/mL mirrored the effects of both primary osteocyte and MLO-Y4 CM by inducing nuclear translocation of β-catenin with myogenic differentiation, suggesting that Wnts might be potential factors secreted by osteocytes that signal to muscle cells. Knocking down Wnt3a in MLO-Y4 osteocytes inhibited the effect of CM on C2C12 myogenic differentiation. Sclerostin (100ng/mL) inhibited both the effects of MLO-Y4 CM and WNT3a on C2C12 cell differentiation. RT-PCR array results supported the activation of the Wnt/β-catenin pathway by MLO-Y4 CM and WNT3a. These results were confirmed by qPCR showing up-regulation of myogenic markers and two Wnt/β-catenin downstream genes, Numb and Flh1 . We postulated that MLO-Y4 CM/WNT3a could modulate intracellular calcium homeostasis as the trigger mechanism for the enhanced myogenesis and contractile force. MLO-Y4 CM and WNT3a increased caffeine-induced Ca 2+ release from the sarcoplasmic reticulum (SR) of C2C12 myotubes and the expression of genes directly associated with intracellular Ca 2+ signaling and homeostasis. Together, these data show that in vitro and ex vivo , osteocytes can stimulate myogenesis and enhance muscle contractile function and suggest that Wnts could be mediators of bone to muscle signaling, likely via modulation of intracellular Ca 2+ signaling and the Wnt/β-Catenin pathway.

The Wnt/β-catenin pathway is deregulated in cemento-ossifying fibromas.

PubMed

Pereira, Thaís Dos Santos Fontes; Diniz, Marina Gonçalves; França, Josiane Alves; Moreira, Rennan Garcias; Menezes, Grazielle Helena Ferreira de; Sousa, Sílvia Ferreira de; Castro, Wagner Henriques de; Gomes, Carolina Cavaliéri; Gomez, Ricardo Santiago

2018-02-01

The molecular pathogenesis of cemento ossifying fibroma (COF) is unclear. The purpose of this study was to investigate mutations in 50 oncogenes and tumor suppressor genes, including APC and CTNNB1, in which mutations in COF have been previously reported. In addition, we assessed the transcriptional levels of the Wnt/β-catenin pathway genes in COF. We used a quantitative polymerase chain reaction array to evaluate the transcriptional levels of 44 Wnt/β-catenin pathway genes in 6 COF samples, in comparison with 6 samples of healthy jaws. By using next-generation sequencing (NGS) in 7 COF samples, we investigated approximately 2800 mutations in 50 genes. The expression assay revealed 12 differentially expressed Wnt/β-catenin pathway genes in COF, including the upregulation of CTNNB1, TCF7, NKD1, and WNT5 A, and downregulation of CTNNBIP1, FRZB, FZD6, RHOU, SFRP4, WNT10 A, WNT3 A, and WNT4, suggesting activation of the Wnt/β-catenin signaling pathway. NGS revealed 5 single nucleotide variants: TP53 (rs1042522), PIK3 CA (rs2230461), MET (rs33917957), KIT (rs3822214), and APC (rs33974176), but none of them was pathogenic. Although NGS detected no oncogenic mutation, deregulation of key Wnt/β-catenin signaling pathway genes appears to be relevant to the molecular pathogenesis of COF. Copyright © 2017 Elsevier Inc. All rights reserved.

Different populations of Wnt-containing vesicles are individually released from polarized epithelial cells

PubMed Central

Chen, Qiuhong; Takada, Ritsuko; Noda, Chiyo; Kobayashi, Satoru; Takada, Shinji

2016-01-01

Accumulating evidence suggests that exosomes are heterogeneous in molecular composition and physical properties. Here we examined whether epithelial cells secrete a heterogeneous population of exosomes, and if that is the case, whether epithelial cell polarity affects release of different populations of exosomes, especially that of those carrying Wnt. Sucrose-density ultracentrifugation and molecular marker analysis revealed that different populations of exosomes or exosome-like vesicles were released from MDCK cells depending on the cell polarity. Wnt3a associated with these vesicles were detectable in culture media collected from both apical and basolateral sides of the cells. Basolaterally secreted Wnt3a were co-fractionated with a typical exosomal protein TSG101 in fractions having typical exosome densities. In contrast, most of apically secreted Wnt3a, as well as Wnt11, were co-fractionated with CD63 and Hsp70, which are also common to the most exosomes, but recovered in higher density fractions. Wnt3a exhibiting similar floatation behavior to the apically secreted ones were also detectable in the culture media of Wnt3a-expressing L and HEK293 cells. The lipidation of Wnt3a was required for its basolateral secretion in exosomes but was dispensable for the apical one. Thus, epithelial cells release Wnt via distinct populations of vesicles differing in secretion polarity and lipidation dependency. PMID:27765945

Expression of Glycogen synthase kinase 3-β (GSK3-β) gene in azoospermic men.

PubMed

Nazarian, Hamid; Ghaffari Novin, Marefat; Jalili, Mohammad Reza; Mirfakhraie, Reza; Heidari, Mohammad Hassan; Hosseini, Seyed Jalil; Norouzian, Mohsen; Ehsani, Nahid

2014-05-01

The Wnt/β- The Wnt/β-catenin signaling pathway is involved in many developmental processes in both fetal and adult life; its abnormalities can lead to disorders including several types of cancers and malfunction of specific cells and tissues in both animals and humans. Its role in reproductive processes has been proven. This study was designed to evaluate the expression of the key regulator of this signaling pathway GSK3-β and its presumed role in azoospermia. WNT3a protein concentration and GSK3-β gene expression levels were measured and compared between two groups of infertile men. The test groups consisted of 10 patients with obstructive and 10 non-obstructive azoospermia. The control group was selected among healthy men after vasectomies that were willing to conceive a child using a testicular biopsy technique. Samples were obtained by testicular biopsy and screened for the most common mutations (84, 86 and 255) in the SRY region before analyzing. GSK3-β gene expression was assessed quantitatively by real time-PCR. The WNT3a protein concentration had no significant difference between the two test groups and controls. Expression of GSK3-β was down-regulated in non-obstructive azoospermia (3.10±0.19) compared with normal (7.12±0.39) and obstructive azoospermia (6.32±0.42) groups (p=0.001). Down-regulation of GSK-3β may cause to non-obstructive azoospermia. Regulation and modification of GSK-3β gene expression by drugs could be used as a therapeutic solution.

Extracellular Matrix Metalloproteinase Inducer (EMMPRIN) promotes lung fibroblast proliferation, survival and differentiation to myofibroblasts.

PubMed

Hasaneen, Nadia A; Cao, Jian; Pulkoski-Gross, Ashleigh; Zucker, Stanley; Foda, Hussein D

2016-02-17

Idiopathic pulmonary fibrosis (IPF) is a chronic progressively fatal disease. Extracellular Matrix Metalloproteinase Inducer (EMMPRIN) is a glycosylated transmembrane protein that induces the expression of some matrix metalloproteinase (MMP) in neighboring stromal cells through direct epithelial-stromal interactions. EMMPRIN is highly expressed in type II alveolar epithelial cells at the edges of the fibrotic areas in IPF lung sections. However, the exact role of EMMPRIN in IPF is unknown. To determine if EMMPRIN contributes to lung fibroblast proliferation, resistance to apoptosis, and differentiation to myofibroblasts, normal Human lung fibroblasts (NHLF) transiently transfected with either EMMPRIN/GFP or GFP were treated with TGF- β1 from 0 to 10 ng/ml for 48 h and examined for cell proliferation (thymidine incorporation), apoptosis (FACS analysis and Cell Death Detection ELISA assay), cell migration (Modified Boyden chamber) and differentiation to myofibroblasts using Western blot for α-smooth actin of cell lysates. The effect of EMMPRIN inhibition on NHLF proliferation, apoptosis, migration and differentiation to myofibroblasts after TGF- β1 treatment was examined using EMMPRIN blocking antibody. We examined the mechanism by which EMMPRIN induces its effects on fibroblasts by studying the β-catenin/canonical Wnt signaling pathway using Wnt luciferase reporter assays and Western blot for total and phosphorylated β-catenin. Human lung fibroblasts overexpressing EMMPRIN had a significant increase in cell proliferation and migration compared to control fibroblasts. Furthermore, EMMPRIN promoted lung fibroblasts resistance to apoptosis. Lung fibroblasts overexpressing EMMPRIN showed a significantly increased expression of α- smooth muscle actin, a marker of differentiation to myofibroblasts compared to control cells. TGF-β1 increased the expression of EMMPRIN in lung fibroblasts in a dose-dependent manner. Attenuation of EMMPRIN expression with the use of an EMMPRIN blocking antibody markedly inhibited TGF-β1 induced proliferation, migration, and differentiation of fibroblasts to myofibroblasts. EMMPRIN overexpression in lung fibroblasts was found to induce an increase in TOPFLASH luciferase reporter activity when compared with control fibroblasts. These findings indicate that TGF-β1 induces the release of EMMPRIN that activates β-catenin/canonical Wnt signaling pathway. EMMPRIN overexpression induces an anti-apoptotic and pro-fibrotic phenotype in lung fibroblasts that may contribute to the persistent fibro-proliferative state seen in IPF.

Wnt some lose some: transcriptional governance of stem cells by Wnt/β-catenin signaling

PubMed Central

Lien, Wen-Hui; Fuchs, Elaine

2014-01-01

In mammals, Wnt/β-catenin signaling features prominently in stem cells and cancers, but how and for what purposes have been matters of much debate. In this review, we summarize our current knowledge of Wnt/β-catenin signaling and its downstream transcriptional regulators in normal and malignant stem cells. We centered this review largely on three types of stem cells—embryonic stem cells, hair follicle stem cells, and intestinal epithelial stem cells—in which the roles of Wnt/β-catenin have been extensively studied. Using these models, we unravel how many controversial issues surrounding Wnt signaling have been resolved by dissecting the diversity of its downstream circuitry and effectors, often leading to opposite outcomes of Wnt/β-catenin-mediated regulation and differences rooted in stage- and context-dependent effects. PMID:25030692

RSPO–LGR4 functions via IQGAP1 to potentiate Wnt signaling

PubMed Central

Carmon, Kendra S.; Gong, Xing; Yi, Jing; Thomas, Anthony; Liu, Qingyun

2014-01-01

R-spondins (RSPOs) and their receptor leucine-rich repeat-containing G-protein coupled receptor 4 (LGR4) play pleiotropic roles in normal and cancer development as well as the survival of adult stem cells through potentiation of Wnt signaling. Current evidence indicates that RSPO–LGR4 functions to elevate levels of Wnt receptors through direct inhibition of two membrane-bound E3 ligases (RNF43 and ZNRF3), which otherwise ubiquitinate Wnt receptors for degradation. Whether RSPO–LGR4 is coupled to intracellular signaling proteins to regulate Wnt pathways remains unknown. We identified the intracellular scaffold protein IQ motif containing GTPase-activating protein 1 (IQGAP1) as an LGR4-interacting protein that mediates RSPO–LGR4’s interaction with the Wnt signalosome. IQGAP1 binds to and modulates the activities of a plethora of signaling molecules, including MAP kinases, Rho GTPases, and components of the Wnt signaling pathways. Interaction of LGR4 with IQGAP1 brings RSPO–LGR4 to the Wnt signaling complex through enhanced IQGAP1–DVL interaction following RSPO stimulation. In this configuration, RSPO–LGR4–IQGAP1 potentiates β-catenin–dependent signaling by promoting MEK1/2-medidated phosphorylation of LRP5/6 as well as β-catenin–independent signaling through regulation of actin dynamics. Overall, these findings reveal that RSPO–LGR4 not only induces the clearance of RNF43/ZNRF3 to increase Wnt receptor levels but also recruits IQGAP1 into the Wnt signaling complex, leading to potent and robust potentiation of both the canonical and noncanonical pathways of Wnt signaling. PMID:24639526

Wnt5a Increases Properties of Lung Cancer Stem Cells and Resistance to Cisplatin through Activation of Wnt5a/PKC Signaling Pathway

PubMed Central

Yang, Jiali; Zhang, Kangjian; Wu, Jing; Shi, Juan; Xue, Jing; Li, Jing; Zhu, Yongzhao; Wei, Jun

2016-01-01

The development of chemoresistance to cisplatin regimens causes a poor prognosis in patients with advanced NSCLC. The role of noncanonical Wnt signaling in the regulation of properties of lung cancer stem cells and chemoresistance was interrogated, by accessing capacities of cell proliferation, migration, invasion, and clonogenicity as well as the apoptosis in A549 cell lines and cisplatin-resistant A549 cells treated with Wnt5a conditional medium or protein kinase C (PKC) inhibitor GF109203X. Results showed that the noncanonical Wnt signaling ligand, Wnt5a, could promote the proliferation, migration, invasion, and colony formation in A549 lung adenocarcinoma cells and cisplatin-resistant A549/DDP cells and increase the fraction of ALDH-positive cell in A549/DDP cells. An exposure of cells to Wnt5a led to a significant reduction of A549/DDP cell apoptosis but not A549 cells. An addition of GF109203X could both strikingly increase the baseline apoptosis and resensitize the Wnt5a-inhibited cell apoptosis. Interestingly, an inhibition of Wnt/PKC signaling pathway could reduce properties of lung cancer stem cells, promote cell apoptosis, and resensitize cisplatin-resistant cells to cisplatin via a caspase/AIF-dependent pathway. These data thus suggested that the Wnt5a could promote lung cancer cell mobility and cisplatin-resistance through a Wnt/PKC signaling pathway and a blockage of this signaling may be an alternative therapeutic strategy for NSCLC patients with resistance to chemotherapies. PMID:27895670

LGR4 and LGR5 are R-spondin receptors mediating Wnt/β-catenin and Wnt/PCP signalling.

PubMed

Glinka, Andrei; Dolde, Christine; Kirsch, Nadine; Huang, Ya-Lin; Kazanskaya, Olga; Ingelfinger, Dierk; Boutros, Michael; Cruciat, Cristina-Maria; Niehrs, Christof

2011-09-30

R-spondins are secreted Wnt signalling agonists, which regulate embryonic patterning and stem cell proliferation, but whose mechanism of action is poorly understood. Here we show that R-spondins bind to the orphan G-protein-coupled receptors LGR4 and LGR5 by their Furin domains. Gain- and loss-of-function experiments in mammalian cells and Xenopus embryos indicate that LGR4 and LGR5 promote R-spondin-mediated Wnt/β-catenin and Wnt/PCP signalling. R-spondin-triggered β-catenin signalling requires Clathrin, while Wnt3a-mediated β-catenin signalling requires Caveolin-mediated endocytosis, suggesting that internalization has a mechanistic role in R-spondin signalling.

LGR4 and LGR5 are R-spondin receptors mediating Wnt/β-catenin and Wnt/PCP signalling

PubMed Central

Glinka, Andrei; Dolde, Christine; Kirsch, Nadine; Huang, Ya-Lin; Kazanskaya, Olga; Ingelfinger, Dierk; Boutros, Michael; Cruciat, Cristina-Maria; Niehrs, Christof

2011-01-01

R-spondins are secreted Wnt signalling agonists, which regulate embryonic patterning and stem cell proliferation, but whose mechanism of action is poorly understood. Here we show that R-spondins bind to the orphan G-protein-coupled receptors LGR4 and LGR5 by their Furin domains. Gain- and loss-of-function experiments in mammalian cells and Xenopus embryos indicate that LGR4 and LGR5 promote R-spondin-mediated Wnt/β-catenin and Wnt/PCP signalling. R-spondin-triggered β-catenin signalling requires Clathrin, while Wnt3a-mediated β-catenin signalling requires Caveolin-mediated endocytosis, suggesting that internalization has a mechanistic role in R-spondin signalling. PMID:21909076

The Transcription Factor Foxg1 Promotes Optic Fissure Closure in the Mouse by Suppressing Wnt8b in the Nasal Optic Stalk

PubMed Central

Smith, Rowena; Huang, Yu-Ting; Tian, Tian; Vojtasova, Dominika; Mesalles-Naranjo, Oscar; Price, David J.

2017-01-01

During vertebrate eye morphogenesis, a transient fissure forms at its inferior part, known as the optic fissure. This will gradually close, giving rise to a healthy, spherical optic cup. Failure of the optic fissure to close gives rise to an ocular disorder known as coloboma. During this developmental process, Foxg1 is expressed in the optic neuroepithelium, with highest levels of expression in the nasal optic stalk. Foxg1−/− mutant mice have microphthalmic eyes with a large ventral coloboma. We found Wnt8b expression upregulated in the Foxg1−/− optic stalk and hypothesized that, similar to what is observed in telencephalic development, Foxg1 directs development of the optic neuroepithelium through transcriptional suppression of Wnt8b. To test this, we generated Foxg1−/−;Wnt8b−/− double mutants of either sex and found that the morphology of the optic cup and stalk and the closure of the optic fissure were substantially rescued in these embryos. This rescue correlates with restored Pax2 expression in the anterior tip of the optic fissure. In addition, although we do not find evidence implicating altered proliferation in the rescue, we observe a significant increase in apoptotic cell density in Foxg1−/−;Wnt8b−/− double mutants compared with the Foxg1−/− single mutant. Upregulation of Wnt/β-catenin target molecules in the optic cup and stalk may underlie the molecular and morphological defects in the Foxg1−/− mutant. Our results show that proper optic fissure closure relies on Wnt8b suppression by Foxg1 in the nasal optic stalk to maintain balanced apoptosis and Pax2 expression in the nasal and temporal edges of the fissure. SIGNIFICANCE STATEMENT Coloboma is an ocular disorder that may result in a loss of visual acuity and accounts for ∼10% of childhood blindness. It results from errors in the sealing of the optic fissure (OF), a transient structure at the bottom of the eye. Here, we investigate the colobomatous phenotype of the Foxg1−/− mutant mouse. We identify upregulated expression of Wnt8b in the optic stalk of Foxg1−/− mutants before OF closure initiates. Foxg1−/−;Wnt8b−/− double mutants show a substantial rescue of the Foxg1−/− coloboma phenotype, which correlates with a rescue in molecular and cellular defects of Foxg1−/− mutants. Our results unravel a new role of Foxg1 in promoting OF closure providing additional knowledge about the molecules and cellular mechanisms underlying coloboma formation. PMID:28729440

Wnt and lithium: a common destiny in the therapy of nervous system pathologies?

PubMed

Meffre, Delphine; Grenier, Julien; Bernard, Sophie; Courtin, Françoise; Dudev, Todor; Shackleford, Ghjuvan'Ghjacumu; Jafarian-Tehrani, Mehrnaz; Massaad, Charbel

2014-04-01

Wnt signaling is required for neurogenesis, the fate of neural progenitors, the formation of neuronal circuits during development, neuron positioning and polarization, axon and dendrite development and finally for synaptogenesis. This signaling pathway is also implicated in the generation and differentiation of glial cells. In this review, we describe the mechanisms of action of Wnt signaling pathways and their implication in the development and correct functioning of the nervous system. We also illustrate how a dysregulated Wnt pathway could lead to psychiatric, neurodegenerative and demyelinating pathologies. Lithium, used for the treatment of bipolar disease, inhibits GSK3β, a central enzyme of the Wnt/β-catenin pathway. Thus, lithium could, to some extent, mimic Wnt pathway. We highlight the possible dialogue between lithium therapy and modulation of Wnt pathway in the treatment of the diseases of the nervous system.

The canonical Wnt signaling pathway in autism.

PubMed

Zhang, Yinghua; Yuan, Xiangshan; Wang, Zhongping; Li, Ruixi

2014-01-01

Mounting attention is being focused on the canonical Wnt signaling pathway which has been implicated in the pathogenesis of autism in some our and other recent studies. The canonical Wnt pathway is involved in cell proliferation, differentiation and migration, especially during nervous system development. Given its various functions, dysfunction of the canonical Wnt pathway may exert adverse effects on neurodevelopment and therefore leads to the pathogenesis of autism. Here, we review human and animal studies that implicate the canonical Wnt signal transduction pathway in the pathogenesis of autism. We also describe the crosstalk between the canonical Wnt pathway and the Notch signaling pathway in several types of autism spectrum disorders, including Asperger syndrome and Fragile X. Further research on the crosstalk between the canonical Wnt signaling pathway and other signaling cascades in autism may be an efficient avenue to understand the etiology of autism and ultimately lead to alternative medications for autism-like phenotypes.

Wnt, Ptk7, and FGFRL expression gradients control trunk positional identity in planarian regeneration.

PubMed

Lander, Rachel; Petersen, Christian P

2016-04-13

Mechanisms enabling positional identity re-establishment are likely critical for tissue regeneration. Planarians use Wnt/beta-catenin signaling to polarize the termini of their anteroposterior axis, but little is known about how regeneration signaling restores regionalization along body or organ axes. We identify three genes expressed constitutively in overlapping body-wide transcriptional gradients that control trunk-tail positional identity in regeneration. ptk7 encodes a trunk-expressed kinase-dead Wnt co-receptor, wntP-2 encodes a posterior-expressed Wnt ligand, and ndl-3 encodes an anterior-expressed homolog of conserved FGFRL/nou-darake decoy receptors. ptk7 and wntP-2 maintain and allow appropriate regeneration of trunk tissue position independently of canonical Wnt signaling and with suppression of ndl-3 expression in the posterior. These results suggest that restoration of regional identity in regeneration involves the interpretation and re-establishment of axis-wide transcriptional gradients of signaling molecules.

Gpr124 controls CNS angiogenesis and blood-brain barrier integrity by promoting ligand-specific canonical wnt signaling.

PubMed

Zhou, Yulian; Nathans, Jeremy

2014-10-27

Canonical Wnt signaling in endothelial cells (ECs) is required for vascularization of the central nervous system (CNS) and for formation and maintenance of barrier properties unique to CNS vasculature. Gpr124 is an orphan member of the adhesion G protein-coupled receptor family that is expressed in ECs and is essential for CNS angiogenesis and barrier formation via an unknown mechanism. Using canonical Wnt signaling assays in cell culture and genetic loss- and gain-of-function experiments in mice, we show that Gpr124 functions as a coactivator of Wnt7a- and Wnt7b-stimulated canonical Wnt signaling via a Frizzled receptor and Lrp coreceptor and that Gpr124-stimulated signaling functions in concert with Norrin/Frizzled4 signaling to control CNS vascular development. These experiments identify Gpr124 as a ligand-specific coactivator of canonical Wnt signaling.

Wnt Protein-mediated Satellite Cell Conversion in Adult and Aged Mice Following Voluntary Wheel Running

PubMed Central

Fujimaki, Shin; Hidaka, Ryo; Asashima, Makoto; Takemasa, Tohru; Kuwabara, Tomoko

2014-01-01

Muscle represents an abundant, accessible, and replenishable source of adult stem cells. Skeletal muscle-derived stem cells, called satellite cells, play essential roles in regeneration after muscle injury in adult skeletal muscle. Although the molecular mechanism of muscle regeneration process after an injury has been extensively investigated, the regulation of satellite cells under steady state during the adult stage, including the reaction to exercise stimuli, is relatively unknown. Here, we show that voluntary wheel running exercise, which is a low stress exercise, converts satellite cells to the activated state due to accelerated Wnt signaling. Our analysis showed that up-regulated canonical Wnt/β-catenin signaling directly modulated chromatin structures of both MyoD and Myf5 genes, resulting in increases in the mRNA expression of Myf5 and MyoD and the number of proliferative Pax7+Myf5+ and Pax7+ MyoD+ cells in skeletal muscle. The effect of Wnt signaling on the activation of satellite cells, rather than Wnt-mediated fibrosis, was observed in both adult and aged mice. The association of β-catenin, T-cell factor, and lymphoid enhancer transcription factors of multiple T-cell factor/lymphoid enhancer factor regulatory elements, conserved in mouse, rat, and human species, with the promoters of both the Myf5 and MyoD genes drives the de novo myogenesis in satellite cells even in aged muscle. These results indicate that exercise-stimulated extracellular Wnts play a critical role in the regulation of satellite cells in adult and aged skeletal muscle. PMID:24482229

Kinase cogs go forward and reverse in the Wnt signaling machine.

PubMed

Dale, Trevor

2006-01-01

An important link between Wnt binding at the cell surface and nuclear -catenin-TCF-dependent transcription has been made with the identification of kinases that promote the association of the Wnt receptor and -catenin turnover complexes. Surprisingly, the enzymes implicated had previously been suggested to inhibit rather than promote Wnt signaling.

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.

Wnt signaling regulates pulp volume and dentin thickness

PubMed Central

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

2015-01-01

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

A truncated Wnt7a retains full biological activity in skeletal muscle

NASA Astrophysics Data System (ADS)

von Maltzahn, Julia; Zinoviev, Radoslav; Chang, Natasha C.; Bentzinger, C. Florian; Rudnicki, Michael A.

2013-11-01

Wnt signaling has essential roles during embryonic development and tissue homoeostasis. Wnt proteins are post-translationally modified and the attachment of a palmitate moiety at two conserved residues is believed to be a prerequisite for the secretion and function of Wnt proteins. Here we demonstrate that a mammalian Wnt protein can be fully functional without palmitoylation. We generate a truncated Wnt7a variant, consisting of the C-terminal 137 amino acids lacking the conserved palmitoylation sites and show that it retains full biological activity in skeletal muscle. This includes binding to and signaling through its receptor Fzd7 to stimulate symmetric expansion of satellite stem cells by activating the planar-cell polarity pathway and inducing myofibre hypertrophy by signaling through the AKT/mTOR pathway. Furthermore, this truncated Wnt7a shows enhanced secretion and dispersion compared with the full-length protein. Together, these findings open important new avenues for the development of Wnt7a as a treatment for muscle-wasting diseases and have broad implications for the therapeutic use of Wnts as biologics.

Derivation of sarcomas from mesenchymal stem cells via inactivation of the Wnt pathway

PubMed Central

Matushansky, Igor; Hernando, Eva; Socci, Nicholas D.; Mills, Joslyn E.; Matos, Tulio A.; Edgar, Mark A.; Singer, Samuel; Maki, Robert G.; Cordon-Cardo, Carlos

2007-01-01

Malignant fibrous histiocytoma (MFH), now termed high-grade undifferentiated pleomorphic sarcoma, is a commonly diagnosed mesenchymal tumor, yet both the underlying molecular mechanisms of tumorigenesis and cell of origin remain unidentified. We present evidence demonstrating that human mesenchymal stem cells (hMSCs) are the progenitors of MFH. DKK1, a Wnt inhibitor and mediator of hMSC proliferation, is overexpressed in MFH. Using recombinant proteins, antibody depletion, and siRNA knockdown strategies of specific Wnt elements, we show that DKK1 inhibits hMSC commitment to differentiation via Wnt2/β-catenin canonical signaling and that Wnt5a/JNK noncanonical signaling regulates a viability checkpoint independent of Dkk1. Finally, we illustrate that hMSCs can be transformed via inhibition of Wnt signaling to form MFH-like tumors in nude mice, and conversely, MFH cells in which Wnt signaling is appropriately reestablished can differentiate along mature connective tissue lineages. Our results provide mechanistic insights regarding the cell of origin of MFH, establish what we believe is a novel tumor suppressor role for Wnt signaling, and identify a potential therapeutic differentiation strategy for sarcomas. PMID:17948129

Mindbomb 1, an E3 ubiquitin ligase, forms a complex with RYK to activate Wnt/β-catenin signaling

PubMed Central

Berndt, Jason D.; Aoyagi, Atsushi; Yang, Peitzu; Anastas, Jamie N.; Tang, Lan

2011-01-01

Receptor-like tyrosine kinase (RYK) functions as a transmembrane receptor for the Wnt family of secreted protein ligands. Although RYK undergoes endocytosis in response to Wnt, the mechanisms that regulate its internalization and concomitant activation of Wnt signaling are unknown. We discovered that RYK both physically and functionally interacts with the E3 ubiquitin ligase Mindbomb 1 (MIB1). Overexpression of MIB1 promotes the ubiquitination of RYK and reduces its steady-state levels at the plasma membrane. Moreover, we show that MIB1 is sufficient to activate Wnt/β-catenin (CTNNB1) signaling and that this activity depends on endogenous RYK. Conversely, in loss-of-function studies, both RYK and MIB1 are required for Wnt-3A–mediated activation of CTNNB1. Finally, we identify the Caenorhabditis elegans orthologue of MIB1 and demonstrate a genetic interaction between ceMIB and lin-18/RYK in vulva development. These findings provide insights into the mechanisms of Wnt/RYK signaling and point to novel targets for the modulation of Wnt signaling. PMID:21875946

Osteoblast-derived WNT16 represses osteoclastogenesis and prevents cortical bone fragility fractures

PubMed Central

Movérare-Skrtic, Sofia; Henning, Petra; Liu, Xianwen; Nagano, Kenichi; Saito, Hiroaki; Börjesson, Anna E; Sjögren, Klara; Windahl, Sara H; Farman, Helen; Kindlund, Bert; Engdahl, Cecilia; Koskela, Antti; Zhang, Fu-Ping; Eriksson, Emma E; Zaman, Farasat; Hammarstedt, Ann; Isaksson, Hanna; Bally, Marta; Kassem, Ali; Lindholm, Catharina; Sandberg, Olof; Aspenberg, Per; Sävendahl, Lars; Feng, Jian Q; Tuckermann, Jan; Tuukkanen, Juha; Poutanen, Matti; Baron, Roland; Lerner, Ulf H; Gori, Francesca; Ohlsson, Claes

2015-01-01

The WNT16 locus is a major determinant of cortical bone thickness and nonvertebral fracture risk in humans. The disability, mortality and costs caused by osteoporosis-induced nonvertebral fractures are enormous. We demonstrate here that Wnt16-deficient mice develop spontaneous fractures as a result of low cortical thickness and high cortical porosity. In contrast, trabecular bone volume is not altered in these mice. Mechanistic studies revealed that WNT16 is osteoblast derived and inhibits human and mouse osteoclastogenesis both directly by acting on osteoclast progenitors and indirectly by increasing expression of osteoprotegerin (Opg) in osteoblasts. The signaling pathway activated by WNT16 in osteoclast progenitors is noncanonical, whereas the pathway activated in osteoblasts is both canonical and noncanonical. Conditional Wnt16 inactivation revealed that osteoblast-lineage cells are the principal source of WNT16, and its targeted deletion in osteoblasts increases fracture susceptibility. Thus, osteoblast-derived WNT16 is a previously unreported key regulator of osteoclastogenesis and fracture susceptibility. These findings open new avenues for the specific prevention or treatment of nonvertebral fractures, a substantial unmet medical need. PMID:25306233

Wnt signaling inhibits CTL memory programming

PubMed Central

Xiao, Zhengguo; Sun, Zhifeng; Smyth, Kendra; Li, Lei

2013-01-01

Induction of functional CTLs is one of the major goals for vaccine development and cancer therapy. Inflammatory cytokines are critical for memory CTL generation. Wnt signaling is important for CTL priming and memory formation, but its role in cytokine-driven memory CTL programming is unclear. We found that wnt signaling inhibited IL-12-driven CTL activation and memory programming. This impaired memory CTL programming was attributed to up-regulation of eomes and down-regulation of T-bet. Wnt signaling suppressed the mTOR pathway during CTL activation, which was different to its effects on other cell types. Interestingly, the impaired memory CTL programming by wnt was partially rescued by mTOR inhibitor rapamycin. In conclusion, we found that crosstalk between wnt and the IL-12 signaling inhibits T-bet and mTOR pathways and impairs memory programming which can be recovered in part by rapamycin. In addition, direct inhibition of wnt signaling during CTL activation does not affect CTL memory programming. Therefore, wnt signaling may serve as a new tool for CTL manipulation in autoimmune diseases and immune therapy for certain cancers. PMID:23911398

Canonical WNT signalling determines lineage specificity in Wilms tumour.

PubMed

Fukuzawa, R; Anaka, M R; Weeks, R J; Morison, I M; Reeve, A E

2009-02-26

Wilms tumours (WTs) have two distinct types of histology with or without ectopic mesenchymal elements, suggesting that WTs arise from either the mesenchymal or epithelial nephrogenic lineages. Regardless of the presence or absence of CTNNB1 mutations, nuclear accumulation of beta-catenin is often observed in WTs with ectopic mesenchymal elements. Here, we addressed the relationship between the WNT-signalling pathway and lineage in WTs by examining CTNNB1 and WT1 mutations, nuclear accumulation of beta-catenin, tumour histology and gene expression profiles. In addition, we screened for mutations in WTX, which has been proposed to be a negative regulator of the canonical WNT-signalling pathway. Unsupervised clustering analysis identified two classes of tumours: mesenchymal lineage WNT-dependent tumours, and epithelial lineage WNT-independent tumours. In contrast to the mesenchymal lineage specificity of CTNNB1 mutations, WTX mutations were surprisingly observed in both lineages. WTX-mutant WTs with ectopic mesenchymal elements had nuclear accumulation of beta-catenin, upregulation of WNT target genes and an association with CTNNB1 mutations in exon 7 or 8. However, epithelial lineage WTs with WTX mutations had no indications of active WNT signalling, suggesting that the involvement of WTX in the WNT-signalling pathway may be lineage dependent, and that WTX may have an alternative function to its role in the canonical WNT-signalling pathway.

HECT Domain-containing E3 Ubiquitin Ligase NEDD4L Negatively Regulates Wnt Signaling by Targeting Dishevelled for Proteasomal Degradation*

PubMed Central

Ding, Yi; Zhang, Yan; Xu, Chao; Tao, Qing-Hua; Chen, Ye-Guang

2013-01-01

Wnt signaling plays a pivotal role in embryogenesis and tissue homeostasis. Dishevelled (Dvl) is a central mediator for both Wnt/β-catenin and Wnt/planar cell polarity pathways. NEDD4L, an E3 ubiquitin ligase, has been shown to regulate ion channel activity, cell signaling, and cell polarity. Here, we report a novel role of NEDD4L in the regulation of Wnt signaling. NEDD4L induces Dvl2 polyubiquitination and targets Dvl2 for proteasomal degradation. Interestingly, the NEDD4L-mediated ubiquitination of Dvl2 is Lys-6, Lys-27, and Lys-29 linked but not typical Lys-48-linked ubiquitination. Consistent with the role of Dvl in both Wnt/β-catenin and Wnt/planar cell polarity signaling, NEDD4L regulates the cellular β-catenin level and Rac1, RhoA, and JNK activities. We have further identified a hierarchical regulation that Wnt5a induces JNK-mediated phosphorylation of NEDD4L, which in turn promotes its ability to degrade Dvl2. Finally, we show that NEDD4L inhibits Dvl2-induced axis duplication in Xenopus embryos. Our work thus demonstrates that NEDD4L is a negative feedback regulator of Wnt signaling. PMID:23396981

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

Liu, Ru-Ming; Sun, Ren-Gang; Zhang, Ling-Tao

This study investigated the pro-proliferative effect of hyaluronic acid (HA) on human amniotic mesenchymal stem cells (hAMSCs) and the underlying mechanisms. Treatment with HA increased cell population growth in a dose- and time-dependent manner. Analyses by flow cytometry and immunocytochemistry revealed that HA did not change the cytophenotypes of hAMSCs. Additionally, the osteogenic, chondrogenic, and adipogenic differentiation capabilities of these hAMSCs were retained after HA treatment. Moreover, HA increased the mRNA expressions of wnt1, wnt3a, wnt8a, cyclin D1, Ki-67, and β-catenin as well as the protein level of β-catenin and cyclin D1 in hAMSCs; and the nuclear localization of β-cateninmore » was also enhanced. Furthermore, the pro-proliferative effect of HA and up-regulated expression of Wnt/β-catenin pathway-associated proteins - wnt3a, β-catenin and cyclin D1 in hAMSCs were significantly inhibited upon pre-treatment with Wnt-C59, an inhibitor of the Wnt/β-catenin pathway. These results suggest that HA may positively regulate hAMSCs proliferation through regulation of the Wnt/β-catenin signaling pathway. - Highlights: • Hyaluronic acid (HA) could promote the proliferation of hAMSCs. • HA treatment dose not affect the pluripotency of hAMSCs. • HA increases hAMSCs proliferation through activation of Wnt/β-catenin signaling.« less

Differential Regulation of Disheveled in a Novel Vegetal Cortical Domain in Sea Urchin Eggs and Embryos: Implications for the Localized Activation of Canonical Wnt Signaling

PubMed Central

Peng, ChiehFu Jeff; Wikramanayake, Athula H.

2013-01-01

Pattern formation along the animal-vegetal (AV) axis in sea urchin embryos is initiated when canonical Wnt (cWnt) signaling is activated in vegetal blastomeres. The mechanisms that restrict cWnt signaling to vegetal blastomeres are not well understood, but there is increasing evidence that the egg’s vegetal cortex plays a critical role in this process by mediating localized “activation” of Disheveled (Dsh). To investigate how Dsh activity is regulated along the AV axis, sea urchin-specific Dsh antibodies were used to examine expression, subcellular localization, and post-translational modification of Dsh during development. Dsh is broadly expressed during early sea urchin development, but immunolocalization studies revealed that this protein is enriched in a punctate pattern in a novel vegetal cortical domain (VCD) in the egg. Vegetal blastomeres inherit this VCD during embryogenesis, and at the 60-cell stage Dsh puncta are seen in all cells that display nuclear β-catenin. Analysis of Dsh post-translational modification using two-dimensional Western blot analysis revealed that compared to Dsh pools in the bulk cytoplasm, this protein is differentially modified in the VCD and in the 16-cell stage micromeres that partially inherit this domain. Dsh localization to the VCD is not directly affected by disruption of microfilaments and microtubules, but unexpectedly, microfilament disruption led to degradation of all the Dsh pools in unfertilized eggs over a period of incubation suggesting that microfilament integrity is required for maintaining Dsh stability. These results demonstrate that a pool of differentially modified Dsh in the VCD is selectively inherited by the vegetal blastomeres that activate cWnt signaling in early embryos, and suggests that this domain functions as a scaffold for localized Dsh activation. Localized cWnt activation regulates AV axis patterning in many metazoan embryos. Hence, it is possible that the VCD is an evolutionarily conserved cytoarchitectural domain that specifies the AV axis in metazoan ova. PMID:24236196

Circadian rhythms, Wnt/beta-catenin pathway and PPAR alpha/gamma profiles in diseases with primary or secondary cardiac dysfunction

PubMed Central

Lecarpentier, Yves; Claes, Victor; Duthoit, Guillaume; Hébert, Jean-Louis

2014-01-01

Circadian clock mechanisms are far-from-equilibrium dissipative structures. Peroxisome proliferator-activated receptors (PPAR alpha, beta/delta, and gamma) play a key role in metabolic regulatory processes, particularly in heart muscle. Links between circadian rhythms (CRs) and PPARs have been established. Mammalian CRs involve at least two critical transcription factors, CLOCK and BMAL1 (Gekakis et al., 1998; Hogenesch et al., 1998). PPAR gamma plays a major role in both glucose and lipid metabolisms and presents circadian properties which coordinate the interplay between metabolism and CRs. PPAR gamma is a major component of the vascular clock. Vascular PPAR gamma is a peripheral regulator of cardiovascular rhythms controlling circadian variations in blood pressure and heart rate through BMAL1. We focused our review on diseases with abnormalities of CRs and with primary or secondary cardiac dysfunction. Moreover, these diseases presented changes in the Wnt/beta-catenin pathway and PPARs, according to two opposed profiles. Profile 1 was defined as follows: inactivation of the Wnt/beta-catenin pathway with increased expression of PPAR gamma. Profile 2 was defined as follows: activation of the Wnt/beta-catenin pathway with decreased expression of PPAR gamma. A typical profile 1 disease is arrhythmogenic right ventricular cardiomyopathy, a genetic cardiac disease which presents mutations of the desmosomal proteins and is mainly characterized by fatty acid accumulation in adult cardiomyocytes mainly in the right ventricle. The link between PPAR gamma dysfunction and desmosomal genetic mutations occurs via inactivation of the Wnt/beta-catenin pathway presenting oscillatory properties. A typical profile 2 disease is type 2 diabetes, with activation of the Wnt/beta-catenin pathway and decreased expression of PPAR gamma. CRs abnormalities are present in numerous pathologies such as cardiovascular diseases, sympathetic/parasympathetic dysfunction, hypertension, diabetes, neurodegenerative diseases, cancer which are often closely inter-related. PMID:25414671

1α,25-Dihydroxyvitamin D3 Modulates the Hair-Inductive Capacity of Dermal Papilla Cells: Therapeutic Potential for Hair Regeneration

PubMed Central

Aoi, Noriyuki; Inoue, Keita; Chikanishi, Toshihiro; Fujiki, Ryoji; Yamamoto, Hanako; Kato, Harunosuke; Eto, Hitomi; Doi, Kentaro; Itami, Satoshi; Kato, Shigeaki

2012-01-01

Dermal papilla cells (DPCs) have the potential to induce differentiation of epithelial stem cells into hair, and Wnt signaling is deeply involved in the initiation process. The functional limitation of expanded adult DPCs has been a difficult challenge for cell-based hair regrowth therapy. We previously reported that 1α,25-dihydroxyvitamin D3 (VD3) upregulates expression of transforming growth factor (TGF)-β2 and alkaline phosphatase (ALP) activity, both features of hair-inducing human DPCs (hDPCs). In this study, we further examined the effects and signaling pathways associated with VD3 actions on DPCs. VD3 suppressed hDPC proliferation in a dose-dependent, noncytotoxic manner. Among the Wnt-related genes investigated, Wnt10b expression was significantly upregulated by VD3 in hDPCs. Wnt10b upregulation, as well as upregulation of ALPL (ALP, liver/bone/kidney) and TGF-β2, by VD3 was specific in hDPCs and not detected in human dermal fibroblasts. Screening of paracrine or endocrine factors in the skin indicated that all-trans retinoic acid (atRA) upregulated Wnt10b gene expression, although synergistic upregulation (combined atRA and VD3) was not seen. RNA interference with vitamin D receptor (VDR) revealed that VD3 upregulation of Wnt10b, ALPL, and TGF-β2 was mediated through the genomic VDR pathway. In a rat model of de novo hair regeneration by murine DPC transplantation, pretreatment with VD3 significantly enhanced hair folliculogenesis. Specifically, a greater number of outgrowing hair shafts and higher maturation of regenerated follicles were observed. Together, these data suggest that VD3 may promote functional differentiation of DPCs and be useful in preserving the hair follicle-inductive capacity of cultured DPCs for hair regeneration therapies. PMID:23197867

Differential Responses to Wnt and PCP Disruption Predict Expression and Developmental Function of Conserved and Novel Genes in a Cnidarian

PubMed Central

Lapébie, Pascal; Ruggiero, Antonella; Barreau, Carine; Chevalier, Sandra; Chang, Patrick; Dru, Philippe; Houliston, Evelyn; Momose, Tsuyoshi

2014-01-01

We have used Digital Gene Expression analysis to identify, without bilaterian bias, regulators of cnidarian embryonic patterning. Transcriptome comparison between un-manipulated Clytia early gastrula embryos and ones in which the key polarity regulator Wnt3 was inhibited using morpholino antisense oligonucleotides (Wnt3-MO) identified a set of significantly over and under-expressed transcripts. These code for candidate Wnt signaling modulators, orthologs of other transcription factors, secreted and transmembrane proteins known as developmental regulators in bilaterian models or previously uncharacterized, and also many cnidarian-restricted proteins. Comparisons between embryos injected with morpholinos targeting Wnt3 and its receptor Fz1 defined four transcript classes showing remarkable correlation with spatiotemporal expression profiles. Class 1 and 3 transcripts tended to show sustained expression at “oral” and “aboral” poles respectively of the developing planula larva, class 2 transcripts in cells ingressing into the endodermal region during gastrulation, while class 4 gene expression was repressed at the early gastrula stage. The preferential effect of Fz1-MO on expression of class 2 and 4 transcripts can be attributed to Planar Cell Polarity (PCP) disruption, since it was closely matched by morpholino knockdown of the specific PCP protein Strabismus. We conclude that endoderm and post gastrula-specific gene expression is particularly sensitive to PCP disruption while Wnt-/β-catenin signaling dominates gene regulation along the oral-aboral axis. Phenotype analysis using morpholinos targeting a subset of transcripts indicated developmental roles consistent with expression profiles for both conserved and cnidarian-restricted genes. Overall our unbiased screen allowed systematic identification of regionally expressed genes and provided functional support for a shared eumetazoan developmental regulatory gene set with both predicted and previously unexplored members, but also demonstrated that fundamental developmental processes including axial patterning and endoderm formation in cnidarians can involve newly evolved (or highly diverged) genes. PMID:25233086

Differential responses to Wnt and PCP disruption predict expression and developmental function of conserved and novel genes in a cnidarian.

PubMed

Lapébie, Pascal; Ruggiero, Antonella; Barreau, Carine; Chevalier, Sandra; Chang, Patrick; Dru, Philippe; Houliston, Evelyn; Momose, Tsuyoshi

2014-09-01

We have used Digital Gene Expression analysis to identify, without bilaterian bias, regulators of cnidarian embryonic patterning. Transcriptome comparison between un-manipulated Clytia early gastrula embryos and ones in which the key polarity regulator Wnt3 was inhibited using morpholino antisense oligonucleotides (Wnt3-MO) identified a set of significantly over and under-expressed transcripts. These code for candidate Wnt signaling modulators, orthologs of other transcription factors, secreted and transmembrane proteins known as developmental regulators in bilaterian models or previously uncharacterized, and also many cnidarian-restricted proteins. Comparisons between embryos injected with morpholinos targeting Wnt3 and its receptor Fz1 defined four transcript classes showing remarkable correlation with spatiotemporal expression profiles. Class 1 and 3 transcripts tended to show sustained expression at "oral" and "aboral" poles respectively of the developing planula larva, class 2 transcripts in cells ingressing into the endodermal region during gastrulation, while class 4 gene expression was repressed at the early gastrula stage. The preferential effect of Fz1-MO on expression of class 2 and 4 transcripts can be attributed to Planar Cell Polarity (PCP) disruption, since it was closely matched by morpholino knockdown of the specific PCP protein Strabismus. We conclude that endoderm and post gastrula-specific gene expression is particularly sensitive to PCP disruption while Wnt-/β-catenin signaling dominates gene regulation along the oral-aboral axis. Phenotype analysis using morpholinos targeting a subset of transcripts indicated developmental roles consistent with expression profiles for both conserved and cnidarian-restricted genes. Overall our unbiased screen allowed systematic identification of regionally expressed genes and provided functional support for a shared eumetazoan developmental regulatory gene set with both predicted and previously unexplored members, but also demonstrated that fundamental developmental processes including axial patterning and endoderm formation in cnidarians can involve newly evolved (or highly diverged) genes.

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

Sominsky, Sophia, E-mail: sophia.tab@gmail.com; Kuslansky, Yael, E-mail: ykuslansky@gmail.com; Shapiro, Beny, E-mail: benyshap@gmail.com

The present study investigated the roles of E6 and E6AP in the Wnt pathway. We showed that E6 levels are markedly reduced in cells in which Wnt signaling is activated. Coexpression of wild-type or mutant E6AP (C820A) in Wnt-activated cells stabilized E6 and enhanced Wnt/β-catenin/TCF transcription. Expression of E6AP alone in nonstimulated cells elevated β-catenin level, promoted its nuclear accumulation, and activated β-catenin/TCF transcription. A knockdown of E6AP lowered β-catenin levels. Coexpression with E6 intensified the activities of E6AP. Further experiments proved that E6AP/E6 stabilize β-catenin by protecting it from proteasomal degradation. This function was dependent on the catalytic activitymore » of E6AP, the kinase activity of GSK3β and the susceptibility of β-catenin to GSK3β phosphorylation. Thus, this study identified E6AP as a novel regulator of the Wnt signaling pathway, capable of cooperating with E6 in stimulating or augmenting Wnt/β-catenin signaling, thereby possibly contributing to HPV carcinogenesis. - Highlights: • The roles of E6 and E6AP in the Wnt pathway were investigated. • E6AP stabilizes E6 and enhances E6 activity in augmentation of Wnt signaling. • E6AP cooperates with E6 to stabilize β-catenin and stimulate Wnt/β-catenin signaling. • E6AP and E6 act through different mechanisms to augment or stimulate Wnt signaling.« less

R-spondin 3 regulates dorsoventral and anteroposterior patterning by antagonizing Wnt/β-catenin signaling in zebrafish embryos.

PubMed

Rong, Xiaozhi; Chen, Chen; Zhou, Pin; Zhou, Yumei; Li, Yun; Lu, Ling; Liu, Yunzhang; Zhou, Jianfeng; Duan, Cunming

2014-01-01

The Wnt/β-catenin or canonical Wnt signaling pathway plays fundamental roles in early development and in maintaining adult tissue homeostasis. R-spondin 3 (Rspo3) is a secreted protein that has been implicated in activating the Wnt/β-catenin signaling in amphibians and mammals. Here we report that zebrafish Rspo3 plays a negative role in regulating the zygotic Wnt/β-catenin signaling. Zebrafish Rspo3 has a unique domain structure. It contains a third furin-like (FU3) domain. This FU3 is present in other four ray-finned fish species studied but not in elephant shark. In zebrafish, rspo3 mRNA is maternally deposited and has a ubiquitous expression in early embryonic stages. After 12 hpf, its expression becomes tissue-specific. Forced expression of rspo3 promotes dorsoanterior patterning and increases the expression of dorsal and anterior marker genes. Knockdown of rspo3 increases ventral-posterior development and stimulates ventral and posterior marker genes expression. Forced expression of rspo3 abolishes exogenous Wnt3a action and reduces the endogenous Wnt signaling activity. Knockdown of rspo3 results in increased Wnt/β-catenin signaling activity. Further analyses indicate that Rspo3 does not promote maternal Wnt signaling. Human RSPO3 has similar action when tested in zebrafish embryos. These results suggest that Rspo3 regulates dorsoventral and anteroposterior patterning by negatively regulating the zygotic Wnt/β-catenin signaling in zebrafish embryos.

R-Spondin 3 Regulates Dorsoventral and Anteroposterior Patterning by Antagonizing Wnt/β-Catenin Signaling in Zebrafish Embryos

PubMed Central

Zhou, Pin; Zhou, Yumei; Li, Yun; Lu, Ling; Liu, Yunzhang; Zhou, Jianfeng; Duan, Cunming

2014-01-01

The Wnt/β-catenin or canonical Wnt signaling pathway plays fundamental roles in early development and in maintaining adult tissue homeostasis. R-spondin 3 (Rspo3) is a secreted protein that has been implicated in activating the Wnt/β-catenin signaling in amphibians and mammals. Here we report that zebrafish Rspo3 plays a negative role in regulating the zygotic Wnt/β-catenin signaling. Zebrafish Rspo3 has a unique domain structure. It contains a third furin-like (FU3) domain. This FU3 is present in other four ray-finned fish species studied but not in elephant shark. In zebrafish, rspo3 mRNA is maternally deposited and has a ubiquitous expression in early embryonic stages. After 12 hpf, its expression becomes tissue-specific. Forced expression of rspo3 promotes dorsoanterior patterning and increases the expression of dorsal and anterior marker genes. Knockdown of rspo3 increases ventral-posterior development and stimulates ventral and posterior marker genes expression. Forced expression of rspo3 abolishes exogenous Wnt3a action and reduces the endogenous Wnt signaling activity. Knockdown of rspo3 results in increased Wnt/β-catenin signaling activity. Further analyses indicate that Rspo3 does not promote maternal Wnt signaling. Human RSPO3 has similar action when tested in zebrafish embryos. These results suggest that Rspo3 regulates dorsoventral and anteroposterior patterning by negatively regulating the zygotic Wnt/β-catenin signaling in zebrafish embryos. PMID:24918770

Controlled levels of canonical Wnt signaling are required for neural crest migration.

PubMed

Maj, Ewa; Künneke, Lutz; Loresch, Elisabeth; Grund, Anita; Melchert, Juliane; Pieler, Tomas; Aspelmeier, Timo; Borchers, Annette

2016-09-01

Canonical Wnt signaling plays a dominant role in the development of the neural crest (NC), a highly migratory cell population that generates a vast array of cell types. Canonical Wnt signaling is required for NC induction as well as differentiation, however its role in NC migration remains largely unknown. Analyzing nuclear localization of β-catenin as readout for canonical Wnt activity, we detect nuclear β-catenin in premigratory but not migratory Xenopus NC cells suggesting that canonical Wnt activity has to decrease to basal levels to enable NC migration. To define a possible function of canonical Wnt signaling in Xenopus NC migration, canonical Wnt signaling was modulated at different time points after NC induction. This was accomplished using either chemical modulators affecting β-catenin stability or inducible glucocorticoid fusion constructs of Lef/Tcf transcription factors. In vivo analysis of NC migration by whole mount in situ hybridization demonstrates that ectopic activation of canonical Wnt signaling inhibits cranial NC migration. Further, NC transplantation experiments confirm that this effect is tissue-autonomous. In addition, live-cell imaging in combination with biophysical data analysis of explanted NC cells confirms the in vivo findings and demonstrates that modulation of canonical Wnt signaling affects the ability of NC cells to perform single cell migration. Thus, our data support the hypothesis that canonical Wnt signaling needs to be tightly controlled to enable migration of NC cells. Copyright © 2016 Elsevier Inc. All rights reserved.

ME-143 Is Superior to Genistein in Suppression of WNT Signaling in Colon Cancer Cells.

PubMed

Pintova, Sofya; Planutis, Kestutis; Planutiene, Marina; Holcombe, Randall F

2017-04-01

This study tested the effect of the soy isoflavones genistein and ME-143, and two chemotherapeutic agents, 5-fluorouracil (5FU) and oxaliplatin, on WNT signaling. Colon cancer cell lines RKO (hereditary nonpolyposis colorectal cancer type) and DLD1 (most common colorectal cancer type driven by a mutation in WNT pathway) were utilized. WNT throughput was measured using a β-catenin-responsive SuperTopFlash luciferase assay. A stabilized β-catenin construct was employed to test β-catenin involvement in the mechanism of drug activity. ME-143 was a more than 10-fold potent inhibitor of DLD1 proliferation than genistein at 3.125 μM. Genistein alone did not inhibit WNT signaling in either cell line. In RKO cells, oxaliplatin and its combination with 5FU significantly inhibited WNT throughput. Neither 5FU, oxaliplatin nor their combination inhibited WNT signaling in DLD1 cells. In both the RKO and DLD1 cell lines, ME-143 significantly reduced WNT throughput by 65-75%. The introduction of stabilized β-catenin attenuated the ME-143-dependent inhibition of the WNT/β-catenin pathway. ME-143 alone and in combination with 5FU and oxaliplatin effectively inhibits the WNT/β-catenin pathway in colorectal cancer cells of diverse genetic background. β-Catenin is directly involved in the mechanism of inhibition, and clinical studies are warranted. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Genomic response to Wnt signalling is highly context-dependent - Evidence from DNA microarray and chromatin immunoprecipitation screens of Wnt/TCF targets

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

Railo, Antti; Pajunen, Antti; Itaeranta, Petri

2009-10-01

Wnt proteins are important regulators of embryonic development, and dysregulated Wnt signalling is involved in the oncogenesis of several human cancers. Our knowledge of the downstream target genes is limited, however. We used a chromatin immunoprecipitation-based assay to isolate and characterize the actual gene segments through which Wnt-activatable transcription factors, TCFs, regulate transcription and an Affymetrix microarray analysis to study the global transcriptional response to the Wnt3a ligand. The anti-{beta}-catenin immunoprecipitation of DNA-protein complexes from mouse NIH3T3 fibroblasts expressing a fusion protein of {beta}-catenin and TCF7 resulted in the identification of 92 genes as putative TCF targets. GeneChip assays ofmore » gene expression performed on NIH3T3 cells and the rat pheochromocytoma cell line PC12 revealed 355 genes in NIH3T3 and 129 genes in the PC12 cells with marked changes in expression after Wnt3a stimulus. Only 2 Wnt-regulated genes were shared by both cell lines. Surprisingly, Disabled-2 was the only gene identified by the chromatin immunoprecipitation approach that displayed a marked change in expression in the GeneChip assay. Taken together, our approaches give an insight into the complex context-dependent nature of Wnt pathway transcriptional responses and identify Disabled-2 as a potential new direct target for Wnt signalling.« less

Acute WNT signalling activation perturbs differentiation within the adult stomach and rapidly leads to tumour formation

PubMed Central

Radulescu, S; Ridgway, R A; Cordero, J; Athineos, D; Salgueiro, P; Poulsom, R; Neumann, J; Jung, A; Patel, S; Woodgett, J; Barker, N; Pritchard, D M; Oien, K; Sansom, O J

2013-01-01

A role for WNT signalling in gastric carcinogenesis has been suggested due to two major observations. First, patients with germline mutations in adenomatous polyposis coli (APC) are susceptible to stomach polyps and second, in gastric cancer, WNT activation confers a poor prognosis. However, the functional significance of deregulated WNT signalling in gastric homoeostasis and cancer is still unclear. In this study we have addressed this by investigating the immediate effects of WNT signalling activation within the stomach epithelium. We have specifically activated the WNT signalling pathway within the mouse adult gastric epithelium via deletion of either glycogen synthase kinase 3 (GSK3) or APC or via expression of a constitutively active β-catenin protein. WNT pathway deregulation dramatically affects stomach homoeostasis at very short latencies. In the corpus, there is rapid loss of parietal cells with fundic gland polyp (FGP) formation and adenomatous change, which are similar to those observed in familial adenomatous polyposis. In the antrum, adenomas occur from 4 days post-WNT activation. Taken together, these data show a pivotal role for WNT signalling in gastric homoeostasis, FGP formation and adenomagenesis. Loss of the parietal cell population and corresponding FGP formation, an early event in gastric carcinogenesis, as well as antral adenoma formation are immediate effects of nuclear β-catenin translocation and WNT target gene expression. Furthermore, our inducible murine model will permit a better understanding of the molecular changes required to drive tumourigenesis in the stomach. PMID:22665058

Cytotoxicity and activation of the Wnt/beta-catenin pathway in mouse embryonic stem cells treated with four GSK3 inhibitors

PubMed Central

2014-01-01

Background Small membrane-permeable molecules are now widely used during maintenance and differentiation of embryonic stem cells of different species. In particular the glycogen synthase kinase 3 (GSK3) is an interesting target, since its chemical inhibition activates the Wnt/beta-catenin pathway. In the present comparative study four GSK3 inhibitors were characterized. Methods Cytotoxicity and potential to activate the Wnt/beta-catenin pathway were tested using the commonly used GSK3 inhibitors BIO, SB-216763, CHIR-99021, and CHIR-98014. Wnt/beta-catenin-dependent target genes were measured by quantitative PCR to confirm the Wnt-reporter assay and finally EC50-values were calculated. Results CHIR-99021 and SB-216763 had the lowest toxicities in mouse embryonic stem cells and CHIR-98014 and BIO the highest toxicities. Only CHIR-99021 and CHIR-98014 lead to a strong induction of the Wnt/beta-catenin pathway, whereas BIO and SB-216763 showed a minor or no increase in activation of the Wnt/beta-catenin pathway over the natural ligand Wnt3a. The data from the Wnt-reporter assay were confirmed by gene expression analysis of the TCF/LEF regulated gene T. Conclusions Out of the four tested GSK3 inhibitors, only CHIR-99021 and CHIR-98014 proved to be potent pharmacological activators of the Wnt/beta-catenin signaling pathway. But only in the case of CHIR-99021 high potency was combined with very low toxicity. PMID:24779365

Cytotoxicity and activation of the Wnt/beta-catenin pathway in mouse embryonic stem cells treated with four GSK3 inhibitors.

PubMed

Naujok, Ortwin; Lentes, Jana; Diekmann, Ulf; Davenport, Claudia; Lenzen, Sigurd

2014-04-29

Small membrane-permeable molecules are now widely used during maintenance and differentiation of embryonic stem cells of different species. In particular the glycogen synthase kinase 3 (GSK3) is an interesting target, since its chemical inhibition activates the Wnt/beta-catenin pathway. In the present comparative study four GSK3 inhibitors were characterized. Cytotoxicity and potential to activate the Wnt/beta-catenin pathway were tested using the commonly used GSK3 inhibitors BIO, SB-216763, CHIR-99021, and CHIR-98014. Wnt/beta-catenin-dependent target genes were measured by quantitative PCR to confirm the Wnt-reporter assay and finally EC50-values were calculated. CHIR-99021 and SB-216763 had the lowest toxicities in mouse embryonic stem cells and CHIR-98014 and BIO the highest toxicities. Only CHIR-99021 and CHIR-98014 lead to a strong induction of the Wnt/beta-catenin pathway, whereas BIO and SB-216763 showed a minor or no increase in activation of the Wnt/beta-catenin pathway over the natural ligand Wnt3a. The data from the Wnt-reporter assay were confirmed by gene expression analysis of the TCF/LEF regulated gene T. Out of the four tested GSK3 inhibitors, only CHIR-99021 and CHIR-98014 proved to be potent pharmacological activators of the Wnt/beta-catenin signaling pathway. But only in the case of CHIR-99021 high potency was combined with very low toxicity.

TCDD Inhibition of Canonical Wnt Signaling Disrupts Prostatic Bud Formation in Mouse Urogenital Sinus

PubMed Central

Peterson, Richard E.

2013-01-01

In mice, in utero exposure to 2,3,7,8-tetrachlorodibenzo-p- dioxin (TCDD) reduces the number of dorsolateral prostatic buds resulting in a smaller dorsolateral prostate and prevents formation of ventral buds culminating in ventral prostate agenesis. The genes and signaling pathways affected by TCDD that are responsible for disrupting prostate development are largely unknown. Here we show that treatment of urogenital sinus (UGS) organ cultures with known inhibitors of canonical Wnt signaling also inhibits prostatic bud formation. In support of the hypothesis that TCDD decreases canonical Wnt signaling, we identify inhibitory effects of TCDD on multiple components of the canonical Wnt signaling pathway in the UGS that temporally coincide with the inhibitory effect of TCDD on prostatic bud formation: (1) expression of R-spondins (Rspo2 and Rspo3) that promote canonical Wnt signaling is reduced; (2) expression of Lef1, Tcf1, and Wif1, established canonical Wnt target genes, is decreased; (3) expression of Lgr5, a RSPO receptor that activates canonical Wnt signaling, is reduced; and (4) expression of Dickkopfs (Dkks), inhibitors of canonical Wnt signaling, is not increased by TCDD. Thus, the TCDD-induced reduction in canonical Wnt signaling is associated with a decrease in activators (Rspo2 and Rspo3) rather than an increase in inhibitors (Dkk1 and Dkk2) of the pathway. This study focuses on determining whether treatment of TCDD-exposed UGS organ cultures with RSPO2 and/or RSPO3 is capable of rescuing the inhibitory effects of TCDD on canonical Wnt signaling and prostatic bud formation. We discovered that each RSPO alone or in combination partially rescues TCDD inhibition of both canonical Wnt signaling and prostatic bud formation. PMID:23429912

TCDD inhibition of canonical Wnt signaling disrupts prostatic bud formation in mouse urogenital sinus.

PubMed

Branam, Amanda M; Davis, Nicole M; Moore, Robert W; Schneider, Andrew J; Vezina, Chad M; Peterson, Richard E

2013-05-01

In mice, in utero exposure to 2,3,7,8-tetrachlorodibenzo-p- dioxin (TCDD) reduces the number of dorsolateral prostatic buds resulting in a smaller dorsolateral prostate and prevents formation of ventral buds culminating in ventral prostate agenesis. The genes and signaling pathways affected by TCDD that are responsible for disrupting prostate development are largely unknown. Here we show that treatment of urogenital sinus (UGS) organ cultures with known inhibitors of canonical Wnt signaling also inhibits prostatic bud formation. In support of the hypothesis that TCDD decreases canonical Wnt signaling, we identify inhibitory effects of TCDD on multiple components of the canonical Wnt signaling pathway in the UGS that temporally coincide with the inhibitory effect of TCDD on prostatic bud formation: (1) expression of R-spondins (Rspo2 and Rspo3) that promote canonical Wnt signaling is reduced; (2) expression of Lef1, Tcf1, and Wif1, established canonical Wnt target genes, is decreased; (3) expression of Lgr5, a RSPO receptor that activates canonical Wnt signaling, is reduced; and (4) expression of Dickkopfs (Dkks), inhibitors of canonical Wnt signaling, is not increased by TCDD. Thus, the TCDD-induced reduction in canonical Wnt signaling is associated with a decrease in activators (Rspo2 and Rspo3) rather than an increase in inhibitors (Dkk1 and Dkk2) of the pathway. This study focuses on determining whether treatment of TCDD-exposed UGS organ cultures with RSPO2 and/or RSPO3 is capable of rescuing the inhibitory effects of TCDD on canonical Wnt signaling and prostatic bud formation. We discovered that each RSPO alone or in combination partially rescues TCDD inhibition of both canonical Wnt signaling and prostatic bud formation.

The non-canonical BMP and Wnt/β-catenin signaling pathways orchestrate early tooth development

PubMed Central

Yuan, Guohua; Yang, Guobin; Zheng, Yuqian; Zhu, Xiaojing; Chen, Zhi; Zhang, Zunyi; Chen, YiPing

2015-01-01

BMP and Wnt signaling pathways play a crucial role in organogenesis, including tooth development. Despite extensive studies, the exact functions, as well as if and how these two pathways act coordinately in regulating early tooth development, remain elusive. In this study, we dissected regulatory functions of BMP and Wnt pathways in early tooth development using a transgenic noggin (Nog) overexpression model (K14Cre;pNog). It exhibits early arrested tooth development, accompanied by reduced cell proliferation and loss of odontogenic fate marker Pitx2 expression in the dental epithelium. We demonstrated that overexpression of Nog disrupted BMP non-canonical activity, which led to a dramatic reduction of cell proliferation rate but did not affect Pitx2 expression. We further identified a novel function of Nog by inhibiting Wnt/β-catenin signaling, causing loss of Pitx2 expression. Co-immunoprecipitation and TOPflash assays revealed direct binding of Nog to Wnts to functionally prevent Wnt/β-catenin signaling. In situ PLA and immunohistochemistry on Nog mutants confirmed in vivo interaction between endogenous Nog and Wnts and modulation of Wnt signaling by Nog in tooth germs. Genetic rescue experiments presented evidence that both BMP and Wnt signaling pathways contribute to cell proliferation regulation in the dental epithelium, with Wnt signaling also controlling the odontogenic fate. Reactivation of both BMP and Wnt signaling pathways, but not of only one of them, rescued tooth developmental defects in K14Cre;pNog mice, in which Wnt signaling can be substituted by transgenic activation of Pitx2. Our results reveal the orchestration of non-canonical BMP and Wnt/β-catenin signaling pathways in the regulation of early tooth development. PMID:25428587

Prostaglandin E2 alters Wnt-dependent migration and proliferation in neuroectodermal stem cells: implications for autism spectrum disorders

PubMed Central

2014-01-01

Prostaglandin E2 (PGE2) is a natural lipid-derived molecule that is involved in important physiological functions. Abnormal PGE2 signalling has been associated with pathologies of the nervous system. Previous studies provide evidence for the interaction of PGE2 and canonical Wnt signalling pathways in non-neuronal cells. Since the Wnt pathway is crucial in the development and organization of the brain, the main goal of this study is to determine whether collaboration between these pathways exists in neuronal cell types. We report that PGE2 interacts with canonical Wnt signalling through PKA and PI-3K in neuroectodermal (NE-4C) stem cells. We used time-lapse microscopy to determine that PGE2 increases the final distance from origin, path length travelled, and the average speed of migration in Wnt-activated cells. Furthermore, PGE2 alters distinct cellular phenotypes that are characteristic of Wnt-induced NE-4C cells, which corresponds to the modified splitting behaviour of the cells. We also found that in Wnt-induced cells the level of β-catenin protein was increased and the expression levels of Wnt-target genes (Ctnnb1, Ptgs2, Ccnd1, Mmp9) was significantly upregulated in response to PGE2 treatment. This confirms that PGE2 activated the canonical Wnt signalling pathway. Furthermore, the upregulated genes have been previously associated with ASD. Our findings show, for the first time, evidence for cross-talk between PGE2 and Wnt signalling in neuronal cells, where PKA and PI-3K might act as mediators between the two pathways. Given the importance of PGE2 and Wnt signalling in prenatal development of the nervous system, our study provides insight into how interaction between these two pathways may influence neurodevelopment. PMID:24656144

Prostaglandin E2 alters Wnt-dependent migration and proliferation in neuroectodermal stem cells: implications for autism spectrum disorders.

PubMed

Wong, Christine T; Ahmad, Eizaaz; Li, Hongyan; Crawford, Dorota A

2014-03-23

Prostaglandin E2 (PGE2) is a natural lipid-derived molecule that is involved in important physiological functions. Abnormal PGE2 signalling has been associated with pathologies of the nervous system. Previous studies provide evidence for the interaction of PGE2 and canonical Wnt signalling pathways in non-neuronal cells. Since the Wnt pathway is crucial in the development and organization of the brain, the main goal of this study is to determine whether collaboration between these pathways exists in neuronal cell types. We report that PGE2 interacts with canonical Wnt signalling through PKA and PI-3K in neuroectodermal (NE-4C) stem cells. We used time-lapse microscopy to determine that PGE2 increases the final distance from origin, path length travelled, and the average speed of migration in Wnt-activated cells. Furthermore, PGE2 alters distinct cellular phenotypes that are characteristic of Wnt-induced NE-4C cells, which corresponds to the modified splitting behaviour of the cells. We also found that in Wnt-induced cells the level of β-catenin protein was increased and the expression levels of Wnt-target genes (Ctnnb1, Ptgs2, Ccnd1, Mmp9) was significantly upregulated in response to PGE2 treatment. This confirms that PGE2 activated the canonical Wnt signalling pathway. Furthermore, the upregulated genes have been previously associated with ASD. Our findings show, for the first time, evidence for cross-talk between PGE2 and Wnt signalling in neuronal cells, where PKA and PI-3K might act as mediators between the two pathways. Given the importance of PGE2 and Wnt signalling in prenatal development of the nervous system, our study provides insight into how interaction between these two pathways may influence neurodevelopment.

Wnt5a / planar cell polarity signaling pathway in urothelial carcinoma, a potential prognostic biomarker

PubMed Central

Saling, Mark; Duckett, Jordan K; Ackers, Ian; Coschigano, Karen; Jenkinson, Scott; Malgor, Ramiro

2017-01-01

Bladder cancer is the fourth most common cancer in men and the most common malignancy of the urinary tract. Bladder cancers detected at an early stage have a very high five-year survival rate, but when detected after local metastasis the rate is only about 50%. Our group recently reported a positive correlation between the expression of Wnt5a, a member of the Wnt proteins family, and histopathological grade and stage of urothelial carcinoma (UC). The objective of this study was to analyze UC cases reported in Athens, Ohio and investigate the major components of Wnt5a / planar cell polarity (PCP) signaling pathway in UC human tissue samples and UC cell lines. Formalin fixed and paraffin embedded transurethral resection tissues were immunostained for Wnt5a, Ror-2, CTHRC1 and E-cadherin. In addition, in vitro studies using UC cell lines were investigated for Wnt5a/PCP signaling and epithelial mesenchymal transition (EMT) gene expression. The IHC results showed a correlation between the expression of Wnt5a, Ror2 and CTHRC1 with high histological grade of the tumor, while E-cadherin showed an opposite trend of expression. Real time RT-PCR results showed that RNA expression of the Wnt5a/ PCP pathway genes vary in low and high grade UC cell lines and that the high grade cell lines exhibited signs of EMT. These findings support that Wnt5a-Ror2 signaling plays a role in UC, support the potential use of Wnt5a as a prognostic marker and provide evidence that Wnt5a signaling may be used as an effective molecular target for novel therapeutic tools. PMID:28427201

New advances of TMEM88 in cancer initiation and progression, with special emphasis on Wnt signaling pathway.

PubMed

Ge, Yun-Xuan; Wang, Chang-Hui; Hu, Fu-Yong; Pan, Lin-Xin; Min, Jie; Niu, Kai-Yuan; Zhang, Lei; Li, Jun; Xu, Tao

2018-01-01

Transmembrane protein 88 (TMEM88), a newly discovered protein localized on the cell membrane. Recent studies showed that TMEM88 was involved in the regulation of several types of cancer. TMEM88 was expressed at significantly higher levels in breast cancer (BC) cell line than in normal breast cell line with co-localized with Dishevelled (DVL) in the cytoplasm of BC cell line. TMEM88 silencing in the ovarian cancer cell line CP70 resulted in significant upregulation of Wnt downstream genes (c-Myc, cyclin-D1) and other Wnt target genes including JUN, PTIX2, CTNNB1 (β-catenin), further supporting that TMEM88 inhibits canonical Wnt signaling pathway. Wnt signaling pathway has been known to play important roles in many diseases, especially in cancer. For instance, hepatocellular carcinoma (HCC) has become one of the most common tumors harboring mutations in the Wnt signaling pathway. As the inhibitor of Wnt signaling, TMEM88 has been considered to act as an oncogene or a tumor suppressor. Up-regulated TMEM88 or gene therapy approaches could be an effective therapeutic approach against tumor as TMEM88 inhibits Wnt signaling through direct interaction with DVL. Here, we review the current knowledge on the functional role and potential clinical application of TMEM88 in the control of various cancers. Highlights Wnt signaling displays an important role in several pathogenesis of cancer. Wnt signaling pathway is activated during cancer development. TMEM88 has an impact on cancer by inhibiting canonical Wnt signaling. We discuss the importance and new applications of TMEM88 in cancer therapy. © 2017 Wiley Periodicals, Inc.

Wnt signalling pathway parameters for mammalian cells.

PubMed

Tan, Chin Wee; Gardiner, Bruce S; Hirokawa, Yumiko; Layton, Meredith J; Smith, David W; Burgess, Antony W

2012-01-01

Wnt/β-catenin signalling regulates cell fate, survival, proliferation and differentiation at many stages of mammalian development and pathology. Mutations of two key proteins in the pathway, APC and β-catenin, have been implicated in a range of cancers, including colorectal cancer. Activation of Wnt signalling has been associated with the stabilization and nuclear accumulation of β-catenin and consequential up-regulation of β-catenin/TCF gene transcription. In 2003, Lee et al. constructed a computational model of Wnt signalling supported by experimental data from analysis of time-dependent concentration of Wnt signalling proteins in Xenopus egg extracts. Subsequent studies have used the Xenopus quantitative data to infer Wnt pathway dynamics in other systems. As a basis for understanding Wnt signalling in mammalian cells, a confocal live cell imaging measurement technique is developed to measure the cell and nuclear volumes of MDCK, HEK293T cells and 3 human colorectal cancer cell lines and the concentrations of Wnt signalling proteins β-catenin, Axin, APC, GSK3β and E-cadherin. These parameters provide the basis for formulating Wnt signalling models for kidney/intestinal epithelial mammalian cells. There are significant differences in concentrations of key proteins between Xenopus extracts and mammalian whole cell lysates. Higher concentrations of Axin and lower concentrations of APC are present in mammalian cells. Axin concentrations are greater than APC in kidney epithelial cells, whereas in intestinal epithelial cells the APC concentration is higher than Axin. Computational simulations based on Lee's model, with this new data, suggest a need for a recalibration of the model.A quantitative understanding of Wnt signalling in mammalian cells, in particular human colorectal cancers requires a detailed understanding of the concentrations of key protein complexes over time. Simulations of Wnt signalling in mammalian cells can be initiated with the parameters measured in this report.

Inhibition of Ape1 Redox Activity Promotes Odonto/osteogenic Differentiation of Dental Papilla Cells.

PubMed

Chen, Tian; Liu, Zhi; Sun, Wenhua; Li, Jingyu; Liang, Yan; Yang, Xianrui; Xu, Yang; Yu, Mei; Tian, Weidong; Chen, Guoqing; Bai, Ding

2015-12-07

Dentinogenesis is the formation of dentin, a substance that forms the majority of teeth, and this process is performed by odontoblasts. Dental papilla cells (DPCs), as the progenitor cells of odontoblasts, undergo the odontogenic differentiation regulated by multiple cytokines and paracrine signal molecules. Ape1 is a perfect paradigm of the function complexity of a biological macromolecule with two major functional regions for DNA repair and redox regulation, respectively. To date, it remains unclear whether Ape1 can regulate the dentinogenesis in DPCs. In the present study, we firstly examed the spatio-temporal expression of Ape1 during tooth germ developmental process, and found the Ape1 expression was initially high and then gradually reduced along with the tooth development. Secondly, the osteo/odontogenic differentiation capacity of DPCs was up-regulated when treated with either Ape1-shRNA or E3330 (a specific inhibitor of the Ape1 redox function), respectively. Moreover, we found that the canonical Wnt signaling pathway was activated in this process, and E3330 reinforced-osteo/odontogenic differentiation capacity was suppressed by Dickkopf1 (DKK1), a potent antagonist of canonical Wnt signaling pathway. Taken together, we for the first time showed that inhibition of Ape1 redox regulation could promote the osteo/odontogenic differentiation capacity of DPCs via canonical Wnt signaling pathway.

Using Ambystoma mexicanum (Mexican axolotl) embryos, chemical genetics, and microarray analysis to identify signaling pathways associated with tissue regeneration.

PubMed

Ponomareva, Larissa V; Athippozhy, Antony; Thorson, Jon S; Voss, S Randal

2015-12-01

Amphibian vertebrates are important models in regenerative biology because they present exceptional regenerative capabilities throughout life. However, it takes considerable effort to rear amphibians to juvenile and adult stages for regeneration studies, and the relatively large sizes that frogs and salamanders achieve during development make them difficult to use in chemical screens. Here, we introduce a new tail regeneration model using late stage Mexican axolotl embryos. We show that axolotl embryos completely regenerate amputated tails in 7days before they exhaust their yolk supply and begin to feed. Further, we show that axolotl embryos can be efficiently reared in microtiter plates to achieve moderate throughput screening of soluble chemicals to investigate toxicity and identify molecules that alter regenerative outcome. As proof of principle, we identified integration 1 / wingless (Wnt), transforming growth factor beta (Tgf-β), and fibroblast growth factor (Fgf) pathway antagonists that completely block tail regeneration and additional chemicals that significantly affected tail outgrowth. Furthermore, we used microarray analysis to show that inhibition of Wnt signaling broadly affects transcription of genes associated with Wnt, Fgf, Tgf-β, epidermal growth factor (Egf), Notch, nerve growth factor (Ngf), homeotic gene (Hox), rat sarcoma/mitogen-activated protein kinase (Ras/Mapk), myelocytomatosis viral oncogene (Myc), tumor protein 53 (p53), and retinoic acid (RA) pathways. Punctuated changes in the expression of genes known to regulate vertebrate development were observed; this suggests the tail regeneration transcriptional program is hierarchically structured and temporally ordered. Our study establishes the axolotl as a chemical screening model to investigate signaling pathways associated with tissue regeneration. Copyright © 2015 Elsevier Inc. All rights reserved.

Bone Morphogenetic Protein 15 (BMP15) Acts as a BMP and Wnt Inhibitor during Early Embryogenesis*

PubMed Central

Di Pasquale, Elisa; Brivanlou, Ali H.

2009-01-01

Bone morphogenetic protein 15 (BMP15) belongs to an unusual subgroup of the transforming growth factor β (TGFβ) superfamily of signaling ligands as it lacks a key cysteine residue in the mature region required for proper intermolecular dimerization. Naturally occurring BMP15 mutation leads to early ovarian failure in humans, and BMP15 has been shown to activate the Smad1/5/8 pathway in that context. Despite its important role in germ cell specification, the embryological function of BMP15 remains unknown. Surprisingly, we find that during early Xenopus embryogenesis BMP15 acts solely as an inhibitor of the Smad1/5/8 pathway and the Wnt pathway. BMP15 gain-of-function leads to embryos with secondary ectopic heads and to direct neural induction in intact explants. BMP15 inhibits BMP4-mediated epidermal induction in dissociated explants. BMP15 strongly inhibits BRE response induced by BMP4 and blocks phosphorylation and activation of Smad1/5/8 MH2-domain. Mechanistically, BMP15 protein specifically interacts with BMP4 protein, suggesting inhibition upstream of receptor binding. Loss-of-function experiments using morpholinos or a naturally occurring human BMP15 dominant-negative mutant (BMP15-Y235C) leads to embryos lacking head. BMP15-Y235C also eliminates the inhibitory activity of BMP15 on BRE (BMP-responsive element). Finally, we show that BMP15 inhibits the canonical branch of the Wnt pathway, upstream of β-catenin. We, thus, demonstrate that BMP15 is necessary and sufficient for the specification of dorso-anterior structures and highlight novel mechanisms of BMP15 function that strongly suggest a reinterpretation of its function in ovaries specially for ovarian failure. PMID:19553676

Homeobox protein MSX-1 inhibits expression of bone morphogenetic protein 2, bone morphogenetic protein 4, and lymphoid enhancer-binding factor 1 via Wnt/β-catenin signaling to prevent differentiation of dental mesenchymal cells during the late bell stage.

PubMed

Feng, Xiao-Yu; Wu, Xiao-Shan; Wang, Jin-Song; Zhang, Chun-Mei; Wang, Song-Lin

2018-02-01

Homeobox protein MSX-1 (hereafter referred to as MSX-1) is essential for early tooth-germ development. Tooth-germ development is arrested at bud stage in Msx1 knockout mice, which prompted us to study the functions of MSX-1 beyond this stage. Here, we investigated the roles of MSX-1 during late bell stage. Mesenchymal cells of the mandibular first molar were isolated from mice at embryonic day (E)17.5 and cultured in vitro. We determined the expression levels of β-catenin, bone morphogenetic protein 2 (Bmp2), Bmp4, and lymphoid enhancer-binding factor 1 (Lef1) after knockdown or overexpression of Msx1. Our findings suggest that knockdown of Msx1 promoted expression of Bmp2, Bmp4, and Lef1, resulting in elevated differentiation of odontoblasts, which was rescued by blocking the expression of these genes. In contrast, overexpression of Msx1 decreased the expression of Bmp2, Bmp4, and Lef1, leading to a reduction in odontoblast differentiation. The regulation of Bmp2, Bmp4, and Lef1 by Msx1 was mediated by the Wnt/β-catenin signaling pathway. Additionally, knockdown of Msx1 impaired cell proliferation and slowed S-phase progression, while overexpression of Msx1 also impaired cell proliferation and prolonged G1-phase progression. We therefore conclude that MSX-1 maintains cell proliferation by regulating transition of cells from G1-phase to S-phase and prevents odontoblast differentiation by inhibiting expression of Bmp2, Bmp4, and Lef1 at the late bell stage via the Wnt/β-catenin signaling pathway. © 2017 Eur J Oral Sci.

AURKA induces EMT by regulating histone modification through Wnt/β-catenin and PI3K/Akt signaling pathway in gastric cancer

PubMed Central

Liu, Xi; Li, Zhaoxia; Song, Yue; Wang, Rui; Han, Lei; Wang, Qixue; Jiang, Kui; Kang, Chunsheng; Zhang, Qingyu

2016-01-01

Gastric cancer, a highly invasive and aggressive malignancy, is the third leading cause of death from cancer worldwide. Genetic association studies have successfully revealed several important genes consistently associated with gastric cancer to date. However, these robust gastric cancer-associated genes do not fully elucidate the mechanisms underlying the development and progression of the disease. In the present study, we performed an alternative approach, a gene expression-based genome-wide association study (eGWAS) across 13 independent microarray experiments (including 251 gastric cancer cases and 428 controls), to identify top candidates (p<0.00001). Additionally, we conducted gene ontology analysis, pathway analysis and network analysis and identified aurora kinase A (AURKA) as our candidate. We observed that MLN8237, which is a specific inhibitor of AURKA, decreased the β-catenin and the phosphorylation of Akt1 and GSK-3β, as well as blocked the Akt and Wnt signaling pathways. Furthermore, MLN8237 arrested the cells in the G2/M phase. The activity of Wnt and Akt signaling pathways affected the level of histone methylation significantly, and we supposed that MLN8237 affected the level of histone methylation through these two signaling pathways. Additionally, the treatment of MLN8237 influenced the level of H3K4 me1/2/3 and H3K27 me1/2/3. Chip data on cell lines suggested that MLN8237 increases the level of H3K27 me3 on the promoter of Twist and inhibits EMT (epithelial-mesenchymal transition). In summary, AURKA is a potential therapeutic target in gastric cancer and induces EMT through histone methylation. PMID:27121204

DKK1 and Kremen Expression Predicts the Osteoblastic Response to Bone Metastasis.

PubMed

Clines, Katrina L; Clines, Gregory A

2018-05-14

Bone metastasis is a complication of advanced breast and prostate cancer. Tumor-secreted Dickkopf homolog 1 (DKK1), an inhibitor of canonical Wnt signaling and osteoblast differentiation, was proposed to regulate the osteoblastic response to metastatic cancer in bone. The objectives of this study were to compare DKK1 expression with the in vivo osteoblastic response in a panel of breast and prostate cancer cell lines, and to discover mechanisms that regulate cancer DKK1 expression. DKK1 expression was highest in MDA-MB-231 and PC3 cells that produce osteolytic lesions, and hence a suppressed osteoblastic response, in animal models of bone metastasis. LnCaP, C4-2B, LuCaP23.1, T47D, ZR-75-1, MCF-7, ARCaP and ARCaP M cancer cells that generate osteoblastic, mixed or no bone lesions had the lowest DKK1 expression. The cell lines with negligible expression, LnCaP, C4-2B and T47D, exhibited methylation of the DKK1 promoter. Canonical Wnt signaling activity was then determined and found in all cell lines tested, even in the MDA-MB-231 and PC3 cell lines despite sizeable amounts of DKK1 protein expression expected to block canonical Wnt signaling. A mechanism of DKK1 resistance in the osteolytic cell lines was investigated and determined to be at least partially due to down-regulation of the DKK1 receptors Kremen1 and Kremen2 in the MDA-MB-231 and PC3 cell lines. Combined DKK1 and Kremen expression in cancer cells may serve as predictive markers of the osteoblastic response of breast and prostate cancer bone metastasis. Published by Elsevier Inc.

Natural product (-)-gossypol inhibits colon cancer cell growth by targeting RNA-binding protein Musashi-1.

PubMed

Lan, Lan; Appelman, Carl; Smith, Amber R; Yu, Jia; Larsen, Sarah; Marquez, Rebecca T; Liu, Hao; Wu, Xiaoqing; Gao, Philip; Roy, Anuradha; Anbanandam, Asokan; Gowthaman, Ragul; Karanicolas, John; De Guzman, Roberto N; Rogers, Steven; Aubé, Jeffrey; Ji, Min; Cohen, Robert S; Neufeld, Kristi L; Xu, Liang

2015-08-01

Musashi-1 (MSI1) is an RNA-binding protein that acts as a translation activator or repressor of target mRNAs. The best-characterized MSI1 target is Numb mRNA, whose encoded protein negatively regulates Notch signaling. Additional MSI1 targets include the mRNAs for the tumor suppressor protein APC that regulates Wnt signaling and the cyclin-dependent kinase inhibitor P21(WAF-1). We hypothesized that increased expression of NUMB, P21 and APC, through inhibition of MSI1 RNA-binding activity might be an effective way to simultaneously downregulate Wnt and Notch signaling, thus blocking the growth of a broad range of cancer cells. We used a fluorescence polarization assay to screen for small molecules that disrupt the binding of MSI1 to its consensus RNA binding site. One of the top hits was (-)-gossypol (Ki = 476 ± 273 nM), a natural product from cottonseed, known to have potent anti-tumor activity and which has recently completed Phase IIb clinical trials for prostate cancer. Surface plasmon resonance and nuclear magnetic resonance studies demonstrate a direct interaction of (-)-gossypol with the RNA binding pocket of MSI1. We further showed that (-)-gossypol reduces Notch/Wnt signaling in several colon cancer cell lines having high levels of MSI1, with reduced SURVIVIN expression and increased apoptosis/autophagy. Finally, we showed that orally administered (-)-gossypol inhibits colon cancer growth in a mouse xenograft model. Our study identifies (-)-gossypol as a potential small molecule inhibitor of MSI1-RNA interaction, and suggests that inhibition of MSI1's RNA binding activity may be an effective anti-cancer strategy. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Norrin mediates angiogenic properties via the induction of insulin-like growth factor-1.

PubMed

Zeilbeck, Ludwig F; Müller, Birgit B; Leopold, Stephanie A; Senturk, Berna; Langmann, Thomas; Tamm, Ernst R; Ohlmann, Andreas

2016-04-01

Norrin is an angiogenic signaling molecule that activates canonical Wnt/β-catenin signaling, and is involved in capillary formation in retina and brain. Moreover, Norrin induces vascular repair following an oxygen-induced retinopathy (OIR), the model of retinopathy of prematurity in mice. Since insulin-like growth factor (IGF)-1 is a very potent angiogenic molecule, we investigated if IGF-1 is a downstream mediator of Norrin's angiogenic properties. In retinae of transgenic mice with an ocular overexpression of Norrin (βB1-Norrin), we found at postnatal day (P)11 a significant increase of IGF-1 mRNA compared to wild-type littermates. In addition, after treatment of cultured Müller cells or dermal microvascular endothelial cells with Norrin we observed an increase of IGF-1 and its mRNA, an effect that could be blocked with DKK-1, an inhibitor of Wnt/β-catenin signaling. When OIR was induced, the expression of IGF-1 was significantly suppressed in both transgenic βB1-Norrin mice and wild-type littermates when compared to wild-type animals that were housed in room air. Furthermore, at P13, one day after the mice had returned to normoxic conditions, IGF-1 levels were significantly higher in transgenic mice compared to wild-type littermates. Finally, after intravitreal injections of inhibitory α-IGF-1 antibodies at P12 or at P12 and P14, the Norrin-mediated vascular repair was significantly attenuated. We conclude that Norrin induces the expression of IGF-1 via an activation of the Wnt/β-catenin signaling pathway, an effect that significantly contributes to the protective effects of Norrin against an OIR. Copyright © 2015 Elsevier Ltd. All rights reserved.

Wnt/β-catenin signaling stimulates the expression and synaptic clustering of the autism-associated Neuroligin 3 gene.

PubMed

Medina, Matías A; Andrade, Víctor M; Caracci, Mario O; Avila, Miguel E; Verdugo, Daniela A; Vargas, Macarena F; Ugarte, Giorgia D; Reyes, Ariel E; Opazo, Carlos; De Ferrari, Giancarlo V

2018-03-05

Synaptic abnormalities have been described in individuals with autism spectrum disorders (ASD). The cell-adhesion molecule Neuroligin-3 (Nlgn3) has an essential role in the function and maturation of synapses and NLGN3 ASD-associated mutations disrupt hippocampal and cortical function. Here we show that Wnt/β-catenin signaling increases Nlgn3 mRNA and protein levels in HT22 mouse hippocampal cells and primary cultures of rat hippocampal neurons. We characterized the activity of mouse and rat Nlgn3 promoter constructs containing conserved putative T-cell factor/lymphoid enhancing factor (TCF/LEF)-binding elements (TBE) and found that their activity is significantly augmented in Wnt/β-catenin cell reporter assays. Chromatin immunoprecipitation (ChIP) assays and site-directed mutagenesis experiments revealed that endogenous β-catenin binds to novel TBE consensus sequences in the Nlgn3 promoter. Moreover, activation of the signaling cascade increased Nlgn3 clustering and co- localization with the scaffold PSD-95 protein in dendritic processes of primary neurons. Our results directly link Wnt/β-catenin signaling to the transcription of the Nlgn3 gene and support a functional role for the signaling pathway in the dysregulation of excitatory/inhibitory neuronal activity, as is observed in animal models of ASD.

Drosophila nemo is an essential gene involved in the regulation of programmed cell death.

PubMed

Mirkovic, Ivana; Charish, Kristi; Gorski, Sharon M; McKnight, Kristen; Verheyen, Esther M

2002-11-01

Nemo-like kinases define a novel family of serine/threonine kinases that are involved in integrating multiple signaling pathways. They are conserved regulators of Wnt/Wingless pathways, which may coordinate Wnt with TGFbeta-mediated signaling. Drosophila nemo was identified through its involvement in epithelial planar polarity, a process regulated by a non-canonical Wnt pathway. We have previously found that ectopic expression of Nemo using the Gal4-UAS system resulted in embryonic lethality associated with defects in patterning and head development. In this study we present our analyses of the phenotypes of germline clone-derived embryos. We observe lethality associated with head defects and reduction of programmed cell death and conclude that nmo is an essential gene. We also present data showing that nmo is involved in regulating apoptosis during eye development, based on both loss of function phenotypes and on genetic interactions with the pro-apoptotic gene reaper. Finally, we present genetic data from the adult wing that suggest the activity of ectopically expressed Nemo can be modulated by Jun N-terminal kinase (JNK) signaling. Such an observation supports the model that there is cross-talk between Wnt, TGFbeta and JNK signaling at multiple stages of development. Copyright 2002 Elsevier Science Ireland Ltd.

An electrochemical biosensor for double-stranded Wnt7B gene detection based on enzymatic isothermal amplification.

PubMed

Li, Junlong; Chen, Zhongping; Xiang, Yu; Zhou, Lili; Wang, Ting; Zhang, Zhang; Sun, Kexin; Yin, Dan; Li, Yi; Xie, Guoming

2016-12-15

Wnt7B gene plays an important role in the development and progression of breast cancer, gastric cancer, esophageal cancer and pancreatic cancer. While, the natural state of DNA is double stranded, which makes it difficult to be directly detected. Here, we develop an electrochemical biosensor method for Wnt7B gene detection without the need to denature the target. This method firstly used nicking enzyme for exploiting in the double-stranded DNA (dsDNA). Then, long single-stranded DNA (ssDNA) was generated from the cutting site through polymerase extension reaction. Whereafter, the long ssDNA triggered a hairpin self-assembly recycling reaction, which gave rise to another isothermal amplification reaction. Last, short ssDNA was formed after the this amplification process, which could hybridize with the capture probe immobilized on Au electrode and result in signal variation. This method showed excellent analytical performance for dsDNA, of which the linear range was 2fM to 500pM and the detection limit was 1.6fM (S/N=3). It also showed an good results when applied to the real sample of Wnt7B gene detection. Copyright © 2016 Elsevier B.V. All rights reserved.

Trypanosoma cruzi Exploits Wnt Signaling Pathway to Promote Its Intracellular Replication in Macrophages.

PubMed

Volpini, Ximena; Ambrosio, Laura F; Fozzatti, Laura; Insfran, Constanza; Stempin, Cinthia C; Cervi, Laura; Motran, Claudia Cristina

2018-01-01

During the acute phase of Trypanosoma cruzi infection, macrophages can act as host cells for the parasites as well as effector cells in the early anti-parasitic immune response. Thus, the targeting of specific signaling pathways could modulate macrophages response to restrict parasite replication and instruct an appropriate adaptive response. Recently, it has become evident that Wnt signaling has immunomodulatory functions during inflammation and infection. Here, we tested the hypothesis that during T. cruzi infection, the activation of Wnt signaling pathway in macrophages plays a role in modulating the inflammatory/tolerogenic response and therefore regulating the control of parasite replication. In this report, we show that early after T. cruzi infection of bone marrow-derived macrophages (BMM), β-catenin was activated and Wnt3a, Wnt5a, and some Frizzled receptors as well as Wnt/β-catenin pathway's target genes were upregulated, with Wnt proteins signaling sustaining the activation of Wnt/β-catenin pathway and then activating the Wnt/Ca +2 pathway. Wnt signaling pathway activation was critical to sustain the parasite's replication in BMM; since the treatments with specific inhibitors of β-catenin transcriptional activation or Wnt proteins secretion limited the parasite replication. Mechanistically, inhibition of Wnt signaling pathway armed BMM to fight against T. cruzi by inducing the production of pro-inflammatory cytokines and indoleamine 2,3-dioxygenase activity and by downregulating arginase activity. Likewise, in vivo pharmacological inhibition of the Wnts' interaction with its receptors controlled the parasite replication and improved the survival of lethally infected mice. It is well established that T. cruzi infection activates a plethora of signaling pathways that ultimately regulate immune mediators to determine the modulation of a defined set of effector functions in macrophages. In this study, we have revealed a new signaling pathway that is activated by the interaction between protozoan parasites and host innate immunity, establishing a new conceptual framework for the development of new therapies.