Roles of Wnt/β-catenin signaling in epithelial differentiation of mesenchymal stem cells

Bone marrow-derived mesenchymal stem cells (MSCs) have been demonstrated to be able to differentiate into epithelial lineage, but the precise mechanisms controlling this process are unclear. Our aim is to explore the roles of Wnt/β-catenin in the epithelial differentiation of MSCs. Using indirect co-culture of rat MSCs with rat airway epithelial cells (RTE), MSCs expressed several airway epithelial markers (cytokeratin 18, tight junction protein occudin, cystic fibrosis transmembrance regulator). The protein levels of some important members in Wnt/β-catenin signaling were determined, suggested down-regulation of Wnt/β-catenin with epithelial differentiation of MSCs. Furthermore, Wnt3α can inhibit the epithelial differentiation of MSCs. A loss of β-catenin induced by Dickkopf-1 can enhance MSCs differentiation into epithelial cells. Lithium chloride transiently activated β-catenin expression and subsequently decreased β-catenin level and at last inhibited MSCs to differentiate into airway epithelium. Taken together, our study indicated that RTE cells can trigger epithelial differentiation of MSCs. Blocking Wnt/β-catenin signaling may promote MSCs to differentiate towards airway epithelial cells.     

Chronic WNT/β-catenin signaling induces cellular senescence in lung epithelial cells

The rapid expansion of the elderly population has led to the recent epidemic of age-related diseases, including increased incidence and mortality of chronic lung diseases, such as Idiopathic Pulmonary Fibrosis (IPF). Cellular senescence is a major hallmark of aging and has a higher occurrence in IPF. The lung epithelium represents a major site of tissue injury, cellular senescence and aberrant activity of developmental pathways such as the WNT/β-catenin pathway in IPF. The potential impact of WNT/β-catenin signaling on alveolar epithelial senescence in general as well as in IPF, however, remains elusive. Here, we characterized alveolar epithelial cells of aged mice and assessed the contribution of chronic WNT/β-catenin signaling on alveolar epithelial type (AT) II cell senescence. Whole lungs from old (16–24 months) versus young (3 months) mice had relatively less epithelial (EpCAM⁺) but more inflammatory (CD45⁺) cells, as assessed by flow cytometry. Compared to young ATII cells, old ATII cells showed decreased expression of the ATII cell marker Surfactant Protein C along with increased expression of the ATI cell marker Hopx, accompanied by increased WNT/β-catenin activity. Notably, when placed in an organoid assay, old ATII cells exhibited decreased progenitor cell potential. Chronic canonical WNT/β-catenin activation for up to 7 days in primary ATII cells as well as alveolar epithelial cell lines induced a robust cellular senescence, whereas the non-canonical ligand WNT5A was not able to induce cellular senescence. Moreover, chronic WNT3A treatment of precision-cut lung slices (PCLS) further confirmed ATII cell senescence. Simultaneously, chronic but not acute WNT/β-catenin activation induced a profibrotic state with increased expression of the impaired ATII cell marker Keratin 8. These results suggest that chronic WNT/β-catenin activity in the IPF lung contributes to increased ATII cell senescence and reprogramming. In the fibrotic environment, WNT/β-catenin signaling thus might lead to further progenitor cell dysfunction and impaired lung repair.     

IL1β-mediated Stromal COX-2 signaling mediates proliferation and invasiveness of colonic epithelial cancer cells

COX-2 is a major inflammatory mediator implicated in colorectal inflammation and cancer. However, the exact origin and role of COX-2 on colorectal inflammation and carcinogenesis are still not well defined. Recently, we reported that COX-2 and iNOS signalings interact in colonic CCD18Co fibroblasts. In this article, we investigated whether activation of COX-2 signaling by IL1β in primary colonic fibroblasts obtained from normal and cancer patients play a critical role in regulation of proliferation and invasiveness of human colonic epithelial cancer cells. Our results demonstrated that COX-2 level was significantly higher in cancer associated fibroblasts than that in normal fibroblasts with or without stimulation of IL-1β, a powerful stimulator of COX-2. Using in vitro assays for estimating proliferative and invasive potential, we discovered that the proliferation and invasiveness of the epithelial cancer cells were much greater when the cells were co-cultured with cancer associated fibroblasts than with normal fibroblasts, with or without stimulation of IL1β. Further analysis indicated that the major COX-2 product, prostaglandin E(2), directly enhanced proliferation and invasiveness of the epithelial cancer cells in the absence of fibroblasts. Moreover, a selective COX-2 inhibitor, NS-398, blocked the proliferative and invasive effect of both normal and cancer associate fibroblasts on the epithelial cancer cells, with or without stimulation of IL-1β. Those results indicate that activation of COX-2 signaling in the fibroblasts plays a major role in promoting proliferation and invasiveness of the epithelial cancer cells. In this process, PKC is involved in the activation of COX-2 signaling induced by IL-1β in the fibroblasts.     

TLR3 promotes MMP-9 production in primary human airway epithelial cells through Wnt/β-catenin signaling

Background

Airway epithelial cells (AEC) act as the first line of defence in case of lung infections. They constitute a physical barrier against pathogens and they participate in the initiation of the immune response. Yet, the modalities of pathogen recognition by AEC and the consequences on the epithelial barrier remain poorly documented.

Method

We investigated the response of primary human AEC to viral (polyinosinic-polycytidylic acid, poly(I:C)) and bacterial (lipopolysaccharide, LPS) stimulations in combination with the lung remodeling factor Transforming Growth Factor-β (TGF-β).

Results

We showed a strong production of pro-inflammatory cytokines (Interleukin (IL)-6, Tumor Necrosis Factor α, TNFα) or chemokines (CCL2, CCL3, CCL4, CXCL10, CXCL11) by AEC stimulated with poly(I:C). Cytokine and chemokine production, except CXCL10, was Toll Like Receptor (TLR)-3 dependent and although they express TLR4, we found no cytokine production after LPS stimulation. Poly(I:C), but not LPS, synergised with TGF-β for the production of matrix metalloproteinase-9 (MMP-9) and fibronectin. Mechanistic analyses suggest the secretion of Wnt ligands by AEC along with a degradation of the cellular junctions after poly(I:C) exposure, leading to the release of β-catenin from the cell membrane and stimulation of the Wnt/β-catenin pathway.

Conclusion

Our results highlight the cross talk between TGF-β and TLR signaling in bronchial epithelium and its impact on the remodeling process.

     

WISP1 silencing confers protection against epithelial–mesenchymal transition of renal tubular epithelial cells in rats via inactivation of the wnt/β-catenin signaling pathway in uremia

Uremia can affect hepatic metabolism of drugs by regulating the clearance of drugs, but it has not been clarified whether gene silencing could modulate the epithelial–mesenchymal transition (EMT) process in uremia. Hence, we investigated the effect of WISP1 gene silencing on the renal tubular EMT in uremia through the wnt/β-catenin signaling pathway. Initially, microarray-based gene expression profiling of uremia was used to identify differentially expressed genes. Following the establishment of uremia rat model, serum creatinine, and urea nitrogen of rats were detected. Renal tubular epithelial cells (TECs) were transfected with shRNA-WISP1 lentivirus interference vectors and LiCI (the wnt/β-catenin signaling pathway activator) to explore the regulatory mechanism of WISP1 in uremia in relation to the wnt/β-catenin signaling pathway. Then, expression of WISP1, wnt2b, E-cadherin, α-SMA, c-myc, Cyclin D1, MMP-2, and MMP-9 was determined. Furthermore, TEC migration and invasion were evaluated. Results suggested that WISP1 and the wnt/β-catenin signaling pathway were associated with uremia. Uremic rats exhibited increased serum creatinine and urea nitrogen levels, upregulated WISPl, and activated wnt/β-catenin signaling pathway. Subsequently, WISP1 silencing decreased wnt2b, c-myc, Cyclin D1, α-SMA, MMP-2, and MMP-9 expression but increased E-cadherin expression, whereas LiCI treatment exhibited the opposite trends. In addition, WISP1 silencing suppressed TEC migration and invasion, whereas LiCI treatment promoted TEC migration and invasion. The findings indicate that WISP1 gene silencing suppresses the activation of the wnt/β-catenin signaling pathway, thus reducing EMT of renal TECs in uremic rats.     

The β-catenin/Tcf4/survivin signaling maintains a less differentiated phenotype and high proliferative capacity of human corneal epithelial progenitor cells

It is clear that the microenvironment or niche plays an important role in determining the fate of stem cells: being stem cells or differentiated. However, the intrinsic pathways controlling the fate of adult stem cells in different niches are largely unknown. This study was to explore the role of β-catenin/Tcf4/survivin signaling in determining the fate of human corneal epithelial stem cells in different media. We observed that the low calcium serum-free media, especially CnT-20, promoted proliferative capacity, colony forming efficiency and stem cell-like phenotype of human corneal epithelial cells (HCECs) when compared with the cells cultured in a high calcium serum-containing medium SHEM. Three key factors in Wnt signaling, β-catenin, Tcf4 and survivin, were found to be expressed higher by HCECs grown in CnT-20 than those cultured in SHEM, as evaluated by real-time PCR, Western blotting and immunostaining. Transfection of siRNA-Tcf4 at 10-50nM knocked down Tcf4, and also significantly suppressed its down stream molecule survivin at both mRNA and protein levels in HCECs. Furthermore, Tcf4 silencing significantly suppressed the proliferative capacity of HCECs, measured by WST-1 assay, compared with the control groups, untreated or transfected with non-coding sequence siRNA-fluorescein. These findings demonstrate that low calcium serum free media promote ex vivo expansion of corneal epithelial progenitor cells that retain a less differentiated phenotype and high proliferative capacity via β-catenin/Tcf4/survivin signaling, a novel intrinsic pathway. This study may have high impact and clinic implication on the expansion of corneal epithelial stem cells in regenerative medicine, especially for ocular surface reconstruction.     

SETDB1 confers colorectal cancer metastasis by regulation of WNT/β-catenin signaling

BackgroundMetastasis is a critical step in tumor development; however, its specific molecular mechanism is still not fully understood. SETDB1 overexpression is associated with tumor progression and poor prognosis. Here, we explored a novel mechanism by which SETDB1 promotes tumor metastasis in colorectal cancer.MethodsWe conducted database and clinical specimen analysis to determine the expression level of SETDB1 in colorectal cancer, as well as the prognosis of colorectal cancer with overexpressed SETDB1. We used wound healing assays, Transwell assays, and animal studies to study the effect of SETDB1 on colorectal cancer. We performed western blotting, qRT–PCR, immunofluorescence, and co-immunoprecipitation to explore the underlying associations between SETDB1 and β-catenin. We further used wound healing assays, Transwell assays, and animal studies to verify the relationship between SETDB1 and Wnt/β-catenin.ResultsSETDB1 expression was upregulated in colorectal cancer and correlated with poor prognosis. Low expression of SETDB1 decreased invasion and metastasis in colorectal cancer. Low-expression of SETDB1 in colorectal tumor cells decreased β-catenin expression and its nuclear import. We also found that SETDB1 can bind and directly methylate β-catenin, Lastly, we discovered that this metastatic ability could be decreased by activating the Wnt/β-catenin pathway with SETDB1 knock-down.ConclusionSETDB1 is highly expressed in colorectal cancer and plays an important role in the invasion and metastasis through the Wnt/β-catenin pathway. It does so by direct methylation of β-catenin. This novel SETDB1/Wnt/β-catenin pathway provides a new strategy for the treatment of colorectal cancer.     

Sensitization of glioma cells to tamoxifen-induced apoptosis by Pl3-kinase inhibitor through the GSK-3β/β-catenin signaling pathway

Malignant gliomas represent one of the most aggressive types of cancers and their recurrence is closely linked to acquired therapeutic resistance. A combination of chemotherapy is considered a promising therapeutic model in overcoming therapeutic resistance and enhancing treatment efficacy. Herein, we show by colony formation, Hochest 33342 and TUNEL staining, as well as by flow cytometric analysis, that LY294002, a specific phosphatidylinositide-3-kinase (PI3K) inhibitor, enhanced significantly the sensitization of a traditional cytotoxic chemotherapeutic agent, tamoxifen-induced apoptosis in C6 glioma cells. Activation of PI3K signaling pathway by IGF-1 protected U251 cells from apoptosis induced by combination treatment of LY294002 and tamoxifen. Interference of PI3K signaling pathway by PI3K subunit P85 siRNA enhanced the sensitization of U251 glioma cells to tamoxifen -induced apoptosis. By Western blotting, we found that combination treatment showed lower levels of phosphorylated Akt(Ser473) and GSK-3β(Ser9) than a single treatment of LY294002. Further, we showed a significant decrease of nuclear β-catenin by combination treatment. In response to the inhibition of β-catenin signaling, mRNA and protein levels of Survivin and the other three antiapoptotic genes Bcl-2, Bcl-xL, and Mcl-1 were significantly decreased by combination treatment. Our results indicated that the synergistic cytotoxic effect of LY294002 and tamoxifen is achieved by the inhibition of GSK-3β/β-catenin signaling pathway.     

Nucleoredoxin promotes adipogenic differentiation through regulation of Wnt/β-catenin signaling

Nucleoredoxin (NRX) is a member of the thioredoxin family of proteins that controls redox homeostasis in cell. Redox homeostasis is a well-known regulator of cell differentiation into various tissue types. We found that NRX expression levels were higher in white adipose tissue of obese ob/ob mice and increased in the early adipogenic stage of 3T3-L1 preadipocyte differentiation. Knockdown of NRX decreased differentiation of 3T3-L1 cells, whereas overexpression increased differentiation. Adipose tissue-specific NRX transgenic mice showed increases in adipocyte size as well as number compared with WT mice. We further confirmed that the Wingless/int-1 class (Wnt)/β-catenin pathway was also involved in NRX-promoted adipogenesis, consistent with a previous report showing NRX regulation of this pathway. Genes involved in lipid metabolism were downregulated, whereas inflammatory genes, including those encoding macrophage markers, were significantly upregulated, likely contributing to the obesity in Adipo-NRX mice. Our results therefore suggest that NRX acts as a novel proadipogenic factor and controls obesity in vivo.     

Bone morphogenetic protein 2 induces the activation of WNT/β-catenin signaling and human trophoblast invasion through up-regulating BAMBI

Both bone morphogenetic protein 2 (BMP2) and WNT/β-catenin signaling promote human trophoblast cell invasion. BMP2 has been shown to up-regulate bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI) in granulosa cells. Besides, studies indicate BAMBI is a positive regulator for WNT/β-catenin signaling. However, whether BMP2 can increase BAMBI expression to induce WNT/β-catenin signaling for trophoblast cell invasion is still unknown. To study the roles of BAMBI in BMP2-induced activation of WNT/β-catenin signaling and human trophoblast invasion, we used immortalized human extravillous trophoblast (EVT) cell line (HTR8/SVneo) and primary human EVT cells as study models. Messenger RNA and protein levels of target genes were checked with RT-qPCR and Western blot assay respectively. The function of target proteins was studied via small interfering RNA (siRNA)-mediated knockdown. In addition, trophoblast cell invasiveness was examined by matrigel-coated transwell assays. Our results demonstrate that BMP2 treatment increased BAMBI mRNA levels and the activation of WNT/β-catenin signaling including the raised phosphorylation of GSK3β, the up-regulation of active (non-phosphorylated) β-catenin as well as its downstream target molecule cyclin D1, all of which were totally blocked by the activin receptor-like kinases (ALK) 2/3 inhibitor DMH1 or siRNA-mediated knockdown of BAMBI in HTR8/SVneo cells. Consistently, in primary human EVT cells, BMP2 also induced the up-regulation of BAMBI and the activation of WNT/β-catenin signaling indicated by the increased levels of active β-catenin and cyclin D1, which were completely blocked by BAMBI knockdown. In conclusion, BMP2 promotes the activation of canonical WNT/β-catenin signaling and human trophoblast cell invasion by up-regulating BAMBI.     

Transglutaminase 2 as a novel activator of LRP6/β-catenin signaling

The β-catenin signaling axis is critical for normal embryonic development and tissue homeostasis in adults. We have previously shown that extracellular enzyme transglutaminase 2 (TG2) activates β-catenin signaling in vascular smooth muscle cells (VSMCs). In this study, we provide several lines of evidence that TG2 functions as an activating ligand of the LRP5/6 receptors. Specifically, we show that TG2 synergizes with LRP6 in the activation of β-catenin-dependent gene expression in Cos-7 cells. Interfering with the LRP5/6 receptors attenuates TG2-induced activation of β-catenin in Cos-7 cells. Further, we show that TG2 binds directly to the extracellular domain of LRP6, which is also able to act as a substrate for TG2-mediated protein cross-linking. Furthermore, inhibitors of TG2 protein cross-linking quench the observed TG2-induced β-catenin activation, implicating protein cross-linking as a novel regulatory mechanism for this pathway. Together, our findings identify and characterize a new activating ligand of the LRP5/6 receptors and uncover a novel activity of TG2 as an agonist of β-catenin signaling, contributing to the understanding of diverse developmental events and pathological conditions in which transglutaminase and β-catenin signaling are implicated.     

MiR-499 induces cardiac differentiation of rat mesenchymal stem cells through wnt/β-catenin signaling pathway

ObjectiveTo test the hypothesis that over-expressing miR-499 in rat bone marrow-derived mesenchymal stem cells (BM-MSCs) induces them to differentiate into cardiomyocyte-like cells through the wnt/β-catenin signaling pathway.MethodsRat BM-MSCs were infected with lentiviral vectors bearing miR-499. The expression of cardiac-specific markers, NKx2.5, GATA4, MEF2C, and cTnI in these cells were examined by rtPCR or Western blot analysis and the activity of the wnt/β-catenin signaling pathway was evaluated by measuring the phosphorylation status of β-catenin.ResultsOver-expression of miR-499 in rat BM-MSCs increased the expression of cardiac-specific genes, such as NKx2.5, GATA4, MEF2C, and cTnI and decreased the ratio of phosphorylated/dephosphorylated β-catenin in the wnt/β-catenin signaling pathway, thus activating the pathway. Knocking down the expression of Dvl, an adaptor molecule in the wnt/β-catenin signaling, partially blocked the role of the miR-499 and decreased those cardiac-specific genes.ConclusionOver-expression of miR-499 in rat BM-MSCs induces them toward cardiac differentiation through the activating the wnt/β-catenin signal pathway.     

Wnt/β-catenin signaling regulates neuronal differentiation of mesenchymal stem cells

Mesenchymal stem cells (MSCs) have been demonstrated to be able to differentiate into neuron-like cells, but the precise mechanisms controlling this process are unclear. Using neuron-specific enolase (NSE) and nestin as neuronal markers, we examined the role of Wnt/β-catenin signaling in MSC neuronal differentiation in present study. The results indicated that the expression of β-catenin increased markedly during the neuronal differentiation of MSCs. Blocking Wnt signaling by treating MSCs with β-catenin siRNA could decrease the differentiation of MSCs into neuron-like cells and up-regulation of Wnt signaling by treating MSCs with Wnt-3a could promote neuronal differentiation of MSCs. Above results suggest that Wnt/β-catenin signaling may play a pivotal role in neuronal differentiation of MSCs. Our data broaden the knowledge of molecular mechanisms involved in the neuronal differentiation of MSCs and provide a potential target for directing differentiation of MSCs for clinical application.     

On the role of Wnt/β-catenin signaling in stem cells

Background

Stem cells are mainly characterized by two properties: self-renewal and the potency to differentiate into diverse cell types. These processes are regulated by different growth factors including members of the Wnt protein family. Wnt proteins are secreted glycoproteins that can activate different intracellular signaling pathways.

Scope of review

Here we summarize our current knowledge on the role of Wnt/β-catenin signaling with respect to these two main features of stem cells.

Major conclusions

A particular focus is given on the function of Wnt signaling in embryonic stem cells. Wnt signaling can also improve reprogramming of somatic cells towards iPS cells highlighting the importance of this pathway for self-renewal and pluripotency. As an example for the role of Wnt signaling in adult stem cell behavior, we furthermore focus on intestinal stem cells located in the crypts of the small intestine.

General significance

A broad knowledge about stem cell properties and the influence of intrinsic and extrinsic factors on these processes is a requirement for the use of these cells in regenerative medicine in the future or to understand cancer development in the adult. This article is part of a Special Issue entitled Biochemistry of Stem Cells.     

Reprogramming adult dermis to a neonatal state through epidermal activation of β-catenin

Hair follicle formation depends on reciprocal epidermal-dermal interactions and occurs during skin development, but not in adult life. This suggests that the properties of dermal fibroblasts change during postnatal development. To examine this, we used a PdgfraEGFP mouse line to isolate GFP-positive fibroblasts from neonatal skin, adult telogen and anagen skin and adult skin in which ectopic hair follicles had been induced by transgenic epidermal activation of β-catenin (EF skin). We also isolated epidermal cells from each mouse. The gene expression profile of EF epidermis was most similar to that of anagen epidermis, consistent with activation of β-catenin signalling. By contrast, adult dermis with ectopic hair follicles more closely resembled neonatal dermis than adult telogen or anagen dermis. In particular, genes associated with mitosis were upregulated and extracellular matrix-associated genes were downregulated in neonatal and EF fibroblasts. We confirmed that sustained epidermal β-catenin activation stimulated fibroblasts to proliferate to reach the high cell density of neonatal skin. In addition, the extracellular matrix was comprehensively remodelled, with mature collagen being replaced by collagen subtypes normally present only in developing skin. The changes in proliferation and extracellular matrix composition originated from a specific subpopulation of fibroblasts located beneath the sebaceous gland. Our results show that adult dermis is an unexpectedly plastic tissue that can be reprogrammed to acquire the molecular, cellular and structural characteristics of neonatal dermis in response to cues from the overlying epidermis.