Wnt-10b, uniquely among Wnts, promotes epithelial differentiation and shaft growth

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 epithelial cells in the hair follicle.     

Effects of Wnt-10b on hair shaft growth in hair follicle cultures

Wnts are deeply involved in the proliferation and differentiation of skin epithelial cells. We previously reported the differentiation of cultured primary skin epithelial cells toward hair shaft and inner root sheath (IRS) of the hair follicle via beta-catenin stabilization caused by Wnt-10b, however, the effects of Wnt-10b on cultured hair follicles have not been reported. In the present study, we examined the effects of Wnt-10b on shaft growth using organ cultures of whisker hair follicles in serum-free conditions. No hair shaft growth was observed in the absence of Wnt-10b, whereas its addition to the culture promoted elongation of the hair shaft, intensive incorporation of BrdU in matrix cells flanking the dermal papilla (DP), and beta-catenin stabilization in DP and IRS cells. These results suggest a promoting effect of Wnt-10b on hair shaft growth that is involved with stimulation of the DP via Wnt-10b/beta-catenin signalling, proliferation of matrix cells next to the DP, and differentiation of IRS cells by Wnt-10b.     

Wnts differentially regulate colony growth and differentiation of chondrogenic rat calvaria cells

The wingless- and int-related proteins (Wnts) have an important role during embryonic development and limb patterning. To investigate their function during chondrocyte differentiation, we used NIH3T3 cells producing seven members of the Wnt family and secreted frizzled-related protein (sFRP-2) for co-culture experiments with the rat chondrogenic cell line pColl(II)-EGFP-5. Pilot experiments showed a negative effect of Wnt-7a on the proliferation of three rodent chondrogenic cell lines, RCJ3.1(C5.18), CFK-2, and C1. To establish a reporter system for chondrogenic differentiation we then produced a stably transfected chondrogenic cell line based on RCJ3.1(C5.18) for further experiments, which expresses green fluorescence protein (EGFP) under the collagen type II promoter (pColl(II)-EGFP-5). This cell line permits convenient observation of green fluorescence as a marker for differentiation in life cultures. The colony size of this cell line in agarose suspension cultures was reduced to 20-40% of control, when exposed to Wnt-1, 3a, 4, 7a, and 7b for 14 days. Similarly, reporter gene expression and the synthesis of cartilage-specific proteoglycans were inhibited by this group of Wnts. In contrast, pColl(II)-EGFP-5 cells exposed to Wnt-5a and Wnt-11 reached 140% of control, and reporter gene expression and proteoglycan synthesis were stimulated. The effects of Wnt-7a and Wnt-5a were additive in pColl(II)-EGFP-5 cells and some but not all Wnt effects were antagonized by the inhibition of proteoglycan sulfation with chlorate, by sFRP-2, which may modulate Wnt receptor binding, or by inhibitors of protein kinase C. These results suggest two functional Wnt subclasses that differentially regulate proliferation and chondrogenic differentiation in vitro which may have implications for cartilage differentiation in vivo. Since some, but not all Wnt effects were sensitive to inhibitors of proteoglycan synthesis or protein kinase C, multiple modes of signal transduction may be involved.     

Wnt-10b promotes differentiation of skin epithelial cells in vitro

To evaluate the role of Wnt-10b in epithelial differentiation, we investigated the effects of Wnt-10b on adult mouse-derived primary skin epithelial cells (MPSEC). Recombinant Wnt-10b protein (rWnt-10b) was prepared using a gene engineering technique and MPSEC were cultured in its presence, which resulted in morphological changes from cuboidal to spindle-shaped and inhibited their proliferation. Further, involvement of the canonical Wnt signal pathway was also observed. MPSEC treated with rWnt-10b showed characteristics of the hair shaft and inner root sheath of the hair follicle, in results of Ayoub Shklar staining and immunocytochemistry. Further, the cells expressed mRNA for differentiated epithelial cells, including keratin 1, keratin 2, loricrin, mHa5, and mHb5, in association with a decreased expression of the basal cell marker keratin 5. These results suggest that Wnt-10b promotes the differentiation of MPSEC.     

Activities of the Wnt-1 class of secreted signaling factors are antagonized by the Wnt-5A class and by a dominant negative cadherin in early Xenopus development

When overexpressed in Xenopus embryos, Xwnt-1, -3A, -8 and -8b define a functional class of Wnts (the Wnt-1 class) that promotes duplication of the embryonic axis, whereas Xwnt-5A, -4, and -11 define a distinct class (the Wnt-5A class) that alters morphogenetic movements (Du, S., S. Purcell, J. Christian, L. McGrew, and R. Moon. 1995. Mol. Cell. Biol. 15:2625-2634). Since come embryonic cells may be exposed to signals from both functional classes of Wnt during vertebrate development, this raises the question of how the signaling pathways of these classes of Wnts might interact. To address this issue, we coexpressed various Xwnts and components of the Wnt-1 class signaling pathway in developing Xenopus embryos. Members of the Xwnt-5A class antagonized the ability of ectopic Wnt-1 class to induce goosecoid expression and a secondary axis. Interestingly, the Wnt-5A class did not block goosecoid expression or axis induction in response to overexpression of cytoplasmic components of the Wnt-1 signaling pathway, beta-catenin or a kinase-dead gsk-3, or to the unrelated secreted factor, BVg1. The ability of the Wnt-5A class to block responses to the Wnt-1 class may involve decreases in cell adhesion, since ectopic expression of Xwnt-5A leads to decreased Ca2+-dependent cell adhesion and the activity of Xwnt-5A to block Wnt-1 class signals is mimicked by a dominant negative N-cadherin. These data underscore the importance of cell adhesion in modulating the responses of embryonic cells to signaling molecules and suggest that the Wnt-5A functional class of signaling factors can interact with the Wnt-1 class in an antagonistic manner.     

Increase in Proliferation and Differentiation of Neural Progenitor Cells Isolated from Postnatal and Adult Mice Brain by Wnt-3a and Wnt-5a

Wnt signaling is implicated in the control of cell growth and differentiation during CNS development. These findings are based on studies of mouse and chick models. However, the action of Wnt signaling, at the cellular level, is poorly understood. In this study, we investigated the roles of Wnt-3a and Wnt-5a on differentiation and proliferation of postnatal neural progenitor cells (NPCs) in mice.NPCs were isolated from the subventricular zone (SVZ) of PN-1 and adult ICR mice. Plasmids containing active Wnt-3a or Wnt-5a were transfected to NPCs; their effects on the formation of neurospheres and differentiation into neuronal cells were then determined. Transfection of Wnt-3a and Wnt-5a plasmids promoted regeneration of neurospheres and differentiation into Map2-positive cells, and decreased differentiation into GFAP-positive cells. The conditioned media obtained from Wnt-3a or Wnt-5a transfected NPCs showed similar effects on differentiation of NPCs with cDNA transfection, although the magnitude of stimulatory effect was less than that by plasmid transfection. Wnt-3a and Wnt-5a transfection did not affect Brdu incorporation of neuronal or glial progenitors in differentiation media. Wnt-3a and Wnt-5a plasmid transfection and the treatment of Wnt-3a and Wnt-5a conditioned media increased $β −cateninlevelsinNPCs. Wnt−3ahadagreatereffecton β $-catenin levels than Wnt-5a. The PKC inhibitor completely blocked the Wnt-5a effect on neuronal differentiation in NPCs. These findings suggest that Wnt-3a and Wnt-5a each have distinct effects on the proliferation and differentiation of NPCs in postnatal mice.     

Expression of Wnt signalling pathway genes during tooth development.

We have carried out comparative in situ hybridisation analysis of six Wnt genes Wnts-3, -4, -5a, -6, -7b, and 10b together with Wnt receptor MFz6 and receptor agonist/antagonists MFrzb1 and Mfrp2 during murine odontogenesis from the earliest formation of the epithelial thickening to the early bell stage. Expression of Wnt-4, Wnt-6, and one Wnt receptor MFz6 was observed in the facial, oral and dental epithelium. Wnt10b was localised specifically to the presumptive dental epithelium. Wnts-3 and -7b were expressed in oral epithelium but showed no expression in the presumptive dental epithelium. Wnt-3 also showed no expression in the epithelial cells of the molar bud stage tooth germs, but showed restricted expression in the enamel knots which are signalling centres believed to be involved in regulating tooth shape. Wnts -6, -10b and MFz6 were also detected in the primary and secondary enamel knots. Wnt-5a and agonist/antagonists MFrzb1 and Mfrp2 were expressed in a graded proximo-distal (P-D) manner in mesenchymal cells during the early stages of tooth development with no overlying expression in the oral or dental epithelium. Wnt-5a and MFrzb1 show strong expression in the dental papilla mesenchyme.     

Biochemical Analysis of Murine Wnt Proteins Reveals both Shared and Distinct Properties

The murine Wnt family of proteins consists of at least 12 members that possess significant amino acid homology. Current evidence suggests that these proteins are secreted cell-signaling molecules which are likely to have multiple roles during both embryonic development and oncogenesis. Although the biochemical properties of Wnt-1 have been thoroughly examined, less is known about the characteristics of other Wnt family members. We have compared the properties of six murine Wnt proteins (Wnt-1, Wnt-3a, Wnt-5a, Wnt-5b, Wnt-6, and Wnt-7b) transiently expressed in COS cells. All members enter the endoplasmic reticulum (ER) and are glycosylated. However, all six Wnt proteins are primarily retained in the ER in association with BiP, a resident ER protein that binds to improperly folded proteins and prevents their secretion and/or promotes proper folding. Although all Wnt family members examined are similarly processed, one notable difference was identified. Whereas addition of suramin to COS cell cultures significantly increases the levels of all six Wnts in the medium, the addition of heparin only influences the levels of Wnt-1, Wnt-6, and Wnt-7b.     

Islet cells are the source of Wnts that can induce beta-cell proliferation in vitro

Wnt proteins act mainly as paracrine signals regulating cell proliferation and differentiation. The canonical Wnt pathway has recently been associated with pancreas development and the onset of type 2 diabetes in rodent and human but the underlying mechanisms are still unclear. The aim of this work was threefold: (a) to screen for Wnt expressed by murine pancreas/islet cells, (b) to investigate whether the Wnt gene expression profile can be changed in hyperplastic islets from type 2 prediabetic mice (fed a high-fat diet), and (c) to verify whether soluble factors (namely Wnts) released by pancreatic islets affect insulin secretion and proliferation of a beta-cell line in vitro condition. The majority of the Wnt subtypes are expressed by islet cells, such as Wnts 2, 2b, 3, 3a, 4, 5a, 5b, 6, 7a, 7b, 8a, 8b, 9a, 9b, and 11, while in the whole pancreas homogenates were found the same subtypes, except Wnts 3, 6, 7a, and 7b. Among all the Wnts, the Wnts 3a and 5b showed a significantly increased gene expression in hyperplastic islets from prediabetic mice compared with those from control mice. Furthermore, we observed that coculture with hyperplastic or nonhyperplastic islets did not change the secretory function of the mouse insulinoma clone 6 (MIN6) beta cells but induced a significant increase in cell proliferation in this lineage, which was partially blocked by the IWR-1 and IWP-2 Wnt inhibitors. In conclusion, we demonstrated that murine pancreas/islet cells can secrete Wnts, and that islet-released Wnts may participate in the regulation of beta-cell mass under normal and prediabetic conditions.     

Wnt-10b secreted from lymphocytes promotes differentiation of skin epithelial cells

Wnt-10b was originally isolated from lymphoid tissue and is known to be involved in a wide range of biological actions, while recently it was found to be expressed early in the development of hair follicles. However, few studies have been conducted concerning the role of Wnt-10b with the differentiation of skin epithelial cells. To evaluate its role in epithelial differentiation, we purified Wnt-10b from the supernatant of a concanavalin A-stimulated lymphocyte culture using an affinity column and investigated its effects on the differentiation of adult mouse-derived primary skin epithelial cells (MPSEC). MPSEC cultured with Wnt-10b showed morphological changes from cuboidal to spindle-shaped with inhibited proliferation, and also obtained characteristics of the hair shaft and inner root sheath of the hair follicle, represented by red-colored Ayoub Shklar staining, and reactions to AE-13 and AE-15 as seen with immunocytology. Further, RT-PCR analysis demonstrated the expression of mRNA for keratin 1, keratin 2, loricrin, mHa5, and mHb5, in association with a decreased expression of the basal cell marker keratin 5, in Wnt-10b-treated MPSEC. In addition, involvement of the canonical Wnt signal pathway was demonstrated by a TCF reporter (pTOPFLASH) assay. These results suggest that Wnt-10b promotes the differentiation of MPSEC and may play an important role in hair follicle development by promoting differentiation of epithelial cells.     

MMP-10/stromelysin-2 promotes invasion of head and neck cancer

Background

Periostin, IFN-induced transmembrane protein 1 (IFITM1) and Wingless-type MMTV integration site family, member 5B (Wnt-5b) were previously identified as the invasion promoted genes of head and neck squamous cell carcinoma (HNSCC) by comparing the gene expression profiles between parent and a highly invasive clone. We have previously reported that Periostin and IFITM1 promoted the invasion of HNSCC cells. Here we demonstrated that Wnt-5b overexpression promoted the invasion of HNSCC cells. Moreover, stromelysin-2 (matrix metalloproteinase-10; MMP-10) was identified as a common up-regulated gene among Periostin, IFITM1 and Wnt-5b overexpressing HNSCC cells by using microarray data sets. In this study, we investigated the roles of MMP-10 in the invasion of HNSCC.

Methods and Findings

We examined the expression of MMP-10 in HNSCC cases by immunohistochemistry. High expression of MMP-10 was frequently observed and was significantly correlated with the invasiveness and metastasis in HNSCC cases. Next, we examined the roles of MMP-10 in the invasion of HNSCC cells in vitro. Ectopic overexpression of MMP-10 promoted the invasion of HNSCC cells, and knockdown of MMP-10 suppressed the invasion of HNSCC cells. Moreover, MMP-10 knockdown suppressed Periostin and Wnt-5b-promoted invasion. Interestingly, MMP-10 overexpression induced the decreased p38 activity and MMP-10 knockdown induced the increased p38 activity. In addition, treatment with a p38 inhibitor SB203580 in HNSCC cells inhibited the invasion.

Conclusions

These results suggest that MMP-10 plays an important role in the invasion and metastasis of HNSCC, and that invasion driven by MMP-10 is partially associated with p38 MAPK inhibition. We suggest that MMP-10 can be used as a marker for prediction of metastasis in HNSCC.     

Endogenous protein kinase CK2 participates in Wnt signaling in mammary epithelial cells

Protein kinase CK2 (formerly casein kinase II) is a serine/threonine kinase overexpressed in many human tumors, transformed cell lines, and rapidly proliferating tissues. Recent data have shown that many cancers involve inappropriate reactivation of Wnt signaling through ectopic expression of Wnts themselves, as has been seen in a number of human breast cancers, or through mutation of intermediates in the Wnt pathway, such as adenomatous polyposis coli or beta-catenin, as described in colon and other cancers. Wnts are secreted factors that are important in embryonic development, but overexpression of certain Wnts, such as Wnt-1, leads to proliferation and transformation of cells. We report that upon stable transfection of Wnt-1 into the mouse mammary epithelial cell line C57MG, morphological changes and increased proliferation are accompanied by increased levels of CK2, as well as of beta-catenin. CK2 and beta-catenin co-precipitate with the Dvl proteins, which are Wnt signaling intermediates. A major phosphoprotein of the size of beta-catenin appears in in vitro kinase reactions performed on the Dvl immunoprecipitates. In vitro translated beta-catenin, Dvl-2, and Dvl-3 are phosphorylated by CK2. The selective CK2 inhibitor apigenin blocks proliferation of Wnt-1-transfected cells, abrogates phosphorylation of beta-catenin, and reduces beta-catenin and Dvl protein levels. These results demonstrate that endogenous CK2 is a positive regulator of Wnt signaling and growth of mammary epithelial cells.     

Exendin-4 upregulates the expression of Wnt-4, a novel regulator of pancreatic β-cell proliferation

The Wnt-signaling pathway regulates β-cell functions. It is not known how the expression of endogenous Wnt-signaling molecules is regulated in β-cells. Therefore, we investigated the effect of antidiabetic drugs and glucose on the expression of Wnt-signaling molecules in β-cells. Primary islets were isolated and cultured. The expression of Wnt-signaling molecules (Wnt-4, Wnt-10b, Frizzled-4, LRP5, TCF7L2) and TNFα was analyzed by semiquantitative PCR and Western blotting. Transient transfections were carried out and proliferation assays of INS-1 β-cells performed using [(3)H]thymidine uptake and BrdU ELISA. Insulin secretion was quantified. A knockdown (siRNA) of Wnt-4 in β-cells was carried out. Exendin-4 significantly increased the expression of Wnt-4 in β-cells on the mRNA level (2.8-fold) and the protein level (3-fold) (P < 0.001). The effect was dose dependent, with strongest stimulation at 10 nM, and it was maintained after long-term stimulation over 4 wk. Addition of exd-(9-39), a GLP-1 receptor antagonist, abolished the effect of exendin-4. Treatment with glucose, insulin, or other antidiabetic drugs had no effect on the expression of any of the examined Wnt-signaling molecules. Functionally, Wnt-4 antagonized the activation of canonical Wnt-signaling in β-cells. Wnt-4 had no effect on glucose-stimulated insulin secretion or insulin gene expression. Knocking down Wnt-4 decreased β-cell proliferation to 45% of controls (P < 0.05). In addition, Wnt-4 and exendin-4 treatment decreased the expression of TNFaα mRNA in primary β-cells. These data demonstrate that stimulation with exendin-4 increases the expression of Wnt-4 in β-cells. Wnt-4 modulates canonical Wnt signaling and acts as regulator of β-cell proliferation and inflammatory cytokine release. This suggests a novel mechanism through which GLP-1 can regulate β-cell proliferation.     

Murine Wnt-1 with an internal c-myc tag recombinantly produced in Escherichia coli can induce intracellular signaling of the canonical Wnt pathway in eukaryotic cells

Wnt-1 belongs to the Wnt family of secreted glycoproteins inducing an intracellular signaling pathway involved in cell proliferation, differentiation, and pattern formation. The canonical branch is one of three known branches. This is also valid in vitro, and Wnts can be considered beneficial for culturing primary cells from organs, provided Wnts are available and applicable even with cells of different species. It was shown here that internally c-myc-tagged murine Wnt-1 produced in the heterologous host Escherichia coli was appropriate for inducing intracellular signaling of the canonical Wnt pathway in eukaryotic cells via stabilization of cytosolic beta-catenin. The pioneering injection of the protein into the blastocoels of Xenopus laevis embryos led to axis duplication and suppression of head formation. Applying the recombinant murine Wnt-1 to metanephric mesenchyme activated the tubulogenic program. The signal-inducing activity of the recombinant protein was also positively demonstrated in the TOP-flash reporter assay. Although Wnts were purified recently from the growth media of stably transfected eukaryotic cell lines, the production of active Wnt proteins in pro- or eukaryotic microorganisms reportedly has never been successful. Here soluble production in E. coli and translocation into the oxidizing environment of the periplasm were achieved. The protein was purified using the internal c-myc tag. The effect on the eukaryotic cells implies that activity was retained. Thus, this approach could make recombinant murine Wnt-1 available as a good starting point for other Wnts needed, for example, for maintaining and differentiating stem cells, organ restoration therapy, and tissue engineering.     

Wnt and Frizzled RNA expression in human mesenchymal and embryonic (H7) stem cells

Background

Wnt signals are important for embryonic stem cells renewal, growth and differentiation. Although 19 Wnt, 10 Frizzled genes have been identified in mammals, their expression patterns in stem cells were largely unknown.

Results

We conducted RNA expression profiling for the Wnt ligands, their cellular receptors "Frizzleds" and co-receptors LRP5/6 in human embryonic stem cells (H7), human bone marrow mesenchymal cells, as well as mouse totipotent F9 teratocarcinoma embryonal cells. Except failing to express Wnt2 gene, totipotent F9 cells expressed RNA for all other 18 Wnt genes as well as all 10 members of Frizzled gene family. H7 cells expressed RNA for each of the 19 Wnt genes. In contrast, human mesenchymal cells did not display detectable RNA expression of Wnt1, Wnt8a, Wnt8b, Wnt9b, Wnt10a, and Wnt11. Analysis of Frizzled RNAs in H7 and human mesechymal cells revealed expression of 9 members of the receptor gene family, except Frizzled8. Expression of the Frizzled co-receptor LRP5 and LRP6 genes were detected in all three cell lines. Human H7 and mouse F9 cells express nearly a full complement of both Wnts and Frizzleds genes. The human mesenchymal cells, in contrast, have lost the expression of six Wnt ligands, i.e. Wnt1, 8a, 8b, 9b, 10a and 11.

Conclusion

Puripotent human H7 and mouse F9 embryonal cells express the genes for most of the Wnts and Frizzleds. In contrast, multipotent human mesenchymal cells are deficient in expression of Frizzled-8 and of 6 Wnt genes.     

Wnt-5a inhibits the canonical Wnt pathway by promoting GSK-3-independent beta-catenin degradation

Wnts are secreted signaling molecules that can transduce their signals through several different pathways. Wnt-5a is considered a noncanonical Wnt as it does not signal by stabilizing beta-catenin in many biological systems. We have uncovered a new noncanonical pathway through which Wnt-5a antagonizes the canonical Wnt pathway by promoting the degradation of beta-catenin. This pathway is Siah2 and APC dependent, but GSK-3 and beta-TrCP independent. Furthermore, we provide evidence that Wnt-5a also acts in vivo to promote beta-catenin degradation in regulating mammalian limb development and possibly in suppressing tumor formation.     

A Synthetic dl-Nordihydroguaiaretic acid (Nordy), Inhibits Angiogenesis,Invasion and Proliferation of Glioma Stem Cells within a Zebrafish Xenotransplantation Model

The zebrafish (Danio rerio) and their transparent embryos represent a promising model system in cancer research. Compared with other vertebrate model systems, we had previously shown that the zebrafish model provides many advantages over mouse or chicken models to study tumor invasion, angiogenesis, and tumorigenesis. In this study, we systematically investigated the biological features of glioma stem cells (GSCs) in a zebrafish model, such as tumor angiogenesis, invasion, and proliferation. We demonstrated that several verified anti-angiogenic agents inhibited angiogenesis that was induced by xenografted-GSCs. We next evaluated the effects of a synthetic dl-nordihydroguaiaretic acid compound (dl-NDGA or “Nordy”), which revealed anti-tumor activity against human GSCs in vitro by establishing parameters through studying its ability to suppress angiogenesis, tumor invasion, and proliferation. Furthermore, our results indicated that Nordy might inhibit GSCs invasion and proliferation through regulation of the arachidonate 5-lipoxygenase (Alox-5) pathway. Moreover, the combination of Nordy and a VEGF inhibitor exhibited an enhanced ability to suppress angiogenesis that was induced by GSCs. By contrast, even following treatment with 50 µM Nordy, there was no discernible effect on zebrafish embryonic development. Together, these results suggested efficacy and safety of using Nordy in vivo, and further demonstrated that this model should be suitable for studying GSCs and anti-GSC drug evaluation.     

Wnt-3A enhances bone morphogenetic protein-2-mediated chondrogenesis of murine C3H10T1/2 mesenchymal cells

We have recently reported the chondrogenic effect of bone morphogenetic protein-2 (BMP-2) in high density cultures of the mouse multipotent mesenchymal C3H10T1/2 cell line and have shown the functional requirement of the cell-cell adhesion molecule N-cadherin in BMP-2-induced chondrogenesis in vitro (Denker, A. E., Nicoll, S. B., and Tuan, R. S. (1995) Differentiation 59, 25-34; Haas, A. R., and Tuan, R. S. (1999) Differentiation 64, 77-89). Furthermore, BMP-2 treatment also results in an increased protein level of beta-catenin, a known N-cadherin-associated Wnt signal transducer (Fischer, L., Haas, A., and Tuan, R. S. (2001) Signal Transduction 2, 66-78), suggesting functional cross-talk between the BMP-2 and Wnt signaling pathways. We have observed previously that BMP-2 treatment up-regulates expression of Wnt-3A in high density cultures of C3H10T1/2 cells. To assess the contribution of Wnt-3A to BMP-2-mediated chondrogenesis, we have generated C3H10T1/2 cell lines overexpressing Wnt-3A and various forms of glycogen synthase kinase-3beta (GSK-3beta), an immediate cytosolic component of the Wnt signaling pathway, and examined their response to BMP-2. We show that overexpression of either Wnt-3A or kinase-dead GSK-3beta enhances BMP-2-mediated chondrogenesis. Furthermore, Wnt-3A overexpression results in decreases in both N-cadherin and GSK-3beta protein levels, whereas Wnt-3A as well as kinase-dead GSK-3beta overexpression increase total and nuclear levels of both beta-catenin and LEF-1. Direct cross-talk between Wnts and BMP-2 was also indicated by the up-regulated interaction between beta-catenin and SMAD-4 in response to BMP-2. These results suggest that Wnt-3A acts in a manner opposite to that of other Wnts, such as Wnt-7A, which were previously identified as inhibitory to chondrogenesis, and is the first BMP-2-regulated, chondrogenesis-enhancing member of the Wnt family.     

Wnt-3a-dependent cell motility involves RhoA activation and is specifically regulated by dishevelled-2

Wnts stimulate cell migration, although the mechanisms responsible for this effect are not fully understood. To investigate the pathways that mediate Wnt-dependent cell motility, we treated Chinese hamster ovary cells with Wnt-3a-conditioned medium and monitored changes in cell shape and movement. Wnt-3a induced cell spreading, formation of protrusive structures, reorganization of stress fibers and migration. Although Wnt-3a stabilized beta-catenin, two inhibitors of the beta-catenin/canonical pathway, Dickkopf-1 and a dominant-negative T cell factor construct, did not reduce motility. The small GTPase RhoA also was activated by Wnt-3a. In contrast to beta-catenin signaling, inhibition of Rho kinase partially blocked motility. Because Dishevelled (Dvl) proteins are effectors of both canonical and noncanonical Wnt signaling, we used immunofluorescent analysis and small interference RNA technology to evaluate the role of Dvl in cell motility. Specific knock-down of Dvl-2 expression markedly reduced Wnt-3a-dependent changes in cell shape and movement, suggesting that this Dvl isoform had a predominant role in mediating Wnt-3a-dependent motility in Chinese hamster ovary cells.