Rho GTPase activity modulates Wnt3a/beta-catenin signaling

Abstract

Wnt proteins constitute a family of secreted signaling molecules that regulate highly conserved pathways essential for development and, when aberrantly activated, drive oncogenesis in a number of human cancers. A key feature of the most widely studied Wnt signaling cascade is the stabilization of cytosolic beta-catenin, resulting in beta-catenin nuclear translocation and transcriptional activation of multiple target genes. In addition to this canonical, beta-catenin-dependent pathway, Wnt3A has also been shown to stimulate RhoA GTPase. While the importance of activated Rho to non-canonical Wnt signaling is well appreciated, the potential contribution of Wnt3A-stimulated RhoA to canonical beta-catenin-dependent transcription has not been examined and is the focus of this study. We find that activated Rho is required for Wnt3A-stimulated osteoblastic differentiation in C3H10T1/2 mesenchymal stem cells, a biological phenomenon mediated by stabilized beta-catenin. Using expression microarrays and real-time RT-PCR analysis, we show that Wnt3A-stimulated transcription of a subset of target genes is Rho-dependent, indicating that full induction of these Wnt targets requires both beta-catenin and Rho activation. Significantly, neither beta-catenin stabilization nor nuclear translocation stimulated by Wnt3A is affected by inhibition or activation of RhoA. These findings identify Rho activation as a critical element of the canonical Wnt3A-stimulated, beta-catenin-dependent transcriptional program.

Figure 1

Characterization of Wnt3A signaling in C3H10T1/2 cells. (A) C3H10T1/2 cells were grown to 75% confluence in 6-well dishes and then treated with Wnt3A conditioned media (CM) or control CM for 3 h prior to lysis and isolation of the cytosolic fractions. 10 µg of protein from these fractions were subjected to SDS-PAGE and immunoblot analysis to detect endogenous β– catenin. The data is from a single experiment that is representative of at least five experiments. (B) C3H10T1/2 cells were grown to 75% confluence and transfected with Super8X TOPFlash and phRG internal control reporter. 5 h after transfection, cells were treated with control CM or Wnt3A–M and incubated a further 18 h prior to lysis and measurement of luciferase activity. The data shown are the mean and standard error from four separate determinations in a single experiment and are representative of at least four experiments. (C) C3H10T1/2 cells were grown to 75% confluence in 10 cm plates, starved overnight, and then treated with 100 ng/ml recombinant purified Wnt3A or vehicle alone for 5 minutes. Activated RhoA was isolated using GST-rhotekin pull down assay. The immunoblot is representative of two independent experiments and the data shown in the graph represent the mean and standard error of these experiments.

Figure 2

Inhibition of Rho by C3 exoenzyme blocks Wnt3A–stimulated differentiation of C3H10T1/2 mesenchymal stem cells. (A) C3H10T1/2 cells were treated either with 2.5 mg/ml C3 exoenzyme in the presence of 20 U/ml tetanolysin (for cellular membrane permeabilization) or with tetanolysin alone (non-C3-treated cells). Following this pretreatment, cells were incubated with Wnt3A CM or control conditioned CM as indicated and then fixed and stained as described in Materials and Methods. A representative image for each condition is shown. (B) C3H10T1/2 cells were treated either with C3/ tetanolysin or tetanolysin alone (non-C3-treated cells) for 3 h prior to incubation with control CM or Wnt3A CM for a further 96 h incubation. Alkaline phosphatase activity was measured as described in Materials and Methods and is reported as arbitrary units/µg total protein. The data shown are the mean and standard error from three separate determinations from a single experiment and are representative of at least 4 different experiments.

Figure 3

Expression microarray analysis of Wnt3A–stimulated C3H10T1/2 cells reveals subset of Rho-sensitive genes. C3H10T1/2 cells were grown to 75% confluence in 10 cm plates and treated either with C3/tetanolysin or tetanolysin alone followed by a further incubation in either control CM or Wnt3A CM for 24 h as described in Materials and Methods. RNA was then harvested, and cDNA was generated, labeled, and hybridized to custom microarrays. (A) Venn diagram indicating total number of gene probes detectable with signal above background under all conditions (outer circle), the number from that group showing ≥ 2-fold increase in signal in the Wnt3A–reated group relative to control (middle circle), and the number from the Wnt3A–stimulated group showing sensitivity to C3 exoenzyme, defined as Wnt3A fold-induction ≥ 1.5X induction of the same probe following treatment with Wnt3A plus C3. (B) For each of the 337 probes showing induction by Wnt3A, the fold-induction in the presence of Wnt3A is plotted versus fold-induction with Wnt3A + C3. The outer bold lines represent where Wnt3A induction would be either 1.5 times induction with Wnt3A + C3 or 1.5 times less; the middle thin line represents Wnt3A–stimulated genes that are unaffected by C3 treatment. The Rho sensitive gene probes are represented by open circles (ο); the Rho-insensitive gene probes are represented by closed circles (•). The data are representative of two independent experiments. (C) Genes represented by probes (62 genes total) showing sensitivity to C3 were classified into the functional groups indicated in the figure based on the individual gene ontology entry for each gene. The percent of the total Rho-sensitive genes in each functional category is listed below the category. (D) C3H10T1/2 cells were grown to 75% confluence in 6-well dishes and treated either with C3/tetanolysin or tetanolysin alone, followed by further incubation with 100 ng/ml purified Wnt3A or vehicle for 24 h. RNA was harvested and quantitative RT-PCR was performed using primers for the genes designated in the figure as described in Materials and Methods. Results were calculated as fold induction relative to the reference gene (Gapdh) and normalized, with 100% defined as maximal induction by Wnt3A, which was typically 7, 3, and 5 for Ctgf, Nedd4, and Igfbp4, respectively. The data represent the mean and standard error of two independent experiments.

Figure 4

Induction of the endogenous Wnt3A target gene, Ctgf, is blocked by inhibition of both TCF/LEF signaling and Rho activation. (A and B) C3H10T1/2 cells were transfected with either empty vector, DN-TCF4, or p190RhoGAP constructs as indicated. 5 h after transfection, cells were treated with control CM or Wnt3A CM and incubated for an additional 24 h. Cells were then harvested, RNA was extracted, and quantitative RT-PCR was performed for each condition using primers specific for Ctgf, as described in Materials and Methods. (C) C3H10T1/2 cells were treated for 24 h in control CM or Wnt3A CM plus either 10 µM Y-27632 or DMSO vehicle, as indicated. Quantitative RT-PCR was performed as above. All results are reported as percentage of Wnt3A CM stimulated induction in the presence of vector (A & B) or vehicle (C), relative to the reference gene (Gapdh). One hundred percent induction was typically ~7–10 fold. The data represent the mean and standard error from three separate determinations and are representative of at least 3 independent experiments (A and C) or 2 independent experiments (B).

Figure 5

RhoA activation or inhibition does not affect cytoplasmic stabilization or nuclear translocation of β–catenin. (A) Cells were transfected with the constructs indicated in B, as described in Materials and Methods. After 24 h, cells were treated with control CM or Wnt3A CM for 4 h prior to lysis and isolation of the cytosolic fractions. 10 µg of protein from these fractions were subjected to SDS-PAGE and immunoblot analysis to detect endogenous β– catenin. (B) Cytosolic β–catenin detected by immunoblot in panel A was quantified using the Odyssey® system from Li-Cor as described in Materials and Methods. Results shown are normalized to RhoGDI, a protein loading control, with 100% defined as the stabilization after Wnt3A treatment. The data are the mean and standard error from three separate determinations from a single experiment and are representative of at least three experiments. (C) Cells were transfected with the vectors indicated in D, then, after 5 h, treated as indicated with control CM or Wnt3A CM for an additional 16 h. Cell nuclei were isolated then extracted in high salt buffer as described in Materials and Methods. 25 µg of protein from each of the indicated nuclear fractions were analyzed by SDS-PAGE and subsequent immunoblot to detect endogenous β– catenin. (D) Nuclear β–catenin detected by immunoblot in panel C was quantified and normalized to CREB levels as in (B) and are the mean and standard error from three separate determinations from a single experiment and are representative of at least three experiments. (E) Cells were transfected with the constructs indicated in F, then, after 5 h, treated as indicated with control CM or Wnt3A CM for an additional 16 h. Cell nuclei were isolated then extracted in high salt buffer as described in Materials and Methods. 25 µg of protein from each of the indicated nuclear fractions was analyzed by SDS-PAGE and subsequent immunoblot to detect endogenous β–catenin. (F) Nuclear β–catenin was quantified as in (D) and the data represent the mean and standard error from two separate experiments.

Figure 6

Isolating the RhoA sensitive element in the Ctgf promoter. (A) Diagram representing the pCtgf reporter (top) and the truncated reporter (pCtgf-trunc, bottom), from which ~1.5 kb of the hCtgf regulatory element was removed by MluI digestion, as described in Materials and Methods. The arrowheads represent the approximate position of TCF/LEF binding sites (see text). (B) Cells were transfected with the pCtgf or pCtgf-trunc reporters, phRG internal control reporter, and RhoA(GV) constructs as indicated in the figure, lysed 18 h later, and luciferase activity was measured as described in Materials and Methods. Results are reported as fold change relative to vector. The data shown are the mean and standard error from four separate determinations in a single experiment and are representative of at least six independent experiments. (C) Cells were transfected with the pCtgf or pCtgf-trunc reporters, along with phRG internal control reporter. 5 h after transfection, cells were incubated with control CM or Wnt3A CM for 18 h prior to lysis and the measurement of luciferase activity. Results are reported as fold change relative to control CM treatment. The data shown are the mean and standard error from seven separate determinations. *P<0.03 (D) Cells were co-transfected with the pCtgf reporter, phRG internal control reporter and the indicated constructs, lysed 18 h later, and luciferase activity was measured as described above. Luciferase results are reported as RLU as described in Materials and Methods. Western blots were performed on lysates used to measure luciferase activity. The data are the mean and standard error from six separate determinations from a single experiment and are representative of at least two independent experiments. ** P<0.0001

Figure 7

Contribution of Rho to canonical Wnt3A signaling. Cytosolic β–catenin is continuously degraded in unstimulated cells via a process initiated by its binding to a complex of proteins that includes axin, the adenomatous polyposis coli (APC) protein and glycogen synthase kinase 3β (GSK3β), termed the β–catenin destruction complex. This complex interacts with β– catenin to induce its phosphorylation, which targets it for ubiquitination and degradation by the proteosome. Wnt binding by Fzd and a co-receptor, LRP5/6, activates a positive regulator of the pathway, disheveled (Dvl), which in turn inactivates the destruction complex and allows the accumulation of cytoplasmic β–catenin [52]. Stabilized β–catenin can then shuttle into the nucleus and bind to members of the T-cell factor (TCF)/lymphoid enhancing factor (LEF) family of transcription factors to induce expression of target genes [53]. Wnt3A binding to Fzd and LRP5/6 also leads to the activation of RhoA GTPase in a manner that depends, at least in part, upon Dvl and possibly other pathways yet to be defined. Our results show that activated RhoA, through the stimulation of ROCK, potentiates the β–catenin-dependent induction of target genes via unknown intermediates indicated by the dashed line. The full activation of these genes, which comprise a subset of the β–catenin targets, is dependent on both β–catenin and Rho and is required for Wnt3A–driven cellular consequences, including osteoblastic differentiation.