Transcription Of Wnt Target Genes Research Articles

P15RS Attenuates Wnt/β-Catenin Signaling by Disrupting β-Catenin·TCF4 Interaction

The formation of a β-catenin·TCF4 complex in the nucleus of cells is well known as a prerequisite for the transcription of Wnt target genes. Although many co-factors have been identified to regulate the activity of the β-catenin·TCF4 complex, it remains unclear how the complex association is negatively regulated. In this study, we report that p15RS, a negative regulator of the cell cycle, blocks β-catenin·TCF4 complex formation and inhibits Wnt signaling. We observed that p15RS interacts with β-catenin and TCF4. Interestingly, whereas the interaction of p15RS with β-catenin is increased, its interaction with TCF4 is decreased upon Wnt1 stimulation. Moreover, overexpression of p15RS reduces the interaction of β-catenin with TCF4, whereas the depletion of p15RS enhances their interaction. We further demonstrate that overexpression of p15RS suppresses canonical Wnt signaling and results in retarded cell growth, whereas depletion of p15RS shows an enhanced effect on Wnt signaling. We analyzed that inhibition of Wnt signaling by p15RS leads to decreased expression of CYCLIN D1 and c-MYC, two Wnt targeted genes critical for cell growth. Our data suggest that p15RS inhibits Wnt signaling by interrupting β-catenin·TCF4 complex formation and that Wnt signaling initiates downstream gene expression by removing p15RS from promoters.

Differential Modulation of TCF/LEF-1 Activity by the Soluble LRP6-ICD

The canonical Wnt/β-catenin (Wnt) pathway is a master transcriptional regulatory signaling pathway that controls numerous biological processes including proliferation and differentiation. As such, transcriptional activity of the Wnt pathway is tightly regulated and/or modulated by numerous proteins at the level of the membrane, cytosol and/or nucleus. In the nucleus, transcription of Wnt target genes by TCF/LEF-1 is repressed by the long Groucho/TLE co-repressor family. However, a truncated member of the Groucho/TLE family, amino terminal enhancer of Split (AES) can positively modulate TCF/LEF-1 activity by antagonizing long Groucho/TLE members in a dominant negative manner. We have previously shown the soluble intracellular domain of the LRP6 receptor, a receptor required for activation of the Wnt pathway, can positively regulate transcriptional activity within the Wnt pathway. In the current study, we show the soluble LRP6 intracellular domain (LRP6-ICD) can also translocate to the nucleus in CHO and HEK 293T cells and in contrast to cytosolic LRP6-ICD; nuclear LRP6-ICD represses TCF/LEF-1 activity. In agreement with previous reports, we show AES enhances TCF/LEF-1 mediated reporter transcription and further we demonstrate that AES activity is spatially regulated in HEK 293T cells. LRP6-ICD interacts with AES exclusively in the nucleus and represses AES mediated TCF/LEF-1 reporter transcription. These results suggest that LRP6-ICD can differentially modulate Wnt pathway transcriptional activity depending upon its subcellular localization and differential protein-protein interactions.

Abstract LB-147: Characterization of a Kaiso overexpressing transgenic mouse model

Abstract Introduction: The WNT signaling pathway plays critical roles during embryogenesis and maintaining homeostasis of adult tissues. However constitutive WNT signaling leads to tumorigenesis. Upon activation of the WNT pathway, the main downstream effector of the WNT pathway, ß-catenin, stabilizes and translocates to the nucleus where it activates transcription of WNT target genes. Recently, our lab along with others identified the novel transcription factor Kaiso as a negative regulator of WNT target genes in mammalian cell lines and Xenopus models. However a study using a Kaiso depleted mouse model implicated Kaiso as a positive regulator of WNT signaling. Kaiso is a member of the BTB/POZ (broad-complex, tramtrack and bric-à-brac/poxvirus and zinc finger) protein family. The goal of this study is to elucidate the role of Kaiso in WNT signaling using the ApcMin/+ mouse model of intestinal cancer. Methodology: To assess the role of Kaiso in colon cancer in vivo, Kaiso overexpressing transgenic mice were created (KaisoTg). Targeted overexpression of Kaiso in KaisoTg mice was obtained by pronuclear injection of Kaiso cDNA under the control of the intestine-specific villin promoter. Reverse transcription PCR (RT-PCR) and Western Blot analyses were used to determine tissue specific Kaiso overexpression in KaisoTg mice. Immunohistochemisty (IHC) was performed on mouse intestinal tissues to examine the subcellular localization of Kaiso and assess developmental defects. KaisoTg mice were then mated with APCMin/+ mice models of intestinal cancer that develop polyps in the small intestine due to constitutively active -catenin. KaisoTg-APCMin/+ mice were examined for gross morphological phenotypes and IHC was performed on mouse intestinal tissues for Kaiso and other WNT target genes (e.g. cyclin D1) to elucidate the effects of Kaiso on the WNT pathway. Results: Western Blot analysis revealed high, moderate and low levels of Kaiso overexpression in three founder lines of the KaisoTg mice. RT-PCR analysis showed Kaiso overexpression limited to villin positive tissues including kidney, small and large intestines. IHC analysis of KaisoTg tissues showed no gross morphological differences compared to non-transgenic mice. However KaisoTg-APCMin/+ displayed decreased body size and weight, and had decreased life span when compared to the parentals (i.e. KaisoTg and APCmin/+). IHC analysis of KaisoTg-APCMin/+ mouse intestines revealed increased polyp numbers with Kaiso staining concentrated near the basal surface of the polyps. These data support Kaiso's potential role as an oncogene in mouse intestinal tissues. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-147.

Abstract 2685: The intratumoral expression of survivin associated with Wnt1 expression, not Wnt5a expression in non-small cell lung cancer

Abstract Introduction: Survivin is a member of the inhibitor of apoptosis protein (IAP) family. Recent studies have revealed that survivin is associated not only with apoptosis but also with cell proliferation. As a result, the survivin expression is considered to be involved in tumorigenesis, including non-small cell lung cancer (NSCLC). On the other hand, the Wnt genes are involved in embryogenesis and tumorigenesis. Among them, Wnt1 stimulates the canonical Wnt signaling pathway, which regulates the transcription of various tumor-associated Wnt-target genes with TCF/LEF1 motif. We have reported that Wnt1 overexpression promotes tumorigenesis in NSCLC through the induction of c-Myc, cyclin D1, VEGF-A (Huang C, et al. Eur J Cancer 2008). Recently, survivin is also reported to be a target gene of the canonical Wnt signaling pathway. Therefore, we performed a clinical study on survivin expression in relation to the expression of Wnt1 (canonical Wnt) and Wnt5a (non-canonical Wnt) in NSCLC. Method: One hundred and ten NSCLC patients were investigated. The expression of wild-type survivin (survivin) was evaluated by semi-quantitative RT-PCR because real-time RT-PCR is not an appropriate method to discriminate between wild-type survivin and other splice variants, such as survivin-2B and survivin-deltaEx3. The intratumoral expressions of survivin, Wnt1 and Wnt5a were evaluated by immunohistochemistry. The Ki-67 proliferation index was also investigated by immunohistochemistry, and the apoptotic index was evaluated by TUNEL method. Results: (1) Sixty carcinomas (54.5%) had an overexpression of survivin. The apoptotic index was significantly lower in survivin-high tumors than in survivin-low tumors (P=0.003). In addition, the Ki-67 proliferation index was significantly higher in survivin-high tumors than in survivin-low tumors (P=0.014). (2) The percentage of Wnt1-positive tumor cells significantly correlated with the survivin gene expression (P<0.001). The survivin gene expression was significantly higher in Wnt1-positive tumors than in Wnt1-negative tumors (P=0.002). (3) The percentage of Wnt5a-positive tumor cells did not correlate with the survivin gene expression (P=0.155). There was no difference in the survivin gene expression according to the Wnt5a expression. (4) As a result, the apoptotic index was significantly lower in Wnt1-high tumors than in Wnt1-low tumors (P=0.032). In addition, the Ki-67 proliferation index was significantly higher in Wnt1-high tumors than in Wnt1-low tumors (P=0.001). Conclusions: The intratumoral Wnt1 expression affects the expression of wild-type survivin in NSCLC to produce more aggressive tumors with low tumor apoptosis and high tumor proliferation rate. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2685.

Coordination of dorsoventral axis formation by multiple heparan sulfate proteoglycans

Heparan sulfate proteoglycans are essential mediators of multiple cell‐cell signaling pathways during embryonic development. To better understand how glypicans (GPCs) ‐ a membrane anchored subfamily of heparan sulfate core proteins ‐ function in zebrafish development, we used antisense morpholinos to individually and specifically knock down the expression of all ten GPC genes in zebrafish. Knockdown of GPC2, GPC6a, or GPC6b uniquely resulted in strong dorsoventral phenotypes between gastrulation and 24hpf. To better understand the molecular pathways in which GPCs are involved, we used a candidate gene approach to study changes in transcription of BMP, Wnt and FGF target genes because these cell‐cell signaling pathways are known to regulate dorsoventral patterning. We identified gene expression patterns that were different between each GPC knockdown, with GPC2 having a distinct affect on FGF target genes. These results were confirmed using several complimentary approaches, suggesting that GPC2, GPC6a, and GPC6b regulate distinct steps in dorsoventral patterning, an observation that adds a new dimension to our understanding of how proteoglycans work together to mediate early development.

Abstract A75: Retinoblastoma protein promotes Wnt signaling pathway activation in MC3T3 osteoblasts

Abstract Disruption of the retinoblastoma (pRb) pathway is observed in most cancers. Mutational inactivation of pRb itself is observed at high frequencies in retinoblastomas, osteosarcomas, and small cell lung carcinoma. pRb blocks cell cycle progression by repressing E2F-dependent transcription of S-phase-related genes inducing cellular differentiation by serving as co-activator of tissue-specific transcription factors. Another pathway frequently targeted in human cancers is the Wnt pathway, which regulates a variety of processes such as chondrocyte and osteoblast differentiation. Wnt activation hinders β-catenin degradation by proteosomes with consequent β-catenin nuclear accumulation and transcription of Wnt target genes. In contrast, the absence of the Wnt signal allows the recruitment of CK1, APC, GSK3-β, and β-catenin by Axin. This complex phosphorylates β-catenin at specific residues serving as a tag for recognition and degradation. Given that inactivation of both pRb and Wnt pathways is commonly observed in osteosarcomas, we sought to determine if these pathways are functionally linked in such a manner that inactivation of one of them leads to deregulation of the other. To test this, we performed qRT-PCR of pRb-expressing and pRb-deficient osteoblasts (MC3T3 Rb+/+ and MC3T3 Rb−/−, respectively) in order to compare the mRNA steady state levels of the Wnt pathway components. We found a 1.8 fold increase (p<0.05) in the levels of PP2, an inducer of β-catenin degradation, in MC3T3 Rb-deficient relative to MC3T3 Rb-expressing. Whereas, we observed a 0.4 fold decrease (p<0.005) in Dvl, an inhibitor of β-catenin degradation, in MC3T3 Rb-deficient relative to MC3T3 Rb-expressing. Taken together these results indicate that pRb promotes Wnt signaling by inhibiting β-catenin's degradation. Wnt activation requires β-catenin's nuclear relocalization, a process blocked in part by phosphorylation of β-catenin at Serine 33 (pSer33) by GSK3-β. To test if pRb regulates β-catenin's relocalization we performed immunocytochemistry assay. Our immunocytochemistry studies showed an increase in nuclear localization of β-catenin and β-catenin pSer33 in the pRb-expressing compared to the pRb-deficient osteoblasts. Upon nuclear relocalization, β-catenin must bind to the Tcf/Lef transcriptional activators forming a complex capable of transcribingWnt targets. To determine if the nuclear β-catenin observed in the pRb-expressing cell line can induce expression of Wnt target genes, we transfected osteoblasts with a TopFlash construct consisting of a luciferase reporter under the control of a β-catenin/Tcf/Lef responsive promoter. Using Dual-Luciferase Assay we demonstrated a 2.8 increase in reporter activity of the pRb-expressing over the pRb-deficient cell line. This increase in luciferase activity of the pRb-expressing indicates an increase in β-catenin Tcf/lef dependent transcription. Our data show that, in the presence of a functional pRb, β-catenin degradation is blocked, proposing a novel role for pRb as a promoter of the Wnt signaling pathway in osteoblasts. This project was supported by PSM Institutional Funds, American Cancer Society's Institutional Research Grant #93-032-13. Citation Information: Cancer Res 2009;69(23 Suppl):A75.

Interactions between SOX factors and Wnt/β‐catenin signaling in development and disease

The SOX family of transcription factors have emerged as modulators of canonical Wnt/beta-catenin signaling in diverse development and disease contexts. There are over 20 SOX proteins encoded in the vertebrate genome and recent evidence suggests that many of these can physically interact with beta-catenin and modulate the transcription of Wnt-target genes. The precise mechanisms by which SOX proteins regulate beta-catenin/TCF activity are still being resolved and there is evidence to support a number of models including: protein-protein interactions, the binding of SOX factors to Wnt-target gene promoters, the recruitment of co-repressors or co-activators, modulation of protein stability, and nuclear translocation. In some contexts, Wnt signaling also regulates SOX expression resulting in feedback regulatory loops that fine-tune cellular responses to beta-catenin/TCF activity. In this review, we summarize the examples of Sox-Wnt interactions and examine the underlying mechanisms of this potentially widespread and underappreciated mode of Wnt-regulation.

The Wnt Target Jagged-1 Mediates the Activation of Notch Signaling by Progastrin in Human Colorectal Cancer Cells

The Wnt and Notch signaling pathways are both abnormally activated in colorectal cancer (CRC). We recently showed that progastrin depletion inhibited Wnt signaling and increased goblet cell differentiation of CRC cells. Here, we show that progastrin down-regulation restores the expression by CRC cells of the early secretory lineage marker Math-1/Hath-1 due to an inhibition of Notch signaling. This effect is mediated by a decreased transcription of the Notch ligand Jagged-1, downstream of beta-catenin/Tcf-4. Accordingly, recombinant progastrin sequentially activated the transcription of Wnt and Notch target genes in progastrin-depleted cells. In addition, restoration of Jagged-1 levels in these cells is sufficient to activate Tcf-4 activity, demonstrating the occurrence of a feedback regulation from Notch toward Wnt signaling. These results suggest that progastrin could be instrumental in maintaining the concomitant activation of Wnt and Notch pathways in CRC cells, further highlighting the interest of progastrin targeting for the clinical management of CRC.

β-Catenin hits chromatin: regulation of Wnt target gene activation

The canonical Wnt pathway has gathered much attention in recent years owing to its fundamental contribution to metazoan development, tissue homeostasis and human malignancies. Wnt target gene transcription is regulated by nuclear beta-catenin, and genetic assays have revealed various collaborating protein cofactors. Their daunting number and diverse nature, however, make it difficult to arrange an orderly picture of the nuclear Wnt transduction events. Yet, these findings emphasize that beta-catenin-mediated transcription affects chromatin. How does beta-catenin cope with chromatin regulation to turn on Wnt target genes?

T-Cell Factor 4 (tcf7l2) Is the Main Effector of Wnt Signaling During Zebrafish Intestine Organogenesis

The Wnt pathway orchestrates cell fate decisions during embryonic development, organogenesis, and adult tissues homeostasis. T-cell factor (Tcf )/lymphoid enhancer-binding factor (Lef) transcription factors are the downstream effectors of canonical Wnt signaling. Upon Wnt signal activation, beta-catenin stabilizes and translocates to the nucleus, where it interacts with Tcfs activating the transcription of Wnt target genes. In the absence of Wnt, levels of stable beta-catenin are reduced by the action of adenomatous polyposis coli (Apc) and other cytoplasmic proteins. Mutations in Apc cause constitutive accumulation of beta-catenin and inappropriate activation of the Wnt pathway. apc(mcr/mcr) fish embryos show absence of expression of tissue-specific differentiation markers in the intestine, suggesting that inappropriate activation of Wnt signaling abrogates gut organogenesis. Which Tcf transcription factor mediates Wnt signaling during zebrafish gut organogenesis remains unclear. We studied the combined effect of loss of Tcf family members and Apc in the developing embryo. Tcf4 (tcf7l2) loss rescues the apc(mcr/mcr) phenotype in the intestine. Single depletion of Tcf1 (tcf7) and Tcf3 (tcf7l1a) function in an Apc mutant background had no effect on endoderm development. This study reveals that Tcf4 (tcf7l2) is the major effector of Wnt signaling in the intestine during zebrafish organogenesis.

Rac1 GTPase and the Rac1 exchange factor Tiam1 associate with Wnt-responsive promoters to enhance beta-catenin/TCF-dependent transcription in colorectal cancer cells

Backgroundβ-catenin is a key mediator of the canonical Wnt pathway as it associates with members of the T-cell factor (TCF) family at Wnt-responsive promoters to drive the transcription of Wnt target genes. Recently, we showed that Rac1 GTPase synergizes with β-catenin to increase the activity of a TCF-responsive reporter. This synergy was dependent on the nuclear presence of Rac1, since inhibition of its nuclear localization effectively abolished the stimulatory effect of Rac1 on TCF-responsive reporter activity. We hypothesised that Rac1 plays a direct role in enhancing the transcription of endogenous Wnt target genes by modulating the β-catenin/TCF transcription factor complex.ResultsWe employed chromatin immunoprecipitation studies to demonstrate that Rac1 associates with the β-catenin/TCF complex at Wnt-responsive promoters of target genes. This association served to facilitate transcription, since overexpression of active Rac1 augmented Wnt target gene activation, whereas depletion of endogenous Rac1 by RNA interference abrogated this effect. In addition, the Rac1-specific exchange factor, Tiam1, potentiated the stimulatory effects of Rac1 on the canonical Wnt pathway. Tiam1 promoted the formation of a complex containing Rac1 and β-catenin. Furthermore, endogenous Tiam1 associated with endogenous β-catenin, and this interaction was enhanced in response to Wnt3a stimulation. Intriguingly, Tiam1 was recruited to Wnt-responsive promoters upon Wnt3a stimulation, whereas Rac1 was tethered to TCF binding elements in a Wnt-independent manner.ConclusionTaken together, our results suggest that Rac1 and the Rac1-specific activator Tiam1 are components of transcriptionally active β-catenin/TCF complexes at Wnt-responsive promoters, and the presence of Rac1 and Tiam1 within these complexes serves to enhance target gene transcription. Our results demonstrate a novel functional mechanism underlying the cross-talk between Rac1 and the canonical Wnt signalling pathway.

Importance of Wnt Signaling in the Tumor Stroma Microenvironment

Wnt signaling plays an important role in cancer. Signaling is initiated by binding of Wnt ligands to Frizzled cell surface receptors and results in signaling via one of three pathways, the canonical Wnt pathway, which is the best characterized in both normal tissues and in cancer, and two non-canonical Wnt pathways, the Ca(2+)-dependent and the PCP pathways. Canonical Wnt signaling results in beta-catenin accumulation in the cytoplasm, translocation into the nucleus and activation of transcription of Wnt target genes including the c-Myc oncogene. Some cancer types, including colorectal cancer, have mutations in APC and Axin, which are involved in beta-catenin phosphorylation, such that the canonical pathway is constitutively active. Few studies have investigated the role non-canonical Wnt signaling in cancer, or of Wnt signaling on tumor stromal cells. Wnt overexpression is observed in tumor stroma, as is overexpression of the Wnt pathway inhibitors, secreted Frizzled-related proteins and Dickkopf proteins. Interactions between epithelial cells and stromal cells have been observed to activate Wnt signaling in both cell types. Wnt signaling is also observed in tumor blood vessels and is likely to be activated by signals from tumor cells. Current cancer therapies focus on interfering with canonical Wnt signaling in the tumor cells. Future therapeutic targets for interfering with Wnt signaling include cell surface receptors such as the RYK and Ror2 receptors and secreted signaling molecules, which mediate signaling between cancer cells and the stromal environment.

Frizzled‐1 is involved in the neuroprotective effect of Wnt3a against Aβ oligomers

The activation of the canonical Wnt signaling pathway protects hippocampal neurons against the toxicity of Alzheimer's amyloid-beta-peptide (Abeta), however, the role played by the Wnt receptors Frizzleds, has not been studied. We report here that Frizzled-1 mediates the activation of the canonical Wnt/beta-catenin pathway by Wnt3a in PC12 cells. In addition, the protective effect of Wnt3a against the toxicity of Abeta oligomers was modulated by Frizzled-1 expression levels in both PC12 cells and hippocampal neurons. Over-expression of Frizzled-1 significantly increased cell survival induced by Wnt3a and diminished caspase-3 activation, while knocking-down Frizzled-1 expression by antisense oligonucleotides decreased the Wnt3a protection. Over-expression of wild-type beta-catenin, but not a transcriptionally inactive mutated version, prevented the toxicity of Abeta suggesting that the transcription of Wnt target genes may be involved in these events. This was confirmed by co-transfecting both Frizzled-1 and the inactive form of beta-catenin, which does not elicited protection levels similar to those showed with endogenous beta-catenin. Our results indicate that Wnt3a protects from Abeta-oligomers toxicity by activating the canonical Wnt signaling pathway through the Frizzled-1 receptor, suggesting a therapeutic potential for this signaling pathway in the treatment of Alzheimer's disease.

Pygopus activates Wingless target gene transcription through the mediator complex subunits Med12 and Med13

Wnt target gene transcription is mediated by nuclear translocation of stabilized beta-catenin, which binds to TCF and recruits Pygopus, a cofactor with an unknown mechanism of action. The mediator complex is essential for the transcription of RNA polymerase II-dependent genes; it associates with an accessory subcomplex consisting of the Med12, Med13, Cdk8, and Cyclin C subunits. We show here that the Med12 and Med13 subunits of the Drosophila mediator complex, encoded by kohtalo and skuld, are essential for the transcription of Wingless target genes. kohtalo and skuld act downstream of beta-catenin stabilization both in vivo and in cell culture. They are required for transcriptional activation by the N-terminal domain of Pygopus, and their physical interaction with Pygopus depends on this domain. We propose that Pygopus promotes Wnt target gene transcription by recruiting the mediator complex through interactions with Med12 and Med13.

Wnt signaling and stem cell control

Wnt signaling has been implicated in the control over various types of stem cells and may act as a niche factor to maintain stem cells in a self-renewing state. As currently understood, Wnt proteins bind to receptors of the Frizzled and LRP families on the cell surface. Through several cytoplasmic relay components, the signal is transduced to ss-catenin, which then enters the nucleus and forms a complex with TCF to activate transcription of Wnt target genes. Wnts can also signal through tyrosine kinase receptors, in particular the ROR and RYK receptors, leading to alternative modes of Wnt signaling. During the growth of tissues, these ligands and receptors are dynamically expressed, often transcriptionally controlled by Wnt signals themselves, to ensure the right balance between proliferation and differentiation. Isolated Wnt proteins are active on a variety of stem cells, including neural, mammary and embryonic stem cells. In general, Wnt proteins act to maintain the undifferentiated state of stem cells, while other growth factors instruct the cells to proliferate. These other factors include FGF and EGF, signaling through tyrosine kinase pathways.

Trabid, a new positive regulator of Wnt-induced transcription with preference for binding and cleaving K63-linked ubiquitin chains

A key effector of the canonical Wnt pathway is beta-catenin, which binds to TCF/LEF factors to promote the transcription of Wnt target genes. In the absence of Wnt stimulation, beta-catenin is phosphorylated constitutively, and modified with K48-linked ubiquitin for subsequent proteasomal degradation. Here, we identify Trabid as a new positive regulator of Wnt signaling in mammalian and Drosophila cells. Trabid show a remarkable preference for binding to K63-linked ubiquitin chains with its three tandem NZF fingers (Npl4 zinc finger), and it cleaves these chains with its OTU (ovarian tumor) domain. These activities of Trabid are required for efficient TCF-mediated transcription in cells with high Wnt pathway activity, including colorectal cancer cell lines. We further show that Trabid can bind to and deubiquitylate the APC tumor suppressor protein, a negative regulator of Wnt-mediated transcription. Epistasis experiments indicate that Trabid acts below the stabilization of beta-catenin, and that it may affect the association or activity of the TCF-beta-catenin transcription complex. Our results indicate a role of K63-linked ubiquitin chains during Wnt-induced transcription.

Nuclear Stabilization of β-Catenin and Inactivation of Glycogen Synthase Kinase-3β by Gonadotropin-Releasing Hormone: Targeting Wnt Signaling in the Pituitary Gonadotrope

The GnRH receptor is a G protein-coupled receptor (GPCR), and its ligand GnRH is the central regulator of the reproductive system. GnRH receptors are known to target a wide variety of signal transduction pathways. Several recent studies have shown that activation of GPCRs can impact on beta-catenin signaling. beta-Catenin is the main effecter of the Wnt signaling pathway where it acts with the transcription factors T cell factor/lymphoid enhancer factor to mediate the transcription of Wnt target genes. We show that GnRH treatment promotes the nuclear accumulation of beta-catenin, activation of T cell factor-dependent transcription, and up-regulation of Wnt target genes, c-Jun, Fra-1, and c-Myc. These results are observed in human embryonic kidney 293/GnRH receptor-expressing cells and have been recapitulated in LbetaT2 and alphaT3-1 mouse gonadotrope cells. In addition to these findings, we show that GnRH treatment mediates the inactivation of glycogen synthase kinase-3, a protein serine/threonine kinase that regulates beta-catenin degradation within the Wnt signaling pathway. Our findings extend the number of GPCRs that can target beta-catenin signaling through diverse pathways. Furthermore, this is the first demonstration of the targeting of Wnt/beta-catenin signaling by a peptide hormone GPCR.

R-spondins Signal Like Wnts, Sort Of

Wnt proteins are lipid-modified secreted molecules that initiate signaling by interacting with a receptor complex that includes the serpentine receptor Frizzled and low-density lipoprotein receptor—related protein 5 (LRP5) or LRP6. Wnt receptor activation leads to stabilization of β-catenin, which accumulates in the nucleus and activates transcription of Wnt target genes. Roof plate-specific spondins (R-spondins) are proteins unrelated in sequence to Wnts that, like Wnts, are implicated in regulation of development and that also activate signaling through β-catenin. Conflicting reports have been published as to whether R-spondins use the same receptors as Wnts and whether they bind to Frizzled or LRP6, both, or neither. Wei et al . present data showing that human R-spondin1 (hRspo1) binds to cultured human embryonic kidney (HEK293T) cells transfected with LRP6. Solid-phase binding assays showed that hRspo1 bound to the extracellular domain of LRP6 with high affinity (K d , 1.2 nM) but showed little if any binding to Frizzled. The hRspo1 protein also induced phosphorylation of LRP6 (as does Wnt). hRspo1 had synergistic effects with Wnt3A on phosphorylation of LRP6 in HEK293T cells and on phosphorylation of endogenous LRP6 in mouse embryo fibroblasts. R-spondins thus appear to work differently from Wnts in some ways (Wnts bind with high affinity to Frizzled, with weak or no binding to LRP6), but both ligands activate similar downstream signaling events. Furthermore, expression of R-spondins is stimulated by Wnts, leading the authors to propose that R-spondins may act in a positive feedback loop that reinforces Wnt signaling during development. Q. Wei, C. Yokota, M. V. Semenov, B. Doble, J. Woodgett, X. He, R-spondin1 is a high affinity ligand for LRP6 and induces LRP6 phosphorylation and β-catenin signaling. J. Biol. Chem. 282 , 15903-15911 (2007). [Abstract] [Full Text]

Increased pontin expression in human colorectal cancer tissue

Wnt signaling plays a fundamental role in the control of cell proliferation and differentiation and is frequently deregulated in colorectal carcinoma leading to an enhanced expression of Wnt target genes. Pontin, a member of the AAA(+) superfamily, has previously been shown to interact with beta-catenin and to enhance TCF/beta-catenin-mediated transcription of Wnt target genes and thus may contribute to carcinogenesis. Here, we studied the expression of pontin in 34 patients with histologically proven colorectal cancer by immunohistochemistry on paraffin-embedded colorectal cancer samples using the monoclonal mouse anti-pontin (5G3-11) antibody. Cytoplasmic pontin staining of tumor cells was present in all cases and was stronger in 84.6% and equal in 15.4% of the cases compared with normal mucosa. In 50% of tumor specimens, an additional nuclear pontin staining pattern was noted with positivity ranging from 10% to 60% of the nuclei. Interestingly, all cases with nuclear pontin expression also revealed nuclear beta-catenin localization. Furthermore, pontin staining was stronger at the invasive margin and in tumor buds than in the tumor center in 41.2% and 37.9% of the cases, respectively. In this context, 66.7% and 64.7% of the cases with enhanced beta-catenin staining at the invasive margin and in tumor buds, respectively, also revealed stronger pontin expression. Analysis of pontin expression in 8 patients by Western blotting confirmed the histologic results. These data suggest that upregulation and nuclear localization of pontin together with beta-catenin may contribute to progression of colorectal carcinoma.

Wnt-mediated Down-regulation of Sp1 Target Genes by a Transcriptional Repressor Sp5

Wnt/beta-catenin signaling regulates many processes during vertebrate development. To study transcriptional targets of canonical Wnt signaling, we used the conditional Cre/loxP system in mouse to ectopically activate beta-catenin during central nervous system development. We show that the activation of Wnt/beta-catenin signaling in the embryonic mouse telencephalon results in the up-regulation of Sp5 gene, which encodes a member of the Sp1 transcription factor family. A proximal promoter of Sp5 gene is highly evolutionarily conserved and contains five TCF/LEF binding sites that mediate direct regulation of Sp5 expression by canonical Wnt signaling. We provide evidence that Sp5 works as a transcriptional repressor and has three independent repressor domains, called R1, R2, and R3, respectively. Furthermore, we show that the repression activity of R1 domain is mediated through direct interaction with a transcriptional corepressor mSin3a. Finally, our data strongly suggest that Sp5 has the same DNA binding specificity as Sp1 and represses Sp1 target genes such as p21. We conclude that Sp5 transcription factor mediates the downstream responses to Wnt/beta-catenin signaling by directly repressing Sp1 target genes.