TCF-4 mediates cell type-specific regulation of proglucagon gene expression by beta-catenin and glycogen synthase kinase-3beta

The proglucagon gene (glu) encodes glucagon, expressed in pancreatic islets, and the insulinotropic hormone GLP-1, expressed in the intestines. These two hormones exert critical and opposite effects on blood glucose homeostasis. An intriguing question that remains to be answered is whether and how glu gene expression is regulated in a cell type-specific manner. We reported previously that the glu gene promoter in gut endocrine cell lines was stimulated by beta-catenin, the major effector of the Wnt signaling pathway, whereas glu mRNA expression and GLP-1 synthesis were activated via inhibition of glycogen synthase kinase-3beta, the major negative modulator of the Wnt pathway (Ni, Z., Anini, Y., Fang, X., Mills, G. B., Brubaker, P. L., & Jin, T. (2003) J. Biol. Chem. 278, 1380-1387). We now show that beta-catenin and the glycogen synthase kinase-3beta inhibitor lithium do not activate glu mRNA or glu promoter expression in pancreatic cell lines. In the intestinal GLUTag cell line, but not in the pancreatic InR1-G9 cell line, the glu promoter G2 enhancer-element was activated by lithium treatment via a TCF-binding motif. TCF-4 is abundantly expressed in the gut but not in pancreatic islets. Furthermore, both TCF-4 and beta-catenin bind to the glu gene promoter, as detected by chromatin immunoprecipitation. Finally, stable introduction of dominant-negative TCF-4 into the GLUTag cell line repressed basal glu mRNA expression and abolished the effect of lithium on glu mRNA expression and GLP-1 synthesis. We have therefore identified a unique mechanism that regulates glu expression in gut endocrine cells only. Tissue-specific expression of TCF factors thus may play a role in the diversity of the Wnt pathway.     

PTH/cAMP/PKA signaling facilitates canonical Wnt signaling via inactivation of glycogen synthase kinase-3beta in osteoblastic Saos-2 cells

Although the intermittent administration of PTH is known to stimulate the bone formation, the underlying mechanisms are not fully understood. Here we investigated the crosstalk between PTH/cAMP signaling and canonical Wnt signaling using the human osteoblastic cell line Saos-2. Treatment with PTH or forskolin, an activator of adenylate cyclase, facilitated T-cell factor (TCF)-dependent transactivation in a dose-dependent manner, which was abolished by pre-treatment with a PKA inhibitor, H89. Wnt3a and forskolin synergistically increased the TCF-dependent transactivation. Interestingly, intermittent treatment with PTH enhanced the TCF-dependent transactivation more profoundly than continuous treatment. In addition to the effects on TCF-dependent reporter activity, treatment with PTH or forskolin resulted in the increased expression of endogenous targets of Wnts, Wnt-induced secreted protein 2 (WISP2) and naked cuticle 2 (NKD2). We then investigated the convergence point of PTH/cAMP signaling and the canonical Wnt pathway. Western blotting demonstrated that GSK-3beta was rapidly phosphorylated at Ser(9) on treatment with PTH or forskolin, leading to its inactivation. Moreover, overexpression of a constitutively active mutant of GSK-3beta abolished the TCF-dependent transactivation induced by forskolin. On the other hand, overexpression of the Wnt antagonist Dickkopf-1 (DKK1) failed to cancel the effects of forskolin on the canonical Wnt pathway. Interestingly, treatment with Wnt3a markedly reduced the forskolin-induced expression of receptor activator of NF-kappaB ligand (RANKL), a target gene of PTH/cAMP/PKA. These results suggest that cAMP/PKA signaling activates the canonical Wnt pathway through the inactivation of GSK-3beta, whereas Wnt signaling might inhibit bone resorption through a negative impact on RANKL expression in osteoblasts.     

Differentiation-inducing factor-1 suppresses gene expression of cyclin D1 in tumor cells

To determine the mechanism by which differentiation-inducing factor-1 (DIF-1), a morphogen of Dictyostelium discoideum, inhibits tumor cell proliferation, we examined the effect of DIF-1 on the gene expression of cyclin D1. DIF-1 strongly reduced the expression of cyclin D1 mRNA and correspondingly decreased the amount of beta-catenin in HeLa cells and squamous cell carcinoma cells. DIF-1 activated glycogen synthase kinase-3beta (GSK-3beta) and inhibition of GSK-3beta attenuated the DIF-1-induced beta-catenin degradation, indicating the involvement of GSK-3beta in this effect. Moreover, DIF-1 reduced the activities of T-cell factor (TCF)/lymphoid enhancer factor (LEF) reporter plasmid and a reporter gene driven by the human cyclin D1 promoter. Eliminating the TCF/LEF consensus site from the cyclin D1 promoter diminished the effect of DIF-1. These results suggest that DIF-1 inhibits Wnt/beta-catenin signaling, resulting in the suppression of cyclin D1 promoter activity.     

Glucagon-like peptide-1 activation of TCF7L2-dependent Wnt signaling enhances pancreatic beta cell proliferation

The insulinotropic hormone GLP-1 (glucagon-like peptide-1) is a new therapeutic agent that preserves or restores pancreatic beta cell mass. We report that GLP-1 and its agonist, exendin-4 (Exd4), induce Wnt signaling in pancreatic beta cells, both isolated islets, and in INS-1 cells. Basal and GLP-1 agonist-induced proliferation of beta cells requires active Wnt signaling. Cyclin D1 and c-Myc, determinants of cell proliferation, are up-regulated by Exd4. Basal endogenous Wnt signaling activity depends on Wnt frizzled receptors and the protein kinases Akt and GSK3beta but not cAMP-dependent protein kinase. In contrast, GLP-1 agonists enhance Wnt signaling via GLP-1 receptor-mediated activation of Akt and beta cell independent of GSK3beta. Inhibition of Wnt signaling by small interfering RNAs to beta-catenin or a dominant-negative TCF7L2 decreases both basal and Exd4-induced beta cell proliferation. Wnt signaling appears to mediate GLP-1-induced beta cell proliferation raising possibilities for novel treatments of diabetes.     

Glucagon-like peptide-2 receptor activation engages bad and glycogen synthase kinase-3 in a protein kinase A-dependent manner and prevents apoptosis following inhibition of phosphatidylinositol 3-kinase

Activation of glucagon-like peptide-2 receptor (GLP-2R) signaling promotes expansion of the mucosal epithelium indirectly via activation of growth and anti-apoptotic pathways; however, the cellular mechanisms coupling direct GLP-2R activation to cell survival remain poorly understood. We now demonstrate that GLP-2, in a cycloheximide-insensitive manner, enhanced survival in baby hamster kidney cells stably transfected with the rat GLP-2R; reduced mitochondrial cytochrome c efflux; and attenuated the caspase-dependent cleavage of Akt, poly(ADP-ribose) polymerase, and beta-catenin following inhibition of phosphatidylinositol 3-kinase (PI3K) by LY294002. The prosurvival effects of GLP-2 on LY294002-induced cell death were independent of Akt, p90(Rsk), or p70 S6 kinase activation; were mimicked by forskolin; and were abrogated by inhibition of protein kinase A (PKA) activity. GLP-2 inhibited activation of glycogen synthase kinase-3 (GSK-3) through phosphorylation at Ser(21) in GSK-3alpha and at Ser(9) in GSK-3beta in a PI3K-independent, PKA-dependent manner. GLP-2 reduced LY294002-induced mitochondrial association of endogenous Bad and Bax and stimulated phosphorylation of a transfected Bad fusion protein at Ser(155) in a PI3K-independent, but H89-sensitive manner, a modification known to suppress Bad pro-apoptotic activity. These results suggest that GLP-2R signaling enhances cell survival independently of PI3K/Akt by inhibiting the activity of a subset of pro-apoptotic downstream targets of Akt in a PKA-dependent manner.     

Anti-apoptotic action of Wnt5a in dermal fibroblasts is mediated by the PKA signaling pathways

Wnts are secreted glycoproteins that control diverse biological processes, such as proliferation, differentiation, and apoptosis. We here found that Wnt5a inhibited apoptosis induced by serum deprivation in primary-cultured human dermal fibroblasts. Anti-apoptotic activity of Wnt5a was not inhibited by a dickkopf-1 (DKK), which blocks the canonical Wnt pathway. On the other hand, loss of function of protein kinase A (PKA), induced by treatment with PKA inhibitors, siRNA-mediated knocking down of endogenous PKA catalytic subunits, or enforced expression of dominant-negative PKA inhibited the Wnt5a anti-apoptotic activity, indicating the involvement of PKA in the Wnt5a anti-apoptotic activity. In agreement, phosphorylation levels of a cAMP response element binding protein (CREB), a representative downstream effector of PKA, the activation of which is known to lead to the pro-survival effects, was elevated by Wnt5a. In addition, Wnt5a increased the nuclear beta-catenin level and treatment with imatinib or ionomycin, either of which blocks the beta-catenin pathway, reduced the anti-apoptotic activity of Wnt5a, together suggesting the simultaneous involvement of the beta-catenin-mediated pathway in the Wnt5a anti-apoptotic activity. Based on another finding indicating that Wnt5a upregulated PKA-mediated phosphorylation of glycogen synthase kinase-3beta (GSK-3beta) at serine 9 that caused inactivation of GSK-3beta and subsequently resulted in activation of the beta-catenin pathway, we have speculated that the Wnt5a anti-apoptotic activity may be partially mediated by PKA-mediated phosphorylation of GSK-3beta and subsequent activation of the beta-catenin pathway.     

The effect of pleiotrophin signaling on adipogenesis

Pleiotrophin (PTN) plays diverse roles in cell growth and differentiation. In this investigation, we demonstrate that PTN plays a negative role in adipogensis and that glycogen synthase kinase 3beta (GSK-3beta) and beta-catenin are involved in the regulation of PTN-mediated preadipocyte differentiation. Knocking down the expression of PTN using siRNA resulted in an increase in phospho-GSK-3beta expression, and the accumulation of nuclear beta-catenin, which are critical downstream signaling proteins for both the PTN and Wnt signaling pathways. Our investigation suggests that there is a PTN/PI3K/AKT/GSK-3beta/beta-catenin signaling pathway, which cross-talks with the Wnt/Fz/GSK-3beta/beta-catenin pathway and negatively regulates adipogenesis.     

Wnt signaling during BMP-2 stimulation of mesenchymal chondrogenesis

Members of both the Wnt and bone morphogenetic protein (BMP) families of signaling molecules have been implicated in the regulation of cartilage development. A key component of the Wnt signaling pathway is the cytosolic protein, beta-catenin. We have recently shown that the chondrogenic activity of BMP-2 in vitro involves the action of the cell-cell adhesion protein, N-cadherin, which functionally complexes with beta-catenin. The aim of this study is to test the hypothesis that Wnts may be involved in BMP-2 induced chondrogenesis, using an in vitro model of high-density micromass cultures of the murine multipotent mesenchymal cell line, C3H10T1/2. Expression of a number of Wnt members was detected in these cultures, including Wnt-3A and Wnt-7A, whose levels were up- and downregulated, respectively, by BMP-2. To assess the functional involvement of Wnt signaling in BMP-2 induced chondrogenesis, cultures were treated with lithium chloride, a Wnt-7A mimetic that acts by inhibiting the serine/threonine phosphorylation activity of glycogen synthase kinase-3beta (GSK-3beta). Lithium treatment significantly inhibited BMP-2 stimulation of chondrogenesis as well as GSK-3beta enzymatic activity, and decreased the levels of N-cadherin protein and mRNA. Furthermore, lithium decreased BMP-2 upregulation of total and nuclear levels of LEF-1 and beta-catenin as well as their interaction during later chondrogenesis; similarly, the interaction of beta-catenin with N-cadherin was also decreased. Interestingly, lithium treatment did not affect the ability of BMP-2 to decrease ubiquitination of beta-catenin, although it did reduce the interaction of beta-catenin with GSK-3beta during late chondrogenesis (days 9-13). We suggest that the chondro-inhibitory effect of lithium on BMP-2 induced chondrogenesis indicates antagonism between lithium-like Wnts and BMP-2 during mesenchymal condensation.     

Wnt signaling enhances FGF2-triggered lens fiber cell differentiation

Wnt signaling is implicated in many developmental processes, including cell fate changes. Several members of the Wnt family, as well as other molecules involved in Wnt signaling, including Frizzled receptors, LDL-related protein co-receptors, members of the Dishevelled and Dickkopf families, are known to be expressed in the lens during embryonic or postembryonic development. However, the function of Wnt signaling in lens fiber differentiation remains unknown. Here, we show that GSK-3beta kinase is inactivated and that beta-catenin accumulates during the early stages of lens fiber cell differentiation. In an explant culture system, Wnt conditioned medium (CM) induced the accumulation of beta-crystallin, a marker of fiber cell differentiation, without changing cell shape. In contrast, epithelial cells stimulated with Wnt after priming with FGF elongated, accumulated beta-crystallin, aquaporin-0, p57kip2, and altered their expression of cadherins. Treatment with lithium, which stabilizes beta-catenin, induced the accumulation of beta-crystallin, but explants treated with lithium after FGF priming did not elongate as they did after Wnt application. These results show that Wnts promote the morphological aspects of fiber cell differentiation in a process that requires FGF signaling, but is independent of beta-catenin. Wnt signaling may play an important role in lens epithelial-to-fiber differentiation.     

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

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

Inhibition of Wnt signaling pathway by a novel axin-binding protein

Axin forms a complex with adenomatous polyposis coli gene product, glycogen synthase kinase-3beta (GSK-3beta), beta-catenin, Dvl, and protein phosphatase 2A and functions as a scaffold protein in the Wnt signaling pathway. In the Axin complex, GSK-3beta efficiently phosphorylates beta-catenin, which is then ubiquitinated and degraded by proteasome. We isolated a novel protein that binds to Axin and named it Axam (for Axin associating molecule). Axam formed a complex with Axin in intact cells and bound directly to Axin. Axam inhibited the complex formation of Dvl with Axin and the activity of Dvl to suppress GSK-3beta-dependent phosphorylation of Axin. Furthermore, Axam induced the degradation of beta-catenin in SW480 cells and inhibited Wnt-dependent axis duplication in Xenopus embryos. These results suggest that Axam regulates the Wnt signaling pathway negatively by inhibiting the binding of Dvl to Axin.