Regulating the balance between peroxisome proliferator-activated receptor gamma and beta-catenin signaling during adipogenesis. A glycogen synthase kinase 3beta phosphorylation-defective mutant of beta-catenin inhibits expression of a subset of adipogenic genes

The differentiation of preadipocytes into adipocytes requires the suppression of canonical Wnt signaling, which appears to involve a peroxisome proliferator-activated receptor gamma (PPARgamma)-associated targeting of beta-catenin to the proteasome. In fact, sustained activation of beta-catenin by expression of Wnt1 or Wnt 10b in preadipocytes blocks adipogenesis by inhibiting PPARgamma-associated gene expression. In this report, we investigated the mechanisms regulating the balance between beta-catenin and PPARgamma signaling that determines whether mouse fibroblasts differentiate into adipocytes. Specifically, we show that activation of PPARgamma by exposure of Swiss mouse fibroblasts to troglitazone stimulates the degradation of beta-catenin, which depends on glycogen synthase kinase (GSK) 3beta activity. Mutation of serine 37 (a target of GSK3beta) to an alanine renders beta-catenin resistant to the degradatory action of PPARgamma. Ectopic expression of the GSK3beta phosphorylation-defective S37A-beta-catenin in Swiss mouse fibroblasts expressing PPARgamma stimulates the canonical Wnt signaling pathway without blocking their troglitazone-dependent differentiation into lipid-laden cells. Analysis of protein expression in these cells, however, shows that S37A-beta-catenin inhibits a select set of adipogenic genes because adiponectin expression is completely blocked, but FABP4/aP2 expression is unaffected. Furthermore, the mutant beta-catenin appears to have no affect on the ability of PPARgamma to bind to or transactivate a PPAR response element. The S37A-beta-catenin-associated inhibition of adiponectin expression coincides with an extensive decrease in the abundance of C/EBPalpha in the nuclei of the differentiated mouse fibroblasts. Taken together, these data suggest that GSKbeta is a key regulator of the balance between beta-catenin and PPARgamma activity and that activation of canonical Wnt signaling downstream of PPARgamma blocks expression of a select subset of adipogenic genes.     

Initiation of Wnt signaling: control of Wnt coreceptor Lrp6 phosphorylation/activation via frizzled, dishevelled and axin functions

Canonical Wnt/beta-catenin signaling has central roles in development and diseases, and is initiated by the action of the frizzled (Fz) receptor, its coreceptor LDL receptor-related protein 6 (Lrp6), and the cytoplasmic dishevelled (Dvl) protein. The functional relationships among Fz, Lrp6 and Dvl have long been enigmatic. We demonstrated previously that Wnt-induced Lrp6 phosphorylation via glycogen synthase kinase 3 (Gsk3) initiates Wnt/beta-catenin signaling. Here we show that both Fz and Dvl functions are critical for Wnt-induced Lrp6 phosphorylation through Fz-Lrp6 interaction. We also show that axin, a key scaffolding protein in the Wnt pathway, is required for Lrp6 phosphorylation via its ability to recruit Gsk3, and inhibition of Gsk3 at the plasma membrane blocks Wnt/beta-catenin signaling. Our results suggest a model that upon Wnt-induced Fz-Lrp6 complex formation, Fz recruitment of Dvl in turn recruits the axin-Gsk3 complex, thereby promoting Lrp6 phosphorylation to initiate beta-catenin signaling. We discuss the dual roles of the axin-Gsk3 complex and signal amplification by Lrp6-axin interaction during Wnt/beta-catenin signaling.     

The effects of myostatin on adipogenic differentiation of human bone marrow-derived mesenchymal stem cells are mediated through cross-communication between Smad3 and Wnt/beta-catenin signaling pathways

The effects of myostatin on adipogenic differentiation are poorly understood, and the underlying mechanisms are unknown. We determined the effects of human recombinant myostatin protein on adipogenesis of bone marrow-derived human mesenchymal stem cells (hMSCs) and adipose tissue-derived preadipocytes. For both progenitor cell types, differentiation in the presence of myostatin caused a dose-dependent reduction of lipid accumulation and diminished incorporation of exogenous fatty acid into cellular lipids. Myostatin significantly down-regulated the expression of adipocyte markers PPARgamma, C/EBPalpha, leptin, and aP2, but not C/EBPbeta. Overexpression of PPARgamma, but not C/EBPbeta, blocked the inhibitory effects of myostatin on adipogenesis. Myostatin induced phosphorylation of Smad3 in hMSCs; knockdown of Smad3 by RNAi or inhibition of its upstream kinase by an Alk5 inhibitor blocked the inhibitory effect of myostatin on adipogenesis in hMSCs, implying an important role of Smad3 activation in this event. Furthermore, myostatin enhanced nuclear translocation of beta-catenin and formation of the Smad3-beta-catenin-TCF4 complex, together with the altered expression of a number of Wnt/beta-catenin pathway genes in hMSCs. The inhibitory effects of myostatin on adipogenesis were blocked by RNAi silencing of beta-catenin and diminished by overexpression of dominant-negative TCF4. The conclusion is that myostatin inhibited adipogenesis in human bone marrow-derived mesenchymal stem cells and preadipocytes. These effects were mediated, in part, by activation of Smad3 and cross-communication of the TGFbeta/Smad signal to Wnt/beta-catenin/TCF4 pathway, leading to down-regulation of PPARgamma.     

LDL receptor-related proteins 5 and 6 in Wnt/beta-catenin signaling: arrows point the way

Wnt signaling through the canonical beta-catenin pathway plays essential roles in development and disease. Low-density-lipoprotein receptor-related proteins 5 and 6 (Lrp5 and Lrp6) in vertebrates, and their Drosophila ortholog Arrow, are single-span transmembrane proteins that are indispensable for Wnt/beta-catenin signaling, and are likely to act as Wnt co-receptors. This review highlights recent progress and unresolved issues in understanding the function and regulation of Arrow/Lrp5/Lrp6 in Wnt signaling. We discuss Arrow/Lrp5/Lrp6 interactions with Wnt and the Frizzled family of Wnt receptors, and with the intracellular beta-catenin degradation apparatus. We also discuss the regulation of Lrp5/Lrp6 by other extracellular ligands, and LRP5 mutations associated with familial osteoporosis and other disorders.     

Calcineurin-independent inhibition of 3T3-L1 adipogenesis by Pasteurella multocida toxin: suppression of Notch1, stabilization of beta-catenin and pre-adipocyte factor 1

Pasteurella multocida toxin (PMT) is a potent mitogen and a specific activator of Gq-dependent signalling pathways. PMT impairs osteoblast differentiation and causes bone loss and fat reduction in vivo. We examined the effect of PMT on cell signalling pathways involved in 3T3-L1 adipocyte differentiation. We demonstrate that PMT treatment before or together with differentiation induction factors inhibits adipogenesis and prevents upregulation of important adipocyte markers - peroxisome-proliferator-activated receptor gamma (PPARgamma) and CAATT enhancer-binding protein alpha (C/EBPalpha). Moreover, PMT completely downregulates PPARgamma and C/EBPalpha expression in mature adipocytes. Differentiation of pre-adipocytes into adipocytes requires the suppression of pre-adipocyte factor 1 (Pref1) and Wnt signalling, along with the degradation of beta-catenin. PMT prevents downregulation of Pref1 and beta-catenin under differentiation-inducing conditions. In addition, PMT treatment downregulates expression of Notch1, a protein responsible for cell fate decision and implicated in regulation of adipogenesis in 3T3-L1 cells. PMT action on adipogenesis was not reversed by cyclosporin A, an inhibitor of Galphaq-PLC-calcium-dependent calcineurin activation. Our results reveal new pathways involved in PMT action on cellular physiology and differentiation. Our study further demonstrates that the effect of PMT on Pref1/PPARgamma/C/EBPalpha expression and adipogenesis does not occur just through activation of the Galphaq-calcium-calcineurin pathway, but involves Wnt/beta-catenin and Notch1 signalling pathways, two signalling pathways strongly linked to cancer predisposition, neurological and immunological dysfunctions, and fat and bone development.     

Role of Wnt10b and C/EBPalpha in spontaneous adipogenesis of 243 cells

This report examines the balance of positive and negative adipogenic factors in a line of immortalized 243 embryonic fibroblasts that undergo spontaneous preadipocyte differentiation. Control of adipogenesis reflects the interplay of factors that promote or inhibit expression of C/EBPalpha and PPARgamma. The 243 cells express C/EBPalpha early and at elevated levels compared to 3T3-F442A preadipocytes or adipocytes. Cell clones were derived from the heterogeneous 243 population for ability or inability to differentiate into adipocytes. Wnt10b, a secreted protein that inhibits adipogenesis, is expressed at high levels in cells with low adipogenic potential and is undetectable in preadipocytes that spontaneously differentiate. In contrast, C/EBPalpha is expressed at reduced levels in cells with low adipogenic potential, and is expressed at high levels in preadipocytes that spontaneously differentiate. These data are consistent with a model in which decreased Wnt10b, coupled with increased C/EBPalpha, results in induction of PPARgamma and spontaneous adipogenesis of 243 cells.     

Regulation of Wnt signaling during adipogenesis

We have identified Wnt10b as a potent inhibitor of adipogenesis that must be suppressed for preadipocytes to differentiate in vitro. Here, we demonstrate that a specific inhibitor of glycogen synthase kinase 3, CHIR 99021, mimics Wnt signaling in preadipocytes. CHIR 99021 stabilizes free cytosolic beta-catenin and inhibits adipogenesis by blocking induction of CCAAT/enhancer-binding protein alpha and peroxisome proliferator-activated receptor gamma. Preadipocyte differentiation is inhibited when 3T3-L1 cells are exposed to CHIR 99021 for any 24 h period during the first 3 days of adipogenesis. Consistent with this time frame of inhibition, expression of Wnt10b mRNA is suppressed upon induction of differentiation, with a 50% decline by 6 h and complete inhibition by 36 h. Of the agents used to induce differentiation, exposure of 3T3-L1 cells to methyl-isobutylxanthine or cAMP is sufficient to suppress expression of Wnt10b mRNA. Inhibition of adipogenesis by Wnt10b is likely mediated by Wnt receptors, Frizzled 1, 2, and/or 5, and co-receptors low density lipoprotein receptor-related proteins 5 and 6. These receptors, like Wnt10b, are highly expressed in preadipocytes and stromal vascular cells. Finally, we demonstrate that disruption of extracellular Wnt signaling by expression of secreted Frizzled related proteins causes spontaneous adipocyte conversion.     

Wnt signaling inhibits adipogenesis through beta-catenin-dependent and -independent mechanisms

Wnt signaling has been reported to block apoptosis and regulate differentiation of mesenchymal progenitors through inhibition of glycogen synthase kinase 3 and stabilization of beta-catenin. The effects of Wnt in preadipocytes may be mediated through Frizzled (Fz) 1 and/or Fz2 as these Wnt receptors are expressed in preadipocytes and their expression declines upon induction of differentiation. We ectopically expressed constitutively active chimeras between Wnt8 and Fz1 or Fz2 in preadipocytes and mesenchymal precursor cells. Our results indicated that activated Fz1 increases stability of beta-catenin, inhibits apoptosis, induces osteoblastogenesis, and inhibits adipogenesis. Although activated Fz2 does not influence apoptosis or osteoblastogenesis, it inhibits adipogenesis through a mechanism independent of beta-catenin. An important mediator of the beta-catenin-independent pathway appears to be calcineurin because inhibitors of this serine/threonine phosphatase partially rescue the block to adipogenesis caused by Wnt3a or activated Fz2. These data supported a model in which Wnt signaling inhibits adipogenesis through both beta-catenin-dependent and beta-catenin-independent mechanisms.     

Modulation of beta-catenin by cyclin-dependent kinase 6 in Wnt-stimulated cells

Beta-catenin is implicated in quite different cellular processes, which require a fine-tuned regulation of its function. Here we demonstrate that cyclin-dependent kinase 6 (CDK6), in association with cyclin D1 (CCND1), directly binds to beta-catenin. We showed that CCND1-CDK6 phosphorylates beta-catenin on serine 45 (S45). This phosphorylation creates a priming site for glycogen synthase kinase 3beta (GSK3beta) and is both necessary and sufficient to initiate the beta-catenin phosphorylation-degradation cascade. Moreover, co-immunoprecipitation assays using Wnt3a-conditioned medium reveals that while Wnt stimulation leads to the dissociation of beta-catenin from axin and casein kinase Ialpha (CKIalpha), Wnt treatment promotes an increase in CCND1 level and the association of beta-catenin with CCND1-CDK6. Furthermore, Wnt3a-stimulated cytosolic beta-catenin levels were higher in CDK6 knockout mouse embryonic fibroblasts (CDK6-/- MEFs) compared to wild-type MEFs. Thus, the CCND1-CDK6 complex is like to negatively regulate Wnt signaling by mediating beta-catenin phosphorylation and its subsequent degradation in Wnt-stimulated cells.     

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.     

C/EBPalpha-dependent induction of glutathione S-transferase zeta/maleylacetoacetate isomerase (GSTzeta/MAAI) expression during the differentiation of mouse fibroblasts into adipocytes

Western blot analysis of 3T3-L1 adipocyte proteins using an anti-C/EBPalpha antibody detected a 24kD polypeptide in addition to the expected 42 and 30kD isoforms of C/EBPalpha. Mass spectrometric sequencing of the protein following its purification by HPLC and preparative 2D gel electrophoresis identified it as glutathione S-transferase zeta/maleylacetoacetate isomerase (GSTzeta/MAAI). Expression of GSTzeta/MAAI mRNA and protein was induced during the terminal phase of adipogenesis in 3T3-L1 preadipocytes. Ectopic expression of PPARgamma2 in NIH-3T3 fibroblasts exposed to insulin and troglitazone-induced perilipin production, but was incapable of activating GSTzeta/MAAI unless C/EBPalpha was also expressed. Similarly, ectopic expression of C/EBPalpha in PPARgamma +/- or PPARgamma -/- MEFs demonstrated that the C/EBPalpha-dependent induction of GSTzeta/MAAI production was dependent on expression of endogenous PPARgamma. These data suggest a role for GSTzeta/MAAI in mature adipocytes that may be responsive to the thiazolidinedione class of insulin sensitizing PPARgamma ligands.     

敲低低密度脂蛋白受体相关蛋白1抑制C3H10T1/2多潜能干细胞定向成脂

C3H10T1/2多潜能干细胞成脂过程分为定向和分化两个阶段,骨形成蛋白4(BMP4)可以诱导其定向成前脂肪细胞．已有的研究表明,脂肪组织特异性敲除低密度脂蛋白受体相关蛋白1(Lrp1)的小鼠体重减轻,脂肪组织含量减少,揭示此基因对成脂具有重要作用．然而,目前尚不清楚Lrp1是否在成脂定向过程中发挥作用．采用小干扰RNA技术(RNAi),在体外水平研究低密度脂蛋白Lrp1对C3H10T1/2多潜能干细胞成脂定向的作用．分别在C3H10T1/2成脂的定向期和脂滴成熟期敲低Lrp1,通过显微镜下观察、油红O染色、Western blotting等实验证实,定向期而非脂滴成熟期敲低Lrp1显著抑制C3H10T1/2多潜能干细胞成脂．BMP4通过激活下游Smad1/5/8信号通路发挥作用,而敲低Lrp1显著抑制BMP4诱导的Smad1/5/8磷酸化．这些结果说明：敲低Lrp1通过下调Smad信号通路,抑制BMP4诱导的C3H10T1/2多潜能干细胞成脂定向．     

Lrp5 and Lrp6 play compensatory roles in mouse intestinal development

Low-density lipoprotein receptor-related proteins 5 and 6 (Lrp5 and Lrp6) are co-receptors of Wnt ligands and play important roles in Wnt/β-catenin signal transduction. Mice homozygous for a germline deletion of Lrp6 die at birth with several associated defects, while Lrp5-deficient mice are viable. Here, we conditionally deleted Lrp5 and/or Lrp6 in the mouse gut ((gut-/-)) by crossing mice carrying floxed alleles of Lrp5 and Lrp6 to a strain expressing Cre recombinase from the villin promoter (villin-Cre). The changes in morphology, differentiation, and Wnt signal transduction were validated using immunohistochemistry and other staining. Consistent with observations in mice carrying a homozygous germline deletion in Lrp5, intestinal development in Lrp5(gut-/-) mice was normal. In addition, mice homozygous for villin-Cre-induced deletion of Lrp6 (Lrp6(gut-/-)) were viable with apparently normal intestinal differentiation and function. However, mice homozygous for villin-Cre inactivated alleles of both genes (Lrp5(gut-/-) ; Lrp6(gut-/-)) died within 1 day of birth. Analysis of embryonic Lrp5(gut-/-); Lrp6(gut-/-) intestinal epithelium showed a progressive loss of cells, an absence of proliferation, and a premature differentiation of crypt stem/precursor cells; no notable change in differentiation was observed in the embryos lacking either gene alone. Further immunohistochemical studies showed that expression of the Wnt/β-catenin target, cyclin D1, was specifically reduced in the intestinal epithelium of Lrp5(gut-/-); Lrp6(gut-/-) embryos. Our data demonstrate that Lrp5 and Lrp6 play redundant roles in intestinal epithelium development, and that Lrp5/6 might regulate intestinal stem/precursor cell maintenance by regulating Wnt/β-catenin signaling.