Wnt/Lrp/beta-catenin signaling suppresses adipogenesis by inhibiting mutual activation of PPARgamma and C/EBPalpha

Wnt/beta-catenin signaling has been implicated in repressing adipogenesis. Several lines of evidence show that the possible mechanism is blockade of PPARgamma induction. However, the precise mechanisms remain to be elucidated. In this study, we demonstrated that Wnt3a conditioned medium suppresses C/EBPbeta/delta-induced adipogenesis of 3T3-L1 cells by inhibiting PPARgamma induction. In addition, the mutual activation of PPARgamma and C/EBPalpha was also repressed in the presence of Wnt3a. To further investigate the role of the canonical Wnt pathway in adipogenesis, we used mouse embryonic fibroblasts (MEFs) isolated from Lrp6-deficient embryos. Contrary to wild-type MEFs, Lrp6-deficient MEFs showed spontaneous adipogenesis and escaped the suppressive effect of exogenous Wnt3a. These findings suggest a critical role of Wnt/Lrp6/beta-catenin signaling in adipogenesis and cell fate decision of mesenchymal stem cells.     

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.     

猪脂肪组织发育过程中Wnt/β-catenin信号通路相关基因及脂肪转录因子的时序表达

为研究Wnt/b-catenin信号通路对猪脂肪组织发育的影响, 并探讨其可能的作用机制, 以1~120日龄长白猪脂肪组织为试验材料, 采用SQ RT-PCR法检测Wnt信号通路中相关基因β-catenin、GSK3β、Fz1及主要的脂肪转录因子PPARγ、C/EBPα 和分化早期标志基因LPL mRNA的表达变化; 石蜡切片免疫组化方法定性检测β-catenin在脂肪组织发育中的时序表达变化。SQ RT-PCR结果显示, β-catenin mRNA在猪出生第1天有高水平表达, 随着个体日龄的增长, 其表达量逐渐降低, 60日龄后维持在一个较低水平。GSK3β和Fz1 mRNA的表达量也伴随脂肪组织的发育而逐渐降低。而LPL、PPARγ和C/EBPα的 mRNA表达量随着个体日龄的增长而逐渐升高, 60日龄后仍保持高水平的表达。石蜡切片免疫组化结果显示, 随着脂肪组织的发育, β-catenin蛋白的表达也随之降低, 并且β-catenin蛋白的表达部位逐渐由细胞核和细胞质共表达变为只在细胞质中表达。上述基因表达的时序变化规律提示, β-catenin在维持前体脂肪细胞的未分化状态, 抑制脂肪组织发育中具有重要作用, 其作用机制可能是通过对脂肪细胞转录因子PPARγ、C/EBPα及分化早期标志基因LPL mRNA的调控来进行的。     

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.     

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.     

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.     

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.     

Molecular mechanism of 1,25-dihydroxyvitamin D3 inhibition of adipogenesis in 3T3-L1 cells

We have investigated the molecular mechanism whereby 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] inhibits adipogenesis in vitro. 1,25(OH)2D3 blocks 3T3-L1 cell differentiation into adipocytes in a dose-dependent manner; however, the inhibition is ineffective 24-48 h after the differentiation is initiated, suggesting that 1,25(OH)2D3 inhibits only the early events of the adipogenic program. Treatment of 3T3-L1 cells with 1,25(OH)2D3 does not block the mitotic clonal expansion or C/EBPbeta induction; rather, 1,25(OH)2D3 blocks the expression of C/EBPalpha, peroxisome proliferator-activated receptor-gamma (PPARgamma), sterol regulatory element-binding protein-1, and other downstream adipocyte markers. The inhibition by 1,25(OH)2D3 is reversible, since removal of 1,25(OH)2D3 from the medium restores the adipogenic process with only a temporal delay. Interestingly, although the vitamin D receptor (VDR) protein is barely detectable in 3T3-L1 preadipocytes, its levels are dramatically increased during the early phase of adipogenesis, peaking at 4-8 h and subsiding afterward throughout the rest of the differentiation program; 1,25(OH)2D3 treatment appears to stabilize the VDR protein levels. Consistently, adenovirus-mediated overexpression of human (h) VDR in 3T3-L1 cells completely blocks the adipogenic program, confirming that VDR is inhibitory. Inhibition of adipocyte differentiation by 1,25(OH)2D3 is ameliorated by troglitazone, a specific PPARgamma antagonist; conversely, hVDR partially suppresses the transacting activity of PPARgamma but not of C/EBPbeta or C/EBPalpha. Moreover, 1,25(OH)2D3 markedly suppresses C/EBPalpha and PPARgamma mRNA levels in mouse epididymal fat tissue culture. Taken together, these data indicate that the blockade of 3T3-L1 cell differentiation by 1,25(OH)2D3 occurs at the postclonal expansion stages and involves direct suppression of C/EBPalpha and PPARgamma upregulation, antagonization of PPARgamma activity, and stabilization of the inhibitory VDR protein.     

Dexamethasone shifts bone marrow stromal cells from osteoblasts to adipocytes by C/EBPalpha promoter methylation

Dexamethasone (Dex)-induced osteoporosis has been described as the most severe side effect in long-term glucocorticoid therapy. The decreased bone mass and the increased marrow fat suggest that Dex possibly shifts the differentiation of bone marrow stromal cells (BMSCs) to favor adipocyte over osteoblast, but the underlying mechanisms are still unknown. In this paper, we established a Dex-induced osteoporotic mouse model, and found that BMSCs from Dex-treated mice are more likely to differentiate into adipocyte than those from control mice, even under the induction of bone morphogenetic protein-2 (BMP2). We also discovered both in vitro and in vivo that the expression level of adipocyte regulator CCAAT/enhancer-binding protein alpha (C/EBPalpha) is significantly upregulated in Dex-induced osteoporotic BMSCs during osteoblastogenesis by a mechanism that involves inhibited DNA hypermethylation of its promoter. Knockdown of C/EBPalpha in Dex-induced osteoporotic cells rescues their differentiation potential, suggesting that Dex shifts BMSC differentiation by inhibiting C/EBPalpha promoter methylation and upregulating its expression level. We further found that the Wnt/beta-catenin pathway is involved in Dex-induced osteoporosis and C/EBPalpha promoter methylation, and its activation by LiCl rescues the effect of Dex on C/EBPalpha promoter methylation and osteoblast/adipocyte balance. This study revealed the C/EBPalpha promoter methylation mechanism and evaluated the function of Wnt/beta-catenin pathway in Dex-induced osteoporosis, providing a useful therapeutic target for this type of osteoporosis.