C/EBPalpha regulation of the growth-arrest-associated gene gadd45.

CCAAT/enhancer-binding protein alpha (C/EBPalpha) is expressed in postmitotic, differentiated adipocytes and is required for adipose conversion of 3T3-L1 cells in culture. Temporal misexpression of C/EBPalpha in undifferentiated adipoblasts leads to mitotic growth arrest. We report here that growth arrest- and DNA damage-inducible gene 45 (gadd45) is preferentially expressed in differentiated 3T3-L1 adipocytes similar to phenotype-associated genes. Furthermore, C/EBPalpha transactivates a reporter plasmid containing 1.5 kb of the gadd45 promoter region. The proto-oncogene myc, which inhibits adipocyte differentiation, abrogates C/EBPalpha activation of gadd45. gadd45 is known to be a target of the tumor suppressor p53 in a G1 checkpoint activated by DNA damage. Immunoprecipitation of radiolabeled proteins with conformation-specific antibodies revealed that wild-type p53 is expressed throughout 3T3-L1 adipocyte development, including the postmitotic period characterized by the accumulation of gadd45 and C/EBPalpha. A stable 3T3-L1 subline was engineered to express a dominant negative p53, human p53(143ala). The p53(143ala) subline differentiated to adipocytes and showed appropriate developmental expression of gadd45. These findings suggest that postmitotic growth arrest is coupled to adipocyte differentiation via C/EBPalpha stimulation of growth arrest-associated and phenotype-associated genes.     

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.     

Inhibition of the terminal stages of adipocyte differentiation by cMyc

The nuclear oncoprotein Myc is a pivotal regulator of several important biological processes, including cellular proliferation, differentiation, and apoptosis. Deregulated Myc expression is incompatible with terminal differentiation in a variety of cell types, including adipocytes. To understand how Myc inhibits adipogenesis, we analyzed the effect of Myc on the expression of genes characteristic of distinct phases of the hormonally induced adipogenic differentiation program in 3T3-L1 preadipocytes. We show that the early regulators, C/EBPbeta and C/EBPdelta, are induced normally in response to hormone in 3T3-L1 preadipocytes constitutively expressing Myc, but that expression of the downstream regulators, C/EBPalpha and PPARgamma2, and later markers of differentiation is suppressed. These data demonstrate that Myc specifically inhibits the terminal stages of the adipogenic program and suggest that Myc may act by blocking C/EBPbeta- and C/EBPdelta-directed activation of C/EBPalpha and PPARgamma2 expression, although the precise molecular mechanism is not understood. Surprisingly, a serum component(s) could override the Myc-induced differentiation block, suggesting that the ability of a cell to undergo terminal differentiation is governed by the action of both positive and negative factors. Since differentiation and proliferation are mutually exclusive events, this has important implications since it may be possible to force malignant cells along a differentiation pathway, thereby curbing their proliferative potential.     

Coculture with BJ fibroblast cells inhibits the adipogenesis and lipogenesis in 3T3-L1 cells

Mouse or human fibroblasts are commonly used as feeder cells to prevent differentiation in stem or primary cell culture. In the present study, we addressed whether fibroblasts can affect the differentiation of adipocytes. We found that the differentiation of 3T3-L1 preadipocytes was strongly suppressed when the cells were cocultured with human fibroblast (BJ) cells. BrdU incorporation analysis indicated that mitotic clonal expansion, an early event required for 3T3-L1 cell adipogenesis, was not affected by BJ cells. The 3T3-L1 cell expression levels of peroxisome proliferator-activated receptor γ2, CCAAT/enhancer-binding protein alpha (C/EBPα), sterol regulatory element binding protein-1c, and Krüppel-like factor 15, but not those of C/EBPβ or C/EBPδ, were decreased by coculture with BJ cells. When mature 3T3-L1 adipocytes were cocultured with BJ cells, their lipid contents were significantly reduced, with decreased fatty acid synthase expression and increased phosphorylated form of acetyl-CoA carboxylase 1. Our data indicate that coculture with BJ fibroblast cells inhibits the adipogenesis of 3T3-L1 preadipocytes and decreases the lipogenesis of mature 3T3-L1 adipocytes.     

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 reverses TGF-beta-mediated inhibition of primary rat preadipocyte differentiation

Dexamethasone and transforming growth factor-beta (TGF-beta) show contrary effects on differentiation of adipocytes. Dexamethasone stimulates adipocyte differentiation whereas TGF-beta inhibits it. In the present study, we investigated whether dexamethasone could reverse the TGF-beta-mediated inhibition of preadipocyte differentiation. Primary rat preadipocytes, obtained from Sprague-Dawley rats, were pretreated with dexamethasone in the presence or absence of TGF-beta, prior to the induction of differentiation. Co-treatment of dexamethasone and TGF-beta before inducing differentiation reversed the TGF-beta-mediated inhibition of preadipocyte differentiation. In order to elucidate the mechanism by which dexamethasone reversed the effect of TGF-beta on the inhibition of preadipocyte differentiation, the expression of CCAAT/enhancer binding protein-alpha (C/EBPalpha) and peroxisome proliferator-activated receptor gamma (PPARgamma) was examined. Dexamethasone increased C/EBPalpha and PPARgamma expression in the absence of TGF-beta and also recovered the TGF-beta-mediated suppression of C/EBPalpha expression in preadipocytes. Its effect was sustained in differentiated adipocytes as well. However, those effects were not observed in 3T3-L1 preadipocytes or differentiated adipocytes. These results indicate that dexamethasone reverses the TGF-beta-mediated suppression of adipocyte differentiation by regulating the expression of C/EBPalpha and PPARgamma, which is dependent on the cellular context.