Peroxisome proliferator activated receptor gamma, CCAAT/enhancer-binding protein alpha, and cell cycle status regulate the commitment to adipocyte differentiation.
ABSTRACT Terminal differentiation of stem cells is characterized by cessation of cell proliferation as well as changes in cell morphology associated with the differentiated state. For adipocyte differentiation, independent lines of evidence show that the transcription factors peroxisome proliferator activated receptor gamma (PPARgamma) and CCAAT/enhancer-binding protein alpha (C/EBPalpha) as well as the tumor suppressor retinoblastoma (Rb) protein are essential. How these proteins promote adipocyte conversion and how they function cooperatively during the differentiation process remain unclear. We have used retinoic acid (RA) inhibition of adipogenesis to investigate these issues. RA blocked adipogenesis of 3T3-L1 cells induced to differentiate by ectopic expression of PPARgamma and C/EBPalpha independently or together. However, under these circumstances RA was only effective at preventing adipogenesis when added prior to confluence, suggesting that factors involved in regulation of the cell cycle might play a role in establishing the commitment state of adipogenesis that is insensitive to RA. During differentiation of wild type 3T3 L1 preadipocytes, we found that Rb protein is hyperphosphorylated early in adipogenesis, corresponding to previously quiescent cells re-entering the cell cycle, and later becomes hypophosphorylated. The data suggest that, together with the coexpression of PPARgamma and C/EBPalpha, permanent exit from the cell cycle establishes the irreversible commitment to adipocyte differentiation.
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ABSTRACT: The aim of this study was to identify the adipocyte-specific gene expression patterns in chorion-derived mesenchymal stem cells during adipogenic differentiation. Chorionic cells were isolated from the third trimester chorions from human placenta at birth and identified morphologically and by fluorescence-activated cell sorting analysis. After inducing adipogenic differentiation for 28 days, cells at days 3, 10, 21 and 28 were analyzed by Oil red O staining and RNA extraction in order to assess the expression levels of adipocyte marker genes, including CCAAT-enhancer binding protein α (C/EBPα), peroxisome proliferator- activated receptor γ (PPARγ), fatty acid binding protein 4 (FABP4) and Glycerol-3-phosphate dehydrogenase (GPD2). Cells not induced for differentiation were compared with the induced cells as a control group. Chorion-derived cells showed the same pattern as fibroblasts, and expressed CD73, CD105, and CD166 antigens, but not CD45, CD34, and HLA-DR antigens. On day 3 after differentiation, cells began to stain positively upon Oil red O staining, and continuously increased in lipid granules for 4 weeks. The expression level of C/EBPα increased 4.6 fold on day 3 after induction, and continued to increase for 4 weeks. PPARγ was expressed at a maximum of 2.9 fold on day 21. FABP4 and GPD2 were significantly expressed at 4.7- and 3.0-fold, respectively, on day 21, compared to controls, and further increased thereafter. Human chorion-derived mesenchymal stem cells exhibited the sequential expression pattern of adipocyte marker genes during differentiation, corresponding to adipogenesis.Yonsei medical journal 09/2012; 53(5):1036-44. · 0.77 Impact Factor
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ABSTRACT: The development of adipocytes from their progenitor cells requires the action of growth factors signaling to transcription factors to induce the expression of adipogenic proteins leading to the accumulation of lipid droplets, induction of glucose transport, and secretion of adipokines signaling metabolic events throughout the body. Murine 3T3-L1 pre-adipocytes sequentially express all the proteins necessary to become mature adipocytes throughout an 8-10 day process initiated by a cocktail of hormones. We examined the role of Clk/STY or Clk1, a cdc2-like kinase, in adipogenesis since it is known to be regulated by Akt, a pivotal kinase in development. Inhibition of Clk1 by a specific inhibitor, TG003, blocked alternative splicing of PKCβII and expression of PPARγ1 and PPARγ2. SiRNA depletion of Clk1 resulted in early expression of PKCβII and sustained PKCβI expression. Since Clk1 is a preferred Akt substrate, required for phosphorylation of splicing factors, mutation of Clk1 Akt phosphorylation sites was undertaken. Akt sites on Clk1 are in the serine/arginine-rich domain and not the kinase domain. Mutation of single and multiple sites resulted in dysregulation of PKCβII, PKCβI, and PPARγ1&2 expression. Additionally, adipogenesis was blocked as assessed by Oil Red O staining, adiponectin, and Glut1 and 4 expression. Immunofluorescence microscopy revealed that Clk1 triple mutant cDNA, transfected into pre-adipocytes, resulted in excluding SRp40 (SFSR6) from co-localizing to the nucleus with PFS, a perispeckle specific protein. This study demonstrates the role of Akt and Clk1 kinases in the early differentiation of 3T3-L1 cells to adipocytes.PLoS ONE 01/2013; 8(1):e53268. · 3.73 Impact Factor
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ABSTRACT: Brown adipocytes are a primary site of energy expenditure and reside not only in classical brown adipose tissue but can also be found in white adipose tissue. Here we show that microRNA 155 is enriched in brown adipose tissue and is highly expressed in proliferating brown preadipocytes but declines after induction of differentiation. Interestingly, microRNA 155 and its target, the adipogenic transcription factor CCAAT/enhancer-binding protein β, form a bistable feedback loop integrating hormonal signals that regulate proliferation or differentiation. Inhibition of microRNA 155 enhances brown adipocyte differentiation and induces a brown adipocyte-like phenotype ('browning') in white adipocytes. Consequently, microRNA 155-deficient mice exhibit increased brown adipose tissue function and 'browning' of white fat tissue. In contrast, transgenic overexpression of microRNA 155 in mice causes a reduction of brown adipose tissue mass and impairment of brown adipose tissue function. These data demonstrate that the bistable loop involving microRNA 155 and CCAAT/enhancer-binding protein β regulates brown lineage commitment, thereby, controlling the development of brown and beige fat cells.Nature Communications 04/2013; 4:1769. · 10.02 Impact Factor