Growth-arrested 3T3-L1 preadipocytes rapidly express CCAAT/enhancer-binding protein-␤ (C/EBP␤) upon hormonal induction of differentiation. However, the DNA binding activity of C/EBP␤ is not activated until the cells synchronously reenter S phase during the mitotic clonal expansion (MCE) phase of differentiation. In this period, C/EBP␤ is sequentially phosphorylated by MAPK and glycogen synthase kinase-3␤, inducing C/EBP␤ DNA binding activity and transcription of its target genes. Because the DNA binding activity of C/EBP␤ is further enhanced by oxidation in vitro, we investigated how redox state affects C/EBP␤ DNA binding and MCE during adipogenesis. When 3T3-L1 cells were treated with H 2 O 2 and hormonal stimuli, differentiation was accelerated with increased expression of peroxisome proliferator-activated receptor ␥. Interestingly, cell cycle progression (S to G 2 /M phase) was markedly enhanced by H 2 O 2 , whereas antioxidants caused an S phase arrest during the MCE. H 2 O 2 treatment resulted in the early appearance of a punctate pattern observed by immunofluorescent staining of C/EBP␤, which is a hallmark for C/EBP␤ binding to regulatory elements, whereas a short antioxidant treatment rapidly dispersed the centromeric localization of C/EBP␤. Consistently, reactive oxygen species production was increased during 3T3-L1 differentiation. Our results indicate that redox-induced C/EBP␤ DNA binding activity, along with the dual phosphorylation of C/EBP␤, is required for the MCE and terminal differentiation of adipocytes.

CCAAT enhancer-binding protein (C͞EBP)␤, C͞EBP␣, and peroxisome proliferator activated receptor (PPAR)␥ act in a cascade where C͞EBP␤ activates expression of C͞EBP␣ and PPAR␥, which then function as pleiotropic activators of genes that produce the adipocyte phenotype. When growth-arrested 3T3-L1 preadipocytes are induced to differentiate, C͞EBP␤ is rapidly expressed but still lacks DNA-binding activity. After a long (14-hour) lag, glycogen synthase kinase 3␤ enters the nucleus, which correlates with hyperphosphorylation of C͞EBP␤ and acquisition of DNA-binding activity. Concurrently, 3T3-L1 preadipocytes synchronously enter S phase and undergo mitotic clonal expansion, a prerequisite for terminal differentiation. Ex vivo and in vitro experiments with C͞EBP␤ show that phosphorylation of Thr-188 by mitogen-activating protein kinase ''primes'' C͞EBP␤ for subsequent phosphorylation on Ser-184 and Thr-179 by glycogen synthase kinase 3␤, acquisition of DNA-binding function, and transactivation of the C͞EBP␣ and PPAR␥ genes. The delayed transactivation of the C͞EBP␣ and PPAR␥ genes by C͞EBP␤ appears necessary to allow mitotic clonal expansion, which would otherwise be prevented, because C͞EBP␣ and PPAR␥ are antimitotic.3T3-L1 preadipocyte ͉ cell cycle ͉ differentiation ͉ mitotic clonal expansion C CAAT enhancer-binding protein ␤ (C͞EBP␤) is expressed early in the adipocyte differentiation program, first initiating mitotic clonal expansion (MCE) (1, 2) and later activating expression of C͞EBP␣ and peroxisome proliferator-activated receptor ␥ (PPAR␥) (3-6), pleiotropic activators of adipocyte genes (3,4,7,8). Both MCE and expression of C͞EBP␣ and PPAR␥ are required for differentiation (1, 2, 9). When treated with differentiation inducers, growth-arrested 3T3-L1 preadipocytes synchronously reenter the cell cycle, undergo approximately two rounds of mitosis, then exit the cell cycle and enter the terminal stages of differentiation. Transcriptional activation of the C͞EBP␣ and PPAR␥ genes is induced by the interaction of C͞EBP␤ with C͞EBP regulatory elements in these gene promoters (3-6).Although expression of C͞EBP␤ occurs within 2 h of induction of differentiation, acquisition of DNA-binding activity and thus transcription of the C͞EBP␣ and PPAR␥ genes are delayed (10). Acquisition of DNA-binding activity begins after a long lag (Ϸ14 h), concurrent with the entry of S phase at the onset of MCE and transcription of the C͞EBP␣ and PPAR␥ genes (ref. 10 and Fig. 1). This lag appears necessary, because C͞EBP␣ and PPAR␥ are antimitotic (11-15), and their premature expression would otherwise prevent the MCE required for differentiation. To elucidate the mechanism by which C͞EBP␤ acquires DNAbinding activity, we considered the possibility that covalent modification of C͞EBP␤ occurs during this time window.Several lines of evidence indicated that C͞EBP␤ can be phosphorylated in vitro by a variety of kinases, including PKA (16), PKC (16), mitogen-activated protein kinase (MAPK) (17), and Ca 2ϩ -calmodulin-dependent kinase II (18). ...

Previous studies showed that exposure of C3H10T1͞2 stem cells to bone morphogenetic protein-4 (BMP-4) produced cells that convert into adipocytes at high frequency when treated with differentiation inducers. In the present investigation, an independent approach shows that BMP-4 is required for stable commitment of pluripotent stem cells to the adipocyte lineage. Exposure of proliferating 10T1͞2 stem cells to 5-azacytidine, a potent DNA methylation inhibitor, gave rise to a subpopulation of cells that can be cloned and that have the capacity to undergo conversion into adipocytes upon treatment with terminal differentiation inducers. 5-azacytidine ͉ adipogenesis ͉ determination ͉ mesenchymal stem cell ͉ preadipocyte

The adipose tissue has important secretory and endocrine functions in humans. The regulation of adipocyte differentiation has been actively pursued using transcriptomic methods over the last several years. Quantitative proteomics has emerged as a promising approach to obtain temporal profiles of biological processes such as differentiation. Stable isotope labeling with amino acids in cell culture (SILAC) is a simple and robust method for labeling proteins in vivo. Here, we describe the development and application of a five-plex SILAC experiment using four different heavy stable isotopic forms of arginine to study the nuclear proteome and the secretome during the course of adipocyte differentiation. Tandem mass spectrometry analysis using a quadrupole time-of-flight instrument resulted in identification of a total 882 proteins from these two proteomes. Of these proteins, 427 were identified on the basis of one or more arginine containing peptides that allowed quantitation. In addition to previously reported molecules that are differentially expressed during the process of adipogenesis (e.g. adiponectin and lipoprotein lipase), we identified several proteins whose differential expression during adipocyte differentiation has not been documented previously. For example, THO complex 4, a context-dependent transcriptional activator in the T-cell receptor alpha enhancer complex, showed highest expression at middle stage of adipogenesis while SNF2 alpha, a chromatin remodeling protein, was downregulated upon initiation of adipogenesis and remained so during subsequent time points. This study using a 5-plex SILAC to investigate dynamics illustrates the power of this approach to identify differentially expressed proteins in a temporal fashion.