Hormone induction of growth-arrested preadipocytes triggers mitotic clonal expansion followed by expression of CCAAT/enhancer- binding protein (C/EBP)α and differentiation into adipocytes. The order of these events is critical because C/EBPα is antimitotic and its expression prematurely would block the mitotic clonal expansion required for differentiation. C/EBPβ, a transcriptional activator of the C/EBPα gene, is expressed early in the differentiation program, but lacks DNA-binding activity and fails to localize to centromeres until preadipocytes traverse the G1-S checkpoint of mitotic clonal expansion. Evidence is presented that dominantnegative CHOP-10 expressed by growth-arrested preadipocytes transiently sequesters C/EBPβ by heterodimerization. As preadipocytes reach S phase, CHOP-10 is down-regulated, apparently releasing C/EBPβ from inhibitory constraint and allowing transactivation of the C/EBPα gene. In support of these findings, up-regulation of CHOP-10 with the protease inhibitor N-acetyl-Leu-Leu-norleucinal prevents activation of C/EBPβ, expression of C/EBPα, and adipogenesis.

Expression of C/EBPα is required for differentiation of 3T3-L1 preadipocytes into adipocytes. Previous investigations indicated that transcription of the C/EBPα gene is sequentially activated during differentiation, initially by C/EBPβ and C/EBPδ and later by C/EBPα (autoactivation). These events are mediated by a C/EBP regulatory element in the promoter of the C/EBPα gene. This article presents evidence that members of the Sp family, notably Sp1, act repressively on the C/EBPα promoter prior to the induction of differentiation. Sp1 was shown to bind to a GC box at the 5' end of the C/EBP regulatory element in the C/EBPα promoter and, in so doing, to competitively prevent binding to and transactivation of the promoter by the C/EBPs. One of the differentiation inducers methylisobutylxanthine (a cAMP phosphodiesterase inhibitor) or Forskolin, both of which increase the cellular cAMP level, causes down-regulation of Sp1. This decrease in Sp1 level early in the differentiation program appears to facilitate access of C/EBPβ and/or C/EBPδ to the C/EBP regulatory element and, thereby, derepression of the C/EBPα gene.

ABSTRACT During adipocyte differentiation, the expression of CyEBPα is activated, which in turn serves to transcriptionally activate numerous adipocyte genes. A previous search for cis elements that regulate transcription of the CyEBPagene led to the identification of a potential repressive element within the proximal 5' flanking region of the gene. Nuclear extracts from 3T3-L1 preadipocytes, but not adipocytes, were found to contain a factor, CUP (C/EBPα undifferentiated protein), that binds to this site (the CUP-1 site). In the present investigation, we show that C/EBPα promoterluciferase constructs containing both the proximal 5' flanking and the entire 5' untranslated regions of the gene exhibit an expression pattern during adipocyte differentiation comparable to that of the endogenous C/EBPα gene. Mutation of the CUP-1 site in these constructs had little effect on reporter gene expression; however, when this mutation was combined with deletion of the 5' untranslated region, reporter gene expression by preadipocytes was dramatically up-regulated. Consistent with this finding, a second CUP binding site (the CUP-2 site) was identified in the 5' untranslated region. Although mutation of either CUP element in constructs containing both the 5' flanking and 5' untranslated region had little effect on reporter gene transcription, mutation of both CUP elements markedly activated transcription. Thus, it appears that dual CUP regulatory elements repress transcription of the C/EBPa gene prior to induction of the adipocyte differentiation program.

When induced to differentiate, growth-arrested 3T3-L1 preadipocytes synchronously reenter the cell cycle and undergo mitotic clonal expansion (MCE) followed by expression of genes that produce the adipocyte phenotype. The preadipocytes traverse the G1/S checkpoint synchronously as evidenced by the expression activation of cdk2-cyclin-E/A, turnover of p27 kip1, hyperphosphorylation of Rb, translocation of cyclin D1 from nuclei to cytoplasm and GSK-3β from cytoplasm to nuclei, and incorporation of [3H] thymidine into DNA. As the cells cross the G1/S checkpoint, C/EBPβ acquires DNA-binding activity, initiating a cascade of transcriptional activation that culminates in the expression of adipocyte proteins. The mitogen-activated protein kinase extracellular signal-regulated kinase kinase (MEK) inhibitor PD98059 delays, but does not block, MCE and differentiation, the extent of the delay causing a comparable delay in the expression of cell-cycle markers, MCE, and adipogenesis. The more potent and specific MEK inhibitor UO126 and the cyclin-dependent kinase inhibitor roscovitine, which inhibit the cell cycle at different points, block MCE, expression of cell cycle and adipocyte markers, as well as adipogenesis. These results show that MCE is a prerequisite for differentiation of 3T3-L1 preadipocytes into adipocytes.

Kaposi sarcoma-associated herpesvirus (KSHV) is an oncogenic DNA virus that causes Kaposi sarcoma and AIDS-related primary effusion lymphoma (PEL). Here we show that KSHV lytic cycle replication in PEL cells induces G1 cell cycle arrest, presumably to facilitate the progression of viral DNA replication. Expression of a KSHV-encoded early lytic protein referred to as RAP or K8 is induced within 12-2h after the onset of lytic cycle induction in host PEL cells, and coincides with increased levels of both the endogenous C/EBPα and p21CIP-1 proteins in the nucleus of the same cells. The KSHV RAP protein binds to C/EBPα in vitro and stimulates C/EBPα-induced expression from both the C/EBPα and p21 promoters in cotransfected cells. A recombinant adenovirus expressing the RAP protein induced the expression of both the C/EBPα and p21 proteins in primary human fibroblasts, and flow cytometric analysis revealed a dramatic inhibition of G1 to S cell cycle progression in the same cells. All of these effects were abolished in cells that lack C/EBPα or by deletion of the basic leucine zipper region in RAP that interacts with C/EBPα. Therefore, C/EBPα is essential for the p21-mediated inhibition of G1 to S-phase progression by RAP in KSHV-infected host cells.