LONG-TERM GOAL: to characterize the events/mechanisms of the adipocyte differentiation program and verify these events in physiological context. The immediate goal is to understand """"""""mitotic clonal expansion"""""""" (MCE), which is required for adipogenesis. The well-characterized 3T3-L1 preadipocyte --) adipocyte differentiation system will serve as primary model. Wild-type and mutant/knockout mouse embryo fibroblasts (MEFs) and a preadipocyte """"""""in vivo implantation"""""""" system will test ex vivo results in physiological context.
SPECIFIC AIMS : to determine the: 1) roles of C/EBPbeta/delta and Calpain: C/EBPbeta/delta lack DNA binding activity early in the program. After a long lag this function is acquired concomitant with phosphorylation and release from inhibitory constraint. As the cells synchronously enter S-phase of MCE. Once C/EBPbeta/delta acquire DNA binding activity, they transcriptionally activate the C/EBPalpha and PPARgamma genes which transcriptionally activate genes that produce the adipocyte phenotype. Relevant phosphorylation sites on C/EBPbeta/delta will be identified, the responsible kinase(s) and interaction partners identified and effects on C/EBPbeta/delta function determined. MCE will be characterized along with converging events at the G1-S checkpoint that are responsible for progression of MCE and differentiation. Among these is the activation of Calpain which we showed plays a crucial role in initiating p27/KIP1 turnover, an essential event in the activation of Cdk2-cyclinA/entry of S-phase. The mechanism(s) by which calpain activation is triggered and its possible role in the transcriptionally activating the Cdk2 gene, a unique feature of adipogenic MCE, will be explored, and 2) roles of CUP/AP-2alpha and Spl: Previous studies identified repressive regulatory elements for, i.e. CUP/AP-2(_and Spl, in the C/EBP( gene promoter. Down-regulation of these repressors occurs concomitant with the onset of MCE just prior to the transcriptional activation of the C/EBPalpha gene. The mechanisms by which these repressors are down-regulated and the effects of forced expression and """"""""knockdown"""""""" on MCE/differentiation and/or de-differentiation will be determined. The 32kDa CUP/AP-2alpha- interacting protein, p32, previously identified in our lab, will be characterized and its role determined.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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Physiological Chemistry Study Section (PC)
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Haft, Carol R
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Johns Hopkins University
Schools of Medicine
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