HORMONAL CONTROL OF ADIPOSE GENE EXPRESSION

描述(由申请人提供)： 长期目标：描述脂肪细胞分化计划的事件/机制，并在生理环境中验证这些事件。近期的目标是了解脂肪形成所需的“有丝分裂克隆扩张”(MCE)。成熟的3T3-L1前脂肪细胞--)脂肪细胞分化系统将作为主要模型。野生型和突变/基因敲除的小鼠胚胎成纤维细胞(MEF)和前脂肪细胞“体内植入”系统将在生理环境下测试体外结果。具体目的：确定：1)C/EBPbeta/Delta和calain的作用：C/EBPbeta/Delta在项目早期缺乏DNA结合活性。在长时间滞后后，这个功能是伴随着磷酸化而获得的，并从抑制约束中释放出来。随着细胞同步进入细胞周期的S期。一旦C/EBPbeta/Delta获得DNA结合活性，它们就转录激活C/EBPalpha和PPARGamma基因，这两个基因转录激活产生脂肪细胞表型的基因。将确定C/EBPbeta/Delta上的相关磷酸化位点，确定负责的激酶(S)和相互作用伙伴，并确定对C/EBPbeta/Delta功能的影响。在G1-S检查点的会聚事件将被描述为MCE的特征，这些事件负责MCE的进展和分化。其中，钙蛋白的激活在启动p27/KIP1的转换中起着至关重要的作用，而p27/KIP1的转换是CDK2-周期蛋白A激活/进入S时相的重要事件。我们将探讨Calain被激活的机制(S)及其在转录激活CDK2基因中的可能作用，这是成脂MCE的一个独特特征，以及2)CUP/AP-2α和Sp1的作用：先前的研究发现，C/EBP(基因启动子)中的抑制性调控元件，即CUP/AP-2(和Sp1)。在C/EBPalpha基因转录激活之前，这些抑制子的下调伴随着MCE的发生。这些抑制子下调的机制以及强迫表达和“敲除”对MCE/分化和/或去分化的影响将被确定。我们实验室以前发现的32 kDa CUP/AP-2α相互作用蛋白p32将被鉴定并确定其作用。

DESCRIPTION (provided by applicant): 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.