HORMONAL CONTROL OF ADIPOSE GENE EXPRESSION

描述（由申请人提供）： 长期目标：表征脂肪细胞分化程序的事件/机制，并在生理背景下验证这些事件。目前的目标是了解"有丝分裂克隆扩增"（MCE），这是脂肪形成所必需的。充分表征的3T3-L1前脂肪细胞-）脂肪细胞分化系统将用作主要模型。野生型和突变/敲除小鼠胚胎成纤维细胞（MEF）和前脂肪细胞"体内植入"系统将在生理背景下测试离体结果。具体目的：确定：1）C/EBP β/δ和钙蛋白酶的作用：C/EBP β/δ在程序早期缺乏DNA结合活性。经过一段长时间的滞后，这一功能的获得伴随着磷酸化和抑制约束的释放。细胞同步进入MCE的S期。一旦C/EBP β/δ获得DNA结合活性，它们转录激活C/EBP α和PPARgamma基因，其转录激活产生脂肪细胞表型的基因。将鉴定C/EBP β/δ上的相关磷酸化位点，鉴定负责的激酶和相互作用伴侣，并确定对C/EBP β/δ功能的影响。MCE将沿着G1-S检查点的会聚事件进行表征，这些事件负责MCE的进展和分化。其中之一是钙蛋白酶的激活，我们发现它在启动p27/KIP1周转中起着至关重要的作用，这是Cdk2-cyclinA激活/进入S期的重要事件。将探索钙蛋白酶激活的触发机制及其在转录激活Cdk2基因（脂肪形成MCE的独特特征）中的可能作用，以及2）CUP/AP-2 α和Spl的作用：先前的研究确定了C/EBP（基因启动子）中的抑制性调节元件，即CUP/AP-2 α和Spl。这些阻遏物的下调伴随着MCE的发作而发生，就在C/EBPalpha基因的转录激活之前。将确定这些阻遏物被下调的机制以及强制表达和"敲低"对MCE/分化和/或去分化的影响。本实验室先前鉴定的32kDa CUP/AP-2 α相互作用蛋白p32将被表征并确定其作用。

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.