Identification of novel modulators of adipocyte differentiation

核受体PPARγ被认为是脂肪细胞分化的中央调节因子。通过天然和人工合成的激动剂激活这种转录因子，可以诱导与脂肪分化和胰岛素增敏相关的基因。尽管自90年代初发现PPARGamma依赖配体的转录激活机制以来，人们对该因子的转录激活机制进行了广泛的研究，但PPARGamma的配体非依赖功能尚未完全阐明。该实验室的主要重点之一是识别能够调节PPARGamma的非配体活性的新分子和途径。为了确定新的PPAR伽马调制器，我们采取了以下方法： 1)我们分析了在脂肪细胞分化过程中似乎表达的几个候选辅助因子，并评估了它们作为PPAR-γ潜在转录辅助因子的能力； 2)我们已经构建了几个表达PPARGamma不同结构域的PPARGamma-GST融合构建体。这些在细菌中产生的融合蛋白已被用于生化分析，以纯化潜在的新型N-末端相互作用。对从前脂肪细胞和完全分化的脂肪细胞中提取的核提取物进行了分析，并用质谱仪鉴定了新的相互作用蛋白。 除了新的PPAR-伽马相互作用分子的特征外，我们还专注于新的信号通路的特征，这些信号通路可以增强对地塞米松刺激的分化。为此，我们分析了几种激酶在脂肪分化不同阶段的表达模式，并确定了几种可能参与脂肪形成过程调节的潜在激酶，特别是对地塞米松刺激的反应。 我们目前正在对前脂肪细胞进行功能增益实验，以评估这些激酶调节脂肪分化的能力。此外，我们正在通过专门在脂肪组织中进行的功能丧失实验，测试这些激酶在体内的作用。 该项目的第三个重点是识别控制脂肪细胞分化早期阶段的新因素，这些因素先于PPAR伽马表达。我们目前正在测试几个新的候选转录因子作为脂肪细胞谱系的早期决定因素的作用。

The nuclear receptor PPARgamma is considered the central regulator of adipocyte differentiation. Activation of this transcription factor via natural and synthetic agonists leads to the induction of genes involved in fat differentiation and insulin sensitization. Although the mechanisms of PPARgamma's ligand-dependent transcriptional activation have been studied extensively since the discovery of this factor in the early nineties, the ligand-independent function of PPARgamma has not yet been fully elucidated. One of the main focus of the lab is the identification of novel molecules and pathways that can modulate PPARgamma's ligand-independent activity. In order to identify novel PPARgamma modulators we have taken the following approaches: 1) We have analyzed several cofactors candidates that appear to be expressed during adipocyte differentiation and assessed their ability to function as potential transcriptional cofactors for PPARgamma; 2) We have generated several PPARgamma-GST fusion constructs that express distinct domains of PPARgamma. These fusion proteins produced in bacteria have been utilized in biochemical assays to purify potential novel N-terminal interactors. Nuclear extracts obtained from preadipocytes and fully differentiated adipocytes have been analyzed and novel interacting proteins have been identified by mass spectrometry. In addition to the characterization of novel PPARgamma-interacting molecules, we are focusing on the characterization of novel signaling pathways that can enhance differentiation in response to dexamethasone stimuli. For this purpose we have analyzed the pattern of expression of several kinases during different phases of fat differentiation and have identified several potential kinases that could be involved in the regulation of the adipogenic process specifically in response to dexamethasone stimulation. We are currently performing gain-of-function experiments in preadipocytes to assess the ability of these kinases to modulate fat differentiation. In addition we are testing the role of these kinases in vivo, via loss-of-function experiments carried out specifically in adipose tissue. The third focus of this project is the identification of novel factors that control early phases of adipocyte differentiation that precede PPARgamma expression. We are currently testing the role of several new candidate transcription factors as early determinants of the adipocyte cell lineage.