Novel molecules and pathways that modulate adipogenesis

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The transcription factor PPARgamma, a member of the nuclear receptor superfamily, is a key regulator of adipogenesis. It has been demonstrated that ligand activation of PPARgamma leads to activation of genes involved in fat differentiation and insulin sensitization. Although PPARgamma?s ability to activate transcription upon ligand binding has been studied extensively since 1994, the ligand-independent function of this transcription factor has not been fully elucidated. One of the goals of the lab is the identification of novel molecules and pathways that can modulate the ligand-independent activity of this transcription factor. We are currently taking different approaches to study how PPARgamma is regulated and how it functions in the absence of ligand. 1)We are interested in the identification of specific cofactors that are able to bind to PPARgamma and modify its transcriptional activity in a ligand-independent manner. For this purpose, we are in the process of generating PPARgamma ?GST fusion constructs expressing different domains of PPARgamma. These constructs will be utilized in biochemical assays to potentially purify novel cofactors, from nuclear extracts of preadipocytes and fully differentiated adipocytes, that are able to interact with unliganded PPARgamma. 2) We are currently determining whether various signaling pathways can alter PPARgamma function independently of the presence of ligand. We are assessing the ability of a number of kinases to directly phosphorylate PPARgamma and its coactivators and potentially alter their adipogenic capacity. We are in the process of setting up in vitro kinase assays coupled to transient transfection and differentiation assays to determine the ability of these candidate kinases to directly influence the basal PPARgamma transcriptional activity in the absence of ligand.