Identification of novel molecules and pathways that modulate adipogenesis

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The nuclear receptor PPARgamma is a master regulator of adipogenesis. Activation of this transcription factor through specific 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 projects in the lab is the identification of novel molecules and pathways that can modulate PPARgamma's ligand-independent activity. In order to identify novel PPARgamma interacting molecules 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. We are currently confirming that these factors interact directly with PPARgamma and that they can modulate PPARgamma's transcriptional activity and adipogenic function, both in vitro and in vivo. In addition to the characterization of novel PPARgamma interacting molecules, we are interested in defining novel signaling pathways that can modify adipogenesis. 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. We are currently performing gain-of-function experiments in preadipocytes to assess the capacity of these kinases to affect PPARgamma function and, overall, adipocyte differentiation.