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. 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 two new transcriptional regulators, ZNF638 and Foxa3, as early determinants of the adipocyte cell lineage and are currently generating animal models to assess the biological function of these factors in vivo.
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