Our long-term goal is to determine the mechanisms regulating smooth muscle development. Unraveling these mechanisms is crucial to our understanding of the pathology of many different, intestinal, urogenital vascular and pulmonary diseases that are associated with altered contractile protein expression and contractility. Experiments are proposed to use the smooth muscle-specific telokin promoter to examine the molecular pathways controlling visceral smooth muscle development. We have previously identified several transcription factors that regulate the telokin promoter activity in vitro. In this proposal we will determine the role played by these factors in regulating telokin expression during intestinal development. In addition, experiments will begin to investigate the signaling pathways that regulate the expression and activity of these proteins. These studies will test the hypothesis that visceral smooth muscle-specific expression of telokin is modulated in part by signals derived from intestinal epithelial cells that regulate smooth muscle differentiation. In this proposal experiments will specifically focus on the role of SRF, Hox and Fox proteins in regulating telokin expression during gut development. Co-transfection studies together with DNA binding experiments and in vivo analysis of mutant promoters that are unable to bind Hox and Fox genes will be used to elucidate the role of these proteins in regulating telokin expression during gut development. SRF is known to be a key regulator of all smooth muscle genes thus far examined, although it is not known if it is required for basal expression of smooth muscle genes or if it is involved in mediating their tissue specific expression. Experiments using mutant chimeric promoters harnessed to beta-galactosidase transgenes will allow us to determine if SRF is required for the visceral smooth muscle-specific expression of telokin. It is likely that the tissue specific functions of SRF result from its interaction with other cell-type specific factors, hence the ability of several cell-restricted, SRF associated transcription factors to regulate telokin expression will be examined. Preliminary results have also suggested that duplin, a beta-catenin binding protein, can interact with SRF. These data suggest that wnt signaling may regulate expression of smooth muscle specific genes through SRF via its interaction with duplin and beta-catenin. The importance of this regulatory pathway will be evaluated in vitro by transfection assays in cultured cells and direct in vitro protein and DNA binding experiments.
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