Overproduction of nitric oxide by inducible NO synthase (iNOS) in vascular smooth muscle cells (VSMC) has been implicated in the hypotension of septic shock, a cytokine-mediated disease for which there is no effective treatment. The PI has shown that TGFb1 inhibits iNOS gene transcription after is it induced by IL-1 in rat VSMC in culture, and that TGFb1 prevents hypotension in endotoxin-treated rats. Deletion analysis of the mouse iNOS promoter revealed that an NF-kB sequence and an AT-rich/octamer (OCT) site are both important for iNOS induction in VSMC by IL-1 and endotoxin. The PI has found that high mobility group (HMG)-I(Y) protein binds to the AT-rich/OCT site and facilitates NF-kB binding and transactivation of the iNOS promoter. HMG-I(Y) does not trigger transactivation by itself; rather, it assembles transcriptional factors into a nucleoprotein complex (an enhanceosome) by altering chromatin structure. The expression of HMG-I(Y) in VSMC is upregulated by both cytokines and endotoxin. The PI hypothesizes that HMG-I(Y) functions as a regulator that orchestrates the assembly of transcriptional factors important in the induction of iNOS. The goals of the proposed work are: 1) To study how HMG-I(Y) interacts with NF-kB and AT-rich/OCT binding proteins to regulate iNOS transcription in vitro and in vivo; 2) To test the importance of NF-kB and OCT sites in transgenic mice. The PI proposes four aims: 1) Determine whether additional nuclear proteins bind to the AT-rich/OCT site in the iNOS promoter and characterize their interaction with HMG-I(Y) and NF-kB; 2) To determine whether TGFb1 inhibits the iNOS promoter by suppressing induction of HMG-I(Y); 3) To determine whether inhibition of HMG-I(Y) with antisense or dominant negative HMG-I(Y) proteins prevents iNOS induction in vitro and in vivo; and 4) To test the activity of the iNOS flanking regions in transgenic mice. Information about the molecular mechanisms that regulate VSMC gene induction should point to targets such as HMG-I(Y) that can be used in the design and screening of agents to inhibit VSMC iNOS gene expression. These agents may provide effective treatments for septic shock.
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