Endotoxic shock is a life-threatening consequence of severe gram- negative infection characterized by vascular smooth muscle cell relaxation and severe hypotension. Nitric oxide (NO), through the inducible NO synthase (iNOS) pathway, is a major contributor in this process. However, recent investigations from our laboratory suggest that heme oxygenase (HO), an enzyme that generates carbon monoxide (CO) in the course of heme metabolism, may also be involved in the hemodynamic compromise of endotoxic shock. Inducible HO (HO-1) is dramatically increased in vascular tissue from rats receiving endotoxin, and non-selective inhibitors of HO activity abrogate endotoxin-induced hypotension. In addition, the induction of HO-1 mRNA is at the level of gene transcription. Beyond an interaction of transcription factors with their DNA binding domains, an appropriate chromatin environment is important for gene transcription. By changing chromatin structure, architectural proteins assemble transcription factors into an enhancer complex. One such architectural protein, high mobility groups protein (HMG)-I(Y), has been shown to promote cooperative binding of transcription factors into a complex that efficiently and synergistically increases gene transcription. Preliminary data from our laboratory show that like HO-1, HMG-I(Y) mRNA and protein is induced in vascular smooth muscle cells by IL-1 beta in vitro and by endotoxin in vivo. Furthermore, HMG-I(Y) binds to 5' flanking sequence of the HO-1 gene. In this application we propose to test the role of HO-1 in endotoxic shock, and to study the molecular mechanisms regulating the transcriptional induction of HO-1 in vascular smooth muscle cells.
Three specific aims are proposed.
Aim 1 : To determine the role of HO-1 in endotoxin-induced hypotension and death by generating mice deficient in the HO-1 gene. We hypothesize that deletion of the HO-1 gene will prevent endotoxin-induced hypotension and death in vivo.
Aim 2 : To identify the specific DNA sequences (cis-acting elements) that are important for HO-1 gene induction in vascular smooth muscle cells, and to determine the role of chromatin in regulating HO-1 gene transcription. We hypothesize that the interaction of cis-acting elements with trans-acting factors, in an integrated setting, mediates HO-1 gene transcription. Finally, Aim 3: To identify and characterize those DNA-binding proteins (trans-acting factors), that in conjunction with HMG-I(Y), are important for regulating the induction of HO-1 gene transcription by IL-1 beta in vascular smooth muscle cells. These studies should provide new insight into the regulation of HO-1, and the pathogenesis of endotoxic shock.
Showing the most recent 10 out of 42 publications
