Sepsis, trauma, and infection initiate systemic responses regulated by cytokines and other inflammatory mediators. In extreme conditions, this response can progress to multiple organ failure, a major cause of mortality in surgical patients. Advances in our understanding of this syndrome are based on the discovery that the inducible nitric oxide synthase (iNOS) gene is expressed in nearly every organ and tissue during the septic response. While NO synthesis has beneficial effects during acute inflammation, over-production of NO during sepsis can be detrimental with massive vasodilation and hypotension. Chronic expression of iNOS has been implicated in NO-mediated cytotoxicity leading to diabetes, arthritis, neurode generative disorders, and certain cancers. Our laboratory has cloned the human iNOS gene from cytokine-stimulated human hepatocytes. We then isolated the promoter region of the human iNOS gene and have shown that the cytokine-responsive DNA elements are located upstream from -4.7 kb. Recently we have characterized a novel NF-kappaB enhancer region that regulates iNOS transcription in response to TNFalpha or IL-1beta, and have shown a role for STAT1alpha in mediating IFNgamma induction. We have also identified extracellular signals that down-regulate iNOS expression including p53 tumor suppressor protein, steroids, heat shock, certain growth factors, and NO itself. Since the human iNOS gene is tightly regulated, we hypothesize that several mechanisms are working jointly to control the expression of this gene. We predict that this will include cytokine-stimulated nuclear factors that exert either positive or negative control over transcription, as well as post-transcriptional mechanisms that regulate iNOS mRNA stability and translational efficiency. In this proposal, we will pursue two interrelated specific aims.
AIM I : TO DEFINE THE TRANSCRIPTIONAL MECHANISMS AND FUNCTIONAL PROMOTER ELEMENTS RESPONSIBLE FOR INDUCTION AND SUPPRESSION OF THE HUMAN iNOS GENE. Additional promoter regions will be fully sequenced and characterized in transfection experiments. DNA elements will be analyzed that are required for cytokine- responsiveness, with an initial focus on the interactions between NF-kappaB and STAT1alpha. Specific mechanisms for gene suppression by p53, steroids, heat shock, TGF-beta, and NO will be sought. Positive and negative transcription will be identified by gel shifts and in vivo footprinting assays.
AIM II . TO DETERMINE THE POST-TRANSCRIPTIONAL MECHANISMS INVOLVED IN REGULATION OF THE HUMAN iNOS GENE. Post-transcriptional changes in mRNA stability or translational efficiency can also regulate gene expression. Cytokines will be tested for effects on human iNOS mRNA stability. Changes in translational efficiency in response to these agents will be measured by pulse-chase experiments. The 3'-untranslated region of the human iNOS gene will be analyzed for elements that mediate these effects. At the completion of our studies, we will have characterized the molecular regulation of the human iNOS gene. The information gained will increase our understanding of the control of iNOS transcription, describe novel mechanisms of cytokine-synergy in signal transduction, and help in designing therapeutic strategies for pathophysiological disease states where cytokine expression is relevant.
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