Sepsis, trauma, and injury complicated by infection all initiate systemic responses mediated by cytokines and other inflammatory hormones released following interaction of host cells with agents such as gram negative bacterial lipopolysaccharide (LPS). In extreme conditions, this response can progress to cell dysfunction and multiple organ failure, a major cause of mortality in critically ill surgical patients. Recent advances in our understanding of this syndrome are based on the discovery that nitric oxide (NO) synthesis is induced in a number of tissues during the septic response. The inducible nitric oxide synthase (iNOS) gene is only expressed in cells after exposure to certain combinations of cytokines and/or LPS. Induced NO synthesis can have both beneficial and detrimental consequences. We have cloned the iNOS gene from stimulated human hepatocytes and found that it is maximally expressed in response to the synergistic actions of LPS and cytokines TNFa, IL-1b, and IFNg. We hypothesize that the molecular regulation of the human iNOS gene is complex and will involve transcriptional as well as post-transcriptional mechanisms. In this proposal, we will pursue two interrelated specific aims to study the regulated expression of the human iNOS gene.
AIM I : TO DEFINE THE TRANSCRIPTIONAL MECHANISMS AND FUNCTIONAL PROMOTER ELEMENTS RESPONSIBLE FOR CYTOKINE AND LIPOPOLYSACCHARIDE INDUCTION OF THE HUMAN iNOS GENE. We will begin by performing nuclear run-on experiments in LPS and cytokine-stimulated human hepatocytes to confirm and quantify transcriptional induction. The human iNOS gene promoter region will isolated, sequenced, and functionally characterized in transfection experiments. Specific DNA elements will be analyzed that are required for LPS and cytokine-responsiveness. Positive and negative transcription factors will be identified in gel shift binding assays. Finally, characterization of the inducible promoter elements in vivo will be carried out by constructing transgenic mice expressing a human iNOS promoter fusion gene.
AIM II : TO DETERMINE THE POST-TRANSCRIPTIONAL MECHANISMS INVOLVED IN THE REGULATION OF THE HUMAN iNOS GENE. Post- transcriptional mechanisms can also regulate gene expression by changes in mRNA stability or translational efficiency. We will determine if LPS or cytokines increase steady state iNOS mRNA levels by increasing mRNA stability. Changes in translational efficiency in response to these agents will be measured by pulse-chase experiments. Inhibitors of induced NO synthesis will be tested for effects on mRNA stability and translation. Finally, if changes in mRNA stability or translational efficiency are observed, we will analyze the 3'-untranslated region of the iNOS gene for elements that mediate these effects. At the completion of our studies, we will have characterized the molecular regulation of the human iNOS gene in response to LPS and/or cytokines. This information will provide important insights into the mechanisms of how cytokines and other inflammatory mediators direct the expression of highly regulated genes in injured and septic surgical patients.
