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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM052021-06
Application #
6127489
Study Section
Surgery, Anesthesiology and Trauma Study Section (SAT)
Program Officer
Somers, Scott D
Project Start
1995-06-01
Project End
2004-05-31
Budget Start
2000-06-01
Budget End
2001-05-31
Support Year
6
Fiscal Year
2000
Total Cost
$230,729
Indirect Cost
Name
University of Pittsburgh
Department
Surgery
Type
Schools of Medicine
DUNS #
053785812
City
Pittsburgh
State
PA
Country
United States
Zip Code
15213
Cardinal, J S; Reddy, S K; Tsung, A et al. (2013) Laparoscopic major hepatectomy: pure laparoscopic approach versus hand-assisted technique. J Hepatobiliary Pancreat Sci 20:114-9
Du, Qiang; Zhang, Xinglu; Liu, Quan et al. (2013) Nitric oxide production upregulates Wnt/?-catenin signaling by inhibiting Dickkopf-1. Cancer Res 73:6526-37
Klune, John R; Dhupar, Rajeev; Kimura, Shoko et al. (2012) Interferon regulatory factor-2 is protective against hepatic ischemia-reperfusion injury. Am J Physiol Gastrointest Liver Physiol 303:G666-73
Li, Peiyuan; Du, Qiang; Cao, Zongxian et al. (2012) Interferon-ýý induces autophagy with growth inhibition and cell death in human hepatocellular carcinoma (HCC) cells through interferon-regulatory factor-1 (IRF-1). Cancer Lett 314:213-22
Du, Qiang; Geller, David A (2010) Cross-Regulation Between Wnt and NF-?B Signaling Pathways. For Immunopathol Dis Therap 1:155-181
Cao, Zongxian; Dhupar, Rajeev; Cai, Changchun et al. (2010) A critical role for IFN regulatory factor 1 in NKT cell-mediated liver injury induced by alpha-galactosylceramide. J Immunol 185:2536-43
Ueki, Shinya; Dhupar, Rajeev; Cardinal, Jon et al. (2010) Critical role of interferon regulatory factor-1 in murine liver transplant ischemia reperfusion injury. Hepatology 51:1692-701
Du, Qiang; Zhang, Xinglu; Cardinal, Jon et al. (2009) Wnt/beta-catenin signaling regulates cytokine-induced human inducible nitric oxide synthase expression by inhibiting nuclear factor-kappaB activation in cancer cells. Cancer Res 69:3764-71
Kim, Kee-Hwan; Dhupar, Rajeev; Ueki, Shinya et al. (2009) Donor graft interferon regulatory factor-1 gene transfer worsens liver transplant ischemia/reperfusion injury. Surgery 146:181-9
Park, Kyung Soo; Guo, Zhong; Shao, Lifang et al. (2009) A far-upstream Oct-1 motif regulates cytokine-induced transcription of the human inducible nitric oxide synthase gene. J Mol Biol 390:595-603

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