Sepsis and endotoxemia are associated with increased production of IL-6 in intestinal mucosa and enterocytes, a response that is augmented by the heat shock response. IL-6, locally produced in the mucosa, may regulate the synthesis of acute phase proteins in enterocytes and IgA in inflammatory cells. In addition, IL-6 may increase intestinal permeability in critical illness and IL-6 released from the gut may have systemic effects. The molecular regulation of mucosal/ enterocyte IL-6 production, as well as the influence of the heat shock response, are not well understood. The purpose of this project is to test the hypotheses that: 1) C/EBP upregulates transcription of the IL-6 gene and increases IL-6 production in IL-1beta-stimulated Caco-2 cells and that C/EBP exerts its effect by interacting with nuclear co-factors, including CBP and p300; 2) sepsis and endotoxemia in mice increase C/EBP activity in intestinal mucosa in vivo; 3) the heat shock response increases C/EBP activity in mucosa of septic and endotoxemic mice and IL-1beta-treated Caco-2 cells; 4) treatment of Caco-2 cells with proteasome inhibitors induces the heat shock response and activates C/EBP activity and IL-6 production. Caco-2 cells, a human intestinal epithelial ceil line, are treated with IL-1beta and C/EBP activity is determined by EMSA. Supershift analysis is performed to determine the activation of individual members of the C/EBP family of transcription factors. To determine the role of C/EBP in enterocyte IL-6 production, cells are transfected with wild-type or C/EBP mutated IL-6 promoter luciferase reporter plasmid and stimulated with IL-1beta. In other experiments, cells are transfected with expression plasmids for C/EBP-beta or -delta. The role of the nuclear co-factors CBP/p300 is examined by performing co-immunoprecipitation and supershift analysis of C/EBP EMSAs. For in vivo experiments, sepsis is induced by cecal ligation and puncture and endotoxemia by subcutaneous injection of LPS in mice. C/EBP activity is determined in mucosa from different regions of the gastrointestinal tract (stomach, jejunum, ileum and colon). In other experiments, the heat shock response is induced in mice by hyperthermia or injection of sodium arsenite before induction of sepsis or endotoxemia. In the in vitro experiments, heat shock response will also be induced by treating Caco-2 cells with proteasome inhibitors (MG132 or lactacystin). The proposed experiments are important because they will determine the influence of sepsis and endotoxemia on C/EBP activity in intestinal mucosa and define the role of C/EBP in enterocyte IL-6 production. The experiments will also elucidate the interaction between heat shock response and enterocyte IL-6 production. Because mucosal IL-6 may have important local as well as systemic effects during sepsis and endotoxemia, a better understanding of the transcriptional regulation of IL-6 in the intestine and the possibility of influencing this regulation with the heat shock response will have significant clinical implications.

National Institute of Health (NIH)
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Research Project (R01)
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Surgery, Anesthesiology and Trauma Study Section (SAT)
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Hamilton, Frank A
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Beth Israel Deaconess Medical Center
United States
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