Major traumatic injuries can lead to the development of sepsis and multiple organ failure. The liver is an important target of the inflammatory mediators that are released after injury, and responds by altering its program of gene expression. A set of proliferation-associated (immediate early [IE] genes (c-fos, c-jun, jun-B, Zif268 and Nup475) that encode putative transcriptional regulatory proteins are induced during hepatic regeneration is also rapidly induced after endotoxin challenge. It is hypothesized that the early systemic response to acute inflammation is mediated, in part, through the induction of these IE genes. A major goal is to identify novel signal transduction components that control early gene expression after systemic inflammatory challenge. The long-term goal is to determine the mechanisms that regulate the responses to systemic injury. The principal model chosen to reproduce the inflammatory response is endotoxin challenge which has important clinical relevance.
The specific aims for this proposal are: 1) to determine whether LPS or macrophage- derived cytokines directly induce IE gene and delayed response gene (TGF- beta1, TGF-alpha and fibronectin) expression in target cells. We will examine induction of IE gene mRNA by Northern blot analysis. 2) To determine whether changes in IE gene mRNA after LPS treatment are associated with similar changes in the levels of the respective transcription factor proteins (in vivo and in cell culture). Transcription factors will be examined by electrophoretic mobility shift assays, immunoblotting, immunohistochemistry and in situ hybridization. 3) To determine whether LPS injection initiates IE gene and delayed response gene mRNA and protein expression in organs other than liver. Major target organs of the systemic inflammatory response (lungs, kidneys, heart, gut and brain) will be examined. 4) To determine whether remote systemic injury can induce the expression of hepatic IE genes and compare effects in endotoxin-resistant mice. Systemic injuries will be surgical injury, acute pancreatitis and subcutaneous injection of turpentine in mice. 5) To determine the molecular mechanisms of hepatic IE gene induciton by LPS. Transcriptional regulation will be examined by nuclear run-on assay of cultured cells and of freshly isolated hepatocytes after LPS challenge and by transient expression assay. The jun-B and Nup475 promoter and 5' flanking DNA regions have been linked to the chloramphenicol acetyltransferase (CAT) gene and transient CAT expression will be examined after transfection with BWTG3 cells to determine responsiveness of inflammatory mediators. The goal is to determine the mechanisms by which inflammatory signals alter gene expression and to develop novel therapy to block harmful responses while preserving beneficial responses. This should lead to novel approaches to modulate the inflammatory responses to major injury or the septic complications of injury.
| Sheng, H; Shao, J; Morrow, J D et al. (1998) Modulation of apoptosis and Bcl-2 expression by prostaglandin E2 in human colon cancer cells. Cancer Res 58:362-6 |
| Chinery, R; Brockman, J A; Peeler, M O et al. (1997) Antioxidants enhance the cytotoxicity of chemotherapeutic agents in colorectal cancer: a p53-independent induction of p21WAF1/CIP1 via C/EBPbeta. Nat Med 3:1233-41 |
| Sheng, H; Shao, J; Kirkland, S C et al. (1997) Inhibition of human colon cancer cell growth by selective inhibition of cyclooxygenase-2. J Clin Invest 99:2254-9 |
| DuBois, R N; Shao, J; Tsujii, M et al. (1996) G1 delay in cells overexpressing prostaglandin endoperoxide synthase-2. Cancer Res 56:733-7 |
