PhD

dissertationEndothelial cells mediate the inflammatory response by coordinating localization and activation of leukocytes via a concerted expression of adhesion molecules and cytokines. Adhesion molecule and cytokine gene expression occurs following activation of specific signaling pathways by endothelial cell agonists. The MAP kinase signaling pathways consist of a cascade of kinases that phosphorylate transcription factors to activate specific genes. The major MAP kinase pathways are the 'ERK' or 'growth' pathway and the 'stress' pathway. NF-?B, another mediator of endothelial gene expression, is normally sequestered by the I?B in the cytoplasm. However, certain stimuli cause degradation of I?B that results in NF-?B nuclear translocation. In the nucleus NF-?B activates transcription from NF-?B dependent promoters of adhesion molecule and cytokine genes. The major aim of this study was to describe signaling mechanisms involved in the induction of adhesion molecules and cytokines by the use of receptor and nonreceptor endothelial cell agonists. The nonreceptor agonists brown recluse spider venom and bacterial sphingomyelinase induced a pattern of gene expression distinct from tumor necrosis factor (TNF). Since TNF activation of NF-?B and MAP kinase was theorized to be downstream of sphingomyelin breakdown due to a TNF receptor associated sphingomyelinase, the results with nonreceptor sphingomyelin breakdown were intriguing. On elucidation of signaling pathways, TNF sends at least two signals. Sphingomyelin breakdown to ceramide activates the ERK MAP kinase pathway, and ceramide-independent activation of stress MAP kinases and NF-?B nuclear translocation. Another novel receptor agonist oncostatin M (OSM) was found to induce a pattern of gene expression distinct from both TNF and bacterial sphingomyelinase. At low concentrations, OSM caused potent ERK activation, but at higher concentrations it caused NF?B nuclear translocation in addition to ERK activation. This unmasked NF-?B-dependent and -independent mechanisms of endothelial cell adhesion molecule and cytokine expression. Furthermore, the above agonists did not induce the same characteristic responses in gene induction and signaling in transformed endothelial cells, showing that alteration in signaling pathways associated with cell transformation alters gene expression. In conclusion, integration of different signals at the promoters of adhesion molecule and cytokine genes mediates a coordinated inflammatory response by endothelial cells