In this proposal, we will investigate the role of heat shock proteins (HSPs) in promoting intestinal mucosal cytoprotection and wound healing in normal and IBD tissues, a hypothesis not previously explored, out supported by recent data from our laboratory. First, we will determine whether inducible HSPs in intestinal IEC-18 cells and REF52 fibroblasts, stimulated by conditions associated with inflammation, have a role in epithelial and mesenchymal cell cytoprotection. These studies will largely focus on hsp70, the major inducible HSP form in these cells. The degree of cytoprotection will be determined by 51Cr release and by measuring specific cellular functions, including protein synthesis, RNA stability, tight junction integrity (transmembrane resistance), Na pump activity, and the activities of transfected """"""""reporter' proteins, beta- galactosidase and luciferase (which require HSPs to preserve their enzymatic activities). We will define the mechanisms involved in the induction process by assessing transcriptional and post-transcriptional processes, the former by gel mobility shift assays. To more directly determine if hsp 70 plays a role in cytoprotection to inflammation- associated conditions such as heat (fever) stress, acidosis, and oxidants (NH2CI), we will selectively induce its expression by transfecting cells with a beta-actin promoter-hsp70 construct. Finally, we will determine if the hsp70 response to injury can be pharmacologically augmented or induced, and, if so, define the mechanisms underlying their actions. In the second phase of the proposal we will investigate the in vivo role of HSPs by studying the expression and induction of hsp70 in clinical specimens and in intestinal tissues of DSS-treated mice developing acute and chronic colitis. Hsp70 will be assessed by MRNA abundance, immunoreactivity, immunolocalization and in situ hybridization in order to quantitate and localize expression relative to areas of inflammation and zones of normal, bordering and healing tissue. Based on outcome of the in vitro experiments which will identify agents that induce hsp70, we will determine if intestinal hsp70 can be pharmacologically-induced in vivo. Additionally, hsp70 expression in colonic mucosal biopsies from patients with chronic ulcerative colitis, being treated on protocol with oral or rectally-administered salicylates, will be compared to that in patients receiving placebo. These data will be correlated with clinical outcome, disease activity, normal and non-involved areas, and therapeutic and endoscopic responses to salicylates. Finally, we will determine if colonic HSPs can be pharmacologically induced or augmented, and if so, see if this confers additional cytoprotection and wound healing in DSS- treated animals with acute or chronic colitis. In summary, the induction of HSPs may represent a previously unexplored and potentially important mechanism for mucosal cytoprotection and wound healing in IBD. Their pharmacological induction may therefore be useful as a new therapeutic strategy.
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