Recent data accumulated from numerous models of inflammatory bowel disease (IBD) continue to reinforce the importance of defining the contribution of luminal bacteria to intestinal inflammation. The pathognomonic feature of salmonellosis, as well as IBD, which is correlated with patient symptomatology, is the transepithelial migration of neutrophils (PMN) across intestinal epithelia and into the luminal space to form crypt abscesses. We have shown that this final step of PMN movement across the epithelium to be directed by nuclear factor kappaB (NF-kappaB)-independent release of a soluble bioactivity from the apical surface of the epithelium - previously termed PEEC (pathogen-elicited epithelial chemoattractant), which we have recently identified as the eicosinoid hepoxilin A3 (hepA3). Our studies investigating the mechanism underlying the release of PEEC/hepA3 from intestinal epithelia have revealed the involvement of the Salmonella typhimurium type III secreted effector protein SipA. SipA activates a novel lipid signaling cascade whereby protein kinase C (PKC-alpha) is activated resulting in secretion of hepA3. Our overall hypothesis is that the signaling pathway resulting in the secretion of hepA3 is important not only during pathogen-induced inflammation (i.e. S. typhimurium) but that this pathway may be dis-regulated during intestinal inflammation. Understanding the mechanism by which epithelial cells are induced by bacteria to orchestrate this response may provide key mechanistic insights into how events occur in an idiopathic disease such as IBD. Thus, the long-term objective of this proposal is to understand the molecular mechanisms underlying SipA regulation of hepA3 release by the intestinal epithelia.
Specific Aim 1 is designed to characterize the molecular interaction that occurs between the S. typhimurium effector molecule, SipA, and the intestinal epithelial cell that triggers the activation of the signal transduction pathway leading to hepA3 secretion. The goals of this aim are to (i) determine the functional domain of SipA that regulates the signaling event leading to hepA3 release, and (ii) to identify and functionally characterize a putative SipA receptor.
Specific Aim 2 is designed to identify the signal transduction events, which lead to the apical release of hepA3. Thus, we plan to (i) map the signal transduction cascade leading to hepA3 production, (ii) examine the molecular mechanism of hepA3 efflux, and (iii) examine the importance of the signaling events in vivo models of intestinal inflammation.
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