Broad, long-term goal: To prevent or reverse the acute attack in inflammatory bowel disease (IBD). Strategy: If we knew the mechanism by which endogenous growth factors (GF) prevent oxidant-induced intestinal injury, we could develop GF-mimetics or enhancers for prevention/treatment of IBD. Background: Disruption of barrier integrity by oxidative stress is a major contributor to intestinal inflammation. Using monolayers of intestinal cells, we established a novel disruptive and protective mechanism-cytoskeletal damage and stability. We further reported that i) damage is caused by nitration of tubulin and actin and disassembly of cytoskeletal filaments by oxidative products of iNOS / NO upregulation; ii) pretreatment with GF (e.g., EGF) prevented damage via activation of PKC-beta1 and PKC- zeta (zetu) isoforms. Our recent Preliminary Data suggest a unifying fundamental mechanism: a) oxidants may upregulate iNOS by activating NF-kappaB (oxidants degraded NF-kappaB's inhibitor, I-kappaBalpha, and translocated NF-kappaB to the nucleus; transfection of a mutant I-kappaBalpha prevented oxidant effects on I-kappaBalpha, NF-kappaB, NO and barrier function); b) GFs protect via PKC isoforms inhibiting NF-kappaB activation (early pilot data suggest that transfection of anti-sense to PKC-beta1 prevents GF's effects on I-kappaBalpha and NF-kappaB). This shared mechanism of damage and protection-modulation of NF-kappaB activation-led to our working Hypothesis: Growth factors in GI epithelial cells protect against oxidant-induced cytoskeletal disruption and loss of barrier integrity by preventing oxidant- induced upregulation of iNOS. Protection is mediated by activation of specific PKC isoforms that stabilize 1-kappaBalpha, prevent translocation and activation of NF-kappaB, and inhibit iNOS upregulation.
Aim 1. To determine whether NF-kappaB inactivation is a key molecular event mediating GF-induced prevention of oxidant-induced effects: a) iNOS upregulation, b) microtubule disassembly; c) barrier disruption.
Aim 2. To determine whether protection of cytoskeletal and barrier integrity by GF is mediated by increased signaling via PKC isoforms that is linked to downstream stabilization of I-kappaB- alpha, inactivation of NF-kappaB and down-regulation of iNOS.
Aims will involve strategies from both pharmacology (activators/ inhibitors) and molecular biology (transfection); monolayers will be used under conditions in which a) oxidants cause iNOS upregulation and cytoskeletal nitration and disassembly and barrier disruption, and b) preincubation with GF prevents this oxidative damage. Significance. Our studies will: i) yield new insights into fundamental protective mechanisms by GF against the oxidative stress of proinflammatory conditions; ii) substantially improve understanding of IBD pathophysiology and suggest new targets for novel anti-IBD drugs; iii) explain intestinal barrier instability during inflammation; iv) establish PKC isoform signaling as crucial in protection against inflammation.
