EGF, Oxidants & Intestinal Barrier Integrity

EGF、氧化剂

• 批准号: 6541168
• 负责人: ALI BANAN
• 金额: $ 28.13万
• 依托单位: RUSH UNIVERSITY MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-06-01 至 2007-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6541168
• 关键词: chemoprevention; cytoprotection; enzyme induction /repression; epidermal growth factor; gastrointestinal epithelium; inflammation; inflammatory bowel diseases; intracellular transport; isozymes; microtubules; nitric oxide; nitric oxide synthase; nuclear factor kappa beta; oxidative stress; oxidizing agents; phosphorylation; protein kinase C; protein transport; tissue /cell culture; transfection

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.

广泛的长期目标：防止或扭转炎症性肠病（IBD）的急性发作。 策略：如果我们知道内源性生长因子（GF）阻止氧化剂引起的肠道损伤的机制，则可以开发GF-Mimetics或增强子来预防/治疗IBD。 背景：氧化应激对屏障完整性的破坏是引起肠道炎症的主要原因。 使用肠细胞的单层，我们建立了一种新型的破坏性和保护性机制 - 骨骼损伤和稳定性。 我们进一步报道，i）损害是由微管蛋白和肌动蛋白硝化以及通过iNOS /无上调的氧化产物对细胞骨骼细丝的脱氮造成的； ii）用GF（例如EGF）预处理通过激活PKC-BETA1和PKC- Zeta（Zetu）同工型来防止损坏。 我们最近的初步数据提出了一种统一的基本机制：a）氧化剂可以通过激活NF-kappab（氧化剂降解NF-kappab的抑制剂I-kappabalpha，氧化剂降解的NF-kappaba，nf-kappabalpha，并转移NF-kappab转移到原子核中； nf-kappab，否和障碍功能）； b）通过抑制NF-kappab激活的PKC同工型保护GFS（早期的初步数据表明，将抗敏感性转染到PKC-BETA1中可以防止GF对I-Kappabalpha和NF-Kappab的影响）。 NF-kappab激活的损伤和保护调节的共同机制指出了我们的工作假设：胃肠道上皮细胞中的生长因子可以防止氧化剂诱导的细胞骨架破坏和屏障完整性的丧失，从而防止氧化剂诱导的INOS上调。 保护是通过激活稳定1-kappabalpha，防止NF-kappab的易位和激活并抑制iNOS上调的特定PKC同工型介导的。 目的1。确定NF-kappab灭活是否是介导GF诱导的氧化剂诱导效应的关键分子事件：a）iNOS上调，b）微管拆卸； c）障碍中断。 目的2。确定GF对细胞骨架和屏障完整性的保护是否是通过通过PKC同工型增加的信号传导来介导的，PKC同工型与I-kappab- alpha的下游稳定相关，NF-kappab失活和INOS下调。目标将涉及药理学（激活剂/抑制剂）和分子生物学（转染）的策略；单层将在a）氧化剂引起iNOS上调和细胞骨架硝化以及拆卸和屏障破坏的条件下使用单层，b）与GF预孵育可防止这种氧化损伤。意义。我们的研究将：i）GF对促炎条件的氧化应激产生对基本保护机制的新见解； ii）实质上提高了对IBD病理生理学的理解，并提出了新型抗IBD药物的新靶标； iii）解释炎症期间肠道障碍的不稳定； iv）建立PKC同工型信号传导对于防止炎症至关重要。