Intestinal Mucosal Protection by Epidermal Growth Factor

表皮生长因子对肠粘膜的保护

• 批准号: 8994319
• 负责人: RADHAKRISHNA RAO
• 金额: $ 6.03万
• 依托单位: UNIVERSITY OF TENNESSEE HEALTH SCI CTR
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-01-13 至 2016-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8994319
• 关键词: Actins; Affinity; Amino Acids; Attenuated; Binding; Biopsy; Cells; Data; Development; Disease; Down-Regulation; Enterocolitis; Epidermal Growth Factor; Epidermal Growth Factor Receptor; Epithelial; Epithelial Cells; Functional disorder; Future; Gastrointestinal Diseases; Gastrointestinal tract structure; Goals; Growth Factor; Health; Human; Hydrogen Peroxide; Inflammatory Bowel Diseases; Injury; Intercellular Junctions; Intestinal Diseases; Intestines; Ischemia; Knowledge; Maintenance; Mediating; Mitogen-Activated Protein Kinases; Molecular; Mus; Necrotizing Enterocolitis; Outcome Study; Oxidative Stress; PTK2 gene; Pathogenesis; Permeability; Phosphorylation; Play; Prevention; Probiotics; Process; Protein Dephosphorylation; Protein Isoforms; Protein Kinase C; Protein Phosphatase 2A Regulatory Subunit PR53; Proteins; Reactive Oxygen Species; Regulation; Reperfusion Therapy; Research; Role; SRC gene; Signaling Molecule; Testing; Threonine; Tight Junctions; Tissues; Transactivation; Tyrosine; Up-Regulation; attenuation; base; cell motility; citrate carrier; claudin 3; claudin 4; gastrointestinal; intestinal epithelium; migration; novel therapeutics; occludin; prevent; protective effect; public health relevance

DESCRIPTION (provided by applicant): Oxidative stress-induced intestinal epithelial barrier dysfunction has been implicated in the pathogenesis of a wide spectrum of intestinal inflammatory diseases including necrotizing enterocolitis, inflammatory bowel disease and infectious enterocolitis. A significant body of evidence indicates that barrier function of GI tract is disrupted in various GI diseases. Our studies during the past several years have demonstrated that oxidative stress disrupts intestinal epithelial tight junction by phosphorylation of occludin on tyrosine residues and dephosphorylation on threonine residues; these processes involve the intracellular signaling molecules such as c-Src, FAK, PI3K, PP2A, PKC7, PKC6 and PP1. Our studies also demonstrated that EGF, a GI mucosal protective factor, prevents the oxidative stress-induced disruption of tight junctions; this process involved signaling molecules, PLC3, PKC2I, PKC5 and MAP kinases. Our preliminary data have identified two tyrosine residues and two threonine residues in occludin that lie in a highly conserved motif of occludin and suggested that phosphorylation of these residues regulates the integrity of tight junctions. Therefore, we plan to further continue our effort to understand the regulation of phosphorylation of these amino acids in the oxidative stress- induced disruption of intestinal epithelial barrier function and EGF-mediated prevention of oxidative stress effects. On the basis of our results it is hypothesized that: a) c-Src-mediated phosphorylation of occludin on Y398 and Y402 mediates the H2O2-induced destabilization of intestinal epithelial tight junctions, b) H2O2- induced disruption of tight junctions involves of dephosphorylation of occludin on T403 and T404 by down regulation of PKC7, and c) EGF and probiotics prevent oxidative stress-induced tight junction disruption by a MAP kinase-dependent mechanism. We propose to determine that: 1) c-Src directly interacts with occludin and phosphorylates Y398/Y402 in H2O2-treated cells. 2) H2O2-induced Tyr-phosphorylation of claudin-3 plays a role in the disruption of tight junctions. 3) c-Src plays a role in H2O2-induced attenuation of steady state dynamics of tight junction proteins. 4) H2O2 accelerates cell spreading and migration by c-Src-mediated phosphorylation of occludin and claudin-3. 5) PKC6 regulates tight junction integrity by Ser/Thr- phosphorylation of tight junction proteins. 6) Oxidative stress disrupts tight junctions by down regulation of PKC7. 7) Phosphorylation of tight junction proteins on specific Ser/Thr residues regulates their self-association and actin binding affinity. 8) Probiotics protect tight junctions from H2O2 by inducing EGF receptor transactivation. 9) EGF and probiotics ameliorate ischemia/reperfusion-induced tight junction disruption in mouse intestine. 10) EGF and probiotics attenuate the H2O2-induced PKC7 down regulation, alteration of occludin phosphorylation and tight junction disruption in human colonic biopsies. The outcome of these studies has a direct relevance to our understanding of the pathogenesis of many GI diseases and has the potential to contribute to the future development of new therapeutic strategies. PUBLIC HEALTH RELEVANCE: Intestinal diseases such as inflammatory bowel disease and infectious enterocolitis are associated with tissue damage caused by oxidative stress and intestinal epithelial barrier dysfunction. On the basis of our research during the past several years we hypothesized that oxidative stress disrupts intestinal epithelial barrier function by inducing phosphorylation of tight junction proteins, and the protective growth factors such as epidermal growth factor prevent such cellular damage by oxidative stress. We propose to conduct studies to uncover the cellular and molecular mechanisms involved in these processes; and the outcome of these studies are expected to provide knowledge to develop new therapies in the treatment of different intestinal diseases.

描述（由申请人提供）：氧化应激诱导的肠上皮屏障功能障碍与多种肠道炎症性疾病的发病机制有关，包括坏死性小肠结肠炎、炎症性肠病和感染性小肠结肠炎。大量证据表明，胃肠道屏障功能在各种胃肠道疾病中受到破坏。我们过去几年的研究表明，氧化应激通过酪氨酸残基上的occludin磷酸化和苏氨酸残基上的去磷酸化来破坏肠上皮紧密连接；这些过程涉及细胞内信号分子，例如 c-Src、FAK、PI3K、PP2A、PKC7、PKC6 和 PP1。我们的研究还表明，EGF（一种胃肠道粘膜保护因子）可以防止氧化应激引起的紧密连接破坏；这个过程涉及信号分子、PLC3、PKC2I、PKC5 和 MAP 激酶。我们的初步数据已经鉴定出occludin中的两个酪氨酸残基和两个苏氨酸残基，它们位于occludin高度保守的基序中，并表明这些残基的磷酸化调节紧密连接的完整性。因此，我们计划进一步继续努力了解这些氨基酸磷酸化在氧化应激诱导的肠上皮屏障功能破坏中的调节以及EGF介导的氧化应激效应的预防。根据我们的结果，假设：a) c-Src 介导的 Y398 和 Y402 上的 occludin 磷酸化介导 H2O2 诱导的肠上皮紧密连接不稳定，b) H2O2 诱导的紧密连接破坏涉及c) EGF 和益生菌通过下调 PKC7 来抑制 T403 和 T404 上的 Occludin 预防氧化应激诱导MAP 激酶依赖性机制导致紧密连接破坏。我们建议确定：1) c-Src 直接与 Occludin 相互作用，并在 H2O2 处理的细胞中磷酸化 Y398/Y402。 2) H2O2 诱导的claudin-3 酪氨酸磷酸化在紧密连接的破坏中发挥作用。 3) c-Src 在 H2O2 诱导的紧密连接蛋白稳态动力学减弱中发挥作用。 4) H2O2 通过 c-Src 介导的 occludin 和 claudin-3 磷酸化加速细胞扩散和迁移。 5) PKC6 通过紧密连接蛋白的 Ser/Thr 磷酸化来调节紧密连接的完整性。 6) 氧化应激通过下调 PKC7 来破坏紧密连接。 7) 紧密连接蛋白在特定 Ser/Thr 残基上的磷酸化可调节其自身缔合和肌动蛋白结合亲和力。 8) 益生菌通过诱导 EGF 受体反式激活来保护紧密连接免受 H2O2 的影响。 9) EGF 和益生菌可改善小鼠肠道缺血/再灌注引起的紧密连接破坏。 10) EGF 和益生菌可减弱 H2O2 诱导的 PKC7 下调、occludin 磷酸化的改变以及人类结肠活检中紧密连接的破坏。这些研究的结果与我们对许多胃肠道疾病发病机制的理解直接相关，并有可能为未来新治疗策略的发展做出贡献。 公共卫生相关性：炎症性肠病和传染性小肠结肠炎等肠道疾病与氧化应激和肠上皮屏障功能障碍引起的组织损伤有关。根据我们过去几年的研究，我们假设氧化应激通过诱导紧密连接蛋白的磷酸化来破坏肠上皮屏障功能，而表皮生长因子等保护性生长因子可以防止氧化应激引起的细胞损伤。我们建议进行研究以揭示这些过程中涉及的细胞和分子机制；这些研究的结果预计将为开发治疗不同肠道疾病的新疗法提供知识。