Intestinal Mucosal Protection by Epidermal Growth Factor

描述(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，在各种胃肠道疾病中，胃肠道屏障功能被破坏。我们在过去几年的研究表明，氧化应激通过酪氨酸残基上阻滞素的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的信号分子，如c-Src、FAK、PI3K、PP2A、PKC7、PKC6和PP1。我们的研究还表明，胃肠道粘膜保护因子EGF可以阻止氧化应激引起的紧密连接的破坏；这一过程涉及信号分子PLC3、PKC2I、PKC5和MAP激酶。我们的初步数据已经在occludin中确定了两个酪氨酸残基和两个苏氨酸残基，它们位于一个高度保守的occludin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，我们计划进一步努力了解这些氨基酸的磷酸化在氧化应激诱导的肠上皮屏障功能破坏和EGF介导的预防氧化应激作用中的调节作用。根据我们的结果，我们假设：a)c-Src介导的Y398和Y402上的occludin的磷酸化介导了H_2O_2诱导的肠上皮紧密连接的不稳定，b)H_2O_2诱导的紧密连接的破坏涉及通过下调PKC7使T403和T404上的occludin去磷酸化，以及c)EGF和益生菌通过MAP依赖的机制防止氧化应激诱导的紧密连接的破坏。我们建议确定：1)在H_2O_2处理的细胞中，c-Src直接与occludin相互作用并磷酸化Y398/Y402。2)H_2O_2诱导的Claudin-3酪氨酸磷酸化在紧密连接的破坏中起作用。3)c-Src在H_2O_2诱导的紧密连接蛋白稳态动力学衰减中起作用。4)H_2O_2通过c-Src介导的occludin和claudin-3的磷酸化，加速细胞的伸展和迁移。5)PKC6通过紧密连接蛋白的丝氨酸/苏氨酸磷酸化来调节紧密连接的完整性。6)氧化应激通过下调PKC7的表达来破坏紧密连接。7)紧密连接蛋白在特定的Ser/Thr残基上的磷酸化调节它们的自结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H_2O_2的影响。9)EGF和益生菌可改善缺血/再灌流所致的小鼠肠紧密连接断裂。10)EGF和益生菌可减轻H_2O_2诱导的人结肠活检组织中PKC7的下调、封闭蛋白磷酸化的改变和紧密连接的破坏。这些研究的结果直接关系到我们对许多胃肠道疾病的发病机制的理解，并有可能为未来新的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道上皮屏障功能障碍造成的组织损伤有关。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，而保护性生长因子如表皮生长因子通过氧化应激来阻止这种细胞损伤。我们建议进行研究，以揭示参与这些过程的细胞和分子机制；这些研究的结果有望为开发治疗不同肠道疾病的新疗法提供知识。

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.