Targeting Tight Junctions To Treat Mucositis

描述（由申请人提供）：我们已经鉴定了一种新的肽，其通过靶向连接相邻上皮细胞的紧密连接（TJ），从而保护粘膜屏障，在放射诱导的口腔粘膜炎的小鼠和仓鼠模型中充当有效的治疗剂。该肽来源于在人胃上皮细胞中表达的18 kD胃窦粘液蛋白（AMP-18）的中心结构域。合成的21-mer肽刺激用于模拟口腔粘膜的人角质形成细胞（HaCaT系）的生长，并且在细胞培养物中还具有保护性和运动原性。重组人AMP-18与正常人口腔粘膜组织中角质形成细胞的质膜结合，表明其作用是受体介导的。AMP肽和重组蛋白都保护氧化剂、吲哚美辛或低钙应激损伤的人上皮细胞培养物的屏障功能和结构。AMP-18似乎通过限制损伤后TJ蛋白的损失、增强这些蛋白组装成新的TJ以及稳定连接周围肌动蛋白来发挥其保护作用。在细胞培养中，AMP-18激活Akt和蛋白激酶C-ζ（PKC 6），其可以介导观察到的胞质蛋白如Par 6和Cdc 42易位到连接结构域中，从而促进TJ破坏后新的或受损的连接的蛋白质组装。在小鼠体内粘膜和人上皮细胞培养物中，AMP肽增加TJ蛋白（occludin，ZO-1）的积累，并刺激p38 MAP激酶和热休克蛋白25的磷酸化，从而稳定肌动蛋白丝。为了确定AMP肽是否可以在体内保护口腔粘膜，在口腔粘膜炎模型中研究了小鼠舌和仓鼠颊粘膜的辐射诱导的损伤。AMP肽处理保护了小鼠舌的表面上皮和结缔组织，并延迟了仓鼠颊粘膜中溃疡形成的出现并降低了溃疡形成的程度。该项目的目标是找出AMP-18如何发挥其新的作用，因为它是我们所知道的唯一一种增加特定TJ蛋白积累和促进其组装的药物，并防止其在损伤后丢失。为了确定AMP-18如何靶向口腔和胃肠道（GI）粘膜中的上皮细胞TJ，我们有两个具体目标：（1）鉴定和表征口腔粘膜细胞中的细胞表面AMP-18受体，以及（2）鉴定TJ蛋白组装所需的AMP-18受体活化的下游靶标。一个下拉（亲和力）的策略已被用来确定一个推定的80 kD AMP-18结合蛋白，和人噬菌体展示cDNA文库的生物淘选已经确定了一个候选序列，可以代表AMP-18受体内的配体结合结构域。我们将确定这些候选受体中的一个或两个是否可以接受AMP-18作为配体。实现我们的目标将为开发一种使用AMP肽来保护和修复在放疗和化疗后发生粘膜炎的癌症患者的口腔粘膜中的TJ的策略提供理论基础。 公共卫生相关性：癌症患者的放疗和化疗通常会损害胃肠道（GI）的粘膜内层-特别是口腔和咽喉，这是一种称为粘膜炎的并发症。我们已经确定并表征了由GI细胞产生的蛋白质片段，该蛋白质片段靶向并加强了口腔和胃肠道细胞之间的连接。该项目旨在了解这种蛋白质片段如何发挥其生物学作用，因为当它被给予患有实验性口腔粘膜炎的小鼠或仓鼠时，它可以防止并加速损伤的恢复。

DESCRIPTION (provided by applicant): We have identified a novel peptide that acts as an effective therapeutic agent in mouse and hamster models of radiation-induced oral mucositis by targeting tight junctions (TJs) that connect adjacent epithelial cells, thereby protecting the mucosal barrier. This peptide is derived from a central domain of an 18 kD Antrum Mucosal Protein (AMP-18) that is expressed in human gastric epithelial cells. The synthetic 21-mer peptide stimulates growth of human keratinocytes (HaCaT line) used to model the oral mucosa, and also has protective, and motogenic properties in cell culture. Recombinant human AMP-18 binds to the plasma membrane of keratinocytes in normal human oral mucosal tissue suggesting that its effects are receptor mediated. Both AMP peptide and the recombinant protein protect barrier function and structure in human epithelial cell cultures injured by an oxidant, indomethacin, or low-calcium stress. AMP-18 appears to exert its protective effects by limiting the loss of TJ proteins after injury, enhancing assembly of these proteins into new TJs, and also stabilizing perijunctional actin. In cell culture, AMP-18 activates Akt and protein kinase C-zeta (PKC6) which could mediate the observed translocation of cytosolic proteins such as Par6 and Cdc42 into junctional domains thereby facilitating protein assembly for new or damaged junctions after TJ disruption. In murine mucosa in vivo and human epithelial cell cultures, AMP peptide increased accumulation of TJ proteins (occludin, ZO-1), and stimulated the phosphorylation of p38 MAP kinase and heat shock protein 25 which could stabilize actin filaments. To determine if AMP peptide could protect the oral mucosa in vivo, radiation- induced injuries of the mouse tongue and hamster buccal mucosa were studied in models of oral mucositis. AMP peptide treatment protected the surface epithelium and connective tissue of the mouse tongue, and delayed the appearance and reduced the extent of ulcer formation in the buccal mucosa of hamsters. The goal of this project is to find out how AMP-18 exerts its novel effects, as it is the only agent of which we are aware that increases accumulation and facilitates assembly of specific TJ proteins, and protects against their loss following injury. To determine how AMP-18 targets epithelial cell TJs in the oral and gastrointestinal (GI) mucosa we have two specific aims: (1) identify and characterize the cell surface AMP-18 receptor in oral mucosal cells, and (2) identify down stream targets of AMP-18 receptor activation required for assembly of TJ proteins. A pull down (affinity) strategy has been used to identify a putative 80 kD AMP-18 binding protein, and biopanning of a human phage-display cDNA library has identified a candidate sequence that could represent a ligand-binding domain within the AMP-18 receptor. We will determine if one or both of these candidate receptors can accept AMP-18 as a ligand. Achieving our aims will provide a rationale for developing a strategy using AMP peptide to protect and repair TJs in the oral mucosa of cancer patients who develop mucositis following radiation and chemotherapy. PUBLIC HEALTH RELEVANCE: Radiation therapy and chemotherapy in cancer patients often damages the mucosal lining of the gastrointestinal (GI) tract - especially the mouth and throat, a complication called mucositis. We have identified and characterized a fragment of a protein made by GI cells that targets and strengthens the connections between cells that line the mouth and GI tract. This project is aimed at learning how this protein fragment exerts its biological effects because when it is given to mice or hamsters with experimental oral mucositis, it protects against and speeds recovery from injury.