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Targeting Tight Junctions To Treat Mucositis

针对紧密连接治疗粘膜炎

基本信息

批准号：
7817005
负责人：
FREDERICK Gary TOBACK
金额：
$ 23.4万
依托单位：
UNIVERSITY OF CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-05-01 至 2011-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7817005
关键词：
Actins Adherent Culture Affinity Amino Acids Animals Antineoplastic Agents Appearance Area Bacteremia Bacteria Binding Binding Proteins Biological Boston Calcium Cancer Patient Cell Culture Techniques Cell Line Cell Surface Receptors Cell membrane Cell surface Cells Clinical Complication Connective Tissue Cytoskeleton Development Dose-Limiting Epithelial Epithelial Cells Epithelium Exhibits Figs - dietary Gastrointestinal tract structure Goals Growth Hamsters Healed Heat shock proteins Hospitals Human Indigenous Indomethacin Injury Learning Length Lesion Letters Ligand Binding Domain Ligands Mediating Microfilaments Modeling Molecular Mucositis Mucous Membrane Mus Oral Oral Ulcer Oral cavity Oral mucous membrane structure Oxidants Pain Pathway interactions Patients Peptides Phage Display Pharyngeal structure Phosphorylation Property Protein Binding Protein Fragment Proteins Proto-Oncogene Proteins c-akt Publishing Pyloric antrum Radiation Radiation therapy Receptor Activation Recombinant Proteins Recombinants Recovery Regimen Reporting Research Sepsis Speed Stomach Stratified Squamous Epithelium Stream Stress Structure Surface Testing Therapeutic Therapeutic Agents Tight Junctions Tongue Toxin Ulcer Woman cDNA Library cell injury chemotherapy effective therapy gastrointestinal healing in vivo injured insight keratinocyte mitogen-activated protein kinase p38 novel novel therapeutics occludin oral cavity epithelium oral mucositis prevent protective effect protein kinase C zeta public health relevance receptor repaired seal tissue culture

项目摘要

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.

描述（由申请人提供）：我们已经确定了一种新的肽，它通过靶向连接邻近上皮细胞的紧密连接（TJs），从而保护粘膜屏障，在辐射诱导的口腔黏膜炎小鼠和仓鼠模型中作为有效的治疗剂。该肽来源于在人胃上皮细胞中表达的18kd胃窦粘膜蛋白（AMP-18）的中心结构域。合成的21-mer肽刺激用于口腔粘膜建模的人角化细胞（HaCaT系）的生长，并且在细胞培养中也具有保护和致动特性。重组人AMP-18与正常人口腔黏膜组织角质形成细胞的质膜结合，表明其作用是受体介导的。AMP肽和重组蛋白都能保护被氧化剂、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，促进这些蛋白组装成新的TJ，并稳定周围结膜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-zeta (PKC6)，这可以介导观察到的细胞质蛋白（如Par6和Cdc42）易位到连接域，从而促进TJ破坏后新连接或受损连接的蛋白质组装。在小鼠体内粘膜和人上皮细胞培养中，AMP肽增加TJ蛋白（occludin, ZO-1）的积累，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肽在体内是否对口腔黏膜具有保护作用，我们在口腔黏膜炎模型中研究了辐射对小鼠舌和仓鼠颊黏膜的损伤。AMP肽处理能保护小鼠舌表面上皮和结缔组织，延缓仓鼠颊黏膜溃疡的出现，降低溃疡形成程度。该项目的目标是找出AMP-18如何发挥其新作用，因为它是我们所知道的唯一一种增加特定TJ蛋白的积累和促进其组装，并防止其在损伤后损失的药物。为了确定AMP-18如何靶向口腔和胃肠道（GI）粘膜上皮细胞TJs，我们有两个特定的目的：(1)鉴定和表征口腔粘膜细胞表面AMP-18受体，(2)鉴定TJ蛋白组装所需的AMP-18受体激活的下游靶点。采用下拉（亲和）策略鉴定了一个假定的80 kD的AMP-18结合蛋白，并对人类噬菌体展示cDNA文库进行生物筛选，鉴定了一个候选序列，该序列可能代表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利用AMP肽保护和修复放射和化疗后发生粘膜炎的癌症患者口腔黏膜TJs的策略提供理论依据。