The Myosin Light Chain Kinase-Phosphatase Axis in GI Homeostasis and Disease

胃肠道稳态和疾病中的肌球蛋白轻链激酶-磷酸酶轴

基本信息

批准号：
8725914
负责人：
JERROLD R. TURNER
金额：
$ 47.01万
依托单位：
UNIVERSITY OF CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2005
资助国家：
美国
起止时间：
2005-07-01 至 2019-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8725914
关键词：
Actomyosin Adherent Culture Animals Apoptosis Award Binding Catalytic Domain Cell Adhesion Chronic Disease Data Development Disease Disease Progression Dominant-Negative Mutation Enterocolitis Epithelial Epithelium Event Exons Foundations Functional disorder Gastrointestinal Diseases Goals Health Homeostasis Human Hypotension Immunoglobulins Impaired wound healing In Vitro Inflammatory Bowel Diseases Intestinal Diseases Intestines Knock-out Knowledge Link Mediating Mission Molecular Molecular Chaperones Muscle Myosin Light Chain Kinase National Institute of Diabetes and Digestive and Kidney Diseases Organ Pathway interactions Patients Permeability Pharmaceutical Preparations Phosphoric Monoester Hydrolases Phosphorylation Phosphotransferases Physiological Process Public Health Publishing RNA Splicing Regulation Research Role Severity of illness Signal Transduction Smooth Muscle Specificity Sterility Systemic disease Tacrolimus Binding Proteins Therapeutic Therapeutic Intervention Tight Junctions Tissues Toxic effect Translations Tumor Necrosis Factor-alpha United States National Institutes of Health Variant Work Wound Healing base chronic graft versus host disease cytokine defined contribution graft vs host disease human disease improved in vivo inhibitor/antagonist innovation insight intestinal epithelium intestinal homeostasis myosin phosphatase novel novel therapeutic intervention prevent programs public health relevance small molecule tool trafficking

项目摘要

DESCRIPTION (provided by applicant): Intestinal barrier dysfunction contributes to progression of gastrointestinal and systemic disease. Over the previous two cycles of this award we have i) discovered mechanisms by which myosin light chain kinase (MLCK) regulates intestinal epithelial tight junction barrier function, in vitro and in vivo; ii) developed tools to prevent this regulation in cultured monolayers and experimental animals; and iii) demonstrated that increasing or reducing intestinal epithelial MLCK activity can enhance or reduce, respectively, progression of both experimental inflammatory bowel disease (IBD) and graft versus host disease (GVHD). Although the tools developed have been extremely informative, they are not suitable for translation to human patients. This is primarily because it is not possibe to specifically inhibit intestinal epithelial MLCK enzymatic activity without also inhibiting smoot muscle MLCK which results in severe, sometimes fatal, toxicities. Further, MLCK serves important functions other than tight junction regulation in intestinal epithelia, including promotin of wound healing. Thus, there is a fundamental gap that separates our previous elucidation of mechanisms and clinicopathologic significance of barrier regulation in disease from development of strategies that can be used to modulate intestinal epithelial tight junction function for therapeutic purposes. This proposal seeks to bridge that gap by building on our recent observations regarding regulation of the MLCK-myosin phosphatase axis in disease. Specifically, we will focus on understanding trafficking of the MLCK1 splice variant. We have shown that tumor necrosis factor (TNF) or chronic disease cause MLCK1 recruitment to the perijunctional actomyosin ring (PAMR), to regulate tight junction permeability. Moreover, we have developed a small molecule inhibitor that blocks this trafficking and is remarkably effective in experimental IBD. Here we propose to define the molecular mechanisms of basal and TNF-induced MLCK1 trafficking and to characterize the therapeutic potential of newly-discovered trafficking inhibitors in experimental IBD and GVHD. Our preliminary data also demonstrate an unexpected, essential, in vivo role of the myosin phosphatase regulatory subunit MYPT1 in mucosal homeostasis. MYPT1 regulates MLC phosphatase activity and specificity and thereby opposes MLCK function. Thus, understanding the means by which MYPT1 loss becomes catastrophic is expected to provide additional new insights into the functions of the MLCK-myosin phosphatase axis in homeostasis and disease. The proposal is innovative because it will define novel regulatory mechanisms and will result in a major shift in our understanding of means to correct barrier function and actomyosin contractile status for therapeutic benefit. The proposed research is significant because it will link specific mechanisms of barrier loss to disease and identify novel therapeutic approaches. Finally, in addition to benefitting diseases associated with intestinal barrier loss, the concepts and tools developed will be applicable to barrier restorative therapy for diseases of other organs that are driven by epithelial or endothelial barrier dysfunction.

描述（由申请人提供）：肠屏障功能障碍有助于胃肠道和全身性疾病的进展。在该奖项的前两个周期中，我们有I）发现了肌球蛋白轻链激酶（MLCK）调节肠上皮紧密屏障功能，体外和体内的机制； ii）开发了工具防止在培养的单层和实验动物中进行调节； iii）证明，增加或减少肠上皮MLCK活性可以分别增强或减少实验性炎症性肠病（IBD）和移植物与宿主疾病（GVHD）的进展。尽管开发的工具非常有用，但它们不适合转化为人类患者。这主要是因为没有可能专门抑制肠上皮MLCK酶活性而不会抑制Smoot肌肉MLCK，从而导致严重的，有时是致命的毒性。此外，MLCK具有重要功能，而不是肠上皮的紧密连接调节，包括促进伤口愈合的促进素。因此，存在一个基本差距，它将我们以前阐明疾病中障碍调节机制的临床病理和临床病理学意义与可用于治疗目的调节肠上皮上皮紧密连接功能的策略的临床病理学意义。该提议试图通过基于我们最近关于调节疾病中MLCK摩西蛋白磷酸酶轴调节的观察来弥合差距。具体而言，我们将专注于了解MLCK1剪接变体的运输。我们已经表明，肿瘤坏死因子（TNF）或慢性疾病会导致MLCK1募集到骨外肌球蛋白环（PAMR），以调节紧密的连接渗透性。此外，我们已经开发了一种小分子抑制剂，该抑制剂可以阻止这种运输，并且在实验性IBD中非常有效。在这里，我们建议定义基底和TNF诱导的MLCK1运输的分子机制，并表征实验性IBD和GVHD中新发现的运输抑制剂的治疗潜力。我们的初步数据还表明，肌球蛋白磷酸酶调节性亚基MYPT1在粘膜稳态中的体内作用出乎意料，必不可少的体内作用。 MYPT1调节MLC磷酸酶活性和特异性，从而相反的MLCK功能。因此，理解MyPT1损失变为灾难性的手段有望提供有关MLCK-肌球蛋白磷酸酶轴在稳态和疾病中的功能的更多新见解。该提案具有创新性，因为它将定义新的调节机制，并将导致我们对纠正障碍功能和肌动菌素收缩状态的手段的理解有重大的转变。拟议的研究很重要，因为它将将障碍丧失的特定机制与疾病联系起来，并确定新颖的治疗方法。最后，除了受益于与肠道障碍丧失相关的疾病外，开发的概念和工具还适用于其他由上皮或内皮屏障功能障碍驱动的其他器官疾病的障碍疗法。