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

描述（由申请方提供）：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i）发现了肌球蛋白轻链激酶（MLCK）在体外和体内调节肠上皮紧密连接屏障功能的机制; ii）开发了工具， 在培养的单层和实验动物中阻止这种调节;和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）和移植物抗宿主病（GVHD）的进展。尽管开发的工具信息量很大，但它们不适合翻译给人类患者。这主要是因为它不可能特异性抑制肠上皮MLCK酶活性而不抑制平滑肌MLCK，这会导致严重的，有时是致命的毒性。此外，MLCK在肠上皮中除了调节紧密连接之外还具有重要功能，包括促进伤口愈合。因此，有一个根本的差距，分离我们以前的阐明机制和疾病中的屏障调节的临床病理意义的发展战略，可用于调节肠上皮细胞紧密连接功能的治疗目的。这项建议旨在弥合这一差距的基础上，我们最近的观察MLCK-肌球蛋白磷酸酶轴的调节疾病。具体来说，我们将重点了解MLCK 1剪接变体的贩运。我们已经表明，肿瘤坏死因子（TNF）或慢性疾病导致MLCK 1募集到连接周围肌动球蛋白环（PAMR），以调节紧密连接的通透性。此外，我们已经开发了一种小分子抑制剂，可以阻断这种运输，并且在实验性IBD中非常有效。在这里，我们建议定义的基础和TNF诱导MLCK 1贩运的分子机制，并表征新发现的贩运抑制剂在实验性IBD和GVHD的治疗潜力。我们的初步数据也表明了一个意想不到的，必不可少的，在体内的作用，肌球蛋白磷酸酶调节亚基MYPT 1在粘膜内稳态。MYPT 1调节MLC磷酸酶活性和特异性，从而对抗MLCK功能。因此，了解MYPT 1损失成为灾难性的手段，预计将提供更多的MLCK-肌球蛋白磷酸酶轴的功能，在稳态和疾病的新见解。该提案是创新的，因为它将定义新的调节机制，并将导致我们对纠正屏障功能和肌动球蛋白收缩状态以获得治疗益处的理解发生重大转变。这项研究意义重大，因为它将把屏障丧失的具体机制与疾病联系起来，并确定新的治疗方法。最后，除了有益于与肠道屏障丧失相关的疾病外，所开发的概念和工具将适用于由上皮或内皮屏障功能障碍驱动的其他器官疾病的屏障恢复治疗。

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.