Unconventional myosins and the regulation of gut barrier integrity and restitution during inflammation

非常规肌球蛋白以及炎症期间肠道屏障完整性和恢复的调节

基本信息

批准号：
10443882
负责人：
Andrei Ivanovich Ivanov
金额：
$ 45.26万
依托单位：
CLEVELAND CLINIC LERNER COM-CWRU
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-09-15 至 2024-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10443882
关键词：
3-Dimensional Actomyosin Acute Address Adherens Junction Adhesions Affect Air Apical Basal Cell Binding Biochemical Biological Markers Biosensor CRISPR/Cas technology Celiac Disease Cell Culture Techniques Cell Death Cell Differentiation process Cell Line Cell Polarity Cell membrane Cells Chronic Phase Colitis Complex Crohn&apos s disease Cytoskeleton Data Defect Defense Mechanisms Destinations Detergents Development Digestive System Disorders Disease Disease remission Down-Regulation Epithelial Epithelial Attachment Epithelial Cells Event Extracellular Matrix Fluorescence Resonance Energy Transfer Fractionation Gastrointestinal Diseases Genes Genetic Gut Mucosa Health Human Image Immunoprecipitation Impairment In Vitro Incidence Infectious colitis Inflammation Inflammatory Inflammatory Bowel Diseases Injury Intestinal Diseases Intestinal Mucosa Intestinal permeability Knock-out Knockout Mice Knowledge Leaky Gut Link Liquid substance Malignant Neoplasms Measurement Mediating Microfilaments Microscopy Modeling Molecular Morbidity - disease rate Morphogenesis Motor Mucositis Mucous Membrane Muscle Myosin ATPase Myosin Type II Neurons Organoids Outcome Pathogenesis Patients Periodicity Permeability Pharmacology Physiology Prevention Process Public Health Recovery Regulation Resolution Role Scaffolding Protein Signal Transduction Structure Testing Therapeutic Intervention Tight Junctions Ulcerative Colitis Western Blotting attenuation cell motility cohort cytokine disorder control epithelial injury experimental study gut homeostasis gut inflammation healing improved in vivo injured innovation insight intestinal barrier intestinal epithelium knockout animal monolayer mortality motor control mouse model mutant new therapeutic target non-muscle myosin novel overexpression pathogenic bacteria prevent repaired restoration targeted treatment tumor growth wound wound healing

项目摘要

PROJECT SUMMARY: Disruption of the intestinal epithelial barrier in patients with inflammatory bowel diseases (IBD) involves disassembly of epithelial tight junctions (TJ) and adherens junctions (AJ), along with formation of mucosal wounds due to excessive epithelial cell death. Restoration of the gut barrier (epithelial restitution) is critical for achieving remission of the disease and it involves mucosal wound healing and AJ/TJ reassembly. Both processes are mediated by common molecular events, most notably, dramatic remodeling of the epithelial actomyosin cytoskeleton. An unconventional myosin 18A (Myo18A) is a unique PDZ-domain containing scaffolding protein that acts as an essential regulator of actin filaments and their motor, non-muscle myosin II (NM II). Myo18A is known to control neuronal and muscle morphogenesis, as well as tumor growth and dissemination, however its functions in the gut have not been previously investigated. Our preliminary data suggests that Myo18A is abundantly expressed in normal human intestinal epithelium and it is a novel component of epithelial junctions, Furthermore, Myo18A depletion leads to various functional defects, including disruption of the epithelial barrier, attenuation of wound healing and impairment of 3-D epithelial morphogenesis. Importantly, we observed a marked downregulation of Myo18A expression in the intestinal epithelium of ulcerative colitis patients. This exciting preliminary data provides a strong scientific premise for the following innovative hypothesis: Myo18A is a novel regulator of the intestinal epithelial barrier integrity and restitution, and its depletion promotes gut leakiness and inhibits mucosal healing in IBD patients. This hypothesis will be tested in the following Aims: (1) to determine the roles of Myo18A in the regulation of intestinal epithelial cell migration and ECM adhesion; (2) to delineate the mechanisms of Myo18A-dependent assembly of the epithelial barrier and establishment of the apico-basal cell polarity; 3) to examine the roles of Myo18A in regulating disruption and restitution of the intestinal epithelial barrier in vivo. The Aims will be accomplished by studies utilizing in vitro intestinal epithelial cell monolayers, ex vivo colonic organoids generated from IBD mucosa and in vivo murine models of epithelial injury and restitution. Roles of Myo18A will be examined by a combination of functional (permeability measurements, wound healing), biochemical (immunoblotting, detergent fractionation), imaging (FRET biosensors, super-resolution microscopy), and genetic (CRISPR/Cas9 gene editing, overexpression of deletion mutants, knockout mice) approaches. Significance: the proposed study will yield novel insights into the mechanisms that regulate intestinal epithelial injury and restitution during inflammation. It will also identify new therapeutic targets to prevent breakdown and enhance reparation of the gut barrier in patients with digestive diseases.

项目摘要：炎症患者肠上皮屏障的破坏疾病（IBD）涉及上皮紧密连接（TJ）和粘附连接（AJ）的拆卸，以及由于上皮细胞死亡过多而导致的粘膜伤。修复肠道障碍（上皮恢复原状）对于实现疾病的缓解至关重要，它涉及粘膜伤口愈合和AJ/TJ 重新组装。这两个过程均由常见分子事件介导，最值得注意的是，上皮性肌球蛋白细胞骨架。非常规肌球蛋白18a（myo18a）是独特的PDZ域含有脚手架蛋白，可作为肌动蛋白丝及其电动机的必不可少的调节剂肌球蛋白II（NM II）。已知MyO18a可以控制神经元和肌肉形态发生，以及肿瘤的生长但是，以前尚未研究其在肠道中的功能。我们的初步数据表明Myo18a在正常的人类肠上皮中大量表达，这是一种新颖上皮连接的组成部分，此外，Myo18a耗竭会导致各种功能缺陷，包括上皮屏障的破坏，伤口愈合的衰减和3D上皮的损害形态发生。重要的是，我们观察到肠道中肌18a表达的明显下调溃疡性结肠炎患者上皮。这个令人兴奋的初步数据为科学的前提提供了强大的前提以下创新假设：Myo18a是肠上皮屏障的新型调节剂完整性和恢复原状及其耗竭会促进肠道泄漏，并抑制IBD中的粘膜愈合患者。该假设将在以下目的中进行检验：（1）确定myo18a在调节中的作用肠上皮细胞迁移和ECM粘附；（2）描述依赖MyO18A的机制上皮屏障的组装和Apico-Basal细胞极性的建立； 3）检查 Myo18a在体内调节肠上皮屏障的破坏和恢复。目的将是通过使用体外肠上皮细胞单层的研究完成由IBD粘膜和上皮损伤和恢复原状的体内鼠模型产生。 Myo18a的角色将通过功能（渗透率测量，伤口愈合），生化的结合来检查（免疫印迹，洗涤剂分馏），成像（FRET生物传感器，超分辨率显微镜）和遗传（CRISPR/CAS9基因编辑，缺失突变体的过表达，敲除小鼠）方法。意义：拟议的研究将对调节肠上皮的机制产生新的见解炎症期间的伤害和恢复原状。它还将确定新的治疗靶标，以防止细分和增加消化疾病患者的肠道屏障的赔偿。

项目成果

期刊论文数量（3）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Inflammation modulates intercellular adhesion and mechanotransduction in human epidermis via ROCK2.

DOI：
10.1016/j.isci.2023.106195
发表时间：
2023-03-17
期刊：
ISCIENCE
影响因子：
5.8
作者：
Shutova, Maria S.;Borowczyk, Julia;Russo, Barbara;Sellami, Sihem;Drukala, Justyna;Wolnicki, Michal;Brembilla, Nicolo C.;Kaya, Gurkan;Ivanov, Andrei I.;Boehncke, Wolf-Henning
通讯作者：
Boehncke, Wolf-Henning

Unique and redundant functions of cytoplasmic actins and nonmuscle myosin II isoforms at epithelial junctions.