粘合蛋白β4 表达调节机理和在急性肺损伤中的作用

Acute lung injury (ALI) is a challenging clinical complication, characterized by increased lung vascular permeability and inflammation, associated with significant morbidity and mortality. With over 100,000 cases of severe ALI occurring annually in the United States and even more ALI cases occurring in China, improving our understanding of ALI would constitute a major advance in clinical practice and health care. A primary goal in the prevention and treatment of ALI is the enhancement of endothelial junction integrity, potentially by increasing junction protein expression and reorganizing endothelial cell (EC) junctions..Integrins, transmembrane receptors bridging cell-extracellular matrix (ECM) interactions, comprise of 18 integrin α subunits and 8 integrin β subunits, where one α unit associates with one β unit to form an integrin αβ complex. Once an integrin αβ complex binds with a ligand, such as collagen, fibronectin or laminin, it induces tyrosine phosphorylation of the integrin β intracellular domain, triggering downstream signaling pathways. Integrin β4 has an unique cytoplasmic domain (1072 amino acids), which contains one CALX-B domain, four fibronectin-like domains, and a TAM subdomain and proximity link sequence. To date, the importance of the integrin β4 in the normal cellular function and in the process of inflammatory response remains largely undefined. We previously reported the protective effects of simvastatin, an HMG CoA-reductase inhibitor, in a murine model of LPS-induced ALI, accompanied by a dramatic induction of integrin β4. Moreover, silencing integrin β4 enhanced EC inflammatory response to lipopolysaccharide, indicating the importance of the association of integrin β4 with α6. We also identified integrin β4 as a novel target of microRNA-9 by screening the 3’UTR sequence of integrin β4 mRNA. More recently, we have determined the critical role of integrin β4 in EC inflammatory responses, and the importance of integrin β4 tyrosine phosphorylation to integrin β4 function in both in vivo and in vitro models. .In the proposed project, using a combination of molecular biology, cell biology and animal model strategies, we will: (1) breed endothelial integrin β4 knockout mice; (2) isolation of pulmonary endothelial cells from flox/flox control mice and integrin β4 knockout mice and sets up stable cell line; (3) clarify the important role of integrin β4 in acute lung injury-mediated inflammatory response on In Vivo and In Vitro model; (4) Identity the genetic and epigenetic regulatory mechanism of integrin β4 expression in endothelial cells; (5) clarify the importance of PI3K-AKT signal pathway for integrin β4 expression regulation. This proposal is aimed at understanding the regulatory mechanisms responsible for integrin β4 expression in acute lung injury. These studies collectively provide the basis for ongoing research, by our lab and others, focused on potential novel therapeutic targets and strategies for patients with acute lung injury.

保护或修复因急性肺损伤破坏的内皮屏障一直是临床科研的重点。我们的前期研究发现粘合蛋白β4 (Integrin β4)可能是急性肺损伤的重要媒介。粘合蛋白β4和其他的蛋白一起在斑点联接中形成复合物，接受外界刺激信号，通过自身氨基酸的磷酸化，激活胞内下游信号通路；通过和内皮细胞的骨架蛋白相连，调节细胞骨架形态，影响内皮细胞屏障的完整性。但是，粘合蛋白β4的表达调节机制和它在急性肺损伤中的作用知之甚少。因此在本项目中，通过分子生物学，细胞生物学技术，在细胞和动物模型上，（1）培育内皮细胞特异敲除粘合蛋白β4小鼠；（2）分离并建立稳定的内皮细胞特异敲除粘合蛋白β4的细胞系；（3）在载体和离体模型上，明确粘合蛋白β4在急性肺损伤炎症中的重要性；(4)明确表观基因调节在粘合蛋白β4表达过程中的作用；（5） 明确PI3K-AKT 信号通路对粘合蛋白β4的基因和表观基因调节的重要性。通过粘合蛋白β4的功能研究，为急性肺损伤提供理论支持和新的临床治疗方案。

Integrin β4广泛存在于上皮，内皮和癌症细胞中。integrin β4的功能在上皮细胞和癌症细胞中重要性被广泛的研究过。在正常的上皮中，integrin β4通过和配体laminin-5结合，是固定上皮细胞的重要结构。由于integrin β4的胞内独特的功能域，它又参与了许多细胞的其他功能，如机械刺激感受，细胞生长的调节等。在癌症细胞中，integrin β4的异常表达和癌症的恶化程度密切相关。 和integrin β4在上皮细胞和癌症细胞中重要性研究相比，integrin β4在内皮细胞中的重要性知之甚少。在本项目中，我们在分子生物水平和细胞水平上鉴定integrin β4胞外区域的功能以及它对内皮细胞的命运的影响。除了特异性敲除内皮细胞的integrin β4的老鼠模型外，我们同时克隆integrin β4的胞外区和包内区段的基因，在细胞和动物模型上探讨integrin β4在内皮细胞中的重要性。.（1）我们首次阐明integrin β4在内皮细胞中自然条件下的表达机制。该发现第一次清晰阐明驱动子，表观基因调节在integrin β4表达中的重要性。并在此基础上，探索一些相关药物对integrin β4的调节作用，为临床应用打下基础。（2）我们成功培养出内皮细胞特异敲除 integrin β4小鼠，并在此基础上，成功分离肺内皮细胞。通过细胞和在体实验，详细和清晰阐明了integrin β4在炎症刺激过程中的作用。（3）在人肺动脉内皮细胞中，通过高表达integrin β4的不同功能区，探索integrin β4对内皮细胞的生理功能和分化的影响。研究显示integrin β4是控制内皮细胞的分化和成熟的关键蛋白。（4）在阐明integrin β4的表达机制基础上，我们发现一系列新的调节药物，如TSA, ABO, simvastatin, atorvastatin等。这些药物对急性肺损伤的治疗研究，特别是在当前新冠肺炎肆虐的情况下，有重要的临床意义和紧迫性。（5）在阐明integrin β4的表达机制基础上，我们鉴定wwox，一种抑癌基因，在吸烟导致的肺癌和炎症过程中与integrin β4的相互调节作用和相关临床后果。.通过该项目的实施，科学家对integrin β4在内皮细胞中的功能有更深刻的了解和开发出一系列相关药物；在此基础上，我们开拓出新的项目。