Role of Connexin47 mutations in primary lymphedema

描述(申请人提供)：这个项目的目标是探索某些形式的原发淋巴水肿是由连接蛋白(Cx)基因突变导致的瓣膜形成缺陷引起的。淋巴(Ly)系统功能对正常的组织液平衡、免疫功能以及膳食脂肪的吸收和运输至关重要，瓣膜确保淋巴向前运动，而不是异常聚集或回流。Ly系统的缺陷，包括那些可能涉及瓣膜缺陷或功能不全的缺陷，会导致一些先天性和获得性疾病。淋巴水肿尤其是一种潜在的衰弱疾病，在这种疾病中，间质液体异常积聚，估计会影响 全球有多达1.4亿至2.5亿人。尽管在识别与Ly发育有关的关键基因方面取得了进展，但控制Ly瓣膜形态发生的分子机制仍然知之甚少。因此，对Ly瓣膜发育过程中分子通路功能的了解的增加为预防或治疗淋巴疾病(如淋巴水肿)提供了一个极好的机会。最近，在瓣膜开发中发现了一个新的关键角色：CXS--一系列相关蛋白质，它们组装成缝隙连接细胞间通道，这种结构允许小分子在相邻细胞之间直接转移。以前的血管工作主要集中在CXS在动脉和小动脉的发育、重塑和生理中的作用。然而，我们发现三种CXs(Cx37、Cx43和Cx47)在发育中的Ly血管的Ly内皮细胞中表达，并且这些蛋白在Ly瓣膜(和静脉瓣膜)中逐渐丰富。使用全球Cx基因敲除小鼠，Cx37和Cx43被证明对Ly瓣膜的形成至关重要。虽然Cx47在一些有原发淋巴水肿的家系中被证明是突变的，但关于Cx47在Ly的发育或功能中的作用尚不清楚。基于Cx47错义突变在高度保守的Cx细胞外环中的位置，我们认为这些突变本质上是显性负性的，并通过联合抑制野生型Cx47和Cx43(位于瓣膜)而导致原发性淋巴水肿，从而导致瓣膜发育缺陷。在这项建议中，我们将在小鼠模型中比较Cx47零突变和假定的Cx47显性负突变导致人类淋巴水肿的影响。这些突变对Ly瓣膜形成、Ly功能和淋巴水肿的发展的影响将在小鼠中进行评估。此外，Cx47和Cx43的联合零突变的影响将被确定为对Cx47和Cx43都必须抑制严重疾病表型这一理论的另一种检验。该项目中产生的小鼠系将为研究原发淋巴水肿提供有价值的新的动物模型，并允许更好地了解Ly功能障碍如何启动或促成疾病。此外，小鼠模型将对未来旨在确定导致继发性淋巴水肿的局部生理条件的研究有用。

DESCRIPTION (provided by applicant): The goal of this project is to explore the idea that some forms of primary lymphedema are caused specifically by defective valve formation resulting from mutations in Connexin (Cx) genes. Lymphatic (Ly) system function is essential for normal tissue fluid balance, immune function, and absorption and transport of dietary fat, and valves ensure that lymph moves forward rather than abnormally collecting or refluxing. Defects of the Ly system, including those that may involve defective or incompetent valves, lead to a number of congenital and acquired disorders. Lymphedema, in particular, is a potentially debilitating disorder in which interstitial fluid abnormally accumulates and is estimated to affect as many as 140-250 million people worldwide. Despite progress in identifying key genes involved in Ly development, the molecular mechanisms controlling the morphogenesis of Ly valves remain poorly understood. Thus, increased knowledge about the molecular pathways functioning during Ly valve development provides an excellent opportunity for new approaches to prevent or treat lymphatic disorders such as lymphedema. Recently, a new key player in valve development was discovered: Cxs - a family of related proteins which assemble into gap junction intercellular channels, structures that allow for the direct transfer of small molecules between adjacent cells. Previous vascular work focused on the role of Cxs in the development, remodeling, and physiology of arteries and arterioles. However, we have discovered that three Cxs (Cx37, Cx43, and Cx47) are expressed in the Ly endothelial cells of developing Ly vessels and that these proteins become progressively enriched at Ly valves (and venous valves). Using global Cx knockout mice, Cx37 and Cx43 were shown to be critical for Ly valve formation. Although Cx47 was shown to be mutated in some families with primary lymphedema, nothing is known about the role of Cx47 in Ly development or function. Based on the location of the Cx47 missense mutations in highly conserved Cx extracellular loops, we propose that the mutations are dominant negative in nature and result in primary lymphedema through a combined inhibition of wild-type Cx47 and Cx43 (which colocalize at valves), leading to defective valve development. In this proposal, we will compare the effects, in mouse models, of a Cx47 null mutation and a putative Cx47 dominant negative mutation that causes lymphedema in humans. The effects of these mutations on Ly valve formation, Ly function, and development of lymphedema will be assessed in the mice. In addition, the effects of a combined null mutation of Cx47 and Cx43 will be determined as another test of the theory that both Cx47 and Cx43 must be inhibited for a severe disease phenotype. The mouse lines generated in this project will provide valuable new animal models for the study of primary lymphedema and allow for a better understanding of how Ly dysfunction initiates or contributes to disease. In addition, the mouse models will be useful for future studies aimed at identifying the local physiological conditions that contribute to secondary lymphedema.