Role of the S100 Family of Proteins in Lens Physiology and Cataract

项目摘要 眼透镜在将入射光聚焦到视网膜上方面起着至关重要的作用，其中透镜透明度中存在像差 以及导致视力受损的机械性能。尽管不断努力，我们对 控制透镜细胞结构、稳态、变形性、透明度和晶状体内分泌的各种分子途径， 白内障的病因机制仍不完全。我们最近发现S100的几个成员 钙结合蛋白家族在透镜中大量表达并显示出不同的分布特征。 S100 A6和S100 A10的表达分布于透镜上皮和纤维，而S100 A4的表达分布于晶状体上皮和纤维。 离散地到透镜纤维细胞。不存在S100 A4但不存在S100 A6或S100 A10导致乳光迟发型 小鼠模型中的白内障形成，以及S100 A5的强烈上调， 在野生型透镜中是检测不到的。除了S100 A4的这些新数据，我们目前没有 了解S100蛋白家族在透镜功能中的作用。S100 A4在细胞中表达， 组织特异性方式，并参与各种疾病的病理生理学。的生理功能 然而，S100 A4却鲜为人知。S100 A4的一个很好理解的分子功能是它与 非肌肉肌球蛋白II（NM II），特别是NM IIA，以及NM II组装，肌动蛋白聚合， 收缩特性、细胞迁移和可塑性。NM II在透镜细胞结构中起关键作用， 机械和功能。尽管NM IIA突变与人类白内障的发生有关， 已知在透镜功能的背景下调节NM II活性。虽然镜片来自成熟的 S100 A4 null小鼠晶状体（2至6个月大）保持清晰，并保持正常的生长和大小， 一个月大的小鼠表现出NM IIA的组装和磷酸化、肌动蛋白的聚合和 水通道蛋白-5、CLIC 5、连接蛋白-50、细胞粘附蛋白（整合素-β1、NrCAM、ZO-1）水平的改变 和α-晶状体蛋白，最终在八个月大的小鼠中形成乳白色白内障。基于这些 初步和新的发现，我们假设S100 A4在调节NM II组装中起着至关重要的作用， 透镜，并且S100 A4的缺乏破坏NM II组装和磷酸化，肌动蛋白聚合， 机械性能和透镜中的渗透稳态，导致迟发性白内障形成。为了验证这一 假设，在三个相互关联的具体目标，我们将探讨：1）在调节机制中的作用S100 A4 透镜NM II组装和磷酸化、肌动蛋白聚合、细胞粘附和透镜拉伸性能的影响，2） 白内障发生的基础是晶状体调节和溶质载体转运机制的破坏， S100 A4无效晶状体，和3）拯救S100 A4无效小鼠透镜纤维中S100 A4表达的作用（使用 转基因方法）对细胞骨架完整性和透镜表型的影响。这些新的和相互关联的研究是 有望揭示S100 A4和S100家族蛋白的其他成员在透镜功能中的重要性， 已知其参与多种疾病的病理生理学。

PROJECT SUMMARY The ocular lens plays a crucial role in focusing incident light on to the retina, with aberrations in lens transparency and mechanical properties leading to impairment of vision. Despite continuing effort, our understanding of the various molecular pathways governing lens cytoarchitecture, homeostasis, deformability, clarity, and the etiological mechanisms of cataract remains incomplete. We recently identified that several members of the S100 family of calcium binding proteins are abundantly expressed and exhibit distinct distribution profiles in the lens. While expression of S100A6 and S100A10 distributed to both the lens epithelium and fibers, S100A4 distributes discretely to lens fiber cells. Absence of S100A4 but not S100A6 or S100A10 leads to opalescent late-onset cataract formation in a mouse model, together with the robust upregulation of S100A5, the expression of which was otherwise undetectable in the wild type lens. Other than these new data on S100A4, we currently have no knowledge of the role played by the S100 family of proteins in lens function. S100A4 is expressed in a cell and tissue specific manner and is involved in the pathophysiology of various diseases. The physiological function of S100A4 however, is poorly understood. A well-understood molecular function of S100A4 is its interaction with non-muscle myosin II (NM II), especially NM IIA, and regulation of NM II assembly, actin polymerization, contractile characteristics, cell migration, and plasticity. NM II plays a crucial role in lens cytoarchitecture, mechanics, and function. Though NM IIA mutations are associated with cataract development in humans, little is known about regulation of NM II activity in the context of lens function. While lenses derived from mature S100A4 null mouse lenses (2 to 6 month old) remain clear and maintain normal growth and size, those from six month-old mice exhibit impairments in assembly and phosphorylation of NM IIA, polymerization of actin, and alterations in the levels of aquaporin-5, CLIC5, connexin-50, cell adhesive proteins (integrin-β1, NrCAM, ZO-1) and α-crystallin, culminating in opalescent cataract formation in eight month-old mice. Based on these preliminary and novel findings, we hypothesize that S100A4 plays a crucial role in regulating NM II assembly in the lens, and that the absence of S100A4 disrupts NM II assembly and phosphorylation, actin polymerization, mechanical properties, and osmotic homeostasis in the lens, leading to late onset cataract formation. To test this hypothesis, in three interrelated specific aims, we will investigate: 1) the mechanistic role of S100A4 in regulation of lens NM II assembly and phosphorylation, actin polymerization, cell adhesion, and lens tensile properties, 2) the disruption of osmoregulation and solute carrier transport mechanisms underlying cataractogenesis in S100A4 null lenses, and 3) the effects of rescuing expression of S100A4 in S100A4 null mouse lens fibers (using a transgenic approach) on cytoskeletal integrity and lens phenotype. These novel and interrelated studies are expected to uncover the significance of S100A4 and other members of the S100 family proteins in lens function, which are known to participate in the pathophysiology of several diseases.