Cell-Matrix Interactions in Epithelial Polarization

描述（由申请人提供）：上皮细胞形成肾脏和大多数其他实体器官的实质。在肾小管中，它们负责吸收和分泌，从过滤后的血液中回收液体和离子，并将废物排放到形成的尿液中。上皮细胞最重要的特性是它们的极性。上皮细胞的质膜分为生物化学和形态不同的顶域和底外侧域。蛋白质在这些膜结构域中的不对称分布使得定向运输成为可能。相反，这一极性的破坏既助长了疾病，也是疾病的后果。先前的大量工作，主要是在体外极化上皮模型中，已经确定上皮细胞的极化是由细胞与细胞外基质蛋白之间的粘附引起的。其中，基底膜蛋白层粘连蛋白的异构体可能起着至关重要的作用。在本项目中，我们将在先前关于肾上皮细胞中整合素和层粘连蛋白的研究基础上，研究特定层粘连蛋白同工型在上皮细胞极化中的作用。特别是，我们将研究laminin-5 （LN5）和laminin-10 （LN10）在Madin-Darby犬肾细胞极化中的协同作用。在Specific Aims 1和2中，我们将分别使用RNA干扰来抑制LN5和LN10的合成，并确定这对可渗透支架和三维胶原凝胶的二维培养中的粘附、极化和信号传导的影响。在Specific Aim 3中，我们将通过使用功能阻断抗体、突变整合素的表达和RNA干扰来确定层粘连蛋白的哪些整合素受体参与极化过程，并将绘制从整合素连接到负责胶原凝胶极化的Rac1激活的信号事件。从这些研究中获得的知识将扩大我们对胚胎分化和缺血或肾毒性损伤后肾脏再生过程中肾上皮细胞极化的理解。

DESCRIPTION (provided by applicant): Epithelial cells form the parenchyma of the kidney and most other solid organs. In kidney tubules they are responsible for absorptive and secretory events that recover fluid and ions from filtered blood and discharge waste products into the forming urine. The key property of epithelial cells that underlies all others is their polarity. The plasma membrane of epithelial cells is organized into apical and basolateral domains which are biochemically and morphologically distinct. Asymmetric distribution of proteins in these membrane domains makes directional transport possible. Conversely, breakdown of this polarity both contributes to and is a consequence of disease. Extensive previous work, primarily with in vitro models of polarized epithelia, has established that polarization of epithelial cells is initiated by adhesion between cells and proteins of the extracellular matrix. Of these, isoforms of the basement membrane protein laminin are likely to play a crucial role. In this project, we will build on our previous work on integrins and laminins in kidney epithelial cells to investigate the involvement of specific laminin isoforms in epithelial cell polarization. In particular, we will investigate the cooperative involvement of laminin-5 (LN5) and laminin-10 (LN10) in the polarization of Madin-Darby canine kidney cells. In Specific Aims 1 and 2, we will use RNA interference to suppress LN5 and LN10 synthesis, respectively, and determine the effects of this on adhesion, polarization, and signaling in both two-dimensional cultures on permeable supports and in three-dimensional collagen gels. In Specific Aim 3 we will determine which integrin receptors for laminin are involved in the polarization process through the use of function-blocking antibodies, expression of mutant integrins, and RNA interference, and will map the signaling events leading from integrin ligation to Rac1 activation that are responsible for polarization in collagen gels. Knowledge gained from these studies will broaden our understanding of kidney epithelial cell polarization both during embryonic differentiation and during regeneration of the kidney following ischemic or nephrotoxic injury.