Cell-Matrix Interactions in Epithelial Polarization

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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.