Steroid control of epithelial cell-cell interactions

University Of California Berkeley, Berkeley CA

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Abstract

We have discovered a previously uncharacterized glucocorticoid-regulated cell signaling cascade and identified steroid-regulated target genes that control cell-cell interactions and polarization of cultured monolayers of tumorigenic and nontumorigenic mammary epithelial cells. The mammary epithelial cell system we have developed is unique because hormone signals can be used to acutely control, in a reversible manner, apical junction organization and tight junction dynamics in a physiologically appropriate manner. The overall goal of this proposal is to test the hypothesis that beta-catenin phosphorylation, stability and accessibility to the adherens junction is controlled by the coordinated glucocorticoid induced ubiquitinproteosome mediated degradation of GSK-3 (Glycogen Synthase Kinase-3), a multifunctional serine/threonine kinase, and of CAS (Cellular Apoptosis Susceptibility gene), a component of the nuclear transport machinery that binds to the importin-alpha nuclear receptor. We propose that the glucocorticoidinduced degradation of CAS directly triggers the cytoplasmic localization of importin-alpha and of the Serumand Glucocorticoid-induced Kinase (Sgk), which we have shown to a primary steroid induced gene and an importin-alpha cargo protein. As a result, Sgk protein is accessible in the cytoplasm to phosphorylate GSK-3 and thereby target GSK-3 for degradation. One goal of this proposal is to use mutagenic and protein biochemical approaches to define the structural domains in GSK-3 and CAS that control their glucocorticoid regulated degradation and to functionally characterize components of this regulated degradative pathway in vitro and in transfected cells. A complementary goal is to use mutagenesis along with in vitro and intact cell nuclear/cytoplasmic trafficking assays to examine how the CAS/importin-alpha interaction regulates Sgk localization. A final goal is to functionally characterize the roles of GSK-3 and CAS in the steroid dependent control of beta-catenin dynamics, apical junction organization and tight junction formation. Cell-cell interactions play a critical role in controlling the growth and differentiation of normal tissue. Information from our proposed studies has the potential to be clinically useful in the development of new therapeutic strategies to control physiologic disorders and neoplasias with apparent alterations in plasma membrane function, adhesion and intercellular communication.