Preliminary studies have indicated that activation of the protein kinase C signal transduction system (with diacylglycerols or phorbol ester tumor promoters) in a renal epithelial cell culture (LLC-PKi) causes the tight junctional (zonula occludens) band around the cells to become leaky. Molecules as large as epidermal growth factor (EGF) have exhibited 40-fold increases in their transepithelial flux. We have also shown EGF to be mitogenic in this cell system, have characterized the cell kinetics, and have demonstrated that EGF is mitogenic only when in contact with the basolateral cell surface. The objective of the proposed study is to systematically investigate the relationship between action of phorbol esters and diacylglycerols and the breakdown of epithelial barriers by weakening the tight junctional region, thereby increasing growth factor/hormone flow between cells. The following areas will address these goals: 1) Is the increased transepithelial flux of these proteins due solely to increased (paracellular) flow across the junctions or does a transcytotic pathway also contribute; 2) Will the junctions of treated cells allow increased passage to growth factors of only certain size and charge; 3) Can this effect be demonstrated using cell lines from different tissues and species; 4) Is there a morphological alteration of the tight junctional region as seen by transmission and freeze fracture electron microscopy; 5) When the junctional region is made leaky by these means, does the polar localization of certain membrane proteins in the apical or basolateral domain break down; 6) What are the implications of this induced junctional leakiness on transport and other functions of these renal epithelia, especially growth regulation, and its control from apical and basolateral receptors.
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