REGULATION OF RETINAL PIGMENTED EPITHELIAL ECMS

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As a step in unraveling the functions of the extracellular matricies (ECMs) produced by the retina pigment epithelium (RPE) studies of the proteoglycans and matrix metalloproteinases (MMPs) of cultured human RPE cells are proposed. RPE cells are involved in the production and maintenance of Bruch's membrane and the interphotoreceptor matrix. These ECMs, which have been implicated in several retinal pathologies, have been subjected to moderate scrutiny and several components have been identified. The studies presented in this proposal are focused on 1) the RPE proteoglycans, 2) their biosynthesis and turnover and 3) the regulation of these processes. Direct biochemical analysis, supplemented by studies with protein and anti- peptide antibodies and oligonucleotide probes, will be used to characterize RPE proteoglycan core proteins and their glycosaminoglycan sidechains. Biochemical analysis, immunoprecipitation, immunoblots of Western transfers, Northern analysis and dot blots will be used to study the localization and interaction of these proteoglycans with RPE cells and to establish their basal biosynthesis and turnover patterns. To investigate the initiation of ECM turnover, a family of MMPs will be studied. RPE cells were shown to secrete MMP-1 (interstitial collagenase), MMP-2 (gelatinase), MMP-3 (stromelysin) and the tissue inhibitor of metalloproteinases (TIMP). Purification and characterization of the MMPs including their substrate specificities, metal requirements, kinetics, multiple glycosylation and activation forms, interactions with TIMPs and activity upon RPE ECM components are proposed. These biochemical methods, antibodies and oligonucleotide probes will then be used to investigate the regulation of the biosynthesis and turnover of these and other ECM components by RPE cells. In addition to select cellular modulators and growth factors, intact and degraded fragments of several ECM components, particularly those containing "growth factor-like" motifs, will be evaluated as potential regulators of the RPE and of its ECMs. Understanding the nature and behavior of RPE ECMs, is an important prerequisite for determining the functions and behaviors of these ECMs in the normal and in the pathological retina.