The control of proliferation of corneal endothelial cells by EGF will be analyzed. The relevance of EGF binding, internalization, and degradation for the induction of cell division will be defined. We will also study the possible binding of EGF to nuclear receptor sites versus cell surface receptor sites. The effect of EGF on the synthesis of cytoplasmic proteins, which may be involved in supporting the progression of the cells through the G1 phase of the cell cycle, will be studied as a function of the cell cycle and of cell density. The possibility that, at confluence, a restriction in the cell surface receptor lateral mobility could prevent the internalization of EGF receptor complexes and their subsequent triggering of cell division will be investigated. The role of the extracellular matrix (ECM) in events controlling corneal endothelial cell proliferation and differentiation will be analyzed using two cell strains of corneal endothelial cells. One expresses its normal phenotype in vitro when maintained in the presence of FGF, while the other, maintained in the absence of FGF, no longer expresses it. The composition, distribution, and polarity of secretion of collagen types produced by sparse versus confluent cultures of corneal endothelial cells maintained in the presence or absence of FGF or EGF will be analyzed using indirect immunofluorescence techniques. This will be followed by the biochemical analysis of the composition and production of collagen produced by sparse versus confluent cultures of corneal endothelial cells maintained in the presence or absence of EGF or FGF. The direct modulation of collagen synthesis by FGF and EGF when added to cultures which no longer express their normal phenotype at confluence will be analyzed. The nature of the permissive effect of the ECM on cell proliferation and differentiation will be studied by investigating the proliferation and differentiation on dishes coated with an ECM produced by bovine corneal endothelial cells of cell types of the ocular system which do not proliferate (human corneal endothelial cells) or differentiate (corneal epithelial cells) on plastic.
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