Work in this research group is aimed at elucidating the mechanism of action of pigment epithelium-derived factor (PEDF). PEDF, an extracellular glycoprotein of the interphotoreceptor matrix, vitreous and aqueous, has neuronal differentiation and survival in vitro and in vivo activities on cells derived from the CNS and the retina. It also exhibits antiangiogenic activity. By sequence homology PEDF is a serpin, but it has no known inhibitory activity against serine proteases. The first step in the biological events of PEDF is the binding to cell-surface receptors on target cells. A region from the human PEDF spanning positions 44-121, 44-mer, represents the receptor binding domain of PEDF. Using classical radioligand binding assays, we found that PEDF binds to high-affinity receptors on plasma membranes of the bovine neural retina and that peptide 44-mer competes for this binding with similar kinetics as that of unlabeled PEDF. Ligand blot and PEDF-affinity chromatography reveal a specific PEDF binding protein of 85-kDa in bovine retina plasma membranes. Specific binding sites for fluoresceinated-PEDF are distributed in a discrete fashion in photoreceptor neurons and selected ganglion cells, and represent the PEDF receptors in the neural retina. These observations are in agreement with PEDF's neuroprotective in vitro and in vivo effects on the retina. We started the characterization of the signal transduction events mediating the PEDF neurotrophic activity. PEDF is an extracellular neuronal survival factor for primary cultures of rat cerebellar granule (CG) cells. We found that PEDF mediated the phosphorylation of extracellular regulated kinases (ERK-1 and -2) and that the selective and potent inhibitor of the MEK-ERK cascade, PD98059, greatly reduced this phosphorylation, suggesting an interaction with the MEK-ERK pathway. We continued the studies on the interactions of PEDF with components of extracellular matrixes and investigated the effects of extracellular matrix on PEDF-receptor interactions. Exogenous additions of heparin moderately enhanced the 125I-PEDF binding to detergent-soluble receptors, while heparitinase- and chlorate-treatment of cultures reduced the 125I-PEDF binding to cell-surface receptors, suggesting an important role for heparin/heparan sulfate in efficient cell-surface receptor binding. To study the interactions of PEDF with collagen, a major component of the vitreous and extracellular matrixes, we developed binding assays and found that PEDF binds specifically to collagen type I, II and III, with type I having the highest affinity. To study the regulation of PEDF by oxygen in the retina, we exposed monkey retinal pigment epithelial cells, the main source of PEDF in the eye, to different concentrations of oxygen and to chemical agents for the induction of hypoxia, CoCl2 and deferoxamine mesyalate(in collaboration with Dr. Lois Smith). The amount of PEDF produced in the cell media was lower under hypoxic conditions, and higher under hyperoxia, than in normoxia. However, quantitative RT-PCR did not show regulation of PEDF mRNA by oxygen.
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