Work in this research group is aimed at elucidating the mechanism of action of pigment epithelium-derived factor (PEDF). PEDF, an extracellular glycoprotein, acts in neuronal differentiation and survival in cells derived from the retina and CNS. It has been reported that it has antiangiogenic effects and that its expression is down-regulated over the replicative lifespan of mammals. 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's neurotrophic activity is the binding to receptors on the surface of target cells. Using radiolabeled and fluoresceinated ligands in radioligand binding assays, ligand blotting, ligand affinity column chromatography and confocal microscopy, we collected data that provide physico-chemical, pharmacological, and biochemical evidence for PEDF binding to cell-surface receptors distributed discretely in the neural retina of adult steers. The collected data correlate with PEDF effects on the survival and morphogenesis of photoreceptor cells in vivo and retina cells in culture suggesting that the PEDF-binding sites on photoreceptor cells correspond to functional receptors. The data also reveal that ganglion cells may be targets for PEDF activity. It has been shown before that PEDF protects motor neurons from chronic glutamate-mediated neurodegeneration. In collaboration with Drs. Ralph Kuncl and Masako Bilak, we have been studying the mechanisms for motor neuron protection by PEDF. A small peptide from human PEDF containing the receptor binding site and termed 44-mer protected motor neurons from chronic glutamate-mediated degeneration, as has been shown for the full length PEDF. We found that 125I-PEDF and fluoresceinated-PEDF bind to spinal cord motor neurons in a specific fashion revealing the presence of PEDF receptors on motor neurons. We continued the studies on the interactions of PEDF with components of extracellular matrixes and investigated those with collagen, a major component of the vitreous, cornea and extracellular matrixes. Using binding assays based in ultrafiltration, we found that PEDF binds specifically to collagen type I, II and III, with type I having the highest affinity. Modified PEDF with 125I and fluorescein retained the affinity for binding collagen I. The binding was sensitive to increasing concentrations of NaCl, suggesting an ionic nature for the collagen-PEDF interactions. Radioligand competition assays showed that unlabeled PEDF and a recombinant fragment of PEDF comprising amino acid positions 44-418 competed with 125I-PEDF for the binding to collagen I, however, the peptide 44-mer (a peptide containing a neurotrophic active sitefor PEDF) and angiostatin and endostatin (other antiangiogenic factors) did not. Using size-exclusion ultrafiltration, Fl-PEDF ligand blotting and PEDF-affinity column chromatography, we found that PEDF can interact with corneal and vitreal proteins that immunoreact with antibodies to collagen I, II and III. The PEDF-collagen interactions may occur in vivo and play a role in PEDF function in the eye. We continued the study on the regulation of PEDF by oxygen in the retina. Monkey retinal pigment epithelial cells were exposed to different concentrations of oxygen and to chemical agents for the induction of hypoxia, CoCl2 and deferoxamine mesyalate. Although, quantitative RT-PCR did not show regulation of PEDF mRNA by oxygen, western blotting demonstrated that the amount of PEDF produced in the cell media was lower under hypoxic conditions, and higher under hyperoxia, than in normoxia. Zymograms, protease solution assays and western blotting revealed that metalloproteinase activities were regulated inversely in the media. The data suggests that the levels of PEDF in cultures exposed to different concentrations of oxygen can be regulated extracellularly by degrading metalloproteinase activities.
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