Pigment Epithelium–Derived Factor Is a Substrate for Matrix Metalloproteinase Type 2 and Type 9: Implications for Downregulation in Hypoxia

Abstract

PURPOSE: Pigment epithelium-derived factor (PEDF), a protein secreted by the retinal pigment epithelium (RPE), acts on retinal survival and angiogenesis. Because hypoxia and VEGF regulate matrix metalloproteinases (MMPs), their effects on PEDF proteolysis were explored. METHODS: Mouse models for retinopathy of prematurity (ROP) were used. Cultured monkey RPE cells were exposed to low oxygen and chemical hypoxia mimetics. PEDF and VEGF mRNA levels in RPE were determined by RT-PCR. MMPs were assessed by zymography, DQ-gelatin degradation solution assays, and MMP immunostaining. PEDF proteolysis was assayed in solution and followed by SDS-PAGE and immunostaining. MMP induction by VEGF was performed in baby hamster kidney (BHK) cells. Retinal R28 cell survival, ex vivo chick embryonic aortic vessel sprouting, and directed in vivo angiogenesis assays were performed. RESULTS: Levels of PEDF in RPE/choroid significantly decreased in the ROP model. Hypoxia decreased PEDF levels in the media conditioned by RPE cells, with no significant change in PEDF mRNA. Conversely, PEDF proteolysis, gelatinolytic activities of approximately 57-kDa and approximately 86-kDa zymogens, and MMP-2 immunoreactivities increased with hypoxia. Addition of VEGF to BHK cells caused a time and dose-related upregulation of approximately 57-kDa zymogens and of DQ-gelatinolytic and PEDF-degrading activity. The PEDF-degrading activity and approximately 57-kDa zymogens in the BHK media shared MMP protease inhibition patterns and MMP-2 immunoreactivities with those in the vitreous. Limited proteolysis with MMP-2 and -9 degraded PEDF in a Ca(+2)-dependent fashion. MMP-mediated proteolysis of PEDF abolished the retinal survival and antiangiogenic activities of the PEDF protein. CONCLUSIONS: Hypoxia and VEGF can downregulate PEDF through proteolytic degradation. PEDF is a novel substrate for MMP-2 and -9. These results reveal a novel posttranslational mechanism for downregulating PEDF, and provide an explanation for hypoxia-provoked increases in VEGF/PEDF ratios, in angiogenesis and/or in neuronal death.