MMP-14 and ADAM-9 regulates FGFR2 level in corneal fibroblasts and endothelial cells

Abstract

Purpose : We aim to elucidate the regulation of FGFR2 protein level through the two enzymatic activities of matrix metalloproteinase-14 (MMP-14) and A disintegrin and a metalloprotease-9 (ADAM-9) in corneal neovascularization.

Methods : Endothelial cells were transduced with short hairpin RNA (shRNA) lentiviral particles to knockdown MMP-14 and were selected through puromycin selection. Corneal fibroblasts wild type (WT) and MMP-14 knockout (KO) cells were isolated from WT and MMP-14 knockout mice, respectively. Protein levels of MMP-14, ADAM-9, FGFR2, and MAPK proteins were compared between WT and MMP-14 KO cells using western blot analysis. In vitro proteolysis assay was performed to investigate the degradation of ADAM-9 and FGFR2 by MMP-14 and ADAM-9, respectively. The Edman degradation analysis was performed to identify the cleavage sites. Surface Plasmon Resonance (SPR) analysis was conducted to determine binding affinities among MMP-14, FGFR2, and ADAM-9.

Results : We found that MMP-14 regulates FGFR2 indirectly through mediation by ADAM-9 in corneal fibroblasts and endothelial cells. Both MMP14 suppression through shRNA and knockout led to an increase in ADAM-9 and a decrease in FGFR2, as compared to the WT. We also exhibited the degradation of ADAM-9 by MMP-14 and the degradation of FGFR2 by ADAM-9 in proteolysis assays. We identified that MMP-14 cleaved ADAM-9 at amino acid serine⁶³⁸, and ADAM-9 digested FGFR2 at amino acid glutamic acid³⁷⁸. Through SPR studies, the binding affinities for cat-MMP14, FGFR2, and ADAM-9 were determined to be 0.099 μM and 0.17 μM, respectively. The dissociation rate of the MMP-14 from ADAM-9 was ~10 times slower than that of FGFR2.

Conclusions : Our findings demonstrate that the regulation of FGFR2 levels in keratocytes and endothelial cells depends on MMP-14 and ADAM-9. In the absence of MMP-14, ADAM-9 degrades FGFR2. Conversely, in the presence of MMP-14, a cascade is observed where MMP-14 degrades ADAM-9, resulting in an increase in FGFR2. Therefore, the two-enzyme cascade involving MMP-14 and ADAM-9 may regulate FGF2/FGFR2-mediated corneal neovascularization.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.