The Control of Matrix Metalloproteinase-2 Expression in Normal and Keratoconic Corneal Keratocyte Cultures

Abstract

Purpose

Early phase keratoconic comeas and their cultured keratocytes abnormally produce the M_r 62,000 form of the matrix metalloproteinase-2 (MMP-2). It is known that platelet derived growth factor (PDGF) and transforming growth factor-β (TGF-β) are involved in the regulation of MMP activity and tissue inhibitor of metalloproteinase (TIMP) production in non-ocular tissues. The purpose of this enquiry was to determine whether these growth factors also play a role in the activity and/or production of corneal MMP-2 and TIMP, and whether their activity could account for the existence of the M_r 62,000 form of MMP-2 in keratoconic corneas.

Methods

Confluent cultures of normal and early-phase keratoconic corneal keratocytes were established and incubated in serum-free media in the presence or absence of PDGF and TGF-β. The proteins secreted by these cells over periods of 7 days were harvested for analysis. The total protein produced was determined spectrophotometrically. MMP-2 was visualised by SDS-gelatin polyacrylamide gel electrophoresis and assayed using radiolabelled type IV collagen as substrate. The enzyme inhibitors, TIMP-1 and TIMP-2, were quantified by dot blot immunoassay.

Results

The addition of PDGF or TGF-β to the culture medium of keratoconic corneal keratocytes had no significant effect on overall protein production, MMP-2 activity or on the amounts of TIMP-1 and TIMP-2 secreted. These observations also applied to normal corneal keratocytes, with the exception that PDGF induced expression of the M_r 62,000 species of MMP-2.

Conclusions

PDGF may be involved in the production of the M_r 62,000 species of MMP-2 that is abnormally produced by early-phase keratoconic corneal keratocytes.