The control of matrix metalloproteinase-2 expression in normal and keratoconic corneal keratocyte cultures

Eur J Ophthalmol. Oct-Dec 2000;10(4):276-85. doi: 10.1177/112067210001000402.

Abstract

Purpose: Early phase keratoconic corneas and their cultured keratocytes abnormally produce the Mr 62,000 form of the matrix metalloproteinase-2 (MMP-2). It is known that platelet derived growth factor (PDGF) and transforming growth factor-beta (TGF-beta) 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 Mr 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-beta. 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-beta 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 Mr 62,000 species of MMP-2.

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

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cells, Cultured
  • Cornea / cytology
  • Cornea / enzymology*
  • Culture Media, Serum-Free
  • Electrophoresis, Polyacrylamide Gel
  • Fibroblasts / drug effects
  • Fibroblasts / enzymology*
  • Humans
  • Keratoconus / enzymology*
  • Keratoconus / pathology
  • Matrix Metalloproteinase 2 / biosynthesis*
  • Molecular Weight
  • Platelet-Derived Growth Factor / pharmacology
  • Tissue Inhibitor of Metalloproteinase-1 / biosynthesis
  • Tissue Inhibitor of Metalloproteinase-2 / biosynthesis
  • Transforming Growth Factor beta / pharmacology

Substances

  • Culture Media, Serum-Free
  • Platelet-Derived Growth Factor
  • Tissue Inhibitor of Metalloproteinase-1
  • Transforming Growth Factor beta
  • Tissue Inhibitor of Metalloproteinase-2
  • Matrix Metalloproteinase 2