The control of matrix metalloproteinase-2 expression in normal and keratoconic corneal keratocyte cultures.
OBJECTIVE
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.
Parkin
B T
BT
Department of Ophthalmology, University of Bristol, Bristol Eye Hospital, UK. benparkin@hotmail.com
Smith
V A
VA
Easty
D L
DL
D002478:Cells, Cultured; D003315:Cornea / Q000166:cytology / Q000201:enzymology*; D016895:Culture Media, Serum-Free; D004591:Electrophoresis, Polyacrylamide Gel; D005347:Fibroblasts / Q000187:drug effects / Q000201:enzymology*; D006801:Humans; D007640:Keratoconus / Q000201:enzymology* / Q000473:pathology; D020778:Matrix Metalloproteinase 2 / Q000096:biosynthesis*; D008970:Molecular Weight; D010982:Platelet-Derived Growth Factor / Q000494:pharmacology; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000096:biosynthesis; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000096:biosynthesis; D016212:Transforming Growth Factor beta / Q000494:pharmacology
D016895:Culture Media, Serum-Free; D010982:Platelet-Derived Growth Factor; D019715:Tissue Inhibitor of Metalloproteinase-1; D016212:Transforming Growth Factor beta; D019716:Tissue Inhibitor of Metalloproteinase-2; D020778:Matrix Metalloproteinase 2