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Published on: November 12, 2015
The control of matrix metalloproteinase-2 expression in normal and keratoconic corneal keratocyte cultures
B T Parkin1, V A Smith, D L Easty
1Department of Ophthalmology, University of Bristol, Bristol Eye Hospital, UK. benparkin@hotmail.com
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.
Insights
Platelet-derived growth factor (PDGF) may contribute to the abnormal production of a specific form of matrix metalloproteinase-2 (MMP-2) in early keratoconus. This finding sheds light on potential mechanisms in this corneal disease.
Area of Science:
- Ophthalmology
- Cell Biology
- Biochemistry
Background:
- Early-phase keratoconic corneas exhibit abnormal production of the Mr 62,000 matrix metalloproteinase-2 (MMP-2).
- Platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGF-beta) are known regulators of MMP activity and tissue inhibitor of metalloproteinase (TIMP) in non-ocular tissues.
Purpose of the Study:
- To investigate the role of PDGF and TGF-beta in regulating corneal MMP-2 activity and TIMP production.
- To determine if these growth factors contribute to the abnormal Mr 62,000 MMP-2 form in keratoconus.
Main Methods:
- Keratocytes from normal and early keratoconic corneas were cultured and treated with PDGF and/or TGF-beta.
- Secreted proteins were analyzed over 7 days for total protein, MMP-2 activity, TIMP-1, and TIMP-2 levels.
Main Results:
- PDGF and TGF-beta did not significantly alter protein production, MMP-2 activity, or TIMP levels in keratoconic keratocytes.
- In normal corneal keratocytes, PDGF induced the expression of the Mr 62,000 MMP-2 species.
Conclusions:
- PDGF may play a role in the production of the abnormal Mr 62,000 MMP-2 species observed in early keratoconus.
- Further research is warranted to elucidate the precise mechanisms involving PDGF in keratoconus pathogenesis.
