Overexpression of MMP-1 and MMP-3 by cultured conjunctivochalasis fibroblasts

Invest Ophthalmol Vis Sci. 2000 Feb;41(2):404-10.

Abstract

Purpose: To determine whether conjunctivochalasis, denoting redundant, loose, nonedematous inferior bulbar conjunctiva, is associated with increased expression and activity of matrix metalloproteinases (MMPS) over their tissue inhibitors (TIMPs).

Methods: Expression of transcripts and proteins of MMPs, TIMPs, and urokinase plasminogen activator (uPA) by cultured normal human conjunctival and conjunctivochalasis fibroblasts was determined by Northern hybridization, enzyme-linked immunosorbent assay (ELISA), and Western blot analysis, respectively. Gelatin and casein zymography and quantitative collagenase activity assay were performed in the serum-free conditioned media.

Results: Compared with normal conjunctival fibroblasts from six subjects, conjunctivochalasis fibroblasts from eight patients showed markedly increased transcript expression of MMP-1 (5- to 32-fold) and MMP-3 (4 to 30-fold), whereas that of MMP-2, TIMP-1, TIMP-2, and uPA was similar between the two groups. Protein levels were increased in the serum-free conditioned media of conjunctivochalasis fibroblasts for MMP-1 (3.5- to 7.6-fold) and MMP-3 (2.3- to 13-fold), determined by ELISA and Western blot analysis. There was increased caseinolytic activity of MMP-3 and collagenolytic activity of MMP-1 (2.2-fold) by conjunctivochalasis fibroblasts, whereas no difference was noted between these two types of fibroblasts in the protein and gelatinolytic activity of MMP-2 or expression of TIMP-1 and TIMP-2 proteins, although that of TIMP-1 transcript was slightly higher in some conjunctivochalasis fibroblasts. No expression of MMP-9 was detected.

Conclusions: Overexpression of MMP-1 and MMP-3 mRNA by conjunctivochalasis fibroblasts is correlated with their increased protein levels and proteolytic activities. Collectively, these data help explain how conjunctivochalasis manifests excessive degradation of the conjunctival matrix and Tenon's capsule.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Blotting, Northern
  • Blotting, Western
  • Cells, Cultured
  • Conjunctiva / enzymology*
  • Conjunctiva / pathology
  • Conjunctival Diseases / enzymology*
  • Conjunctival Diseases / pathology
  • DNA Probes
  • Enzyme-Linked Immunosorbent Assay
  • Fibroblasts / enzymology*
  • Fibroblasts / pathology
  • Humans
  • Matrix Metalloproteinase 1 / biosynthesis*
  • Matrix Metalloproteinase 1 / genetics
  • Matrix Metalloproteinase 2 / biosynthesis
  • Matrix Metalloproteinase 2 / genetics
  • Matrix Metalloproteinase 3 / biosynthesis*
  • Matrix Metalloproteinase 3 / genetics
  • RNA / isolation & purification
  • Tissue Inhibitor of Metalloproteinases / biosynthesis
  • Tissue Inhibitor of Metalloproteinases / genetics
  • Urokinase-Type Plasminogen Activator / biosynthesis
  • Urokinase-Type Plasminogen Activator / genetics

Substances

  • DNA Probes
  • Tissue Inhibitor of Metalloproteinases
  • RNA
  • Urokinase-Type Plasminogen Activator
  • Matrix Metalloproteinase 3
  • Matrix Metalloproteinase 2
  • Matrix Metalloproteinase 1