Regulated expression of collagenases MMP-1, -8, and -13 and stromelysins MMP-3, -10, and -11 by human corneal epithelial cells

Invest Ophthalmol Vis Sci. 2003 Jul;44(7):2928-36. doi: 10.1167/iovs.02-0874.

Abstract

Purpose: This study investigated the regulated expression of collagenases (MMP-1, -8, and -13) and stromelysins (MMP-3, -10, and -11) by human corneal epithelial cells treated with IL-1 beta, TNF-alpha, and doxycycline, a medication used to treat ocular surface diseases.

Methods: Primary human corneal epithelial cell cultures were treated with IL-1 beta or TNF-alpha, with or without their corresponding inhibitors. Total RNA extracted from cells treated for 4 to 24 hours was subjected to semiquantitative RT-PCR and Northern hybridization. Conditioned media from 24-hour-treated cultures were evaluated for MMP production by ELISA and activity assays.

Results: Semiquantitative RT-PCR and Northern hybridization revealed that the mRNAs of MMP-1, -13, -3, -10, and -11 were dose dependently upregulated by IL-1 beta and TNF-alpha, whereas MMP-8 and -14 and tissue inhibitor of metalloproteinase (TIMP)-1 were not altered, in corneal epithelial cells. MMP ELISA and activity assays confirmed this dose-dependent increase in MMP-1, -13, -3, and -10 protein production in conditioned media by IL-1 beta and TNF-alpha. This stimulated production was inhibited by their neutralizing antibodies and by IL-1 receptor antagonist. Doxycycline suppressed stimulated MMP-1, -10, and -13 production at both the mRNA and protein levels.

Conclusions: This study demonstrated that IL-1 beta and TNF-alpha upregulate collagenases (MMP-1, -13) and stromelysins (MMP-3, -10, and -11) in human corneal epithelial cells. Doxycycline suppresses stimulated MMP-1, -13, and -10 at the mRNA and protein levels, which suggests that collagenases and stromelysins may play a role in the pathogenesis of sterile corneal ulceration and other ocular surface diseases.

Publication types

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

MeSH terms

  • Blotting, Northern
  • Cells, Cultured
  • Collagenases / genetics
  • Collagenases / metabolism
  • Dose-Response Relationship, Drug
  • Doxycycline / pharmacology*
  • Enzyme-Linked Immunosorbent Assay
  • Epithelium, Corneal / drug effects
  • Epithelium, Corneal / enzymology*
  • Gene Expression Regulation, Enzymologic / drug effects*
  • Humans
  • Interleukin-1 / pharmacology*
  • Matrix Metalloproteinase 1 / genetics
  • Matrix Metalloproteinase 1 / metabolism
  • Matrix Metalloproteinase 10
  • Matrix Metalloproteinase 11
  • Matrix Metalloproteinase 13
  • Matrix Metalloproteinase 3 / genetics
  • Matrix Metalloproteinase 3 / metabolism
  • Matrix Metalloproteinase 8 / genetics
  • Matrix Metalloproteinase 8 / metabolism
  • Metalloendopeptidases / genetics*
  • Metalloendopeptidases / metabolism
  • RNA, Messenger / metabolism
  • Reverse Transcriptase Polymerase Chain Reaction
  • Tumor Necrosis Factor-alpha / pharmacology*
  • Up-Regulation

Substances

  • Interleukin-1
  • RNA, Messenger
  • Tumor Necrosis Factor-alpha
  • Collagenases
  • MMP13 protein, human
  • Matrix Metalloproteinase 11
  • Matrix Metalloproteinase 13
  • Metalloendopeptidases
  • Matrix Metalloproteinase 3
  • Matrix Metalloproteinase 10
  • Matrix Metalloproteinase 8
  • Matrix Metalloproteinase 1
  • Doxycycline