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Attachment to laminin-111 facilitates transforming growth factor β-induced expression of matrix metalloproteinase-3 in synovial fibroblasts

Abstract

Background: In the synovial membrane of patients with rheumatoid arthritis (RA), a strong expression of laminins and matrix degrading proteases was reported.

Aim: To investigate the regulation of matrix metalloproteinases (MMPs) in synovial fibroblasts (SFs) of patients with osteoarthritis (OA) and RA by attachment to laminin-1 (LM-111) and in the presence or absence of costimulatory signals provided by transforming growth factor β (TGFβ).

Methods: SFs were seeded in laminin-coated flasks and activated by addition of TGFβ. The expression of genes was investigated by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), immunocytochemistry and ELISA, and intracellular signalling pathways by immunoblotting, and by poisoning p38MAPK by SB203580, MEK-ERK by PD98059 and SMAD2 by A-83-01.

Results: Attachment of SF to LM-111 did not activate the expression of MMPs, but addition of TGFβ induced a fivefold higher expression of MMP-3. Incubation of SF on LM-111 in the presence of TGFβ induced a significant 12-fold higher expression of MMP-3 mRNA, and secretion of MMP-3 was elevated 20-fold above controls. Functional blocking of LM-111–integrin interaction reduced the laminin-activated MMP-3 expression significantly. Stimulation of SF by LM-111 and TGFβ activated the p38MAPK, ERK and SMAD2 pathways, and inhibition of these pathways by using SB203580, PD98059 or A-83-01 confirmed the involvement of these pathways in the regulation of MMP-3.

Conclusion: Attachment of SF to LM-111 by itself has only minor effects on the expression of MMP-1 or MMP-3, but it facilitates the TGFβ-induced expression of MMP-3 significantly. This mode of MMP-3 induction may therefore contribute to inflammatory joint destruction in RA independent of the proinflammatory cytokines interleukin (IL)1β or tumour necrosis factor (TNF)α.

  • JNK, jun-N kinase-1
  • LM, laminin
  • mAb, monoclonal antibody
  • MAP, mitogen-activated protein
  • MMP, matrix metalloproteinase
  • OA, osteoarthritis
  • qRT-PCR, quantitative reverse transcriptase-polymerase chain reaction
  • RA, rheumatoid arthritis
  • SAPK, stress-activated protein kinase
  • SF, synovial fibroblast
  • SMAD, transcription factor (homologue to mothers against DPP and SMA genes)
  • TGF, transforming growth factor
  • TIMP, tissue inhibitor of metalloproteinase
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