EXTENDED REPORT

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

Maik Hoberg¹, Maximilian Rudert¹, Thomas Pap², Gerd Klein³, Steffen Gay⁴, Wilhelm K Aicher⁵

¹ Department of Orthopedic Surgery, CRONA University Hospital, Tübingen, Germany
² Division of Molecular Medicine of Musculoskeletal Tissue, Department of Orthopedics, Münster University Hospital, Münster, Germany
³ Center for Medical Research (ZMF), Section for Transplantation Immunology, University of Tübingen, Tübingen, Germany
⁴ WHO Center for Exp. Rheumatology, University Hospital Zürich, and Zürich Center for Integrative Human Physiology (ZIHP), Zürich, Switzerland
⁵ Center for Medical Research (ZMF), Department of Orthopaedic Surgery, Eberhard-Karls-University Medical School, Tübingen, Germany

Correspondence to:
W K Aicher
ZMF, Center for Medical Research, Eberhard-Karls-University Medical School, Waldhoernle Strasse 22, D 72072 Tuebingen, Germany; aicher{at}uni-tuebingen.de

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 ß (TGFß).

Methods: SFs were seeded in laminin-coated flasks and activated by addition of TGFß. 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 TGFß induced a fivefold higher expression of MMP-3. Incubation of SF on LM-111 in the presence of TGFß 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 TGFß 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 TGFß-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)1ß or tumour necrosis factor (TNF).

Abbreviations: 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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