Laminin type 1 augments the transforming growth factor beta-induced expression of matrix metalloproteinase 3 in synovial fibroblasts

Elevated expression of laminins (LN) and integrins in the synovial membrane of rheumatoid arthritis (RA) versus osteoarthritis patients has been reported but metabolic effects of attachment of synovial fibroblasts (SF) to LN are not well studied. We therefore investigated gene expression patterns in SF upon attachment to LN1 (EHS laminin) in comparison with LN10.

Expression of IL-1α, IL-1β, IL-6, IL-8, IL-16, IL-18 as well as matrix metalloproteinase (MMP)-1 and MMP-3 were investigated in RA SF (n = 6) or osteoarthritis SF (n = 7) in primary or early passage cultures. Fibroblasts were seeded onto LN1-coated vessels (BD BioCoat^®) for 24–72 hours and cells attached to cell culture vessels served as controls in all experiments. In addition, cells were activated with cytokines and growth factors such as transforming growth factor beta (TGF-β). After activation, transcript amounts of individual genes were enumerated by quantitative RT-PCR. A recombinant cytokine standard and GAPDH RT-PCR served as controls in each sample. Expression of the α1, β1 and γ1 chains of LN1 laminin by SF was investigated by quantitative RT-PCR and immunocytochemistry. The secretion of MMPs was enumerated by ELISA in SF supernatants.

Growth of SF on LN1-coated surfaces without additional stimuli induced a significant IL-8 mRNA response (3.1-fold, ± 1.24, P ≤ 0.001) and lower responses for IL-1β, IL-16, IL-1α, IL-18 and IL-6. MMP-1 mRNA was upregulated 2.3-fold (± 0.79, P ≤ 0.001), and MMP-3 mRNA only 1.5-fold. Upon incubated of SF on LN10, cytokine and MMP expressions were not changed. Addition of TGF-β (10 ng/ml, 24 hours) to SF attached to tissue culture vessels showed a different induction profile. Here IL-6, IL-1β, IL-1α, IL-8, IL-16 and MMP-1 mRNAs were induced to some degree, IL-18 mRNA was reduced whereas MMP-3 mRNA was induced (3.15-fold, ± 0.7, P ≤ 0.04), when compared with controls. Next combinations of activation by TGF-β and laminin signaling were investigated. For cytokine expressions no additive effects of combining these signals were seen and MMP-1 mRNA was induced to some extent (threefold, ± 1.76). In contrast, MMP-3 mRNA was induced more that 10-fold (12.8 ± 8.7, P ≤ 0.025) and MMP-3 secretion was elevated almost 20-fold (19.67 ± 6.9, P ≤ 0.06). In SF, mRNAs encoding α1, β1 and γ1 laminin – which encode the proteins for LN1 – were detected by quantitative RT-PCR and transcript amounts encoding the α1 and β1 chains of LN1 were higher than mRNA encoding the LN1 γ1 chain. Using an anti-EHS serum, LN1 was detected on SF by immunocytochemistry. However, using monoclonal antibodies to laminin α1 or β1 proteins, staining signals were very weak.

Attachment to LN1-laminin in the presence of TGF-β induces elevated MMP-3 expression in SF. However, an autocrine stimulation of MMP-3 expression by SF via TGF-β and LN1 seems rather unlikely, as LN1 is not expressed in high amounts in the adult synovial membrane. Still, activation of SF by LN1 may serve as a model for activation of fibroblasts by extracellular matrix compounds in the presence of growth factors or cytokines, and both pathways contribute to the aggressive invasive growth of SF in the course of RA.

Affiliations

1. Department of Orthopedic Surgery, University Medical Center, Tuebingen, Germany

   • M Hoberg
   • , M Rudert
   •  & WK Aicher

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