Attachment of synovial fibroblasts to type 1 laminin boosts the transforming growth factor beta-induced expression of stromelysin-1 (MMP-3)

Background and objective

In previous studies, the effects of attachment of synovial fibroblasts (SF) to different matrix compounds such as type I collagen or fibronectin on IL-16 expression were investigated. The focus of this study is the analysis of gene expression in SF upon stimulation with transforming growth factor beta (TGF-β) after attachment to LN1-laminin (EHS laminin).

Methods

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 SF from rheumatoid arthritis patients (n = 6) or osteoarthritis patients (n = 5) in primary or early passage cultures. Cells were derived from biopsies and expanded in DMEM + 10% FCS medium. Fibroblasts were seeded onto LN1-laminin-coated vessels (BD BioCoat^®) for 24–72 hours, and cells attached to cell culture vessels served as controls in all experiments. After these incubations, 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 LN-1 laminin by SF was investigated by RT-PCR and immunocytochemistry.

Results

Growth of SF on LN1-laminin coated surfaces without additional stimuli induced a significant IL-8 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 was upregulated 2.3-fold (± 0.79, P ≤ 0.001), MMP-3 only 1.5-fold. Upon incubation of SF on LN10-laminin, the cytokine and MMP expression 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 showed the most prominent induction (4.1-fold, ± 3.6; P ≤ 0.21), IL-1β, IL-α, IL-8, IL-16 and MMP-1 were induced to a lesser degree, and IL-18 mRNA was lower whereas MMP-3 was induced (3.15-fold, ± 0.7; P ≤ 0.04), when compared with controls. Next, the combination 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 expression was induced only to some extent (3-fold, ± 1.76). In contrast, MMP-3 was induced more than 10-fold. In SF mRNA encoding α1, β1 and γ1 laminin, which encode the proteins for LN1-laminin, were detected by RT-PCR – whereas α5 laminin mRNA, encoding the α-chain of LN10-laminin, remained undetectable by RT-PCR. Using an anti-EHS serum, LN1-laminin was detected on SF by immunocytochemistry. However, using monoclonal antibodies to laminin α1 or γ1 proteins, staining signals were very weak.

Conclusions

Attachment to LN1-laminin in the presence of TGF-β may induce elevated MMP-3 expression in SF. An autocrine stimulation of MMP-3 expression by SF via TGF-β and LN1-laminin seems rather unlikely, as LN1-laminin is not expressed in high amounts in the adult synovial membrane. Still, activation of SF by LN1-laminin 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 rheumatoid arthritis.