Arthritis & Rheumatism, Volume 60,
October 2009 Abstract Supplement
The 2009 ACR/ARHP Annual Scientific Meeting
Philadelphia October 16-21, 2009.
The Fibronectin III 1314 Domains Activate Catabolic Pathways in Joint Tissue Via TLR4
Sofat1, Nidhi, Wait2, Robin, Nagase3, Hideaki
Fibronectin is an extracellular matrix (ECM) molecule implicated in a number of processes during physiology and inflammation. Expression of full-length FN is upregulated in chronic conditions such as rheumatoid arthritis and osteoarthritis and fibronectin fragments induce specific biological effects. The aggrecanases (ADAMTSs) and MMPs are believed to be the key enzymes mediating joint damage in arthritis. The C-terminal heparin-binding region of fibronectin has been implicated in regulating aggrecanase activity in vitro, but the activation of aggrecanase activity by this region has not been investigated in experimental models of arthritis.
The expression of fibronectin and its fragments was investigated in normal and arthritic tissue by Western blotting. Normal porcine articular cartilage was then used to test recombinant fragments of fibronectin, either alone, or in combination with the cytokines IL-1 and TNF, for their ability to induce aggrecanase and MMP activity. Enzyme activity was measured by RT-PCR and neoepitope assays by Western blotting. The ability of recombinant fragments to regulate the production of other proteins in chondrocytes was established by metabolic labelling using cysteine and methionine-free medium and then probing for newly synthesised proteins using one-dimensional gels analysed by mass spectrometry. The receptor for the most active fibronectin fragment was established using cultures from hip explants of TLR-4 knockout mice.
Our data showed that full-length fibronectin and its fragmented forms are highly upregulated in arthritic cartilage from subjects with rheumatoid arthritis and osteoarthritis. Here, we show for the first time, that the fibronectin III 1314 domains in the C-terminal heparin-binding region are potent inducers of aggrecanase activity at micromolar concentrations in articular cartilage. The FN III 1314 domains also induced aggrecananase activity in a synergistic manner with the cytokines IL-1 and TNF, which was 56 fold higher than each of these molecules alone. We also show that the FN III 1314 domains induce release of MMP-1, MMP-3, hsp 70, gp 38 and serum amyloid A-like protein in chondrocytes, an effect that was not observed for full-length FN. Furthermore, the III 1314 domains increased IL-6, IL-8 and PGE2 production in monocytes. In studies using knockout mice, wild-type, but not TLR4 knockout murine cartilage showed the induction of aggrecanase activity by fibronectin domains III 1314.
Our data describes a new mechanism of activation of TLR4 in articular cartilage which may be a significant activation pathway during joint damage. These findings show how the release of endogenous matrix proteins such as fibronectin maintains the persistence of chronic inflammation by the activation of pro-inflammatory pathways in diseases such as rheumatoid arthritis and osteoarthritis. Targeting pathways involving the imbalance of ECM proteins may provide new therapeutic options for the treatment of arthritis in the future.
To cite this abstract, please use the following information:
Sofat, Nidhi, Wait, Robin, Nagase, Hideaki; The Fibronectin III 1314 Domains Activate Catabolic Pathways in Joint Tissue Via TLR4 [abstract]. Arthritis Rheum 2009;60 Suppl 10 :2039