A fibronectin fragment induces type II collagen degradation by collagenase through an interleukin-1-mediated pathway.
OBJECTIVE
To examine the effects of a fibronectin (FN) fragment containing the COOH-terminal heparin-binding domain (HBFN-f) on chondrocyte-mediated type II collagen (CII) cleavage by collagenase and proteoglycan (PG) degradation in articular cartilage in explant culture.


METHODS
Intact FN or HBFN-f was added to explant cultures of mature bovine articular cartilage. We investigated collagenase-mediated cleavage of CII caused by HBFN-f in explant cultures using a new immunoassay for detection and measurement of the primary collagenase cleavage site of CII. CII denaturation in cartilage was also measured using a specific enzyme-linked immunosorbent assay. Degradation of PG (principally aggrecan) was analyzed by a dye-binding assay. APMA and/or a matrix metalloproteinase 13 (MMP-13) preferential inhibitor or interleukin-1 receptor antagonist (IL-1Ra) were added to some cultures to examine the presence of latent procollagenase or the involvement of MMP-13 or IL-1, respectively, in cartilage breakdown induced by HBFN-f. Secretion of MMP-3 and MMP-13 into media was detected by immunoblotting.


RESULTS
In contrast to intact FN, HBFN-f was shown to stimulate CII cleavage by collagenase in a dose-dependent manner following PG degradation, similar to cartilage breakdown induced by IL-1. Treatment with HBFN-f also resulted in elevated denaturation of CII. Immunoblotting demonstrated that HBFN-f enhanced pro-matrix metalloproteinase 13 (proMMP-13) production as well as that of proMMP-3. APMA, which activates latent proMMPs, enhanced the HBFN-f-mediated cleavage of CII by collagenase. An MMP-13 preferential inhibitor or IL-1Ra suppressed HBFN-f-induced collagen cleavage to control levels.


CONCLUSIONS
Our data demonstrate that HBFN-f can induce early PG degradation and subsequent CII cleavage. The latter is probably mediated by early proMMP-13 induction involving an IL-1-dependent pathway. Activation of latent collagenase is delayed. This new information, together with existing data on other FN fragments, reveals that increased levels of these fragments, found in diseased joints such as in osteoarthritis and rheumatoid arthritis, may stimulate cartilage breakdown by mechanisms of the kind demonstrated in the present study.
Yasuda
Tadashi
T
Department of Orthopaedics, Kyoto University Graduate School, Japan.
Poole
A Robin
AR
D000818:Animals; D001172:Arthritis, Rheumatoid / Q000276:immunology / Q000378:metabolism; D002358:Cartilage, Articular / Q000166:cytology / Q000201:enzymology* / Q000276:immunology; D002417:Cattle; D002478:Cells, Cultured; D024043:Collagen Type II / Q000378:metabolism*; D017364:Collagenases / Q000378:metabolism*; D004792:Enzyme Precursors / Q000378:metabolism; D005353:Fibronectins / Q000737:chemistry / Q000378:metabolism*; D006493:Heparin / Q000378:metabolism; D006801:Humans; D053590:Interleukin 1 Receptor Antagonist Protein; D007375:Interleukin-1 / Q000378:metabolism*; D053509:Matrix Metalloproteinase 13; D019278:Matrix Metalloproteinase 3 / Q000378:metabolism; D061965:Matrix Metalloproteinase Inhibitors; D010003:Osteoarthritis / Q000276:immunology / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012795:Sialoglycoproteins / Q000494:pharmacology
D024043:Collagen Type II; D004792:Enzyme Precursors; D005353:Fibronectins; C507763:IL1RN protein, human; D053590:Interleukin 1 Receptor Antagonist Protein; D007375:Interleukin-1; D061965:Matrix Metalloproteinase Inhibitors; D010446:Peptide Fragments; D012795:Sialoglycoproteins; D006493:Heparin; D017364:Collagenases; C507079:MMP13 protein, human; D053509:Matrix Metalloproteinase 13; C022440:procollagenase; D019278:Matrix Metalloproteinase 3