Relaxin's induction of metalloproteinases is associated with the loss of collagen and glycosaminoglycans in synovial joint fibrocartilaginous explants

Abstract

Diseases of specific fibrocartilaginous joints are especially common in women of reproductive age, suggesting that female hormones contribute to their etiopathogenesis. Previously, we showed that relaxin dose-dependently induces matrix metalloproteinase (MMP) expression in isolated joint fibrocartilaginous cells. Here we determined the effects of relaxin with or without beta-estradiol on the modulation of MMPs in joint fibrocartilaginous explants, and assessed the contribution of these proteinases to the loss of collagen and glycosaminoglycan (GAG) in this tissue. Fibrocartilaginous discs from temporomandibular joints of female rabbits were cultured in medium alone or in medium containing relaxin (0.1 ng/ml) or beta-estradiol (20 ng/ml) or relaxin plus beta-estradiol. Additional experiments were done in the presence of the MMP inhibitor GM6001 or its control analog. After 48 hours of culture, the medium was assayed for MMPs and the discs were analyzed for collagen and GAG concentrations. Relaxin and beta-estradiol plus relaxin induced the MMPs collagenase-1 and stromelysin-1 in fibrocartilaginous explants--a finding similar to that which we observed in pubic symphysis fibrocartilage, but not in articular cartilage explants. The induction of these proteinases by relaxin or beta-estradiol plus relaxin was accompanied by a loss of GAGs and collagen in joint fibrocartilage. None of the hormone treatments altered the synthesis of GAGs, suggesting that the loss of this matrix molecule probably resulted from increased matrix degradation. Indeed, fibrocartilaginous explants cultured in the presence of GM6001 showed an inhibition of relaxin-induced and beta-estradiol plus relaxin-induced collagenase and stromelysin activities to control baseline levels that were accompanied by the maintenance of collagen or GAG content at control levels. These findings show for the first time that relaxin has degradative effects on non-reproductive synovial joint fibrocartilaginous tissue and provide evidence for a link between relaxin, MMPs, and matrix degradation.

Figure 1

Relaxin induces collagenase-1 and stromelysin-1 in fibrocartilaginous explants from temporomandibular joint. Disc hemisections were exposed for 48 hours to basal control medium (Ct), β-estradiol (Es, 20 ng/ml), or relaxin (R, 0.1 ng/ml) or to β-estradiol plus relaxin (Es+R). Conditioned medium, standardized by tissue weight, was subjected to SDS–polyacrylamide-gel electrophoresis and transferred to membranes for western immunoblots for collagenase-1 (a) or stromelysin-1 (d) or assayed in gels containing gelatin (b) or α-casein (e). Images of the substrate gels were digitized, and the 53/58 kDa and 43 kDa gelatinase activities (collagenase and active collagenase, respectively) (c) and the 51/54 kDa caseinolytic activity (stromelysin) (f) were quantified by videodensitometry. The samples used in lane 6 of panels (b) and (e) are positive controls for collagenase-1 and stromelysin-1. P, gels incubated in buffer containing the metalloproteinase inhibitor 1,10-phenanthroline; Cl-1, collagenase-1; ACl-1, active collagenase-1; Sl-1, stromelysin-1; α-Cl, anti-collagenase-1 antibody; α-Sl, anti-stromelysin-1 antibody. * P < 0.05.

Figure 2

Relaxin induces collagenase-1 and stromelysin-1 in pubic symphysis fibrocartilage but not in articular cartilage. Pubic symphysis fibrocartilage or knee articular cartilage explants were exposed for 48 hours to basal control medium (Ct), β-estradiol (Es, 20 ng/ml), or relaxin (R, 0.1 ng/ml) or to β-estradiol plus relaxin (Es+R). Conditioned medium, standardized by tissue weight, was subjected to western blotting for collagenase-1 (a, b) or stromelysin-1 (c, d). Cl-1, collagenase-1; Sl-1, stromelysin-1.

Figure 3

Induction of matrix metalloproteinases by relaxin but not by estrogen is accompanied by loss of glycosaminoglycans (GAGs). (a) Disc explants were cultured for 48 hours in basal control medium (Ct), β-estradiol (Es, 20 ng/ml), or relaxin (R, 0.1 ng/ml) or in β-estradiol plus relaxin (Es+R), then sectioned and stained with Safranin-O for GAGs. The percentage area staining positive for GAGs was determined histomorphometrically and plotted. Values are means ± SD. (b) Hormone-mediated changes in GAG synthesis were assessed by ³⁵S-labeling of fibrocartilaginous disc explants. The explants were washed and digested with papain, and the radioactivity was measured. Fold changes (means ± SD) in ³⁵S incorporated into the explants incubated with hormones relative to that in control discs were determined and plotted. (c) To evaluate the modulation of tissue inhibitor of metalloproteinases-1 (TIMP-1) by hormones, the conditioned medium, standardized dry tissue weight (mg), was resolved electrophoretically and transferred to nitrocellulose membranes, and the membranes were probed with anti-TIMP-1 antibody. (d) The bands were quantified by videodensitometry, and the fold induction (mean ± SD) of TIMP-1 by various hormone treatments relative to untreated control explants was plotted. T-1, TIMP-1. * P < 0.05, ** P < 0.01 by Fisher's test.

Figure 4

Inhibition of matrix metalloproteinase (MMP) activity prevents relaxin-mediated loss of glycosaminoglycans (GAGs). Conditioned medium from disc hemisections incubated with β-estradiol (Es), relaxin (R), or β-estradiol plus relaxin (Es+R) in the presence of the MMP inhibitor GM6001 or its control analog was assayed by casein substrate zymograms (a, b). Disc digests from these experiments were assayed for GAGs with the 1,9-dimethylmethylene blue assay, and the results were standardized to tissue dry weight (mg). Fold changes in GAG concentration (mean ± SD) were calculated and plotted (c, d). The untreated control (Ct) discs used in all experiments were exposed to control analog only. * P < 0.05, ** P < 0.01, *** P < 0.001 by Fisher's test.

Figure 5

Relaxin-induced collagenase activity contributes to loss of disc collagen. Conditioned medium from disc incubated with control medium (Ct), β-estradiol (Es), relaxin (R), or β-estradiol plus relaxin (Es+R) in the presence of the matrix metalloproteinase inhibitor GM6001 or its control analog was subjected to fluorescein isothiocyanate-labelled collagen degradation assay. The collagenase activity (relative fluorescence units [RFU]/ml) was standardized by the dry weight of the tissue (mg), and fold changes (means ± SD) were plotted (a, b). Disc digests from these experiments were assayed for collagen with the Sircol assay, and the results were standardized to tissue dry weight (mg). Fold changes in collagen concentration (means ± SD) were calculated and plotted (c, d). The untreated control (Ct) discs used in all experiments were exposed to control analog only. ** P < 0.01, *** P < 0.0001 by Fisher's test.