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

Naqvi T, Duong TT, Hashem G, Shiga M, Zhang Q, Kapila S - Arthritis Res. Ther. (2004)

Bottom Line: 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.None of the hormone treatments altered the synthesis of GAGs, suggesting that the loss of this matrix molecule probably resulted from increased matrix degradation.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: University of California, San Francisco, USA. sheema@hotmail.com

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.

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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.
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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.

Mentions: To establish an association between the increased MMP activity and the loss of GAGs in explants treated with relaxin or β-estradiol plus relaxin, we cultured the explants with the MMP inhibitor GM6001 or its control analog. Western blot analysis showed a higher expression of stromelysin-1 in hormone-treated than untreated disc explants in the presence of GM6001 or its control analog (data not shown). However, zymography showed increased 51/54 kDa caseinolytic activity (stromelysin-1) only in hormone-treated explants incubated with the control analog (Fig. 4a), and not in those incubated with GM6001 (Fig. 4b).


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

Naqvi T, Duong TT, Hashem G, Shiga M, Zhang Q, Kapila S - Arthritis Res. Ther. (2004)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC1064880&req=5

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.
Mentions: To establish an association between the increased MMP activity and the loss of GAGs in explants treated with relaxin or β-estradiol plus relaxin, we cultured the explants with the MMP inhibitor GM6001 or its control analog. Western blot analysis showed a higher expression of stromelysin-1 in hormone-treated than untreated disc explants in the presence of GM6001 or its control analog (data not shown). However, zymography showed increased 51/54 kDa caseinolytic activity (stromelysin-1) only in hormone-treated explants incubated with the control analog (Fig. 4a), and not in those incubated with GM6001 (Fig. 4b).

Bottom Line: 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.None of the hormone treatments altered the synthesis of GAGs, suggesting that the loss of this matrix molecule probably resulted from increased matrix degradation.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: University of California, San Francisco, USA. sheema@hotmail.com

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.

Show MeSH
Related in: MedlinePlus