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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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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.
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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.

Mentions: Explanted discs constitutively expressed collagenase-1 (MMP-1) (Fig. 1a, lane 1), and the expression of this proteinase was increased by exposure to relaxin alone or to β-estradiol plus relaxin (Fig. 1a, lanes 3 and 4). Gelatin substrate zymograms confirmed the induction of 53/58 kDa proteinase by these hormones and, because this assay is more sensitive than western blots, showed an additional 43 kDa gelatinolytic enzyme (Fig. 1b). Because the gelatinolytic enzymes were inhibited by 1,10-phenanthroline (Fig. 1b, lane 5), these proteinases were characterized as MMPs, most probably procollagenase-1 and active collagenase-1. Western blots with conditioned medium from a disc explant exposed to relaxin showed that the 53/58 kDa and 43 kDa activities corresponded to procollagenase-1 and collagenase-1, respectively (Fig. 1b, lane 6). Proteinase expression was about 1.7-fold higher in relaxin-treated and β-estradiol plus relaxin-treated discs than in control cultures (P < 0.05) and was not potentiated by β-estradiol (Fig. 1c).


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)

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.
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Related In: Results  -  Collection

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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.
Mentions: Explanted discs constitutively expressed collagenase-1 (MMP-1) (Fig. 1a, lane 1), and the expression of this proteinase was increased by exposure to relaxin alone or to β-estradiol plus relaxin (Fig. 1a, lanes 3 and 4). Gelatin substrate zymograms confirmed the induction of 53/58 kDa proteinase by these hormones and, because this assay is more sensitive than western blots, showed an additional 43 kDa gelatinolytic enzyme (Fig. 1b). Because the gelatinolytic enzymes were inhibited by 1,10-phenanthroline (Fig. 1b, lane 5), these proteinases were characterized as MMPs, most probably procollagenase-1 and active collagenase-1. Western blots with conditioned medium from a disc explant exposed to relaxin showed that the 53/58 kDa and 43 kDa activities corresponded to procollagenase-1 and collagenase-1, respectively (Fig. 1b, lane 6). Proteinase expression was about 1.7-fold higher in relaxin-treated and β-estradiol plus relaxin-treated discs than in control cultures (P < 0.05) and was not potentiated by β-estradiol (Fig. 1c).

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