Matrix metalloproteinase induction by relaxin causes cartilage matrix degradation in target synovial joints

Abstract

Our long-term goal is to understand the mechanisms by which relaxin and estrogen potentially contribute to joint diseases, particularly those afflicting the fibrocartilaginous temporomandibular joint (TMJ). Previously, we showed that relaxin produces a dose-dependent induction of tissue-degrading enzymes of the matrix metalloproteinase (MMP) family, specifically MMP-1 (collagenase-1), MMP-3 (stromelysin-1), MMP-9 (92-kDa gelatinase), and MMP-13 (collagenase-3) in cell isolates and tissue explants from TMJ fibrocartilage. The induction of these MMPs is accompanied by loss of collagen and glycosaminoglycans (GAGs), which was blocked by a pan-MMP inhibitor. We also found the targeted in vivo loss of collagen and GAGs in TMJ discs of ovariectomized rabbits treated with beta-estradiol, relaxin, or both hormones together. Progesterone attenuated the induction of MMPs and matrix loss by relaxin and estrogen. The modulation of matrix composition in TMJ fibrocartilage by these hormones was similar to that observed in the pubic symphysis and differed from that of the knee meniscus. The two target tissues showing the greatest modulation of MMPs and matrix loss, namely, the TMJ disc and pubic symphysis, had similar expression profiles of the estrogen receptors alpha and beta, relaxin-1 receptor (RXFP1, LGR7), and insulin-like peptide 3 receptor (RXFP2, LGR8) and these profiles differed from those in cells from the knee meniscus. These findings suggest a novel model for targeted tissue turnover of cartilage of specific joints through hormone-mediated induction of select MMPs.

Figure 1

Dose-dependent modulation of 52/55-kDa gelatinolytic proteinase by relaxin (R) in the absence or presence of β-estradiol (E) or progesterone (P) in mice TMJ disc, knee meniscus and pubic symphysis fibrochondrocytes. Early passage cells plated at 10⁵/cm² in serum-free medium were exposed to increasing concentrations of relaxin (0 to 10 ng/ml) in the absence or presence of β-estradiol (0.1 ng/ml) or progesterone (10 ng/ml) for 48 hours. The cell-conditioned medium, standardized by total protein, was analyzed by gelatin substrate zymography. Relaxin produced a bimodal dose-dependent increase in expression of the 52/55-kDa-gelatinolytic proteinase (presumably MMP-13) in the TMJ disc and pubic symphysis but very minimally in the knee meniscus. The activity of this proteinase band was inhibited in the presence of the MMP inhibitor 1,10 phenanthroline (data not shown) characterizing this enzyme as a metalloproteinase. In TMJ disc and pubic symphysis cells the induction of this proteinase by relaxin was potentiated by β-estradiol and attenuated by progesterone.

Figure 2

β-estradiol (E), relaxin (R) and progesterone (P) modulate MMP-3 (A), -9 (B) and -13 (C) gene expression in mouse TMJ disc fibrochondrocytes. Early passage fibrochondrocytes were treated as described in figure 1 except that only a single concentration of relaxin (0.1 ng/ml) was used. Total RNA was extracted and subjected to qRT-PCR for the MMPs following standardization by total RNA concentration. 18s RNA was used as an internal control. All three MMPs were significantly up-regulated by E or R or E+R treatments. E increased the induction of MMP-9 and -13 by relaxin, while P attenuated the induction of MMP-3, -9, and -13 by relaxin or β-estradiol. Data represents means ± SD fold increase relative to controls from 3 different experiments. (Ct=control).