This study shows that relaxin induced the expression of collagenase-1 and stromelysin-1 in rabbit TMJ disc explants, accompanied by a loss of GAGs and collagen, but did not affect GAG synthesis. In explants cultured with the MMP inhibitor GM6001, collagenase-1 and stromelysin-1 activities in hormone-treated discs were inhibited to baseline levels, and collagen and GAG content were maintained at control levels. These findings show that relaxin has degradative effects on nonreproductive synovial joint fibrocartilaginous tissue and provide evidence that increases in MMP activity mediated by relaxin and β-estradiol plus relaxin contribute directly to the loss of disc collagen and GAGs. The lack of effect on GAG synthesis further validates the importance of the degradative component of the remodeling cycle in relaxin's modulation of matrix loss in fibrocartilage.
Because the MMP inhibitor used in our studies is not specific for collagenase-1 and stromelysin-1, the hormone-induced loss of collagen and GAGs cannot be specifically linked to those two proteinases. Rather, our findings implicate MMPs in general in this response. However, because GM6001 has a low dissociation constant for both collagenase-1 and stromelysin-1 [30
], and their induction by relaxin was accompanied by a loss of their matrix substrates, collagenase-1 and stromelysin-1 are probably involved in the relaxin-mediated loss of collagen and GAGs, respectively.
In contrast to the results obtained with relaxin and β-estradiol plus relaxin, the induction of collagenase-1 and stromelysin-1 by β-estradiol alone was not accompanied by changes in GAG or collagen content within the disc. How can we explain this apparent discrepancy? β-Estradiol had little effect on GAG synthesis, as measured by 35S incorporation, but it produced a statistically significant increase in TIMP-1 expression that could have counteracted any increases in degradative activity due to increased expression of collagenase-1 and stromelysin-1. Indeed, the results of the collagen degradation assay lend credence to this hypothesis. These findings imply that relaxin and β-estradiol selectively contribute to the degeneration of fibrocartilaginous tissue by differentially modulating MMP expression, matrix synthesis, and net matrix content.
The potential similarities in the responsiveness of TMJ fibrocartilaginous explants and the pubic symphysis fibrocartilage to relaxin are reflected not only by the relaxin's induction of collagenase but also by the comparable loss of collagen on the exposure of these tissues to the hormone [4
]. Thus, the extent of collagen loss in fibrocartilaginous disc explants exposed to relaxin (40%) or β-estradiol plus relaxin (60%) was similar to that in the pubic symphysis of unprimed and β-estradiol-primed ovariectomized nonpregnant rats (64 ± 4% and 68 ± 6%, respectively) [4
]. Similarly, in pregnant ovariectomized rats, relaxin decreased collagen to 39% of the levels in nonpregnant animals [9
]. Additionally, β-estradiol alone had minimal effects on the collagen content of the fibrocartilaginous TMJ disc, which is also similar to observations on the pubic symphysis [4
]. Thus, relaxin with or without β-estradiol, but not β-estradiol alone, has a potent effect on the amount of collagen in fibrocartilaginous tissues from different sites, including the pubic symphysis and synovial joints. These findings also suggest that in fibrocartilaginous tissues, including the TMJ disc and possibly the pubic symphysis, relaxin decreases collagen and GAG content primarily by inducing MMP expression.
The response of articular cartilage to relaxin or β-estradiol plus relaxin was substantially different from that of the TMJ disc and pubic symphysis fibrocartilages. Although the reasons for these differences remain to be determined, it is well accepted that articular cartilage is a cartilaginous tissue containing chondrocytic cells, whereas fibrocartilage is a heterogenous tissue composed of cartilage and fibrous tissue that contains cells of fibroblastic, chondrocytic, and fibrochondocytic phenotypes. It is plausible that of these cells, the fibroblastic and/or fibrochondrocytic cells found in fibrocartilage, rather than the chondrocytic cells, are those that produce the observed responses to relaxin and β-estradiol plus relaxin. Indeed, previous findings on both dermal fibroblasts showing a potent induction of MMP-1 [18
] and on articular chondrocytes that show minimal modulation of total collagen synthesis by relaxin [31
] lend credence to this hypothesis. Additional studies are indicated to address the mechanistic basis for the differences in responsiveness of fibrocartilaginous versus cartilaginous cells to relaxin.
Our findings are consistent with emerging data suggesting that the mechanisms for the loss of matrix macromolecules caused by relaxin are tissue-specific [32
]. Thus, for example whereas relaxin increases collagenase-1 expression in TMJ disc and pubic symphyseal [6
] fibrocartilages, it had minimal effects on its expression in articular cartilage explants. In monolayer articular or multilayer growth plate rabbit chondrocytes, relaxin produces no net change in collagen synthesis and no alterations in type II collagen mRNA levels, but increases the expression of types I and III collagen mRNA, thereby amplifying the dedifferentiation process [31
]. In contrast, relaxin downregulates collagen expression by up to 40% and induces collagenase expression in cultured dermal fibroblasts [18
]. As in our study, relaxin increases collagenase activity in human cervical stromal cells; however, in contrast to our findings, it also increases GAG synthesis [15
MMPs contribute substantially to tissue degeneration in inflammatory joint diseases, including rheumatoid arthritis and osteoarthritis [33
]. Our findings show that relaxin directly modulates MMP expression and probably causes matrix loss in fibrocartilaginous tissues from a synovial joint. Although the effects of relaxin on loss of matrix macromolecules, particularly collagen, have been demonstrated in the fibrocartilaginous pubic symphysis [4
], this is the first study to demonstrate a similar targeting of fibrocartilaginous tissues from the synovial TMJ, and may implicate this hormone in the pathogenesis of TMJ disease in a subset of women with these disorders. Because even subtle alterations in collagen and GAG composition can affect the structural properties and the ability of joint tissues to function normally, this modulation of MMPs and resulting matrix loss in the fibrocartilaginous TMJ disc by relaxin might explain the distinct age and gender distribution of TMJ diseases. Furthermore, these findings have potential physiologic relevance because the induction of collagenase-1 and stromelysin-1 and the loss of collagen and GAGs occurred at concentrations of relaxin found systemically in cycling women [36
]. Although the ability of systemic relaxin to access the TMJ and reach the avascular disc remains to be determined, our recent findings in vivo
showing relaxin-mediated decreases in GAG concentration in the TMJ discs of ovariectomized rabbits suggest that this systemic hormone can indeed access the TMJ disc and contribute to its degradation [39