Introduction
In certain sites in and around joints, ligaments and tendons subjected to complex tensile and compressive loading specialize into fibrocartilaginous tissues [
1‐
3] containing types I and II collagens and cartilage-specific proteoglycans. These tissues include specific regions of the metacarpophalangeal ligament and the deep flexor tendon, the temporomandibular joint (TMJ) disc, and the pubic symphysis. Within the pubic symphysis of several species, the reproductive hormone relaxin induces matrix remodeling activity during pregnancy and parturition, causing a marked decrease in collagen content through partly characterized mechanisms that transform this tissue into a ligamentous structure [
4‐
9]. The relaxin-mediated loss of matrix macromolecules in the pubic symphysis and other tissues is exacerbated by estrogen [
4,
7,
8,
10]. The relative contribution of matrix synthesis and degradation to these relaxin-mediated changes is not clear, although collagen loss through increased proteolysis has been suggested [
4], and studies in relaxin-knockout mice have implicated increased collagenase activity [
11].
To understand the potential basis for relaxin and estrogen's modulation of the composition of fibrocartilaginous tissues, we previously studied cells isolated from rabbit TMJ discs. Relaxin induced the expression of the matrix metalloproteinases (MMPs) collagenase-1 (MMP-1) and stromelysin-1 (MMP-3) in a dose-dependent fashion but had little effect on the expression of tissue inhibitor of metalloproteinase-1 (TIMP-1) or TIMP-2 [
12]. In cells primed with β-estradiol, however, the relaxin concentration required for maximal induction of collagenase-1 and stromelysin-1 was 90–99% lower than in unprimed cells. Notably, the MMP response to relaxin was specific to fibrocartilaginous cells and was not observed in TMJ synoviocytes. These findings suggest that relaxin, by targeting fibrocartilage, might predispose women to musculoskeletal diseases of fibrocartilaginous joints.
One such disease is TMJ disorders, which affect some 11 million adults in the USA [
13,
14], predominantly women, with a female : male ratio of 2:1 to 6:1 [
14]. Unlike similar diseases of other joints, TMJ disorders occur primarily in women of reproductive age [
14]. Given the gender and age distribution of these disorders and the relaxin-induced loss of matrix macromolecules in the pubic symphysis fibrocartilage [
4,
6,
7,
9] and isolated TMJ fibrocartilaginous cells [
12], we have proposed that relaxin compromises the integrity of fibrocartilaginous tissues by enhancing the degradation of their matrices directly through the induction of specific MMPs. However, although relaxin causes a loss of collagens and proteoglycans in reproductive organs [
6,
7] and also increases MMP expression in specific tissues [
6,
12,
15‐
21], the induction of MMPs by relaxin has not been demonstrated in joint fibrocartilaginous tissues or its induction of MMPs has not been linked to the loss of matrix macromolecules in any tissue.
In this study we determined the effects of relaxin with or without β-estradiol on the modulation of MMPs, and assessed the contribution of these proteinases to the changes in collagen and glycosaminoglycan (GAG) content in fibrocartilaginous disc explants. Our findings are consistent with the hypothesis that relaxin-mediated induction of MMPs is associated with the loss of matrix macromolecules that could compromise tissue function and biomechanics and might lead to joint disease.
Materials and methods
Materials
Twenty-week-old female New Zealand white rabbits were obtained from Nita Bell Laboratories (Hayward, California, USA). Ketamine hydrochloride was from Parke Davis (Morris Plains, New Jersey, USA), and xylazine was from Rugby Lab (Rockville Center, New York, USA). Lactalbumin hydrolysate, α-casein, β-estradiol-17-valerate, pepsin, papain, chondroitin sulfate A sodium from bovine trachea, Safranin-O, Fast Green, cetylpyridinium chloride, and other reagents were from Sigma (St Louis, Missouri, USA). 1,9-Dimethylmethylene blue (DMMB) was from Molecular Probes (Eugene, Oregon, USA), and 35S was from Amersham (Arlington Heights, IL, USA). Protein assay kits, gelatin (EIA grade), and nitrocellulose membrane were from Bio-Rad (Hercules, California). α-Minimal essential medium, trypsin, penicillin–streptomycin, and Fungizone® were from Gibco (Grand Island, New York, USA). All other standard chemicals were from Sigma or Fisher Scientific (Pittsburg, Pennsylvania, USA).
Rabbit anti-human collagenase-1 polyclonal antibody and rabbit anti-mouse stromelysin-1 monoclonal antibody, horseradish peroxidase-conjugated secondary antibodies, and the MMP inhibitor GM6001 and its control analog were from Chemicon International (Temecula, California, USA). Rabbit anti-human-TIMP-1 antibody that cross-reacts with the rabbit inhibitor [
12] was from Triple Point Biologics (Forest Grove, Oregon, USA). Enhanced chemiluminescence reagent for western blotting was from Amersham International (Little Chalfont, Bucks., UK). Sircol collagen assay kit was from Accurate Chemical and Scientific Corporation (Westbury, New York, USA), and fluorescein isothiocyanate (FITC)-labelled collagen was from Chondrex (Seattle, Washington, USA). Recombinant human relaxin was kindly provided by Connetics Corporation (Palo Alto, California, USA).
Retrieval and culturing of TMJ discs, pubic symphysis, and articular cartilage
All procedures on rabbits were approved by the Committee on Animal Research of the University of California, San Francisco, and conducted in accord with accepted standards of humane animal care. Rabbits were anesthetized with ketamine hydrochloride (40 mg/kg) and xylazine (3–5 mg/kg), and the TMJ discs were harvested bilaterally under sterile conditions and immediately placed in calcium-free and magnesium-free phosphate-buffered saline (PBS) containing antibiotics (100 U/ml penicillin, 100 mg/ml streptomycin, and 100 U/ml Fungizone). After removal of the synovium under a dissecting microscope, each disc was washed three times in PBS and bisected longitudinally such that four samples from each rabbit were available (three for hormone treatments and one for control). The hemisections were weighed, placed in wells of a 24-well culture plate, covered with 1 ml of serum-free medium (phenol-free α-minimal essential medium with 0.2% lactalbumin hydrolysate, glutamine, nonessential amino acids, 100 U/ml penicillin, and 100 mg/ml streptomycin) with or without hormones, and cultured at 37°C in air containing 5% CO2.
For determination of MMPs and GAG staining, 32 hemisections from eight rabbits were exposed to medium alone, β-estradiol (20 ng/ml), relaxin (0.1 ng/ml), or both hormones at the same doses for 48 hours. The conditioned medium was collected and stored for MMP assays, and the discs were processed for GAG staining. To assess the contribution of relaxin-induced MMPs to the loss of collagen and GAGs, 24 hemisections from six rabbits were cultured with the MMP inhibitor GM6001 or its control analog 2 hours before and during the hormone treatments. The inhibitor was used at 10 μM, because this concentration was shown to inhibit collagenase activity induced by 0.1 ng/ml relaxin in dose–response experiments to baseline levels. The conditioned medium was collected and stored at -70°C for total protein and MMP assays. The discs were dried in a SpeedVac®, weighed, digested, and used for the determination of GAG and collagen content.
To determine whether the observed induction of collagenase by relaxin is specific to fibrocartilage, experiments were performed with pubic symphysis fibrocartilage, which is a known target site for β-estradiol and relaxin as a positive control, and with articular cartilage from the knee. For retrieval of articular cartilage, the joint was shaved, the articular surfaces were exposed, and the cartilage was scraped from the articular surfaces of the femur and tibia and incubated in PBS with antibiotic as described above. Similarly, the pubic bones and symphyseal areas were exposed under sterile conditions and the pubic symphysis (fibrocartilaginous tissues between the pubic bones) was dissected, removed, and incubated in PBS with antibiotics. The tissues were weighed, placed in wells of a 24-well culture plate, and studied as described above.
Western blotting
Hormone-induced changes in collagenase-1, stromelysin-1, and TIMP-1 were determined by western blotting. Disc-conditioned medium was mixed with 4 × sample buffer and subjected to SDS–polyacrylamide-gel electrophoresis with 10% or 18% gels. Equal amounts of protein (determined with a bicinchoninic acid protein assay kit) were loaded in each lane. The proteins were transferred to nitrocellulose membranes, which were blocked, washed, and incubated for 1 hour with antibodies against TIMP-1 (1:250 dilution), collagenase-1 (1:250 dilution in Tris-buffered saline), or stromelysin-1 (1:500 dilution). The membranes were then washed, incubated with horseradish peroxidase-conjugated goat anti-rabbit antibody (1:1000 dilution), and washed again. Bands were revealed by incubation with enhanced chemiluminescence reagent and exposure to radiographic film. The bands for TIMP-1 western blots were quantified by videodensitometry as described [
22]. Conditioned medium from pubic symphysis and articular cartilage explants was similarly subjected to western blot analysis for collagenase-1 and stromelysin-1.
Substrate zymography
Enzyme activities were quantified by substrate zymography of conditioned media from 32 hemisections (mean wet weight 13 ± 9 mg). The samples were standardized by total protein and subjected to SDS–polyacrylamide-gel electrophoresis with 10% gels containing 2 mg/ml gelatin or casein at 15°C as described [
22]. The gels were washed in 2.5% Triton X-100 for 30 min with one change of wash buffer, incubated at 37°C for 60–72 hours in incubation buffer (50 mM Tris-HCl buffer pH 8, 5 mM CaCl
2, 0.02% NaN
3), stained with 5% Coomassie blue, and destained in 10% acetic acid and 40% methanol until proteinase bands were clearly visible. Images of the gels were captured with a charge-coupled device camera and NIH image software. The levels of 53/58 kDa gelatinolytic and 51/54 kDa caseinolytic enzymes and their low-molecular-mass activated forms were quantified by videodensitometry [
22]. The substrate zymograms rather than western blots were used to quantify hormone-mediated increases in proteinase levels because zymograms are more sensitive, often display both pro-forms and active forms of proteinases, show a greater linear range of densitometric values and have good reproducibility that together enable a reliable quantification of the enzymes from these gels [
23‐
25]. In addition, gelatin zymograms selectively detect proteinase activity at 53/58 kDa and at 43 kDa attributable primarily to collagenase rather than stromelysin because gelatin is a poor substrate for stromelysin [
25,
26].
Histochemical staining and quantification of GAGs
To assess changes in GAG levels, the discs were washed three times in PBS, frozen in OCT compound, and sectioned with a cryostat. The section were defrosted for 30 min, fixed for 10 min in methanol, air-dried for 15 min, stained with 1% Fast Green solution for 3 min, placed in 1% acetic acid for 1 min, stained with 2% Safranin-O for 2 min, dehydrated through successive ethanol and xylene washes, and mounted with coverslips. Ten sections of each hemisection were analyzed by an examiner blinded to the hormone treatment. The stained discs were videodigitized and analyzed with a software program that automatically outlined the total and Safranin-O-stained areas with threshold settings (Photoshop 4.0; Adobe, San Jose, California, USA). These areas were then quantified with NIH Image 1.62, and the percentage of disc staining positive for GAGs was calculated from the ratio of the stained area to the total area in each section. The average of the 10 values for each half disc was used for analysis.
Determination of GAG synthesis by 35S radiolabeling
To quantify GAG biosynthesis, 32 disc hemisections (mean weight 14 ± 4 mg) were incubated at 37°C for 6 hours in 1 ml of phenol-free and serum-free medium with or without hormones and 165 kBq (0.0044 mCi) of
35S as described [
27]. The discs were washed three times with medium containing 1 mg/ml sodium sulfate and digested for 24 hours with 20 U/ml papain. The digest (500 μl) was incubated for 30 min with 100 μl of 5% cetyl pyridiuium chloride in 0.3 M potassium chloride at room temperature (20–22°C) to precipitate GAGs. After centrifugation (3000
g for 20 min), the supernatant was removed and the precipitate was dissolved in 600 μl of concentrated formic acid by heating to 70°C for 10 min. Aliquots (20 μl) of this solution were added to 3 ml of scintillation fluid and subjected to liquid scintillation counting. The radioactivity (counts/min) was standardized to the total dry disc weight.
Quantification of GAGs and collagen
Each disc hemisection was digested in 600 μl of 3 mg/ml pepsin in 0.05 M acetic acid and incubated at 37°C for 18–20 hours in a dry bath. DMMB binding assays for GAGs, and Sircol assays for collagen content, were performed in triplicate on 24 disc hemisections. The DMMB reagent was prepared as described [
28]. Pepsin digests (200 μl) from each treatment group (GM6001 or analog control) were mixed with 1 ml of DMMB reagent, and absorbance at 525 nm was determined with a spectrophotometer. The GAG concentration (μg/ml) was determined by comparing the absorbance of the sample against a standard curve prepared from bovine chondroitin sulfate A, and the disc GAG content was standardized to the total dry tissue weight.
For the collagen assay, 200 μl of pepsin digest was mixed with 1 ml of Sircol dye reagent, incubated for 30 min at room temperature, and centrifuged at 10,000 g to separate the unbound dye from the collagen-bound dye. After removal of the unbound dye, 1 ml of the alkali reagent was added to the collagen–dye complex and vortex-mixed to dissolve the collagen-bound dye completely. Aliquots (200 μl) were transferred to the 96-well plates, and absorbance at 550 nm was determined with a microtiter plate reader (Molecular Devices, Sunnyvale, California, USA). The collagen concentration (μg/ml) was determined against a collagen standard curve, and the disc collagen content was standardized to the total disc dry weight.
Quantification of collagenase activity
Collagenase activity in conditioned medium from discs cultured with GM6001 or control analog was assessed by FITC–collagen assay. A 96-well plate was coated with FITC–collagen (10 μg per well) overnight at 4°C and washed twice with PBS. Disc-conditioned medium (100 μl) was added to the wells, and the plate was incubated at 35°C for 1 hour. As a reference, 100 μl of blank medium containing 3000 ng of bacterial collagenase was added to one set of wells for complete digestion of FITC–collagen. After incubation, 90 μl from each well was transferred to another 96-well plate, and the fluorescence intensity of degraded FITC–collagen products was determined with a microplate spectrofluorometer (Spectramax Gemini XS; Molecular Devices) with excitation at 494 nm and emission at 518 nm. The data were converted to relative fluorescence units of collagenase activity as described by the manufacturer and standardized to the dry weight of each half disc. The fold differences in collagenase activity in medium from control and hormone-treated discs were determined for each experiment. All assays were performed in duplicate.
Statistical analysis
Because of inherent variability in matrix content and proteinase activity in discs from different rabbits, three disc hemisections from each rabbit were treated with hormones and one served as control. MMP levels and the GAG and collagen content in each hormone-treated disc hemisection were standardized to the values of the control hemisection within each animal and the fold changes were plotted as histograms. The statistical significance of differences was determined by single-factorial analysis of variance (ANOVA). Intergroup differences were analyzed by Fisher's multiple comparisons test; P < 0.05 was considered statistically significant. Values are expressed as means ± SD.
Discussion
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,
9]. 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,
9]. 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,
16].
MMPs contribute substantially to tissue degeneration in inflammatory joint diseases, including rheumatoid arthritis and osteoarthritis [
33‐
35]. 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‐
7], 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‐
38]. 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].
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
TN performed all experiments, assays and analysis in which MMP inhibitors were used. TTD performed all experiments to characterize the changes in MMPs and GAGs in joint fibrocartilage in response to relaxin and β-estradiol. GH and QZ characterized the responses of the pubic symphysis fibrocartilage and articular cartilage to the hormones. MS retrieved tissues from animals and assisted in several MMP assays. SK conceived the study, participated in its design and coordination, supervised the statistical analysis, and wrote the manuscript. All authors read and approved the final manuscript.