Modulation Of Matrix Metalloproteinases Research Articles

Modulation of matrix metalloproteinase and TIMP-1 expression by TGF-β1 in cultured human RPE cells

In order to investigate the effects of TGF-beta1 on the expression of MMP-2, -9 and TIMP-1 in human retinal pigment epithelial (RPE) cells, the third-sixth passage cultured RPE cells were treated with TGF-beta1 at different concentrations (0.01, 0.1, 1.0, 10 ng/mL), the expression of MMP-2, -9 and TIMP-1 mRNA was detected by semi-quantitative RT-PCR assays. MMP-2, -9 and TIMP-1 mRNA were expressed in the cultured RPE cells. The values of MMP-2/beta-actin in the cells treated with 0.1, 1.0, 10 ng/mL TGF-beta1 were 1.04 +/- 0.04, 1.07 +/- 0.02 and 1.11 +/- 0.03, respectively, significantly higher than in the control group (0.96 +/- 0.03, P < 0.05-0.01). The expression of MMP-2 mRNA could be up-regulated by TGF-beta1, in a dose-dependent manner. The expression of MMP-9 mRNA in the cultured RPE cells was slightly up-regulated by various TGF-beta1 concentrations treatment. The values of TIMP-1/beta-actin in the cells treated with 0.01 and 0.1 ng/ mL TGF-beta1 were 0.85 +/- 0.01 and 0.97 +/- 0.02 respectively, significantly lower than in the control group (1.07 +/- 0.04, P < 0.01), indicating that the expression of TIMP-1 mRNA was down-regulated by TGF-beta1 at low concentrations. But along with the increase of TGF-beta1 concentrations (1.0 and 10 ng/mL), the expression of TIMP-1 mRNA was slightly up-regulated, not significantly different from that in the control group (P > 0.05). It was concluded that TGF-beta1 might play an important role in the up-regulation of the expression of MMP-2 in RPE cells and result in a directional shift in the balance between MMP and TIMP. This may be facilitated for RPE cells to migrate in the pathogenesis of vitreoretinopathy.

Cytokine-mediated modulation of MMPs and TIMPs in multipotential neural precursor cells

Recent studies have implicated the inflammatory process during experimental allergic encephalomyelitis (EAE) in triggering migration and differentiation of transplanted neural precursors cells (NPCs) into the inflamed white matter. The pro-inflammatory cytokines tumor necrosis factor (TNF)-α and interferon (IFN)-γ are key factors in the pathogenesis of brain inflammation in EAE and were shown to enhance NPCs migration in vitro. As cell migration is dependent on extracellular matrix remodeling, involving proteolytic enzyme members of the matrix metalloproteinase (MMPs) family, we characterized the profile of expression of MMPs and their endogenous inhibitors (TIMPs) in rat NPCs, and evaluated the effects of TNF-α, IFN-γ and IFN-β, a clinically proven modulator of brain inflammation, on the expression of these molecules. Newborn rat striatal NPCs were expanded in spheres as nestin+, PSA-NCAM+ and NG2(−) cells, which can differentiate into astrocytes, oligodendrocytes and neurons. NPCs' gelatinase activities of MMP-2 and MMP-9, as determined by zymography, were increased by TNF-α, and to a lesser extent by IFN-γ. Semi-quantitative RT-PCR indicated that TNF-α also upregulated MMP-9 mRNA levels. IFN-β suppressed the TNF-α-induced levels of secreted MMP-9 and MMP-2, while enhancing the expression of TIMP-1 and TIMP-2 mRNA. These results suggest that MMPs activity is induced in NPCs by pro-inflammatory cytokines to mobilize them for promoting reparative processes. IFN-β, on the other hand, appears to have an anti-proteolytic influence that may attenuate such NPC-mediated repair processes.

Inhibition of matrix metalloproteinase‐2 secretion and invasion by human ovarian cancer cell line SK‐OV‐3 with lysine, proline, arginine, ascorbic acid and green tea extract

Based on the poor prognosis associated with ovarian cancer and reported anticancer properties of specific nutrients, we investigated the effect of a nutrient mixture (NM) containing lysine, proline, arginine, ascorbic acid and epigallocatechin gallate on human ovarian cancer cells SK-OV-3 by measuring: cell proliferation, modulation of matrix metalloproteinase (MMP)-2 and -9 expression, and cancer cell invasive potential. Cell proliferation was evaluated by MTT assay, MMP activity by gelatinase zymography, and invasion through Matrigel. Human ovarian cancer cell growth was not significantly affected by the NM. Zymography demonstrated inhibition of MMP-2 secretion in a dose-dependent fashion with virtual total inhibition at 50 microg/mL NM concentration. Invasion of human ovarian cancer cells through Matrigel decreased in a dose-dependent fashion, with 90% inhibition at 500 microg/mL NM and 100% inhibition at 1000 microg/mL NM (P < 0.0001). The combination of lysine, proline, arginine, ascorbic acid and green tea extract tested inhibited critical steps in cancer development and spread, such as MMP expression and invasion, indicating its potential as a treatment modality against ovarian cancer.

Electroporative α-MSH gene transfer attenuates thioacetamide-induced murine hepatic fibrosis by MMP and TIMP modulation

Hepatic fibrosis represents a process of healing and scarring in response to chronic liver injury. alpha-Melanocyte-stimulating hormone (alpha-MSH) is a 13-amino-acid peptide with potent anti-inflammatory effects. We have previously demonstrated that alpha-MSH gene therapy protects against thioacetamide (TAA)-induced acute liver failure. Therefore, the aim of this study is to investigate whether alpha-MSH gene therapy possesses antihepatic fibrogenic effect. Liver fibrosis was induced by long-term TAA administration in mice. alpha-Melanocyte-stimulating hormone expression plasmid was delivered via electroporation after liver fibrosis was established. Our results showed that alpha-MSH gene therapy attenuated liver fibrosis in TAA-treated mice. Reverse transcription polymerase chain reaction revealed that alpha-MSH gene therapy attenuated the liver transforming growth factor-beta1, collagen alpha1 and cell adhesion molecule mRNA upregulation. Following gene transfer, the expression of alpha-smooth muscle actin and cyclooxygenase-2 were both significantly attenuated. Further, alpha-MSH significantly increased matrix metalloproteinase (MMP), while tissue inhibitors of matrix metalloproteinase (TIMPs) were inactivated. In summary, alpha-MSH gene therapy reversed established liver fibrosis in mice and prevented the upregulated fibrogenic and pro-inflammatory gene responses after TAA administration. Its collagenolytic effect might be attributed to MMP and TIMP modulation. Hence, alpha-MSH gene therapy may be an effective therapeutic modality against liver fibrosis with potential clinical use.

α‐Melanocyte‐stimulating hormone gene therapy reverses carbon tetrachloride induced liver fibrosis in mice

Hepatic fibrosis represents a process of healing and scarring in response to chronic liver injury. Effective therapies are lacking. We have previously demonstrated that alpha-melanocyte-stimulating hormone (alpha-MSH) gene therapy protects against thioacetamide-induced acute liver failure in mice. Recent reports showed that collagen metabolism is a novel target of alpha-MSH. Therefore, the aim of this study is to investigate whether alpha-MSH gene therapy possesses anti-hepatic fibrogenic effect in mice. Liver fibrosis was induced in mice by administering carbon tetrachloride (CCl4) continuously for 10 weeks. Alpha-MSH expression plasmid was delivered via electroporation after liver fibrosis had been established. Histopathology, reverse-transcription polymerase chain reaction (RT-PCR), immunoblotting, and gelatin zymography were used to investigate its possible mechanisms of action. Alpha-MSH gene therapy reversed established liver fibrosis in CCl4-treated mice. RT-PCR revealed that alpha-MSH gene therapy attenuated the liver TGF-beta1, collagen alpha1, and cell adhesion molecule mRNA upregulation. Following gene transfer, both the activation of alpha-smooth muscle actin (alpha-SMA) and cyclooxygenase-2 (COX-2) was significantly attenuated. Further, alpha-MSH significantly increased matrix metalloproteinase (MMP) activity with tissue inhibitors of matrix metalloproteinase (TIMP) inactivation. We have demonstrated that alpha-MSH gene therapy reversed established liver fibrosis in mice. It also prevented the upregulated fibrogenic and proinflammatory gene response after CCl4 administration. Its collagenolytic effect may be attributed to MMP and TIMP modulation. In summary, alpha-MSH gene therapy may be an effective therapeutic modality against liver fibrosis with potential clinical use.

The role of DNA hypomethylation in the control of stromelysin gene expression

Genome-wide DNA hypomethylation is a critical mechanism underlying neoplastic transformation. Thus, genes that are suppressed in normal tissues or in specific cell types may become aberrantly expressed in neoplasia. To determine whether DNA methylation can modulate matrix metalloproteinase (mmp) gene expression, we have used a genetically engineered cell line in which both key DNA methyltransferase genes, Dnmt- 1 and Dnmt- 3b, were removed by homologous recombination. We found that cells bearing a dual knock-out of both Dnmt- 1 and Dnmt- 3b genes induced de novo expression of mmp- 3 gene, but not that of mmp- 1 and mmp- 2. Furthermore, treatment of the wild-type cells with DNA methylase inhibitors 5-aza-dC and zebularine also induced mmp- 3 gene expression. On the other hand, in vitro methylation of the mmp-3 promoter suppressed its transcriptional activity. Finally, we found that induction of mmp- 3 and mmp- 10 gene expression by hypomethylation was cell-specific, suggesting that epigenetic changes may predispose cells to express stromelysin genes.

Cytokine pattern determines the progression of experimental periodontal disease induced by Actinobacillus actinomycetemcomitans through the modulation of MMPs, RANKL, and their physiological inhibitors

Inflammatory and immune reactions raised in response to periodontopathogens are thought to trigger periodontal tissue destruction. We therefore investigated the expression of matrix metalloproteinases (MMPs) and the osteoclastogenic factor RANKL (receptor activator of nuclear factor-kappaB ligand), their respective inhibitors TIMPs (tissue inhibitors of metalloproteinases) and OPG (osteoprotegerin) and their possible correlation with the expression of inflammatory and regulatory cytokines in the course of experimental periodontal disease in mice. We characterized the time course of leukocyte migration and alveolar bone loss in C57BL/6 mice infected with Actinobacillus actinomycetemcomitans. Quantitative polymerase chain reaction (RealTime PCR) and ELISA were performed to determine the expression of MMPs, TIMPs, RANKL, OPG and cathepsin K, interleukin-1beta, tumor necrosis factor-alpha, interferon-gamma, interleukin-12, interleukin-4 and interleukin-10 in periodontal tissue samples harvested throughout the course of experimental disease. Oral inoculation of A. actinomycetemcomitans results in an intense and widespread migration of leukocytes to the gingival tissues, besides marked alveolar bone resorption. Our data also demonstrate two distinct patterns of MMP/TIMP and RANKL/OPG expression in the course of experimental periodontal disease. The expression of MMPs (MMP-1, 2 and 9) and RANKL was correlated with the expression of interleukin-1beta, tumor necrosis factor-alpha and interferon-gamma, in a time period characterized by the intense increase of inflammatory reaction and alveolar bone loss. On the other hand, interleukin-4 and interleukin-10 were associated with higher expression of TIMPs (TIMP 1, 2 and 3) and OPG, with a lower expression of MMPs and RANKL, and with reduced rates of increase of cellular infiltration in periodontal tissues and alveolar bone loss. It is possible that the pattern of cytokines produced in periodontal tissues determines the progression and the severity of experimental periodontal disease, controlling the breakdown of soft and bone tissues through the balance between MMPs/TIMP and RANKL/OPG expression in gingival tissues.

Suppression of human cervical cancer cell lines Hela and DoTc2 4510 by a mixture of lysine, proline, ascorbic acid, and green tea extract

Cervical cancer, the second most common cancer in women, once metastasized, leads to poor prognosis. We investigated the antitumor effect of a nutrient mixture (NM) containing lysine, proline, arginine, ascorbic acid, and green tea extract on human cervical cancer cells Hela (CCL-2) and DoTc2 4510 by measuring cell proliferation (MTT assay), modulation of matrix metalloproteinases (MMP)–2 and MMP-9) expression (gelatinase zymography), and cancer cell invasive potential (Matrigel). NM showed significant antiproliferative effect on CCL-2 and DoTc2 4510 cancer cells. The NM inhibited CCL-2 expression of MMP-2 and MMP-9 in a dose-dependent fashion, with virtual total inhibition of MMP-2 at 1000 μg/mL and MMP-9 at 500 μg/mL NM. Untreated DoTc2 4510 cells showed MMP-9 expression, which was enhanced with phorbol 12-myristate 13-acetate treatment. NM inhibited MMP-9 expression in a dose-dependent fashion, with virtual inhibition at 500 μg/mL. Invasion of human cervical cancer cells CCL-2 and DoTc2 4510 through Matrigel decreased in a dose-dependent fashion, with 100% inhibition at 500 μg/mL NM (P &lt; 0.0001) and 1000 μg/mL NM (P &lt; 0.0001), respectively. Our results suggest that the mixture of lysine, proline, arginine, ascorbic acid, and green tea extract has potential in the treatment of cervical cancer by inhibiting critical steps in cancer development and spread.

Culture of Human Fibrosarcoma HT-1080 Cells in Presence of Fibronectin Activates MMP-2

The importance of tumor cell surface integrin receptors in regulation of matrix metalloproteinase (MMP) expression and function has been reported. Integrin-ECM ligand interaction leads to phosphorylation of focal adhesion kinase (FAK) and activation of mitogen activated protein kinase (MAPK) pathways. In this present study, we cultured human fibrosarcoma cells, HT-1080, in presence of fibronectin to study fibronectin-integrin mediated modulation of MMP activity. HT-1080 cells were cultured in serum free medium (SFCM) in presence of fibronectin, SFCM was collected, and gelatin zymography was performed. Western blot and immunocytochemistry were performed with HT-1080 cells cultured in presence of fibronectin. Culture of HT-1080 cells in presence of 50 microg/1.5 ml fibronectin led to expression of pro-MMP-9 and activation of MMP-2 within 1 hr. When HT-1080 cells were treated with PI-3K inhibitor (LY294002) and grown in presence of fibronectin, MMP-2 activation was partially inhibited, but when cells were treated with ERK inhibitor (PD98059) and grown in presence of fibronectin, MMP-2 activation was almost completely inhibited. Tyrosine phosphorylation of FAK and ERK were increased in HT-1080 cells grown in presence of fibronectin. Processing of MT1-MMP was also observed in HT-1080 cells grown in presence of fibronectin. The reorganization of actin filaments in fibronectin treated HT-1080 cells was also noticeable. Our findings indicate that culture of HT-1080 cells in SFCM in presence of fibronectin perhaps generates a signaling cascade that leads to expression of pro-MMP-9 and activation of MMP-2 within 1 hr. The signaling pathway activated seems to be the FAK/ERK pathway.

Matrix metalloproteinase 2 secretion in WEHI 164 fibrosarcoma cells is nitric oxide-related and modified by morphine

Matrix metalloproteinases (MMP) are ubiquitous enzymes involved in extracellular matrix remodeling, and as a consequence in a number of physiological and pathological states, including development, wound healing and cancer. A crucial feature of cancer progression and metastasis is the disruption of extracellular matrix, and spreading of proliferating cancer cells. Modulation of MMP is a main target of cancer research. Using the mouse fibrosarcoma cell line WEHI 164, producing high amounts of MMP-2, we investigated whether we could modulate its production. We report that MMP-2 is under the control of nitric oxide (NO)/nitric oxide synthase (NOS) system. In addition, we show that NOS activity is controlled by opioids in a non-opioid receptor-related manner. Finally, we provide evidence that morphine, when administrated at low, non-toxic concentrations (< 10 − 9 M) attenuates MMP-2 activity. We conclude that, as morphine is able to decrease metalloproteinase activity via the NO/NOS system, it may have a place in the treatment of several sarcomas including fibrosarcoma.

Retinoids and steroids in bovine mammary gland immunobiology

Major variations in systemic retinoid and steroid levels during the periparturient period suggest that these potent signaling molecules play a role in the decreased innate immune response observed at early lactation. This period coincides with a marked increase in the incidence of coliform mastitis. Two critical parameters have been identified within the cow's first line of defense during acute coliform mastitis: efficient mammary epithelium remodeling and functional polymorphonuclear leukocytes. The overall aim of this review is to relate the actual knowledge on retinoids and steroids to bovine mammary gland immunobiology. Recent insights on the potential role of retinoic acid, estradiol and progesterone in the development and maintenance of a normal glandular epithelial architecture are provided. In parallel, their influence on myelopoiesis and PMN functionality is also discussed. Retinoic acid and steroid hormones predominantly serve as ligands that activate specific transcription factors belonging to a nuclear receptor superfamily and thereby control gene expression. Additionally, both retinoids and steroids may act via non-nuclear receptor mediated pathways. There are also indications on a subtle interplay between retinoic acid and estrogen pathways. These messengers probably affect cell proliferation and apoptosis through interaction with local growth factors e.g., the insulin-like growth factor /insulin-like growth factor binding protein-axis, and with the extracellular matrix e.g., by modulating matrix metalloproteinase activity. In conclusion, arguments are provided supporting the importance of retinoids and sex steroid hormones in the mammary gland innate immune defense.

Phorbol ester induced short- and long-term permeabilization of the blood–CSF barrier in vitro

Interconnected by tight junctions, the epithelial cells of the choroid plexus form a barrier separating the cerebrospinal fluid (CSF) from blood. Using an in vitro model based on porcine choroid plexus epithelial cells (PCPEC), we investigated the influence of PKC activating phorbol 12-myristate 13-acetate (PMA) on barrier properties and analyzed mechanisms involved in the regulation of barrier tightness. Applied in concentrations of 5–25 nM, PMA induced a fast and lasting decrease of the transepithelial electrical resistance (TER), which could be blocked by rottlerin, indicating the involvement of PKCδ in signal transduction. The immediate impairment of barrier integrity was accompanied by dephosphorylation of occludin and formation of actin bundles. Moreover, in the presence of at least 25 nM PMA, changes of cell shape as well as discontinuities of tight junction strands were observed, suggesting the disruption of cell–cell contacts. Exposure to PMA for 1–2 days additionally induced down-regulation of claudin-2 and up-regulation of barrier modulating matrix metalloproteinase (MMP)-9, respectively. The results show that different interconnected mechanisms directly and indirectly targeting at the tight junctions are released by PMA contributing to the short-term and long-term decrease of TER and opening of the blood–CSF barrier in vitro.

Adhesion of elastomeric impression materials to trays.

The tensile and shear adhesive bond strengths of two addition cured silicones (Provil and Express) and a polyether (Impregum) impression material to brass, poly(methylmethacrylate) and visible light-cured (VLC) tray resin were determined. Adhesive application significantly increased the bond strength; Provil and Express adhered most strongly to brass; whilst the Impregum-VLC combination produced the strongest bond. Indeed, VLC resin generated greater adhesion than acrylic resin. Exchanging the adhesives specified for each silicone material generally resulted in higher bond strengths. No correlation was established between speed of separation of the test surfaces and bond strength. For optimum clinical performance, the impression material (adhesive) tray material giving the highest bond strength should be utilized.

Divergent effects of oncostatin M on astroglioma cells: Influence on cell proliferation, invasion, and expression of matrix metalloproteinases

Oncostatin M (OSM), a cytokine of the interleukin-6 (IL-6) family, can either promote or inhibit cell growth in various normal and tumor cells. We addressed the effects of exogenous OSM on the proliferation and invasion of human astroglioma cells. In addition, we investigated one of the possible mechanisms involved: modulation of matrix metalloproteinase (MMP) expression and enzymatic activity. We found that OSM inhibited the proliferation of two human astroglioma cell lines (CH235-MG and U87-MG), and that this effect was not due to apoptosis. The inhibitory effect of OSM on proliferation was mediated through the gp130/OSMRbeta receptor complex. To extend these findings, we analyzed the effects of OSM on primary tumor cells from glioblastoma patients. OSM suppressed the proliferation of primary glioblastoma cells, but not that of normal astrocytes. Interestingly, OSM did not suppress astroglioma cell invasion. This may be due to the differential regulation of MMPs by OSM. We found that OSM inhibited the constitutive expression of MMP-2, while MMP-9 expression was enhanced in astroglioma cell lines. We conclude that OSM inhibits proliferation of human astroglioma cells and primary glioblastoma cells via the gp130/OSMRbeta receptor complex. However, OSM does not affect the invasive capacity of the astroglioma cells, which may be due to the divergent effects of OSM on MMP-2 and MMP-9 expression. Collectively, these findings suggest a complex role for OSM in astroglioma biology.

What role do extracellular matrix changes contribute to the cardiovascular disease burden of diabetes mellitus?

Matrix metalloproteinases (MMP) and their inhibitors (TIMP) are central factors in the control of extracellular matrix turnover. They are important in normal physiology and also during a range of pathological states. In this review, we have systematically identified clinical articles relevant to cardiovascular disease in diabetes from the last 10 years. Our aim was to outline the structure, function and regulation of metalloproteinases and their key roles in cardiomyopathy and vasculopathy in diabetes. We also explore the effects of drug intervention on both human subjects with diabetes and experimental animal models. The modulation of MMP and TIMP activity using drugs that affect the expression and function of these proteins may provide us with new ways to treat this serious and disabling disease, and we explore potential mechanisms and treatments.

VEGF-Induced BBB Permeability is Associated with an MMP-9 Activity Increase in Cerebral ischemia: Both Effects Decreased by ANG-1

After cerebral ischemia, angiogenesis, by supplying for the deficient perfusion, may be a beneficial process for limiting neuronal death and promoting tissue repair. In this study, we showed that the combination of Ang-1 and vascular endothelial growth factor (VEGF) provides a more adapted therapeutic strategy than the use of VEGF alone. Indeed, we showed on a focal ischemia model that an early administration of VEGF exacerbates ischemic damage, because of its effects on blood-brain barrier (BBB) permeability. In contrast, a coapplication of Ang-1 and VEGF leads to a significant reduction of the ischemic and edema volumes by 50% and 42%, respectively, in comparison with VEGF-treated mice. We proposed that Ang-1 blocks the BBB permeability effect of VEGF in association with a modulation of matrix metalloproteinase (MMP) activity. Indeed, we showed on both ischemic in vivo and BBB in vitro models that VEGF enhances BBB damage and MMP-9 activity and that Ang-1 counteracts both effects. However, we also showed a synergic angiogenic effect of Ang-1 and VEGF in the brain. Taken together, these results allow to propose that, in cerebral ischemia, the combination of Ang-1 and VEGF could be used early to promote the formation of mature neovessels without inducing side effects on BBB permeability.

Matrix Metalloproteinases in Coronary Artery Disease:Clinical and Therapeutic Implications and Pathological Significance

Matrix metalloproteinases (MMP) and their inhibitors (TIMP) are central factors in the control of extracellular matrix turnover. They are important in normal physiology and also during a range of pathological states. Only recently has their role in cardiovascular disease been explored and their analysis through measurements in blood been studied. We have systematically identified clinical articles relevant to coronary artery disease from the last 10 years using MEDLINE. In this review we outline the structure, function and regulation of metalloproteinases and their key roles in angiogenesis, stable and unstable coronary artery disease. Metalloproteinases and their inhibitors are fundamental mediators of change in aging and atherosclerosis, the cell membrane, and in myocardial and vascular tissue. Defining their overall importance and understanding their complex interrelationships with pressure, thrombosis and local neural and hormonal tone will require detailed clinical study. The modulation of MMP and TIMP activity using drugs that affect the expression and function of these proteins will provide us with new ways to treat these serious and disabling diseases, and we explore potential mechanisms and treatments.

Cyclic strain of adult stem cells modulates matrix metalloproteinase activity: Mechanism for promoting cell-based cardiac remodeling

Myocardial infarction leads to loss of cardiomyocytes and slippage due to disorganization of the extracellular matrix (ECM), contributing to the loss of heart pump function. Adult human mesenchymal stem cell (hMSC) transplantation is under investigation for cardiac repair and regeneration. We hypothesize that hMSCs exposed to mechanical strain, undergo alteration of phenotype that can promote both myocyte and ECM restoration.

Collagen degradation products modulate matrix metalloproteinase expression in cultured articular chondrocytes

Destruction of collagen within osteoarthritic cartilage depends in part on collagen-degrading matrix metalloproteases (MMP). Degradative fragments of type II collagen (Col II) occur in normal and in osteoarthritic cartilage, and may contribute to regulation of matrix turnover by interfering with normal cell-matrix communication pathways. Therefore, the effects of different types of collagen fragments on mRNA and protein levels of MMP-2, MMP-3, MMP-9, and MMP-13 in cultured bovine articular knee chondrocytes and explants were examined. Primary chondrocytes and explants were incubated with fragments from whole cartilage collagen matrix (Colf) and from purified type II collagen (Col2f), or with a synthetic 29-mer peptide representing the amino-terminal domain of type II collagen (Ntelo). Gelatin zymography revealed increases of proMMP-2, a shift towards active MMP-2 and increases in proMMP-9, depending on the type of fragment. In situ hybridization of cartilage sections displayed MMP-3 mRNA in virtually all cells. Moderate to strong increases in MMP-2, MMP-3, MMP-9, and MMP-13 mRNA levels were detected by quantitative PCR. The results demonstrate stimulating effects of collagen fragments on both mRNA and/or protein from MMP -2, -3, -9, and -13, and suggest a novel mechanism of MMP induction and activation that includes a particular role for N-telo in controlling catabolic pathways of matrix turnover.

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

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 β-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 β-estradiol (20 ng/ml) or relaxin plus β-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 β-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 β-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 β-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.