The role of matrix metalloproteinase-2 and matrix metalloproteinase-9 in antibody-induced arthritis

Matrix metalloproteinases (MMPs) are a large group of enzymes responsible for matrix degradation. Among them, the family of gelatinases (MMP-2/gelatinase A and MMP-9/gelatinase B) is overproduced in the joints of patients with rheumatoid arthritis. Because of their degradative effects on the extracellular matrix, gelatinases have been believed to play an important role in progression and cartilage degradation in this disease, although their precise roles are yet to be defined. To clarify these roles, we investigated the development of Ab-induced arthritis, one of the murine models of rheumatoid arthritis, in MMP-2 or MMP-9 knockout (KO) mice. Surprisingly, the MMP-2 KO mice exhibited severe clinical and histologic arthritis than wild-type mice. The MMP-9 KO mice displayed milder arthritis. Recovery from exacerbated arthritis in the MMP-2 KO mice was possible by injection of wild-type fibroblasts. These results indicated a suppressive role of MMP-2 and a pivotal role of MMP-9 in the development of inflammatory joint disease.     

Immunocharacterization of matrix metalloproteinase-2 and matrix metalloproteinase-9 in canine transmissible venereal tumors

Matrix metalloproteases (MMPs) are endogenous proteases that are responsible for degradation of extracellular matrix (ECM) proteins and cell surface antigens. The breakdown of ECM participates in the local invasion and distant metastases of malignant tumors. Canine transmissible venereal tumor (CTVT) is a naturally occurring contagious round cell neoplasm of dogs that affects mainly the external genitalia of both sexes. CTVT generally is a locally invasive tumor, but distant metastases also are common in puppies and immunocompromised dogs. We investigated the immune expressions and activities of MMP-2 and MMP-9 in CTVT. The presence of these enzymes in tumor cells and tissue homogenates was demonstrated by immunohistochemistry and western blotting. We used gelatin substrate zymography to evaluate the activities of MMP-2 and MMP-9 enzymes in tumor homogenates. We found that tumor cells expressed both MMP-2 and MMP-9. Electrophoretic bands corresponding to MMP-9 and MMP-2 were identified in immunoblots and clear bands that corresponded to the active forms of MMP-2 and MMP-9 also were detected in gelatin zymograms. Our study is the first detailed documentation of MMPs in CTVT.     

Selective spatiotemporal induction of matrix metalloproteinase-2 and matrix metalloproteinase-9 transcription after myocardial infarction

Myocardial remodeling after myocardial infarction (MI) is associated with increased levels of the matrix metalloproteinases (MMPs). Levels of two MMP species, MMP-2 and MMP-9, are increased after MI, and transgenic deletion of these MMPs attenuates post-MI left ventricular (LV) remodeling. This study characterized the spatiotemporal patterns of gene promoter induction for MMP-2 and MMP-9 after MI. MI was induced in transgenic mice in which the MMP-2 or MMP-9 promoter sequence was fused to the beta-galactosidase reporter, and reporter level was assayed up to 28 days after MI. Myocardial localization with respect to cellular sources of MMP-2 and MMP-9 promoter induction was examined. After MI, LV diameter increased by 70% (P < 0.05), consistent with LV remodeling. beta-Galactosidase staining in MMP-2 reporter mice was increased by 1 day after MI and increased further to 64 +/- 6% of LV epicardial area by 7 days after MI (P < 0.05). MMP-2 promoter activation occurred in fibroblasts and myofibroblasts in the MI region. In MMP-9 reporter mice, promoter induction was detected after 3 days and peaked at 7 days after MI (53 +/- 6%, P < 0.05) and was colocalized with inflammatory cells at the peri-infarct region. Although MMP-2 promoter activation was similarly distributed in the MI and border regions, activation of the MMP-9 promoter was highest at the border between the MI and remote regions. These unique findings visually demonstrated that activation of the MMP-2 and MMP-9 gene promoters occurs in a distinct spatial relation with reference to the MI region and changes in a characteristic time-dependent manner after MI.     

Substrate hydrolysis by matrix metalloproteinase-9

The catalytic clefts of all matrix metalloproteinases (MMPs) have a similar architecture, raising questions about the redundancy in substrate recognition across the protein family. In the present study, an unbiased phage display strategy was applied to define the substrate recognition profile of MMP-9. Three groups of substrates were identified, each occupying a distinct set of subsites within the catalytic pocket. The most prevalent motif contains the sequence Pro-X-X-Hy-(Ser/Thr) at P(3) through P(2'). This sequence is similar to the MMP cleavage sites within the collagens and is homologous to substrates the have been selected for other MMPs. Despite this similarity, most of the substrates identified here are selective for MMP-9 over MMP-7 and MMP-13. This observation indicates that substrate selectivity is conferred by key subsite interactions at positions other than P(3) and P(1'). This study shows that MMP-9 has a unique preference for Arg at both P(2) and P(1), and a preference for Ser/Thr at P(2'). Substrates containing the consensus MMP-9 recognition motif were used to query the protein data bases. A surprisingly limited list of putative physiologic substrates was identified. The functional implications of these proteins lead to testable hypotheses regarding physiologic substrates for MMP-9.     

A residue in the S2 subsite controls substrate selectivity of matrix metalloproteinase-2 and matrix metalloproteinase-9

Matrix metalloproteinase (MMP)-2 and MMP-9 are closely related metalloproteinases that are implicated in angiogenesis. The two proteins have a similar domain structure and highly homologous catalytic domains, making them an excellent comparative model for understanding the structural basis of substrate recognition by the MMP family. Although the two MMPs exhibit some overlap in substrate recognition, our recent work showed that MMP-2 can cleave a set of peptide substrates that are only poorly recognized by MMP-9 (Chen, E. I., Kridel, S. J., Howard, E. W., Li, W., Godzik, A., and Smith, J. W. (2002) J. Biol. Chem. 277, 4485-4491). Mutations at the P(2) position of these peptide substrates dramatically reduced their selectivity for MMP-2. Inspection of the corresponding S(2) pocket of the substrate-binding cleft of the protease reveals that MMP-9 contains an Asp, whereas MMP-2 contains Glu. Here, we test the hypothesis that this conservative substitution has a role in substrate selectivity. Mutation of Glu(412) in MMP-2 to Asp significantly reduced the hydrolysis of selective substrates, with only a minor effect on hydrolysis of non-selective substrates. The predominant effect of the mutation is at the level of k(cat), or turnover rate, with reductions reaching as high as 37-fold. The residues that occupy this position in other MMPs are highly variable, providing a potential structural basis for substrate recognition across the MMP family.     

Role of matrix metalloproteinase-3 in neurodegeneration

Matrix metalloproteinase-3 (MMP-3) is a member of the class of zinc-dependent proteases known to degrade the extracellular matrix. MMP-3 activity is regulated at three different levels: gene expression, proteolytic activation of the zymogen, and inhibition by the endogenous tissue inhibitors of metalloproteinase. A line of evidence indicates a role of MMP-3 in neurodegeneration. In neuronal cells, MMP-3 expression is increased in response to cell stress, and the cleaved, active MMP-3 participates in apoptotic signaling. In the extracellular space, MMP-3 triggers microglia to produce proinflammatory and cytotoxic molecules as well as MMP-3, which in turn contribute to neuronal damage. MMP-3 is increased in various experimental models of Parkinson's disease that are produced by selective toxins and by inflammagen, and the neuronal death is attenuated by various ways that inhibit MMP-3. α-Synuclein, whose gene mutations are associated with familial forms of Parkinson's disease, is proteolyzed by MMP-3. Contribution of MMP-3 toward the pathogenesis of Alzheimer's disease and other neurodegenerative diseases has also been suggested. Thus, modulation of MMP-3 expression and/or activity could be of therapeutic value for neurodegenerative diseases.     

Inhibition of matrix metalloproteinase-2 by PARP inhibitors

Matrix metalloproteinase-2 (MMP-2), a ubiquitously expressed zinc-dependent endopeptidase, and poly(ADP-ribosyl) polymerase (PARP), a nuclear enzyme regulating DNA repair, are activated by nitroxidative stress associated with various pathologies. As MMP-2 plays a detrimental role in heart injuries resulting from enhanced nitroxidative stress, where PARP and MMP inhibitors are beneficial, we hypothesized that PARP inhibitors may affect MMP-2 activity. Using substrate degradation assays to determine MMP-2 activity we found that four PARP inhibitors (3-AB, PJ-34, 5-AIQ, and EB-47) inhibited 64 kDa MMP-2 in a concentration-dependent manner. The IC₅₀ values of PJ-34 and 5-AIQ were in the high micromolar range and comparable to those of known MMP-2 inhibitors doxycycline, minocycline or o-phenanthroline, whereas those for 3-AB and EB-47 were in the millimolar range. Co-incubation of PARP inhibitors with doxycycline showed an additive inhibition of MMP-2 that was significant for 3-AB alone. These data demonstrate that the protective effects of some PARP inhibitors may include inhibition of MMP-2 activity.     

Thyroid hormone induces myocardial matrix degradation by activating matrix metalloproteinase-1.

Hyperthyroid patients develop left ventricular hypertrophy associated with alterations of several cardiac parameters such as heart rate, cardiac output, cardiac contraction and hemodynamic overload leading to cardiac complications. Although cardiac hypertrophy and contractile abnormality occur, interstitial fibrosis in the heart usually does not take place in hyperthyroid condition. Therefore, in the present study, the mechanism regulating myocardial extracellular matrix (ECM) remodeling in hyperthyroid condition was investigated. Cardiac hypertrophy was developed in Sprague-Dawley rats by administration of 3,5,3'-triiodo-L-thyronine (triiodothyronine, 8 microg/100g BW, ip, SID) and glucocorticosteroid, dexamethasone (DEX, 35 microg/100g BW, po, SID), which is also an inducer of hypertrophy for 15 days. Heart/Body weight ratio and atrial and brain natriuretic peptide mRNAs were significantly increased in both triiodothyronine- and DEX-treated rats compared to control. Collagens-I and -III deposition in the left ventricular sections was reduced in triiodothyronine-treated rats, whereas in DEX-treated animals those were increased compared to control. While mRNA and protein levels of procollagens-I and -III were increased with triiodothyronine (p<0.01), the levels of mature collagens-I and -III were decreased. The levels of the mature collagens were increased with DEX compared to control. MMP-1 activity in the serum and left ventricle was higher with reduced levels of TIMPs-3 and -4 in the left ventricle of triiodothyronine-treated rats. The results suggest that accelerated breakdown of collagens-I and -III by MMP-1 due to suppression of the endogenous TIMPs plays an important role in regulating the ECM in myocardium of hyperthyroid rat.     

Osteopontin, a novel substrate for matrix metalloproteinase-3 (stromelysin-1) and matrix metalloproteinase-7 (matrilysin)

Osteopontin (OPN) is a secreted phosphoprotein shown to function in wound healing, inflammation, and tumor progression. Expression of OPN is often co-localized with members of the matrix metalloproteinase (MMP) family. We report that OPN is a novel substrate for two MMPs, MMP-3 (stromelysin-1) and MMP-7 (matrilysin). Three cleavage sites were identified for MMP-3 in human OPN, and two of those sites were also cleaved by MMP-7. These include hydrolysis of the human Gly166-Leu167, Ala201-Tyr202 (MMP-3 only), and Asp210-Leu211 peptide bonds. Only the N-terminal Gly-Leu cleavage site is conserved in rat OPN (Gly151-Leu152). These sites are distinct from previously reported cleavage sites in OPN for the proteases thrombin or enterokinase. We found evidence for the predicted MMP cleavage fragments of OPN in vitro in tumor cell lines, and in vivo in remodeling tissues such as the postpartum uterus, where OPN and MMPs are co-expressed. Furthermore, cleavage of OPN by MMP-3 or MMP-7 potentiated the function of OPN as an adhesive and migratory stimulus in vitro through cell surface integrins. We predict that interaction of MMPs with OPN at tumor and wound healing sites in vivo may be a mechanism of regulation of OPN bioactivity.     

Mechanisms of cytosolic targeting of matrix metalloproteinase-2

Matrix metalloproteinase-2 (MMP-2) is best understood for its biological actions outside the cell. However, MMP-2 also localizes to intracellular compartments and the cytosol where it has several substrates, including troponin I (TnI). Despite a growing list of cytosolic substrates, we currently do not know the mechanism(s) that give rise to the equilibrium between intracellular and secreted MMP-2 moieties. Therefore, we explored how cells achieve the unique distribution of this protease. Our data show that endogenous MMP-2 targets inefficiently to the endoplasmic reticulum (ER) and shows significant amounts in the cytosol. Transfection of canonical MMP-2 essentially reproduces this targeting pattern, suggesting it is the quality of the MMP-2 signal sequence that predominantly determines MMP-2 targeting. However, we also found that human cardiomyocytes express an MMP-2 splice variant which entirely lacks the signal sequence. Like the fraction of ER-excluded, full-length MMP-2, this variant MMP-2 is restricted to the cytosol and specifically enhances TnI cleavage upon hypoxia-reoxygenation injury in cardiomyocytes. Together, our findings describe for the first time a set of mechanisms that cells utilize to equilibrate MMP-2 both in the extracellular milieu and intracellular, cytosolic locations. Our results also suggest approaches to specifically investigate the overlooked intracellular biology of MMP-2.     

Type I collagen stabilization of matrix metalloproteinase-2

The activity of matrix metalloproteinase-2 (MMP-2) is regulated stringently on the posttranslational level. MMP-2 efficiently undergoes autolysis into inactive polypeptides in vitro, prompting the hypothesis that MMP-2 autolysis may function as an alternative mechanism for posttranslational control of MMP-2 in vivo. Moreover, MMP-2 binds to intact type I collagen fibrils; however, the functional consequences of this interaction have not been fully elucidated. To test the hypothesis that MMP-2 binding to type I collagen functions as a positive regulator of MMP-2 proteolytic potential, the effect of type I collagen on MMP-2 activity, inhibition by tissue inhibitor of metalloproteinase-2 (TIMP-2), and enzyme stability was examined. Here, we report that purified MMP-2 binds but does not cleave intact type I collagen. The presence of type I collagen affects neither enzymatic activity against a quenched fluorescent peptide substrate nor the kinetics of inhibition by TIMP-2. However, MMP-2 is stabilized from autolysis in the presence of type I collagen, but not by elastin, fibrinogen, or laminin. These data provide biochemical evidence that MMP-2 exosite interactions with type I collagen may function in the posttranslational control of MMP-2 activity by reducing the rate of autolytic inactivation.     

Genetic variants in matrix metalloproteinase-9 gene modify metalloproteinase-9 levels in black subjects

Altered matrix metalloproteinases (MMPs) levels are involved in cardiovascular diseases and increased MMP-9 levels enhance the cardiovascular risk in apparently healthy subjects. We investigated the effects of MMP-9 gene polymorphisms and haplotypes on the circulating MMP-9 levels in healthy black subjects and the effects of an MMP-2 polymorphism on the plasma MMP-2 concentrations. We studied 190 healthy subjects, nonsmokers, self-reported as blacks (18-63 years). Genotypes for the MMP-2 C(-1306)T polymorphism and the MMP-9 C(-1562)T, 90(CA)(14-24) and Q279R polymorphisms (rs243865, rs3918242, rs2234681, and rs17576, respectively) were determined by TaqMan(?) Allele Discrimination assay and real-time polymerase chain reaction or restriction fragment length polymorphism. Alleles for the 90(CA)(14-24) polymorphism were grouped as low (L) when there were <21 and high (H) when there were ≥21 CA repeats. The plasma levels of MMP-2 and MMP-9 were determined by gelatin zymography. The software PHASE 2.1 was used to estimate the haplotypes frequencies. Although we found no effects of the MMP-9 C(-1562)T or the Q279R polymorphisms on MMP-9 levels, higher MMP-9 levels were associated with the HH genotype for the -90(CA)(14-24) polymorphism compared with the HL or LL genotypes. Lower MMP-9 levels were found in carriers of the CRL haplotype (combining the C, R, and L alleles for the MMP-9 polymorphisms) compared with the CRH haplotype. Consistent with this finding, the CRL haplotype was more commonly found in subjects with low MMP-9 levels. The MMP-2 C(-1306)T polymorphism had no effects on the plasma MMP-2 levels. Our results show that MMP-9 genetic variations modify MMP-9 levels in black subjects and may offer biochemical evidence implicating MMP-9 in the pathogenesis of cardiovascular diseases in blacks.     

Dynamic alterations of connexin43, matrix metalloproteinase-2 and tissue inhibitor of matrix metalloproteinase-2 during ventricular fibrillation in canine

The aim of this study is to investigate the dynamic alterations of cardiac connexin 43 (Cx43), matrix metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-2 (TIMP-2) in the setting of different ventricular fibrillation (VF) duration. In this study, thirty-two dogs were randomly divided into sham control group, 8-min VF group, 12-min VF group, and 30-min VF group. Cx43 and phosphorylated Cx43 (p-Cx43) in tissues were detected by western blot and immunofluorescence analysis. MMP-2 and TIMP-2 were detected by western blot and immunohistochemistry analysis. The results showed that Cx43 levels in three VF groups were significantly decreased compared with sham control group. p-Cx43 levels in 12-min and 30-min VF groups were significantly reduced compared with sham control group. The ratio of p-Cx43/Cx43 was also decreased in VF groups. Compared with sham controls, no significant difference was observed between the sham control group and 8-min VF group in MMP-2 level, but MMP-2 level increased in 12-min and 30-min VF groups. The ratios of MMP-2/TIMP-2 were higher in VF groups, and were correlated with the duration of VF. A remarkable correlation was observed between the ratio of p-Cx43/Cx43 and MMP-2/TIMP-2 (r = ?0.93, P < 0.01). In conclusion, the alteration of Cx43 and/or p-Cx43 levels and the imbalance of MMP-2 and TIMP-2 may contribute to the initiation and/or persistence of VF. Maneuvers managed to modulate Cx43 level or normalize the balance of MMP-2/TIMP-2 are promising to ameliorate prognosis of VF.     

Thrombin regulates matrix metalloproteinase-9 expression in human monocytes

We investigated whether thrombin, the final activator of coagulation cascade, regulates expression of matrix metalloproteinases (MMP)-9 in human monocytes.We show that thrombin stimulation induced MMP-9 secretion of monocytes dose- and time-dependently as revealed by gelatin zymography. Real-time RT-PCR and Western blot analysis demonstrated that thrombin up-regulated mRNA and protein levels of MMP-9. Pre-incubation with anti-protease-activated receptor (PAR)-1 or anti-PAR-3 antibody partially inhibited the thrombin-induced MMP-9 secretion. Simultaneous incubation with both showed synergistic effect, indicating the involvement of both receptors in this thrombin effect. BAPTA, a Ca²⁺ chelator, abolished the thrombin-induced MMP-9 secretion, indicating the requirement of Ca²⁺ mobilization in this process. Inhibition of thrombin-induced MMP-9 secretion by either MEK inhibitor or p38 kinase inhibitor revealed that the thrombin effect was mediated by both ERK1/2 and p38 pathways. The activation of NFκB by thrombin as demonstrated by electromobility shift assay was also shown to be critical to the thrombin-induced MMP-9 up-regulation.     

Elevated matrix metalloproteinase-9 in patients with systemic sclerosis

Matrix metalloproteinase-9 (MMP-9) has been implicated in the pathogenesis of cancer, autoimmune disease, and various pathologic conditions characterized by excessive fibrosis. In this study, we investigated the expression of MMP-9 and its clinical significance in systemic sclerosis (SSc). The patients (n = 42) with SSc had higher concentrations of MMP-9 and of tissue inhibitor of metalloproteinase-1 (TIMP-1) and a higher ratio of MMP-9 to TIMP-1 in sera than healthy controls (n = 32). Serum MMP-9 concentrations were significantly higher in the diffuse type (n = 23) than the limited type of SSc (n = 19). Serum concentrations of MMP-9 correlated well with the degree of skin involvement, as determined by the Rodnan score and with serum concentrations of transforming growth factor β. Moreover, dermal fibroblasts from patients with SSc produced more MMP-9 than those from healthy controls when they were stimulated with IL-1β, tumor necrosis factor α, or transforming growth factor β. Such an increase in MMP-9 production was partially blocked by treatment with cyclosporin A. In summary, the serum MMP-9 concentrations were elevated in SSc patients and correlated well with skin scores. The increased MMP-9 concentrations may be attributable to overproduction by dermal fibroblasts in SSc. These findings suggest that the enhanced production of MMP-9 may contribute to fibrogenic remodeling during the progression of skin sclerosis in SSc.     

Lack of matrix metalloproteinase-9 worsens ventilator-induced lung injury

Matrix metalloproteinase-9 (MMP-9) is released by neutrophils at the sites of acute inflammation. This enzyme modulates matrix turnover and inflammatory response, and its activity has been found to be increased after ventilator-induced lung injury. To clarify the role of MMP-9, mice lacking this enzyme and their wild-type counterparts were ventilated for 2 h with high- or low-peak inspiratory pressures (25 and 15 cmH2O, respectively). Lung injury was evaluated by gas exchange, respiratory mechanics, wet-to-dry weight ratio, and histological analysis. The activity of MMP-9 and levels of IL-1beta, IL-4, and macrophage inflammatory protein (MIP-2) were measured in lung tissue and bronchoalveolar lavage fluid (BALF). Cell count and myeloperoxidase activity were measured in BALF. There were no differences between wild-type and Mmp9-/- animals after low-pressure ventilation. After high-pressure ventilation, wild-type mice exhibited an increase in MMP-9 in tissue and BALF. Mice lacking MMP-9 developed more severe lung injury than wild-type mice, in terms of impaired oxygenation and lung mechanics, and higher damage in the histological study. These effects correlated with an increase in both cell count and myeloperoxidase activity in the BALF, suggesting an increased neutrophilic influx in response to ventilation. An increase in IL-1beta and IL-4 in the BALF only in knockout mice could be responsible for the differences. There were no differences between genotypes in MMP-2, MMP-8, or tissue inhibitors of metalloproteinases. These results show that MMP-9 protects against ventilator-induced lung injury by decreasing alveolar neutrophilic infiltration, probably by modulation of the cytokine response in the air spaces.