Expression and function of matrix metalloproteinase (MMP)-28

Matrix metalloproteinase-28 (MMP-28, epilysin) is highly expressed in the skin by keratinocytes, the developing and regenerating nervous system and a number of other normal human tissues. In epithelial cells, over-expression of MMP-28 mediates irreversible epithelial to mesenchymal transition concomitant with loss of E-cadherin from the cell surface and an increase in active transforming growth factor beta. We recently reported the expression of MMP-28 in both cartilage and synovium where expression is increased in patients with osteoarthritis.In human chondrosarcoma cells MMP-28 was activated by proprotein convertases and the active form of the enzyme preferentially associated with the extracellular matrix in a C-terminal independent manner. over-expression of MMP-28 in chondrosarcoma cells led to altered cell morphology with increased organisation of actin. Adhesion to type II collagen and fibronectin was increased, and migration across the former was decreased. MMP-28 was localised to the cell surface, at least transiently, in a C-terminal dependent manner. Heparin prevented both extracellular matrix association and cell surface binding of MMP-28 suggesting that both are via heparan sulphate proteoglycans. Over-expression of activatable MMP-28, but not catalytically inactive EA mutant increased the expression and activity of MMP-2, and all forms of MMP-28 tested increased expression of MMP19 and TIMP3 mRNA.These data demonstrate that expression of MMP28 alters cell phenotype towards a more adhesive, less migratory behaviour. Further, MMP-28 activity may reside predominantly in the extracellular matrix, and we are currently searching for substrates in this compartment.     

Detection of matrix metalloproteinase (MMP)-2 and MMP-9 in canine seminal plasma

Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes that play a central role in degradation of protein components of the extracellular matrix and basement membrane. Previous studies have shown that MMP-2 and MMP-9 are present in human seminal plasma, but there is little information available on the presence of MMPs in canine seminal plasma. This study aims to investigate the presence of MMPs in canine seminal plasma and their clinical manifestation at the level of various semen parameters in canine species. Latent and active forms of MMP-2 and MMP-9 were evaluated using gelatin zymography and their association with semen parameters was examined. Results demonstrate that both latent and active forms of MMP-2 and MMP-9 are present in canine seminal plasma and the latent forms are predominant. The latent and active MMP-9 activities were elevated in the semen with unsatisfactory quality traits and proMMP-2 was inversely correlated with semen quality whereas, MMP-2 was positively correlated with semen quality traits. These findings suggest that proMMP-9 and MMP-9 activation contributes to the variation in semen, while the activation of MMP-2 improves the sperm functionality.     

Expression of matrix metalloproteinase (MMP)-2, MMP-14 and tissue inhibitor of matrix metalloproteinase (TIMP)-2 during bovine placentation and at term with or without placental retention

Matrix metalloproteinases (MMPs) and counteracting tissue inhibitors of metalloproteinases (TIMPs) are balancing extracellular matrix (ECM) formation and degradation. The latter is believed to be an important aspect for the detachment of fetal membranes postpartum when loosening the feto-maternal connection which is a prerequisite to avoid placental retention a common disease in cows leading to considerable economic loss. Membrane-type (MT) MMPs have been suggested as potential activators controlling ECM remodelling. In particular, MT1-MMP (MMP-14) is able to degrade ECM substrates and activate MMP-2 through binding TIMP-2 at the cell surface. Since the connection between the trophoblast and the maternal caruncular epithelium is supported by integrin receptors bound to ECM, we hypothesize that impaired modulation of the ECM by TIMPs/MMPs participates in the aetiology of bovine retained fetal membranes. To analyse this involvement, placentomes were collected from cows after term parturition and timely release of fetal membranes (n = 4) and cows with retained fetal membranes after various treatments for the induction of parturition using progesterone antagonist (aglepristone), PGF_2α analogue, glucocorticoid, and after elective caesarean sections (each group n = 3). The expression of MMP-14, MMP-2 and of TIMP-2 was examined by real-time-PCR, immunohistochemistry, Western blot and zymography. The relative mRNA expression levels of MMP-14 remained unchanged, while the expression levels of TIMP-2 and MMP-2 partly increased in animals with induced parturition and retention of fetal membranes compared to animals without placental retention. MMP-14 protein was expressed in cells of the uninucleated trophoblast, the fetal mesenchyme and maternal stroma. TIMP-2 was present exclusively in trophoblast giant cells, while MMP-2 could be detected in uninucleated trophoblast cells and the fetal mesenchyme. The presence of the activated enzyme was confirmed by zymography. In conclusion, MMP-14, MMP-2 and TIMP-2 are co-localized in the fetal compartment and therefore could influence the timely release of fetal membranes in cattle.     

Catalytic activities of membrane-type 6 matrix metalloproteinase (MMP25)

This study describes the biochemical characterisation of the catalytic domain of membrane-type 6 matrix metalloproteinase (MT6-MMP, MMP25, leukolysin). Its activity towards synthetic peptide substrates, components of the extracellular matrix and inhibitors of MMPs was studied and compared with MT1-MMP, MT4-MMP and stromelysin-1. We have found that MT6-MMP is closer in function to stromelysin-1 than MT1 and MT4-MMP in terms of substrate and inhibitor specificity, being able to cleave type-IV collagen, gelatin, fibronectin and fibrin. However, it differs from stromelysin-1 and MT1-MMP in its inability to cleave laminin-I, and unlike stromelysin-1 cannot activate progelatinase B. Our findings suggest that MT6-MMP could play a role in cellular migration and invasion of the extracellular matrix and basement membranes and its activity may be tightly regulated by all members of the TIMP family.     

Synthesis and matrix metalloproteinase (MMP)-12 inhibitory activity of ageladine A and its analogs

Ageladine A (1) and its analogs 2-10 were expeditiously synthesized by featuring the biosynthetic route proposed for 1 (for 1-10) and by employing 2-(N-t-butoxycarbonylamino)imidazol-4-carbaldehyde as the starting material (for 1-8). From MMP-12 inhibitory activity assay, it appeared evident that the two bromine atoms and the three NH groups (1-NH, 14-NH, and 15-NH2) were indispensable for 1 to exhibit excellent activity.     

Catalytic domain of MMP20 (Enamelysin) - the NMR structure of a new matrix metalloproteinase

The solution structure of the catalytic domain of MMP-20, a member of the matrix metalloproteinases family not yet structurally characterized, complexed with N-Isobutyl-N-(4-methoxyphenylsulfonyl)glycyl hydroxamic acid (NNGH), is here reported and compared with other MMPs-NNGH adducts. The backbone dynamic has been characterized as well. We have found that, despite the same fold and very high overall similarity, the present structure experiences specific structural and dynamical similarities with some MMPs and differences with others, around the catalytic cavity. The present solution structure, not only contributes to fill the gap of structural knowledge on human MMPs, but also provides further information to design more selective and efficient inhibitors for a specific member of this class of proteins.     

Expression and characterization of recombinant osteopontin peptides representing matrix metalloproteinase proteolytic fragments.

Osteopontin (OPN) is a secreted, arginine-glycine-aspartic acid (RGD)-containing phosphoprotein proteolytically modified by members of the matrix metalloproteinase (MMP) family. We previously defined the MMP-3 and MMP-7 cleavage sites in OPN and found increased adhesive and migratory activity of a pool of MMP-cleaved fragments compared to full-length OPN. In the present study, we performed mutational analysis of recombinant full-length OPN and generated recombinant OPN fragments corresponding to the MMP-cleaved fragments, which have apparent molecular weights of 40, 32, and 25 kD by SDS-PAGE. Single residue mutations in 167L and 211L do not abrogate MMP cleavage although processing of the putative C-terminal fragment appears to be affected by a 167L to 167A mutation. The N-terminal 40-kD fragment was a stronger adhesive substrate compared to full-length OPN despite the observation that full-length OPN displayed greater binding in soluble phase to endothelial cell surfaces. While the 32-kD fragment showed significant binding to endothelial cell surfaces, the C-terminal 25-kD fragment did not interact with cell surface. Our data indicate that the increased adhesive activity of MMP-cleaved OPN was accountable by the N-terminal 40-kD fragment. We further analyzed receptor binding, using competition with peptides representing the alpha4beta1 and alpha9beta1 binding sites in the 40-kD N-terminal fragment. Using Jurkat cells, we found that a peptide corresponding to 131ELVTDFPTDLPATE144 had no effect on cell adhesion, whereas the peptide SVVYGLR competitively inhibited cell adhesion. These results suggest that a shorter motif that is found in MMP-cleaved OPN, 162SVVYG166, is sufficient to mediate cell adhesion of Jurkat cells to receptors, including the beta1 integrins, which have been previously characterized to bind the SVVYGLR sequence.     

Progesterone inhibits activation of latent matrix metalloproteinase (MMP)-2 by membrane-type 1 MMP: enzymes coordinately expressed in human endometrium

Matrix metalloproteinases (MMP) have specific spatial and temporal expression patterns in human endometrium and are critical for menstruation. Expression and activation mechanisms for proMMP-2 differ from other MMPs; in many cells proMMP-2 is specifically activated by membrane-type (MT)-MMPs. We examined the expression and localization of proMMP-2, MT1-MMP, and MT2-MMP in human endometrium across the menstrual cycle; and we examined the expression of MT1-MMP and activation of proMMP-2 in cultured endometrial stromal cells and their regulation by progesterone. MMP-2 was immunolocalized in 25 of 32 endometrial samples in all cellular compartments but with greatest intensity in degrading menstrual tissue. MT1-MMP mRNA was present throughout the cycle, and immunoreactive protein was detected in 24 of 32 samples, with the strongest staining in subsets of macrophages, neutrophils, and granular lymphocytes (but not mast cells or eosinophils) during the menstrual, mid-proliferative and mid-secretory phases. Patchy epithelial staining and staining of decidual cells, often periglandular in menstrual tissue, were also seen. MT2-MMP was more widespread than MT1-MMP without apparent cyclical variation and with maximal intensity in glandular epithelium. Cultured endometrial stromal cells released proMMP-2, and progesterone treatment significantly reduced the percentage level of its active (62 kDa) form (22.5 +/- 1.8% vs. 3.0 +/- 1.3%, without and with treatment, respectively, mean +/- SEM, P < 0.0001). This activation was blocked by a specific MMP inhibitor and restored following inhibitor removal. Progesterone also attenuated cell expression of MT1-MMP mRNA. We postulate that MT1-MMP activates proMMP-2 in endometrium, this activity being increased at the end of the cycle when progesterone levels fall, thus contributing to menstruation.     

Toll-like receptor (TLR) and matrix metalloproteinase (MMP) polymorphisms and periodontitis susceptibility: a meta-analysis

The aim of this study was to determine whether the toll-like receptor (TLR) and matrix metalloproteinase (MMP) polymorphisms confers susceptibility to periodontitis in ethnically different populations. A literature search using PubMed and Embase provided the data to conduct a meta-analysis on the associations between the TLR2 Arg753Gln, TLR4 Asp299Gly, Thr399Ile, MMP-1 ?1607 G1/G2 and MMP-9 ?1562 C/T polymorphisms and periodontitis. A total of 32 studies (14 on TLR polymorphisms and 18 on MMP polymorphisms) were considered in the meta-analysis. The meta-analysis showed no association between periodontitis and the TLR2 753Arg allele (Odds ratio [OR] = 0.962, 95 % confidence interval [CI] = 0.662–1.400, p = 0.841). Meta-analysis of the TLR4 Asp299Gly and Thr399Ile polymorphisms showed no association between periodontitis and the TLR4 299Asp allele in all study subjects (OR = 0.984, 95 % CI = 0.761–1.271, p = 0.900; OR = 1.030, 95 % CI = 0.748–1.418, p = 0.854). Meta-analysis showed an association between the MMP-1 ?1607 G2G2 genotype and periodontitis in Asians (OR = 3.778, 95 % CI = 1.210–11.80, p = 0.022). Meta-analysis containing only studies in Hardy–Weinberg equilibrium revealed no association between chronic periodontitis and the MMP-9 ?1562TT genotype (OR = 0.638, 95 % CI = 0.265–1.533, p = 0.315). This meta-analysis demonstrates a lack of association between the TLR2 Arg753Gln, TLR4 Asp299Gly, Thr399Ile, MMP-9 ?1562 C/T polymorphisms and periodontitis, but shows an association between the MMP-1 ?1607 G2G2 genotype and periodontitis in Asians.     

Expression and localization of immunoreactive-sialomucin complex (Muc4) in salivary glands

Sialomucin Complex (SMC; Muc4) is a heterodimeric glycoprotein consisting of two subunits, the mucin component ASGP-1 and the transmembrane subunit ASGP-2. Northern blot and immunoblot analyses demonstrated the presence of SMC/Muc4 in submaxillary, sublingual and parotid salivary glands of the rat. Immunocytochemical staining of SMC using monoclonal antisera raised against ASGP-2 and glycosylated ASGP-1 on paraffin-embedded sections of parotid, submaxillary and sublingual tissues was performed to examine the localization of the mucin in the major rat salivary glands. Histological and immunocytochemical staining of cell markers showed that the salivary glands consisted of varying numbers of serous and mucous acini which are drained by ducts. Parotid glands were composed almost entirely of serous acini, sublingual glands were mainly mucous in composition and a mixture of serous and mucous acini were present in submaxillary glands. Since immunoreactive (ir)-SMC was specifically localized to the serous cells, staining was most abundant in parotid glands, intermediate levels in submaxillary glands and least in sublingual glands. Ir-SMC in sublingual glands was localized to caps of cells around mucous acini, known as serous demilunes, which are also present in submaxillary glands. Immunocytochemical staining of SMC in human parotid glands was localized to epithelial cells of serous acini and ducts. However, the staining pattern of epithelial cells was heterogeneous, with ir-SMC present in some acinar and ductal epithelial cells but not in others. This report provides a map of normal ir-SMC/Muc4 distribution in parotid, submaxillary and sublingual glands which can be used for the study of SMC/Muc4 expression in salivary gland tumors.     

Endogenous nitric oxide formation correlates negatively with circulating matrix metalloproteinase (MMP)-2 and MMP-9 levels in black subjects

Deficient formation of endogenous nitric oxide (NO) contributes to cardiovascular diseases, and this may be associated with increased circulating levels of matrix metalloproteinase-9 (MMP-9), as previously shown in white subjects. Because interethnic differences exist with respect to risk factors, prevalence, and severity of cardiovascular diseases, we designed this study to examine whether the circulating levels of nitrites (a marker of endogenous NO formation) are associated with the plasma levels of MMP-9 and MMP-2 in healthy black subjects. We studied 198 healthy subjects self-reported as blacks not taking any medications. Venous blood samples were collected and plasma and whole blood nitrite levels were measured using an ozone-based chemiluminescence assay. Plasma MMP-2 and MMP-9 levels were determined by gelatin zymography. We found a positive correlation between plasma MMP-9 and MMP-2 levels (P < 0.0001, rs = 0.556). Interestingly, we found a negative relationship between the plasma MMP-9 levels and the plasma or whole blood nitrites levels (P = 0.04, rs = -0.149; and P < 0.0001, rs = -0.349, respectively). In parallel, we found similar negative relationships between plasma MMP-2 levels and plasma or whole blood nitrites levels (P = 0.02, rs = -0.172; and P < 0.0001, rs = -0.454, respectively). This is the first study to show that endogenous nitric oxide formation correlates negatively with the circulating levels of both MMP-2 and MMP-9 in black subjects. Our findings suggest a mechanistic link between deficient NO formation and increased MMPs levels, which may promote cardiovascular diseases.     

Differential inhibition of membrane type 3 (MT3)-matrix metalloproteinase (MMP) and MT1-MMP by tissue inhibitor of metalloproteinase (TIMP)-2 and TIMP-3 rgulates pro-MMP-2 activation

The membrane type (MT)-matrix metalloproteinases (MMPs) constitute a subgroup of membrane-anchored MMPs that are major mediators of pericellular proteolysis and physiological activators of pro-MMP-2. The MT-MMPs also exhibit differential inhibition by members of the tissue inhibitor of metalloproteinase (TIMP) family. Here we investigated the processing, catalytic activity, and TIMP inhibition of MT3-MMP (MMP-16). Inhibitor profile and mutant enzyme studies indicated that MT3-MMP is regulated on the cell surface by autocatalytic processing and ectodomain shedding. Inhibition kinetic studies showed that TIMP-3 is a high affinity inhibitor of MT3-MMP when compared with MT1-MMP (K(i) = 0.008 nm for MT3-MMP versus K(i) = 0.16 nm for MT1-MMP). In contrast, TIMP-2 is a better inhibitor of MT1-MMP. MT3-MMP requires TIMP-2 to accomplish full pro-MMP-2 activation and this process is enhanced in marimastatpretreated cells, consistent with regulation of active enzyme turnover by synthetic MMP inhibitors. TIMP-3 also enhances the activation of pro-MMP-2 by MT3-MMP but not by MT1-MMP. TIMP-4, in contrast, cannot support pro-MMP-2 activation with either enzyme. Affinity chromatography experiments demonstrated that pro-MMP-2 can assemble trimolecular complexes with a catalytic domain of MT3-MMP and TIMP-2 or TIMP-3 suggesting that pro-MMP-2 activation by MT3-MMP involves ternary complex formation on the cell surface. These results demonstrate that TIMP-3 is a major regulator of MT3-MMP activity and further underscores the unique interactions of TIMPs with MT-MMPs in the control of pericellular proteolysis.     

Expression in Escherichia coli, refolding, and purification of the recombinant mature form of human matrix metalloproteinase 7 (MMP-7)

In the latent pro-form of matrix metalloproteinase 7 (MMP-7), the cysteine residue in the pro-peptide binds the active-site zinc ion. Hence, recombinant active MMP-7 was prepared from pro-MMP-7 by modification of this cysteine residue with a mercuric reagent. In this study, mature MMP-7 was expressed in Escherichia coli as inclusion bodies, solubilized, and refolded with 1 M L-arginine. The purified product was indistinguishable from the one prepared from pro-MMP-7 as assessed by hydrolysis of (7-methoxycoumarin-4-yl)acetyl-L-Pro-L-Leu-Gly-L-Leu-[N(3)-(2,4-dinitrophenyl)-L-2,3-diaminopropionyl]-L-Ala-L-Arg-NH(2).