Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.24.17 (MMP-3)
 3,419 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The mammary gland, during post-lactational involution, is subjected to extensive tissue reconstruction. This process is governed by the concerted expression of extracellular-matrix-degrading enzymes and their inhibitors. During carcinogenesis, the invasive growth of tumor cells is characterized by the penetration of the basement membrane and stromal invasion. We compared the expression of the tissue-remodeling enzymes stromelysin-1, a matrix metalloproteinase, and its inhibitor, tissue inhibitor of metalloproteinase-1 (TIMP-1), during mammary gland involution and carcinogenesis in mouse. In involuting mammary glands, stromelysin-1 was expressed in myoepithelial cells, whereas TIMP-1 was confined to the stromal tissue. To analyze the involvement of these tissue-remodeling genes in tumor development, we examined mammary tumors of transgenic mice expressing either the activated Ha-ras or c-myc oncogene under the control of a milk-protein gene promoter. In the undifferentiated and metastasizing Ha-ras-induced tumors, stromelysin-1 expression was comparable to that seen in involution, whereas TIMP-1 expression was greatly elevated. During Ha-ras-induced carcinogenesis, stromelysin-1 expression was first detected in the myo-epithelial cells surrounding preneoplastic lesions. In contrast, in the well-differentiated and non-metastatic mammary tumors induced by c-myc, no expression of either gene was observed. Thus, expression of stromelysin-1 and TIMP-1 is confined to the aggressively growing tumors and is induced in the earliest stages of carcinogenesis.
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PMID:Expression of stromelysin-1 and TIMP-1 in the involuting mammary gland and in early invasive tumors of the mouse. 796 Feb 27

Stromelysin, a member of the matrix metalloproteinase family of enzymes, has been implicated in the pathogenesis of tumor metastasis and inflammatory diseases such as rheumatoid arthritis. To screen prospective inhibitors of this protease, we developed a fluorogenic substrate with excitation and emission spectra compatible with commercially available 96-well plate readers. The substrate is based on the addition of 6-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino] hexanoic acid (NBD) (EX467/EM534) and 7-dimethylaminocoumarin-4-acetate (DMC) (EX368/EM459) to the previously reported peptide substrate for stromelysin, Arg-Pro-Lys-Pro-Leu-Ala-Nva-Trp-NH2. The new substrate, NBD-Arg-Pro-Lys-Pro-Leu-Ala-Nva-Trp-Lys-(DMC)-NH2 is 95% quenched and the fluorescent product, Nva-Trp-Lys(DMC)-NH2 is easily detected (EX350/EM465). In competition assays the new fluorogenic substrate has a relative kcat/Km that is one half that of the parent peptide. The fluorophores NBD and DMC were chosen based on the high fluorescence yield of DMC and the overlap of the emission spectrum of DMC and excitation spectrum of NBD which results in an efficient energy transfer system in the intact substrate. These characteristics make this an excellent substrate for routine determination of in vitro activities of stromelysin inhibitors.
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PMID:A high throughput fluorogenic substrate for stromelysin (MMP-3). 797 5

Dysregulated extracellular matrix (ECM) metabolism may contribute to vascular remodeling during the development and complication of human atherosclerotic lesions. We investigated the expression of matrix metalloproteinases (MMPs), a family of enzymes that degrade ECM components in human atherosclerotic plaques (n = 30) and in uninvolved arterial specimens (n = 11). We studied members of all three MMP classes (interstitial collagenase, MMP-1; gelatinases, MMP-2 and MMP-9; and stromelysin, MMP-3) and their endogenous inhibitors (TIMPs 1 and 2) by immunocytochemistry, zymography, and immunoprecipitation. Normal arteries stained uniformly for 72-kD gelatinase and TIMPs. In contrast, plaques' shoulders and regions of foam cell accumulation displayed locally increased expression of 92-kD gelatinase, stromelysin, and interstitial collagenase. However, the mere presence of MMP does not establish their catalytic capacity, as the zymogens lack activity, and TIMPs may block activated MMPs. All plaque extracts contained activated forms of gelatinases determined zymographically and by degradation of 3H-collagen type IV. To test directly whether atheromata actually contain active matrix-degrading enzymes in situ, we devised a method which allows the detection and microscopic localization of MMP enzymatic activity directly in tissue sections. In situ zymography revealed gelatinolytic and caseinolytic activity in frozen sections of atherosclerotic but not of uninvolved arterial tissues. The MMP inhibitors, EDTA and 1,10-phenanthroline, as well as recombinant TIMP-1, reduced these activities which colocalized with regions of increased immunoreactive MMP expression, i.e., the shoulders, core, and microvasculature of the plaques. Focal overexpression of activated MMP may promote destabilization and complication of atherosclerotic plaques and provide novel targets for therapeutic intervention.
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PMID:Increased expression of matrix metalloproteinases and matrix degrading activity in vulnerable regions of human atherosclerotic plaques. 798 8

Vascular matrix remodeling occurs during development, growth, and several pathological conditions that affect blood vessels. We investigated the capacity of human smooth muscle cells (SMCs) to express matrix metalloproteinases (MMPs), enzymes that selectively digest components of the extracellular matrix (ECM), in the basal state or after stimulation with certain cytokines implicated in vascular homeostasis and pathology. Enzymatic activity associated with various proteins secreted in the culture media was detected by gelatin or casein sodium dodecyl sulfate-polyacrylamide gel electrophoresis zymography. Proteins were identified by immunoprecipitation and mRNA by Northern blotting. SMCs constitutively secreted a 72-kD gelatinase and the tissue inhibitors of MMPs (TIMPs) types 1 and 2. SMCs stimulated with interleukin-1 or tumor necrosis factor-alpha synthesized de novo 92-kD gelatinase, interstitial collagenase, and stromelysin. Several lines of evidence suggest that when stimulated by cytokines, SMCs produce activated forms of MMPs. Together, the constitutive and the cytokine-induced enzymes can digest all the major components of the vascular ECM. Moreover, since these mediators augment the production of MMPs without appreciably affecting the synthesis of TIMPs, locally secreted cytokines may tip the regional balance of MMP activity in favor of vascular matrix degradation.
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PMID:Cytokine-stimulated human vascular smooth muscle cells synthesize a complement of enzymes required for extracellular matrix digestion. 801 77

We have examined the correlation between matrix metalloproteinase (MMP) expression and metastatic properties of a low metastatic osteosarcoma cell line, osteosarcoma takase (OST), under stimulation by tumour necrosis factor alpha (TNF alpha). In vivo, OST cells exhibited significantly increased colonization in the lungs of nude mice in a dose-dependent manner when they were treated by TNF alpha prior to injection. In vitro, TNF alpha enhanced tumour cell invasion through the reconstituted basement membrane in a transwell chamber up to 2.5-fold. Gelatin zymography and sandwich enzyme immunoassays demonstrated marked production of MMP-9 [92-kDa gelatinase/type IV collagenase (gelatinase B)] but not MMP-2 [72-kDa gelatinase/type IV collagenase (gelatinase A)], MMP-3 (stromelysin-1) or MMP-7 (matrilysin). Motility of the tumour cells and adhesion to cultured endothelial cells were slightly increased by the TNF alpha treatment up to 1.6-fold and 1.4-fold, respectively, while the growth rate was decreased. These results suggest that upregulation of MMP-9 together with enhanced motility and endothelial adhesion contribute to the increased metastatic ability of OST cells induced by TNF alpha treatment.
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PMID:Expression of matrix metalloproteinase 9 (92-kDa gelatinase/type IV collagenase) induced by tumour necrosis factor alpha correlates with metastatic ability in a human osteosarcoma cell line. 803 35

A prominent metalloproteinase activity with an apparent molecular mass of 80 kD and additional activities at 67 through 70, 50, and 32 kD have been observed on casein, gelatin, and elastin gel zymography in extracts from abdominal aortic aneurysms (AAAs). The forms at 80, 50, and 32 kD were isolated by affinity to recombinant tissue inhibitor of metalloproteinases, and the 80-kD and 50-kD components were shown to be derived from matrix metalloproteinase-9 (MMP-9). The relative electrophoretic mobility of these forms under reducing and nonreducing conditions corresponds to those of MMP-9 generated by MMP-3 (stromelysin-1) cleavage, and the active forms of MMP-3 at 45 and 35 kD were detected in aneurysmal extracts under reducing conditions by using specific antibody. Confirmation that the major proteolytic activity observed at 80 kD is MMP-9 was also demonstrated by immunoprecipitation of the activity with specific antibody. Comparative immunoblots of tissue extracts from 10 typical AAA patients, using specific antibody against MMP-9, revealed bands at 92, 82, 67, 51 through 53, 27, 23, and 20 kD under reducing conditions; six aortic control specimens displayed negligible immunoreactivity. This report is the first to show that known activated forms of MMP-3 and MMP-9 are present in the aneurysmal aortic wall and that they may play a role in the destruction of aortic matrix in AAA disease.
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PMID:Identification of matrix metalloproteinases 3 (stromelysin-1) and 9 (gelatinase B) in abdominal aortic aneurysm. 804 93

Members of the matrix metalloproteinase (MMP) family have been implicated in disease states such as arthritis, periodontal disease, and tumor cell invasion and metastasis. Stromelysin 1 (MMP-3) has a broad substrate specificity and participates in the activation of several MMP zymogens. We examined known sequences of MMP-3 cleavage sites in natural peptides and proteins and compared sequence specificities of MMP-3 and interstitial collagenase (MMP-1) in order to design fluorogenic substrates that (i) would be hydrolyzed rapidly by MMP-3, (ii) would discriminate between MMP-3 and MMP-1, and (iii) could be monitored continuously without interference from MMP amino acid residues. Designed substrates were then screened for activity toward MMP-1, gelatinase A (MMP-2), MMP-3, and gelatinase B (MMP-9). The first of these substrates, NFF-1 (Mca-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Lys-(Dnp)-Gly, where Mca is (7-methoxycoumarin-4-yl)acetyl and Dnp is 2,4-dinitrophenyl), was hydrolyzed equally well by MMP-3 and MMP-2 (kcat/Km approximately 11,000 s-1 M-1). MMP-1 had 25% of the activity of MMP-3 toward NFF-1. The second substrate, NFF-2 (Mca-Arg-Pro-Lys-Pro-Tyr-Ala-Nva-Trp-Met-Lys(Dnp)-NH2, where Nva is norvaline), was hydrolyzed 60 times more rapidly by MMP-3 (kcat/Km = 59,400 s-1 M-1) than MMP-1. Unfortunately, NFF-2 showed little discrimination between MMP-3, MMP-2 (kcat/Km = 54,000 s-1 M-1), and MMP-9 (kcat/Km = 55,300 s-1 M-1). The third substrate, NFF-3 (Mca-Arg-Pro-Lys-Pro-Val-Glu-Nva-Trp-Arg-Lys(Dnp)-NH2), was hydrolyzed rapidly by MMP-3 (kcat/Km = 218,000 s-1 M-1) and very slowly by MMP-9 (kcat/Km = 10,100 s-1 M-1), but there was no significant hydrolysis by MMP-1 and MMP-2. NFF-3 is the first documented synthetic substrate hydrolyzed by only certain members of the MMP family and thus has important application for the discrimination of MMP-3 activity from that of other MMPs. Although NFF-3 was designed by assuming that substrate subsites were independent and hence free energy changes derived from single mutation experiments were additive, we found discrepancies between predicted and experimental kcat/Km values, one on the order of 2000-5000. Thus, the design of additional discriminatory MMP substrates may require approaches other than assuming additive free energy changes, such as screening synthetic libraries and consideration of secondary and tertiary structures of substrates and the enzyme.
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PMID:Design and characterization of a fluorogenic substrate selectively hydrolyzed by stromelysin 1 (matrix metalloproteinase-3). 806 13

Matrix metalloproteinases are secreted enzymes important in inflammation and tumor invasion. Earlier, we demonstrated that in normal human FS-4 fibroblasts, collagenase and stromelysin mRNA levels are increased not only after treatment with known matrix metalloproteinase inducers such as tumor necrosis factor (TNF), interleukin-1, and 12-O-tetradecanoylphorbol-13-acetate, but also with interferon-beta (IFN-beta). In this study, we compared the regulation of these matrix metalloproteinase genes by TNF and IFN-beta. We show that both TNF and IFN-beta increase steady-state levels of collagenase and stromelysin mRNAs with similar slow kinetics. The glucocorticoid dexamethasone blocked matrix metalloproteinase induction by both cytokines. The protein synthesis inhibitor cycloheximide inhibited collagenase mRNA induction by TNF or IFN-beta, suggesting that induction by both agents is indirect. Consistent with these observations, both TNF and IFN-beta increased c-fos and c-jun mRNA levels. Furthermore, treatment with TNF or IFN-beta increased the transcriptional activity of activator protein-1-responsive chloramphenicol acetyltransferase reporter gene constructs, including a native collagenase promoter-driven chloramphenicol acetyltransferase construct. These findings show that regulation of matrix metalloproteinase gene expression by both TNF and IFN-beta involves the transcription factor activator protein-1 and demonstrate a novel indirect mechanism of type I IFN-induced gene expression.
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PMID:Interferon-beta induces metalloproteinase mRNA expression in human fibroblasts. Role of activator protein-1. 806 4

We have investigated the inhibition of the human matrix metalloproteinase stromelysin (SLN) by the peptide phosphonamidate, phthaloyl-N-(CH2)4-P(O2-)-Ile-(beta-naphthyl)Ala-NH-CH3, and find that it is a potent, slow-binding inhibitor of SLN with kon = 2.7 x 10(4) M-1 sec-1, koff = 1.9 x 10(-4) sec-1, and Ki = 7 nM (pH 5.0, 25 degrees C). To probe the mechanism of inhibition, we determined pH-dependencies and solvent deuterium isotope effects. pH-dependencies of the kinetic parameters for inhibition are complex but reflect greater inhibitory potency at lower pH and suggest a mechanism for inhibition that involves the same active site groups as are involved in catalysis. The solvent isotope on kon (kon, H2O/Kon,D2O) is normal and equals 1.5 +/- 0.1. Together with the pH-dependence of inhibition, this value suggests that kon is rate-limited by a process that involves general-acid/general-base catalysis. We propose that kon is rate-limited by general-acid catalyzed ligand exchange of inhibitor for the zinc-bound water molecule.
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PMID:Mechanistic studies on the inhibition of stromelysin by a peptide phosphonamidate. 808 34

The aim of this study is to examine the transcriptional regulation of matrix metalloproteinase transin in glomerular mesangial cells responding to inflammatory cytokines and heparin. Northern blot analysis revealed that IL-1 beta preferentially induced transin mRNA. The stimulatory effect was not specific to transin, and upregulation of procollagen alpha 1(IV), laminin B2 and tissue inhibitor of metalloproteinases-1 (TIMP-1) mRNAs was also observed. After IL-1 stimulation, expression of the transin transcript increased progressively for up to 48 hours, differing from the limited induction of procollagen alpha 1(IV) or TIMP-1. When mesangial cells were stimulated by IL-1 beta in the presence of heparin, transin expression was markedly suppressed in a dose-dependent manner. The inhibitory effect of heparin was specific to transin, and induction of procollagen alpha 1(IV), laminin B2 or TIMP-1 by IL-1 beta was not affected. These findings revealed the selective counter regulation by IL-1 beta and heparin of the transin expression in mesangial cells.
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PMID:Heparin selectively inhibits gene expression of matrix metalloproteinase transin in cultured mesangial cells. 809 49


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