Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.24.35 (matrix metalloproteinase 9)
 2,207 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The precursor of matrix metalloproteinase 9 (proMMP-9), also known as '92 kDa progelatinase/type IV procollagenase', was purified from the conditioned medium of U937 monocytic leukaemia and HT1080 fibrosarcoma cell lines stimulated with phorbol 12-myristate 13-acetate. ProMMP-9 in these culture media is non-covalently complexed with the 29 kDa tissue inhibitor of metalloproteinases (TIMP), but free proMMP-9 was separated from the TIMP-proMMP-9 complex by chromatography on Green A Dyematrex gel. The final product was homogeneous on SDS/PAGE, with a molecular mass of 88 kDa without reduction and 92 kDa with reduction. Treatment of proMMP-9 with 4-aminophenylmercuric acetate converted the 88 kDa precursor into 80 kDa and 68 kDa forms. Gelatin-containing zymographic analysis showed zones of lysis associated with all three species. However, only the 68 kDa species was shown to be catalytically active by its ability to bind to alpha 2-macroglobulin. In the presence of an equimolar amount of TIMP, only the 80 kDa species was generated by treatment with 4-aminophenylmercuric acetate, but no enzyme activity was detected. This indicates that TIMP binds to the 80 kDa intermediate and inhibits the generation of the active 68 kDa species. Eight endopeptidases (trypsin, chymotrypsin, plasmin, plasma kallikrein, thrombin, cathepsin G, neutrophil elastase and thermolysin) were tested for their ability to activate proMMP-9. Of them, trypsin was the most effective activator of proMMP-9. Only partial activation (10-30%) was observed with plasmin, cathepsin G and chymotrypsin. The active forms generated by trypsin were identified as 80 kDa, 74 kDa and 66 kDa by their abilities to bind to alpha 2-macroglobulin. In the presence of an equimolar amount of TIMP, proMMP-9 was also converted into the same molecular-mass species by trypsin, but they were not proteolytically active. This suggests activated MMP-9 is inhibited by TIMP. Activated MMP-9 digested gelatin, type-V collagen, reduced carboxymethylated transferrin and, to a lesser extent, type-IV collagen and laminin A chain. The specific activity against gelatin was estimated to be 15,000 units/mg (1 unit = 1 microgram of gelatin degraded/min at 37 degrees C) by titration with alpha 2-macroglobulin. Comparative studies on digestion of gelatin and collagen types IV and V by MMP-9 and MMP-2 indicated that both enzymes degrade these substrates into similar fragments. However, the susceptibilities of laminin, fibronectin and reduced carboxymethylated transferrin to these two MMPs were sufficiently different to indicate differences in substrate specificities between these two closely related proteinases.
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PMID:Purification and characterization of matrix metalloproteinase 9 from U937 monocytic leukaemia and HT1080 fibrosarcoma cells. 137 48

Matrix metalloproteinase 9 (MMP-9) has been purified as an inactive zymogen of M(r) 92,000 (proMMP-9) from the culture medium of HT 1080 human fibrosarcoma cells. The NH2-terminal sequence of proMMP-9 is Ala-Pro-Arg-Gln-Arg-Gln-Ser-Thr-Leu-Val-Leu-Phe-Pro, which is identical to that of the 92-kDa type IV collagenase/gelatinase. The zymogen can be activated by 4-aminophenylmercuric acetate, yielding an intermediate form of M(r) 83,000 and an active species of M(r) 67,000, the second of which has a new NH2 terminus of Met-Arg-Thr-Pro-Arg-(Cys)-Gly-Val-Pro-Asp-Leu-Gly-Arg-Phe-Gln-Thr- Phe-Glu. Immunoblot analyses demonstrate that this activation process is achieved by sequential processing of both NH2- and COOH-terminal peptides. TIMP-1 complexed with proMMP-9 inhibits the conversion of the intermediate form to the active species of M(r) 67,000. The proenzyme is fully activated by cathepsin G, trypsin, alpha-chymotrypsin, and MMP-3 (stromelysin 1) but not by plasmin, leukocyte elastase, plasma kallikrein, thrombin, or MMP-1 (tissue collagenase). During the activation by MMP-3, proMMP-9 is converted to an active species of M(r) 64,000 that lacks both NH2- and COOH-terminal peptides. In addition, HOCl partially activates the zymogen by reacting with an intermediate species of M(r) 83,000. The enzyme degrades type I gelatin rapidly and also cleaves native collagens including alpha 2 chain of type I collagen, collagen types III, IV, and V at undenaturing temperatures. These results indicate that MMP-9 has different activation mechanisms and substrate specificity from those of MMP-2 (72-kDa gelatinase/type IV collagenase).
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PMID:Matrix metalloproteinase 9 (92-kDa gelatinase/type IV collagenase) from HT 1080 human fibrosarcoma cells. Purification and activation of the precursor and enzymic properties. 140 Apr 81

We investigated the ability of murine bone organ cultures and osteoblast-like bone cells to produce 72- and 92-kDa gelatinase. 4-6 day newborn mouse calvaria cultures were found to release gelatinase activity into their conditioned medium (CM). This activity was increased by four stimulators of resorption, tumor necrosis factor alpha (TNF), interleukin-1 alpha (IL-1), parathyroid hormone (PTH) and the active phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA). Both the 72- and 92-kDa forms of gelatinase were produced by murine bone cultures. In unstimulated bones 72-kDa gelatinase activity was approximately equal to that of the 92-kDa enzyme. IL-1, TNF, PTH and TPA all increased 92-kDa gelatinase activity in the CM of the bone cultures by about 2- to 2.5-fold. In addition TPA and IL-1 also increased 72-kDa gelatinase activity. In unstimulated osteoblast-like MC3T3-E1 cell cultures 72-kDa gelatinase enzyme activity was much greater than 92-kDa activity and was not substantially regulated (less than 40% change) by IL-1, TNF or PTH. In contrast, these agents stimulated 92-kDa gelatinase activity by 2- to 5-fold. As with the MC3T3-E1 cells, primary cells constitutively produced both 72-kDa and 92-kDa gelatinase. This was true for cells with both the most differentiated osteoblast-like phenotype (populations 3 and 4) and the least osteoblast-like phenotype (populations 1 and 2). In unstimulated cultures of all 4-primary populations, 92-kDa gelatinase production was less than 72-kDa and IL-1, TNF and PTH had only small effects on 72-kDa production in any of the populations (less than 60% change).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Production of both 92- and 72-kDa gelatinases by bone cells. 143 12

Neutrophils synthesize and store intracellularly a 92-kDa type IV collagenase (gelatinase), the primary structure of which is unknown. We designed a primer based on the highly conserved cysteine-switch region of metalloproteinases and employed the polymerase chain reaction to generate a probe of the human neutrophil gelatinase (HNG) gene. This probe was used to clone the cDNA encoding HNG by screening a chronic granulocytic leukemia cDNA library. In vitro translation of the cDNA-derived HNG mRNA yielded a major product of 78 kDa and smaller autolytically activated or degraded products, all of which were recognized by anti-HNG antibody. The HNG cDNA sequence is nearly identical to that encoding a 92-kDa gelatinase secreted by HT1080 cells. In addition, primer extension and S1 analysis reveal that the above two gelatinase transcripts have similar initiation sites. The HNG cDNA hybridized to a 2.8-kilobase mRNA from chronic granulocytic leukemia cells. HNG mRNA expression was absent from uninduced HL60 cells and from HL60 cells induced to granulocytic maturation with Me2SO. However, unlike other neutrophil secondary granule genes, HNG mRNA was detected in HL60 cells induced to monocytic maturation with 12-O-tetradecanoylphorbol 13-acetate. This suggests that the HNG gene may be subject to differential control pathways in two related but distinct hematopoietic lineages.
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PMID:Structure and expression of neutrophil gelatinase cDNA. Identity with type IV collagenase from HT1080 cells. 146 22

Explants of human endometrium were cultured to study the release of matrix metalloproteinases (MMPs). Analysis of conditioned media by zymography revealed latent and active forms of collagenase (MMP-1, EC 3.4.24.7), 72-kDa gelatinase A (MMP-2, EC 3.4.24.24), and 92-kDa gelatinase B (MMP-9, EC 3.4.24.35). These proteinases were identified by their M(r), their inhibition by tissue inhibitor of metalloproteinases, and the activation of their zymogens by trypsin or aminophenylmercuric acetate. In the absence of sex hormone, explants released large amounts of enzyme activities, as measured by densitometry of zymograms or in soluble assays. Physiological concentrations of progesterone (10-200 nM) almost totally abolished the release of collagenase, of total gelatinase activity, and of the active form of gelatinase B and largely inhibited the release of the active form of gelatinase A. These effects, which were antagonized by mifepristone (RU 38486), suggest that progesterone restrains endometrial tissue breakdown by blocking the secretion and activation of MMPs.
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PMID:Progesterone regulates the activity of collagenase and related gelatinases A and B in human endometrial explants. 146

Human neutrophils were found to release a 91-kDa gelatinase that is serologically related to tumor-derived gelatinolytic enzymes, as evidenced by immunoprecipitation. In order to identify the neutrophil gelatinase, the activity in conditioned medium from human neutrophil suspensions was purified by affinity chromatography on a gelatin substrate. The 91-kDa active enzyme was further separated from other stainable protein bands by classical SDS PAGE and blotting to a solid support. Amino-terminal sequence analysis of blotted proteins showed that the 91-kDa enzyme is a truncated form of tumor-derived 92-kDa gelatinase (type IV collagenase), lacking eight residues at the NH2-terminus. Sequence analysis of enzymatically inactive cleavage products of this neutrophil gelatinase demonstrated that the gelatin-binding part of the molecule is restricted to the amino-terminal third. Exocytosis of gelatinase-containing granules from neutrophils occurred spontaneously within 6 h after neutrophil plating. When the cells were triggered with the phorbol ester phorbol 12-myristate 13-acetate, a strong secretagogue, rapid gelatinase release was observed. When granulocytes were stimulated with the neutrophil-activating peptide interleukin-8, maximal exocytosis occurred within 1 h. The almost immediate release of neutrophil gelatinase after stimulation of the cells with a chemotactic factor might play a key role in remodeling of the extracellular matrix during granulocyte movement in response to chemotactic stimuli.
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PMID:Purification and identification of 91-kDa neutrophil gelatinase. Release by the activating peptide interleukin-8. 164 57

Transformed human fibroblasts secrete two structurally and functionally related inhibitors of matrix metalloproteinases, tissue inhibitor of metalloproteinases (TIMP) 1 and 2. In assays measuring the relative inhibitory capability of TIMP-1 and TIMP-2 against autoactivated 72-kDa gelatinase, which consists of two major active peptides and several inactive fragments, TIMP-2 was more effective than TIMP-1. The isolated 42.5-kDa active fragment that formed as a result of the autoactivation of 72-kDa gelatinase showed the greatest preference for TIMP-2; at half-maximal inhibition, TIMP-2 was greater than 10-fold more effective than TIMP-1. TIMP-2 was also greater than 2-fold more effective than TIMP-1 at inhibiting 72-kDa gelatinase-TIMP-2 complexes activated with 4-aminophenylmercuric acetate, and greater than 7-fold more effective than TIMP-1 at inhibiting 92-kDa gelatinase activated with 4-aminophenylmercuric acetate. Furthermore, these active gelatinases preferentially bound 125I-TIMP-2 when incubated with equal amounts of radiolabeled TIMP-1 and TIMP-2. The ratios of 125I-TIMP-2/125I-TIMP-1 binding to 92-kDa gelatinase, autoactivated 72-kDa gelatinase, and 42.5-kDa fragment were 4.4, 10, and 33, respectively. On the other hand, interstitial collagenase was inhibited by TIMP-1 greater than 2-fold more effectively than TIMP-2 in assays measuring cleavage of loose collagen fibrils.
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PMID:Preferential inhibition of 72- and 92-kDa gelatinases by tissue inhibitor of metalloproteinases-2. 164 75

The complete structure of the human gene for 92-kDa type IV collagenase was determined. Two overlapping genomic clones spanning 26 kilobases (kb) of genomic DNA were shown to contain the entire 7.7-kb structural gene together with 15 and 3.5 kb of 5'-end and 3'-end flanking regions, respectively. The 92-kDa type IV collagenase gene contains 13 exons as does the 72-kDa type IV collagenase gene. All intron locations of the 92-kDa enzyme gene coincided with intron locations in the 72-kDa enzyme gene. Exons 5, 6, and 7 which were 174, 174, and 177 base pairs long, respectively, each encoded one complete internal repeat which resembles the collagen-binding domains of fibronectin. The sequence coding for a unique 48-residue segment in the 92-kDa type IV collagenase that has no counterpart in other metalloproteinases was not present in a separate exon, but was contained in exon 9 which also codes for sequences with homology to the other metalloproteinases. The initiation site for transcription was determined by primer extension analysis. Sequencing analysis of 599 base pairs of the 5'-end flanking region showed that the promoter does not have a TATA motif, but a TTAAA sequence at position -29 to -25. A CAAT motif was not observed but there was one GC box. Two putative 12-O-tetradecanoyl-phorbol-13-acetate (TPA) response elements, that might serve as binding sites for the transcription factor AP-1 and a consensus sequence of a transforming growth factor beta 1 (TGF-beta 1) inhibitory element were found in the promoter region. Gelatinase assay of enzyme secreted by cultured human fibrosarcoma cells (HT-1080) revealed only low levels of 92-kDa type IV collagenase activity, whereas considerable activity of the 72-kDa enzyme was present. Northern hybridization analysis confirmed these findings. Treatment of the HT-1080 cells with TPA resulted in induction of the secretion of 92-kDa type IV collagenase activity. This induction could not be significantly inhibited by concomitant incubation with TGF-beta 1. TPA and TGF-beta 1 did not markedly affect the activities of the 72-kDa enzyme. The activities of the secreted 92- and 72-kDa enzymes by HT-1080 cells correlated with the amounts of mRNA as estimated by Northern analyses.
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PMID:Complete structure of the human gene for 92-kDa type IV collagenase. Divergent regulation of expression for the 92- and 72-kilodalton enzyme genes in HT-1080 cells. 165 38

The effect of the phorbol ester tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) on cell invasion was studied using an in vitro assay for cell invasion through a reconstituted basement membrane matrix (Matrigel). TPA inhibited the invasiveness of malignant human fibrosarcoma HT1080 cells. In contrast, WI-38 lung fibroblasts, which show a very low invasive capacity, were stimulated (3-fold) to invade Matrigel after exposure to TPA for 48 hours. The inhibitory or stimulatory effects of TPA on cell invasion were correlated with a decrease or an increase in cell motility and collagenase IV activity, respectively. Synthetic diacylglycerols partially mimicked the inhibitory action of TPA on HT1080 cells but failed to stimulate WI-38 cell invasion. Immunoblots demonstrated that in both cell lines the alpha and beta isoforms of protein kinase C were equally down-regulated after a 5 hour exposure to TPA despite the basal low level of protein kinase C polypeptide in the malignant cells. Thus, whereas in WI-38 cells induction of an invasive behavior could be observed in the absence of protein kinase C, in the malignant cells disappearance of the kinase was associated with a non-invasive phenotype.
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PMID:Differential effects of phorbol ester on the in vitro invasiveness of malignant and non-malignant human fibroblast cells. 215 89

To study the effect of mononuclear cell differentiation on metalloproteinase production, the human monocytic cell lines U937 and THP-1 were exposed to two well known differentiating agents, the phorbol esters (phorbol myristate acetate (PMA)) and 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). With U937 cells, PMA-induced differentiation increased the production of both interstitial collagenase and 92-kDa gelatinase, whereas exposure to 1,25-(OH)2D3 induced full interstitial collagenase expression in the absence of any detectable 92-kDa gelatinase production. In fact, when U937 cells were differentiated with PMA and then exposed to vitamin D3, the hormone actually suppressed phorbol-induced 92-kDa gelatinase biosynthesis. With THP-1 cells, PMA also induced the production of 92-kDa gelatinase fully, but unlike U937 cells, the combination of PMA and 1,25-(OH)2D3 was required for substantial interstitial collagenase biosynthesis. As with U937 cells, the addition of 1,25-(OH)2D3 to PMA-differentiated THP-1 cells caused a dose-dependent inhibition of 92-kDa gelatinase production. Northern hybridizations demonstrated that both phorbol esters and vitamin D3 act on monocytic cell lines at a pretranslational level. To determine whether metalloproteinase biosynthesis in normal differentiated mononuclear phagocytes was also modified by 1,25-(OH)2D3, human blood monocytes and alveolar macrophages were exposed to this hormone. In both cell types, basal and Staphylococcal-stimulated 92-kDa gelatinase production was markedly inhibited by 1,25-(OH)2D3. In contrast, interstitial collagenase production was completely unaffected by the hormone. In summary, the two major metalloproteinases produced by monocytic cells are regulated via distinct molecular pathways by the action of PMA and 1,25-(OH)2D3. Furthermore, vitamin D3 completely dissociates the production of 92-kDa gelatinase and interstitial collagenase in human mononuclear phagocytes.
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PMID:1,25-dihydroxyvitamin D3 dissociates production of interstitial collagenase and 92-kDa gelatinase in human mononuclear phagocytes. 750 4


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