Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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 gelatinases (type IV collagenases) are members of the matrix metalloproteinase family that not only have a high degree of structural homology but are known to be nearly identical in their digestion profile against macromolecular substrates. We have shown previously that the preferred cleavage sites in the hydrolysis of type I gelatin, catalyzed by gelatinase A (72 kDa type IV collagenase), are bracketed by hydroxyproline in the P5 and P5' positions. In this report, a kinetic investigation using a series of collagenous dodecylpeptides in which the P5 and P5' hydroxyprolines were systematically varied and used as substrates for recombinant human gelatinase A, we show that replacement with either proline or alanine always resulted in increased Km. In contrast, substitution of the hydroxylated amino acids tyrosine and serine at P5 and P5' reduced the Km significantly, indicating that the hydroxyl moiety of the hydroxyproline is the functional group responsible for favorable enzyme-substrate affinity. This was shown by the kcat/Km ratio, which was doubled by the substitution of serine in that site. Cleavage of the same series of dodecylpeptides by recombinant human gelatinase B (92 kDa type IV collagenase) showed a very different kinetic profile for which no patterns were discernible. In subsequent comparisons of the two enzymes, it was found that gelatinase B cleaved the thiopeptolide substrate AcProLeuGly-S-LeuGly-OC2H5 at double the velocity of gelatinase A. In contrast, gelatinase A digested type I gelatin about 2.5-times faster than gelatinase B. SDS-PAGE analysis of gelatin cleavage products showed different patterns of product peptides for each enzyme. Further comparisons of the proteinases using synthetic peptide substrates with variations in size and in substituents at the P2' site again showed marked kinetic differences. Although these two matrix metalloproteinases seem similar in that they are both gelatinolytic and can degrade a nearly identical battery of macromolecular matrix components including type IV collagen, it is clear from these results that they are very different enzymatically. Since the regulatory portions of gelatinases A and B differ markedly, it has been assumed that the enzymes serve the same function, but respond to different stimuli. The differences in substrate specificity described herein suggest that their proposed physiological roles may require reevaluation.
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PMID:Comparison of cleavage site specificity of gelatinases A and B using collagenous peptides. 862 38

Periodontal ligament (PDL) cell motility and the passage of PDL cells along a root surface are important components of tissue remodeling during periodontal regeneration. Proteolytic enzymes, including fibroblast collagenase, have been demonstrated to play an important role in tissue remodeling. Previous studies have shown that PDL cells chemotactically respond to a variety of matrix and growth factors. We therefore studied the effects of type I collagen fragments and fibroblast collagenase on PDL cell migration, since PDL cells have been shown to adhere preferentially to partially demineralized root surfaces with exposed type I collagen. Gingival epithelial cells were used as a control cell population. We report that PDL cells but not gingival epithelial cells preferentially migrate in a dose-dependent manner to both fibroblast collagenase and to type I collagen degradation products. Epithelial cell migration to fibroblast collagenase and type I collagen fragments was observed. Antibody to type I collagen inhibited the type I collagen fragment-mediated migration. Collagenase pre-treatment of PDL cells enhanced PDL cell migration to type I collagen fragments. In other assays, enzyme inhibitors were shown to decrease the collagenase-mediated PDL cell motility. Epithelial cells were shown to migrate preferentially to 92-kDa type IV collagenase and type IV collagen degradation products. Antibody to type IV collagen inhibited type IV collagen-induced epithelial cell migration. Taken together, these data suggest a role for collagenase in the fine control of PDL cell migration in tissue remodeling during periodontal regeneration.
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PMID:Periodontal ligament cells are chemotactic to fibroblast collagenase. 870 41

Members of the metalloproteinase family (MMPs) are known to play a crucial role in the metastatic cascade. Here, we report some investigations about the synthesis of interstitial and type-IV collagenases (gelatinases A and B) in a model of coculture of human fibroblasts and HT 1080 fibrosarcoma cells. The interstitial collagenase activity, mainly found in the conditioned medium of fibroblasts, and its mRNA level were increased in the in vitro coculture model. In contrast, gelatinase A was produced by both cell types. The HT 1080 cells additionally synthesised gelatinase B. In coculture, an enhancement of gelatinase A and the presence of its activated form were observed. Northern blot analysis demonstrated that this enzymatic enhancement occurred at a pretranslational level. The stimulation of the interstitial collagenase activity was partially mediated through soluble factor(s), whereas increased gelatinase A appeared to require direct cell-cell interactions. The extracellular matrix component, type-I collagen, stimulated the enzymatic activities released by the individual cells, but it did not modulate the synthesis of interstitial collagenase in coculture. Our results demonstrate that distinct MMPs are modulated by distinct mechanisms, all depending on specific interactions between tumour cells and host fibroblasts.
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PMID:Modulation of the expression of interstitial and type-IV collagenases in coculture of HT1080 fibrosarcoma cells and fibroblasts. 876 91

The rabbit colon was used to establish an in vitro model for examining development-related cellular changes in colonocyte function. Colonic epithelia from newborn, weanling, and adult animals were separated from the muscle and subjected to enzymatic digestion. A mixture of 0.05% Pronase, 0.015% collagenase IV, and 0.023% DTT was determined to be optimal for the isolation of newborn and weanling colonocytes. This solution yielded significantly more cells and of greater viability than a 0.1% Pronase, 0.03% collagenase IV, 0.07% DTT mixture that is optimal for adult colonocytes. The epithelial origin of the colonocytes was confirmed by immunofluorescent staining of cytokeratins. The isolation procedure resulted in a crypt-enriched population and the cell yield/g of mucosa increased with age as did the crypt depth. Colonocyte viability of adults but not of newborns and weanlings, declined from 24 to 72 h. When grown on plastic, the newborn and weanling colonocytes show a approximately 2-fold increase in number, DNA and protein content over 48 h. In contrast, for all three parameters the adult colonocytes revealed only a approximately 10% increase. The colonocytes also showed an age-related decline in attachment to extracellular matrices. Colonocytes showed maximal attachment to Matrigel and collagen IV; newborn and weanling colonocytes show > 80% attachment, whereas adult colonocytes showed only a 45% attachment. The efficacy of attachment to Matrigel compared with that on plastic also differed with age, representing 9.3-, 5.5-, and 4.4-fold increase in adult, weanling, and newborn colonocytes, respectively. Newborn and weanling colonocytes grown on Matrigel for 48 h, showed a significant, 15% increase in cell number, DNA, and protein content compared with those grown on plastic. There was no difference in these parameters when adult colonocytes grown on Matrigel were compared with those grown on plastic. In summary, we have established an in vitro model for studying colonic epithelial cells at different stages of development.
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PMID:Altered growth and attachment of rabbit crypt colonocytes isolated from different developmental stages. 882 2

Implantation and placental development are dependent upon trophoblast invasion of the endometrium. While the villous trophoblast does not display invasive behavior, the extravillous cytotrophoblast is highly invasive. By cloning BeWo choriocarcinoma cells, we have isolated two distinct clones that share similarities with villous and extravillous cytotrophoblasts. When cultured at the surface of a type I collagen gel, BeWo MC-1 cells were not invasive, whereas BeWo MC-2 cells rapidly invaded this matrix. When injected subcutaneously in nude mice, BeWo MC-1 cells developed a localized tumor and BeWo MC-2 cells developed larger tumors with micrometastases. Gelatinase A expression and minute amounts of gelatinase B were detected in the parental cell line and in both clones. However, the parental and the BeWo MC-2 cells secreted 5-to 10-fold more gelatinase A than the BeWo MC-1 cells. Laminin and matrigel stimulated the production of gelatinase A in BeWo MC-2 cells. Type I collagen promoted the conversion of the 72-kDa progelatinase A in an active enzyme only in parental BeWo and in BeWo MC-2 cells. These clones provide an interesting model for studying the complex mechanisms regulating implantation as well as the controlled invasiveness during implantation compared to tumor invasion.
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PMID:Cloning of choriocarcinoma cells shows that invasion correlates with expression and activation of gelatinase A. 883 62

Collagen tissue content and both interstitial (MMP-1) and type IV collagenases (also known as gelatinases) activity within the normal human ovarian capsule were investigated. The apical tunica albuginea (n = 10) displayed a lower mean total collagen concentration than the ovarian capsule areas (n = 9) with no follicles underneath them (137.8 +/- 36.1 vs. 176.6 +/- 23.1 micrograms/mg wet weight tissue (ww), P = 0.004). This was accompanied by higher net interstitial collagenase activity (12.96 +/- 2.26 vs. 5.97 +/- 1.9 U/g ww, P = 0.016) which was present within the ovarian capsule in active form only. Zymography revealed the dominance of the 72-kDa over the 92-kDa gelatinase form regardless of the capsule area investigated. Our results indicate that extracellular matrix remodelling within human tunica albuginea is more strongly pronounced in the apical region.
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PMID:Extracellular matrix remodelling within the normal human ovarian capsule. 884 8

Human heart matrix metalloproteinases (MMP) are present in the latent form and activated in the failing heart. To examine whether the MMP activation was due to gene and/or post-translational modification, we analysed tissue from 10 explanted hearts due to coronary heart disease (CHD) and five normal left atrial tissue from donor hearts. Based on in situ immunolabeling MMP-1, tissue inhibitor of metalloproteinase (TIMP-1) and collagen were co-localized in the interstitial tissue. Based on sandwich ELISA, TIMP-1 and MMP-1 levels were 37 +/- 8 ng/mg and 9 +/- 2 ng/mg in normal tissue (P < 0.01) and 12 +/- 5 ng/mg and 75 +/- 11 ng/mg in the infarcted tissue (P < 0.01), respectively. These levels suggest repression of TIMP-1 during myocardial infarction. Northern blot analysis indicated that the mRNAs for both MMP-1 and TIMP-1 were increased three-to four-fold in the infarcted tissue as compared to the normal tissue, suggesting upregulation of MMP and TIMP gene transcription following infarction. Based on in situ tissue overlay zymography, the generalized activation of MMP was observed in the interstitium of the infarcted heart. Zymographic and immunoblot analysis demonstrated the presence of one band at 66 kDa (MMP-2) in the normal tissue and several bands at 92 (MMP-9), 66 (MMP-2) and 54 kDa (MMP-1) in the infarcted heart. Incubation of the zymographic gel with metal chelator (phenanthroline) abolished bands at 92 kDa and 54 kDa but phenanthroline did not abolish the lytic band at 66 kDa. The 66 kDa band was completely abolished in the presence of phenanthroline and phenyl methyl sulfonyl fluoride (PMSF). 2D-zymographic analysis suggested that the lytic band at 66 kDa was a mixture of two neutral proteinases with different isoelectric point. Plasminogen/gelatin zymographic analysis of infarcted tissue extract indicated that the band at 66 kDa was plasmin generated due to increased expression of tissue plasminogen activator (tPA) activity. In relation to increased expression of gelatinase in the infarcted tissue, our data suggest that gelatinase B (92 kDa) is induced in diseased heart. The results suggest that tPA converts plasminogen to plasmin which, in turn, activates MMPs and inactivates TIMP-1 post-translationally following ischemic cardiomyopathy.
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PMID:Post-transcriptional regulation of extracellular matrix metalloproteinase in human heart end-stage failure secondary to ischemic cardiomyopathy. 884 29

Polymorphonuclear neutrophil (PMN) migration across basement membrane is thought to be dependent on the degradation of membrane constituents. PMN gelatinase B, a metalloproteinase able to degrade type IV collagen, may be involved in this phenomenon. PMN gelatinase B is released in the extracellular medium as a latent proform and then activated, mainly by PMN elastase. We investigated the role of gelatinase B in PMN migration across a Matrigel basement membrane matrix coated onto a filter, in a Boyden chamber. The effects of gelatinase and elastase inhibitors on PMN migration in this system were tested. Chemokinesis of PMN was tested in the same Boyden chamber across a filter free of basement membrane. The agarose method was used to test the same inhibitors for effects on PMN chemotaxis. In both systems, FMLP 10(-7)M was used as a chemoattractant. Addition of 10(-8)M TIMP-1 (the preferential gelatinase B inhibitor) inhibited trans-basement membrane PMN migration by 52 +/- 6% (P<0.05), without affecting PMN chemokinesis, chemotaxis, or degranulation. Also, (Ala)(2) Pro Val chloromethyl ketone (AAPVCK) 100 micron, a specific elastase inhibitor, inhibited trans-basement membrane PMN migration by 51 +/- 8% (P<0.05), without affecting PMN chemokinesis, chemotaxis, or degranulation. The AAPVCK-TIMP combination led to a decrease in migration across Matrigel basement membrane (46 +/- 2%, P,0.05)similar to that seen with TIMP alone. AAPVCK was responsible for inhibition of gelatinase B activation, leading to a decrease in activated gelatinase from 14% to 2% of total gelatinase release (P<0.05). All these results strongly suggest that gelatinase B is a major factor of PMN migration across basement membrane and that elastase may contribute to this process by activating pro-gelatinase B.
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PMID:Role of gelatinase B and elastase in human polymorphonuclear neutrophil migration across basement membrane. 884 80

In neurodegenerative disease or after brain injury, parenchymal cells in the central nervous system are activated to produce inflammatory mediators, mainly consisting of cytokine-induced factors, in a manner similar to, but clearly different from a peripheral inflammatory response. The upregulated expression of several extracellular matrix proteins in astrocytes located surrounding a neuritic plaque in Alzheimer's disease is a good example of such a response. A family of mediators which is cytokine-induced during an inflammatory response in the periphery are the matrix metalloproteinases. Matrix metalloproteinases are calcium-requiring, zinc-containing endopeptidases that constitute a major component of the enzyme cascade responsible for degradation of extracellular matrix proteins such as collagen, proteoglycan and laminin. Little is known about the cellular source or the function of matrix metalloproteinases in the central nervous system or how their expression is regulated in brain. Thus, it was of interest to determine which factors of the so-called 'brain inflammatory response' regulate the expression of these proteases in the nervous system. To this end, we measured the expression of matrix metalloproteinases in cultured rat astrocytes and microglia after treatment with various cytokines. Interleukin-1 beta, tumor necrosis factor-alpha and lipopolysaccharide were potent stimulators of matrix metalloproteinase-2 (gelatinase A) and matrix metalloproteinase-9 (gelatinase B) in cultured rat astrocytes; the effect of each secretagogue was inhibited in the presence of glucocorticoid. Interleukin-1 beta and lipopolysaccharide also stimulated the production of matrix metalloproteinase-3 (stromelysin-1) in astrocytes. In addition, activated microglia release matrix metalloproteinase-9. The 'coactivator' of monocytic phagocytes, interferon-gamma, rather than augmenting the response to lipopolysaccharide, inhibited it. Thus, cytokines appear to be potent regulators of matrix metalloproteinase production in astrocytes and microglia. The presence of these enzymes in 'inflamed' central nervous system may suggest their involvement in the pathogenesis or progression of neurodegenerative diseases which are associated with an inflammatory component. Much remains to be learned about the potential substrates for these enzymes and the mechanism of their activation in the central nervous system.
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PMID:Regulation of matrix metalloproteinase expressions in astrocytes, microglia and neurons. 894 20

Although heart attack is caused by occlusion of a major coronary artery, some patients have occlusion without heart attack because these patients have sufficient collateral circulation to provide an alternate pathway for blood supply to the myocardium at ischemic risk. The growth of new capillary vessels (angiogenesis) and enlargement of preexisting vessels play an important role in the collateral development. We evaluated the hypothesis that extracellular matrix metalloproteinase (MMP) expression is altered in coronary collateral arteries (0.5-1 mm o.d.) isolated from canine hearts 2-4 months after surgical placement of an ameroid occluder around the proximal left circumflex artery (n = 4), during the development of collateral vessels and restructuring new vessels. Histologic studies (hematoxylin and eosin, trichrome, and van Gieson stains) indicated cellular proliferation and increased collagen and elastin content in collateral vessels compared with comparable-sized unoccluded arterial segments of the left anterior descending (LAD) artery. In situ MMP activity of collateral vessels, measured using denatured collagen in the gel matrix, indicated an increase in total MMP activity in the intima of collateral vessels compared with normal LAD vessels. To further identify the type of MMP, tissue homogenates were prepared from collateral and LAD vessels and analyzed by SDS-PAGE zymography. The results suggest induction of gelatinase A and gelatinase B expression in collateral vessels compared with normal LAD tissue, when identical amounts of total protein were loaded onto each lane in the gel. Based on plasminogen-casein zymography, we observed the tissue plasminogen activator level to be increased in collateral vessels. On the basis of immunoblot and mRNA (Northern blot) analyses, we determined that the MMP-1 level was induced in collateral vessels 2 and 4 months after ameroid occlusion. In contrast with MMP-1, the level of TIMP-1 (tissue inhibitor of metelloproteinases) was decreased significantly (p < 0.001) in collateral compared with LAD vessels, suggesting a role for arterial TIMP in anti-angiogenic activity. Collectively, these results suggest that chronic occlusion of a major coronary artery induces upregulation of vascular remodeling mechanisms subserving collateral development. Increased MMP-2 activity in collaterals may be associated with decreased levels of tissue inhibitor of metalloproteinases and fibrous tissue remodeling following angiogenic and (or) adaptive responses of the myocardium to chronic ischemia.
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PMID:Temporal expression of extracellular matrix metalloproteinases and tissue plasminogen activator in the development of collateral vessels in the canine model of coronary occlusion. 896 Mar 89


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