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:3.4.24.3 (collagenase)
18,340 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The present communication reports the effects of N-nitroso-pyrrolidine (NPYR) on some essential metabolic pathways in isolated hepatocytes. Isolated cells prepared by the collagenase-perfusion technique are incubated for 4 hours in suspension in a Waymouth medium, either in the absence or in the presence of NPYR (20 to 40 mmol/l). Under these conditions, NPYR, even at 40 mmol/l, has no effect on the vital staining of the cells by erythrosin B and does not increase the leakage of LDH, indicating no lethal effect. However, the glycogen synthesis which occurs in control cells is inhibited in presence of NPYR and the intracellular glycogen is even degraded. This effect is accompanied by reactivation of phosphorylase 'a'. NPYR also affects the protein synthesis capacity of the isolated hepatocytes. Incubation in the presence of non-metabolizable labelled amino acids has demonstrated that such an effect could be explained by a specific action of NPYR on the L-transport system for amino acids through the hepatic plasma membrane.
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PMID:Biochemical effects of nitrosopyrrolidine on isolated hepatocytes. 714 61

Insoluble collagen fibrils were obtained from Cysticercus cellulosae after homogenization and treatment with NaCl/mercaptoethanol solutions and were solubilized after limited pepsin digestion. Solubilized Cysticercus collagen shows two different alpha subunits in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 100,000 and is readily degraded by bacterial collagenase. The amino acid composition is characteristic of collagen except that it contains no hydroxyproline. Segment long spacing crystallites measuring 280 nm in length were prepared. These segments showed a band pattern different from that of vertebrate and other invertebrate collagens. The denaturation temperature at neutral pH was 35 degrees C and correlated with the total pyrrolidine content as observed for other collagens. An intrinsic viscosity value of 15.3 dl/g was obtained for this collagen. Its possible evolutionary relationship with other collagens is discussed.
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PMID:The isolation, purification, and characterization of the collagen of Cysticercus cellulosae. 724 Jan 81

Tumor necrosis factor alpha (TNF-alpha) has been shown to induce the production of interstitial collagenase by fibroblasts and chondrocytes. We investigated the role of TNF-alpha in collagenase gene expression by U937 monocyte/macrophage cells. Transcription of the TNF-alpha gene was observed after 0.5 h of phorbol myristate acetate (PMA) stimulation. Collagenase mRNA expression was not observed until 5-7 h of activation with PMA. TNF-alpha was detected in the culture supernatants 2-3 h before transcription of the collagenase gene. Neutralization of TNF-alpha protein with anti-TNF-alpha antibodies significantly reduced collagenase mRNA expression. Protein kinase C (PKC) and protein tyrosine kinase (PTK) inhibition essentially abolished both PMA-induced TNF-alpha protein secretion and collagenase mRNA expression. Collagenase gene expression induced by exogenous TNF-alpha in U937 cells stimulated with a suboptimal concentration of PMA was suppressed by PTK, but not PKC, inhibition. The pyrrolidine derivative of dithiocarbamate, a potent inhibitor of nuclear factor-kappa B (NF-kappa B) activation, resulted in a marked reduction in collagenase gene transcription, however, no reduction of TNF-alpha secretion was noted. Anti-TNF-alpha antibodies inhibited PMA-induced NF-kappa B activation. These observations demonstrate an important role for TNF-alpha in the autocrine regulation of collagenase gene expression by U937 cells. Additionally, TNF-alpha-induced PTK and NF-kappa B activation were important in collagenase gene expression in this cell line.
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PMID:Autocrine regulation of collagenase gene expression by TNF-alpha in U937 cells. 855 60

Latent matrix metalloproteinases (MMPs) in normal myocardium are activated in end-stage heart failure. In vitro oxidized glutathione (GSSG) activates myocardial MMPs which contains a cysteine residue. In vivo GSSG induce the collagen lysis and cardiac dilatation. To assess whether thiol and non-thiol reducing agents have direct effect on the interstitial human heart fibroblast (HHF) proliferation and MMP expression, HHF and polyoma virus transformed fibroblast cells were cultured with or without the thiol-containing reduced (GSH) or oxidized (GSSG) glutathiones, pyrrolidine dithiocarbamate (PDTC) and N-acetylcysteine (NAC), and non-thiol ascorbic acid. After 100 micrograms/ml (approximately 0.3 mM) GSH or PDTC treatment the proliferative (synthetic) phenotype of transformed fibroblast cells was changed to quiescent (contractile) phenotype. Also, after GSH, PDTC, and ascorbic acid treatment the medium was then analyzed for MMP activity by zymography. The results indicate reduction in MMP expression in transformed fibroblast cells after GSH and PDTC treatments and no effect after ascorbic acid treatment. Based on reverse zymography, we observed the level of tissue inhibitor of metalloproteinase (TIMP) at a decreased level in transformed cells. The effect of the reducing agent at the gene transcription was measured by estimating mRNA (Northern blot analysis) of MMP and of TIMP in the cells that were cultured in medium in the presence and absence of GSH. These results indicate that GSH induces MMP-2 and MMP-1 expression in normal HHF and that GSH reduces MMP-2 and MMP-1 in transformed fibroblast cells. After the treatment, the TIMP-2 level was repressed in normal HHF and TIMP-2 level increased in transformed fibroblast cells. These events are dependent on the nuclear transcription factor activity on the collagenase promoter in normal HHF cells. On the other hand, in polyoma transform fibroblast cells these events are not dependent on this collagenase promoter. These results suggest that oxidative environment induces normal HHF cell proliferation, and the reducing agent decreases normal HHF cell proliferation by inducing MMP and repressing TIMP gene transcription. In transformed cells reducing agents inhibit MMP expression and increase TIMP levels, which suggests a role of antioxidants in preventing tumorigenesis.
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PMID:Reduction-oxidation (redox) state regulation of extracellular matrix metalloproteinases and tissue inhibitors in cardiac normal and transformed fibroblast cells. 872 63

Cross-coupling of active protein-1 (AP-1) and nuclear factor (NF)-kappaB has been reported. In the present study, we investigated the possibility that both of these two transcription factors might contribute to the process of tumor promoter-induced transformation. To establish a stable reporter cell system, two reporter genes were stably transfected into a JB6 mouse tumor promotion-sensitive (P+) cell line: a luciferase reporter controlled by a collagenase AP-1 sequence and a chloramphenicol acetyltransferase reporter controlled by an interleukin 6 NF-kappaB sequence. This double-reporter cell line maintained the phenotype of tumor promotion sensitivity and was able to report basal or induced AP-1 and NF-kappaB transactivation. The cytokine tumor promoter tumor necrosis factor (TNF)-alpha transactivated NF-kappaB and AP-1 for both DNA binding and transcriptional activity. Pyrrolidine dithiocarbamate, an antioxidant that acts as an NF-kappaB inhibitor, efficiently inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA) or TNF-alpha induced NF-kappaB as well as AP-1 transactivation and cell transformation, suggesting dependency of transformation on both transcription factors. The AP-1 transrepressing-retinoid SR11302 transrepressed AP-1 and cell transformation when these were TPA induced but not when TNF-alpha induced, indicating different signaling pathways for TNF-alpha and TPA. Supershift electrophoresis mobility shift assay revealed that Jun B and c-Jun were absent from the AP-1/DNA complex following TNF-alpha but present following TPA treatment. Together, these results suggest that both AP-1 and NF-kappaB activation may be required for transformation whether induced by TPA or by TNF, and the differential sensitivity of TPA and TNF-alpha-induced transformation to inhibition by a retinoid might be explained by differences in the composition of the DNA-bound AP-1 complexes.
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PMID:Inhibitors of both nuclear factor-kappaB and activator protein-1 activation block the neoplastic transformation response. 927 30

The traditional two-step EGTA/collagenase method is widely used in studying nitric oxide (NO) production in hepatocytes. The present study first revealed that hepatocytes isolated by this method spontaneously express an iNOS mRNA. Thereafter, based on this novel finding, we characterized the expression and regulation of the gene in primary cultured hepatocytes. Using Northern blot analysis, the iNOS mRNA was observed 4 h after isolation, reached peak at 8 h, and declined to an undetectable level after 24 h. iNOS gene expression was shown to be serum-independent and not due to lipopolysaccharide contamination. Time-course analysis of the effects of actinomycin D demonstrated that the increase in iNOS transcripts is the result of an accompanying great increase in iNOS gene transcription and lower iNOS mRNA stability; also blockage by cycloheximide suggests that it is dependent on de novo protein synthesis. Inhibition by pyrrolidine dithiocarbamate, a NF-kappaB/c-rel inhibitor, further implies the involvement of NF-kappaB/c-rel. To clarify reason(s) for the induction, hepatocytes were isolated with the collagenase buffer perfusion step omitted. As a consequence, iNOS mRNA was undetectable in the hepatocytes. These findings show that the traditional hepatocyte-isolation culture does indeed transiently express a serum-independent but de novo protein synthesis-dependent iNOS mRNA due to collagenase (type IV) buffer perfusion.
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PMID:Post-isolation inducible nitric oxide synthase gene expression due to collagenase buffer perfusion and characterization of the gene regulation in primary cultured murine hepatocytes. 979 10

Chemokines are a superfamily of structurally related chemoattractant cytokines. JE (monocyte chemoattractant protein-1) and IP-10 (interferon-inducible protein-10) have been detected in the diseased liver. However the in vitro expression is unclear. In this report, we revealed that JE, KC (melanoma growth-stimulating activity gene), and IP-10 mRNAs are not expressed in the normal liver but spontaneously and time-dependently expressed in the primary hepatocytes. The serum-independent gene expression of both JE and KC lasted over 72 h, but that of IP-10 became undetectable 24 h after isolation with collagenase perfusion method. The induction of the genes' expression was not due to LPS contamination but nevertheless was associated with isolation procedure. Actinomycin D blocked their expression. The increase of their transcripts resulted from greater increase in gene transcription and lower mRNA stability. Consistent with c-jun, their mRNA expressions were simultaneously superinduced by cycloheximide (1 microg/ml), suggesting that de novo protein synthesis is involved their transcriptions. Inhibition by pyrrolidine dithiocarbamate (PDTC), a NF-kappaB/c-rel inhibitor, and EMSA imply that NF-kappaB/c-rel is important in their expressions. Of particular interest is that dexamethasone upregulated the spontaneous expression of KC, but suppressed that of JE and IP-10. LPS upregulated the mRNA levels of JE and KC but did not affect that of IP-10. IFN-gamma induced the expression of IP-10; however unlike in macrophages, it did not selectively inhibit that of JE and KC. Our data demonstrated the existence and differential gene expression of JE, KC, and IP-10 in primary cultured hepatocytes, and these are considered to be a reflex of the alteration of hepatocyte cellular physiology during and after isolation.
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PMID:Differential expression and regulation of chemokines JE, KC, and IP-10 gene in primary cultured murine hepatocytes. 1049 15

In this study, we determined whether the proinflammatory cytokines tumor necrosis factor (TNF)-alpha and interleukin-1 beta contribute to the regulation of matrix metalloproteinase (MMP)-9 in human bronchial epithelial cells and whether the induction of MMP-9 is regulated by the transcription factor nuclear factor (NF)-kappa B. We demonstrated that TNF-alpha induced MMP-9 at both the protein and mRNA levels in human bronchial epithelial cells and that interleukin-1 beta did not. In contrast, induction of the tissue inhibitor of metalloproteinase-1 by TNF-alpha was less than that of interleukin-1 beta. Increased expression of MMP-9 and NF-kappa B activation induced by TNF-alpha were inhibited by pyrrolidine dithiocarbamate and N-acetyl-L-cysteine but were not inhibited by curcumin. These results suggest that TNF-alpha induces the expression of MMP-9 in human bronchial epithelial cells and that this induction is mediated via the NF-kappa B-mediated pathway.
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PMID:Induction of MMP-9 in normal human bronchial epithelial cells by TNF-alpha via NF-kappa B-mediated pathway. 1170 41

The renin-angiotensin system contributes to atherogenesis. Matrix metalloproteinases (MMP) are thought to participate in plaque destabilization through degradation of extracellular matrix. This study tested whether angiotensin II (ANG II) induces MMP in human vascular smooth muscle cells (SMC). ANG II induced expression of MMP-1, -3, and -9, but not of MMP-2 in SMC. The expression of MMP-1, a key enzyme for collagen degradation, was studied in detail. SMC stimulated with ANG II concentration-dependently released enzymatically active MMP-1. The ANG II type 1 receptor antagonists losartan and candesartan blocked ANG-II-induced MMP-1 release. Inhibition experiments with actinomycin D suggest ANG-II-induced MMP-1 mRNA regulation at the transcriptional level. Decoy oligodeoxynucleotides against nuclear factor-kappaB and activator protein 1 inhibited MMP-1 secretion, demonstrating participation of these transcription factors in MMP-1 transcription. Stimulation of MMP-1 by ANG II depended on cyclooxygenase 2. The antioxidants pyrrolidine dithiocarbamate and N-acetylcysteine, the flavin protein inhibitor diphenylene iodonium, and the NADP(H) oxidase inhibitor apocynin blocked MMP-1 release, suggesting a redox-sensitive mechanism involving NADP(H) oxidase. The reactive oxygen species (ROS) donor 2,3-dimethoxy-1,4-naphthoquinone induced MMP-1 secretion and enhanced ANG-II-stimulated MMP-1 expression. These findings indicate that ROS may increase their own production by activation of NADP(H) oxidase. The capability of ANG II to induce functionally active MMP in human SMC may contribute to the altered plaque composition seen in complicated stages of atherosclerosis.
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PMID:Angiotensin II stimulates matrix metalloproteinase secretion in human vascular smooth muscle cells via nuclear factor-kappaB and activator protein 1 in a redox-sensitive manner. 1610 92

Activated NF-kappaB plays an important role in the expression of matrix metalloproteinase (MMP)-1 and MMP-13 in rheumatoid arthritis and osteoarthritis. The objective of this study was to determine the effects of the NF-kappaB inhibitor pyrrolidine dithiocarbamate (PDTC) on the expression of MMPs in IL-1beta-stimulated fibroblast-like synoviocytes (FLSs) of rheumatoid arthritis patients. FLSs were treated with IL-1beta (10 ng/ml) for 24 h in the presence or absence of PDTC. The level of MMP-1 and MMP-13 increased in response to PDTC in time- and dose-dependent manners in IL-1beta-stimulated FLSs; the expressions of IL-6 and vascular endothelial growth factor (VEGF) decreased in a PDTC concentration-dependent manner. However, PDTC-mediated repression of IL-6 and VEGF expression was not observed in TNF-alpha-stimulated rheumatoid arthritis FLSs. In contrast, other NF-kappaB inhibitors, such as fenofibrate, N-acetylcysteine and MG132, decreased MMP expression in IL-1beta-stimulated FLSs. The stimulatory effect of PDTC on MMP expression was not mimicked by specific inhibitors of the mitogen-activated protein kinase (MAPK) signaling pathway. Treatments with 100 muM PDTC did not inhibit the phosphorylation of p-ERK1/2, p-P38, and p-JNK, or the transnuclear migration of NF-kappaB through degradation of IkappaB-alpha in IL-1beta-stimulated FLSs. These results suggest that the increase of MMP expression may occur in a stimuli-specific manner or by an NF-kappaB independent mechanism. Therefore, therapeutic NF-kappaB inhibitors should be thoroughly studied before their clinical use in treating rheumatoid arthritis, as undesirable genes may be upregulated through unknown mechanisms, possibly resulting in worse symptoms.
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PMID:Pyrrolidine dithiocarbamate, a NF-kappaB inhibitor, upregulates MMP-1 and MMP-13 in IL-1beta-stimulated rheumatoid arthritis fibroblast-like synoviocytes. 1937 26


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