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)

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

Physical disruption of an atheromatous lesion often underlies acute coronary syndromes. Matrix-degrading enzymes, eg, matrix metalloproteinases (MMPs), may cause loss in mechanical integrity of plaque tissue that favors rupture. T lymphocytes accumulate at sites where atheromata rupture, but the mechanisms by which these immune cells may contribute to plaque destabilization are unknown. This study tested the hypothesis that the T-lymphocyte surface molecule CD40 ligand (CD40L), recently localized in atherosclerotic plaques, regulates the expression of MMPs in human vascular smooth muscle cells (SMCs), the most numerous cell type in arteries. We report here that stimulated human T lymphocytes induced the expression of the matrix-degrading enzymes, ie, interstitial collagenase (MMP-1), stromelysin (MMP-3), gelatinase B (MMP-9), and activated gelatinase A (MMP-2), in human vascular SMCs by cell contact via CD40 ligation, as demonstrated by Western blot analysis, zymography, and antibody neutralization. Recombinant human CD40L (rCD40L) induced de novo synthesis of MMP-1, MMP-3, and MMP-9 on vascular SMCs and stimulated the expression of these enzymes to a greater extent than did maximally effective concentrations of tumor necrosis factor-alpha or interleukin-1beta, established agonists of MMP expression. Interferon gamma, another T-lymphocyte- derived cytokine, inhibited the induction of MMPs by rCD40L. Immunohistochemical analysis of human coronary atheromata colocalized MMP-1 and MMP-3 with CD40-positive SMCs. These results demonstrated that CD40 ligand, expressed on T lymphocytes, promoted the expression of matrix-degrading enzymes in vascular SMCs and thus established a new pathway of immune-modulated destabilization in human atheromata.
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PMID:Regulation of matrix metalloproteinase expression in human vascular smooth muscle cells by T lymphocytes: a role for CD40 signaling in plaque rupture? 928 47

A rapid, reproducible method for the isolation of murine endothelial cells (ECs) has been developed. Murine ECs were highly enriched by collagenase digestion of mechanically minced lung and subcutaneous sponge implants followed by specific selection with rat anti-mouse CD31 (i.e., PECAM-1) monoclonal antibody-coated magnetic beads (Dynabeads). Pure EC populations were isolated from primary cultures by a second cycle of immunomagnetic selection. The cells from the lung were then cloned by a limiting-dilution method to exclude the possibility of nonendothelial cell contamination. Of the 300 cells plated, 29 clones (approximately 10%) were obtained. The clones were positive for CD31 as measured by flow cytometry, and one clone from the lungs (1G11) and the cells from sponge implants (designated as SIECs) were then subjected to subsequent culture in vitro for 40 and 30 passages (up to 5 months), respectively. Characterization was performed on cells between passage 3 and 10. Both cell types formed contact-inhibited monolayers on gelatin and capillary-like "tubes" on Matrigel. However, 1G11 cells exhibited a "cobblestone" morphology, whereas SIECs had a fibroblast-like appearance at confluence. By flow cytometry and enzyme-linked immunosorbent assay, these cells constitutively expressed CD31, VE-cadherin (cadherin-5), CD34, ICAM-1, VCAM-1, and P-selectin. After stimulation with 30 ng/mL of tumor necrosis factor-alpha, the cells became positive for E-selectin (at 4 hours poststimulation) and the expression of ICAM-1, VCAM-1, and P-selectin was upregulated (after 24 hours of stimulation). The presence of VE-cadherin in 1G11 cells and SIECs was confirmed by fluorescence microscopy and Northern blot analysis. The phenotype and morphology of both cell types were stable during 5 months of culture, and there was no evidence of overgrowth by contaminating cells. Taken together, the approach outlined herein may provide a general strategy for the isolation and culture of ECs from a variety of murine tissues. The general strategy outlined here is simple, effective, and flexible, allowing the inclusion of further positive or negative selection steps.
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PMID:A general strategy for isolation of endothelial cells from murine tissues. Characterization of two endothelial cell lines from the murine lung and subcutaneous sponge implants. 930 41

Glomerulosclerosis is the final outcome of a number of different causes of glomerular injury, during which the structures of the glomerulus are obliterated by extracellular matrix. Accumulating evidence suggests that infiltrating macrophages play a pivotal role in the progression to glomerulosclerosis. The present study defines the role played by macrophages at both cellular and molecular levels in the initiation of the sclerotic process in cultured rat mesangial cells. Macrophage-conditioned medium (MPCM) generated from thioglycollate-elicited, lipopolysaccharide-stimulated macrophages upregulated mesangial cell fibronectin production in a dose- and time-dependent manner, independently of cell proliferation. Immunoprecipitation of metabolically labeled 35S-fibronectin confirmed that the matrix protein was synthesized de novo. The genes for fibronectin and the matrix proteins laminin and collagen IV were also found to be upregulated 2.86 +/- 0.24-, 4.94 +/- 0.17-, and 3.03 +/- 0.31-fold over controls, respectively (P < 0.001). Macrophage modulation of matrix turnover was suggested by an upregulation of both transin and tissue inhibitor of metalloproteinase-1 gene transcription. Transforming growth factor (TGF) beta1, platelet-derived growth factor, tumor necrosis factor (TNF) alpha, or interleukin (IL)-1beta could not be detected in the MPCM per se; however, TGFbeta1 and platelet-derived growth factor AB were found to be secreted into mesangial cell culture supernatants. Secretion was augmented 1.69 +/- 0.16- and 2.28 +/- 0.28-fold, respectively (both P < 0.001), in response to MPCM. Northern blot analysis demonstrated that protein secretion had been preceded by upregulation of the genes for these cytokines (2.2 +/- 0.4-fold [P < 0.001] and 5.7 +/- 1.2-fold [P < 0.004], respectively). Incubation of MPCM with either neutralizing antibody or the growth factor receptor antagonist suramin demonstrated that TGFbeta1 played a significant, although minor, role in MPCM-stimulated fibronectin production. In conclusion, this study provides compelling evidence for a direct role of macrophages in the progression to glomerulosclerosis.
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PMID:Macrophages promote prosclerotic responses in cultured rat mesangial cells: a mechanism for the initiation of glomerulosclerosis. 933 80

Kupffer cells are sessile tissue macrophages that have a role in liver defense against endogenous endotoxins. Because little information is available on the role of bovine Kupffer cells, we developed a primary culture method to investigate the function of bovine Kupffer cells. Kupffer cells were isolated from the caudate lobe of calf liver by perfusion with collagenase and pronase. Then, the cells were purified by gradient centrifugation followed by counterflow centrifugal elutriation. With the methods, a mean number of 1.5 x 10(6) Kupffer cells with a final viability of over 98% was obtained from 1 g of the liver. Over 95% of the isolated cells were positive for non-specific esterase activity and had surface molecule of CD68. The cultured Kupffer cells expressed mRNAs of tumor necrosis factor-alpha, interleukin (IL)-1 alpha, IL-1 beta and IL-6 by stimulation for 3 h with lipopolysaccharide. The primary culture of bovine Kupffer cells could be useful to investigate the systemic inflammatory response in bovine liver.
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PMID:Primary culture and expression of cytokine mRNAs by lipopolysaccharide in bovine Kupffer cells. 933 83

We previously reported the isolation of zeaxanthin and zeaxanthin dipalmitate using bioactivity-guided fractionation to discover hepatoprotective components of Lycium chinense against carbon tetrachloride induced hepatotoxicity. The present study was designed to uncover the effects of zeaxanthin dipalmitate on hepatic parenchymal and nonparenchymal cells in vitro. Uptake of [3H]thymidine by cultured rat Ito cells in response to zeaxanthin dipalmitate was measured. Collagen synthesis was assessed by the collagenase digestion method. The effects of zeaxanthin dipalmitate on the formation of nitric oxide (NO) and the release of tumor necrosis factor-alpha (TNF-alpha) from Kupffer cells and peritoneal macrophages were also assayed. Zeaxanthin dipalmitate showed a significant hepatoprotective activity against carbon tetrachloride toxicity. Cellular malondialdehyde (MDA) levels declined significantly with the treatment of the compound in a concentration dependent manner. Zeaxanthin dipalmitate significantly inhibited the uptake of [3H]thymidine by Ito cells. Zeaxanthin dipalmitate also reduced collagen synthesis in Ito cells by 65.1% (p < 0.05) as compared to untreated controls. The formation of NO in either Kupffer cells or in peritoneal macrophages was significantly decreased by zeaxanthin dipalmitate in a concentration dependent manner. The release of TNF-alpha was somewhat less affected by the compound. From these results, we conclude that zeaxanthin dipalmitate exerts a potent hepatoprotective activity by inhibiting Ito cell proliferation, collagen synthesis and by inhibiting certain biochemical functions of Kupffer cells.
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PMID:Zeaxanthin dipalmitate from Lycium chinense has hepatoprotective activity. 938 90

Neutrophil collagenase (matrix metalloproteinase-8 or MMP-8) is regarded as being synthesized exclusively by polymorphonuclear neutrophils (PMN). However, in vivo MMP-8 expression was observed in mononuclear fibroblast-like cells in the rheumatoid synovial membrane. In addition, we detected MMP-8 mRNA expression in cultured rheumatoid synovial fibroblasts and human endothelial cells. Up-regulation of MMP-8 was observed after treatment of the cells with either tumor necrosis factor-alpha (10 ng/ml) or phorbol 12-myristate 13-acetate (10 nM). Western analysis showed a similar regulation at the protein level. The size of secreted MMP-8 was 50 kDa, which is about 30 kDa smaller than MMP-8 from PMN. Conditioned media from rheumatoid synovial fibroblasts contained both type I and II collagen degrading activity. However, degradation of type II collagen, but not that of type I collagen, was completely inhibited by 50 microM doxycycline, suggesting specific MMP-8 activity. In addition, doxycycline down-regulated MMP-8 induction, at both the mRNA and protein levels. Thus MMP-8 exerts markedly wider expression in human cells than had been thought previously, implying that PMN are not the only source of cartilage degrading activity at arthritic sites. The inhibition of both MMP-8 activity and synthesis by doxycycline provides an incentive for further studies on the clinical effects of doxycycline in the treatment of rheumatoid arthritis.
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PMID:Matrix metalloproteinase-8 is expressed in rheumatoid synovial fibroblasts and endothelial cells. Regulation by tumor necrosis factor-alpha and doxycycline. 939 86

Activated hepatic stellate cells (HSC) participate in matrix remodeling and deposition in liver fibrosis. The present study demonstrates that interleukin (IL)-10 is expressed by HSC upon activation in vitro or in vivo and that autocrine effects of this cytokine include inhibition of collagen production. Culture activation of HSC caused a distinct increase in IL-10 mRNA level compared with freshly isolated quiescent HSC. Treatment of cultured HSC with tumor necrosis factor-alpha, transforming growth factor-beta, or lipopolysaccharide further increased IL-10 mRNA by 2-fold and resulted in the release of IL-10 protein into the medium. HSC isolated from rats after bile duct ligation (BDL) showed prominent increases in IL-10 mRNA (x 100) and protein (x 30) levels at 7 days after BDL, but such induction disappeared in advanced liver fibrosis (19 days after BDL). IL-10 expression correlated positively with mRNA expression of interstitial collagenase and inversely with that of alpha1(I) collagen. Addition of anti-IL-10 IgG to cultured HSC caused enhanced collagen production under a basal or stimulated condition with TGF-beta, tumor necrosis factor-alpha, or lipopolysaccharide. These effects were associated with increased alpha1(I) collagen mRNA and reciprocally reduced collagenase mRNA levels. Co-transfection of HSC with an IL-10 expression vector and collagen reporter genes showed a 40% inhibition of alpha1(I) collagen promoter activity. These results demonstrate that activation of HSC causes enhanced autocrine expression of IL-10 which possesses a negative autoregulatory effect on HSC collagen production mediated at least in part by alpha1(I) collagen transcriptional inhibition and stimulation of collagenase expression. These findings, along with the demonstrated early induction of HSC IL-10 expression and its late disappearance during biliary liver fibrosis, suggest its in vivo role in matrix remodeling and a possibility that failure for HSC to sustain IL-10 expression underlies pathologic progression to liver cirrhosis.
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PMID:Expression of interleukin-10 by in vitro and in vivo activated hepatic stellate cells. 941 80

Inflammatory cytokines tumor necrosis factor-alpha and interleukin-1 trigger the ceramide signaling pathway, initiated by neutral sphingomyelinase-elicited hydrolysis of cell membrane phospholipid sphingomyelin to ceramide, a new lipid second messenger. Here, we show that triggering the ceramide pathway by sphingomyelinase or C2- and C6-ceramide enhances collagenase-1 (matrix metalloproteinase-1; MMP-1) gene expression by fibroblasts. C2-ceramide activates three distinct mitogen-activated protein kinases (MAPKs) in dermal fibroblasts, i.e. extracellular signal-regulated kinase 1/2 (ERK1/2), stress-activated protein kinase/Jun N-terminal-kinase (SAPK/JNK), and p38. Stimulation of MMP-1 promoter activity by C2-ceramide is dependent on the presence of a functional AP-1 cis-element and is entirely inhibited by overexpression of MAPK inhibitor, dual specificity phosphatase CL100 (MAPK phosphatase-1). Activation of MMP-1 promoter by C2-ceramide is also effectively inhibited by kinase-deficient forms of ERK1/2 kinase (MEK1/2) activator Raf-1, ERK1 and ERK2, SAPK/JNK activator SEK1, or SAPKbeta. In addition, ceramide-dependent induction of MMP-1 expression is potently prevented by PD 98059, a selective inhibitor of MEK1 activation, and by specific p38 inhibitor SB 203580. These results show that triggering the ceramide signaling pathway activates MMP-1 gene expression via three distinct MAPK pathways, i.e. ERK1/2, SAPK/JNK, and p38, and suggest that targeted modulation of the ceramide signaling pathway may offer a novel therapeutic approach for inhibiting collagenolytic activity, e.g. in inflammatory disorders.
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PMID:Enhancement of fibroblast collagenase (matrix metalloproteinase-1) gene expression by ceramide is mediated by extracellular signal-regulated and stress-activated protein kinase pathways. 947 67

Mechanical forces and biochemical stimuli may interact to regulate cellular responses. In this study, we tested the hypothesis that very small mechanical strains interact with growth factors in the regulation of matrix metalloproteinase (MMP)-1. Human vascular smooth muscle cells (VSMCs) were cultured on a precoated silicone membrane in a device that imposes a highly uniform biaxial strain. VSMCs cultured on fibronectin were treated with cyclic 1-Hz strains of 0, 1, or 4%, and MMPs were assayed by Western analysis or gelatin zymography. Small strains did not induce MMP-1 in VSMCs, but strain was a potent inhibitor of platelet-derived growth factor (PDGF)- or tumor necrosis factor-alpha-induced synthesis of MMP-1. In contrast, MMP-2 and TIMP-2 levels were not changed by PDGF and/or mechanical strain. VSMCs strained on the 120-kDa chymotryptic fragment of fibronectin or RGD peptides suppressed PDGF-induced expression of MMP-1, indicating that this effect is not mediated by the heparin-binding domain or connecting segment-1 of fibronectin. Northern analysis of ets-1, a transcriptional activator of MMP-1 expression, showed that strain down-regulated ets-1 expression, whereas c-fos expression was augmented. Thus, small deformations can selectively suppress MMP-1 synthesis by VSMCs, demonstrating the exquisite sensitivity of the cell to mechanical stimuli.
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PMID:Small mechanical strains selectively suppress matrix metalloproteinase-1 expression by human vascular smooth muscle cells. 949 91


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