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)

Phorbol esters stimulate and glucocorticoid hormones down-regulate a variety of promoters such as that of the collagenase gene through the transcription factor AP-1 (Fos/Jun). We now show by genomic footprinting of the collagenase promoter that phorbol ester treatment of cells results in the binding of AP-1 to its cognate DNA binding site in vivo. The DNA-protein contacts obtained in living cells are also found in vitro using cloned DNA and purified AP-1. Although in vitro synthesized glucocorticoid receptor can disturb the DNA binding of Jun homodimers, it does not interfere with the binding of Fos-Jun heterodimers or of purified AP-1 in vitro. Consistently, fully inhibitory doses of glucocorticoid hormone cause no change in apparent occupation of the AP-1 binding site in vivo. The hormone receptor acts without itself binding to DNA.
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PMID:Interference between pathway-specific transcription factors: glucocorticoids antagonize phorbol ester-induced AP-1 activity without altering AP-1 site occupation in vivo. 131 96

Transcription of the human collagenase I gene is induced by phorbol esters and repressed by glucocorticoids. Both types of regulation are mediated by the major enhancer element of the gene, which is localized between positions -73 and -65. The enhancer suffices to transmit positive and negative signals to a heterologous promoter or to a minimal promoter carrying only the TATA-box, both detected by the appearance of chloramphenicol-acetyl-transferase transcribed from the reporter gene linked to the promoters. Sequences 5' of the enhancer modulate its activity. Up- and down-regulation of gene constructs which contain only the collagenase enhancer linked to a heterologous promoter, are independent of ongoing protein synthesis, suggesting posttranslational modification of the transcription factor binding to the enhancer. Repression by glucocorticoids depends on an activated glucocorticoid receptor; a tenfold lower glucocorticoid concentration is needed for repression of the collagenase gene as compared to the activation of the mouse mammary tumor virus long terminal repeat. Immunoprecipitates of the dexamethasone receptor contain AP-1, suggesting a direct interaction of both transcription factors; this interaction may lead to the inactivation of AP-1. The action mechanism of phorbol esters and dexamethasone confirms the central role of AP-1 in proliferation control and tumor promotion. It appears that the most effective tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and the most effective anti-tumor promoter dexamethasone exert their action through the modulation of the same transcription factor.
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PMID:Positive and negative regulation of collagenase gene expression. 148 17

Glucocorticoids are potent inhibitors of collagenase induction by phorbol esters and inflammatory mediators. The target for this negative effect is the AP-1 site within the collagenase promoter, which also mediates its induction. Negative regulation is due to repression of AP-1 activity by the glucocorticoid receptor (GCR). While the GCR is a potent inhibitor of AP-1 activity (Jun/Fos), both c-Jun and c-Fos are potent repressors of GCR activity. In vitro experiments using purified GCR and c-Jun proteins suggest that mutual repression is due to direct interaction between the two. Direct interaction between GCR and either c-Jun or c-Fos is demonstrated by cross-linking and coimmunoprecipitation. These findings reveal a cross talk between two major signal transduction systems used to control gene transcription in response to extracellular stimuli, and a novel mechanism for transcriptional repression.
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PMID:Transcriptional interference between c-Jun and the glucocorticoid receptor: mutual inhibition of DNA binding due to direct protein-protein interaction. 216 52

Connective tissue metabolism was studied in detail in three human skin fibroblast lines, demonstrating low, medium, or high levels of glucocorticoid receptor densities. In the cell lines with low and medium receptor density, dexamethasone, in the range of 10(-5)-10(-9) M, had no effect on collagen production, using short incubation time periods and high (20%) fetal calf serum concentration, while in the cells with highest receptor density, a slight stimulation of collagen synthesis was noted in the concentration range 10(-6)-10(-9) M. In the presence of low concentration (0.5%) of serum, dexamethasone markedly inhibited collagen production. The production of collagenase, assayed by degradation of 3H-labelled type I collagen substrate with a brief trypsin activation of the enzyme, was reduced in a dose dependent manner in all 3 cell lines, the inhibition with 10(-5) dexamethasone being up to 56% of the control. Similarly, the activity of an elastase-like neutral protease was decreased in the presence of dexamethasone. Thus the results indicate that glucocorticoids may have profound effects on the degradation of connective tissue components, while the effects on collagen synthesis may be more variable depending on the environmental milieu.
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PMID:Modulation of collagen metabolism in cultured human skin fibroblasts by dexamethasone: correlation with glucocorticoid receptor density. 243 73

The ability of polycyclic aromatic hydrocarbons and glucocorticoids to regulate monooxygenase activity of human fetal liver has been studied using hepatocytes prepared by collagenase digestion of liver samples from human abortuses of 13 to 19 weeks of gestational age, and maintained in primary monolayer culture for periods up to 5 days. Addition of 1,2-benzanthracene to the cells caused an increase in monooxygenase activity (3-hydroxylation of benzo[a]pyrene and O-deethylation of 7-ethoxycoumarin) in a time-and concentration-dependent fashion. The concentration of 1,2-benzanthracene required to achieve half-maximal induction was 5 microM. The inductive effect of the polycyclic hydrocarbon was potentiated approximately 2.5-fold when dexamethasone (250 nM) or other glucocorticoids were included in the culture medium. Dexamethasone alone had little or no effect on the induction of monooxygenase activity. The concentration of dexamethasone required for half-maximal stimulation of monooxygenase activity in the presence of 1,2-benzanthracene was 5-10 nM, and the action of dexamethasone was reversed by the addition of cortisol 21-mesylate, consistent with the concept that the action of dexamethasone was mediated by binding to a glucocorticoid receptor. These results are suggestive that glucocorticoids, which are produced by the fetal adrenal and have an important role in the regulation of fetal development, act synergistically with polycyclic aromatic hydrocarbons to induce the activity of liver monooxygenases in the human fetus.
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PMID:Synergistic induction of monooxygenase activity by glucocorticoids and polycyclic aromatic hydrocarbons in human fetal hepatocytes in primary monolayer culture. 375 39

We have isolated epithelial cell clusters from mammary glands of pregnant and lactating rats by collagenase-hyaluronidase-deoxyribonuclease digestion, followed by Ficoll density-gradient centrifugation. Clusters of greater than 90% viable cells were identified by light microscopy as essentially devoid of other cell types; the integrity of their subcellular organelles verified by electron microscopy. Binding characteristics of the synthetic glucocorticoid [3H]dexamethasone were studied in cytosols prepared from isolated cell clusters. Cytosols from both pregnant and lactating rats bound [3H]dexamethasone with high affinity to a single class of low capacity binding sites. In both types of cytosol the dissociation constant (Kd 4 degrees C approximately/nM) of the binding was similar; the number of sites per cell in lactating rats was approximately double that in pregnant rats. The specificity of binding was typical of a classical glucocorticoid receptor, with a hierarchy of affinity by competition studies dexamethasone greater than progesterone greater than aldosterone much much greater than testosterone = estradiol. In particular, no difference in progesterone affinity for these glucocorticoid receptors was seen between pregnancy and lactation. This suggests that reported differences in inhibitory action of progesterone, pregnancy versus post-partum, are not glucocorticoid-receptor mediated.
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PMID:Glucocorticoid receptors in epithelial cells isolated from the mammary glands of pregnant and lactating rats. 705 35

The neutral metalloproteinase collagenase is known to be, among others, one of the key enzymes promoting joint destruction in patients with rheumatoid arthritis. Because inflammatory cytokines, e.g., interleukin-1 and tumor necrosis factor-alpha, are considered to activate collagenase gene expression through activation of the transcription factor activator protein-1, we examined whether the water-soluble gold compound aurothiomalate (AuTM) influenced collagenase gene expression, using phorbol ester-treated human fibroblasts. However, AuTM did not prevent phorbol ester-mediated activation of activator protein-1 DNA-binding activity and subsequent induction of collagenase gene expression. In contrast, AuTM counteracted the repressive effects of glucocorticoids on collagenase gene expression and restored collagenase mRNA levels. The molecular target of this paradoxical AuTM action was suggested to be the glucocorticoid receptor.
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PMID:Paradoxical derepression of collagenase gene expression by the antirheumatic gold compound aurothiomalate. 780 28

The role of the ligand in glucocorticoid receptor-mediated transactivation and transrepression of gene expression was investigated. Half-maximal transactivation of a mouse mammary tumor virus-chloramphenicol acetyltransferase reporter gene in transfected cells expressing the human glucocorticoid receptor mutant GRL753F, from which the rate of ligand dissociation is four to five times higher than the rate of dissociation from normal receptors, required a 200- to 300-fold-higher concentration of dexamethasone than was required in cells expressing the normal receptor. Immunocytochemical analysis demonstrated that this difference was not the result of a failure of the mutant receptor to accumulate in the nucleus after steroid treatment. In contrast, in cells cotransfected with a reporter gene containing the AP-1-inducible collagenase gene promoter, the concentration of dexamethasone required for 50% transrepression was the same for mutant and normal receptors. Efficient receptor-mediated transrepression was also observed with the double mutant GRL753F/C421Y, in which the first cysteine residue of the proximal zinc finger has been replaced by tyrosine, indicating that neither retention of the ligand nor direct binding of the receptor to DNA is required. RU38486 behaved as a full agonist with respect to transrepression. In addition, receptor-dependent transrepression, but not transactivation, was observed in transfected cells after heat shock in the absence of the ligand. Taken together, these results suggest that unlike transactivation, transrepression of AP-1 activity by the nuclear glucocorticoid receptor is ligand independent.
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PMID:Hormone-independent repression of AP-1-inducible collagenase promoter activity by glucocorticoid receptors. 782 16

Short-term culture of rainbow trout (Onchorhynchus mykiss) hepatocytes was used to examine the effect of dexamethasone (DEX) on microsomal CYP 1A1 protein content and 7-ethoxyresorufin-O-deethylase (EROD) activity in vitro. Hepatocytes prepared by controlled collagenase digestion and plated at a density of 0.25 x 10(6) cells/cm2 in plastic culture dishes precoated with trout skin extract (7.6 micrograms skin protein/cm2) to facilitate cell attachment were maintained at 16 degrees C. Cells were treated with DEX (10(-9) to 10(-7) M) or vehicle (dimethyl sulfoxide, DMSO) at 24 h. Microsomal CYP 1A1 protein content and EROD activities were measured at 72 h. Both CYP 1A1 protein as measured by Western blots using CYP 1A1 specific anti-sera and EROD activity were significantly lower in DEX (10(-8) to 10(-7) M)-treated hepatocytes compared to untreated (control) or DMSO-treated cells. The effect was dose dependent in that a gradual decrease of CYP 1A1 protein and EROD activities were seen with increasing doses of DEX (10(-8) to 10(-7) M). DEX at 10(-9) M was ineffective. Concomitant addition of 10(-6) M RU486, a type II specific glucocorticoid receptor antagonist, to hepatocytes treated with 10(-7) M DEX abolished the DEX effect. RU486 at 10(-8) M was ineffective. Spironolactone (10(-8) to 10(-6) M), a type I specific glucocorticoid receptor antagonist, did not counteract the DEX effect. RU486 or spironolactone (10(-6) M) alone had no effect on CYP 1A1 under similar conditions. DEX thus down regulates CYP 1A1 in fish cultured hepatocytes and this regulation is mediated through the type II glucocorticoid receptor(s).
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PMID:Down regulation of CYP 1A1 by glucocorticoids in trout hepatocytes in vitro. 839 84

Glucocorticoid occupancy of a large percentage of glucocorticoid receptor (GR) is necessary for the suppression of matrix metalloprotease synthesis by human articular chondrocytes. In this study, we evaluated the levels of GR binding, cellular GR protein, and messenger RNA expression in both normal and osteoarthritic (OA) human articular chondrocytes and compared the degree of suppression of collagenase synthesis by glucocorticoids in cultures of the two cell types in order to investigate whether or not the GR system played an important role in the pathophysiology of OA. By radioreceptor binding assay, we recorded 56,320 +/- 8,230 sites per cell (mean +/- SE, n = 9) in primary cultures of normal chondrocytes and 27,480 +/- 14,240 sites in OA cells (n = 10, P < 0.0001). Equilibrium dissociation constant (Kd) values did not vary between normal (12.4 +/- 1.4 nmol/L) and OA (13.0 +/- 1.8 nmol/L). Subculturing of primary OA chondrocytes resulted in the up-regulation of the number of GR binding sites per cell to values comparable to those obtained in normal chondrocytes. Analysis of protein-immuno dot-blots of cytosolic extracts from normal (n = 4) and OA chondrocytes (n = 4) revealed that the former cytosols contained a 1.9 +/- 0.2 (P < 0.05) higher relative density of GR protein than the latter. By comparing the optical densities of GR-polymerase chain reaction products generated from normal (n = 6) and OA (n = 9) chondrocyte total RNA (normalized using an internal standard, glyceraldehyde phosphate dehydrogenase), we established a relative ratio, normal/OA, of 1.4. Experiments comparing the biological responsiveness of normal and OA chondrocytes to glucocorticoid suppression of interleukin-1-stimulated metalloprotease synthesis showed that dexamethasone inhibited collagenase synthesis in a dose-dependent manner with an IC50 of 6.3 +/- 1.2 x 10(-10) mol/L (n = 5) in normal cells while an IC50 of 5.0 +/- 0.4 x 10(-9) mol/L (P < 0.05) was recorded using OA (n = 5) chondrocytes. The results suggest that OA chondrocytes express fewer GR than normal cells as a result of a decrease in specific gene expression. The decreased responsiveness of OA cells to circulating glucocorticoids may be among the factors responsible for an increased level of metalloprotease synthesis by chondrocytes in OA cartilage.
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PMID:Reduced expression of glucocorticoid receptor levels in human osteoarthritic chondrocytes. Role in the suppression of metalloprotease synthesis. 849 2


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