Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.24.3 (collagenase)
 18,340 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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

The abundance of 12 cytochrome P-450 (CYP) mRNAs was quantified in the caudate lobe of rat livers before dissociation of the organ into single cells by perfusion with 0.025% (w/v) collagenase. Comparison of the initial abundance of CYP-1A1, -1A2, -2A subfamily, -2B1/2, -2C7, -2C11, -2D subfamily, -2E1, -3A1/2 and -4A1 transcripts in the caudate lobe of the intact liver with the values found in freshly isolated hepatocytes demonstrated that the relatively brief (1 h) cell isolation and washing procedures routinely caused 2-3-fold increases in the mRNAs encoding CYP-1A2, -2B1/2, -3A1/2, and -4A1, concomitant with a 50% decline in CYP2C11 mRNA. Further changes in the expression of CYP mRNAs occurred when the hepatocytes were cultured. Thus CYP1A1 mRNA, which is not constitutively expressed in rat liver, became detectable in hepatocytes cultured for 1 h, and after 6 h CYP3A1/2 mRNA levels began to increase. In contrast, levels of all other CYP mRNAs studied had declined after 24 h of culture concomitant with the loss of total cytochrome P-450 content. Culture of hepatocytes with 0.5 mM metyrapone (which prevents the loss of total P-450 content) increased CYP1A1 and CYP3A1/2 mRNA levels still further, such that after 72 h of culture these transcripts were conservatively 10-18-fold higher than in hepatocytes prior to culture. This suggests that these two isoenzymes comprise the bulk of the total cytochrome P-450 content maintained by metyrapone. Collectively, these results demonstrate that the technique commonly used to isolate rat hepatocytes alters hepatic gene expression, as illustrated by the elevation of the mRNAs encoding CYP-1A2, -2B1/2, -3A1/2 and -4A1, and that such perturbations are exacerbated during culture under standard conditions by the loss of the constitutive CYP2C11 and the precocious induction of CYP1A1 and CYP3A1/2 mRNAs.
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PMID:Altered expression of cytochrome P-450 mRNAs, and potentially of other transcripts encoding key hepatic functions, are triggered during the isolation of rat hepatocytes. 843 60

An hepatocyte culture system was developed for potential use in toxicological studies in vitro. Rat hepatocytes were isolated by two-step collagenase perfusion and cultured on Vitrogen-coated Permanox dishes in a modified Chee's medium containing 1 microM dexamethasone and 1% dimethylsulfoxide. The cells remained highly viable for at least 10 d as determined by lactate dehydrogenase release and total protein levels. Albumin secretion into the medium, as a measure of differentiated function, was maintained at elevated levels over the course of 10 d in culture. A number of CYP activities were determined by the analysis of testosterone metabolism in freeze-thawed cells, diazepam metabolism in live cells, and specific assays for CYP 1A1/2, 2B1/2, 2E1, and 3A. Results of these assays indicated that a wide range of CYP isozymes were maintained, some activities were enhanced under the conditions of culture and some activities were inducible. Activities of the phase II enzymes, glutathione S-transferase and UDP-glucuronosyltransferase, and glutathione levels were also maintained in the cultured hepatocytes for at least 6 d. These results strongly support the use of this hepatocyte culture system for in vitro toxicological studies.
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PMID:Characterization of a primary hepatocyte culture system for toxicological studies. 872 45

The objective of this study was to determine the acute effects of glucocorticoids on in vivo levels of bone collagen synthesis in neonatal mice. Mice were injected with vehicle or dexamethasone at the start of the experiment. At 22 h, mice were given a 10 microCi injection of [3H]proline. At 24 h, the mice were sacrificed and the incorporation of [3H]proline into collagenase-digestible CDP labeling) and noncollagen (NCP labeling) protein in calvariae were determined by digestion with bacterial collagenase. Calvarial RNA was analyzed for COL 1A1 and osteocalcin mRNA levels by Northern blotting. After 24 h, vehicle-treated mice showed a 9.8 +/- 1.0% weight gain while dexamethasone-treated mice (1 mg/kg) had a 7.4 +/- 0.8% weight loss. Dexamethasone (1 mg/kg) decreased CDP and NCP labeling in calvariae by 51 +/- 4% and 17 +/- 4%, respectively (13 experiments). The inhibitory effect on protein labeling was selective for collagen since dexamethasone decreased the percent collagen synthesis from 25.4 +/- 1.6% to 16.6 +/- 1.0% (13 experiments). Dexamethasone at 3 mg/kg also decreased CDP labeling and the percent collagen synthesis in calvariae. There was a 30% reduction in COL1A1 mRNA levels and a 67% decrease in osteocalcin mRNA levels. To determine the reversibility of the inhibition of collagen synthesis, mice were given a single injection of dexamethasone (1 mg/kg) and then injected with [3H]proline 2 h prior to sacrifice at 24, 48, or 72 h. The reduction in CDP labeling observed at 24 h was fully reversed by 48-72 h. Moreover, by 72 h, the-rate of weight gain by dexamethasone-treated mice was similar to vehicle-treated controls. These data show that administration of dexamethasone to neonatal mice leads to a selective decrease in bone collagen synthesis within 24 h that is accompanied by down-regulation of osteocalcin and COL1A1 mRNA levels. This model will be useful in determining mechanisms by which high dose glucocorticoids inhibit bone formation in vivo.
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PMID:Dexamethasone suppresses in vivo levels of bone collagen synthesis in neonatal mice. 898 46

Oral submucous fibrosis (OSF) is a precancerous condition of the oral cavity. It is a collagen-related disorder induced by betel quid chewing, a habit that is common in Taiwan. However, the cumulative exposure to betel quids varies in OSF patients. It seems that there is individual susceptibility to betel quid-induced OSF. This study compared the association of OSF and polymorphisms of six collagen-related genes, collagen 1A1 and 1A2 (COL1A1 and COL1A2), collagenase-1 (COLase), transforming growth factor beta1 (TGF-beta1), lysyl oxidase (LYOXase), and cystatin C (CST3), between patients with low and high exposure to betel quids. A total of 166 patients with OSF from a medical center and 284 betel quid chewers who were free of OSF and oral cancer, from the same hospital and five townships, were recruited. PCR-based restriction fragment length polymorphism assays were used to determine the genotypes of the six collagen-related genes situated on different chromosomes. We found that the genotypes associated with the highest OSF risk for collagen 1A1, collagen 1A2, collagenase-1, transforming growth factor beta1, lysyl oxidase, and cystatin C were CC, AA, TT, CC, AA, and AA, respectively, for the low-exposure group, and TT, BB, AA, CC, GG, and AA, respectively, for the high-exposure group. A trend was noted for an increased risk of OSF with increasing number of high-risk alleles for those with both high and low exposures for betel quid. The cell selection mechanism of oral fibroblasts is proposed to explain the effect of the modification of cumulative betel quid exposure on the risk profiles of collagen-related genes. These results imply that susceptibility to OSF could involve multigenic mechanisms modified by the betel quid-exposure dose.
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PMID:Interaction of collagen-related genes and susceptibility to betel quid-induced oral submucous fibrosis. 1210 Nov 12

The present work describes an isozyme-related effect of collagenase perfusion on hepatocyte microsomal cytochrome (CYP)-dependent monooxygenase activities: CYP 1A1/2-, 2B1/2-, 3A1/2- and 2E1-dependent activities in microsomes from rat hepatocytes after isolation were about 60% of that of liver microsomes, and CYP 4A1-dependent activity was equivalent to liver microsomes. In contrast, the microsomal protein content of the various CYP isoforms was not affected by hepatocyte isolation. This is in accordance with the hypothesis of CYP inactivation during the process of hepatocyte isolation by collagenase digestion. L-NAME (1 mM) was found unable to protect from the decline of CYP-dependent monooxygenase activities following hepatocyte isolation. It is possible that the decrease in glutathione peroxidase activity observed in the presence of L-NAME, namely depression of defense against peroxynitrite, could counteract the beneficial effect of L-NAME on nitric oxide synthesis inhibition. The present work also shows that L-NAME could not avoid the progressive, isoform-specific, most probably turnover-related, decline of CYP proteins and related monooxygenase activities in cultured hepatocytes. Dysregulations in the mechanisms of CYP expression in rat hepatocyte cultures, presently unknown but nitric oxide independent, thus appear to occur in cultured rat hepatocytes.
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PMID:Time course of cytochromes P450 decline during rat hepatocyte isolation and culture: effect of L-NAME. 1253 63

It was previously reported that in vivo exposure of fish to combined aryl hydrocarbon receptor agonist (AhR; 3,3',4,4'-tetrachlorobiphenyl, PCB-77) and estrogen receptor agonist (ER; nonylphenol, NP) resulted in potentiation and inhibition (depending on dose ratio, sequential order of exposure, and seasonal changes) of NP-induced responses by PCB-77. The experiments described in this report extend this study by testing whether the effects of PCB-77 on NP-induced ER signaling are mediated through AhR-induced transcriptional suppression of target genes. Trout hepatocytes were isolated by a two-step collagenase perfusion method. After 48-h culture, hepatocytes were exposed to 5 or 10 microM nonylphenol (NP) singly and in combination with PCB-77 at 0.1, 1, and 10 microM. Cells were harvested after 96-h exposure and processed for RNA isolation. Gene expression patterns were quantified using real-time polymerase chain reaction (PCR) with specific primer sets and by Northern blot. Exposure of cells to NP caused significant elevation of ERalpha, ERbeta, Vtg, and Zrp mRNA expressions, while combined exposure with PCB-77 concentration inhibited NP-induced ERs and their target gene expressions. Exposure of trout hepatocytes to PCB-77 alone caused a rapid induction of cytochrome P-450 (CYP) 1A1 mRNA, and combined exposure with NP caused significant reduction in PCB-77 induced CYP1A1 gene expression. Exposure of cells to PCB-77 concentrations induced significant reduction in AhRalpha mRNA (except 1 microM PCB-77, which caused the induction of AhRalpha mRNA levels). AhRbeta mRNA levels in the cells were inhibited after 96-h exposure to PCB-77, while combined exposure with 5 microM NP restored the PCB-77-inhibited AhRbeta mRNA levels to baseline. Taken together, the overall results in this study show that PCB-77 suppresses the gene expression of the ERs and their target genes by transcription mechanism(s). The roles of AhRs in mediating these responses seem to involve the ligand-activated AhR transcriptional induction of CYP1A1. In addition to their frequently described functions as activators of metabolic potentiation and detoxification of various foreign chemicals, data presented in the present study point to other endogenous functions of AhRs that need to be studied further.
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PMID:Gene expression patterns in estrogen (nonylphenol) and aryl hydrocarbon receptor agonists (PCB-77) interaction using rainbow trout (Oncorhynchus Mykiss) primary hepatocyte culture. 1629 59

Bathing in the Blue Lagoon, a specific geothermal biotope in Iceland has been known for many years to be beneficial for human skin in general and for patients with psoriasis and atopic dermatitis in particular. The scientific rationale for this empirical observation, however has remained elusive. We now report that extracts prepared from silica mud and two different microalgae species derived from the Blue Lagoon are capable of inducing involucrin, loricrin, transglutaminase-1 and filaggrin gene expression in primary human epidermal keratinocytes. The same extracts also affects primary human dermal fibroblasts, because extracts from silica mud and one type of algae inhibited UVA radiation-induced upregulation of matrix metalloproteinase-1 expression and both algae, as well as silica mud extracts induced collagen 1A1 and 1A2 gene expression in this cell type. These effects were not restricted to the in vitro situation because topical treatment of healthy human skin (n = 20) with a galenic formulation containing all three extracts induced identical gene regulatory effects in vivo, which were associated with a significant reduction of transepidermal water loss. In aggregate, these results suggest that the bioactives in Blue Lagoon have the capacity to improve skin barrier function and to prevent premature skin ageing. These observations explain at least some of the beneficial effects of bathing in the Blue Lagoon and provide a scientific basis for the use of Blue Lagoon extracts in cosmetic and/or medical products.
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PMID:Bioactive molecules from the Blue Lagoon: in vitro and in vivo assessment of silica mud and microalgae extracts for their effects on skin barrier function and prevention of skin ageing. 1831 88

Photoaged skin is characterized by a decrease of dermal collagen fibers, resulting from an increased breakdown and a diminished de novo synthesis. The increased breakdown results from an increased expression of matrix metalloproteinases (MMPs). The main building blocks involved in de novo synthesis of collagen fibers are collagen 1A1 and 1A2, the expression of which is reduced in photoaged skin. We studied the effect of topical application of vitamins, phytosterols and ceramides on UV-induced up-regulation of the expression of MMP-1 and on UV-induced down-regulation of COL1A1 and COL1A2. The study was conducted with 10 subjects with healthy skin who were comparatively treated for 10 days with (i) a basic preparation containing jojoba oil, (ii) the basic preparation supplemented with vitamins, (iii) the basic preparation supplemented with phytosterols and ceramides, and (iv) the basic preparation supplemented with vitamins, phytosterols and ceramides. All four preparations inhibited the UV induced up-regulation of MMP-1. Neither the basic product nor that supplemented with vitamins inhibited down-regulation of COL1A1 and COL1A2, but addition of phytosterols and ceramides caused a decreased down-regulation of the expression of these genes. Our results indicate that phytosterols and ceramides are effective in blocking the reduced collagen synthesis after UV irradiation and even stimulating synthesis. They may be useful additions to anti-aging products.
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PMID:[Topical application of vitamins, phytosterols and ceramides. Protection against increased expression of interstital collagenase and reduced collagen-I expression after single exposure to UVA irradiation]. 1852 70

Aryl hydrocarbon receptor (AhR) and the activation of the AhR pathway are involved in xenobiotic-induced toxicity and carcinogenesis. Although xenobiotics, such as cigarette smoke, contribute to the development of urothelial carcinoma (UC), the relationship between AhR and UC is unclear. In the present study, we investigated AhR expression in 209 patients with upper urinary tract UC. The nuclear expression of AhR was significantly associated with histological grade, pathological T stage, lymphovascular invasion and lymph node involvement. A multivariate Cox analysis revealed that nuclear AhR expression was a significant and independent predictor for disease-specific survival (hazard ratio = 2.469, P = 0.013). To determine whether the AhR pathway can be activated in the T24 UC cell line, we examined the expression of cytochrome P450 (CYP) 1A1 and CYP1B1, which are target genes of the AhR pathway, following exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a ligand of AhR. TCDD treatment upregulated the expression levels of AhR, CYP1A1 and CYP1B1. TCDD enhanced T24 cell invasion associated with the upregulation of matrix metalloproteinase (MMP)-1 and MMP-9. Furthermore, targeting AhR messenger RNA (mRNA) expression in T24 cells with small interfering RNA (siRNA) downregulated the mRNA expression of AhR, CYP1A1, CYP1B1, MMP-1, MMP-2 and MMP-9; furthermore, the cells transfected with siRNA for AhR showed decreased invasion activity in comparison with the cells transfected with a non-targeting siRNA. Our results therefore suggest that AhR plays a role in the invasiveness of UC cells and can serve as a marker for the prognosis of upper urinary tract UC.
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PMID:Activation of the aryl hydrocarbon receptor pathway enhances cancer cell invasion by upregulating the MMP expression and is associated with poor prognosis in upper urinary tract urothelial cancer. 1975 61


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