Autocrine control of vitamin D metabolism in synovial cells from arthritic patients.
(9/1365)
OBJECTIVE: This study was designed to investigate whether 1, 25-dihydroxyvitamin D3 (1,25-(OH)2D3), produced by activated synovial fluid macrophages, promotes its own catabolism by upregulating vitamin D-24-hydroxylase (24-OHase) in synovial fibroblasts through a vitamin D receptor (VDR) mediated mechanism. METHODS: Synovial macrophages and fibroblasts were derived from patients with rheumatoid arthritis. Expression of VDR and 24-OHase mRNAs was determined using in situ hybridisation. Vitamin D hydroxylase activity was determined by incubating cells with [3H]-25-(OH)D3, or [3H]-1,25-(OH)2D3, and metabolite synthesis quantified using high performance liquid chromatography. RESULTS: 1, 25-(OH)2D3 increased expression of mRNA for both VDR and 24-OHase in fibroblasts by approximately threefold over 24 hours. 1,25-(OH)2D3 increased fibroblast 24-OHase activity, yielding 24-hydroxylated, and more polar, metabolites. In co-culture, fibroblasts were able to catabolise macrophage derived 1,25-(OH)2D3. CONCLUSIONS: 1, 25-(OH)2D3 is produced by macrophages in vitro at biologically relevant concentrations and can increase its own catabolism by synovial fibroblasts; this effect is probably mediated via upregulation of both synovial fibroblast VDR and 24-OHase. (+info)
Differential regulation of direct repeat 3 vitamin D3 and direct repeat 4 thyroid hormone signaling pathways by the human TR4 orphan receptor.
(10/1365)
In situ hybridization analysis demonstrated that abundant testicular orphan receptor (TR4) transcripts were detected in kidney, intestine, and bone, which are vitamin D3 target organs. Cell transfection studies also demonstrated that the expression of the vitamin D3 target gene, 25-hydroxyvitamin D3 24-hydroxylase, can be repressed by TR4 through high affinity binding (Kd = 1.32 nM) to the direct repeat 3 vitamin D3 receptor response element (DR3VDRE). This TR4-mediated repression of DR3VDRE is in contrast to our earlier report that TR4 could induce thyroid hormone target genes containing a direct repeat 4 thyroid hormone response element (DR4T3RE). Electrophoretic mobility shift assay using several TR4 monoclonal antibodies when combined with either TR4-DR3VDRE or TR4-DR4T3RE showed a distinct supershifted pattern, and proteolytic analysis further demonstrated distinct digested peptides with either TR4-DR3VDRE or TR4-DR4T3RE. These results may therefore suggest that TR4 can adapt to different conformations once bound to DR3VDRE or DR4T3RE. The consequence of these different conformations of TR4-DR3VDRE and TR4-DR4T3RE may allow each of them to recruit different coregulators. The differential repression of TR4-mediated DR3VDRE and DR4T3RE transactivation by the receptor interacting protein 140, a TR4 coregulator, further strengthens our hypothesis that the specificity of gene regulation by TR4 can be modulated by protein-DNA and protein-protein interactions. (+info)
Antagonistic action of novel 1alpha,25-dihydroxyvitamin D3-26, 23-lactone analogs on differentiation of human leukemia cells (HL-60) induced by 1alpha,25-dihydroxyvitamin D3.
(11/1365)
We examined the effects of two novel 1alpha,25-dihydroxyvitamin D3-26,23-lactone (1alpha,25-lactone) analogues on human promyelocytic leukemia cell (HL-60) differentiation using the evaluation system of the vitamin D nuclear receptor (VDR)/vitamin D-responsive element (DRE)-mediated genomic action stimulated by 1alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3) and its analogues. We found that the 1alpha,25-lactone analogues (23S)-25-dehydro-1alpha-hydroxyvitamin-D3-26,23-lactone (TEI-9647), and (23R)-25-dehydro-1alpha-hydroxyvitamin-D3-26,23-lactone (TEI-9648) bound much more strongly to the VDR than the natural (23S, 25R)-1alpha,25(OH)2D3-26,23-lactone, but did not induce cell differentiation even at high concentrations (10(-6) M). Intriguingly, the differentiation of HL-60 cells induced by 1alpha,25(OH)2D3 was inhibited by either TEI-9647 or TEI-9648 but not by the natural lactone. In contrast, retinoic acid or 12-O-tetradecanoylphorbol-13-acetate-induced HL-60 cell differentiation was not blocked by TEI-9647 or TEI-9648. In separate studies, TEI-9647 (10(-7) M) was found to be an effective antagonist of both 1alpha,25(OH)2D3 (10(-8) M) mediated induction of p21(WAF1, CIP1) in HL-60 cells and activation of the luciferase reporter assay in COS-7 cells transfected with cDNA containing the DRE of the rat 25(OH)D3-24-hydroxylase gene and cDNA of the human VDR. Collectively the results strongly suggest that our novel 1alpha,25-lactone analogues, TEI-9647 and TEI-9648, are specific antagonists of 1alpha, 25(OH)2D3 action, specifically VDR/DRE-mediated genomic action. As such, they represent the first examples of antagonists, which act on the nuclear VDR. (+info)
A case of aldosterone-producing adenoma with severe postoperative hyperkalemia.
(12/1365)
It is known that some patients with primary aldosteronism show postoperative hyperkalemia, which is due to inability of the adrenal gland to secrete sufficient amounts of aldosterone. However, hyperkalemia is generally neither severe nor prolonged, in which replacement therapy with mineralocorticoid is seldom necessary. We report a case of a 46-year-old woman with an aldosterone-producing adenoma associated with severe postoperative hyperkalemia. After unilateral adrenalectomy, the patient showed episodes of severe hyperkalemia for four months, which required not only cation-exchange resin, but also mineralocorticoid replacement. Plasma aldosterone concentration (PAC) was low, although PAC was increased after rapid ACTH test. Histological examination indicated the presence of adrenocortical tumor and paradoxical hyperplasia of zona glomerulosa in the adjacent adrenal. Immunohistochemistry demonstrated that the enzymes involved in aldosterone synthesis, such as cholesterol side chain cleavage (P-450scc), 3beta-hydroxysteroid dehydrogenase (3beta-HSD), and 21-hydroxylase (P-450c21), or the enzyme involved in glucocorticoid synthesis, 11beta-hydroxylase (P-450c11beta), were expressed in the tumor, but they were completely absent in zona glomerulosa of the adjacent adrenal. These findings were consistent with the patterns of primary aldosteronism. Serum potassium level was gradually decreased with concomitant increase in PAC. These results suggest that severe postoperative hyperkalemia of the present case was attributable to severe suppression of aldosterone synthesis in the adjacent and contralateral adrenal, which resulted in slow recovery of aldosterone secretion. It is plausible that aldosterone synthesis of adjacent and contralateral adrenal glands is severely impaired in some cases with primary aldosteronism, as glucocorticoid synthesis in Cushing syndrome. (+info)
Lack of single-dose disulfiram effects on cytochrome P-450 2C9, 2C19, 2D6, and 3A4 activities: evidence for specificity toward P-450 2E1.
(13/1365)
Disulfiram and its primary metabolite diethyldithiocarbamate are effective mechanism-based inhibitors of cytochrome P-450 2E1 (CYP2E1)1 in vitro. Single-dose disulfiram diminishes CYP2E1 activity in vivo and has been used to identify CYP2E1 participation in human drug metabolism and prevent CYP2E1-mediated toxification. Specificity of single-dose disulfiram toward CYP2E1 in vivo, however, remains unknown. This investigation determined single-dose disulfiram effects on human CYP 2C9, 2C19, 2D6, and 3A4 activities in vivo. In four randomized crossover experiments, volunteers received isoform-selective probes (oral tolbutamide, mephenytoin, dextromethorphan, or i.v. midazolam) on two occasions, 10 h after oral disulfiram or after no pretreatment (controls). Plasma and/or urine parent and/or metabolite concentrations were measured by HPLC or gas chromatography-mass spectrometry. CYP2C9, 2C19, 2D6, and 3A4 activities were determined from the tolbutamide metabolic ratio, 4'-hydroxymephenytoin excretion, and dextromethorphan/dextrorphan ratios in urine and midazolam systemic clearance, respectively. Midazolam clearance (670 +/- 190 versus 700 +/- 240 ml/min, disulfiram versus controls), dextromethorphan/dextrorphan metabolic ratio (0.013 +/- 0.033 versus 0.015 +/- 0.035), 4'-hydroxymephenytoin excretion (122 +/- 22 versus 128 +/- 25 micromol), and tolbutamide metabolite excretion (577 +/- 157 versus 610 +/- 208 micromol) were not significantly altered by disulfiram pretreatment, although the tolbutamide metabolic ratio was slightly diminished after disulfiram (60 +/- 17 versus 81 +/- 40, p <.05). Results show that single-dose disulfiram does not cause clinically significant inhibition of human CYP2C9, 2C19, 2D6, and 3A4 activities in vivo. When single-dose disulfiram is used as an in vivo probe for P-450, inhibition of drug metabolism suggests selective involvement of CYP2E1. Single-dose disulfiram should not cause untoward drug interactions from inhibition of other P-450 isoforms. (+info)
Analyses of bronchial bulky DNA adduct levels and CYP2C9, GSTP1 and NQO1 genotypes in a Hungarian study population with pulmonary diseases.
(14/1365)
Carcinogen-DNA adducts may represent an intermediate end-point in the carcinogenic cascade and may reflect exposure to chemical carcinogens, as well as susceptibility and, ultimately, cancer risk. Interindividual variability in activity of enzymes involved in the metabolism of polycyclic aromatic hydrocarbons to mutagenic diol epoxides may predict adduct levels and, indirectly, lung cancer risk. Using 32P-postlabeling methods, the levels of bulky DNA adducts were determined in macroscopically normal bronchial tissues obtained from resected lobes of 143 Hungarian patients with lung malignancy and other pulmonary conditions. DNA from normal tissue was also evaluated for polymorphisms in cytochrome P450 2C9 (CYP2C9) at two sites, codons 144 (Arg/Cys) and 359 (Ile/Leu), for glutathione S-transferase P1 (GSTP1) at codon 105 and for NAD(P)H:quinone oxidoreductase (NQO1) at codon 187 (Pro/Ser). Using the Mann-Whitney U-test and analysis of variance, levels of adducts were evaluated in relation to variant genotypes, separately for smokers and non-smokers. As previously reported, bulky DNA adduct levels in smokers (n = 104) were estimated to be 54% higher than in non-smokers (n = 39) (8.6 +/- 4.2 versus 5.6 +/- 3.3 per 10(8) nucleotides, respectively, P < 0.01). Adduct levels were 16-29% higher in individuals with the homozygous Ile359/Ile359 CYP2C9 allele than in those heterozygous for the variant allele (Ile359/Leu359) [8.8 +/- 4.3 (n = 84) versus 7.6 +/- 3.5 (n = 20) for smokers and 5.8 +/- 3.5 (n = 32) versus 4.5 +/- 1.3 (n = 7) for non-smokers], although differences were not statistically significant. There were no clear differences in adduct levels in relation to genotypes of NQO1 or GSTP1. Although numbers of patients in this study are large in relation to many studies of carcinogen-DNA adducts, it is still possible that significant differences were not noted for polymorphisms in xenobiotic metabolizing enzymes due to relatively small numbers in stratified data. (+info)
Molecular cloning, characterization, and promoter analysis of the human 25-hydroxyvitamin D3-1alpha-hydroxylase gene.
(15/1365)
The human 25-hydroxyvitamin D3-1alpha-hydroxylase (1alpha-OHase) gene has been cloned. It contained nine exons and eight introns spanning approximately 6.5 kb and a 1.4-kb 5'-flanking region. The 5'-flanking region contains consensus or highly conserved sequences for TATA, Pu, and CCAAT boxes, four cAMP response elements, two activator protein-1 (AP-1) response elements, two AP-2 response elements, three specific protein-1 (Sp1) response elements, and four NF-kappaB binding sites, but no vitamin D response element. By using luciferase reporter gene constructs of truncated forms of the 1alpha-OHase promoter transfected into a modified pig kidney cell line, AOK-B50, we identified regulatory regions of the 1.4-kb 1alpha-OHase promoter for parathyroid hormone 1-34 [PTH(1-34)], forskolin, and 1,25-hydroxyvitamin D3 [1,25(OH)2D3]. The 1.4-kb 1alpha-OHase promoter (AN1) modestly (1.7-fold) induced luciferase activity, whereas 1,100- (AN2), 827- (AN3), 672- (AN4), 463-(AN5), and 363-bp (AN6)-truncated promoters greatly stimulated luciferase activity by 494-fold, 18.4-fold, 55.3-fold, 643-fold, and 56.4-fold, respectively. PTH(1-34) and forskolin stimulated the activity of all constructs to varying degrees with significantly greater responsiveness for both compounds on AN2 and AN5. 1,25(OH)2D3 suppressed PTH(1-34)-induced activity on AN2 and AN5 constructs by 58% and 52%, respectively, but had no effect on the other constructs. These studies characterize the regulatory regions of the human 1alpha-OHase gene and provide insight into the physiologic basis for regulation of the expression of this gene by PTH and 1,25(OH)2D3. (+info)
RNA molecules containing exons originating from different members of the cytochrome P450 2C gene subfamily (CYP2C) in human epidermis and liver.
(16/1365)
Reverse transcription-PCR analysis in human epidermis, using primers from CYP2C18 and CYP2C19, revealed products containing combinations between canonically defined exons of these two genes. The major RNA species identified contained 2C18 exon 8 spliced with 2C19 exon 2. However, the terminal exons 1 and 9 were never detected in any of these composite molecules. When similar experiments were performed with liver RNA, exons 1 and 9 of both 2C18 and 2C19 were readily identified in composite 2C18/2C19 RNAs. Moreover, molecules containing 2C9 sequences spliced with 2C18 exons were also detected. These findings suggest that during the process of RNA splicing of the 2C transcripts, various exon juxtaposition events may occur, including combinations between exons of distinct genes. However, the frequency of these events is quite low and the levels of the composite RNA molecules are generally estimated at less than one molecule per cell. Since the order of these genes on chromosome 10q24 is CYP2C18 - CYP2C19 - CYP2C9, it is conceivable that the composite RNAs may result from multiple canonical and inverse splicing events of a long pre-mRNA that encompasses the three genes. However, these molecules could also be rationalized as being the products of trans splicing phenomena between distinct pre-mRNAs. (+info)