The effect of uremic plasma ultrafiltrate on calcitriol synthesis was investigated. Renal 1 alpha-hydroxylase activity was measured in normal rats infused for 20 h with 20 mL of normal or uremic plasma ultrafiltrate. Renal 1 alpha-hydroxylase activity was determined by the generation of calcitriol measured 5, 10, 20, and 30 min after the reaction was initiated by the addition of cold 25(OH)D3. The activity was significantly lower in rats infused with uremic plasma ultrafiltrate. Kidney homogenates preincubated for 3 h with uremic plasma ultrafiltrate also had significantly lower renal 1 alpha-hydroxylase activity than did those preincubated with normal plasma ultrafiltrate. In addition, the effect of the putative uremic toxin, guanidinosuccinic acid (GSA), on renal 1 alpha-hydroxylase activity was studied. Normal rats infused for 20 h with 20 mL of saline solution containing 1.5 mg/dL of GSA had significantly lower renal 1 alpha-hydroxylase activity than did rats infused with normal saline. The enzyme activity was also lower in kidney homogenates preincubated for 3 h with 4 mg/dL of GSA. Enzyme kinetic analysis revealed that the inhibition of renal 1 alpha-hydroxylase by GSA was noncompetitive. It was concluded that uremic plasma contains substances that directly inhibit renal 1 alpha-hydroxylase activity. (+info)
Cholesterol feeding of mice expressing cholesterol 7alpha-hydroxylase increases bile acid pool size despite decreased enzyme activity.
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Dietary cholesterol regulation of cholesterol 7alpha-hydroxylase (Cyp7a1), the rate-limiting enzyme in the classical pathway of bile acid synthesis, has been implicated in plasma cholesterol responsiveness. In the current study, the effects of 0.0% and 0.5% cholesterol diets were examined in Cyp7a1 knockout (KO), heterozygous Cyp7a1 KO (Het), and human Cyp7a1 transgenic mice on the mouse Cyp7a1 KO background (Tg+KO). We confirmed previous findings that dietary cholesterol increased mouse Cyp7a1 activity in Het mice but decreased human Cyp7a1 activity in Tg+KO mice. However, in both Het and Tg+KO mice, dietary cholesterol increased bile acid pool size (36% and 72%, respectively) and fecal bile acid excretion (2.2- and 3.6-fold, respectively). The expression of cholesterol 27-hydroxylase (Cyp27), the major enzyme of the alternative pathway of bile acid synthesis, was not significantly different in cholesterol-fed KO, Het, or Tg+KO mice. Furthermore, dietary cholesterol had comparable effects on total plasma cholesterol and non-high-density lipoprotein cholesterol in KO, Het, and Tg+KO mice. Thus, in Tg+KO mice, dietary cholesterol regulates bile acid pool size, fecal bile acid excretion, and plasma cholesterol independently of Cyp7a1 activity. These results challenge the notion that dietary cholesterol regulation of Cyp7a1 is a major determinant of plasma cholesterol responsiveness. (+info)
Identification of a novel rat microsomal vitamin D3 25-hydroxylase.
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Vitamin D3 requires the 25-hydroxylation in the liver and the subsequent 1alpha-hydroxylation in the kidney to exert its biological activity. Vitamin D3 25-hydroxylation is hence an essential modification step for vitamin D3 activation. Until now, three cytochrome P450 molecular species (CYP27A1, CYP2C11, and CYP2D25) have been characterized well as vitamin D3 25-hydroxylases. However, their physiological role remains unclear because of their broad substrate specificities and low activities toward vitamin D3 relative to other substrates. In this study, we purified vitamin D3 25-hydroxylase from female rat liver microsomes. The activities of the purified fraction toward vitamin D3 and 1alpha-hydroxyvitamin D3 were 1.1 and 13 nmol/min/nmol of P450, respectively. The purified fraction showed a few protein bands in a 50-60-kDa range on SDS-PAGE, typical for a cytochrome P450. The tryptic peptide mass fingerprinting of a protein band (56 kDa) with matrix-assisted laser desorption ionization/time of flight mass spectrometry identified this band as CYP2J3. CYP2J3 was heterologously expressed in Escherichia coli. Purified recombinant CYP2J3 showed strong 25-hydroxylation activities toward vitamin D3 and 1alpha-hydroxyvitamin D3 with turnover numbers of 3.3 and 22, respectively, which were markedly higher than those of P450s previously characterized as 25-hydroxylases. Quantitative PCR analysis showed that CYP2J3 mRNA is expressed at a level similar to that of CYP27A1 without marked sexual dimorphism. These results strongly suggest that CYP2J3 is the principal P450 responsible for vitamin D3 25-hydroxylation in rat liver. (+info)
Analysis of the vitamin D system in basal cell carcinomas (BCCs).
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Using real-time PCR (LightCycler) and immunohistochemistry, we have analyzed expression of key components of the vitamin D system in basal cell carcinomas (BCCs) and normal human skin (NS). Increased VDR-immunoreactivity was demonstrated in BCCs using a streptavidin-peroxidase technique. RNA expression of vitamin D receptor (VDR) and of main enzymes involved in synthesis and metabolism of calcitriol (vitamin D-25-hydroxylase [25-OHase], 25-hydroxyvitamin D-1alpha-hydroxylase [1alpha-OHase], 1,25-dihydroxyvitamin D-24-hydroxylase [24-OHase]) was detected in BCCs and NS. Expression levels were determined as ratios between target genes (VDR, 1alpha-OHase, 25-OHase, 24-OHase) and the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as internal control. Median of mRNA ratios for VDR/GAPDH (BCCs: 16.54; NS: 0.00021), 1alpha-OHase/GAPDH (BCCs: 0.739; NS 0.000803) and 24-OHase/GAPDH (BCCs: 0.00585; NS 0.000000366) was significantly (Wilcoxon-Mann-Whitney U-test) elevated in BCCs. In contrast, median of mRNA ratio for 25-OHase/GAPDH (BCCs: 0.17; NS: 0.016) was not significantly altered in BCCs as compared to NS. Additionally, we report for the first time expression of 1alpha-OHase splice variants in BCCs and NS, that were detected using conventional RT-PCR. In conclusion, our findings provide supportive evidence for the concept that endogeneous synthesis and metabolism of vitamin D metabolites as well as VDR expression may regulate growth characteristics of BCCs. New vitamin D analogs that exert little calcemic side effects, their precursors, or inhibitors of 24-OHase may offer a new approach for the prevention or therapy of BCCs. The function of alternative transcripts of 1alpha-OHase that we describe here for the first time in BCCs and NS and their effect on activity level has to be investigated in future experiments. (+info)
Genetic evidence that the human CYP2R1 enzyme is a key vitamin D 25-hydroxylase.
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The synthesis of bioactive vitamin D requires hydroxylation at the 1 alpha and 25 positions by cytochrome P450 enzymes in the kidney and liver, respectively. The mitochondrial enzyme CYP27B1 catalyzes 1 alpha-hydroxylation in the kidney but the identity of the hepatic 25-hydroxylase has remained unclear for >30 years. We previously identified the microsomal CYP2R1 protein as a potential candidate for the liver vitamin D 25-hydroxylase based on the enzyme's biochemical properties, conservation, and expression pattern. Here, we report a molecular analysis of a patient with low circulating levels of 25-hydroxyvitamin D and classic symptoms of vitamin D deficiency. This individual was found to be homozygous for a transition mutation in exon 2 of the CYP2R1 gene on chromosome 11p15.2. The inherited mutation caused the substitution of a proline for an evolutionarily conserved leucine at amino acid 99 in the CYP2R1 protein and eliminated vitamin D 25-hydroxylase enzyme activity. These data identify CYP2R1 as a biologically relevant vitamin D 25-hydroxylase and reveal the molecular basis of a human genetic disease, selective 25-hydroxyvitamin D deficiency. (+info)
Differential expression of cholesterol hydroxylases in Alzheimer's disease.
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Cholesterol is eliminated from neurons by oxidization, which generates oxysterols. Cholesterol oxidation is mediated by the enzymes cholesterol 24-hydroxylase (CYP46A1) and cholesterol 27-hydroxylase (CYP27A1). Immunocytochemical studies show that CYP46A1 and CYP27A1 are expressed in neurons and some astrocytes in the normal brain, and CYP27A1 is present in oligodendrocytes. In Alzheimer's disease (AD), CYP46A1 shows prominent expression in astrocytes and around amyloid plaques, whereas CYP27A1 expression decreases in neurons and is not apparent around amyloid plaques but increases in oligodendrocytes. Although previous studies have examined the effects of synthetic oxysterols on the processing of amyloid precursor protein (APP), the actions of the naturally occurring oxysterols have yet to be examined. To understand the role of cholesterol oxidation in AD, we compared the effects of 24(S)- and 27-hydroxycholesterol on the processing of APP and analyzed the cell-specific expression patterns of the two cholesterol hydroxylases in the human brain. Both oxysterols inhibited production of Abeta in neurons, but 24(S)-hydroxycholesterol was approximately 1000-fold more potent than 27-hydroxycholesterol. The IC(50) of 24(S)-hydroxycholesterol for inhibiting Abeta secretion was approximately 1 nm. Both oxysterols induced ABCA1 expression with IC(50) values similar to that for inhibition of A beta secretion, suggesting the involvement of liver X receptor. Oxysterols also inhibited protein kinase C activity and APP secretion following stimulation of protein kinase C. The selective expression of CYP46A1 around neuritic plaques and the potent inhibition of APP processing in neurons by 24(S)-hydroxycholesterol suggests that CYP46A1 affects the pathophysiology of AD and provides insight into how polymorphisms in the CYP46A1 gene might influence the pathophysiology of this prevalent disease. (+info)
Adenosine A2A receptor occupancy stimulates expression of proteins involved in reverse cholesterol transport and inhibits foam cell formation in macrophages.
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Transport of cholesterol out of macrophages is critical for prevention of foam cell formation, the first step in the pathogenesis of atherosclerosis. Proteins involved in this process include cholesterol 27-hydroxylase and adenosine 5'-triphosphate-binding cassette transporter A1 (ABCA1). Proinflammatory cytokines and immune complexes (IC) down-regulate cholesterol 27-hydroxylase and impede cholesterol efflux from macrophages, leading to foam cell formation. Prior studies have suggested occupancy of the anti-inflammatory adenosine A2A receptor (A2AR) minimizes early atherosclerotic changes in arteries following injury. We therefore asked whether A2AR occupancy affects macrophage foam cell formation in response to IC and the cytokine interferon-gamma. We found that the selective A2AR agonist 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamido-adenosine (CGS-21680) inhibited foam cell formation in stimulated THP-1 human macrophages, and the effects of CGS-21680 were reversed by the selective A2AR antagonist 4-(2-[7-amino-2-(2-furyl) [1, 2, 4]triazolo[2,3-a] [1, 3, 5]triazin-5-ylamino]ethyl)phenol. In confirmation of the role of A2AR in prevention of foam cell formation, CGS-21680 also inhibited foam cell formation in cultured murine peritoneal macrophages but did not affect foam cell formation in A2AR-deficient mice. Agents that increase foam cell formation also down-regulate cholesterol 27-hydroxylase and ABCA1 expression. Therefore, we determined the effect of A2AR occupancy on expression of these reverse cholesterol transport (RCT) proteins and found that A2AR occupancy stimulates expression of message for both proteins. These results indicate that one mechanism for the antiatherogenic effects of adenosine is stimulation of the expression of proteins involved in RCT. These findings suggest a novel approach to the development of agents that prevent progression of atherosclerosis. (+info)
Transcriptional regulation of human CYP27 integrates retinoid, peroxisome proliferator-activated receptor, and liver X receptor signaling in macrophages.
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Cholesterol uptake and efflux are key metabolic processes associated with macrophage physiology and atherosclerosis. Peroxisome proliferator-activated receptor gamma (PPARgamma) and liver X receptor alpha (LXRalpha) have been linked to the regulation of these processes. It remains to be identified how activation of these receptors is connected and regulated by endogenous lipid molecules. We identified CYP27, a p450 enzyme, as a link between retinoid, PPARgamma, and LXR signaling. We show that the human CYP27 gene is under coupled regulation by retinoids and ligands of PPARs via a PPAR-retinoic acid receptor response element in its promoter. Induction of the enzyme's expression results in an increased level of 27-hydroxycholesterol and upregulation of LXR-mediated processes. Upregulated CYP27 activity also leads to LXR-independent elimination of CYP27 metabolites as an alternative means of cholesterol efflux. Moreover, human macrophage-rich atherosclerotic lesions have an increased level of retinoid-, PPARgamma-, and LXR-regulated gene expression and also enhanced CYP27 levels. Our findings suggest that nuclear receptor-regulated CYP27 expression is likely to be a key integrator of retinoic acid receptor-PPARgamma-LXR signaling, relying on natural ligands and contributing to lipid metabolism in macrophages. (+info)