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Beumer JH Parise RA Kanterewicz B Petkovich M D'Argenio DZ Hershberger PA 《Steroids》2012,77(5):477-483
The vitamin D(3) catabolizing enzyme, CYP24, is frequently over-expressed in tumors, where it may support proliferation by eliminating the growth suppressive effects of 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)). However, the impact of CYP24 expression in tumors or consequence of CYP24 inhibition on tumor levels of 1,25(OH)(2)D(3)in vivo has not been studied due to the lack of a suitable quantitative method. To address this need, an LC-MS/MS assay that permits absolute quantitation of 1,25(OH)(2)D(3) in plasma and tumor was developed. We applied this assay to the H292 lung tumor xenograft model: H292 cells eliminate 1,25(OH)(2)D(3) by a CYP24-dependent process in vitro, and 1,25(OH)(2)D(3) rapidly induces CYP24 expression in H292 cells in vivo. In tumor-bearing mice, plasma and tumor concentrations of 1,25(OH)(2)D(3) reached a maximum of 21.6 and 1.70ng/mL, respectively, following intraperitoneal dosing (20μg/kg 1,25(OH)(2)D(3)). When co-administered with the CYP24 selective inhibitor CTA091 (250μg/kg), 1,25(OH)(2)D(3) plasma levels increased 1.6-fold, and tumor levels increased 2.6-fold. The tumor/plasma ratio of 1,25(OH)(2)D(3) AUC was increased 1.7-fold by CTA091, suggesting that the inhibitor increased the tumor concentrations of 1,25(OH)(2)D(3) independent of its effects on plasma disposition. Compartmental modeling of 1,25(OH)(2)D(3) concentration versus time data confirmed that: 1,25(OH)(2)D(3) was eliminated from plasma and tumor; CTA091 reduced the elimination from both compartments; and that the effect of CTA091 on tumor exposure was greater than its effect on plasma. These results provide evidence that CYP24-expressing lung tumors eliminate 1,25(OH)(2)D(3) by a CYP24-dependent process in vivo and that CTA091 administration represents a feasible approach to increase tumor exposure to 1,25(OH)(2)D(3). 相似文献
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Omdahl JL Swamy N Serda R Annalora A Berne M Rayb R 《The Journal of steroid biochemistry and molecular biology》2004,(1-5):159-162
25-hydroxyvitamin D(3)- or 1alpha,25-dihydroxyvitamin D(3)-24R-hydroxylase (cytochromeP450C24 or CYP24) has a dual role of removing 25-OH-D(3) from circulation and excess 1,25(OH)(2)D(3) from kidney. As a result, CYP24 is an important multifunctional regulatory enzyme that maintains essential tissue-levels of Vitamin D hormone. As a part of our continuing interest in structure-function studies characterizing various binding proteins in the Vitamin D endocrine system, we targeted recombinant rat CYP24 with a radiolabeled 25-OH-D(3) affinity analog, and showed that the 25-OH-D(3)-binding site was specifically labeled by this analog. An affinity labeled sample of CYP24 was subjected to MS/MS analysis, which identified Ser57 as the only amino acid residue in the entire length of the protein that was covalently modified by this analog. Site-directed mutagenesis was conducted to validate the role of Ser57 towards substrate-binding. S57A mutant displayed significantly lower binding capacity for 25-OH-D(3) and 1,25(OH)(2)D(3). On the other hand, S57D mutant strongly enhanced binding for the substrates and conversion of 1,25(OH)(2)D(3) to calcitroic acid. The affinity probe was anchored via the 3-hydroxyl group of 25-OH-D(3). Therefore, these results suggested that the 3-hydroxyl group (of 25-OH-D(3) and 1,25(OH)(2)D(3)) in the S57D mutant could be stabilized by hydrogen bonding or a salt bridge leading to enhanced substrate affinity and metabolism. 相似文献
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1alpha-Hydroxy-23 carboxy-24,25,26,27-tetranorvitamin D(3) (calcitroic acid) is known to be the major water-soluble metabolite produced during the deactivation of 1,25-(OH)(2)D(3). This deactivation process is carried out exclusively by the multicatalytic enzyme CYP24 and involves a series of oxidation reactions at C(24) and C(23) leading to side-chain cleavage and, ultimately, formation of the calcitroic acid. Like 1,25-(OH)(2)D(3), 1alpha,25-1,25-(OH)(2)D(2) is also known to undergo side-chain oxidation and side-chain cleavage to form calcitroic acid (Zimmerman et al. [2001]. 1,25-(OH)(2)D(2) differs from 1,25-(OH)(2)D(3) by the presence of a double bond at C(22) and a methyl group at C(24). To date, there have been no studies detailing the participation of CYP24 in the production of calcitroic acid from 1,25-(OH)(2)D(2). We, therefore, studied the metabolism of 1,25-(OH)(2)D(3) and 1,25-(OH)(2)D(2) using a purified rat CYP24 system. Lipid and aqueous-soluble metabolites were prepared for characterization. Aqueous-soluble metabolites were subjected to reverse-phase high-pressure liquid chromatography (HPLC) analysis. As expected, 1,23(OH)(2)-24,25,26,27-tetranor D and calcitroic acid were the major lipid and aqueous-soluble metabolites, respectively, when 1,25-(OH)(2)D(3) was used as substrate. However, when 1,25-(OH)(2)D(2) was used as substrate, 1,24(R),25-(OH)(3)D(2) was the major lipid-soluble metabolite with no evidence for the production of either 1,23(OH)(2)-24,25,26,27-tetranor D or calcitroic acid. Apparently, the CYP24 was able to 24-hydroxylate 1,25-(OH)(2)D(2), but was unable to effect further changes, which would result in side-chain cleavage. These data suggest that the presence of either the double bond at C(22) or the C(24) methyl group impedes the metabolism of 1,25-(OH)(2)D(2) to calcitroic acid by CYP24 and that enzymes other than CYP24 are required to effect this process. 相似文献
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Schuster I Egger H Bikle D Herzig G Reddy GS Stuetz A Stuetz P Vorisek G 《Steroids》2001,66(3-5):409-422
Human keratinocytes convert 25(OH)D(3) to hormonally active 1alpha,25(OH)(2)D(3) and respond to its antiproliferative/prodifferentiating action in vitro and in vivo. Levels and activity of 1alpha,25(OH)(2)D(3) are short-lived. 1alpha,25(OH)(2)D(3) induces 24-hydroxylase (CYP24) that rapidly metabolizes the hormone, yielding a cascade of side-chain oxidized products and this eventually results in the loss of activity. Aiming at stabilizing the levels of active hormone, we have searched for potent, selective inhibitors of CYP24. Selective inhibition was crucial in order to avoid impairment of 1alpha,25(OH)(2)D(3) synthesis, catalyzed by 1alpha-hydroxylase - a related member of cytochrome P-450 (CYP) superfamily. We describe here the testing protocol, using primary human keratinocyte cultures as an appropriate source of CYP24 and 1alpha-hydroxylase, (3)H-25(OH)D(3) (at physiological concentrations) as substrate and sensitive HPLC techniques to analyze the complex metabolite profiles. Four hundred potential inhibitors were screened by this method; most of them were synthesized in our laboratory. These compounds (entitled azoles) were capable of direct binding to the heme iron and of additional interactions with other parts of the enzyme. In this paper, we present VID400 and SDZ 89-443, as first examples of powerful selective CYP24 inhibitors. As anticipated, these compounds increased the levels of 1alpha-hydroxylated products generated from (3)H-25(OH)D(3) and extended their lifetime. Importantly, blocking of 24-hydroxylation led to a switch in metabolism, namely to preferential conversion of 1alpha,25(OH)(2)D(3) to 1alpha,25(OH)(2)-3epi-D(3). As spin-off from our program, selective inhibitors of 1alpha-hydroxylase were also found (e.g. SDZ 88-357). Using (3)H-25(OH)D(3) as substrate in the absence of SDZ 88-357, CYP24 showed high preference for freshly generated 1alpha-hydroxylated metabolites over abundant 25(OH)D(3). In the presence of SDZ 88-357, we noticed a great increase in 24-hydroxylation of (3)H-25(OH)D(3). Besides their use as valuable tools in elucidating regulatory mechanisms, inhibitors of VD hydroxylases may give rise to novel therapeutic strategies, especially in defects of cell growth and differentiation. 相似文献
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Clinckspoor I Hauben E Verlinden L Van den Bruel A Vanwalleghem L Vander Poorten V Delaere P Mathieu C Verstuyf A Decallonne B 《The journal of histochemistry and cytochemistry》2012,60(7):502-511
1,25-Dihydroxyvitamin D(3) (1,25(OH)(2)D(3)), the active form of vitamin D, mediates antitumor effects in various cancers. The expression of key players in vitamin D signaling in thyroid tumors was investigated. Vitamin D receptor (VDR) and CYP27B1 and CYP24A1 (respectively activating and catabolizing vitamin D) expression was studied (RT-PCR, immunohistochemistry) in normal thyroid, follicular adenoma (FA), differentiated thyroid cancer (DTC) consisting of the papillary (PTC) and follicular (FTC) subtype, and anaplastic thyroid cancer (ATC). VDR, CYP27B1, and CYP24A1 expression was increased in FA and DTC compared with normal thyroid. However, in PTC with lymph node metastasis, VDR and CYP24A1 were decreased compared with non-metastasized PTC. In ATC, VDR expression was often lost, whereas CYP27B1/CYP24A1 expression was comparable to DTC. Moreover, ATC with high Ki67 expression (>30%) or distant metastases at diagnosis was characterized by more negative VDR/CYP24A1/CYP27B1 staining. In conclusion, increased expression of key players involved in local 1,25(OH)(2)D(3) signaling was demonstrated in benign and differentiated malignant thyroid tumors, but a decrease was observed for local nodal and especially distant metastasis, suggesting a local antitumor response of 1,25(OH)(2)D(3) in early cancer stages. These findings advocate further studies with 1,25(OH)(2)D(3) and analogs in persistent and recurrent iodine-refractory DTC. 相似文献
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Ren S Nguyen L Wu S Encinas C Adams JS Hewison M 《The Journal of biological chemistry》2005,280(21):20604-20611
Synthesis of the active form of vitamin D, 1,25-dihydroxyvitamin D (1,25-(OH)(2)D), by renal epithelial cells is tightly controlled during normal calcium homeostasis. By contrast, macrophage production of 1,25-(OH)(2)D is often dysregulated with potential hypercalcemic complications. We have postulated that this is due to abnormal catabolism of 1,25-(OH)(2)D by the feedback control enzyme, vitamin D-24-hydroxylase (CYP24). Using chick HD-11 and human THP-1 myelomonocytic cell lines, we have shown that macrophage-like cells express a splice variant of the CYP24 gene (CYP24-SV), which encodes a truncated protein. Compared with the holo-CYP24 gene product in chick and human cells (508 and 513 amino acids, respectively), the truncated CYP24-SV versions consisted of 351 and 372 amino acids. These CYP24-SV proteins retained intact substrate-binding domains but lacked mitochondrial targeting sequences and were therefore catalytically inactive. In common with CYP24, expression of the CYP24 variants was induced by 1,25-(OH)(2)D but without a concomitant rise in 24-hydroxylase activity. However, overexpression of CYP24-SV in HD-11 and THP-1 cells reduced synthesis of 1,25-(OH)(2) D (40-50%), whereas antisense CYP24-SV expression increased 1,25-(OH)(2)D production by 2-7-fold. These data suggest that alternative splicing of CYP24 leads to the generation of a dominant negative-acting protein that is catalytically dysfunctional. We theorize that expression of the CYP24-SV may contribute to the extracellular accumulation of 1,25(OH)(2)D in human health and disease. 相似文献
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Ramos-Lopez E Brück P Jansen T Pfeilschifter JM Radeke HH Badenhoop K 《The Journal of steroid biochemistry and molecular biology》2007,103(3-5):807-810
1,25(OH)(2)D(3) and 25(OH)D(3) have been associated with type 1 diabetes. Diverse enzymes are involved in the synthesis of these metabolites: the 25-Vitamin-D-hydroxylase (CYP2R1), the 25-hydroxyvitamin-D(3)-1-alpha-hydroxylase (CYP27B1) and the 25(OH)D(3)-24-hydroxylase (CYP24) among others. Serum levels of 25(OH)D(3) and 1,25(OH)(2)D(3) were investigated in type 1 diabetes patients (n=173) and the mRNA expression of the CYP2R1, CYP27B1 and CYP24 genes in type 1 diabetes patients (n=33) and healthy controls (n=23). These parameters were correlated with the -1260 (C/A) polymorphism in the CYP27B1 gene. Lower expression of CYP27B1 mRNA in comparison with healthy controls (1.7165 versus 1.7815, P=0.0268) was found. Additionally, patients carrying the genotype CC possessed a reduced amount of CYP27B1 mRNA compared to healthy controls (1.6855 versus 1.8107, respectively, P=0.0220). The heterozygosity rate of the -1260 C/A polymorphism was more frequent in patients with normal levels of 1,25(OH)(2)D(3) (> or =19.9 pmol/ml) than in whose with a level of less than 19.9 pmol/ml (46.7% versus 22.2%, P=0.0134). No correlation with serum levels of 25(OH)D(3) was found. Thus, CYP27B1 gene could play a functional role in the pathogenesis of type 1 diabetes through modulation of its mRNA expression and influence serum levels of 1,25(OH)(2)D(3) via the -1260 C/A polymorphism. 相似文献
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Nguyen TM Lieberherr M Fritsch J Guillozo H Alvarez ML Fitouri Z Jehan F Garabédian M 《The Journal of biological chemistry》2004,279(9):7591-7597
If both rapid and genomic pathways may co-exist in the same cell, the involvement of the nuclear vitamin D receptor (VDR) in the rapid effects of 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) remains unclear. We therefore studied rapid and long term effects of 1,25-(OH)(2)D(3) in cultured skin fibroblasts from three patients with severe vitamin D-resistant rickets and one age-matched control. Patients bear homozygous missense VDR mutations that abolished either VDR binding to DNA (patient 1, mutation K45E) or its stable ligand binding (patients 2 and 3, mutation W286R). In patient 1 cells, 1,25-(OH)(2)D(3) (1 pm-10 nm) had no effect on either intracellular calcium or 24-hydroxylase (enzyme activity and mRNA expression). In contrast, cells bearing the W286R mutation had calcium responses to 1,25-(OH)(2)D(3) (profile and magnitude) and 24-hydroxylase responses to low (1 pm-100 pm) 1,25-(OH)(2)D(3) concentrations (activity, CYP24, and ferredoxin mRNAs) similar to those of controls. The blocker of Ca(2+) channels, verapamil, impeded both rapid (calcium) and long term (24-hydroxylase activity, CYP24, and ferredoxin mRNAs) responses in patient and control fibroblasts. The MEK 1/2 kinase inhibitor PD98059 also blocked the CYP24 mRNA response. Taken together, these results suggest that 1,25-(OH)(2)D(3) rapid effects require the presence of VDR and control, in part, the first step of 1,25-(OH)(2)D(3) catabolism via increased mRNA expression of the CYP24 and ferredoxin genes in the 24-hydroxylase complex. 相似文献
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Astecker N Bobrovnikova EA Omdahl JL Gennaro L Vouros P Schuster I Uskokovic MR Ishizuka S Wang G Reddy GS 《Archives of biochemistry and biophysics》2004,431(2):261-270
Recently, 25-hydroxyvitamin D3-24-hydroxylase (CYP24A1) has been shown to catalyze not only hydroxylation at C-24 but also hydroxylations at C-23 and C-26 of the secosteroid hormone 1alpha, 25-dihydroxyvitamin D3 (1alpha,25(OH)2D3). It remains to be determined whether CYP24A1 has the ability to hydroxylate vitamin D3 compounds at C-25. 1alpha,24(R)-dihydroxyvitamin D3 (1alpha,24(R)(OH)2D3) is a non-25-hydroxylated synthetic vitamin D3 analog that is presently being used as an antipsoriatic drug. In the present study, we investigated the metabolism of 1alpha,24(R)(OH)2D3 in human keratinocytes in order to examine the ability of CYP24A1 to hydroxylate 1alpha,24(R)(OH)2D3 at C-25. The results indicated that keratinocytes metabolize 1alpha,24(R)(OH)2D3 into several previously known both 25-hydroxylated and non-25-hydroxylated metabolites along with two new metabolites, namely 1alpha,23,24(OH)3D3 and 1alpha,24(OH)2-23-oxo-D3. Production of the metabolites including the 25-hydroxylated ones was detectable only when CYP24A1 activity was induced in keratinocytes 1alpha,25(OH)2D3. This finding provided indirect evidence to indicate that CYP24A1 catalyzes C-25 hydroxylation of 1alpha,24(R)(OH)2D3. The final proof for this finding was obtained through our metabolism studies using highly purified recombinant rat CYP24A1 in a reconstituted system. Incubation of this system with 1alpha,24(R)(OH)2D3 resulted in the production of both 25-hydroxylated and non-25-hydroxylated metabolites. Thus, in our present study, we identified CYP24A1 as the main enzyme responsible for the metabolism of 1alpha,24(R)(OH)2D3 in human keratinocytes, and provided unequivocal evidence to indicate that the multicatalytic enzyme CYP24A1 has the ability to hydroxylate 1alpha,24(R)(OH)2D3 at C-25. 相似文献
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CYP24A1 is the cytochrome P450 component of the 25-hydroxyvitamin D(3)-24-hydroxylase enzyme that catalyzes the conversion of 25-hydroxyvitamin D(3) (25-OH-D(3)) and 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)) into 24-hydroxylated products, which constitute the degradation of the vitamin D molecule. This review focuses on recent data in the CYP24A1 field, including biochemical, physiological and clinical developments. Notable among these are: the first crystal structure for rat CYP24A1; mutagenesis studies which change the regioselectivity of the enzyme; and the finding that natural inactivating mutations of CYP24A1 cause the genetic disease idiopathic infantile hypercalcemia (IIH). The review also discusses the emerging correlation between rising serum phosphate/FGF-23 levels and increased CYP24A1 expression in chronic kidney disease, which in turn underlies accelerated degradation of both serum 25-OH-D(3) and 1,25-(OH)(2)D(3) in this condition. This review concludes by evaluating the potential clinical utility of blocking this enzyme with CYP24A1 inhibitors in various disease states. 相似文献
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Puri S Bansal DD Uskoković MR MacGregor RR 《American journal of physiology. Endocrinology and metabolism》2000,278(2):E293-E301
We investigated the effects of 1,25-dihydroxyvitamin D(3) [25(OH)(2)D(3)] on tissue plasminogen activator (tPA) secretion from primary cultures of rat heart microvascular cells. After an initial 5-day culture period, cells were treated for 24 h with 1,25(OH)(2)D(3) and several of its analogs. The results showed that 1,25(OH)(2)D(3) induced tPA secretion at 10(-10) to 10(-16) M. A less calcemic analog, Ro-25-8272, and an analog that binds the vitamin D receptor but is ineffective at perturbing Ca(2+) channels, Ro-24-5531, were approximately 10% as active as 1,25(OH)(2)D(3). An analog that binds the vitamin D receptor poorly but is an effective Ca(2+) channel agonist, Ro-24-2287, required approximately 10(-13) M to induce tPA secretion. Combinations of Ro-24-5531 and Ro-24-2287 were approximately as potent as 1,25(OH)(2)D(3). Treatment of the cells with BAY K 8644 or thapsigargin also increased tPA secretion, suggesting that increased cytosolic calcium concentration ([Ca(2+)]) induces tPA secretion. The results suggested that the sensitivity of the tPA secretory response of microvascular cells to 1,25(OH)(2)D(3) was due in part to generation of a vitamin D-depleted state in vitro and in part to synergistic effects of 1,25(OH)(2)D(3) on two different induction pathways of tPA release. 相似文献