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Glenville Jones - One of the best experts on this subject based on the ideXlab platform.
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CYP24A1 and slc34a1 pathogenic variants are uncommon in a canadian cohort of children with hypercalcemia or hypercalciuria
Hormone Research in Paediatrics, 2021Co-Authors: Isabelle Rousseaunepton, Glenville Jones, Martin Kaufmann, Karlpiet Schlingmann, Caroline Zuijdwijk, Karine Khatchadourian, Indra R Gupta, Daniele Pacaud, Maury Pinsk, Arati MokashiAbstract:OBJECTIVES Biallelic pathogenic variants in CYPA24A1 and SLC34A1 are causes of idiopathic infantile hypercalcemia. Pathogenic variants in both may also give rise to hypercalciuria with nephrocalcinosis or nephrolithiasis without previous hypercalcemia (renal group). Our objective was to examine the frequency of CYP24A1 or SLC34A1 variants in children with early hypercalcemia or late-onset hypercalciuria. METHOD Forty-one children from 7 centers across Canada were recruited. Local investigations were undertaken. The serum was evaluated by liquid chromatography tandem-mass spectrometry for the ratio of 25-hydroxyvitamin D3 to 24,25-dihydroxyvitamin D3, (25-OH-D3:24,25-(OH)2D3), an elevation pathognomonic for the loss of function of the CYP24A1 enzyme. Mutational analyses were undertaken. Family cascade screening was performed if pathogenic variants were detected in probands. RESULTS Twenty-nine children had early-onset hypercalcemia; none had elevated 25-OH-D3:24,25-(OH)2D3 or variants. Interestingly, 2 of 12 in the renal group had elevated 25-OH-D3:24,25-(OH)2D3 and presented as preadolescents. In case 1, cascade testing revealed a sibling and parent with asymptomatic pathogenic variants in CYP24A1. Four CYP24A1 pathogenic variants were identified in these 2 probands: 3 have been described in European populations, and 1 is a rare variant in exon 7 (c931delC) that is likely pathogenic. No SLC34A1 pathogenic variants were detected. CONCLUSION In Canada, pathogenic variants in CYP24A1 appear to manifest with late-onset hypercalciuria and its sequelae. The 25-OH-D3:24,25-(OH)2D3 ratio is an excellent tool for screening for biallelic pathogenic variants in CYP24A1. We confirm that cascade testing is important for these variants.
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a chromatin based mechanism controls differential regulation of the cytochrome p450 gene CYP24A1 in renal and non renal tissues
Journal of Biological Chemistry, 2019Co-Authors: Mark B Meyer, Glenville Jones, Martin Kaufmann, Seong Min Lee, Alex H Carlson, Nancy A Benkusky, Wesley J PikeAbstract:Cytochrome P450 family 27 subfamily B member 1 (CYP27B1) and CYP24A1 function to maintain physiological levels of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in the kidney. Renal Cyp27b1 and CYP24A1 expression levels are transcriptionally regulated in a highly reciprocal manner by parathyroid hormone (PTH), fibroblast growth factor 23 (FGF23), and 1,25(OH)2D3. In contrast, CYP24A1 regulation in nonrenal target cells (NRTCs) is limited to induction by 1,25(OH)2D3. Herein, we used ChIP-Seq analyses of mouse tissues to identify regulatory regions within the CYP24A1 gene locus. We found an extended region downstream of CYP24A1 containing a cluster of sites, termed C24-DS1, binding PTH-sensitive cAMP-responsive element–binding protein (CREB) and a cluster termed C24-DS2 binding the vitamin D receptor (VDR). VDR-occupied sites were present in both the kidney and NRTCs, but pCREB sites were occupied only in the kidney. We deleted each segment in the mouse and observed that although the overt phenotypes of both cluster deletions were unremarkable, RNA analysis in the C24-DS1–deleted strain revealed a loss of basal renal CYP24A1 expression, total resistance to FGF23 and PTH regulation, and secondary suppression of renal Cyp27b1; 1,25(OH)2D3 induction remained unaffected in all tissues. In contrast, loss of the VDR cluster in the C24-DS2–deleted strain did not affect 1,25(OH)2D3 induction of renal CYP24A1 expression yet reduced but did not eliminate CYP24A1 responses in NRTCs. We conclude that a chromatin-based mechanism differentially regulates CYP24A1 in the kidney and NRTCs and is essential for the specific functions of CYP24A1 in these two tissue types.
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25 hydroxyvitamin d 24 hydroxylase CYP24A1 its important role in the degradation of vitamin d
Archives of Biochemistry and Biophysics, 2012Co-Authors: Glenville Jones, David E. Prosser, Martin KaufmannAbstract:CYP24A1 is the cytochrome P450 component of the 25-hydroxyvitamin D3-24-hydroxylase enzyme that catalyzes the conversion of 25-hydroxyvitamin D3 (25-OH-D3) and 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) 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-D3 and 1,25-(OH)2D3 in this condition. This review concludes by evaluating the potential clinical utility of blocking this enzyme with CYP24A1 inhibitors in various disease states.
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CYP24A1 and kidney disease
Current Opinion in Nephrology and Hypertension, 2011Co-Authors: Martin Petkovich, Glenville JonesAbstract:Purpose of review Patients with chronic renal disease have elevated serum phosphate levels, elevated fibroblast-like growth factor 23 (FGF-23), and declining vitamin D status. These changes are related and may be responsible for elevated 25-hydroxyvitamin D-24-hydroxylase (CYP24A1) and dysfunctional vitamin D metabolism. This review focuses on the biochemistry and pathophysiology of CYP24A1 and the utility of blocking this enzyme with CYP24A1 inhibitors in chronic kidney disease (CKD) patients. Recent findings CYP24A1 is the cytochrome P450 enzyme that catalyzes the conversion of 25-hydroxyvitamin D3 (25-OHD3) and its hormonal form, 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], into 24-hydroxylated products targeted for excretion. The CYP24A1-null phenotype is consistent with the catabolic role of CYP24A1. A number of polymorphisms of CYP24A1 have recently been identified. New data from the uremic rat and humans suggest that dysfunctional vitamin D metabolism is due to changes in CYP24A1 expression caused by phosphate and FGF-23 elevations. Summary Changes in serum phosphate and FGF-23 levels in the CKD patient increase CYP24A1 expression resulting in decreased vitamin D status. Vitamin D deficiency may exacerbate defective calcium and phosphate homeostasis causing renal osteodystrophy and contribute to the other complications of renal disease. These findings argue for increased focus on correcting vitamin D deficiency in CKD patients by blocking CYP24A1 activity.
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Bioengineering Anabolic Vitamin D-25-Hydroxylase Activity into the Human Vitamin D Catabolic Enzyme, Cytochrome P450 CYP24A1, by a V391L Mutation
The Journal of biological chemistry, 2011Co-Authors: Martin Kaufmann, David E. Prosser, Glenville JonesAbstract:CYP24A1 is a mitochondrial cytochrome P450 (CYP) that catabolizes 1α,25-dihydroxyvitamin D3 (1α,25-(OH)2D3) to different products: calcitroic acid or 1α,25-(OH)2D3-26,23-lactone via multistep pathways commencing with C24 and C23 hydroxylation, respectively. Despite the ability of CYP24A1 to catabolize a wide range of 25-hydroxylated analogs including 25-hydroxyvitamin D3, the enzyme is unable to metabolize the synthetic prodrug, 1α-hydroxyvitamin D3 (1α-OH-D3), presumably because it lacks a C25-hydroxyl. In the current study we show that a single V391L amino acid substitution in the β3a-strand of human CYP24A1 converts this enzyme from a catabolic 1α,25-(OH)2D3-24-hydroxylase into an anabolic 1α-OH-D3-25-hydroxylase, thereby forming the hormone, 1α,25-(OH)2D3. Furthermore, because the mutant enzyme retains its basal ability to catabolize 1α,25-(OH)2D3 via C24 hydroxylation, it can also make calcitroic acid. Previous work has shown that an A326G mutation is responsible for the regioselectivity differences observed between human (primarily C24-hydroxylating) and opossum (C23-hydroxylating) CYP24A1. When the V391L and A326G mutations were combined (V391L/A326G), the mutant enzyme continued to form 1α,25-(OH)2D3 from 1α-OH-D3, but this initial product was diverted via the C23 hydroxylation pathway into the 26,23-lactone. The relative position of Val-391 in the β3a-strand of a homology model and the crystal structure of rat CYP24A1 is consistent with hydrophobic contact of Val-391 and the substrate side chain near C21. We interpret that the substrate specificity of V391L-modified human CYP24A1 toward 1α-OH-D3 is enabled by an altered contact with the substrate side chain that optimally positions C25 of the 1α-OH-D3 above the heme for hydroxylation.
Nithya Ramnath - One of the best experts on this subject based on the ideXlab platform.
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the cytochrome p450 enzyme CYP24A1 increases proliferation of mutant kras dependent lung adenocarcinoma independent of its catalytic activity
Journal of Biological Chemistry, 2020Co-Authors: Wei Huang, David G. Beer, Zhuwen Wang, Guoan Chen, Paramita Ray, Derek J Nancarrow, Stefanie Galban, Theodore S Lawrence, Alnawaz Rehemtulla, Nithya RamnathAbstract:We previously reported that overexpression of cytochrome P450 family 24 subfamily A member 1 (CYP24A1) increases lung cancer cell proliferation by activating RAS signaling and that CYP24A1 knockdown inhibits tumor growth. However, the mechanism of CYP24A1-mediated cancer cell proliferation remains unclear. Here, we conducted cell synchronization and biochemical experiments in lung adenocarcinoma cells, revealing a link between CYP24A1 and anaphase-promoting complex (APC), a key cell cycle regulator. We demonstrate that CYP24A1 expression is cell cycle-dependent; it was higher in the G2-M phase and diminished upon G1 entry. CYP24A1 has a functional destruction box (D-box) motif that allows binding with two APC adaptors, CDC20-homologue 1 (CDH1) and cell division cycle 20 (CDC20). Unlike other APC substrates, however, CYP24A1 acted as a pseudo-substrate, inhibiting CDH1 activity and promoting mitotic progression. Conversely, overexpression of a CYP24A1 D-box mutant compromised CDH1 binding, allowing CDH1 hyperactivation, thereby hastening degradation of its substrates cyclin B1 and CDC20, and accumulation of the CDC20 substrate p21, prolonging mitotic exit. These activities also occurred with a CYP24A1 isoform 2 lacking the catalytic cysteine (Cys-462), suggesting that CYP24A1's oncogenic potential is independent of its catalytic activity. CYP24A1 degradation reduced clonogenic survival of mutant KRAS-driven lung cancer cells, and calcitriol treatment increased CYP24A1 levels and tumor burden in Lsl-KRASG12D mice. These results disclose a catalytic activity-independent growth-promoting role of CYP24A1 in mutant KRAS-driven lung cancer. This suggests that CYP24A1 could be therapeutically targeted in lung cancers in which its expression is high.
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oncogenic potential of CYP24A1 in lung adenocarcinoma
Journal of Thoracic Oncology, 2017Co-Authors: Hiroe Shiratsuchi, David G. Beer, Zhuwen Wang, Guoan Chen, Paramita Ray, Jules Lin, Zhuo Zhang, Lili Zhao, Dipankar Ray, Nithya RamnathAbstract:Abstract Introduction We have previously demonstrated that a subset of lung cancer cells express higher CYP24A1 mRNA, a metabolizing enzyme for 1,25-D3, compared to benign tumors or surrounding normal lung and that high CYP24A1 mRNA expression is associated with poor prognosis in resected lung adenocarcinoma (AC). We hypothesized that CYP24A1 has oncogenic potential and increased CYP24A1 expression may contribute to tumor growth, whereas, CYP24A1 targeting may reduce tumor burden. Methods Two low CYP24A1 expressing human lung cancer cell lines (SK-LU-1 and Calu-6) were stably transfected either with an empty lentiviral vector or with the CYP24A1 expressing vector. Over-expression of mRNA and protein levels of CYP24A1 in SK-LU-1 and Calu-6 were confirmed using qRT-PCR and immunoblotting respectively. Next, effects of targeting CYP24A1 were examined in lung cancer cells (A549 and H441), which express higher basal levels of CYP24A1. Finally, we studied the effects of stable knockdown of CYP24A1 in xenograft models. Results Over-expression of CYP24A1 correlated with accelerated cell growth and invasion compared to control vector-transfected cells. CYP24A1 over-expression also increased RAS protein expression. Knockdown of CYP24A1 using either si- or shRNA reduced CYP24A1 mRNA and protein expression and significantly decreased cell proliferation (30-60%) and reduced mitochondrial DNA content compared to non-targeting (NT) si-/shRNA transfected/transduced cells. Transfection with CYP24A1 siRNA also decreased total RAS protein, thus reducing phosphorylated AKT. Importantly, stable knockdown of CYP24A1 in A549 and H441 lung tumor xenograft models resulted in tumor growth delay and smaller tumor size as evident from tumor bioluminescence and tumor volume measurement studies. Such observations were correlated with decreased tumor cell proliferation as evidenced by reduced Ki67 and Cyclin D staining. Conclusions Our data suggest that CYP24A1 has oncogenic properties mediated by increasing RAS signaling, targeting of which may provide an alternate strategy to treat a subset of lung AC.
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epigenetic regulation of vitamin d metabolism in human lung adenocarcinoma
Journal of Thoracic Oncology, 2014Co-Authors: Paul J. Christensen, Nithya Ramnath, Ernest Nadal, Chae Kyung Jeon, Juan Sandoval, Justin A Colacino, Laura S Rozek, Manel Esteller, David G. BeerAbstract:analysis revealed specific histone modifications within the CYP24A1 promoter region. Treatment with TSA increased H3K4me2 and H3K9ac and simultaneously decreased H3K9me2 at the CYP24A1 promoter and treatment with 5-Aza and/or TSA increased the recruit- ment of vitamin D receptor (VDR) to vitamin D response elements (VDRE) of the CYP24A1 promoter. Conclusions: The expression of CYP24A1 gene in human lung AC is in part epigenetically regulated by promoter DNA methylation and repressive histone modifications. These findings should be taken into consideration when targeting CYP24A1 to optimize antiproliferative effects of 1,25-D 3 in lung AC.
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The Vitamin D/CYP24A1 Story in Cancer
Anti-cancer agents in medicinal chemistry, 2010Co-Authors: Amanda N. King, David G. Beer, Paul J. Christensen, Robert U. Simpson, Nithya RamnathAbstract:There is increasing evidence linking the incidence of certain cancers to low serum Vitamin D levels. The active metabolite of Vitamin D, calcitriol (1, 25-Dihydroxyvitamin D3, 1,25(OH)2D3) apart from a crucial role in maintaining mineral homeostasis and skeletal functions, has antiproliferative, apoptosis and differentiation inducing as well as immunomodulatory effects in cancer. In studying the role of 1,25(OH)2D3 in cancer, it is imperative to examine the potential pathways that control local tissue levels of 1,25(OH)2D3. The enzyme CYP24A1 or 24-hydroxylase converts 1,25(OH)2D3 to inactive calcitroic acid. Extra-renal production of this enzyme is observed and has been increasingly recognized as present in cancer cells. This enzyme is rate limiting for the amount of local 1,25(OH)2D3 in cancer tissues and elevated expression is associated with an adverse prognosis. The gene that encodes CYP24A1 has been reported as an oncogene and may contribute to tumor aggressiveness by abrogating local anti-cancer effects of 1,25(OH)2D3. It is imperative to study the regulation of CYP24A1 in cancer and especially the local metabolism of 1,25(OH)2D3 in cancer cells. CYP24A1 may be a predictive marker of 1,25(OH)2D3 efficacy in patients with cancer as an adjunctive therapy. The following review summarizes the available literature on CYP24A1 as it relates to 1,25(OH)2D3 in cancer and outlines potential ways to inhibit CYP24A1 in an effort to improve the efficacy of exogenous 1,25(OH)2D3.
Martin Kaufmann - One of the best experts on this subject based on the ideXlab platform.
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CYP24A1 and slc34a1 pathogenic variants are uncommon in a canadian cohort of children with hypercalcemia or hypercalciuria
Hormone Research in Paediatrics, 2021Co-Authors: Isabelle Rousseaunepton, Glenville Jones, Martin Kaufmann, Karlpiet Schlingmann, Caroline Zuijdwijk, Karine Khatchadourian, Indra R Gupta, Daniele Pacaud, Maury Pinsk, Arati MokashiAbstract:OBJECTIVES Biallelic pathogenic variants in CYPA24A1 and SLC34A1 are causes of idiopathic infantile hypercalcemia. Pathogenic variants in both may also give rise to hypercalciuria with nephrocalcinosis or nephrolithiasis without previous hypercalcemia (renal group). Our objective was to examine the frequency of CYP24A1 or SLC34A1 variants in children with early hypercalcemia or late-onset hypercalciuria. METHOD Forty-one children from 7 centers across Canada were recruited. Local investigations were undertaken. The serum was evaluated by liquid chromatography tandem-mass spectrometry for the ratio of 25-hydroxyvitamin D3 to 24,25-dihydroxyvitamin D3, (25-OH-D3:24,25-(OH)2D3), an elevation pathognomonic for the loss of function of the CYP24A1 enzyme. Mutational analyses were undertaken. Family cascade screening was performed if pathogenic variants were detected in probands. RESULTS Twenty-nine children had early-onset hypercalcemia; none had elevated 25-OH-D3:24,25-(OH)2D3 or variants. Interestingly, 2 of 12 in the renal group had elevated 25-OH-D3:24,25-(OH)2D3 and presented as preadolescents. In case 1, cascade testing revealed a sibling and parent with asymptomatic pathogenic variants in CYP24A1. Four CYP24A1 pathogenic variants were identified in these 2 probands: 3 have been described in European populations, and 1 is a rare variant in exon 7 (c931delC) that is likely pathogenic. No SLC34A1 pathogenic variants were detected. CONCLUSION In Canada, pathogenic variants in CYP24A1 appear to manifest with late-onset hypercalciuria and its sequelae. The 25-OH-D3:24,25-(OH)2D3 ratio is an excellent tool for screening for biallelic pathogenic variants in CYP24A1. We confirm that cascade testing is important for these variants.
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a chromatin based mechanism controls differential regulation of the cytochrome p450 gene CYP24A1 in renal and non renal tissues
Journal of Biological Chemistry, 2019Co-Authors: Mark B Meyer, Glenville Jones, Martin Kaufmann, Seong Min Lee, Alex H Carlson, Nancy A Benkusky, Wesley J PikeAbstract:Cytochrome P450 family 27 subfamily B member 1 (CYP27B1) and CYP24A1 function to maintain physiological levels of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in the kidney. Renal Cyp27b1 and CYP24A1 expression levels are transcriptionally regulated in a highly reciprocal manner by parathyroid hormone (PTH), fibroblast growth factor 23 (FGF23), and 1,25(OH)2D3. In contrast, CYP24A1 regulation in nonrenal target cells (NRTCs) is limited to induction by 1,25(OH)2D3. Herein, we used ChIP-Seq analyses of mouse tissues to identify regulatory regions within the CYP24A1 gene locus. We found an extended region downstream of CYP24A1 containing a cluster of sites, termed C24-DS1, binding PTH-sensitive cAMP-responsive element–binding protein (CREB) and a cluster termed C24-DS2 binding the vitamin D receptor (VDR). VDR-occupied sites were present in both the kidney and NRTCs, but pCREB sites were occupied only in the kidney. We deleted each segment in the mouse and observed that although the overt phenotypes of both cluster deletions were unremarkable, RNA analysis in the C24-DS1–deleted strain revealed a loss of basal renal CYP24A1 expression, total resistance to FGF23 and PTH regulation, and secondary suppression of renal Cyp27b1; 1,25(OH)2D3 induction remained unaffected in all tissues. In contrast, loss of the VDR cluster in the C24-DS2–deleted strain did not affect 1,25(OH)2D3 induction of renal CYP24A1 expression yet reduced but did not eliminate CYP24A1 responses in NRTCs. We conclude that a chromatin-based mechanism differentially regulates CYP24A1 in the kidney and NRTCs and is essential for the specific functions of CYP24A1 in these two tissue types.
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CYP24A1 mutations in a cohort of hypercalcemic patients evidence for a recessive trait
The Journal of Clinical Endocrinology and Metabolism, 2015Co-Authors: Arnaud Molin, Martin Kaufmann, R Baudoin, Jeanclaude Souberbielle, Amelie Ryckewaert, M C Vantyghem, P Eckart, Justine Bacchetta, Georges Deschenes, G KeslerrousseyAbstract:Context: Loss-of-function mutations of CYP24A1 (which encodes the 25-OH-D3-24-hydroxylase) have recently been reported to cause hypercalcemia. Objectives: The aims of this study were: 1) to evaluate the frequency of CYP24A1 mutations in patients with medical history of hypercalcemia; 2) to show the clinical utility of a simultaneous assay of serum 25-hydroxyvitamin D3 (25-OH-D3) and 24,25-dihydroxyvitamin D3 (24,25-[OH]2D3) by liquid chromatography tandem mass spectrometry (LC-MS/MS); and 3) to investigate biochemical parameters in heterozygous gene carriers with CYP24A1 mutations. Patients and Methods: We screened for CYP24A1 mutations in 72 patients with serum calcium levels > 2.6 mmol/L and PTH levels < 20 pg/mL and recruited 24 relatives after genetic counseling for subsequent investigations. Vitamin D metabolite concentrations were assessed in a subset of patients by LC-MS/MS and results expressed as a ratio (R) of 25-OH-D3:24,25-(OH)2D3. Results: Twenty-five patients with hypercalcemia (35%) harbore...
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25 hydroxyvitamin d 24 hydroxylase CYP24A1 its important role in the degradation of vitamin d
Archives of Biochemistry and Biophysics, 2012Co-Authors: Glenville Jones, David E. Prosser, Martin KaufmannAbstract:CYP24A1 is the cytochrome P450 component of the 25-hydroxyvitamin D3-24-hydroxylase enzyme that catalyzes the conversion of 25-hydroxyvitamin D3 (25-OH-D3) and 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) 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-D3 and 1,25-(OH)2D3 in this condition. This review concludes by evaluating the potential clinical utility of blocking this enzyme with CYP24A1 inhibitors in various disease states.
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Bioengineering Anabolic Vitamin D-25-Hydroxylase Activity into the Human Vitamin D Catabolic Enzyme, Cytochrome P450 CYP24A1, by a V391L Mutation
The Journal of biological chemistry, 2011Co-Authors: Martin Kaufmann, David E. Prosser, Glenville JonesAbstract:CYP24A1 is a mitochondrial cytochrome P450 (CYP) that catabolizes 1α,25-dihydroxyvitamin D3 (1α,25-(OH)2D3) to different products: calcitroic acid or 1α,25-(OH)2D3-26,23-lactone via multistep pathways commencing with C24 and C23 hydroxylation, respectively. Despite the ability of CYP24A1 to catabolize a wide range of 25-hydroxylated analogs including 25-hydroxyvitamin D3, the enzyme is unable to metabolize the synthetic prodrug, 1α-hydroxyvitamin D3 (1α-OH-D3), presumably because it lacks a C25-hydroxyl. In the current study we show that a single V391L amino acid substitution in the β3a-strand of human CYP24A1 converts this enzyme from a catabolic 1α,25-(OH)2D3-24-hydroxylase into an anabolic 1α-OH-D3-25-hydroxylase, thereby forming the hormone, 1α,25-(OH)2D3. Furthermore, because the mutant enzyme retains its basal ability to catabolize 1α,25-(OH)2D3 via C24 hydroxylation, it can also make calcitroic acid. Previous work has shown that an A326G mutation is responsible for the regioselectivity differences observed between human (primarily C24-hydroxylating) and opossum (C23-hydroxylating) CYP24A1. When the V391L and A326G mutations were combined (V391L/A326G), the mutant enzyme continued to form 1α,25-(OH)2D3 from 1α-OH-D3, but this initial product was diverted via the C23 hydroxylation pathway into the 26,23-lactone. The relative position of Val-391 in the β3a-strand of a homology model and the crystal structure of rat CYP24A1 is consistent with hydrophobic contact of Val-391 and the substrate side chain near C21. We interpret that the substrate specificity of V391L-modified human CYP24A1 toward 1α-OH-D3 is enabled by an altered contact with the substrate side chain that optimally positions C25 of the 1α-OH-D3 above the heme for hydroxylation.
Eniko Kallay - One of the best experts on this subject based on the ideXlab platform.
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epigenetic regulation of the 1 25 dihydroxyvitamin d3 24 hydroxylase CYP24A1 in colon cancer cells
The Journal of Steroid Biochemistry and Molecular Biology, 2013Co-Authors: Julia Hobaus, Irfete S Fetahu, Maya Khorchide, Teresa Manhardt, Eniko KallayAbstract:Calcitriol is the hormonally active form of vitamin D and has anti-proliferative and pro-apoptotic effects. Calcitriol and its precursor calcidiol (25(OH)D3) are degraded by the 1,25-dihydroxyvitamin D3 24-hydroxylase (CYP24A1). This enzyme is overexpressed in colorectal tumors, however, the mechanisms of this overexpression remain to be elucidated. CYP24A1 mRNA level differs among colorectal cancer cell lines and range from almost undetectable to high. Since DNA methylation and histone acetylation regulate CYP24A1 gene expression in prostate cancer cell lines, we investigated whether epigenetic mechanisms could explain the differences in basal expression of CYP24A1 in colon cancer cells. Methyltransferase inhibitor 5-aza-2'-deoxycytidine (DAC) treatment resulted in an over 50-fold induction of CYP24A1 mRNA expression in Coga1A and HT-29 cells but in no response in Caco2/AQ and Coga13 cells. This finding is supported by a strong increase in CYP24A1 activity after DAC treatment in Coga1A (35%). In addition, calcitriol and DAC had synergistic effects on CYP24A1 gene transcription. Interestingly, the CYP24A1 promoter was not methylated in Coga1A and HT-29 (<5%), while in Caco2/AQ it was 62% methylated. This suggests that DNA demethylation must activate genes upstream of CYP24A1 rather than act on the gene itself. However, transcriptional regulators of CYP24A1 such as vitamin D receptor (VDR), retinoid X receptor (RXR), specificity protein 1 (SP1), or mediator complex subunit 1 (MED1) were not upregulated. We conclude that in colon cancer cells, CYP24A1 gene expression is inducible by methyltransferase and some histone deacetylase inhibitors in a cell line-dependent manner. This effect does not correlate with the methylation state of the promoter and therefore must affect genes upstream of CYP24A1. This article is part of a Special Issue 'Vitamin D Workshop'.
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the candidate oncogene CYP24A1 a potential biomarker for colorectal tumorigenesis
Journal of Histochemistry and Cytochemistry, 2010Co-Authors: Henrik Csaba Horvath, Janos P Kosa, Thomas Nittke, Gabor Speer, Peter L Lakatos, Katalin Borka, Krisztian Bacsi, Giovanna Bises, Pamela A Hershberger, Eniko KallayAbstract:The main autocrine/paracrine role of the active metabolite of vitamin D3, 1α,25-dihydroxyvitamin D3 (1,25-D3), is inhibition of cell growth and induction of cell differentiation and/or apoptosis. Synthesis and degradation of the secosteroid occurs not only in the kidney but also in normal tissue or malignant extrarenal tissues such as the colon. Because 25-hydroxyvitamin D3 24-hydroxylase (CYP24A1) is considered to be the main enzyme determining the biological half-life of 1,25-D3, we have examined expression of the CYP24A1 mRNA (by real-time RT-PCR) and protein (by immunohistochemistry) in normal human colon mucosa, colorectal adenomas, and adenocarcinomas in 111 patients. Although 76% of the normal and benign colonic tissue was either completely devoid of or expressed very low levels of CYP24A1, in the majority of the adenocarcinomas (69%), the enzyme was present at high concentrations. A parallel increased expression of the proliferation marker Ki-67 in the same samples suggests that overexpression of CYP24A1 reduced local 1,25-D3 availability, decreasing its antiproliferative effect. (J Histochem Cytochem 58:277–285, 2010)
Toshiyuki Sakaki - One of the best experts on this subject based on the ideXlab platform.
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CYP24A1 as a potential target for cancer therapy
Anti-cancer Agents in Medicinal Chemistry, 2014Co-Authors: Toshiyuki Sakaki, Keiko Yamamoto, Atsushi Kittaka, Kaori Yasuda, Tai C ChenAbstract:Increasing evidence has accumulated to suggest that vitamin D may reduce the risk of cancer through its biologically active metabolite, 1α,25(OH)2D3, which inhibits proliferation and angiogenesis, induces differentiation and apoptosis, and regulates many other cellular functions. Thus, it is plausible to assume that rapid clearance of 1α,25(OH)2D3 by highly expressed CYP24A1 could interrupt the normal physiology of cells and might be one cause of cancer initiation and progression. In fact, enhancement of CYP24A1 expression has been reported in literature for many cancers. Based on these findings, CYP24A1-specific inhibitors and vitamin D analogs which are resistant to CYP24A1-dependent catabolism might be useful for cancer treatment. CYP24A1-specific inhibitor VID400, which is an azole compound, markedly enhanced and prolonged the antiproliferative activity of 1α,25(OH)2D3 in the human keratinocytes. Likewise, CYP24A1-resistant analogs such as 2α-(3-hydroxypropoxy)-1α,25(OH)2D3 (O2C3) and its C2-epimer ED-71 (Eldecalcitol), and 19nor- 2α-(3-hydroxypropyl)-1α,25(OH)2D3 (MART-10) showed potent biological effects. Our in vivo studies using rats revealed that MART-10 had a low calcemic effect, which is a suitable property as an anticancer drug. Much lower affinity of MART-10 for vitamin D binding protein (DBP) as compared with 1α,25(OH)2D3 may be related to its more potent cellular activities. Based on these results, we conclude that (1) high affinity for VDR, (2) resistance to CYP24A1-dependent catabolism, (3) low affinity for DBP, and (4) low calcemic effect may be required for designing potent vitamin D analogs for cancer treatment.
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metabolism of 2α propoxy 1α 25 dihydroxyvitamin d3 and 2α 3 hydroxypropoxy 1α 25 dihydroxyvitamin d3 by human cyp27a1 and CYP24A1
Drug Metabolism and Disposition, 2005Co-Authors: Daisuke Abe, Yoshitomo Suhara, Toshiyuki Sakaki, Tatsuya Kusudo, Atsushi Kittaka, Nozomi Saito, Toshie Fujishima, Hiroaki Takayama, Hiromi Hamamoto, Masaki KamakuraAbstract:Recently, we demonstrated that some A-ring-modified vitamin D3 analogs had unique biological activity. Of these analogs, 2alpha-propoxy-1alpha,25(OH)2D3 (C3O1) and 2alpha-(3-hydroxypropoxy)-1alpha,25(OH)2D3 (O2C3) were examined for metabolism by CYP27A1 and CYP24A1. Surprisingly, CYP27A1 catalyzed the conversion from C3O1 to O2C3, which has 3 times more affinity for vitamin D receptor than C3O1. Thus, the conversion from C3O1 to O2C3 by CYP27A1 is considered to be a metabolic activation process. Five metabolites were detected in the metabolism of C3O1 and O2C3 by human CYP24A1 including both C-23 and C-24 oxidation pathways. On the other hand, three metabolites of the C-24 oxidation pathway were detected in their metabolism by rat CYP24A1, indicating a species-based difference in the CYP24A1-dependent metabolism of C3O1 and O2C3 between humans and rats. Kinetic analysis revealed that the Km and kcat values of human CYP24A1 for O2C3 are, respectively, approximately 16 times more and 3 times less than those for 1alpha,25(OH)2D3. Thus, the catalytic efficiency, kcat/Km, of human CYP24A1 for O2C3 is only 2% of 1alpha,25(OH)2D3. These results and a high calcium effect of C3O1 and O2C3 in animal experiments using rats suggest that C3O1 and O2C3 are promising for clinical treatment of osteoporosis.
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metabolism of vitamin d3 by cytochromes p450
Frontiers in Bioscience, 2005Co-Authors: Toshiyuki Sakaki, Norio Kagawa, Keiko Yamamoto, Kuniyo InouyeAbstract:The vitamin D3 25-hydroxylase (CYP27A1), 25-hydroxyvitamin D3 1alpha-hydroxylase (CYP27B1) and 1alpha,25-dihydroxyvitamin D3 24-hydroxylase (CYP24A1) are members of the cytochrome P450 superfamily, and key enzymes of vitamin D3 metabolism. Using the heterologous expression in E. coli, enzymatic properties of the P450s were recently investigated in detail. Upon analyses of the metabolites of vitamin D3 by the reconstituted system, CYP27A1 surprisingly produced at least seven forms of minor metabolites including 1alpha,25(OH)2D3 in addition to the major metabolite 25(OH)D3. These results indicated that human CYP27A1 catalyzes multiple reactions involved in the vitamin D3 metabolism. In contrast, CYP27B1 only catalyzes the hydroxylation at C-1alpha position of 25(OH)D3 and 24R,25(OH)2D3. Enzymatic studies on substrate specificity of CYP27B1 suggest that the 1alpha-hydroxylase activity of CYP27B1 requires the presence of 25-hydroxyl group of vitamin D3 and is enhanced by 24-hydroxyl group while the presence of 23-hydroxyl group greatly reduced the activity. Eight types of missense mutations in the CYP27B1 gene found in vitamin D-dependent rickets type I (VDDR-I) patients completely abolished the 1alpha-hydroxylase activity. A three-dimensional model of CYP27B1 structure simulated on the basis of the crystal structure of rabbit CYP2C5 supports the experimental data from mutagenesis study of CYP27B1 that the mutated amino acid residues may be involved in protein folding, heme-propionate binding or activation of molecular oxygen. CYP24A1 expressed in E. coli showed a remarkable metabolic processes of 25(OH)D3 and 1alpha,25(OH)2D3. Rat CYP24A1 catalyzed six sequential monooxygenation reactions that convert 1alpha,25(OH)2D3 into calcitroic acid, a known final metabolite of C-24 oxidation pathway. In addition to the C-24 oxidation pathway, human CYP24A1 catalyzed also C-23 oxidation pathway to produce 1alpha,25(OH)2D3-26,23-lactone. Surprisingly, more than 70 % of the vitamin D metabolites observed in a living body were found to be the products formed by the activities of CYP27A1, CYP27B1 and CYP24A1. The species-based difference was also observed in the metabolism of vitamin D analogs by CYP24A1, suggesting that the recombinant system for human CYP24A1 may be of great use for the prediction of the metabolism of vitamin D analogs in humans.
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metabolism of a ring diastereomers of 1α 25 dihydroxyvitamin d3 by CYP24A1
Biochemical and Biophysical Research Communications, 2004Co-Authors: Tatsuya Kusudo, Toshiyuki Sakaki, Daisuke Abe, Atsushi Kittaka, Toshie Fujishima, Hiroaki Takayama, Susumi Hatakeyama, Miho Ohta, Kuniyo InouyeAbstract:Abstract The metabolism of 1α,25(OH) 2 D 3 (1α,3β) and its A-ring diastereomers, 1β,25(OH) 2 D 3 (1β,3β), 1α,25(OH) 2 -3-epi-D 3 (1α,3α), and 1β,25(OH) 2 -3-epi-D 3 (1β,3α), was examined to compare the substrate specificity and reaction specificity of CYP24A1 between humans and rats. The ratio between C-23 and C-24 oxidation pathways in human CYP24A1-dependent metabolism of (1α,3α) and (1β,3α) was 1:1, although the ratio for (1α,3β) and (1β,3β) was 1:4. These results indicate that the orientation of the hydroxyl group at the C-3 position determines the ratio between C-23 and C-24 oxidation pathways. A remarkable increase of metabolites in the C-23 oxidation pathway was also observed in rat CYP24A1-dependent metabolism. The binding affinity of human CYP24A1 for A-ring diastereomers was (1α,3β) > (1α,3α) > (1β,3β) > (1β,3α), indicating that both hydroxyl groups at C-1 and C-3 positions significantly affect substrate-binding. The information obtained in this study is quite useful for understanding substrate recognition of CYP24A1 and designing new vitamin D analogs.
