THU398 Variants In The Vitamin D Receptor, 1alpha-hydroxylase Enzyme Genes In Cholecalciferol Therapy
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PROVIDER: S-EPMC10554864 | biostudies-literature | 2023 Oct
REPOSITORIES: biostudies-literature
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Project description:IntroductionThe cytochrome P450 mitochondrial enzyme 25-hydroxyvitamin D3 1alpha-hydroxylase (1alpha-hydroxylase) of renal tubule cells hydroxylates the major circulating form of vitamin D (25(OH)D3) to the active systemic hormone 1,25(OH)2D3. Local production of 1,25(OH)2D3 appears to occur also at other sites where 1alpha-hydroxylase is expressed for autocrine/paracrine regulation. To reduce risks of hypercalcemia during treatment with vitamin D, we have previously suggested use of non-1alpha-hydroxylated vitamin D analogues to target tissues where 1alpha-hydroxylase is expressed, including the parathyroid glands in secondary hyperparathyroidism. The present study was undertaken to examine expression of 1alpha-hydroxylase in breast cancer and to investigate whether a non-1alpha-hydroxylated vitamin D analogue displayed biological function. In addition, expression of the 25-hydroxyvitamin D3 24-hydroxylase (24-hydroxylase) and the vitamin D receptor (VDR) was investigated.MethodsThe expression of 1alpha-hydroxylase, 24-hydroxylase and VDR was investigated in breast cancer specimens (n = 19) and normal breast tissues (n = 10) by immunohistochemistry and/or RT-PCR. Consecutive cryosections of 6 mum essentially free of immune cells were used in the analyses. The effect of vitamin D analogues on transcriptional activation was analyzed in transiently transfected MCF-7 breast cancer cells.Results1alpha-hydroxylase protein was demonstrated in 79% and 100% of breast cancer specimens and normal breast, respectively. The overall relative mRNA levels of 1alpha-hydroxylase and 24-hydroxylase in normal breast compared to breast tumors were: 1alpha-hydroxylase, 1 +/- 0.07 versus 0.7 +/- 0.05, respectively (p < 0.001); 24-hydroxylase, 1 +/- 0.08 verus 2.1 +/- 0.2, respectively (p < 0.001). The VDR was expressed in 95% of the tumors as expected, with mRNA levels of 1 +/- 0.09 and 1.4 +/- 0.12 (p < 0.05) in breast cancer and normal breast, respectively. The ketoconazole-sensitive transcription activation potential of the non-1alpha-hydroxylated vitamin D analogue prodrug of EB1089 (EB1285) was demonstrated in MCF-7 cells, which express 1alpha-hydroxylase. The activity of EB1285 was about 20% of 1,25(OH)2D3.ConclusionThese results demonstrate nearly normal expression levels of 1alpha-hydroxylase, 24-hydroxylase and VDR in the majority of investigated breast cancer specimens. A non-1alpha-hydroxylated vitamin D analogue displayed activity in breast cancer cells. Such analogues may present future therapeutic options for proliferative disorders where 1alpha-hydroxylase is expressed.
| S-EPMC1410765 | biostudies-literature
Project description:The human vitamin D receptor (hVDR) is a member of the nuclear receptor superfamily, involved in calcium and phosphate homeostasis; hence implicated in a number of diseases, such as Rickets and Osteoporosis. This receptor binds 1α,25-dihydroxyvitamin D(3) (also referred to as 1,25(OH)(2)D(3)) and other known ligands, such as lithocholic acid. Specific interactions between the receptor and ligand are crucial for the function and activation of this receptor, as implied by the single point mutation, H305Q, causing symptoms of Type II Rickets. In this work, further understanding of the significant and essential interactions between the ligand and the receptor was deciphered, through a combination of rational and random mutagenesis. A hVDR mutant, H305F, was engineered with increased sensitivity towards lithocholic acid, with an EC(50) value of 10 μM and 40±14 fold activation in mammalian cell assays, while maintaining wild-type activity with 1,25(OH)(2)D(3). Furthermore, via random mutagenesis, a hVDR mutant, H305F/H397Y, was discovered to bind a novel small molecule, cholecalciferol, a precursor in the 1α,25-dihydroxyvitamin D(3) biosynthetic pathway, which does not activate wild-type hVDR. This variant, H305F/H397Y, binds and activates in response to cholecalciferol concentrations as low as 100 nM, with an EC(50) value of 300 nM and 70±11 fold activation in mammalian cell assays. In silico docking analysis of the variant displays a dramatic conformational shift of cholecalciferol in the ligand binding pocket in comparison to the docked analysis of cholecalciferol with wild-type hVDR. This shift is hypothesized to be due to the introduction of two bulkier residues, suggesting that the addition of these bulkier residues introduces molecular interactions between the ligand and receptor, leading to activation with cholecalciferol.
| S-EPMC3105188 | biostudies-literature
Project description:Vitamin D-dependent rickets type I (VDDR-I), also known as pseudo-vitamin D-deficiency rickets, appears to result from deficiency of renal vitamin D 1alpha-hydroxylase activity. Prior work has shown that the affected gene lies on 12q13.3. We recently cloned the cDNA and gene for this enzyme, mitochondrial P450c1alpha, and we and others have found mutations in its gene in a few patients. To determine whether all patients with VDDR-I have mutations in P450c1alpha, we have analyzed the P450c1alpha gene in 19 individuals from 17 families representing various ethnic groups. The whole gene was PCR amplified and subjected to direct sequencing; candidate mutations were confirmed by repeat PCR of the relevant exon from genomic DNA from the patients and their parents. Microsatellite haplotyping with the markers D12S90, D12S305, and D12S104 was also done in all families. All patients had P450c1alpha mutations on both alleles. In the French Canadian population, among whom VDDR-I is common, 9 of 10 alleles bore the haplotype 4-7-1 and carried the mutation 958DeltaG. This haplotype and mutation were also seen in two other families and are easily identified because the mutation ablates a TaiI/MaeII site. Six families of widely divergent ethnic backgrounds carried a 7-bp duplication in association with four different microsatellite haplotypes, indicating a mutational hot spot. We found 14 different mutations, including 7 amino acid replacement mutations. When these missense mutations were analyzed by expressing the mutant enzyme in mouse Leydig MA-10 cells and assaying 1alpha-hydroxylase activity, none retained detectable 1alpha-hydroxylase activity. These studies show that most if not all patients with VDDR-I have severe mutations in P450c1alpha, and hence the disease should be referred to as "1alpha-hydroxylase deficiency."
| S-EPMC1377641 | biostudies-other
Project description:ContextSingle-nucleotide polymorphisms (SNPs) in genes related to vitamin D metabolism have been associated with serum 25-hydroxyvitamin D [25(OH)D] concentration, but these relationships have not been examined following antenatal cholecalciferol supplementation.ObjectiveTo determine whether SNPs in DHCR7, CYP2R1, CYP24A1, and GC are associated with the response to gestational cholecalciferol supplementation.DesignWithin-randomization group analysis of the Maternal Vitamin D Osteoporosis Study trial of antenatal cholecalciferol supplementation.SettingHospital antenatal clinics.ParticipantsIn total, 682 women of white ethnicity (351 placebo, 331 cholecalciferol) were included. SNPs at rs12785878 (DHCR7), rs10741657 (CYP2R1), rs6013897 (CYP24A1), and rs2282679 (GC) were genotyped.Interventions1000 IU/d cholecalciferol from 14 weeks of gestation until delivery.Main outcome measure25(OH)D at randomization and 34 weeks of gestation were measured in a single batch (Liaison; Diasorin, Dartford, UK). Associations between 25(OH)D and the SNPs were assessed by linear regression using an additive model [β represents the change in 25(OH)D per additional common allele].ResultsOnly rs12785878 (DHCR7) was associated with baseline 25(OH)D [β = 3.1 nmol/L; 95% confidence interval (CI), 1.0 to 5.2 nmol/L; P < 0.004]. In contrast, rs10741657 (CYP2R1) (β = -5.2 nmol/L; 95% CI, -8.2 to -2.2 nmol/L; P = 0.001) and rs2282679 (GC) (β = 4.2 nmol/L; 95% CI, 0.9 to 7.5 nmol/L; P = 0.01) were associated with achieved 25(OH)D status following supplementation, whereas rs12785878 and rs6013897 (CYP24A1) were not.ConclusionsGenetic variation in DHCR7, which encodes 7-dehyrocholesterol reductase in the epidermal vitamin D biosynthesis pathway, appears to modify baseline 25(OH)D. In contrast, the response to antenatal cholecalciferol supplementation was associated with SNPs in CYP2R1, which may alter 25-hydroxylase activity, and GC, which may affect vitamin D binding protein synthesis or metabolite affinity.
| S-EPMC5546866 | biostudies-literature
Project description:The active form of vitamin D, 1alpha,25-dihydroxyvitamin D [1alpha,25(OH)2D], is synthesized from its precursor 25 hydroxyvitamin D [25(OH)D] via the catalytic action of the 25(OH)D-1alpha-hydroxylase [1alpha(OH)ase] enzyme. Many roles in cell growth and differentiation have been attributed to 1,25(OH)2D, including a central role in calcium homeostasis and skeletal metabolism. To investigate the in vivo functions of 1,25(OH)2D and the molecular basis of its actions, we developed a mouse model deficient in 1alpha(OH)ase by targeted ablation of the hormone-binding and heme-binding domains of the 1alpha(OH)ase gene. After weaning, mice developed hypocalcemia, secondary hyperparathyroidism, retarded growth, and the skeletal abnormalities characteristic of rickets. These abnormalities are similar to those described in humans with the genetic disorder vitamin D dependent rickets type I [VDDR-I; also known as pseudovitamin D-deficiency rickets (PDDR)]. Altered non-collagenous matrix protein expression and reduced numbers of osteoclasts were also observed in bone. Female mutant mice were infertile and exhibited uterine hypoplasia and absent corpora lutea. Furthermore, histologically enlarged lymph nodes in the vicinity of the thyroid gland and a reduction in CD4- and CD8-positive peripheral T lymphocytes were observed. Alopecia, reported in vitamin D receptor (VDR)-deficient mice and in humans with VDDR-II, was not seen. The findings establish a critical role for the 1alpha(OH)ase enzyme in mineral and skeletal homeostasis as well as in female reproduction and also point to an important role in regulating immune function.
| S-EPMC34697 | biostudies-literature
Project description:ObjectiveDiabetic impaired angiogenesis is associated with impairment of hypoxia-inducible factor-1alpha (HIF-1alpha) as well as vasculature maturation. We investigated the potential roles and intracellular mechanisms of angiopoietin-1 (Ang-1) gene therapy on myocardial HIF-1alpha stabilization and vascular maturation in db/db mice.Research design and methodsdb/db mice were systemically administrated adenovirus Ang-1 (Ad-CMV-Ang-1). Myocardial HIF-1alpha, vascular endothelial growth factor (VEGF), hemeoxygenase-1 (HO-1), endothelial nitric oxide synthase (eNOS), Akt, and HIF-1alpha-prolyl-4-hydroxylase-2 (PHD)2 expression were measured. Vasculature maturation, capillary and arteriole densities, and cardiac interstitial fibrosis were analyzed in the border zone of infarcted myocardium.ResultsSystemic administration of Ad-CMV-Ang-1 results in overexpression of Ang-1 in db/db mice hearts. Ang-1 gene therapy causes a significant increase in Akt and eNOS expression and HIF-1alpha stabilization. This is accompanied by a significant upregulation of VEGF and HO-1 expression. Intriguingly, Ang-1 gene therapy also leads to a significant inhibition of PHD2 expression. Smooth muscle recruitment and smooth muscle coverage in the neovessels of the border zone of infarcted myocardium are severely impaired in db/db mice compared with wild-type mice. Ang-1 gene therapy rescues these abnormalities, which leads to a dramatic increase in capillary and arteriole densities and a significant reduction of cardiac hypertrophy and interstitial fibrosis at 14 days after ischemia. Taken together, our data show that Ang-1 increases myocardial vascular maturation and angiogenesis together with suppression of PHD2 and the upregulation of HIF-1alpha signaling.ConclusionsNormalization of immature vasculature by Ang-1 gene therapy may represent a novel therapeutic strategy for treatment of the diabetes-associated impairment of myocardial angiogenesis.
| S-EPMC2584141 | biostudies-literature
Project description:Kinetics of vitamin D-depleted and -repleted rat liver microsomal cholecalciferol 25-hydroxylase were studied. Anaerobiosis, CO, omission of a NADPH-generating system and addition of detergents all decreased the activities, showing that the hydroxylase behaves like a cytochrome P-450-dependent enzyme. An apparent Km of 0.18 micrometer and Vmax. of 32pmol/min per g of tissue were found for vitamin D-deficient animals. Although both apparent Km and Vmax. were significantly altered in vitamin D-repleted animals no inhibition of the enzyme was elicited. These latter results show that at normal vitamin D intake, rat liver cholecalciferol 25-hydroxylase is not feedback-inhibited.
| S-EPMC1185715 | biostudies-other
Project description:Objectives:The objective of the present study was to evaluate the pattern of genetic expression of vitamin D receptor (VDR), 1 alpha-hydroxylase (1?-OHase) enzyme and chemokine regulated on activation normal T-cell expressed and secreted (RANTES) in peripheral blood of Holstein dairy cows during transition period. Methods:Blood samples were collected from 16 Holstein dairy cows at 3 weeks prior expected date of delivery (EDD), at the day of parturition, and 3 weeks post-partum for assessment of expression profile of studied genes using real-time PCR and measurement of glucose, ionized calcium (Ca), parathyroid hormone (PTH), inorganic phosphorous (P), sodium (Na), potassium (K), chloride (Cl), and magnesium (Mg) levels. Results:Compared with 3 weeks prior EDD, VDR gene expression decreased significantly at the day of parturition then increased significantly at 3 weeks post-partum. The genetic expression of 1?-OHase enzyme as well as PTH, K, Na and Cl levels increased significantly at the day of parturition. The Ca level decreased significantly at the day of parturition then increased significantly at 3 weeks post-partum. The P level increased significantly at the day of parturition then decreased significantly at 3 weeks post-partum. Glucose level decreased significantly at the day of parturition and at 3 weeks post-partum. RANTES gene expression showed non-significant changes among the three different time points. The expression of VDR gene had a negative correlation with the expression of 1?-OHase enzyme gene, and serum levels of glucose, PTH, P and K, but had a positive correlation with the serum Ca level. The expression of 1?-OHase enzyme gene had a positive correlation with serum levels of PTH, P and K, but had a negative correlation with the serum Ca level. Conclusions:Results of the current study indicate the importance of monitoring the genetic expression of VDR and 1?-OHase enzyme as indicators of metabolic changes during transition period, suggesting that they are candidate genes to judge the health status of dairy cows during such period.
| S-EPMC7045247 | biostudies-literature
Project description:Cholecalciferol (D(3)) supplementation results in variable increases in serum 25(OH)D(3) levels, however, the influence of genetic polymorphisms on these variable responses is unclear. We measured serum 25(OH)D(3), 24,25(OH)(2)D(3), 1,25(OH)2D(3) and VDBP levels in 50 colorectal cancer (CRC) patients before and during 2,000 IU daily oral D(3) supplementation for six months and in 263 archived CRC serum samples. Serum PTH levels and PBMC 24-OHase activity were also measured during D(3) supplementation. TagSNPs in CYP2R1, CYP27A1, CYP27B1, CYP24A1, VDR, and GC genes were genotyped in all patients, and the association between these SNPs and serum vitamin D(3) metabolites levels before and after D(3) supplementation was analyzed. The mean baseline serum 25(OH)D(3) level was less than 32 ng/mL in 65 % of the 313 CRC patients. In the 50 patients receiving D(3) supplementation, serum levels of 25(OH)D(3) increased (p = 0.008), PTH decreased (p = 0.036) and 24,25(OH)(2)D(3), 1,25(OH)(2)D(3), VDBP levels and PBMC 24-OHase activity were unchanged. GC SNP rs222016 was associated with high 25(OH)D(3) and 1,25(OH)(2)D(3) levels at baseline while rs4588 and rs2282679 were associated with lower 25(OH)D(3) and 1,25(OH)(2)D(3) levels both before and after D(3) supplementation. CYP2R1 rs12794714 and rs10500804 SNPs were significantly associated with low 25(OH)D(3) levels after supplementation but not with baseline 25(OH)D(3). Our results show that D(3) supplementation increased 25(OH)D(3) levels in all patients. GC rs4588 and rs2283679 SNPs were associated with increased risk of vitamin D(3) insufficiency and suboptimal increase in 25(OH)D(3) levels after D(3) supplementation. Individuals with these genotypes may require higher D(3) supplementation doses to achieve vitamin D(3) sufficiency.
| S-EPMC3689467 | biostudies-literature
Project description:Background/objectivesSeveral studies have shown a relationship between vitamin D and migraine, including the association between decreased serum 25-hydroxyvitamin D in patients with migraine and the positive effects of vitamin D supplementations in the therapy of this disease. Two single-nucleotide variants (SNVs) vitamin D receptor (VDR) gene, VDR rs2228570, and VDR rs731236 have shown an association with migraine risk in a previous case-control association study, while an exome sequencing study identified a rare variant in GC vitamin D binding protein gene. This study aims to look for the association between several common variants in these two genes and the risk for migraine.MethodsWe genotyped 290 patients diagnosed with migraine and 300 age-matched controls using specific TaqMan assays for VDR rs2228570, VDR rs731236, VDR rs7975232, VDR rs739837, VDR rs78783628, GC rs7041, and GC rs4588 SNVs.ResultsWe did not find an association between these SNVs and the risk for migraine. None of these SNVs were related to the positivity of a family history of migraine or with the presence of aura. The VDR rs731236A allele showed a significant association with the triggering of migraine attacks by ethanol (Pc = 0.007).ConclusionsIn summary, the results of the current study suggest a lack of association between common SNVs in the VDR and GC gene and the risk of developing migraine. The possible relationship between VDR rs731236 and the triggering of migraine episodes with ethanol deserves future studies.
| S-EPMC10578880 | biostudies-literature