25-hydroxyvitamin D3 24-hydroxylase Research Articles

Nephrolithiasis and Multicystic Kidneys in a Young Patient: A Quiz

Nephrolithiasis and Multicystic Kidneys in a Young Patient: A Quiz

Sterol regulatory element binding protein 1 trans-activates 25-hydroxy vitamin D3 24-hydroxylase gene expression in renal proximal tubular cells

The physiological activity of the steroid derived hormone vitamin D is regulated by several enzymatic steps. Both 25-hydroxy vitamin D3 1α-hydroxylase (CYP27B1) and 25-hydroxyvitamin D3 24-hydroxylase (CYP24A1) modulate blood levels of 1,25-dihydroxyvitamin D3, an activated form of vitamin D. We previously demonstrated that CYP27B1 expression was trans-activated by sterol regulatory element binding protein 1 (SREBP1), although whether SREBP1 also regulates CYP24A1 transcription was unclear. Here we investigated the ability of SREBP1 to affect CYP24A1 transcription. In a luciferase reporter assay, mouse and human CYP24A1 promoter activity was strongly activated by SREBP1 in opossum kidney proximal tubular cells (OK-P). Three putative SREs (pSREs) were found in the mouse Cyp24a1 gene promoter and the SREBP1 protein showed specific binding to the pSRE1 element in EMSAs. Site-directed mutagenesis of the pSRE1 element strongly decreased SREBP1-mediated Cyp24a1 gene transcription. Moreover, siRNA-mediated SREBP1 knock-down repressed CYP24A1 expression in human renal proximal tubular epithelial cells (HKC-8). In animal studies, mice given various doses of thyroid hormone (T3) showed dose-dependent reductions in renal Srebp1c and Cyp24a1 mRNA levels. Taken together, our results suggest that SREBP1 trans-activates CYP24A1 expression through SREBP binding elements present in the promoter.

VDR SUPPRESSION AND CYP24 INDUCTION BY AMYLOID BETA MIGHT INVOLVE MIR-27B-3P, LET-7A-5P, MIR-125B-5P IN AMYLOID BETA-TREATED CORTICAL NEURON CULTURES

In 2011, a study by our group demonstrated that Aβ induces neurodegeneration also by significantly suppressing the expression of VDR. We also reported that the expression of mRNA for 25-hydroxyvitaminD3–24-hydroxylase (24OHase) encoded by CYP24, the enzyme that accelerates vitamin D catabolism, is induced by Aβ in hippocampal neurons. This finding suggests that the involvement of vitamin D with AD includes also the disruption of vitamin D metabolism and action. The present study investigated the expression levels of miRNAs that were suggested to regulate VDR (hsa-mir-27b-3p, hsa-let-7a-5p, hsa-mir-125b-5p), CYP24 (hsa-mir-125b-5p) in order to evaluate whether alteration of the expressions of VDR or 24(OH)ase by amyloid beta involve these miRNAs or not? 24, 48 and 72 hours of Aβ, vitamin D, vitamin D+Aβ and Aβ+vitamin D treated groups and untreated control groups were established in the primary cortical neuron cultures of Sprague Dawley rat embryos. Amyloid pathology was established with the treatment of 10 μM of Aβ1–42. Vitamin D groups were treated with 10-8M 1α-25dihydroxyvitamin D3 (1,25(OH)2D3). The expressions of let-7a, miR-27b-3p, miR-125b-5p, miR-26b-5p, miR-192–5p, were investigated with qRT-PCR. Our results indicated that only let7a-5p expression was significantly increased in amyloid beta treated cortical neurons at 24 hours of treatments. miR125b-5p expression decreased in both amyloid beta or vitamin D treated ones, yet the attenuations were not statistically significant. miR125b-5p expression also decreased in amyloid beta, amyloid beta+vitamin D, vitamin D+amyloid beta treated groups in 48 hours of treatments compared to control groups. miR27b-3p expression induced in vitamin D treated neurons at the 24 and 48 hours of treatments compared with the control groups. Consequently, our preliminary results indicate that miR27b-3p might be involved in regulation of vitamin D action, by regulating VDR expression. The more likely small RNA that is involved in VDR suppression by amyloid beta might be let7a-5p. CYP24 induction by amyloid beta might involve the suppression of miR125b-5p by amyloid beta. (The present work was supported by the Research Fund of Istanbul University, Project no:21585 and by the Scientific and Technological Research Council of Turkey-TUBITAK, Project no:214S585.)

25-Hydroxyvitamin D₃ 24-Hydroxylase: A Key Regulator of 1,25(OH)₂D₃ Catabolism and Calcium Homeostasis.

One of the most pronounced effects of the hormonally active form of vitamin D, 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), is increased synthesis of 25-hydroxyvitamin D3 24-hydroxylase (CYP24A1), the enzyme responsible for the catabolism of 1,25(OH)2D3. Thus, 1,25(OH)2D3 regulates its own metabolism, protecting against hypercalcemia and limiting the levels of 1,25(OH)2D3 in cells. This chapter summarizes the catalytic properties of CYP24A1, the recent data related to the crystal structure of CYP24A1, the findings obtained from the generation of mice deficient for the Cyp24a1 gene as well as recent data identifying a causal role of a genetic defect in CYP24A1 in certain patients with idiopathic infantile hypercalcemia. This chapter also reviews the regulation of renal and placental CYP24A1 as well as the genomic mechanisms, including coactivators, repressors, and epigenetic modification, involved in modulating 1,25(OH)2D3 regulation of CYP24A1. We conclude with future research directions related to this key regulator of 1,25(OH)2D3 catabolism and calcium homeostasis.

Novel Mechanism of Negative Regulation of 1,25-Dihydroxyvitamin D3-induced 25-Hydroxyvitamin D3 24-Hydroxylase (Cyp24a1) Transcription: EPIGENETIC MODIFICATION INVOLVING CROSS-TALK BETWEEN PROTEIN-ARGININE METHYLTRANSFERASE 5 AND THE SWI/SNF COMPLEX

The SWI/SNF chromatin remodeling complex facilitates gene transcription by remodeling chromatin using the energy of ATP hydrolysis. Recent studies have indicated an interplay between the SWI/SNF complex and protein-arginine methyltransferases (PRMTs). Little is known, however, about the role of SWI/SNF and PRMTs in vitamin D receptor (VDR)-mediated transcription. Using SWI/SNF-defective cells, we demonstrated that Brahma-related gene 1 (BRG1), an ATPase that is a component of the SWI/SNF complex, plays a fundamental role in induction by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) of the transcription of Cyp24a1 encoding the enzyme 25-hydroxyvitamin D3 24-hydroxylase involved in the catabolism of 1,25(OH)2D3. BRG1 was found to associate with CCAAT-enhancer-binding protein (C/EBP) β and cooperate with VDR and C/EBPβ in regulating Cyp24a1 transcription. PRMT5, a type II PRMT that interacts with BRG1, repressed Cyp24a1 transcription and mRNA expression. Our findings indicate the requirement of the C/EBP site for the inhibitory effect of PRMT5 via its methylation of H3R8 and H4R3. These findings indicate that the SWI/SNF complex and PRMT5 may be key factors involved in regulation of 1,25(OH)2D3 catabolism and therefore in the maintenance of calcium homeostasis by vitamin D. These studies also define epigenetic events linked to a novel mechanism of negative regulation of VDR-mediated transcription.

The vitamin D receptor in the proximal renal tubule is a key regulator of serum 1α,25-dihydroxyvitamin D₃.

It is well established that the mitochondria of proximal convoluted tubule cells of the kidney are the site of production of circulating 1α,25-dihydroxyvitamin D3 [1,25(OH)2D3]. The production of 1,25(OH)2D3 at this site is tightly regulated. Parathyroid hormone markedly stimulates 1,25(OH)2D3 production, whereas 1,25(OH)2D3 itself suppresses production. The mechanism of suppression by 1,25(OH)2D3 has not yet been elucidated. We have now found that in the absence of vitamin D (vitamin D deficiency), the vitamin D receptor (VDR) is found in the interior of the apical brush border of the proximal tubule cells. This is unique for the proximal tubule cells, since this has not been observed in the distal tubule cells or in other epithelial cells, such as intestinal mucosa. Administration of 1,25(OH)2D3 to vitamin D-deficient rats results in the movement of VDR from the brush border to the cytoplasm and nucleus presumably bound to reabsorbed 1,25(OH)2D3. The VDR bound to 1,25(OH)2D3 suppresses expression of 25-hydroxyvitamin D3 1α-hydroxylase and stimulates the 25-hydroxyvitamin D3 24-hydroxylase. Thus, VDR in the apical brush border of the proximal convoluted tubule cells serves to "sense" the level of circulating 1,25(OH)2D3 and modulates the activity of the 1α-hydroxylase and the 24-hydroxylase accordingly.

1,25-Dihydroxyvitamin D3 Suppresses Telomerase Expression and Human Cancer Growth through MicroRNA-498

Telomerase is an essential enzyme that counteracts the telomere attrition accompanying DNA replication during cell division. Regulation of the promoter activity of the gene encoding its catalytic subunit, the telomerase reverse transcriptase, is established as the dominant mechanism conferring the high telomerase activity in proliferating cells, such as embryonic stem and cancer cells. This study reveals a new mechanism of telomerase regulation through non-coding small RNA by showing that microRNA-498 (miR-498) induced by 1,25-dihydroxyvitamin D3 (1,25(OH)(2)D(3)) decreases the mRNA expression of the human telomerase reverse transcriptase. MiR-498 was first identified in a microarray analysis as the most induced microRNA by 1,25(OH)(2)D(3) in ovarian cancer cells and subsequently validated by quantitative polymerase chain reaction assays in multiple human cancer types. A functional vitamin D response element was defined in the 5-prime regulatory region of the miR-498 genome, which is occupied by the vitamin D receptor and its coactivators. Further studies showed that miR-498 targeted the 3-prime untranslated region of human telomerase reverse transcriptase mRNA and decreased its expression. The levels of miR-498 expression were decreased in malignant human ovarian tumors as well as human ovarian cancer cell lines. The ability of 1,25(OH)(2)D(3) to decrease human telomerase reverse transcriptase mRNA and to suppress ovarian cancer growth was compromised when miR-498 was depleted using the sponges in cell lines and mouse tumor models. Taken together, our studies define a novel mechanism of telomerase regulation by small non-coding RNAs and identify miR-498 as an important mediator for the anti-tumor activity of 1,25(OH)(2)D(3).

Severe hypercalcemic crisis in an infant with idiopathic infantile hypercalcemia caused by mutation in CYP24A1 gene

We report on a male infant presenting at 4 months of age with failure to thrive, dehydration, hypotonia, lethargy, and vomiting. Laboratory and imaging tests revealed severe hypercalcemia (5.8 mmol/l), suppressed parathyroid hormone (0.41 pmol/l), hypercalciuria (8.0 mmol/mmol creatinine), elevated 25-hydroxyvitamin D3 (over 600 nmol/l), and nephrocalcinosis. These symptoms are characteristic of idiopathic infantile hypercalcemia (IIH, MIM 143880). Conservative therapy (parenteral rehydration, diuretics, corticosteroids, bisphosphonates, and vitamin D prophylaxis withdrawal) was not able to improve the symptoms and laboratory values, and acute hemodiafiltration was necessary to normalize hypercalcemia. Clinical symptoms resolved rapidly after normalization of serum calcium levels. Molecular genetic testing revealed a homozygous mutation (R396W) in the CYP24A1 gene (MIM 126065) encoding 25-hydroxyvitamin D3 24-hydroxylase, which is the key enzyme responsible for 1,25-dihydroxyvitamin D3 degradation. The CYP24A1 gene mutation leads to the increased sensitivity of the patients to even prophylactic doses of vitamin D and to the development of severe symptomatic hypercalcemia in patients with IIH. Our patient is only the thirteenth patient with IIH caused by mutation in the CYP24A1 gene and the first one needing acute hemodiafiltration for severe symptomatic hypercalcemic crisis. In all patients with suspected IIH the DNA analysis for CYP24A1 gene mutations should be performed regardless of the type of vitamin D supplementation and serum levels of vitamin D.

The serum 24,25-dihydroxyvitamin D concentration, a marker of vitamin D catabolism, is reduced in chronic kidney disease

Chronic kidney disease is characterized, in part, as a state of decreased production of 1,25-dihydroxyvitamin D (1,25(OH)2D); however, this paradigm overlooks the role of vitamin D catabolism. We developed a mass spectrometric assay to quantify serum concentration of 24,25-dihydroxyvitamin D (24,25(OH)2D), the first metabolic product of 25-hydroxyvitamin D (25(OH)D) by CYP24A1, and determined its clinical correlates and associated outcomes among 278 participants with chronic kidney disease in the Seattle Kidney Study. For eGFRs of 60 or more, 45–59, 30–44, 15–29, and under 15 ml/min/1.73m2, the mean serum 24,25(OH)2D concentrations significantly trended lower from 3.6, 3.2, 2.6, 2.6, to 1.7 ng/ml, respectively. Non-Hispanic Black race, diabetes, albuminuria, and lower serum bicarbonate were also independently and significantly associated with lower 24,25(OH)2D concentrations. The 24,25(OH)2D concentration was more strongly correlated with that of parathyroid hormone than was 25(OH)D or 1,25(OH)2D. A 24,25(OH)2D concentration below the median was associated with increased risk of mortality in unadjusted analysis, but this was attenuated with adjustment for potential confounding variables. Thus, chronic kidney disease is a state of stagnant vitamin D metabolism characterized by decreases in both 1,25(OH)2D production and vitamin D catabolism.

A role for the phosphatidylinositol 3-kinase – protein kinase C zeta – Sp1 pathway in the 1,25-dihydroxyvitamin D3 induction of the 25-hydroxyvitamin D3 24-hydroxylase gene in human kidney cells

The molecular mechanisms that underlie non-genomic induction of the 25-hydroxyvitamin D3 24-hydroxylase (CYP24) gene promoter by the steroid hormone, 1,25-Dihydroxyvitamin D3 (1,25D), are poorly understood. Although we have previously identified a functional inverted GC-box in the early promoter at −113/−105bp, it is not known whether this site is important for 1,25D induction of the promoter. Using transfected human embryonic kidney (HEK) 293T cells, we now report the functional characterisation of the GC-box and that 1,25D induction of the promoter requires PI3-kinase, PKCζ and Sp1 but not Sp3. The data show that 1,25D rapidly stimulates PI3-kinase activity which is required for the activation of PKCζ and the phosphorylation of Sp1. The effects of the PI3-kinase inhibitor, LY294002, and a dominant negative PKCζ mutant on 1,25D induction of wild-type and a GC-box mutated CYP24 promoter constructs are consistent with the Sp1 site being the target of both kinases. However, these kinases are not required for basal expression of the CYP24 promoter. The data establish a novel non-genomic mechanism which couples 1,25D to the induction of CYP24 gene transcription via the PI3-kinase – PKCζ – Sp1 pathway acting through the GC-box.

The Candidate Oncogene CYP24A1: A Potential Biomarker for Colorectal Tumorigenesis

The main autocrine/paracrine role of the active metabolite of vitamin D(3), 1alpha,25-dihydroxyvitamin D(3) (1,25-D(3)), 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 D(3) 24-hydroxylase (CYP24A1) is considered to be the main enzyme determining the biological half-life of 1,25-D(3), 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-D(3) availability, decreasing its antiproliferative effect.

Antineoplastic agents target the 25-hydroxyvitamin D3 24-hydroxylase messenger RNA for degradation: implications in anticancer activity

Calcitriol or 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] has antitumor activity and hence its levels in patients may play an important role in disease outcome. Here, we report that the antineoplastic agents, daunorubicin hydrochloride, etoposide, and vincristine sulfate inhibited the ability of 1,25(OH)(2)D(3) to cause the accumulation of mRNA for kidney 25-hydroxyvitamin D(3) 24-hydroxylase (CYP24), an enzyme which catabolizes this hormone. This was not due to a drug-induced cytotoxic effect, reduction in the expression of the vitamin D receptor or inhibition of the vitamin D receptor-mediated activation of the mitogen-activated protein kinases or CYP24 promoter activity. Interestingly, there was selective degradation of CYP24 mRNA in the presence of the drugs. This was accompanied by an enhancement in the levels of 1,25(OH)(2)D(3) in cells incubated with 25-hydroxy vitamin D(3). These data identify a novel mechanism of action of some commonly used antineoplastic agents which by decreasing the stability of CYP24 mRNA would prolong the bioavailability of 1,25(OH)(2)D(3) for anticancer actions.

25-Hydroxyvitamin D31α-hydroxylase expression in breast cancer and use of non-1α-hydroxylated vitamin D analogue

IntroductionThe cytochrome P450 mitochondrial enzyme 25-hydroxyvitamin D3 1α-hydroxylase (1α-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 1α-hydroxylase is expressed for autocrine/paracrine regulation. To reduce risks of hypercalcemia during treatment with vitamin D, we have previously suggested use of non-1α-hydroxylated vitamin D analogues to target tissues where 1α-hydroxylase is expressed, including the parathyroid glands in secondary hyperparathyroidism. The present study was undertaken to examine expression of 1α-hydroxylase in breast cancer and to investigate whether a non-1α-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 1α-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 μm 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.Results1α-hydroxylase protein was demonstrated in 79% and 100% of breast cancer specimens and normal breast, respectively. The overall relative mRNA levels of 1α-hydroxylase and 24-hydroxylase in normal breast compared to breast tumors were: 1α-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-1α-hydroxylated vitamin D analogue prodrug of EB1089 (EB1285) was demonstrated in MCF-7 cells, which express 1α-hydroxylase. The activity of EB1285 was about 20% of 1,25(OH)2D3.ConclusionThese results demonstrate nearly normal expression levels of 1α-hydroxylase, 24-hydroxylase and VDR in the majority of investigated breast cancer specimens. A non-1α-hydroxylated vitamin D analogue displayed activity in breast cancer cells. Such analogues may present future therapeutic options for proliferative disorders where 1α-hydroxylase is expressed.

Molecular action of 1,25-dihydroxyvitamin D3 and phorbol ester on the activation of the rat cytochrome P450C24 (CYP24) promoter: role of MAP kinase activities and identification of an important transcription factor binding site

Although investigations of the transcriptional regulation of the rat cytochrome P450C24 [CYP24 (25-hydroxyvitamin D3 24-hydroxylase)] gene by 1,25D (1,25-dihydroxyvitamin D3) at either the genomic, or more recently at the non-genomic, level have provided insight into the mechanism of control of 1,25D levels, this regulation is still poorly characterized. Using HEK-293T cells (human embryonic kidney 293T cells), we reported that 1,25D induction of CYP24 requires JNK (c-Jun N-terminal kinase) but not the ERK1/2 (extracellular-signal-regulated kinase 1/2). The phenomenon of synergistic up-regulation of CYP24 expression by PMA and 1,25D is well known and was found to be protein kinase C-dependent. Whereas ERK1/2 was not activated by 1,25D alone, its activation by PMA was potentiated by 1,25D also. The importance of ERK1/2 for transcriptional synergy was demonstrated by transfection of a dominant-negative ERK1(K71R) mutant (where K71R stands for Lys71-->Arg), which resulted in a reduced level of synergy on a CYP24 promoter-luciferase construct. JNK was also shown to be required for synergy. We report, in the present study, the identification of a site located at -171/-163, about 30 bp upstream of the vitamin D response element-1 in the CYP24 proximal promoter. This sequence, 5'-TGTCGGTCA-3', is critical for 1,25D induction of CYP24 and is therefore termed the vitamin D stimulatory element. The vitamin D stimulatory element, a target for the JNK module, and an Ets-1 binding site were shown to be vital for synergy between PMA and 1,25D. This is the first report to identify the DNA binding sequences required for the synergy between PMA and 1,25D and a role for JNK on the CYP24 gene promoter.

5α‐dihydrotestosterone inhibits 1α,25‐dihydroxyvitamin D3‐induced expression of CYP24 in human prostate cancer cells

A cross-talk between 1alpha,25-dihydroxyvitamin D(3) [1alpha,25-(OH)(2)D(3)] and 5alpha-dihydrotestosterone (DHT) in the growth inhibition has been demonstrated, but the mechanism is unknown. The expression of 25-hydroxyvitamin D(3) 24-hydroxylase (24-hydroxylase) was measured using a real-time quantitative RT-PCR assay and the catabolism of 1alpha,25-(OH)(2)D(3) was measured using a radioreceptor assay. Real-time RT-PCR showed that DHT at 1-100 nM significantly inhibited 1alpha,25-(OH)(2)D(3)-induced expression of 24-hydroxylase in LNCaP cells. Furthermore, the catabolism of 1alpha,25-(OH)(2)D(3) was decreased by 10 nM DHT. An androgen receptor (AR) antagonist, Casodex antagonized the DHT effect, whereas an AR agonist (due to the mutant AR in LNCaP cells) hydroxyflutamide did not. We demonstrated, for the first time, that DHT reduces the ability of 1alpha,25-(OH)(2)D(3) to induce 24-hydroxylase expression. Our results not only support the earlier finding of a cross-talk between androgen and vitamin D in human prostate cancer cells but also provide a possible mechanism how androgen and vitamin D signaling pathways may interact.

Functional Cooperation between CCAAT/Enhancer-Binding Proteins and the Vitamin D Receptor in Regulation of 25-Hydroxyvitamin D3 24-Hydroxylase

1,25-Dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] induces the synthesis of 25-hydroxyvitamin D(3) 24-hydroxylase [24(OH)ase], an enzyme involved in its catabolism, thereby regulating its own metabolism. Here we demonstrate that CCAAT enhancer binding protein beta (C/EBPbeta) is induced by 1,25(OH)(2)D(3) in kidney and in osteoblastic cells and is a potent enhancer of vitamin D receptor (VDR)-mediated 24(OH)ase transcription. Transfection studies indicate that 1,25(OH)(2)D(3) induction of 24(OH)ase transcription is enhanced a maximum of 10-fold by C/EBPbeta. Suppression of 1,25(OH)(2)D(3)-induced 24(OH)ase transcription was observed with dominant negative C/EBP or osteoblastic cells from C/EBPbeta(-/-) mice. A C/EBP site was identified at positions -395 to -388 (-395/-388) in the rat 24(OH)ase promoter. Mutation of this site inhibited C/EBPbeta binding and markedly attenuated the transcriptional response to C/EBPbeta. We also report the cooperation of CBP/p300 with C/EBPbeta in regulating VDR-mediated 24(OH)ase transcription. We found that not only 1,25(OH)(2)D(3) but also parathyroid hormone (PTH) can induce C/EBPbeta expression in osteoblastic cells. PTH potentiated the induction of C/EBPbeta and 24(OH)ase expression in response to 1,25(OH)(2)D(3) in osteoblastic cells. Data with the human VDR promoter (which contains two putative C/EBP sites) indicate a role for C/EBPbeta in the protein kinase A-mediated induction of VDR transcription. From this study a fundamental role has been established for the first time for cooperative effects and cross talk between the C/EBP family of transcription factors and VDR in 1,25(OH)(2)D(3)-induced transcription. These findings also indicate a novel role for C/EBPbeta in the cross talk between PTH and 1,25(OH)(2)D(3) that involves the regulation of VDR transcription.

25-hydroxyvitamin D3 is an active hormone in human primary prostatic stromal cells.

According to the present paradigm, 1alpha,25-dihydroxyvitamin D3 [1alpha,25-(OH)2D3] is a biologically active hormone; whereas 25-hydroxyvitamin D3 (25OHD3) is regarded as a prohormone activated through the action of 25-hydroxyvitamin D3 1alpha-hydroxylase (1alpha-hydroxylase). Although the role of vitamin D3 in the regulation of growth and differentiation of prostatic epithelial cells has been well studied, its action and metabolism in prostatic stroma are still largely unknown. We investigated the effects of 25OHD3 and 1alpha,25-(OH)2D3 on two human stromal primary cultures termed P29SN and P32S. In a cell proliferation assay, 25OHD3 was found at physiological concentrations of 100-250 nM to inhibit the growth of both primary cultures, whereas 1alpha,25-(OH)2D3 at a pharmacological concentration of 10 nM exhibited the growth-inhibitory effects on P29SN cells but not on P32S cells. Quantitative real-time RT-PCR analysis revealed that both 25OHD3 and 1alpha,25-(OH)2D3 induced 25-hydroxyvitamin D3 24-hydroxylase (24-hydroxylase) mRNA in a dose- and time-dependent manner. By inhibiting 1alpha-hydroxylase and/or 24-hydroxylase enzyme activities, the induction of 24-hydroxylase mRNA by 250 nM 25OHD3 was clearly enhanced, suggesting that 1alpha-hydroxylation is not a prerequisite for the hormonal activity of 25OHD3. Altogether our results suggest that 25OHD3 at a high but physiological concentration acts as an active hormone with respect to vitamin D3 responsive gene regulation and suppression of cell proliferation.

2-Methylene-19-nor-(20S)-1,25-dihydroxyvitamin D3 Potently Stimulates Gene-specific DNA Binding of the Vitamin D Receptor in Osteoblasts

2-Methylene-19-nor-(20S)-1,25-dihydroxyvitamin D3 (2MD) is a highly potent analog of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) whose actions are mediated through the vitamin D receptor (VDR). In this report, we have replicated this increased potency of 2MD in vitro using osteoblastic cells and explored its underlying molecular mechanism. 2MD stimulates the expression of several vitamin D-sensitive genes including 25-hydroxyvitamin D3-24 hydroxylase (Cyp24), osteopontin and receptor activator of NF kappa B ligand and suppresses osteoprotegerin at concentrations two logs lower than that for 1,25(OH)2D3. 2MD is also more potent in stimulating transfected chimeric reporter genes under either Cyp24 or the osteocalcin promoter control. Enhanced potency is retained regardless of medium serum content. Interestingly, the uptake of both 1,25(OH)2D3 and 2MD into cells is similar, as is their rapid association with the VDR. This indicates that comparable levels of occupied VDR do not elicit equivalent levels of transactivation. Using chromatin immunoprecipitation (ChIP), however, we observed a strong correlation between DNA-bound receptor and the level of induced transcription suggesting a 2MD-induced increase in affinity of the VDR for DNA. Additional studies using a mammalian two-hybrid system and ChIP indicate that 2MD is also more potent in promoting interaction with RXR and the coactivators SRC-1 and DRIP205. Finally, protease digestion studies revealed a unique VDR conformation in the presence of 2MD. These studies suggest that the molecular mechanism of 2MD potency is due to its ability to promote enhanced levels of specific DNA binding by the VDR and could suggest possible explanations for the tissue- and gene-selective actions of 2MD.

Increased 25-hydroxyvitamin D3 1alpha-hydroxylase and reduced 25-hydroxyvitamin D3 24-hydroxylase expression in parathyroid tumors--new prospects for treatment of hyperparathyroidism with vitamin d.

Vitamin D analogues are in clinical use for prevention and treatment of secondary hyperparathyroidism (HPT) in chronic renal failure. Despite recent advances there is a need for vitamin D derivatives with maintained parathyroid hormone suppressive activity and less hypercalcemic and hyperphosphatemic toxicity. Here we show coincident increased expression of the vitamin D activating enzyme 25-hydroxyvitamin D(3) 1alpha-hydroxylase (1alpha-hydroxylase) and reduced expressions of the 1,25(OH)(2)D(3) catabolizing enzyme 25-hydroxyvitamin D(3) 1alpha-hydroxylase (1alpha-hydroxylase) in the majority of investigated parathyroid adenomas and secondary hyperplastic glands. In addition, this relationship was found for the mitochondrial CYP27A enzyme (25-hydroxylase), a potential physiological vitamin D(3) 25-hydroxylase. These findings should be considered in future development of vitamin D analogues for treatment of HPT.

YY1 Represses Vitamin D Receptor-Mediated 25-Hydroxyvitamin D3 24-Hydroxylase Transcription: Relief of Repression by CREB-Binding Protein

YY1 Represses Vitamin D Receptor-Mediated 25-Hydroxyvitamin D3 24-Hydroxylase Transcription: Relief of Repression by CREB-Binding Protein