24-hydroxylase Gene Research Articles

Importance of apical membrane delivery of 1,25-dihydroxyvitamin D3 to vitamin D-responsive gene expression in the colon

Synthetic conjugation of a glucuronide to 1,25-dihydroxyvitamin D3 (1,25D3) to produce β-25-monoglucuronide-1,25D3 (βGluc-1,25D3) renders the hormone biologically inactive and resistant to mammalian digestive enzymes. However, β-glucuronidase produced by bacteria in the lower intestinal tract can cleave off the glucuronide, releasing the active hormone. In mice given a single oral dose of 1,25D3, 24-hydroxylase (Cyp24a1) gene expression was strongly enhanced in the duodenum, but not in the colon, despite circulating concentrations of 1,25D3 that peaked at ∼3.0 nmol/l. In contrast, in mice treated with an equimolar dose of βGluc-1,25D3, Cyp24a1 gene expression increased 700-fold in the colon but was significantly weaker in the duodenum compared with mice treated with 1,25D3. Similar results were observed with another vitamin D-dependent gene. When administered subcutaneously, 1,25D3 weakly stimulated colon Cyp24a1 gene expression while βGluc-1,25D3 again resulted in strong enhancement. Surgical ligation to block passage of ingesta beyond the upper intestinal tract abolished upregulation of colon Cyp24a1 gene expression by orally and subcutaneously administered βGluc-1,25D3. Feeding βGluc-1,25D3 for 5 days revealed a linear, dose-dependent increase in colon Cyp24a1 gene expression but did not significantly increase plasma 1,25D3 or calcium concentrations. This study indicates that the colon is relatively insensitive to circulating concentrations of 1,25D3 and that the strongest gene enhancement occurs when the hormone reaches the colon via the lumen of the intestinal tract. These findings have broad implications for the use of vitamin D compounds in colon disorders and set the stage for future therapeutic studies utilizing βGluc-1,25D3 in their treatment.

On the formation and possible biological role of 25-hydroxycholesterol

The oxysterol 25-hydroxycholesterol is a widely used compound displaying an array of pharmacological actions in invitro systems and cell based experimental systems. In spite of the frequent use of this compound over the last few decades and a large number of studies invitro and invivo, its mechanism of formation invivo is still not well understood. Cholesterol autoxidation does not seem to be an important contributor to invivo formation of 25-hydroxycholesterol. A number of different cytochrome P450 enzymes such as CYP27A1 and CYP3A4 have been reported to catalyze the conversion of cholesterol to 25-hydroxycholesterol invitro, but the importance of these reactions invivo remains unclear. The dioxygenase enzyme cholesterol 25-hydroxylase has been shown to generate 25-hydroxycholesterol, but in cholesterol 25-hydroxylase knockout mice there are still significant levels of 25-hydroxycholesterol in several tissues. This suggests that cholesterol 25-hydroxylase is not the sole producer of 25-hydroxycholesterol. The relative importance of different mechanisms of formation of 25-hydroxycholesterol invivo have still to be elucidated. The maintenance of cholesterol homeostasis is of great importance to supply tissues with the appropriate amount of cholesterol and prevent accumulation that may affect health. Numerous articles mention 25-hydroxycholesterol as an important regulator of cholesterol metabolism. However, mice with a disruption of the cholesterol 25-hydroxylase gene regulate cholesterol metabolism normally and patients with highly elevated levels of 25-hydroxycholesterol also display normal cholesterol and bile acid levels. These reports challenge the hypothesis that 25-hydroxycholesterol is an important regulator of cholesterol metabolism. Recent reports suggest that 25-hydroxycholesterol and one of its metabolites may have functions in regulation of humoral immunity. Thus, 25-hydroxycholesterol may be more important as a regulator of immunity than as a regulator of cholesterol metabolism.

Polymorphisms Related to the Serum 25-Hydroxyvitamin D Level and Risk of Myocardial Infarction, Diabetes, Cancer and Mortality. The Tromsø Study

ObjectiveLow serum 25(OH)D levels are associated with cardiovascular risk factors, and also predict future myocardial infarction (MI), type 2 diabetes (T2DM), cancer and all-cause mortality. Recently several single nucleotide polymorphisms (SNPs) associated with serum 25-hydroxyvitamin D (25(OH)D) level have been identified. If these relations are causal one would expect a similar association between these SNPs and health.MethodsDNA was prepared from subjects who participated in the fourth survey of the Tromsø Study in 1994–1995 and who were registered with the endpoints MI, T2DM, cancer or death as well as a randomly selected control group. The endpoint registers were complete up to 2007–2010. Genotyping was performed for 17 SNPs related to the serum 25(OH)D level.ResultsA total of 9528 subjects were selected for genetic analyses which were successfully performed for at least one SNP in 9471 subjects. Among these, 2025 were registered with MI, 1092 with T2DM, 2924 with cancer and 3828 had died. The mean differences in serum 25(OH)D levels between SNP genotypes with the lowest and highest serum 25(OH)D levels varied from 0.1 to 7.8 nmol/L. A genotype score based on weighted risk alleles regarding low serum 25(OH)D levels was established. There was no consistent association between the genotype score or individuals SNPs and MI, T2DM, cancer, mortality or risk factors for disease. However, for rs6013897 genotypes (located at the 24-hydroxylase gene (CYP24A1)) there was a significant association with breast cancer (P<0.05).ConclusionOur results do not support nor exclude a causal relationship between serum 25(OH)D levels and MI, T2DM, cancer or mortality, and our observation on breast cancer needs confirmation. Further genetic studies are warranted, particularly in populations with vitamin D deficiency.Trial RegistrationClinicalTrials.gov NCT01395303

Abstract 1653: Genetic variation in the vitamin D pathway and risk for colorectal cancer in African Americans

Abstract Background: Colorectal cancer (CRC) is the third leading cause of cancer-related death in both sexes in the United States. There is a large disparity in both CRC incidence and survival between African Americans (AAs) and all other US racial groups. Differences in serum vitamin D levels could contribute to this disparity because vitamin D is thought to protect against CRC and AAs have lower serum vitamin D levels. In addition, genetic variants in vitamin D-related genes could modify the association between vitamin D levels and CRC risk. Methods: We tested for associations between CRC and 39 single nucleotide polymorphisms (SNPs) in vitamin D-related genes (CYP27A1, GC, CYP3A4, CYP2R1, DHCR7/NADSYN1, VDR, CYP27B1 and CYP24A1). We genotyped 357 AAs cases and 443 AA controls from the North Carolina Colorectal Cancer Study. We calculated odds ratios (OR) using logistic regression, assuming an additive genetic model and controlling for age, gender, and ancestry. We also analyzed SNP associations for colon and rectal cancer cases separately. We analyzed linkage disequilibrium and haplotype association using Haploview. Results: Associations (p&amp;lt;0.05) were detected between CRC and two SNPs in the vitamin D binding protein gene GC, rs3755967 (OR, 0.69; P = .039,) and rs16847024 (OR, 1.50; P = .047,). We also detected an association with a SNP in the 24-hydroxylase gene CYP24A1, rs73913755 (OR, 0.75; P = .034,), which degrades vitamin D. A trend for association was detected in the 25-hydroxylase gene CYP2R1, rs10741657 (OR, 0.79; P = .051), which converts cholecalciferol into calcidiol. When we analyzed colon and rectal cancers separately, only one SNP was associated with colon cancer whereas 9 SNPs were associated with rectal cancer. In particular, rs73913755 (OR, 0.39; P = 3 x 10-7) was statistically significant even after adjustment for multiple testing. Conclusion: We tested vitamin D-related SNPs and found suggestive evidence that they influence the risk of developing CRC in AAs, with differences in risk between the colon and rectum. Comparison of these data with data from US whites could shed light on disparities in these different US populations. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1653. doi:1538-7445.AM2012-1653

Effect of phorbol 12‐myristate 13‐acetate activated signaling pathways on 1α, 25 dihydroxyvitamin D3 Regulated Human 25‐hydroxyvitamin D3 24‐hydroxylase Gene Expression in Differentiated Caco‐2 Cells

Phorbol-12-myristate-13-acetate (PMA), a protein kinase C(PKC) activator, can modulate 1α, 25 dihydroxyvitamin D(3) (1,25(OH)(2)D(3))-induced expression of the 24-hydroxylase (CYP24A1) gene but this has not been studied in differentiated enterocytes, a primary 1,25(OH)(2) D(3) target cell. We found that in differentiated Caco-2 cells, an established model of the mature absorptive epithelial cell, PMA significantly enhanced 1,25(OH)(2)D(3)-induced human CYP24A1 (hCYP24A1) mRNA accumulation and hCYP24A1 promoter-luciferase reporter gene activation by 150%. Reporter gene studies further identified the region between -298 and +74 bp in the hCYP24A1 promoter as critical for the PMA enhancing effect and chromatin immunoprecipitation (ChIP) analysis showed that PMA enhanced 1,25(OH)(2)D(3)-induced binding of vitamin D receptor to this region. PMA can activate PKC, ERK1/2, and p38 MAP kinases and inhibition of these signaling pathways reduced both 1,25(OH)(2)D(3)-induced hCYP24A1 gene transcription and the enhancing effect of PMA. The PMA enhancing effect on 1,25(OH)(2)D(3) action was evident in a minimal promoter with three osteocalcin VDREs and was reduced after mutation of a putative vitamin D stimulatory site in the hCYP24A1 promoter. In contrast, mutation of a Ets binding site (EBS) in the hCYP24A1 promoter had no impact on 1,25(OH)(2)D(3) action or the PMA enhancing effect. These data suggest that in the differentiated enterocyte PMA-induced activation of several signaling pathways contribute to 1,25(OH)(2)D(3)-induced hCYP24A1 gene expression through multiple regulatory motifs within the proximal hCYP24A1 promoter.

Genetic Defect inCYP24A1, the Vitamin D 24-Hydroxylase Gene, in a Patient with Severe Infantile Hypercalcemia

Idiopathic infantile hypercalcemia (IIH) is a disorder the genetic etiology and physiological basis of which are not well understood. The objective of the study was to describe the underlying physiology and genetic cause of hypercalcemia in an infant with severe IIH and to extend these genetic findings into an additional cohort of children with IIH. This was an inpatient study of a single patient with consanguineous parents at an academic medical center with follow-up in a specialty clinic cohort. The patient population was one patient with severe IIH for gene discovery and physiological testing and 27 patients with idiopathic infantile hypercalcemia in the replication cohort. Interventions included a calcium isotopic absorption study as well as homozygosity mapping and whole-exome sequencing in a single patient followed up by gene sequencing in replication cohort. Fractional absorption of calcium and genetic variants causing hypercalcemia were measured. Intestinal calcium absorption was extremely elevated (∼90%). A rare homozygous deletion in the CYP24A1 gene was found, leading to the loss of a single highly conserved amino acid. In vivo functional studies confirmed decreased 24-hydroxylase activity because the subject had undetectable levels of 24,25-dihydroxyvitamin D. No coding variants in CYP24A1 were found in the 27 additional patients with IIH. Our study confirms that CYP24A1 plays a causal role in some but not all cases of IIH via markedly increased intestinal absorption of calcium, suggesting that genetic diagnosis could be helpful in a subset of IIH patients. This case demonstrates the power of an unbiased, genome-wide approach accompanied by informative physiological studies to provide new insights into human biology.

Targeted delivery of vitamin D to the colon using β-glucuronides of vitamin D: therapeutic effects in a murine model of inflammatory bowel disease

1,25-Dihydroxyvitamin D(3) [1,25(OH)(2)D] has been shown to inhibit development of dextran sodium sulfate (DSS)-induced colitis in mice but can also cause hypercalcemia. The aim of this study was to evaluate whether β-glucuronides of vitamin D could deliver 1,25(OH)(2)D to the colon to ameliorate colitis while reducing the risk of hypercalcemia. Initial studies demonstrated that bacteria residing in the lower intestinal tract were capable of liberating 1,25(OH)(2)D from 1,25-dihydroxyvitamin D(3)-25-β-glucuronide [β-gluc-1,25(OH)(2)D]. We also determined that a much greater upregulation of the vitamin D-dependent 24-hydroxylase gene (Cyp24) was induced in the colon by treatment of mice with an oral dose of β-gluc-1,25(OH)(2)D than 1,25(OH)(2)D, demonstrating targeted delivery of 1,25(OH)(2)D to the colon. We then tested β-glucuronides of vitamin D in the mouse DSS colitis model in two studies. In mice receiving DSS dissolved in distilled water and treated with 1,25(OH)(2)D or β-gluc-1,25(OH)(2)D, severity of colitis was reduced. Combination of β-gluc-1,25(OH)(2)D with 25-hydroxyvitamin D(3)-25-β-glucuronide [β-gluc-25(OH)D] resulted in the greatest reduction of colitis lesions and symptoms in DSS-treated mice. Plasma calcium concentrations were lower in mice treated with β-gluc-1,25(OH)(2)D alone or in combination with β-gluc-25(OH)D than in mice treated with 1,25(OH)(2)D, which were hypercalcemic at the time of death. β-Glucuronides of vitamin D compounds can deliver 1,25(OH)(2)D to the lower intestine and can reduce symptoms and lesions of acute colitis in this model.

Coping strategies adopted by pregnant women in Pakistan to resist spousal violence

[1] Novakovic B, Sibson M, Ng HK, Manuelpillai U, Rakyan V, Down T, et al. Placentaspecific methylation of the vitamin D 24-hydroxylase gene: implications for feedback autoregulation of active vitamin D levels at the fetomaternal interface. J Biol Chem 2009;284(22):14838–48. [2] Scholl TO, Chen X. Vitamin D intake during pregnancy: association with maternal characteristics and infant birth weight. Early Hum Dev 2009;85(4):231–4. [3] Van der Meer IM, Karamali NS, Boeke AJ, Lips P, Middelkoop BJ, Verhoeven I, et al. High prevalence of vitamin D deficiency in pregnant non-Western women in The Hague, Netherlands. Am J Clin Nutr 2006;84(2):350–3. [4] McCullough ML. Vitamin D deficiency in pregnancy: bringing the issues to light. J Nutr 2007;137(2):305–6.

In Vivo Activation of the Intracrine Vitamin D Pathway in Innate Immune Cells and Mammary Tissue during a Bacterial Infection

Numerous in vitro studies have shown that toll-like receptor signaling induces 25-hydroxyvitamin D3 1α-hydroxylase (1α-OHase; CYP27B1) expression in macrophages from various species. 1α-OHase is the primary enzyme that converts 25-hydroxyvitamin D3 to 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). Subsequently, synthesis of 1,25(OH)2D3 by 1α-OHase in macrophages has been shown to modulate innate immune responses of macrophages. Despite the numerous in vitro studies that have shown 1α-OHase expression is induced in macrophages, however, evidence that 1α-OHase expression is induced by pathogens in vivo is limited. The objective of this study was to evaluate 1α-OHase gene expression in macrophages and mammary tissue during an in vivo bacterial infection with Streptococcus uberis. In tissue and secreted cells from the infected mammary glands, 1α-OHase gene expression was significantly increased compared to expression in tissue and cells from the healthy mammary tissue. Separation of the cells by FACS9 revealed that 1α-OHase was predominantly expressed in the CD14+ cells isolated from the infected mammary tissue. The 24-hydroxylase gene, a gene that is highly upregulated by 1,25(OH)2D3, was significantly more expressed in tissue and cells from the infected mammary tissue than from the healthy uninfected mammary tissue thus indicating significant local 1,25(OH)2D3 production at the infection site. In conclusion, this study provides the first in vivo evidence that 1α-OHase expression is upregulated in macrophages in response to bacterial infection and that 1α-OHase at the site of infection provides 1,25(OH)2D3 for local regulation of vitamin D responsive genes.

Abstract 4944: Regulation of vitamin D 24-hydroxylase gene expression in human prostate cancer by epigenetic mechanisms

Abstract 1,25-dihydroxycholecalciferol (calcitriol), the active form of vitamin D3, plays a major role in regulating calcium homeostasis and bone mineralization. Calcitriol also modulates cellular proliferation and differentiation in a variety of cell types. Furthermore, calcitriol has an anti-tumor effect in several tumor models. 1,25-(OH)2D-hydroxylase (24-hydroxylase), encoded by the CYP24A1 gene, plays a key role in attenuation of calcitriol effects by degrading calcitriol to a much less active vitamin D metabolite. CYP24A1 is dysregulated in wide range of tumors. The over-expression of CYP24A1 is associated with poor prognosis of some human cancers. Interestingly, CYP24A1 is down-regulated in human prostate cancer. Little is known about the mechanism(s) underlying the regulation of CYP24A1 expression in human prostate cancer. We have analyzed 3 prostate cancer cell lines (LNCaP, DU-145 and PC3) which express variable levels of CYP24A1. We have found that CYP24A1 gene expression is inversely correlated with promoter DNA methylation in human prostate cell lines. Treatment with the DNA methyltransferase inhibitor, 5-aza-2′-deoxycytidine (DAC), activates CYP24A1 gene expression in prostate cancer cells. In vitro methylation of the CYP24A1 promoter by methylases SssI, HpaII and HhaI represses its promoter activity. Furthermore, inhibition of histone deacetylases by trichostatin A (TSA) enhances the expression of CYP24A1 in prostate cancer cells. ChIP-qPCR reveals that histone modifications are associated with the CYP24A1 promoter region. Treatment with TSA increases acetylated H3K9 and dimethylated H3K4 and simultaneously decreases dimethylated H3K9 at the CYP24A1 promoter. ChIP-qPCR assay also shows that treatment of prostate cancer cells with DAC and TSA increases the recruitment of VDR to the CYP24A1 promoter. We have also collected paired samples of benign and malignant tissue from 30 human prostatectomy samples. RT-PCR analysis of paired human prostate samples reveals that CYP24A1 expression is down-regulated in prostate malignant lesions compared to matched benign lesions (p&amp;lt;0.001). Bisulfite DNA pyrosequencing reveals that CYP24A1 gene is hypermethylated in malignant lesions compared to matched benign lesions. Taken together, these data indicate that the repression of CYP24A1 gene expression in human prostate cancer cells is mediated in part by promoter DNA methylation and repressive histone modifications. Epigenetic repression of CYP24A1 may involve blocked recruitment of VDR to CYP24A1 regulatory regions, and nucleation of repressive chromatin structures at the CYP24A1 promoter. This study was supported by NIH/NCI grants CA067267, CA095045 and CA085142. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4944.

The antifungal drug voriconazole is an efficient inhibitor of brain cholesterol 24S-hydroxylase in vitro and in vivo

Cholesterol 24S-hydroxylase (CYP46A1) is of key importance for cholesterol homeostasis in the brain. This enzyme seems to be resistant toward most regulatory factors and at present no drug effects on its activity have been described. The crystal structures of the substrate-free and substrate-bound CYP46A1 were recently determined (Mast et al., Crystal structures of substrate-bound and substrate-free cytochrome P450 46A1, the principal cholesterol hydroxylase in the brain. Proc. Natl. Acad. Sci. USA. 2008. 105: 9546-9551). These structural studies suggested that ligands other than sterols can bind to CYP46A1. We show here that the antifungal drug voriconazole binds to the enzyme in vitro and inhibits CYP46A1-mediated cholesterol 24-hydroxylation with a Ki of 11 nM. Mice treated with daily intraperitoneal injections of voriconazole for 5 days had high levels of voriconazole in the brain and significantly reduced brain levels of 24S-hydroxycholesterol. The levels of squalene, lathosterol, and HMG-CoA reductase mRNA were reduced in the brain of the voriconazole-treated animals as well, indicating a reduced cholesterol synthesis. Most of this effect may be due to a reduced utilization of cholesterol by CYP46A1. One of the side-effects of voriconazole is visual disturbances. Because CYP46A1 is also expressed in the neural retina, we discuss the possibility that the inhibition of CYP46A1 by voriconazole contributes to these visual disturbances.

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.

25-Hydroxycholesterol secreted by macrophages in response to Toll-like receptor activation suppresses immunoglobulin A production

25-Hydroxycholesterol is produced in mammalian tissues. The function of this oxysterol is unknown. Here we describe a central role for 25-hydroxycholesterol in regulating the immune system. In initial experiments, we found that stimulation of macrophage Toll-like receptors (TLR) induced expression of cholesterol 25-hydroxylase and the synthesis of 25-hydroxycholesterol. Treatment of naïve B cells with nanomolar concentrations of 25-hydroxycholesterol suppressed IL-2-mediated stimulation of B cell proliferation, repressed activation-induced cytidine deaminase (AID) expression, and blocked class switch recombination, leading to markedly decreased IgA production. Consistent with these findings, deletion of the mouse cholesterol 25-hydroxylase gene caused an increase in serum IgA. Conversely, inactivation of the CYP7B1 oxysterol 7alpha-hydroxylase, which degrades 25-hydroxycholesterol, decreased serum IgA. The suppression of IgA class switching in B cells by a macrophage-derived sterol in response to TLR activation provides a mechanism for local and systemic negative regulation of the adaptive immune response by the innate immune system.

Cholesterol 24-Hydroxylase: An Enzyme of Cholesterol Turnover in the Brain

Cholesterol 24-hydroxylase is a highly conserved cytochrome P450 that is responsible for the majority of cholesterol turnover in the vertebrate central nervous system. The enzyme is expressed in neurons, including hippocampal and cortical neurons that are important for learning and memory formation. Disruption of the cholesterol 24-hydroxylase gene in the mouse reduces both cholesterol turnover and synthesis in the brain but does not alter steady-state levels of cholesterol in the tissue. The decline in synthesis reduces the flow of metabolites through the cholesterol biosynthetic pathway, of which one, geranylgeraniol diphosphate, is required for learning in the whole animal and for synaptic plasticity in vitro. This review focuses on how the link between cholesterol metabolism and higher-order brain function was experimentally established.

Are side-chain oxidized oxysterols regulators also in vivo?

Oxsterols are oxygenated metabolites of cholesterol that are short-lived intermediates or end products in cholesterol excretion pathways. They are present in very low concentrations in mammalian systems, always accompanied by a high excess of cholesterol. According to current concepts, side-chain oxidized oxysterols may be mediators of many cholesterol-induced regulatory effects. When added to cultured cells in vitro, side-chain oxidized oxysterols limit intracellular cholesterol levels by at least three different mechanisms: 1) binding to Insig with subsequent block of the sterol regulatory element-binding proteins (SREBP)-mediated mechanism for regulation of sterol sensitive genes; 2) increasing degradation of hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase, eventually by a mechanism involving binding of Insig to the enzyme; 3) activation of LXR-mediated stimulation of cholesterol transporters and cholesterol metabolism. Addition of pure unesterified oxysterols to cell cultures is however highly unphysiological, and the in vivo relevance of such experiments is questionable. Transgenic mouse models with markedly reduced or increased concentration of some specific oxysterols do not present marked disturbances in cholesterol turnover and homeostasis. Oxysterol-binding proteins such as LXR have been conclusively shown to be of importance for cholesterol turnover in vivo, but their physiological ligands have not yet been defined with certainty. During the last few years, new experimental data has accumulated supporting the contention that side-chain oxysterols are involved in some LXR-mediated regulation in vivo, at least in some biological systems. The new findings will be critically reviewed here.

Cholesterol 24‐hydroxylase (CYP46A1) polymorphisms are associated with faster cognitive deterioration in Chinese older persons: a two‐year follow up study

We previously found that the polymorphisms of cholesterol 24-hydroxylase (CYP46A1) gene were associated with the risk of Alzheimer's disease (AD) in Chinese. However, its effect in predicting progression of cognitive decline remains unknown. Two hundred and eighty-one Chinese subjects (121 cognitively intact, 101 with mild cognitive impairment and 59 with mildly dementia) were followed-up with a mean (SD) duration of 25.22(5.74) months. Association between the CYP46A1 gene polymorphisms and 2-year cognitive deterioration were evaluated. At follow-up, 225(80.0%) subjects were reassessed. Sixty-three subjects were diagnosed as AD, 68 were MCI and 94 were cognitively intact. Among them, 158 had improved or remained stable while 67 deteriorated. The 'deteriorated' group was older than 'improved or stable' group (t-test, t = -2.87, p < 0.001). IVS2-150 polymorphism was associated with a higher risk of cognitive deterioration. Subjects with T allele were more likely to deteriorate compared with those without T allele (Pearson chi(2) = 8.98, df 2, p = 0.011). IVS3-128 CC genotype was higher in 'improved or stable' group (Likelihood Ratio = 6.55, df 2, p = 0.038), suggesting a protective role for this allele. The two other polymorphisms, IVS1-192 and IVS4-122, did not show any significant association with cognitive function. CYP46A1 gene may act to modulate the course of cognitive deterioration in late life.

Effects of MAPK signaling on 1,25‐dihydroxyvitamin D‐mediated CYP24 gene expression in the enterocyte‐like cell line, Caco‐2

We examined the role of the extracellular signal regulated kinases (ERK) in 1,25-dihydroxyvitamin D (1,25(OH)(2)D(3))-induced gene expression in the differentiated Caco-2 cells. 1,25(OH)(2)D(3)-regulated expression of the 25-hydroxyvitamin D, 24-hydroxylase (CYP24) gene (both natural gene and promoter construct) was strongly modulated by altering ERK activity (i.e., reduced by MEK inhibitors and dominant negative (dn) ERK1 and ERK2, activated by epidermal growth factor) but ERK inhibition had no effect on 1,25(OH)(2)D(3)-regulated expression of the transient receptor potential cation channel, subfamily V, member 6 (TRPV6). ERK5-mediated phosphorylation of the transcription factor Ets-1 enhanced 1,25(OH)(2)D(3)-mediated CYP24 gene transcription in proliferating but not differentiated Caco-2 cells due to reduced levels of ERK5 and Ets-1 (total and phosphoprotein levels) in differentiated cells. MEK inhibition reduced 1,25(OH)(2)D(3)-induced 3X-VDRE promoter activity but had no impact on the association of vitamin D receptor (VDR) with chromatin suggesting a role for co-activator recruitment in ERK-modulation of vitamin D-regulated CYP24 gene activation. Chromatin immunoprecipitation assays revealed that the ERK1/2 target, mediator 1 (MED1), is recruited to the CYP24, but not the TRPV6, promoter following 1,25(OH)(2)D(3) treatment. MED1 phosphorylation was sensitive to activators and inhibitors of the ERK1/2 signaling and MED1 siRNA reduced 1,25(OH)(2)D(3)-regulated human CYP24 promoter activity. This suggests ERK1/2 signaling enhances 1,25(OH)(2)D(3) effects on the CYP24 promoter by MED1-mediated events. Our data show that there are both promoter-specific and cell stage-specific roles for the ERK signaling pathway on 1,25(OH)(2)D(3)-mediated gene induction in enterocyte-like Caco-2 cells.

Nuclear Xenobiotic Receptor Pregnane X Receptor Locks Corepressor Silencing Mediator for Retinoid and Thyroid Hormone Receptors (SMRT) onto the CYP24A1 Promoter to Attenuate Vitamin D3 Activation

We have studied the molecular mechanism by which the nuclear xenobiotic receptors pregnane X receptor (PXR) and constitutive active/androstane receptor (CAR) regulate transcription of the vitamin D(3) 24-hydroxylase (CYP24A1) gene. In the absence of vitamin D(3), PXR activates the CYP24A1 gene by directly binding to and transactivating vitamin D-response elements (VDREs) within its promoter. Vitamin D(3) activates the CYP24A1 promoter by dissociating the corepressor silencing mediator for retinoid and thyroid hormone receptors (SMRT) from the vitamin D receptor (VDR) on those VDREs. PXR strongly represses vitamin D(3) activation of the CYP24A1 gene, in which PXR indirectly binds to and prevents vitamin D(3)-dependent dissociation of SMRT from the CYP24A1 promoter. The degree of the PXR-mediated locking of SMRT depends on the relative concentration of vitamin D(3) to the human PXR activator rifampicin; SMRT increased its dissociation as this ratio increased. CAR is also found to prevent dissociation of SMRT from the CYP24A1 promoter. Thus, our present study defines the novel molecular mechanism by which PXR and CAR mediate drug interactions with vitamin D(3) to regulate the CYP24A1 gene. Pxr(+/+) and Pxr(-/-) mice were continuously treated with mouse PXR activator PCN to evaluate the hypothesis that induction of the Cyp24a1 gene is responsible for the loss of bone mineral density often observed in patients treated continuously with PXR-activating drugs. PCN-dependent loss of mineral density is observed in the metaphyseal bones of only the Pxr(+/+) mice. This loss, however, does not correlate with the expression levels of the Cyp24a1 gene in these mice.

1α,25-Dihydroxyvitamin D 3-26,23-lactam analogues function as vitamin D receptor antagonists in human and rodent cells

(23 S,25 S)- N-Benzyl-1α,25-dihydroxyvitamin D 3-26,23-lactam ((23 S,25 S)- N-benzyl-1α,25-(OH) 2D 3-26,23-lactam, (23 S,25 S)-DLAM-1P) antagonizes nuclear vitamin D receptor (VDR)-mediated differentiation of human promyelocytic leukemia (HL-60) cells [Y. Kato, Y. Nakano, H. Sano, A. Tanatani, H. Kobayashi, R. Shimazawa, H. Koshino, Y. Hashimoto, K. Nagasawa, Synthesis of 1α,25-dihydroxy vitamin D 3-26,23-lactams (DLAMs), a novel series of 1α,25-dihydroxy vitamin D 3 antagonist, Bioorg. Med. Chem. Lett. 14 (2004) 2579–2583]. To enhance its VDR antagonistic actions, we synthesized multiple analogues of 1α,25-(OH) 2D 3-26,23-lactam. Among these analogues, (23 S,25 S)- N-phenetyl-1α,25-(OH) 2D 3-26,23-lactam, ((23 S,25 S)-DLAM-2P) had the strongest VDR binding affinity, which was 3 times higher than that of (23 S,25 S)-DLAM-1P. The 1α,25-(OH) 2D 3-26,23-lactam analogues never induced HL-60 cell differentiation even at 10 −6 M, but (23 S,25S)-DLAM-1P and (23 S,25 S)-DLAM-2P significantly and dose-dependently inhibited HL-60 differentiation induced by 10 −8 M 1α,25-dihydroxyvitamin D 3 (1α,25-(OH) 2D 3). These compounds also inhibited human and mouse cultures of osteoclast formation by marrow cells treated with 1α,25-(OH) 2D 3. Moreover, the 1α,25-(OH) 2D 3-26,23-lactam analogues minimally induced 25-hydroxyvitamin D 3-24-hydroxylase gene expression in HL-60 cells and human and mouse osteoblastic cells, but 10 −6 M (23 S,25 S)-DLAM-1P or (23 S,25 S)-DLAM-2P significantly blocked 24-hydroxylase gene expression induced by 10 −8 M 1α,25-(OH) 2D 3. (23 S,25 S)-DLAM-2P was 5–12 times more potent as a vitamin D antagonist than (23 S,25 S)-DLAM-1P in HL-60 cells, human and mouse bone marrow cultures. These results demonstrate that (23 S,25 S)-DLAM-1P and (23 S,25 S)-DLAM-2P antagonize HL-60 cell differentiation and osteoclast formation by human and mouse osteoclast precursors induced by 1α,25-(OH) 2D 3 through blocking VDR-mediated gene transcription. In contrast, (23 S)-25-deoxy-1α-hydroxyvitamin D 3-26,23-lactone, which only blocks human VDR, these vitamin D antagonists can block VDR in human cells and rodent cells.

Correlation of cholesterol 24-hydroxylase and ATP-binding cassette transporter A1 polymorphisms with Alzheimer's disease

To investigate the association of the single nucleotide polymorphisms (SNPs) A-->G in the intron 2 of cholesterol 24-hydroxylase (CYP46) gene and G-->A (R219K) in the exon 6 of ATP-binding cassette transporter A1 (ABCA1) gene with sporadic Alzheimer's disease (SAD) in the Han Chinese population. Peripheral blood samples were collected from 168 SAD patients, 74 males and 94 females, aged 74 +/- 8 (52 - 95), with an average age at onset of 69 +/- 7 (47 - 86), and sex-, and age-matched 215 healthy persons. PCR-restriction fragment length polymorphism (PCR-RFLP) was used to detect the genotypes. Apolipoprotein E (ApoE) genotyping was conducted. The strength of association between the polymorphisms and AD was estimate. The frequency of ApoE genotype of the SAD patients was 14%, significantly higher than that of the controls (5.1%, chi(2) = 19.060, P < 0.01). The risk of SAD in those with at least one epsilon 4 allele was 2.8 times that in those without epsilon 4 allele (OR = 2.82, 95% CI = 1.54 - 5.17, P = 0.001). There was no significant difference in the genotype or allele frequencies for CYP46 gene between the SAD patients and controls. However, there was an obvious association between the polymorphism of ABCA1 gene and SAD (chi(2) = 8.230, P = 0.016). The frequency of G/A + A/A genotypes in the SAD patients was 61.3%, significantly lower than that of the controls (73.5%, chi(2) = 6.444, P = 0.011). The risk of SAD in the carriers of A alleles (G/A + A/A genotypes) was significantly lower than those with GG genotype by 43% (adjusted OR = 0.57, 95% CI = 0.36 - 0.91, P = 0.019), and the risk of SAD in the AA homozygote carriers was significantly lower than that of the GG genotype carriers by 60% (adjusted OR = 0.40; 95% CI = 0.21 - 0.77, P = 0.006). The CYP46 intron 2 polymorphism may not be associated with the risk of AD, but AA genotype or A allele of ABCA1 gene may have a protective effect for AD in Han Chinese.