CYP11B2 Expression Research Articles

Vitamin D, the placenta and early pregnancy: effects on trophoblast function.

Pregnancy is associated with significant changes in vitamin D metabolism, notably increased maternal serum levels of active vitamin D, 1,25-dihydroxyvitamin (1,25(OH)2D). This appears to be due primarily to increased renal activity of the enzyme 25-hydroxyvitamin D-1α-hydroxylase (CYP27B1) that catalyzes synthesis of 1,25(OH)2D, but CYP27B1 expression is also prominent in both the maternal decidua and fetal trophoblast components of the placenta. The precise function of placental synthesis of 1,25(OH)2D remains unclear, but is likely to involve localized tissue-specific responses with both decidua and trophoblast also expressing the vitamin D receptor (VDR) for 1,25(OH)2D. We have previously described immunomodulatory responses to 1,25(OH)2D by diverse populations of VDR-expressing cells within the decidua. The aim of the current review is to detail the role of vitamin D in pregnancy from a trophoblast perspective, with particular emphasis on the potential role of 1,25(OH)2D as a regulator of trophoblast invasion in early pregnancy. Vitamin D deficiency is common in pregnant women, and a wide range of studies have linked low vitamin D status to adverse events in pregnancy. To date, most of these studies have focused on adverse events later in pregnancy, but the current review will explore the potential impact of vitamin D on early pregnancy, and how this may influence implantation and miscarriage.

Intravitreal enzyme replacement therapy attenuates retinal disease progression in a canine model of neuronal ceroid lipofuscinosis type 2 (CLN2)

Mineralocorticoid receptor activation of the epithelial sodium channel in endothelial cells (ECs) (EnNaC) is accompanied by aldosterone induced endothelial stiffening and impaired nitric oxide (NO)-mediated arterial relaxation. Recent data support enhanced activity of the alpha subunit of EnNaC (αEnNaC) mediates this aldosterone induced endothelial stiffening and associated endothelial NO synthase (eNOS) activation. There is mounting evidence that diet induced obesity diminishes expression and activation of AMP-activated protein kinase α (AMPKα), sirtuin 1 (Sirt1), which would be expected to lead to impaired downstream eNOS activation. Thereby, we posited that enhanced EnNaC activation contributes to diet induced obesity related increases in stiffness of the endothelium and diminished NO mediated vascular relaxation by increasing oxidative stress and related inhibition of AMPKα, Sirt1, and associated eNOS inactivation.Sixteen to twenty week-old αEnNaC knockout (αEnNaC−/−) and wild type littermate (EnNaC+/+) female mice were fed a mouse chow or an obesogenic western diet (WD) containing excess fat (46%) and fructose (17.5%) for 16 weeks. Sodium currents of ECs, endothelial stiffness and NO mediated aortic relaxation were examined along with indices of aortic oxidative stress, vascular remodeling and fibrosis.Enhanced EnNaC activation-mediated WD-induced increases in sodium currents in isolated lung ECs, increased endothelial stiffness and impaired aortic endothelium-dependent relaxation to acetylcholine (10−9–10−4 mol/L). These abnormalities occurred in conjunction with WD-mediated aortic tissue oxidative stress, inflammation, and decreased activation of AMPKα, Sirt1, and downstream eNOS were substantially mitigated in αEnNaC−/− mice. Importantly, αEnNaC−/− prevented WD induced increases in endothelial stiffness and related impairment of endothelium-dependent relaxation as well as aortic fibrosis and remodeling. However, EnNaC signaling was not involved in diet-induced abnormal expression of adipokines and CYP11b2 in abdominal aortic perivascular adipose tissue.These data suggest that endothelial specific EnNaC activation mediates WD-induced endothelial stiffness, impaired eNOS activation, aortic fibrosis and remodeling through increased aortic oxidative stress and increased inflammation related to a reduction of AMPKα and Sirt 1 mediated eNOS phosphorylation/activation and NO production.

Age-associated expression of vitamin D receptor and vitamin D-metabolizing enzymes in the male reproductive tract and sperm of Hu sheep

The cellular response to 1,25-dihydroxyvitamin D3 (Vit D3; biologically active form of Vitamin D) is complex and depends not only on Vitamin D receptor (VDR) expression but also on cellular uptake of circulating Vit D3 and the presence and activity of Vitamin D-metabolizing enzyme. This study evaluated the expression of VDR and Vitamin D-metabolizing enzymes in the ram reproductive tract at different developmental stages and in spermatozoa. Nearly all cell types in the testes and epithelial cells of the caput, corpus, and cauda expressed VDR, CYP27B1, and CYP24A1 proteins. The mRNA and protein expression of CYP2R1, CYP27A1, and CYP27B1 in the testes and cauda increased significantly with increasing age (P < 0.05). However, epididymal VDR mRNA and protein expression showed no significant difference (P < 0.05) between adult (9- and 24-month-old) and prepubertal (3-month-old) rams. Furthermore, VDR and CYP24A1 were mainly concentrated in the mid-piece of ejaculated or cauda epididymis spermatozoa or both. Additionally, VDR and CYP27B1 mRNA and protein expression levels were significantly higher in ejaculated spermatozoa than in cauda epididymal spermatozoa (P < 0.05). Moreover, VDR and CYP24A1 expression was significantly higher in high-motility than in low-motility spermatozoa (P < 0.05). The diverse expression patterns of VDR and Vitamin D-metabolizing enzymes in the ram reproductive tract at different developmental stages and spermatozoa suggest it plays a potential role in spermatogenesis.

Effects of Cholecalciferol on Key Components of Vitamin D-Endo/Para/Autocrine System in Experimental Type 1 Diabetes.

Objectives Recent prospective studies have found the associations between type 1 diabetes (T1D) and vitamin D deficiency. We investigated the role of vitamin D in the regulation of 25OHD-1α-hydroxylase (CYP27B1) and VDR expression in different tissues of T1D rats. Design T1D was induced in male Wistar rats by streptozotocin (55 mg/k b.w.). After 2 weeks of T1D, the animals were treated orally with or without vitamin D3 (cholecalciferol; 100 IU/rat, 30 days). Methods Serum 25-hydroxyvitamin D (25OHD) was detected by ELISA. CYP27A1, CYP2R1, CYP27B1, and VDR were assayed by RT-qPCR and Western blotting or visualized by immunofluorescence staining. Results We demonstrated that T1D led to a decrease in blood 25OHD, which is probably due to the established downregulation of CYP27A1 and CYP2R1 expression. Vitamin D deficiency was accompanied by elevated synthesis of renal CYP27B1 and VDR. Conversely, CYP27B1 and VDR expression decreased in the liver, bone tissue, and bone marrow. Cholecalciferol administration countered the impairments of the vitamin D-endo/para/autocrine system in the kidneys and extrarenal tissues of diabetic rats. Conclusions T1D-induced vitamin D deficiency is associated with impairments of renal and extrarenal CYP27B1 and VDR expression. Cholecalciferol can be effective in the amelioration of diabetes-associated abnormalities in the vitamin D-endo/para/autocrine system.

Targeting CXCR4 (CXC Chemokine Receptor Type 4) for Molecular Imaging of Aldosterone-Producing Adenoma.

Primary aldosteronism is the most frequent cause of secondary hypertension and is associated with increased morbidity and mortality compared with hypertensive controls. The central diagnostic challenge is the differentiation between bilateral and unilateral disease, which determines treatment options. Bilateral adrenal venous sampling, currently recommended for differential diagnosis, is an invasive procedure with several drawbacks, making it desirable to develop novel noninvasive diagnostic tools. When investigating the expression pattern of chemokine receptors by quantitative real-time polymerase chain reaction and immunohistochemistry, we observed high expression of CXCR4 (CXC chemokine receptor type 4) in aldosterone-producing tissue in normal adrenals, adjacent adrenal cortex from adrenocortical adenomas, and in aldosterone-producing adenomas (APA), correlating strongly with the expression of CYP11B2 (aldosterone synthase). In contrast, CXCR4 was not detected in the majority of nonfunctioning adenomas that are frequently found coincidently. The specific CXCR4 ligand 68Ga-pentixafor has recently been established as radiotracer for molecular imaging of CXCR4 expression and showed strong and specific binding to cryosections of APAs in our study. We further investigated 9 patients with primary aldosteronism because of APA by 68Ga-pentixafor-positron emission tomography. The tracer uptake was significantly higher on the side of increased adrenocortical aldosterone secretion in patients with APAs compared with patients investigated by 68Ga-pentixafor-positron emission tomography for other causes. Molecular imaging of aldosterone-producing tissue by a CXCR4-specific ligand may, therefore, be a highly promising tool for noninvasive characterization of patients with APAs.

Endogenous Calcitriol Synthesis Controls the Humoral IgE Response in Mice

The vitamin D receptor participates in the control of IgE class-switch recombination in B cells. The physiologic vitamin D receptor agonist, 1,25(OH)2D3 (calcitriol), is synthesized by the essential enzyme 25-hydroxyvitamin D3-1α-hydroxylase (CYP27B1), which can be expressed by activated immune cells. The role of endogenous calcitriol synthesis for the regulation of IgE has not been proven. In this study, we investigated IgE-responses in Cyp27b1-knockout (KO) mice following sensitization to OVA or intestinal infection with Heligmosomoides polygyrus Specific Igs and plasmablasts were determined by ELISA and ELISpot, Cyp27b1 expression was measured by quantitative PCR. The data show elevated specific IgE and IgG1 concentrations in the blood of OVA-sensitized Cyp27b1-KO mice compared with wild-type littermates (+898 and +219%). Accordingly, more OVA-specific IgG1-secreting cells are present in spleen and fewer in the bone marrow of Cyp27b1-KO mice. Ag-specific mechanisms are suggested as the leucopoiesis is in general unchanged and activated murine B and T lymphocytes express Cyp27b1 Accordingly, elevated specific IgE concentrations in the blood of sensitized T cell-specific Cyp27b1-KO mice support a lymphocyte-driven mechanism. In an independent IgE-inducing model, i.e., intestinal infection with H. polygyrus, we validated the increase of total and specific IgE concentrations of Cyp27b1-KO compared with wild-type mice, but not those of IgG1 or IgA. We conclude that endogenous calcitriol has an impact on the regulation of IgE in vivo. Our data provide genetic evidence supporting previous preclinical and clinical findings and suggest that vitamin D deficiency not only promotes bone diseases but also type I sensitization.

Disordered CYP11B2 Expression in Primary Aldosteronism.

Primary aldosteronism is the most common type of secondary hypertension affecting 6-10% of patients with primary hypertension. PA is mainly caused by unilateral hyperaldosteronism due to an aldosterone-producing adenoma, unilateral hyperplasia with or without micronodules or bilateral zona glomerulosa hyperplasias with or without macro or micronodules. The development of antibodies against the terminal enzyme of aldosterone biosynthesis (CYP11B2) has permitted the further characterization of normal adrenals and resected adrenals from patients with primary aldosteronism. Normal adrenals exhibit two different patterns of cellular expression of CYP11B2: young individuals display a relatively uniform expression of the enzyme throughout the zona glomerulosa while the adrenals of older individuals have dispersed CYP11B2-expressing cells but have more groups of cells called aldosterone-producing cell clusters (APCC). APAs exhibit different patterns of CYP11B2 staining that vary from uniform to homogeneous. There are also a proportion of cells within the APA that co-express different enzymes that are not normally co-expressed in normal individuals. Approximately 30% of patients with unilateral hyperaldosteronism do not have an APA, but either have an increased number of CYP11B2 expressing micronodules or hyperplasia of the zona glomerulosa. In summary, the studies reported in this review are shedding new light on the pathophysiology of primary aldosteronism. The wide variation in histopathological features of the adenomas and concurrent presence of APCCs raises the possibility that most cases of unilateral production of aldosterone actually might represent bilateral asymmetric hyperplasia with nodules frequently due to the development of somatic aldosterone-driving mutations.

Effects of 1,25 and 24,25 Vitamin D on Corneal Epithelial Proliferation, Migration and Vitamin D Metabolizing and Catabolizing Enzymes

This study investigated the effects of 1,25(OH)2D3 and 24R,25(OH)2D3 on corneal epithelial cell proliferation, migration, and on the vitamin D activating enzyme CYP27B1 (produces 1,25(OH)2D3) and inactivating enzyme CYP24A1 (produces 24R,25(OH)2D3). The role of the vitamin D receptor (VDR) was also examined. In VDR wildtype mouse corneal epithelial cells (WT), 1,25(OH)2D3 increased CYP24A1 protein expression and decreased CYP27B1 expression. In VDR knockout mouse epithelial cells (KO), 1,25(OH)2D3 increased CYP24A1 and CYP27B1 protein expression. 1,25(OH)2D3 did not affect WT cell proliferation, but did stimulate VDR KO cell proliferation. In a human corneal epithelial cell line (HCEC), 1,25(OH)2D3 increased CYP24A1 mRNA and protein expression. 1,25(OH)2D3 increased CYP27B1 mRNA levels in HCEC, but had no effect on CYP27B1 protein levels. 1,25(OH)2D3 inhibited HCEC proliferation and stimulated cell migration in primary human epithelial cells. 24,25(OH)2D3, on the other hand, increased both CYP24A1 and CYP27B1 protein expression in WT and VDR KO cells, and stimulated cell proliferation in both WT and KO cells. In HCEC, 24,25(OH)2D3 increased CYP24A1 and CYP27B1 mRNA and protein expression, and stimulated cell migration. In human primary corneal epithelial cells, 24,25(OH)2D3 stimulated migration. We conclude that 24R,25(OH)2D3 is likely involved in corneal epithelial cell regulation independent of 1,25(OH)2D3 or VDR.

The age-related changes of dietary phosphate responsiveness in plasma 1,25-dihydroxyvitamin D levels and renal Cyp27b1 and Cyp24a1 gene expression is associated with renal α-Klotho gene expression in mice.

In this study, we investigated the relationship between age-related changes in renal α-Klotho gene expression, vitamin D metabolism and the responsiveness of dietary phosphate in 1, 2 and 13 month-old mice fed a high phosphate (phosphate 1.2%) diet or low phosphate (phosphate 0.02%) diet for 5 days. We found that 1,25-dihydroxyvitamin D levels in plasma were significantly lower in the high phosphate group than the low phosphate group for 1 and 2 month-old mice, but not 13 month-old mice. In addition, in the high phosphate group plasma 1,25-dihydroxyvitamin D levels were decreased in 2 month-old mice relative to 1 month-old mice, but 13 month-old mice had higher levels than 2 month-old mice. In fact, plasma 1,25-dihydroxyvitamin D levels showed a significant correlation with vitamin D metabolism gene Cyp27b1 and Cyp24a1 mRNA expression in the high phosphate group. Interestingly, renal α-Klotho mRNA and protein levels were significant change with age. Furthermore, α-Klotho mRNA expression showed a significant negative correlation with plasma 1,25-dihydroxyvitamin D levels in the high phosphate group. Our results suggest that age-related alterations in renal α-Klotho expression could affect the responsiveness of dietary phosphate to vitamin D metabolism.

Central and peripheral slow-pressor mechanisms contributing to Angiotensin II-salt hypertension in rats.

High salt intake markedly enhances hypertension induced by angiotensin II (Ang II). We explored central and peripheral slow-pressor mechanisms which may be activated by Ang II and salt. In protocol I, Wistar rats were infused subcutaneously with low-dose Ang II (150 ng/kg/min) and fed regular (0.4%) or high salt (2%) diet for 14 days. In protocol II, Ang II-high salt was combined with intracerebroventricular infusion of mineralocorticoid receptor (MR) blockers (eplerenone, spironolactone), epithelial sodium channel (ENaC) blocker (benzamil), angiotensin II type 1 receptor (AT1R) blocker (losartan) or vehicles. Ang II alone raised mean arterial pressure (MAP) ∼10 mmHg, but Ang II-high salt increased MAP ∼50 mmHg. Ang II-high salt elevated plasma corticosterone, aldosterone and endogenous ouabain but not Ang II alone. Both Ang II alone and Ang II-high salt increased mRNA and protein expression of CYP11B2 (aldosterone synthase gene) in the adrenal cortex but not of CYP11B1 (11-β-hydroxylase gene). In the aorta, Ang II-high salt increased sodium-calcium exchanger-1 (NCX1) protein. The Ang II-high salt induced increase in MAP was largely prevented by central infusion of MR blockers, benzamil or losartan. Central blockades significantly lowered plasma aldosterone and endogenous ouabain and markedly decreased Ang II-high salt induced CYP11B2 mRNA expression in the adrenal cortex and NCX1 protein in the aorta. These results suggest that in Ang II-high salt hypertension, MR-ENaC-AT1R signalling in the brain increases circulating aldosterone and endogenous ouabain, and arterial NCX1. These factors can amplify blood pressure responses to centrally-induced sympatho-excitation and thereby contribute to severe hypertension.

Calneuron 1 Increased Ca2+ in the Endoplasmic Reticulum and Aldosterone Production in Aldosterone-Producing Adenoma.

Aldosterone production is initiated by angiotensin II stimulation and activation of intracellular Ca2+ signaling. In aldosterone-producing adenoma (APA) cells, the activation of intracellular Ca2+ signaling is independent of the renin-angiotensin-aldosterone systems. The purpose of our study was to clarify molecular mechanisms of aldosterone production related to Ca2+ signaling. Transcriptome analysis revealed that the CALN1 gene encoding calneuron 1 had the strongest correlation with CYP11B2 (aldosterone synthase) among genes encoding Ca2+-binding proteins in APA. CALN1 modulation and synthetic or fluorescent compounds were used for functional studies in human adrenocortical carcinoma (HAC15) cells. CALN1 expression was 4.4-fold higher in APAs than nonfunctioning adrenocortical adenomas. CALN1 expression colocalized with CYP11B2 expression as investigated using immunohistochemistry in APA and zona glomerulosa of male rats fed by a low-salt diet. CALN1 expression was detected in the endoplasmic reticulum (ER) by using GFP-fused CALN1, CellLight ER-RFP, and the corresponding antibodies. CALN1-overexpressing HAC15 cells showed increased Ca2+ in the ER and cytosol fluorescence-based studies. Aldosterone production was potentiated in HAC15 cells by CALN1 expression, and dose-responsive inhibition with TMB-8 showed that CALN1-mediated Ca2+ storage in ER involved sarcoendoplasmic reticulum calcium transport ATPase. The silencing of CALN1 decreased Ca2+ in ER, and abrogated angiotensin II- or KCNJ5 T158A-mediated aldosterone production in HAC15 cells. Increased CALN1 expression in APA was associated with elevated Ca2+ storage in ER and aldosterone overproduction. Suppression of CALN1 expression prevented angiotensin II- or KCNJ5 T158A-mediated aldosterone production in HAC15 cells, suggesting that CALN1 is a potential therapeutic target for excess aldosterone production.

Study of gene expression alteration in male androgenetic alopecia: evidence of predominant molecular signalling pathways.

Male androgenetic alopecia (AGA) is the most common form of hair loss in men. It is characterized by a distinct pattern of progressive hair loss starting from the frontal area and the vertex of the scalp. Although several genetic risk loci have been identified, relevant genes for AGA remain to be defined. To identify biomarkers associated with AGA. Molecular biomarkers associated with premature AGA were identified through gene expression analysis using cDNA generated from scalp vertex biopsies of hairless or bald men with premature AGA, and healthy volunteers. This monocentric study reveals that genes encoding mast cell granule enzymes, inflammatory mediators and immunoglobulin-associated immune mediators were significantly overexpressed in AGA. In contrast, underexpressed genes appear to be associated with the Wnt/β-catenin and bone morphogenic protein/transforming growth factor-β signalling pathways. Although involvement of these pathways in hair follicle regeneration is well described, functional interpretation of the transcriptomic data highlights different events that account for their inhibition. In particular, one of these events depends on the dysregulated expression of proopiomelanocortin, as confirmed by polymerase chain reaction and immunohistochemistry. In addition, lower expression of CYP27B1 in patients with AGA supports the notion that changes in vitamin D metabolism contributes to hair loss. This study provides compelling evidence for distinct molecular events contributing to alopecia that may pave the way for new therapeutic approaches.

Associations between abnormal vitamin D metabolism pathway function and non-small cell lung cancer.

Lung cancer is a type of malignant tumor derived from the respiratory system, which is the leading cause of cancer-associated mortality worldwide, of which ~80% of cases are attributable to non-small cell lung cancer (NSCLC). A previous study demonstrated that 1α,25-Dihydroxyvitamin D3 (1α,25(OH)2D3), derived from the vitamin D metabolic pathway contributes an antitumor effect. Aberrant expression of the essential enzyme encoding genes, Cytochrome P450 Family 27 Subfamily A Member 1 (CYP27A1), Cytochrome P450 Family 27 Subfamily B Member 1 (CYP27B1), and Cytochrome P450 Family 24 Subfamily A Member 1 (CYP24A1) may be associated with lung cancer. However, a lack of evidence exists concerning the association between CYP27A1, CYP27B1, CYP24A1 expression and NSCLC. The aim of the present study was to investigate the functions of CYP27A1, CYP27B1 and CYP24A1 expression in NSCLC. Lung cancer tissue and para-carcinoma control tissue were collected from patients with NSCLC. Reverse transcription-quantitative polymerase chain reaction was applied to analyze CYP27A1, CYP27B1 and CYP24A1 mRNA expression in lung cancer tissues. An association analysis was performed between the aforementioned metabolic enzymes and patients with NSCLC age, gender, tumor node metastasis (TNM) stage, pathological type, differentiation and prognosis. CYP27B1 and CYP24A1 mRNA were upregulated in NSCLC compared with controls (P<0.05). However, no significant differences in CYP27A1 expression were observed between NSCLC and control. In addition, CYP24A1 expression was not associated with age, sex, smoking or TNM stage, but was associated with pathological type, differentiation and prognosis (P<0.05). CYP27B1 expression was significantly associated with TNM stage, differentiation, and prognosis, but not age, sex, smoking or pathological type. In conclusion, CYP27B1 and CYP24A1 may be considered as independent prognostic factors of NSCLC and may be novel therapeutic targets to assist clinical diagnosis, treatment and prognosis of the disease.

Molecular profiles of oxyphilic and chief cell parathyroid adenoma.

Parathyroid adenomas may be composed of chief cells (conventional or water-clear), oxyphilic cells or a mixture of both cells. The molecular background is rarely studied. To molecularly characterize parathyroid adenomas of different cell type composition. Chief and oxyphilic cell adenomas were compared in a cohort of 664 sporadic cases. Extensive analyses of parathyroid tissues were performed in subgroup. Gene expressions of known parathyroid-related genes were quantified by qRT-PCR. Protein expression profiles determined by liquid chromatography - tandem mass spectrometry (LC-MS/MS) were compared between each type of parathyroid adenomas. Selected proteins were analysed by Western blot and immunohistochemistry. Patients with oxyphilic cell adenoma were found to be older at the time of operation than chief cell adenoma cases but did not differ in gender, serum calcium or tumor weight. The gene expression of CASR, VDR, FGFR1, CYP27B1, CYP24A1, PTHLH, GCM2, NDUFA13, CDKN1B, MEN1 and CNND1 did not differ between the groups. VDR protein levels were weaker in oxyphilic adenomas. The proteomic studies identified a set of novel dysregulated proteins of interest such as nuclear receptor subfamily 2 group C member 2 (TR4), LIM domain only protein 3 (LMO3) and calcium-binding protein B (S100B). LMO3 and S100B showed higher expression in oxyphilic adenoma and may be involve in parathyroid tumorgenesis through the p53 pathway. TR4 showed different subcellular localisation between adenoma and normal rim. Chief and oxyphilic cell parathyroid adenomas have partly overlapping but also distinct molecular profiles. The calmodulin-eEF2K, TR4 and p53 pathways may be involved in the tumor development.

A kidney-specific genetic control module in mice governs endocrine regulation of the cytochrome P450 gene Cyp27b1 essential for vitamin D3 activation

The vitamin D endocrine system regulates mineral homeostasis through its activities in the intestine, kidney, and bone. Terminal activation of vitamin D3 to its hormonal form, 1α,25-dihydroxyvitamin D3 (1,25(OH)2D3), occurs in the kidney via the cytochrome P450 enzyme CYP27B1. Despite its importance in vitamin D metabolism, the molecular mechanisms underlying the regulation of the gene for this enzyme, Cyp27b1, are unknown. Here, we identified a kidney-specific control module governed by a renal cell-specific chromatin structure located distal to Cyp27b1 that mediates unique basal and parathyroid hormone (PTH)-, fibroblast growth factor 23 (FGF23)-, and 1,25(OH)2D3-mediated regulation of Cyp27b1 expression. Selective genomic deletion of key components within this module in mice resulted in loss of either PTH induction or FGF23 and 1,25(OH)2D3 suppression of Cyp27b1 gene expression; the former loss caused a debilitating skeletal phenotype, whereas the latter conferred a quasi-normal bone mineral phenotype through compensatory homeostatic mechanisms involving Cyp24a1 We found that Cyp27b1 is also expressed at low levels in non-renal cells, in which transcription was modulated exclusively by inflammatory factors via a process that was unaffected by deletion of the kidney-specific module. These results reveal that differential regulation of Cyp27b1 expression represents a mechanism whereby 1,25(OH)2D3 can fulfill separate functional roles, first in the kidney to control mineral homeostasis and second in extra-renal cells to regulate target genes linked to specific biological responses. Furthermore, we conclude that these mouse models open new avenues for the study of vitamin D metabolism and its involvement in therapeutic strategies for human health and disease.

Cortisol overproduction results from DNA methylation of CYP11B1 in hypercortisolemia

Adrenocortical hormone excess, due to primary aldosteronism (PA) or hypercortisolemia, causes hypertension and cardiovascular complications. In PA, hypomethylation of aldosterone synthase (CYP11B2) is associated with aldosterone overproduction. However, in hypercortisolemia, the role of DNA methylation of 11β-hydroxylase (CYP11B1), which catalyzes cortisol biosynthesis and is highly homologous to CYP11B2, is unclear. The aims of our study were to determine whether the CYP11B1 expression was regulated through DNA methylation in hypercortisolemia with cortisol-producing adenoma (CPA), and to investigate a possible relationship between DNA methylation and somatic mutations identified in CPA. Methylation analysis showed that the CYP11B1 promoter was significantly less methylated in CPA than in adjacent unaffected adrenal tissue and white blood cells. Furthermore, in CPA with somatic mutations in either the catalytic subunit of protein kinase A (PRKACA) or the guanine nucleotide-binding protein subunit alpha (GNAS) gene, the CYP11B1 promoter was significantly hypomethylated. In addition, DNA methylation reduced CYP11B1 promoter activity using a reporter assay. Our study results suggest that DNA methylation at the CYP11B1 promoter plays a role in the regulation of CYP11B1 expression and cortisol production in CPA, and that somatic mutations associated with CPA reduce DNA methylation at the CYP11B1 promoter.

Differential expression of vitamin D-associated enzymes and receptors in brain cell subtypes

Accumulating evidence indicates that the active form of vitamin D, 1,25(OH)2D3, can be considered as a neurosteroid. However, the cerebral expression of vitamin D-associated enzymes and receptors remains controversial. With the idea of carrying out a comparative study in mind, we compared the transcript expression of Cyp27a1, Cyp27b1, Cyp24a1, Vdr and Pdia3 in purified cultures of astrocytes, endothelial cells, microglia, neurons and oligodendrocytes. We observed that endothelial cells and neurons can possibly transform the inactive cholecalciferol into 25(OH)D3. It can then be metabolised into 1,25(OH)2D3, by neurons or microglia, before being transferred to astrocytes where it can bind to VDR and initiate gene transcription or be inactivated when in excess. Alternatively, 1,25(OH)2D3 can induce autocrine or paracrine rapid non-genomic actions via PDIA3 whose transcript is abundantly expressed in all cerebral cell types. Noticeably, brain endothelial cells appear as a singular subtype as they are potentially able to transform cholecalciferol into 25(OH)D3 and exhibit a variable expression of Pdia3, according to 1,25(OH)2D3 level. Altogether, our data indicate that, within the brain, vitamin D may trigger major auto-/paracrine non genomic actions, in addition to its well documented activities as a steroid hormone.

Abstract 057: Cellular and Genetic Causes of Idiopathic Hyperaldosteronism

Background: Primary aldosteronism (PA) affects ~10% of hypertensive patients and has unilateral and bilateral forms (30%:70%). Unilateral PA is caused by aldosterone-producing adenomas (APA), which express CYP11B2 (aldosterone synthase) and frequently harbor somatic mutations in aldosterone regulating genes ( KCNJ5&gt;&gt;CACNA1D ). We recently demonstrated that adrenals from normotensive patients present with pockets of cell expressing aldosterone synthase (CYP11B2). These aldosterone-producing foci (APF) have somatic gene mutations similar to those found in APA ( CACNA1D &gt;&gt; KCNJ5 ). Bilateral PA, which is typically treated by mineralocorticoid receptor (MR) blockade rather than surgery, is termed idiopathic hyperaldosteronism (IHA). Its pathobiology is largely unknown but has been thought to be due to zona glomerulosa (ZG) hyperplasia. Methods: We studied 11 IHA patients (7 males, 4 females) who had unilateral adrenalectomy. Immunohistochemistry for CYP11B2 and next generation sequencing (NGS) targeting genes found in APA were performed on formalin fixed paraffin embedded adrenal tissue. Results were compared to previously described cohorts of 53 age-matched normotensive patients (29 males, 24 females) which were evaluated similarly. Results: CYP11B2 expression was absent from intervening ZG cells in 8/11 (73%) IHA adrenals, but all adrenals harbored at least one APF. The median number and size of APF per case were significantly larger in IHA than normotensive controls (6.1 vs 0 APF/cm 2 of adrenal cortex and 0.25 vs. 0.16 mm 2 , respectively; p&lt;0.0001 and p&lt;0.006). In this IHA cohort, NGS identified CACNA1D and KCNJ5 somatic mutations in 44/71 (62%) and 1/71 (1%) of APF, respectively. Interpretations. Diffuse CYP11B2 expression in adrenal ZG cells was only observed in 3/11 IHA cases, arguing against ZG hyperplasia as the major underlying pathobiology. Rather, we demonstrated increased and enlarged APF in IHA adrenals compared to normotensive controls, supporting potential contribution to the clinical manifestations of hyperaldosteronism. The frequent occurrence of aldosterone-dysregulating CACNA1D somatic mutations in APF support CACNA1D as a potential therapeutic target in IHA to complement current MR blockade approaches.

High glucose stimulates expression of aldosterone synthase (CYP11B2) and secretion of aldosterone in human adrenal cells.

Aldosterone synthase is the key rate‐limiting enzyme in adrenal aldosterone production, and induction of its gene (CYP11B2) results in the progression of hypertension. As hypertension is a frequent complication among patients with diabetes, we set out to elucidate the link between diabetes mellitus and hypertension. We examined the effects of high glucose on CYP11B2 expression and aldosterone production using human adrenal H295R cells and a stable H295R cell line expressing a CYP11B2 5′‐flanking region/luciferase cDNA chimeric construct. d‐glucose (d‐glu), but not its enantiomer l‐glucose, dose dependently induced CYP11B2 transcription and mRNA expression. A high concentration (450 mg·dL−1) of d‐glu time dependently induced CYP11B2 transcription and mRNA expression. Moreover, high glucose stimulated secretion of aldosterone into the media. Transient transfection studies using deletion mutants/nerve growth factor‐induced clone B (NGFIB) response element 1 (NBRE‐1) point mutant of CYP11B2 5′‐flanking region revealed that the NBRE‐1 element, known to be activated by transcription factors NGFIB and NURR1, was responsible for the high glucose‐mediated effect. High glucose also induced the mRNA expression of these transcription factors, especially that of NURR1, but NURR1 knockdown using its siRNA did not affect high glucose‐induced CYP11B2 mRNA expression. Taken together, it is speculated that high glucose may induce CYP11B2 transcription via the NBRE‐1 element in its 5′‐flanking region, resulting in the increase in aldosterone production although high glucose‐induced NURR1 is not directly involved in the effect. Additionally, glucose metabolism and calcium channels were found to be involved in the high glucose effect. Our observations suggest one possible explanation for the high incidence of hypertension in patients with diabetes.

Suppressive effects of RXR agonist PA024 on adrenal CYP11B2 expression, aldosterone secretion and blood pressure.

The effects of retinoids on adrenal aldosterone synthase gene (CYP11B2) expression and aldosterone secretion are still unknown. We therefore examined the effects of nuclear retinoid X receptor (RXR) pan-agonist PA024 on CYP11B2 expression, aldosterone secretion and blood pressure, to elucidate its potential as a novel anti-hypertensive drug. We demonstrated that PA024 significantly suppressed angiotensin II (Ang II)-induced CYP11B2 mRNA expression, promoter activity and aldosterone secretion in human adrenocortical H295R cells. Human CYP11B2 promoter functional analyses using its deletion and point mutants indicated that the suppression of CYP11B2 promoter activity by PA024 was in the region from -1521 (full length) to -106 including the NBRE-1 and the Ad5 elements, and the Ad5 element may be mainly involved in the PA024-mediated suppression. PA024 also significantly suppressed the Ang II-induced mRNA expression of transcription factors NURR1 and NGFIB that bind to and activate the Ad5 element. NURR1 overexpression demonstrated that the decrease of NURR1 expression may contribute to the PA024-mediated suppression of CYP11B2 transcription. PA024 also suppressed the Ang II-induced mRNA expression of StAR, HSD3β2 and CYP21A2, a steroidogenic enzyme group involved in aldosterone biosynthesis. Additionally, the PA024-mediated CYP11B2 transcription suppression was shown to be exerted via RXRα. Moreover, the combination of PPARγ agonist pioglitazone and PA024 caused synergistic suppressive effects on CYP11B2 mRNA expression. Finally, PA024 treatment significantly lowered both the systolic and diastolic blood pressure in Tsukuba hypertensive mice (hRN8-12 x hAG2-5). Thus, RXR pan-agonist PA024 may be a candidate anti-hypertensive drugs that acts via the suppression of aldosterone synthesis and secretion.