Renal CYP27B1 Research Articles

A New Look at Vitamin D Metabolism and “Idiopathic” Hypercalcemia

A New Look at Vitamin D Metabolism and “Idiopathic” Hypercalcemia

Vitamin D metabolism and signaling in the immune system

Vitamin D has emerged as a pleiotropic regulator of human physiology, and recent work has revealed that it has several roles in control of human immune system function. Vitamin D was originally characterized for its role in calcium homeostasis, and the active form, 1,25-dihydroxyvitamin D (1,25D), can be produced in the kidney by 1α-hydroxylation of circulating 25-hydroxyvitamin D catalyzed by the enzyme CYP27B1. Renal CYP27B1 expression is regulated by calcium regulatory inputs, and 1,25D produced in the kidney was thought to function largely as an endocrine hormone. However, it is now clear that CYP27B1 is expressed in numerous tissues, and that 1,25D acts at several sites in the body in an intracrine or paracrine manner. In particular, both CYP27B1 and the vitamin D receptor (VDR) are expressed in several cell types in the immune system, where CYP27B1 production is controlled by a number of immune-specific inputs. Recent research has opened several windows on the molecular mechanisms by which 1,25D signaling regulates both innate and adaptive immune responses in humans. Moreover, intervention trials are beginning to provide evidence that vitamin D supplementation can bolster clinical responses to infection. This review will discuss recent developments in our understanding of how immune signaling controls local vitamin D metabolism and how, in turn, the 1,25D-bound VDR modulates immune system function. A particular emphasis will be placed on the interplay between vitamin D signaling and signaling through different classes of pattern recognition receptors in the production of antimicrobial peptides during innate immune responses to microbial infection.

MafB interacts with Gcm2 and regulates parathyroid hormone expression and parathyroid development

Serum calcium and phosphate homeostasis is critically regulated by parathyroid hormone (PTH) secreted by the parathyroid glands. Parathyroid glands develop from the bilateral parathyroid-thymus common primordia. In mice, the expression of transcription factor Glial cell missing 2 (Gcm2) begins in the dorsal/anterior part of the primordium on embryonic day 9.5 (E9.5), specifying the parathyroid domain. The parathyroid primordium then separates from the thymus primordium and migrates to its adult location beside the thyroid gland by E15.5. Genetic ablation of gcm2 results in parathyroid agenesis in mice, indicating that Gcm2 is essential for early parathyroid organogenesis. However, the regulation of parathyroid development at later stages is not well understood. Here we show that transcriptional activator v-maf musculoaponeurotic fibrosarcoma oncogene homologue B (MafB) is developmentally expressed in parathyroid cells after E11.5. MafB expression was lost in the parathyroid primordium of gcm2 null mice. The parathyroid glands of mafB(+/-) mice were mislocalized between the thymus and thyroid. In mafB(-/-) mice, the parathyroid did not separate from the thymus. Furthermore, in mafB(-/-) mice, PTH expression and secretion were impaired; expression levels of renal cyp27b1, one of the target genes of PTH, was decreased; and bone mineralization was reduced. We also demonstrate that although Gcm2 alone does not stimulate the PTH gene promoter, it associates with MafB to synergistically activate PTH expression. Taken together, our results suggest that MafB regulates later steps of parathyroid development, that is, separation from the thymus and migration toward the thyroid. MafB also regulates the expression of PTH in cooperation with Gcm2.

A Downstream Intergenic Cluster of Regulatory Enhancers Contributes to the Induction of CYP24A1 Expression by 1α,25-Dihydroxyvitamin D3

CYP24A1 expression is up-regulated by 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) via a vitamin D receptor (VDR)/retinoid X receptor (RXR) heterodimer that binds to two vitamin D response elements (VDREs) located near the proximal promoter. Interestingly, although 1,25(OH)(2)D(3) induced VDR/RXR binding to the VDRE-containing proximal promoter, the VDR/RXR heterodimer also localized to a cluster of at least four potential enhancers located in intergenic regions 50-69 kb downstream of the human CYP24A1 gene and 35-45 kb downstream of the mouse Cyp24a1 gene as revealed by ChIP-chip and ChIP-seq analyses. To address whether this downstream region and potential VDREs located within mediated CYP24A1 induction, we constructed recombinant wild-type and mutant bacterial artificial chromosome clones that spanned mouse and human loci and contained luciferase reporters inserted into their 3'-untranslated regions. The activity of these clones in stably transfected cells revealed that both the proximal and the putative downstream elements contributed to CYP24A1 up-regulation by 1,25(OH)(2)D(3). Further analysis using transfected enhancer fragments led to the identification of contributing regulatory elements in several of these downstream regions. Additional studies of coregulator recruitment using ChIP-chip analysis revealed both similarities and differences between the region located proximal to and those located downstream of the promoter. Recruitment of these coregulators was likely responsible for the increase in RNA polymerase II and histone H4 acetylation, which was also observed in response to 1,25(OH)(2)D(3) at the enhancer sites across the locus. We conclude that a more complex mechanism is responsible for the striking CYP24A1 up-regulation induced by the vitamin D hormone in target cells.

Supraphysiological testosterone administration alters renal 25‐hydroxyvitamin D‐3 1α‐hydroxylase protein expression in female rodents

The enzyme 25‐hydroxyvitamin D 1α‐hydroxylase (CYP27B1) converts circulating 25‐hydroxyvitamin D to its active form, 1,25‐dihydroxyvitamin D. The effects of testosterone (T) on CYP27B1 expression within the kidney, an androgen sensitive organ, remain unclear. The purpose of this study was to determine whether gonadectomy (GNX) or T alter renal CYP27B1 protein expression. 3 month old male and female F344 rats (n = 5/group) received SHAM surgery + vehicle (V), GNX + V, or GNX + supraphysiological T‐enanthate (GNX+TE) for 28 days. The kidney was removed at sacrifice and CYP27B1 expression was measured in the renal cortex by Western blot. GNX+TE increased kidney mass by 19% and 28% in males and females compared with Shams and by 29% and 26% compared with GNX + V (p < 0.05). No differences in renal CYP27B1 expression were present between intact males and females. GNX did not alter CYP27B1 protein expression in either sex. No change in CYP27B1 protein expression was observed in GNX+TE males. However, renal cortex CYP27B1 protein expression was reduced by 28% in GNX+TE females compared with SHAMs (p < 0.05). Our results indicate that supraphysiological TE down‐regulates renal cortex CYP27B1 protein expression in female rodents, through a yet to be identified mechanism. Future studies examining the effects of T on the activity and expression of CYP27B1 should account for androgen induced alterations in kidney mass.

The effect of dietary calcium on 1,25(OH) 2D 3 synthesis and sparing of serum 25(OH)D 3 levels

Vitamin D depletion in rats causes osteopenia in at least three skeletal sites. However it is unclear whether modulation of dietary calcium intake impacts on the relationship between the level of serum 25-hydroxyvitamin D (25D) and bone loss. Nine-month-old female Sprague–Dawley rats ( n = 5–6/group) were pair-fed a semi-synthetic diet containing either 0 or 20 IU vitamin D3/day with either low (0.1%) or high (1%) dietary Ca for 6 months. At 15 months of age, fasting bloods were collected for biochemical analyses. Serum 25D levels were lowest in the animals fed 0 IU vitamin D and 0.1% Ca. The animals fed 1% Ca had significantly higher serum 25D levels when compared to animals fed 0.1% Ca ( P < 0.05). The major determinants of serum 25D were dietary vitamin D and dietary calcium (Multiple R = 0.75, P < 0.05). Animals fed 0.1% Ca had higher renal CYP27B1 mRNA expression and 12–18-fold increased levels of serum 1,25D. Hence, the reported effects of low calcium diets on bone loss may be, in part, due to the subsequent effects of 25D metabolism leading to reduction in vitamin D status. Such an interaction has significant implications, given the recent evidence for local synthesis of active vitamin D in bone tissue.

Hypergravity modulates vitamin D receptor target gene mRNA expression in mice

The possibility of pathological calcium metabolism is a critical health concern introduced by long-term space travel. Because vitamin D plays an important role in calcium homeostasis, we evaluated the effects of hypergravity on the expression of genes involved in vitamin D and calcium metabolism in ICR mice. When exposed to 2G hypergravity for 2 days, the mRNA expression of renal 25-hydroxyvitamin D 24-hydroxylase (Cyp24a1) was increased and that of 25-hydroxyvitamin D 1alpha-hydroxylase (Cyp27b1) was decreased. Although hypergravity decreased food intake and increased the expression of starvation-induced genes, the changes in Cyp24a1 and Cyp27b1 expression were not due to starvation, suggesting that hypergravity affects these genes directly. Hypergravity decreased plasma 1alpha,25-dihydroxyvitamin D(3) levels in ICR mice, suggesting a consequence of decreased Cyp27b1 and increased Cyp24a1 expression. Although 1alpha-hydroxyvitamin D(3) [1alpha(OH)D(3)] treatment induced the expression of vitamin D receptor (VDR) target genes in the kidney of 2G-exposed ICR mice to similar levels as controls, 1alpha(OH)D(3) increased the intestinal expression of Cyp24a1 in ICR mice. Hypergravity-dependent changes of Cyp24a1 and Cyp27b1 expression were diminished in mice exposed to hypergravity for 14 days, which may represent an adaptation to hypergravity stress. Hypergravity exposure also increased Cyp24a1 expression in the kidney of C57BL/6J mice. We examined the effects of hypergravity on VDR-null mice and found that renal Cyp27b1 expression in VDR-null mice was decreased by hypergravity while renal Cyp24a1 expression was not detected in VDR-null mice. Thus hypergravity modifies the expression of genes involved in vitamin D metabolism.

Reduction of the vitamin D hormonal system in kidney disease is associated with increased renal inflammation

To examine any potential role for 1,25-dihydroxyvitamin D (1,25(OH)2D) in inflammation associated with chronic kidney disease we measured vitamin D metabolites, markers of inflammation and gene expression in 174 patients with a variety of kidney diseases. Urinary MCP-1 protein and renal macrophage infiltration were each significantly but inversely correlated with serum 1,25(OH)2D levels. Logistic regression analysis with urinary MCP-1 as binary outcome showed that a 10-unit increase in serum 1,25(OH)2D or 25OHD resulted in lower renal inflammation. Analysis of 111 renal biopsies found that renal injury was not associated with a compensatory increase in mRNA for the vitamin D-activating enzyme 25-hydroxyvitamin D-1alpha-hydroxylase (CYP27B1), its catabolic counterpart 24-hydroxylase, or the vitamin D receptor. There was, however, a significant association between tissue MCP-1 and CYP27B1. Patients with acute renal inflammation had a significant increase in urinary and tissue MCP-1, macrophage infiltration, and macrophage and renal epithelial CYP27B1 expression but significantly lower levels of serum 1,25(OH)2D in comparison to patients with chronic ischemic disease despite similar levels of renal damage. In vitro, 1,25(OH)2D attenuated TNFalpha-induced MCP-1 expression by human proximal tubule cells. Our study indicates that renal inflammation is associated with decreased serum vitamin D metabolites and involves activation of the paracrine/autocrine vitamin D system.

Specific regulation of CYP27B1 and VDR in proximal versus distal renal cells

In this study, we utilized murine renal proximal (MPCT-G) and distal (DKC-8) tubular epithelial cell lines to compare the gene expressions and promoter activities of 1,25(OH) 2D 3 receptor (VDR) and 25-hydroxyvitamin D-1α-hydroxylase (CYP27B1) in response to 50 nM of parathyroid hormone (PTH) and changes in extracellular calcium (Ca 2+) concentration. In MPCT-G cells, VDR gene expression was suppressed by PTH, whereas CYP27B1 gene expression was elevated in response to PTH. In DKC-8 cells, treatment of PTH significantly increased the relative gene expression of VDR by 6.5-fold while CYP27B1 gene expression was unchanged. High Ca 2+ exposure stimulated VDR gene expression and repressed CYP27B1 gene expression in both dose and time-dependent fashion in MPCT-G but not DKC-8 cells. The analysis of promoter activities and VDR protein levels corresponded with the gene expression data. We conclude that PTH-mediated decrease in VDR and increase in renal CYP27B1 is proximal cell-specific.

Serum Metabolite Profiles and Target Tissue Gene Expression Define the Effect of Cholecalciferol Intake on Calcium Metabolism in Rats and Mice

We studied the effect of cholecalciferol (VD3) intake on VD3 status and markers of calcium (Ca) homeostasis in mice and rats. Serum 25 hydroxycholecalciferol (25OH-VD3) concentrations were increased in animals fed diets containing 400–20,000 international units (IU) VD3/kg (37 nmol·L−1·1000 IU VD3 −1), but body weight, serum Ca, and duodenal gene expression were not altered. High-VD3 intake decreased serum 1, 25-dihydroxycholecalciferol [1,25(OH)2-VD3] and renal 25 hydroxycholecalciferol-1 αhydroxylase (CYP27B1) mRNA, suggesting that rodents tolerate high-VD3 intake by suppressing the activity of the VD3 endocrine system. Serum 25OH-VD3 declined when animals were fed diets containing 1000 to 25 IU VD3/kg (9–11 wk, inflection at 200 IU/kg, 4-fold steeper slope below this). Neither body weight nor serum Ca were influenced by low-VD3 intake. However, mice fed the 25-IU/kg diet had lower serum 1,25(OH)2-VD3, duodenal calbindin D9k mRNA, bone mineral density, and renal 25 hydroxycholecalciferol-24 hydroxylase mRNA, whereas renal CYP27B1 mRNA was elevated when rodents were fed <200 IU VD3/kg. These data reveal a stress on VD3 and Ca metabolism at low dietary VD3 intake. Dietary Ca restriction (0.25 vs. 0.5%, 9 wk) increased serum 1,25(OH)2-VD3 and was 30% greater in rats fed a 10,000-IU VD3/kg diet. High-VD3 intake did not prevent Ca restriction-induced bone loss. Our data show that modeling human VD3 status requires lower intake than the current NRC rodent requirement (1000-IU/kg diet). Also, although rodents are very tolerant of high-VD3 intake, it cannot compensate for moderate Ca restriction.

Chronic Ethanol Consumption Leads to Disruption of Vitamin D3 Homeostasis Associated with Induction of Renal 1,25 Dihydroxyvitamin D3-24-Hydroxylase (CYP24A1)

Bone loss resulting from chronic ethanol (EtOH) abuse is frequently accompanied by altered vitamin D3 homeostasis. In the current study, we examined EtOH effects in a female rat model in which control or EtOH-containing diets were infused intragastrically. EtOH treatment reduced plasma 1,25-dihydroxycholecalciferol (1,25 (OH)2 D3) coincident with a decrease in renal CYP27B1 (25(OH)D3 1alpha-hydroxylase) mRNA and an increase in expression of renal CYP24A1 (1,25 (OH)2 D3- 24-hydroxylase). EtOH induction of CYP24A1 occurred as a result of increased transcription and was also observed in vitro in primary cultures of rat renal proximal tubule cells (RPTCs) and in NRK-52E cells. Synergistic induction of CYP24A1 by EtOH in combination with 1,25 (OH)2 D3 was observed. The major EtOH metabolizing enzymes, alcohol dehydrogenase-1 and CYP2E1, were induced by EtOH in RPTCs. Inhibition of EtOH metabolism by 4-methylpyrazole inhibited the induction of CYP24A1 mRNA. CYP24A1 mRNA induction in RPTCs was also inhibited by the protein synthesis inhibitor cycloheximide. CYP24A1 was also induced after hydrogen peroxide treatment, and EtOH treatment of RPTCs resulted in production of reactive oxygen species as measured by flow cytometry using the fluorescent probe dichlorofluorescin acetate. In addition, inhibition of MAPK signaling pathways with the MAPK kinase inhibitor U0126 or the p38 inhibitor SB203580 inhibited EtOH induction of CYP24A1. Our data suggest that EtOH reduces circulating 1,25 (OH)2 D3 concentrations as the result of CYP24A1 induction that is mediated via MAPK activation resulting from renal oxidative stress produced by local metabolism of EtOH via CYP2E1 and antidiuretic hormone-1.

Modeling human vitamin D status in experimental rodents

We studied the effect of vitamin D3 (VD3) intake on VD status and markers of calcium (Ca) homeostasis. Experiment 1: 10 week old Sprague Dawley rats raised on a 400 IU/kg VD3 diet were fed one of five diets containing 400, 1,000, 5,000, 10,000, or 20,000 IU VD3/kg for 4 weeks (n=10 per diet). C57/BL6 mice were fed these diets until 10 wks of age (n=6 per diet). Serum 25 hydroxyvitamin D (25OH D) was directly related to VD3 intake in rats and mice (15 or 7.2 ng/ml per 1,000 IU VD3). Body weight (BW), serum Ca, and duodenal gene expression were not altered but high VD3 decreased serum 1, 25-dihydroxyvitamin D (1,25 D) and renal 1alpha-hydroxylase (CYP27B1) mRNA (−75% and −88% at 20,000 IU/kg). Thus, rodents tolerate high VD intake by balancing elevated 25OH D levels and the VD endocrine system. Experiment 2: Rats and mice were fed AIN93G diets containing 25, 50, 100, 200, 400, or 1,000 IU VD3/kg from weaning until 12 weeks of age (n=6 group). Serum 25OH D declined with lower VD3 following a split-line regression (break point at 200 IU/kg, 4-fold steeper slope below this point). BW and serum Ca were normal, however, renal CYP27B1 mRNA increased 2-fold in rats fed < 200 IU VD3/kg suggesting stimulation of the VD endocrine system. For scientists who wish to model the suboptimal vitamin D status common in humans, dietary VD3 levels below the NRC requirement for rodents of 1000 IU/kg are required. Supported by NIH awards DK054111 and CA101113 to JCF

Determinants of circulating 1,25-dihydroxyvitamin D3 levels: the role of renal synthesis and catabolism of vitamin D*1

Details of the molecular mechanisms determining levels of the secosteroid, 1,25-dihydroxyvitamin D 3 (1,25D) remain to be elucidated. The current paradigm for the control of serum 1,25D levels is the tight regulation of renal 25-hydroxyvitamin D-1α-hydroxlase (CYP27B1) activity by a number of physiological factors. 1,25D production is also regulated by the cytochrome P450 enzyme, 25-hydroxyvitamin D-24-hydroxylase (CYP24), which through side chain hydroxylation reactions, inactivates 1,25D. We have recently demonstrated that renal CYP27B1 and CYP24 expression contribute equally to regulating serum 1,25D levels. We now describe the contribution of renal Vitamin D receptor (VDR) expression in determining serum 1,25D levels. Serum 1,25D levels were decreased when the dietary calcium intake was increased. We measured mRNA levels for CYP27B1, CYP24 and VDR receptor in kidney RNA extracts from animals fed diets containing different levels of calcium, ranging from 0.05 to 1%. Serum 1,25D levels were negatively correlated with renal CYP24 mRNA levels ( R 2 =0.35, P <0.01) while renal VDR is positively correlated with renal CYP24 mRNA ( R 2 =0.80, P <0.001). However, only renal VDR mRNA remained a significant determinant of renal CYP24 expression when both these variables were included in multiple linear regression analysis (multiple R 2 =0.89, P <0.001). These findings suggest that kidney CYP24 activity acts in concert with kidney CYP27B1 to control serum 1,25D levels and that serum 1,25D stimulates renal CYP24 expression by acting through the renal VDR.

Determinants of circulating 1,25-dihydroxyvitamin D 3 levels: the role of renal synthesis and catabolism of vitamin D

Details of the molecular mechanisms determining levels of the secosteroid, 1,25-dihydroxyvitamin D 3 (1,25D) remain to be elucidated. The current paradigm for the control of serum 1,25D levels is the tight regulation of renal 25-hydroxyvitamin D-1α-hydroxlase (CYP27B1) activity by a number of physiological factors. 1,25D production is also regulated by the cytochrome P450 enzyme, 25-hydroxyvitamin D-24-hydroxylase (CYP24), which through side chain hydroxylation reactions, inactivates 1,25D. We have recently demonstrated that renal CYP27B1 and CYP24 expression contribute equally to regulating serum 1,25D levels. We now describe the contribution of renal Vitamin D receptor (VDR) expression in determining serum 1,25D levels. Serum 1,25D levels were decreased when the dietary calcium intake was increased. We measured mRNA levels for CYP27B1, CYP24 and VDR receptor in kidney RNA extracts from animals fed diets containing different levels of calcium, ranging from 0.05 to 1%. Serum 1,25D levels were negatively correlated with renal CYP24 mRNA levels ( R 2=0.35, P<0.01) while renal VDR is positively correlated with renal CYP24 mRNA ( R 2=0.80, P<0.001). However, only renal VDR mRNA remained a significant determinant of renal CYP24 expression when both these variables were included in multiple linear regression analysis (multiple R 2=0.89, P<0.001). These findings suggest that kidney CYP24 activity acts in concert with kidney CYP27B1 to control serum 1,25D levels and that serum 1,25D stimulates renal CYP24 expression by acting through the renal VDR.

Calcitonin is a major regulator for the expression of renal 25-hydroxyvitamin D 3 -1α-hydroxylase gene in normocalcemic rats

Regulation of vitamin D metabolism has long been examined by using vitamin D-deficient hypocalcemic animals. We previously reported that, in a rat model of chronic hyperparathyroidism, expression of 25-hydroxyvitamin D3-1alpha-hydroxylase (CYP27B1) mRNA was markedly increased in renal proximal convoluted tubules. It is believed that the major regulator for the expression of renal CYP27B1 is parathyroid hormone (PTH). However, in the normocalcemic state, the mechanism to regulate the renal CYP27B1 gene could be different, since plasma levels of PTH are very low. In the present study, the effect of PTH and calcitonin (CT) on the expression of renal CYP27B1 mRNA was investigated in normocalcemic sham-operated rats and normocalcemic thyroparathyroidectomized (TPTX) rats generated by either PTH or CaCl2 infusion. A single injection of CT dose-dependently decreased the expression of vitamin D receptor mRNA in the kidney of normocalcemic sham-TPTX rats. Concomitantly, CT greatly increased the expression of CYP27B1 mRNA in the kidney of normocalcemic sham-TPTX rats. CT also increased the expression of CYP27B1 mRNA in the kidney of normocalcemic TPTX rats. Conversion of serum [3H]1alpha,25(OH)2D3 from 25-hydroxy[3H]vitamin D3 in vivo was also greatly increased by the injection of CT into sham-TPTX rats and normocalcemic TPTX rats, but not into hypocalcemic TPTX rats. In contrast, administration of PTH did not induce the expression of CYP27B1 mRNA in the kidney of vitamin D-replete sham-TPTX rats and hypocalcemic TPTX rats. PTH increased the expression of renal CYP27B1 mRNA only in vitamin D-deficient hypocalcemic TPTX rats. These results suggest that CT plays an important role in the maintenance of serum 1alpha,25(OH)2D3 under normocalcemic physiological conditions, at least in rats.