Cytochrome P450 2R1 Research Articles

Anterior Cruciate Ligament Reconstruction Upregulates Vitamin D Activating Pathways within the Quadriceps

Anterior cruciate ligament (ACL) tears are prevalent sport‐related injuries that result in protracted quadriceps atrophy and weakness despite surgical reconstruction and physical therapy. Vitamin D and its receptor are integral for skeletal muscle development and homeostasis. In addition, vitamin D status is positively associated with muscle size and strength. If the vitamin D receptor and related enzymes are upregulated with ACL injury and reconstruction, then overall vitamin D status may be an important effector of recovery. Concentrations of vitamin D needed to support optimal recovery may differ from typical clinical cut points used to diagnose vitamin D deficiency, which could have powerful therapeutic implications following ACL injury and reconstruction. This is especially important given recent research showing over 50% of athletes have vitamin D inadequacy. We hypothesized that subjects with circulating vitamin D sufficient to suppress parathyroid hormone secretion (≥ 40 ng/mL) would be better protected against quadriceps atrophy at the conclusion of rehabilitation. Muscle biopsies (vastus lateralis) were obtained from ACL‐injured and Healthy limbs of 16 participants (6M,10F; 19±5yr, body mass index: 25.1±3.7kg/m2) prior to surgical reconstruction, 1 week following reconstruction (Recon), and following the completion of post‐surgical rehabilitation (6 months following surgery, Post‐Rehab). RNA was isolated from all quadriceps biopsies, followed by library construction, sequencing, and bioinformatics analysis. Transcript abundance of vitamin D activating pathway regulators (vitamin D receptor [VDR], and Cytochrome P450 2R1 [CYP2R1] and Family 27 Subfamily B Member 1 [CYP27B1]) was assessed following analysis using DESeq2 and false discovery rate (FDR) adjustment of p‐values. Additionally, 25‐hydroxy vitamin D (25(OH)D) was assessed in serum samples obtained at the time of reconstruction surgery. Quadriceps fiber cross‐sectional area was measured via laminin immunohistochemistry. Recon (1 week post‐surgery) muscle showed increased transcript abundance of the vitamin D receptor, VDR(elevated 7.8 fold, FDR<0.05), and vitamin D‐activating pathway regulators CYP2R1 (elevated 1.5 fold, FDR<0.05) and CYP27B1 (elevated 2.6 fold, FDR<0.05) when compared to the Healthy limb. Following the completion of rehabilitation at the 6‐month follow‐up, participants with 25(OH)D above 40 ng/mL showed 6.4% atrophy of quadriceps fiber size compared to 22.8% atrophy in participants with 25(OH)D below 40 ng/mL (p=0.06). These findings provide evidence for surgically induced elevations in the vitamin D activating pathway within skeletal muscle. Upregulation of the vitamin D activating pathway may support quadriceps mitochondrial health and satellite cell activity and self‐renewal capacity, both of which are compromised following ACL reconstruction. In addition, elevated vitamin D concentrations may protect against protracted quadriceps atrophy. Future research should investigate if manipulation of vitamin D concentrations can enhance quadriceps functional recovery following ACL reconstruction.

New Variants of the Cytochrome P450 2R1 (CYP2R1) Gene in Individuals with Severe Vitamin D-Activating Enzyme 25(OH)D Deficiency.

Background: Vitamin D is a fat-soluble cholesterol derivative found in two forms, vitamin D2, and vitamin D3. Cytochrome P450 2R1 (CYP2R1) encoded by the CYP2R1 gene is the major hydroxylase that activates vitamin D by catalyzing the formation of 25-hydroxyvitamin D (25(OH)D). Methods: We collected 89 (100%) subjects, 46 of which (51.69%) had a documented severe deficiency of 25(OH)D (<10 ng/mL) and 43 (48.31%) in the control group with documented optimum levels of 25(OH)D (>30 ng/mL). We performed Sanger sequencing of three selected fragments of the CYP2R1 gene (Ch11: 14878000–14878499; Ch11: 14880058–14880883 and Ch11: 14885321–14886113) that affect the binding of substrates to this enzyme and analyzed the possible involvement of genetic variation in these regions with an increased risk of vitamin D deficiency in healthy Polish individuals. Results: Two substitutions were found within the three fragments. Bioinformatic analysis suggested that one of these (NC_000011.10: g.14878291G>A) may influence the structure and function of CYP2R1. Conclusions: Variant NC_000011.10: g.14878291G>A may have a perturbing effect on heme binding in the active site of CYP2R1 and on the function of 25-hydroxylase and probably affects the concentration of 25(OH)D in vivo. We intend to perform functional verification in a larger patient population to confirm and extend these results.

The CYP2R1 Enzyme: Structure, Function, Enzymatic Properties and Genetic Polymorphism.

Since the discovery of its role in vitamin D metabolism, significant progress has been made in the understanding of gene organisation, protein structure, catalytic function, and genetic polymorphism of cytochrome P450 2R1 (CYP2R1). Located on chromosome 11p15.2, CYP2R1 possesses five exons, unlike most other CYP isoforms that carry nine exons. CYP2R1 crystal structure displays a fold pattern typical of a CYP protein, with 12 a-helices as its structural core, and b-sheets mostly arranged on one side, and the heme buried in the interior part of the protein. Overall, CYP2R1 structure adopts a closed conformation with the B' helix serving as a gate covering the substrate access channel, with the substrate vitamin D3 occupying a position with the side chain pointing toward the heme group. In liver, CYP2R1 25-hydroxylates vitamin D and serves as an important determinant of 25(OH)D level in the tissue and in circulation. While substrate profile has been well studied, inhibitor specificity for CYP2R1 requires further investigation. Both exonic and non-exonic single nucleotide polymorphisms (SNPs) have been reported in CYP2R1, including the CYP2R1*2 carrying Leu99Pro exchange, and a number of non-exonic SNPs with variable functional consequences in gene regulation. A non-exonic SNP, rs10741657, has its causal relationship with diseases established, including that of rickets, ovarian cancer, and multiple sclerosis. The role of other CYP2R1 SNPs in vitamin D deficiency and their causal link to other traits however remain uncertain currently and more studies are warranted to help identify possible physiological mechanisms underlying those complex traits.

Genetic Variation in Cytochrome P450 2R1 and Vitamin D Binding Protein Genes are associated with Vitamin D Deficiency in Adolescents.

Background: Genome Wide Association Studies (GWAS) have evaluated several genes related to vitamin D synthesis, metabolism and transport. They have proposed a genetic basis for low levels of vitamin D in the blood. The current study aims to investigate the relationship between certain vitamin D-associated gene variants and vitamin D deficiency in Iranian adolescents. Methods: In this case-control study, the genomic DNA was extracted by Real Time PCR High Resolution Melt(HRM). All measurements were carried out with triple repetition. The following factors were assessed: single nucleotide polymorphisms (SNPs) in Vitamin D binding protein (DBP, rs2282679), 7-Dehydrocholesterol reductase (DHCR7, rs12785878) and Cytochrome P450 2R1 (CYP2R1, rs10741657). Results: the genomic DNA of blood samples obtained from 481 adolescents. Participants with hypovitaminosis D were compared with a control group. The average vitamin D level of sufficient subjects (controls) was 44.88±14.01ng/mL, while subjects who were insufficient (cases) had an average vitamin D level of 7.03±1.24ng/mL. No statistically significant differences were found in the allelic and genotypic distributions between genders. The SNP frequency in CYP2R1 (rs10741657) and DBP (rs2282679) in the vitamin D deficient group was significantly higher than in the control group (p-values <0.001 and 0.01 respectively). There were no statistically significant differences in the DHCR7 SNP (rs12785878) distributions between the Vitamin D deficient group and control group. Conclusion: The present study demonstrated evidence of the ability of the SNPs under investigation to predict circulating vitamin D concentration. Further study is needed to better understand if and how genetic factors contribute to vitamin D levels, and certain skeletal-associated disorders in adolescents.

Genetically determined vitamin D levels and change in bone density during a weight-loss diet intervention: the Preventing Overweight Using Novel Dietary Strategies (POUNDS Lost) Trial

Obesity is closely associated with bone health. Although diet and weight loss produce many metabolic benefits, studies of weight loss diets on bone health are conflicting. Genetic variations, such as vitamin D levels, may partly account for these conflicting observations by regulating bone metabolism. We investigated whether the genetic variation associated with vitamin D concentration affected changes in bone mineral density (BMD) in response to a weight-loss diet intervention. In the 2-y Preventing Overweight Using Novel Dietary Strategies (POUNDS Lost) trial, BMD was measured for 424 participants who were randomly assigned to 1 of 4 diets varying in macronutrient intakes. A genetic risk score (GRS) was calculated based on 3 genetic variants [i.e., 7-dehydrocholesterol reductase (DHCR7) rs12785878, cytochrome P450 2R1 (CYP2R1) rs10741657 and group-specific component globulin (GC) rs2282679] related to circulating vitamin D levels. A dual-energy X-ray absorptiometry scan was performed to assess changes in whole-body BMD over 2 y. The final analysis included 370 participants at baseline. We found a significant interaction between dietary fat intake and vitamin D GRS on 2-y changes in whole-body BMD (P-interaction=0.02). In the high-fat diet group, participants with higher GRS showed significantly less reduction in whole-body BMD than those with lower GRS, whereas the genetic associations were not significant in the low-fat diet group. We also found a significant interaction between dietary fat intake and the GRS on 6-mo change in femur neck BMD (P-interaction=0.02); however, the interaction became nonsignificant at 2 y. Our data indicate that dietary fat intake may modify the effect of vitamin D-related genetic variation on changes in BMD. Overweight or obese patients predisposed to sufficient vitamin D may benefit more in maintaining BMD along with weight loss by eating a low-fat diet. This trial was registered at clinicaltrials.gov as NCT03258203.

Effects of Gene Variants Controlling Vitamin D Metabolism and Serum Levels on Hepatic Steatosis

Background/Aims: Common genetic variations in vitamin D metabolism are associated with liver stiffness. Whether these genes are implicated in hepatic steatosis remains unclear. Here we aimed to analyse the association of common vitamin D pathway gene variants with liver steatosis. Methods: Liver steatosis was assessed non-invasively in 241 patients with chronic liver conditions by controlled attenuation parameter (CAP). The following polymorphisms were genotyped using TaqMan assays: group-specific component (GC) rs7041, 7-dehydrocholesterol reductase (DHCR7) rs12785878, cytochrome P450 2R1 (CYP2R1) rs10741657, ­vitamin D receptor (VDR) rs7974353. Chemiluminescence immunoassay determined serum 25-hydroxyvitamin D (25(OH) D) concentrations. Results: Vitamin D deficiency (defined by 25(OH)D concentrations <20 ng/mL) occurred in 66% of patients. Median CAP was 296 (100–400) dB/m. Patients with advanced steatosis (CAP ≥280 dB/m) had significantly (p = 0.033) lower 25(OH)D levels as compared to patients with CAP <280 dB/m. Moreover, the rare allele [T] in GC rs7041 was significantly (p = 0.018) associated with higher 25(OH)D levels in patients with CAP <280 dB/m. However, GC, DHCR7, CYP2R1, and VDR polymorphisms were not related to liver steatosis and obesity traits. Conclusions: Higher CAP values are associated with low serum 25(OH)D concentrations but not with common vitamin D pathway gene variants.

Fatty Acid Oxidation in Zebrafish Adipose Tissue Is Promoted by 1α,25(OH)2D3

1α,25(OH)2D3 (vitamin D3) is crucial for mineral homeostasis in mammals, but the precise effects of 1α,25(OH)2D3 in adipose tissue remain to be clarified invivo. The initial 25-hydroxylation is catalyzed by liver microsomal cytochrome P450 2R1 (CYP2R1), which is conserved in vertebrates. To probe the physiological function(s) of 1α,25(OH)2D3 in teleosts, we generated two independent cyp2r1-deficient zebrafish lines. These mutants exhibit retarded growth and increased obesity, especially in the visceral adipose tissue (VAT). These defects could be rescuedwith 25(OH)D3 treatments. ChIP-PCR analyses demonstrated that pgc1a is the target of the vitamin D receptor in the liver and VAT of zebrafish. Significantly decreased protein levels of Pgc1a, impaired mitochondrial biogenesis, and free fatty acid oxidation are also observed in the cyp2r1 mutant VAT. Our results demonstrate that regulation of 1α,25(OH)2D3 during lipid metabolism occurs through the regulation of Pgc1a for mitochondrial biogenesis and oxidative metabolism within zebrafish VAT.

Genetic Variation in CYP2R1 and GC Genes Associated With Vitamin D Deficiency Status

This cross-sectional study enrolled 180 patients at a private family practice in Virginia. Total serum vitamin D concentrations were obtained weekly from January 30, 2013, through March 30, 2013, in consecutive patients regularly scheduled for laboratory work at the practice. Patients were categorized into 2 groups and analyzed for variant alleles in vitamin D receptor (VDR; rs2228570), cytochrome P450 2R1 (CYP2R1; rs10741657), 7-dehydrocholesterol reductase (DHCR7; rs12785878), and group-specific component (GC; rs2282679) to determine whether variants of those alleles influenced total serum 25(OH)D concentrations. One-hundred and eighty patients were enrolled, with 40 (22%) being sufficient, 25-hydroxy vitamin D level 25(OH)D ≥ 30 ng/mL, and 140 (78%) being insufficient, 25(OH)D < 30 ng/mL. Of the 4 genes, 2 genes, CYP2R1 (rs10741657) and GC (rs2282679), demonstrated a significant association related to vitamin D status. Subjects with 1 or more variant alleles at rs10741657 were almost 3.7 (odds ratio [OR] 3.67; 95% confidence interval [CI]: 1.35-9.99) times more likely be insufficient in vitamin D and subjects with 1 or more variant alleles at rs2282679 were about half (OR 0.42; 95% CI: 0.18-0.93) as likely to be insufficient in vitamin D. Allelic variations in CYP2R1 (rs10741657) and GC (rs2282679) affect vitamin D levels, but variant alleles on VDR (rs2228570) and DHCR7 (rs12785878) were not correlated with vitamin D deficiency, 25(OH)D < 30 ng/mL.

High fat diet enriched with saturated, but not monounsaturated fatty acids adversely affects femur, and both diets increase calcium absorption in older female mice

Diet induced obesity has been shown to reduce bone mineral density (BMD) and Ca absorption. However, previous experiments have not examined the effect of high fat diet (HFD) in the absence of obesity or addressed the type of dietary fatty acids. The primary objective of this study was to determine the effects of different types of high fat feeding, without obesity, on fractional calcium absorption (FCA) and bone health. It was hypothesized that dietary fat would increase FCA and reduce BMD. Mature 8-month-old female C57BL/6J mice were fed one of three diets: a HFD (45% fat) enriched either with monounsaturated fatty acids (MUFAs) or with saturated fatty acids (SFAs), and a normal fat diet (NFD; 10% fat). Food consumption was controlled to achieve a similar body weight gain in all groups. After 8wk, total body bone mineral content and BMD as well as femur total and cortical volumetric BMD were lower in SFA compared with NFD groups (P<.05). In contrast, femoral trabecular bone was not affected by the SFAs, whereas MUFAs increased trabecular volume fraction and thickness. The rise over time in FCA was greater in mice fed HFD than NFD and final FCA was higher with HFD (P<.05). Intestinal calbindin-D9k gene and hepatic cytochrome P450 2r1 protein levels were higher with the MUFA than the NFD diet (P<.05). In conclusion, HFDs elevated FCA overtime; however, an adverse effect of HFD on bone was only observed in the SFA group, while MUFAs show neutral or beneficial effects.

Update on the relationship between testicular function and bone metabolism

It has been well known that testosterone is the only connection between testis and bone as it plays an important role in skeletal growth and bone mass accrual. In the past few years, studies showed that factors other than testosterone could also be responsible for bone health. The Leydig cells in the testis produce insulin-like 3 and express cytochrome P450 2R1(CYP2R1)gene which encodes the major enzyme involved in 25-hydroxylation of vitamin D. The understanding of the crosstalk between testis and bone could contribute to defining an improved clinical approach to the biochemical diagnosis and therapeutic management of hypogonadism and male osteoporosis. (Chin J Endocrinol Metab, 2015, 31: 372-374) Key words: Testosterone; Insulin-like 3; Cytochrome P450 2R1; Bone metabolism

1α,25-dihydroxyvitamin D3 modulates CYP2R1 gene expression in human oral squamous cell carcinoma tumor cells.

Oral squamous cell carcinomas (OSCC) are the most common malignant neoplasms associated with mucosal surfaces of the oral cavity and oropharynx. 1α,25-Dihydroxyvitamin D3 (1,25(OH)2D3) is implicated as an anticancer agent. Cytochrome P450 2R1 (CYP2R1) is a microsomal vitamin D 25-hydroxylase which plays an important role in converting dietary vitamin D to active metabolite, 25-(OH)D3. We identified high levels of CYP2R1 expression using tissue microarray of human OSCC tumor specimens compared to normal adjacent tissue. Therefore, we hypothesize that 1,25(OH)2D3 regulates CYP2R1 gene expression in OSCC tumor cells. Interestingly, real-time RT-PCR analysis of total RNA isolated from OSCC cells (SCC1, SCC11B, and SCC14a) treated with 1,25(OH)2D3 showed a significant increase in CYP2R1 and vitamin D receptor (VDR) mRNA expression. Also, Western blot analysis demonstrated that 1,25(OH)2D3 treatment time-dependently increased CYP2R1 expression in these cells. 1,25(OH)2D3 stimulation of OSCC cells transiently transfected with the hCYP2R1 promoter (-2kb)-luciferase reporter plasmid demonstrated a 4.3-fold increase in promoter activity. In addition, 1,25(OH)2D3 significantly increased c-Fos, p-c-Jun expression, and c-Jun N-terminal kinase (JNK) activity in these cells. The JNK inhibitor suppresses 1,25(OH)2D3, inducing CYP2R1 mRNA expression and gene promoter activity in OSCC cells. Furthermore, JNK inhibitor significantly decreased 1,25(OH)2D3 inhibition of OSCC tumor cell proliferation. Taken together, our results suggest that AP-1 is a downstream effector of 1,25(OH)2D3 signaling to modulate CYP2R1 gene expression in OSCC tumor cells, and vitamin D analogs could be potential therapeutic agents to control OSCC tumor progression.

OC002—Cyp2r1 Genetic Polymorphisms Are Associated With Lower 25-Hydroxy Vitamin D Levels In Lebanese Subjects

Despite plentiful sunshine in the Middle East, populations in general, and in Lebanon in particular, have some of the lowest levels of 25-hydroxy vitamin D (25-OHD) worldwide. Our group has demonstrated such findings across the lifespan; predictors include gender, season, and clothing style (http://staff.aub.edu.lb/~webcmop/publications.html). However, the possibility of an underlying genetic modulation of circulating 25-OHD levels remains unexplored. 25-Hydroxylation is mainly driven by cytochrome P-450 2R1 (CYP2R1) drug metabolizing enzyme; we therefore hypothesized that carriage of CYP2R1 single nucleotide polymorphisms (SNPs) may be associated with variability in 25-OHD levels.

Vitamin D and the Parenteral Nutrition Patient

Vitamin D is a prohormone produced in the skin epidermis when irradiated with sunlight or ultraviolet light B. It is also absorbed from food or supplements. Vitamin D must be converted to 25-hydroxyvitamin D3 (circulating form) and finally to the hormone, 1a,25-dihydroxyvitamin D3, before it can function. This hormone acts through a single nuclear receptor. The details of these conversions and molecular biology of the action of vitamin D3 are summarized. The physiologic functions of vitamin D have been expanded beyond the mineralization of the skeleton to include modulation of the immune system, terminal differentiation in several tissues, suppression of malignant cells, and anabolic activity in the skeleton and in the renal-cardiovascular system. Epidemiologic studies have associated vitamin D deficiency with an increased risk of colorectal and breast cancers and an increased risk of autoimmune diseases, such as multiple sclerosis, type 1 diabetes, and cardiovascular events. Thus, vitamin D is essential not only for the skeleton but also many other organ systems. Recommendations for 25-hydroxyvitamin D3 levels for PN patients are presented.