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BACKGROUND Prostate specific membrane antigen (PSMA) expression correlates with prostate cancer grade and is increased in hormone-refractory prostate cancer. The increased expression of PSMA following androgen deprivation therapy may be a consequence of the down-regulation of PSMA expression by androgen. Moreover, 1α,25-dihydroxyvitamin D3 (1,25-VD) has been shown to suppress prostate cancer progression as well as cell motility and invasion. Since PSMA is positively correlated with both of these characteristics, we hypothesized that 1,25-VD would regulate PSMA expression. METHODS LNCaP prostate cancer cells were treated with 1,25-VD, followed by analysis of cell surface PSMA expression. The PSMA enhancer, located within the third intron of the PSMA gene, was cloned into a reporter vector and regulation by 1,25-VD was investigated. The role of the androgen receptor (AR) in 1,25-VD mediated suppression of PSMA expression was examined using Casodex and AR specific siRNA. RESULTS Surface expression of PSMA was significantly decreased in a dose-dependent manner by 10 nM 1,25-VD or greater. Regulation by 1,25-VD occurred at the level of the PSMA enhancer. Over-expression of the vitamin D receptor (VDR) also decreased expression of PSMA. Additionally, suppression of AR translation using siRNA technology blocked the suppressive effect of 1,25-VD on PSMA expression, however inhibition of PSMA expression by 1,25-VD occurred in the absence of androgens. CONCLUSIONS Suppression of PSMA by 1,25-VD occurs at the level of the PSMA enhancer and is elevated by over-expression of the VDR. This regulation involves the AR, but is not dependent on the presence of androgens. Prostate 68: 773–783, 2008. © 2008 Wiley-Liss, Inc.

Cytochrome P450C24A1 (CYP24A1), a peripheral inner mitochondrial membrane hemoprotein and candidate oncogene, regulates the side-chain metabolism and biological function of vitamin D and many of its related analog drugs. Rational mutational analysis of rat CYP24A1 based on hybrid (2C5/BM-3) homology modeling and affinity labeling studies clarified the role of key domains (N-terminus, A', A, and F-helices, beta3a strand, and beta5 hairpin) in substrate binding and catalysis. The scope of our study was limited by an inability to purify stable mutant enzyme targeting soluble domains (B', G, and I-helices) and suggested greater conformational flexibility among CYP24A1's membrane-associated domains. The most notable mutants developed by modeling were V391T and I500A, which displayed defective-binding function and profound metabolic defects for 25-hydroxylated vitamin D3 substrates similar to a non-functional F-helix mutant (F249T) that we previously reported. Val-391 (beta3a strand) and Ile-500 (beta5 hairpin) are modeled to interact with Phe-249 (F-helix) in a hydrophobic cluster that directs substrate-binding events through interactions with the vitamin D cis-triene moiety. Prior affinity labeling studies identified an amino-terminal residue (Ser-57) as a putative active-site residue that interacts with the 3beta-OH group of the vitamin D A-ring. Studies with 3-epi and 3-deoxy-1,25(OH)2D3 analogs confirmed interactions between the 3beta-OH group and Ser-57 effect substrate recognition and trafficking while establishing that the trans conformation of A-ring hydroxyl groups (1alpha and 3beta) is obligate for high-affinity binding to rat CYP24A1. Our work suggests that CYP24A1's amphipathic nature allows for monotopic membrane insertion, whereby a pw2d-like substrate access channel is formed to shuttle secosteroid substrate from the membrane to the active-site. We hypothesize that CYP24A1 has evolved a unique amino-terminal membrane-binding motif that contributes to substrate specificity and docking through coordinated interactions with the vitamin D A-ring.

The aim of this study was to investigate effects of 1,25(OH)(2)D(3) (calcitriol), 25OHD(3), and EB1089 on cell growth and on Vitamin D receptor (VDR) mRNA and 1alpha-hydroxylase (1alpha-OHase) mRNA expression in normal canine prostatic primary cultures. Canine prostatic epithelial cells were isolated, cultured, and treated with vehicle (ethanol), calcitriol, 25OHD(3), and EB1089 at 10(-9) and 10(-7)M. The VDR was present in epithelial and stromal cells of the canine prostate gland. 1,25(OH)(2)D(3), 25OHD(3), and EB1089 inhibited epithelial cell growth at 10(-7)M compared to vehicle-treated controls [calcitriol (P < 0.01), EB1089 (P < 0.01), and 25OHD(3) (P < 0.05)]. Epithelial cells treated with calcitriol and EB1089 at 10(-7)M had slightly increased VDR mRNA expression (0.2-0.3-fold) at 6 and 12h compared to controls. There was no difference in 1alpha-OHase mRNA expression in epithelial cells treated with these three compounds. 1,25(OH)(2)D(3) and its analogs may be effective antiproliferative agents of epithelial cells in certain types of prostate cancer.

Although vitamin D(3) is a natural product of a sunlight-mediated process in the skin, the secosteroid's biological function is dependent upon specific cytochrome P450 enzymes that mediate the parent vitamin's bioactivation and inactivation. Cytochrome P450C1 (CYP27B1) is the regulatory rate-limiting enzyme that directs the bioactivation process through introduction of a C-1alpha hydroxyl group. The resultant 1,25-dihydroxyvitamin D(3) (1,25D) is the biologically active secosteroid hormone that directs the multitude of vitamin D-dependent actions involved with calcium homeostasis, cellular differentiation and growth, and the immune response. The circulating and cellular level of 1,25D is regulated through a coordinated process involving the hormone's synthesis and degradation. Central to the degradation and turnover of 1,25D is the regulatory multi-catalytic cytochrome P450C24 (CYP24) enzyme that directs the introduction of C-24R groups onto targeted 25-hydroxy substrates. Discussed in this article is the action of the rat CYP24 to catalyze the side-chain oxidation and cleavage of 25-hydroxylated vitamin D metabolites. Expression and characterization of purified recombinant rat CYP24 is discussed in light of mutations directed at the enzyme's active site.

The current study investigated the action of 1,25-dihydroxyvitamin D3 (1,25D) at the genomic and signal transduction levels to induce rat cytochrome P450C24 (CYP24) gene expression. A rat CYP24 promoter containing two vitamin D response elements and an Ets-1 binding site was used to characterize the mechanism of actions for the 1,25D secosteroid hormone. The Ets-1 binding site was determined to function cooperatively with the most proximal vitamin D response element in a hormone-dependent fashion. Evidence was obtained for distinct roles of ERK1/ERK2 and ERK5 in the 1,25D-inductive actions. Specifically, 1,25D stimulated the activities of ERK1/ERK2 and ERK5 in a Ras-dependent manner. Promoter induction was inhibited by mitogen-activated protein (MAP) kinase inhibitors (PD98059 and U0126) and a dominant-negative Ras mutant (Ras17N). Induction ofCYP24 by 1,25D was also inhibited by overexpression of dominant-negative mutants of ERK1 and MEK5 (ERK1K71R and MEK5(A)). The p38 and JNK MAP kinases were not required for the action of 1,25D. 9-cis retinoid X receptor α (RXRα) interacted with ERK2 but not ERK5 in intact cells, whereas Ets-1 interacted preferentially with ERK5. Increased phosphorylation of RXRα and Ets-1 was detected in response to 1,25D. Activated ERK2 and ERK5 specifically phosphorylated RXRα and Ets-1, respectively. Mutagenesis of Ets-1 (T38A) reduced CYP24 promoter activity to levels observed with the dominant-negative MEK5(A) and inhibited ERK5-directed phosphorylation. Mutated RXRα (S260A) inhibited 1,25D-induced CYP24 promoter activity and abolished phosphorylation by activated ERK2. The 1,25D-inductive action through ERK5 involved Ets-1 phosphorylation at threonine 38, whereas hormone stimulation of ERK1/ERK2 required RXRα phosphorylation on serine 260. The ERK1/ERK2 and ERK5 modules provide a novel mechanism for linking the rapid signal transduction and slower transcription actions of 1,25D to induce CYP24 gene expression.

Transcription of the rat CYP24 gene is induced by 1,25-dihydroxyvitamin D3(1,25-(OH)2D3) through two vitamin D response elements (VDREs). A functional Ras-dependent Ets-binding site (EBS) was located downstream from the proximal VDRE and was critical to 1,25(OH)2D3-mediated induction. Cotransfection of Ets-1 and Ets-2 stimulated induction, which was lost when the EBS was mutated. Multiple nuclear-protein complexes from COS-1 cells bound to the EBS in which three complexes were immunologically related to Ets-1. Transcriptional synergy was observed between the proximal VDRE and adjacent EBS as was the attendant formation of a ternary complex between vitamin D receptor- retinoid X receptor (VDR·RXR) and Ets-1. In the absence of 1,25-(OH)2D3 or in the presence of an inactive proximal VDRE, the EBS failed to respond to exogenous Ets-1. However, Ets-1 increased basal expression when cotransfected with a mutant VDR. The inductive action of 1,25-(OH)2D3 was substantially increased by Ras, which was ablated by mutagenesis of the EBS or by expression of a mutated Ets-1 protein (T38A). EBS contribution to hormone induction was prevented by manumycin A, an inhibitor of Ras farnesylation. A fundamental role was established for transcriptional cooperation between Ras-activated Ets proteins and the VDR·RXR complex in mediating 1,25-(OH)2D3action on the CYP24 promoter.

Background: Monitoring CYP2E1 levels in alcoholic individuals holds inherent appeal because such determinations might indicate individuals at increased risk for alcoholic liver disease. We previously demonstrated that lymphocyte CYP2E1 expression reflects in vivo activity of the hepatic enzyme. Methods: To further validate this approach, the current investigation compared lymphocyte CYP2E1 content and chlorzoxazone pharmacokinetics in 51 alcoholic and nonalcoholic White, Navajo, and Mexican American subjects. After an oral dose of chlorzoxazone, blood samples were collected and lymphocytes isolated. Results: Alcoholics exhibited a 2-fold clevation in lymphocyte CYP2E1 messenger ribonucleic acid (mRNA) and protein compared to nonalcoholics. Chlorzoxazone clearance rates were 1.9-fold higher and area under the concentration curve (AUC) values 1.8-fold lower in alcoholic individuals compared to nonalcoholics. Furthermore, chlorzoxazone clearance rates correlated (r= 0.55, p < 0.01, n= 38) with lymphocyte CYP2E1 mRNA content, and transcript levels further correlated (r= 0.52, p < 0.001, n= 38) with CYP2E1 protein content. To compare phenotype with genotype, restriction fragment length polymorphism analyses on deoxyribonuclcic acid samples were performed to identify polymorphisms in the CYP2E1 gene. No subjects were homozygous for rare alleles c2 or C. Nonetheless, 27% of the Navajos and 15% of the Mexican Americans were heterozygous for the c2 allele. Two White subjects appeared heterozygous (c1/c2) when RsaI was used to characterize CYP2E1 genotype but homozygous (c1/c1) at the PstI locus. Fifteen percent of Mexican American subjects, 20% of Navajo subjects, and 6% of White subjects were heterozygous for the C allele. Neither CD nor c1/c2 genotypes were associated with alcoholism. Conclusions: Human lymphocyte CYP2E1 mRNA levels may be useful predictors of alcohol-mediated alterations in hepatic CYP2E1 activity. Moreover, ethnicity does not appear to play a major role in the levels of expression of lymphocyte CYP2E1.

We previously demonstrated augmented endothelium-derived nitric oxide (EDNO)-dependent pulmonary arterial dilation and increased arterial endothelial nitric oxide synthase (eNOS) levels in chronic hypoxic (CH) and monocrotaline (nonhypoxic) models of pulmonary arterial hypertension. Therefore, we hypothesized that the long-term elevation of arterial eNOS levels associated with CH is related to pulmonary hypertension or some factor(s) associated with hypertension and not directly to hypoxia. To test this hypothesis, we examined responses to the EDNO-dependent dilator ionomycin in U-46619-constricted, isolated, saline-perfused lungs from control rats, CH (4 wk at 380 mmHg) rats, and rats previously exposed to CH but returned to normoxia for 4 days or 2 wk. Microvascular pressure was assessed by double-occlusion technique, allowing calculation of segmental resistances. In addition, vascular eNOS immunoreactivity was assessed by quantitative immunohistochemistry, and eNOS mRNA abundance was determined by RT-PCR assays. Our findings indicate that 4-day and 2-wk posthypoxic rats exhibit persistent pulmonary hypertension, likely due to maintained arterial remodeling and polycythemia associated with prior exposure to CH. Furthermore, arterial dilation to ionomycin was augmented in lungs from each experimental group compared with controls. Finally, arterial eNOS immunoreactivity and whole lung eNOS mRNA levels remained elevated in posthypoxic animals. These findings suggest that altered vascular mechanical forces or vascular remodeling contributes to enhanced EDNO-dependent arterial dilation and upregulation of arterial eNOS in various models of established pulmonary hypertension.

Mitochondrial cytochrome P450(24) expression in the vitamin D-degradation pathway is induced by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. The molecular basis of this enzyme regulation was investigated by isolating the rat P450(24) gene and examining the 5'-flanking region for possible cis-acting regulatory elements involved in the induction process. Constructs containing different lengths of 5'-flanking region of the gene were linked to a luciferase reporter gene and transiently co-transfected with a human vitamin D receptor (hVDR) expression vector (pRSV-hVDR) into COS-1 cells. These experiments showed that the flanking region from -298 to -122 directed a 24-fold increase in luciferase activity in response to 1,25-(OH)2D3 provided that the cells were co-transfected with pRSV-hVDR. Within this region, the sequence from position -171 to -123 conferred 1,25-(OH)2D3 responsiveness to both the native P450(24) promoter and the heterologous thymidine kinase promoter. Mutagenesis revealed that the sequence from position -150 to -136 is required for induction by 1,25-(OH)2D3 and that this sequence shares similarity to other vitamin D responsive elements (VDREs) reported for other genes. Gel shift mobility assays showed this region specifically bound a nuclear protein complex from 1,25-(OH)2D3 treated COS-1 cells that had been co-transfected with pRSV-hVDR. The retarded band was specifically competed with the well characterized VDRE from the mouse osteopontin gene. A VDRE at position -150 to -136 in the promoter of the rat P450(24) gene is identified in this study and found to be important in mediating the enhanced expression of the gene by 1,25-(OH)2D3.

Porcine renodoxon is a kidney mitochondrial iron-sulfur protein (ISP) that functions to transfer electron to cytochromes P450 of the vitamin D pathway. A full-length cDNA clone to porcine renodoxin was isolated in the current investigation and used to study the protein's primary structure and immunological properties. The cysteine ligands for the iron-sulfur center, and the surface protein-binding and phosphorylation sites occupied identical positions in both porcine renodoxin and bovine adrenodoxin. Furthermore, porcine renodoxin was functionally indistinguishable from bovine adrenodoxin and the mature forms of both proteins had the same encoded length and shared approximately 91% sequence similarity. A synthetic peptide to the surface protein-binding region was used to demonstrate the antigenicity of the domain in both the porcine and the bovine ISPs. However, porcine renodoxin displayed only limited immunological identity to other regions of bovine adrenodoxin as measured by competitive enzyme-linked immunosorbent assay. Part of this immunological distinction was attributed to the COOH-terminal processing of porcine renodoxin, an action which negated expression of a COOH-terminal antigenic site that is present in bovine adrenodoxin. Other antigenic differences were linked to charged-residue substitutions that were located in predicted surface domains. The highest frequency of surface-residue substitutions in ferredoxin proteins was predicted for porcine renodoxin, which could provide a basis for understanding why the pig protein appears more antigenically divergent than other ferredoxins.

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

The hormone 1,25-dihydroxyvitamin D (1,25D) may play a protective role in prostate cancer. 25-hydroxyvitamin D 1-alpha hydroxylase (CYP27B1) is the enzyme responsible for the regulation of cellular 1,25D levels. CYP27B1 is substantially repressed in prostate cancer cells. We have investigated the molecular basis for this inhibition. First, we identify a repressive region between -997 and -1200 in the human CYP27B1 promoter following transient transfection analysis in the prostate cancer cell lines DU145, PC3 and LNCaP. Next, we demonstrate a role for the transcription factor growth factor independent-1 (GFI1) in the repression of CYP27B1. Electrophoretic mobility assays with nuclear extracts from prostate cancer cell lines established binding of GFI1 to the sequence 5'-TGGTACAATCATAACTCACTGCAG-3' present at -997 to -1200 in the repressive region. Site directed mutagenesis of the core GFI1 binding sequence (5'-AATC-3') substantially increased while forced expression of GFI1 decreased the expression of the CYP27B1 reporter construct. Importantly, GFI1 repression is dependent on an intact GFI1 binding site in the -997 to -1200 region. GFI1 is an oncoprotein known to form a large protein complex with co-repressors that recruit histone deacetylases. We propose that the formation of such a repressive complex on the inhibitory domain of the CYP27B1 gene in prostate cancer cells could lead to silencing of either the nearby enhancer or proximal promoter domains and lead to cancer progression by reducing local production of 1,25D. These studies provide the basis for a more detailed understanding of CYP27B1 repression in prostate cancer cells and could provide a novel insight in future diagnosis and treatment.

We evaluated the prevalence, trends, outcomes and the general experience of physicians performing atrial fibrillation ablation (AF-ABL) in the United States (US).AF-ABL is a non-pharmacological and potentially curative therapy for AF. Success rates for AF-ABL have been reported to be between 80 and 90%. Although there are numerous clinical trial addressing this therapy little is known about the general status of AF-ABL in clinical practice.We administered a mailed survey to the physician members of a professional arrhythmia society (Heart Rhythm Society, formerly known as the North American Society of Pacing and Electrophysiology) who practiced in the US (n = 1843).There were 304 responses, 66% (n = 204) performed ABL and 30% (n = 92) performed AF-ABL. The study group performed a total of 5,592 AF-ABL from 2000 to 2003, out of 72,575 total ABL procedures during the same time period. There was a four-fold increase in the number of AF-ABL between 2000 and 2003 (2000: 628 vs. 2003: 2,575). In the same period, the self-reported short and long-term success rates of AF-ABL improved an average of 18 +/- 4% (por = 0.001). In 2003 the average self-reported one-month, one-year, and two-year success rates were: 71 +/- 4%, 66 +/- 5%, 63 +/- 6% respectively. The predicted five-year success was 60 +/- 4%. The average procedure took 4.5 +/- 0.4 hours. Physicians reported that approximately 29 +/- 4% of their patents were potential candidates for AF-ABL.AF-ABL is becoming a much more common procedure in the US. Over the last four years the perceived short and long term success rates of AF-ABL have improved. Success rates in this survey are 10 to 20% lower than those reported in the recent clinical trials.

Recently, 25-hydroxyvitamin D3-24-hydroxylase (CYP24A1) has been shown to catalyze not only hydroxylation at C-24 but also hydroxylations at C-23 and C-26 of the secosteroid hormone 1alpha, 25-dihydroxyvitamin D3 (1alpha,25(OH)2D3). It remains to be determined whether CYP24A1 has the ability to hydroxylate vitamin D3 compounds at C-25. 1alpha,24(R)-dihydroxyvitamin D3 (1alpha,24(R)(OH)2D3) is a non-25-hydroxylated synthetic vitamin D3 analog that is presently being used as an antipsoriatic drug. In the present study, we investigated the metabolism of 1alpha,24(R)(OH)2D3 in human keratinocytes in order to examine the ability of CYP24A1 to hydroxylate 1alpha,24(R)(OH)2D3 at C-25. The results indicated that keratinocytes metabolize 1alpha,24(R)(OH)2D3 into several previously known both 25-hydroxylated and non-25-hydroxylated metabolites along with two new metabolites, namely 1alpha,23,24(OH)3D3 and 1alpha,24(OH)2-23-oxo-D3. Production of the metabolites including the 25-hydroxylated ones was detectable only when CYP24A1 activity was induced in keratinocytes 1alpha,25(OH)2D3. This finding provided indirect evidence to indicate that CYP24A1 catalyzes C-25 hydroxylation of 1alpha,24(R)(OH)2D3. The final proof for this finding was obtained through our metabolism studies using highly purified recombinant rat CYP24A1 in a reconstituted system. Incubation of this system with 1alpha,24(R)(OH)2D3 resulted in the production of both 25-hydroxylated and non-25-hydroxylated metabolites. Thus, in our present study, we identified CYP24A1 as the main enzyme responsible for the metabolism of 1alpha,24(R)(OH)2D3 in human keratinocytes, and provided unequivocal evidence to indicate that the multicatalytic enzyme CYP24A1 has the ability to hydroxylate 1alpha,24(R)(OH)2D3 at C-25.

25-hydroxyvitamin D(3)- or 1alpha,25-dihydroxyvitamin D(3)-24R-hydroxylase (cytochromeP450C24 or CYP24) has a dual role of removing 25-OH-D(3) from circulation and excess 1,25(OH)(2)D(3) from kidney. As a result, CYP24 is an important multifunctional regulatory enzyme that maintains essential tissue-levels of Vitamin D hormone. As a part of our continuing interest in structure-function studies characterizing various binding proteins in the Vitamin D endocrine system, we targeted recombinant rat CYP24 with a radiolabeled 25-OH-D(3) affinity analog, and showed that the 25-OH-D(3)-binding site was specifically labeled by this analog. An affinity labeled sample of CYP24 was subjected to MS/MS analysis, which identified Ser57 as the only amino acid residue in the entire length of the protein that was covalently modified by this analog. Site-directed mutagenesis was conducted to validate the role of Ser57 towards substrate-binding. S57A mutant displayed significantly lower binding capacity for 25-OH-D(3) and 1,25(OH)(2)D(3). On the other hand, S57D mutant strongly enhanced binding for the substrates and conversion of 1,25(OH)(2)D(3) to calcitroic acid. The affinity probe was anchored via the 3-hydroxyl group of 25-OH-D(3). Therefore, these results suggested that the 3-hydroxyl group (of 25-OH-D(3) and 1,25(OH)(2)D(3)) in the S57D mutant could be stabilized by hydrogen bonding or a salt bridge leading to enhanced substrate affinity and metabolism.

A high level of functional recombinant rat cytochrome P450C24 enzyme (CYP24A1) was obtained (40-50mg/L) using an Escherichia coli expression system. Purified enzyme was stable with retention of spectral and catalytic activity. The rate of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] side-chain oxidation and cleavage to the end-product calcitroic acid was directly related to the rate of electron transfer from the ferredoxin redox partner. It was determined from substrate-induced spectral shifts that the 1 alpha- and 25-hydroxyl groups on vitamin D(3) metabolites and analogs were the major determinants for high-affinity binding to CYP24A1. Lowest K(d) values were obtained for 1 alpha-vitamin D(3) (0.06 microM) and 1,25-dihydroxyvitamin D(3) (0.05 microM) whereas unmodified parental vitamin D(3) and the non-secosteroid 25-hydroxycholesterol had lower affinities with K(d) values of 1.3 and 1.9 microM, respectively. The lowest binding affinity for natural vitamin D metabolites was observed for 24,25-dihydroxyvitamin D(3) [24,25(OH)(2)D(3)] (0.43 microM). Kinetic analyses of the two natural substrates 25-hydroxyvitamin D(3) [25(OH)D(3)] and 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] revealed similar K(m) values (0.35 and 0.38 microM, respectively), however, the turnover number was higher for 25(OH)D(3) compared to 1,25(OH)(2)D(3) (4.2 and 1 min(-1), respectively). Mutagenesis of F249 within the F-helix of CYP24A1 altered substrate binding and metabolism. Most notable, the hydrophobic to polar mutant F249T had a strong impact on lowering substrate-binding affinity and catalysis of the final C(23) oxidation sequence from 24,25,26,27-tetranor-1,23-dihydroxyvitamin D(3) to calcitroic acid. Two other hydrophobic 249 mutants (F249A and F249Y) also lowered substrate binding and expressed metabolic abnormalities that included the C(23)-oxidation defect observed with mutant F249T plus a similar defect involving an earlier pathway action for the C(24) oxidation of 1,24,25-trihydroxyvitamin D(3). Therefore, Phe-249 within the F-helix was demonstrated to have an important role in properly binding and aligning substrate in the CYP24A1 active site for C(23) and C(24) oxidation reactions.

The molecular basis for pseudo vitamin D deficiency rickets (PDDR) in the Hannover pig model was determined in the current study. Consistent with the inability of Hannover PDDR pigs to maintain ambient levels of 1,25-dihydroxyvitamin D (i.e., 1,25D), the bioactivation enzyme cytochrome P450C1 (or CYP27B1) was determined to contain coding-region deletions that rendered the enzyme ineffective due to frame-shift mutations and expression of a premature termination codon. Expression levels of P450C1mRNA were up-regulated in response to the low-1,25D high-parathyroid hormone state of the PDDR animals. In a complementary manner, cytochrome P450C24 mRNA was not detectable in PDDR pigs. Two different deletions were detected within the Hannover pig strain in which the P450C1 coding region contained either 173 bp or 329 bp deletions that resulted in the expression of non-sense products beginning within the I-helix region and extending through the truncated C-terminal domains. The boundaries for the deletion segments aligned with derived mRNA processing sites. This observation was consistent with an mRNA processing error as the causative factor for the coding-region deletions. Based upon the expression of a non-functional P450C1 enzyme, the Hannover pig model for PDDR was determined to be identical to the human disease in which enzyme-inhibitory mutations are the molecular basis for the calcium disorder.

The regulation of the gene for renal 25-hydroxyvitamin D 1alpha- hydroxylase (1alpha(OH)ase; CYP27B1) by parathyroid hormone (PTH) under hypocalcemic conditions is fundamentally important for the maintenance of calcium and phosphate homeostasis. The molecular mechanism that underlies this hormonal response is of current interest and has been investigated in the present study by transfection analysis of the human 1alpha(OH)ase promoter in kidney AOK-B50 cells. We have shown that the first 305 bp of promoter can be induced by hormone in transient transfection assays and also within a chromatin environment when stably integrated. Mutagenesis of possible transcription factor binding sites within this promoter length has shown that three sites clustered within the region from -66 to -135 contribute to basal expression. A likely Sp1 and a CCAAT box site are particularly important for basal expression although these sites are not likely to functionally cooperate in a major way. Mutagenesis of the CCAAT box site consistently reduced PTH induction although mutagenesis of the Sp1, Ets and other possible binding sites in the 305 bp of promoter has no significant effect on the level of PTH induction. Other experiments showed that PTH induction but not basal expression was sensitive to the protein kinase inhibitor H89. We have therefore identified for the first time the sites in the 1alpha(OH)ase promoter responsible for basal expression and provide evidence for the role of a CCAAT box binding protein in a PTH mechanism of induction that involves an H89 sensitive step.

Cytochromes P450c1 and P450c24 are regulated hydroxylase enzymes that direct the bioactivation and metabolic degradation of vitamin D. The bioactivation pathway is regulated by cytochrome P450c1 through its synthesis of 1alpha,25(OH)(2)D(3), the hormonally active form of the vitamin. Expression of the P450c1 gene is regulated at the transcription level. Promoter regions within the P450c1 gene have been identified that respond to cAMP and 1alpha,25(OH)(2)D(3) during the respective up- and down-regulation of P450c1 gene expression. The diametric action of 1alpha,25(OH)(2)D(3) to up-regulate P450c24 gene expression is discussed in the context of two vitamin D response elements (VDREs) that are linked functionally to an adjoining Ets-binding site. It is apparent from sequence-derived data that the P450c1 and P450c24 enzymes share only 10-25% sequence identity, yet they display functionally similar domains that are conserved across the family of cytochrome P450 enzymes. Expression of E. coli recombinant P450c1 and P450c24 enzymes, and the substrate-binding parameters for P450c24 are discussed. Finally, the natural point mutations in human P540c1 from patients with pseudovitamin D-deficiency rickets (PDDR) are discussed in the context of the enzyme's structure and function.

Previous studies using microdissected nephron segments reported that the exclusive site of renal 25-hydroxyvitamin D3-24-hydroxylase (24OHase) activity is the renal proximal convoluted tubule (PCT). We now report the presence of 24OHase mRNA, protein, and activity in cells that are devoid of markers of proximal tubules but express characteristics highly specific for the distal tubule. 24OHase mRNA was undetectable in vehicle-treated mouse distal convoluted tubule (DCT) cells but was markedly induced when DCT cells were treated with 1,25 dihydroxyvitamin D3[1,25(OH)2D3]. 24OHase protein and activity were also identified in DCT cells by Western blot analysis and HPLC, respectively. 8-Bromo-cAMP (1 mM) or parathyroid hormone [PTH-(1—34); 10 nM] was found to potentiate the effect of 1,25(OH)2D3on 24OHase mRNA. The stimulatory effect of cAMP or PTH on 24OHase expression in DCT cells suggests differential regulation of 24OHase expression in the PCT and DCT. In the presence of cAMP and 1,25(OH)2D3, a four- to sixfold induction in vitamin D receptor (VDR) mRNA was observed. VDR protein, as determined by Western blot analysis, was also enhanced in the presence of cAMP. Transient transfection analysis in DCT cells with rat 24OHase promoter deletion constructs demonstrated that cAMP enhanced 1,25(OH)2D3-induced 24OHase transcription but this enhancement was not mediated by cAMP response elements (CREs) in the 24OHase promoter. We conclude that 1) although the PCT is the major site of localization of 24OHase, 24OHase mRNA and activity can also be localized in the distal nephron; 2) both PTH and cAMP modulate the induction of 24OHase expression by 1,25(OH)2D3in DCT cells in a manner different from that reported in the PCT; and 3) in DCT cells, upregulation of VDR levels by cAMP, and not an effect on CREs in the 24OHase promoter, is one mechanism involved in the cAMP-mediated modulation of 24OHase transcription.

Pseudovitamin D-deficiency rickets (PDDR) is an autosomal recessive disorder that may be due to impaired activity of 25-hydroxyvitamin D-1alpha-hydroxylase, a renal cytochrome P450 enzyme (P450[1alpha]) of the vitamin D pathway. The disease locus for PDDR has been mapped by linkage analysis to 12q13-q14, but the molecular defect underlying the enzyme dysfunction has remained elusive due to the lack of sequence information for the P450(1alpha) gene (hereafter referred to as 1alpha-OHase). We have used a probe derived from the rat 25-hydroxyvitamin D-24-hydroxylase (CYP24; 24-OHase) sequence to identify and clone the 1alpha-OHase cDNA. The full-length 1alpha-OHase clone of 2.4 kb codes for a protein of predicted Mr 55 kDa. Functional activity of the cloned sequence was assessed using transient transfection, and the production of authentic 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] was confirmed using high performance liquid chromatography fractionation and time-of-flight mass spectrometry. The expression of the gene was analyzed in vitamin D-replete animals; treatment with 1alpha,25(OH)2D3 reduced 1alpha-OHase transcript levels by 70%, while administration of parathyroid hormone led to a 2-fold increase in the expression of the gene, thus confirming the hormonal regulation previously described using biochemical methods. The rat cDNA was used to obtain a human genomic clone. Interestingly, the human 1alpha-OHase gene mapped to 12q13.1-q13.3, providing strong evidence that a mutation in the 1alpha-OHase gene is responsible for the PDDR phenotype. The availability of a cloned sequence for 1alpha-OHase generates novel tools for the study of the molecular etiology of PDDR, and will allow the investigation of other disturbances of vitamin D metabolism.

Transcription of the CYP24 gene is induced by 1,25-(OH)2D3 through a vitamin D receptor-dependent process. The functional activities of three possible vitamin D response elements (VDREs), located on the antisense strand of the rat CYP24 promoter, were investigated by transient expression of native and mutant promoter constructs in COS-1, JTC-12, and ROS 17/2.8 cells. A putative VDRE with a half-site spacing of 6 base pairs at −249/−232 (VDRE-3) did not contribute to 1,25-(OH)2D3 induced expression in the native promoter, although activity has been reported when the element was fused to the heterologous thymidine kinase promoter. Two VDREs with half-site spacings of 3 base pairs at −150/−136 and −258/−244 (VDRE-1 and VDRE-2, respectively), showed transcriptional synergism in COS-1 cells when treated with 1,25-(OH)2D3 (10−7 to 10−11 M). The contribution of both VDREs was hormone-concentration dependent from 10−10 to 10−12 M, with VDRE-1 demonstrating greatest sensitivity to 1,25-(OH)2D3. Transactivation by VDRE-1 was always greater than VDRE-2, but the converse was observed for the binding of vitamin D receptor-retinoid X receptor complex by each VDRE in gel mobility shift assays. The synergy observed between VDRE-1 and VDRE-2 may have important implications in cellular responses to different circulating levels of 1,25-(OH)2D3.

Vitamin D-depleted chicks which were repleted with 25-hydroxycholecalciferol adapted to the feeding of a low or high calcium diet by respectively increasing or decreasing their rate of intestinal calcium absorption. The synthesis of 1,25-dihydroxycholecalciferol in chicks fed differing calcium diets correlated directly with intestinal calcium absorption activity, suggesting that the intestinal calcium adaptation response is medicated by the modulation of kidney 25-hydroxycholecalciferol-1-hydroxylase activity. This view was strongly supported by the demonstration that repletion of the chicks with 1,25-dihydroxycholecalciferol eliminated the ability of the chicks to adjust their rates of intestinal calcium absorption to dietary levels of calcium.

An acute chick kidney tubule model was used to evaluate the molecular specificity of steroids which act to inhibit kidney 25-hydroxyvitamin D3-1-hydroxylase and induce 25-hydroxyvitamin D3-24-hydroxylase enzyme activities. Such hydroxylase-regulatory activity was confined to the vitamin D family of secosteroids. Vitamin D3 per se was not active. Rather, expression of hydroxylase-regulatory activity required acquisition of a C-25 hydroxyl grouping. Of the metabolites tested, 25-hydroxyvitamin D3 demonstrated the greatest hydroxylase-regulatory activity. Metabolites of 25-hydroxyvitamin D3 which contained hydroxyl groups at carbon atoms 1, or 24, or both, were also active in regulating the kidney hydroxylase enzymes. The hydroxylase-regulatory action of 1,25-dihydroxyvitamin D3 was blocked by inhibitors of RNA or protein synthesis, implicating a requirement for de novo protein synthesis. It was suggested that the regulatory process involves both hydroxylase-enzyme synthesis and turnover as well as enzyme-level modulation of endogenous enzyme activities.

The action of parathyroid extract (PTE) on the renal metabolism of 25-hydroxyvitamin D 3 (25-OHD 3 ) was evaluated in rat models for strontium rickets and hypoparathyroidism. PTE elevated the production of 1α,25-(OH) 2 D 3 and suppressed the synthesis of 24,25-(OH) 2 D 3 in both animal models. Part of strontium's action in suppressing 1α,25-(OH) 2 D 3 and stimulating 24,25-(OH) 2 D 3 synthesis in strontium rickets appears to involve a decrease in parathyroid hormone (PTH) secretion and/or action. Calcitonin (CT) was not implicated in the cation's action. Thyroparathyroidectomized rats showed a low level of 1α,25-(OH) 2 D 3 production which increased four- to eightfold following chronic PTE treatment. PTH appears to be the major calcium regulatory hormone involved in modulation of renal 25-OHD 3 metabolism.

Oxidative phosphorylation and 1 α,25-dihydroxyvitamin D3 [lα,25-(OH)2D3]synthesis in isolated mitochondria were decreased by the addition of strontium. Calcium effected a similar inhibition of 1α,25-(OH)2D3 synthesis which correlated with cation-induced mitochondrial swelling. The ultrastructural changes were found to be a consequence of experimental conditions and not a prerequisite for suppressed 1α,25-(OH)2D3 synthesis. Dietary administration of strontium or calcium also resulted in a decreased rate of 1α,25-(OH)2D3 synthesis; however, the decrease in 1-hydroxylase activity was accompanied by an induction of mitochondrial 25-hydroxyvitamin D3 24-hydroxylase activity. Such an in vivo-prompted mitochondrial response occurred in the absenee of morphological changes or extensive loss of oxidative phosphorylation activity. In contrast, no induction of 24-hydroxylase activity could be observed in acute studies using isolated mitochondria. Therefore, the in vitro action of calcium and strontium does not appear to reflect the in vivo mechanism whereby the cations act to change renal 25-hydroxyvitamin D3 (25-OHD3) hydroxylation. Results from in vitro studies corcerning the action of calcium to alter renal 25-OHD3 metabolism should be interpreted in light of the cation's capacity to decrease oxidative phosphorylation and the subsequent intramitochondrial generation of NADPH.

SummaryChicks regulated to a lowcalcium diet exhibited the expected facultative process regarding the intestinal absorption of calcium. Augmentation of the facultative process was noted when such low-calcium adapted chicks were given a high-calcium diet. This response was transitory, becoming evident 72 hr after initiation of the high-calcium feeding and declining rapidly thereafter. The potentiative response was actinomycin sensitive and could not be produced by the molar substitution of strontium for calcium. Vitamin D was essential for development of the facultative process in lowcalcium fed chicks and the potentiative effect noted in such animals given a high-calcium diet. It is suggested that the additional calcium could have acted on the intestinal cellular processes to accentuate the facultative response by increasing mitotic activity or by inhibiting ribonuclease. It is also theorized that calcium could have effected the facultative response indirectly by controlling synthesis of an active vitamin...

Inhibition of calcium absorption by dietary strontium is the result of a block in the renal synthesis of 1,25-dihydroxycholecalciferol (1,25-(OH)2CC) from 25-hydroxycholecalciferol (25-OHCC). The administration of 1,25-(OH)2CC but not 25-OHCC restores to normal calcium absorption in strontium-fed chicks. Kidney mitochondria from strontium-fed chicks do not metabolize 25-OHCC to 1,25-(OH)2CC, but rather to a metabolite designated Peak Va. The function of Peak Va is unknown while it is believed that 1,25-(OH)2CC is the metabolically active form of vitamin D in the stimulation of calcium absorption from intestine and mobilization from bone. The changes in the kidney metabolism of 25-OHCC in response to dietary strontium occur over a period of several days and correlate closely with changes in intestinal calcium absorption. Feeding a normal calcium diet can correct the strontium-induced inhibition of intestinal calcium absorption. This restoration of calcium absorption correlates with an increased rate of in vitro 1,25-(OH)2CC synthesis. The exact mechanism whereby strontium alters the renal metabolism of 25-OHCC is unknown. However, indirect evidence suggests that it may operate through humoral agents such as parathyroid hormone, or calcitonin, or both, which are responsible for maintenance of plasma calcium concentration.

The subcellular distribution and properties of an enzyme system responsible for hydroxylation of 25-hydroxycholecalciferol (25-OHD3) to 1,25-dihydroxycholecalciferol (1,25-(OH)2D3) in chicken kidney have been described. The enzyme, which is highly specific for 25-OHD3 since it does not hydroxylate cholecalciferol or dihydrotachysterol, is located in the mitochondria and has a pH optimum of 7.4 and an apparent Km of 2.2 x 10-6 m. At 8.34 x 10-7 m 25-OHD3, 1,25-(OH)2D3 production is linear with mitochondrial protein concentrations of up to 8 mg per ml. Enzyme activity is maximal in Tris-acetate buffer and requires Mg2+, oxygen, and a source of reduced pyridine nucleotide, which in intact mitochondria is provided by malate or succinate. The succinate-supported hydroxylation is dependent upon electron transport and oxidative phosphorylation. The 25-OHD3-1-hydroxylase is inhibited by carbon monoxide, metapyrone, p-chloromercuribenzoate, and diphenyl-p-phenylenediamine. The enzyme is also inhibited by its product, 1,25-(OH)2D3. It is, however, much more strongly inhibited competitively by 25-hydroxydihydrotachysterol, a close analog of 1,25-(OH)2D3.

24,25-Dihydroxyvitamin D3 (24,25-(OH)2D3) gives a marked increase in intestinal calcium transport and serum calcium concentration of vitamin D-deficient rats fed a diet containing adequate calcium and phosphorus. These responses occur after a considerable time lag following administration of the metabolite. The 24,25-(OH)2D3 on a long term basis and at low doses has striking activity in the stimulation of intestinal calcium transport but not bone calcium mobilization. Radioactive 24,25-(OH)2D3 is metabolized to a more polar compound which increases in concentration in serum, intestinal mucosa, and bone over a 48-hour period. Nephrectomy or the feeding of a high calcium diet abolishes the stimulation of intestinal calcium transport by 24,25-(OH)2D3 and the in vivo genesis of the more polar compound from tritiated 24,25-(OH)2D3. It is concluded that 24,25-(OH)2D3, the usual dihydroxy metabolite of vitamin D3 in normal rats, has potential physiological importance through its conversion to a more polar metabolite. This metabolite possibly acts to facilitate intestinal calcium absorption without stimulating bone calcium mobilization.

Vitamin D depleted rats which are given 25-hydroxyvitamin D 3 and fed a high calcium-low phosphorus diet produce 1α,25-dihydroxyvitamin- 3 H-D 3 from the precursor 25-hydroxyvitamin- 3 H-D 3 . Supplementation of these rats with 1α,25-dihydroxyvitamin D 3 results in an induced synthesis of 24,25-dihydroxyvitamin- 3 H-D 3 and a nearly total suppression of 1α,25-dihydroxyvitamin- 3 H-D 3 synthesis. The inductive action of 1α,25-dihydroxyvitamin D 3 can be effected in approximately 3 hours, occurs in the absence of thyroid and parathyroid tissue and is inhibited by actinomycin D. It appears that 1α,25-dihydroxyvitamin D 3 effects its action via a transcriptional process which can occur independently of parathyroid hormone's and calcitonin's influence on kidney 25-hydroxyvitamin D 3 metabolism.

Two separate ferredoxins that differ in molecular mass by about 1.5 kDa were isolated from both pig kidney and bovine adrenal mitochondria. The proteins had different biochemical and immunological properties and appeared to be distinct gene products. The smaller ferredoxin from pig kidney (renodoxin, Mr approximately equal to 13,500) was very similar to bovine adrenodoxin (Mr = 14,048). Both proteins had nearly identical N-terminal amino acid sequences and electron-transfer activities. However, renodoxin and adrenodoxin expressed distinct antigenic determinants, although they were immunologically cross-reactive. The larger kidney (approximately equal to 15-kDa) and adrenal (approximately equal to 15.3-kDa) ferredoxins were biochemically similar to each other but they had lower specific activities and their N-terminal sequences were different when compared to renodoxin and adrenodoxin. Each of the four ferredoxins had a visible absorption spectrum characteristic of a [2Fe-2S] chromophore. But in addition, the larger ferredoxins displayed a prominent A276 peak due to their higher tyrosine content and the presence of tryptophan, which is absent in adrenodoxin and renodoxin. Consequently, the larger ferredoxins were termed Trp-ferredoxin. Using antibody to pig kidney Trp-ferredoxin, the larger adrenal and kidney ferredoxins were found to be very similar immunologically; however, the Trp-containing and adrenodoxin-type ferredoxins did not cross-react in immunoblot analysis. Nevertheless, it was shown from competition ELISA and activity-inhibition analysis that the two ferredoxin types had limited common antigenic determinants. Trp-ferredoxin was the major iron-sulfur protein in kidney whereas adrenodoxin was the dominant molecular form in adrenal gland.

Summary1,25-Dihydroxycholecaliferol (1,25-DHCC) is a metabolite of vitamin D produced in the kidney from the major circulating biologically active form of the vitamin, 25-hydroxycholecalciferol (25-HCC) and has been demonstrated to be the derivative of cholecalciferol (CC) primarily responsible for the initiation of calcium transport from the gut contents. Because previous studies have shown that both CC and 25-HCC act acutely to enhance phosphate, sodium and calcium reabsorption in the kidney, the effect of 1,25-DHCC was evaluated in clearance studies performed in mildly expanded thyroparathyroidectomized dogs. In a dose of 25 U (0.625 μg), 1,25-DHCC induced an invariable fall in both fractional and absolute phosphate excretion (mean declines of 40 and 36%, respectively, p < .05 and < .01). Fractional sodium and calcium excretion also deccreased by mean values of 26 and 36%, respectively (p < .02). Renal hemodynamic changes and alterations in serum ultrafilterable calcium and phosphate concentrations did...

IT is now recognized that hydroxylated metabolites of vitamin D (that is, cholecalciferol) function as effectors of the physiological actions originally attributed to the unaltered vitamin1. The activation of vitamin D by specific hydroxylation reactions and sequestration of the resultant metabolites by target tissues represents a hormonal control loop which is feed-back sensitive. 25-Hydroxycholecalciferol (25-HCC) and 1,25-dihydroxycholecalciferol (1,25-DHCC) have been shown to be participants in the control loop, vitamin D being first metabolized in the liver to 25-HCC2 which in turn is hydroxylated in the C-1 position to 1,25-DHCC in the kidney3,4. The metabolically active form in the intestine appears to be 1,25-DHCC5,6.

This chapter discusses the experimental study made in both vitamin D-deficient and vitamin D-supplemented rats of the effect of varying the dietary calcium content on the in vivo production of 1,25-DHCC from 25-hydroxycholecalciferol (25-HCC). The profiles of radioactive metabolites separated on Sephadex LH-20 from serum extracts were obtained from rats fed the various diets with and without vitamin D. The small dose of vitamin D 3 is chosen to minimize dilution of radioactive metabolites and to provide a small amount of 25-HCC to induce the factor(s) necessary for 1-hydroxylation in the kidney. A consideration of the serum calcium values and rat illustrates a growth effect of the vitamin even at the low level although vitamin D deficiency persisted. In vitamin D-deficient rats fed diets with 0–02% Ca through 2–0% Ca, 14 to 18% of the total radioactivity in the serum is recovered consistently as 1,25-DHCC. No correlation is found between the serum inorganic phosphorus levels and production of the metabolites.