1alpha,25-Dihydroxyvitamin D3 down-regulates expression of prostate specific membrane antigen in prostate cancer cells.

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, 1alpha,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.

Hybrid homology modeling and mutational analysis of cytochrome P450C24A1 (CYP24A1) of the Vitamin D pathway: insights into substrate specificity and membrane bound structure-function.

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.

23-carboxy-24,25,26,27-tetranorvitamin D3 (calcioic acid) and 24-carboxy-25,26,27-trinorvitamin D3 (cholacalcioic acid): end products of 25-hydroxyvitamin D3 metabolism in rat kidney through C-24 oxidation pathway.

During the past two and half decades the elucidation of the metabolic pathways of 25OHD(3) and its active metabolite 1alpha,25(OH)(2)D(3) progressed in parallel. In spite of many advances in this area of vitamin D research, the unequivocal identification of the end products of 25OHD(3) metabolism through C-24 oxidation pathway has not been achieved. It is now well established that both 25OHD(3) and 1alpha,25(OH)(2)D(3) are metabolized through the same C-24 oxidation pathway initiated by the enzyme 24-hydroxylase (CYP24A1). Based on the information that the end product of 1alpha,25(OH)(2)D(3) metabolism through C-24 oxidation pathway is 1alpha-OH-23- COOH-24,25,26,27-tetranor D(3) or calcitroic acid; the metabolism of 25OHD(3) into 23-COOH-24,25,26,27-tetranor D(3) has been assumed. Furthermore, a previous study indicated 24-COOH-25,26,27-trinor D(3) as a water soluble metabolite of 24R,25(OH)(2)D(3) produced in rat kidney homogenates. Therefore, 24-COOH-25,26,27-trinor D(3) was also assumed as another end product of 25OHD(3) metabolism through C-24 oxidation pathway. We embarked on our present study to provide unequivocal proof for these assumptions. We first studied the metabolism of 25OHD(3) at low substrate concentration (3x10(-10)M) using [1,2-(3)H]25OHD(3) as the substrate in the perfused rat kidneys isolated from both normal and vitamin D(3) intoxicated rats. A highly polar water soluble metabolite, labeled as metabolite X was isolated from the kidney perfusate. The amount of metabolite X produced in the kidney of a vitamin D intoxicated rat was about seven times higher than that produced in the kidney of a normal rat. We then produced metabolite X in a quantity sufficient for its structure identification by perfusing kidneys isolated from vitamin D intoxicated rats with high substrate concentration of 25OHD(3) (5x10(-6)M). Using the techniques of electron impact and thermospray mass spectrometry, we established that the metabolite X contained both 23-COOH-24,25,26,27-tetranor D(3) and 24-COOH-25,26,27-trinor D(3) in a ratio of 4:1. The same metabolite X containing both acids in the same ratio of 4:1 was also produced when 24R,25(OH)(2)D(3) was used as the starting substrate. Previously, the trivial name of cholacalcioic acid was assigned to 24-COOH-25,26,27-trinorvitamin D(3). Using the same guidelines, we now assign the trivial name of calcioic acid to 23-COOH-24,25,26,27-tetranor D(3). In summary, for the first time our study provides unequivocal evidence to indicate that both calcioic and cholacalcioic acids as the end products of 25OHD(3) metabolism in rat kidney through C-24 oxidation pathway.

Identification of growth factor independent-1 (GFI1) as a repressor of 25-hydroxyvitamin D 1-alpha hydroxylase (CYP27B1) gene expression in human prostate cancer cells.

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.

Molecular action of 1,25-dihydroxyvitamin D3 and phorbol ester on the activation of the rat cytochrome P450C24 (CYP24) promoter: role of MAP kinase activities and identification of an important transcription factor binding site.

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.

C-25 hydroxylation of 1alpha,24(R)-dihydroxyvitamin D3 is catalyzed by 25-hydroxyvitamin D3-24-hydroxylase (CYP24A1): metabolism studies with human keratinocytes and rat recombinant CYP24A1.

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.

Rat cytochrome P450C24 (CYP24A1) and the role of F249 in substrate binding and catalytic activity.

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.

Role of MAP kinases in the 1,25-dihydroxyvitamin D3-induced transactivation of the rat cytochrome P450C24 (CYP24) promoter. Specific functions for ERK1/ERK2 and ERK5.

The current study investigated the action of 1,25-dihydroxyvitamin D(3) (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 of CYP24 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 alpha (RXR alpha) interacted with ERK2 but not ERK5 in intact cells, whereas Ets-1 interacted preferentially with ERK5. Increased phosphorylation of RXR alpha and Ets-1 was detected in response to 1,25D. Activated ERK2 and ERK5 specifically phosphorylated RXR alpha 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 RXR alpha (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 RXR alpha 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.

Hydroxylase enzymes of the vitamin D pathway: expression, function, and regulation.

Vitamin D is a secosteroid that is metabolically activated and degraded through the actions of three cytochrome P450 hydroxylase enzymes. Bioactivation occurs through the sequential actions of cytochromes P450C25 and P450C1, resulting in synthesis of the pleiotropic hormone 1,25-dihydroxyvitamin D (1,25VD), which regulates over 60 genes whose actions include those associated with calcium homeostasis and immune responses as well as cellular growth, differentiation, and apoptosis. Inactivation of 1,25VD occurs by C23/C24 oxidation pathways that are catalyzed by the multifunctional cytochrome P450C24 enzyme. Both P450C1 and P450C24 are highly regulated enzymes whose differential expression is controlled in response to numerous cellular modulatory agents such as parathyroid hormone (PTH), calcitonin, interferon gamma, calcium, phosphorus, and pituitary hormones as well as the secosteroid hormone 1,25VD. Most thoroughly studied at the molecular level are the actions of PTH to upregulate P450C1 gene expression and 1,25VD to induce the expression of P450C24. The regulatory action of PTH is mediated through the protein kinase A pathway and involves the phosphorylation of transcription factors that function at the proximal promoter of the P450C1 gene. The upregulation of P450C24 by 1,25VD has both a rapid nongenomic and a slower genomic component that are functionally linked. The rapid response involves protein kinase C and mitogen-activated protein kinase (MAPK) pathways that direct the phosphorylation of nuclear transcription factors. The slower genomic actions are linked to the binding of 1,25VD to the vitamin D receptor (VDR) and the interaction of the VDR-1,25VD complex with its heterodimer partner retinoid-X-receptor and associated coactivators. The regulatory complex is assembled on vitamin D response elements in the proximal promoter of the P450C24 gene and functions to increase the transcription rate.

Basal and parathyroid hormone induced expression of the human 25-hydroxyvitamin D 1alpha-hydroxylase gene promoter in kidney AOK-B50 cells: role of Sp1, Ets and CCAAT box protein binding sites.

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.