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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.

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.

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.

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.

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.