Essential role of constitutive androstane receptor in Ginkgo biloba extract induced liver hypertrophy and hepatocarcinogenesis.

Ginkgo biloba extract (GBE) is commonly used as a herbal supplement. The National Toxicology Program (NTP) study of GBE reported clear evidence of hepatocarcinogenicity in mice. To clarify the mode of action (MOA) for hepatocarcinogenesis by GBE, we investigated the involvement of the constitutive androstane receptor (CAR) in hepatocarcinogenesis induced by GBE using CAR-knockout (CARKO) and wild type (WT) mice. We used the same lot of GBE that was used for the NTP study. In 1-week GBE dietary treatment, hepatocellular DNA replication was increased in WT mice but not in CARKO mice. In 4- or 13-week treatment, greater hepatic Cyp2b10 induction and hepatocellular hypertrophy were observed in WT mice, whereas these effects of GBE were much smaller in CARKO mice. In a two-stage hepatocarcinogenesis model initiated by diethylnitrosamine, 27-week treatment with GBE resulted in an increase of eosinophilic altered foci and adenomas in WT mice. By contrast, foci and adenomas were clearly less evident in CARKO mice. These results indicate that GBE-induced hepatocarcinogenesis is mainly CAR-mediated. Since CAR-mediated MOA for hepatocarcinogenesis in rodents is considered to be qualitatively implausible for humans, our findings would be helpful to evaluate the carcinogenic characterization of GBE to humans.

1α,25-Dihydroxyvitamin D3-3ß-bromoacetate, a potential cancer therapeutic agent: synthesis and molecular mechanism of action.

Synthesis of 1α,25-dihydroxyvitamin D(3)-3ß-bromoacetate (1,25(OH)(2)D(3)-3-BE), a potential anti-cancer agent is presented. We also report that mechanism of action of 1,25(OH)(2)D(3)-3-BE may involve reduction of its catabolism, as evidenced by the reduced and delayed expression of 1α,25-dihydroxyvitamin D(3)-24-hydroxylase (CYP24) gene in cellular assays.

Vitamin D analogues targeting CYP24 in chronic kidney disease.

The cytochrome P450 enzyme 24-hydroxylase (CYP24) plays a critical role in regulating levels of vitamin D hormone. Aberrant expression of CYP24 has been implicated in vitamin D insufficiency and resistance to vitamin D therapy. We have demonstrated amplified CYP24 expression in uremic rats, suggesting that CYP24 has an etiological role in vitamin D insufficiency commonly associated with chronic kidney disease (CKD). We have designed two new analogues of 1alpha,25-dihydroxyvitamin D3 (1alpha,25(OH)2D3), namely CTA091 and CTA018/MT2832, which are potent inhibitors of CYP24. In vitro studies with CTA091 show that it enhances the potency of 1alpha,25(OH)2D3. In vivo studies demonstrate that CTA091 decreases serum intact parathyroid hormone (iPTH) levels and increases circulating 1alpha,25(OH)2D3. CTA091 increases both Cmax and AUC of co-administered 1alpha,25(OH)2D3. These studies indicate that CYP24 inhibition can increase cellular responsiveness to vitamin D hormone and potentiate vitamin D therapy. CTA018/MT2832 differs from CTA091 in that it also has the ability to activate vitamin D receptor-mediated transcription. CTA018/MT2832 effectively suppresses elevated iPTH secretion at doses which do not affect serum calcium or phosphorus levels in a rodent model of CKD. Studies with both new analogues underscore the potential utility of CYP24 inhibition in the treatment of secondary hyperparathyroidism in CKD.

Enhancement of ligand-dependent vitamin D receptor transactivation by the cardiotonic steroid bufalin.

Bufalin, a bufadienolide type cardiotonic steroid that is one of the major components of the toad venom-prepared traditional Chinese medicine called Ch'an Su or Senso, exhibits a cardiotonic action by inhibiting the membranous Na(+),K(+)-ATPase. Bufalin also induces differentiation of leukemia cells alone or in combination with other differentiation inducers including 1alpha,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)]. In this study, we performed a transient cotransfection assay using a vitamin D receptor (VDR) expression vector and a luciferase reporter and found that although bufalin did not transactivate the VDR, it effectively enhanced VDR activity induced by 1,25(OH)(2)D(3). Bufalin also augmented VDR activation by bile acid ligands, such as lithocholic acid and 3-ketocholanic acid. Other cardiotonic steroids including ouabain, digitoxigenin and cinobufagin did not enhance VDR activation. Bufalin did not bind directly to VDR but did modulate the interaction of VDR and cofactors, such as steroid receptor coactivator-1 and nuclear receptor corepressor. Bufalin treatment significantly increased the expression of an endogenous VDR target gene, CYP24, in kidney- and monocyte-derived cell lines treated with 1,25(OH)(2)D(3). The data indicate that bufalin-mediated cellular mechanisms such as interaction with Na(+), K(+)-ATPase may affect VDR transcriptional activity. Bufalin may be a useful tool in the investigation of VDR regulation by membrane-originating cellular signals and of pathophysiological mechanisms linking VDR to cardiovascular dysfunction.

Alternative splicing of vitamin D-24-hydroxylase: a novel mechanism for the regulation of extrarenal 1,25-dihydroxyvitamin D synthesis.

Synthesis of the active form of vitamin D, 1,25-dihydroxyvitamin D (1,25-(OH)(2)D), by renal epithelial cells is tightly controlled during normal calcium homeostasis. By contrast, macrophage production of 1,25-(OH)(2)D is often dysregulated with potential hypercalcemic complications. We have postulated that this is due to abnormal catabolism of 1,25-(OH)(2)D by the feedback control enzyme, vitamin D-24-hydroxylase (CYP24). Using chick HD-11 and human THP-1 myelomonocytic cell lines, we have shown that macrophage-like cells express a splice variant of the CYP24 gene (CYP24-SV), which encodes a truncated protein. Compared with the holo-CYP24 gene product in chick and human cells (508 and 513 amino acids, respectively), the truncated CYP24-SV versions consisted of 351 and 372 amino acids. These CYP24-SV proteins retained intact substrate-binding domains but lacked mitochondrial targeting sequences and were therefore catalytically inactive. In common with CYP24, expression of the CYP24 variants was induced by 1,25-(OH)(2)D but without a concomitant rise in 24-hydroxylase activity. However, overexpression of CYP24-SV in HD-11 and THP-1 cells reduced synthesis of 1,25-(OH)(2) D (40-50%), whereas antisense CYP24-SV expression increased 1,25-(OH)(2)D production by 2-7-fold. These data suggest that alternative splicing of CYP24 leads to the generation of a dominant negative-acting protein that is catalytically dysfunctional. We theorize that expression of the CYP24-SV may contribute to the extracellular accumulation of 1,25(OH)(2)D in human health and disease.

Characterization of mixtures of recombinant human cytochrome p450s as a screening model for metabolic stability in drug discovery.

1. Recombinant human cytochrome p450 (rhCYP) has become an important screening model in drug metabolism studies due to the high cost of human and animal hepatic tissue. Until now, rhCYPs have been evaluated and used as separate forms, but a mixture of CYP forms comparable with the human liver could be of value in early drug discovery. 2. In the present study, rhCYP2C9, rhCYP2D6 and rhCYP3A4 co-expressed with reductase in Escerichia coli were mixed and evaluated with regards to kinetic properties (K(m) and V(max)). Furthermore, antioxidant was added to investigate whether a free radical scavenger would affect the kinetic parameters. Results were compared with data obtained in human liver microsomes (HLM). 3. Results showed a good correlation between mixed rh CYP data and HLM data for K(m) and V(max). K(m) varied < 3-fold between matrices for CYP2C9 and CYP3A4, whereas the K(m) for CYP2D6 varied up to 4.5-fold. V(max) differed up to 3-fold between matrices for the CYP forms investigated. However, the discrepancy in V(max) may depend on the anticipated level of each form in HLM. The addition of antioxidant increased V(max) for CYP2C9 and CYP2D6 by 75 and 50%, respectively, whereas V(max) for CYP3A4 was unchanged. 4. In conclusion, the rhCYP mixture shows promising results as a predictor of CYP kinetic parameters. Furthermore, addition of antioxidant can in certain cases increase catalytic activity.

Oxysterol biosynthetic enzymes.

Oxysterols, herein defined as derivatives of cholesterol with a hydroxyl group on the side chain, play several roles in lipid metabolism. Members of this class regulate the expression of genes that participate in both sterol and fat metabolism, serve as substrates for the synthesis of bile acids, and are intermediates in the transfer of sterols from the periphery to the liver. Three abundant naturally occurring oxysterols are 24-hydroxycholesterol, 25-hydroxycholesterol, and 27-hydroxycholesterol. The cholesterol hydroxylase enzymes that synthesize each of these have been isolated over the last several years and their study has produced insight into the biology of oxysterols. This article focuses on the properties of these enzymes.

Identification of key residues in rabbit liver microsomal cytochrome P450 2B4: importance in interactions with NADPH-cytochrome P450 reductase.

A cytochrome P450 2B4 (CYP2B4) model was used to select key residues supposed to serve in interactions with NADPH-cytochrome P450 reductase (P450R). Eight amino acid residues located on the surface of the hemoprotein were chosen for mutagenesis experiments with CYP2B4(Delta2-27) lacking the NH(2)-terminal signal anchor sequence. The mutated proteins were expressed in Escherichia coli, purified, and characterized by EPR- and CD-spectral analysis. Replacement of histidine 226 with alanine caused a 3.8-fold fall in the affinity for P450R with undisturbed reductive capacity of the system. Similarly, the K225A, R232A, and R253A variants exhibited P450R-directed activity that was depressed to about half that of the control enzyme, suggesting that the deletion of positive charges on the surface of CYP2B4(Delta2-27) resulted in impaired electrostatic contacts with complementary amino acids on the P450R protein. While the Y235A mutant did not show appreciably perturbed reduction activity, the conservative substitution with alanine of the phenylalanine residues at positions 223 and 227 gave a 2.1- to 6. 1-fold increase in the K(m) values with unchanged V(max); this was attributed to the disruption of hydrophobic forces rather than to global structural rearrangement(s) of the engineered pigments. Measurement of the stoichiometry of aerobic NADPH consumption and H(2)O(2) formation revealed the oxyferrous forms of the F223A, H226A, and F227A mutants to autoxidize more readily owing to less efficient coupling of the systems. Noteworthy, the F244A enzyme did not exhibit significant reduction activity, suggesting a pivotal role of Phe-244 in the functional coupling of P450R. The residue was predicted to constitute part of an obligatory electron transfer conduit through pi-stacking with Phe-296 located close to the heme unit. All of the residues examined reside in the putative G helix of CYP2B4, so that this domain obviously defines part of the binding site for P450R.

Interaction of catalytic domains in cytochrome P450scc--adrenodoxin reductase--adrenodoxin fusion protein imported into yeast mitochondria.

We have constructed plasmids for yeast expression of the fusion protein pre-cytochrome P450scc--adrenodoxin reductase-adrenodoxin (F2) and a variant of F2 with the yeast CoxIV targeting presequence. Mitochondria isolated from transformed yeast cells contained the F2 fusion protein at about 0.5% of total protein and showed cholesterol hydroxylase activity with 22(R)-hydroxycholesterol. The activity increased 17- or 25-fold when sonicated mitochondria were supplemented with an excess of purified P450scc or a mixture of adrenodoxin (Adx) and adrenodoxin reductase (AdxRed), respectively. These data suggest that, at least in yeast mitochondria, the interactions of the catalytic domains of P450scc, Adx, and AdxRed in the common polypeptide chain are restricted.

Enzymatic determinants of the substrate specificity of CYP2C9: role of B'-C loop residues in providing the pi-stacking anchor site for warfarin binding.

Previous modeling efforts have suggested that coumarin ligand binding to CYP2C9 is dictated by electrostatic and pi-stacking interactions with complementary amino acids of the protein. In this study, analysis of a combined CoMFA-homology model for the enzyme identified F110 and F114 as potential hydrophobic, aromatic active-site residues which could pi-stack with the nonmetabolized C-9 phenyl ring of the warfarin enantiomers. To test this hypothesis, we introduced mutations at key residues located in the putative loop region between the B' and C helices of CYP2C9. The F110L, F110Y, V113L, and F114L mutants, but not the F114Y mutant, expressed readily, and the purified proteins were each active in the metabolism of lauric acid. The V113L mutant metabolized neither (R)- nor (S)-warfarin, and the F114L mutant alone displayed altered metabolite profiles for the warfarin enantiomers. Therefore, the effect of the F110L and F114L mutants on the interaction of CYP2C9 with several of its substrates as well as the potent inhibitor sulfaphenazole was chosen for examination in further detail. For each substrate examined, the F110L mutant exhibited modest changes in its kinetic parameters and product profiles. However, the F114L mutant altered the metabolite ratios for the warfarin enantiomers such that significant metabolism occurred for the first time on the putative C-9 phenyl anchor, at the 4'-position of (R)- and (S)-warfarin. In addition, the Vmax for (S)-warfarin 7-hydroxylation decreased 4-fold and the Km was increased 13-fold by the F114L mutation, whereas kinetic parameters for lauric acid metabolism, a substrate which cannot interact with the enzyme by a pi-stacking mechanism, were not markedly affected by this mutation. Finally, the F114L mutant effected a greater than 100-fold increase in the Ki for inhibition of CYP2C9 activity by sulfaphenazole. These data support a role for B'-C helix loop residues F114 and V113 in the hydrophobic binding of warfarin to CYP2C9, and are consistent with pi-stacking to F114 for certain aromatic ligands.

cDNA cloning of mouse and human cholesterol 25-hydroxylases, polytopic membrane proteins that synthesize a potent oxysterol regulator of lipid metabolism.

Oxysterols regulate the expression of genes involved in cholesterol and lipid metabolism and serve as intermediates in cholesterol catabolism. Among the most potent of regulatory oxysterols is 25-hydroxycholesterol, whose biosynthetic enzyme has not yet been isolated. Here, we report the cloning of cholesterol 25-hydroxylase cDNAs from the mouse and human. The encoded enzymes are polytopic membrane proteins of 298 and 272 amino acids, respectively, which contain clusters of histidine residues that are essential for catalytic activity. Unlike most other sterol hydroxylases, cholesterol 25-hydroxylase is not a cytochrome P450, but rather it is a member of a small family of enzymes that utilize diiron cofactors to catalyze the hydroxylation of hydrophobic substrates. The cholesterol 25-hydroxylase gene lacks introns, and in the human it is located on chromosome 10q23. The murine gene is expressed at low levels in multiple tissues. Expression of cholesterol 25-hydroxylase in transfected cells reduces the biosynthesis of cholesterol from acetate and suppresses the cleavage of sterol regulatory element binding protein-1 and -2. The data suggest that cholesterol 25-hydroxylase has the capacity to play an important role in regulating lipid metabolism by synthesizing a co-repressor that blocks sterol regulatory element binding protein processing and ultimately leads to inhibition of gene transcription.

Cloning and expression of the chicken 25-hydroxyvitamin D3 24-hydroxylase cDNA.

Using a cDNA probe from the rat 24-hydrovitamin D3 24-hydroxylase, the chicken 25-hydroxyvitamin D3 24-hydroxylase cDNA has been isolated from a chicken kidney lambda gt11 library. The high degree of similarity with the mammalian 24-hydroxylase cDNAs strongly supports the belief that it is the chicken 25-hydroxyvitamin D3 24-hydroxylase cDNA. The deduced amino acid sequences are also very well conserved and 325 of them are identical among the four known 25-hydroxyvitamin D3 24-hydroxylases. This cDNA expressed in E. coli produces 24-hydroxylase activity.

The 25-hydroxyvitamin D 1-alpha-hydroxylase gene maps to the pseudovitamin D-deficiency rickets (PDDR) disease locus.

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.

Mouse vitamin D-24-hydroxylase: molecular cloning, tissue distribution, and transcriptional regulation by 1alpha,25-dihydroxyvitamin D3.

Vitamin D-24-hydroxylase (24-OHase) is a cytochrome P-450 enzyme that catalyzes the conversion of 25-hydroxyvitamin D3 (25OHD3) and 1alpha,25-dihydroxyvitamin D3 [1,25-(OH)2D3] to 24,25-dihydroxyvitamin D3 and 1,24,25-trihydroxyvitamin D3, respectively. A full-length complementary DNA for mouse 24-OHase has now been characterized. The complementary DNA consists of 3309 bp and encodes a protein of 514 amino acids that shows 82% and 95% sequence identity with the human and rat enzymes, respectively. Northern blot analysis of tissues from mice injected with 1,25-(OH)2D3 (24 pmol/g) revealed that the 3.4-kb 24-OHase messenger RNA (mRNA) is most abundant in kidney and intestine, with smaller amounts present in skin, thymus, and bone. RT-PCR and Southern blot analysis detected 24-OHase mRNA in several other tissues including lung, testis, spleen, pancreas, and heart. Intraperitoneal injection of 1,25-(OH)2D3 induced dose- and time-dependent increases in both 24-OHase mRNA abundance and enzyme activity in mouse kidney. Similarly, 1,25-(OH)2D3-induced increases in both 24-OHase mRNA and activity were apparent in the duodenum. Although 1,25-(OH)2D3 increased the amount of 24-OHase mRNA in skin, enzyme activity was not detected in this tissue. Pretreatment of mice with cycloheximide (400 microg/g), an inhibitor of protein synthesis, potentiated the increase in 24-OHase mRNA abundance, but blocked the increase in 24-OHase activity, induced by 1,25-(OH)2D3 in kidney and duodenum, suggesting that 24-OHase gene expression may be regulated not only by the vitamin D receptor but also by a short-lived repressor protein.

Partial deletion of the gene encoding sterol 27-hydroxylase in a subject with cerebrotendinous xanthomatosis.

An Italian subject with cerebrotendinous xanthomatosis (CTX) was found to have a partial deletion of the gene encoding the enzyme sterol 27-hydroxylase (CYP27 gene). Southern blot analysis revealed that this deletion (approximately 2 kb) spans from intron 6 to the 3' flanking (3'FLK) region, eliminating exons 7-9, the last three exons of CYP27 gene. No sterol 27-hydroxylase mRNA was detected in proband cells, either by Northern blot analysis or by reverse transcription polymerase chain reaction (PCR). This suggests that the mutant mRNA devoid of the exon encoding the whole untranslated sequence (exon 9) might be rapidly degraded in the cytoplasm. We used inverse PCR to obtain a partial sequence of the 3'FLK region of the normal CYP27 gene; this allowed us to define the mechanism underlying the deletion. The established sequence was used to design suitable primers to perform step-wise sequences of a 1.7 kb segment of the 3'FLK region of the normal gene and of the deletion joint in the CTX patient. The analysis of the sequence data indicate that the deletion might result from a complex mechanism involving two intragenic recombinations between a) two 14 nucleotide complementary sequences, one in intron 6 and the other in the 3'FLK region: and b) AT-rich complementary sequences of the 3'FLK region, and a slipped mispairing between two 6 nucleotide direct repeats, one in intron 6 and the other in the 3'FLK region. Such repeats are brought close to each other by the formation of the stem-loops induced by the two intragenic recombinations. This is the first example of CTX caused by a rearrangement of CYP27 gene.

cDNA cloning and characterization of a vitamin D3 hydroxylase-associated protein.

We previously reported the generation of monoclonal antibodies which immunoprecipitate a fraction of the total chick kidney 1,25-dihydroxyvitamin D3-24R-hydroxylase activity. These antibodies were used to screen a chick kidney lambda gt11 cDNA library resulting in the isolation of a full-length cDNA encoding a protein that is not the 1,25-dihydroxyvitamin D3-24R-hydroxylase but another protein we term the vitamin D3 hydroxylase-associated protein (VDHAP). The deduced amino acid sequence agreed with an NH2-terminal amino acid sequence from the isolated VDHAP. Gene and protein bank search did not identify homology to known sequences or functional domains in the VDHAP cDNA. VDHAP mRNA levels are not altered by conditions which either induce 1,25-dihydroxyvitamin D3-24R-hydroxylase activity (78-fold) or 25-hydroxyvitamin D3-1 alpha-hydroxylase activity (30-fold). Northern analysis of poly(A)+ RNA from chick tissues revealed VDHAP only in kidney. Cellular fractionation experiments demonstrated that VDHAP and the 25-hydroxyvitamin D3-1 alpha-hydroxylase are colocalized in the inner membrane of mitochondria. The VDHAP antibody immunoprecipitates 14% of the total 1,25-dihydroxyvitamin D3-24R-hydroxylase activity (7-fold over background) and immunoprecipitates 21% of the total 25-hydroxyvitamin D3-1 alpha-hydroxylase activity (2-fold over background). VDHAP is a novel chick kidney-specific inner membrane protein of mitochondria, which associates with a fraction of the 1,25-dihydroxyvitamin D3-24R-hydroxylase and 25-hydroxyvitamin D3-1 alpha-hydroxylase.

Dissociation of phosphaturia and 25(OH)D-1 alpha-hydroxylase trophism using a novel analogue of parathyroid hormone.

Certain parathyroid hormone (PTH) analogues have been shown to selectively impair some but not all physiological actions of PTH. In this study, transaminated rat (r) PTH [TA-rPTH-(1-34)], a PTH analogue that differs from the rPTH-(1-34) fragment in that the NH2-terminal alanine is converted to pyruvate, was infused into mice to determine its properties in vivo and specifically to determine whether stimulation of 25-hydroxyvitamin D-1 alpha-hydroxylase (1 alpha-hydroxylase) activity was more dependent on concomitant renal handling of phosphate or on generation of adenosine 3',5'-cyclic monophosphate (cAMP). High-performance liquid chromatography-purified TA-rPTH-(1-34) was infused into C57BL mice at 10 or 30 pmol/h for 24 h. At 30 pmol/h, TA-rPTH-(1-34) was comparable with rPTH-(1-34) in its hypophosphatemic and phosphaturic effects but was less potent than rPTH-(1-34) in raising serum calcium. TA-rPTH-(1-34) was markedly less effective in stimulating renal 1 alpha-hydroxylase than rPTH-(1-34). Stimulation of urinary cAMP excretion occurred after infusion with TA-rPTH-(1-34), but this effect was significantly less than that seen with rPTH-(1-34). These findings indicate that PTH-induced hypophosphatemia and phosphaturia can be uncoupled from PTH stimulation of 1 alpha-hydroxylase. Furthermore, cAMP-related signal transduction appears to be more significant in regulation of 1 alpha-hydroxylase than mechanisms that mediate PTH-sensitive phosphate transport, independent of cAMP.