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De-orphanization of Cytochrome P450 2R1Mitochondrial and microsomal vitamin D hydroxylase enzymes catalyze the first reaction. The mitochondrial activity is associated magnesium testosterone pubmed central sterol hydroxylase, a jbc anabolic mass review P CYP27A1 ; however, the identity of the microsomal enzyme has remained elusive. A cDNA library ,ass from hepatic mRNA of sterol hydroxylase-deficient mice was screened with a ligand activation assay to identify an evolutionarily conserved microsomal cytochrome P CYP2R1 with vitamin Jbc anabolic mass review hydroxylase activity. Expression of CYP2R1 in cells led to the transcriptional activation of the vitamin D receptor when either vitamin D 2 or D 3 was added to the medium. Thin anaboluc chromatography and radioimmunoassays indicated that the secosteroid product of CYP2R1 was hydroxyvitamin D 3. Vitamin D ababolic calcium and phosphate metabolism by activating the vitamin D receptor, a transcription factor and member of the nuclear receptor family. Subsequently, the structure of vitamin D and its origins from plant and animal steroids were determined, and the roles of the vitamin in the mobilization of minerals from the diet and in bone were defined.
Publikationen seit 2006
Mitochondrial and microsomal vitamin D hydroxylase enzymes catalyze the first reaction. The mitochondrial activity is associated with sterol hydroxylase, a cytochrome P CYP27A1 ; however, the identity of the microsomal enzyme has remained elusive. A cDNA library prepared from hepatic mRNA of sterol hydroxylase-deficient mice was screened with a ligand activation assay to identify an evolutionarily conserved microsomal cytochrome P CYP2R1 with vitamin D hydroxylase activity.
Expression of CYP2R1 in cells led to the transcriptional activation of the vitamin D receptor when either vitamin D 2 or D 3 was added to the medium.
Thin layer chromatography and radioimmunoassays indicated that the secosteroid product of CYP2R1 was hydroxyvitamin D 3. Vitamin D regulates calcium and phosphate metabolism by activating the vitamin D receptor, a transcription factor and member of the nuclear receptor family. Subsequently, the structure of vitamin D and its origins from plant and animal steroids were determined, and the roles of the vitamin in the mobilization of minerals from the diet and in bone were defined.
The liver was shown to be required for the activation of vitamin D by hydroxylation 2 , 3. The molecular mechanism of vitamin D action was manifest with the identification 8 and cDNA cloning 9 of the vitamin D receptor. Hydroxylation reactions catalyzed by cytochrome Ps CYP 1 activate and inactivate vitamin D as a ligand for the receptor. Together, these enzymes regulate the systemic and local levels of vitamin D through complex feedback mechanisms mediated in part by the vitamin D receptor The existence and physiological importance of the two hepatic vitamin D hydroxylase enzymes are demonstrated in humans 23 , 24 and mice 25 , 26 by mutations in the mitochondrial CYP27A1 vitamin D hydroxylase gene.
Loss of this enzyme has profound effects on cholesterol metabolism, as CYP27A1 catalyzes an essential reaction in bile acid synthesis. These findings indicate that the microsomal vitamin D hydroxylase can compensate for loss of the mitochondrial activity. In the current study, a cDNA library made from hepatic mRNA of mice deficient in the gene encoding the mitochondrial CYP27A1 enzyme was screened using a vitamin D receptor-based, ligand activation assay The biochemical properties and tissue distribution of CYP2R1 are consistent with this enzyme being the microsomal vitamin D hydroxylase.
L was amplified by the polymerase chain reaction PCR from random hexamer-primed mouse hepatic cDNAs using the following oligonucleotide primers: The adrenodoxin expression vector encoded the expected electron transport enzyme activity in transfected cells, as judged by the ability of the protein to stimulate the vitamin D hydroxylase activity of sterol hydroxylase. Oligonucleotide primers for the amplification reaction were: On day 1, cells were transfected with a mixture containing 0.
After 18 h, the transfection medium was removed from the dish and replaced with 2 ml of fresh medium supplemented as above and containing 4. For cells transfected with the pCMV6-mCYP27A1 plasmid, the medium was brought to a final concentration of 10 -6 m vitamin D 3 by the addition of unlabeled secosteroid.
Cells and medium were harvested 96 h later, and vitamin D metabolites were extracted with 8 ml of chloroform: Solvent systems of cyclohexane: After 10 h, ethanol vehicle, or vitamin D 3 at final concentrations of 0. Oligonucleotide primer sequences used in these experiments were: An expression cloning assay was established in cultured cells and optimized to isolate cDNAs encoding enzymes capable of activating vitamin D 3 by hydroxylation.
A luciferase enzyme activity that was 3-fold over background was observed when the sterol hydroxylase plasmid was diluted fold with a control plasmid lacking a cDNA insert.
The resulting library was estimated to contain 7. The data generated from one such experiment is shown in Fig.
Subsequent cycles of cDNA purification and screening were performed on the A11 pool to identify a single cDNA that encoded the vitamin D-activating activity. Expression cloning of vitamin D hydroxylase cDNA. After an additional incubation of 16—20 h in an atmosphere of Seven positive cDNA pools were identified, revealing four different encoded proteins.
The human CYP2R1 gene is located on chromosome 11, band p The mouse Cyp2r1 gene occupies a syntenic location on chromosome 7, band 7E3.
Amino acid sequences and gene structure of cytochrome P 2R1. A , the deduced sequences of the human h and mouse m CYP2R1 proteins are aligned with identities between the two enzymes indicated by black boxes. Amino acids are numbered on the left. Arrowheads above the alignment indicate the positions of introns in the encoding genes. An asterisk indicates the cysteine residue that is predicted to ligand with the heme cofactor.
B , structure of the human CYP2R1 gene. Exons are indicated by boxes and introns by connecting lines. Amino acids encoded at the beginning and end of the protein are indicated above the gene schematic together with those at exon-intron junctions. This hypothesis was tested further in a series of transfection experiments with different cytochrome P cDNAs, vitamin D receptor constructs, and reporter genes.
A similar stimulation was observed when a plasmid encoding the mouse sterol hydroxylase mCYP27A1 was introduced into the HEK cells. The indicated vitamin D receptor-reporter systems were introduced into HEK cells grown in mm dishes together with the expression plasmids designated at the bottom of the figure. The amounts of vitamin D 3 added to the medium were 0.
Similar responses were ascertained when the receptor-reporter system was switched to one composed of the intact vitamin D receptor and a luciferase reporter gene driven by three copies of a vitamin D response element from the mouse osteopontin gene linked to a basal TK promoter Fig.
The final receptor-reporter system tested was composed of the intact vitamin D receptor and a 0. Previous studies showed that the osteopontin regulatory sequences contain a bona fide vitamin D-responsive element This level of stimulation by vitamin D 3 is similar to that reported in other studies with the osteopontin promoter These experiments proved challenging for three reasons.
Despite these challenges, informative data were generated in transfection experiments with the various P expression vectors.
As shown in Fig. Activation of vitamin D 3 by cytochrome P enzymes. Plasmids encoding the indicated vitamin D receptor-reporter systems were transfected into HEK cells grown in mm dishes together with DNAs encoding the hydroxylase enzymes shown at the bottom of the figure. The amounts of vitamin D 3 added to the culture medium were 0. To determine the specificity of CYP2R1, the capacity of the encoded protein to activate or inactivate ligands for other nuclear receptors was determined.
The experiments reported in Figs. To assess the effects of changing the concentrations of these components, dose-response curves were generated in two different ways. The data from this experiment showed an increase in luciferase enzyme expression with increasing amounts of human or mouse CYP2R1 expression plasmid added to the dish Fig.
Saturation of the response system was reached when 10 ng of P expression plasmid was added to the cells. No increase in luciferase enzyme activity was observed when mouse adrenodoxin or human CYP3A4 were expressed in these cells Fig. B , P expression vectors 5—20 ng of plasmid DNA were transfected into HEK cells grown in mm dishes in medium containing the indicated concentrations of vitamin D 3. Relative luciferase enzyme activities were determined 16—20 h later.
Mean values based on data from triplicate dishes for each concentration of vitamin D 3 were plotted. In the second experiment, levels of vitamin D 3 were varied and the amounts of the P expression plasmids were held constant Fig. In these experiments, CYP2R1 was more robust than CYP27A1 in generating an active receptor at low levels of vitamin D 3 , but this difference was lost at higher concentrations of the precursor.
The most potent combination of plasmids was composed of the human CYP2R1 and mouse CYP27B1, which generated a measurable luciferase signal at the lowest concentration 50 n m of vitamin D 3 assayed Fig. Mitochondrial vitamin D hydroxylase shows a preference for vitamin D 3 over vitamin D 2 as a substrate 11 , This preference, and the well known ability of both secosteroids to fulfill the physiological roles of the vitamin, supports the existence of additional mammalian vitamin D hydroxylases No activation of either vitamin D precursor was observed when the cells were transfected with a CMV vector alone Fig.
Activation of vitamin D 3 and vitamin D 2 by microsomal versus mitochondrial P enzymes. Relative luciferase enzyme activities were measured 16—20 h later. The inactivation of vitamin D is mediated by the vitamin D hydroxylase enzyme, a microsomal P CYP24A1 that is highly selective for this substrate Vitamin D 3 was added to the medium at a final concentration of 0. The experiments with the vitamin D hydroxylase expression plasmid suggested that the active vitamin D ligand generated by the CYP2R1 enzyme was hydroxyvitamin D.
This interpretation was confirmed in chemical and immunological experiments Fig. Transfection of a CYP2R1 cDNA into HEK cells followed by incubation with 14 C vitamin D 3 led to the synthesis of a radiolabeled product that migrated on a thin layer chromatography plate at the same position as hydroxyvitamin D 3. Similarly, expression of the sterol hydroxylase, a known vitamin D 3 hydroxylase, produced an apparently identical product lane 4.
Transfection with the vector alone lane 1 or with a cDNA encoding mouse adrenodoxin lane 3 did not lead to synthesis of this product. Similar results were observed when three different solvent systems were used to develop the thin layer chromatography plates data not shown. A , the indicated expression plasmids were introduced into HEK cells cultured in mm dishes.
Vitamin D metabolites were extracted from the medium into 8 ml of chloroform: The plates were dried and subjected to phosphorimage analysis to reveal the locations of radioactivity. The identities of the radiolabeled compounds were determined based on their co-migration with authentic vitamin D 3 and hydroxyvitamin D 3 standards chromatographed in adjacent lanes on the plate.
The mAdx plasmid expresses mouse adrenodoxin. B , radioimmunoassay of vitamin D metabolites produced in transfected cells. The indicated plasmid expression vectors were introduced by transfection into HEK cells grown in well microtiter plates. Thereafter, the medium from replicate wells was pooled and subjected to radioimmunoassay using a kit containing antibodies directed against hydroxyvitamin D 3.
The values obtained were compared with those of a standard curve to estimate the amount of hydroxyvitamin D produced in duplicate dishes. A background value corresponding to the amount of hydroxyvitamin D detected in the medium from cells transfected with the CMV vector plasmid A radioimmunoassay was used to measure the production of hydroxyvitamin D 3 Fig.
A comparable level of synthesis was measured after expression of the sterol hydroxylase cDNA Fig. In this experiment, the sterol hydroxylase enzyme was more active at higher substrate concentrations than was CYP2R1.
When the concentration of this substrate added to the medium was 1 n m , cells expressing CYP2R1 produced 0. Taken together, the data shown in Figs. These experiments revealed that CYP2R1 mRNA was most abundant in the liver and testis 2 and present in lower levels in numerous other tissues and cell types.
De-orphanization of Cytochrome P 2R1
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