©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Different Mechanisms of Hydroxylation Site Selection by Liver and Kidney Cytochrome P450 Species (CYP27 and CYP24) Involved in Vitamin D Metabolism (*)

F. Jeffrey Dilworth (1), Ian Scott (1), Andrew Green (1), Stephen Strugnell (1), Yu-Ding Guo (1), Eve A. Roberts (2), Richard Kremer (3), Martin J. Calverley (4), Hugh L. J. Makin (5), Glenville Jones(§) (1)

From the (1)Department of Biochemistry, Queen's University, Botterell Hall, Kingston, Ontario, Canada K7L 3N6, the (2)Department of Pharmacology, University of Toronto, Toronto, Ontario, Canada M5G 1X8, the (3)Department of Biochemistry, McGill University, Montreal, Quebec, Canada H3A 1A1, the (4)Chemical Research Department, Leo Pharmaceutical Products, Ballerup, Denmark DK-2750, and the (5)Department of Chemical Pathology, London Hospital Medical College, University of London, London, United Kingdom E1 2AD

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

A series of homologated 1-hydroxyvitamin D and 1,25-dihydroxyvitamin D molecules with one to three extra carbons in the side chain were used to examine the substrate preferences and hydroxylation site selection mechanisms of the liver vitamin D-25-hydroxylase (CYP27) and the target cell 25-hydroxyvitamin D-24-hydroxylase (CYP24). Cultured and transfected cell models, used as sources of these hydroxylases, gave 23-, 24-, 25-, and 27-hydroxylated metabolites which were identified by their high performance liquid chromatography and GC-MS characteristics. Lengthening the side chain is tolerated by each cytochrome P450 isoform such that 25-hydroxylation or 24-hydroxylation continues to occur at the same rate as in the native side chain, while the site of hydroxylation remains the same for the liver enzyme in that CYP27 continues to hydroxylate at C-25 and C-27 (minor) despite the two-carbon-atom extension. Somewhat surprising is the finding that C-24 and C-23 (minor) hydroxylations also do not change as the side chain is extended by as much as three carbons. We conclude that CYP24 must be directed to its hydroxylation site(s) by the distance of carbon 24 from the vitamin D ring structure and not as in CYP27 by the distance of the hydroxylation site from the end of the side chain.


INTRODUCTION

In recent years there has been an explosion in the number of novel analogs of vitamin D chemically synthesized for the purpose of developing drugs for the treatment of metabolic bone disease, cancer, and psoriasis(1) . In large part, this recent interest in vitamin D analogs arose from the finding that the hormonal form, 1,25-dihydroxyvitamin D (1,25-(OH)D),()stimulates differentiation of certain cells in the skin and myeloid lineages as well as performing its classical roles in Ca and PO homeostasis(2) . Despite the fact that many of these analogs have been screened for biological activity in a variety of assays (calcemic activity, differentiation activity, vitamin D receptor binding, vitamin D binding globulin binding), few have been subjected to pharmacokinetic analysis (3). Even fewer have been subjected to detailed metabolic studies. Exceptions include MC903 (calcipotriol) (4, 5) and 22-oxa-calcitriol(6) ,()but in these studies the principal focus has been defining the nature of the metabolic products for governmental drug approval. We know of no example where a series of analogs have been used to define the substrate requirements of the hydroxylase enzymes involved in vitamin D metabolism.

Homologated vitamin D metabolites with a single extra carbon (C-24a) between carbons C-24 and C-25 or on the C-26 and/or C-27 were first synthesized by Ikekawa and co-workers and tested biologically by DeLuca and co-workers (8, 9, 10) and by Stern's group(11) . It was quickly recognized that these homologated analogs might offer potential as drugs because of increased biological activity in several in vitro assays. However, the exact molecular basis for this increased biological activity was never fully elucidated, and the metabolic fate of these homologated analogs never studied. The availability of a series of such homologated vitamin D analogs together with much biological data made them particularly attractive for metabolic study using a systematic approach. Furthermore, the recent clinical research interest in the use of more complex homologated vitamin Ds (e.g. EB1089 and KH1060, Leo Pharmaceutical Products) made the choice of metabolism of homologated vitamin D analogs a topical subject for investigation.

The vitamin D hydroxylases constitute a family of mixed-function oxidases which contain as their specific component, a cytochrome P450 isoform. Two such cytochrome P450 isoforms have been cloned, one putatively representing the liver vitamin D-25-hydroxylase (CYP27) and the other that of the kidney 25-OH-D-24-hydroxylase (CYP24)(12, 13) . Few experiments have been carried out using the expression of the cDNAs of these cytochrome P450s to determine their specificity, but other studies have shown that CYP27 has broad substrate specificity, hydroxylating bile acid intermediates and a range of vitamin D compounds(14, 15) . Furthermore, our own laboratory has shown CYP27 to lack regiospecificity, placing hydroxyl groups into a number of carbon positions at C-24, C-25, and C-27 of the classical D and D side chains(16) . No studies have been performed with the newer analogs to probe the structural requirements of these cytochrome P450 isoforms.

Thus, in these studies, we set out to use the homologated series of analogs as tools in the study of the two best known hydroxylases, the liver vitamin D-25-hydroxylase and the target cell 25-OH-D-24-hydroxylase. We chose to study metabolism in vitro using cultured cell systems we have established for this purpose (17, 5) and, in the case of CYP27, confirmed cell culture findings with cDNA transfection studies(16) . Our findings show that the homologated vitamin D analogs are extremely valuable in that they reveal important information about how the two enzymes interact with their substrates.


EXPERIMENTAL PROCEDURES

Materials

The human keratinocyte cell line, HPK1A (18) transformed with the ras oncogene, HPK1A-ras(19) was developed previously in collaboration with Dr. Johng Rhim (National Cancer Institute). Similarly, the development of the human liver cell line, HH01, by one of us (E. R.) has been described previously(20) . The SV-40 transformed African Green Monkey kidney cell line, COS-1, was purchased from the American Tissue Culture Collection. All solvents used were of HPLC grade and were obtained from Caledon Laboratories (Georgetown, ON, Canada). The bovine adrenodoxin expression plasmid pBAdx-4 (21) was a gift of Dr. M. Waterman (University of Texas Medical School, Dallas, TX).

Synthesis of Vitamin D Analogs

The vitamin D analogs: 24a,24b-dihomo-1-(OH)D (MC1281), 24a-homo-1,25-(OH)D (MC1127), 24a,24b-dihomo-1,25-(OH)D (MC1147), and 24a,24b,24c-trihomo-1,25-(OH)D (MC1179) (Fig. 1) were synthesized as described previously(22) . Their 23- and 24-hydroxylated derivatives were synthesized by methods to be described elsewhere. The 23-hydroxylated compounds were available separately with known configuration, whereas the 24-hydroxylated compounds were available as 1:1 diastereoisomeric mixtures. The radioactive hormone, [1-H]1,25-dihydroxyvitamin D ([H]1,25-(OH)D) was synthesized as described previously(23) .


Figure 1: Structures of homologated vitamin D analogs. Structures of the side chains of 1,25-(OH)D analogs and 1-OH-D analogs, together with the ring structure of both types of vitamin D analogs.



Generation of Metabolites of MC1127, MC1147, and MC1179 Using HPK1A-ras Cells

Metabolites were generated from HPK1A-ras cells as described previously(5, 24) . Cells were maintained in 150-mm plates using Dulbecco's culture medium (Dulbecco's modified Eagle's medium). Near confluence, cells were treated with 1,25-(OH)D (10 nM) to induce transcription of catabolic enzymes. After 18 h, the medium was replaced by Dulbecco's modified Eagle's medium supplemented with 1% BSA and 100 µM DPPD (Sigma). Cells were then incubated for 48 h in the presence of vehicle (0.01% EtOH) or a 10 µM concentration of analog in vehicle.

Generation of Metabolites of MC1281 Using HH01 Cells

HH01 were maintained in -MEM supplemented as described previously(20) . Near confluence, the medium was replaced with -MEM supplemented with 1% BSA and 100 µM DPPD. Cells were incubated in the presence of vehicle (0.01% EtOH) or 25 µM MC1281 in vehicle for 20 h.

Incubations of MC1281 with CYP27-transfected COS-1 Cells

COS-1 were transfected with the expression vectors pSG5-CYP27 and pBAdx (adrenodoxin) using the standard DEAE-Dextran method (25) as described previously(16) . Forty-eight hours post-transfection, cells were washed twice with phosphate-buffered saline, and the medium was replaced with Dulbecco's MEM supplemented with 1% BSA and 100 µM DPPD. Cells were then incubated for 30 h in the presence of vehicle (0.01% EtOH) or MC1281 (25 µM) in vehicle.

Purification of Metabolites

Cells and medium were extracted as described previously(24) . HPLC separation of metabolites was achieved using a Zorbax-SIL (0.62 8 cm; 3 µ) column eluted with HIM 91:7:2 at a flow rate of 1 ml/min. Metabolites were identified based on their characteristic vitamin D chromophore (UV = 265, UV = 228, UV/UV = 1.75).

Further steps of HPLC were performed on the crude peaks isolated from the first step of purification, using a Zorbax-CN (0.46 25 cm; 6 µ) column and 91:7:2 (HIM) solvent system at a flow rate of 1 ml/min. Two rounds of HPLC yielded metabolites which were pure on the basis of the homogeneity of the collected peak.

Co-chromatography of each purified biologically derived metabolite with its chemically synthesized counterpart was attempted using one of the two HPLC column systems described above. The inclusion of 1-OH-D as an internal standard allowed us to convert retention times of metabolites into relative retention times, and mean relative retention times were calculated from triplicate HPLC runs. Mean relative retention times were considered identical if they were within two standard deviations of each other.

Chemical Modification of Metabolites

Purified metabolites were subject to chemical modification using sodium metaperiodate or sodium borohydride as described previously(24) . Modified metabolites were subject to HPLC analysis using the system described above.

GC-MS

Purified metabolites were derivatized to pertrimethylsilyl ethers, then analyzed by gas chromatography-mass spectrometry (GC-MS) as described previously(26) . Injection of metabolites of analogs described in this paper into the high temperature injection zone of the GC causes B ring closure producing pyro- and isopyro- isomers. Mass spectra were obtained only from the pyro- isomer, and, for simplicity, in discussion of fragmentation and in figures illustrating spectra obtained, the uncyclized metabolite structure is used rather than that of the correct pyro- isomer. Mass spectra were obtained by averaging each peak and subtracting the background.

Catabolism Rate Assay

The rate of metabolism of the 1,25-(OH)D analogs was measured as described previously(24) . HPK1A-ras cells were incubated with [H]1,25-(OH)D (23 nM) in the presence or absence of varying concentrations of analog (0 to 23 µM) for 3 h at 37 °C. Triplicate 500-µl aliquots of aqueous fraction from the cell/medium extract were mixed with aqueous scintillation mixture, and the radioactivity was measured using a scintillation counter.

25-Hydroxylation Rate Assay

The rate of metabolism of 1-(OH)D and MC1281 were compared using a time course study. The cell line HH01 was grown in 6-well plates as described above. Near confluence, the medium was replaced with -MEM supplemented with 1% BSA and 100 µM DPPD. Cells were incubated with 25 µM analog for 0, 6, 12, or 18 h, then extracted, and purified as described above. Both the amount of substrate remaining and metabolites produced were determined by integrating the area under peak of UV absorbance at 265 nm. Each data point is the average of three incubations.


RESULTS

Generation of Metabolites from 1,25-(OH)Din HPK1A-ras Cells

Incubation of 1,25-(OH)D with HPK1A-ras cells resulted in the formation of the four lipid-soluble metabolites of the hormone (Fig. 2D). The four metabolites generated include 1,24,25(OH)D (peak D), 24-oxo-1,25-(OH)D (peak A), 24-oxo-1,23,25(OH)D (peak C), and 24,25,26,27-tetranor-1,23(OH)D (peak B) as determined by co-migration with standard compounds (data not shown). These metabolites correspond to the intermediates which have previously been reported to be a part of the C-24 oxidation pathway of vitamin D(27) . This confirms that the Ha-ras transformed cell line, HPK1A-ras, expresses functional enzymes involved in the side chain oxidation pathway for inactivation of 1,25-(OH)D.


Figure 2: HPLC profiles of lipid extracts from HPK1A-ras cells incubated with homologated vitamin D analogs. Total lipid extracts were separated on a Zorbax-SIL column using the solvent HIM 91:7:2 at a flow rate of 1 ml/min. Metabolites of MC1127 (A), MC1147 (B), MC1179 (C), and 1,25-(OH)D (D) were identified based on their characteristic vitamin D chromophore (UV = 265 nm, UV = 228 nm, UV/UV = 1.75) and have been shaded. Only the region of the chromatogram containing vitamin D metabolites is depicted. The inset in B shows the subsequent separation of metabolites MC1147-MetC and MC1147-MetD during Zorbax-CN rechromatography of the 19.8-22-min peak isolated from B.



Generation of Metabolites from Homologated Analogs in HPK1A-ras Cells

Analytical HPLC separation of lipid soluble HPK1A-ras cell extracts taken from cells incubated with homologated analogs (10 µM) indicated that we were able to generate three (or more) metabolites from MC1127 (Fig. 2A) and MC1147 (Fig. 2B), while only two metabolites were generated from MC1179 (Fig. 2C). Rechromatography of the peak labeled MC1147-MetC (see inset in Fig. 2B) indicated that this peak was a mixture of two metabolites which could be resolved on Zorbax-CN (termed peaks C and D). As we were able to generate only two metabolites from MC1179, a second incubation was performed in which the ratio of cultured cell density:concentration of substrate (MC1179) was increased in order to generate a greater amount of less abundant metabolites. In doing so, we were able to generate sufficient quantities of this third metabolite, termed MC1179-MetC (peak not apparent in the chromatogram shown in Fig. 2C), to be identified later by GC-MS. The chromatographic properties of the purified metabolites generated from all three analogs are summarized in . The availability of reference standards of both R and S forms allowed the exclusion of the presence of other isomers in the extracts.

The major metabolites were identified by GC-MS and direct comparison with chemically synthesized standards (see ). All three putative 23-hydroxylated metabolites exhibited identical relative retention times on HPLC to one of the two 23(R)- or 23(S)- chemically synthesized epimeric forms. The metabolite, MC1127-MetC (RRT = 3.52 ± 0.02) co-migrated with 23(R)-OH-MC1127 (RRT = 3.56 ± 0.01) but not 23(S)-OH-MC1127 (RRT = 3.44 ± 0.01); the metabolite MC1147-MetC (RRT = 3.50 ± 0.02) co-migrated with 23(S)-OH-MC1147 (RRT = 3.59 ± 0.04) but not 23(R)-OH-MC1147 (RRT = 3.75 ± 0.03); the metabolite MC1179-MetC (RRT = 4.41 ± 0.02) co-migrated with 23(R)-OH-MC1179 (RRT = 4.43 ± 0.03) but not 23(S)-OH-MC1179 (RRT = 4.23 ± 0.06) (see for column and solvent systems used). Since the synthetic reference 24-hydroxylated standards ran as single peaks on HPLC and GC, identification of R/S isomers was not possible. However, relative retention times on HPLC of MC1127-MetA, MC1147-MetD, and MC1179-MetB were similar to those of the diastereoisomeric mixtures of their respective 24-hydroxylated standards. These were as follows: MC1127-MetA (RRT = 2.97 ± 0.01 min) compared with 24-OH-MC1127 (RRT = 2.99 ± 0.01); MC1147-MetD (RRT = 3.22 ± 0.02) compared with 24-OH-MC1147 (RRT = 3.21 ± 0.06); MC1179-MetB (RRT = 3.59 ± 0.04) compared with 24-OH-MC1179 (RRT = 3.65 ± 0.02) (see for column and solvent systems used).

The identity of the 23-hydroxylated metabolites MC1127-MetC, MC1147-MetC, and MC1179-MetC were confirmed by comparing the mass spectra of pertrimethylsilylated ether derivatives of the putative metabolites with those of chemically synthesized standards (Fig. 3). In each case, the mass spectrum obtained from the biologically generated metabolite is very similar to that of its chemically synthesized standard (essentially no differences between R/S versions were observed). Molecular ions of m/z 734, 748, and 762 were observed for the pertrimethylsilylated ether derivatives of MC1127-MetC, MC1147-MetC, and MC1179-MetC, respectively, as expected for hydroxylation of the starting compounds. Ions of m/z 171, 185, and 199 result from the loss of a single silanol group from side chain fragments m/z 261, 275, and 289 for MC1127-MetC, MC1147-MetC, and MC1179-MetC, respectively, which arise from cleavage of the C-22-C-23 bond and suggest C-22-C-23 fragility. The mass spectra of all three 23-hydroxylated metabolites contained ions at m/z 395 and 305 due to the sequential loss of two and three silanol groups from the predicted ion m/z 575 (M - 159, M - 173, or M - 187, respectively) common to all 23-hydroxylated metabolites. This fragmentation pattern again suggests C-23-C-24 fragility. As with MC1147-MetB, an ion of m/z 278 was observed in all three spectra. This ion probably arises from a cleavage of the bond between C-22 and C-23 resulting in the loss of 261, 275, or 289 mass units from the molecular ion of each 23-hydroxylated metabolite, respectively. The resultant fragment of m/z 473 loses a methyl group and 2 silanols to give rise to the fragment at m/z 278. These results further confirm the identity of MC1127-MetC, MC1147-MetC, and MC1179-MetC as 23(R)-OH-MC1127, 23(S)-OH-MC1147, and 23(R)-OH-MC1179, respectively.


Figure 3: Mass spectra of putative 23-hydroxylated metabolites. TMSi derivatives of the purified metabolites MC1127-MetC (top left), MC1147-MetC (top middle), and MC1179-MetC (top right) and the synthetic standards 23(R)-OH-MC1127 (bottom left), 23(S)-OH-MC1147 (bottom middle), and 23(R)-OH-MC1179 (bottom right) were analyzed by GC-MS under conditions described in the text. In each case, spectra represent the pyro- isomer derived from each metabolite and for convenience spectral interpretation is given with reference to the parent metabolite.



To confirm the identities of MC1127-MetA, MC1147-MetD, and MC1179-MetB as the 24-hydroxylated metabolites, the mass spectra of pertrimethylsilylated ether derivatives of the purified metabolites were again compared to those of the chemically synthesized standards (Fig. 4). The mass spectra of the pertrimethylsilylated ether derivatives of the putative 24-hydroxylated metabolites are very similar to those of their corresponding chemically synthesized standards. In each case, molecular ions at m/z 734, 748, and 762 for the derivatives of MC1127-MetA, MC1147-MetD, and MC1179-MetB are consistent with the hydroxylation of the substrate. Ions of m/z 157, 171, and 185 due to the loss of a single silanol from side chain fragments of m/z 247, 261, and 275 are observed for MC1127-MetA, MC1147-MetD, and MC1179-MetB, respectively, and indicate C-23-C-24 bond fragility. The mass spectra of all three 24-hydroxylated metabolites contained ions of m/z 499 and 409 due to the sequential loss of one and two silanol groups, respectively, from the predicted m/z 589 (M - 145, M - 159, or M - 173) fragment common to all 24-hydroxylated metabolites. This fragmentation pattern suggests C-24-C-24a bond fragility. Also observed in the mass spectra of all three 24-hydroxylated metabolites was an ion at m/z 292, which is probably generated by cleavage of the bond between C-23 and C-24 resulting in the loss of 247, 261, or 275 mass units from the molecular ion of each 24-hydroxylated metabolite, respectively. The resultant fragment of m/z 487 loses a methyl group and 2 silanols to give the fragment at m/z 292. We conclude on the basis of these results that MC1127-MetA, MC1147-MetD, and MC1179-MetB are the 24-hydroxylated metabolites of their respective analogs.


Figure 4: Mass spectra of putative 24-hydroxylated metabolites. TMSi derivatives of the purified metabolites MC1127-MetA (top left), MC1147-MetD (top middle), and MC1179-MetB (top right) and the synthetic standards 24-OH-MC1127 (bottom left), 24-OH-MC1147 (bottom middle), and 24-OH-MC1179 (bottom right) were analyzed by GC-MS under conditions described in the text. In each case, spectra represent the pyro- isomer derived from each metabolite and for convenience spectral interpretation is given with reference to the parent molecule.



The minor metabolites illustrated in Fig. 2and listed in , MC1127-MetB, MC1147-MetB, and MC1179-MetA were all found to be sodium borohydride-sensitive, and their mass spectral and chromatographic properties (see ) are consistent with the presence of oxo-groups at C-23 or C-24. One other metabolite, MC1147-MetA, was studied but not conclusively identified on the basis of the information gathered (see ).

Rate of 24-Hydroxylation of Homologated Analogs

The effect of lengthening the vitamin D side chain on the rate of 24-hydroxylation was also examined using HPK1A-ras cells (Fig. 5). Measuring the ability of the analogs to compete with [1-H]1,25-(OH)D for the enzymes of the side chain oxidation pathway, we observe that extending the vitamin D side chain does not significantly alter the rate of 24-hydroxylation.


Figure 5: Competitive inhibition of [1-H]calcitroic acid production using nonradioactive homologated 1,25-(OH)D analogs in HPK1A-ras cells. [1-H]1,25-(OH)D was incubated in HPK1A-ras cells in the presence of vehicle alone or varying concentrations of MC1127 (▾), MC1147 (), MC1179 (), or 1,25-(OH)D (). Incubations were performed as outlined under ``Experimental Procedures.'' Each point in the figure is the mean ± S.E. of three flasks counted in triplicate.



Generation of Metabolites from Homologated 1-OH-DAnalog

Northern analysis of HH01 cells using a CYP27 cDNA probe (16) indicated that CYP27 mRNA was expressed in this cell line (data not shown). Incubation of HH01 cells with either 1-OH-D or MC1281 in each case resulted in the generation of two metabolites (Fig. 6, A and B). Other studies have shown that the vitamin D metabolites formed by HH01 cells are generated by cytochrome P450-dependent steps as judged by their sensitivity to heat inactivation and their total inhibition by the anti-fungal agent ketoconazole (used at a concentration of 5 µM).()The two products of 1-OH-D (MetA and MetB) have been previously identified as 1,25-(OH)D and 1,26(27)-(OH)D, respectively(16) . In the case of MC1281, the less polar of the two metabolites (MC1281-MetA) co-migrated with the synthetic standard 24a,24b-dihomo-1,25-(OH)D (MC1147) on two separate HPLC systems ().


Figure 6: HPLC profiles of lipid extracts from HH01 cells incubated with 1-OH-D or MC1281. Total lipid extracts were separated on a Zorbax-SIL column using the solvent HIM 91:7:2 at a flow rate of 1 ml/min. Metabolites of 1-OH-D (A) or MC1281 (B) were identified based on their characteristic vitamin D chromophore (UV = 265 nm, UV = 228 nm, UV/UV = 1.75) and have been shaded.



The metabolites generated in HH01 cells were further characterized by GC-MS analysis. The mass spectra of the pertrimethylsilylated ether derivatives of MC1281-MetA and the chemically synthesized standard 24a,24b-dihomo-1,25-(OH)D (MC1147) are shown in Fig. 7. Both spectra show a molecular ion at m/z 660 and a major fragment at m/z 131 corresponding to [(CH)C=OTMS] produced by cleavage of the bond between C-24 and C-25. Such a fragment at m/z 131 is characteristic of most 25-hydroxylated vitamin D derivatives (e.g. 25-OH-D). This evidence confirms the identify of MC1281-MetA as the 25-hydroxylated derivative. The mass spectrum of the pertrimethylsilylated ether derivative of MC1281-MetB also showed a molecular ion of m/z 660, again suggesting hydroxylation of MC1281. However, the appearance of a fragment at m/z 103 and none at m/z 131 indicates that hydroxylation has occurred at a different site, probably C-27 in this case, the fragment at m/z 103 representing [C(27)-OTMS] and being derived from C-25-C-27 cleavage. Thus the data are consistent with the minor metabolite, MC1281-MetB being the 27-OH derivative.


Figure 7: Mass spectra of the putative 25-hydroxylated metabolite MC1281-MetA formed in HH01 cells and its chemically synthesized standard. TMSi derivatives of the purified HH01 metabolite MC1281-MetA from Fig. 6B was compared to synthetic 25-OH-MC1281 (also known as MC1147) on GC-MS as described in the text. In each case, spectra represent the pyro- isomer derived from each metabolite, and, for convenience, spectral interpretation is given with reference to the parent molecule.



The rate of metabolism of the side-chain-lengthened analog was compared to that of 1-OH-D in HH01 cells. Measuring either the generation of metabolites (Fig. 8) or the disappearance of substrate (Fig. 8, inset), we observe that the rate of metabolism of MC1281 is not significantly different from that of 1-OH-D.


Figure 8: Time course of the metabolism of 1-OH-D and MC1281 in HH01 cells. Cells were incubated with 15 µg of analog for various times between 0 and 18 h. The graph depicts the amount of product (25- and 27-hydroxylated) formed; the inset shows the amount of substrate metabolized versus time. Metabolites were quantitated based on integration of UV peaks at an absorbance of 265 nm. Each point is the average ± S.E. of three incubations.



To confirm that the two metabolites generated from MC1281 in HH01 cells were oxidation products mediated by the cytochrome P450 CYP27, MC1281 was incubated with COS-1 cells transfected with the cDNAs encoding CYP27 and adrenodoxin. Incubation of MC1281 with the transfected COS-1 cells resulted in the generation of two metabolite peaks that were absent in control transfected COS-1 cells. On HPLC, the two putative CYP27-generated products possessed similar retention times to metabolites MC1281-MetA and MC1281-MetB generated in HH01 cells (data not shown).


DISCUSSION

In this paper we have used two in vitro cultured cell models to compare and contrast the substrate preferences of the cytochromes P450 responsible for 25-hydroxylation of vitamin D in the liver and the 23- and 24-hydroxylation of 1,25-(OH)D in vitamin D target cells. Specifically, we have shown that lengthening the side chain of the vitamin D molecule is tolerated quite well in that each enzyme is able to continue to hydroxylate efficiently. However, most interestingly, our results reveal that the two cytochromes P450 must recognize their respective sites of hydroxylation by different mechanisms: the 25-hydroxylase appears to be directed to its terminal hydroxylation site by the distance from the end of side chain, whereas the 23- and 24-hydroxylases appear to be positioned at their hydroxylation site by the distance from the vitamin D nucleus. As a result and in accord with the recent studies of the liver side-chain hydroxylation of a variety of vitamin D compounds with the conventional D and D side chains(16) , 25- and 27-hydroxylation of 24a,24b-dihomo-1-OH-D can be demonstrated to occur in the HH01 cell line. However, using the target cell side-chain hydroxylation system (HPKIA-ras), we continued to observe 23- and 24-hydroxylation of 24a-homo, 24a,24b-dihomo, and 24a,24b,24c-trihomo-1,25-(OH)D derivatives even with the addition of 1-, 2-, and 3-carbon extensions to the side chain. In other words, the 23- and 24-hydroxylases did not move up the side chain to C-24 and C-24a of MC1127 or C-24a and C-24b of MC1147; or C-24b and C-24c of MC1179 as might have been expected if the 23- and 24-hydroxylase enzyme(s) had recognized its (their) site of hydroxylation based upon the distance from the end of the side chain. The experimental basis for these conclusions are well supported by careful and rigorous identification of side-chain-hydroxylated metabolites by HPLC, GC-MS, and chemical derivatization techniques. The identifications are further reinforced by comparisons to a series of 23- and 24-hydroxylated standards chemically synthesized for this purpose. It is clear that the 23- and 24-positions remain the primary hydroxylation sites for all members of the homologated series.

Although the cytochromes P450 involved in these liver and target cell systems are consistent in their site of hydroxylation and are tolerant of small changes in the vitamin D side chain, we did observe some modification in their efficiency. The ratio of 25:27 hydroxylation changes as the side chain is extended. Since these two hydroxylations are carried out by the same cytochrome, CYP27, and since this ratio is altered when other changes are made to the substrate (e.g. using a bile acid precursor with an intact steroidal ring structure or using a vitamin D side chain), this finding is not surprising. Transfection studies reported here lend support to the concept that CYP27 is responsible for most 25- and 27-hydroxylations of steroidal substrates in HH01 liver cells. CYP27 gives a pattern of products similar to that seen in HH01 cells. The efficiency of 23-/24-hydroxylation of homologated 1,25-(OH)D analogs is similarly well maintained despite a greater degree of chain extension. However, at the extreme of C addition to the side chain in 24a-, 24b-, and 24c-trihomo-1,25-(OH)D, we observe reduced 23-hydroxylation of the side chain and a much increased ratio of 24:23-hydroxylation suggesting that this side chain is less well accommodated by the active site of the enzyme or enzymes involved. It is now well established that 24-hydroxylation is carried out in vitamin D target cells by CYP24, a cytochrome P450 distinct from the other families(13) . Not so obvious is whether 23-hydroxylation can be carried out by a separate cytochrome P450 or is another product of CYP24. Early studies of CYP24 did not reveal catalytic properties in addition to 24-hydroxylation(28) . However, more recently, Akiyoshi-Shibata et al.(29) showed that a bacterially expressed CYP24 was active in a reconstitution system and was capable of the generation of 24-oxo metabolites and 24-oxo-23-hydroxylated metabolites as well as 24-hydroxylated metabolites from vitamin D substrates. These same products of CYP24 were previously proposed to lie on a C-24 oxidation pathway purported to function in the catabolism of 1,25-(OH)D to calcitroic acid(23) . In their work on CYP24, Akiyoshi-Shibata et al.(29) did not observe the direct 23-hydroxylation of 25-OH-D or 1,25-(OH)D, although this may be the result of the assay conditions used rather than the fact that CYP24 is incapable of direct 23-hydroxylation of vitamin D substrates. We observed here both the formation of 24-oxo metabolites as well as 24-oxo-23-hydroxylated metabolites from side-chain-extended analogs. Consistent with CYP24 being multifunctional and being responsible for the enzyme activities observed in HPK1A-ras cells here, were the observations that: 1) the 23- and 24-hydroxylase activities are present together; 2) the activities are both vitamin D-inducible; 3) and yet there appears only one mRNA species in Northern blots from such vitamin D-inducible HPK1A-ras cell total RNA when hybridized with fragments or full-length probes of CYP24()suggesting that even if a separate 23-hydroxylase exists it is only distantly related. Thus, in the data reported here, it is not clear if the change in the ratio of 23:24-hydroxylation is a result of differences in the substrate preferences of two different cytochromes P450 or is a result of constraints of the substrate pocket of a single cytochrome with dual properties, namely a CYP24 capable of both 23- and 24-hydroxylation.

These findings have implications for modelling of the substrate binding pocket of the vitamin D-related cytochrome P450 isoforms and for the physiological role of these enzymes. The liver mitochondrial cytochrome P450, CYP27, seems to be a broad specificity enzyme tolerating not only extension of the side chain as we have observed here, but also greatly modified steroidal/vitamin D nucleus (15, 16, 30) and 20-epimerization of the side chain(31) . Taken together, these data suggest that the substrate binding pocket of CYP27 can tolerate a variety of steroidal shapes either by accommodating only the terminal carbons of the side chain or by having a specific cleft for the side chain within a broader pocket for the vitamin D/steroidal ring structure (Fig. 9).


Figure 9: Model for the interaction of the cytochromes P450, CYP27 and CYP24, with their respective substrates. A two-chamber model is proposed which allows for accommodation of the vitamin D nucleus as well as its side chain. The model for CYP27 comprises a large nonspecific pocket for different sterol/secosterol ring structures and a more specific adjoining pocket for cholesterol-type side chains of varying length. The model for CYP24 comprises a smaller, secosterol-specific binding pocket and a longer, open-ended cylindrical chamber to accommodate side chains of varying length. The relative positions of the heme group of each cytochrome is illustrated by the porphyrin ring structure, each coordinating an iron atom.



On the other hand, the HPK1A-ras 23- and 24-hydroxylases (which could be the same enzyme) are specific for vitamin D substrates, but seem to ignore lengthening of the side chain, continuing to insert a hydroxyl group at C-23 or C-24. Although the distance from the C-23 or C-24 to the terminal carbons changes by as much as three carbons in our experiments here, it is worth noting that the distance from C-23 and C-24 to the vitamin D nucleus remains approximately the same. It is thus attractive to hypothesize that the structural feature used by the 24- (and 23-) hydroxylase(s) to locate its hydroxylation site is the distance between the ring structure (possibly the D ring because this is closest) and the side chain carbon. Recently, Reddy et al.(32) reported the lack of 23-hydroxylation of analogs with a 16-ene in the D ring of 1,25-(OH)D implying that the 23-hydroxylase activity is sensitive to such changes. Similarly, we have also shown that another analog 20-epi-1,25-(OH)D is not readily 23-hydroxylated(31) . We conclude that the substrate binding pocket of CYP24 must be cylindrical, permitting the entry of a specific cholesterol-like side chain in addition to recognizing structural features of the D ring and performing hydroxylation at a site (or sites) part way down the cylinder. One would hypothesize that the cylinder must be open at both ends in order to allow entry of the side chain while continuing to facilitate hydroxylation of homologated derivatives with up to three carbon atom extensions. These crude ideas of the shapes of substrate binding pockets are certain to be substantially refined in the foreseeable future as the deduced amino acid structures of CYP24 and CYP27 are now available and recent advances have occurred in the computerized molecular modelling of other related steroidal cytochrome P450 isoforms (33) based upon x-ray crystallographic data obtained from prokaryotic enzymes.

Whereas in this discussion we have concentrated on the investigation of the regioselectivity of the hydroxylating enzymes, the question of their stereoselectivity remains to be addressed. The availability of both 23R- and 23S-hydroxylated standards has enabled us to make the inference that only one of the two isomers is formed in substantial amounts in each case. However, the alternating pattern that begins to emerge (23R-hydroxylation of MC1127, 23S-hydroxylation of MC1147, 23R-hydroxylation of MC1179) is a puzzling result but is reminiscent of the 23S, 24R, 25S pattern pointed out by Ikekawa(7) . Identification of the 24-configuration of the 24-OH metabolites and the 25-configuration of the 27-OH metabolites awaits further work.

This study has firmly established the value of performing metabolic studies of vitamin D analogs in vitro using cultured cells. The convenient incubation of cells with vitamin D analogs permits the isolation of nanogram to microgram quantities of metabolic products which can be rigorously identified with the aid of GC-MS techniques and comparison to chemically synthesized standards.()The experiments involving transfection of cDNAs for vitamin D-related cytochromes performed here is an extension of the cultured cell technology and also allows for the dissection of the cytochrome P450 isoforms involved in these hydroxylations in the cultured cells. With the broadening of this approach to include site-directed mutagenesis of the putative residues involved in the substrate binding pockets, the models built from substrate studies performed here can be tested and refined.

  
Table: 0p4in HPLC conditions: Zorbax-CN (0.46 25 cm) column eluted with HIM 91/7/2 at a flow rate of 2 ml/min.

  
Table: 0p4in HPLC conditions: Zorbax-CN (0.46 25 cm) column eluted with HIM 91/7/2 at a flow rate of 1 ml/min.


FOOTNOTES

*
This work was supported by grants from the Medical Research Council of Canada (to G. J.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed. Tel.: 613-545-2498; Fax: 613-545-2987.

The abbreviations used are: D or D, vitamin D or vitamin D; OH or (OH), hydroxy or dihydroxy, e.g. 1,25-(OH)D = 1,25-dihydroxyvitamin D (note that in the nomenclature used, no distinction between C-26 and C-27 is implied); BSA, bovine serum albumin; DPPD, N,N`-diphenylethylenediamine; HIM, hexane/isopropyl alcohol/methanol; RRT, relative retention time (compared to 1-OH-D); -MEM, -minimal essential medium; HPLC, high performance liquid chromatography.

S. Masuda, V. Byford, R. Kremer, H. L. J. Makin, N. Kubodera, Y. Nishii, T. Okano, T. Kobayashi, and G. Jones, submitted for publication.

F. J. Dilworth and G. Jones, unpublished observations.

D. Prosser and G. Jones, unpublished results.

In fact, recent experiments allowed the generation of sufficient quantities of MC1127-MetA (25 µg) to allow direct comparison with the chemically synthesized 24-hydroxy-MC1127, by one- and two-dimensional proton NMR analysis.


ACKNOWLEDGEMENTS

We acknowledge the important contribution of Dr. Johng Rhim, NCI, National Institutes of Health, Bethesda, MD, in the development of the HPK1A-ras cell line used in some aspects of this work.


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