Production of 22-Hydroxy Metabolites of Vitamin D₃ by Cytochrome P450scc (CYP11A1) and Analysis of Their Biological Activities on Skin Cells

Abstract

Cytochrome P450scc (CYP11A1) can hydroxylate vitamin D₃, producing 20S-hydroxyvitamin D₃ [20(OH)D₃] and 20S,23-dihydroxyvitamin D₃ [20,23(OH)₂D₃] as the major metabolites. These are biologically active, acting as partial vitamin D receptor (VDR) agonists. Minor products include 17-hydroxyvitamin D₃, 17,20-dihydroxyvitamin D₃, and 17,20,23-trihydroxyvitamin D₃. In the current study, we have further analyzed the reaction products from cytochrome P450scc (P450scc) action on vitamin D₃ and have identified two 22-hydroxy derivatives as products, 22-hydroxyvitamin D₃ [22(OH)D₃] and 20S,22-dihydroxyvitamin D₃ [20,22(OH)₂D₃]. The structures of both of these derivatives were determined by NMR. P450scc could convert purified 22(OH)D₃ to 20,22(OH)₂D₃. The 20,22(OH)₂D₃ could also be produced from 20(OH)D₃ and was metabolized to a trihydroxyvitamin D₃ product. We compared the biological activities of these new derivatives with those of 20(OH)D₃, 20,23(OH)₂D₃, and 1α,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃]. 1,25(OH)₂D₃, 20(OH)D₃, 22(OH)D₃, 20,23(OH)₂D₃, and 20,22(OH)₂D₃ significantly inhibited keratinocyte proliferation in a dose-dependent manner. The strongest inducers of involucrin expression (a marker of keratinocyte differentiation) were 20,23(OH)₂D₃, 20,22(OH)₂D₃, 20(OH)D₃, and 1,25(OH)₂D₃, with 22(OH)D₃ having a heterogeneous effect. Little or no stimulation of CYP24 mRNA expression was observed for all the analogs tested except for 1,25(OH)₂D₃. All the compounds stimulated VDR translocation from the cytoplasm to the nucleus with 22(OH)D₃ and 20,22(OH)₂D₃ having less effect than 1,25(OH)₂D₃ and 20(OH)D₃. Thus, we have identified 22(OH)D₃ and 20,22(OH)₂D₃ as products of CYP11A1 action on vitamin D₃ and shown that, like 20(OH)D₃ and 20,23(OH)₂D₃, they are active on keratinocytes via the VDR, however, showing a degree of phenotypic heterogeneity in comparison with other P450scc-derived hydroxy metabolites of vitamin D₃.

Introduction

Cytochrome P450scc catalyzes the three-step cleavage of the side chain of cholesterol to produce pregnenolone as the first enzymatic step of steroid biosynthesis (Tuckey, 2005). The reaction that occurs in mitochondria involves sequential hydroxylation at C22 and C20 and then oxidative cleavage of the bond between C20 and C22. Besides cholesterol, P450scc can also metabolize vitamins D₂ and D₃ and their precursors, ergosterol and 7-dehydrocholesterol, respectively (Guryev et al., 2003; Slominski et al., 2004, 2005a,b, 2006; Tuckey et al., 2008a,b, 2009; Nguyen et al., 2009). Metabolism of 7-dehydrocholesterol and vitamin D₃ has been observed for purified bovine P450scc and also for P450scc in rat adrenal mitochondria (Slominski et al., 2005b, 2009). For vitamin D₃, the favored hydroxylation position is at C20 of the side chain so that 20-hydroxyvitamin D₃ [20(OH)D₃] is the major product of the reaction (Guryev et al., 2003; Slominski et al., 2005b; Tuckey et al., 2008a,b). Our recent chemical synthesis of this compound and a reevaluation of the NMR data for the P450scc-derived product strongly indicate that the product is the S-epimer, 20S(OH)D₃ (Li et al., 2010). As well as C20, P450scc can hydroxylate vitamin D₃ at C17 and C23, potentially producing seven different products. Three of these products, 20(OH)D₃, 20,23-dihydroxyvitamin D₃ [20,23(OH)₂D₃] and 17,20,23-trihydroxyvitamin D₃ [17,20,23(OH)₃D₃] have been purified and their identity established from UV, mass, and NMR spectra (Guryev et al., 2003; Slominski et al., 2005b; Tuckey et al., 2008a). 20,23(OH)₂D₃ was originally designated as 20,22-dihydroxyvitamin D₃ [20,22(OH)₂D₃] (Guryev et al., 2003; Slominski et al., 2005b), but improved NMR procedures resulted in its designation as 20,23(OH)₂D₃ (Tuckey et al., 2008a). Another three products of P450scc action on vitamin D₃, namely 23-hydroxyvitamin D₃ [23(OH)D₃], 17-hydroxyvitamin D₃, and 17,20-dihydroxyvitamin D₃ [17,20(OH)₂D₃] have been purified and tentatively identified from their UV and mass spectra and from their further metabolism by P450scc to known products. For example, 23(OH)D₃ was identified from the evidence that it is a monohydroxy derivative, distinct from 20(OH)D₃, that P450scc converts to a product with an HPLC retention time identical to that of 20,23(OH)₂D₃ (Tuckey et al., 2008a).

The two major products of vitamin D₃ metabolism by P450scc, 20(OH)D₃ and 20,23(OH)₂D₃, have been tested on a range of skin and other cell types, and they have many but not all of the properties of the hormonally active form of vitamin D₃, 1,25(OH)₂D₃, acting as partial receptor agonists (Zbytek et al., 2008; Janjetovic et al., 2009, 2010; Slominski et al., 2010; Tang et al., 2010a). Properties in common with 1,25(OH)₂D₃ on skin cells include inhibition of proliferation and stimulation of differentiation of epidermal keratinocytes, and inhibition of nuclear factor-κB activity via the stimulation of the expression of inhibitor of nuclear factor-κB α. They also inhibit the proliferation and stimulate the differentiation of human and mouse leukemia cells (Slominski et al., 2010). The calcemic toxicity of 20(OH)D₃ was tested in rats, and it did not raise serum calcium at a dose as high as 3 μg/kg, in contrast to the strong calcemic effect seen with an equivalent dose of 1,25(OH)₂D₃ (Slominski et al., 2010). 1,25(OH)₂D₃ is a very strong inducer of the expression of CYP24, the enzyme that catalyzes vitamin D inactivation (Prosser and Jones, 2004; Schuster, 2011). This induction forms part of the tight endocrine control over the levels of 1,25(OH)₂D₃ in the bloodstream. In contrast, both 20(OH)D₃ and 20,23(OH)₂D₃ are poor inducers of CYP24 expression (Zbytek et al., 2008; Janjetovic et al., 2009; Li et al., 2010; Tang et al., 2010a). For example, with human neonatal keratinocytes, 1,25(OH)₂D₃ stimulated CYP24 expression at the mRNA level more than 4000-fold in 24 h, whereas 20,23(OH)₂D₃ was without significant effect (Tang et al., 2010a). The lack of calcemic toxicity and relatively poor induction of CYP24 by the P450scc-derived hydroxyvitamin D₃ compounds makes them attractive candidates for possible therapeutic use in hyperproliferative and inflammatory/autoimmune diseases. Both 20(OH)D₃ and 20,23(OH)₂D₃ are poor substrates for 1α-hydroxylation by CYP27B1 compared with 25-hydroxyvitamin D₃ (Tang et al., 2010a,b).

In the present study, we identified and defined the structure of two additional P450scc-derived vitamin D₃ derivatives, 22-hydroxyvitamin D₃ [22(OH)D₃] and 20,22(OH)₂D₃, and report on their biological activity in epidermal keratinocytes. We also clarify previous reports by our laboratories (Slominski et al., 2005b; Tuckey et al., 2008a) and others (Guryev et al., 2003) on the production of both 22-hydroxyderivatives and 23-hydroxyderivatives by the action of P450scc on vitamin D₃.

Materials and Methods

Materials.

2-Hydroxypropyl-β-cyclodextrin, vitamin D₃, dioleoyl phosphatidylcholine, bovine heart cardiolipin, and NADPH were from Sigma-Aldrich Pty. Ltd. (Sydney, Australia). Other biochemicals and materials used were pGro7 plasmid (Takara Bio Inc., Shiga, Japan), silica gel plates (Alugram Sil G; Macherey-Nagel, Inc., Easton, PA), penicillin-streptomycin-amphotericin B-antibiotic solution (Sigma-Aldrich, St. Louis, MO), [³H]thymidine (Moravek Biochemicals Inc., Brea, CA), Absolutely RNA Miniprep Kit (Stratagene, La Jolla, CA), TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA), and fluorescence mounting medium (Dako, Glostrup, Denmark).

Preparation of Enzymes.

Adrenodoxin was expressed in Escherichia coli and purified as described previously (Woods et al., 1998). Adrenodoxin reductase was expressed in E. coli and purified as described before (Tuckey and Sadleir, 1999), except that E. coli (JM109) containing the pGro7 plasmid (encoding chaperones GroEL/GroES under the control of the araB promoter) were used (Tang et al., 2010a). This markedly increased the expression level from 2 to 200 nM in the culture. Bovine cytochrome P450scc was expressed similarly to our previous report (Headlam et al., 2003) but also with pGro7 present, which doubled the P450scc expression level to approximately 400 nM. The P450scc from a 1-liter culture was extracted from the bacterial membrane fraction as described before (Woods et al., 1998) except that 1% sodium cholate was used. The extract was centrifuged at 107,000g for 60 min to remove insoluble debris, and the supernatant applied to a 6 × 2.5 cm hydroxylapatite column equilibrated with buffer comprising 20 mM potassium phosphate (pH 7.4), 0.1 mM dithiothreitol, 0.1 mM EDTA, and 20% glycerol. The column was washed with 100 ml of the same buffer containing 0.25% sodium cholate and then the P450scc was eluted by including 500 mM potassium phosphate in the wash buffer. The P450scc was dialyzed against 1 liter of 20 mM potassium phosphate (pH 7.4), 0.1 mM dithiothreitol, 0.1 mM EDTA, and 20% glycerol, concentrated to 40 μM and stored at −80°C until use. This scheme produced a large amount of partially pure enzyme with high activity (k_cat = 100 mol of pregnenolone/min/mol P450scc with cholesterol as substrate) and was used for the large-scale synthesis of vitamin D₃ derivatives. Highly purified P450scc was used for small-scale metabolic and kinetic studies and was isolated from bovine adrenal glands in a procedure involving extraction of the enzyme from a mitochondrial fraction with sodium cholate and then purification by ammonium sulfate fractionation and octyl Sepharose chromatography (Tuckey and Stevenson, 1984).

Large-Scale Incubations of Vitamin D₃ with Cytochrome P450scc and Preliminary Purification of Products by TLC.

Stock vitamin D₃ was prepared by dissolving it in 45% 2-hydroxypropyl-β-cyclodextrin (cyclodextrin) to a final concentration of 10 mM, by stirring in the dark for 3 days at room temperature (De Caprio et al., 1992; Tuckey et al., 2008b). Incubations (12.5 ml) were performed in buffer comprising 20 mM HEPES (pH 7.4), 100 mM NaCl, 0.1 mM dithiothreitol, 0.1 mM EDTA, 2 μM bovine cytochrome P450scc, 15 μM adrenodoxin, 0.3 μM adrenodoxin reductase, 2 mM glucose 6-phosphate, 2 U/ml glucose-6-phosphate dehydrogenase, and 50 μM NADPH. Stock vitamin D₃ in cyclodextrin (0.25 ml) was added to the incubation mixture to give a final vitamin D₃ concentration of 200 μM and a cyclodextrin concentration of 0.9%. Samples were preincubated for 8 min, reactions were started by the addition of NADPH, and incubations were performed for 3 h at 37°C with shaking. Reactions were stopped by the addition of 20 ml of ice-cold dichloromethane. After shaking and centrifugation, the lower phase was retained, and the upper aqueous phase was extracted another three times with 20 ml of dichloromethane. The combined dichloromethane extracts were dried under nitrogen at 30°C, and the combined sample from two incubations was applied as a band to a 20 cm × 20 cm × 0.2 mm Silica Gel 60 plate using three 300-μl aliquots of chloroform. Authentic standards and 1% of the extract were run separately as spots on either side of the plate, using a procedure similar to that described before (Slominski et al., 2005b). Plates were developed three times in hexane-ethyl acetate (3:1, v/v), and sections of the plate containing standards and the 1% of the reaction mixture were removed, sprayed with a solution of 2 mM FeSO₄ containing 5% concentrated sulfuric acid and 5% glacial acetic acid, and then charred by heating to reveal the position of standards and the major products from the reaction. These strips were then aligned with the remainder of the plate, and the positions of the major products in the unstained section of the plate were marked (Fig. 1). These areas were removed, and the products were eluted from the silica gel with three 15-ml aliquots of CHCl₃-CH₃OH (1:1, v/v). The solvent was removed under nitrogen at 30°C, and samples were dissolved in 4 ml of methanol and filtered through a 0.1-μm filter to remove any remaining silica particles. The solvent was again removed under nitrogen gas, and the samples were dissolved in the solvent required for HPLC (see below).

Fig. 1.

Thin-layer chromatogram illustrating the metabolism of vitamin D₃ by cytochrome P450scc. Vitamin D₃ (100 μM) in cyclodextrin (0.9%) was incubated with 2 μM bovine P450scc for 3 h at 37°C in medium containing adrenodoxin reductase, adrenodoxin, and NADPH. The dichloromethane extract was subjected to TLC as described under Materials and Methods. Lane A, control incubation with adrenodoxin excluded; lane B, test incubation.

HPLC Purification of Hydroxyvitamin D₃ Products.

Initial HPLC purifications were performed using an HPLC system (PerkinElmer Life and Analytical Sciences, Waltham, MA) equipped with a UV monitor set at 265 nm. The filtered extract from the TLC plate containing the 22(OH)D₃ was chromatographed on a C18 column (GraceSmart, 15 cm × 4.6 mm, particle size 5 μm) using an isocratic mobile phase of 76% methanol in water at a flow rate of 0.5 ml/min. TLC-purified 20(OH)D₃ was further purified using a Brownlee Aquapore column (25 cm × 10 mm, particle size 20 μm) with an isocratic mobile phase of 85% methanol in water at a flow rate of 1.5 ml/min. 20,23(OH)₂D₃ was purified on the same column using an isocratic mobile phase of 80% methanol in water. The purified product contained some 20,22(OH)₂D₃ (see Results). The 20,22(OH)₂D₃ and 20,23(OH)₂D₃ were separated using an Agilent 1200 system and a Luna C18 column (15 cm × 4.6 mm, particle size 3 μm; Phenomenex, Torrance, CA). An isocratic mobile phase of 45% acetonitrile in water was used at a flow rate of 1 ml/min. Initial NMR analysis showed that the resulting 20,22(OH)₂D₃ sample contained impurities. These were removed by HPLC using an Alure Biphenyl column (25 cm × 4.5 mm) with isocratic 75% methanol in water for 2 min followed by a linear gradient to 97% methanol in water over 25 min and then 97% methanol for 20 min, at a flow rate of 1 ml/min.

NMR Spectroscopy.

NMR measurements were performed using an inverse triple-resonance 3-mm probe on a Varian Unity INOVA 500 MHz spectrometer (Agilent Technologies Inc., Santa Clara, CA) or using a 1.7-mm cryogenic probe on a Bruker 600-MHz spectrometer (Bruker Biospin, Billerica, MA). Samples were dissolved in CD₃OD and transferred to 3-mm NMR tubes (Shigemi Inc., Allison Park, PA) or 1.7-mm NMR tubes. Temperature was regulated at 22°C and was controlled with an accuracy of ±0.1°C. Chemical shifts were referenced to residual solvent peaks for CD₃OD (3.31 ppm for proton and 49.15 ppm for carbon). Standard two-dimensional NMR experiments [¹H-¹H correlation spectroscopy (COSY), ¹H-¹H total correlation spectroscopy (TOCSY), ¹H-¹³C heteronuclear single quantum correlation spectroscopy (HSQC), and ¹H-¹³C heteronuclear multiple bond correlation spectroscopy (HMBC)] were performed to fully elucidate the structures of the metabolites. All data were processed using ACD software (Advanced Chemistry Development, Toronto, ON, Canada) with zero-filling in the direct dimension and linear prediction in the indirect dimension.

Small-Scale Incubations of Hydroxyvitamin D₃ with Cytochrome P450scc to Determine Kinetic Constants.

Vesicles were prepared from dioleoyl phosphatidylcholine and bovine heart cardiolipin in the ratio 85:15 (moles per mole). Hydroxyvitamin D₃ substrates were added to the phospholipid as required (see Results). Buffer comprising 20 mM HEPES (pH 7.4), 100 mM NaCl, 0.1 mM dithiothreitol, and 0.1 mM EDTA was added to 1.25 μmol of phospholipid, and the mixture was sonicated for 10 min in a bath-type sonicator (Tuckey and Kamin, 1982). Purified bovine adrenal P450scc was added to the vesicles, and incubations were performed at 37°C for 3 min as described in detail before (Tuckey et al., 2008b). HPLC analysis of products and calculation of kinetic constants were also performed as described before (Tuckey et al., 2008b).

Cell Proliferation Assay.

Immortalized human epidermal keratinocytes (HaCaT) were grown in Dulbecco's modified Eagle's medium containing 5% fetal bovine serum and 1% penicillin-streptomycin-amphotericin B antibiotic solution at 37°C. Confluent cells were detached with trypsin and collected by centrifugation. Cells were seeded in 24-well plates and treated with vitamin D₃ derivatives (or ethanol for controls) diluted in Dulbecco's modified Eagle's medium media supplemented with 5% charcoal-stripped serum, 500 μl/well, at concentrations ranging from 0.1 to 100 nM. After 48 or 72 h of incubation, [³H]thymidine at a concentration of 1 μCi/ml was added to the cells and incubated for 4 h. Cells were harvested, and [³H]thymidine incorporation into the DNA was measured as before (Janjetovic et al., 2010).

Measurement of Involucrin Expression.

HaCaT cells were plated in chamber slides and hydroxy derivatives of vitamin D₃ were added at a final concentration of 100 nM. After 24 h of culture at 37°C, cells were fixed and treated with anti-involucrin antibody followed by secondary antibody (nucleolin-fluorescein isothiocyanate-anti-mouse) as described before (Slominski et al., 2011). Cell nuclei were stained with propidium iodide, and cells were viewed with a fluorescent microscope and images taken under 20× magnification. Images were analyzed for the number of green cells per area and the intensity of immunofluorescence per area using ImageJ Launcher (http://rsb.info.nih.gov/ij/).

Measurement of CYP24 and VDR mRNA Levels.

HaCaT keratinocytes were treated with the hydroxyvitamin D₃ derivatives at a final concentration of 100 nM. After 24 h of culture, cells were collected and subjected to RNA isolation using an Absolutely RNA Miniprep Kit. The RNA was reverse-transcribed, and real-time PCR was performed using TaqMan Universal PCR Master Mix and primers, as described previously (Janjetovic et al., 2010). The data were collected with a Roche Light Cycler 480, and signals were normalized by the comparative C_t method, using β-actin as a housekeeping gene.

VDR Translocation.

To measure VDR translocation to the nucleus induced by hydroxyvitamin D₃ derivatives, SKMEL-188 cells transfected with pLenti-CMV-VDR-EGFP-pgk-puro (VDR and enhanced green fluorescent protein expressed as a fusion protein) (Slominski et al., 2011) were incubated with the derivatives at concentrations ranging from 10⁻¹⁰ to 10⁻⁷ M, overnight. Cells were then fixed with 4% paraformaldehyde, mounted with fluorescence mounting medium, and analyzed with a fluorescent microscope. At least six pictures were taken from different fields, and the cells containing fluorescent nuclei were counted. Data are presented as a percentage of cells with nuclear staining relative to the total cell number.

Other Procedures.

The concentration of cytochrome P450scc was determined from the CO-reduced minus reduced difference spectrum using an extinction coefficient of 91,000 M⁻¹ cm⁻¹ for the absorbance difference between 450 and 490 nm (Omura and Sato, 1964). The concentrations of vitamin D₃ and its hydroxy derivatives were quantitated using an extinction coefficient of 18,000 M⁻¹ cm⁻¹ at 263 nm (Hiwatashi et al., 1982). Mass spectra of 22(OH)D₃, 20,22(OH)₂D₃, and 20,23(OH)₂D₃ were acquired in a Bruker Esquire-LC/MS system (Bruker Daltonics, Billerica, MA) using the ionization source of electrospray ionization. Data were collected and processed by ACD mass processor. The mass spectrum of the product of P450scc action on 20,22(OH)₂D₃ was acquired using a Waters LCT Premier XE LC/MS system (Waters, Milford, MA) using atmospheric pressure chemical ionization.

Results

Identification of Two Products Present in 20,23(OH)₂D₃ Samples.

Figure 1 shows the typical TLC profile of the reaction products from a large-scale reaction of vitamin D₃ with P450scc. Products were identified from authentic standards analyzed previously by NMR (Tuckey et al., 2008a) or in the case of 22(OH)D₃ from the NMR performed in this study (see below). The corresponding HPLC profile of the reaction mixture run with a methanol-water mobile phase is shown in Fig. 2A. When the profile was determined using an acetonitrile-water mobile phase, it was noted that the peak corresponding to 20,23(OH)₂D₃ was partially split into two. Baseline separation of these two peaks was obtained when a column with higher resolving power was used with an acetonitrile-water mobile phase (Fig. 2B). Our standard 20,23(OH)₂D₃, purified by TLC and isocratic HPLC on a C18 column using a mobile phase of 85% methanol in water and previously identified by NMR was similarly split into two peaks in this HPLC system (Fig. 2B, inset). UV analysis revealed that both products had the same UV spectrum with the expected peak at 264 nm, characteristic of the vitamin D₃ chromophore. Both compounds were identified as dihydroxy derivatives of vitamin D₃ from their electrospray mass spectra, which both showed the abundant ion at m/z = 439 (416 + 23), corresponding to the mass of dihydroxyvitamin D₃ complexed to Na⁺ (Supplemental Fig. 1, A and B). Subsequent NMR analysis (see below) showed that the major peak was, as expected, 20,23(OH)₂D₃, and the minor peak was 20,22(OH)₂D₃. The 20,23(OH)₂D₃ represented 75% of the sample and the smaller peak corresponding to 20,22(OH)₂D₃ represented 25%.

Fig. 2.

HPLC analysis of the products of vitamin D₃ metabolism by cytochrome P450scc. Incubations were carried out as described in the legend to Fig. 1. A, HPLC was performed on a C18 GraceSmart column (15 cm × 4.6 mm, particle size 5 μm). The samples were applied in 64% methanol and eluted with a 64 to 100% methanol gradient in water for 15 min and then 100% methanol for 30 min, all at a flow rate of 0.5 ml/min. B, HPLC was performed with an Grace Alltima C18 column (25 cm × 4.6 mm, particle size 5 μm). The mobile phase was 55 to 100% acetonitrile in water for 15 min at 0.5 ml/min and 100% acetonitrile for 30 min at 0.5 ml/min followed by100% acetonitrile at 1.5 ml/min for 20 min. Inset, separation of standard 20,23(OH)₂D₃ into two peaks under similar HPLC conditions (see text for identifications).

NMR Analysis of 20,22(OH)₂D₃ and 20,23(OH)₂D₃.

The two compounds present in the 20,23(OH)₂D₃ samples prepared by isocratic HPLC using methanol-water were separated using 45% acetonitrile under isocratic conditions (see Materials and Methods). This resulted in 104 nmol of 20,22(OH)₂D₃ and 125 nmol of 20,23(OH)₂D₃ being available for NMR. The proton NMR spectrum of 20,22(OH)₂D₃ is shown in Fig. 3A. The sites of hydroxylation were confirmed to be at 20- and 22-positions based on the analysis of two-dimensional NMR spectra for this metabolite (Fig. 3, B–E). We first confirmed the hydroxylation site at the 20-position; the doublet of 21-CH₃ in vitamin D₃ became a singlet in the metabolite (¹H at 1.19 ppm, ¹³C at 20.8 ppm) (Fig. 3B, projection), indicating the loss of scalar coupling from 20-CH. All four methyl groups (18, 21, 26, and 27) were intact (Fig. 3B). ¹H-¹H COSY indicates that scalar coupling between 21-CH₃ and 20-CH does not exist (Fig. 3C). By taking together all these changes, the presence of a 20-OH group in the metabolite is established. The assignment of the second hydroxylation at the 22-position is mainly based on ¹H-¹³C HSQC and ¹H-¹³C HMBC. ¹H-NMR and ¹H-¹³C HSQC revealed the presence of a methine (CH) peak at 3.32 ppm (¹³C at 77.7 ppm) (Fig. 3B, inset). ¹H-¹H TOCSY indicated that this methine (¹H at 3.32 ppm) is in the same spin system as 26/27-CH₃ (¹H at 0.93 ppm) (Fig. 3E), indicating the hydroxylation is in the side chain. ¹H-¹³C HMBC showed that 21-CH₃ (1.19 ppm) has correlations with the 17-CH (¹³C at 56.6 ppm), 20-C (¹³C at 77.6 ppm), and 22-C (¹³C at 77.7 ppm) (Fig. 3D). The coincident overlaps of 20-C (¹³C at 77.6 ppm) and 22-C (¹³C at 77.7 ppm) were confirmed by the ¹H-¹³C HMBC correlation from the 17-CH (¹H at 1.74 ppm) to 20-C (¹³C at 77.6 ppm) (Fig. 3D, inset). From all these correlations taken together, the second hydroxylation site was assigned to be at C22 and thus gave the 20,22(OH)₂D₃. The full assignments for this metabolite are summarized in Table 1 and expanded NMR spectra are shown in Supplemental Fig. 1D.

Fig. 3.

NMR spectra of 20,22(OH)₂D₃. A, one-dimensional proton. B, ¹H-¹³C HSQC. C, ¹H-¹H COSY. D, ¹H-¹³C HMBC. E, ¹H-¹H TOCSY. Full spectra are shown in supplemental data.

TABLE 1.

NMR assignments for 22(OH)D₃, 20,23(OH)₂D₃, and 20,22 (OH)₂D₃

Atom	22(OH)D3		20,23(OH)2D3		20,22(OH)2D3
	1H	13C	1H	13C	1H	13C
1	2.11α, 2.41β	33.4	2.12α, 2.41β	33.4	2.14α, 2.42β	33.5
2	1.98α, 1.53β	36.4	1.97α, 1.54β	36.5	1.98α, 1.55β	36.5
3	3.76	70.4	3.76	70.5	3.80	70.4
4	2.54α, 2.20β	46.9	2.54α, 2.20β	46.8	2.54α, 2.21β	47.0
5	N.A.	*N.A.	N.A.	*N.A.	N.A.	137.2
6	6.23	122.4	6.22	122.5	6.23	122.5
7	6.04	118.8	6.03	119.3	6.03	119.3
8	N.A.	*N.A.	N.A.	147.2	N.A.	147.4
9	1.71α, 2.87β	29.8	1.68α, 2.85β	29.7	1.74α, 2.87β	29.8
10	N.A.	*N.A.	N.A.	*N.A.	N.A.	147.1
11	1.56α, 1.68β	24.4	1.54α, 1.67β	24.2	1.59α, 1.69β	24.3
12	1.37α, 2.03β	41.7	1.46α, 2.19β	41.0	1.41α, 2.14β	42.1
13	N.A.	46.9	N.A.	47.1	N.A.	47.4
14	2.02	57.0	2.01	57.7	2.02	57.5
15	1.50	23.1	1.50	22.7	1.52	23.0
16	1.38α, 1.79β	27.8	1.32α, 1.80β	23.1	1.60α, 1.90β	22.3
17	1.35	54.9	1.65	62.1	1.74	56.6
18	0.57	12.1	0.70	14.1	0.75	13.8
19	4.75, 5.04	112.5	4.75, 5.04	112.5	4.77, 5.06	112.5
20	1.67	43.8	N.A.	77.0	N.A.	77.6
21	0.92	23.2	1.37	26.3	1.19	20.8
22	3.57	74.5	1.64, 1.47	48.1	3.32	77.7
23	1.38,1.21	28.1	4.03	68.0	1.52, 1.22	30.2
24	1.46, 1.17	37.2	1.40, 1.16	48.1	1.42, 1.22	37.4
25	1.54	29.1	1.74	25.2	1.58	29.0
26, 27	0.91	22.8	0.93	23.5	0.93	23.2

N.A., not applicable (ternary carbons);

^* N.A., not available because of low resolution.

The proton NMR spectrum of 20,23(OH)₂D₃ is shown in Fig. 4A. Confirmation of the hydroxylation site at C20 followed exactly the same rationale as that for the identification of the 20-OH in 20,22(OH)₂D₃ described above. The determination of 23-OH was established according to the following analysis. First, ¹H-¹³C HSQC indicated that all four methyl groups (18, 21, 26, and 27) (Fig. 4B) are intact and the presence of a methine peak at 4.03 ppm implied that this methine is connected with a hydroxy group (Fig. 4C). Second, from ¹H-¹H TOCSY, the methine at 4.03 ppm has correlations to 26/27-CH₃ (¹H at 0.93 ppm), suggesting that this methine is in the same spin system as 26/27-CH₃ (Fig. 4D), and thus the hydroxylation occurred in the side chain. Third, ¹H-¹³C HMBC showed the correlations from 21-CH₃ (¹H at 1.37 ppm) to 17-CH (¹³C at 62.1 ppm), 20-C (¹³C at 77.0 ppm), and 22-C (¹³C at 48.1 ppm) (Fig. 4E); meanwhile, the correlations from 26/27-CH₃ (¹H at 0.93 ppm) to 25-CH (¹³C at 25.2 ppm) and 24-CH₂ (¹³C at 48.1 ppm) can also be identified. Thus, the only position that is left unassigned in the side chain is position 23. From all these correlations, this metabolite can be unambiguously assigned as 20,23(OH)₂D₃. The full assignments for this compound are summarized in Table 1, and expanded NMR spectra are shown in Supplemental Fig. 1D. It should be noted that we have very strong evidence that the 20-OH group in 20(OH)D₃ is the S-isomer (Li et al., 2010). Because, as shown below, both 20,22(OH)₂D₃ and 20,22(OH)₂D₃ can be made by P450scc action on 20S(OH)D₃, the above two dihydroxyvitamin D₃ compounds can be designated as 20S,22(OH)₂D₃ and 20S,23(OH)₂D₃.

Fig. 4.

NMR spectra of 20,23(OH)₂D₃. A, one-dimensional proton. B, ¹H-¹³C HSQC. C, ¹H-¹H COSY. D, ¹H-¹³C HMBC. E, ¹H-¹H TOCSY. Full spectra are shown in supplemental data.

Identification of 22-Hydroxyvitamin D₃ as a Product of P450scc Action on Vitamin D₃.

The vitamin D₃ spot running just below the major 20(OH)D₃ product by TLC [now identified as 22(OH)D₃] (Fig. 1) was previously tentatively identified as 23(OH)D₃ (Tuckey et al., 2008a). That identification was based on its conversion by P450scc to a product with a retention time identical to that of 20,23(OH)₂D₃ when subjected to HPLC with a methanol-water gradient, as in Fig. 1. From 16 large-scale incubations (12.5 ml each) of vitamin D₃ with P450scc, performed in part to produce 20(OH)D₃ and 20,23(OH)₂D₃ for biological testing, we were able to purify 125 nmol of 22(OH)D₃ for identification by NMR. Electrospray mass spectrometry of this compound showed the predominant ion at m/z 423 (400 + 23), corresponding to the mass of monohydroxyvitamin D₃ complexed to Na⁺ (Supplemental Fig. 1C). This finding is consistent with our previous designation of this compound as a monohydroxy derivative of vitamin D₃ (Tuckey et al., 2008a).

The proton NMR spectrum of 22(OH)D₃ is shown in Fig. 5A. For 22(OH)D₃, the site of hydroxylation was determined on the basis of two-dimensional NMR spectra. ¹H-¹³C HSQC indicated that 20-CH (¹H at 1.67 ppm and ¹³C at 43.8 ppm) (Fig. 5B, inset) and all four methyl groups (18, 21, 26, and 27) (Fig. 5B) are intact. The presence of a methine peak at 3.57 ppm (¹³C at 74.5 ppm) can be identified easily (Fig. 5C), suggesting that the hydroxylation occurred at this methine position. ¹H-¹H TOCSY shows that this methine is in the same spin system as 26/27-CH₃ (¹H at 0.91 ppm) (Fig. 5D), indicating that the hydroxylation is in the side chain. ¹H-¹³C HMBC revealed the correlation from 21-CH₃ (¹H at 0.92 ppm) to 17-CH (¹³C at 54.9 ppm), 20-CH (¹³C at 43.8 ppm), and 22-C (¹³C at 74.5 ppm) (Fig. 5E). Collectively, the above analysis unambiguously assigned the hydroxylation site to the 22-position and thus gives 22(OH)D₃. The full assignments of this compound are summarized in Table 1.

Fig. 5.

NMR spectra of 22(OH)D₃. A, one-dimensional proton. B, ¹H-¹³C HSQC. C, ¹H-¹H COSY. D, ¹H-¹³C HMBC. E, ¹H-¹H TOCSY. Full spectra are shown in supplemental data.

20,22(OH)₂D₃ Can Be Produced from Both 22(OH)D₃ and 20(OH)D₃.

When 22(OH)D₃ incorporated into phospholipid vesicles was incubated with cytochrome P450scc, it was converted to 20,22(OH)₂D₃ (Fig. 6A). This product was identified from its retention time, which was identical to that of the authentic standard under the HPLC conditions listed for Fig. 6, A and B (data not shown). There were also two unidentified products with retention times slightly longer than that for the substrate (Fig. 6A). Incubation of 20(OH)D₃ with P450scc resulted in the formation of three dihydroxyvitamin D₃ products, 17,20(OH)₂D₃, 20,22(OH)₂D₃, and 20,23(OH)₂D₃, as well as minor products, which included 17,20,23(OH)₃D₃ (Fig. 6B). Table 2 lists the kinetic constants for 20-hydroxylation of 22(OH)D₃ and for 17-hydroxylation, 22-hydroxylation, and 23-hydroxylation of 20(OH)D₃. K_m values for metabolism of 20(OH)D₃ were reasonably similar, regardless of the hydroxylation site, consistent with these values reflecting the strength of 20(OH)D₃ binding to the P450scc. The k_cat value for 23-hydroxylation of 20(OH)D₃ was 3.2-fold higher than the k_cat for 22-hydroxylation, explaining the relative proportion of products (approximately 3:1) seen in large-scale 3-h incubations of P450scc with vitamin D₃ (Fig. 2). The k_cat values for hydroxylation of 20(OH)D₃ at C17 and C22 were similar. It should be noted that the kinetic data in Table 2 do not take into account the further metabolism of a small amount of the dihydroxy products [for example, to form 17,20,23(OH)₃D₃] in the 3-min reaction time used, and, therefore, k_cat values are slightly underestimated. The k_cat for 20-hydroxylation of 22(OH)D₃ was higher than that seen for 22-hydroxylation of 20(OH)D₃, consistent with 20-hydroxylation being favored whether the substrate is vitamin D₃ (Tuckey et al., 2008b) or 22(OH)D₃.

Fig. 6.

Production of 20,22(OH)₂D₃ from both 22(OH)D₃ and 20(OH)D₃. Substrates were incorporated into phospholipids vesicles at a molar ratio of secosteroid to phospholipid of 0.05 and incubated with 2 μM P450scc for 3 min. A, reaction of 22(OH)D₃. Products were separated on a 15-cm GraceSmart C18 column with a gradient of 64 to 100% methanol in water for 15 min and then 100% methanol for 20 min at 0.5 ml/min. B, reaction with 20(OH)D₃. Products were separated on a 25-cm Grace Alltima C18 column with a gradient of 45 to 100% acetonitrile in water for 40 min and then 100% acetonitrile for 20 min at 0.5 ml/min. Insets show control reactions for which adrenodoxin was omitted. (OH)₄D₃, tetrahydroxyvitamin D₃.

TABLE 2.

Kinetic parameters for hydroxylation of 22(OH)D₃ and 20(OH)D₃ by P450scc

Kinetic constants were determined for substrates incorporated into phospholipid vesicles prepared from dioleoyl phosphatidylcholine and cardiolipin. Substrate concentrations are expressed as moles of substrate per mole of phospholipid (PL). Values for K_m and k_cat are means ± S.E. from the hyperbolic curve fitted to the data by least-squares nonlinear regression using KaleidaGraph 4.1.

Substrate	Hydroxylation Site	Km	kcat
		mol/mol PL	min−1
22(OH)D3	C20	0.15 ± 0.08	2.09 ± 0.90
20(OH)D3	C17	0.27 ± 0.04	0.47 ± 0.04
20(OH)D3	C22	0.16 ± 0.01	0.43 ± 0.01
20(OH)D3	C23	0.25 ± 0.01	1.55 ± 0.06

The K_m values for hydroxylation of both 22(OH)D₃ and 20(OH)D₃ are considerably lower than those for hydroxylation of vitamin D₃ (Tuckey et al., 2008b). This finding is consistent with the data for metabolism of cholesterol by P450scc in which the 20- and 22-hydroxycholesterol intermediates bind much more tightly to the active site of the enzyme and display lower K_m values than those seen for cholesterol (Lambeth et al., 1982; Tuckey and Cameron, 1993a,b). The crystal structure of P450scc bound to 22R-hydroxycholesterol indicates direct coordination of the 22-OH group to the heme iron (Mast et al., 2011; Strushkevich et al., 2011).

20,22(OH)₂D₃ Is Slowly Metabolized to a More Polar Derivative.

We have previously reported that the original 20,22(OH)₂D₃/20,23(OH)₂D₃ mixture obtained by TLC and isocratic HPLC purification using 85% methanol in water is slowly metabolized by P450scc to a major metabolite identified as 17,20,23(OH)₃D₃ (Tuckey et al., 2008a,b). We therefore tested the ability of P450scc to further metabolize pure 20,22(OH)₂D₃ and 20,23(OH)₂D₃. 20,23(OH)₂D₃ was metabolized to the expected 17,20,23(OH)₃D₃ product, identified from its identical retention time to authentic standard (Fig. 7B). 20,22(OH)₂D₃ was slowly converted to a major product with a retention time longer that of 17,20,23(OH)₃D₃ (Fig. 7). Analysis of this product by HPLC/mass spectrometry with chemical ionization gave the parent ion (M + 1)⁺ at m/z = 433.3 and major ions with m/z values of 415.3, 397.3, and 379.3, corresponding to the loss of one, two, and three water molecules, respectively. Thus, the product is trihydroxyvitamin D₃.

Fig. 7.

Metabolism of 20,22(OH)₂D₃ and 20,23(OH)₂D₃ by P450scc. Substrates were incorporated into phospholipid vesicles at a molar ratio of secosteroid to phospholipid of 0.05 and incubated with 2 μM P450scc for 30 min. Products were separated on a 25-cm Grace Alltima C18 column with a mobile phase comprising 45 to 100% acetonitrile in water for 40 min at 0.5 ml/min and then 100% acetonitrile for 20 min at 0.5 ml/min. A, reaction with 20,22(OH)₂D₃. B, reaction with 20,23(OH)₂D₃. Insets show control reactions for which adrenodoxin was omitted.

Effects of 22-Hydroxyvitamin D₃ Derivatives on Keratinocytes.

We compared the biological activities of these new enzymatically produced 22-hydroxy derivatives of vitamin D₃ with those of 20(OH)D₃ and 20,23(OH)₂D₃ as well as with those of the 25(OH)D₃ and 1,25(OH)₂D₃ controls. After 48 h of culture, only 1,25(OH)₂D₃, 20(OH)D₃, and 20,23(OH)₂D₃ significantly inhibited keratinocyte proliferation as evaluated by one-way ANOVA (data not shown). After 72 h, a significant inhibitory effect was seen not only for 1,25(OH)₂D₃, 20(OH)D₃, and 20,23(OH)₂D₃ but also for 22(OH)D₃ and 20,22(OH)₂D₃ (Fig. 8). 25(OH)D₃ which requires activation via 1α-hydroxylation by CYP27B1, showed a similar trend, which was not significant (p > 0.05) as evaluated by one-way ANOVA (Fig. 8).

Fig. 8.

The effects of P450scc-generated hydroxyderivatives of vitamin D₃ on HaCaT keratinocyte proliferation. HaCaT keratinocytes were incubated with the hydroxy derivatives of vitamin D₃ for 72 h and [³H]thymidine incorporation into DNA was measured as described under Materials and Methods. The values represent means ± S.D. (n = 4). Data were analyzed with GraphPad Prism 4 software using one-way ANOVA with a Dunnett post-test. Statistical significance: *, p < 0.05; **, p < 0.01.

Next, we compared the ability of 22-hydroxyvitamin D₃ derivatives and the other vitamin D₃ compounds to stimulate keratinocyte differentiation. As assessed from the number of cells expressing the differentiation marker, involucrin (Fig. 9A) and from the intensity of the total cellular immunofluorescent staining (Fig. 9B), the strongest inducers of differentiation were 20(OH)D₃, 20,22(OH)₂D₃, 20,23(OH)₂D₃, and 1,25(OH)₂D₃. 22(OH)D₃ showed a weaker effect (Fig. 9A) or no effect (Fig. 9B).

Fig. 9.

The effects of P450scc-generated hydroxy derivatives of vitamin D₃ on involucrin expression. HaCaT cells were treated with the hydroxy derivatives (100 nM) and involucrin expression was measured by immunofluorescent staining. Results are expressed as the number of involucrin-positive cells (A) and the intensity or immunofluorescent (IF) staining (B). Data were analyzed using GraphPad Prism and Student's t test. Statistical significance: *, p < 0.05; **, p < 0.01; ***, p < 0.001. Data represent means ± S.D. (n = 4).

All of the compounds tested, including 22(OH)D₃ and 20,22(OH)₂D₃, significantly increased the expression of the vitamin D receptor at the mRNA level (Fig. 10A), with the largest stimulation seen for 1,25(OH)₂D₃. The expression of mRNA for the vitamin D-inactivating enzyme, CYP24, was dramatically stimulated by 1,25(OH)₂D₃ and only weakly stimulated by 20(OH)D₃ (Fig. 10B). The other hydroxy derivatives, including 22(OH)D₃ and 20,22(OH)₂D₃, were without effect.

Fig. 10.

The effects of P450scc-generated hydroxy derivatives of vitamin D₃ on VDR and CYP24 expression. HaCaT keratinocytes were treated with the hydroxyvitamin D₃ derivatives for 24 h before RNA extraction and real-time RT-PCR to measure VDR mRNA (A) and CYP24 (B) mRNA levels. Data represent means ± S.D. (n = 4), and they were analyzed using GraphPad Prism and Student's t test. Statistical significance: *, p < 0.05; **, p < 0.01; ***, p < 0.001. The difference between 1,25(OH)₂D₃-treated cells and cells treated with other compounds is p < 0.001.

We investigated the ability of the P450scc-derived hydroxyvitamin D₃ compounds to induce VDR translocation from the cytoplasm to nucleus (Fig. 11), using the VDR-green fluorescent protein construct described previously (Slominski et al., 2011). The most optimal concentration of the ligand to use for comparative studies was established to be 10⁻⁷ M using 1,25(OH)₂D₃ (Fig. 11A). Although all the compounds stimulated VDR translocation from the cytoplasm to the nucleus (Fig. 11C), we found that 22(OH)D₃ and 20,22(OH)₂D₃ caused significantly less translocation than 1,25(OH)₂D₃ and 20(OH)D₃ (Fig. 11B). There was no significant difference in effects between 1,25(OH)₂D₃ and 20,23(OH)₂D₃.

Fig. 11.

The effects of P450scc-generated hydroxy derivatives of vitamin D₃ on the translocation of VDR from the cytoplasm to the nucleus. A, dose-response curve for 1,25(OH)₂D₃. B, stimulation of translocation by 10⁻⁷ M 20(OH)D₃, 22(OH)D₃, 20,23(OH)₂D₃, and 20,22(OH)₂D₃ compared with 1,25(OH)₂D₃. C, fluorescent nuclei in representative translocation studies. Data are presented as means ± S.D. (n ≥ 6). The differences between groups were analyzed with Student's t test: *. p < 0.05; **, p < 0.01; ***, p < 0.001. EtOH, ethanol.

Discussion

In the present study, we clearly show that C22 is a site of vitamin D₃ hydroxylation by cytochrome P450scc with the identification of both 22(OH)D₃ and 20,22(OH)₂D₃ as reaction products. We have clarified past confusion over the identity of the major dihydroxyvitamin D₃ product, which was initially designated as 20,22(OH)₂D₃ by NMR analysis (Guryev et al., 2003; Slominski et al., 2005b), but subsequently corrected to 20,23(OH)₂D₃ when increased amounts of the product were available for NMR and the NMR conditions were improved (Tuckey et al., 2008a). We now show that the preparation used in those studies was a 3:1 mixture of 20,23(OH)₂D₃ and 20,22(OH)₂D₃, with both silica gel TLC and reverse-phase HPLC with an isocratic mobile phase of methanol in water being unable to resolve these two compounds. Separation was achieved with a reverse-phase HPLC system using acetonitrile in water as the mobile phase, enabling us to purify the individual compounds for NMR analysis, providing unequivocal identification.

Consistent with our identification of 20,22(OH)₂D₃ as a product of vitamin D₃ metabolism by P450scc, we have also identified 22(OH)D₃ as one of the monohydroxy products. This product had previously been tentatively identified as 23(OH)D₃ (Tuckey et al., 2008a). Enzymatic production of more of this derivative enabled us to use NMR to solve its structure. The purified 22(OH)D₃ was efficiently converted to 20,22(OH)₂D₃ by P450scc. The k_cat value for the reaction (2.1 min⁻¹) was comparable to that for the overall metabolism of 20(OH)D₃ by P450scc (2.4 min⁻¹) and 4.8-fold higher than that for the 22-hydroxylation of 20(OH)D₃. This relatively high rate of metabolism, plus its slow rate of formation from vitamin D₃, results in 22(OH)D₃ being only a minor product of the reaction of vitamin D₃ with P450scc. The current study also shows that 22-hydroxylation of vitamin D₃ can occur either before or after 20-hydroxylation, but in both cases 20-hydroxylation is favored. The recently published crystal structure of human P450scc with cholesterol bound in the active site provides some structural basis for the preferential hydroxylation of vitamin D at C20 (Strushkevich et al., 2011). For cholesterol, C22 is the closest carbon to the heme iron and 22R-hydroxycholesterol is the initial hydroxylation product. The docking of vitamin D in its extended trans conformation into the substrate binding site predicts that C20 rather than C22 is closest to the heme iron, thus favoring 20-hydroxylation (Strushkevich et al., 2011).

The ability of P450scc to further metabolize 20,22(OH)₂D₃ was tested, and it was slowly converted to a trihydroxyvitamin D₃ derivative, distinct from the 17,20,23(OH)₃D₃ product arising from the further metabolism of 20,23(OH)₂D₃. The 20,22-dihydroxy derivatives produced as intermediates of P450scc action on cholesterol and 7-dehydrocholesterol both undergo cleavage between C20 and C22, producing pregnenolone and 7-dehydropregnenolone, respectively (Tuckey, 2005; Slominski et al., 2009). In contrast, we could not detect any appreciable cleavage of 20,22(OH)₂D₃, suggesting that it does not sit in the P450scc active site in a suitable position to permit cleavage. The stereochemistry for hydroxylation of vitamin D₃ at C22 remains to be established. It is likely that the product is the R-isomer by analogy with 22R-hydroxycholesterol, an intermediate in the metabolism of cholesterol by P450scc (Burstein et al., 1975).

A summary of the pathways for the metabolism of vitamin D₃ by P450scc, deduced from the current and previous studies (Tuckey et al., 2008a), is shown in Fig. 12. Hydroxylation at C17, C20, C22, and/or C23 could produce 15 possible products. The pathways that we have identified to date involve 8 of these possible metabolites. The site of the third hydroxyl group in the product of P450scc action on 20,22(OH)₂D₃ [20,22,X(OH)₃D₃ in Fig. 12] remains to be established, because we were not able to make enough of this derivative for NMR analysis. The only monohydroxy vitamin D₃ derivative that we have not characterized is 23(OH)D₃. This is likely to be one of the minor unidentified products seen in the HPLC chromatogram of vitamin D₃ metabolites produced by P450scc (Fig. 2B). We have not seen hydroxyl groups at both C22 and C23 in any of the products of P450scc action on vitamin D₃ identified to date, suggesting that once a 22- or 23-hydroxyl group is present, the other hydroxylation sites at C20 and C17 are favored. Hydroxylation at all sites that P450scc can target would result in the formation of 17,20,22,23-tetrahydroxy vitamin D₃. Although such a product has not been identified, the chromatogram of products (Fig. 2B) does reveal a minor peak with a shorter retention time (19 min) than 17,20,23(OH)₃D₃, which could correspond to this metabolite.

Fig. 12.

Pathways for metabolism of vitamin D₃ by cytochrome P450scc. The major pathway is shown by the bold arrows. X represents an unknown hydroxylation site.

The products of P450scc action on vitamin D are of particular interest because both 20(OH)D₃ (Slominski et al., 2010) and 20(OH)D2 (Slominski et al., 2011) are noncalcemic at concentrations as high as 3 or 4 μg/kg in rats, which is in contrast to the strong calcemic activity seen at even lower doses of 1,25(OH)₂D₃. They also display strong antiproliferative and prodifferentiation effects on a range of cells, including keratinocytes and melanoma and leukemia cells, and an inhibitory action on nuclear factor-κB activity (Zbytek et al., 2008; Janjetovic et al., 2009, 2010; Slominski et al., 2010, 2011), identifying 20(OH)D as a potential therapeutic or adjuvant agent for a number of hyperproliferative and inflammatory diseases. In this study, we have found that 22(OH)D₃ acting through the VDR also shows antiproliferative and, to a limited degree, prodifferentiation (Fig. 9) activity; however, these effects were slightly weaker than those of 20(OH)D₃.

We have previously found that the second major product of P450scc action on vitamin D₃, 20,23(OH)₂D₃, shares the potency and many of the effects of 20(OH)D₃ on skin and leukemia cells, although some variations between these analogs were seen, depending on the parameter measured and the cell type used (Janjetovic et al., 2010; Slominski et al., 2010). Because the purified peak of 20,23(OH)₂D₃ used in those studies contained some 20,22(OH)₂D₃, we measured the activities of the purified individual components. Pure 20,22(OH)₂D₃ and 20,23(OH)₂D₃ generally exhibit similar biological properties on keratinocytes to each other and to those we have previously reported for the less pure preparation of 20,23(OH)₂D₃ (Janjetovic et al., 2010; Slominski et al., 2010). Comparable effects of 20,22(OH)₂D₃ and 20,23(OH)₂D₃ were seen on the inhibition of proliferation with both compounds causing significant inhibition at 72 h or treatment at a concentration of 10 nM. Both compounds caused similar stimulation of differentiation using involucrin expression as a marker and on the stimulation of VDR expression at the mRNA level. Both dihydroxy compounds also stimulated VDR translocation from the cytoplasm to the nucleus with 20,23(OH)₂D₃ causing stimulation comparable to that seen for 1,25(OH)₂D₃. Both 20,22(OH)₂D₃ and 20,23(OH)₂D₃ were without effect on the expression of the 1,25(OH)₂D₃-inactivating enzyme, CYP24, in contrast to the 1600-fold stimulation of CYP24 mRNA expression seen with 1,25(OH)₂D₃.

In conclusion, we have identified C22 as a site of hydroxylation of vitamin D₃ by P450scc using NMR to identify 22(OH)D₃ and 20,22(OH)₂D₃ as new reaction products. 20,22(OH)₂D₃ can be produced from both 20(OH)D₃ and 22(OH)D₃ and can be metabolized to a trihydroxy product, distinct from 17,20,23(OH)₃D₃ made from 20,23(OH)₂D₃. Although 22(OH)D₃ and 20,22(OH)₂D₃ displayed biological activity on HaCaT epidermal keratinocytes, they showed some heterogeneity that included slightly weaker effects than 20(OH)D₃ and 20,23(OH)₂D₃, indicating the importance of the 20-hydroxyl group for strong biological activity.

Authorship Contributions

Participated in research design: Tuckey, Li, Shehabi, Janjetovic, Nguyen, Kim, Chen, Howell, Benson, and Slominski.

Conducted experiments: Tuckey, Li, Shehabi, Janjetovic, Nguyen, Kim, Chen, Howell, Sweatman, Baldisseri, and Slominski.

Contributed new reagents or analytic tools: Tuckey, Li, Benson, Sweatman, Baldisseri, and Slominski.

Performed data analysis: Tuckey, Li, Shehabi, Janjetovic, Nguyen, Kim, Chen, Howell, Benson, Sweatman, Baldisseri, and Slominski.

Wrote or contributed to the writing of the manuscript: Tuckey, Li, Janjetovic, Nguyen, Kim, and Slominski.