The Mechanism of 1,25-Dihydroxyvitamin D3Autoregulation in Keratinocytes*

The synthesis of 1,25-dihydroxyvitamin D3(1,25(OH)2D3) from its precursor, 25-dihydroxyvitamin D3 (25(OH)D3), is catalyzed by the mitochondrial cytochrome P450 enzyme 25-hydroxyvitamin D3-1α-hydroxylase (1α-hydroxylase). It has been generally assumed that 1,25(OH)2D3inhibits the activity of this enzyme by regulating its expression at the genomic level. We confirmed that 1,25(OH)2D3 reduced the apparent conversion of 25(OH)D3 to 1,25(OH)2D3 while stimulating the conversion of 1,25(OH)2D3 and 25(OH)D3 to 1,24,25(OH)3D3 and 24,25(OH)2D3, respectively. However, 1,25(OH)2D3 failed to reduce the abundance of its mRNA or its encoded protein in human keratinocytes. Instead, when catabolism of 1,25(OH)2D3 was blocked with a specific inhibitor of the 25-hydroxyvitamin D3-24-hydroxylase (24-hydroxylase) all apparent inhibition of 1α-hydroxylase activity by 1,25(OH)2D3 was reversed. Thus, the apparent reduction in 1α-hydroxylase activity induced by 1,25(OH)2D3 is due to increased catabolism of both substrate and product by the 24-hydroxylase. We believe this to be a unique mechanism for autoregulation of steroid hormone synthesis.


EXPERIMENTAL PROCEDURES
Cell Culture-Normal human keratinocytes were isolated from neonatal human foreskins and grown in serum-free keratinocyte growth medium (KGM, Clonetics, San Diego, CA) as previously described (19). Briefly, keratinocytes were isolated from newborn human foreskins by trypsinization (0.25% trypsin, 4°C, overnight), and primary cultures were established in KGM containing 0.07 mM calcium. First and second passage keratinocytes were plated with KGM containing 0.03 mM calcium and used in the subsequent experiments described.
Enzyme Activity Assays-To measure 1␣-hydroxylase and 24-hydroxylase activities, we used the method of Bikle et al. (8). Briefly, 0.05 Ci of [ 3 H]25(OH)D 3 or [ 3 H]1,25(OH) 2 D 3 (Amersham Biosciences) was added to cultured normal human keratinocytes in 6-well plates. Following 1 h of incubation at 37°C, the reaction was stopped with 1 ml of methanol. Both cells and medium were extracted by the method of Bligh and Dyer (20). Metabolites in the chloroform extract were separated and quantitated by a Waters high performance liquid chromatography (HPLC) system (Waters Associates, Milford, MA) linked to a Flow Scintillation Analyzer (Packard, Meriden, CT). HPLC utilized a DuPont Zorbax Sil column (4.6 ϫ 25 cm) and a non-linear concave gradient from 97:3 to 90:10 hexane:isopropanol for 1␣-hydroxylated steroids or 90:10 hexane:isopropanol for 24-hydroxylated steroids. Output was monitored by radioactivity with a Flow Scintillation Analyzer (Packard, Downers Grove, IL). Chemically synthesized standards were used to determine the elution volumes of the metabolites.
RNA Analysis-Total RNA was isolated from the keratinocytes using the STAT-60 kit (Tel-Test "B", Inc., Friendswood, TX), according to the procedures recommended by the manufacturer. The isolated RNA (20 mg per lane) was electrophoresed through a 0.8% agarose-formaldehyde gel, transferred to a nylon membrane (Hybond-Nϩ; Amersham * This work was supported by Grants PO1AR39448, RO1AR38386, and DK37922 from National Institutes of Health and Grant 98A079 from the American Institute for Cancer Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Biosciences) using PosiBlot 30 -30 Pressure Blotter (Stratagene, La Jolla, CA), and immobilized by baking the membrane at 80°C for 2 h. Complementary DNA probes for human 1␣-hydroxylase (4) and human involucrin (gift from Dr. Howard Green, Harvard Medical School) were labeled with [ 32 P]dCTP (Amersham Biosciences) by Random Prime-IT, II labeling kit (Stratagene), and purified by NucTrap Probe Purification Columns (Stratagene). The membrane was prehybridized and hybridized in 5ϫ SSC, 5ϫ Denhardt's solution, 0.5% SDS, and 20 mg/ml salmon sperm DNA with the 32 P-labeled human 1␣-hydroxylase and involucrin cDNAs. After hybridization at 65°C overnight, the membrane was washed in solutions with decreasing ionic strength and increasing temperature to a final stringency of 0.1ϫ SSC and 0.1% SDS at 65°C. The [ 32 P]cDNA-mRNA hybrids were visualized by exposing to x-ray film. The 18 S ribosomal RNA on the same RNA blot hybridized with a 32 P-labeled cDNA for 18 S RNA was used as a control.
Immunoblotting-Keratinocytes were washed twice with PBS and then incubated in lysis buffer containing 50 mM HEPES, pH 7.4, 1% Triton X-100, 0.1% SDS, 150 mM NaCl, 1 mM EDTA, 20 mg/ml phenylmethylsulfonyl fluoride, 1 mg/ml leupeptin, 1 mg/ml pepstatin A, and 2 mg/ml aprotinin for 5 min. Cells were scraped into microcentrifuge tubes, incubated on ice for 15 min, and pelleted by centrifugation. The supernatant was collected. The protein concentration of the lysate was measured by the BCA Protein Assay Kit (Pierce, Rockford, IL). Equal amounts of protein were then electrophoresed through 7.5% polyacrylamide gels at 200 V for 30 min and electroblotted onto polyvinylidene difluoride membranes (0.2 micron, Bio-Rad Laboratories, Hercules, CA) in an electroblotting buffer (25 mM Tris, 192 mM glycine, 5% methanol) at 130 V for 2 h. After incubation in blocking buffer (100 mM Tris base, 150 mM NaCl, 5% non-fat milk, and 0.5% Tween 20), the blot was incubated with the appropriate primary antibodies overnight at 4°C. 1␣-hydroxylase protein was detected with a polyclonal rabbit antihuman antibody (21) at a dilution of 1:20,000 in blocking buffer. Involucrin protein was detected with a monoclonal mouse anti-human involucrin antibody (Sigma) at a dilution of 1:2000 in blocking buffer. After washes in the blocking buffer, the membranes were incubated for 1 h with the appropriate anti-IgG secondary antibody conjugated to horseradish peroxidase (Amersham Biosciences) diluted 1:5000 in the blocking buffer. After a second series of washes, bound antibody complexes were visualized using the SuperSignal Ultra Chemiluminescent Kit (Pierce) and subsequent exposure to x-ray film.

RESULTS AND DISCUSSION
To determine the ability of 1,25(OH) 2 D 3 to inhibit its own production, we incubated keratinocytes overnight with varying concentrations (from 10 Ϫ12 to 10 Ϫ8 M) of 1,25(OH) 2  These results indicate that 1,25(OH) 2 D 3 inhibited 1␣-hydroxylase activity and stimulated 24-hydroxylase activity. We then determined whether the abundances of 1␣-hydroxylase mRNA and protein were regulated by 1,25(OH) 2 D 3 . A 2.4-kb mRNA transcript and a 56-kDa protein were detected by Northern and Western analysis, respectively. The levels of 1␣-hydroxylase mRNA and its protein in human keratinocytes were not regulated by 10 Ϫ11 to 10 Ϫ8 M 1,25(OH) 2 D 3 . In contrast, the level of mRNA and protein for involucrin (22), a marker for keratinocyte differentiation and analyzed on the same blots, were stimulated in a dose-dependent manner by 1,25(OH) 2 D 3 (Fig. 2, A and B). These data indicate that 1,25(OH) 2 D 3 does not regulate 1␣-hydroxylase mRNA or protein expression.
These results led us to hypothesize that the apparent inhibition of 1,25(OH) 2 D 3 production by 1,25(OH) 2 D 3 is due to increased catabolism of 1,25(OH) 2 (Fig. 3). These data demonstrate that exogenous 1,25(OH) 2 D 3 stimulates the catabolism of endogenously produced 1,25(OH) 2 D 3 in human keratinocytes. To test this hypothesis further, we examined the effect of an inhibitor of the 24-hydroxylase (VID400) (18, 23) on 1␣-hydroxylase and 24-hydroxylase activities in keratinocytes. Cells were preincubated overnight with either 1,25(OH) 2  ity by more than 30% (Fig. 4B). In contrast, 100 nM VID400 inhibited 24-hydroxylase activity by more than 90% (Fig. 4A) but only slightly inhibited 1␣-hydroxylase activity (Fig. 4B). Thus, we used 100 nM VID400 to determine the degree to which induction of 24-hydroxylation contributed to the apparent inhibition of 1␣-hydroxylase activity by 1,25(OH) 2 D 3 . Keratinocytes were preincubated for 24 h with 1,25(OH) 2 D 3 , and the conversion of [ 3 H]25(OH)D 3 to [ 3 H]1,25(OH) 2 D 3 was measured with and without the presence of VID400. Preincubation with 10 Ϫ9 M 1,25(OH) 2 D 3 reduced the apparent 1␣-hydroxylase activity by 90%. However, addition of 100 nM VID400 restored most of the apparent reduction in 1␣-hydroxylase activity (Fig.  5). When the concentration of VID400 was increased to 200 nM, no reduction in apparent 1␣-hydroxylase activity was observed in the cells treated with 1,25(OH) 2 D 3 , although basal activity was reduced (data not shown). In agreement with recent data (18), these results indicate that the 1␣-hydroxylase activity is not regulated by 1,25(OH) 2 D 3 . The apparent reduction in 1,25(OH) 2 D 3 production is due to increased catabolism of The intracellular concentration of 1,25(OH) 2 D 3 in keratinocytes is controlled by 1␣-hydroxylase and 24-hydroxylase, which are responsible for 1,25(OH) 2 D 3 synthesis and degradation, respectively. Transcription of the gene encoding 24-hydroxylase is induced by 1,25(OH) 2 D 3 , and the promoter of this gene contains two vitamin D-responsive elements (24) that mediate the ability of 1,25(OH) 2 D 3 to induce this gene in keratinocytes (25) and other cells. Studies purporting to demonstrate negative regulation of 1␣-hydroxylase by 1,25(OH) 2 D 3 need to be reconsidered in light of our observations. For example, mice lacking the vitamin D receptor have increased amounts of 1␣-hydroxylase mRNA (3), suggesting a genomic mechanism of regulation by 1,25(OH) 2 D 3 . However, parathyroid hormone can induce transcription of the gene for 1␣hydroxylase (26), and parathyroid hormone is quite elevated in mice lacking the vitamin D receptor. Thus, increased serum concentrations of parathyroid hormone, rather than decreased 1,25(OH) 2 D 3 -regulated genomic activity, could well be responsible for the increased abundance of 1␣-hydroxylase mRNA in these animals. The present data support the conclusion that the regulation of 1,25(OH) 2 D 3 levels by 1,25(OH) 2 D 3 itself involves induction of its catabolism rather than inhibition of its production. This mechanism appears to be a unique mode for autoregulation of steroid hormone synthesis.