INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

THE CLASSICAL FUNCTION OF vitamin D is to regulate mineral ion homeostasis. In addition to these effects, vitamin D mediates diverse cellular processes, including effects on cell growth and differentiation, on the immune system and on hormone secretion (see Ref. 15 for review). 1,25-Dihydroxyvitamin D₃ [1,25(OH)₂D₃] is the most active metabolite of vitamin D, and vitamin D receptors (VDRs) mediate its effects by affecting the transcription of 1,25(OH)₂D₃-responsive genes (15). 1,25(OH)₂D₃ is produced by two sequential hydroxylations of vitamin D by 25-hydroxylase in the liver and by 25-hydroxyvitamin D₃ 1-hydroxylase (1-hydroxylase) in the kidney (18). 1,25(OH)₂D₃ can be further hydroxylated in the kidney by 25-hydroxyvitamin D₃ 24hydroxylase (24OHase). 24-Hydroxylation of 1,25(OH)₂D₃ is believed to be the first step in the catabolism of the hormonally active form of vitamin D (46). Intestine, bone, and kidney are the three major target tissues involved in the regulation by 1,25(OH)₂D₃ of mineral homeostasis. 1,25(OH)₂D₃ has been reported to act at the kidney to enhance calcium transport in the distal nephron (7, 21, 28), the site of parathyroid hormone (PTH) modulation of renal calcium absorption. [³H]1,25(OH)₂D₃ uptake (54) and vitamin D-dependent calcium binding proteins (14, 50) are localized predominantly in the distal convoluted tubule (DCT), additionally suggesting that the DCT is the site of 1,25(OH)₂D₃ action on calcium conservation. Besides the suggested role of 1,25(OH)₂D₃ in the tubular reabsorption of calcium, another important effect of 1,25(OH)₂D₃ in the kidney is the regulation of the 25(OH)D₃ hydroxylases. 1,25(OH)₂D₃ has been shown to self-induce its deactivation by inhibiting the 1-hydroxylase enzyme and by inducing the 24OHase enzyme, ultimately limiting its own biological activity (27, 47). It is believed that the effect of 1,25(OH)₂D₃ on the renal 1- and 24-hydroxylases is mediated by the VDR (55). Recently, the cloning of the human and rat 24OHase genes (11, 42) as well as the mouse 1-hydroxylase gene (55) has been reported. The promoter of the 24OHase gene, the most transcriptionally responsive vitamin D-inducible gene identified to date, has been sequenced and two functional vitamin D response elements (VDREs) have been mapped in the 5' flanking region (10, 34). Although the effect of 1,25(OH)₂D₃ on calcium conservation is in the DCT (7, 21, 28), previous studies using microdissected nephron segments reported that the exclusive site of renal 1-hydroxylase (9, 33) and 24OHase (33) activities is in the renal proximal tubule (PCT). However, recent studies using antibodies against 24OHase by Kumar et al. (38) showed that epitopes for 24OHase are present in both proximal and distal tubules of the human kidney. In addition, 24OHase protein was identified in both proximal and distal tubules of rat kidney using quantitative immunoelectron microscopic analysis (32). 24OHase mRNA, although predominantly in the PCT, was also recently observed under certain conditions in microdissected cortical collecting ducts (CCD) of the rat distal nephron (29). 24OHase mRNA in the CCD was reported to be induced in intact rats fed low dietary calcium, a condition that resulted in a striking suppression of 24OHase mRNA in the PCT (29). These findings strongly suggest that the proximal tubules are not the sole site of localization of 24OHase in the kidney.

Although the major site of localization of 24OHase is the proximal tubule, in this study we report that 24OHase can also be localized in the distal nephron, consistent with recent immunolocalization studies (32, 38) and recent studies localizing 24OHase mRNA using microdissected nephron segments (29). Because 1,25(OH)₂D₃ and PTH are major regulators of vitamin D metabolism (18, 27), we examined the effect of 1,25(OH)₂D₃ and PTH or cAMP (a second messenger of PTH action) on 24OHase expression as well as on the mechanisms involved in the coordinate regulation by 1,25(OH)₂D₃ and cAMP or PTH of 24OHase transcription. Our findings suggest differential regulation of the 24OHase enzyme in the PCT and DCT and therefore different biological effects of PTH and cAMP on 1,25(OH)₂D₃ action in these different nephron segments. It is also evident from this study that in the distal nephron, upregulation of VDR levels by cAMP, and not an effect on CREs in the 24OHase promoter, is one mechanism by which cAMP modulates 1,25(OH)₂D₃-induced transcription of 24OHase.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Materials. [¹⁴C]chloramphenicol (50 mCi/mmol) and [³²P]deoxycytidine triphosphate (3,000 Ci/mmol, 370 MBq/ml) were obtained from DuPont-New England Nuclear (Boston, MA). RadPrime DNA labeling system and all restriction enzymes were purchased from GIBCO BRL-Life Technologies (Gaithersburg, MD). Biotrans nylon membranes were obtained from ICN Biochemicals (Costa Mesa, CA). Oligo (dT) cellulose was purchased from Boehringer Mannheim (Indianapolis, IN). 8-Bromo-cAMP, acetyl coenzyme A, phorbol 12-myristate 13-acetate, and human PTH fragment (134) were obtained from Sigma (St. Louis, MO). Phenol, formamide, and guanidinium isothiocyanate were purchased from International Biotechnologies (New Haven, CT). Rat anti-vitamin D receptor antibody was from Affinity BioReagents (Neshanic Station, NJ). This antibody has been reported to cross-react with avian and mammalian VDR (43). Polyclonal antibody against rat 24OHase was generated and characterized in the laboratory of Dr. R. Kumar (38). Mouse 24OHase has been reported to share 94.7% amino acid identity with rat 24OHase (31). Thus the rat antibody cross-reacts with mitochondrial protein prepared from mouse cells. Polyclonal antibody against rat thiazide-sensitive NaCl transporter was provided by S. Hebert of Vanderbilt University School of Medicine (Nashville, TN). The specificity of this antibody has been characterized previously (45). Immunocytochemical studies have indicated that the rat NaCl cotransporter antibody cross-reacts with mouse kidney and specifically labels the apical membrane of the mouse distal convoluted tubule and early collecting duct (personal communication, Dr. S. Hebert). Chemically synthesized 1,25(OH)₂D₃ was provided by Dr. M. Uskokovic of Hoffmann-LaRoche (Nutley, NJ). 1,25(OH)₂[23,24-N-³H]vitamin D₃ (95 Ci/mmol) and 25(OH)[26(27)-methyl-³H]vitamin D₃ (21 Ci/mmol) were purchased from Amersham Life Science (Arlington Heights, IL).

Cell culture. The preparation, culture conditions, and characterization of immortalized mouse DCT cells have been described previously (22, 44). These cells, which are derived from both DCT and cortical thick ascending limbs of Henle's loop, are devoid of markers of proximal tubules (such as alkaline phosphatase and Na⁺-glucose cotransport) but express characteristics of DCT cells, including thiazide-inhibitable but not bumetanide-inhibitable sodium transport. DCT cells were maintained in Dulbecco's modified Eagle's medium plus Ham's F12 nutrient mixture (DMEM-F12, GIBCO BRL-Life Technologies) supplemented with 5% heat-inactivated fetal bovine serum (FBS) (Gemini, Calabasas, CA) in a humidified atmosphere of 95% O₂-5% CO₂ at 37°C. Cells were grown to 60% confluence, and 24 h before the start of experiments, medium was changed to serum-free DMEM-F12 medium. Cells were treated with the vehicle or the compounds noted at the concentrations and times indicated.

Studies were also done using primary cultures derived from either mouse DCT or PCT prepared with a double-antibody separation procedure as previously described (44). Primary cultures of these cells have been previously characterized and shown to exhibit a phenotype specific to their site of origin in the nephron (44). Cells were initially plated at a density of 3 × 10⁴ cells/cm² in 100-mm tissue culture plates in Optimem (GIBCO BRL-Life Technologies) and allowed to attach and grow for 4-5 days at 37°C in a humidified atmosphere of 95% O₂-5% CO₂. After 5 days, media were changed. At 7-8 days in culture, cells were treated with vehicle or 1,25(OH)₂D₃.

RNA isolation and Northern blot hybridization analysis. Total RNA was prepared from DCT cells by the guanidinium thiocyanate-phenol chloroform method described by Chomczynski and Sacchi (13). Polyadenylated [poly(A)⁺] mRNA was prepared by two cycles of oligo (deoxythymidine)-cellulose chromatography. Northern blot analysis was performed as previously described (58). Labeled probes were prepared according to the random prime method (19) using the Random Primers DNA Labeling System (GIBCO BRL-Life Technologies). The blots were hybridized to specific ³²P-labeled cDNA probes for 16 h at 42°C, washed, and subjected to autoradiography as previously described (58). To detect any problems with transfer or differences in loading, blots were probed with ³²P-labeled -actin cDNA and/or ³²P-labeled 18S rRNA cDNA. All autoradiograms were analyzed by densitometric scanning using the Dual-Wavelength Flying Spot Scanner (Shimadzu Scientific Instruments, Princeton, NJ). The relative optical densities obtained using the test probes were divided by the relative optical density obtained after probing with the control probe to normalize for sample variation.

Measurement of 24OHase activity. DCT cells were subcultured into 9.6-cm² wells and maintained in 2 ml DMEM-F12 medium supplemented with 5% FBS. Twenty-four hours before the experiments, the incubation media was changed to DMEM-F12 medium containing 2% charcoal-dextran stripped FBS to ensure vitamin D-free conditions. The experiments were initiated under induced conditions by treatment with unlabeled 1,25(OH)₂D₃ (10⁷ M in 0.1% ethanol) or 1,25(OH)₂D₃ + 1 mM 8-bromo-cAMP and under basal conditions by treatment with vehicle alone (0.1% ethanol). At the end of 24 h, the cells were rinsed with serum-free media and 0.05 µCi [26,27-methyl-³H]25(OH)D₃ (1 nM) was added to each well containing 2 ml serum-free DMEM-F12 medium with 0.2% BSA. The incubations were terminated at 1 h with 1 ml methanol. HPLC was performed as described by Siu-Caldera et al. (53). Lipids from both cells and media were extracted using the procedure described by Reddy and Tserng (46). All samples were spiked with 1 µg unlabeled 25(OH)D₃ before lipid extraction to assess the recovery of tritiated metabolites. Before HPLC analysis, 1 µg of unlabeled 24(R)25-dihydroxyvitamin D₃ [24(R),25(OH)₂D₃] and 23(S)25-dihydroxy-24-oxo-vitamin D₃ [24(S),25(OH)₂-24-oxo-D₃] standards were added to each lipid extract sample. HPLC analysis was performed with a Waters System Controller (model 600E) equipped with a photodiode array detector (model PDA 900) to monitor ultraviolet-absorbing material at 265 nm (Waters, Milford, MA). The samples were analyzed using a Zorbax-SIL column (4.6 mm × 25 cm; DuPont, Wilmington, DE) eluted with isopropanol-hexane (3:97, vol/vol) at a flow rate of 2 ml/min. One-minute fractions were collected, and the radioactivity in each vial was measured in a scintillation counter (Beta Trac; TM Analytic, Elk Grove Village, IL) after the addition of 4 ml Scintilene (Fisher Scientific, Pittsburgh, PA). The identity of individual radioactive peaks of both 24(R),25(OH)₂D₃ and 23(S),25(OH)₂-24-oxo-D₃ from the first HPLC system were further verified by comigration with their respective synthetic standards on a second HPLC system.

Western blot analysis of VDR, 24OHase, and NaCl transporter proteins. For Western blot analysis of VDR, DCT cells were processed for preparation of chromatin as previously described (59). Aliquots of the KCl-extracted chromatin preparation were assayed for protein concentration by the method of Bradford (8), and 20 µg of protein from each sample were used for Western blot analysis. For Western blot analysis of 24OHase, mitochondrial protein was prepared from 1,25(OH)₂D₃-treated DCT cells as described by Iwata et al. (32). Membrane protein was isolated from DCT cells for Western analysis of the NaCl transporter. Membrane protein was prepared by homogenization of harvested DCT cell in 0.32 M sucrose, 5 mM Tris · HCl (pH 7.5), and 2 mM EDTA and centrifugation at 3,000 g for 10 min. The resulting supernatant was removed and centrifuged at 100,000 g at 4°C for 30 min. The pellet was resuspended in buffer containing 5 mM Tris · HCl (pH 7.5) and 2 mM EDTA. Protein was measured by the method of Bradford (8), and 30 µg of protein were used for Western analysis. DCT cell protein (mitochondrial protein for 24OHase and membrane protein for the NaCl transporter or KCl-extracted chromatin preparation for VDR) was used for electrophoresis either on a 12% SDS polyacrylamide gel for VDR or on 9% SDS gel for 24OHase and NaCl transporter. After electrophoresis, proteins were transferred electrophoretically to a nitrocellulose membrane (Hybond-ECL; Amersham) that was incubated with antibody [anti-VDR monoclonal antibody 9A7, 1:2,000 dilution; anti-rat 24OHase polyclonal antibody (38), 1:400 dilution; or anti-NaCl transporter protein, 1:1,000 dilution] in Tris-buffered saline, pH 7.5 (TBS) for 12 h at 4°C. After washing with TBS, the membrane was incubated with secondary antibody [for anti-VDR, goat anti-rat IgG conjugated to horseradish peroxidase (HRP) was used, and for the polyclonal antibodies goat anti-rabbit IgG conjugated to HRP (Sigma) was used] for 1 h at room temperature. After washing with TBS, the antigen-antibody complex was detected using the electrochemiluminescent Western blotting detection system (Amersham) according to the manufacturer's protocol.

Complementary DNA probes, 24OHase-chloramphenicol acetyltransferase constructs, cell transfection, and assay of chloramphenicol acetyltransferase activity. The 3.2-kb rat 24OHase cDNA was obtained by EcoR I digestion and was a generous gift from K. Okuda (Hiroshima University School of Dentistry, Hiroshima, Japan) (42). The rat 24OHase cDNA has previously been shown to hybridize with mRNA prepared from mouse kidney (55). A 1.7-kb rat VDR cDNA was obtained by digestion of pIBI76 with EcoR I (43). Hybridization of the rat VDR cDNA with mRNA prepared from mouse cell lines has previously been reported (36). The 18S rRNA cDNA was obtained from R. Guntaka (University of Missouri, Columbia, MO), and the 2.1-kb chicken -actin cDNA was obtained from M. W. Kirschner (17).

For transfection studies, constructs of a chimeric gene in which the rat 24OHase promoter (1,367/+74) was linked to the chloramphenicol acetyltransferase (CAT) gene and deletion mutant constructs (671/+74 and 291/+74) as previously described (34) were used. A 160/+3 fragment was isolated using the rat 24OHase promoter sequence 1,367/+74 as a template. Two 20-bp primers corresponding to region 161/141 of the 24OHase promoter and to 3/+17 of its complementary strand were synthesized and used for amplification by PCR. The size of the amplified product, 161/+3 of the rat 24OHase promoter, was confirmed by agarose gel electrophoresis. This product was purified by the "crush and soak" method described by Maxam and Gilbert (41). After phosphorylation of the PCR product with T4 polynucleotide kinase in the presence of 50 mM Tris · HCl, pH 8.0, 10 mM MgCl₂, 15 mM dithiothreitol (DTT), and 0.33 mM ATP, the PCR fragment was ligated into the Sma I site of phCAT (which is derived from pSV2CAT by deleting the simian virus 40 promoter; phCAT was a gift from M. Tocci, Merck, Sharpe and Dohme). The orientation of the inserts was determined by DNA sequencing. Within 161/+3 of the 24OHase promoter is the proximal VDRE as well as a putative cAMP response element (CRE) (TGACTCCA) (132/125). To mutate the putative CRE, 132/125 was replaced by a random sequence GACTCATG by PCR as described above using a sequence corresponding to the region 160/97 with the putative CRE replaced by the random sequence and a 20-bp sequence corresponding to region +3/17 of the complementary strand as primers. The mutation construct was sequenced and the base substitution was confirmed. To construct a proximal rat 24OHase VDRE-thymidine kinase (tk) CAT reporter plasmid, two complementary oligonucleotides containing the proximal VDRE of the rat 24OHase promoter (5'-CTAGGAGGCCCCCACTCGCGACTCATGTCCT-3' and 5'CTAGAGGACATGAGTCGCCGGGGCCTC-3') were synthesized with Xba I half-site overhangs at their 5' end. One hundred micrograms of each oligonucleotide were mixed with 7 µl medium salt buffer (10× medium salt buffer: 100 mM Tris · HCl, 100 mM magnesium acetate, 500 mM NaCl, 10 mM DTT, pH 7.5; Boehringer Mannheim), and the final volume was adjusted to 70 µl with H₂O. The two complementary oligonucleotides providing the 5' overhang of the Xba I half-site were allowed to anneal by heating at 100°C for 5 min and cooling to room temperature. After separation of the annealed double-stranded DNA from oligonucleotides by agarose gel electrophoresis, the annealed product was excised from the gel and purified (41). The purified DNA fragment was phosphorylated by T4 polynucleotide kinase as described above. Phosphorylated double-stranded DNA fragments were ligated into the Xba I site of the tk promoter CAT reporter gene construct by T4 DNA ligase in the presence of 50 mM Tris · HCl (pH 7.6), 10 mM MgCl₂, 1 mM ATP, 1 mM DTT, and 5% polyethylene glycol-8,000. The number of inserts was checked by Pst I digestion, and the orientation was confirmed by DNA sequencing. The VDRE multimers were inserted 105 bp upstream of the tk transcription start site. The pBLCAT2 contains the tk promoter inserted upstream of the reporter CAT gene (from J. W. Pike, University of Cincinnati, Cincinnati, OH).

For transfections, cells were plated at a density of 1 × 10⁶ cells/100 mm plate in serum-free media 24 h before transfection. DCT cells were cotransfected with reporter plasmid (8 µg) and the -galactosidase expression vector pCH110 (4 µg; from Pharmacia, Piscataway, NJ), an internal control for transfection efficiency, using the calcium phosphate DNA precipitation method (5). Cells were transfected for 16 h, shocked for 1 min with 10% dimethyl sulfoxide-PBS, washed with PBS, and treated for 24 h with vehicle (0.1% ethanol) or test compound at the concentrations indicated. After 24-h incubation, cells were harvested and cell extracts were prepared by freeze (80°C)-thaw (37°C) three times, 5 min each. The CAT assay was performed at constant -galactosidase activity following standard protocols (5, 23). To verify that regions in the promoter other than the 24OHase VDRE were not involved in the cAMP enhancement, additional studies were done using mutant 24OHase promoter luciferase constructs with mutations introduced into the proximal VDRE (M1) or in the proximal and distal VDREs (M4). The M1 and M4 mutations have been described previously (34). Luciferase activity, performed at constant -galactosidase activity, was determined using a luciferase assay system (Promega). Results were quantitated as relative light units using a luminometer.

Statistical analysis. Data were tested for significance by Student's t-test for two-group comparison or analysis of variance for multiple-group comparison. Differences <0.05 were assumed to be significant.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Expression of 24OHase mRNA, protein, and activity in DCT cells. Because of the discrepancy between immunolocalization and PCR studies, which showed that epitopes and mRNA for 24OHase are present in both proximal and distal tubules (29, 32, 38), and previous studies that reported that the exclusive site of 24OHase activity is in the proximal tubule (33), we tested the recently available DCT cell line (22, 44) (which is devoid of markers of proximal tubules but which expresses characteristics consistent with the distal tubule) for the presence of 24OHase mRNA, protein, and activity. With the use of Northern blot analysis, 24OHase mRNA was observed in DCT cells after 1,25(OH)₂D₃ treatment (10⁷ M, 24 h); however, basal levels were undetectable. Although 8-bromo-cAMP (1 mM) or 12-O-tetradecanoylphorbol 13-acetate (TPA; 100 mM) alone had no effect on 24OHase mRNA expression, cAMP or TPA enhanced 1,25(OH)₂D₃-dependent upregulation of 24OHase mRNA by 10- and 6-fold, respectively (Fig. 1). To confirm that the mouse DCT indeed contains 24OHase mRNA, Northern blot analysis was carried out using primary cultures derived specifically from the distal nephron (cortical thick ascending limb plus DCT) or from proximal tubules. Previous studies characterizing the cells derived from the distal nephron in primary culture have indicated, similar to the immortalized cells, that they are devoid of markers of proximal tubules (for example alkaline phosphatase and Na⁺-glucose cotransporter) but express characteristics of the distal tubule (44). Basal levels of 24OHase mRNA were undetectable in primary cultures derived from both the PCT and DCT. However, 1,25(OH)₂D₃ treatment induced the expression of 24OHase mRNA derived from both nephron segments. Primary cultures derived from the PCT exhibited fivefold higher levels of 24OHase mRNA than cells derived from the DCT (Fig. 2). Besides 24OHase mRNA, 24OHase protein was also identified in DCT cells by Western blot analysis using a polyclonal antibody against rat 24OHase. Western blot analysis also indicated the presence of the highly specific distal tubule marker, thiazide-sensitive NaCl transporter, in DCT cells (Fig. 3). In addition, as determined by HPLC, DCT cells were found to metabolize [³H]25(OH)D₃ to polar metabolites produced by 24OHase. After pretreatment of DCT cells with 1,25(OH)₂D₃ (10⁷ M, 24 h), the two major metabolites detected at 1 h comigrated with authentic 24(R),25(OH)₂D₃ and 23(S),25(OH)24-oxo-D₃ (Fig. 4, Table 1), both of which are products of the 24OHase enzyme (1, 6).

View larger version (55K): [in this window] [in a new window]   Fig. 1.   Effects of 1,25 dihydroxyvitamin D3 [1,25(OH)2D3] and 1,25(OH)2D3 + cAMP or 12-O-tetradecanoylphorbol 13-acetate (TPA) on 25-hydroxyvitamin D3-24-hydroxylase (24OHase) mRNA expression in mouse distal convoluted tubule (DCT) cells. Poly(A)+ RNA was prepared from the DCT cells treated with vehicle (0.1% ethanol), 1,25(OH)2D3 (107 M), 8-bromo-cAMP (1 mM), TPA (100 nM), 1,25(OH)2D3 + cAMP, or 1,25(OH)2D3 + TPA for 24 h. DCT cell poly(A)+ RNA was fractionated on a formaldehyde-agarose gel and transferred to a nylon membrane. Filter was hybridized with rat 24OHase and 18S rRNA cDNAs. Similar results were observed in 4 additional experiments.

View larger version (91K): [in this window] [in a new window]   Fig. 2.   Induction of 24OHase mRNA by 1,25(OH)2D3 in primary cultures derived from mouse proximal convoluted tubule (PCT) or DCT. Primary cultures derived from either mouse DCT or PCT were prepared with a double-antibody procedure previously described (44). Renal cells were treated with vehicle (0.1% ethanol; D) or 1,25(OH)2D3 (107 M; +D) for 24 h. Northern blot was performed using 30 µg of total RNA as described in MATERIALS AND METHODS. For each lane of Northern blot, total RNA was prepared from primary culture prepared from 16-18 kidneys dissected from 4-wk-old mice. Blot was probed for expression of 24OHase and rehybridized with 32P-labeled -actin cDNA as a control for RNA loading. A duplicate experiment yielded similar results.

View larger version (29K): [in this window] [in a new window]   Fig. 3.   Identification of 24OHase and NaCl transporter proteins in mouse DCT cells. Western blot analysis was performed using 30 µg protein from DCT cells and DCT cells treated with 1,25(OH)2D3 (107 M, 24 h) and probed with antibody against thiazide-sensitive NaCl transporter (A) or rat 24OHase (B), respectively.

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Analysis of 24OHase activity in control and 1,25(OH)2D3-treated DCT cells. A: HPLC profile of authentic standards of 25(OH)D3 (1), 23(S),25(OH)2-24-oxo-D3 (4), and 24(R),25(OH)2D3 (5). B and C: HPLC profile of [3H]25(OH)D3 (substrate) and major metabolites produced by DCT cells after 1-h incubation with substrate after 24-h pretreatment with ethanol (B) or 1,25(OH)2D3 (107M) (C). Similar results were observed in 2 additional experiments. Arrows and numbers indicate elution position of standards shown in A. Other metabolites of 25(OH)D3 produced by 24OHase enzyme, namely 25(OH)-24-oxo-D3 (2) and 23(OH)-24,25,26,27-tetranor-D3 (3), were not evaluated in this study because 25 (OH)-24-oxo-D3 is a minor metabolite that elutes very closely to the substrate, and 23(OH)-24,25,26,27-tetranor-D3 is not detectable because of loss of tritium label on carbons 26 and 27 during side-chain cleavage. UV, ultraviolet.

Effect of cAMP on the induction of 24OHase mRNA by 1,25(OH)₂D₃ and on VDR mRNA in DCT cells. In previous in vivo studies in rats fed a diet low in calcium, which results in an increase in circulating levels of the PTH and 1,25(OH)₂D₃, 24OHase mRNA was reported to be expressed in the distal nephron but strikingly suppressed in the PCT (29). To obtain a better understanding of the regulation of 24OHase mRNA in the distal nephron, we analyzed the time course of 24OHase mRNA expression in vitro in DCT cells in the presence of 1,25(OH)₂D₃ or 1,25(OH)₂D₃ + cAMP, a second messenger of PTH action. Because it had been reported that regulation of VDR in the PCT plays an important role in the reciprocal regulation of 24OHase mRNA in these different nephron segments (29), we also examined the regulation of VDR mRNA in DCT cells.

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View larger version (41K): [in this window] [in a new window]   Fig. 5.   Time-dependent effect of 1,25(OH)2D3 or 1,25(OH)2D3 + cAMP on levels of vitamin D receptor (VDR) mRNA and 24OHase mRNA in DCT cells. A: Northern analysis was performed using 8 µg poly(A)+ RNA per lane from DCT cells that had been treated with 107 M 1,25(OH)2D3 or vehicle control and harvested at various times after 1,25(OH)2D3 administration. Filter was hybridized with 32P-labeled rat VDR; then blots were stripped and rehybridized with 32P-rat 24OHase and mouse -actin cDNAs sequentially. B: quantification of results obtained by Northern blot analysis. Data from 3 independent experiments (mean ± SE) are expressed as percentage of maximal response. Quantitation of VDR mRNA included both transcripts. Data were normalized on basis of results obtained on rehybridization with -actin cDNA. C: Northern analysis was performed using 8 µg poly (A)+ RNA per lane from mouse DCT cells that had been treated with 1,25(OH)2D3 (107 M) + 8-bromo-cAMP (1 mM) and harvested at various times after treatment. Filter was hybridized as described above for experiments done in absence of cAMP. D: quantification of results obtained by Northern blot analysis. Data from 3 independent experiments (mean ± SE) are expressed as percentage of maximal response. Quantitation of VDR mRNA and normalization of data were done as described above.

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View larger version (37K): [in this window] [in a new window]   Fig. 6.   Dose-dependent effect of 1,25(OH)2D3 in presence or absence of cAMP on levels of VDR mRNA and 24OHase mRNA in DCT cells. A: Northern analysis was performed using 8 µg poly(A)+ RNA per lane from mouse DCT cells that had been treated for 24 h with indicated concentrations of 1,25(OH)2D3 in presence or absence of 8-bromo-cAMP (1 mM). Filter was hybridized with 32P-labeled rat VDR cDNA; then blots were stripped and rehybridized with 32P-rat 24OHase and mouse -actin cDNAs sequentially. B: quantification of results obtained by Northern blot analysis. Data from 3 independent experiments (mean ± SE) are expressed as percentage of maximal response. Quantitation of VDR mRNA included both transcripts. Data were normalized on basis of results obtained on rehybridization with -actin cDNA. In presence of 8-bromo-cAMP, VDR and 24OHase mRNAs were significantly induced at all concentrations of 1,25(OH)2D3 [P < 0.05 compared with treatment with 1,25(OH)2D3 alone]. [It should be noted that 8-bromo-cAMP (1 mM) alone, in absence of 1,25(OH)2D3, was able to induce VDR mRNA 5.5 ± 1-fold (not shown), equivalent to induction observed with 1,25(OH)2D3 + cAMP].

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Effect of cAMP and 1,25(OH)₂D₃ on VDR protein levels. It was suggested from the time course of VDR mRNA expression in the presence of 8-bromo-cAMP and 1,25(OH)₂D₃ that cAMP may mediate the enhanced induction of 24OHase mRNA by upregulating VDR levels. Thus the effect of 1,25(OH)₂D₃ and 8-bromo-cAMP on the levels of VDR protein was examined. After DCT cells were treated with vehicle, 1,25(OH)₂D₃, or 1,25(OH)₂D₃ + cAMP for 24 h, the cells were harvested and chromatin-associated proteins were isolated and analyzed for VDR by Western blot analysis. Interestingly, 1,25(OH)₂D₃ enhanced the level of VDR protein (Fig. 7), although 1,25(OH)₂D₃ had no effect on VDR mRNA (Fig. 5, A and B), suggesting that 1,25(OH)₂D₃-induced upregulation of VDR protein is mediated by a posttranscriptional mechanism. cAMP further enhanced the expression of 1,25(OH)₂D₃-induced VDR (Fig. 7). Densitometric analysis of the VDR protein band obtained from five separate determinations indicated a 6.8 ± 1.3-fold induction of VDR in the presence of 1,25 (OH)₂D₃ and a 12.5 ± 2.0-fold induction in the presence of 1,25 (OH)₂D₃ + cAMP (P < 0.01). These results indicate that the enhancement of 1,25(OH)₂D₃-induced 24OHase mRNA expression by cAMP is due, at least in part, to the upregulation of VDR.

View larger version (72K): [in this window] [in a new window]   Fig. 7.   Effect of 1,25(OH)2D3 and cAMP on VDR protein content. DCT cells were treated with vehicle 0.1% ethanol (D), 107 M 1,25(OH)2D3 (+D), or 1,25(OH)2D3 + 1 mM 8-bromo-cAMP (cAMP + D) for 24 h. Nuclear associated proteins were prepared as described in MATERIALS AND METHODS. Western analysis was performed using 30 µg protein and probed with polyclonal antibody against rat VDR. Molecular size markers are indicated at right. VDR is visualized as a 50-kDa immunoreactive band. Nature of highest molecular mass protein cross-reacting with VDR antibody [which has been observed by others (36)] is not known.

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View larger version (31K): [in this window] [in a new window]   Fig. 8.   Effects of cAMP or parathyroid hormone (PTH) on 1,25(OH)2D3-induced transcription on rat 24OHase promoter chloramphenicol acetyltransferase (CAT) construct 671/+74. A: DCT cells were treated with CAT construct of rat 24OHase promoter 671/+74 [which contains both vitamin D response elements (VDREs) at 258/244 and 150/136], using calcium phosphate DNA precipitation method described in MATERIALS AND METHODS. Transfected cells were treated with vehicle (0.1% ethanol; D) or 107 M 1,25(OH)2D3 in presence (cAMP + D) or absence (+D) of 1 mM 8-bromo-cAMP for 24 h. CAT assay was performed and -galactosidase activity was used for normalization. B: DCT cells were transfected with 671/+74 phCAT. Transfected cells were treated with indicated concentrations of 1,25(OH)2D3 in presence () or absence () of 1 mM 8-bromo-cAMP for 24 h. CAT assay was performed, and data from 3 independent experiments (mean ± SE) are expressed as percentage of maximal response. Cells treated with 1,25(OH)2D3 + cAMP had increased CAT activity at all doses of 1,25(OH)2D3 (P < 0.01). Cells treated with 107 M 1,25(OH)2D3 exhibited 36 ± 2-fold induction of CAT activity over control. C: DCT cells were transfected with 671/+74 phCAT. Transfected cells were treated with indicated concentrations of 1,25(OH)2D3 in presence () or absence () of PTH (10 nM) for 24 h. CAT assay was performed, and data are expressed as percentage of maximal response. Note for CAT assays done using 671/+74 24OHase promoter construct and transfection in DCT cells, basal levels were undetectable.

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View larger version (39K): [in this window] [in a new window]   Fig. 9.   A: nucleotide sequence of 5'-flanking region (400/110) of rat 24OHase gene. VDREs are underlined; putative CREs are in bold. B: CAT constructs of 24OHase promoter (1,367/+74) and deletion mutant used in transient transfection experiments. Putative CREs are indicated as solid squares; proximal and distal CREs were also identified by Zierold et al. (62) by computational analysis as putative CREs in rat 24OHase promoter; sequence of middle putative CRE is similar to sequence of novel CRE identified in rat osteocalcin promoter (57); solid triangle represents mutated putative CRE sequence. Two VDREs are indicated as open squares.

View larger version (18K): [in this window] [in a new window]   Fig. 10.   Effect of cAMP on 1,25(OH)2D3-induced transcription of rat 24OHase promoter CAT constructs 160/+3, 160m/+3, or rat 24OHase VDRE (151/137) thymidine kinase (tk) CAT construct. A: DCT cells were transfected with rat 24OHase promoter CAT construct 160/+3. Transfected cells were treated with indicated concentrations of 1,25(OH)2D3 in presence () or absence () of 1 mM 8-bromo-cAMP for 24 h. CAT assays were performed, and results are expressed as percentage of maximal response (mean of duplicate experiments). Cells treated with 107 M 1,25(OH)2D3 exhibited average 4.8-fold induction in CAT activity. B: DCT cells were transfected with rat 24OHase promoter CAT construct 160m/+3 (mutated putative CRE at 132/125). Transfected cells were treated with indicated concentrations of 1,25(OH)2D3 in presence () or absence () of 8-bromo-cAMP for 24 h. CAT assays were performed, and results are expressed as percentage of maximal response (mean of duplicate experiments). Cells transfected with 160m/+3 phCAT and treated with 107 M 1,25(OH)2D3 exhibited an average 4.2-fold induction in CAT activity. C: 3 copies of proximal VDRE (151/137) were linked to tkCAT reporter gene construct. MDCT cells were transfected with 8 µg VDRE-tkCAT. Cells transfected with VDRE-tkCAT construct and treated with 107 M 1,25(OH)2D3 exhibited a 9.3 ± 2-fold induction in CAT activity. Transfected cells were treated with indicated concentrations of 1,25(OH)2D3 in presence () or absence () of 1 mM 8-bromo-cAMP for 24 h. CAT assay was performed and data from 3 independent experiments (mean ± SE) are expressed as percentage of maximal response. Cells treated with 1,25(OH)2D3 + cAMP had increased CAT activity at all doses of 1,25(OH)2D3 (P < 0.01).

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	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Although 24OHase is primarily localized in the proximal tubule, recent immunocytochemical studies (32, 38) as well as studies localizing 24OHase mRNA using microdissected nephron segments (29) and the findings we report in this study strongly suggest that the proximal tubule is not the exclusive site of localization of 24OHase in the kidney. It is possible in previous studies, because of the difficulty of microdissecting DCT, which is of very short length and comprises only a small fraction of the tubular structure of the renal cortex, that low levels of 24OHase activity in the distal tubule went undetected. In addition, low levels of 24OHase activity may only be detected in the microdissected distal nephron under conditions in which it may be maximally induced at this site, for example under low dietary calcium conditions, as was suggested by studies of 24OHase mRNA in discrete nephron segments (29).

In our studies in DCT cells, we found that PTH or cAMP potentiated rather than inhibited the induction of 24OHase mRNA by 1,25(OH)₂D₃. The effect of cAMP and PTH in DCT cells is in contrast to the effect in cells that have characteristics of the proximal tubule (40), suggesting differential regulation of 24OHase in different sites of the nephron. Previous studies of PTH effects using kidney homogenates (52, 56), primary renal cultures from total kidney (24, 26), or renal slices (4) would not reflect differences in hormonal regulation of 24OHase between the proximal and distal tubule particularly because markedly lower levels of 24OHase protein and mRNA have been reported to be present in the distal nephron compared with the levels in the proximal tubule (29, 32). The biological significance of the specific action of PTH in vitamin D metabolism in the DCT may be related to the interaction between 1,25(OH)₂D₃ and PTH on calcium transport in the DCT. The major effect of 1,25(OH)₂D₃ at concentrations of 10⁹ and 10¹⁰ M is to shorten the time course of PTH-dependent Ca²⁺ uptake (21). 1,25(OH)₂D₃ alone was reported to have no effect on distal tubular Ca²⁺ uptake (21). Assuming a biological function of 24OHase is to control the intracellular level of 1,25(OH)₂D₃, it is reasonable to hypothesize that 1,25(OH)₂D₃ and PTH may work cooperatively first to enhance calcium uptake at the distal tubule and then to catabolize 1,25(OH)₂D₃ at higher concentrations of the hormone (>10⁹ M; Fig. 6), preventing a hypercalcemic effect by resulting in a reduction of cellular 1,25(OH)₂D₃ and therefore a suppression of the enhanced calcium uptake. Consistent with this hypothesis, higher concentrations of 1,25(OH)₂D₃ were found to have decreased effects on calcium uptake (21). Taken together, these data suggest that the different biological functions of PTH in the DCT and the PCT result in discrete interactions between PTH and 1,25(OH)₂D₃ on 24OHase in the different nephron segments.

Although a cAMP response region was reported in the promoter of the vitamin D-responsive osteocalcin gene (57), and cAMP response regions have been identified in the promoters that direct the expression of enzymes important in steroidogenesis [steroid 21-hydroxylase (61), aromatase (20), and cholesterol side chain cleavage enzyme (16)], we did not find that the cAMP modulation of 1,25(OH)₂D₃ induction of 24OHase expression in DCT cells was mediated via CREs in the 24OHase promoter. It is possible that CREs in the 24OHase promoter may be important for the inhibitory effect of PTH in the proximal tubule and that the mechanisms involved in the regulation of 24OHase by cAMP may be cell type specific. Further studies using cell lines derived from the proximal tubule would be needed to examine the molecular mechanisms involved in a negative regulation of 24OHase by PTH. Thus far, however, very few studies have been done related to the regulation of 24OHase as well as VDR expression using cell lines derived from kidney tubules. Besides our study using the distal tubular cell line, previous studies have been done using two cell lines of proximal tubular origin, the opossum kidney cell line OK (30) and the monkey kidney cell line JTC-12 (40). Using JTC-12 cells, a 30% reduction in 1,25(OH)₂D₃-induced 24OHase activity was observed in the presence of PTH (40). The effect of PTH or cAMP on 24OHase expression was not studied in OK cells (30). However, Northern analysis of mRNA from OK cells or JTC-12 cells treated with 1,25(OH)₂D₃ indicated only very weak hybridization with the rat VDR and 24OHase cDNAs (Yang and Christakos, unpublished observation). In more recent studies by Reinholz and DeLuca (49) using the AOK-B50 cell line (a subset of LLC-PK₁ porcine renal epithelial cell line that stably expresses opossum receptors for PTH), PTH inhibited 24OHase mRNA in a dose-dependent manner. The authors suggest that the AOK-B50 cell line is the first cell line in which regulation of 24OHase mRNA by PTH resembles in vivo regulation in the proximal tubule. Thus this cell line should be useful in future studies characterizing the molecular mechanisms involved in the regulation of 24OHase by PTH in the proximal tubule, which may involve, in part, transcriptional regulation via CREs in the 24OHase promoter.

In our study using DCT cells derived from the distal tubule, we found that regulation of VDR levels by cAMP, and not an effect on CREs in the 24OHase promoter, is one mechanism by which cAMP and PTH may modulate 1,25(OH)₂D₃-induced transcription of 24OHase. It has been reported that VDR abundance is one of the major factors determining the level of response to 1,25(OH)₂D₃ and that homologous regulation of VDR, as well as regulation of VDR by signal transduction pathways, may play an important role in modulating target cell responsiveness to 1,25(OH)₂D₃ (36). In our study we found, similar to reports in other cell lines and in transformed yeast cells (2, 37, 51, 60), that 1,25(OH)₂D₃ treatment resulted in an upregulation of VDR protein levels, but VDR mRNA was not affected by 1,25(OH)₂D₃, suggesting that induction of VDR protein by 1,25(OH)₂D₃ is due to altered stability of the occupied receptor. 8-Bromo-cAMP treatment, however, resulted in an increase in VDR mRNA, as well as an enhancement of 1,25(OH)₂D₃ induction of VDR protein levels and a potentiation of the effect of 1,25(OH)₂D₃ on 24OHase mRNA and transcription. In previous studies, elevation of intracellular cAMP levels in NIH-3T3 mouse fibroblasts (36), mouse osteoblasts (MC3T3-E1 cells) (35), or in rat osteosarcoma cells (UMR106-01 cells) (35) was also reported to result in an induction in VDR mRNA and protein. Similar to our studies, the change in VDR by agents that raise intracellular cAMP in NIH-3T3 cells (36) and in UMR cells (3, 35) corresponded to an enhanced functional response (3, 35, 36). However, it should be noted that opposite findings concerning the effect of activation of protein kinase A (PKA) on VDR have been reported by others. In rat osteosarcoma cells (ROS17/2.8), which exhibit a more osteoblastic phenotype than MC3T3-E1 or UMR106 cells, PTH or forskolin has been shown to downregulate VDR (48), suggesting that cell type, proliferation state, and stage of differentiation may affect the interaction between 1,25(OH)₂D₃ and the PKA pathway. Cell type specificity of VDR regulation under conditions that result in an elevation of PTH was also noted in in vivo studies reported by Iida et al. (29) using microdissected rat nephron segments. In PCTs, VDR mRNA was found to be markedly downregulated to barely detectable levels under conditions of low dietary calcium, resulting in a marked inhibition of 24OHase mRNA. However, in the distal nephron under low dietary calcium conditions, VDR mRNA was not downregulated and 24OHase mRNA was found to be induced. Although the exact mechanism of regulation of VDR mRNA in the microdissected nephron segments was not clearly defined, the authors suggested that intracellular signaling caused by PTH in response to hypocalcemia may be an important determining factor involved. It will be of interest in future studies to examine the promoter of the VDR gene for CREs and to determine the role of coactivators, such as CRE-binding protein, and the role of phosphorylation of VDR and other transcriptional coactivators that may be involved as part of the mechanism underlying the interaction between 1,25(OH)₂D₃ and the PKA signaling pathway. The role of cell type-specific factors that may be involved in the downregulation of VDR mRNA in the PCT but not in the DCT also needs to be considered. In addition, the interaction between 1,25(OH)₂D₃ and the PKA pathway may not only be cell type specific but may also be gene specific and may involve effects not only on VDR levels but also on the promoter of certain target genes in specific cell types.

It should be noted that besides modulation via the cAMP-dependent PKA signal pathway, evidence also exists for protein kinase C involvement in the regulation of renal 24OHase (12, 25, 26, 39). The phorbol ester TPA has been reported to increase the production of 24,25(OH)₂D₃ and to decrease the production of 1,25(OH)₂D₃ (12, 25, 39). It has been suggested that PKC and PKA act through independent mechanisms to alter the production of 1,25(OH)₂D₃ (25). In our studies in DCT cells we found, similar to the report of Chen et al. (12) using primary cultures of rat kidney cells, that TPA enhanced the 1,25(OH)₂D₃-induced increase in 24OHase mRNA (Fig. 1). TPA increased the rapidity of the response to 1,25(OH)₂D₃, and dose-response studies indicated a shift to the left in the presence of TPA. Unlike studies with 8-bromo-cAMP or PTH, TPA resulted in a significant decrease in VDR mRNA at 1, 3, and 6 h after treatment, and Western blot analysis did not indicate an upregulation of VDR in the presence of TPA (Yang and Christakos, unpublished observations). These findings suggest that the effect of TPA on 24OHase is not mediated by an effect on new receptor synthesis but rather may be due to effects on regulatory regions in the 24OHase promoter and/or an effect on DNA binding to transcription factors.

In summary, although the PCT is the major site of localization of 24OHase, our findings provide evidence for the first time that 24OHase mRNA, protein, and activity can be localized in the distal nephron. In DCT cells both cAMP and PTH modulate the 1,25(OH)₂D₃-induced expression of 24OHase in a manner different from that reported in the PCT, suggesting different roles for PKA activation in the DCT and PCT. We suggest that 24OHase in the DCT may play a role in modulating 1,25(OH)₂D₃ action on calcium transport by controlling cellular 1,25(OH)₂D₃ levels. In DCT cells, regulation of VDR levels by cAMP, and not an effect on CREs in the 24OHase promoter, is one mechanism by which cAMP modulates 1,25(OH)₂D₃-induced transcription of 24OHase.