Expression of 25(OH)D3
24-hydroxylase in distal nephron: coordinate regulation by
1,25(OH)2D3
and cAMP or PTH
Wen
Yang,
Peter A.
Friedman,
Rajiv
Kumar,
John L.
Omdahl,
Brian K.
May,
Mei-Ling
Siu-Caldera,
G. Satyanarayana
Reddy, and
Sylvia
Christakos
Department of Biochemistry and Molecular Biology, University of
Medicine and Dentistry of New Jersey-New Jersey Medical School and
Graduate School of Biomedical Sciences, Newark, New Jersey 07103;
Department of Pharmacology and Toxicology, Dartmouth Medical School,
Hanover, New Hampshire 03755; Department of Medicine, Mayo Clinic and
Foundation, Mayo Medical School, Rochester, Minnesota 55905;
Departments of Medicine and Biochemistry, University of New Mexico,
Albuquerque, New Mexico 87131; Department of Biochemistry, University
of Adelaide, Adelaide, South Australia 5025; and Department of
Pediatrics, Women and Infants Hospital of Rhode Island, Providence,
Rhode Island 02905
 |
ABSTRACT |
Previous studies
using microdissected nephron segments reported that the exclusive site
of renal 25-hydroxyvitamin
D3-24-hydroxylase (24OHase)
activity is the renal proximal convoluted tubule (PCT). We now report
the presence of 24OHase mRNA, protein, and activity in cells that are
devoid of markers of proximal tubules but express characteristics
highly specific for the distal tubule. 24OHase mRNA was undetectable in
vehicle-treated mouse distal convoluted tubule (DCT) cells but was
markedly induced when DCT cells were treated with 1,25 dihydroxyvitamin
D3
[1,25(OH)2D3].
24OHase protein and activity were also identified in DCT cells by
Western blot analysis and HPLC, respectively. 8-Bromo-cAMP (1 mM) or
parathyroid hormone [PTH-(1
34); 10 nM] was found to
potentiate the effect of
1,25(OH)2D3
on 24OHase mRNA. The stimulatory effect of cAMP or PTH on 24OHase
expression in DCT cells suggests differential regulation of 24OHase
expression in the PCT and DCT. In the presence of cAMP and
1,25(OH)2D3,
a four- to sixfold induction in vitamin D receptor (VDR) mRNA was
observed. VDR protein, as determined by Western blot analysis, was also
enhanced in the presence of cAMP. Transient transfection analysis in
DCT cells with rat 24OHase promoter deletion constructs demonstrated
that cAMP enhanced
1,25(OH)2D3-induced 24OHase transcription but this enhancement was not mediated by cAMP
response elements (CREs) in the 24OHase promoter. We conclude that
1) although the PCT is the major
site of localization of 24OHase, 24OHase mRNA and activity can also be
localized in the distal nephron; 2)
both PTH and cAMP modulate the induction of 24OHase expression by
1,25(OH)2D3
in DCT cells in a manner different from that reported in the PCT; and
3) in DCT cells, upregulation of VDR
levels by cAMP, and not an effect on CREs in the 24OHase promoter, is
one mechanism involved in the cAMP-mediated modulation of 24OHase transcription.
vitamin D regulation; parathyroid hormone
 |
INTRODUCTION |
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 |
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 (1
34) 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
(10
7 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'-CTAGGAGGCCCC
CACT
CGCGACTCATGTCCT-3' and
5'CTAGAGGACATGAGTCG
CCGGGGCCTC-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 |
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 (10
7 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
(10
7 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).

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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
(10 7 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.
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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
(10 7 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.
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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
(10 7 M, 24 h) and probed
with antibody against thiazide-sensitive NaCl transporter
(A) or rat 24OHase
(B), respectively.
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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
(10 7M)
(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.
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Table 1.
3H-labeled metabolites of 25(OH)D3 produced by
DCT cells incubated with [3H]25(OH)D3 after
pretreatment with 1,25(OH)2D3 or
1,25(OH)2D3 + 8-bromo cAMP
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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.
DCT cells were treated with
10
7 M
1,25(OH)2D3
or 10
7 M
1,25(OH)2D3 + 1 mM 8-bromo-cAMP. Results of Northern blot analysis are shown in
Fig. 5. Marked induction in 24OHase mRNA by
1,25(OH)2D3 alone was observed at 12 h, although quantitatively minor induction was
observed after longer autoradiographic exposure as early as 6 h. The
maximal response was observed at 48 h. However, VDR mRNA levels
remained unchanged during this period (Fig. 5,
A and
B). In the presence of both
1,25(OH)2D3
and cAMP, 24OHase mRNA was induced as early as 3 h, reached a plateau
from 6 h to 24 h, and then decreased at 48 h (Fig. 5,
C and
D). Thus cAMP enhanced the rapidity
of 24OHase mRNA expression induced by
1,25(OH)2D3.
HPLC analysis indicated that pretreatment of DCT cells with
10
7 M
1,25(OH)2D3 + 1 mM 8-bromo-cAMP for 24 h also resulted in an induction in the
production of metabolites of
[3H]25(OH)D3
produced by 24OHase over the levels observed in the presence of
1,25(OH)2D3
alone
[1,25(OH)2D3 + cAMP/1,25(OH)2D3 = 1.8-fold for the production of
23(S),25(OH)2-24-oxo-D3
and 2.4-fold for the production of
24(R),25(OH)2D3
(Table 1)]. In contrast to the treatment with
1,25(OH)2D3
alone, cotreatment with cAMP resulted in an upregulation of VDR mRNA
expression (Fig. 5, C and
D). In the presence of cAMP, the
first significant induction in VDR mRNA was at 3 h. VDR mRNA levels
reached a plateau after 6 h. The effect of cAMP on VDR mRNA expression
suggests that cAMP may mediate the enhanced induction of 24OHase mRNA
by
1,25(OH)2D3 through upregulation of VDR levels.

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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
10 7 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
(10 7 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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The induction of 24OHase mRNA by
1,25(OH)2D3
was dose dependent, and cAMP potentiated the effect of
1,25(OH)2D3
(Fig. 6). At
10
8 M
1,25(OH)2D3,
24OHase mRNA was clearly observed in the cells treated with
1,25(OH)2D3 + cAMP but was detectable in the cells treated with
1,25(OH)2D3
alone only after longer autoradiographic exposure. Increasing the
concentration of
1,25(OH)2D3
from 10
9 M to
10
6 M did not significantly
affect the induction of VDR mRNA by cAMP (Fig. 6). Further Northern
analyses performed using poly(A)+
RNA from DCT cells treated for 24 h in the presence of 1 mM
8-bromo-cAMP alone indicated that cAMP alone was able to induce VDR
mRNA 5.5 ± 1-fold over basal levels (data not shown), equivalent to
the induction observed with
1,25(OH)2D3 + cAMP (Fig. 6B). These findings suggest that the effect of cAMP on VDR mRNA expression is independent of the
1,25(OH)2D3
concentration. In summary, cAMP not only accelerated 24OHase mRNA
expression induced by
1,25(OH)2D3,
but it also shifted the dose-response curve to the left so that
1,25(OH)2D3
was effective at lower concentrations.

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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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Similar to the effect of 8-bromo-cAMP, when DCT cells were treated with
1,25(OH)2D3
alone (10
7 M) or
1,25(OH)2D3
in the presence of PTH (10 nM, 1
34) for 12 h, PTH upregulated VDR
mRNA by 3.3-fold and also potentiated
1,25(OH)2D3 induction of 24OHase mRNA expression by 6.4-fold (results are the mean
of two separate experiments; data not shown).
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.

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Fig. 7.
Effect of
1,25(OH)2D3
and cAMP on VDR protein content. DCT cells were treated with vehicle
0.1% ethanol ( D),
10 7 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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Effect of cAMP on transcriptional activity of 24OHase
gene. When a series of deletion mutant constructs that
contained the fragments of the 5'-flanking region of rat 24OHase
gene (
1,367/+74,
671/+74,
291/+74) were
transfected into DCT cells, CAT expression was stimulated significantly
by
1,25(OH)2D3
(10
7 M, 24 h). All the
constructs responded to a threefold potentiation of the
1,25(OH)2D3
effect in the presence of 8-bromo-cAMP (1 mM). However, cAMP (1 mM
8-bromo-cAMP) or PTH (10 nM) alone was found to have no effect on the
transcription of the rat 24OHase gene [CAT activity was not
significantly different from basal CAT activity (not shown)]. The
effect of cAMP on
1,25(OH)2D3-induced
transcription using
671/+74 phCAT is shown in Fig.
8, A and
B. These results demonstrate that
modulation of the
1,25(OH)2D3
induction of 24OHase mRNA by cAMP is at the transcriptional level.
Similarly, PTH was observed to potentiate the dose-dependent activation
of transcription of the 24OHase gene by
1,25(OH)2D3
(Fig. 8C).

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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
10 7 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
10 7 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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Towler and Rodan (57) have reported that cAMP upregulated the
transcription of osteocalcin, a vitamin D-dependent gene, through a
novel CRE. Whether the 24OHase promoter contains a functional CRE,
which mediates cAMP action on 24OHase expression, was not known. We
examined the rat 24OHase promoter for consensus CREs. Three putative
CREs were found by computational analysis (Fig. 9A; Refs.
57, 62). To evaluate the functional activity of each CRE, a series of
deletion mutant fragments of the 5'-flanking region of rat
24OHase gene were linked to a CAT reporter gene, which resulted in the
elimination of the first two CREs. Because the third possible CRE was
located between the proximal VDRE and the start site of transcription,
random mutagenesis of this site was done to determine whether this site
was involved in the enhancement of
1,25(OH)2D3-induced
transcription by cAMP. The deleted fragment and the mutated fragment
were obtained by PCR as described in MATERIALS AND
METHODS section. All the putative CREs were present in
671/+74 phCAT. The chimeric constructs
291/+74
phCAT and
160/+3 phCAT do not include the distal and middle
CREs, respectively, and the proximal putative CRE sequence was mutated
in the construct
160m/+3 phCAT (Fig.
9B). The synthesized constructs were
transfected into DCT cells and treated with
1,25(OH)2D3
(10
10-10
7
M) in the presence or absence of cAMP (1 mM), and transcriptional activity was assessed using the CAT assay.

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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.
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Using
160/+3 phCAT (only one putative CRE) and
160m/+3
phCAT (the one putative CRE mutated), at all concentrations of
1,25(OH)2D3, treatment with
1,25(OH)2D3 + cAMP exhibited a three- to fourfold higher CAT activity than
1,25(OH)2D3
alone (Fig. 10,
A and
B). Both constructs retained the
cAMP effect, similar to what was observed using
1,367/+74 phCAT
(not shown),
671/+74 phCAT (Fig. 8), and
291/+74 phCAT
(not shown), resulting in a potentiation of
1,25(OH)2D3-induced
transcriptional activity by a similar degree (three- to fourfold).
These results suggest that the putative CREs are not primarily involved
in the enhancement of
1,25(OH)2D3-induced 24OHase mRNA expression by cAMP.

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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
10 7 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
10 7 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
10 7 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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To rule out the possibility that other sequences in the rat 24OHase
gene may mediate the cAMP effect, we prepared a VDRE/tkCAT construct by
introducing multiple copies of the proximal VDRE (
150/
136) of the rat 24OHase promoter into the CAT
plasmid. Figure 10C shows that this
construct responded to
1,25(OH)2D3
and cAMP, indicating that the VDRE of the rat 24OHase gene, in the absence of other sequences in the 24OHase promoter, is able to confer
cAMP enhancement. In addition, when a construct (
186/+74) was
used with the
150/
136 VDRE mutated (M1) as well
as an additional construct (
298/+74) with the proximal as well
as the distal VDRE mutated (M4; see Ref. 34), no increase in
transcription greater than 1.6-fold over basal levels was observed in
the presence of 1,25(OH)2D3
alone (10
8 M), 8-bromo-cAMP
alone (1 mM), or
1,25(OH)2D3 + cAMP [M1:
1,25(OH)2D3 (1.6 ± 0.2-fold), cAMP (1.6 ± 0.3-fold),
1,25(OH)2D3 + cAMP (1.4 ± 0.2-fold over basal); M4:
1,25(OH)2D3
(1.5 ± 0.1-fold), cAMP (1.3 ± 0.1-fold),
1,25(OH)2D3 + cAMP (1.4 ± 0.1-fold over basal)]. The results of these
experiments provide additional evidence suggesting that regions in the
24OHase promoter other than the VDRE are not primarily involved in the
marked enhancement of
1,25(OH)2D3-induced 24OHase transcription by cAMP.
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DISCUSSION |
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 10
9
and 10
10 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 (>10
9 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.
 |
ACKNOWLEDGEMENTS |
We acknowledge the secretarial assistance of Connie Sheffield and
Sharon Washington. We also gratefully acknowledge the assistance of
Bonita A. Coutermarsh in certain aspects of this investigation.
 |
FOOTNOTES |
This study was supported in part by National Institutes of Health
grants to S. Christakos (DK-38961) and P. A. Friedman (DK-54171), by a
grant from the Australian Research Council to B. K. May, and by a grant
from the University of New Mexico Medical Trust Funds to J. L. Omdahl.
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: S. Christakos,
Dept. of Biochemistry and Molecular Biology, UMDNJ-New Jersey Medical
School, 185 South Orange Ave., Newark, NJ 07103-2714
(E-mail:christak{at}umdnj.edu).
Received 14 August 1998; accepted in final form 5 January 1999.
 |
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