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-Hydroxylase in a Transformed Human Proximal Tubule Cell Line: Evidence for Direct Regulation of Vitamin D Metabolism by Calcium1
Department of Medicine, Institute of Clinical Research, University of Birmingham, Birmingham, United Kingdom B15 2TT
Address all correspondence and requests for reprints to: Dr. M. Hewison, Department of Medicine, Queen Elizabeth Hospital, Edgbaston, Birmingham, United Kingdom B15 2TH. E-mail: m.hewison{at}bham.ac.uk
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-hydroxylase (1-hydroxylase). Production of
1,25-(OH)2D3 occurs predominantly in the renal
proximal tubule, with 1-hydroxylase activity being impaired in renal
insufficiency and renal disease. The expression and activity of
1-hydroxylase are tightly regulated in response to serum levels of
PTH, calcium, phosphate, and 1,25-(OH)2D3
itself. As a consequence of this, the characterization of
1-hydroxylase in human renal tissue has proved difficult. In this
study we have characterized constitutive 1-hydroxylase expression in
a simian virus 40-transformed human proximal tubule cell line, HKC-8.
Initial analyses of [3H]25-hydroxyvitamin D3
(25OHD3) metabolism in these cells using straight and
reverse phase HPLC revealed product peaks that coincided with authentic
1,25-(OH)2D3 as well as 24,25-dihydroxyvitamin
D3 (24,25-(OH)2D3). Enzyme kinetic
studies indicated that the Km for synthesis of
1,25-(OH)2D3 in HKC-8 cells was 120 nmol/liter
25OHD3, with a maximum velocity of 21 pmol/h/mg protein.
This activity was inhibited by treatment with ketoconazole, but not
diphenyl phenylenediamine. RT-PCR analysis of RNA from HKC-8 cells
revealed a transcript similar in size to that observed in keratinocytes
and primary cultures of human proximal tubule cells, and protein was
detected by Western blot analysis. Synthesis of
1,25-(OH)2D3 was up regulated by treatment with
forskolin (10 µmol/liter, 24 h) and was down-regulated by
1,25-(OH)2D3 (10 nmol/liter, 24 h).
1-Hydroxylase activity in HKC-8 cells was also sensitive to the
concentration of calcium. Cells grown in low calcium (0.5 mmol/liter)
showed a 4.8-fold induction of 1-hydroxylase, whereas treatment with
medium containing high levels of calcium (2 mmol/liter) significantly
inhibited 1,25-(OH)2D3 production. These data
suggest that direct effects of calcium on proximal tubule cells may be
an important feature of the regulation of renal
1,25-(OH)2D3 production. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-hydroxylase (1-hydroxylase)
(1). Although 1-hydroxylase activity has been demonstrated at
several ectopic sites (2), circulating levels of
1,25-(OH)2D3 appear to be dependent
on the expression of this enzyme in the proximal tubules of the kidney
(3, 4). The recent cloning of mouse (5), rat (6, 7), and human (8, 9)
complementary DNAs (cDNAs) for 1-hydroxylase has revealed
homology with the liver enzyme 25-hydroxylase (10), and the
ubiquitously expressed 24-hydroxylase (11). Mutations causing the
inherited disorder vitamin D-dependent rickets type 1, also known as
pseudovitamin D deficiency rickets have been described for the
1-hydroxylase gene, and these have been mapped to chromosome 12q14
by linkage analysis (8, 12).
Renal production of 1,25-(OH)2D3
plays a pivotal role in maintaining serum calcium homeostasis by
enhancing intestinal calcium and phosphate absorption. Consequently,
the expression and activity of 1-hydroxylase are tightly regulated.
Peptide factors such as PTH (13, 14) and insulin-like growth factor I
(15) have been shown to enhance 1-hydroxylase activity, whereas
1,25-(OH)2D3 itself has inhibitory
effects (16). The metabolism of 25OHD3 is also modulated by
serum calcium and phosphate (17). Regulation of 1-hydroxylase by
calcium is mediated at least in part via stimulation of PTH secretion
by the parathyroid glands (18). However, previous studies in
vitro and in vivo have suggested that calcium may also
exert direct regulatory effects on vitamin D metabolism by the kidney
(14, 19, 20). Furthermore, the membrane calcium-sensing receptor (CaR),
which mediates calcium effects on the parathyroid chief cells, is also
expressed by renal cells, including proximal tubules (21).
To date, analysis of the mechanisms that control renal
1,25-(OH)2D3 production has proved
difficult as a result of the apparent low expression and activity of
1-hydroxylase in normal tissue. In vitro model systems
have been described for normal mouse (14) as well as rachitic chick
kidney cells (22, 23). Most recently, the cloning of a cDNA for mouse
1-hydroxylase was achieved using renal tissue isolated from vitamin
D receptor (VDR) null mice, which have high endogenous levels of
1-hydroxylase activity (5). In contrast, the human gene was
initially isolated from keratinocytes, a nonclassical source of
1-hydroxylase activity (8). Keratinocytes produce significant levels
of 1,25-(OH)2D3 during early stages
of development, and this appears to be a function of cell
differentiation, VDR levels, and coexpression of 24-hydroxylase.
However, it seems likely that the regulation of 1-hydroxylase in
keratinocytes (as well as other nonclassical sites) will be different
from that observed in the kidney (2).
Relatively few human model systems exist for the study of renal vitamin
D and mineral homeostasis in vitro. However, in a recent
report Racusen and colleagues have described simian virus 40 viral
immortalization of renal tubule epithelial cells isolated from normal
kidney cortex (24). Several cell lines were isolated that expressed
markers of the renal proximal tubular epithelium and were biochemically
comparable to other widely used proximal tubular cell lines, including
LLC-RK1, OK, and HK-2. In studies presented here we have used one of
these cell lines (HKC-8) as an in vitro model for renal
vitamin D metabolism. Data confirm the presence of messenger RNA (mRNA)
and protein for 1-hydroxylase in HKC-8 cells and indicate that the
activity of this enzyme is responsive to changes in calcium levels
in vitro.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Measurement of 1- and 24-hydroxylase activities
HPLC. Cells were cultured to 80% confluence in defined
medium as described above and washed twice with serum-free DMEM/F12
medium. Assays were then carried out by incubating the cell monolayers
with 5 nmol/liter [3H]25OHD3 (180 Ci/mmol;
Amersham, Aylesbury, UK) in 400 µl of serum-free
DMEM/F12 for 5 h at 37 C. Dose-dependency studies were carried out
using varying concentrations of [3H]25OHD3
(5–500 nmol/liter). Time-course studies were carried out using
incubation periods of 1–8 h. Preliminary analyses of the inhibition of
1,25-(OH)2D3 production were carried
out by preincubating cells for 2 h with the antioxidant diphenyl
phenylenediamine (DPPD) or the cytochrome P450 inhibitor, ketoconazole.
Each of these agents was added in 0.1% ethanol at 10 µmol/liter, and
each was included in the final substrate incubation mix at the same
concentration. All assays were terminated by freezing cells at -20 C.
Metabolites of [3H]25OHD3 were extracted from
assay mixtures (both medium and cell monolayers) by addition of 2.5 ml
chloroform-methanol (4:1, vol/vol) and vigorous vortexing. Solvent
fractions were separated, dried under nitrogen, and resuspended in 20
µl HPLC running solvent (see below). Vitamin D metabolites were
initially separated by straight phase HPLC using a Zorbax-sil column
(4.6 x 250 mm) eluted with a mixture of
hexane-methanol-isopropanol (92:4:4, vol/vol/vol) at 2 ml/min.
Fractions were collected at 30-sec intervals, and aliquots of each
fraction were assessed for radioactivity by scintillation counting.
Fractional counts per min were then plotted against an elution profile
for unlabeled 25OHD3,
24,25-(OH)2D3,
25,26-(OH)2D3, and
1,25-(OH)2D3. Production of
radiolabeled vitamin D metabolites was measured as picomoles per h/mg
cellular protein. To confirm the authenticity of
1,25-(OH)2D3 produced by HKC-8 cells,
reverse phase HPLC separation of appropriate straight phase fractions
was performed on a Zorbax-ODS column (6.2 x 250 mm) eluted with a
mixture of methanol and water (4:1, vol/vol) at 2 ml/min.
TLC. TLC analysis of hydroxylated metabolites of vitamin D3 was performed as described previously (25) using an adapted method. For analysis of enzyme activity studies, HKC-8 cells were seeded in 24-well plates (5 x 104/well) and grown to 50% confluence before transfer to defined medium (400 µl). Cells were incubated with 3.75 nmol/liter [3H]25OHD3 (normal and high calcium concentrations) or 11.25 nmol/liter 25OHD3 (1:2, [3H]25OHD3-25OHD3; for low calcium concentrations) for 4 h at 37 C. The reaction was terminated by freezing at -20 C. Cell extracts and medium were combined, and vitamin D metabolites were extracted in 2.5 ml chloroform-methanol (4:1, vol/vol). After evaporation of the organic phase, steroids were resuspended in 50 µl dichloromethane and separated on silica TLC plates in dichloromethane-isopropanol (9:1, vol/vol). Standard lanes were included that contained only [3H]25OHD3 or [3H]1,25-(OH)2D3. Conversion of tritiated steroid was measured on a Bioscan, Inc. System 200 imaging TLC plate scanner (Bioscan, Inc., Edmonds, WA). Residual cell monolayers from parallel wells were lysed and assayed for total cellular protein using a Bio-Rad Laboratories, Inc., protein assay (Bio-Rad Laboratories, Inc., Melville, NY).
Experimental treatments. Cells maintained in the presence of 5% FCS were transferred to defined medium 24 h before treatments. Cells were the treated with forskolin (10 µmol/liter), 1,25-(OH)2D3 (10 nmol/liter), or PTH (100 ng/ml) for 24 h or with varying medium calcium concentrations (0–2 mmol/liter) for 4–24 h. [3H]25OHD3 was included for the final 4 h. Vitamin D3 metabolites were extracted as described above.
Analysis of mRNA expression
Preparation of RNA. RNA was prepared from 80% confluent
cells by acid-guanidinium extraction (26)
RT-PCR. RT of 1 µg RNA was performed using a Promega Corp. Reverse Transcription System (Madison, WI), using a gene-specific downstream primer (5 µmol/liter) following the manufacturer’s protocol. An aliquot of 5 µl of this reaction was then used in subsequent (PCR) reactions.
PCR. Analysis of 1-hydroxylase mRNA expression was
carried out using the following primers: 5',5'-ACGCTGTTGACCATGGC-3';
and 3',5'-GTGACACAGAGTGACCAGCATAT-3'. PCR reactions (20 µl) were set
up using 1 x (final concentration) PCR buffer containing 50
mmol/liter KCl, 10 mmol/liter Tris-HCl (pH 9.0), 0.1% Triton X-100,
1.5 mmol/liter MgCl2, 0.2 mmol/liter of each deoxy-NTP, 0.5
µmol/liter of each primer, and 1 U of Taq DNA polymerase
(Promega Corp.). Amplification of samples was performed
using an initial denaturation step of 95 C for 4 min, followed by 35
cycles of 95 C (1 min), 60 C (1.5 min), and 72 C (2 min). A final
elongation step of 72 C for 7 min was also included. Purified PCR
products were sequenced by direct chain termination sequencing on an
automatic DNA sequencer (Alta Bioscience, Birmingham, UK).
Northern analysis. Total RNA (20 µg/lane) was loaded into a denaturing 1.5% formaldehyde-agarose gel and resolved by electrophoresis (100 V; 3–4 h) before capillary transfer to Hybond N+ nylon filters (Amersham) overnight (27). Filters were prehybridized (3 h) and hybridized (18 h) at 65 C to 32P-labeled probes in a phosphate buffer containing 0.77 M NaH2PO4-Na2HPO4 (pH 7.2), 5 mmol/liter EDTA, 7% SDS, and 100 µg/ml sonicated salmon sperm DNA. After hybridization filters were washed to a final stringency of 0.1 x SSC (sodium chloride-sodium citrate)-0.1% SDS for 30 min at 65 C and then autoradiographed at -70 C for 30 min (18S RNA) or 3 days (CaR). Autoradiographs were quantified by laser densitometry (LKB 2202 Ultrascan laser densitometer, LKB, Bromma, Sweden; Hewlett-Packard Co./LKB reporting integrator 3390A Hewlett-Packard Co., Avondale, PA), and mRNA expression was standardized relative to the expression of 18S ribosomal RNA (ribosomal RNA). Probes, which included a BfaI fragment of the CaR (NPS Pharmaceuticals, Inc., Salt Lake City, UT) and a BamHI/HindIII genomic fragment specific for 18S ribosomal RNA, were labeled with [32P]deoxy-CTP (3000 Ci/mmol; Amersham) by nick translation (CaR; Amersham) or the random primer method (18S RNA; Pharmacia Biotech, St. Albans, UK).
Protein preparation
For VDR analysis, nuclear proteins were prepared as described
previously (28). For CaR expression, total cell lysates were prepared
as described below. Cells were subcultured into 75-cm2
flasks and grown until 80% confluent. Cells were removed by scraping
and were washed in PBS. Cell pellets were resuspended in 0.6 ml 0.25
M Tris-HCl (pH 7.8) containing 0.5% Nonidet P-40, 1
mmol/liter phenylmethylsulfonylfluoride (Sigma Chemical Co.) and 5 mmol/liter dithiothreitol. The cellular suspension
was vortexed briefly, then agitated at 4 C for 20 min, and the cellular
membranes were pelleted (15,000 x g at 4 C for 5 min).
Supernatants containing CaR protein were aliquoted and stored at -70
C. For 1-hydroxylase expression, cells were subcultured into
25-cm2 flasks. After treatments, cells were removed by
scraping and washed in PBS. Cell lysate protein was prepared by freeze
thawing (three times) in 0.2 ml PBS containing 0.5 mmol/liter
phenylmethylsulfonylfluoride. Cell membranes were pelleted (2,900
x g at 4 C for 5 min), and supernatants containing the
1-hydroxylase protein were aliquoted and stored at -70 C.
Western analysis
Proteins were subjected to SDS-PAGE (3 or 10 µg/lane) and
electroblotted onto an Immobilon P membrane as described previously
(28), except for CaR detection, where proteins were separated in a
7.5% resolving gel (pH 8.8) and diluted 1:1 in sample buffer. Filters
were analyzed with specific monoclonal antibodies against the human VDR
(29) (Cambridge BioScience, Cambridge, UK), and the human CaR
(NPS Pharmaceuticals, Inc.). Additional Western blots were
carried out using a polyclonal 1-hydroxylase antibody raised against
an antigenic region of the reported mouse amino acid sequence (5) (The
Binding Site Ltd., University of Birmingham, Birmingham, U.K).
Membranes were blocked (1 h; 25 C) in PBS-T (PBS plus 0.1% Tween-20;
Sigma Chemical Co.) containing 20% (wt/vol) nonfat milk
powder (Marvel, Premier Brands, Stafford, UK) and rinsed twice in PBS,
followed by an additional wash in PBS-T for 15 min. Filters were
incubated overnight at 4 C with the primary antibody diluted 1:500 (VDR
and 1-hydroxylase) in PBS-T (0.05%) and 160 ng/ml (CaR) in PBS-T
(0.05%) containing 0.1% nonfat milk (wt/vol). After three 10-min
washes in PBS-T, filters were incubated with the secondary antibody
(horseradish peroxidase conjugated, VDR and CaR, Amersham;
1-hydroxylase, The Binding Site Ltd.) diluted 1:50,000 (VDR) or
1:75,000 (1-hydroxylase) in PBS-T (0.05%) and 1:60,000 (CaR) in
PBS-T (0.05%) containing 0.1% nonfat milk (wt/vol) for 90 min at 25 C
(60 min, 1-hydroxylase) and washed for three 10-min periods in
PBS-T. Specific receptor proteins were detected by the enhanced
chemiluminescent assay (ECL, Amersham) after exposure of
filters to x-ray film for 1–20 min (VDR and CaR) and 10–30 sec
(1-hydroxylase). Autoradiographs were quantified by laser
densitometry, as described above. Control experiments were included
where primary antibody was omitted, and filters were exposed to
secondary antibody and ECL detection. An additional control was
included for the 1-hydroxylase antibody, where primary antibody was
preabsorbed with an excess of immunizing peptide. No protein bands were
detected in these controls (data not shown).
Data analysis
Assays for 1- or 24-hydroxylase were carried out in
triplicate and are reported as the mean ± SEM of at
least three experiments. Enzyme activities are quoted either as
picomoles per h/mg protein or as mean enzyme induction relative to
control values derived from untreated cells. Statistical analysis of
data was performed using one-way ANOVA linked to Tukey-Kramer multiple
comparison posttests (Instat version 2.04a computer program,
GraphPad Software, Inc., San Diego, CA).
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Results |
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). HPLC fractions that corresponded to
[3H]1,25-(OH)2D3 were
pooled, concentrated, and analyzed further by an additional separation
using reverse phase HPLC (Fig. 1B). Results confirmed that the product
obtained using nonpolar straight phase conditions continued to
comigrate with authentic 1,25-(OH)2D3
under polar reverse phase conditions. To assess more rapid methods for
analysis of [3H]25OHD3 metabolism, HKC-8
incubation mixtures were also subjected to TLC analysis, followed by
quantitative scanning TLC plate analysis. A representation of a typical
TLC separation of [3H]25OHD3 metabolism by
HKC-8 cells is shown in Fig. 1C, which shows a pattern of separation
similar to that seen with straight phase HPLC with less polar
metabolites (e.g. 25OHD3) having a greater
mobility than more polar metabolites (e.g.
1,25-(OH)2D3). Further
characterization of the kinetics of 1-hydroxylase activity in HKC-8
cells is shown in Fig. 2. Data indicated
firstly that metabolism of 25OHD3 to
1,25-(OH)2D3 in HKC-8 cells was
saturable. Analysis of the kinetics of 1-hydroxylase activity using
Lineweaver-Burk plots indicated that the Km for synthesis
of 1,25-(OH)2D3 in HKC-8 cells was
120 nmol/liter 25OHD3, with a maximum velocity of 21
pmol/h/mg protein (data not shown). The synthesis of
1,25-(OH)2D3 was unaffected by
treatment with the antioxidant DPPD, but was inhibited by coincubation
with ketoconazole. Production of
1,25-(OH)2D3 was significantly
up-regulated by treatment with forskolin (10 µmol/liter, 24 h)
and PTH (100 ng/ml, 24 h) and was down-regulated by
1,25-(OH)2D3 (10 nmol/liter, 24
h; Fig. 3A). In contrast, the activity of
24-hydroxylase was significantly stimulated by
1,25-(OH)2D3 (10 nmol/liter, 24
h), but unaffected by forskolin or PTH (Fig. 3B).
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-hydroxylase activity in HKC-8
cells, RT-PCR analysis of mRNA expression was carried out using primers
specific for human 1-hydroxylase (Fig. 4A). The data indicated the presence of a
1-hydroxylase transcript in HKC-8 cells that was similar in size to
that observed in keratinocytes and primary cultures of proximal tubule
cells. Sequence analysis of the PCR product confirmed its identity to
the published human 1-hydroxylase sequence (8). To determine whether
HKC-8 cells expressed VDR, which would be necessary for
1,25-(OH)2D3 responses, basal
expression of VDR protein was determined. Western blotting analysis
using a monoclonal antibody specific for VDR detected a single band of
57 kDa (Fig. 4B).
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-hydroxylase activity (normal medium calcium
concentration, 1 mmol/liter), leading to a significant reduction in
1,25-(OH)2D3 production at 10 and
6 h (Fig. 5A). Incubation in medium
containing 4 mmol/liter calcium completely abolished 1-hydroxylase
activity (data not shown). In contrast, incubation in medium containing
a low concentration of calcium (0.5 mmol/liter) significantly increased
1,25-(OH)2D3 synthesis (Fig. 5B). The
increase in 1-hydroxylase activity was greatest at 4 h and had
returned to control levels by 24 h. Incubation in medium
containing no calcium maintained, but did not further stimulate,
1-hydroxylase activity (data not shown). Regulation of
24-hydroxylase activity by calcium was similar to that observed for
1-hydroxylase. Incubation in medium containing 2 mmol/liter calcium
caused a small, but nonsignificant, decrease in
24,25-(OH)2D3 production (Fig. 5C).
In contrast, incubation in medium containing 0.5 mmol/liter calcium
significantly stimulated synthesis of
24,25-(OH)2D3 (Fig. 5D).
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-hydroxylase protein
in HKC-8 cells. A single band of approximately 56 kDa, the predicted
molecular mass of the 1-hydroxylase protein (8), was detected (Fig. 6). Densitometric analysis of band
intensity for the 1-hydroxylase Western blots indicated that protein
expression was decreased by 23% in the presence of 2 mmol/liter
calcium (10 h) and was decreased by 35% after treatment with
1,25-(OH)2D3 (10 nmol/liter, 24
h). Incubation with forskolin (10 µmol/liter, 24 h) produced a
47% increase in protein levels. PTH (100 ng/ml, 24 h) and low
calcium (0.5 mmol/liter, 10 h) treatments had no apparent
effect.
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). Two transcripts were detected;
a predominant transcript of approximately 2.5 kb and a much fainter
transcript of approximately 3.8 kb. Although these are smaller than
previously reported transcript species (30, 31), CaR protein of an
appropriate size (
120 kDa) was detected (Fig. 4C
). The expression of
CaR mRNA did not vary after treatment with either inhibitors
(1,25-(OH)2D3 or high calcium) or
stimulators (forskolin or low calcium) of 1-hydroxylase activity
(Fig. 7).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-hydroxylase and 24-hydroxylase. The
former is expressed predominantly in the proximal tubule of the kidney,
but is also present in several nonclassical tissues (1, 2). The latter
is inducible in all
1,25-(OH)2D3-responsive tissues,
although circulating levels of 24-hydroxylated vitamin D metabolites
appear to be a reflection of renal 24-hydroxylase activity (32). The
expression of 1-hydroxylase is an activation step, catalyzing the
synthesis of active vitamin D
[1,25-(OH)2D3], whereas
24-hydroxylase can function as a negative feedback enzyme by
inactivating 1,25-(OH)2D3 to
1,24,25-trihydroxvitamin D3 in
1,25-(OH)2D3 target tissues. However,
more recent studies of 24-hydroxylase indicate that its activity may
not simply be restricted to the modulation of
1,25-(OH)2D3 availability. It would
appear that an important component of 24-hydroxylase endocrinology is
its ability to metabolize 25OHD3 to
24,25-(OH)2D3. Although the precise
function of 24-hydroxylase in this context remains unclear, several
recent reports have suggested that synthesis of
24,25-(OH)2D3 is an important
component in the mechanisms controlling bone turnover (33). Studies of
the 24-hydroxylase knockout mouse have highlighted a possible role for
24,25-(OH)2D3 in directing
intramembrane epiphyseal ossification (34), but
24,25-(OH)2D3 may also be involved in
the early processes of fracture repair (35, 36).
Modulation of renal 1- and 24-hydroxylase activities is therefore
likely to be central to the control of both bone and mineral
metabolism. This has been demonstrated in part by studies of mineral
homeostasis and renal disease. The normal maintenance of vitamin D
metabolism by 1- and 24-hydroxylase is perturbed by disorders such
as chronic renal insufficiency, renal tubular disease, and the
autosomal recessive disorder pseudovitamin D deficiency rickets
(37, 38, 39, 40, 41). Analysis of patients with chronic renal failure has
highlighted the association between decreased
1,25-(OH)2D3 production during early
stages of renal disease and subsequent secondary hyperparathyroidism
(38). The resulting increase in PTH levels acts in a compensatory
fashion to maintain circulating calcium and phosphate levels, and
consequently, serum calcium and phosphate levels may remain normal in
all but severe cases of renal failure. This link between
1-hydroxylase activity and PTH secretion not only results in
possible deleterious effects on bone turnover, but also complicates
analysis in vivo of the roles of calcium and phosphate as
modulators of renal vitamin D metabolism. To determine the possible
direct influence of calcium on renal synthesis of
1,25-(OH)2D3, we have studied a novel
human proximal tubule cell line that expresses both 1-hydroxylase
and 24-hydroxylase activities. Analysis of the regulation of
25OHD3 metabolism by these cells indicates that calcium can
have a direct regulatory effect on renal synthesis of
1,25-(OH)2D3.
Previous in vitro studies of renal 1-hydroxylase have
mainly been carried out using primary cultures of rachitic chick kidney
cells (22, 23). Using these model systems it was possible to confirm
in vivo experiments that indicated that
1,25-(OH)2D3 itself is a potent
inhibitor of 1-hydroxylase activity but stimulates 24-hydroxylase
activity (23). Conversely, removal of
1,25-(OH)2D3 from culture medium
results in increased 1-hydroxylase activity. This latter feature has
been used to describe 1-hydroxylase activity in normal and
hypophosphatemic (Hyp) mice. Using serum-free [and
therefore 1,25-(OH)2D3-free] medium
preparations, it was possible to stimulate 1-hydroxylase activity in
primary cell cultures of renal cortex from hypophosphatemic mice (42).
After the recent cloning of mouse and rat 1-hydroxylase cDNAs it
would appear that the effects of
1,25-(OH)2D3 on the activity of
1-hydroxylase activity are due to decreased expression of mRNA for
this enzyme (6). RT-PCR and sequence analysis of mRNA for
1-hydroxylase in HKC-8 cells indicated that this was the same
transcript as that observed in human keratinocytes (8), and modulation
of 1,25-(OH)2D3 production in HKC-8
cell by agents such as forskolin and
1,25-(OH)2D3 was similar to that
observed in primary cultures (13, 16). In particular, it is important
to note that the induction of 1-hydroxylase activity in HKC-8 cells
by forskolin could also be achieved by treatment with PTH, which
correlates with the stimulation of 1-hydroxylase activity by PTH
in vivo (1).
In contrast to 1-hydroxylase, the 24-hydroxylase enzyme appears to
be widely expressed throughout the kidney (43, 44). Furthermore, unlike
1-hydroxylase, 24-hydroxylase mRNA expression and activity are
stimulated by 1,25-(OH)2D3 (16, 45)
and are unaffected or inhibited by forskolin (13, 46). The calcium
sensitivity of renal 24-hydroxylase activity appears to be inversely
linked to 1-hydroxylase. Studies in vivo have shown that
elevated or normal levels of calcium induce 24-hydroxylase activity,
whereas low concentrations suppress this activity (47). The in
vitro experiments presented here indicate that both 1- and
24-hydroxylase show similar responses to changes in calcium
concentration, particularly after relatively short term exposure to low
calcium. These data emphasize the difficulties in relating in
vivo and in vitro analyses of vitamin D metabolism, but
also highlight a common mechanism for induction of 1- and
24-hydroxylase activities. In particular, our observations suggest that
induction of 24-hydroxylase activity in response to decreased calcium
concentration occurs as a specific response, as opposed to indirect
stimulation by increased levels of
1,25-(OH)2D3. Recent studies in bone
indicate that synthesis of
24,25-(OH)2D3 by local 24-hydroxylase
activity may play an important role in modulating normal bone
development (34, 35, 36). Our data support a physiological role for
24-hydroxylase and suggest that 24-hydroxylated vitamin D metabolites
may be involved in specific autocrine or paracrine responses in the
kidney.
The direct effects of calcium on kidney cells and the role of the CaR
in this process have been reviewed previously (48). Specifically,
studies in vivo using parathyroidectomized, PTH-replete rats
have shown that elevated calcium levels are able to inhibit circulating
levels of 1,25-(OH)2D3, highlighting
a direct mechanism for modulation of 1-hydroxylase activity (20).
Data to date suggest that the production of
1,25-(OH)2D3 occurs exclusively in
the proximal tubules (3). In contrast, in situ hybridization
and immunocytochemistry studies indicate that CaR may be expressed in
all segments of the nephron (48). Sizes of CaR transcripts have been
shown to vary both within and between species (48). Two human CaR mRNA
species of 4.0 and 5.2 kb, respectively, have been isolated from
parathyroid (30) and renal (31) cDNA libraries. In contrast, the
predominant mRNA in HKC-8 cells is considerably smaller, but Western
blot analysis detected a protein of approximately 120 kDa, indicating
translation of the mRNA into an appropriately sized protein. Thus,
clearly, renal expression of CaR is likely to serve several purposes,
including maintenance of mineral homeostasis and modulation of renal
PTH function. The coexistence of CaR with 1-hydroxylase in HKC-8
proximal tubule cells suggests that calcium sensing may also influence
renal production of 1,25-(OH)2D3.
However, further studies are required to determine the precise
interaction between these two proteins.
Analysis of HKC-8 cells provides further in vitro evidence
for mineral-sensitive regulation of
1,25-(OH)2D3 production in renal
proximal tubules. Synthesis of active vitamin D within the proximal
tubule may serve several purposes, as this is the major renal site of
calcium and phosphate reabsorption. However, it is also important to
note that previous studies have shown that calcium reabsorption in the
proximal tubule is not influenced by agents such as PTH or
1,25-(OH)2D3 (49). Similarly, recent
reports indicate that proteins associated with calcium transport, such
as calbindin D28K and the membrane calcium pump, as well as
the VDR are present in higher amounts in the distal rather than the
proximal tubule (50). It is therefore clear that our understanding of
the link between renal 1- and 24-hydroxylase expression and activity
and calcium and phosphate handling is far from complete. Further
analysis of the HKC-8 cell line will provide a valuable insight into
the contribution of proximal tubule cells to this aspect of mineral
homeostasis.
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Acknowledgments |
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Footnotes |
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2 Medical Research Council Senior Clinical Fellow.
Received June 23, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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