Calreticulin Inhibits Vitamin Ds Action on the PTH Gene in Vitro and May Prevent Vitamin Ds Effect in Vivo in Hypocalcemic Rats
Alin Sela-Brown,
John Russell,
Nicholas J. Koszewski,
Marek Michalak,
Tally Naveh-Many and
Justin Silver
Minerva Center for Calcium and Bone Metabolism (A.S-B.,
T.N-M, J.S.) Nephrology Services Hadassah University
Hospital and Hebrew University Medical School Jerusalem, Israel
91120
Department of Medicine (J.R.) Albert Einstein
College of Medicine Bronx, New York 10461
Department of
Medicine (N.J.K.) University of Kentucky Medical School
Lexington, Kentucky 40536
Molecular Biology of Membranes
Research Group (M.M.) University of Alberta Edmonton, Alberta
T6G 2S2, Canada
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ABSTRACT
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1,25-dihydroxyvitaminD3
[1,25-(OH)2D3] and
PTH both act to increase serum calcium. In addition,
1,25-(OH)2D3 decreases
PTH gene transcription, which is relevant both to the physiology of
calcium homeostasis and to the management of the secondary
hyperparathyroidism of patients with chronic renal failure. In chronic
hypocalcemia there is secondary hyperparathyroidism with increased
levels of PTH mRNA and serum PTH despite markedly increased levels of
1,25-(OH)2D3. We have
studied the role of calreticulin in this resistance to
1,25-(OH)2D3. Weanling
rats fed a low-calcium diet were hypocalcemic and had increased PTH
mRNA levels despite high serum
1,25-(OH)2D3 levels.
1,25-(OH)2D3 given by
continuous minipump infusion to normal rats led to the expected
decrease in PTH mRNA. The hypocalcemic rats had an increased
concentration of calreticulin in the nuclear fraction of their
parathyroids, but not in other tissues. Gel shift assays showed that a
purified vitamin D receptor and retinoid X receptor-ß bound to the
PTH promoters chicken and rat vitamin D response element (VDRE), and
this binding was inhibited by added pure calreticulin. Transfection
studies with a PTH VDRE-chloramphenicol acetyltransferase (CAT)
construct showed that
1,25-(OH)2D3 decreased
CAT transcription. Cotransfection of PTH VDRE-CAT with a calreticulin
expression vector in the sense orientation prevented the
transcriptional effect of
1,25-(OH)2D3, but a
calreticulin vector in the antisense orientation had no effect. These
results show that calreticulin prevents the binding of vitamin D
receptor-retinoid X receptor-ß to the PTH VDRE in gel retardation
assays and prevents the transcriptional effect of
1,25-(OH)2D3 on the PTH
gene. This is the first report of calreticulin inhibiting a
down-regulatory function of a sterol hormone and may help explain the
refractoriness of the secondary hyperparathyroidism of many chronic
renal failure patients to
1,25-(OH)2D3.
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INTRODUCTION
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Calreticulin is a calcium-binding protein mainly present in the
lumen of the endoplasmic reticulum of the cell that also has functions
in other cellular compartments (1). Calreticulins cytoplasmic role
was shown in fibroblasts from mice with homozygous deletions in the
calreticulin gene (2). These mice had defects in integrin-mediated cell
adhesion to extracellular matrix as well as the transient elevation in
cytoplasmic calcium after integrin engagement (2). This cytoplasmic
function is related to calreticulins interaction with the
conserved amino acid sequence KLGFFKR found in the
-subunit of
integrins (3, 4). Calreticulin may also have a nuclear function in that
it modulates transcriptional activity of steroid hormone nuclear
receptors (5, 6). It binds the same conserved protein motif KXFF(K/R)R
found in the DNA-binding domain of nuclear hormone receptors of sterol
hormones. In this way it prevents them from interacting with their
DNA-responsive elements. There is recent evidence that the nuclear
function may be dependent upon a calreticulin-dependent signaling from
the endoplasmic reticulum (7), in addition to the sterol
hormone-dependent enhanced nuclear localization of calreticulin (8).
Calreticulin inhibited glucocorticoid- and androgen-mediated gene
transcription (5, 6). In addition, it prevented the binding and action
of 1,25-dihydroxyvitamin D3
[1,25-(OH)2D3] on the osteopontin gene
in vitro (9).
The chicken PTH vitamin D3 response element (cPTH VDRE) is
located between -74 and -60 of the cPTH gene 5'-flanking region. It
is composed of two imperfect direct repeats, GGGTCA and GGGTGT, that
are separated by a 3-bp interval (10). Vitamin D receptor (VDR) binds
to this responsive element primarily as a heterodimer, in the presence
of retinoid X receptor-ß (RXRß) (10). In the rat PTH promoter there
are two VDREs, of different affinities (11). The rat high PTH
high-affinity VDRE (VDRE1) is located at -793 to -779 and is GGTTCA
GTG AGGTAC (11). In transfection experiments the two negative VDREs
were required for maximal inhibition of CAT transcription by
1,25-(OH)2D3 (11). In bovine parathyroid cells
there is an additive response of retinoic acid with
1,25-(OH)2D3 to decrease PTH mRNA levels (12).
In addition, Farrow and co-workers (13, 14) have identified DNA
sequences upstream of the bovine PTH gene that bind the
1,25-(OH)2D3 receptor. Demay et al.
(15) identified DNA sequences in the human PTH gene that bound the
1,25-(OH)2D3 receptor. When placed upstream to
a heterologous viral promoter, the sequences contained in this 25-bp
oligonucleotide mediated transcriptional repression in response to
1,25-(OH)2D3 in GH4C1 cells but not in ROS
17/2.8 cells. The down-regulatory element in the PTH promoter differs
from up-regulatory elements both in sequence composition and in the
requirement for particular cellular factors other than the VDR for
repressing PTH transcription (15, 16, 17). The transcription of the PTH
gene is markedly decreased by 1,25-(OH)2D3 both
in vitro (18, 19) and in vivo (20, 21). This
effect of 1,25-(OH)2D3 is used clinically in
patients with chronic renal failure to decrease the synthesis and
secretion of PTH (22, 23).
Therefore, the regulation of PTH gene transcription is relevant not
only in understanding the physiology of the parathyroid gland, but also
in the pathophysiology of diseases involving the parathyroid in which
there is parathyroid hyperplasia such as chronic renal failure. There
are many patients (>40% in some series) with chronic renal failure in
whom administration of 1,25-(OH)2D3 does not
decrease serum PTH levels (24). This may be related to a number of
factors, such as decreased parathyroid levels of the VDR (25) and the
calcium-sensing receptor (26, 27, 28), monoclonality of the parathyroid
hyperplasia (29), hyperphosphatemia (23), or other factors that have
not yet been defined.
Chronic hypocalcemia is an experimental model that simulates the
resistance of the parathyroid to 1,25-(OH)2D3
(30). Chronic hypocalcemia leads to increased PTH gene expression
despite very high serum 1,25-(OH)2D3 levels,
which would be expected to decrease PTH mRNA levels (30). We have
studied the role of calreticulin in this model in vitro and
in vivo. We show in vivo that hypocalcemic rats
had increased amounts of calreticulin protein in the nuclear fraction
of their parathyroids and not in other tissues. In vitro,
calreticulin blocked DNA binding of VDR-RXRß heterodimers to the cPTH
VDRE. Overexpression of calreticulin in transiently transfected opossum
kidney (OK) cells inhibited the effect of
1,25-(OH)2D3 on the cotransfected PTH promoter.
These results suggest that calreticulin may prevent the transcriptional
effect of 1,25-(OH)2D3 on the PTH promoter.
This effect of calreticulin may help explain why
1,25-(OH)2D3 is ineffective in controlling the
secondary hyperparathyroidism of many patients with chronic renal
failure.
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RESULTS AND DISCUSSION
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Hypocalcemia Increases Calreticulin Protein in the Parathyroid
Weanling rats were fed a low-calcium diet for 2 weeks. This led to
a marked hypocalcemia (6.5 ± 0.6: 11 ± 1.1 mg/dl (n =
4); P < 0.01) and increased serum
1,25-(OH)2D3 levels (845 ± 56: 145
± 26 pg/ml; P < 0.01) as compared with controls.
There was a marked increase in PTH mRNA levels in these hypocalcemic
rats (Fig. 1A
) despite the increased
levels of endogenous 1,25-(OH)2D3 similar to
our previous results (31). However,
1,25-(OH)2D3 given to normal rats as a single
injection ip (20) or as a continuous dose by osmotic minipumps for 7
days resulted in a decrease in serum PTH and PTH mRNA levels (Fig. 1B
).
It is not known what prevents the parathyroid cell from responding to
the high levels of endogenous 1,25-(OH)2D3 in
the situation of hypocalcemia (30). Calreticulin has been shown to
inhibit the binding of sterol hormone receptors to their DNA responsive
elements and prevent their action on their target genes. We therefore
studied whether calreticulin was involved in the paradoxical increase
in PTH mRNA by a low serum calcium despite the high levels of
1,25-(OH)2D3.

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Figure 1. PTH mRNA Levels Are Increased by Hypocalcemia and
Decreased by 1,25-(OH)2D3
A, PTH mRNA levels are increased by dietary hypocalcemia. Rats were
maintained on a control diet or a low-calcium diet for 2 weeks after
weaning. RNA was run on gels for Northern blots, and each lane
represents thyroparathyroid RNA from a single rat. Hybridization was
performed for PTH mRNA and 18S RNA as a control gene. B, PTH mRNA
levels are decreased by 1,25-(OH)2D3.
1,25-(OH)2D3 was given to normal rats by
constant infusion by minipumps (50 pmol/day) for 7 days. PTH mRNA is
shown, and the ethidium bromide-stained membrane shows the 28S and 18S
rRNA. Each lane represents thyroparathyroid RNA from a single rat.
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We studied the levels of calreticulin protein in parathyroid protein
extracts from rats fed a normal or a low-calcium diet. A Western blot
of nuclear and cytoplasmic proteins showed that in the nuclear fraction
of the parathyroids of rats fed a low-calcium diet, there was a 2-fold
increase in calreticulin level (Fig. 2
).
There was no change in the cytoplasmic calreticulin in these rats.
Similar results were obtained in two repeat experiments of different
parathyroid protein preparations. Other tissues, such as liver, kidney,
and brain showed no differences in calreticulin concentration in their
nuclear fractions between control and hypocalcemic rats (not shown).
Because of this increase in calreticulin concentration in the nuclear
fraction of the parathyroids of hypocalcemic rats, we studied
whether calreticulin affected the binding of the VDR to the
PTH-VDRE.

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Figure 2. Nuclear Calreticulin Protein Is Increased in
Parathyroids of Hypocalcemic Rats
Western blot for calreticulin protein in rat parathyroids fed a normal
or a low-calcium diet. Calreticulin protein was increased in the
nuclear fraction (N) of hypocalcemic rats compared with control rats,
but there was no change in calreticulin protein in the cytoplasmic
fraction (C) of these rats.
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Calreticulin Prevents Binding of the VDR-RXRß Heterodimer to the
PTH VDRE Oligonucleotide
The VDR binds as a heterodimer with the RXRß to VDREs (10). We
therefore studied the effect of calreticulin on VDR-RXRß heterodimer
binding to the VDRE of the cPTH promoter. In gel shift assays VDR and
RXRß bound an oligonucleotide corresponding to the chicken VDRE
(cVDRE) (Fig. 3
) or rat VDRE (not shown)
forming two complexes (Fig. 3
). VDR added to the cVDRE displayed
binding (not shown) that was enhanced when the VDR was added together
with RXRß (Fig. 3
). Surprisingly, RXRß alone also bound the VDRE.
MacDonald et al. (12) showed that when
1,25-(OH)2D3 was added to bovine parathyroid
cells in primary culture, there was an additive effect of
9-cis-retinoic acid to decrease PTH mRNA levels, which would
be compatible with the enhanced binding of the combined VDR and RXRß
to the cVDRE. The binding of the VDR-RXRß heterodimer to the
cPTH-VDRE was inhibited by pure calreticulin in a dose-dependent manner
(Fig. 3
). Calreticulin has three distinct structural domains, referred
to as the N, P, and C domains (5). Burns et al. (5) showed
that calreticulin prevented the glucocorticoid receptor binding to the
glucocorticoid response element, and that this was mediated by the N
domain and not the P domain of calreticulin. The C domain of
calreticulin led to enhanced binding of the glucocorticoid receptor to
the glucocorticoid response element (5) and the reason for that finding
was not clear. In the present study, the addition of both the
glutathione-S-transferase-N (GST-N) or GST-P fusion proteins
(Fig. 3
) or the GST protein (not shown) did not affect the binding of
VDR-RXRß to the cVDRE oligonucleotide in mobility shift assays (Fig. 3
). The reason why both the N and the P domains of the calreticulin
protein did not inhibit binding of the VDR-RXRß to the VDRE while the
full-length calreticulin did inhibit binding is not clear. It may be
because of S-S bridges or folding differences between the full-length
calreticulin and its separate domains. Our results, therefore, do not
allow us to draw a conclusion as to which domains in calreticulin
interact with the VDR-RXRß complex.

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Figure 3. Calreticulin Inhibits Binding of the VDR-RXRß
Heterodimer to the cVDRE Oligonucleotide
VDR-RXRß was incubated without or with increasing amounts of intact
calreticulin or the calreticulin N and P domains linked to GST after
which the cVDRE probe was added. VDR-RXRß formed a complex with the
cVDRE resulting in two bands indicated by the arrows. Added
calreticulin, in increasing concentrations, inhibited the binding of
the heterodimer to the cVDRE. Addition of GST-N domain of calreticulin
or GST-P domain of calreticulin did not affect the binding.
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We have shown that in vitro calreticulin interferes with the
binding of the VDR-RXRß heterodimer to the VDRE oligonucleotide. A
similar effect had been shown for the osteopontin gene shown by Wheeler
et al. (9) who demonstrated that calreticulin prevented
binding of the VDR-RXR heterodimer to the mouse osteopontin VDRE.
Together with the present results, this shows that calreticulin
inhibits DNA binding by the VDR in vitro. This effect may be
due to binding of the calreticulin protein to the KXFF(K/R)R-like motif
present in the DNA-binding domain of the receptor. This is the first
demonstration that calreticulin can interfere with a down-regulatory
effect of a sterol hormone on gene transcription. To determine a
functional role for the effect of calreticulin on the response of the
PTH promoter to 1,25-(OH)2D3, we performed
transfection experiments.
Calreticulin Inhibits the Transcriptional Effect of
1,25-(OH)2D3 on the PTH
VDRE
Opossum kidney (OK) cells were transiently transfected with the
cVDRE-CAT construct. In the presence of
1,25-(OH)2D3 (1 x 10-8
M), CAT expression, as assessed by ribonuclease (RNase)
protection assay, was reduced by 75% compared with cells that were not
treated with 1,25-(OH)2D3 (Fig. 4
, A and C) and (10). Overexpression of
calreticulin, by using a sense orientation expression vector
cotransfected with the cPTH gene promoter-CAT construct, completely
abolished the effect of 1,25-(OH)2D3 on the PTH
promoter (Fig. 4
, B and C). Cotransfection of the VDRE-CAT plasmid with
a plasmid transcribing antisense calreticulin had no effect on the
down-regulation of CAT transcription by
1,25-(OH)2D3 (Fig. 4
, B and C). Similar results
were obtained with the rat PTH VDRE (not shown). In all experiments,
expression of a cotransfected cytomegalovirus (CMV)-ß-galactosidase
plasmid was used as a control, and the results were normalized for
ß-galactosidase expression (Fig. 4
). To verify that the calreticulin
cDNA was being expressed, a portion of the RNA from the transfected
cells was analyzed by Northern blots. A calreticulin transcript of the
correct size was easily detectable in cells with the overexpressed
calreticulin plasmids, whereas it was undetectable in nontransfected
cells (not shown). These results show that overexpression of
calreticulin prevents the effect of
1,25-(OH)2D3 to down-regulate CAT expression
from a PTH-VDRE promoter. This is the first demonstration that
calreticulin can prevent a down-regulatory effect of
1,25-(OH)2D3. It has been shown that
calreticulin prevents the up-regulation by
1,25-(OH)2D3 of the osteopontin gene
transcription (9). Calreticulins effect on the VDRE has been shown to
be specific to the VDRE as it had no effect on the transcription of
other promoter elements, such as the cAMP response element (9). There
are other examples in which calreticulin overexpression has been shown
to inhibit a biological effect, such as the decreased expression of
dexamethazone-sensitive cytochrome P450 protein and mRNA (5). In
addition, the overexpression of calreticulin and androgen receptor in
Vero fibroblasts transfected with the androgen-responsive element-CAT
construct, inhibited CAT activity induced by androgens (6). The present
results, therefore, support the notion that calreticulin can modify the
action of sterol hormones on their target genes.

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Figure 4. Calreticulin Inhibits the Transcriptional Effect of
1,25-(OH)2D3 on the PTH Promoter
A, Ribonuclease protection analysis of gene transcripts for CAT and
ß-galactosidase of total RNA from OK cells that were transiently
transfected with cPTH VDRE-CAT plasmid and the ß-galactosidase
plasmid and then treated without (-D3) or with
(+D3) 1,25-(OH)2D3 (1 x
10-8 M). B, The same as in panel A but in
addition the cells were cotransfected with a plasmid that expressed the
antisense transcript for calreticulin (lanes 1 and 2) or cotransfected
with a plasmid that expressed the sense orientation of the calreticulin
cDNA (lanes 3 and 4). The labels -D and +D indicate the absence or
presence of 10-8 M
1,25-(OH)2D3 in the cell incubation medium,
respectively. C, Quantification of the effect of calreticulin antisense
and sense expression on the regulation of cPTH VDRE CAT gene
transcription by 1,25-(OH)2D3. The results are
expressed as CAT expression normalized for ß-galactosidase expression
measured by RNase protection assays. The mean ± SEM
are shown for four separate experiments in which each experimental
condition was run in triplicate. Significant differences
(P < 0.05) between experimental and control levels
are denoted by an asterisk.
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The effect of hypocalcemia to increase PTH mRNA levels in
vivo is mainly posttranscriptional (31), while the effect of
1,25-(OH)2D3 is transcriptional (20).
Therefore, there must be a mechanism whereby the effect of hypocalcemia
dominates over that of 1,25-(OH)2D3.
Hypocalcemic rats have an increase in their PTH mRNA levels despite
increased levels of 1,25-(OH)2D3 that would
have been expected to decrease PTH gene transcription. This paradox
implies that there is a factor that prevents the effect of
1,25-(OH)2D3 on the PTH gene. It is possible
that calreticulin might be that factor. In the present study rats with
hypocalcemia had an increased calreticulin concentration in the nuclear
fraction of their parathyroids. This result is given a functional
relevance by the findings in vitro in the present studies in
which calreticulin prevented binding of the VDR-RXRß to the PTH-VDRE
and where cotransfected sense calreticulin prevented the effect of
1,25-(OH)2D3 to decrease VDRE-CAT
transcription.
Many patients with chronic renal failure treated by chronic dialysis do
not respond as expected to administration of
1,25-(OH)2D3 (24). In some patients this may be
related to monoclonal replication of the parathyroids (29) or
down-regulation of the VDR (25), but this certainly does not apply to
all such resistant patients. It is possible that in this subset of
patients there is an increase in the calreticulin protein in their
parathyroids. The model used in the present study was secondary
hyperparathyroidism due to hypocalcemia. This model simulates many of
the features of secondary hyperparathyroidism of renal failure such as
increased levels of PTH mRNA and serum PTH (30) as well as increased
parathyroid cell proliferation (32). In addition, the high levels of
serum 1,25-(OH)2D3 provide an opportunity to
understand the resistance of the parathyroid to
1,25-(OH)2D3 in many patients with secondary
hyperparathyroidism. We have shown that in vivo these
hypocalcemic rats have an increase in calreticulin protein in their
nuclear fraction. In addition, calreticulin inhibited VDR-RXRß
binding to the PTH VDRE. Overexpression of calreticulin inhibited the
effect of 1,25-(OH)2D3 on VDRE-CAT
expression in transfected cells. These results suggest a functional
role for calreticulin in the down-regulation of the PTH gene
transcription by 1,25-(OH)2D3.
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MATERIALS AND METHODS
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Animals
Weanling male Hebrew University strain rats were maintained on
either a normal calcium (0.6%) diet or a low-calcium (0.02%) (-Ca)
diet (Teklad, Madison, WI) for 2 weeks. For the experiment with
1,25-(OH) 2D3, rats of 150 g body weight
were implanted with osmotic minipumps sc and received
1,25-(OH)2D3 (50 pmol/day) or propylene glycol
vehicle for 7 days. The thyroparathyroid tissue was removed under
pentobarbital anesthesia and blood samples were taken for serum calcium
and 1,25-(OH)2D3. All animal studies were
conducted in accord with the principles and procedures outlined in the
"Guidelines for Care and Use of Experimental Animals." For RNA
extractions, thyroparathyroid tissue from individual rats was frozen in
liquid nitrogen and stored at -70 C until analysis. RNA was extracted
from the thyroparathyroids of each rat and analyzed by Northern blots
for PTH mRNA and 18S ribosomal RNA as previously described (33). For
protein extraction, pooled microdissected parathyroids from 10 rats in
each group, or other tissues from individual rats, were removed, and
proteins from the nuclear and cytoplasmic fractions were extracted
immediately by the method of Dignam et al. (34).
Western Blot Analysis
Forty micrograms of the nuclear and cytosolic extracts were run
on 10% SDS-polyacrylamide gel. The gels were transferred to
nitrocellulose membrane and incubated with polyclonal goat
anticalreticulin as first antibody (dilution of 1:300 in 1% BSA-1x
PBS) and then with horseradish peroxidase-rec Protein G (Zymed
Laboratories, South San Francisco, CA) as second antibody. The
peroxidase reaction was developed using an ECL detection system (New
England Biolabs, Beverly, MA).
DNA Mobility Shift Assay
Recombinant human (h) VDR and hRXRß were diluted 1:10 in
ice-cold KTEDG buffer (400 mM KCl, 20 mM Tris,
pH 7.5, 1 mM EDTA, 2 mM EDTA, 10% glycerol, 2
mM dithiothreitol), 1.6 µl of the diluted proteins were
used in 1x binding buffer (100 mM KCl, 20 mM
Tris, pH 7.5, 1.5 mM EDTA, 5% glycerol, 0.1% NP 40, 2
mM dithiothreitol), at a final volume of 20 µl, which
included 1 µg deoxyinosinic-deoxycytidylic acid and the end-labeled
VDRE DNA fragment probe (30,000 cpm). The reaction was incubated on ice
for 1 h, loaded on a cooled prerun 5% nondenaturating
polyacrylamide gel and run for 3 h in 0.5x Tris-borate-EDTA
buffer at 4 C. The gels were dried and exposed to x-ray film. The
chicken cPTH VDRE (bold letters) consisted of
5'-gatcGGAAGCTTACATGAGGGTCAGGAGGGTGTGCCT-GCAGG-3'
3'-CCTTCGAATGTACTCCCAGTCCTCCCACACGG-ACGTCCctag-5'
and was end-labeled with Klenow large-fragment enzyme using
[
-32P[

ß]dGTP.
When the binding of the VDR-RXRß heterodimer to the VDRE was
performed with increasing concentrations of calreticulin and its N and
P domains or the GST protein, they were incubated at 4 C for 15 min
before addition of the VDRE probe.
Plasmid Constructions
The cPTH promoter sequence, -55 to +20 was synthesized by PCR
and ligated into a commercially available p-CAT expression vector
(Promega, Madison WI) using the SalI and XbaI
restriction sites present in the multiple cloning site. An
oligonucleotide containing the cPTH VDRE, GGGTCA GGA GGGTGT, was placed
upstream from the cPTH promoter using the HindIII and
SalI sites that were available. The calreticulin expression
plasmid in the sense orientation was prepared by subcloning the
XhoI-SmaI fragment of the pSVL-CRT plasmid (5)
into pMCC-ZP plasmid containing a CMV promoter. The calreticulin
expression plasmid in the antisense orientation was prepared as
described (5). Plasmids that expressed either the sense or antisense
transcripts for the calreticulin gene were synthesized as described
previously (5).
Transfection Studies
Opossum kidney (OK) cells were cotransfected by the method of
lipofection (35) with the cPTH VDRE-CAT plasmid (2.5 µg),
CMV-ß-galactosidase (1 µg) plasmid, and either the sense or
antisense plasmid for calreticulin (1 µg). Analysis of CAT and
ß-galactosidase gene transcripts after treatment with hormone (1
x 10-8 M) for 24 h were quantified by
ribonuclease protection assay using a commercially available kit
(Ambion, Austin TX). Total RNA from OK cells transfected with the
cPTH-CAT and CMV-ß-galactosidase plasmid constructs was prepared by
extraction with phenol-guanidinium thiocyanate. The RNA extracts were
incubated with RNase-free DNase I (GIBCO-BRL, Bethesda MD) for 5 min at
room temperature and ethanol precipitated. The RNA pellets were
incubated with 32P-labeled RNA probes (158 bases for CAT
and 370 bases for ß-galactosidase) in 20 µl of buffer containing
80% formamide, 100 mM sodium citrate, pH 6.4, 300
mM sodium acetate, pH 6.4, and 1 mM EDTA,
overnight at 43 C. The hybridization mixtures were digested with a
combination of RNase A and RNase T1 at 37 C for 30 min. The protected
RNA hybrids were precipitated with propanol-guanidinium thiocyanate and
separated on 6% nondenaturing polyacrylamide gels. The gels were dried
and exposed to x-ray film for 6 h at -80 C. The resulting
autoradiograms were quantified by densitometric scanning, and values
for the CAT gene transcripts were normalized with respect to the values
for ß-galactosidase gene transcripts. To verify that the calreticulin
genes were being expressed in the experiments where calreticulin
expression vectors were used, a portion of the RNA from the transfected
cells was analyzed by Northern blots. In all cases a calreticulin
transcript of the correct size was observed.
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ACKNOWLEDGMENTS
|
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We thank Ms. Miriam Offner for excellent technical assistance.
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FOOTNOTES
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Address requests for reprints to: Justin Silver, M.D., Ph.D., Nephrology Services, P.O. Box 12000, Jerusalem, Israel 91120.
Research support was provided for these studies by the Israel Academy
of Sciences (to T.N-M), NIH Grant RO1-DK38422 (to J.R.), University of
Kentucky Medical Center Research Fund (to N.J.K.), and the Medical
Research Council of Canada (M.M.).
Received for publication September 3, 1997.
Revision received March 9, 1998. Revision received April 22, 1998.
Accepted for publication April 23, 1998.
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REFERENCES
|
---|
-
Krause KH, Michalak M 1997 Calreticulin. Cell 88:439443[Medline]
-
Coppolino MG, Woodside MJ, Demaurex N, Grinstein S, St-Arnaud
R, Dedhar S 1997 Calreticulin is essential for integrin-mediated
calcium signalling and cell adhesion. Nature 386:843847[CrossRef][Medline]
-
Coppolino M, Leung-Hagesteijn C, Dedhar S, Wilkins J 1995 Inducible interaction of integrin alpha 2 beta 1 with calreticulin.
Dependence on the activation state of the integrin. J Biol Chem 270:2313223138[Abstract/Free Full Text]
-
Opas M, Szewczenko-Pawlikowski M, Jass GK, Mesaeli N,
Michalak M 1996 Calreticulin modulates cell adhesiveness via regulation
of vinculin expression. J Cell Biol 135:19131923[Abstract]
-
Burns K, Duggan B, Atkinson EA, Famulski KS, Nemer M,
Bleakley RC, Michalak M 1994 Modulation of gene expression by
calreticulin binding to the glucocorticoid receptor. Nature 367:476480[CrossRef][Medline]
-
Dedhar S, Rennie PS, Shago M, Leung-Hagestein C, Yang H,
Hilmus J, Hawley RG, Bruchovsky N, Cheng H, Matusik RJ, Giguere V 1994 Inhibition of nuclear hormone receptor activity by
calreticulin. Nature 367:480483[CrossRef][Medline]
-
Michalak M, Burns K, Andrin C, Mesaeli N, Jass GH, Busaan JL,
Opas M 1996 Endoplasmic reticulum form of calreticulin modulates
glucocorticoid-sensitive gene expression. J Biol Chem 271:2943629445[Abstract/Free Full Text]
-
Roderick HL, Campbell AK, Llewellyn DH 1997 Nuclear
localisation of calreticulin in vivo is enhanced by its
interaction with glucocorticoid receptors. FEBS Lett 405:181185[CrossRef][Medline]
-
Wheeler DG, Horsford J, Michalak M, White JH, Hendy GN 1995 Calreticulin inhibits vitamin D3 signal transduction. Nucleic Acids Res 23:32683274[Abstract]
-
Liu SM, Koszewski N, Lupez M, Malluche HH, Olivera A, Russell
J 1996 Characterization of a response element in the 5'-flanking region
of the avian (chicken) parathyroid hormone gene that mediates negative
regulation of gene transcription by 1,25-dihydroxyvitamin
D3 and binds the vitamin D3 receptor. Mol
Endocrinol 10:206215[Abstract]
-
Russell J, Ashok S, Koszewski NJ Vitamin D receptor
interactions with the rat parathyroid hormone gene: cooperative effects
between two negative vitamin D response elements. [Abstract] J
Bone Miner Res 1997 12:[Suppl 1]S122
-
MacDonald PN, Ritter C, Brown AJ, Slatopolsky E 1994 Retinoic
acid suppresses parathyroid hormone (PTH) secretion and PreproPTH mRNA
levels in bovine parathyroid cell culture. J Clin Invest 93:725730[Medline]
-
Farrow SM, Hawa NS, Karmali R, Hewison M, Walters JC,
ORiordan JL 1990 Binding of the receptor for 1,25-dihydroxyvitamin
D3 to the 5'-flanking region of the bovine parathyroid
hormone gene. J Endocrinol 126:355359[Abstract]
-
Hawa NS, ORiordan JL, Farrow SM 1994 Binding of
1,25-dihydroxyvitamin D3 receptors to the 5'-flanking
region of the bovine parathyroid hormone gene. J Endocrinol 142:5360[Abstract]
-
Demay MB, Kiernan MS, DeLuca HF, Kronenberg HM 1992 Sequences
in the human parathyroid hormone gene that bind the
1,25-dihydroxyvitamin D3 receptor and mediate
transcriptional repression in response to 1,25-dihydroxyvitamin
D3. Proc Natl Acad Sci USA 89:80978101[Abstract]
-
Mackey SL, Heymont JL, Kronenberg HM, Demay MB 1996 Vitamin D binding to the negative human parathyroid hormone vitamin D
response element does not require the retinoid X receptor. Mol
Endocrinol 10:298305[Abstract]
-
Masuyama H, Jefcoat SCJ, MacDonald PN 1997 The N-terminal
domain of transcription factor IIB is required for direct interaction
with the vitamin D receptor and participates in vitamin D-mediated
transcription. Mol Endocrinol 11:218228[Abstract/Free Full Text]
-
Okazaki T, Igarashi T, Kronenberg HM 1988 5'-flanking region
of the parathyroid hormone gene mediates negative regulation by
1,25-(OH)2 vitamin D3. J Biol Chem 263:22032208[Abstract/Free Full Text]
-
Russell J, Lettieri D, Sherwood LM 1986 Suppression by
1,25(OH)2D3 of transcription of the
pre-proparathyroid hormone gene. Endocrinology 119:28642866[Abstract]
-
Silver J, Naveh-Many T, Mayer H, Schmelzer HJ, Popovtzer
MM 1986 Regulation by vitamin D metabolites of parathyroid hormone gene
transcription in vivo in the rat. J Clin Invest 78:12961301[Medline]
-
Naveh-Many T, Marx R, Keshet E, Pike JW, Silver J 1990 Regulation of 1,25-dihydroxyvitamin D3 receptor gene
expression by 1,25-dihydroxyvitamin D3 in the parathyroid
in vivo. J Clin Invest 86:19681975[Medline]
-
Slatopolsky E, Weerts C, Thielan J, Horst R, Harter H, Martin
KJ 1984 Marked suppression of secondary hyperparathyroidism by
intravenous administration of 1,25-dihydroxy-cholecalciferol in uremic
patients. J Clin Invest 74:21362143[Medline]
-
Quarles LD, Yohay DA, Carroll BA, Spritzer CE, Minda SA,
Bartholomay D, Lobaugh BA 1994 Prospective trial of pulse oral vs.
intravenous calcitriol treatment of hyperparathyroidism in ESRD. Kidney
Int 45:17101721[Medline]
-
Indridason OS, Quarles LD 1995 Oral vs. intravenous
calcitriol: is the route of administration really important? Curr Opin
Nephrol Hypertens 4:307312[Medline]
-
Fukuda N, Tanaka H, Tominaga Y, Fukagawa M, Kurokawa K, Seino
Y 1993 Decreased 1,25-dihydroxyvitamin D3 receptor density
is associated with a more severe form of parathyroid hyperplasia in
chronic uremic patients. J Clin Invest 92:14361443[Medline]
-
Juhlin C, Klareskog L, Nygren P, Ljunghall S, Gylfe E, Rastad
J, Akerstrom G 1988 Hyperparathyroidism is associated with reduced
expression of a parathyroid calcium receptor mechanism defined by
monoclonal antiparathyroid antibodies. Endocrinology 122:29993001[Abstract]
-
Kifor O, Moore FDJ, Wang P, Goldstein M, Vassilev P, Kifor I,
Hebert SC, Brown EM 1996 Reduced immunostaining for the extracellular
Ca2+-sensing receptor in primary and uremic secondary
hyperparathyroidism. J Clin Endocrinol Metab 81:15981606[Abstract]
-
Gogusev J, Duchambon P, Hory B, Giovannini M, Goureau Y,
Sarfati E, Drueke TB 1997 Depressed expression of calcium receptor in
parathyroid gland tissue of patients with hyperparathyroidism. Kidney
Int 51:328336[Medline]
-
Arnold A, Brown MF, Urena P, Gaz RD, Sarfati E, Drueke TB 1995 Monoclonality of parathyroid tumors in chronic renal failure and in
primary parathyroid hyperplasia. J Clin Invest 95:20472053[Medline]
-
Naveh-Many T, Silver J 1990 Regulation of parathyroid hormone
gene expression by hypocalcemia, hypercalcemia, and vitamin D in the
rat. J Clin Invest 86:13131319[Medline]
-
Moallem E, Silver J, Kilav R, Naveh-Many T 1998 RNA protein
binding and post-transcriptional regulation of PTH gene expression by
calcium and phosphate. J Biol Chem 273:52535259[Abstract/Free Full Text]
-
Naveh-Many T, Rahamimov R, Livni N, Silver J 1995 Parathyroid
cell proliferation in normal and chronic renal failure rats: the
effects of calcium, phosphate and vitamin D. J Clin Invest 96:17861793[Medline]
-
Kilav R, Silver J, Naveh-Many T 1995 Parathyroid hormone gene
expression in hypophosphatemic rats. J Clin Invest 96:327333[Medline]
-
Dignam JD, Lebovitz RM, Roeder RG 1983 Accurate transcription
initiation by RNA polymerase II in a soluble extract from isolated
mammalian nuclei. Nucleic Acids Res 11:14751489[Abstract]
-
Felgner PL, Ringold GM 1989 Cationic liposome-mediated
transfection. Nature 337:387388[CrossRef][Medline]