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THE
JOURNAL
OF
BIOLOGICAL
CHEMISTRY
0
1988
by
The
American Society
for
Biochemistry and Molecular Biology, Inc.
Vol
,263,
No.
Issue
of
September
15,
pp.
13112-13116,1988
Printed in
U.S.A.
Tissue-specific Regulation
of
Avian Vitamin D-dependent Calcium-
binding Protein 28-kDa mRNA by 1,25-Dihydroxyvitamin D3*
(Received for publication, March 9, 1988)
Thomas
L.
ClemensSO, Susan
A.
McGlade$, Karla
P.
Garrett$, Noboru HoriuchiS, and
Geoffrey
N.
Hendyllll
From the $Regional Bone Center, Helen Hayes Hospital, West Haverstraw, New
York
10993, the Department
of
Pathology,
Columbia Universitv. New
York.
New
York
10023.
and
the TDepartment
of
Medicine, McGill University and Royal Victoria
Hospital, Montrea1,"Quebec H3A'lAl, Canada
I
We have studied the regulation, by 1,25-dihydroxy-
vitamin D3 (1,25-(OH)2D3), of vitamin D-dependent
calcium-binding protein (28-kDa CaBP) mRNA in
chick tissues
in
vivo.
Northern analysis of poly(A)+
RNA was carried out using,
as
hybridization probes,
synthetic oligonucleotides complementary to chick
28-kDa CaJ3P mRNA. In vitamin D-deficient chicks,
28-kDa CaBP mRNA was virtually undetectable in
intestine, was clearly detectable in kidney, and
present at the highest levels in cerebellum. After
a
single intravenous dose of
500
ng of 1,25-(OH)2D3,
intestinal 28-kDa CaBP mRNA levels were increased
50-fold, kidney levels were increased 4-fold, and cer-
ebellum levels were unchanged. Increased levels of
28-
kDa CaBP mRNA were appreciated
2
h after induction
and were maximal
at
12 h. Pretreatment of vitamin D-
deficient chicks with actinomycin D had little effect on
the acute phase of the 1,25-(OH)& induction of
28-
kDa CaBP mRNA in intestine but blunted the induction
in kidney. Pretreatment with cycloheximide caused
a
delayed response to 1,25-(OH)2D3 in the intestine, al-
though control (noninhibition) levels of 28-kDa CaBP
mRNA were present 12 h after hormone administra-
tion. By contrast, in the kidney, cycloheximide
pre-
treatment resulted in an increased steady-state (vita-
min D-deficient) level of 28-kDa CaBP mRNA, but
completely abolished the induction of 1,25-(OH)2D3.
Our studies indicate that, whereas 1,25-(OH)2D3 does
not regulate 28-kDa CaBP mRNA levels in the brain,
the hormone modulates 28-kDa CaBP gene expression
in intestine and kidney in
a
tissue-specific manner, by
acting through both transcriptional and post-tran-
scriptional mechanisms.
The vitamin D-dependent
calcium-binding protein
(CaBP)'
was first isolated from chick intestine
(1)
and is the best
characterized vitamin D-responsive protein
(2).
At
least two
different types of vitamin D-dependent CaBPs exist: an
M,
*
This work was supported in part by National Institutes of Health
grants AR 36446 and AR 39191 and Grant MA 9315 from the Medical
Research Council of Canada. A preliminary report of this work was
presented at the Ninth Annual Meeting of the American Society for
Bone and Mineral Research, Indianapolis, IN, 1987.
§To whom correspondence and reprint requests should be ad-
dressed Regional Bone Center, Helen Hayes Hospital, Route 9W,
West Haverstraw, NY 10993.
11
Recipient
of
a scholarship from the Medical Research Council of
Canada.
The abbreviations used are: CaBP, calcium-binding protein; 1,25-
(OH)zD3, 1,25-dihydroxyvitamin D3; MOPS, 4-morpholinepropane-
sulfonic acid.
9,000 protein is present in mammalian intestine (3, 4), pla-
centa (5), and yolk sac (6); a larger
M,
28,000 vitamin D-
inducible CaBP has been identified in avian intestine
(I),
kidney, and other tissues
(7).
Subsequent studies have shown
that mammalian kidney (8-lo), brain (11, 12), and other
tissues
(2)
also express a 28-kDa CaBP which is immunolog-
ically similar but not identical to the avian 28-kDa CaBP.
Although there is considerable evidence that 1,25-(OH)2&
regulates both chick and rat intestinal CaBPs, the precise
cellular functions of these proteins, aside from their ability to
bind calcium (2), have not yet been established. The finding
of 1,25-(OH)zD3 receptors in many of the same tissues which
contain CaBP (13, 14) suggested that CaBP might function
by mediating the action of 1,25-(OH)2D3 in vitamin D-respon-
sive tissue. Indeed, it has been proposed that all tissues
expressing CaBP are targets for 1,25-(OH)2D3 (11), but recent
evidence suggests that CaBP expression in some tissues is
regulated differently
or
not influenced at all by vitamin D.
For
example, vitamin D deficiency in rats and chicks results
in a virtual elimination of CaBP from the intestine
(7,
E),
whereas kidney CaBP levels are reduced but clearly detectable
(7,
8,
16), and brain CaBP levels are unchanged (8, 17).
The recent cloning of the chick intestinal 28-kDa CaBP
(18, 20), as well as the intestinal rat (21,
22)
9-kDa CaBPs,
has permitted the first studies of the regulation of the mRNA
for these proteins in the intestine
(18-21).
To study further
the regulation of CaBP, especially in tissues other than intes-
tine, we have synthesized oligonucleotides complementary to
the mRNA sequences of chick intestinal 28-kDa CaBP and
rat intestinal 9-kDa CaBP. Using these oligomers as hybrid-
ization probes for their respective mRNAs, we have measured
28-kDa CaBP mRNA in vitamin D-deficient chick tissues
before and after administration of 1,25-(OH)2D3. Examination
of the effect of actinomycin and cycloheximide on the induc-
tion of 28-kDa CaBP mRNA in intestine and kidney suggests
that there are tissue-specific differences in the mechanisms
for hormone-induced changes in CaBP gene expression.
EXPERIMENTAL PROCEDURES
Materials-Radioisotopes and "multi-prime" labeling kits were
purchased from Amersham Corp. (Arlington Heights, IL). Synthetic
1,25-(OH)2D3 was generously provided by Dr. Milan Uskokovic (Hoff-
mann-La Roche, Nutley, NJ). Polynucleotide kinase and oligo(dT)-
cellulose were purchased from Sigma.
Animals and
Diets-White Leghorn cockerels were purchased from
Spafas (Norfolk, CT) and raised on a vitamin D-deficient diet
(No.
170245, Teklad, Madison, WI) for 4 weeks in an ultraviolet light-free
environment. These chicks were hypocalcemic and had undetectable
(6
pg/ml) circulating 1,25-(OH)zD3 concentrations. Weanling male
Holtzman rats
(Holtzman-Sprague-Dawley,
Madison, WI) were
raised on a vitamin D-deficient diet (No. TD
85013,
Teklad, Madison,
WI) for 7 weeks. In these animals, circulating 1,25-(OH)2D3 levels
13112
Tissue-specific Regulation of Avian CaBP 28-kDa
13113
were measurable (20-40 pg/ml) but were subnormal (normal
=
50-
90 pg/ml). All the experiments were approved by the Institutional
Animal Care and Use Committee at Helen Hayes Hospital.
Induction with 1,25-(0H)2D3 and Time Course Experiments-Vi-
tamin D-deficient chicks and rats were killed before and 24 h after a
single intravenous dose of 500 ng of 1,25-(OH)2& in 0.05 ml of 100%
ethanol. Chick intestine, kidney, and cerebellum, and rat intestine
and kidney tissues were collected and immediately frozen in liquid
nitrogen for subsequent mRNA isolation. In additional time course
studies, vitamin D-deficient chicks received 500 ng of 1,25-(OH)zDs
and pairs of chicks were killed at 0,2,4,6,12,24, and 48 h. Intestine,
kidney, and cerebellum were collected for mRNA isolation.
Metabolic Inhibitor Experiments-Groups of vitamin D-deficient
chicks were injected intraperitoneally with either actinomycin D (50
pg/lOO
g of body weight) or cycloheximide (25 pg/lOO g of body
weight) in 0.15 ml of 0.9% NaCl or vehicle alone. Two hours later all
chicks received 500 ng of 1,25-(OH)2D3. Inhibitor injections were
repeated every 4 h. Pairs of chicks were killed at
0,
2,
4,
and 12 h
after 1,25-(OH)2& administration, and intestine and kidney collected
for mRNA isolation. These doses and regimen for treatment with
inhibitors were selected based on previous experiments in which they
were used effectively as inhibitors of RNA and protein synthesis in
studies of the regulation of vitamin D metabolism (26, 27) and rat
intestinal CaBP 9-kDa mRNA (24).
Oligonucleotide Synthesis and Structure-Oligonucleotides were
42-mer of sequence
5'-ATTTTCCTCAGCACAGAGAATGAGAG-
synthesized by the phosphoramidite technique (28). CaBP-28K1, a
CCAGTTCTGCTCGGTA-3', is complementary to the mRNA se-
quence encoding amino acids 249-262 at the carboxy terminus of the
vitamin D-dependent chick intestinal 28-kDa CaBP
(18).
CaBP-
28K2, a 42-mer of sequence of 5"AAGTTCATTTTCATCTATA-
TATCCATTGCCATCTTGATCGTA-3',
is complementary to the
mRNA sequence encoding amino acids 199-212, which corresponds
to one of the calcium-binding regions of the chick intestinal
28-kDa CaBP
(18).
CaBP-SKI, a 45-mer of sequence 5'-GT-
CTT-3', is complementary to the mRNA sequence encoding amino
acids 7-21 of the vitamin D-dependent, intestinal 9-kDa CaBP (29).
Oligonucleotide Purification and Labeling-Unpurified oligonucle-
otides were dissolved in
80%
formamide, 50 mM Tris borate, pH 8.3,
2 mM EDTA and heated at 100 "C for 5 min. Bromphenol blue marker
dye was added, and the samples were electrophoresed on a 20%
acrylamide gel at
10
mA constant current for several hours in 50 mM
Tris borate, pH
8.3,
2 mM EDTA. Oligomers were visualized using
UV shadowing by placing the gel on a TLC plate containing an
activated zinc silicate fluorescent indicator and exposed briefly to
short-wave UV (254 nm) light. The most prominent upper band was
cut out and diced with a razor blade, and the pieces were added to a
microcentrifuge tube containing
1
ml of 10 mM Tris-HCI,
1
mM
EDTA, pH 8.0. After heating at 100 "C for 5 min, the aqueous layer
was collected and the concentration of oligomer was determined by
measurement of the absorbance at 260 nm. Purified oligonucleotides
(100-200 ng) were end-labeled with [32P]ATP and polynucleotide
kinase to specific activities of 5-10
X
10' cpm/pg.
Preparation and Analysis
of
mRNA-Total RNA was extracted
from all tissues by the guanidine thiocyanate method (30) followed
by cesium chloride gradient centrifugation
(31).
Enrichment for
poly(A)' RNA was achieved by a single passage of each RNA sample
through oligo(dT)-cellulose (32). Concentrations of RNA were deter-
mined by measurement of the absorbance at 260 nm. Ten-microgram
aliquots of each poly(A)' RNA were denatured in formaldehyde and
formamide and size-separated by electrophoresis on 1.1% agarose
formaldehyde gels in 20 mM MOPS, 5 mM sodium acetate,
1
mM
EDTA, pH
7.0
(32). Sample integrity was assessed by visualization
of RNA by transillumination of the gel at 302 nm (Fotodyne Inc.,
New Berlin,
WI)
after staining in 0.5 pg/ml ethidium bromide and
destaining with 10% glycine. Prior to transfer of RNA to nitrocellu-
lose by blotting (33). gels were equilibrated with 20
X
SSC
(1
X
SSC
is 0.15
M
NaCI, 0.015
M
trisodium citrate) for at least 40 min.
Nitrocellulose filters were baked at
80
"C for 2 h, and prehybridization
and hybridization to 32P-labeled oligomer were performed according
to Thomas (34). Filters were washed to a stringency of 0.1
X
SSC,
0.1% sodium dodecyl sulfate at 50 "C and exposed to Kodak X-Omat
x-ray film with intensifying screens at
-70
"C. Quantitative com-
parisons of hybridization signals were made by densitometry (G3-300
scanning densitometer, Hoefer, San Francisco, CA) of autoradi-
ographs of varying exposures. Oligonucleotide probes were removed
from the filters (34) that were then rehybridized with the insert from
TTGGATCGCCTTCTTTGGCTGCATATTTTTGAAAAATGCT-
plasmid HM A-PX (a human 0-actin cDNA (35)), which had been
labeled with ["PIdCTP to a specific activity of
IO9
cpm/pg by the
random primer method (36).
RESULTS
Evaluation
of
the Specificity of Oligonucleotide Probes-
Intestinal poly(A)+ RNA from either vitamin D-deficient
or
1,25-(OH)2D3-dosed chicks and rats was subjected to North-
ern blot analysis using either 32P-labeled CaBP-28K1, CaBP-
28K2,
or
CaBP-SKI as the probe. CaBP-28K1 (Fig.
1)
hybrid-
ized strongly with the mRNA from 1,25-(OH)2D3-treated
chicks, whereas this signal was absent in the intestinal RNA
from vitamin D-deficient birds. Three species of mRNA were
identified: a major species of 2.0 kilobases and two minor
species of 2.8 and 3.0 kilobases. Identical results were obtained
when CaBP-28K2 was used as probe (data not shown). Neither
CaBP-28K probe hybridized with intestinal and kidney
poly(A)+ RNA from rats which had been treated with
1,25-(OH)&. When this blot was probed with 32P-labeled
CaBP-9K1,
it
strongly hybridized with a single mRNA species
of
500
bases from rat intestine, which was reduced, but not
eliminated, in intestine from vitamin D-deficient rats (Fig.
1).
No hybridization was obtained with CaBP-9KI and rat
kidney mRNA (Fig.
1).
In a separate experiment, it was clearly
shown that probe CaBP-SKI did not hybridize with chick
intestinal poly(A)+ RNA (data not shown). These studies
established the specificity of the oligomer probes and showed
that CaBP-28K1 and CaBP-28K2 behaved in an identical
fashion. In the remaining studies, only results using CaBP-
28K2 as probe are shown.
Induction of
28-kDa
CaBP
mRNA
by
1,25-(0H)2D3--Analy-
sis of 28-kDa CaBP mRNA levels in vitamin D-deficient
chick tissues before and 24 h after intravenous administration
of 500 ng of 1,25-(OH)2D3 is shown in Fig. 2. In vitamin D-
deficient chicks, the highest level of mRNA was observed in
the cerebellum, a reduced (but detectable) level was present
in the kidney, and the mRNA was virtually absent from the
intestine. Twenty-four hours after 1,25-(OH)2& treatment,
cerebellum levels were unchanged, whereas kidney levels had
increased approximately 3-4-fold, and intestinal levels were
increased more than 50-fold. All three mRNA species were
induced to the same extent.
28s-
I
18s-
I,
-4380
-2320
-2020
-580
-0
*D
*D
+D
-0
+D +D
-D
FIG.
1.
Evaluation
of
the specificity
of
the oligonucleotide
probes.
Northern blot analysis of chick intestinal and rat intestinal
(Int) and kidney (Kid) poly(A)' RNAs obtained before (-D) and
after
(+D)
treatment with 1,25-(OH)2D3 was carried out as described
under "Experimental Procedures." "P-labeled oligomer CaBP-28K1
was used as probe for chick intestinal 28-kDa CaBP mRNA and
32P-
labeled oligomer CaBP-9K1 was used as probe for rat intestinal 9-
kDa CaBP mRNA. The migration positions of 28
S
and
18
S
ribo-
somal RNA as well as DNA restriction fragment markers are shown.
13114
Tissue-specific Regulation of Avian CaBP 28-kDa
28s-
18s-
-D
4
-D
+D
-D
+D
FIG.
2.
Effect of 1,25-(OH)2D3 treatment on intestinal
(Znt),
kidney
(Kid),
and cerebellar
(CB)
28-kDa CaBP mRNA in
vitamin D-deficient chicks.
Northern blot analysis
of
vitamin D-
deficient chick tissue poly(A)+ RNA samples obtained before
(-D)
and after
(+D)
1,25-(OH)2D~ administration was carried out as
described under “Experimental Procedures.” ”P-labeled oligomer
CaBP-28K2 was used as probe.
Int.
1
1-
28s-
18s-
Hours+
0
2
6
12 24
48
0
2
6
12
20
40
-m-
Intestine
*
Kidney
.=
4
0
0
c
co
6
12 18 24 48
Time
(hours)
FIG.
3.
Time course of 1,25-(OH)zD3 induction of 28-kDa
CaBP mRNA in vitamin D-deficient chick intestine and kid-
ney.
Northern blot analysis of vitamin D-deficient chick intestinal
and kidney poly(A)+ RNA before
(0)
and at various times after 1,25-
(OH)?D3 treatment was carried out as described under “Experimental
Procedures.” ”P-labeled oligomer CaBP-28K2 was used
as
probe
(upper panel)
and then removed and the blot rehybridized with
‘*P-
labeled HM A-PX to assess n-actin mRNA levels
(middle panel).
The
lower pane/
shows the relative quantitative changes in 28-kDa
CaBP mRNA levels determined by densitometry of the autoradi-
ograph in the
upper panel.
Time Course of
1,25-(0f&D3
Induction of
28-kDa
CaBP
mRNA-The levels of 28-kDa CaBP mRNA in intestine and
kidney of vitamin D-deficient chicks at vzrious times after
1,25-(OH)2D3 administration are shown in Fig.
3.
The time
course of induction was similar in intestine and kidney, with
h
++
r-7
Cyclo Hex
Hours-
0
2 4 12
0
2 4 12
0
2 4 12
Intestine
c
v)
100-
.-
C
=
80-
??
2
60-
40
-
c
.-
L
ln
0
>
20
0
.-
L
-
m
a,
-
E
0-
I
0
5
10 15
Time
(hours)
FIG.
4.
Effect
of
metabolic inhibitors on the 1,25-(OH)2Ds
induction of 28-kDa CaBP mRNA levels in vitamin D-defi-
cient chick intestine.
Upperpanel
shows Northern blot analysis of
poly(A)+ RNA before
(0)
and at various times after 1,25-(OH)~D3
treatment of vitamin D-deficient chicks, carried out as described
under “Experimental Procedures.” Co, without inhibitor;
Act
D,
with
actinomycin D pretreatment; Cyclo
Hex,
with cycloheximide pretreat-
ment.
Lower panel
shows relative quantitative changes in 28-kDa
CaBP mRNA levels determined by densitometry of the autoradi-
ograph in the
upper panel.
0,
without inhibitor;
+,
actinomycin D;
W,
cycloheximide.
elevated levels first being observed at 2 h and a maximum
level at 12 h. Elevated levels were still present at 48 h after
1,25-(OH)2D3 treatment, in both tissues. In the intestine the
28-kDa CaBP mRNA disappeared with a half-life greater
than 12 h, whereas in the kidney the mRNA was apparently
even more stable.
Effect of Metabolic Inhibitors on
I,25-(oH)2D3
Induction of
28-kDa
CaBP
mRNA Levels-The effect of actinomycin
D
and cycloheximide on the steady-state (vitamin D-deficient)
and 1,25-(OH)2Ds-induced 28-kDa CaBP mRNA levels in
intestine and kidney is shown in Figs. 4 and
5.
Actinomycin
D
treatment had little effect on the acute rise of 28-kDa CaBP
mRNA levels in the intestine, with concentrations being
comparable to those observed in chicks that did not receive
inhibitor, at 2 and 4 h after 1,25-(OH)2D3 treatment. At 12 h,
however, there was a reduction in mRNA levels. In contrast,
cycloheximide markedly blunted the acute induction phase of
the intestinal 28-kDa CaBP mRNA; mRNA levels were
30%
of control (no inhibitor) in cycloheximide-treated chicks
4
h
after induction, but had increased to control levels
at
12 h. In
the kidney, treatment with actinomycin D reduced the steady-
state level and blunted (but did not abolish) the hormone-
induced increase in 28-kDa CaBP mRNA levels. The level at
12 h after induction was less than 20% of that in the control.
Cycloheximide treatment resulted in increased steady-state
mRNA to levels that approached the maximum induced value
(12 h) in the control group. In addition, cycloheximide abol-
ished the induction of 28-kDa CaBP mRNA by 1,25-(OH)&
All experiments with the metabolic inhibitors were repeated
Tissue-specific
Regulation
of
Avian
CaBP
28-kDa
13115
co Act
D
Cyclo
Hex
24120241202412
Kidney
60
40
20
“I
0
5
10
1s
Time
(hours)
FIG.
5.
Effect of metabolic inhibitors on
the
1,25-(OH)zDs
induction
of
28-kDa CaBP mRNA
levels
in vitamin D-defi-
cient chick kidney.
Upper panel
shows Northern blot analysis
of
poly(A)’ RNA before
(0)
and at various times after 1,25-(OH)’D3
treatment
of
vitamin D-deficient chicks, carried out as described
under “Experimental Procedures.”
Co,
without inhibitor;
Act
D,
with
actinomycin D pretreatment;
Cyclo
Hex,
with cycloheximide pretreat-
ment.
Lower panel
shows relative quantitative changes in mRNA
levels determined by densitometry
of
the autoradiograph in the
upper
panel.
Symbols
are the same as
for
Fig.
4.
in a second group of chicks, and identical results were ob-
tained.
DISCUSSION
In this study we examined CaBP mRNA expression in
chick tissues both in the vitamin D-deficient state, and fol-
lowing administration of 1,25-(OH)2D3. Synthetic oligonucle-
otides complementary to portions of the 28-kDa CaBP mRNA
sequence were found to be useful
as
probes for the measure-
ment of CaBP mRNA by hybridization analysis. In agreement
with previous studies (19, 20, 23), three species of 28-kDa
CaBP mRNA were identified with the smallest, of 2.0 kilo-
bases, being the most abundant. Identical results were ob-
tained with two different oligomers complementary to differ-
ent domains of the coding region of the chick 28-kDa CaBP
mRNA. Hunziker (23) has shown that these species share
a
common
5’
end, but differ in the lengths of their
3‘
noncoding
regions. The fact that neither of our 28-kDa probes hybridized
to rat intestinal
or
kidney mRNA suggests there are structural
differences in the mammalian and avian CaBPs, even among
those having similar molecular weights (28 kDa). Previous
studies showed significant hybridization of
a
chick intestinal
28-kDa CaBP cDNA with rat renal 28-kDa CaBP mRNA
(23), suggesting some homology in the mRNAs encoding these
two proteins. In addition, it is well known that common
antigenic determinants exist among avian and mammalian
28-kDa CaBPs (37). Therefore, it is likely that the oligomer
probes used in the present study specifically recognize non-
homologous regions of the mRNAs for these different 28-kDa
proteins. In this regard the recent cloning of the 28-kDa rat
renal CaBP
(38)
should clarify the structural differences
among these related proteins.
In vitamin D-deficient chicks, 28-kDa CaBP mRNA was
barely detectable in intestine, reduced but clearly detectable
in kidney, and present at the highest level in cerebellum. After
1,25-(OH)2D3 administration, intestinal and renal 28-kDa
CaBP mRNA levels were increased, whereas cerebellum levels
were unchanged. It is noteworthy that vitamin D deprivation
in the chick, which produces marked hypocalcemia and un-
detectable circulating 1,25-(OH)2& concentrations within 2
weeks,2 has no effect on cerebellar CaBP mRNA measured
after 4 weeks (2 weeks after 1,25-(OH)@3 concentrations are
undetectable). The lack of effect of 1,25-(OH)&, on brain
cerebellum 28-kDa CaBP mRNA provides further evidence
that 28-kDa CaBP in this tissue is not regulated by this sterol
(16, 17).
The time course of accumulation of 28-kDa CaBP mRNA
following 1,25-(OH)2D3 administration was similar in intes-
tine and kidney; the mRNA was cleared slowly in both tissues.
Although this could suggest that different mechanisms are
operating to remove the mRNA, it may also be due, in part,
to the rapid turnover of intestinal epithelial cells. The most
striking difference between these two tissues with regard to
the mRNA expression is that the renal CaBP mRNA con-
tinues to be expressed in the vitamin D-deficient state,
whereas intestinal CaBP mRNA displays an absolute depend-
ence on 1,25-(OH)& This observation prompted us to study
the effect of metabolic inhibitors on the 1,25-(OH)~D3 induc-
tion of 28-kDa CaBP mRNA in the vitamin D-deficient chick.
In the intestine the transcriptional inhibitor actinomycin
D had little effect on the acute 1,25-(0H)& induction of 28-
kDa CaBP mRNA, suggesting that the hormone initially acts
at
post-transcriptional sites. Repeated administration of ac-
tinomycin D, however, reduced intestinal 28-kDa CaBP
mRNA levels by 12 h, indicating that the hormone did affect
transcription as well. This suggests that 1,25-(OH)2& may
be acting in a manner analogous to that shown, for example,
for
prolactin stimulation of casein mRNA in the mammary
gland (39), where
a
modest increase in casein gene transcrip-
tion is coupled with a marked enhancement in mRNA stabi-
lization. In contrast to the effect of actinomycin D, cyclohex-
imide inhibited the acute rise in CaBP mRNA following 1,25-
(OH)2D3 administration, but did not prevent the induction of
28-kDa CaBP mRNA, since control levels were present at 12
h. This observation is consistent with previous observations
made by Theofan and Norman (25), who demonstrated that,
in vitamin D-replete chicks, cycloheximide reduced 28-kDa
CaBP mRNA levels. The apparent requirement for continued
protein synthesis suggested the existence in the chick intes-
tine of
a
short-lived protein that stabilizes the 28-kDa CaBP
mRNA. Our data would support this concept. In contrast,
Dupret
et
al.
(24) found no effect of cycloheximide on the
lr25-(OH)2D3 induction of rat intestinal 9-kDa CaBP mRNA
in vitamin D-deficient rats
in
vivo.
Subsequent studies by this
group (40), however, using nuclear run-on assays to assess
CaBP gene transcriptional activity, suggested that newly
transcribed mRNA is stabilized by 1,25-(OH)&, which again
supports the concept of a vitamin D-dependent stabilizing
protein in intestinal cells.
In the kidney the responses to treatment with the metabolic
inhibitors were clearly different from those in the intestine.
In contrast to the lack of effect of actinomycin D on acute
intestinal 28-kDa CaBP mRNA, this transcriptional inhibitor
blunted the lr25-(OH)2D3 induction of mRNA in kidney.
Therefore, continuous gene transcription appears to be re-
’
K.
P. Garrett and
T.
L.
Clemens, unpublished results.
13116
Tissue-specific Regulation
of
Avian
CaBP
28-kDa
quired for hormone-induced 28-kDa CaBP mRNA expression
in the kidney. In support of this we have recently found that
actinomycin D also blocks 1,25-(OH)zD3-induced 28-kDa
CaBP mRNA production in cultured chick kidney cells
in
vitro?
In the present
in
vivo
study, the effect of cycloheximide
in the kidney was different from that in the intestine. Cyclo-
heximide pretreatment resulted in an increased steady-state
level
of
28-kDa CaBP mRNA in chick kidney, but completely
abolished the response to 1,25-(OH)zD3. One potential mech-
anism suggested by this result would involve the presence of
a short-lived protein that acts at the level of transcription.
Thus, under vitamin D-deficient conditions, this protein
would act to inhibit 28-kDa CaBP gene transcription, but in
the presence of 1,25-(OH)~Da it would be destabilized, and
transcription of the gene would be stimulated. Alternatively,
it is also possible that the hormone acts at other post-tran-
scriptional sites to modulate CaBP mRNA expression.
These studies illustrate the complexity of the regulation of
28-kDa CaBP synthesis and indicate that it is achieved by
different mechanisms in different tissues. Steroid hormones
such as estrogen and progesterone are known to stabilize
mRNA by acting through post-transcriptional mechanisms
(41, 42) and by affecting gene transcription. Our results
suggest that 1,25-(OH)2D3 likewise is capable of acting at both
transcriptional and post-transcriptional levels. Further stud-
ies, using an
in vitro
chick kidney cell culture system (43) in
conjunction with transcriptional assays, are now underway,
which should help elucidate the mechanisms involved in the
regulation of CaBP synthesis in the kidney.
Acknowledgments-We
thank Dr. James Romano and Richard
Birchman for assistance in the preparation of the manuscript.
REFERENCES
1. Wasserman, R. H., and Taylor,
A.
N. (1966)
Science
152, 791-
2. Norman, A. W., Roth, J., and Orci, L. (1982)
Endocr. Reu.
3,
3. Kallfelz, F. A., Taylor, A.
N.,
and Wasserman, R. H. (1967)
Proc.
SOC. Exp. Biol. Med.
125,54-58
4.
Hofmann, T., Kawakami, M., Hitchman, A.
J.
W.,
Harrison,
J.
E.,
and Dorrington, K.
J.
(1979)
Can.
J.
Biochem.
57, 737-748
5. Bruns, M.
E.
H., Fausto, A., and Avioli, L. V. (1978)
J.
Biol.
Chem.
253,3186-3190
6. Bruns, M. E., Kleeman, E., andBruns, D.
E.
(1986)
J.
Biol.
Chem.
7. Christakos.
S.,
and Norman, A. W. (1980)
Arch. Biochem. Bio-
793
331-366
261, 7485-7490
phys.
203,809-815
8.
Pansani.
A.
R..
and Christakos.
S.
(1984)
J.
Biol. Chem.
259.
,.,
9735-9741
9. Taylor,
A.
N., McIntosh,
J.
E., and Bourdeau,
J.
E.
(1982)
Kidney
10. Delorme, A. C., Danan,
J.
L., and Mathieu, H. (1983)
J.
Biol.
11. Jande,
S.
S.,
Maler,
L.,
and Lawson, D. E. M. (1981)
Nature
294,
Int.
21,765-773
Chem.
258,1878-1884
765-767
T.
L. Clemens, K.
P.
Garrett,
S.
McGlade, G. L. Craviso, and G.
N. Hendy, unpublished results.
12, Baudier, J., Glasser, N., Strid,
L.,
Brehier, A., Thomasset, M.,
and Gerard, D. (1985)
J.
Biol. Chem.
260,10662-10670
13. Stumpf, W.
E.,
Sar,
M.,
Reid, F. A., Tanaka, Y., and DeLuca,
H.
F. (1979)
Science
206,
1188-1190
14. Haussler, M. R. (1986)
Annu. Rev. Nutr.
6,527-562
15. Taylor, A.
N.
(1983)
J.
Histochem. Cytochem.
31,426-432
16. Sonnenberg, J., Pansini, A. R., and Christakos,
S.
(1984)
Endo-
crinology
116.640-648
17. Clemens,
T.
L.,
Christakos,
S.,
and Sloviter, R.
S.
(1984)
Cakif.
Tissue
Int.
63, A
18. Wilson,
P.
W., Harding, M., and Lawson, D.
E.
M. (1985)
Nucleic
Acids Res.
13,8867-8881
19. Hunziker, W., Siebert,
P.
D., King, M. W., Stucki, P., Dugaiczyk,
A., and Norman, A.
W.
(1983)
Proc. Natl. Acad.
Sci.
U.
S.
A.
80,4228-4232
20. Theofan, G., Hall, A. K., King, M. W., and Norman, A. W. (1985)
in
Vitamin
D:
Chemical, Biochemical, and Clinical Update
(Nor-
man, A. W., Schaefer, K., Grigoleit, H. G., and Von Herrath,
D., eds) pp. 332-342, de Gruyter, Berlin
21. Desplan, C., Heidmann,
O.,
Lillie,
J.
W., Auffray, C., and Tho-
masset, M. (1983)
J.
Biol.
Chem.
258, 13502-13505
22. Darwish, H. M., Kritsinger, J., Strom, M., and DeLuca, H. F.
(1987)
Proc. Natl. Acud. Sci.
U.
S.
A.
84,6108-6111
23. Hunziker, W. (1986) Proc.
Natl. Acad. Sci.
U.
S.
A.
83, 7578-
7582
24. Dupret, J.-M., Brun,
P.,
and Thomasset, M. (1986)
Endocrinology
25. Theofan, G., and Norman,
A.
W. (1986)
J.
Biol. Chem.
261,
26. Henry, H., Midgett, R.
J.,
and Norman, A. W. (1974)
J.
Biol.
27. Tanaka,
Y.,
and DeLuca, H. F. (1971)
Proc. Natl. Acad. Sci.
U.
28. Sinha, N. D., Biernat, J., McManus, J., and Koster, H. (1984)
29. Perret, C., Desplan, C., and Thomasset, M. (1985)
Eur.
J.
30. Chirgwin,
J.
M., Przybyla, A.
E.,
MacDonald, R.
J.,
and Rutter,
31. Glisin. V.. Crkveniakov. R., and Byus,
C.
(1974)
Biochemistry,
119,2476-2483
7311-7315
Chem.
249,7584-7592
S.
A.
66,605-608
Nucleic Acids Res.
12,4539-4557
Biochem.
150,211-217
W.
J.
(1979)
Biochemistry
18,5294-5299
13,'2633-2637
-
..
32. Maniatis.
T..
Fritsch.
E.
F.. and Sambrook.
J.
(1982)
Molecular
Cloning:
A
Laboratory
Manual,
pp. 197-198, Cold Spring
Harbor Laboratory, Cold Spring Harbor, NY
33. Southern,
E.
(1975)
J.
Mol. Biol.
98,503-517
34. Thomas,
P.
S.
(1980)
Proc. Natl. Acad. Sci.
U.
S.
A.
77,
5201-
35. Gunning, P., Ponte,
P.,
Okayama, H., Engel,
J.,
Blau, H., and
36. Feinberg, A. P., and Vogelstein, B. (1983)
Anal. Biochem.
137,
37.
Wasserman, R. H., Fullmer, C.
S.,
and Taylor, A.
N.
(1978) in
Vitamin
D
(Lawson,
D.
E.
M., ed) pp. 133-166, Academic Press,
New York
38.
Wood,
I.,
Tobin, A. J., Varghese,
S.,
and Christakos,
S.
(1986)
J.
Bone
Min. Res.
1,433A
39. Guyette, W. A,, Matusik, R. J., and Rosen,
J.
M. (1979)
Cell
17,
40. Dupret, J.-M., Brun,
P.,
Perret, C., Lomri, N., Thomasset, M.,
and Cuisinier-Gleizes,
P.
(1987)
J.
Biol. Chem.
262, 16553-
16557
41. McKnight, G.
S.,
and Palmiter, R. D. (1979)
J.
Biol. Chem.
254,
42. Brock, M.
L.,
and Shapiro, D.
J.
(1983)
Cell
34,207-214
43. Craviso, G. L., Garrett,
K.
P., and Clemens,
T.
L.
(1987)
Endo-
5205
Kedes,
L.
(1983)
Mol. Cell. Biol.
3, 787-795
266-267
1013-1023
9050-9058
crinology
120,894-902
