From the Laboratory of Endocrinology and Molecular Metabolism, Graduate School of Nutritional Sciences, University of Shizuoka, 52-1 Yada, Shizuoka City 422-8526, Japan
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ABSTRACT |
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To understand the mechanism underlying the
regulation of the Ca2+-binding protein regucalcin
gene expression, we characterized the 5'-flanking region of the rat
regucalcin gene. The transcriptional start site of the rat regucalcin
gene was determined by the cap site hunting method with rat liver cap
site cDNA. The 5'-flanking region of the rat regucalcin gene
ligated to a luciferase reporter gene possessed functional promoter
activity in rat H4-II-E hepatoma cells. 3'- and 5'-deletion analyses
indicated the sequence required for basal functional promoter activity
of the rat regucalcin gene. The promoter activity of the rat regucalcin
gene was enhanced by treatment with Bay K 8644, dibutyryl cAMP, phorbol
esters, insulin, and dexamethasone. Using gel mobility shift assays, we found that nuclear proteins from H4-II-E cells specifically bind to the
5'-flanking region of the rat regucalcin gene. Moreover, gel mobility
shift assays revealed that Bay K 8644, dibutyryl cAMP, phorbol esters,
and insulin stimulated the binding of nuclear factors to the
5'-flanking region of the rat regucalcin gene in H4-II-E cells. These
results suggest that Bay K 8644-, dibutyryl cAMP-, phorbol ester-, and
insulin-inducible nuclear factors mediate the stimulatory effect of
each regulator on promoter activity of the rat regucalcin gene.
Ca2+ plays an important role in the regulation of many
cell functions. Ca2+ signals are partly transmitted to
intracellular responses, which are mediated through a family of
Ca2+-binding proteins (1). We have reported previously that
the novel Ca2+-binding protein regucalcin, which differs
from calmodulin, is distributed in the cytoplasm of hepatocytes in rats
(2). This protein has a reversible effect on the activation and
inhibition of various enzymes by Ca2+ in liver cells
(2-4). Regucalcin may play a regulatory role in liver cell functions
related to Ca2+.
The rat regucalcin gene consists of seven exons and six introns, and
several consensus regulatory elements have been found in the
5'-flanking region of the gene (5). The regucalcin gene is localized on
the proximal end of rat chromosome Xqll.1-12 (6), and has been
demonstrated in human, mouse, bovine, monkey, dog, rabbit, and chicken
but not yeast (7). We have previously reported that rat regucalcin
mRNA is mainly present in the liver and only to a small extent in
the kidney, as assayed by Northern blotting analysis (8), suggesting
that it is expressed in a highly tissue-specific manner. In fact, it
has been demonstrated that tissue-specific nuclear factor-DNA complexes
are formed on the 5'-flanking region of the rat regucalcin gene in gel
mobility shift assays (9).
The expression of hepatic regucalcin mRNA is induced by various
factors. We have shown that the expression of regucalcin mRNA in
the liver is markedly stimulated by administration of CaCl2 to rats; the expression may be mediated through
Ca2+/calmodulin (8, 10). With respect to this regulatory
mechanism by which Ca2+ administration stimulates the
expression of hepatic regucalcin mRNA, it has been demonstrated
that Ca2+ administration stimulates the additional binding
of AP-1 factor to the 5'-flanking region of the rat regucalcin gene,
and that this binding may be mediated through a
Ca2+/calmodulin-dependent pathway (11). The
expression of hepatic regucalcin mRNA is clearly stimulated by a
single subcutaneous administration of insulin to fasted rats (12). In
addition, we reported that the expression of regucalcin mRNA in
human HepG2 hepatoma cells is stimulated by insulin treatment (13), and that 17 Regulation of gene expression at the transcription level is mediated by
the interaction of a trans-acting factor with a
cis-acting DNA sequence in genes (15). Accordingly, the
interaction between a trans-acting regulatory factor and a
cis-acting regulatory element may be important for
regucalcin gene expression. However, the molecular mechanism of the
transcriptional regulation of regucalcin gene has not been clarified.
Therefore, identification of basal and regulatory DNA elements in the
5'-flanking region of the rat regucalcin gene will provide important
insight into the molecular mechanisms underlying regulation of
expression of this gene.
The aim of the present study was to examine the characteristics of the
functional promoter of the rat regucalcin gene. To examine regulation
of the rat regucalcin gene promoter, chimeric constructs containing
serial deletions of the 5'-flanking region of the rat regucalcin gene
ligated to the luciferase reporter gene were prepared and transfected
into rat H4-II-E hepatoma cells. We have identified the region that
plays an important role in determining basal promoter activity of the
gene. Moreover, it was found that Bay K 8644, dibutyryl cAMP, phorbol
ester, insulin, and dexamethasone response sequences are located within
the 5'-flanking region of the rat regucalcin gene. In addition, gel
mobility shift assays indicated that Bay K 8644-, dibutyryl cAMP-,
phorbol ester-, and insulin-inducible nuclear proteins from H4-II-E
cells bind specifically to the 5'-flanking region of the rat regucalcin gene.
Materials--
pBluescript II SK+ vector was
obtained from Stratagene (La Jolla, CA). Leupeptin, aprotinin,
dithiothreitol, phenylmethylsulfonyl fluoride, bovine serum albumin,
N6,2'-dibutyryl cyclic adenosine
3',5'-monophosphate (dibutyryl cAMP), phorbol 12-myristate 13-acetate
(PMA),1 insulin, and
dexamethasone were purchased from Sigma. S( Determination of the Transcriptional Start Site--
The
transcriptional start site of the rat regucalcin gene was determined by
the cap site hunting method with rat liver cap site cDNA (Nippon
Gene, Toyama, Japan) according to the manufacturer's instructions.
This method consists of removing the cap with tobacco acid
pyrophosphatase and ligating r-oligos to decapped mRNAs with T4 RNA
ligase. This reaction was made cap-specific by removing 5'-phosphates
of non-capped RNAs with alkaline phosphatase prior to tobacco acid
pyrophosphatase treatment. Unlike the conventional methods that label
the 5' end of cDNAs, this method specifically labels the capped
ends of mRNAs with a synthetic r-oligo prior to first-strand
cDNA synthesis. The linked mRNA was used as template to
synthesize the first-strand cDNA by reverse transcriptase in the
presence of random primers. The cap site region of the mRNA was
identified simply by reverse transcription-polymerase chain reaction (PCR).
The first round of PCR was performed using a sense DNA primer
complementary to r-oligo (1RC, 5'-CAAGGTACGCCACAGCGTATG-3', provided by
the supplier) paired with the gene-specific antisense primer 3 (GSAP3,
5'-CACCAACTCGCTGCACTCGATTG-3', see Fig. 2). Samples were amplified for
35 cycles under the following conditions: denaturation for 30 s at
94 °C, annealing for 1 min at 55 °C, and extension for 1 min at
72 °C. Aliquots of the first PCR reaction were used as the template
in the second round of PCR reaction (nested PCR). Nested PCR was
performed using a nested sense DNA primer complementary to r-oligo
(2RC, 5'-GTACGCCACAGCGTATGATGC-3', provided by the supplier) paired
with the nested gene-specific antisense primer 1 or 2 (GSAP1,
5'-TTGAAGGGATGTCTACAAACAGCA-3; GSAP2, 5'-GATCGAATCCCATCGGCAGACAG-3', see Fig. 2). Samples were amplified for 20 cycles under the following conditions: denaturation for 30 s at 94 °C, annealing for 1 min at 55 °C, and extension for 1 min at 72 °C. The PCR products were excised from the 2% low melting temperature agarose gel, cloned into
TA vector, and sequenced using a DNA sequencer (Applied Biosystems Inc.).
Construction of the Reporter Gene Plasmid--
The reporter gene
plasmids were generated by cloning restriction fragments isolated from
the 5'-flanking region of the rat regucalcin gene. PCR was performed
using pBluescript SK+ containing the 5.5-kilobase pair
EcoRI-XhoI fragment of genomic Cell Culture and Transfection--
Rat H4-II-E hepatoma cells
were maintained in Preparation of Nuclear Extracts--
All steps were carried out
at 4 °C or on ice. The cells were grown on 35-mm dishes to
approximately 80% confluence, washed twice with ice-cold
phosphate-buffered saline, and harvested by scraping into 1 ml of
ice-cold phosphate-buffered saline containing 0.5 mM
dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, and 2 µg/ml aprotinin. The cells were pelleted by
centrifugation at 500 × g for 5 min, gently
resuspended in 1 ml of hypotonic buffer (10 mM HEPES-NaOH,
pH 7.9, 10 mM KCl, 1.5 mM MgCl2,
0.1 mM EGTA, 0.5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 2 µg/ml leupeptin, and
2 µg/ml aprotinin), and incubated on ice for 10 min. The cells were
lysed by homogenization, and the nuclei were collected by
centrifugation for 5 min at 1000 × g. Nuclear extracts
were prepared by a modification of the method of Dignam et
al. (16). The nuclei were resuspended in 150 µl of ice-cold
extraction buffer (10 mM HEPES-NaOH, pH 7.9, 420 mM NaCl, 1.5 mM MgCl2, 0.1 mM EGTA, 0.5 mM dithiothreitol, 5% glycerol, and 1 mM phenylmethylsulfonyl fluoride). After slowly
mixing for 30 min at 4 °C, the suspensions were centrifuged at
13,000 × g for 15 min. The supernatant was dialyzed
against dialysis buffer (20 mM HEPES-NaOH, pH 7.9, 75 mM NaCl, 0.1 mM EDTA, 0.5 mM
dithiothreitol, 5% glycerol, and 1 mM phenylmethylsulfonyl
fluoride) for 2 h. The dialysate was then centrifuged at
13,000 × g for 15 min, divided into aliquots, and
stored at DNA Fragments for Gel Mobility Shift Assays--
The
radiolabeled probes and competitor fragments used in the binding assays
are shown in Fig. 5A. Fragment Gel Mobility Shift Assays--
Gel mobility shift assays were
carried out according to the method of Garner and Revzin (18). Nuclear
extracts (1-6 µg of protein) were preincubated in 20 µl of binding
buffer (10 mM Tris-HCl, pH 7.5, 50 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol, 150 µg/ml poly(dI-dC)·(dI-dC), and
5% glycerol) for 15 min at 24 °C. The labeled probes (0.15 ng) were
then added and incubated at 24 °C for an additional 30 min. The
reaction mixtures were loaded onto 4% nondenaturing polyacrylamide
gels (acrylamide/bisacrylamide ratio, 30:1) and electrophoresed at 10 V/cm for 90 min in 0.5× TBE (45 mM Tris, 45 mM
boric acid, and 1 mM EDTA). The gels were dried and
analyzed by autoradiography on x-ray film. The density of the
autoradiographic data was quantified by densitometry (Dual-wavelength Flying-spot Scanner, CS-9000, Shimadzu Co., Tokyo, Japan). For the
competition experiments, preincubation was performed in the presence of
unlabeled competitor DNA fragment at the indicated molar excess.
Statistical Analysis--
The significance of difference between
values was estimated by Student's t-test. A P
value of less than 0.05 was considered significant.
Mapping of the Transcriptional Start Site--
As the first step
toward characterizing the promoter of the rat regucalcin gene, the
transcriptional start site was mapped by the cap site hunting method
with rat liver cap site cDNA. In the first amplification, no
specific PCR products were detectable using 1RC as the sense primer
complementary to r-oligo and GSAP3 as the gene-specific antisense
primer (data not shown). The resulting cDNA extending to the cap
site was then amplified by nested PCR in the presence of 2RC as the
sense primer complementary to r-oligo and either GSAP1 or GSAP2 as the
gene-specific antisense primer. The amplified products were
electrophoresed in agarose gels and stained with ethidium bromide (Fig.
1). Products of approximately 220 and 250 bp were obtained using 2RC/GSAP1 and 2RC/GSAP2, respectively. Two
specific PCR products were cloned and sequenced. The results of
sequencing analysis demonstrated that all 17 selected clones terminated
at the same nucleotide (designated as +1) at their 5' ends (Fig.
2), indicating that this nucleotide is
the major transcriptional initiation site of the rat regucalcin gene.
Several putative DNA-binding elements that may play roles in basal and regulated regucalcin transcriptional activity were identified (Fig. 2).
The TATA-like sequence and CCAAT box were located at nucleotide Basal Promoter Region of the Rat Regucalcin Gene--
To determine
whether the 5'-flanking sequence of the rat regucalcin gene possesses
functional promoter activity, we directionally subcloned an amplified
DNA fragment at KpnI/XhoI sites of the pGL3
promoterless luciferase plasmid. The
To identify the region regulating basal promoter activity of the rat
regucalcin gene, a series of 3'- and 5'-deletion constructions were
transiently transfected into H4-II-E cells and assayed for luciferase
activity. 3'-Deletion construct deleted to Regulation of the Regucalcin Promoter by Bay K 8644, Dibutyryl cAMP, PMA, Insulin, Dexamethasone, and Estradiol--
To
identify regions involved in the physiologically regulated expression
of regucalcin gene, H4-II-E cells transfected with either Binding of Nuclear Proteins from H4-II-E Cells to the 5'-Flanking
Region of the Rat Regucalcin Gene--
To investigate the contribution
of nuclear factors to the transcriptional activity of the rat
regucalcin gene, gel mobility shift assays using nuclear extracts
obtained from H4-II-E cells were employed. Fragments Effects of Bay K 8644, Dibutyryl cAMP, PMA, Insulin, Dexamethasone,
and Estradiol on Nuclear Protein Binding Activity to the 5'-Flanking
Region of the Rat Regucalcin Gene--
Gel mobility shift assays were
performed to detect the presence of nuclear proteins responsive to
various physiological mediators. When the radiolabeled fragment
When radiolabeled fragment To characterize the 5'-flanking region of the rat regucalcin gene,
we employed reporter plasmids consisting of the 3'- or 5'-deletion
fragment linked to the luciferase reporter gene. As Northern blotting
analysis showed detectable amounts of regucalcin mRNA in rat
H4-II-E hepatoma cells,2
H4-II-E hepatoma cells were used in transfection experiments. In the
present study, we demonstrated that the 5'-flanking region of the rat
regucalcin gene possesses functional promoter activity when transfected
into H4-II-E cells. The results of 3'- and 5'-deletion analyses
indicated that the region A previous study suggested that the induction of hepatic regucalcin
mRNA expression by Ca2+ administration is attributable
to the activation of a
Ca2+/calmodulin-dependent pathway (10). The
elevation of intracellular Ca2+ level may lead to the
formation of Ca2+/calmodulin complex and the activation of
Ca2+/calmodulin-dependent protein kinases.
Therefore, it was possible that an increase in intracellular-free
Ca2+ concentration triggers the induction of hepatic
regucalcin mRNA expression. To assess whether the DNA sequence of
the 5'-flanking region of the rat regucalcin gene can functionally
respond to elevation of intracellular Ca2+ level, we
transiently transfected H4-II-E cells with chimeric constructs
containing serial deletions of the 5'-flanking region of the regucalcin
gene and examined the effects of Bay K 8644, a Ca2+-channel
agonist, on promoter activity. Analysis of this deletion series
suggested that the cis-acting Ca2+ response
sequence(s) is located within region It is also interesting to note that PMA stimulated luciferase activity
from The expression of hepatic regucalcin mRNA is clearly stimulated by
a single subcutaneous administration of insulin to fasted rats (12).
Additionally, the expression of regucalcin mRNA in human HepG2
hepatoma cells is stimulated by insulin treatment (13). In the present
study, we demonstrated that the stimulatory effect of insulin on rat
regucalcin promoter activity in H4-II-E cells is mediated through a
cis-acting sequence(s) located between nucleotides We have recently reported that Ca2+ administration
stimulates the additional binding of AP-1 to region There is evidence that treatment with dibutyryl cAMP increases
luciferase activity in H4-II-E cells transfected with the It has been reported that dexamethasone administration causes a marked
increase in regucalcin mRNA levels in the kidney cortex of rats
(28). Although this report was based on data from the kidney, it is
possible that the positive regulation of regucalcin mRNA by
dexamethasone also occurs in hepatoma cells. Our results indicated that
the promoter activity of regucalcin gene in H4-II-E cells is enhanced
by dexamethasone treatment. 5'-Deletion analysis indicated that the
dexamethasone response sequence(s) may be located within region
17 In conclusion, it has been demonstrated that the 5'-flanking region of
the rat regucalcin gene ligated to the luciferase reporter gene
possesses promoter activity in H4-II-E hepatoma cells. We have
identified the region regulating basal promoter activity of the
regucalcin gene. Moreover, it was found that Ca2+, cAMP,
phorbol ester, insulin, and dexamethasone response sequences are
located within the 5'-flanking region of the rat regucalcin gene. We
have identified the existence of trans-acting factors responsible for Ca2+, cAMP, phorbol ester, and insulin
responses of the rat regucalcin gene. These results provide a basis for
examining the nature of both cis- and
trans-acting factors involved in the transcriptional regulation of the rat regucalcin gene.
INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References
-estradiol stimulates the expression of hepatic regucalcin mRNA in rats (14). These results suggest that the regucalcin promoter contains sequence elements that are targets for regulatory transcription factors.
EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References
)-Bay K 8644 was obtained
from Research Biochemicals International (Natick, MA). The TA cloning
vector pCRII was purchased from Invitrogen (San Diego, CA). pUC18
vector, restriction enzymes, and T4 polynucleotide kinase were obtained
from Takara Shuzo Co. (Shiga, Japan). Poly(dI-dC)·(dI-dC) was
purchased from Pharmacia Biotech (Uppsala, Sweden). A 100-base pair DNA
ladder was obtained from New England Biolabs (Beverly, MA). Adenosine
5'-[
-32P]triphosphate ([
-32P]ATP; 111 TBq/mmol) was purchased from Amersham (Buckinghamshire, UK). Working
stocks of PMA (1 mM), S(
)-Bay K 8644 (5 mM),
dexamethasone (2 mM), and estradiol (2 mM) were
prepared in dimethyl sulfoxide (Me2SO). Working stocks of
dibutyryl cAMP (200 mM) and insulin (20 µM)
were freshly prepared in water and phosphate buffer, respectively. pGL3-Basic vector, pRL-TK vector, AP-1 consensus oligonucleotide (5'-CGCTTGATGAGTCAGCCGGAA-3'), and AP-2 consensus oligonucleotide (5'-GATCGAACTGACCGCCCGCGGCCCGT-3') were obtained from Promega (Madison,
WI). Fetal bovine serum,
-minimum essential medium (
-MEM),
penicillin, and streptomycin were purchased from Life Technologies,
Inc. Reagents (analytical grade) were obtained from Sigma and Wako Pure
Chemical Co. (Osaka, Japan).
RCB2 (5) as the
substrate to obtain DNA fragments
710/+157,
710/+18,
710/
223,
710/
343,
582/+157,
462/+157,
342/+157,
222/+157, and
102/+157 using the following primer pairs: fragment
710/+157, 5'-ACAGGTACCGAATTCCTGACTGATCTTT-3' and
5'-ACACTCGAGAAGAAAGAGCTGATAAGAC-3'; fragment
710/+18,
5'-ACAGGTACCGAATTCCTGACTGATCTTT-3' and
5'-ACACTCGAGGGTTGTAATGACTCCTGGC
3'; fragment
710/
223,
5'- ACAGGTACCGAATTCCTGACTGATCTTT-3' and
5'-ACACTCGAGGTATATGGCTGAGGTTGAA-3'; fragment
710/
343,
5'-ACAGGTACCGAATTCCTGACTGATCTTT-3' and
5'-ACACTCGAGGAAGGGCAATTTCCCTGGG-3'; fragment
582/+157,
5'-ACAGGTACCCCAGTTCACTGGTCTTTGG-3' and
5'-ACACTCGAGAAGAAAGAGCTGATAAGAC-3'; fragment
462/+157,
5'-ACAGGTACCTCATCCACTGCAGTGGAGC-3' and
5'-ACACTCGAGAAGAAAGAGCTGATAAGAC-3'; fragment
342/+157,
5'-ACAGGTACCACACCTGCCATTGTCCGAA-3' and
5'-ACACTCGAGAAGAAAGAGCTGATAAGAC-3'; fragment
222/+157,
5'-ACAGGTACCCAAGCCTCTGGCTGTTAAC-3' and
5'-ACACTCGAGAAGAAAGAGCTGATAAGAC-3'; and fragment
102/+157,
5'-ACAGGTACCGGGTAACCTGCAGACACCC-3' and 5'-ACACTCGAGAAGAAAGAGCTGATAAGAC-3'. Samples were amplified for 30 cycles under the following conditions: denaturation for 1 min at
94 °C, annealing for 1 min at 55 °C, and extension for 1 min at
72 °C. A DNA fragment was then separated by electrophoresis on a 2%
low melting temperature agarose gel, cloned into TA vector, and
sequenced using a DNA sequencer (Applied Biosystems Inc.). The DNA
fragments
710/+157,
710/+18,
710/
223,
710/
343,
582/+157,
462/+157,
342/+157,
222/+157, and
102/+157 were prepared from each vector by KpnI/XhoI restriction digestion. A
series of DNA fragments with different 3' and 5' ends were cloned into
the pGL3-Basic promoterless plasmid containing the firefly luciferase gene.
-MEM supplemented with 5 mM glucose,
10% heat-inactivated fetal bovine serum, 50 units/ml penicillin, and
50 µg/ml streptomycin in humidified 5% CO2, 95% air at
37 °C. For the transfection experiments, the cells were grown on
15-mm dishes to approximately 70% confluence and washed once with
serum-free
-MEM. Either 2 µg of pGL3-Basic plasmid or an
equivalent molar amount of test plasmid was co-transfected into H4-II-E
cells along with 0.5 µg of pRL-TK plasmid using the synthetic
cationic lipid component, Tfx-20 reagent, according to the
manufacturer's instructions (Promega). The pRL-TK vector containing
the Renilla luciferase gene under control of the herpes simplex virus thymidine kinase promoter (Promega) was used as an
internal control for differences in transfection efficiency and cell
number. For functional analysis of the basal promoter region of the rat
regucalcin gene, the transfected cells were maintained for 48 h in
serum-supplemented medium before harvesting. For analysis of regulation
of the regucalcin promoter by signaling factors, the transfected cells
were maintained for 24 h in serum-supplemented medium and
preincubated for 14 h in serum-free
-MEM supplemented with
0.1% bovine serum albumin, 50 units/ml penicillin, and 50 µg/ml
streptomycin. After preincubation, the transfectants were incubated for
16-20 h in the same medium supplemented with or without Bay K 8644 (2.5 µM), dibutyryl cAMP (0.5 mM), PMA (1 µM), insulin (10 nM), dexamethasone (1 µM), and estradiol (1 µM) before harvesting. At the end of the culture period, the transfectants were
lysed, and the luciferase activity in the cell lysates was measured by
dual-luciferase reporter assay system (Promega).
80 °C. The protein concentration was determined by the
method of Bradford (17) with a kit from Bio-Rad and bovine serum
albumin as a standard.
710/
343 was prepared by
digesting
710/
343 TA vector with KpnI and
XhoI. Fragment
342/+58 was prepared by digesting
342/+157 TA vector with KpnI and DraI. The
double-stranded DNA probes were end-labeled with [
-32P]ATP and T4 polynucleotide kinase. The labeled
DNA fragments were separated by electrophoresis through 4%
nondenaturing polyacrylamide gels (acrylamide/bisacrylamide ratio, 30:
1), eluted with a high salt buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, and 1 M NaCl) overnight at room
temperature, and purified.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
25
and
69, respectively. Putative binding sites for AP-1, GATA-1, and
AP-2 were also found.
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Fig. 1.
Gel electrophoresis of PCR products of
oligo-capped regucalcin mRNA. Rat liver cap site cDNA was
used as a PCR template to identify the transcriptional start site of
the rat regucalcin gene. The first round of PCR was performed using 1RC
as the sense DNA primer complementary to r-oligo and GSAP3 as the
gene-specific antisense primer. An aliquot of the initial PCR reaction
served the template for nested PCR. The resulting cDNA extending to
the cap site was amplified by nested PCR using 2RC as the nested sense
DNA primer complementary to r-oligo and either GSAP1 or GSAP2 as the
nested gene-specific antisense primer. The nested PCR products were
electrophoresed in 2% agarose gels and stained with ethidium bromide.
The positions of GSAP1, -2, and -3 are indicated in Fig. 2. A 100-bp
DNA ladder was used as the molecular size marker. Lane
1, 100-bp ladder; lane 2, nested PCR
product (approximately 220 bp) using 2RC/GSAP1; lane
3, nested PCR product (approximately 250 bp) using
2RC/GSAP2.
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Fig. 2.
Nucleotide sequence of the 5'-flanking region
of the rat regucalcin gene. The transcriptional initiation site
determined by the cap site hunting was assigned as +1. The positions of
gene-specific antisense primers used for the cap site hunting method
are underlined. The translation initiator codon ATG is
marked by a double underline. The arrows indicate
the deletion sites of the constructs used in the reporter assays.
Potential regulatory cis-elements are indicated by
boxes. The intron sequences are indicated by lowercase
letters. These genomic sequences have been deposited in the
GenBank data base under accession numbers D67069 and D67071.
710/+157 region of the rat
regucalcin gene ligated to the luciferase reporter gene showed promoter
activity in H4-II-E hepatoma cells.
223 did not show promoter
activity (Fig. 3), suggesting that the
sequence between nucleotides
223 and +18 contributes to basal
promoter activity. 5'-Deletion constructs deleted to
102 retained the promoter activity (Fig. 4), suggesting
that the region between nucleotides
102 and +157 contributes to basal
promoter activity. The results of 3'- and 5'-deletion analyses
suggested that the region between nucleotides
102 and +18 is needed
for basal activity of the regucalcin promoter in H4-II-E cells.
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Fig. 3.
Functional analysis of 3'-deletion constructs
of the rat regucalcin gene. A series of DNA fragments with
different 3' ends (nucleotides +157, +18, 223, and
343) and a
common 5' end (nucleotide
710) were ligated into the pGL3-Basic
promoterless plasmid. H4-II-E hepatoma cells were transiently
co-transfected with test plasmid and pRL-TK internal control plasmid.
3'-Deletion constructs are schematically shown on the left.
Luciferase activity in the cell lysates was measured 48 h after
transfection. The firefly luciferase activity of the test plasmid was
corrected for Renilla luciferase activity of the pRL-TK
plasmid. Luciferase activity of each construct is graphically shown in
the right panel. Results are expressed as means ± S.D.
from four to seven independent experiments.
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Fig. 4.
Functional analysis of 5'-deletion constructs
of the rat regucalcin gene. A series of DNA fragments with
different 5' ends (nucleotides 710,
582,
462,
342,
222, and
102) and a common 3' end (nucleotide +157) were ligated into the
pGL3-Basic promoterless plasmid. H4-II-E hepatoma cells were
transiently co-transfected with test plasmid and pRL-TK internal
control plasmid. 5'-Deletion constructs are schematically shown on the
left. Luciferase activity in the cell lysates was measured
48 h after transfection. The firefly luciferase activity of the
test plasmid was corrected for Renilla luciferase activity
of the pRL-TK plasmid. Luciferase activity of each construct is
graphically shown in the right panel. Results are expressed
as means ±S.D. from four to seven independent experiments.
710/+157
LUC construct or
342/+157 LUC construct were treated with Bay K 8644, dibutyryl cAMP, PMA, insulin, dexamethasone, and estradiol. Treatment
with Bay K 8644, PMA, and insulin significantly increased luciferase
activity in H4-II-E cells transfected with the
710/+157 LUC
construct, but
342/+157 LUC construct was completely unresponsive
(Tables I and
II). These results suggested that the
cis-acting DNA sequences that mediate Bay K 8644, PMA, and insulin responsiveness in H4-II-E cells are located in the region
710/
343 of the rat regucalcin gene. Treatment with dibutyryl cAMP
and dexamethasone increased luciferase activity in H4-II-E cells
transfected with either
710/+157 LUC construct or
342/+157 LUC
construct (Tables I and II). These results suggested that regulatory
elements that are essential for the stimulation of the regucalcin
promoter activity by dibutyryl cAMP and dexamethasone are located
between nucleotides
342 and +157. Luciferase activity in H4-II-E
cells transfected with either
710/+157 LUC construct or
342/+157
LUC construct was not affected by estradiol (Tables I and II).
Effects of Bay K 8644, dibutyryl cAMP, PMA, insulin, dexamethasone, and
estradiol on promoter activity from 710/+157 LUC in H4-II-E hepatoma
cells
710/+157 was ligated into the pGL3-Basic promoterless
plasmid (Basic LUC). H4-II-E hepatoma cells were transiently
co-transfected with test plasmid and pRL-TK internal control plasmid
and maintained in serum-supplemented medium for 24 h. The cells
were preincubated in serum-free medium for 14 h, and then
incubated for 20 h in the same medium supplemented with or without
2.5 µM Bay K 8644, 0.5 mM dibutyryl cAMP, 1 µM PMA, 10 nM insulin, 1 µM
dexamethasone, and 1 µM estradiol. Luciferase
activity was measured by the dual-luciferase reporter assay system. The
firefly luciferase activity of the test plasmid was corrected for
Renilla luciferase activity of the pRL-TK plasmid. The
results are expressed as -fold stimulation compared to the luciferase
activity measured after transfection with Basic LUC, which was set as
1.0. All values represent the means ± S.D. of six independent
experiments, which were all carried out in triplicate. *,
p < 0.05 compared with
710/+157 LUC alone.
Effects of Bay K 8644, dibutyryl cAMP, PMA, insulin, dexamethasone, and
estradiol on promoter activity from 342/+157 LUC in H4-II-E hepatoma
cells
342/+157 was ligated into the pGL3-Basic promoterless
plasmid (Basic LUC). H4-II-E hepatoma cells were transiently
co-transfected with test plasmid and pRL-TK internal control plasmid,
and maintained in serum-supplemented medium for 24 h. The cells
were preincubated in serum-free medium for 14 h, and then
incubated for 20 h in the same medium supplemented with or without
2.5 µM Bay K 8644, 0.5 mM dibutyryl cAMP, 1 µM PMA, 10 nM insulin, 1 µM
dexamethasone, and 1 µM estradiol. Luciferase
activity was measured by the dual-luciferase reporter assay system. The
firefly luciferase activity of the test plasmid was corrected for
Renilla luciferase activity of the pRL-TK plasmid. The
results are expressed as -fold stimulation compared to the luciferase
activity measured after transfection with Basic LUC, which was set as
1.0. All values represent the means ± S.D. of six independent
experiments, which were all carried out in triplicate. *,
p < 0.05 compared with
342/+157 LUC alone.
710/
343 and
342/+58 were used as radiolabeled probes in gel mobility shift assays
(Fig. 5A). When radiolabeled fragment
710/
343 was incubated with nuclear extracts from H4-II-E cells, gel mobility shift assays revealed the formation of complexes I
and II, which were shifted upward from the free DNA (Fig.
5B, lanes 1 and 2). The
presence of unlabeled fragment
710/
343 prevented the formation of
the indicated complexes when the competition reactions were performed
following a 15-min preincubation with a 10-100-fold molar excess of
unlabeled fragment
710/
343 (Fig. 5B, lanes
2-5). The nonspecific oligonucleotides were unable to displace any of the complexes I and II (Fig. 5B,
lanes 2, 6, and 7). These
results indicated that complexes I and II are specifically formed by
interaction of nuclear proteins to fragment
710/
343. When
radiolabeled fragment
342/+58 was incubated with nuclear extracts
obtained from H4-II-E cells, gel mobility shift assays revealed the
formation of complexes III and IV, which were shifted upward from the
free DNA (Fig. 5C, lanes 1 and
2). The presence of unlabeled fragment
342/+58 prevented
the formation of the indicated complexes when the competition reactions
were performed following a 15-min preincubation with a 10-100-fold
molar excess of unlabeled fragment
342/+58 (Fig. 5C,
lanes 2-5). The nonspecific oligonucleotides did
not diminish formation of these two complexes (Fig. 5C,
lanes 2, 6, and 7). These
results indicated that complexes III and IV are formed due to
sequence-specific binding.
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Fig. 5.
Binding profile of nuclear proteins from
H4-II-E cells to the 5'-flanking region of the rat regucalcin
gene. A, fragments 710/
343 and
342/+58 of the
5'-flanking region of the rat regucalcin gene used in gel mobility
shift assays. These fragments were produced from the 5'-flanking region
of the rat regucalcin gene as described under "Experimental
Procedures." B, gel retardation analysis of fragment
710/
343 with nuclear proteins extracted from H4-II-E cells. An
end-labeled fragment
710/
343 was incubated with nuclear extracts (1 µg of protein) prepared from H4-II-E cells. Competition assays were
performed in the presence of a 10-100-fold molar excess of unlabeled
DNA fragment as a competitor. Lane 1, no
extracts; lane 2, no competitor; lane
3, 10-fold molar excess of fragment
710/
343 as
competitor; lane 4, 50-fold molar excess of
fragment
710/
343 as competitor; lane 5,
100-fold molar excess of fragment
710/
343 as competitor;
lane 6, 100-fold molar excess of pUC18 DNA
fragment (a 322-bp PvuII restriction fragment) as
nonspecific competitor; lane 7, 100-fold molar
excess of regucalcin gene fragment (a 314-bp
HincII-SacI restriction fragment in Ref. 11) as
nonspecific competitor. C, gel retardation analysis of
fragment
342/+58 with nuclear proteins extracted from H4-II-E cells.
An end-labeled fragment
342/+58 was incubated with nuclear extracts
(4 µg of protein) prepared from H4-II-E cells. Competition assays
were performed in the presence of a 10-100-fold molar excess of
unlabeled DNA fragment as a competitor. Lane 1,
no extracts; lane 2, no competitor;
lane 3, 10-fold molar excess of fragment
342/+58 as competitor; lane 4, 50-fold molar
excess of fragment
342/+58 as competitor; lane
5, 100-fold molar excess of fragment
342/+58 as
competitor; lane 6, 100-fold molar excess of
pUC18 DNA fragment (a 322-bp PvuII restriction fragment) as
nonspecific competitor; lane 7, 100-fold molar
excess of regucalcin gene fragment (a 314-bp
HincII-SacI restriction fragment in Ref. 11) as
nonspecific competitor.
710/
343 was incubated with nuclear extracts obtained from control
and mediator-treated cells, the formation of complex I, which appeared
as a single band, was clearly increased in Bay K 8644-, PMA-, and
insulin-treated cells (Bay K 8644-, PMA-, and insulin-treated: 3.9 ± 0.3-, 3.7 ± 0.5-, and 3.2 ± 0.4-fold, respectively,
compared with control cells; p < 0.05; Fig.
6, lanes 1-3,
5, and 6). Whether these inducible nuclear proteins are common or distinct trans-acting factors is
unclear. A comparison of nuclear extracts obtained from control cells
and dibutyryl cAMP-, dexamethasone-, and estradiol-treated cells
revealed no changes in the binding pattern of complex I (dibutyryl
cAMP-, dexamethasone-, and estradiol-treated: 1.1 ± 0.3-, 1.0 ± 0.4-, and 1.2 ± 0.6-fold, respectively compared with
control cells; not significant; Fig. 6, lanes 1,
2, 4, 7, and 8). In
contrast, the formation of complex II was not affected by any of these
six mediators (Fig. 6, lanes 1-8).
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Fig. 6.
Effects of Bay K 8644, dibutyryl cAMP, PMA,
insulin, dexamethasone, and estradiol on nuclear protein binding
activity to region 710/
343 of the rat regucalcin gene. H4-II-E
cells were deprived of serum for 14 h and then treated with
control solution (water and 0.05% Me2SO), 2.5 µM Bay K 8644, 0.5 mM dibutyryl cAMP, 1 µM PMA, 10 nM insulin, 1 µM
dexamethasone, or 1 µM estradiol for 16 h. In the
gel mobility shift experiments, end-labeled fragment
710/
343 was
incubated with nuclear extracts (1 µg of protein) obtained from
control cells (lanes 1 and 2), Bay K
8644-treated cells (lane 3), dibutyryl
cAMP-treated cells (lane 4), PMA-treated cells
(lane 5), insulin-treated cells (lane
6), dexamethasone-treated cells (lane
7), or estradiol-treated cells (lane
8). The figure shows representative results of four separate
experiments. The density of the autoradiographic data was quantified by
densitometry. The band intensities of complex I in Bay K 8644-, PMA-,
and insulin-treated cells were significantly increased 3.9 ± 0.3-, 3.7 ± 0.5-, and 3.2 ± 0.4-fold (mean ± S.D.,
p < 0.05, n = 4), respectively, as
compared with control cells.
342/+58 was incubated with nuclear
extracts obtained from control and mediator-treated cells, the
formation of complex III, which appeared as a single band, was clearly
increased in dibutyryl cAMP-treated cells (dibutyryl cAMP-treated,
4.1 ± 0.6-fold compared with control cells; p < 0.05; Fig. 7, lanes
1 and 4). A comparison of nuclear extracts obtained from control cells and Bay K 8644-, PMA-, insulin-,
dexamethasone-, and estradiol-treated cells revealed no changes in the
binding pattern of complex III (Bay K 8644-, PMA-, insulin-,
dexamethasone-, and estradiol-treated: 1.1 ± 0.3-, 1.0 ± 0.5-, and 1.0 ± 0.6-fold, respectively compared with control; not
significant; Fig. 7, lanes 1-3 and
5-8). There were no differences in binding pattern or intensity of complex IV between nuclear extracts from control cells and
those treated with any of these six mediators (Fig. 7, lanes
1-8).
View larger version (47K):
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Fig. 7.
Effects of Bay K 8644, dibutyryl cAMP, PMA,
insulin, dexamethasone, and estradiol on nuclear protein binding
activity to region 342/+58 of the rat regucalcin gene. H4-II-E
cells were deprived of serum for 14 h and then treated with
control solution (water and 0.05% Me2SO), 2.5 µM Bay K 8644, 0.5 mM dibutyryl cAMP, 1 µM PMA, 10 nM insulin, 1 µM
dexamethasone, or 1 µM estradiol for 16 h. In the
gel mobility shift experiments, end-labeled fragment
342/+58 was
incubated with nuclear extracts (4 µg of protein) obtained from
control cells (lanes 1 and 2), Bay K
8644-treated cells (lane 3), dibutyryl
cAMP-treated cells (lane 4), PMA-treated cells
(lane 5), insulin-treated cells (lane
6), dexamethasone-treated cells (lane
7), or estradiol-treated cells (lane
8). The figure shows representative results of four separate
experiments. The density of the autoradiographic data was quantified by
densitometry. The band intensities of complex III were significantly
increased 4.1 ± 0.6-fold (mean ± S.D., p < 0.05, n = 4) as compared with control cells.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
102/+18 is essential for basal functional
promoter activity of the rat regucalcin gene. A TATA-like sequence
(
30ATAAA
25) and CCAAT box
(
73CCAAT
69) were found at appropriate
positions relative to the start site. These results suggested that both
a TATA-like sequence and a CCAAT box located between nucleotides
102
and +18 play an important role in determining basal promoter activity
of the rat regucalcin gene.
710/
343 of the rat regucalcin
gene. Also, gel mobility shift assays indicated that the amount of
complex formed on the regucalcin gene sequence
710/
343 was
increased in Bay K 8644-treated cells. The agreement between the
results of functional and binding analyses suggested that the
Ca2+-inducible nuclear factor(s) is involved in positive
regulation of regucalcin gene transcription by Ca2+.
710/+157 LUC plasmid. The deletion of fragment
710/
343
abolished PMA responses. This suggested that the stimulatory effect of
PMA on rat regucalcin gene expression is mediated through a
cis-acting sequence(s) located between nucleotides
710 and
343. It has also been demonstrated in electrophoretic mobility shift
assays that PMA-inducible nuclear factor(s) bound to region
710/
343
of the rat regucalcin gene is present in nuclear extracts from
PMA-stimulated cells. These results suggest that PMA-sensitive nuclear
factor(s) plays a crucial role in PMA-regulated expression of the
regucalcin gene. PMA has been demonstrated to activate transcription by
regulating AP-1 binding (19). Further evidence that AP-1 is not
involved in PMA-induced formation of complexes in the region
710/
343 was obtained in mobility shift experiments using a
consensus oligonucleotide for this factor (data not shown). It is
generally accepted that changes in the activity of protein kinase C
mediated by PMA initiate a cascade of events which ultimately affects
the action of specific transcription factors (20). It is possible,
therefore, that the activity of identified PMA-inducible nuclear
factor(s) may be modulated by the protein kinase C signaling pathway.
710 and
343 of the rat regucalcin gene. In addition, we identified the
insulin-inducible nuclear protein(s) that binds to region
710/
343
of the rat regucalcin gene. These results suggest that
insulin-inducible nuclear protein(s) mediates the stimulatory effect of
insulin on the regucalcin promoter activity.
710/
575 of the
rat regucalcin gene, suggesting that this factor participates in the regulation of regucalcin gene expression (11). In fact, the nucleotide
sequence matching a potential AP-1-binding site was found in the region
710/
575 of the gene (5). In many cells, AP-1, which consists of
homo- and/or heterodimers of the c-jun and c-fos
gene products (21, 22), regulates the expression of some genes that
contain specific AP-1 binding sites, PMA-responsive elements. In
addition, it has been reported that haloperidol stimulates the DNA
binding activity of AP-1 in PC12 pheochromocytoma cells and that its
effect is dependent on calcium influx (23), suggesting that the
elevation of intracellular Ca2+ leads to the activation of
DNA binding activity of AP-1. Furthermore, insulin has been shown to
stimulate AP-1-mediated gene expression and the phosphorylation of AP-1
transcription factor and several Fos-related proteins, suggesting that
the phosphorylation of AP-1 by insulin plays an important role in the
hormonal regulation of gene expression (24). In view of these results,
endogenous AP-1 may also mediate the effects of Bay K 8644, PMA, and
insulin on the regucalcin promoter activity in intact cells.
710/+157 LUC plasmid. When regucalcin sequences were deleted to nucleotide
342, no change in reporter gene activity was observed in dibutyryl cAMP-treated H4-II-E cells. These results suggest that the
cis-acting element(s) that mediates cAMP responsiveness is
located within the region
342/+157 of the rat regucalcin gene. Using
gel mobility shift assays, we found the cAMP-sensitive nuclear
protein(s) which specifically binds to the region
342/+58 of the rat
regucalcin gene. Therefore, the identified trans-acting
factors may be involved in the induction of regucalcin gene promoter
activity by cAMP. Transcriptional responses to cAMP are most commonly
mediated by cAMP response element (25) and AP-2 element (26). The
region between nucleotides
342 and +58 of the rat regucalcin gene
contains the potential AP-2-binding sites (consensus sequence,
5'-CCC(A/C)N(G/C)(G/C)(G/C)-3' (27);
150CCCACCCC
143,
58CCCGCCCC
51), whereas a sequence
homologous to the consensus cAMP response element is not present in the
region. Further evidence that AP-2 is not involved in dibutyryl
cAMP-induced formation of complex in region
342/+58 was obtained from
mobility shift experiments using a consensus oligonucleotide for this
factor (data not shown). Presumably, binding of the identified
cAMP-inducible nuclear protein(s) to the regucalcin gene is mediated
through cAMP-dependent protein kinase signaling pathway.
342/+157 of the rat regucalcin gene, although a potential
glucocorticoid response element (29) is not present in this region.
Thus, as dexamethasone-inducible nuclear protein bound to region
342/+58 was not detected by gel mobility shift experiments, the
results of promoter analysis suggested that an endogenous regulatory
mechanism may exist in the stimulation of regucalcin gene promoter by dexamethasone.
-Estradiol has been demonstrated to stimulate the expression of
hepatic regucalcin mRNA in rats (14). It was suggested that an
estradiol response sequence(s) may be localized within the 5'-flanking
region of the rat regucalcin gene. However, region
710/+157 of the
rat regucalcin gene ligated to the luciferase reporter gene did not
show estradiol responsiveness. In gel mobility shift experiments, there
was no difference in the binding pattern between nuclear extracts from
control or estradiol-treated cells. These results suggested that the
cis-acting element for estradiol is localized in the
sequence upstream from the position
710 of the regucalcin gene.
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FOOTNOTES |
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* This work was supported in part by Grant-in-aid 08672922 from the Ministry of Education, Science and Culture, Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) D67069 and D67071.
To whom correspondence should be addressed. Tel./Fax:
81-54-264-5580; E-mail: yamaguch{at}fns1.u-shizuoka-ken.ac.jp.
The abbreviations used are:
PMA, phorbol
12-myristate 13-acetate; -MEM,
-minimum essential medium; bp, base pair(s); PCR, polymerase chain reaction; r-oligo, oligoribonucleotide.
2 M. Yamaguchi and T. Murata, unpublished data.
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REFERENCES |
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