Download Thyroid Hormone Stimulates Renin Gene Expression Through the

Thyroid Hormone Stimulates Renin Gene Expression
Through the Thyroid Hormone Response Element
Hiroyuki Kobori, Matsuhiko Hayashi, Takao Saruta
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Abstract—We previously reported that thyroid hormone stimulates renin synthesis in vivo and in vitro. Here, we analyzed
the 5⬘-flanking sequence of the human renin gene for promoter activity responsive to thyroid hormone using Calu-6
cells, which secrete renin endogenously and express thyroid hormone receptor-␤. The luciferase reporter gene was
cloned together with 5⬘-flanking portions of the human renin gene of various lengths into the pGL3-Basic vector.
Luciferase activity assays were performed using the Dual Luciferase Reporter Assay System. 3,3⬘,5-Triiodo-L-thyronine
stimulated the promoter activity of pGL3-Basic⫺1111/⫹12 and pGL3-Basic⫺1298/⫹12 by 2.3⫾0.1- and 1.7⫾0.1fold, respectively. Shorter constructs (pGL3-Basic⫺144/⫹12, pGL3-Basic⫺226/⫹12, pGL3-Basic⫺452/⫹12, and
pGL3-Basic⫺953/⫹12) were not stimulated by thyroid hormone. These results suggest that there is a possible thyroid
hormone response element (5⬘-AGG TCA GGT CAc aat GTT CCT-3⬘) between nucleotides ⫺1111 and ⫺953. In 3
constructs with site-directed mutations in this sequence, basal promoter activities were significantly increased, whereas
promoter activation by thyroid hormone was abolished. Electrophoretic mobility shift assays showed that the
⫺1111/⫺953 DNA fragment of the intact human renin gene was bound to nuclear proteins of Calu-6 cells; however,
none of the 3 mutant probes were bound to any nuclear proteins. These results suggest that thyroid hormone stimulates
the promoter activity of the human renin gene through thyroid hormone response element– dependent mechanisms in
Calu-6 cells. (Hypertension. 2001;37:99-104.)
Key Words: human 䡲 renin 䡲 gene expression 䡲 hormones, thyroid 䡲 response elements
T
he renin-angiotensin system plays a pivotal role in the
long-term regulation of blood pressure and electrolyte
homeostasis.1 Although elevated blood pressure should suppress renin release from renal juxtaglomerular cells, high
levels of plasma renin are often found in hypertensive
patients.2 Increased plasma renin levels are related to the
incidences of stroke, myocardial infarction, and other vascular injuries.3 Understanding the mechanism that regulates
renin gene expression may provide better approaches to the
treatment of hypertension.
Renin and renin mRNA are expressed in many cell types,
such as in the placental, ovarian, testicular,4 adrenal,5 and
pituitary6,7 cells of a number of species. The expression of
renin is particularly high in juxtaglomerular cells.8 Juxtaglomerular cells are difficult to isolate and culture, and they lose
their ability to produce renin after a single passage.9 Thus,
because of the lack of any established renin-producing cell
lines, very little is known about the mechanisms that control
human renin gene expression.
Recently, Lang et al10 showed that the cell line Calu-6,
derived from human lung cancer, endogenously produces
human renin and renin mRNA. Successive studies have
demonstrated that renin synthesis in Calu-6 cells, like juxtaglomerular cells, is activated by cAMP11 and inhibited by
mechanical stretch.12 Therefore, the Calu-6 cell line is a
potentially useful model for studying transcriptional regulation of the human renin gene.
We recently reported that thyroid hormone directly stimulates renin mRNA in vivo13 and in vitro.14 In the present
study, we examined 2 hypotheses: (1) that thyroid hormone
increases renin mRNA in Calu-6 cells and (2) that thyroid
hormone stimulates the promoter activity of the human renin
gene in Calu-6 cells.
Methods
Cell Cultures
The Calu-6 cell line was obtained from American Type Culture
Collection. Cells were grown in minimum essential medium with
Earle’s salt (GIBCO BRL) supplemented with nonessential amino
acids (GIBCO BRL), sodium pyruvate (GIBCO BRL), and 10% fetal
bovine serum (GIBCO BRL) as according to the supplier’s protocol.
Cells were incubated at 37°C in 95% air-5% CO2 to 90% confluence.
The cells were passaged; 48 hours later, they were inspected for
Received June 15, 2000; first decision July 10, 2000; revision accepted July 12, 2000.
From the Department of Internal Medicine, Keio University School of Medicine, Tokyo, Japan.
Presented in part in abstract form at the 31st Annual Meeting of the American Society of Nephrology (J Am Soc Nephrol. 1998;9:309A), the 53rd Annual
Fall Conference and Scientific Sessions of the Council for High Blood Pressure Research (Hypertension. 1999;34:364), and the 32nd Annual Meeting
of the American Society of Nephrology (J Am Soc Nephrol. 1999;10:348A).
Correspondence to Hiroyuki Kobori, MD, PhD, Department of Physiology, SL39, Tulane University School of Medicine, 1430 Tulane Ave, New
Orleans, LA 70112-2699. E-mail [email protected]
© 2001 American Heart Association, Inc.
Hypertension is available at http://www.hypertensionaha.org
99
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January 2001
attachment to the culture dishes. The culture medium was changed,
and Calu-6 cells were treated with or without 10⫺7 mol/L 3,3⬘,5triiodo-L-thyronine (T3; Aldrich) for 24 hours, because this concentration and incubation time induced the maximum stimulation of
renin synthesis in a preliminary study.
Expression of Human Thyroid
Hormone Receptor-␤
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Calu-6 cells were rinsed 3 times with PBS, collected by scraping,
resuspended in 500 ␮L guanidine isothiocyanate, and homogenized
with a Polytron homogenizer (Kinematica) for 30 seconds at 4°C.
Total RNA from cultured Calu-6 cells was extracted using a total
RNA extraction kit (Clontech) according to the manufacturer’s
protocol.
RT-PCR was performed with the GeneAmp RNA PCR core kit
(Perkin–Elmer Cetus). Sense (5⬘-GAG GAG AAG AAA TGT AAA
GG-3⬘) and antisense (5⬘-TGT TGC CAT GCC AAC ATA GAT
GC-3⬘) primers for PCR amplification of human thyroid hormone
receptor-␤ (hTR-␤) cDNA corresponded to hTR-␤ gene nucleotides
⫹250 through ⫹269 and ⫹491 through ⫹513, respectively.15 PCR
was performed for 30 cycles with a 30-second denaturation step at
94°C, a 60-second annealing step at 50°C, and a 75-second extension
step at 72°C. The PCR product was sequenced to confirm that it was
hTR-␤ cDNA through dideoxy sequencing with the ABI PRISM
cycle sequencing kit (Perkin–Elmer Cetus). The sequences obtained
were identical to those previously reported.15
Expression Analysis
Total RNA from cultured Calu-6 cells was extracted as described
earlier. Quantitative RT-PCR was performed precisely as previously
described.16 –18 Sense (5⬘-AAT GAA GGG GGT GTC TGT GG-3⬘)
and antisense (5⬘-CAC GAC ATA ATC AAA CAG CC-3⬘) primers
for PCR amplification of human renin cDNA corresponded to human
renin gene nucleotides ⫹812 through ⫹831 and ⫹952 through
⫹971, respectively.19 Sense (5⬘-CGG ATT TGG TCG TAT TGG
GC-3⬘) and antisense (5⬘-AAA TGA GCC CCA GCC TTC TCC-3⬘)
primers for PCR amplification of human glyceraldehyde-3phosphate-dehydrogenase (GAPDH) cDNA corresponded to human
GAPDH gene nucleotides ⫹87 through ⫹106 and ⫹375 through
⫹395, respectively.20 PCR was performed for 30 cycles with a
30-second denaturation step at 94°C, a 60-second annealing step at
57°C, and a 75-second extension step at 72°C. After coamplification
of total RNA with each sense and antisense primer, gel electrophoresis exhibited 2 clear bands at 160 and 309 bp that represent the
human renin and GAPDH genes, respectively. GAPDH mRNA was
used as an internal standard, because thyroid hormone treatment did
not alter the quantity of this mRNA in Calu-6 cells. Human renin
mRNA expression was determined as the ratio of the human renin to
GAPDH band density. The PCR products were sequenced to confirm
that they were human renin and human GAPDH cDNA with the ABI
PRISM cycle sequencing kit. The sequences obtained were identical
to those previously reported.19,20
Figure 1. A, Restriction enzyme sites in 5⬘-flanking region of the
human renin gene and schema of the construction of luciferase
reporter plasmid. B, Effect of various construct lengths on the
promoter activity of the human renin gene. Values are presented
as mean⫾SEM. *P⬍0.02 vs pGL3-Basic (1155⫾219 luminometric units). †P⬍0.05 vs pGL3-Basic⫺144/⫹12. ‡P⬍0.04 vs
pGL3-Basic⫺226/⫹12. §P⬍0.02 vs pGL3-Basic⫺452/⫹12.
¶P⬍0.02 vs pGL3-Basic⫺953/⫹12. n⫽6. C, Effect of thyroid
hormone on the promoter activity of the human renin gene. Data
are expressed as fold change in comparison to that without T3.
Values shown are mean⫾SEM. *P⬍0.01 between with and without T3. n⫽6.
Constructs
Human genomic DNA was isolated with a GenTLE kit (Takara)
from peripheral blood of a healthy volunteer. With this genomic
DNA used as a template, overlapping 5⬘-flanking intervals of the
human renin gene were isolated by PCR with an LA PCR kit version
2 (Takara). Sense (5⬘-GCC AAG CAT TCA TTA ACC C-3⬘) and
antisense (5⬘-GCT GAT CGT AAG GAC TGA ATG AC-3⬘) primers
for PCR amplification of human renin 5⬘-flanking area corresponded
to human renin gene nucleotides ⫺2754 through ⫺2736 (relative to
the start site of human gene transcription) and ⫹226 through ⫹248,
respectively.19 Each PCR cycle consisted of a 30-second denaturation step at 94°C, a 60-second annealing step at 54°C, and a
75-second extension step at 72°C, for a total of 30 cycles. The PCR
product was cloned into the pT7Blue(R) T-Vector (Novagen) to
generate the construct pT7Blue(R)⫹248/⫺2754. Fusions between
the 5⬘-flanking overlapping intervals and the luciferase reporter gene
were made with the pGL3-Basic vector (Promega). The plasmids
pGL3-Basic⫺1298/⫹12, pGL3-Basic⫺1111/⫹12, pGL3-Basic⫺953/⫹12, pGL3-Basic⫺452/⫹12, pGL3-Basic⫺226/⫹12, and
pGL3-Basic⫺144/⫹12 have a common 3⬘ end at ⫹12 and promoters
of varying lengths and were cloned as shown in Figure 1A.
Sequences of all constructs were confirmed through dideoxy sequencing with an ABI PRISM cycle sequencing kit.
Transfection Studies
Plasmid DNAs were purified with a commercial kit (Qiagen).
Transient DNA transfection was performed with Tfx-20 Reagent
(Promega). The culture medium was replaced with serum-free
medium shortly before the transfection. Each luciferase reporter
plasmid (0.5 ␮g) was cotransfected with 0.5 ␮g of the pRL-TK
vector (Promega). When needed, T3 (10⫺7 mol/L) was added immediately. Twenty-four hours after transfection, the cells were lysed by
Kobori et al
Thyroid Hormone and the Human Renin Gene
101
the addition of 100 ␮L passive lysis buffer (Promega). Luciferase
activity assays were performed with the Dual Luciferase Reporter
Assay System (Promega) and were read in a TD-20/20 automatic
luminometer (Turner Designs). The pRL-TK luciferase activity was
used to correct for transfection efficiency, sample loss, and other
factors that could affect the results. Experiments in which pRL-TK
luciferase activity varied by ⬎2-fold were discarded. Consequently,
when luciferase activities of pRL-TK were evaluated as the transfection efficiency, the efficiency varied within 1.9-fold (maximum
59 840, minimum 32 070, and average 45 694⫾1915 luminometric
units). In a preliminary study, we examined the relation between
transfection time and luciferase activity by using pGL-Basic⫺144/
⫹12 without T3. When cells were harvested 12 hours after transfection, luciferase activity was 25 008⫾1124 luminometric units (n⫽6).
Luciferase activity significantly increased at 24 and 36 hours
(47 308⫾3121 and 42 206⫾3065 luminometric units, respectively)
and then notably decreased at 48 hours to 26 990⫾1039 luminometric units. Therefore, as noted above, we harvested cells 24 hours after
transfection in the following protocols.
Mutant Study
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Domain mutants were generated from pGL3-Basic⫺1111/⫹12 plasmid with a QuickChange site-directed mutagenesis kit (Stratagene)
according to the manufacturer’s recommendations. Mutants-1, -2,
and -3 (M1, M2, and M3) were changed from 5⬘-AGG TCA-3⬘
(⫺1066 to ⫺1061) to 5⬘-TCA AGG-3⬘, from 5⬘-AGG TCA-3⬘
(⫺1061 to ⫺1056) to 5⬘-TCA AGG-3⬘, and from 5⬘-GTT CCT-3⬘
(⫺1051 to ⫺1046) to 5⬘-TCC TTG-3⬘, respectively (Figure 2A). The
transfection study was performed as described earlier.
Electrophoretic Mobility Shift Assays
Nuclear extracts were prepared from 108 confluent Calu-6 cells
according to the Hennighausen method.21 The ⫺1111/⫺953 DNA
fragment of the intact human renin gene was 5⬘-end labeled with
␥-32P-ATP (Amersham Pharmacia Biotech) with T4 polynucleotide
kinase (Takara). Ten micrograms of nuclear protein from Calu-6
cells or bovine serum albumin was incubated for 30 minutes at room
temperature in the presence of 0.3 U of poly(dI/dC) in 20 mmol/L
Tris-glycine (pH 7.6) and 1 mmol/L EDTA. The 5⬘-labeled probe
(0.1 pmol) was added, and the preparation was further incubated for
30 minutes at room temperature. After chilling on ice, the mixture
was run on an 8% nondenaturing polyacrylamide gel and exposed for
autoradiography. In competition assays, a 200-fold excess of unlabeled ⫺1111/⫺953 DNA fragment of the intact human renin gene or
the 3 human renin gene mutants was added to the reaction mixture
and incubated for 30 minutes at room temperature before incubation
with the labeled probe.
RNase Protection Assays
The levels of hTR-␤, renin, and GAPDH mRNA were also measured
by RNase protection of specific oligonucleotide probes using the
Multi-NPA Kit (Ambion) according to the manufacturer’s protocol.
For hTR-␤ mRNA detection, a 53-mer oligonucleotide antisense to
the hTR-␤ mRNA with 10 nucleotides mismatched at its 3⬘-end was
used. The sequence of this oligomer is 5⬘-CGT GAT ACA GCG
GTA GTG ATA CCC GGT GGC TTT GTC ACC ACA Ctc tgt gta
gt-3⬘, complementary to nucleotides ⫹312 through ⫹354 of the
hTR-␤ gene.15 To detect renin mRNA, a 51-mer oligonucleotide
complementary to the human renin mRNA with 9 nucleotides
mismatched at its 3⬘-ends was used. The sequence of this oligomer
(5⬘-GGA GCT GGT AGA ACC TGA GAT GTA GGA TGC ACC
GGT GTC TAC gtg gca ttg-3⬘) is complementary to nucleotides
⫹873 through ⫹914 of the human renin gene.19 The lowercase
letters of both oligomers represent the mismatched bases. The levels
of human GAPDH mRNA were measured in these studies with a
commercially available 44-mer (Ambion) that gives a 36-mer
nuclease-protected fragment. These probes were 5⬘-end labeled as
described earlier. The radiolabeled probe (300 pg ⬇ 4⫻104 cpm) was
hybridized to 10 ␮g of sample total RNA and subjected to nuclease
Figure 2. A, Schema of 3 mutants of the human renin gene. B,
Effects of mutations in the putative THRE motifs. Data are
expressed as the ratio to the average luminometric units of
pGL3-Basic⫺1111/⫹12. Values are presented as mean⫾SEM.
*P⬍0.0001, †P⬍0.01, ‡P⬍0.03 vs pGL3-Basic⫺1111/⫹12. n⫽6.
C, Electrophoretic mobility shift assay of the radioactive labeled
⫺1111/⫺953 DNA fragment of the intact human renin gene with
bovine serum albumin (lane 1) or the nuclear extract from
Calu-6 cells (lanes 2 to 6). No slow migrating band was
observed when the labeled DNA was incubated with bovine
serum albumin (lane 1); however, a single specific band
appeared with retarded mobility when the labeled DNA was
incubated with nuclear extract from Calu-6 cells (lane 2). Furthermore, the binding of labeled putative THRE to the nuclear
proteins was displaced by the excessive unlabeled DNA fragment of the intact human renin gene (lane 3) but not by the
excessive unlabeled DNA fragments of the 3 mutants of the
human renin gene (lanes 4 to 6). Similar observations were
obtained from 2 other experiments.
treatment. The nuclease-protected oligomers were analyzed by 15%
polyacrylamide, 7 mol/L urea gel (Novex) electrophoresis.
Statistical Analysis
Statistical analysis was performed with an unpaired t test or 1-way
ANOVA with the post hoc Schéffe F test. All data are presented as
mean⫾SEM. P⬍0.05 was considered significant.
Results
After RT-PCR with total RNA from Calu-6 cells with
hTR-␤–specific primers, gel electrophoresis exhibited 1 clear
band at 264 bp that represents the hTR-␤ gene (Figure 3A)
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Figure 3. A, Determination of hTR-␤ expression in Calu-6 cells
by RT-PCR. The PCR product was sequenced and confirmed to
be identical to hTR-␤ cDNA. B, The expression of hTR-␤ in
Calu-6 cells was also confirmed with RNase protection assay.
When sample RNA and RNase treatments were omitted, 2
bands for full-length probes were shown (lane 1). When sample
RNA was excluded, no band was seen, because RNase treatment digested both probes (lane 2). When RNase protection
assay was performed with 2 kinds of probe and sample RNA
including RNase treatments, 2 shorter fragments that were protected from RNase treatment were confirmed (lane 3).
and confirmed the expression of hTR-␤ in Calu-6 cells. The
expression of hTR-␤ was also confirmed with RNase protection assay (Figure 3B).
The effect of thyroid hormone on the expression of renin
mRNA is depicted in Figure 4A. According to quantitative
RT-PCR, thyroid hormone significantly increased human
renin mRNA levels by 92% (2.30⫾0.20 versus 1.20⫾0.10,
densitometric ratio to GAPDH, Figure 4B). The enhancement
of the expression of the human renin gene by thyroid
hormone treatment was also confirmed with RNase protection assay (Figure 4C). Densitometric analysis showed that
thyroid hormone significantly increased human renin mRNA
levels by 98% (2.58⫾0.21 versus 1.30⫾0.02, densitometric
ratio to GAPDH, Figure 4D).
The effect of varying lengths of the 5⬘-flanking sequence
on the promoter activity of the human renin gene is shown in
Figure 1B. Under basal conditions, pGL3-Basic⫺144/⫹12
showed more reporter activity than the parent pGL3-Basic
plasmid (1155⫾219 luminometric units). Less reporter activ-
Figure 4. Effect of thyroid hormone on renin mRNA expression.
A, Representative blots of renin and GAPDH mRNA levels in
Calu-6 cells by RT-PCR. The PCR products were sequenced
and were confirmed to be human renin and human GAPDH
cDNA. B, Densitometric analysis of the bands shows that thyroid hormone increases renin mRNA expression by 92%. C,
Representative blots of renin and GAPDH mRNA levels in
Calu-6 cells by RNase protection assay. When RNase protection
assay was performed with 2 kinds of probe and sample RNA
including RNase treatments, 2 shorter fragments that were protected from RNase treatment were confirmed (lanes 3 to 6). D,
Densitometric analysis of the bands shows that thyroid hormone
increases renin mRNA expression by 98%. Values shown are
mean⫾SEM. *P⬍0.01 vs without T3. n⫽12.
ity was observed from extracts of cells transfected with
pGL3-Basic⫺266/⫹12 than from those transfected with
pGL3-Basic⫺144/⫹12. pGL3-Basic⫺452/⫹12 showed more
activity than pGL3-Basic⫺226/⫹12. pGL3-Basic⫺953/⫹12
demonstrated less activity than pGL3-Basic⫺452/⫹12. pGL3Basic⫺1111/⫹12 and pGL3-Basic⫺1298/⫹12 exhibited less
activity than pGL3-Basic⫺953/⫹12.
The effect of thyroid hormone on the promoter activity of
the human renin gene is shown in Figure 1C. In comparison
with basal activity, thyroid hormone stimulated the promoter
activity of pGL3-Basic⫺1111/⫹12 and pGL3-Basic⫺1298/
⫹12 by 2.3⫾0.1- and 1.7⫾0.1-fold, respectively. Other
shorter constructs did not exhibit stimulation by thyroid
hormone.
The effects of mutations in the putative thyroid hormone
(THRE) motifs are shown in Figure 2B. Basal promoter
activities were significantly increased in M1, M2, and M3
compared with pGL3-Basic⫺1111/⫹12 (lanes 2 to 4),
whereas thyroid hormone did not stimulate promoter activities in mutated constructs (lanes 5 to 7).
Kobori et al
The interaction of the putative human renin gene THRE
with the nuclear proteins of Calu-6 cells was examined with
electrophoretic mobility shift assay. Figure 2C shows the
binding of Calu-6 cell nuclear extract with the labeled
putative THRE. A single specific band appeared with retarded mobility (lane 2). No specific band was observed when
the labeled DNA was incubated with bovine serum albumin
alone (lane 1). Furthermore, the binding of labeled putative
THRE to the nuclear proteins was displaced by the excessive
unlabeled DNA fragment of the intact human renin gene (lane
3). The excessive unlabeled DNA fragments of the 3 mutants
of the human renin gene did not show any obvious changes in
electrophoretic mobility (lanes 4 to 6).
Discussion
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Expression of renin mRNA in Calu-6 cells was increased by
thyroid hormone in the same manner as in primary cultured
juxtaglomerular cells, suggesting that Calu-6 cells may provide a good model to study the effect of thyroid hormone on
transcriptional changes in the renin gene. The 5⬘-flanking
DNA sequence ⫺144/⫹12 appears to contain the human
renin gene promoter. The same region (⫺149/⫹13,10 ⫺148/
⫹11,22 and ⫺148/⫹18,23 and ⫺137/⫹1624 ) was also identified as the human renin gene promoter using Calu-6, GC rat
lactotrope precursor, and JEG-3 human choriocarcinoma cell
lines. These consistent findings among different cell lines
support the notion that this 150- to 160-nucleotide stretch in
the 5⬘ area of the human renin gene does contain the
promoter.
Our results also suggested that the 5⬘-flanking DNA
sequences, ⫺226/⫺144, ⫺953/⫺452, and ⫺1111/⫺953,
contain a negative regulatory element (Figure 1B), which is
consistent with the findings of Lang et al ,10 Sun et al,22,23 and
Borensztein et al.24 The 5⬘-flanking DNA sequence ⫺452/
⫺226 appears to contain a positive regulatory element (Figure 1B), which is also consistent with the findings of Sun et
al.22 However, pGL3-Basic⫺1111/⫹12 and pGL3-Basic⫺1298/⫹12 repressed promoter activity (Figure 1B).
The sequences ⫺1111/⫹12 and ⫺1298/⫹12 had significantly increased promoter activity with thyroid hormone
treatment; constructs with sequences ⫺144/⫹12, ⫺226/⫹12,
⫺452/⫹12, and ⫺953/⫹12 did not show any significant
changes. These stimulatory effects of T3 on luciferase activity
with constructs pGL3-Basic⫺1111/⫹12 and ⫺1298/⫹12
suggest that hTR-␤ bound to THRE and activated basal
promoter activity in the presence of thyroid hormone. Because the shorter construct did not show any response to T3,
the sequence between ⫺1111 and ⫺953 may contain a
THRE.
We found 3 candidate THRE motifs (5⬘-AGG TCA-3⬘) in
sequence ⫺1111/⫺953: (1) 5⬘-AGG TCA-3⬘ (⫺1066 to
⫺1061), (2) 5⬘-AGG TCA-3⬘ (⫺1061 to ⫺1056), and (3)
5⬘-GTT CCT-3⬘ (⫺1051 to ⫺1046). The most common
motifs of THRE are an inverted palindrome without spaces
(AGG TCA TGA CCT)25 and a direct repeat with 4 spaces
(AGG TCA (n)4 AGG TCA).26 However, other patterns have
been recently reported to be THREs.27,28 When variation in
the THRE motif is taken into account, it is possible that the
perfect direct repeat without space (5⬘-AGG TCA GGT
Thyroid Hormone and the Human Renin Gene
103
CA-3⬘, ⫺1066 to ⫺1056) and the nearly perfect inverted
palindrome spaced by 4 bp (5⬘-AGG TCA caa tGT TCC T-3⬘,
⫺1061 to ⫺1046) are putative THREs.
Nucleotides 5⬘-AGG TCA GGT CAc aat GTT CCT-3⬘
(⫺1066 to ⫺1046) inhibited renin gene promoter activity. It
has been reported that hTR-␤ constitutively binds to THRE
and represses basal promoter activity in the absence of
thyroid hormone. It is possible that hTR-␤ could not bind the
mutated THRE motif sequences in the present study and
therefore could not repress basal promoter activity. In addition, the stimulatory effect of thyroid hormone was abolished
by each mutation, indicating that thyroid hormone and hTR-␤
cannot form a complex on the mutated THRE.
The electrophoretic mobility shift assay data (Figure 2C)
further confirmed that the intact putative THRE is important
for binding with Calu-6 cell nuclear extract and that all 3
putative motifs are essential for the interaction of the putative
THRE of the human renin gene with Calu-6 cell nuclear
proteins. This result is consistent with the luciferase assay
studies shown in Figure 2B. In Figure 2B, each mutated
construct lost all thyroid hormone responsiveness, whereas
each construct had intact 2 putative motifs. It is, however, still
not clear why the remaining 2 motifs in each mutant DNA
fragment of the human renin gene, M1, M2, and M3, did not
compete with intact DNA probe. It is possible that the binding
ability of DNA with 3 intact motifs is so potent that the
remaining 2 motifs in mutated DNA cannot replace the
binding between wild-type DNA and the nuclear proteins.
Previous reports showed that 3 motifs worked as 1 THRE in
the ␣-myosin heavy chain,29 growth hormone,30 and malic
enzyme31 genes. Sugawara et al32 examined the role of 3
THRE motifs in the growth hormone gene and, consistent
with our present data, found that the interaction of THRE and
hTR-␤ was lost when 1 of 3 putative motifs was mutated,
suggesting that all 3 half-sites are necessary.
In conclusion, we present the following findings. (1)
Thyroid hormone stimulated renin mRNA expression in
Calu-6 cells. (2) Thyroid hormone stimulated the promoter
activity of the pGL3-Basic⫺1111/⫹12 and the pGL3-Basic⫺1298/⫹12 by 2.3⫾0.1- and 1.7⫾0.1-fold over basal
levels, respectively; however, other shorter constructs (pGL3Basic⫺144/⫹12, pGL3-Basic⫺226/⫹12, pGL3-Basic⫺452/
⫹12, and pGL3-Basic⫺953/⫹12) were not stimulated by
thyroid hormone. (3) In sequence ⫺1111/⫺953, there is a
5⬘-AGG TCA GGT CAc aat GTT CCT-3⬘ sequence that may
be a THRE. Mutant studies indicated that hTR-␤ could not
repress the basal promoter activity in the absence of thyroid
hormone. (4) The intact motif of the putative THRE is
important for binding to Calu-6 cell nuclear proteins. These
results suggest that thyroid hormone stimulates the promoter
activity of the human renin gene through a THRE-dependent
mechanism in Calu-6 cells.
Acknowledgments
This work was supported in part by the Japanese Ministry of
Education, Science, Sports and Culture; Grant-in-Aid for Encouragement of Young Scientists 08770511 (1996) and 09770500 (1997–
1998); a Young Investigator Prize for Encouragement from Keio
University (1996 and 1998); a grant-in-aid from Keio University for
the Promotion of Science (1997); research fellowships of the
104
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January 2001
Yokoyama Clinical Pharmacology Foundation (1998); and the Uehara Memorial Foundation (1999).
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Thyroid Hormone Stimulates Renin Gene Expression Through the Thyroid Hormone
Response Element
Hiroyuki Kobori, Matsuhiko Hayashi and Takao Saruta
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Hypertension. 2001;37:99-104
doi: 10.1161/01.HYP.37.1.99
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