© 1999 American Heart Association, Inc.
Original Contribution |
Angiotensinogen Gene–Activating Elements Regulate Blood Pressure in the Brain
From the Departments of Geriatric Medicine (T.N., A.M., R.M., K.Y., S.N., N.T., H.M., J.H., T.O.) and Gene Therapy Science (R.M., Y.K.), Osaka University Medical School, and Department of Applied Biochemistry (A.F.), University of Tsukuba, Tsukuba, Japan.
Correspondence to Toshio Ogihara, MD, Department of Geriatric Medicine, Osaka University Medical School, 2-2 Yamada-oka, Suita 565-0821, Japan. E-mail moriguch{at}geriat.med.osaka-u.ac.jp
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Abstract—Although the angiotensinogen gene is a possible candidate as a determinant of hypertension, the molecular mechanisms of tissue angiotensinogen gene regulation have yet to be clarified. We identified essential transcription regulators of angiotensinogen production in the central nervous system using synthetic double-stranded oligodeoxynucleotides (ODNs) as "decoy" cis elements to block the binding of nuclear factors to promoter regions of the targeted gene. Using a gel mobility shift assay, angiotensinogen gene–activating element (AGE) 2 binding protein was detected in the brain nuclear extracts of both spontaneously hypertensive rats (SHRs) and normotensive Wistar Kyoto rats (WKYs). Importantly, the binding activity of AGE 2 and angiotensinogen mRNA level were significantly higher in the brain of SHRs than in that of WKYs. Using the decoy approach, we demonstrated a significant decrease in the blood pressure of SHRs by transfection of AGE 2 decoy, but not mismatched, ODNs into the lateral cerebroventricle, accompanied by a significant decrease in brain angiotensinogen concentration and mRNA, and angiotensin II level. That these effects, demonstrated herein, are due to central effects is confirmed by the fact that no changes in circulating levels of angiotensinogen or angiotensin II concentrations were observed. Notably, AGE 2 decoy ODNs did not decrease the blood pressure of WKYs. We conclude that the abnormal expression of AGE 2 binding protein in the central nervous system plays a crucial role in high blood pressure of a genetically hypertensive rat model.
Key Words: transcriptional cis element • central nervous system • HVJ-liposome method • renin-angiotensin system • decoy oligodeoxynucleotide
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Introduction |
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Angiotensinogen, which is mainly produced in the liver, plays important roles in the renin-angiotensin system in the pathogenesis of essential hypertension. Results of recent genetic studies suggest that the angiotensinogen gene is a possible determinant of hypertension.1 2 3 4 The role of angiotensinogen in the regulation of blood pressure was previously demonstrated by the observation that anti-angiotensinogen antibody administration resulted in a reduction in blood pressure.5 In contrast, in another study acute administration of pure rat angiotensinogen to rats resulted in an increase in blood pressure.6 Using a "loss-of-function" approach, we and others have reported that generation of circulating angiotensinogen is a rate-limiting step in blood pressure regulation.7 8 9 Given the importance of angiotensinogen regulation in the pathogenesis of hypertension, understanding how the angiotensinogen gene itself is regulated is essential. One study suggests that the angiotensinogen gene is regulated by novel transcriptional factors such as angiotensinogen gene–activating factor (AGF) 1 to 3 in cultured human hepatocytes (HepG2 cells) in vitro.10 Previously, we demonstrated that angiotensinogen gene–activating element 2 (AGE 2), which is the proximal promoter element extending from –96 to –52, has a pivotal role in the regulation of hepatic angiotensinogen production in vivo.11
On the other hand, accumulating evidence has indicated the presence of
a tissue renin-angiotensin system that involves the central
nervous system.12 13 14 15 However, the molecular mechanisms of
angiotensinogen gene regulation in the brain remain
unknown. Moreover, no reports have described the importance of brain
angiotensinogen in blood pressure control. Therefore, in
this study, we examined (1) how brain angiotensinogen is
regulated in vivo and (2) whether brain angiotensinogen
regulates blood pressure. To answer these questions, we used synthetic
double-stranded oligodeoxynucleotides (ODNs) as "decoy"
cis elements to block the binding of nuclear factors to
promoter regions of the targeted gene, resulting in the inhibition of
gene transactivation16 17 18 19 (Figure 1
).
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Synthesis of ODNs and Selection of Sequence Targets
The sequences of phosphorothioate double-stranded ODNs complementary to AGF 2 and AGF 3 binding sites and mismatched ODNs used in the present study have been previously described.10 11 The sequences of the phosphorothioate ODNs were as follows: AGE 2 decoy ODNs (core sequences are underlined; 2-bp mismatches are included in 10-bp consensus sequences):
- 5'-CCCAGCCTCTGTACAGAGTAGCCCA-3'
3'-GGGTCGGAGACATGTCTCATCGGGT-5' Mismatched AGE 2 decoy ODNs (mismatched sequences are in bold):
5'-CCCAGCTGAGGTTTAGAGTAGCCCA-3'
3'-GGGTCGACTCCAAATCTCATCGGGT-5' AGE 3 decoy ODNs (core sequences are underlined):
5'-AGGGGATAGCTGTGCTTGTCTAGGTT-3'
3'-TCCCCTATCGACACGAACAGATCCAA-5' Mismatched AGE 3 decoy ODNs (mismatched sequences are in bold):
5'-AGGGGATGTTGGATGCGAGCCTAGGTT-3'
3'-TCCCCTACAACCTACGCTCGGATCCAA-5'
Decoy ODNs have been shown to bind to AGF 2 and AGF 3.10 11 20 Synthetic ODNs were washed with 70% ethanol, dried, and dissolved in sterile Tris-EDTA buffer (10 mmol/L Tris, 1 mmol/L EDTA). The supernatant was purified using a NICK column (Pharmacia) and quantified with a spectrophotometer. ODNs were annealed for 40 minutes while the temperature decreased from 85°C to 25°C.
Preparation of HVJ–Liposome Complex
Hemagglutinating virus of Japan (HVJ, Z strain) was propagated
in chorioallantonic fluid of embryonated eggs, as previously
described.7 8 11 20 HVJ was collected by
centrifugation at 27 000g for 40 minutes
and suspended in balanced salt solution (BSS, in mmol/L, NaCl 137,
KCl 5.4, and Tris-HCl 13, pH 7.6) overnight. The resuspended HVJ was
stored at 4°C and used within 1 week after purification. The
hemagglutinating activity of HVJ was determined as described
previously.7 8 11 20 A 540-nm optical density
unit of HVJ suspension contained 1 mg/mL protein and was equivalent to
15 000 hemagglutinating activity units/mL. Three types of lipids
(phosphatidylcholine, phosphatidylserine, and
cholesterol) were mixed at a ratio of 4.8:1:2 (wt/wt/wt),
and the lipid mixture (10 mg) in tetrahydrofuran was deposited in a
rotary evaporator. The dried lipid residue was hydrated in 200 µL of
BSS containing decoy ODNs (400 nmol), and decoy ODNs were then
incorporated into the liposomes by shaking and sonication. After
adjusting the total volume to 0.5 mL with BSS, the ODN liposomes were
incubated at 37°C for 30 minutes with gentle shaking (2 strokes per
second). The liposome suspension and purified HVJ (30 000
hemagglutinating units), which had been inactivated by
ultraviolet irradiation (110 erg/mm2 per second)
for 3 minutes just before use, were mixed and incubated at 4°C for 10
minutes. After adjusting the volume to 2 mL BSS, the mixture was
incubated at 37°C for 60 minutes with shaking. Free HVJ was removed
from the HVJ liposomes by sucrose density gradient
centrifugation. The top layer of the sucrose gradient
containing the HVJ liposomes was collected for use. The final
concentration of decoy ODNs was 20 µmol/L. We also prepared a
lower dose (5 µmol/L) or higher dose (40 µmol/L) of decoy
ODNs.
In Vivo Transfection of HVJ–Liposome Complex
All animal studies were approved by the Osaka University
Animal Use Review Board. Male spontaneously hypertensive rats (SHRs)
and Wistar Kyoto rats (WKYs) (20 weeks old, 250 to 300 g; SHRs,
n=32; WKYs, n=20) obtained from closed colonies at Charles River
Breeding Laboratories (Osaka, Japan) were anesthetized
with an intraperitoneal injection of pentobarbital
(50 mg/kg). Anesthetized rats were then placed in a
stereotaxic frame (Narishige Scientific Instrument
Laboratory), and the skull was exposed. A stainless steel injection
needle (23 gauge, Terumo Inc) with a specially designed
polytetrafluoroethylene connector
(FEP tube, Bioanalytical Systems) was inserted into the right
lateral cerebroventricle, as previously described.21 The
stereotaxic coordinates were the following: lateral brain
ventricle, 1.3 mm posterior to bregma, 2.1 mm lateral to the
midline, and 3.6 mm below the skull surface. A total volume of 10
µL of final HVJ-liposome complex containing ODNs (20 µmol/L in
liposomes) was then injected into the right lateral cerebroventricle.
This procedure was completed by administering a
prophylactic antibiotic (penicillin G, 30 000 units IM).
After infusion of the HVJ-liposome complex, the injection needle was
removed. Systolic blood pressure was then measured using the
tail-cuff method (BP-98A, Softron) until 14 days after transfection. In
a separate group, rats (SHRs, n=8; WKYs, n=8) were decapitated without
use of anesthesia at 3 days after transfection. Brain
tissue was carefully and promptly removed, washed with PBS (in
mmol/L, NaCl 137, KCl 3,
Na2HPO4 8, and
KH2PO4 1), and immediately
frozen in liquid nitrogen for use in the gel mobility shift assay and
measurement of brain angiotensinogen and
angiotensin (Ang) II concentrations. All brain tissue was
stored at -80°C before use. Blood samples were also collected in
prechilled tubes containing EDTA-2Na (1 mg/mL whole blood) and 2.5
mmol/L phenylmethylsulfonyl fluoride (PMSF) and
centrifuged at 4°C. To measure Ang II, plasma was stored at
-80°C before assay.
Gel Mobility Shift Assay
Nuclear extracts were prepared from transfected brain as
described previously.10 11 17 In brief, rat brains were
homogenized in a Wheaton all-glass Dounce
homogenizer in 2 volumes of ice-cold
homogenization buffer (in mmol/L, HEPES (pH
7.9) 10, PMSF 0.5, MgCl2 1.5, KCl 10, and DTT
0.5. After centrifugation at 12 000g for 15
minutes at 4°C, each pellet was lysed in 1 volume of ice-cold
homogenization buffer (in mmol/L, PMSF 0.5,
HEPES [pH 7.9] 20, MgCl2 1.5, DTT 0.5, and EDTA
0.2; 0.42 mol/L NaCl; and 25% [vol/vol] glycerol) by
homogenization in the Dounce
homogenizer. The suspension was rocked gently for 30
minutes at 4°C and centrifuged at 12 000g for 15
minutes at 4°C, and the supernatant was dialyzed overnight at 4°C
(Dialysis Tubing, GIBCO) against at least 50 volumes of
dialyzing buffer (in mmol/L, PMSF 0.5, HEPES 20, DTT 0.5, and EDTA
0.2; 0.1 mol/L KCl; and 20% [vol/vol] glycerol) with one change
after 2 hours. After centrifugation at
12 000g for 15 minutes at 4°C, the protein concentration
of the supernatant was determined. Aliquots containing protein were
snap frozen in liquid nitrogen and stored at –80°C.
Decoy ODNs were labeled as probes at the 5' end using a 5' end-labeling
kit (KinaseMax, Ambion). Binding reactions containing nuclear extract
(50 µg), poly(dI-dC) (2 µg) (Pharmacia Biotech), 10x binding
reaction buffer (40% glycerol and [in mmol/L], EDTA 10,
b-mercaptoethanol 10, Tris-HCl (pH 7.5) 100, and NaCl 400) and
32P-labeled ODNs (
10 000 cpm) were incubated
for 30 minutes at room temperature and then loaded onto a 5% PAGE and
dried. For the control, samples were incubated with an excess (2x,
4x) of unlabeled AGE 2 and 3 ODNs, which completely abolished binding.
Gels were analyzed by autoradiography. The
activity of binding protein was quantified and compared by
densitometric analysis.
Rat Angiotensinogen and Ang II Assay
The brain or plasma angiotensinogen level was
determined indirectly by the measurement of Ang I generated after
incubation with excess recombinant human active renin (kindly donated
by Dr Fumiaki Suzuki, Gifu University, Japan), as described
previously.22 23 Samples were homogenized
using a Polytron in angiotensinogen RIA buffer
(in mmol/L, Na2HPO4
150, NaCl 160, and EDTA 3, and 5% BSA [pH 6.5]) and
centrifuged at 12 000g for 30 minutes at 4°C. The
supernatant or 1-mL sample of plasma was incubated with 1 mL (Goldblatt
units) recombinant human active renin and 1 mmol/L PMSF for 2
hours at 37°C. During the incubation, angiotensinogen was
completely converted to Ang I.22 23 The Ang I level
of tissue sample was determined by RIA, and the results were expressed
as nanograms of Ang I per gram tissue.
To measure Ang II in the brain samples, the brain was homogenized using a Polytron in 0.1N HCl (2 mL/1 g tissue), boiled in a waterbath for 7 minutes, and centrifuged at 12 000g for 30 minutes at 4°C. The supernatant was applied to an octyl minicolumn (Amprep C8, Amersham), which had been prewashed with 3 mL of 100% methanol and 3 mL of 0.1% trifluoroacetic acid (TFA).24 25 After washing the column with 3 mL of 0.1% of TFA, Ang II was eluted with 2 mL of ethanol:water:TFA (80:19.9:0.1, vol/vol). The eluant was dried under vacuum in a centrifugal concentrator (CC-181, Tommy) and the resultant Ang II–containing residue resuspended in 100 µL of 0.1% TFA. HPLC characterization was then performed as previously described.24 25 In the appropriate peak fractions, samples were collected and dried in a vacuum centrifuge and redissolved in 0.1 mol/L Tris acetate, pH 7.4, containing 2.6 mmol/L EDTA-2Na, 1 mmol/L PMSF, and 0.1% BSA. The immunoreactive Ang II was measured using RIA with a specific Ang II antibody. A 1-mL sample of each plasma specimen was promptly concentrated in an Amprep C8 minicolumn and quantified using the same procedure.24 25 The recovery of Ang II was 98±2% (n=5), which was verified by 125I-labeled Ang II detection. The data were not corrected for this recovery, because the variation of recovery was negligible. The elution times for Ang II, Ang III, and Ang I were 19.0, 20.7, and 23.7 minutes, respectively. The sensitivity of this immunoassay was 0.1 pg/tube. The recovery of Ang II after HPLC was 85±5%. The cross-reactivity of the Ang II antibody was 100% for Ang II and <0.1% for Ang I, Ang III, or Ang-(1–7).
RNA Analysis
For Northern blot analysis, the brain was promptly
removed from rats before transfection (SHRs, n=5; WKYs, n=5), and SHRs
were transfected with decoy ODNs 3 days after transfection (AGE 2 decoy
ODNs, n=5; mismatch decoy ODNs, n=5). All brain tissues were frozen in
liquid nitrogen and stored at –80°C before RNA extraction. The whole
brain (except cerebellum) was homogenized with RNAzol B (2
mL/100 mg tissue, Tel-Test, Inc) using a Polytron, chloroform added to
extract the RNA, and the mix centrifuged at 12 000g
for 15 minutes at 4°C. The aqueous phase was collected, and
isopropanol was added to precipitate the RNA. After
centrifugation, the RNA pellet was washed in 75%
ethanol and dissolved in Tris-EDTA. For Northern blot analysis,
20 µg of total RNA was electrophoresed through a 1.5%
agarose-formaldehyde denaturing gel and transferred to nitrocellulose
membrane (Amersham International). The filter was baked, prehybridized,
and hybridized to full-length cDNA for rat
angiotensinogen22 25 and human GAPDH cDNA
control probe (Clontech Laboratories, Inc); both were labeled with
32P, with the Rad Prime DNA Labeling System (Life
Technologies, Inc), for Northern blotting. The filter was then washed
and exposed to x-ray film. The expression of mRNA was quantified and
compared by densitometric analysis.
Statistical Analysis
The results are expressed as mean±SEM. Statistical
analysis was performed using ANOVA followed by multiple
comparisons. When appropriate, the repeated measures option was applied
to the ANOVA. The Duncan multiple range test was applied to assess
differences whenever statistical significance was found. The Student
t test was also used for paired and unpaired observations.
For statistical purposes, values below the detection levels of an assay
were recorded as the detectable level. Statistical significance was
established at the P<0.05 level.
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Role of AGE 2 Binding Protein in Brain Angiotensinogen Production and High Blood Pressure Regulation in SHRs but Not WKYs
To detect AGE binding proteins, gel mobility shift assay was performed using nuclear extracts and synthetic double-stranded ODNs that had sequence homology with AGE 2. Similar to the liver,10 11 AGE 2 binding protein was detected in brain tissue (Figure 2A
and 2B). Moreover,
complete competition for the increased binding of AGE 2 by an excess
amount of AGE 2 decoy ODNs, but not mismatched AGE 2 decoy ODNs, was
observed (Figure 2A), which was consistent with a
previous report.10 11 Importantly, the binding activity of
AGE 2 was significantly higher in SHRs than WKYs (P<0.01,
Figure 2B and 2C).
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Given the significant activation of AGE 2 binding protein, we
hypothesized that the activation of AGE 2 binding activity may
contribute to higher upregulation of angiotensinogen gene
expression in SHRs than WKYs, thereby resulting in the high blood
pressure of SHRs. Indeed, the mRNA level of angiotensinogen
was significantly higher in the brains of SHRs than WKYs
(P<0.05, Figure 3).
Therefore, we examined the inhibitory effects of decoy ODNs
against AGE 2 on the high blood pressure of SHRs. Because we previously
reported the highly efficient ODNs transfection into the brain using
the HVJ-liposome method,26 we first examined the
physiological effect of AGE 2 decoy ODN
transfection by the HVJ-liposome method into the lateral
cerebroventricle. As shown in Figure 4A, transfection of AGE 2 decoy ODNs (20 µmol/L) into the
intracerebroventricle resulted in a significant transient decrease in
systolic blood pressure in SHRs at 1 day after transfection
through 7 days. On the other hand, transfection of mismatched decoy
ODNs did not change blood pressure. Decrease in systolic blood
pressure observed in AGE 2 decoy ODN-transfected rats reached
statistical significance at 1, 2, 4, and 7 days after transfection as
compared with blood pressure in mismatched decoy ODN-transfected rats.
No changes in systolic blood pressure were observed in response
to either transfection of AGE 2 decoy ODNs or mismatched decoy ODNs in
WKYs (data not shown). As shown in Figure 4B, a lower
concentration of ODNs (5 µmol/L) did not decrease blood pressure
in SHRs (mismatched decoy ODNs, 229±3 mm Hg; 5 µmol/L of
AGE 2 decoy ODNs, 225±4 mm Hg; NS) and a higher concentration of
ODNs (20 and 40 µmol/L) elicited significant decrease in
systolic blood pressure in SHRs (20 µmol/L, 213±6
mm Hg; 40 µmol/L, 201±4 mm Hg; P<0.05 versus
mismatched decoy ODNs, respectively), although the statistical
significance between 20 and 40 µmol/L was not observed.
Interestingly, Northern blot analysis showed that brain
angiotensinogen mRNA in SHRs was significantly decreased by
the transfection of AGE 2 decoy at 3 days after transfection
(P<0.05, Figure 5). In
contrast, there was no change in the GAPDH mRNA level between SHRs
treated with either AGE 2 decoy ODNs or mismatched decoy ODNs.
Consistent with the mRNA data, brain
angiotensinogen concentration as well as brain Ang II
concentration was significantly lower in SHRs transfected with AGE 2
decoy ODNs than in those treated with mismatched AGE 2 decoy ODNs
(Figure 6A and 6B,
angiotensinogen, -32±5%; Ang II, -25±6%;
P<0.05 versus mismatched). The significant contribution of
AGE 2 to high blood pressure in SHRs was also supported by the
observation that transfection of AGE 2 decoy ODNs did not decrease
blood pressure in WKYs, unlike in SHRs. This observation was further
confirmed by the fact that there was no significant change in brain
angiotensinogen and brain Ang II concentrations in WKYs
transfected with AGE 2 decoy ODNs (data not shown). Plasma
concentrations of either angiotensinogen or Ang II showed
comparable levels in SHRs or WKYs between decoy-transfected and control
animals (data not shown).
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P<0.05 vs day 0, *P<0.05 vs
mismatch decoy ODNs, **P<0.01 vs mismatch decoy ODNs.
B, Systolic blood pressure observed in SHRs at 3 days after
transfection with either mismatched decoy ODNs or 5 µL, 20 µL, or
40 µL of AGE 2 decoy ODNs. *P<0.05 vs mismatched
decoy ODNs.
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Role of AGE 3 Binding Protein
As mentioned above, AGE 3 (from -6 to +22), which is located
directly around the transcriptional start site in the core promoter
region, may also regulate angiotensinogen
production. Similar to AGE 2 binding protein, AGE 3 binding
protein was also detected in the brain tissue (Figure 7A and 7B). Moreover, complete
competition for the increased binding of AGE 3 by an excess amount of
AGE 3 decoy ODNs, but not mismatched AGE 3 decoy ODNs, was observed
(Figure 7A), consistent with a previous
report.10 11 The binding activity of AGE 3 was also
significantly higher in SHRs than in WKYs (P<0.01, Figure 7B and 7C). However, transfection of AGE 3 decoy ODNs into the
brain did not induce any significant change in high blood pressure in
SHRs (data not shown). Brain angiotensinogen and Ang II
concentrations were also not significantly different between SHRs
transfected with AGE 3 decoy ODNs and those transfected with mismatched
AGE 3 decoy ODNs (data not shown). Similar to WKYs, transfection of AGE
3 decoy ODNs into the brain failed to induce a change in blood
pressure. There were no significant differences in brain
angiotensinogen and Ang II concentrations between WKYs
transfected with AGE 3 decoy ODNs and those transfected with mismatched
AGE 3 decoy ODNs (data not shown).
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Discussion |
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Angiotensinogen is the precursor of the vasoactive peptide Ang II and is thought to be an important determinant of blood pressure and electrolyte homeostasis.27 28 Recent genetic linkage studies suggest that angiotensinogen is one of the candidate genes for hypertension.1 2 3 4 Indeed, recent findings suggest the pivotal role of angiotensinogen in high blood pressure regulation.7 8 9 Therefore, it is becoming increasingly important to study the molecular mechanism(s) involved in the regulation of the angiotensinogen gene. As previously described, the 5'-flanking region of the human angiotensinogen gene is important for tissue- and cell type–specific expression of the gene in vivo as well as in vitro.10 11 29 30 In human hepatocytes in vitro, cell type–specific activation of angiotensinogen gene transcription results from the cooperative interaction of a proximal promoter element (AGE 2; from –96 to –52) with AGE 3 (from –6 to + 22), which is located around the transcriptional start site in the core promoter region.10 However, little is known about the molecular mechanism(s) of angiotensinogen gene regulation in vivo. Therefore, using a decoy strategy, we examined this issue and found that the transcriptional cis element AGE 2, rather than AGE 3, plays an important role in the control of hepatic angiotensinogen gene expression using a decoy strategy.
Currently, the decoy strategy is considered a useful tool in a new
class of anti-gene strategies.16 17 18 19 Transfection of
double-stranded ODNs corresponding to the cis sequence will
result in the attenuation of the authentic cis-trans
interaction, leading to the removal of the trans factors
from the endogenous cis element, with subsequent
modulation of gene expression16 17 18 19 (see Figure 1
). Therefore, the decoy approach enables investigation of gene
regulation in vivo as well as in vitro by modulation of
endogenous transcriptional regulation. Previously, we
proved that the HVJ-liposome method prolonged the half-life of ODNs and
concentrated them in the nuclei of cultured neuronal cells in vitro and
in rat brains in vivo.26 We also confirmed the safety of
this method by histological examination and behavioral
observation. In this kinetic study,
intracerebroventricular administration
of FITC-labeled ODNs with HVJ-liposome produced a wider distribution of
ODNs in the central nervous system in vivo than did administration of
ODNs alone.26 This important experiment showed the broad
distribution of FITC-labeled ODNs in the neuronal cells of the
hypothalamic nuclei, subfornical organ, and some other
periventricular regions, which are considered important
areas in blood pressure regulation and which may be responsible
for the decrease in blood pressure via angiotensinogen gene
suppression.
Recent progress in molecular biology clearly indicates the existence of the components of a renin-angiotensin system in several tissues, including brain. Angiotensinogen, renin, angiotensin-converting enzyme (ACE), and the Ang II receptor have all been detected in brain.13 14 15 Furthermore, the intracerebroventricular infusion of angiotensin-converting enzyme inhibitors lowers the high blood pressure of SHRs.31 32 In addition, the neurons in the anteroventral third ventricular regions are rich in immunoreactive Ang II.33 This raises the possibility that the brain is involved in blood pressure control by the regulation of angiotensinogen production. Thus, it is noteworthy to clarify the detailed molecular mechanisms of angiotensinogen production in the brain of a genetically hypertensive model animal.
AGE is the proximal promoter element and a binding site for AGF. This transcriptional factor was detected by Tamura et al10 in the HepG2 cell line and by us11 in rat liver. The present results are the first observations to detect this transcriptional factor in the rat brain tissue. To our knowledge, no report has been published that AGE 2 binding factor in the brain regulates other genes in addition to angiotensinogen. However, this transcriptional factor may induce activation of some other genes. Further experiments using molecular biological techniques are required to clarify this possibility and unknown gene activation.
The palindromic sequences of AGE 2 are well conserved between the rat and mouse angiotensinogen gene, and a previous study has shown that the palindromic sequence of the rat angiotensinogen gene is important for the formation of a specific complex with HepG2 nuclear extract.10 This palindromic region (AGE 2) and AGF 2 appear to be directly involved in dictating the hepatocyte-specific expression of the angiotensinogen promoter. In contrast, AGE 3 may be involved in basal level expression in extrahepatic tissues, because AGF 3 is a ubiquitous nuclear factor in various cells.10 Therefore, we hypothesized that brain angiotensinogen expression may depend on AGE 3, whereas hepatic angiotensinogen expression is dependent on AGE 2. Indeed, the present study detected significantly stronger induction of binding activity of AGE 2 binding protein and AGE 3 binding protein in SHRs than WKYs. Unexpectedly, the regulation of angiotensinogen in brain is similar to that in liver, given that our present study demonstrated that only transfection of AGE 2 decoy ODNs can reduce high blood pressure in SHRs, accompanied by a significant decrease in brain angiotensinogen mRNA, brain angiotensinogen concentration, and brain Ang II concentration. These results indicate that, unexpectedly, AGE 2 rather than AGE 3 plays a pivotal role in angiotensinogen production in brain, as in the liver.
Previous reports have also suggested the potential contribution of AGE 3 binding protein in angiotensinogen production in extrahepatic tissue. In addition, the present data revealed the critical role of the specific AGE 2 binding protein on brain angiotensinogen expression. More importantly, transfection of AGE 2 decoy ODNs had little effect on blood pressure in WKYs, accompanied by no change in brain angiotensinogen or Ang II concentrations. These data suggest that increased angiotensinogen may be due to the induction of AGE 2 binding protein in the brain of SHRs. Yang and Sigmund34 recently reported that upstream (AGE 2) and downstream (d61–2) of the human angiotensinogen gene were not required for expression of angiotensinogen gene using transgenic mice carrying a double-mutant construct. In the present study, decoy ODNs against AGE 2 did not change expression of angiotensinogen gene in the brain of the normotensive animal. Inhibitory responses of angiotensinogen gene expression and high blood pressure by the introduction of decoy ODNs against AGE 2 in SHRs suggest that genetic abnormality of this regulatory element may exist in the brain of the hypertensive animal. Thus, high blood pressure in SHRs may be due to AGE 2 binding protein–driven angiotensinogen gene induction, although AGE 2 binding protein has yet to be cloned.
Overall, our results demonstrate that transfection of decoy ODNs against AGE 2, but not against AGE 3, of the angiotensinogen gene results in a transient decrease in the high blood pressure of SHRs, which suggests that the transcriptional cis element AGE 2, rather than the AGE 3 binding site, plays an important role in high blood pressure through the control of brain angiotensinogen production in SHRs. This study also revealed the utility of gene transfer and decoy technology for hypertension research, especially for evaluation of the tissue-specific functions of transcriptional factors of target gene regulation.
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Acknowledgments |
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This work was partially supported by the following grants to A.M.: a grant from the Japan Heart Foundation, a grant for research on the sympathetic nervous system and hypertension (Kimura Memorial Heart Foundation/Pfizer Pharmaceuticals Inc), and grants from the Ministry of Education, Science and Culture of Japan (C-08770455). The work was also supported by the following grants to R.M.: grants from the Japan Society for the Promotion of Science, Ono Medical Research Foundation, and the Uehara Memorial Foundation, as well as the Japan Heart Foundation-Pfizer Pharmaceuticals Grant for Research on Coronary Artery Disease.
Received March 29, 1999; accepted May 18, 1999.
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