Role of Transcriptional cis-Elements, Angiotensinogen Gene–Activating Elements, of Angiotensinogen Gene in Blood Pressure Regulation
Abstract Results of recent genetic studies suggest that the angiotensinogen gene is a possible determinant of hypertension. Using antisense technology, we demonstrated that generation of circulating angiotensinogen is a rate-limiting step in blood pressure regulation. In the present study, we examined how the angiotensinogen gene is regulated in vivo. The transcriptional cis-elements, angiotensinogen gene–activating elements (AGE) 2 and 3, have been reported to regulate angiotensinogen production in human hepatocytes in vitro. To determine the critical transcriptional regulator of angiotensinogen production in vivo, we used synthetic double-stranded oligodeoxynucleotides (ODN) as “decoy” cis-elements to block the binding of nuclear factors to promoter regions of the targeted gene, resulting in the inhibition of gene transactivation. Here we examined whether AGE 2 and AGE 3 in the promoter region of the angiotensinogen gene have a pivotal role in hepatic angiotensinogen production in vivo. Hepatic angiotensinogen mRNA was decreased by the transfection of AGE 2 but not mismatched decoy ODN. Transfection of decoy but not mismatched ODN against AGE 2 resulted in a transient decrease in blood pressure of spontaneously hypertensive rats (SHR), accompanied by a reduction in plasma angiotensinogen and angiotensin II levels. In contrast, transfection of AGE 3 decoy ODN had little effect on blood pressure. Overall, our results demonstrate that transfection of decoy ODN against AGE 2, but not against AGE 3, of the angiotensinogen gene resulted in a transient decrease in high blood pressure of SHR, suggesting that the transcriptional cis-element AGE 2, rather than AGE 3, has an important role in blood pressure regulation through the control of circulating angiotensinogen.
- transcription, genetic
- parainfluenza virus type 1
- blood pressure
- angiotensinogen promoter
- gene expression regulation
Angiotensinogen, which is mainly produced in the liver, is a unique component of the RAS because it is the only known substrate for Ang I generation.1 2 Recent findings of genetic studies suggest that the angiotensinogen gene is a possible determinant of hypertension.3 4 5 6 To clarify the role of angiotensinogen in blood pressure regulation, we previously demonstrated with the use of antisense technology that generation of circulating angiotensinogen is a rate-limiting step in blood pressure regulation.7 Given the importance of angiotensinogen regulation in the pathogenesis of hypertension, the manner in which the angiotensinogen gene is regulated is also important. The angiotensinogen gene has been suggested to be regulated by novel transcriptional factors such as AGF 1 and AGF 3 in cultured HepG2 cells in vitro.8 However, the molecular mechanism or mechanisms of angiotensinogen regulation in vivo have not yet been clarified. In the present study, we examined how hepatic angiotensinogen gene expression is regulated in vivo. To determine the critical transcriptional regulator of hepatic angiotensinogen production in vivo, we used synthetic double-stranded ODN as “decoy” cis-elements to block the binding of nuclear factors to promoter regions of the targeted gene, resulting in the inhibition of gene transactivation.9 10 11 12 Using this strategy, we examined whether AGE 2 and AGE 3 in the promoter region of the angiotensinogen gene have a pivotal role in the regulation of circulating angiotensinogen production in vivo. Here we addressed two specific questions: Do transcriptional cis-elements AGE 2 and AGE 3 have a role in the regulation of the angiotensinogen gene in vivo? And is it possible to change blood pressure level by manipulating transcriptional factors?
All studies were performed with the approval of the Ethics Committee for Animal Research, Osaka University Medical School.
Synthesis of Oligonucleotides and Selection of Sequence Targets
The sequences of phosphorothioate double-stranded ODN against AGF 2 and AGF 3 binding sites and mismatched ODN used in the study were as previously reported.8 The sequences of the phosphorothioate ODN were as follows: AGE 2 decoy ODN (core sequences are underlined), 5′-CCCAGCCTCTGTACAGAGTAGCCCA-3′ and 3′-GGGTCGGAGACATGTCTCATCGGGT-5′; mismatched AGE 2 decoy ODN (mismatched sequences are in bold), 5′-CCCAGCTGAGGTTTAGAGTAGCCCA-3′ and 3′-GGGTCGACTCCAAATCTCATCGGGT-5′; AGE 3 decoy ODN (core sequences are underlined), 5′-AGGGGATAGCTGT GCTTGT CTAGGTT-3′ and 3′-T CCCCT ATCGACACGAACAGATCCAA-5′; and mismatched AGE 3 decoy ODN (mismatched sequences are in bold), 5′-AGGGGATGTTGGATGCGAGC CTAGGTT-3′ and 3′-T CCCCT ACAACCTACGCTCGGATCCAA-5′.
Decoy ODN have been shown to bind the AGF 2 and AGF 3 transcriptional factors, respectively.8 Synthetic ODN 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 with an NAP 10 column (Pharmacia) and quantified with spectrophotometry. ODN were annealed for 2 hours while the temperature descended from 80°C to 25°C.9
Preparation of HVJ/Liposome Solution
HVJ (Z strain) was propagated in chorioallantonic fluid of embryonated eggs, as previously described.7 11 12 13 14 15 16 17 18 Briefly, HVJ was collected by centrifugation at 27 000g for 40 minutes and suspended in balanced salt solution overnight. This procedure was repeated at least twice. 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 11 12 13 14 15 16 17 18 One absorbance at 540 nm of HVJ suspension contained 1 mg/mL protein and was equivalent to 15 000 hemagglutinating activity units/mL. Lipids (phosphatidylcholine, phosphatidylserine, and cholesterol) were mixed at a ratio of 4.8:1:2 (wt/wt/wt), as described previously.7 11 12 13 14 15 16 17 18 The lipid mixture (10 mg) in tetrahydrofuran was deposited in a rotary evaporator. Decoy ODN were incorporated into liposomes by shaking and sonication. The liposomes and HVJ, inactivated by ultraviolet irradiation (110 erg·[mmol/L]−2·s−1) for 3 minutes just before use, were incubated at 4°C for 10 minutes and then at 37°C for 30 minutes with gentle shaking (two strokes per second). This solution was centrifuged by sucrose gradient. The top layer was collected for use. As previously described, we used HMG-1 for gene transfer. In contrast, in antisense ODN transfer conducted in this study, HMG-1 was not used for ODN transfection.
In Vivo Introduction of HVJ/Liposome Solution
Eight- or 20-week-old male SHR obtained from closed colonies at Charles River Japan, Inc were anesthetized with an intraperitoneal injection of pentobarbital (50 mg/kg). The abdomen was opened with a median incision, and the liver and portal vein were exposed. Then a total volume of 2.5 mL of final HVJ/liposome solution containing ODN (3 μmol/L in liposomes) was injected into the liver via the portal vein.7 18
The liver was promptly removed, immediately frozen in liquid nitrogen, and stored at −80°C before RNA extraction. Total RNA was extracted from total liver with guanidine thiocyanate by ultracentrifugation through a dense cushion of CsCl.19 20 For Northern blot analysis, 20 μg of total RNA was subjected to electrophoresis on 1.5% agarose-formaldehyde denaturing gel and transferred to a nitrocellulose membrane (Amersham International). The filter was baked, prehybridized, and hybridized to full-length cDNA for rat angiotensinogen (provided by Dr Mori, Kyoto University) and rat β-actin probe (both labeled with 32P). Then the filter was washed and exposed to x-ray film.
Rat Angiotensinogen Assay
The levels of angiotensinogen in each medium, tissue, and plasma were determined indirectly by the measurement of Ang I generated after incubation with excess recombinant human active renin, as described previously.21 Samples were resuspended in angiotensinogen RIA buffer (150 mmol/L Na2HPO4, 160 mmol/L NaCl, 3 mmol/L EDTA, 5% bovine serum albumin) and incubated with 1 mL (Goldblatt Unit) 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.21 22 The Ang I level of each sample was determined by RIA, and the results were expressed as nanograms of Ang I.
Blood Pressure and Ang II Measurement
Systolic blood pressure was measured by the tail-cuff method, and then the rats were killed. Blood samples were collected by decapitation without anesthesia in prechilled tubes containing EDTA-2Na (1 mg/mL whole blood) and 2.5 mmol/L PMSF and centrifuged at 4°C. To measure Ang II, plasma was stored at −70°C before assay. Then 1 mL of each freshly separated plasma sample was promptly concentrated in an Amprep C8 minicolumn (Amersham) as previously reported.21 23
After the column was washed with 10 mL of 0.1% TFA, Ang II was eluted with 2 mL of ethanol/water/TFA (80:19.9:0.1, v/v/v). The eluate was dried with a centrifugal concentrator in a vacuum (CC-181, Tomy). The recovery of Ang II with this procedure was 98±2% (n=5), which was examined with [125I]Ang II. The data were not corrected for this recovery because its variation was negligible. The resultant residue was resuspended in 100 μL of 0.1% TFA. HPLC characterization was performed as previously described.21 23 Samples were collected from the appropriate fraction, dried in a vacuum centrifuge, and redissolved in 0.1 mmol/L Tris acetate, pH 7.4, containing 2.6 mmol/L EDTA-2Na, 1 mmol/L PMSF, and 0.1% bovine serum albumin. Elution times of Ang II, Ang III, and Ang I were 19.0, 20.7, and 23.7 minutes, respectively. Immunoreactive Ang II was measured with RIA using specific Ang II antibody (donated by Kazuaki Shimamoto, MD, Sapporo Medical College24 ). The sensitivity of this assay was 0.1 pg/tube. The recovery of Ang II after HPLC was 85±5%. The cross-reactivity was 100% for Ang III and <0.1% for Ang I.24
The results are expressed as mean±SEM. Statistical analysis was performed with ANOVA followed by multiple comparisons. Differences were considered statistically significant at P<.05. All experiments were repeated at least three times.
Initially, we examined the transfection of AGE 2 decoy ODN with HVJ/liposome complex via the portal vein to deliver ODN into the whole liver. Fig 1⇓ shows a typical example of Northern blot analyses of hepatic angiotensinogen mRNA in SHR treated with either AGE 2 decoy or mismatched AGE 2 decoy ODN. A reduction in hepatic angiotensinogen mRNA was observed on day 2 after transfection with AGE 2 decoy ODN. There was no difference in hepatic angiotensinogen mRNA level between mismatched AGE 2 decoy ODN-treated and untreated groups (data not shown). There was no change in β-actin mRNA level between SHR treated with AGE 2 decoy ODN and mismatched decoy ODN (data not shown).
Given that transfection of AGE 2 decoy ODN decreased hepatic angiotensinogen mRNA level, we evaluated the effect of transfection of AGE 2 decoy ODN via the portal vein on blood pressure level of SHR. As shown in Fig 2⇓, transfection of AGE 2, but not mismatched, decoy ODN resulted in a transient decrease in high blood pressure of SHR at the developmental stage (8 week-old) on days 2 and 3 after transfection. There was no significant difference in blood pressure between mismatched decoy ODN-treated and untreated groups (data not shown). Next we examined the effect of AGE 2 decoy ODN at the maintenance stage of hypertension in SHR (20 weeks old). Systolic blood pressure began to decrease at 1 day after injection and decreased from 204 mm Hg on day 0 to 160 mm Hg on day 2 after the injection of HVJ/liposome solution (P<.01, Fig 3⇓). Transfection of AGE 2 decoy ODN resulted in a significant decrease in blood pressure level from day 1 to day 6 after injection compared with mismatched decoy ODN treatment (P<.05, Fig 3⇓). There was no significant difference in blood pressure between mismatched decoy ODN-treated and untreated groups (data not shown).
To investigate whether the transfection of AGE 2 decoy ODN affects angiotensinogen production, we measured plasma angiotensinogen concentration after transfection of decoy ODN to SHR (20 weeks old). Our results documented a decrease in plasma angiotensinogen level on day 3 after AGE 2 decoy ODN treatment (Fig 4⇓). In contrast, there was no significant difference on day 7 after transfection between AGE 2 and mismatched decoy ODN treatment groups. Next we measured plasma Ang II level after transfection of ODN to study whether the reduction in angiotensinogen affects plasma Ang II level. As shown in Fig 5⇓, our data showed a decrease in plasma Ang II level on day 3 after AGE 2 decoy ODN treatment, which was parallel with the reduction of plasma angiotensinogen level. On the other hand, there was no significant difference on day 7 after transfection between AGE 2 decoy ODN and mismatched decoy ODN treatment groups. These results were not due to hepatic dysfunction because no apparent liver toxicity was observed throughout the experiments (data not shown). Finally, we tested whether AGE 3 decoy ODN has an effect on blood pressure level of SHR (20 weeks old). As shown in Fig 6⇓, there was no significant decrease in blood pressure among vehicle-treated, AGE 3 decoy ODN-treated, and mismatched decoy ODN-transfected rats.
Angiotensinogen is the precursor of the vasoactive peptide Ang II and is therefore speculated to be an important determinant of blood pressure and electrolyte homeostasis.1 2 Recent linkage genetic studies, including ours, suggest that angiotensinogen is one of the candidate genes for hypertension.3 4 5 6 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.25 In contrast, it was also reported that acute administration of pure rat angiotensinogen to rats resulted in an increase in blood pressure.26 We have also reported that transfection of antisense ODN against the angiotensinogen gene resulted in a transient decrease in blood pressure in SHR and normotensive rats.7 These findings indicate that circulating angiotensinogen has an important role in the pathogenesis of hypertension. Therefore, the molecular mechanism or mechanisms in the regulation of the angiotensinogen gene are of interest. 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.27 28 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 the novel cis-acting element AGE 3 (from −6 to +22) that resides directly around the transcriptional start site in the core promoter region.8 However, little is known about the molecular mechanism or mechanisms of angiotensinogen gene regulation in vivo. Therefore, it is important to examine how the angiotensinogen gene is regulated in vivo, especially in the liver. As mentioned earlier, we addressed two specific questions. Do the transcriptional cis-elements AGE 2 and AGE 3 have a role in the regulation of the angiotensinogen gene in vivo? And is it possible to change blood pressure level by manipulating the transcriptional factors AGF 2 and AGF 3?
With these aims, we used molecular biological techniques with a decoy strategy that is considered a useful tool in a new class of antigene strategies.9 10 11 12 Transfection of double-stranded ODN 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 expression (Fig 7⇓9 10 11 12 ). Therefore, the decoy approach enabled us to study gene regulation in vivo as well as in vitro through modulation of endogenous transcriptional regulation. In previous studies, we used antisense technology as a “loss of function” approach at the transcriptional and translational levels.7 15 16 On the other hand, in the present study we used cis-element decoy strategy as a loss of function approach at the pretranscriptional and transcriptional levels to study transcriptional factors. Alternatively, another tool for the loss of function approach is transgenic/gene-targeting technology, which provides many advantages, such as the ability to study the specific gene function as systemic and developmental effects and the ability to test a specific gene function on a long-term basis. Nevertheless, this technology has several disadvantages: it is time consuming and costly, the effect of the overexpressed transgene throughout development results in the difficulty to separate out gene effects at special times, and it is very difficult to target transgenic expression in a tissue-specific manner. If the targeted gene can cause a lethal effect, it is impossible to test specific functions by transgenic or gene-targeting techniques. Thus, in the present study, cis-element decoy strategy was used to study the role of novel transcriptional factors, AGF, on the regulation of hepatic angiotensinogen gene expression.
We initially examined how AGF 2 affects the gene regulation of hepatic angiotensinogen in vivo. As shown in Fig 1⇑, transfection of AGE 2, but not mismatched, decoy ODN decreased hepatic angiotensinogen mRNA level at 2 days after injection, whereas no significant change was observed in β-actin mRNA level after transfection of AGE 2 decoy ODN. Therefore, we next evaluated the biological effects of AGE 2 decoy ODN transfection in SHR. Interestingly, transfection of AGE 2 decoy ODN resulted in a transient decrease in high blood pressure of SHR at both 8 weeks (developmental stage) and 20 weeks of age (maintenance stage), whereas mismatched decoy ODN showed no effect. This reduction of high blood pressure was due to the transient reduction of hepatic angiotensinogen mRNA, plasma angiotensinogen, and angiotensin II levels (Figs 1⇑, 5⇑, and 6⇑). These results are consistent with our previous observation that transfection of antisense angiotensinogen ODN resulted in a transient decrease in blood pressure of SHR and normotensive rats.7 Interestingly, the pattern of the transient decrease in blood pressure was similar in antisense and decoy studies.
A classic approach to defining the role of transcriptional factors in the regulation of genes is to use promoter-reporter gene transfection experiments with CAT and luciferase constructs. This approach is very useful for identifying cis- and trans-acting element interactions, but it has some disadvantages: it is costly and time consuming to make a series of constructs, it cannot be used to analyze endogenous gene regulation, and it is hard to determine the specific elements. In contrast, the decoy approach has many advantages, as we presented: decoys are easily synthesized; it can be used to study endogenous gene regulation and pathophysiological roles; and it can be used to determine the specific cis-elements, even if the specific regulatory cis-elements have not been clarified. In the present study, our results demonstrated that AGE 2, but not AGE 3, has an important role in the regulation of hepatic angiotensinogen gene expression in the liver because transfection of AGE 2, but not AGE 3, decoy ODN decreased high blood pressure of SHR. This is the first report that cis-element AGE 2 has a pivotal role in the regulation of hepatic angiotensinogen of SHR in vivo. Our conclusion is supported by the previous observation that the AGE 2–driven CAT construct caused a marked activation of CAT activity compared with the AGE 3–driven CAT construct in HepG2 cells.8 Thus, AGE 3 probably acts synergistically with AGE 2 in HepG2 cells. Alternatively, because AGF 3 is a ubiquitous nuclear factor in various cells,8 AGE 2 may be important for high-level expression in the liver, and AGE 3 may be involved in basal-level expression in extrahepatic tissues. On the other hand, the palindromic sequences of AGE 2 are well conserved between the rat and mouse angiotensinogen gene,29 and a previous study showed that the palindromic sequence of the rat angiotensinogen gene is important for the formation of a specific complex with the HepG2 nuclear extract.30 Therefore, this palindromic region (AGE 2) and AGF 2 appear to be directly involved in dictating the hepatocyte-specific expression of the angiotensinogen promoter.
Overall, our results demonstrate that transfection of decoy ODN against AGE 2, but not AGE 3, of the angiotensinogen gene resulted in a transient decrease in high blood pressure of SHR, suggesting that the transcriptional cis-element AGE 2, rather than AGE 3, has an important role in blood pressure regulation through the control of circulating angiotensinogen. The present study also revealed the utility of gene transfer and decoy technology for hypertension research, especially to evaluate the specific functions of transcriptional factors of target gene regulation.
Selected Abbreviations and Acronyms
|AGE||=||angiotensinogen gene-activating element(s)|
|AGF||=||angiotensinogen gene-activating factor(s)|
|HepG2 cells||=||human hepatocytes|
|HPLC||=||high-performance liquid chromatography|
|HVJ||=||hemagglutinating virus of Japan|
|SHR||=||spontaneously hypertensive rat(s)|
This work was supported in part by grants from the Japan Research Foundation for Clinical Pharmacology, Kanae Foundation of Research for New Medicine, Osaka Kidney Foundation (OKF 95-0002), and Research Foundation for Pharmaceutical Sciences. Dr Morishita is the recipient of a Japan Vascular Disease Research Foundation Award and a Research Fellow of the Japan Society for the Promotion of Science. We thank Misako Mashimoto and Keiko Zaitsu for their technical assistance. We also thank Drs Keiji Tanimoto and Akiyoshi Fukamizu for their advice.
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