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Circulation Research. 1995;77:249-257

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(Circulation Research. 1995;77:249-257.)
© 1995 American Heart Association, Inc.

Articles

Identification of a cis-Acting Glucocorticoid Responsive Element in the Rat Angiotensin II Type 1A Promoter

Deng-Fu Guo, Shusei Uno, Akira Ishihata, Norifumi Nakamura, Tadashi Inagami

From the Department of Biochemistry (D.-F.G., S.U., A.I., T.I.), Vanderbilt University, School of Medicine, Nashville, Tenn, and the Research Division (N.N.), The Green Cross Corp, Osaka, Japan.

Correspondence to Department of Biochemistry, Vanderbilt University, School of Medicine, Nashville, TN 37232.


*    Abstract
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 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
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Abstract Enhanced vascular responsiveness to angiotensin II at the AT1 receptor has been considered one of the major contributing factors to vascular hypertrophy and high blood pressure. The transcription of the rat angiotensin II type 1A receptor gene is stimulated by glucocorticoids. To clarify the molecular mechanism for glucocorticoid action in rat vascular smooth muscle cells, we investigated the effects of dexamethasone on the promoter activity of the angiotensin II type 1A receptor by using promoter/luciferase reporter gene constructs and heterologous context constructs (containing the thymidine kinase promoter) in transfected vascular smooth muscle cells (<12 passages). There are three putative glucocorticoid responsive elements (GREs) in the promoter. However, only one GRE was found to respond to dexamethasone (1 µmol/L) and was located at positions -756 to -770 bp upstream from the transcription initiation site. When compared with the consensus sequence of GRE, 9 of 12 bases were identical. RU38486, a glucocorticoid antagonist, completely blocked the induction by dexamethasone, suggesting that the GRE was functional through a specific glucocorticoid receptor. The response to dexamethasone was lost in vascular smooth muscle cells at higher passage numbers (>8 passages) but was restored when the cells were transfected with a glucocorticoid-receptor expression construct. This finding provided additional support that the response to dexamethasone was mediated by the glucocorticoid receptor. The gel mobility supershift assay showed that the GRE binds in vitro–translated rat glucocorticoid receptors in a specific manner. Compared with the angiotensin II type 1A receptor promoter, no effect by dexamethasone was observed in vascular smooth muscle cells transfected with the angiotensin II type 1B receptor promoter/luciferase reporter gene constructs. We conclude from these experiments that the dexamethasone-induced increase in the transcription of the angiotensin II type 1A receptor gene occurred through the binding of GRE to the glucocorticoid-specific receptor.


Key Words: angiotensin II • glucocorticoid responsive element • luciferase • dexamethasone • type 1A receptor


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Ang II plays important roles in the regulation of the cardiovascular system by diverse mechanisms,1 which include vasoconstriction, stimulation of aldosterone production, vascular smooth muscle hypertrophy, hyperplasia, and facilitation of adrenergic release.2 At least two major isoforms of the Ang II receptor, AT1 and AT2, have been identified by nonpeptidic isoform-specific antagonists3 and molecular cloning.4 5 6 7 8 The AT1 receptor mediates many of the classic functions assigned to Ang II to date, whereas the functions of the AT2 receptor have yet to be established. In the rat, AT1 has two subtypes, termed AT1A and AT1B.6 9 10 11 12 The expression of the rat AT1A and AT1B receptor genes is tissue dependent. AT1A is abundant in smooth muscle, kidney, and heart, whereas AT1B is more abundant in the pituitary and adrenal glands.12 13 14

AT1 is regulated by various vasoactive substances, growth factors, and steroids that have either enhancing or suppressive effects on the transcriptional activity of this gene. Aldosterone and glucocorticoids elevate the expression of the AT1A, whereas Ang II, phorbol esters, and estrogens suppress it.15 16 17 18 19 20 21 22 23 In rat glomerular mesangial cells, the mRNA level of the rat AT1 receptor is downregulated by Ang II, dibutyryl cAMP, forskolin, and cholera toxin.24 In human adrenocortical carcinoma H295 cells, the transcription of the AT1 gene is inhibited by Ang II, phorbol esters, and forskolin.25 In rat VSMCs, where the AT1A is the major Ang II receptor, the transcription of this gene is induced by glucocorticoid hormones.16 17 18 19 Recently, Matsubara et al19 have reported a dexamethasone-induced increase in the mRNA level and receptor number of AT1A, but not AT1B, in rat cardiac fibroblasts and cardiomyocytes. The genomic DNA for rat AT1A has been cloned by us26 and others10 11 27 and has been found to contain three introns and four exons. It has been demonstrated that the promoter of this gene was highly functional in VSMCs, A10, and glial cells but poorly functional in PC12 cells.27 28

In general, glucocorticoid hormones increase the mRNA level of a given gene via two mechanisms. The first is through enhancing the transcriptional rate via GRE(s) in the gene promoter. The other is via a glucocorticoid-stabilizing element(s), which increases the stability of mRNA.29 It has been reported that the glucocorticoid-induced increase in the mRNA level of rat AT1A was caused by an increase in the transcription rate but not by mRNA stabilization. However, the precise mechanism of the glucocorticoid-induced increase in the mRNA level of the AT1A gene has not been determined in the 5'-flanking region of the rat AT1A gene. To clarify the molecular basis for response to glucocorticoids in VSMCs, we generated a series of the promoter/luciferase reporter gene and heterologous context constructs and used these to identify a functional regulatory site in the rat AT1A receptor promoter. The effect of glucocorticoid in the AT1A receptor expression in different passages of VSMCs was examined. Response to dexamethasone was also determined in adrenal cortex–derived Y-1 cells and compared with VSMCs for a possible tissue-specific difference in the mechanism of AT1A expression.


*    Materials and Methods
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 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Materials
Dexamethasone and BSA were obtained from Sigma Chemical Co, and DMEM, PBS, FCS, Ham's F-10 medium, and horse serum were from GIBCO/BRL. RU38486, a glucocorticoid antagonist, was a gift from Roussel-UCLAF. Monoclonal (mouse) anti–glucocorticoid receptor antibody (IgG2) was purchased from Affinity Bioreagents, Inc. The in vitro–translated products of rat glucocorticoid receptor (plasmid T7X556 contains amino acid residues 407 to 556, including the DNA binding domain of the rat glucocorticoid receptor) were kindly supplied by Keith Yamamoto, University of California, San Francisco.30

Cell Culture
VSMCs were isolated from adult male SHR purchased from Charles River Laboratories, Wilmington, Mass, by the method of Gunther et al.31 Briefly, the thoracic aorta of 12-week-old rats was dissected from the surrounding tissues. After the adipose tissues around the aorta were removed, the aorta was digested in DMEM containing 0.7 mg/mL collagenase (type IA, Sigma), 0.25 mg/mL elastase (type III, Sigma), 0.4 mg/mL soybean trypsin inhibitor, and 1 mg/mL BSA for 30 minutes at 37°C. Dissociated VSMCs were seeded into conventional plastic tissue culture plates and grown in DMEM containing 10% FCS, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37°C under 5% CO2/95% air. The medium was changed every 3 or 4 days. Cells between passages 3 and 14 were used.

Y-1 cells, which were derived from a mouse adrenal cortical tumor, were obtained from American Type Culture Collection. These cells were grown in Ham's F-10 medium containing 15% horse serum, 2.5% FCS, 100 U/mL penicillin, and 100 µg/mL streptomycin at 37°C under 5% CO2/95% air.

Plasmid Constructs
A 980-bp EcoRI-BstXI fragment in the 5'-flanking region of the rat AT1A receptor gene was blunted with the Klenow fragment and inserted into the pBluescript KS(+) vector. It was then cleaved by Sac I–Xho I digestion and inserted into the same sites of the pGL2 basic vector (Promega). This plasmid was named pLuc1. From plasmid pLuc1, two additional constructs were generated by using Nsi I and Xho I restriction enzymes, respectively. Resultant constructs with 799- and 560-bp sequences upstream from the transcription initiation site of the AT1A promoter were named pLuc2 and pLuc3, respectively.

For heterologous experiments, two fragments digested with either EcoRI–Nsi I (123 bp) or Nsi I–Xho I (230 bp) were blunted with the Klenow fragment and inserted into the EcoRV site of the pBluescript KS(+) vector, and then the Sac I–Xho I fragments were recloned into the same restriction enzyme sites of pTKLuc basic vector (which contains a segment of the herpes simplex virus thymidine kinase promoter between -105 and +51 bp) and named pTKLucF1 and pTKLucF2, respectively. Four 25-mers (sense and antisense) of oligonucleotides (corresponding to -751 to -775 bp and -851 to -875 bp, respectively) prepared in an Applied Biosystems 380A DNA synthesizer were gel-purified. The annealed double-strand oligomers were inserted into the pBluescript KS(+) vector, then cleaved by Sac I–Xho I digestion, inserted into the pTKLuc basic vector, and named pTKLucG1 and pTKLucG2, respectively. For deletion mutant experiments of GRE2, the fragments of EcoRI–Nsi I (123 bp) were blunted and inserted into the Sma I site of pLuc3 (named {Delta}pLuc.4). Ligation, transformation, and plasmid DNA preparation were performed by use of standard techniques.32 The DNA sequence of all constructs was verified by using a USB sequencing kit.33

To test the effect of dexamethasone on the rat AT1B promoter, four AT1B promoter/luciferase reporter gene constructs were generated. A 1514-bp EcoRI–BstXI fragment in the 5'-flanking region of the rat AT1B receptor gene was blunted with the Klenow fragment and inserted into the pBluescript KS(+) vector. It was cleaved by Kpn I–Sca I digestion and inserted into the same sites of the pGL2 basic vector. This plasmid was named pLucB1. From plasmid pLucB1, three additional constructs were generated by using Acc I, Nhe I, and EcoNI restriction enzymes, respectively. Resultant constructs with 700-, 381-, and 250-bp sequences upstream from the transcription initiation site of the rat AT1B promoter were named pLucB2, pLucB3, and pLucB4, respectively.

Transient Transfections and Luciferase Assays
VSMCs were seeded at a density of 5x105 cells per 60-mm dish and grown in DMEM containing 10% FCS overnight. Transient transfections were performed by using the DEAE-dextran method as recommended by the manufacturer (Promega). For luciferase assays, cells were cotransfected with 6 µg of plasmid DNA and 2 µg of a pSV–ß-galactosidase vector (Promega) to allow for normalization for transfection efficiency. After the transfection, cells were grown in DMEM containing 10% FCS for 2 days, washed, and then exposed to DMEM containing 0.2% BSA with or without dexamethasone (1 µmol/L). After stimulation for 16 hours, cells were washed twice with PBS and harvested with lysis buffer (25 mmol/L Tris-HCl at pH 7.8, 2 mmol/L dithiothreitol, 2 mmol/L 1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid, 10% glycerol, and 1% Triton X-100) for the luciferase and ß-galactosidase assay. Cells were scraped with a rubber policeman, transferred to 1.5 mL microcentrifuge tubes, and spun at 12 000 rpm for 5 minutes. Supernatant was transferred to new tubes and directly used for luciferase and ß-galactosidase assays. A glass tube containing 20 µL of supernatant was placed in a luminometer (Optocomp I, MGM Instruments), 100 µL of 470 µmol/L luciferin was added automatically, and integrated peak luminescence was measured over a 45-second window after a 5-second delay. The ß-galactosidase activity was determined by absorbance at 405 nm in a spectrophotometer after a 3-hour incubation of 100 µL supernatant with the same volume of 2x assay buffer (200 nmol/L Na2PO4, 90 mmol/L ß-mercaptoethanol, and 8 mg/mL O-nitrophenol-ß-D-galactopyranoside) and was used to normalize for differences in transfection efficiency. All transfections were replicated at least three times with similar results.

Y-1 cells were seeded at a density of 1x106 cells per 60-mm dish and grown in Ham's F-10 medium containing 15% horse serum and 2.5% FCS overnight. Transfection, hormone treatment, and luciferase activity were performed as described above.

VSMCs of higher passages (>10) were depleted of the glucocorticoid receptor. The glucocorticoid receptor was restored by transfecting the cells with pRSVGR, a glucocorticoid receptor expression vector kindly supplied by Keith Yamamoto, University of California, San Francisco (Meisfeld et al34 ). VSMCs (>8 passages) were cotransfected with 2 µg pRSVGR, 6 µg pTKLucG2 construct, and 2 µg pSV–ß-galactosidase vector. To inhibit the effect of glucocorticoid, 1 µmol/L RU38486, a glucocorticoid antagonist, was added to the dexamethasone-containing medium.

Aldosterone can stimulate gene expression with a GRE sequence. To test whether aldosterone affects the AT1A promoter function, VSMCs transfected with 6 µg pTKLucG2 and 2 µg pRSVGR were treated with 100 nmol/L aldosterone for 16 hours.

Northern Hybridization Analysis
VSMCs and Y-1 cells were seeded at a density of 2x106 cells per 100-mm dish and grown in the media containing 10% FCS or 15% horse serum and 2.5% FCS, respectively, for 3 to 5 days. Cells were maintained in FCS-free medium containing 0.2% BSA for 2 days and then exposed to fresh FCS-free medium containing dexamethasone (1 µmol/L) for 8 hours before harvesting for preparation of total RNA. Total RNA was isolated from VSMCs of SHR at passage numbers 4, 8, and 12 and from Y-1 cells (65th passages) by the acid guanidinium thiocyanate–phenol–chloroform extraction method.35 Total cellular RNA (20 µg) was separated on a 1% agarose formaldehyde gel and transferred onto a nylon membrane. 32P-labeled probes (random primer method) were used: a 750-bp Ava I fragment cleaved from the pRSVGR plasmid (which is located 927 to 1677 bp downstream from the translation start codon) was used as a specific probe for glucocorticoid receptor, a 235-bp fragment from AT1A (which is located 63 to 298 bp downstream from translation termination codon) was amplified by polymerase chain reaction and used as a specific probe for AT1A, and a 1287-bp Pst I fragment of GAPDH (which contains the entire coding region) was used for the hybridization as control. Prehybridization was in 50% formamide, 6x SSC (1x SSC consists of 0.15 mol/L NaCl and 0.015 mol/L sodium citrate, pH 7.0), 2x Denhardt's solution (0.1% Ficoll, 0.1% BSA, and 0.1% polyvinylpyrrolidone), 1% SDS, 10 mmol/L sodium phosphate buffer, and 0.2 mg/mL denatured salmon sperm DNA for 4 hours, and then hybridization was performed in the same buffer with a specific probe for 16 to 24 hours at 42°C. High-stringency washes were performed twice in 0.1x SSC and 0.2% SDS at 65°C for 30 minutes each. The membrane was exposed to Kodak XAR 5 film. The autoradiograms were scanned by an ES-800C imaging scanner (Epson America Inc) and quantified by using the IMAGE 1.49 program. The same filter was rehybridized with the control probe (GAPDH).

Gel Mobility Shift Assay
A 25-mer oligonucleotide (corresponding to -751 to -775 bp) as mentioned above was used in gel mobility shift assays. The oligomer was radiolabeled with [{gamma}32P]ATP by use of T4 polynucleotide kinase. The end-labeled oligonucleotide (0.1 ng) was incubated with 1 mg of in vitro–translated rat glucocorticoid receptor (plasmid T7X556, which contains amino acid residues 407 to 556 of the rat glucocorticoid receptor, including the DNA binding domain30 ) in the presence of 10 mmol/L HEPES, pH 7.9, 5 mmol/L spermidine, 2.5 mmol/L dithiothreitol, 50 mmol/L NaCl, 2 µg poly(dI-dC)–poly(dI-dC), and 10% (vol/vol) glycerol in a final volume of 20 µL for 30 minutes at 4°C. Where indicated, unlabeled competitor DNA (50-fold molar excess) was mixed with the radiolabeled oligomer before the in vitro–translated rat glucocorticoid receptor was added. In supershift experiments, anti-glucocorticoid receptor antibody raised against the DNA binding domain and in vitro–translated rat glucocorticoid receptor (1 mg) were added into the reaction buffer and incubated for 30 minutes; then the end-labeled oligonucleotide (0.1 ng) was added, and incubation continued for 30 minutes. The mixture was loaded onto a 5% acrylamide gel and electrophoresed for 3 to 4 hours at 150 V in a buffer containing 25 mmol/L Tris-HCl at pH 7.8, 190 mmol/L glycine, and 1 mmol/L EDTA at 4°C. Gels were dried and exposed to Kodak XAR 5 film (Eastman Kodak Co).


*    Results
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
Rat AT1A Receptor Gene Expression in Transfected Cells
As shown in Fig 1ADown, three putative GREs were found by homology search. These elements were located at positions -856 to -870 bp, -756 to -770 bp, and -393 to -407 bp upstream from the transcription initiation site of the rat AT1A receptor gene.27



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  		Figure 1. Chimeric constructs generated from 5'-deleted mutants of the rat AT1A receptor promoter and the luciferase reporter gene and their promoter activity in VSMCs. The GREs in the rat AT1A promoter are illustrated (A). The chimeric plasmid (pLuc1) consists of the rat AT1A promoter region between -980 and +1 bp relative to the transcription initiation site,27 the luciferase reporter gene, and the simian virus 40 splice and polyadenylation signals (SV40). The structure of the constructs and their designations are shown (B). The insertion of a 980-bp fragment into the Sac I–Xho I sites of the pLuc basic plasmid created pLuc1. The deletion mutant of GRE2 ({Delta}pLuc.4) is shown (C). The passage number of the VSMCs was <5. Transfection, hormone treatment, and luciferase assays were performed as described in "Materials and Methods." Data are presented as fold induction (mean±SEM) of four independent transfections.

To clarify whether these three putative GREs are functional, we generated three deletion mutants of the AT1A promoter/luciferase constructs. Among them, pLuc1 contains all of the three putative GREs, pLuc2 contains two putative GREs in the upstream region, and pLuc3 contains only the proximal putative GRE. To test the effect of dexamethasone on the transcription of the rat AT1A receptor gene, rat VSMCs transfected with each of the three constructs were incubated with dexamethasone (1 µmol/L) for 16 hours, and the luciferase activity was determined. Plate-to-plate differences in transfection efficiency were normalized by comparing the ß-galactosidase activity arising from the cotransfected reporter plasmid pSV–ß-galactosidase. As shown in Fig 1BUp, pLuc1 and pLuc2 showed significant induction by dexamethasone. As discussed below, it was noted that pLuc2 produced a greater induction than did pLuc1. pLuc3 did not show a recognizable response to dexamethasone. Experiments with shorter constructs further confirmed the absence of response to dexamethasone of GRE3. Thus, neither a 489-bp promoter/luciferase (containing GRE3) nor a 331-bp construct without any GRE responded to dexamethasone. These results suggest that GRE1 or GRE2 or both are functionally active but GRE3 is not. To identify the active GRE(s), heterologous context constructs were generated.

Analysis of GRE1 and GRE2 in a Heterologous Context Construct
As shown in Fig 2BDown, fragments containing GRE1 or GRE2 were constructed into a heterologous context construct. VSMCs transfected with pTKLucF1 or pTKLucF2 were stimulated by dexamethasone (1 µmol/L) for 16 hours, and the luciferase activity was measured. The induction by dexamethasone was found only with pTKLucF2 but not with pTKLucF1. These data suggest that GRE2 may be functional in VSMCs. To further ascertain this postulate, two 25-mer of double-strand oligomers were inserted into the heterolo-gous pTKLuc basic vector. VSMCs transfected with pTKLucG1 or pTKLucG2 were incubated with dexamethasone (1 µmol/L) for 16 hours, and then the luciferase activity was determined as above. Induction by dexamethasone was seen only with pTKLucG2 but not with pTKLucG1 (Fig 2CDown). No induction was observed with the pTKLuc basic vector alone. Together, both longer and shorter constructs containing GRE2 responded to dexamethasone, but those containing GRE1 did not. Additionally, when we created a deletion mutant of GRE2 in the native promoter (named {Delta}pLuc.4), no induction by dexamethasone was detected in VSMCs transfected with this plasmid (Fig 1CUp). Thus, it is safe to conclude that only the second GRE of the rat AT1A receptor gene is functional.



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  		Figure 2. Function analysis of GRE1 and GRE2 elements in heterologous context constructs. Tested vectors are illustrated (A). The pTKLuc basic plasmid consists of a polylinker (5' to 3') that includes Sac I (S) and Xho I (X) restriction sites, the herpes simplex virus thymidine kinase promoter (TK, -105 to +51 bp relative to the transcription initiation site), the luciferase reporter gene (Luc), and the simian virus 40 splice and polyadenylation signals (SV). Two fragments that were digested with EcoRI–Nsi I and Nsi I–Xho I (-980 to -799 bp and -798 to -560 bp upstream from the transcription initiation site) were blunted and inserted into the EcoRV site of pBluescript KS(+) and then inserted into the pTKLuc basic vector (B). Two oligomers that correspond to GRE1 (-851 to -875 bp) and GRE2 (-751 to -775 bp) were inserted into the EcoRV site of the pBluescript vector and then resubcloned into Sac I–Xho I sites of the pTKLuc vector individually (C). Transfection, hormone treatment, and luciferase assays were performed as described in "Materials and Methods." The data are presented as fold induction (mean±SEM) of four independent transfections. The passage number of the VSMCs was <6.

Effect of the Glucocorticoid Receptor on the GRE2 Element
Northern blot analysis of rat VSMC mRNA revealed the presence of two transcription products of the rat AT1A gene presumably due to alternative splicing (Fig 3Down). The molecular size of the major band was {approx}2.3 kb, and the minor band was 3.2 kb. The AT1A mRNA expression was increased 2-fold in the minor 3.2-kb band and 1.6-fold in the major 2.3-kb band in VSMCs of passage 4 as shown in Fig 3Down. The response was completely lost after passage 8. Examination of the promoter activity of the AT1A gene with the luciferase reporter gene construct showed a 2.0-fold induction in cells below passage 5, but no induction was seen with cells over passage 10 (Fig 4Down). This loss of induction seemed to be due to the loss of the glucocorticoid receptor in cells of high passage numbers. Representative Northern blot analysis of glucocorticoid receptor mRNA revealed that it decreased with passage, as shown in Fig 5Down. Two transcription products of glucocorticoid receptor by alternative splicing were seen, in agreement with an earlier report.34 A 12-fold reduction in glucocorticoid receptor mRNA was observed in VSMCs at passage 12 (lane 2) compared with passage 4 (lane 4). To obtain a reproducible response to glucocorticoid, the receptor level was restored by cotransfecting cells with a glucocorticoid receptor expression vector (pRSVGR) and heterologous context constructs. As shown in Fig 4Down, the induction was observed in the heterologous context constructs (pTKLucG2) but not in pTKLucG1 in pRSVGR-cotransfected VSMCs of high passage number. Equipped with information that the glucocorticoid receptor is depleted steadily in VSMCs over passage, we attempted to maximize the response of early-passage cells to dexamethasone by cotransfection with pRSVGR. However, no further increase in the response of the heterologous context construct was seen, indicating that the cells contain sufficient receptor for inducing the maximal responses of the expression of the construct. The maximal response to dexamethasone was between 1.8- and 2.1-fold.



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  		Figure 3. Northern blot analysis of the effect of dexamethasone on AT1A expression in VSMCs of different passages. A 235-bp polymerase chain reaction–amplified fragment was used as a specific probe for rat AT1A receptor. GAPDH and ethidium bromide staining are shown. The passage number of VSMCs and the presence or absence of dexamethasone are also shown. This is representative of three experiments.



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  		Figure 4. Effect of passage number of VSMCs on the gene induction by dexamethasone. The plasmid, passage number of VSMCs, and the fold induction by dexamethasone are shown. In the case of VSMCs at higher passage numbers, a glucocorticoid receptor expression vector was cotransfected, and the luciferase activity was measured as described in "Materials and Methods."



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  		Figure 5. Northern blot analysis of glucocorticoid receptor (GR) expression in Y-1 cells and VSMCs at different passage numbers. A 750-bp Ava I fragment digested from pRSVGR35 was used as a specific probe. GAPDH and ethidium bromide staining are shown. Lanes are as follows: 1, Y-1 cells (passage 64); lanes 2 through 4, VSMCs at passages 12, 8, and 4, respectively.

Analysis of GRE1 and GRE2 in Different Types of Cells
As shown in Fig 5Up, Y-1 cells, which were derived from a tumor of the mouse adrenal cortex, expressed sufficient numbers of the glucocorticoid receptors (lane 1) even after 64 passages, thus providing a convenient system for testing glucocorticoid effects. As shown in Fig 6ADown, the induction by dexamethasone was observed with pLuc1 and pLuc2 but not pLuc3. Furthermore. the induction by dexamethasone was limited to pTKLucF2 and pTKLucG2 and not seen with pTKLucF1 or pTKLucG1. The results obtained in Y-1 cells were in close agreement with those obtained with VSMCs.



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  		Figure 6. A, The promoter function of chimeric constructs and heterologous context constructs in Y-1 cells. Plasmid, fold induction, and cell types are shown. The data are presented as fold induction (mean±SEM) of four independent transfections. The passage numbers of Y-1 cells are 62 to 68. B, The effect of the glucocorticoid antagonist RU38486 in transfected VSMCs and Y-1 cells. Plasmid, fold induction, and cell types are indicated. VSMCs at passages 3 to 6 and Y-1 cells (passages 64 to 66) were used. The data are presented as fold induction of two independent transfections.

Evidence that the effects of dexamethasone on the AT1A gene expression are mediated by the specific glucocorticoid receptor was obtained by the inhibition of the effects by RU38486, a specific receptor antagonist. Cells transfected with pTKLucG2 were incubated with 1 µmol/L each of RU38486 and dexamethasone for 16 hours; the induction of luciferase activity by dexamethasone was completely inhibited by RU38486 both in VSMCs and in Y-1 cells (Fig 6BUp).

Analysis of Rat AT1B Promoter Function by Dexamethasone in Transfected Cells
As shown in Fig 7ADown, two putative GREs were found by homology search. They were located at positions -424 to -438 bp and -1208 to -1222 bp upstream from the transcription initiation site of the rat AT1B receptor gene.36 Four deletion mutants of the AT1B promoter/luciferase reporter gene construct were generated. VSMCs transfected with pLucB2 gave the highest luciferase activity; therefore, its luciferase level was used as a reference (set as 100%) in each series of experiments. The promoter/luciferase constructs and relative luciferase activity of each construct are shown in Fig 7BDown. Compared with the highest luciferase expression in pLucB2, pLucB1 and pLucB3 gave 84% and 90% of pLucB2, respectively. The transfectant with pLucB4 that contains a GC box gave <1% luciferase activity. The sequence of the first construct, pLucB1, contains both GREs, and pLucB2 contains the second GRE only, but pLucB3 and pLucB4 do not contain GRE. No significant induction by dexamethasone (1 µmol/L) for 16 hours was observed in VSMCs transfected with each of pLucB1 to pLucB3 constructs (Fig 7BDown). This is in accord with the finding that dexamethasone did not affect the transcription level of the AT1B receptor gene in cardiomyocytes and cardiac fibroblasts.19



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  		Figure 7. Analysis of rat AT1B receptor promoter/luciferase reporter gene constructs in transfected VSMCs. The GREs in the rat AT1B promoter are illustrated (A). The chimeric plasmid (pLucB1) consists of the rat AT1B promoter region between -1459 and +55 bp relative to the transcription initiation site,36 the luciferase reporter gene (Luc), and the simian virus 40 splice and polyadenylation signals (SV). The structure of the constructs and their relative luciferase activity are shown (B). The insertion of a 1514-bp fragment into the Kpn I–Sac I sites of the pLuc basic plasmid created pLucB1. Values are expressed as percentages of activity obtained with the construct pLucB2 (100%), which gave the highest activity, and presented as the means of three experiments. Hatched column indicates presence of dexamethasone treatment; stippled column, absence of dexamethasone treatment. The passage number of the VSMCs was <6.

Gel Mobility Shift Assay
To investigate whether GRE2 binds rat glucocorticoid receptors, the in vitro–translated rat glucocorticoid receptor (plasmid T7X556) was used in either gel mobility shift or supershift experiments. The presence of an in vitro–translated rat glucocorticoid receptor that binds to GRE2 was demonstrated by gel mobility shift assay by using a radiolabeled GRE2 oligomer as a probe. Incubation of the GRE2 probe with the in vitro–translated rat glucocorticoid receptor produced a pattern characteristic of the protein-DNA complex (Fig 8Down, lane 2). The band seems to represent specific binding, since the unlabeled GRE2 oligonucleotide competed for binding (lane 3). However, either GRE1 or GRE3 did not show binding activity by same experiment (authors' unpublished data, 1994). This result suggests that the GRE2 of the rat AT1A promoter is functional and binds the rat glucocorticoid receptor. The pattern of the GRE2 DNA-protein complex was similar to that of the consensus GRE DNA-protein complex (authors' unpublished data, 1994). The DNA binding protein was further examined in supershift experiments carried out with monoclonal anti-glucocorticoid receptor antibody. The anti-glucocorticoid receptor shifted all of the observed protein-DNA complex in lane 2 (Fig 8Down, lane 4). Control antibody (anti-STAT p91, Santa Cruz Biotechnology, Inc) had no effect on band shift mobility.



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  		Figure 8. Gel mobility shift assay with the GRE2 oligonucleotide. The GRE2 oligonucleotide (5'-AAGCTTGTACACTATTGTCTGAGTT-3') and in vitro–translated rat glucocorticoid receptor (plasmid T7X556, which contains 407 to 556 amino acid residues including the DNA binding domain of rat glucocorticoid receptor) were used. One microgram protein of in vitro–translated rat glucocorticoid receptor was preincubated with the molar excesses (0, lane 2; 50, lane 3) of unlabeled GRE2 oligonucleotide for 10 minutes at 4°C before the addition of 32P-labeled GRE2 oligonucleotide. After another 30 minutes of incubation, the protein-DNA complex was resolved on 5% acrylamide gels. In supershift experiments, gel shift assay analysis was performed as previously described, but monoclonal anti-mouse glucocorticoid receptor antibody was substituted for the competing cold oligonucleotide in the first 30-minute incubation period. Lane 1 indicates probe alone; lane 2, noncompeting cold GRE2; lane 3, 50-fold competing cold GRE2; and lane 4, supershift assay. SS indicates a supershift DNA-rat glucocorticoid receptor and antibody complex.


*    Discussion
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
In the present study, we identified a functionally active cis-acting GRE in the promoter region of the rat AT1A receptor gene and investigated the effect of glucocorticoid on AT1A receptor gene expression in VSMCs and Y-1 cells derived from mouse adrenal cortex. Dexamethasone induced the gene expression of the AT1A receptor through the glucocorticoid-specific receptor binding to GRE in the AT1A promoter in VSMCs and Y-1 cells.

Adrenal steroids as well as Ang II itself are well-known stimulators of hepatic angiotensinogen production.37 Furthermore, it has been reported that Ang II exerts a positive-feedback effect on the adrenal AT1 receptor.38 Recent cloning of the Ang II receptor and sequence analysis of the promoter sequences of AT1 showed the presence of several potential GREs. Measurements of AT1 mRNA in cultured cells confirmed a positive effect of dexamethasone on AT1 expression in cardiomyocytes and VSMCs.18 However, it is not clear which of the potential GREs is functionally active. The intriguing upregulation of AT1 by Ang II38 in the adrenal gland is in contrast to the downregulation observed in other tissues. The clarification of such a mechanism also requires understanding of the precise mechanism of regulation of receptor expression by adrenal steroids.

A number of circulating factors were shown to modulate AT1 receptor binding. These include Ang II,20 24 39 40 aldosterone,15 41 potassium,42 estrogen,43 catecholamines,44 glucocorticoids,16 17 18 19 45 testosterone,46 and mineralocorticoids.47 The role of glucocorticoids in regulating AT1 binding may be of particular importance in relation to actions of aldosterone-glucocorticoids bound to specific receptors as they interact with the same GRE sequence. The transcription level of the AT1 receptor is increased by aldosterone and glucocorticoids but is inhibited by phorbol esters, estrogens, and Ang II itself. Effects of glucocorticoid on the regulation of the Ang II receptor have been demonstrated in studies in vivo and in vitro. Although Chappell et al48 showed that glucocorticoid downregulated the AT1 receptor in pancreatic acinar cells, many other studies have demonstrated that the same steroid upregulates the AT1 receptor in VSMCs,18 cardiac fibroblasts, and cardiomyocytes.19 However, in studies in vivo, Douglas45 reported that dexamethasone infusion produced downregulation of glomerular Ang II receptors obtained from sodium-loaded rats. The disparity may be due to the difference in the dose of dexamethasone infused in the sodium-loaded rats in other in vitro studies. Although exact reasons for the difference between studies in pancreatic acinar cells and VSMCs are not clear, it is also possible that glucocorticoid has tissue- and cell-specific regulation. Interestingly, it has been reported that the AT1A receptor gene responds to dexamethasone at the transcription level in cultured VSMCs and rat cardiac fibroblasts and cardiomyocytes, whereas the AT1B receptor was not affected by dexamethasone in these cells.19

Nucleotide sequence analyses have suggested the presence of three putative GRE-like sequences, GRE1, GRE2, and GRE3, in the rat AT1A promoter. The present studies produced evidence that only GRE2 is functionally active, responding positively to dexamethasone. This conclusion is supported by three layers of experimental evidence. Deletion mutants of the promoter/luciferase constructs produced results indicating that GRE1 or GRE2 or both are responsive to dexamethasone, whereas GRE3 is inactive. Heterologous context constructs showed that GRE1 was not active but that GRE2 was clearly responsive to dexamethasone. As shown in Fig 9Down, the sequence of GRE3 differs significantly from the consensus GRE sequence, whereas in GRE1 and GRE2, 9 of 12 nucleotides are identical with the consensus sequence. In GRE3, only 5 of 12 are identical with the consensus sequence. A detailed analysis of GRE in mouse mammary tumor virus, in which all possible substitutions were made at each of the 12 consensus positions, suggests that five positions (indicated by underlines in Fig 9Down) are critical for optimal function.49 In GRE1 and GRE2, three nucleotides are identical with the consensus sequence. The 11th base in GRE2 is cytosine (in agreement with the consensus sequence), whereas it is not in GRE1. It may be that this cytosine base is critical for the GRE function. In the rat AT1B, two putative GRE-like GRE sequences were found at positions -424 to -438 bp and -1208 to -1222 bp upstream from the transcription initiation site. As shown in Fig 9Down, the sequence of two AT1B GREs differs significantly from the consensus GRE sequence: only 3 and 4 of 12 nucleotides are identical with the consensus GRE sequence. They also showed that 4 and 5 of 12 nucleotides are identical with the AT1A GRE2 sequence. As shown in Fig 7Up, no significant induction by dexamethasone was observed in VSMCs transfected with each of three rat AT1B promoter/luciferase reporter genes. Taken together, these are in accord with the finding that dexamethasone did not affect the transcription level of AT1B receptor in cardiomyocytes and cardiac fibroblasts.19



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  		Figure 9. Comparison of the sequence of three putative AT1A gene GREs with the consensus sequence. The consensus nucleotide sequence of GRE, the three candidate glucocorticoid receptor binding sites (GRE1, GRE2, and GRE3) in the rat AT1A receptor gene promoter, and two glucocorticoid receptor binding sites (GREB1 and GREB2) in the rat AT1B receptor gene promoter are compared. The most critical nucleotides in the consensus GRE for a maximal response to glucocorticoids as defined by Nordeen et al49 are underlined. The two columns on the right show the ratio of base matches by position for the putative rat AT1A GREs compared with the consensus sequence and with the critical nucleotides defined by Nordeen et al.

We have compared the sequence of the AT1A promoter region among rat strains. It should be noted that no difference in the sequence of AT1A GREs was found among Sprague-Dawley rats, SHR, and Wistar-Kyoto rats.

In most steroid-regulated genes, their expression is influenced by a change in the transcription rate. Several recent studies have investigated the effect of glucocorticoid on the mRNA concentration of seven transmembrane domain receptors, such as the {alpha}1B-adrenergic receptor. Glucocorticoid regulated the expression of this receptor by increasing the rate of transcription. Sato et al18 have reported that glucocorticoid induced the expression of the AT1 receptor gene, resulting in an increase in the number of the receptor. They have further shown that the glucocorticoid did not exert a significant effect on the postreceptor signaling pathway in VSMCs. The pattern of change in expression of AT1A receptor mRNA by dexamethasone was similar to that of the {alpha}1B-adrenergic receptor mRNA.50 The transcriptional level of AT1A was increased by dexamethasone 2.2-fold in VSMCs and 2.1-fold in cardiac fibroblasts and cardiomyocytes.18 19 In the present study, the extent of stimulation of AT1A by dexamethasone ranged from 1.8- to 2.1-fold. Aldosterone production is sensitively controlled by Ang II. This steroid hormone bound to its receptor can stimulate a gene expression with a GRE sequence. In experiments using pTKLucG2 in rat VSMCs, aldosterone was found to increase promoter activity 1.5-fold (authors' unpublished data, 1994). In conclusion, we have demonstrated that a functionally active GRE is present in the AT1A promoter. It is involved in the glucocorticoid- and aldosterone-induced expression of the AT1A receptor gene.


*    Selected Abbreviations and Acronyms
 
Ang II = angiotensin II
BSA = bovine serum albumin
FCS = fetal calf serum
GRE = glucocorticoid responsive element
SHR = spontaneously hypertensive rat(s)
SSC = standard saline citrate
VSMC = vascular smooth muscle cell


*    Acknowledgments
 
This study was supported in part by research grants HL-14192 and HL-35323 from the National Institutes of Health. We wish to thank Tririta Fitzgerald for superb technical assistance in cell culturing. The authors thank Dr Erwin J. Landon for critical reading of the manuscript.

Received September 8, 1994; accepted May 18, 1995.


*    References
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