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(Hypertension. 1995;25:720-725.)
© 1995 American Heart Association, Inc.

Articles

Transcriptional Regulation of the Mouse Angiotensin II Type 2 Receptor Gene

Toshihiro Ichiki; Tadashi Inagami

From the Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tenn.

Correspondence to Tadashi Inagami, Dept of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN 37232.

	Abstract

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Abstract The promoter region of the mouse angiotensin II type 2 receptor gene was cloned, and the nucleotide sequences were determined. A computer homology search for a 1.5-kb promoter region showed that there are several consensus cis DNA elements such as C/EBP, NF-IL6, and AP-1 in this region. Primer extension experiments showed that there are two transcription initiation sites 16 bp apart in the mouse type 2 receptor gene. Deletion mutants of this 1.5-kb segment were prepared and fused to a luciferase reporter gene. These type 2 receptor promoter–luciferase constructs were introduced into PC12W cells, which are from a pheochromocytoma cell line expressing the type 2 receptor, and luciferase activity was measured. It showed that a DNA segment between nucleotides -1497 and -874 suppresses the promoter activity of the type 2 receptor gene and that a DNA segment between nucleotides -47 and +56 is important for the basal promoter activity of the type 2 receptor gene. This proximal segment showed very weak promoter activity when introduced into vascular smooth muscle cells. Gel mobility shift assay with nuclear extracts from PC12W cells showed the presence of three DNA binding proteins that bound to a DNA probe between nucleotides -47 and +8. One DNA binding protein was only very weakly expressed in nuclear extracts from vascular smooth muscle cells, which do not express the type 2 receptor. Two other DNA binding proteins were not observed in nuclear extracts from vascular smooth muscle cells. These data suggest that this DNA segment between nucleotides -47 and +56 is important not only for basal transcription but for differential expression of the type 2 receptor gene in PC12W cells and vascular smooth muscle cells.

Key Words: angiotensin II • type 2 receptor (AT₂) • promoter regions (genetics) • DNA binding protein

	Introduction

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Angiotensin II (Ang II) plays a pivotal role not only in blood pressure regulation, fluid homeostasis, and drinking behavior but in cell growth and proliferation as well.^{1 2} The physiological effects of Ang II are mediated by Ang II receptors on target cells such as vascular smooth muscle cells (VSMC), adrenal cortex cells, and renal mesangial cells.^{1 2} It is known that there are two isoforms of the Ang II receptor on the basis of the differential sensitivity of ligand-receptor binding to a reducing agent.³ The isoform-specific antagonists have clearly shown that at least two isoforms of Ang II receptor are present.^{4 5} The type 1 receptor (AT₁) belongs to a family of seven-transmembrane domain-type receptors^{6 7} and has proved to mediate practically all known biological effects of Ang II such as vasoconstriction, aldosterone release, and the facilitation of adrenergic nerve activity.^{1 2} The type 2 receptor (AT₂) was also shown to have the putative seven-transmembrane domain structure.^{8 9} However, definitive signaling mechanisms mediated by this receptor have not been established.^{10 11 12 13}

In contrast to the elusiveness of the function of the AT₂ receptor, the gene expression of the AT₂ receptor is well characterized. Binding studies using ¹²⁵I-labeled Ang II and isoform-specific antagonists showed that the AT₂ receptor is highly expressed in rat fetal tissues, most conspicuously in mesenchymal tissues¹⁴ and various brain nuclei.¹⁵ This expression is decreased or shut off rapidly after birth. In adult rat, the AT₂ receptor shows a unique tissue distribution. It is expressed in some brain nuclei,¹⁶ heart,^{17 18} myometrium,⁴ adrenal medulla,⁵ and ovarian granulosa cells.¹⁹ The tissue-specific and ontogeny-dependent expression suggests possible developmental, neurological, and reproductive roles of Ang II effected by means of the AT₂ receptor.

In vitro binding studies using cell lines that express the AT₂ receptor, such as R3T3 cells²⁰ and PC12W cells,²¹ showed that the expression of the AT₂ receptor is suppressed by growth factors. The expression in R3T3 cells is downregulated by fibroblast growth factor and serum.²⁰ Nerve growth factor suppressed the AT₂ receptor expression in PC12W cells.²¹ These in vivo and in vitro binding studies suggest that expression of the AT₂ receptor is tightly controlled and closely related to its biological roles. To clarify this unique tissue-specific and ontogeny-dependent expression, we cloned the promoter region of the mouse AT₂ gene and examined its promoter function.

	Methods

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Reagent
A luciferase assay kit was purchased from Promega. Fetal calf serum and Dulbecco's modified Eagle medium (DMEM) were obtained from GIBCO BRL, and other tissue culture supplies were from Sigma.

DNA Cloning and Nucleotide Sequencing
A genomic DNA library of 129 SVJ mouse was purchased from Stratagene. By use of a conventional plaque hybridization method,²² 500 000 phages were screened, with ³²P-labeled full-length complementary DNA of the mouse AT₂ receptor²³ used as a probe. Eight positive clones were obtained and a BamHI–Sac I fragment (Fig 1A) that contains the first exon of mouse AT₂ gene in its 3' region was subcloned into pBluescript (Stratagene). Deletion mutants of the inserted BamHI–Sac I fragment were prepared by using an Erase-A-Base kit (Promega). Nucleotide sequences were determined by a dideoxy chain termination method with a Sequenase kit (United States Biochemicals) in both the sense and antisense directions.

View larger version (39K): [in this window] [in a new window]   Figure 1. Schematic representation shows genomic organization, restriction map, and promoter activity of the mouse angiotensin II type 2 receptor (AT2) gene. A, Three boxes indicate the exons of the mouse AT2 gene.23 Shaded region indicates the coding region. Arrows shown below the first exon indicate two transcription initiation sites. Double-headed arrows indicate the length of gene sequenced. Restriction map was deduced from the restriction sites of the genomic DNA clone of the AT2 gene and Southern blot analysis. Restriction endonucleases are BamHI (B), EcoRI (E), Kpn I (K), and Sac I (S). Promoter region of the nucleotide sequences determined is shown above by an arrow. B, A detailed restriction map shows the promoter region of the mouse AT2 gene. P indicates PvuII; A, Acc I. Some consensus cis DNA elements (C/EBP, NF-IL6, AP-1, and PEA 3) are shown below. The upstream transcription initiation site is designated as +1. C, Design of deletion mutant constructs of the promoter region of the mouse AT2 gene is shown. DNA fragments comprising nucleotides -1497 through +56, -874 through +56, -427 through +56, -284 through +56, and -47 through +56 were fused to a luciferase gene and designated D1, D2, D3, D4, and D5, respectively. D, Relative luciferase activities in PC12W cells of the deletion mutants shown in C are indicated. Five micrograms of the AT2 promoter–luciferase constructs and 2 µg pSVß-galactosidase were introduced into PC12W cells or vascular smooth muscle cells (VSMC) (D5 construct) by DOTAP (N-[1-(2,3-dioleoyloxy) propyl]-N,N,N-trimethyl-ammonium methylsulfate) liposome transfection reagents. After 48 hours of the transfection, cells were lysed and luciferase and ß-galactosidase activities were measured. The luciferase activity was normalized in reference to the ß-galactosidase activity. Luciferase activity in the D1 construct was set to 100%. A mock transfection was performed using the same amount of a promoterless luciferase gene. Data are mean±SEM of six independent experiments. *P<.05 compared with D1.

Primer Extension Experiment
A 20-mer primer specific for the first exon of the AT₂ gene (Fig 2; 5'-GCAGGCTGAAGTAAGCTTTC-3', antisense strand) was end-labeled with [³²P]-ATP and T4 polynucleotide kinase and then purified by an ammonium acetate–ethanol precipitation. Poly(A)⁺ RNA was prepared from 18–gestation day mouse fetus using a Fast Track kit (In Vitrogen). One microgram of poly(A)⁺ RNA or 50 µg of tRNA was reverse transcribed using the ³²P-labeled primer and Moloney's murine leukemia virus reverse transcriptase (NEB). The resultant product was phenol extracted, ethanol precipitated, and resuspended in 4 µL loading buffer (95% formamide, 20 mmol/L EDTA) and electrophoresed in 6% acrylamide–8 mol/L urea gel after heat denaturation. Sequencing ladders were obtained by the same primer with a Sequenase kit (United States Biochemicals).

View larger version (53K): [in this window] [in a new window]   Figure 2. Diagram shows the nucleotide sequences of the promoter region of the mouse angiotensin II type 2 receptor (AT2) gene. Nucleotide sequences of the BamHI–Sac I fragment were determined by a dideoxy chain termination method with a Sequenase kit in both the sense and antisense directions. Consensus cis DNA elements, some restriction sites, and the location of primer used in primer extension experiments (see Fig 3) are indicated by underlines. Two transcription initiation sites are indicated in bold type and by arrows. The DNA segment used as a probe in the gel mobility shift assay (see Fig 4) is indicated in the box.

Preparation of AT₂ Promoter–Luciferase Construct
Five deletion fragments of the promoter region of the AT₂ receptor gene were prepared by digestion with restriction endonuclease, as shown in Fig 1B, except for D4, which was prepared by an Erase-A-Base deletion mutant kit (Promega). These fragments were cloned into the pGL2E (Promega) luciferase reporter vector. Plasmid DNA was prepared with a plasmid kit (Qiagen Inc) and purified once by centrifugation over a cesium chloride cushion followed by dialysis against TE buffer (10 mmol/L Tris, pH 7.5, 1 mmol/L EDTA) and ethanol precipitation.

Cell Culture
PC12W cells were maintained in DMEM supplemented with 2.5% fetal calf serum, 25 µg/mL insulin, transferrin, and sodium serenate, 0.05% bovine serum albumin, 5 µg/mL linoleic acid, and 50 µg/mL gentamycin. Cells were cultured in the presence of 5% CO₂ at 37°C. VSMC from Wistar-Kyoto rats were prepared as described previously.²⁴ They were maintained in DMEM supplemented with 10% fetal calf serum and 50 µg/mL gentamycin.

Transfection to PC12W Cells
The day before transfection, 5x10⁵ PC12W cells or VSMC were prepared in a 6-cm tissue culture dish. On the day of transfection, the medium was changed to fresh medium and incubated for 1 hour at 37°C. The cells were then transfected with 5 µg AT₂ promoter–luciferase construct and 2 µg pSVß-galactosidase (Promega). The transfection was performed using DOTAP (N-[1-(2,3-dioleoyloxy) propyl]-N,N,N-trimethyl-ammonium methylsulfate) liposome transfection agent according to the manufacturer's instructions (Boehringer Mannheim). Cells were incubated with the AT₂ promoter–luciferase and pSVß-galactosidase DNAs and DOTAP for 24 hours and then with the fresh medium alone for an additional 24 hours. The cells were washed twice with Hanks' balanced salt solution and lysed in 200 µL lysis buffer (25 mmol/L Tris, pH 7.8, 2 mmol/L EDTA, 2 mmol/L DL-dithiothreitol, 10% glycerol, and 1% Triton X-100). Fifty microliters of lysate was used for luciferase activity assay in an Opticomp I luminometer (MGM Instruments Inc). The assay was started by adding 100 µL of 470 mmol/L luciferin to cell lysate, and integrated peak luminescence was determined over a 45-second window after a 5-second delay. The ß-galactosidase activity in the same sample was measured spectrophotometrically according to Sambrook et al²⁵ and used to normalize the luciferase activity.

Gel Mobility Shift Assay
A 55-bp DNA fragment between nucleotides -47 and +8 (Fig 2) was end-labeled by [³²P]-ATP and T4 polynucleotide kinase. The labeled probe was purified by a Sephadex G-50 Quick Spin Column (Boehringer Mannheim). Nuclear extracts from PC12W cells and VSMC were prepared according to the method of Dignam et al.²⁶ About 10 000 cpm of the labeled probe (approximately equal to 1 ng DNA) and 2 µg of nuclear extracts were incubated on ice in a buffer containing 12 mmol/L HEPES, pH 7.9, 60 mmol/L KCl, 1 mmol/L EDTA, 1 mmol/L dithiothreitol, 12% glycerol, 0.5 mmol/L phenylmethylsulfonyl fluoride, and 3 µg polydeoxyinosinic-deoxycytidylic acid (Pharmacia) for 30 minutes. The nuclear extracts–DNA mixture was then electrophoresed in 4% acrylamide gel in a low-concentration TAE buffer (6.7 mmol/L Tris-HCl, pH 7.5, 3.3 mmol/L sodium acetate, and 1 mmol/L EDTA) at 4°C. The gel was dried and exposed to Kodak X-OMAT film for 16 hours at -70°C.

Statistics
Data are given as mean±SEM. Statistical analysis was performed with ANOVA and Duncan's test. Values of P<.05 were considered statistically significant.

	Results

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Cloning and Nucleotide Sequence of the Promoter Region of Mouse AT₂ Gene
We reported previously that the mouse AT₂ gene is composed of three exons.²³ We extended the restriction map of the mouse AT₂ gene on the basis of the nucleotide sequences of the 4.5-kb EcoRI fragment that contains all three exons and Southern blot analysis of the genomic DNA and genomic DNA clones. The restriction map and exon-intron organization is shown in Fig 1A. A 1.5-kb BamHI–Sac I segment that is located in the upstream region of the 4.5-kb EcoRI segment was subcloned from the genomic DNA clone. Its detailed restriction map is shown in Fig 1B. The nucleotide sequence of this upstream region was determined (Fig 2). A search for consensus cis DNA elements in the database TFD 7.3 revealed the presence of potential sites for C/EBP, AP-1, NF-IL6, and PEA3, as shown in Fig 1B and Fig 2.

Transcription Initiation Site of the Mouse AT₂ Gene
To determine the transcription initiation site of the mouse AT₂ gene, primer extension experiments were performed. One microgram of poly(A)⁺ RNA from eviscerated mouse fetus (Fig 3, lane 1) and 50 µg of tRNA (lane 2) were reverse transcribed with a ³²P-labeled 20-mer oligonucleotide specific for the first exon of the mouse AT₂ gene (Fig 2, nucleotides +64 to +83). The resultant products were electrophoresed in acrylamide-urea gel. Two bands, 16 bp apart, were observed (Fig 3, lane 1). These initiation sites are indicated in bold type and by arrows in Fig 2 (nucleotides +1 and +17). No bands were observed when tRNA was reverse transcribed (Fig 3, lane 2).

View larger version (50K): [in this window] [in a new window]   Figure 3. Photograph shows results of primer extension experiments done to determine the transcription initiation sites of the mouse angiotensin II type 2 receptor (AT2) gene. Poly(A)+ RNA from eviscerated carcasses of mouse fetus 18 days into gestation (lane 1) and transfer RNA (lane 2) were reverse transcribed with a 32P-labeled primer specific for the first exon of the AT2 gene (see Fig 2, nucleotides +64 to +83). The products were electrophoresed in 6% acrylamide–8 mol/L urea gel. Nucleotide sequences were obtained by use of the same primer as was used for the determination of the transcription initiation site. Two arrows indicate the 5' end (transcription initiation site) of the messenger RNA of the AT2 gene. The same results were obtained in two independent additional experiments.

Promoter Activity of the Mouse AT₂ Gene
We examined the promoter activity of the 1.5-kb BamHI–Sac I fragment and its deletion mutants. Five DNA fragments were prepared and fused to a luciferase reporter gene (Fig 1C). These AT₂ promoter–luciferase constructs were introduced into PC12W cells, which express the AT₂ receptor.¹⁰ The luciferase activity was normalized in reference to ß-galactosidase activity expressed by cotransfected pSVß-galactosidase DNA. Results are shown in Fig 1D. The luciferase activity of the D1 construct was set to 100%, and this activity is approximately 4% of the promoter activity of SV40. Deletion of the DNA segment between nucleotides -1497 and -874 increased relative luciferase activity by about 70%. The relative luciferase activity of the shortest deletion mutant, D5 (nucleotides -47 to +56), showed approximately 130% of that of the D1 construct. The luciferase activity of the D5 construct in VSMC was about 5% of that in PC12W cells (Fig 1D).

DNA Binding Protein Bound to Proximal Promoter Region
On the basis of the data for promoter activity of the AT₂ gene in PC12W cells, we hypothesized that the DNA segment between nucleotides -47 and +56 is important for the basal promoter activity and used a gel mobility shift assay to study the possibility of there being DNA binding protein bound to this segment. A 55-bp DNA fragment between nucleotides -47 and +8 was end-labeled by ³²P and used as a probe. Three bands of DNA binding proteins were observed in nuclear extracts from PC12W cells (Fig 4, lane 1, arrows 1 through 3). By addition of 100-mol/L (Fig 4, lane 2) and 200-mol/L (lane 3) excesses of the cold DNA probe, these bands were competed out, indicating the specific binding of nuclear proteins and probe DNA. Nuclear extracts from VSMC, which do not express the AT₂ receptor,⁴ were also examined (Fig 4, lanes 4 and 5). Although the DNA binding protein 2 was expressed very weakly in nuclear extracts from VSMC (Fig 4, lane 4), the two other DNA binding proteins were not observed.

View larger version (49K): [in this window] [in a new window]   Figure 4. Photograph shows gel mobility shift assay using nuclear extracts from PC12W cells and vascular smooth muscle cells (VSMC). A 32P-labeled DNA fragment between nucleotides -47 and +8 (see Fig 2) was incubated with 2 µg nuclear extracts from PC12W cells (lanes 1 through 3) or VSMC (lanes 4 and 5) in the absence (lanes 1 and 4) or presence of unlabeled DNA probe added to 100-mol/L (lanes 2 and 5) and 200-mol/L (lane 3) excesses. The mixture of nuclear extracts and DNA was electrophoresed in 4% acrylamide gel in a low-concentration TAE buffer (6.7 mmol/L Tris-HCl, pH 7.5, 3.3 mmol/L sodium acetate, and 1 mmol/L EDTA). Gel was dried and exposed to x-ray film at -70°C. Arrows (1 through 3) indicate DNA binding proteins bound to this probe.

	Discussion

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In contrast to the elusiveness of the function of the AT₂ receptor, its expression is characterized well by binding studies that use radiolabeled Ang II.^{4 5 16 17 18 19} In rat fetus, the AT₂ receptor is highly expressed in mesenchymal tissues¹⁴ and certain brain nuclei such as the hypoglossal nucleus and the paratrigeminal nucleus.¹⁵ This expression is decreased rapidly after birth. In adult rat, the AT₂ receptor is known to be expressed in several brain nuclei (eg, the inferior olive, thalamic nuclei, and locus ceroleus),¹⁶ adrenal medulla,⁵ heart,^{17 18} myometrium,⁴ and ovarian granulosa cells.¹⁹ The tissue-specific and ontogeny-dependent expression of the AT₂ receptor gene suggests possible developmental, neurological, and reproductive roles of Ang II effected by means of the AT₂ receptor and indicates that the biological roles of this receptor are closely related to its unique expression pattern.

Because the expression patterns of the AT₂ receptor in fetal and adult tissues are different and most of the AT₂ sites expressed in the fetus are decreased rapidly after birth, the regulatory mechanism of the AT₂ receptor gene expression could be different between the adult and fetal tissues. To clarify this unique tissue-specific and ontogeny-dependent AT₂ receptor expression, we cloned the promoter region of the mouse AT₂ gene. The nucleotide sequence of a 1.5-kb BamHI–Sac I fragment showed that there are several consensus cis DNA elements in this region, such as AP-1, C/EBP, NF-IL6, and PEA3. However, roles of these cis DNA elements, as in the promoter of the AT₂ gene, have not been determined yet. Some of these cis DNA elements are clustered in the DNA segment between nucleotides -1497 and -874 (Fig 1B), and deletion of this segment increased relative luciferase activity by about 70% (Fig 1C and 1D, deletion mutants D1 and D2). Therefore, this upstream segment (-1497 to -874) may negatively regulate the promoter activity of the AT₂ gene by these cis DNA elements. The deletion of the DNA segment between nucleotides -874 and -427 reduced the relative luciferase activity to a baseline level, as shown in Fig 1D. Relative luciferase activity of the D3 construct was approximately equal to that of the D1 construct. In this region a PEA3 consensus sequence is present.

The shortest deletion mutant, D5, showed significantly higher relative luciferase activity than that of the D1 construct (Fig 1D). Therefore, the DNA segment between nucleotides -1497 and -48 appears to suppress most of the promoter activity of the AT₂ gene.

In the DNA segment between nucleotides -47 and +56, there is a TATA box consensus sequence (Figs 1B and 2) that may be important for the basal promoter activity of the AT₂ gene. Although this segment seems to be important for the promoter activity of the AT₂ gene in PC12W cells, its activity was very weak in VSMC, which do not express the AT₂ receptor.⁴ This result suggests that this segment may be responsible for the differential expression of the AT₂ gene. The absence or presence of the DNA binding protein bound to this region is probably responsible for the differential promoter activity of this segment in PC12W cells and VSMC.

We found three DNA binding proteins that bound to the DNA segment between nucleotides -47 and +8. Addition of 100-mol/L (Fig 4, lane 2) and 200-mol/L (lane 3) excesses of a cold DNA probe competed out these three bands in a gel mobility shift assay, indicating that the binding of proteins is specific. Although DNA binding protein 2 (Fig 4, arrow 2) was very weakly expressed in nuclear extracts from VSMC (lane 4), two other DNA binding proteins (arrows 1 and 3) were not observed in nuclear extracts from VSMC. Therefore, it is likely that the latter two DNA binding proteins may be responsible for the differential expression of the AT₂ receptor gene in PC12W cells and VSMC. As mentioned above, there is a TATA box in the DNA segment between nucleotides -47 and +56. Thus, one of these DNA binding proteins bound to this region may be a TATA box binding protein.²⁷ The identification of a cis-acting element specifically bound by these nuclear proteins may reveal the control mechanism of the tissue-specific expression of the AT₂ receptor gene in PC12W cells.

PC12W cells, from a rat pheochromocytoma cell line, have been reported to constitutively express the AT₂ receptor.²¹ They are derived from the adrenal medulla, which expresses AT₂ sites in the adult rat. Therefore, the results of deletion analysis of the promoter region of the AT₂ receptor gene in the present study probably reflect the transcriptional control mechanism of this gene in the adult tissue and may be different from that in the fetal tissue. Comparison of the results of deletion analysis of the promoter region of the AT₂ receptor gene in PC12W cells with that in fetus-derived cells expressing AT₂ sites may clarify the mechanisms of the differential regulation of AT₂ receptor expression in the fetal and adult tissues.

Although we examined the promoter activity of the mouse AT₂ gene with PC12W cells, which are from a rat cell line, the DNA sequences of the promoter region are highly conserved between mouse and rat AT₂ genes. In particular, only 3 nucleotides of 55 are different in the DNA segment between nucleotides -47 and +8, and a TATA box is conserved (Y. Kambayashi et al, unpublished data, 1994). Therefore, it is safe to assume that these two rodent species share a common regulatory mechanism for the AT₂ receptor gene expression, at least in this proximal promoter region.

	Acknowledgments

 
This work was supported in part by research grants from the US Public Health Service and grants HL-14192 and HL-35323 from the National Institutes of Health. We especially thank Trinita Fitzgerald for excellent technical assistance in cell culture.

	References

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