©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Growth-dependent Expression of Angiotensin II Type 2 Receptor Is Regulated by Transcription Factors Interferon Regulatory Factor-1 and -2 (*)

(Received for publication, May 11, 1995)

Masatsugu Horiuchi George Koike Takehiko Yamada Masashi Mukoyama Masatoshi Nakajima Victor J. Dzau (§)

From the Division of Cardiovascular Medicine, Falk Cardiovascular Center, Stanford University School of Medicine, Stanford, California 94305-5246

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Angiotensin II type 2 (AT(2)) receptor is abundantly and widely expressed in fetal tissues but present only in restricted tissues in the adult such as brain and atretic ovary. This receptor is speculated to be involved in tissue growth and/or differentiation. To elucidate the molecular mechanism of growth-regulated AT(2) receptor expression, we cloned the mouse AT(2) receptor genomic DNA and studied its promoter function in mouse fibroblast-derived R3T3 cells, which express AT(2) receptor in the confluent, quiescent state but very low levels of the receptor in actively growing state. Promoter/luciferase reporter deletion analysis of AT(2) receptor in R3T3 cells showed that the putative negative regulatory region is located between the positions -453 and -225, which plays an important role in the transcriptional control of AT(2) receptor gene expression along with the cell growth. We identified the interferon regulatory factor (IRF) binding motif in this region using DNase footprinting analysis and demonstrated that IRF binding oligonucleotide treatment increased the AT(2) receptor expression in growing R3T3 cells but not in confluent cells. Furthermore, by antisense treatment, we demonstrated that IRF-2 attenuated the AT(2) receptor expression in both growing and confluent R3T3 cells, whereas IRF-1 enhanced AT(2) receptor expression in the confluent cells only. Consistent with this result, gel mobility shift assay demonstrated that growing R3T3 cells exhibited only IRF-2 binding, whereas confluent cells exhibited both IRF-1 and IRF-2 binding. Furthermore, we observed using reverse transcription-polymerase chain reaction that the IRF-1 mRNA expression was more abundant in confluent cells than growing cells, whereas IRF-2 expression did not change with R3T3 cell growth. We conclude that, in confluent cells, the enhanced expression of IRF-1 antagonizes the IRF-2 effect and increases the AT(2) receptor expression. We speculate that these transcriptional factors influence cell growth in part by regulating AT(2) receptor expression.


INTRODUCTION

Angiotensin II (Ang II), (^1)a key regulator of cardiovascular homeostasis, exerts various actions in its diverse target tissues controlling vascular tone, hormone secretion, tissue growth, and neuronal activities. Multiple lines of evidence have suggested the existence of Ang II receptor subtypes, but it was only recently that at least two distinct receptor subtypes were defined. Based on their differential pharmacological and biochemical properties, the subtypes have been designated as type 1 (AT(1)) and type 2 (AT(2)) receptors(1, 2) . To date, extensive pharmacological evidence indicates that most of the known effects of Ang II in adult tissues are attributable to the AT(1) receptor. In contrast, much less is known about the regulation and function of the AT(2) receptor. It is abundantly and widely expressed in fetal tissues and immature brain (3, 4, 5) but present only in scant levels in tissues in the adult(1, 2, 3, 5, 6, 7) , suggesting that this receptor may be involved in tissue growth and/or differentiation. Using the expression cloning strategy in COS-7 cells from a rat fetus expression library, we have reported the cloning of the rat AT(2) receptor(8) ; Kambayashi et al.(9) also cloned rat AT(2) receptor from rat pheochromocytoma cell line PC12W cells. Hydropathy analysis indicates that it belongs to a seven-transmembrane receptor family. It is 34% identical in sequence to AT(1) receptor. The signal mechanism of the AT(2) receptor is still poorly defined.

To understand the molecular mechanism of the developmental and growth regulation of AT(2) receptor expression, we cloned, in this study, mouse AT(2) receptor gene, analyzed its structure, and examined the promoter activity. We then employed R3T3 cells, a mouse fibroblast cell line, as model to study the AT(2) receptor promoter activity since these cells express the only AT(2) subtype binding sites, and the expression of AT(2) receptor sites in these cells are modulated by the growth state of the cells (10, 11) . This characteristic of this cell line provides us an excellent model for studying the growth regulation of the AT(2) receptor.

Interferon regulatory factor (IRF)-1 and related IRF-2 protein have been shown to recognize same DNA sequence elements(12) . IRF-1 activates transcription of interferon (IFN)-inducible genes, whereas IRF-2 inhibits it(12, 13, 14, 15, 16, 17) . Recently, competitive binding of IRF-1 and IRF-2 was reported to play critical roles in determining cell growth, transformation, and apoptosis. Furthermore, IRF-1 has been implicated to be a tumor suppresser gene and IRF-2 as an oncogene(18, 19, 20) . Our results show that the expression of AT(2) receptor in R3T3 cells is transcriptionally regulated by the competitive binding of IRF-1 and IRF-2, suggesting that AT(2) receptor is one of the target genes of IRF system and that IRF system may regulate the cell growth via the AT(2) receptor.


EXPERIMENTAL PROCEDURES

Cloning of Mouse AT Receptor Gene

Mouse genomic library prepared from BALB/c male liver (Clontech) were screened by a PstI-NsiI fragment of mouse AT(2) receptor cDNA (21) as probe.

Luciferase Reporter Vector Construction and R3T3 Cell Transfection

A series of nested restriction fragment deletions of AT(2) receptor gene promoter region were subcloned into pGL2-basic luciferase vector (Promega). The following restriction fragments from 5`-end of MG1 (Fig. 1) were subcloned by direct ligation: XhoI-HindIII (-1081/+52), HaeIII-HindIII (-892/+52), PvuII-HindIII (-802/+52), and DraIHindIII (-453/+52 and -224/+52). R3T3 cells were cultured as described (10) in a 10-cm dish (Falcon) for transfection. Cells were seeded at 7 10^4/dish. 1 day after seeding, cells were used as growing cells, and cells 2 days after reaching confluent state were used as confluent cells. 2 µg of construct/10^6 cells was transfected by modified DEAE-dextran transfection method(8) . pSV-beta-galactosidase control vector (Promega) was cotransfected to standardize the transfection efficiency. After a 48-h culture, the cells were harvested, and cell lysate was prepared. Luciferase assays were performed as previously described (22) using luciferase assay system (Promega). beta-Galactosidase activity was used to normalize variations in transfection efficiency.


Figure 1: Sequence analysis of promoter region of mouse AT(2) receptor (A) and putative transcription factor binding sites (B). Restriction enzyme sites for making AT(2) receptor promoter-luciferase expression vectors (XhoI, HaeIII, PvuII, DraI, and HindIII) and transcription initiation sites (P1, P1`, and P2) are shown. The transcription initiation site P1 is presented as position 0. Bold and italic nucleotides indicate the sequence of primer for primer extension analysis.



DNase I Footprinting Analysis

Nuclear extract was prepared by the method of Dignam et al.(23) . DNase I footprinting analysis was performed essentially as previously described (24) . Probe DNAs were AccI-DraI (-443/-224) and HinfI-DraI (-340/-224) fragments. AccIHindIII (-443/+52) and HinfI-HindIII (-340/+52) fragments were, respectively, labeled by [-P]ATP using T4 kinase, cut with DraI, and purified by 10% polyacrylamide gel electrophoresis.

Oligonucleotides

Oligonucleotides used for treating cultured R3T3 cells include the following: octamer binding, 5`-AATAGGAGTAAAAATTCAAAATCTGCTAAAAG-3`; IRF binding, 5`-GAAAAAGAGAAAGAGAAAATTCTGCTAAAAATGATA-3`; and mutant IRF binding, 5`-GAAAAAAGGAAAAGGAAAATTCTGCTAAAAAGGATA-3`.

IRF binding oligonucleotide was also used in gel mobility shift assay. IRF and octamer binding sequences were designed according to the footprinted sequences (Fig. 3, A and B). Three repeats of putative IRF binding hexamer motif, as shown in Fig. 1B, were replaced with C4 oligomer (AAAGGA), which does not bind to IRFs(14) , and this oligonucleotide was used as mutant IRF binding oligonucleotide. Oligonucleotides were annealed to complementary sequences and used as double-stranded oligonucleotides. Oligonucleotides for treating cells as antisense DNA for IRF-1 and IRF-2 were as follows: IRF-1 antisense, 5`-GAAAGATGCCCGAGATGC-3` (-111/-94)(25) ; IRF-2 antisense, 5`-GTGTGAGTGTTGTTAGGG-3` (-71/-54)(14) .


Figure 3: DNA binding properties of negative regulatory regions by DNase I footprinting (A and B), the effect of oligonucleotides spanning two distinct protected regions observed by DNase I footprinting on AT(2) receptor binding (C and D), and expression in R3T3 cells (E). A and B, probe DNAs AccI-DraI (-443/-224) (A) and HinfI-DraI (-340/-224) (B) fragments were subjected to DNase I footprinting analysis. Lanes1 and 2, probe DNAs were reacted with nuclear extracts prepared from growing cells (15 and 30 µg, respectively); lanes3 and 4, probe DNAs were reacted with nuclear extracts prepared from confluent cells (15 and 30 µg, respectively) and digested by DNase I. LanesG and G+A, probe DNA cleaved by G and G + A reactions(41) . The protected regions are depicted on the leftsides of the panel, and the non-protected region is underlined. C and D, three double-stranded oligonucleotides (octamer binding, IRF binding, and mutant IRF binding oligonucleotides) (see ``Experimental Procedures'') were transfected into growing (C) and confluent (D) R3T3 cells. The AT(2) receptor binding in untreated cells is taken as 100%. The values were expressed as mean ± S.D. obtained from eight different cell culture wells. E, total RNA was prepared from cultured R3T3 cells 24 h after oligonucleotide treatment. RNA (20 µg/lane) was separated by electrophoresis, and hybridization was carried with a probe for the AT(2) receptor or for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a control. The result is a representative of data obtained in three different experiments.



Transfection of Hemagglutinating Virus of Japan (HVJ)/Liposomes/Oligonucleotide into R3T3 Cells

Mixture of lipids, oligonucleotides, and UV-inactivated virus was prepared as previously described(26, 27) . Cells were plated on 24-well plates (Falcon) at the concentration of 2.5 10^3 cells/well; the cells 2 days after seeding were used as growing cells, and cells 8 days after seeding were used as confluent cells. Cells were incubated with HVJ/liposome/oligonucleotides solution (200 µl; final oligonucleotide concentration, 10 µM) at 4 °C for 10 min, and further incubation was done at 37 °C for 30 min. Solution was aspirated and incubated with fresh medium. Cells were harvested 3 days after transfection, and AT(2) receptor was measured. For RNA preparation, we used a 75-cm^2 tissue culture flask (Corning).

Receptor Binding Assay

Cells were washed twice with phosphate-buffered saline containing 0.1% bovine serum albumin. Reactions were performed with 0.2 nMI-labeled CGP42112A (2,176 Ci mmol, Peptide Radioiodination Center, Washington State University) at 37 °C for 1 h. After incubation, cells were washed with ice-cold phosphate-buffered saline twice, treated with 0.5 M NaOH, and counted for radioactivity. Nonspecific binding was defined in the presence of 1 µM unlabeled CGP42112A.

Northern Blot Analysis

Total RNA was prepared from cultured R3T3 cells using RNAzol (Tel-Test) 24 h after oligonucleotide treatment. RNA (20 µg/lane) was separated by electrophoresis and transferred onto a nylon membrane (Amersham); hybridization was carried out using a P-labeled HindIII-Nsi1 fragment of mouse cDNA (21) or 0.78-kilobase PstI-XbaI fragment of a human glyceraldehyde-3-phosphate dehydrogenase clone.

Gel Mobility Shift Assay

IRF binding oligonucleotide and C1 oligomer (AAGTGA)(4)(25) were labeled by [-P]ATP using T4 kinase. Gel mobility shift assay was performed essentially as previously described(24, 28) .

Reverse Transcription-PCR (RT-PCR) for IRF-1 and IRF-2

Total RNA was prepared from cultured R3T3 cells using RNAzol (Tel-Test). PCR primers for identifying IRF-1 used were as follows: 5`- CCTGATGACCACAGCAGTTAC-3` and 5`- CTTCATCTCCGTGAAGACATG-3`. For identifying IRF-2, PCR primers were 5`-GCGGTCCTGACTTCAGCTATA-3` and 5`-CTTCTTGATGACACTGGCCGG-3`. PCR reaction was carried out with 30 cycles of 1 min of denaturation at 94 °C, 1 min of annealing at 55 °C, and 3 min of extension at 72 °C followed by 15 min of final extension step. Expression of beta-actin was determined using Amplimer Sets (Clontech) by RT-PCR.

Statistical Analysis

All values are expressed as mean ± S.D. Statistical significance was assessed by ANOVA (analysis of variance) followed by Scheffe's test. p < 0.01 was considered significant.


RESULTS AND DISCUSSION

We screened mouse liver genomic libraries by PstI-NsiI fragment of mouse AT(2) receptor cDNA (21) and obtained three independent positive clones. Restriction enzyme mapping revealed that these three clones overlapped each other, and each clone contained full-length mouse cDNA. One of these (MG1, 14.3 kilobases) was further analyzed. Sequence comparison between genomic DNA and cDNA reveals that there are two introns in the 5`-untranslated region. Coding region in the third exon is not interrupted by any intron, and there is no intron in the 3`-untranslated region. Similar observation was reported by Ichiki et al.(29) using 129SV mouse strain. To investigate further the role of the promoter region in controlling AT(2) receptor gene expression, we sequenced the upstream promoter region (Fig. 1) and performed primer extension analysis using mouse fetus RNA (data not shown). Primer was designed in the upstream of the first intron as shown in Fig. 1. Primer extension products were observed at two distinct sites (P2 and P1, P1`) (Fig. 1A). P1 and P1` corresponds to T and G, and there are 3 bp between these initiation sites. Adjacent to these initiation sites, a TATA motif is observed in the upstream sequence. Another primer extension product (P2) is observed at position -67. In the following experiments, we assigned P1 site as the initiation site to the sequence.

To investigate the growth-dependent AT(2) receptor expression, we used R3T3 cells. Specifically, the expression of AT(2) binding sites is very low in actively growing cells but markedly increased in confluent, quiescent cells(11) . We subcloned HindIII-XhoI fragment (-1081/+52) of MG1 into the enhancerless luciferase expression plasmid (p0Luc) and transfected this construct into the R3T3 cells. Luciferase activity obtained in experiment with p(-1081/+52)Luc using confluent R3T3 cells was taken as 100% in each experiment. Transfection with p0Luc gave a background luciferase activity. As shown in Fig. 2, A and B, luciferase expression by this AT(2) receptor-luciferase expression vector construct was 5-fold higher in the confluent R3T3 cells than that in the rapidly growing cells. Transfection with pSVLuc, which contains p0Luc plus the heterologous SV40 virus early promoter/enhancer region driving luciferase expression, did not show the difference in luciferase activity between growing and quiescent R3T3 cells. These results suggest that the AT(2) receptor promoter region (-1081/+52) contains specific cis DNA elements that determine cell growth-regulated AT(2) receptor gene expression.


Figure 2: Transcriptional activity in R3T3 from deletions of the mouse AT(2) receptor promoter region. The chimeric mouse AT(2) receptor promoter-luciferase constructs, p(-1081/+52)Luc, p(-892/+52)Luc, p(-802/+52)Luc, p(-453/+52)Luc, p(-224/+52)Luc, p0Luc, and pSVLuc were transfected in growing R3T3 cells (A) and confluent, quiescent R3T3 cells (B). Luciferase activity obtained in experiment with p(-1081/+52)Luc using confluent R3T3 cells was taken as 100%. pSV-beta-galactosidase control vector was cotransfected to normalize variations in transfection efficiency. The values (mean ± S.D.) are obtained from eight individual experiments (chimeric constructs), four experiments (p0Luc), and five experiments (pSVLuc).



We made five different AT(2) receptor-luciferase expression vectors and transfected these into R3T3 cells. As shown in Fig. 2, A and B, deletion of -453/-225 fragment enhanced luciferase activity 4-fold in growing R3T3 cells when compared to other constructs. This result suggests that the sequence between -453 and -225 may contain functional negative regulatory elements for AT(2) receptor gene expression during cell growth. However, in confluent R3T3 cells, we could not observe any significant differences in the expressed luciferase activities. Interestingly, we found in this region the several putative consensus sequences such as two repeats (5`-AAGAGAAAGAGA-3`) of IRF binding sequence (30) at the position -282 bp and this hexamer motif (5`-AAATGA3-`) at the position -258 bp (Fig. 1B). We also found protein kinase C-responsive element (AP1) (31) at position -340 bp and octamer binding sequence (5`-ATTCAAAAT-3`) for POU domain family of transcriptional factors (32) at position -394 bp (Fig. 1B). In addition, we also demonstrated that luciferase activity caused by p(-224/+52)Luc transfection in growing R3T3 cells was still lower (approximately 80%, p < 0.05) than that of confluent R3T3 cells transfected by same expression vector construct (Fig. 2, A and B). This result suggests the existence of an additional mechanism for positive regulation of the AT(2) receptor gene expression in these cells from growing to quiescent state. Alternatively, there exists another weak negative regulatory element between the positions -224 and +52.

We focused on the molecular mechanism of negative regulation of AT(2) receptor expression during R3T3 cell growth by the putative negative regulatory elements located between -453 and -225 bp. To examine which sequences are the specific cis DNA elements involved in the negative regulation, we performed DNase footprinting analysis. We observed two distinct protected regions as shown in Fig. 3, A and B, and more clear protected regions were obtained with a higher concentration of nuclear extracts, suggesting that these sites are specifically protected by nuclear proteins. However, there were no significant differences in protected regions and their intensities between growing and confluent cells. Interestingly, these regions contain putative octamer binding motif and IRF binding sequence, respectively.

To examine these protected regions, which are in fact involved in the negative regulation of AT(2) receptor expression in growing R3T3 cells, we made double-stranded oligonucleotides corresponding to these protected sequences (see ``Experimental Procedures'') and delivered these oligonucleotides to R3T3 cells to act as ``decoy.'' This assay is based on the competition between the cis elements present in an endogenous target gene and exogenously added oligonucleotides corresponding to the cis sequence. This method seems advantageous compared to performing the further deletion of the promoter region and examining the effects of putative sequences on luciferase or CAT expression because we can observe the direct effects of candidate cis elements on the AT(2) receptor binding and expression. To deliver the oligonucleotides efficiently into R3T3 cells, we employed the HVJ-liposome delivery method(26, 27) . By this transfer method, we observed that nuclei of more than 90% of the cells were stained with transfected fluorescein isothiocyanate-labeled oligonucleotides 1 h after transfection, and the stained cells continued up to at least 3 days.

Three days after transfection of oligonucleotides, AT(2) receptor binding was measured. As shown in Fig. 3, C and D, octamer binding oligonucleotide transfection did not affect AT(2) receptor binding both in growing and confluent R3T3 cells, suggesting that this sequence alone may not play an important role in AT(2) receptor expression in R3T3 cells. On the other hand, IRF binding oligonucleotide treatment increased the AT(2) receptor binding in growing R3T3 cells but not in confluent cells. However, mutant IRF binding decoy oligonucleotide treatment did not affect the AT(2) receptor binding, supporting the notion that this effect is specific for IRF binding. We also measured AT(2) receptor mRNA levels at 24 h after oligonucleotide treatment and observed that IRF binding decoy oligonucleotide increased AT(2) receptor mRNA in growing R3T3 cells only, whereas octamer binding oligonucleotide did not affect AT(2) receptor mRNA level, supporting the contention that the changes of AT(2) receptor expression are transcriptionally regulated (Fig. 3E). These results indicate that IRF binding sequence may play a critical role in the negative regulation of AT(2) receptor in growing R3T3 cells.

Next, we examined whether specific nuclear transcriptional proteins recognize this sequence. Gel mobility shift assay using IRF binding oligonucleotide showed a specific retarded band both in growing and confluent R3T3 cells (Fig. 4A). This specific binding was abolished by the addition of C1 oligomer(25) , which is known to bind specifically to IRFs. Furthermore, as shown in Fig. 4B, we demonstrated the specific binding to P-labeled C1 oligomer that was competed by the IRF binding oligonucleotide, supporting that IRFs recognize this region. IRF-1 and IRF-2 are structurally related, particularly in their amino-terminal regions, which confer DNA binding specificity, and they recognize the same DNA sequence elements(12, 14) . IRF-1 and IRF-2 bind to the promoters of IFN-alpha, IFN-beta, and several IFN-inducible genes and modulate these gene expressions(12, 13, 14, 15, 16, 17) . Gene transfection studies have demonstrated that these two factors are mutually antagonistic; IRF-1 activates transcription whereas IRF-2 inhibits it(14, 15) .


Figure 4: IRF binding to AT(2) receptor promoter and expression in R3T3 cells. A, gel shift analysis of IRF to P-labeled IRF binding oligonucleotide with the nuclear extracts prepared from R3T3 cells in growing (lanes2-5, 5, 10, 15, and 15 µg of protein, respectively) and confluent states (lanes6-9, 5, 10, 15, and 15 µg of protein, respectively). Lanes5 and 9 were carried out with 100-fold molar excess of C1 oligomer (AAGTGA)(4)(25) as competitor. Lane1, without nuclear extract. B, gel shift analysis of IRF to P-labeled C1 oligomer with the nuclear extracts prepared from R3T3 cells in growing (lanes2-4, 5, 10, and 10 µg of protein, respectively) and confluent states (lanes5-7, 5, 10, and 10 µg of protein, respectively). Lanes4 and 7 were carried out with 100-fold molar excess of IRF binding oligonucleotide as competitor. Lane1, without nuclear extract. C, RT-PCR analysis of IRF-1 (leftpanel), IRF-2 (middlepanel), and beta-actin expressions (rightpanel) in R3T3 cells. Total RNA (lanes1 and 4, 0.12 µg; lanes2 and 4, 0.24 µg; lanes3 and 5, 0.36 µg) prepared from growing (lanes1-3) and confluent (lanes4-6) R3T3 cells was subjected to RT-PCR. The lanes on the rightsides show HaeIII-digested X marker. D, gel shift analysis of IRF to P-labeled IRF binding oligonucleotide with the nuclear extracts (15 µg) prepared from R3T3 cells in growing and confluent states with 2 µg of IRF-1 or IRF-2 antibody (Santa Cruz Biotechnology). Arrow indicates IRF binding.



Accordingly, we hypothesized that IRF-2 may act as silencer for AT(2) receptor expression in R3T3 cells, and IRF-1 enhances AT(2) receptor expression by interacting with the AT(2) receptor promoter region. To test this hypothesis, we transfected antisense oligonucleotides for IRF-1 and IRF-2 into R3T3 cells and observed that IRF-2 antisense enhanced the AT(2) receptor binding both in growing and confluent R3T3 cells (Fig. 5, C and D), and sense oligonucleotide for IRF-2 did not influence the AT(2) receptor binding. On the other hand, IRF-1 antisense oligonucleotide attenuated the AT(2) receptor binding in confluent R3T3 cells but had no effect in growing cells (Fig. 5, A and B). These changes in AT(2) receptor binding levels in response to antisense treatment were accompanied by changes in the AT(2) receptor mRNA levels (Fig. 5E), supporting further that IRF-1 and IRF-2 act as transcription regulators for AT(2) receptor expression. These results suggest that IRF-2 binding decreases the AT(2) receptor level both in growing and confluent R3T3 cells, whereas IRF-1 binding counteracts IRF-2 effect by competing the binding of IRF-2 in the confluent R3T3 cells. Consistent with this result, we observed that IRF-1 mRNA expression was more abundant in confluent cells than growing cells, whereas IRF-2 expression did not change with R3T3 cell growth (Fig. 4C). We also demonstrated that IRF binding in growing cells was mainly with IRF-2, whereas the IRF binding in confluent cells was with both IRF-1 and IRF-2 (Fig. 4D). These data suggest that the ratio of IRF-1 and IRF-2 may determine the AT(2) receptor expression associated with the growth of R3T3 cells. In addition to IRF family proteins such as IRF-1 and IRF-2, elucidation of the roles of other transcription factors such as interferon consensus sequence binding protein (20) may provide further understanding of the mechanism of growth-regulated AT(2) receptor expression.


Figure 5: AT(2) receptor binding (A-D) and expression (E) by the treatment of antisense oligonucleotides for IRF-1 and IRF-2. Antisense oligonucleotides for IRF-1 (A and B) and IRF-2 (C and D) were transfected into R3T3 cells in growing state (A and C) and confluent state (B, D). The AT(2) receptor binding in untreated cells is taken as 100%, respectively. The values were expressed as mean ± S.D. obtained from eight different cell culture wells. E, total RNA was prepared from cultured R3T3 cells 24 h after oligonucleotide treatment. RNA (20 µg/lane) was separated by electrophoresis, and hybridization was carried with a probe for the AT(2) receptor or for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a control. The result is a representative of data obtained in three different experiments.



IRF-1 manifests antiproliferative properties(33, 34) . Taniguchi and colleagues (33) have shown that the IRF-1/IRF-2 expression ratio oscillates throughout the cell cycle of NIH 3T3 cells, with IRF-1 expression at its highest when cells are growth-arrested owing to serum starvation. Furthermore, overexpression of the repressor IRF-2 in these cells leads to cell transformation and increased tumorgenicity, and this phenotype can be reverted by the concomitant overexpression of IRF-1(35) . IRF-2 is also reported to be more abundant in growing cells than IRF-1, but after stimulation by IFN or viruses, the amount of IRF-1 increases relative to IRF-2(36) . Interestingly, in addition to the IFN system, IRF binding consensus element was recently identified in inducible nitric oxide synthase gene promoter region and IRF-1 is essential for inducible nitric oxide synthase activation in murine macrophages(19) . In this paper, we report that IRFs may regulate the AT2 receptor expression during cell growth and that AT(2) receptor is one of the target genes of IRF system.

Janiak et al.(37) has shown that CGP42112A, an AT(2) receptor ligand suspected to be an AT(2) receptor agonist, attenuates neointimal development after vascular injury. We also observed that overexpression of AT(2) receptor in balloon-injured rat carotid artery attenuates the neointimal formation(38) . Daud et al.(39) reported that the granulosa cells of the atretic ovarian follicles, with an event dependent on apoptosis, express high levels of Ang II receptors, and Pucell et al.(6) also reported that the follicular granulosa cell Ang II receptors are the AT(2) receptor. Our preliminary results demonstrated that Ang II enhanced apoptotic changes in confluent R3T3 cells(40) . These results suggest that the AT(2) receptor exerts antigrowth effects on cell growth and may induce apoptosis in some cells. Tanaka et al.(18) recently reported that IRF-1 may be a critical determinant of oncogene-induced cell transformation or apoptosis in mouse embryonic fibroblast. Taken together, these results provide us the hypothesis that IRFs regulate cell growth and/or differentiation by modulating AT(2) receptor expression, thereby supporting a role for this receptor in these processes. Our results also will give further insights into the role of IRF-1/IRF-2 in the regulation of cell growth.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants HL46631, HL35252, HL35610, HL48638, and HL07708, by the American Heart Association Bugher Foundation Center for Molecular Biology in the Cardiovascular System, and by a grant from Ciba-Geigy, Basel Switzerland. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank[GenBank].

§
Recipient of National Institutes of Health MERIT Award HL35610. To whom correspondence should be addressed: Falk Cardiovascular Research Center, Stanford University School of Medicine, 300 Pasteur Dr., Stanford, CA 94305-5246. Tel.: 415-723-5013; Fax: 415-725-2178.

(^1)
The abbreviations used are: Ang II, angiotensin II; AT(1) and AT(2), angiotensin II type 1 and type 2; IRF, interferon regulatory factor; IFN, interferon; HVJ, hemagglutinating virus of Japan; bp, base pair(s); RT-PCR, reverse transcription-polymerase chain reaction.


ACKNOWLEDGEMENTS

We gratefully acknowledge Dr. M. de Gasparo for CGP42112A, Dr. D. Dudley for R3T3 cells, and M. Hing for secretarial assistance.


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