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(Circulation Research. 2000;86:233.)
© 2000 American Heart Association, Inc.

Molecular Medicine

Interferon- Induces AT₂ Receptor Expression in Fibroblasts by Jak/STAT Pathway and Interferon Regulatory Factor-1

Masatsugu Horiuchi, Wataru Hayashida, Masahiro Akishita, Seiichiro Yamada, Jukka Y. A. Lehtonen, Kouichi Tamura, Laurent Daviet, Yuquing E. Chen, Meiko Hamai, Tai-Xing Cui, Masaru Iwai, Yasuhiko Minokoshi

From Cardiovascular Research (M. Horiuchi, J.Y.A.L., K.T., L.D., Y.E.C.), Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, Mass; the Department of Medical Biochemistry (M. Horiuchi, S.Y., M. Hamai, T.-X.C., M.I., Y.M.), Ehime University School of Medicine, Ehime, Japan; the Department of Medicine (W.H.), Graduate School of Medicine, Kyoto University, Kyoto, Japan; and the Department of Geriatric Medicine (M.A.), Graduate School of Medicine, University of Tokyo, Tokyo, Japan.

Correspondence to Masatsugu Horiuchi, MD, PhD, Department of Medical Biochemistry, Ehime University School of Medicine, Shitsukawa, Shigenobu, Onsen-gun, Ehime 791-0295, Japan. E-mail horiuchi{at}m.ehime-u.ac.jp

	Abstract

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Abstract—The expression of angiotensin II type 2 (AT₂) receptor is closely associated with cell growth, differentiation, and/or injury. We examined the effect of interferon (IFN)- on AT₂ receptor expression in mouse fibroblast R3T3 cells and demonstrated that IFN- treatment increased the expression of AT₂ receptor mRNA as well as its binding. Interferon regulatory factor (IRF)-1 was induced in mouse fibroblast R3T3 cells after IFN- stimulation, and electrophoretic mobility shift assay showed an increase in IRF-1 binding with the IRF-specific binding sequence in the AT₂ receptor gene promoter region after IFN- stimulation. The IRF-1 gene promoter contains an IFN-–activated sequence (GAS) motif for possible binding of signal transducer(s) and activator(s) of transcription (STAT). Indeed, in R3T3 cells, IFN- treatment resulted in rapid activation of Janus kinase (Jak) 1, Jak2, and STAT1 via tyrosine phosphorylation. Electrophoretic mobility shift assay with the GAS probe revealed increased STAT1 binding to the IRF-1 gene promoter in response to IFN- stimulation. Transfection of GAS-binding oligonucleotides inhibited the effect of IFN- on IRF-1 production, resulting in the AT₂ receptor trans-activation. Taken together, our data show that IFN- upregulates AT₂ receptor expression in R3T3 cells via the activation of the intracellular Jak/STAT pathway and production of IRF-1.

Key Words: angiotensin • cytokine • receptor • transcription

	Introduction

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Since the successful cloning of the second type of angiotensin (Ang) II receptor, designated the AT₂ receptor,^{1 2} the function and cellular signaling of this receptor have been under active investigation. Accumulating evidence reveals that this receptor acts as an antagonist to the classic Ang II type 1 (AT₁) receptor^{3 4} ; ie, the AT₂ receptor exerts antigrowth, antihypertrophic, and proapoptotic effects.^{5 6 7 8 9 10 11 12 13} Because of advances in our understanding of the in vitro and in vivo functions of the AT₂ receptor, it can be hypothesized that the effect of Ang II is regulated by the balance of the expression of AT₁ and AT₂ receptors in target tissues, thereby contributing to the pathogenesis of cardiovascular diseases and consequent remodeling.¹⁴

The expression of AT₂ receptors in vasculature is enhanced in certain pathological conditions, such as vascular injury and vascular inflammation, including the inflamed tissue surrounding the injured artery.^{5 15 16} Myocardial AT₂ receptor expression has also been shown to be increased in experimental myocardial infarction in both infarcted and noninfarcted areas¹⁷ and in the rat hypertrophied heart¹⁸ as well as in the failing human heart^{19 20} ; these findings suggest that the AT₂ receptor plays an important role in cardiovascular remodeling. Moreover, in failing Bio14.6 cardiomyopathic hamster hearts, AT₂ receptor expression has been reported to be increased in cardiac fibroblasts in fibrous regions.²¹ AT₂ receptor expression is also enhanced in skin wounds.²² These results suggest that AT₂ receptor expression is closely associated with cell growth and differentiation and, furthermore, that the expression of this receptor is regulated by growth factors and cytokines.

To elucidate the molecular mechanism of growth-regulated AT₂ receptor expression, we cloned the mouse AT₂ receptor genomic DNA and studied its promoter function in mouse fibroblast R3T3 cells.²³ We have identified the interferon regulatory factor (IRF) binding motif in a negative regulatory region between positions -453 and -225 and demonstrated that the expression of AT₂ receptors in these cells is transcriptionally regulated by the competitive binding of 2 related IRFs (IRF-1 and IRF-2).²³ Ichiki et al²⁴ have also examined the effects of several growth factors on the expression of AT₂ receptor mRNA in R3T3 cells and observed that serum (10%), fibroblast growth factor, phorbol ester, or lysophosphatidic acid reduced AT₂ receptor mRNA expression, whereas interleukin-1ß or insulin enhanced the expression, thereby suggesting that AT₂ receptor expression is modulated by multiple growth factors in both positive and negative directions.

The genes for IRF-1 and IRF-2 are both virus and interferon (IFN) inducible.²⁵ The IRF-1 gene is also induced by other cytokines, such as tumor necrosis factor-, interleukin-1, interleukin-6, leukemia inhibitory factor, and prolactin.^{26 27 28} IFNs are well known as regulators of cell growth and differentiation. These results led us to examine the effect of IFN on AT₂ receptor expression. We focused on IFN-, because it is known to be involved in the pathogenesis and consequent remodeling of cardiovascular diseases,^{29 30 31 32 33} and we also examined the enhanced AT₂ receptor expression in these diseased states,^{5 14 15 16 17 18 19 20 21} which suggests a close causal association of IFN- with the AT₂ receptor. We investigated the effect of IFN- on AT₂ receptor expression in R3T3 cells and demonstrated that this cytokine upregulates AT₂ receptor expression by activating the Janus kinase (Jak)/signal transducer(s) and activator(s) of transcription (STAT) pathway and, thereby, inducing the transcriptional factor IRF-1.

	Materials and Methods

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Cell Culture
Mouse R3T3 cells were cultured as previously described.^{11 23 34 35} Vascular smooth muscle cells (VSMCs) were isolated from adult Sprague-Dawley rat thoracic aortas.^{10 13}

Transfection of Oligonucleotides to Cultured Cells
Oligonucleotides used as "decoys" include the following: IFN-–activated sequence (GAS) binding,³⁶ 5'-AGCCTGATTTCCCCG-AAATGA-3'; scrambled GAS binding, 5'-GACGTCGACCTA-TCAGATACT-3'. Oligonucleotides were annealed to complementary sequences and used as double-stranded oligonucleotides. Transfection was performed with LipofectAMINE Reagent (GIBCO-BRL, Life Technologies), 3:1 (wt/wt) liposome formulation of the polycationic lipid 2,3-dioleoyloxy-N-[2(sperminecarboxamide)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate and the neutral lipid dioleoyl phosphatidylethanolamine as previously described.¹¹ One day after transfection, the transfected R3T3 cells and VSMCs were used for the following experiments.

AT₂ Receptor Whole-Cell Binding Assay
AT₂ receptor binding was assessed as previously described.^{2 11 23}

Northern Blot Analysis
RNA (20 µg) was separated by 1% formaldehyde–agarose gel electrophoresis and transferred onto a nylon membrane (Hybond N⁺, Amersham Life Sciences). Hybridization with ³²P-labeled probes was carried out with (1) a HindIII-NsiI fragment of mouse AT₂ receptor cDNA,³⁷ (2) mouse IRF-1 and IRF-2 cDNA,¹¹ or (3) a 0.78-kb PstI-XbaI fragment of human GAPDH in Rapid-hyb buffer (Amersham Life Sciences).

Preparation of Nuclear Extracts
Nuclear extracts were prepared as previously reported.²³

Electrophoretic Mobility Shift Assay
Electrophoretic mobility shift assay (EMSA) was performed as previously described.^{11 23} We used the following oligonucleotides: IRF-binding oligonucleotide probe, 5'-GAAAAAGAGAAAGAAA-AAAGGAAAAGGAAAATTCTGCTAAAAAGGATA-3'^{11 23} ; C1 oligomer probe, 5'-(AAGTGA)₄-3'³⁸ ; and GAS oligonucleotide probe, 5'-AGCCTGATTTCCCCGAAATGA-3'.³⁶ Oligonucleotides were annealed to complementary sequences and used as double-stranded oligonucleotides. To examine the specificity of the binding, the nuclear extract was preincubated for 30 minutes with 5 µg of antibody to IRF-1 or STAT1 (Santa Cruz Biotechnology Inc) before the reaction with the ³²P-labeled probe.

Analysis of Activation of Jak/STAT Pathway
For the analysis of Jak/STAT pathway activation, the R3T3 cells and VSMCs, after 12 hours of serum removal, were stimulated with IFN- (100 U/mL), and the cell lysates were prepared as previously described.⁴ The cell lysate (200 µg) was incubated with 10 µg of anti-phosphotyrosine antibody (clone 4G10, Upstate Biotechnology) at 4°C overnight and precipitated by the addition of 20 µL of protein A/G-agarose (Santa Cruz Biotechnology Inc) for 2 hours. The immunoprecipitate was run on 8% SDS-PAGE, transferred to a nitrocellulose membrane (Hybond-ECL, Amersham Life Sciences), blotted with antibody to Jak1, Jak2, or STAT1/ß (Santa Cruz Biotechnology Inc), and detected by the enhanced chemiluminescence (ECL) method (Amersham Life Sciences).

Western Blot Analysis
The cell lysate was run on SDS-PAGE and blotted with antibody to IRF-1, nuclear factor-B (NF-B), or actin (Santa Cruz Biotechnology Inc). In addition, to assess nuclear translocation of STAT1, the nuclear extract was run on SDS-PAGE and blotted with antibody to STAT1 or NF-B (Santa Cruz Biotechnology Inc).

Statistical Analysis
ANOVA was used for statistical comparison of receptor binding. A value of P<0.05 was considered significant.

An expanded Materials and Methods section is available online at http://www.circresaha.org.

	Results

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AT₂ Receptor Expression After IFN- Stimulation
We initially examined the effect of IFN- on AT₂ receptor expression in R3T3 cells. As shown in Figure 1A, Northern blot analysis revealed an increase in AT₂ receptor mRNA after IFN- (100 U/mL) stimulation, and this increase persisted after 48 hours. Moreover, using a whole-cell receptor binding assay, we confirmed the increased density of AT₂ receptors in R3T3 cells 48 hours after IFN- treatment (Figure 1B).

View larger version (23K): [in this window] [in a new window]   Figure 1. Effect of IFN- on AT2 receptor mRNA expression (A) and AT2 receptor binding (B). Confluent R3T3 cells were stimulated with 100 U/mL of IFN-. A, Total RNA was prepared from cultured R3T3 cells at 6, 24, and 48 hours after IFN- treatment. RNA (20 µg/lane) was separated by electrophoresis, and hybridization was carried out with a probe for the AT2 receptor or for GAPDH as an internal control to standardize the amount of total RNA actually blotted onto the membrane. The result is representative of data obtained in 3 different experiments. B, AT2 receptor binding used 125I-labeled CGP 42112A, an AT2 receptor–specific ligand, 2 days after IFN- treatment [IFN- (+)]. Values are expressed as mean±SD obtained from 8 different cell culture wells. *P<0.01 compared with the value obtained from the cells without the treatment of IFN- [IFN- (-)].

IRF Binding to AT₂ Receptor Gene Promoter
We previously reported that IRF-1 binding to the IRF binding sequence in the AT₂ receptor-promoter region enhances AT₂ receptor expression.^{1 23} To examine whether IRF-1 is involved in this AT₂ receptor upregulation by IFN-, we studied IRF expression after IFN- treatment. Total RNA was prepared from the IFN-–treated cells, and mRNA expression of IRFs was analyzed by Northern blotting. IRF-1 mRNA expression increased after IFN- stimulation, whereas the level of IRF-2 mRNA did not change up to 24 hours after IFN- treatment (Figure 2A). This result was confirmed by an increase in IRF-1 protein level on Western blotting (Figure 2B). We next performed EMSA with the use of nuclear extracts prepared from IFN-–treated R3T3 cells and a ³²P-labeled IRF-binding oligonucleotide probe, which spans the IRF binding sequence in the AT₂ receptor-promoter region,²³ and observed a time-dependent increase in the complex formation (Figure 3). The addition of 50-fold excess unlabeled IRF-binding oligonucleotide and C1 consensus oligomer as competitors abolished the IFN-–induced IRF binding. Incubation of nuclear extract with antibody to IRF-1 decreased the binding. These results suggest that IFN- increases IRF-1 binding to the IRF binding element in the AT₂ receptor-promoter region.

View larger version (28K): [in this window] [in a new window]   Figure 2. Effect of IFN- on IRF mRNA expression (A) and IRF-1 protein level (B). A, Total RNA was prepared from cultured R3T3 cells 6 or 24 hours after IFN- (100 U/mL) treatment. RNA (20 µg/lane) was separated by electrophoresis and hybridized sequentially with a probe for IRF-1 and IRF-2 and also for GAPDH as internal control to standardize the amount of total RNA actually blotted onto the membrane. The figure is representative of data obtained in 4 different experiments. IRF-1 indicates 1-day autoradiographic exposure; IRF-2, 2-day autoradiographic exposure. B, Cells were harvested 6, 12, 24, or 48 hours after IFN- treatment, and cell lysates were prepared. Cell lysates (30 µg) were resolved by 12% SDS-PAGE, electroblotted onto nitrocellulose membrane, and immunoblotted with antibody to IRF-1 (top) or actin (bottom). Antibodies were detected by horseradish peroxidase–linked secondary antibody with use of an ECL system.

View larger version (91K): [in this window] [in a new window]   Figure 3. Effect of IFN- on IRF-1 binding to IRF binding sequence in the AT2 receptor-promoter region. EMSA was performed with 32P-labeled IRF-binding oligonucleotide with the nuclear extracts (10 µg) prepared from R3T3 cells treated with IFN- (100 U/mL) for 6, 12, 24, or 48 hours. EMSA was also carried out with 50-fold molar excess of IRF-binding oligonucleotides (IRFEs) or C1 oligomer (AAGTGA)4 as competitor or with 5 µg of anti–IRF-1 antibody. Left lane shows the result without nuclear extract.

Activation of Jak/STAT Pathway and STAT1 Binding to IRF-1 Gene Promoter
One potential GAS was found within the IRF-1 promoter.³⁶ IFN- is known to activate Jak1, Jak2, and, subsequently, the STAT1 transcriptional factor.³⁹ We postulated that IFN-–activated STAT1 acts as an activator of the transcription of IRF-1 binding with the GAS motif in the IRF-1 gene. To explore this possibility, we studied the activation of the Jak/STAT signaling pathway. Figure 4A shows tyrosine phosphorylation of Jak1, Jak2, and STAT1/ß at baseline and after IFN- stimulation. Both Jak and STAT1 were activated rapidly by IFN- as a result of tyrosine phosphorylation. Figure 4B shows Western blot analysis of the nuclear translocation of STAT1/ß in response to IFN- stimulation. STAT1 was shown to translocate into nuclei 30 minutes after stimulation and remained detectable 24 hours later. The NF-B binding sequence was also reported in the IRF-1 promoter region.³⁶ Therefore, we examined the effect of IFN- on NF-B but found no significant increase in this transcriptional factor in the nucleus or in the cytosol (Figure 4C).

View larger version (30K): [in this window] [in a new window]   Figure 4. Effect of IFN- on tyrosine phosphorylation of Jak1, Jak2, and STAT (A), nuclear translocation of STAT1 (B), and NF-B (C). A, R3T3 cells were treated with IFN- (100 U/mL) for 5, 15, or 30 minutes. Cell lysates were prepared from these cells, immunoprecipitated with anti-phosphotyrosine antibody, separated by 8% SDS-PAGE, transferred to nitrocellulose, and immunoblotted with antibody to Jak1, Jak2 or STAT1. IP indicates immunoprecipitation. B, Nuclear extracts were prepared from the IFN- (100 U/mL)–treated R3T3 cells for the indicated times. Nuclear extract (20 µg) was separated by 8% SDS-PAGE, transferred to nitrocellulose, and immunoblotted with antibody to STAT1. C, Nuclear extract (30 µg) or cell lysate (30 µg) was separated by 10% SDS-PAGE, transferred to nitrocellulose, and immunoblotted with antibody to p65 NF-B. D, R3T3 cells were treated with IFN- (100 U/mL). Cell lysates were prepared from these cells, separated by 8% SDS-PAGE, transferred to nitrocellulose, and immunoblotted with antibody to actin.

We then performed EMSA with the use of the ³²P-labeled GAS oligonucleotide probe to study STAT1 binding with the GAS motif in the IRF-1 gene promoter. As shown in Figure 5A, IFN- stimulation induced nuclear protein binding to the GAS probe, which was diminished by the addition of monoclonal antibody to STAT1. This finding documents an interaction between STAT1 and the IRF-1 gene promoter.

View larger version (64K): [in this window] [in a new window]   Figure 5. Effect of IFN- on GAS binding (A) and specificity of GAS-binding oligonucleotide (B). A, EMSA was performed with 32P-labeled GAS-binding oligonucleotide with the nuclear extracts (20 µg) prepared from R3T3 cells treated with IFN- (100 U/mL) for 0.5, 1, or 3 hours. EMSA was also carried out with 5 µg of antibody to STAT1. Left lane shows the result without nuclear extract. B, EMSA was performed with 32P-labeled GAS-binding oligonucleotide with the nuclear extract (20 µg) prepared from R3T3 cells treated with IFN- (100 U/mL) for 3 hours with 100-fold molar excess of GAS oligonucleotide or scrambled GAS oligonucleotide as a competitor. Left lane shows the result without nuclear extract.

Role of STAT and IRF-1 in AT₂ Receptor trans-Activation by IFN-
We have demonstrated that IFN- stimulation enhanced STAT1 binding to GAS in the IRF-1 promoter and consequently enhanced IRF-1 production. To confirm that this pathway indeed trans-activates AT₂ receptor expression, we tried to inhibit the endogenous GAS binding with IFN-–activated STAT1 and examine AT₂ receptor binding. For this purpose, we transfected a double-stranded GAS-binding oligonucleotide, which spans GAS in the IRF-1 promoter region, as a decoy. As a control, we transfected R3T3 cells with scrambled double-stranded GAS oligonucleotides. To examine the specificity of these oligonucleotides, we performed EMSA with 100-fold excess of these oligonucleotides as competitors. Addition of scrambled GAS oligonucleotides to nuclear extracts prepared from IFN-–treated cells did not influence the binding of STAT1 to ³²P-labeled GAS oligonucleotides, whereas the addition of GAS oligonucleotides inhibited the binding of STAT1 to ³²P-labeled GAS probes (Figure 5B).

One day after GAS decoy or scrambled oligonucleotide transfection, the transfected cells were stimulated with IFN- (100 U/mL), and the cell lysates were prepared 12 hours after the stimulation. Western blotting showed that GAS decoy oligonucleotide transfection diminished IFN-–induced IRF-1 production, whereas scrambled GAS oligonucleotide transfection did not influence IRF-1 production (Figure 6A). We prepared nuclear extracts 12 hours after IFN- stimulation and examined their binding with ³²P-labeled IRF-1 probe. Consistent with the result of Western blotting, we demonstrated that GAS decoy oligonucleotide transfection diminished IRF-1 binding, whereas scrambled GAS oligonucleotide did not (Figures 7). Densitometric analysis using an image scanner (Arcus II, Agfa) and NIH image software revealed that IRF-1 binding after GAS decoy treatment is 15% of that without GAS decoy treatment (Figure 7B). On the other hand, nuclear translocation of STAT1 measured 3 hours after IFN- stimulation was not affected by the oligonucleotide transfection (Figure 6B). Finally, we examined the effect of IFN- on AT₂ receptor binding in these transfected cells and found a similar increase in AT₂ receptor binding in untransfected and scrambled GAS oligonucleotide–transfected cells (Figure 8A). In contrast, the IFN-–mediated AT₂ receptor increase was diminished in GAS oligonucleotide–transfected cells. Moreover, we examined the effect of IFN- on AT₂ receptor binding in adult rat VSMCs, which express very low levels of endogenous AT₂ receptors, and observed that IFN- treatment increased the expression of AT₂ receptor binding in these cells (Figure 8B). Transfection of GAS-binding oligonucleotide inhibited the effect of IFN- on the increase in AT₂ receptor binding in VSMCs. The effects of IFN- on tyrosine phosphorylation of Jak1, Jak2, and STAT1 and on the expression of IRF-1 in VSMCs are shown in Figure 8C.

View larger version (23K): [in this window] [in a new window]   Figure 6. Effect of the transfection of decoy oligonucleotide that spans GAS present in the IRF-1 promoter region on IRF-1 protein level (A) and the nuclear translocation of STAT1 (B). Two double-stranded oligonucleotides (GAS and scrambled GAS-binding "decoy" oligonucleotides) (see Materials and Methods) were transfected into confluent R3T3 cells; 1 day later, the cells were stimulated with IFN- (100 U/mL) for 12 hours (for IRF-1 production) or 3 hours (for STAT1 nuclear translocation). A, Cell lysates (30 µg) were resolved by 12% SDS-PAGE, electroblotted onto nitrocellulose membrane, and immunoblotted with antibody to IRF-1. B, Nuclear extract (20 µg) was separated by 8% SDS-PAGE, transferred to nitrocellulose, and immunoblotted with antibody to STAT1.

View larger version (43K): [in this window] [in a new window]   Figure 7. Effect of the transfection of GAS oligonucleotide as decoy on IRF-1 binding to IRF binding sequence in the AT2 receptor-promoter region. GAS and scrambled GAS-binding "decoy" oligonucleotides were transfected into confluent R3T3 cells 1 day before IFN- treatment. A, EMSA was performed with 32P-labeled GAS-binding oligonucleotide with the nuclear extracts (20 µg) prepared from R3T3 cells treated with IFN- (100 U/mL) for 12 hours. Left lane shows the result without nuclear extract. Representative data are from 4 separate experiments. B, The values of densitometric measurements of binding are expressed as mean±SEM of 4 different experiments. *P<0.01 compared with the value obtained from the cells without the treatment of GAS decoy and with the treatment of IFN-.

View larger version (27K): [in this window] [in a new window]   Figure 8. Effect of the transfection of GAS oligonucleotide decoy on AT2 receptor binding in R3T3 cells (A) and VSMCs (B) and the effect of IFN- on tyrosine phosphorylation of Jak1, Jak2, and STAT and the expression of IRF-1 in VSMCs (C). A and B, R3T3 cells (A) or VSMCs (B) were stimulated with IFN- 1 day after oligonucleotide transfection. AT2 receptor binding was determined 2 days after IFN- (100 U/mL) treatment. Values are expressed as mean±SD obtained from 6 different culture wells. *P<0.01 compared with the value obtained from the cells without IFN- treatment. C, VSMCs were treated with IFN- (100 U/mL) for 5, 15, or 30 minutes. Cell lysates were prepared from these cells, immunoprecipitated with anti-phosphotyrosine antibody, separated by 8% SDS-PAGE, transferred to nitrocellulose, and immunoblotted with antibody to Jak1, Jak2, or STAT1. The values of densitometric measurements of tyrosine phosphorylation of Jak1, Jak2, or STAT1 and the IRF-1 expression are expressed as mean±SEM of 4 different experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Expression of the AT₂ receptor is known to be growth and developmentally regulated.^{40 41} The possible positive and negative regulation of AT₂ receptor expression by several growth factors and the cytokines has been suggested^{24 42 43 44 45} ; however, the molecular and cellular mechanisms of the regulation of this receptor gene are unclear. We have shown that the growth-dependent regulation of AT₂ receptor expression in mouse fibroblast R3T3 cells is regulated by an interplay of the transcriptional factors IRF-1 and IRF-2,²³ which have been shown to recognize the same DNA sequence elements.^{46 47} IRF-1 activates transcription of IFN-inducible genes, whereas IRF-2 inhibits it.^{25 26 27 47 48 49 50} Recently, competitive binding of IRF-1 and IRF-2 was reported to play a critical role in determining growth, transformation, and apoptosis of cells.^{51 52 53 54 55} We have demonstrated, in R3T3 cells, that IRF-2 is constitutively expressed in the proliferating state and suppresses AT₂ receptor gene expression. However, in confluent R3T3 cells, IRF-1 is rapidly induced and upregulates AT₂ receptor expression.²³ We also showed that the increase in the IRF-1 in apoptotic R3T3 cells, after serum withdrawal, trans-activates the AT₂ receptor and results in enhanced AT₂ receptor–mediated apoptosis.¹² IRF-1 also stimulates interleukin-1ß–converting enzyme⁵¹ and inducible nitric oxide synthase,^{55 56} which are known to be apoptosis mediators. These results support the notion that IRF-1 plays physiological roles in "growth"-regulated AT₂ receptor expression.

The increase in AT₂ receptor expression has been reported in some disease states.^{5 14 15 16 17 18 19 20 21 22 40 41} To define the role of the endogenous AT₂ receptor in vascular disease, we applied the mouse model of vascular disease induced by polyethylene cuff placement¹⁶ by using wild-type and AT₂ receptor knockout mice.⁵⁷ Our preliminary experiments with this model of cuff-wrapped mouse femoral artery revealed upregulation of AT₂ receptor expression preceded by an increase in inflammatory cytokines and IRF-1 and showed that whereas AT₂ receptor knockout mice and wild-type mice both developed neointima in the femoral artery, the lesion was twice the size in knockout mice as in wild-type mice.¹⁶ We hypothesize that the inflammatory response to vascular pathological stimuli is the release of cytokines, which induces the expression of IRF-1 in cytokine-responsive cells. IFN- is one of the well-known cytokines that play important roles in the pathogenesis of cardiovascular diseases and in the subsequent remodeling process.^{29 30 31 32 33}

On the basis of these previous observations, we studied the effect of IFN- on AT₂ receptor expression. Our data have demonstrated that IFN- enhances IRF-1 mRNA and protein expression in R3T3 cells. Moreover, we have demonstrated increased binding of IRF-1 to the IRF binding site in the AT₂ receptor-promoter region as well as an increase in AT₂ receptor mRNA and AT₂ receptor binding. We have also examined the effect of IFN- on AT₂ receptor binding in adult rat VSMCs and observed that IFN- treatment increases the expression of AT₂ receptor binding in these cells, suggesting that similar pathways are involved in rat adult VSMCs. IRFs are known to be induced by IFNs and other cytokines and also by viral infection.^{25 26} A previous study found that the IRF gene promoter contains highly GC-rich sequences and consensus binding sequences for several transcriptional factors.³⁶ Sequence comparison between human and mouse IRF-1 promoters reveals extensive homology from -228 to +38,³⁸ which suggests that this is an important region in virus or IFN stimulation.³⁶ In fact, one potential GAS was found at -122 to -112.³⁶ IFN- is known to activate Jak and STAT1 by tyrosine phosphorylation.³⁹ In the present study, we examined the activation of the Jak/STAT pathway after IFN- stimulation in R3T3 cells and observed that IFN- activates Jak1, Jak2, and, consequently, STAT1/ß by phosphorylating their tyrosine residues. The activated STAT1 then translocates into the nuclei of the R3T3 cells. A gel-shift assay using the GAS probe revealed increased binding of nuclear protein, which was abolished by addition of the competitor and antibody to STAT1. These results suggest that increased GAS-STAT1 binding contributes to IRF-1 production and results in the trans-activation of the AT₂ receptor. Indeed, our experiment revealed that transfection of GAS-binding oligonucleotides attenuated the IFN-–mediated increase in IRF-1 production and IRF-1 binding to the AT₂ receptor promoter region and inhibited the upregulation of AT₂ receptor expression via IFN- without affecting the IFN-–induced nuclear translocation of STAT1. Moreover, we could not find a potential GAS motif in the mouse AT₂ receptor-promoter region, which suggests that IFN-–activated STAT does not directly bind with the AT₂ receptor-promoter and does not trans-activate the AT₂ receptor.

The promoters of the IRF-1 gene possess NF-B binding elements.¹⁸ The role of NF-B in the expression of a wide range of signal-responsive genes has been well documented. Therefore, we examined the possibility that IFN- activates NF-B. We observed that IFN- did not influence nuclear and cytosolic NF-B contents. Moreover, Sims et al⁵⁸ have provided evidence that an IFN-– and IFN-–inducible novel element is located at -130, which suggests multiple regulatory elements induced by IFN within the promoter. The participation of these elements in IFN-–induced IRF-1 production in R3T3 cells has not been addressed in the present study. However, our results clearly support the notion that IFN-–activated STAT1 plays a pivotal role in IRF-1 production in R3T3 cells and contributes to the upregulation of AT₂ receptor expression.

IFN- has been shown not only to be involved in immunomodulatory and antiviral activities but also to exert antiproliferative effects on the cells.⁵⁹ In addition to the direct effect of IFN- on the cell growth-promoting signaling pathways, the present data suggest that the effect of IFN- may also be exerted via its modulation of the expression of other growth-regulatory receptors, such as the AT₂ receptor. Indeed, a recent study⁶⁰ reported that IFN- affects the mitogenic response to growth factors, such as epidermal growth factor, in certain types of the cells. Our results suggest that the complex interactions of IFN- with the expression of other growth-regulatory receptors, eg, the AT₂ receptor, are important for the integrated regulation of cell growth.

	Acknowledgments

 
This study was supported by National Institutes of Health grants HL-46631, HL-35252, HL-35610, HL-48638, HL-07708, and HL-58616; a grant from America Heart Association; a grant from the Longwood Foundation for Translational Research; and a grant from the Ministry of Education, Science, Sports, and Culture of Japan. We acknowledge M. Nomura for secretarial assistance.

Received August 2, 1999; accepted November 3, 1999.

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