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(Circulation Research. 1999;84:876-882.)
© 1999 American Heart Association, Inc.

Original Contribution

Stimulation of Different Subtypes of Angiotensin II Receptors, AT₁ and AT₂ Receptors, Regulates STAT Activation by Negative Crosstalk

Masatsugu Horiuchi, Wataru Hayashida, Masahiro Akishita, Kouichi Tamura, Laurent Daviet, Jukka Y. A. Lehtonen, Victor J. Dzau

From Cardiovascular Research, Department of Medicine, Harvard Medical School, Brigham and Women's Hospital, Boston, Mass.

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

	Abstract

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Abstract—Angiotensin II type 2 (AT₂) receptor exerts an inhibitory action on cell growth. In the present study, we report that the stimulation of AT₂ receptor in AT₂ receptor cDNA–transfected rat adult vascular smooth muscle cells (VSMCs) inhibited angiotensin II type 1 (AT₁) receptor–mediated tyrosine phosphorylation of STAT (signal transducers and activators of transcription) 1/ß, STAT2, and STAT3 without influence on Janus kinase. AT₂ receptor activation also inhibited the tyrosine phosphorylation of STAT1/ß induced by interferon-, epidermal growth factor, and platelet-derived growth factor. Similar effects of AT₂ receptor were observed in R3T3 fibroblast and mouse fetal VSMCs, which express endogenous AT₂ receptor. Moreover, AT₂ receptor inhibited serine phosphorylation of STAT1 and STAT3 via the inhibition of extracellular signal–regulated kinase (ERK) activation. Stimulation of AT₂ receptor inhibited the binding of STATs with sis-inducing element in c-fos promoter, resulting in decreased c-fos expression. Taken together, our results suggest that AT₂ receptor can crosstalk negatively with multiple families of growth receptors by inhibiting ERK and STAT activation.

Key Words: angiotensin • receptor • STAT • vascular smooth muscle cell

	Introduction

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The peptide angiotensin (Ang) II exerts hemodynamic, renal, and electrolyte as well as cardiovascular structural effects. Two distinct types of Ang II receptors have been cloned and are designated as type 1 (AT₁)^{1 2} and type 2 (AT₂) receptors.^{3 4} Most of the known effects of Ang II in adult tissues are attributable to the AT₁ receptor. In contrast, the AT₂ receptor is abundantly and widely expressed in fetal tissues and reexpressed in certain pathological conditions such as ovarian atresia, myocardial infarction, and vascular injury,^{5 6 7 8} suggesting that this receptor plays a pivotal role in differentiation and growth. Although both AT₁ and AT₂ receptors belong to the 7-transmembrane, G protein–coupled receptor family, recent evidences reveal that the functions of AT₁ and AT₂ receptors are antagonistic. AT₁ receptor promotes cell growth whereas AT₂ receptor mediates growth inhibition,^{7 9 10 11} differentiation,^{12 13} and/or apoptosis^{14 15 16 17} in vascular smooth muscle cells (VSMCs), cardiomyocytes, endothelial cells, neuronal cells (PC12W cells), and fibroblasts (R3T3 cells). Growth inhibitory effects of AT₂ receptor are unique in that this is a 7-transmembrane, G protein–coupled receptor that counteracts the growth action of other 7-transmembrane, G protein–coupled receptors as well as other classes of growth factor receptors.

It has been reported that these effects of AT₂ receptor are, at least partly, mediated by the activation of protein tyrosine phosphatase (PTPase), which results in the inactivation AT₁ receptor– and/or growth factor–activated mitogen-activated protein (MAP) kinase (p42 and p44 MAP kinases are known as extracellular signal–regulated kinases [ERKs]).^{7 11 14 16 18 19 20 21} Given the breadth of its interaction with multiple growth factor receptors, we hypothesize that the AT₂ receptor signaling must interact with other cellular signal transduction pathways in which tyrosine phosphorylation is involved.

STATs (signal transducers and activators of transcription) are initially identified as the primary mediators of interferon (IFN)-dependent signaling and now are known to be activated by a number of cytokines^{22 23} and growth factors,^{24 25} as well as AT₁ receptor,^{26 27 28} mediating their effects on cell growth.^{29 30 31} The binding of ligands to their receptors triggers tyrosine phosphorylation of STATs via activation of receptor-associated Jak (Janus kinase) family of tyrosine kinases (Jak1, Jak2, Jak3, and Tyk2).^{23 32 33} The function of STAT is also influenced by serine phosphorylation. Wen et al³⁴ reported that the phosphorylation of serine residues, potential ERK phosphorylation sites in STAT1 and STAT3, is induced by IFN- and platelet-derived growth factor (PDGF) and that maximal activation of transcription by STAT1 and STAT3 requires both tyrosine and serine phosphorylation of these proteins. Interestingly, recent studies have revealed that AT₁ receptor stimulation activates STAT transcription factor via tyrosine and serine phosphorylation through the activation of Jak^{28 31 35} and ERK,³⁶ respectively. Accordingly, we postulated in the present study that AT₂ receptor stimulation inactivates STAT transcription factors through tyrosine dephosphorylation as well as through inhibiting serine phosphorylation by the inhibition of ERK activation.

	Materials and Methods

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Cell Culture
Adult rat aortic VSMCs were isolated from Sprague-Dawley rat thoracic aorta, cultured, and used at passage 4 to 8 as previously reported.^{7 11} The AT₂ receptor cDNA was constructed into a plasmid pUC-CAGGS that has the cytomegalovirus enhancer and the chicken ß-actin promoter.^{3 7 11} AT₂ receptor expression vector or control vector (parental pUC-CAGGS) was transfected into the subconfluent VSMCs using cationic liposome-mediated gene transfer as previously reported.¹¹ After the transfection, the cells were fed with the medium containing 10% calf serum for 24 hours and then maintained in the defined serum-free medium containing 0.5 µmol/L insulin, 5 µg/mL transferrin, and 0.2 mmol/L ascorbate for an additional 2 days. The quiescent cells thus obtained were used for the subsequent experiments. Thoracic aortas were removed from mouse fetuses (FVB/N strain) (embryonic day 20) and microdissected free from the surrounding adventitial tissues. The aortas from individual animals were placed into separate individual test tubes and then digested in collagenase (1 mg/mL, type II; Sigma Chemical Co) in DMEM (GIBCO/BRL) for 30 minutes at 37°C. After trituration and centrifugation twice, the cells were seeded in 10-cm culture dishes and cultivated in DMEM supplemented with 10% FBS. Culture medium was changed every other day. At 10 to 14 days after primary culture, the cells were trypsinized and seeded for the following experiments. All experimental procedures were approved and carried out in accordance with the guideline of the Harvard Medical Area Standing Committee on Animals. R3T3 fibroblast cells were cultured as described.³⁷

Immunoprecipitation and Western Blot Analysis
The cells were stimulated with Ang II (Sigma), DuP753 (Merck & Co), PD123319 (Research Biochemicals International), IFN-, epidermal growth factor (EGF), or PDGF (GIBCO/BRL). In some experiments, the VSMCs were treated with PD98059 (New England Biolabs) 60 minutes before Ang II treatment. At the end of the stimulation, the cells were quickly washed twice with HEPES-buffered saline and frozen in liquid nitrogen. The cells were lysed in the lysis buffer (20 mmol/L Tris-HCl [pH 7.4], 150 mmol/L NaCl, 2.5 mmol/L EDTA, 1% (vol/vol) Triton X-100, 0.1% [wt/vol] sodium deoxycolate, 0.1% [wt/vol] SDS, 50 mmol/L NaF, 10 mmol/L Na₃P₂O₇, 10% glycerol, 1 mmol/L sodium orthovanadate, 10 µg/mL aprotinin, and 1 mmol/L phenylmethylsulfonyl fluoride), and the supernatant fraction was obtained as cell lysate by centrifugation at 12 000 rpm for 25 minutes at 4°C. The cell lysate was incubated with 10 µg of antiphosphotyrosine monoclonal antibody (Upstate Biotechnology) or antiphosphoserine antibody (ZYMED) at 4°C for 12 hours and precipitated by addition of 25 µL of protein A/G-agarose (Santa Cruz Biotechnology). The immunoprecipitate was resuspended in 2x Laemli sample buffer and run on the 8% SDS/PAGE. The proteins were then transferred to nitrocellulose membrane (Amersham), blotted with anti-STAT1, STAT2, STAT3, Jak2, or Tyk2 antibody (Santa Cruz Biotechnology), and detected by the enhanced chemiluminescence method (Amersham). Densitometric analysis was performed using an image scanner (Arcus II, Agfa) and NIH image software. All values are expressed as mean±SEM. Statistical significance was assessed by ANOVA followed by Scheffé test. P<0.05 was considered significant.

Preparation and Analyses of Nuclear Extract
Nuclear extract was prepared from VSMCs as previously reported.³⁷ For immunoblotting for STAT in the nuclear extract, nuclear protein (100 µg) was immunoprecipitated with 10 µg of antiphosphotyrosine or antiphosphoserine antibody and immunoblotted with anti-STAT antibodies. For electrophoretic gel mobility shift assay, the oligonucleotide probes for sis-inducing element (SIE) and mutant SIE were synthesized as follows^{26 27} : SIE, 5'-CAGTTCCCGTCAATC-3'; mutant SIE, 5'-CAGCCACCGTCAATC-3'. In addition, the AP-1 consensus oligonucleotide, 5'-CGCTTGATGAGTCAGCCGGAA-3', was also used for a competition assay. Complementary oligonucleotides were annealed for 2 hours, while the temperature descended from 80°C to 25°C. The double-stranded SIE and mutant SIE probes were labeled with [³²P]-ATP (3,000 Ci/mmol) (Amersham) and T4 polynucleotide kinase.³² P-labeled probe (30 000 cpm) was incubated for 30 minutes at room temperature with 10 µg of nuclear proteins and 1 µg of poly(dI:dC/dI:dC) and electrophoresed on 5% PAGE as previously described.³⁷ The gel was dried and exposed for autoradiography.

Northern Blot Analysis
RNA was extracted from the harvested cells by RNAzol B reagent (Tel-Test). Total RNA (20 µg) was electrophoresed on 1% formaldehyde-agarose gel and transferred to a nylon membrane (Amersham). The membrane was then blotted with ³²P-labeled 1.0-kb PstI fragment of murine c-fos probe and 0.78-kb PstI-XbaI fragment of a human GAPDH.

	Results

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Tyrosine Dephosphorylation of STAT by AT₂ Receptor Stimulation
We transfected the rat AT₂ receptor cDNA expression vector into cultured VSMCs, because VSMCs prepared from adult rat aorta showed high levels of AT₁ receptor binding but minimally detectable levels of AT₂ receptor.^{7 11} Three days after transfection, the density of AT₂ receptor increased in these cells (AT₁ receptor, 8.89±0.44 fmol/10⁶ cells; AT₂ receptor, 5.84±0.32 fmol/10⁶ cells; n=6, mean±SE). Lysates were prepared from Ang II (10^-7 mol/L)–treated VSMCs, immunoprecipitated with antiphosphotyrosine antibody, and immunoblotted with STAT antibodies. In control vector–transfected VSMCs, Ang II stimulated tyrosine phosphorylation of STAT1/ß, STAT2, and STAT3 (Figure 1A) as previously reported.^{26 27 28} However, in AT₂ receptor–transfected cells, the effect of Ang II on the tyrosine phosphorylation of these STATs was attenuated (Figure 1A and Figure 2), and the addition of PD123319 (an AT₂ receptor–specific antagonist) restored phosphorylation of STATs, whereas the addition of DuP753 (an AT₁ receptor–specific antagonist) attenuated the effect of Ang II (Figure 1A, middle and bottom panels). These results suggest that AT₂ receptor stimulation inhibits the tyrosine phosphorylation of STATs and antagonizes the effect of AT₁ receptor.

View larger version (26K): [in this window] [in a new window]   Figure 1. Ang II–mediated tyrosine phosphorylation and dephosphorylation of STATs and Jak. A, Control vector–transfected VSMCs (CV) or AT2 receptor cDNA–transfected VSMCs (AT2R) were treated with Ang II (10-7 mol/L) (top panel). AT2 receptor–transfected VSMCs were also treated with Ang II (10-7 mol/L), DuP753 (10-7 to 10-5 mol/L), and/or PD123319 (10-5 mol/L) for 15 minutes. Cell lysates were immunoprecipitated with antiphosphotyrosine antibody and immunoblotted with STAT antibodies. B and C, AT2 receptor–transfected VSMCs were treated with IFN- (1 µg/mL) or PDGF (10 ng/mL) with or without Ang II (10-7 mol/L) plus DuP753 (10-5 mol/L). Cell lysates were immunoprecipitated with antiphosphotyrosine antibody and immunoblotted with STAT1 antibody. D, VSMCs were treated with Ang II (10-7 mol/L), and cell lysates were immunoprecipitated with antiphosphotyrosine antibody and Western blotted with Jak2 or Tyk2 antibody. Data are representative of 6 separate experiments.

View larger version (13K): [in this window] [in a new window]   Figure 2. Densitometric measurements of tyrosine phosphorylation of STAT1 (A), STAT2 (B), and STAT3 (C) 15 minutes after Ang II (10-7 mol/L) stimulation in control vector–transfected VSMCs (CV) or AT2 receptor cDNA–transfected VSMCs (AT2R). Values expressed are mean±SEM of 6 different experiments.

To further confirm that AT₂ receptor stimulation dephosphorylates STAT, we next examined the possibility that AT₂ receptor stimulation also inhibits the tyrosine phosphorylation of STAT induced by other families of receptors. As shown in Figure 1B and 1C, IFN- or PDGF phosphorylated STAT1/ß in AT₂ receptor–transfected VSMCs. Selective stimulation of AT₂ receptor (Ang II plus DuP753) inhibited the phosphorylation of STAT1/ß.

The AT₂ receptor is abundantly and widely expressed in fetal vasculature but is present only at low levels in adult tissues, leading to the hypothesis that this receptor is involved in growth, development, and/or differentiation. To examine the role of endogenous AT₂ receptor on STATs, we used VSMCs prepared from mouse fetal aorta. VSMCs were cultured from the aorta of fetal mouse at embryonic day 20, which expresses both AT₁ and AT₂ receptor (AT₁ receptor, 4.72±0.81 fmol/10⁶ cells; AT₂ receptor, 2.35±0.81 fmol/10⁶ cells; n=4, mean±SE). Stimulation with Ang II (10^-7 mol/L) in the presence of the AT₂ receptor–specific antagonist (PD123319, 10^-5 mol/L) enhanced further the tyrosine phosphorylation of STAT1/ß and STAT3 compared with stimulation with Ang II alone (Figure 3A). The effect of Ang II on tyrosine phosphorylation of STATs was attenuated with the addition of the AT₁ receptor–specific antagonist DuP753 (10^-5 mol/L) (Figure 3A). To confirm further the observation in other cells, we studied mouse fibroblast R3T3 cells, which express abundant AT₂ receptor but not AT₁ receptor.³⁷ As shown in Figure 3B, Ang II inhibited the tyrosine phosphorylation of STAT1/ß by IFN-, EGF, and PDGF, supporting our notion that AT₂ receptor stimulation dephosphorylates STAT.

View larger version (42K): [in this window] [in a new window]   Figure 3. AT2 receptor–mediated tyrosine dephosphorylation of STATs in mouse fetal VSMCs and R3T3 cells. A, Cultured VSMCs were prepared from the fetal mouse aorta (embryonic day 20) and stimulated with Ang II (10-7 mol/L), DuP753 (10-5 mol/L), and/or PD123319 (10-5 mol/L) for 15 minutes. Cell lysates were immunoprecipitated with antiphosphotyrosine antibody, separated by SDS/PAGE, transferred to nitrocellulose, and immunoblotted with STAT1 or STAT3 antibody. B, Confluent R3T3 fibroblasts were treated with IFN- (1 µg/mL), EGF (100 ng/mL), or PDGF (10 ng/mL) with or without Ang II (10-7 mol/L) for 15 minutes. Cell lysates were immunoprecipitated with antiphosphotyrosine antibody and immunoblotted with STAT1 antibody. Data are representative of 6 separate experiments.

It has been reported that the AT₁ receptor stimulation activates STAT via tyrosine phosphorylation through the activation of Jak.^{28 30 31 35} To explore the possibility that AT₂ receptor inhibits Jak activation and results in STAT inactivation, we studied the effect of AT₂ receptor on the tyrosine phosphorylation of Jak2 and Tyk2 (Figure 1D). There was no difference in Ang II–stimulated tyrosine phosphorylation of Jak2 and Tyk2 between control vector– and AT₂ receptor–transfected VSMCs, suggesting that AT₂ receptor stimulation does not affect the activation of Jak2 and Tyk2.

Serine Dephosphorylation of STAT by AT₂ Receptor Stimulation
Recently, it has been reported that the function of STAT is also enhanced by serine phosphorylation.^{34 36 38 39} We examined the effect of AT₂ receptor stimulation on serine phosphorylation of STAT. AT₂ receptor–transfected VSMCs were treated with Ang II (10^-7 mol/L) for 15 minutes, and the cell lysates were immunoprecipitated with antiphosphoserine antibody and immunoblotted with STAT antibodies. As shown in Figure 4A and 4C, Ang II stimulation increased the serine phosphorylation of STAT1 and STAT3. Ang II plus PD123319 treatment further enhanced serine phosphorylation of STAT1 and STAT3, whereas the addition of DuP753 to Ang II attenuated the effect of Ang II on serine phosphorylation (Figure 4A and 4C). STAT1ß and STAT2 lack putative serine phosphorylation sites for ERK, and we did not observe the serine phosphorylation of STAT1ß and STAT2 by Ang II (data not shown).

View larger version (26K): [in this window] [in a new window]   Figure 4. Effect of Ang II on serine phosphorylation and dephosphorylation of STATs. A, AT2 receptor–transfected VSMCs were treated with Ang II (10-7 mol/L), DuP753 (10-5 mol/L), and/or PD123319 (10-5 mol/L) for 15 minutes. Cell lysates were immunoprecipitated with antiphosphoserine antibody and immunoblotted with STAT1 or STAT3 antibody. Densitometric measurements of serine phosphorylation of STAT1 (a) and STAT3 (b) 15 minutes after Ang II (10-7 mol/L) stimulation is shown in panel C. Values expressed are mean±SEM of 6 different experiments. B, Effect of MEK-1 inhibitor (PD98059) on Ang II–mediated serine phosphorylation of STATs. Control vector–transfected VSMCs were treated with 50 µmol/L of PD98059 60 minutes before the addition of Ang II (10-7 mol/L) and stimulated with Ang II for 15 minutes. Cell lysates were immunoprecipitated with antiphosphoserine antibody and immunoblotted with STAT antibodies. Data are representative of 6 separate experiments.

To examine the effect of ERK on serine phosphorylation on STAT1 and STAT3 via AT₁ receptor, we treated control VSMCs with a specific inhibitor to MAP kinase kinase 1 (also known as MEK-1 inhibitor), PD98059 (50 µmol/L), 60 minutes before the addition of Ang II (10^-7 mol/L). As shown in Figure 4B, pretreatment with PD98059 inhibited the stimulatory effect of the AT₁ receptor on serine phosphorylation of STAT1 and STAT3.

We next examined the protein levels of STATs, Jak2, and Tyk2 3 days after AT₂ receptor cDNA transfection into the VSMCs, and we did not observe any detectable changes in these protein levels (Figure 5).

View larger version (71K): [in this window] [in a new window]   Figure 5. Effect of AT2 receptor transfection on the protein levels of STATs, Jak2, and Tyk2. Three days after AT2 receptor cDNA (AT2R) or control vector (CV) transfection into VSMCs, cell lysates were prepared, separated on SDS/PAGE, and immunoprecipitated with STAT1, STAT2, STAT3, Jak2, or Tyk2 antibody.

Effect of AT₂ Receptor on Nuclear Translocation of STAT
We next examined the possibility that AT₂ receptor–mediated dephosphorylation of STATs influences the nuclear translocation of STATs. Tyrosine- and serine-phosphorylated STAT1 and STAT3 were shown to increase in the control VSMCs nuclei after Ang II stimulation (Figure 6). In the AT₂ receptor–transfected cells, however, accumulation of nuclear phosphorylated STAT1 and STAT3 was attenuated.

View larger version (31K): [in this window] [in a new window]   Figure 6. Nuclear translocation of STAT1 and STAT3. Nuclear extracts were prepared from control vector (CV) or AT2 receptor cDNA (AT2R)–transfected VSMCs, which were treated with Ang II (10-7 mol/L) for 30 minutes. Nuclear extract (100 µg) was immunoprecipitated with antiphosphotyrosine (A) or antiphosphoserine antibody (B) and Western blotted with STAT1 or STAT3 antibody. Data are representative of 3 separate experiments.

Induction and DNA binding of the sis-inducing factor (SIF) complex were assessed by electrophoretic gel mobility shift assay using the radiolabeled probe for SIE in the c-fos gene promoter region. As shown in Figure 7A, Ang II induced the formation of the SIF complex in the control vector–transfected cells within 30 minutes. In contrast, the Ang II–induced formation of the SIF complex was attenuated in the AT₂ receptor cDNA–transfected cells. The addition of 100-fold excess of unlabeled SIE as a competitor abolished the SIF complex (lanes 2 and 5, Figure 7B), whereas the unlabeled mutant SIE had no effect (lanes 3 and 6, Figure 7B). In addition, the ³²P-labeled mutant SIE probe did not show any specific binding (lanes 7 and 8, Figure 7B). The addition of AP-1 oligonucleotide competitor did not affect the formation of SIF (Figure 7C), indicating that the SIF is distinct from classical AP-1 components such as the serum response factor. When STAT1 or STAT3 was removed from nuclear extract by immunoprecipitation, the SIF complex was not detected (Figure 7D), suggesting that both STAT1 and STAT3 are components of the SIF complex. Next, we studied the expression of c-fos mRNA by Northern blot and observed that Ang II induces c-fos mRNA expression after 30 minutes of stimulation in the control- and the AT₂ receptor expression vector–transfected VSMCs (Figure 7E). However, its expression was attenuated markedly in the AT₂ receptor–transfected cells.

View larger version (62K): [in this window] [in a new window]   Figure 7. Effect of Ang II on SIF binding with SIE and c-fos mRNA expression. Nuclear extracts were prepared from the control vector–transfected (CV) or AT2 receptor expression vector (AT2R)–transfected VSMCs, which were treated with Ang II (10-7 mol/L). 32P-labeled SIE probe was incubated with nuclear extract (10 µg) and electrophoresed. A, Time course of SIE binding. Left lane, No SIF binding without the incubation of nuclear extract. B, Specificity of the SIF-SIE interaction was shown by the use of competitors: 32P-labeled SIE probe was incubated with nuclear extract with 100-fold excess of unlabeled double-stranded SIE oligonucleotide or mutant SIE (mSIE) oligonucleotide. The binding of 32P-labeled mutant SIE probe with nuclear extract was also examined. C, Addition of 100-fold excess of double-stranded AP-1 oligonucleotide did not affect the formation of SIF. D, Nuclear extract (100 µg) was immunoprecipitated with 10 µg of anti-STAT1 (lane 2) or anti-STAT3 antibody (lane 3). As control, nuclear extract was also treated with nonimmune rabbit serum (lane 1). Top panel, IP-Blot (immunoprecipitation): Immunoprecipitated nuclear extracts were immunoblotted with anti-STAT antibodies. Bottom panel, EMSA (electrophoresis mobility shift assay): Supernatant fraction after removal of STAT1 or STAT3 could not form the SIF complex (lanes 2 and 3). E, Total RNA (20 µg/lane) prepared from the control vector–transfected or AT2 receptor cDNA expression vector–transfected VSMCs treated with Ang II (10-7 mol/L) for indicated times was separated by electrophoresis and hybridized sequentially with a probe for the c-fos and for GAPDH as internal control. Data are representative of 3 separate experiments.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
STATs are now known to be activated by many different extracellular signaling proteins including cytokines, growth factors such as EGF and PDGF, and Ang II via the AT₁ receptor, and the mechanism of STAT activation is well defined. In contrast, the inactivation mechanism(s) of the Jak/STAT pathway is not well known. Recent evidences suggest that phosphatases may play important roles in regulating Jak/STAT function. Indeed, the use of phosphatase inhibitors can lead to constitutive activation of some STAT pathways,^{40 41} and SHPTP1 has been implicated in the downregulation of IFN signaling through STAT.⁴² We have demonstrated that AT₂ receptor activates PTPase, antagonizes the effects of growth factor and AT₁ receptors, and exerts antigrowth effects in several types of cells such as VSMCs.^{7 11 14 16} PTPase activation by the AT₂ receptor has also been demonstrated in rat adrenal glomerulosa and PC12W cells,¹⁸ neuronal NG 108-15 cells,¹⁹ and R3T3 cells.²⁰ These results have led us to examine the possibility that AT₂ receptor stimulation dephosphorylates the tyrosine residues of Jak and/or STATs.

The data obtained from the present study have demonstrated that AT₂ receptor stimulation inhibits the AT₁ receptor–mediated tyrosine phosphorylation of STAT1/ß, STAT2, and STAT3, whereas AT₂ receptor does not affect the tyrosine phosphorylation of Jak2 and Tyk2. The fact that STAT, but not Jak, is dephosphorylated via AT₂ receptor is interesting. These results also suggest that AT₂ receptor–activated PTPases may directly dephosphorylate the tyrosine residues of STAT1/ß, STAT2, and STAT3. Identification of specific phosphatase(s), which is activated by AT₂ receptor stimulation and inhibits STAT phosphorylation, is intriguing and may provide new insights into the regulatory mechanism of the function of STATs as well as the signaling mechanism of the AT₂ receptor. Recently, the activation of SHPTP1 via AT₂ receptor was reported,²¹ suggesting that SHPTP1 is one of the phosphatases that mediates dephosphorylation of STATs by AT₂ receptor stimulation. Moreover, evaluation of the mechanism of dephosphorylation of Jak may reveal further the mechanism of the distinct dephosphorylation of STAT and Jak via AT₂ receptor.

Recent evidences have revealed that serine phosphorylation and tyrosine phosphorylation are required for the maximal activation of STATs. It has been reported that phosphorylation of serine residues is induced by IFN- and PDGF³⁴ and that direct association of ERK2 with IFN receptor and STAT1 occurs after IFN stimulation.³⁸ AT₁ receptor stimulation has been also reported to phosphorylate serine residue of STAT3 in rat neonatal cardiac fibroblasts and CHO-K1 cells by ERK activation.³⁶ We have also confirmed that AT₁ receptor stimulation phosphorylates the serine residues of STAT1 and STAT3 in VSMCs via ERK activation. As expected, on the basis of the previous observation that AT₂ receptor stimulation inhibits ERK,^{7 11 14 16 19 21} we have demonstrated in the present study that AT₂ receptor stimulation inhibits serine phosphorylation of STAT1 and STAT3.

To examine further the mechanism of STAT inactivation by AT₂ receptor on VSMC growth, we examined the binding of STATs with SIE in the c-fos gene promoter. We first demonstrated that in response to AT₁ receptor stimulation, tyrosine- and serine-phosphorylated STAT1 and STAT3 accumulate in the nuclei of VSMCs and become a component of the nuclear SIF complex. In contrast, in AT₂ receptor–transfected VSMCs, AT₁ receptor–mediated SIF complex binding with SIE in the c-fos gene promoter was attenuated. This decrease was associated with the reduction of the levels of phosphorylated STAT1 and STAT3. Consistent with these findings, the AT₁ receptor–induced c-fos gene expression was decreased significantly by the concomitant stimulation of the AT₂ receptor in these cells. The c-fos gene expression is regulated by the net interaction with different transcriptional factors.⁴³ Partial inhibition of c-fos could be mediated by other mechanisms. The inactivation of ERK by AT₂ receptor may also result in a decreased production of serum response factor, and this may act in concert with the inactivation of STAT via the inhibition of serine phosphorylation, thereby resulting in the decrease of c-fos transcription.

The AT₂ receptor is abundantly expressed in the fetal vasculature with rapid decline after birth and reexpression in vascular injury, suggesting an important role of the AT₂ receptor in vascular development and vascular remodeling. In the present study, we examined the interaction of AT₁ and AT₂ receptors in fetal VSMCs, which express both receptors. As expected, we demonstrated that AT₂ receptor stimulation inhibited AT₁ receptor–mediated STAT phosphorylation, suggesting that the negative regulation of STAT activation may also be involved in the AT₂ receptor–mediated vasculogenesis in the fetus.

The present study suggests that AT₁ receptor activates (1) STAT1/ß, STAT2, and STAT3 by tyrosine phosphorylation via Jak2 and Tyk2 activation and (2) STAT1 and STAT3 by serine phosphorylation via ERK activation. In contrast, the AT₂ receptor stimulation inactivates STATs by tyrosine dephosphorylation without affecting Jak as well as by serine dephosphorylation via the inhibition of ERK, thereby antagonizing the AT₁ receptor–mediated upregulation of the c-fos gene. Moreover, AT₂ receptor stimulation also dephosphorylates STAT, which is activated by IFN-, PDGF, and EGF, in VSMCs as well as in R3T3 cells. These results suggest that the AT₂ receptor is capable of exerting broad inhibitory effects on STAT activation induced by multiple families of receptors (growth factor, cytokine, and 7-transmembrane receptors). In summary, our data provide evidence for negative crosstalk of the 7- transmembrane-spanning AT₂ receptor with multiple families of receptors via the inactivation of ERK and STAT pathways.

	Acknowledgments

 
This work was supported by National Institutes of Health grants HL46631, HL35252, HL35610, HL48638, HL07708, and HL58616 and a grant from Longwood Foundation for Translational Research. Victor J. Dzau is a recipient of National Institutes of Health Merit Award HL35610.

Received December 16, 1998; accepted January 25, 1999.

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