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(Circulation Research. 1997;81:611-617.)
© 1997 American Heart Association, Inc.

Articles

Role of Angiotensin II in Activation of the JAK/STAT Pathway Induced by Acute Pressure Overload in the Rat Heart

Jing Pan, Keiichi Fukuda, Hiroaki Kodama, Shinji Makino, Toshiyuki Takahashi, Motoaki Sano, Shingo Hori, , Satoshi Ogawa

From the Cardiopulmonary Division (J.P., K.F., H.K., S.M., T.T., M.S., S.O.), Department of Internal Medicine, and the Department of Emergency Medicine (S.H.), Keio University, Tokyo, Japan.

Correspondence to Keiichi Fukuda, Cardiopulmonary Division, Department of Internal Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160, Japan. E-mail kfukuda{at}mc.med.keio.ac.jp

	Abstract

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Abstract This study was designed to determine whether the JAK/STAT (indicating just another kinase/signal transducer and activator of transcription) pathway is activated in cardiac hypertrophy induced in vivo by pressure overload in rats and to demonstrate whether angiotensin II is involved in the activation of the JAK/STAT pathway. Acute pressure overload was produced by constricting the abdominal aorta of Wistar rats. Immunoprecipitation–Western blot analysis revealed that pressure overload activated JAK1, JAK2, and Tyk2 as early as 5 minutes and that STAT1, STAT2, and STAT3 were tyrosine-phosphorylated rapidly after exposure to the pressure overload. Phosphorylation of STAT1 and STAT2 peaked in the early stage at 5 to 15 minutes, whereas that of STAT3 peaked in the late stage at 60 minutes. Gel mobility shift of the interferon gamma activation site/interferon alpha–stimulating response element was observed immediately after the aortic banding, whereas the band of sis-inducing element was shifted in the late stage at 60 minutes. Both cilazapril (angiotensin II–converting enzyme inhibitor) and E4177 (angiotensin II type 1 [AT₁] receptor antagonist) significantly suppressed the phosphorylation of Tyk2 and partially inhibited the phosphorylation of JAK2, but neither affected JAK1. Coimmunoprecipitation of the AT₁ receptor with JAK2 or Tyk2 was clearly observed at 5 minutes and peaked at 15 minutes (20-fold the control value). These results indicate that the JAK/STAT pathway is activated by acute pressure overload in rats and that angiotensin II is involved in activating Tyk2, and partially activating JAK2, via the AT₁ receptor. Both angiotensin II–dependent and –independent pathways take part in activating the JAK/STAT pathway in the pressure-overloaded rat heart.

Key Words: myocardial hypertrophy • pressure overload • signal transduction • JAK/STAT pathway • angiotensin II

	Introduction

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Cardiac hypertrophy is not only an adaptive response of the heart to several pathological situations aimed at maintaining adequate cardiac contractile function but also an important cause of increased morbidity and mortality.^{1 2 3} The mechanisms governing the development of cardiac hypertrophy, however, are not completely understood. Evidence has accumulated over many years indicating that cardiac hypertrophy is induced by mechanical load^{4 5 6 7} and humoral factors, such as Ang II,^{8 9 10} endothelin,^{11 12} ₁- and ß-adrenergic agonists,^{13 14} transforming growth factor-ß,¹⁵ insulin, and insulin-like growth factor.^{16 17} A number of studies have shown that the renin-angiotensin system plays an important role in modulating the adaptive growth pattern in cardiac hypertrophy^{18 19 20} in response to pressure overload. All components of the renin-angiotensin system have been demonstrated in the myocardium,^{21 22} and both ACE inhibitors^{23 24 25} and AT₁ receptor antagonists^{26 27} are very effective in inducing regression of cardiac hypertrophy, not only in animals but also in humans. Recent studies have also shown that Ang II is secreted by neonatal cardiomyocytes in response to passive stretch^{28 29} and acts as a growth-promoting factor directly on cardiac myocytes. Ang II secreted from a stretched cardiomyocyte has been shown to stimulate the cardiomyocyte itself in an autocrine/paracrine manner and to activate a variety of protein kinases and signaling molecules to induce various genes that promote cardiac hypertrophy.^{30 31 32 33}

JAK/STAT is a newly discovered intracellular signal transduction pathway^{34 35} that is activated by many cytokines and growth factors.^{36 37 38 39 40} Binding of ligands to receptors leads to the activation of the JAK tyrosine kinase family, and the activated receptor-kinase complexes recruit members of the STAT family and activate them by phosphorylation. As a result, the phosphorylated STAT proteins dimerize, translocate into the nucleus, and bind response elements in the promoters of target genes to stimulate transcription.⁴¹ Although much progress has recently been made in elucidating the intracellular signal transduction pathway for hypertrophy-inducing stimuli, much less is known about whether the JAK/STAT signal transduction pathway is involved in mechanical load–induced cardiac hypertrophy. Recently, Marrero and colleagues^{42 43 44} have reported that the JAK/STAT pathway is directly activated by Ang II in rat aortic smooth muscle cells, and we have found that JAK2, Tyk2, STAT1, and STAT2 are tyrosine-phosphorylated by Ang II in rat neonatal cardiomyocytes.⁴⁵ Therefore, we hypothesized that the JAK/STAT pathway may also play a crucial role in the induction of cardiac hypertrophy in vivo. On the basis of this hypothesis, in the present study we investigated whether activation of the JAK/STAT pathway is involved in the process of cardiac hypertrophy induced by pressure overload and whether blockade of the renin-angiotensin system by an ACE inhibitor (cilazapril) and an AT₁ receptor antagonist (E4177) activates the JAK/STAT pathway in the pressure-overloaded rat heart.

	Materials and Methods

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Immunoprecipitation and Immunoblotting
Polyclonal antibodies to JAK1, JAK2, Tyk2, and STAT3 and monoclonal antibodies to STAT1 and STAT2 were purchased from Santa Cruz Laboratory. Polyclonal antibody to the AT₁ receptor was purchased from Chemicon International Inc. Monoclonal antibody to anti-phosphotyrosine (4G10) was purchased from Upstate Biotechnology Inc.

Wistar rats (8 weeks) were anesthetized with ether, and the abdominal aorta was ligated distal to the renal arteries. A total of 146 animals were used in the experiments. The left ventricular myocardium was excised at 0, 5, 15, 30, and 60 minutes, homogenized with a Polytron homogenizer (Niti-on), and lysed in lysis buffer containing 20 mmol/L Tris-HCl (pH 7.4), 150 mmol/L NaCl, 2.5 mmol/L EDTA, 50 mmol/L NaF, 0.1 mmol/L Na₄P₂O₇, 1 mmol/L Na₃VO₄, 1 mmol/L phenyl-methylsulfonyl fluoride, 1% Triton X-100, 10% glycerol, 0.1% SDS, 1% deoxycholic acid, 1 µg/mL aprotinin, and 1 µg/mL leupeptin. After centrifugation, cell lysates containing equal amounts (5 mg) of protein were incubated with 1 µg of antibodies to JAK1, JAK2, Tyk2, STAT1, STAT2, or STAT3 for 2 hours at 4°C. The immune complexes were precipitated with protein A or G Sepharose. The immunoprecipitates were separated by electrophoresis on 6% to 10% SDS-polyacrylamide gel and transferred to reinforced nitrocellulose membranes (Schleicher & Schuell). The membranes were then blocked with 5% bovine serum albumin in TBST (20 mmol/L Tris-HCl [pH 7.4], 150 mmol/L NaCl, and 0.05% Tween 20) solution for 1 hour at room temperature. To detect phosphotyrosine, membranes were incubated with anti-phosphotyrosine antibody, followed by incubation with peroxidase-conjugated goat anti-mouse IgG antibody and chemiluminescence detection (ECL, Amersham). For protein binding analysis, immunoprecipitation was performed using the same lysis buffer without Na₄P₂O₇ or deoxycholic acid. The proteins associated with JAK kinases were detected with anti–AT₁ receptor antibody, followed by incubation with peroxidase-conjugated goat anti-rabbit IgG.

Drug Administration
Similar experiments were performed on rats pretreated with ACE inhibitor (cilazapril^{25 46} ) or AT₁ receptor–specific antagonist (E4177⁴⁷ ). Cilazapril and E4177 were obtained from Eisai Pharmaceutical Co, Ltd. E4177 was administered at a dose of 30 mg · kg^-1 · d^-1 PO and cilazapril at a dose of 10 mg · kg^-1 · d^-1 PO beginning 7 days before aortic ligation at 10:00 AM.

Preparation of Nuclear Extracts
The left ventricle was excised at 0, 5, 15, 30, 60, and 120 minutes after ligation of the abdominal aorta and washed with precooled PBS, and nuclear extracts were prepared according to the standard method.⁴⁸ Briefly, the myocardium was rinsed in 5 vol of hypotonic buffer (10 mmol/L HEPES-KOH [pH 7.9], 10 mmol/L KCl, and 1.5 mmol/L MgCl₂) supplemented with protease and phosphatase inhibitors (0.5 mmol/L phenylmethylsulfonyl fluoride and 1 mmol/L NaF), Dounce-homogenized, and sedimented at 3300g for 15 minutes at 4^oC, and the pelleted nuclei were collected. The pelleted nuclei were resuspended with the low salt buffer (20 mmol/L HEPES [pH 7.9], 25% glycerol, 20 mmol/L KCl, 1.5 mmol/L MgCl₂, and 0.2 mmol/L EDTA) supplemented with proteinase and phosphatase inhibitors (see above) and then incubated with high salt buffer (same as low salt buffer except that 1.2 mol/L KCl was used) for 30 minutes at 4°C. The supernatant was used as the nuclear extract, and the nuclear extracts were dialyzed against dialysis buffer (20 mmol/L HEPES-KOH [pH 7.9], 0.2 mmol/L EDTA, 100 mmol/L KCl, and 20% glycerol) for 12 hours at 4°C. The protein concentration was determined by Bradford assay, and the nuclear extracts were stored at -80°C.

Gel Mobility Shift Assays
Nuclear extracts (10 µg each) were incubated with 2 µg of poly(dI-dC)–poly(dI-dC) (Pharmacia Biotech) with or without competitor oligonucleotide for 15 minutes at 30°C, and the samples were incubated with 1 or 2 fmol of ³²P-labeled probes (10 000 cpm) for 15 minutes at 30°C. The sequences of the probes used in gel shift analysis were as follows: SIE-DNA,^{49 50} 5'-GTGCATTTCCCGTAAATCTTGTCTACA-3'; mutant SIE-DNA, 5'-GTGCATCCACCGTAAATCTTGTCTACA3'; GAS/ISRE-DNA,^{51 52} 5'-AAGTACTTTCAGTTTCATATTACTCTA-3'; and mutant-GAS/ISRE-DNA, 5'-AAGTACTTTCAGTGGTCTATTACTCTA-3'. The oligonucleotides were labeled with [-³²P]ATP by using T4 polynucleotide kinase. Binding reactions were resolved on a 4% native polyacrylamide gel containing 1x TAE buffer (40 mmol/L Tris [pH 7.5], 40 mmol/L sodium acetate, and 1 mmol/L EDTA) for 2 to 3 hours at 150 V in a cold room (4°C) and dried, and x-ray film was exposed to the gel for 12 to 24 hours.

Statistical Analysis
All values shown are mean±SD. The differences among mean values were determined by ANOVA for repeated measurements. Student's t test was used when two values were compared. Statistical significance was considered to exist at a value of P<.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Pressure Overload Activated JAK Family Kinases
To determine whether the JAK family kinases are involved in cardiac hypertrophy induced in vivo by pressure overload, we performed immunoprecipitation–Western blot analysis to detect tyrosine phosphorylation of JAK kinases in this model.

The results are shown in Fig 1. In sham-operated (control or time 0) rats, JAK1, JAK2, and Tyk2 were slightly phosphorylated, but the levels of phosphorylation were very low. JAK1, JAK2, and Tyk2 were rapidly tyrosine-phosphorylated at 5 minutes after ligation, and their phosphorylation peaked at 30 to 60 minutes (Fig 1a). Five separate experiments yielded similar results. Densitometric analysis of the tyrosine phosphorylation are shown in Fig 1b.

View larger version (15K): [in this window] [in a new window]   Figure 1. Effect of pressure overload on tyrosine phosphorylation of JAK family kinases in rat heart. a, Extracts from rat hearts were immunoprecipitated with antibodies to JAK1, JAK2, and Tyk2 and immunoblotted with anti-phosphotyrosine antibody. Pressure overload induced rapid tyrosine phosphorylation of JAK1, JAK2, and Tyk2. All experiments were performed at least five times. IP indicates immunoprecipitation. b, Densitometric analysis was performed in five separate experiments.

Effect of Pressure Overload on STAT Phosphorylation
Activated JAK kinases phosphorylate various combinations of STAT transcription factors, which form dimers, translocate into the nucleus, and transactivate responsive elements in the promoters. In order to identify which STAT was activated by pressure overload, we detected tyrosine-phosphorylated members of the STAT family. STAT1, STAT2, and STAT3 were tyrosine-phosphorylated 5 minutes after aortic banding, but no phosphorylation of STAT1ß was observed. Tyrosine phosphorylation of STAT1 and STAT2 peaked at 5 to 15 minutes, whereas that of STAT3 peaked at 60 minutes (Fig 2a). Similar results were obtained in five separate experiments. Densitometric analysis of the phosphorylation of STAT1, STAT2, and STAT3 were shown in Fig 2b. These results indicate that STAT1 and STAT2 were activated in the pressure-overloaded rat heart in the early stage, whereas STAT3 was activated in the late stage.

View larger version (13K): [in this window] [in a new window]   Figure 2. Tyrosine phosphorylation of STAT1/ß, STAT2, and STAT3 in response to acute pressure overload. a, Extracts were immunoprecipitated with antibodies to STAT1 and STAT3 and immunoblotted with anti-phosphotyrosine antibody. To test for tyrosine phosphorylation of STAT2, extracts were immunoprecipitated with anti-phosphotyrosine and immunoblotted with anti-STAT2 antibody. STAT1, STAT2, and STAT3 were phosphorylated immediately after the aortic ligation. IP indicates immunoprecipitation. b, Densitometric analysis was performed in five separate experiments.

Gel Mobility Shift Assay of GAS/ISRE and SIE in Pressure-Overload Model
ISGF complex is formed by a heterodimer of STAT1 and STAT2 that activates the GAS/ISRE element,⁵³ and the SIF complex is formed by homodimers or heterodimers of STAT1 and STAT3 that activate the SIE element in the promoter of genes to induce expression.^{54 55} To determine whether pressure overload induces ISGF-like activity or SIF-like activity in the rat heart, a gel mobility shift assay was performed using ³²P-labeled oligonucleotides representing GAS/ISRE and SIE in nuclear extracts from the hearts of rats at the times indicated after sham operation or aortic banding (Fig 3).

View larger version (53K): [in this window] [in a new window]   Figure 3. Gel mobility shift assay of GAS/ISRE and SIE in pressure-overload hearts. a and b, Nuclear extracts were prepared and analyzed by electrophoretic mobility shift assay by using 32P-labeled GAS/ISRE and SIE. The positions of the ISGF (a) and SIF (b) complexes are indicated. c, Results of the supershift assay of SIE are shown. Samples were preincubated with anti-STAT1 (-STAT1) and anti-STAT3 (-STAT3) antibody before electrophoresis. Four separate experiments showed similar results.

DNA-protein complexes with GAS/ISRE increased immediately (5 minutes after aortic ligation), remained elevated until 60 minutes, and decreased at 120 minutes. No band shifts were observed with mutant GAS/ISRE (Fig 3a, right two lanes), and addition of higher concentrations of unlabeled GAS/ISRE completely competed away the ISGF complex (data not shown). These results indicated that this band was specific to GAS/ISRE.

In contrast, no gel mobility shift of SIE was observed for the first 15 minutes after aortic ligation. Interestingly, we found that the DNA-protein complex with SIE was observed at 30 minutes, peaked at 60 minutes, and decreased at 120 minutes. The time course of the activation of SIF complex was clearly delayed compared with that of the ISGF complex. No band mobility shift was observed with mutant SIE (Fig 3b, right two lanes), and the addition of higher concentrations of unlabeled SIE also competed with the SIF complex (data not shown), showing that this band corresponds to SIE. We used anti-STAT1 and anti-STAT3 antibodies to determine whether the pressure overload–induced SIF complex contained STAT1 and STAT3. As shown in Fig 3c, incubation of the DNA-protein complex with anti-STAT1 antibody inhibited formation of the SIF complexes, and incubation with anti-STAT3 antibody also inhibited binding of the SIF complexes. These results demonstrated that the SIF complexes activated by pressure overload contained both STAT1 and STAT2. The time course of the activation of GAS/ISRE and SIE corresponded to the time course of phosphorylation of STAT1, STAT2, and STAT3.

AT₁ Receptor Antagonist and ACE Inhibitor Block Pressure Overload–Induced JAK Kinase Activation
Previous studies have reported that the local renin-angiotensin system may play a critical role in cardiac hypertrophy induced by pressure overload^{18 19 20} and that Ang II may act to promote the growth of cardiac myocytes by autocrine/paracrine mechanisms. To demonstrate whether Ang II is involved in the tyrosine phosphorylation of the JAK/STAT signaling pathway in the in vivo pressure-overloaded rat heart, we performed similar experiments using rats pretreated with either AT₁ receptor blocker (E4177) or ACE inhibitor (cilazapril).

Immunoprecipitation–Western blot analysis was performed at 0, 5, and 30 minutes after aortic banding of rats pretreated with E4177 or cilazapril for 7 days. Interestingly, as shown in Fig 4a and Fig 5a, pretreatment with E4177 and cilazapril inhibited tyrosine phosphorylation of Tyk2 induced by pressure overload. E4177 decreased the tyrosine phosphorylation of Tyk2 by 89.3±5.3% (P<.01, n=6, Fig 4b). Cilazapril similarly inhibited the phosphorylation of Tyk2 by 78.8±6.8% (P<.01, n=6, Fig 5b). Tyrosine phosphorylation of JAK2 was partially suppressed by E4177 (32.8±9.3%, P<.05, n=6, Fig 4b) and cilazapril (21.2±7.6%, P<.05, n=6, Fig 5b), but neither E4177 nor cilazapril affected the tyrosine phosphorylation of JAK1 (P=NS, n=6, Fig 4b and 5b). These results indicate that Ang II plays an important role in the activation of Tyk2 kinase induced by pressure overload but that factors other than Ang II are required for activation of JAK1 and JAK2 kinases.

View larger version (25K): [in this window] [in a new window]   Figure 4. Effect of AT1 receptor blocker on the tyrosine phosphorylation of JAK kinases induced by pressure overload. a, Representative trace showing inhibition by E4177 pretreatment of Tyk2 phosphorylation induced by pressure overload. JAK1 phosphorylation was unaffected by E4177 pretreatment. b, Rats were pretreated with the AT1 receptor–specific antagonist E4177 for 7 days before the experiments. Tyrosine phosphorylation of Tyk2 was significantly suppressed by E4177 (P<.01). C indicates control (n=6); E, E4177 (n=6). PY indicates phosphotyrosine.

View larger version (19K): [in this window] [in a new window]   Figure 5. Effect of cilazapril (CP, n=6) on the tyrosine phosphorylation of JAK kinases induced by pressure overload. a, Representative trace shows inhibition by CP pretreatment of Tyk2 phosphorylation induced by pressure overload. IP indicates immunoprecipitation; C, control (n=6). b, Rats were pretreated with CP for 7 days before the experiments. Tyrosine phosphorylation of Tyk2 was significantly suppressed by the ACE inhibitor CP (P<.01). Suppression of phosphorylation of JAK2 was also observed by CP pretreatment, but the level of suppression was weaker than that of Tyk2.

Coimmunoprecipitation of AT₁ Receptor With JAK2 and Tyk2
To further demonstrate whether Ang II directly activates JAK2 and Tyk2 through AT₁ receptors in the pressure-overloaded rat heart, we investigated direct binding of JAK2 and Tyk2 kinases to the AT₁ receptor. We immunoprecipitated JAK2 and Tyk2 and then performed Western blot analysis with polyclonal anti–AT₁ receptor antibody. The results are shown in Fig 6. Slight association between the AT₁ receptor and JAK2 or Tyk2 was observed even in the control condition. The association of the AT₁ receptor with JAK2 and Tyk2 was markedly increased within 5 minutes after ligation, peaked at 15 minutes (20-fold of the control value), and returned to the control level at 60 minutes. The association between the AT₁ receptor and JAK2 or Tyk2 suggested that JAK2 and Tyk2 might be activated by autocrine/paracrine-secreted Ang II induced by pressure overload.

View larger version (54K): [in this window] [in a new window]   Figure 6. Time course of the pressure overload–induced association of JAK2 and Tyk2 with AT1 receptor. Lysates from rat hearts were immunoprecipitated with anti-JAK2 or anti-Tyk2 antibody and immunoblotted with anti-AT1 receptor antibody. The results indicate that the association of JAK2 and Tyk2 with the AT1 receptor increased immediately after ligation. Similar results were obtained in three separate experiments. IP indicates immunoprecipitation.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the present study, we demonstrated the following: (1) Acute pressure overload activates JAK1, JAK2, and Tyk2 kinases in rat heart immediately after aortic banding. (2) STAT1, STAT2, and STAT3 are tyrosine-phosphorylated after pressure overload, but phosphorylation of STAT1ß does not occur. Phosphorylation of STAT1 and STAT2 peaked in the early phase at 5 to 15 minutes, whereas that of STAT3 peaked in the late stage at 60 minutes. (3) Gel mobility shift of GAS/ISRE was observed as early as 5 minutes, but the band shift of SIE peaked in the late stage at 60 minutes, corresponding to the late phosphorylation of STAT1 and STAT3. (4) Both cilazapril and E4177 significantly suppressed the tyrosine phosphorylation of Tyk2 and partially inhibited that of JAK2 but did not affect JAK1. (5) Coimmunoprecipitation of the AT₁ receptor with JAK2 and Tyk2 markedly increased at 15 minutes after aortic banding.

JAK/STAT was first found to be a major signaling pathway of cytokine superfamilies. Different cytokines and growth factors activate different combinations of JAK kinases and STAT transcription factors.^{34 35} We⁵⁶ and others⁵⁷ have observed that LIF causes cardiac hypertrophy and markedly activates JAK1, JAK2, STAT1, and STAT3 via gp130 in neonatal rat cardiomyocytes. Cardiotrophin-1,^{58 59 60} a member of the interleukin-6 cytokine family, was recently cloned and was also shown to cause cardiac hypertrophy and to activate the JAK/STAT pathway in in vitro studies. These findings suggested that the JAK/STAT pathway may play an important role in the process of cardiac hypertrophy.

Various lines of evidence suggest that Ang II may be a crucial factor in mediating cardiac hypertrophy.^{18 19 20 21 22 23 24 25 26 27} The signal transduction pathway of Ang II–induced cardiac hypertrophy has been studied for many years, and Ang II has been found to stimulate multiple intracellular second-messenger systems, such as phospholipases C, D, and A2, Src family kinases, p21^ras, mitogen-activated protein kinase cascades, ribosomal S6 kinases, and protein kinase C in neonatal rat cardiomyocytes.^{30 31 32 33} Recent studies have reported that Ang II directly activates the JAK/STAT pathway via the AT₁ receptor, a G protein–coupled seven transmembrane–spanning receptor, in vascular smooth muscle cells.^{42 43 44} We have found that Ang II induces tyrosine phosphorylation of JAK2, Tyk2, STAT1, and STAT2 in neonatal rat cardiac myocytes.⁴⁵ However, the relationship between mechanical load–induced cardiac hypertrophy and the JAK/STAT pathway remained unknown. Therefore, we investigated whether the JAK/STAT pathway is activated by pressure overload in vivo in rats and whether Ang II is involved in the activation of the JAK/STAT pathway in this model. The findings in the present study have shown that JAK1, JAK2, and Tyk2 are rapidly tyrosine-phosphorylated after the heart is exposed to pressure overload. Subsequently, STAT1, STAT2, and STAT3 are also tyrosine-phosphorylated, form ISGF or SIF complexes to translocate into the nucleus, and bind to the GAS/ISRE or SIE element. The heart is not a homogeneous tissue, being composed of multiple cell types. The activation of the JAK/STAT pathway observed in the present study is mainly occurring in the cardiomyocytes; however, it should be considered that other cell types may partly affect the result.

We have also demonstrated that JAK2 and Tyk2 are physically associated with the AT₁ receptor after pressure overload. Marrero and colleagues⁴² have suggested that the physical association of JAK2 with the AT₁ receptor in response to ligand occupancy may cause JAK2 activation in Ang II signaling. According to their hypothesis, our findings suggest that autocrine/paracrine-secreted Ang II induced by pressure overload is at least partially involved in the activation of JAK2 and Tyk2 through the AT₁ receptor.

In an in vitro study using neonatal rat cardiomyocytes, we observed that Ang II–induced tyrosine phosphorylation of JAK2 and Tyk2 was suppressed by an AT₁ receptor antagonist, cv11974.⁴⁵ In the present study, phosphorylation of Tyk2 was significantly inhibited by both E4177 and cilazapril. As other investigators have demonstrated, Ang II is rapidly released or secreted from cardiomyocytes after myocardial stretch²⁸ or acute pressure overload, and autocrine/paracrine-secreted Ang II should play an important role in JAK/STAT activation. The rapid activation of the ISGF complex (dimer of STAT1 and STAT2) could be explained by Ang II via STAT1 and STAT2 activation. In the present study, activation of the SIF complex (dimer of STAT1 and STAT3) was also observed at 60 minutes. Marrero and colleagues⁴² reported that Ang II induced phosphorylation of STAT3 at 1 hour. Thus, it is possible that Ang II could (directly or indirectly) induce the STAT3 phosphorylation observed in the present study. However, activation of JAK1 was unaffected by either cilazapril or E4177, and JAK2 was only slightly inhibited by these agents. Moreover, it should be mentioned that the time course for JAK and STAT tyrosine phosphorylation did not indicate a simple precursor-product relationship. Maximal phosphorylation of STAT1 and STAT2 was observed before maximal phosphorylation of JAKs. It is difficult to explain the late-phase activation of JAK1, JAK2, Tyk2, STAT1, and STAT3 as being caused by Ang II alone. These findings suggest that factors other than Ang II play an important role in the activation of the JAK/STAT pathway in the pressure-overloaded rat heart. Further study is needed to clarify the mechanism of delayed phosphorylation of the JAK/STAT pathway.

On the basis of these findings, we have concluded that the JAK/STAT pathway is activated by acute pressure overload in rats and that Ang II is involved in activating Tyk2, and partially activating JAK2, via the AT₁ receptor. We also hypothesize that (1) the mechanical load directly activates the JAK/STAT pathway through a mechanosensitive receptor, and/or (2) autocrine/paracrine-secreted Ang II in combination with other growth factors activates the JAK/STAT pathway in response to pressure overload. Further studies are needed to clarify the mechanism of activation of the JAK/STAT pathway induced by pressure overload.

	Selected Abbreviations and Acronyms

 

ACE = angiotensin II–converting enzyme Ang II = angiotensin II AT1 = angiotensin II type 1 GAS/ISRE = interferon gamma activation site/interferon alpha–stimulating response element ISGF = interferon-stimulating gene factor JAK = just another kinase, Janus kinase LIF = leukemia inhibitory factor SIE = sis-inducing element SIF = sis-inducing factor STAT = signal transducer and activator of transcription

	Acknowledgments

 
This study was supported in part by research grants from the Ministry of Education, Science and Culture, Japan, and the Japan Owner's Association. Dr Pan is a recipient of a fellowship from the Japan Society for the Promotion of Science. The authors wish to acknowledge Yoshiko Kurokawa and Rie Inaba for their technical assistance.

Received May 30, 1997; accepted August 14, 1997.

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