Angiotensin II–Stimulated Induction of sis-Inducing Factor Is Mediated by Pertussis Toxin–Insensitive Gq Proteins in Cardiac Myocytes
Abstract—The Janus kinase–signal transducers and activators of transcription (JAK-STAT) pathway is stimulated by angiotensin II (Ang II) via the type 1 receptor after acute pressure overload in the heart. The purpose of this study was to determine whether activation of the JAK-STAT pathway by Ang II is dependent on G proteins. Ang II (100 nmol/L for 120 minutes) caused formation of sis-inducing factor (SIF) complexes and tyrosine phosphorylation of STAT proteins in neonatal rat ventricular myocytes. The percentage of change in Ang II–stimulated SIF induction was not affected by pertussis toxin (PTX) or GP antagonist-2A, compounds that inhibit activation of Gi and Go proteins. In contrast, GP antagonist-2A, a peptide that selectively inhibits activation of Gq proteins, completely abolished Ang II–stimulated SIF induction and STAT3 tyrosine phosphorylation. Pretreatment of cardiac myocytes with U73122, an inhibitor of phosphatidylinositol-specific phospholipase C (PLC) activity, decreased Ang II–stimulated SIF induction and STAT3 tyrosine phosphorylation in a dose-dependent manner. Chelation of intracellular Ca2+ with BAPTA-AM did not alter Ang II–stimulated SIF induction. In contrast, pretreatment of cardiac myocytes with Ro-31-8220, a potent and specific inhibitor of protein kinase C (PKC), decreased Ang II–stimulated SIF induction in a dose-dependent manner. Ang II–stimulated SIF induction was abolished in cardiac myocytes after downregulation of PKC by treatment with PMA. From these data, we conclude that Ang II–stimulated SIF induction and STAT3 tyrosine phosphorylation is mediated by PTX-insensitive G proteins through a Gq-PLC-PKC–mediated pathway in neonatal rat ventricular myocytes.
The Janus kinase–signal transducers and activators of transcription (JAK-STAT) pathway was initially described as a pathway activated by ligand binding to cytokine receptors.1 2 This pathway is composed of STAT proteins, a family of latent transcription factors present in the cytoplasm. After ligand binding to cytokine receptors, STAT proteins become tyrosine phosphorylated on a single residue and form dimers, which then translocate to the nucleus and bind to specific sequences present in DNA to alter gene transcription. JAKs are a family of nonreceptor tyrosine kinases that associate directly with cytokine receptors and become activated by reciprocal tyrosine phosphorylation after ligand-induced receptor oligomerization. Activated JAKs phosphorylate residues in the cytoplasmic domain of cytokine receptors, which then serve as docking sites for STAT proteins. Recent evidence suggests that Src kinases, as well as JAKs, are capable of tyrosine phosphorylation of STAT proteins.2 Regardless of the identity of the tyrosine kinase involved, phosphorylation is an obligatory step in the formation of transcriptionally active complexes of STAT proteins.
The JAK-STAT pathway is activated by numerous cytokine receptors and represents a direct link between ligand binding to cell-surface receptors and changes in gene transcription.1 2 Recently, our group described activation of this pathway by ligand binding to a guanine nucleotide–binding regulatory protein (G protein)–coupled receptor (GPCR), the angiotensin II (Ang II) type 1 (AT1) receptor.3 The AT1 receptor contains 7 transmembrane spanning regions and, like cytokine receptors, lacks intrinsic tyrosine kinase activity. Until recently, it was thought that the AT1 receptor initiates signal transduction pathways only through activation of heterotrimeric G proteins. However, there is evidence to suggest that Ang II–stimulated, G protein–independent signal transduction pathways exist. Nonreceptor tyrosine kinases such as JAKs coimmunoprecipitate with the AT1 receptor,4 5 6 raising the possibility that this receptor-enzyme complex can initiate activation of the JAK-STAT pathway in a manner analogous to that described for cytokine receptors.
Ang II–stimulated activation of the JAK-STAT pathway has been demonstrated in cardiac myocytes,5 7 cardiac fibroblasts,3 and vascular smooth muscle cells.4 Ang II stimulates tyrosine phosphorylation of all ubiquitously expressed STAT proteins.3 4 5 7 8 9 The sequence of the AT1 receptor contains tyrosine-based motifs that may act as binding sites for STAT proteins,5 8 as described previously in cytokine receptors.10 Recent data from our group shows a ligand-dependent association of STAT proteins with the AT1 receptor.5 8 Little is known about the role of G proteins, if any, in the process of recruitment, and subsequent activation, of STAT proteins after binding of Ang II to the AT1 receptor.
Activation of STAT proteins may play an important role in modulating growth of cardiac and vascular smooth muscle cells. Ang II–induced proliferation of vascular smooth muscle cells is abolished by electroporation of antibodies to either STAT1 or STAT3.11 Activation of STAT1 and STAT3 proteins by tyrosine phosphorylation results in the formation of sis-inducing factor (SIF) complexes, which bind to the sis-inducing element (SIE) present in several genes, such as c-fos.3 Cytokines that induce SIF complex formation, such as leukemia inhibitory factor (LIF), are potent hypertrophic stimuli in cardiac myocytes.12 Ang II–stimulated SIF induction occurs in an in vitro,5 as well as in an in vivo,13 model of pressure-overload cardiac hypertrophy. DNA binding activity of STAT3 is increased in genetically hypertensive, compared with age-matched normotensive, rats.9 The results of these studies strongly suggest that activation of the JAK-STAT pathway by Ang II and other ligands plays a role in modulating physiological and/or pathophysiological growth of the heart.
Although significant progress has been made in understanding the mechanisms of activation of the JAK-STAT pathway by ligand binding to cytokine receptors, relatively little is known about the mechanisms of activation used by GPCR. In the study described here, we tested the hypothesis that activation of the JAK-STAT pathway by the AT1 receptor is mediated by G proteins. We also report the initial characterization of G proteins involved in Ang II–stimulated SIF induction and STAT3 tyrosine phosphorylation in neonatal rat ventricular myocytes.
GP antagonist-2A (H-Arg-Pro-Lys-Pro-Gln-Gln-D-Trp-Phe-D-Trp-D-Trp-Met-NH2), U73122, U73343, PMA, and Ro-31-8220 were purchased from Calbiochem. Pertussis toxin (PTX), GP antagonist-2 (Pyr-Gln-D-Trp-Phe-D-Trp-D-Trp-Met-NH2), and ionomycin were obtained from Biomol. BAPTA-AM was purchased from RBI. Additional reagents, unless otherwise noted, were purchased from Fisher Scientific or Sigma Chemical Company.
Preparation of Neonatal Rat Ventricular Myocytes
Primary cultures of ventricular myocytes were obtained from 1- to 2-day-old Sprague-Dawley rat pups as described previously (rats were raised in our facility).14 Approximately 24 hours after plating, myocytes were switched to a defined, serum-free Dulbecco modified Eagle medium:F-12 (Ham) (1:1) medium with 15 mmol/L HEPES (Gibco BRL) and supplemented with antibiotic/antimycotic solution (Gibco BRL), 10 ng/mL sodium selenate, 1 μg/mL transferrin, 3 mmol/L pyruvic acid, 100 μmol/L ascorbic acid, and 1 μg/mL insulin.15 The medium was replaced after 48 hours. After an additional 36 hours, neonatal rat ventricular myocytes were “starved” for 12 hours before the start of an experiment in the same medium as described above, minus ascorbic acid and insulin. By use of these methods, cultures that contained ≈90% to 95% myocytes were obtained.14 15
Experiments involving administration of GP antagonist-2A were performed in cardiac myocytes that were transiently permeabilized according to published methods.16 In brief, cardiac myocytes were gradually cooled and placed on ice. Cells were incubated for 10 minutes with ice-cold permeabilization buffer (20 mmol/L HEPES [pH 7.4], 10 mmol/L EGTA, 140 mmol/L KCl, 50 μg/mL saponin, 5 mmol/L NaN3, and 5 mmol/L oxalic acid dipotassium salt), with or without GP antagonist-2A. In addition, 200 mmol/L ATP (pH 7.4) was added (30 μL/ml permeabilization buffer) just before use. Cells were rinsed several times and incubated with PBS for 20 minutes on ice. Cardiac myocytes were gradually warmed, and the original medium was replaced. Cells were returned to the incubator and allowed to recover for 30 minutes before the start of an experiment.
Preparation of Nuclear Extracts
After treatment of myocytes with various pharmacological agents, cells were rinsed with ice-cold PBS and nuclear extracts were prepared.3 Myocytes were scraped and resuspended in 5 volumes of hypotonic buffer (10 mmol/L Tris-HCl [pH 7.5], 1.5 mmol/L MgCl2, 10 mmol/L KCl, 0.5 mmol/L PMSF, 0.5 mmol/L dithiothreitol [DTT], and 1 mmol/L Na3VO4), incubated on ice for 10 minutes, and sedimented. Cells were resuspended in 2 volumes of hypotonic buffer, Dounce homogenized, and sedimented. Pelleted nuclei were resuspended and incubated for 30 minutes on ice in high-salt buffer (20 mmol/L Tris-HCl [pH 7.5], 400 mmol/L NaCl, 1 mmol/L EDTA, 25% glycerol, 0.5 mmol/L PMSF, 0.5 mmol/L DTT, and 1 mmol/L Na3VO4). The supernatant was dialyzed for 8 to 12 hours against a low-salt buffer (20 mmol/L Tris-HCl [pH 7.5], 50 mmol/L KCl, 0.2 mmol/L EDTA, 20% glycerol, 0.5 mmol/L PMSF, 0.5 mmol/L DTT, and 1 mmol/L Na3VO4) and sedimented for 10 minutes at 4°C. Extracts were stored at −80°C before assay.
Electrophoretic Mobility Shift Assays
Electrophoretic mobility shift assays were performed as described previously.3 Five micrograms of nuclear extract proteins were incubated with 2 μg of poly(dI-dC) (Pharmacia Biotech) in cocktail buffer (10 mmol/L Tris-HCl [pH 7.5], 50 mmol/L KCl, 1 mmol/L EDTA, 5% glycerol, and 1 mmol/L DTT) for 10 minutes at room temperature. Samples were incubated with radiolabeled SIE oligonucleotide (5′-CAGTTCCCGTCAATC-3′) for 10 minutes at room temperature and resolved on a 4% native polyacrylamide gel. SIF induction was quantified on a STORM model 840 PhosphorImager with ImageQuaNT software (Molecular Dynamics).
Immunoblotting was performed with STAT3 pTyr(705) phosphospecific (Quality Controlled Biochemicals) or anti-ACTIVE mitogen-activated protein kinase (MAPK; Promega) polyclonal antibodies. For Western blotting of tyrosine-phosphorylated STAT3, 5 μg of nuclear extract proteins was resolved on an 8% SDS-polyacrylamide gel and transferred to nitrocellulose. For immunoblotting of the active form of MAPK, myocytes were lysed with radioimmunoprecipitation buffer (50 mmol/L Tris-HCl [pH 7.4], 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L PMSF, 1 mmol/L Na3VO4, and 1 mmol/L NaF). Cell lysates were incubated on ice for 15 minutes and then sedimented. Five micrograms of this whole-cell lysate were resolved on a 10% SDS-polyacrylamide gel and transferred to nitrocellulose. Signals were visualized with enhanced chemiluminescence (NEN Dupont).
Values are expressed as mean±SEM. Statistical analysis of dose-response experiments was performed by ANOVA, followed by Tukey-Kramer multiple comparisons tests. Statistical analysis of other experiments was performed with paired Student t tests. P<0.05 was considered statistically significant.
Ang II–Stimulated SIF Induction Is Mediated by PTX-Insensitive G Proteins in Neonatal Rat Ventricular Myocytes
The AT1 receptor couples to both PTX-sensitive and -insensitive G proteins.17 As a first step in determining the type of G proteins involved in SIF induction, we incubated myocytes with PTX (150 ng/mL for 24 hours)18 before stimulation with Ang II (100 nmol/L for 120 minutes). This treatment results in complete ADP ribosylation and inactivation of Gi and Go proteins in neonatal rat ventricular myocytes.18 The percentage of change in Ang II–stimulated SIF induction was not affected by PTX (control, 418.1%±86.8%; PTX, 404.0%±72.4%; n=5; P<0.84), suggesting that PTX-sensitive G proteins were not involved in Ang II–stimulated SIF induction.
We performed additional experiments to test the involvement of PTX-sensitive G proteins in Ang II–stimulated SIF induction. Nuclear extracts were prepared from cardiac myocytes pretreated with GP antagonist-2 (10 μmol/L for 15 minutes), a peptide derived from substance P that inhibits activation of Gi and Go proteins by blocking receptor–G protein interactions.19 GP antagonist-2 had no effect on the percentage of change in SIF induction in response to Ang II (100 nmol/L for 120 minutes; control, 239.1±44.3%; GP antagonist-2, 202.5±42.1%; n=3; P<0.59). Activation of MAPK by lysophosphatidic acid (LPA) in cardiac myocytes is reported to be mediated by PTX-sensitive G proteins.18 For this reason, we assessed the ability of GP antagonist-2 to inhibit LPA-stimulated MAPK activity in cardiac myocytes using an antibody specific for the dually phosphorylated, or active, form of MAPK.20 As expected, GP antagonist-2 significantly inhibited LPA-stimulated MAPK activity by ≈65% (control, 11.1±2.1-fold increase at 5 minutes; GP antagonist-2, 3.5±1.5-fold increase at 5 minutes, n=4; P<0.05). Thus, incubation of neonatal rat ventricular myocytes with GP antagonist-2 was effective in inhibiting signaling by PTX-sensitive G proteins. Together, these results confirm those obtained with PTX, indicating that Ang II–stimulated SIF induction in cardiac myocytes is not mediated by PTX-sensitive G proteins.
Gq Proteins Mediate Ang II–Stimulated SIF Induction and STAT3 Tyrosine Phosphorylation in Cardiac Myocytes
Our data strongly suggest the involvement of PTX-insensitive G proteins in mediation of SIF induction by the AT1 receptor. To test this hypothesis, cardiac myocytes were pretreated with GP antagonist-2A, a peptide derived from substance P that inhibits activation of Gq proteins by blocking receptor–G protein interactions.19 Because GP antagonist-2A is unable to cross the cell membrane, cardiac myocytes were transiently permeabilized to allow delivery of this peptide.16 GP antagonist-2A completely abolished Ang II–stimulated SIF induction (Figures 1A⇓ and 1B⇓). The principal complex formed by Ang II was identified as SIF-A, because this complex was supershifted by the addition of antibodies to STAT3 (data not shown). Because SIF-A complex is composed of a homodimer of tyrosine-phosphorylated STAT3,21 we measured the amount of this protein in nuclear extracts with a phosphospecific antibody. Results obtained by immunoblotting corroborated those obtained by electrophoretic mobility shift assay (Figure 1C⇓). In contrast, pretreatment of cardiac myocytes with this inhibitor had no effect on LIF-stimulated SIF induction (data not shown), suggesting that the effect of GP antagonist-2A on Ang II–stimulated SIF induction was due to inhibition of signaling by Gq proteins. These data support the hypothesis that Ang II–stimulated SIF induction and STAT3 tyrosine phosphorylation are mediated by Gq proteins in neonatal rat ventricular myocytes.
Phosphatidylinositol-Specific Phospholipase C Mediates SIF Induction and STAT3 Tyrosine Phosphorylation After Ang II Treatment in Neonatal Rat Ventricular Myocytes
Activation of phosphatidylinositol-specific phospholipase C (PLC) is mediated by Gq proteins.22 For this reason, we tested the effect of U73122, a phosphatidylinositol-specific PLC antagonist,23 on Ang II–stimulated SIF induction. Pretreatment of cardiac myocytes with U73122 for 15 minutes24 decreased Ang II–stimulated SIF induction in a dose-dependent manner (Figures 2A⇓ and 2B⇓). Complete inhibition of Ang II–stimulated SIF induction after pretreatment with U73122 (10 μmol/L for 15 minutes) correlated well with complete inhibition of phosphatidylinositol-specific PLC activity determined previously in neonatal rat ventricular myocytes.25 In contrast, pretreatment with U73343 (10 μmol/L for 15 minutes), an inactive (but structurally similar) derivative of U73122,23 had no effect on Ang II–stimulated SIF induction (control, 237.7%±19.33%; U73343, 266.7%± 45.8%; n=3; P<0.59). U73122 decreased the amount of tyrosine-phosphorylated STAT3 present in cardiac myocytes treated with Ang II (Figure 2C⇓). These data demonstrate that phosphatidylinositol-specific PLC plays a role in AT1 receptor–mediated activation of the JAK-STAT pathway. These results confirm and extend those obtained with GP antagonist-2A and support the hypothesis that Ang II–stimulated SIF induction and STAT3 tyrosine phosphorylation in cardiac myocytes is mediated by Gq proteins.
Protein Kinase C, but Not Intracellular [Ca2+], Mediates SIF Induction After Ang II Treatment in Cardiac Myocytes
The products of phosphatidylinositol-specific PLC activity, inositol-1,4,5-trisphosphate and sn-1,2-diacylglycerol, result in increased intracellular [Ca2+] and protein kinase C (PKC) activity, respectively. To assess the role of intracellular [Ca2+] in Ang II–stimulated SIF induction, neonatal rat ventricular myocytes were pretreated with BAPTA-AM (10 μmol/L for 30 minutes), a calcium chelator. This treatment was demonstrated to block Ang II–mediated increases in intracellular [Ca2+] (Sadoshima et al26 and data not shown). In the present study, chelation of intracellular [Ca2+] had no effect on Ang II–stimulated SIF induction (control, 281.5±35.1%; BAPTA-AM, 299.0±50.3%; n=6; P<0.78). In addition, administration of ionomycin (2 μmol/L), a calcium ionophore, did not increase SIF induction (97.7±7.4%; n=5) compared with controls. These results suggest that intracellular [Ca2+] does not mediate Ang II–stimulated SIF induction in cardiac myocytes.
To assess the role of PKC in Ang II–stimulated SIF induction, we treated cardiac myocytes with the phorbol ester PMA (200 nmol/L for 120 minutes). PMA caused a significant increase in SIF induction at 120 minutes (240.9%±43.3%; n=4) similar to that observed with Ang II. Pretreatment of cardiac myocytes with Ro-31-8220, a potent and specific PKC inhibitor,27 decreased Ang II–stimulated SIF induction in a dose-dependent manner (Figure 3A⇓). Finally, cardiac myocytes were pretreated with PMA (500 nmol/L for 48 hours) to downregulate PKC.14 As shown in Figure 3B⇓, downregulation of PKC completely abolished Ang II–stimulated SIF induction in cardiac myocytes. Collectively, these data strongly suggest a role of PKC in mediating Ang II–stimulated SIF induction in neonatal rat ventricular myocytes.
Nonreceptor tyrosine kinases such as JAKs coimmunoprecipitate with the AT1 receptor,4 5 6 raising the possibility that this receptor-enzyme complex can initiate activation of the JAK-STAT pathway in a manner analogous to that described for cytokine receptors (ie, independent of G proteins). The results of this study, however, argue against this possibility. Ang II activates both PTX-sensitive and -insensitive G proteins by binding to the AT1 receptor.17 We demonstrated the involvement of PTX-insensitive G proteins in AT1 receptor–mediated activation of the JAK-STAT pathway. These results are the first to describe involvement of G proteins in activation of this pathway by a member of the GPCR family.
Our results demonstrate the involvement of PTX-insensitive G proteins in Ang II–stimulated SIF induction and STAT3 tyrosine phosphorylation in neonatal rat ventricular myocytes. PTX-resistant G proteins include members of the Gq and G12 as well as Gz subfamilies.28 Expression of Gz is extremely limited.28 G12 proteins, however, are ubiquitously expressed,28 and one subfamily member (Gα13) has recently been reported to couple to the AT1 receptor in vascular myocytes.29 Two lines of evidence argue against involvement of G12 proteins in Ang II–stimulated SIF induction in ventricular myocytes. First, SIF induction was completely abolished by GP antagonist-2A, a peptide that selectively inhibits activation of Gq proteins.19 Second, pretreatment with U73122, a phosphatidylinositol-specific PLC antagonist,23 decreases SIF induction in a dose-dependent manner. Activation of phosphatidylinositol-specific PLC is mediated by Gq,22 not G12, proteins. These data provide strong evidence that Gq proteins mediate Ang II–stimulated SIF induction and STAT3 tyrosine phosphorylation in cardiac myocytes.
Ang II-stimulated SIF induction in neonatal rat ventricular myocytes is mediated by JAK activation.5 Indeed, pretreatment of cardiac myocytes with a selective JAK inhibitor completely abolishes STAT tyrosine phosphorylation.5 8 One of the most important questions in the field of Ang II–stimulated signal transduction is whether activation of nonreceptor tyrosine kinases (such as JAKs) occurs upstream of activation of G proteins. Studies are currently under way in our laboratory to determine whether activation of JAKs occurs before activation of Gq proteins by the AT1 receptor in cardiac myocytes.
Activation of the JAK-STAT pathway was described initially in cells after ligand binding to cytokine receptors.1 2 Shortly after the discovery of this pathway, our group and others3 4 demonstrated activation of the JAK-STAT pathway by Ang II via the AT1 receptor, a member of the GPCR family. Since then, ligands binding to additional members of this family have been shown to activate the JAK-STAT pathway, including endothelin-1,30 α-thrombin,31 and serotonin.32 The results of these studies suggest the importance of this pathway in GPCR-mediated signal transduction. Additional studies are needed to determine whether these ligands also use Gq proteins to activate the JAK-STAT pathway or whether additional G proteins are involved.
In summary, we conclude that Ang II–stimulated SIF induction and STAT3 tyrosine phosphorylation are mediated by PTX-insensitive G proteins in neonatal rat ventricular myocytes. In addition, we demonstrated that these G proteins are members of the Gq subfamily. Our results provide insight into the mechanism of activation of the JAK-STAT pathway by the AT1 receptor. Given the importance of this pathway in cell growth and gene expression, our results may contribute to our understanding of the signaling events mediating cardiac hypertrophy.
This work was supported by grants from the National Institutes of Health (HL-60529 and HL-58439 to K.M.B.), the American Heart Association (9650210N to K.M.B.), and the Geisinger Foundation. R.A.H. is the recipient of an Individual National Research Service Award (HL-09484). K.M.B. is an Established Investigator of the American Heart Association. We thank George W. Booz for his valuable comments on a draft of this manuscript as well as Lois L. Carl and Thomas J. Motel for their expert technical assistance.
The present address of G. Jayarama Bhat is University of Texas Health Center at Tyler, Medical Research Building C-6, PO Box 2003, Tyler, TX 75710.
- Received February 10, 1999.
- Revision received April 12, 1999.
- Accepted June 8, 1999.
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