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(Circulation Research. 2002;91:961.)
© 2002 American Heart Association, Inc.

Integrative Physiology

G_12/13 Mediates ₁-Adrenergic Receptor–Induced Cardiac Hypertrophy

Yoshiko Maruyama, Motohiro Nishida, Yoshiyuki Sugimoto, Shihori Tanabe, Justin H. Turner, Tohru Kozasa, Teiji Wada, Taku Nagao, Hitoshi Kurose

From the Laboratory of Pharmacology and Toxicology (Y.M., M.N., Y.S., S.T., T.N., H.K.) and the Laboratory of Physiological Chemistry (T.W.), Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo, Japan; the Department of Medicine (J.H.T.), Medical University of South Carolina, Charleston; and the Department of Pharmacology (T.K.), University of Illinois at Chicago. Dr Wada is now at Amgen Institute, Ontario Cancer Institute, and the Department of Medical Biophysics and Immunology, University of Toronto, Toronto, Ontario, Canada; Dr Sugimoto is at Kyowa Hakko Inc, Shizuoka, Japan; Dr Nagao is at the National Institute of Health Sciences, Tokyo, Japan; and Dr Kurose is at the Laboratory of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka, Japan.

Correspondence to Hitoshi Kurose, PhD, Laboratory of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan. E-mail kurose{at}phar.kyushu-u.ac.jp

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
In neonatal cardiomyocytes, activation of the G_q-coupled ₁-adrenergic receptor (₁AR) induces hypertrophy by activating mitogen-activated protein kinases, including c-Jun NH₂-terminal kinase (JNK). Here, we show that JNK activation is essential for ₁AR-induced hypertrophy, in that ₁AR-induced hypertrophic responses, such as reorganization of the actin cytoskeleton and increased protein synthesis, could be blocked by expressing the JNK-binding domain of JNK-interacting protein-1, a specific inhibitor of JNK. We also identified the classes and subunits of G proteins that mediate ₁AR-induced JNK activation and hypertrophic responses by generating several recombinant adenoviruses that express polypeptides capable of inhibiting the function of specific G-protein subunits. ₁AR-induced JNK activation was inhibited by the expression of carboxyl terminal regions of G_q, G₁₂, and G₁₃. JNK activation was also inhibited by the G_q/11- or G_12/13-specific regulator of G-protein signaling (RGS) domains and by C3 toxin but was not affected by treatment with pertussis toxin or by expression of the carboxyl terminal region of G protein–coupled receptor kinase 2, a polypeptide that sequesters Gß. ₁AR-induced hypertrophic responses were inhibited by G_q/11- and G_12/13-specific RGS domains, C3 toxin, and the carboxyl terminal region of G protein–coupled receptor kinase 2 but not by pertussis toxin. Activation of Rho was inhibited by carboxyl terminal regions of G₁₂ and G₁₃ but not by G_q. Our findings suggest that ₁AR-induced hypertrophic responses are mediated in part by a G_12/13-Rho-JNK pathway, in part by a G_q/11-JNK pathway that is Rho independent, and in part by a Gß pathway that is JNK independent.

Key Words: c-Jun NH₂-terminal kinase • G₁₂ family G proteins • ₁-adrenergic receptors • hypertrophy

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
In cardiomyocytes, stimulation of G-protein–coupled receptors activates mitogen-activated protein kinases, including c-Jun NH₂-terminal kinase (JNK), which can subsequently trigger hypertrophic responses.¹ ₁-Adrenergic receptors (₁ARs) are G_q-coupled receptors expressed in the heart, and they are thought to play a role in cardiac hypertrophy. Significant evidence points to an important role for G_q in receptor-induced hypertrophic responses. For example, ₁AR-induced hypertrophy can be inhibited by neutralizing antibodies that recognize G_q. In addition, a mutated M1 muscarinic acetylcholine receptor with impaired coupling to G_q, in contrast to the wild-type receptor, is unable to induce hypertrophy.^2,3 It has also been reported that cardiac hypertrophy can be induced by expressing a constitutively active mutant of G_q.⁴ In total, these results suggest that G_q plays an essential role in G_q-coupled receptor–mediated hypertrophy, a conclusion strengthened by evidence that overexpression of protein kinase Cß, which is activated by diacylglycerol, can induce hypertrophy in a transgenic animal model.⁵ Recent reports have demonstrated that various proteins acting downstream from G_q-coupled receptors, including JNK and RhoA, are also involved in ₁AR-induced hypertrophy.^6,7 ₁AR-induced JNK activation and hypertrophic responses can be inhibited by the expression of dominant-negative RhoA and Clostridium botulinum C3 toxin,⁶ whereas transgenic overexpression of wild-type G_q in the heart has been shown to induce hypertrophy.⁷ Consequently, it is generally thought that ₁AR-induced hypertrophy is mediated, in part, by G_q, RhoA, and JNK. The mechanism through which G_q activates Rho has yet to be fully established. In COS-7 cells, expression of G_q leads to a direct interaction between G_q and the Rho guanine nucleotide exchange factor (RhoGEF) but does not result in an associated increase in activated Rho.⁸ Much about the signal transduction mechanisms mediating G_q-dependent activation of JNK and Rho remain unresolved in cardiomyocytes. Also yet to be described is whether pertussis toxin (PTX)-insensitive G proteins other than G_q, such as G₁₂ and G₁₃, play a role in ₁AR-induced JNK activation and hypertrophy.

Many G_q-coupled receptors, including those for thrombin, thromboxane A₂, and lysophosphatidic acid, also couple to and activate G₁₂ and G₁₃.^9–11 These are members of a distinct subfamily of G-protein subunits; they are insensitive to PTX¹² and have previously been shown to regulate stress fiber formation, cell transformation, apoptosis, and activation of the Na⁺-H⁺ exchanger.¹³ GTPase-deficient mutants of G₁₂ and G₁₃ are capable of activating JNK in many cell types.^14,15 In rat neonatal cardiomyocytes, expression of a constitutively active mutant of G₁₃ induces hypertrophic responses, including increased gene expression of atrial natriuretic factor and ß-myosin heavy chain.¹⁶ A recent report establishing a link between G_12/13 and Rho, a small G protein, in an in vitro system showed that G₁₃ can directly stimulate p115-RhoGEF, thereby triggering Rho activation,^17,18 and that although p115-RhoGEF does not appear to be involved in activation of Rho by G₁₂, the regulator of the G-protein signaling (RGS) domain of p115-RhoGEF (p115-RGS) can bind G₁₂ as well.

There are a few available tools to probe the functions of PTX-insensitive G proteins, such as G_q and G_12/13. Among the more promising approaches are the RGS domains, which are polypeptides of 125 amino acids that interact with and inhibit activated G subunits.¹⁹ For example, the RGS domain of G-protein–coupled receptor kinase 2 (GRK2) interacts with G_q and blocks its activity.²⁰ Likewise, the RGS domain of p115-RhoGEF interacts with both G₁₂ and G₁₃ but does not bind G_q or G_i.¹⁸ Another promising tool is the carboxyl terminal portion of G subunits. Several groups have reported that carboxyl terminal fragments of G_q and G₁₃ can inhibit the interaction of receptors with G_q and G₁₃, respectively.^21–23 Therefore, we have used RGS domains and carboxyl terminal fragments of G subunits as inhibitors for analyzing G_q-, G₁₂-, and G₁₃-mediated signaling pathways. We have also constructed a carboxyl terminal region of GRK2 (GRK2-ct) that can bind to and block the function of Gß.

In the present study, we demonstrate that ₁AR stimulation activates JNK in both a G_12/13-Rho–dependent and G_q-Rho–independent manner. We also demonstrate that G_12/13 and Gß, in addition to G_q, are involved in ₁AR-induced hypertrophy.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials
DMEM, penicillin/streptomycin, PTX, and Thermoscript were purchased from Invitrogen. A DNA thermal cycler sequencing kit was purchased from Pharmacia Biotech. L-Phenylephrine hydrochloride (PE) and (±)-propranolol hydrochloride were from Sigma-Aldrich, and collagenase A was from Roche Molecular Biochemicals. Anti-JNK1 (SC-474 for immunoprecipitation), anti-JNK1 (SC-571 for Western blotting), anti-RhoA (SC-418), anti-G_q (SC-393), anti-G₁₂ (SC-409), anti-G₁₃ (SC-410), and horseradish peroxidase–conjugated anti-rabbit and anti-mouse IgG antibodies were purchased from Santa Cruz Biotechnology. Anti-G_q, anti-G₁₂, and anti-G₁₃ antibodies show the expected specificity. [-³²P]ATP and [³H]leucine were from Perkin-Elmer Life Sciences, the RNeasy kit was from Qiagen, and Alexa Fluor 594-phalloidin was from Molecular Probes.

Cloning of cDNA and Production of Recombinant Adenoviruses
Details of the primers used to amplify regions of G_q (G_q-ct, amino acids 305 to 359), G₁₂ (G₁₂-ct, amino acids 325 to 379), G₁₃ (G₁₃-ct, amino acids 322 to 378), p115-RhoGEF (p115-RGS, amino acids 1 to 252), RGS4 (entire coding region), and the RGS domain of GRK2 (GRK2-RGS, amino acids 1 to 188) have been described previously.²⁴ The entire coding region of C3 toxin was amplified, and its sequence was confirmed by a thermal cycler sequencing kit. We identified a sequence error at position 325 (G to A transition) in the original plasmid, which resulted in an amino acid change (Thr to Ala) at position 109. The carboxyl end of the p115-RGS construct was supplemented with a geranylgeranylation signal (Cys-Val-Leu-Leu) to facilitate translocation to the membrane.²⁴ Each polymerase chain reaction (PCR) product was inserted into the appropriate sites of pAdTrack-CMV or pShuttle-CMV, and recombinant adenoviruses were prepared according to the method of He et al.²⁵ Recombinant adenoviruses expressing GRK2-ct (amino acids 542 to 685) were produced as previously described.²⁶ Recombinant adenovirus expressing LacZ was provided by RIKEN DNA Bank and was amplified as previously described.²⁶ The JNK-binding domain of JNK-interacting protein-1 (JIP1-JBD, amino acids 127 to 281) was amplified by PCR with two primers (forward primer, 5'-GCGCGAATTCATCCTGGAGGACCTCAATATG-3'; reverse primer, 5'-CGCGCTCGAGTCAGCCTGTCTTCTCCAGCAC-CTGGGC-3'). After confirming the sequence, JIP1-JBD was inserted into KpnI and HindIII sites of pShuttle-CMV. To pull down activated Rho, a glutathione S-transferase (GST)-Rho–binding domain (RBD) fusion protein was constructed. The RBD of Rhotekin (amino acids 7 to 89) was PCR-amplified from cDNA freshly prepared from mouse brain RNA and confirmed by DNA sequencing. The resulting product was inserted into EcoRI and XhoI sites of pGEX-4T1, and the GST fusion protein (GST-RBD) was prepared by standard methods.

Preparation of Rat Neonatal Cardiomyocytes, Adenoviral Infection, and JNK Kinase Assay
Ventricular cardiomyocytes were isolated from 1-day-old Sprague-Dawley rat hearts and cultured as described previously.^24,26 Cardiomyocytes were plated on 1% gelatin-coated plates and cultured in DMEM supplemented with 5% FBS. Myocytes were seeded at a density of 7x10⁶ cells per 6-cm dish for JNK kinase assay, 3.5x10⁶ cells per well of a 6-well plate for Rho activation assay and intracellular cAMP measurement, 5x10⁵ cells per well of a 12-well plate for [³H]leucine incorporation, and 3x10⁵ cells per well of a 4-well slide chamber for actin staining. After 24 hours, the cells were infected with recombinant adenoviruses at a multiplicity of infection of 100 or 300 for 2 hours at 37°C. Cells were then starved in serum-free DMEM containing 10 nmol/L insulin and 5 mmol/L taurine and cultured for an additional 48 hours before treatment. Under these conditions, infection with adenoviruses coding for LacZ or green fluorescent protein resulted in almost 100% of cells that were LacZ or green fluorescent protein positive. JNK kinase activity in an immunocomplex prepared from stimulated or unstimulated cells was determined as previously described.²⁴

Rho Activation Assay
Rho activation was determined by a pull-down assay using GST-RBD.²⁷ Forty-eight hours after adenovirus infection, cardiomyocytes were treated with 30 µmol/L PE (PE is always supplemented with 1.5 µmol/L propranolol to block the ß-stimulating activity of PE) for 1 minute and harvested in lysis buffer (in mmol/L: NaCl 150, MgCl₂ 30, Tris-HCl [pH 7.5] 50, dithiothreitol 1, and phenylmethylsulfonyl fluoride 1) supplemented with 0.1% Triton X-100, 10% glycerol, 1 µg/mL leupeptin, and 1 µg/mL aprotinin. Cell lysates were homogenized and centrifuged at 25 000g for 10 minutes at 4°C. The supernatant was then incubated with 12 µg of GST-RBD and glutathione-Sepharose beads for 1 hour at 4°C. The beads were washed twice with lysis buffer, and proteins were eluted with SDS-sample buffer, boiled, and subjected to Western blot analysis as previously described.^24,26 Densitometry was analyzed using Image Gauge (FUJIFILM), an image analysis program . Aliquots of cell lysates from each sample were subjected to Western blot analysis to confirm that equal amounts of RhoA were present under each condition.

Hypertrophic Responses
Protein synthesis and actin reorganization were monitored by [³H]leucine incorporation into trichloroacetic acid–precipitable materials and by staining with Alexa Fluor 594-phalloidin, respectively.

Intracellular cAMP Measurement
Twenty-four hours after infection, rat neonatal cardiomyocytes were labeled with [³H]adenine for 24 hours, washed with serum-free DMEM, and stimulated by each agonist for 15 minutes. Intracellular cAMP accumulation was determined as previously described.²⁸

Statistical Analysis
Statistical significance was evaluated by ANOVA, followed by the Dunnett test.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
JNK Activation and Hypertrophic Responses
To delineate the signal transduction cascades mediating ₁AR-induced JNK activation in rat neonatal cardiomyocytes, it is necessary to introduce foreign cDNAs or cDNA fragments into the cells. However, rat neonatal cardiomyocytes resist typical transfection procedures, such as calcium phosphate precipitation and lipid-mediated transfection. As an alternative, we decided to produce various recombinant adenoviruses that express polypeptides that inhibit protein-protein interactions between different cellular signaling components. To determine whether JNK mediates ₁AR-induced hypertrophic responses, we expressed JIP1-JBD, a specific inhibitor of JNK. ₁AR stimulation by PE increased JNK activity by 5-fold (Figure 1A), which was blocked almost completely by the expression of JIP1-JBD. H₂O₂ also produced a marked increase in JNK activation, which was inhibited by the expression of JIP1-JBD. Prolonged ₁AR-stimulation induced hypertrophic responses such as increased protein synthesis and reorganization of the actin cytoskeleton (Figures 1B and 1C). These responses were also inhibited by JIP1-JBD, suggesting that JNK activation is essential for the hypertrophic responses induced by the ₁AR.

View larger version (23K): [in this window] [in a new window]   Figure 1. Effects of JIP1-JBD on PE-induced JNK activation and hypertrophic responses. Cardiomyocytes were infected with adenoviruses coding either LacZ or JIP1-JBD. A, Forty-eight hours after infection, cells were stimulated with 30 µmol/L PE for 30 minutes or 5 µmol/L H2O2 for 15 minutes, and JNK activities were determined. Representative autoradiogram of JNK kinase assay is shown (top). Data are shown as mean±SE of at least 3 independent experiments (graph). Western blot analysis confirms that equal amounts of JNK were used for immunoprecipitation under each condition (p54 JNK, p46 JNK). *P<0.01 vs LacZ+PE; **P<0.01 vs LacZ+H2O2. B, Twenty-four hours after infection, myocytes were stimulated with 30 µmol/L PE for 48 hours. [3H]Leucine (2 µCi/mL) was added to the culture medium 8 hours before harvest. [3H]Leucine incorporation into cells was determined as described in Materials and Methods. Data are shown as mean±SE of at least 3 independent experiments. *P<0.01 vs LacZ+PE. C, Twenty-four hours after infection, cardiomyocytes were stimulated with 30 µmol/L PE for 48 hours. The cells were then washed, fixed, and stained with Alexa Fluor 594-phalloidin to visualize actin filaments. Bar=50 µm.

Regulation of ₁AR-Induced JNK Activation by G_q, G₁₂, and G₁₃
We have produced recombinant adenoviruses coding the carboxyl terminal regions of Gq (G_q-ct), G₁₂ (G₁₂-ct), and G₁₃ (G₁₃-ct). Previously, we demonstrated that these G-ct constructs can inhibit receptor–G-protein coupling with subunit-specific selectivity.²⁴ In CHO-K1 cells, adenovirus-mediated expression of G_q-ct, but not G₁₂-ct and G₁₃-ct, inhibited the ATP-induced increase in [Ca²⁺] by activation of phospholipase C. In contrast, ATP-induced actin polymerization, possibly mediated through Rho, was inhibited by G₁₂-ct and G₁₃-ct, but not G_q-ct. The expression of G-ct constructs was previously confirmed by reverse transcription–PCR. Before further examining the selectivity of these constructs, we also confirmed the expression of G₁₂, G₁₃, and G_q in membranes of neonatal cardiomyocytes by Western blot (Figure 2A. These results indicate that these constructs are capable of competing with endogenously expressed G₁₂, G₁₃, and G_q. We next examined whether G_q-ct, G₁₂-ct, and G₁₃-ct could affect G_s-mediated, G_i-mediated, or receptor-independent signaling pathways. Figure 2B shows that isoproterenol-stimulated (G_s-mediated) cAMP accumulation and carbachol-stimulated (G_i-mediated) inhibition of isoproterenol-induced cAMP accumulation were not affected by G_q-ct, G₁₂-ct, or G₁₃-ct. H₂O₂ has previously been shown to strongly activate JNK in neonatal cardiomyocytes, although the precise mechanism through which this occurs remains unknown. Again, expression of G_q-ct, G₁₂-ct, and G₁₃-ct did not affect H₂O₂-induced (receptor-independent) JNK activation (Figure 2C). These and previous results demonstrate that G_q-ct, G₁₂-ct, and G₁₃-ct selectively inhibit receptor-G_q, -G₁₂, and -G₁₃ coupling, respectively.^21,24 As shown in Figure 3, rat neonatal cardiomyocytes expressing G_q-ct, G₁₂-ct, or G₁₃-ct were stimulated with 30 µmol/L PE for 30 minutes, and JNK activity was determined. Overexpression of G_q-ct strongly inhibited PE-induced JNK activation, as did overexpression of G₁₂-ct and G₁₃-ct, suggesting that JNK activation by ₁AR stimulation is mediated by G_q, G₁₂, and G₁₃.

View larger version (42K): [in this window] [in a new window]   Figure 2. Failure of carboxyl terminal regions of G-protein subunits to affect intracellular cAMP production and H2O2-induced JNK activation. A, Expression of endogenous Gq, G12, and G13 in rat neonatal cardiomyocytes was examined by Western blotting. Cytosolic fraction was prepared from cardiomyocytes and subjected to SDS-polyacrylamide gel electrophoresis.24 The proteins were transferred to polyvinylidine difluoride membrane by a semidry method. Gq, G12, and G13 were detected by specific antibody. Cardiomyocytes were infected with adenoviruses coding LacZ, Gq-ct, G12-ct, or G13-ct. B, Twenty-four hours after infection, 2 µCi/mL [3H]adenine was added to the culture medium, and the cells were incubated for 24 hours. Cells were then stimulated with 10 µmol/L isoproterenol (Iso) or 10 µmol/L Iso and 1 mmol/L carbachol (CCh) for 15 minutes, and intracellular cAMP production was determined as described in Materials and Methods. cAMP production was expressed as a percentage relative to cells overexpressing LacZ and treated with 100 µmol/L forskolin. Data represent mean±SE of 3 independent experiments. C, Forty-eight hours after infection, cells were stimulated with 5 µmol/L H2O2 for 15 minutes, and JNK activities were determined. Representative autoradiogram of JNK kinase assay is shown (top). Data are shown as mean±SE of at least 3 independent experiments (graph). Western blot analysis confirms that equal amounts of JNK were used for immunoprecipitation under each condition (p54 JNK, p46 JNK).

View larger version (36K): [in this window] [in a new window]   Figure 3. Effects of carboxyl terminal regions of G-protein subunits on PE-induced JNK activation. Cardiomyocytes were infected with adenoviruses coding LacZ, Gq-ct, G12-ct, or G13-ct. Forty-eight hours after infection, cells were stimulated with 30 µmol/L PE for 30 minutes and harvested. JNK activity was determined by immune complex kinase assay using GST-c-Jun as described in Materials and Methods. Representative result of kinase assay is shown (top). JNK activity is expressed as fold increase relative to unstimulated control cells. Data represent mean±SE of 3 independent experiments (graph). Western blot analysis confirms that equal amounts of JNK were used for immunoprecipitation under each condition (p54 JNK, p46 JNK). *P<0.01 vs LacZ+PE.

We next determined which G-protein subunits may be involved in PE-induced JNK activation. To examine the involvement of subunits of G_q, G₁₂, and G₁₃, we produced adenoviruses coding RGS domains of GRK2 (GRK2-RGS) and p115-RhoGEF (p115-RGS), which function as specific inhibitors of G_q and G₁₂/G₁₃, respectively. We also produced adenoviruses coding RGS4, which inhibits both G_q and G_i. JNK activation after PE stimulation was strongly inhibited by p115-RGS, RGS4, and GRK2-RGS (Figures 4A and 4B). Pretreatment of cells with 100 ng/mL PTX, which ADP-ribosylates G_i and uncouples receptors from G_i, had no effect on PE-induced JNK activation (Figure 4C). We also examined the role of Gß using GRK2-ct, which binds Gß and blocks Gß-mediated signaling pathways. GRK2-ct did not affect PE-induced JNK activation (Figure 4D), suggesting that a Gß-mediated signal is not required for JNK activation by the ₁AR. These results indicate that in cardiomyocytes JNK activation by the ₁AR is mediated by the activation of G_q, G₁₂, and G₁₃, but not Gß or G_i.

View larger version (49K): [in this window] [in a new window]   Figure 4. Effects of various RGS constructs, PTX treatment, and GRK2-ct on PE-induced JNK activation. A and B, Cardiomyocytes were infected with adenoviruses coding LacZ, p115-RGS, or RGS4 (A) or LacZ or GRK2-RGS (B). C, Cardiomyocytes were pretreated with PTX for 16 hours. D, Cardiomyocytes were infected with adenoviruses coding LacZ or GRK2-ct. Forty-eight hours after infection or 16 hours after PTX pretreatment, cells were stimulated with 30 µmol/L PE for 30 minutes, and JNK activities were determined. Representative autoradiogram of JNK kinase assay is shown (top). JNK activity is expressed as fold increase relative to unstimulated control cells expressing LacZ. Data represent mean±SE of at least 3 independent experiments (graph). Western blot analysis confirms that equal amounts of JNK were used for immunoprecipitation under each condition (p54 JNK, p46 JNK). *P<0.01 vs LacZ+PE.

Role of Rho in ₁AR-Induced JNK Activation
Present knowledge suggests that Rho, a small G protein, is activated downstream from G₁₂ and G₁₃. In the present study, we have examined the contribution of Rho to ₁AR-induced JNK activation. C3 toxin is frequently used to demonstrate an involvement of Rho in cellular functions, inasmuch as it has the ability to ADP-ribosylate and inactivate Rho. In cardiomyocytes, the expression of C3 toxin inhibited JNK activation after stimulation by PE (Figure 5A) and completely inhibited PE-induced Rho activation (Figure 5B), suggesting that Rho is involved in ₁AR-induced JNK activation. We next examined whether G_q, G₁₂, or G₁₃ contributes to the PE-induced activation of Rho. ₁AR stimulation increased the amount of activated Rho that was pulled down by GST-RBD by 4-fold, suggesting that ₁AR stimulation activates Rho in neonatal cardiomyocytes (Figure 6). This activation could be inhibited by G₁₂-ct and G₁₃-ct, but not G_q-ct. These results suggest that JNK is activated through G_12/13-Rho–dependent and G_q-Rho–independent pathways in a concerted manner.

View larger version (30K): [in this window] [in a new window]   Figure 5. Involvement of Rho in PE-induced JNK activation. Cardiomyocytes were infected with adenoviruses coding LacZ or C3 toxin. A, Forty-eight hours after infection, cells were stimulated with 30 µmol/L PE for 30 minutes, and JNK activity was determined. Representative result of JNK kinase assay is shown (top). Data are shown as mean±SE of 3 independent experiments (graph). Western blot analysis confirms that equal amounts of JNK were used for immunoprecipitation under each condition (p54 JNK, p46 JNK). *P<0.01 vs LacZ+PE. B, Forty-eight hours after infection, cells were stimulated with 30 µmol/L PE for 1 minute, and Rho activity was determined by pull-down assay with GST-RBD as described in Materials and Methods. Representative result of Rho activation assay is shown (top). Rho activity is expressed as fold stimulation relative to unstimulated control cells. Data are shown as mean±SE of at least 3 independent experiments (graph). Western blot analysis confirms that equal amounts of Rho were used for pull-down assay under each condition (bottom of panel). *P<0.01 vs LacZ+PE.

View larger version (33K): [in this window] [in a new window]   Figure 6. Effects of carboxyl terminal regions of G-protein subunits on PE-induced Rho activation. Cardiomyocytes were infected with adenoviruses coding LacZ, Gq-ct, G12-ct, or G13-ct. Forty-eight hours after infection, cells were stimulated with 30 µmol/L PE for 1 minute and harvested. Rho activity was determined as described in Materials and Methods. Representative result of Rho activation assay is shown (top). Rho activity is expressed as fold stimulation relative to unstimulated control cells. Data are shown as mean±SE of at least 3 independent experiments (graph). Western blot analysis confirms that equal amounts of Rho were used for pull-down assay under each condition (bottom of panel). *P<0.01 vs LacZ+PE.

Involvement of G_12/13 in Hypertrophic Response Induced by ₁AR
We next investigated the roles of individual G-protein subunits in ₁AR-induced hypertrophic responses. Measures of hypertrophy, including reorganization of the actin cytoskeleton and changes in protein synthesis, were evaluated by staining actin filaments with phalloidin and by measuring the incorporation of ³H into cellular proteins, respectively. Compared with no stimulation of cardiomyocytes, stimulation of the ₁AR with 30 µmol/L PE for 48 hours induced drastic reorganization of actin filaments (Figures 7A and 7B). ₁AR-induced reorganization of actin filaments was not inhibited by PTX treatment (Figure 7A), consistent with previous results showing that PTX does not affect ₁AR-induced JNK activation. In contrast, ₁AR-induced actin reorganization was inhibited by p115-RGS, GRK2-RGS, and C3 toxin (Figure 7B). Figure 7B also shows that GRK2-ct could inhibit ₁AR-induced actin reorganization, indicating that Gß contributes to actin reorganization through a signaling pathway that does not involve JNK activation. Measurements of protein synthesis rate produced similar results. ₁AR stimulation increased the rate of protein synthesis by 2-fold (Figures 8A and 8B). This increase in protein synthesis was not affected by PTX treatment (Figure 8A) but was inhibited by p115-RGS, GRK2-RGS, GRK2-ct, and C3 toxin (Figure 8B).

View larger version (43K): [in this window] [in a new window]   Figure 7. PE-induced actin reorganization. A, Cardiomyocytes were pretreated with 100 ng/mL PTX for 16 hours. B, Cardiomyocytes were infected with adenoviruses coding LacZ, p115-RGS, GRK2-RGS, GRK2-ct, or C3 toxin. Sixteen hours after PTX pretreatment or 24 hours after infection, cells were stimulated with 30 µmol/L PE for 48 hours. After stimulation, cells were washed, fixed, and stained with Alexa Fluor 594-phalloidin to visualize actin filaments. Bar=50 µm.

View larger version (31K): [in this window] [in a new window]   Figure 8. Incorporation of [3H]leucine induced by PE. A, Cardiomyocytes were pretreated with 100 ng/mL PTX. B, Cardiomyocytes were infected with adenoviruses coding LacZ, p115-RGS, GRK2-RGS, GRK2-ct, or C3 toxin. Sixteen hours after PTX pretreatment or 24 hours after infection, cells were stimulated with 30 µmol/L PE for 48 hours. [3H]Leucine (2 µCi/mL) was added to the culture medium 8 hours before harvest. [3H]Leucine incorporation was then determined as described in Materials and Methods. Data are shown as mean±SE of at least 3 independent experiments. *P<0.01 vs LacZ+PE; #P<0.05 vs LacZ+PE.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the present study, we demonstrated that ₁AR stimulation activates JNK via G_q- and G_12/13-mediated pathways in rat neonatal cardiomyocytes. This finding is consistent with previous reports indicating that transient expression of wild-type or constitutively active mutants of G_q and G₁₃ can induce JNK activation.^14–16,29 To demonstrate an involvement of G_q, G₁₂, and G₁₃ in ₁AR-induced JNK activation in cardiomyocytes, we used unique reagents, such as RGS domains and carboxyl terminal fragments of G subunits. Expression of G_q-ct, G₁₂-ct, and G₁₃-ct inhibited JNK activation, indicating that G_q, G₁₂, and G₁₃ are involved in ₁AR-induced JNK activation. This conclusion is further supported by data showing that overexpression of RGS domains specific for G_q, G₁₂, or G₁₃ also significantly or almost completely inhibited ₁AR-induced JNK activation. The RGS domains inhibited JNK activation with a rank order of potency of p115-RGS>GRK2-RGS>RGS4. Whether these differences reflect the differential contributions of the various G subunits to ₁AR-mediated JNK activation remains unclear, inasmuch as the expression levels and K_i values of the various inhibitory peptides could not be determined. Nonetheless, the present study clearly demonstrates that RGS domains and G-ct are useful tools for delineating the role of individual PTX-insensitive G subunits in intracellular signal transduction pathways.

The inhibition of JNK activation by GRK2-RGS, RGS4, and p115-RGS suggests that G_q or G_12/13 alone is not sufficient for ₁AR-induced JNK activation and that a concerted contribution by G_q, G₁₂, and G₁₃ may instead be necessary. Activation of Rho by the ₁AR was inhibited by G₁₂-ct and G₁₃-ct, but not G_q-ct. Therefore, G_q- and G₁₂/G₁₃-derived signals converge at a point after Rho activation but preceding JNK activation. In the future, it will be necessary to determine the specific target molecule(s) of G_q to precisely delineate the JNK activation pathway. We also showed that Gß is not involved in ₁AR-induced JNK activation in rat neonatal cardiomyocytes. The inability of GRK2-ct and PTX to affect JNK activation cannot be explained by either low expression of GRK2-ct or insufficient ADP-ribosylation of G_i by PTX, because under the same conditions, we observed that GRK2-ct and PTX treatment can block endothelin-1–induced ERK activation.²⁴ Two groups have previously reported a role for Gß in the activation of JNK by G_q-coupled receptors in HEK293 cells.^29,30 It is possible that various cells may express different signaling components at different levels and that the relative contributions of these signaling components to JNK activation may differ in both a cell-type–specific and receptor-dependent manner.

The present study also demonstrated an essential role for G_12/13 in ₁AR-induced hypertrophy. Finn et al¹⁶ observed in rat neonatal cardiomyocytes that the transient overexpression of a constitutively active mutant of G₁₃ causes hypertrophic responses, such as increased gene expression of atrial natriuretic factor and ß-myosin heavy chain, and an increase in cell size. Consistent with that report, we have shown that inhibition of G_12/13 by p115-RGS dramatically decreases ₁AR-induced hypertrophic responses. Many groups have also reported that G_q is essential for ₁AR-mediated hypertrophy.^2–4,7 Likewise, we have shown that inhibition of G_q by GRK2-RGS blocks ₁AR-induced hypertrophic responses. These results suggest an important role for G₁₂ and G₁₃, in addition to G_q, in ₁AR-induced hypertrophy. Furthermore, these results suggest that G_q-, G₁₂-, and G₁₃-mediated signal transduction pathways are all necessary to fully induce hypertrophic responses by ₁AR stimulation. However, inasmuch as G₁₂-ct and G₁₃-ct, but not G_q-ct, inhibited Rho activation, whereas G₁₂-ct, G₁₃-ct, and G_q-ct inhibited JNK activation, it appears that G_q activates JNK in a Rho-independent manner.

We have also demonstrated that Gß inhibition by GRK2-ct can inhibit the PE-induced reorganization of the actin cytoskeleton and the increased protein synthesis, suggesting that Gß also plays a role in ₁AR-induced cardiac hypertrophy. The inhibitory action of GRK2-ct may be due to an inhibition of ERK activation. Yue et al³¹ reported that inactivation of ERK by U0126 can inhibit the hypertrophic responses induced by PE. Conversely, Gß could target a protein involved in Rho activation. In HeLa but not Swiss 3T3 cells, expression of Gß induces stress fiber formation in a Rho-dependent manner.³² In rat neonatal cardiomyocytes, expression of a dominant-negative mutant of Rho inhibits the hypertrophic response induced by PE.⁶ Therefore, it is possible that Gß may induce hypertrophic responses through the activation of Rho.

Few reports have investigated the role of Gß in ₁AR-induced hypertrophy. A recent study showed that in vivo pressure-overload hypertrophy activates phosphatidylinositol 3-kinase and Akt through G_q-coupled receptor stimulation and subsequent Gß dissociation in the heart.³³ However, blockade of Gß function by GRK2-ct in a transgenic mouse heart fails to affect pressure overload–induced cardiac hypertrophy.³³ However, this pressure overload–induced hypertrophy could be attenuated by cardiac-specific overexpression of G_q-ct, which inhibits receptor-G_q coupling.^21,33 Further studies will be necessary to elucidate the role of Gß in the induction of hypertrophic responses and to define the relationship between Gß and Rho activation in cardiomyocytes.

In summary, we have demonstrated in cardiomyocytes that the ₁AR activates G_q, G₁₂, and G₁₃ and that ₁AR-induced JNK activation is mediated via the activation of G_q, G₁₂, and G₁₃, but not G_i or Gß. We have also shown that G_12/13 and G_q are required for ₁AR-induced hypertrophy.

	Acknowledgments

 
This study was supported by a grant from the Ministry of Education, Science, Sports, and Culture of Japan (to T.N. and H.K.). We thank Dr Melvin I. Simon for mouse G₁₂ and G₁₃ cDNAs and Dr Robert J. Lefkowitz for rat GRK2 cDNA. We also thank the RIKEN DNA Bank for adenovirus expressing LacZ.

Received March 28, 2002; revision received September 9, 2002; accepted October 14, 2002.

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