G (cid:1) 12/13 - and Reactive Oxygen Species-dependent Activation of c-Jun NH 2 -terminal Kinase and p38 Mitogen-activated Protein Kinase by Angiotensin Receptor Stimulation in Rat Neonatal Cardiomyocytes*

In the present study, we examined signal transduction mechanism of reactive oxygen species (ROS) production and the role of ROS in angiotensin II-induced activation of mitogen-activated protein kinases (MAPKs) in rat neonatal cardiomyocytes. Among three MAPKs, c-Jun NH 2 -terminal kinase (JNK) and p38 MAPK required ROS production for activation, as an NADPH oxidase inhibitor, diphenyleneiodonium, inhibited the activation. The angiotensin cod- ing PrxII The sequence coding Cdc42/Rac interactive binding domain of PAK was cloned from mouse brain, sequenced, and ligated into pGEX-4T-1 to make GST fusion protein construct. GST-TPR construct was GST fusion proteins were expressed at room temperature and purified using glutathione-Sepharose as described (28, 29). Rat AT1R was cloned from rat heart, and the sequence was confirmed. Anti-phospho-ERK and anti-ERK antibodies were purchased from Cell Signaling. Anti-phos-pho-p38 MAPK and anti-p38 MAPK antibodies were from Promega. Anti-G (cid:2) protein GRK2-ct domain

Ang II 1 is a bioactive peptide involved in cardiac hypertrophy (1). Receptor stimulation by Ang II is assumed to activate G q and G i and turns on various signaling cascades dependent on cell types. Many groups have reported the regulation of MAPKs including JNK (2,3), ERK (4,5), and p38 MAPK (6) in a variety of cells (7). MAPKs are thought to be key intracellular transducers of mitogenic stimulation and have been implicated in the signaling pathways leading to cardiac hypertrophy (8,9). An earlier study demonstrated that Ang II-induced JNK activation is dependent on extracellular calcium and protein kinase C and partially on a tyrosine kinase (2). On the other hand, Ang II-induced ERK activation is mediated by the Ras pathway or protein kinase C pathway (10). A recent study showed that ␤-arrestin-mediated internalization of ATR is involved in ERK activation (11). ERK or p38 MAPK activation by Ang II requires the EGF receptor transactivation, whereas JNK activation is regulated by other signaling proteins (7). These results indicate that the signal transduction mechanism of MAPK activation depends on the types of MAPKs and cellular contexts that are analyzed.
ROS such as hydrogen peroxide and oxygen radicals play various roles in living cells as a second messenger to elicit physiological responses or as a toxic intermediate leading to cellular damage (12). Recent studies suggest that ROS work as regulators of signal transduction (13,14). We have reported that heterotrimeric G i/o proteins are putative target molecules of ROS (15,16). Although Ang II produces ROS in vascular smooth muscle cells and cardiac myocytes (6,17,18), the molecular mechanism of ROS production and the identification of ROS target molecules are largely unknown.
Rac, one of the small GTP-binding proteins, is believed to participate in the production of ROS by activating NADPH oxidase in neutrophil (19). Previous findings have demonstrated that one of the Rac effector PAKs mediates JNK activation by Ang II, and PAK phosphorylates a subunit of the NADPH oxidase complex (20). MAPKs are activated by Rac as well as RhoA and Raf-1, and the resulting activation of MAPKs induces hypertrophic responses through the activation of intracellular signaling cascades (21)(22)(23)(24). However, upstream molecules of and the relationship between these intracellular signaling molecules are not fully determined. G 12 family G proteins, G 12 and G 13 , couple with various G protein-coupled receptors and mediate physiological responses by interacting with different signaling proteins (25). The role of G 12/13 in the heart, however, has not been revealed because of the unavailability of a specific inhibitor. Recent studies showed that p115RhoGEF has an RGS domain for G␣ 12/13 (26,27). We examined by using the RGS domain of p115RhoGEF whether G 12/13 is involved in Ang II-mediated signal transduction pathway and ROS production and whether ROS work as a mediator in cardiac myocytes. To demonstrate the signal transduction cascade, we constructed various recombinant adenoviruses coding G␣ 12/13 -or G␣ q -specific RGS domains and DN-Rac. We demonstrate in the present study that Ang II-induced JNK and p38 MAPK activation requires ROS, and Ang II-induced ROS production is mediated by sequential activation of G 12/13 , Rho, and Rac.

EXPERIMENTAL PROCEDURES
Materials and Plasmid Construction-AT1R blocker CV11974 was provided from Takeda Chemical Industries Ltd. (Osaka, Japan). AG1478, PTX, and Y27632 were purchased from Calbiochem. DPI, N-acetyl-L-cysteine, catalase, and PD123319 were from Sigma. Fura-2/AM was from Dojindo (Kumamoto, Japan). Collagenase and FuGENE 6 were from Roche Applied Science. 2Ј,7Ј-Dichlorofluorescein diacetate was from Molecular Probe. Glutathione-Sepharose beads were from Amersham Biosciences. The cDNA coding DN-Rac1 was provided by Dr. Kozo Kaibuchi (Nagoya University, Nagoya, Japan). The plasmid coding PrxII was provided by Dr. Sue Goo Rhee (National Institutes of Health). The sequence coding Cdc42/Rac interactive binding domain of PAK was cloned from mouse brain, sequenced, and ligated into pGEX-4T-1 to make GST fusion protein construct. GST-TPR construct was provided by Dr. Manabu Negishi (Kyoto University, Kyoto, Japan). GST fusion proteins were expressed at room temperature and purified using glutathione-Sepharose as described (28,29). Rat AT1R was cloned from rat heart, and the sequence was confirmed. Anti-phospho-ERK and anti-ERK antibodies were purchased from Cell Signaling. Anti-phospho-p38 MAPK and anti-p38 MAPK antibodies were from Promega. Anti-G␣ 13 , anti-RhoA, horseradish peroxidase-conjugated anti-rabbit IgG, and anti-mouse IgG antibodies were from Santa Cruz Biotechnology. Anti-Rac1 antibody was from Transduction Laboratories.
Determination of JNK, ERK, and p38 MAPK Activation-The rat neonatal cardiac myocytes were isolated from 1-2-day-old Sprague-Dawley rat pups as previously described (15). The activity of JNK was determined as described (32,33). ERK and p38 MAPK activation were determined by Western blot analysis using phospho-specific antibodies (32,33). The optical density of the film was scanned and measured with Scion Image Software.
Quantification of Intracellular ROS Produced by Ang II Receptor Stimulation-ROS production was measured with the fluorescent dye DCF as described (34). The cardiomyocytes were plated on glass-bottomed 35-mm dishes and incubated for 10 min with 5 M DCF. The DCF fluorescence at an emission wavelength of 510 nm was observed at room temperature by exciting DCF at 488 nm using a video image analysis system (Aquacosmos, Hamamatsu Photonics).
Measurement of Rac Activity-Activation of Rac was measured by the method of Ren and Schwartz (35) with a slight modification. Forty-eight h after adenovirus infection, the cardiomyocytes were stimulated by Ang II and lysed in buffer containing 50 mM Tris (pH 7.2), 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS, 500 mM NaCl, 10 mM MgCl 2 , 10 g/ml leupeptin, 10 g/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, and 4 g of GST-PAK-Cdc42/Rac interactive binding domain. Supernatant was incubated with glutathione-Sepharose beads for 2 h at 4°C. The beads were washed and finally suspended in SDS sample buffer. Pulled down Rac was detected with anti-Rac1 antibody.
Measurement of Rho Activity-Rho activation was determined by the method of Maruyama et al. (32). Cells were stimulated by Ang II (100 nM) for 1 min, and lysed in buffer containing 50 mM Tris (pH 7.5), 0.1% Triton X-100, 10% glycerol, 150 mM NaCl, 30 mM MgCl 2 , 1 mM dithiothreitol, 10 g/ml leupeptin, 10 g/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride. The supernatant of cell lysates was incubated with 12 g of GST-Rho-binding domain and glutathione-Sepharose beads for 120 min at 4°C. The bead was washed, and finally suspended in SDS sample buffer. Pulled down Rho was detected with anti-Rho antibody.
Measurement of G␣ 13 Activity-HEK293 cells in six-well dishes were transfected with rat wild type AT1R and G␣ 13 with or without respective RGS domains, using FuGENE 6 reagent. Cardiomyocytes in 60-mm dishes were infected with LacZ, p115-RGS, or GRK2-RGS at 100 MOI or with CA-G␣ 13 at 30 MOI. Forty-eight h after transfection, activation of G␣ 13 was measured by the method of Yamaguchi et al. (36). After Ang II stimulation, the cells were harvested with 500 l of ice-cold lysis buffer containing 20 mM Hepes (pH 8.0), 2 mM MgCl 2 , 1 mM EDTA, 1 mM dithiothreitol, 0.5% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 10 g/ml aprotinin, 10 g/ml leupeptin, and 10 g of GST-TPR. The cell lysates were then centrifuged for 5 min at 12,000 ϫ g, and the supernatants were incubated with glutathione-Sepharose bead for 120 min at 4°C. The bead was washed and finally suspended in SDS sample buffer. Pulled down G␣ 13 was detected with anti-G␣ 13 antibody.
Intracellular Ca 2ϩ Measurement-The intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ) of cardiomyocytes was determined as described previously (21,33). Briefly, the cells (1 ϫ 10 6 ) were plated on gelatin-coated glass-bottomed 35-mm dishes and were loaded with 2.5 M Fura-2/AM in the cultured medium at 37°C for 30 min. The cells were washed with Tyrode solution containing 118 mM NaCl, 5.4 mM KCl, 2 mM CaCl 2 , 2 mM MgCl 2 , 10 mM Hepes (pH 7.4), 0.33 mM NaH 2 PO 4 , 10 mM glucose, and 30 mM taurine. Fluorescence images of green fluorescent proteinpositive cells were recorded and analyzed with a video image analysis system (Aquacosmos, Hamamatsu Photonics). All of the ratio data were calculated for determination of [Ca 2ϩ ] i with in vivo calibration method.
Statistical Analysis-The results are shown as the means Ϯ S.E. The mean values were compared with control by Student's t test (for two groups) or one-way analysis of variance followed by Dunnett's test (for three or more groups).

RESULTS
Ang II-induced MAPK Activation-To delineate the signal transduction cascade leading to MAPK activation by Ang II stimulation, the concentration dependence and time course of MAPK activation were determined. Stimulation with Ang II resulted in a dose-dependent activation of ERK, JNK, and p38 MAPK (Fig. 1A). The EC 50 values of Ang II to activate ERK, JNK, or p38 MAPK were different between three MAPKs. ERK, JNK, and p38 MAPK activation peaked at 5, 20, and 20 min, respectively (Fig. 1B). These results demonstrate that Ang II stimulation activated three MAPKs in cardiac myocytes.
ROS Mediate Ang II-induced JNK and p38 MAPK Activation-We examined first which MAPK requires ROS for their activation. One of ROS producing systems is NADPH oxidase complex, which is activated by receptor stimulation. Thus, we examined the effects of chemical antioxidants, such as N-acetyl-L-cysteine (a radical scavenger), DPI (an NADPH oxidase inhibitor), and catalase (an H 2 O 2 -degrading enzyme), on MAPK activation. Ang II-induced ERK activation was not affected by these antioxidants (Fig. 1C). However, Ang II-induced activation of p38 MAPK and JNK was inhibited by all three reagents (Fig. 1, D and E). We have also found that the expression of Prx II (a radical scavenging enzyme) significantly inhibited Ang II-induced JNK activation but not ERK activation (data not shown). These results indicate that JNK and p38 MAPK activation are sensitive to ROS and suggest that ROS produced by NADPH oxidase is necessary for Ang II-induced JNK and p38 MAPK activation.
Selective Inhibition of G␣ 12/13 by the Expression of p115-RGS-To investigate which G protein is involved in Ang IIinduced MAPK activation, a variety of adenoviruses that specifically inhibit G protein-mediated signaling were produced. Among them, p115-RGS or GRK2-RGS was used to specifically inhibit G␣ 12/13 or G␣ q , respectively. To delineate the selectivity of GRK2-RGS and p115-RGS, two RGS domains were expressed, and Ang II-stimulated increase in [Ca 2ϩ ] i was determined in the absence of extracellular Ca 2ϩ . Ang II stimulation increased [Ca 2ϩ ] i (Fig. 2). Because [Ca 2ϩ ] i was measured in the absence of extracellular Ca 2ϩ , the increase in [Ca 2ϩ ] i represents the activation of G␣ q -phospholipase C pathway. GRK2-RGS completely blocked the increase in [Ca 2ϩ ] i , but p115-RGS did not (Fig. 2, A and B). GRK2-RGS did not affect the caffeineinduced increase in [Ca 2ϩ ] i . Because caffeine promotes Ca 2ϩ release from intracellular Ca 2ϩ pool, the effect of GRK2-RGS is specific for the receptor-stimulated increase in [Ca 2ϩ ] i . These results suggest that GRK2-RGS but not p115-RGS blocks ATRmediated phospholipase C activation through G␣ q . We have also examined the specificity of p115-RGS. Because signaling molecule downstream of G␣ 12/13 such as [Ca 2ϩ ] i and cAMP has not been firmly established, we directly measured the activation of G␣ 12/13 by Ang II stimulation. It has been reported that G␣ 12/13 activation can be detected by selective pull-down of activated G␣ 12/13 using the TPR domain of protein phosphatase type 5 (36). Then we examined whether p115-RGS selectively inhibits Ang II receptor-stimulated G␣ 12/13 activation by pulldown assay, using CA-G␣ 13 as a positive control. Expression of CA-G␣ 13 increased the amount of CA-G␣ 13 pulled down by GST-TPR (Fig. 2C). The amount of pulled down G␣ 13 was completely abolished by p115-RGS, but not by GRK2-RGS. Because p115-RGS encodes RGS domain specific for G␣ 12/13 and the RGS domain can bind G␣ in active conformation, the mechanism of this inhibition by p115-RGS is competition between p115-RGS and GST-TPR for the binding of CA-G␣ 13 . By considering that p115-RGS did not affect the Ang II-induced increase in [Ca 2ϩ ] i , it is concluded that p115-RGS specifically binds the active form of G␣ 13 .
Activation of G 12/13 by ATR Stimulation-In AT1R-and G␣ 13 -expressing HEK293 cells, AT1R stimulation rapidly activated G␣ 13 (Fig. 3A). The activation reached a maximum at 3 min and sustained more than 2-fold for about 10 min. The Ang II-induced G␣ 13 activation was completely inhibited by p115-RGS, but not by GRK2-RGS (Fig. 3B). Because the RGS domain of p115-RGS has the ability to accelerate GTPase activity of G␣ 12/13 , the mechanism of this inhibition by p115-RGS is the inactivation of GTP-bound G␣ 13 . Fig. 3B also shows that AG1478 treatment did not inhibit Ang II-stimulated G␣ 13 activation (Fig. 3B). This result suggests that the EGF receptor is not involved in AT1R-mediated G␣ 13 activation. Furthermore, stimulation of endogenous ATR also activated G␣ 13 in rat cardiac myocytes (Fig. 3C). The activation reached a maximum at 1 min and gradually decreased to the basal level within 5 min. This Ang II-induced G␣ 13 activation was completely inhibited by p115-RGS but not by GRK2-RGS (Fig. 3D). These results indicate that stimulation of AT1R activates not only G q but also G 13 and that p115-RGS selectively inhibits Ang II-induced G␣ 13 activation.
G 12/13 Mediates Ang II-induced JNK and p38 MAPK Activation-We examined whether G␣ 12/13 is involved in Ang IIinduced MAPK activation. Ang II-induced ERK activation was not affected by p115-RGS, but the activation of JNK and p38 MAPK was significantly inhibited by p115-RGS (Fig. 4, A-C). We also used PTX to block ATR-G i coupling. PTX treatment and expression of GRK2-RGS did not affect Ang II-induced JNK activation (Fig. 4D). We also confirmed that Ang II-induced p38 MAPK activation was insensitive to PTX or GRK2-RGS (n ϭ 2; data not shown). These results suggest that the activation of JNK and p38 MAPK is mainly mediated by G␣ 12/13 but not G␣ q and G i . These results indicate that ATR couples with G 12/13 , and G␣ 12/13 activates signal transduction cascade, leading to JNK and p38 MAPK activation.
ROS-dependent Activation of JNK and p38 MAPK Induced by G␣ 12/13 Activation-We next determined the relationship between G␣ 12/13 and ROS in Ang II-induced JNK and p38 MAPK activation. Expression of CA-G␣ q , CA-G␣ 12 , or CA-G␣ 13 resulted in activation of all three MAPKs (Fig. 5). This result indicates that activated G␣ q , G␣ 12 , or G␣ 13 can induce activation of MAPKs in neonatal cardiac myocytes. Furthermore, G␣ 12 -or G␣ 13 -induced activation of p38 MAPK (Fig. 5B) and JNK (Fig. 5C), but not ERK (Fig. 5A), was sensitive to DPI. In contrast, G␣ q -induced JNK and p38 MAPK activation was not affected by DPI. These results are consistent with the fact that activation of G␣ 12/13 by ATR stimulation activates JNK and p38 MAPK through ROS production.
Rac-dependent ROS Production by ATR Stimulation-We examined whether ATR stimulation actually produces ROS in rat neonatal cardiomyocytes. Fig. 6A shows that exposure of Ang II increased intracellular concentration of ROS. The concentration of ROS by Ang II stimulation was compared with the amount of fluorescence generated by exogenously added H 2 O 2 . Ang II stimulation increased ROS to about 4 M, and p115-RGS and PrxII inhibited ROS production (Fig. 6B). However, the Ang II-induced ROS production was not affected by GRK2-RGS. These results indicate that G␣ 12/13 but not G␣ q mediates Ang II-induced ROS production. Furthermore, expression of DN-Rac or treatment with DPI inhibited Ang II-induced ROS production, suggesting that NADPH oxidase mediates Ang IIinduced ROS production.

FIG. 3. Activation of G␣ 13 by AT1R stimulation in HEK293 cells and cardiac myocytes. Selectivity of p115-RGS in HEK293 cells was determined by pull-down assay.
A, time-dependent activation of G␣ 13 by AT1R stimulation in wild type AT1R-and G␣ 13 -expressing HEK293 cells. The cell lysates were incubated with GST-TPR, and bound G␣ 13 was detected by immunoblotting with anti-G␣ 13 antibody. B, effects of p115-RGS, GRK2-RGS or AG1478 on Ang II-stimulated activation of G␣ 13 . HEK293 cells were transfected with p115-RGS or GRK2-RGS, with AT1R and G␣ 13 . The cells were treated with AG1478 (500 nM) for 20 min before the addition of Ang II (100 nM, 3 min). The experiments were repeated five times. *, p Ͻ 0.05 versus Ang II stimulation of pcDNA3-transfected cells. C, time-dependent activation of G␣ 13 by ATR stimulation in neonatal cardiac myocytes. The cardiac myocytes were stimulated with Ang II (100 nM) for the indicated times. For positive control, the cells were infected with CA-G␣ 13 (30 MOI). D, effect of p115-RGS or GRK2-RGS on Ang II-induced activation of G␣ 13 in cardiac myocytes. The cells were stimulated with Ang II (100 nM) for 1 min. The experiments were repeated five to six times. *, p Ͻ 0.05, as statistically analyzed by Student's t test. G␣ 12/13 Activates Rac Leading to JNK and p38 MAPK Activation-We examined the involvement of Rac in Ang II-induced MAPK activation. Expression of DN-Rac inhibited Ang II-induced activation of JNK and p38 MAPK, but not ERK (Fig. 7,  A-C). Because DN-Rac inhibited Ang II-induced ROS production (Fig. 6B), Rac may mediate Ang II-induced JNK and p38 MAPK activation through ROS production. Rac activation can be determined by selective pull-down assay using the Cdc42/ Rac interactive binding domain of PAK that is one of the Rac effectors. Rac activation was detected by angiotensin II stimulation, which peaked at 1 min and quickly returned to the basal state (data not shown). As expected, the expression of DN-Rac completely inhibited Ang II-induced activation of JNK and p38 MAPK but not ERK (Fig. 7, A-C). Rac activation was inhibited by p115-RGS, but not by PTX, GRK2-ct (a G␤␥-sequestering polypeptide), and GRK2-RGS (Fig. 7D). The basal Rac activity of PTX-treated cells was increased for unknown reasons. Similar high basal Rac binding activity with PTX treatment has been reported by another group (37). These results suggest that G 12/13 mediate Ang II-induced Rac activation.
Rho Mediates Ang II-induced Rac Activation-Because p115-RGS inhibited JNK, p38 MAPK, and Rac activation, we speculated that G␣ 12/13 may activate JNK, p38 MAPK, and Rac through Rho activation. Rho is specifically inactivated by C3 toxin that ADP-ribosylates Asn at position 41 of Rho. The expression of C3 toxin inhibited Ang II-stimulated JNK and Rac activation (Fig. 8, A and B). These results indicate that Rho mediates Rac and JNK activation by ATR stimulation. Because Rho regulates various kinases including ROCK, we next examined whether ROCK mediates Ang II-induced Rac activation (38). A ROCK inhibitor, Y27632, inhibited Rac activation (Fig. 8C). These results suggest that Ang II-induced Rac activation is mediated by Rho and consequent activation of ROCK. Furthermore, stimulation of ATR with Ang II activated Rho by 3-fold, which was completely inhibited by p115-RGS, but not GRK2-RGS and DN-Rac (Fig. 8D). Because ROS production is inhibited by DN-Rac, Ang II-induced Rho/ROCK activation could participate in Rac-dependent ROS production. These results also suggest that Rac activation is downstream of Rho activation.
ATR Subtype and Role of EGF Receptor Transactivation for Ang II-induced JNK Activation-Ang II-induced JNK activa- tion was significantly inhibited by CV11974 (a selective AT1R blocker), but not by PD123319 (a selective AT2R blocker), indicating that JNK was activated by type 1 subtype of ATR (Fig.  9A). It has been reported that EGF receptor transactivation plays an important role in G protein-coupled receptor-induced MAPK activation including AT1R (7). To examine the involvement of EGF receptor transactivation, we used an inhibitor of EGF receptor kinase AG1478. AG1478 did not affect Ang IIstimulated G␣ 13 activation (Fig. 3B), JNK activation, ROS production, Rho activation, and Rac activation (Fig. 9). DN-Rac inhibited Rac activation, validating the assay method (Fig. 9D). These results suggest that EGF receptor is not involved in Ang II-induced JNK activation through G␣ 12/13 -mediated Rho/ ROCK activation in cardiac myocytes. DISCUSSION We demonstrated in the present study that Ang II-induced JNK/p38 MAPK activation was mediated by a ROS-dependent signal transduction pathway: Ang II 3 AT1R 3 G␣ 12/13 3 Rho 3 ROCK 3 Rac 3 ROS 3 JNK/p38 MAPK (Fig. 10). We clearly demonstrate that ROS are produced by G␣ 12/13 -mediated Rac activation, and ROS participate in JNK and p38 MAPK but not ERK activation. Previous findings indicated that Ang II stimulation produces ROS in vascular smooth muscle cells (6,17) and suggested the role of ROS as a mediator of Ang II action (6,14). The present study is consistent with the reports that ROS are mediators of Ang II action. We further demonstrated that the possible origin of Ang II-induced ROS production in neonatal cardiac myocytes is NADPH oxidase, because a selective inhibitor of NADPH oxidase could inhibit ROS-dependent JNK and p38 MAPK activation by Ang II stimulation.
The expression of CA-G␣ 12 and CA-G␣ 13 activates all three MAPKs in rat cardiac myocytes (Fig. 5). This result is partly supported by the report that G␣ 12 activates JNK specifically through the stimulation of MKK7, an upstream kinase of JNK (39). Furthermore, CA-G␣ 12 -or CA-G␣ 13 -induced activation of JNK and p38 MAPK was inhibited by DPI (an NADPH oxidase inhibitor). Because Ang II-induced JNK and p38 MAPK activation was significantly inhibited by catalase (an H 2 O 2 -degrading enzyme), we speculate the activation scheme that NADPH  oxidase-generated superoxide anion is converted to H 2 O 2 , and H 2 O 2 forms more reactive species that participate in JNK and p38 MAPK activation machinery.
A selective EGF receptor kinase blocker AG1478 demonstrates that AT1R-stimulated JNK activation does not requires EGF receptor transactivation (Fig. 9). With cardiac fibroblasts, Murasawa et al. (40) reported that Ang II activates JNK through Pyk, Src, Rac, and PAK. In contrast with JNK activation, ERK activation in cardiac fibroblasts requires EGF receptor transactivation through Pyk2 and Src activation. The present study using cardiac myocytes demonstrated that Ang II-induced JNK activation was not inhibited by AG1478. We further demonstrated that Ang II-induced G␣ 13 activation, Rho activation, Rac activation, and ROS production were not inhibited by AG1478 (Figs. 3B and 9). These results suggest that EGF receptor transactivation is not necessary for Ang II-induced JNK and p38 MAPK activation through G␣ 12/13 -mediated ROS production.
The present study demonstrated that Rho and ROCK positively regulate Rac activation. On the other hand, the negative regulation of Rac by Rho and ROCK has been observed by Hirose et al. (41). They reported that Rho/ROCK stimulated neurite retraction, and Cdc42/Rac stimulated neurite outgrowth in NIE-115 cells. They suggested that Rho/ROCK negatively regulated the Cdc42 and Rac pathway. The differential regulatory mechanism of Rac by Rho/ROCK is unknown. However, different types of RacGEF that are regulated by ROCK may express in NIE-115 and cardiac myocytes. Because we also found that a ROCK inhibitor Y27632 inhibited Ang II-induced ROS production (data not shown), the present study also suggests that ROCK mediates Ang II-induced JNK and p38 MAPK activation through Rac activation.
When cells were treated with PTX, the active form of Rac was increased even in the absence of agonist stimulation (Fig. 7D). The mechanism of high Rac activity by PTX treatment is unknown. One of possibilities may be mitogenic action of PTX. PTX is an A-B toxin; A-protomer is the entity that ADP-ribosylates G␣ i , and B-oligomer consists of several subunits that bind to the plasma membrane. PTX binds to cells via B-oligomer, and Boligomer has a mitogenic activity. Therefore, the increased basal Rac activation may be caused by the mitogenic action of PTX.
DN-Rac inhibited Ang II-induced ROS production and activation of JNK and p38 MAPK. The reagents that inhibit ROS production blocked JNK and p38 MAPK activation. Thus, Racmediated JNK and p38 MAPK activation requires ROS. However, Rac is believed to be involved in various signaling pathways other than ROS production system (NADPH oxidase). For instance, Rac is an activator of PAK leading to JNK activation (20). We speculate that Rac activates at least two signaling pathways such as ROS production and PAK activation, and concomitant activation of two pathways is required for JNK and p38 MAPK activation.
The idea that G 12/13 proteins are involved in Ang II-mediated signaling has been also supported by some previous reports (42,43). For example, Gohla et al. (42) have reported that angiotensin II stimulation increases the incorporation of azidoanilido [␣-32 P]GTP into G␣ 12 and G␣ 13 in membranes of aortic smooth muscle cells. The present study substantiated their result that AT1R can directly activate G␣ 13 . We further demonstrated that inhibition of EGF receptor or G␣ q did not affect Ang II-stimulated G␣ 13 activation. Macrez et al. (43) also reported that a ␤␥ dimmer derived from G 13 transduces AT1R signaling. Because the ␤␥-sequestering peptide GRK2-ct did not affect Ang II-induced Rac activation (Fig. 7D), ␤␥ derived from G 12/13 may not participate in JNK and p38 MAPK activation induced by Ang II. G␣ q is generally thought to mediate Ang II-induced responses. We demonstrated that expression of CA-G␣ q activates three MAPKs in rat neonatal cardiomyocytes. However, the present study did not reveal any role of G␣ q in Ang II receptor-stimulated JNK and p38 MAPK activation. Zou et al. (44) have reported that G␣ q and protein kinase C mediate Ang II-induced ERK activation in rat cardiac myocytes. Therefore, it is reasonable to assume that the mechanism of Ang II-induced MAPK activation is different between three MAPKs; G␣ q mediates mainly ERK activation, and G␣ 12/13 mediates JNK and p38 MAPK activation. The blockade of JNK or p38 MAPK activation resulted in complete inhibition of Ang II-induced hypertrophic responses (32,45). Therefore, it may be necessary to turn on multiple pathways at the same time for the full induction of hypertrophic responses upon Ang II receptor stimulation.
In summary, we have demonstrated a new signal transduction pathway of Ang II-induced JNK and p38 MAPK activation: AT1R 3 G 12/13 3 Rho/ROCK 3 Rac 3 ROS 3 JNK and p38 MAPK. The signaling connection between G␣ 12/13 and ROS in cardiac myocytes will provide a new direction of Ang II receptor-mediated signaling pathway.