Rho Is Required for Gαq and α1-Adrenergic Receptor Signaling in Cardiomyocytes DISSOCIATION OF Ras AND Rho PATHWAYS

G protein-coupled receptor agonists initiate a cascade of signaling events in neonatal rat ventricular myocytes that culminates in changes in gene expression and cell growth characteristic of hypertrophy. These responses have been previously shown to be dependent on Gq and Ras. Rho, a member of the Ras superfamily of GTPases, regulates cytoskeletal rearrangement and transcriptional activation of the c-fos serum response element. Immunofluorescence staining of cardiomyocytes shows that Rho is present and predominantly cytosolic. We used two inhibitors of Rho function, dominant negative N19RhoA and Clostridium botulinum C3 transferase, to examine the possible requirement for Rho in α1-adrenergic receptor-mediated hypertrophy. Both inhibitors markedly attenuated atrial natriuretic factor (ANF) reporter gene expression induced by α1-adrenergic receptor stimulation with phenylephrine, and virtually abolished the increase in ANF reporter gene expression induced by GTPase-deficient Gαq. These effects were reproduced with the myosin light chain-2 reporter gene. Notably, N19RhoA did not block the ability of activated Ras to induce ANF and myosin light chain-2 reporter gene expression. Furthermore, activation of the extracellular signal-regulated kinase by phenylephrine was not blocked by N19RhoA, nor was it stimulated by an activated mutant of RhoA. Since activated RhoA and Ras produce a large synergistic effect on ANF-luciferase gene expression, we conclude that Rho functions in a pathway separate from but complementary to Ras. Our results provide direct evidence that Rho is an effector of Gαq signaling and suggest for the first time that a low molecular weight GTPase other than Ras is involved in regulating myocardial cell growth and gene expression in response to heterotrimeric G protein-linked receptor activation.

While many of the features of myocardial hypertrophy can be reproduced in vitro, the precise molecular signaling pathways regulating these hypertrophic responses have not yet been fully elucidated. Several lines of evidence indicate that ␣ 1 -adrenergic receptor-induced hypertrophy is mediated by both G q -and Ras-dependent pathways (8,9), and it has been suggested that G␣ q and Ras regulate independent pathways leading from ␣ 1adrenergic receptor activation to ANF gene expression (9). The Ras/mitogen-activated protein (MAP) kinase cascade can mediate downstream responses to ␣ 1 -adrenergic agonists (10 -15), but MAP kinase/ERK is not required for the morphological changes induced by PE (11) and ERK activation is not sufficient to induce hypertrophic responses (14). Likewise, expression of constitutively activated G␣ q alone is not sufficient to induce the typical changes in cell size and cellular organization seen with ␣ 1 -adrenergic receptor stimulation or Ras (9). Thus the activation of both Ras and G q signaling pathways may be required to induce the genetic and morphological responses associated with hypertrophy.
Recently, the Rho family of low molecular weight GTP-binding proteins (Cdc42, Rac, Rho) has been shown to participate in the regulation of various kinase (16 -18) and cytoskeletal pathways (19 -21). These proteins are well accepted as regulators of the actin cytoskeleton and are involved in the formation of filopodia, lamellipodia, stress fibers, and focal adhesions. In addition to these cytoskeletal effects, Rho has recently been demonstrated to regulate activation of the serum response element (SRE) in the c-fos promoter (22). Given the prominent effects of Rho on both gene expression and morphology in other systems, we hypothesized that this low molecular weight GTPase might also have a functional role in myocardial cell regulation.
In this study we examined the involvement of Rho in the ␣ 1 -adrenergic receptor-activated hypertrophic responses. We show that Rho is required for PE-induced ANF and MLC-2 gene expression. Furthermore, while the induction of ANF and MLC-2 gene expression by activated G␣ q is demonstrated to be dependent on Rho function, the response to oncogenic Ras is not. Our data suggest that the ␣ 1 -adrenergic receptor and heterotrimeric G q protein transduce signals through Rho, and that Rho functions in a pathway separate from but complementary to Ras in mediating genetic responses in myocardial hypertrophy.

EXPERIMENTAL PROCEDURES
Cell Culture-For all transfection experiments, neonatal rat ventricular myocytes were isolated and cultured as described previously (23,24). Briefly, hearts were obtained from 1-2-day-old Sprague-Dawley rat pups, digested with collagenase, and myocytes purified by passage through a Percoll gradient. Cells were plated onto tissue culture dishes precoated with 1% gelatin and maintained overnight in 4:1 Dulbecco's modified Eagle's medium/medium 199, supplemented with 10% horse serum, 5% fetal calf serum, and antibiotics (100 units/ml penicillin and 100 g/ml streptomycin).
Plasmid Constructs and Reagents-Reporter gene constructs used include a 638-base pair fragment of the rat ANF promoter or a 2700base pair fragment of the MLC-2 promoter fused to firefly luciferase cDNA (6,26). Expression plasmids encoding dominant-negative (N19), and activated (L63) RhoA were provided by Dr. G. Bokoch (The Scripps Research Institute); plasmid encoding C3 transferase (EFC3) was provided by Dr. R. Treisman (Imperial Cancer Research Foundation, London); plasmid encoding GTPase-deficient mutant of G␣ q (R183C) was provided by Dr. M. Simon (California Institute of Technology); plasmid encoding oncogenic V12Ras was from Dr. M. Wigler (Cold Spring Harbor Laboratory); plasmids encoding oncogenic L61Ras, HA-tagged ERK2, and HA-JNK1 were provided by Dr. M. Karin (University of California, San Diego). A C3-related exoenzyme producing strain of Clostridium limosum was generously provided by Dr. K. Aktories (Albert-Ludwigs-Universitä t, Freiburg, Germany). This C3-related exoenzyme, which specifically ADP-ribosylates Rho proteins, was prepared as described previously (27).
Transient Transfection-Myocytes in six-well plates were transfected in serum-containing medium using a modified calcium phosphate transfection technique as described previously (24). Following transfection, cells were washed extensively and cultured for 48 h in serum-free medium in the presence or absence of 100 M PE and 2 M propranolol. To determine reporter gene activity, cells were lysed in lysis buffer containing 1% Triton X-100 and luciferase activity assayed and quantitated using a Berthold luminometer as described (28). Luciferase data were normalized to protein concentration or coexpressed ␤-galactosidase activity and used as an index of gene expression.
Immunofluorescence Analysis-Cells on coverslips were fixed in 2% paraformaldehyde and permeabilized in 0.05% Triton X-100. Cells were stained for RhoA using a polyclonal rabbit anti-RhoA antibody (Santa Cruz Biotechnology, Inc.) and fluorescein isothiocyanate-conjugated donkey anti-rabbit secondary antibody (Amersham), or for F-actin filaments using tetramethylrhodamine isothiocyanate-conjugated phalloidin (Sigma). Subcellular Fractionation and ADP-ribosylation Assay-Myocytes were homogenized using a Polytron in ice-cold buffer containing 20 mM Tris-HCl (pH 7.4), 1 mM dithiothreitol, 1 M (4-amidino-phenyl)methanesulfonyl fluoride, and 0.25 M sucrose. The homogenate was separated into cytosolic and membrane fractions by centrifugation at 100,000 ϫ g for 30 min. Protein (50 g) from each fraction was then subjected to ADP-ribosylation using 30 ng of the C3-related exoenzyme from C. limosum as described previously (29).
Kinase Assays-Myocytes plated on 100-mm plates were cotransfected with pCMV5, N19RhoA, or L63RhoA and HA-ERK2 or HA-JNK1, and assayed as described previously (17,30). Briefly, following transfection, cells were washed and cultured in serum-free medium for 48 h and then mock-stimulated or stimulated with 100 M PE and 2 M propranolol (5 min for ERK or 20 min for JNK). Cells were harvested in lysis buffer containing 0.5% Nonidet P-40, immunoprecipitated with 12CA5 anti-HA antibody (Boehringer Mannheim) conjugated to protein A-Sepharose (Pharmacia Biotech Inc.), and assayed (at 30°C for 15 min for ERK or 20 min for JNK) in kinase buffer containing [␥-32 P]ATP and substrate (myelin basic protein (Sigma) for ERK or glutathione Stransferase-c-Jun for JNK). Phosphorylated substrates were analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography. 32 P incorporation was quantitated by radioanalytic scanning (AMBIS).

Rho Is Endogenously Expressed in Neonatal Cardiomyocytes, Does Not Colocalize with Actin Filaments, and Is Predomi-
nantly Cytosolic-We determined whether Rho was endogenously expressed in neonatal rat cardiomyocytes because Fritz et al. had previously noted that Rho was expressed only at low levels in the adult rabbit heart (31). Immunofluorescence staining using an anti-RhoA antibody clearly demonstrated that Rho was present in myocytes (Fig. 1A). Its localization was predominantly cytosolic, and it was not colocalized with actin filaments (Fig. 1, compare A and B). C3-related exoenzymecatalyzed ADP-ribosylation of membrane and cytosolic fractions from neonatal rat cardiac myocytes confirmed that most of the Rho protein was in the cytosolic rather than the membrane fraction (Fig. 1C).

FIG. 1. Rho is expressed in neonatal cardiomyocytes.
Cardiomyocytes were prepared as described (25) and cultured in Dulbecco's modified Eagle's medium/F-12 medium containing 5% calf serum. Cells were stained using an antibody directed against RhoA (A) and with phalloidin (B). C, cytosolic (Cytosol) and membrane (Memb) fractions of cardiomyocytes were prepared and subjected to ADP-ribosylation using 30 ng of C3-related exoenzyme from C. limosum and [␣-32 P]NAD.

C3 Transferase Inhibits PE-induced ANF-Luciferase Gene
Expression-One of the most prominent features of myocardial hypertrophy is the induction of ANF gene expression. PE is a highly effective activator of ANF gene expression in the cell culture model of hypertrophy, and a 638-base pair fragment of the ANF promoter fused to luciferase has been found to be PE-inducible (6). Botulinum C3 transferase specifically ADPribosylates and hence inactivates Rho (32). In order to determine if Rho regulates PE-induced ANF gene expression, an expression plasmid encoding C3 transferase, EFC3 (22), was transfected into myocytes along with an ANF-luciferase reporter gene, and the cells stimulated with PE. Expression of EFC3 decreased the basal level of ANF-luciferase expression, but most notably led to a pronounced inhibition of PE-induced activation of the ANF-luciferase reporter gene (Fig. 2). In support of these data, the PE-induced increase in ANF protein was also markedly decreased by pretreatment of cardiomyocytes with Botulinum C3 toxin. 2 Dominant Negative RhoA Inhibits PE-induced ANF-and MLC-2-Luciferase Gene Expression-As further evidence that Rho is involved in transcriptional activation by PE, the effect of dominant-negative N19RhoA, which acts by competitively inhibiting the interaction of endogenous Rho with its exchange factors, was examined. Activation of ANF-luciferase as well as the MLC-2 reporter gene, which is also up-regulated in hypertrophy, were examined. As shown in Fig. 3, N19RhoA reduced PE-induced activation of the ANF-luciferase and MLC-2-luciferase reporters by 50 -60%. At similar concentrations, N19RhoA had no inhibitory effect either on the basal level of ANF-luciferase expression or on RSV-luciferase or CMV-␤-galactosidase reporter gene expression (data not shown), thus nonspecific disruption of reporter gene expression by N19RhoA appears unlikely. These results suggest that Rho function is required for ␣ 1 -adrenergic receptor-induced ANF and MLC-2 gene expression.
Dominant Negative RhoA Blocks G q -induced, but Not Rasinduced, ANF-and MLC-2-Luciferase Expression-The activation of ANF and MLC-2 gene expression by PE has been shown to require both G q and Ras function (8,9). As shown in Fig. 4, cells transfected with constitutively activated mutants of either G␣ q (panel A) or Ras (panel B) displayed 8 -10-fold increases in ANF-and MLC-2-luciferase reporter gene activities. Coexpression of N19RhoA markedly suppressed G␣ q -induced reporter gene expression (Fig. 4A). However, expression of the same amount of N19RhoA was ineffective at blocking the activation of either reporter gene by Ras (Fig. 4B). Two different oncogenic Ras mutants (L61Ras and V12Ras) were used, each yielding comparable levels of reporter gene activation, and both resistant to blockade by N19RhoA. We conclude from these data that G␣ q -induced ANF and MLC-2 gene expression require Rho function, whereas Ras-mediated activation of these genes appears to be independent of Rho function.

Rho Does Not Regulate Activation of MAP Kinases-The
ERK family of MAP kinases are downstream effectors of Ras (14,33). We asked whether the activation of ERKs could also be regulated by Rho. An epitope (HA)-tagged ERK2 (p42 MAP kinase) construct was coexpressed in cardiac myocytes with N19RhoA or its backbone vector. In control-transfected cells, treatment with PE for 5 min increased ERK activity 2-2.5-fold. Expression of N19RhoA caused no significant inhibition of PEinduced ERK activation. Furthermore, L63RhoA, which is activated by a mutation that decreases its intrinsic GTPase activity, was unable to induce activation of ERK (Fig. 5). Activation of JNK was also unaffected by changes in Rho function (data not shown). These data are consistent with the results shown in Fig. 4B, indicating that Rho does not function in a Ras/MAP kinase pathway coupled to generation of hypertrophic responses.
Activated RhoA Synergizes with Activated Ras to Stimulate ANF-Luciferase Gene Expression-Since our results suggested that Rho does not function downstream of Ras, we hypothesized that Rho and Ras function in independent but complementary pathways. To test this, activated mutants of Rho and Ras were expressed and their combined effect on ANF-luciferase expression examined. As shown in Fig. 6, both activated mutants, L63RhoA and L61Ras, stimulated ANF reporter activity. A striking synergistic increase in ANF-luciferase gene expression was seen when the two activated small G proteins were coexpressed. Similar results were obtained when GTPasedeficient G␣ q and L61Ras were coexpressed (data not shown). Thus, both Rho and Ras activation can stimulate pathways leading to transcriptional activation of the ANF promoter, and they appear to define independent pathways as depicted schematically in Fig. 7.

DISCUSSION
Evidence implicating Rho in G protein-coupled receptor-induced gene expression was recently presented in a study published by Treisman's laboratory. These authors demonstrated that in NIH 3T3 cells a constitutively activated Rho mutant induced transcriptional activation of the c-fos SRE; in addition lysophosphatidic acid-and AlF 4 Ϫ -induced signaling to c-fos SRE activation was inhibited by C3 transferase (22). Our work in cardiomyocytes further implicates Rho in mediating the effects of G protein-coupled receptor agonists on transcriptional activation and, more specifically, identifies the cardiac-specific ANF and MLC-2 genes as targets for Rho-dependent activation.
Our finding that inhibition of Rho function blocks PE-induced activation of the ANF-and MLC-2-luciferase reporter genes implicates Rho in mediating genetic responses to ␣ 1adrenergic receptor stimulation. The fact that the inhibition was incomplete could reflect insufficient levels of expression of N19RhoA, or alternatively, PE signaling through an additional pathway that is independent of Rho function. The latter alternative is consistent with data from previous studies, which suggested the existence of two distinct pathways mediating PE-induced gene expression (9). One of these pathways is mediated by G␣ q , and the other by Ras. Interestingly, the effect of coexpressing N19RhoA with activated G␣ q or oncogenic Ras differentiated these pathways. Whereas N19RhoA blocked G␣ q -induced ANF-and MLC-2-luciferase gene expression, it had no apparent inhibitory effect on Ras-induced gene expression. In addition, PE-induced activation of kinase cascades downstream of Ras (ERK and JNK) was unaffected by N19RhoA. Our data therefore suggest that Rho is required in G␣ q -mediated signaling, but not in Ras-mediated events. Further support for the existence of two distinct pathways come from our demonstration that there is a synergistic effect of oncogenic L61Ras and either activated G␣ q or L63RhoA on ANF-luciferase gene expression.
The level at which the separate Ras and Rho signaling pathways converge to give a synergistic increase in ANF-luciferase expression is not yet clear. Microinjection studies in fibroblasts suggest an ordered set of interactions between Ras and the Rho family GTPases, namely that Ras is upstream of and required for activation of Rac, which in turn activates Rho (21,34). Coordination of Ras-and Rho-mediated signaling pathways could occur through the interaction between p120RasGAP and p190RhoGAP proteins (35,36), through RalGDS (37,38), through Ras guanine nucleotide exchange proteins (39), or through phosphatidylinositol 3-kinase (40 -42). However, our results suggest that Rho is activated by PE and G␣ q independent of Ras. In addition, Rho does not interact with the Ras signaling pathway via activation of MAP kinases.
Where in the signaling pathway Rho functions is under current investigation. One mechanism by which Rho might affect G q -dependent hypertrophic responses is by regulating the cellular level of phosphatidylinositol 4,5-bisphosphate (PIP 2 ). This possibility is suggested by the finding that in fibroblasts Rho regulates the activity of phosphatidylinositol 4-phosphate 5-kinase (43), an enzyme that is critical for the production of PIP 2 , the substrate for G q -activated phospholipase C. If RhoA regulates the production of PIP 2 in cardiac myocytes, the amount of substrate available for phospholipase C hydrolysis and consequent protein kinase C activation and Ca 2ϩ mobilization initiated by G q -linked ␣ 1 -adrenergic receptor activation might be regulated by this pathway. Alterations in PIP 2 levels may also affect the activity of other phospholipid-metabolizing enzymes, such as phospholipase D, which has been shown to be regulated by Rho (44,45).
In addition, Rho has been postulated to be regulated by and/or to modulate tyrosine kinase activities (46,47). Targets of Rho-dependent tyrosine phosphorylation include the p125 focal adhesion kinase (p125 FAK ), which is itself a tyrosine kinase, and paxillin (48,49). Several serine-threonine protein kinases have also recently been cloned and characterized as Rho effectors (50 -52), and the study of their activation and possible kinase cascades that they influence will further aid our understanding of the role of Rho in regulating the hypertrophic response.
It is well established that, in selected systems, Rho function is required to effect changes in cellular morphology induced by G protein-coupled receptors. Jalink et al. (53) demonstrated that ADP-ribosylation of Rho inhibits neurite retraction and neuronal cell rounding induced by lysophosphatidic acid and thrombin. In addition, Tigyi et al. (54) recently reported that lysophosphatidic acid-induced neurite retraction in PC12 cells is regulated by phosphoinositide-Ca 2ϩ signaling and is blocked by ADP-ribosylation of Rho. These effects are pertussis toxininsensitive, suggesting the involvement of a G q pathway. Rhodependent cytoskeletal responses might also be activated through G q -coupled receptors in cardiac myocytes and serve as triggers for subsequent events involved in the establishment of a hypertrophic phenotype. For example, activation of tyrosine kinases associated with the actin cytoskeleton (e.g. p125 FAK ) may serve to signal from G protein-coupled receptors to downstream kinase cascades, as observed in integrin signaling pathways (55).
Another pertussis toxin-insensitive protein, G 12 , has been previously demonstrated to possess transforming activity (56 -58). We recently presented evidence that G 12 can couple to low molecular weight GTPases and participates in thrombin-stimulated growth responses (30,59,60). In addition, activated G␣ 12 and/or the related G␣ 13 stimulate stress fiber formation, focal adhesion assembly and Na ϩ -H ϩ exchange through Rho-dependent mechanisms (61,62). Thus, G 12 is another possible transducer of information from the ␣ 1 -adrenergic receptor to the small G protein Rho.
In this study we provide the first direct evidence that Rho is required for G q -mediated signaling. Our findings demonstrate a specific role for Rho in regulating ␣ 1 -adrenergic receptormediated activation of ANF and MLC-2 gene expression, and show that in cardiac myocytes, Rho function is required for G␣ q but not for Ras-induced responses. These results suggest that growth regulation through G q -coupled receptors involves activation of Rho-dependent as well as Ras-dependent processes.