Cell Type–Specific Angiotensin II–Evoked Signal Transduction Pathways

Critical Roles of G_ß Subunit, Src Family, and Ras in Cardiac Fibroblasts

From the Department of Medicine III (Y.Z., I.K., T.Y., S.K., R.A., W.Z., I.S., Y.H., K.T., T.K., Y.Y.), University of Tokyo School of Medicine, and the Health Service Center (T.Y.), University of Tokyo (Japan).

Correspondence to Issei Komuro, MD, PhD, Department of Medicine III, University of Tokyo School of Medicine, 7–3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan. E-mail komuro-tky{at}umin.u-tokyo.ac.jp

	Abstract

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Abstract—Angiotensin II (Ang II) induces hypertrophy of cardiac myocytes and hyperplasia of cardiac fibroblasts. To determine the molecular mechanism by which Ang II displayed different effects on cardiac myocytes and fibroblasts, we examined signal transduction pathways leading to activation of extracellular signal–regulated kinases (ERKs). Ang II–induced ERK activation was abolished by pretreatment with pertussis toxin and by overexpression of the G_ß subunit–binding domain of the ß-adrenergic receptor kinase 1 in cardiac fibroblasts but not in cardiac myocytes. Inhibition of protein kinase C strongly inhibited activation of ERKs by Ang II in cardiac myocytes, whereas inhibitors of tyrosine kinases but not of protein kinase C abolished Ang II–induced ERK activation in cardiac fibroblasts. Overexpression of C-terminal Src kinase (Csk), which inactivates Src family tyrosine kinases, suppressed the activation of transfected ERK in cardiac fibroblasts. Ang II rapidly induced phosphorylation of Shc and association of Shc with Grb2. Cotransfection of the dominant-negative mutant of Ras or Raf-1 kinase abolished Ang II–induced ERK activation in cardiac fibroblasts. Overexpression of Csk or the dominant-negative mutant of Ras had no effects on Ang II–induced ERK activation in cardiac myocytes. These findings suggest that Ang II–evoked signal transduction pathways differ among cell types. In cardiac fibroblasts, Ang II activates ERKs through a pathway including the G_ß subunit of G_i protein, tyrosine kinases including Src family tyrosine kinases, Shc, Grb2, Ras, and Raf-1 kinase, whereas G_q and protein kinase C are important in cardiac myocytes.

Key Words: angiotensin II • cardiac fibroblast • extracellular signal–regulated kinase • G protein • Src

	Introduction

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The heart is composed of cardiac myocytes and several kinds of nonmyocytes, including fibroblasts, smooth muscle cells, and endothelial cells. Nonmyocytes, most of which are fibroblasts, constitute two thirds of the cell population of the heart.^{1 2} Although cardiac myocytes lose their proliferative ability soon after birth, nonmyocytes can still proliferate even in the adult heart. When the heart is exposed to various stresses, such as hemodynamic overload and myocardial infarction, cardiac myocytes increase in size, whereas cardiac fibroblasts increase in number and produce ECM proteins, such as collagens and fibronectins.^{3 4} The abnormal proliferation of cardiac fibroblasts with excessive accumulation of ECM proteins is one of the features of left ventricular remodeling, which leads ultimately to cardiac dysfunction.^{5 6} To elucidate the molecular mechanism of proliferation of cardiac fibroblasts is thus very important.

Ang II is a potent inducer of the cardiac hypertrophy and remodeling following sustained hypertension or myocardial infarction.^{5 7 8 9 10 11 12 13} Many laboratories, including our own, have reported that Ang II induces cardiomyocyte hypertrophy in vitro and in vivo and that it also plays a critical role in mechanical stress–induced cardiac hypertrophy.^{14 15 16 17} In cardiac fibroblasts, Ang II has also been demonstrated to stimulate proliferation and induce the expression of genes for collagens, fibronectin, and integrins.^{12 18 19} Ang II exerts its hypertrophic and hyperplastic effects by activating a number of intracellular signal transduction pathways through AT1Rs.^{12 17 18 20} Many studies have indicated that Ang II stimulates phosphatidylinositol-specific PLC through a heterotrimeric guanine nucleotide–binding protein (G protein). The activation of PLC induces the generation of DG and IP3, which, respectively, induce activation of PKC and the release of Ca²⁺ from intracellular stores in many cell types.^{9 12 21} It has recently been reported that Ang II also activates tyrosine kinases, including Src family tyrosine kinases, and Ras in cardiac myocytes,²² cardiac fibroblasts,²⁰ and smooth muscle cells.^{23 24 25} Many lines of evidence have suggested that ERKs function as integrators for mitogenic and differentiation signals originating from several distinct classes of cell surface receptors, such as receptor tyrosine kinases and G protein–coupled receptors.^{26 27 28} In cardiac myocytes, activation of ERKs is also required for phenylephrine-induced expression of specific genes, such as the atrial natriuretic factor, c-fos, and myosin light chain 2 genes.²⁹ Although ERK activation is not sufficient for full promotion of cardiac hypertrophy,³⁰ a recent study using an antisense oligodeoxynucleotide has shown that ERKs are necessary for phenylephrine-induced sarcomerogenesis and increase in cardiac cell size.³¹ We have recently demonstrated that in cardiac myocytes, PKC, but not tyrosine kinases or Ras, plays an essential role in Ang II–induced activation of ERKs.³² On the other hand, in smooth muscle cells, Ang II has been shown to activate ERKs through Src/Ras-dependent pathways.²⁵ These observations suggest that Ang II–induced signal transduction pathways may differ among cell types.

To clarify the molecular mechanism of proliferation of cardiac fibroblasts, we determined the Ang II–evoked signal transduction pathways leading to activation of ERKs, which are required for DNA synthesis in cardiac fibroblasts. In the present study, we show that in cardiac fibroblasts, Ang II activates ERKs through a pathway consisting of the G_ß subunit of the G_i protein, tyrosine kinases including Src family tyrosine kinases, Shc, Grb2, Ras, and Raf-1, which differs considerably from the pathway in cardiac myocytes.³²

	Materials and Methods

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Materials
[-³²P]ATP was purchased from Du Pont-New England Nuclear Co. DMEM, FBS, tyrphostin (A25), and genistein were from GIBCO-BRL Co. Pertussis toxin was from List Biological Laboratories, Inc. Calphostin C was from BIOMOL. Anti-HA polyclonal antibody was from Mitsubishi Biochemical Laboratories. Anti-Shc and anti-Grb2 polyclonal antibodies and anti-phosphotyrosine monoclonal antibody (PY20) were from Santa Cruz Biotechnology, Inc. Anti-rabbit IgG conjugated with horseradish peroxidase and the enhanced chemiluminescence reaction system were from Amersham. CV-11974, an active form of TCV-116, was a gift from Takeda Chemical Industries, Ltd (Osaka, Japan). PD123319 was a gift from Parke-Davis (Ann Arbor, Mich). PD98059 was purchased from New England Biolabs, Inc. Ang II, TPA, MBP, and other reagents were from Sigma Chemical Co.

cDNA Plasmids
HA-tagged ERK2 (HA-ERK2) in the SV40 promoter was a kind gift from M. Karin, School of Medicine, University of California at San Diego, La Jolla.³³ The cDNA encoding carboxyl-terminal ß subunit of the G_i protein (G_ß subunit)–binding domain of the bovine ßARK1 residues Gly-495 to Leu-689 was provided by K. Touhara, Howard Hughes Medical Institute, Duke University Medical Center, Durham, NC.³⁴ The dominant-negative mutant (Asn-17) of Ras (D.N.Ras) and the dominant-negative mutant (Ala-375) of Raf-1 kinase (D.N.Raf-1), both of which are driven by the cytomegalovirus promoter, were provided by Y. Takai, Kobe (Japan) University School of Medicine,³⁵ and T. Kadowaki, Faculty of Medicine, University of Tokyo (Japan),³⁶ respectively. Wild-type Csk and the kinase-negative mutant of Csk (Csk^-) were provided by H. Sabe, Rockefeller University, New York, NY.³⁷ All plasmid DNA was prepared using QIAGEN plasmid DNA preparation kits.

Cell Culture
Cardiac myocytes and fibroblasts from ventricles of 1-day-old Wistar rats were isolated as previously described.³⁸ To selectively enrich cardiac myocytes, dissociated cells were preplated onto 100-mm culture dishes for 30 minutes, which permitted preferential attachment of fibroblasts to the bottom of the dish. Nonadherent cells were plated at a field density of 1x10⁵ cells/cm² on 35-mm culture dishes with 2 mL of culture medium (DMEM with 10% FBS) as a cardiomyocyte-rich culture. Cardiac fibroblasts were obtained from adherent cells on the preplating dish and split twice before use. Cells were grown to subconfluence in DMEM+10% FBS, and the culture medium was changed to serum-free DMEM at 48 hours before treatment.

[³H]Thymidine Incorporation
DNA synthesis was assessed by measuring [³H]thymidine incorporation as previously described.³⁹ Cardiac fibroblasts were serum-deprived for 2 days and then incubated for 24 hours with Ang II or vehicle. [³H]Thymidine (1.25 µCi/mL) was added 2 hours before the harvest. Plates were then placed on ice, quickly washed three times with 1 mL of ice-cold PBS, incubated 10 minutes with 1 mL of 10% trichloroacetic acid, and washed twice with 1 mL of 10% trichloroacetic acid and three times with 1 mL of 95% ethanol. Precipitates were solubilized for 1.5 hours in 800 µL of 0.2N NaOH and neutralized, and radioactivity was measured by liquid scintillation spectroscopy.

Transfection
Twenty-four hours after plating the cardiac myocytes or fibroblasts on 35-mm culture dishes, DNA was transfected by the calcium phosphate method as previously described.³² For each dish, 2.5 µg of HA-ERK2 plasmid DNA was transfected with or without 7.5 µg of other relevant plasmids such as ßARK1, Csk, D.N.Ras, or D.N.Raf-1. After 20 hours of transfection, the culture medium was removed, and cells were washed with PBS twice and then maintained in serum-free DMEM for 48 hours before stimulation with Ang II. The transfection efficiency of each experiment was 1% in cardiac myocytes and 5% to 10% in fibroblasts as assessed by LacZ staining after transfection of a LacZ-containing expression plasmid.

Assay of ERK Activity
ERK activities were measured using MBP-containing gel as previously described.⁴⁰ In brief, cell lysates were electrophoresed on an SDS-polyacrylamide gel containing 0.5 mg/mL MBP. ERKs in the gel were denatured in guanidine HCl and renatured in Tris-HCl containing Triton X-100 and 2-mercaptoethanol. Phosphorylation activities of ERKs were assayed by incubating the gel with [-³² P]ATP. After incubation, the gel was washed extensively and subjected to autoradiography.

Kinase Assay of Transfected HA-Tagged ERK (MBP Assay)
The activity of transfected HA-tagged ERK2 was assayed as previously described.³² In brief, after transfection of HA-tagged ERK2, cell lysates were incubated with anti-HA polyclonal antibody for 1 hour at 4°C. After incubation, the immunocomplex was precipitated using protein A–Sepharose beads, washed, resuspended, and incubated with MBP and [-³² P]ATP for 10 minutes. The sample was subjected to SDS-PAGE, and the phosphorylated MBP band was visualized by autoradiography.

Phosphorylation of Shc and Its Association With Grb2
Tyrosine phosphorylation of Shc was examined by Western blot analysis. After treatment, cell lysates were incubated with anti-Shc polyclonal antibody (1:1000) for 1 hour at 4°C. After precipitation with protein A–Sepharose beads, the immunocomplex was subjected to SDS-PAGE and transferred to Immobilon-P membrane (Millipore). Association of Shc with Grb2 was examined by incubating cell lysates with anti-Grb2 antibody (1:1000) for 1 hour at 4°C. After blocking with 30% nonfat dry milk, immunoblots were incubated with anti-phosphotyrosine monoclonal antibody (PY20) (1:1000) or anti-Shc polyclonal antibody (1:1000) for 1 hour at 37°C. After it was washed, the membrane was incubated with horseradish peroxidase–conjugated anti-mouse or anti-rabbit IgG, and immunoreactivity was detected using the enhanced chemiluminescence system according to the manufacture's direction.

Statistics
Statistical comparison of the control group with treated groups was carried out using the paired-sample t test with P values corrected by the Bonferroni method. The accepted level of significance was P<.05.

	Results

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Activation of ERKs Is Required for Ang II–Induced DNA Synthesis in Cardiac Fibroblasts Through AT1R
When 10^-6 mol/L Ang II was added to the culture medium of cardiac fibroblasts obtained from neonatal rats, thymidine incorporation into cells was increased by 40% (Fig 1). This Ang II–induced increase in thymidine incorporation was completely inhibited by an AT1R antagonist, CV-11974, but not by an AT2R antagonist, PD123319 (Fig 1), consistent with the findings of previous studies.^{12 18} A growing body of evidence has suggested that ERKs play key roles in transducing cytoplasmic signals to the nucleus and in proliferation and differentiation of many types of cells.^{26 27 28} Therefore, we examined whether ERKs are required for Ang II–induced DNA synthesis in cardiac fibroblasts with the use of a MEK1 inhibitor, PD98059, which specifically inhibits activation of ERKs.⁴¹ When 5x10^-5 mol/L PD98059 was added to the culture medium at 30 minutes before the addition of Ang II, the Ang II–induced increase in thymidine incorporation was completely suppressed (Fig 1), suggesting that MEK and thus probably ERKs play a pivotal role in Ang II–induced DNA synthesis in cardiac fibroblasts.

View larger version (49K): [in this window] [in a new window]   Figure 1. Ang II–induced thymidine incorporation into DNA of cardiac fibroblasts. Serum-deprived (48 hours) cardiac fibroblasts were incubated with 10-6 mol/L Ang II for 24 hours and over the final 2 hours were pulse-labeled with [3H]thymidine (1.25 µCi/mL). Some dishes were pretreated with 10-6 mol/L CV-11974 (CV119), 10–6 mol/L PD123319 (PD123), and 5x10-5 mol/L PD98059 (PD980) for 30 minutes and by 100 ng/mL islet-activating protein (IAP) for 1 day before stimulation with Ang II. Incorporation of [3H]thymidine into acid-precipitated cellular fraction was determined. The data are presented as mean±SE of three independent experiments and compared with untreated control (=100%, solid bar). *P<.05 vs control.

Ang II Activates ERKs Through AT1R in Cardiac Fibroblasts
To determine the molecular mechanism of Ang II–induced proliferation of cardiac fibroblasts, we examined the signal transduction pathways leading to the activation of ERKs. We first examined whether Ang II activates ERKs in cardiac fibroblasts. When 10^-6 mol/L Ang II was added to the culture medium, ERKs were strongly activated at 2 minutes, and activity reached a peak at 8 minutes (Fig 2A and 2B). Activity decreased sharply thereafter and returned to unstimulated levels by 30 minutes after stimulation with Ang II. The time course of ERK activation by Ang II in cardiac fibroblasts was almost the same as that previously reported for cardiac myocytes.¹⁷

View larger version (38K): [in this window] [in a new window]   Figure 2. Time course of Ang II–induced ERK activation in cardiac fibroblasts. A, Cultured cardiac fibroblasts were exposed to 10-6 mol/L Ang II for various periods of time. Activities of ERKs were assayed by the "in-gel" method. Cell lysates were electrophoresed on an SDS-polyacrylamide gel containing 0.5 mg/mL MBP. ERKs in the gel were denatured in guanidine HCl and renatured in Tris-HCl containing Triton X-100 and 2-mercaptoethanol. Phosphorylation activities of ERKs were assayed by incubating the gel with [-32P]ATP. After incubation, the gel was washed extensively and subjected to autoradiography. A representative autoradiogram is shown. B, Relative kinase activities were determined by scanning each band with a densitometer. Results are presented as mean±SE of four independent experiments. Activities of ERK1 (solid bar) and ERK2 (hatched bar) are expressed relative to those of ERK1 obtained in unstimulated control (=1). C, Cardiac fibroblasts were pretreated with either AT1R antagonist (10-6 mol/L CV-11974) or AT2R antagonist (10-6 mol/L PD123319) for 30 minutes and stimulated with 10-6 mol/L Ang II for 8 minutes. Activities of ERKs were assayed by the "in-gel" method as described above.

It has been reported that two subtypes of Ang II receptors, AT1R and AT2R, are present in cardiac fibroblasts of neonatal rats⁴² and that both receptors are involved in proliferative responses to Ang II.^{18 43} To determine which receptor is involved in Ang II–induced ERK activation in cardiac fibroblasts, we stimulated cardiac fibroblasts with Ang II (10^-6 mol/L) after pretreatment with CV-11974 (10^-6 mol/L) or PD123319 (10^-6 mol/L) for 30 minutes. As was the case for DNA synthesis, Ang II–induced ERK activation was completely suppressed by pretreatment with CV-11974 but not by that with PD123319 (Fig 2C), suggesting that Ang II–induced ERK activation in cardiac fibroblasts is mediated through AT1R.

G_i Protein and G_ß Subunit Are Required for Ang II–Induced ERK Activation in Cardiac Fibroblasts
AT1R is a prototypical G protein–coupled receptor with seven transmembrane–spanning regions.^{20 44} Which G proteins are coupled to AT1R depends on cell type. It has been reported that G_q couples to AT1R in cardiac myocytes²² and vascular smooth muscle cells²⁵ and that G_i couples to AT1R in hepatocytes.⁴⁵ To examine whether Ang II–induced ERK activation occurs via G_i protein, we preincubated cardiac fibroblasts and myocytes with 100 ng/mL pertussis toxin for 24 hours and then treated them with 10^-6 mol/L Ang II for 8 minutes. Activation of ERKs by Ang II was completely suppressed by pretreatment with pertussis toxin in cardiac fibroblasts (Fig 3A) but not in cardiac myocytes (Fig 3B). Pertussis toxin also suppressed Ang II–induced thymidine incorporation into cardiac fibroblasts (Fig 1). These findings suggest that pertussis toxin–sensitive G_i protein mediates Ang II–induced activation of ERKs and an increase in DNA synthesis in cardiac fibroblasts, whereas in cardiac myocytes, a pertussis toxin–insensitive G protein, possibly G_q protein, is involved in Ang II–induced ERK activation.

View larger version (44K): [in this window] [in a new window]   Figure 3. The effect of pertussis toxin on Ang II–induced ERK activation in cardiac fibroblasts and myocytes. Cardiac fibroblasts (A) or myocytes (B) were preincubated with 100 ng/mL pertussis toxin (islet-activating protein [IAP]) for 24 hours. The cells were then treated with 10-6 mol/L Ang II for 8 minutes. Activities of ERKs were assayed as described in Fig 2. Representative autoradiograms from three independent experiments are shown.

It has recently been reported that stimulation of G_i protein–coupled receptors activates ERKs by the ß subunit complex (G_ß subunit) but not by the subunit.^{46 47 48 49} The carboxyl-terminal 195 amino acids of ßARK1 (Gly-495 to Leu-689), which include pleckstrin homology domains, have been reported to bind G_ß subunit and inhibit G_ß subunit–dependent activation of a wide variety of cell regulatory processes.^{50 51} We therefore examined the role of the G_ß subunit in Ang II–induced ERK activation by overexpressing a minigene construct encoding ßARK1^495–689 polypeptides with HA-ERK2 in cultured cardiac fibroblasts or myocytes. Ang II increased the activity of the transfected ERK2 in both cardiac fibroblasts (Fig 4A) and cardiac myocytes (Fig 4B) to almost the same extent. Although overexpression of a vector alone did not affect Ang II–induced ERK2 activation in either cell type (data not shown), expression of ßARK1^495–689 polypeptide abolished Ang II–induced activation of ERK2 in cardiac fibroblasts (Fig 4A) but not in cardiac myocytes (Fig 4B). These findings suggest that Ang II activates ERKs via the G_ß subunit–dependent pathway in cardiac fibroblasts but that in cardiac myocytes, Ang II–induced activation of ERKs is independent of the G_ß subunit.

View larger version (37K): [in this window] [in a new window]   Figure 4. The effect of overexpression of ßARK1495–689 on transfected ERK2 activation by Ang II in cardiac fibroblasts and myocytes. cDNA encoding ßARK1495–689 polypeptide was cotransfected with cDNA encoding HA-ERK2 into cardiac fibroblasts (A) and myocytes (B). Eight minutes after the addition of 10-6 mol/L Ang II, HA-ERK2 was immunoprecipitated with anti-HA polyclonal antibody, and the immunocomplex was incubated with 25 µg MBP as a substrate and 2 µCi[-32P]ATP for 10 minutes. Aliquots of the reaction mixture were subjected to SDS-PAGE, and the gel was washed, dried, and subjected to autoradiography. A representative autoradiogram from three independent experiments is shown.

Ang II–Induced Activation of ERKs Requires Tyrosine Kinases but Not PKC in Cardiac Fibroblasts
We have recently demonstrated that Ang II activates ERKs through PKC-dependent pathways in cultured cardiac myocytes.³² We therefore examined the role of PKC in Ang II–induced ERK activation in cardiac fibroblasts. When PKC was downregulated by incubation with 10^-7 mol/L TPA for 24 hours, readdition of 10^-7 mol/L TPA did not activate ERKs in cardiac fibroblasts (data not shown). With the same pretreatment, however, Ang II strongly activated ERKs at levels almost the same as without pretreatment (Fig 5A). A highly specific PKC inhibitor, calphostin C (10^-6 mol/L), also had no effects on Ang II–induced ERK activation in cardiac fibroblasts (Fig 5A), suggesting that unlike cardiac myocytes, in cardiac fibroblasts, Ang II activates ERKs through PKC-independent pathways.

View larger version (51K): [in this window] [in a new window]   Figure 5. The role of PKC and tyrosine kinase in Ang II–induced activation of ERKs in cardiac fibroblasts. Cardiac fibroblasts were pretreated with 10-6 mol/L calphostin C (calp. C) (A) for 60 minutes, 10-7 mol/L TPA (A) for 24 hours, 5x10-5 mol/L tyrphostin (B), or 2x10-5 mol/L genistein (B) for 30 minutes and stimulated with 10-6 mol/L Ang II for 8 minutes. ERK activities were assayed as described in Fig 2. A representative autoradiogram from three independent experiments is shown.

Activation of receptor and nonreceptor tyrosine kinases activates ERKs in many types of cells,^{26 27} and it has recently been reported that Ang II activates tyrosine kinases in several cell types, including hepatocytes,⁴⁵ smooth muscle cells,^{20 23 25} cardiac myocytes,²² and cardiac fibroblasts.²⁰ We therefore examined whether tyrosine kinases are involved in the activation of ERKs induced by Ang II in cardiac fibroblasts. Cultured cardiac fibroblasts were pretreated with two chemically and mechanistically dissimilar tyrosine kinase inhibitors, tyrphostin A25 (5x10^-5 mol/L), which is a potent inhibitor of the epidermal growth factor receptor–associated protein tyrosine kinase, and genistein (2x10^-5 mol/L), for 30 minutes and then stimulated with Ang II (10^-6 mol/L) for 8 minutes. In cardiac fibroblasts, unlike cardiac myocytes,³² ERK activation by Ang II was completely suppressed by pretreatment with tyrphostin or genistein (Fig 5B). These findings suggest that Ang II activates ERKs through tyrosine kinase–dependent pathways in cardiac fibroblasts.

Src Family Protein Tyrosine Kinases Are Required for Ang II–Induced Activation of ERKs in Cardiac Fibroblasts
It has been reported that Ang II activates Src family protein kinases in cardiac myocytes²² and smooth muscle cells.^{23 24} We therefore examined the role of Src family protein tyrosine kinases in Ang II–induced ERK activation by overexpressing Csk in cardiac fibroblasts and myocytes. Csk has been reported to phosphorylate the tyrosine residue in the carboxyl terminus of Src family protein kinases and thereby inactivate their function.^{37 52} We transfected Csk or kinase domain–deleted Csk (Csk^-) with HA-ERK2 into cells and determined the activity of transfected ERK2 after stimulation with Ang II. Although cotransfection of Csk^-, which has no effect on Src family tyrosine kinases, did not affect Ang II–induced ERK2 activation (Fig 6A), overexpression of Csk completely inhibited activation of ERK2 by Ang II in cardiac fibroblasts (Fig 6A). In contrast, in cardiac myocytes, overexpression of Csk had no effects on Ang II–induced ERK activation (Fig 6B). Overexpression of Csk, however, partially suppressed insulin-induced ERK activation in cardiac myocytes (data not shown). These findings suggest that Src family tyrosine kinases play a pivotal role in Ang II–induced ERK activation in cardiac fibroblasts but not in cardiac myocytes.

View larger version (50K): [in this window] [in a new window]   Figure 6. The effects of Csk on transfected ERK2 activation by Ang II in cardiac fibroblasts and myocytes. HA-ERK2 was transfected into cardiac fibroblasts (A) or cardiac myocytes (B) with Csk or Csk-. Eight minutes after the addition of 10-6 mol/L Ang II, the transfected ERK2 activity was measured using MBP as a substrate as described in Fig 4. A representative autoradiogram is shown. Similar results were obtained from three independent experiments.

Ang II Induces Tyrosine Phosphorylation of Shc and Association of Shc With Grb2 in Cardiac Fibroblasts
It has been shown that adapter proteins containing Src homology 2 domains, such as Shc and Grb2, transduce activation of tyrosine kinases to the Ras/ERK pathway via the guanine nucleotide exchange factor Sos.^{53 54 55 56} We therefore examined whether Shc is activated by Ang II in cardiac fibroblasts. The cell lysates from stimulated or unstimulated fibroblasts were immunoprecipitated with an anti-Shc polyclonal antibody. The immunoprecipitates were subjected to SDS-PAGE followed by immunoblotting with an anti-phosphotyrosine antibody. Ang II (1 µmol/L) rapidly (within 1 minute) increased tyrosine phosphorylation of 52-kD Shc in cardiac fibroblasts (Fig 7A) but not in cardiac myocytes (Fig 7B). A band corresponding to 46-kD Shc was also observed with long exposure (data not shown). Phosphorylation levels rapidly decreased from 4 minutes and returned to the basal levels by 15 minutes (Fig 7A). We next examined the association of Shc with another adapter protein, Grb2. The cell lysates were immunoprecipitated with an anti-Grb2 polyclonal antibody, and the immunoprecipitates were subjected to SDS-PAGE followed by immunoblotting with an anti-Shc antibody. As shown in Fig 7C, the intensities of bands around 52 and 46 kD were enhanced by Ang II stimulation. These bands comigrated with the 52- and 46-kD Shc detected when Shc immunoprecipitates were immunoblotted with the anti-Shc antibody (data not shown). These findings indicate that the 52- and 46-kD forms of Shc form a complex with Grb2 after Ang II stimulation.

View larger version (55K): [in this window] [in a new window]   Figure 7. Phosphorylation and association with Grb2 of Shc by Ang II. Cardiac fibroblasts (A and C) and myocytes (B) were stimulated with 10-6 mol/L Ang II for the indicated periods of time. The extracts of cells were incubated with anti-Shc (A and B) or anti-Grb2 (C) polyclonal antibody, and the immunocomplex was subjected to SDS-PAGE. The blotted membrane was incubated with anti-phosphotyrosine monoclonal antibody (PY20) (A and B) or anti-Shc antibody (C). A representative autoradiogram is shown. Three independent experiments showed similar results.

Ras and Raf-1 Are Required for Ang II–Induced ERK Activation in Cardiac Fibroblasts
Association of Grb2 with Shc usually results in the recruitment of a Ras activator, Sos, to the membrane fraction, resulting in activation of Ras.^{53 54 55 56} We therefore next examined whether Ras is involved in Ang II–induced ERK activation in cardiac fibroblasts. Although Ras is not required for Ang II–induced ERK activation in cardiac myocytes,³² Ang II–induced activation of transfected ERK2 was completely suppressed by overexpression of D.N.Ras in cardiac fibroblasts (Fig 8A), suggesting that activation of Ras is required for ERK activation by Ang II in cardiac fibroblasts.

View larger version (74K): [in this window] [in a new window]   Figure 8. The role of Ras and Raf-1 in Ang II–induced activation of transfected ERK2 in cardiac fibroblasts. HA-ERK2 was transfected with D.N.Ras (A) or D.N.Raf-1 (B) into cardiac fibroblasts, and cells were stimulated with 10-6 mol/L Ang II for 8 minutes. The activity of transfected ERK2 was assessed by measuring MBP phosphorylation as described in Fig 4. A representative autoradiogram from three independent experiments is shown.

Raf-1 has been reported to be activated by Ras and to activate dual protein kinase MEK.⁵⁷ MEK in turn activates ERKs by phosphorylating their threonine and tyrosine residues.^{26 27} We finally examined whether Raf-1 is required for Ang II–induced ERK activation in cardiac fibroblasts. After transfection of HA-ERK2 with or without D.N.Raf-1, cardiac fibroblasts were exposed to Ang II (10^-6 mol/L) for 8 minutes. Activation of transfected ERK2 by Ang II was abolished by cotransfection of D.N.Raf-1 (Fig 8B), indicating that activation of Raf-1 is critical for activation of ERKs by Ang II in cardiac fibroblasts as well as in cardiac myocytes.³²

	Discussion

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Two-thirds of the cells in the heart are nonmyocytes, the vast majority of which (>90%) are fibroblasts.^{1 2} Cardiac fibroblasts enhance the production of ECM proteins when the heart is exposed to a variety of injuries, such as myocardial infarction and myocarditis. Increase in the number of cardiac fibroblasts and the content of ECM proteins during cardiac remodeling is one of the major causes of cardiac dysfunction.^{5 6} Therefore, elucidation of the mechanism of proliferation of cardiac fibroblasts is of great importance. Ang II has been reported to be a potent inducer of both cardiac fibroblast hyperplasia and cardiomyocyte hypertrophy.^{12 13 17 18} In the present study, we compared the Ang II–induced signal transduction pathways leading to proliferation of cardiac fibroblasts with the pathways leading to cardiomyocyte hypertrophy. Ang II activated ERKs and enhanced DNA synthesis in cardiac fibroblasts of neonatal rats. The activation of ERKs was critical for the enhancement of DNA synthesis in cardiac fibroblasts, as is the case for many other types of cells.^{26 27 28} In cardiac fibroblasts, Ang II activated ERKs via a pathway through an AT1R, a G_ß subunit derived from G_i, Src family tyrosine kinases, Shc, Ras, and Raf-1; this pathway differs considerably from the pathway in cardiac myocytes, in which G_q and PKC play critical roles.³²

Two major subtypes of the Ang II receptor, AT1R and AT2R, are each seven-membrane serpentine receptors and elicit intracellular signals through heterotrimeric G proteins. Ang II activated ERKs and enhanced DNA synthesis through AT1R in cardiac fibroblasts, as previously reported.^{12 18} It has also been reported that AT1R couples to G_q in cardiac myocytes and smooth muscle cells^{22 25} and G_i in hepatocytes.⁴⁶ In cardiac fibroblasts, pretreatment with pertussis toxin abolished the Ang II–induced activation of ERKs and the increase in DNA synthesis, whereas pertussis toxin had no effects on cardiac myocytes, indicating that AT1R may couple to G_i in cardiac fibroblasts and to G_q in cardiac myocytes. Thus, which G proteins couple to AT1R depends on cell type, according to these findings.⁵⁸ However, there is another possibility that there are unidentified factors determining signal transduction pathways downstream from G proteins and leading to ERK activation. It has been reported that although lysophosphatidic acid receptors couple to both pertussis toxin–sensitive and –insensitive G proteins and that the latter G protein induces phosphoinositide hydrolysis with subsequent Ca²⁺ mobilization and stimulation of PKC, the pertussis toxin–insensitive G protein does not activate ERKs in fibroblasts.⁵⁹ Thus, we cannot rule out the possibility that AT1R couples to both G_q and G_i proteins in both cardiac myocytes and fibroblasts, but the G_q predominantly leads to ERK activation in cardiac myocytes, whereas the AT1R signals to ERKs predominantly via G_i in the cardiac fibroblasts.

A growing body of evidence has suggested that the G_ß subunit derived from pertussis toxin–sensitive G_i protein regulates many effectors within the cell.^{44 46 47 49} Stimulation of many receptors, such as _2A-adrenergic, M₂ muscarinic acetylcholine, D₂ dopamine, and A₁ adenosine receptors, induces Ras-dependent ERK activation by G_ß subunits in COS-7 cells.⁴⁶ In addition, overexpression of G_ß subunits, but not of constitutively activated G_i1 or G_i2 mutants, has been demonstrated to be sufficient for activation of ERKs in COS-7 cells.⁴⁷ By overexpressing the ßARK1^495–689 polypeptide minigene, we showed that the G_ß subunit is required for Ang II–induced ERK activation in cardiac fibroblasts. Although the precise mechanism of ERK activation by the G_ß subunit remains to be determined, the G_ß subunit has been reported to activate ERKs through Src family tyrosine kinases.^{60 61} It has also been reported that the G_ß subunit activates PI3K, which in turn activates Ras through Src and Shc.^{34 58 62 63 64} Quite recently, it has been proposed that PI3K mediates signals from G_ß to Src family protein tyrosine kinases.⁶⁵ We have recently observed that PI3K plays a critical role in the Ang II–induced activation of p70 S6 kinase and the increase in protein synthesis in cardiac myocytes (authors' unpublished data, 1997). Further studies will be needed to determine whether PI3K is involved in G_ß subunit–induced Src activation by Ang II in cardiac fibroblasts.

AT1R stimulates PLC, thereby generating two major second messengers, IP3 and DG.²¹ IP3 leads to the release of Ca²⁺ from intracellular Ca²⁺ stores, and DG activates PKC. In cardiac myocytes, Ang II activates ERKs through the PKC-dependent pathway.³² In cardiac fibroblasts, however, tyrosine kinases, but not PKC, play a critical role in Ang II–induced ERK activation. Since AT1R itself does not have tyrosine kinase activity, nonreceptor-type tyrosine kinases might play roles in Ang II–induced ERK activation. It has been reported that Ang II activates Src family protein kinases in cardiac myocytes²² and smooth muscle cells.^{23 24} We therefore examined whether Src family protein kinases are involved in Ang II–induced ERK activation in cardiac fibroblasts by expressing Csk, a negative regulator of Src family. Csk is a unique tyrosine kinase, in that it phosphorylates the tyrosine residue in the carboxyl terminus of Src family tyrosine kinases and thereby inactivates them.^{37 52} Overexpression of Csk completely inhibited the activation of ERKs by Ang II in cardiac fibroblasts, but not in cardiac myocytes. These findings suggest that Src family proteins play a critical role in Ang II–induced ERK activation in cardiac fibroblasts and that even though Fyn, an Src family protein, has been reported to be activated by Ang II in cardiac myocytes,²² Src family proteins are not required for Ang II–induced ERK activation in cardiac myocytes.

Activation of tyrosine kinases, including Src family tyrosine kinases, leads to activation of Ras through adapter proteins such as Shc and Grb2 and the guanine exchange factor Sos.^{20 53 54 55 56} It has been demonstrated that Shc is tyrosine-phosphorylated in response to Ang II in cardiac fibroblasts, and evidence has been obtained that Shc may serve as a converging target in the growth factor–and G protein–coupled receptor-stimulated signal transduction events resulting in activation of Ras protein.²⁰ We demonstrated that Ang II rapidly induced phosphorylation of Shc and association of Shc with Grb2, possibly resulting in the translocation of the Ras guanine nucleotide exchange factor Sos to the membrane fraction. We also examined whether Src family tyrosine kinases are involved in the Ang II–induced activation of Shc in fibroblasts using CHO fibroblasts permanently transfected by Csk. Although Shc was phosphorylated by Ang II in parental CHO cells, phosphorylation levels of Shc were not elevated in Csk-overexpressing CHO fibroblasts by Ang II treatment for up to 10 minutes (data not shown). These findings suggest that Ang II–induced Shc phosphorylation in fibroblasts requires activation of Src family tyrosine kinases. Taken together, these findings suggest that Ang II may activate Ras through Src, Shc, Grb2, and Sos in cardiac fibroblasts. In contrast, Ang II did not enhance phosphorylation of Shc in cardiac myocytes. It has been reported that Ang II activates Ras via phosphorylation of Shc in cardiac myocytes.²² The reason for this discrepancy in findings is unknown but may be due to differences in culture conditions. In primary cultures of cardiac myocytes, some contaminating nonmyocytes are always included. We have recently reported that Ang II–induced ERK activation is completely dependent on PKC and that tyrosine kinase inhibitors had no effects on Ang II–induced ERK activation in cardiac myocytes.³² Collectively, these findings suggest that Ang II–induced activation of tyrosine kinases is weak, if it occurs at all, and does not play a major role in activating ERKs in cardiac myocytes.

Many lines of evidence have suggested that Ras plays a key role in a variety of cell functions through sequential activation of Raf-1 and ERKs.^{66 67} In some types of cells, however, PKC, but not Ras, activates ERKs through Raf-1.⁶⁸ Whether the Raf-1/ERK cascade is activated by PKC or Ras may depend on cell type and G protein. In cardiac myocytes, in which AT1R couples to G_q, PKC, but not Ras or tyrosine kinases, is critical for Ang II–induced Raf-1/ERK activation.³² On the other hand, in cardiac fibroblasts, in which AT1R couples to G_i, Ras, but not PKC, is a key component of signal transduction pathways leading to the activation of ERKs induced by Ang II. In vascular smooth muscle cells, AT1R has been reported to couple to pertussis toxin–insensitive G protein (G_q) and activate ERKs through Ca²⁺, tyrosine kinases, and the Ras-dependent pathway.^{24 25} These results collectively suggest that AT1R-evoked signal transduction pathways are quite different among cell types. G proteins coupling to AT1R may be different, or signal transduction pathways downstream from G proteins leading to ERK activation may be different among cell types. These distinctive signaling pathways may result in different effects of Ang II: hypertrophy in cardiac myocytes, hyperplasia in cardiac fibroblasts, and hypertrophy and hyperplasia in smooth muscle cells.

	Selected Abbreviations and Acronyms

 

ßARK1 = ß-adrenergic receptor kinase 1 Ang II = angiotensin II Anti-HA = anti-hemagglutinin AT1R, AT2R = Ang II type-1 and -2 receptor Csk = C-terminal Src kinase D.N.Raf-1, D.N.Ras = dominant-negative mutants of Raf-1 and Ras DG = diacylglycerol ECM = extracellular matrix ERK = extracellular signal–regulated kinase HA = hemagglutinin IP3 = inositol trisphosphate MBP = myelin basic protein MEK = mitogen-activated protein kinase/ERK kinase PI3K = phosphatidylinositol-3 kinase PKC = protein kinase C PLC = phospholipase C TPA = 12-O-tetradecanoylphorbol 13-acetate

	Acknowledgments

 
This study was supported by a Grant-in-Aid for Scientific Research, Developmental Scientific Research, and Scientific Research on Priority Areas from the Ministry of Education, Science, Sports, and Culture of Japan and by a Japan Heart Foundation/Pfizer Pharmaceuticals grant for Research on Coronary Artery Diseases, Tanabe Medical Frontier Conference (Dr Komuro). We wish to thank Drs M. Karin, Y. Takai, H. Sabe, and K. Touhara for plasmids.

	Footnotes

 
This manuscript was sent to Robert J. Lefkowitz, Consulting Editor, for review by expert referees, editorial decision, and final disposition.

Received September 2, 1997; accepted November 7, 1997.

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