Angiotensin II Type 1 Receptor–Induced Extracellular Signal–Regulated Protein Kinase Activation Is Mediated by Ca²⁺/Calmodulin-Dependent Transactivation of Epidermal Growth Factor Receptor

From the Department of Medicine II (S.M., Y.M., H. Masaki, K.M., Y. Tsutsumi, Y.M., Y.S., Y. Tanaka, T.I., M.I., H. Matsubara), Kansai Medical University, Osaka, Japan; the Pharmacological Laboratory (Y.N.), Taiho Pharmaceutical Co, Ltd, Tokushima, Japan; and the Department of Genetics (N.G., M.S.), Institute of Medical Science, University of Tokyo, Tokyo, Japan.

Correspondence to Hiroaki Matsubara, MD, Division of Endocrine Hypertension and Metabolism, Department of Medicine II, Kansai Medical University, Fumizonocho 10–15, Moriguchi, Osaka 570, Japan. E-mail matsubah{at}takii.kmu.ac.jp

	Abstract

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Abstract—The signaling cascade elicited by angiotensin II (Ang II) resembles that characteristic of growth factor stimulation, and recent evidence suggests that G protein–coupled receptors transactivate growth factor receptors to transmit mitogenic effects. In the present study, we report the involvement of epidermal growth factor receptor (EGF-R) in Ang II–induced extracellular signal–regulated kinase (ERK) activation, c-fos gene expression, and DNA synthesis in cardiac fibroblasts. Ang II induced a rapid tyrosine phosphorylation of EGF-R in association with phosphorylation of Shc protein and ERK activation. Specific inhibition of EGF-R function by either a dominant-negative EGF-R mutant or selective tyrphostin AG1478 completely abolished Ang II–induced ERK activation. Induction of c-fos gene expression and DNA synthesis were also abolished by the inhibition of EGF-R function. Calmodulin or tyrosine kinase inhibitors, but not protein kinase C (PKC) inhibitors or downregulation of PKC, completely abolished transactivation of EGF-R by Ang II or the Ca²⁺ ionophore A23187. Epidermal growth factor (EGF) activity in concentrated supernatant from Ang II–treated cells was not detected, and saturation of culture media with anti-EGF antibody did not affect the Ang II–induced transactivation of EGF-R. Conditioned media in which cells were incubated with Ang II could not induce phosphorylation of EGF-R on recipient cells. Platelet-derived growth factor-ß receptor was not phosphorylated on Ang II stimulation, and Ang II–induced c-jun gene expression was not affected by tyrphostin AG1478. Our results demonstrated that in cardiac fibroblasts Ang II–induced ERK activation and its mitogenic signals are dominantly mediated by EGF-R transactivated in a Ca²⁺/calmodulin-dependent manner and suggested that the effects of Ang II on cardiac fibroblasts should be interpreted in association with the signaling pathways regulating cellular proliferation and/or differentiation by growth factors.

Key Words: angiotensin II receptor • angiotensin II • Ca²⁺ • G protein–coupled receptor • epidermal growth factor receptor

	Introduction

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Angiotensin II has been shown to act as a cellular growth factor and to be involved in the remodeling of the heart after chronic hypertension and myocardial infarction.^{1 2} Neonatal rat cardiac fibroblasts have abundant high-affinity G_q protein–linked Ang II receptors, which are classified pharmacologically as belonging to the AT1-R subtype.^{3 4} AT1-R stimulation was found to stimulate DNA synthesis and cell proliferation³ and also increase the synthesis of extracellular matrix proteins.⁵ These results, as well as in vivo study,⁶ support the idea that cardiac fibroblasts are a target for Ang II in situ, explaining how Ang II may contribute to remodeling of the cardiac interstitium in a variety of physiological and pathological conditions.

Ang II, acting via AT1-R, initiates early biochemical events, including rapid production of diacylglycerol and inositol 1,4,5-trisphosphate by PLC-mediated hydrolysis of inositol phospholipids and activation of PKC.^{7 8 9} Ang II also induces an increase in expression of the growth-associated nuclear proto-oncogenes and stimulates tyrosine phosphorylation of multiple substrates, including mitogen-activated protein/ERKs.^{8 10 11 12 13 14} The ERK is activated by phosphorylation on threonine and tyrosine residues catalyzed by MEK, and the MEK is in turn regulated by serine phosphorylation by several MEK kinases, including Raf-1.¹⁵

Recently, the signaling pathway from growth factor receptor tyrosine kinases to ERKs has been elucidated^13,16; adapter proteins containing the SH2 domain, such as Grb2 or Shc (known to exist in 3 forms: p46, p52, and p66),¹⁷ link tyrosine-phosphorylated receptor tyrosine kinases with the guanine nucleotide exchange factor Sos to activate Ras. Ras recruits Raf to the membrane for activation, possibly by Src kinases. Certain aspects of signal transduction characteristic of Ang II stimulation resemble those evoked by growth factors. Activation of phospholipase C, tyrosine kinases, and ERK and the expression of nuclear proto-oncogenes exemplify phenomena common to Ang II and growth factor signaling (see References 8⁸ [review] and 11). Recently, cross talk between G protein–coupled receptors and growth factor receptors with intrinsic tyrosine kinases was shown. In VSMCs, stimulation by Ang II resulted in phosphorylation of PDGF-R¹⁸ or EGF-R¹⁹ associated with formation of the Shc/Grb2 complex. Stimulation of Rat-1 cells with endothelin-1, LPA, or thrombin induced a rapid increase in tyrosine phosphorylation of EGF-R and p185^neu, leading to activation of the ERK.²⁰ Stimulation of Cos-7 cells with G_i- or G_q-coupled receptors caused phosphorylation of EGF-R that was associated with assembly of Shc and Grb2,^{21 22} and Ca²⁺-dependent EGF-R activation by stimulation of voltage-sensitive Ca²⁺ channel was also found in PC12 cells.^{23 24} Ang II activated tyrosine phosphorylation of the insulin-like growth factor-1 receptor and insulin receptor substrate-1 in VSMCs.²⁵ In addition, the m1 muscarinic acetylcholine receptor was also shown to transactivate EGF-R and its downstream signaling, resulting in modulation of the K⁺ channel in human embryonic kidney 293 cells.²⁶ Thus, additional tyrosine kinases that phosphorylate receptor tyrosine kinases appear to contribute (independent of ligand) in a general or cell type–specific way to mitogenic signaling mediated through G protein–coupled receptors. In the present study, we demonstrated for the first time that in cardiac fibroblasts the signal transduction from AT1-R to ERK activation, c-fos expression, and DNA synthesis is mainly mediated through tyrosine phosphorylation of EGF-R transactivated in a Ca²⁺/calmodulin-dependent manner.

	Materials and Methods

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Materials
Genistein, ST638, BAPTA-AM, W7, and tyrphostin AG1478 were purchased from Calbiochem. A23187 and PMA were from Sigma Chemical Co. Losartan was provided by DuPont Merck Pharmaceutical. PD123319 was provided by Parke-Davis and Warner-Lambert Co. c-Fos and c-Jun cDNA were kind gifts from Dr Komuro, Tokyo University, Tokyo, Japan. Antibodies were purchased from the following vendors: Upstate Biotechnology (4G10-HRP, anti-Shc, anti-EGF, anti-human–specific EGF-R), Santa Cruz (anti-human EGF-R, anti–PDGF-R, anti-FAK, anti-paxillin, and normal rabbit IgG), and New England Bio Labs (phosphospecific ERK). Protein A/G agarose was from Santa Cruz. Recombinant human BB-PDGF and mouse EGF were from Upstate Biotechnology and Toyobo, respectively. Anti-Shc, anti–EGF-R, anti-FAK antisera were anti-rabbit antibodies.

Cell Culture
Cardiac fibroblasts were prepared from ventricles of 1- to 2-day-old Wistar rats and grown as previously described.^{27 28} Subcultured fibroblasts from passages 4 to 6, used in this experiment, were >99% positive for immunostaining with vimentin antibody and were negative for desmin (for myocytes), smooth muscle -actin (for VSMCs), and a polyclonal antibody against von Willebrand factor (for endothelial cells).^{27 28} Subconfluent cells were serum-starved for 24 hours and used for the experiments.

ERK Activity
Fibroblasts grown on 24-well plates were stimulated with agonists at 37°C in serum-free DMEM for specified durations. The reaction was terminated by replacement of medium with ice-cold lysis buffer (10 mmol/L Tris-HCl [pH 7.4], 20 mmol/L NaCl, 2 mmol/L EGTA, 2 mmol/L dithiothreitol, 1 mmol/L Na₃VO₄, 1 mmol/L phenylmethylsulfonyl fluoride, 10 µg/mL leupeptin, and 10 µg/mL aprotinin). After brief sonication, the samples were centrifuged for 5 minutes at 14 000g, and the supernatant was assayed for ERK activity with an assay kit (Amersham) that measured the incorporation of [-³³P]ATP into synthetic peptide (KRELVEPLTPAGEAPNQALLR) as a specific ERK substrate. The reaction was carried out with the cell lysate in 75 mmol/L HEPES (pH 7.4) containing 1.2 mmol/L MgCl₂, 2 mmol/L substrate peptide, and 1.2 mmol/L ATP, along with 1 µCi of [-³³P]ATP for 30 minutes at 30°C as described.¹⁴ The resultant solution was applied to a phosphocellulose membrane and extensively washed in 1% acetic acid and then in H₂O. The radioactivity was measured by liquid scintillation counting.

Immunoprecipitation and Immunoblot Analysis
Cells were lysed in lysis buffer containing 20 mmol/L Tris-HCl, 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1% Triton X-100, 2.5 mmol/L sodium pyrophosphate, 1 mmol/L ß -glycerophosphate, 1 mmol/L Na₃VO₄, 1 µg/mL leupeptin, 1 µg/mL antipain, 0.2% (wt/vol) aprotinin, 1 µg/mL chymostatin, and 1 µg/mL phenylmethylsulfonyl fluoride. After they were incubated for 30 minutes at 4°C, the cell lysates were centrifuged at 12 000g for 10 minutes, and the supernatant was collected. In determining cellular phosphotyrosine proteins, proteins were resolved by SDS-PAGE, transferred to PVDF membrane, and immunoblotted with 4G10-HRP. For immunoprecipitation experiments, proteins were precleared with protein A/G agarose for 30 minutes at 4°C. Then appropriate antibodies were added to the precleared samples and incubated for 6 hours at 4°C, followed by the addition of protein A/G agarose for 2 hours at 4°C. Immune complexes were boiled in SDS-sample buffer, subjected to SDS-PAGE, transferred to PVDF membrane, and immunoblotted with the desired antibody. After incubation with secondary antibodies, immunoreactive proteins were detected by the enhanced chemiluminescence reaction (ECL, Amersham). When appropriate, the PVDF membranes were stripped and reprobed with another antibody.

Determination of DNA Synthesis and Northern Blotting
Relative rates of DNA synthesis were determined as previously reported.⁵ Fibroblasts were rendered quiescent by serum deprivation for 24 hours and cultured for a further 24 hours in serum-free medium with 5 µCi/mL of [³H]thymidine (NEN) in the presence or absence of Ang II (0.1 µmol/L). Tyrphostin AG1478 was added 15 minutes before the addition of Ang II. At the end of the labeling period, medium was aspirated off, and the cells were washed twice with PBS and then incubated (30 minutes at 4°C) with 10% perchloric acid. Cell precipitates were solubilized in 0.3N NaOH/1% SDS for 2 hours and examined with a liquid scintillation counter. For Northern analysis, total RNA was extracted by guanidinium isothiocyanate–cesium chloride centrifugation, fractionated on 1% agarose/formaldehyde gels, and transferred to nylon membranes as previously reported.^{27 28 29 30} Blots were then hybridized with random-primed ³²P-labeled cDNA probes consisting of rat c-fos and GAPDH as an internal control. The c-fos and GAPDH mRNA signals were stripped by boiling the hybridized membrane and were then used for the detection of c-jun mRNA signals. Hybridized signals were measured by scanning densitometry.

Generation of HEGFR 533del and Transfection of DNA
For construction of HEGFR 533del, an XbaI fragment of the full-length human EGF-R³¹ was subcloned into pBluescript. Site-directed mutagenesis was performed with a QuickChange site-directed mutagenesis kit (Stratagene) using the oligonucleotides 5'-CGTTCGGAAGCGCTAGCTGCGGAGGCTGC and 5'-GCAGCCTCCGCAGCTAGCGCTTCCGAACG (654-Thr changed to termination codon). The mutated region was sequenced to ensure that only substituted positions were modified. This HEGFR 533del was subcloned into the expression vector pcDNA (Invitrogen) and transfected to cardiac fibroblasts as previously reported.²⁸ In brief, cells were incubated with HEGFR 533del or pcDNA plasmid alone using Lipofectamine Plus reagent according to the manufacturer's instruction (GIBCO BRL) and selected with geneticin.³²

Binding Assay of EGF-R, PDGF-R, and AT1-R
The binding of growth factors to cardiac fibroblast cells was performed as described previously.³³ Briefly, quiescent cardiac fibroblast cells in 24-well tissue culture dishes were washed once with 1.0 mL of ice-cold binding buffer (PBS with 1 mmol/L CaCl₂ and 1% BSA [pH 7.4]). After removing this buffer, the cells were incubated at 4°C for 4 hours in 0.5 mL binding medium (Ham's medium F12 buffered at pH 7.4 with 25 mmol/L HEPES and 0.1% BSA) containing a constant amount of ¹²⁵I-labeled growth factors (PDGF or EGF) (0.1 to 20 ng/mL). Nonspecific binding was determined using 200 ng/mL of PDGF-BB or 1 mg/mL of EGF. At the end of the binding period, the cells were washed (3 times) at 4°C with 1.0 mL PBS. For the determination of cell-attached ¹²⁵I-labeled growth factor, the wells were extracted by adding 1 mL/well buffer (0.5 mol/L acetic acid and 150 mmol/L NaCl) to count the bound radioactivity using a gamma counter. Binding was expressed as femtomoles of growth factor bound per 10⁵ cells. To determine cell number, parallel culture wells were incubated in binding medium without ¹²⁵I-labeled growth factor. We found that EGF-R densities in cardiac fibroblasts were stable before passage 7 but moderately decreased after passage 8, whereas AT1-R densities were at least stable before passage 8. The measurement of AT1-R densities was performed using membrane fractions as previously described.²⁸

Measurement of Dehydrogenase Activities (LDH)
A spectrophotometric enzyme assay (DRI-CHEM slide LDH-P, Fuji Film) was performed to measure LDH release in the medium. The procedure followed was the same as described by the manufacturer. In brief, 10 µL of triplicate medium and reagent were mixed for 2 minutes at room temperature. Absorbance at 540 nm was determined, and LDH activity (in units per liter) was automatically calculated by FD3030 analyzer. One unit of LDH activity (U/L) is defined as that amount of enzyme that will catalyze the formation of 1 µmol of NADH per minute under the condition of the assay procedure. The LDH activity determined by this assay method had a high relationship (r=0.995) with that determined by the autoanalyzer.

EGF Detection
To detect the presence in the culture medium, cultured supernatant from serum-starved cells treated with EGF (50 ng/mL, 10 minutes at 37°C), Ang II (100 nmol/L, 10 minutes at 37°C), and untreated cells (control) was collected and concentrated in a Centricon 3 concentrator (Amicon). The concentrated samples were subsequently resolved in 12% SDS-PAGE, transferred to PVDF membrane, and immunoblotted with an anti-EGF antibody. The sensitivity of the anti-EGF antibody was determined by resolving known amounts of EGF by 12% SDS-PAGE, transferring the samples to PVDF membrane, and immunoblotting with the anti-EGF antibody.

Medium Transfer or Neutralizing Experiments
Conditioned medium from cells exposed to Ang II (100 nmol/L, 10 minutes at 37°C) was transferred onto serum-starved recipient cells and incubated for 10 minutes at 37°C in the absence or presence of losartan (10 µmol/L). In certain experiments, the medium was saturated with an anti-EGF antibody (20 µg/mL) capable of neutralizing at least 10 ng of EGF/mL at 37°C for 15 minutes and then incubated with Ang II (100 nmol/L, 10 minutes at 37°C). Cells were lysed, and ERK activities were determined.

Statistical Analysis
The results are expressed as mean±SE. ANOVA and the Fisher least significant difference test were used for multigroup comparisons, with P<0.05 considered significant.

	Results

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AT1-R–Mediated Transactivation of EGF-R
Accumulated evidence demonstrated that stimulation with heterotrimeric G protein–coupled receptor agonists, including endothelin-1, Ang II, 2A adrenaline, LPA, thrombin, and m1 muscarinic acetylcholine, resulted in tyrosine phosphorylation of EGF-R and subsequent complex formation of Shc/Grb2 and stimulation of ERK activity.^{19 20 21 22 26} Thus, since the Shc adaptor protein is an important component of the pathways linking receptor tyrosine kinases and G protein–coupled receptors to activation of the Ras cascade, we examined whether Shc isoforms become tyrosine-phosphorylated in cardiac fibroblasts. We immunoprecipitated the Shc protein from cells that had been treated with EGF (10 ng/mL) or Ang II (100 nmol/L) and analyzed the immunoprecipitates using anti-phosphotyrosine antibody. As expected, the control treatment with EGF caused a robust increase in the phosphotyrosine content of p66, p52, and p46 Shc (Figure 1A). Similarly, Ang II evoked a potent increase in tyrosine-phosphorylated Shc (Figure 1A). Interestingly, we also observed two higher molecular mass bands of 170 and 120 kDa that coimmunoprecipitated with Shc and became tyrosine-phosphorylated in response to either EGF or Ang II treatment. Since Shc was identified originally as a substrate for the 170-kDa EGF-R, we reprobed the same filter with an anti–EGF-R antibody and found that the 170-kDa band became labeled and that association with Shc was increased after EGF or Ang II treatment (Figure 1A). In addition, pretreatment of cells with a specific inhibitor of the EGF-R kinase activity, tyrphostin AG1478 (250 nmol/L),³⁵ blocked both the EGF-stimulated and Ang II–stimulated tyrosine phosphorylation of Shc as well as the Shc-associated 170-kDa protein identified as the EGF-R (Figure 1A). This transactivation by Ang II in cardiac fibroblasts was completely blocked by pretreatment with 1 µmol/L of the AT1-R antagonist losartan (data not shown). The control study indicated that the same amount of all Shc isoforms (p66, p52, and p46) was precipitated in each lane (Figure 1B). We also found that the p66 Shc showed a reduction in mobility after treatment with EGF or Ang II (Figure 1B). Since it is assumed that the reduced mobility is due to Ser/Thr phosphorylation, this is an EGF-R–mediated effect and may be important in negative regulation of the ERK activation by the EGF-R downstream signaling, as previously suggested.^{36 37}

View larger version (44K): [in this window] [in a new window]   Figure 1. Ang II–induced tyrosine phosphorylation of the EGF-R and Shc. Cardiac fibroblasts were serum-starved for 24 hours. Unstimulated cells (control), cells stimulated with 10 ng/mL EGF for 5 minutes (EGF) or with 100 nmol/L Ang II for 5 minutes (Ang II), cells pretreated with 250 nmol/L AG1478 for 20 minutes and challenged with 10 ng/mL EGF for 5 minutes (EGF/AG1478) or with 100 nmol/L Ang II for 5 minutes (Ang II/AG1478) were lysed and incubated with anti-Shc rabbit antibody (-Shc). The immunoprecipitates were resolved by 10% SDS-PAGE, transferred to membranes, and immunoblotted with an anti-phosphotyrosine antibody (PY, panel A, top). The same membranes were reprobed and blotted with anti–EGF-R antibody (EGFR, panel A, bottom) or -Shc (panel B). As the control experiment, lysates from control cells and cells stimulated with EGF (10 ng/mL) or Ang II (100 nmol/L) were immunoprecipitated with the control rabbit IgG and blotted with PY (panel C). IP indicates immunoprecipitation.

To examine whether the protein precipitated with the rabbit anti-Shc antibody represents each respective target in the immunoprecipitation/immunoblotting experiments, we used the control rabbit IgG to precipitate the protein and blotted with anti-phosphotyrosine antibody. The result revealed that no specific bands were detected in nonstimulated cells or cells exposed to EGF and Ang II (Figure 1C), suggesting that the precipitated protein in Figure 1A represents each respective target. Although in this study we could not identify a protein with a molecular mass of 120 kDa associated with Shc (Figure 1), these findings indicated that activation of the AT1-R induces tyrosine phosphorylation of the EGF-R, resulting in association with activated Shc protein.

We next confirmed the selectivity of tyrphostin AG1478 by testing its effect on Ang II–induced tyrosine phosphorylation of FAK and paxillin. The effect by this drug on PDGF- or PMA-induced ERK activation was also examined. These experiments demonstrated that cells pretreated with AG1478 failed to inhibit Ang II–induced tyrosine phosphorylation of FAK (Figure 2A) and paxillin (Figure 2B) and that activity and phosphorylation of ERK induced by PDGF or PMA were not affected by AG1478 (Figure 2C). Although a single band was detected in immunoblotting with an anti–phospho-ERK antibody (Figure 2C), we found in the study using an anti-ERK antibody that p42 ERK is dominantly phosphorylated by PDGF, EGF, or Ang II in cardiac fibroblasts, resulting in a single phosphorylated ERK band. Since it is assumed that AG1478 did not influence endothelin-1–induced phosphorylation of FAK and paxillin in rat-1 fibroblasts²⁰ and that carbachol-dependent phosphorylation of p60 Src and FAK or activation of insulin receptors by insulin was not affected by AG1478 in kidney 293 cells,²⁶ these findings establish selective inhibition of EGF-R activation by tyrphostin AG1478, which does not interfere with functional coupling to AT1-R–mediated downstream signaling in cardiac fibroblasts.

View larger version (52K): [in this window] [in a new window]   Figure 2. Effects of tyrphostin AG1478 on tyrosine phosphorylation of FAK (A) and paxillin (B) and on PDGF- or PMA-induced ERK activation (C). In panel A, cardiac fibroblasts were serum-starved for 24 hours. Unstimulated cells (control), cells stimulated with 100 nmol/L Ang II for 2 minutes (Ang II), and cells pretreated with 250 nmol/L AG1478 for 20 minutes (AG1478) and challenged with 100 nmol/L Ang II for 2 minutes (Ang II/AG1478) were lysed and incubated with anti-FAK antibody. The immunoprecipitates were resolved by 10% SDS-PAGE, transferred to membranes, and immunoblotted with an anti-phosphotyrosine antibody (PY, panel A, top). The same membranes were reprobed and blotted with anti-FAK antibody (panel A, bottom). To detect paxillin, immunoprecipitation (IP) was performed with PY and blotted with anti-paxillin, because available anti-paxillin antibody was not good for IP (panel B, top). The same amount of protein was resolved by SDS-PAGE without IP and blotted with anti-paxillin antibody (panel B, bottom), in which slowly migrating phosphorylated bands were detected with Ang II or Ang II/AG1478 treatment. In panel C, serum-starved cells were stimulated with PDGF (10 ng/mL) or PMA (1 µmol/L) for 8 minutes in the presence or absence of AG1478, and ERK activities were examined as described in Materials and Methods. Cell lysates were resolved by 9% SDS-PAGE and immunoblotted with anti–phospho-ERK antibody. Results shown are means±SE of 3 separate experiments. *P<0.01 vs the ERK activity of the control.

Specificity of EGF-R Transactivation in AT1-R–Mediated Signaling
Time course experiments showed rapid tyrosine phosphorylation of the EGF-R, reaching a maximum within 2 minutes after stimulation of AT1-R (Figure 3A), consistent with the finding that maximal activation of Ras was observed at 5 minutes after Ang II stimulation in cardiomyocytes¹³ and VSMCs.¹⁴ We also examined the involvement of the PDGF-R after stimulation by Ang II or the Ca²⁺ ionophore A23187. Although the ligand binding assay revealed the presence of a single class of PDGF-Rs with high-affinity binding sites (Table 1) and PDGF-BB–induced tyrosine phosphorylation of the PDGF-R, the PDGF-R was not phosphorylated by Ang II or A23187 (Figure 3B).

View larger version (51K): [in this window] [in a new window]   Figure 3. Time course of Ang II–induced phosphorylation of EGF-R, lack of Ang II–induced PDGF-R phosphorylation, and specific inhibition of Ang II–induced EGF-R phosphorylation by dominant-negative EGF-R mutants. Cardiac fibroblasts were serum-starved for 24 hours and stimulated with Ang II (100 nmol/L) for indicated durations (panel A) or with PDGF-BB (20 ng/mL), Ang II (100 nmol/L), and A23187 (1 µmol/L) for 5 minutes (panel B). Expression of the dominant-negative EGF-R mutants 533del-1 and del-2 was confirmed by labeling cells overnight with [35S]methionine, immunoprecipitation (IP) with anti-human EGF-R antibody (human EGF-R), which selectively recognizes human EGF-R but not rat EGF-R, and SDS-PAGE followed by autoradiography (panel C). Cardiac fibroblasts stably transfected with pcDNA expression vector alone (control) or pcDNA containing HEGFR 533del were serum-starved for 12 hours and stimulated with Ang II (100 nmol/L) or EGF (2 and 50 ng/mL) for 2 minutes (panel D). The immunoprecipitates using anti–EGF-R antibody (EGF-R), which recognizes both rat and human EGF-R, were resolved by 7.5% SDS-PAGE, transferred to membranes, and immunoblotted with either the same anti-phosphotyrosine antibody (PY) or EGF-R as used in immunoprecipitation (IP). The experiments were separately repeated 3 times with comparable results, and the representative data are shown.

The role of EGF-R in AT1-R signaling was further analyzed by specific inhibition of the EGF-R signal. The dominant-negative EGF-R mutant, which lacks the cytoplasmic domain of human EGF-R (HEGFR 533del), was constructed and stably transfected in cardiac fibroblasts. This mutant was shown to inhibit the downstream signaling of rat EGF-R by formation of signaling-defective heterodimers with the wild-type receptor.³⁴ Expression of the HEGFR 533del was confirmed by metabolic labeling, followed by immunoprecipitation with an anti-human EGF-R antibody, which selectively recognizes the human but not rat EGF-R (Figure 3C). We obtained several cloned cells expressing HEGFR 533del and selected 2 clones that most abundantly (HEGFR 533del-1) or ito a lesser extent (HEGFR 533del-2) expressed the mutated EGF-R (Figure 3C). AT1-R numbers in these clones were examined by the ligand binding assay using the membrane fraction, and its expression level was found to be comparable to that in the control cells (Table 2). Ang II–induced elevation of the intracellular Ca²⁺ level in these cloned cells was also comparable to that in the control cells (data not shown). Using these cloned cells, we examined Ang II–induced or EGF-induced effects on tyrosine phosphorylation of the EGF-R. In HEGFR 533del-1 cells, receptor activation at low ligand concentration (2 ng/mL EGF) was attenuated compared with the control transfected cells, whereas tyrosine phosphorylation of the EGF-R after Ang II stimulation was completely blocked (Figure 3D). In HEGFR 533del-2 cells, which expressed the mutated EGF-R to a lesser extent than did the HEGFR 533del-1 cells, Ang II–induced or EGF-induced EGF-R phosphorylation was more moderately inhibited than that observed in HEGFR 533del-1 cells (Figure 3D).

We next investigated the effect of EGF-R inhibition on Ang II–induced ERK activation. As shown in Figure 4, Ang II (100 nmol/L) stimulates ERK activity with a maximal increase (11-fold) around 10 minutes, followed by a gradual decline. Ang II–induced ERK activation was increased dose-dependently, with a maximal peak at 100 nmol/L (data not shown). In HEGFR 533del-1 transfectants in which Ang II–induced transactivation of EGF-R was abolished, ERK activation after Ang II treatment was completely blocked, whereas PMA-induced ERK activation that uses upstream pathways different from AT1-R–mediated ERK signaling (Figure 2C) was preserved in the transfectants (Figure 4B), suggesting that EGF-R must be considered to be an integral and essential element of the Ang II signaling pathway, leading to ERK activation in cardiac fibroblasts.

View larger version (37K): [in this window] [in a new window]   Figure 4. Effects of dominant-negative EGF-R mutant on Ang II–induced ERK activation. Cells stably transfected with expression vector alone (control) or dominant-negative mutant HEGFR 533del-1 were stimulated with Ang II (100 nmol/L) or PMA (1 µmol/L), and time-dependent changes in ERK activities and phosphorylation were examined as described in Figure 2. The experiments were repeated separately 3 times with comparable results, and the representative data are shown. Results shown in panel A are the means of 4 separate experiments.

EGF-R Transactivation Is a Ca²⁺/Calmodulin-Dependent PKC-Insensitive Response
Since it was reported that PLC activation through AT1-R–coupled G_q protein played a critical role in Ang II–induced Ras activation in VSMCs,¹⁴ we examined the effects of Ca²⁺ mobilization and PKC activation after PLC activation on Ang II–induced EGF-R transactivation. As shown in Figure 5, AT1-R stimulation or addition of A23187 caused phosphorylation of EGF-R, and this transactivation was completely inhibited by tyrphostin AG1478. To define the roles of protein tyrosine kinases, intracellular Ca²⁺ mobilization, Ca²⁺/calmodulin kinases, G_i protein, and PKC activation on Ang II–induced phosphorylation of EGF-R, the effects of genistein and ST638 (tyrosine kinase inhibitors), chelation of intracellular Ca²⁺ by BAPTA-AM, reduction of extracellular Ca²⁺ by EGTA, W7, and calmidazolium (Ca²⁺/calmodulin kinase inhibitors) and by PTX, and the downregulation of PKC by 24-hour incubation with 1 µmol/L PMA or PKC inhibitors, such as GF109203X and calphostin C, were examined (Figure 5). We found that Ang II, A23187, and EGF stimulated phosphorylation of EGF-R by 6.4±0.3-fold, 6.7±0.4-fold, and 7.0±0.3-fold (n=4 each), respectively, compared with the basal phosphorylation level of EGF-R. Pretreatment with genistein, ST638, W7, calmidazolium, AG1478, and BAPTA-AM completely (P<0.0001) inhibited the Ang II–induced phosphorylation of EGF-R to the control level. EGTA, calphostin C, GF109203X, PKC depletion, and PTX did not have any significant effect on Ang II–induced phosphorylation levels of EGF-R. All inhibitors tested here did not significantly affect the basal phosphorylation levels of EGF-R. Similar inhibitory effects were also observed on A23187-induced phosphorylation of EGF-R, and EGF-induced phosphorylation of its receptor was completely abolished by pretreatment with genistein, whereas W7 did not affect the ligand-induced autophosphorylation of EGF-R (bottom panel of Figure 5; data for genistein, W7, and AG1478 are shown). These findings demonstrate that EGF-R transactivation by Ang II is mediated in a Ca²⁺/calmodulin-dependent PKC-independent manner and that a PTX-insensitive G protein plays a critical role in the transduction pathway.

View larger version (66K): [in this window] [in a new window]   Figure 5. Ang II–induced tyrosine phosphorylation of EGF-R in a Ca2+/calmodulin-dependent, PKC-independent, and PTX-insensitive manner. Cells were pretreated with Ang II (100 nmol/L), A23187 (1 µmol/L), or EGF (2 ng/mL) in the absence or presence (30-minute pretreatment) of genistein (10 µmol/L), ST638 (1 µmol/L), W7 (10 µmol/L), calmidazolium (10 µmol/L), tyrphostin AG1478 (250 nmol/L), BAPTA-AM (10 µmol/L), EGTA (5 mmol/L), calphostin C (100 nmol/L), and GF109203X (1 µmol/L). PKC depletion was performed by incubating cells with PMA (1 µmol/L) for 24 hours, and the effect of PTX was examined by incubating cells with 1 µg/mL PTX for 24 hours. Phosphorylation of EGF-R was examined as in Figure 3. IP indicates immunoprecipitation; Ab, antibody; EGF-R, anti–EGF-R Ab; and PY, anti-phosphotyrosine Ab. The experiments were separately repeated 4 times with comparable results. The representative data are shown here, and statistical analyses are described in the text.

We next examined the effects of these kinase inhibitors on cell toxicity. Ca²⁺/calmodulin kinase inhibitors (W7 and calmidazolium) did not influence EGF-induced ERK activation, and tyrosine kinase inhibitors (genistein and ST638) failed to inhibit PMA-induced ERK activation (data not shown). Although we also measured LDH in culture medium from cells treated with these kinase inhibitors, no LDH activities were detected, suggesting that complete inhibition of Ang II–induced or A23187-induced phosphorylation of EGF-R by these kinase inhibitors less likely results from cell toxicity.

Autocrine Release of EGF in Response to Ang II Is Not Involved in Transactivation of EGF-R
It is possible that Ang II binding to AT1-R triggers a signaling pathway that induces the release of EGF, which then binds and activates the EGF-R through an autocrine mechanism. Therefore, we investigated whether Ang II induced the release of EGF from these cells. Culture supernatant from 50 ng/mL EGF–or 100 nmol/L Ang II–treated cells was collected, concentrated, and subjected to immunoblot analysis with an anti-EGF antibody. We found that 2 ng/mL of EGF was required to stimulate the phosphotyrosine content of EGF-R in a manner similar to 100 nmol/L Ang II under the conditions used for all experiments described in this study (Figure 3D). However, we were unable to detect the presence of EGF in the concentrated supernatants (10 mL) collected from Ang II–treated cells (Figure 6A, top), even though the anti-EGF antibody was able to detect >10 ng of EGF in this assay (Figure 6A, bottom). Furthermore, culture medium was saturated with the anti-EGF antibody and then transferred onto fresh cultures. Although control experiments demonstrated that preincubation of cells with medium containing the antibody could neutralize at least 10 ng of EGF/mL, Ang II treatment was fully effective in eliciting EGF-R phosphorylation (data not shown).

View larger version (46K): [in this window] [in a new window]   Figure 6. EGF activity in culture supernatant from Ang II– treated cells and its effect on EGF-R phosphorylation Culture supernatants from unstimulated cells (control), cells stimulated with 2 ng/mL EGF for 8 minutes, and cells stimulated with Ang II (100 nm/L) for 8 minutes were concentrated. To detect EGF in the culture supernatants, samples were resolved by 12% SDS-PAGE, transferred to membranes, and immunoblotted with an anti-EGF antibody (EGF, panel A, top). The sensitivity of EGF was tested by blotting defined amounts of EGF (panel A, bottom). Conditioned media in which cells were incubated with Ang II (100 nmol/L) for 8 minutes were transferred onto recipient cells in the presence of losartan (10 µmol/L), and the effect on EGF-R tyrosine phosphorylation was examined as described in Figure 3. For comparison, the effect of unstimulated cells (control) and cells stimulated with 100 nmol/L Ang II for 8 minutes are also shown (panel B). PY indicates anti-phosphotyrosine antibody; EGF-R, anti–EGF-R antibody. The experiments were separately repeated 3 times with comparable results, and the representative data are shown.

Although autocrine release of EGF failed to account for Ang II–induced phosphorylation of EGF-R, this did not rule out autocrine release of a factor different from EGF. To examine this possibility, we tested whether medium transferred from cells stimulated with Ang II could activate the signaling pathway in the presence of losartan. As shown in Figure 6B, the conditioned medium in which cells were incubated with Ang II (100 nmol/L) did not induce phosphorylation of EGF-R on recipient cells in the presence of losartan (10 µmol/L), suggesting a lack of autocrine release of factors other than EGF.

EGF-R Signaling Is Required for Induction of a Full Response in Ang II–Mediated c-fos Expression and DNA Synthesis
We studied the contribution of EGF-R activation to Ang II–induced c-fos gene expression. AG1478 pretreatment effectively reduced c-fos mRNA levels by 88% on Ang II stimulation, whereas EGF-induced c-fos mRNA levels were completely inhibited by AG1478 (Figure 7A). The lack of complete inhibition on Ang II–induced c-fos expression may have been due to synergistic signals involving, to different extents, Jak-STAT^{38 39} or Rho-dependent⁴⁰ pathways, JNK,⁴¹ or other as-yet-undefined parallel systems. The presence of these synergistic pathways may reflect the finding that c-fos mRNA levels stimulated with Ang II were significantly greater (34%, P<0.05) than EGF-induced c-fos mRNA levels (Figure 7A). We also examined the effect of AG1478 on Ang II–induced c-jun mRNA expression (Figure 7B). Ang II and EGF similarly stimulated c-jun mRNA expression, and AG1478 pretreatment completely blocked EGF-induced c-jun expression, whereas no inhibitory effect was observed in Ang II–stimulated c-jun mRNA levels (Figure 7B). These findings demonstrate that Ang II–induced c-fos gene expression is mainly mediated through EGF-R transactivation, whereas EGF-R–mediated downstream signaling is not involved in Ang II–induced c-jun gene expression.

View larger version (38K): [in this window] [in a new window]   Figure 7. Involvement of EGF-R transactivation in Ang II–induced increase in c-fos or c-jun expression and DNA synthesis. A, Serum-starved cells (passage 4) were pretreated with or without tyrphostin AG1478 (250 nmol/L) for 20 minutes and stimulated with either Ang II (100 nmol/L) or EGF (10 ng/mL) for 30 minutes. Aliquots of 20 µg of total RNA were separated on agarose formaldehyde gels and transferred onto nylon membranes for hybridization with 32P-labeled c-fos or GAPDH cDNA probes. Exposure time was 24 hours with the BAS 2000 (Fuji) detection system. Densities of c-fos mRNA signals were measured by densitometer and normalized with those in GAPDH mRNA signals. The results were shown as percent changes relative to Ang II–induced c-fos expression in the absence of AG1478 (n=4 for each). The c-fos mRNA signals in AG1478/EGF-treated cells were undetectable. *P<0.01 vs Ang II–induced c-fos mRNA levels in the absence of AG1478. B, The same filter was reprobed and rehybridized with c-jun probes. C, After serum deprivation for 24 hours, cells were subjected to 20-minute preincubation with or without AG1478 (250 nmol/L) and cultured for a further 24 hours in serum-free medium with 5 µCi/mL of [3H]thymidine in the presence or absence of Ang II (100 nmol/L) or EGF (10 ng/mL) (n=4 for each). The results were shown as percent changes relative to EGF-induced incorporation rates of [3H]thymidine after normalization with basal incorporation rates in untreated cells. *P<0.01 vs the data of EGF-induced incorporation in the absence of AG1478.

For quantification of EGF-R–transmitted mitogen signaling, we also examined Ang II–induced DNA synthesis in the presence or absence of AG1478. Incubation with this agent completely blocked EGF-mediated induction of DNA synthesis, whereas Ang II stimulation was reduced by 74±3% compared with DNA synthesis in the absence of AG1478 (Figure 7C). These observations suggest that for the induction of a full proliferative response to Ang II, functional EGF-R signaling is required. The incomplete inhibition of Ang II–induced DNA synthesis by AG1478 may have been due to mitogen pathways other than the AT1-R/EGF-R/ERK cascade or induction of secondary proliferative stimuli bypassing the blocked EGF-R during the 24-hour assay.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The main findings in the present study indicate that signal transduction from AT1-R to the ERK activation in cardiac fibroblasts is mainly mediated by tyrosine phosphorylation of EGF-R transactivated by the PKC-independent Ca²⁺/calmodulin-dependent pathway. It was also shown that transactivation of EGF-R after AT1-R stimulation plays a central role in both mitogenic signals, leading to DNA synthesis and an increase in c-fos gene expression. Transactivation of EGF-R has been reported in endothelin-1 and LPA and in thrombin signaling in rat-1 fibroblasts,²⁰ bombesin signaling in Cos cells,²² and m1 muscarinic acetylcholine signaling in embryonic kidney 293 cells.²⁶ Intracellular Ca²⁺ mobilization induced by stimulation of the voltage-sensitive Ca²⁺ channel is also known to activate EGF-R in PC12 cells.^{23 24} Linseman et al reported Ang II–induced transactivation of PDGF-R in rat VSMCs,¹⁸ whereas Eguchi et al¹⁹ found the involvement of EGF-R but not PDGF-R in Ang II signaling in the same rat VSMCs. At present, we could not clearly explain why in cardiac fibroblasts Ang II transactivates only EGF-R, although the PDGF-R is present in the cells (Table 1). Because EGF-R is the prototype of the growth factor receptor and because the C-terminal domain (including autophosphorylation sites) greatly differs between EGF-R and PDGF-R,¹⁶ a common mechanism that specifically phosphorylates EGF-R in a ligand-independent manner (such as activation of a nonreceptor tyrosine kinase or inhibition of a phosphotyrosine phosphatase) might contribute in a general way to mitogenic signaling mediated through G protein–coupled receptors. Booz et al⁹ reported that in cardiac fibroblasts the Ang II analogue [Sar¹]Ang II stimulated ERK activity by a Ca²⁺-dependent and phorbol ester–insensitive pathway, in good agreement with the present observations. We further extended their study by examining transduction pathways from AT1-R to ERK in more detail and found a key role of the EGF-R–mediated ERK pathway transactivated in a Ca²⁺/calmodulin-dependent manner. Eguchi et al¹⁴ also reported that AT1-R signaling to the Ras/ERK cascade in VSMCs was dominantly activated by Ca²⁺/calmodulin-dependent tyrosine kinases.

How increases in cytosolic Ca²⁺ stimulate tyrosine phosphorylation on EGF-R has not been determined. Booz et al⁹ reported that in cardiac fibroblasts an increase in intracellular Ca²⁺ levels after Ang II treatment was completely blocked by pretreatment with the intracellular Ca²⁺ chelator BAPTA-AM. In the preliminary study, we also confirmed this finding and found that this increase was not blocked by EGTA or by pretreatment with tyrphostin AG1478 (authors' unpublished data, 1998). These findings indicate that in cardiac fibroblasts Ca²⁺ mobilization after AT1-R stimulation is mainly caused by the release of Ca²⁺ from intracellular stores (possibly by inositol trisphosphate stimulation of Ca²⁺ release from endoplasmic reticulum) and that this Ca²⁺ release does not result from downstream signaling after transactivation of EGF-R. In the present study, we found that both Ang II–induced and A23187-induced EGF-R phosphorylation was completely blocked by tyrosine kinase inhibitors, suggesting that tyrosine kinases activated by Ca²⁺/calmodulin-dependent pathways play a central role on transactivation of EGF-R after AT1-R stimulation. However, tyrosine kinase inhibitors inhibited not only the proposed tyrosine kinases upstream from the EGF-R but also any autophosphorylation by the EGF-R tyrosine kinase (Figure 5). Furthermore, they will inhibit the Src kinases, which have been proposed to be involved in Raf activation.^{42 43} Thus, it remains to be determined whether Ca²⁺/calmodulin-dependent transactivation of EGF-R is due to activation of nonreceptor tyrosine kinases, inhibition of a phosphotyrosine phosphatase, or an unidentified mechanism. In cultured rat liver epithelial cells⁴⁴ and VSMCs,^{45 46} Ang II–induced activation of tyrosine kinases has been shown to be dependent on Ca²⁺, and such a Ca²⁺/calmodulin-activated tyrosine kinase has been recently purified from the bovine uterus.⁴⁷ Tyrosine kinase, such as Ca²⁺-dependent tyrosine kinase PYK2,⁴⁸ might be involved in this pathway. However, PYK2, a member of the FAK family of nonreceptor tyrosine kinases, which has been shown to transmit the Ca²⁺ signal from a G protein–coupled receptor to the formation of Shc-Grb2-Sos complex, lacks a calmodulin-binding motif and is not directly activated by Ca²⁺.⁴⁹ Thus, it is unlikely that PKY2 is a direct candidate, and further studies are required to identify a Ca²⁺/calmodulin-dependent pathway leading to phosphorylation of EGF-R.

Ang II increases the PDGF A chain, transforming growth factor-ß1, and basic fibroblast growth factor expression.⁵⁰ In view of this, we evaluated whether autocrine release of EGF or a factor different from EGF stimulated by the AT1-R–mediated Ca²⁺/calmodulin system accounted for phosphorylation of EGF-R. Although we quantified EGF activity in concentrated supernatant from Ang II–treated cells and also blocked the effect of released EGF using anti-EGF antibody, we were unable to detect the presence of EGF in incubation medium from Ang II–treated cells. Furthermore, conditioned medium in which cells were incubated with Ang II could not induce the expected biological response on recipient cells. However, it might be possible that EGF is released locally and achieves high concentrations at adjacent cells and that the released EGF immediately binds to its receptor before reacting with anti-EGF antibody. Thus, although these findings suggest that Ang II–induced transactivation of EGF-R is unlikely because of the autocrine release of EGF, an involvement of locally released EGF was not completely ruled out in the present study.

We found that in cardiac fibroblasts Ang II stimulated c-fos mRNA expression and that this induction was mainly mediated through EGF-R transactivation (Figure 7A). The c-fos promoter contains serum response element, and induction of c-fos expression occurs on the formation of a ternary complex factor, p62^TCF, at the serum response element.⁵¹ ERK was shown to phosphorylate p62^TCF (also known as elk-1 or SAP-1), resulting in enhanced ternary complex formation.⁵² Thus, it is likely that Ang II–stimulated c-fos gene expression is mainly regulated by phosphorylated p62^TCF after EGF-R–mediated ERK activation. However, we found that Ang II–induced c-fos expression was not completely blocked by specific inhibition of EGF-R signaling and that c-fos mRNA levels stimulated with Ang II were significantly greater than EGF-induced c-fos mRNA levels (Figure 7A), suggesting that there are synergistic signals involving, to different extents, Jak-STAT^{38 39} or Rho-dependent pathways,⁴⁰ JNK,⁴¹ or other as-yet-undefined parallel systems. On the other hand, we found that Ang II–induced expression of the c-jun gene was not mediated through EGF-R transactivation. c-Jun is one of the major components of the transcriptional factor, activator protein-1, which regulates the expression of many genes having a TPA-responsive element in their promoter regions.⁵³ In cardiac myocytes, it was shown that Ang II stimulated c-jun mRNA expression, in which activation of JNK was closely involved.⁵⁴ JNK was reported to phosphorylate 2 serine residues in the presumptive activation domain of c-Jun and to increase its transcriptional activity.^{55 56} JNK is weakly activated by growth factors but markedly activated in response to the inflammatory cytokine, tumor necrosis factor-, ultraviolet irradiation, and a variety of cellular stress.^{55 56} Recently, it has been reported that MEK kinase activates JNK through stress-activated protein kinase/ERK kinase-1⁵⁷ and that small GTP binding proteins of the Rho family (Rac 1 and Cdc 42, which were hitherto thought to function in the regulation of cell morphology)⁵⁸ regulate the activity of JNK.⁵⁹ Given that JNK also phosphorylates and activates p62^TCF and can upregulate the expression of c-fos,⁴¹ JNK activation may therefore at least partially contribute to the Ang II induction of c-fos and be the major route for c-jun expression. Taken together, these findings indicate that in cardiac fibroblasts Ang II mainly activates c-jun gene expression through pathways different from downstream signaling of EGF-R, resulting in increased expression of TPA-responsive element–containing genes, such as atrial natriuretic peptide⁶⁰ or endothelin-1.⁵⁴

Interstitial fibroblast proliferation and collagen accumulation is associated with compensatory remodeling of the hypertrophic myocardium,^{1 2} and the process of structural remodeling leads to diastolic and systolic dysfunction.⁶¹ Ang II is closely involved in the cardiac remodeling process by stimulating hyperplastic growth of cardiac fibroblasts^{3 4} and synthesis of extracellular matrix proteins.^{2 5} The direct involvement of the EGF-R in this process presents a novel paradigm for cross talk between AT1-R and growth factor receptor signaling pathways; therefore, it is important to interpret cardiac effects of Ang II in association with the signaling cascade regulating cellular proliferation and/or differentiation by growth factors.

	Selected Abbreviations and Acronyms

 

Ang II = angiotensin II AT1-R = Ang II type 1 receptor EGF = epidermal growth factor EGF-R = EGF receptor ERK = extracellular signal–regulated kinase FAK = focal adhesion kinase HEGFR = human EGF-R Jak/STAT = Janus kinase/signal transducer and activator of transcription JNK = c-jun N-terminal kinase LPA = lysophosphatidic acid MEK = ERK kinase PDGF = platelet-derived growth factor PDGF-R = PDGF-ß receptor PKC = protein kinase C PLC = phospholipase C PMA = phorbol 12-myristate 13-acetate PTX = pertussis toxin PVDF = polyvinylidene fluoride TPA = 12-O-tetradecanoylphorbol 13-acetate VSMC = vascular smooth muscle cell

	Acknowledgments

 
This study was supported in part by research grants from the Ministry of Education, Science, and Culture, Japan; the Study Group of Molecular Cardiology, Naito Foundation; the Clinical Pharmacology Foundation; the Japan Medical Association; and the Japan Smoking Foundation, Japan Heart Foundation. Dr Murasawa is a Research Fellow of the Japan Society for the Promotion of Science.

Received January 7, 1998; accepted April 27, 1998.

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