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Inhibitory crosstalk between ERK and AMPK in the growth and proliferation of cardiac fibroblasts
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Biochemical and Biophysical Research Communications 368 (2008) 402–407 www.elsevier.com/locate/ybbrc
Inhibitory crosstalk between ERK and AMPK in the growth and proliferation of cardiac fibroblasts Jianhai Du, Tongju Guan, Hui Zhang, Yi Xia, Fei Liu, Youyi Zhang * Institute of Vascular Medicine, Peking University Third Hospital and Key Laboratory of Molecular Cardiovascular Sciences, Ministry of Education, Beijing 100083, PR China Received 31 December 2007 Available online 1 February 2008
Abstract Extracellular signal-regulated kinase (ERK) is one of the key protein kinases that regulate the growth and proliferation in cardiac fibroblasts (CFs). As an energy sensor of cellular metabolism, AMP-activated protein kinase (AMPK) is found recently to be involved in myocardial remodeling. In this study, we investigated the crosstalk between ERK and AMPK in the growth and proliferation of CFs. In neonatal rat cardiac fibroblasts (NRCFs), we found that serum significantly inhibited basal AMPK phosphorylation between 10 min and 24 h and also partially inhibited AMPK phosphorylation by AMPK activator, 5-aminoimidazole-4-carboxamide-ribonucleoside (AICAR). Furthermore, ERK inhibitor could greatly reverse the inhibition of AMPK by serum. Conversely, activation of AMPK by AICAR also showed a significant inhibition of basal and serum-induced ERK phosphorylation but it showed a delayed and steadfast inhibition which appeared after 60 min and lasted until 12 h. Moreover, inhibition of ERK could repress the activation of p70S6K, an important kinase in cardiac proliferation, and AICAR could also inhibit p70S6K phosphorylation. In addition, under both serum and serum-free medium, AICAR significantly inhibited the DNA synthesis and cell numbers, and reduced cells at S phase. In conclusion, AMPK activation with AICAR inhibited growth and proliferation in cardiac fibroblasts, which involved inhibitory interactions between ERK and AMPK. This is the first report that AMPK could be a target of ERK in growth factors-induced proliferation, which may give a new mechanism that growth factors utilize in their promotion of proliferation in cardiac fibroblasts. Ó 2008 Elsevier Inc. All rights reserved. Keywords: AMPK; ERK; Cardiac fibroblasts
Cardiac fibroblasts (CFs) play a central role in the maintenance of extracellular matrix in the normal heart and the mediation of inflammatory and fibrotic myocardial remodeling in the injured and failing heart. An array of evidence suggests that ERK activation is required for cell proliferation to proceed [1]. In CFs, the application of ERK inhibitors substantially inhibited the proliferation by various stimulators such as growth factors [2], Angiotensin II [3] and mechanical force [4]. AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase that has emerged as a key regulator of cellular energy homeostasis and coordinates multiple cata*
Corresponding author. Fax: +86 10 82802306. E-mail address: [email protected] (Y. Zhang).
0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.01.099
bolic and anabolic pathways in the heart [5]. In neonatal rat cardiomyocytes, AMPK activation could inhibit the activation of p70S6K and the cardiac hypertrophy induced by phenylephrine or overexpressed AKT [6]. Through activating AMPK, adiponectin inhibited pressure-overload or angiotensin II-induced cardiac hypertrophy in mice [7]. Injection of AMPK activator, 5-aminoimidazole-4-carboxamide-ribonucleoside (AICAR) attenuated pressure-overload-induced cardiac hypertrophy in rats [8]. We hypothesized that in the growth and proliferation of CFs, it required not only the enhancement of positive signals like ERK, but also the lessening of some negative signals like AMPK. Therefore, we explored, in this study: (1) whether activation of ERK by growth factors repressed AMPK activation; (2) the effect of AMPK activation on ERK
J. Du et al. / Biochemical and Biophysical Research Communications 368 (2008) 402–407
phosphorylation; and (3) the effect of AMPK activation on proliferation in NRCFs by growth factors. Materials and methods Reagents. AICAR was obtained from Toronto Research Chemicals (Toronto, Canada). Fetal bovine serum (FBS) was from Hyclone (Logan, UT). Antibodies against phospho-AMPK alpha (Thr172), phospho-ERK (phospho Thr202/Tyr204), phospho-p70S6K (Thr389), and p70S6K were from Cell Signaling Technology (Beverly, MA). Antibodies against ERK, and eukaryotic translation initiation factor (eIF5) were from Santa Cruz Biotechnology (Santa Cruz, CA). [3H]Thymidine was from Amersham bioscience (Bucks, UK). U0126 and rapamycin were from Sigma (St. Louis, MO). Fetal bovine serum (FBS) was from Hyclone (Logan, UT). Cell culture. This study was conducted in accordance with the Guide for the Care and Use of Laboratory Animals (National Academic Press, Washington, DC, 1996). Cultured neonatal rat cardiac fibroblasts were isolated from 1 day-old Sprague–Dawley rats as we previously described [2]. After digestion of minced ventricles with 0.1% trypsin, the cells were collected and plated for 60 min at 37 °C to allow fibroblasts to attach to the 10-cm cell culture plates in 10 ml of Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 1% penicillin/streptomycin and 10% FBS. After rinsed with DMEM twice and discarded, the NRCFs were cultured in DMEM with 10% FBS at 37 °C for 24–48 h until they reached confluence. NRCFs were passaged into 24-well or 60 mm plates for experiment. In all experiment, only the second passage was used. Western blot analysis. Western blot was performed as previously described [2]. After the cell samples were lysed in 150 ll lysis buffer containing protease and phosphatase inhibitors, the protein concentrations were measured by BCA protein assay kit (Pierce Biotechology, IL). Samples (30 lg) were loaded onto 8–10% SDS–polyacrylamide gel and electrophoretically transferred to nitrocellulose membranes (Pall, NY, US). The membranes were probed with various antibodies according to the supplier’s protocol and visualized with peroxidase and an enhancedchemiluminescence’s system (Pierce Biotechnology, IL). Cell proliferation assay. After synchronization by serum deprivation for 24 h, NRCFs were treated with or without AICAR and incubated for 24 h in medium with or without 10% FBS for 24 h. Cell proliferation was assayed by cell count with a hemocytometer and DNA synthesis by [3H]thymidine incorporation as described [2]. Five hours prior to the end of the treatment, 0.5 lCi/ml of [3H]thymidine was added to the medium. The cells then were precipitated with 5% trichloroacetic acid and solubilized in 0.1 M NaOH before measured by scintillation counter. Flow cytometry assessment of cell cycle. NRCFs were incubated with 10% FBS or serum-free medium for 24 h with or without AICAR. Cells were trypsinized and washed twice with cold PBS, then fixed with 70% ethanol at 4 °C overnight. Ethanol-fixed cells were then resuspended with PBS containing 0.1 mg/ml RNase and incubated at 37 °C for 30 min. The pelleted cells were suspended in 0.5 ml of 50 lg/ml propidium iodide and analyzed with flow cytometer (FACScan, Becton-Dickinson). The cell cycle distribution was determined by ModFit LT cell cycle analysis software (Verity Software House, Inc.). Statistical analysis. Data are expressed as mean ± SEM. The statistical significance of the differences was determined by ANOVA or unpaired two-tailed t-tests; a value of P < 0.05 was considered significant.
Results Serum inhibited AMPK via ERK in NRCFs To determine whether AICAR could activate AMPK in NRCFs, we assessed the AMPK phosphorylation at Thr172 since the phosphorylation of this site is essential for AMPK activity. After incubation with AICAR of 1 mM in NRCFs from 10 min to 24 h, the AMPK phos-
403
phorylation at Thr172 dramatically increased over control at all time points treated as shown in Fig. 1A. In accordance with AMPK activation, ACC, a downstream target of AMPK, was also phosphorylated by AICAR in the same time course. To examine whether serum could affect AMPK activation, we challenged NRCFs with 10% FBS. Surprisingly, the results in Fig. 1B showed that serum repressed AMPK phosphorylation. The inhibition started at 10 min, increased at 60 min and 3 h, and came back toward normal level at 12 and 24 h. Serum could also partially inhibit the activated phosphorylation of AMPK (Fig. 1C). Moreover, inhibition of ERK by U0126 could greatly cancel the inhibitory effect by serum at 10 min. These results imply that that AMPK may be an important downstream target of ERK in NRCFs. AMPK activation inhibited both basal and serum-induced ERK phosphorylation To explore the effect of AMPK activation on the phosphorylation of ERK, we pretreated cells with 1 mM AICAR for 2 h and then stimulated with 10% FBS for different time, since FBS is a potent stimulator of ERK phosphorylation. Within 60 min, serum activated ERK in a sustainable manner by elevating phosphorylated ERK, but pretreatment with AICAR did not affect the ERK phosphorylation at 10, 30, and 60 min (Fig. 2A). Interestingly, AICAR began to inhibit the ERK phosphorylation by serum stimulation at 90 min, and the inhibition became obvious at 3 h and remained at 6 and 12 h (Fig. 2A). Furthermore, incubation with AICAR also inhibited the basal phosphorylation level of ERK at 3, 6, and 12 h (Fig. 2B). Therefore, the inhibition of both basal and induced ERK phosphorylation may indicate that AMPK activation will not only inhibit the stimulated proliferation but the basal growth as well in NRCFs. p70S6K is downstream of ERK and activation of AMPK suppressed its phosphorylation Previously, we found both ERK and P70S6K are essential in serum-induced proliferation [2]. To further study the crosstalk between these two pathways, we inhibited ERK and p70S6K using specific inhibitors, respectively. Pretreatment with 10 nM rapamycin, the p70S6K inhibitor, obviously inhibited the phosphorylation of p70S6K by serum at 10, 30, and 60 min, but had no effect on ERK phosphorylation (Fig. 3A). On the other hand, specific ERK inhibitor, U0126 at 10 lM not only blocked the ERK phosphorylation but also inhibited the phosphorylation of p70S6K at 10, 30, and 60 min (Fig. 3B). These data suggest that ERK could effect on p70S6K but not vice versa in NRCFs. As AMPK activation inhibited ERK phosphorylation (Fig. 2A) which could trigger the phosphorylation of
J. Du et al. / Biochemical and Biophysical Research Communications 368 (2008) 402–407
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Fig. 1. Serum inhibited the activation AMPK. (A) NRCFs were exposed to AICAR (1 mM) alone from 10 min to 24 h, (B) incubated with or without AICAR and stimulated with 10% FBS for different time as indicated, (C) treated with AICAR, 10% FBS or both for 3 h, or pretreated with U0126 for 30 min and then stimulated with 10% serum for 10 min. The lysates were immunoblotted with antibodies against phospho-AMPK (Thr172), and eIF5 antibodies which served as an internal control for normalization. Results are representative three blots. The upper panels of (A), (C), and (D) are the normalized densitometer, respectively. *P < 0.05 and **P < 0.01 compared with control; and #P < 0.05 compared with AICAR alone in (C) or serum alone in (D).
p70S6K (Fig. 3B), we asked whether activation of AMPK would inhibit p70S6K phosphorylation. From 3 to 24 h the phosphorylation of p70S6K by serum was gradually attenuated but still higher than basal level (Fig. 3C). Though no inhibition at 3 h was observed, AICAR still vigorously inhibited p70S6K phosphorylation at 6, 12, and 24 h (Fig. 4D). Since the basal phosphorylated levels of p70S6K were very low, we could not measure the effect of AICAR on its phosphorylation without serum stimulation. There were reports that AMPK could inhibit p70S6K through AKT/mTOR pathway [9] but we found no inhibition of AKT and mTOR between 3 and 6 h by AICAR upon serum stimulation (data not shown here). These data demonstrated
that AMPK activation could inhibit ERK and its downstream substrate p70S6K in NRCFs. AMPK activation inhibited cell growth and proliferation in NRCFs To explore the role of AMPK in the cell growth and proliferation in NRCFs, DNA synthesis and cell numbers were determined by [3H]thymidine incorporation and cell counts in basal and mitogen-induced growth upon activation with AICAR. Under unstimulated serum-free conditions, incubation with 1 mM AICAR for 24 h inhibited the basal DNA synthesis, but AICAR at 250 and 500 lM had no effect (Fig. 4A). In response to 10% FBS stimula-
J. Du et al. / Biochemical and Biophysical Research Communications 368 (2008) 402–407
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Fig. 2. AMPK activation inhibited both serum-induced and basal ERK phosphorylation. NRCFs were incubated with or without AICAR (1 mM) and 10% FBS (A) from 10 min to 12 h; (B) or they were incubated with AICAR alone for 3, 6, and 12 h. Cell lysates were immunoblotted with the antiphosphospecific ERK antibody (P-ERK). The membranes were stripped off and reprobed with the anti-ERK antibody (ERK) and the anti-eIF antibody. The data shown here are representatives of three separated experiments.
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Fig. 3. AICAR repressed the phosphorylation of p70S6K. NRCFs were (A) pretreated with or without rapamycin for 30 min, (B) with or without U0126, (C) or with or without AICAR (1 mM) followed by the administration of 10% FBS for different time as indicated. The activation of p70S6K was determined by immunoblotted with antibodies against phospho-p70S6K, p70S6K or anti-eIF5. All of the results were repeated for three times.
tion for 24 h, the [3H]thymidine uptake increased about fourfold over control, and AICAR had a dose-dependent inhibition on serum-induced DNA synthesis (Fig. 4B). AICAR at 500 lM and 1 mM significantly inhibited the
induction of growth by serum (P < 0.05 and P < 0.001). Similarly, AICAR at 1 mM significantly reduced the cell numbers under both serum and serum-free conditions (Fig. 4C). To address whether AICAR would affect the cell
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Fig. 4. AMPK activation inhibited cell proliferation in NRCFs. Serum-deprived NRCFs were incubated with AICAR alone and medium containing 10% FBS with or without AICAR for 24 h. Then cells were (A and B) precipitated for DNA synthesis by [3H]thymidine incorporation or (C) trypsinized for cell counts. (D) NRCFs were maintained in serum-free medium with or without 10% FBS and AICAR or without AICAR (1 mM) for 24 h. Cells were fixed, stained with propidium iodide, and analyzed for DNA content by flow cytometry. Data were obtained from 15,000 events and represented as percentage of cells in. *P < 0.05 and **P < 0.01 compared with control; #P < 0.05 and ##P < 0.01 compared with serum (N = 4).
cycles, we treated the cells in the same conditions like that in the proliferation assay and analyzed cell cycles using the flow cytometer. For cells in S phase, AICAR reduced not only cells with serum stimulation (from 8.6 ± 1.1% to 3.2 ± 0.9%), but cells with serum-free medium as well (from 5.3 ± 0.6% to 2.1 ± 0.5%) (Fig. 4D). These data showed that AMPK activation not only inhibited growth factors induce proliferation, but also suppressed the cell growth in NRCFs. Discussion In this study, we found for the first time that growth factors could inhibit AMPK activation via ERK, in return, activation of AMPK could also inhibit ERK phosphorylation. Furthermore, AMPK activation suppressed the phosphorylation of p70S6K, a downstream target of ERK, and inhibited both basal cell growth and serum-induced proliferation in NRCFs. Therefore, there may be an inhibitory regulatory loop between AMPK and ERK in the growth and proliferation of cardiac fibroblasts. As a well-known pharmacological activator of AMPK, AICAR has been widely used as an effective tool to study the pathophysiological role of AMPK. In NRCFs AICAR
strongly increased the phosphorylation of AMPK on Thr172, which is similar to what we reported in CFs from adult mice. Although serum could also partly lessen the activation of AMPK by AICAR (Fig. 1C), AICAR is strong enough to override the inhibitory effect by serum, indicating that growth factors may regulate AMPK less when it is vigorously activated. In CFs, ERK could be activated by multiple stimuli such as growth factors [2], mechanical forces [4], angiotensin II [10], beta-adrenergic receptor agonist [11] and high glucose [12] mediating proliferation, collagen deposition, and myofibroblast differentiation. In HEK293 cells, AICAR inhibited the ERK activation by insulin-like growth factor 1 but not epidermal grow factor [13]; while in L6 myotubes AICAR elevated ERK phosphorylation [14]. In this study, we found AMPK activation by AICAR inhibited not only serum-induced but also basal ERK phosphorylation. In our previous study we found that ERK and p70S6K were essential in serum-induced proliferation [2]. In adult cardiac muscle cells, ERK inhibitor could inhibit PKC-mediated p70S6K phosphorylation [15]. Also, in human hepatocarcinoma cells, ERK dependent uPAR expression was via phosphorylation of p70S6K [16]. Therefore, we hypothesized that ERK might be the upstream of
J. Du et al. / Biochemical and Biophysical Research Communications 368 (2008) 402–407
p70S6K in CFs and AICAR might also inhibit p70S6K. We found that rapamycin strongly inhibited the phosphorylation of p70S6K, but it did not affect ERK activation. This indicates that p70S6K is a downstream target of ERK in CFs. We also found that AICAR could obviously inhibit p70S6K upon serum stimulation and the time pattern is similar to ERK inhibition. However, there might be also some other factors that regulate p70S6K, since the inhibited p70S6K phosphorylation was reversed at 3 h. In agreement with the inhibition of ERK phosphorylation, AICAR significantly lowered the DNA synthesis, cell numbers, and cells at S phase in both serum-free and 10% serum conditions, demonstrating that AMPK is a negative regulator of the proliferation in NRCFs. Our finding that AICAR is anti-proliferative is consistent with some previous studies in other cellular context [9]. However, in contrast with our study, Hattori et al. [17] reported that AICAR could further enhance the proliferation by angiotensin II in NRCFs. It may be due to different cell conditions. They used 4–6 passages of cardiac fibroblasts which probably had partially been transformed into myofibroblasts. Furthermore, a latest report on cardiomyocytes was also in agreement with our study. They found that AICAR inhibited angiotensin II-induced protein synthesis [8]. In conclusion, we confirmed here that activation of AMPK with AICAR in NRCFs inhibited growth and serum-induced proliferation. There is an inhibitory crosstalk between ERK and AMPK in the growth and proliferation which may offer a mechanism that growth factors utilize in their promotion of proliferation in cardiac fibroblasts.
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
Acknowledgments This work was supported by the National Key Basic Research Program (NKBRP) of People’s Republic of China (2006CB503806) and the Natural Science Foundation of China (30672466, 30570716). References [1] J.C. Chambard, R. Lefloch, J. Pouyssegur, P. Lenormand, ERK implication in cell cycle regulation, Biochim. Biophys. Acta 1773 (2007) 1299–1310. [2] J. Du, W. Liao, Y. Wang, C. Han, Y. Zhang, Inhibitory effect of 14-33 proteins on serum-induced proliferation of cardiac fibroblasts, Eur. J. Cell Biol. 84 (2005) 843–852. [3] S. Murasawa, Y. Mori, Y. Nozawa, N. Gotoh, M. Shibuya, H. Masaki, K. Maruyama, Y. Tsutsumi, Y. Moriguchi, Y. Shibazaki, Y. Tanaka, T. Iwasaka, M. Inada, H. Matsubara, Angiotensin II type 1 receptor-induced extracellular signal-regulated protein kinase activa-
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Biochemical and Biophysical Research Communications 368 (2008) 402–407 www.elsevier.com/locate/ybbrc
Inhibitory crosstalk between ERK and AMPK in the growth and proliferation of cardiac fibroblasts Jianhai Du, Tongju Guan, Hui Zhang, Yi Xia, Fei Liu, Youyi Zhang * Institute of Vascular Medicine, Peking University Third Hospital and Key Laboratory of Molecular Cardiovascular Sciences, Ministry of Education, Beijing 100083, PR China Received 31 December 2007 Available online 1 February 2008
Abstract Extracellular signal-regulated kinase (ERK) is one of the key protein kinases that regulate the growth and proliferation in cardiac fibroblasts (CFs). As an energy sensor of cellular metabolism, AMP-activated protein kinase (AMPK) is found recently to be involved in myocardial remodeling. In this study, we investigated the crosstalk between ERK and AMPK in the growth and proliferation of CFs. In neonatal rat cardiac fibroblasts (NRCFs), we found that serum significantly inhibited basal AMPK phosphorylation between 10 min and 24 h and also partially inhibited AMPK phosphorylation by AMPK activator, 5-aminoimidazole-4-carboxamide-ribonucleoside (AICAR). Furthermore, ERK inhibitor could greatly reverse the inhibition of AMPK by serum. Conversely, activation of AMPK by AICAR also showed a significant inhibition of basal and serum-induced ERK phosphorylation but it showed a delayed and steadfast inhibition which appeared after 60 min and lasted until 12 h. Moreover, inhibition of ERK could repress the activation of p70S6K, an important kinase in cardiac proliferation, and AICAR could also inhibit p70S6K phosphorylation. In addition, under both serum and serum-free medium, AICAR significantly inhibited the DNA synthesis and cell numbers, and reduced cells at S phase. In conclusion, AMPK activation with AICAR inhibited growth and proliferation in cardiac fibroblasts, which involved inhibitory interactions between ERK and AMPK. This is the first report that AMPK could be a target of ERK in growth factors-induced proliferation, which may give a new mechanism that growth factors utilize in their promotion of proliferation in cardiac fibroblasts. Ó 2008 Elsevier Inc. All rights reserved. Keywords: AMPK; ERK; Cardiac fibroblasts
Cardiac fibroblasts (CFs) play a central role in the maintenance of extracellular matrix in the normal heart and the mediation of inflammatory and fibrotic myocardial remodeling in the injured and failing heart. An array of evidence suggests that ERK activation is required for cell proliferation to proceed [1]. In CFs, the application of ERK inhibitors substantially inhibited the proliferation by various stimulators such as growth factors [2], Angiotensin II [3] and mechanical force [4]. AMP-activated protein kinase (AMPK) is a serine/threonine protein kinase that has emerged as a key regulator of cellular energy homeostasis and coordinates multiple cata*
Corresponding author. Fax: +86 10 82802306. E-mail address: [email protected] (Y. Zhang).
0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.01.099
bolic and anabolic pathways in the heart [5]. In neonatal rat cardiomyocytes, AMPK activation could inhibit the activation of p70S6K and the cardiac hypertrophy induced by phenylephrine or overexpressed AKT [6]. Through activating AMPK, adiponectin inhibited pressure-overload or angiotensin II-induced cardiac hypertrophy in mice [7]. Injection of AMPK activator, 5-aminoimidazole-4-carboxamide-ribonucleoside (AICAR) attenuated pressure-overload-induced cardiac hypertrophy in rats [8]. We hypothesized that in the growth and proliferation of CFs, it required not only the enhancement of positive signals like ERK, but also the lessening of some negative signals like AMPK. Therefore, we explored, in this study: (1) whether activation of ERK by growth factors repressed AMPK activation; (2) the effect of AMPK activation on ERK
J. Du et al. / Biochemical and Biophysical Research Communications 368 (2008) 402–407
phosphorylation; and (3) the effect of AMPK activation on proliferation in NRCFs by growth factors. Materials and methods Reagents. AICAR was obtained from Toronto Research Chemicals (Toronto, Canada). Fetal bovine serum (FBS) was from Hyclone (Logan, UT). Antibodies against phospho-AMPK alpha (Thr172), phospho-ERK (phospho Thr202/Tyr204), phospho-p70S6K (Thr389), and p70S6K were from Cell Signaling Technology (Beverly, MA). Antibodies against ERK, and eukaryotic translation initiation factor (eIF5) were from Santa Cruz Biotechnology (Santa Cruz, CA). [3H]Thymidine was from Amersham bioscience (Bucks, UK). U0126 and rapamycin were from Sigma (St. Louis, MO). Fetal bovine serum (FBS) was from Hyclone (Logan, UT). Cell culture. This study was conducted in accordance with the Guide for the Care and Use of Laboratory Animals (National Academic Press, Washington, DC, 1996). Cultured neonatal rat cardiac fibroblasts were isolated from 1 day-old Sprague–Dawley rats as we previously described [2]. After digestion of minced ventricles with 0.1% trypsin, the cells were collected and plated for 60 min at 37 °C to allow fibroblasts to attach to the 10-cm cell culture plates in 10 ml of Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 1% penicillin/streptomycin and 10% FBS. After rinsed with DMEM twice and discarded, the NRCFs were cultured in DMEM with 10% FBS at 37 °C for 24–48 h until they reached confluence. NRCFs were passaged into 24-well or 60 mm plates for experiment. In all experiment, only the second passage was used. Western blot analysis. Western blot was performed as previously described [2]. After the cell samples were lysed in 150 ll lysis buffer containing protease and phosphatase inhibitors, the protein concentrations were measured by BCA protein assay kit (Pierce Biotechology, IL). Samples (30 lg) were loaded onto 8–10% SDS–polyacrylamide gel and electrophoretically transferred to nitrocellulose membranes (Pall, NY, US). The membranes were probed with various antibodies according to the supplier’s protocol and visualized with peroxidase and an enhancedchemiluminescence’s system (Pierce Biotechnology, IL). Cell proliferation assay. After synchronization by serum deprivation for 24 h, NRCFs were treated with or without AICAR and incubated for 24 h in medium with or without 10% FBS for 24 h. Cell proliferation was assayed by cell count with a hemocytometer and DNA synthesis by [3H]thymidine incorporation as described [2]. Five hours prior to the end of the treatment, 0.5 lCi/ml of [3H]thymidine was added to the medium. The cells then were precipitated with 5% trichloroacetic acid and solubilized in 0.1 M NaOH before measured by scintillation counter. Flow cytometry assessment of cell cycle. NRCFs were incubated with 10% FBS or serum-free medium for 24 h with or without AICAR. Cells were trypsinized and washed twice with cold PBS, then fixed with 70% ethanol at 4 °C overnight. Ethanol-fixed cells were then resuspended with PBS containing 0.1 mg/ml RNase and incubated at 37 °C for 30 min. The pelleted cells were suspended in 0.5 ml of 50 lg/ml propidium iodide and analyzed with flow cytometer (FACScan, Becton-Dickinson). The cell cycle distribution was determined by ModFit LT cell cycle analysis software (Verity Software House, Inc.). Statistical analysis. Data are expressed as mean ± SEM. The statistical significance of the differences was determined by ANOVA or unpaired two-tailed t-tests; a value of P < 0.05 was considered significant.
Results Serum inhibited AMPK via ERK in NRCFs To determine whether AICAR could activate AMPK in NRCFs, we assessed the AMPK phosphorylation at Thr172 since the phosphorylation of this site is essential for AMPK activity. After incubation with AICAR of 1 mM in NRCFs from 10 min to 24 h, the AMPK phos-
403
phorylation at Thr172 dramatically increased over control at all time points treated as shown in Fig. 1A. In accordance with AMPK activation, ACC, a downstream target of AMPK, was also phosphorylated by AICAR in the same time course. To examine whether serum could affect AMPK activation, we challenged NRCFs with 10% FBS. Surprisingly, the results in Fig. 1B showed that serum repressed AMPK phosphorylation. The inhibition started at 10 min, increased at 60 min and 3 h, and came back toward normal level at 12 and 24 h. Serum could also partially inhibit the activated phosphorylation of AMPK (Fig. 1C). Moreover, inhibition of ERK by U0126 could greatly cancel the inhibitory effect by serum at 10 min. These results imply that that AMPK may be an important downstream target of ERK in NRCFs. AMPK activation inhibited both basal and serum-induced ERK phosphorylation To explore the effect of AMPK activation on the phosphorylation of ERK, we pretreated cells with 1 mM AICAR for 2 h and then stimulated with 10% FBS for different time, since FBS is a potent stimulator of ERK phosphorylation. Within 60 min, serum activated ERK in a sustainable manner by elevating phosphorylated ERK, but pretreatment with AICAR did not affect the ERK phosphorylation at 10, 30, and 60 min (Fig. 2A). Interestingly, AICAR began to inhibit the ERK phosphorylation by serum stimulation at 90 min, and the inhibition became obvious at 3 h and remained at 6 and 12 h (Fig. 2A). Furthermore, incubation with AICAR also inhibited the basal phosphorylation level of ERK at 3, 6, and 12 h (Fig. 2B). Therefore, the inhibition of both basal and induced ERK phosphorylation may indicate that AMPK activation will not only inhibit the stimulated proliferation but the basal growth as well in NRCFs. p70S6K is downstream of ERK and activation of AMPK suppressed its phosphorylation Previously, we found both ERK and P70S6K are essential in serum-induced proliferation [2]. To further study the crosstalk between these two pathways, we inhibited ERK and p70S6K using specific inhibitors, respectively. Pretreatment with 10 nM rapamycin, the p70S6K inhibitor, obviously inhibited the phosphorylation of p70S6K by serum at 10, 30, and 60 min, but had no effect on ERK phosphorylation (Fig. 3A). On the other hand, specific ERK inhibitor, U0126 at 10 lM not only blocked the ERK phosphorylation but also inhibited the phosphorylation of p70S6K at 10, 30, and 60 min (Fig. 3B). These data suggest that ERK could effect on p70S6K but not vice versa in NRCFs. As AMPK activation inhibited ERK phosphorylation (Fig. 2A) which could trigger the phosphorylation of
J. Du et al. / Biochemical and Biophysical Research Communications 368 (2008) 402–407
AMPK phosphorylation (fold increase)
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Fig. 1. Serum inhibited the activation AMPK. (A) NRCFs were exposed to AICAR (1 mM) alone from 10 min to 24 h, (B) incubated with or without AICAR and stimulated with 10% FBS for different time as indicated, (C) treated with AICAR, 10% FBS or both for 3 h, or pretreated with U0126 for 30 min and then stimulated with 10% serum for 10 min. The lysates were immunoblotted with antibodies against phospho-AMPK (Thr172), and eIF5 antibodies which served as an internal control for normalization. Results are representative three blots. The upper panels of (A), (C), and (D) are the normalized densitometer, respectively. *P < 0.05 and **P < 0.01 compared with control; and #P < 0.05 compared with AICAR alone in (C) or serum alone in (D).
p70S6K (Fig. 3B), we asked whether activation of AMPK would inhibit p70S6K phosphorylation. From 3 to 24 h the phosphorylation of p70S6K by serum was gradually attenuated but still higher than basal level (Fig. 3C). Though no inhibition at 3 h was observed, AICAR still vigorously inhibited p70S6K phosphorylation at 6, 12, and 24 h (Fig. 4D). Since the basal phosphorylated levels of p70S6K were very low, we could not measure the effect of AICAR on its phosphorylation without serum stimulation. There were reports that AMPK could inhibit p70S6K through AKT/mTOR pathway [9] but we found no inhibition of AKT and mTOR between 3 and 6 h by AICAR upon serum stimulation (data not shown here). These data demonstrated
that AMPK activation could inhibit ERK and its downstream substrate p70S6K in NRCFs. AMPK activation inhibited cell growth and proliferation in NRCFs To explore the role of AMPK in the cell growth and proliferation in NRCFs, DNA synthesis and cell numbers were determined by [3H]thymidine incorporation and cell counts in basal and mitogen-induced growth upon activation with AICAR. Under unstimulated serum-free conditions, incubation with 1 mM AICAR for 24 h inhibited the basal DNA synthesis, but AICAR at 250 and 500 lM had no effect (Fig. 4A). In response to 10% FBS stimula-
J. Du et al. / Biochemical and Biophysical Research Communications 368 (2008) 402–407
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Fig. 2. AMPK activation inhibited both serum-induced and basal ERK phosphorylation. NRCFs were incubated with or without AICAR (1 mM) and 10% FBS (A) from 10 min to 12 h; (B) or they were incubated with AICAR alone for 3, 6, and 12 h. Cell lysates were immunoblotted with the antiphosphospecific ERK antibody (P-ERK). The membranes were stripped off and reprobed with the anti-ERK antibody (ERK) and the anti-eIF antibody. The data shown here are representatives of three separated experiments.
P-p85S6K P-p70S6K
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Fig. 3. AICAR repressed the phosphorylation of p70S6K. NRCFs were (A) pretreated with or without rapamycin for 30 min, (B) with or without U0126, (C) or with or without AICAR (1 mM) followed by the administration of 10% FBS for different time as indicated. The activation of p70S6K was determined by immunoblotted with antibodies against phospho-p70S6K, p70S6K or anti-eIF5. All of the results were repeated for three times.
tion for 24 h, the [3H]thymidine uptake increased about fourfold over control, and AICAR had a dose-dependent inhibition on serum-induced DNA synthesis (Fig. 4B). AICAR at 500 lM and 1 mM significantly inhibited the
induction of growth by serum (P < 0.05 and P < 0.001). Similarly, AICAR at 1 mM significantly reduced the cell numbers under both serum and serum-free conditions (Fig. 4C). To address whether AICAR would affect the cell
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Fig. 4. AMPK activation inhibited cell proliferation in NRCFs. Serum-deprived NRCFs were incubated with AICAR alone and medium containing 10% FBS with or without AICAR for 24 h. Then cells were (A and B) precipitated for DNA synthesis by [3H]thymidine incorporation or (C) trypsinized for cell counts. (D) NRCFs were maintained in serum-free medium with or without 10% FBS and AICAR or without AICAR (1 mM) for 24 h. Cells were fixed, stained with propidium iodide, and analyzed for DNA content by flow cytometry. Data were obtained from 15,000 events and represented as percentage of cells in. *P < 0.05 and **P < 0.01 compared with control; #P < 0.05 and ##P < 0.01 compared with serum (N = 4).
cycles, we treated the cells in the same conditions like that in the proliferation assay and analyzed cell cycles using the flow cytometer. For cells in S phase, AICAR reduced not only cells with serum stimulation (from 8.6 ± 1.1% to 3.2 ± 0.9%), but cells with serum-free medium as well (from 5.3 ± 0.6% to 2.1 ± 0.5%) (Fig. 4D). These data showed that AMPK activation not only inhibited growth factors induce proliferation, but also suppressed the cell growth in NRCFs. Discussion In this study, we found for the first time that growth factors could inhibit AMPK activation via ERK, in return, activation of AMPK could also inhibit ERK phosphorylation. Furthermore, AMPK activation suppressed the phosphorylation of p70S6K, a downstream target of ERK, and inhibited both basal cell growth and serum-induced proliferation in NRCFs. Therefore, there may be an inhibitory regulatory loop between AMPK and ERK in the growth and proliferation of cardiac fibroblasts. As a well-known pharmacological activator of AMPK, AICAR has been widely used as an effective tool to study the pathophysiological role of AMPK. In NRCFs AICAR
strongly increased the phosphorylation of AMPK on Thr172, which is similar to what we reported in CFs from adult mice. Although serum could also partly lessen the activation of AMPK by AICAR (Fig. 1C), AICAR is strong enough to override the inhibitory effect by serum, indicating that growth factors may regulate AMPK less when it is vigorously activated. In CFs, ERK could be activated by multiple stimuli such as growth factors [2], mechanical forces [4], angiotensin II [10], beta-adrenergic receptor agonist [11] and high glucose [12] mediating proliferation, collagen deposition, and myofibroblast differentiation. In HEK293 cells, AICAR inhibited the ERK activation by insulin-like growth factor 1 but not epidermal grow factor [13]; while in L6 myotubes AICAR elevated ERK phosphorylation [14]. In this study, we found AMPK activation by AICAR inhibited not only serum-induced but also basal ERK phosphorylation. In our previous study we found that ERK and p70S6K were essential in serum-induced proliferation [2]. In adult cardiac muscle cells, ERK inhibitor could inhibit PKC-mediated p70S6K phosphorylation [15]. Also, in human hepatocarcinoma cells, ERK dependent uPAR expression was via phosphorylation of p70S6K [16]. Therefore, we hypothesized that ERK might be the upstream of
J. Du et al. / Biochemical and Biophysical Research Communications 368 (2008) 402–407
p70S6K in CFs and AICAR might also inhibit p70S6K. We found that rapamycin strongly inhibited the phosphorylation of p70S6K, but it did not affect ERK activation. This indicates that p70S6K is a downstream target of ERK in CFs. We also found that AICAR could obviously inhibit p70S6K upon serum stimulation and the time pattern is similar to ERK inhibition. However, there might be also some other factors that regulate p70S6K, since the inhibited p70S6K phosphorylation was reversed at 3 h. In agreement with the inhibition of ERK phosphorylation, AICAR significantly lowered the DNA synthesis, cell numbers, and cells at S phase in both serum-free and 10% serum conditions, demonstrating that AMPK is a negative regulator of the proliferation in NRCFs. Our finding that AICAR is anti-proliferative is consistent with some previous studies in other cellular context [9]. However, in contrast with our study, Hattori et al. [17] reported that AICAR could further enhance the proliferation by angiotensin II in NRCFs. It may be due to different cell conditions. They used 4–6 passages of cardiac fibroblasts which probably had partially been transformed into myofibroblasts. Furthermore, a latest report on cardiomyocytes was also in agreement with our study. They found that AICAR inhibited angiotensin II-induced protein synthesis [8]. In conclusion, we confirmed here that activation of AMPK with AICAR in NRCFs inhibited growth and serum-induced proliferation. There is an inhibitory crosstalk between ERK and AMPK in the growth and proliferation which may offer a mechanism that growth factors utilize in their promotion of proliferation in cardiac fibroblasts.
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
Acknowledgments This work was supported by the National Key Basic Research Program (NKBRP) of People’s Republic of China (2006CB503806) and the Natural Science Foundation of China (30672466, 30570716). References [1] J.C. Chambard, R. Lefloch, J. Pouyssegur, P. Lenormand, ERK implication in cell cycle regulation, Biochim. Biophys. Acta 1773 (2007) 1299–1310. [2] J. Du, W. Liao, Y. Wang, C. Han, Y. Zhang, Inhibitory effect of 14-33 proteins on serum-induced proliferation of cardiac fibroblasts, Eur. J. Cell Biol. 84 (2005) 843–852. [3] S. Murasawa, Y. Mori, Y. Nozawa, N. Gotoh, M. Shibuya, H. Masaki, K. Maruyama, Y. Tsutsumi, Y. Moriguchi, Y. Shibazaki, Y. Tanaka, T. Iwasaka, M. Inada, H. Matsubara, Angiotensin II type 1 receptor-induced extracellular signal-regulated protein kinase activa-
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