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5-Hydroxytryptamine augments migration of human aortic smooth muscle cells through activation of RhoA and ERK
BBRC Biochemical and Biophysical Research Communications 337 (2005) 916–921 www.elsevier.com/locate/ybbrc
5-Hydroxytryptamine augments migration of human aortic smooth muscle cells through activation of RhoA and ERK Satoshi Matsusaka, Ichiro Wakabayashi * Department of Hygiene and Preventive Medicine, Yamagata University School of Medicine, Yamagata 990-9585, Japan Received 20 September 2005 Available online 30 September 2005
Abstract The purpose of this study was to elucidate the mechanism of 5-hydroxytryptamine (5-HT, serotonin) action on migration of vascular smooth muscle cells. Migration of cultured human aortic smooth muscle cells (HASMCs), evaluated using time-lapse microscopy, was significantly enhanced by 5-HT at concentrations of 1–100 nM. The enhancing effect of 5-HT on cell migration was markedly inhibited in the presence of ketanserin, a 5-HT2 receptor antagonist, but not by GR 55562, a 5-HT1 receptor antagonist. Activities of RhoA and ERK were increased by 5-HT, and the increase in cell migration by 5-HT was abolished in the presence of U0126, a MEK1/2 inhibitor, or Y-27632, a Rho-kinase inhibitor. Activation of ERK was strongly inhibited by Y-27632. 5-HT-induced formation of stress fiber and detachment of uropod (trailing edge) were abolished by Y-27632. Thus, 5-HT has a potent enhancing action on migration of HASMCs due to an increase in stress fiber formation by 5-HT2 receptor stimulation followed by activation of the Rho-kinase and ERK pathways. Ó 2005 Elsevier Inc. All rights reserved. Keywords: Atherosclerosis; Cytoskeleton; 5-Hydroxytryptamine; MAP kinase; Serotonin; Signal transduction
Atherosclerosis—a progressive disease characterized by accumulation of lipids and fibrous elements in large arteries—involves several highly interrelated processes. Multiple steps, including platelet activation and migration of vascular smooth muscle cells (VSMCs), are involved in the pathogenesis of atherosclerosis [1]. Migration of VSMCs play a crucial role in the formation and progression of atherosclerotic lesions. Increased VSMC migration activity has been reported to be the main cause of intimal hyperplasia [2]. HDL has been reported to inhibit platelet-derived growth factor (PDGF)-induced migration of VSMCs, and this may be a novel anti-atherogenic mechanism of HDL [3]. Thus, migration of VSMCs into the tunica intima and their accumulation within the intima have been recognized as key events in atherosclerosis. Platelet activation is an important step of atherosclerosis. 5-Hydroxytryptamine (5-HT, serotonin) and throm*
Corresponding author. Fax: +81 23 628 5255. E-mail address: [email protected] (I. Wakabayashi). 0006-291X/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.09.129
boxane A2, potent vasoconstrictors released from platelets, play crucial roles in hemostasis under physiological conditions as well as pathological conditions such as ischemia due to thrombosis. 5-HT has been reported to induce proliferation of canine aortic and porcine coronary smooth muscle cells via 5-HT2 receptors [4,5]. In addition, 5-HT2 receptor antagonists have been reported to retard progression of atherosclerosis [6]. Thus, 5-HT is also involved in the formation of atherosclerotic lesions. Although migration of VSMCs is a key event in atherosclerosis, there have been, to the best of our knowledge, only two primitive studies on 5-HT action on migration of vascular cells: 5-HT enhanced migration of bovine and rat aortic smooth muscle cells [7,8]. Moreover, the mechanism of 5-HT-induced migration of VSMCs still remains to be clarified. The present study was therefore undertaken to determine the effects of 5-HT on migration of human aortic smooth muscle cells (HASMCs) and to elucidate the mechanism involving in 5-HT action on migration. We found that 5-HT enhances migration of HASMCs via 5-HT2 receptor stimulation through
S. Matsusaka, I. Wakabayashi / Biochemical and Biophysical Research Communications 337 (2005) 916–921
activation of the Rho-kinase and extracellular signal-regulated kinase (ERK) pathways. Materials and methods Cell culture. Human aortic smooth muscle cells (HASMCs) (KURABO, Osaka, Japan) were cultured in HuMedia-SG2 medium (KURABO) in CO2 (5%)-containing humidified air at 37 °C. All HASMC cultures used in this study were between the 5th and 9th passages. Cell migration assay. The cells were incubated in RPMI1640 medium (Sigma, St. Louis, MO, USA) containing 5% fetal bovine serum (FBS) under CO2 (5%)-containing humidified air at 37 °C for 24 h before each experiment in a 35-mm plastic plate. Cell movement on the plate was recorded using a time-lapse microscope for 120 min after stimulation with 5-HT and analyzed by an image analyzing software, MetaVue (Universal Imaging, PA, USA). The results shown are representative results for the largest field of the cells, and experiments were repeated at least three times for each assay condition. The mean migration velocity was compared under different conditions. Western blotting. The adhering cells, incubated in RPMI1640 medium containing FBS (5%) under CO2 (5%)-containing humidified air at 37 °C for 24 h before each experiment, were suspended in 0.1 ml of a lysis buffer [1% Triton X-100, 50 mM Tris–HCl (pH 7.2), 100 mM NaCl, 5 mM
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EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1 mM Na3VO4]. Cell lysates were separated by SDS–PAGE and transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA). The membrane was blocked with 5% skim milk in phosphate-buffered saline (PBS) containing 0.1% Tween 20 (T–PBS) for 30 min at room temperature and then incubated for 60 min with anti-phospho-p44/p42 MAPK polyclonal antibody or anti-p44/p42 MAPK polyclonal antibody (Cell Signaling Technology, MA, USA). The membrane was then washed three times with T–PBS and incubated with horseradish peroxidase-conjugated goat anti-rabbit antibodies for 30 min, and specific proteins were detected using an enhanced chemiluminescence immunoblotting system. GST pull-down assay for activated RhoA. GST pull-down assay was performed as previously described [9]. Briefly, the adhering cells, incubated in RPMI1640 medium containing FBS (5%) under CO2 (5%)-containing humidified air at 37 °C for 24 h before each experiment in the presence or absence of 5-HT (100 nM), were lysed in a buffer [50 mM Tris– HCl (pH 7.2), 1% Triton X-100, 100 mM NaCl, 10 mM MgCl2, and 1 mM PMSF]. After centrifugation at 15,000 rpm for 15 min at 4 °C, cell lysates were incubated with GST-rhotekin Rho-binding domain (RBD) immobilized on glutathione agarose (Upstate, NY, USA) for 60 min at 4 °C. The beads were incubated with the cell lysates, and the proteins on the beads after washing were run on SDS–PAGE. GTP-bound RhoA was detected by immunoblotting with anti-RhoA polyclonal antibody (Santa Cruz Biotechnology, CA, USA) and anti-GST-Tag antibody.
Fig. 1. (A) Time-lapse images of migration of HASMCs. The migration of vehicle (top)-treated or 5-hydroxytryptamine (5-HT) (100 nM)-stimulated HASMCs and 5-HT (100 nM)-stimulated HASMCs in the presence of ketanserin (100 nM) or GR 55562 (100 nM) was recorded at the indicated times. Bars indicate a length of 50 lm. (B) Time course of HASMC migration velocity. HASMCs were incubated with 5-HT (100 nM) or a vehicle for 120 min. The mean 5-min interval velocity with SE is shown. Asterisks denote significant difference (*p < 0.01; **p < 0.05) from the control without 5-HT stimulation. N = 17. (C) Migration velocity of HASMCs for 30 min stimulated with 5-HT (1–100 nM) alone or 5-HT (100 nM) in the presence of ketanserin (100 nM) or GR 55562 (100 nM). The mean velocity with SE is shown. Asterisks denote significant difference (p < 0.01) from the control without 5-HT stimulation. N = 20.
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Immunostaining. After incubation on the culture slides (Becton–Dickinson, CA, USA) at 37 °C overnight, HASMCs were stimulated in the presence or absence of 5-HT (100 nM) for 30 min. Then the cells were fixed with 3.3% paraformaldehyde for 15 min at room temperature, treated with 0.2% Triton X-100 for 2 min, and blocked with 5% bovine serum albumin for 30 min. The cells were stained with Alexa Fluor 488conjugated phalloidin (Molecular Probes, OR, USA) for 30 min. After washing, the stained cells were mounted with antifade-containing glycerol/ PBS, and cell movement was observed under a confocal laser-scanning microscope (LSM510, Zeiss, Oberkochen, Germany). Drugs. 5-HT (Sigma, St. Louis, MO, USA), Y-27632 (Calbiochem, La Jolla, CA, USA), and GR 55562 (Tocris Coolson, UK) were dissolved in distilled water to make stock solutions of 10 mM, 1 mM, and 10 lM, respectively. U0126 (Cell Signaling Technology, Beverly, MA, USA) and ketanserin (Sigma, St. Louis, MO, USA) were dissolved in dimethyl sulfoxide (DMSO) to make stock solutions of 100 mM and 1 mM, respectively. Stock solutions of 5-HT, ketanserin, and GR 55562 were kept at 4 °C and those of Y-27632 and U0126 were kept at 20 °C. Statistical analysis. Statistical analysis was done using analysis of variance followed by Scheffe´Õs F test. P values less than 0.05 were regarded as significant.
Results Enhancement of cell migration by 5-HT Migration velocity of HASMCs was significantly increased by 5-HT (1–100 nM) in a concentration-dependent manner (Figs. 1A–C). Fig. 1B displays time course of mean 5-min interval migration velocity. Cell migration was increased only at an early time (within 30 min) after 5-HT stimulation, and cell migration velocity from 30 to 120 min after 5-HT stimulation was not significantly different from that in the control cells without 5-HT stimulation. The action of 5-HT (100 nM) on cell migration was markedly attenuated in the presence of ketanserin (100 nM) but was not affected in the presence of GR 55562 (100 nM) (Figs. 1A and C). Involvement of ERK MAPK activation in enhancing action of 5-HT on cell migration Slight expression of phosphorylated ERK was detected in the basal condition without 5-HT stimulation (Fig. 2A). 5-HT stimulation increased ERK activity timedependently until 5 min after the onset of stimulation. Then ERK activity decreased to the basal level over the next 10 min (Fig. 2A). The increase in cell migration induced by stimulation with 5-HT (100 nM) was abolished in the presence of U0126, a MEK1/2 inhibitor (Fig. 2B).
Fig. 2. (A) 5-Hydroxytryptamine (5-HT)-induced ERK MAPK activation in HASMCs. HASMCs were treated with 5-HT (100 nM) for the indicated times (min). Expression of phospho-ERK (top) and total ERK (bottom) is shown. (B) Effect of ERK MAPK inhibition on migration of HASMCs. Migration velocity of HASMCs stimulated with 5-HT (100 nM) for 30 min was compared in the presence and absence of U0126 (10 lM). The mean velocity with SE is shown. An asterisk denotes significant difference (p < 0.01) from the control without 5-HT stimulation. N = 20.
tion (Fig. 3B). The increase in 5-HT-induced ERK phosphorylation was also abolished in the presence of Y27632 (Fig. 3C). 5-HT-induced rearrangement of actin cytoskeleton Thick actin, which is associated with rapid cell migration, was observed in cells stimulated with 5-HT (Fig. 4A). Inhibition of ERK activation (Fig. 4B) or Rho kinase (Fig. 4C) blocked stress fiber formation. Imaging of cell detachment In response to 5-HT stimulation, the uropod, defined as the trailing edge, was detached in most of the cells (Fig. 5A), resulting in a higher migration speed. However, in the presence of Y-27632, 5-HT-induced uropod detachment was abolished (Fig. 5B). On the other hand, U0126 treatment did not affect uropod detachment following 5HT stimulation (data not shown). Discussion
Involvement of RhoA activation in enhancing action of 5-HT on cell migration Similar to ERK activity, slight activation of RhoA was also detected in the absence of 5-HT (Fig. 3A). RhoA activity was increased at 2.5 min after the onset of 5-HT stimulation and then decreased to the basal level (Fig. 3A). Y-27632, a Rho kinase (ROCK) inhibitor, abolished the enhancing action of 5-HT (100 nM) on cell migra-
The present study clearly demonstrated that 5-HT enhances migration of HASMCs. 5-HT, released from activated platelets, plays a major role in ischemia in thrombotic diseases through a potent vasocontractile action. In addition to this action, 5-HT also has an enhancing action on migration of arterial smooth muscle cells, and the action of 5-HT on migration may accelerate the formation of an atherosclerotic lesion. In fact, 5-HT antagonists have been
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Fig. 5. Time-lapse images of migration. The migration of 5-hydroxytryptamine (5-HT) (100 nM)-stimulated HASMCs in the absence (A) and presence (B) of Y-27632 (10 lM) was recorded at the indicated times. The arrows indicate uropods. Bars indicate a length of 50 lm.
Fig. 3. (A) 5-Hydroxytryptamine (5-HT)-induced RhoA activation in HASMCs. HASMCs were treated with 5-HT (100 nM) for the indicated times (min). GTP-bound RhoA was detected by pull-down assays using GST-rhotekin Rho-binding domain (top). (B) Effect of RhoA inhibition on the migration of HASMCs. Migration velocity of HASMCs stimulated with 5-HT (100 nM) for 30 min was compared in the presence and absence of Y-27632 (10 lM). The mean velocity with SE is shown. An asterisk denotes significant difference (p < 0.01) from the control without 5-HT stimulation. N = 20. (C) Effects of Y-27632, a Rho-kinase inhibitor, on 5hydroxytryptamine (5-HT)-induced ERK activation in human aortic smooth muscle cells (HASMCs). After pretreatment with Y-27632 (10 lM) for 30 min or a vehicle, the cells were stimulated with 5-HT (100 nM) for the indicated times (min). Expression of phospho-ERK (top) and total ERK (bottom) is shown.
Fig. 4. Actin cytoskeletal structure of HASMCs stimulated with 5hydroxytryptamine (5-HT, 100 nM) in the presence or absence of U0126 (10 lM) or Y-27632 (10 lM). The cells were grown on coverslips, washed, fixed, and processed for visualization of F-actin with Alexa 488conjugated phalloidin. HASMCs stimulated with 5-HT displayed an array of thick bundled actin stress fibers (A). In contrast, the actin filaments in HASMCs in the presence of U0126 (B) or Y-27632 (C) were notably less bundled and had an indistinct and less organized appearance.
reported to suppress atherosclerotic progress [6]. 5-HT-induced enhancement of HASMC migration was mediated through activation of 5-HT2 receptors, which are known to be involved in arterial smooth muscle proliferation, as well as contraction [10]. The present results also agree with the results of a study demonstrating that 5-HT enhanced growth factor-stimulated migration of cultured rat aortic smooth muscle cells (RASMCs) [8]. The mechanism of 5-HT-induced migration of vascular smooth muscle cells remained unknown. On the other hand, 5-HT-induced proliferation of bovine pulmonary arterial smooth muscle cells has recently been reported to be dependent on activation of ERK [11]. The present study demonstrated that ERK activity was greatly increased by 5-HT, and the enhancement of cell migration by 5-HT was abolished by U0126, an ERK inhibitor. Thus, ERK activation plays an important role in regulation of the cellular signaling required for migration as well as proliferation of VSMCs. Rho-kinase has also recently been reported to be involved in 5-HT-induced proliferation of VSMCs [11]. Rho-kinase has been demonstrated to be involved in contraction of human internal thoracic arteries [12]. It has been demonstrated in experiments using Y-27632 that activation of the Rho/ROCK pathway is involved in migration of vascular smooth muscle cells in response to urokinase, thrombin, and PDGF [13–16]. The present study clearly demonstrated that 5-HT increased RhoA activity and that Y-27632 abolished 5-HT-induced enhancement of cell migration, indicating that the Rho-kinase pathway also plays an important role in 5-HT-induced migration of HASMCs. 5-HT-induced enhancement of HASMC migration at an early time after stimulation indicates its association with activation of early signaling molecules such as RhoA and ERK. In the former studies, migration of VSMCs was analyzed by a conventional method using Boyden chambers. However, the detailed features of individual VSMC migration have not been known. In the present study, 5-HT-induced VSMC migration was analyzed by using time-lapse microscopy, a recently developed sensitive method for
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evaluation of single cell migration. Cell migration depends on reorganization of the actin cytoskeleton and development of cell polarity, enabling cells to make new adhesive contacts at their leading edges and to break contacts existing at their trailing edges (uropods). Activated ERK has been reported to phosphorylate myosin light-chain kinase and thereby to increase its kinase activity, resulting in enhancement of migration in COS cells [17]. A major function of Rho family GTPases in VSMCs is to regulate the organization of the actin cytoskeleton, and the Rho/ ROCK pathway is required for the formation of stress fibers and focal adhesion. The absence of dense actin bundles and failure in reorganization into actin stress fibers are reflected by inhibition of migration. The present study demonstrated the presence of thick actin filament following 5-HT stimulation that increases the velocity of HASMC migration. Furthermore, inhibition of ERK activation by U0126 or inhibition of Rho-kinase by Y-27632 blocked 5-HT-induced stress-fiber formation, resulting in a decrease in migration velocity of HASMCs. Recent studies have shown that Rho-kinase is an upper-stream signal for ERK activation in fibroblasts, NIH 3T3 cells, rat kidney cells, and RASMCs [18–22]. This agrees with the present finding that 5-HT-induced ERK activation in HASMCs was abolished by Y-27632. Activation of the Rho/ROCK pathway has been suggested to be essential for regulation of the process of uropod detachment and rear release in migrating leukocytes [23]. Furthermore, activation of Rho-kinase has been reported to be associated with the formation of uropod in BAF (pro B cells) [24]. Therefore, regulatory mechanism of uropod detachment differs by the kind of cell. However, the intracellular signaling mechanism involved in cytoskeletal changes related to migration of VSMCs remained to be clarified. In the present study, 5-HT stimulation of the cells induced rear release, and detachment of the uropod in HASMCs was abolished by Y-27632 but not affected by U0126. These results suggest that Rho-kinase, but not ERK, is involved in detachment of the uropod, while activation of both Rho-kinase and ERK pathways regulates 5-HT-induced formation of stress fibers. The present study is the first study that demonstrated the mechanism of 5-HT-induced migration of VASMCs. In conclusion, 5-HT has a potent enhancing action on migration of HASMCs due to an increase in stress fiber formation through stimulation of 5-HT2 receptors followed by activating the ERK and Rho-kinase pathways. Our study suggests that inhibitors targeting ERK and Rho/ ROCK pathways as well as anti-5-HT2 drugs may provide new therapeutic approaches for the treatment of atherosclerotic vascular diseases. Acknowledgment This work was supported by a grant for scientific research from the Grant of the Yamagata University 21st Century COE program.
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5-Hydroxytryptamine augments migration of human aortic smooth muscle cells through activation of RhoA and ERK Satoshi Matsusaka, Ichiro Wakabayashi * Department of Hygiene and Preventive Medicine, Yamagata University School of Medicine, Yamagata 990-9585, Japan Received 20 September 2005 Available online 30 September 2005
Abstract The purpose of this study was to elucidate the mechanism of 5-hydroxytryptamine (5-HT, serotonin) action on migration of vascular smooth muscle cells. Migration of cultured human aortic smooth muscle cells (HASMCs), evaluated using time-lapse microscopy, was significantly enhanced by 5-HT at concentrations of 1–100 nM. The enhancing effect of 5-HT on cell migration was markedly inhibited in the presence of ketanserin, a 5-HT2 receptor antagonist, but not by GR 55562, a 5-HT1 receptor antagonist. Activities of RhoA and ERK were increased by 5-HT, and the increase in cell migration by 5-HT was abolished in the presence of U0126, a MEK1/2 inhibitor, or Y-27632, a Rho-kinase inhibitor. Activation of ERK was strongly inhibited by Y-27632. 5-HT-induced formation of stress fiber and detachment of uropod (trailing edge) were abolished by Y-27632. Thus, 5-HT has a potent enhancing action on migration of HASMCs due to an increase in stress fiber formation by 5-HT2 receptor stimulation followed by activation of the Rho-kinase and ERK pathways. Ó 2005 Elsevier Inc. All rights reserved. Keywords: Atherosclerosis; Cytoskeleton; 5-Hydroxytryptamine; MAP kinase; Serotonin; Signal transduction
Atherosclerosis—a progressive disease characterized by accumulation of lipids and fibrous elements in large arteries—involves several highly interrelated processes. Multiple steps, including platelet activation and migration of vascular smooth muscle cells (VSMCs), are involved in the pathogenesis of atherosclerosis [1]. Migration of VSMCs play a crucial role in the formation and progression of atherosclerotic lesions. Increased VSMC migration activity has been reported to be the main cause of intimal hyperplasia [2]. HDL has been reported to inhibit platelet-derived growth factor (PDGF)-induced migration of VSMCs, and this may be a novel anti-atherogenic mechanism of HDL [3]. Thus, migration of VSMCs into the tunica intima and their accumulation within the intima have been recognized as key events in atherosclerosis. Platelet activation is an important step of atherosclerosis. 5-Hydroxytryptamine (5-HT, serotonin) and throm*
Corresponding author. Fax: +81 23 628 5255. E-mail address: [email protected] (I. Wakabayashi). 0006-291X/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.09.129
boxane A2, potent vasoconstrictors released from platelets, play crucial roles in hemostasis under physiological conditions as well as pathological conditions such as ischemia due to thrombosis. 5-HT has been reported to induce proliferation of canine aortic and porcine coronary smooth muscle cells via 5-HT2 receptors [4,5]. In addition, 5-HT2 receptor antagonists have been reported to retard progression of atherosclerosis [6]. Thus, 5-HT is also involved in the formation of atherosclerotic lesions. Although migration of VSMCs is a key event in atherosclerosis, there have been, to the best of our knowledge, only two primitive studies on 5-HT action on migration of vascular cells: 5-HT enhanced migration of bovine and rat aortic smooth muscle cells [7,8]. Moreover, the mechanism of 5-HT-induced migration of VSMCs still remains to be clarified. The present study was therefore undertaken to determine the effects of 5-HT on migration of human aortic smooth muscle cells (HASMCs) and to elucidate the mechanism involving in 5-HT action on migration. We found that 5-HT enhances migration of HASMCs via 5-HT2 receptor stimulation through
S. Matsusaka, I. Wakabayashi / Biochemical and Biophysical Research Communications 337 (2005) 916–921
activation of the Rho-kinase and extracellular signal-regulated kinase (ERK) pathways. Materials and methods Cell culture. Human aortic smooth muscle cells (HASMCs) (KURABO, Osaka, Japan) were cultured in HuMedia-SG2 medium (KURABO) in CO2 (5%)-containing humidified air at 37 °C. All HASMC cultures used in this study were between the 5th and 9th passages. Cell migration assay. The cells were incubated in RPMI1640 medium (Sigma, St. Louis, MO, USA) containing 5% fetal bovine serum (FBS) under CO2 (5%)-containing humidified air at 37 °C for 24 h before each experiment in a 35-mm plastic plate. Cell movement on the plate was recorded using a time-lapse microscope for 120 min after stimulation with 5-HT and analyzed by an image analyzing software, MetaVue (Universal Imaging, PA, USA). The results shown are representative results for the largest field of the cells, and experiments were repeated at least three times for each assay condition. The mean migration velocity was compared under different conditions. Western blotting. The adhering cells, incubated in RPMI1640 medium containing FBS (5%) under CO2 (5%)-containing humidified air at 37 °C for 24 h before each experiment, were suspended in 0.1 ml of a lysis buffer [1% Triton X-100, 50 mM Tris–HCl (pH 7.2), 100 mM NaCl, 5 mM
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EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1 mM Na3VO4]. Cell lysates were separated by SDS–PAGE and transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA, USA). The membrane was blocked with 5% skim milk in phosphate-buffered saline (PBS) containing 0.1% Tween 20 (T–PBS) for 30 min at room temperature and then incubated for 60 min with anti-phospho-p44/p42 MAPK polyclonal antibody or anti-p44/p42 MAPK polyclonal antibody (Cell Signaling Technology, MA, USA). The membrane was then washed three times with T–PBS and incubated with horseradish peroxidase-conjugated goat anti-rabbit antibodies for 30 min, and specific proteins were detected using an enhanced chemiluminescence immunoblotting system. GST pull-down assay for activated RhoA. GST pull-down assay was performed as previously described [9]. Briefly, the adhering cells, incubated in RPMI1640 medium containing FBS (5%) under CO2 (5%)-containing humidified air at 37 °C for 24 h before each experiment in the presence or absence of 5-HT (100 nM), were lysed in a buffer [50 mM Tris– HCl (pH 7.2), 1% Triton X-100, 100 mM NaCl, 10 mM MgCl2, and 1 mM PMSF]. After centrifugation at 15,000 rpm for 15 min at 4 °C, cell lysates were incubated with GST-rhotekin Rho-binding domain (RBD) immobilized on glutathione agarose (Upstate, NY, USA) for 60 min at 4 °C. The beads were incubated with the cell lysates, and the proteins on the beads after washing were run on SDS–PAGE. GTP-bound RhoA was detected by immunoblotting with anti-RhoA polyclonal antibody (Santa Cruz Biotechnology, CA, USA) and anti-GST-Tag antibody.
Fig. 1. (A) Time-lapse images of migration of HASMCs. The migration of vehicle (top)-treated or 5-hydroxytryptamine (5-HT) (100 nM)-stimulated HASMCs and 5-HT (100 nM)-stimulated HASMCs in the presence of ketanserin (100 nM) or GR 55562 (100 nM) was recorded at the indicated times. Bars indicate a length of 50 lm. (B) Time course of HASMC migration velocity. HASMCs were incubated with 5-HT (100 nM) or a vehicle for 120 min. The mean 5-min interval velocity with SE is shown. Asterisks denote significant difference (*p < 0.01; **p < 0.05) from the control without 5-HT stimulation. N = 17. (C) Migration velocity of HASMCs for 30 min stimulated with 5-HT (1–100 nM) alone or 5-HT (100 nM) in the presence of ketanserin (100 nM) or GR 55562 (100 nM). The mean velocity with SE is shown. Asterisks denote significant difference (p < 0.01) from the control without 5-HT stimulation. N = 20.
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Immunostaining. After incubation on the culture slides (Becton–Dickinson, CA, USA) at 37 °C overnight, HASMCs were stimulated in the presence or absence of 5-HT (100 nM) for 30 min. Then the cells were fixed with 3.3% paraformaldehyde for 15 min at room temperature, treated with 0.2% Triton X-100 for 2 min, and blocked with 5% bovine serum albumin for 30 min. The cells were stained with Alexa Fluor 488conjugated phalloidin (Molecular Probes, OR, USA) for 30 min. After washing, the stained cells were mounted with antifade-containing glycerol/ PBS, and cell movement was observed under a confocal laser-scanning microscope (LSM510, Zeiss, Oberkochen, Germany). Drugs. 5-HT (Sigma, St. Louis, MO, USA), Y-27632 (Calbiochem, La Jolla, CA, USA), and GR 55562 (Tocris Coolson, UK) were dissolved in distilled water to make stock solutions of 10 mM, 1 mM, and 10 lM, respectively. U0126 (Cell Signaling Technology, Beverly, MA, USA) and ketanserin (Sigma, St. Louis, MO, USA) were dissolved in dimethyl sulfoxide (DMSO) to make stock solutions of 100 mM and 1 mM, respectively. Stock solutions of 5-HT, ketanserin, and GR 55562 were kept at 4 °C and those of Y-27632 and U0126 were kept at 20 °C. Statistical analysis. Statistical analysis was done using analysis of variance followed by Scheffe´Õs F test. P values less than 0.05 were regarded as significant.
Results Enhancement of cell migration by 5-HT Migration velocity of HASMCs was significantly increased by 5-HT (1–100 nM) in a concentration-dependent manner (Figs. 1A–C). Fig. 1B displays time course of mean 5-min interval migration velocity. Cell migration was increased only at an early time (within 30 min) after 5-HT stimulation, and cell migration velocity from 30 to 120 min after 5-HT stimulation was not significantly different from that in the control cells without 5-HT stimulation. The action of 5-HT (100 nM) on cell migration was markedly attenuated in the presence of ketanserin (100 nM) but was not affected in the presence of GR 55562 (100 nM) (Figs. 1A and C). Involvement of ERK MAPK activation in enhancing action of 5-HT on cell migration Slight expression of phosphorylated ERK was detected in the basal condition without 5-HT stimulation (Fig. 2A). 5-HT stimulation increased ERK activity timedependently until 5 min after the onset of stimulation. Then ERK activity decreased to the basal level over the next 10 min (Fig. 2A). The increase in cell migration induced by stimulation with 5-HT (100 nM) was abolished in the presence of U0126, a MEK1/2 inhibitor (Fig. 2B).
Fig. 2. (A) 5-Hydroxytryptamine (5-HT)-induced ERK MAPK activation in HASMCs. HASMCs were treated with 5-HT (100 nM) for the indicated times (min). Expression of phospho-ERK (top) and total ERK (bottom) is shown. (B) Effect of ERK MAPK inhibition on migration of HASMCs. Migration velocity of HASMCs stimulated with 5-HT (100 nM) for 30 min was compared in the presence and absence of U0126 (10 lM). The mean velocity with SE is shown. An asterisk denotes significant difference (p < 0.01) from the control without 5-HT stimulation. N = 20.
tion (Fig. 3B). The increase in 5-HT-induced ERK phosphorylation was also abolished in the presence of Y27632 (Fig. 3C). 5-HT-induced rearrangement of actin cytoskeleton Thick actin, which is associated with rapid cell migration, was observed in cells stimulated with 5-HT (Fig. 4A). Inhibition of ERK activation (Fig. 4B) or Rho kinase (Fig. 4C) blocked stress fiber formation. Imaging of cell detachment In response to 5-HT stimulation, the uropod, defined as the trailing edge, was detached in most of the cells (Fig. 5A), resulting in a higher migration speed. However, in the presence of Y-27632, 5-HT-induced uropod detachment was abolished (Fig. 5B). On the other hand, U0126 treatment did not affect uropod detachment following 5HT stimulation (data not shown). Discussion
Involvement of RhoA activation in enhancing action of 5-HT on cell migration Similar to ERK activity, slight activation of RhoA was also detected in the absence of 5-HT (Fig. 3A). RhoA activity was increased at 2.5 min after the onset of 5-HT stimulation and then decreased to the basal level (Fig. 3A). Y-27632, a Rho kinase (ROCK) inhibitor, abolished the enhancing action of 5-HT (100 nM) on cell migra-
The present study clearly demonstrated that 5-HT enhances migration of HASMCs. 5-HT, released from activated platelets, plays a major role in ischemia in thrombotic diseases through a potent vasocontractile action. In addition to this action, 5-HT also has an enhancing action on migration of arterial smooth muscle cells, and the action of 5-HT on migration may accelerate the formation of an atherosclerotic lesion. In fact, 5-HT antagonists have been
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Fig. 5. Time-lapse images of migration. The migration of 5-hydroxytryptamine (5-HT) (100 nM)-stimulated HASMCs in the absence (A) and presence (B) of Y-27632 (10 lM) was recorded at the indicated times. The arrows indicate uropods. Bars indicate a length of 50 lm.
Fig. 3. (A) 5-Hydroxytryptamine (5-HT)-induced RhoA activation in HASMCs. HASMCs were treated with 5-HT (100 nM) for the indicated times (min). GTP-bound RhoA was detected by pull-down assays using GST-rhotekin Rho-binding domain (top). (B) Effect of RhoA inhibition on the migration of HASMCs. Migration velocity of HASMCs stimulated with 5-HT (100 nM) for 30 min was compared in the presence and absence of Y-27632 (10 lM). The mean velocity with SE is shown. An asterisk denotes significant difference (p < 0.01) from the control without 5-HT stimulation. N = 20. (C) Effects of Y-27632, a Rho-kinase inhibitor, on 5hydroxytryptamine (5-HT)-induced ERK activation in human aortic smooth muscle cells (HASMCs). After pretreatment with Y-27632 (10 lM) for 30 min or a vehicle, the cells were stimulated with 5-HT (100 nM) for the indicated times (min). Expression of phospho-ERK (top) and total ERK (bottom) is shown.
Fig. 4. Actin cytoskeletal structure of HASMCs stimulated with 5hydroxytryptamine (5-HT, 100 nM) in the presence or absence of U0126 (10 lM) or Y-27632 (10 lM). The cells were grown on coverslips, washed, fixed, and processed for visualization of F-actin with Alexa 488conjugated phalloidin. HASMCs stimulated with 5-HT displayed an array of thick bundled actin stress fibers (A). In contrast, the actin filaments in HASMCs in the presence of U0126 (B) or Y-27632 (C) were notably less bundled and had an indistinct and less organized appearance.
reported to suppress atherosclerotic progress [6]. 5-HT-induced enhancement of HASMC migration was mediated through activation of 5-HT2 receptors, which are known to be involved in arterial smooth muscle proliferation, as well as contraction [10]. The present results also agree with the results of a study demonstrating that 5-HT enhanced growth factor-stimulated migration of cultured rat aortic smooth muscle cells (RASMCs) [8]. The mechanism of 5-HT-induced migration of vascular smooth muscle cells remained unknown. On the other hand, 5-HT-induced proliferation of bovine pulmonary arterial smooth muscle cells has recently been reported to be dependent on activation of ERK [11]. The present study demonstrated that ERK activity was greatly increased by 5-HT, and the enhancement of cell migration by 5-HT was abolished by U0126, an ERK inhibitor. Thus, ERK activation plays an important role in regulation of the cellular signaling required for migration as well as proliferation of VSMCs. Rho-kinase has also recently been reported to be involved in 5-HT-induced proliferation of VSMCs [11]. Rho-kinase has been demonstrated to be involved in contraction of human internal thoracic arteries [12]. It has been demonstrated in experiments using Y-27632 that activation of the Rho/ROCK pathway is involved in migration of vascular smooth muscle cells in response to urokinase, thrombin, and PDGF [13–16]. The present study clearly demonstrated that 5-HT increased RhoA activity and that Y-27632 abolished 5-HT-induced enhancement of cell migration, indicating that the Rho-kinase pathway also plays an important role in 5-HT-induced migration of HASMCs. 5-HT-induced enhancement of HASMC migration at an early time after stimulation indicates its association with activation of early signaling molecules such as RhoA and ERK. In the former studies, migration of VSMCs was analyzed by a conventional method using Boyden chambers. However, the detailed features of individual VSMC migration have not been known. In the present study, 5-HT-induced VSMC migration was analyzed by using time-lapse microscopy, a recently developed sensitive method for
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evaluation of single cell migration. Cell migration depends on reorganization of the actin cytoskeleton and development of cell polarity, enabling cells to make new adhesive contacts at their leading edges and to break contacts existing at their trailing edges (uropods). Activated ERK has been reported to phosphorylate myosin light-chain kinase and thereby to increase its kinase activity, resulting in enhancement of migration in COS cells [17]. A major function of Rho family GTPases in VSMCs is to regulate the organization of the actin cytoskeleton, and the Rho/ ROCK pathway is required for the formation of stress fibers and focal adhesion. The absence of dense actin bundles and failure in reorganization into actin stress fibers are reflected by inhibition of migration. The present study demonstrated the presence of thick actin filament following 5-HT stimulation that increases the velocity of HASMC migration. Furthermore, inhibition of ERK activation by U0126 or inhibition of Rho-kinase by Y-27632 blocked 5-HT-induced stress-fiber formation, resulting in a decrease in migration velocity of HASMCs. Recent studies have shown that Rho-kinase is an upper-stream signal for ERK activation in fibroblasts, NIH 3T3 cells, rat kidney cells, and RASMCs [18–22]. This agrees with the present finding that 5-HT-induced ERK activation in HASMCs was abolished by Y-27632. Activation of the Rho/ROCK pathway has been suggested to be essential for regulation of the process of uropod detachment and rear release in migrating leukocytes [23]. Furthermore, activation of Rho-kinase has been reported to be associated with the formation of uropod in BAF (pro B cells) [24]. Therefore, regulatory mechanism of uropod detachment differs by the kind of cell. However, the intracellular signaling mechanism involved in cytoskeletal changes related to migration of VSMCs remained to be clarified. In the present study, 5-HT stimulation of the cells induced rear release, and detachment of the uropod in HASMCs was abolished by Y-27632 but not affected by U0126. These results suggest that Rho-kinase, but not ERK, is involved in detachment of the uropod, while activation of both Rho-kinase and ERK pathways regulates 5-HT-induced formation of stress fibers. The present study is the first study that demonstrated the mechanism of 5-HT-induced migration of VASMCs. In conclusion, 5-HT has a potent enhancing action on migration of HASMCs due to an increase in stress fiber formation through stimulation of 5-HT2 receptors followed by activating the ERK and Rho-kinase pathways. Our study suggests that inhibitors targeting ERK and Rho/ ROCK pathways as well as anti-5-HT2 drugs may provide new therapeutic approaches for the treatment of atherosclerotic vascular diseases. Acknowledgment This work was supported by a grant for scientific research from the Grant of the Yamagata University 21st Century COE program.
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