Differential Regulation of Airway Smooth Muscle Cell ... - ATS Journals

Differential Regulation of Airway Smooth Muscle Cell Migration by E-Prostanoid Receptor Subtypes Hiromichi Aso1*, Satoru Ito1*, Akemi Mori1, Nobukazu Suganuma1, Masataka Morioka1, Norihiro Takahara1, Masashi Kondo1, and Yoshinori Hasegawa1 1

Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, Nagoya, Japan

Migration of airway smooth muscle (ASM) cells plays an important role in the pathophysiology of airway hyperresponsiveness and remodeling in asthma. It has been reported that prostaglandin (PG)E2 inhibits migration of ASM cells. Although PGE2 regulates cellular functions via binding to distinct prostanoid EP receptors, the role of EP receptor subtypes in mechanisms underlying cell migration has not been fully elucidated. We investigated the role of EP receptors in the inhibitory effects of PGE2 on the migration of human ASM cells. Migration induced by platelet-derived growth factor (PDGF)-BB (10 ng/ml, 6 h) was assessed by a chemotaxis chamber assay. PDGF-BB–induced cell migration was inhibited by PGE2, the specific EP2 agonist ONO-AE1-259–01, the specific EP4 agonist ONO-AE1-329, and cAMP-mobilizing agents. The inhibition of cell migration by PGE2 was significantly reversed by a blockade of EP2 and EP4 receptors using antagonists or transfection with siRNAs. Moreover, PGE2, the EP2 agonist, and the EP4 agonist significantly increased phosphorylation of small heat shock protein 20, one of the protein substrates for protein kinase A (PKA), with depolymerization of actin. In contrast, the EP3 agonist ONO-AE-248 significantly promoted baseline cell migration without affecting PDGF-BB–induced cell migration. In summary, activation of EP2 and EP4 receptors and subsequent activation of the cAMP/PKA pathway are the main mechanisms of inhibition of ASM cell migration by PGE2. HSP20 phosphorylation by PKA is possibly involved in this mechanism. Conversely, EP3 is potent in promoting cell migration. EP receptor subtypes may be novel therapeutic target molecules in airway remodeling and asthma. Keywords: asthma; airway smooth muscle; cAMP; prostaglandin E2; remodeling

Airway remodeling is characterized by structural changes in the airway walls that limit airflow in patients with chronic asthma (1). The increase in airway smooth muscle (ASM) mass due to proliferation and hypertrophy of ASM cells is one of the major causes of airway wall thickening and remodeling (2). Moreover, ASM cell migration toward the epithelium has been implicated in the pathogenesis of airway remodeling (3–5). Prostaglandin (PG)E2, one of the arachidonic acid cyclooxygenase metabolites, has multiple physiological roles in the lung (6–8). It is well established that PGE2 inhibits contraction, proliferation, and migration of ASM cells via increases in cytosolic cAMP levels (9–15). Yan and colleagues demonstrated that the antimitogenic effects of PGE2 and b-agonists are mostly dependent on activation (Received in original form April 29, 2012 and in final form October 14, 2012) * These authors contributed equally to this work. This work was supported by Grants-in-Aid no. 22890837 from the Ministry of Education, Culture, Sports, Science and Technology of Japan (S.I.).

CLINICAL RELEVANCE It is known that cytosolic cAMP elevation by b2-adrenoceptor activation and prostaglandin (PG)E 2 has a bronchoprotective effect. PGE2 inhibits migration of airway smooth muscle (ASM) cells, but the mechanisms involved are unclear. This study addresses the role of E-prostanoid (EP) receptors in regulation of human ASM cell migration. Because the migrating property of ASM cells contributes to airway hyperresponsiveness and remodeling, activation of EP2 and EP4 receptors could be beneficial in the management of asthma and chronic obstructive pulmonary disease.

of protein kinase A (PKA), a major cAMP effector (12). Thus, it is considered that PGE2 has a bronchoprotective role similar to that of b2-agonists, which are widely used to relieve bronchoconstriction in acute asthma attacks (16–18). PGE2 mediates physiological functions by binding to four different prostanoid EP receptor subtypes, termed EP1, EP2, EP3, and EP4 receptors, in target organs (19, 20). Specifically, stimulation of Gs-coupled EP2 and EP4 receptors by PGE2 increases intracellular cAMP via adenylyl cyclase activation (19). We recently reported that human ASM cells express EP2, EP3, and EP4 receptor mRNAs (11), consistent with a previous finding by Clarke and colleagues (21). There are no reports demonstrating functional expression of EP1 receptor in human ASM cells. It was demonstrated that activation of EP2 and EP4 receptors and subsequent cAMP elevation is involved in the mechanisms of inhibitions of contraction and proliferation by PGE2 in human ASM cells (11, 22, 23). Conversely, EP3 receptor activation elicits intracellular Ca21 concentrations ([Ca21]i) without affecting cytosolic cAMP levels in human ASM cells (11). However, little is known about the role of these EP receptor subtypes in the regulation of ASM cell migration. This study was designed to investigate the regulation of migration by different EP receptor subtypes in human ASM cells. Specific agonists for their respective EP receptors (6, 11) and short interfering (si)RNAs targeting EP receptors were used to assess the role of EP receptors. We demonstrated that activation of EP2 and EP4 receptors are involved in the mechanisms underlying the inhibition of platelet-derived growth factor (PDGF)-BB–evoked cell migration by PGE2. In contrast to EP2 and EP4 receptors, EP3 receptor activation is potent in promoting ASM cell migration.

Correspondence and requests for reprints should be addressed to Satoru Ito, M.D., Ph.D., Assistant Professor, Department of Respiratory Medicine, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan. E-mail: [email protected]

MATERIALS AND METHODS

This article has an online supplement, which is accessible from this issue’s table of contents at www.atsjournals.org

Reagents

Am J Respir Cell Mol Biol Vol 48, Iss. 3, pp 322–329, Mar 2013 Copyright ª 2013 by the American Thoracic Society Originally Published in Press as DOI: 10.1165/rcmb.2012-0158OC on December 6, 2012 Internet address: www.atsjournals.org

A detailed description of the methods is provided in the online supplement.

Specific EP receptor agonists (ONO-DI-004 [EP1 receptor], ONO-AE1-259– 01 [EP2 receptor], ONO-AE-248 [EP3 receptor], and ONO-AE1-329 [EP4 receptor]) and a specific EP4 receptor antagonist (ONO-AE2–227) were gifts from Ono Pharmaceutical Co. (Osaka, Japan) (6, 11). A potent

Aso, Ito, Mori, et al.: EP Receptors in Airway Smooth Muscle Cell Migration

EP2 receptor antagonist (AH6809) was from Cayman (Ann Arbor, MI). PGE2 was from Wako (Osaka, Japan). Forskolin, PDGF-BB, and procaterol were from Sigma-Aldrich (St. Louis, MO). 6-Bnz-cAMP, a potent PKA activator, was from Biolog (Berlin, Germany).

Cell Culture Primary cultures of normal human bronchial smooth muscle cells were obtained from Lonza (Walkersville, MD) as described previously (24, 25).

Quantitative Real-Time PCR Total cellular RNA was extracted using an RNeasy Mini Kit (Qiagen, Hilden, Germany). RNA was reverse transcribed to cDNA using Superscript III kit (Invitrogen, Carlsbad, CA). TaqMan Gene Expression Assays for EP2 receptor (Hs00168754_m1), EP4 receptor (Hs00168761_m1), and glyceraldehyde 3-phosphate dehydrogenase (Hs99999905_m1) genes were purchased from Applied Biosystems (Foster City, CA). Quantitative PCR was performed as described previously (6, 26).

Transfection of siRNA RNA interference was performed using siRNAs specific for EP2 and EP4 receptors (SMART Pool; Dharmacon, Lafayette, CO) and the scrambled siRNA (Invitrogen, Paisley, UK). Transfection reagent-siRNA complexes were prepared by using Lipofectamine2000 and Opti-MEM (Invitrogen) (27). Fifty percent confluent cells cultured for 24 hours without antibiotics were incubated with the siRNA (200 nM) or scrambled siRNA in culture medium without antibiotics for 48 hours at 378 C.

Cell Migration Assay Cell migration was assessed by a chemotaxis assay using a modified Boyden chamber (8-mm pore filter, 24-well cell clusters) (Chemotaxicell; Kurabo, Osaka, Japan) (27). Cell culture membrane inserts were coated by collagen type I (Nitta Gelatin Inc., Osaka, Japan). Cells (2 3 104 cells) in DMEM/F12 cell culture medium (Invitrogen) containing 0.1% FBS were placed into the upper well of the chamber and pretreated with PGE2, EP receptor agonists, forskolin, procaterol, or 6-Bnz-cAMP for 30 minutes. Then PDGF-BB (10 ng/ml) was placed in the bottom chamber for 6 hours at 378 C in a 5% CO2 incubator. The migrated cells on the lower surface of the membrane were fixed, stained with Diff-Quick (Sysmex, Kobe, Japan), and mounted onto glass slides. Cells in five different randomly selected fields per chamber were counted under a light microscope (3200). Each experimental condition was tested in duplicate.

Western Blotting Methods are essentially similar to those as described previously (6, 28). The detailed explanation of the technique is provided in the online supplement.

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cell migration compared with time-matched control cell cultures (n ¼ 4; P , 0.001). The PDGF-BB–induced cell migration was effectively inhibited by PGE2 in a concentration-dependent manner (n ¼ 4; P , 0.05) (Figure 1). Effects of Specific EP Receptor Agonists on PDGF-BB–Induced Migration

The effects of specific agonists for their respective EP receptors on PDGF-BB–induced cell migration were investigated. The concentration (1 mM) of each agonist used in the present study was specific for its respective EP receptor according to previous studies (6, 11, 23, 31). Application of the EP2 agonist ONOAE1-259–01 (1 mM) or the EP4 agonist ONO-AE1-329 (1 mM) significantly inhibited cell migration induced by PDGF-BB (10 ng/ml; 6 h) (n ¼ 6; P , 0.001) (Figure 2A). In contrast, the EP1 agonist ONO-DI-004 (1 mM) or the EP3 agonist ONO-AE248 (1 mM) did not inhibit PDGF-BB–induced cell migration (n ¼ 6) (Figure 2A). There was no statistically significant difference between the groups treated with PGE2 (1 mM) and the EP2 agonist ONO-AE1-259–01 or the EP4 agonist ONO-AE1-329. EP3 Receptor Agonist Enhances Baseline Cell Migration

We further examined whether PGE2 and EP receptor agonists act as chemoattractants and enhance baseline cell migration. To test this, the lower chamber was treated with PGE2 or either EP receptor agonist (1 mM) in DMEM/F-12 medium containing 0.1% FBS without PDGF-BB. Application of the EP3 agonist ONO-AE-248 (1 mM) modestly but significantly increased migrating cell numbers compared with time-matched control cell cultures (n ¼ 6; P , 0.05) (Figure 2B). In contrast, PGE2 (1 mM), the EP2 agonist ONO-AE1–259–01 (1 mM), or the EP4 agonist ONOAE1–329 (1 mM) slightly decreased the baseline cell migration, but the differences were not statistically significant (n ¼ 6) (Figure 2B). The EP1 agonist ONO-DI-004 (1 mM) did not affect cell migration (n ¼ 6) (Figure 2B). Effects of EP2 and EP4 Receptor Antagonists on Cell Migration Reduced by PGE2

To confirm the role of EP2 and EP4 receptors in cell migration reduced by PGE2, the effects of EP receptor–specific antagonists were assessed. The cells were pretreated with 10 mM AH6809, a potent

Fluorescent F-actin Staining The cells grown on glass coverslips (Lab-Tek; Nunc, Rochester, NY) coated with type I collagen were fixed with 4% formaldehyde in PBS and permeabilized with Triton X-100. F-actin was stained with fluorochrome-conjugated phalloidin (Alexa488-Phalloidin; Molecular Probes, Eugene, OR) and visualized as described previously (29, 30).

Statistical Analysis Data are expressed as means 6 SD. ANOVA followed by the Bonferroni test for post hoc analysis or t test was used to evaluate the statistical significance (SigmaPlot11.0; Systat Software Inc., San Jose, CA). P , 0.05 was considered statistically significant.

RESULTS Inhibition of PDGF-BB–Induced Cell Migration by PGE2

Concentration-dependent effects of PGE2 (10 nM to 10 mM) on ASM cell migration stimulated by PDGF-BB (10 ng/ml; 6 h) were examined. PDGF-BB treatment significantly enhanced

Figure 1. Concentration-dependent effects of prostaglandin (PG)E2 (10 nM to 10 mM) on migration of human airway smooth muscle (ASM) cells after stimulation by 10 ng/ml platelet-derived growth factor (PDGF)-BB for 6 hours (n ¼ 4). *Significantly different from the values for PDGF-BB alone (P , 0.05). Values are means 6 SD.

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Figure 3. Effects of a potent EP2 receptor antagonist, AH6809, and the specific EP4 receptor antagonist ONO-AE3-208 on the PGE2-induced inhibition of cell migration. The cells migrated in PDGF-BB (10 ng/ml, 6 h) with 1 mM PGE2 and 1 mM PGE2 plus 10 mM AH6809, 1 mM ONOAE2-227, or both (n ¼ 6). *Significantly different between the groups (P , 0.05). Values are means 6 SD.

Figure 2. (A) Effects of PGE2 (1 mM) and specific E-prostanoid (EP) receptor agonists (1 mM), ONO-DI-004 (EP1 agonist), ONO-AE1-259–01 (EP2 agonist), ONO-AE-248 (EP3 agonist), and ONO-AE1-329 (EP4 agonist) on cell migration induced by PDGF-BB (n ¼ 6). The cells migrated in PDGF-BB (10 ng/ml, 6 h) with 1 mM PGE2 or 1 mM EP receptor agonist. *Significantly different from the values for PDGF-BB alone (P , 0.05). (B) Effects of EP receptor agonists ONO-DI-004 (1 mM), ONO-AE1-259–01 (1 mM), ONOAE-248 (1 mM), and ONO-AE1-329 (1 mM) on basal cell migration in DMEM/F12 with 0.1% FBS without PDGF-BB (n ¼ 6). *Significantly different from the values of the baseline (control) (P , 0.05). Values are means 6 SD.

EP2 receptor antagonist, or with 1 mM ONO-AE2–227, a specific EP4 receptor antagonist (32), for 30 minutes before application of 1 mM PGE2. Figure 3 shows inhibitions of PDGF-BB–induced cell migration by 1 mM PGE2 alone or 1 mM PGE2 plus 10 mM AH6809 or 1 mM ONO-AE2–227. The inhibitory effects of PGE2 were partially but significantly reversed by the addition of AH6809 (n ¼ 6; P , 0.05). The effects of AH6809 and ONO-AE2227 were additive, and the inhibition of PDGF-BB cell migration by PGE2 was significantly reversed by AH6809 plus ONO-AE2227 (n ¼ 6; P , 0.001) (Figure 3). However, migrating cell numbers by PDGF-BB were still significantly more than those by PDGF-BB with PGE2 plus both AH6809 and ONO-AE2-227 (Figure 3). Neither AH6809 nor ONO-AE2-227 affected PDGF-BB–induced cell migration in the absence of PGE2 (data not shown). Transfection of siRNA Targeting EP2 and EP4 Receptor Antagonists on Reduced Cell Migration by PGE2

Cells were transfected with siRNA sequences for EP2 receptor, EP4 receptor, or the negative control (scrambled siRNA). Real-time

quantitative PCR data show that siRNA for EP2 receptor but not for EP4 receptor or scrambled siRNA almost completely inhibited the EP2 receptor mRNA levels (n ¼ 3; P , 0.001) (Figure 4A). Similarly, EP4 receptor mRNA levels were largely decreased by siRNA for EP4 receptor (n ¼ 3; P , 0.001) but not by siRNA for EP2 receptor or scrambled siRNA (Figure 4B). EP2 and EP4 receptor mRNA levels were also largely decreased by simultaneous transfection of siRNAs for the EP2 and EP4 receptors (n ¼ 3; P , 0.001) (Figures 4A and 4B). Three different siRNAs targeting the same gene were tested and gave similar mRNA expression results (data not shown). Cell viability as assessed by trypan blue exclusion was not affected by either siRNA transfection. The effects of transfection with siRNAs for EP2 and EP4 receptors on cell migration were investigated. The cells transfected with siRNA for EP2, EP4, or scrambled siRNA were stimulated by PDGF-BB (10 ng/ml, 6 h) in the presence of 1 mM PGE2. Transfection with scrambled siRNA did not affect cell migration reduced by PGE2 (n ¼ 5) (Figure 4C). Migrating cell numbers were significantly higher in the groups transfected with siRNA for EP2 or EP4 receptors in the presence of PGE2 than those with scrambled siRNA (n ¼ 5; P , 0.05) (Figure 4C). Moreover, simultaneous knockdown of EP2 and EP4 receptors with siRNAs had additive effects and significantly increased migrating cell numbers compared with the EP2 siRNA transfection alone (n ¼ 5; P , 0.05) (Figure 4C). Role of cAMP in PGE2 Effects

We further examined the role of cAMP on cell migration. Goncharova and colleagues have demonstrated that activation of PKA inhibits PDGF-induced migration of human ASM cells (10, 15). Forskolin (1 mM), the short-acting b2-agonist procaterol (1 mM), and a potent PKA activator 6-Bnz-cAMP (100 mM) effectively inhibited the PDGF-BB–induced migration (n ¼ 4; P , 0.05) in a manner similar to 1 mM PGE2 (Figure 5). These findings indicate that cytosolic cAMP elevation and subsequent PKA activation are involved in the reduction of cell migration by PGE2.


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Figure 4. Effects of siRNA-targeted knockdown of EP2 (siEP2) and EP4 (siEP4) receptor mRNAs on the change in mRNA expression of EP2 (A) and EP4 (B) receptors over control normalized to the reference gene glyceraldehyde 3-phosphate dehydrogenase are shown (n ¼ 3). Changes in mRNA expression were assessed by quantitative realtime PCR. *Significantly different from the values of the scrambled siRNA condition (P , 0.05). (C) Effects of siRNA treatment targeting EP2 and EP4 receptor on cell migration induced by PDGF-BB (n ¼ 5). *Significantly different between the groups (P , 0.05). Values are means 6 SD.

Phosphorylation of HSP20 by Activation of EP2 and EP4 Receptors

Small heat shock protein (HSP)20 was identified as a substrate protein of PKA (33). PKA activation induces phosphorylation of HSP20 on serine (Ser)16 (34), leading to ASM relaxation by modulating actin dynamics (35). We examined whether PGE2 and EP receptor activation increase the phosphorylation of HSP20 on Ser16 in the downstream pathways of cAMP/PKA. Figure 6A shows a time-dependent effect of PGE2 (1 mM; z 12 h) on phosphorylation of HSP20. Application of PGE2 increased HSP20 phosphorylation within 15 minutes, followed by a plateau phase up to 12 hours (Figure 6A). In contrast, HSP20 phosphorylation was not induced in the time-matched control cells (data not shown). The phospho-HSP20/total HSP20 ratio was significantly increased by treatment with PGE2 compared with the control (n ¼ 3; P , 0.05) (Figure 6A). Next, the effects of EP receptor agonists and cAMP/PKArelated agents on HSP20 phosphorylation were examined. The cells were treated with PGE2, the EP receptor agonists, forskolin, procaterol, and a potent PKA activator (6-Bnz-cAMP) for 15 minutes to assess HSP20 phosphorylation. The phospho-HSP20/ total HSP20 ratio was significantly enhanced by treatment with PGE2, ONO-AE1-259–01 (1 mM), and ONO-AE1-329 (1 mM) (n ¼ 3; P , 0.05) (Figure 6B). The EP1 agonist ONO-DI-004 (1 mM) or the EP3 agonist ONO-AE-248 (1 mM) did not induce HSP20 phosphorylation (n ¼ 3) (Figure 6B). Similar to PGE2, forskolin (1 mM), procaterol (1 mM), and 6-Bnz-cAMP (100 mM) significantly increased HSP20 phosphorylation (n ¼ 3; P , 0.05) (Figure 6C).

Effects of PGE2 on Actin Stress Fiber Formation

It has been reported that HSP20 phosphorylation by PKA activation leads to depolymerization of actin stress fibers in smooth muscle cells (33, 35, 36). Therefore, the effects of PGE2 and EP receptor agonists on F-actin formation by human ASM cells

Figure 5. Effects of pretreatment with1 mM PGE2, 1 mM forskolin, 1 mM procaterol, and 100 mM 6-Bnz-cAMP, a potent protein kinase A activator, on cell migration induced by PDGF-BB (10 ng/ml, 6 h) (n ¼ 4). *Significantly different from the values for PDGF-BB alone (P , 0.05). Values are means 6 SD.

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Figure 6. (A) Time-dependent effects of PGE2 (1 mM) on phosphorylation of heat shock protein (HSP)20 were assessed by Western blotting. (B) Effects of 1 mM PGE2 and 1 mM EP receptor agonists, ONO-DI-004 (EP1 agonist), ONO-AE1–259–01 (EP2 agonist), ONO-AE-248 (EP3 agonist), and ONO-AE1–329 (EP4 agonist) on HSP20 phosphorylation are shown. (C) Effects of 1 mM forskolin, 1 mM procaterol, and 100 mM 6-Bnz-cAMP on HSP20 phosphorylation are shown. HSP20 activations expressed as the phosphorylated (p)-HSP20/total (t)-HSP20 ratio are compared (n ¼ 3). *Significantly different from the values of control cells without agents (control) (P , 0.05). Values are means 6 SD.

treated by PDGF-BB (10 ng/ml for 1 h) were examined. Representative images of F-actin stress fiber formation stained with Alexa488-phalloidin are shown in Figure 7. Compared with control cells (Figure 7A), pretreatment of the cells with PGE2 (1 mM), the EP2 agonist ONO-AE1-259–01 (1 mM), or the EP4 agonist ONO-AE1-329 (1 mM) for 30 minutes inhibited net F-actin stress fiber formation (Figures 7B–7D).

with the increase in phosphorylation of HSP20, a substrate of PKA. Thus, we demonstrated for the first time that activation of EP2 and EP4 receptors inhibits PDGF-BB–stimulated cell migration in human ASM cells. It has been established that cytosolic cAMP elevation is a negative regulator of ASM cell migration. Indeed, forskolin and the

Effects of PDGF-BB on Cofilin Phosphorylation

We examined the effects of PDGF-BB on phosphorylation of cofilin, a protein belonging to the actin depolarizing factor family, in human ASM cells. PDGF-BB treatment (10 ng/ml, 15 min) significantly reduced the phospho-cofilin/total cofilin ratio (n ¼ 4; P , 0.05) (see Figure S1 in the online supplement).

DISCUSSION It has been demonstrated that PGE2 is a negative regulator of ASM cell migration (10, 14, 15). Our study extends these previous findings by assessing different EP receptors in human ASM cells. The role of these individual receptors was demonstrated by the use of selective EP receptor agonists and antagonists and by transfection of siRNAs for EP2 and EP4 receptors. The main findings of the present study were that (1) specific EP2 and EP4 agonists attenuated migration of human ASM cells stimulated by PDGF-BB, (2) the EP3 receptor agonist enhanced baseline cell migration, (3) the reduction of PDGFBB–evoked cell migration by PGE2 was significantly reversed by an EP2 receptor or EP4 receptor blockade using pharmacological antagonists or siRNA transfection, and (4) PGE2, EP2 agonist, and EP4 agonist disrupted F-actin stress fiber formation

Figure 7. Representative images of the F-actin visualized using Alexa488-conjugated phalloidin in control cells (A) and cells pretreated with 1 mM PGE2 (B), 1 mM ONO-AE1–259–01 (EP2 agonist) (C), and 1 mM ONO-AE1–329 (EP4 agonist) (D). Cells were stimulated by PDGFBB (10 ng/ml, 1 h) after pretreatment with one of the agents.


b2-agonist procaterol significantly inhibited the PDGF-BB–induced migration similarly to PGE2 (Figure 5). We previously reported that the EP2 receptor agonist ONO-AE1-259–01 and the EP4 receptor agonist ONO-AE1-329 increased the cytosolic cAMP levels in human ASM cells (11). In the present study, these EP agonists significantly inhibited the PDGF-BB–induced migration (Figure 2A). Moreover, the inhibition of PDGF-BB–evoked cell migration by PGE2 was additively reversed by the EP2 and EP4 antagonists (Figure 3) or by siRNA transfection (Figure 4). PKA is one of main effectors of cAMP in regulating ASM functions (37). Using an effective molecular strategy for inhibiting PKA, Yan and colleagues provided direct evidence that the inhibitory effect of PGE2 on human ASM cell proliferation is dependent on PKA activation (12). Recently, Goncharova and colleagues demonstrated that PGE2 and the b2-agonist albuterol inhibit the PDGF-induced migration of human ASM cells associated with vasodilator-stimulated phosphoprotein phosphorylation at Ser157 via PKA activation (10). They also found that phosphorylation of vasodilator-stimulated phosphoprotein by PKA is required for the PGE2–mediated inhibition of ASM cell migration. Taken together, our results show that the inhibitory effect of PGE2 on the PDGF-BB–induced ASM cell migration was mediated by EP2 and EP4 receptors and by subsequent activation of the cAMP/PKA pathway. Only the EP3 agonist ONO-AE-248 significantly promoted basal cell migration without PDGF-BB treatment (Figure 2B). We and other laboratories have found expression of EP3 receptor in human ASM cells (11, 17, 21, 38). We previously reported that activation of the EP3 receptor by ONO-AE-248 induced oscillatory increases in [Ca21]i (11). Intracellular Ca21 mobilization is the second messenger for mediating contraction of ASM cells (39). It has been proposed that cell migration and motility are regulated by [Ca21]i (27, 40, 41). Thus, it is suggested that activation of the EP3 receptor promotes ASM cell migration via elevation of [Ca21]i. Furthermore, Yan and colleagues reported that EP3 receptor activation has a promitogenic role in human ASM cells different from EP2 receptor activation (12). These findings indicate that the roles of EP3 receptors in regulation of proliferation and migration are opposite to those of EP2 and EP4 receptors. Nevertheless, cell migration promoted by EP3 receptor activation was much less than that by PDGF-BB stimulation (Figure 2). Moreover, EP3 receptor– promoted cell migration is relatively small compared with the reduced cell migration mediated via EP2 and EP4 receptor activation in PGE2 effects. In the present study, activation of the cAMP/PKA pathway that led to inhibition of ASM cell migration was associated with phosphorylation of HSP20 on Ser16, whereas neither the EP1 nor the EP3 receptor agonist affected it (Figure 6). A previous study indicated that phosphorylation of HSP20 inhibits migration in vascular smooth muscle cells (42). HSP20 is a substrate protein of PKA and is constitutively expressed in muscle tissues (33, 43). It has been demonstrated that phosphorylation of HSP20 on Ser16 by cAMP/PKA activation leads to ASM relaxation and loss of actin stress fibers (33, 35, 36, 43). Regardless of the cell type, reorganization of the actin cytoskeleton is essential for motility and migration. Activation of the cAMP/PKA pathway has been implicated in inhibiting the actin cytoskeleton in ASM cells (35, 44). Consistent with these previous findings, treatment of human ASM cells with PGE2, EP2, and EP4 receptor agonists inhibited net F-actin stress fiber formation (Figure 7). Actin depolymerization followed by PKA-mediated phosphorylation of HSP20 may be one of the mechanisms underlying the inhibition of ASM cell migration by EP2 and EP4 receptor activation. Komalavilas and colleagues demonstrated that phosphorylation of HSP20 by PKA activation decreases phosphorylation of cofilin

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in human ASM cells (35). Cofilin is a protein belonging to the actin depolarizing factor family and regulates actin polymerization and depolymerization (33, 45, 46). Cofilin plays a critical role in directional cell migration and serves as a dynamic component of the cell (47). It has been demonstrated that PDGF-BB decreases cofilin phosphorylation levels and promotes cell migration in vascular smooth muscle cells (48, 49). We also found that PDGF-BB treatment (10 ng/ml for 15 min) reduced phosphorylation of cofilin in human ASM cells (Figure S1). Activation of PKA/HSP20 phosphorylation and PDGF-BB triggers cofilin dephosphorylation (34–36), although these pathways play opposite roles in the regulation of ASM cell migration. One of the possible reasons is that cofilin regulates cell migration not simply via polymerization and depolymerization of actin fibers but also by affecting intracellular actin dynamics and turnover (47, 50). Primary cultures of ASM cells isolated from biopsies, autopsies, or lung resection specimens have been widely used to investigate cell functions in vitro because of their availability (51–54). In the present study, commercially available primary ASM cells were used (25, 27, 30). However, the possibility that precultured cells may lose some features of the differentiated ASM cell due to phenotypic changes cannot be ruled out. It was shown that ASM cells isolated from subjects with asthma proliferate more than those from control subjects (55). However, it is unclear whether ASM cell migration is increased in subjects with asthma. Burgess and colleagues demonstrated that the ASM cells isolated from patients with asthma had more expression of EP2 and EP4 receptors than those from normal subjects (17). They also found that antimitogenic effects of PGE2 were enhanced in patients with asthma compared with control subjects. Thus, studies are needed to determine the differences in PGE2–induced inhibition of ASM cell migration between subjects with asthma and control subjects. In summary, PGE2 inhibited PDGF-BB–induced migration via EP2 and EP4 receptors and subsequent cAMP/PKA pathway activation in human ASM cells. In contrast, EP3 receptor activation promoted basal cell migration without affecting the migration response to PDGF-BB. Moreover, the PGE2 effects were associated with HSP20 activation and depolymerization of actin stress fibers. Taken together, in conjunction with its antimitogenic and broncho-relaxing effects, PGE2 could protect against airway remodeling in patients with asthma via EP2 and EP4 receptor stimulation. Research on EP receptors may lead to novel therapeutic strategies for the treatment of airway remodeling in asthma. Author disclosures are available with the text of this article at www.atsjournals.org. Acknowledgments: The authors thank the Ono Pharmaceutical Co. for generous gifts of specific EP receptor agonists and EP4 antagonist and Ms. Katherine Ono for providing language help.

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