Experimental Lung Research, 40, 251–258, 2014 Copyright © 2014 Informa Healthcare USA, Inc. ISSN: 0190-2148 print / 1521-0499 online DOI: 10.3109/01902148.2014.913092
ORIGINAL ARTICLE
Activation of AMPK inhibits pulmonary arterial smooth muscle cells proliferation Yuanyuan Wu, Lu Liu, Yonghong Zhang, Guizuo Wang, Dong Han, Rui Ke, Shaojun Li, Wei Feng, and Manxiang Li
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Department of Respiratory Medicine, Respiratory Diseases Research Center, The Second Affiliated Hospital of Medical College, Xi’an Jiaotong University, Xi’an, Shaanxi, PR China A B STRA CT The aims of the present study were to examine the effect of AMPK activation on pulmonary arterial smooth muscle cells (PASMCs) proliferation and to address its potential mechanisms. ET-1 dose and time-dependently induced PASMCs proliferation, and this effect was suppressed by a selective AMPK activator metformin. The results of the study further indicated that the proliferation of PASMCs stimulated by ET-1 was associated with the increase of Skp2 and decrease of p27, and metformin reversed ET-1-induced Skp2 elevation and raised p27 protein level. Our study suggests that activation of AMPK suppresses PASMCs proliferation and has potential value in negatively modulating pulmonary vascular remodeling and therefore could prevent or treat the development of pulmonary arterial hypertension (PAH). KEYWORDS AMPK, p27, proliferation, pulmonary arterial smooth muscle cells, Skp2
INTRODUCTION Pulmonary arterial hypertension (PAH) is a common clinical syndrome, which is characterized by increased pulmonary vascular resistance and elevated pulmonary artery pressure, frequently leading to right-sided heart failure, and ultimately death. The pathological mechanisms underlying the development of pulmonary hypertension include persistent pulmonary vasoconstriction, vascular remodeling, and thrombosis in situ [1, 2]. Vascular remodeling is extremely critical among these pathogeneses, which is commonly caused by intima thickness and media hyperplasia, and pulmonary arterial smooth muscle cells (PASMCs) proliferation/migration plays a prominent role in this process [3,4]. Therefore, it is important to explore the molecular mechanisms responsible for PASMCs proliferation to prevent or
Received 29 November 2013; accepted 4 April 2014. Address correspondence to Dr. Manxiang Li, Department of Respiratory Medicine, the Second Affiliated Hospital of Medical College, Xi’an JiaoTong University, No.157, West 5th Road, Xi’an, Shaanxi, P.R. China 710004. E-mail: [email protected]
to reverse pulmonary vascular remodeling and thus to treat PAH. AMPK has been shown to be a cellular metabolic sensor with serine/threonine kinase activity, composes a catalytic α subunit and regulatory β and γ subunits [5]. Pathological changes causing cellular depletion of ATP or elevation of AMP, such as glucose deprivation, hypoxia, ischemia, and heat shock, induce AMPK activation [6]. Further study has indicated that AMPK is also activated by berberine [7] and resveratrol [8] independent of energy crisis [9]. Activation of AMPK regulates a wide variety of pathophysiological processes such as substrate metabolism, protein synthesis, cell proliferation and apoptosis [10]. Studies have found that activation of AMPK suppress the proliferation of a variety of tumor cells as well as nonmalignant cells [11–13], which has potential value in the treatment of cancer [14], as well as vascular diseases, such as atherosclerosis [15], cardiac hypertrophy and heart failure [16]. Recent studies have shown that AMPK phosphorylation is reduced in pulmonary artery endothelial cells (PAEC) of utero pulmonary hypertension (IPH) [17] and in PASMCs of idiopathic PAH (IPAH) [18]. Further studies indicated that enhancing AMPK 251
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phosphorylation improves IPH-PAEC angiogenesis [17] and suppresses PASMCs proliferation in IPAH [18]. Metformin has also been shown to inhibit the development of PAH in animal model by suppressing vascular remodeling via activation of AMPK [19]. Yet, the detailed molecular mechanisms underlying AMPK inhibition of pulmonary vascular remodeling are still unclear. To clarify this, primary cultured PASMCs were stimulated with ET-1, and the effects of metformin on PASMCs proliferation were examined, and its underlying molecular mechanisms were also explored.
MATERIALS AND METHODS Cell Preparation and Culture PASMCs from pulmonary artery were prepared from Sprague-Dawley rats (70–80 g). Pulmonary arteries were rapidly removed from sacrificed rats, washed in phosphate-buffered saline (4◦ C), and then dipped into Dulbecco’s Modified Eagle Medium (DMEM, Gibco) with 10% fetal bovine serum (FBS, Sijiqing, Hangzhou, China), 100 U/mL penicillin, and 100 μg/mL streptomycin (complete DMEM). A thin layer of the adventitia was carefully stripped off with a forceps and the endothelium was removed by gently scratching the intima surface with an elbow tweezers. Next, the arteries were cut into 1-mm pieces and placed into a culture flask and incubated at 37◦ C in an atmosphere of 95% air and 5% CO2 till cells reaching 80% confluence. Then cells were passaged using 0.25% trypsin. All experiments were performed using cells between passages 4 and 8. The purity of PASMCs was determined by immunostaining with α-actin. Before each experiment, cells were incubated in 1% FBS-DMEM overnight to minimize serum-induced effect on the cells. ET-1 (Alexis) was used to stimulate cell proliferation. Compound C (Calbiochem) was applied to inhibit the activity of AMPK.
Immunofluorescent Staining Assay To determine the purity of PASMCs, immunofluorescent staining against α-SM-actin was used. Briefly, Cells were fixed with 4% paraformaldehyde (20 minutes) and permeabilized in 0.25% Triton X-100 prior to blocking in 1% BSA for 30 minutes at room temperature. Cells were incubated with a rabbit monoclonal antibody against a-SM-actin (Abcam) at 4◦ C overnight and then fluorescent-conjugated secondary antibodies (Rhodamine (TRITC)-conjugated AffiniPure Goat Anti-rabbit IgG, Beijing Zhongshan
Golden Bridge Biological Technology Company, Beijing, China) for 1 hour at room temperature. The nucleus was stained with 4 6 -diamidino-2-phenylindole (DAPI, Invitrogen). Fluorescence was examined using an immunofluorescent microscope (Olympus, Japan).
Brdu Incorporation Assay To examine PASMCs proliferation, the rate of BrdU incorporation was determined using BrdU ELISA Kit (Maibio, Shanghai, China) according to the manufacturer’s instructions. Briefly, cells were seeded into 96-well plate at a density of 5 × 103 cells /well, allowing to adhere for at least 24 hours, and serum starved overnight (1% FBS in DMEM) before the start of experiments. Cells were treated with ET-1 or vehicle for 24 hours, metformin was added 6 hours before ET-1 stimulation. Then, BrdU labeling reagent was added to the wells and incubated for 2 hours at 37◦ C. Next, Cells were denatured with FixDenat solution for 30 minutes at room temperature, and followed by incubating with anti-BrdU mAbs conjugated to peroxidase for 90 minutes at room temperature. After removing antibody conjugate, substrate solution was added for reaction of 10 minutes. The absorbance at 370 nm was determined with a microplate reader (Bio-Rad). The blank corresponded to 100 μL of culture medium with or without BrdU.
Immunoblotting Cells were lysed in RIPA Lysis Buffer (50 mM Tris PH 7.4, 150 mM NaCl, 1% NP40, 0.5% Sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na3 VO4 , 1 mM NaF, and proteinase inhibitors). Lysates were centrifuged at 13,000 rpm at 4◦ C for 15 minutes, and supernatant was collected as total protein. Protein was separated on an SDS–PAGE gel and transferred to a TransBlot Nitrocellulose membrane (Bio-Rad). Polyclonal antibodies against total- AMPK, phosphor-AMPK, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Cell Signaling Technology) and monoclonal antibodies against p27 and Skp2 (Cell Signaling Technology) were used following manufacturer’s protocols. Horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse IgG was used as the secondary antibody (Sigma). Reactions were developed with SuperSignal West Pico Chemiluminescent Substrate (Pierce Inc) and exposure to autoradiographic film. Signaling was quantified from scanned films using Quality One software (Bio-Rad). Experimental Lung Research
AMPK Inhibition of Smooth Muscle Cells Proliferation
Statistical Analysis Values are presented as mean ± S.D. Data were analyzed using one-way ANOVA followed by a posthoc Student’s t-test. P < .05 was considered to represent significant differences between groups.
RESULTS
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Characteristics of PASMCs Cultured cells had a “ribbon-like” or “spindle” shape. Cells show a typical “hills and valleys” morphology at confluence (Figure 1A). Immune-staining for αactin showed that more than 90% of cells are smooth muscle cells (Figure 1B).
ET-1 Stimulates PASMCs Proliferation To examine whether ET-1 induces PASMCs proliferation, cells were treated with different concentrations of ET-1 for different times, and cell proliferation was determined by BrdU incorporation assay. As shown in Figure 2A, ET-1 dose-dependently stimulated PASMCs proliferation, 100 nM ET-1 triggered 1.97-fold increase in BrdU incorporation in 24 hours compared with control (P < .05). Figure 2B demonstrates that ET-1 stimulated PASMCs proliferation in a time-dependent manner, 100 nM ET-1 caused a 2.39-fold increase in BrdU incorporation over control at the time of 72 hours (P < .01).
Metformin Inhibits PASMCs Proliferation via AMPK Activation To examine the effect of metformin on ET-1-induced PASMCs proliferation, cells were stimulated with 100 nM ET-1 for 24 hours with or without preincubation of cells with metformin (4 mM) for 6 hours. Cell proliferation was determined using BrdU incorporation assay. Figure 3A shows that metformin dramatically suppressed ET-1-induced PASMCs proliferation (P < .01 versus ET-1-stimulated cells). BrdU incorporation rate decreased from 1.97- fold increase to 1.12-fold increase over control. To investigate the mechanisms of metformin inhibition of ET-1-stimulated PASMCs proliferation, cells were treated with metformin (4 mM) for different times, phorsphorylation of AMPK was determined using western blotting. As shown in Figure 3B, metformin induced AMPK phosphorylation in a time dependent manner with a maximal 3.29-fold increase over control at the peak time of 6 hours. Figure 3C demonstrates that pretreatment of cells with compound C blocked metformin-induced phosphorylation of AMPK (6 hours), the phosphorylation level of AMPK declined from 3.29-fold to 1.16-fold increase over control (P < .05 versus metformin-stimulated cells), indicating that compound C selectively suppresses AMPK phosphorylation. To verify the involvement of AMPK in the inhibitory effect of metformin on PASMCs proliferation, a selective AMPK inhibitor compound C (10 μM) was applied simultaneously with metformin (4 mM) for 6 hours before ET-1 stimulation (100 nM) for 24 hours, cell proliferation was assayed by BrdU incorporation. Figure 3D indicates
FIGURE 1. Characterization of PASMCs. A: Characteristic “hills and valleys” morphology of PASMCs under light microscopy, ×100. B: Immunofluorescent staining of PASMCs with α-smooth muscle actin with a magnification of 100. C
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FIGURE 2. Effect of ET-1 on PASMCs proliferation. A: PASMCs were stimulated with
different concentration of ET-1 ranging from 0 to 100 nM for 24 hours, the rate of BrdU incorporation in cells was determined by BrdU ELISA assay Kit (n = 4 each group). B: Cells were exposed with 100 nM ET-1 for the indicated times, BrdU incorporation in cells was measured (n = 4 each group). ∗ P < .05 versus control; # P < .01 versus control.
that the presence of compound C dramatically reversed the inhibitory effect of metformin on ET-1induced PASMCs proliferation, the rate of BrdU incorporation raised to 1.71-fold again compared with control cells (P < .05 versus metformin and ET-1treated cells).
Activation of AMPK Increases Skp2 and Decreases p27 It has been shown that inhibition of Skp2-mediated p27 degradation underlies AMPK suppression of several types of non-pulmonary smooth muscle cells proliferation [20, 21]. Here, we examined
whether the changes of Skp2 and p27 also associated with the inhibitory effect of activation of AMPK on PASMCs proliferation. PASMCs were prior treated with metformin (4 mM) with or without compound C (10 μM) for 6 hours before stimulation with 100 nM ET-1 for 24 hours, protein level of p27 and Skp2 were measured using western blotting. Figure 4A shows that ET-1 significantly reduced p27 protein level, which declined to 0.74-fold compared with control (P < .05), while preincubation of cells with metformin increased p27 protein level to 1.32-fold compared with control cells (P < .05 versus ET-1-treated cells and control cells), and the presence of compound C reversed the effect of metformin on p27 expression, Experimental Lung Research
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FIGURE 3. Metformin inhibition of ET-1-stimulated PASMCs proliferation. A: cells were pretreated with metformin (4 mM) for
6 hours before stimulation with ET-1(100 nM) for 24 hours, BrdU incorporation in cells was measured (n = 4 each group). B: PASMCs were incubated with metformin (4 mM) for the indicated times, and phosphorylation of AMPK was determined by immunoblotting (n = 3 each group). C: PASMCs were treated with metformin (4 mM) for 6 hours with or without compound C (CC,10 μM), phosphorylation of AMPK was measured by immunoblotting (n = 3 each group). D: PASMCs were treated with metformin (4 mM) for 6 hours with or without compound C (CC, 10 μM) before ET-1 stimulation (100 nM) for 24 hours, the rate of BrdU incorporation in cells was detected (n = 4 each group). ∗ P < .05 versus control, & P < .01 versus control, # P < .05 versus ET-1-treated cells, † P < .05 versus metformin-treated cells, ‡ P < .05 versus metformin and ET-1-treated cells.
protein level declined to 0.79-fold again compared with control (P < .05 versus metformin and ET-1treated cells). Figure 4B indicates that ET-1 dramatically increased Skp2 protein level, which raised to 2.21-fold compared to control (P < .01), while preincubation of cells with metformin reduced Skp2 protein level from 2.21-fold to 1.13-fold increase over control (P < .01 versus ET-1-treated cells). The presence of compound C reversed the effect of metformin on Skp2 level, which increased to 1.97-fold again compared with control (P < .05 versus metformin and ET-1-treated cells). These results suggested that ET-1 stimulated PASMCs proliferation by upregulating Skp2 and reducing p27, and activation of AMPK by metformin inhibited Skp2 expression and raised p27 to suppress the proliferation of PASMCs. C
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DISCUSSION The present study provides direct evidence that ET-1 stimulates PASMCs proliferation by increasing Skp2 and decreasing p27, and activation of AMPK reversed ET-1-induced elevation of Skp2 and reduction of p27, therefore suppressed PASMCs proliferation. Our study indicates that enhancing the activity of AMPK might have potential value in the treatment of PAH by modulation of vascular remodeling. ET-1 plays an important role in the development of PAH by stimulating vascular constriction and vascular remodeling (largely due to PASMCs proliferation). Activation of several pro-proliferation signaling pathways is responsible for ET-1 stimulation of PASMCs proliferation [22–25]. It has
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FIGURE 4. Effects of ET-1 and metformin on the expression of p27 and Skp2 protein in PASMCs. PASMCs were preincubated with metformin (4 mM) for 6 hours with or without the presence of compound C (CC, 10 μM) before stimulation with ET-1 for 24 hours. A: Protein level of p27 was determined using immunoblotting, GAPDH served as loading control. A representative blot and quantification of bands are shown (n = 3 each group). B: Protein level of Skp2 was measured using immunoblotting, GAPDH served as loading control (n = 3 each group). ∗ p < .05 versus control, & P < .01 versus control, # P < .05 versus ET-1-treated cells, ## P < .01 versus ET-1-treated cells, ‡ P < .05 versus metformin and ET-1-treated cells.
been demonstrated that p27 plays a critical role in regulating cell cycle progression in mammalian cells [26, 27]. p27 as a cyclin-dependent kinases (CDK) inhibitor binds to and inhibits the function of cyclin E/CDK2 complex, and further blocks cell cycle progression from G1 into S phase and suppresses cell proliferation [28]. Study in human mesangial cells suggested that ET-1 reduces protein level of p27 contribution to cells proliferation [29]. The results of the present study indicated that ET-1 also reduced p27 level and contributed to PASMCs proliferation. Studies have shown that activation of AMPK signaling pathway benefits a variety of types of malignant tumors and atherosclerosis by inhibiting the proliferation of tumor cells and artery vascular smooth cells [11,13, 18]. Here, we confirmed that activation of AMPK also suppressed the proliferation of primary cultured pulmonary artery smooth muscle cells. Inhibition of pro-proliferation signaling cascades, such as PI3K/Akt signaling, underlies the effect of activation of AMPK inhibiting cell proliferation [30]. Activation of mTOR by PI3K/Akt is associated with proteins synthesis required for cell proliferation [11], while AMPK inhibits mTOR activity by activation of tuberous sclerosis (TSC) and further suppresses cells proliferation [31]. The present study found that activation of AMPK by metformin reversed ET-1 induced p27 reduction, this was accompanied with the
increase of S phase kinase-associated protein 2 (Skp2) protein level. It has been shown that mTOR promotes cell cycle progression by proteasome-dependent p27 degradation which is associated with the increase of one subunit of ubiquitin protein ligase complex SCFs (SKP1-cullin-F-box), Skp2, Skp2 is a F-box protein acts as the substrate recognition factor [32, 33]. It is known that the level of p27 protein is correlated with its expression and phosphorylation [20, 34]. AMPK inhibits p27 degradation by suppressing mTOR-dependent Skp2 expression and by increasing phosphorylation of p27 at site of threonine 197 which makes it resistant to degradation [6, 34]. Metformin is an in vitro synthetic AMPK agonist which has been commonly used in clinic to treat type 2 diabetes with wide clinical experience and safety record [35]. Results of the present study indicate that activation of AMPK by metformin suppresses PASMCs proliferation, suggesting that metformin might inhibit pulmonary vascular remodeling and has potential value in the treatment of pulmonary artery hypertension. Yet, this needs to be verified in clinical trial.
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. Experimental Lung Research
AMPK Inhibition of Smooth Muscle Cells Proliferation
This work was supported by Chinese National Science Foundation (No. 81070045 and No. 81330002).
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Experimental Lung Research
ORIGINAL ARTICLE
Activation of AMPK inhibits pulmonary arterial smooth muscle cells proliferation Yuanyuan Wu, Lu Liu, Yonghong Zhang, Guizuo Wang, Dong Han, Rui Ke, Shaojun Li, Wei Feng, and Manxiang Li
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Department of Respiratory Medicine, Respiratory Diseases Research Center, The Second Affiliated Hospital of Medical College, Xi’an Jiaotong University, Xi’an, Shaanxi, PR China A B STRA CT The aims of the present study were to examine the effect of AMPK activation on pulmonary arterial smooth muscle cells (PASMCs) proliferation and to address its potential mechanisms. ET-1 dose and time-dependently induced PASMCs proliferation, and this effect was suppressed by a selective AMPK activator metformin. The results of the study further indicated that the proliferation of PASMCs stimulated by ET-1 was associated with the increase of Skp2 and decrease of p27, and metformin reversed ET-1-induced Skp2 elevation and raised p27 protein level. Our study suggests that activation of AMPK suppresses PASMCs proliferation and has potential value in negatively modulating pulmonary vascular remodeling and therefore could prevent or treat the development of pulmonary arterial hypertension (PAH). KEYWORDS AMPK, p27, proliferation, pulmonary arterial smooth muscle cells, Skp2
INTRODUCTION Pulmonary arterial hypertension (PAH) is a common clinical syndrome, which is characterized by increased pulmonary vascular resistance and elevated pulmonary artery pressure, frequently leading to right-sided heart failure, and ultimately death. The pathological mechanisms underlying the development of pulmonary hypertension include persistent pulmonary vasoconstriction, vascular remodeling, and thrombosis in situ [1, 2]. Vascular remodeling is extremely critical among these pathogeneses, which is commonly caused by intima thickness and media hyperplasia, and pulmonary arterial smooth muscle cells (PASMCs) proliferation/migration plays a prominent role in this process [3,4]. Therefore, it is important to explore the molecular mechanisms responsible for PASMCs proliferation to prevent or
Received 29 November 2013; accepted 4 April 2014. Address correspondence to Dr. Manxiang Li, Department of Respiratory Medicine, the Second Affiliated Hospital of Medical College, Xi’an JiaoTong University, No.157, West 5th Road, Xi’an, Shaanxi, P.R. China 710004. E-mail: [email protected]
to reverse pulmonary vascular remodeling and thus to treat PAH. AMPK has been shown to be a cellular metabolic sensor with serine/threonine kinase activity, composes a catalytic α subunit and regulatory β and γ subunits [5]. Pathological changes causing cellular depletion of ATP or elevation of AMP, such as glucose deprivation, hypoxia, ischemia, and heat shock, induce AMPK activation [6]. Further study has indicated that AMPK is also activated by berberine [7] and resveratrol [8] independent of energy crisis [9]. Activation of AMPK regulates a wide variety of pathophysiological processes such as substrate metabolism, protein synthesis, cell proliferation and apoptosis [10]. Studies have found that activation of AMPK suppress the proliferation of a variety of tumor cells as well as nonmalignant cells [11–13], which has potential value in the treatment of cancer [14], as well as vascular diseases, such as atherosclerosis [15], cardiac hypertrophy and heart failure [16]. Recent studies have shown that AMPK phosphorylation is reduced in pulmonary artery endothelial cells (PAEC) of utero pulmonary hypertension (IPH) [17] and in PASMCs of idiopathic PAH (IPAH) [18]. Further studies indicated that enhancing AMPK 251
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phosphorylation improves IPH-PAEC angiogenesis [17] and suppresses PASMCs proliferation in IPAH [18]. Metformin has also been shown to inhibit the development of PAH in animal model by suppressing vascular remodeling via activation of AMPK [19]. Yet, the detailed molecular mechanisms underlying AMPK inhibition of pulmonary vascular remodeling are still unclear. To clarify this, primary cultured PASMCs were stimulated with ET-1, and the effects of metformin on PASMCs proliferation were examined, and its underlying molecular mechanisms were also explored.
MATERIALS AND METHODS Cell Preparation and Culture PASMCs from pulmonary artery were prepared from Sprague-Dawley rats (70–80 g). Pulmonary arteries were rapidly removed from sacrificed rats, washed in phosphate-buffered saline (4◦ C), and then dipped into Dulbecco’s Modified Eagle Medium (DMEM, Gibco) with 10% fetal bovine serum (FBS, Sijiqing, Hangzhou, China), 100 U/mL penicillin, and 100 μg/mL streptomycin (complete DMEM). A thin layer of the adventitia was carefully stripped off with a forceps and the endothelium was removed by gently scratching the intima surface with an elbow tweezers. Next, the arteries were cut into 1-mm pieces and placed into a culture flask and incubated at 37◦ C in an atmosphere of 95% air and 5% CO2 till cells reaching 80% confluence. Then cells were passaged using 0.25% trypsin. All experiments were performed using cells between passages 4 and 8. The purity of PASMCs was determined by immunostaining with α-actin. Before each experiment, cells were incubated in 1% FBS-DMEM overnight to minimize serum-induced effect on the cells. ET-1 (Alexis) was used to stimulate cell proliferation. Compound C (Calbiochem) was applied to inhibit the activity of AMPK.
Immunofluorescent Staining Assay To determine the purity of PASMCs, immunofluorescent staining against α-SM-actin was used. Briefly, Cells were fixed with 4% paraformaldehyde (20 minutes) and permeabilized in 0.25% Triton X-100 prior to blocking in 1% BSA for 30 minutes at room temperature. Cells were incubated with a rabbit monoclonal antibody against a-SM-actin (Abcam) at 4◦ C overnight and then fluorescent-conjugated secondary antibodies (Rhodamine (TRITC)-conjugated AffiniPure Goat Anti-rabbit IgG, Beijing Zhongshan
Golden Bridge Biological Technology Company, Beijing, China) for 1 hour at room temperature. The nucleus was stained with 4 6 -diamidino-2-phenylindole (DAPI, Invitrogen). Fluorescence was examined using an immunofluorescent microscope (Olympus, Japan).
Brdu Incorporation Assay To examine PASMCs proliferation, the rate of BrdU incorporation was determined using BrdU ELISA Kit (Maibio, Shanghai, China) according to the manufacturer’s instructions. Briefly, cells were seeded into 96-well plate at a density of 5 × 103 cells /well, allowing to adhere for at least 24 hours, and serum starved overnight (1% FBS in DMEM) before the start of experiments. Cells were treated with ET-1 or vehicle for 24 hours, metformin was added 6 hours before ET-1 stimulation. Then, BrdU labeling reagent was added to the wells and incubated for 2 hours at 37◦ C. Next, Cells were denatured with FixDenat solution for 30 minutes at room temperature, and followed by incubating with anti-BrdU mAbs conjugated to peroxidase for 90 minutes at room temperature. After removing antibody conjugate, substrate solution was added for reaction of 10 minutes. The absorbance at 370 nm was determined with a microplate reader (Bio-Rad). The blank corresponded to 100 μL of culture medium with or without BrdU.
Immunoblotting Cells were lysed in RIPA Lysis Buffer (50 mM Tris PH 7.4, 150 mM NaCl, 1% NP40, 0.5% Sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM Na3 VO4 , 1 mM NaF, and proteinase inhibitors). Lysates were centrifuged at 13,000 rpm at 4◦ C for 15 minutes, and supernatant was collected as total protein. Protein was separated on an SDS–PAGE gel and transferred to a TransBlot Nitrocellulose membrane (Bio-Rad). Polyclonal antibodies against total- AMPK, phosphor-AMPK, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Cell Signaling Technology) and monoclonal antibodies against p27 and Skp2 (Cell Signaling Technology) were used following manufacturer’s protocols. Horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse IgG was used as the secondary antibody (Sigma). Reactions were developed with SuperSignal West Pico Chemiluminescent Substrate (Pierce Inc) and exposure to autoradiographic film. Signaling was quantified from scanned films using Quality One software (Bio-Rad). Experimental Lung Research
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Statistical Analysis Values are presented as mean ± S.D. Data were analyzed using one-way ANOVA followed by a posthoc Student’s t-test. P < .05 was considered to represent significant differences between groups.
RESULTS
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Characteristics of PASMCs Cultured cells had a “ribbon-like” or “spindle” shape. Cells show a typical “hills and valleys” morphology at confluence (Figure 1A). Immune-staining for αactin showed that more than 90% of cells are smooth muscle cells (Figure 1B).
ET-1 Stimulates PASMCs Proliferation To examine whether ET-1 induces PASMCs proliferation, cells were treated with different concentrations of ET-1 for different times, and cell proliferation was determined by BrdU incorporation assay. As shown in Figure 2A, ET-1 dose-dependently stimulated PASMCs proliferation, 100 nM ET-1 triggered 1.97-fold increase in BrdU incorporation in 24 hours compared with control (P < .05). Figure 2B demonstrates that ET-1 stimulated PASMCs proliferation in a time-dependent manner, 100 nM ET-1 caused a 2.39-fold increase in BrdU incorporation over control at the time of 72 hours (P < .01).
Metformin Inhibits PASMCs Proliferation via AMPK Activation To examine the effect of metformin on ET-1-induced PASMCs proliferation, cells were stimulated with 100 nM ET-1 for 24 hours with or without preincubation of cells with metformin (4 mM) for 6 hours. Cell proliferation was determined using BrdU incorporation assay. Figure 3A shows that metformin dramatically suppressed ET-1-induced PASMCs proliferation (P < .01 versus ET-1-stimulated cells). BrdU incorporation rate decreased from 1.97- fold increase to 1.12-fold increase over control. To investigate the mechanisms of metformin inhibition of ET-1-stimulated PASMCs proliferation, cells were treated with metformin (4 mM) for different times, phorsphorylation of AMPK was determined using western blotting. As shown in Figure 3B, metformin induced AMPK phosphorylation in a time dependent manner with a maximal 3.29-fold increase over control at the peak time of 6 hours. Figure 3C demonstrates that pretreatment of cells with compound C blocked metformin-induced phosphorylation of AMPK (6 hours), the phosphorylation level of AMPK declined from 3.29-fold to 1.16-fold increase over control (P < .05 versus metformin-stimulated cells), indicating that compound C selectively suppresses AMPK phosphorylation. To verify the involvement of AMPK in the inhibitory effect of metformin on PASMCs proliferation, a selective AMPK inhibitor compound C (10 μM) was applied simultaneously with metformin (4 mM) for 6 hours before ET-1 stimulation (100 nM) for 24 hours, cell proliferation was assayed by BrdU incorporation. Figure 3D indicates
FIGURE 1. Characterization of PASMCs. A: Characteristic “hills and valleys” morphology of PASMCs under light microscopy, ×100. B: Immunofluorescent staining of PASMCs with α-smooth muscle actin with a magnification of 100. C
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FIGURE 2. Effect of ET-1 on PASMCs proliferation. A: PASMCs were stimulated with
different concentration of ET-1 ranging from 0 to 100 nM for 24 hours, the rate of BrdU incorporation in cells was determined by BrdU ELISA assay Kit (n = 4 each group). B: Cells were exposed with 100 nM ET-1 for the indicated times, BrdU incorporation in cells was measured (n = 4 each group). ∗ P < .05 versus control; # P < .01 versus control.
that the presence of compound C dramatically reversed the inhibitory effect of metformin on ET-1induced PASMCs proliferation, the rate of BrdU incorporation raised to 1.71-fold again compared with control cells (P < .05 versus metformin and ET-1treated cells).
Activation of AMPK Increases Skp2 and Decreases p27 It has been shown that inhibition of Skp2-mediated p27 degradation underlies AMPK suppression of several types of non-pulmonary smooth muscle cells proliferation [20, 21]. Here, we examined
whether the changes of Skp2 and p27 also associated with the inhibitory effect of activation of AMPK on PASMCs proliferation. PASMCs were prior treated with metformin (4 mM) with or without compound C (10 μM) for 6 hours before stimulation with 100 nM ET-1 for 24 hours, protein level of p27 and Skp2 were measured using western blotting. Figure 4A shows that ET-1 significantly reduced p27 protein level, which declined to 0.74-fold compared with control (P < .05), while preincubation of cells with metformin increased p27 protein level to 1.32-fold compared with control cells (P < .05 versus ET-1-treated cells and control cells), and the presence of compound C reversed the effect of metformin on p27 expression, Experimental Lung Research
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FIGURE 3. Metformin inhibition of ET-1-stimulated PASMCs proliferation. A: cells were pretreated with metformin (4 mM) for
6 hours before stimulation with ET-1(100 nM) for 24 hours, BrdU incorporation in cells was measured (n = 4 each group). B: PASMCs were incubated with metformin (4 mM) for the indicated times, and phosphorylation of AMPK was determined by immunoblotting (n = 3 each group). C: PASMCs were treated with metformin (4 mM) for 6 hours with or without compound C (CC,10 μM), phosphorylation of AMPK was measured by immunoblotting (n = 3 each group). D: PASMCs were treated with metformin (4 mM) for 6 hours with or without compound C (CC, 10 μM) before ET-1 stimulation (100 nM) for 24 hours, the rate of BrdU incorporation in cells was detected (n = 4 each group). ∗ P < .05 versus control, & P < .01 versus control, # P < .05 versus ET-1-treated cells, † P < .05 versus metformin-treated cells, ‡ P < .05 versus metformin and ET-1-treated cells.
protein level declined to 0.79-fold again compared with control (P < .05 versus metformin and ET-1treated cells). Figure 4B indicates that ET-1 dramatically increased Skp2 protein level, which raised to 2.21-fold compared to control (P < .01), while preincubation of cells with metformin reduced Skp2 protein level from 2.21-fold to 1.13-fold increase over control (P < .01 versus ET-1-treated cells). The presence of compound C reversed the effect of metformin on Skp2 level, which increased to 1.97-fold again compared with control (P < .05 versus metformin and ET-1-treated cells). These results suggested that ET-1 stimulated PASMCs proliferation by upregulating Skp2 and reducing p27, and activation of AMPK by metformin inhibited Skp2 expression and raised p27 to suppress the proliferation of PASMCs. C
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DISCUSSION The present study provides direct evidence that ET-1 stimulates PASMCs proliferation by increasing Skp2 and decreasing p27, and activation of AMPK reversed ET-1-induced elevation of Skp2 and reduction of p27, therefore suppressed PASMCs proliferation. Our study indicates that enhancing the activity of AMPK might have potential value in the treatment of PAH by modulation of vascular remodeling. ET-1 plays an important role in the development of PAH by stimulating vascular constriction and vascular remodeling (largely due to PASMCs proliferation). Activation of several pro-proliferation signaling pathways is responsible for ET-1 stimulation of PASMCs proliferation [22–25]. It has
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FIGURE 4. Effects of ET-1 and metformin on the expression of p27 and Skp2 protein in PASMCs. PASMCs were preincubated with metformin (4 mM) for 6 hours with or without the presence of compound C (CC, 10 μM) before stimulation with ET-1 for 24 hours. A: Protein level of p27 was determined using immunoblotting, GAPDH served as loading control. A representative blot and quantification of bands are shown (n = 3 each group). B: Protein level of Skp2 was measured using immunoblotting, GAPDH served as loading control (n = 3 each group). ∗ p < .05 versus control, & P < .01 versus control, # P < .05 versus ET-1-treated cells, ## P < .01 versus ET-1-treated cells, ‡ P < .05 versus metformin and ET-1-treated cells.
been demonstrated that p27 plays a critical role in regulating cell cycle progression in mammalian cells [26, 27]. p27 as a cyclin-dependent kinases (CDK) inhibitor binds to and inhibits the function of cyclin E/CDK2 complex, and further blocks cell cycle progression from G1 into S phase and suppresses cell proliferation [28]. Study in human mesangial cells suggested that ET-1 reduces protein level of p27 contribution to cells proliferation [29]. The results of the present study indicated that ET-1 also reduced p27 level and contributed to PASMCs proliferation. Studies have shown that activation of AMPK signaling pathway benefits a variety of types of malignant tumors and atherosclerosis by inhibiting the proliferation of tumor cells and artery vascular smooth cells [11,13, 18]. Here, we confirmed that activation of AMPK also suppressed the proliferation of primary cultured pulmonary artery smooth muscle cells. Inhibition of pro-proliferation signaling cascades, such as PI3K/Akt signaling, underlies the effect of activation of AMPK inhibiting cell proliferation [30]. Activation of mTOR by PI3K/Akt is associated with proteins synthesis required for cell proliferation [11], while AMPK inhibits mTOR activity by activation of tuberous sclerosis (TSC) and further suppresses cells proliferation [31]. The present study found that activation of AMPK by metformin reversed ET-1 induced p27 reduction, this was accompanied with the
increase of S phase kinase-associated protein 2 (Skp2) protein level. It has been shown that mTOR promotes cell cycle progression by proteasome-dependent p27 degradation which is associated with the increase of one subunit of ubiquitin protein ligase complex SCFs (SKP1-cullin-F-box), Skp2, Skp2 is a F-box protein acts as the substrate recognition factor [32, 33]. It is known that the level of p27 protein is correlated with its expression and phosphorylation [20, 34]. AMPK inhibits p27 degradation by suppressing mTOR-dependent Skp2 expression and by increasing phosphorylation of p27 at site of threonine 197 which makes it resistant to degradation [6, 34]. Metformin is an in vitro synthetic AMPK agonist which has been commonly used in clinic to treat type 2 diabetes with wide clinical experience and safety record [35]. Results of the present study indicate that activation of AMPK by metformin suppresses PASMCs proliferation, suggesting that metformin might inhibit pulmonary vascular remodeling and has potential value in the treatment of pulmonary artery hypertension. Yet, this needs to be verified in clinical trial.
Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. Experimental Lung Research
AMPK Inhibition of Smooth Muscle Cells Proliferation
This work was supported by Chinese National Science Foundation (No. 81070045 and No. 81330002).
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