International Journal of
Molecular Sciences Article
Knockdown of AMPKα2 Promotes Pulmonary Arterial Smooth Muscle Cells Proliferation via mTOR/Skp2/p27Kip1 Signaling Pathway Rui Ke 1 , Lu Liu 1 , Yanting Zhu 1 , Shaojun Li 1 , Xinming Xie 1 , Fangwei Li 1 , Yang Song 1 , Lan Yang 1 , Li Gao 2 and Manxiang Li 1, * 1
2
*
Department of Respiratory and Critical Care Medicine, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an 710061, China; [email protected] (R.K.); [email protected] (L.L.); [email protected] (Y.Z.); [email protected] (S.L.); [email protected] (X.X.); [email protected] (F.L.); [email protected] (Y.S.); [email protected] (L.Y.) Division of Allergy and Clinical Immunology, Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD 21224, USA; [email protected] Correspondence: [email protected]; Tel./Fax: +86-29-8532-3850
Academic Editor: Atsushi Matsuzawa Received: 4 March 2016; Accepted: 24 May 2016; Published: 31 May 2016
Abstract: It has been shown that activation of adenosine monophosphate-activated protein kinase (AMPK) suppresses proliferation of a variety of tumor cells as well as nonmalignant cells. In this study, we used post-transcriptional gene silencing with small interfering RNA (siRNA) to specifically examine the effect of AMPK on pulmonary arterial smooth muscle cells (PASMCs) proliferation and to further elucidate its underlying molecular mechanisms. Our results showed that knockdown of AMPKα2 promoted primary cultured PASMCs proliferation; this was accompanied with the elevation of phosphorylation of mammalian target of rapamycin (mTOR) and S-phase kinase-associated protein 2 (Skp2) protein level and reduction of p27Kip1 . Importantly, prior silencing of mTOR with siRNA abolished AMPKα2 knockdown-induced Skp2 upregulation, p27Kip1 reduction as well as PASMCs proliferation. Furthermore, pre-depletion of Skp2 by siRNA also eliminated p27Kip1 downregulation and PASMCs proliferation caused by AMPKα2 knockdown. Taken together, our study indicates that AMPKα2 isoform plays an important role in regulation of PASMCs proliferation by modulating mTOR/Skp2/p27Kip1 axis, and suggests that activation of AMPKα2 might have potential value in the prevention and treatment of pulmonary arterial hypertension. Keywords: AMPK; pulmonary arterial smooth muscle cells; proliferation; mTOR; Skp2
1. Introduction Pulmonary arterial hypertension (PAH) is a common clinical syndrome characterized by sustained elevation of pulmonary vascular resistance and increased pulmonary arterial pressure, leading to right heart failure and ultimately death [1,2]. Pulmonary vascular remodeling characterized by thickening of all layers of vascular wall is a hallmark of all types of PAH [3]. Pulmonary arterial smooth muscle cells (PASMCs) proliferation in situ and migration to intima is critical to the pathogenesis of pulmonary vascular remodeling [4]. The deregulation of multiple signaling pathways in PASMCs is convinced to be associated with excessive proliferation of PASMCs [5,6]. Therefore, strategies either to enhance the function of anti-proliferative cascades or to inhibit pro-proliferative signaling pathways are important in the amelioration of pulmonary vascular remodeling and therefore the management of PAH. Adenosine monophosphate-activated protein kinase (AMPK) is a key metabolic and redox sensor that has emerged as a critical regulator of cell proliferation and apoptosis [7]. AMPK exists as a heterotrimeric complex with a catalytic subunit α and two regulatory subunits β and γ [8]. Int. J. Mol. Sci. 2016, 17, 844; doi:10.3390/ijms17060844
www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2016, 17, 844
2 of 11
Adenosine monophosphate-activated protein kinase (AMPK) is a key metabolic and redox sensor that has emerged as a critical regulator of cell proliferation and apoptosis [7]. AMPK exists as Int. J. Mol. Sci. 2016, 17, 844 2 of 11 a heterotrimeric complex with a catalytic subunit α and two regulatory subunits β and γ [8]. Pathological changes associated with increased AMP to ATP ratio, such as nutrient deprivation, hypoxia, ischemia, heat shock and oxidative stress, AMPK [8,9]. AMPK is also Pathological changes associated with increased AMPcause to ATP ratio,activation such as nutrient deprivation, found toischemia, be activated by several chemical compounds [10,11] and [8,9]. signaling pathways [12] hypoxia, heat shock and oxidative stress, cause AMPK activation AMPK is also found independent of energy imbalance. Activation of AMPK regulates diverse cellular processes to be activated by several chemical compounds [10,11] and signaling pathways [12] independent of including cell migration, proliferation, survival [13]. Studies including have shown AMPK energy imbalance. Activation of AMPKapoptosis regulates and diverse cellular processes cellthat migration, activation suppresses proliferation of [13]. a variety of tumor as that well AMPK as nonmalignant which proliferation, apoptosis and survival Studies have cells shown activation cells, suppresses indicates that activation of AMPK is a promising strategy for the treatment of cancer as well as other proliferation of a variety of tumor cells as well as nonmalignant cells, which indicates that activation human diseases [14–16]. strategy for the treatment of cancer as well as other human diseases [14–16]. of AMPK is a promising Recent studies have employed employed AMPK AMPK activators activators such such as as 5-aminoimidazole-4-carboxamide 5-aminoimidazole-4-carboxamide Recent studies have ribonucleoside (AICAR) and metformin to demonstrate that activation of suppresses AMPK suppresses ribonucleoside (AICAR) and metformin to demonstrate that activation of AMPK PASMCs PASMCs proliferation [17,18]. in animal model have also thatof activation ofmetformin AMPK by proliferation [17,18]. Studies in Studies animal model have also found that found activation AMPK by metformin the development PAH by suppressing vascular remodeling [19]. inhibits the inhibits development of PAH by of suppressing pulmonarypulmonary vascular remodeling [19]. However, However, downstream targets regulated by AMPK to confer its roles in PAH are not fully clarified downstream targets regulated by AMPK to confer its roles in PAH are not fully clarified yet; yet; in addition, pharmacological modulation of AMPK is not entirely specific forand AMPK and has in addition, pharmacological modulation of AMPK is not entirely specific for AMPK has off-target off-target effects [20,21]. RNA interference (RNAi) is a simple and effective method for silencing effects [20,21]. RNA interference (RNAi) is a simple and effective method for silencing gene expression genehas expression and has great advantagesapproaches over traditional approaches examining themolecule role of a and great advantages over traditional for examining thefor role of a specific specific molecule in the development of a particular disease [22]. In the present study, we used in the development of a particular disease [22]. In the present study, we used post-transcriptional post-transcriptional gene silencing with small interfering RNA (siRNA) to selectively inhibit AMPK gene silencing with small interfering RNA (siRNA) to selectively inhibit AMPK in primary cultured in primary PASMCs, and examined the proliferation, effect of AMPK on molecular PASMCs PASMCs, andcultured specifically examined the specifically effect of AMPK on PASMCs and the proliferation, and the molecular mechanisms underlying this effect were further explored. mechanisms underlying this effect were further explored. 2. Results 2. Results 2.1. 2.1. Knockdown of Adenosine Monophosphate-Activated Monophosphate-Activated Protein Protein Kinase Kinase α2 α2 (AMPKα2) (AMPKα2) Promotes Promotes Pulmonary Pulmonary Arterial Smooth Muscle Cells (PASMCs) Proliferation Arterial Smooth Muscle Cells (PASMCs) Proliferation To To specifically specifically examine examine the the effect effect of of AMPK AMPK on on PASMCs PASMCs proliferation, proliferation, siRNA-mediated siRNA-mediated knockdown of either AMPKα1 or AMPKα2 was performed. As depicted knockdown of either AMPKα1 or AMPKα2 was performed. As depicted in in Figure Figure 1A,B, 1A,B, transfection transfection of the corresponding specific siRNA reduced the levels of AMPKα1 and AMPKα2 protein of the corresponding specific siRNA reduced the levels of AMPKα1 and AMPKα2 protein by by 78% 78% and 79%, respectively (p < 0.01 versus control). Non-targeting siRNA transfection did not and 79%, respectively (p < 0.01 versus control). Non-targeting siRNA transfection did not reduce reduce AMPKα1 Figure 1C 1C indicates AMPKα1 and and AMPKα2 AMPKα2 protein protein expression. expression. Figure indicates that that PASMCs PASMCs lacking lacking AMPKα2 AMPKα2 protein exhibited an elevated cells proliferation assessed by BrdU incorppration assay, protein exhibited an elevated cells proliferation assessed by BrdU incorppration assay, which which was was 1.46-fold 1.46-fold increase increase over over control control (p (p
Molecular Sciences Article
Knockdown of AMPKα2 Promotes Pulmonary Arterial Smooth Muscle Cells Proliferation via mTOR/Skp2/p27Kip1 Signaling Pathway Rui Ke 1 , Lu Liu 1 , Yanting Zhu 1 , Shaojun Li 1 , Xinming Xie 1 , Fangwei Li 1 , Yang Song 1 , Lan Yang 1 , Li Gao 2 and Manxiang Li 1, * 1
2
*
Department of Respiratory and Critical Care Medicine, The First Affiliated Hospital of Xi’an Jiaotong University, Xi’an 710061, China; [email protected] (R.K.); [email protected] (L.L.); [email protected] (Y.Z.); [email protected] (S.L.); [email protected] (X.X.); [email protected] (F.L.); [email protected] (Y.S.); [email protected] (L.Y.) Division of Allergy and Clinical Immunology, Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD 21224, USA; [email protected] Correspondence: [email protected]; Tel./Fax: +86-29-8532-3850
Academic Editor: Atsushi Matsuzawa Received: 4 March 2016; Accepted: 24 May 2016; Published: 31 May 2016
Abstract: It has been shown that activation of adenosine monophosphate-activated protein kinase (AMPK) suppresses proliferation of a variety of tumor cells as well as nonmalignant cells. In this study, we used post-transcriptional gene silencing with small interfering RNA (siRNA) to specifically examine the effect of AMPK on pulmonary arterial smooth muscle cells (PASMCs) proliferation and to further elucidate its underlying molecular mechanisms. Our results showed that knockdown of AMPKα2 promoted primary cultured PASMCs proliferation; this was accompanied with the elevation of phosphorylation of mammalian target of rapamycin (mTOR) and S-phase kinase-associated protein 2 (Skp2) protein level and reduction of p27Kip1 . Importantly, prior silencing of mTOR with siRNA abolished AMPKα2 knockdown-induced Skp2 upregulation, p27Kip1 reduction as well as PASMCs proliferation. Furthermore, pre-depletion of Skp2 by siRNA also eliminated p27Kip1 downregulation and PASMCs proliferation caused by AMPKα2 knockdown. Taken together, our study indicates that AMPKα2 isoform plays an important role in regulation of PASMCs proliferation by modulating mTOR/Skp2/p27Kip1 axis, and suggests that activation of AMPKα2 might have potential value in the prevention and treatment of pulmonary arterial hypertension. Keywords: AMPK; pulmonary arterial smooth muscle cells; proliferation; mTOR; Skp2
1. Introduction Pulmonary arterial hypertension (PAH) is a common clinical syndrome characterized by sustained elevation of pulmonary vascular resistance and increased pulmonary arterial pressure, leading to right heart failure and ultimately death [1,2]. Pulmonary vascular remodeling characterized by thickening of all layers of vascular wall is a hallmark of all types of PAH [3]. Pulmonary arterial smooth muscle cells (PASMCs) proliferation in situ and migration to intima is critical to the pathogenesis of pulmonary vascular remodeling [4]. The deregulation of multiple signaling pathways in PASMCs is convinced to be associated with excessive proliferation of PASMCs [5,6]. Therefore, strategies either to enhance the function of anti-proliferative cascades or to inhibit pro-proliferative signaling pathways are important in the amelioration of pulmonary vascular remodeling and therefore the management of PAH. Adenosine monophosphate-activated protein kinase (AMPK) is a key metabolic and redox sensor that has emerged as a critical regulator of cell proliferation and apoptosis [7]. AMPK exists as a heterotrimeric complex with a catalytic subunit α and two regulatory subunits β and γ [8]. Int. J. Mol. Sci. 2016, 17, 844; doi:10.3390/ijms17060844
www.mdpi.com/journal/ijms
Int. J. Mol. Sci. 2016, 17, 844
2 of 11
Adenosine monophosphate-activated protein kinase (AMPK) is a key metabolic and redox sensor that has emerged as a critical regulator of cell proliferation and apoptosis [7]. AMPK exists as Int. J. Mol. Sci. 2016, 17, 844 2 of 11 a heterotrimeric complex with a catalytic subunit α and two regulatory subunits β and γ [8]. Pathological changes associated with increased AMP to ATP ratio, such as nutrient deprivation, hypoxia, ischemia, heat shock and oxidative stress, AMPK [8,9]. AMPK is also Pathological changes associated with increased AMPcause to ATP ratio,activation such as nutrient deprivation, found toischemia, be activated by several chemical compounds [10,11] and [8,9]. signaling pathways [12] hypoxia, heat shock and oxidative stress, cause AMPK activation AMPK is also found independent of energy imbalance. Activation of AMPK regulates diverse cellular processes to be activated by several chemical compounds [10,11] and signaling pathways [12] independent of including cell migration, proliferation, survival [13]. Studies including have shown AMPK energy imbalance. Activation of AMPKapoptosis regulates and diverse cellular processes cellthat migration, activation suppresses proliferation of [13]. a variety of tumor as that well AMPK as nonmalignant which proliferation, apoptosis and survival Studies have cells shown activation cells, suppresses indicates that activation of AMPK is a promising strategy for the treatment of cancer as well as other proliferation of a variety of tumor cells as well as nonmalignant cells, which indicates that activation human diseases [14–16]. strategy for the treatment of cancer as well as other human diseases [14–16]. of AMPK is a promising Recent studies have employed employed AMPK AMPK activators activators such such as as 5-aminoimidazole-4-carboxamide 5-aminoimidazole-4-carboxamide Recent studies have ribonucleoside (AICAR) and metformin to demonstrate that activation of suppresses AMPK suppresses ribonucleoside (AICAR) and metformin to demonstrate that activation of AMPK PASMCs PASMCs proliferation [17,18]. in animal model have also thatof activation ofmetformin AMPK by proliferation [17,18]. Studies in Studies animal model have also found that found activation AMPK by metformin the development PAH by suppressing vascular remodeling [19]. inhibits the inhibits development of PAH by of suppressing pulmonarypulmonary vascular remodeling [19]. However, However, downstream targets regulated by AMPK to confer its roles in PAH are not fully clarified downstream targets regulated by AMPK to confer its roles in PAH are not fully clarified yet; yet; in addition, pharmacological modulation of AMPK is not entirely specific forand AMPK and has in addition, pharmacological modulation of AMPK is not entirely specific for AMPK has off-target off-target effects [20,21]. RNA interference (RNAi) is a simple and effective method for silencing effects [20,21]. RNA interference (RNAi) is a simple and effective method for silencing gene expression genehas expression and has great advantagesapproaches over traditional approaches examining themolecule role of a and great advantages over traditional for examining thefor role of a specific specific molecule in the development of a particular disease [22]. In the present study, we used in the development of a particular disease [22]. In the present study, we used post-transcriptional post-transcriptional gene silencing with small interfering RNA (siRNA) to selectively inhibit AMPK gene silencing with small interfering RNA (siRNA) to selectively inhibit AMPK in primary cultured in primary PASMCs, and examined the proliferation, effect of AMPK on molecular PASMCs PASMCs, andcultured specifically examined the specifically effect of AMPK on PASMCs and the proliferation, and the molecular mechanisms underlying this effect were further explored. mechanisms underlying this effect were further explored. 2. Results 2. Results 2.1. 2.1. Knockdown of Adenosine Monophosphate-Activated Monophosphate-Activated Protein Protein Kinase Kinase α2 α2 (AMPKα2) (AMPKα2) Promotes Promotes Pulmonary Pulmonary Arterial Smooth Muscle Cells (PASMCs) Proliferation Arterial Smooth Muscle Cells (PASMCs) Proliferation To To specifically specifically examine examine the the effect effect of of AMPK AMPK on on PASMCs PASMCs proliferation, proliferation, siRNA-mediated siRNA-mediated knockdown of either AMPKα1 or AMPKα2 was performed. As depicted knockdown of either AMPKα1 or AMPKα2 was performed. As depicted in in Figure Figure 1A,B, 1A,B, transfection transfection of the corresponding specific siRNA reduced the levels of AMPKα1 and AMPKα2 protein of the corresponding specific siRNA reduced the levels of AMPKα1 and AMPKα2 protein by by 78% 78% and 79%, respectively (p < 0.01 versus control). Non-targeting siRNA transfection did not and 79%, respectively (p < 0.01 versus control). Non-targeting siRNA transfection did not reduce reduce AMPKα1 Figure 1C 1C indicates AMPKα1 and and AMPKα2 AMPKα2 protein protein expression. expression. Figure indicates that that PASMCs PASMCs lacking lacking AMPKα2 AMPKα2 protein exhibited an elevated cells proliferation assessed by BrdU incorppration assay, protein exhibited an elevated cells proliferation assessed by BrdU incorppration assay, which which was was 1.46-fold 1.46-fold increase increase over over control control (p (p
