International Journal of Cardiology 175 (2014) 515–521
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RORα suppresses proliferation of vascular smooth muscle cells through activation of AMP-activated protein kinase Eun-Jin Kim a,b, Young-Keun Choi c, Yong-Hyun Han a,b, Hyeon-Ji Kim a,b, In-Kyu Lee c, Mi-Ock Lee a,b,⁎ a b c
College of Pharmacy, Seoul National University, Seoul, South Korea Bio-MAX Institute, Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul, South Korea World Class University Program, Research Institute of Aging and Metabolism, Kyungpook National University School of Medicine, Daegu, South Korea
a r t i c l e
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Article history: Received 29 January 2014 Received in revised form 5 May 2014 Accepted 24 June 2014 Available online 2 July 2014 Keywords: AMPK Atherosclerosis RORα vSMC proliferation
a b s t r a c t Background: Retinoic acid-related orphan receptor α (RORα) has been implicated in the progression of atherosclerosis, but its role in the proliferation of vascular smooth muscle cells (vSMCs) has not been fully examined. We previously reported that RORα activates AMP-activated protein kinase (AMPK), which is associated with the suppression of vSMC proliferation. Therefore, we investigated the suppressive function of RORα on the proliferation of vSMCs and the molecular mechanisms involved. Results: First, RORα and its activator, cholesterol sulfate (CS), induced the activation of AMPK in both human aortic SMCs and rat A7r5 cells, which was accompanied by the suppression of mammalian target of rapamycin (mTOR) and p70 ribosomal protein S6 kinase 1. Second, RORα and CS modulated the expression of cell-cycleregulating factors, such as p53, p27, and cyclin D in vSMCs. Consistent with this, the overexpression of RORα or CS treatment suppressed the proliferation of human aortic SMCs and rat A7r5 cells, possibly through G1 arrest. RORα and CS also inhibited the migration of A7r5 cells in two-dimensional and three-dimensional cell migration assays. Finally, we demonstrated that the infusion of adenovirus encoding RORα into arteries suppressed neointima formation after balloon injury in rats. Conclusion: These results demonstrate that RORα inhibits vSMC proliferation through AMPK-induced mTOR suppression, and suggest that RORα is a therapeutic target for the cardiovascular diseases associated with vSMC proliferation. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Atherosclerosis is a chronic immunoinflammatory and fibroproliferative disease of the arteries. A key process of atherosclerosis involves the proliferation of vascular smooth muscle cells (vSMCs), which is linked to other cellular processes such as inflammation, lipoprotein retention, and matrix alterations [1]. The proliferation of vSMCs is also a critical event in the development of in-stent restenosis, transplant vasculopathy, and vein bypass graft failure [2]. Therefore, the identification of factors that control the proliferation of vSMCs has been a major focus of atherosclerosis research, especially in the development of new therapeutic strategies. Recently, AMP activated protein kinase (AMPK), a metabolite-sensing protein kinase that acts as a cellular fuel sensor, has emerged as a therapeutic target for atherosclerosis, as well as for restenosis [3]. AMPK is considered a key molecule linking energy metabolism and atherosclerotic changes in the vascular system. 5-Aminoimidazole-4carboxamide-1-β-D-ribofuranoside (AICAR), an activator of AMPK, ⁎ Corresponding author at: College of Pharmacy, Seoul National University, Seoul 151742, South Korea. Tel.: +82 2 880 9221; fax: +82 2 880 9122. E-mail address: [email protected] (M.-O. Lee).
http://dx.doi.org/10.1016/j.ijcard.2014.06.043 0167-5273/© 2014 Elsevier Ireland Ltd. All rights reserved.
suppresses human aortic SMC proliferation through the activation of the p53–p21 axis and subsequent cell-cycle arrest in G1/S phase [4]. In particular, the deletion of AMPKα2 reduces the level of p27, probably through S-phase kinase-associated protein 2-mediated degradation, and enhances neointimal hyperplasia in response to wire injury [5]. AMPK regulates the protein synthetic pathway essential for cell growth; AICAR inhibits the phosphorylation of mTOR and reduces protein synthesis through the action of p70S6K and eukaryotic translation initiation factor 4E-binding protein 1 [6]. The AMPK signaling network that inhibits the proliferation of vSMCs is composed of a number of tumor suppressor proteins, including retinoblastoma (RB), liver kinase B1 (LKB1), tuberous sclerosis 1 (TSC1), and TSC2 [3]. In addition to AICAR, several chemical activators of AMPK exhibit strong inhibitory effects on the proliferation of vSMCs, further supporting the role of AMPK in the regulation of vSMC growth. β-Lapachone, a potent antitumor agent that stimulates NAD(P)H:quinone oxidoreductase 1, inhibits the proliferation of vSMCs in vitro and in vivo via the LKB1-dependent activation of AMPK [7]. A natural product, berberine, inhibits the platelet-derived growth-factor-induced growth of vSMCs, partly through the activation of the AMPK–p53–p21 signaling axis with the inactivation of the cyclin D/cyclin-dependent kinases (CDKs) [8]. Natural and synthetic polyphenols that stimulate AMPK have shown protective and antiatherogenic
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properties in aortic atherosclerotic lesions in type 1 diabetic lowdensity lipoprotein (LDL)-receptor-deficient mice [9]. Retinoic acid receptor-related orphan receptor α (RORα1; NR1F1) is a member of the steroid/thyroid hormone receptor superfamily of transcription factors [10]. Cholesterol derivatives, including cholesterol sulfate (CS) fit in the ligand-binding pocket of RORα and act as endogenous ligands [11,12]. RORα plays an important role in the regulation of the metabolic pathways of lipids and atherogenesis [13]. Staggerer mice, which carry a deletion in the carboxy terminus of RORα, develop severe atherosclerosis [14]. RORα expression levels are significantly reduced in the atherosclerotic plaques of patients [15]. Recently, we and others reported that RORα signaling is associated with the regulation of the AMPK signaling pathway, which is associated with the expression of v-akt murine thymoma viral oncogene homolog 2 (AKT2) or LKB1 [16,17]. Because AMPK is one of the major signaling pathways controlling the growth of vSMCs in response to nutrient status, we asked whether RORα suppresses the proliferation of vSMCs in this study. Here, we report that RORα induced the activation of AMPK in vSMCs and the suppression of vSMC growth, thereby ultimately protecting against restenosis after balloon injury in rats.
by intraperitoneal injection of sodium pentobarbital (50 mg/kg), and a balloon catheter was introduced through the right external carotid artery into the aortic arch. The carotid artery was damaged by passing the inflated balloon catheter back and forth through the lumen. After the balloon injury, Ad-GFP or Ad-RORα1 (1 × 1010 plaque-forming units/mL) was infused into the ligated segment of the common carotid artery for 30 min. At 14 days after surgery, the right and left common carotid arteries were harvested, fixed overnight in 4% formaldehyde in phosphate-buffered saline, and paraffin embedded. The euthanasia was performed by exsanguination with anesthesia induced by intraperitoneal injection of urethane (1.25 g/kg). Serial cross-sections (4 μm thick) of the femoral arteries were stained with hematoxylin and eosin (H&E) and used for histological analysis. For morphometric analysis, neointimal area was analyzed using iSolution DT Ver 7.7 imaging software (IMT i-Solution Inc. Inc., Coquitlam, Canada). Neointimal proliferation was estimated by intima/media ratio which was measured as follows: Intimal area = internal elastic lamina − end of neointima; Medial area = external elastic lamina − internal elastic lamina. Viral transduction was evaluated by examining cryosections for GFP expression using immunohistochemistry with anti-GFP antibodies (Abcam, Cambridge, UK). Expression of GFP, Ki67, RORα, p27, and pAMPK was analyzed by immunohistochemistry, as previously described [21].
2.5. Statistics All values were expressed as means ± SEM. Statistical analysis was performed using nonparametric Mann–Whitney U test (Fig. 5) or two-way ANOVA followed by Bonferroni posttest (Fig. 4). P b 0.05 was considered as statistically different.
2. Materials and methods 2.1. Cells culture and materials A7r5 cells were obtained from the American Type Culture Collection (Rockville, MD) and cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS). Human aortic SMCs (hvSMCs) were purchased from Lonza (Basel, Switzerland) and cultured in smooth muscle basal medium (Lonza) supplemented with 1% human epidermal growth factor, 1% insulin, 0.2% human fibroblast growth factor B, and 5% FBS. When treated with CS (Sigma-Aldrich, St. Louis, MO), the cells were cultured in medium supplemented with charcoal-stripped FBS. AICAR, a nucleoside converted within the cell to an AMP mimetic, and compound C (6-[4-(2-Piperidin-1-yl-ethoxy)phenyl)]-3-pyridin-4-yl-pyrrazolo[1,5-a]-pyrimidine), an ATP-competitive inhibitor of AMPK, were purchased from Toronto Research Chemicals Inc. (Toronto, Canada) and Sigma-Aldrich (St. Louis, MO), respectively [18]. 2.2. Plasmids, recombinant adenovirus, small interfering RNAs (si-RNAs), transient transfection, western blotting, and immunoprecipitation Eukaryotic expression vectors including the FLAG-tagged full-length RORα1 and their transient transfection have been described previously [16]. Recombinant adenoviruses encoding RORα1 and RORα4, pAdTrack-CMV and pShuttle-IRES-hrGFP-2, respectively, were previously reported [16,19]. The target sequences for si-RORα were 5′-TACGTGTGAAGGCTGCAAGGGC-3′. The si-RNA duplex targeting AMPKα1 and LKB1 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Western blotting and immunoprecipitation were basically performed using specific antibodies including anti-AMPKα antibody which detects both α1 and α2 (Cell Signaling, Danvers, MA), as previously described [16]. 2.3. Cell proliferation, cell cycle, and migration assay Cell proliferation was estimated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. For cell cycle analysis, A7r5 cells were seeded and cultured overnight. At 50% to 60% confluency of plates, serum was removed for starvation for 24 h, and then the cells were infected with Ad-GFP or Ad-RORα (MOI = 10) or treated with CS in complete media for 24 h. Cell-cycle phase was analyzed by flow cytometry (Becton Dickinson, Franklin Lakes, NJ). Two-dimensional cell migration was analyzed with the scratch wound assay. A7r5 cells were seeded in culture-insert dishes (ibidi, Martinsried, Germany) and incubated overnight. After the cells had attached, the culture insert was removed and the adenoviruses were added for infection for 24 h. Cell mobility was estimated from the wound size that was analyzed by ImageJ software (http://rsb.info.nih. gov). Three-dimensional cell migration was examined using a modified Boyden Transwell® culture chamber with a gelatin-coated polycarbonate membrane (Corning Life Sciences, Tewksbury, MA). Virus-infected or CS-treated A7r5 cells were seeded in the upper chamber and incubated for 8 h. The migrated cells to the membranes were counted under microscope. 2.4. Rat carotid artery balloon injury and histological analysis The animal care was performed according to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. All experiments were conducted by the guidelines of Seoul National University Institutional Animal Care and Use Committee. Carotid artery balloon injury was performed in seven-week-old male Sprague–Dawley rats, as described previously [20]. Briefly, the animals were anesthetized
3. Results 3.1. RORα induces the activation of the AMPK–mTOR–S6K1 signaling pathway Previously, we demonstrated that RORα activates AMPK and enhances fatty acid β-oxidation, resulting in the attenuation of hepatic steatosis [16]. Because AMPK is one of the major signaling molecules controlling the growth of vSMCs, we asked whether RORα is associated with the proliferation of vSMCs. First, we examined whether RORα induces the activation of AMPK in human primary vSMCs and in rat A7r5 cells. Of four different isoforms of RORα, RORα1 and RORα4 were expressed in both vSMCs and A7r5 cells, similarly to the previous observation (Fig. 1A) [15]. When either RORα1 or RORα4 was exogenously introduced by adenoviral transduction, the phosphorylation at Thr172 of AMPK increased. This increase was ascertained by the enhancement of the phosphorylation at Ser79 of acetyl-CoA carboxylase 1, a downstream target of activated AMPK (Fig. 1A). Because mTOR signaling is suppressed by AMPK in vSMCs, we examined whether the activity of mTOR was altered by RORα [3]. The phosphorylation of mTOR, but not its expression, was greatly reduced after the cells were infected with Ad-RORα1 or Ad-RORα4 (Fig. 1B). The phosphorylation of 70-kDa ribosomal protein S6 kinase 1 (p70S6K1 or S6K1), the most characterized downstream effector of mTOR, was also reduced (Fig. 1B). Treatment with putative ligands of RORα, such as melatonin, 22(R)-hydroxycholesterol, CS, and 7-dehydrocholesterol, increased the levels of pAMPK (Supplementary Fig. IA). CS induced the activation of AMPK at concentrations as low as 0.1 μM in a time-dependent manner (Fig. 1C and Supplementary Fig. IB). CS- or melatonin-elevated pAMPK was abolished after knockdown of RORα using si-RNA, indicating that the effect of these ligands was mediated by RORα (Fig. 1D and Supplementary Fig. IC). The CS-mediated reductions in pmTOR and p70S6K1 were reversed by treatment with compound C, an inhibitor of AMPK, or silencing of AMPK by si-RNA, demonstrating the involvement of AMPK (Fig. 1E and F). The overexpression of RORα or treatment with CS increased the amount of pLKB1, an upstream kinase of AMPK, indicating that the RORα-induced activation of AMPK may be mediated by the activation of LKB1 (Fig. 2A and B). Indeed, silencing of LKB1 repressed the CS-induced AMPK activation (Fig. 2C). Infection of human vSMCs with the Ad-RORα virus or treatment with CS led to a significant decrease in ATP levels, which may cause the elevation of pLKB1 and the subsequent activation of AMPK after activation of RORα (Supplementary Fig. II).
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Fig. 1. RORα induces activation of AMPK-mTOR signaling pathway in vSMCs. (A and B) Human vSMCs or A7r5 cells were infected by Ad-GFP, Ad-RORα1, or Ad-RORα4 for 24 h. (C) A7r5 cells were treated with the indicated concentrations of CS for 24 h. (D) A7r5 cells were transfected with 50 nmol/L non-specific si-RNA (NS) or si-RORα. After 48 h of transfection, the cells were treated with 20 μmol/L CS for 24 h. (E) A7r5 cells were treated with vehicle or 20 μmol/L CS. After 24 h of treatment, the cells were treated with or without 10 μmol/L compound C (Comp C) for 4 h. (F) A7r5 cells were transfected with 50 nmol/L non-specific si-RNA (NS) or si-AMPK. After 48 h of transfection, the cells were treated with 20 μmol/L CS for 24 h. The expression of protein was analyzed by western blot analysis. One representative of at least three independent experiments with similar results is shown.
3.2. RORα modulates the expression of cell cycle control factors and induces cell cycle arrest
Fig. 2. RORα-induced AMPK activation is LKB1-dependent. (A) A7r5 cells were infected by Ad-GFP, Ad-RORα1, or Ad-RORα4 for 24 h. (B) A7r5 cells were treated with the indicated concentrations of CS for 24 h. (C) A7r5 cells were transfected with 50 nmol/L non-specific si-RNA (NS) or si-LKB1. After 48 h of transfection, the cells were treated with 20 μmol/L CS for 24 h. One representative of at least three independent experiments with similar results is shown.
Recently, it was reported that AMPK activation involves the accumulation and activation of tumor suppressor p53 and CDK inhibitors, such as p21 and p27, in vSMCs [3,4]. The expression of the cell-cycle control factors such as p53, p27, and p21 increased dramatically in vSMCs after the adenoviral transduction of RORα1 or RORα4, whereas the expression of cyclin D decreased (Fig. 3A). Treatment with RORα ligands increased the expression of p53 and p27, whereas they reduced the levels of pRB (Supplementary Fig. III). The effect of CS was time dependent; it was seen as early as 1 h after treatment and continued for at least 24 h (Fig. 3B). The CS-induced alterations were abolished by siRORα, si-AMPK, or treatment with compound C, indicating that the effects of CS are RORα-mediated and AMPK-dependent (Fig. 3C–E). When hvSMCs were infected with Ad-RORα1 or Ad-RORα4, the number of cells decreased as the incubation time increased. CS also suppressed the proliferation of hvSMCs by about 40% after six days of treatment (Fig. 4A). A similar pattern of decline was observed in the A7r5 cells. A cell cycle analysis showed that infection with Ad-RORα1 or Ad-RORα4, or CS treatment increased G1 phase in A7r5 cells, indicating that RORα mediates G1 arrest (Fig. 4B). Because the migration of vSMCs plays an important role in the formation of atherosclerotic plaques, we analyzed whether RORα affects migration capacity of vSMCs. The migration of A7r5 cells infected with Ad-RORα1 into scratched areas did not differ markedly from that of control cells. However, the migration of cells that were stimulated with tumor necrosis factor α (TNFα) was clearly inhibited after Ad-RORα1 infection (Fig. 4C). To examine whether the wound is closed due to increased cell motility, we performed migration assay using Boyden chamber. Infection with AdRORα or treatment with CS greatly inhibited the movement of TNFαstimulated cells across the gelatin layer in the three-dimensional cell migration assay (Fig. 4D).
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Fig. 3. RORα modulates expression of p53, CDK inhibitors, and cyclin D. (A) Human vSMCs or A7r5 cells were infected by Ad-GFP, Ad-RORα1, or Ad-RORα4 for 24 h. (B) A7r5 cells were treated with 20 μmol/L CS during the indicated time. (C and D) A7r5 cells were transfected with 50 nmol/L non-specific si-RNA (NS), si-RORα (C), or si-AMPK (D). After 48 h of transfection, the cells were treated with 20 μmol/L CS for 24 h. (E) A7r5 cells were treated with 20 μmol/L CS. After 24 h of treatment, the cells were treated or without 10 μmol/L compound C (Comp C) for 4 h. The expression of protein was analyzed by western blot analysis. One representative of at least three independent experiments with similar results is shown.
3.3. RORα suppresses neointima formation in the rat carotid artery after balloon injury Finally, we examined the effect of RORα on neointima formation in the rat carotid artery in vivo. When a balloon-induced injury was created in the carotid artery, neointimal hyperplasia was induced. However, the infusion of Ad-RORα1 into the artery significantly reduced the intima:media ratio in the injured region (Fig. 5A). The expression of Ki67, a marker of cell proliferation, was reduced significantly in the αSMA stained neointimal region; however, apoptotic cell death was not increased in the Ad-RORα1 infused arteries when examined by TUNEL assay (Fig. 5B and Supplementary Fig. IV) [22]. The expression of pAMPK and p27 was increased significantly in the neointimal region after infection with Ad-RORα1, further supporting the inhibitory function of RORα in the proliferation of vSMCs (Fig. 5C).
4. Discussion In this study, we demonstrated for the first time that RORα suppresses neointima formation through the activation of AMPK-induced pathways including inhibition of protein synthesis and regulation of cell cycle in vSMCs (Fig. 5D). We found that RORα decreased the intracellular ATP level in hvSMCs, which may explain how this receptor activates LKB1-AMPK pathway (Supplementary Fig. II). Activation of AMPK induced phosphorylation of RORα backward, which leads transcriptional activation of this receptor as shown in our previous report (Supplementary Fig. V) [11]. Indeed AMPK increased the expression level as well as transcriptional activity of RORα in vSMC (Supplementary Fig. VI). Together these data suggest a positive activating circuit between AMPK and RORα, that plays an important role in the RORαmediated activation of AMPK for the regulation of vSMC proliferation.
However, currently it is not clear how RORα increases the intracellular AMP:ATP ratio. Whether RORα increases ATP consumption or decreases ATP production in conjunction with mitochondrial functions will be an interesting question to be addressed in future. Downstream target genes of RORα might be involved in the balancing intracellular energy status that affects cellular proliferation of vSMCs. Identification of such RORα downstream genes would help further understanding of role of the positive feedforward activation of RORα and AMPK in neointima formation. Interestingly, RORα suppressed the TNFα-induced proliferation of vSMC (Fig. 4C), which largely involves Rb-E2F transcriptional regulatory pathway [23,24]. As shown in Supplementary Fig. III, activators of RORα such as melatonin and CS decreased the level of pRB, indicating that RORα may inhibit the TNFα-induced vSMC proliferation through blocking of phosphorylation of RB. Involvement of AMPK and its contribution to the RORα-induced repression of pRB in vSMC needs to be investigated in future. The suppressive function of AMPK on cellular proliferation also contributes to the inhibition of tumor cell growth [3]. Therefore, RORαinduced AMPK activation may be associated with the development and progression of human cancers. Indeed, it has been reported that RORα is downregulated at the mRNA level in certain types of cancerous tissues and tumor cell lines [25]. Interestingly, RORα is phosphorylated at Ser35 by protein kinase Cα, which inhibits canonical Wnt5a/PKCα signaling, contributing to the development of colon cancer [26]. RORα is also induced in a p53-dependent manner in response to DNA damage and activates p53, leading to the activation of the specific genes involved in apoptosis [27]. RORα protein levels are markedly reduced in breast cancer, which might be attributable to its methylationdependent degradation caused by the elevated EZH2 protein levels in these cancer tissues [28]. In contrast, a recent report demonstrated a role for RORα in cellular responses to genotoxicity in the lung, which
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Fig. 4. RORα inhibits proliferation of vSMC and induces cell cycle arrest. (A) Human vSMCs or A7r5 cells were infected by Ad-GFP, Ad-RORα1, or Ad-RORα4. Or the cells were treated with 20 μmol/L CS. After treatment, cell proliferation was assessed by MTT assay. 100% represents the OD570 nm of Ad-GFP infected or vehicle treated wells at the indicated time. The numbers represent mean ± SEM (n = 3). *P b 0.05, **P b 0.01, and ***P b 0.001 vs Ad-GFP or vehicle at each time; ###P b 0.001 vs Ad-GFP or vehicle group. (B) A7r5 cells were infected by Ad-GFP, Ad-RORα1, or Ad-RORα4. Or the cells were treated with 20 μmol/L CS. After treatment, the cells were stained with propidium iodide and analyzed by flow cytometry. Representatives of three independent experiments with similar results are shown. (C) A7r5 cells were infected by Ad-GFP or Ad-RORα1, and then treated with or without 10 ng/mL TNFα for indicated time periods. The migrated cells were imaged under a light microscope (left). Cell mobility was estimated from the wound size that is indicated by dotted lines and analyzed by ImageJ software. The graph shows the relative cell mobility. Ad-GFP without TNFα group at 24 h is defined as 1. #P b 0.05 vs Ad-GFP with vehicle; *P b 0.05 vs Ad-GFP with TNFα (n = 3) (right). (D) A7r5 cells were infected by Ad-GFP, Ad-RORα1, or Ad-RORα4. Or the cells were treated with 20 μmol/L CS. Then the cells were added to the upper chamber, while 10 ng/mL TNFα or vehicle was placed in the lower chamber. The migrated cells were stained with hematoxylin and imaged under a light microscope (top). Black bars represent 100 μm. The numbers of migrated cells in the field were counted. ##P b 0.01 and ###P b 0.001 vs Ad-GFP with vehicle; *P b 0.05 and **P b 0.01 vs Ad-GFP with TNFα (n = 3) (bottom).
may promote pulmonary emphysema [29]. Therefore, in future studies, attention should be directed to the roles of RORα in the pathophysiology of diverse chronic metabolic diseases, including cancer. Various nuclear receptors, including the peroxisome proliferatoractivated receptor (PPAR) and NUR77, play important roles in the regulation of neointima formation and plaque growth in atherosclerotic lesions. Troglitazone and rosiglitazone, high affinity ligands of PPARγ, suppress vSMC proliferation and prevent rat aortic neointima formation after balloon injury through a PPARγ-mediated pathway [30,31]. Moreover, PPARα inhibits vSMC proliferation through the induction of p16, a CDK inhibitor that blocks G1/S cell-cycle progression [32]. Inducers of Nur77, 6-mercaptopurine and α-lipoic acid, protect against excessive vSMC proliferation and neointima formation [20,33]. Interestingly, Nur77 inhibits AMPK activation via its interaction with LKB1 [34]. Because a complicated network of nuclear receptors is involved in controlling the proliferation of vSMCs and lipid metabolism in the development and progression of atherosclerosis, specific ligands of these receptors
are promising potential drug candidates for atherosclerosis. Recently, we and others developed a synthetic RORα-activating ligands that activates RORα-driven transcription [16,35]. Further researches into the role of these RORα ligands in atherosclerotic lesion formation could extend our understanding of the biological functions of RORα in neointimal hyperplasia and suggest therapeutic strategies against atherosclerosis. In conclusion, this is the first study to show that RORα effectively suppressed cell proliferation and cell cycle progression of vSMC through activation of AMPK. Our conclusion suggests that RORα could be a valuable target for the treatment of atherosclerosis and its ligands could provide a potential remedy against vascular proliferative disorders.
Conflict of interest None declared.
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Fig. 5. RORα suppresses neointima formation in rat carotid artery after balloon injury. (A) Representative photomicrographs of cross-sections of the left common carotid arteries obtained from the balloon injured rats with infusion of either Ad-GFP or Ad-RORα1. Black bars represent 200 μm (left). Neointima formation was analyzed by the ratio of intima/media that calculated by computer-based morphometric analyzer. Values represent means ± SEM (n = 5). *P b 0.05 vs Ad-GFP (right). (B) Immunohistochemistry was carried out using antibodies against Ki67, GFP, or αSMA. Black bars represent 20 μm (left). The Ki67 positive cells in the section were counted under a microscope and represented as % of total cells. αSMA was stained as a marker of vSMC in the injured arteries. Values represent means ± SEM (n = 6 for Ad-GFP and n = 5 for Ad-RORα1). *P b 0.05 vs Ad-GFP (right). Black bars represent 20 μm. (C) Immunohistochemistry was carried out using antibodies against pAMPK, p27, or RORα. Representative photomicrographs from five samples from each group were shown (left). Values represent means ± SEM (n = 5). *P b 0.05 vs Ad-GFP (right). (D) Schematic model for mechanism of the RORα-induced suppression of vSMC proliferation.
Acknowledgment This work was supported by grants from the NRF (2012M3A9B6055338) and the ERC programs (R11-2007-107-01001-0). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.ijcard.2014.06.043. References [1] Dzau VJ, Braun-Dullaeus RC, Sedding DG. Vascular proliferation and atherosclerosis: new perspectives and therapeutic strategies. Nat Med 2002;8:1249–56. [2] Owens GK, Kumar MS, Wamhoff BR. Molecular regulation of vascular smooth muscle cell differentiation in development and disease. Physiol Rev 2004;84:s767–801. [3] Motoshima H, Goldstein BJ, Igata M, Araki E. AMPK and cell proliferation-AMPK as a therapeutic target for atherosclerosis and cancer. J Physiol 2006;574:63–71. [4] Igata M, Motoshima H, Tsuruzoe K, et al. Adenosine monophosphate-activated protein kinase suppresses vascular smooth muscle cell proliferation through the inhibition of cell cycle progression. Circ Res 2005;97:837–44. [5] Song P, Wang S, He C, et al. AMPKα2 deletion exacerbates neointima formation by upregulating Skp2 in vascular smooth muscle cells. Circ Res 2011;109:1230–9.
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Contents lists available at ScienceDirect
International Journal of Cardiology journal homepage: www.elsevier.com/locate/ijcard
RORα suppresses proliferation of vascular smooth muscle cells through activation of AMP-activated protein kinase Eun-Jin Kim a,b, Young-Keun Choi c, Yong-Hyun Han a,b, Hyeon-Ji Kim a,b, In-Kyu Lee c, Mi-Ock Lee a,b,⁎ a b c
College of Pharmacy, Seoul National University, Seoul, South Korea Bio-MAX Institute, Research Institute of Pharmaceutical Sciences, Seoul National University, Seoul, South Korea World Class University Program, Research Institute of Aging and Metabolism, Kyungpook National University School of Medicine, Daegu, South Korea
a r t i c l e
i n f o
Article history: Received 29 January 2014 Received in revised form 5 May 2014 Accepted 24 June 2014 Available online 2 July 2014 Keywords: AMPK Atherosclerosis RORα vSMC proliferation
a b s t r a c t Background: Retinoic acid-related orphan receptor α (RORα) has been implicated in the progression of atherosclerosis, but its role in the proliferation of vascular smooth muscle cells (vSMCs) has not been fully examined. We previously reported that RORα activates AMP-activated protein kinase (AMPK), which is associated with the suppression of vSMC proliferation. Therefore, we investigated the suppressive function of RORα on the proliferation of vSMCs and the molecular mechanisms involved. Results: First, RORα and its activator, cholesterol sulfate (CS), induced the activation of AMPK in both human aortic SMCs and rat A7r5 cells, which was accompanied by the suppression of mammalian target of rapamycin (mTOR) and p70 ribosomal protein S6 kinase 1. Second, RORα and CS modulated the expression of cell-cycleregulating factors, such as p53, p27, and cyclin D in vSMCs. Consistent with this, the overexpression of RORα or CS treatment suppressed the proliferation of human aortic SMCs and rat A7r5 cells, possibly through G1 arrest. RORα and CS also inhibited the migration of A7r5 cells in two-dimensional and three-dimensional cell migration assays. Finally, we demonstrated that the infusion of adenovirus encoding RORα into arteries suppressed neointima formation after balloon injury in rats. Conclusion: These results demonstrate that RORα inhibits vSMC proliferation through AMPK-induced mTOR suppression, and suggest that RORα is a therapeutic target for the cardiovascular diseases associated with vSMC proliferation. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Atherosclerosis is a chronic immunoinflammatory and fibroproliferative disease of the arteries. A key process of atherosclerosis involves the proliferation of vascular smooth muscle cells (vSMCs), which is linked to other cellular processes such as inflammation, lipoprotein retention, and matrix alterations [1]. The proliferation of vSMCs is also a critical event in the development of in-stent restenosis, transplant vasculopathy, and vein bypass graft failure [2]. Therefore, the identification of factors that control the proliferation of vSMCs has been a major focus of atherosclerosis research, especially in the development of new therapeutic strategies. Recently, AMP activated protein kinase (AMPK), a metabolite-sensing protein kinase that acts as a cellular fuel sensor, has emerged as a therapeutic target for atherosclerosis, as well as for restenosis [3]. AMPK is considered a key molecule linking energy metabolism and atherosclerotic changes in the vascular system. 5-Aminoimidazole-4carboxamide-1-β-D-ribofuranoside (AICAR), an activator of AMPK, ⁎ Corresponding author at: College of Pharmacy, Seoul National University, Seoul 151742, South Korea. Tel.: +82 2 880 9221; fax: +82 2 880 9122. E-mail address: [email protected] (M.-O. Lee).
http://dx.doi.org/10.1016/j.ijcard.2014.06.043 0167-5273/© 2014 Elsevier Ireland Ltd. All rights reserved.
suppresses human aortic SMC proliferation through the activation of the p53–p21 axis and subsequent cell-cycle arrest in G1/S phase [4]. In particular, the deletion of AMPKα2 reduces the level of p27, probably through S-phase kinase-associated protein 2-mediated degradation, and enhances neointimal hyperplasia in response to wire injury [5]. AMPK regulates the protein synthetic pathway essential for cell growth; AICAR inhibits the phosphorylation of mTOR and reduces protein synthesis through the action of p70S6K and eukaryotic translation initiation factor 4E-binding protein 1 [6]. The AMPK signaling network that inhibits the proliferation of vSMCs is composed of a number of tumor suppressor proteins, including retinoblastoma (RB), liver kinase B1 (LKB1), tuberous sclerosis 1 (TSC1), and TSC2 [3]. In addition to AICAR, several chemical activators of AMPK exhibit strong inhibitory effects on the proliferation of vSMCs, further supporting the role of AMPK in the regulation of vSMC growth. β-Lapachone, a potent antitumor agent that stimulates NAD(P)H:quinone oxidoreductase 1, inhibits the proliferation of vSMCs in vitro and in vivo via the LKB1-dependent activation of AMPK [7]. A natural product, berberine, inhibits the platelet-derived growth-factor-induced growth of vSMCs, partly through the activation of the AMPK–p53–p21 signaling axis with the inactivation of the cyclin D/cyclin-dependent kinases (CDKs) [8]. Natural and synthetic polyphenols that stimulate AMPK have shown protective and antiatherogenic
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properties in aortic atherosclerotic lesions in type 1 diabetic lowdensity lipoprotein (LDL)-receptor-deficient mice [9]. Retinoic acid receptor-related orphan receptor α (RORα1; NR1F1) is a member of the steroid/thyroid hormone receptor superfamily of transcription factors [10]. Cholesterol derivatives, including cholesterol sulfate (CS) fit in the ligand-binding pocket of RORα and act as endogenous ligands [11,12]. RORα plays an important role in the regulation of the metabolic pathways of lipids and atherogenesis [13]. Staggerer mice, which carry a deletion in the carboxy terminus of RORα, develop severe atherosclerosis [14]. RORα expression levels are significantly reduced in the atherosclerotic plaques of patients [15]. Recently, we and others reported that RORα signaling is associated with the regulation of the AMPK signaling pathway, which is associated with the expression of v-akt murine thymoma viral oncogene homolog 2 (AKT2) or LKB1 [16,17]. Because AMPK is one of the major signaling pathways controlling the growth of vSMCs in response to nutrient status, we asked whether RORα suppresses the proliferation of vSMCs in this study. Here, we report that RORα induced the activation of AMPK in vSMCs and the suppression of vSMC growth, thereby ultimately protecting against restenosis after balloon injury in rats.
by intraperitoneal injection of sodium pentobarbital (50 mg/kg), and a balloon catheter was introduced through the right external carotid artery into the aortic arch. The carotid artery was damaged by passing the inflated balloon catheter back and forth through the lumen. After the balloon injury, Ad-GFP or Ad-RORα1 (1 × 1010 plaque-forming units/mL) was infused into the ligated segment of the common carotid artery for 30 min. At 14 days after surgery, the right and left common carotid arteries were harvested, fixed overnight in 4% formaldehyde in phosphate-buffered saline, and paraffin embedded. The euthanasia was performed by exsanguination with anesthesia induced by intraperitoneal injection of urethane (1.25 g/kg). Serial cross-sections (4 μm thick) of the femoral arteries were stained with hematoxylin and eosin (H&E) and used for histological analysis. For morphometric analysis, neointimal area was analyzed using iSolution DT Ver 7.7 imaging software (IMT i-Solution Inc. Inc., Coquitlam, Canada). Neointimal proliferation was estimated by intima/media ratio which was measured as follows: Intimal area = internal elastic lamina − end of neointima; Medial area = external elastic lamina − internal elastic lamina. Viral transduction was evaluated by examining cryosections for GFP expression using immunohistochemistry with anti-GFP antibodies (Abcam, Cambridge, UK). Expression of GFP, Ki67, RORα, p27, and pAMPK was analyzed by immunohistochemistry, as previously described [21].
2.5. Statistics All values were expressed as means ± SEM. Statistical analysis was performed using nonparametric Mann–Whitney U test (Fig. 5) or two-way ANOVA followed by Bonferroni posttest (Fig. 4). P b 0.05 was considered as statistically different.
2. Materials and methods 2.1. Cells culture and materials A7r5 cells were obtained from the American Type Culture Collection (Rockville, MD) and cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (FBS). Human aortic SMCs (hvSMCs) were purchased from Lonza (Basel, Switzerland) and cultured in smooth muscle basal medium (Lonza) supplemented with 1% human epidermal growth factor, 1% insulin, 0.2% human fibroblast growth factor B, and 5% FBS. When treated with CS (Sigma-Aldrich, St. Louis, MO), the cells were cultured in medium supplemented with charcoal-stripped FBS. AICAR, a nucleoside converted within the cell to an AMP mimetic, and compound C (6-[4-(2-Piperidin-1-yl-ethoxy)phenyl)]-3-pyridin-4-yl-pyrrazolo[1,5-a]-pyrimidine), an ATP-competitive inhibitor of AMPK, were purchased from Toronto Research Chemicals Inc. (Toronto, Canada) and Sigma-Aldrich (St. Louis, MO), respectively [18]. 2.2. Plasmids, recombinant adenovirus, small interfering RNAs (si-RNAs), transient transfection, western blotting, and immunoprecipitation Eukaryotic expression vectors including the FLAG-tagged full-length RORα1 and their transient transfection have been described previously [16]. Recombinant adenoviruses encoding RORα1 and RORα4, pAdTrack-CMV and pShuttle-IRES-hrGFP-2, respectively, were previously reported [16,19]. The target sequences for si-RORα were 5′-TACGTGTGAAGGCTGCAAGGGC-3′. The si-RNA duplex targeting AMPKα1 and LKB1 was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Western blotting and immunoprecipitation were basically performed using specific antibodies including anti-AMPKα antibody which detects both α1 and α2 (Cell Signaling, Danvers, MA), as previously described [16]. 2.3. Cell proliferation, cell cycle, and migration assay Cell proliferation was estimated with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. For cell cycle analysis, A7r5 cells were seeded and cultured overnight. At 50% to 60% confluency of plates, serum was removed for starvation for 24 h, and then the cells were infected with Ad-GFP or Ad-RORα (MOI = 10) or treated with CS in complete media for 24 h. Cell-cycle phase was analyzed by flow cytometry (Becton Dickinson, Franklin Lakes, NJ). Two-dimensional cell migration was analyzed with the scratch wound assay. A7r5 cells were seeded in culture-insert dishes (ibidi, Martinsried, Germany) and incubated overnight. After the cells had attached, the culture insert was removed and the adenoviruses were added for infection for 24 h. Cell mobility was estimated from the wound size that was analyzed by ImageJ software (http://rsb.info.nih. gov). Three-dimensional cell migration was examined using a modified Boyden Transwell® culture chamber with a gelatin-coated polycarbonate membrane (Corning Life Sciences, Tewksbury, MA). Virus-infected or CS-treated A7r5 cells were seeded in the upper chamber and incubated for 8 h. The migrated cells to the membranes were counted under microscope. 2.4. Rat carotid artery balloon injury and histological analysis The animal care was performed according to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. All experiments were conducted by the guidelines of Seoul National University Institutional Animal Care and Use Committee. Carotid artery balloon injury was performed in seven-week-old male Sprague–Dawley rats, as described previously [20]. Briefly, the animals were anesthetized
3. Results 3.1. RORα induces the activation of the AMPK–mTOR–S6K1 signaling pathway Previously, we demonstrated that RORα activates AMPK and enhances fatty acid β-oxidation, resulting in the attenuation of hepatic steatosis [16]. Because AMPK is one of the major signaling molecules controlling the growth of vSMCs, we asked whether RORα is associated with the proliferation of vSMCs. First, we examined whether RORα induces the activation of AMPK in human primary vSMCs and in rat A7r5 cells. Of four different isoforms of RORα, RORα1 and RORα4 were expressed in both vSMCs and A7r5 cells, similarly to the previous observation (Fig. 1A) [15]. When either RORα1 or RORα4 was exogenously introduced by adenoviral transduction, the phosphorylation at Thr172 of AMPK increased. This increase was ascertained by the enhancement of the phosphorylation at Ser79 of acetyl-CoA carboxylase 1, a downstream target of activated AMPK (Fig. 1A). Because mTOR signaling is suppressed by AMPK in vSMCs, we examined whether the activity of mTOR was altered by RORα [3]. The phosphorylation of mTOR, but not its expression, was greatly reduced after the cells were infected with Ad-RORα1 or Ad-RORα4 (Fig. 1B). The phosphorylation of 70-kDa ribosomal protein S6 kinase 1 (p70S6K1 or S6K1), the most characterized downstream effector of mTOR, was also reduced (Fig. 1B). Treatment with putative ligands of RORα, such as melatonin, 22(R)-hydroxycholesterol, CS, and 7-dehydrocholesterol, increased the levels of pAMPK (Supplementary Fig. IA). CS induced the activation of AMPK at concentrations as low as 0.1 μM in a time-dependent manner (Fig. 1C and Supplementary Fig. IB). CS- or melatonin-elevated pAMPK was abolished after knockdown of RORα using si-RNA, indicating that the effect of these ligands was mediated by RORα (Fig. 1D and Supplementary Fig. IC). The CS-mediated reductions in pmTOR and p70S6K1 were reversed by treatment with compound C, an inhibitor of AMPK, or silencing of AMPK by si-RNA, demonstrating the involvement of AMPK (Fig. 1E and F). The overexpression of RORα or treatment with CS increased the amount of pLKB1, an upstream kinase of AMPK, indicating that the RORα-induced activation of AMPK may be mediated by the activation of LKB1 (Fig. 2A and B). Indeed, silencing of LKB1 repressed the CS-induced AMPK activation (Fig. 2C). Infection of human vSMCs with the Ad-RORα virus or treatment with CS led to a significant decrease in ATP levels, which may cause the elevation of pLKB1 and the subsequent activation of AMPK after activation of RORα (Supplementary Fig. II).
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Fig. 1. RORα induces activation of AMPK-mTOR signaling pathway in vSMCs. (A and B) Human vSMCs or A7r5 cells were infected by Ad-GFP, Ad-RORα1, or Ad-RORα4 for 24 h. (C) A7r5 cells were treated with the indicated concentrations of CS for 24 h. (D) A7r5 cells were transfected with 50 nmol/L non-specific si-RNA (NS) or si-RORα. After 48 h of transfection, the cells were treated with 20 μmol/L CS for 24 h. (E) A7r5 cells were treated with vehicle or 20 μmol/L CS. After 24 h of treatment, the cells were treated with or without 10 μmol/L compound C (Comp C) for 4 h. (F) A7r5 cells were transfected with 50 nmol/L non-specific si-RNA (NS) or si-AMPK. After 48 h of transfection, the cells were treated with 20 μmol/L CS for 24 h. The expression of protein was analyzed by western blot analysis. One representative of at least three independent experiments with similar results is shown.
3.2. RORα modulates the expression of cell cycle control factors and induces cell cycle arrest
Fig. 2. RORα-induced AMPK activation is LKB1-dependent. (A) A7r5 cells were infected by Ad-GFP, Ad-RORα1, or Ad-RORα4 for 24 h. (B) A7r5 cells were treated with the indicated concentrations of CS for 24 h. (C) A7r5 cells were transfected with 50 nmol/L non-specific si-RNA (NS) or si-LKB1. After 48 h of transfection, the cells were treated with 20 μmol/L CS for 24 h. One representative of at least three independent experiments with similar results is shown.
Recently, it was reported that AMPK activation involves the accumulation and activation of tumor suppressor p53 and CDK inhibitors, such as p21 and p27, in vSMCs [3,4]. The expression of the cell-cycle control factors such as p53, p27, and p21 increased dramatically in vSMCs after the adenoviral transduction of RORα1 or RORα4, whereas the expression of cyclin D decreased (Fig. 3A). Treatment with RORα ligands increased the expression of p53 and p27, whereas they reduced the levels of pRB (Supplementary Fig. III). The effect of CS was time dependent; it was seen as early as 1 h after treatment and continued for at least 24 h (Fig. 3B). The CS-induced alterations were abolished by siRORα, si-AMPK, or treatment with compound C, indicating that the effects of CS are RORα-mediated and AMPK-dependent (Fig. 3C–E). When hvSMCs were infected with Ad-RORα1 or Ad-RORα4, the number of cells decreased as the incubation time increased. CS also suppressed the proliferation of hvSMCs by about 40% after six days of treatment (Fig. 4A). A similar pattern of decline was observed in the A7r5 cells. A cell cycle analysis showed that infection with Ad-RORα1 or Ad-RORα4, or CS treatment increased G1 phase in A7r5 cells, indicating that RORα mediates G1 arrest (Fig. 4B). Because the migration of vSMCs plays an important role in the formation of atherosclerotic plaques, we analyzed whether RORα affects migration capacity of vSMCs. The migration of A7r5 cells infected with Ad-RORα1 into scratched areas did not differ markedly from that of control cells. However, the migration of cells that were stimulated with tumor necrosis factor α (TNFα) was clearly inhibited after Ad-RORα1 infection (Fig. 4C). To examine whether the wound is closed due to increased cell motility, we performed migration assay using Boyden chamber. Infection with AdRORα or treatment with CS greatly inhibited the movement of TNFαstimulated cells across the gelatin layer in the three-dimensional cell migration assay (Fig. 4D).
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Fig. 3. RORα modulates expression of p53, CDK inhibitors, and cyclin D. (A) Human vSMCs or A7r5 cells were infected by Ad-GFP, Ad-RORα1, or Ad-RORα4 for 24 h. (B) A7r5 cells were treated with 20 μmol/L CS during the indicated time. (C and D) A7r5 cells were transfected with 50 nmol/L non-specific si-RNA (NS), si-RORα (C), or si-AMPK (D). After 48 h of transfection, the cells were treated with 20 μmol/L CS for 24 h. (E) A7r5 cells were treated with 20 μmol/L CS. After 24 h of treatment, the cells were treated or without 10 μmol/L compound C (Comp C) for 4 h. The expression of protein was analyzed by western blot analysis. One representative of at least three independent experiments with similar results is shown.
3.3. RORα suppresses neointima formation in the rat carotid artery after balloon injury Finally, we examined the effect of RORα on neointima formation in the rat carotid artery in vivo. When a balloon-induced injury was created in the carotid artery, neointimal hyperplasia was induced. However, the infusion of Ad-RORα1 into the artery significantly reduced the intima:media ratio in the injured region (Fig. 5A). The expression of Ki67, a marker of cell proliferation, was reduced significantly in the αSMA stained neointimal region; however, apoptotic cell death was not increased in the Ad-RORα1 infused arteries when examined by TUNEL assay (Fig. 5B and Supplementary Fig. IV) [22]. The expression of pAMPK and p27 was increased significantly in the neointimal region after infection with Ad-RORα1, further supporting the inhibitory function of RORα in the proliferation of vSMCs (Fig. 5C).
4. Discussion In this study, we demonstrated for the first time that RORα suppresses neointima formation through the activation of AMPK-induced pathways including inhibition of protein synthesis and regulation of cell cycle in vSMCs (Fig. 5D). We found that RORα decreased the intracellular ATP level in hvSMCs, which may explain how this receptor activates LKB1-AMPK pathway (Supplementary Fig. II). Activation of AMPK induced phosphorylation of RORα backward, which leads transcriptional activation of this receptor as shown in our previous report (Supplementary Fig. V) [11]. Indeed AMPK increased the expression level as well as transcriptional activity of RORα in vSMC (Supplementary Fig. VI). Together these data suggest a positive activating circuit between AMPK and RORα, that plays an important role in the RORαmediated activation of AMPK for the regulation of vSMC proliferation.
However, currently it is not clear how RORα increases the intracellular AMP:ATP ratio. Whether RORα increases ATP consumption or decreases ATP production in conjunction with mitochondrial functions will be an interesting question to be addressed in future. Downstream target genes of RORα might be involved in the balancing intracellular energy status that affects cellular proliferation of vSMCs. Identification of such RORα downstream genes would help further understanding of role of the positive feedforward activation of RORα and AMPK in neointima formation. Interestingly, RORα suppressed the TNFα-induced proliferation of vSMC (Fig. 4C), which largely involves Rb-E2F transcriptional regulatory pathway [23,24]. As shown in Supplementary Fig. III, activators of RORα such as melatonin and CS decreased the level of pRB, indicating that RORα may inhibit the TNFα-induced vSMC proliferation through blocking of phosphorylation of RB. Involvement of AMPK and its contribution to the RORα-induced repression of pRB in vSMC needs to be investigated in future. The suppressive function of AMPK on cellular proliferation also contributes to the inhibition of tumor cell growth [3]. Therefore, RORαinduced AMPK activation may be associated with the development and progression of human cancers. Indeed, it has been reported that RORα is downregulated at the mRNA level in certain types of cancerous tissues and tumor cell lines [25]. Interestingly, RORα is phosphorylated at Ser35 by protein kinase Cα, which inhibits canonical Wnt5a/PKCα signaling, contributing to the development of colon cancer [26]. RORα is also induced in a p53-dependent manner in response to DNA damage and activates p53, leading to the activation of the specific genes involved in apoptosis [27]. RORα protein levels are markedly reduced in breast cancer, which might be attributable to its methylationdependent degradation caused by the elevated EZH2 protein levels in these cancer tissues [28]. In contrast, a recent report demonstrated a role for RORα in cellular responses to genotoxicity in the lung, which
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Fig. 4. RORα inhibits proliferation of vSMC and induces cell cycle arrest. (A) Human vSMCs or A7r5 cells were infected by Ad-GFP, Ad-RORα1, or Ad-RORα4. Or the cells were treated with 20 μmol/L CS. After treatment, cell proliferation was assessed by MTT assay. 100% represents the OD570 nm of Ad-GFP infected or vehicle treated wells at the indicated time. The numbers represent mean ± SEM (n = 3). *P b 0.05, **P b 0.01, and ***P b 0.001 vs Ad-GFP or vehicle at each time; ###P b 0.001 vs Ad-GFP or vehicle group. (B) A7r5 cells were infected by Ad-GFP, Ad-RORα1, or Ad-RORα4. Or the cells were treated with 20 μmol/L CS. After treatment, the cells were stained with propidium iodide and analyzed by flow cytometry. Representatives of three independent experiments with similar results are shown. (C) A7r5 cells were infected by Ad-GFP or Ad-RORα1, and then treated with or without 10 ng/mL TNFα for indicated time periods. The migrated cells were imaged under a light microscope (left). Cell mobility was estimated from the wound size that is indicated by dotted lines and analyzed by ImageJ software. The graph shows the relative cell mobility. Ad-GFP without TNFα group at 24 h is defined as 1. #P b 0.05 vs Ad-GFP with vehicle; *P b 0.05 vs Ad-GFP with TNFα (n = 3) (right). (D) A7r5 cells were infected by Ad-GFP, Ad-RORα1, or Ad-RORα4. Or the cells were treated with 20 μmol/L CS. Then the cells were added to the upper chamber, while 10 ng/mL TNFα or vehicle was placed in the lower chamber. The migrated cells were stained with hematoxylin and imaged under a light microscope (top). Black bars represent 100 μm. The numbers of migrated cells in the field were counted. ##P b 0.01 and ###P b 0.001 vs Ad-GFP with vehicle; *P b 0.05 and **P b 0.01 vs Ad-GFP with TNFα (n = 3) (bottom).
may promote pulmonary emphysema [29]. Therefore, in future studies, attention should be directed to the roles of RORα in the pathophysiology of diverse chronic metabolic diseases, including cancer. Various nuclear receptors, including the peroxisome proliferatoractivated receptor (PPAR) and NUR77, play important roles in the regulation of neointima formation and plaque growth in atherosclerotic lesions. Troglitazone and rosiglitazone, high affinity ligands of PPARγ, suppress vSMC proliferation and prevent rat aortic neointima formation after balloon injury through a PPARγ-mediated pathway [30,31]. Moreover, PPARα inhibits vSMC proliferation through the induction of p16, a CDK inhibitor that blocks G1/S cell-cycle progression [32]. Inducers of Nur77, 6-mercaptopurine and α-lipoic acid, protect against excessive vSMC proliferation and neointima formation [20,33]. Interestingly, Nur77 inhibits AMPK activation via its interaction with LKB1 [34]. Because a complicated network of nuclear receptors is involved in controlling the proliferation of vSMCs and lipid metabolism in the development and progression of atherosclerosis, specific ligands of these receptors
are promising potential drug candidates for atherosclerosis. Recently, we and others developed a synthetic RORα-activating ligands that activates RORα-driven transcription [16,35]. Further researches into the role of these RORα ligands in atherosclerotic lesion formation could extend our understanding of the biological functions of RORα in neointimal hyperplasia and suggest therapeutic strategies against atherosclerosis. In conclusion, this is the first study to show that RORα effectively suppressed cell proliferation and cell cycle progression of vSMC through activation of AMPK. Our conclusion suggests that RORα could be a valuable target for the treatment of atherosclerosis and its ligands could provide a potential remedy against vascular proliferative disorders.
Conflict of interest None declared.
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Fig. 5. RORα suppresses neointima formation in rat carotid artery after balloon injury. (A) Representative photomicrographs of cross-sections of the left common carotid arteries obtained from the balloon injured rats with infusion of either Ad-GFP or Ad-RORα1. Black bars represent 200 μm (left). Neointima formation was analyzed by the ratio of intima/media that calculated by computer-based morphometric analyzer. Values represent means ± SEM (n = 5). *P b 0.05 vs Ad-GFP (right). (B) Immunohistochemistry was carried out using antibodies against Ki67, GFP, or αSMA. Black bars represent 20 μm (left). The Ki67 positive cells in the section were counted under a microscope and represented as % of total cells. αSMA was stained as a marker of vSMC in the injured arteries. Values represent means ± SEM (n = 6 for Ad-GFP and n = 5 for Ad-RORα1). *P b 0.05 vs Ad-GFP (right). Black bars represent 20 μm. (C) Immunohistochemistry was carried out using antibodies against pAMPK, p27, or RORα. Representative photomicrographs from five samples from each group were shown (left). Values represent means ± SEM (n = 5). *P b 0.05 vs Ad-GFP (right). (D) Schematic model for mechanism of the RORα-induced suppression of vSMC proliferation.
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