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PPARγ activation abolishes LDL-induced proliferation of human aortic smooth muscle cells via SOD-mediated down-regulation of superoxide
Biochemical and Biophysical Research Communications 359 (2007) 1017–1023 www.elsevier.com/locate/ybbrc
PPARc activation abolishes LDL-induced proliferation of human aortic smooth muscle cells via SOD-mediated down-regulation of superoxide Kyung-Sun Heo a,c, Dong-Uk Kim a, Sungwoo Ryoo a, Miyoung Nam a, Seung Tae Baek a, Lila Kim a, Song-Kyu Park b, Chang-Seon Myung c, Kwang-Lae Hoe a,* a
Functional Genomics Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Yuseong, Daejeon, Republic of Korea b Bio-Evaluation Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Yuseong, Daejeon, Republic of Korea c Department of Pharmacy, Chungnam National University, Yuseong, Daejeon, Republic of Korea Received 23 May 2007 Available online 8 June 2007
Abstract Native LDL would be a mitogenic and chemotactic stimulus of VSMC proliferation and differentiation in the atherosclerotic lesion where endothelial disruption occurred. In previous studies, our group investigated the molecular mechanisms by which LDL induces IL-8 production and by which PPARa activation abolishes LDL effects in human aortic SMCs (hAoSMCs). Herein is the first report of PPARc activation by troglitazone (TG) exerting its inhibitory effects on LDL-induced cell proliferation via generation not of H2O2, but of O2 , and the subsequent activation of Erk1/2 in hAoSMCs. Moreover, in this study TG abolished the LDL-accelerated G1–S progression to control levels via down-regulation of active cyclinD1/CDK4 and cyclinE/CDK2 complexes and up-regulation of p21Cip1 expression. TG exerted its anti-proliferative effects through the up-regulation of basal superoxide dismutase (SOD) expression. This data suggests that the regulation of O2 is located at the crossroads between LDL signaling and cell proliferation. 2007 Elsevier Inc. All rights reserved. Keywords: Atherosclerosis; Low-density lipoproteins; MAP kinase; PPAR; Smooth muscle cell; Superoxide; Troglitazone
Proliferation and differentiation of vascular smooth muscle cells (VSMCs) play key roles in the development of restenosis and in the progression of atherosclerosis [1], which are hallmarks of the pathogenesis of atherosclerotic lesions [2,3]. In addition to many VSMC growth factors, including platelet-derived growth factor (PDGF) [4,5], LDL would be a mitogenic and chemotactic regulator of VSMCs in the lesions where endothelial disruption occurred due to injury or angioplasty. LDL has been reported to induce proliferation of human VSMCs via generation of ROS and activation of Erk1/2 [6].
*
Corresponding author. Fax: +82 2 860 4594. E-mail address: [email protected] (K.-L. Hoe).
0006-291X/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.06.006
Reactive oxygen species (ROS) are recognized as intracellular second messengers [7]. Fibroblasts, endothelial cells (ECs), and SMCs produce ROS in response to various activation signals. The type of ROS produced and their subsequent effects may differ depending on the type of stimulus. Many proliferation stimuli, such as PDGF, thrombin, and oxidized LDL, induce proliferation of VSMCs via generation of ROS, which subsequently activate redox-sensitive Erk1/2 [8,9]. In contrast, activation of p38, rather than Erk1/2, is responsible for Angiotensin II-induced proliferation of VSMCs [10]. The PPAR subfamily consists of three distinct subtypes, denoted as a, b/d, and c [11]. PPARs are biologically relevant to metabolic disorders associated with the development of atherosclerosis, such as hyperlipidemia and diabetes, as they link lipid metabolism and inflammation
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[12]. PPAR activation interferes with atherosclerotic processes by inhibiting proliferation of vessel cells induced by chemokines [13], angiotensin II [14], and injury [15]. Recently, there is an increasing body of evidence that PPARs exert their beneficial effects via inhibition of ROS production and of cell cycle progression. PPARc activation induces an increase in the level of the superoxide-scavenger enzyme [16–18], Cu2+/Zn2+-superoxide dismutase (CuZn– SOD), or SOD via binding of activated receptor complexes to specific recognition sites on DNA and PPREs [19]. Furthermore, PPAR activation inhibits the G1–S phase progression of the cell cycle via down-regulation of cyclins and up-regulation of cyclin-dependent kinase inhibitors (CKIs) such as p21Cip1 or p27 [20–22]. Although PPARs play beneficial roles by interfering with atherosclerotic progression in VSMCs, the type of PPARs and specific signaling pathways involved in LDL-induced activation remain unidentified. In previous studies, we reported that PPARa activation suppresses LDL-stimulated IL-8 up-regulation in hAoSMCs [23]. The present study investigated the specific molecular mechanism by which PPARc activation exerts its beneficial effects on LDL-induced cell proliferation of hAoSMCs. Materials and methods Chemicals and antibodies. PD98059, dihydroethidine (HE), and 2 0 ,7 0 dichlorodihydro fluorescein diacetate (H2DCF-DA) were purchased from Calbiochem. Troglitazone (TG) was obtained from Sankyo. All chemicals and enzymes, such as fenofibrate (FF), diphenylene iodinium (DPI), 4,5dihydroxyl-1,3-benzenedisulphonic acid (Tiron), LY83583 (LY), hydrogen peroxide, and glycol-conjugated superoxide dismutase (PEG-SOD), catalase (CAT), were purchased from Sigma, unless otherwise stated. PCR primers and PCR-premix were purchased from Bioneer. Rabbit polyclonal antibodies specific for Erk1/2 (#9102), phospho-Erk1/2 (#9101), and phospho-Rb (#9308) were obtained from Cell Signaling. Mouse monoclonal antibodies for p21Cip1 (#556430) and p27 (#554069) were purchased from BD Biosciences. Mouse monoclonal antibody against cyclin D1 (SC-450) and rabbit polyclonal antibodies against cyclin A (SC-751), cyclin E (SC-481), CDK2 (SC-163), and CDK4 (SC-749) were purchased from Santa Cruz Biotechnology. Isolation of LDL and cell cultures. LDL (density 1.019–1.063 g/ml) were isolated from plasma of normocholesterolemic subjects (serum cholesterol <6.2 mM) by differential ultracentrifugation as previously described [24]. No oxidation was observed in isolated LDL for periods of up to 3 weeks, as determined by the thiobarbiturate method. Human AoSMCs (Clonetics) were cultured in SmGM-2 Bullet kit medium (Clonetics) at 37 C in 5% CO2. Cells were used at passages 3 through 8. For all experiments, the cells were grown to 80–90% confluence and made quiescent by starvation in a maintenance medium (DMEM containing 0.1% FBS, Invitrogen) for at least 24 h. Cells were pretreated with TG or FF for 30 min before LDL treatment. Cell proliferation assays. BrdU incorporation and WST-1 methods. Approximately, 104 cells per well were incubated in 96-well plates overnight at 37 C, starved for 24-h, and pretreated with various inhibitors in the presence or absence of LDL. After an additional 24-h incubation, cell proliferation then was measured by two different methods—BrdU incorporation and/or WST-1 assays (Roche). BrdU incorporation into DNA was evaluated by photometric analysis (A450) using a microplate reader (Emax, Molecular devices) [15]. For the WST-1 assay, cells were incubated with 10 ll of WST-1 reagent for 45 min, and the A450 then was measured using a microplate reader.
Western blot analysis. Cells were lysed in SDS sample buffer (62.5 mM Tris, pH 6.8, 2% SDS, and 10% glycerol). Each sample was resolved by 10% SDS–PAGE, transferred onto PVDF membranes (Pall), analyzed with antibodies according to the supplier’s protocol, and visualized with peroxidase and an enhanced-chemiluminescence system (ECL kit, Amersham). Normalization was performed with the SMC-specific a-actin antibody (M0851, BD Sciences). The Western blot figure represents a typical example of three independent experiments. Estimation of intracellular O2 and H2O2. Cells treated with various chemicals were washed three times with DPBS, incubated with 5 lM HE or H2DCF-DA in serum-free medium at 37 C for 30 min, according to the manufacturer’s instructions. HE was used to determine the amount of superoxide anion (O2 ). Superoxide anion oxidizes HE into ethidium bromide (EtBr), which emits red fluorescence (Ex: 535 nm; Em: 610 nm). H2DCF-DA was used to measure the amounts of hydrogen peroxide (H2O2) (Ex: 504 nm; Em: 530 nm) [25]. Their fluorescent nature was analyzed using fluorescence-activated cell sorting (FACS) using a FACScalibur (Becton & Dickinson). For each run, 10,000 events were collected. Measurement of cellular superoxide dismutase activity. Cells treated with various chemicals were sonicated in ice-cold RIPA buffer (25 mM Tris–HCl, pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, and 0.1% SDS supplemented with PMSF) and centrifuged at 15,000g for 5 min at 4 C. The SOD activity of supernatants (each 20 lg protein) was calculated from the absorbance at 450 nm using the SOD Assay Kit-WST (Dojindo Molecular Technologies). The SOD activity was represented as U/mg protein and compared with the standard curve of 1 U of bovine liver SOD (Sigma) used as a control. Statistical analysis. Experimental data were analyzed with the GraphPad Prism program, version 2.00 (GraphPad Software). All values are reported as means ± SD of at least three independent experiments. The unpaired Student’s t-test was used to assess the significance in the differences between the two groups. A value of P < 0.05 was accepted as significant.
Results and discussion Superoxide anion (O2 ) played a critical role in Erk1/2 activation, which was essential for LDL-induced hAoSMC proliferation As far as cell proliferation and differentiation are concerned, LDL would a mitogenic and chemotactic regulator of VSMCs in the artherosclerotic lesion where endothelial disruption occurred. According to the results of the supplementary data, treatment of hAoSMCs with LDL resulted in an increase in cell proliferation by 36.5% and 175% as determined by WST-1 and BrdU incorporation assays, respectively. The activation of Erk1/2 was responsible for the proliferative effects of LDL. Previously reported results by other research groups suggest that activation of Erk1/2 may be mediated by ROS [6]. Therefore, the type of ROS related to the phosphorylation of Erk1/2 in response to LDL activation was identified. As judged by the BrdU incorporation and Western blot assays (Fig. 1A), both the O2 -scavenging enzyme, SOD, and the NADPH oxidase inhibitors, DPI and Tiron, resulted in complete blockade of Erk1/2 phosphorylation and nearly complete inhibition of LDL-induced cell proliferation. In contrast, the H2O2 scavenging enzyme, CAT, showed a slight
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Fig. 1. Superoxide anion (O2 ) contributed to LDL-stimulated hAoSMC proliferation via a decrease in Erk1/2 phosphorylation. (A) hAoSMCs were pretreated with the ROS-inhibitors, SOD (500 U/ml), DPI (10 lM), Tiron (10 lM), or catalase (CAT, 500 U/ml) for 30 min. In addition, cells were pretreated with exogenous ROS, LY (1 lM) or H2O2 (10 lM), for 5 min. Erk1/2 phosphorylation then was measured by Western analysis after 5 min incubation in the presence or absence of LDL (100 lg/ml) using un-phosphoryrlated Erk1/2 as a normalization control. Cell proliferation was assayed after 24-h incubation in the presence or absence of LDL (100 lg/ml) using two different methods, BrdU incorporation (n = 7, A) and WST-1 assay (n = 11, B). *P < 0.01 versus control, #P < 0.05, ##P < 0.01 versus LDL.
blockade of Erk1/2 phosphorylation and cell proliferation. Furthermore, the effects of LY (an O2 -generating chemical) and H2O2 on cell proliferation was investigated. As expected, only LY was able to activate Erk1/ 2 to the levels observed following LDL treatment. However, addition of exogenous H2O2 showed no effects on cell proliferation. Similar results were observed for the WST-1 assay (Fig. 1B). Taken together, these results suggest that the generation of O2 through NADPH oxidase and the subsequent ROS-mediated activation of Erk1/2 are critical for LDL-induced cell proliferation. The results are inconsistent with those observed in the case of PDGF-BB, in which generated O2 induces inflammatory reactions [26]. Taking into account our previous study, which demonstrated that LDL induces IL-8 expression via the generation of H2O2 [23], this is the first report, to the best of our knowledge, showing that two different ROS families, generated in response to a single stimulus, exert different effects in the same cell—O2 induces cell proliferation, whereas H2O2 induces inflammatory reactions.
PPARc activation completely abolished the effects of LDL on cell proliferation via deactivation of Erk1/2 Our previous studies have shown that PPARa activation abolishes LDL-induced IL-8 expression in hAoSMCs [23]. However, the type of PPARs and the specific signaling pathways related to LDL-induce cell proliferation in hAoSMCs have yet to be identified. Therefore, this study assessed which PPAR activator is related to LDL-induced cell proliferation using proliferation and Western-blot assays. According to the results of the BrdU assay (Fig. 2A), treatment of hAoSMCs with TG or FF alone affected neither cell proliferation nor Erk1/2 phosphorylation, suggesting that TG or FF alone is not harmful to hAoSMCs. However, the PPARc agonist (TG) completely abolished both activation of Erk1/2 and subsequent LDLinduced cell proliferation. In contrast, the PPARa agonist (FF) showed no effects on proliferation nor on Erk1/2 phosphorylation. The WST-1 assay showed a pattern similar to that observed in the BrdU assay (Fig. 2B). Treatment with TG decreased LDL-induced cell proliferation
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Fig. 2. The PPARc agonist (TG) abolished the effects of LDL on cell proliferation via deactivation of Erk1/2. (A) hAoSMCs were pretreated with PPAR agonists, TG for PPARc (20 lM) and FF for PPARa (20 lM) for 30 min, and their inhibitory effects on DNA synthesis then were measured by BrdU incorporation assay after an additional 24-h incubation in the presence or absence of LDL (100 lg/ml) (upper). At the same time, Erk1/2 phosphorylation was assessed by Western analysis (lower). n = 6, *P < 0.01 versus control, #P < 0.01 versus LDL. (B) Cells were pretreated with PPARc agonists at the indicated concentrations of FF or TG, and their effects on cell proliferation were measured by WST-1 assay after an additional 24-h incubation in the presence or absence of LDL (100 lg/ml). n = 10, *P < 0.01 versus control, #P < 0.05, ##P < 0.01 versus LDL.
in a dose-dependant manner, whereas FF had no effect on LDL-induced cell proliferation. These results suggest that activation of PPARc, but not of PPARa, plays a beneficial role in LDL-induced proliferation of hAoSMCs via deactivation of Erk1/2. PPARc activation abolished the generation of superoxide(O2 ) evoked by LDL via up-regulation of SOD The findings that PPARc activation completely abolished LDL-induced cell proliferation via deactivation of Erk1/2 prompted us to investigate the molecular mechanisms by which TG exerts its inhibitory effects on LDL-induced Erk1/2 phosphorylation. Therefore, the relationship between ROS and PPARc activation was closely investigated in the present study. The effects of TG or FF on LDL-induced ROS generation were assessed using FACS analysis, measurement of intracellular concentrations of O2 and H2O2 (Fig. 3A, upper), and intracellular SOD activity was assessed following TG treatment
(Fig. 3A, lower). According to the FACS analysis (Fig. 3A, upper), LDL treatment resulted in the generation of both O2 and H2O2. However, only TG specifically inhibited LDL-induced O2 production as much as SOD, which served as a positive control (left). In contrast, FF specifically inhibited the generation of H2O2 as much as CAT, which was used as a positive control (right). When the effects of TG or FF on intracellular concentrations of ROS induced by LDL were assessed (Fig. 3A, lower), similar results were obtained. LDL increased generation of O2 and H2O2 by 4.7- and 7-fold, respectively. Both SOD and TG down-regulated LDL-induced O2 production, whereas CAT and FF down-regulated LDL-induced H2O2 production. In other words, treatment with TG and FF mimicked the effects of SOD and CAT, respectively. Recently, it has been reported that activated PPARs bind to their specific PPREs in the promoter region of target genes, especially the SOD gene [16], implying that PPARc activation may exert its beneficial effects via an
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Fig. 3. The PPARc agonist (TG), not the PPARa agonist (FF), abolished LDL-induced generation of O2 via an increase in SOD activity. (A) hAoSMCs were pretreated with TG (20 lM) or FF (20 lM) 30 min before 24-h LDL treatment (100 lg/ml). Intracellular levels of O2 (left) or H2O2 (right) were measured by both FACS (upper) and spectrophotometry (lower) methods using HE (5 lM) and H2DCF-DA (5 lM) as fluorescent dyes. SOD (500 U/ml) and CAT (500 U/ml) were used as specific scavengers of O2 and H2O2, respectively. n = 10, *P < 0.01 versus control, #P < 0.01 versus LDL. (B) Cells were pretreated with TG in the presence or absence of LDL, and SOD activity in cell lysates then were determined. Exogenous SOD was used as a positive control. Experiments were repeated three times. n = 5, *P < 0.01 versus control, #P < 0.01 versus LDL.
increase in cellular concentrations of SOD. These findings led us to examine whether the down-regulation of O2 , induced by PPARc activation, is related to the up-regulation of SOD. As shown in Fig. 3B, the activity of SOD was decreased by LDL treatment compared with the untreated control, which may explain why LDL increases the level of O2 . However, the activity of SOD was increased by TG treatment as much as by exogenous PEG-SOD, despite LDL treatment. These results suggest that TG exerts its inhibitory effects on LDL-induced cell proliferation via an increase in basal SOD activity. These results are consistent with previous reports concluding that PPARc activation is anti-atherogenic via vascular
CuZn–SOD in hypercholesterolemia [18] and regulates endothelial function via CuZn–SOD in HUVEC [17]. PPARc activation inhibited LDL-induced G1–S phase progression of the cell cycle via down-regulation of active cyclins/CDK complexes and up-regulation of p21 Although beneficial roles of PPARc are well-known, its effects on cell-cycle modulators differ depending on the type of cells and on the stimulus [27]. Therefore, the issue of which cell cycle regulatory molecules are modulated by LDL was examined using Western blot analysis (Fig. 4A, upper). Treatment of hAoSMCs with
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Fig. 4. The PPARc agonist (TG) inhibited LDL-induced G1–S phase progression of the cell cycle via down-regulation of active cyclins/CDK complexes and up-regulation of p21. (A) Cells were treated with LDL (100 lg/ml), and protein levels of cell cycle regulatory molecules, including cyclins, CDKs, and CKIs, then were assessed by Western blot analysis at the indicated times after exposure to LDL. (B) Cells were pretreated with SOD (500 U/ml), TG (20 lM), or PD (20 lM) for 30 min. Protein levels of the indicated cell-cycle modulators then were measured by Western blot analysis 24-h after exposure to LDL.
LDL resulted in a significant increase in the protein levels of cyclinD1/CDK4 and cyclinE/CDK2 complexes in a time-dependent manner, which has been reported to be related to the G1–S progression of the cell cycle [15]. Furthermore, the activity of the up-regulated CDK complexes was tested by measuring phosphorylation of Rb, a target of active CDK. As expected, LDL enhanced phosphorylation of the Rb protein in a time-dependent manner, suggesting that that the up-regulated CDK complexes were functionally active. In addition, the issue of which type of CKI is related to LDL stimulation was assessed (Fig. 4A, lower). Only the levels of p21Cip1 were affected by LDL. The levels of p21Cip1 gradually decreased from 1-h after LDL stimulation. Taken together, these results suggest that LDL induces cell proliferation of hAoSMCs by stimulating the progression of G1–S phase via up-regulation of active cyclinD1/CDK4 and cyclinE/CDK2 complexes and down-regulation of p21Cip1. The results shown Fig. 4A led to the examination of which cell-cycle regulators are affected by PPARc activation. As shown in Fig. 4B, TG, as well as SOD and PD, abolished the up-regulation of active cyclinD1/CDK4 and cyclinE/CDK2 complexes induced by LDL; the amounts of the complexes after treatment with TG, SOD and PD were the same as the controls that were not treated with LDL. Furthermore, TG negated the LDL-induced reduction in p21Cip1; TG co-treatment increased p21Cip1 to levels exceeding those observed for the LDL-untreated control. Taken together, the results of the present study suggest that PPARc activation with TG abolished the effects of LDL via down-regulation of
active CDK complexes and up-regulation of CKI (p21Cip1). These results are consistent with previous reports, which asserted that PPARc ligands affect the proliferation of VSMCs via down-regulation of both cyclin D1 and cyclin E [22] and up-regulation of p21Cip1 [21]. However, the results are inconsistent with those observed in the case of PDGF-induced proliferation of rat AoSMCs, which is caused by up- rather than down-regulation of p21Cip1 [28]. The discrepancy may be due to different mechanisms of cell proliferation between PDGF-BB and LDL. In conclusion, the results of the present study provide a previously unrecognized mechanism for the anti-proliferative effects of PPARc ligands against LDL-induced proliferation in atherosclerotic lesions and support the concept that PPARc ligands may constitute a novel therapeutic approach for the treatment of proliferative cardiovascular diseases. Acknowledgments This work was supported by the intramural Mission 2007 research program of KRIBB and by the 21st century Frontier R&D Program and Chemical Genomics Research Program from the Ministry of Science and Technology of Korea. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc. 2007.06.006.
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PPARc activation abolishes LDL-induced proliferation of human aortic smooth muscle cells via SOD-mediated down-regulation of superoxide Kyung-Sun Heo a,c, Dong-Uk Kim a, Sungwoo Ryoo a, Miyoung Nam a, Seung Tae Baek a, Lila Kim a, Song-Kyu Park b, Chang-Seon Myung c, Kwang-Lae Hoe a,* a
Functional Genomics Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Yuseong, Daejeon, Republic of Korea b Bio-Evaluation Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Yuseong, Daejeon, Republic of Korea c Department of Pharmacy, Chungnam National University, Yuseong, Daejeon, Republic of Korea Received 23 May 2007 Available online 8 June 2007
Abstract Native LDL would be a mitogenic and chemotactic stimulus of VSMC proliferation and differentiation in the atherosclerotic lesion where endothelial disruption occurred. In previous studies, our group investigated the molecular mechanisms by which LDL induces IL-8 production and by which PPARa activation abolishes LDL effects in human aortic SMCs (hAoSMCs). Herein is the first report of PPARc activation by troglitazone (TG) exerting its inhibitory effects on LDL-induced cell proliferation via generation not of H2O2, but of O2 , and the subsequent activation of Erk1/2 in hAoSMCs. Moreover, in this study TG abolished the LDL-accelerated G1–S progression to control levels via down-regulation of active cyclinD1/CDK4 and cyclinE/CDK2 complexes and up-regulation of p21Cip1 expression. TG exerted its anti-proliferative effects through the up-regulation of basal superoxide dismutase (SOD) expression. This data suggests that the regulation of O2 is located at the crossroads between LDL signaling and cell proliferation. 2007 Elsevier Inc. All rights reserved. Keywords: Atherosclerosis; Low-density lipoproteins; MAP kinase; PPAR; Smooth muscle cell; Superoxide; Troglitazone
Proliferation and differentiation of vascular smooth muscle cells (VSMCs) play key roles in the development of restenosis and in the progression of atherosclerosis [1], which are hallmarks of the pathogenesis of atherosclerotic lesions [2,3]. In addition to many VSMC growth factors, including platelet-derived growth factor (PDGF) [4,5], LDL would be a mitogenic and chemotactic regulator of VSMCs in the lesions where endothelial disruption occurred due to injury or angioplasty. LDL has been reported to induce proliferation of human VSMCs via generation of ROS and activation of Erk1/2 [6].
*
Corresponding author. Fax: +82 2 860 4594. E-mail address: [email protected] (K.-L. Hoe).
0006-291X/$ - see front matter 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2007.06.006
Reactive oxygen species (ROS) are recognized as intracellular second messengers [7]. Fibroblasts, endothelial cells (ECs), and SMCs produce ROS in response to various activation signals. The type of ROS produced and their subsequent effects may differ depending on the type of stimulus. Many proliferation stimuli, such as PDGF, thrombin, and oxidized LDL, induce proliferation of VSMCs via generation of ROS, which subsequently activate redox-sensitive Erk1/2 [8,9]. In contrast, activation of p38, rather than Erk1/2, is responsible for Angiotensin II-induced proliferation of VSMCs [10]. The PPAR subfamily consists of three distinct subtypes, denoted as a, b/d, and c [11]. PPARs are biologically relevant to metabolic disorders associated with the development of atherosclerosis, such as hyperlipidemia and diabetes, as they link lipid metabolism and inflammation
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[12]. PPAR activation interferes with atherosclerotic processes by inhibiting proliferation of vessel cells induced by chemokines [13], angiotensin II [14], and injury [15]. Recently, there is an increasing body of evidence that PPARs exert their beneficial effects via inhibition of ROS production and of cell cycle progression. PPARc activation induces an increase in the level of the superoxide-scavenger enzyme [16–18], Cu2+/Zn2+-superoxide dismutase (CuZn– SOD), or SOD via binding of activated receptor complexes to specific recognition sites on DNA and PPREs [19]. Furthermore, PPAR activation inhibits the G1–S phase progression of the cell cycle via down-regulation of cyclins and up-regulation of cyclin-dependent kinase inhibitors (CKIs) such as p21Cip1 or p27 [20–22]. Although PPARs play beneficial roles by interfering with atherosclerotic progression in VSMCs, the type of PPARs and specific signaling pathways involved in LDL-induced activation remain unidentified. In previous studies, we reported that PPARa activation suppresses LDL-stimulated IL-8 up-regulation in hAoSMCs [23]. The present study investigated the specific molecular mechanism by which PPARc activation exerts its beneficial effects on LDL-induced cell proliferation of hAoSMCs. Materials and methods Chemicals and antibodies. PD98059, dihydroethidine (HE), and 2 0 ,7 0 dichlorodihydro fluorescein diacetate (H2DCF-DA) were purchased from Calbiochem. Troglitazone (TG) was obtained from Sankyo. All chemicals and enzymes, such as fenofibrate (FF), diphenylene iodinium (DPI), 4,5dihydroxyl-1,3-benzenedisulphonic acid (Tiron), LY83583 (LY), hydrogen peroxide, and glycol-conjugated superoxide dismutase (PEG-SOD), catalase (CAT), were purchased from Sigma, unless otherwise stated. PCR primers and PCR-premix were purchased from Bioneer. Rabbit polyclonal antibodies specific for Erk1/2 (#9102), phospho-Erk1/2 (#9101), and phospho-Rb (#9308) were obtained from Cell Signaling. Mouse monoclonal antibodies for p21Cip1 (#556430) and p27 (#554069) were purchased from BD Biosciences. Mouse monoclonal antibody against cyclin D1 (SC-450) and rabbit polyclonal antibodies against cyclin A (SC-751), cyclin E (SC-481), CDK2 (SC-163), and CDK4 (SC-749) were purchased from Santa Cruz Biotechnology. Isolation of LDL and cell cultures. LDL (density 1.019–1.063 g/ml) were isolated from plasma of normocholesterolemic subjects (serum cholesterol <6.2 mM) by differential ultracentrifugation as previously described [24]. No oxidation was observed in isolated LDL for periods of up to 3 weeks, as determined by the thiobarbiturate method. Human AoSMCs (Clonetics) were cultured in SmGM-2 Bullet kit medium (Clonetics) at 37 C in 5% CO2. Cells were used at passages 3 through 8. For all experiments, the cells were grown to 80–90% confluence and made quiescent by starvation in a maintenance medium (DMEM containing 0.1% FBS, Invitrogen) for at least 24 h. Cells were pretreated with TG or FF for 30 min before LDL treatment. Cell proliferation assays. BrdU incorporation and WST-1 methods. Approximately, 104 cells per well were incubated in 96-well plates overnight at 37 C, starved for 24-h, and pretreated with various inhibitors in the presence or absence of LDL. After an additional 24-h incubation, cell proliferation then was measured by two different methods—BrdU incorporation and/or WST-1 assays (Roche). BrdU incorporation into DNA was evaluated by photometric analysis (A450) using a microplate reader (Emax, Molecular devices) [15]. For the WST-1 assay, cells were incubated with 10 ll of WST-1 reagent for 45 min, and the A450 then was measured using a microplate reader.
Western blot analysis. Cells were lysed in SDS sample buffer (62.5 mM Tris, pH 6.8, 2% SDS, and 10% glycerol). Each sample was resolved by 10% SDS–PAGE, transferred onto PVDF membranes (Pall), analyzed with antibodies according to the supplier’s protocol, and visualized with peroxidase and an enhanced-chemiluminescence system (ECL kit, Amersham). Normalization was performed with the SMC-specific a-actin antibody (M0851, BD Sciences). The Western blot figure represents a typical example of three independent experiments. Estimation of intracellular O2 and H2O2. Cells treated with various chemicals were washed three times with DPBS, incubated with 5 lM HE or H2DCF-DA in serum-free medium at 37 C for 30 min, according to the manufacturer’s instructions. HE was used to determine the amount of superoxide anion (O2 ). Superoxide anion oxidizes HE into ethidium bromide (EtBr), which emits red fluorescence (Ex: 535 nm; Em: 610 nm). H2DCF-DA was used to measure the amounts of hydrogen peroxide (H2O2) (Ex: 504 nm; Em: 530 nm) [25]. Their fluorescent nature was analyzed using fluorescence-activated cell sorting (FACS) using a FACScalibur (Becton & Dickinson). For each run, 10,000 events were collected. Measurement of cellular superoxide dismutase activity. Cells treated with various chemicals were sonicated in ice-cold RIPA buffer (25 mM Tris–HCl, pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, and 0.1% SDS supplemented with PMSF) and centrifuged at 15,000g for 5 min at 4 C. The SOD activity of supernatants (each 20 lg protein) was calculated from the absorbance at 450 nm using the SOD Assay Kit-WST (Dojindo Molecular Technologies). The SOD activity was represented as U/mg protein and compared with the standard curve of 1 U of bovine liver SOD (Sigma) used as a control. Statistical analysis. Experimental data were analyzed with the GraphPad Prism program, version 2.00 (GraphPad Software). All values are reported as means ± SD of at least three independent experiments. The unpaired Student’s t-test was used to assess the significance in the differences between the two groups. A value of P < 0.05 was accepted as significant.
Results and discussion Superoxide anion (O2 ) played a critical role in Erk1/2 activation, which was essential for LDL-induced hAoSMC proliferation As far as cell proliferation and differentiation are concerned, LDL would a mitogenic and chemotactic regulator of VSMCs in the artherosclerotic lesion where endothelial disruption occurred. According to the results of the supplementary data, treatment of hAoSMCs with LDL resulted in an increase in cell proliferation by 36.5% and 175% as determined by WST-1 and BrdU incorporation assays, respectively. The activation of Erk1/2 was responsible for the proliferative effects of LDL. Previously reported results by other research groups suggest that activation of Erk1/2 may be mediated by ROS [6]. Therefore, the type of ROS related to the phosphorylation of Erk1/2 in response to LDL activation was identified. As judged by the BrdU incorporation and Western blot assays (Fig. 1A), both the O2 -scavenging enzyme, SOD, and the NADPH oxidase inhibitors, DPI and Tiron, resulted in complete blockade of Erk1/2 phosphorylation and nearly complete inhibition of LDL-induced cell proliferation. In contrast, the H2O2 scavenging enzyme, CAT, showed a slight
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Fig. 1. Superoxide anion (O2 ) contributed to LDL-stimulated hAoSMC proliferation via a decrease in Erk1/2 phosphorylation. (A) hAoSMCs were pretreated with the ROS-inhibitors, SOD (500 U/ml), DPI (10 lM), Tiron (10 lM), or catalase (CAT, 500 U/ml) for 30 min. In addition, cells were pretreated with exogenous ROS, LY (1 lM) or H2O2 (10 lM), for 5 min. Erk1/2 phosphorylation then was measured by Western analysis after 5 min incubation in the presence or absence of LDL (100 lg/ml) using un-phosphoryrlated Erk1/2 as a normalization control. Cell proliferation was assayed after 24-h incubation in the presence or absence of LDL (100 lg/ml) using two different methods, BrdU incorporation (n = 7, A) and WST-1 assay (n = 11, B). *P < 0.01 versus control, #P < 0.05, ##P < 0.01 versus LDL.
blockade of Erk1/2 phosphorylation and cell proliferation. Furthermore, the effects of LY (an O2 -generating chemical) and H2O2 on cell proliferation was investigated. As expected, only LY was able to activate Erk1/ 2 to the levels observed following LDL treatment. However, addition of exogenous H2O2 showed no effects on cell proliferation. Similar results were observed for the WST-1 assay (Fig. 1B). Taken together, these results suggest that the generation of O2 through NADPH oxidase and the subsequent ROS-mediated activation of Erk1/2 are critical for LDL-induced cell proliferation. The results are inconsistent with those observed in the case of PDGF-BB, in which generated O2 induces inflammatory reactions [26]. Taking into account our previous study, which demonstrated that LDL induces IL-8 expression via the generation of H2O2 [23], this is the first report, to the best of our knowledge, showing that two different ROS families, generated in response to a single stimulus, exert different effects in the same cell—O2 induces cell proliferation, whereas H2O2 induces inflammatory reactions.
PPARc activation completely abolished the effects of LDL on cell proliferation via deactivation of Erk1/2 Our previous studies have shown that PPARa activation abolishes LDL-induced IL-8 expression in hAoSMCs [23]. However, the type of PPARs and the specific signaling pathways related to LDL-induce cell proliferation in hAoSMCs have yet to be identified. Therefore, this study assessed which PPAR activator is related to LDL-induced cell proliferation using proliferation and Western-blot assays. According to the results of the BrdU assay (Fig. 2A), treatment of hAoSMCs with TG or FF alone affected neither cell proliferation nor Erk1/2 phosphorylation, suggesting that TG or FF alone is not harmful to hAoSMCs. However, the PPARc agonist (TG) completely abolished both activation of Erk1/2 and subsequent LDLinduced cell proliferation. In contrast, the PPARa agonist (FF) showed no effects on proliferation nor on Erk1/2 phosphorylation. The WST-1 assay showed a pattern similar to that observed in the BrdU assay (Fig. 2B). Treatment with TG decreased LDL-induced cell proliferation
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Fig. 2. The PPARc agonist (TG) abolished the effects of LDL on cell proliferation via deactivation of Erk1/2. (A) hAoSMCs were pretreated with PPAR agonists, TG for PPARc (20 lM) and FF for PPARa (20 lM) for 30 min, and their inhibitory effects on DNA synthesis then were measured by BrdU incorporation assay after an additional 24-h incubation in the presence or absence of LDL (100 lg/ml) (upper). At the same time, Erk1/2 phosphorylation was assessed by Western analysis (lower). n = 6, *P < 0.01 versus control, #P < 0.01 versus LDL. (B) Cells were pretreated with PPARc agonists at the indicated concentrations of FF or TG, and their effects on cell proliferation were measured by WST-1 assay after an additional 24-h incubation in the presence or absence of LDL (100 lg/ml). n = 10, *P < 0.01 versus control, #P < 0.05, ##P < 0.01 versus LDL.
in a dose-dependant manner, whereas FF had no effect on LDL-induced cell proliferation. These results suggest that activation of PPARc, but not of PPARa, plays a beneficial role in LDL-induced proliferation of hAoSMCs via deactivation of Erk1/2. PPARc activation abolished the generation of superoxide(O2 ) evoked by LDL via up-regulation of SOD The findings that PPARc activation completely abolished LDL-induced cell proliferation via deactivation of Erk1/2 prompted us to investigate the molecular mechanisms by which TG exerts its inhibitory effects on LDL-induced Erk1/2 phosphorylation. Therefore, the relationship between ROS and PPARc activation was closely investigated in the present study. The effects of TG or FF on LDL-induced ROS generation were assessed using FACS analysis, measurement of intracellular concentrations of O2 and H2O2 (Fig. 3A, upper), and intracellular SOD activity was assessed following TG treatment
(Fig. 3A, lower). According to the FACS analysis (Fig. 3A, upper), LDL treatment resulted in the generation of both O2 and H2O2. However, only TG specifically inhibited LDL-induced O2 production as much as SOD, which served as a positive control (left). In contrast, FF specifically inhibited the generation of H2O2 as much as CAT, which was used as a positive control (right). When the effects of TG or FF on intracellular concentrations of ROS induced by LDL were assessed (Fig. 3A, lower), similar results were obtained. LDL increased generation of O2 and H2O2 by 4.7- and 7-fold, respectively. Both SOD and TG down-regulated LDL-induced O2 production, whereas CAT and FF down-regulated LDL-induced H2O2 production. In other words, treatment with TG and FF mimicked the effects of SOD and CAT, respectively. Recently, it has been reported that activated PPARs bind to their specific PPREs in the promoter region of target genes, especially the SOD gene [16], implying that PPARc activation may exert its beneficial effects via an
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Fig. 3. The PPARc agonist (TG), not the PPARa agonist (FF), abolished LDL-induced generation of O2 via an increase in SOD activity. (A) hAoSMCs were pretreated with TG (20 lM) or FF (20 lM) 30 min before 24-h LDL treatment (100 lg/ml). Intracellular levels of O2 (left) or H2O2 (right) were measured by both FACS (upper) and spectrophotometry (lower) methods using HE (5 lM) and H2DCF-DA (5 lM) as fluorescent dyes. SOD (500 U/ml) and CAT (500 U/ml) were used as specific scavengers of O2 and H2O2, respectively. n = 10, *P < 0.01 versus control, #P < 0.01 versus LDL. (B) Cells were pretreated with TG in the presence or absence of LDL, and SOD activity in cell lysates then were determined. Exogenous SOD was used as a positive control. Experiments were repeated three times. n = 5, *P < 0.01 versus control, #P < 0.01 versus LDL.
increase in cellular concentrations of SOD. These findings led us to examine whether the down-regulation of O2 , induced by PPARc activation, is related to the up-regulation of SOD. As shown in Fig. 3B, the activity of SOD was decreased by LDL treatment compared with the untreated control, which may explain why LDL increases the level of O2 . However, the activity of SOD was increased by TG treatment as much as by exogenous PEG-SOD, despite LDL treatment. These results suggest that TG exerts its inhibitory effects on LDL-induced cell proliferation via an increase in basal SOD activity. These results are consistent with previous reports concluding that PPARc activation is anti-atherogenic via vascular
CuZn–SOD in hypercholesterolemia [18] and regulates endothelial function via CuZn–SOD in HUVEC [17]. PPARc activation inhibited LDL-induced G1–S phase progression of the cell cycle via down-regulation of active cyclins/CDK complexes and up-regulation of p21 Although beneficial roles of PPARc are well-known, its effects on cell-cycle modulators differ depending on the type of cells and on the stimulus [27]. Therefore, the issue of which cell cycle regulatory molecules are modulated by LDL was examined using Western blot analysis (Fig. 4A, upper). Treatment of hAoSMCs with
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Fig. 4. The PPARc agonist (TG) inhibited LDL-induced G1–S phase progression of the cell cycle via down-regulation of active cyclins/CDK complexes and up-regulation of p21. (A) Cells were treated with LDL (100 lg/ml), and protein levels of cell cycle regulatory molecules, including cyclins, CDKs, and CKIs, then were assessed by Western blot analysis at the indicated times after exposure to LDL. (B) Cells were pretreated with SOD (500 U/ml), TG (20 lM), or PD (20 lM) for 30 min. Protein levels of the indicated cell-cycle modulators then were measured by Western blot analysis 24-h after exposure to LDL.
LDL resulted in a significant increase in the protein levels of cyclinD1/CDK4 and cyclinE/CDK2 complexes in a time-dependent manner, which has been reported to be related to the G1–S progression of the cell cycle [15]. Furthermore, the activity of the up-regulated CDK complexes was tested by measuring phosphorylation of Rb, a target of active CDK. As expected, LDL enhanced phosphorylation of the Rb protein in a time-dependent manner, suggesting that that the up-regulated CDK complexes were functionally active. In addition, the issue of which type of CKI is related to LDL stimulation was assessed (Fig. 4A, lower). Only the levels of p21Cip1 were affected by LDL. The levels of p21Cip1 gradually decreased from 1-h after LDL stimulation. Taken together, these results suggest that LDL induces cell proliferation of hAoSMCs by stimulating the progression of G1–S phase via up-regulation of active cyclinD1/CDK4 and cyclinE/CDK2 complexes and down-regulation of p21Cip1. The results shown Fig. 4A led to the examination of which cell-cycle regulators are affected by PPARc activation. As shown in Fig. 4B, TG, as well as SOD and PD, abolished the up-regulation of active cyclinD1/CDK4 and cyclinE/CDK2 complexes induced by LDL; the amounts of the complexes after treatment with TG, SOD and PD were the same as the controls that were not treated with LDL. Furthermore, TG negated the LDL-induced reduction in p21Cip1; TG co-treatment increased p21Cip1 to levels exceeding those observed for the LDL-untreated control. Taken together, the results of the present study suggest that PPARc activation with TG abolished the effects of LDL via down-regulation of
active CDK complexes and up-regulation of CKI (p21Cip1). These results are consistent with previous reports, which asserted that PPARc ligands affect the proliferation of VSMCs via down-regulation of both cyclin D1 and cyclin E [22] and up-regulation of p21Cip1 [21]. However, the results are inconsistent with those observed in the case of PDGF-induced proliferation of rat AoSMCs, which is caused by up- rather than down-regulation of p21Cip1 [28]. The discrepancy may be due to different mechanisms of cell proliferation between PDGF-BB and LDL. In conclusion, the results of the present study provide a previously unrecognized mechanism for the anti-proliferative effects of PPARc ligands against LDL-induced proliferation in atherosclerotic lesions and support the concept that PPARc ligands may constitute a novel therapeutic approach for the treatment of proliferative cardiovascular diseases. Acknowledgments This work was supported by the intramural Mission 2007 research program of KRIBB and by the 21st century Frontier R&D Program and Chemical Genomics Research Program from the Ministry of Science and Technology of Korea. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc. 2007.06.006.
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