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International Journal of Cardiology 158 (2012) 54–58
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International Journal of Cardiology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j c a r d
Rosiglitazone suppresses lipopolysaccharide-induced matrix metalloproteinase-2 activity in rat aortic endothelial cells via Ras-MEK1/2 signaling Xianghong Wu ⁎, Lang Li Department of Cardiology, the First Affiliated Hospital of Guangxi Medical University, Nanning 530021, China
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Article history: Received 1 September 2010 Received in revised form 13 December 2010 Accepted 30 December 2010 Available online 17 January 2011 Keywords: PPARγ MMP-2 Aortic endothelial cells Ras-MEK1/2 signaling pathways NF-κB
a b s t r a c t Objectives: Matrix metalloproteinases (MMPs) play a key role in the pathogenesis of chronic inflammatory disease, such as atherosclerosis. Among MMPs, MMP-2 is regarded as a major proteinase in atherosclerotic plaque lesions. Peroxisome proliferator activated receptor-gamma (PPARγ) ameliorates oxidative stress and the inflammatory response. The aim of the present study was to evaluate the effect of Rosiglitazone on lipopolysaccharide (LPS)-induced MMP-2 activation as well as its possible mechanism. Methods: Primary culture of rat aortic endothelial cells (RAEC) was derived from male Sprague–Dawley rat. MMP-2 activity was assayed by gelatin zymography. Protein expressions were determined by Western Blotting. DNA binding activity of NF-κB was studied with electrophoretic mobility shift assay. Results: LPS-induced MMP-2 activity was inhibited by Rosiglitazone (PPARγ agonist) in the rat aortic endothelial cells (RAEC). LPS-induced MMP-2 activation was diminished due to exposure to NF-κB Activation Inhibitor II (JSH-23) or Ras inhibitor, farnesylthiosalicylic acid (FTS). Further study shows that LPS-induced activation of Phospho-Ras homologue gene family, member A (Rho A) and Phospho-mitogen-activated protein kinase kinase 1/2 (MEK1/2) were significantly inhibited by Rosiglitazone. The activation of NF-κB p65 in the nuclear extract of cells was also significantly suppressed by Rosiglitazone, moreover, the expression of NF-κB p65 was partly activated by GW9662 (PPARγ antagonist). NF-κB DNA binding activity was also demolished by Rosiglitazone. Conclusions: Our data shows that PPARγ agonist, Rosiglitazone suppresses LPS-activated MMP-2 secretion via Ras-MEK1/2 signaling pathways and NF-κB activation. PPARγ agonist and Ras-MEK1/2 pathway may be another potential therapeutic target for the disease induced by chronic inflammation. © 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Extracellular matrix (ECM) plays a key role in the pathogenesis of atherosclerosis, which is known as a chronic inflammatory disease [1]. Matrix metalloproteinases (MMPs) influence the remodeling of the ECM, such as atherosclerotic lesion formation [2]. MMP/Tissue Inhibitor of Metalloproteinases (TIMP) imbalance and cytokine expression are present in patients with heart failure [3]. The pathogenetic role of MMPs in the process of atherosclerosis includes increasing the migration of vascular smooth muscle cells through the internal elastic lamina into the intimal space, where they proliferate and contribute to plaque formation [2]. Moreover, inhibition of MMPs has been shown to decrease venous neointimal hyperplasia in a porcine model [4]. Circulating MMP-1 levels in patients with chest pain have been associated with atherosclerotic plaque inflammation [5]. Evidence indicates that several MMPs participate in different steps of the ⁎ Corresponding author. Department of Cardiology, The First Affiliated Hospital of Guangxi Medical University, #6 Shuangyong Road, Nanning, Guangxi, 530021, China. Tel.: +86 771 535 6520; fax: +86 771 5350031. E-mail address: [email protected] (X. Wu). 0167-5273/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijcard.2010.12.105
angiogenic response, including MMP-2, MMP-3, MMP-7, MMP-9 and MMP-13 [6,7], especially, MMP-2 plays an important role in angiogenesis and vasculogenesis [8]. Under hypoxic conditions, fibroblasts will convert to myofibroblasts through an MMP-2-mediated pathway [9]. In addition to ECM substrates, gelatinases (including MMP-2 and MMP-9) cleave different inactive bio-molecules to active types, which also play a role in the inflammatory modulation of atherosclerosis, such as cytokines (pro-Tumor growth factor-β1 [10], pro-Tumor necrosis factor-α [11], and pro-Interleukin-1β [12]), chemokines (Interleukin8 [13]), and the vasoconstrictor endothelin-1 [14]. Peroxisome proliferator activated receptor-gamma (PPARγ) is a nuclear receptor highly expressed in the gastrointestinal tract and plays a key role in inflammation [15]. PPAR ameliorates oxidative stress and the inflammatory response [16]. One of PPARγ agonist, Rosiglitazone has beneficial effect on post-infarct ventricular remodeling [17]. Rosiglitazone also reduces neointimal hyperplasia via activation of glycogen synthase kinase-3 beta followed by inhibition of MMP-9 [18]. Another PPARγ agonist Ciglitazon mitigated the MMP2, MMP-9 and MMP-13 protein levels of left ventricular in chronic pressure overload myocardium [19]. However, the effect of PPARγ on MMP-2 activity as well as its possible mechanism in the aortic
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endothelial cells is not fully understood. MMPs expression is regulated at the transcriptional level by modulation of the activation of transcription factors such as activator protein 1 (AP-1) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) [20–22]. Lipopolysaccharide (LPS), which is released from Gramnegative bacteria, aggravates atherosclerosis in humans and rodents by inducing inflammatory response in the arterial vascular wall [23,24]. Because MMP-2 is a major proteinase in atherosclerotic plaque lesions [25], therefore, the aim of this study was to evaluate the effect of Rosiglitazone on LPS-induced MMP-2 activation as well as its possible mechanism. Our data showed that PPARγ agonist, Rosiglitazone suppresses LPS-activated MMP-2 secretion via Ras-MEK1/2 signaling pathways.
Protein Extraction Kit (Novagen) and analyzed by EMSA as described [28]. Briefly, the double-stranded oligonucleotides (Santa Cruz Biotechnology) containing the consensus sequences of the binding sites for NF-κB (5′-AGT TGA GGG GAC TTT CCC AGG C-3′) was labeled with biotin at 5′ end. Binding reactions were performed in a 20 μl volume containing 2 μg of nuclear extracts, 1 μl of 1 μg/μl Poly (dI.dC), 1 μl of 100 mM MgCl2, 1 μl of 50% Glycerol, 1 μl of 1% NP-40 and 2 μl of 20 fM biotin-labeled probe following the instruction of LightShift chemiluminescent EMSA kit (Thermo Scientific Pierce Protein Research Products). The binding reactions were incubated at room temperature for 20 min before loading. Protein–DNA complexes were analyzed on a non-denaturing 5% polyacrylamide gel using 0.5× TBE (Tris, boric acid, ethylenediaminetetraacetic acid) buffer for 60 min. The binding reactions were transferred to Nylon Membrane at 380 mA for 60 min. Cross-linking transferred DNA to membrane for 20 s with a UV Stratalinker (Stratagene). The biotin-labeled DNA in the membrane was blocked for 15 min and detected by Chemiluminescent Nucleic Acid Detection Module (Thermo Scientific Pierce Protein Research Products). The images were taken by using Kodak Image Station 2000R.
2. Methods
2.5. Statistical analysis
2.1. Primary culture of rat aortic endothelial cells
The intensity of the bands corresponding to specific protein or DNA binding was determined by Image J software (NIH, Baltimore, MD). Routine statistical analysis was completed using Sigma-Stat 2.03 (SPSS, Chicago, IL, USA). One-way ANOVA was used to compare mean responses among the treatments. Statistical probability of p b 0.05 was considered to be significant.
Rat aortic endothelial cells (RAEC) were derived from male Sprague–Dawley rat (180–200 g, Guangxi Medical University, China) aortic endothelium. RAEC were isolated as described earlier [26]. In brief, segments of thoracic aortae (18–24 mm) were excised and immediately put in cold Hanks’ Balanced Salt Solution (HBSS). The blood residues in the lumen of the vessels were flushed with HBSS. 1 mg/ml collagenase (Sigma) was used to fill the lumen of vessels and incubated in HBSS at 37 °C for 20 min. The effluent from the lumen of vessels was collected and centrifuged at 2800 rpm for 5 min. The pellet was washed and suspended in RPMI 1640 (Invitrogen) with 20% fetal bovine serum (FBS) and 1% penicillin–streptomycin (Invitrogen) at 37 °C in a 95% air/5% CO2 incubator. Experiments were performed on cells at passages 5–8. All dishes were coated with 3% Collagen Type I (BD Biosciences) during the RAEC cultures. 2.2. MMP-2 activity assay MMP-2 activity was detected by gelatin zymography [27] on pre-made 10% polyacrylamide gels containing 0.1% gelatin using 10 μl serum-free media from treated cultures according to the instruction provided by the manufacturer (Invitrogen). In brief, Confluent RAEC cells in 6-well were pretreated with Rosiglitazone (10 μM; Alexis Biochemicals), or NF-κB Activation Inhibitor II (JSH23, 30 μM, Santa Cruz), or Ras inhibitor, farnesylthiosalicylic acid (FTS, 20 μM, Sigma) in serum-free media for 2 h, followed by co-exposure to 1 μg/ml Lipopolysaccharide (Sigma) and Rosiglitazone/JSH-23/FTS for 12 h or 24 h. After electrophoresis, the gel was removed and incubated in 1× Zymogram Renaturing Buffer for 30 min at room temperature with gentle agitation. The gel was equilibrated for 30 min with 1× Zymogram Developing Buffer, and then incubated with fresh 1× Zymogram Developing Buffer overnight. The bands were visualized by staining for 30–60 min with a solution containing 0.1% Coomassie R-250 in 40% ethanol and 10% acetic acid; followed by destaining for 2 h at room temperature in a solution containing 10% ethanol and 7.5% acetic acid. The images were taken by using Kodak Image Station 2000R. 2.3. Western blotting Confluent RAEC cells cultured on 6-well plates were pretreated to Rosiglitazone (10 μM; Alexis Biochemicals) or GW9662 (1 μM; Alexis Biochemicals) for 2 h, and then co-exposure to 1 μg/ml Lipopolysaccharide and Rosiglitazone/GW9662 for 3 h. Protein was extracted using the RIPA lysis buffer (Santa Cruz Biotechnology) and centrifuged at 15,000 g for 15 min. The supernatants were collected and protein concentrations were determined using BCA™ Protein Assay Kit (Pierce,). An aliquot of 30 μg protein from each sample was separated on 10% Tris–HCL Ready sodium dodecyl sulfate (SDS)polyacrylamide gel (Bio-Rad Laboratories), transferred onto nitrocellulose membrane (Bio-Rad Laboratories), which was incubated with 3% skimmed milk in Tris-buffered saline solution for 1 h, subsequently incubated with respective primary antibodies at 4 °C overnight, then with a peroxidase-conjugated anti-rabbit secondary antibody (Sigma) at room temperature for 120 min, finally visualized using a chemiluminescence kit (Amersham Biosciences). Anti-total- Ras homologue gene family, member A (Rho A), anti-Phospho-mitogen-activated protein kinase kinase 1/2 (p-MEK 1/2), and anti-total-MEK 1/2 were from Cell Signaling Technology. Monoclonal Anti-β-Actin– Peroxidase antibody was purchased from Sigma. All secondary antibodies, anti-NF-κB (Anti-p65) antibody, anti-Phospho-Rho A (p-Rho A) and NF-κB Activation Inhibitor II (JSH-23) were from Santa Cruz Biotechnology. The images were taken by using Kodak Image Station 2000R. 2.4. Electrophoretic mobility shift assay (EMSA) Confluent RAEC cells cultured on 6-well plates were pretreated to Rosiglitazone (10 μM; Alexis Biochemicals) for 2 h, and then co-exposure to 1 μg/ml Lipopolysaccharide and Rosiglitazone for 3 h. Nuclear extracts were prepared using NucBuster™
3. Results 3.1. PPARγ agonist suppresses LPS-induced-MMP-2 activities in the RAEC cells To determine the activity of secreted MMP-2 in the culture media, zymography was performed. As indicated in Fig. 1, MMP-2 activities were activated by LPS in the RAEC cells in the time-dependent experiments. MMP-2 activity increased with the time of exposure to LPS, and reached maximum at 24 h. However, MMP-2 activation induced by LPS was suppressed by PPARγ agonist (Rosiglitazone), especially at 24 h. 3.2. Ras-MEK1/2 signaling is involved in the LPS-induced MMP-2 activity To study whether Ras-MEK1/2 signaling is involved in the LPSinduced MMP-2 activity, NF-κB Activation Inhibitor II (JSH-23, 30 μM) and Ras inhibitor (FTS, 20 μM) were applied in the experiment. MMP2 activity was measured by zymography after exposure to 1 μg/ml LPS for 24 h with serum-free media. LPS-induced MMP-2 activation was almost diminished with the treatment of JSH-23 as well as FTS (Fig. 2). 3.3. PPARγ agonist attenuates LPS-induced MMP-2 activation via the Ras-MEK1/2 signaling Based on our results from zymography and the data from other group [29], the Ras-MEK1 pathway plays a key role in the activation of MMPs secretion. To further address the mechanism of PPARγ inhibiting LPS-induced MMP-2 activities, Ras-MEK1/2 signaling was studied. LPS-induced activation of Phospho-Rho A (p-Rho A) was significantly inhibited by Rosiglitazone (Fig. 3A). We next studied the role of MEK1, a downstream effector for Ras in the regulation of MMPs secretion. As shown in Fig. 3B, Phospho-MEK1/2 (p-MEK1/2) activated by LPS was also suppressed predominantly by Rosiglitazone. 3.4. PPARγ agonist inhibits LPS-induced NF-κB activity Activation of Ras-MEK1 pathway regulates subsequently the activities of nuclear transcription factors, such as NF-κB in MMPs transcription [30]. NF-κB is the main transcription factor that regulates inflammation. MMP-9 is crucially regulated at the transcriptional level by important transcription factors such as NF-κB [31]. Based on these data, we hypotheses that NF-κB is involved in LPS-induced MMP-2 activation and PPARγ agonist suppresses LPS-induced MMP-2 by inhibiting the activation of NF-κB. Therefore, p65, the NF-κB binding
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Fig. 1. LPS time-dependently increases MMP-2 activity in Rat aortic endothelial cells (RAEC). Confluent RAEC cells in 6-well were pretreated with 10 μM Rosiglitazone in serum-free media for 2 h, followed by co-exposure to 1 μg/ml LPS and 10 μM Rosiglitazone for 12 h or 24 h. The changes in MMP-2 activity were demonstrated in zymography. The blots represent MMP-2 activity, expressed as percentages of the amount of control (12 h). Results represent the mean ± SEM of 3 separate experiments. **, p b 0.001 vs. control. #, p b 0.05 vs. LPS treatment (24 h); ##, p b 0.001 vs. LPS treatment (48 h).
subunit in the nuclear extracts of cells was used as evaluating the NFκB- activity. As indicated in Fig. 4, NF-κB was induced by LPS in the RAEC cells and inhibited by Rosiglitazone. Moreover, NF-κB activity was partly activated by PPARγ antagonist of GW9662. To further study the role of NF-κB in the regulation of MMPs expression, DNA binding activity of NF-κB was studied with EMSA. The DNA binding activity of NF-κB was significantly activated by LPS, but co-treatment with LPS and Rosiglitazone completely demolished this effect (Fig. 5).
MMP-1 secretion; however, troglitazone at high concentrations (N=30 mmol/l) inhibited MMP-1 protein synthesis [39]. This indicates different PPARγ agonists with different concentrations have different effects on MMP-1 expression. The effect of PPARγ agonist on the activity of MMP-2 as well as its mechanism is not fully understood.
4. Discussion MMP-2 or MMP-9 levels in plasma/serum are increased after acute cardiovascular events and have been suggested to be useful biomarkers to detect the group with high risk of atherosclerotic disease. This includes the evaluation of lesions and identification those at highest risk of a heart or neurologic event [32,33]. PPARs have been reported to regulate inflammatory responses [34]. PPARγ agonists may demonstrate anti-inflammatory action [35,36] by down-regulating NF-kB transcription [36,37]. Therefore, inhibition of NF-kB activation by PPARγ agonist may well reduce the expression of pro-inflammatory mediators, including tumor necrosis factor α, Interleukin-1, etc. Previous data indicate that one of the natural ligands for PPARγ, 15-deoxyprostaglandin J2 can inhibit MMP-13 synthesis [38]. Another PPARγ agonist, Troglitazone at physiological concentrations (5–15 mmol/l), but not pioglitazone and rosiglitazone, stimulated
Fig. 2. Ras-MEK1/2 signaling is involved in the MMP-2 secretion. Confluent RAEC cells in 6-well were pretreated with 30 μM JSH-23 (NF-κB Activation Inhibitor II), or 20 μM FTS (Ras inhibitor) in serum-free media for 2 h, followed by co-exposure to 1 μg/ml LPS and JSH-23 or FTS for 24 h. LPS-induced MMP-2 activation measured by zymography was almost diminished with the treatment of JSH-23 as well as FTS. The blots represent MMP-2 activity, expressed as percentages of the amount of control. Results represent the mean ± SEM of 3 separate experiments. **, p b 0.001 vs. control; ## p b 0.001 vs. LPS treatment.
Fig. 3. PPARγ agonist attenuates LPS-induced MMP-2 activation via the Ras-MEK1/2 signaling. Confluent RAEC cells in 6-well were pretreated with 10 μM Rosiglitazone in serum-free media for 2 h, followed by co-exposure to 1 μg/ml LPS and 10 μM Rosiglitazone for 3 h. LPS-induced activation of p-Rho A was significantly inhibited by Rosiglitazone (A). p-MEK1/2 activated by LPS was also suppressed predominantly by Rosiglitazone (B). The blots represent protein expressions, expressed as percentages of the amount of control. Results represent the mean ± SEM of 3 separate experiments. **, p b 0.001 vs. control. #, p b 0.05 vs. LPS treatment.
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Therefore, we firstly studied the relation of PPARγ and the MMP-2 activity activated by LPS, and found that LPS-induced MMP-2 activation was inhibited by Rosiglitazone (Fig. 1). Transcriptional regulation is important in the synthesis and release of MMPs. Two major cis-acting elements are found in most MMP promoters: AP-1 and Polyomavirus Enhancer Activator-3 (PEA-3) which interact with the Fos, Jun and Ets family. The activity of Ets transcription factors was regulated by extracellular signal activated protein Kinase (ERK) 1/2 / c-Jun activated protein kinase (JNK) / stress activated protein kinase (SAPK) and p38 mitogen-activated protein kinase pathways [40–42]. Activation of Ras has also been shown to trans-activate ETS-1 and ETS-2, and ERK1/2 and JNK / SAPK pathways [43]. Activation of Jak/Vav/Rho GTPase pathway by chemokine (C-X-C motif) ligand 12 is a key signaling event for membrane-type (MT) 1-MMP/MMP-2-dependent melanoma cell invasion [44]. Both MEK and ERK are mitogen-activated kinases. So secondly, we studied whether Ras-MEK1/2 signaling is involved in the LPS-induced MMP-2 activity. As shown in Fig. 2, LPS-induced MMP-2 activation was almost diminished no matter the exposure to JSH-23 or FTS. Further study shows that LPS-induced activation of p-Rho A and p-MEK1/2 were significantly inhibited by Rosiglitazone (Fig. 3). Similarly, another PPARγ agonist, Troglitazone inhibited vascular endothelial growth factor (VEGF)-induced MMP-2 and MT1-MMP expression through the suppression of VEGF-induced ROS production and ERK phosphorylation [45]. Since NF-κB activation is required for the expression of MMP-1 and MMP-13, as well as inflammatory stimuli such as Interleukin-1, Interleukin-6 and tumor necrosis factor α [46,47]. Activation of RasMEK1 pathway regulates subsequently the activities of NF-κB in MMPs transcription [30], so the expression of NF-κB in the nuclear of RAEC cells was studied. The gene expression and activity of MMP-2 and MMP-9 in macrophages are reduced through PPARγ-dependent inhibition of NF-κB [48]. Our results are in accordance to this finding. The protein expression of NF-κB major binding subunit (p65) in the nuclear of RAEC cells was significantly inhibited by Rosiglitazone. Moreover, the activity of NF-κB was partly activated by PPARγ antagonist of GW9662 (Fig. 4). In line with data of protein expression, the result from EMSA also showed that Rosiglitazone downregulated the DNA binding activity of NF-κB induced by LPS (Fig. 5). In summary, LPS significantly activated MMP-2 secretion in the RAEC cells. Co-treatment of LPS and Rosiglitazone indicates the
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Fig. 5. PPARγ agonist inhibits NF-κB DNA binding activity. Confluent RAEC cells in 6well were pretreated with 10 μM Rosiglitazone in serum-free media for 2 h, followed by co-exposure to 1 μg/ml LPS and Rosiglitazone for 3 h. The DNA binding activity of NF-κB detected by EMSA was significantly activated by LPS, but was completely demolished by Rosiglitazone. The blots represent protein expressions, expressed as percentages of the amount of control. Results represent the mean ± SEM of 3 separate experiments. **, p b 0.001 vs. control, ## p b 0.001 vs LPS treatment.
inhibitory role of Rosiglitazone in MMP-2 activation. Moreover, both inhibitors of NF-κB and Ras respectively attenuated MMP-2 secretion induced by LPS in the RAEC cells. Further study showed that LPSinduced activation of p-Rho A and p-MEK1/2 was significantly inhibited by Rosiglitazone. The protein expression of NF-κB p65 was significantly suppressed by PPARγ agonist, and partly activated by PPARγ antagonist. In consistent with these result, LPS-induced NF-κB DNA binding activity was also demolished by Rosiglitazone. Taken together, downregulation of Ras-MEK1/2 and NF-κB activation indicates the possible pathway by which Rosiglitazone exerts its inhibitory effect on MMP-2 regulation in the RAEC cells, leading to the decreased DNA binding activity of NF-κB, disrupting the transcription of the MMP-2 gene. PPARγ agonist and Ras-MEK1/2 pathway may be another potential therapeutic target for the disease induced by chronic inflammation, such as atherosclerosis. Acknowledgements This work was supported by the Nature Science Foundation of Guangxi Province, China (GKQ 0542049). The authors of this manuscript have certified that they comply with the Principles of Ethical Publishing in the International Journal of Cardiology [49]. References
Fig. 4. PPARγ agonist inhibits NF-κB protein expression. Confluent RAEC cells in 6-well were pretreated with 10 μM Rosiglitazone or 1 μM GW9662 in serum-free media for 2 h, followed by co-exposure to 1 μg/ml LPS and Rosiglitazone or GW9662 for 3 h. NFκB was induced by LPS in the RAEC cells and inhibited by Rosiglitazone . Moreover, NFκB activity was partly induced by PPARγ antagonist of GW9662. The blots represent protein expressions, expressed as percentages of the amount of control. Results represent the mean ± SEM of 3 separate experiments. **, p b 0.001 vs. control. #, p b 0.05 vs. LPS treatment, ## p b 0.001 vs LPS treatment.
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International Journal of Cardiology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / i j c a r d
Rosiglitazone suppresses lipopolysaccharide-induced matrix metalloproteinase-2 activity in rat aortic endothelial cells via Ras-MEK1/2 signaling Xianghong Wu ⁎, Lang Li Department of Cardiology, the First Affiliated Hospital of Guangxi Medical University, Nanning 530021, China
a r t i c l e
i n f o
Article history: Received 1 September 2010 Received in revised form 13 December 2010 Accepted 30 December 2010 Available online 17 January 2011 Keywords: PPARγ MMP-2 Aortic endothelial cells Ras-MEK1/2 signaling pathways NF-κB
a b s t r a c t Objectives: Matrix metalloproteinases (MMPs) play a key role in the pathogenesis of chronic inflammatory disease, such as atherosclerosis. Among MMPs, MMP-2 is regarded as a major proteinase in atherosclerotic plaque lesions. Peroxisome proliferator activated receptor-gamma (PPARγ) ameliorates oxidative stress and the inflammatory response. The aim of the present study was to evaluate the effect of Rosiglitazone on lipopolysaccharide (LPS)-induced MMP-2 activation as well as its possible mechanism. Methods: Primary culture of rat aortic endothelial cells (RAEC) was derived from male Sprague–Dawley rat. MMP-2 activity was assayed by gelatin zymography. Protein expressions were determined by Western Blotting. DNA binding activity of NF-κB was studied with electrophoretic mobility shift assay. Results: LPS-induced MMP-2 activity was inhibited by Rosiglitazone (PPARγ agonist) in the rat aortic endothelial cells (RAEC). LPS-induced MMP-2 activation was diminished due to exposure to NF-κB Activation Inhibitor II (JSH-23) or Ras inhibitor, farnesylthiosalicylic acid (FTS). Further study shows that LPS-induced activation of Phospho-Ras homologue gene family, member A (Rho A) and Phospho-mitogen-activated protein kinase kinase 1/2 (MEK1/2) were significantly inhibited by Rosiglitazone. The activation of NF-κB p65 in the nuclear extract of cells was also significantly suppressed by Rosiglitazone, moreover, the expression of NF-κB p65 was partly activated by GW9662 (PPARγ antagonist). NF-κB DNA binding activity was also demolished by Rosiglitazone. Conclusions: Our data shows that PPARγ agonist, Rosiglitazone suppresses LPS-activated MMP-2 secretion via Ras-MEK1/2 signaling pathways and NF-κB activation. PPARγ agonist and Ras-MEK1/2 pathway may be another potential therapeutic target for the disease induced by chronic inflammation. © 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Extracellular matrix (ECM) plays a key role in the pathogenesis of atherosclerosis, which is known as a chronic inflammatory disease [1]. Matrix metalloproteinases (MMPs) influence the remodeling of the ECM, such as atherosclerotic lesion formation [2]. MMP/Tissue Inhibitor of Metalloproteinases (TIMP) imbalance and cytokine expression are present in patients with heart failure [3]. The pathogenetic role of MMPs in the process of atherosclerosis includes increasing the migration of vascular smooth muscle cells through the internal elastic lamina into the intimal space, where they proliferate and contribute to plaque formation [2]. Moreover, inhibition of MMPs has been shown to decrease venous neointimal hyperplasia in a porcine model [4]. Circulating MMP-1 levels in patients with chest pain have been associated with atherosclerotic plaque inflammation [5]. Evidence indicates that several MMPs participate in different steps of the ⁎ Corresponding author. Department of Cardiology, The First Affiliated Hospital of Guangxi Medical University, #6 Shuangyong Road, Nanning, Guangxi, 530021, China. Tel.: +86 771 535 6520; fax: +86 771 5350031. E-mail address: [email protected] (X. Wu). 0167-5273/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.ijcard.2010.12.105
angiogenic response, including MMP-2, MMP-3, MMP-7, MMP-9 and MMP-13 [6,7], especially, MMP-2 plays an important role in angiogenesis and vasculogenesis [8]. Under hypoxic conditions, fibroblasts will convert to myofibroblasts through an MMP-2-mediated pathway [9]. In addition to ECM substrates, gelatinases (including MMP-2 and MMP-9) cleave different inactive bio-molecules to active types, which also play a role in the inflammatory modulation of atherosclerosis, such as cytokines (pro-Tumor growth factor-β1 [10], pro-Tumor necrosis factor-α [11], and pro-Interleukin-1β [12]), chemokines (Interleukin8 [13]), and the vasoconstrictor endothelin-1 [14]. Peroxisome proliferator activated receptor-gamma (PPARγ) is a nuclear receptor highly expressed in the gastrointestinal tract and plays a key role in inflammation [15]. PPAR ameliorates oxidative stress and the inflammatory response [16]. One of PPARγ agonist, Rosiglitazone has beneficial effect on post-infarct ventricular remodeling [17]. Rosiglitazone also reduces neointimal hyperplasia via activation of glycogen synthase kinase-3 beta followed by inhibition of MMP-9 [18]. Another PPARγ agonist Ciglitazon mitigated the MMP2, MMP-9 and MMP-13 protein levels of left ventricular in chronic pressure overload myocardium [19]. However, the effect of PPARγ on MMP-2 activity as well as its possible mechanism in the aortic
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endothelial cells is not fully understood. MMPs expression is regulated at the transcriptional level by modulation of the activation of transcription factors such as activator protein 1 (AP-1) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) [20–22]. Lipopolysaccharide (LPS), which is released from Gramnegative bacteria, aggravates atherosclerosis in humans and rodents by inducing inflammatory response in the arterial vascular wall [23,24]. Because MMP-2 is a major proteinase in atherosclerotic plaque lesions [25], therefore, the aim of this study was to evaluate the effect of Rosiglitazone on LPS-induced MMP-2 activation as well as its possible mechanism. Our data showed that PPARγ agonist, Rosiglitazone suppresses LPS-activated MMP-2 secretion via Ras-MEK1/2 signaling pathways.
Protein Extraction Kit (Novagen) and analyzed by EMSA as described [28]. Briefly, the double-stranded oligonucleotides (Santa Cruz Biotechnology) containing the consensus sequences of the binding sites for NF-κB (5′-AGT TGA GGG GAC TTT CCC AGG C-3′) was labeled with biotin at 5′ end. Binding reactions were performed in a 20 μl volume containing 2 μg of nuclear extracts, 1 μl of 1 μg/μl Poly (dI.dC), 1 μl of 100 mM MgCl2, 1 μl of 50% Glycerol, 1 μl of 1% NP-40 and 2 μl of 20 fM biotin-labeled probe following the instruction of LightShift chemiluminescent EMSA kit (Thermo Scientific Pierce Protein Research Products). The binding reactions were incubated at room temperature for 20 min before loading. Protein–DNA complexes were analyzed on a non-denaturing 5% polyacrylamide gel using 0.5× TBE (Tris, boric acid, ethylenediaminetetraacetic acid) buffer for 60 min. The binding reactions were transferred to Nylon Membrane at 380 mA for 60 min. Cross-linking transferred DNA to membrane for 20 s with a UV Stratalinker (Stratagene). The biotin-labeled DNA in the membrane was blocked for 15 min and detected by Chemiluminescent Nucleic Acid Detection Module (Thermo Scientific Pierce Protein Research Products). The images were taken by using Kodak Image Station 2000R.
2. Methods
2.5. Statistical analysis
2.1. Primary culture of rat aortic endothelial cells
The intensity of the bands corresponding to specific protein or DNA binding was determined by Image J software (NIH, Baltimore, MD). Routine statistical analysis was completed using Sigma-Stat 2.03 (SPSS, Chicago, IL, USA). One-way ANOVA was used to compare mean responses among the treatments. Statistical probability of p b 0.05 was considered to be significant.
Rat aortic endothelial cells (RAEC) were derived from male Sprague–Dawley rat (180–200 g, Guangxi Medical University, China) aortic endothelium. RAEC were isolated as described earlier [26]. In brief, segments of thoracic aortae (18–24 mm) were excised and immediately put in cold Hanks’ Balanced Salt Solution (HBSS). The blood residues in the lumen of the vessels were flushed with HBSS. 1 mg/ml collagenase (Sigma) was used to fill the lumen of vessels and incubated in HBSS at 37 °C for 20 min. The effluent from the lumen of vessels was collected and centrifuged at 2800 rpm for 5 min. The pellet was washed and suspended in RPMI 1640 (Invitrogen) with 20% fetal bovine serum (FBS) and 1% penicillin–streptomycin (Invitrogen) at 37 °C in a 95% air/5% CO2 incubator. Experiments were performed on cells at passages 5–8. All dishes were coated with 3% Collagen Type I (BD Biosciences) during the RAEC cultures. 2.2. MMP-2 activity assay MMP-2 activity was detected by gelatin zymography [27] on pre-made 10% polyacrylamide gels containing 0.1% gelatin using 10 μl serum-free media from treated cultures according to the instruction provided by the manufacturer (Invitrogen). In brief, Confluent RAEC cells in 6-well were pretreated with Rosiglitazone (10 μM; Alexis Biochemicals), or NF-κB Activation Inhibitor II (JSH23, 30 μM, Santa Cruz), or Ras inhibitor, farnesylthiosalicylic acid (FTS, 20 μM, Sigma) in serum-free media for 2 h, followed by co-exposure to 1 μg/ml Lipopolysaccharide (Sigma) and Rosiglitazone/JSH-23/FTS for 12 h or 24 h. After electrophoresis, the gel was removed and incubated in 1× Zymogram Renaturing Buffer for 30 min at room temperature with gentle agitation. The gel was equilibrated for 30 min with 1× Zymogram Developing Buffer, and then incubated with fresh 1× Zymogram Developing Buffer overnight. The bands were visualized by staining for 30–60 min with a solution containing 0.1% Coomassie R-250 in 40% ethanol and 10% acetic acid; followed by destaining for 2 h at room temperature in a solution containing 10% ethanol and 7.5% acetic acid. The images were taken by using Kodak Image Station 2000R. 2.3. Western blotting Confluent RAEC cells cultured on 6-well plates were pretreated to Rosiglitazone (10 μM; Alexis Biochemicals) or GW9662 (1 μM; Alexis Biochemicals) for 2 h, and then co-exposure to 1 μg/ml Lipopolysaccharide and Rosiglitazone/GW9662 for 3 h. Protein was extracted using the RIPA lysis buffer (Santa Cruz Biotechnology) and centrifuged at 15,000 g for 15 min. The supernatants were collected and protein concentrations were determined using BCA™ Protein Assay Kit (Pierce,). An aliquot of 30 μg protein from each sample was separated on 10% Tris–HCL Ready sodium dodecyl sulfate (SDS)polyacrylamide gel (Bio-Rad Laboratories), transferred onto nitrocellulose membrane (Bio-Rad Laboratories), which was incubated with 3% skimmed milk in Tris-buffered saline solution for 1 h, subsequently incubated with respective primary antibodies at 4 °C overnight, then with a peroxidase-conjugated anti-rabbit secondary antibody (Sigma) at room temperature for 120 min, finally visualized using a chemiluminescence kit (Amersham Biosciences). Anti-total- Ras homologue gene family, member A (Rho A), anti-Phospho-mitogen-activated protein kinase kinase 1/2 (p-MEK 1/2), and anti-total-MEK 1/2 were from Cell Signaling Technology. Monoclonal Anti-β-Actin– Peroxidase antibody was purchased from Sigma. All secondary antibodies, anti-NF-κB (Anti-p65) antibody, anti-Phospho-Rho A (p-Rho A) and NF-κB Activation Inhibitor II (JSH-23) were from Santa Cruz Biotechnology. The images were taken by using Kodak Image Station 2000R. 2.4. Electrophoretic mobility shift assay (EMSA) Confluent RAEC cells cultured on 6-well plates were pretreated to Rosiglitazone (10 μM; Alexis Biochemicals) for 2 h, and then co-exposure to 1 μg/ml Lipopolysaccharide and Rosiglitazone for 3 h. Nuclear extracts were prepared using NucBuster™
3. Results 3.1. PPARγ agonist suppresses LPS-induced-MMP-2 activities in the RAEC cells To determine the activity of secreted MMP-2 in the culture media, zymography was performed. As indicated in Fig. 1, MMP-2 activities were activated by LPS in the RAEC cells in the time-dependent experiments. MMP-2 activity increased with the time of exposure to LPS, and reached maximum at 24 h. However, MMP-2 activation induced by LPS was suppressed by PPARγ agonist (Rosiglitazone), especially at 24 h. 3.2. Ras-MEK1/2 signaling is involved in the LPS-induced MMP-2 activity To study whether Ras-MEK1/2 signaling is involved in the LPSinduced MMP-2 activity, NF-κB Activation Inhibitor II (JSH-23, 30 μM) and Ras inhibitor (FTS, 20 μM) were applied in the experiment. MMP2 activity was measured by zymography after exposure to 1 μg/ml LPS for 24 h with serum-free media. LPS-induced MMP-2 activation was almost diminished with the treatment of JSH-23 as well as FTS (Fig. 2). 3.3. PPARγ agonist attenuates LPS-induced MMP-2 activation via the Ras-MEK1/2 signaling Based on our results from zymography and the data from other group [29], the Ras-MEK1 pathway plays a key role in the activation of MMPs secretion. To further address the mechanism of PPARγ inhibiting LPS-induced MMP-2 activities, Ras-MEK1/2 signaling was studied. LPS-induced activation of Phospho-Rho A (p-Rho A) was significantly inhibited by Rosiglitazone (Fig. 3A). We next studied the role of MEK1, a downstream effector for Ras in the regulation of MMPs secretion. As shown in Fig. 3B, Phospho-MEK1/2 (p-MEK1/2) activated by LPS was also suppressed predominantly by Rosiglitazone. 3.4. PPARγ agonist inhibits LPS-induced NF-κB activity Activation of Ras-MEK1 pathway regulates subsequently the activities of nuclear transcription factors, such as NF-κB in MMPs transcription [30]. NF-κB is the main transcription factor that regulates inflammation. MMP-9 is crucially regulated at the transcriptional level by important transcription factors such as NF-κB [31]. Based on these data, we hypotheses that NF-κB is involved in LPS-induced MMP-2 activation and PPARγ agonist suppresses LPS-induced MMP-2 by inhibiting the activation of NF-κB. Therefore, p65, the NF-κB binding
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Fig. 1. LPS time-dependently increases MMP-2 activity in Rat aortic endothelial cells (RAEC). Confluent RAEC cells in 6-well were pretreated with 10 μM Rosiglitazone in serum-free media for 2 h, followed by co-exposure to 1 μg/ml LPS and 10 μM Rosiglitazone for 12 h or 24 h. The changes in MMP-2 activity were demonstrated in zymography. The blots represent MMP-2 activity, expressed as percentages of the amount of control (12 h). Results represent the mean ± SEM of 3 separate experiments. **, p b 0.001 vs. control. #, p b 0.05 vs. LPS treatment (24 h); ##, p b 0.001 vs. LPS treatment (48 h).
subunit in the nuclear extracts of cells was used as evaluating the NFκB- activity. As indicated in Fig. 4, NF-κB was induced by LPS in the RAEC cells and inhibited by Rosiglitazone. Moreover, NF-κB activity was partly activated by PPARγ antagonist of GW9662. To further study the role of NF-κB in the regulation of MMPs expression, DNA binding activity of NF-κB was studied with EMSA. The DNA binding activity of NF-κB was significantly activated by LPS, but co-treatment with LPS and Rosiglitazone completely demolished this effect (Fig. 5).
MMP-1 secretion; however, troglitazone at high concentrations (N=30 mmol/l) inhibited MMP-1 protein synthesis [39]. This indicates different PPARγ agonists with different concentrations have different effects on MMP-1 expression. The effect of PPARγ agonist on the activity of MMP-2 as well as its mechanism is not fully understood.
4. Discussion MMP-2 or MMP-9 levels in plasma/serum are increased after acute cardiovascular events and have been suggested to be useful biomarkers to detect the group with high risk of atherosclerotic disease. This includes the evaluation of lesions and identification those at highest risk of a heart or neurologic event [32,33]. PPARs have been reported to regulate inflammatory responses [34]. PPARγ agonists may demonstrate anti-inflammatory action [35,36] by down-regulating NF-kB transcription [36,37]. Therefore, inhibition of NF-kB activation by PPARγ agonist may well reduce the expression of pro-inflammatory mediators, including tumor necrosis factor α, Interleukin-1, etc. Previous data indicate that one of the natural ligands for PPARγ, 15-deoxyprostaglandin J2 can inhibit MMP-13 synthesis [38]. Another PPARγ agonist, Troglitazone at physiological concentrations (5–15 mmol/l), but not pioglitazone and rosiglitazone, stimulated
Fig. 2. Ras-MEK1/2 signaling is involved in the MMP-2 secretion. Confluent RAEC cells in 6-well were pretreated with 30 μM JSH-23 (NF-κB Activation Inhibitor II), or 20 μM FTS (Ras inhibitor) in serum-free media for 2 h, followed by co-exposure to 1 μg/ml LPS and JSH-23 or FTS for 24 h. LPS-induced MMP-2 activation measured by zymography was almost diminished with the treatment of JSH-23 as well as FTS. The blots represent MMP-2 activity, expressed as percentages of the amount of control. Results represent the mean ± SEM of 3 separate experiments. **, p b 0.001 vs. control; ## p b 0.001 vs. LPS treatment.
Fig. 3. PPARγ agonist attenuates LPS-induced MMP-2 activation via the Ras-MEK1/2 signaling. Confluent RAEC cells in 6-well were pretreated with 10 μM Rosiglitazone in serum-free media for 2 h, followed by co-exposure to 1 μg/ml LPS and 10 μM Rosiglitazone for 3 h. LPS-induced activation of p-Rho A was significantly inhibited by Rosiglitazone (A). p-MEK1/2 activated by LPS was also suppressed predominantly by Rosiglitazone (B). The blots represent protein expressions, expressed as percentages of the amount of control. Results represent the mean ± SEM of 3 separate experiments. **, p b 0.001 vs. control. #, p b 0.05 vs. LPS treatment.
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Therefore, we firstly studied the relation of PPARγ and the MMP-2 activity activated by LPS, and found that LPS-induced MMP-2 activation was inhibited by Rosiglitazone (Fig. 1). Transcriptional regulation is important in the synthesis and release of MMPs. Two major cis-acting elements are found in most MMP promoters: AP-1 and Polyomavirus Enhancer Activator-3 (PEA-3) which interact with the Fos, Jun and Ets family. The activity of Ets transcription factors was regulated by extracellular signal activated protein Kinase (ERK) 1/2 / c-Jun activated protein kinase (JNK) / stress activated protein kinase (SAPK) and p38 mitogen-activated protein kinase pathways [40–42]. Activation of Ras has also been shown to trans-activate ETS-1 and ETS-2, and ERK1/2 and JNK / SAPK pathways [43]. Activation of Jak/Vav/Rho GTPase pathway by chemokine (C-X-C motif) ligand 12 is a key signaling event for membrane-type (MT) 1-MMP/MMP-2-dependent melanoma cell invasion [44]. Both MEK and ERK are mitogen-activated kinases. So secondly, we studied whether Ras-MEK1/2 signaling is involved in the LPS-induced MMP-2 activity. As shown in Fig. 2, LPS-induced MMP-2 activation was almost diminished no matter the exposure to JSH-23 or FTS. Further study shows that LPS-induced activation of p-Rho A and p-MEK1/2 were significantly inhibited by Rosiglitazone (Fig. 3). Similarly, another PPARγ agonist, Troglitazone inhibited vascular endothelial growth factor (VEGF)-induced MMP-2 and MT1-MMP expression through the suppression of VEGF-induced ROS production and ERK phosphorylation [45]. Since NF-κB activation is required for the expression of MMP-1 and MMP-13, as well as inflammatory stimuli such as Interleukin-1, Interleukin-6 and tumor necrosis factor α [46,47]. Activation of RasMEK1 pathway regulates subsequently the activities of NF-κB in MMPs transcription [30], so the expression of NF-κB in the nuclear of RAEC cells was studied. The gene expression and activity of MMP-2 and MMP-9 in macrophages are reduced through PPARγ-dependent inhibition of NF-κB [48]. Our results are in accordance to this finding. The protein expression of NF-κB major binding subunit (p65) in the nuclear of RAEC cells was significantly inhibited by Rosiglitazone. Moreover, the activity of NF-κB was partly activated by PPARγ antagonist of GW9662 (Fig. 4). In line with data of protein expression, the result from EMSA also showed that Rosiglitazone downregulated the DNA binding activity of NF-κB induced by LPS (Fig. 5). In summary, LPS significantly activated MMP-2 secretion in the RAEC cells. Co-treatment of LPS and Rosiglitazone indicates the
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Fig. 5. PPARγ agonist inhibits NF-κB DNA binding activity. Confluent RAEC cells in 6well were pretreated with 10 μM Rosiglitazone in serum-free media for 2 h, followed by co-exposure to 1 μg/ml LPS and Rosiglitazone for 3 h. The DNA binding activity of NF-κB detected by EMSA was significantly activated by LPS, but was completely demolished by Rosiglitazone. The blots represent protein expressions, expressed as percentages of the amount of control. Results represent the mean ± SEM of 3 separate experiments. **, p b 0.001 vs. control, ## p b 0.001 vs LPS treatment.
inhibitory role of Rosiglitazone in MMP-2 activation. Moreover, both inhibitors of NF-κB and Ras respectively attenuated MMP-2 secretion induced by LPS in the RAEC cells. Further study showed that LPSinduced activation of p-Rho A and p-MEK1/2 was significantly inhibited by Rosiglitazone. The protein expression of NF-κB p65 was significantly suppressed by PPARγ agonist, and partly activated by PPARγ antagonist. In consistent with these result, LPS-induced NF-κB DNA binding activity was also demolished by Rosiglitazone. Taken together, downregulation of Ras-MEK1/2 and NF-κB activation indicates the possible pathway by which Rosiglitazone exerts its inhibitory effect on MMP-2 regulation in the RAEC cells, leading to the decreased DNA binding activity of NF-κB, disrupting the transcription of the MMP-2 gene. PPARγ agonist and Ras-MEK1/2 pathway may be another potential therapeutic target for the disease induced by chronic inflammation, such as atherosclerosis. Acknowledgements This work was supported by the Nature Science Foundation of Guangxi Province, China (GKQ 0542049). The authors of this manuscript have certified that they comply with the Principles of Ethical Publishing in the International Journal of Cardiology [49]. References
Fig. 4. PPARγ agonist inhibits NF-κB protein expression. Confluent RAEC cells in 6-well were pretreated with 10 μM Rosiglitazone or 1 μM GW9662 in serum-free media for 2 h, followed by co-exposure to 1 μg/ml LPS and Rosiglitazone or GW9662 for 3 h. NFκB was induced by LPS in the RAEC cells and inhibited by Rosiglitazone . Moreover, NFκB activity was partly induced by PPARγ antagonist of GW9662. The blots represent protein expressions, expressed as percentages of the amount of control. Results represent the mean ± SEM of 3 separate experiments. **, p b 0.001 vs. control. #, p b 0.05 vs. LPS treatment, ## p b 0.001 vs LPS treatment.
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