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RelA subunit of NF-κB in TNF-α-induced SIRT1 expression in vascular smooth muscle cells
Biochemical and Biophysical Research Communications 397 (2010) 569–575
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Involvement of the p65/RelA subunit of NF-jB in TNF-a-induced SIRT1 expression in vascular smooth muscle cells Hui-Na Zhang, Li Li, Peng Gao, Hou-Zao Chen, Ran Zhang, Yu-Sheng Wei, De-Pei Liu *, Chih-Chuan Liang National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, No. 5 Dong Dan San Tiao, Beijing 100005, China
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Article history: Received 24 May 2010 Available online 4 June 2010 Keywords: Regulation of SIRT1 expression p65/RelA NF-jB TNF-a Vascular smooth muscle cell
a b s t r a c t The proinflammatory cytokine TNF-a plays an important role in stimulating inflammatory responses of vascular smooth muscle cells (VSMCs). The anti-inflammatory function of Sirtuin 1 (SIRT1), a NADdependent class III histone/protein deacetylase, has been well documented, but how SIRT1 is regulated under inflammatory conditions is largely unknown. In the present research, we showed that levels of SIRT1 mRNA and protein expression increased in TNF-a-treated VSMCs. Overexpression of the p65/RelA subunit of NF-jB, a TNF-a-activated inflammatory transcription factor, in A7r5 cells, upregulated SIRT1 mRNA and protein expression as well as SIRT1 promoter activity, while knockdown of endogenous p65/ RelA expression by RNAi not only led to a decrease in SIRT1’s basal protein expression and promoter activity, but almost abolished the TNF-a-induced elevation of SIRT1 protein expression and SIRT1 promoter activity. Furthermore, using promoter deletion analysis and chromatin immunoprecipitation assays, we found that p65/RelA bound to the SIRT1 promoter at a consensus NF-jB binding site. Our study indicates that p65/RelA mediates the TNF-a-induced elevated expression of SIRT1 in VSMCs, shedding new light on the regulation of SIRT1 under inflammatory conditions. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Vascular smooth muscle cells (VSMCs) are sensitive to inflammatory lesions, such as balloon injury and atherosclerosis [1]. Notable responses of VSMCs to inflammatory lesions such as proliferation and migration, are always accompanied by markedly induced expression of proinflammatory cytokines, especially TNF-a [2,3]. VSMCs are not only a source of TNF-a [4], but also an important target of TNF-a in injured vasculature. TNF-a per se and a number of TNF-a-induced genes are implicated in the process of VSMC migration and proliferation [5–8]. TNF-a exerts its inflammatory function through many downstream factors, especially the inflammation-related transcriptional factor NF-jB [9,10]. The predominant form of NF-jB is the p50/p65/RelA heterodimer, which is sequestered in the cytoplasm by its inhibitory protein, IjB, under basal conditions. Under inflammatory stimuli, activated NF-jB translocates from the cytoplasm to the nucleus, binds to its consensus sequences in the promoter and initiates the transcription of target genes [11]. Studies show that the p65/RelA subunit, an important transcriptional binding and activation component of NF-jB, has the ability to regulate gene expression indepen-
* Corresponding author. Fax: +86 10 65296415. E-mail address: [email protected] (D.-P. Liu). 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.05.160
dently. However, the p50 subunit, which only has DNA binding activity, cannot initiate gene transcription [12]. Moreover, p50/ p50 homodimers can even inhibit the binding of the p50/p65/RelA complex and subsequently result in a blockade of gene expression [12,13]. Sirtuin 1 (SIRT1), a NAD-dependent class III histone/protein deacetylase, is expressed in most mammalian cells including VSMCs [14]. The anti-inflammatory properties of SIRT1 are closely related to its negative regulation of NF-jB by physical interaction with and deacetylation of the NF-jB p65/RelA subunit at lysine 310 [15]. Through the mediation of NF-jB, SIRT1 broadly suppresses the production of a range of proinflammatory cytokines, such as TNF-a, and IL-6 [16–18]. Knockdown of endogenous SIRT1 leads to a significant increase in mRNA expression for TNF-a, IL-6, MCP-1, VCAM-I, MMP-9, CRP, COX-2 and IL-1 in 3T3-L1 adipocytes [19]. Furthermore, studies have shown that activation or overexpression of SIRT1 directly ameliorates inflammation-associated diseases. Zhang et al. reported that overexpression of SIRT1 significantly reduces atherosclerotic plaque lesions in the SIRT1-Tg/ apoE/ mouse model [20]. Pfluger et al. further demonstrated that activation of SIRT1 protects mice from high fat diet-induced hepatic inflammation through decreasing the NF-jB-mediated inflammatory cytokines TNF-a and IL-6 [21]. Activation of SIRT1 by the polyphenolic compound resveratrol attenuates the cigarette smoke-induced vascular inflammatory phenotype and ameliorates
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dextran sodium sulphate-induced colitis in mice [17,22]. The above studies demonstrate the critical anti-inflammatory role played by SIRT1 in various kinds of cells and tissues, but do not address the regulation of its expression under inflammatory stimulation. Investigating the regulation of SIRT1 expression will help provide a more thorough understanding of its role in inflammatory reactions. In the present research, we observed that TNF-a increased SIRT1 mRNA and protein expression as well as SIRT1 promoter activity in VSMCs. The p65/RelA subunit of NF-jB upregulated SIRT1 mRNA and protein expression, while knockdown of endogenous p65/RelA expression by RNAi led to a decrease in basal and TNF-a-induced SIRT1 protein expression and SIRT1 promoter activity in A7r5 cells. Furthermore, luciferase analysis and ChIP assays demonstrated that p65/RelA could be recruited to the putative NF-jB binding site of the SIRT1 promoter. These data indicate that TNF-a stimulates SIRT1 transcription and protein expression via a p65/RelA-dependent mechanism in VSMCs.
2. Materials and methods 2.1. Cell culture and treatment A7r5 and 293A cells were purchased from the American Type Culture Collection. Human VSMCs were obtained from ScienCell (USA). Rat aortic VSMCs were isolated from the thoracic aorta of male Sprague–Dawley rats by enzymatic digestion. All cells were cultured in Dulbecco’s modified Eagle medium with antibiotics (100 U/ml penicillin, 100 lg/ml streptomycin) and 10% FBS. Cells
(approximately 80% confluent) were treated with or without TNF-a for different lengths of time before the start of experiments. 2.2. Plasmid construction and transfection Full-length cDNAs including human p65/RelA, p50 (from HepG2), A20 (a gift from Prof. Jingbo Zhang) [23], and SIRT1 (a gift from Dr. Ishikawa) [24] were sequenced and subcloned into a pcDNA3.1 expression vector. Full-length human c-Fos and c-Jun cDNAs were also subcloned into pcDNA3.1 expression vectors as described previously [25]. The rat p65/RelA RNAi plasmid was constructed using a pSIREN-RetroQ vector. The sense primer 50 gatccGGACCTACGAGACCTTCAATTCAAGAGATTGAAGGTCTCGTAGGTCC TTT TTTg-30 (the rat p65/RelA interference sequence and its complementary sequence are italics) and the antisense primer 50 aattcAAAAAAGGACCTACGAGACCTTCAATCTCTTGAATTGAAGGTCTC GTAGGTCCg-30 , or a scrambled sequence (the control, named U6), were annealed and inserted into the pSIREN-RetroQ vector between the BamH1 and EcoR1 sites. pGL3-MMP-9-luc was kindly provided by Prof. Zhiping Liu (UT Southwestern Medical Center, USA); pGL3-NF-jB-luc was constructed by inserting four tandem copies of the NF-jB binding sequence into a pGL3-basic vector; a pTA-luc plasmid containing a 2825 bp fragment of the SIRT1 promoter was kindly provided by Prof. Toren Finkel (Bethesda campus, NHLBI). A series of deletion constructs based on the pTA-luc SIRT1 promoter were generated by PCR amplification of the desired portions followed by insertion into a pGL3-basic plasmid. These deletion constructs were named pGL3-SIRT1-2685-luc, pGL3SIRT1-800-luc and pGL3-SIRT1-309-luc. All the above plasmids were transfected into A7r5 or 293A cells using Lipofectamine
Fig. 1. TNF-a elevates SIRT1 expression in a time- and dose-dependent manner in VSMCs. (A) VSMCs were treated with 30 ng/ml TNF-a for different lengths of time (0, 1, 8 and 16 h) and Western blotting was performed to detect SIRT1 protein expression. (B, C) VSMCs were treated with the indicated concentrations of TNF-a for 8 h. Western blotting (B) and RT-PCR (C) were carried out to detect SIRT1 protein and mRNA expression. Quantification of SIRT1 expression was normalized to b-actin (lower panel). (D) A7r5 cells were co-transfected with pGL3-SIRT1-2685-luc and Renilla luciferase plasmid (pRL-TK) for 36 h, followed by TNF-a (30 ng/ml) treatment for 8 h. A luciferase reporter assay was carried out to detect SIRT1 promoter activity. Luciferase activity results are displayed as the mean ± SE. *P < 0.05, **P < 0.01 versus the control.
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2000 reagent according to the manufacturer’s instructions (Invitrogen). 2.3. Western blotting Cells were extracted with RIPA buffer (25 mM Tris–HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, and 0.1% SDS). After complete homogenization on ice, cell lysates were centrifuged and the supernatants obtained were fractionated by 10% SDS–PAGE and electro-transferred onto a PVDF membrane. After blocking with Tris buffered saline (TBS) containing 5% non-fat milk, the membranes were probed with primary antibodies for SIRT1 (1:2000 dilution, Millipore), p65/RelA (1:1000 dilution, Chemicon) or p50 (1:500 dilution, Santa Cruz) at 4 °C overnight. Horseradish peroxidase-conjugated secondary antibody was used for ECL detection. Band intensities were quantified by densitometry. The results were normalized to b-actin (1:10000, Sigma-Aldrich). 2.4. RT-PCR Total RNA was extracted with TRIzol reagent and reverse-transcribed into cDNA using the manufacturer’s protocol (Invitrogen).
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Then the cDNA was amplified by polymerase chain reaction (PCR). Primers used for PCR were: rat SIRT1 primer: sense 50 -CAGAGCAT CACACGCAAGC-30 , antisense 50 -CAGGAAACAG AAACCCCAG C-30 ; rat b-actin primer: sense 50 -GAGAGGGAAATCGTGCGTGAC-30 , antisense 50 -TAGAGCCACCAATCCACA CAGAG-30 . 2.5. Luciferase reporter assay A7r5 cells were cultured in triplicate to 80% confluence in 24well plates and co-transfected with the indicated reporter constructs (0.2 lg), expression vectors (0.2 lg) and the internal control plasmid pRL-TK-Renilla-luc (30 ng). Luciferase activity was assessed with the Dual-Luciferase Reporter Assay System (Promega). 2.6. ChIP assay Human VSMCs were cultured to 80% confluence and then treated with TNF-a for 8 h. ChIP experiments were performed essentially the same way as previously reported [26]. The cells were lysed and immunoprecipitated using p65/RelA antibody. Extracted DNA was PCR amplified using specific primers for the SIRT1 pro-
Fig. 2. p65/RelA promotes SIRT1 expression at the transcriptional level. (A) Alignment of the proximal core region of human, mouse and bovine SIRT1 promoter sequences. The underlined bases indicate the conserved sequence in the putative NF-jB binding site, located between 339 and 328 in the 50 -flanking region of the human SIRT1 gene. (B) The p65/RelA and p50 expression plasmids were co-transfected into A7r5 cells for 36 h. pcDNA3.1 was transfected as a control. The level of SIRT1 mRNA was detected by RT-PCR. (C, D) The pGL3-SIRT1-2685-luc and Renilla luciferase (pRL-TK) plasmids were co-transfected with the p65/RelA expression plasmid (C, D), p50 expression plasmid (C) or A20 expression plasmid (D) into A7r5 cells for 36 h and luciferase activity was measured. pcDNA3.1 was transfected as a control. pGL3-NF-jB-luc and pGL3-MMP-9-luc were transfected as positive controls. (E) pGL3-SIRT1-promoter-luc plasmids containing 2685 bp, 800 bp, or 309 bp promoter sequences were separately co-transfected with either pcDNA3.1 or pcDNA3.1-p65/RelA into A7r5 cells for 36 h, and luciferase activity was measured. Luciferase activity results are presented as the mean ± SE. **P < 0.01 versus controls. (F) Human VSMCs were treated with TNF-a (30 ng/ml, 8 h) and a ChIP assay was performed to detect the recruitment of p65/RelA to the SIRT1 promoter. Binding of p65/RelA to the TNF-a promoter was used as a positive control. PCR amplification of the GAPDH promoter was used to demonstrate background binding of p65/ RelA, and functioned as a negative control.
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moter (encompassing the putative NF-jB binding site), the sense primer was 50 -TCCTTTTGCCTCTCTTCCTAC-30 , and the antisense primer was 50 -GCCGCTTTCTCAACTTCTC-30 . TNF-a promoter and GAPDH promoter amplification products acted as positive and negative controls respectively. Primers used were described previously [15,27]. An equal volume of non-precipitated (input) genomic DNA at a 1:45 dilution was used to correct for the differences in PCR amplification efficiencies and amounts of DNA.
activity also increased in a dose-dependent manner (Fig. 1C and D). These results suggest that TNF-a upregulates SIRT1 expression in VSMCs and that the increase in the level of SIRT1 protein induced by TNF-a could be due to the increase in the level of SIRT1 mRNA.
2.7. Statistical analysis
To identify potential transcription factors responsible for the upregulation of SIRT1 by TNF-a, we predicted putative transcription factor binding elements in the SIRT1 promoter using rVISTA2.0. The NF-jB binding element consensus sequence -339TGGAAATT CCCA-328 was found in the human SIRT1 promoter 50 -flanking region (Fig. 2A). The crucial function of NF-jB in the TNF-a-induced inflammatory response has been well established [9]. To determine whether NF-jB is involved in transcriptional regulation of SIRT1, we co-expressed both the p65/RelA and p50 subunits of NF-jB in A7r5. As expected, overexpression of both p65/RelA and p50 markedly increased SIRT1 mRNA expression (Fig. 2B). Further investigation was carried out using a reporter construct containing a 2685 bp region of the SIRT1 promoter to check the role of the p65/RelA and p50 subunits in SIRT1 regulation. Consistent with the RT-PCR results described above, overexpression of p65/RelA and p50 significantly upregulated SIRT1 promoter activity. We noted that p65/ RelA alone upregulated SIRT1 promoter activity more than 4-fold, an effect which was even more marked than that of the p65/RelA and p50 overexpression group (2.7-fold). However, p50 had almost no significant influence on SIRT1 promoter activity (Fig. 2C).
All of the numerical results were expressed as the mean ± SE derived from three independent experiments. Statistical analyses were performed using a one-factor ANOVA or paired-samples ttests. P-values less than 0.05 were considered statistically significant.
3. Results 3.1. TNF-a upregulates SIRT1 mRNA and protein expression and the activity of the SIRT1 promoter in VSMCs To explore whether TNF-a influenced SIRT1 protein expression, we treated rat VSMCs with 30 ng/ml TNF-a. The level of SIRT1 protein increased significantly with treatment time (Fig. 1A). A gradual elevation of SIRT1 protein expression was also observed when VSMCs were exposed to increasing concentrations of TNFa for 8 h (Fig. 1B). In addition, SIRT1 mRNA and SIRT1 promoter
3.2. Overexpression of the p65/RelA subunit of NF-jB upregulates SIRT1 expression at the transcriptional level
Fig. 3. p65/RelA upregulates SIRT1 protein expression. (A) pcDNA3.1-p65/RelA was co-transfected with pcDNA3.1-p50 into A7r5 cells for 36 h, and pcDNA3.1 was transfected as a control. The SIRT1 protein expression level was determined by Western blotting. (B, C) A7r5 cells were transfected with pcDNA3.1-p65/RelA or pcDNA3.1-p50 for 36 h. The level of SIRT1 and p65/RelA (C) or p50 (B) protein expression was detected by Western blotting. (D) 293A cells were transfected with pcDNA3.1-p65/RelA alone or cotransfected with pcDNA3.1-p65/RelA and pcDNA3.1-p50 for 36 h, followed by Western blotting to detect SIRT1 expression. Quantitative analysis (lower panel). *P < 0.05, **P < 0.01 versus the control.
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When we used pGL3-MMP-9-luc and pGL3-NF-jB-luc as positive controls, we noted that the promoter activity of all three samples, including pGL3-SIRT1-2685-luc, could be activated by p65/RelA overexpression. In addition, we observed that A20, an inhibitor of NF-jB, markedly decreased p65/RelA-induced SIRT1 promoter activity (Fig. 2D). All the above data suggest that NF-jB, particularly the p65/RelA subunit, upregulates SIRT1 expression at the transcriptional level. We hypothesized that p65/RelA might have the capacity to bind to the SIRT1 promoter. Both luciferase reporter assays and ChIP assays were used to verify this possibility. As shown in Fig. 2E, luciferase reporter assays indicated that activity of the SIRT1-309bp promoter (without the putative NF-jB binding site) was not affected by p65/RelA. However, the activities of pGL3SIRT1-2685-luc and pGL3-SIRT1-800-luc, both containing the predicted NF-jB binding site, were greatly elevated by p65/RelA. To test in vivo binding of p65/RelA to the predicted NF-jB binding site in the SIRT1 promoter, we performed a ChIP assay with an anti-p65/ RelA antibody. As shown in Fig. 2F, the SIRT1 promoter fragment containing the putative NF-jB binding site coprecipitated with endogenous p65/RelA in human VSMCs treated with TNF-a. No chromatin fragments were precipitated by the control IgG. The TNF-a (containing the NF-jB binding site) and GAPDH promoters were amplified as positive and negative controls. These results sug-
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gest that p65/RelA is recruited to the NF-jB binding site in the SIRT1 promoter, presumably leading to the up regulation of SIRT1 expression. 3.3. p65/RelA promotes SIRT1 protein expression To further confirm the effect of p65/RelA on SIRT1 expression in VSMCs, plasmids encoding p65/RelA and p50 were co-transfected into A7r5. As shown in Fig. 3A, overexpression of p65/RelA and p50 increased the level of SIRT1 protein. However, consistent with the luciferase reporter assay results described above, when we overexpressed p65/RelA or p50 in A7r5 cells separately, p65/RelA significantly upregulated SIRT1 protein expression, but p50 had little effect (Fig. 3B and C). We obtained similar results in 293A cells, and verified that NF-jB or p65/RelA significantly elevated SIRT1 protein expression (Fig. 3D). Since activator protein-1 (AP-1) is another important TNF-a-induced transcriptional factor [10], we coexpressed the AP-1 subunits c-Jun and c-Fos in A7r5 to determine whether AP-1 could also upregulate SIRT1. However, there was no significant difference in SIRT1 expression between the control and AP-1 groups (Supplementary Fig. 1). The above results indicate that p65/RelA plays a key role in promoting SIRT1 protein expression in VSMCs.
Fig. 4. p65/RelA is involved in the elevated expression of SIRT1 induced by TNF-a in A7r5. (A and B) p65/RelA RNAi or a U6-scrambled control was transfected into A7r5 cells for 36 h with (B) or without (A) TNF-a (30 ng/ml) treatment for another 8 h. Western blotting was used to detect SIRT1 and endogenous p65/RelA expression. Quantitative analysis (lower panel). (C) p65/RelA RNAi or a U6-scrambled control was co-transfected with pGL3-SIRT1-2685-luc into A7r5 cells for 36 h, and luciferase reporter assays were performed. pGL3-NF-jB-luc and pGL3-MMP-9-luc were transfected as positive controls. (D) p65/RelA RNAi or a U6-scrambled control was co-transfected with SIRT12685-luc into A7r5 cells for 36 h, followed by TNF-a (30 ng/ml) treatment for another 8 h. Luciferase reporter assays were carried out. Luciferase activity results are displayed as the mean ± SE. *P < 0.05, **P < 0.01 versus the control.
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3.4. p65/RelA plays a pivotal role in mediating the upregulation of SIRT1 by TNF-a To determine whether p65/RelA is implicated in the process of TNF-a-induced SIRT1 expression, p65/RelA RNAi was transfected into A7r5 for 36 h. Western blotting showed that SIRT1 protein expression was markedly suppressed (Fig. 4A). Luciferase reporter assays indicate that p65/RelA RNAi attenuates SIRT1 promoter activity by half, (MMP-9-luc and NF-jB-luc acted as positive controls) (Fig. 4C). When A7r5 cells were transfected with p65/RelA RNAi for 36 h, followed by TNF-a (30 ng/mL) treatment for another 8 h, both the expression of SIRT1 protein and the activity of the SIRT1 promoter induced by TNF-a were almost abolished by inhibiting endogenous p65/RelA (Fig. 4B and D). These results indicate that p65/RelA plays a pivotal role in mediating the elevated expression of SIRT1 by TNF-a. 4. Discussion The function of SIRT1 as a versatile subtle tuner of multiple physiological and pathological processes has been extensively investigated. Recent studies have demonstrated that SIRT1 expression is tightly regulated. The regulation of SIRT1 by transcriptional factors such as p53, HIC1, E2F1, and C/EBPa has been reported [28– 31]. In the present study, we found that SIRT1 mRNA and protein expression were induced by TNF-a in a time- and dose-dependent manner (Fig. 1). In addition, we observed that the p65/RelA subunit of the inflammatory transcriptional factor NF-jB mediated the TNF-a-induced elevated expression of SIRT1 and that when p65/ RelA was knocked down using p65/RelA RNAi in A7r5, TNF-a-induced SIRT1 expression was almost abolished (Fig. 4). Furthermore, using bioinformatics analysis we found that there was a putative NF-jB binding site in the 50 -flanking region of the SIRT1 promoter, from 339 to 328. Luciferase activity assays, with or without the putative NF-jB binding site, confirmed our expectation that p65/RelA is implicated in the transcriptional regulation of SIRT1. ChIP assays further confirmed that p65/RelA is recruited to the putative NF-jB binding region in the SIRT1 promoter (Fig. 2). These results suggest that TNF-a upregulates SIRT1 expression via p65/RelA at the transcriptional level. Multiple inflammatory mediators have been reported to be suppressed by SIRT1. Attenuation of NF-jB activity by SIRT1 is known to result in the suppression of TNF-a [16–18]. Since our results showed that TNF-a upregulates SIRT1 expression in VSMCs (Fig. 1), we hypothesize that there may be a potential negative feedback loop controlling TNF-a production in VSMCs. Such a loop would play an important role because the elevation of TNF-a by many inflammatory stimuli is concomitant with the upregulation of SIRT1. This loop would ameliorate the harmful accumulation of TNF-a and orchestrate an appropriate inflammatory response. SIRT1 inhibition of NF-jB activity through deacetylation of p65/ RelA at lysine 310 has been well documented in previous reports [15]. This, together with our results indicating that p65/RelA upregulates SIRT1 expression (Fig. 3), lead us to speculate that there is another autoregulated feedback loop between SIRT1 and p65/RelA to balance both SIRT1 and NF-jB activity appropriately for the specific physiological or pathological needs of VSMCs. Taken together, our data demonstrate that the p65/RelA subunit of NF-jB mediates elevated expression of SIRT1 by TNF-a in VSMCs. Given that TNF-a is a major inflammatory mediator and is induced by a wide range of pathogenic stimuli in vasculature, our data suggest that the upregulation of SIRT1 by TNF-a may be a ubiquitous auto-protective response of VSMCs to inflammatory events. Further studies are needed to elucidate the potential physiological and pathological significance of TNF-a-induced SIRT1 expression in VSMCs.
Acknowledgments We thank Zhu-Qin Zhang, Rui-Feng Yang, Guo-Wei Zhao, Shuang Zhou, Zhi-Gang She and Qing-Jun Zhang for technical assistance. This work was supported by the National Basic Research Program of China (Grant No. 2006CB503801), the Special Fund of the National Laboratory of China (Grant No. 2060204), the National Natural Science Foundation of China (Grant No. 30721063), and the National 863 Project (Grant NO. 2006AA02A406). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.bbrc.2010.05.160. References [1] V.J. Dzau, R.C. Braun-Dullaeus, D.G. Sedding, Vascular proliferation and atherosclerosis: new perspectives and therapeutic strategies, Nat. Med. 8 (2002) 1249–1256. [2] S. Jovinge, A. Hultgardh-Nilsson, J. Regnstrom, J. Nilsson, Tumor necrosis factor-alpha activates smooth muscle cell migration in culture and is expressed in the balloon-injured rat aorta, Arterioscler. Thromb. Vasc. Biol. 17 (1997) 490–497. [3] H. Tanaka, G. Sukhova, D. Schwartz, P. Libby, Proliferating arterial smooth muscle cells after balloon injury express TNF-alpha but not interleukin-1 or basic fibroblast growth factor, Arterioscler. Thromb. Vasc. Biol. 16 (1996) 12– 18. [4] S. Bacci, L. Pieri, A.M. Buccoliero, A. Bonelli, G. Taddei, P. Romagnoli, Smooth muscle cells, dendritic cells and mast cells are sources of TNFalpha and nitric oxide in human carotid artery atherosclerosis, Thromb. Res. 122 (2008) 657– 667. [5] S. Yoshida, M. Ono, T. Shono, H. Izumi, T. Ishibashi, H. Suzuki, M. Kuwano, Involvement of interleukin-8, vascular endothelial growth factor, and basic fibroblast growth factor in tumor necrosis factor alpha-dependent angiogenesis, Mol. Cell. Biol. 17 (1997) 4015–4023. [6] T. Couffinhal, C. Duplaa, L. Labat, J.M. Lamaziere, C. Moreau, O. Printseva, J. Bonnet, Tumor necrosis factor-alpha stimulates ICAM-1 expression in human vascular smooth muscle cells, Arterioscler. Thromb. 13 (1993) 407–414. [7] J.L. Barks, J.J. McQuillan, M.F. Iademarco, TNF-alpha and IL-4 synergistically increase vascular cell adhesion molecule-1 expression in cultured vascular smooth muscle cells, J. Immunol. 159 (1997) 4532–4538. [8] Z.S. Galis, M. Muszynski, G.K. Sukhova, E. Simon-Morrissey, P. Libby, Enhanced expression of vascular matrix metalloproteinases induced in vitro by cytokines and in regions of human atherosclerotic lesions, Ann. NY Acad. Sci. 748 (1995) 501–507. [9] Z. Wang, M.R. Castresana, W.H. Newman, NF-kappaB is required for TNFalpha-directed smooth muscle cell migration, FEBS Lett. 508 (2001) 360–364. [10] H. Wajant, K. Pfizenmaier, P. Scheurich, Tumor necrosis factor signaling, Cell Death Differ. 10 (2003) 45–65. [11] M.S. Hayden, S. Ghosh, Signaling to NF-kappaB, Genes Dev. 18 (2004) 2195– 2224. [12] M.L. Schmitz, P.A. Baeuerle, The p65 subunit is responsible for the strong transcription activating potential of NF-kappa B, EMBO J. 10 (1991) 3805– 3817. [13] S. Kastenbauer, H.W. Ziegler-Heitbrock, NF-kappaB1 (p50) is upregulated in lipopolysaccharide tolerance and can block tumor necrosis factor gene expression, Infect. Immun. 67 (1999) 1553–1559. [14] R. Miyazaki, T. Ichiki, T. Hashimoto, K. Inanaga, I. Imayama, J. Sadoshima, K. Sunagawa, SIRT1, a longevity gene, downregulates angiotensin II type 1 receptor expression in vascular smooth muscle cells, Arterioscler. Thromb. Vasc. Biol. 28 (2008) 1263–1269. [15] F. Yeung, J.E. Hoberg, C.S. Ramsey, M.D. Keller, D.R. Jones, R.A. Frye, M.W. Mayo, Modulation of NF-kappaB-dependent transcription and cell survival by the SIRT1 deacetylase, EMBO J. 23 (2004) 2369–2380. [16] Z. Shen, J.M. Ajmo, C.Q. Rogers, X. Liang, L. Le, M.M. Murr, Y. Peng, M. You, Role of SIRT1 in regulation of LPS- or two ethanol metabolites-induced TNF-a production in cultured macrophage cell lines, Am. J. Physiol. Gastrointest. Liver Physiol. 296 (2009) G1047–G1053. [17] A. Csiszar, N. Labinskyy, A. Podlutsky, P.M. Kaminski, M.S. Wolin, C. Zhang, P. Mukhopadhyay, P. Pacher, F. Hu, R. de Cabo, P. Ballabh, Z. Ungvari, Vasoprotective effects of resveratrol and SIRT1: attenuation of cigarette smoke-induced oxidative stress and proinflammatory phenotypic alterations, Am. J. Physiol. Heart Circ. Physiol. 294 (2008) H2721–2735. [18] J.H. Lee, M.Y. Song, E.K. Song, E.K. Kim, W.S. Moon, M.K. Han, J.W. Park, K.B. Kwon, B.H. Park, Overexpression of SIRT1 protects pancreatic beta-cells against cytokine toxicity by suppressing the nuclear factor-kappaB signaling pathway, Diabetes 58 (2009) 344–351. [19] T. Yoshizaki, J.C. Milne, T. Imamura, S. Schenk, N. Sonoda, J.L. Babendure, J.C. Lu, J.J. Smith, M.R. Jirousek, J.M. Olefsky, SIRT1 exerts anti-inflammatory effects
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Involvement of the p65/RelA subunit of NF-jB in TNF-a-induced SIRT1 expression in vascular smooth muscle cells Hui-Na Zhang, Li Li, Peng Gao, Hou-Zao Chen, Ran Zhang, Yu-Sheng Wei, De-Pei Liu *, Chih-Chuan Liang National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, No. 5 Dong Dan San Tiao, Beijing 100005, China
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Article history: Received 24 May 2010 Available online 4 June 2010 Keywords: Regulation of SIRT1 expression p65/RelA NF-jB TNF-a Vascular smooth muscle cell
a b s t r a c t The proinflammatory cytokine TNF-a plays an important role in stimulating inflammatory responses of vascular smooth muscle cells (VSMCs). The anti-inflammatory function of Sirtuin 1 (SIRT1), a NADdependent class III histone/protein deacetylase, has been well documented, but how SIRT1 is regulated under inflammatory conditions is largely unknown. In the present research, we showed that levels of SIRT1 mRNA and protein expression increased in TNF-a-treated VSMCs. Overexpression of the p65/RelA subunit of NF-jB, a TNF-a-activated inflammatory transcription factor, in A7r5 cells, upregulated SIRT1 mRNA and protein expression as well as SIRT1 promoter activity, while knockdown of endogenous p65/ RelA expression by RNAi not only led to a decrease in SIRT1’s basal protein expression and promoter activity, but almost abolished the TNF-a-induced elevation of SIRT1 protein expression and SIRT1 promoter activity. Furthermore, using promoter deletion analysis and chromatin immunoprecipitation assays, we found that p65/RelA bound to the SIRT1 promoter at a consensus NF-jB binding site. Our study indicates that p65/RelA mediates the TNF-a-induced elevated expression of SIRT1 in VSMCs, shedding new light on the regulation of SIRT1 under inflammatory conditions. Ó 2010 Elsevier Inc. All rights reserved.
1. Introduction Vascular smooth muscle cells (VSMCs) are sensitive to inflammatory lesions, such as balloon injury and atherosclerosis [1]. Notable responses of VSMCs to inflammatory lesions such as proliferation and migration, are always accompanied by markedly induced expression of proinflammatory cytokines, especially TNF-a [2,3]. VSMCs are not only a source of TNF-a [4], but also an important target of TNF-a in injured vasculature. TNF-a per se and a number of TNF-a-induced genes are implicated in the process of VSMC migration and proliferation [5–8]. TNF-a exerts its inflammatory function through many downstream factors, especially the inflammation-related transcriptional factor NF-jB [9,10]. The predominant form of NF-jB is the p50/p65/RelA heterodimer, which is sequestered in the cytoplasm by its inhibitory protein, IjB, under basal conditions. Under inflammatory stimuli, activated NF-jB translocates from the cytoplasm to the nucleus, binds to its consensus sequences in the promoter and initiates the transcription of target genes [11]. Studies show that the p65/RelA subunit, an important transcriptional binding and activation component of NF-jB, has the ability to regulate gene expression indepen-
* Corresponding author. Fax: +86 10 65296415. E-mail address: [email protected] (D.-P. Liu). 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.05.160
dently. However, the p50 subunit, which only has DNA binding activity, cannot initiate gene transcription [12]. Moreover, p50/ p50 homodimers can even inhibit the binding of the p50/p65/RelA complex and subsequently result in a blockade of gene expression [12,13]. Sirtuin 1 (SIRT1), a NAD-dependent class III histone/protein deacetylase, is expressed in most mammalian cells including VSMCs [14]. The anti-inflammatory properties of SIRT1 are closely related to its negative regulation of NF-jB by physical interaction with and deacetylation of the NF-jB p65/RelA subunit at lysine 310 [15]. Through the mediation of NF-jB, SIRT1 broadly suppresses the production of a range of proinflammatory cytokines, such as TNF-a, and IL-6 [16–18]. Knockdown of endogenous SIRT1 leads to a significant increase in mRNA expression for TNF-a, IL-6, MCP-1, VCAM-I, MMP-9, CRP, COX-2 and IL-1 in 3T3-L1 adipocytes [19]. Furthermore, studies have shown that activation or overexpression of SIRT1 directly ameliorates inflammation-associated diseases. Zhang et al. reported that overexpression of SIRT1 significantly reduces atherosclerotic plaque lesions in the SIRT1-Tg/ apoE/ mouse model [20]. Pfluger et al. further demonstrated that activation of SIRT1 protects mice from high fat diet-induced hepatic inflammation through decreasing the NF-jB-mediated inflammatory cytokines TNF-a and IL-6 [21]. Activation of SIRT1 by the polyphenolic compound resveratrol attenuates the cigarette smoke-induced vascular inflammatory phenotype and ameliorates
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dextran sodium sulphate-induced colitis in mice [17,22]. The above studies demonstrate the critical anti-inflammatory role played by SIRT1 in various kinds of cells and tissues, but do not address the regulation of its expression under inflammatory stimulation. Investigating the regulation of SIRT1 expression will help provide a more thorough understanding of its role in inflammatory reactions. In the present research, we observed that TNF-a increased SIRT1 mRNA and protein expression as well as SIRT1 promoter activity in VSMCs. The p65/RelA subunit of NF-jB upregulated SIRT1 mRNA and protein expression, while knockdown of endogenous p65/RelA expression by RNAi led to a decrease in basal and TNF-a-induced SIRT1 protein expression and SIRT1 promoter activity in A7r5 cells. Furthermore, luciferase analysis and ChIP assays demonstrated that p65/RelA could be recruited to the putative NF-jB binding site of the SIRT1 promoter. These data indicate that TNF-a stimulates SIRT1 transcription and protein expression via a p65/RelA-dependent mechanism in VSMCs.
2. Materials and methods 2.1. Cell culture and treatment A7r5 and 293A cells were purchased from the American Type Culture Collection. Human VSMCs were obtained from ScienCell (USA). Rat aortic VSMCs were isolated from the thoracic aorta of male Sprague–Dawley rats by enzymatic digestion. All cells were cultured in Dulbecco’s modified Eagle medium with antibiotics (100 U/ml penicillin, 100 lg/ml streptomycin) and 10% FBS. Cells
(approximately 80% confluent) were treated with or without TNF-a for different lengths of time before the start of experiments. 2.2. Plasmid construction and transfection Full-length cDNAs including human p65/RelA, p50 (from HepG2), A20 (a gift from Prof. Jingbo Zhang) [23], and SIRT1 (a gift from Dr. Ishikawa) [24] were sequenced and subcloned into a pcDNA3.1 expression vector. Full-length human c-Fos and c-Jun cDNAs were also subcloned into pcDNA3.1 expression vectors as described previously [25]. The rat p65/RelA RNAi plasmid was constructed using a pSIREN-RetroQ vector. The sense primer 50 gatccGGACCTACGAGACCTTCAATTCAAGAGATTGAAGGTCTCGTAGGTCC TTT TTTg-30 (the rat p65/RelA interference sequence and its complementary sequence are italics) and the antisense primer 50 aattcAAAAAAGGACCTACGAGACCTTCAATCTCTTGAATTGAAGGTCTC GTAGGTCCg-30 , or a scrambled sequence (the control, named U6), were annealed and inserted into the pSIREN-RetroQ vector between the BamH1 and EcoR1 sites. pGL3-MMP-9-luc was kindly provided by Prof. Zhiping Liu (UT Southwestern Medical Center, USA); pGL3-NF-jB-luc was constructed by inserting four tandem copies of the NF-jB binding sequence into a pGL3-basic vector; a pTA-luc plasmid containing a 2825 bp fragment of the SIRT1 promoter was kindly provided by Prof. Toren Finkel (Bethesda campus, NHLBI). A series of deletion constructs based on the pTA-luc SIRT1 promoter were generated by PCR amplification of the desired portions followed by insertion into a pGL3-basic plasmid. These deletion constructs were named pGL3-SIRT1-2685-luc, pGL3SIRT1-800-luc and pGL3-SIRT1-309-luc. All the above plasmids were transfected into A7r5 or 293A cells using Lipofectamine
Fig. 1. TNF-a elevates SIRT1 expression in a time- and dose-dependent manner in VSMCs. (A) VSMCs were treated with 30 ng/ml TNF-a for different lengths of time (0, 1, 8 and 16 h) and Western blotting was performed to detect SIRT1 protein expression. (B, C) VSMCs were treated with the indicated concentrations of TNF-a for 8 h. Western blotting (B) and RT-PCR (C) were carried out to detect SIRT1 protein and mRNA expression. Quantification of SIRT1 expression was normalized to b-actin (lower panel). (D) A7r5 cells were co-transfected with pGL3-SIRT1-2685-luc and Renilla luciferase plasmid (pRL-TK) for 36 h, followed by TNF-a (30 ng/ml) treatment for 8 h. A luciferase reporter assay was carried out to detect SIRT1 promoter activity. Luciferase activity results are displayed as the mean ± SE. *P < 0.05, **P < 0.01 versus the control.
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2000 reagent according to the manufacturer’s instructions (Invitrogen). 2.3. Western blotting Cells were extracted with RIPA buffer (25 mM Tris–HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, and 0.1% SDS). After complete homogenization on ice, cell lysates were centrifuged and the supernatants obtained were fractionated by 10% SDS–PAGE and electro-transferred onto a PVDF membrane. After blocking with Tris buffered saline (TBS) containing 5% non-fat milk, the membranes were probed with primary antibodies for SIRT1 (1:2000 dilution, Millipore), p65/RelA (1:1000 dilution, Chemicon) or p50 (1:500 dilution, Santa Cruz) at 4 °C overnight. Horseradish peroxidase-conjugated secondary antibody was used for ECL detection. Band intensities were quantified by densitometry. The results were normalized to b-actin (1:10000, Sigma-Aldrich). 2.4. RT-PCR Total RNA was extracted with TRIzol reagent and reverse-transcribed into cDNA using the manufacturer’s protocol (Invitrogen).
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Then the cDNA was amplified by polymerase chain reaction (PCR). Primers used for PCR were: rat SIRT1 primer: sense 50 -CAGAGCAT CACACGCAAGC-30 , antisense 50 -CAGGAAACAG AAACCCCAG C-30 ; rat b-actin primer: sense 50 -GAGAGGGAAATCGTGCGTGAC-30 , antisense 50 -TAGAGCCACCAATCCACA CAGAG-30 . 2.5. Luciferase reporter assay A7r5 cells were cultured in triplicate to 80% confluence in 24well plates and co-transfected with the indicated reporter constructs (0.2 lg), expression vectors (0.2 lg) and the internal control plasmid pRL-TK-Renilla-luc (30 ng). Luciferase activity was assessed with the Dual-Luciferase Reporter Assay System (Promega). 2.6. ChIP assay Human VSMCs were cultured to 80% confluence and then treated with TNF-a for 8 h. ChIP experiments were performed essentially the same way as previously reported [26]. The cells were lysed and immunoprecipitated using p65/RelA antibody. Extracted DNA was PCR amplified using specific primers for the SIRT1 pro-
Fig. 2. p65/RelA promotes SIRT1 expression at the transcriptional level. (A) Alignment of the proximal core region of human, mouse and bovine SIRT1 promoter sequences. The underlined bases indicate the conserved sequence in the putative NF-jB binding site, located between 339 and 328 in the 50 -flanking region of the human SIRT1 gene. (B) The p65/RelA and p50 expression plasmids were co-transfected into A7r5 cells for 36 h. pcDNA3.1 was transfected as a control. The level of SIRT1 mRNA was detected by RT-PCR. (C, D) The pGL3-SIRT1-2685-luc and Renilla luciferase (pRL-TK) plasmids were co-transfected with the p65/RelA expression plasmid (C, D), p50 expression plasmid (C) or A20 expression plasmid (D) into A7r5 cells for 36 h and luciferase activity was measured. pcDNA3.1 was transfected as a control. pGL3-NF-jB-luc and pGL3-MMP-9-luc were transfected as positive controls. (E) pGL3-SIRT1-promoter-luc plasmids containing 2685 bp, 800 bp, or 309 bp promoter sequences were separately co-transfected with either pcDNA3.1 or pcDNA3.1-p65/RelA into A7r5 cells for 36 h, and luciferase activity was measured. Luciferase activity results are presented as the mean ± SE. **P < 0.01 versus controls. (F) Human VSMCs were treated with TNF-a (30 ng/ml, 8 h) and a ChIP assay was performed to detect the recruitment of p65/RelA to the SIRT1 promoter. Binding of p65/RelA to the TNF-a promoter was used as a positive control. PCR amplification of the GAPDH promoter was used to demonstrate background binding of p65/ RelA, and functioned as a negative control.
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moter (encompassing the putative NF-jB binding site), the sense primer was 50 -TCCTTTTGCCTCTCTTCCTAC-30 , and the antisense primer was 50 -GCCGCTTTCTCAACTTCTC-30 . TNF-a promoter and GAPDH promoter amplification products acted as positive and negative controls respectively. Primers used were described previously [15,27]. An equal volume of non-precipitated (input) genomic DNA at a 1:45 dilution was used to correct for the differences in PCR amplification efficiencies and amounts of DNA.
activity also increased in a dose-dependent manner (Fig. 1C and D). These results suggest that TNF-a upregulates SIRT1 expression in VSMCs and that the increase in the level of SIRT1 protein induced by TNF-a could be due to the increase in the level of SIRT1 mRNA.
2.7. Statistical analysis
To identify potential transcription factors responsible for the upregulation of SIRT1 by TNF-a, we predicted putative transcription factor binding elements in the SIRT1 promoter using rVISTA2.0. The NF-jB binding element consensus sequence -339TGGAAATT CCCA-328 was found in the human SIRT1 promoter 50 -flanking region (Fig. 2A). The crucial function of NF-jB in the TNF-a-induced inflammatory response has been well established [9]. To determine whether NF-jB is involved in transcriptional regulation of SIRT1, we co-expressed both the p65/RelA and p50 subunits of NF-jB in A7r5. As expected, overexpression of both p65/RelA and p50 markedly increased SIRT1 mRNA expression (Fig. 2B). Further investigation was carried out using a reporter construct containing a 2685 bp region of the SIRT1 promoter to check the role of the p65/RelA and p50 subunits in SIRT1 regulation. Consistent with the RT-PCR results described above, overexpression of p65/RelA and p50 significantly upregulated SIRT1 promoter activity. We noted that p65/ RelA alone upregulated SIRT1 promoter activity more than 4-fold, an effect which was even more marked than that of the p65/RelA and p50 overexpression group (2.7-fold). However, p50 had almost no significant influence on SIRT1 promoter activity (Fig. 2C).
All of the numerical results were expressed as the mean ± SE derived from three independent experiments. Statistical analyses were performed using a one-factor ANOVA or paired-samples ttests. P-values less than 0.05 were considered statistically significant.
3. Results 3.1. TNF-a upregulates SIRT1 mRNA and protein expression and the activity of the SIRT1 promoter in VSMCs To explore whether TNF-a influenced SIRT1 protein expression, we treated rat VSMCs with 30 ng/ml TNF-a. The level of SIRT1 protein increased significantly with treatment time (Fig. 1A). A gradual elevation of SIRT1 protein expression was also observed when VSMCs were exposed to increasing concentrations of TNFa for 8 h (Fig. 1B). In addition, SIRT1 mRNA and SIRT1 promoter
3.2. Overexpression of the p65/RelA subunit of NF-jB upregulates SIRT1 expression at the transcriptional level
Fig. 3. p65/RelA upregulates SIRT1 protein expression. (A) pcDNA3.1-p65/RelA was co-transfected with pcDNA3.1-p50 into A7r5 cells for 36 h, and pcDNA3.1 was transfected as a control. The SIRT1 protein expression level was determined by Western blotting. (B, C) A7r5 cells were transfected with pcDNA3.1-p65/RelA or pcDNA3.1-p50 for 36 h. The level of SIRT1 and p65/RelA (C) or p50 (B) protein expression was detected by Western blotting. (D) 293A cells were transfected with pcDNA3.1-p65/RelA alone or cotransfected with pcDNA3.1-p65/RelA and pcDNA3.1-p50 for 36 h, followed by Western blotting to detect SIRT1 expression. Quantitative analysis (lower panel). *P < 0.05, **P < 0.01 versus the control.
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When we used pGL3-MMP-9-luc and pGL3-NF-jB-luc as positive controls, we noted that the promoter activity of all three samples, including pGL3-SIRT1-2685-luc, could be activated by p65/RelA overexpression. In addition, we observed that A20, an inhibitor of NF-jB, markedly decreased p65/RelA-induced SIRT1 promoter activity (Fig. 2D). All the above data suggest that NF-jB, particularly the p65/RelA subunit, upregulates SIRT1 expression at the transcriptional level. We hypothesized that p65/RelA might have the capacity to bind to the SIRT1 promoter. Both luciferase reporter assays and ChIP assays were used to verify this possibility. As shown in Fig. 2E, luciferase reporter assays indicated that activity of the SIRT1-309bp promoter (without the putative NF-jB binding site) was not affected by p65/RelA. However, the activities of pGL3SIRT1-2685-luc and pGL3-SIRT1-800-luc, both containing the predicted NF-jB binding site, were greatly elevated by p65/RelA. To test in vivo binding of p65/RelA to the predicted NF-jB binding site in the SIRT1 promoter, we performed a ChIP assay with an anti-p65/ RelA antibody. As shown in Fig. 2F, the SIRT1 promoter fragment containing the putative NF-jB binding site coprecipitated with endogenous p65/RelA in human VSMCs treated with TNF-a. No chromatin fragments were precipitated by the control IgG. The TNF-a (containing the NF-jB binding site) and GAPDH promoters were amplified as positive and negative controls. These results sug-
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gest that p65/RelA is recruited to the NF-jB binding site in the SIRT1 promoter, presumably leading to the up regulation of SIRT1 expression. 3.3. p65/RelA promotes SIRT1 protein expression To further confirm the effect of p65/RelA on SIRT1 expression in VSMCs, plasmids encoding p65/RelA and p50 were co-transfected into A7r5. As shown in Fig. 3A, overexpression of p65/RelA and p50 increased the level of SIRT1 protein. However, consistent with the luciferase reporter assay results described above, when we overexpressed p65/RelA or p50 in A7r5 cells separately, p65/RelA significantly upregulated SIRT1 protein expression, but p50 had little effect (Fig. 3B and C). We obtained similar results in 293A cells, and verified that NF-jB or p65/RelA significantly elevated SIRT1 protein expression (Fig. 3D). Since activator protein-1 (AP-1) is another important TNF-a-induced transcriptional factor [10], we coexpressed the AP-1 subunits c-Jun and c-Fos in A7r5 to determine whether AP-1 could also upregulate SIRT1. However, there was no significant difference in SIRT1 expression between the control and AP-1 groups (Supplementary Fig. 1). The above results indicate that p65/RelA plays a key role in promoting SIRT1 protein expression in VSMCs.
Fig. 4. p65/RelA is involved in the elevated expression of SIRT1 induced by TNF-a in A7r5. (A and B) p65/RelA RNAi or a U6-scrambled control was transfected into A7r5 cells for 36 h with (B) or without (A) TNF-a (30 ng/ml) treatment for another 8 h. Western blotting was used to detect SIRT1 and endogenous p65/RelA expression. Quantitative analysis (lower panel). (C) p65/RelA RNAi or a U6-scrambled control was co-transfected with pGL3-SIRT1-2685-luc into A7r5 cells for 36 h, and luciferase reporter assays were performed. pGL3-NF-jB-luc and pGL3-MMP-9-luc were transfected as positive controls. (D) p65/RelA RNAi or a U6-scrambled control was co-transfected with SIRT12685-luc into A7r5 cells for 36 h, followed by TNF-a (30 ng/ml) treatment for another 8 h. Luciferase reporter assays were carried out. Luciferase activity results are displayed as the mean ± SE. *P < 0.05, **P < 0.01 versus the control.
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3.4. p65/RelA plays a pivotal role in mediating the upregulation of SIRT1 by TNF-a To determine whether p65/RelA is implicated in the process of TNF-a-induced SIRT1 expression, p65/RelA RNAi was transfected into A7r5 for 36 h. Western blotting showed that SIRT1 protein expression was markedly suppressed (Fig. 4A). Luciferase reporter assays indicate that p65/RelA RNAi attenuates SIRT1 promoter activity by half, (MMP-9-luc and NF-jB-luc acted as positive controls) (Fig. 4C). When A7r5 cells were transfected with p65/RelA RNAi for 36 h, followed by TNF-a (30 ng/mL) treatment for another 8 h, both the expression of SIRT1 protein and the activity of the SIRT1 promoter induced by TNF-a were almost abolished by inhibiting endogenous p65/RelA (Fig. 4B and D). These results indicate that p65/RelA plays a pivotal role in mediating the elevated expression of SIRT1 by TNF-a. 4. Discussion The function of SIRT1 as a versatile subtle tuner of multiple physiological and pathological processes has been extensively investigated. Recent studies have demonstrated that SIRT1 expression is tightly regulated. The regulation of SIRT1 by transcriptional factors such as p53, HIC1, E2F1, and C/EBPa has been reported [28– 31]. In the present study, we found that SIRT1 mRNA and protein expression were induced by TNF-a in a time- and dose-dependent manner (Fig. 1). In addition, we observed that the p65/RelA subunit of the inflammatory transcriptional factor NF-jB mediated the TNF-a-induced elevated expression of SIRT1 and that when p65/ RelA was knocked down using p65/RelA RNAi in A7r5, TNF-a-induced SIRT1 expression was almost abolished (Fig. 4). Furthermore, using bioinformatics analysis we found that there was a putative NF-jB binding site in the 50 -flanking region of the SIRT1 promoter, from 339 to 328. Luciferase activity assays, with or without the putative NF-jB binding site, confirmed our expectation that p65/RelA is implicated in the transcriptional regulation of SIRT1. ChIP assays further confirmed that p65/RelA is recruited to the putative NF-jB binding region in the SIRT1 promoter (Fig. 2). These results suggest that TNF-a upregulates SIRT1 expression via p65/RelA at the transcriptional level. Multiple inflammatory mediators have been reported to be suppressed by SIRT1. Attenuation of NF-jB activity by SIRT1 is known to result in the suppression of TNF-a [16–18]. Since our results showed that TNF-a upregulates SIRT1 expression in VSMCs (Fig. 1), we hypothesize that there may be a potential negative feedback loop controlling TNF-a production in VSMCs. Such a loop would play an important role because the elevation of TNF-a by many inflammatory stimuli is concomitant with the upregulation of SIRT1. This loop would ameliorate the harmful accumulation of TNF-a and orchestrate an appropriate inflammatory response. SIRT1 inhibition of NF-jB activity through deacetylation of p65/ RelA at lysine 310 has been well documented in previous reports [15]. This, together with our results indicating that p65/RelA upregulates SIRT1 expression (Fig. 3), lead us to speculate that there is another autoregulated feedback loop between SIRT1 and p65/RelA to balance both SIRT1 and NF-jB activity appropriately for the specific physiological or pathological needs of VSMCs. Taken together, our data demonstrate that the p65/RelA subunit of NF-jB mediates elevated expression of SIRT1 by TNF-a in VSMCs. Given that TNF-a is a major inflammatory mediator and is induced by a wide range of pathogenic stimuli in vasculature, our data suggest that the upregulation of SIRT1 by TNF-a may be a ubiquitous auto-protective response of VSMCs to inflammatory events. Further studies are needed to elucidate the potential physiological and pathological significance of TNF-a-induced SIRT1 expression in VSMCs.
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