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ORIGINAL ARTICLE Q1
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Sirtuin-6 inhibits cardiac fibroblast differentiation into myofibroblasts via inactivation of nuclear factor kB signaling KUNMING TIAN1, ZHIPING LIU1, JIAOJIAO WANG1, SUOWEN XU2, TIANHUI YOU, and PEIQING LIU GUANGZHOU, CHINA
Differentiation of cardiac fibroblasts (CFs) into myofibroblasts represents a key event in cardiac fibrosis that contributes to pathologic cardiac remodeling. However, regulation of this phenotypic transformation remains elusive. Here, we show that sirtuin-6 (SIRT6), a member of the sirtuin family of NAD1-dependent histone deacetylase, plays a role in the regulation of myofibroblast differentiation. SIRT6 expression was upregulated under pathologic conditions in angiotensin II (Ang II)-stimulated CFs and in myocardium of rat subject to abdominal aortic constriction surgery. SIRT6 depletion by RNA interference (small interfering RNA [siRNA]) in CFs resulted in increased cell proliferation and extracellular matrix deposition. Further examination of SIRT6-depleted CFs demonstrated significantly higher expression of a-smooth muscle actin (a-SMA), the classical marker of myofibroblast differentiation, and increased formation of focal adhesions. Notably, SIRT6 depletion further exacerbated Ang II–induced myofibroblast differentiation. Overexpression of SIRT6 restored a-SMA expression in SIRT6-depleted or Ang II–treated CFs. Moreover, SIRT6 depletion induced the DNA binding activity and transcriptional activity of nuclear factor kB (NFkB). Importantly, using an NF-kB p65 siRNA or pyrrolidine dithiocarbamate, a specific inhibitor of NF-kB activity, reversed the expression of phenotypic markers of myofibroblasts. Our findings unravel a novel role of SIRT6 as a key modulator in the phenotypic conversion of CFs to myofibroblasts. (Translational Research 2014;-:1–12)
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INTRODUCTION
H
eart failure is a major cause of death in patients with coronary heart disease in the developed world. The development of heart failure is invariably associated with extensive fibrosis, which
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Kunming Tian, Zhiping Liu, and Jiaojiao Wang contributed equally to this work. 2 Present address: Aab Cardiovascular Research Institute, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Box CVRI, Rochester, NY 14642. From the School of Nursing, Guangdong Pharmaceutical University, Guangzhou, China; Department of Pharmacology and Toxicology, School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou, China.
aggravates diastolic dysfunction and predisposes the heart to arrhythmias and sudden cardiac death.1,2 A critical event in the progression of cardiac fibrosis is the differentiation of cardiac fibroblasts (CFs) into myofibroblasts, characterized by the expression of Submitted for publication May 25, 2014; revision submitted August 25, 2014; accepted for publication August 28, 2014. Reprint requests: Peiqing Liu, Department of Pharmacology and Toxicology, School of Pharmaceutical Sciences, Sun Yat-Sen University (Higher Education Mega Center), 132 East Wai-Huan Road, Guangzhou 510006, Guangdong, China; e-mail: [email protected]. 1931-5244/$ - see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.trsl.2014.08.008
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AT A GLANCE COMMENTARY Tian K, et al. Background
Sirtuin-6 (SIRT6), an NAD1-dependent histone deacetylase, has been implicated in the regulation of genomic stability, cellular metabolism, stress responses, and life-span. However, whether SIRT6 plays a role in the regulation of myofibroblast differentiation and cardiac fibrosis remains largely unknown. Translational Significance
The present study has demonstrated that SIRT6 depletion induces cardiac fibroblast differentiation into myofibroblasts. Previous study has shown that SIRT6 expression was markedly reduced in failing human hearts. Thus, development of specific activators of SIRT6 to increase the levels or activity of this protein may be beneficial for the prevention and/or therapeutic treatment of cardiac fibrosis and heart failure.
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a-smooth muscle actin (a-SMA) and production of extracellular matrix (ECM) components such as fibronectin (FN), collagen type I, and collagen type III.3,4 Myofibroblast differentiation is induced by several profibrotic factors, such as angiotensin II (Ang II), endothelin-1, platelet-derived growth factor, and transforming growth factor b (TGF-b).5-7 Ang II can also stimulate the expression of TGF-b and endothelin-1,8 additionally amplifying in the pathogenesis of cardiac fibrosis. Sirtuins (SIRTs), a family of evolutionary conserved NAD1-dependent histone deacetylases, are implicated in the regulation of multiple pathophysiological processes, including DNA damage repair, metabolic homeostasis, apoptosis, tumorigenesis, neurodegeneration, and aging.9,10 So far, 7 SIRT genes have been found in mammal genomes, named as SIRT1–7. Resveratrol, a well-known SIRT agonist, has been shown to inhibit Ang II and TGF-b–induced CF proliferation and myofibroblast differentiation.11 In the present study, we screened the messenger RNA (mRNA) expressions of all SIRT isoforms in Ang II–induced CFs and found the SIRT1 and SIRT6 were upregulated in response to Ang II stimulation. Importantly, change in the expression of SIRT6 was the most significant, indicating that SIRT6 may play a role in Ang II–treated CFs. Recently, mounting evidence suggests that SIRT6 exerts protective effects in
multiple disease models, including obesity,12 liver cancer,13 fatty liver,14 cardiac hypertrophy,15,16 and chronic liver inflammation.17 However, the precise role of SIRT6 in myofibroblast differentiation and cardiac fibrosis remains unknown. This study aims to investigate the possible roles of SIRT6 in the regulation of myofibroblast differentiation and to further explore the potential mechanisms. MATERIALS AND METHODS Chemicals and reagents. Rabbit anti–nuclear factor kB (anti–NF-kB) p65 antibody was purchased from Abcam (Cambridge, Massachusetts). Rabbit anti–IkB-a, a-SMA, paxillin, vinculin, FN, plasminogen activator inhibitor 1 (PAI-1), and collagen type I antibodies were from Santa Cruz Biotechnology (Santa Cruz, California). Rabbit anti-phospho-p65 antibody was Q5 obtained from Cell Signaling Technology (Danvers, Massachusetts). Mouse anti-a-tubulin antibody, rabbit anti-SIRT6, histone H1 antibodies, and pyrrolidine dithiocarbamic acid ammonium salt were purchased from Sigma-Aldrich (St. Louis, Missouri). Other reagents were from Sigma-Aldrich unless otherwise specified. Cell culture. Adult rat left ventricular CFs were isolated as previously described.18 In brief, 8- to 10-week-old (150–200 g) male Sprague-Dawley rats were anaesthetized with an intraperitoneal injection of sodium pentobarbitone (150 mg/kg). Hearts were then excised from anesthetized rats and rinsed in cold phosphate-buffered saline (PBS). The ventricles were Q6 minced and digested in Dulbecco’s modified Eagle’s medium containing 0.1% collagenase type II (Gibco BRL, Grand Island, New York) at 37 C with continuous shaking for 60 minutes. The dissociated cells were collected and plated for 1 hour at 37 C. After preplating, the unattached cells (including myocytes and endothelial cells) were removed and the CFs were cultured in Dulbecco’s modified Eagle’s medium containing 10% new born calf serum (Gibco), 100 U/mL penicillin, and 100 mg/mL streptomycin. The purity of CFs was more than 95% as determined by positive staining for vimentin and negative staining for a-actin and von Willebrand factor. The cells in the second to fourth passage were used in this study. All experimental procedures were conducted as per the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996) and were approved by the Institutional Ethics Review Board of Sun Yat-Sen University. Real-time polymerase chain reaction. RNA isolation Q7 and real-time polymerase chain reaction (PCR) was
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conducted as previously described in detail.19 Rat-specific primers for SIRT1–7, a-SMA, paxillin, vinculin, FN, PAI-1, collagen type I, and GAPDH were used (see Supplemental Table 1). Western blot. Western blot analyses were performed as previously described.19 For analysis of p65 protein expression, cytoplasmic and nuclear proteins were extracted with the CelLytic NuCLEAR Extraction Kit (Sigma). Histone H1 and a-tubulin were used to normalize nuclear and cytosolic protein loading. Plasmid transfection. CFs were transiently transfected with SIRT6 (EGFP-SIRT6) or empty vector (EGFP-N3) using Lipofectamine 2000 (Invitrogen, Carlsbad, California) according to the manufacturer’s instructions. After incubation for 24 hours, the cells were treated with or without Ang II for indicated time. RNA interference. Three different duplex small interfering RNAs (siRNAs) for SIRT6, p65, and negative control siRNAs were purchased from GenePharma (Shanghai, China). The sequences of siRNAs are shown in the Supplemental material (Supplemental Tables 2 and 3). Subconfluent CFs (70% confluence) were transfected either with SIRT6 siRNAs or with negative control siRNA using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. In shortterm silencing experiments, once the transfection complexes were removed, the cultures were maintained in a normal fresh medium for further 48 hours before analysis. In long-term silencing experiments, the cells were harvested by trypsinization after 48 hours of transfection, then seeded at a density of 5 3 106 cells/mL, and maintained in fresh medium for a further 24-hour period before analysis. Real-time PCR and Western blot were used at 48 hours after transfection to compare silencing efficiency of different duplex siRNAs (see Supplemental Figs 1 and 2). In all our siRNA experiments, we did not observe any significant difference between negative siRNA and untreated cells, eliminating the possibility that siRNA has a nonspecific effect on CFs. CFs proliferation assay. Cell Counting Kit-8 (Dojindo, Shanghai, China) was used in CF proliferation assay. Cells were seeded at 5 3 104 cells per well into 96-well plates in a final volume of 100 mL and transfected with negative control siRNA or S6 siRNA. After 72 hours of transfection, cells were subjected to assay by adding 10 mL of Cell Counting Kit-8 solution to each well, and the plate was further incubated for 4 hours at 37 C. The absorbance at 450 nm was measured with a microplate reader. The experiment was performed in 10 duplicates. Hydroxyproline measurement. The content of hydroxyproline in CFs was assayed by a commercial kit (Nanjing Jiancheng Bioengineering Institute,
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Nanjing, China) according to the manufacturer’s instructions. Immunofluorescence staining. CFs cultured on glass coverslips were fixed in 4% paraformaldehyde for 30 minutes, washed with PBS 3 times, and permeabilized with 0.1% Triton X-100 in PBS for 20 minutes. After rinsing with PBS 3 times, the slides were blocked with 5% goat serum at room temperature for 30 minutes. Next, the cells were incubated with a rabbit polyclonal anti-a-SMA primary antibody (1:100 dilution) overnight at 4 C. The slides were then rinsed and incubated with a goat anti-rabbit secondary antibody conjugated to Alexa Fluor 594 (1:1, 000 dilution; Invitrogen) for 1 hour at room temperature. The slides were counterstained with DAPI (5 mg/mL, Sigma) and mounted in a glycerin jelly medium, Q10 then subjected to confocal microscopy (LSM710; Carl Zeiss). Control coverslips were processed the same way, except for omission of the primary antibody or secondary antibody. Luciferase reporter gene assay. NF-kB–dependent transcriptional activity was conducted as previously described.16 In brief, CFs were cotransfected with NF-kB reporter plasmid and pRL-TK as an internal Q11 control. After 8 hours of incubation, the cells were subjected to Ang II stimulation, RNA interference, or transfection with SIRT6 or empty vector. The luciferase activity was measured using the DualLuciferase Reporter Assay System (Promega, Madison, Wisconsin) according to the manufacturer’s protocol. Relative luciferase activity was calculated as the ratio of firefly luciferase activity to Renilla luciferase activity. Electrophoretic mobility shift assay. Electrophoretic mobility shift assay (EMSA) was performed using a LightShift Chemiluminescent EMSA kit (Pierce) ac- Q12 cording to our established procedure.19,20 Briefly, CFs were washed with ice-cold PBS and scrapped off with a cell scraper. Nuclear extracts were prepared using the Nuclear Extract Kit (Active Motif) according to the manufacturer’s instructions. Two micrograms of nuclear protein was then used to assess DNA binding activity using a 30 -biotin–labeled oligonucleotide probes containing the consensus sequence (underlined) of the NF-kB: 50 -tgg aaa tgg gaa gtc tca tag gac-30 (sense) and 50 -gtc cta tga gac ttc cca ttt cca-30 (antisense). Each sample was electrophoresed in a 6% nondenaturing polyacrylamide gel in 0.53 TBE buffer at 100 V for 60 minutes. For cold competition experiments, 50-fold molar excess unlabeled duplex oligonucleotides containing NF-kB consensus sequence was added to the nuclear extracts before incubation with the biotin-labeled oligonucleotides. Abdominal aortic constriction surgery. SpragueDawley rats (male, weighing 180–200 g, SPF grade,
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certification no. 44008500003151) were supplied by the Experimental Animal Center of Sun Yat-Sen University (Guangzhou, China). Pressure overload was induced by suprarenal abdominal aortic constriction (AAC) as previously described.21 The rats were anaesthetized by an intraperitoneal injection of sodium pentobarbitone (50 mg/kg) before AAC surgery. Loss of reflex was detected by pricking the rats’s feet and legs with forceps. At the end of experiment, rats were sacrificed by CO2 inhalation and then hearts were harvested and stored at 280 C for Western blot analysis. The investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). The animal experiments were approved by the Institutional Ethics Review Board of Sun Yat-Sen University. Statistical analysis. Data are presented as the mean 6 standard error of the mean. Statistical analysis was performed with t test or by 1- or 2-way analysis of variance followed by Bonferroni’s post hoc tests as appropriate. In all cases, differences were considered statistically significant with P , 0.05. RESULTS Expression of SIRT6 was upregulated in the in vitro and in vivo model of cardiac fibrosis. To assess whether
SIRT6 might play a relevant role in CFs, we first compared the mRNA expression profile of SIRT1–7 in CFs, using the expression of SIRT1 as a reference. As shown in Supplemental Fig 3, SIRT1, SIRT5, and SIRT6 were highly expressed in CFs, and the mRNA expressions of SIRT1 and SIRT6 were almost equal. Ang II has been well recognized as a potent stimulator of cardiac fibrotic response by promoting CF proliferation and myofibroblast differentiation.5,11 We thus examined the effects of Ang II on mRNA levels of SIRT1–7 in CFs. Fig 1, A shows that the mRNA levels of SIRT1 and SIRT6 were increased in response to Ang II stimulation, and the increase in SIRT6 was the most prominent. However, we did not observe significant alterations in the mRNA expression of other SIRTs. Moreover, Western blot analysis revealed that Ang II in a time- and concentration-dependent manner increased the protein expression of SIRT6 (Fig 1, B and C). Comparing with the untreated group, treatment with 100 nM of Ang II for 24 hours resulted in the increase in protein level of SIRT6 by 3.05 6 0.36 fold (P , 0.05). To further examine whether there was a similar alteration of SIRT6 in pressure overload-induced cardiac fibrosis of rats, we analyzed SIRT6 expression in the hearts from AAC or sham-operated rats at the age of 8 weeks. Collagen deposition and the expression of
fibrotic markers including a-SMA, collagen type I, and FN were significantly increased in AAC rats (Supplemental Fig 4), suggesting the successful induction of cardiac fibrosis by AAC surgery. In addition, the protein expression of SIRT6 was markedly upregulated in pressure-overloaded hearts induced by AAC (Fig 1, D) and in CFs isolated from the hearts of AAC rat (Supplemental Fig 5). These results raised the possibility that SIRT6 may play a critical role in the development of cardiac fibrosis. Silencing of SIRT6-induced CF proliferation and ECM deposition. To explore the potential role of SIRT6 in
cardiac fibrosis, we first knocked down SIRT6 in CFs using siRNA. Real-time PCR and Western blot were performed to evaluate the effect of 3 independent siRNAs, marked si1, si2, and si3, respectively. As shown in Supplemental Fig 1, si3 reduced the mRNA and protein expressions of SIRT6 by 85% and 92%, respectively, without affecting other SIRT isoforms (Supplemental Fig 6). Therefore, si3 was used in the following experiments. CFs transfected with the negative control siRNA were well spread and displayed few plasma membrane protrusions (Fig 2, A). In contrast, SIRT6-silenced CFs exhibited an irregular cell shape, which was characterized by numerous protrusions of the cytoplasm (Fig 2, B). SIRT6-silenced cells showed increased rates of proliferation (Fig 2, C), suggesting a proproliferative phenotype of SIRT6 depletion. Cardiac fibrosis leads to ECM deposition and changes in gene expression reminiscent of a pathologic phenotype, such as FN, collagen type I, and PAI-1. Consistent with this notion, Fig 2, D shows that in SIRT6-depleted CFs there is a significant increase in hydroxyproline content. In addition, real-time PCR analysis showed that SIRT6-silenced CFs displayed higher mRNA levels of FN, collagen type I, and PAI-1 (Fig 2, E). Similarly, testing for these fibrosis-related genes by Western blot showed paralleling changes at the protein level (Fig 2, F). Silencing of SIRT6 induced CF differentiation into myofibroblasts. To examine the potential effect of
SIRT6 on myofibroblast differentiation, CFs were transfected with SIRT6 siRNA or/and treated with Ang II. Q13 SIRT6 depletion could mimic the effects of Ang II to markedly induce mRNA (Supplemental Fig 7) and protein (Fig 3, A) levels of a-SMA. Importantly, SIRT6 siRNA transfection exacerbated the effect of Ang II-mediated upregulation of a-SMA mRNA and protein expression. This effect was confirmed by immunofluorescence analysis (Fig 3, B). Furthermore, immunofluorescent staining assay also revealed increased cell size of CFs and a-SMA containing stress-fibers indicative of myofibroblasts formation in
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Fig 1. SIRT6 was upregulated in Ang II–treated CFs and in AAC rats. (A) Adult rat left ventricular CFs were treated with 100 nM of Ang II for 24 hours, and real-time polymerase chain reaction was performed to screen the mRNA expression profile of SIRT1–7. Data are presented as the mean 6 SEM (*P , 0.05 vs control, n 5 5). (B and C) CFs were treated with 100 nM of Ang II for indicated time (left panel) or with indicated concentrations of Ang II for 24 hours (right panel). The levels of mRNA (B) and protein (C) of SIRT6 were detected and normalized (mRNA by GAPDH and protein by a-tubulin) and then presented as fold of control level. Data are presented as the mean 6 SEM (*P , 0.05 vs control, n 5 6). (D) Sprague-Dawley rats were subjected to AAC surgery for 8 weeks. Hearts were collected from sham rats or AAC rats, and subjected to Western blot to examine the expression of SIRT6 and a-tubulin. Data are presented as the mean 6 SEM (*P , 0.05 vs sham group, n 5 8). AAC, abdominal aortic constriction; CF, cardiac fibroblast; Ang II, angiotensin II; SIRT, sirtuin; SEM, standard error of the mean; mRNA, messenger RNA.
SIRT6-silenced cells. In contrast, overexpression of SIRT6 partially reversed the increase in a-SMA protein expression in cells treated with Ang II (Fig 3, C) or SIRT6 siRNA (Fig 3, D). Silencing of SIRT6 increased focal adhesions in CFs. Another morphologic hallmark of myofibroblasts
is focal adhesions. We therefore examined the impact of depletion of SIRT6 on paxillin and vinculin. Realtime PCR (Fig 4, A) and Western blot (Fig 4, B) analysis showed that SIRT6-silenced CFs displayed higher levels of mRNA and protein expressions of both genes, mimicking the effects of Ang II stimulation. Furthermore, our results also demonstrated a significant trend toward the increase in protein expression of both genes in the SIRT6 siRNA
plus Ang II-treated cells compared with SIRT6 siRNA and Ang II alone, suggesting SIRT6 depletion aggravates Ang II–induced CF phenotypic conversion. Silencing of SIRT6 activated NF-kB signaling. Previous studies have demonstrated that NF-kB activity is increased in multiple cardiovascular diseases and its signaling is critically implicated in the development of cardiac fibrosis.22-24 Kawahara et al25 found that SIRT6 deacetylates histone H3 lysine 9 (H3K9) and acts as a transcriptional corepressor of NF-kB. Therefore, we supposed that NF-kB signaling might be involved in SIRT6-mediated protection against cardiac fibrosis in CFs. To test this hypothesis, we first examined the effects of SIRT6 depletion on the activation of NF-kB. EMSA assay demonstrated that
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Fig 2. Depletion of SIRT6 induced CF proliferation and ECM deposition. (A and B) Morphology of CFs transfected with NC siRNA (A) or S6 siRNA (B), 3200 magnification. (C) Cell proliferation was measured by CCK-8 assay after 72 hours of transfection with NC siRNA or S6 siRNA under standard culture conditions (10% FCS). *P , 0.05 vs NC siRNA, n 5 5. (D) Collagen synthesis was determined by hydroxyproline assay in CFs after 72 hours of transfection with NC siRNA or S6 siRNA, *P , 0.05 vs NC siRNA, n 5 6. (E and F) Real-time polymerase chain reaction (E) and Western blot (F) analysis of mRNA and protein levels of PAI-1, collagen type I, and FN in CFs transfected with NC or S6 siRNA. Data are expressed as the mean 6 SEM, *P , 0.05 vs NC siRNA, n 5 6. CF, cardiac fibroblast; SIRT, sirtuin; SEM, standard error of the mean; NC, negative control; siRNA, small interfering RNA; ECM, extracellular matrix; CCK-8, Cell Counting Kit-8; PAI-1, plasminogen activator inhibitor 1; FN, fibronectin.
silencing of SIRT6 induced a significant increase in NF-kB DNA binding activity (Fig 5, A). We also examined the effect of SIRT6 on NF-kB–dependent
transcriptional activity by using dual-luciferase reporter gene assay. As shown in Fig 5, B, SIRT6 depletion led to increased NF-kB–luciferase reporter gene
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Fig 3. Depletion of SIRT6 induced CF differentiation into myofibroblasts. (A) Representative Western blot (upper panel) and quantitative analysis (lower panel) of a-SMA and SIRT6 protein expression in CFs transfected with NC siRNA or S6 siRNA for 48 hours in the presence or absence of Ang II (100 nM). Data are presented as the mean 6 SEM (*P , 0.05 vs NC siRNA, n 5 5). (B) Representative images of immunofluorescence staining for a-SMA (red) in CFs treated as mentioned previously. Nuclei were stained with DAPI (blue). Scale bar, 20 mm. (C) Representative Western blot (upper panel) and quantitative analysis (lower panel) of the protein levels of a-SMA and SIRT6 in CFs transfected with empty vector (EGFP-N3) or SIRT6 plasmid for 48 hours in the presence or absence of Ang II (100 nM). Data are presented as the mean 6 SEM (n 5 6). ***P , 0.001; #P , 0.05. (D) Representative Western blot (upper panel) and quantitative analysis (lower panel) of the protein levels of a-SMA and SIRT6 in CFs treated with S6 siRNA, with or without SIRT6 overexpression. Data are presented as the mean 6 SEM (n 5 6). ***P , 0.001; #P , 0.05. CF, cardiac fibroblast; SIRT, sirtuin; SEM, standard error of the mean; NC, negative control; a-SMA, a-smooth muscle actin; siRNA, small interfering RNA; Ang II, angiotensin II. For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.
activity, mimicking the effect of Ang II stimulation. On the contrary, SIRT6 overexpression repressed both basal and Ang II–induced NF-kB transcriptional activity. SIRT6 inhibited myofibroblast differentiation via inactivation of NF-kB–dependent transcriptional activity. To further investigate the potential role of
NF-kB in SIRT6-depleted CFs during myofibroblast transformation, CFs were transfected with p65 siRNA or treated with pyrrolidine dithiocarbamate (PDTC), a specific inhibitor of NF-kB activity.26 As shown in Fig 6, A and B, SIRT6 depletion increased the mRNA
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Fig 4. Depletion of SIRT6 increased focal adhesions in CFs. (A and B) Real-time polymerase chain reaction (A) and Western blot (B) analysis of mRNA and protein levels of paxillin and vinculin in CFs transfected with NC siRNA or S6 siRNA for 48 hours in the presence or absence of Ang II (100 nM). Data are presented as the mean 6 SEM (*P , 0.05 vs NC siRNA, n 5 6). CF, cardiac fibroblast; SEM, standard error of the mean; NC, negative control; Ang II, angiotensin II; siRNA, small interfering RNA.
and protein expressions of a-SMA, which can be abrogated by p65 siRNA or PDTC. This finding was also confirmed by confocal microscopy (Fig 6, C). Similarly, the effects of SIRT6 depletion on paxillin (Fig 7, A) and vinculin (Fig 7, B) were also abolished by PDTC. Taken together, these results indicate that the effect of SIRT6 on myofibroblast transformation required the participation of NF-kB. DISCUSSION
Emerging evidence suggests that SIRTs are involved in the onset and progression of cardiovascular diseases.
For example, SIRT1 protects the heart from aging, stress, inflammation, and hypertrophy27,28; SIRT3 and SIRT7 also play important roles in the regulation of stress responses in the heart.29,30 We have previously showed that SIRT6 acts as a negative regulator of cardiac hypertrophy,16 indicating its potential cardioprotective role; however, whether SIRT6 plays a role in myofibroblast transformation and cardiac fibrosis is not clear. Here, we have provided the first evidence that SIRT6 negatively regulates CF differentiation into myofibroblasts. Additionally, our data show that SIRT6 depletion increases CF proliferation, ECM deposition, and upregulates focal adhesion-related genes
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Fig 5. Depletion of SIRT6 activated NF-kB signaling in CFs. (A) CFs were transfected with NC siRNA or S6 siRNA, or treated with Ang II (100 nM) or pyrrolidine dithiocarbamate (PDTC). ESMA assays were performed to evaluate the influence of SIRT6 depletion on DNA binding activity. (B) Dual-luciferase reporter assays were performed to evaluate the influence of SIRT6 depletion, overexpression, and/or Ang II (100 nM) treatment on NF-kB–dependent transcriptional activity. Data are presented as the mean 6 SEM (*P , 0.05 vs control, # P , 0.05 vs Ang II treatment, n 5 6). CF, cardiac fibroblast; SIRT, sirtuin; SEM, standard error of the mean; NF-kB, nuclear factor kB; NC, negative control; siRNA, small interfering RNA; Ang II, angiotensin II.
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(paxillin and vinculin) and fibrosis-related genes (FN, collagen type I, and PAI-1). Mechanistically, we have documented a strong correlation between SIRT6 and NF-kB signaling in cardiac fibrotic response. In the first set of the experiments, we have revealed that SIRT6 is upregulated in Ang II–stimulated CFs and pressure overload–induced cardiac fibrosis. It seems paradoxical that SIRT6 is upregulated in the pressure-overloaded myocardium and inhibits fibroblast activation. In previous studies, Mao et al31 found that SIRT6 is upregulated in response to oxidative stress and promotes DNA repair; Minagawa et al32 also showed that SIRT6 expression is increased in idiopathic pulmonary fibrosis lung and inhibits senescence of human bronchial epithelial cells. They speculated that increased expression of SIRT6 might be an insufficient compensatory mechanism against stress-induced cellular senescence. Similarly, upregulation of SIRT6 in the pressure-overloaded myocardium might also be a compensatory mechanism against stress-induced fibroblast activation. Another potential explanation is that SIRT6 upregulation in the pressure-overloaded heart may act as a STOP signal limiting the fibrotic process. CF proliferation is vital for pathologic cardiac remodeling. In the present study, we showed a proproliferative phenotype of SIRT6-silenced CFs. Previous study has shown that depletion of SIRT6 reduces endothelial cell proliferation33; it was also demonstrated that depletion of SIRT6 had no significant effect on U2OS cell proliferation.34 Therefore, it appears that the effect of STRT6 on proliferation may be cell type-specific. One of the key features of cardiac fibrosis is the increased deposition of the ECM. The expression of
FN and collagen in CFs has been well established in de novo biosynthesis of ECM components and the fibrotic response. Consistent with this notion, our work showed that depletion of SIRT6 led to an increase in the mRNA and protein levels of the factor FN, collagen type I, and PAI-1. Furthermore, depletion of SIRT6 resulted in an increase in the content of hydroxyproline, the most specific amino acid in collagen and its change in concentration indicates a change in collagen levels.35 Taken together, these findings suggest that SIRT6 plays a prominent role in regulating collagen synthesis during pathologic cardiac remodeling. A major finding of this study is that SIRT6 is a negative regulator of myofibroblast differentiation, another key event in the cardiac remodeling processes. Myofibroblasts are highly active cells characterized by augmented expression of a-SMA, production of ECM components, and formation of focal adhesions. Upon injury, myofibroblast differentiation is induced by profibrotic factors such as Ang II/TGF-b and plays a key role in wound healing and remodeling processes.36,37 However, persistent myofibroblasts expedite scar formation and fibrosis, eventually leading to myocardial stiffness and impairment of cardiac function.38,39 Prevention of myofibroblast differentiation might, therefore, be a relevant approach to delay fibrosis in the heart. In the present study, we found that depletion of SIRT6 led to a significant increase in the mRNA and protein levels of a-SMA. Furthermore, overexpression of SIRT6 partially reversed the increase in a-SMA protein expression, in SIRT6-depleted or Ang II–treated CFs. In line with these findings, increased formation of focal adhesions occurred in SIRT6-depleted
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Fig 6. Depletion of SIRT6 induced myofibroblast differentiation by activating NF-kB. (A and B) Real-time polymerase chain reaction (A) and Western blot (B) analysis of mRNA and protein levels of a-SMA in SIRT6-depleted CFs treated with or without PDTC, or transfected with or without p65 siRNA. Data are presented as the mean 6 SEM (*P , 0.05 vs NC siRNA, #P , 0.05 vs S6 siRNA, n 5 6). (C) Representative images of immunofluorescent staining for a-SMA (red) in CFs treated as mentioned previously. Nuclei were stained with DAPI (blue). Scale bar, 20 mm. CF, cardiac fibroblast; SIRT, sirtuin; SEM, standard error of the mean; NF-kB, nuclear factor kB; a-SMA, a-smooth muscle actin; NC, negative control; siRNA, small interfering RNA; PDTC, pyrrolidine dithiocarbamate. For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.
CFs. Taken together, these data strongly suggested that SIRT6 plays a crucial role in the phenotypic transformation of CFs to myofibroblasts. Mechanistically, SIRT6 inhibits myofibroblast differentiation through its ability to suppress the transcriptional activity of NF-kB. NF-kB is a key regulator of inflammatory reaction. Activation of NF-KB signaling results in the synthesis and release of numbers of cytokine and chemotactic factors, which can induce the expression of a-SMA and other fibrosis-related genes, eventually contribute to myofibroblast differentiation and cardiac fibrosis.40 Accumulating evidence suggests that NF-kB is involved in the development of pathologic
cardiac remodeling. NF-kB activation leads to myocardial hypertrophy, myocardial fibrosis, and subsequent heart failure.22,23,41,42 In this study, we revealed that NF-kB DNA binding activity and transcriptional activity were increased in SIRT6-depleted CFs. In addition, overexpression of SIRT6 repressed both basal and Ang II–induced NF-kB transcriptional activity. Most importantly, inhibition of NF-kB signaling was sufficient to block myofibroblast differentiation in SIRT6-depleted CFs, suggesting that NF-kB signaling has a major role in this phenotypic transformation. SIRT6 is a nuclear histone deacetylase, and it is generally accepted that the biological functions of SIRT6
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Fig 7. Depletion of SIRT6 increased focal adhesions in CFs by activating NF-kB. (A and B) Representative Western blot (upper panel) and quantitative analysis (lower panel) of protein expression of paxillin (A) and vinculin (B) in CFs treated with or without PDTC. Data are presented as the mean 6 SEM (*P , 0.05 vs NC siRNA, #P , 0.05 vs S6 siRNA, n 5 5). CF, cardiac fibroblast; SIRT, sirtuin; SEM, standard error of the mean; NF-kB, nuclear factor kB; NC, negative control; siRNA, small interfering RNA; PDTC, pyrrolidine dithiocarbamate.
have been linked to its deacetylase activity on H3K9 or H3K56.43,44 Therefore, SIRT6 might negatively regulate NF-kB–dependent transcription via histone deacetylation. In fact, Kawahara et al25 have shown a model in which SIRT6 was recruited by transcription factor NF-kB to the promoter regions of target genes and deacetylates H3K9, thereby suppressing the target gene expression and leading to NF-kB signal termination. Q15
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CONCLUSIONS
Our study suggests that SIRT6 protects CFs from differentiation into myofibroblasts via inhibition of NF-kB–dependent transcriptional activation. Previous study has shown that SIRT6 is deficient in failing human hearts and in mouse hearts undergoing pressure overload.15 In this context, our findings that depletion of SIRT6 in CFs induces a pathologic phenotype (CF proliferation, ECM deposition, and myofibroblast differentiation) indicate that therapeutic approaches by increasing SIRT6 expression/activity may be a promising strategy for the prevention/treatment of cardiac fibrosis. ACKNOWLEDGMENTS
Conflicts of Interest: The authors declare no conflict of interest. All authors have read the journal’s policy on disclosure of potential conflicts of interest and the journal’s authorship agreement. The authors gratefully acknowledge financial support from the National Natural Science Foundation of China
(Nos. 81072641 and 81273499), the National Science and Technology Major Project of China ‘‘Key New Drug Creation and Manufacturing Program’’ (No. 2011ZX09401-307), Team Item of the Natural Science Foundation of Guangdong Province (No. S201103 0003190), Natural Science Foundation of Guangdong Province (No. S2013010016210), Major Project of Guangdong Province (Nos. 2008A030201013 and 2012A080201007), Major Project of the Department of Education of Guangdong Province (No. CXZ D1006), and the fostering discipline construction projects of Guangdong Pharmaceutical University. Supplementary Data
Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.trsl.2014.08.008.
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25. Kawahara TL, Michishita E, Adler AS, et al. SIRT6 links histone H3 lysine 9 deacetylation to NF-kappaB-dependent gene expression and organismal life span. Cell 2009;136:62–74. 26. Mahmoodzadeh S, Fritschka S, Dworatzek E, et al. Nuclear factor-kappaB regulates estrogen receptor-alpha transcription in the human heart. J Biol Chem 2009;284:24705–14. 27. Alcendor RR, Gao S, Zhai P, et al. Sirt1 regulates aging and resistance to oxidative stress in the heart. Circ Res 2007;100: 1512–21. 28. Planavila A, Iglesias R, Giralt M, Villarroya F. Sirt1 acts in association with PPARalpha to protect the heart from hypertrophy, metabolic dysregulation, and inflammation. Cardiovasc Res 2011;90:276–84. 29. Sundaresan NR, Gupta M, Kim G, Rajamohan SB, Isbatan A, Gupta MP. Sirt3 blocks the cardiac hypertrophic response by augmenting Foxo3a-dependent antioxidant defense mechanisms in mice. J Clin Invest 2009;119:2758–71. 30. Vakhrusheva O, Smolka C, Gajawada P, et al. Sirt7 increases stress resistance of cardiomyocytes and prevents apoptosis and inflammatory cardiomyopathy in mice. Circ Res 2008;102: 703–10. 31. Mao Z, Hine C, Tian X, et al. SIRT6 promotes DNA repair under stress by activating PARP1. Science 2011;332:1443–6. 32. Minagawa S, Araya J, Numata T, et al. Accelerated epithelial cell senescence in IPF and the inhibitory role of SIRT6 in TGF-betainduced senescence of human bronchial epithelial cells. Am J Physiol Lung Cell Mol Physiol 2011;300:L391–401. 33. Cardus A, Uryga AK, Walters G, Erusalimsky JD. SIRT6 protects human endothelial cells from DNA damage, telomere dysfunction, and senescence. Cardiovasc Res 2013;97:571–9. 34. Kaidi A, Weinert BT, Choudhary C, Jackson SP. Human SIRT6 promotes DNA end resection through CtIP deacetylation. Science 2010;329:1348–53. 35. Wu G, Bazer FW, Burghardt RC, et al. Proline and hydroxyproline metabolism: implications for animal and human nutrition. Amino Acids 2011;40:1053–63. 36. Sun Y, Weber KT. Angiotensin converting enzyme and myofibroblasts during tissue repair in the rat heart. J Mol Cell Cardiol 1996; 28:851–8. 37. Gurtner GC, Werner S, Barrandon Y, Longaker MT. Wound repair and regeneration. Nature 2008;453:314–21. 38. Frangogiannis NG. Targeting the inflammatory response in healing myocardial infarcts. Curr Med Chem 2006;13:1877–93. 39. Sun Y, Weber KT. Infarct scar: a dynamic tissue. Cardiovasc Res 2000;46:250–6. 40. Hall G, Hasday JD, Rogers TB. Regulating the regulator: NF-kappaB signaling in heart. J Mol Cell Cardiol 2006;41: 580–91. 41. Purcell NH, Molkentin JD. Is nuclear factor kappaB an attractive therapeutic target for treating cardiac hypertrophy? Circulation 2003;108:638–40. 42. Zou J, Le K, Xu S, et al. Fenofibrate ameliorates cardiac hypertrophy by activation of peroxisome proliferator-activated receptoralpha partly via preventing p65-NFkappaB binding to NFATc4. Mol Cell Endocrinol 2013;370:103–12. 43. Michishita E, McCord RA, Berber E, et al. SIRT6 is a histone H3 lysine 9 deacetylase that modulates telomeric chromatin. Nature 2008;452:492–6. 44. Michishita E, McCord RA, Boxer LD, et al. Cell cycle-dependent deacetylation of telomeric histone H3 lysine K56 by human SIRT6. Cell Cycle 2009;8:2664–6.
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Sirtuin-6 inhibits cardiac fibroblast differentiation into myofibroblasts via inactivation of nuclear factor kB signaling KUNMING TIAN1, ZHIPING LIU1, JIAOJIAO WANG1, SUOWEN XU2, TIANHUI YOU, and PEIQING LIU GUANGZHOU, CHINA
Differentiation of cardiac fibroblasts (CFs) into myofibroblasts represents a key event in cardiac fibrosis that contributes to pathologic cardiac remodeling. However, regulation of this phenotypic transformation remains elusive. Here, we show that sirtuin-6 (SIRT6), a member of the sirtuin family of NAD1-dependent histone deacetylase, plays a role in the regulation of myofibroblast differentiation. SIRT6 expression was upregulated under pathologic conditions in angiotensin II (Ang II)-stimulated CFs and in myocardium of rat subject to abdominal aortic constriction surgery. SIRT6 depletion by RNA interference (small interfering RNA [siRNA]) in CFs resulted in increased cell proliferation and extracellular matrix deposition. Further examination of SIRT6-depleted CFs demonstrated significantly higher expression of a-smooth muscle actin (a-SMA), the classical marker of myofibroblast differentiation, and increased formation of focal adhesions. Notably, SIRT6 depletion further exacerbated Ang II–induced myofibroblast differentiation. Overexpression of SIRT6 restored a-SMA expression in SIRT6-depleted or Ang II–treated CFs. Moreover, SIRT6 depletion induced the DNA binding activity and transcriptional activity of nuclear factor kB (NFkB). Importantly, using an NF-kB p65 siRNA or pyrrolidine dithiocarbamate, a specific inhibitor of NF-kB activity, reversed the expression of phenotypic markers of myofibroblasts. Our findings unravel a novel role of SIRT6 as a key modulator in the phenotypic conversion of CFs to myofibroblasts. (Translational Research 2014;-:1–12)
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Abbreviations: --- ¼ ---
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INTRODUCTION
H
eart failure is a major cause of death in patients with coronary heart disease in the developed world. The development of heart failure is invariably associated with extensive fibrosis, which
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Kunming Tian, Zhiping Liu, and Jiaojiao Wang contributed equally to this work. 2 Present address: Aab Cardiovascular Research Institute, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Box CVRI, Rochester, NY 14642. From the School of Nursing, Guangdong Pharmaceutical University, Guangzhou, China; Department of Pharmacology and Toxicology, School of Pharmaceutical Sciences, Sun Yat-Sen University, Guangzhou, China.
aggravates diastolic dysfunction and predisposes the heart to arrhythmias and sudden cardiac death.1,2 A critical event in the progression of cardiac fibrosis is the differentiation of cardiac fibroblasts (CFs) into myofibroblasts, characterized by the expression of Submitted for publication May 25, 2014; revision submitted August 25, 2014; accepted for publication August 28, 2014. Reprint requests: Peiqing Liu, Department of Pharmacology and Toxicology, School of Pharmaceutical Sciences, Sun Yat-Sen University (Higher Education Mega Center), 132 East Wai-Huan Road, Guangzhou 510006, Guangdong, China; e-mail: [email protected]. 1931-5244/$ - see front matter Ó 2014 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.trsl.2014.08.008
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AT A GLANCE COMMENTARY Tian K, et al. Background
Sirtuin-6 (SIRT6), an NAD1-dependent histone deacetylase, has been implicated in the regulation of genomic stability, cellular metabolism, stress responses, and life-span. However, whether SIRT6 plays a role in the regulation of myofibroblast differentiation and cardiac fibrosis remains largely unknown. Translational Significance
The present study has demonstrated that SIRT6 depletion induces cardiac fibroblast differentiation into myofibroblasts. Previous study has shown that SIRT6 expression was markedly reduced in failing human hearts. Thus, development of specific activators of SIRT6 to increase the levels or activity of this protein may be beneficial for the prevention and/or therapeutic treatment of cardiac fibrosis and heart failure.
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a-smooth muscle actin (a-SMA) and production of extracellular matrix (ECM) components such as fibronectin (FN), collagen type I, and collagen type III.3,4 Myofibroblast differentiation is induced by several profibrotic factors, such as angiotensin II (Ang II), endothelin-1, platelet-derived growth factor, and transforming growth factor b (TGF-b).5-7 Ang II can also stimulate the expression of TGF-b and endothelin-1,8 additionally amplifying in the pathogenesis of cardiac fibrosis. Sirtuins (SIRTs), a family of evolutionary conserved NAD1-dependent histone deacetylases, are implicated in the regulation of multiple pathophysiological processes, including DNA damage repair, metabolic homeostasis, apoptosis, tumorigenesis, neurodegeneration, and aging.9,10 So far, 7 SIRT genes have been found in mammal genomes, named as SIRT1–7. Resveratrol, a well-known SIRT agonist, has been shown to inhibit Ang II and TGF-b–induced CF proliferation and myofibroblast differentiation.11 In the present study, we screened the messenger RNA (mRNA) expressions of all SIRT isoforms in Ang II–induced CFs and found the SIRT1 and SIRT6 were upregulated in response to Ang II stimulation. Importantly, change in the expression of SIRT6 was the most significant, indicating that SIRT6 may play a role in Ang II–treated CFs. Recently, mounting evidence suggests that SIRT6 exerts protective effects in
multiple disease models, including obesity,12 liver cancer,13 fatty liver,14 cardiac hypertrophy,15,16 and chronic liver inflammation.17 However, the precise role of SIRT6 in myofibroblast differentiation and cardiac fibrosis remains unknown. This study aims to investigate the possible roles of SIRT6 in the regulation of myofibroblast differentiation and to further explore the potential mechanisms. MATERIALS AND METHODS Chemicals and reagents. Rabbit anti–nuclear factor kB (anti–NF-kB) p65 antibody was purchased from Abcam (Cambridge, Massachusetts). Rabbit anti–IkB-a, a-SMA, paxillin, vinculin, FN, plasminogen activator inhibitor 1 (PAI-1), and collagen type I antibodies were from Santa Cruz Biotechnology (Santa Cruz, California). Rabbit anti-phospho-p65 antibody was Q5 obtained from Cell Signaling Technology (Danvers, Massachusetts). Mouse anti-a-tubulin antibody, rabbit anti-SIRT6, histone H1 antibodies, and pyrrolidine dithiocarbamic acid ammonium salt were purchased from Sigma-Aldrich (St. Louis, Missouri). Other reagents were from Sigma-Aldrich unless otherwise specified. Cell culture. Adult rat left ventricular CFs were isolated as previously described.18 In brief, 8- to 10-week-old (150–200 g) male Sprague-Dawley rats were anaesthetized with an intraperitoneal injection of sodium pentobarbitone (150 mg/kg). Hearts were then excised from anesthetized rats and rinsed in cold phosphate-buffered saline (PBS). The ventricles were Q6 minced and digested in Dulbecco’s modified Eagle’s medium containing 0.1% collagenase type II (Gibco BRL, Grand Island, New York) at 37 C with continuous shaking for 60 minutes. The dissociated cells were collected and plated for 1 hour at 37 C. After preplating, the unattached cells (including myocytes and endothelial cells) were removed and the CFs were cultured in Dulbecco’s modified Eagle’s medium containing 10% new born calf serum (Gibco), 100 U/mL penicillin, and 100 mg/mL streptomycin. The purity of CFs was more than 95% as determined by positive staining for vimentin and negative staining for a-actin and von Willebrand factor. The cells in the second to fourth passage were used in this study. All experimental procedures were conducted as per the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996) and were approved by the Institutional Ethics Review Board of Sun Yat-Sen University. Real-time polymerase chain reaction. RNA isolation Q7 and real-time polymerase chain reaction (PCR) was
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conducted as previously described in detail.19 Rat-specific primers for SIRT1–7, a-SMA, paxillin, vinculin, FN, PAI-1, collagen type I, and GAPDH were used (see Supplemental Table 1). Western blot. Western blot analyses were performed as previously described.19 For analysis of p65 protein expression, cytoplasmic and nuclear proteins were extracted with the CelLytic NuCLEAR Extraction Kit (Sigma). Histone H1 and a-tubulin were used to normalize nuclear and cytosolic protein loading. Plasmid transfection. CFs were transiently transfected with SIRT6 (EGFP-SIRT6) or empty vector (EGFP-N3) using Lipofectamine 2000 (Invitrogen, Carlsbad, California) according to the manufacturer’s instructions. After incubation for 24 hours, the cells were treated with or without Ang II for indicated time. RNA interference. Three different duplex small interfering RNAs (siRNAs) for SIRT6, p65, and negative control siRNAs were purchased from GenePharma (Shanghai, China). The sequences of siRNAs are shown in the Supplemental material (Supplemental Tables 2 and 3). Subconfluent CFs (70% confluence) were transfected either with SIRT6 siRNAs or with negative control siRNA using Lipofectamine 2000 (Invitrogen) according to the manufacturer’s instructions. In shortterm silencing experiments, once the transfection complexes were removed, the cultures were maintained in a normal fresh medium for further 48 hours before analysis. In long-term silencing experiments, the cells were harvested by trypsinization after 48 hours of transfection, then seeded at a density of 5 3 106 cells/mL, and maintained in fresh medium for a further 24-hour period before analysis. Real-time PCR and Western blot were used at 48 hours after transfection to compare silencing efficiency of different duplex siRNAs (see Supplemental Figs 1 and 2). In all our siRNA experiments, we did not observe any significant difference between negative siRNA and untreated cells, eliminating the possibility that siRNA has a nonspecific effect on CFs. CFs proliferation assay. Cell Counting Kit-8 (Dojindo, Shanghai, China) was used in CF proliferation assay. Cells were seeded at 5 3 104 cells per well into 96-well plates in a final volume of 100 mL and transfected with negative control siRNA or S6 siRNA. After 72 hours of transfection, cells were subjected to assay by adding 10 mL of Cell Counting Kit-8 solution to each well, and the plate was further incubated for 4 hours at 37 C. The absorbance at 450 nm was measured with a microplate reader. The experiment was performed in 10 duplicates. Hydroxyproline measurement. The content of hydroxyproline in CFs was assayed by a commercial kit (Nanjing Jiancheng Bioengineering Institute,
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Nanjing, China) according to the manufacturer’s instructions. Immunofluorescence staining. CFs cultured on glass coverslips were fixed in 4% paraformaldehyde for 30 minutes, washed with PBS 3 times, and permeabilized with 0.1% Triton X-100 in PBS for 20 minutes. After rinsing with PBS 3 times, the slides were blocked with 5% goat serum at room temperature for 30 minutes. Next, the cells were incubated with a rabbit polyclonal anti-a-SMA primary antibody (1:100 dilution) overnight at 4 C. The slides were then rinsed and incubated with a goat anti-rabbit secondary antibody conjugated to Alexa Fluor 594 (1:1, 000 dilution; Invitrogen) for 1 hour at room temperature. The slides were counterstained with DAPI (5 mg/mL, Sigma) and mounted in a glycerin jelly medium, Q10 then subjected to confocal microscopy (LSM710; Carl Zeiss). Control coverslips were processed the same way, except for omission of the primary antibody or secondary antibody. Luciferase reporter gene assay. NF-kB–dependent transcriptional activity was conducted as previously described.16 In brief, CFs were cotransfected with NF-kB reporter plasmid and pRL-TK as an internal Q11 control. After 8 hours of incubation, the cells were subjected to Ang II stimulation, RNA interference, or transfection with SIRT6 or empty vector. The luciferase activity was measured using the DualLuciferase Reporter Assay System (Promega, Madison, Wisconsin) according to the manufacturer’s protocol. Relative luciferase activity was calculated as the ratio of firefly luciferase activity to Renilla luciferase activity. Electrophoretic mobility shift assay. Electrophoretic mobility shift assay (EMSA) was performed using a LightShift Chemiluminescent EMSA kit (Pierce) ac- Q12 cording to our established procedure.19,20 Briefly, CFs were washed with ice-cold PBS and scrapped off with a cell scraper. Nuclear extracts were prepared using the Nuclear Extract Kit (Active Motif) according to the manufacturer’s instructions. Two micrograms of nuclear protein was then used to assess DNA binding activity using a 30 -biotin–labeled oligonucleotide probes containing the consensus sequence (underlined) of the NF-kB: 50 -tgg aaa tgg gaa gtc tca tag gac-30 (sense) and 50 -gtc cta tga gac ttc cca ttt cca-30 (antisense). Each sample was electrophoresed in a 6% nondenaturing polyacrylamide gel in 0.53 TBE buffer at 100 V for 60 minutes. For cold competition experiments, 50-fold molar excess unlabeled duplex oligonucleotides containing NF-kB consensus sequence was added to the nuclear extracts before incubation with the biotin-labeled oligonucleotides. Abdominal aortic constriction surgery. SpragueDawley rats (male, weighing 180–200 g, SPF grade,
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certification no. 44008500003151) were supplied by the Experimental Animal Center of Sun Yat-Sen University (Guangzhou, China). Pressure overload was induced by suprarenal abdominal aortic constriction (AAC) as previously described.21 The rats were anaesthetized by an intraperitoneal injection of sodium pentobarbitone (50 mg/kg) before AAC surgery. Loss of reflex was detected by pricking the rats’s feet and legs with forceps. At the end of experiment, rats were sacrificed by CO2 inhalation and then hearts were harvested and stored at 280 C for Western blot analysis. The investigation conformed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). The animal experiments were approved by the Institutional Ethics Review Board of Sun Yat-Sen University. Statistical analysis. Data are presented as the mean 6 standard error of the mean. Statistical analysis was performed with t test or by 1- or 2-way analysis of variance followed by Bonferroni’s post hoc tests as appropriate. In all cases, differences were considered statistically significant with P , 0.05. RESULTS Expression of SIRT6 was upregulated in the in vitro and in vivo model of cardiac fibrosis. To assess whether
SIRT6 might play a relevant role in CFs, we first compared the mRNA expression profile of SIRT1–7 in CFs, using the expression of SIRT1 as a reference. As shown in Supplemental Fig 3, SIRT1, SIRT5, and SIRT6 were highly expressed in CFs, and the mRNA expressions of SIRT1 and SIRT6 were almost equal. Ang II has been well recognized as a potent stimulator of cardiac fibrotic response by promoting CF proliferation and myofibroblast differentiation.5,11 We thus examined the effects of Ang II on mRNA levels of SIRT1–7 in CFs. Fig 1, A shows that the mRNA levels of SIRT1 and SIRT6 were increased in response to Ang II stimulation, and the increase in SIRT6 was the most prominent. However, we did not observe significant alterations in the mRNA expression of other SIRTs. Moreover, Western blot analysis revealed that Ang II in a time- and concentration-dependent manner increased the protein expression of SIRT6 (Fig 1, B and C). Comparing with the untreated group, treatment with 100 nM of Ang II for 24 hours resulted in the increase in protein level of SIRT6 by 3.05 6 0.36 fold (P , 0.05). To further examine whether there was a similar alteration of SIRT6 in pressure overload-induced cardiac fibrosis of rats, we analyzed SIRT6 expression in the hearts from AAC or sham-operated rats at the age of 8 weeks. Collagen deposition and the expression of
fibrotic markers including a-SMA, collagen type I, and FN were significantly increased in AAC rats (Supplemental Fig 4), suggesting the successful induction of cardiac fibrosis by AAC surgery. In addition, the protein expression of SIRT6 was markedly upregulated in pressure-overloaded hearts induced by AAC (Fig 1, D) and in CFs isolated from the hearts of AAC rat (Supplemental Fig 5). These results raised the possibility that SIRT6 may play a critical role in the development of cardiac fibrosis. Silencing of SIRT6-induced CF proliferation and ECM deposition. To explore the potential role of SIRT6 in
cardiac fibrosis, we first knocked down SIRT6 in CFs using siRNA. Real-time PCR and Western blot were performed to evaluate the effect of 3 independent siRNAs, marked si1, si2, and si3, respectively. As shown in Supplemental Fig 1, si3 reduced the mRNA and protein expressions of SIRT6 by 85% and 92%, respectively, without affecting other SIRT isoforms (Supplemental Fig 6). Therefore, si3 was used in the following experiments. CFs transfected with the negative control siRNA were well spread and displayed few plasma membrane protrusions (Fig 2, A). In contrast, SIRT6-silenced CFs exhibited an irregular cell shape, which was characterized by numerous protrusions of the cytoplasm (Fig 2, B). SIRT6-silenced cells showed increased rates of proliferation (Fig 2, C), suggesting a proproliferative phenotype of SIRT6 depletion. Cardiac fibrosis leads to ECM deposition and changes in gene expression reminiscent of a pathologic phenotype, such as FN, collagen type I, and PAI-1. Consistent with this notion, Fig 2, D shows that in SIRT6-depleted CFs there is a significant increase in hydroxyproline content. In addition, real-time PCR analysis showed that SIRT6-silenced CFs displayed higher mRNA levels of FN, collagen type I, and PAI-1 (Fig 2, E). Similarly, testing for these fibrosis-related genes by Western blot showed paralleling changes at the protein level (Fig 2, F). Silencing of SIRT6 induced CF differentiation into myofibroblasts. To examine the potential effect of
SIRT6 on myofibroblast differentiation, CFs were transfected with SIRT6 siRNA or/and treated with Ang II. Q13 SIRT6 depletion could mimic the effects of Ang II to markedly induce mRNA (Supplemental Fig 7) and protein (Fig 3, A) levels of a-SMA. Importantly, SIRT6 siRNA transfection exacerbated the effect of Ang II-mediated upregulation of a-SMA mRNA and protein expression. This effect was confirmed by immunofluorescence analysis (Fig 3, B). Furthermore, immunofluorescent staining assay also revealed increased cell size of CFs and a-SMA containing stress-fibers indicative of myofibroblasts formation in
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Fig 1. SIRT6 was upregulated in Ang II–treated CFs and in AAC rats. (A) Adult rat left ventricular CFs were treated with 100 nM of Ang II for 24 hours, and real-time polymerase chain reaction was performed to screen the mRNA expression profile of SIRT1–7. Data are presented as the mean 6 SEM (*P , 0.05 vs control, n 5 5). (B and C) CFs were treated with 100 nM of Ang II for indicated time (left panel) or with indicated concentrations of Ang II for 24 hours (right panel). The levels of mRNA (B) and protein (C) of SIRT6 were detected and normalized (mRNA by GAPDH and protein by a-tubulin) and then presented as fold of control level. Data are presented as the mean 6 SEM (*P , 0.05 vs control, n 5 6). (D) Sprague-Dawley rats were subjected to AAC surgery for 8 weeks. Hearts were collected from sham rats or AAC rats, and subjected to Western blot to examine the expression of SIRT6 and a-tubulin. Data are presented as the mean 6 SEM (*P , 0.05 vs sham group, n 5 8). AAC, abdominal aortic constriction; CF, cardiac fibroblast; Ang II, angiotensin II; SIRT, sirtuin; SEM, standard error of the mean; mRNA, messenger RNA.
SIRT6-silenced cells. In contrast, overexpression of SIRT6 partially reversed the increase in a-SMA protein expression in cells treated with Ang II (Fig 3, C) or SIRT6 siRNA (Fig 3, D). Silencing of SIRT6 increased focal adhesions in CFs. Another morphologic hallmark of myofibroblasts
is focal adhesions. We therefore examined the impact of depletion of SIRT6 on paxillin and vinculin. Realtime PCR (Fig 4, A) and Western blot (Fig 4, B) analysis showed that SIRT6-silenced CFs displayed higher levels of mRNA and protein expressions of both genes, mimicking the effects of Ang II stimulation. Furthermore, our results also demonstrated a significant trend toward the increase in protein expression of both genes in the SIRT6 siRNA
plus Ang II-treated cells compared with SIRT6 siRNA and Ang II alone, suggesting SIRT6 depletion aggravates Ang II–induced CF phenotypic conversion. Silencing of SIRT6 activated NF-kB signaling. Previous studies have demonstrated that NF-kB activity is increased in multiple cardiovascular diseases and its signaling is critically implicated in the development of cardiac fibrosis.22-24 Kawahara et al25 found that SIRT6 deacetylates histone H3 lysine 9 (H3K9) and acts as a transcriptional corepressor of NF-kB. Therefore, we supposed that NF-kB signaling might be involved in SIRT6-mediated protection against cardiac fibrosis in CFs. To test this hypothesis, we first examined the effects of SIRT6 depletion on the activation of NF-kB. EMSA assay demonstrated that
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Fig 2. Depletion of SIRT6 induced CF proliferation and ECM deposition. (A and B) Morphology of CFs transfected with NC siRNA (A) or S6 siRNA (B), 3200 magnification. (C) Cell proliferation was measured by CCK-8 assay after 72 hours of transfection with NC siRNA or S6 siRNA under standard culture conditions (10% FCS). *P , 0.05 vs NC siRNA, n 5 5. (D) Collagen synthesis was determined by hydroxyproline assay in CFs after 72 hours of transfection with NC siRNA or S6 siRNA, *P , 0.05 vs NC siRNA, n 5 6. (E and F) Real-time polymerase chain reaction (E) and Western blot (F) analysis of mRNA and protein levels of PAI-1, collagen type I, and FN in CFs transfected with NC or S6 siRNA. Data are expressed as the mean 6 SEM, *P , 0.05 vs NC siRNA, n 5 6. CF, cardiac fibroblast; SIRT, sirtuin; SEM, standard error of the mean; NC, negative control; siRNA, small interfering RNA; ECM, extracellular matrix; CCK-8, Cell Counting Kit-8; PAI-1, plasminogen activator inhibitor 1; FN, fibronectin.
silencing of SIRT6 induced a significant increase in NF-kB DNA binding activity (Fig 5, A). We also examined the effect of SIRT6 on NF-kB–dependent
transcriptional activity by using dual-luciferase reporter gene assay. As shown in Fig 5, B, SIRT6 depletion led to increased NF-kB–luciferase reporter gene
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Fig 3. Depletion of SIRT6 induced CF differentiation into myofibroblasts. (A) Representative Western blot (upper panel) and quantitative analysis (lower panel) of a-SMA and SIRT6 protein expression in CFs transfected with NC siRNA or S6 siRNA for 48 hours in the presence or absence of Ang II (100 nM). Data are presented as the mean 6 SEM (*P , 0.05 vs NC siRNA, n 5 5). (B) Representative images of immunofluorescence staining for a-SMA (red) in CFs treated as mentioned previously. Nuclei were stained with DAPI (blue). Scale bar, 20 mm. (C) Representative Western blot (upper panel) and quantitative analysis (lower panel) of the protein levels of a-SMA and SIRT6 in CFs transfected with empty vector (EGFP-N3) or SIRT6 plasmid for 48 hours in the presence or absence of Ang II (100 nM). Data are presented as the mean 6 SEM (n 5 6). ***P , 0.001; #P , 0.05. (D) Representative Western blot (upper panel) and quantitative analysis (lower panel) of the protein levels of a-SMA and SIRT6 in CFs treated with S6 siRNA, with or without SIRT6 overexpression. Data are presented as the mean 6 SEM (n 5 6). ***P , 0.001; #P , 0.05. CF, cardiac fibroblast; SIRT, sirtuin; SEM, standard error of the mean; NC, negative control; a-SMA, a-smooth muscle actin; siRNA, small interfering RNA; Ang II, angiotensin II. For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.
activity, mimicking the effect of Ang II stimulation. On the contrary, SIRT6 overexpression repressed both basal and Ang II–induced NF-kB transcriptional activity. SIRT6 inhibited myofibroblast differentiation via inactivation of NF-kB–dependent transcriptional activity. To further investigate the potential role of
NF-kB in SIRT6-depleted CFs during myofibroblast transformation, CFs were transfected with p65 siRNA or treated with pyrrolidine dithiocarbamate (PDTC), a specific inhibitor of NF-kB activity.26 As shown in Fig 6, A and B, SIRT6 depletion increased the mRNA
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Fig 4. Depletion of SIRT6 increased focal adhesions in CFs. (A and B) Real-time polymerase chain reaction (A) and Western blot (B) analysis of mRNA and protein levels of paxillin and vinculin in CFs transfected with NC siRNA or S6 siRNA for 48 hours in the presence or absence of Ang II (100 nM). Data are presented as the mean 6 SEM (*P , 0.05 vs NC siRNA, n 5 6). CF, cardiac fibroblast; SEM, standard error of the mean; NC, negative control; Ang II, angiotensin II; siRNA, small interfering RNA.
and protein expressions of a-SMA, which can be abrogated by p65 siRNA or PDTC. This finding was also confirmed by confocal microscopy (Fig 6, C). Similarly, the effects of SIRT6 depletion on paxillin (Fig 7, A) and vinculin (Fig 7, B) were also abolished by PDTC. Taken together, these results indicate that the effect of SIRT6 on myofibroblast transformation required the participation of NF-kB. DISCUSSION
Emerging evidence suggests that SIRTs are involved in the onset and progression of cardiovascular diseases.
For example, SIRT1 protects the heart from aging, stress, inflammation, and hypertrophy27,28; SIRT3 and SIRT7 also play important roles in the regulation of stress responses in the heart.29,30 We have previously showed that SIRT6 acts as a negative regulator of cardiac hypertrophy,16 indicating its potential cardioprotective role; however, whether SIRT6 plays a role in myofibroblast transformation and cardiac fibrosis is not clear. Here, we have provided the first evidence that SIRT6 negatively regulates CF differentiation into myofibroblasts. Additionally, our data show that SIRT6 depletion increases CF proliferation, ECM deposition, and upregulates focal adhesion-related genes
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Fig 5. Depletion of SIRT6 activated NF-kB signaling in CFs. (A) CFs were transfected with NC siRNA or S6 siRNA, or treated with Ang II (100 nM) or pyrrolidine dithiocarbamate (PDTC). ESMA assays were performed to evaluate the influence of SIRT6 depletion on DNA binding activity. (B) Dual-luciferase reporter assays were performed to evaluate the influence of SIRT6 depletion, overexpression, and/or Ang II (100 nM) treatment on NF-kB–dependent transcriptional activity. Data are presented as the mean 6 SEM (*P , 0.05 vs control, # P , 0.05 vs Ang II treatment, n 5 6). CF, cardiac fibroblast; SIRT, sirtuin; SEM, standard error of the mean; NF-kB, nuclear factor kB; NC, negative control; siRNA, small interfering RNA; Ang II, angiotensin II.
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(paxillin and vinculin) and fibrosis-related genes (FN, collagen type I, and PAI-1). Mechanistically, we have documented a strong correlation between SIRT6 and NF-kB signaling in cardiac fibrotic response. In the first set of the experiments, we have revealed that SIRT6 is upregulated in Ang II–stimulated CFs and pressure overload–induced cardiac fibrosis. It seems paradoxical that SIRT6 is upregulated in the pressure-overloaded myocardium and inhibits fibroblast activation. In previous studies, Mao et al31 found that SIRT6 is upregulated in response to oxidative stress and promotes DNA repair; Minagawa et al32 also showed that SIRT6 expression is increased in idiopathic pulmonary fibrosis lung and inhibits senescence of human bronchial epithelial cells. They speculated that increased expression of SIRT6 might be an insufficient compensatory mechanism against stress-induced cellular senescence. Similarly, upregulation of SIRT6 in the pressure-overloaded myocardium might also be a compensatory mechanism against stress-induced fibroblast activation. Another potential explanation is that SIRT6 upregulation in the pressure-overloaded heart may act as a STOP signal limiting the fibrotic process. CF proliferation is vital for pathologic cardiac remodeling. In the present study, we showed a proproliferative phenotype of SIRT6-silenced CFs. Previous study has shown that depletion of SIRT6 reduces endothelial cell proliferation33; it was also demonstrated that depletion of SIRT6 had no significant effect on U2OS cell proliferation.34 Therefore, it appears that the effect of STRT6 on proliferation may be cell type-specific. One of the key features of cardiac fibrosis is the increased deposition of the ECM. The expression of
FN and collagen in CFs has been well established in de novo biosynthesis of ECM components and the fibrotic response. Consistent with this notion, our work showed that depletion of SIRT6 led to an increase in the mRNA and protein levels of the factor FN, collagen type I, and PAI-1. Furthermore, depletion of SIRT6 resulted in an increase in the content of hydroxyproline, the most specific amino acid in collagen and its change in concentration indicates a change in collagen levels.35 Taken together, these findings suggest that SIRT6 plays a prominent role in regulating collagen synthesis during pathologic cardiac remodeling. A major finding of this study is that SIRT6 is a negative regulator of myofibroblast differentiation, another key event in the cardiac remodeling processes. Myofibroblasts are highly active cells characterized by augmented expression of a-SMA, production of ECM components, and formation of focal adhesions. Upon injury, myofibroblast differentiation is induced by profibrotic factors such as Ang II/TGF-b and plays a key role in wound healing and remodeling processes.36,37 However, persistent myofibroblasts expedite scar formation and fibrosis, eventually leading to myocardial stiffness and impairment of cardiac function.38,39 Prevention of myofibroblast differentiation might, therefore, be a relevant approach to delay fibrosis in the heart. In the present study, we found that depletion of SIRT6 led to a significant increase in the mRNA and protein levels of a-SMA. Furthermore, overexpression of SIRT6 partially reversed the increase in a-SMA protein expression, in SIRT6-depleted or Ang II–treated CFs. In line with these findings, increased formation of focal adhesions occurred in SIRT6-depleted
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Fig 6. Depletion of SIRT6 induced myofibroblast differentiation by activating NF-kB. (A and B) Real-time polymerase chain reaction (A) and Western blot (B) analysis of mRNA and protein levels of a-SMA in SIRT6-depleted CFs treated with or without PDTC, or transfected with or without p65 siRNA. Data are presented as the mean 6 SEM (*P , 0.05 vs NC siRNA, #P , 0.05 vs S6 siRNA, n 5 6). (C) Representative images of immunofluorescent staining for a-SMA (red) in CFs treated as mentioned previously. Nuclei were stained with DAPI (blue). Scale bar, 20 mm. CF, cardiac fibroblast; SIRT, sirtuin; SEM, standard error of the mean; NF-kB, nuclear factor kB; a-SMA, a-smooth muscle actin; NC, negative control; siRNA, small interfering RNA; PDTC, pyrrolidine dithiocarbamate. For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.
CFs. Taken together, these data strongly suggested that SIRT6 plays a crucial role in the phenotypic transformation of CFs to myofibroblasts. Mechanistically, SIRT6 inhibits myofibroblast differentiation through its ability to suppress the transcriptional activity of NF-kB. NF-kB is a key regulator of inflammatory reaction. Activation of NF-KB signaling results in the synthesis and release of numbers of cytokine and chemotactic factors, which can induce the expression of a-SMA and other fibrosis-related genes, eventually contribute to myofibroblast differentiation and cardiac fibrosis.40 Accumulating evidence suggests that NF-kB is involved in the development of pathologic
cardiac remodeling. NF-kB activation leads to myocardial hypertrophy, myocardial fibrosis, and subsequent heart failure.22,23,41,42 In this study, we revealed that NF-kB DNA binding activity and transcriptional activity were increased in SIRT6-depleted CFs. In addition, overexpression of SIRT6 repressed both basal and Ang II–induced NF-kB transcriptional activity. Most importantly, inhibition of NF-kB signaling was sufficient to block myofibroblast differentiation in SIRT6-depleted CFs, suggesting that NF-kB signaling has a major role in this phenotypic transformation. SIRT6 is a nuclear histone deacetylase, and it is generally accepted that the biological functions of SIRT6
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Fig 7. Depletion of SIRT6 increased focal adhesions in CFs by activating NF-kB. (A and B) Representative Western blot (upper panel) and quantitative analysis (lower panel) of protein expression of paxillin (A) and vinculin (B) in CFs treated with or without PDTC. Data are presented as the mean 6 SEM (*P , 0.05 vs NC siRNA, #P , 0.05 vs S6 siRNA, n 5 5). CF, cardiac fibroblast; SIRT, sirtuin; SEM, standard error of the mean; NF-kB, nuclear factor kB; NC, negative control; siRNA, small interfering RNA; PDTC, pyrrolidine dithiocarbamate.
have been linked to its deacetylase activity on H3K9 or H3K56.43,44 Therefore, SIRT6 might negatively regulate NF-kB–dependent transcription via histone deacetylation. In fact, Kawahara et al25 have shown a model in which SIRT6 was recruited by transcription factor NF-kB to the promoter regions of target genes and deacetylates H3K9, thereby suppressing the target gene expression and leading to NF-kB signal termination. Q15
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CONCLUSIONS
Our study suggests that SIRT6 protects CFs from differentiation into myofibroblasts via inhibition of NF-kB–dependent transcriptional activation. Previous study has shown that SIRT6 is deficient in failing human hearts and in mouse hearts undergoing pressure overload.15 In this context, our findings that depletion of SIRT6 in CFs induces a pathologic phenotype (CF proliferation, ECM deposition, and myofibroblast differentiation) indicate that therapeutic approaches by increasing SIRT6 expression/activity may be a promising strategy for the prevention/treatment of cardiac fibrosis. ACKNOWLEDGMENTS
Conflicts of Interest: The authors declare no conflict of interest. All authors have read the journal’s policy on disclosure of potential conflicts of interest and the journal’s authorship agreement. The authors gratefully acknowledge financial support from the National Natural Science Foundation of China
(Nos. 81072641 and 81273499), the National Science and Technology Major Project of China ‘‘Key New Drug Creation and Manufacturing Program’’ (No. 2011ZX09401-307), Team Item of the Natural Science Foundation of Guangdong Province (No. S201103 0003190), Natural Science Foundation of Guangdong Province (No. S2013010016210), Major Project of Guangdong Province (Nos. 2008A030201013 and 2012A080201007), Major Project of the Department of Education of Guangdong Province (No. CXZ D1006), and the fostering discipline construction projects of Guangdong Pharmaceutical University. Supplementary Data
Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.trsl.2014.08.008.
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