The International Journal of Biochemistry & Cell Biology 49 (2014) 98–104
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Short communication
The role of SIRT6 in the differentiation of vascular smooth muscle cells in response to cyclic strain Qing-Ping Yao, Ping Zhang, Ying-Xin Qi, Si-Guo Chen, Bao-rong Shen, Yue Han, Zhi-Qiang Yan ∗ , Zong-Lai Jiang Institute of Mechanobiology & Medical Engineering, School of Life Sciences & Biotechnology, Shanghai Jiao Tong University, Shanghai, China
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Article history: Received 12 September 2013 Received in revised form 7 January 2014 Accepted 20 January 2014 Available online 2 February 2014 Keywords: SIRT6 Cyclic strain Differentiation Vascular smooth muscle cell Transforming growth factor-1
a b s t r a c t Vascular smooth muscle cells (VSMCs) may switch their phenotype between a quiescent contractile phenotype and a synthetic phenotype in response to cyclic strain, and this switch may contribute to hypertension, atherosclerosis, and restenosis. SIRT 6 is a member of the sirtuin family, and plays an important role in different cell processes, including differentiation. We hypothesized that cyclic strain modulates the differentiation of VSMCs via a transforming growth factor-1 (TGF-1)-Smad-SIRT6 pathway. VSMCs were subjected to cyclic strain using a Flexercell strain unit. It was demonstrated that the strain stimulated the secretion of TGF-1 into the supernatant of VSMCs. After exposed to the strain, the expressions of contractile phenotype markers, including smooth muscle protein 22 alpha, alpha-actin, and calponin, and phosphorylated Smad2, phosphorylated Smad5, SIRT6 and c-fos were up-regulated in VSMCs by western blot and immunofluorescence. And the expression of intercellular-adhesion molecule1 (ICAM-1) was also increased detected by flow cytometry. The strained-induced up-regulation of SIRT6 was blocked by a TGF-1 neutralizing antibody. Furthermore, the effects of strain on VSMCs were abrogated by SIRT6-specific siRNA transfection via the suppression c-fos and ICAM-1. These results suggest that SIRT6 may play a critical role in the regulation of VSMC differentiation in response to the cyclic strain. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Vascular smooth muscle cells (VSMCs) are a highly differentiated cell type, and subjected to cyclic mechanical strain due to pulsating blood flow in the media of arteries. Unlike the majority of differentiated cells, VSMCs maintain phenotypic plasticity. VSMCs can switch between a differentiated (also termed contractile) state, which is characterized by a spindle-shaped morphology; contractile gene expression; a low rate of proliferation; and a dedifferentiated (also termed synthetic) phenotype in response to various stimuli, including cytokines, cell adhesions, extracellular matrix interactions, injury stimuli, and mechanical force (Owens and Kumar, 2004). Dysregulation of phenotype switching is associated with vascular disorders, including hypertension, atherosclerosis and restenosis (Rzucidlo et al., 2007). An increasing number of studies have demonstrated that the cyclic strain can modulate VSMC morphology, migration, proliferation and apoptosis through
∗ Corresponding author at: Institute of Mechanobiology & Medical Engineering, School of Life Sciences & Biotechnology, P.O. Box 888, Shanghai Jiao Tong University, 800 Dongchuan Road, Minhang, Shanghai 200240, China. Tel.: +86 21 34204863; fax: +86 21 34204118. E-mail address: [email protected] (Z.-Q. Yan). 1357-2725/$ – see front matter © 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biocel.2014.01.016
various signaling pathways. In particular, the cyclic strain can induce many growth factor/cytokines, such as: vascular endothelial growth factor (VEGF) (Schad et al., 2011), platelet-derived growth factor (PDGF) (Yung et al., 2009), angiotensin II (Ang II) and TGF (Li et al., 1998). It has been showed that cytokine signaling can dramatically affect the differentiation status of VSMCs. For example, PDGF promotes multiple aspects of the synthetic VSMC phenotype (Tallquist and Kazlauskas, 2004); Conversely, TGF- inhibits VSMC proliferation and migration and promotes increased expression of VSMC contractile genes (Li et al., 2010). TGF- phosphorylates receptor-activated Smads (R-Smads), including Smad1, Smad2, Smad3, Smad5, and Smad8. The phosphorylated R-Smads then form a complex with the common Smad, Smad4, and translocate into the nucleus to regulate target gene expression through interactions with other cofactors (Heldin and Moustakas, 2012). Until now, how the cyclic strain induces TGF- in VSMCs to mediate the VSMC phenotype switching has remained unclear. SIRT6 is a member of sirtuin sub-class IV and is located in the nucleus, where it acts as both a deacetylase and a mono-ADP-ribosyl-transferase to regulate metabolism, inflammation, aging, transcription, and stress resistance. The highest concentrations of SIRT6 have been detected in muscle, brain, heart, liver, and the thymus, where SIRT6 affects transcriptional regulation in a tissue-specific manner (Beauharnois et al., 2013).
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In the cardiovascular system, growing evidence demonstrates that SIRT6 participates in many biological processes via many molecules. For examples, SIRT6-depleted human umbilical vein ECs (HUVECs) expresses higher levels of intercellular-adhesion molecule-1 (ICAM-1) (Lappas, 2012). ICAM-1 is highly expressed in flow-induced vascular remodeling (Chiang et al., 2009) and induced by cyclic strain in periodontal ligament cells (Saminathan et al., 2012). SIRT6 protects cardiomyocytes from hypertrophy via NF-Bdependent transcriptional activity (Yu et al., 2013) and insulin-like growth factor (IGF)-Akt signaling by targeting c-jun (Sundaresan et al., 2012). C-fos/c-jun control liver cancer initiation through SIRT6-dependent inhibition of surviving (Min et al., 2012). Hence, ICAM-1 and c-fos/c-jun may be involved in the VSMC differentiation induced by cyclic strain. However, no reports have addressed the expression of SIRT6 in VSMCs. Therefore, the aims of this study are to examine the role of SIRT6 in the regulation of VSMC differentiation in response to the cyclic strain and to elaborate the functional mechanism of the possible signaling way that underlies this process. 2. Materials and methods 2.1. Cell culture and cyclic strain loading The animal care and experimental protocols conformed to the Animal Management Rules of China (Documentation 55, 2001, Ministry of Health, China). VSMCs were isolated from the thoracic aortas of male Sprague–Dawley rats, 220–260 g, by an explant method as previously described (Qi et al., 2008). VSMCs between passages 4 and 8, with cell populations of more than 95% purity, were used in all experiments. For cyclic strain loading, VSMCs were seeded in gelatin-coated (Sigma, 0.1%) 6-well Bioflex plates (Flexcell International) at a density of 2 × 105 cells/well. After achieving 70% confluence, cells were serum-starved in DMEM for 24 h, and then subjected to the cyclic strain of 10% elongation at 1.25 Hz for 24 h using a Flexercell Tension Plus system (FX-4000 T, Flexcell International, USA). Unstretched VSMCs were served as a static control. For antibody-neutralizing studies, VSMCs were incubated with TGF-1 antibody (R&D Systems, 5 g/ml) prior to strain loading. VSMCs treated without TGF-1 antibody and loaded with the same strain were used as the control.
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2.4. SDS-PAGE and western blot analysis Protein extracts were separated by 10% SDS-PAGE. The proteins were detected using primary antibodies specific for Sm-22 (Abcam, 1:500) ␣-actin (Sigma, 1:1000), calponin (Sigma, 1:1000), p-Smad2 (Signalway Antibody, 1:500), Smad2 (Proteintech, 1:500), p-Smad5 (Epitomics, 1:500), Smad5 (Proteintech, 1:500), Smad4 (Epitomics, 1:500), SIRT6 (Abcam, 1:500), c-fos (BioWorld 1:500), c-jun (BioWorld 1:500), and GAPDH (Proteintech, 1:500). After incubation with alkaline phosphatase-conjugated secondary antibodies (Jackson ImmunoResearch), the signals were visualized with nitroblue tetrazolium-5 bromo-4 chloro-3 indolyl phosphate (Bio Basic, Inc.) and quantified with Quantity One software (Bio-Rad). 2.5. Flow cytometry For the flow cytometry (FCM) assay, the cells with and without stretch were treated with a 0.125% trypsin solution and digestion was stopped by adding PBS with 1% BSA; then centrifuged at 1000 rpm for 5 min and rinsed with PBS with 1% BSA. The cells were incubated with a PE-conjugated ICAM-1 antibody (Santa Cruz, 1 g per 1 × 106 cells) for 30 min at a room temperature and rinsed with PBS twice. An isotype PE-conjugated mouse IgG without ICAM-1 antibody served as the negative control. The cells were resuspended in 300 L of PBS and analyzed with a FACScan flow cytometer (BD Biosciences FACSCaliburTM ). 2.6. Immunofluorescence staining The attached VSMCs were fixed with 4% paraformaldehyde for 20 min, permeabilized for 5 min in 0.5% Triton X-100, and blocked with 1% BSA in PBS for 30 min. Then, cells were incubated with primary antibodies against p-Smad2 (1:50), p-Smad5 (1:50) and SIRT6 (1:100) at 4 ◦ C over night. VSMCs incubated with 1% BSA in PBS served as the negative control. All samples were washed in PBS for 30 min and incubated with an Alexa Fluor 555-conjugated secondary antibody (Cell Signaling Technology, 1:1000) along with DAPI for 1 h at room temperature. The samples were examined with a laser scanning confocal microscope (Olympus, LV1000). 2.7. siRNA transfection
2.2. Vascular smooth muscle cell treatment with recombinant human TGF-ˇ1 Cells were seeded in 10% FBS DMEM in 6-well plates. 24 h prior to treatment, the existing medium was replaced with DMEM. Recombinant TGF-1 (Pepro Tech Inc. TGF-1 reconstituted in vehicle (4 mM HCl/0.1% BSA)) was added at a final concentration of 1, 2, 5 or 10 ng/ml, and the cells were incubated for 24 h. Corresponding controls were treated with DMEM plus vehicle only. 2.3. ELISA The concentration of TGF-1 was analyzed with a Quantikine ELISA Kit (R&D Systems). After the application of cyclic strain, 100 l cell culture supernatant was activated by adding 0.01 mol acid. According to the manufacturer’s instructions, TGF-1 in the activated sample, the negative control and a standard TGF-1 control was detected by solid phase sandwich ELISA. After the chromogenic reaction, the samples were read at 450 nm within 30 min. TGF-1 in cell culture supernatant was quantified in parallel with the standard curves.
For the RNA interference experiment, VSMCs were transfected with 100 nmol/L of TGF-1, SIRT6 siRNA or control non-silencing siRNA (Gene-Pharma, China) for 8 h using lipofectamine 2000 (Invitrogen) in Opti-MEM medium (Gibco) according to the manufacturer’s instructions. The sequences of the siRNA oligos are as follows: TGF-1 siRNA: sense 5 CCAGAAAUAUAGCAACAAUTT-3 and antisense 5 -AUUGUUGCUAUAUUUCUGGTA-3 . SIRT6 siRNA: sense 5 -GCCGUCUGGUCAUUGUCAATT-3 and antisense 5 -UUGACAAUGACCAGACGGCTT-3 . non-silencing siRNA: sense 5 -UUCUCCGA ACGUGUCACGUTT-3 and anti-sense 5 -ACGUGACACGUUCGGAGAATT -3 . 2.8. Statistical analysis Each experiment was performed at least in triplicate, and all values are expressed as the mean ± SD. The student’s t-test was used to compare two groups, and multiple comparisons were performed by one-way ANOVA. Values of p < 0.05 were accepted as significant.
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3. Results 3.1. Cyclic strain induced VSMC differentiation and increased the expression of the TGF-ˇ1/SIRT6/c-fos ICAM-1 signal pathway The effect of cyclic strain on the secretion of TGF-1 was examined by ELISA first. As shown in Fig. 1A, the expression level of TGF-1 was increased in the supernatant from VSMCs subjected to the cyclic strain. Based on the ELISA analysis, the concentration of TGF-1 was nearly 2-fold greater in the strained group (867.6 ± 50.6 pg/ml) than in the control (460.4 ± 87.9 pg/ml). To evaluate the VSMC phenotype transformation, contractile phenotype markers were examined. Western blot analysis showed that the expressions of Sm-22, ␣-actin and calponin in VSMCs were increased compared with the static (Fig. 1B), which means the cyclic strain induced VSMC differentiation. Then, the effects of cyclic strain on the expression of p-Smad 2, pSmad5, SIRT6 and c-fos in VSMCs were analyzed. Western blotting showed that p-Smad2, p-Smad5, SIRT6 and c-fos were up-regulated by the cyclic strain. However, the total Smad2, Smad5, Smad4 and c-jun levels did not change (Fig. 1C and D). In addition, FCM analysis demonstrated that cyclic strain upregulated the expression of ICAM-1 in VSMCs (Fig. 1E). 3.2. Cyclic strain increased p-Smad2, p-Smad5, and SIRT6 Having demonstrated that p-Smad2, p-Smad5 and SIRT6 protein expressions were markedly induced by the cyclic strain in VSMCs, then their distributions were analyzed by immunostaining subsequently. The left panel of figures showed the stained proteins as red. The middle panel of figures showed the nucleus as blue. The right
panel of figures was merged. Fig. 2 demonstrated that p-Smad2, p-Smad5 and SIRT6 are all located in the nucleus. In addition, the intensity of immunostaining in the strained group is much stronger than in the static control group. These results further confirm that the cyclic strain increase the expression of these proteins in VSMCs. 3.3. Recombinant TGF-ˇ1 increased the expression of SIRT6 Because the cyclic strain increased TGF-1 secretion and SIRT6 expression, we tested whether TGF-1 can directly induce the expression of SIRT6. VSMCs were incubated with recombinant TGF1 at concentrations of 0, 1, 2, 5 or 10 ng/ml in the absence of the cyclic strain for 24 h. As shown in Fig. 3, 1, 2, 5 or 10 TGF1 increased the expression of SIRT6 compared with the “0 ng/ml” group. Further, the “1 ng/ml” TGF-1 treatment is approximately the concentration of TGF-1 that is induced by the cyclic strain. 3.4. A TGF-ˇ1 neutralizing antibody blocked the activation of SIRT6 and TGF-ˇ1-specific siRNA transfection inhibited the effect of cyclic strain on VSMC differentiation markers To determine whether TGF-1 in the supernatant from VSMCs induces the upregulation of SIRT6 caused by the cyclic strain, a TGF-1 neutralizing antibody (R&D, 5 g/ml) was used. As shown in Fig. 4A, the addition of the TGF-1 neutralizing antibody depressed the phosphorylation of Smad2 and Smad5 and attenuated the expression of SIRT6. This result means that the cyclic strain increases SIRT6 via TGF-1/Smad signaling. To determine whether TGF-1 in the supernatant from VSMCs induces the VSMC differentiation, another method were used
Fig. 1. The cyclic strain induced VSMC differentiation and increased the expression of TGF-1/SIRT6/c-fos ICAM-1 signaling pathway. VSMCs were subjected to 10% elongation strain for 24 h. Static control was served as control. (A) The concentration of TGF-1 with or without cyclic strain was determined by ELISA. Cyclic strain increased the concentration of TGF-1 in culture medium. n = 5. (B) Cyclic strain increased the contractile phenotype markers as determined by western blot. n = 8. (C) Cyclic strain increased p-Smad2 and p-Smad5 as determined by western blot. n = 8. (D) Cyclic strain increased SIRT6 and c-fos as determined by western blot. n = 8. (E) Cyclic strain increased ICAM-1 expression, as determined by FCM. n = 4. Values shown are the mean ± SD for each group from at least three independent experiments, *p < 0.05.
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Fig. 3. Recombinant TGF-1 increased the expression of SIRT6. Quiescent VSMCs were incubated with recombinant TGF-1 (Peprotech, USA) at concentration of 0, 1, 2, 5 or 10 ng/ml for 24 h. Compared with the 0 ng/ml control group, the 1, 2, 5 or 10 ng/ml treatment all increased the expression of SIRT6 as determined by western blot. Values shown are the mean ± SD for each group from at least three independent experiments, *p < 0.05. n = 6.
to inhibit TGF-1.VSMCs were transfected with nonspecific control siRNA or a siRNA specific for TGF-1 (100 nmol/mL) for 8 h and then subjected to cyclic strain for 24 h. Fig. 4B showed that TGF-1-specific siRNA transfection inhibited the effect of cyclic strain on VSMC differentiation markers compared with the NC group.
3.5. SIRT6-specific siRNA transfection inhibited the effect of cyclic strain on VSMC differentiation via c-fos and ICAM-1
Fig. 2. Cyclic strain modulates the distribution of p-Smad2, p-Smad5 and SIRT6. VSMCs were cultured on the silica membrane of Bioflex plates. After subjecting the cells to cyclic strain or static conditions as a control group for 24 h, cells were fixed incubated with primary antibodies against p-Smad2, p-Smad5 and SIRT6 and incubated with an Alexa Fluor 555-conjugated secondary antibody along with DAPI. The left panel of figures showed the stained proteins as red. The middle panel of figures showed the nucleus as blue. The right panel of figures was merged. The samples were examined with a laser scanning confocal microscope (Olympus, LV1000). Scale bar = 40 m. n = 3.
Whether SIRT6 was responsible for the cyclic strain-mediated modulation of VSMC differentiation was examined using SIRT6specific siRNA transfection. VSMCs were transfected with the SIRT6 siRNA for 24 h prior to loading the cyclic strain. As shown in Fig. 5A, silencing SIRT6 resulted in a significant decrease in SIRT6 expression and attenuated the expression of differentiation markers (Fig. 5B). In addition silencing SIRT6 also decreased the expression of Smad4 (Fig. 5C), c-fos (Fig. 5D) and ICAM-1 (Fig. 5E). However, “knockdown of” SIRT6 did not affect the expression of p-Smad2 (Fig. 5C), p-Smad5 (Fig. 5C) and c-jun (Fig. 5D) or TGF-1 secretion (Fig. 5F). These results demonstrate that SIRT6 plays a crucial role in the cyclic strain-induced VSMC differentiation. C-fos and ICAM-1 may participate in the downstream signal transmission. In contrast, the TGF-1/Smad may act upstream of SIRT6.
Fig. 4. (A) showed a TGF-1 neutralizing antibody reversed the cyclic strain-induced activation of p-Smad2, p-Smad5 and SIRT6. VSMCs were pretreated with a TGF-1 neutralizing antibody (R&D, 5 g/ml) or pretreated with mouse IgG as the control group, then both groups were subjected to cyclic strain for 24 h. Compared with the control group, TGF-1 neutralizing antibody decreased the expression of p-Smad2, p-Smad5 and SIRT6. (B) showed that TGF-1-specific siRNA transfection inhibited the effect of cyclic strain on VSMC differentiation markers compared with the NC group. VSMCs were transfected with control siRNA or an siRNA specific for TGF-1 (100 nmol/mL) for 8 h and then subjected to cyclic strain for 24 h. Values shown are the mean ± SD for each group from at least three independent experiments, *p < 0.05. n = 4.
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Fig. 5. SIRT6-specific siRNA transfection inhibited the effect of cyclic strain on VSMC differentiation via c-fos and ICAM-1. VSMCs were transfected with nonspecific control (NC) siRNA or a siRNA specific for SIRT6 (100 nmol/mL) for 8 h and then subjected to cyclic strain for 24 h. In all figures the NC group is as control. (A) siRNA specific for SIRT6 decreased the expression of SIRT6. The interference efficiency is about 65%. n = 3. (B) siRNA specific for SIRT6 decreased the expression of contractile phenotype markers as determined by western blot. n = 5. (C) siRNA specific for SIRT6 decreased the expression of Smad4 as determined by western blot. n = 6. (D) siRNA specific for SIRT6 decreased the expression of c-fos as determined by western blot. n = 6. (E) siRNA specific for SIRT6 decreased the expression of ICAM-1 as determined by FCM. n = 3. (F) The concentration of TGF-1 was determined by ELISA. n = 6. Values shown are the mean ± SD for each group from at least three independent experiments, *p < 0.05.
4. Discussions The present study shows that SIRT6 may play a crucial role in the cyclic strain-induced differentiation of VSMCs. The data suggest that the cyclic strain induces SIRT6 protein expression in VSMCs through TGF-1 in an autocrine and paracrine manner. Importantly, we determined that silencing SIRT6 leads to the suppression of c-fos, Smad4 and ICAM-1, thus resulting in the dedifferentiation of VSMCs (Fig. 6). VSMCs are primarily subjected to the cyclic strain resulting from pulsatile blood pressure in vivo (Haga et al., 2007). Numerous studies have elucidated the diverse effect of cyclic strain on SMC phenotype modulation at elongation magnitude from 0% to 24% and frequency from 0.5 Hz to 2 Hz (Liu et al., 2010; Mills et al., 1997; Qu et al., 2007). Based on our previous work (Qu et al., 2007; Liu et al., 2008), VSMCs were subjected to the cyclic strain at 10% and 1.25 Hz, which led to the up-regulation of differentiation markers at protein level; these changes are consistent with our previous study. The cyclic strain is also generally accepted as an
important factor that induces VSMC differentiation from stem cells. For example cyclic strain induces VSMC differentiation from murine embryonic mesenchymal progenitor cells (Riha et al., 2007). Identifying the ideal parameters of cyclic strain to induce VSMC differentiation is an essential goal of tissue engineering. TGF- is a member of the TGF- super-family, a group of highly conserved cytokines, that includes bone morphogenic proteins, activins and inhibins. TGF-1 is the most described of the three TGF- isoforms (TGF-1–3), and this protein is involved in embryonic development, fibrosis, inflammation and carcinoma. Recent findings highlight the complex tissue-specific and cancer-stagespecific regulation of the TGF-1/Smad signaling (Patil et al., 2011). Some of these effects are bi-directional. For example, low concentrations of TGF-1 stimulate cell proliferation (Ikeda et al., 2013), whereas concentrations within the nanomolar range inhibit proliferation (Bellone et al., 1997). Our study determined that nearly 1 ng/ml active TGF-1 is secreted by strained-VSMCs; this concentration induces SIRT6 expression, and mediates VSMC differentiation. Similarly, TGF- also induces SIRT6 in human bronchial
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Fig. 6. Schematic drawing outlines the possible signaling pathway of TGF-1-SmadSIRT6. 10% cyclic strain loading on the VSMCs activates the secretion of TGF-1, induces phosphorylation of Smad2/5 and Smad4, then translocate into nucleus together to affect SIRT6, which induce VSMC differentiation with the up-regulation of c-fos and ICAM-1.
epithelial cells (Minagawa et al., 2011). After TGF-1 stimulation, both the Smad2/3 and the Smad1/5/8 are phosphorylated in SMCs (Tang et al., 2011). TGF- also increases the phosphorylation of Smads 1/5 without depleting levels of Smad 5 in human pulmonary microvascular ECs (Star et al., 2009). Consistent with these results, the Smad signaling cascade is initiated by phosphorylation of Smad2/5 when VSMCs were subjected to cyclic strain for 24 h in the present study, while the Smad2/5 remains stable. Smad4 forms complexes with R-Smads to translocate into the nucleus. We find that the expression of Smad4 in VSMCs remains stable at 24 h and increase at 48 h after stretch (Supplementary Fig. 1), which indicates that the activation of Smad4 requires a longer time than the phosphorylation of Smad2/5 and the activation of SIRT6. It is well known the stability of Smad is regulated by ubiquitin-mediated degradation, with Smad7 acting as a negative feedback factor. We observe that Smad4 is decreased, but not p-Smad2/5 following the SIRT6 down-regulation, which indicates that SIRT6 may not involve in the initiation of TGF-1/Smad signaling, but in feedback mechanism to regulate TGF-1/Smad signaling via Smad4. Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biocel. 2014.01.016. Sirtuins are conserved across specials and are found in a variety of organisms. Because of their important roles in aging, the stress response and carcinoma, sirtuins have attracted increasing attention. SIRT6, which is similar to but distinct from the other sirtuin structures, contains a single helix that interacts with ADPr and NAADPr. As an NAD+-dependent deacetylase, SIRT6 deacetylates histone H3K56 and H3K9 and selected specific target genes. For example, SIRT6 binds to NF-kB subunit RelA to attenuate NF-B signaling (Kawahara et al., 2009) and also binds to HIF1-␣regulated genes. Recently, transgenic mice overexpressing SIRT6 shows a longer lifespan with lower serum level of IGF1 (Kanfi et al., 2012). SIRT6 blocks IGF-Akt signaling and the development of cardiac hypertrophy by targeting c-jun (Sundaresan et al., 2012). Transcription factor activator protein-1 (AP-1) is composed of the Jun and Fos families. In neonatal rat aortic SMCs, c-jun but not cfos mRNA expression is dramatically up-regulated by exposure to mechanical stimuli (Morawietz et al., 1999). However, 20% fixed stretch results in a rapid induction of c-fos mRNA in rat mesenteric artery SMCs (Lyall et al., 1994). Our study demonstrates that
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c-fos but not c-jun is highly expressed in VSMCs by stretch and is decreased by SIRT6 knockdown, which may be explained by cfos, but not other AP-1 proteins, increasing the transcription from the SIRT6 reporter and directly activating SIRT6 transcription (Min et al., 2012). The contradictionary role of AP-1 in the effect of cyclic strain on VSMC and the relationship with SIRT6 are worthy to be further investigated. ICAM-1 is an immunoglobulin (Ig)-like cell adhesion molecule expressed on several cell types including ECs and VMSCs (Lee et al., 2008). ICAM-1 expression is increased by various stimuli including cytokines and mechanical stress. For example, TGF-1 (1 ng/ml) causes increased ICAM-1expression in HUVECs (Suzuki et al., 1994). Cyclic strain increases the expression of ICAM-1 in periodontal ligament cells (Saminathan et al., 2012). However, little information is available concerning the effect of stretch on the expression of ICAM-1 in VSMCs. In the present study, we demonstrate that cyclic strain induces expression of ICAM-1, and SIRT6 inhibition resulted in decreased ICAM-1. Previous observation shows low density lipoprotein induces ICAM-1 correlate with AP-1 (c-fos/cjun) (Verna et al., 2006). Therefore, the relationship among SIRT6, ICAM-1and c-fos/c-jun during mechanical stretch should be investigated in the future. Taken together, our findings elucidate the effects of SIRT6 induced by the cyclic strain on regulating differentiation of VSMCs, and indicate that the cyclic strain-induced activation of SIRT6 is mediated through the TGF-1/Smad signaling pathway. In addition, the regulation of SIRT6 may function as a key point that determines VSMC differentiation in response to the cyclic strain via c-fos and ICAM-1. Conflict of interest statement No conflicts of interest, financial or otherwise, are declared by the authors. Acknowledgments This research was supported by grants from the National Natural Science Foundation of China, Nos. 11232010, 10972141, 11229202 and 11202133. References Beauharnois JM, Bolívar BE, Welch JT. Sirtuin 6: a review of biological effects and potential therapeutic properties. Molecular Biosystem 2013;9(7):1789–806. Bellone G, Silvestri S, Artusio E, Tibaudi D, Turletti A, Geuna M, et al. Growth stimulation of colorectal carcinoma cells via the c-kit receptor is inhibited by TGF-beta 1. Journal of Cellular Physiology 1997;172(1):1–11. Chiang HY, Korshunov VK, Serour A, Shi F, Sottile J. Fibronectin is important regulator of flow-induced vascular remodeling. Arteriosclerosis Thrombosis and Vascular Biology 2009;29:1074–9. Haga JH, Li YS, Chien S. Molecular basis of the effects of mechanical stretch on vascular smooth muscle cells. Journal of Biomechanics 2007;40(5):947–60. Heldin CH, Moustakas A. Role of Smads in TGF signaling. Cell Tissue Research 2012;347(1):21–36. Ikeda K, Nakajima T, Yamamoto Y, Takano N, Tanaka T, Kikuchi H, et al. Roles of transient receptor potential canonical (TRPC) channels and reversemode Na+ /Ca2+ exchanger on cell proliferation in human cardiac fibroblasts: effects of transforming growth factor 1. Cell Calcium 2013;54(3):213–25, http://dx.doi.org/10.1016/j.ceca.2013. 06.005. Kanfi Y, Naiman S, Amir G, Peshti V, Zinman G, Nahum L, et al. The sirtuin SIRT6 regulates lifespan in male mice. Nature 2012;483(7388):218–21. Kawahara TL, Michishita E, Adler AS, Damian M, Berber E, Lin M, et al. SIRT6 links histone H3 lysine 9 deacetylation to NF-kappaB-dependent gene expression and organismal life span. Cell 2009;136(1):62–74. Lappas M. Anti-inflammatory properties of sirtuin 6 in human umbilvein endothelial cells. Mediators Inflammation 2012;2012., ical http://dx.doi.org/10.1155/2012/597514, Article ID 597514, 11 pages. Lee HM, Kim HJ, Won HJ, Choi WS, Lee KY, Bae YM, et al. Contribution of soluble intercellular adhesion molecule-1 to the migration of vascular smooth muscle cells. European Journal of Pharmacology 2008;579:260–8.
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Short communication
The role of SIRT6 in the differentiation of vascular smooth muscle cells in response to cyclic strain Qing-Ping Yao, Ping Zhang, Ying-Xin Qi, Si-Guo Chen, Bao-rong Shen, Yue Han, Zhi-Qiang Yan ∗ , Zong-Lai Jiang Institute of Mechanobiology & Medical Engineering, School of Life Sciences & Biotechnology, Shanghai Jiao Tong University, Shanghai, China
a r t i c l e
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Article history: Received 12 September 2013 Received in revised form 7 January 2014 Accepted 20 January 2014 Available online 2 February 2014 Keywords: SIRT6 Cyclic strain Differentiation Vascular smooth muscle cell Transforming growth factor-1
a b s t r a c t Vascular smooth muscle cells (VSMCs) may switch their phenotype between a quiescent contractile phenotype and a synthetic phenotype in response to cyclic strain, and this switch may contribute to hypertension, atherosclerosis, and restenosis. SIRT 6 is a member of the sirtuin family, and plays an important role in different cell processes, including differentiation. We hypothesized that cyclic strain modulates the differentiation of VSMCs via a transforming growth factor-1 (TGF-1)-Smad-SIRT6 pathway. VSMCs were subjected to cyclic strain using a Flexercell strain unit. It was demonstrated that the strain stimulated the secretion of TGF-1 into the supernatant of VSMCs. After exposed to the strain, the expressions of contractile phenotype markers, including smooth muscle protein 22 alpha, alpha-actin, and calponin, and phosphorylated Smad2, phosphorylated Smad5, SIRT6 and c-fos were up-regulated in VSMCs by western blot and immunofluorescence. And the expression of intercellular-adhesion molecule1 (ICAM-1) was also increased detected by flow cytometry. The strained-induced up-regulation of SIRT6 was blocked by a TGF-1 neutralizing antibody. Furthermore, the effects of strain on VSMCs were abrogated by SIRT6-specific siRNA transfection via the suppression c-fos and ICAM-1. These results suggest that SIRT6 may play a critical role in the regulation of VSMC differentiation in response to the cyclic strain. © 2014 Elsevier Ltd. All rights reserved.
1. Introduction Vascular smooth muscle cells (VSMCs) are a highly differentiated cell type, and subjected to cyclic mechanical strain due to pulsating blood flow in the media of arteries. Unlike the majority of differentiated cells, VSMCs maintain phenotypic plasticity. VSMCs can switch between a differentiated (also termed contractile) state, which is characterized by a spindle-shaped morphology; contractile gene expression; a low rate of proliferation; and a dedifferentiated (also termed synthetic) phenotype in response to various stimuli, including cytokines, cell adhesions, extracellular matrix interactions, injury stimuli, and mechanical force (Owens and Kumar, 2004). Dysregulation of phenotype switching is associated with vascular disorders, including hypertension, atherosclerosis and restenosis (Rzucidlo et al., 2007). An increasing number of studies have demonstrated that the cyclic strain can modulate VSMC morphology, migration, proliferation and apoptosis through
∗ Corresponding author at: Institute of Mechanobiology & Medical Engineering, School of Life Sciences & Biotechnology, P.O. Box 888, Shanghai Jiao Tong University, 800 Dongchuan Road, Minhang, Shanghai 200240, China. Tel.: +86 21 34204863; fax: +86 21 34204118. E-mail address: [email protected] (Z.-Q. Yan). 1357-2725/$ – see front matter © 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biocel.2014.01.016
various signaling pathways. In particular, the cyclic strain can induce many growth factor/cytokines, such as: vascular endothelial growth factor (VEGF) (Schad et al., 2011), platelet-derived growth factor (PDGF) (Yung et al., 2009), angiotensin II (Ang II) and TGF (Li et al., 1998). It has been showed that cytokine signaling can dramatically affect the differentiation status of VSMCs. For example, PDGF promotes multiple aspects of the synthetic VSMC phenotype (Tallquist and Kazlauskas, 2004); Conversely, TGF- inhibits VSMC proliferation and migration and promotes increased expression of VSMC contractile genes (Li et al., 2010). TGF- phosphorylates receptor-activated Smads (R-Smads), including Smad1, Smad2, Smad3, Smad5, and Smad8. The phosphorylated R-Smads then form a complex with the common Smad, Smad4, and translocate into the nucleus to regulate target gene expression through interactions with other cofactors (Heldin and Moustakas, 2012). Until now, how the cyclic strain induces TGF- in VSMCs to mediate the VSMC phenotype switching has remained unclear. SIRT6 is a member of sirtuin sub-class IV and is located in the nucleus, where it acts as both a deacetylase and a mono-ADP-ribosyl-transferase to regulate metabolism, inflammation, aging, transcription, and stress resistance. The highest concentrations of SIRT6 have been detected in muscle, brain, heart, liver, and the thymus, where SIRT6 affects transcriptional regulation in a tissue-specific manner (Beauharnois et al., 2013).
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In the cardiovascular system, growing evidence demonstrates that SIRT6 participates in many biological processes via many molecules. For examples, SIRT6-depleted human umbilical vein ECs (HUVECs) expresses higher levels of intercellular-adhesion molecule-1 (ICAM-1) (Lappas, 2012). ICAM-1 is highly expressed in flow-induced vascular remodeling (Chiang et al., 2009) and induced by cyclic strain in periodontal ligament cells (Saminathan et al., 2012). SIRT6 protects cardiomyocytes from hypertrophy via NF-Bdependent transcriptional activity (Yu et al., 2013) and insulin-like growth factor (IGF)-Akt signaling by targeting c-jun (Sundaresan et al., 2012). C-fos/c-jun control liver cancer initiation through SIRT6-dependent inhibition of surviving (Min et al., 2012). Hence, ICAM-1 and c-fos/c-jun may be involved in the VSMC differentiation induced by cyclic strain. However, no reports have addressed the expression of SIRT6 in VSMCs. Therefore, the aims of this study are to examine the role of SIRT6 in the regulation of VSMC differentiation in response to the cyclic strain and to elaborate the functional mechanism of the possible signaling way that underlies this process. 2. Materials and methods 2.1. Cell culture and cyclic strain loading The animal care and experimental protocols conformed to the Animal Management Rules of China (Documentation 55, 2001, Ministry of Health, China). VSMCs were isolated from the thoracic aortas of male Sprague–Dawley rats, 220–260 g, by an explant method as previously described (Qi et al., 2008). VSMCs between passages 4 and 8, with cell populations of more than 95% purity, were used in all experiments. For cyclic strain loading, VSMCs were seeded in gelatin-coated (Sigma, 0.1%) 6-well Bioflex plates (Flexcell International) at a density of 2 × 105 cells/well. After achieving 70% confluence, cells were serum-starved in DMEM for 24 h, and then subjected to the cyclic strain of 10% elongation at 1.25 Hz for 24 h using a Flexercell Tension Plus system (FX-4000 T, Flexcell International, USA). Unstretched VSMCs were served as a static control. For antibody-neutralizing studies, VSMCs were incubated with TGF-1 antibody (R&D Systems, 5 g/ml) prior to strain loading. VSMCs treated without TGF-1 antibody and loaded with the same strain were used as the control.
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2.4. SDS-PAGE and western blot analysis Protein extracts were separated by 10% SDS-PAGE. The proteins were detected using primary antibodies specific for Sm-22 (Abcam, 1:500) ␣-actin (Sigma, 1:1000), calponin (Sigma, 1:1000), p-Smad2 (Signalway Antibody, 1:500), Smad2 (Proteintech, 1:500), p-Smad5 (Epitomics, 1:500), Smad5 (Proteintech, 1:500), Smad4 (Epitomics, 1:500), SIRT6 (Abcam, 1:500), c-fos (BioWorld 1:500), c-jun (BioWorld 1:500), and GAPDH (Proteintech, 1:500). After incubation with alkaline phosphatase-conjugated secondary antibodies (Jackson ImmunoResearch), the signals were visualized with nitroblue tetrazolium-5 bromo-4 chloro-3 indolyl phosphate (Bio Basic, Inc.) and quantified with Quantity One software (Bio-Rad). 2.5. Flow cytometry For the flow cytometry (FCM) assay, the cells with and without stretch were treated with a 0.125% trypsin solution and digestion was stopped by adding PBS with 1% BSA; then centrifuged at 1000 rpm for 5 min and rinsed with PBS with 1% BSA. The cells were incubated with a PE-conjugated ICAM-1 antibody (Santa Cruz, 1 g per 1 × 106 cells) for 30 min at a room temperature and rinsed with PBS twice. An isotype PE-conjugated mouse IgG without ICAM-1 antibody served as the negative control. The cells were resuspended in 300 L of PBS and analyzed with a FACScan flow cytometer (BD Biosciences FACSCaliburTM ). 2.6. Immunofluorescence staining The attached VSMCs were fixed with 4% paraformaldehyde for 20 min, permeabilized for 5 min in 0.5% Triton X-100, and blocked with 1% BSA in PBS for 30 min. Then, cells were incubated with primary antibodies against p-Smad2 (1:50), p-Smad5 (1:50) and SIRT6 (1:100) at 4 ◦ C over night. VSMCs incubated with 1% BSA in PBS served as the negative control. All samples were washed in PBS for 30 min and incubated with an Alexa Fluor 555-conjugated secondary antibody (Cell Signaling Technology, 1:1000) along with DAPI for 1 h at room temperature. The samples were examined with a laser scanning confocal microscope (Olympus, LV1000). 2.7. siRNA transfection
2.2. Vascular smooth muscle cell treatment with recombinant human TGF-ˇ1 Cells were seeded in 10% FBS DMEM in 6-well plates. 24 h prior to treatment, the existing medium was replaced with DMEM. Recombinant TGF-1 (Pepro Tech Inc. TGF-1 reconstituted in vehicle (4 mM HCl/0.1% BSA)) was added at a final concentration of 1, 2, 5 or 10 ng/ml, and the cells were incubated for 24 h. Corresponding controls were treated with DMEM plus vehicle only. 2.3. ELISA The concentration of TGF-1 was analyzed with a Quantikine ELISA Kit (R&D Systems). After the application of cyclic strain, 100 l cell culture supernatant was activated by adding 0.01 mol acid. According to the manufacturer’s instructions, TGF-1 in the activated sample, the negative control and a standard TGF-1 control was detected by solid phase sandwich ELISA. After the chromogenic reaction, the samples were read at 450 nm within 30 min. TGF-1 in cell culture supernatant was quantified in parallel with the standard curves.
For the RNA interference experiment, VSMCs were transfected with 100 nmol/L of TGF-1, SIRT6 siRNA or control non-silencing siRNA (Gene-Pharma, China) for 8 h using lipofectamine 2000 (Invitrogen) in Opti-MEM medium (Gibco) according to the manufacturer’s instructions. The sequences of the siRNA oligos are as follows: TGF-1 siRNA: sense 5 CCAGAAAUAUAGCAACAAUTT-3 and antisense 5 -AUUGUUGCUAUAUUUCUGGTA-3 . SIRT6 siRNA: sense 5 -GCCGUCUGGUCAUUGUCAATT-3 and antisense 5 -UUGACAAUGACCAGACGGCTT-3 . non-silencing siRNA: sense 5 -UUCUCCGA ACGUGUCACGUTT-3 and anti-sense 5 -ACGUGACACGUUCGGAGAATT -3 . 2.8. Statistical analysis Each experiment was performed at least in triplicate, and all values are expressed as the mean ± SD. The student’s t-test was used to compare two groups, and multiple comparisons were performed by one-way ANOVA. Values of p < 0.05 were accepted as significant.
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3. Results 3.1. Cyclic strain induced VSMC differentiation and increased the expression of the TGF-ˇ1/SIRT6/c-fos ICAM-1 signal pathway The effect of cyclic strain on the secretion of TGF-1 was examined by ELISA first. As shown in Fig. 1A, the expression level of TGF-1 was increased in the supernatant from VSMCs subjected to the cyclic strain. Based on the ELISA analysis, the concentration of TGF-1 was nearly 2-fold greater in the strained group (867.6 ± 50.6 pg/ml) than in the control (460.4 ± 87.9 pg/ml). To evaluate the VSMC phenotype transformation, contractile phenotype markers were examined. Western blot analysis showed that the expressions of Sm-22, ␣-actin and calponin in VSMCs were increased compared with the static (Fig. 1B), which means the cyclic strain induced VSMC differentiation. Then, the effects of cyclic strain on the expression of p-Smad 2, pSmad5, SIRT6 and c-fos in VSMCs were analyzed. Western blotting showed that p-Smad2, p-Smad5, SIRT6 and c-fos were up-regulated by the cyclic strain. However, the total Smad2, Smad5, Smad4 and c-jun levels did not change (Fig. 1C and D). In addition, FCM analysis demonstrated that cyclic strain upregulated the expression of ICAM-1 in VSMCs (Fig. 1E). 3.2. Cyclic strain increased p-Smad2, p-Smad5, and SIRT6 Having demonstrated that p-Smad2, p-Smad5 and SIRT6 protein expressions were markedly induced by the cyclic strain in VSMCs, then their distributions were analyzed by immunostaining subsequently. The left panel of figures showed the stained proteins as red. The middle panel of figures showed the nucleus as blue. The right
panel of figures was merged. Fig. 2 demonstrated that p-Smad2, p-Smad5 and SIRT6 are all located in the nucleus. In addition, the intensity of immunostaining in the strained group is much stronger than in the static control group. These results further confirm that the cyclic strain increase the expression of these proteins in VSMCs. 3.3. Recombinant TGF-ˇ1 increased the expression of SIRT6 Because the cyclic strain increased TGF-1 secretion and SIRT6 expression, we tested whether TGF-1 can directly induce the expression of SIRT6. VSMCs were incubated with recombinant TGF1 at concentrations of 0, 1, 2, 5 or 10 ng/ml in the absence of the cyclic strain for 24 h. As shown in Fig. 3, 1, 2, 5 or 10 TGF1 increased the expression of SIRT6 compared with the “0 ng/ml” group. Further, the “1 ng/ml” TGF-1 treatment is approximately the concentration of TGF-1 that is induced by the cyclic strain. 3.4. A TGF-ˇ1 neutralizing antibody blocked the activation of SIRT6 and TGF-ˇ1-specific siRNA transfection inhibited the effect of cyclic strain on VSMC differentiation markers To determine whether TGF-1 in the supernatant from VSMCs induces the upregulation of SIRT6 caused by the cyclic strain, a TGF-1 neutralizing antibody (R&D, 5 g/ml) was used. As shown in Fig. 4A, the addition of the TGF-1 neutralizing antibody depressed the phosphorylation of Smad2 and Smad5 and attenuated the expression of SIRT6. This result means that the cyclic strain increases SIRT6 via TGF-1/Smad signaling. To determine whether TGF-1 in the supernatant from VSMCs induces the VSMC differentiation, another method were used
Fig. 1. The cyclic strain induced VSMC differentiation and increased the expression of TGF-1/SIRT6/c-fos ICAM-1 signaling pathway. VSMCs were subjected to 10% elongation strain for 24 h. Static control was served as control. (A) The concentration of TGF-1 with or without cyclic strain was determined by ELISA. Cyclic strain increased the concentration of TGF-1 in culture medium. n = 5. (B) Cyclic strain increased the contractile phenotype markers as determined by western blot. n = 8. (C) Cyclic strain increased p-Smad2 and p-Smad5 as determined by western blot. n = 8. (D) Cyclic strain increased SIRT6 and c-fos as determined by western blot. n = 8. (E) Cyclic strain increased ICAM-1 expression, as determined by FCM. n = 4. Values shown are the mean ± SD for each group from at least three independent experiments, *p < 0.05.
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Fig. 3. Recombinant TGF-1 increased the expression of SIRT6. Quiescent VSMCs were incubated with recombinant TGF-1 (Peprotech, USA) at concentration of 0, 1, 2, 5 or 10 ng/ml for 24 h. Compared with the 0 ng/ml control group, the 1, 2, 5 or 10 ng/ml treatment all increased the expression of SIRT6 as determined by western blot. Values shown are the mean ± SD for each group from at least three independent experiments, *p < 0.05. n = 6.
to inhibit TGF-1.VSMCs were transfected with nonspecific control siRNA or a siRNA specific for TGF-1 (100 nmol/mL) for 8 h and then subjected to cyclic strain for 24 h. Fig. 4B showed that TGF-1-specific siRNA transfection inhibited the effect of cyclic strain on VSMC differentiation markers compared with the NC group.
3.5. SIRT6-specific siRNA transfection inhibited the effect of cyclic strain on VSMC differentiation via c-fos and ICAM-1
Fig. 2. Cyclic strain modulates the distribution of p-Smad2, p-Smad5 and SIRT6. VSMCs were cultured on the silica membrane of Bioflex plates. After subjecting the cells to cyclic strain or static conditions as a control group for 24 h, cells were fixed incubated with primary antibodies against p-Smad2, p-Smad5 and SIRT6 and incubated with an Alexa Fluor 555-conjugated secondary antibody along with DAPI. The left panel of figures showed the stained proteins as red. The middle panel of figures showed the nucleus as blue. The right panel of figures was merged. The samples were examined with a laser scanning confocal microscope (Olympus, LV1000). Scale bar = 40 m. n = 3.
Whether SIRT6 was responsible for the cyclic strain-mediated modulation of VSMC differentiation was examined using SIRT6specific siRNA transfection. VSMCs were transfected with the SIRT6 siRNA for 24 h prior to loading the cyclic strain. As shown in Fig. 5A, silencing SIRT6 resulted in a significant decrease in SIRT6 expression and attenuated the expression of differentiation markers (Fig. 5B). In addition silencing SIRT6 also decreased the expression of Smad4 (Fig. 5C), c-fos (Fig. 5D) and ICAM-1 (Fig. 5E). However, “knockdown of” SIRT6 did not affect the expression of p-Smad2 (Fig. 5C), p-Smad5 (Fig. 5C) and c-jun (Fig. 5D) or TGF-1 secretion (Fig. 5F). These results demonstrate that SIRT6 plays a crucial role in the cyclic strain-induced VSMC differentiation. C-fos and ICAM-1 may participate in the downstream signal transmission. In contrast, the TGF-1/Smad may act upstream of SIRT6.
Fig. 4. (A) showed a TGF-1 neutralizing antibody reversed the cyclic strain-induced activation of p-Smad2, p-Smad5 and SIRT6. VSMCs were pretreated with a TGF-1 neutralizing antibody (R&D, 5 g/ml) or pretreated with mouse IgG as the control group, then both groups were subjected to cyclic strain for 24 h. Compared with the control group, TGF-1 neutralizing antibody decreased the expression of p-Smad2, p-Smad5 and SIRT6. (B) showed that TGF-1-specific siRNA transfection inhibited the effect of cyclic strain on VSMC differentiation markers compared with the NC group. VSMCs were transfected with control siRNA or an siRNA specific for TGF-1 (100 nmol/mL) for 8 h and then subjected to cyclic strain for 24 h. Values shown are the mean ± SD for each group from at least three independent experiments, *p < 0.05. n = 4.
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Fig. 5. SIRT6-specific siRNA transfection inhibited the effect of cyclic strain on VSMC differentiation via c-fos and ICAM-1. VSMCs were transfected with nonspecific control (NC) siRNA or a siRNA specific for SIRT6 (100 nmol/mL) for 8 h and then subjected to cyclic strain for 24 h. In all figures the NC group is as control. (A) siRNA specific for SIRT6 decreased the expression of SIRT6. The interference efficiency is about 65%. n = 3. (B) siRNA specific for SIRT6 decreased the expression of contractile phenotype markers as determined by western blot. n = 5. (C) siRNA specific for SIRT6 decreased the expression of Smad4 as determined by western blot. n = 6. (D) siRNA specific for SIRT6 decreased the expression of c-fos as determined by western blot. n = 6. (E) siRNA specific for SIRT6 decreased the expression of ICAM-1 as determined by FCM. n = 3. (F) The concentration of TGF-1 was determined by ELISA. n = 6. Values shown are the mean ± SD for each group from at least three independent experiments, *p < 0.05.
4. Discussions The present study shows that SIRT6 may play a crucial role in the cyclic strain-induced differentiation of VSMCs. The data suggest that the cyclic strain induces SIRT6 protein expression in VSMCs through TGF-1 in an autocrine and paracrine manner. Importantly, we determined that silencing SIRT6 leads to the suppression of c-fos, Smad4 and ICAM-1, thus resulting in the dedifferentiation of VSMCs (Fig. 6). VSMCs are primarily subjected to the cyclic strain resulting from pulsatile blood pressure in vivo (Haga et al., 2007). Numerous studies have elucidated the diverse effect of cyclic strain on SMC phenotype modulation at elongation magnitude from 0% to 24% and frequency from 0.5 Hz to 2 Hz (Liu et al., 2010; Mills et al., 1997; Qu et al., 2007). Based on our previous work (Qu et al., 2007; Liu et al., 2008), VSMCs were subjected to the cyclic strain at 10% and 1.25 Hz, which led to the up-regulation of differentiation markers at protein level; these changes are consistent with our previous study. The cyclic strain is also generally accepted as an
important factor that induces VSMC differentiation from stem cells. For example cyclic strain induces VSMC differentiation from murine embryonic mesenchymal progenitor cells (Riha et al., 2007). Identifying the ideal parameters of cyclic strain to induce VSMC differentiation is an essential goal of tissue engineering. TGF- is a member of the TGF- super-family, a group of highly conserved cytokines, that includes bone morphogenic proteins, activins and inhibins. TGF-1 is the most described of the three TGF- isoforms (TGF-1–3), and this protein is involved in embryonic development, fibrosis, inflammation and carcinoma. Recent findings highlight the complex tissue-specific and cancer-stagespecific regulation of the TGF-1/Smad signaling (Patil et al., 2011). Some of these effects are bi-directional. For example, low concentrations of TGF-1 stimulate cell proliferation (Ikeda et al., 2013), whereas concentrations within the nanomolar range inhibit proliferation (Bellone et al., 1997). Our study determined that nearly 1 ng/ml active TGF-1 is secreted by strained-VSMCs; this concentration induces SIRT6 expression, and mediates VSMC differentiation. Similarly, TGF- also induces SIRT6 in human bronchial
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Fig. 6. Schematic drawing outlines the possible signaling pathway of TGF-1-SmadSIRT6. 10% cyclic strain loading on the VSMCs activates the secretion of TGF-1, induces phosphorylation of Smad2/5 and Smad4, then translocate into nucleus together to affect SIRT6, which induce VSMC differentiation with the up-regulation of c-fos and ICAM-1.
epithelial cells (Minagawa et al., 2011). After TGF-1 stimulation, both the Smad2/3 and the Smad1/5/8 are phosphorylated in SMCs (Tang et al., 2011). TGF- also increases the phosphorylation of Smads 1/5 without depleting levels of Smad 5 in human pulmonary microvascular ECs (Star et al., 2009). Consistent with these results, the Smad signaling cascade is initiated by phosphorylation of Smad2/5 when VSMCs were subjected to cyclic strain for 24 h in the present study, while the Smad2/5 remains stable. Smad4 forms complexes with R-Smads to translocate into the nucleus. We find that the expression of Smad4 in VSMCs remains stable at 24 h and increase at 48 h after stretch (Supplementary Fig. 1), which indicates that the activation of Smad4 requires a longer time than the phosphorylation of Smad2/5 and the activation of SIRT6. It is well known the stability of Smad is regulated by ubiquitin-mediated degradation, with Smad7 acting as a negative feedback factor. We observe that Smad4 is decreased, but not p-Smad2/5 following the SIRT6 down-regulation, which indicates that SIRT6 may not involve in the initiation of TGF-1/Smad signaling, but in feedback mechanism to regulate TGF-1/Smad signaling via Smad4. Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biocel. 2014.01.016. Sirtuins are conserved across specials and are found in a variety of organisms. Because of their important roles in aging, the stress response and carcinoma, sirtuins have attracted increasing attention. SIRT6, which is similar to but distinct from the other sirtuin structures, contains a single helix that interacts with ADPr and NAADPr. As an NAD+-dependent deacetylase, SIRT6 deacetylates histone H3K56 and H3K9 and selected specific target genes. For example, SIRT6 binds to NF-kB subunit RelA to attenuate NF-B signaling (Kawahara et al., 2009) and also binds to HIF1-␣regulated genes. Recently, transgenic mice overexpressing SIRT6 shows a longer lifespan with lower serum level of IGF1 (Kanfi et al., 2012). SIRT6 blocks IGF-Akt signaling and the development of cardiac hypertrophy by targeting c-jun (Sundaresan et al., 2012). Transcription factor activator protein-1 (AP-1) is composed of the Jun and Fos families. In neonatal rat aortic SMCs, c-jun but not cfos mRNA expression is dramatically up-regulated by exposure to mechanical stimuli (Morawietz et al., 1999). However, 20% fixed stretch results in a rapid induction of c-fos mRNA in rat mesenteric artery SMCs (Lyall et al., 1994). Our study demonstrates that
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c-fos but not c-jun is highly expressed in VSMCs by stretch and is decreased by SIRT6 knockdown, which may be explained by cfos, but not other AP-1 proteins, increasing the transcription from the SIRT6 reporter and directly activating SIRT6 transcription (Min et al., 2012). The contradictionary role of AP-1 in the effect of cyclic strain on VSMC and the relationship with SIRT6 are worthy to be further investigated. ICAM-1 is an immunoglobulin (Ig)-like cell adhesion molecule expressed on several cell types including ECs and VMSCs (Lee et al., 2008). ICAM-1 expression is increased by various stimuli including cytokines and mechanical stress. For example, TGF-1 (1 ng/ml) causes increased ICAM-1expression in HUVECs (Suzuki et al., 1994). Cyclic strain increases the expression of ICAM-1 in periodontal ligament cells (Saminathan et al., 2012). However, little information is available concerning the effect of stretch on the expression of ICAM-1 in VSMCs. In the present study, we demonstrate that cyclic strain induces expression of ICAM-1, and SIRT6 inhibition resulted in decreased ICAM-1. Previous observation shows low density lipoprotein induces ICAM-1 correlate with AP-1 (c-fos/cjun) (Verna et al., 2006). Therefore, the relationship among SIRT6, ICAM-1and c-fos/c-jun during mechanical stretch should be investigated in the future. Taken together, our findings elucidate the effects of SIRT6 induced by the cyclic strain on regulating differentiation of VSMCs, and indicate that the cyclic strain-induced activation of SIRT6 is mediated through the TGF-1/Smad signaling pathway. In addition, the regulation of SIRT6 may function as a key point that determines VSMC differentiation in response to the cyclic strain via c-fos and ICAM-1. Conflict of interest statement No conflicts of interest, financial or otherwise, are declared by the authors. Acknowledgments This research was supported by grants from the National Natural Science Foundation of China, Nos. 11232010, 10972141, 11229202 and 11202133. References Beauharnois JM, Bolívar BE, Welch JT. Sirtuin 6: a review of biological effects and potential therapeutic properties. Molecular Biosystem 2013;9(7):1789–806. Bellone G, Silvestri S, Artusio E, Tibaudi D, Turletti A, Geuna M, et al. Growth stimulation of colorectal carcinoma cells via the c-kit receptor is inhibited by TGF-beta 1. Journal of Cellular Physiology 1997;172(1):1–11. Chiang HY, Korshunov VK, Serour A, Shi F, Sottile J. Fibronectin is important regulator of flow-induced vascular remodeling. Arteriosclerosis Thrombosis and Vascular Biology 2009;29:1074–9. Haga JH, Li YS, Chien S. Molecular basis of the effects of mechanical stretch on vascular smooth muscle cells. Journal of Biomechanics 2007;40(5):947–60. Heldin CH, Moustakas A. Role of Smads in TGF signaling. Cell Tissue Research 2012;347(1):21–36. Ikeda K, Nakajima T, Yamamoto Y, Takano N, Tanaka T, Kikuchi H, et al. Roles of transient receptor potential canonical (TRPC) channels and reversemode Na+ /Ca2+ exchanger on cell proliferation in human cardiac fibroblasts: effects of transforming growth factor 1. Cell Calcium 2013;54(3):213–25, http://dx.doi.org/10.1016/j.ceca.2013. 06.005. Kanfi Y, Naiman S, Amir G, Peshti V, Zinman G, Nahum L, et al. The sirtuin SIRT6 regulates lifespan in male mice. Nature 2012;483(7388):218–21. Kawahara TL, Michishita E, Adler AS, Damian M, Berber E, Lin M, et al. SIRT6 links histone H3 lysine 9 deacetylation to NF-kappaB-dependent gene expression and organismal life span. Cell 2009;136(1):62–74. Lappas M. Anti-inflammatory properties of sirtuin 6 in human umbilvein endothelial cells. Mediators Inflammation 2012;2012., ical http://dx.doi.org/10.1155/2012/597514, Article ID 597514, 11 pages. Lee HM, Kim HJ, Won HJ, Choi WS, Lee KY, Bae YM, et al. Contribution of soluble intercellular adhesion molecule-1 to the migration of vascular smooth muscle cells. European Journal of Pharmacology 2008;579:260–8.
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