The Role Of Sirt6 In Epithelial To Mesenchymal Transition And The Underlying Mechanisms

Part Ⅰ Sirt6 inhibits multi-walled carbon nanotube or TGF-β1-induced epithelial to mesenchymal transition phenotypeObjective:Sirt6 is a member of NAD+ dependent deacetylase Ⅲ which plays a key role in aging,cancer,and metabolism,but whether Sirt6 plays a potential role in pulmonary epithelial to mesenchymal transition(EMT)process has been poorly explored.Methods:Male C57BL mice(8 weeks old)were divided into control,multi-walled carbon nanotube(MWCNT),and bleomycin groups,10 mice per group.The mice were administrated with 50 μl saline or 60 μg MWCNT for 56 days and 2 mg/kg bleomycin for 21 days,respectively.Lung tissue was harvested and subjected to immunohistochemisty(IHC)and Western blot analysis to measuere the protein level of Sirt6.A549 cells were transfected with Sirt6 and Sirt6 siRNA and then subjected to 16μg/cm2 MWCNT or 5 ng/ml TGF-β1(Transformed growth factor-beta 1)treatment for 24 h.Then,Western blot,Real-time PCR,and immunofluorescencent staining were used to examine the expression of E-cadherin,vimentin,and α-SMA.The protein and the mRNA levels of FN and CTGF were examined by Western blot and Real-time PCR,respectively.Transwell assay was employed to assess the migratory ability of A549 cells.Results:Sirt6 was upregulated in bleomycin or MWCNT-induced lung fibrosis model.Additionally,MWCNT or TGF-β1 time and dose-dependently increased the protein level of Sirt6(P<0.05).Importantly,MWCNT or TGF-β1 elicited EMT phenotype,which could be restored by Sirt6 overexpression(P<0.05).Unexpectedly,loss of Sirt6 failed to impact EMT markers with or without TGF-β1 treatment.Moreover,TGF-β1 dramatically upregulated the mRNA levels of FN,CTGF,COL3A1,MMP-2,MMP-9,and TGF-β1,which were ameliorated by overexpression of Sirt6(P<0.05 or P<0.01).Futhernnore,there was a significant reduction in the protein levels of FN and CTGF in Sirt6 forced expression cells compared with that of the control cells(P<0.05).Consistent with ECM synthesis,the increased content of hydroxyproline elicited by TGF-β1 was also reversed by Sirt6 overexpression(P<0.05).In addition,forced expression of Sirt6 almost completely abolished TGF-(31-induced migratory behavior.Conclusions:Overexpression of Sirt6 but not knockdown of Sirt6 significantly blocked EMT phenotype.In addition,EMT-associated cell behaviors were also blunted by gain of Sirt6.Part Ⅱ The mechanisms involved in epithelial to mesenchymal transition inhibition by Sirt6Objective:To investigate the mechanisms by which Sirt6 modulates EMT process.Methods:A549 cells were transfected with Sirt6 followed by 5 ng/ml TGF-β1 treatment for 60 min or 24 h.Total protein were extracted and then subjected to Western blot to measure the protein levels of snaill,TGF-β1,Smad2,Smad3,Smad4,p-Smad2,and p-Smad3.The nuclear translocation of Smad3 and co-location of Sirt6 and Smad3 were determined by immunofluorescencent staining.The binding between Sirt6 and Smad3 or snail1 were examined by co-immunoprecipitation.Real-time PCR was used to determine the mRNA levels of snaill,slug,twistl,ZEB1,and ZEB2.Results:The marked increase of TGF-β1 protein level elicited by TGF-β1 treatment was blunted by Sirt6 overexpression(P<0.05).Interestingly,there was no significant difference in the protein expression of Smad2,Smad3,and Smad4 between ad-GFP and ad-Sirt6 transfected cells in the presence or absence of TGF-β1.Sirt6 overexpression severely impaired phosphorylation of Smad3 but not Smad2 in response to TGF-β1 stimulation(P<0.05).TGF-β1-induced Smad3 translocation into the nucleus in control cells,wherea Smad3 primarily maintained in the cytoplasm in ad-Sirt6 transfected cells.We observed that Smad3 translocated into the nucleus of the cells and co-located with Sirt6 in the nucleus upon TGF-β1 treatment,as shown by double immunostaining.The interaction between Sirt6 and Smad3 was augmented by TGF-β1 stimulation.Additionally,we employed co-immunoprecipitation analysis and found that administration of A549 cells with TGF-β1 significantly increased snail 1-Smad3 interaction,which was blocked by Sirt6 overexpression.Finally,Real-time PCR analysis demonstrated that the transcription levels of snaill,slug,twist1,ZEB1,and ZEB2 were significantly lower in A549 cells that transfected with ad-Sirt6 compared to those of the cells with ad-GFP under TGF-β1 administration(P<0.05 or P<0.01).Conclusions:Sirt6 may inhibit EMT during idiopathic pulmonary fibrosis via inactivating TGF-β1/Smad3 signaling.The blunted EMT-related transcription factors may also contribute to dimininshed EMT phenotype by Sirt6.Part Ⅲ Lung targeted overexpression of Sirt6 reverses bleomycin-induced pulmonary epithelial to mesenchymal transition and lung fibrosisObjective:To confirm the anti-EMT effect of Sirt6 in vivo.Methods:C57BL mice were divided four goups(GFPAAV,Sirt6AAV,GFPAAV+bleomycin,and Sirt6AAV+bleomycin,15 mice per group).Adeno-associated virus(AAV)carrying Sirt6 gene was delivered to mice lung by intratracheal injection with 50 μl PBS containing 2×1011 vg per mouse.Bleomycin(2 mg/kg)was intratracheally injected one week later.Mice were sacrificed 3 weeks after bleomycin administration.Results:Bleomycin markedly promoted EMT phenotype in GFPAAV mice,as shown by enhanced vimentin and a-SMA staining and diminished E-cadherin in alveolar epithelial cells by IHC staining.However,the above effects were significantly blunted in Sirt6AAV mice.In addition,there were an increase in proSPC,a type Ⅱalveolar epithelial cells marker,and a decrease in vimentin and a-SMA staining in Sirt6AAV mice compared with GFPAAV littermate mice in response to bleomycin treatment.After bleomycin instillation,mice displayed pulmonary parenchymal damage,collapsed alveoli,and thicker alveolar membrane,as determined by Hematoxylin-eosin staining.Masson’s trichrome staining of collagen showed severe collagen deposition in lung mesenchyme.These bleomycin-induced lung damage was ameliorated in Sirt6AAV mice.In line with the histopathological changes,IHC analysis revealed that FN and CTGF were also ameliorated in Sirt6AAV mice.Conclusions:Taken together,delivery of Sirt6 to lung abrogated bleomycin-induced EMT-like phenotype and pulmonary fibrosis.