The Role And Mechanisms Of SIRT6 In Myofibroblast Differentiation In Human Fetal Lung Fibroblasts

Objective:Idiopathic pulmonary fibrosis(IPF)is a chronic,progressive,irreversible,and usually lethal lung disease and myofibroblasts activated by lung fibroblasts are the major responsible cells.Myofibroblast is characterized by increased expression of?-smooth muscle actin(?-SMA)and their high capacity of extracellular matrix including fibronectin and collagens.Sirtuin 6(SIRT6),a member of the sirtuin family of nicotinamide adenine dinucleotide(NAD~+)-dependent protein deacetylases,is involved in DNA repair,inflammation,cancer and life span.SIRT6 has been proved to inhibit epithelial to mesenchymal transition during IPF.However,the function of SIRT6 in lung myofibroblast differentiation is still obscure.Human fetal lung fibroblast(HFL1)was used as a cellular model in our study.We aimed to explore the role of SIRT6 in fibroblast-to-myofibroblast differentiation induced by TGF-?1 in HFL1 cells and the possible molecular mechanisms,which can provide a theoretical basis for finding effective therapeutic targets for IPF.Methods:We measured the protein level of SIRT6 in HFL1 cells treated with TGF-?1 at 0,1,2,5,10,and 15 ng/ml for 24 h or at 5 ng/ml for 0,6,12,24,48,and 72 h respectively by performing Western blotting.HFL1 cells were transfected with an adenoviral vector encoding SIRT6 or knockdown by an adenoviral vector encoding SIRT6 shRNA and then serum-deprived for 12 h,subsequently treated with with 5ng/ml TGF-?1 for 24 h.Western blotting analysis of?-SMA,collagen I,fibronectin and SIRT6 was performed.HFL1 cells were transfected with wild-type SIRT6 or the H133 Y mutant and then serum-deprived for 12 h,subsequently treated with with 5 ng/ml TGF-?1 for 30 min.Western blotting analysis of the protein levels of p-Smad2,Smad2,p-Smad3 and Smad3 were performed and the nuclear translocation of Smad2 were examined by confocal immunofluorescence microscopy.HFL1 cells were treated with 5ng/ml TGF-?1 for 30 and 60 min,and the interaction of SIRT6 and p65 was analyzed by co-IP.NF-?B-dependent transcriptional activity was evaluated by dual luciferase reporter assay and the mRNA levels of NF-?B target genes including IL-1?,IL-6 and MMP9 were analyzed by real-time PCR.Results:1.The expression of SIRT6 in TGF-?1-treated HFL1 cells:The protein expression of SIRT6 was significantly upregulated at the doses of 2 and 5 ng/ml of TGF-?1 for 24 h(P<0.05)and at the time points of 12 and 24 h with 5 ng/ml TGF-?1(P<0.05 or P<0.01)in HFL1 cells.2.The effect of SIRT6 in TGF-?1-induced myofibroblast differentiation:After treatment with 5 ng/ml TGF-?1 for 24 h,the protein expression of?-SMA,collagen I,and fibronectin were significantly upregulated in HFL1 cells(P<0.01).Overexpression of SIRT6 alone had no effect on fibroblast-to-myofibroblast differentiation.However,overexpression of SIRT6 significantly diminished the protein expression of?-SMA,collagen I,and fibronectin induced by TGF-?1(P<0.05 or P<0.01).SIRT6 knockdown only significantly increased the protein level of fibronectin induced by TGF-?1(P<0.05),but didn't change the protein levels of?-SMA and collagen I.3.The effect of SIRT6 in TGF-?1/Smad2 signal pathway:The protein levels of p-Smad2 and p-Smad3 were all markedly increased after 5 ng/ml TGF-?1 stimulation for 30 min.SIRT6 overexpression significantly ameliorated phosphorylation of Smad2(P<0.01),but not Smad3 in HFL1 cells treated with TGF-?1.The SIRT6(H133Y)mutant without histone deacetylase activity failed to inhibit phosphorylation of Smad2 induced by TGF-?1.Wild-type SIRT6 not the H133 Y mutant inhibited TGF-?1-induced nuclear translocation of Smad2.4.The effect of SIRT6 in NF-?B signal pathway:SIRT6 interacted with the NF-?B subunit p65 and repressed TGF-?1-induced NF-?B-dependent transcriptional activity and the expression of NF-?B-dependent genes including IL-1?,IL-6 and MMP9,which is also dependent on its deacetylase activity.Conclusions:After treatment with TGF-?1 in HFL1 cells,the protein expression of SIRT6 was increased in a dose-and time-dependent manner.SIRT6 overexpression attenuated fibroblast-to-myofibroblast differentiation in HFL1 cells treated with TGF-?1by inactivation of TGF-?1/Smad2 and NF-?B signaling pathways,all of which depended on its deacetylase activity.