mTORC1 Signaling Pathway Mediated SIRT6 Overexpression in TGF-β1-Induced Pulmonary Fibrosis

Fibroblast-to-myobroblast transdifferentiation and myobroblast hyperproliferation play a major role in Idiopathic pulmonary brosis (IPF). It was also reported that mTOR signaling pathway and SIRT6 have a critical role in pulmonary brosis. However, the mechanisms whether mTOR signaling pathway and SIRT6 affect the myobroblasts differentiation in IPF remain unclear. The results show that SIRT6 is signicantly upregulated by TGF-β1 with a time and concentration-dependent manner in MRC5 line and primary lung broblasts isolated from IPF patients. SIRT6 protein is also increased in IPF brotic lung tissues and bleomycin-challenged mice lung tissues. Also, the activity of mTOR signaling is activated in MRC5 and primary lung broblasts. Furthermore, the inhibitor of mTOR, rapamycin treatment signicantly suppress mTORC1 pathway activity and SIRT6 protein expression. SIRT6 siRNA failed to mediate the activity of mTORC1 pathway and autophagy induction. Finally, deciency of SIRT6 could promote TGF-β1 induced pro-brotic cytokines. In summary, the study have suggested that SIRT6 is a downstream of mTORC1 signaling pathway in the pulmonary brosis caused by TGF-β1-induced. Deciency of SIRT6 mediated myobroblasts differentiation through induced pro-brotic cytokines production but not induced-autophagy. It was indicated that manipulations of SIRT6 expression may provide a new therapeutic strategy to reverse the progression of pulmonary brosis. SIRT6 by TGF-β1 in lung of SIRT6 signicantly suppresses TGF-β1 ‐ induced myobroblast in HFL1 cells et also that SIRT6 was upregulated in TGF-β1-treated bleomycin-injured mice, in which have reversed TGF-β1-induced epithelial to mesenchymal transition (EMT) in duct SIRT6 activation liver damage and regulating Orphan nuclear receptor estrogen-related receptor γ (ERRγ) 28 . Other ndings suggested that SIRT6-decient broblasts to myobroblasts of transforming growth factor β1 (TGF-β1) signaling in a cell-autonomous manner HFL1:human broblasts, CMECs: Cardiac microvascular endothelial IRS-1: Insulin receptor substrate 1, Estrogen-related receptor γ BDL: bile duct ligation.


Introduction
Idiopathic pulmonary brosis (IPF) is a chronic, brosing interstitial lung pneumonia, characterized by abnormal accumulation of extracellular matrix (ECM) proteins by lung broblasts, leading to respiratory failure and affecting adults over 50 years old [1][2][3] . The signs and symptoms are of insidious onset and are characterized by progressive dyspnea and persistent dry cough 4,5 . Increasing evidence has demonstrated that myo broblasts are primary effector cells for matrix deposition and tissue remodeling in IPF patients 6,7 . Lung broblasts differentiate into contractile myo broblasts that secrete excessive extracellular matrix (ECM) proteins and pro brotic factors 8, 9 . Although Nintedanib and Pirfenidone signi cantly reduced forced vital capacity (FVC) decline and prolonged time to rst acute exacerbation, failed to improve respiratory failure [10][11][12] . Therefore, the pathogenesis of IPF still need to further understand.
The PI3K/Akt/mTOR signaling pathway is an essential signaling regulator of cell metabolism, proliferation, differentiation, and survival 13,14 . Mammalian target of rapamycin (mTOR) is a serine/threonine kinase in the PI3K family that is an central regulator of protein and lipid biosynthesis, cell cycle progression, proliferation, survival, and senescence 15,16 . While mTORC1 controls cell growth and metabolism and is highly sensitive to rapamycin, mTORC2 regulates cell proliferation and survival and is relatively insensitive to rapamycin 14,17 . PI3K/Akt/mTOR inhibition attenuates transforming growth factor (TGF)-β1-induced collagen production in human lung broblasts (LFs), and collagen formation markers in IPF lung tissue 18 . Previous study showed that the aberrant PTEN/AKT/mTOR axis desensitizes IPF broblasts from collagen matrix-driven stress by suppressing autophagy, which produces a viable IPF broblast phenotype on collagen 19 . However, the mechanism of mTOR signaling regulated myo broblasts differentiation is still unclear.
Sirtuins are a family of nicotinamide adenine dinucleotide-dependent deacetylases that are involved in regulating stress resistance, metabolism, and organismal life span 20  Immunohistochemical (IHC) analysis showed that SIRT6 localized to areas of active brosis, the broblastic foci, and sub-epithelial and sub-endothelial regions within IPF lung (Fig. 1a). We also examined SIRT6 expression in normal lung broblasts and those derived from patients with IPF. The expression of SIRT6 was also con rmed in primary pulmonary broblasts by Western blotting. Compared with control, the presence of higher levels of SIRT6 and α-SMA were observed in IPF patient with the dosedependent of TGF-β1 (Fig. 1b). Moreover, we found that the expression of SIRT6 in the advanced IPF was higher than in health control (P=0.032) by R2 platform ( (Fig. 1c).
Also, in order to understand the role of SIRT6 in bleomycin-induced pulmonary brosis, we established pulmonary brosis mice (N=5) treated with 5U/kg BLM-induced. Lung tissues were collected at 21 days, and lung sections were stained with H&E staining and Masson's Trichrome. It was demonstrated that the alveolar structure in bleomycin-treated mice was destroyed with hyperplasia of collagen deposition from on days 21 (Fig. 2a). IHC analysis demonstrated diffuse staining for SIRT6 protein expression within the lungs of bleomycin-induced mice (Fig. 2b). Furthermore, human fetal lung broblasts (MRC5) were cultured with 10ng/mL TGF-β1 for 72 hours to induce myo broblast differentiation. It was showed that the expression of SIRT6 and α-SMA were also upregulated by TGF-β1 induced in dose-dependent manner (Fig. 2c). Taken together, these results con rmed that SIRT6 was overexpressed in pulmonary brosis.
2. Activity of mTOR signaling pathway is up-regulated in myo broblast differentiation. Having investigated the activity of mTOR signaling pathway in lung myo broblast differentiation, we measured mTOR kinase activity with Western botting. In MRC5 line, the phosphorylated mTORC1 protein level was signi cantly high with increasing dose of TGF-β1 using phosphorylated S6 level, a direct substrate of mTORC1. Additionally, the phosphorylated-Akt ser473, which was a maker of mTORC2 kinase, and Thr308 level were decreased in MRC5 due to feedback loop of up-regulated level of phosphorylated S6 ( Fig. 3a). We next examined mTOR kinase activity in IPF broblasts. There were also a signi cant increasing in the activity of phosphorylated S6 kinase, when TGF-β1 induced pulmonary myo broblasts differentiation in a dose dependent pattern. In contrast, TGF-b1 treatment decreased the level of phosphorylated-Akt ser473, Thr308 in a dose-dependent manner (Fig. 3b). It was indicated that activated mTOR signaling involved in mediating the potent brotic effects of TGF-β1.
mTORC1 inhibitor suppress SIRT6 protein in pulmonary brosis. To further determine the regulation relationship between SIRT6 and mTOR signaling pathway in lung myo broblast differentiation, we assessed the SIRT6 expression with rapamycin treatment. Using TGF-β1 (20ng/mL) induced MRC5 line differentiation model, the expression of SIRT6 was inhibited by rapamycin treatment in the dose dependent manner (Fig. 4a). Furthermore, it was also found that rapamycin markedly suppressed SIRT6 protein with the dose dependent manner in IPF myo broblasts (Fig. 4b). We next also measured the activity of mTOR pathway with rapamycin treatment in pulmonary myo broblasts differentiation. The results showed that the phosphorylated S6 protein was markedly suppressed with increasing the dose of rapamycin in MRC5. There was a different increasing in the expression of phosphorylated-Akt ser473 and Thr308 with different concentration of rapamycin treatment (Fig. 4c). After exposure to TGF-β1 (20ng/mL) in present of rapamycin for 48h, the level of phosphorylated S6 protein and Akt were measured in primary IPF patient. We found rapamycin signi cantly suppressed the expression of phosphorylation of S6, and phosphorylated Akt (Ser473 and Thr 308) were up-regulated with various concentrations of rapamycin, shown by Western blotting (Fig 4d). It was suggested that activated mTORC1 promoted the SIRT6 expression in pulmonary brosis.
4. Knockdown SIRT6 failed to regulated mTORC1 but upregulated activity of phosphorylated Akt. We next explored whether mTORC1 modulated the expression of SIRT6, in which broblasts were treated with SIRT6 siRNA. Treatment with SIRT6 siRNA dramatically down-regulated the SIRT6 protein in MRC5 line ( Fig. 5a). In human lung broblasts treated with SIRT6 siRNA, SIRT6 expression was also decreased compared with control by Western blotting (Fig. 5b). Further, the level of SIRT6 mRNA was markedly decreased in MRC5 treated with SIRT6 siRNA (Fig. 5c). These results suggested that the expression level of SIRT6 was markedly downregulated in SIRT6 siRNA knockdown model.
We also evaluated the activity of mTOR signaling and SIRT6 by TGF-β1 induction in MRC5. The result indicated that SIRT6 siRNA failed to regulate the level of phosphorylated S6 protein. The phosphorylated-Akt (Ser473, Thr308) by TGF-β1 induction was increased in MRC5 cells when treated with SIRT6 siRNA (Fig. 5d). Moreover, the knockdown of the expression of SIRT6 by siRNA did not signi cantly affect the activity of mTORC1 signaling whether induced by TGF-β1 or not, as indicated by the levels of phosphorylated S6 protein in IPF patient. The level of phosphorylated serine 473 and thr 308 of Akt were increased in human lung broblasts treated by SIRT6 siRNA (Fig. 5e). These results suggested that silencing SIRT6 failed to regulated mTORC1 but upregulated activity of phosphorylated Akt.
5. TGF-b1 inhibits autophagy during myo broblast differentiation through activated mTORC1 signaling not SIRT6. Considering that insu cient autophagy has a role in pulmonary brosis and mTOR signaling moderate the autophagy, whether SIRT6 regulates myo broblast differentiation by inducing autophagy.
Western blotting con rmed that TGF-β1 reduced autophagy in IPF broblasts, using the ratio of LC3 II/I, a widely recognized autophagy biomarker (Fig. 6a). We further measured the effect of TGF-β upon autophagy in human lung broblasts and MRC5 by treating with TGF-β1 (10, 20 ng/mL) for 72h. The results indicated that LC3II levels was decreased upon addition of TGF-β1 in a dose-dependent manner after 48h whether in human lung broblasts (Fig. 6b). And according with results, TGF-β1-mediated decline in LC3II was also observed in MRC5 (Fig. 6c).
To demonstrate the mTORC1 signaling was involved in the regulation of autophagy, human lung broblasts was treated by rapamycin. It was reported that rapamycin signi cantly increased the protein of LC3II with dose-dependent manner in IPF lung tissue with or without TGF-β1 treatment (Fig. 6d). Moreover, in order to determine whether SIRT6 also regulated the autophagy via mTORC1 pathway. We subsequently silenced SIRT6 with siRNA transfection in IPF broblasts and MRC5. The data indicated that the expression of LC3II was inhibited by TGF-β1 in human lung broblasts, but no signi cant difference was noticed by SIRT6 siRNA treatment whether TGF-β1-inducing or not after 48h (Fig. 6e). In MRC5, blocking SIRT6 with siRNA could not decreased the level of LC3II in the present of TGF-b1 or not after 48h (Fig. 6f). These ndings suggest that rapamycin enhanced induced-autophagy in presence of TGF-b1 and SIRT6 failed to regulate TGF-β1mediated autophagy.
6. De ciency of SIRT6 have promoted the expression of brosis-related factors. In order to con rm the pro-brotic role of SIRT6 in TGF-b1-induced myo broblast differentiation and extracellular matrix (ECM) accumulation, we next examined whether SIRT6 modulated the expression of brotic gene. MRC5 was incubated with SIRT6 siRNA for 12h before TGF-b1 treatment. As shown in Fig 7a-

Discussion
To our acknowledge, TGF-β1 has an important role in brotic processes via effectively promoting the differentiation of broblasts into myo broblasts and increase the secretion of ECM components 26 . In the study, we have established TGF-β1-induced myo broblast differentiation model in vitro in order to investigate the role of SIRT6 in pulmonary brogenesis ( Supplementary Fig. 1). The protein level of SIRT6 was signi cantly upregulated in the MRC5 and primary IPF broblasts treated with TGF-β1. Additionally, it was found that SIRT6 protein level was increased in the bleomycin mouse model of pulmonary brosis and IPF lung tissues. The results indicated that the protein of SIRT6 was enhanced in pulmonary brosis.
Previous studies have also demonstrated that SIRT6 has increased by TGF-β1 in human fetal lung broblasts (HFL1) and overexpression of SIRT6 signi cantly suppresses TGF-β1-induced myo broblast differentiation in HFL1 cells 27 . Tian et al also found that SIRT6 was upregulated in TGF-β1-treated A549 cells and bleomycin-injured mice, in which have reversed TGF-β1-induced epithelial to mesenchymal transition (EMT) in A549 cells (24). Moreover, studies have explored that bile duct ligation (BDL)-induced liver injury was aggravated by SIRT6 de ciency. SIRT6 activation alleviated cholestatic liver damage and brosis through regulating Orphan nuclear receptor estrogen-related receptor γ (ERRγ) 28 . Other ndings suggested that SIRT6-de cient broblasts transform spontaneously to myo broblasts through hyperactivation of transforming growth factor β1 (TGF-β1) signaling in a cell-autonomous manner 29 .
Overall, the de ciency of SIRT6 has resulted in progressive renal in ammation and brosis 30 . It was also reported that SIRT6 de ciency increased expression of brosis-associated markers in myo broblasts.
Although emerging evidence have indicated the importance of mTOR signaling pathway on the regulation of pulmonary brosis, current understanding of mechanism of mTOR pathway in brosis is still need to be elucidated. Previous study demonstrated the abnormal mTOR activity was found in lung tissue from IPF patients 32 . It has been reported that a novel dual mTOR/PI3K inhibitor (GSK2126458) inhibited PI3K signaling and functional response in IPF broblasts derived from patients with brotic foci in phase clinical trial 18 . Furthermore, another study have showed that a dual mTORC1 and mTORC2 inhibitor (MLN0128) exhibited potent anti-brotic activity in both in vitro and in vivo models 33 . Although mounting evidence have indicated that the inhibition of mTOR is effective to suppress the brotic process, serious adverse event could be occurred in clinical trials 15,[34][35][36][37] . Other studies also showed that the effect of rapamycin has been limited due to the inhibition of mTORC1 may remove the negative feedback loop on mTORC2/Akt 38 . mTOR is present in cells as a member of 2 complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2), which have different subunits and substrate speci city. The PI3K-mTORC1 complex is regulated by negative feedback loops, including the reduction of Akt activity and mTORC1 signaling in response to S6K phosphorylation of insulin receptor substrate 1 (IRS-1), which leads to IRS-1 degradation 39 . The present study showed that TGF-β1-induced phosphorylation of the mTORC1 substrates pS6 in MCR5 and IPF broblasts with a concentration-dependent manner, but the phosphorylation of mTORC2 substrates Akt (Ser473) and Akt (Thr308) were attenuated due to negative feedback loop of mTORC1. In our results, mTORC1 pathway was activated by TGF-β1-induced broblasts differentiation. Moreover, treatment with rapamycin markedly attenuated mTORC1-dependent phosphorylation pS6 in MCR5 and IPF broblasts. In contrast, rapamycin increased Akt phosphorylation at both Thr308 and Ser473 sites in addition to downstream Akt signaling in TGF-β1-induced MRC5 and IPF broblasts.
We next identi ed the relationship between mTOR pathway and SIRT6 in regulating myo broblasts differentiation. Recent article has shown that SIRT6 is able to control protein synthesis through the mTOR signaling 40 . Our current ndings demonstrated that rapamycin signi cantly attenuated the SIRT6 expression with TGF-β1-induced broblasts differentiation. Interesting, knockdown of SIRT6 failed to mediate mTORC1 regardless of presence or absence of TGF-β1 in MRC5 or IPF broblasts. Additionally, phosphorylation of Akt on ser473 was increased in SIRT6 knockdown lysates indicating upregulated mTORC2 activity. It was indicated that activated mTORC1 modulated SIRT6 expression whereas SIRT6 knockdown enhanced activity of phosphorylation of Akt (ser473), a maker of mTORC2 pathway.
On the other hand, it was demonstrated that mTOR pathway plays a critical role in autophagy regulation. mTOR activity may be deregulated in IPF broblasts, leading to the proliferative and apoptosis-resistant broblast phenotype through inducing autophagy 41 . Previous data showed that LC3B-expression was relatively reduced in IPF broblasts, a marker of autophagy when compared to control broblasts 19,42 . Consistent with our studies, TGF-β1 signi cantly inhibited autophagy in MRC5 and IPF broblasts, which was shown by decreased conversion to LC3-II from LC3-I. Rapamycin clearly increased autophagy induction in dose dependent manner with or without TGF-β1treatment. Intriguingly, no signi cance was found in autophagy induction with SIRT6 siRNA intervention Together, these studies illustrated that de ciency of SIR6 failed to mediate autophagy with TGF-β1 treatment. Finally, we investigated the effects of SIRT6 on the expression of brosis-associated transcription factors. SIRT6 knockdown signi cantly increased the expression of brosis-related factors. It may be one of reason for the effects of mTOR inhibitors on pulmonary brosis was limited.
In summary, we identi ed that mTORC1 was activated in brosis models whether vivo or vitro ( Supplementary Fig. 3). Abnormal mTORC1 mediated myo broblasts differentiation and regulated SIRT6 overexpression. Effect of mTORC1 inhibition was limited in anti-brosis due to negative feedback loop on mTORC2 and suppressed SIRT6 accelerated the expression of brosis-related transcription factors.
Although rapamycin could effectively inhibited the protein of α-SMA expression, it could decrease SIRT6 level to promote the pro brotic genes expression. Future study aims to develop effective and safe agonist or target gene inhibitior for mTORC1 to treat IPF.

Methods
Ethics statement. This study involves the analysis of human IPF patient specimens. Primary broblast lines were obtained from unused, existing pathological human tissue samples, and therefore is exempt.
Tissue samples were stripped of all identi ers and designated as waste. All patients underwent procedures for diagnostic or therapeutic procedures. Written informed consent was obtained on all patients prior to the procedure being performed. Use of human tissues was approved by the Xiamen University and Wuxi Lung Transplant Center. All methods were performed in accordance with the relevant guidelines and regulations laid down by the Committee.
Human subjects. Cell lines were derived from lungs removed at the time of transplantation. The diagnosis of IPF was supported by history, physical examination, pulmonary function tests, and typical high resolution chest computed tomography ndings of IPF. In all cases, the diagnosis of IPF was con rmed by microscopic analysis of lung tissue and demonstrated the characteristic morphological ndings of usual interstitial pneumonia. All patients ful lled the criteria for the diagnosis of IPF as established by the American Thoracic Society (ATS) and the European Respiratory Society (ERS) 43  University School of Medicine. Cells were characterized as broblasts as previously described 44 . The levels of SIRT6 in IPF were derived from Meltzer in R2 Genomics Analysis and Visualization Platform.
Histological and Masson's trichrome analysis. Left lungs were xed in 4% neutral buffered paraformaldehyde, pH 7.4, for 24 hours at room temperature, dehydrated in a series of ethanol and subsequently xylol and embedded in para n. Sections of the left lungs were cut at a thickness of 4 μm, rehydrated and stained with H&E (hematoxylin and eosin) and Masson's Trichrome. Each successive eld was individually assessed for the severity of interstitial brosis in a blinded method by two pathologists using the Ashcroft scoring system. All slides were evaluated histopathologically applying a semiquantitative grading: 1=minimal, 2=slight, 3= marked, 4=severe, 5= massive.
Immunohistochemistry. Immunostaining was done on formalin-xed, para n-embedded mice lung tissue specimens, and 4 mm thick para n sections were cut. These sections were incubated overnight Cell Proliferation Assay. MRC5 was plated at a density of 1.0 ×10 3 cells/well in 96-well plates. The cells were cultured in the corresponding serum-free medium for 24 h. Cell viability was examined using10 mL/well CCK-8 solution (Dojindo, Kumamoto, Japan). After incubating for 2 h, the absorbance was measured at 450 nm.
Quantitative Real-Time PCR (qRT-PCR). Total RNA was extracted using the TRIzol reagent (Invitrogen, Carlsbad, CA) according to manufacturer's instructions. Then, reverse transcription was performed to get the rst strand cDNA by using the PrimeScript® RT reagent kit (TaKaRa, Dalian, China). The expression level of a-SMA, CTGF were determined by qPCR reactions and were performed by using the ABI 7500 Fast system (Applied Biosystems, CA) with SYBR green (TaKaRa). The 2 −ΔΔCt method was used for quanti cation. All reactions were triplicated. The relative expression of Collagen1, 3, Fibronectin, MMP3 were respectively normalized to GAPDH.
Statistical Analysis. Results are presented as means ± SEM. Signi cance of the differences between means was assessed using one-way analysis of variance or a two-tailed Student's t-test. Values of P less than 0.05 were considered signi cant.

Declarations
Availability of data and materials All data generated or analyzed during this study are included in this published article. Figure 1 SIRT6 expression was up-regulated in IPF lung tissues. a. Immunohistochemistry staining (IHC) was performed with lung tissues from idiopathic pulmonary brosis (IPF) patient of from histologically normal lungs (both N=1) using SIRT6 antibody. Shown was SIRT6 expression in cells within the broblastic foci of IPF patients (right panel) is higher than normal lung alveoli tissue specimens (left panel), Scale bar: 100μm, **, P< 0.01. b. Primary human lung broblasts isolated from control subject (N=1) or IPF patient (N=1) were treated with 10, 20ng/mL of TGF-b1. SIRT6 and a-SMA level were measured by Western blotting. Shown was SIRT6 and a-SMA expression in lung myo broblasts from IPF increased in the dose-dependent of TGF-β1. c. The expression of SIRT6 (reporter: 219613_s_at) in health control (n=6) versus the advanced IPF (n=9) in R2 Genomics Analysis and Visualization Platform (http://r2.amc.nl). P=0.032, signi cance was determined by unpaired Student's test.

Figure 2
Overexpression of SIRT6 was observed in pulmonary brosis models. a. Lung sections of C57BL/6J mice was collected at 21days after intratracheal bleomycin or saline injection. Mason's trichrome staining demonstrates increased lung matrix deposition in bleomycin-administered mice (Fibrosis) (N=5) compared to saline-treated (Control) (N=5) mice. Scale bar: 100μm, **, P< 0.01. b. IHC analysis demonstrated a signi cant increase in levels of SIRT6 production in the lungs of bleomycin-induced mice as compared to saline induced controls on 21 days. Scale bar: 100μm, *, P< 0.05. c. MRC5 line was cultured for 72 hours with TGF-β1-induced myo broblast differentiation in a concentration-dependent manner (0, 10, 20ng/mL). Expression of SIRT6 and α-SMA protein induced by TGF-β1 were increased in a concentration-dependent manner. Figure 3 mTOR signaling pathway is highly activated in TGF-b1 induced lung broblast differentiation. a. Immunoblots of MRC5 cell lysates, treated as indicated, showed that TGF-β1-induced phosphorylation of S6 was also upregulated and phosphorylated p-Akt (Ser473 and Thr308) were reduced. b. Western blotting showed the mTOR activity in primary human lung broblasts from patient with IPF (N=1) after incubation with TGF-b1 (0, 10, 20ng/mL) for 24 hours. The expression of phosphorylated S6 level was increased induced by TGF-b1 in a-concentration-dependent manner. Phosphorylated p-Akt (Ser473 and Thr308) were down-regulated by TGF-b1-induced in a concentration-dependent manner.

Figure 4
Rapamycin suppresses the expression of SIRT6 in myo broblast differentiation. a. Western blotting showed that SIRT6 protein expression was reduced in MRC5 after TGF-b1 (20ng/mL) incubation for 48 hours in the presence of rapamycin (0, 1, 10mm). b. In primary myo broblast from IPF patient (N=1), the level of SIRT6 was dramatically reduced by rapamycin (0, 1, 10mm) for 48 hours in a dose-dependent manner. c. In MRC5 line, the activity of mTOR pathway was also assessed after 48 hours incubation with 0, 1, 10mm rapamycin and 20ng/mL TGF-β1. Rapamycin (1, 10mm) signi cantly inhibited TGF-β1induced phosphorylation of S6 level in the MRC5. While rapamycin increased TGF-β1-induced the level of phosphorylation of Akt (Ser473 and Thr308) in a dose dependent manner. d. Primary pulmonary myo broblast was stimulated with rapamycin (0, 1, 10mm) for 48h and mTOR pathway was assessed by Western blotting. Phosphorylation of S6 was signi cantly inhibited with a concentration-dependent manner of rapamycin. An increased trend was found in phosphorylation of Akt (Ser473 and Thr308) between different concentration groups of rapamycin. Figure 5 siRNA SIRT6 was unable to modulate mTORC1 signaling pathway. a. SIRT6 protein was also signi cantly decreased in MRC5 transduced with SIRT6 siRNA. b. Primary pulmonary myo broblasts were transduced with SIRT6 siRNA showed marked decrease in the expression of SIRT6. c. Real-time qPCR analysis showed that SIRT6 siRNA treatment signi cantly decreased the SIRT6 mRNA level in MRC5 (n=3), **, P< 0.01. d. Western blotting showed that knocking down SIRT6 by siRNA in MRC5 cell did not affect phosphorylation levels of S6. The levels of the phosphorylated-Akt of Ser473 and Thr308 were reduce by TGF-β1 stimulated, while SIRT6 siRNA was able to increase phosphorylation (Ser473, Thr308) without or with TGF-β1. e. Primary pulmonary myo broblasts were transduced with SIRT6 siRNA or control siRNA followed by treatment with TGF-b1. SIRT6 de ciency could not inhibit phosphorylation of pS6 but increase the level of the Akt phosphorylation (Ser473, Thr308). SIRT6 failed to mediate the autophagy activity in pulmonary brosis. a. The conversion of LC3 from LC3-I (free form) to LC3-II (phosphatidylethanolamine-conjugated form) was con rmed by western blotting in primary lung broblasts. Autophagy inhibition was observed in IPF lung tissue (N=1). b. In primary lung broblast, TGF-b1 stimulation reduced autophagy induction as determined by decreased conversion of LC3 from LC3-I to LC3-II in a concentration-dependent manner. c. TGF-b1 stimulation further inhibited MRC5 autophagy by means of decreased LC3-II conversion. d. Although TGF-b1 suppress autophagy production, increasing concentrations of rapamycin markedly enhanced autophagy induction as determined by increased conversion of LC3 from LC3-I to LC3-II in IPF. e. Primary lung broblasts were transfected with siRNA SIRT6 and followed by treatment with TGF-b1. Western blotting showed that siRNA SIRT6 had no effect on decreasing the level of LC3-II/ I protein whether stimulation with TGF-β1 or not. f. Knockdown of SIRT6 was con rmed by Western blotting, and no signi cance difference was observed in LC3-II conversion following TGF-b1 treatment or not in MRC5.