Mediation of Anti-Keloid Effects of mTOR Inhibitors by Autophagy-Independent Machinery

Meng Jiang, Wen-Bo Bu, Yu-Jie Chen, Li Li, Ta Xiao, Heng Gu,

1Department of Physiotherapy, Hospital for Skin Diseases (Institute of Dermatology), Chinese Academy of Medical Sciences and Peking Union Medical, Nanjing, Jiangsu 210042, China;2Department of Dermatologic Surgery, Hospital for Skin Diseases (Institute of Dermatology), Chinese Academy of Medical Sciences and Peking Union Medical, Nanjing, Jiangsu 210042, China;3Jiangsu Key Laboratory of Molecular Biology for Skin Diseases and STIs, Hospital for Skin Disease (Institute of Dermatology), Chinese Academy of Medical Sciences and Peking Union Medical College, Nanjing, Jiangsu 210042, China.

Abstract Objective: Blocking mechanistic target of rapamycin (mTOR) activation with mTOR inhibitors has promising therapeutic potential for keloids.However,the precise mechanism of mTOR inhibitors remains unclear.This study was aimed to investigate the role of autophagy machinery in the anti-keloid effects of mTOR inhibitors.Methods:We first validated the biological effects induced by the mTOR inhibitors rapamycin(100 nmol/L)and KU-0063794(5 μmol/L)on the proliferation,apoptosis,migration,and collagen synthesis of keloid fibroblasts(KFs)derived from Han Chinese persons through a Cell Counting Kit-8 assay, 5-Bromo-2’-deoxyuridine incorporation, Annexin V/propidium iodide staining,migration,and western blotting.To explore whether autophagy machinery is involved in the anti-keloid effects of mTOR inhibitors, we first blocked the autophagy activation induced by rapamycin and KU-0063794 with a pharmacological autophagy inhibitor(wortmannin)or by silencing the key autophagy gene(ATG5),and we then re-evaluated these biological effects on KFs.Results:Blocking mTOR activation with either rapamycin or KU-0063794 completely inhibited proliferation,migration,and collagen synthesis of primary KFs but did not affect apoptosis.Incubating KFs with the autophagy inhibitor wortmannin or performing ATG5 silencing abrogated the subsequent activation of autophagic activity induced by rapamycin (rapamycin+E-64d+pepstatin vs. rapamycin+wortmannin+E-64d+pepstatin: 1.88±0.38 vs. 1.02±0.35,F=6.86,P=0.013),(non-sense control+rapamycin vs.ATG5 small interfering RNA+rapamycin:1.46±0.18 vs.0.75±0.20, respectively; F=7.68, P=0.01) or KU-0063794 (KU-0063794+E-64d+pepstatin vs. KU-0063794+wortmannin+E-64d+pepstatin: 1.65±0.35 vs. 0.76±0.17, F=10.01, P=0.004), (NC+KU-0063794 vs.ATG5 small interfering RNA+KU-0063794: 1.59±0.50 vs. 0.77±0.09, F=5.93, P=0.02) as evidenced by decreased accumulation of LC3-II.However,blockage of autophagy induction in mTOR inhibitor-treated KFs with both methods did not disturb their anti-keloid effects,such as inhibition of cell viability,cell migration,and collagen synthesis(P>0.05 each).Conclusion:Blocking mTOR activation with the mTOR inhibitors rapamycin and KU-0063794 showed anti-keloid effects in KFs.Restoration of autophagy inhibition by mTOR inhibitors does not contribute to their anti-keloid effects.

Keywords: autophagy, fibroblast, keloid, KU-0063794, mTOR inhibitor, rapamycin

Introduction

Keloids are caused by an abnormal fibroproliferative wound healing reaction.The pathogenic factors are poorly defined but are known to include mechanical forces, hormones,inflammation, and genetics.1-2The currently available treatment options include silicone films,topical ointments,intralesional corticosteroids, and surgical removal combined with radiotherapy;however, the clinical outcomes remain unsatisfactory.3-4Because the pathogenesis at the molecular level is only partially understood, the clinical recurrence rate of keloids is still high when nontargeted therapy is used.Thus,further mechanistic research is needed to improve the clinical outcomes of keloids.

Autophagy is a complex lysosomal degradation process that provides an alternative energy sourceviaselfdigestion or removal of aggregated proteins, superfluous organelles, and intracellular pathogens to maintain structural and metabolic homeostasis.5Although autophagy is considered a cell survival mechanism, studies also implicate the role of autophagy in cell death.6-7Defective autophagy is implicated in various pathological conditions such as neurodegenerative diseases,infections,cancer,and fibrosis, indicating that targeting mediators of autophagy could be a promising therapeutic strategy for specific diseases.8–10Importantly, our previous studies have also confirmed that mechanistic target of rapamycin(mTOR)-dependent autophagy machinery is inhibited in keloids.11mTOR signaling has been proven to be overactivated in the pathogenesis of keloids.12Moreover, Syedet al.13-14found that the mTORC1 inhibitor rapamycin and the dual mTORC1/2 inhibitors KU-0063794 and KU-0068650 exhibited effects against the abnormal biology of fibroblasts, indicating therapeutic potential in keloids.Therefore,it is reasonable to speculate that defective autophagy capacity may be involved in the uncontrolled proliferation of fibroblasts and pose an obstacle to matrix degradation.

Our previous studies have confirmed that use of the mTOR inhibitors rapamycin and KU-0063794 can result in activation of autophagy in keloid-derived fibroblasts(KFs).11The dual roles of mTOR inhibitors allow them to achieve their functions by regulating the biosynthesis of effector proteins and by mediating the autophagic process as inducers.Theoretically,the anti-keloid effects of mTOR inhibitors can be achieved by inducing autophagy to promote fibroblast autophagic death and to accelerate collagen catabolism.This may suggest that the anti-keloid effects of mTOR inhibitors are realizedviainduction of autophagy activation.

To support this notion,we first confirmed the anti-keloid effects of rapamycin (100 nmol/L) and KU-0063794 (5 μ mol/L) on KFs from Han Chinese individuals, including cell proliferation, apoptosis, cell migration, and collagen synthesis.We then blocked mTOR inhibitor-induced autophagy activation with an autophagy inhibitor (wortmannin) orATG5silencing to explore whether the autophagic mechanism was involved in mitigating keloid progression by mTOR inhibitors.This study was performed based on the negative regulation of mTOR/autophagy signaling in the pathogenesis of keloids.We confirmed the biological effects on keloids by inhibiting the activation of mTOR and explored whether autophagy can serve as a drug target for keloid treatment.

Materials and methods

Reagents and antibodies

The following reagents were used in this study:rapamycin,KU-0063794, dimethyl sulfoxide (DMSO), E-64d, pepstatin (all from Sigma-Aldrich, St.Louis, MO, USA), and wortmannin (MedChemExpress, Monmouth Junction,NJ, USA).With the exception of anti-collagen I (R&D Systems, Minneapolis, MN, USA) and anti-collagen III(Boster Biological Technology, Pleasanton, CA, USA), all primary antibodies used for western blotting assays,including anti-GAPDH, anti-LC3 A/B, anti-caspase 3,anti-PARP, and anti-ATG5, were purchased from Cell Signaling Technology (Danvers, MA, USA).Secondary antibodies included goat anti-rabbit antibody (Cell Signaling Technology)and sheep IgG horseradish peroxidase-conjugated antibody (R&D Systems).

Tissue specimens

The KFs for this experiment were randomly chosen from a specimen bank derived from excised keloid specimens diagnosed by dermatologists.They were isolated from a sample obtained from the anterior chest of a 39-year-old man who had received no treatment and who had provided written informed consent for collection of specimens before surgical excision.This study was approved by the Ethics Committee of the Hospital for Skin Diseases (Institute of Dermatology), Chinese Academy of Medical Sciences, and Peking Union Medical College (2015-KY-019).

Isolation and culture of KFs

The method of establishing primary KFs from surgically removed fresh tissue was performed as previously described.15Briefly, excised sterile tissues were digested with 5 mg/mL Dispase II (Sigma-Aldrich)overnight at 4°C.The specimens were replaced with 3 mg/mL collagenase type I(Thermo Fisher Scientific)for 2 hours at 37°C.They were then transferred to a cell strainer and filtered to a centrifuge tube.After centrifugation at 200gfor 5 minutes, the precipitate was resuspended and cultured in sterile flasks with Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA).KFs from passages 2 to 5 were used for subsequent experiments.

Treatment of KF cultures

KFs were cultured in vitro as described above.KFs in the exponential growth phase were seeded into cell culture plates at the specific density for different experiments.All reagents, including rapamycin, KU-0063794, wortmannin, E-64d, and pepstatin, were dissolved in DMSO.To explore the biological effects of mTOR inhibitors, KFs were treated with the final concentration of 100 nM rapamycin or 5 μmol/L KU-0063794 for the corresponding time.To detect the inhibitory effect of wortmannin(1 μmol/L) on autophagic flux, KFs were pretreated with wortmannin for 4 hours before the mTOR inhibitor treatment, and the lysosomal inhibitors E-64d and pepstatin (10 μg/mL) were added for 4 hours before protein collection.Vehicle controls were treated with DMSO (<0.1%) solvent at the same volume in the presence or absence of lysosomal inhibitors.

Cell viability

The cell viability of fibroblasts was determined by the Cell Counting Kit-8 (CCK-8;4A Biotech, Beijing, China)according to the manufacturer’s instructions.We seeded KFs in 96-well plates at a density of 2×103/well.The KFs were incubated for 24 hours and then continued to be incubated in the presence or absence of 100 nmol/L rapamycin or 5 μmol/L KU-0063794 for 0, 24, 48, and 72 hours.DMSO (<0.1%) served as the solvent control.Each group was assessed in triplicate replications.After incubation with the CCK-8 reagent for 1 hour at 37°C,the optical density at 450 nm was determined by a microplate reader (NanoDrop ND-1000, Thermo Fisher Scientific).

5-Bromo-2’-deoxyuridine (BrdU) cell proliferation

BrdU labeling and detection kits (Roche Applied Science,Basel,Switzerland)were used to assess the DNA synthesis for quantifying cell proliferation according to the manufacturer’s instructions.At 48 hours after incubation with rapamycin or KU-0063794, the cells were cultured with BrdU labeling reagent for 6 hours and then fixed in FixDenat solution (100 μL) (Sigma-Aldrich) for 20 minutes.Next,anti-BrdU-peroxidase working solution was used to incubate the fixed cells for 90 minutes at room temperature.After washing and incubation with the substrate for 5 minutes, absorbance was determined at 372 and 490 nm by a microplate reader (Thermo Fisher Scientific).

Annexin V/propidium iodide (PI) assay

Annexin V–enhanced green fluorescent protein(EGFP)/PI detection kits (4A Biotech) were used to detect apoptosis according to the manufacturer’s instructions.KFs were seeded into six-well plates at a density of 1.5×105/well.After incubation for 24 hours,the cells were incubated in the presence or absence of rapamycin or KU-0063794 for 48 hours.After harvesting, neutralization, centrifugation,and washing,we resuspended the cell precipitates in 100 μL 1×binding buffer.Next, we stained the samples with Annexin V-EGFP for 5 minutes at room temperature in the dark, followed by staining with 10 μL PI.Apoptosis was analyzed by flow cytometry in a BD FACSVerse system (FACSVerse, Becton Dickinson,Franklin Lakes, NJ, USA).

Two-dimensional migration in vitro

Cell migration was assayed by Oris Cell Migration Assay Kits(Cambridge Bioscience,Cambridge,UK)according to the manufacturer’s instructions.Cells were seeded at a density of 1×104/well and cultured at 37°C overnight.Next,we removed the cell-seeding stoppers from the wells and replaced the medium with fresh medium in the presence or absence of the indicated inhibitors.The cells were free to migrate into the migration zone (2-mm diameter) for 36 hours after removal of the cell-seeding stoppers.Calcein-AM(Dojindo Laboratories,Kumamoto,Japan)was used to visualize the live cells.The images of the cells were recorded by an inverted fluorescence microscope(Nikon,Tokyo,Japan).We calculated the number of cells that migrated into the migration zone from three independent samples using ImageJ software (National Institutes of Health, Bethesda, MD, USA).

Western blotting

The protocol of the western blotting assay was followed as previously described.16KFs were lysed by RIPA lysis buffer (Beyotime Biotechnology,Jiangsu,China)containing a phosphatase inhibitor and a protease inhibitor cocktail (Roche Applied Science) for 10 minutes on ice.The lysates were then collected and centrifuged at 7500gfor 15 minutes at 4°C.The supernatant was collected and the protein concentration was quantified by BCA assay kits (Beyotime Biotechnology).Equal amounts of protein lysates (20 μg) were loaded into 4%–15% precast SDSPAGE gels (Bio-Rad, Hercules, CA, USA) and then transferred to polyvinylidene fluoride membranes by the semi-dry transfer method.The membranes were incubated with 3% bovine serum albumin for 2 hours to block nonspecific protein binding and then with the corresponding primary antibodies overnight at 4°C.The membranes transferred with protein samples were washed with TBST and incubated with appropriate horseradish peroxidaseconjugated secondary antibodies for 2 hours.They were washed again with TBST and visualized using enhanced chemiluminescence substrate kits in a chemiluminescence imaging system (Bio-Rad).The band intensity was quantified by Quantity One software (Bio-Rad).

Small RNA interference

BothATG5small interfering RNA(siRNA)and non-sense control (NC) siRNA were synthesized by GenePharma(Shanghai, China).We seeded and incubated KFs in sixwell plates at 37°C overnight.After the cell confluence reached 60% to 70%,ATG5or NC siRNA was transiently transfected into the cells by Lipofectamine RNAiMAX(Invitrogen,Carlsbad,CA,USA)according to the manufacturer’s instructions.After incubation for 24 or 48 hours, we detected the gene silencing efficiency using quantitative real-time polymerase chain reaction (qRTPCR) and western blotting for the levels of mRNA and protein, respectively.At 6 hours after transfection of siRNA, the cell culture medium was replaced with fresh medium in the presence or absence of rapamycin or KU-0063794 for the indicated times.The respective 5’ to 3’sense and antisense sequences of siRNA were as follows:GUGAUGAUUCAUGGAAUUGTT and CAAUUCCA UGAAUCAUCACTT forATG5and UUCUCCGAACGU GUCACGUTT and ACGUGACACGUUCGGAGAATT for NC.

RNA isolation and qRT-PCR

We seeded the cells in six-well plates at a density of 1.5×105/well.After transfection withATG5or NC siRNA for 24 hours, total RNA was isolated in Trizol reagent(Invitrogen).We measured the concentration and purity of RNA using a NanoDrop ND-1000 spectrophotometer(Thermo Fisher Scientific).Total RNA(1 μg in 20 μL)was reverse-transcribed by SuperScript III RT-PCR Kits(Invitrogen).Amplified target genes were determined by qRT-PCR using PreAmp Supermix (Bio-Rad) on a Light-Cycler 480 system (Roche).GAPDHserved as the housekeeping gene for the normalization ofATG5mRNA levels.This experiment was assessed in triplicate replications.The relative mRNA levels of the target genes were quantified by the 2－ΔΔCTmethod.

The respective 5’ to 3’ forward and reverse sequences were as follows: GCCATCAATCGGAAACTCA and CAGCCACAGGACGAAACAG forATG5and AAAATC AAGTGGGGCGATGC and GATGACCCTTTTGGCT CCCC forGAPDH.

Statistical analysis

Data are presented as the mean ± standard deviation of at least three independent experiments.Differences between two groups and among multiple groups were compared using Student’st-test and one-way analysis of variance,followed by least significant difference analysis.Data were analyzed using SPSS software 20.0 (IBM Corp., Armonk, NY, USA), and data graphs were created using GraphPad Prism software 6.0 (GraphPad Software, San Diego, CA, USA).Statistical significance was defined asP<0.05.

Results

mTOR inhibitors rapamycin and KU-0063794 exert anti-keloid effects on KFs

We first detected the effects of the mTOR inhibitors rapamycin and KU-0063794 on cell viability of KFs.The CCK-8 assay showed that both rapamycin and KU-0063794 continuously inhibited cell viability after 24 hours of incubation (Controlvs.rapamycinvs.KU-0063794: 0.36±0.01vs.0.31±0.02vs.0.26±0.03,F=35.92,P=0.01) (Fig.1A).These results were validated by BrdU incorporation after 48 hours of incubation (Controlvs.rapamycinvs.KU-0063794:0.52±0.04vs.0.32±0.05vs.0.19±0.03,F=104.06,P=0.01) (Fig.1B).

Next, we investigated whether apoptosis is involved in the effects of the two mTOR inhibitors.Annexin V-EGFP/PI staining with flow cytometry showed that incubation with both rapamycin and KU-0063794 for 48 h did not activate apoptosis of KFs (P>0.05 each) (Fig.1C).We further confirmed these findings through detection of cleavage of caspase 3 and PARP by western blotting assay because neither mTOR inhibitor induced their cleavage(Fig.1D).

To assess the anti-fibrosis property of the two mTOR inhibitors,we detected the protein levels of collagen I and collagen III in KFs in the presence or absence of rapamycin or KU-0063794 for 48 hours.We found that treatment with both mTOR inhibitors decreased the protein levels of collagen I and collagen III,indicating inhibition of collagen synthesis (Fig.1E).

We used the Oris two-dimensional migration assays to detect the migration capacity of KFs after treatment with the two mTOR inhibitors.We found that rapamycin treatment inhibited cell migration in KFs compared with control (Controlvs.rapamycinvs.KU-0063794: 334.67±27.50vs.183.67±71.43vs.269.17±95.83,F=11.70,P=0.03) (Fig.1F).

Autophagy induction triggered by mTOR inhibitors can be inhibited by using wortmannin and via ATG5 silencing

We first validated whether transfection ofATG5siRNA inhibited the gene expression ofATG5at both the mRNA and protein levels through qRT-PCR (NCvs.ATG5 siRNA: 1.00±0.12vs.0.36±0.15,t=9.28,P=0.001)(Fig.2A) and western blotting (NCvs.si-ATG5: 1.14±0.35vs.0.52±0.19,F=14.89,P=0.01) (Fig.2B).As shown in Figure 2C,rapamycin and KU-0063794 induced the autophagic flux response of KFs in the presence of lysosomal protease inhibitors as evidenced by greater accumulation of LC3-II than in the E-64d+pepstatin group (NCvs.NC+rapamycin: 0.85±0.27vs.1.46±0.18,F=7.68,P=0.01;NCvs.NC+KU-0063794:0.74±0.29vs.1.59±0.50,F=5.93,P=0.02).Moreover, we observed that the induction of autophagic flux caused by rapamycin(NC+rapamycinvs.ATG5siRNA+rapamycin: 1.46±0.18vs.0.75±0.20,F=7.68,P=0.01) or KU-0063794 (NC+KU-0063794vs.ATG5siRNA+KU-0063794: 1.59±0.50vs.0.77±0.09,F=5.93,P=0.02) could also be inhibited in KFs transfected withATG5siRNA as shown by less LC3-II accumulation than in the NC group treated with both mTOR inhibitors(Fig.2C).Furthermore, we found that treatment with wortmannin (1 μmol/L) inhibited the increase of LC3-II accumulation in KFs after treatment with rapamycin(rapamycin+E-64d+pepstatinvs.rapamycin+wortmannin+E-64d+pepstatin: 1.88±0.38vs.1.02±0.35,F=6.86,P=0.013) or KU-0063794 (KU-0063794+E-64d+pepstatinvs.KU-0063794+wortmannin+E-64d+pepstatin: 1.65±0.35vs.0.76±0.17,F=10.01,P=0.004) in the presence of E-64d and pepstatin, indicating that wortmannin treatment can inhibit the induction of autophagic flux in mTOR inhibitor-treated KFs (Fig.2D).These data demonstrate that the induction of autophagic flux by the mTOR inhibitors rapamycin and KU-0063794 can be inhibited in KFs.

Figure 1. Effects of rapamycin and KU-0063794 on KF behavior.KFs were treated with rapamycin(100 nmol/L)or KU-0063794(5 μmol/L)for the indicated time prior to the corresponding experiments.DMSO-treated (<0.1%) cells were used as vehicle controls (Con).A and B: Cell proliferation was detected by the CCK-8 for 0,24,48,72 hours and by BrdU incorporation for 48 hours.C and D:Cell apoptosis was tested by Annexin V-EGFP/PI staining with flow cytometry, and caspase 3 and PARP cleavage were detected by western blotting after 48 hours of treatment.E:Collagen synthesis was measured by western blotting after 48 hours of treatment.F:Cell migration was detected by the Oris twodimensional migration assay stained by Calcein-AM after 36 hours of treatment.All data are presented as mean±standard deviation from three independent experiments, and panels A and B are representative of three independent experiments.?P<0.05.BrdU: 5-Bromo-2’-deoxyuridine;CCK-8:cell counting kit-8;DMSO:dimethyl sulfoxide;EGFP/PI:enhanced green fluorescent protein/propidium iodide;KFs:keloid fibroblasts.

Figure 2. Wortmannin treatment or ATG5 silencing inhibited the autophagic flux induced by rapamycin and KU-0063794.A and B:Verification of ATG5 silencing efficiency.KFs were transfected with non-sense control siRNA (si-NC) or ATG5 siRNA (si-ATG5) using Lipofectamine RNAiMAX for 6 hours,and quantitative real-time polymerase chain reaction was performed after 24 hours of cultivation.Western blotting was conducted under the detection system of the autophagic flux after 48 hours of cultivation.C:Following pre-transfection with si-NC or si-ATG5 for 6 hours, rapamycin or KU-0063794 was applied to the KFs for 24 hours.D: Following pretreatment with wortmannin (1 μmol/L) for 4 hours,rapamycin or KU-0063794 was applied to the KFs for 24 hours in the presence of wortmannin or vehicle.Finally,all groups were treated with lysosomal protease inhibitors(E-64d+pepstatin,10 μg/mL each)for 4 hours,and western blotting was performed to assess the accumulation of LC3-II.GAPDH was used as the loading control.All data are presented as mean±standard deviation from three independent experiments.?P<0.05.KFs: keloid fibroblasts;siRNA: small interfering RNA.

Figure 3. Re-examination of cellular functions after autophagy inhibition.KFs were treated with rapamycin or KU-0063794 for the indicated time after blocking autophagy with wortmannin or ATG5 silencing.Cell proliferation (A and B), cell migration (C), and collagen synthesis (D) were retested in KFs as described above.All data are presented as mean±standard deviation from three independent experiments,and panels(A and B) are representative of three independent experiments.?P<0.05.KFs: keloid fibroblasts.

Autophagy might not be involved in the anti-keloid effects of rapamycin and KU-0063794

To investigate whether autophagy regulation is involved in the anti-keloid effects of rapamycin or KU-0063794,wortmannin orATG5silencing was used to inhibit the autophagy induction in mTOR inhibitor-treated KFs for inducing anti-keloid effects.We found that wortmannin orATG5silencing did not disturb the inhibition of cell viability (assessedviathe CCK-8 assay and BrdU incorporation) by treatment with rapamycin or KU-0063794 (P>0.05 each) (Fig.3A and 3B).Furthermore,we found that the rapamycin- or KU-0063794-induced inhibition of the migration capacity of KFs was not affected even after subjection to wortmannin treatment orATG5siRNA transfection (P>0.05 each) (Fig.3C).Moreover, neither wortmannin treatment norATG5silencing disturbed the inhibitory effects of rapamycin or KU-0063794 treatment on the synthesis of collagen I and collagen III in KFs (Fig.3D).

Discussion

Cancer and fibrotic diseases are closely related to autophagy deficiency because autophagic death cannot be initiated in tumor cells, and redundant intracellular components cannot be degraded.17–19Clinically, keloids are defined as raised scar tissues that overgrow and invasively exceed the original wound boundary.Histopathologically, these features are based on the abnormal proliferation of dermal fibroblasts and the excessive deposition of collagen, manifesting as the dual characteristics of tumor formation and fibrosis.1,20The present study verified that mTOR-dependent autophagy activity is decreased in KFs.However, the actual role of autophagy inhibition in keloids remains unclear.It has been reported that ubiquitin accumulation might indicate a block in the autophagy process or inhibition of degradation of the proteasomal pathway.21We observed the accumulation of ubiquitin in keloid tissues.This demonstrates that autophagy inhibition might contribute the impaired degradation mechanism in KFs.Considering that autophagy machinery is extensively involved in cellular biology,autophagy may be intimately involved in the development of keloids.

mTOR kinases are structurally formed by two multiprotein complexes,namely mTORC1 and mTORC2,and these complexes have distinct substrates.The typical target of mTORC1 is S6K and that of mTORC2 is AKT, and both participate in the regulation of many cellular functions.22Rapamycin establishes an interaction with mTOR kinase,leading to selective inhibition of mTORC1.However, prolonged exposure to rapamycin disrupts mTORC2 signaling.23KU-0063794 is a highly selective ATP-competitive inhibitor of mTOR kinase activity,inhibiting both mTORC1 and mTORC2.24Previous studies have shown that mTOR inhibitors can be considered promising modulators against certain related human disorders, and their induction of autophagy may contribute to their pharmaceutical effects.25–27We have observed that the mTOR inhibitors rapamycin and KU-0063794 not only inhibit the activation of mTOR signaling but also restore the autophagy inhibition in KFs, which might be involved in mitigating keloid progression.Therefore, promoting autophagy might be considered a promising strategy to improve the prognosis of diseases related to autophagy deficiency.Interestingly,the present study showed that treatment with the mTOR inhibitor rapamycin or KU-0063794 exhibited significant anti-keloid effectsin vitro, including suppression of cell proliferation, inhibition of cell migration, and decreased collagen synthesis;these findings are consistent with previously reported results.13-14Given that the autophagy process is extensively related to cellular biological mechanisms,we doubt whether these anti-keloid effects of mTOR inhibitors are induced by activated autophagy in KFs.

Most information in previous studies of autophagy was obtained through autophagic inhibition.Blockage of class III PI3-kinase and knockdown of autophagy-related genes are the most common methods of inhibiting autophagy,functioning by blocking the formation of autophagosomes.28-29Use of the pharmacological inhibitor wortmannin andATG5knockdown are traditionally used for this purpose.30-31To support our hypothesis that activated autophagy machinery plays roles in the effects of mTOR inhibitors, we re-evaluated the biological effects after blocking autophagy activation using wortmannin orvia ATG5silencing.We found that both methods(wortmannin andATG5silencing) could successfully inhibit autophagy activation induced by rapamycin and KU-0063794 as evidenced by the decreased accumulation of LC3-II.However, blocking rapamycin- or KU-0063794-induced autophagy activation could not reverse their biological effects on KFs.

There are several limitations in this study.First,inconsistent with previous research, the apoptosis-inducing effects of rapamycin and KU 0063794 were not observed in our study.We speculated that differences in race or genetic background might lead to this difference.Finally,although our study did not clarify the actual role of autophagy impairment in the pathogenesis and development of keloids, the recovery effect on autophagy machinery is worthy of investigation.

In conclusion, our study has demonstrated that treatment targeting mTOR signaling can lead to effective inhibition of some crucial biological events in KFs,such as cell proliferation, cell migration, and collagen synthesis.Despite the availability of several clinical therapies, no single treatment for keloids has been proven successful.In the present study,we found that mTOR inhibitors exerted effects on both cell proliferation and collagen synthesis,indicating the therapeutic potential of mTOR inhibitors for the prevention and intervention of keloids and other fibrotic diseases.Although mTOR inhibitors are involved in the activation of KF autophagy, the autophagy machinery does not mediate their anti-keloid effects.

Source of funding

This work was supported by the CAMS Innovation Fund for Medical Sciences (No.CIFMS-2017-I2M-1-017) and Nanjing Incubation Program for National Clinical Research Center (No.2019060001).