mTOR-Dependent Autophagy Machinery Is Inhibited in Fibroblasts of Keloid

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

Jiangsu Key Laboratory of Molecular Biology for Skin Diseases and STIs,Hospital for Skin Diseases(Institute of Dermatology),Chinese Academy of Medical Sciences and Peking Union Medical College,Nanjing,Jiangsu210042,China.

Abstract

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

Introduction

Keloidisabenignproliferativedisorderofdermalfibroblasts,and abnormal tissue repair after various damages is involved initspathogenesis.1-2Keloidhasuniqueclinicalpresentations and its growth pattern is beyond the original wound margins which is used to distinguish it from hypertrophic scar.The pathogenesis of keloid is complicated and has not yet been fully clarified.Although multiple clinical therapies are available,the clinical prognosis for keloid is still a challenge due to the high recurrence.3Thus,in order to improve the outcomes of clinical interventions,the pathogenesis of keloid needs more investigations.

Autophagy is one evolutionarily conserved cellular process that conserves homeostasis through complex selfdegradation mechanism associated with lysosome.4-5Aberrant autophagy machinery is implicated in many kinds of disorders such as neurodegeneration,cancer,and fibrosis,which are closely related to autophagy deficiency for its failure of initiating autophagic death of tumor cells and degrading redundant intracellular components.6-7Therefore,to understand the role of autophagy and the regulated signaling pathways involved in diseases treatment is critical.In addition,it may provide new opportunities for therapeutic advantage.The main histopathological characteristics of keloid are abnormal proliferation of dermal fibroblasts and excessive deposition of extracellular matrix which combines the properties oftumor andfibrosis.2These evidences indicate that an aberrant autophagy machinery may participate in the pathogenesis of keloid.

The autophagic process is strictly regulated by autophagy-related genes(ATGs)and associated signaling pathways precisely coordinate the autophagy process.In mammalian cells,Beclin1initiates phagophore formation.Microtubule-associated protein1light chain3(LC3),a protein marker of autophagy,is converted from its cytosolic(LC3-I)to its phosphatidylethanolamine-conjugated form(LC3-II)as autophagy is initiated.In the final stages of autophagic process,the autophagosomes fuse with lysosome for degradation of their“cargo”which require the ubiquitin–proteasome system participation.4Among all modulatory elements,the activation of mechanistic target of rapamycin(mTOR)signaling plays a crucial negative role in autophagy machinery,which is achieved by inhibiting autophagosome formation at an early step.Thus,the regulatory method of mTORmediated autophagy is nominated as mTOR-dependent autophagy.8

mTOR,structured by two distinct complexes:mTOR complex1(MTORC1)and mTOR complex2(MTORC2),is an evolutionarily conserved serine/threonine kinase.MTORC1can be inhibited by rapamycin,an inducer of autophagy in mammals both in vitro and in vivo,and MTORC2is not a direct regulator of autophagy.As a central controller in regulating cell growth and metabolism,activated mTOR signaling works primarily through positive regulation of protein synthesis and inhibition of autophagy.9Studies have found that dysregulation of mTOR signaling has been correlated with many diseases involving in cancer,metabolic and fibrotic diseases et al.10–12In addition,over activated mTOR signaling has been proved in the pathogenesis of keloid in previous studie.13

Considered the crucial biological function of mTOR in keloid and in cellular autophagy machinery,we first investigated the baseline levels of mTOR,S6ribosomal protein,and their activated forms in keloid tissues(KTs)and extra-lesional tissues obtained from the same patient,as well as the baseline autophagy levels marked by LC3,Beclin1,and Ubiquitin.In addition,corresponding detection were also carried out at the level of keloid fibroblasts(KFs)derived from Han Chinese individuals.Finally,by inhibiting mTOR activity,we confirmed our hypothesis that mTOR-dependent autophagy inhibition is involved in the pathogenesis of keloid which can be explained from the perspective of autophagy machinery.

Materials and methods

Reagents and antibodies

The compounds in our study were sourced as stated:dimethylsulphoxide(DMSO),rapamycin,KU-0063794,acridine orange(AO),dispase II,E-64d,and pepstatin(all from Sigma-Aldrich,St.Louis,MO,USA),and collagenase type I(from Thermo Fisher Scientific,Waltham,MA,USA).Primary antibodies for immunohistochemistry(IHC)included anti-mTOR,anti-phospho-mTOR Ser2448,anti-S6ribosomal protein,anti-Beclin1(all from Abcam,Cambridge,UK)and anti-phospho-S6ribosomal protein Ser235/236,anti-LC3A/B,anti-Ubiquitin(all from Cell Signaling Technology,Danvers,MA,USA).Primary antibodies including anti-ULK1,anti-phospho-ULK1,anti-ATG7,anti-ATG9A,anti-ATG12,and anti-LC3A/B,and secondary antibodies(goat anti-rabbit IgG)for western blotting assays were all purchased from Cell Signaling Technology.

Tissue specimens

The tissue samples of KTs and extra-lesional tissues(ELTs)were harvested from excisional surgery for patients with keloid diagnosed by dermatologists.The diagnosis of keloid was further validated by the histopathology examination after surgery.The patients in this study included7men and6women(age range,19–59years).All patients or their authorized representatives provided written informed consent for collection of specimens(Table1).Total protein was extracted from the KTs1-4,and primary KFs were isolated and cultured from the KTs 5-7.Paraffin-embedded specimens(KTs8-13)were assessed by IHC study,and extra-lesional tissues served as the controls.This study was approved by the Ethics Committee of the Institute of Dermatology,Chinese Academy of Medical Sciences,and Peking Union Medical College(2015-KY-019).

Culture of keloid fibroblasts

We isolated the normal fibroblasts(NFs)from healthy men who had accepted circumcision.The establish of primary KFs were described as previously.14Briefly,excised tissues were first digested with5mg/mL dispase II(Sigma-Aldrich)overnight at4°C,followed by3mg/mL collagenase type I(Thermo Fisher Scientific)for2hours at37°C,then filtered through a cell strainer.After centrifugation,the precipitate was resuspended and cultured in sterile flasks with Dulbecco modified Eagle medium containing10% fetal bovine serum(Thermo Fisher Scientific).In this study,passage2to passage5cells were used.In this study,we used the passage2to5cells.

mTOR inhibitors Rapamycin and KU-0063794 treatment of keloid derived fibroblasts cultures

Keloid-derived fibroblasts were set up as above mentioned.The experimental group was treated with either rapamycin(100nmol/L)or KU-0063794(5μmol/L)with DMSO as solvent,and KFs in the normal control group was given of DMSO(＜0.1%)solvent at the same volume.Cell cultures were collected for western blotting analysis after24hours incubation.

Table1 Baseline data of keloid patients.

Acridine orange staining

Due to AO’s staining properties for acidic vesicular,autophagosomes fused with lysosomes can be visualized as the formation of acidic vesicular organelles(AVOs).The protocol of AO staining was described as previously.15The acidic compartments showed red fluorescence and the cytosolic and nuclear compartments showed green fluorescence.Three lines of primary keloid-derived fibroblasts isolated from the patients and one line of primary NFs were seeded in confocal culture dishes.The cells were fixed with4%paraformaldehyde for15minutes at37°C when they reached80% confluence.The cells were washed with PBS,stained with5μg/mL AO for10 minutes in the dark at37°C,washed thrice with PBS,then visualized using an FV1000laser scanning confocal microscope(Olympus Corporation,Tokyo,Japan).

Immunohistochemistry study

Excised skin specimens were immediately fixed in10%formaldehyde,routinely embedded in paraffin,and cut into4-μm sections.Embedded sections were dewaxed and rehydrated;then,antigen was retrieved,and endogenous peroxidase was blocked using3% H2O2.Nonspecific antigens were blocked by incubation with5%goat serum for1hour;then,the sections were incubated with specific primary antibodies at4°C overnight.Antigens were probed with biotinylated secondary antibody(1:1,000 dilution),and avidin-biotin peroxidase complex(Dako,Glostrup,Denmark)was applied per manufacturer instructions.Objective antigens were visualized after the staining of diaminobenzidine and counterstaining of hematoxylin.

Western blotting

The protocol of western blotting was described as previously.15Cells were lysed by RIPA lysis buffer(Beyotime Biotechnology,Jiangsu,China)containing a protease inhibitor cocktail and a phosphatase inhibitor(Roche Applied Science,Basel,Switzerland)for10minutes on ice.Lysates were centrifuged at7500g for15minutes at 4°C.Protein concentration in the supernatant was quantified using BCA assay kits(Beyotime Biotechnology).Equal amounts of protein lysates(20–40μg)were loaded into4%–15%precast SDS-PAGE gels(Bio-Rad,Hercules,CA,USA),then transferred to PVDF membranes.Nonspecific protein binding was blocked with3% BSA for2hours,and the membranes were incubated with the corresponding primary antibodies overnight at4°C.The membranes were washed with TBST and incubated with appropriate horseradish peroxidase-conjugated secondary antibodies for2hours.The membranes transferred with protein samples were visualized by ECL substrate kits in the chemiluminescence imaging system(Bio-Rad).Band intensity was quantified using Quantity One software(Bio-Rad).

The extraction of total tissue proteins from KTs and ELTs were conducted using the MinuteTMTotal Protein Extraction Kit(Invent Biotechnologies,Eden Prairie,MN,USA)from the spin-column technology according to the instructions of manufacturer.First,the native cell lysis buffers were used to lyse the tissues for5minutes.The tissue samples were grinded repeatedly in the centrifugal column.Then the lysates were collected in a receiver tube by centrifugation(7,500g;5minutes;4°C),and processed for Western blotting assay.

Statistical analysis

The data are exhibited as the means±SD from three independent experiments at least.The effect of mTOR inhibitors on mTOR signaling was evaluated within groups by the unpaired Student t-test and the paired t test was used to compare the expression of key labeling proteins between KTs and ELTs.The effect of mTOR inhibitors on autophagic flux in KFs was analyzed by the one-way analysis of variance,followed by LSD(Least Significant Difference)analysis.SPSS software20.0(IBM Corp.,Armonk,NY,USA)was used to analyze the data,and GraphPad Prism software6.0(GraphPad Software,San Diego,CA)was used to present the graphs.P＜0.05 was defined as the statistical significance.

Results

mTOR activity is enhanced in keloid tissues and keloid derived fibroblasts

In first,we detected the protein and phosphorylated levels of two core proteins of mTOR signaling,mTOR and S6 ribosomal protein,in KTs and ELTs through the assays of IHC(n=6,Fig.1A)and western blotting(n=4,Fig.1B).We observed the increase in both protein levels and phosphorylation of mTOR(and detecting the phosphorylation at Ser2448)and S6ribosomal protein(and detecting the phosphorylation at Ser235/236)in the KTs compared with ELTs(Fig.1A and1B).Although we had not observed the increase in the protein level and phosphorylation of mTOR in KFs,the S6ribosomal protein phosphorylation at Ser235/236which reflects mTOR activation signaling,was increased in KFs compared with NFs(Fig.1C).These data suggested the activated mTOR signaling in keloid.

Inhibition of autophagy activity in keloid tissues and keloid derived fibroblasts

Figure1.MTOR activity analysis by detecting the expression of total-MTOR,phosphorylated-MTOR(p-MTOR),total-S6and phosphorylated-S6(p-S6).(A)Representative photomicrographs of immunohistochemical staining with specific antibody against MTOR signaling in keloid tissues(KTs)and its extra-lesional tissues(ELTs)(n=6).Scale bar indicates200μm.(B)Western blotting analysis of MTOR signaling and the conversion of LC3-I to LC3-II in4cases of KTs and ELTs.(C)Western blotting analysis in three cases of primary keloid fibroblasts(KFs)from persons of Han Chinese ancestry compared with normal fibroblasts(NFs).GAPDH was used as loading control.MTOR:mechanistic target of rapamycin.

We first detected the expression level in situ of some crucial autophagy-related molecules including LC3,Beclin1,and Ubiquitin in KTs and ELTs through the assays of IHC(n=6).We observed a reduced expression of LC3and Beclin1 and an increased expression of Ubiquitin in the cytoplasm of KFs than that in fibroblasts from extra-lesional tissues(Fig.2A).Importantly,the conversion of LC3-I to LC3-II was impeded in KTs compared with extra-lesional tissues as shown by western blotting assay(n=4),indicating the inhibited autophagy activity(Fig.1B).Furthermore,we determined some crucial ATGs LC3,ULK1(and its phosphorylation at Ser757),ATG7,ATG9A,ATG12by western blotting assay in KFs and NFs.Although the labeled ATGs including ATG7,ATG12,ATG9A,and ULK1expression did not differ compared with that in NFs,we still found an impeded conversion from LC3-I to LC3-II in KFs compared with NFs(Fig.2B),suggesting an inhibition on autophagy activity.According to the previous study,15we validated the autophagy inhibition in KFs through the assay of AO staining which can marked with acid vesicles.We found the red fluorescence intensity was decreased in KFs compared with NFs(Fig.2C),indicating that the defective autophagy capacity led to reduced AVOs formation.These findings indicated that autophagic activity is inhibited in keloids.

mTOR inhibitors rapamycin and KU-0063794can inhibit mTOR activity in keloid derived fibroblasts

We evaluated the effects on mTOR activity in KFs treated with rapamycin(100nmol/L)and KU-0063794(5μmol/L)for24hours by western blotting assay,and DMSO(＜0.1%)was used as solvent control.By detecting the expression of downstream effectors,we found that the treatment of both mTOR inhibitors decreased the protein levels of p-mTOR Ser2448and p-S6Ser235/236,indicating inhibition on mTOR activity(Fig.3A).

mTOR inhibitors activate autophagy in keloid derived fibroblasts

To monitor whether the treatment of rapamycin and KU-0063794affects autophagic flux in KFs,KFs were treated with rapamycin or KU-0063794in presence or absence of lysosomal inhibitors E-64d and pepstatin(10μg/mL)for4 hours,and DMSO(＜0.1%)was used as solvent control.Moreover,we determined the LC3-II accumulation(the ratios of LC3-II/GAPDH)by western blotting assay.The results showed that compared with the control group,treatment with E-64d and pepstatin could increase the accumulation of LC3-II to some extent which reflected the basic autophagic flux.The data also showed that LC3-II accumulation was remarkably increased in KFs treated with rapamycin(E-64d+pepstatin vs.rapamycin+E-64d+pepstatin:[0.88±0.35]vs.[1.56±0.46],F=5.56,P=0.049)or KU-0063794(E-64d+pepstatin vs.KU-0063794+E-64d+pepstatin:[0.92±0.22]vs.[1.51±0.25],F=25.88,P=0.011)in the presence of E-64d and pepstatin compared with KFs treated with E-64d and pepstatin,suggesting the increase of autophagic flux(Fig.3B).Therefore,the above findings demonstrate that activation of mTOR activity led to autophagy inhibition in KFs.

Figure3.Effects of MTOR inhibitors,rapamycin and KU-0063794on MTOR signaling and autophagic flux in KFs.KFs were treated with rapamycin(100nmol/L)or KU-0063794(5μmol/L)for the indicated time prior to the corresponding experiments.DMSO-treated(＜0.1%)cells used as vehicle controls(Con).(A)Western blotting analysis of MTOR activity in KFs treated with MTOR inhibitors for24hours by detecting its downstream molecules,p-MTOR and p-S6.(B)KFs were treated with rapamycin or KU-0063794for4hours in the presence or absence of E-64d and pepstatin and tested by western blotting to assess the autophagic flux reflected by accumulation of LC3-II.GAPDH was used as loading control.The data are exhibited as the means±SD from three independent experiments.?P＜0.05.KFs:keloid fibroblasts;MTOR:mechanistic target of rapamycin;p-MTOR:phosphorylated-MTOR.

Discussion

The dysregulation of mTOR signaling pathway is involved in the imbalance between catabolism and anabolism,contributing to various human disorders’development.16-17Our study demonstrated that the protein level and phosphorylation of mTOR and its key substrate S6 ribosomal protein were overexpressed in KTs compared with extra-lesional tissues.Importantly,keloid-derived fibroblasts exhibit the increase of S6ribosomal protein phosphorylation compared with NFs in culture in vitro.These findings further validated the previous studies that mTOR signaling is involved in the pathogenesis of keloid which played important roles in promoting fibroblast proliferation and extracellular matrix deposition.6

Autophagy machinery plays a crucial role in catabolic process and provides an alternative energy source via selfdigestion through the complex process of lysosomal degradation to maintain homeostasis.It functions both as a scavenger for dysfunctional endogenous components and as a recycler for nutrients to protect cells from death under stresses and changes in intracellular and extracellular conditions.4-5Previous studies reported that deficiency of autophagy machinery is closely associated with fibrotic disorders and cancer because the property of autophagic cell death and degraded mechanism for redundant and abnormal intracellular components are impaired in the tumor cells.18–20These evidences indicate that targeting mediators of autophagy will be a promising strategy for treatment of related diseases.Autophagy is strictly regulated by ATGs and associated signaling pathways,especially the mTOR signaling pathway negatively modulates the autophagic process.21-22Our study indicates that abnormal activation of mTOR is accompanied with the autophagy inhibition in keloid.Thus,we speculate that the abnormal autophagy regulation might contribute to the pathogenesis and development of keloid.A few recent studies reported that the roles of autophagic mechanism in keloids are controversial.23–25Our study found a reduced expression of LC3and Beclin1in the cytoplasm of KFs than that in fibroblasts from extralesional tissues.Importantly,there was accumulation of Ubiquitin protein in the tissue section,suggesting damage to ubiquitinated proteins in the autophagic degradation pathway.Then,we detected autophagic markers in KTs and keloid derived fibroblasts by western blotting assay,and found reduced autophagic activity in keloid which was reflected as an impeded conversion from LC3-I to LC3-II.Monitoring AVOs formation is a supplementary means of detecting autophagy.26Therefore,we also assessed the formation of acid vesicles in KFs by AO staining.Finally,our findings of AO staining showed that AVOs accumulation were also decreased in keloid-derived fibroblasts compared with NFs.Therefore,we concluded that autophagy is inhibited in keloid.However,the precise mechanism by which the defective autophagy activity leads to keloid is still not entirely clear.

Autophagy is a dynamic process controlled by various regulators,including ATGs,both mTOR-dependent and alternative mTOR-independent pathways.4,27-28We have labeled some ATGs expression in keloid-derived fibroblasts and found that ATG7,ATG12,ATG9A,and ULK1 expression did not differ compared with that in NFs.As a central controller in regulating cell growth and metabolism,mTOR serves to promote protein translation and inhibit autophagy process.9,29-30To further confirm that the defective autophagy activity in keloid is regulated by mTOR-dependent pathway,we applied the mTORC1 inhibitor,rapamycin,and the dual mTORC1/2inhibitors,KU-0063794to inhibit mTOR activity.We verified an inhibition on mTOR activity in keloid-derived fibroblasts treated with mTOR inhibitors,rapamycin,and KU-0063794,which could significantly reduce the protein levels of p-mTOR Ser2448and p-S6Ser235/236.Furthermore,mTOR inhibitors,could reverse the inhibitory effect on autophagy of keloid-derived fibroblasts as evidenced by an increased LC3-II accumulation,suggesting the increase of autophagic flux.

There are several limitations in this study.First,more biological replicates should be set in NFs to ensure the stability of the results.In addition,the effect of mTOR inhibitors on autophagic activity of NFs was not discussed in this study,which is worthy of further study.

In conclusion,our study demonstrates that the abnormal activation of mTOR is accompanied with the autophagy inhibition in keloid.In addition,we verified that mTOR activation contributes to autophagy inhibition due to the treatment of mTOR inhibitors activate autophagy in KFs;that is,the mechanism of autophagy defect in keloid is regulated by mTOR-dependent pathway.

Source of funding

This work was supported by CAMS Innovation Fund for Medical Sciences(CIFMS-2017-I2M-1-017).