Ski promotes proliferation and inhibits apoptosis in fibroblasts under high‐glucose conditions via the FoxO1 pathway

Abstract

Objectives
The present study clarified the role and signalling pathway of Ski in regulating proliferation and apoptosis in fibroblasts under high‐glucose (HG) conditions.

Materials and Methods
The proliferation and apoptosis of rat primary fibroblasts were assessed using EdU incorporation and TUNEL assays. The protein and phosphorylation levels of the corresponding factors were measured using immunofluorescence staining and Western blotting. Immunoprecipitation was used to determine the interactions between Ski and FoxO1 or Ski and HDAC1. The Ski protein was overexpressed via recombinant adenovirus transfection, and FoxO1 and HDAC1 were knocked down using targeted small‐interfering RNA.

Results
The present study found that HG inhibited fibroblast proliferation, increased apoptosis and reduced Ski levels in rat primary fibroblasts. Conversely, increasing Ski protein levels alleviated HG‐induced proliferation inhibition and apoptosis promotion. Increasing Ski protein levels also increased Ski binding to FoxO1 to decrease FoxO1 acetylation, and interfering with FoxO1 caused loss of the regulatory effect of Ski in fibroblasts under HG. Increasing Ski protein levels decreased FoxO1 acetylation via HDAC1‐mediated deacetylation.

Conclusions
Therefore, these findings confirmed for the first time that Ski regulated fibroblast proliferation and apoptosis under HG conditions via the FoxO1 pathway.

Full text

Cell Proliferation. 2021;54:e12971.  
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https://doi.org/10.1111/cpr.12971
wileyonlinelibrary.com/journal/cpr
1 | INTRODUCTION
Impaired or delayed wound healing is the main secondar y complica-
tion of diabetes, and it is very har mful and often leads to limb loss and
disability.1,2 The impaired healing of diabetic wounds is the result of
a combination of many factors, such as persistent inflammation of
the wound, impaired angiogenesis and dysfunction of repair cells.1,2
Among these factors, the change in fibroblast function is an import-
ant reason for the damage of epithelialisation and delayed healing of
diabetic wounds,3,4 and this function is related to the decreased pro-
liferation and increased apoptosis of fibroblasts in diabetic wounds.3,4
Therefore, targeted therapy to improve the function of fibroblasts is
an important method to promote the healing of refractory diabetic
wounds.4 -7 The regulation of fibroblast proliferation and apoptosis is
primarily achieved by improving the environment, such as increas-
ing growth factors.4,5,7 The direct targeting of fibroblasts to improve
their proliferation and anti-apoptotic effects may become a new and
more effective way to treat refractor y diabetic wounds.
ski is an intracellular homologue of the viral oncogene v-ski. Its
protein product Ski (called Ski protein in humans and c-Ski protein in
animals) is a multifunctional transcription regulator that participates in
many physiological and pathological processes, such as hematopoietic
Received:11September2020
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Revised:16November2020
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Accepted:7Decemb er2020
DOI:10.1111/cpr.12971
ORIGINAL ARTICLE
Ski promotes proliferation and inhibits apoptosis in fibroblasts
under high-glucose conditions via the FoxO1 pathway
Yan Peng | Ren-Ping Xiong | Zhuo-Hang Zhang | Ya-Lei Ning | Yan Zhao |
Si-Wei Tan | Yuan-Guo Zhou | Ping Li
This is an op en access article under t he terms of the Creat ive Commons Attributio n License, which permits use, dist ribution and reproduc tion in any medium,
provide d the orig inal work is proper ly cited .
© 2020 The Authors . Cell Proliferation Published by John Wiley & Sons Ltd.
Depar tmentofA rmyOccupationalDisease,
State Key Laboratory of Trauma , Burn and
CombinedInjur y,DapingHospital,Army
Medical University (Third Military Medical
University), Chongqing, China
Correspondence
Yuan-guoZhouandPingLi,Department
ofArmyOccupationalDisease,State
Key Laboratory of Trauma, Bur n and
CombinedInjur y,DapingHospital,Army
Medical University (Third Military Medical
University), 10 Changjiang Zhilu, Chongqing
400042, China.
Emails: ygzhou@tmmu.edu.cn;
ping_ping0074@sina.com
Funding information
Nationa lNaturalScienceFoundationof
China,G rant/AwardNumber :81701915.
Abstract
Objectives: The present study clarified the role and signalling pathway of Ski in regu-
latingproliferationandapoptosisinfibroblastsunderhigh-glucose(HG)conditions.
Materials and Methods: The proliferation and apoptosis of rat primary fibroblasts
were assessedusing EdU incorporation and TUNEL assays.The proteinand phos-
phorylation levels of the corresponding factors were measured using immunofluo-
rescence staining and Western blotting. Immunoprecipitation was used to determine
theinteraction sb etweenSk iandFoxO1orSkia ndHDAC1.Th eS kiprote inwasover-
expresse d via recombinant ade novirus transfe ction, and FoxO1 and H DAC1 were
knockeddownusingtargetedsmall-interferingRNA.
Results: ThepresentstudyfoundthatHGinhibitedfibroblastproliferation,increased
apoptosis and reduced Ski levels in rat primary fibroblasts. Conversely, increasing Ski
proteinlevelsalleviated HG-induced proliferation inhibition and apoptosispromo-
tion. Increasing Skiprotein levelsalso increased Skibinding to FoxO1todecrease
FoxO1acetylation, andinterferingwithFoxO1causedlossoftheregulatoryeffect
ofS kiinfib ro blast sunde rH G.Incre asingSkiproteinlevelsde creasedFoxO1acety la-
tionviaHDAC1-mediateddeacetylation.
Conclusions: Therefore, these findings confirmed for the first time that Ski regulated
fibroblastproliferationandapoptosisunderHGconditionsviatheFoxO1pathway.

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cell proliferation, muscle regeneration, bone and nervous system
development, synaptic projection, wound healing, fibrosis, tumori-
genesis and proliferation.8 -11However,therearefewreportsonthe
role of Ski in the HG environment.BecauseSki promotesfibroblast
proliferation12 and inhibits apoptosis by inhibiting the transforming
growth f actor β1(TGF-β1)/Smad signalling pathway.13,14 This suggest s
that Ski, as an important regulator of the biological behaviour of fi-
broblasts, may also promote the proliferation and anti-apoptosis of
fibroblastsindiabeticwoundsorunderHGconditions.
BasedonthefindingthatHGinhibitedfibroblastproliferation,
promoted fibroblast apoptosis and reduced Ski protein levels, the
present study found that increasing Ski protein levels via trans-
fectionof therecombinantadenovirus Adeno-MCMV-SKI-3Flag-
P2A-EGFP(Ad-Ski)alleviatedHG-inducedfibroblastproliferation
inhibition and apoptosis promotion without affecting the level of
Smad2/3. Then,FoxO1siRNA was used to verif ythatincreasing
Ski protein levels alleviated HG-induced fibroblast proliferat ion
inhibitionandapoptosis promotion throughtheFoxO1signalling
pathway. Finally,co-immunoprecipitation (IP) andWestern blot-
ting were used to confirm that increasing Ski protein levels under
HG condit ions increased t he interact ion of Ski with FoxO1 and
reducedtheacetylationlevelofFoxO1,whichwasrealisedbythe
recruitmentofHDAC1viaSki.
2 | MATERIALS AND METHODS
2.1 | Animals
MaleSprague-Dawleyrats,weighingapproximately100-120g,were
provided bythe Experimental AnimalCentre of the Army Medical
Centre [CertificateNo.SYXK( Yu)2012-0 010].Allproceduresused
inthisstudywereperformedinstrictaccordancewiththeNational
Institutes of Health Guide for the Care and Use of Laboratory
Animals (N IH Publicatio n No. 86-23, Revised 1985) and were ap -
proved by the Institutional Animal Care and Use Committee of the
Third Military Medical University.
2.2 | Antibodies and reagents
Dulbecc o'smodifiedE ag le'sm edium(D MEM)a ndfoetalb ovinese ru m
(FBS)wereobtainedfromGibco.D-glucose(G7021),mannitol(M1902)
and anti-acetyl-lysine (05-515) were purchased from Sigma-Aldrich
(Sigma-Aldrich®, Merck KG). Antibodies against p27kip1 (C67H9,
No.3686), Smad3 (C67H9,No.9524),p-Smad3(Ser423/Ser425,No.
9520),Smad2(No.5339),p-Smad2(S465/S467,No.18338),Bim(No.
2933),cleavedcaspase-3(No.9661),FoxO1(No.2880), andtheirre-
spectivehorseradishperoxidase-coupledsecondaryantibodieswere
purchased from Cell Signaling Technologies. Antibodies against Ski
(sc-914 0),PCNA(sc-56)andGAPDH(sc-56)wereobta inedfromSanta 
Cruz Biotechnolog y. Protein A/G Plus A garose beads (No. 20423)
wereobtainedfromThermoScientific(ThermoFisherScientific).
2.3 | Primary fibroblast culture
Primar y fibrobla sts were ex tracted f rom the dors al skin of male
Sprague-Dawley rats using the explant technique, as previously
described.FibroblastswereculturedinDMEMsupplementedwith
10%FBS,100U/mLpenicillin and 100 μg/mL streptomycin in an
incubator at 37°C and 5% CO2. Fibroblasts from passages 3-5
were used in our study. Before treatment, the cells were precul-
tured in serum-free medium for 24 hour. The medium was changed
to DMEM supplemented with 10% FBS containing 5.0 mmol/L
D- glu cos e( NG,n orm alcon d it i on s),25mm o l/ LD- glu co s e( HG,hig h
glucose)or 5.5 mmol/L D-glucose, and 19.5mmol/L D-mannitol
(HM, high ma nnitol) was us ed as an osmotic co ntrol to maint ain
osmolarity.
2.4 | Construction, amplification and purification of
Ad-Ski and transfections
The Ski-overexpressing recombinant adenovirus Adeno-MCMV-
SKI-3Flag-P2A-EGFP (Ad-Ski) and the empty control recombi-
nant adenovirus Adeno-MCMV-3Flag-P2A-EGFP (Ad-EGFP) were
purchased from Obio Technology Corp., Ltd. Human Ski cDNA
(GenBankaccessionnumberNM_003036.3)waspackaged intothe
adenovirus vector. The virus was amplified in human embryo kidney
293(HEK293)cells,purifiedbyVivapureAdenoPACK20(Sartorius),
and titrated as previously described.15 The efficiency of Ad-Ski in-
fection in fibroblasts was demonstrated by Western blotting and im-
munoflu orescence. Pri mary fibro blasts were t reated with HG an d
transientlytransfectedwithAd-EGFPorAd-Skifor48hour,follow-
ingthemanufacturer'sinstructions.
2.5 | siRNA transfection
Specific small-interfering RNAs (siRNAs) targeting rat mRNA
FoxO1(5′- GGACAGCAAATCAAGTTAT-3′), siRNAs targeting rat
HDAC1 mRNA (5′- GGCCTGCACCATGCGAAGA-3′), and the cor-
respondingscrambledsiRNAusedasanegativecontrol(NCsiRNA)
wereallchemicallysynthesisedbyRiboBioCo.,Ltd.ThesiRNAwas
diluted in r iboFECTTM C P Buffer and mi xed with riboFEC TM CP
Reagent for20minutes. FibroblastsweretransfectedwithHDAC1
siRNAorFoxO1siRNAfor24hoursfollowedbyanother48hoursin
thepresenceofNGorHGconditions.
2.6 | Cell Counting Kit-8 assay
TheCellCounting Kit (CCK)-8assay(Beyotime)wasusedtoas-
sess cell viability. Briefly, fibroblasts (2 × 10 3/well) were seeded
in96-wellplateswith100μL of medium. After various cell treat-
ments, 10 µL of CCK-8( Beyotime) solut ion was added to e ach
wellandincubated for 2hourat37°Cand5%CO2.Theoptical

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densit y (OD) values (8 wells/group) we re measured at 450 nm
using a microplate reader (Bio-Tek Instruments). Wells without
cells served as blankcontrols.Each experiment wasperformed
in triplicate.
2.7 | EdU incorporation assay
We assessed fibroblast proliferation using a Cell-Light 5-ethynyl-
2-deoxyuridine (EdU) Apollo567 In Vitro Kit (Ribobio Co., Ltd.)
FIGURE 1 EffectofHGonfibroblast
proliferationandapoptosis.Fibroblasts
weretreatedwithNG,HGorHMfor24
or48hours,respectively.A,ACCK-8
assay was used to evaluate fibroblast
viabilit y. n =8,Theresultsrepresentthe
mean ±SDofatleastthreeindependent
experiments.*P < .05, **P < .01,
***P < .001 compared to corresponding
NGgroup,one-wayANOVA,Dunnett's
posthoctests.Fibroblastproliferation
was assessed by an EdU assay (B), and
fibroblast apoptosis was tested by a
TUNELassay(D).n=8,Corresponding
quantitative analyses are shown (C) and
(E).EdUandTUNELstaining,red;nuclei,
blue. *P < .05, **P < .01, ***P < .001,
Student'sttestcomparingtheNGgroup
withtheHGgroup.Scalebars= 10 0 µm.
F,Immunofluorescencestainingwas
conductedtodetectSkiexpression.
NucleiareindicatedwithDAPIstaining
(blue),andSkiexpressionisindicatedby
red fluorescence. G , Quantification of
immunofluorescence staining; *P < .05,
**P < .01, ***P <.001,Student'st test
comparingtheNGgroupwiththeHG
group. scale bars = 10 0 µm.H-I,Western
blotanalysisofSki,PCNA,p27,cleaved
caspase-3andBimproteinexpression
levels. The results were obtained
fromthreeindependentexperiments.
*P < .05, **P < .01, ***P < .001 compared
tocorrespondingNGgroup,one-way
ANOVA,Dunnett'sposthoctests

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according to the manufacturer's instructions. Briefly, fibroblasts
(2 × 104/well) were cultured in a Mi llicell EZ SLIDE 8-Well glas s
slide (Merck Millipore Ltd.) and then incubated with 50 μmol/L EdU
(1:1000)for12 hour.Fibroblasts werefixed with 4%formaldehyde
for 20 minutes at 37°C, followed by permeabilisation in 0.5% Triton
X-100at37°C.Then,100μL of Apollo® reaction cocktail was added
to each well and incubated for 30 minutes under light-shading condi-
tions. Af ter three washes with PBS, the nuclei were counterstained
with4′,6′-diamidino-2-phenylindole(DAPI)for20 minutesat 37°C,
and the EdU-labelled cells were observed by a laser scanning confo-
cal micro scopy (Leica S P8) and normal ised to the tota l number of
DAPI-stainedcells.
FIGURE 2 Increasing Ski protein
levelsalleviatedHG-inducedproliferation
inhibition and apoptosis promotion in
fibroblasts without affecting the level of
Smad2/3phosphorylation.Fibroblasts
wereinfectedwithAd-SkiorAd-EGFP
for48hour,combinedwithHGtreatment
for48hour.A,Immunofluorescence
stainingforSkiexpression.Nucleiare
indicatedwithDAPIstaining(blue),
andSkiexpressionisindicatedbyred
fluorescence. The left panels show high-
magnificationimagesoftheboxedregions
in the right panels. scale bar = 10 0 µm.
B,FibroblastswereinfectedwithAd-Ski
orAd-EGFPfor48hour.TheSkilevels
wereexaminedbyWesternblotanalysis.
The results were obtained from three
independentexperiments.*P < .05,
**P < .01, ***P < .001 compared to
theDMEMgroup,one-wayANOVA,
Dunnett'sposthoctests.C,CCK-8assay
was used to evaluate fibroblast viability.
n =8,*P < .05, **P < .01, ***P < .001
comparedtotheNG+Ad-EGFPgroup;
#P < .05, ##P < .01, ###P < .001 compared
totheHG+Ad-EGFPgroup,one-way
ANOVA,Dunnett'sposthoctests.
D,Fibroblastproliferationabilitywas
assessed by an EdU assay, and fibroblast
apoptosiswastestedbyaTUNELassay,
n =8.Correspondingquantitative
analysesareshown(E)and(F).EdUand
TUNELstaining,red;nuclei,blue.*P < .05,
**P < .01, ***P < .001 compared to the
NG+Ad-EGFPgroup;#P < .05, ##P < .01,
###P <.001comparedtotheHG+ Ad-
EGFPgroup,one-wayANOVA,Dunnett's
post hoc tests. scale bars = 100 µm.
G-H:Westernblotanalysisofp-Smad2
andp-Smad3proteinexpressionlevels.
The results were obtained from three
independentexperiments.*P < .05,
**P < .01, ***P < .001 compared to
correspondingNG+Ad-EGFPgroup,one-
wayANOVA,Dunnett'sposthoctests

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2.8 | TUNEL assay
A TUNEL ass ay was performe d using an In Situ Cel l Detection Ki t,
TMRred(12156792910)accordingtothemanufacturer'sinstructions.
Briefly, fibroblasts (2 × 104/well) wereplated ina Millicell EZ SLIDE
8-Wellglassslide (MerckMillipore Ltd.). Af ter fixationwith 4%para-
formaldehyde, the fibroblasts were treated with a permeabilisation
solution(0.1%TritonX-100in0.1%sodium citrate) for 2 minutes on
ice and then incubated with 100 µLo fTUNELre ac tionm ix tu reat37 °C 
for 1 hourin the dark. Nuclei werelabelled with DAPI, and TUNEL-
positive cells were analysed by a laser scanning confocal microscopy
(LeicaSP8).Theapoptoticindex(AI)wasdefinedasthepercentageof
TUNEL-positivecellsamongthetotalnumberofDAPI-stainedcells.
2.9 | Immunofluorescence staining
The fibroblast s were fixed with 4%paraformaldehyde for 20 min-
utesat37°C,permeabilised with 0.3% TritonX-100,andincubated
with primary antibodies against Ski, p-Smad2 and p-Smad3 (1:100) at
4°C overnight. After several washes with PBS, the cells were treated
with a Cy3-conjugated goat anti-rabbit antibody (1:500, Abcam) for
1hourat37°C.ThenucleiwerestainedwithDAPI.Theresultswere
captured by a laser sc anning confocal microscopy and analysed using
Image-ProPlus6.0.
2.10 | Western blotting and immunoprecipitation
(IP)
The lysates were immunoprecipitated with specific primary antibod-
ies overnight and incubated with Protein A/G Plus A garose beads
for 4 hours at 4°C. Total cell lysates or immunoprecipitates were
subjectedtoSDS-polyacrylamidegelelectrophoresis,electrotrans-
ferredonto polyvinylidenefluoride(PVDF)membranes (Millipore),
and probed with the indicated antibodies. The membranes were
washed with TBST and incubated with a goat anti-rabbit second-
aryantibodyconjugated with horseradishperoxidase anddetected
with a chemiluminescence substrate (ECL, Amersham Biosciences).
GAPDH ex pression was mea sured in each sam ple to verify eq ual
protein loading.
2.11 | Statistical analysis
Each experiment was repeated at leastthree times, and for all the
quantit ative analyses re presented in hist ograms, the dat a are ex-
pressed as the mean values ±SDThesignificanceofdifferencesbe-
tweentreatedanduntreatedgroupswasassessedusingStudent'st
test.One-wayanalysisofvariance(ANOVA)wasusedfordatawith
onevariableandmultipleconditions,followedbyDunnett'sposthoc
tests. Differenceswereconsideredstatistically significant when P
values were less than 0.05 (P < .05).
3 | RESULTS
3.1 | HG inhibited the proliferation and promoted
apoptosis of rat primary fibroblasts, decreased Ski
and PCNA protein levels, and increased p27, cleaved
caspase-3 and Bim protein levels
To examine the ef fects of HG on t he proliferat ion and apopto-
sis of fibroblasts, we first used theCCK-8kit to detect that HG
significantly reduced primary fibroblast viability compared with
NGandHMcontrols(Figure1A).Second,EdUandTUNELassays
found thatHG not onlyinhibitedthe proliferation of fibroblasts
(Figure 1B an d C) but also prom oted their apo ptosis (Figure 1D
andE).WBfoundthatHGsignificantlyreducedtheproteinlevels
ofSkiprotein(Figure1F-I)andPCNA(Figure1HandI)butsignifi-
cantly increased the protein levels of p27, cleaved caspase-3 and
Bim(Figure1HandI).
3.2 | Increasing Ski protein levels alleviated HG-
induced proliferation inhibition and apoptosis
promotion in fibroblasts without affecting the level of
Smad2/3 phosphorylation
Toexaminetheroleofincreasing Ski protein levelsintheinhibi-
tion of fibroblast proliferation and the increase in apoptosis caused
by HG, we use d an adenovir us transfec tion metho d. The result s
showed that Ad-Ski transfection significantly increased Ski protein
levelsinadose-dependentmannerunderHGconditions(Figure2A
and B), and significantly increased the viability and proliferation ca-
pacity of fibroblasts under SG conditions. Ad-Ski transfection also
significantly improved the inhibitory effect of HG on fibroblast
viability and proliferation capacity (Figure 2C-E)andsignificantly
reduced t he HG-induce d pro-apoptoti c effect (F igure 2D and F).
HGdid notaffecttotalSmad2/3proteinlevels,but itsignificantly
increased the levels of p-Smad2/3. Ad-Ski transfection had no sig-
nificanteffectontotalSmad2/3proteinorp-Smad2/3(Figure2G
andH,FigureS1).
3.3 | FoxO1 inhibition abolished the improvement
observed with increased Ski protein levels on HG-
induced proliferation inhibition and apoptosis
promotion in fibroblasts
We examined whether increasing Ski protein levels, which sig-
nificantly improved the proliferation inhibition and pro-apop-
totic eff ect of HG on fibr oblasts , was related to FoxO1. FoxO1
siRNA inhibited FoxO1protein levels in a dose-dependent man-
ner(Figure3A).Theinhibitionof FoxO1proteinlevelsdid notaf-
fecttheviabilityorproliferationcapacityoffibroblastsunderNG
conditions, but it significantly improved the viability and prolif-
erationcapacityoffibroblast sunderHGconditions(Figure3B-D).

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FIGURE 3 AfterinhibitionofFoxO1,
the improvement observed with increased
SkiproteinlevelsonHG-induced
proliferation inhibition and apoptosis
promotion in fibroblasts was lost.
FibroblastsweretransfectedwithFoxO1
siRNAorNCsiRNAfor24hoursand
theninfectedwithAd-SkiorAd-EGFP,
asindicated,foranother48hoursinthe
presenceofNGorHGconditions.A ,
FoxO1levelsweredetectedbyWestern
blot analysis. The data are presented as
the mean ±SEM,andeachexperiment
was repeated three times. *P < .05,
**P < .01, ***P <.001comparedtotheNC
siRNAgroup,one-wayANOVA,Dunnett's
posthoctests.B,ACCK-8assaywas
used to evaluate fibroblast viability. n =8,
*P < .05, **P < .01, ***P < .001 compared
totheNG+Ad-EGFP+NCsiRNA
group; #P < .05, ##P < .01, ###P < .001
comparedtotheHG+Ad-EGFP+NC
siRNAgroup,one-wayANOVA,Dunnett's
posthoctests.Fibroblastproliferation
ability was assessed by an EdU assay (C),
and fibroblast apoptosis was tested by a
TUNELassay(D).n=8,Corresponding
quantitativeanalysesareshown(D)and
(E).EdUandTUNELstaining,red;nuclei,
blue. *P < .05, **P < .01, ***P < .001
comparedtotheNG+Ad-EGFP+NC
siRNAgroup;#P < .05, ##P < .01,
###P <.001comparedtotheHG+ Ad-
EGFP+NCsiRNAgroup;&P < .01
comparedtotheHG+ Ad-Ski +NCsiRNA
group,one-wayANOVA,Dunnett'spost
hoc test s. scale bars = 10 0 µm

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Ad-Ski transfection had no significant effect on the viability or
proliferationcapacityoffibroblastscausedbytheFoxO1interfer-
enceunder HG conditions (Figure3B-D).Similarly,FoxO1 siRNA
inhibitedtheHG-inducedpro-apoptoticeffectsonfibroblasts,but
increasing Ski levels had no significant effect on HG-inducedfi-
broblastapoptosis(Figure3CandE).
3.4 | Increasing Ski protein levels under HG
conditions increased the binding of Ski to FoxO1 and
thus inhibited FoxO1 acetylation
AcetylationisanimportantactivationformofFoxO1,anditsacetylation
levelsincreaseunderHGconditions.16WefoundthattotalFoxO1lev-
elswerenotsignificantlychangedunderHGconditions,andincreasing
SkiproteinlevelsdidnotsignificantlyaffectFoxO1 levels (Figure4A).
Co-IP experiment s showed an interaction between Ski and FoxO1
after Ad-Ski transfection under SG conditions, and the combination of
SkiandFoxO1increasedsignificantlyunderHGconditions(Figure4B
andC).WefoundthatAc-FoxO1wassignificantlyincreasedunderHG
conditions, but its acetylation level was significantly decreased after
Ad-SkitransfectionunderNGandHGconditions(Figure4A).
3.5 | HG conditions increased Ski protein
levels and promoted the binding of Ski to HDAC1,
while inhibition of HDAC1 significantly reduced the
inhibitory effect of high Ski protein expression on
Ac-FoxO1
HDAC1 deacetylation of nuclear transcription factors is a clas-
sic regulation mode of Ski.Wefound that Ski and HDAC1 mutu-
allyboundafter Ad-Ski transfectionunderNGconditions, and the
combin ationofthet wosignificant lyi mprovedunderHGcondi tions
(Figure 5A and B). We confirmed that FoxO1 siRNA inhibited
FoxO1proteinlevelsinadose-dependentmanner(Figure 5C),but
HDAC1 siRNA tra nsfection alon e or co-transfect ion with Ad-Ski
had no signi ficant effec t on fibroblast v iability under H G condi-
tions(Figure5D).WBresultsshowedthatHDAC1siRNAtransfec-
tionhadnosignificanteffectonAc-FoxO1protein,butitreversed
thealleviatingef fect of Ad-Ski onAc-FoxO1underHGconditions
(Figure5E).Therewasnosignificantef fectonthetotalSkiprotein
levelunder NG orHGconditionsandregardlessof HDAC1siRNA
transfection(Figure5F).
4 | DISCUSSION
The present study found that the proliferation of primary fi-
broblasts from the skin was significantly reduced under HG
conditions, and apoptosis was significantly increased, which
is consiste nt with a previous rep ort indicati ng that HG caused
a decrease in fibroblast proliferation and an increase in apop-
tosis.4,17,18WealsofoundforthefirsttimethatSkiproteinex-
pressio nw as significant ly decreased un der HG conditio ns. Our
results, in combination with previous findings that Ski promoted
fibroblast proliferation and inhibited apoptosis,13,14 suggest that
this protein may be one cause of decreased fibroblast prolifera-
tion and in creased apopto sis under HG condi tions and why di-
abetic wounds are difficult to heal. This result is similar to our
previousfindingthatreducedSkiproteinexpressioninfibroblasts
was the main reason for slow wound healing in combined radia-
tion injury.19 In contrast, adenovirus transfection significantly
increasedSkiprotein expression andalsosignificantlyimproved
theeffectof HG onthe inhibitionoffibroblastproliferationand
theinductionofapoptosis.Especiallycomparedwith NGcondi-
tions, the proliferation efficiency of Ski increased from 25% to
37%und erHGcon ditio ns,wh ic hindi cates thatthe de cre aseinSk i
FIGURE 4 IncreasingSkiproteinlevelsunderHGconditionsincreasedthebindingofSkitoFoxO1andthusinhibitedacet ylationof
FoxO1.FibroblastswereinfectedwithAd-SkiorAd-EGFPfor48hours,combinedwithHGtreatmentfor48hours.A ,IPwithananti-FoxO1
antibodyfollowedbyimmunoblottingwithanti-acetyl-lysine(ac-lysine)ortotalFoxO1antibodies.B,C ,IPofSkiandFoxO1usingananti-Ski
oranti-FoxO1antibodyastheIPantibodyandWesternblottingwithananti-FoxO1oranti-Skiantibody.RabbitIgGservedasanegative
control.Inputisthetotalcelllysateofthecontrolwithoutanti-FoxO1oranti-Skiimmunoprecipitation.Thedataarepresentedasthe
mean ±SEM,andeachexperimentwasrepeatedthreetimes.*P < .05, **P < .01, ***P <.001comparedtotheDMEM+NGgroup;#P < .05,
##P < .01, ###P <.001comparedtotheDMEM+HGgroup,one-wayANOVA,Dunnett'sposthoctest s

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levelsunderthepathologicalconditionofHGisanimport antrea-
son for the decrease in fibroblast proliferation and the increase
inapoptosis.TheseresultsalsosuggestthathighSki expression
has a power ful effec t on the HG-indu ced changes in fi broblast
proliferation and apoptosis, which may become a new target and
newstrategyforthetreatmentofdiabeticrefractorywounds.Of
course, the actual effects of this hypothesis must be confirmed in
furtherexperiments.
Previous studies showed that the ability of Ski to promote fibro-
blast proliferation and inhibit apoptosis was associated with the inhi-
bition of TGF-β1/Smad signalling.13,14 In contrast, the present study
found thatSmad2/3 activityin skin fibroblasts underHG conditions
wassignificantly dif ferent fromNGconditions,but the increased Ski
lev elsunderH Gcondit io nshadno significantef fe ctonSmad2/3activ-
ity, which sug gests that the ef fect of increasing Ski levels on promoting
proliferation and inhibiting apoptosis in fibroblasts may not be medi-
atedbytheTGF-β1/SmadsignallingpathwayunderHGconditions.
InadditiontoTGF-β1/Smad playing a role in the regulation of fi-
broblast proliferation and apoptosis, an increasing number of studies
showedthatthetranscriptionfactorforkheadboxO1(FoxO1)was
also involved in the regulation of fibroblast proliferation and apop-
tosis.20 FoxO1, as a mul tifunctio nal transcri ption facto r, is closely
related tocellapoptosis,proliferation,oxidative stress,energyme-
tabolism, etc21,22Increased FoxO1activityin diabetic mice23 plays
a key role in wound healing.24 Therefore, regulating the effects of
FoxO1oncellproliferationandapoptosisisanimportantwaytopro-
mote diabetic wound healing.25
We found for the first time that increasing Ski levels promoted
thebindingofFoxO1anddecreasedtheacetylationlevelofFoxO1.
The cell proliferation rate increases and the apoptosis rate decreases
FIGURE 5 IncreasingSkiproteinlevelsunderHGconditionspromotedthebindingofSkitoHDAC1,andtheinhibitionofHDAC1
significantlyreducedtheinhibitor yeffectofhighSkiproteinexpressiononAc-FoxO1.FibroblastsweretransfectedwithHDAC1siRNA
orNCsiRNAfor24hoursandtheninfectedwithAd-SkiorAd-EGFP,asindicated,foranother48hoursinthepresenceofNGorHG
conditions.A,B,IPofSkiandHDAC1usingananti-Skioranti-HDAC1antibodyastheIPantibodyandWesternblottingwithananti-HDAC1
oranti-Skiantibody.RabbitIgGser vedasanegativecontrol,andinputisthetotalcelllysateofthecontrolwithoutanti-HDAC1oranti-
Skiimmunoprecipitation.C,HDAC1levelsweredetectedbyWesternblotanalysis.n= 3, *P < .05, **P < .01, ***P < .001 compared to the
NCsiRNAgroup,one-wayANOVA,Dunnett'sposthoctests.D,ACCK-8assaywasusedtoevaluatefibroblastviability.n=8,*P < .05,
**P < .01, ***P <.001comparedtotheDMEM+NGgroup;#P < .05, ##P < .01, ###P <.001comparedtotheDMEM+NCsiRNA+HGgroup,
one-wayANOVA,Dunnett'sposthoctests.E,IPwithananti-FoxO1antibodyfollowedbyimmunoblottingwithanti-acetyl-lysine(ac-lysine)
ortotalFoxO1antibodies.F,WesternblotanalysisofSkiproteinexpressionlevels.Thedataarepresentedasthemean± SEM, and each
experimentwasrepeatedthreetimes.*P < .05, **P < .01, ***P <.001comparedtotheDMEM+NGgroup;#P < .05, ##P < .01, ###P < .001
comparedtotheDMEM+NCsiRNA+HGgroup,one-wayANOVA,Dunnett'sposthoctest s

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after FoxO1 acetylat ion decreases und er HG conditions, 25 which
may underlie the role of Ski in promoting proliferation and inhibiting
apoptosisinskinfibroblastsunderHGconditions.Notably,theinhi-
bition ofFoxO1withRNAi alone is equivalentto,orevenstronger
than that of increasing Ski protein levels, which improved the effec ts
ofHG-inducedpromotionofproliferationandinhibitionofapoptosis
in skin fibroblasts. It is suggested that increasing the Ski protein level
may promote proliferation and inhibit apoptosis in skin fibroblasts by
decreasingFoxO1acetylationlevels.Thishypothesiswassupported
by one stud y that reported that the cell apoptosis rate decreased and
thecellproliferationrateincreasedafterHGdeacetylatedFoxO1.25
FoxO1isgenerallyacet ylated bylysineacetyltransferases(KATs)
and deacetylated by histone deacetylases (HDACs).26,27 Histone
dea cet yl ase1(HDAC1)isamemberofth ehistonedeacet yl ationf am-
ily,whichregulatesthelevelofFoxO1.28,29HDAC1preventsth egene
transcription protein complex from entering the promoter binding
site by removing the acetyl group on histones, which inhibits gene
transcription and participates in the regulation of cell growth, differ-
entiation, proliferation and apoptosis. The present study found that
increasing the Ski level significantly increased the binding of Ski to
HDAC1under HG conditions and significantlyreduced the acetyla-
tion level of FoxO1.It is suggested thatSki formed a complex with
FoxO1andHDAC1toenhancethedeacet ylationeffectofHDAC1on
FoxO1,whichissimilartoSkicooperationwithHDAC1toplayitsclas-
sicauxiliaryregulatoryrole.30AlthoughthereisnoreportofHDAC1
regulating the proliferation or apoptosis of diabetic skin fibroblasts,
HDACinhibitorsareoftenusedasatreatmentfo rcardiacf ibrosisand
tumours,31,32 which also shows that it is possible for Ski to regulate
FoxO1throughHDAC1.Inaddition,thisstudyfoundthatHDAC1in-
hibitionalonehadlittleeffectontheacetylationofFoxO1,whichmay
berelated tothe factthatthedeacetylation ofHDAC1requires the
cooperation of the nuclear cofactor Ski.33 The present study found
asignificant reduction inSkiexpressionunderHGconditions, which
providedthebasisforHDAC1'sinabilitytoperformdeacetylation.
The present study did not find that Ski interacted with FoxO1
orHDAC1whenthe Ski levelwasnotincreased. In contrast, after
increasi ng Ski protein leve ls, Ski inter acted with FoxO1or HDAC1
under SG a nd HG conditions. B ecause Ski is gene rally express ed
primarily in the nucleus, and the cytoplasmic distribution of Ski in-
creased w hen it was expr essed at high l evels (Figure 2 A). HDAC1
is generally distributed in the cytoplasm, which indicates that the
binding abilityofSkito FoxO1orHDAC1is notincreasedafterthe
increase in Ski protein level, but the distribution of Ski in fibroblasts
changes after high expressionofSki,whichprovidesthepossibilit y
ofSkibindingwithFoxO1orHDAC1.
Overall,the present studyfound for thefirsttime that HGre-
duced Ski protein levels and increasing Ski protein levels significantly
improved the fibroblast proliferation inhibition and apoptosis induc-
tion observed under HG conditions. The underlying mechanism of
action is notrelated to the Smad2/3pathway.However,this func-
tion may be ac hieved by the direc t combination wit h FoxO1 and
HDAC1toformaheteromertopromotethedeacetylationofFoxO1
andreduceitsactivity(Figure6).Thisresulthasguidingsignificance
for direct intervention in fibroblast proliferation and apoptosis by
regulating Ski to promote the healing of diabetic refractory wounds,
and it reveals a new pathway and mechanism for Ski to regulate cell
proliferation and apoptosis.
CONFLICTS OF INTEREST
The authors declare no competing financial interests.
AUTHOR CONTRIBUTIONS
PLandYGZdesignedthestudy.YP,RPX,SWTandZHZresearched
thedata.YP,YLNandYZanalysedthedata.PLand YPdraftedthe
manuscript. The manuscript was reviewed by all authors.
DATA AVAIL ABI LIT Y S TATEM ENT
The data that support the findings of this study are available from
the corresponding author upon reasonable request.
ORCID
Ping Li https://orcid.org/0000-0002-4926-3835
FIGURE 6 SchematicrepresentationofSkiinhibitionoftheroleofFoxO1transcriptionfactorthroughFoxO1deacetylationmediatedby
HDAC1underHGconditions.UnderHGconditions,FoxO1acetylationlevelwasincreased.Asatranscriptionfac tor,FoxO1involvedinthe
induction of a special subset of genes that regulate cellular proliferation or apoptosis, etc Increased Ski protein increased the binding of Ski
toFoxO1,whichmediateddeacetylationthroughSkibindingwithHDAC1andresultedinareductioninFoxO1acetylationlevel.Therefore,
theroleofFoxO1transcriptionfactorwasinhibited

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SUPPORTING INFORMATION
Additional suppor ting information may be found online in the
Supporting Information section.
How to cite this article:PengY,XiongR-P,ZhangZ-H,etal.Ski
promotes proliferation and inhibits apoptosis in fibroblasts
underhigh-glucoseconditionsviatheFoxO1pathway.Cell
Prolif. 2021;54:e12971. https ://doi.or g/10.1111/cpr.12971