Role of glucose as a modulator of anabolic and catabolic gene expression in normal and ...

12 Pages·2011·1.44 MB·English

Cellular
A
RTICLE
Journal of
Biochemistry
JournalofCellularBiochemistry112:2813–2824(2011)
Role of Glucose as a Modulator of Anabolic and
Catabolic Gene Expression in Normal and
Osteoarthritic Human Chondrocytes
Susana C. Rosa,1,2 Ana T. Rufino,1,2 Fernando M. Judas,3,4 Carlos M. Tenreiro,5
Maria C. Lopes,1,2 and Alexandrina F. Mendes1,2*
1Center for Neuroscience and CellBiology, Universityof Coimbra, LargoMarques de Pombal, 3004-517 Coimbra,
Portugal
2Faculty of Pharmacy, Universityof Coimbra, Po´lodas Cieˆncias da Sau´de,Azinhaga de Santa Comba,
3000-548Coimbra, Portugal
3OrthopedicsDepartment,University Hospital of Coimbra, Avenida Bissaya Barreto,Bloco de Celas,
3000-075Coimbra, Portugal
4Faculty of Medicine,University of Coimbra,RuaLarga,3004-504 Coimbra,Portugal
5CMUC, Departmentof Mathematics,University of Coimbra,Apartado 3008, 3001-454 Coimbra,Portugal
ABSTRACT
Cartilagematrixhomeostasisinvolvesadynamicbalancebetweennumeroussignalsthatmodulatechondrocytefunctions.Thisstudyaimed
at elucidating the role of the extracellular glucose concentration in modulating anabolic and catabolic gene expression in normal and
osteoarthritic(OA)humanchondrocytesanditsabilitytomodifythegeneexpressionresponsesinducedbypro-anabolicstimuli,namely
TransformingGrowthFactor-b(TGF).Forthis,weanalyzedbyrealtimeRT-PCRtheexpressionofarticularcartilagematrix-specificandnon-
specificgenes,namelycollagentypesIIandI,respectively.Theexpressionofthematrixmetalloproteinases(MMPs)-1and-13,whichplaysa
majorroleincartilagedegradationinarthriticconditions,andoftheirtissueinhibitors(TIMP)wasalsomeasured.Theresultsshowedthat
exposure to high glucose (30mM) increased the mRNA levels of both MMPs in OA chondrocytes, whereas in normal ones only MMP-1
increased.CollagenIImRNAwassimilarlyincreasedinnormalandOAchondrocytes,buttheincreaselastedlongerinthelater.Exposureto
high glucose for 24h prevented TGF-induced downregulation of MMP-13 gene expression in normal and OA chondrocytes, while the
inhibitoryeffectofTGFonMMP-1expressionwasonlypartiallyreduced.Otherresponseswerenotsignificantlymodified.Inconclusion,
exposureofhumanchondrocytestohighglucose,asoccursinvivoindiabetesmellituspatientsandinvitrofortheproductionofengineered
cartilage,favorsthechondrocytecatabolicprogram.Thismaypromotearticularcartilagedegradation,facilitatingOAdevelopmentand/or
progression,aswellascompromisethequalityandconsequentinvivoefficacyoftissueengineeredcartilage.J.Cell.Biochem. 112:2813–
2824, 2011. (cid:1)2011Wiley-Liss,Inc.
KEY WORDS:
COLLAGEN;GENEEXPRESSION;GLUCOSE;HUMANCHONDROCYTE;MMP;OSTEOARTHRITIS;TIMP
A
rticular cartilage is a specialized connective tissue that maintainingitshomeostasisbyensuringthesynthesisandturnover
supportsanddistributesloadsandensuresanear-friction- ofitscomponents[Martel-Pelletieretal.,2008;GoldringandMarcu,
less motion in joints. These unique properties are due to the 2009].
structural organization of the main macromolecules that compose Cartilage matrix homeostasis involves a dynamic balance
thecartilageextracellularmatrix,namelycollagensandproteogly- betweenavarietyofsignalsthatmodulatechondrocytefunctions,
cans.Chondrocytes,theonlycelltypepresentinarticularcartilage, namelymechanicalforces,cytokinesandgrowthfactorsandcell–
are embedded in the extracellular matrix and are responsible for matrixinteractions,somefavoringananabolicprogramandothers
Grantsponsor:PortugueseFoundationforScienceandTechnology(FCT);Grantnumber:PTDC/SAU-OSM/67936/
2006;Grantsponsor:FCT;Grantnumbers:SFRH/BD/19763/2004,SFRH/BD/47470/2008.
*Correspondenceto:AlexandrinaF.Mendes,CenterforNeuroscienceandCellBiology,LargoMarquesdePombal,
2813
3004-517Coimbra,Portugal.E-mail:[email protected]
Received16September2010;Accepted13May2011 (cid:1)DOI10.1002/jcb.23196 (cid:1)(cid:1)2011Wiley-Liss,Inc.
Publishedonline23May2011inWileyOnlineLibrary(wileyonlinelibrary.com).
stimulating catabolic responses. Aging and mechanical stress of canactinarticularchondrocytesinanautocrineand/orparacrine
joints are major risk factors for osteoarthritis (OA), but growing way, inducing anabolic effects and inhibiting catabolic responses.
evidenceindicatesthatmetabolicfactorsplayanimportantrolein Morespecifically,TGFhasbeenshowntostimulateproteoglycans,
disease development and progression. For instance, a significant collagentypeII[Miyazakietal.,2000;Martel-Pelletieretal.,2008]
positivecorrelationwasfoundbetweenelevatedbloodglucoseand andTIMPsexpression[Wangetal.,2002],whilereversingsomeof
radiologicalevidenceofOA[Hartetal.,1995].Accordingly,OAis theIL-1-induced catabolic effects [Seifarth et al., 2009].
increasinglyenvisagedasa‘‘metabolicdisorder’’linkedtoobesity, Besidesthesewell-characterizedstimuli,chondrocytesrespondto
diabetesmellitus,dyslipidemia,hypertension,andinsulinresistance andareinfluencedbyothersignals.Amongthese,theextracellular
[Sturmer et al., 2001; Burner and Rosenthal, 2009; Siviero et al., glucose concentration, either increased or decreased, has been
2009; Palluet al., 2010; Velasquez andKatz, 2010]. showntodirectlyaffectsomechondrocytefunctions[Kelleyetal.,
Theseconditionsseemtofavortheinitiationandprogressionof 1999;Richardsonetal.,2003;McNultyetal.,2005].Inparticular,
arthritic diseases, such as OA, by providing catabolic signals that culture of rabbit chondrocytes in either low (<5mM) or high
lead,amongotherresponses,toincreasedproductionofproteolytic (25mM) glucose concentrations decreased proteoglycan synthesis
enzymes.Thesedegradethematrixcomponentscausingprogressive induced by the anabolic growth factor, IGF-I [Kelley et al., 1999].
cartilagedestruction,themainfeatureofOA[Martel-Pelletieretal., Exposure to increasing glucose concentrations, dose-dependently
2008; GoldringandMarcu, 2009]. decreased dehydroascorbate uptake by human chondrocytes
Among the proteolytic enzymes, aggrecanases and colagenases [McNulty et al., 2005], which may have functional implications
which belongto the Matrix MetalloProteases (MMPs)family are of as dehydroascorbate is essential for collagen synthesis. Moreover,
special importance [Arner, 2002; Nagase and Kashiwagi, 2003; ourpreviousstudyshowedthatnormalhumanchondrocytesadjust
Burrageetal.,2006].Collagenasescleavecollagentriplehelixfibers, tochangesintheextracellularglucoseconcentrationbyregulating
leaving them susceptible to further degradation by other enzymes. glucoseuptake,atleastinpart,throughmodulationofthecellular
Human articular chondrocytes have been shown to express several content of the Glucose Transporter (GLUT)-1 [Rosa et al., 2009], a
collagenases,amongwhichcollagenase-1(MMP-1),-2(MMP-8),and memberoftheGLUT/SLC2Afacilitativeglucosetransporterfamily.
-3 (MMP-13) are especially relevant since their expression is Moreover,thatstudyalsoshowedthatOAchondrocytesexposedto
increased by pro-inflammatory and catabolic stimuli [Arner, 2002; a high, hyperglycemia-like extracellular glucose concentration
NagaseandKashiwagi,2003;Burrageetal.,2006].MMP-13,whose (30mM) were unable to reduce glucose transport and GLUT-1
expression has been shown to be augmented in OA cartilage [Bau contentwhichledtoincreasedglucoseaccumulationandprolonged
etal.,2002;Kevorkianetal.,2004],hasasignificantroleincartilage Reactive Oxygen Species (ROS) production [Rosa et al., 2009].
degradationandOAprogressionsinceitpreferentiallycleavestypeII Increased oxidative stress has been pointed out as a major
collagenfibers[Takaishietal.,2008].MMP-1,alsoparticipatesinthe pathogenic mechanism of OA [Maneiro et al., 2003; Grishko
initialcleavageofcollagenII,butwithaloweractivitythanMMP-13 etal.,2009],andatthecellularlevel,increasedROSproductionhas
[Mitchelletal.,1996].TypeIIcollagenisamajormacromoleculeof been showntomediate manycatabolic responses in chondrocytes
theextracellularmatrixthatconfersstructuralsupporttocartilage.Its [Lo et al.,1998; Mendes et al., 2003a,b].
enhanceddegradationandconsequentlosswithassociatedcartilage Thus,thisstudywasundertakentodeterminewhetherexposure
erosion is a key characteristic of OA pathophysiology. Allied to toahighextracellularglucoseconcentrationcanshiftnormaland
increased matrix degradation, OA is characterized by a shift in the OA chondrocyte functions towards a pro-catabolic and/or anti-
chondrocyteanabolicactivity,sothatcollagenIIsynthesisisreplaced anabolic gene expression profile, either direct or indirectly by
byincreasedproductionofthenon-cartilagematrixspecificcollagen modifying the responses induced by pro-anabolic stimuli, namely
typeI.Thisleadstotheformationofafibrocartilaginoustissuewhich TGF.
isbiomechanicallylesseffectivethanarticularcartilage[Miosgeetal., Forthispurpose,weexaminedbyrealtimeRT-PCR(qRT-PCR)the
2004;TescheandMiosge,2005]. expressionofgenesthatareimportantforcartilagehomeostasisand
The activity of MMPs can be negatively regulated at the post- OApathogenesisandthatinothercelltypeshavebeenreportedtobe
transcriptionallevelbymembersofthefamilyofTissueInhibitorsof modulatedbyexposuretohighglucose[Deathetal.,2003;Hoetal.,
MetalloProteases (TIMPs) [Baker et al., 2002]. Among the TIMPs 2007;Andreeaetal.,2008;Kimetal.,2008].Thosegenesincludethe
identifiedinhumanchondrocytes,TIMP-2hasbeenreportedtobe MMPs-1 and -13 due to their major role in cartilage matrix
constitutively expressed and therefore suggested to have a role in degradation,andcollagentypeII,TIMP-1andTIMP-2asimportant
themaintenanceofcartilageintegrityinnormalconditions,while anabolicandanti-catabolicgenes,respectively.CollagentypeIwas
TIMP-1 and TIMP-3 seem to have a more important role in alsoassessed since itisfrequently reportedtobeincreased inOA,
pathological conditions [Zafarullah et al., 1996; Su et al., 1999]. representing the inability of OA chondrocytes to promote an
Highratiosofproteinase/inhibitorsfavorproteolysisandhavebeen adequatecartilage matrixrepair response [Miosge et al., 2004].
found in thesynovial fluidof OApatients [Ishiguro et al., 1999].
MATERIALS AND METHODS
MembersoftheMMPandTIMPfamiliesareinverselyregulatedat
the transcriptional level by catabolic stimuli, such as the pro-
inflammatorycytokines,Interleukin-1b(IL-1)andTumorNecrosis CARTILAGESAMPLES ANDCHONDROCYTECULTURE
Factor-a(TNF),andbyanabolicgrowthfactors,namelyTransform- Humankneecartilagewascollected,within12hofdeath,fromthe
ingGrowthFactor-b(TGF).Thelastisespeciallyimportantsinceit distal femoral condyles of multi-organ donors (18–40 years old,
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mean¼32,n¼7,normalcartilage)orwithinformedconsentfrom andanelongationstepat728Cfor30s.Fluorescencemeasurements
patients (50–71 years old, mean¼64, n¼11, OA cartilage) were taken every cycle at the end of the annealing step and the
undergoing total knee replacement surgery at the Orthopedic specificityoftheamplificationproductswascheckedbyanalysisof
DepartmentoftheUniversityHospitalofCoimbra(HUC).TheEthics the melting curve. The efficiency of theamplification reaction for
CommitteeofHUCapprovedallprocedures.Cartilagefromallmulti- each gene was calculated by running a standard curve of serially
organ donors appeared grossly normal without macroscopic dilutedcDNAsample.Ineachassay,acontrolreactionwithoutthe
changes other than some yellowish discoloration and, in a few cDNA wasalsosubjected toPCR amplification.
samples (aged >35 years old), small areas of slightly roughened Gene expression changes were analyzed using the built-in iQ5
surface. Chondrocytes were isolated by enzymatic digestion as Opticalsystemsoftwarev2,whichenablestheanalysisoftheresults
described previously [Rosa et al., 2009]. Non-proliferating mono- withthePfafflmethod,avariationoftheDDCTmethodcorrectedfor
layercultureswereestablishedfromeachcartilagesample,allowed gene-specific efficiencies.
to recover in medium containing 5% fetal bovine serum for 24h,
serum-starved overnight and maintained thereafter in serum-free STATISTICAL ANALYSIS
culture medium. The cells were subsequently cultured, for the In Figure 1, the nonparametric Mann–Whitney test was used to
periods indicated in figures and figure legends, in Ham’s F-12 comparethedistributionsofthebasalexpressionofcatabolicand
(RegularGlucoseMedium,whichcontains10mMglucose)orinthe anabolicgenesinnormalandOAhumanchondrocytes.Inallother
samemediumsupplementedwith20mMD-glucosetoyieldafinal figures,theresultsarepresentedasthegeneexpressionlevelrelative
glucoseconcentrationof30mM(HighGlucoseMedium).Inselected tothecontrolsituation(normalorOAchondrocytesmaintainedin
experiments,recombinanthumanTGF-b(Peprotech,RockyHill,NJ) RGM).Thestatisticalanalysiswasperformedusingthepairedtwo-
wasusedin thefinal concentration of10ng/ml. tailedStudentt-testtocomparedifferencesbetweenthecontroland
thetestsituationorbetweentheresponsesinducedbytreatmentof
TOTALRNA EXTRACTION ANDcDNA PREPARATION chondrocyteswithTGFinRGMandinHGM,forthesameincubation
Total RNA was extracted with TRIzol (Invitrogen, Paisley, UK), period.Toassessnormalityfortheobserveddifferences,agraphical
analyzed using Experion RNA StdSens Chip (Bio-Rad) and analysis based on normal quantile plots was used in all cases,
quantifiedinaNanoDropND-1000Spectrophotometer(NanoDrop showing no strong departure from normality, thus supporting the
Technologies, Inc., Wilmington, DE) at 260nm. The cDNA was use of the Student t-test. Results were considered statistically
reversetranscribedfrom1mgoftotalRNA,usingiScriptTMSelect significant for P<0.05.
cDNA Synthesis Kit (Bio-Rad), according to the manufacturer’s
instructions.ThecDNAobtainedwasstoredat(cid:2)208Cuntilfurther RESULTS
analysis.
BASAL EXPRESSIONOF ANABOLIC ANDCATABOLICGENES IN
REAL-TIMEREVERSE TRANSCRIPTASE-PCR(qRT-PCR) NORMAL ANDOAHUMANCHONDROCYTES
Specific sets of primers were designed using Beacon Designer The expression of MMPs-1and-13, TIMPs-1and -2andcollagen
software(PREMIERBiosoftInternational,PaloAlto,CA).Detailsof typesIandIIwasanalyzedbyqRT-PCRacrosscDNAstranscribed
theforwardandreverseprimersforthehumangenesevaluatedare fromtotalRNAextractedfromchondrocyteculturesobtainedfrom
presented in Table I. qRT-PCR was performed with iTaqTM DNA 7normaland11OAhumankneecartilagesamples.Previousstudies
polymerase using iQTM SYBR Green Supermix (BioRad). Thermal showedthatHPRT-1wasthecandidatehousekeepinggene,among
cycling conditions included 3min at 958C to activate the iTaqTM seventested,whoseexpressionvariedleastbetweennormalandOA
DNA polymerase, followed by 45 cycles, each consisting of a chondrocytes.Accordingly,HPRT-1wasusedasreferencegenefor
denaturationstepat958Cfor10s,anannealingstepat548Cfor30s comparisonsofmRNAlevelsbetweennormalandOAchondrocytes.
TABLEI. Oligonucleotide Primer PairsUsed for qRT-PCR
Gene name Primer sequences (50–30) RefSeq ID
Hypoxanthinephosphoribosyltransferase-1(HPRT-1) F:TGACACTGGCAAAACAAT NM_000194
R:GGCTTATATCCAACACTTCG
MatrixMetalloprotease-1(MMP-1) F:GACTCTCCCATTCTACTG NM_002421
R:TTATAGCATCAAAGGTTAGC
MatrixMetalloprotease-13(MMP-13) F:GTTTCCTATCTACACCTACAC NM_002427
R:CTCGGAGACTGGTAATGG
Tissueinhibitorofmetalloproteinase-1(TIMP-1) F:TGTTGCTGTGGCTGATAG NM_003254
R:CTGGTATAAGGTGGTCTGG
Tissueinhibitorofmetalloproteinase-2(TIMP-2) F:ACGATATACAGGCACATTATG NM_003255
R:GGTCAGGAGTCTTAACAGG
CollagenTypeI(COL1A1) F:GGAGGAGAGTCAGGAAGG NM_000088
R:GCAACACAGTTACACAAGG
CollagentypeII(COL2A1) F:GGCAGAGGTATAATGATAAGG NM_001844
R:ATTATGTCGTCGCAGAGG
F:Forwardsequence;R:Reversesequence.
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Fig.1. BasalexpressionofcatabolicandanabolicgenesinnormalandOAhumanchondrocytes.BoxandwhiskersplotsofthemRNAlevelsofMMPs-1(A)and-13(B),TIMPs-
1(C)and-2(D)andcollagensI(E)andII(F)innormal(n¼7)andOA(n¼11)chondrocytes.TheboxesrepresentthedistributionofmRNAlevelsbetweenthe25thand75th
percentiles,whilethebars(whiskers)representextremevaluesoutsidethatrange.Thelinedividingeachboxrepresentsthemedianofthedistribution,thatis,thecentralmRNA
valueforwhichthereisanequalnumberofsampleswithmRNAvaluesaboveandbelowthatone.(cid:4)P<0.05and(cid:4)(cid:4)P<0.01.
TheboxandwhiskersplotspresentedinFigure1AandBshow On the other hand, the expression of TIMP-1 was significantly
that, despite considerable interindividual variability and a few higher (approximately fourfold in average) in OA than in
outliers in both groups, basal MMP-1 and -13 mRNA levels were normal chondrocytes (Fig. 1C, P¼0.04). TIMP-2 mRNA levels,
significantlyhigherinOAthaninnormalchondrocytes.Onaverage, however, were only 1.7-fold higher in OA than in normal
MMP-1 and -13 mRNA levels were approximately five and eight chondrocytes and the difference did not reach statistical signifi-
times higher in OA (118,011.7(cid:3)39,681.4 and 11,926.9(cid:3)3,215.4) cance(Fig.1D, P¼0.54).
than in normal (24,243.6(cid:3)10,100.4 and 1,469.7(cid:3)694.9) chon- ConcerningthemRNAexpressionofcollagensIandII,atendency
drocytes, respectively. In all samples tested, either normal or OA, toincreasedlevelsofcollagenIIinnormalandtoelevatedlevelsof
MMP-1 was always detected at an earlier mean threshold cycle collagen I in OA chondrocytes was observed (Fig. 1E and F),
than MMP-13, indicating a higher basal expression level of the although those differences did not reach statistical significance.
former. Moreover,theratioofcollagenIIovercollagenImRNAlevels(Col
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II/Col I) was on average 3.5-fold higher in normal than in OA MMP-1 (Fig. 2B) and -13 (Fig. 2D) mRNA levels were similarly
chondrocytes, but again the difference did not reach statistical increasedbycultureinHGMfor24,48,and72h.Nonetheless,the
significance (P¼0.07). differences observed for MMP-1 at 24 and 48h did not reach
statistical significance.
ROLEOFHIGHGLUCOSEINMODULATINGTHEEXPRESSIONOFTHE
CATABOLICGENES, MMP-1 AND-13 ROLE OF HIGH GLUCOSEON TIMP-1AND-2EXPRESSION
The results obtained show that in normal chondrocytes, MMP-1 TheresultsshowninFigure3AindicatethattheexpressionofTIMP-
mRNAlevels(Fig.2A)relativetochondrocytesculturedinregular 1wasdecreasedbycultureofnormalchondrocytesinHGMfor48
glucosemedium(RGM,10mMglucose)decreaseduponexposureto and 72h, while no significant changes were found in OA
highglucose(HGM,30mMglucose)for48and72h,whileMMP-13 chondrocytes (Fig. 3B). TIMP-2 mRNA levels were significantly
mRNA levels (Fig. 2C) remained unchanged regardless of the upregulated in normal (Fig. 3C), but not in OA (Fig. 3D)
duration of exposure to high glucose. In OA chondrocytes, both chondrocytesuponexposuretohighglucosefor24h.Nosignificant
Fig.2. RoleofhighglucoseinmodulatingtheexpressionofMMP-1andMMP-13genesinnormalandOAchondrocytes.RelativeMMP-1(A,B)andMMP-13(C,D)mRNA
levelsinnormal(n¼5)andOA(n¼6)chondrocytesculturedinmediumwithanelevated(30mM)glucoseconcentration(HGM)for24,48,and72hormaintainedinmedium
withtheregularglucoseconcentration(RGM)fortheentireexperiment(normalandOAcontrols).Eachbarrepresentsthemean(cid:3)SD.(cid:4)P<0.05and(cid:4)(cid:4)(cid:4)P<0.001relativetothe
respectivecontrol.
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Fig.3. RoleofhighglucoseinmodulatingtheexpressionofTIMP-1andTIMP-2genesinnormalandOAchondrocytes.RelativeTIMP-1(A,B)andTIMP-2(C,D)mRNAlevels
innormal(n¼6)andOA(n¼6)chondrocytesculturedinmediumwithanelevated(30mM)glucoseconcentration(HGM)for24,48,and72hormaintainedinmediumwith
theregularglucoseconcentration(RGM)fortheentireexperiment(normalandOAcontrols).Eachbarrepresentsthemean(cid:3)SD.(cid:4)P<0.05relativetotherespectivecontrol.
differences relative to cells maintained in RGM were found at the normal and OA chondrocytes had returned to those found in the
other time points, either in normal (Fig. 3C) or OA chondrocytes respective control cellsmaintainedin RGM.
(Fig.3D).
ROLEOFHIGHGLUCOSEINMODULATINGMMP-1AND-13GENE
EXPRESSIONPROFILES INDUCED BYTGF INNORMALANDOA
ROLEOFHIGHGLUCOSEONCOLLAGENTYPEIANDIIEXPRESSION CHONDROCYTES
Figure4AandBshowsthatcultureofnormalandOAchondrocytes, To determine whether exposure to high glucose can modify
respectively,inHGMfor24,48,or72hdidnotsignificantlyaffect chondrocyte responses to TGF, the gene expression profiles of
collagen type I expression relative to the respective control cells MMP-1 and -13 were evaluated in normal and OA chondrocytes
maintainedin RGM. treatedwithTGFforvarioustimeperiodsinthepresenceorabsence
On the other hand, culture in HGM for 24h similarly increased ofhighglucose(HGM).TheresultswerenormalizedusingHPRT-1as
collagen II mRNA levels in normal (1.3(cid:3)0.07, Fig. 4C) and OA the housekeeping gene since its expression was not significantly
(1.3(cid:3)0.13, Fig. 4D) chondrocyte cultures. This increase, although affected bytreatment of normalandOAchondrocytes with TGF.
lower,wasstillsignificantafterincubationofOAchondrocytesin TreatmentwithTGFfor24hinducedamarkeddecreaseinMMP-1
HGMfor48h.After72h,however,collagenIImRNAlevelsbothin mRNAlevelsinnormal(0.31(cid:3)0.015,Fig.5A)andOA(0.38(cid:3)0.05,
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Fig.4. RoleofhighglucoseinmodulatingtheexpressionofcollagentypeIandIIgenesinnormalandOAchondrocytes.RelativecollagentypeI(A,B)andcollagentypeII
(C,D)mRNAlevelsinnormal(n¼5)andOA(n¼5)chondrocytesculturedinmediumwithanelevated(30mM)glucoseconcentration(HGM)for24,48,and72hor
maintainedinmediumwiththeregularglucoseconcentration(RGM)fortheentireexperiment(normalandOAcontrols).Eachbarrepresentsthemean(cid:3)SD.(cid:4)P<0.05and
(cid:4)(cid:4)P<0.01relativetotherespectivecontrol.
Fig.5B)chondrocytesrelativetotherespectivecontrolsmaintainedin identical.Indeed,innormalchondrocytes,exposuretohighglucose
RGM, which persisted up to, at least, 72h. Yet, exposure to high for 24hcompletely reversedtheeffect ofTGF onMMP-13mRNA
glucosefor24and72hsignificantlyreducedtheinhibitoryeffectof levels, but more prolonged culture periods under high glucose,
TGF on MMP-1 expression, either in normal or OA chondrocytes namely48and72h,nolongersignificantlyaffectedtheresponseto
(Fig. 5A and B, respectively), which corresponds to higher MMP-1 TGF.InOAchondrocytesculturedinHGMfor24h,theresponseto
mRNAlevels.At48h,nosignificantdifferenceswerefoundbetween TGF was completely abolished and still significant at 48h. Full
theresponsestoTGFtreatmentinthepresenceorabsenceofHGM, recoveryoftheabilityofTGFtoreduceMMP-13mRNAlevelsinthe
eitherinnormalorinOAchondrocytes. presence of HGMoccurred onlyuponculture for72h.
Moreover,treatmentofnormal(Fig.5C)andOA(Fig.5D)human
chondrocytes with TGF in RGM induced a marked and sustained ROLE OF HIGH GLUCOSEIN MODULATINGTGF-INDUCED TIMP-1
decrease in MMP-13 expression that was maximal at 72h AND -2GENE EXPRESSION INNORMALAND OACHONDROCYTES
(0.33(cid:3)0.11 and 0.25(cid:3)0.09, respectively). Under high glucose Figure6Ashowsthatinnormalchondrocytes,TIMP-1expressionwas
(HGM), however, the inhibitory effect of TGF treatment was onlyslightandtransientlyincreasedbytreatmentwithTGFfor48hin
abolished,butthekineticsinnormalandOAchondrocyteswerenot RGM and was not modified by exposure to high glucose. In OA
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Fig.5. RoleofhighglucoseinmodulatingMMP-1and-13geneexpressioninducedbyTGFinnormalandOAchondrocytes.RelativemRNAlevelsofMMP-1(A,B)andMMP-
13(C,D)innormal(n¼5)andOA(n¼6)chondrocytes.ChondrocytesweretreatedwithTGF10ng/ml,for24,48,and72hinmediumwithahigh(30mM,HGM)orregular
(10mM,RGM)glucoseconcentrationorleftuntreatedunderRGM(normalandOAcontrols).(cid:4)(cid:4)P<0.01and(cid:4)(cid:4)(cid:4)P<0.001relativetotherespectivecontrol;#P<0.05,
##P<0.01and###P<0.001betweenTGFinRGMandinHGMforeachtimepoint.
chondrocytes,however,TIMP-1expression(Fig.6B)wassignificantly chondrocytesandindependentlyoftheglucoseconcentrationinthe
induced in a time-dependent manner upon TGF treatment, but culture media. Interestingly, the relative increase in collagen I
independentlyoftheglucoseconcentrationintheculturemedium. expression was significantly higher in OA than in normal
In contrast, TGF induced TIMP-2 mRNA expression in normal chondrocytes. Indeed, treatment of this group with TGF for 24
chondrocytes (Fig. 6C) relative to the respective control cells. The and48hincreasedcollagenImRNAlevelsto2.0(cid:3)0.2and2.1(cid:3)0.5
observed increase was time-dependent and maximal at 72h (Fig. 7A), respectively, whereas in OA chondrocytes the increases
(2.7(cid:3)0.26). As shown in Figure 6D, in OA chondrocytes, TGF- were 3.8(cid:3)0.8 and 2.5(cid:3)0.5 (Fig. 7B), respectively, relative to the
induced TIMP-2 mRNA levels were similar and significantly corresponding cells maintained in RGM in the absence of TGF
augmented at the three time points, but the relative increase was treatment. Additionally, collagen I mRNA levels remained signifi-
lowerthanthatobservedinnormalchondrocytes.InductionofTIMP- cantly elevated in OA chondrocytes treated with TGF for 72h
2mRNAlevelsbyTGFwasnotaffectedbytheglucoseconcentration (2.7(cid:3)0.8), while in normal chondrocytes those levels (1.6(cid:3)0.5)
intheculturemedium,eitherinnormalorOAchondrocytes. were notsignificantly different from the respective control group.
Treatment of normal chondrocytes with TGF for 24 and 48h,
ROLEOFHIGHGLUCOSEINMODULATINGCOLLAGENIANDIIGENE regardless of the presence or absence of high glucose, increased
EXPRESSION INDUCED BYTGFIN NORMALANDOA collagenIImRNAlevels,butthedifferencesdidnotreachstatistical
CHONDROCYTES significance (Fig. 7C). In contrast, collagen II expression in OA
TheresultspresentedinFigure7AandBshowthattreatmentwith chondrocytes was significantly increased by TGF treatment
TGF increased collagen I mRNA levels both in normal and OA (Fig. 7D). The relative increase in collagen II expression was
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GLUCOSEMODULATIONOFCHONDROCYTEGENEEXPRESSION JOURNALOFCELLULARBIOCHEMISTRY
Fig.6. RoleofhighglucoseinmodulatingTIMP-1andTIMP-2geneexpressioninducedbyTGFinnormalandOAchondrocytes.RelativemRNAlevelsofTIMP-1(A,B)and
TIMP-2(C,D)innormal(n¼6)andOA(n¼6)chondrocytes.ChondrocytesweretreatedwithTGF10ng/mlfor24,48,and72hinmediumwithahigh(30mM,HGM)orregular
(10mM,RGM)glucoseconcentrationorleftuntreatedunderRGM(normalandOAcontrols).(cid:4)P<0.05,(cid:4)(cid:4)P<0.01,and(cid:4)(cid:4)(cid:4)P<0.001relativetotherespectivecontrol;
#P<0.05and##P<0.01betweenTGFinRGMandinHGMforeachtimepoint.
maximalat24h(2.2(cid:3)0.6),slightlydecreasingthereafter(1.7(cid:3)0.3 eachgroupthatonlymuchlargercohortswouldminimize.TheCol
and 2.1(cid:3)0.6 at 48 and 72h, respectively). Culture under high II/ColIratiohasbeendefinedasachondrocytedifferentiationindex
glucosedidnotsignificantlyaltertheresponsetoTGF,exceptat72h and used to characterize normal and OA cartilage and the
when a small, but statistically significant increase was found differentiation state of monolayer chondrocyte cultures, where
(2.5(cid:3)0.6, P¼0.008). lower ratios correspond to a less differentiated phenotype [Martin
et al., 2001; Marlovits et al., 2004]. Even so, since we could not
obtainage-matchednormalandOAcartilagesamplesandgiventhe
DISCUSSION
relatively large age range in each group, the possibility that the
differences observed areage-relatedcannot be excluded.
The results presented in Figure 1 are in agreement with previous The major purpose of this study was to determine whether
reports showing that MMP-1 and -13 expression is higher in OA variations in the extracellular glucose concentration affect
than in normal chondrocytes [Bau et al., 2002; Kevorkian et al., chondrocyte anabolic and catabolic gene expression. The results
2004]. Moreover, the average ratio of collagen type II expression obtainedshowthathumanchondrocytesrespondtotheextracellu-
overcollagentypeI(ColII/ColI)wasfoundtobehigherinnormal lar glucose concentration which can modulate anabolic and
thaninOAchondrocytes,likelyduetotheshifttowardsincreased catabolic responses, both directly and by modifying the response
collagenIanddecreasedcollagenIIexpressioninOAchondrocytes to anabolic growth factors, namely TGF. In particular, this study
(Fig.1EandF).Nonetheless,thisdifferencedidnotreachstatistical shows that exposure of OA chondrocytes to high glucose (30mM)
significance, reflecting a great variability between individuals in increased MMP-1 and -13 mRNA expression and slight and
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Fig.7. RoleofhighglucoseinmodulatingCollagenstypeIandIIgeneexpressioninducedbyTGFinnormalandOAchondrocytes.RelativemRNAlevelsofcollagenI(A,B)and
collagenII(C,D)innormal(n¼5)andOA(n¼5)chondrocytestreatedwithTGF,10ng/ml,for24,48,and72hinmediumwithahigh(30mM,HGM)orregular(10mM,RGM)
glucoseconcentrationorleftuntreatedunderRGM(normalandOAcontrols).(cid:4)P<0.05and(cid:4)(cid:4)P<0.01relativetotherespectivecontrol;#P<0.01betweenTGFinRGMandin
HGMforeachtimepoint.
transientlyaugmentedcollagenIIexpression,whileTIMP-1,TIMP-2 proteinlevelthealterationslastlonger.Althoughbeyonditsscope,
and collagen I were not affected. In contrast, the expression of this study paves the way for future research on the role of high
MMP-13 and collagen I was not affected by high glucose in glucose in modulating the protein levels of the genes whose
normal chondrocytes, whereas MMP-1 and TIMP-1 expression expression was found to be altered, as well as of other proteins
weredecreasedandTIMP-2andcollagenIIlevelsweretransitorily relevantin chondrocyte biologyandarthritis pathology.
increased. These results indicate that exposure to high glucose Moreover,theobservationthatinOAchondrocyteshighglucose-
can promote both catabolic and anabolic responses in human inducedMMP-1and-13mRNAexpressionwasnotaccompaniedby
chondrocytes, but OA chondrocytes are more susceptible to increased TIMP-1 and -2 expression, suggests that the uncoordi-
deleterious effects than their normal counterparts. This is in nated expression of the MMP/TIMP system can lead to a greater
agreementwithourpreviousstudyshowingthatOAchondrocytes imbalanceintheactivityoftheformer,favoringmatrixdegradation
failtodownregulateglucosetransportandproducemoreROSover and further compromising OA cartilage integrity. Therefore, this
longer periods whenexposed tohigh glucose [Rosa et al.,2009]. may represent a relevant pathological mechanism by which high
On the other hand, most of the effects induced by exposure of glucoseconcentrationscancontributetoOApathology.Ontheother
either normal or OA chondrocytes to high glucose were transient. hand,exposuretoahighglucoseconcentrationdidnotaffectTGF-
Twomechanismsmaycontributetothereversibilityofthoseeffects. inducedanabolicresponses,exceptforaslightincreaseincollagen
Ononehand,glucoseconcentrationintheculturemediumdecreases II mRNA levels (Fig. 7D), but it did reverse TGF-induced anti-
overtimeduetocellconsumption,whichmaycausetheinitialeffect catabolicgeneexpression.Inparticular,elevatedglucoseinhibited
to gradually wear out. On the other hand, it is also possible that TGF-induced decrease in MMP-13 expression both in normal and
theeffectsatthelevelofgeneexpressionaretransient,whileatthe OA chondrocytes, although the effect was more prolonged in OA
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