Co-distribution
of
cysteine
cathepsins
and
matrix
metalloproteases
in
human
dentin
Polliana
Mendes
Candia
Scaffa
a,b,*
,
Lorenzo
Breschi
c,
Annalisa
Mazzoni
d,
Cristina
de
Mattos
Pimenta
Vidal
a,e,
Rosa
Curci
f,
Fabianni
Apolonio
g,
Pietro
Gobbi
h,
David
Pashley
i,
Leo
Tjäderhane
j,k,
Ivarne
Luis
dos
Santos
Tersariol
l,m,
Fábio
Dupart
Nascimento
m,
Marcela
Rocha
Carrilho
n,o,*
a
FacultyofDentistryatPiracicaba,UniversityofCampinas,Piracicaba,13414-903,Brazil
b
DepartmentofBiologicalScience,BauruSchoolofDentistry,UniversityofSaoPaulo,Bauru,SP,17012-901,Brazil
c
DepartmentofBiomedicalandNeuromotorSciences,UniversityofBologna,Bologna,40126,Italy
d
DepartmentofMedicalSciences,UniversityofTrieste,Trieste,34128,Italy
e
DepartmentofOperativeDentistry,CollegeofDentistry,UniversityofIowa,801NewtonRd,IowaCity,IA,52246,USA
fIGM-CNR,UnitofBolognac/oIOR,Bologna,40136,Italy
gDepartmentofPharmacy,DentistryandNursing,FederalUniversityofCeara,Fortaleza,60020-181,Brazil h
DepartmentofSteVA,University“CarloBo”,Urbino,61029,Italy
i
EmeritusProfessorofOralBiology,DentalCollegeofGeorgia,GeorgiaHealthSciencesUniversity,Augusta,Georgia,30912,USA
j
InstituteofDentistry,UniversityofOulu,andMedicalResearchCenterOulu,OuluUniversityHospitalandUniversityofOulu,OuluFI-90014,Finland
k
DepartmentofOralandMaxillofacialDiseases,UniversityofHelsinki,andHelsinkiUniversityHospital,HelsinkiFI-00014,Finland
l
DepartmentofBiochemistry,FederalUniversityofSãoPaulo,SãoPaulo,04021-001,Brazil
mCentroInterdisciplinardeInvestigaçãoBioquímica,UniversityofMogidasCruzes,MogidasCruzes,08773-520,Brazil nBiomaterialsinDentistryProgram,AnhangueraUniversitySãoPaulo(UNIAN-SP),SãoPaulo,05145-200,Brazil o
BiotechnologyandInnovationinHealthProgram,AnhangueraUniversitySãoPaulo(UNIAN-SP),SãoPaulo,05145-200,Brazil
ARTICLE INFO
Articlehistory: Received21March2016
Receivedinrevisedform26October2016 Accepted20November2016 Keywords: Cysteinecathepsins Matrixmetalloproteases Proteolyticactivity Dentin Immunohistochemistry ABSTRACT
Ithasbeenhypothesizedthatcysteinecathepsins(CTs)alongwithmatrixmetalloproteases(MMPs)may workinconjunctionintheproteolysisofmaturedentinmatrix.Theaimofthisstudywastoverify simultaneously the distribution and presence of cathepsins B (CT-B) and K (CT-K) in partially demineralizeddentin;andfurthertoevaluatetheactivityofCTsandMMPsinthesametissue.The distributionofCT-BandCT-Kinsoundhumandentinwasassessedbyimmunohistochemistry.A double-immunolabelingtechniquewasusedtoidentify,atonce,theoccurrenceofthoseenzymesindentin. ActivitiesofCTsandMMPsindentinextractswereevaluatedspectrofluorometrically.Inaddition,insitu gelatinolyticactivityofdentinwasassayedbyzymography.TheresultsrevealedthedistributionofCT-B andCT-Kalongthedentinorganicmatrixandalsoindicatedco-occurrenceofMMPsandCTsinthat tissue.Theenzymekineticsstudiesshowedproteolyticactivityindentinextractsforbothclassesof proteases.Furthermore,itwasobservedthat,atleastforsoundhumandentinmatrices,theactivityof MMPsseemstobepredominantovertheCTsone.
©2016ElsevierLtd.Allrightsreserved.
1.Introduction
Dentinmatrixhasendogenousproteolyticactivity,whichhas been attributed to the collagenolytic/gelatinolytic function of enzymessuchasmatrixmetalloproteases(MMPs)(Martin-DeLas
Herasetal.,2000;Mazzonietal.,2007;Sulkalaetal.,2007)and cysteinecathepsins(CTs)(Nascimentoetal.,2011;Tersarioletal., 2010). More recently, we have also identified in dentin the presence ofcysteinecathepsin K(CT-K)(Vidaletal.,2014), the mostpotentmammaliancollagenase(Fonovic&Turk,2014).
ThegeneexpressionsofMMPs(Palosaarietal.,2003)andCTs (Tersarioletal.,2010)inmaturehumanodontoblastssuggestthat these enzymes could share multiple functions in physio-pathological processes occurring in dentin matrix (Dickinson 2002; Hannaset al., 2007). The co-expression of two distinct
* Correspondingauthorsat:AnhangueraUniversityofSãoPauloUNIAN-SP,Rua Girassol584–ap301A,05433001SãoPaulo,SP,Brazil.
E-mailaddresses:marcelacarrilho@gmail.com,carrilhomarcela@gmail.com (M.R.Carrilho).
http://dx.doi.org/10.1016/j.archoralbio.2016.11.011 0003-9969/©2016ElsevierLtd.Allrightsreserved.
ContentslistsavailableatScienceDirect
Archives
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familiesofextracellularmatrix–degradingenzymesinaspecific tissueisinfrequent,particularlybecausethecellsinquestion(i.e. odontoblasts)arenotthoughttobeverymuchactiveaftertissue (i.e. dentin) maturation and under physiologic conditions. However, in pathological conditions, the collagen degrading activityof theseenzymes hasbeenwidelyrecognized (Hannas etal.,2007;Tjäderhaneetal.,2013).Ourpreviousdatasuggested that CTs might be responsible for activating dentin-bound or salivaryMMPs,establishingasynergybetweenthesetwoclasses of enzymes acting in different stages in caries progression (Nascimentoetal.,2011).EventhoughtheinvolvementofMMPs indentinpathologies wassuggestedalreadyaboutfifteenyears ago(Tjäderhaneetal.,1998a,1998b),CTswereonlydetectedin dentin recently(Nascimento et al., 2011; Tersariolet al., 2010; Vidaletal.,2014).Therefore,nodetailedinformation aboutthe individualorsynergisticrolesoftheseenzymesondentinmatrix remodeling/proteolysisprocessesis available (Tjäderhaneet al., 2013).
Wehypothesizethat,toworksynergistically,thesetwogroups of enzymesshould belocalized veryclose togetherand in the vicinitiesoftheirtargetsubstrates.Thisissupportedsomehowby ourpreviousstudieswhereinwefoundtheexistenceofatleast some MMPs and CTs members distributed in the same space occupiedbycollagen,bothinsoundandcariousteeth(Vidaletal., 2014),whichinturnindicatesapossibleinterplaybetweenthese differentclasses ofproteasesastheycanbefoundactivewhen extractedfromdentin (Nascimentoet al.,2011; Tersariolet al., 2010).ThepresentstudyinvestigatedthedistributionofCT-Band Kindentinmatrixandevaluatedwhetherco-occurrenceofCTsand MMPscanbeobservedinthistissue. Additionally,itwastested whethertheproteolyticactivityofdentinwouldbepredominantly duetoactivityofeitherCTsorMMPs.
2.Material&methods 2.1.Material
Fifteenfreshlyextractedhumanmolars,obtainedwithpatient’s informedconsentunderaprotocolapprovedbyIRBCommitteeof PiracicabaSchoolofDentistry/UNICAMPwereusedinthisstudy. Reagents werepurchased from Sigma Chemical (St. Louis, MO, USA)unlessotherwisespecified.
2.2.Pre-embeddingtechnique:FEI-SEManalysis
Specimens wereprocessedfora pre-immunolabelling proce-dureasdescribedbyMazzonietal.,2009.Briefly,cryofractured dentinfragmentswerepartiallydemineralizedin0.5Methylene diaminetetraceticacid(EDTA;pH7.4)for30min,immersedina 0.05MTrisHClbuffersolution(TBS)atpH7.6,with0.15MNaCl and0.1%bovineserumalbuminandthenpre-incubatedinnormal goatserumin0.05MTBSatpH7.6for30min.Specimens were then incubated overnight with one of the primary antibodies: rabbit IgG anti-human cathepsin B (Calbiochem, Billerica, MA, USA) or mouse IgG anti-human cathepsin K (Biovendor, Brno, CzechRepublic)(dilution1:100in0.05MTBS,pH7.6,37C).Gold labelingwasperformedusingasecondaryantibody,agoat anti-mouse or anti-rabbitIgG conjugated with15nm colloidalgold particles(BritishBioCellInternational,Cardiff,UK,dilution1:20)in 0.02MTBS(pH8.2)for90min.
All specimens were fixed in 2.5% glutaraldehyde in 0.15M cacodylatebufferpH7.2for4h,rinsedanddehydratedingraded concentrationsofethanol.Thesampleswerecriticallypointdried and coated withcarbon using a Balzers Med 010 Multicoating System(Bal-TecAG,Liechtenstein).Observationswereperformed usingFEI-SEM(JSM890,JEOL,Tokyo,Japan)at7kVand110 12A.
Imageswereobtainedusingacombinationofbackscatteredand secondaryelectrondetectors(Breschietal.,2003).
2.3.Post-embeddingtechnique:TEManalysis
Dentinfragmentssubmittedtothepost-embeddingtechnique wereimmediatelyfixedin4%paraformaldehyde–0.1% glutaralde-hydein0.15Mcacodylatebuffer(pH7.2,for4h)anddemineralized in0.5MEDTAfor3months.Then,thespecimensweredehydrated ingradedconcentrationsofethanolandembeddedinLRWhite resin (London Resin, Berkshire, UK). Ultrathin sections (80nm thick) were obtained, mounted on formvar nickel grids and processed for immunohistochemical labelling (Breschi et al., 2003).
Immunolabelingwasperformedasdescribedaboveusingthe same primary anti-CT-B, anti-CT-K and secondary antibodies. Additionally, a double-labeling technique was performed to identify,simultaneously,CT-BandMMP-2(mouseIgGanti-human MMP-2,Abcam,Cambridge,UK)ondentinmatrix.Afterincubation withtheprimaryantibodies,sampleswererinsedandincubated withtwocolloidalgold-conjugatedsecondaryantibodies,agoat anti-rabbitIgGconjugatedwith30-nmcolloidalgoldparticlesfor theidentificationofCT-Bandagoatanti-mouseIgGconjugated with15-nmcolloidalgoldparticlesforMMP-2identification.Grids were then stained with 3% uranyl acetate and lead citrate for examinationinaZEISS109TEMoperatedat60kV(Zeiss,EM109, CarlZeiss,Oberkochen,Germany).Controlsconsistedofsections incubatedwithsecondaryantibodiesonly.
2.4.Insituzymography
Two freshly extracted third molars were used to obtain longitudinalslicesof 200
m
mthickness(Isomet–Buehler Ltda, LakeBluff,IL,USA).Toverifythespecificityofenzymesactivity,the sliceswerepreviouslyincubatedindeionizedwater(control),E-64 (specificinhibitorofCTs)or1,10-Phenanthroline(MMPsinhibitor) for 30min at room temperature. In situ zymography was performed using a quenched fluorescein-conjugated gelatin as substrate (E-12055, Molecular Probes, Eugene, OR, USA), as described by Mazzoni et al., 2012. The gelatin stock solution wasdiluted1:8ina buffer(NaCl150mM,CaCl2 5mM,Tris-HCl50mM,pH8.0)with10
m
Lofanti-fadingagent(MountingMedium with Dapi H-1200, Vectashield, Vector Laboratories LTD, Cam-bridgeshire,UK).A80-m
Lofthismixturewasplacedontopofeach slabandincubatedinadarkhumidchamberat37Cfor24h.The hydrolysis of quenched fluorescein-conjugated gelatin substratewas assessed bya multi-photon confocalmicroscope, excitation: 488nmand emission: 530nm (Zeiss, LSM780, Carl Zeiss,Oberkochen,Germany).Opticalsectionsof85
m
mthickwere acquiredand thestackedimageswereanalysed,quantified,and processedwithZEN2010software(CarlZeiss).2.5.Monitoringoftheprotelotyticactivityindentinextract Dentin powder of 10 freshly extracted human molars was obtainedasdescribed previously(Scaffaetal.,2012).Aliquotof 1.2gwasusedforenzymeextraction(Martin-DeLasHerasetal., 2000).Briefly,dentinpowderwastreatedwith4Mguanidine-HCl; thendemineralizedwith0.5MEDTAandtreatedagainwith4M guanidine-HCl(G2-extract).TheamountofproteininG2-extract was determined according toLowry et al. (1951), and specific activitieswerecalculatedwithreferencetoproteinconcentration. ThetotalCTsactivitywasmonitoredspectrofluorometricallyby usingtheCT-specificfluorogenicsubstrateZ-FR-MCA (carboben-zoxy-phenylalanine-arginine-7-amido-4-methyl coumarin). For theassay,10
m
Lofthedentinextractwasaddedto50mMsodiumphosphatebuffer(pH5.7)containing10
m
Mofthesubstratewith 1mMDTTandincubatedat37Cfor24h.Excitationandemission wavelengthsweresetat380and460nm,respectively.Thetotal collagenolytic/gelatinolytic MMP activity in dentin extract was measuredwiththeMMP-specificinternallyquenchedfluorescent peptide substrate Abz-GPQGLAGQ-EDDnp [ortho-aminobenzoic acid– Gly-Pro-Gln-Gly-Leu-Ala-Gly-Gln-N-(2,4-dinitrophenyl)-ethylenediamine]. For MMPs,10m
L of the dentin extract was addedto50mMHEPESbuffer(pH7.5)containing10m
Mof the substrate and incubated at 37C for 24h. The excitation andemission wavelengths were 320 and 420nm. The substrate’s hydrolysis were checked using 2mmol/L 1,10 phenanthroline (specificMMPinhibitor)or10
m
ME-64(specificCTinhibitor).The assays were performed in duplicate, in a 96-well plate by a fluorometer(TecanGENiosProMultifunctionMicroplateReader; Tecan,Mannedorf,Switzerland).Thespecificenzymaticactivity, forbothCTsandMMPswascalculatedasthedifferencebetween final and initial fluorescence readings (delta fluorescence) and expressed in arbitrary units of fluorescence per microgram of proteinintheextract(AUF/m
g).3.Results
Positive immunolabeling patterns for CT-B and CT-K were observedinpartiallydemineralizeddentinsurfacesunderFEI-SEM examinationasindicatedbythepresenceofthegoldnanoparticles,
whichappearassphericalspotsof15nmindiameter(Fig.1).Both CT-B and CT-K were localized along the collagen fibrils. High-magnificationimagesshowglobularaggregatesbyaclusterof2–3 goldnanoparticlesalongthebranchingpointsoncollagen(Fig. 1B). TEM images of demineralized dentin revealed different distributionoftheCT-Band CT-Klabeling(Fig.2).Theintensity ofimmunoreactionforCT-BwasscatteredandhigherthanCT-Kin dentinmatrix. LabelingforCT-Kshowed weakdistributionover thecollagenfibrils(Fig.2).Similarlabelingpatternswereobtained forCT-BandCT-KunderbothFEI-SEMandTEMobservations.The double-labelingofCT-BandMMP-2wasperformedwithtwosizes of colloidal gold particles (black spots) conjugated with the antibodies,inwhichCT-Bisrepresentedbythelarger30nmand MMP-2bythesmaller15nmparticles.TEMimagesrevealedthe co-occurrence of CT-B and MMP-2 in dentin matrix, but with differentdistribution(Fig.2E,F).LabelingforMMP-2wasrandomly distributedoverthecollagenmatrix,whileCT-Btendedtolocalize inaggregates(Fig.2E,F).
Theinsituzymographyrevealedtherelativeeffectsofdifferent familiesofenzymesonthetotalenzymaticactivityofdentin.For thecontrols,anintensegreenfluorescenceindicatinggelatinolytic activitywasobservedinmineralizeddentin,locatedespeciallyin dentinaltubules(Fig.3B,C). ThetreatmentwithE-64and 1,10-phenanthroline inhibited the gelatinolytic activity, seen as decreaseingreenfluorescence(Fig.3E,F,H,I)withdifferentrates of specificity. Phenanthroline inhibition of MMPs resulted in
Fig.1.FEI-SEMmicrographsofpartiallydemineralizeddentinafterapre-embeddingimmunolabelingprocedure.Labelingcanbeidentifiedaselectron-densewhitespots undertheelectronbeam(arrows).(a,c)Lowmagnificationview(30,000)ofthepartiallydemineralizeddentinsurfaceshowingamildCT-BandCT-Klabelingsignal.(b)A highermagnificationview(50,000)ofthepartiallydemineralizedsurface:positiveimmunohistochemicalstainingidentifyingCT-Blocatedalongthecollagenfibrils.(d) HighmagnificationFEI-SEMmicrographs(100,000)revealingamoderatelabelingforCT-Kstaining.
strongerreductioninfluorescence(Fig.3J)indicatingthatintrinsic MMPsgelatinolyticactivitywashigherthanthatofCTs.
ProteolyticactivityinG2-dentinextractwasdetectedusing CT-and MMP-specific fluorogenic substrates (Fig. 4). The activity analysisforCT-substratedemonstratedstronginhibitionbyE-64 (Fig.4A)andalsoadecreaseintherateofhydrolysisofthe MMP-substrateinthepresenceofMMPinhibitor1,10-phenanthroline (Fig.4B).Resultsshowed, therefore,thepresenceofbothactive MMPsandCTsindentin.
4.Discussion
The proteolyticenzymespotentiallyinvolvedin thecollagen metabolismcanbeexaminedbyimmunocytochemistry,a power-fulimagingtechniqueforcorrelatingcompositionwithstructure (Nanci,1999).Tothebestofourknowledge,thisisthefirststudy thatevaluates theinsitu co-occurrence/distribution of CTs and
MMPsinhumandentinmatrixbyimmunogoldlabelingcorrelative FEI-SEMandTEMmicroscopies.
FEI-SEMdemonstratedthepresenceofCT-BandCT–Kindentin surfaceafterbriefdemineralizationwithEDTA.Calciumchelation by EDTA provides a mild process of tissue demineralization, preservingtheantigenicbindingsitesbetterthanstrongacidsdo (Breschietal.,2003)andithasbeenthereforeusedtorevealthe presenceofvariousproteinswithFEI-SEM,includingMMP-2,-3 and-9(Mazzonietal.,2009,2011).EventhoughFEI-SEMimages indicatedthelocalizationofCTsapparentlyontocollagenfibrils,it must be taken in consideration that SEM images represent, at maximum,abidimensionaldepictionofsurfacetopography,hence theactualinteractionbetweentheenzymesand dentinorganic matrixcannotbefullyconfirmedthroughthismethod.Therefore, TEM analyses with completely demineralized dentin were performed, confirming CT-B and CT-K binding along collagen fibrils.Thebindingmightoccurdirectlytocollagenor
collagen-Fig.2.TEMmicrographsofdemineralizeddentinafterapost-embeddingimmunolabelingprocedure.Labelingcanbeidentifiedasblackspots.(a,b)showsimmunoreaction forCT-B(15nm);(c,d)showsimmunoreactionforCT-K.(e,f)showsthedoublelabelingforCT-B(30nm)andMMP-2(15nm).(a)LowmagnificationTEMimage(30,000) showingCT-Blabelingovertheintertubulardentin.(b)Ahighmagnificationimage(50,000)showsmoderatelabelingsignalforCT-B.(c)LowmagnificationTEMimage showingCT-Klabelingoverthecollagen.(d)Ahighmagnificationimage(85,000)showssparselabelingsignalforCT-K.(e)Double-labellingwithMMP-2andCT-B antibodies.LowmagnificationTEMimage(20,000)showingmoresparseCT-Blabeling(30nmgoldparticles;widearrow)associatedwithmoderateandevenlydistributed MMP-2(15nmparticles;arrows)overtheintertubulardentin.(f)AhighmagnificationimageshowssparseglobularlabelingsignalforCT-Bandmoderateregularly distributedMMP-2labelingovertheintertubulardentin.
boundproteoglycans(PGs),sinceithasbeenalreadydemonstrated thatCTsreadilyformcomplexeswithPGglycosaminoglycan(GAG) sidechains(Fonovic&Turk,2014;Lietal.,2004).GAGsmayhavea significant effectonCT activities,asthey differentiallyregulate their activities (Fonovic & Turk, 2014; Li et al., 2004). This is especiallytrueforCT-K,whichishighlydependentonforminga complex with chondroitin-4-sulfate GAG for its collagenolytic activity(Lietal.,2004),butnotagainstnon-helicalsubstratessuch asgelatin(Selentetal.,2007).
Theproteolyticactivityofproteasescanbeverifiedindifferent ways (Martin-De Las Heras et al., 2000; Mazzoni et al., 2012;
Tersarioletal.,2010).Inthisstudy,thecollagenolytic/gelatinolytic activities were demonstrated by substrate hydrolysis at the presence/absence ofenzymesinhibitors.In addition,the gelati-nolytic activity of enzymes was further assessed by in situ zymography. Both MMPs and CTs activities were observed spectrofluorometrically using fluorogenic substrates. Using this technique, once the peptide is enzymatically hydrolyzed, an increase in the fluorescence can be detected. Although these enzymesmaybeinvolvedinenamelanddentinformation,their fateaftermineralizationisnotknown.TheG2-extractrepresents theproteolyticactivityofenzymesthatwereextractedwithEDTA
Fig.3.Dentinslicespre-incubatedwithoutinhibitors(control)orwithE-64and1,10-phenanthrolineandincubatedfor24hwithquenchedfluorescein-labeledgelatin;(A,D, G):Differentialinterferencecontrast(DIC),showingtheopticaldensityofthedentinsurface;(B,E,H):Acquiredimageatgreen-channel,showingfluorescenceinmineralized dentin;(C,F,I):Mergedimages.(A–C)controlgroupindicatingthatthefluoresceinconjugatedgelatinwasstronglyhydrolyzed;(D–F)dentinsliceincubatedwithE-64 inhibitor,showingpartialinhibitionofgelatinolyticactivityindentin;(G–I)dentinsliceincubatedwith1,10-phenanthrolineinhibitor,showingstrongerinhibitionof gelatinolyticactivity;(J)Specificfluorescenceemissionintensityincontrolandcathepsin-inhibited(withE-64)orMMP-inhibited(with1,10-phenanthroline)samples.
whilewerefoundoriginallydeeplyembeddedinmineral( Martin-DeLasHerasetal.,2000).TheproteolyticactivityintheG2extract suggeststhattheseenzymesweredepositedinthedentinmatrix duringmineralizationandweremostprobablytightlyboundtothe collagen.Dentinenzymeactivityanalysisagainstenzyme-specific syntheticsubstratesdemonstratedthat bothCTs andMMPsare activeindentinmatrixandfullycapabletodegradetheirtarget substrates.
To better determine the role of each class of enzymes in proteolytic degradation of collagen, we performed in situ zymographyusingspecificenzymesinhibitors.Gelatinwasused hereinasasubstrate,ascommonlyreportedinliteraturefordental andcraniofacialtissues(DeVito-Moraesetal.,2016;Mazzonietal., 2012;Pessoaetal.,2013).Althoughotherdye-quenched fluoro-genic substrates such as collagen and casein might result in differentpatternsofproteolytic/gellatinolytic activity(Sakuraba etal.,2006),thedramaticreductioninfluorescencebyadditionof E-64and1,10-Phenanthrolinesupportourresults.Indeed,thein situ zymography demonstrated more intrinsic MMP activity, indicatingstrongerroleindentincollagenousmatrixdegradation inpathologies.Tothebestofourknowledge,thisisthefirststudy todirectlycomparedentinalCT-andMMP-activities.Previously,
Tezvergil-Mutluay et al. (2013) demonstrated 90% lower CT-dependentreleaseofCTX(C-terminalcrosslinkedtelopeptideof typeI collagen)compared toMMP-dependentICTP (C-terminal crosslinked telopeptide of type I collagen). Even though these telopeptidaseactivitiesmaynotdirectlyreflectthetotalamounts of different enzyme groups in dentin, together with our
immunolabelingresults,theysuggestthatMMPsmaybepresent inmarkedlyhigherquantitiesindentin.
ThereasonforthehigherMMPsthanCTsactivityindentinis not well understood,but maybe related to the role of theses enzymesincollagenmetabolismprocesses.Thesetwofamiliesof proteasesplaydifferentcoordinatedrolesinmatrixdegradation andboneresorption.CT-Kplaysauniqueroleamongthevarious protease, since its collagenolytic activity does not depend on destabilizationofthetriplehelixincontrasttotheotherenzymes (Garneroetal.,1998).Thus,oncecollageniscleaved,thetriplehelix would unwind and become available for degradation by other gelatinases(Delaisséetal.,2003).
Inothertissues,MMPsandCTsareknowntobeabletoregulate theactivitiesofeachother,andpossibleCT-MMPinteractionshave alsobeensuggestedtooccurindentin(Nascimentoetal.,2011; Tjäderhaneetal., 2013).Inprinciple,this wouldrequirea close proximitywitheachotherandthesubstrate,i.e.collagen.Although double-immunolabelingwithMMP-2andCT-Bantibodiesshowed co-occurrence of the enzymes, which is suggestive of an interaction, it could not be confirmed by this method. Some degreeofjointactioninenzymeactivitiesissupportedbythein situ zymography findings. CT-specific inhibition withE-64 and MMP-specific inhibition with 1,10-phenanthroline maintained approximately50%and20%ofgelatinolysiscomparedtountreated controls,respectively.Therefore,itappearsthatthetotalactivityis notonlyadditive,butinhibitionofoneenzymefamilyhassome inhibition onother, too.Thus, even though the results do not support the hypothesis of CT-MMP interplay in dentin matrix degradation,thepossibilityofinteractionsofotherenzymesstill remains. Furtherstudies withinhibitorsthat specifically inhibit certainmembersofMMP-orCT-familiesareneededtoexamine thispossibility.
ThelatentformsofCTsandMMPsenzymescanbeactivatedin acidicconditions.CTsareauto-activatedinlowpH,andmostare consideredunstableandinactiveinneutralpH(Turk,Turk,&Turk, 2000).ConverselyMMPsareneutralproteinases,butlatentforms can be activated with acidic pH followed by neutralization (Tjäderhaneetal.,1998a).CT-BenhancestheactivityoftheMMPs by shifting the balance between MMPs and TIMPs through inactivation of the MMP-specific tissue inhibitors TIMP-1 and TIMP-2 (Nagase 1997; Kostoulas et al., 1999). Therefore, CTs activated at the acidic conditions may furtheractivate dentin-boundMMPs,which inturnwouldbecomefunctional afterpH neutralization,resultingindentinmatrixenzymaticdegradation. ThissuggeststhepotentialpH-relatedinteractionsandsynergistic effectsofCTsandMMPsincollagendegradation.
Inconclusion,MMPsandCTsareco-distributedwithinsound dentinmatrix,whichsupportstheinteractionofbothfamiliesof enzymesfortheproteolyticdegradationofdentininpathological conditions. While active enzymes were extracted from dentin, MMPsactivityispredominantoverCTs.Furtherinvestigationson proteolytic activity associated with a proteomicapproach may showtheinvolvementandinterplaybetweenthemainproteolytic enzymesin dentin. In this way,theactualrole of thedifferent classes ofenzymesparticipatingin themechanismsofcollagen degradationinphysiologicalandpathologicalconditionscouldbe established.
Conflictofinterests None.
Funding
FAPESP (2007/54618-4 P.I. Carrilho and 2009/14005-9 P.I. Scaffa)andCNPq(312768/2013-3P.I.Carrilho),Brazil.
Fig.4.EnzymesactivityinG2extractovertheenzyme-specificsubstrateswithand without(control) respectivespecificinhibitors.(A)Activity against cathepsin-specificZFR-MCApeptide.E-64resultedwith93%inhibitioncomparedtountreated control.(B)ActivityagainstMMP-specificAbz-GPQGLAGQ-EDDnppeptide. MMP-specificinhibitor1,10-phenatrolineinhibited76%oftheactivity.
Ethicalapproval
The manuscript was approved by the Ethics Committee in ResearchoftheFacultyofDentistryatPiracicaba-Universityof Campinas(117/2009).
Acknowledgments
TheauthorsthankthefundingagenciesFAPESP(2016/07996-2 –P.I:M.R.Carrilho)andCNPq(312768/2013-3–P.I:M.R.Carrilho), Brazil.
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