Are Osteoclasts Needed for the Bone Anabolic Response to

12 Pages·2010·1.54 MB·English

THEJOURNALOFBIOLOGICALCHEMISTRYVOL.285,NO.36,pp.28164–28173,September3,2010
©2010byTheAmericanSocietyforBiochemistryandMolecularBiology,Inc. PrintedintheU.S.A.
Are Osteoclasts Needed for the Bone Anabolic Response
to Parathyroid Hormone?
ASTUDYOFINTERMITTENTPARATHYROIDHORMONEWITHDENOSUMABOR
ALENDRONATEINKNOCK-INMICEEXPRESSINGHUMANIZEDRANKL*□S
Receivedforpublication,January7,2010,andinrevisedform,June1,2010Published,JBCPapersinPress,June17,2010,DOI10.1074/jbc.M110.101964
DominiqueD.Pierroz‡,NicolasBonnet‡,PaulA.Baldock§,MichaelS.Ominsky¶,MarinaStolina¶,PaulJ.Kostenuik¶,
andSergeL.Ferrari‡1
Fromthe‡ServiceofBoneDiseases,DepartmentofRehabilitationandGeriatrics,GenevaUniversityHospitalandFacultyof
Medicine,1211Geneva14,Switzerland,the§BoneandMineralProgram,GarvanInstituteofMedicalResearch,St.Vincent’s
Hospital,Sydney,NewSouthWales2010,Australia,andthe¶MetabolicDisordersResearch,AmgenInc.,ThousandOaks,California91320
PTH stimulates osteoblastic cells to form new bone and to bonemicroarchitectureandstrength(1).PTHinducesarapid
produceosteoblast-osteoclastcouplingfactorssuchasRANKL. increaseinmarkersofboneformationbeforeitalsoincreases
WhetherosteoclastsortheiractivityareneededforPTHanab- boneresorption,whichhasbeendescribedasan“anabolicwin-
olismremainsuncertain.WetreatedovariectomizedhuRANKL dow”(2).Inthiswindow,PTHhasbeensuggestedtodirectly
knock-in mice with a human RANKL inhibitor denosumab activateboneformationviaboneliningcellsonquiescentbone
(DMAb),alendronate(Aln),orvehiclefor4weeks,followedby surfaces (i.e. modeling surfaces), as indicated by an increase
co-treatmentwithintermittentPTHfor4weeks.Lossofbone indoubletetracycline-labeledsurfacesandasmoothcement
massandmicroarchitecturewaspreventedbyAlnandfurther line(3).PTHsubsequentlystimulatesboneremodeling,first
significantly improved by DMAb. PTH improved bone mass, through activation of osteoclastic bone resorption in the
microstructure,andstrength,andwasadditivetoAlnbutnot basicmulticellullarunits(BMU),followedbynewbonefor-
toDMAb.Alninhibitedbiochemicalandhistomorphometri- mation by osteoblasts. These remodeling-based BMUs are
calindicesofboneturnover,-i.e.osteocalcinandboneforma- associated with scalloped cement lines by microscopy. Data
tionrate(BFR)oncancellousbonesurfaces-,andDmabinhib- from untreated adult rodents indicate that the ratio of bone
ited them further. However Aln increased whereas Dmab
modeling:remodeling surfaces is less than 1:5 and further
suppressedosteoclastnumberandsurfaces.PTHsignificantly
decreases with age (4). Although PTH has been shown to
increased osteocalcin and bone formation indices, in the
increase bone modeling in rodents, the vast majority of bone
absence or presence of either antiresorptive, although BFR
formationinhumansappearstoberemodeling-based(5).Data
remainedlowerinpresenceofDmab.TofurtherevaluatePTH
fromhumanstreatedwithPTHindicatethatboneformation
effectsinthecompleteabsenceofosteoclasts,highdosePTH
onmodelingsurfacesaccountsforonly5–30%ofPTHanabolic
wasadministeredtoRANK(cid:1)/(cid:1)mice.PTHincreasedosteocalcin
effects(6).WhileintermittentPTHcanpartiallyrestorebone
similarlyinRANK(cid:1)/(cid:1)andWTmice.ItalsoincreasedBMDin
microarchitecture by increasing bone formation in modeling
RANK(cid:1)/(cid:1)mice,althoughlessthaninWT.Theseresultsfurther
andremodelingosteons(5),italsostimulatesHaversianbone
indicatethatosteoclastsarenotstrictlyrequiredforPTHanab-
remodelingleadingtocorticalporosity(7,8).Incontrast,anti-
olism,whichpresumablystilloccursviastimulationofmodel-
resorptivetherapies,suchasbisphosphonates(e.g.alendronate
ing-basedboneformation.HoweverthemagnitudeofPTHana-
(Aln)), inhibit bone remodeling and maintain, but do not
bolic effects on the skeleton, in particular its additive effects
restore, bone microarchitecture. These attributes have led to
withantiresorptives,dependsontheextentoftheremodeling
significantinterestinthecombinationofPTHwithantiresorp-
space,asdeterminedbythenumberandactivityofosteoclasts
tiveagents.However,someclinicalstudieshavefailedtodem-
onbonesurfaces.
onstrate an additive effect of PTH plus Aln on bone, at least
whenPTHtreatmentwasinitiatedtogetherwithAln(9–11).
This outcome might suggest that when remodeling is sup-
Parathyroidhormone(PTH),2whenadministeredintermit-
pressedbyantiresorptiveagents,theanabolicpotentialofinter-
tently, increases bone mineral density (BMD) and improves
mittentPTHtherapyisrelatedtotheextentofresidualremod-
*ThisworkwassupportedbyAmgenInc.
□S Theon-lineversionofthisarticle(availableathttp://www.jbc.org)contains
supplementalTableS1. merichuman/mouseRANKL;TRAP,tartrate-resistantacidphosphatase;
1Towhomcorrespondenceshouldbeaddressed:ServiceofBoneDiseases, DMAb,denosumab;OVX,ovariectomized;SC,subcutaneous;Veh,vehicle;
Dept.ofRehabilitationandGeriatrics,HUGs,4,rueGabrielle-Perret-Gentil, micro-CT, micro-computed tomography; vBMD, trabecular volumetric
1211 Geneva 14. Tel.: [phone]; Fax: [phone]; E-mail: BMD;pDXA,peripheraldual-energyx-rayabsorptiometry;TBBMD,total
[email protected]. bodyBMD;TRACP5b,serumtartrate-resistantacidphosphataseform5b;
2Theabbreviationsusedare:PTH,parathyroidhormone;BMD,bonemineral MS,mineralizingsurface;BS,bonesurface;MAR,mineralappositionrate;
density;BMU,basicmulticellullarunit;Aln,alendronate;BMP,bonemor- BFR,boneformationrate;BV/TV,bonevolumefraction;TbN,trabecular
phogeneticprotein;M-CSF,macrophagecolonystimulatingfactor;OPG, number;TbTh,thickness;OcS/BS,osteoclastsurface;OcN,osteoclastnum-
osteoprotegerin;rhOPG,recombinantformofhumanOPG;huRANKL,chi- ber;Sh,sham.
28164 JOURNALOFBIOLOGICALCHEMISTRY VOLUME285•NUMBER36•SEPTEMBER3,2010
This is an Open Access article under the CC BY license.
DenosumabandAlendronateinhuRANKLMice
eling space, and to the ability of PTH to stimulate modeling- against RANKL, denosumab (31). In this model, denosumab
basedboneformation. wasexpectedtomarkedlyreduceosteoclastnumbersandbone
Although still incompletely understood, physiological cou- remodeling, while Aln was expected to inhibit remodeling
pling between osteoclasts and osteoblasts occurs through the withoutreducingosteoclastnumbers.IntroducingPTHunder
complexactionsofsystemichormones(12,13),factorsreleased theseconditionscouldhelptodeterminewhetherPTHanabo-
byosteoclastsand/orfromthebonematrixbyresorption(e.g. lismismorerelatedtoosteoclastnumbersortotheiractivity.
TGF-(cid:1),IGF-IandII,andbonemorphogeneticproteins(BMP)) However, PTH proved capable of somewhat increasing oste-
(14),anddirectcell-to-cellcontactthroughephrinsandephrin oclastnumbersandboneformationparameterseveninpres-
receptorsonthecellsurface(15–17).Pharmacologiccoupling enceofhigh-dosedenosumab.Wethereforefurtherexamined
ofosteoclaststoosteoblastsisalsoapparentwithintermittent whether PTH could stimulate bone formation and increase
PTH administration and some evidence suggests that active bonemassinmicethatwerevirtuallydevoidofosteoclasts,i.e.
resorptionplaysanimportantrole(13–16).Forexample,PTH RANK knock-out (RANK(cid:1)/(cid:1)) mice (19, 32). Altogether our
hadnoeffectsonbonemicroarchitectureinapatientwithpyc- dataindicatethatPTHisstillcapabletostimulateboneforma-
nodysostosis,adiseasecharacterizedbythepresenceofoste- tionwhenboneresorptionisinhibited,however,themagnitude
oclasts but suppressed bone resorption due to mutations in ofPTHanaboliceffectsontheskeleton,inparticularitsaddi-
cathepsin K (18). Aln might provide a useful tool to test the tiveeffectswithantiresorptives,isdeterminedbythenumberof
hypothesis that active bone-resorbing osteoclasts are more osteoclastsonbonesurfaces.
importantthanthemerepresenceofosteoclastsforPTHanab-
EXPERIMENTALPROCEDURES
olism because Aln can potently suppress osteoclast activity
withoutnecessarilyreducingosteoclastnumbers(12). MiceandMaterials—FemalehuRANKLmice,maleRANK(cid:1)/(cid:1)
Osteoclastformation,activation,andsurvivalrequireRANK mice, and their respective wild-type (WT) littermates (C57/
andarestimulatedbyRANKligand(RANKL)andmacrophage 129/Black Swiss background) were provided by Amgen Inc.
colony-stimulating factor (M-CSF). Abundant evidence from (ThousandOaks,CA)(31).RecombinanthumanPTH-(1–34),
knock-out and transgenic rodent models have demonstrated Aln (Calbiochem, San Diego, CA), and denosumab (DMAb)
thatinhibitionoftheRANK/RANKLpathwayincreasesbone weregenerouslyprovidedbyAmgenInc.
mass (19, 20), structure (21), and strength (22). Osteoblasts/ InVivoExperiments—HuRANKLmicewereovariectomized
stromal cells also produce osteoprotegerin (OPG), a soluble (OVX) at 18 weeks of age or sham-operated under ketamine
decoyreceptorthatpreventsRANKactivationbybindingand (120mg/kg)/xylazine(16mg/kg)anesthesia(Drs.Graub,Bern,
sequestering RANKL. OPG negatively regulates osteoclasto- and Provet, Lyssach, Switzerland, respectively). Mice had
genesis, promotes apoptosis of mature osteoclasts, and ulti- accesstomousechowRM3(SDS,Betchworth,Surrey,UK)and
mately inhibits bone resorption (23). Hence, OPG-deficient wateradlibitumandwereexposedto12hlight,12hdarkcycle.
miceexhibitincreasedboneturnoverandreductionsincortical ThedayafterOVX,micewereassignedto3differentgroups
and trabecular bone volume, and they develop spontaneous andeachpretreatedgroupreceivedeitherAln(100(cid:2)g/kg,sub-
fractures (24). In contrast, overexpression of OPG or soluble cutaneous (sc), twice a week), DMAb (10 mg/kg, sc, twice a
RANK-Fc (which functions as a RANKL inhibitor) in trans- week),orvehicle(Veh,PBS)for4weeks(n(cid:2)6–8/group).The
genicrodentsinhibitedboneremodelingandresultedinhigh denosumabdosingregimenwasselectedforitsabilitytoover-
bone mass (23, 25). Concomitant administration of OPG-Fc comepotentialimmuneresponsesintheseimmunocompetent
andPTHinrodentshasshownadditiveeffectsonBMD(26,27) micetowardthefullyhumandenosumabprotein.Ineachpre-
despiteevidenceofbluntingofboneformationrelativetoPTH treatedgroup,micewerethenfurtherassignedtoPTH(1–34)
alone(28).Importantly,afteraccountingforitsantiremodeling (80 (cid:2)g/kg/d, sc) or Veh for 4 weeks (n (cid:2) 6–8/group) while
effect, OPG did not reduce the magnitude by which PTH continuing their original treatments. Sham mice (n (cid:2) 6)
increasedtheboneformationmarkerosteocalcin(29).More- receivedVehfor8weeks(Fig.1).Micereceivedascinjectionof
over, OPG did not directly inhibit differentiation of cultured calcein(20mg/kg,Sigma)at7and2daysbeforesacrifice,then
osteoblasts (28). These observations suggest that RANKL were sacrificed at week 8, and blood was collected for serum
inhibitorsmayrestrictPTHanabolismthroughtheireffectson measurements.Lumbarspineandtherightfemurwereexcised
bone remodeling rather than on bone-forming cells them- formicro-computedtomography(micro-CT)analysisandthe
selves. The effects of antiresorptives and PTH might further tibiae processed for histomorphometry. The left femur was
dependonthesequenceanddurationofexposuretotheagents. excised and frozen at (cid:1)20°C for biomechanical testing. The
PTHcombinedwithAlninrodentshasgenerallyledtoadditive animalprotocolforhuRANKLmicewasapprovedbytheEth-
effects on bone mass and structure, whereas bone formation icalCommitteeoftheUniversityofGenevaSchoolofMedicine
parameters appear suppressed relative to PTH alone (27, 28, andStateofGenevaVeterinarianOffice.
30).Theseresultsfurtherhighlightthelackofclarityregarding Male RANK(cid:1)/(cid:1) mice and WT littermates, aged to 7 to 8
theroleofosteoclastsforPTHanaboliceffectsonbone. weeksofage,weretreatedbydailyscinjectionfor14dayswith
Were-evaluatedtheroleofosteoclastsandboneresorption eitherVehorwithhumanPTHat0.4mg/kg/day(n(cid:2)9–10/
onintermittentPTHeffectsontheskeletonin2mousemodels. genotype/group).RANK(cid:1)/(cid:1)mice,whichareseverelyosteope-
The first was a knock-in mouse model expressing a chimeric troticandtoothless,weremaintainedonapowderedversionof
human/mouseRANKLprotein(huRANKL),whichwasdevel- standard mouse chow (Harlan Teklad, Madison, WI). Mice
opedtorespondtotheneutralizinghumanmonoclonalIgG2 weresacrificedafter2weeksoftreatmentandthedistalfemur
SEPTEMBER3,2010•VOLUME285•NUMBER36 JOURNALOFBIOLOGICALCHEMISTRY 28165
DenosumabandAlendronateinhuRANKLMice
Biochemical Determinations—Serum tartrate resistant acid
phosphataseform5b(TRACP5b),osteocalcin,andPTHwere
measured by ELISA according to manufacturer instructions
(IDSLtd,UK;BiomedicalTechnologiesInc.,Stoughton,MA;
and Immunotopics Inc, San Clemente, CA, respectively). In
RANK(cid:1)/(cid:1) mice, osteocalcin was measured by a Luminex
microbeadassay(Milipore/Linco,St.Charles,MO).
Biomechanical Testing—The night before mechanical test-
ing,femurswerethawedslowlyat7°Candthenmaintainedat
room temperature. The length of the femur was measured
using a digital caliper and the middle of the shaft was deter-
mined.Thefemurwasplacedinthematerialtestingmachine
on2supportsseparatedbythedistanceof9.9mm,andload
wasappliedtothemiddleoftheshaft,thuscreatinga3-point
bending test. Between the different steps of preparation,
each specimen was kept immersed in physiological solution.
Themechanicalresistancetofailurewastestedusingaservo-
controlled electromechanical system (Instron 1114, Instron
Corp.,HighWycombe,UK).Thecross-headspeedforalltests
was2mm/minandtheupperrollercontactedthefemuratthe
FIGURE 1. Schematic representation of the experimental design. anteriormid-diaphysiswiththeloaddirectionperpendicularto
huRANKLmicewereovariectomized(OVX)ornot(Sham)at18weeksofage
themedio-lateraldiameter.Bothdisplacementandloadwere
andassignedtoreceiveeitherVeh,Aln,orDMAbtreatment.After4weeks,
micewerefurtherassignedtointermittentPTH(1–34)(80(cid:2)g/kg/day)orVeh recorded. Maximal load (N) and stiffness (slope of the linear
administration,inadditiontoAln,DMAb,orVeh. part of the curve representing elastic deformation [N/mm]),
were calculated from the load-displacement curves. Ultimate
washarvestedformicro-CTanalysisoftrabecularvolumetric stress((cid:3),MPa)andYoung’smodulus(E,modulusofelasticity;
u
BMD (vBMD). The protocol and procedures for RANK(cid:1)/(cid:1) MPa)weredeterminedbytheequationspreviouslydescribed
micewereapprovedbytheInstitutionalAnimalCareandUse byTurnerandBurr(35).
CommitteeofAmgenInc. Histology—Tibiae of huRANKL knock-in mice were
BoneMineralDensityandBoneMorphology—InhuRANKL embeddedinmethylmethacrylate(Merck),and5-(cid:2)msagit-
mice,totalbody,spine,andfemoralBMD(g/cm2)wasevalu- tal sections of the proximal third of the tibiae were stained
ated in vivo using dual-energy x-ray absorptiometry (DXA, and histomorphometric measurements were performed on
PIXImus, GE Lunar, Madison, WI). Total body BMD (TB the secondary spongiosa, as previously described (36). To
BMD)valuesweresimilarbetweengroupsatbaseline,anddata measure dynamic indices of bone formation, 8 (cid:2)m sagittal
oftotalbody,spine,andfemoralBMDareexpressedaspercent sections were cut and mounted unstained for evaluation of
changefrombaseline.Weassessedtrabecularandcorticalbone
the calcein labels, as previously described (36). In the
architectureatweek8usingmicro-CT((cid:2)CT40,ScancoMedi-
DMAb-treatedmice,nosingle-ordouble-labeledbonesur-
calAG,Basserdorf,Switzerland),employinga12(cid:2)misotropic
faceswerepresentinthesectionsandthedifferentparame-
voxelsize.Specifically,trabecularbonearchitectureaswellas
tersofboneformationweredescribedas“notdetectable”in
corticalwidth(asthemeanofthewholesection)wereevaluated
Table2.Theseparametersofboneformationincludedmin-
at the 5th lumbar vertebra and distal femoral metaphysis,
eralizingsurfaces(MS/BS),mineralappositionrate(MAR),
whereascorticalbonemorphologywasevaluatedatthefemoral
andBFR.
mid-shaft,aspreviouslydescribed(33,34).
InRANK(cid:1)/(cid:1)mice,vBMDwasdeterminedusingadesktop Statistical Analysis—For the huRANKL mice, a 2-factor
analysisofvariancewasusedtoassesstheeffectofAln/DMAb
micro-CTsystem(GEeXploreLocusSPSpecimenScanner;GE
and PTH on skeletal morphology. As appropriate, post-hoc
Healthcare). The whole left femur was scanned and images
reconstructedwitha13(cid:2)misotropicvoxelsize.Contourswere testing was performed using Fisher’s Protected Least Squares
Difference(PLSD).Alltestswere2-tailed,withdifferencescon-
drawntoselectatrabecularregion(10%offemurlength)begin-
sideredsignificantatp(cid:3)0.05.Dataarepresentedasmeans(cid:4)
ning at 30% of the femur length from the distal end in
RANK(cid:1)/(cid:1) mice and 15% in WT mice. These regions were S.E.ofthemean(S.E.),unlessotherwisenoted.
selected to avoid the distal growth plate, which was wider in FortheRANK(cid:1)/(cid:1)mousestudy,1-wayANOVAfollowedby
RANK(cid:1)/(cid:1)mice,andthereforepenetratedmoredeeplyintothe Dunnett’stestwasusedtocomparedifferencesbetweenVeh-
metaphysis. Cortical regions (10% of femur length) were also treatedWTversusRANK(cid:1)/(cid:1)knock-outmice,andtocompare
selecteddistalfromthefemurmidpointforassessmentofcor- differences between Veh- and PTH-treated mice within the
ticalvBMD,aspreviouslydescribed(22).Becauseofmajordif- samegenotype.TestswererunusingJMPsoftware(SASInsti-
ferencesinBMDbetweenthegenotypes,trabecularvBMDwas tute, Cary, NC), with p (cid:3) 0.05 used to determine significant
measuredwithoutapplyingthresholds. differences.
28166 JOURNALOFBIOLOGICALCHEMISTRY VOLUME285•NUMBER36•SEPTEMBER3,2010
DenosumabandAlendronateinhuRANKLMice
TABLE1
EffectsofAln,DMAb,AlnplusPTH,orDMAbplusPTHonbonemassandvertebralandfemoralmicroarchitectureinhuRANKLmice
Inparenthesesarethenumberofmicepergroup.Conndens,connectivitydensity.
OVX
Sham(6) NoPTH PTH
Veh(6) Aln(8) DMAb(8) Veh(6) Aln(7) DMAb(8)
BMDchange(%,week8-week0)
Totalbody (cid:1)1.0(cid:4)1.2 (cid:1)4.1(cid:4)1.9 2.9(cid:4)1.0b 8.1(cid:4)1.1b,c 1.0(cid:4)0.8d 9.2(cid:4)1.2d,f 5.2(cid:4)1.9f,g
Lumbarspine (cid:1)6.8(cid:4)3.1 (cid:1)12.5(cid:4)5.6 7.6(cid:4)4.0b 7.8(cid:4)4.5b (cid:1)12.1(cid:4)2.5 11.6(cid:4)4.3f 6.3(cid:4)4.1f
Femoralshaft (cid:1)3.2(cid:4)1.3 (cid:1)1.6(cid:4)2.8 3.4(cid:4)1.9 5.5(cid:4)1.8b (cid:1)6.1(cid:4)1.1 4.1(cid:4)2.0f 4.8(cid:4)3.6f
Vertebraltrabecularbone
BV/TV(%) 21.8(cid:4)1.6 15.7(cid:4)1.3a 22.6(cid:4)0.7b 23.7(cid:4)1.6b 18.7(cid:4)1.2 28.8(cid:4)1.8d,f 24.1(cid:4)1.9f,g
TbN(/mm) 3.4(cid:4)0.1 2.9(cid:4)0.2a 3.6(cid:4)0.2b 3.7(cid:4)0.2b 3.0(cid:4)0.1 3.6(cid:4)0.1f 3.6(cid:4)0.1f
TbTh((cid:2)m) 62.5(cid:4)2.9 55.2(cid:4)1.7a 59.0(cid:4)0.9 62.5(cid:4)2.b 59.0(cid:4)0.8 69.3(cid:4)1.5d,f 62.9(cid:4)2.5g
Conndens(mm(cid:1)3) 84.1(cid:4)4.1 89.0(cid:4)9.4 85.2(cid:4)6.4 97.2(cid:4)6.5 112.6(cid:4)15.9 81.3(cid:4)5.4f 98.2(cid:4)9.7
Corticalwidth((cid:2)m) 95.3(cid:4)1.7 83.3(cid:4)2.1a 100.3(cid:4)2.1b 123.4(cid:4)5.8b,c 92.1(cid:4)2.0 114.0(cid:4)3.5d,f 114.5(cid:4)6.3f
Distalfemurtrabecularbone
BV/TV(%) 5.0(cid:4)0.8 1.3(cid:4)0.3a 4.4(cid:4)0.5 8.5(cid:4)1.4b,c 2.3(cid:4)0.4 8.6(cid:4)1.4d,f 7.0(cid:4)1.3f
TbN(/mm) 2.7(cid:4)0.2 1.6(cid:4)0.1a 2.8(cid:4)0.1b 3.9(cid:4)0.4b,c 1.9(cid:4)0.2 3.6(cid:4)0.3d,f 3.6(cid:4)0.4f
TbTh((cid:2)m) 42.4(cid:4)1.9 37.4(cid:4)2.3a 38.5(cid:4)0.9 43.2(cid:4)1.3b,c 42.1(cid:4)0.9d 45.3(cid:4)0.8d 40.8(cid:4)1.5g
Conndens(mm(cid:1)3) 25.1(cid:4)6.6 2.5(cid:4)0.7 30.6(cid:4)4.6 66.8(cid:4)14.3b,c 7.4(cid:4)1.6 72.4(cid:4)19.2d,f 52.0(cid:4)12.3f
Corticalwidth((cid:2)m) 133.4(cid:4)3.6 108.7(cid:4)5.4a 122.3(cid:4)3.3b 131.8(cid:4)5.4b 117.4(cid:4)3.4 126.2(cid:4)3.2 129.4(cid:4)4.7f
Femoralcorticalbone
Crosssectionalarea(mm2) 0.272(cid:4)0.005 0.260(cid:4)0.006 0.279(cid:4)0.008 0.283(cid:4)0.014 0.279(cid:4)0.007 0.286(cid:4)0.006 0.272(cid:4)0.007
Bonearea(mm2) 0.164(cid:4)0.002 0.140(cid:4)0.002a 0.160(cid:4)0.005b 0.153(cid:4)0.003b 0.151(cid:4)0.003d 0.167(cid:4)0.004f 0.157(cid:4)0.003
Medullaryarea(mm2) 0.108(cid:4)0.003 0.120(cid:4)0.005 0.119(cid:4)0.005 0.130(cid:4)0.013 0.128(cid:4)0.005 0.119(cid:4)0.003 0.114(cid:4)0.005
Corticalwidth((cid:2)m) 274(cid:4)1 239(cid:4)4a 262(cid:4)7b 251(cid:4)5 246(cid:4)4 267(cid:4)6f 262(cid:4)5f
ap(cid:3)0.05comparedwithsham.
bp(cid:3)0.05comparedwithOVXVeh.
cp(cid:3)0.05comparedwithOVXAln.
dp(cid:3)0.05comparedwithnon-PTHinthesamepretreatmentgroup.
fp(cid:3)0.05comparedwithOVXVeh-PTH.
gp(cid:3)0.05comparedwithOVXAln-PTH.
RESULTS contrast,PTHexertednoadditionaleffectsonmicroarchitec-
Effects of Aln, DMAb, and Combination with PTH on Bone turewhencombinedwithDMAb(Table1andFig.2,B–E).
MassandMicroarchitectureinOVXhuRANKLMice—InOVX Effects of Aln, DMAb, and Combination with PTH on Bone
huRANKLmice,AlnandDMAbbothpreventedbonelossat StrengthinOVXhuRANKLMice—Treatmenteffectsonbone
thelumbarspineandfemur,althoughDMAbcausedasignifi- strength were further evaluated at the midshaft femur by
cantlygreaterincreaseinTBBMDcomparedwithAlnafter4 3-pointbendingtests.PTHwasmosteffectiveatimprovingthe
and8weeks(Table1andFig.2A).OVXdecreasedcancellous momentofinertiaandthecorticalstructuralproperties,suchas
bonevolumefraction(BV/TV),trabecularnumber(TbN)and stiffness,comparedwithVeh(supplementalTableS1).Alnand
thickness (Tb Th), and cortical width at the distal femur and DMAb both prevented the decrease in Young’s modulus but
vertebrae,aswellascorticalwidthatthemidshaftfemur(p(cid:3) didnotimprovecorticalstiffnesscomparedwithVeh(Fig.2D
0.05 compared with sham). Aln and DMAb maintained bone andsupplementalTableS1).TheeffectsofPTHonthestruc-
microstructureattheseskeletalsites(Table1andFig.2,B–E), turalproperties(maximalloadandstiffness)weremaintained
but DMAb caused significantly greater increases than Aln in in combination with Aln, although they were not better than
microarchitecturalparametersatthedistalfemurandvertebral PTHalone.InDMAb-treatedmice,PTHshowedanon-signif-
corticalthickness(Table1andFig.2B). icanttrendtoimprovefemurstrengthcomparedDMAbalone
DailyPTHadministeredbetween4and8weekspost-OVX (Fig. 2D and supplemental Table S1), suggesting that PTH
improved TB BMD compared with OVX, but did not impact effects on bone-modeling surfaces could be maintained (see
BMDatthelumbarspineorfemoralshaft.Incombinationwith below,RANK(cid:1)/(cid:1)mice).
Aln,PTHmarkedlyimprovedBMDatallsitesandwassignifi- BiochemicalMarkersofBoneTurnoverandTheirRelation-
cantlybetterthanAlnalone.Incontrast,PTHcombinedwith ship to Bone Mass and Structure in huRANKL Mice—OVX
DMAb did not further improve TB BMD compared with increasedTRACP5bandtransientlyincreasedosteocalcincom-
DMAbalone(Table1andFig.2A).ComparedwithVeh,PTH paredwithbaseline.ThesechangeswerepreventedbyAln,how-
alonesignificantlyincreasedTbThatthedistalfemurandbone everDMAbfurtherdecreasedmarkersofboneturnoverandthere
area at the femoral midshaft (Table 1). PTH in combination wasasignificantandpersistentincreaseofserumPTHinDMAb-
withAln,comparedwithAlnalone,significantlyincreasedcan- treated mice (Fig. 3A). We next correlated TRACP5b values at
cellousboneparametersindistalfemurandvertebrae,includ- sacrifice with parameters of bone mass and structure in OVX
ingBV/TVandTbTh(Table1andFig.2B).ThePTHandAln Veh-,Aln-,andDMAb-treatedmice.TBBMD,vertebralBV/TV,
combination also improved cancellous and cortical bone andcorticalthickness,aswellasserumPTHlevelswereallne-
parameters compared with PTH alone, confirming that the 2 gatively and significantly correlated with post-treatment
treatments exerted additive effects (Table 1 and Fig. 2B). In TRACP5b levels (Fig. 3B). This indicates that the greater the
SEPTEMBER3,2010•VOLUME285•NUMBER36 JOURNALOFBIOLOGICALCHEMISTRY 28167
DenosumabandAlendronateinhuRANKLMice
FIGURE2.BMD,bonemicroarchitecture,andstrengthinOVXhuRANKLmicetreatedwithAlnorDMAb,withorwithoutPTH.A,effectsofthedifferent
notedtherapiesonTBBMD.Dataareexpressedasthepercentagechangefrombaseline(0week),DXA((cid:4)S.E.).Vertebralandfemoralcancellous(B)andcortical
(C)microarchitectureatthemidshaftfemur,andfemoralstiffness(D)inOVXhuRANKLmicetreatedwithAlnorDMAb,withorwithoutPTH,for8weeks
post-OVX.E,twodimensionalmicro-CTrepresentativeimagesofthefemoralmetaphysisanddiaphysisforeachtreatmentgroup.a,p(cid:3)0.05comparedwith
Sham;b,p(cid:3)0.05comparedwithOVXVeh;c,p(cid:3)0.05comparedwithOVXAln;d,p(cid:3)0.05comparedwithnon-PTHinthesamepretreatmentgroup;e,p(cid:3)0.05
comparedwithbaseline;f,p(cid:3)0.05comparedwithOVXVeh-PTH;g,p(cid:3)0.05comparedwithOVXAln-PTH.
suppressionofosteoclastsandboneresorption,thebetterthe Veh,buthadnosignificanteffectsonMAR(Table2).DMAb
bonemassandstructureinamonotherapysetting. suppressed all bone forming indices, as shown by the lack of
TheadditionofintermittentPTHincreasedosteocalcininall single- or double-labeled surfaces. Intermittent PTH alone
groupscomparedwiththeothernon-PTHtreatmentgroups, clearlyincreasedbone-formingindices,aswellasincombina-
reachingsignificanceintheDMAb-PTHversusDMAbgroup. tionwithAlnorDMAb.Inparticular,MARwassimilarinthe
However,meanosteocalcinlevelsremainedsignificantlylower DMAb-PTH and Aln-PTH groups, and only 25–35% lower
inDMAb-PTHthanintheVeh-PTHorAln-PTHgroups. compared with PTH alone. In contrast, PTH effects on BFR
TreatmentEffectsonBoneRemodelingIndicesinhuRANKL were more markedly affected by the antiresorptives, as it
Mice—Asevaluatedbyhistomorphometryofcancellousbone remained40%lowerinpresenceofAln,and75%lowerinpres-
attheproximaltibiametaphysis,osteoclastsurface(OcS/BS), ence of DMAb, compared with PTH alone (Table 2 and Fig.
andosteoclastnumber(OcN)weredecreasedwithDMAband 4A). BFR however was not correlated to osteoclast surfaces/
increasedwithAln(Table2).TheadditionofPTHresultedina numbersinthePTH-treatedgroups(Fig.4B),suggestingthat
significant increase in OcS/BS and OcN in DMAb-treated osteoclasts were not the most important determinants of the
mice, although these values remained lower than in the Aln- bone-formingresponsetoPTH.
treatedgroup.Boneformingindicesalsodifferedbetweenanti- However, PTH had induced the appearance of some oste-
resorptives. Aln decreased MS/BS and BFR compared with oclastsinDMAb-treatedmice,whichmighthavecontributed
28168 JOURNALOFBIOLOGICALCHEMISTRY VOLUME285•NUMBER36•SEPTEMBER3,2010
DenosumabandAlendronateinhuRANKLMice
tothePTHanabolismtheyexhibitedbyhistomorphometryand EffectsofIntermittentPTHinRANK(cid:1)/(cid:1)Mice—Forthispur-
byserumosteocalcin.Wethereforeexaminedthepotentialfor posewetreatedosteoclast-lessRANK(cid:1)/(cid:1)miceandWTcon-
PTHanabolisminRANK(cid:1)/(cid:1)mice,whicharevirtuallydevoid trolswithhigh-dosePTH(400(cid:2)g/kg/d)for2weeks.PTHsig-
ofosteoclastsevenwhenstimulatedwithPTH(31). nificantly increased osteocalcin and TRACP5b levels in WT
mice(Fig.5,AandB).InRANK(cid:1)/(cid:1)
mice, TRACP5b levels were 91%
lowerthaninWTmiceanddidnot
change with PTH. Nevertheless,
PTHincreasedserumosteocalcinin
RANK(cid:1)/(cid:1) mice similarly to WT,
indicating that increased osteocal-
cininducedbyPTHcanoccurinde-
pendently of osteoclasts or bone
resorption. Distal femur trabecular
vBMD was markedly increased in
Veh-treated RANK(cid:1)/(cid:1) mice com-
pared with Veh-treated WT con-
trols(Fig.5C).Twoweeksofinter-
mittent PTH increased trabecular
vBMDby66%inWTandby15%in
RANK(cid:1)/(cid:1) mice compared with
Veh.PTHalsosignificantlyincreased
cortical vBMD in the femoral mid-
shaftofRANK(cid:1)/(cid:1)mice,from900.0
mg/cm3 (Veh controls) to 981.5
mg/cm3 (p (cid:3) 0.05). In contrast,
PTHcausedanexpectedandmod-
est reduction in cortical vBMD in
WT mice, from 1086.8 mg/cm3
(Veh)to1061.7mg/cm3(non-signif-
icant). Representative micro-CT
imagesoftrabecularbonefromthe
distal femoral metaphysis of each
groupareshowninFig.5D.
DISCUSSION
FIGURE3.BoneserummarkersofremodelingandcorrelationtobonemassandstructureinhuRANKL
micetreatedwithAlnorDMAb,withorwithoutPTH,8weekspost-OVX.A,serumTRACP5b,osteocalcin, In this study, we examined the
andPTHweremeasuredattime0,week4,andweek8(sacrifice).B,correlationbetweenTRACP5blevels roleofosteoclastsandboneremod-
(markerofboneturnover)andbonemass,architecture,andPTH.Allparametersweremeasured8weeks
post-OVXinOVXVeh-,Aln-,andDMAb-treatedhuRANKLmice.a,p(cid:3)0.05comparedwithSham;b,p(cid:3)0.05 elingintheboneanabolicresponse
comparedwithOVXVeh;c,p(cid:3)0.05comparedwithOVXAln;d,p(cid:3)0.05comparedwithnon-PTHinthesame to intermittent PTH. More specifi-
pretreatmentgroup;e,p(cid:3)0.05comparedwithbaseline;f,p(cid:3)0.05comparedwithOVXVeh-PTH;g,p(cid:3)0.05
cally, we evaluated whether PTH
comparedwithOVXAln-PTH.
TABLE2
BoneremodelingindicesoncancellousbonesurfacesinOVXhuRANKLmicetreatedwithAln,DMAb,AlnplusPTH,orDMAbplusPTH
Inparenthesisarethenumberofmicepergroup.
OVX
Sham(6) NoPTH PTH
Veh(6) Aln(8) DMAb(6) Veh(3) Aln(5) DMAb(5)
Single-labeledbonesurface/BS(%) 0.928(cid:4)0.074 1.580(cid:4)0.119a 1.150(cid:4)0.111b n.d. 0.653(cid:4)0.080d 0.810(cid:4)0.118d,f 0.497(cid:4)0.149d,g
Double-labeledbonesurface/BS(%) 0.652(cid:4)0.088 0.751(cid:4)0.297 0.069(cid:4)0.012b n.d. 1.117(cid:4)0.117d 0.591(cid:4)30.050d,f 0.338(cid:4)0.206d,f
MS/BS(%) 22.78(cid:4)2.06 22.84(cid:4)2.82 12.16(cid:4)0.9b n.d. 31.18(cid:4)1.02d 26.24(cid:4)0.83d 11.51(cid:4)4.57d,f,g
MAR((cid:2)m/day) 1.71(cid:4)0.17 1.50(cid:4)0.07 1.30(cid:4)0.17 n.d. 1.99(cid:4)0.03 1.50(cid:4)0.24 1.31(cid:4)0.20d,f
BFR/BS((cid:2)m3/(cid:2)m2/day) 0.38(cid:4)0.05 0.35(cid:4)0.06 0.16(cid:4)0.03b n.d. 0.62(cid:4)0.01d 0.39(cid:4)0.06d,f 0.17(cid:4)0.08d,f,g
OcS/BS(%) 8.87(cid:4)1.28 10.29(cid:4)0.92 16.11(cid:4)1.58b 0.78(cid:4)0.10b,c 8.37(cid:4)0.29 13.056(cid:4)2.05f 5.12(cid:4)1.29d,g
OcN(/mm) 50.7(cid:4)6.2 29.2(cid:4)6.7 134.4(cid:4)13.2b 8.1(cid:4)1.3c 35.0(cid:4)3.1 91.4(cid:4)17.8d,f 39.8(cid:4)9.7d,g
ap(cid:3)0.05comparedwithsham.
bp(cid:3)0.05comparedwithOVXVeh.
cp(cid:3)0.05comparedwithOVXAln.
dp(cid:3)0.05comparedwithnon-PTHinthesamepretreatmentgroup.
fp(cid:3)0.05comparedwithOVXVeh-PTH.
gp(cid:3)0.05comparedwithOVX-Veh-Aln.
n.d.,notdetectable.
SEPTEMBER3,2010•VOLUME285•NUMBER36 JOURNALOFBIOLOGICALCHEMISTRY 28169
DenosumabandAlendronateinhuRANKLMice
contrasttotheeffectsofDMAb,Alnmonotherapyresultedina
paradoxicandsignificantincreaseinosteoclastsurfacesandan
expected reduction in bone formation parameters that was
consistentwiththediminutionofboneremodeling.Histologic
andbiochemicalremodelingparametersweremoresuppressed
withDMAbthanwithAln,andBMDincreasesweregenerally
greaterwithDMAbthanwithAln.Thesefindingsareconsis-
tent with previous head-to-head comparisons in human and
non-humanprimatestreatedwiththeseagents(40,41).
In huRANKL mice, administration of intermittent PTH
increasedBMD,corticalbonearea,andstiffness,inadditionto
itseffectsonBFRatcancellousbonesurfaces,comparedwith
OVX. When PTH was added to Aln, it still increased bone-
formingindicesandexertedclearadditiveeffectsonBMDand
microarchitecture,consistentwithpreviousdata(27,28).Some
clinical trials have reported that in patients treated with Aln,
beforeaswellasduringexposuretoPTH,thecombinedther-
apywassuperiortoAlnalone(42,43).However,ithasnotbeen
demonstratedthattheBMDresponsetoPTHplusantiresorp-
tivesissuperiortoPTHaloneinhumans.Whencombinedwith
DMAb,PTHwasabletosomewhatreactivateosteoclastogen-
esis,asshownbyanincreaseofosteoclastnumbersoncancel-
lous bone. PTH also increased bone-forming indices in the
presence of DMAb, indicating that the suppression of bone
turnover with DMAb was at least partially reversible, even
when DMAb treatment was continued at a dose level that
greatlyexceedsthoseusedclinically.Mostimportantly,PTH-
stimulated MAR reached similar levels in presence of DMAb
andAln,despitelargedifferencesinthenumberofosteoclasts,
indicatingthatstimulationofosteoblastactivityislargelyinde-
pendent of osteoclast number and activity. However, MS/BS
andBFRremainedmarkedlyreducedwhenPTHwasadminis-
teredwithDMAbcomparedwithintermittentPTHalone,indi-
cating that a critical reduction in osteoclast numbers, and
therebyofremodelingsurfaces,maycompromisetheextentof
FIGURE4.TibialhistologyforhuRANKLmice.A,representativehistologic
images(sagittalsection)oftibialcancellousboneofeachtreatmentgroup PTH anabolic effects, which could explain why PTH and
aftertreatmentwithAlnorDMAb,withorwithoutPTH,8weekspost-OVX. DMAb failed to exert additional effects on bone mass and
B,correlationbetweencancellousOcS/BSversusBFRforPTH-treatedanimals.
microarchitecture.
There are other potential explanations for the lack of an
couldexertadditiveeffectsonbonemassinthemodelofthe additiveBMDeffectwhenPTHwasaddedtoDMAb-treated
OVX huRANKL mouse treated with the potent osteoclast animals.The4-weekperiodofPTHco-administrationwith
inhibitor DMAb. At the doses tested in this study, DMAb DMAb might not have been sufficient to observe additive
causedgreatersuppressionofboneturnoverthanAln,resulting effectsonBMD.InanagedOVXratstudy,theadditiveeffectof
infurtherimprovementsinBMDandcancellousmicroarchi- PTHplusOPGonvertebralBMDcomparedwithPTHalone
tecture.Inturn,intermittentPTHexertedadditiveeffectswith wasnotapparentuntil3monthsaftertheirco-administration
Aln,butnotwithDMAb. (28).However,4weekswasadequatetoobserveadditiveeffects
DMAbandAlnexerttheirantiresorptiveactionthroughdif- ofPTHplusAlnonBMDundertheconditionsofthecurrent
ferent mechanisms. DMAb selectively binds to RANKL and study.AnotherpossibilityrelatestotheDMAbdosingregimen
inhibitsosteoclast-mediatedboneresorption,osteoclastmatu- (10mg/kg,twiceweekly),whichgreatlyexceedsthedoseused
ration, and survival, while Aln binds to the mineralized bone clinically for bone loss (1 mg/kg, twice yearly). This DMAb
matrix and decreases osteoclast activity, without necessarily dosingregimen,whichwasusedtoensureadequatedrugexpo-
reducingthenumberofosteoclasts(37–39).TreatmentofOVX sure in the face of potential immune responses to this fully
huRANKL mice with DMAb alone was associated with a humanprotein,mightlimittheabilityofthesefindingstopre-
markedreductionofosteoclastsurfacesandwithhistological dictclinicaloutcomesinhumans.Finally,animalspre-treated
parametersofboneformationthatwerebarelydetectable.The withDMAbhadhigherBMDthanthosepretreatedwithAlnat
latter finding indicates that remodeling is the predominant the time PTH was introduced, which could limit the former
physiologicalformofboneturnoverinthesemiceatthisage, grouppotentialforfurtherBMDincrementswithPTH.Con-
and that remodeling was markedly suppressed by DMAb. In sistentwiththisnotion,PTHwasabletoincreaseBMDmuch
28170 JOURNALOFBIOLOGICALCHEMISTRY VOLUME285•NUMBER36•SEPTEMBER3,2010
DenosumabandAlendronateinhuRANKLMice
thelocalinjectionofanevenhigher
concentration ((cid:5)1,400 (cid:2)g/kg/day)
of parathyroid hormone-related
protein (PTHrP), which activates
the same receptor as PTH, was
unabletoinducetheappearanceof
any osteoclasts in the calvaria of
RANK(cid:1)/(cid:1)mice(32).Inthecurrent
study,short-termadministrationof
high-dose PTH (400 (cid:2)g/kg/day)
resulted in an increase of serum
osteocalcinthatwassimilartoWT
mice, indicating that both geno-
types could exhibit bone-forming
responsestoPTH.
PTH-mediated increases in se-
rum osteocalcin in aged OVX rats
were previously shown to be un-
affected by co-administration of
recombinant OPG (29), suggesting
that this phenomenon is not re-
strictedtoyoungosteopetroticmice
and does not require high doses of
PTH. Also, PTH significantly
FIGURE5.Effectsof2weeksofintermittentPTHtreatmentonserumTRACP5b,osteocalcin,andtrabe-
cularBMDinWTandRANK(cid:1)/(cid:1)mice.A,serumTRACPinWTandRANK(cid:1)/(cid:1)miceinresponsetoPTH.B,serum increased trabecular vBMD in
osteocalcininPTH-treatedWTandRANK(cid:1)/(cid:1)mice.C,effectsofPTHontrabecularvBMDinthedistalfemur RANK(cid:1)/(cid:1)mice,eventhoughtheir
metaphysisofWTandRANK(cid:1)/(cid:1)mice.D,representativemicro-CTimagesofthedistalfemurmetaphysisofWT
andRANK(cid:1)/(cid:1)micetreatedwithVehorwithintermittentPTH.Selectedimagesrepresentthemediantrabec- baseline trabecular vBMD was
ularbonevolumefractionvaluefromeachgroup.a,p(cid:3)0.05comparedwithVeh-treatedWT;b,p(cid:3)0.05 already extremely high. This high
comparedwithVeh-treatedRANK(cid:1)/(cid:1).
bone mass phenotype probably
explainswhytheBMDresponseto
more in WT mice compared with osteopetrotic RANK(cid:1)/(cid:1) PTH in RANK(cid:1)/(cid:1) mice was somewhat lower than the
mice. Nonetheless, the overall data are consistent with the response observed in WT controls, which had much more
hypothesis that trabecular bone remodeling is an important marrowspaceavailablefornewboneformation.Theosteo-
attributeofPTHanabolisminhuRANKLmice. calcin and BMD responses to PTH in RANK(cid:1)/(cid:1) mice
If remodeling is indeed important for PTH anabolism, it is occurred despite very low basal levels of serum TRACP5b,
importanttounderstandwhy,undercertainconditions,PTHis and despite a complete lack of response of this osteoclast
capableofrobustlyincreasingBMDinmiceorratsco-treated marker to PTH. It is also interesting to note that PTH also
withpotentantiresorptivessuchasAln(28,29,30)orOPG(28, increasedcorticalvBMDinRANK(cid:1)/(cid:1)mice,butnotinWT
30).Thisisaparticularlyimportantquestionbecausetheaddi- mice.IncreasedcorticalBMDisnottypicallyobservedwith
tiveeffectsofPTHandAlnonvertebralBMDinrodentsstands PTH administration, primarily due to increases in oste-
incontrasttothebluntedvertebralBMDresponsetoPTHplus oclast-mediatedcorticalporosity(44).Henceseverallinesof
AlnversusPTHaloneinhumanstudies(9,10).Onehypothesis evidenceindicatethatboneformationcouldbestimulatedin
isthatmiceandratshaveagreatercapacitythanhumansfor RANK(cid:1)/(cid:1) mice in the absence of basal or stimulated bone
PTH stimulation of modeling-based bone formation, which resorption.ThefactthatPTHincreasedBMDinRANK(cid:1)/(cid:1)
occursintheabsenceofpriorosteoclastactivation. micebutnotinDMAb-treatedhuRANKLmicecouldrelate
TheextenttowhichPTHcanincreaseBMDinmiceviapure tothehigherdoseofPTH.
modeling-basedboneformationwasexaminedbyadminister- Erben showed that in young rats, most of the cancellous
ingPTHtoRANK(cid:1)/(cid:1)mice,whicharedevoidofosteoclastsand bone forming sites in the untreated condition were model-
arethereforeincapableoftrueboneremodeling(32).Ashort ing-based(4).Comparedwithyoungratsormice,theextent
(2-week)durationwaschosentominimizehandlingandstress of modeling-based bone formation in the untreated adult
totheseverelyosteopetroticandtoothlessRANK(cid:1)/(cid:1)mice.A humanosteoporoticskeletonislikelytobemuchlower(45).
high dose of PTH was used to produce a sufficiently robust The extent to which PTH can stimulate modeling-based
BMDresponseofWTmicetoPTH,overthisbrieftreatment bone formation in this population is unclear. Current evi-
period,todeterminewhetherornotRANK(cid:1)/(cid:1)miceweretruly denceindicatesthatthisextentisverylowinthesteady-state
resistanttoPTH.Itwasnotpossibletoperformreliablehisto- situation after long-term PTH therapy (5), but could be
morphometryontrabecularsurfacesinRANK(cid:1)/(cid:1)mice,dueto higher during the earlier phase of treatment (6). Based on
theirlackofmarrowspaceandabsenceofclearlydefinedtra- theseobservationsandthecurrentresults,itseemsreason-
becular surfaces (32). However, it was previously shown that abletohypothesizethattheneteffectsofPTHontrabecular
SEPTEMBER3,2010•VOLUME285•NUMBER36 JOURNALOFBIOLOGICALCHEMISTRY 28171
DenosumabandAlendronateinhuRANKLMice
BMDinpostmenopausalwomenreceivingDMAbwouldbe
Acknowledgments—WethankFannyCavatandMadeleineLachize
greatestintheearlyperiodofconcomitanttherapy,priorto
ofGeneva,andFrankAsuncion,DeniseDwyer,andSeanMoronyof
the refilling of remodeling space (i.e. short-lived). Over the
Amgen Inc. for their skillful technical expertise. Erica Rockabrand,
long term, the overall BMD response to PTH in DMAb-
PhDofAmgenInc.providedassistancewitheditingandformatting.
treatedsubjectsmightbedeterminedprimarilybythecapac-
ityofPTHtopromotetrabecularmodeling-basedbonefor-
REFERENCES
mation, and hence remain quite modest. At cortical sites,
antiresorptiveco-therapywithAlndoesnotappeartosignif- 1. Cosman,F.(2008)Curr.Opin.Endocrinol.DiabetesObes.15,495–501
icantly blunt PTH-mediated BMD increases (9, 10). This 2. Girotra,M.,Rubin,M.R.,andBilezikian,J.P.(2006)Rev.Endocr.Metab.
Disord.7,113–121
outcomemightreflectthepotentialforAlntoreducecorti-
3. Hodsman,A.B.,andSteer,B.M.(1993)Bone14,523–527
calporosityandremodeling-basedcorticalboneformation,
4. Erben,R.G.(1996)Anat.Rec.246,39–46
whichcouldhaveoffsettingeffectsoncorticalBMDinthat 5. Ma, Y. L., Zeng, Q., Donley, D. W., Ste-Marie, L. G., Gallagher, J. C.,
population. Similarly, DMAb significantly reduced cortical Dalsky,G.P.,Marcus,R.,andEriksen,E.F.(2006)J.BoneMiner.Res.21,
bone remodeling and cortical porosity in adult OVX cyno- 855–864
6. Lindsay,R.,Cosman,F.,Zhou,H.,Bostrom,M.P.,Shen,V.W.,Cruz,J.D.,
molgus monkeys (46) and RANKL inhibition by OPG
Nieves,J.W.,andDempster,D.W.(2006)J.BoneMiner.Res.21,366–373
reduced cortical porosity in mice with constitutively active
7. Sato,M.,Westmore,M.,Ma,Y.L.,Schmidt,A.,Zeng,Q.Q.,Glass,E.V.,
PTH receptors (47). The ability of PTH to increase cortical
Vahle,J.,Brommage,R.,Jerome,C.P.,andTurner,C.H.(2004)J.Bone
vBMD in RANK(cid:1)/(cid:1) mice, in which bone resorption was at Miner.Res.19,623–629
undetectablelevels,suggeststhatintermittentPTHtherapy 8. Dempster,D.W.,Parisien,M.,Silverberg,S.J.,Liang,X.G.,Schnitzer,M.,
mighthavepositiveeffectsoncorticalBMDinpatientsco- Shen,V.,Shane,E.,Kimmel,D.B.,Recker,R.,Lindsay,R.,andBilezikian,
J.P.(1999)J.Clin.Endocrinol.Metab.84,1562–1566
treatedwithDMAb.
9. Black,D.M.,Greenspan,S.L.,Ensrud,K.E.,Palermo,L.,McGowan,J.A.,
Insummary,DMAbreducedboneremodelingandincreased
Lang,T.F.,Garnero,P.,Bouxsein,M.L.,Bilezikian,J.P.,andRosen,C.J.
BMDinOVXhuRANKLmicetoagreaterextentthanAlnatthe (2003)N.Engl.J.Med.349,1207–1215
dosestested.WhileBMDtendedtobeincreasedbytheadditionof 10. Finkelstein,J.S.,Hayes,A.,Hunzelman,J.L.,Wyland,J.J.,Lee,H.,and
PTHtreatmentinanimalspretreatedwithAln,thisadditiveeffect Neer,R.M.(2003)N.Engl.J.Med.349,1216–1226
wasnotapparentinmicepre-treatedwithDMAb.Theseresults 11. Finkelstein,J.S.,Leder,B.Z.,Burnett,S.M.,Wyland,J.J.,Lee,H.,delaPaz,
A.V.,Gibson,K.,andNeer,R.M.(2006)J.Clin.Endocrinol.Metab.91,
are consistent with the hypothesis that the capacity for bone
2882–2887
remodeling,andtheavailabilityofremodelingspace,aregenerally
12. Martin,T.J.,Quinn,J.M.,Gillespie,M.T.,Ng,K.W.,Karsdal,M.A.,and
importantattributesofPTHanabolism.Whetherthisremodeling Sims,N.A.(2006)Ann.N.Y.Acad.Sci.1068,458–470
capacityisadvantageousintermsoftheoverallpotentialforBMD 13. Henriksen,K.,Neutzsky-Wulff,A.V.,Bonewald,L.F.,andKarsdal,M.A.
gainsinresponsetoPTHisunclear,asBMDinanimalstreated (2009)Bone44,1026–1033
14. Martin,T.J.,andSims,N.A.(2005)TrendsMol.Med.11,76–81
with Aln plus PTH was generally similar to BMD in animals
15. Zhao,C.,Irie,N.,Takada,Y.,Shimoda,K.,Miyamoto,T.,Nishiwaki,T.,
treatedwithDMAbalone,inwhichremodelingspacewasmini-
Suda,T.,andMatsuo,K.(2006)CellMetab.4,111–121
mal.ThefactthatPTHincreasedbonemassinRANK(cid:1)/(cid:1)mice,
16. Allan,E.H.,Ha¨usler,K.D.,Wei,T.,Gooi,J.H.,Quinn,J.M.,Crimeen-
whicharedevoidofosteoclastsandwhichshowednoevidenceof Irwin,B.,Pompolo,S.,Sims,N.A.,Gillespie,M.T.,Onyia,J.E.,andMar-
bone resorption with PTH administration, demonstrates that tin,T.J.(2008)J.BoneMiner.Res.23,1170–1181
micealsohaveasignificantcapacityforPTH-stimulatedmodel- 17. Pierroz,D.D.,Rufo,A.,Bianchi,E.N.,Glatt,V.,Capulli,M.,Rucci,N.,
Cavat,F.,Rizzoli,R.,Teti,A.,Bouxsein,M.L.,andFerrari,S.L.(2009)
ing-basedboneformation.Thebalanceofmodeling-andremod-
J.BoneMiner.Res.24,775–784
eling-based bone formation might ultimately dictate skeletal
18. Chavassieux,P.,AsserKarsdal,M.,Segovia-Silvestre,T.,Neutzsky-Wulff,
responsivenesstoPTHandtheabilityofPTHtoincreasebone A.V.,Chapurlat,R.,Boivin,G.,andDelmas,P.D.(2008)J.BoneMiner.Res.
massinthepresenceofantiresorptiveco-therapy.ThehuRANKL 23,1076–1083
mousestudyalsoprovidesthefirstevidencethatPTHcansignifi- 19. Dougall,W.C.,Glaccum,M.,Charrier,K.,Rohrbach,K.,Brasel,K.,De
cantlyincreasehistologicparametersofboneresorptionandbone Smedt,T.,Daro,E.,Smith,J.,Tometsko,M.E.,Maliszewski,C.R.,Arm-
strong,A.,Shen,V.,Bain,S.,Cosman,D.,Anderson,D.,Morrissey,P.J.,
formationdespitetheco-administrationofDMAb.Thisobserva-
Peschon,J.J.,andSchuh,J.(1999)GenesDev.13,2412–2424
tionsuggeststhatDMAbmightstillpermitthelocalactivationof
20. Kong,Y.Y.,Yoshida,H.,Sarosi,I.,Tan,H.L.,Timms,E.,Capparelli,C.,
boneremodelingatthetissuelevelundercertainstimulatedcon- Morony,S.,Oliveira-dos-Santos,A.J.,Van,G.,Itie,A.,Khoo,W.,Wake-
ditions,evenwhensystemicturnoverappearstoremainverylow. ham,A.,Dunstan,C.R.,Lacey,D.L.,Mak,T.W.,Boyle,W.J.,andPen-
Future studies will examine whether such local stimulation of ninger,J.M.(1999)Nature397,315–323
remodeling might lead to increased BMD with PTH treatment 21. Min,H.,Morony,S.,Sarosi,I.,Dunstan,C.R.,Capparelli,C.,Scully,S.,
Van,G.,Kaufman,S.,Kostenuik,P.J.,Lacey,D.L.,Boyle,W.J.,andSimo-
after longer periods of co-treatment with DMAb. From these
net,W.S.(2000)J.Exp.Med.192,463–474
observations we conclude that antiresorptives did not preclude
22. Ominsky,M.S.,Stolina,M.,Li,X.,Corbin,T.J.,Asuncion,F.J.,Barrero,
PTHanaboliceffectsontheskeletonandthatneitherboneresorp- M.,Niu,Q.T.,Dwyer,D.,Adamu,S.,Warmington,K.S.,Grisanti,M.,
tionnorosteoclastswerestrictlynecessaryforPTH-inducedbone Tan,H.L.,Ke,H.Z.,Simonet,W.S.,andKostenuik,P.J.(2009)J.Bone
formation.However,areductioninosteoclastnumberand/orsur- Miner.Res.24,1234–1246
23. Simonet,W.S.,Lacey,D.L.,Dunstan,C.R.,Kelley,M.,Chang,M.S.,
face,bylimitingtheextentoftheremodelingspace,willrestrictthe
Lu¨thy,R.,Nguyen,H.Q.,Wooden,S.,Bennett,L.,Boone,T.,Shimamoto,
actionsofPTHtothemodelingspace,whichmaythenrequire
G.,DeRose,M.,Elliott,R.,Colombero,A.,Tan,H.L.,Trail,G.,Sullivan,J.,
higherdosesand/orlongerperiodsofadministrationtobeeffec-
Davy,E.,Bucay,N.,Renshaw-Gegg,L.,Hughes,T.M.,Hill,D.,Pattison,
tiveatincreasingbonemass. W.,Campbell,P.,Sander,S.,Van,G.,Tarpley,J.,Derby,P.,Lee,R.,and
28172 JOURNALOFBIOLOGICALCHEMISTRY VOLUME285•NUMBER36•SEPTEMBER3,2010
DenosumabandAlendronateinhuRANKLMice
Boyle,W.J.(1997)Cell89,309–319 1854–1862
24. Bucay,N.,Sarosi,I.,Dunstan,C.R.,Morony,S.,Tarpley,J.,Capparelli,C., 35. Turner,C.H.,andBurr,D.B.(1993)Bone14,595–608
Scully,S.,Tan,H.L.,Xu,W.,Lacey,D.L.,Boyle,W.J.,andSimonet,W.S. 36. Baldock,P.A.,Sainsbury,A.,Allison,S.,Lin,E.J.,Couzens,M.,Boey,D.,
(1998)GenesDev.12,1260–1268 Enriquez,R.,During,M.,Herzog,H.,andGardiner,E.M.(2005)J.Bone
25. Hsu,H.,Lacey,D.L.,Dunstan,C.R.,Solovyev,I.,Colombero,A.,Timms, Miner.Res.20,1851–1857
E.,Tan,H.L.,Elliott,G.,Kelley,M.J.,Sarosi,I.,Wang,L.,Xia,X.Z.,Elliott, 37. Lacey,D.L.,Timms,E.,Tan,H.L.,Kelley,M.J.,Dunstan,C.R.,Burgess,
R.,Chiu,L.,Black,T.,Scully,S.,Capparelli,C.,Morony,S.,Shimamoto,G., T.,Elliott,R.,Colombero,A.,Elliott,G.,Scully,S.,Hsu,H.,Sullivan,J.,
Bass, M. B., and Boyle, W. J. (1999) Proc. Natl. Acad. Sci. U.S.A. 96, Hawkins,N.,Davy,E.,Capparelli,C.,Eli,A.,Qian,Y.X.,Kaufman,S.,
3540–3545 Sarosi,I.,Shalhoub,V.,Senaldi,G.,Guo,J.,Delaney,J.,andBoyle,W.J.
26. Kostenuik,P.J.,Capparelli,C.,Morony,S.,Adamu,S.,Shimamoto,G., (1998)Cell93,165–176
Shen, V., Lacey, D. L., and Dunstan, C. R. (2001) Endocrinology 142,
38. Rogers,M.J.,Gordon,S.,Benford,H.L.,Coxon,F.P.,Luckman,S.P.,
4295–4304
Monkkonen,J.,andFrith,J.C.(2000)Cancer88,2961–2978
27. Samadfam, R., Xia, Q., and Goltzman, D. (2007) Endocrinology 148,
39. Weinstein,R.S.,Roberson,P.K.,andManolagas,S.C.(2009)N.Engl.
2778–2787
J.Med.360,53–62
28. Samadfam,R.,Xia,Q.,andGoltzman,D.(2007)J.BoneMiner.Res.22,
40. Ominsky, M., Jolette, J., Smith, S. Y., Vlasseros, F., Samadfam, R., and
55–63
Kostenuik,P.J.(2008)J.BoneMin.Res.23,S61
29. Kostenuik,P.J.(2005)Curr.Opin.Pharmacol.5,618–625
41. Brown,J.P.,Prince,R.L.,Deal,C.,Recker,R.R.,Kiel,D.P.,deGregorio,
30. Johnston,S.,Andrews,S.,Shen,V.,Cosman,F.,Lindsay,R.,Dempster,
L.H.,Hadji,P.,Hofbauer,L.C.,Alvaro-Gracia,J.M.,Wang,H.,Austin,M.,
D.W.,andIida-Klein,A.(2007)Endocrinology148,4466–4474
Wagman,R.B.,Newmark,R.,Libanati,C.,SanMartin,J.,andBone,H.G.
31. Kostenuik,P.J.,Nguyen,H.Q.,McCabe,J.,Warmington,K.S.,Kurahara,
(2009)J.BoneMiner.Res.24,153–161
C.,Sun,N.,Chen,C.,Li,L.,Cattley,R.C.,Van,G.,Scully,S.,Elliott,R.,
42. Cosman,F.,Nieves,J.,Zion,M.,Woelfert,L.,Luckey,M.,andLindsay,R.
Grisanti,M.,Morony,S.,Tan,H.L.,Asuncion,F.,Li,X.,Ominsky,M.S.,
(2005)N.Engl.J.Med.353,566–575
Stolina,M.,Dwyer,D.,Dougall,W.C.,Hawkins,N.,Boyle,W.J.,Simonet,
W.S.,andSullivan,J.K.(2009)J.BoneMiner.Res.24,182–195 43. Cosman,F.,Wermers,R.A.,Recknor,C.,Mauck,K.F.,Xie,L.,Glass,E.V.,
32. Li,J.,Sarosi,I.,Yan,X.Q.,Morony,S.,Capparelli,C.,Tan,H.L.,McCabe, andKrege,J.H.(2008)CalcifTissueInt.82,S59
S.,Elliott,R.,Scully,S.,Van,G.,Kaufman,S.,Juan,S.C.,Sun,Y.,Tarpley, 44. Fox,J.,Miller,M.A.,Newman,M.K.,Recker,R.R.,Turner,C.H.,and
J.,Martin,L.,Christensen,K.,McCabe,J.,Kostenuik,P.,Hsu,H.,Fletcher, Smith,S.Y.(2007)Bone41,321–330
F.,Dunstan,C.R.,Lacey,D.L.,andBoyle,W.J.(2000)Proc.Natl.Acad.Sci. 45. Takahashi,H.,Hattner,R.,Epker,B.N.,andFrost,H.M.(1964)Henry
U.S.A.97,1566–1571 FordHosp.Med.Bull.12,359–364
33. Bouxsein,M.L.,Pierroz,D.D.,Glatt,V.,Goddard,D.S.,Cavat,F.,Rizzoli, 46. Ominsky,M.S.,Schroeder,J.,Jolette,J.,Smith,S.Y.,Farell,D.J.,Atkinson,
R.,andFerrari,S.L.(2005)J.BoneMiner.Res.20,635–643 J.E.,andKostenuik,P.J.(2008)J.BoneMin.Res.22,S88
34. Ferrari,S.L.,Pierroz,D.D.,Glatt,V.,Goddard,D.S.,Bianchi,E.N.,Lin, 47. Ohishi,M.,Chiusaroli,R.,Ominsky,M.,Asuncion,F.,Thomas,C.,Khatri,
F. T., Manen, D., and Bouxsein, M. L. (2005) Endocrinology 146, R.,Kostenuik,P.,andSchipani,E.(2009)Am.J.Pathol.174,2160–2171
SEPTEMBER3,2010•VOLUME285•NUMBER36 JOURNALOFBIOLOGICALCHEMISTRY 28173