Withaferin A: a proteasomal inhibitor promotes healing after injury and exerts anabolic effect on

17 Pages·2013·5.35 MB·English

by  V Khedgikar

OPEN Citation:CellDeathandDisease(2013)4,e778;doi:10.1038/cddis.2013.294
&2013MacmillanPublishersLimited Allrightsreserved 2041-4889/13
www.nature.com/cddis
Withaferin A: a proteasomal inhibitor promotes healing
after injury and exerts anabolic effect on osteoporotic
bone
VKhedgikar1,PKushwaha1,JGautam1,AVerma2,BChangkija1,AKumar1,SSharma2,GKNagar1,DSingh1,PKTrivedi3,
NSSangwan4,PRMishra2andRTrivedi*,1
WithaniasomniferaorAshwagandhaisamedicinalherbofAyurveda.Thoughtheextractandpurifiedmolecules,withanolides,
from this plant have been shown to have different pharmacological activities, their effect on bone formation has not been
studied.Here,weshowthatoneofthewithanolide,withaferinA(WFA)actsasaproteasomalinhibitor(PI)andbindstospecific
catalytic b subunit of the 20S proteasome. It exerts positive effect on osteoblast by increasing osteoblast proliferation and
differentiation. WFA increased expression of osteoblast-specific transcription factor and mineralizing genes, promoted
osteoblastsurvivalandsuppressedinflammatorycytokines.Inosteoclast,WFAtreatmentdecreasedosteoclastnumberdirectly
bydecreasingexpressionoftartarate-resistantacidphosphataseandreceptoractivatorofnuclearfactorkappa-B(RANK)and
indirectlybydecreasingosteoprotegrin/RANKligandratio.OurdatashowthatinvitrotreatmentofWFAtocalvarialosteoblast
cells decreased expression of E3 ubiquitin ligase, Smad ubiquitin regulatory factor 2 (Smurf2), preventing degradation of
Runt-related transcription factor 2 (RunX2) and relevant Smad proteins, which are phosphorylated by bone morphogenetic
protein2.IncreasedSmurf2expressionduetoexogenoustreatmentoftumornecrosisfactora(TNFa)toprimaryosteoblastcells
was decreased by WFA treatment. This was corroborated by using small interfering RNA against Smurf2. Further, WFA also
blocked nuclear factor kappa-B (NF-kB) signaling as assessed by tumor necrosis factor stimulated nuclear translocation of
p65-subunit of NF-kB. Overall data show that in vitro proteasome inhibition by WFA simultaneously promoted
osteoblastogenesis by stabilizing RunX2 and suppressed osteoclast differentiation, by inhibiting osteoclastogenesis. Oral
administrationofWFAtoosteopenicovariectomizedmiceincreasedosteoprogenitorcellsinthebonemarrowandincreased
expression of osteogenic genes. WFA supplementation improved trabecular micro-architecture of the long bones, increased
biomechanicalstrengthparametersofthe vertebra andfemur,decreasedboneturnovermarkers(osteocalcinandTNFa)and
expressionofskeletalosteoclastogenicgenes.Italsoincreasednewboneformationandexpressionofosteogenicgenesinthe
femur bone as compared with vehicle groups (Sham) and ovariectomy (OVx), Bortezomib (known PI), injectible parathyroid
hormoneandalendronate(FDAapproveddrugs).WFApromotedtheprocessofcorticalboneregenerationatdrill-holessitein
thefemurmid-diaphysisregionandcorticalgapwasbridgedwithwovenbonewithin11daysofbothestrogensufficientand
deficient(ovariectomized,Ovx)mice.TogetherourdatasuggestthatWFAstimulatesboneformationbyabrogatingproteasomal
machineryandprovidesknowledgebaseforitsclinicalevaluationasaboneanabolicagent.
CellDeathandDisease(2013)4,e778;doi:10.1038/cddis.2013.294;publishedonline22August2013
SubjectCategory:ExperimentalMedicine
Bone mass is dependent on the balance between bone regulated by a number of regulatory factors including bone
formation by osteoblasts and bone resorption by osteo- morphogenetic proteins (BMPs), key signal transduction
clasts.1–3 The differentiation and function of these cells is molecules and critical transcription factors including Runt-
1DivisionofEndocrinologyandCenterforResearchinAnabolicSkeletalTargetsinHealthandIllness(ASTHI),CSIR-CentralDrugResearchInstitute,Lucknow226001,
India;2DivisionofPharmaceutics,CSIR-CentralDrugResearchInstitute,Lucknow226001,India;3PlantGeneExpressionDivision,CSIR-NationalBotanicalResearch
Institute,Lucknow226001,Indiaand4MetabolicandStructuralBiology,PlantBiologyDivision,CSIR-CentralInstituteofMedicinalandAromaticPlants,Lucknow
226001,India
*Correspondingauthor:RTrivedi,DivisionofEndocrinologyandCenterforResearchinAnabolicSkeletalTargetsinHealthandIllness(ASTHI),CSIR-CentralDrug
ResearchInstitute,Lucknow226001,India.Tel:[phone];Fax:[phone];E-mail:[email protected][email protected]
Keywords:osteoporosis;proteasome;boneformation;withaferin;drillhole;bonehealing;micro-CT
Abbreviations: Ald,alendronate;ALP,alkalinephosphataseactivity;AUC,areaunderthecurve;BFR,boneformationrate;BrdU,5-Bromo-20-deoxyuridine;Bzb,
Bortezomib;BMCs,bonemarrowcells;BMP2,bonemorphogeneticprotein2;BV/TV,bonevolume/tissuevolume;ColI,collagentypeI;EBSS,earle’sbalancedsalt
solution; IL-6, interleukin-6; MAR, mineral apposition rate; MCO, mouse calverial osteoblast; m-CT, micro-computed tomography; iPTH, injectible parathyroid
hormone;MCP-1,monocytechemo-attractantprotein-1;MRT,meanresidencetime;NF-kB,nuclearfactork-B;OCN,osteocalcin;OPG,osteoprotegrin;OVx,
ovariectomy;PI,proteasomalinhibitor;PNPP,p-nitrophenylpyro-phosphate;Q-PCR,quantitativerealtimePCR;RunX2,runtrelatedtranscriptionfactor2;RANKL,
receptoractivatorofNF-kBligand;RANK,receptoractivatorofnuclearfactorkappa-B;Smurf2,smadubiquitinregulatoryfactor;SMI,structuralmodelindex;TRAP,
tartarate resistant acid phosphatase; Tb.No, trabecular number; Tb.Th, trabecular thickness; TNF-a, tumor necrosis factor a; Vd, volume of distribution;
WFA,withaferinA
Received06.11.12;revised16.5.13;accepted29.5.13;EditedbyMFederici
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relatedtranscriptionfactor2(RunX2).4Outofthesefactors, a highly oxygenated withanolide, which is the major
signaling by BMP is important in bone formation, which biologically active constituent.23,24 Despite these studies
involvestheubiquitin–proteasomepathway.Inthispathway, on pharmacological activities of WFA, their mechanism of
Smad ubiquitin regulatory factor 1 (Smurf1), which is E3 actionisnotfullyunderstood.
ubiquitinligase,directlyinteractswithRunX2andinducesits We demonstrate that WFA induces osteoblast differen-
degradation in a ubiquitin- and proteasome-dependent tiation by inhibiting the proteasomal machinery decrea-
manner.5 Conversely, BMP2 protects RunX2 from Smurf- singSmurf2expressionanddegradationofRunX2protein.
catalyzedproteolysisbystimulatingRunX2acetylation,which This was validated using small interfering RNA (siRNA)
occursbySmad-dependentmechanism.6Studiesshowthat against Smurf2 and TNFa that increases Smurf2 expre-
under patho-physiological conditions the inflammatory ssion. To mimic post-menopausal condition, we carried
cytokine tumor necrosis factor a (TNFa) increases Smurf1 outinvivoexperiments,byovariectomizingmicethatisan
expression in osteoblasts.7 This results in degradation of equivalent animal model for post-menopausal osteo-
Smad1andRunX2,andsubsequentlyimpairedosteoblasto- porosis.25,26 Overall, our findings point towards a novel
genesis.5Experimentsintransgenicmicesupporttheabove molecular mechanism for WFA on bone. Based on these
findingsasSmurf1knockoutmicearebornnormalbutexhibit preclinical data, we provide a rationale for the use of
age-dependent increases in bone mass.8 At the same time, WFA, a natural PI, in the treatment of post-menopausal
transgenic mice overexpressing Smurf1 in osteoblasts osteoporosis.
have impaired bone formation during postnatal life.7–11
Reports also provide evidence for interaction of Smad Results
ubiquitin regulatory factor 2 (Smurf2) with BMP-activated
Smad1. Smurf2 that is a Smurf1-related member of hect Effect of WFA on osteoblast survival, proliferation and
domainE3ligase,definessubstratespecificitybyrecogniz- mineralization. Figure1ashowschemicalstructureofWFA
ing the target protein and then degradation by 26 S (MW¼470.6). Mice calvarial osteoblasts (MCOs) were
proteasome.12,13 The proteasome is also directly involved tested with increasing concentrations (1pM to 1mM) of
in osteoclastogenesis by directly controlling important WFA(Figure1b).WFAconcentrationdependentlystimulated
signaling pathway like nuclear factor kappa-B (NF-kB) osteoblast proliferation as assessed by direct cell count
pathway.14 (Figure 1bI) and 5-bromo-2-deoxyuridine (BrdU) incorpora-
Clinical and experimental research revealed that tion assay (Figure 1bII). Changes in cell count were
inhibition of proteasome activity is critical for the activation calculated compared with control group set as 100. As
of osteogenic transcription factors and bone metabolism.15 10nM concentration of WFA maximally stimulated MCOs’
Further, preclinical studies on synthetic proteasomal proliferation, this concentration was used in remaining
inhibitor’s (PI’s) like bortezomib (Bzb; anti-myloma agent) experiments. MCOs were treated with WFA and Bzb (1pM
show that it promotes osteoblastogenesis and suppresses to 1mM) and stimulation of alkaline phosphatase (ALP)
osteoclastogenesis by regulating the ubiquitin–proteasome- activity was studied. Dose of 10nM was most effective in
dependent degradationofproteins.16–18 However,nonatural stimulating ALP activity (Po0.01) as compared with control
PIisreportedforenhancedosteogeniceffect. (Figure1d).At10nMALPactivityofWFAwasapproximately
Withaniasomniferaisoneofthemostancientherbused threefoldhigherascomparedwithBzb(Po0.001).However,
asmedicineanddietarysupplement.19,20InAyurveda,the reducedproliferation observedat 1mMmaybedue tosteric
leaves and roots of Withania are prescribed to cure hindranceofWFAinosteoblasts.
inflammation-related disorders.21 This plant has been WFA (10nM) treatment stimulated osteoblast minerali-
studied extensively for its biologically active constituents, zation(Figure1e)(Po0.001)comparedwithcontrolandBzb
steroidal lactones and withanolides.22 The health benefits (Po0.001)(Figure1e).Further,WFAincreasedexpressionof
of Ashwagandha are supported by clinical trials in case of osteogenic genes (Figure 1f) with increased expression of
inflammation, immune modulation and reducing arthritis transcriptionfactorassociatedwithosteoblastdifferentiation.
pain.ThemostabundantconstituentiswithaferinA(WFA), Of the studied genes, RunX2 (2.5-fold, Po0.001), collagen
Figure1 WFAhasosteogeniceffectinvitro.(a)StructureofWFA.(b)EffectofWFAonprimaryosteoblastproliferation.Primaryosteoblastswereculturedinincreasing
concentrationsofWFAandBzbfor24handharvestedforcellproliferationdirectcellcountforcellproliferationassay(I)andBrdUincorporationcellproliferationassay(II).The
resultswereexpressedasrelativecellgrowthinpercentage,whichwascomparedwithcontrolgroup.Wesetthecontrolgroupas100.Valuesrepresentsmean±S.E.ofthree
independentexperiments(n¼3).*Po0.05,**Po0.01whencomparedwithcontrol.(c)WFAtreatmentofMCOsfor48hinosteoblastdifferentiationmediumsignificantly
increasedALPproductioncomparedwithcontrol.Valuesrepresentmean±S.E.ofthreeindependentexperiments(n¼3).**Po0.01,***Po0.001comparedwithcontrol
vehiclegroup.(d)PhotomicrographsshowthattreatmentofMCOswithWFAinosteoblastdifferentiationmediumsignificantlyincreasedmineralizednodulescomparedwith
control,asassessedbyalizarinRed-Sstaining.(e)Quantitationofmineralization(AlizarinRed-Sstain)dataareshownasODat405nm.Valuesrepresentmean±S.E.of
threeindependentexperiments(n¼3).***Po0.001comparedwithcontrolvehiclegroup.(f)WFA(10nM)treatmentofMCOsincreasedexpressionofosteogenicgenes.
FigureshowsincreasedexpressionofRunX2,OCNandColIwithWFAtreatment.ExpressionwasnormalizedtoGAPDHinternalcontrol.Valuesrepresentmean±S.E.of
threeindependentexperiments(n¼3).*Po0.05,***Po0.001comparedwithvehiclecontrolgroup.(g)WFAexertsanti-apoptoticeffectsinosteoblasts.UsingBecton
DickinsonFACSandFL-Hchannel(annexin-V)andFL2-Hchannel(PI)datashowthatWFAtreatmentinhibitedapoptosisofosteoblastcells.Shownarerepresentativedot
plots.(h)Quantificationofflowcytometrydataareshownaspercentoftotalcells.Valuesrepresentsmean±S.E.ofthreeindependentexperiments(n¼3).*Po0.05
comparedwithcontrolforearlyapoptosisand##Po0.01comparedwithcontrolforlateapoptosis
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type I (ColI, 1.5-fold, Po0.05) that is progression marker of WFAactslikePI,inducesBMP2responsivegenestoprevent
mineralization, and osteocalcin (OCN), 3.6-fold, Po0.001) degradation of Smad that interacts with Smurf2 to increase
showedincreaseinexpression.ExpressionofOCNbyWFA boneformationbyincreasingexpressionofRunX2.4,11
wasapproximatelytwofoldmorethanwithBzb.
Apoptosis was induced in MCOs by serum deprivation EffectofWFAonSmurfE3ubiquitinligase. Westernblot
(0.5%FBScontainingmedium),effectofWFAwasanalyzed with anti-Smad1 and anti-RunX2 antibody shows that
by fluorescence-activated cell sorting (FACS) following expression of Smurf2 decreased in presence of WFA or
annexin-V/PI staining (Figure 1g). MCOs cultured in 10% Bzb with concomitant increase in expression of RunX2 and
FBScontained4%totalapoptoticcellscomparedwithB8% Smad1 (Figure 2f). Significant reduction in Smurf2 expre-
under apoptotic (0.5% FBS) conditions (Figure 1h). WFA ssionascomparedwithSmurf1suggeststhatSmad/RunX2
when added to serum-deprived MCOs, only 3% of the cells exhibit greater affinity for Smurf2 in the presence of WFA/
werefoundtobeapoptotic(Figure1h)ascomparedwithBzb. Bzb,asSmad/RunX2arepulleddownwithreducedamounts
ofSmurf2exhibitinggreater affinityforSmurf2protein.
Effect of WFA on proteasome. WFA’s proteasomal effect ToseeifWFApreventsRunX2ubiquitination,osteoblasts
on20SsubunitincalvarialosteoblastsshowsthatWFAacts were treated with TNFa in the presence and absence of
asanaturalPI(Figure2a).WFAinhibitedproteasomal20Sb WFA and endogenous RunX2 was immuno-precipitated
subunit activity within 3h with maximum inhibition at 6h with anti- RunX2 antibody followed by western blot using
(B40–50%) as compared with Bzb (Figure 2a). Lactacystin anti-ubiquitin antibody. WFA treatment revealed reduced
(negativecontrol)significantlyinhibitedthe20Sproteasomal ubiquitinated RunX2 (Figure 2g). Increased ubiquitinated
activity.Consistentwithproteasomeinhibition,treatmentwith proteinsinthepresenceofTNFa,indicatesthatTNFainduces
WFA and Bzb increased accumulation of ubiquitinated ubiquitinationofRunX2proteinleadingtoitsrapidbreakdown
proteinsat6hasobservedbywesternblot(Figure2b). through proteasomal degradation.6 TNFa inhibits bone
WFA’s effect on osteoblast differentiation was examined formationbyupregulatingSmurfanddegradationofRunX2;7
in primary osteoblasts in vitro; pre-treatment with WFA however, WFA treatment decreased Smurf2 expression. To
significantly increased expression of BMP2 (B5.2-fold; authenticate this, TNFa was used to increase Smurf levels
Po0.001).Atthesametime,Smurf1andSmurf2expression (Figure2h)andthenseeexpressionofSmurf2inpresenceof
decreased (Po0.05), (Po0.01), respectively (Figure 2c). WFAorBzb.OsteoblaststreatedwithTNFa(5ng/ml),WFAor
Although data were consistent with Bzb, WFA decreased BzbincreasedexpressionofRunX2,inthepresenceofWFA
expression of Smurf2 significantly. Transcript data corrobo- or Bzb with reduced expression in the presence of TNFa
rated with protein data showing increased expression of (Figure2h).Figure2ishowsB20%reductioninSmurf2inthe
BMP2 and phosphorylation of Smad (Figure 2d). Overall, presenceofWFAplusTNFaascomparedwithTNFaalone.
Figure2 EffectofWFAonosteoblastsignaling.(a)ProteasomaleffectofWFAincalvarialosteoblastcells.20Sproteasomeactivitiesweremeasured1,3,6hafter
treatmentwithWFA.Valuesrepresentmean±S.E.ofthree(n¼3)independentexperiments.***Po0.001,*Po0.001comparedwithcontrol.(b)Lowerpanelshowsthat6h
oftreatmentwithWFAinducesubiquitinationofproteinsthroughwesternblot.Proteinladdersweredetectedbyanti-ubiquitinantibody.(c)qPCRdataoftheosteogenicgenes
afterWFAtreatment.WFAtargetsBMP2signalingbyinducingmRNAlevelsofBMP2responsivegenesinosteoblastcellsafter24hoftreatment.Datarepresentmean±S.E.
ofthreeindependentexperiments(n¼3).*Po0.05,**Po0.01***Po0.001whencomparedwithcontrol.(d)WesternblotanalysisofvariousosteogenicgenesBMP2,
Smad1anditsphosphorylationstateinthepresenceofWFA.ExpressionofSmurf1,Smurf2anegativeregulatorofosteogenesisisalsoshown.Allblotswerenormalizedwith
b-actin.(e)Densitometricanalysesofwesternblot(d)showingfoldchange.Foldincreasewascalculatedrelativetocontrolvehicle-treatedcells.Thevaluesarethe
mean±S.E.ofthreeexperiments.***Po0.001versusvehicle-treatedgroup.(f)InteractionofSmurf2withSmad1andRunX2inthepresenceofWFA.Immuno-precipitation
(IP)assayswereperformedusinganti-Smurfantibodyfollowedbywesternblotusinganti-Smad1andanti-RunX2antibodies.(g)WFApreventsRunX2ubiquitinationand
proteosomal degradation. Primary osteoblast cells were treated with 5ng/ml TNFa for 24h in the presence or absence of WFA, and endogenous RunX2 was
immunoprecipatedbyanti-RunX2antibody.UbiquitinatedRunX2(Ub-RunX2)proteinladdersweredetectedbyanti-ubiquitinantibody(upperpanel).Afterstrippingthe
antibody,totalun-ubiquitinatedRunX2proteinlevelsweredeterminedbyanti-RunX2antibody(lowerpanel).MW,molecularmass.(h)WesternblotofRunX2andSmurf2in
thepresenceofvarioustreatments.Allblotswerenormalizedwithhousekeepinggeneb-actin.(i)Densitometricanalysesofwesternblot(h)showingfoldchange.Fold
increasewascalculatedrelativetocontrolvehicle-treatedcells.Thevaluesarethemean±S.E.ofthreeexperiments.*Po0.05,**Po0.01***Po0.001versusvehicle-
treatedgroup.(j)RelativeexpressionofSmurf2SiRNA.OsteoblastcellsweretreatedwithSmurf2siRNAsandmRNAlevelsweremeasuredbyreal-timeRT-PCR.Thevalues
arethemean±S.E.ofthreeexperiments.**Po0.01versusthevehicle-treatedgroup.(k)Smurf2knockdownreduceddegradationofRunX2.ExpressionofRunX2and
Smurf2wasdeterminedbywesternblotanalysisaftertheosteoblastcellsweretreatedwithSmurf2siRNAinthepresenceandabsenceofWFAandBzb.(l)Densitometric
analysesofwesternblot(k)showingfoldchange.Foldincreasewascalculatedrelativetocontrolvehicle-treatedcells.Thevaluesarethemean±S.E.ofthreeexperiments.
*Po0.05,**Po0.01***Po0.001versusvehicle(vehicle-treated)group.(m)Smurf2siRNAblocksTNF-inducedRunX2degradation.Osteoblastcellsweretreatedwith
Smurf2siRNAandthentreatedwith(5ng/ml)TNFfor24h.Smurf2andRunX2expressionwasdeterminedbywesternblotanalysis.(n)TNF-inducedubiquitinationofRunX2
isreversedbySmurf2knockdown.EndogenousSmurf2wasknockeddowninprimaryosteoblastcellsthentreatedwith5ng/mlTNFafor24handendogenousRunX2was
immunoprecipatedbyanti-RunX2antibody.UbiquitinatedRunX2(Ub-RunX2)proteinladdersweredetectedbyanti-ubiquitinantibody(upperpanel).Afterstrippingthe
antibody,totalun-ubiquitinatedRunX2proteinlevelsweredeterminedbyanti-RunX2antibody(lowerpanel).MW,molecularmass.(o)Smurf2knockdownpreventsRunX2
ubiquitinationandproteosomaldegradationinpresenceofWFA.PrimaryosteoblastcellsweretreatedwithWFAfor24h,andendogenousRunX2immunoprecipatedbyanti-
RunX2antibody.UbiquitinatedRunX2(Ub-RunX2)proteinladdersweredetectedbyanti-ubiquitinantibody.Afterstrippingtheantibody,totalun-ubiquitinatedRunX2protein
levelsweredeterminedbyanti-RunX2antibody(lowerpanel).(p)Smurf2knockdownincreasedALPactivityinthepresenceofWFAandTNFa.WFAtreatmentofMCOsfor
48hinosteoblastdifferentiationmediumsignificantlyincreasedALPproductioncomparedwithcontrolthatwasabrogatedinthepresenceofTNFa.TreatmentwithSmurf2
siRNAinthepresenceofTNFareversedtheeffect.Valuesrepresentmean±S.E.ofthreeindependentexperiments(n¼3).*Po0.05,***Po0.001comparedwithcontrol
vehiclegroup.(q)Smurf2knockdownincreasedexpressionofosteogenicgenes.mRNAexpressionofBMP2andRunX2wasassessedbyreal-timePCR.Thevaluesarethe
mean±S.E.ofthreeexperiments.*Po0.05,***Po0.001versuscontrol(vehicle)group
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WFA or Bzb significantly reduced Smurf1 and Smurf2; treatment to BMSC’s abrogated formation of multinucleated
however, with TNFa the reduction in Smurf2 was more osteoclasts that resorb bone versusmononuclearcells. Multi-
pronounced as compared with Smurf1 (Figure 2i). This nucleated TRAP-positive cells reduced by B30–40%
suggeststhatWFAorBzbseemstoberegulatedbySmurf2. (Po0.01) as compared with mononuclear cells in the
presenceofWFAorBzb(Figure3b)followedbysignificantly
Smurf2 knockdown reduced degradation of RunX2. reduced mRNA expression of TRAP and RANK (Figure 3c).
Reduction in Smurf2-dependent RunX2 degradation by
WFA directly inhibits osteoclast differentiation, and indirectly
WFA was explained using siRNA specific for Smurf2 in
osteoblast-generatedosteoclastogenesis.
osteoblasts.Smurf2siRNAreducedSmurf2mRNAlevelsin Figure 3d shows that whereas TNFa robustly increased
vehicle cells to B50% (Figure 2j). Reduced endogenous
mRNA expression of MCP-1 and IL-6 (osteoclastogenic
Smurf2 due to Smurf2 siRNA led to approximately
cytokines)inosteoblasts,presenceofWFA/Bzbsignificantly
twofoldincreaseinRunX2expression(Figure2k).Likewise, reduced TNFa-stimulated expression. NF-KB p65-subunit
WFA and Bzb in the presence of SiSmurf2 increased
labeling by immunofluorescence shows characteristic cyto-
RunX2 expression (approximately twofold) as shown in
plasmic and perinuclear localization in unstimulated osteo-
Figure2l. blasts(Figure3e).WhereasTNFaresultedinintensenuclear
To see if TNFa regulates Smurf2 expression, osteoblasts labeling of p65-subunit, WFA/Bzb inhibited the nuclear
were treated with TNFa (5ng/ml) for 24h, then Smurf2
labeling(Figure3e).Nucleartranslocationofp65-subunitby
expression was examined (Figure 2m). TNFa increased TNFawasquantifiedbywesternblotnormalizedwithhistone
Smurf2 expression with abrogation of RunX2 expression. 3 antibody for nuclear extract (Figure 3f). TNFa increased
BlockingendogenousSmurf2withSiinpresenceofTNFaled nucleartranslocationofp65-subunitbyBtwofold(Figure3g).
toreversalinTNFa-mediatedresponseforSmurf2andRunX2 WFA/Bzb alone or in the presence of TNFa decreased
(Figure2m).Figure2nshowsincreasedubiquitinatedRunX2 TNFa-induced p65 nuclear translocation lower than the
complexes in TNF-treated cells. SiSmurf2 treatment revealed
control. Altogether, WFA’s proteasomal effect on osteoblast
small amounts of ubiquitinated RunX2 with increased RunX2
and production of osteoclastic factors in osteogenic cells
expression.AssessmentofSiSmurf2onosteoblastfunctionin
indirectlyimpactsosteoclastogenesis.
thepresenceandabsenceofWFAandTNFashowsthatSmurf2
knockdownincreasedALPactivityandexpressionofBMP2and Pharmacokinetic studies of WFA after oral administra-
RunX2 that further increased to approximately twofold in the tion. WFAatanoraldoseof10mg/kgresultedinmaximum
presenceofWFA(Figures2pandq)establishingthatblocking plasmalevelsof8.41±1.4mg/mlafter3h.TheAUC and
0–24
endogenous Smurf2 increases osteoblast differentiation. This t of WFA was found to be 55.01±8.4mgh/ml was
1/2
increasewasmoreascomparedwiththeeffectbyWFAalone. 7.1±1.2h, respectively. The Vd and MRT was found to be
TNFa attenuated ALP activity that was compensated in the 0.043land 6.52h,respectively, asshowninFigure4.
presence of SiSmurf2 (Figure 2p). Expression of RunX2 and
BMP2substantiatedtheALPdata(Figure2q). EffectofWFAonboneregeneration. Invivoregeneration
ofbonetomonitorlocalrepairprocesswasdoneusingm-CT
Effect of WFA on osteoclast. Whether, WFA alters in Sham and ovariectomy (OVx) groups from days 11 to 21
osteoclastfunction,MCOsweretreatedwithWFAandmRNA aftercreationofthehole.28–31Intensityofcalceinlabelingin
expressionofosteoprotegrin(OPG)andreceptoractivatorof the drill hole shows that WFA at 5 and 10mg/kg/day
nuclearfactor kappa-B(RANK)ligand(RANKL) producedby increased mineral deposition (Figures 5a and b). Modest
osteoblasts were determined which stimulate formation of mineralized calluses appeared at defect and intra-medulla
tartarate-resistantacidphosphatase(TRAP)-positivecellsand regionofOVxandShamgroups.Byday11,extentofcallus
thus osteoclast function.27 Data are expressed as ratio of mineralization increased, in the intra-medulla region.
RANKL/OPG,wheredecreasedratio(approximatelythreefold Post drilling 11th day, WFA treatment at 5mg/kg/day dose
at day 9) represents induction of osteoclastogenesis ledtopredominantlyoccupiedmineralizedcallus,anddefect
(Figure 3a). Direct effect of WFA on osteoclasts shows that region was partially bridged, however, the effect was more
Figure3 EffectofWFAonosteoclastogenesisindirectlythroughosteoblasts.(a)WFAincreasesmRNAlevelsofOPGbutdecreasesmRNAlevelsofRANKLinprimary
osteoblasts.RatioofRANKL:OPGweredeterminedandquantifiedwithqPCRandnormalizedwithGAPDH.Valuesrepresentsmean±S.E.***Po0.001and**Po0.01of
threeindependentexperiments(n¼3)whencomparedwithvehicle-treatedcells.(b)WFAtreatmenttoOVxmicereducesosteoclastogenesisandincreasesosteoprogenitor
cellsinbonemarrow.BMCsfrommiceofvariousexperimentalgroupswereseededinto48-wellplatesandosteoclastdifferentiationwasinducedasdescribed.Cellswere
stainedforTRAPactivityforosteoclastformation.FigureshowsquantitativerepresentationofTRAP þivemononuclearandmultinuclearcells.(c)WFAincreasesmRNA
levelsofOPGbutdecreasesmRNAlevelsofTRAPandRANKinosteoclastculture.BMCswereisolatedfrom4-to6-week-oldmice.Afterovernightculturecellswerecultured
for6daysinthepresenceofMCSFandRANKL.mRNAlevelsofTRAPandRANKrelatedtoosteoclastogenesiswasdeterminedbyqPCRfromthetotalRNAmadefrom
BMC’s.Datarepresentmean±S.E.M.;n¼3.**Po0.01and***Po0.001comparedwithvehicle-treatedcells.(d)WFAtreatmentdecreasesexpressionofinflammatory
cytokinesinosteoblasts.qPCRdatashowmRNAlevelsIL-6,MCP-1.Valuesrepresentsmean±S.E.**Po0.01and***Po0.001ofthreeindependentexperiments(n¼3)
whencomparedwithcontrol.(e)WFAinhibitsTNFa-inducedNF-kBnucleartranslocationinosteoblastcells.Representativephotomicrographofsubcellularlocalizationof
p65wasdeterminedbyimmunofluorescence(magnification (cid:2)40)undercontrolandtreatmentconditionsfromthreeindependentexperiments(n¼3).(f)WFAtreatment
abolishedthenucleartranslocationoftheNF-kBinosteoblasts.OsteoblastsweretreatedwithTNFaandWFA.CytoplasmicornuclearextractswerepreparedandNF-kB
proteinlevelsweredetectedbyimmunoblotting.TNFaenhancedthenucleartranslocationofNF-kB,WFAabolishedit.HistoneH3wasusedasaloadingcontrolfornuclear
extract,b-actinwasusedasloadingcontrolsforcytoplasmicfraction.(g)ThisfigureshowsthedensitometricanalysisofNF-kBfromthreeindependentblots.Values
representsmean±S.E.ofthreeindependentexperiments(n¼3),***Po0.001,**Po0.01whencomparedwithcontrol
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significant at 10mg/kg/day with more mineralized callus 10mg/kg/dayhadtheabilityformineraldepositioncompared
within intra-medulla region to be remodeled to completely with OVxþvehicle group.
bridge the defect (Figure 5c). Six weeks post-surgery OVx OvxgroupdisplayedlowerBV/TVcomparedwithSham,from
mice had significantly lower mineral deposition (50% less) day11to21inthedefectregion;however,thiswassignificantly
than shamþvehicle group. However, OvxþWFA at 5 and increased with WFA treatment at 10mg/kg/day opposed to
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5mg/kg/daygroup(Figures5d–f).Inbothgroups,BV/TVinthe WFA, at lower dose, and alendronate (Ald) did not
intra-medullaspacemoderatelyincreasedwithtime,peakedday significantlyinducemineralization.
11,andreturnedtobaselineonday21.Inter-dosecomparisonin Dynamic histomorphometric data of cortical bone
theintra-medullaregionwasnotsignificantintheshamgroup, (Figures 6c and d) show that OVx group had significantly
however, in the OVx group dose of 10mg/kg/day was lowerperiostealMARandBFR compared withshamgroup.
significantlybetter.(Figures5e–g).Datawerecorroboratedby Comparison of WFA (10mg/kg/day) or Ald, Bzb and iPTH
qPCR. After drill-hole injury RunX2, BMP2 and OCN mRNA groups with sham revealed no significant difference in MAR
expressionweresignificantlyhigherinShamthanOVxgroupat andBFR.
day11and21(Figures5h–j).WFAtreatment(5and10mg/kg/
day)increasedexpressionofthesegeneswithrobustchangeat Effect of WFA on bone micro-architecture and biome-
10mg/kg/dayinSham þWFAversusOVx þWFAgroup. chanical -strength. 3D mCT shows deterioration of bone
owing to destruction of the trabecular architecture as
Effect of WFA on bone formation in osteopenic mice. observed in OVxþvehicle group compared with well-devel-
WFA’s pharmacokinetics was studied in osteoporotic mice oped trabeculae in shamþvehicle group (Figure 6e).
after its oral administration in gum acacia. Particle size of Trabecular response to WFA treatment to ovariectomized
WFA in dispersion was 1.023±0.28mm with poly dispersity ratswasquantifiedintibiaproximal(SupplementaryTable2),
index 0.423 of particles. Zeta potential and viscosity was femur epiphysial (Figures 6f–i) and lumbar vertebral L-5
40.7±8.06mVand2.86Pas,respectively(datanotshown). region (Figures 6k–n), and values compared with positive
Post treatment, BMC’s harvested from bones of OVxþ controls.Femoraldatashowthatcomparedwithshamgroup,
vehiclegrouphaddecreasedmineralizednodules.OVxmice theOVxþvehiclegroupexhibitedsignificantlyreducedBV/TV,
treated with WFA at 10mg/kg/day Bzb or injectible para- trabecularnumber(Tb.No),trabecularthickness(Tb.Th)and
thyroid hormone (iPTH) had nodule formation that was increased structure mode index (SMI). Post WFA treatment
comparable with sham group (Figures 6a and b); however, BV/TV, Tb. No was at par to sham group at the dose of
10mg/kg/day. Likewise, SMI increased due to OVx and
decreased with WFA, PTH and Bzb treatment but was
comparable to Sham group. Inter-dose comparison with
shamgrouprevealedthatoutofthethreedosesof1,5and
10mg/kg/day WFA, 10mg/kg/day was most effective.
Vertebral data show the OVxþvehicle group had signifi-
cantly reduced BV/TV, Tb.No, Tb.Th and increased SMI.
WFA treatment at 10mg/kg/day was comparable in BV/TV,
Tb.No, Tb.Th and SMI with Sham group. Bone formed by
WFA at 10mg/kg/day was comparable with the osteogenic
agentiPTH.
Biomechanical strength data suggest that OVxþVehicle
grouphadsignificantlyreducedenergyandstiffness,whereas
WFA treatment increased these parameters. Whereas OVx
group required less compressive energy for breaking LV5
comparedwiththeshamgroup(Table1),WFAtreatmentatall
three doses for 60 days required more compressive energy
thantheOVxgroupwith10mg/kg/daybeingthemostefficient
dose.
Figure4 PlasmaconcentrationtimeprofileofWFAafteroraladministrationat
thedoseof10mg/kg.Thedataarerepresentedasmean±S.D.ofthreeindividual EffectofWFAonboneturnovermarker. SerumOCNwas
experiments elevatedinthe OVxþvehiclegroup comparedwith shamþ
Figure5 WFApromotesboneregenerationinthedrill-holesiteinsham-operated(ovaryintact)andOVxmice.(a)Representativeconfocalimages(magnification¼(cid:2)100)
ofcalceinlabelingshowninthedrill-holesiteofvariousgroups0,11and21daysafterinjurywithoutandwithWFAtreatment(5and10mg/kg/day).(b)Datashowthe
quantification of the mean intensity of calcein labeling. Values represent mean±S.E. **Po0.01, ***Po0.001 compared with Sham vehicle. #Po0.05, ##Po0.01,
###Po0.001comparedwithOVxvehiclegroup.Inter-dosecomparisonshowsthatcPo0.05when10mg/kg/daydosewascomparedwith5mg/kg/daydose.*Po0.05when
OVxþVcomparedwithShamþV.(c)RepresentativemCTimagesfromthecenterofthebonyhole(defectregion)ondays0,11and21intheshamandOVxgroups.(dand
e)GraphrepresentsmCTanalysisinthedefectandintra-medullaregionsofshamandshamþ WFA-treatmentgroup.DatashowBV/TV(%)at5and10mg/kg/daydosein
boththeregions.Valuesrepresentmean±S.E.**Po0.01,***Po0.001comparedwithShamvehicle.##Po0.01comparedwithOVxvehicle.Inter-dosecomparisonshows
aPo0.05when10mg/kg/daydoseiscomparedwith5mg/kg/daydoseinshamgrouponday11.(fandg)GraphrepresentsmCTanalysisinthedefectandintra-medulla
regionsofOVxandOVxþ WFAtreatmentgroups.ValuesrepresentMean±S.E.*Po0.05,***Po.001comparedwithShamvehicle.#Po0.05and##Po0.01compared
withOVxvehicle.Inter-dosecomparisonshowsaPo0.05when10mg/kg/daydoseiscomparedwith5mg/kg/daydoseinshamgrouponday11.(h–j)EffectofWFAon
osteogenicgenesafterdrill-holeinjury.mRNAprofileofRunX2,BMP2andOCNextractedfromcallusofdrillsiteofbone.Valuesrepresentmean±S.E.ofthreeindependent
experimentsn¼3.**Po0.01,***Po0.001comparedwithShamvehicle.#Po0.05,##Po0.01,###Po0.001comparedwithOVxvehicle.Inter-dosecomparisonshows
aPo0.05when10mg/kg/daydoseiscomparedwith5mg/kg/daydoseinshamgrouponday11.bPo0.05when10mg/kg/daydoseiscomparedwith5mg/kg/dayinsham
grouponday21.cPo0.05when10mg/kg/daydoseiseffectivethan5mg/kg/dayinOVxgrouponday11.dPo0.05when10mg/kg/daydoseiseffectivethan5mg/kg/dayin
OVxgroupatday21forRunX2gene
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