The Matricellular Protein Periostin Is Required for SostInhibition and the Anabolic Response to Mechanical Loadingand Physical Activity*□S

Received for publication, August 27, 2009, and in revised form, October 15, 2009 Published, JBC Papers in Press, October 16, 2009, DOI 10.1074/jbc.M109.060335

Nicolas Bonnet‡1, Kara N. Standley§, Estelle N. Bianchi‡, Vincent Stadelmann¶, Michelangelo Foti�,Simon J. Conway§, and Serge L. Ferrari‡

From the ‡Division of Bone Diseases, Department of Rehabilitation and Geriatrics, World Health Organization CollaboratingCenter for Osteoporosis Prevention, and the �Department of Cellular Physiology and Metabolism, Geneva University Hospital,1211 Geneva 14, Switzerland, the §Riley Heart Research Center, Wells Center for Pediatric Research, Indiana University School ofMedicine, Indianapolis, Indiana 46202, and the ¶Laboratory of Biomechanical Orthopedics, Ecole Polytechnique Federale deLausanne, 1015 Lausanne, Switzerland

Periostin (gene Postn) is a secreted extracellular matrix pro-tein involved in cell recruitment and adhesion and plays animportant role in odontogenesis. In bone, periostin is preferen-tially expressed in theperiosteum,but its functional significanceremains unclear. We investigated Postn�/� mice and their wildtype littermates to elucidate the role of periostin in the skeletalresponse tomoderate physical activity anddirect axial compres-sion of the tibia. Furthermore, we administered a sclerostin-blocking antibody to these mice in order to demonstrate theinfluence of sustained Sost expression in their altered bone phe-notypes. Cancellous and cortical bonemicroarchitecture as wellas bending strength were altered in Postn�/� compared withPostn�/� mice. Exercise and axial compression both signifi-cantly increased bonemineral density and trabecular and corti-cal microarchitecture as well as biomechanical properties of thelong bones in Postn�/� mice by increasing the bone formationactivity, particularly at the periosteum. These changes corre-lated with an increase of periostin expression and a consecutivedecrease of Sost in the stimulated bones. In contrast,mechanicalstimuli hadno effect on the skeletal properties ofPostn�/�mice,where base-line expression of Sost levels were higher thanPostn�/� and remained unchanged following axial compres-sion. In turn, the concomitant injection of sclerostin-blockingantibody rescued the bone biomechanical response in Postn�/�

mice. Taken together, these results indicate that thematricellu-lar periostin protein is required for Sost inhibition and therebyplays an important role in the determination of bone mass andmicrostructural in response to loading.

Periostin gene (Postn) expression was first identified usingsubtractive hybridization techniques onMC3T3-E1 osteoblast-like cells and initially named osteoblast-specific factor 2(OSF-2) (1). Subsequently, several developmentally regulatedand differentially spliced isoforms of Postn have been identifiedin both mice and humans and are expressed in many non-skel-etal tissues, including stromal cells from ovary, breast, colon,and brain tumors. Periostin is a 90-kDa secreted extracellularmatrix protein that binds integrins �v�3 and �v�5, therebyregulating cell adhesion and mobility (2, 3). Periostin potentlypromotes metastatic growth of colon cancer cells by augment-ing their survival via the Akt/protein kinase B pathway (4).Besides, periostin is predominantly expressed in tissues subjectto mechanical stress, suggesting a potential function of perios-tin in maintaining the structure and integrity of connective tis-sues. For example, periostin is expressed by cardiac fibroblasts(5–7), where protein expression increases after heart failureand in a model of overload hypertrophy of the heart (8). Peri-ostin is also linked to type I collagen in the periodontal liga-ment, where it regulates fibrillogenesis and consequently thebiomechanical properties of fibrous connective tissues aroundthe tooth (9). Its expression is increased in the periodontal lig-ament upon mechanical loading and is essential for the integ-rity and function of this ligament during occlusal loading (10,11). Moreover, in vitro application of tensional forces to peri-odontal cells increases Postn expression. In contrast, followingin vivomasticatory unloading, PostnmRNA levels decrease (11,12). Postn-deficient mice (Postn�/�) show severe alterations intooth (incisor) eruption, resulting from a failure to digest colla-gen fibers in the shear zone of the periodontal ligament (13, 14).As a consequence, the enamel and dentin of the incisors is com-pressed and disorganized.In MC3T3-E1 osteoblast cells, periostin is secreted into the

collagen matrix and regulates cell adhesion and differentiation(15, 16). Inactivation of Postn using specific periostin-blockingantibodies leads to a severe reduction of osteoblast-specific dif-ferentiation markers, such as type I collagen, osteocalcin,osteopontin, and alkaline phosphatase (16). In the rodent adultskeleton, Postn expression appears to be restricted to the peri-osteum,without any reported expressionwithin the endosteum(15). For this reason, Postn has recently been used to identify

* This work was supported, in whole or in part, by National Institutes of HealthGrants R01HL060714 and R01HL092508 (to S. J. C.) and T32 HL079995 (toK. S.). These studies were also supported by Swiss National Science Foun-dation Grants 3100A0-116633/1 (to S. L. F.) and 310030-120280 (to M. F.),by the seventh Framework program of the European Community(HEALTH-F2–2008-201099) (to S. L. F.), the Riley Children’s Foundation (toS. J. C.), and the Indiana University Department of Pediatrics/Cardiology (toS. J. C.). This work was further supported by a grant from the “Societe Fran-caise de Rhumatologie” and the GRIO (to N. B.).

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Tables S1 and S2.

1 To whom correspondence should be addressed: Geneva University Hospi-tal, Dept. of Rehabilitation and Geriatrics, Service of Bone Diseases, RueMicheli-du-Crest 24, CH-1211 Geneva 14, Switzerland. Tel.: 41-22-382-9968; Fax: 41-22-382-9973; E-mail: Nicolas.bonnet@unige.ch.

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periosteal osteoblasts ex vivo (17). During fracture repair, PostnmRNA is up-regulated 2-fold and localizes to preosteoblasticcells within the periosteum as well as in undifferentiated mes-enchymal cells close to the fracture site (18). Furthermore,Postn�/�mice have shorter long bones, suggesting a disruptionof the cartilaginous growth plate (13). Whether periostin influ-ences bone turnover, however, remains to be elucidated.In young and adult rats, treadmill exercise, typically 1 h/day,

is known to increase cortical and cancellous bone mass of thetibia due to enhanced bone formation and reduced boneresorption (19–21). Cyclic axial compression of the ulna ortibia can also increase bone mass and size in rodents (22). Inthese models, stimulation of bone formation occurs mainly atthe periosteal rather than endocortical surfaces (23–25). Thisbone (re)modeling effect largely depends on the suppression ofsclerostin gene (Sost) expression by osteocytes (26), which thenallows for the stimulation of Wnt/LRP5/�-catenin signalingwithin lining osteoblasts (27). Interestingly, preliminary studiessuggest that sclerostin antagonistic activity on Wnt signalingcan be inhibited by periostin (28). These observations led us tohypothesize that periostin modulates bone turnover, specifi-cally in response tomechanical stimulation. In order to test thishypothesis, we characterized bone mass, microarchitecture,and strength in Postn�/� mice.We show that PostnmRNA andprotein expression in bone is stimulated bymechanical loading,which precedes inhibition of Sost gene expression. Further-more, we report that periostin is required for the complete bio-mechanical responses of the skeleton to both axial compressionand physical activity and that sclerostin-blocking antibodiesrestore the bone biomechanical response in Postn�/� mice.

EXPERIMENTAL PROCEDURES

Animals—Postn�LacZ knock-in mice (Postn�/�) were gener-ated as reported previously (13). Postn�/� mice were subse-quently bred with C57BL/6J mice, and tail DNA analyzed byPCR was used to identify Postn heterozygous mice. We inter-bredmice that were heterozygous carriers of this mutation andobtained wild-type (Postn�/�), heterozygous (Postn�/�), andhomozygousmutant (Postn�/�) offspring in the expectedMen-delian genetic frequencies. These mice were subsequentlyback-crossed for 6 generations, resulting in a genome of 98%C57BL/6J. Mice were housed five per cage, maintained understandard non-barrier conditions, and had access to water andsoft diet ad libitum (2019 Teklad, Harlan Laboratories, Shard-low, UK). A soft diet was chosen to reduce malnutrition in thePostn�/� mice, which was previously observed under a stand-ard diet due to the enamel and dentin defects of the incisors andmolars (13). All of the mice received the same diet throughoutthe experiment. 12- and 14 week-old male mice were used forthe treadmill exercise and the loading studies, respectively. Anadditional group of mice were sacrificed at 12 weeks old todescribe the bone phenotype of the Postn-deficientmice at baseline (n� 10mice/group). Animal procedures were approved bythe University of Geneva School of Medicine Ethical Commit-tee and the State of Geneva Veterinarian Office.In Vivo Axial Compression—The loading apparatus was spe-

cifically adapted for mouse tibiae as previously described (29).Custom molded pads were placed on the axes to apply com-

pression on the bone extremities. The tibiae were then placedon the stimulation machine between the moving pad on theproximal side (the knee) and the fixed pad on the distal side (thefoot). Strain magnitudes were calibrated ex vivo using minia-ture strain gauges bound to the midshaft tibia surface, as previ-ously reported (24, 29). The left tibia of each mouse was sub-jected to dynamic axial stimulation, using the followingparameters: peak load� 12 newtons; peak strain (midshaft cor-tex)� 1500micro strain; pulse period (trapeze-shaped pulse)�0.1 s; rest time between pulses� 10 s; full cycle frequency (pulse� rest)� 0.1 Hz. A total of 40 cycles (�7min) were applied perday. The non-stimulated right tibia served as an internal con-trol. The mice used to study the bone response to direct axialcompression were stimulated on 3 alternate days/week for 2weeks and sacrificed 3 days later. To measure dynamic indicesof bone formation, mice received subcutaneous injections ofcalcein (25 mg/kg; Sigma) 9 and 2 days before euthanasia. Themice used for immunohistochemical staining of periostin werestimulated for 2 consecutive days and then sacrificed on day 3.The mice used for real-time analyses were stimulated for a sin-gle session (1 day) and sacrificed 6 or 24 h later. For the axialcompression procedure, mice were anesthetized by intraperi-toneal injection of ketamin xylasine. The total duration of anes-thesia lasted just a bit longer than the loading period, up to amaximum of 20 min. None of the mice showed signs of lame-ness or decreased activity levels after recovery from anesthesia(n � 10 mice/group).This loading experiment was repeated in Postn�/� and

Postn�/� mice with concomitant intravenous injection of asclerostin-blocking antibody (Sost-Ab2; 12 mg/kg/week) or acontrol antibody (anti-cyclosporin A, Ig-G2A) for 2 weeks (n�6 mice/group/genotype). The anti-sclerostin antibody was iso-lated from a combinatorial antibody library using phage displaytechnology (MorphoSys AG, Martinsried, Germany) andadministered 1 h prior to axial compression at a concentrationpreviously found to have mild anabolic effects on bone (30).Measurements of the Strain Distribution—Nine mice were

sacrificed at the age of 14 weeks, and the tibiae were immedi-ately excised. Soft tissues were removed, and the tibia werecleaned with acetone. The tibiae were separated randomly intothree groups of six corresponding to three zones of tests on thetibia: zone 1, anteroproximal; zone 2, anterotibial crest; zone 3,posterodistal. Each tibia received a single element foil straingauge (EA-06-015LA-120, Vishay Micro-measurements, Raleigh,NC) aligned with the long axis bound to bone surface with cya-noacrylate. The gauge was connected to a tension amplifier anddigital recorder (DAQ NI9215, National Instruments, Switzer-land). Gauge location allowed for the analysis of the whole

2 The abbreviations used are: Sost-Ab, sclerostin-blocking antibody; Ab, anti-body; ANOVA, analysis of variance; 1F-ANOVA, one-factor ANOVA; 2F-ANOVA,two-factor ANOVA; BMD, bone mineral density; microCT, microcomputedtomography; N, newton; PLSD, protected least squares difference; BV, bonevolume; TV, total volume; TbTH, trabecular thickness; TbN, trabecular number;Tb, trabecular; Conn density, connectivity density; SMI, structural model index;CtTV, cortical tissue volume; CtBV, cortical tissue bone volume; BMaV, bonemarrow volume; CtTh, average cortical width; MAR, mineral apposition rate;SLS, single-labeled surface; BS, bone surface; dLS, double-labeled surface; MS,mineralizing surface; BFR, bone formation rate; TbSp, separation between tra-beculae; MPm, mineralization perimeter; BPm, bone perimeter.

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strain submitted to the bone during an axial compression of thetibia without any space left between the gauges.Exercise Training—Male mice (12 weeks old) were randomly

divided into exercise-trained and rested groups for each geno-type, Postn�/�, Postn�/�, and Postn�/� mice. The mice weretrained 5 days/week for 6 weeks. During the first week, thetreadmill speed and the duration of each running session weregradually increased from 8 m/min for 10 min to 16 m/min for40 min. For the last 5 weeks, the running sessions consisted of16 m/min for 40 min with a treadmill inclination of 8°, corre-sponding tomoderate exercise for thesemice at this specific age(31). Restedmice serving as control were handled twice daily at1-h intervals to mimic the stress induced by handling beforeand after running. It should be noted that the above interven-tions do not affect food intake in mice (data not shown). At theend of the study (i.e. at 18 weeks of age), all mice were sacrificedwith an overdose of ketamin xylasine (n � 8 mice/group). Tomeasure dynamic indices of bone formation, mice receivedsubcutaneous injections of calcein 9 and 2 days beforeeuthanasia.In Vivo Measurement of Bone Mineral Density (Exercise

Training)—Total body, femoral, and spinal bone mineral den-sity (BMD; g/cm2) weremeasured in vivo, at 12 and 18 weeks ofage in the exercise experiment and at 13 and 16 weeks of age inthe axial compression experiment, by dual energy x-ray absorp-tiometry (PIXImus2, GE lunar, Madison, WI) (32).In Vivo Measurement of Skeletal Morphology and Microar-

chitecture (Axial Compression)—A high resolution in vivomicrocomputed tomography system (microCT Skyscan 1076,Skyscan,Aartselaar, Belgium)was used to scan the left and righttibiae. The in vivomicroCT system consists of an x-ray sourceand detector that rotates around the animal bed. The machineis equippedwith a 100-kV x-ray source with a spot size of 5�m.Each scan lasted�20min, resulting in shadow projections witha pixel size of 10 �m. A modified Feldkamp algorithm, usingundersampling to reduce noise, was applied to the scan data,resulting in reconstructed three-dimensional data sets with avoxel size of 20 �m. (33). Themice were scanned at 14 weeks ofage before loading and at 16 weeks of age, 3 days after the lastloading. A detailed description and validation of the algorithmis published elsewhere (34). Cortical and trabecular bones wereseparated manually with “CT Analyzer” software (Skyscan,Aartselaar, Belgium). For a description of the bonemicroarchi-tecture parameters analyzed, see below.Ex Vivo Measurement of Morphology and Microarchitecture

(Exercise Training)—Microcomputed tomography (microCTUCT40, ScancoMedical AG, Basserdorf, Switzerland)was usedto assess trabecular bone volume fraction and microarchitec-ture in the excised 5th lumber spine body and distal femur andcortical bone geometry at the midshaft femoral diaphysis asdescribed previously (35). Briefly, trabecular and cortical boneregions were evaluated using isotropic 12-�m voxels. For thevertebral trabecular region, we evaluated 250 transverse CTslices between the cranial and caudal end plates, excluding 100�m near each end plate. For the femoral and tibial trabecularregion, to eliminate the primary spongious, we analyzed 100slices from the 50 slices under the distal growth plate. Femoralcortical geometry was assessed using 50 continuous CT slides

(600 �m) located at the femoral midshaft. Images were seg-mented using an adaptative-iterative thresholding approachrather than a fixed threshold. Morphometric variables werecomputed from binarized images using direct, three-dimen-sional techniques that do not rely on prior assumptions aboutthe underlying structure (36). For the trabecular bone regions,we assessed the bone volume fraction (BV/TV, percentage),trabecular thickness (TbTh, �m), trabecular number (TbN,mm�1), trabecular connectivity density (Tb Conn density,mm�3), and structuralmodel index (SMI). The structuremodelindex wasmeasured to determine the prevalence of platelike orrodlike trabecular structures, where 0 represents “plates” and 3represents “rods” (36). For cortical bone at the femoral andtibial midshaft, we measured the cortical tissue volume (CtTV,mm3), bone volume (CtBV, mm3), marrow volume (BMaV,mm3), and average cortical width (CtTh, �m).RNAExtraction andQuantitative PCR—Thewhole tibia was

excised, and both tibial extremitieswere cut to remove the bonemarrow from the diaphysis, by flushing with cold phosphate-buffered saline. Tibial diaphysis and extremities were immedi-ately pulverized to a fine powder and homogenized in peqGoldTrifast (peQLab Biotechnologie GmbH) using a FastPrep sys-tem apparatus (QBiogene) in order to achieve quantitativeRNA extraction. Total RNAwas extracted and then purified onminicolumns (RNeasy minikit, Qiagen) in combination with adeoxyribonuclease treatment (RNase-free DNase set, Qiagen)to avoid DNA contamination.Single-stranded cDNA templates for quantitative real-time

PCR analyses were carried out using SuperScript III reversetranscriptase (Invitrogen) following the manufacturer’s in-structions. Quantitative real-time PCR was performed usingpredesigned TaqMan� gene expression assays (referencesas follows: B2m, Mm00437762_m1; Sost, Mm00470479_m1;Postn, Mm00450111_m1) (Applied Biosystems, Rotkreuz,Switzerland) consisting of twounlabeled primers and a FAMTM

dye-labeled TaqMan� MGB probe and the correspondentbuffer TaqMan� universal PCR master mix (Applied Biosys-tems, Rotkreuz, Switzerland). A Biomek 2000 robot (BeckmanCoulter, Nyon, Switzerland) was used for liquid handling (10�l) in 384-well plates with 3 replicates/sample. The cDNA wasPCR-amplified in a 7900HT SDS System, and raw thresholdcycle (Ct) values were obtained from SDS version 2.0 software(Applied Biosystems). Relative quantities (RQ) were calculatedwith the formula RQ � E � Ct using an efficiency (E) of 2 bydefault. For each gene, the highest quantity was arbitrarily des-ignated a value of 1.0. The mean quantity was calculated fromtriplicates for each sample, and this quantity was normalized tothe similarly measured mean quantity of the �2-microglobulinnormalization gene. Finally, normalized quantities were aver-aged for 3–4 animals and represented as mean � S.E.Immunohistochemistry—The right and left tibiae were

excised and subsequently fixed in 4% paraformaldehyde over-night at 4 °C. They were then decalcified in 19% EDTA and 4%phosphate-buffered formalin for 3 weeks. The tibiae were thendehydrated in an ascending series of ethanol, cleared in Propar(Anatech LTD, Battle Creek, MI), and embedded in paraffinblocks. 10-�m-thick sections were cut from the blocks at thetibia midshaft level using a RM2155 microtome (Leica, Ger-

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many) andmounted on Superfrost Plus slides (Fisher). Sectionswere air-dried overnight at room temperature. Prior to stain-ing, they were incubated at 60 °C for 1 h, deparaffinized inxylene, and rehydrated in a descending series of ethanol. Depar-affinized slides were pretreated in 3% hydrogen peroxide inmethanol to quench endogenous peroxidase and rinsed in tapwater followed by nonspecific avidin/biotin blocking (VectorLaboratories, Burlingame, CA) according to themanufacturer’sdirections. All incubations took place in a humidified chamber.Additional protein blocking was accomplished with ProteinBlock-Serum Free (DAKO, Carpinteria, CA). Using the Vec-tastain Elite ABC (rabbit IgG) kit (Vector Laboratories), theslides were incubated in 1.5% normal goat serum for 30 min atroom temperature. The primary antibody (rabbit anti-perios-tin) (Ed Krug, Medical University of South Carolina, Charles-ton, SC) was diluted in antibody diluent (DAKO) to a final con-centration of 1:6000 and incubated at 4 °C overnight. Thefollowing day, slides were rinsed inWash Buffer (DAKO) for 15min on a rocker at room temperature and incubated in biotin-ylated goat anti-rabbit (Vectastain kit) secondary antibodydiluted 1:1000 for 30 min at room temperature, followed byanother rinse in Wash Buffer for 15 min on a rocker at roomtemperature. The ABC reagent from the Vector kit was pre-pared according to the manufacturer’s directions at a dilutionof 1:250, and the slides were incubated in it for 30 min at roomtemperature and rinsed, as above, in Wash Buffer. All incuba-tion steps were performed at room temperature, and all rinsesteps employed the DAKO Wash Buffer at room temperatureon a rocker. The following protocol was used: incubation instreptavidin-horseradish peroxidase diluted at 1:100 for 30minand washed for 15 min. Slides were developed in a workingsolution of 3,3�-diaminobenzidine (DAB Substrate Kit for Per-oxidase Kit, Vector Laboratories) prepared according to themanufacturer’s directions for 10min at room temperature. Fol-lowing a final rinse in deionized water, the slides were counter-stained in Weak Methyl Green and mounted in Cytoseal 60(Richard-Allan Scientific, Kalamazoo, MI). Positive periostinstaining was quantified using a microscope interfaced with animage analysis system (Leica Corp.).Histomorphometry—To measure dynamic indices of bone

formation,mice received subcutaneous injections of calcein (10mg/kg; Sigma) 9 and 2 days before euthanasia. Tibiae wereembedded in methyl-methacrylate (Merck), and 8-�m-thicktransversal sections of themidshaft were cut with a Leica Corp.Polycut E microtome (Leica Corp. Microsystems AG, Glatt-burg, Switzerland) and mounted unstained for evaluation offluorescence. 5-�m-thick sagittal sections were stained withmodified Goldner’s trichrome, and histomorphometric meas-urements were performed on the secondary spongiosa of theproximal tibia metaphysis and on the endocortical and perios-teal bone surfaces in the middle of the tibia, using a Leica Corp.Q image analyzer at �40 magnification. All parameters werecalculated and expressed according to standard formulas andnomenclatures (37): mineral apposition rate (MAR, �m/day),single-labeled surface (sLS/BS, percentage), and double-labeledsurface (dLS/BS, percentage). Mineralizing surface per bonesurface (MS/BS, percentage) was calculated by adding dLS/BS

and one-half sLS/BS. Bone formation rate (BFR/BS, �m3/�m2/day) was calculated as the product of MS/BS and MAR.Preparation of Specimens for Transmission Electron Mi-

croscopy—The left femur of three Postn�/� and Postn�/� mice(12 weeks old) was excised, soft tissue was removed, and thebones were prepared for electron microscopy. The tissue wasfixed in 0.1 M sodium cacodylate buffer containing 4%paraformaldehyde and 1% glutaraldehyde at room temperaturefor 24 h. Samples were then washed three times with Tris-HCl,pH 7.4, and decalcified 4 days at 4 °C by incubation in a 10%EDTA/Tris-HCl, pH7.4, solution replaced every 24h.Themid-diaphysis was isolated and postfixed with 1% osmium tetraox-ide and 1.5% potassium ferrocyanide in 0.1 M cacodylate bufferfor 1 h at room temperature. Samples were then dehydrated,embedded in Epon resin, and processed for electron micros-copy as described previously (38). Ultrathin sections werefinally contrasted with uranyl acetate and lead citrate andobservedwith aTechnai 20 electronmicroscope (FEICo., Eind-hoven,Netherlands).Morphometricmeasurementsofthenum-ber and size of collagen fibrils were performedwithMetamorphsoftware on 20 pictures/sample.Testing ofMechanical Resistance—Thenight beforemechan-

ical testing, bones were thawed slowly at 7 °C and then main-tained at room temperature. The fibula was removed, thelength of the tibia (distance from intermalleolar to intercondy-lar region) wasmeasured using calipers with an integrated elec-tronic digital display, and the midpoint of the shaft was deter-mined. The tibia then was placed on the material testingmachine on two supports separated by a distance of 9.9 mm,and load was applied to the midpoint of the shaft, thus creatinga three-point bending test. Between each preparation step, thespecimens were kept immersed in physiological solution. Themechanical resistance to failure was tested using a servo-con-trolled electromechanical system (Instron 1114, Instron Corp.,High Wycombe, UK) with actuator displaced at 2 mm/min.Both displacement and load were recorded. Ultimate force(maximal load, measured in newtons), stiffness (slope of thelinear part of the curve, representing the elastic deformation,N/mm), and energy (surface under the curve, N�mm) were cal-culated. Ultimate stress (N/mm2) and Young’s modulus (mega-pascals)were determined by the equations previously describedby Turner and Burr (39). Reproducibility was 5.8% for proximaltibia and 3.3% formidshaft tibia, and the coefficient of variationof paired sample measurements (left/right) was evaluated.Data Analysis—We first tested the effects of loading or exer-

cise within groups (Postn�/�, Postn�/�, and Postn�/�) bypaired or unpaired t tests. In the mechanical loading experi-ments, we compared stimulated and non-stimulated tibia in thesame animal using a paired t test. For the exercise investigation,we compared rested versus trained animals using unpaired ttests. We then tested the effects of repeated measures withingroups (stimulated/non-stimulated and exercise/sedentary) byone-way ANOVA repeat measurements with the genotypeemployed as a factor. To compare the effect of genotype and theresponse to loading (mechanical loading and exercise), we useda two-way ANOVA. As appropriate, post hoc testing was per-formed using Fisher’s protected least squares difference(PLSD). The p of interaction between the genotype and loading

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(mechanical stimulation or exercise) is only indicated when itwas found to be significant. Differences were considered signif-icant at p � 0.05. Data are presented as mean � S.E.

RESULTS

Exercise Versus Rested Conditions in Postn�/� Mice

We first evaluated the role of periostin during the skeletalresponse tophysiologicalmechanical stimuli inducedbymoderatephysical activity. For this purpose, 12-week-oldmice were trained

5 days/week for 6 weeks on a tread-mill, at 16m/min for 40min/daywitha treadmill inclination of 8° (31).Body Weight, Body Composition,

and Bone Mineral Density—Over-all, body weight tended to be lowerin Postn�/� mice compared withPostn�/� and Postn�/� mice butincreased significantly over 5 weeksin both resting and exercise condi-tions in Postn�/� and Postn�/�

mice (Fig. 1 and supplemental TableS1). The percentages of lean and fatmass were similar in all groups andnot significantly changed bymoder-ate exercise.BMD was similar in all groups

under resting conditions; however,in the exercise groups, BMD at thefemur and tibia were significantlylower in Postn�/� mice comparedwith Postn�/� and Postn�/� (Fig. 1and supplemental Table S1).Indeed, in Postn�/� mice, exerciseincreased BMD �20.2% in femurand �10.5% in tibia above base line(versus �2.2% and �1.8% in therested group, respectively; p �0.05), whereas in Postn�/� andPostn�/� mice, BMD gain did notdiffer between exercise and restinggroups (interaction between Postnand exercise, p � 0.05, by 2F-ANOVA) (supplemental Table S1).Bone Microarchitecture—At 12

weeks of age (base line), bonemicroarchitecture evaluated atdistal femur by ex vivo microCTshowed a lower TbN and a lowerBV/TV, more separation betweentrabeculae (TbSp), and a morerodlike trabecular shape (higherSMI) in Postn�/� compared withPostn�/� mice. Moreover, femurmidshaft size was smaller in Postn�/�

mice (i.e. a lower TV) and containedlower CtBV (Table 1).Compared with resting condi-

tions, exercise significantly in-creased TbN (�18.2%, p � 0.05) and connectivity-density(�52.1%, p � 0.05), decreased TbSp (�14.0%, p � 0.05), andhad borderline effects on BV/TV (�14.90, p � 0.09) inPostn�/� mice (Fig. 1 and supplemental Table S2). In contrast,exercise had no effect on trabecular bone parameters in eitherPostn�/� or Postn�/� mice (interaction between periostinpresence and exercise, p � 0.05 for TbN and connectivity den-sity, by 2F-ANOVA). At the midshaft femur, exercise signifi-cantly increased TV, CtBV, and BMaV in the Postn�/� mice

FIGURE 1. Effects of treadmill exercise on the femur. Bars, mean � S.E. measured after 5 weeks of exercise(closed bars) or rested (open bars) in Postn�/�, Postn�/�, and Postn�/� mice. A, body weight. B, BMD at totalfemur. C, Tb bone microarchitecture at distal femur. D, cortical bone microarchitecture at femur midshaft.E, biomechanical properties of the cortical femur measured by three-point bending. *, p � 0.05, unpaired t testcompared with rested group within periostin group; $, p � 0.05; $$, p � 0.01 unpaired Postn�/� versusPostn�/� mice by post hoc Fisher’s PLSD following 2F-ANOVA. Shown are means � S.E.

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(�23.1, �11.2, and �12.4% versus rest, respectively, all p �0.05) but not in Postn�/� or Postn�/� mice (Fig. 1 and supple-mental Table S2). As a result, the postexercise cortical and tra-becular bone microarchitecture was significantly improved inPostn�/� mice, whereas Postn�/� littermates showed minimalchange.Biomechanical Properties—To evaluate whether differ-

ences in cortical microarchitecture were translated into dif-ferences in bone strength, femurs were tested in bendingassays. At base line, Postn�/� mice had lower ultimate force(�27.4%, p � 0.05), ultimate stress (�28.2%, p � 0.05), andYoung’s modulus (�27.6%, p � 0.05) compared withPostn�/� mice, consistent with their smaller cortical bonevolume and reduced material properties (Table 1), whereasPostn�/� mice exhibited the expected intermediate biome-chanical properties between Postn�/� and Postn�/� mice(Table 1). Exercise significantly increased ultimate force andstiffness (�24.2 and �36.7%, respectively, versus rest, all p �0.05) in Postn�/� mice but not in Postn�/� or Postn�/� mice(Fig. 1). Young’s modulus, or the modulus of elasticity, wasalso significantly higher after 5 weeks of exercise in Postn�/�

compared with Postn�/� mice (37,116 � 3528 versus22,239 � 6363, p � 0.04), with intermediate values inPostn�/� mice (31,192 � 4383).Bone Turnover—We next evaluated the relative indices of

bone turnover in tibia from Postn�/� and Postn�/� mice byhistomorphometry. In resting conditions, few differences wereobserved in bone turnover between Postn�/� and Postn�/�

mice besides a 33% lower MAR on trabecular bone surfaces inthe latter; nor were there any significant differences in osteo-blast and osteoclast number per bone surface (Table 2).Although we did not observe any significant effects of exerciseonosteoblast number or surface, exercise did significantly stim-ulate bone formation indices (MAR, BFR, and MPm/BPm) onperiosteal and, to a lesser extent, trabecular surfaces inPostn�/� mice, with similar trends at the endocortical surface,whereas no significant effect on bone turnover was observed inPostn�/� mice (Table 2).

Skeletal Response to Axial Compression

Having established that periostin modulates the skeletalresponse to moderate physical activity, we next investigatedwhether mechanotransduction of direct mechanical strains onbone also required the presence of periostin. More specifically,we wanted to evaluate whether an intense biomechanical stim-ulus could overcome the effects of deficient Postn expression inbone. For this purpose,micewere subjected to direct axial com-pression of the tibia in vivo for 2 weeks (see “Experimental Pro-cedures” for details), and longitudinal changes in microarchi-tecture were assessed by in vivomicroCT.BoneMicroarchitecture and Strength—Trabecular and corti-

cal bone microarchitectural parameters of the non-stimulatedand stimulated tibiae at base line were significantly lower inPostn�/� mice and intermediate in Postn�/� mice when com-pared with Postn�/� littermates (Table 3). Axial compressionsignificantly increased BV/TV (�58.2% versus �4.0% abovebase line in the stimulated and non-stimulated tibia, respec-tively, p � 0.05), TbN, TbTh, TV, CtBV, and CtTh in Postn�/�

mice (Table 3 and Fig. 2,A–D). Contrasting with their responseto moderate physical activity (above), haploinsufficientPostn�/� mice had changes of bone microarchitecture followingaxial compression that were similar to those in Postn�/� mice(Table 3 and Fig. 2). In contrast, in the Postn�/� mice, no signifi-cantdifferenceswereobserved ineither trabecularorcorticalbonegainbetweenstimulatedandnon-stimulatedbones.Theseperiostin-dependent changes in thecortical bone response toaxial compres-sion were translated into significant differences in bone biome-chanical properties, such as stiffness and Young’s modulus, thatwere significantly higher in Postn�/� and Postn�/� mice com-pared with Postn�/� mice (Fig. 2E).Bone Turnover—Bone histomorphometry confirmed that

Postn�/�mice had increased bone turnover in response to axialcompression (Fig. 3 and supplemental Table S3). Hence, at theperiosteum, loading increased the BFR 15.5-fold and the min-eralization perimeter (MPm/BPm) 8.5-fold compared with thenon-stimulated tibia in Postn�/� mice (p � 0.01, Fig. 3 andsupplemental Table S3). In contrast, compression did not sig-

TABLE 1Characterization of femoral bone microarchitecture and biomechanical properties in periostin-deficient miceCancellous bonemicroarchitecture was evaluated at distal femur and cortical microarchitecture at midshaft femur by ex vivomicroCT at 12 weeks of age (base line, n � 10mice/group). Bending strength of femur midshaft was evaluated by a three-point bending test on excised femur.

Parameters Postn�/� Postn�/� Postn�/� p value

Trabecular BV/TV (%) 21.6 � 1.2 15.2 � 1.1a 11.1 � 1.5a 0.02Conn density (1/mm3) 180.1 � 15.4 129.5 � 10.7a 101.4 � 7.1a 0.02

Tb.Th (�m) 56 � 4 52 � 3 47 � 4 0.39Tb.N 5.4 � 0.1 4.7 � 0.1a 4.4 � 0.04a 0.02

Tb.Sp (�m) 177 � 5 210 � 6a 225 � 2a 0.02SMI 1.5 � 0.09 2.1 � 0.1a 2.5 � 0.1a 0.02

Cortical CtTV (mm3) 1.31 � 0.08 1.30 � 0.03 1.12 � 0.04a,b 0.04CtBV (mm3) 0.55 � 0.04 0.52 � 0.02 0.46 � 0.02a 0.04CtTh (�m) 280 � 15 271 � 8 261 � 11 0.43BMaV (�m3) 0.78 � 0.05 0.77 � 0.02 0.66 � 0.03a,b 0.04

Bending strength Ultimate force (N) 15.7 � 0.8 13.9 � 0.8 11.4 � 1.0a 0.04Ultimate Stress (N/mm²) 546 � 65 537 � 89 392 � 63a 0.05Elastic Energy (N�mm) 1.67 � 0.2 1.30 � 0.2 1.33 � 0.2 0.22Plastic Energy (N�mm) 2.95 � 0.5 2.80 � 0.2 2.60 � 0.4 0.47

Stiffness (N/mm) 64.8 � 6.3 53.8 � 5.4 47.2 � 6.8a 0.04Young’s modulus (megapascals) 37888 � 3284 31457 � 4050 27427 � 3864a 0.04

a p � 0.05 versus Postn�/� mice, by post hoc Fisher’s PLSD. Means � S.E.; p value for differences between periostin groups (1F-ANOVA).b p � 0.05 versus Postn�/� mice, by post hoc Fisher’s PLSD. Means � S.E.; p value for differences between periostin groups (1F-ANOVA).

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nificantly increase periosteal MPm/BPm in Postn�/� mice,whereas BFR/BPm increased 3.5-fold (p� 0.05). Thus, the peri-osteal BFR in the stimulated bone of Postn�/� mice remainedsignificantly lower compared with Postn�/� mice. Similarly, atthe trabecular surfaces, axial compression increased MAR(�41.6%) and BFR (�92.4%) in Postn�/� mice (p � 0.01 versusthe non-stimulated tibia) (Fig. 3), whereas in Postn�/� mice,trabecular bone formation indices did not significantly differbetween stimulated and non-stimulated bones (supplemental

Table S3). Contrary to its prominent effects on periosteal andtrabecular bone turnover, periostin appeared not to influencethe bone forming indices at endocortical surfaces, whichresponded equally in Postn�/� and Postn�/� mice.Effects of Axial Compression on Postn and Sost Expression—

In order to clarify the molecular mechanisms by which perios-tin mediates the skeletal response to mechanical stimulation,we next examined the effects of axial compression on periostinprotein expression in bone.

TABLE 2Influence of periostin and exercise on bone turnover at cortical and trabecular bone surfacesBone histomorphometry performed at tibia metaphysis (trabecular) and diaphysis (periosteal and endocortical) after 5 weeks of exercise or resting conditions. Ps,periosteum; Ec, endocortical; Tb, trabecular.

Parameters Postn�/� Postn�/� p value

RestPeriosteal Ps MAR (�m/day) 0.21 � 0.11 0.22 � 0.08 0.70

Ps BFR/BPm (�m2/�m/day) 0.004 � 0.004 0.01 � 0.005 0.31Ps MPm/BPm (%) 0.19 � 0.1 0.22 � 0.08 0.59

Endocortical Ec MAR (�m/day) 0.15 � 0.15 0.24 � 0.08 0.72Ec BFR/BPm (�m2/�m/day) 0.003 � 0.017 0.01 � 0.006 0.52

Ec MPm/BPm (%) 0.13 � 0.04 0.27 � 0.10 0.20Trabecular Tb MAR (�m/day) 0.60 � 0.04 0.40 � 0.11a 0.02

Tb BFR/BS (�m2/�m3/day) 0.19 � 0.04 0.08 � 0.09 0.08Tb MS/BS (%) 32.8 � 5.3 20.9 � 7.4 0.16OcS/BS (%) 6.5 � 1.0 6.4 � 1.5 0.75

OcN/BPm (mm�1) 3.2 � 0.5 3.2 � 0.7 0.68ObS/BS (%) 10.6 � 1.2 7.4 � 0.7 0.36

ObN/BPm (mm�1) 10.0 � 1.3 6.9 � 0.7 0.10ExercisePeriosteal Ps MAR (�m/day) 0.44 � 0.04b 0.30 � 0.16 0.13

Ps BFR/BPm (�m2/�m/day) 0.03 � 0.008b 0.018 � 0.011 0.22Ps MPm/BPm (%) 0.59 � 0.14b 0.30 � 0.11 0.09

Endocortical Ec MAR (�m/day) 0.28 � 0.10 0.22 � 0.10 0.73Ec BFR/BPm (�m2/�m/day) 0.008 � 0.12 0.006 � 0.004 0.69

Ec MPm/BPm (%) 0.24 � 0.06 0.26 � 0.10 0.54Trabecular Tb MAR (�m/day) 0.82 � 0.15b 0.44 � 0.03a 0.01

Tb BFR/BS (�m2/�m3/day) 0.36 � 0.11 0.13 � 0.10a 0.02Tb MS/BS (%) 43.4 � 8.2 29.8 � 7.3 0.15OcS/BS (%) 4.0 � 1.6 7.8 � 2.3 0.22

OcN/BPm (mm�1) 1.9 � 0.8 3.9 � 1.1 0.17ObS/BS (%) 6.6 � 1.3 9.2 � 1.8 0.53

ObN/BPm (mm�1) 6.2 � 1.2 7.5 � 1.7 0.53a p � 0.05 versus Postn�/� mice (Fisher’s PLSD) (n � 8 mice/group) (means � S.E.).b p � 0.05 versus rested mice (unpaired t test) (n � 8 mice/group) (means � S.E.).

TABLE 3Influence of periostin on changes of tibial bone microarchitecture in response to axial compressionCancellous bone microarchitecture was evaluated at proximal tibia and cortical microarchitecture at midshaft tibia in the axially compressed bone and its controlateralnon-stimulated control bone by in-vivo microCT at base line (14 weeks old) and after 2 weeks (n � 10 mice/group).

ParametersPostn�/� Postn�/� Postn�/�

p valueBaseline 2 weeks Baseline 2 weeks Baseline 2 weeks

Non-stimulatedTrabecular BV/TV (%) 12.5 � 1.4 13.0 � 1.1 11.8 � 1.9 13.3 � 1.6 6.4 � 0.6 10.1 � 0.9a 0.01

TbN (mm�1) 1.95 � 0.19 1.87 � 0.13 1.71 � 0.26 1.80 � 0.18 1.04 � 0.08 1.45 � 0.11a 0.008TbTh (�m) 63.21 � 1.4 69.10 � 1.6 67.3 � 1.6 71.9 � 2.5 61.0 � 1.7 69.0 � 1.6a 0.20TbSp (mm) 234 � 12 262 � 11 282 � 28 291 � 19 363 � 16 335 � 22 0.03

SMI 2.39 � 0.06 2.42 � 0.04 2.43 � 0.09 2.32 � 0.07 2.60 � 0.03 2.45 � 0.04a 0.16Cortical TV (mm3) 0.89 � 0.03 1.19 � 0.03 0.85 � 0.03 1.19 � 0.04 0.66 � 0.03 1.04 � 0.03a 0.01

BV (mm3) 0.54 � 0.02 0.60 � 0.02 0.54 � 0.02 0.60 � 0.02 0.43 � 0.02 0.56 � 0.02a 0.04CTh (�m) 248 � 5 237 � 3 245 � 9 236 � 3 217 � 3 241 � 6a 0.38

BMaV (�m3) 352 � 15 594 � 24 318 � 14 593 � 24a 235 � 10 447 � 16a 0.01StimulatedTrabecular BV/TV (%) 11.0 � 1.2 17.4 � 0.9a,b 10.8 � 1.3 18.2 � 1.1a,b 7.2 � 0.8 11.4 � 0.9a 0.001

TbN (1/mm) 1.77 � 0.17 2.32 � 0.12a,b 1.61 � 0.17 2.33 � 0.13a,b 1.18 � 0.12 1.51 � 0.10a 0.001TbTh (�m) 61.52 � 1.0 74.69 � 1.4a,b 66.0 � 1.7 77.7 � 1.2a 60.1 � 1.5 70.1 � 1.5a 0.03TbSp (mm) 245 � 14 218 � 8a,b 268 � 18 223 � 8a,b 326 � 21 299 � 14 0.01

SMI 2.50 � 0.06 2.40 � 0.06 2.56 � 0.04 2.33 � 0.05a 2.65 � 0.04 2.41 � 0.03a 0.4Cortical TV (mm3) 0.90 � 0.04 1.29 � 0.04a,b 0.85 � 0.02 1.31 � 0.04a,b 0.65 � 0.03 0.97 � 0.03a 0.001

BV (mm3) 0.56 � 0.02 0.67 � 0.01a,b 0.53 � 0.01 0.68 � 0.02a,b 0.42 � 0.02 0.52 � 0.02a 0.001CTh (�m) 240 � 6 257 � 5a,b 240 � 4 253 � 5a,b 220 � 5 235 � 6a 0.05

BMaV (�m3) 346 � 18 624 � 28a 320 � 12 629 � 24a 236 � 9 475 � 18a 0.01a p � 0.05 versus base line (one way ANOVA with repeat measurements); p value for differences between periostin groups (one-way ANOVA with repeat measurements)(means � S.E.).

b p � 0.05 versus non-stimulated (paired t test). p value for differences between periostin groups (one-way ANOVA with repeat measurements) (means � S.E.).

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Immunohistochemistry revealed robustly elevated expres-sion of the periostin in the periosteum 24 h after axial com-pression (Fig. 4A). The specificity of periostin expressionwas established by the lack of staining in Postn�/� bone sec-tions. Periostin deposition along the bone axis indicatesmaximal expression within bone regions supporting the

highest strain, such as the proxi-mal diaphysis, which exhibited rel-atively large peak strains duringaxial compression (zones 1–4) (Fig.4B) and the greatest increase inperiostin staining (�56%) (Fig. 4B),whereas sections from themidshaft,which experience only relativelysmall peak strains during axial com-pression, only exhibited a modestincrease in periostin staining (zones5–7) (Fig. 4B). Consistent with pre-vious reports (13), no periostin de-position was detected at endocorti-cal surfaces in the proximal or distaldiaphysis or in the midshaft region(Fig. 4A). Nevertheless, weak peri-ostin staining was detected in can-cellous bone after axial compression(Fig. 4C).We further evaluated the time

course of Postn and Sost mRNAexpression following axial compres-sion. A single regimen of axial com-pression (12N and 0.1 Hz for 7min)of the tibia induced a rapid (6 h), 2.2-fold increase of Postn expression(relative abundance: 0.42 � 0.1 ver-sus 0.13 � 0.04, p � 0.05 in loadedversus non-loaded; data not shown).After 24 h, increased Postn expres-sion was sustained and accompa-nied by a significant 50% decrease ofSost expression in Postn�/� micebut not in Postn�/� mice. In the lat-ter, Sost expressionwas significantlyhigher than Postn�/� mice in boththe non-loaded and loaded tibia(Fig. 4D). These data suggest thatstimulation of Postn and inhibitionof Sost expression in response tomechanical loading are temporallyrelated.

Skeletal Response to AxialCompression Combined withSclerostin-blocking Antibodies

To directly evaluate the role ofsclerostin in the molecular mecha-nisms by which periostin mediatesthe skeletal response to mechanicalstimulation, we next examined if the

injection of Sost-Ab intoPostn�/�mice could rescue their bonephenotypes and restore their responses to loading.Bone Mineral Density and Architecture—Again, in control

Ab-treated mice, tibia BMD gain in response to axial compres-sion was significantly lower in Postn�/� mice compared withPostn�/� mice (�6.8% versus �20.7%, p � 0.05). In Postn�/�

FIGURE 2. Effects of axial compression on tibia. Bars, mean � S.E. measured after 2 weeks of axial compres-sion in stimulated tibia (closed bars) or non-stimulated tibia (open bars) in Postn�/�, Postn�/�, and Postn�/�

mice. A, trabecular bone microarchitecture of the proximal tibia. B, three-dimensional reconstruction images ofthe proximal tibia metaphysis cancellous bone. C, cortical bone microarchitecture of the midshaft tibia. D, two-dimensional reconstruction of the midshaft tibia. E, biomechanical properties of the cortical tibia measured bythree-point bending. *, p � 0.05 paired t test compared with non-stimulated tibia within the periostin group; $,p � 0.05; $$, p � 0.01; $$$, p � 0.001, genotype effect between Postn�/�, Postn�/�, and Postn�/� mice by post hocFisher’s PLSD following 2F-ANOVA. Shown are means � S.E. S, stimulated tibia; NS, non-stimulated tibia.

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mice receiving Sost-Ab, the BMD gain in the stimulated tibiawas fully rescued (�34.8%) (i.e. not different from the Postn�/�

mice receiving Sost-Ab) (�26%). The cortical response to axialcompression was also restored by Sost-Ab in the Postn�/�

mice, as shown by their higher cortical thickness in the stimu-lated versus the non-stimulated tibia (�6.9% versus�1.5% (p�0.05) in the group receiving control Ab) (Fig. 5A). Meanwhile,in Postn�/� mice, the administration Sost-Ab concomitant toaxial compression did not significantly improve the anaboliceffect of compression alone, which is consistent with the Sost-inhibitory effects of mechanical loading in normal mice. A sim-ilar trend was observed for BV/TV of the trabecular bone com-partment (Fig. 5A).Of note, the lower vertebral BMD and BV/TV in Postn�/�

mice compared with Postn�/� mice was also rescued by shortterm administration of Sost-Ab (Fig. 5B), further demonstrat-ing that the higher expression of Sostwas in part responsible forthe lower bone mass of Postn-deficient mice.Collagen Structure in Postn�/� Mice—Because it has been

reported that the size and structure of collagen fibrils isaltered in Postn�/� alveolar bone, we next hypothesized thatpoor mechanotransduction properties in the cortical bone ofPostn-deficient mice could reflect the altered structure ofthe bone matrix at this site. For this purpose, we analyzedcollagen size and shape at the periosteum by electronmicroscopy. As shown in Fig. 6, the cortical bone of Postn�/�

mice was characterized by more abundant and larger colla-gen fibrils compared with Postn�/� mice (Fig. 6).

DISCUSSION

The main objective of our study was to elucidate the invivo role of periostin in the skeletal response to mechanical

loading. The loss of Postn in miceresulted in altered cancellous bonemicroarchitecture (lower BV/TVand TbN and higher TbSp andSMI), reduced cortical bone vol-ume, and decreased bendingmechanical properties (bonestrength). We specifically demon-strate that periostin regulates theskeletal response to mechanical sig-nals by mediating Sost inhibition.These results confirm and expandthe important role of periostin inthe determination of bonemass andstructure (13) and demonstrate theinfluence of Postn in bone turnover.More broadly, they emphasize therole of periostin in transducingmechanical signals, as previouslyshown in other tissues, includingthe periodontal ligament and heartvalves (5, 7, 9, 15, 40, 41).We provide several lines of evi-

dence indicating that periostin ex-pression is essential for the down-re-gulation of Sost. First, Postn mRNA

and protein expression increased by axial compression prior tothe inhibition of Sost expression in the same bone compart-ment, indicating that these events are both spatially and tem-porally related. Second, in Postn�/� mice, Sost expression wasincreased and was not inhibited following axial compression.Third, administration of a Sost-Ab to Postn�/� miceimproved their low bone mass and trabecular bone volumein vertebrae, a non-loaded site, and rescued the biomechani-cal response of the tibia to axial compression. To potentiallyexplain the absence of Sost inhibition in Postn�/� mice, twomajor hypotheses are suggested. First, the absence of Postncould alter the structure of the bone matrix and thereforeimpair its mechanotransduction properties. Altered shape,structure, and disorganization of type I collagen fibers hadpreviously been reported in the alveolar bone of Postn�/�

mice (14). We now confirm similar alterations in type I col-lagen fibers of the Postn�/� periosteal bone by electronmicroscopy. Second, periostin activates the integrin signal-ing pathways (Akt/phosphatidylinositol 3-kinase) (42, 43),which we also found in UMR-106 osteoblast-like cells (datanot shown). Integrins are known to mediate osteocyteresponse to mechanical stimulation (44, 45), and it is possi-ble therefore that periostin contributes to Sost inhibition byco-activating integrin signaling in these cells. In addition,phosphatidylinositol 3-kinase/Akt signaling may influenceWnt-LRP5 signaling by inhibiting GSK3 kinase and releasing�-catenin from its inhibitory complex (46–48). It is there-fore also possible that periostin directly stimulates canonicalsignaling pathways for bone formation via integrinreceptors.The presence of shorter long bones in Postn-/- mice suggests

a disruption of the cartilaginous growth plate (13). Moreover,

FIGURE 3. Bone remodeling at cortical bone surfaces in response to axial compression. A, immunofluo-rescent sections of midshaft tibia show cortical calcein labels on cortical surfaces of stimulated and non-stimulated bone in Postn�/� and Postn�/� mice. B, BFR on the surfaces of bone. Bars, mean � S.E. measuredafter 2 weeks of axial compression in stimulated tibia (closed bars) or non-stimulated tibia (open bars) inPostn�/� and Postn�/� mice. *, p � 0.05; **, p � 0.01, paired t test compared with non-stimulated tibia withinthe periostin group; $, p � 0.05, Postn�/� versus Postn�/� mice by post hoc Fisher’s PLSD following 2F-ANOVA.Shown are means � S.E.

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due to their periodontal alterations and ensuing difficultieswith feeding, these mice are known to mature slowly and topresent a deficit in body weight. Although we attempted toimprove their feeding and growth rate by giving them a soft dietfrom birth, Postn�/� mice still exhibited a slower growth ratewith a continuous increase of BV/TV up to 16–18 weeks of age(i.e. longer than wild type mice). Nevertheless, Postn�/� mice

never catch up the higher bonemassof Postn�/� mice. In any case, a pro-longed period of bonemass growth inPostn�/� mice could only favor,rather than inhibit, their response tomechanical stimulation. Therefore,the slight growth delay in Postn�/�

mice is very unlikely to explain theirsignificant lack of responses tomechanical stimulation.Pharmacological studies have

previously suggested that periostinmay play a role in bone formation.Horiuchi et al. (15) observed a dose-dependent response of periostinexpression to TGF-� stimulation inprimary osteoblast cells. PTH wasalso shown to up-regulate Postn,together with other cell adhesionproteins, concomitant to its stimu-lation of bone formation on histo-morphometric analyses of rat bones(49, 50). In addition, in 6-week-oldrats,Postn overexpression increasedbone formation and bone mass, asevaluated by microCT and histo-morphometry (51). In vitro, overex-pression of Postn results in a signif-icant increase in primary osteoblastcell proliferation and differentiation(51). Conversely, deletion of Postninduces a defect in the attachmentof bone cells, which affects theirdifferentiation and mineralizationprocesses (16). Our results furtherindicate that, although osteoblastnumbers and bone formation indicesarenotnotablyprominentlyaltered inmice lacking Postn, bone turnover (asevaluated by BFR) in response toloading is clearly diminished in theabsence of periostin, particularlywithin the cortical compartment.Although we did not observe

clear differences of osteoblastnumber or surface betweenPostn�/� and Postn�/� mice sub-jected to physical activity, activityof osteoblast (Tb MAR and TbBFR) was significantly higher inPostn�/� mice at the trabecular

level. This is not unexpected, because osteoblast function,rather than proliferation, is primarily affected by exercise(52). However, we cannot exclude the possibility that histo-morphometry performed at the end of the experiment (5weeks after the first training session) could have missed theanabolic effects of exercise that may have occurred earlier(53).

FIGURE 4. Periostin and Sost expression in bone response to mechanical strain. A, immunohistochemicalanalysis of periostin expression in longitudinal sections of the proximal tibia 24 h after mechanical stimulationor not. Note the brown staining of periostin in the periosteum (Ps) of wild type mice that increased withstimulation, whereas no staining is detectable at endocortical surfaces (Ec) or in Postn�/� mice. B, thickness ofthe periostin staining is shown in relation to the strain generated by axial compression along the tibia; high strainregions are green to red, and low strain regions are blue. Bars, mean measured after 24 h of axial compression instimulated tibia (closed bars) or non-stimulated tibia (open bars) in Postn�/� mice. C, immunohistochemical analysisof periostin expression in longitudinal section of the proximal metaphysis 24 h after mechanical stimulation or not.Note the soft staining of periostin in Tb compared with the periosteum bone (Fig. 5A). D, Postn and Sost geneexpression levels in stimulated and non-stimulated tibia evaluated by quantitative real-time PCR analysis of RNAextracted from the tibia diaphysis. Bars, mean � S.E. measured after 24 h of axial compression in stimulated tibia(closed bars) or non-stimulated tibia (open bars) in Postn�/� mice. *, p � 0.05, paired t test compared with non-stimulated tibia within periostin group. $, p � 0.05, Postn�/� versus Postn�/� mice by post hoc Fisher’s PLSD follow-ing 2F-ANOVA. Data are presented as means � S.E. of n � 4 mice in each genotype.

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Mechanical loading (axial compression or exercise) is aneffective way to increase bone mass and prevent bone loss, andit may reduce fracture risk (54–56). Mechanical properties oflong bones are more profoundly affected by modifications ofthe periosteal surface than the endocortical compartment (57–59). Indeed, mechanical loading induces an increase in bonestrength by targeting new bone formation to skeletal surfacesexperiencing the largest mechanical strain (i.e. the periosteum)(24, 27, 60). Interestingly, periostin is preferentially expressedin the periosteum rather than in the endocortical region, andselective stimulation of periostin expression in the periosteumas well as greater increases of bone formation indices in theperiosteal compartment both occurred in response to mechan-ical stimulation.Our current results therefore suggest that peri-ostin may serve as a molecular conduit to concentrate biome-chanical signals to the outer surfaces of bone (i.e.where they aremost needed). Of note, the skeletal phenotype of Postn haplo-insufficient mice (Postn�/� mice) did not significantly differ

from wild type mice; nor was theirresponse to direct axial compres-sion affected. In contrast, the skele-tal response of Postn�/� mice tomoderate physical activity wasimpaired, as seen in Postn�/� mice.This may suggest that the level ofperiostin expression modulates thesensitivity of the mechanostat (i.e.that periostin may be able todecrease the threshold for a bonebiomechanical response). Thus, inresponse to high mechanical loads(axial compression), a low level ofperiostin expression (as in haploin-sufficient mice) was necessary andsufficient to induce a biomechanicalresponse, whereas with moderatemechanical stimuli (i.e. during exer-cise), even a partial deficiency ofperiostin resulted in a diminishedskeletal response. Another possibleexplanation is that lowperiostin lev-els may disrupt the myotendinousjunction, which is one of the impor-tantmechanisms bywhich exercise-induced strain can be transmitted tothe bone (61). This hypothesis isfurther supported by the recentfindings that periostin regulates col-lagen fibrillogenesis, whereas in theabsence of Postn, the biomechanicalproperties of connective tissues,such as the tendon, are affected (9).Taken together, these data sug-

gest a role for periostin inmediatingbone formation in response tomechanical loading. Specifically,mechanical loading increases peri-ostin expression, which is necessary

to inhibit Sost expression and thereby to up-regulate osteoblastfunctions. In the absence of periostin, bone formation isimpaired, and mechanical loading is unable to effectivelyimprove bone structure and strength, unless sclerostin proteinis antagonized.

Acknowledgments—We thank Goldie Lin for histological assistanceand Dr. Tara Brennan for editorial assistance. We also thank Dr.Michaela Kneissel (Novartis, AG, Basel, Switzerland) for providingthe sclerostin-blocking antibodies and for assistance in designing theseexperiments. We thank Fanny Cavat for technical assistance and thePole Facultaire deMicroscopie ultrastructural at theGenevaMedicalFaculty for access to transmission electron microscopy equipment.

REFERENCES1. Takeshita, S., Kikuno, R., Tezuka, K., and Amann, E. (1993) Biochem. J.

294, 271–2782. Gillan, L., Matei, D., Fishman, D. A., Gerbin, C. S., Karlan, B. Y., and

FIGURE 5. Effects of Sost-Ab on tibia response to axial compression. A, differences in tibia BMD, trabecularbone volume, and cortical thickness between the stimulated and the non-stimulated tibia for the BMD in micereceiving Sost-Ab (gray bars) or control Ab (open bars). B, bone mineral density and three-dimensional trabe-cular bone microarchitecture of the vertebrae in Postn�/� and Postn�/� mice receiving Sost-Ab (closed bars) orcontrol Ab (open bars). Bars, mean � S.E. $, p � 0.05, Postn�/� versus Postn�/� mice; *, Sost-Ab versus control Abby post hoc Fisher’s PLSD following 2F-ANOVA. Shown are means � S.E.

FIGURE 6. Characterization of the collagen fibril at the periosteum in Postn�/� and Postn�/� mice. Elec-tron microscopy shows cross-sectional collagen fibril in 12-week-old Postn�/� mice on the left and in 12-week-old Postn�/� mice on the right. Postn�/� mice shows abundant and bigger collagen fibrils. Means � S.E. **, p �0.01; **, p � 0.001, unpaired t test compared Postn�/� and Postn�/� mice. ROI, region of interest.

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Chang, D. D. (2002) Cancer Res. 62, 5358–53643. Shimazaki, M., Nakamura, K., Kii, I., Kashima, T., Amizuka, N., Li, M.,

Saito, M., Fukuda, K., Nishiyama, T., Kitajima, S., Saga, Y., Fukayama, M.,Sata, M., and Kudo, A. (2008) J. Exp. Med. 205, 295–303

4. Bao, S., Ouyang, G., Bai, X., Huang, Z.,Ma, C., Liu,M., Shao, R., Anderson,R. M., Rich, J. N., and Wang, X. F. (2004) Cancer Cell 5, 329–339

5. Katsuragi, N., Morishita, R., Nakamura, N., Ochiai, T., Taniyama, Y., Ha-segawa, Y., Kawashima, K., Kaneda, Y., Ogihara, T., and Sugimura, K.(2004) Circulation 110, 1806–1813

6. Dorn, G. W., 2nd (2007) N. Engl. J. Med. 357, 1552–15547. Snider, P., Hinton, R. B., Moreno-Rodriguez, R. A., Wang, J., Rogers, R.,

Lindsley, A., Li, F., Ingram, D. A., Menick, D., Field, L., Firulli, A. B., Molk-entin, J. D.,Markwald, R., and Conway, S. J. (2008)Circ. Res. 102, 752–760

8. Litvin, J., Blagg, A., Mu, A., Matiwala, S., Montgomery, M., Berretta, R.,Houser, S., and Margulies, K. (2006) Cardiovasc. Pathol. 15, 24–32

9. Norris, R. A., Damon, B., Mironov, V., Kasyanov, V., Ramamurthi, A.,Moreno-Rodriguez, R., Trusk, T., Potts, J. D., Goodwin, R. L., Davis, J.,Hoffman, S.,Wen, X., Sugi, Y., Kern, C. B.,Mjaatvedt, C. H., Turner, D. K.,Oka, T., Conway, S. J., Molkentin, J. D., Forgacs, G., and Markwald, R. R.(2007) J. Cell. Biochem. 101, 695–711

10. Wilde, J., Yokozeki, M., Terai, K., Kudo, A., and Moriyama, K. (2003) CellTissue Res. 312, 345–351

11. Rios, H. F., Ma, D., Xie, Y., Giannobile, W. V., Bonewald, L. F., Conway,S. J., and Feng, J. Q. (2008) J. Periodontol. 79, 1480–1490

12. Afanador, E., Yokozeki, M., Oba, Y., Kitase, Y., Takahashi, T., Kudo, A.,and Moriyama, K. (2005) Arch. Oral Biol. 50, 1023–1031

13. Rios, H., Koushik, S. V., Wang, H., Wang, J., Zhou, H. M., Lindsley, A.,Rogers, R., Chen, Z., Maeda, M., Kruzynska-Frejtag, A., Feng, J. Q., andConway, S. J. (2005)Mol. Cell. Biol. 25, 11131–11144

14. Kii, I., Amizuka, N., Minqi, L., Kitajima, S., Saga, Y., and Kudo, A. (2006)Biochem. Biophys. Res. Commun. 342, 766–772

15. Horiuchi, K., Amizuka, N., Takeshita, S., Takamatsu, H., Katsuura, M.,Ozawa,H., Toyama, Y., Bonewald, L. F., andKudo,A. (1999) J. BoneMiner.Res. 14, 1239–1249

16. Litvin, J., Selim, A.H.,Montgomery,M.O., Lehmann, K., Rico,M. C., Devlin,H., Bednarik, D. P., and Safadi, F. F. (2004) J. Cell. Biochem. 92, 1044–1061

17. Ogita, M., Rached, M. T., Dworakowski, E., Bilezikian, J. P., and Kousteni,S. (2008) Endocrinology 149, 5713–5723

18. Nakazawa, T., Nakajima, A., Seki, N., Okawa, A., Kato, M., Moriya, H.,Amizuka, N., Einhorn, T. A., and Yamazaki, M. (2004) J. Orthop. Res. 22,520–525

19. Iwamoto, J., Takeda, T., and Ichimura, S. (1998) J. Orthop. Sci. 3, 257–26320. Iwamoto, J., Yeh, J. K., and Aloia, J. F. (1999) Bone 24, 163–16921. Bonnet, N., Laroche, N., Beaupied, H., Vico, L., Dolleans, E., Benhamou,

C. L., and Courteix, D. (2007) J. Appl. Physiol. 103, 524–53322. Robling, A. G., Burr, D. B., and Turner, C. H. (2001) J Musculoskelet.

Neuronal Interact. 1, 249–26223. Lee, K. C., Maxwell, A., and Lanyon, L. E. (2002) Bone 31, 407–41224. de Souza, R. L., Pitsillides, A. A., Lanyon, L. E., Skerry, T.M., andChenu, C.

(2005) J. Bone Miner Res. 20, 2159–216825. Robling, A. G., Li, J., Shultz, K. L., Beamer,W. G., and Turner, C. H. (2003)

FASEB J. 17, 324–32626. Robling, A. G., Niziolek, P. J., Baldridge, L. A., Condon, K.W., Allen,M. R.,

Alam, I., Mantila, S. M., Gluhak-Heinrich, J., Bellido, T. M., Harris, S. E.,and Turner, C. H. (2008) J. Biol. Chem. 283, 5866–5875

27. Sawakami, K., Robling, A.G., Ai,M., Pitner, N. D., Liu, D.,Warden, S. J., Li,J., Maye, P., Rowe, D.W., Duncan, R. L.,Warman,M. L., and Turner, C. H.(2006) J. Biol. Chem. 281, 23698–23711

28. Huang, Q. Y. (2008) International Bone and Mineral Workshops, Davos,SwitzerlandMarch 9–11, 2009

29. Stadelmann, V. A., Hocke, J., Verhelle, J., Forster, V., Merlini, F., Terrier,A., and Pioletti, D. P. (2008) Comput. Methods Biomech. Biomed. Engin.12, 95–100

30. Li, X., Ominsky, M. S., Warmington, K. S., Morony, S., Gong, J., Cao, J.,Gao, Y., Shalhoub, V., Tipton, B., Haldankar, R., Chen, Q., Winters, A.,Boone, T., Geng, Z., Niu, Q. T., Ke, H. Z., Kostenuik, P. J., Simonet, W. S.,

Lacey, D. L., and Paszty, C. (2009) J. Bone Miner. Res. 24, 578–58831. Narkar, V. A., Downes, M., Yu, R. T., Embler, E., Wang, Y. X., Banayo, E.,

Mihaylova, M. M., Nelson, M. C., Zou, Y., Juguilon, H., Kang, H., Shaw,R. J., and Evans, R. M. (2008) Cell 134, 405–415

32. Iida-Klein, A., Lu, S. S., Yokoyama, K., Dempster, D.W., Nieves, J.W., andLindsay, R. (2003) J. Clin. Densitom. 6, 25–33

33. Waarsing, J. H., Day, J. S., van der Linden, J. C., Ederveen, A. G., Spanjers,C., De Clerck, N., Sasov, A., Verhaar, J. A., and Weinans, H. (2004) Bone34, 163–169

34. Waarsing, J. H., Day, J. S., andWeinans, H. (2002)The IXth Congress of theInternational Society of Bone Morphometry, Edingburgh, UK, April, 2002

35. Bouxsein, M. L., Pierroz, D. D., Glatt, V., Goddard, D. S., Cavat, F., Rizzoli,R., and Ferrari, S. L. (2005) J. Bone Miner Res. 20, 635–643

36. Hildebrand, T., and Ruegsegger, P. (1997) J. Microsc. 185, 67–7537. Parfitt, A. M., Drezner, M. K., Glorieux, F. H., Kanis, J. A., Malluche, H.,

Meunier, P. J., Ott, S. M., and Recker, R. R. (1987) J. Bone Miner. Res. 2,595–610

38. Foti, M., Carpentier, J. L., Aiken, C., Trono, D., Lew, D. P., and Krause,K. H. (1997)Mol. Biol. Cell 8, 1377–1389

39. Turner, C. H., and Burr, D. B. (1993) Bone 14, 595–60840. Litvin, J., Zhu, S., Norris, R., andMarkwald, R. (2005) Anat. Rec. A Discov.

Mol. Cell Evol. Biol. 287, 1205–121241. Norris, R. A., Moreno-Rodriguez, R. A., Sugi, Y., Hoffman, S., Amos, J.,

Hart, M. M., Potts, J. D., Goodwin, R. L., and Markwald, R. R. (2008) Dev.Biol. 316, 200–213

42. Ruan, K., Bao, S., and Ouyang, G. (2009) Cell Mol. Life Sci. 66, 2219–223043. Kashima,T.G.,Nishiyama,T., Shimazu,K., Shimazaki,M.,Kii, I.,Grigoriadis,

A. E., Fukayama, M., and Kudo, A. (2009)Hum. Pathol. 40, 226–23744. Miyauchi, A., Gotoh, M., Kamioka, H., Notoya, K., Sekiya, H., Takagi, Y.,

Yoshimoto, Y., Ishikawa, H., Chihara, K., Takano-Yamamoto, T., Fujita,T., and Mikuni-Takagaki, Y. (2006) J. Bone Miner. Metab. 24, 498–504

45. Phillips, J. A., Almeida, E. A., Hill, E. L., Aguirre, J. I., Rivera, M. F., Nach-bandi, I., Wronski, T. J., van der Meulen, M. C., and Globus, R. K. (2008)Matrix Biol. 27, 609–618

46. Baril, P., Gangeswaran, R., Mahon, P. C., Caulee, K., Kocher, H. M.,Harada, T., Zhu, M., Kalthoff, H., Crnogorac-Jurcevic, T., and Lemoine,N. R. (2007) Oncogene 26, 2082–2094

47. Armstrong, V. J., Muzylak, M., Sunters, A., Zaman, G., Saxon, L. K., Price,J. S., and Lanyon, L. E. (2007) J. Biol. Chem. 282, 20715–20727

48. He, X., Semenov, M., Tamai, K., and Zeng, X. (2004) Development 131,1663–1677

49. Onyia, J. E., Helvering, L.M.,Gelbert, L.,Wei, T.,Huang, S., Chen, P., Dow,E. R., Maran, A., Zhang, M., Lotinun, S., Lin, X., Halladay, D. L., Miles,R. R., Kulkarni, N. H., Ambrose, E. M., Ma, Y. L., Frolik, C. A., Sato, M.,Bryant, H. U., and Turner, R. T. (2005) J. Cell. Biochem. 95, 403–418

50. Li, X., Liu, H., Qin, L., Tamasi, J., Bergenstock,M., Shapses, S., Feyen, J. H.,Notterman, D. A., and Partridge, N. C. (2007) J. Biol. Chem. 282,33086–33097

51. Zhu, S., Barbe, M. F., Liu, C., Hadjiargyrou, M., Popoff, S. N., Rani, S.,Safadi, F. F., and Litvin, J. (2008) J. Cell. Physiol. 218, 584–592

52. Barengolts, E. I., Lathon, P. V., Curry, D. J., and Kukreja, S. C. (1994) BoneMiner. 26, 133–140

53. Bourrin, S., Palle, S., Pupier, R., Vico, L., and Alexandre, C. (1995) J. BoneMiner. Res. 10, 1745–1752

54. Zehnacker, C. H., and Bemis-Dougherty, A. (2007) J. Geriatr. Phys. Ther.30, 79–88

55. Sinaki, M. (2007) Phys. Med. Rehabil. Clin. N. Am. 18, 593–60856. Engelke, K., Kemmler, W., Lauber, D., Beeskow, C., Pintag, R., and

Kalender, W. A. (2006) Osteoporos. Int. 17, 133–14257. Ferretti, J. L., Frost, H. M., Gasser, J. A., High,W. B., Jee,W. S., Jerome, C.,

Mosekilde, L., andThompson,D.D. (1995)Calcif. Tissue Int. 57, 399–40458. Seeman, E., and Delmas, P. D. (2006) N. Engl. J. Med. 354, 2250–226159. Seeman, E. (2002) Lancet 359, 1841–185060. Chambers, T. J., Evans, M., Gardner, T. N., Turner-Smith, A., and Chow,

J. W. (1993) Bone Miner. 20, 167–17861. Frost, H. M. (1987) Anat. Rec. 219, 1–9

Role of Periostin in Bone Response to Physical Activity

35950 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 284 • NUMBER 51 • DECEMBER 18, 2009

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Michelangelo Foti, Simon J. Conway and Serge L. FerrariNicolas Bonnet, Kara N. Standley, Estelle N. Bianchi, Vincent Stadelmann,

Anabolic Response to Mechanical Loading and Physical Activity Inhibition and theSostThe Matricellular Protein Periostin Is Required for

doi: 10.1074/jbc.M109.060335 originally published online October 16, 20092009, 284:35939-35950.J. Biol. Chem. 

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