Bone 53 (2013) 87–93

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Original Full Length Article

Impaired osteoclast differentiation and function and mild osteopetrosis developmentin Siglec-15-deficient mice

Yoshiharu Hiruma a,⁎, Eisuke Tsuda b, Naoyuki Maeda c, Akiko Okada a, Noriko Kabasawa b,Megumi Miyamoto c, Hiroyuki Hattori c, Chie Fukuda b

a Frontier Research Laboratories, Daiichi Sankyo Co., Ltd., Tokyo, Japanb Biological Research Laboratories, Daiichi Sankyo Co., Ltd., Tokyo, Japanc Medicinal Safety Research Laboratories, Daiichi Sankyo Co., Ltd., Tokyo, Japan

Abbreviations: BMM, bone marrow-derived moDNAX-activating protein of 12 kDa; DPD, deoxypyridinolireceptors; IL-1, interleukin-1; ITAM, immunoreceptor tyM-CSF, macrophage colony-stimulating factor; pQCT, petomography; RANK, receptor activator of nuclear factor κnuclear factor κB ligand; Siglec-15, sialic acid-binding iTNF-α, tumor necrosis factor-α; TRAP, tartrate-resistantmetric bone mineral content.⁎ Corresponding author at: Frontier Research Laborat

1-16-13, Kitakasai, Edogawa-ku, Tokyo 134-8630, JapanE-mail address: hiruma.yoshiharu.hy@daiichisankyo

8756-3282/$ – see front matter © 2012 Elsevier Inc. Allhttp://dx.doi.org/10.1016/j.bone.2012.11.036

a b s t r a c t

a r t i c l e i n f o

Article history:Received 17 September 2012Revised 12 November 2012Accepted 17 November 2012Available online 11 December 2012

Edited by: J. Aubin

Keywords:OsteopetrosisSiglec-15OsteoclastsKnockout miceOsteoclastogenesis

Sialic acid-binding immunoglobulin-like lectin 15 (Siglec-15) is a cell surface receptor for sialylated glycanligands. Recent in vitro studies revealed upregulated Siglec-15 expression in differentiated osteoclasts and in-hibition of osteoclast differentiation by anti-Siglec-15 polyclonal antibody, demonstrating Siglec-15 involve-ment in osteoclastogenesis. To discern the physiological role of Siglec-15 in skeletal development andosteoclast formation and/or function in vivo, we generated Siglec-15-deficient (siglec-15−/−) mice and ana-lyzed their phenotype. The siglec-15−/− mice developed without physical abnormalities other than increasedtrabecular bone mass in lumbar vertebrae and metaphyseal regions of the femur and tibia, causing mildosteopetrosis. Histological analyses demonstrated that the number of osteoclasts present on the femoraltrabecular bone of the mutant mice was comparable to that of the wild-type mice. However, urinarydeoxypyridinoline, a systemic bone resorption marker, decreased in the siglec-15−/− mice, indicating thatimpaired osteoclast function was responsible for increased bone mass in the mutant mice. In addition, theability of bone marrow-derived monocytes/macrophages from the siglec-15−/− mice to differentiate intoosteoclasts was impaired, as determined in vitro by cellular tartrate-resistant acid phosphatase activity in re-sponse to the receptor activator of nuclear factor-κB ligand or tumor necrosis factor-α. These results revealthe importance of Siglec-15 in the regulation of osteoclast formation and/or function in vivo, providingnew insights into osteoclast biology.

© 2012 Elsevier Inc. All rights reserved.

Introduction

Development andmaintenance of the skeletal systemdepend on thebalance between bone formation by osteoblasts and bone resorption byosteoclasts, and these opposing processes interact, forming a functionalcoupling [1,2]. Osteoblasts, derived frommesenchymal stem cells, existon the bone surface and synthesize bonematrix. In functional coupling,osteoblasts regulate osteoclast formation both by secreting factors, in-cluding macrophage colony-stimulating factor (M-CSF), interleukin-1(IL-1), and IL-6 [3], and by direct interaction with osteoclast precursors

nocytes/macrophages; DAP12,ne; IgLRs, immunoglobulin-likerosine-based activation motif;ripheral quantitative computedB; RANKL, receptor activator ofmmunoglobulin-like lectin 15;acid phosphatase; vBMC, volu-

ories, Daiichi Sankyo Co., Ltd.,. Fax: +81 3 5696 8337..co.jp (Y. Hiruma).

rights reserved.

[4,5]. Conversely, osteoclasts are multinucleated cells positive fortartrate-resistant acid phosphatase (TRAP) generated frommononucle-ar monocyte/macrophage lineage hematopoietic precursors [6], whichresorb bone by secreting protons and proteases into resorptionzones. Accordingly, mice carrying mutated genes responsible forosteoclast formation and/or function exhibit increased bone mass(osteopetrotic phenotype) caused by impaired bone resorption [7,8].During osteoclastogenesis, the receptor activator of nuclear factor-κB li-gand (RANKL)—produced by cells of osteoblastic lineage—binds to itsreceptor, receptor activator of nuclear factor-κB (RANK)—expressedon the cell surface of osteoclast precursors—triggering key intracellularsignaling cascades [9–11].

Togetherwith RANK/RANKL signaling, costimulatory signalsmediat-ed by immunoreceptor tyrosine-based activation motif (ITAM)-bearingadaptor proteins are required for the terminal differentiation of osteo-clasts [12–14]. DNAX-activating protein of 12 kDa (DAP12) is one suchITAM-bearing adaptor; its deficiency is reportedly associated withskeletal abnormalities in mice and humans, suggesting the involvementof this signaling pathway in regulating osteoclast development and/or function [15–19]. ITAM-bearing adaptor proteins have minimal ex-tracellular binding domains and are thought to convey extracellular

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signals through interaction with ligand-binding, immunoglobulin-like receptors (IgLRs). Triggering receptor expressed on myeloidcells 2 (TREM-2), signal-regulatory protein β1, and myeloid DAP12-associating lectin-1 are IgLRs expressed on osteoclasts, and candidateDAP12-associating partners [12,14,19]. However, studies in mutantmice have not yet clarified the relevance of these genes in bone homeo-stasis, implying the existence of undiscovered DAP12-associated recep-tors critical for osteoclastogenesis.

Sialic acid-binding immunoglobulin-like lectin 15 (Siglec-15) is anewly identified member of the Siglec family of cell-surface IgLRsthat recognizes sialylated glycan ligands [20–22]. Siglec-15 reported-ly associates more avidly with DAP12 than other ITAM-bearing sig-naling adaptors, such as DNAX-activating protein of 10 kDa or Fcreceptor γ subunit (FcRγ), in 293 T cells co-transfected with con-structs expressing these genes [22]. Although an immune systemfunction was suggested based on its gene expression in macrophagesand/or dendritic cells [22], the physiological role of Siglec-15 in verte-brates remains unknown. Recently, we reported that Siglec-15 ex-pression is upregulated during osteoclast differentiation in mouseand human osteoclast culture systems. Moreover, functional analysisusing a polyclonal antibody against Siglec-15, demonstrated an inhib-itory effect on osteoclast formation in vitro, suggesting that Siglec-15is critical to osteoclastogenesis [23]. We generated and analyzedSiglec-15-deficient (siglec-15−/−) mice to elucidate the physiologicalrole of this gene in vivo, especially during osteoclastogenesis and skel-etal development.

Materials and methods

Gene targeting and generation of siglec-15−/− mice

Genomic clones encoding the siglec-15 locus were isolated from a129S6/SvEvTac mouse bacterial artificial chromosome library byscreening with a radiolabeled DNA fragment of a 550-base pair regionof exon 1. A targeting vector was constructed by replacing exons 2–4,encoding two immunoglobulin-like domains and a transmembranedomain, with a phosphoglycerate kinase neomycin resistance cas-sette. The linearized targeting vector was electroporated into 129Sv/EvTac-derived embryonic stem cells. G418-resistant colonieswere genotyped by PCR and genomic Southern blot analyses to iden-tify clones carrying a targeted allele. Targeted cells were injected intoC57BL/6 blastocysts, and transferred into pseudopregnant female ICRmice. Resulting chimeric males were bred with C57BL/6 females; het-erozygous offspring were then backcrossed to the C57BL/6 strain for7–8 generations by in vitro fertilization. Following heterozygous mat-ings of backcrossed animals, the siglec-15−/− mice were identified bySouthern blot analysis of genomic DNA cut with KpnI using a specificprobe. All procedures described here were performed at PhoenixBioCo., Ltd (Japan).

Animal experiments

Experimental procedures were performed in accordance with thein-house guidelines of the Institutional Animal Care and Use Commit-tee of Daiichi Sankyo Co., Ltd. Seven-week-old male and femalesiglec-15−/− mice and their wild-type littermates were obtainedfrom PhoenixBio Co., Ltd. The animals were fed a normal diet andgiven tap water ad libitum under controlled room temperature andhumidity. Whole-body microradiographs were performed using aμFX-1000 radiography system (Fujifilm, Japan) and BAS-2500 imag-ing system (Fujifilm) on anesthetized mice 4 days before necropsy.Body weight was measured 1 day before necropsy.

At 18 weeks of age, the mice were euthanized by exsanguinationafter collecting blood samples from the inferior vena cava under anes-thesia. Whole blood samples were used for hematological and bloodchemical analyses. Lumbar vertebrae, femurs, and tibiae were excised

for bone phenotypic analysis. Thoracic and abdominal organs wereobserved macroscopically, and the liver, thymus, spleen, and testeswere weighed. The following organs/tissues were histopathologicallyexamined: sternum (bone marrow), thymus, lung, heart, mesentericlymph node, liver, spleen, kidney, adrenal gland, pancreas, stomach,duodenum, jejunum, ileum, cecum, colon, rectum, thyroid (includingparathyroid), testis (male), ovary (female), uterus (female), tibia(bone marrow), eye, and skin (dorsal region). Testes were fixedwith FSA, eyes with Davidson's fixative, and other organs/tissueswith 10% neutral-buffered formalin. After fixation, histopathologicalsections were prepared and stained with hematoxylin and eosin.

Bone densitometry and micro-CT imaging

After removing connective tissue, excised lumbar vertebrae(L3–6) and paired femurs were defatted and dehydrated by ethanoltreatment, with subsequent air seasoning.

Areal bone mineral density of the lumbar vertebrae and femurswas measured by dual-energy X-ray absorptiometry using aDCS-600EX-IIIR (Aloka, Japan) optimized for small bone specimens.Scan speed was 25 mm/s, and scan width was 1 mm. Bone mineraldensity analysis was performed based on single energy (22 keV)X-ray absorption data for lumbar vertebrae L3–6 and 20 equally di-vided femoral regions.

Right femoral volumetric bone mineral content (vBMC) was mea-sured by peripheral quantitative computed tomography (pQCT) usingXCT Research SA+ (StraTec Medizintechnik, Germany) and XCT6.20analyzing software (Stratec Medizintechnik) at ELK Corporation(Japan). Two slices were measured with an 80×80×460 μm voxelsize at 2 mm proximal to the distal femoral growth cartilage for trabec-ular regions and the femoral midpoint for cortical regions. Trabecularbone in the metaphysis and cortical bone in the diaphysis were ana-lyzed with contour mode 2, with peel mode 20 (trabecular area 35%),and contour mode 1 using a threshold of 690 mg/cm3, respectively.

Micro-computed tomography (micro-CT) imaging of the fifth lum-bar vertebra was performed using ScanXmate-L080H (Comscantecno,Japan), with a voxel size of 14 μm in three dimensions. Three-dimensional images were reconstructed using bone structural analysissoftware, TRI/3D-BON (RATOC System Engineering, Japan).

TRAP staining of bone

Right tibiae were excised and fixed in cold 70% ethanol. Processingof bone specimens was performed at Kureha Special Laboratory Co.,Ltd (Japan). Proximal tibiae were dehydrated in graded ethanol con-centrations and embedded in glycol methacrylate (Wako Pure Chem-ical Industries, Japan) without decalcification. Frontal plane sections(3 μm thick) of the proximal metaphysis were prepared using anRM2255 rotary microtome (Leica Microsystems, Germany) and stainedfor TRAP activity using a method adapted from that previously de-scribed [24]. Hematoxylin and eosin staining was performed usinghistopathological sections of tibiae fixed with 10% neutral-bufferedformalin.

Bone histomorphometry

Mice were subcutaneously injected with calcein (20 mg/kg;Dojindo, Japan) and tetracycline hydrochloride (20 mg/kg; Sigma-Aldrich, USA) 7 and 3 days prior to dissection, respectively. At necropsy,right femurs were excised and fixed in 70% ethanol, and adherentmuscle removed. Bone specimen processing and histomorphometryin cortical and cancellous bone were performed at the Ito BoneHistomorphometry Institute (Japan). Distal and central femurs werestained with Villanueva bone stain for 5 days, dehydrated in gradedethanol concentrations, and embedded in methyl methacrylate (WakoPure Chemical Industries) without decalcification. For cancellous bone

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Fig. 1. Generation of siglec-15−/− mice. (A) Schematic representation of the wild-typeand targeted alleles at the siglec-15 locus. Exons, represented by black boxes, are num-bered. The phosphoglycerate kinase neomycin resistance cassette (PGK-neo) replacedthe genomic region containing exons 2–4, encoding two immunoglobulin-like do-mains and a transmembrane domain of Siglec-15. An unfilled box indicates the probefor Southern blot analysis. K, KpnI. (B) Southern blot analysis of offspring derivedfrom heterozygous matings. Genomic DNA from the wild-type (+/+), heterozygous(+/−), and homozygous (−/−) Siglec-15-deficient mice were digested with KpnIand hybridized with the probe. DNA fragments sizes are indicated.

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histomorphometry, frontal plane sections (5 μm thick) of the distalmetaphysis were prepared using the RM2255 rotary microtome. Theregion of interest was located 0.25–1.00 mm proximal to the distalfemoral growth cartilage/metaphyseal junction, and 0.25 mm fromthe endosteal border of the cortices. Measurements were collected at400× magnification using the semiautomatic image analyzing system,Histometry RT (System Supply, Japan), linked to a BX-51 light/epifluorescentmicroscope (Olympus, Japan). The following parameterswere measured: bone volume per total tissue volume, trabeculae num-ber, and osteoblast surface, osteoclast surface, and bone formation rateper unit bone surface. Cortical bone histomorphometry was performedon femoral midpoint cross-sections ground to 10 to 20-μm-thick usingSpeed Lap ML-521 (Maruto Instrument, Japan). Cortical area, osteoidsurface, eroded surface, and labeled surface were measured per unitperiosteal bone surface. Standard bone histomorphometric nomencla-ture, symbols, and units were used according to the American Societyof Bone and Mineral Research Histomorphometry Nomenclature Com-mittee [25].

Measurement of bone metabolic markers

Urine samples were collected by gently pushing the abdomen of9- and 18-week-old animals. Urinary deoxypyridinoline (DPD) excre-tion was measured using an enzyme-linked immunosorbent assay kitfrom DS Pharma Biomedical Co., Ltd (Japan). To compensate for indi-vidual variation in DPD excretion, urinary creatinine concentrationwas also measured using an assay kit (Kainos, Japan) according tothe manufacturer's instructions. Serum samples prepared at necropsywere used to measure levels of the bone formation marker, osteocalcin(Mouse Osteocalcin EIA kit; Biomedical Technologies, USA). A micro-plate reader, SpectraMax 250 (Molecular Devices, USA), was used foreach assay.

In vitro osteoclast formation assay

Mouse bone marrow-derived monocytes/macrophages (BMM) wereprepared according to previously described methods, with slight modifi-cations [12]. In brief, bone marrow cells were flushed from femurs andtibiae of the 10-week-oldmale siglec-15−/− or wild-typemice, and seed-ed to flasks with α-MEM (Life Technologies, USA), containing 5 ng/mLhuman M-CSF (R&D Systems, USA), 10% fetal bovine serum, 100 μg/mLstreptomycin, and 100 U/mL penicillin. After overnight culture,nonadherent cells were collected and suspended at 1.5×105 cells/mLin medium containing 10 ng/mL human M-CSF alone for the RANKL-induced osteoclast formation assay, or human M-CSF in combinationwith 2 ng/mL human transforming growth factor-β (R&D Systems)for the tumor necrosis factor-α (TNF-α)-induced assay. The cells werethen seeded in 96-well plates (0.2 mL/well), and cultured for 2 daysto generate mouse BMM. After replacing old medium with fresh medi-um, cells were cultured in medium containing 10 ng/mL M-CSF, and30 ng/mL human RANKL (PeproTech, USA) or 30 ng/mL human TNF-α(R&D Systems) for an additional 4 days. For the TRAP activity assay,cells were solubilized with 50 μL of 50 mM sodium citrate buffer (pH6.1) containing 1% Triton™ X-100, and incubated with 50 μL of 50 mMsodium citrate buffer (pH 6.1), containing 5 mg/mL p-nitrophenyl phos-phate (Sigma-Aldrich) and 0.46% sodium tartaric acid, for 20 min atroom temperature. The reaction was stopped by adding 50 μL of 1 NNaOH, and an absorbance at 405 nm was measured using SpectraMax250 to determine the amount of p-nitrophenol as a catabolite.

Statistical analysis

Mean and standard error were calculated using Excel 2003. All sta-tistical analyses were conducted using an in-house program, based onSAS System, Release 8.2, SAS Institute. Comparison between thewild-type and siglec-15−/− littermates was performed by the F-test

of variance, followed by Student's t-test (homogeneous variance) orWelch's t-test (non-homogeneous variance). A p value of b0.05 wasconsidered significant.

Results

Generation of siglec-15−/− mice

To disrupt Siglec-15 in mice, we designed a targeting vector to re-place exons 2–4with a neomycin resistance cassette (Figs. 1A, B), delet-ing most of the extracellular and transmembrane domains of theSiglec-15 protein. Southern blot analysis of 4-week-old offspring of het-erozygousmatings revealed that the siglec-15−/−micewere born in ap-proximatelyMendelian ratios (+/+: 149 [27%];+/−: 280 [51%];−/−:118 [22%]). The siglec-15−/− mice grew normally, without apparentphysical abnormalities except for skeletal changes, according to bodyweight, hematological, blood chemistry, and pathological examinationsperformed on organs and tissues from 18-week-old mice, as describedin the Materials and methods (data not shown). Although highSiglec-15 mRNA expression was reported in the human spleen, testis,and small intestine [22], histopathological examinations detected noobvious abnormalities in these organs in the siglec-15−/− mice.

Mild osteopetrotic phenotype in siglec-15−/− mice

To analyze the phenotypic changes in skeletal tissues, we performedradiography and bone densitometry on 18-week-old siglec-15−/−mice.Whole body radiography demonstrated that the skeletal phenotype inmutantmice resembled to that in the wild-typemice (Fig. 2A), withoutprofound osteopetrotic hallmarks, such as long bone shortening ortooth eruption failure [26–28]. However, increased bone density wasevident, especially in the distal femur and proximal tibia of thesiglec-15−/−mice (Fig. 2B). Measurement of areal bonemineral density

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Fig. 2. Osteopetrotic phenotype in siglec-15−/− mice. (A, B) Representative radiographs of 18-week-old female wild-type and siglec-15−/− mice. Arrows in the magnified picture ofthe siglec-15−/− mouse left hind limb indicate increased radiodensity. (C) Areal bone mineral density (aBMD) was measured in 20 longitudinal divisions of femurs from the femalewild-type and siglec-15−/− mice by dual-energy X-ray absorptiometry. (D) Volumetric bone mineral content (vBMC) of trabecular and cortical bone was analyzed by peripheralquantitative computed tomography at 2 mm proximal to the distal femoral growth cartilage and femoral midpoint, respectively. (E) Representative three-dimensionalmicro-computed tomography images of the fifth lumbar vertebrae of the 18-week-old female wild-type and siglec-15−/− mice. Scale bar: 500 μm. (F) aBMD was measured inthe lumbar vertebrae L3–6 from the wild-type and siglec-15−/− mice. Data represent the mean±standard error from the 18-week-old male wild-type (n=10), malesiglec-15−/− (n=7), female wild-type (n=10) or female siglec-15−/− mice (n=9). *pb0.05, **pb0.01 and ***pb0.001 compared with the wild-type mice of the same sex.

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confirmed this quantitatively, showing a marked increase, especiallyfrom the distal metaphysis to themidshaft of the femur (Fig. 2C). In ad-dition, pQCT demonstrated profoundly increased trabecular vBMC inthe distal femoral metaphysis in the siglec-15−/− mice (Fig. 2D). In-creased vBMC was also observed in the cortical bone of the femoral di-aphysis in the female mutant mice, although the increase was slightlycompared with that in trabecular bone. No vBMC increase was evidentin the cortical bone of the male mutant mice, suggesting sexual dimor-phism of the cortical bone phenotype. Next, we performed micro-CTimaging of lumbar vertebrae to observe the phenotype at sites otherthan long bones. Trabecular bone mass was markedly increased in thefifth lumbar vertebra of the siglec-15−/− mice compared with thewild-type mice (Fig. 2E), consistent with areal bone mineral density oflumbar vertebrae L3–6 (Fig. 2F).

Histological and histomorphometrical analyses of the bone in siglec-15−/−

mice

We performed histological examinations on the hind limbs of themutant mice to confirm an osteopetrotic phenotype and analyze themorphological, quantitative, and dynamic changes in trabecular and cor-tical bone. Hematoxylin and eosin staining of longitudinal sections of theproximal metaphyseal tibia showed increased trabeculae number andtrabecular bone volume, especially in the secondary spongiosa andepiphyseal ossification center, in the siglec-15−/− mice (Fig. 3). No obvi-ous histological changes were observed in the proximal tibial growth

plate, consistent with the normal skeletal growth in the siglec-15−/−

mice (Fig. 2A). Despite the increased bone mass, osteoclasts weredetected by TRAP staining at primary and secondary spongiosa in thesiglec-15−/− mice comparably to the wild-type mice. In addition, bonehistomorphometry of the distal femur demonstrated profound increasesin both trabecular bone volume per total tissue volume and trabeculaenumber, but no substantial changes in the percentage osteoclast surfaceper unit bone surface (Table 1). With regard to bone formation, osteo-blast surface per unit bone surface and the bone formation rate perunit bone surface weremarkedly reduced in the siglec-15−/−mice com-paredwith thewild-typemice, suggesting that trabecular bone turnoverwas suppressed in the siglec-15−/− mice. In cortical bone, the corticalarea was slightly higher in the female mutant mice than in thewild-typemice, while no differencewas evident inmalemice, consistentwith the pQCT-determined vBMC of the femoral diaphysis (Fig. 2D). Inthe female mutant mice, reduced eroded surface per unit periostealbone surface and increased calcein- or tetracycline-labeled periostealsurfacewere evident, suggesting reduced bone resorption and enhancedbone formation. Conversely, in the male mutant mice, there were noclear changes in these parameters. Osteoid surface per unit periostealbone surface did not change in the male or female mutant mice.

Changes in bone metabolic markers in siglec-15−/− mice

To assess systemic bone turnover in the mutant mice, urinary DPDexcretion and serum osteocalcin levels were measured as markers of

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Fig. 3. Histologic evaluation of bone morphology in siglec-15−/− mice. (Left panels) He-matoxylin and eosin staining of the proximal metaphysis of the right tibiae from the18-week-old female wild-type and siglec-15−/− mice. Scale bar: 1 mm. (Right panels)Magnified pictures of the primary spongiosa of proximal tibiae stained fortartrate-resistant acid phosphatase activity to visualize osteoclasts in red. Scale bar:100 μm.

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bone resorption and formation, respectively. Urinary DPD excretionin both male and female mutant mice decreased compared with thewild-type mice at 9 and 18 weeks of age (Fig. 4A), indicating that

Table 1Bone histomorphometrical analysis of femurs in wild-type and siglec-15−/− mice. BV/TV:bone volume/tissue volume; Tb.N: trabecular number; Ob.S/BS: osteoblast surface/bonesurface; Oc.S/BS: osteoclast surface/bone surface; BFR/BS: bone formation rate/bone sur-face; Ct.Ar: cortical area; Ps.OS/Ps.BS: periosteal osteoid surface/bone surface; Ps.ES/Ps.BS: periosteal eroded surface/bone surface; Ps.LS/Ps.BS: labeled periosteal surface/bone surface. Data represent the mean±standard error from 18-week-old malewild-type (n=8), male siglec-15−/− (n=8), female wild-type (n=6) or femalesiglec-15−/−mice (n=9). apb0.05, bpb0.01, and cpb0.001 comparedwithwild-typemice.

Parameters Wild-type siglec-15−/−

MaleDistal femoral metaphysis(cancellous bone)BV/TV (%) 9.66±0.65 20.58±2.19b

Tb.N (#/mm) 3.49±0.15 8.09±0.97b

Ob.S/BS (%) 19.18±1.16 6.71±1.05c

Oc.S/BS (%) 7.702±0.528 7.255±0.860BFR/BS (mm3/mm2/y) 0.1492±0.0100 0.0297±0.0034c

Femoral midshaft(cortical bone)Ct.Ar (mm2) 0.8348±0.0262 0.8537±0.0101Ps.OS/Ps.BS (%) 24.90±2.60 25.65±2.94Ps.ES/Ps.BS (%) 14.98±3.24 22.75±3.09Ps.LS/Ps.BS (%) 43.40±1.83 37.99±3.97

FemaleDistal femoral metaphysis(cancellous bone)BV/TV (%) 6.21±0.60 24.62±1.74c

Tb.N (#/mm) 2.29±0.17 7.71±0.72c

Ob.S/BS (%) 42.80±2.52 13.34±1.24c

Oc.S/BS (%) 9.263±1.002 8.646±0.421BFR/BS (mm3/mm2/y) 0.3475±0.0079 0.0776±0.0094c

Femoral midshaft(cortical bone)Ct.Ar (mm2) 0.8326±0.0145 0.9056±0.0114b

Ps.OS/Ps.BS (%) 37.03±2.77 38.69±3.71Ps.ES/Ps.BS (%) 21.88±2.66 7.58±1.31c

Ps.LS/Ps.BS (%) 46.82±3.39 59.09±3.36a

overall bone resorption activity was reduced in the siglec-15−/−

mice. Conversely, at 18 weeks of age, osteocalcin levels were compa-rable in the male mutant mice and higher in the female mutant mice(Fig. 4B).

Impaired differentiation of siglec-15−/− bone marrow-derivedmonocytes/macrophages into osteoclasts in vitro

We evaluated developmental differences in osteoclasts, induced invitro from BMM from the wild-type and siglec-15−/− mice by RANKLor TNF-α stimulation in the presence of M-CSF [12,29]. Cellular TRAPactivity, a marker of osteoclast differentiation, was upregulated inBMM from the wild-type mice by both RANKL and TNF-α treatment(Fig. 5). Although TRAP activity upregulation was observed insiglec-15−/− BMM, upregulation occurred to a lesser extent than inthe wild-type cells in both osteoclast formation culture systems.These results indicate that osteoclast differentiation is impaired, atleast in vitro, by Siglec-15 deficiency.

Discussion

Osteopetrosis is a heterogeneous group of heritable genetic dis-eases, characterized by increased bone mass due to defective osteo-clastic bone resorption [7,8]. In mice, many genes associated withincreased bone mass have been identified by mutant studies, reveal-ing that osteopetrosis severity depends on genes functional in osteo-clast differentiation and/or function. Osteopetrosis in the siglec-15−/−

mice was mild compared with, for example, c-Fos or RANK knockoutmice, which exhibit a severe osteopetrotic phenotype, including longbone shortening and tooth eruption failure [26–28]. Our observationssuggest that Siglec-15 deficiency causes partially reduced bone re-sorption, leading to mild osteopetrosis in vivo. Reduced bone resorp-tion was evident from lower urinary DPD excretion, while trabecularbone mass increased, despite comparable osteoclast numbers on fem-oral and tibial trabecular bone. These findings suggest that impairedosteoclast function, not reduced osteoclast number, underlies theosteopetrotic phenotype of the siglec-15−/− mice. These observationssupport the development of therapies suppressing Siglec-15 functionto treat bone loss diseases, such as osteoporosis. In addition, a pheno-type restricted to skeletal tissues may mean that such therapies havefewer adverse effects on other organs or tissues.

In femoral trabecular bone, both osteoblast surface and bone forma-tion rate per unit bone surface were reduced by Siglec-15 deficiency,indicating a possible role of the gene in osteoblast regulation as well.However, preliminary RT-PCR examinations detected no Siglec-15 ex-pression in murine osteoblast lineage cells, such as ST2 and MC3T3-E1,evenwhen theywere cultured under differentiation-inducing conditions(unpublished data). In addition, anti-Siglec-15 antibody, which has a po-tential to inhibit osteoclast differentiation, did not affect osteoblastic dif-ferentiation in vitrowhen determined by alkaline phosphatase activity inthe cells (unpublished data). Although we need to assess the osteogeniccapacity of siglec-15−/− osteoprogenitor cells tomake a conclusion, thosepreliminary results suggest a low possibility of a direct involvement ofSiglec-15 in osteoblast regulation. Instead, reduction in osteoblast num-ber and the bone formation rate observed in the siglec-15−/− micecould be accountable by considering the secondary effect resulting fromreduced bone resorption based on the functional coupling concept inbone formation and resorption.

Although the osteopetrotic phenotype of trabecular bone was sim-ilar in the male and female siglec-15−/− mice, increased bone masswas detected by pQCT and histomorphometrical analyses in corticalbone of the femoral midshaft in the female mice. Interestingly, femo-ral labeled periosteal surface per unit bone surface increased in thefemale mutant mice, suggesting that enhanced bone formationpartly contributes to increased cortical bone mass. Increased serumosteocalcin levels in the female siglec-15−/− mice support this idea,

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Fig. 4. Changes in bone turnover markers in siglec-15−/− mice. (A) Urine samples were collected from the wild-type and siglec-15−/− mice at 9 and 18 weeks of age. Urinarydeoxypyridinoline (DPD) excretion was measured, and adjusted for creatinine (Cre) concentration. (B) Serum levels of osteocalcin were determined with samples preparedfrom the 18-week-old wild-type and siglec-15−/− mice. Data represent the mean±standard error from the male wild-type (n=10), male siglec-15−/− (n=7), female wild-type(n=10), and female siglec-15−/− mice (n=9), with the exception of urinary DPD excretion at 9 weeks of age, where male wild-type (n=9), and male siglec-15−/− (n=5).*pb0.05, **pb0.01, and ***pb0.001 compared with the wild-type mice of the same sex.

92 Y. Hiruma et al. / Bone 53 (2013) 87–93

as approximately 80% of bone in the entire body comprises corticalbone [30]. However, enhanced cortical bone formation provokes afurther question when contrasted with trabecular bone, where osteo-blast surface and bone formation rate were reduced. We do not knowwhy morphological changes in cortical bone differed between maleand female mutant mice, nor why bone formation differed betweenfemoral trabecular and cortical bone in female mutants. The molecu-lar mechanisms responsible for the bone-sparing effects of sex ste-roids (estrogens and androgens) vary as a function of gender,skeletal site, and bone compartment [31,32], which could provide in-sight on these differences.

Siglec-15 reportedly associates with the DAP12 adaptor molecule[22], deficiency of which in mice is known to cause mild osteopetrosis[18,19]. Comparing the skeletal phenotype in the siglec-15−/− micewith DAP12-deficient (DAP12−/−) mice, we find many similarities.Both mutant mice grow normally without gross skeletal abnormali-ties, while exhibiting markedly increased trabecular bone. Micro-CTand histological images of trabecular bone in the two mutants arevery alike. Another commonality is that the impact of genetic defi-ciency on osteoclast development differs in vivo and in vitro. Report-edly, osteoclast number and morphology in the DAP12−/− micewere not significantly different from wild type mice, while impairedosteoclast formation was evident in vitro [18]. Similarly, in thesiglec-15−/− mice, TRAP-positive osteoclasts in the tibial metaphysiswere indistinguishable from those of wild type mice, but BMM

0.00

0.05

0.10

0.15ControlRANKL

wt -/-

***

TR

AP

act

ivity

(A

bs 4

05 n

m)

Fig. 5. Osteoclast formation assay using bone marrow-derived monocytes/macrophages derrophages were prepared from the 10-week-old male wild-type and siglec-15−/− mice. Cells30 ng/mL TNF-α, for 4 days in the presence of 10 ng/mL macrophage colony-stimulating facosteoclast differentiation. Data represent the mean±standard error (n=12) from one exp

differentiation into osteoclasts was impaired in vitro, evident in re-duced induction of cellular TRAP activity in response to RANKL orTNF-α. These observations indicate that Siglec-15 mediates cellularsignals through interaction with DAP12 in osteoclasts, positivelyregulating cell differentiation and/or function. Although anotherDAP12-associated receptor, TREM-2, is considered the principal part-ner of DAP12 in osteoclasts, its deficiency in mice reportedly causedaccelerated osteoclastogenesis in vitro without osteopetrosis in vivo,in contrast to DAP12 deficiency [33]. Taken together, our results sug-gest that the Siglec-15/DAP12 complex mediates the signals requiredfor proper osteoclastogenesis more dominantly than the TREM-2/DAP12 complex in mice. Furthermore, the differences between osteo-clasts in vivo and in vitro may be accounted for the ability ofsiglec-15−/− osteoclasts to differentiate in vivo via signaling pathwaysmediated by other ITAM-bearing adaptors, such as FcRγ; it wassuggested that ITAM-bearing adaptors can functionally compensatefor each other [12,13]. Recently, Ishida-Kitagawa et al. demonstratedthe importance of Siglec-15/DAP12-mediated signaling in functionalosteoclast formation using chimeras of these proteins in vitro [34].Their results support the relevance of this signaling pathway to invivo osteoclastogenesis.

In summary, we generated siglec-15−/− mice, which developedmild osteopetrosis resulting from impaired bone resorption. Our find-ings suggest that Siglec-15 functions in the regulation of osteoclastdifferentiation and/or function in vivo.

0.00

0.05

0.10

0.15

0.20

0.25ControlTNF-α

wt -/-

***

ived from the wild-type and siglec-15−/− mice. Bone marrow-derived monocytes/mac-were cultured with 30 ng/mL receptor activator of nuclear factor-κB ligand (RANKL), ortor. Tartrate-resistant acid phosphatase (TRAP) activity was measured as an indicator oferiment. ***pb0.001 compared with wild-type cells.

93Y. Hiruma et al. / Bone 53 (2013) 87–93

Acknowledgments

The authors acknowledge Dr. Seiichiro Kumakura for thoughtfuldiscussion. All authors are employees of Daiichi Sankyo Co., Ltd.

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