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SC I ENCE S I GNAL ING | R E S EARCH ART I C L E
PHYS IOLOGY
1Department of Biochemistry, Matsumoto Dental University, Shiojiri, Nagano399-0781, Japan. 2Biosignal Research Center, Kobe University, Kobe, Hyogo657-8501, Japan. 3Department of Periodontology, Tokyo Medical and DentalUniversity, Tokyo 113-0034, Japan. 4Department of Orthopedic Surgery, The JikeiUniversity School of Medicine, Tokyo 105-8461, Japan. 5Institute for Oral Science,Matsumoto Dental University, Shiojiri, Nagano 399-0781, Japan. 6Department ofPhysiology and Cell Biology, Graduate School of Medicine, Kobe University, Kobe,Hyogo 650-0017, Japan. 7Department of Biochemistry, Tokyo Dental College, Tokyo101-0061, Japan. 8Center for Regional Cooperation, Iwaki Meisei University, Iwaki,Fukushima 970-8551, Japan. 9Research Institute of Innovative Medicine, TokiwaFoundation, Iwaki, Fukushima 972-8322, Japan.*Corresponding author. Email: [email protected]
Uehara et al., Sci. Signal. 10, eaan0023 (2017) 29 August 2017
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Protein kinase N3 promotes bone resorption byosteoclasts in response to Wnt5a-Ror2 signalingShunsuke Uehara,1 Nobuyuki Udagawa,1 Hideyuki Mukai,2 Akihiro Ishihara,3 Kazuhiro Maeda,4
Teruhito Yamashita,5 Kohei Murakami,1 Michiru Nishita,6 Takashi Nakamura,7 Shigeaki Kato,8,9
Yasuhiro Minami,6 Naoyuki Takahashi,5 Yasuhiro Kobayashi5*
Cytoskeletal reorganization in osteoclasts to form actin rings is necessary for these cells to attach to bone andresorb bone matrices. We delineated the pathway through which Wnt5a signaling through receptor tyrosinekinase–like orphan receptor 2 (Ror2) promoted the bone-resorbing activity of osteoclasts. Wnt5a binding toRor2 stimulated Rho, a small GTPase involved in cytoskeletal reorganization. Subsequently, the Rho effectorkinase Pkn3 bound to and enhanced the activity of c-Src, a nonreceptor tyrosine kinase that is critical for actinring formation. Mice with an osteoclast-specific deficiency in Ror2 (Ror2DOcl/DOcl) had increased bone mass. Osteo-clasts derived from these mice exhibited impaired bone resorption and actin ring formation, defects that were res-cued by overexpression of constitutively active RhoA. These osteoclasts also exhibited reduced interaction betweenc-Src and Pkn3 and reduced c-Src kinase activity. Similar to Ror2DOcl/DOcl mice, mice with a global deficiency of Pkn3(Pkn3−/−) had increased bone mass. The proline-rich region and kinase domain of Pkn3 were required to restore thebone-resorbing activity of osteoclasts derived from Pkn3−/−mice. Thus, Pkn3 promotes bone resorption downstreamof Wnt5a-Ror2-Rho signaling, and this pathway may be a therapeutic target for bone diseases such as osteoporosis,rheumatoid arthritis, and periodontal disease.
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INTRODUCTIONThe actin cytoskeleton regulates the shape and polarity of cells and alsomediates various biological functions such as cell movements and divi-sion in all eukaryotic cells (1–3). The cytoskeleton in osteoclasts is highlyorganized to resorb the bone matrix (4, 5). A clearer understanding ofthe regulation of the actin cytoskeleton has important implications fordiseases including osteoporosis. Bone mass is maintained by a balancebetween the activity of bone-resorbing osteoclasts and bone-formingosteoblasts (6, 7). Excessive bone-resorbing activity of osteoclasts causespostmenopausal osteoporosis and inflammatory bone diseases such asrheumatoid arthritis and periodontal disease (8).
The differentiation of osteoclasts is stimulated by receptor activatorof nuclear factor kB ligand (Rankl, encoded by Tnfsf11) and colony-stimulating factor 1 (Csf1, encoded byCsf1), both ofwhich are expressedin osteoblast-lineage cells such as osteoblasts (9, 10) and osteocytes(11, 12). Binding of Rankl and Csf1 to their receptors [Rank (encodedby Tnfrs11a) and Csf1 receptor (Csf1r)] triggers the differentiation ofosteoclast precursors into osteoclasts. Osteoblast-lineage cells alsoproduce osteoprotegerin (encoded by Tnfrsf11b), which inhibits osteo-clast formation by interfering with the Rankl-Rank interaction.
Activated osteoclasts attach to bone surfaces through sealing zones(4, 13, 14), which are ringed-like structures of F-actin dots (also calledactin rings). Protons, chloride ions, and several proteases includingcathepsin K are secreted into the resorption lacunae surrounded by
the sealing zone (4), thereby acidifying the resorption lacunae to resolvemineralized and degrade nonmineralized bonematrices. The formationof the sealing zone in osteoclasts requires cytoskeletal reorganization,which is promoted by small guanosine 5′-triphosphatases (GTPases)such as Rac (15) and Rho (16, 17). Vav3, a Rho family guanine nucleo-tide exchange factor, is involved in the formation of the sealing zone inosteoclasts (18). Vav3-deficient osteoclasts exhibit impaired bone-resorbing activity in vivo and in vitro, a phenotype attributed to defectsin theCsf1-induced activation of Rac and inavb3 integrin–induced c-Srcactivity. These findings suggest that Rac is crucially involved in the bone-resorbing activity of osteoclasts. In addition toRac, Rho is involved in theformation of actin rings in osteoclasts. Addition of the Clostridiumbotulinum C3 exoenzyme, an inhibitor of Rho proteins, in osteoclastcultures disrupts actin rings and impairs bone-resorbing activity ofosteoclasts (19). The transduction of TAT-fusion constitutively active(CA)–RhoA into osteoclasts stimulates their podosome assembly,motility, and bone-resorbing activity (20). In contrast, a microinjectionof CA-RhoA-GFP (green fluorescent protein) complementary DNA(cDNA) into osteoclasts causes podosomes to disappear from the cellperiphery (21). Thus, how these small GTPases, especially Rho, areactivated and form the sealing zone in osteoclasts has not yet beenelucidated.
Wnt proteins promote cell differentiation, migration, and polariza-tion through small GTPases such as Rho, Rac, and Cdc42 (22, 23). Wntproteins bind to a receptor complex of Frizzled and low-density lipo-protein receptor–related protein 5/6 and activateWnt/b-catenin signals.On the other hand, a ligand-receptor complex of Wnt, Frizzled, andreceptor tyrosine kinase–like orphan receptor 1/2 (Ror1/2) activatesb-catenin–independent signaling to promote cell migration and polar-ization (24–26).We have previously shown thatWnt5a, a noncanonicalWnt ligand, enhances Rankl-induced osteoclastogenesis through Ror2(27). However, the role of Wnt5a-Ror2 signaling in the bone-resorbingactivity of osteoclasts remains unclear.
Using genetic approaches, we demonstrated that Wnt5a-Ror2signals promoted osteoclastic bone-resorbing activity through the
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Daam2 [dishevelled (Dvl)–associated ac-tivator of morphogenesis 2]–Rho–Pkn3signaling axis. Pkn3 phosphorylatedthrough Ror2 signaling formed a com-plex of c-Src and proline-rich tyrosine ki-nase 2 (Pyk2). This complex facilitatedthe activation of c-Src, thereby inducingthe formation of actin rings and the bone-resorbing activity of osteoclasts. Thus,this signaling axis not only helps ensureproper bone homeostasis but also repre-sents a therapeutic target for bone diseasessuch as osteoporosis and inflammatorybone diseases.
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RESULTSWnt5a secreted from osteoclastscell autonomously promotesbone-resorbing activityTo further understand the roles of Wntsignaling in bone resorption, we examinedthe expression of genes encoding Wntligands during Rankl-induced osteoclastformation in bone marrow–derived mac-rophage (BMM) cultures using real-timepolymerase chain reaction (RT-PCR)(Fig. 1A). Culturing BMMs in the pres-ence of Rankl and Csf1 increased the ex-pression of the osteoclastmarkerCathepsinK (Ctsk) and of Wnt5a, Wnt6, Wnt10a,Wnt10b, Wnt11, and Wnt16. Wnt5amRNAwas particularly abundant amongthese Wnt ligand–encoding mRNAs inosteoclast cultures. Immunoblottinganalysis confirmed that Wnt5a was abun-dant in BMM-derived osteoclasts (Fig.1B). Because Wnt5a−/− mice died beforebone marrow is formed (27), we culturedmacrophages from the livers of wild-typeandWnt5a−/−mice on dentin slices in thepresence of Rankl and Csf1 to induce dif-ferentiation into osteoclasts in vitro. Nosignificant differences were observed inthe number of osteoclasts between wild-type and Wnt5a−/− macrophage cultures(fig. S1). The area of resorption pits onthe dentin slices was significantly lower inWnt5a−/− osteoclast cultures than in wild-type cultures, suggesting an impairment inthe bone-resorbing activity of Wnt5a−/−
osteoclasts (Fig. 1C). Rhodamine-labeledphalloidin staining revealed that the num-ber of actin rings was lower in Wnt5a−/−
osteoclast cultures than in wild-type cul-tures (Fig. 1D).Theadditionof recombinantWnt5a toWnt5a−/− osteoclast cultures res-cued the impaired actin ring formation andbone-resorbing activity (Fig. 1, C and D).
Uehara et al., Sci. Signal. 10, eaan0023 (2017) 29
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Fig. 1. Wnt5a secreted from osteoclasts regulates osteoclast bone-resorbing activities. (A) RT-PCR analysis ofCtsk and Wnt expression in BMM cultures treated with or without Rankl and Csf1. n = 5 dishes for each time point.BMMs were prepared from five mice. ND, not detected. (B) Immunoblotting analysis of Wnt5a abundance in BMMcultures treated with glutathione S-transferase (GST)–Rankl plus Csf1. n = 3 biological replicates. (C and D) Effects ofrecombinant Wnt5a on the formation of resorption pits (hematoxylin staining) (C) and actin rings (D) by osteoclastsderived from wild-type (WT) and Wnt5a−/− liver macrophages on dentin slices. n = 5 slices for each genotype. Osteo-clasts were prepared from three mice for each genotype. Scale bars, 100 mm. In (A), (C), and (D), error bars represent SD.*P < 0.05, **P < 0.01. n.s., not significant. For statistical analyses, Kruskal-Wallis and Steel-Dwass test (A) or analysis ofvariance (ANOVA) and Scheffé test (C and D) were used.
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These results suggested that Wnt5a secreted from osteoclasts cellautonomously promoted their bone-resorbing activity.
Ror2-mediated signaling induces the bone-resorbingactivity of osteoclasts in vivoWe next examined the expression of Ror1 and Ror2 in osteoclastsusing RT-PCR (Fig. 2A). Ror2, but not Ror1, was strongly expressedin osteoclasts, suggesting that Ror2-mediated signaling may havesome roles in mature osteoclasts. We generated osteoclast-specificRor2 conditional knockout mice (Ror2DOcl/DOcl, Ror2fl/fl: CtskCre/+)by crossing Ror2-floxed mice with CtskCre/+ mice. RT-PCR analysisshowed that Ror2 expression was lower in osteoclasts derived fromBMMs from these mice than in those derived from Ror2fl/fl mice(control) BMMs (Fig. 2B). The abundance of Ror2 transcripts wassignificantly lower in Ror2DOcl/DOcl osteoclasts than in Ror2DOcl/DOcl
BMMs and in osteoclasts derived from control mice. Immunoblottingconfirmed that Ror2 protein abundance was reduced in Ror2DOcl/DOcl
osteoclasts (Fig. 2C).We observed no gross abnormalities in the skeletal development
of Ror2DOcl/DOcl mice. Micro–computed tomography (CT) of distalfemurs frommale and femaleRor2DOcl/DOcl mice showed that the bonevolume, number of trabeculae, and trabecular thickness were higher,but trabecular separation was lower compared to femurs from controlmice (Fig. 2D and fig. S2A). Similar bone phenotypes were observedin the lumbar vertebrae ofmale and femaleRor2DOcl/DOclmice (fig. S2B).Histomorphometric analysis revealed that the numbers of osteoclasts inthe distal femurswere similar inRor2DOcl/DOcl and controlmice (Fig. 2E),but that the depth and surface area of bone erosions were decreasedin Ror2DOcl/DOcl mice (Fig. 2F). Serum collagen type I cross-linkedC-terminal telopeptide (CTX), amarker of bone resorption, was lowerin Ror2DOcl/DOcl mice (Fig. 2G). Serum alkaline phosphatase activity,a marker for bone formation, was normal in these mice (Fig. 2H).
To confirm the bone-resorbing activity of Ror2DOcl/DOcl osteoclasts,BMMs from Ror2DOcl/DOcl mice were cultured on dentin slices in thepresence of Csf1 and Rankl (fig. S2, C and D). No significant differ-ence was observed in the number of osteoclasts between control andRor2DOcl/DOcl osteoclast cultures (fig. S2C), but the area of resorptionpits on dentin slices was smaller in Ror2DOcl/DOcl cultures (fig. S2D).These results suggested that Ror2DOcl/DOcl mice exhibited a high bonemass phenotype due to defects in the bone-resorbing activity of os-teoclasts.
Wnt5a-Ror2 signaling induces the bone-resorbing activityof osteoclasts through RhoThe small GTPases Rac and Rho are involved in osteoclastic boneresorption by promoting actin ring formation (16, 17). Therefore, wedetermined whether Wnt5a-activated Rac and Rho in osteoclastsformed fromRor2DOcl/DOcl and controlmice (Fig. 3A).Wnt5a activatedboth small GTPases in control osteoclasts within 5 min, but not inRor2DOcl/DOcl-derived osteoclasts. This result suggested that Wnt5a ac-tivated Rac andRho in osteoclasts throughRor2-mediated signaling. Toestablish whether Rho or Rac activity was involved in bone-resorbingactivity under Ror2 signaling, we assessed the bone-resorbing activity ofRor2DOcl/DOcl osteoclasts transduced with adenoviruses encoding CA-RhoA or CA-Rac1 (Fig. 3B). The overexpression of CA-RhoA, but notCA-Rac1, rescued the impaired formation of actin rings and resorp-tion pits inRor2DOcl/DOcl osteoclasts (Fig. 3, C andD). These results sug-gested that the Wnt5a-Ror2 signal stimulated osteoclast function byactivating Rho.
Uehara et al., Sci. Signal. 10, eaan0023 (2017) 29 August 2017
Daam2 is involved in the bone-resorbing activityof osteoclastsIn the Wnt signaling pathway, Daam mediates Rho and b-cateninsignaling (28, 29). We investigated whether Daams were involvedin the Wnt5a-induced bone-resorbing activity of osteoclasts. RT-PCR
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Fig. 2. Ror2-mediated signals are required for bone-resorbing activity of osteo-clasts. (A) Reverse transcription PCR analysis of Ror1 and Ror2 in osteoclasts. n = 3biological replicates. (B) RT-PCR analysis of Ror2 mRNA in BMMs and osteoclastsfrom Ror2fl/fl (Control) and Ror2fl/fl: CtskCre/+ (Ror2DOcl/DOcl) mice. n = 5 cultures ofBMMs and osteoclasts for each genotype. (C) Immunoblotting of Ror2 in osteo-clasts. n = 3 biological replicates. (D) Micro-CT of distal femurs from male control(Ror2fl/fl) and Ror2DOcl/DOcl mice. n = 8 mice for each genotype. Scale bar, 1 mm.(E) TRAP and hematoxylin staining of the distal femurs. Scale bar, 50 mm. Osteoclastnumber per bone perimeter from control and Ror2DOcl/DOcl mice. n = 8 mice for eachgenotype. (F) Erosion depth and eroded surface per bone surface. Erosion depth: 120resorption lacunae assessed from seven control and nine Ror2DOcl/DOcl mice. Erodedsurface per bone surface: n = 8 mice for each genotype. (G) Serum collagen type I cross-linked CTX in control and Ror2DOcl/DOcl mice. n = 8 mice for each genotype. (H) Alkalinephosphatase activity in serum. n = 8 mice for each genotype. In (B) and (D) to (H),error bars represent SD. ***P < 0.001, **P < 0.01, *P < 0.05. For statistical analyses,Kruskal-Wallis and Steel-Dwass test (B) or two-tailed Student’s t test (D to H) was used.
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analysis revealed greater expression of Daam2 in osteoclasts than inBMMs (Fig. 3E). The expression of Daam1 was not detected in BMMsor osteoclasts (Fig. 3E). Therefore, we focused on the role of Daam2 inWnt5a-induced Rho activities in osteoclasts. Knockdown of Daam2 byshort hairpin RNA (shRNA) abrogated recombinant Wnt5a-induced
Uehara et al., Sci. Signal. 10, eaan0023 (2017) 29 August 2017
Rho activity in osteoclasts (Fig. 3, F andG, and fig. S3, A and B). The knockdownof Daam2 in osteoclasts suppressed actinring formation and pit-forming activitywithout affecting their differentiation(Fig. 3, H and I, and fig. S3, C to E). Incontrast, the overexpression of CA-RhoArescued the impaired osteoclast functionin Daam2-deficient osteoclasts (Fig. 3, Hand I, and fig. S3, C and D). These resultssuggested that Daam2 mediated Rho ac-tivity under Ror2 signaling in osteoclasts.
Pkn3 is a Rho effector involved inosteoclast functionRho-associated kinase (ROCK) (30) andmDia2 (31) act as Rho effectors in osteo-clast function. RT-PCR analysis of 13 Rhoeffectors (32) demonstrated that the ex-pression of Pkn3 (which encodes proteinkinase N3) in osteoclasts was significantlyhigher than that in BMMs (Fig. 4A). TheshRNA-mediated knockdown of Pkn3,but not that of Pkn1, Pkn2, ormDia2, in-hibited actin ring formation andpit-formingactivity of osteoclasts without affecting os-teoclast differentiation (Fig. 4, B and C,and fig. S4, A to D). We also determinedwhether ROCKwas involved in the bone-resorbing activity (Fig. 4, D and E). Inhibi-tion of ROCK with Y27632 suppressedstress fiber formation in bone marrow–derived stromal cells but did not affectthe formation of actin rings and resorptionpits by osteoclasts. These results suggestedthat Pkn3, but not mDia2 or ROCK, wasthe Rho effector largely involved in osteo-clast function.
To further clarify the roles of Pkn3 inbone resorption in vivo, we examined thebone phenotypes of 8-week-old Pkn3−/−
mice. Pkn3−/− mice were born at theexpected Mendelian ratio, and no grossabnormalities were observed in skeletaldevelopment. Immunoblotting analysisshowed that the production of Pkn3 wasnot detected in osteoclasts formed fromPkn3−/− mice (Fig. 5A). Micro-CT analy-sis revealed that bone volume/tissue vol-ume, trabecular thickness, and trabecularnumber were increased in the distalfemurs and lumbar vertebrae of 8-week-oldmale Pkn3−/− mice, whereas trabecularseparation was lower (Fig. 5B and fig.
S5A). Similar bone phenotypes were observed in femurs as well as lum-bar vertebrae of female Pkn3−/− mice (fig. S5, A and B). Histomorpho-metric analysis showed that eroded surface per bone surface was lowerin Pkn3−/−mice; however, the number of osteoclasts in bone tissues wassimilar in Pkn3−/−mice and wild-type mice (Fig. 5C). Furthermore, the
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Fig. 3. Daam2 is a critical scaffold molecule linking Ror2 and Rho. (A) Wnt5a-induced Rac and Rho activity inosteoclasts. n = 5 dishes for each genotype. a.u, arbitrary units. (B) Immunoblotting of RhoA and Rac1 in osteoclastsexpressing CA-RhoA or CA-Rac1. n = 3 biological replicates. (C and D) Effects of CA-RhoA and CA-Rac1 on actin ringformation (C) and resorbing pits (D). n = 5 dentine slices for each genotype. Scale bars, 100 mm. (E) RT-PCR ofDaam1 and Daam2 expression. n = 5 dishes for each genotype. (F) RT-PCR of Daam2 expression in osteoclaststransfected with shDaam2. n = 5 dishes for each condition. (G) Effects of shRNA-mediated knockdown of Daam2on Wnt5a-induced Rho activity in osteoclasts. n = 5 dishes for each group. (H and I) Effects of the knockdown ofDaam2 and overexpression of CA-RhoA on actin ring formation (H) and resorbing pits (I) in osteoclasts. n = 5 den-tine slices for each group. Scale bars, 100 mm. In (A) and (C) to (I), error bars represent SD. **P < 0.01, *P < 0.05. Forstatistical analyses, Kruskal-Wallis and Steel-Dwass test (A and G), ANOVA and Scheffé test (C, D, H, and I), two-tailedStudent’s t test (F), or two-tailed Welch’s t test (E) was used.
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erosion depthwas shallower in Pkn3−/−mice (Fig. 5D). SerumCTXwaslower in Pkn3−/− mice (Fig. 5E). On the other hand, bone formationparameters including osteoblast numbers remained unchanged inPkn3−/− mice (Fig. 5F).
Ex vivo analysis showed that Rankl-induced osteoclast formationwas normal in BMM cultures from Pkn3−/− mice (fig. S6A). RT-PCRanalysis also confirmed that the expression of osteoclast marker genes
Uehara et al., Sci. Signal. 10, eaan0023 (2017) 29 August 2017
such as Tnfrs11a and Csf1rwas normal inPkn3−/− osteoclasts (fig. S6B). However,the formation of actin rings and resorp-tion pits by Pkn3−/− osteoclasts was im-paired (Fig. 5, G and H). A podosome beltwas observed at the cell periphery of wild-type osteoclasts (fig. S6C). In contrast, podo-some dots, but not a podosome belt, werepresent in Ror2DOcl/DOcl or Pkn3−/− osteo-clasts, suggesting that assembly of podo-somes was impaired in these osteoclasts.The overexpression of CA-RhoA failedto rescue the impaired pit-forming activityof Pkn3−/− osteoclasts (fig. S6D). Further-more, alkaline phosphatase activity andmineralized nodule formation werenormal in calvaria-derived osteoblastic cellcultures from Pkn3−/− mice (fig. S6E).These results suggested that Pkn3 actedas a Rho effector in the bone-resorbingactivity of osteoclasts.
The proline-rich region ofPkn3 is necessary forbone-resorbing activityThe phosphorylation of Thr718 in humanPKN3 is required for full kinase activity(33). Therefore, we investigated the in-volvement of Ror2 signaling in the phos-phorylation of Pkns. Immunoblottinganalysis showed that phosphorylated Pknswere markedly decreased in osteoclastsfrom Ror2DOcl/DOcl mice (Fig. 6A). RT-PCR analysis confirmed that the expres-sion of Pkn3mRNA remained unchangedbetween Ror2DOcl/DOcl and control osteo-clasts (Fig. 6B). These results suggested thattheWnt5a-Ror2 signal was involved in thephosphorylation of Pkn3 in osteoclasts.
We then examined the localization ofF-actin and Pkn3 in osteoclasts on calci-um phosphate–coated plates. DsRed-fusion b-actin and Venus-fusion Pkn3(Venus-Pkn3) were coexpressed in osteo-clasts using adenovirus-mediated genetransfer. Venus-Pkn3 was observed in aperinuclear region and in the cell periph-ery of osteoclasts. A part of Venus-Pkn3was colocalized with actin rings visualizedby DsRed-fusion b-actin (Fig. 6C, whitearrow). Thus, Pkn3 was localized with ac-tin rings.
Pkn2 interacts with the Src family kinase Fyn and plays a role in theRho-induced activation of Fyn in keratinocytes (34). Furthermore, Rhoactivates c-Src in Fyn−/− keratinocytes (34). c-Src is abundant in osteo-clasts, and the activation of c-Src is essential to form actin rings in os-teoclasts (35–37). These findings prompted us to clarify whether theRho-Pkn3 pathway promotes actin ring formation in osteoclaststhrough the activation of c-Src. Immunoprecipitation assays using an
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Fig. 4. Pkn3 acts as a Rho effector for the bone-resorbing activity of osteoclasts. (A) RT-PCR analysis of theexpression of mRNAs encoding Rho effectors in BMMs and osteoclasts. n = 5 dishes of BMM and osteoclast cultures.(B) Effect of shRNA-mediated knockdown of Pkn family members on the bone-resorbing activity of osteoclasts. n =5 slices for each group. Osteoclasts were prepared from five mice. Scale bar, 100 mm. (C) Effects of the shRNA-mediated knockdown of mDia2 on the bone-resorbing activity of osteoclasts. n = 5 slices for each group. Osteoclastswere prepared from three mice. Scale bar, 100 mm. (D) Effects of Y27632 on stress fiber formation in bone marrowstromal cells. n = 5 wells for each treatment. Bone marrow stromal cells were prepared from two mice. Scale bar,100 mm. (E) Effects of Y27632 on the formation of actin rings (the left two images and the left bar graph) and resorbingpits (the right two images and the right bar graph) in osteoclasts cultured on dentin slices. n = 5 slices for each treat-ment. Osteoclasts were prepared from two mice. Scale bars, 100 mm. In (A) to (E), error bars represent SD. **P < 0.01. Forstatistical analyses, Mann-Whitney U test (A), Kruskal-Wallis and Steel-Dwass test (B), or two-tailed Student’s t test (C to E)was used.
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antibody specific for enhancedGFP(EGFP)revealed that in control osteoclasts, Venus-Pkn3 associated with c-Src and Pyk2, akinase that promotes osteoclast adhesionand forms a complexwith c-Src to activateosteoclast function (Fig. 6D) (38, 39). Incontrast, these associations were abol-ished in Ror2DOcl/DOcl osteoclasts. Thekinase activity of c-Src in osteoclasts fromRor2DOcl/DOcl mice was lower than that incontrol osteoclasts (Fig. 6E); however, theabundance of c-Src inRor2DOcl/DOcl osteo-clasts was similar to that in control osteo-clasts (fig. S7A). The kinase activity ofc-Srcwas also lower inPkn3−/− osteoclasts(fig. S7B). Knockdown of Daam2 sup-pressed the phosphorylation of Pkn3and complex formation of Pkn3, c-Src,and Pyk2 (Fig. 6F and fig. S7C). We alsodetermined the domains of Pkn3 thatwereneeded for the interaction between Pkn3and c-Src by overexpressing deletion mu-tant forms of Pkn3 in osteoclasts (fig. S7D).Immunoprecipitation assays showed thatPkn3 lacking the kinase domain (Dkinase),but not the form lacking the proline-richregion (DPRR), interacted with c-Src orPyk2 (Fig. 6G). Notably, the expression offull-length Pkn3 (Pkn3 full) rescued im-paired actin ring formationandpit-formingactivity of Pkn3−/− osteoclasts, whereas thatof Pkn3-DPRR and Pkn3-Dkinase did not(Fig. 6, H and I). Together, these results in-dicated that Pkn3 and its kinase activitypromoted osteoclast function through aninteraction with c-Src.
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DISCUSSIONWnt5a secreted from osteoblast-lineagecells enhances the production of Rankin osteoclast precursors, which, in turn,promotes Rankl-induced osteoclasto-genesis (27). Using genetic and bio-chemical approaches, we showed thatWnt5a also promotes the bone-resorbingactivity of osteoclasts through Ror2-mediated signaling. Although Rho isinvolved in the bone-resorbing activityof osteoclasts as demonstrated by theuse of the C3 exoenzyme and CA-RhoA(19–21), it currently remains unclearhowRho promotes the formation of actinrings in osteoclasts. Rho promotes the for-mation of stress fibers in cells including fi-broblasts. In contrast, transformation offibroblasts by active c-Src disrupts actinstress fibers and triggers the alternativeformation of podosome belts without aAW
ild-ty
pe-1
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)
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)
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asts
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rebmun tsalboets
Ope
r bon
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(%)
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2 6 10 14 18Erosion depth (μm)
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Ser
um C
TX (n
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Fig. 5. Impaired bone-resorbing activity of osteoclasts in Pkn3−/− mice. (A) Immunoblotting of Pkn3 in osteo-clasts formed from Pkn3−/− mice. n = 3 biological replicates. (B) Micro-CT analysis of femurs. n = 7 mice for eachgenotype. Scale bar, 1 mm. (C) TRAP and hematoxylin staining images and bone histomorphometric analysis offemurs. n = 7 mice for each genotype. Scale bar, 50 mm. (D) Erosion depth and the frequency distribution of theerosion depth in femurs. n = 7 mice for each genotype. Erosion depth: 200 resorption lacunae were assessed. (E) SerumCTX in WT and Pkn3−/− mice. n = 7 mice for each genotype. (F) Bone histomorphometric analysis of bone formationparameters in distal femurs. n = 7 mice for each genotype. (G) Ex vivo analysis of actin ring formations in osteo-clasts. (H) Ex vivo analysis of resorbing pits in WT and Pkn3−/− mice–derived osteoclasts. In (G) and (H), n = 5 slicesfor each genotype. Osteoclasts were prepared from three mice for each genotype. Scale bars, 100 mm. In (B) to (H),error bars represent SD. ***P < 0.001, **P < 0.01, *P < 0.05. For statistical analyses, two-tailed Student’s t test (B to F)or two-tailed Welch’s t test (G and H) was used.
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A
Rel
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Pyk2
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Input IP Venus
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Control(Venus)
ΔPRR
ΔKinase
Full
G
J
H
IPkn3
–/–
Control Pkn3-ΔPRR Pkn3 full Pkn3-Δkinase
200
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Control Pkn3 full Pkn3-ΔPRR Pkn3-ΔkinaseWnt5a
Ror2
Rho
Pkn3c-SrcPyk2
Bone resorption
Daam2
Dishevelled
Frizzled
Fig. 6. Pkn3 forms complexes with c-Src and Pyk2 to promote bone-resorbing activity. (A) Immunoblotting of phosphorylated Pkns in osteoclasts. n = 3 biologicalreplicates. (B) RT-PCR of Pkn3 mRNA in osteoclasts. n = 5 dishes for each genotype. (C) Confocal microscopic images in osteoclasts expressing Venus-Pkn3 and DsRed-actin proteins. n = 3 biological replicates. Scale bar, 50 mm. (D) Interactions between Venus-Pkn3 and c-Src in Ror2DOcl/DOcl osteoclasts. n = 3 biological replicates. (E) c-Src kinaseactivity in Ror2DOcl/DOcl osteoclasts. n = 5 dishes of BMM and osteoclast cultures for each genotype. (F) Effects of Daam2 knockdown on interactions between Pkn3, c-Src, andPyk2. n = 3 biological replicates. (G) Immunoprecipitation (IP) analysis of osteoclasts expressing full-length Pkn3-Venus (full), Pkn3-Venus lacking the PRR domain (DPRR), andPkn3-Venus lacking the kinase domain (Dkinase). n = 3 biological replicates. (H and I) Effects of the enforced expression of Pkn3 full, Pkn3-DPRR, and Pkn3-Dkinase on the actinring formation (H) and resorbing pits (I) of Pkn3−/− osteoclasts. n = 5 slices for each group. Osteoclasts were prepared from three mice. Scale bars, 100 mm. In (B), (E), (G), and (H),error bars represent SD. **P < 0.01. (J) Proposed pathway in our current study. For statistical analyses, Mann-Whitney U test (B), Kruskal-Wallis and Steel-Dwass (E), or ANOVAand Scheffé test (H and I) were used.
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decrease in Rho activity, but rather by localizing active Rho in podo-somes (40). Furthermore, inhibition ofRho activity usingC3 exoenzymeand transfection of dominant negative forms of Rho disrupts podo-some belts (40). These findings indicate that Rho cooperates with c-Srcto form podosome belts in c-Src–transformed cells. Therefore, Rhopreferentially forms podosome belts, but not stress fibers in c-Src–positive cells including osteoclasts, supporting our model that Rhoactivated byWnt5a-Ror2 signals and c-Src are involved in actin ringformation in osteoclasts.
Daam1 binds the PDZ and DEP domains of Dvl2 and Rho andmediates theWnt-induced activation of Rho (28). However, Rac direct-ly binds to DEP domains of Dvl2 to activate downstream signaling in aDaam-independentmanner (41). These findings indicate that Dvl playsan important role as a hubmolecule in the activation of Rho and Rac byWnt signals and that Daam1 is required for the activation of Rho byFzd-Dvl2 signaling. Osteoclasts strongly expressed Daam2, but notDaam1 (Fig. 3E), suggesting that Rho was activated by Daam2, ratherthan Daam1. Daam2 reportedly binds not only to the DIX domain ofDvl3 to activate Wnt/b-catenin signaling but also to the PDZ and DEPdomains of Dvl3 (29). On the basis of the knockdown experiments ofDaam2 in osteoclasts and the overexpression of CA-RhoA in Daam2-deficient osteoclasts, we propose thatDaam2 acts as a linkbetweenRor2/Fzd and Rho-mediated signals in osteoclasts.
Rho signaling activates several Rho effectors, including ROCK.The ROCK inhibitor Y27632 did not inhibit the formation of actinrings and resorption activities of osteoclasts under our experimentalconditions, suggesting that ROCK is primarily involved in the forma-tion of stress fibers but not podosomes.AlthoughROCKmaybe activatedin osteoclasts byWnt5a-Ror2-Rho signals, the activated c-Src may inhibitthe stress fiber formations as described above. Further studies areneeded toclarify how c-Src inhibits ROCK activity in osteoclasts.
Our results showed that Pkn3 played a critical role in osteoclasticbone resorption stimulated by Wnt5a-Ror2-Rho signaling. SH3 do-mains bind to proline-rich domains tomediate protein-protein interac-tions (42). Similarly, Pkn3 may associate with the SH3 domain of c-Srcthrough its proline-richdomain. It is unlikely thatPkn3directly associateswith Pyk2 because Pyk2 does not have an SH3 domain (43). c-Src bindsto Pyk2 and phosphorylates tyrosine residues in Pyk2 in osteoclasts in acell adhesion–dependent manner (44). These previous findings and ourpresent study suggest that c-Src primarily forms a complex with Pyk2,and then the complex associates with Pkn3 in response to Wnt5a-Ror2signals. The kinase activity of Pkn2 promotes the activity of Fyn (34) andthat Pkn1 is autophosphorylated in response to RhoA signals (45). Weshowed that Daam2 was required for the phosphorylation of Pkn3 in re-sponse to Wnt5a-Ror2 signals. These findings also suggest that Pkn3 isautophosphorylated, and then phosphorylated Pkn3 promotes the acti-vation of c-Src. Further experiments are needed to determine how Pkn3promotes the c-Src activity.
We found that Pkn3 was abundant in osteoclasts, but not in BMMsas osteoclast precursors. However, Pkn1 expression was higher inBMMs than in osteoclasts (Fig. 4A). These findings suggest thatRankl-Rank signaling could induce Pkn3 expression. However, the ex-pression of Pkn3 was lower than that of Pkn1 in spleen and intestine(46), tissues in which Rankl or Rank are abundant (47–49). Together,these results suggest that Rankl-Rank signals do not directly inducePkn3 expression during osteoclast formation.
Vav3 plays a role in the Csf1-induced activation of Rac and avb3integrin–mediated activation of c-Src. The Csf1-induced activationof Rho is normal in Vav3−/− osteoclasts (18). These findings suggest
Uehara et al., Sci. Signal. 10, eaan0023 (2017) 29 August 2017
thatVav3mainly activates Rac underCsf1r-mediated signals, andRhomay be activated by Vav3-independent signals includingWnt5a-Ror2signals. The activation of c-Src by avb3 integrin–mediated signals is im-paired in Vav3−/− osteoclasts and also in osteoclasts formed fromRor2DOcl/DOcl mice. The overexpression of CA-RhoA, but not CA-Rac1, rescued the impaired pit-forming activity of these osteoclasts.These results indicated that Rac activity in response to Csf1r- and avb3integrin–mediated signals and Rho activity in response to Wnt5a-Ror2signals promote c-Src activity and bone-resorbing activity in osteoclasts.Further studies are needed to clarify the mechanism by which Pkn3activates c-Src.
The importance of Wnt5a-Ror2 signaling in the activation of c-Srcdoes not appear to be limited to bone resorption. Ror2 knockdown inSaOS-2 cells, a human osteosarcoma cell line, suppresses the activationof c-Src and the production of matrix metalloproteinase-13 (MMP-13),thereby limiting their invasive properties (50). The treatment of SaOS-2cells with Wnt5a enhances c-Src activation and MMP-13 production.These findings suggest that Wnt5a-Ror2 signaling is involved in the me-tastasis ofmalignant neoplasms through the activation of c-Src. Thus, theWnt5a-Ror2 signaling axis promotes not only bone resorption but alsotumor invasion.
We showed that Wnt5a-Ror2 signals promoted the bone-resorbingactivity of osteoclasts through aDaam2-Rho-Pkn3 signaling axis (Fig. 6J).This signaling pathwaywas critical for the bonemass under physiologicalconditions. This signaling pathwaymayalso be crucial under pathologicalconditions such as rheumatoid arthritis, a condition in whichWnt5a se-cretion is increased from synovial cells, osteoblast-lineage cells, and osteo-clasts. Thus,Wnt5a-Ror2 signalsmay represent a therapeutic target for bonediseases such as osteoporosis, rheumatoid arthritis, and periodontitis.
MATERIALS AND METHODSAnimals and reagentsWnt5a−/− (51) and CtskCre/+ (52) mice were generated and maintainedas described previously. Mice harboring the floxed Ror2 gene weremaintained as described previously (27). Pkn3−/−mice (46) were gener-ated by H. Mukai and appropriately maintained. All procedures foranimal care were approved by the Animal Management Committeeof Matsumoto Dental University. GST-Rankl and Csf1 were purchasedfrom Oriental Yeast and Kyowa Kirin, respectively. Rhodamine-conjugated phalloidin was from Molecular Probes. All other reagentswere from Sigma.
Analysis of bone phenotypesMicro-CTanalysis (ScanXmate-A080, ComscanTecno)was performedto measure morphological indices in the distal metaphysis of femurs(27). These indices were calculated in trabecular bones located between0.5 and 1.5 mm from the growth plates using image analysis software(TRI/3D-BON, Ratoc System Engineering). Morphological indices offifth lumbar vertebrae were calculated in trabecular bones located be-tween 0.76 and 0.9 mm from the ventral surface of the vertebral body.For histomorphometric analysis, the distal half of the femurs wasstained with Villanueva bone stain and embedded in glycol methacrylate(PolyScience). The frontal plane sections of the femurs were subjected tohistomorphometric examinations as described previously (27). Collagentype I cross-linked CTX and bone-specific alkaline phosphatase activityin serum were measured using enzyme-linked immunosorbent assay(RatLaps, Immunodiagnostic Systems) and a TRACP & ALP Assaykit (Takara Bio), respectively.
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Osteoclast formation and bone-resorbing activityassays in vitroBone marrow cells (1 × 107 cells) were cultured in a-MEM (minimumessentialmedium) containing 10% fetal bovine serum (FBS) in the pres-ence of Csf1 (100 ng/ml) on dishes 60 mm in diameter. Nonadherentcells containing osteoclast precursors were harvested and seeded ondentin slices (5.0 × 104 cells per dentin slice). Cells were cultured for3 days in the presence of Csf1 (50 ng/ml) and cultured further in thepresence of Csf1 (50 ng/ml) and GST-Rankl (200 ng/ml). Macrophagesas osteoclast precursors were prepared from the liver cells ofWnt5a−/−
and wild-type fetuses as described previously (27). After osteoclastformation, the dentin slices were fixed with 3.7% formaldehyde andthen treated with 0.1% Triton X-100 in phosphate-buffered saline(PBS). They were incubated in PBS containing rhodamine-conjugatedphalloidin to visualize F-actin. The number of actin rings was countedand adjusted by the area of each dentin slice (53, 54). To visualize podo-some belts, osteoclasts were cultured on vitronectin-coated cover slide.To count the number of osteoclasts, cells on dentine slices and cultureplates were stained for tartrate-resistant acid phosphatase (TRAP) ac-tivity, and TRAP-positive cells withmore than three nuclei were counted(27). In some experiments, osteoclasts were treated with 10 mMROCKinhibitor Y27632. After removing cells from the dentin slices, the sliceswere stainedwithMayer’s hematoxylin (Sigma) to visualize resorptionpits (53, 54). The area of resorption pits was measured using ImageJ(National Institutes of Health).
Adenovirus gene transferAdenoviruses with CA-Rac1 and CA-RhoA were obtained from CellBiolabs Inc. Adenoviruses expressing shRNA were prepared as follows.Double-stranded oligonucleotides of the target were inserted intoRNAi-Ready pSIREN-Shuttle vectors (Takara Clontech). PCR frag-ments containing a U6 promoter and the target sequences were ligatedinto pAdenoX-PRLS-ZsGreen vectors (Takara Clontech). The linear-ized vectors were transfected into human embryonic kidney (HEK)293T cells to produce adenoviruses according to the manufacturer’s in-structions. The target sequences are as follows: Daam2#1, 5′-GGAT-GAATTGGACCTCACA-3′; Daam2#2, 5′-CTCTCATTGGCTGCATCAA-3′;Daam2#3, 5′-CTCTCATTGGCTGCATCAA-3′; Pkn1#1, 5′-AGGACAG-TAAGACCAAGAT-3′; Pkn1#2, 5′-AGGACAGTAAGACCAAGAT-3′;Pkn2#1, 5′-TCCGGATGCAGATTCTTCA-3′; Pkn2#2, 5′-GTCCAAGTGA-CAACAGATC-3′; Pkn3#1, 5′-TGAGGACTTCCTGGACAAT-3′; Pkn3#2,5′-CGTTGAAGAAGCAGGAAGT-3′; mDia2#1, 5′-GCACAAAGTCATC-CAGTGT-3′; and mDia2#2, 5′-GGCATAACTCAGTGAACCT-3′.Purified adenoviruses were infected into cells at a dose of 50 to 100multiplicity of infection.
Construction of adenovirus vectors for the expression ofDsRed-actin and Venus-Pkn3pDsRed-Monomer-Actin and Adeno-X Adenoviral System 3 werepurchased from Clontech Laboratories. Mouse Pkn3 cDNA was fromThermo Scientific Open Biosystems. The vector for Venus (an EGFPmutant) expressionwas provided byRIKEN (55). DsRed-fusion humanb-actin (DsRed-actin), Pkn3, and Venus were amplified by PCR. Toprepare deletion mutants of Pkn3, PCR fragments containing 1 to1365 nucleotides and fragments containing 1630 to 2637 nucleotidesin the open reading frame of Pkn3 were amplified for Pkn3-DPRR.Similarly, PCR fragments containing 1 to 1641 and 2422 to 2637 nu-cleotides in the open reading frame of Pkn3 were amplified for Pkn3-Dkinase. The fragments were linked with pAdenoX vectors using an
Uehara et al., Sci. Signal. 10, eaan0023 (2017) 29 August 2017
In-Fusion enzyme (Takara Clontech) according to the manufacturer’sinstructions. The ligated plasmid was linearized with Pac I and thentransfected into HEK293T cells using the X-tremeGENE 9 (Roche).After amplification of the adenovirus, the adenoviruses were purifiedusing the Adeno-X Maxi Purification Kit (Clontech) according to themethod described previously (56).
Rac and Rho activitiesOsteoclast precursors (5 × 105 cells) were seeded on 12-well plates anddifferentiated into osteoclasts with Csf1 and GST-Rankl as describedabove. Osteoclasts were starved for 8 hours with 2% FBS containinga-MEM without Csf1 or GST-Rankl. Osteoclasts were then stimulatedwith Wnt5a (100 ng/ml) or vehicle for 5 min and lysed. Lysates werecollected according to the manufacturer’s manual. Active Rac and Rhoin cell lysates were assayed by G-LISA Rac Activation Assay BiochemKit and G-LISA Rho Activation Assay Biochem Kit (Cytoskeleton), re-spectively, according to the manufacturer’s manual.
Src kinase activityCell lysates were immunoprecipitated with an antibody recognizingc-Src (Cell Signaling Technology). Tyrosine kinase activity of precipi-tated proteins was assayed with Universal Tyrosine Kinase Assay Kit(Takara).
RT-PCR analysiscDNA was synthesized from total RNA and amplified using a PCRthermal cycler (TP600, Takara). RT-PCR was performed using SYBRGreen Master Mix (Life Technologies) with the StepOnePlus System(Life Technologies) as described previously (56). Fold-change ratioswere calculated between the test and control samples. PCR primers werepurchased from Takara Bio Inc.
Immunological analysisCell lysates were subjected to an immunoblot analysis using the followingantibodies: goat antibody specific for Wnt5a (R&D Systems, AF645,1:800), mouse antibody specific for RhoA (Millipore, clone 55, 1:1000),mouse antibody specific for Rac1 (Millipore, clone 23A8, 1:1000), rabbitantibody specific for Pkn3 (Abcam, ab155076, 1:1000), rabbit antibodyspecific for phosphorylated Pkns (Abcam, ab124709, 1:1000), goatantibody specific for EGFP (Abcam, ab111258, 1:1000), mouse anti-body specific for Pyk2 (BD Biosciences, 610548, 1:1000), mouse anti-body specific for c-Src (Abcam, ab16885, 1:1000), and goat antibodyspecific for Daam2 (Santa Cruz Biotechnology, sc-68297, 1:500).Antibody specific for Ror2 (57) was from Y. Minami. All immuno-blotting analyses were repeated three times.
For immunoprecipitation assays, cell lysates (250mg)were incubatedwith 10 mg of antibody specific for EGFP (Abcam) and protein Asepharose (Amersham Biosciences) at 4°C for 16 hours. Then, proteinA sepharose was pelleted and samples were eluted by SDS samplebuffer. Twenty micrograms (Fig. 6, D and F) or 10 mg (Fig. 6G) of totalcell lysates was loaded as input.
Osteoblast-lineage cell differentiationOsteoblast-lineage cells were obtained from the calvaria of 1-day-oldPkn3−/− mice and wild-type litters according to a previously describedmethod (56). Cells were cultured in a-MEM containing 10% FBS in thepresence of b-glycerophosphate (10 mM) and ascorbic acid (50 mg/ml)for 2 weeks (for alkaline phosphatase staining) or 5 weeks (for AlizarinRed S staining).
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Statistical analysisStatistical analyses were performed using the two-tailed Student’s t testandWelch’s t test, when the number of groups was two. If the numberof groups was larger than three, an ANOVA and post hoc test (Scheffétest) were used for statistical analyses. Nonparametrical analysis, such asKruskal-Wallis, Mann-Whitney U test, and Steel-Dwass test, was alsoused for statistical analysis of normalized data. Each in vitro experimentwas repeated at least three times, and similar results were obtained. Nosample was excluded from the statistical analysis.
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SUPPLEMENTARY MATERIALSwww.sciencesignaling.org/cgi/content/full/10/494/eaan0023/DC1Fig. S1. Rankl-induced formation of osteoclasts from Wnt5a−/− liver macrophages on dentinslices.Fig. S2. Micro-CT analysis of femurs and lumbar vertebrae in Ror2DOcl/DOcl mice andbone-resorbing activity of osteoclasts formed from Ror2DOcl/DOcl mice.Fig. S3. Effects of suppression of Daam2 on osteoclasts.Fig. S4. Effects of shRNA-mediated knockdown of Pkns and mDia2 on osteoclasts.Fig. S5. Micro-CT analysis of femurs and lumbar vertebrae of Pkn3−/− mice.Fig. S6. Osteoclast and osteoblast differentiation in cultures prepared from Pkn3−/− mice.Fig. S7. The expression of c-Src, phosphorylation of Pkn3, and schematic of Pkn3.
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Acknowledgments: We thank A. Kikuchi (Osaka University) for providing recombinant Wnt5aand reading the manuscript. We also thank A. Miyawaki (RIKEN Brain Science Institute) forproviding pCS2-Venus vectors and T. Ara (Matsumoto Dental University) for providingadvice on statistical analysis. Funding: This study was supported by Japan Society for thePromotion of Science KAKENHI grants JP25462904 and JP16K11494 (S.U.), JP24390417 andJP16H05508 (N.U.), JP25221310 and JP16H05144 (N.T.), and JP25293423 and JP16H02691 (Y.K.)and by the Japanese Association for Oral Biology Grant-in-Aid for Young Scientists (S.U.).Author contributions: S.U. and Y.K. performed experiments and prepared the manuscript.A.I., K. Maeda, T.Y., and K. Murakami contributed to in vitro and in vivo experiments and datainterpretations. H.M., M.N., Y.M., T.N., and S.K. supported the generation of geneticallymodified mice and contributed to data interpretations. N.U. and N.T. contributed to datainterpretations and preparation of the manuscript. Y.K. directed the project and wrote themanuscript. Competing interests: The authors declare that they have no competing financialinterests. Data and materials availability: The use of Pkn3−/− mice requires a materialstransfer agreement (MTA) from Kobe University. The use of Ror2fl/fl mice requires an MTA fromKobe University. The use of CtskCre/+ mice requires an MTA from Institute of Molecular andCellular Biosciences, University of Tokyo. The use of pCS2-Venus vectors requires an MTA fromRIKEN Brain Science Institute.
Submitted 17 February 2017Accepted 8 August 2017Published 29 August 201710.1126/scisignal.aan0023
Citation: S. Uehara, N. Udagawa, H. Mukai, A. Ishihara, K. Maeda, T. Yamashita, K. Murakami,M. Nishita, T. Nakamura, S. Kato, Y. Minami, N. Takahashi, Y. Kobayashi, Protein kinase N3promotes bone resorption by osteoclasts in response to Wnt5a-Ror2 signaling. Sci. Signal.10, eaan0023 (2017).
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signalingProtein kinase N3 promotes bone resorption by osteoclasts in response to Wnt5a-Ror2
Murakami, Michiru Nishita, Takashi Nakamura, Shigeaki Kato, Yasuhiro Minami, Naoyuki Takahashi and Yasuhiro KobayashiShunsuke Uehara, Nobuyuki Udagawa, Hideyuki Mukai, Akihiro Ishihara, Kazuhiro Maeda, Teruhito Yamashita, Kohei
DOI: 10.1126/scisignal.aan0023 (494), eaan0023.10Sci. Signal.
operate in metastasizing cancer cells.authors note that several of these proteins are implicated in tumor metastasis and that this signaling pathway mayRor2, activated the small GTPase Rho, its effector Pkn3, and c-Src, a kinase that is required for actin ring formation. The
characterized a pathway that stimulated bone resorption by osteoclasts. Binding of Wnt5a to one of its receptors,et al.density in diseases such as osteoporosis. Using genetically manipulated mice and cells derived from these mice, Ueharathat control actin ring formation in osteoclasts could reveal potential therapeutic targets for reversing losses in bone
To resorb bone, osteoclasts must remodel the cytoskeleton to form actin rings. Thus, understanding the pathwaysBone breakdown
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