gp130-Mediated Signaling Is Necessary for NormalOsteoblastic Function in Vivo and in Vitro

HONG-IN SHIN, PAOLA DIVIETI, NATALIE A. SIMS, TATSUYA KOBAYASHI, DENGSHUN MIAO,ANDREW C. KARAPLIS, ROLAND BARON, RICHARD BRINGHURST, AND HENRY M. KRONENBERG

Endocrine Unit (H.-I.S., P.D., T.K., R.Br., H.M.K.), Massachusetts General Hospital and Harvard Medical School, Boston,Massachusetts 02114; Departments of Cell Biology and Orthopedics (N.A.S., R.Ba.), Yale University School of Medicine,New Haven, Connecticut 06510; Calcium Research Laboratory (D.M.), McGill University Health Centre, and Department ofMedicine, McGill University, Montreal, Quebec, Canada H3A 1A1; and Lady Davis Research Institute (A.C.K.), SirMortimer B. Davis Jewish General Hospital, and Department of Medicine, McGill University, Montreal, Quebec,Canada H3T 1A1

Previous studies have shown that mice missing gp130, thecommon receptor subunit for many cytokines, die at or beforebirth with multiple skeletal abnormalities. Furthermore, in-teractions between PTH and gp130 signaling have suggestedthat gp130 signaling might influence calcium homeostasis.We, therefore, examined the function of osteoblasts, oste-oclasts, and calcium homeostasis in gp130�/� mice, both in vivoand in vitro. Osteoblasts from these mice exhibit widespreadabnormalities, including decreased alkaline phosphatasemRNA and protein, both in vivo and in osteoblast cultures.Although osteoclast number is increased in gp130�/� fetuses,these osteoclasts exhibit abnormalities in the resorptive or-ganelle and the ruffled border, and the mice are mildly hy-

pocalcemic. Although the hypocalcemia is associated withsecondary hyperparathyroidism, the increase in PTH doesnot explain the increase in osteoclast number because re-moval of the PTH gene in gp130�/� fetuses does not impor-tantly change osteoclast number. Calvarial bone resorption inresponse to PTH is defective, as is the ability of osteoblasticcells from gp130�/� mice to stimulate osteoclastogenesis fromnormal precursors in vitro or to increase receptor activator ofnuclear factor-�B ligand mRNA levels after exposure to PTH.These studies demonstrate the importance of gp130 signalingfor osteoblast function and calcium homeostasis. (Endocri-nology 145: 1376–1385, 2004)

BONE CELLS RESPOND both to local cytokines and sys-temic hormones to accomplish their dual roles of main-

taining the skeleton’s structural integrity and facilitatingmineral ion homeostasis. The cytokine family that uses gp130as a component of its signaling receptors influences the ac-tivity of both osteoblasts and osteoclasts (1), for example, andalso influences the actions of PTH to maintain calciumhomeostasis.

The gp130-using cytokine family has seven mammalianmembers [IL-6, IL-11, leukemia inhibitory factor (LIF), on-costatin M, ciliary neurotrophic factor (CNTF), cardiotrophin1, and cardiotrophin-like cytokine] that signal by activatingthe transmembrane protein, gp130, which binds these li-gands in association with ligand-specific receptor subunits(2). Osteoblasts and osteoblast-like cells express IL-6, IL-11,and LIF as well as gp130 and ligand-specific receptor sub-units for IL-6, IL-11, LIF, oncostatin M, and CNTF (3). Os-teoclasts also express gp130 and ligand-specific receptor sub-units for IL-6 and IL-11 (4, 5).

A large body of literature (1), primarily studying cells inculture, suggests that gp130 signaling influences both osteo-blast development (6) and differentiation (7), osteoclast de-velopment (4, 8), and activity of mature osteoclasts (5, 9).Furthermore, the action of PTH to stimulate osteoclastogen-esis has been shown to partly depend on gp130 signaling.PTH stimulates production of IL-6 (10), IL-11 (8), and LIF (11)by osteoblasts, and antibodies to gp130 (8) or to the IL-6receptor (10) partially block osteoclast development andbone resorption in vitro. Furthermore, when low doses ofPTH are infused in mice, the increase in bone resorption seenin normal mice is greatly blunted in mice null for the IL-6gene (12). Both gp130 activation, acting through signal trans-ducer and activator of transcription 3, and PTH increaseexpression of the ligand for receptor activator of nuclearfactor-�B [receptor activator of nuclear factor-�B ligand(RANKL)], a key activator of osteoclast development andaction (13).

The physiological relevance of gp130 signaling in bone hasbeen strongly supported by studies of mice null for gp130gene expression. These mice die at birth or earlier, dependingon the genetic background, and exhibit abnormalities in car-diac and hematopoietic development (14). At the time ofbirth, these mice exhibit normal growth plates and corticalbone, with a dramatic decrease in the amount of trabecularbone and an increase in number of osteoclasts (15). Micemissing the receptor subunit specific for LIF and CNTF ex-hibit a similar decrease in trabecular bone and increase inosteoclast number (16). These studies raise the possibility

Abbreviations: ALP, Alkaline phosphatase; CNTF, ciliary neurotro-phic factor; dpc, days after conception; E, embryonic day; FBS, fetalbovine serum; LIF, leukemia inhibitory factor; M-CSF, macrophagecolony-stimulating factor; MNC, multinucleated cell; OPG, osteoprote-gerin; RANKL, receptor activator of nuclear factor-�B; sRANKL, solubleRANKL; TRAP, tartrate-resistant acid phosphatase; TRAP�, positiveTRAP.Endocrinology is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving theendocrine community.

0013-7227/04/$15.00/0 Endocrinology 145(3):1376–1385Printed in U.S.A. Copyright © 2004 by The Endocrine Society

doi: 10.1210/en.2003-0839

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that osteoblast development may be abnormal in these miceand raise questions about the regulation of bone resorptionand calcium homeostasis in this model. Therefore, we haveperformed further analyses both in vivo and in vitro to char-acterize the role of gp130 signaling in osteoblast develop-ment and function and analyze the possible interactions ofPTH and gp130 signaling in regulating osteoclasts and cal-cium homeostasis. We show that gp130 signaling is vital fornormal osteoblast function and for calcium homeostasis infetal life.

Materials and MethodsAnimals

Mice carrying a gp130 gene disrupted by homologous recombination,using a targeting vector that inserts a pMC1Neo-poly (A) cassette intothe HindIII site in exon 2 just downstream of the translation initiationcodon of the gp130 gene was generated in a CD-1 genetic backgroundby Yoshida et al. (14). gp130-deficient mice were obtained by crossingheterozygous mice and genotyped by PCR using genomic DNA isolatedfrom tail clips. Due to perinatal lethality of homozygotes, embryos wereobtained before birth by cesarean section from different days of gestationafter timed mating. These studies were approved by the institutionalanimal care committee of Massachusetts General Hospital. All mice weregiven a standard chow diet and water.

Ionized calcium measurement

Individual fetuses were removed from their amniotic sacs at embry-onic day (E)18.5 by cesarean section. Whole blood was collected into50-�l heparinized capillary tubes from cervical vessels cut by transverseincision. All capillary tubes were capped and immediately immersed inice. Each blood sample was analyzed within 20 min of collection usinga CIBA/Corning 634 Ca2�/pH analyzer (Ciba-Corning DiagnosticCorp., Medfield, MA).

Serum PTH measurement

Fetal blood was collected as for ionized calcium measurements into50-�l plain capillary tubes instead of heparinized capillary tubes, andthen the serum was separated by centrifugation at 1300 rpm for 3 min.The separated sera were stored at �20 C until used. After genotyping,the sera from 12 embryos of each genotype were pooled and measuredwith an immunoradiometric assay directed at two sites in rat PTH (1–34)(Immutopics, San Clemente, CA).

Placental calcium transport assay

Pregnant mice at 17.5 dpc (days after conception) were injected withmixture of 50 �Ci 45CaCl2 and 50 �Ci 51Cr-EDTA by intracardiac injec-tion as described (17). At 5 min after injection, each embryo was removedfrom its amniotic sac by cesarean section, weighed, and then placed intoa capped test tube. After measurement of 51Cr activity by a �-counter(Packard Instrument Co., Meriden, CT), the embryos were subsequentlysolubilized with Scintigest at 60 C for 24–48 h and vortexed briefly. 45Caactivity was then counted using a liquid scintillation counter (Beckman,Inc., Fullerton, CA) after equilibration for12–24 h at room temperature.The ratio of 45Ca/51Cr activity was calculated for each fetus. Each valueof 45Ca and 51Cr activities was normalized to the body weight. Tocompare the activity among groups, the mean ratio for heterozygotes ofeach litter was set at 100%.

Calcium release assay

Pregnant mice were injected with 50 �Ci 45CaCl2 sc at d 16.5 dpc. Twodays later the calvarial bones were isolated from each embryo anddivided into four pieces. They were preincubated in 0.5 ml DMEM mediafor 1 d and then subsequently incubated for 3 d with 10�7 m hPTH (1–34)or 20 ng/ml soluble RANKL (sRANKL) (PeproTech, Rocky Hill, NJ). Thebones were dissolved in 2 N HCl, and then aliquots of media and boneswere analyzed for radioactivity by liquid scintillation. The extent of bone

resorption was evaluated by the release of 45Ca from bone to the mediumand expressed as a percentage of initial radioactivity present in bones.

Skeletal preparation and histologic analysis

Skeletons were prepared and stained with Alcian blue and alizarinred as described (18). The tissue of embryos from E15.5 to E18.5 wereharvested and fixed in 10% formalin for routine light microscopy or 2%paraformaldehyde�2% glutaraldehyde solution for electron micros-copy at 4 C for 24 h. Undemineralized paraffin and Epon blocks wereprepared by standard histological procedures. The selected paraffin-embedded sections were stained with hematoxylin and eosin or by thevon Kossa method, and for alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) enzymatic activity, respectively, asdescribed (15). The collected sections for electron microscopy werestained with uranyl acetate and lead citrate and then observed with anH-800 (Hitachi, Tokyo, Japan) electron microscope at 75 Kv. In situhybridization for �1(I) collagen, ALP, and osteocalcin was performedusing 35S RNA probes using standard protocols (19).

Histomorphometry

Whole hind limbs from wild-type and gp130�/� embryos at E18.5were fixed in 3.7% formaldehyde in PBS and embedded in methyl-methacrylate. The undemineralized 5-�m sections stained with tolu-idine blue were analyzed by standard histomorphometric procedures(20) using the Osteomeasure system (OsteoMetrics, Inc., Atlanta, GA).Because there is no trabecular bone in the diaphysis of the gp130�/�mice, histomorphometry was carried out at the diaphysis on the en-docortical surfaces and in a metaphyseal area (total area of 340 �m2) atthe base of the growth plate, including both primary and secondaryspongiosa. Three to four sections from each tibia were measured froma total of four to five embryos per group

PTH: gp130 double-mutant analysis and TRAP staining

PTH� mice (21) were crossed to gp130� to obtain PTH�;gp130�double heterozygous mice. Genotyping for the mutant PTH alleles wasperformed by PCR using primers; common 5�-AAGATGATGTCTG-CAAACACCGTGG-3�, Wt-specific 5�-GGTGTTTGCCCAGGTTGTG-CATAA-3�, and Mut-specific 5�-TCCAGACTGCCTTGGGAAAA-GCGC-3�. The wild-type PTH allele generates a 250-bp-long PCR prod-uct, and the PTH null allele generates a 200-bp-long PCR product.Double-mutant mice were fertile and indistinguishable from wild-typelittermates. Double-mutant mice were subsequently intercrossed to ob-tain PTH�/�;gp130�/� double-nullizygous embryos. Embryos wererecovered at E18.5 by cesarean section. From 12 litters, total three doublemutants were obtained. Comparison was performed using samples fromlittermates.

Induction of TRAP-positive (TRAP�) multinuclearosteoclastic cells

Primary calvarial osteoblastic cells (2 � 104 cells/well) harvestedfrom wild-type and gp130�/� embryos at E18.5 by serial digestion usinga mixture of 0.1% collagenase I and II were cocultured with wild-typespleen cells (5 � 105 cells/well) from 6- to 7-wk-old CD-1 male mice in0.5ml �-MEM supplemented with 10% fetal bovine serum (FBS) using48-well hydroxyapatite-coated plates (OAAS kit, Oscotec, Chenan, Ko-rea). The cells were treated with 10�8 m dexamethasone and 10�7 mhPTH (1–34) or 20 ng/ml sRANKL or 1,25(OH) 2 vitamin D3 for 2 wk.All cultures were replaced by a half-change of fresh medium every 3 d.The cells were then fixed in 10% formalin and stained for TRAP. TRAP�multinucleated cells containing more than three nuclei were countedfrom at least three wells, and the data were represented as mean � se.

Pit formation assay

The cells prepared for study of TRAP� multinuclear osteoclastic cellformation were also cultured on dentine slices in 200 �l media supple-ment with 10�8 m dexamethasone and either 10�7 m hPTH (1–34) or 20ng/ml sRANKL for 2 wk using 98-well plates. After 2 wk of culture, thepits on dentin slices were visualized with 1% toluidine blue after re-

Shin et al. • gp130 Signaling in Osteoblasts in Vivo Endocrinology, March 2004, 145(3):1376–1385 1377

moving attached cells with 1 m NH4OH. The number of pits on six dentinslices was counted, and the data were expressed as the average numberof pits on each dentin slice.

Nodule mineralization assay

Primary calvarial osteoblasts harvested from wild-type and gp130�/�

embryos at E18.5 were plated in 24-well multiplates and cultured for 3wk with mineralizing media (�-MEM supplemented with 10% FBS, 10mm �-glycerol phosphate, 50 �g/ml ascorbic acid, and 10�7 m dexa-methasone). Media were replaced every 3 d. At the end of incubation,mineralization was detected with alizarin red staining.

ALP activity assay

ALP activity was evaluated histochemically and biochemically, usingthe Sigma kits n.85 and 104-LS, respectively, according to the manu-facturer’s instructions (Sigma-Aldrich, St. Louis, MO).

Northern blot hybridization

Total RNA was extracted from cultured primary calvarial osteoblasts,which were harvested from WT and gp130�/� embryos at E18, andcultured for 0.5, 3, 6, 12, and 24 h, respectively, in the presence of 10�7

m hPTH (1–34), using Trizol solution (Life Technologies, Inc., GlandIsland, NY) and quantitated by spectrophotometry. Ten micrograms ofRNA was separated by electrophoresis on a 1.5% formaldehyde agarosegel and transferred to Nytran nylon membrane (Schleicher & Schuell,Keene, NH) by capillary blotting. The 32P-labeled probes for RANKL,osteoprotegerin (OPG), macrophage colony-stimulating factor (M-CSF),and glyceraldehyde-3-phosphate dehydrogenase were prepared using arandom primer DNA labeling kit (Amersham, Arlington, IL). Prehy-bridization and hybridization were carried out using Express Hyb so-lution (Clontech, Palo Alto, CA). After washing with 2 � saline sodiumcitrate, 0.1% sodium dodecyl sulfate at room temperature and then with0.1 � saline sodium citrate, 0.1% sodium dodecyl sulfate at 55 C, mem-branes were subjected to autoradiography at �70 C. The experiment wasperformed three times, with similar results.

Statistics

The experimental data are expressed as mean � se of at least threeindependent experiments. The significance of differences was analyzedby Tukey’s multiple range tests after ANOVA. P � 0.05 was conven-tionally considered statistically significant.

ResultsAbnormal bone development in gp130�/� mice

To determine the development of the abnormalities in theskeleton at birth noted by Kawasaki et al. (15), we examinedboth gp130�/� fetuses and wild-type littermates at earliertimes. gp130�/� embryos developed to term but died within1 d after birth for unknown reasons as previously reported(14, 15). On gross examination, the fetuses appeared normalexcept that they were smaller and had shorter limbs thanthose of heterozygous and wild-type littermates. Develop-ment of epiphyseal cartilage and its mineralization was nor-mal in the gp130�/� mice at all time points. In stained skeletalpreparation, the gp130�/� embryos at E18.5 had small skel-etons and characteristic bending of most limb elements (Fig.1A). In the tibia, vascular invasion into the cartilage corethrough the bone collar was noted in both genotypes at E15.5(Fig. 1B). However, at E16.5 (Fig. 1C, the wild-type tibiaexhibited a well-formed marrow space and diaphysis withscattered thin spicules of primary trabecular bone, whereasequivalent maturation was seen in gp130�/� tibiae only atE17.5 (Fig. 1D). In addition to this delay in bone develop-ment, the gp130�/� mice exhibited poor development of the

primary spongiosa and eccentric thickening of the diaphy-seal cortex. At E18.5 the delay in development of thegp130�/� tibia continued to be apparent, with a relativepaucity of hematopoietic marrow when compared withwild-type littermates (shown more clearly in hematoxylinand eosin sections (data not shown).

Osteoblast abnormalities in gp130�/� mice

To understand better the cellular underpinning of the de-crease in trabecular bone in the gp130�/� fetuses, histomor-phometric analysis was performed on E18.5 gp130�/� miceand their WT littermates. Our findings confirmed and extendthose of Kawasaki et al. (15). Osteoblast number within themetaphyseal area and percentage of osteoblast surface perunit bone surface were significantly reduced in gp130�/�

tibiae (data not shown). Furthermore, the expression of ALPand collagen type I mRNAs was markedly decreased in thegp130�/� fetal tibia at E18.5, but there was only slight de-crease in osteocalcin mRNA expression (Fig. 2A). Like theALP mRNA levels, the level of ALP enzyme activity wasreduced in gp130�/� fetuses, compared with wild type (Fig.2B). Because mineralization of bone was grossly normal (Fig.1), we presumed that the abnormality of ALP expression wasnot sufficient to interfere dramatically with mineralization.To determine whether the abnormalities in osteoblasts notedin vivo were cell autonomous or instead depended on signalsfrom outside the bone microenvironment that might be dis-turbed in gp130�/� mice, primary calvarial osteoblasts wereharvested by serial digestion of calvarial bone from gp130 �/�

embryos at E18.5 and cultured for 2 wk. (Calvariae havenormally mineralized bone at this stage both in wild-typeand gp130�/� embryos.) These cells also revealed lower en-zyme activity, compared with those from wild-type litter-mates (Fig. 2, C and E). Furthermore, the primary calvarialosteoblasts from gp130�/� embryos did not induce noduleformation when cultured for 3 wk with mineralizing media,whereas the cells from wild-type littermates induced nu-merous well-mineralized nodules (Fig. 2D). This strikingabnormality may correlate with the decrease in osteoblastnumber or osteoblast differentiation seen in vivo, but theprecise in vivo correlate of these in vitro findings is difficultto define. These findings demonstrate that osteoblasts fromgp130�/� mice have substantial abnormalities of differenti-ated function.

Osteoclast structure and function

Because activation of gp130 stimulates osteoclastogenesisin vitro, the initial observation (15) that gp130�/� mice havean increase in osteoclast number was surprising. In histo-morphometric analysis of tibia from wild-type and gp130�/�

embryos at E18.5, we confirmed that the osteoclasts numberand percentage of osteoclast surface per bone surface weresignificantly increased in gp130�/� at both the metaphysisand diaphysis (data not shown). The TRAP� multinucleatedcells (MNCs) in the gp130�/� tibia concentrated along thelower border of the growth plate were characteristically largeand ovoid in shape with multinucleation. Only a small num-ber of TRAP� MNCs was noted associated with the poorlydeveloped primary spongiosa, whereas the evenly distrib-

1378 Endocrinology, March 2004, 145(3):1376–1385 Shin et al. • gp130 Signaling in Osteoblasts in Vivo

uted wild-type TRAP� MNCs along the surface of primaryspongiosa revealed small and flat shape with scant cyto-plasm, which was clear at high magnification (Fig. 3A). Ul-trastructurally, the osteoclasts from both wild-type andgp130�/� mice showed normal signs of activity, with clearzones, cytoplasmic vacuolization, ruffled borders, and ex-tended cytoplasmic processes embracing the mineralizedcartilage matrix. However, the osteoclasts in gp130�/� tibia,although characteristically larger than normal with abun-dant cytoplasmic organelles and numerous nuclei, exhibitedpoorly developed ruffled borders. Whereas osteoclasts fromwild-type fetuses exhibited ruffled borders with numerouselongated villi, osteoclasts from gp130�/� fetuses exhibitedruffled borders that were thicker, blunter and fewer in num-ber (Fig. 3B). The decreased number of cytoplasmic processesin the ruffled border was confirmed by histomorphologicanalysis of osteoclasts located in both the metaphyseal anddiaphyseal regions (Fig. 3C). Because the ruffled border is theresorptive organelle of the osteoclast, these observations sug-gest that the osteoclasts of the gp130�/� fetuses may bedysfunctional. Because we could not detect any specific signsof osteopetrosis, such as retained cartilage remnants in thesemice, the functional implications of this morphological ab-normality remain to be defined.

Secondary hyperparathyroidism in gp130�/� mice

These abnormalities in osteoclast architecture led us tomeasure indices of calcium metabolism in the gp130�/� mice.The ionized calcium of gp130�/� fetuses at E18.5 (1.44 � 0.03mol/liter, n � 12) was significantly lower than that of theirheterozygous and wild-type littermates (1.73 � 0.02 mmol/liter, n � 33 and 1.77 � 0.03 mmol/liter, n � 15, respectively)(P � 0.001 vs. heterozygous and wild type). However, bloodcalcium in the homozygotes was still higher than in the dams(1.35 � 0.03 mmol/liter, n � 4). In addition, the blood PTHlevel was strikingly increased in gp130�/� embryos (239 �5.3 pg/ml, n � 4) at E18.5, compared with heterozygous(41.4 � 5.3 pg/ml, n � 4) and wild-type (20.9 � 5.3 pg/ml,n � 4) littermates (P � 0.001 vs. heterozygous and wild type).

Placental calcium transport in gp130�/� mice

These abnormalities in calcium and PTH levels might re-flect abnormal bone metabolism but might also reflect ab-normal transport of calcium across the placenta. To assess theefficiency of placental Ca2� transfer, the transfer of 45Caacross the placenta was measured as a 45Ca/51Cr activityratio to control for variation in blood flow across the placenta(17). At E17.5, gp130�/� embryos showed a significantly

FIG. 1. Abnormal bone development in gp130�/� mice. A, Alcian blue/Alizarin red skeletal staining. gp130�/� embryo at E18.5 exhibitsgeneralized reduction of skeleton size and characteristic bending of long bone elements of limbs. The von Kossa staining of tibiae from wild-typeand gp130�/� fetus at E15.5 (B), E16.5 (C), and E17.5 (D). The von Kossa staining illustrates the poorly developed primary spongiosa andeccentric mineralized cortex in gp130�/� fetal tibiae (original magnification, �100). E, Osteoblast number per unit total tibial metaphyseal area(ObN/TAr). F, Percentage of osteoblast surface per unit bone surface (ObS/BS) are both lower in gp130�/� mice, compared with wild-typelittermates. Bars represent mean � SE from least three tibial sections from four to five mice of each genotype. *, P � 0.05 vs. wild type.

Shin et al. • gp130 Signaling in Osteoblasts in Vivo Endocrinology, March 2004, 145(3):1376–1385 1379

higher accumulated 45Ca/51Cr activity ratio determined 5min after maternal administration of the isotopes than nor-mal littermates. The high ratio was caused by reduced ac-cumulation of 51Cr activity in gp130�/� embryos rather thanincreased accumulation of 45Ca activity. The mean value ofaccumulated 51Cr activity in gp130�/� embryos (1095.7 �268.3 cpm, P � 0.05 vs. wild type and heterozygous, n � 3)was significantly reduced, compared with wild-type andheterozygous littermates (2629.4 � 268.3 cpm, n � 3, and2081.6 � 140.1 cpm, n � 11, respectively), whereas there wereno significant differences in the mean value of accumulated45Ca activity among the groups (wild type, 9740.1 � 1333.7cpm, gp130�/�, 9392.2 � 696.5 cpm and gp130�/�, 7523.7 �1333.7 cpm, respectively) (Fig. 4A). The values for 51Cr trans-fer were proportional to the body weights of each group offetuses (data not shown). When compared with the meanvalue for heterozygous mice, which was set at 100%, the

mean value of accumulated 45Ca/51Cr activity ratio ingp130�/� and wild-type was 148% and 83%, respectively(P � 0.001 vs. wt). This result suggests that the blood flowfrom dam to fetus (51Cr transfer) was reduced in gp130�/�

and that the efficiency of placental Ca2� transfer for theamount of blood flow may be increased (Fig. 4B). The pla-centas of gp130�/� fetuses were smaller than those of wild-type littermates, but there was no remarkable histopatho-logic change. Furthermore, there were no remarkabledifferences in placental expression of Ca2� binding proteinmRNA and PTH/PTHrP receptor mRNA between wild-typeand gp130�/� embryos at E17.5 (data not shown). Thesestudies suggest that the modest (not statistically significant)decrease in calcium transport in gp130�/� can be explainedby lower body and placental size of gp130�/� fetuses andcannot explain the lower blood calcium levels in thegp130�/� than in wild-type littermates.

FIG. 2. Reduced osteoblastic activity in gp130�/� mice. A, In situ hybridization for collagen type I (Col I), ALP, and osteocalcin (OC) mRNAsin wild-type (upper panel) and gp130�/� (lower panel) fetal tibiae at E18.5. The expression of ALP and Col I mRNAs was markedly decreased,but OC mRNA was only slightly decreased in the gp130�/� tibia(original magnification �40). B, Histochemistry for ALP activity in wild-typeand gp130�/� tibia at E18.5. ALP activity stains brown. C, Cytochemistry for ALP in 2-wk cultured wild-type and gp130�/� primary calvarialosteoblastic cells. D, Alizarin red staining for mineralized nodules in primary calvarial osteoblasts from wild-type and gp130�/� fetuses culturedfor 3 wk with �-MEM supplemented with 10% FBS, 10 mM �-glycerophosphate, 50 �g/ml ascorbic acid, and 10�7 M dexamethasone. E, ALPactivity in cell extracts from primary calvarial osteoblastic cells from E18.5 fetuses after culturing for 2 wk. The assay colorimetrically measuresthe conversion of p-nitrophenyl phosphate to p-nitrophenyl (NaPNP) plus inorganic phosphate. *, P � 0.05.

1380 Endocrinology, March 2004, 145(3):1376–1385 Shin et al. • gp130 Signaling in Osteoblasts in Vivo

Osteoclast number and PTH

The dramatic increase in PTH levels in the gp130�/� micepresumably reflects a state of secondary hyperparathyroid-ism in response to the relative fall in blood calcium in thesemice. This hyperparathyroidism might provide an explana-tion for the increase in osteoclasts noted in these mice. Toexplore this possibility, PTH�/�; gp130�/� double-heterozy-gote mice were mated with each other, and the tibiae of theresultant fetuses were examined after TRAP staining. Figure5 shows that the number of osteoclasts in PTH�/�; gp130�/�

fetuses were similar to that in PTH�/�; gp130�/� mice.Therefore, hyperparathyroidism cannot explain the in-crease in osteoclast number in the gp130�/� mice; wecannot rule out modest effects of PTH on osteoclast num-ber, however.

Resorptive activity of osteoclasts in response to PTHand sRANKL

To assess osteoclast function in the context of intactbony structures, E18.5 calvariae, prelabeled with 45Ca invivo, were cultured, and release of 45Ca was assessed. Basal

rates of 45Ca release did not differ among genotypes. How-ever, the 45Ca release in response to 10�7 m PTH (1–34) wassignificantly increased in wild type and heterozygotes,compared with basal rates, whereas there was no signif-icant increase in gp130�/� mice (Fig. 6). The responses ofwild-type and heterozygote fetuses in response to PTH didnot differ significantly, but their responses were both sig-nificantly greater than that of gp130�/� fetuses. Becauseboth PTH/PTHrP receptor and gp130 activation lead toproduction of RANKL (13) and this production might bedefective in gp130�/� fetuses, we determined the responseof the calvariae to added sRANKL. SRANKL was tested at10, 20, and 100 ng/ml, with a dose-dependent increase inresponse with dose using WT calvariae (data not shown).The 45Ca release in response to 20 ng/ml sRANKL isshown in Fig. 6 and was significantly increased over itsbasal rate in wild-type and heterozygote but not ingp130�/� fetuses. Because the response to sRANKL wasnot normal in the gp130�/� calvariae, the interpretation ofthe defective response to PTH in gp130�/� is complicated.We cannot conclude that defective production of RANKL

FIG. 3. gp130�/� osteoclasts in vivo are increased in number with abnormal morphology. A, TRAP staining of tibia from wild-type and gp130�/�

fetuses at E17.5 (original magnification, �100). The characteristically large ovoid multinucleated gp130�/� TRAP� MNCs are concentratedat the lower border of the growth plate, whereas the evenly distributed wild-type TRAP� MNCs along the surface of primary spongiosa aresmall and flat, seen best at high magnification (inset, original magnification, �400). B, Transmission electron micrographs of in vivo osteoclastsfrom wild-type and gp130�/� fetuses at E18.5 reveal clear zone (*), ruffled border (arrows), cytoplasmic vacuolization (v), and embracedmineralized matrix (m). Note the increased osteoclast size with numerous nuclei (N) and abundant cytoplasm but less developed ruffled borderin gp130�/� osteoclast. Bar, 0.5 �m. C, Both epiphyseal and diaphyseal osteoclasts in the gp130�/� fetal tibia have a significantly decreasednumber of cytoplasmic folds in their ruffled border, compared with wild-type osteoclasts (*, P � 0.005, n � 10). The bars represent mean � SE.

FIG. 4. Increased placental calciumtransport in gp130�/� fetuses. A, The ac-cumulated 51Cr and 45Ca activities. B,The mean 45Ca/51Cr activity. The ratio of45Ca/51Cr activity accumulated in fe-tuses was determined 5 min after ma-ternal administration of 51Cr-EDTA and45CaCl2. The mean heterozygote 45Ca/51Cr ratio of each litter was set at 100%to allow the results of multiple litters tobe compared. *, P � 0.05 vs. wild type(Wt) and heterozygous (Het). **, P �0.001 vs. Wt and Het).

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in response to PTH is the sole cause of the decreased Ca45

release from the gp130 �/� calvariae. It is possible that, inaddition, gp130 signaling is required in osteoclasts, thatM-CSF responses to PTH are defective in calvariae ofgp130�/� mice or that some other aspect of the PTH re-sponse is defective in gp130�/� calvariae, in addition to apossible defect in RANKL generation (see below).

Osteoclastogenesis in vitro

To clarify the roles of individual cell types in the bluntedresponses to PTH just noted, primary calvarial osteoblastsand spleen cells from varying genotypes were coculturedand the stimulation of osteoclastogenesis was assessed.Adult wild-type spleen cells were cultured with either wild-type or gp130�/� primary calvarial E18.5 osteoblasts for 2wk. Wild-type primary calvarial osteoblastic cells inducedlarge number of TRAP� osteoclastic cells, whereas the pri-mary calvarial osteoblasts from gp130�/� embryos did not

induce TRAP� multinucleated cells in the presence of 10�7

m hPTH (1–34). In contrast, 10�8 m 1,25(OH)2D3 and 20ng/ml sRANKL treatment of these cells induced large num-bers of TRAP� osteoclastic cells. Even with these effectivestimuli, however, the gp130�/� osteoblasts did not behavenormally. The number of osteoclasts produced in the pres-ence of gp130�/� osteoblasts was significantly decreased,compared with that of wild type (Fig. 7, A and B). This resultsuggests that PTH activation of osteoclastogenesis dependsmore on gp130 signaling than activation of osteoclastogen-esis by 1,25(OH)2D3 or RANKL. The less than normal re-sponse to 1,25(OH)2D3 suggests the possibility that theRANKL response to 1,25(OH)2D3 may not be entirely normalin gp130 �/� calvariae. The less-than-normal response toRANKL suggests that the calvarial cells may make excessOPG or produce less M-CSF than normal.

The osteoclasts formed in response to RANKL were ableto form pits when grown in the presence of wild-type cal-varial osteoblasts but not when grown in the presence ofgp130�/� osteoblasts (Fig. 7, C and D). Whereas we cannotexplain this result on a molecular level, it emphasizes thatgp130�/� calvarial cells have abnormal properties beyondtheir responses to PTH.

To determine why the response of gp130�/� osteoblaststo PTH was so dramatically blunted, the levels of expres-sion of RANKL, OPG, M-CSF, and PTH receptor mRNA incultured calvarial osteoblasts were analyzed. OPG bindsRANKL and blocks the activation of RANK by RANKL.In WT calvarial osteoblasts, RANKL mRNA levels inresponse to 10�7 m hPTH (1–34) were markedly up-regulated after 3 h treatment with no consistent change inlevels of OPG, M-CSF, or PTH receptor mRNA expression.In contrast, the gp130�/� primary calvarial osteoblasticcells showed only a modest increase in RANKL mRNAlevels after PTH and no change in OPG, M-CSF, or PTHreceptor mRNA levels in response to PTH. These resultssuggest that a poor RANKL mRNA response to PTH maycontribute to the poor osteoclastic response to PTH, at leastin vitro (Fig. 8). We cannot eliminate the possibility thatchanges at the protein level occur as well, but attempts tomeasure these parameters were unsuccessful, presumablybecause of the small number of available cells.

FIG. 5. Effect of PTH on osteoclasts ofgp130�/� tibiae. Mice doubly heterozy-gous for knockout of PTH and gp130were mated, and tibiae, right limb radii,and right limb ulnae from progeny wereexamined by TRAP staining at E18.5.Bones from two doubly homozygousmice were compared with those of twogp130�/� littermates. A third doublyhomozygous mouse was compared witha gp130�/� mouse of the same age fromanother litter. Three or more sections ofeach bone were examined. The tibiaeshown here are representative of the 12bones of each genotype examined. Ge-notypes are indicated above each sec-tion.

FIG. 6. Resorptive activity of gp130�/� osteoclasts. The extent of boneresorption was evaluated by the release of prelabeled 45Ca from boneto the medium, expressed as a percentage of initial radioactivitypresent in bones. After sc injection of 45CaCl2 at 16.5 dpc, calvarialbones taken from gp130�/� and wild-type or heterozygous littermatesat E18.5 were cultured in the presence or absence of 10�7 M hPTH(1–34) or 20 ng/ml sRANKL for 3 d. Bars, mean � SE (*, P � 0.001 vs.basal; **, P � 0.05 vs. basal).

1382 Endocrinology, March 2004, 145(3):1376–1385 Shin et al. • gp130 Signaling in Osteoblasts in Vivo

Discussion

These studies establish that loss of signaling by gp130results in multiple abnormalities in osteoblast function. Thedevelopmental sequence demonstrates a general delay inbone development, with apparently normal growth platesbut dramatically decreased amounts of trabecular bone. Insitu hybridization demonstrates a decrease in expression ofcollagen �1(I) mRNA and ALP mRNA as well as decreasedALP activity in bone. Histomorphometric analysis showed adecrease in the number of osteoblasts in trabecular bone aswell. These studies of intact bone are complemented by anal-ysis of calvarial osteoblasts cultured in vitro. These cells, after2 wk in culture, have a substantially lower level of ALPactivity and fail to form the mineralized nodules formed bytheir WT counterparts under the same conditions. Thus, inthe absence of gp130 signaling, osteoblasts reveal a cell-autonomous defect in differentiation. These findings are con-sistent with previous studies showing important effects ofactivation of gp130 for osteoblast development and differ-entiation in vitro (1, 6, 7). We cannot explain the relativesparing of development of cortical bone, compared withtrabecular bone, because the molecular and in vitro analyseswould predict similar defects in both bone compartments.Presumably, some other aspect of gp130 signaling explains

the dramatic loss of trabecular bone. Perhaps gp130 signalinginfluences the entry of osteoblast precursors into the marrowcompartment. Alternatively, an action of gp130, such as thedecreased apoptosis of osteoblast-like cells seen in vitro (22),may be more active in the microenvironment of trabecularbone.

The relative hypocalcemia and secondary hyperparathy-roidism of the gp130�/� mice shows that gp130 signalingcontributes to normal fetal calcium homeostasis. Activetransport of calcium across the placenta contributes impor-tantly to fetal calcium metabolism (23), so this was assessedby measuring 45Ca transport across the placenta, with nor-malization both for placental blood flow and fetal weight.Total calcium transport into gp130�/� fetuses was increased,when normalized for the decrease in placental blood flowmeasured with 51Cr transport or for fetal weight. These find-ings suggest that reduced placental calcium transport cannotexplain the lower blood calcium in the gp130�/� mice. Renalcalcium handling was not measured because such measure-ments are difficult in the fetus. Because the fetal urine isswallowed as amniotic fluid and absorbed by the fetal in-testine, it is unlikely that abnormalities of renal calcium han-dling contribute importantly to the fetal hypocalcemia of thegp130�/� mice. Therefore, we considered possible abnor-

FIG. 7. Osteoclast induction in vitro. A, TRAP� MNCs were induced by cocultures of adult spleen cells and primary calvarial osteoblastic cellsfrom wild-type or gp130�/� fetuses at E18.5 in the presence of 10�7 [scap]m hPTH (1–34), 20 ng/ml sRANKL or 10�8 M 1,25(OH)2 vitamin D3for 2 wk. B, Number of TRAP� MNCs induced in vitro. In vitro induction of TRAP� MNCs by PTH was dramatically blocked and significantlyreduced after induction by both sRANKL and 1,25(OH)2 vitamin D3. The TRAP� cells containing more than three nuclei were counted (*, P �0.001; **, P � 0.05 vs. wild type). No TRAP� cells were found in control wells without inducers. Bars, mean � SE (n � 4). C, Formation ofresorption pits by induced TRAP� MNCs. Primary calvarial osteoblastic cells from gp130�/� and gp130�/� embryos at E18.5 were coculturedwith wild-type spleen cells in the presence of 10�7 M hPTH (1–34) or 20 ng/ml sRANKL for 2 wk on dentin slices in 96-well plates. The pitswere visualized by 1% toluidine staining after removal of attached cells (original magnification, �100). D, Resorption activity of induced TRAP�MNCs by PTH and sRANKL treatment. The number of resorption pits counted on six dentin slices is represented as mean � SE.

Shin et al. • gp130 Signaling in Osteoblasts in Vivo Endocrinology, March 2004, 145(3):1376–1385 1383

malities of bone resorption as a contributor to the relativehypocalcemia.

Although the number of osteoclasts was increased in thegp130�/� bones, these osteoclasts had poorly developed ruf-fled border regions in comparison with their normal coun-terparts. In contrast to the numerous elongated villi in ruffledborder of wild-type osteoclasts, the gp130�/� osteoclasts hadthickened and blunted villi, with marked reduction in num-ber in their ruffled borders. Because the ruffled border is thesite of active bone resorption by osteoclasts, this findingsuggests that the effectiveness of individual osteoclasts maybe diminished. Defective bone resorption was demonstrateddirectly by stimulating calvarial bone resorption in vitro us-ing either PTH or sRANKL. The response of gp130�/� cal-variae to PTH was less than the response of WT calvariae,whereas the response of gp130�/� to sRANKL did not differfrom the basal response. The defective response to PTH couldcertainly explain the relative hypocalcemia in the gp130�/�

mice in the face of hyperparathyroidism. The modestly lowlevel of blood calcium in the gp130�/� fetuses contrasts withthe dramatically lower blood calcium reported in mice nullfor the PTH/PTHrP receptor gene. Those mice have bloodcalcium levels considerably lower than those of their dams(17). Although conclusions can only be tentative because thegenetic background of the PTH/PTHrP receptor�/� mice dif-fered from that of the gp130�/� mice, these results suggestthat the gp130�/� mice respond to PTH but in a bluntedfashion.

To explore further the osteoclast defect in the gp130�/�

mice, the ability of calvarial cells from these mice to supportosteoclastogenesis, when cocultured with osteoclast precur-sors (from adult spleen) from WT mice, was examined. Theresponse to 1,25(OH)2D3 and sRANKL was blunted, and theresponse to PTH was virtually absent. The latter results con-firm and extend previous in vitro studies in which antibodiesto either gp130 (8) or the IL-6 receptor (10) decreased oste-

oclastogenesis after PTH administration. Because osteoblas-tic cells stimulate osteoclastogenesis largely through the pro-duction of RANKL and M-CSF (24), with constraint of theaction of RANKL by production of OPG (25), we examinedthe effect of PTH on the levels of mRNA encoding RANKL,OPG, and M-CSF in osteoblasts from gp130�/� mice. In re-sponse to PTH, WT osteoblasts increased their levels ofRANKL mRNA, as previously reported (26), but thegp130�/� osteoblasts had a considerably weaker response. Incontrast, no substantial changes in OPG or M-CSF mRNAlevels occurred in response to PTH in either WT or gp130�/�

osteoblasts. Furthermore, the WT and gp130�/� osteoblastsexpressed similar levels of PTH receptor mRNA levels, andthese levels did not change after PTH administration. Wecannot rule out possible changes in protein levels or at latertimes. Thus, the abnormalities in supporting osteoclastogen-esis in gp130�/� osteoblasts in vitro probably result, at leastin part, from abnormalities in RANKL mRNA expression.

These findings, of course, fail to explain the actual increasein osteoclast number in the gp130�/� mice. We wonderedwhether this increase might result from the dramatic increasein PTH levels in these mice. However, when PTH was re-moved through appropriate matings with mice missing thePTH gene, the osteoclast number was not substantiallychanged. These findings are, perhaps not surprising becausemice missing the PTH/PTHrP receptor have normal num-bers of osteoclasts, despite their profound hypocalcemia.Presumably, osteoclast development in fetal life is primarilyregulated by signals other than those activated by the PTH/PTHrP receptor or gp130.

These studies suggest that the activity of the osteoclasts inthe gp130�/� mice may be diminished and may explain therelative hypocalcemia in these mice. Because we were unableto develop quantitatively dependable assays for RANKL,M-CSF, and OPG mRNA or protein suitable for use on thebones of fetal gp130�/� mice, we cannot determine whetherabnormalities in these genes occurs in vivo in the gp130�/�

mice (data not shown). Previous studies have shown thatactivation of gp130 on osteoblast-like cells increases oste-oclastogenesis by increasing RANKL production (13).RANKL also stimulates the activity of mature osteoclasts(27). Therefore, the defect in RANKL mRNA production byosteoblasts from gp130�/� mice suggests that a defect inRANKL production may contribute to the abnormal oste-oclast function in the gp130�/� mice. Furthermore, a smallnumber of studies suggest that gp130 on mature osteoclastsmay participate more directly in osteoclast function. Ad-ebanjo et al. (9) showed that IL-6 reverses the inhibition ofbone resorption induced by high extracellular calcium andCappellen et al. (5) showed that IL-6 and IL-11, when addedalong with IL-1 to osteoclasts generated in vitro, increasedbone resorbing activity (pit area) by these osteoclasts. Thus,it is possible that activation of gp130 on osteoclasts mayincrease their bone resorbing properties.

These studies thus establish that bones develop abnor-mally in the absence of gp130 signaling. Osteoblasts arefewer in number in trabecular bone and exhibit widespreadabnormalities of differentiated function. Although osteoclastnumbers are increased, the osteoclasts exhibit abnormal mor-

FIG. 8. Northern blot analysis for RANKL, OPG, M-CSF, PTH re-ceptor 1, and glyceraldehyde-3-phosphate dehydrogenase. TotalRNAs from cultured primary calvarial osteoblastic cells were ex-tracted from WT and gp130�/� embryos at E18.5 after treatment with10�7 M hPTH (1–34) for 3, 6, 12, and 24 h. Ten micrograms of RNAwere electrophoresed, transferred to nylon membrane, and hybrid-ized with 32P-labeled probes.

1384 Endocrinology, March 2004, 145(3):1376–1385 Shin et al. • gp130 Signaling in Osteoblasts in Vivo

phology, and decreased bone resorption contributes to rel-ative hypocalcemia and hyperparathyroidism in these mice.

Acknowledgments

The authors thank Drs. Tetsuya Taga, Kanji Yoshida, and TadamitsuKishimoto for generously providing the gp130 knockout mice for thesestudies.

Received July 7, 2003. Accepted November 4, 2003.Address all correspondence and requests for reprints to: Henry Kro-

nenberg, Endocrine Unit, Massachusetts General Hospital, 50 BlossomStreet, Boston, Massachusetts 02114. E-mail: kronenberg.henry@mgh.harvard.edu.

This work was supported by NIH Grant DK56246 and a grant fromthe Korea Health 21 R&D project, Ministry of Health and Welfare,Republic of Korea (01-PJ3-PG6-01GN11-0002).

Current addresses for H.-I.S.: Department of Oral Pathology, Schoolof Dentistry, Kyungpook National, University, 101 Dongin 2-Ga, Jung-Gu, Daegu 700-422, Korea.

Current address for N.A.S.: St. Vincent’s Institute of Medical Researchand Department of Medicine, St. Vincent’s Hospital Melbourne, Uni-versity of Melbourne, Fitzroy, Australia.

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