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Endocrinology, doi:10.1210/en.2003-0839
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Endocrinology Vol. 145, No. 3 1376-1385
Copyright © 2004 by The Endocrine Society

gp130-Mediated Signaling Is Necessary for Normal Osteoblastic 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 of Medicine, McGill University, Montréal, Québec, Canada H3A 1A1; and Lady Davis Research Institute (A.C.K.), Sir Mortimer B. Davis Jewish General Hospital, and Department of Medicine, McGill University, Montréal, Québec, Canada H3T 1A1

Address all correspondence and requests for reprints to: Henry Kronenberg, Endocrine Unit, Massachusetts General Hospital, 50 Blossom Street, Boston, Massachusetts 02114. E-mail: kronenberg.henry{at}mgh.harvard.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Previous studies have shown that mice missing gp130, the common receptor subunit for many cytokines, die at or before birth with multiple skeletal abnormalities. Furthermore, interactions between PTH and gp130 signaling have suggested that gp130 signaling might influence calcium homeostasis. We, therefore, examined the function of osteoblasts, osteoclasts, and calcium homeostasis in gp130-/- mice, both in vivo and in vitro. Osteoblasts from these mice exhibit widespread abnormalities, including decreased alkaline phosphatase mRNA and protein, both in vivo and in osteoblast cultures. Although osteoclast number is increased in gp130-/- fetuses, these osteoclasts exhibit abnormalities in the resorptive organelle and the ruffled border, and the mice are mildly hypocalcemic. Although the hypocalcemia is associated with secondary hyperparathyroidism, the increase in PTH does not explain the increase in osteoclast number because removal of the PTH gene in gp130-/- fetuses does not importantly change osteoclast number. Calvarial bone resorption in response to PTH is defective, as is the ability of osteoblastic cells from gp130-/- mice to stimulate osteoclastogenesis from normal precursors in vitro or to increase receptor activator of nuclear factor-{kappa}B ligand mRNA levels after exposure to PTH. These studies demonstrate the importance of gp130 signaling for osteoblast function and calcium homeostasis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
BONE CELLS RESPOND both to local cytokines and systemic hormones to accomplish their dual roles of maintaining the skeleton’s structural integrity and facilitating mineral ion homeostasis. The cytokine family that uses gp130 as a component of its signaling receptors influences the activity of both osteoblasts and osteoclasts (1), for example, and also influences the actions of PTH to maintain calcium homeostasis.

The gp130-using cytokine family has seven mammalian members [IL-6, IL-11, leukemia inhibitory factor (LIF), oncostatin M, ciliary neurotrophic factor (CNTF), cardiotrophin 1, and cardiotrophin-like cytokine] that signal by activating the transmembrane protein, gp130, which binds these ligands 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 subunits for IL-6, IL-11, LIF, oncostatin M, and CNTF (3). Osteoclasts also express gp130 and ligand-specific receptor subunits for IL-6 and IL-11 (4, 5).

A large body of literature (1), primarily studying cells in culture, suggests that gp130 signaling influences both osteoblast development (6) and differentiation (7), osteoclast development (4, 8), and activity of mature osteoclasts (5, 9). Furthermore, the action of PTH to stimulate osteoclastogenesis 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-6 receptor (10) partially block osteoclast development and bone resorption in vitro. Furthermore, when low doses of PTH are infused in mice, the increase in bone resorption seen in normal mice is greatly blunted in mice null for the IL-6 gene (12). Both gp130 activation, acting through signal transducer and activator of transcription 3, and PTH increase expression of the ligand for receptor activator of nuclear factor-{kappa}B [receptor activator of nuclear factor-{kappa}B ligand (RANKL)], a key activator of osteoclast development and action (13).

The physiological relevance of gp130 signaling in bone has been strongly supported by studies of mice null for gp130 gene expression. These mice die at birth or earlier, depending on the genetic background, and exhibit abnormalities in cardiac and hematopoietic development (14). At the time of birth, these mice exhibit normal growth plates and cortical bone, with a dramatic decrease in the amount of trabecular bone and an increase in number of osteoclasts (15). Mice missing the receptor subunit specific for LIF and CNTF exhibit a similar decrease in trabecular bone and increase in osteoclast number (16). These studies raise the possibility that osteoblast development may be abnormal in these mice and raise questions about the regulation of bone resorption and calcium homeostasis in this model. Therefore, we have performed further analyses both in vivo and in vitro to characterize the role of gp130 signaling in osteoblast development and function and analyze the possible interactions of PTH and gp130 signaling in regulating osteoclasts and calcium homeostasis. We show that gp130 signaling is vital for normal osteoblast function and for calcium homeostasis in fetal life.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Mice carrying a gp130 gene disrupted by homologous recombination, using a targeting vector that inserts a pMC1Neo-poly (A) cassette into the HindIII site in exon 2 just downstream of the translation initiation codon of the gp130 gene was generated in a CD-1 genetic background by Yoshida et al. (14). gp130-deficient mice were obtained by crossing heterozygous mice and genotyped by PCR using genomic DNA isolated from tail clips. Due to perinatal lethality of homozygotes, embryos were obtained before birth by cesarean section from different days of gestation after timed mating. These studies were approved by the institutional animal care committee of Massachusetts General Hospital. All mice were given a standard chow diet and water.

Ionized calcium measurement
Individual fetuses were removed from their amniotic sacs at embryonic day (E)18.5 by cesarean section. Whole blood was collected into 50-µl heparinized capillary tubes from cervical vessels cut by transverse incision. All capillary tubes were capped and immediately immersed in ice. Each blood sample was analyzed within 20 min of collection using a CIBA/Corning 634 Ca2+/pH analyzer (Ciba-Corning Diagnostic Corp., Medfield, MA).

Serum PTH measurement
Fetal blood was collected as for ionized calcium measurements into 50-µl plain capillary tubes instead of heparinized capillary tubes, and then 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 measured with 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 with mixture of 50 µCi 45CaCl2 and 50 µCi 51Cr-EDTA by intracardiac injection as described (17). At 5 min after injection, each embryo was removed from its amniotic sac by cesarean section, weighed, and then placed into a capped test tube. After measurement of 51Cr activity by a {gamma}-counter (Packard Instrument Co., Meriden, CT), the embryos were subsequently solubilized with Scintigest at 60 C for 24–48 h and vortexed briefly. 45Ca activity 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 value of 45Ca and 51Cr activities was normalized to the body weight. To compare the activity among groups, the mean ratio for heterozygotes of each litter was set at 100%.

Calcium release assay
Pregnant mice were injected with 50 µCi 45CaCl2 sc at d 16.5 dpc. Two days later the calvarial bones were isolated from each embryo and divided into four pieces. They were preincubated in 0.5 ml DMEM media for 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). The bones were dissolved in 2 N HCl, and then aliquots of media and bones were analyzed for radioactivity by liquid scintillation. The extent of bone resorption was evaluated by the release of 45Ca from bone to the medium and expressed as a percentage of initial radioactivity present in bones.

Skeletal preparation and histologic analysis
Skeletons were prepared and stained with Alcian blue and alizarin red as described (18). The tissue of embryos from E15.5 to E18.5 were harvested and fixed in 10% formalin for routine light microscopy or 2% paraformaldehyde+2% glutaraldehyde solution for electron microscopy at 4 C for 24 h. Undemineralized paraffin and Epon blocks were prepared by standard histological procedures. The selected paraffin-embedded sections were stained with hematoxylin and eosin or by the von Kossa method, and for alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) enzymatic activity, respectively, as described (15). The collected sections for electron microscopy were stained with uranyl acetate and lead citrate and then observed with an H-800 (Hitachi, Tokyo, Japan) electron microscope at 75 Kv. In situ hybridization for {alpha}1(I) collagen, ALP, and osteocalcin was performed using 35S RNA probes using standard protocols (19).

Histomorphometry
Whole hind limbs from wild-type and gp130-/- embryos at E18.5 were fixed in 3.7% formaldehyde in PBS and embedded in methylmethacrylate. The undemineralized 5-µm sections stained with toluidine 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 endocortical surfaces and in a metaphyseal area (total area of 340 µm2) at the base of the growth plate, including both primary and secondary spongiosa. Three to four sections from each tibia were measured from a 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 was performed by PCR using primers; common 5'-AAGATGATGTCTGCAAACACCGTGG-3', Wt-specific 5'-GGTGTTTGCCCAGGTTGTGCATAA-3', and Mut-specific 5'-TCCAGACTGCCTTGGGAAAAGCGC-3'. The wild-type PTH allele generates a 250-bp-long PCR product, and the PTH null allele generates a 200-bp-long PCR product. Double-mutant mice were fertile and indistinguishable from wild-type littermates. Double-mutant mice were subsequently intercrossed to obtain PTH-/-;gp130-/- double-nullizygous embryos. Embryos were recovered at E18.5 by cesarean section. From 12 litters, total three double mutants were obtained. Comparison was performed using samples from littermates.

Induction of TRAP-positive (TRAP+) multinuclear osteoclastic cells
Primary calvarial osteoblastic cells (2 x 104 cells/well) harvested from wild-type and gp130-/- embryos at E18.5 by serial digestion using a mixture of 0.1% collagenase I and II were cocultured with wild-type spleen cells (5 x 105 cells/well) from 6- to 7-wk-old CD-1 male mice in 0.5ml {alpha}-MEM supplemented with 10% fetal bovine serum (FBS) using 48-well hydroxyapatite-coated plates (OAAS kit, Oscotec, Chenan, Korea). The cells were treated with 10-8 M dexamethasone and 10-7 M hPTH (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 counted from at least three wells, and the data were represented as mean ± SE.

Pit formation assay
The cells prepared for study of TRAP+ multinuclear osteoclastic cell formation were also cultured on dentine slices in 200 µl media supplement with 10-8 M dexamethasone and either 10-7 M hPTH (1–34) or 20 ng/ml sRANKL for 2 wk using 98-well plates. After 2 wk of culture, the pits on dentin slices were visualized with 1% toluidine blue after removing attached cells with 1 M NH4OH. The number of pits on six dentin slices was counted, and the data were expressed as the average number of 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 3 wk with mineralizing media ({alpha}-MEM supplemented with 10% FBS, 10 mM ß-glycerol phosphate, 50 µg/ml ascorbic acid, and 10-7 M dexamethasone). 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, using the Sigma kits n.85 and 104-LS, respectively, according to the manufacturer’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, and cultured 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., Gland Island, NY) and quantitated by spectrophotometry. Ten micrograms of RNA was separated by electrophoresis on a 1.5% formaldehyde agarose gel 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 a random primer DNA labeling kit (Amersham, Arlington, IL). Prehybridization and hybridization were carried out using Express Hyb solution (Clontech, Palo Alto, CA). After washing with 2 x saline sodium citrate, 0.1% sodium dodecyl sulfate at room temperature and then with 0.1 x saline sodium citrate, 0.1% sodium dodecyl sulfate at 55 C, membranes were subjected to autoradiography at -70 C. The experiment was performed three times, with similar results.

Statistics
The experimental data are expressed as mean ± SE of at least three independent experiments. The significance of differences was analyzed by Tukey’s multiple range tests after ANOVA. P < 0.05 was conventionally considered statistically significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Abnormal bone development in gp130-/- mice
To determine the development of the abnormalities in the skeleton at birth noted by Kawasaki et al. (15), we examined both gp130-/- fetuses and wild-type littermates at earlier times. gp130-/- embryos developed to term but died within 1 d after birth for unknown reasons as previously reported (14, 15). On gross examination, the fetuses appeared normal except that they were smaller and had shorter limbs than those of heterozygous and wild-type littermates. Development of epiphyseal cartilage and its mineralization was normal in the gp130-/- mice at all time points. In stained skeletal preparation, the gp130-/- embryos at E18.5 had small skeletons and characteristic bending of most limb elements (Fig. 1AGo). In the tibia, vascular invasion into the cartilage core through the bone collar was noted in both genotypes at E15.5 (Fig. 1BGo). However, at E16.5 (Fig. 1CGo, the wild-type tibia exhibited a well-formed marrow space and diaphysis with scattered thin spicules of primary trabecular bone, whereas equivalent maturation was seen in gp130-/- tibiae only at E17.5 (Fig. 1DGo). In addition to this delay in bone development, the gp130-/- mice exhibited poor development of the primary spongiosa and eccentric thickening of the diaphyseal cortex. At E18.5 the delay in development of the gp130-/- tibia continued to be apparent, with a relative paucity of hematopoietic marrow when compared with wild-type littermates (shown more clearly in hematoxylin and eosin sections (data not shown).



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  		FIG. 1. Abnormal bone development in gp130-/- mice. A, Alcian blue/Alizarin red skeletal staining. gp130-/- embryo at E18.5 exhibits generalized reduction of skeleton size and characteristic bending of long bone elements of limbs. The von Kossa staining of tibiae from wild-type and gp130-/- fetus at E15.5 (B), E16.5 (C), and E17.5 (D). The von Kossa staining illustrates the poorly developed primary spongiosa and eccentric mineralized cortex in gp130-/- fetal tibiae (original magnification, x100). 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-type littermates. Bars represent mean ± SE from least three tibial sections from four to five mice of each genotype. *, P < 0.05 vs. wild type.

 
Osteoblast abnormalities in gp130-/- mice
To understand better the cellular underpinning of the decrease in trabecular bone in the gp130-/- fetuses, histomorphometric analysis was performed on E18.5 gp130-/- mice and their WT littermates. Our findings confirmed and extend those of Kawasaki et al. (15). Osteoblast number within the metaphyseal area and percentage of osteoblast surface per unit bone surface were significantly reduced in gp130-/- tibiae (data not shown). Furthermore, the expression of ALP and collagen type I mRNAs was markedly decreased in the gp130-/- fetal tibia at E18.5, but there was only slight decrease in osteocalcin mRNA expression (Fig. 2AGo). Like the ALP mRNA levels, the level of ALP enzyme activity was reduced in gp130-/- fetuses, compared with wild type (Fig. 2BGo). Because mineralization of bone was grossly normal (Fig. 1Go), we presumed that the abnormality of ALP expression was not sufficient to interfere dramatically with mineralization. To determine whether the abnormalities in osteoblasts noted in vivo were cell autonomous or instead depended on signals from outside the bone microenvironment that might be disturbed in gp130-/- mice, primary calvarial osteoblasts were harvested by serial digestion of calvarial bone from gp130-/- embryos at E18.5 and cultured for 2 wk. (Calvariae have normally mineralized bone at this stage both in wild-type and gp130-/- embryos.) These cells also revealed lower enzyme activity, compared with those from wild-type littermates (Fig. 2Go, C and E). Furthermore, the primary calvarial osteoblasts from gp130-/- embryos did not induce nodule formation when cultured for 3 wk with mineralizing media, whereas the cells from wild-type littermates induced numerous well-mineralized nodules (Fig. 2DGo). This striking abnormality may correlate with the decrease in osteoblast number or osteoblast differentiation seen in vivo, but the precise in vivo correlate of these in vitro findings is difficult to define. These findings demonstrate that osteoblasts from gp130-/- mice have substantial abnormalities of differentiated function.



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  		FIG. 2. Reduced osteoblastic activity in gp130-/- mice. A, In situ hybridization for collagen type I (Col I), ALP, and osteocalcin (OC) mRNAs in 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 x40). B, Histochemistry for ALP activity in wild-type and gp130-/- tibia at E18.5. ALP activity stains brown. C, Cytochemistry for ALP in 2-wk cultured wild-type and gp130-/- primary calvarial osteoblastic cells. D, Alizarin red staining for mineralized nodules in primary calvarial osteoblasts from wild-type and gp130-/- fetuses cultured for 3 wk with {alpha}-MEM supplemented with 10% FBS, 10 mM ß-glycerophosphate, 50 µg/ml ascorbic acid, and 10-7 M dexamethasone. E, ALP activity in cell extracts from primary calvarial osteoblastic cells from E18.5 fetuses after culturing for 2 wk. The assay colorimetrically measures the conversion of p-nitrophenyl phosphate to p-nitrophenyl (NaPNP) plus inorganic phosphate. *, P < 0.05.

 
Osteoclast structure and function
Because activation of gp130 stimulates osteoclastogenesis in vitro, the initial observation (15) that gp130-/- mice have an increase in osteoclast number was surprising. In histomorphometric analysis of tibia from wild-type and gp130-/- embryos at E18.5, we confirmed that the osteoclasts number and percentage of osteoclast surface per bone surface were significantly increased in gp130-/- at both the metaphysis and diaphysis (data not shown). The TRAP+ multinucleated cells (MNCs) in the gp130-/- tibia concentrated along the lower border of the growth plate were characteristically large and ovoid in shape with multinucleation. Only a small number of TRAP+ MNCs was noted associated with the poorly developed primary spongiosa, whereas the evenly distributed wild-type TRAP+ MNCs along the surface of primary spongiosa revealed small and flat shape with scant cytoplasm, which was clear at high magnification (Fig. 3AGo). Ultrastructurally, the osteoclasts from both wild-type and gp130-/- mice showed normal signs of activity, with clear zones, cytoplasmic vacuolization, ruffled borders, and extended cytoplasmic processes embracing the mineralized cartilage matrix. However, the osteoclasts in gp130-/- tibia, although characteristically larger than normal with abundant cytoplasmic organelles and numerous nuclei, exhibited poorly developed ruffled borders. Whereas osteoclasts from wild-type fetuses exhibited ruffled borders with numerous elongated villi, osteoclasts from gp130-/- fetuses exhibited ruffled borders that were thicker, blunter and fewer in number (Fig. 3BGo). The decreased number of cytoplasmic processes in the ruffled border was confirmed by histomorphologic analysis of osteoclasts located in both the metaphyseal and diaphyseal regions (Fig. 3CGo). Because the ruffled border is the resorptive organelle of the osteoclast, these observations suggest that the osteoclasts of the gp130-/- fetuses may be dysfunctional. Because we could not detect any specific signs of osteopetrosis, such as retained cartilage remnants in these mice, the functional implications of this morphological abnormality remain to be defined.



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  		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, x100). The characteristically large ovoid multinucleated gp130-/- TRAP+ MNCs are concentrated at the lower border of the growth plate, whereas the evenly distributed wild-type TRAP+ MNCs along the surface of primary spongiosa are small and flat, seen best at high magnification (inset, original magnification, x400). B, Transmission electron micrographs of in vivo osteoclasts from wild-type and gp130-/- fetuses at E18.5 reveal clear zone (*), ruffled border (arrows), cytoplasmic vacuolization (v), and embraced mineralized matrix (m). Note the increased osteoclast size with numerous nuclei (N) and abundant cytoplasm but less developed ruffled border in gp130-/- osteoclast. Bar, 0.5 µm. C, Both epiphyseal and diaphyseal osteoclasts in the gp130-/- fetal tibia have a significantly decreased number of cytoplasmic folds in their ruffled border, compared with wild-type osteoclasts (*, P < 0.005, n = 10). The bars represent mean ± SE.

 
Secondary hyperparathyroidism in gp130-/- mice
These abnormalities in osteoclast architecture led us to measure indices of calcium metabolism in the gp130-/- mice. The ionized calcium of gp130-/- fetuses at E18.5 (1.44 ± 0.03 mol/liter, n = 12) was significantly lower than that of their heterozygous 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, blood calcium in the homozygotes was still higher than in the dams (1.35 ± 0.03 mmol/liter, n = 4). In addition, the blood PTH level 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 reflect abnormal bone metabolism but might also reflect abnormal transport of calcium across the placenta. To assess the efficiency of placental Ca2+ transfer, the transfer of 45Ca across the placenta was measured as a 45Ca/51Cr activity ratio to control for variation in blood flow across the placenta (17). At E17.5, gp130-/- embryos showed a significantly higher accumulated 45Ca/51Cr activity ratio determined 5 min after maternal administration of the isotopes than normal littermates. The high ratio was caused by reduced accumulation of 51Cr activity in gp130-/- embryos rather than increased accumulation of 45Ca activity. The mean value of accumulated 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 and heterozygous littermates (2629.4 ± 268.3 cpm, n = 3, and 2081.6 ± 140.1 cpm, n = 11, respectively), whereas there were no significant differences in the mean value of accumulated 45Ca activity among the groups (wild type, 9740.1 ± 1333.7 cpm, gp130+/-, 9392.2 ± 696.5 cpm and gp130-/-, 7523.7 ± 1333.7 cpm, respectively) (Fig. 4AGo). The values for 51Cr transfer were proportional to the body weights of each group of fetuses (data not shown). When compared with the mean value for heterozygous mice, which was set at 100%, the mean value of accumulated 45Ca/51Cr activity ratio in gp130-/- and wild-type was 148% and 83%, respectively (P < 0.001 vs. wt). This result suggests that the blood flow from dam to fetus (51Cr transfer) was reduced in gp130-/- and that the efficiency of placental Ca2+ transfer for the amount of blood flow may be increased (Fig. 4BGo). The placentas of gp130-/- fetuses were smaller than those of wild-type littermates, but there was no remarkable histopathologic change. Furthermore, there were no remarkable differences in placental expression of Ca2+ binding protein mRNA and PTH/PTHrP receptor mRNA between wild-type and gp130-/- embryos at E17.5 (data not shown). These studies suggest that the modest (not statistically significant) decrease in calcium transport in gp130-/- can be explained by lower body and placental size of gp130-/- fetuses and cannot explain the lower blood calcium levels in the gp130-/- than in wild-type littermates.



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  		FIG. 4. Increased placental calcium transport in gp130-/- fetuses. A, The accumulated 51Cr and 45Ca activities. B, The mean 45Ca/51Cr activity. The ratio of 45Ca/51Cr activity accumulated in fetuses was determined 5 min after maternal administration of 51Cr-EDTA and 45CaCl2. The mean heterozygote 45Ca/51Cr ratio of each litter was set at 100% to allow the results of multiple litters to be compared. *, P < 0.05 vs. wild type (Wt) and heterozygous (Het). **, P < 0.001 vs. Wt and Het).

 
Osteoclast number and PTH
The dramatic increase in PTH levels in the gp130-/- mice presumably reflects a state of secondary hyperparathyroidism in response to the relative fall in blood calcium in these mice. This hyperparathyroidism might provide an explanation for the increase in osteoclasts noted in these mice. To explore this possibility, PTH+/-; gp130+/- double-heterozygote mice were mated with each other, and the tibiae of the resultant fetuses were examined after TRAP staining. Figure 5Go shows that the number of osteoclasts in PTH-/-; gp130-/- fetuses were similar to that in PTH+/+; gp130-/- mice. Therefore, hyperparathyroidism cannot explain the increase in osteoclast number in the gp130-/- mice; we cannot rule out modest effects of PTH on osteoclast number, however.



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  		FIG. 5. Effect of PTH on osteoclasts of gp130-/- tibiae. Mice doubly heterozygous for knockout of PTH and gp130 were mated, and tibiae, right limb radii, and right limb ulnae from progeny were examined by TRAP staining at E18.5. Bones from two doubly homozygous mice were compared with those of two gp130-/- littermates. A third doubly homozygous mouse was compared with a gp130-/- mouse of the same age from another litter. Three or more sections of each bone were examined. The tibiae shown here are representative of the 12 bones of each genotype examined. Genotypes are indicated above each section.

 
Resorptive activity of osteoclasts in response to PTH and sRANKL
To assess osteoclast function in the context of intact bony structures, E18.5 calvariae, prelabeled with 45Ca in vivo, were cultured, and release of 45Ca was assessed. Basal rates of 45Ca release did not differ among genotypes. However, the 45Ca release in response to 10-7 M PTH (1–34) was significantly increased in wild type and heterozygotes, compared with basal rates, whereas there was no significant increase in gp130-/- mice (Fig. 6Go). The responses of wild-type and heterozygote fetuses in response to PTH did not differ significantly, but their responses were both significantly greater than that of gp130-/- fetuses. Because both PTH/PTHrP receptor and gp130 activation lead to production of RANKL (13) and this production might be defective in gp130-/- fetuses, we determined the response of the calvariae to added sRANKL. SRANKL was tested at 10, 20, and 100 ng/ml, with a dose-dependent increase in response with dose using WT calvariae (data not shown). The 45Ca release in response to 20 ng/ml sRANKL is shown in Fig. 6Go and was significantly increased over its basal rate in wild-type and heterozygote but not in gp130-/- fetuses. Because the response to sRANKL was not normal in the gp130-/- calvariae, the interpretation of the defective response to PTH in gp130-/- is complicated. We cannot conclude that defective production of RANKL in response to PTH is the sole cause of the decreased Ca45 release from the gp130-/- calvariae. It is possible that, in addition, gp130 signaling is required in osteoclasts, that M-CSF responses to PTH are defective in calvariae of gp130-/- mice or that some other aspect of the PTH response is defective in gp130-/- calvariae, in addition to a possible defect in RANKL generation (see below).



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  		FIG. 6. Resorptive activity of gp130-/- osteoclasts. The extent of bone resorption was evaluated by the release of prelabeled 45Ca from bone to the medium, expressed as a percentage of initial radioactivity present in bones. After sc injection of 45CaCl2 at 16.5 dpc, calvarial bones taken from gp130-/- and wild-type or heterozygous littermates at 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).

 
Osteoclastogenesis in vitro
To clarify the roles of individual cell types in the blunted responses to PTH just noted, primary calvarial osteoblasts and spleen cells from varying genotypes were cocultured and 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 2 wk. Wild-type primary calvarial osteoblastic cells induced large number of TRAP+ osteoclastic cells, whereas the primary 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 20 ng/ml sRANKL treatment of these cells induced large numbers of TRAP+ osteoclastic cells. Even with these effective stimuli, however, the gp130-/- osteoblasts did not behave normally. The number of osteoclasts produced in the presence of gp130-/- osteoblasts was significantly decreased, compared with that of wild type (Fig. 7Go, A and B). This result suggests that PTH activation of osteoclastogenesis depends more on gp130 signaling than activation of osteoclastogenesis by 1,25(OH)2D3 or RANKL. The less than normal response to 1,25(OH)2D3 suggests the possibility that the RANKL response to 1,25(OH)2D3 may not be entirely normal in gp130-/- calvariae. The less-than-normal response to RANKL suggests that the calvarial cells may make excess OPG or produce less M-CSF than normal.



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  		FIG. 7. Osteoclast induction in vitro. A, TRAP+ MNCs were induced by cocultures of adult spleen cells and primary calvarial osteoblastic cells from 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 D3 for 2 wk. B, Number of TRAP+ MNCs induced in vitro. In vitro induction of TRAP+ MNCs by PTH was dramatically blocked and significantly reduced 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 of resorption pits by induced TRAP+ MNCs. Primary calvarial osteoblastic cells from gp130+/+ and gp130-/- embryos at E18.5 were cocultured with 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 pits were visualized by 1% toluidine staining after removal of attached cells (original magnification, x100). 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.

 
The osteoclasts formed in response to RANKL were able to form pits when grown in the presence of wild-type calvarial osteoblasts but not when grown in the presence of gp130-/- osteoblasts (Fig. 7Go, C and D). Whereas we cannot explain this result on a molecular level, it emphasizes that gp130-/- calvarial cells have abnormal properties beyond their responses to PTH.

To determine why the response of gp130-/- osteoblasts to PTH was so dramatically blunted, the levels of expression of RANKL, OPG, M-CSF, and PTH receptor mRNA in cultured calvarial osteoblasts were analyzed. OPG binds RANKL and blocks the activation of RANK by RANKL. In WT calvarial osteoblasts, RANKL mRNA levels in response to 10-7 M hPTH (1–34) were markedly up-regulated after 3 h treatment with no consistent change in levels of OPG, M-CSF, or PTH receptor mRNA expression. In contrast, the gp130-/- primary calvarial osteoblastic cells showed only a modest increase in RANKL mRNA levels after PTH and no change in OPG, M-CSF, or PTH receptor mRNA levels in response to PTH. These results suggest that a poor RANKL mRNA response to PTH may contribute to the poor osteoclastic response to PTH, at least in vitro (Fig. 8Go). We cannot eliminate the possibility that changes at the protein level occur as well, but attempts to measure these parameters were unsuccessful, presumably because of the small number of available cells.



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  		FIG. 8. Northern blot analysis for RANKL, OPG, M-CSF, PTH receptor 1, and glyceraldehyde-3-phosphate dehydrogenase. Total RNAs from cultured primary calvarial osteoblastic cells were extracted from WT and gp130-/- embryos at E18.5 after treatment with 10-7 M hPTH (1–34) for 3, 6, 12, and 24 h. Ten micrograms of RNA were electrophoresed, transferred to nylon membrane, and hybridized with 32P-labeled probes.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
These studies establish that loss of signaling by gp130 results in multiple abnormalities in osteoblast function. The developmental sequence demonstrates a general delay in bone development, with apparently normal growth plates but dramatically decreased amounts of trabecular bone. In situ hybridization demonstrates a decrease in expression of collagen {alpha}1(I) mRNA and ALP mRNA as well as decreased ALP activity in bone. Histomorphometric analysis showed a decrease in the number of osteoblasts in trabecular bone as well. These studies of intact bone are complemented by analysis of calvarial osteoblasts cultured in vitro. These cells, after 2 wk in culture, have a substantially lower level of ALP activity and fail to form the mineralized nodules formed by their WT counterparts under the same conditions. Thus, in the absence of gp130 signaling, osteoblasts reveal a cell-autonomous defect in differentiation. These findings are consistent with previous studies showing important effects of activation of gp130 for osteoblast development and differentiation in vitro (1, 6, 7). We cannot explain the relative sparing of development of cortical bone, compared with trabecular bone, because the molecular and in vitro analyses would predict similar defects in both bone compartments. Presumably, some other aspect of gp130 signaling explains the dramatic loss of trabecular bone. Perhaps gp130 signaling influences the entry of osteoblast precursors into the marrow compartment. Alternatively, an action of gp130, such as the decreased apoptosis of osteoblast-like cells seen in vitro (22), may be more active in the microenvironment of trabecular bone.

The relative hypocalcemia and secondary hyperparathyroidism of the gp130-/- mice shows that gp130 signaling contributes to normal fetal calcium homeostasis. Active transport of calcium across the placenta contributes importantly to fetal calcium metabolism (23), so this was assessed by measuring 45Ca transport across the placenta, with normalization both for placental blood flow and fetal weight. Total calcium transport into gp130-/- fetuses was increased, when normalized for the decrease in placental blood flow measured with 51Cr transport or for fetal weight. These findings suggest that reduced placental calcium transport cannot explain the lower blood calcium in the gp130-/- mice. Renal calcium handling was not measured because such measurements are difficult in the fetus. Because the fetal urine is swallowed as amniotic fluid and absorbed by the fetal intestine, it is unlikely that abnormalities of renal calcium handling contribute importantly to the fetal hypocalcemia of the gp130-/- mice. Therefore, we considered possible abnormalities of bone resorption as a contributor to the relative hypocalcemia.

Although the number of osteoclasts was increased in the gp130-/- bones, these osteoclasts had poorly developed ruffled border regions in comparison with their normal counterparts. In contrast to the numerous elongated villi in ruffled border of wild-type osteoclasts, the gp130-/- osteoclasts had thickened and blunted villi, with marked reduction in number in their ruffled borders. Because the ruffled border is the site of active bone resorption by osteoclasts, this finding suggests that the effectiveness of individual osteoclasts may be diminished. Defective bone resorption was demonstrated directly by stimulating calvarial bone resorption in vitro using either PTH or sRANKL. The response of gp130-/- calvariae to PTH was less than the response of WT calvariae, whereas the response of gp130-/- to sRANKL did not differ from the basal response. The defective response to PTH could certainly explain the relative hypocalcemia in the gp130-/- mice in the face of hyperparathyroidism. The modestly low level of blood calcium in the gp130-/- fetuses contrasts with the dramatically lower blood calcium reported in mice null for the PTH/PTHrP receptor gene. Those mice have blood calcium levels considerably lower than those of their dams (17). Although conclusions can only be tentative because the genetic background of the PTH/PTHrP receptor-/- mice differed from that of the gp130-/- mice, these results suggest that the gp130-/- mice respond to PTH but in a blunted fashion.

To explore further the osteoclast defect in the gp130-/- mice, the ability of calvarial cells from these mice to support osteoclastogenesis, when cocultured with osteoclast precursors (from adult spleen) from WT mice, was examined. The response to 1,25(OH)2D3 and sRANKL was blunted, and the response to PTH was virtually absent. The latter results confirm and extend previous in vitro studies in which antibodies to either gp130 (8) or the IL-6 receptor (10) decreased osteoclastogenesis after PTH administration. Because osteoblastic cells stimulate osteoclastogenesis largely through the production of RANKL and M-CSF (24), with constraint of the action of RANKL by production of OPG (25), we examined the effect of PTH on the levels of mRNA encoding RANKL, OPG, and M-CSF in osteoblasts from gp130-/- mice. In response to PTH, WT osteoblasts increased their levels of RANKL mRNA, as previously reported (26), but the gp130-/- osteoblasts had a considerably weaker response. In contrast, no substantial changes in OPG or M-CSF mRNA levels occurred in response to PTH in either WT or gp130-/- osteoblasts. Furthermore, the WT and gp130-/- osteoblasts expressed similar levels of PTH receptor mRNA levels, and these levels did not change after PTH administration. We cannot rule out possible changes in protein levels or at later times. Thus, the abnormalities in supporting osteoclastogenesis in gp130-/- osteoblasts in vitro probably result, at least in part, from abnormalities in RANKL mRNA expression.

These findings, of course, fail to explain the actual increase in osteoclast number in the gp130-/- mice. We wondered whether this increase might result from the dramatic increase in PTH levels in these mice. However, when PTH was removed through appropriate matings with mice missing the PTH gene, the osteoclast number was not substantially changed. These findings are, perhaps not surprising because mice missing the PTH/PTHrP receptor have normal numbers of osteoclasts, despite their profound hypocalcemia. Presumably, osteoclast development in fetal life is primarily regulated by signals other than those activated by the PTH/PTHrP receptor or gp130.

These studies suggest that the activity of the osteoclasts in the gp130-/- mice may be diminished and may explain the relative hypocalcemia in these mice. Because we were unable to develop quantitatively dependable assays for RANKL, M-CSF, and OPG mRNA or protein suitable for use on the bones of fetal gp130-/- mice, we cannot determine whether abnormalities in these genes occurs in vivo in the gp130-/- mice (data not shown). Previous studies have shown that activation of gp130 on osteoblast-like cells increases osteoclastogenesis by increasing RANKL production (13). RANKL also stimulates the activity of mature osteoclasts (27). Therefore, the defect in RANKL mRNA production by osteoblasts from gp130-/- mice suggests that a defect in RANKL production may contribute to the abnormal osteoclast function in the gp130-/- mice. Furthermore, a small number of studies suggest that gp130 on mature osteoclasts may participate more directly in osteoclast function. Adebanjo et al. (9) showed that IL-6 reverses the inhibition of bone resorption induced by high extracellular calcium and Cappellen et al. (5) showed that IL-6 and IL-11, when added along with IL-1 to osteoclasts generated in vitro, increased bone resorbing activity (pit area) by these osteoclasts. Thus, it is possible that activation of gp130 on osteoclasts may increase their bone resorbing properties.

These studies thus establish that bones develop abnormally in the absence of gp130 signaling. Osteoblasts are fewer in number in trabecular bone and exhibit widespread abnormalities of differentiated function. Although osteoclast numbers are increased, the osteoclasts exhibit abnormal morphology, and decreased bone resorption contributes to relative hypocalcemia and hyperparathyroidism in these mice.


   Acknowledgments
 
The authors thank Drs. Tetsuya Taga, Kanji Yoshida, and Tadamitsu Kishimoto for generously providing the gp130 knockout mice for these studies.


   Footnotes
 
This work was supported by NIH Grant DK56246 and a grant from the 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, School of 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 Research and Department of Medicine, St. Vincent’s Hospital Melbourne, University of Melbourne, Fitzroy, Australia.

Abbreviations: ALP, Alkaline phosphatase; CNTF, ciliary neurotrophic factor; dpc, days after conception; E, embryonic day; FBS, fetal bovine serum; LIF, leukemia inhibitory factor; M-CSF, macrophage colony-stimulating factor; MNC, multinucleated cell; OPG, osteoprotegerin; RANKL, receptor activator of nuclear factor-{kappa}B; sRANKL, soluble RANKL; TRAP, tartrate-resistant acid phosphatase; TRAP+, positive TRAP.

Received July 7, 2003.

Accepted for publication November 4, 2003.


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 Introduction
 Materials and Methods
 Results
 Discussion
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