Sclerostin antibody treatment enhances bone strength but does not prevent growth retardation in young mice treated with dexamethasone.код для вставкиСкачать
ARTHRITIS & RHEUMATISM Vol. 63, No. 8, August 2011, pp 2385–2395 DOI 10.1002/art.30385 © 2011 American College of Rheumatology Sclerostin Antibody Treatment Enhances Bone Strength but Does Not Prevent Growth Retardation in Young Mice Treated With Dexamethasone M. Marenzana, K. Greenslade, A. Eddleston, R. Okoye, D. Marshall, A. Moore, and M. K. Robinson Objective. Exposure to supraphysiologic levels of glucocorticoid drugs is known to have detrimental effects on bone formation and linear growth. Patients with sclerosteosis lack the bone regulatory protein sclerostin, have excessive bone formation, and are typically above average in height. This study was undertaken to characterize the effects of a monoclonal antibody to sclerostin (Scl-AbI) in mice exposed to dexamethasone (DEX). Methods. Young mice were concomitantly treated with DEX (or vehicle control) and Scl-AbI antibody (or isotype-matched control antibody [Ctrl-Ab]) in 2 independent studies. Linear growth, the volume and strength of the bones, and the levels of bone turnover markers were analyzed. Results. In DEX-treated mice, Scl-AbI had no significant effect on linear growth when compared to control treatment (Ctrl-Ab). However, in mice treated with DEX and Scl-ABI, a significant increase in trabecular bone at the femoral metaphysis (bone volume/total volume ⴙ117% versus Ctrl-Ab–treated mice) and in the width and volume of the cortical bone at the femoral diaphysis (ⴙ24% and ⴙ20%, respectively, versus Ctrl- Ab–treated mice) was noted. Scl-AbI treatment also improved mechanical strength (as assessed by 4-point bending studies) at the femoral diaphysis in DEXtreated mice (maximum load ⴙ60% and ultimate strength ⴙ47% in Scl-AbI–treated mice versus Ctrl-Ab– treated mice). Elevated osteocalcin levels were not detected in DEX-treated mice that received Scl-AbI, although levels of type 5b tartrate-resistant acid phosphatase were significantly lower than those observed in mice receiving DEX and Ctrl-Ab. Conclusion. Scl-AbI treatment does not prevent the detrimental effects of DEX on linear growth, but the antibody does increase both cortical and trabecular bone and improves bone mechanical properties in DEX-treated mice. Glucocorticoid (GC)–based drugs have potent immunosuppressive and antiinflammatory properties and have assumed an important role in the treatment of many types of inflammatory and autoimmune conditions. However, drugs of this type are associated with a range of well-known side effects (1). One of the most serious problems associated with GC exposure is a deleterious effect on bone, which leads to a high proportion of patients who, after receiving long-term GC therapy, develop GC-induced osteoporosis and are susceptible to bone fractures (2). The detrimental effect of GCs on bone strength has been reported to involve many different mechanisms, including inhibition of osteoblastic bone formation, increased osteoclastic bone resorption, changes in calcium balance, and inhibition of the osteoanabolic action of sex steroids (3). More recently, it has also been proposed that GC exposure not only may cause changes to bone mass and bone architecture, but also may alter the localized material properties of bone (4). When administered to children or to growing Supported by UCB. M. Marenzana, PhD (current address: Imperial College, London, UK), K. Greenslade, BSc, A. Eddleston, MSc, R. Okoye, MSc, D. Marshall, PhD, A. Moore, PhD, M. K. Robinson, PhD: UCB Celltech, Slough, UK. Ms Okoye and Drs. Marshall, Moore, and Robinson hold stock or stock options in UCB Celltech. Dr. Robinson is a named inventor on a number of UCB patents, including ones related to sclerostin. Address correspondence to M. K. Robinson, PhD, Department of Inflammation Biology, UCB Celltech, 216 Bath Road, Slough SL1 4EN, UK. E-mail: Martyn.Robinson@ucb.com. Submitted for publication August 19, 2010; accepted in revised form March 29, 2011. 2385 2386 animals, GCs not only have detrimental effects on bone strength, but also cause linear growth retardation (5–7). The inhibitory effects of GCs on growth are primarily mediated by disruption of chondrogenesis at the growth plate (8). This occurs via direct effects of GCs on chondrocyte growth and apoptosis (9,10), as well as through changes to factors such as those in the insulin-like growth factor (11) and vascular endothelial growth factor pathways (12), which disturb normal growth plate function. Sclerostin is a glycoprotein that is produced by osteocytes and negatively regulates osteoblastic bone formation (13). The gene encoding sclerostin was identified by molecular and genetic analysis of the rare inherited condition sclerosteosis, a condition in which the gene is known to be defective (14,15). Patients with sclerosteosis are typically of large stature and show excessive bone formation (16). Sclerostin has been reported to antagonize signaling in the bone morphogenetic protein pathway (17), but it is currently believed that inhibition of the Wnt pathway is more important for the activity of sclerostin on bone formation in vivo (18). Antibodies to sclerostin increase bone strength, bone formation, and bone healing in a range of animal models (19–22), and a monoclonal antibody to sclerostin was recently evaluated for its clinical effects (23). Chen et al (24) reported that inhibition of signaling through ␤-catenin (a pivotal component of the Wnt signaling pathway) led to decreased width of the proliferating and hypertrophic zones of the growth plate, and this was associated with decreased skeletal growth. In adolescent humans, sclerostin has been reported to be expressed by both osteocytes and mineralized hypertrophic chondrocytes in the growth plate. It has been speculated that sclerostin may play a role in regulating chondrocyte proliferation, and that this could explain the tall stature of patients with sclerosteosis (25). The negative regulatory role of sclerostin in bone formation and Wnt signaling, coupled with its reported expression close to the growth plate, suggest that its inactivation with a monoclonal antibody might both stimulate bone formation and prevent growth retardation in young, GC-treated animals. In the present study, we describe the effects of a monoclonal antibody to sclerostin (Scl-AbI) in young mice being concomitantly treated with dexamethasone (DEX). The young age of the mice makes this model most relevant to the inhibition of the bone growth (or bone rarefaction) seen in children treated with GCs (5,6). MARENZANA ET AL MATERIALS AND METHODS Antibodies and drugs. DEX and methylcellulose were obtained from Sigma UK. The Scl-AbI antibody and isotypematched control antibody (Ctrl-Ab) have been previously described (19). Animals and experiment outline. BALB/c mice (obtained from Charles River UK) were ⬃7 weeks of age at the start of the studies. Mice were maintained and studied in a manner in compliance with UK Home Office regulations. Two similar experiments were performed with DEX (prepared in a 0.5% methylcellulose solution), which was administered at a daily dose of 3 mg/kg by oral gavage for either 6 weeks or 9 weeks. In both experiments, 4 groups of mice were treated, as follows: 1) vehicle (0.5% methylcellulose solution) and CtrlAb, 2) DEX and Ctrl-Ab, 3) vehicle and Scl-AbI, or 4) DEX and Scl-AbI. The sclerostin or control antibodies were administered subcutaneously at a dosage of 25 mg/kg twice weekly. The body weight of the animals was monitored weekly. Measurement of serum markers. Serum levels of type 5b tartrate-resistant acid phosphatase (TRAP5b) and N-terminal type I procollagen propeptide (PINP) were measured using mouse sandwich enzyme-linked immunosorbent assay kits from ImmunoDiagnostic Systems. Serum levels of osteocalcin were measured using Luminex kits obtained from Millipore. All kits were used according to the manufacturer’s recommendations. Measurement of areal bone mineral density (BMD). Mice were anesthetized with 2% isofluorane inhalation. After being placed under general anesthesia, the mice were scanned on a Lunar PIXImus (GE Medical Systems). BMD measurements were determined at baseline and at 3, 6, and 9 weeks thereafter. Microfocal computed tomography (micro-CT) analysis. Microstructural analysis was performed on bone samples at the end of the 6-week dosing experiment. Dissected femurs were scanned at high resolution (7 m pixel size) in a micro-CT scanner (Scanco CT 35). The Scoutview images were used to measure femur length and to position the imaging window. Analysis was performed in 2 regions: 1) the middiaphysis cortical bone, a 0.7-mm–long segment (or 100 tomograms) centered over the femoral midline, and 2) the distal femoral metaphysis, a region starting 0.29 mm from an anatomic landmark in the growth plate and extending 1.2 mm (or 170 tomograms) proximally. The vertebral bodies of L5 vertebra, including upper and lower disks, were selected from the Scoutview images of the whole mouse spine and scanned at a resolution of 6 m (pixel size). The longitudinal length of the vertebral body was the difference between the top and bottom tomograms. Trabecular bone regions of interest were manually drawn, with exclusion of the cortex. Two- and three-dimensional morphometric measurements were obtained using Scanco analysis software, with a fixed threshold of 500 mg/cm3 to segment bone from the background. Cortical volumetric BMD and bone mineral content were also obtained, after calibrating the absorption values against known density phantoms. The width of the growth plate in the distal femur was measured in reconstructed scans by generating 3 coronal sections (1 central and 2 symmetrically placed 100 m apart) from the stack of cross-sectional tomograms, and then enclosing the growth plate and a thin layer of bone tissue along its length. An inverse threshold was applied EFFECTS OF SCLEROSTIN ANTIBODY ON BONE STRENGTH IN DEX-TREATED MICE to this region (i.e., everything below 400 mg/cm3) to select nonmineralized cartilage and exclude bone. The average width and other geometric features of the segmented regions were obtained using Scanco analysis software. Results from 10 age-matched mice that were killed at the start of the study were used for the baseline values. Immunohistochemistry. Femurs were fixed in neutral buffered formalin before decalcification in EDTA, and were then embedded in paraffin wax. Five-micrometer sections were cut, followed by proteinase K antigen retrieval and blocking of endogenous peroxidase. Sections were incubated with goat polyclonal anti-mouse sclerostin antibody (AF1589, used at 1:100 dilution; R&D Systems) followed by biotinylated donkey anti-goat secondary antibody (used at 1:500 dilution; Stratech Scientific). The signal was visualized using a peroxidase– streptavidin and diaminobenzidine substrate–chromogen system (Dako UK). Sections were washed and counterstained with hematoxylin. Four-point bending test of the femoral shaft. Mechanical testing was performed on the right femurs of the mice. The femurs had been excised from the mice at the termination of the 9-week study, and were kept frozen at ⫺70°C until the day of the test. For mechanical testing, the 4-point bending test was used (26). This test allows the calculation of a number of bone mechanical properties, including resistance to bending under load (stiffness), the maximum load that a bone can sustain (maximum load), and the maximum stress that a bone can sustain and the amount of energy the bone can absorb before failure (toughness). The mechanical testing was carried out at MDS Pharma, as previously described (19). Statistical analysis. Statistical analysis of the data was performed using one-way analysis of variance with a Bonferroni post hoc test to adjust for multiple comparisons. P values less than 0.05 were considered significant. RESULTS Distribution of sclerostin-positive cells in the growing mouse epiphysis. Immunohistochemical analysis of the mouse femurs using Scl-AbI confirmed previ- 2387 ous findings indicating that the osteocyte is the major cellular source of sclerostin in the mouse epiphysis (Figure 1A). It was also clear that the basal layer of the articular cartilage contained a number of sclerostinpositive cells, which appeared to be hypertrophic chondrocytes, similar to observations recently reported in a study by van Bezooijen et al (25). Although the growth plate itself did not contain sclerostin-positive cells, there were nests of cells adjacent to the growth plate that were positive for sclerostin expression (Figure 1B). The results of immunohistochemistry revealed no obvious differences in the distribution of sclerostin expression or the intensity of sclerostin staining when mice were treated with DEX (results not shown). Lack of effect of Scl-AbI treatment on DEXinduced changes in body weight, bone length, or growth plate thickness. As shown in Figures 2A and B, the pattern of changes in body weight in the groups of mice in 2 separate experiments, involving treatment of growing mice with DEX for either 6 weeks or 9 weeks, was very similar in both experiments. In each case, the vehicle/Ctrl-Ab–treated mice showed a normal pattern of weight gain, whereas DEX/Ctrl-Ab–treated mice failed to gain weight over the course of the experiment. The weight of the mice in the DEX/Scl-AbI treatment group was not significantly different from that of those in the DEX/Ctrl-Ab group, indicating that Scl-AbI had no influence on the suppressive effects of DEX on body weight. Femurs from the mice in the 6-week experiment were examined to determine bone length. Micro-CT was used to determine the thickness of the femoral distal epiphyseal growth plate and L5 vertebrae. The vehicle/ Ctrl-Ab–treated mice showed a significant increase in Figure 1. Immunocytochemical localization of sclerostin in the femoral head of mice. The results of immunohistochemistry in a representative mouse femur reveal A, sclerostin-positive cells (in dark brown, with nuclei counterstained in blue) in the articular surface (AS) of the epiphysis, growth plate (GP), and metaphysis, and B, nests of sclerostin-positive cells adjacent to the growth plate (arrowheads). The image in B is a higher-power magnification of the boxed area in A. 2388 MARENZANA ET AL Figure 2. Effects of dexamethasone (DEX) and sclerostin monoclonal antibody (Scl-AbI) treatment on body weight and bone growth of mice. A and B, Body weight was assessed after treatment of mice in 2 separate experiments, with durations of 9 weeks (A) and 6 weeks (B). C and D, Femoral length, as measured from microfocal computed tomography (micro-CT) Scoutview images (C), and growth plate thickness, as measured from micro-CT reconstruction images (D), were assessed in mice after 6 weeks of treatment. Values are the mean with 95% confidence intervals in 5–10 mice per group, including a satellite group of 5–10 age-matched mice from which samples were obtained at baseline (BSLN). ⴱ ⫽ P ⬍ 0.05 versus vehicle/isotype-matched control antibody (Ctrl-Ab); ⴱⴱ ⫽ P ⬍ 0.05 versus DEX/Ctrl-Ab, DEX/Scl-AbI, and BSLN; ⴱⴱⴱ ⫽ P ⬍ 0.05 versus vehicle/Ctrl-Ab and BSLN; † ⫽ P ⬍ 0.05 versus DEX/Ctrl-Ab and DEX/Scl-AbI. both femoral length (Figure 2C) and L5 vertebral length (⫹4.4%) (Table 1) when compared with the baseline values from the satellite group of mice that were killed at the start of the experiment, which was consistent with Table 1. Microstructural parameters of trabecular bone in the distal femur metaphysis and L5 vertebral body of young mice concomitantly treated with dexamethasone (DEX) (or vehicle control) and an antibody to sclerostin (Scl-AbI) (or isotype-matched control antibody [Ctrl-Ab])* Ctrl-Ab Distal femur BV/TV, % TbTh, mm TbSp, mm TbN/mm Vertebra L5 BV/TV, % TbTh, mm TbSp, mm TbN/mm Longitudinal length Scl-AbI Vehicle DEX Vehicle DEX Baseline 13.69 (12.76–14.62) 0.028 (0.027–0.029) 0.179 (0.167–0.191) 4.861 (4.593–5.128) 13.56 (12.71–14.41) 0.025 (0.025–0.026)§ 0.163 (0.154–0.172) 5.344 (5.108–5.581)§ 31.33 (29.50–33.17)† 0.054 (0.051–0.056)† 0.118 (0.113–0.124)† 5.811 (5.655–5.967)¶ 29.38 (26.94–31.82)‡ 0.046 (0.045–0.048)‡ 0.113 (0.104–0.122)‡ 6.304 (6.016–6.592)‡ 12.79 (11.97–13.62) 0.026 (0.026–0.027) 0.180 (0.170–0.190) 4.873 (4.647–5.099) 18.85 (18.15–19.54) 0.033 (0.032–0.034) 0.141 (0.136–0.147) 5.753 (5.562–5.943) 3.385 (3.320–3.450) 18.38 (17.49–19.27) 0.030 (0.030–0.031)§ 0.136 (0.130–0.141) 6.026 (5.841–6.211) 3.191 (3.142–3.240)§ 40.99 (39.98–42.01)† 0.066 (0.064–0.067)† 0.094 (0.092–0.097)† 6.257 (6.170–6.344)§ 3.373 (3.326–3.420)# 28.84 (27.63–30.05)‡ 0.048 (0.048–0.049)‡ 0.120 (0.113–0.126)‡ 5.976 (5.755–6.197) 3.232 (3.163–3.301)§ 17.98 (17.14–18.82) 0.030 (0.029–0.030)§ 0.136 (0.130–0.143) 6.035 (5.813–6.258) 3.241 (3.190–3.292)§ * Values are the mean (95% confidence interval) measurements in 5–10 mice per group. Baseline values were obtained from a group of 5–10 age-matched mice that were killed at the start of the study. BV/TV ⫽ femoral trabecular volume fraction expressed as bone volume/total volume; TbTh ⫽ trabecular bone thickness; TbSp ⫽ trabecular bone spacing; TbN ⫽ trabecular number. † P ⬍ 0.05 versus vehicle/Ctrl-Ab, DEX/Ctrl-Ab, DEX/Scl-AbI, and baseline. ‡ P ⬍ 0.05 versus vehicle/Ctrl-Ab, DEX/Ctrl-Ab, and baseline. § P ⬍ 0.05 versus vehicle/Ctrl-Ab. ¶ P ⬍ 0.05 versus vehicle/Ctrl-Ab and DEX/Ctrl-Ab. # P ⬍ 0.05 versus DEX/Ctrl-Ab, DEX/Scl-AbI, and baseline. EFFECTS OF SCLEROSTIN ANTIBODY ON BONE STRENGTH IN DEX-TREATED MICE 2389 Figure 3. Effects of DEX and Scl-AbI treatment of mice on femoral bone mineral density (BMD) and bone mechanical strength. A and B, Changes to areal BMD over 9 weeks (A) or 6 weeks (B) of treatment were assessed by dual x-ray absorptiometry. C and D, Biomechanical properties of the excised mouse femurs after 9 weeks of treatment were assessed using the 4-point bending test, which tested for maximum load to failure (C) and ultimate strength (D). Values are the mean with 95% confidence intervals in 3–10 mice per group. ⴱ ⫽ P ⬍ 0.05 versus vehicle/Ctrl-Ab; ⴱⴱ ⫽ P ⬍ 0.05 versus vehicle/Ctrl-Ab and DEX/Ctrl-Ab; ⴱⴱⴱ ⫽ P ⬍ 0.05 versus DEX/Ctrl-Ab. See Figure 2 for other definitions. normal growth. Both groups of mice receiving DEX had significantly shorter femurs (Figure 2C) and vertebrae (Table 1) than did those receiving vehicle. There was no significant difference in the values for femoral and vertebral length between the mice in the DEX/Ctrl-Ab and DEX/Scl-AbI treatment groups (Table 1 and Figure 2C). Measurements of the femoral epiphyseal growth plates showed that the thickness of the growth plate in vehicle/Ctrl-Ab–treated mice was significantly less than that in mice at baseline (probably due to a slowing in bone growth as the mice aged). In DEX/Ctrl-Ab–treated mice, the growth plates were significantly thinner than those in vehicle/Ctrl-Ab–treated mice, indicating a negative effect of DEX on the growth plate (Figure 2D). In vehicle/Scl-AbI–treated mice, the thickness of the growth plate was not significantly different from that in the vehicle/Ctrl-Ab group. When Scl-AbI was administered to DEX-treated mice (DEX/Scl-AbI group), the growth plate thickness was not significantly different from that in mice treated with DEX/Ctrl-Ab (Figure 2D). These findings indicate a significant inhibitory effect of DEX on growth in young mice, which is consistent with the results from previous studies (7). Moreover, the findings show that Scl-AbI treatment is unable to counter this effect. Prevention of DEX-induced changes to areal BMD by Scl-AbI. Assessments of areal BMD of the femur in both the 6-week study and the 9-week study indicated that, from the start of the experiment, there was a gradual increase in areal BMD in vehicle/Ctrl-Ab– treated mice (Figures 3A and B). These changes were consistent with the age of the mice and their continuing skeletal growth. In the DEX/Ctrl-Ab treatment group, there was no significant increase in the areal BMD of these mice over the course of the experiments, in either the femur (Figures 3A and B) or the spine (results not shown). By the end of the experiments (6 and 9 weeks), the values for areal BMD in the DEX/Ctrl-Ab group were significantly lower than those in the vehicle/ Ctrl-Ab group. Mice in the vehicle/Scl-AbI treatment group had significantly higher areal BMD after 6 and 9 weeks of treatment, both in the femur (Figures 3A and B) and in the spine (results not shown), when compared with mice in the vehicle/Ctrl-Ab group. Mice in the DEX/Scl-AbI group had areal BMD values that were significantly higher than those in the DEX/Ctrl-Ab group at 6 and 9 weeks, in the femur (Figures 3A and B) as well as in the lumbar spine and caudal vertebrae (6-week mean BMD of lumber spine 0.093 gm/cm2, 95% confidence interval [95% CI] 0.091–0.094 gm/cm2 in DEX/Scl-AbI group 2390 versus 0.074 gm/cm2, 95% CI 0.072–0.075 gm/cm2 in DEX/Ctrl-Ab group; 6-week mean BMD of caudal vertebrae 0.059 gm/cm2, 95% CI 0.057–0.062 gm/cm2 in DEX/Scl-AbI group versus 0.051 gm/cm2, 95% CI 0.049–0.052 gm/cm2 in DEX/Ctrl-Ab group). The areal BMD values observed in the femurs of the DEX/SclAbI–treated mice were not significantly different from those in the vehicle/Ctrl-Ab group, but these levels did not reach the values seen in mice in the vehicle/Scl-AbI group (Figures 3A and B). Prevention of DEX-induced changes in bone mechanical strength by Scl-AbI. High-level DEX exposure is known to reduce both bone formation and bone mechanical properties (4). To investigate the effects of Scl-AbI treatment on bone mechanical properties in DEX-treated mice, the femurs of mice in the 9-week experiment were removed at the end of the experiment and subjected to 4-point bending tests. Data on the mechanical strength of the femurs are shown in Figures 3C and D. Compared with vehicle/Ctrl-Ab–treated mice, mice in the DEX/Ctrl-Ab group had weaker bones, as evidenced by significantly reduced maximum load (Figure 3C) and stiffness (mean 183.4 N/mm, 95% CI 161.9–204.8 N/mm in vehicle/Ctrl-Ab group versus 105 N/mm, 95% CI 77.7–132.4 N/mm in DEX/Ctrl-Ab group) (both are extrinsic bone properties), as well as significantly reduced ultimate strength (Figure 3D) and toughness (mean 8.21 MJ/m3, 95% CI 5.96–10.46 MJ/m3 in vehicle/Ctrl-Ab group versus 3.73 MJ/m3, 95% CI 2.68–4.78 MJ/m3 in DEX/Ctrl-Ab group) (both are intrinsic bone properties). Mice in the vehicle/Scl-AbI group had improved bone mechanical properties, with significantly increased values for maximum load (Figure 3C), stiffness (mean 252.8 N/mm, 95% CI 225.1–280.5 N/mm), toughness (mean 15.93 MJ/m3, 95% CI 12.38–19.48 MJ/m3), and ultimate strength (Figure 3D) in comparison with the values observed in mice in the vehicle/Ctrl-Ab group. Intrinsic and extrinsic mechanical properties of the femurs in the mice in the DEX/Scl-AbI–treated group were significantly improved (maximum load and ultimate strength shown in Figures 3C and D; stiffness and toughness, mean 172.3 N/mm, 95% CI 155.2–189.5 N/mm and mean 8.19 MJ/m3, 95% CI 4.62–11.76 MJ/m3, respectively) when compared with those in the DEX/ Ctrl-Ab group, and the values were not significantly different from those in the vehicle/Ctrl-Ab group. However, the values for intrinsic and extrinsic bone properties of the femurs in the DEX/Scl-AbI–treated mice were not as high as the values in those receiving vehicle/ Scl-AbI. These findings show that the dose of DEX used MARENZANA ET AL in these studies produced profound changes in growth, body weight, areal BMD, and bone mechanical strength in Ctrl-Ab–treated mice. Importantly, Scl-AbI treatment was able to prevent some of the detrimental changes that compromised the bone in DEX-treated mice. Prevention of DEX-induced changes in cortical and trabecular bone by Scl-AbI. Femurs removed from the mice in the 6-week dosing experiment were subjected to further analysis by micro-CT. The cortical bone volume (CtV) and average cortical bone width (CtW) of the femoral mid-shaft diaphysis were significantly higher in the vehicle/Ctrl-Ab–treated group than in mice killed at baseline (Figure 4A). In contrast, mice in the DEX/ Ctrl-Ab group showed no significant change in CtV from baseline over the course of the experiment, whereas there was a small, but significant, reduction in average CtW when compared with baseline values (Figure 4B). Vehicle/Scl-AbI–treated mice showed significant increases in the CtV and CtW as compared with both the baseline and vehicle/Ctrl-Ab groups. When Scl-AbI was administered to DEX-treated mice (DEX/Scl-AbI group), the CtV and CtW were both significantly higher than the values observed in mice in either the DEX/ Ctrl-Ab group or the baseline group (Figures 4A and B). The values for CtV and CtW in the DEX/Scl-AbI– treated group were not significantly different from the values found in mice in the vehicle/Ctrl-Ab group (Figures 4A and B). The changes in cortical thickness seen in mice treated with DEX, Scl-AbI, or their combination were mostly endosteal, since we found no differences in the periosteal perimeter or cross-sectional area of the femoral midshaft. However, we did observe significant changes in the bone marrow area between the groups (mean 0.604 mm2, 95% CI 0.555–0.654 mm2 in vehicle/Ctrl-Ab group versus 0.725 mm2, 95% CI 0.692–0.758 mm2 in DEX/Ctrl-Ab group; mean 0.526 mm2, 95% CI 0.499– 0.554 mm2 in vehicle/Scl-AbI group versus 0.612 mm2, 95% CI 0.567–0.658 mm2 in DEX/Scl-AbI group). The secondary spongiosa of the distal femoral metaphysis was also scanned by micro-CT at the end of the experiment (week 6). The values for femoral trabecular volume fraction (bone volume/total volume [BV/TV]) and trabecular thickness (TbTh) in the vehicle/Ctrl-Ab–treated group were not significantly different from those obtained in mice killed at baseline (Table 1 and Figures 4C and D). Similarly, the femoral BV/TV values at week 6 were not significantly different between the vehicle/Ctrl-Ab group and the DEX/ Ctrl-Ab group; the values for TbTh were marginally lower in the DEX/Ctrl-Ab group compared with the vehicle/Ctrl-Ab group. EFFECTS OF SCLEROSTIN ANTIBODY ON BONE STRENGTH IN DEX-TREATED MICE 2391 Figure 4. Effects of DEX and Scl-AbI treatment of mice on cortical and trabecular bone. Microfocal computed tomography measurements were made after 6 weeks of treatment, to determine the femoral cortical bone volume (A), femoral cortical width (B), trabecular bone fraction (bone volume/total volume [BV/TV]) of the femoral metaphysis (C), and trabecular thickness (TbTh) of the femoral metaphysis (D). Values are the mean with 95% confidence intervals in 9–10 mice per group. ⴱ ⫽ P ⬍ 0.05 versus vehicle/Ctrl-Ab; ⴱⴱ ⫽ P ⬍ 0.05 versus vehicle/Ctrl-Ab, DEX/Ctrl-Ab, DEX/Scl-AbI, and BSLN; ⴱⴱⴱ ⫽ P ⬍ 0.05 versus DEX/Ctrl-Ab and BSLN; † ⫽ P ⬍ 0.05 versus vehicle/Ctrl-Ab and BSLN; †† ⫽ P ⬍ 0.05 versus vehicle/Ctrl-Ab, DEX/Ctrl-Ab, and BSLN. See Figure 2 for other definitions. Mice receiving vehicle/Scl-AbI treatment had significantly higher femoral BV/TV and TbTh values than were found in the vehicle/Ctrl-Ab group (Table 1 and Figures 4C and D). When Scl-AbI administration was combined with DEX treatment (DEX/Scl-AbI), femoral BV/TV and TbTh values were significantly higher than those in mice treated with DEX/Ctrl-Ab, but were not significantly different from the values obtained from the vehicle/Scl-AbI group (Table 1 and Figures 4C and D). Changes in the L5 vertebral trabecular bone showed patterns similar to those found in the trabecular bone of the femoral metaphysis (Table 1). These findings indicate a detrimental effect of DEX on cortical and trabecular bone structure that was prevented by concurrent Scl-AbI treatment. Serum markers of bone turnover. The levels of bone turnover markers in mice from the 6-week experiment are shown in Figure 5. At 3 weeks of treatment, vehicle/Scl-AbI–treated mice, compared to vehicle/Ctrl-Ab–treated mice, had significantly elevated levels of osteocalcin (Figure 5A) and also had elevated levels of PINP, although the difference was not significant (Figure 5C). These findings are consistent with the increased bone found in vehicle/Scl-AbI–treated mice. However, by week 6, neither of these bone formation markers was expressed at higher levels in the vehicle/ Scl-AbI group compared to the vehicle/Ctrl-Ab group (Figure 5B) (for PINP at 6 weeks, mean 41.7 ng/ml, 95% CI 34.6–48.9 ng/ml in vehicle/Ctrl-Ab group versus 44.0 ng/ml, 95% CI 37.0–50.9 ng/ml in vehicle/Scl-Ab group). In DEX/Ctrl-Ab–treated mice, the levels of osteocalcin and PINP were significantly and profoundly depressed relative to the levels in mice treated with vehicle/CtrlAb, at week 3 (Figures 5A and C) and at week 6 (Figure 5B and results not shown). Treatment with Scl-AbI did not elevate the level of either of these markers in mice receiving DEX at either the 3-week or 6-week time point. Treatment with DEX/Ctrl-Ab resulted in significantly higher serum levels of TRAP5b than were found in the vehicle/Ctrl-Ab group at week 6, indicating a higher level of bone resorption in the mice exposed to DEX. At this time point, TRAP5b levels in vehicle/SclAbI–treated mice were not significantly different from those in the vehicle/Ctrl-Ab group. Mice in the DEX/ Scl-AbI group had significantly lower TRAP5b levels than did those in the vehicle/Ctrl-Ab and DEX/Ctrl-Ab groups (Figure 5D), suggesting that Scl-AbI treatment reduced bone resorption in the presence of DEX. DISCUSSION GC treatment is known to have negative effects on both bone strength and linear bone growth, but the 2392 MARENZANA ET AL Figure 5. Effects of DEX and Scl-AbI treatment of mice on bone serologic markers. The markers assessed were the levels of osteocalcin after 3 weeks (A) and 6 weeks (B) of treatment, as well as the levels of N-terminal type I procollagen propeptide (PINP) after 3 weeks of treatment (C) and type 5b tartrate-resistant acid phosphatase (TRAP5b) after 6 weeks of treatment (D). Values are the mean with 95% confidence intervals in 8–10 mice per group. ⴱ ⫽ P ⬍ 0.05 versus vehicle/Ctrl-Ab; ⴱⴱ ⫽ P ⬍ 0.05 versus vehicle/Ctrl-Ab, DEX/Ctrl-Ab, and DEX/Scl-AbI; ⴱⴱⴱ ⫽ P ⬍ 0.05 versus DEX/Ctrl-Ab and DEX/Scl-AbI; † ⫽ P ⬍ 0.05 versus vehicle/Ctrl-Ab and DEX/Ctrl-Ab. See Figure 2 for other definitions. exact mechanism of these detrimental effects remains unclear. One pathway that is believed to play an important role in regulating many aspects of bone formation is the Wnt signaling pathway (27). GC treatment has been shown to up-regulate the expression of a number of molecules involved in inhibiting Wnt signaling (28–31). In addition, Wang et al (31) showed that downregulation of Dkk-1 (a Wnt antagonist) with antisense oligonucleotides protected bone from the effects of GC exposure. Sclerostin is a relatively newly reported regulator of Wnt signaling, whose expression is largely restricted to bone and cartilage tissue (14). The immunohistologic findings described in the present study are consistent with those from previous studies describing sclerostin expression as being primarily osteocytic, but they also confirm a recent report describing sclerostin expression in cells in the vicinity of the growth plate (25). Based on the expression pattern of sclerostin and the large stature of individuals with a defective sclerostin gene, it has been postulated that sclerostin may help to regulate linear growth in humans (25). The results presented herein show that neutralization of sclerostin by monoclonal antibody treatment does not prevent DEXinduced inhibition of linear growth in mice. However, differences in the patterns of sclerostin expression around the growth plate (25) make it difficult to be certain of the relevance of this finding in humans. At least some of the positive effects of parathyroid hormone on bone formation have been attributed to its ability to moderately down-regulate sclerostin (32–34). The inability of Scl-AbI to prevent DEXinduced inhibition of growth is therefore consistent with the findings from a study by van Buul-Offers et al (35), in which they showed that parathyroid hormone was also ineffective at reducing GC-mediated growth inhibition (36). Scl-AbI treatment of mice receiving DEX had beneficial effects on bone volume and bone strength. Mice exposed to DEX that were concomitantly treated with Scl-AbI showed significantly higher areal BMD in both the femur (⫹22% at week 6) and the lumbar spine (⫹26% at week 6) than did those treated with DEX and Ctrl-Ab (Table 1 and Figure 3). In addition, micro-CT analysis showed that mice in the DEX/Scl-AbI group had significantly more cortical bone volume (CtV ⫹20%) and cortical bone width (CtW ⫹24%) than did those receiving DEX/Ctrl-Ab (Figures 4A and B). In DEX-treated mice, the Scl-AbI antibody did not significantly increase the periosteal perimeter but did produce a decrease in marrow area, which is consistent with bone EFFECTS OF SCLEROSTIN ANTIBODY ON BONE STRENGTH IN DEX-TREATED MICE formation occurring predominantly on the endosteal surface. Scl-AbI treatment was less effective at increasing bone in the presence of DEX than was achieved in the absence of DEX, suggesting that the presence of DEX somewhat blunted the effects of Scl-Ab. A similar blunting effect has also been seen on the activity of parathyroid hormone in DEX-treated animals (36). Micro-CT analysis of trabecular bone in the femoral metaphysis showed no significant differences in trabecular bone volume between mice in the DEX/ Ctrl-Ab group and the vehicle/Ctrl-Ab group. DEX/SclAbI treatment had a very positive effect on trabecular bone in DEX-treated mice, with significant increases at the femoral metaphysis in both trabecular thickness (TbTh ⫹84%) and trabecular volume (BV/TV ⫹117%) when compared with mice in the DEX/Ctrl-Ab group (Figures 4C and D). Similar responses were also seen in trabecular bone in the lumbar spine (BV/TV ⫹57% in DEX/Scl-AbI group versus DEX/Ctrl-Ab group) (Table 1). Generally, the changes in trabecular bone in response to Scl-AbI appeared smaller in the presence of DEX than in its absence, again suggesting a slight blunting effect of the Scl-AbI effect by DEX. Scl-AbI treatment of mice dosed with DEX not only had positive effects on areal BMD and bone volume, but also resulted in femurs with significantly better mechanical properties (both intrinsic and extrinsic) than were observed in mice dosed with DEX and Ctrl-Ab. Differences in bone geometry in Scl-AbI– treated mice may have contributed to improvements in the maximum load values, but increases in ultimate strength suggest that improvements in bone quality may also have occurred. Volumetric BMD was not affected by DEX treatment (mean 1,094 mg/cm3, 95% CI 1,078– 1,109 mg/cm3 in vehicle/Ctrl-Ab group versus 1,081 mg/ cm3, 95% CI 1,062–1,099 mg/cm3 in DEX/Ctrl-Ab group), suggesting that changes in mineralization played only a minor role in the DEX-induced decline of bone mechanical properties in this model. It is possible that the reduced body weight of the DEX-treated mice may have contributed to the failure of the antibody treatment to increase areal BMD during the course of the experiments. However, similar trends were seen in the weight-bearing femur and in the non–weight-bearing lumbar spine, as well as in the non–weight-bearing caudal vertebrae. Osteocalcin and PINP are well-recognized markers of osteoblast activity, and the levels of both markers were elevated at week 3 (although the increase in PINP levels did not reach significance) in the mice in the vehicle/Scl-AbI group when compared with mice in the 2393 vehicle/Ctrl-Ab group (Figures 5A and C). Osteocalcin levels (Figure 5B) and PINP levels (results not shown) were measured again at week 6 and, by this point, they were no longer elevated. We have previously noted that the rate at which BMD increases in Scl-AbI–treated animals declines with time (19), which may help explain the failure to see elevated bone formation markers at the later time point in this study. In both DEX treatment groups, osteocalcin and PINP levels were significantly lower than those in mice not receiving DEX. The failure of Scl-AbI to elevate the levels of these osteoblast markers is surprising, in light of our findings showing more trabecular and cortical bone in Scl-AbI–treated mice. It is possible to speculate that the DEX effect on osteocalcin levels is a direct effect on the promoter (37), and that Scl-AbI could stimulate bone formation in the absence of osteocalcin, since this gene product is not essential for bone formation (38). Circulating PINP is derived from both osseous and nonosseous tissues, and this may complicate interpretation of changes in this marker in steroid-treated animals. Further work is needed to investigate the mechanisms of bone formation in animals treated with both DEX and Scl-AbI. Analysis of circulating levels of TRAP5b, a marker of osteoclast numbers, showed that at week 6, TRAP5b levels were elevated in DEX/Ctrl-Ab–treated mice when compared to mice receiving vehicle/Ctrl-Ab, consistent with a GC-induced increase in osteoclast activity. DEX/Scl-AbI–treated mice had significantly lower TRAP5b levels than did DEX/Ctrl-Ab–treated mice. This suggests that one of the positive effects of Scl-AbI in DEX-treated animals is a reduction of bone resorption. It has previously been observed that Scl-AbI can reduce circulating TRAP5b levels in mice (19). This observation was supported by the findings from a recent study in human volunteers (23), in which individuals treated with an antibody to sclerostin showed a dosedependent reduction in serum C-telopeptide (CTX) levels, a marker of bone resorption. In addition, it was recently suggested that sclerostin might have an important role in mediating GC-induced bone resorption. This suggestion was based on measurements of serum CTX in a GC-treated patient with van Buchem’s disease (a condition in which expression of sclerostin is defective in adults) (39). In summary, although Scl-AbI treatment does not prevent the GC-mediated inhibition of linear growth, it does prevent some of the detrimental effects of GCs on bone formation and bone strength. The molecular mechanisms by which Scl-AbI treatment generate improved bone strength and architecture in GCtreated animals will require further investigation. 2394 MARENZANA ET AL ACKNOWLEDGMENTS We thank the sclerostin team members from Amgen Inc. and UCB for their support of these studies. AUTHOR CONTRIBUTIONS All authors were involved in drafting the article or revising it critically for important intellectual content, and all authors approved the final version to be published. Dr. Robinson had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. Study conception and design. Marenzana, Greenslade, Marshall, Moore, Robinson. Acquisition of data. Marenzana, Greenslade, Eddleston, Okoye. Analysis and interpretation of data. Marenzana, Greenslade, Okoye, Marshall, Moore, Robinson. ROLE OF THE STUDY SPONSOR UCB Celltech employed all of the authors at the time of the study. The study design, execution, data analysis, and the decision to submit the manuscript for publication were solely the responsibility of the authors. In addition, the content of the manuscript was decided solely by the authors. Publication of this article was not contingent upon approval by UCB Celltech. REFERENCES 1. Curtis JR, Westfall AO, Allison J, Bijlsma JW, Freeman A, George V, et al. Population-based assessment of adverse events associated with long-term glucocorticoid use. Arthritis Rheum 2006;55:420–6. 2. Canalis E, Mazziotti G, Giustina A, Bilezikian JP. Glucocorticoidinduced osteoporosis: pathophysiology and therapy. Osteoporos Int 2007;18:1319–28. 3. Patschan D, Loddenkemper K, Buttgereit F. Molecular mechanisms of glucocorticoid-induced osteoporosis. Bone 2001;29:498–505. 4. Lane NE, Yao W, Balooch M, Nalla RK, Balooch G, Habelitz S, et al. Glucocorticoid-treated mice have localized changes in trabecular bone material properties and osteocyte lacunas size that are not observed in placebo-treated or estrogen-deficient mice. J Bone Miner Res 2006;21:466–76. 5. Avioli LV. Glucocorticoid effects on statural growth. Br J Rheumatol 1993;32 Suppl 2:27–30. 6. Allen DB. Growth suppression by glucocorticoid therapy. Endocrinol Metab Clin North Am 1996;25:699–717. 7. Atman A, Hochberg Z, Silbermann M. Interactions between growth hormone and dexamethasone in skeletal growth and bone structure of the young mouse. Calcif Tissue Int 1992;51:298–304. 8. Van der Eerden BC, Karperien M, Wit JM. Systemic and local regulation of the growth plate. Endocr Rev 2003;24:782–801. 9. Silvestrini G, Ballanti P, Patacchioli FR, Mocetti P, Di Grezia R, Wedard BM, et al. Evaluation of apoptosis and the glucocorticoid receptor in the cartilage growth plate and metaphyseal bone cells of rats after high-dose treatment with corticosterone. Bone 2000; 26:33–42. 10. Chrysis D, Ritzen EM, Savendahl L. Growth retardation induced by dexamethasone is associated with increased apoptosis of the growth plate chondrocytes. J Endocrinol 2003;176:331–7. 11. Smink JJ, Gresnigt MG, Hamers N, Koedam JA, Berger R, Van Buul-Offers SC. Short-term glucocorticoid treatment of prepubertal mice decreases growth and IGF-I expression in the growth plate. J Endocrinol 2003;177:381–8. 12. Koedam JA, Smink JJ, van Buul-Offers SC. Glucocorticoids inhibit vascular endothelial growth factor expression in growth plate chondrocytes. Mol Cell Endocrinol 2002;29;197:35–44. 13. Paszty C, Turner CH, Robinson MK. Sclerostin: a gem from the genome leads to bone-building antibodies. J Bone Miner Res 2010;25:1897–904. 14. Brunkow ME, Gardner JC, Van Ness J, Paeper BW, Kovacevich BR, Proll S, et al. Bone dysplasia sclerosteosis results from loss of the SOST gene product, a novel cystine knot-containing protein. Am J Hum Genet 2001;68:577–89. 15. Balemans W, Ebeling M, Patel N, Van Hul E, Olson P, Dioszegi M, et al. Increased bone density in sclerosteosis is due to the deficiency of a novel secreted protein (SOST). Hum Mol Genet 2001;10:537–43. 16. Beighton P. Sclerosteosis. J Med Genet 1988;25:200–3. 17. Winkler DG, Sutherland MK, Geoghegan JC, Yu C, Hayes T, Skonier JE, et al. Osteocyte control of bone formation via sclerostin, a novel BMP antagonist. EMBO J 2003;22:6267–76. 18. Van Bezooijen RL, Svensson JP, Eefting D, Visser A, van der Horst G, Karperien M, et al. Wnt but not BMP signaling is involved in the inhibitory action of sclerostin on BMP-stimulated bone formation. J Bone Miner Res 2007;22:19–28. 19. Eddleston A, Marenzana M, Moore AR, Stephens P, Muzylak M, Marshall D, et al. A short treatment with an antibody to sclerostin can inhibit bone loss in an ongoing model of colitis. J Bone Miner Res 2009;24:1662–71. 20. Li X, Ominsky MS, Warmington KS, Morony S, Gong J, Cao J, et al. Sclerostin antibody treatment increases bone formation, bone mass, and bone strength in a rat model of postmenopausal osteoporosis. J Bone Miner Res 2009;24:578–88. 21. Ominsky M, Vlasseros F, Jolette J, Smith S, Stouch B, Doellgast G, et al. Two doses of sclerostin antibody in cynomolgus monkeys increases bone formation, bone mineral density, and bone strength. J Bone Miner Res 2010;25:948–59. 22. Agholme F, Li X, Isaksson H, Ke HZ, Aspenberg P. Sclerostin antibody treatment enhances metaphyseal bone healing in rats. J Bone Miner Res 2010;25:2412–8. 23. Padhi D, Jang G, Stouch B, Fang L, Posvar E. Single-dose, placebo-controlled, randomized study of AMG 785, a sclerostin monoclonal antibody. J Bone Miner Res 2011;26:19–26. 24. Chen M, Zhu M, Awad H, Li TF, Sheu TJ, Boyce BF, et al. Inhibition of ␤-catenin signaling causes defects in postnatal cartilage development. J Cell Sci 2008;1;121:1455–65. 25. Van Bezooijen RL, Bronckers AL, Gortzak RA, Hogendoorn PC, van der Wee-Pals L, Balemans W, et al. Sclerostin in mineralized matrices and van Buchem disease. J Dent Res 2009;88:569–74. 26. Turner CH, Burr DB. Basic biomechanical measurements of bone: a tutorial. Bone 1993;14:595–606. 27. Krishnan V, Bryant HU, MacDougald OA. Regulation of bone mass by Wnt signaling. J Clin Invest 2006;116:1202–9. 28. Yao W, Cheng Z, Pham A, Busse C, Zimmermann EA, Ritchie RO, et al. Glucocorticoid-induced bone loss in mice can be reversed by the actions of parathyroid hormone and risedronate on different pathways for bone formation and mineralization. Arthritis Rheum 2008;58:3485–97. 29. Hayashi K, Yamaguchi T, Yano S, Kanazawa I, Yamauchi M, Yamamoto M, et al. BMP/Wnt antagonists are upregulated by dexamethasone in osteoblasts and reversed by alendronate and PTH: potential therapeutic targets for glucocorticoid-induced osteoporosis. Biochem Biophys Res Commun 2009;379:261–6. 30. Wang FS, Lin CL, Chen YJ, Wang CJ, Yang KD, Huang YT, et al. Secreted frizzled-related protein 1 modulates glucocorticoid attenuation of osteogenic activities and bone mass. Endocrinology 2005;146:2415–23. 31. Wang FS, Ko JY, Yeh DW, Ke HC, Wu HL. Modulation of Dickkopf-1 attenuates glucocorticoid induction of osteoblast apoptosis, adipocytic differentiation, and bone mass loss. Endocrinology 2008;149:1793–801. 32. Bellido T, Ali AA, Gubrij I, Plotkin LI, Fu Q, O’Brien CA, et al. Chronic elevation of parathyroid hormone in mice reduces expression of sclerostin by osteocytes: a novel mechanism for hor- EFFECTS OF SCLEROSTIN ANTIBODY ON BONE STRENGTH IN DEX-TREATED MICE monal control of osteoblastogenesis. Endocrinology 2005;146: 4577–83. 33. Leupin O, Kramer I, Collette NM, Loots GG, Natt F, Kneissel M, et al. Control of the SOST bone enhancer by PTH using MEF2 transcription factors. J Bone Miner Res 2007;22:1957–67. 34. Silvestrini G, Ballanti P, Leopizzi M, Sebastiani M, Berni S, Di Vito M, et al. Effects of intermittent parathyroid hormone (PTH) administration on SOST mRNA and protein in rat bone. J Mol Histol 2007;38:261–9. 35. Van Buul-Offers SC, Smink JJ, Gresnigt R, Hamers N, Koedam J, Karperien M. Thyroid hormone, but not parathyroid hormone, partially restores glucocorticoid-induced growth retardation. Pediatr Nephrol 2005;20:335–41. 2395 36. Oxlund H, Ortoft G, Thomsen JS, Danielsen CC, Ejersted C, Andreassen TT. The anabolic effect of PTH on bone is attenuated by simultaneous glucocorticoid treatment. Bone 2006;39: 244–52. 37. Leclerc N, Noh T, Khokhar A, Smith E, Frenkel B. Glucocorticoids inhibit osteocalcin transcription in osteoblasts by suppressing Egr2/Krox20-binding enhancer. Arthritis Rheum 2005;52:929–39. 38. Ducy P, Desbois C, Boyce B, Pinero G, Story B, Dunstan C, et al. Increased bone formation in osteocalcin-deficient mice. Nature 1996;382:448–52. 39. Van Lierop AH, Hamdy NA, Papapoulos SE. Glucocorticoids are not always deleterious for bone. J Bone Miner Res 2010;25: 2520–4.