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ABSTRACT: Denosumab is a fully human monoclonal antibody against RANK ligand (RANKL), an essential cytokine for the formation, function, and survival of osteoclasts. The role of excessive RANKL as a contributor to conditions characterized by bone loss or bone destruction has been well studied. With its novel mechanism of action, denosumab offers a significant advance in the treatment of postmenopausal osteoporosis; bone loss associated with hormone ablation therapy in women with breast cancer and men with prostate cancer; and the prevention of skeletal-related events in patients with bone metastases from solid tumors by offering clinical benefit to these patients in need.
Annals of the New York Academy of Sciences 07/2012; 1263:29-40. · 3.15 Impact Factor
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ABSTRACT: Bone is a complex tissue that provides mechanical support for muscles and joints, protection for vital organs, a mineral reservoir that is essential for calcium homeostasis, and the environment and niches required for haematopoiesis. The regulation of bone mass in mammals is governed by a complex interplay between bone-forming cells termed osteoblasts and bone-resorbing cells termed osteoclasts, and is guided physiologically by a diverse set of hormones, cytokines and growth factors. The balance between these processes changes over time, causing an elevated risk of fractures with age. Osteoclasts may also be activated in the cancer setting, leading to bone pain, fracture, spinal cord compression and other significant morbidities. This Review chronicles the events that led to an increased understanding of bone resorption, the elucidation of the signalling pathway mediated by osteoprotegerin, receptor activator of NF-κB (RANK) and RANK ligand (RANKL) and its role in osteoclast biology, as well as the evolution of recombinant RANKL antagonists, which culminated in the development of the therapeutic RANKL-targeted antibody denosumab.
dressNature Reviews Drug Discovery 05/2012; 11(5):401-19. · 29.01 Impact Factor
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ABSTRACT: A unilateral injection of botulinum toxin A (BTxA) in the calf induces paralysis and profound loss of ipsalateral trabecular bone within days. However, the cellular mechanism underlying acute muscle paralysis-induced bone loss (MPIBL) is poorly understood. We hypothesized that MPIBL arises via rapid and extensive osteoclastogenesis. We performed a series of in vivo experiments to explore this thesis. First, we observed elevated levels of the proosteoclastogenic cytokine receptor activator for nuclear factor-κB ligand (RANKL) within the proximal tibia metaphysis at 7 d after muscle paralysis (+113%, P<0.02). Accordingly, osteoclast numbers were increased 122% compared with the contralateral limb at 5 d after paralysis (P=0.04) and MPIBL was completely blocked by treatment with human recombinant osteoprotegerin (hrOPG). Further, conditional deletion of nuclear factor of activated T-cells c1 (NFATc1), the master regulator of osteoclastogenesis, completely inhibited trabecular bone loss (-2.2±11.9%, P<0.01). All experiments included negative control assessments of contralateral limbs and/or within-animal pre- and postintervention imaging. In summary, transient muscle paralysis induced acute RANKL-mediated osteoclastogenesis resulting in profound local bone resorption. Elucidation of the pathways that initiate osteoclastogenesis after paralysis may identify novel targets to inhibit bone loss and prevent fractures.
The FASEB Journal 11/2011; 26(3):1110-8. · 5.71 Impact Factor
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ABSTRACT: Denosumab is a fully human monoclonal antibody that inhibits RANKL, a protein essential for osteoclast formation, function, and survival. Osteoclast inhibition with denosumab decreased bone resorption, increased bone mineral density (BMD), and reduced fracture risk in osteoporotic women. The effects of 16months of continuous osteoclast inhibition on bone strength parameters were examined in adult ovariectomized (OVX) cynomolgus monkeys (cynos). One month after surgery, OVX cynos (n=14-20/group) were treated monthly with subcutaneous vehicle (OVX-Veh) or denosumab (25 or 50mg/kg). Sham-operated controls were treated with vehicle (n=17). OVX-Veh exhibited early and persistent increases in the resorption marker CTx, followed by similar increases in the formation marker BSAP, consistent with increased bone remodeling. Denosumab reduced CTx and BSAP throughout the study to levels significantly lower than in OVX-Veh or Sham-Veh, consistent with reduced remodeling. Increased remodeling in OVX-Veh led to absolute declines in areal BMD of 4.3-7.4% at the lumbar spine, total hip, femur neck, and distal radius (all p<0.05 vs baseline). Denosumab significantly increased aBMD at each site to levels exceeding baseline or OVX-Veh controls, and denosumab significantly increased cortical vBMC of the central radius and tibia by 7% and 14% (respectively) relative to OVX-Veh. Destructive biomechanical testing revealed that both doses of denosumab were associated with significantly greater peak load for femur neck (+19-34%), L3-L4 vertebral bodies (+54-55%), and L5-L6 cancellous cores (+69-82%) compared with OVX-Veh. Direct assessment of bone tissue material properties at cortical sites revealed no significant changes with denosumab. For all sites analyzed biomechanically, bone mass (BMC) and strength (load) exhibited strong linear correlations (r(2)=0.59-0.85 for all groups combined). Denosumab did not alter slopes of load-BMC regressions at any site, and denosumab groups exhibited similar or greater load values at given BMC values compared with OVX-Veh or Sham. In summary, denosumab markedly reduced biochemical markers of bone remodeling and increased cortical and trabecular bone mass in adult OVX cynos. Denosumab improved structural bone strength parameters at all sites analyzed, and strength remained highly correlated with bone mass. There was no evidence for reduced material strength properties of cortical bone with denosumab over this time period, which approximates to 4years of remodeling in the slower-remodeling adult human skeleton. These data indicate that denosumab increased bone strength by increasing bone mass and preserving bone quality.
Bone 08/2011; 49(2):162-73. · 4.02 Impact Factor
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Xiaodong Li,
Mario Grisanti,
Wei Fan,
Frank J Asuncion,
Hong-Lin Tan,
Denise Dwyer,
Chun-Ya Han,
Longchuan Yu,
Jae Lee,
Edward Lee, [......],
Ji Li,
Min Liu,
Chaoyang Li,
David L Lacey,
W Scott Simonet,
Hua Zhu Ke,
Philip Babij,
Marina Stolina,
Michael S Ominsky,
William G Richards
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ABSTRACT: The physiological role of Dickkopf-1 (Dkk1) during postnatal bone growth in rodents and in adult rodents was examined utilizing an antibody to Dkk1 (Dkk1-Ab) that blocked Dkk1 binding to both low density lipoprotein receptor-related protein 6 (LRP6) and Kremen2, thereby preventing the Wnt inhibitory activity of Dkk1. Treatment of growing mice and rats with Dkk1-Ab resulted in a significant increase in bone mineral density because of increased bone formation. In contrast, treatment of adult ovariectomized rats did not appreciably impact bone, an effect that was associated with decreased Dkk1 expression in the serum and bone of older rats. Finally, we showed that Dkk1 plays a prominent role in adult bone by mediating fracture healing in adult rodents. These data suggest that, whereas Dkk1 significantly regulates bone formation in younger animals, its role in older animals is limited to pathologies that lead to the induction of Dkk1 expression in bone and/or serum, such as traumatic injury.
Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research 07/2011; 26(11):2610-21. · 6.04 Impact Factor
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Xiaodong Li,
Michael S Ominsky,
Kelly S Warmington,
Qing-Tian Niu,
Franklin J Asuncion,
Mauricio Barrero,
Denise Dwyer,
Mario Grisanti,
Marina Stolina, Paul J Kostenuik,
William S Simonet,
Chris Paszty,
Hua Zhu Ke
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ABSTRACT: Clinical studies have revealed a blunting of the bone anabolic effects of parathyroid hormone treatment in osteoporotic patients in the setting of pre- or cotreatment with the antiresorptive agent alendronate (ALN). Sclerostin monoclonal antibody (Scl-Ab) is currently under clinical investigation as a new potential anabolic therapy for postmenopausal osteoporosis. The purpose of these experiments was to examine the influence of pretreatment or cotreatment with ALN on the bone anabolic actions of Scl-Ab in ovariectomized (OVX) rats. Ten-month-old osteopenic OVX rats were treated with ALN or vehicle for 6 wk, before the start of Scl-Ab treatment. ALN-pretreated OVX rats were switched to Scl-Ab alone or to a combination of ALN and Scl-Ab for another 6 wk. Vehicle-pretreated OVX rats were switched to Scl-Ab or continued on vehicle to serve as controls. Scl-Ab treatment increased areal bone mineral density, volumetric bone mineral density, trabecular and cortical bone mass, and bone strength similarly in OVX rats pretreated with ALN or vehicle. Serum osteocalcin and bone formation rate on trabecular, endocortical, and periosteal surfaces responded similarly to Scl-Ab in ALN or vehicle-pretreated OVX rats. Furthermore, cotreatment with ALN did not have significant effects on the increased bone formation, bone mass, and bone strength induced by Scl-Ab in the OVX rats that were pretreated with ALN. These results indicate that the increases in bone formation, bone mass, and bone strength with Scl-Ab treatment were not affected by pre- or cotreatment with ALN in OVX rats with established osteopenia.
Endocrinology 07/2011; 152(9):3312-22. · 4.46 Impact Factor
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ABSTRACT: This study examined the effects of denosumab, an anti-RANKL antibody that inhibits bone resorption, on bone histomorphometry in adult ovariectomized cynomolgus monkeys (OVX cynos). A month after surgery, OVX cynos were treated with subcutaneous vehicle (OVX-Veh) or denosumab (25 or 50mg/kg/month) for 16months (n=14-20/group). Sham controls were treated with vehicle (Sham-Veh; n=17). Areal and volumetric BMD, urine NTx, and serum osteocalcin were measured at baseline and months 3, 6, 12, and 16. Double fluorochrome labels were injected prior to iliac and rib biopsies at month 6 and month 12, and prior to sacrifice at month 16. Histomorphometry was performed on these biopsies, the tibial diaphysis, the L2 vertebra, and the proximal femur. Strength of humeral cortical beams, femur diaphysis, femur neck, and trabecular cores of L5-L6 vertebrae was determined by destructive biomechanical testing. There was no evidence of woven bone, osteomalacia, or other bone histopathologic changes with OVX or with denosumab. OVX-Veh animals exhibited significantly greater bone remodeling at all skeletal sites relative to Sham-Veh controls. Both doses of denosumab markedly inhibited bone remodeling at all sites, including significant reductions in trabecular eroded surfaces (48-86% lower than OVX-Veh controls), cortical porosity (28-72% lower), and dynamic parameters of bone formation (81-100% lower). Decreased fluorochrome labeling with denosumab was related to reductions in cortical porosity and trabecular eroded surfaces, and regression analyses suggested that these reductions contributed to denosumab-related increments in BMD and bone strength. Denosumab-treated animals with the lowest levels of fluorescent labeling exhibited the greatest structural bone strength values at each site. Intracortical remodeling had no relationship with material properties including ultimate strength, elastic modulus or toughness (r(2)=0.00-0.01). These data suggest that remodeling inhibition with denosumab improved structural strength without altering material properties under these experimental conditions. Greater structural strength in the denosumab-treated animals can be primarily explained by the combined effects of increased trabecular and cortical bone mass, and reductions in trabecular eroded surfaces and cortical porosity.
Bone 03/2011; 49(2):151-61. · 4.02 Impact Factor
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Xiaodong Li,
Kelly S Warmington,
Qing-Tian Niu,
Franklin J Asuncion,
Mauricio Barrero,
Mario Grisanti,
Denise Dwyer,
Brian Stouch,
Theingi M Thway,
Marina Stolina,
Michael S Ominsky, Paul J Kostenuik,
William S Simonet,
Chris Paszty,
Hua Zhu Ke
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ABSTRACT: The purpose of this study was to evaluate the effects of sclerostin inhibition by treatment with a sclerostin antibody (Scl-AbII) on bone formation, bone mass, and bone strength in an aged, gonad-intact male rat model. Sixteen-month-old male Sprague-Dawley rats were injected subcutaneously with vehicle or Scl-AbII at 5 or 25 mg/kg twice per week for 5 weeks (9-10/group). In vivo dual-energy X-ray absorptiometry (DXA) analysis showed that there was a marked increase in areal bone mineral density of the lumbar vertebrae (L(1) to L(5) ) and long bones (femur and tibia) in both the 5 and 25 mg/kg Scl-AbII-treated groups compared with baseline or vehicle controls at 3 and 5 weeks after treatment. Ex vivo micro-computed tomographic (µCT) analysis demonstrated improved trabecular and cortical architecture at the fifth lumbar vertebral body (L(5) ), femoral diaphysis (FD), and femoral neck (FN) in both Scl-AbII dose groups compared with vehicle controls. The increased cortical and trabecular bone mass was associated with a significantly higher maximal load of L(5) , FD, and FN in the high-dose group. Bone-formation parameters (ie, mineralizing surface, mineral apposition rate, and bone-formation rate) at the proximal tibial metaphysis and tibial shaft were markedly greater on trabecular, periosteal, and endocortical surfaces in both Scl-AbII dose groups compared with controls. These results indicate that sclerostin inhibition by treatment with a sclerostin antibody increased bone formation, bone mass, and bone strength in aged male rats and, furthermore, suggest that pharmacologic inhibition of sclerostin may represent a promising anabolic therapy for low bone mass in aged men.
Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research 12/2010; 25(12):2647-56. · 6.04 Impact Factor
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ABSTRACT: PTH stimulates osteoblastic cells to form new bone and to produce osteoblast-osteoclast coupling factors such as RANKL. Whether osteoclasts or their activity are needed for PTH anabolism remains uncertain. We treated ovariectomized huRANKL knock-in mice with a human RANKL inhibitor denosumab (DMAb), alendronate (Aln), or vehicle for 4 weeks, followed by co-treatment with intermittent PTH for 4 weeks. Loss of bone mass and microarchitecture was prevented by Aln and further significantly improved by DMAb. PTH improved bone mass, microstructure, and strength, and was additive to Aln but not to DMAb. Aln inhibited biochemical and histomorphometrical indices of bone turnover,--i.e. osteocalcin and bone formation rate (BFR) on cancellous bone surfaces-, and Dmab inhibited them further. However Aln increased whereas Dmab suppressed osteoclast number and surfaces. PTH significantly increased osteocalcin and bone formation indices, in the absence or presence of either antiresorptive, although BFR remained lower in presence of Dmab. To further evaluate PTH effects in the complete absence of osteoclasts, high dose PTH was administered to RANK(-/-) mice. PTH increased osteocalcin similarly in RANK(-/-) and WT mice. It also increased BMD in RANK(-/-) mice, although less than in WT. These results further indicate that osteoclasts are not strictly required for PTH anabolism, which presumably still occurs via stimulation of modeling-based bone formation. However the magnitude of PTH anabolic effects on the skeleton, in particular its additive effects with antiresorptives, depends on the extent of the remodeling space, as determined by the number and activity of osteoclasts on bone surfaces.
Journal of Biological Chemistry 09/2010; 285(36):28164-73. · 4.77 Impact Factor
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ABSTRACT: PTH stimulates osteoblastic cells to form new bone and to produce osteoblast-osteoclast coupling factors such as RANKL. Whether
osteoclasts or their activity are needed for PTH anabolism remains uncertain. We treated ovariectomized huRANKL knock-in mice
with a human RANKL inhibitor denosumab (DMAb), alendronate (Aln), or vehicle for 4 weeks, followed by co-treatment with intermittent
PTH for 4 weeks. Loss of bone mass and microarchitecture was prevented by Aln and further significantly improved by DMAb.
PTH improved bone mass, microstructure, and strength, and was additive to Aln but not to DMAb. Aln inhibited biochemical and
histomorphometrical indices of bone turnover, -i.e. osteocalcin and bone formation rate (BFR) on cancellous bone surfaces-, and Dmab inhibited them further. However Aln increased
whereas Dmab suppressed osteoclast number and surfaces. PTH significantly increased osteocalcin and bone formation indices,
in the absence or presence of either antiresorptive, although BFR remained lower in presence of Dmab. To further evaluate
PTH effects in the complete absence of osteoclasts, high dose PTH was administered to RANK−/− mice. PTH increased osteocalcin similarly in RANK−/− and WT mice. It also increased BMD in RANK−/− mice, although less than in WT. These results further indicate that osteoclasts are not strictly required for PTH anabolism,
which presumably still occurs via stimulation of modeling-based bone formation. However the magnitude of PTH anabolic effects
on the skeleton, in particular its additive effects with antiresorptives, depends on the extent of the remodeling space, as
determined by the number and activity of osteoclasts on bone surfaces.
Journal of Biological Chemistry 09/2010; 285(36):28164-28173. · 4.77 Impact Factor
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ABSTRACT: Osteoporosis and vascular calcification frequently coincide. A potential mediator of bone metabolism and vascular homeostasis is the triad cytokine system, which consists of receptor activator of nuclear factor-kappaB (RANK) ligand (RANKL), its receptor RANK, and the decoy receptor osteoprotegerin. Unopposed RANKL activity in osteoprotegerin-deficient mice resulted in osteoporosis and vascular calcification. We therefore analyzed the effects of RANKL inhibition by denosumab, a human monoclonal antibody against RANKL, on vascular calcium deposition following glucocorticoid exposure. Prednisolone pellets were implanted into human RANKL knock-in (huRANKL-KI) mice, which unlike wild-type mice are responsive to denosumab. No histomorphological abnormalities or differences in aortic wall thickness were detected between wild-type and huRANKL-KI mice, regardless of treatment with prednisolone, denosumab, or both. However, concurrent treatment with denosumab reduced aortic calcium deposition of prednisolone-treated huRANKL-KI mice by up to 50%, based on calcium measurement. Of note, aortic calcium deposition in huRANKL-KI mice was correlated negatively with bone mineral density at the lumbar spine (P = 0.04) and positively with urinary excretion of deoxypyridinoline, a marker of bone resorption (P = 0.01). In summary, RANKL inhibition by denosumab reduced vascular calcium deposition in glucocorticoid-induced osteoporosis in mice, which is further evidence for the link between the bone and vascular systems. Therefore, the prevention of bone loss by denosumab might also be associated with reduced vascular calcification in certain conditions.
American Journal Of Pathology 09/2009; 175(2):473-8. · 4.89 Impact Factor
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Xiaodong Li,
Michael S Ominsky,
Marina Stolina,
Kelly S Warmington,
Zhaopo Geng,
Qing-Tian Niu,
Frank J Asuncion,
Hong-Lin Tan,
Mario Grisanti,
Denise Dwyer,
Steven Adamu,
Hua Zhu Ke,
W Scott Simonet, Paul J Kostenuik
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ABSTRACT: Orchiectomized (ORX) rats were used to examine the extent to which their increased bone resorption and decreased bone density might relate to increases in RANKL, an essential cytokine for bone resorption. Serum testosterone declined by >95% in ORX rats 1 and 2 weeks after surgery (p<0.05 versus sham controls), with no observed changes in serum RANKL. In contrast, RANKL in bone marrow plasma and bone marrow cell extracts was significantly increased (by approximately 100%) 1 and 2 weeks after ORX. Regression analyses of ORX and sham controls revealed a significant inverse correlation between testosterone and RANKL levels measured in marrow cell extracts (R=-0.58), while marrow plasma RANKL correlated positively with marrow plasma TRACP-5b, an osteoclast marker (R=0.63). The effects of RANKL inhibition were then studied by treating ORX rats for 6 weeks with OPG-Fc (10 mg/kg, twice/week SC) or with PBS, beginning immediately after surgery. Sham controls were treated with PBS. Vehicle-treated ORX rats showed significant deficits in BMD of the femur/tibia and lower trabecular bone volume in the distal femur (p<0.05 versus sham). OPG-Fc treatment of ORX rats increased femur/tibia BMD and trabecular bone volume to levels that significantly exceeded values for ORX or sham controls. OPG-Fc reduced trabecular osteoclast surfaces in ORX rats by 99%, and OPG-Fc also prevented ORX-related increases in endocortical eroded surface and ORX-related reductions in periosteal bone formation rate. Micro-CT of lumbar vertebrae from OPG-Fc-treated ORX rats demonstrated significantly greater cortical and trabecular bone volume and density versus ORX-vehicle controls. In summary, ORX rats exhibited increased RANKL protein in bone marrow plasma and in bone marrow cells, with no changes in serum RANKL. Data from regression analyses were consistent with a potential role for testosterone in suppressing RANKL production in bone marrow, and also suggested that soluble RANKL in bone marrow might promote bone resorption. RANKL inhibition prevented ORX-related deficits in trabecular BMD, trabecular architecture, and periosteal bone formation while increasing cortical and trabecular bone volume and density. These results support the investigation of RANKL inhibition as a strategy for preventing bone loss associated with androgen ablation or deficiency.
Bone 06/2009; 45(4):669-76. · 4.02 Impact Factor
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ABSTRACT: RANKL has been implicated in the pathogenesis of glucocorticoid-induced osteoporosis. This study was undertaken to evaluate the efficacy of denosumab, a neutralizing monoclonal antibody against human RANKL (hRANKL), in a murine model of glucocorticoid-induced osteoporosis.
Eight-month-old male homozygous hRANKL-knockin mice expressing a chimeric RANKL protein with a humanized exon 5 received 2.1 mg/kg of prednisolone or placebo daily over 4 weeks via subcutaneous slow-release pellets and were additionally treated with phosphate buffered saline or denosumab (10 mg/kg subcutaneously twice weekly). Two groups of wild-type mice were also treated with either prednisolone or vehicle.
The 4-week prednisolone treatment induced loss of vertebral and femoral volumetric bone mineral density in the hRANKL-knockin mice. Glucocorticoid-induced bone loss was associated with suppressed vertebral bone formation and increased bone resorption, as evidenced by increases in the number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts, TRAP-5b protein in bone extracts, serum levels of TRAP-5b, and urinary excretion of deoxypyridinoline. Denosumab prevented prednisolone-induced bone loss by a pronounced antiresorptive effect. Biomechanical compression tests of lumbar vertebrae revealed a detrimental effect of prednisolone on bone strength that was prevented by denosumab.
Our findings indicate that RANKL inhibition by denosumab prevents glucocorticoid-induced loss of bone mass and strength in hRANKL-knockin mice.
Arthritis & Rheumatism 05/2009; 60(5):1427-37. · 7.87 Impact Factor
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Michael S Ominsky,
Marina Stolina,
Xiaodong Li,
Timothy J Corbin,
Franklin J Asuncion,
Mauricio Barrero,
Qing‐Tian Niu,
Denise Dwyer,
Steven Adamu,
Kelly S Warmington,
Mario Grisanti,
Hong L Tan,
Hua Z Ke,
William S Simonet, Paul J Kostenuik
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ABSTRACT: RANKL is an essential mediator of bone resorption, and its activity is inhibited by osteoprotegerin (OPG). Transgenic (Tg) rats were engineered to continuously overexpress OPG to study the effects of continuous long-term RANKL inhibition on bone volume, density, and strength. Lumbar vertebrae, femurs, and blood were obtained from 1-yr-old female OPG-Tg rats (n = 32) and from age-matched wildtype (WT) controls (n = 23). OPG-Tg rats had significantly greater serum OPG (up to 260-fold) and significantly lower serum TRACP5b and osteocalcin compared with WT controls. Vertebral histomorphometry showed significant reductions in osteoclasts and bone turnover parameters in OPG-Tg rats versus WT controls, and these reductions were associated with significantly greater peak load in vertebrae tested through compression. No apparent differences in bone material properties were observed in OPG-Tg rat vertebrae, based on their unchanged intrinsic strength parameters and their normal linear relationship between vertebral bone mass and strength. Femurs from OPG-Tg rats were of normal length but showed mild osteopetrotic changes, including reduced periosteal perimeter (−6%) and an associated reduction in bending strength. Serum OPG levels in WT rats showed no correlations with any measured parameter of bone turnover, mass, or strength, whereas the supraphysiological serum OPG levels in OPG-Tg rats correlated negatively with bone turnover parameters and positively with vertebral bone mass and strength parameters. In summary, low bone turnover after 1 yr of OPG overexpression in rats was associated with increased vertebral bone mass and proportional increases in bone strength, with no evidence for deleterious effects on vertebral material properties.
Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research 02/2009; 24(7):1234 - 1246. · 6.04 Impact Factor
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Xiaodong Li,
Michael S Ominsky,
Kelly S Warmington,
Sean Morony,
Jianhua Gong,
Jin Cao,
Yongming Gao,
Victoria Shalhoub,
Barbara Tipton,
Raj Haldankar,
Qing Chen,
Aaron Winters,
Tom Boone,
Zhaopo Geng,
Qing-Tian Niu,
Hua Zhu Ke, Paul J Kostenuik,
W Scott Simonet,
David L Lacey,
Chris Paszty
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ABSTRACT: The development of bone-rebuilding anabolic agents for potential use in the treatment of bone loss conditions, such as osteoporosis, has been a long-standing goal. Genetic studies in humans and mice have shown that the secreted protein sclerostin is a key negative regulator of bone formation, although the magnitude and extent of sclerostin's role in the control of bone formation in the aging skeleton is still unclear. To study this unexplored area of sclerostin biology and to assess the pharmacologic effects of sclerostin inhibition, we used a cell culture model of bone formation to identify a sclerostin neutralizing monoclonal antibody (Scl-AbII) for testing in an aged ovariectomized rat model of postmenopausal osteoporosis. Six-month-old female rats were ovariectomized and left untreated for 1 yr to allow for significant estrogen deficiency-induced bone loss, at which point Scl-AbII was administered for 5 wk. Scl-AbII treatment in these animals had robust anabolic effects, with marked increases in bone formation on trabecular, periosteal, endocortical, and intracortical surfaces. This not only resulted in complete reversal, at several skeletal sites, of the 1 yr of estrogen deficiency-induced bone loss, but also further increased bone mass and bone strength to levels greater than those found in non-ovariectomized control rats. Taken together, these preclinical results establish sclerostin's role as a pivotal negative regulator of bone formation in the aging skeleton and, furthermore, suggest that antibody-mediated inhibition of sclerostin represents a promising new therapeutic approach for the anabolic treatment of bone-related disorders, such as postmenopausal osteoporosis.
Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research 01/2009; 24(4):578-88. · 6.04 Impact Factor
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David L Allen,
Eric R Bandstra,
Brooke C Harrison,
Seiha Thorng,
Louis S Stodieck, Paul J Kostenuik,
Sean Morony,
David L Lacey,
Timothy G Hammond,
Leslie L Leinwand,
W Scott Argraves,
Ted A Bateman,
Jeremy L Barth
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ABSTRACT: Spaceflight results in a number of adaptations to skeletal muscle, including atrophy and shifts toward faster muscle fiber types. To identify changes in gene expression that may underlie these adaptations, we used both microarray expression analysis and real-time polymerase chain reaction to quantify shifts in mRNA levels in the gastrocnemius from mice flown on the 11-day, 19-h STS-108 shuttle flight and from normal gravity controls. Spaceflight data also were compared with the ground-based unloading model of hindlimb suspension, with one group of pure suspension and one of suspension followed by 3.5 h of reloading to mimic the time between landing and euthanization of the spaceflight mice. Analysis of microarray data revealed that 272 mRNAs were significantly altered by spaceflight, the majority of which displayed similar responses to hindlimb suspension, whereas reloading tended to counteract these responses. Several mRNAs altered by spaceflight were associated with muscle growth, including the phosphatidylinositol 3-kinase regulatory subunit p85alpha, insulin response substrate-1, the forkhead box O1 transcription factor, and MAFbx/atrogin1. Moreover, myostatin mRNA expression tended to increase, whereas mRNA levels of the myostatin inhibitor FSTL3 tended to decrease, in response to spaceflight. In addition, mRNA levels of the slow oxidative fiber-associated transcriptional coactivator peroxisome proliferator-associated receptor (PPAR)-gamma coactivator-1alpha and the transcription factor PPAR-alpha were significantly decreased in spaceflight gastrocnemius. Finally, spaceflight resulted in a significant decrease in levels of the microRNA miR-206. Together these data demonstrate that spaceflight induces significant changes in mRNA expression of genes associated with muscle growth and fiber type.
Journal of Applied Physiology 01/2009; 106(2):582-95. · 3.75 Impact Factor
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ABSTRACT: The role of osteoclast-mediated resorption during fracture healing was assessed. The impact of two osteoclast inhibitors with different mechanisms of action, alendronate (ALN) and denosumab (DMAB), were examined during fracture healing. Male human RANKL knock-in mice that express a chimeric (human/murine) form of RANKL received unilateral transverse femur fractures. Mice were treated biweekly with ALN 0.1 mg/kg, DMAB 10 mg/kg, or PBS (control) 0.1 ml until death at 21 and 42 days after fracture. Treatment efficacy assessed by serum levels of TRACP 5b showed almost a complete elimination of TRACP 5b levels in the DMAB-treated animals but only approximately 25% reduction of serum levels in the ALN-treated mice. Mechanical testing showed that fractured femurs from both ALN and DMAB groups had significantly increased mechanical properties at day 42 compared with controls. muCT analysis showed that callus tissues from DMAB-treated mice had significantly greater percent bone volume and BMD than did both control and ALN-treated tissues at both 21 and 42 days, whereas ALN-treated bones only had greater percent bone volume and BMC than control at 42 days. Qualitative histological analysis showed that the 21-and 42-day ALN and DMAB groups had greater amounts of unresorbed cartilage or mineralized cartilage matrix compared with the controls, whereas unresorbed cartilage could still be seen in the DMAB groups at 42 days after fracture. Although ALN and DMAB delayed the removal of cartilage and the remodeling of the fracture callus, this did not diminish the mechanical integrity of the healing fractures in mice receiving these treatments. In contrast, strength and stiffness were enhanced in these treatment groups compared with control bones.
Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research 12/2008; 24(2):196-208. · 6.04 Impact Factor
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ABSTRACT: Rats with collagen-induced arthritis (CIA) were necropsied on 14 occasions from 4 days after induction to 27 days after disease onset to evaluate the kinetics of local (joint protein extracts) and systemic (serum) levels of inflammatory and pro-erosive factors. Systemic increases in alpha1 acid glycoprotein and KC/GRO together with systemic and local enrichment of interleukin (IL)-1beta, IL-6, CCL2, transforming growth factor (TGF)-beta and elevated IL-1alpha and IL-18 in joint extracts preceded the onset of clinical disease. Systemic upregulation of IL-1beta, IL-6, TGF-beta CCL2, RANKL and prostaglandin E(2) (PGE(2)) during acute and/or chronic CIA coincided with systemic leukocytosis and a CD4+ T-cell increase in blood and spleen. In contrast, progression of joint erosions during clinical CIA was associated with intra-articular increases in IL-1alpha/beta, IL-6, IL-18, CCL2, KC/GRO and RANKL, and a dramatic decline in osteoprotegerin (OPG). These data indicate that systemic and local events in inflammatory arthritis can be discrete processes, driven by multiple cellular and humoral mediators with distinct temporospatial profiles.
Biomarkers 12/2008; 13(7):692-712. · 2.21 Impact Factor
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Paul J Kostenuik,
Hung Q Nguyen,
James McCabe,
Kelly S Warmington,
Carol Kurahara,
Ning Sun,
Ching Chen,
Luke Li,
Russ C Cattley,
Gwyneth Van, [......],
Frank Asuncion,
Xiaodong Li,
Michael S Ominsky,
Marina Stolina,
Denise Dwyer,
William C Dougall,
Nessa Hawkins,
William J Boyle,
William S Simonet,
John K Sullivan
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ABSTRACT: RANKL is a TNF family member that mediates osteoclast formation, activation, and survival by activating RANK. The proresorptive effects of RANKL are prevented by binding to its soluble inhibitor osteoprotegerin (OPG). Recombinant human OPG-Fc recognizes RANKL from multiple species and reduced bone resorption and increased bone volume, density, and strength in a number of rodent models of bone disease. The clinical development of OPG-Fc was discontinued in favor of denosumab, a fully human monoclonal antibody that specifically inhibits primate RANKL. Direct binding assays showed that denosumab bound to human RANKL but not to murine RANKL, human TRAIL, or other human TNF family members. Denosumab did not suppress bone resorption in normal mice or rats but did prevent the resorptive response in mice challenged with a human RANKL fragment encoded primarily by the fifth exon of the RANKL gene. To create mice that were responsive to denosumab, knock-in technology was used to replace exon 5 from murine RANKL with its human ortholog. The resulting “huRANKL” mice exclusively express chimeric (human/murine) RANKL that was measurable with a human RANKL assay and that maintained bone resorption at slightly reduced levels versus wildtype controls. In young huRANKL mice, denosumab and OPG-Fc each reduced trabecular osteoclast surfaces by 95% and increased bone density and volume. In adult huRANKL mice, denosumab reduced bone resorption, increased cortical and cancellous bone mass, and improved trabecular microarchitecture. These huRANKL mice have potential utility for characterizing the activity of denosumab in a variety of murine bone disease models.
Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research 11/2008; 24(2):182 - 195. · 6.04 Impact Factor
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Xiaodong Li,
Michael S Ominsky,
Qing-Tian Niu,
Ning Sun,
Betsy Daugherty,
Diane D'Agostin,
Carole Kurahara,
Yongming Gao,
Jin Cao,
Jianhua Gong, [......],
Kelly Warmington,
Denise Dwyer,
Marina Stolina,
Sean Morony,
Ildiko Sarosi, Paul J Kostenuik,
David L Lacey,
W Scott Simonet,
Hua Zhu Ke,
Chris Paszty
[show abstract]
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ABSTRACT: Sclerosteosis is a rare high bone mass genetic disorder in humans caused by inactivating mutations in SOST, the gene encoding sclerostin. Based on these data, sclerostin has emerged as a key negative regulator of bone mass. We generated SOST knockout (KO) mice to gain a more detailed understanding of the effects of sclerostin deficiency on bone.
Gene targeting was used to inactivate SOST and generate a line of SOST KO mice. Radiography, densitometry, microCT, histomorphometry, and mechanical testing were used to characterize the impact of sclerostin deficiency on bone in male and female mice. Comparisons were made between same sex KO and wildtype (WT) mice.
The results for male and female SOST KO mice were similar, with differences only in the magnitude of some effects. SOST KO mice had increased radiodensity throughout the skeleton, with general skeletal morphology being normal in appearance. DXA analysis of lumbar vertebrae and whole leg showed that there was a significant increase in BMD (>50%) at both sites. microCT analysis of femur showed that bone volume was significantly increased in both the trabecular and cortical compartments. Histomorphometry of trabecular bone revealed a significant increase in osteoblast surface and no significant change in osteoclast surface in SOST KO mice. The bone formation rate in SOST KO mice was significantly increased for trabecular bone (>9-fold) at the distal femur, as well as for the endocortical and periosteal surfaces of the femur midshaft. Mechanical testing of lumbar vertebrae and femur showed that bone strength was significantly increased at both sites in SOST KO mice.
SOST KO mice have a high bone mass phenotype characterized by marked increases in BMD, bone volume, bone formation, and bone strength. These results show that sclerostin is a key negative regulator of a powerful, evolutionarily conserved bone formation pathway that acts on both trabecular and cortical bone.
Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research 06/2008; 23(6):860-9. · 6.04 Impact Factor
