Paul J. Kostenuik

Targeted deletion of the sclerostin gene in mice results in increased bone formation and bone strength.

Introduction: 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.

Sclerostin Antibody Treatment Increases Bone Formation, Bone Mass, and Bone Strength in a Rat Model of Postmenopausal Osteoporosis

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 sclerostins 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 sclerostins 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.

Denosumab, a Fully Human Monoclonal Antibody to RANKL, Inhibits Bone Resorption and Increases BMD in Knock‐In Mice That Express Chimeric (Murine/Human) RANKL

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.

Bench to bedside: elucidation of the OPG–RANK–RANKL pathway and the development of denosumab

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.

Effects of Parathyroid Hormone Treatment on Circulating Sclerostin Levels in Postmenopausal Women

CONTEXT Intermittent PTH treatment stimulates bone formation, but the mechanism(s) of this effect remain unclear. Sclerostin is an inhibitor of Wnt signaling, and animal studies have demonstrated that PTH suppresses sclerostin production. OBJECTIVE The objective of the study was to test whether intermittent PTH treatment of postmenopausal women alters circulating sclerostin levels. DESIGN Prospective study. SETTING The study was conducted at a clinical research unit. PARTICIPANTS AND INTERVENTIONS Participants included 27 postmenopausal women treated with PTH (1-34) for 14 d and 28 control women. MAIN OUTCOME MEASURES Serum sclerostin levels were measured. RESULTS Circulating sclerostin levels decreased significantly in the PTH-treated subjects, from (mean ± SEM) 551 ± 32 to 482 ± 31 pg/ml (-12.7%, P < 0.0001) but did not change in the control women (baseline, 559 ± 34 pg/ml; end point, 537 ± 40 pg/ml, P = 0.207; P = 0.017 for difference in changes between groups). Bone marrow plasma was obtained in a subset of the control and PTH-treated subjects (n = 19 each) at the end of the treatment period, and marrow plasma and peripheral serum sclerostin levels were significantly correlated (R = 0.64, P < 0.0001). Marrow plasma sclerostin levels were 24% lower in PTH-treated compared with control women, but perhaps due to the smaller sample size, this difference was not statistically significant (P = 0.173). CONCLUSIONS Circulating sclerostin levels correlate with bone marrow plasma levels and are reduced by intermittent PTH therapy in postmenopausal women. Further studies are needed to assess the extent to which decreases in sclerostin production contribute to the anabolic skeletal response to PTH.

Osteoprotegerin: a physiological and pharmacological inhibitor of bone resorption.

OPG is a new member of the tumor necrosis factor (TNF) receptor family which plays a key role in the physiological regulation of osteoclastic bone resorption. The protein, which is produced by osteoblasts and marrow stromal cells, lacks a transmembrane domain and acts as a secreted decoy receptor which has no direct signaling capacity. OPG acts by binding to its natural ligand OPGL, which is also known as RANKL (receptor activator of NF-kappaB ligand). This binding prevents OPGL from activating its cognate receptor RANK, which is the osteoclast receptor vital for osteoclast differentiation, activation and survival. Overexpression of OPG in transgenic mice leads to profound osteopetrosis secondary to a near total lack of osteoclasts. Conversely, ablation of the OPG gene causes severe osteoporosis in mice. Ablation of OPGL or RANK also produces profound osteopetrosis, indicating the important physiological role of these proteins in regulating bone resorption. The secretion of OPG and OPGL from osteoblasts and stromal cells is regulated by numerous hormones and cytokines, often in a reciprocal manner. The relative levels of OPG and OPGL production are thought to ultimately dictate the extent of bone resorption. Excess OPGL increases bone resorption, whereas excess OPG inhibits resorption. Recombinant OPG blocks the effects of virtually all factors which stimulate osteoclasts, in vitro and in vivo. OPG also inhibits bone resorption in a variety of animal disease models, including ovariectomy-induced osteoporosis, humoral hypercalcemia of malignancy, and experimental bone metastasis. OPG might represent an effective therapeutic option for diseases associated with excessive osteoclast activity.

Inhibition of sclerostin by monoclonal antibody increases bone formation, bone mass, and bone strength in aged male rats

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 (L1 to L5) 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 (L5), 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 L5, 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.

Effects of spaceflight on murine skeletal muscle gene expression

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.

Comparison of Effects of the Bisphosphonate Alendronate Versus the RANKL Inhibitor Denosumab on Murine Fracture Healing

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 ∼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. μCT 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.

Skeletal unloading causes resistance of osteoprogenitor cells to parathyroid hormone and to insulin-like growth factor-I

Skeletal unloading decreases bone formation and osteoblast number in vivo and decreases the number and proliferation of bone marrow osteoprogenitor (BMOp) cells in vitro. We tested the ability of parathyroid hormone (PTH) to stimulate BMOp cells in vivo by treating Sprague Dawley rats (n = 32) with intermittent PTH(1–34) (1 h/day at 8 μ g/100 g of body weight), or with vehicle via osmotic minipumps during 7 days of normal weight bearing or hind limb unloading. Marrow cells were flushed from the femur and cultured at the same initial density for up to 21 days. PTH treatment of normally loaded rats caused a 2.5‐fold increase in the number of BMOp cells, with similar increases in alkaline phosphatase (ALP) activity and mineralization, compared with cultures from vehicle‐treated rats. PTH treatment of hind limb unloaded rats failed to stimulate BMOp cell number, ALP activity, or mineralization. Hind limb unloading had no significant effect on PTH receptor mRNA or protein levels in the tibia. Direct in vitro PTH challenge of BMOp cells isolated from normally loaded bone failed to stimulate their proliferation and inhibited their differentiation, suggesting that the in vivo anabolic effect of intermittent PTH on BMOp cells was mediated indirectly by a PTH‐induced factor. We hypothesize that this factor is insulin‐like growth factor‐I (IGF‐I), which stimulated the in vitro proliferation and differentiation of BMOp cells isolated from normally loaded bone, but not from unloaded bone. These results suggest that IGF‐I mediates the ability of PTH to stimulate BMOp cell proliferation in normally loaded bone, and that BMOp cells in unloaded bone are resistant to the anabolic effect of intermittent PTH therapy due to their resistance to IGF‐I.