Degenerative and inflammatory joint diseases lead to a destruction of the joint architecture. Whereas degenerative osteoarthritis results in the formation of new bone, rheumatoid arthritis leads to bone resorption. The molecular basis of these different patterns of joint disease is unknown. By inhibiting Dickkopf-1 (DKK-1), a regulatory molecule of the Wnt pathway, we were able to reverse the bone-destructive pattern of a mouse model of rheumatoid arthritis to the bone-forming pattern of osteoarthritis. In this way, no overall bone erosion resulted, although bony nodules, so-called osteophytes, did form. We identified tumor necrosis factor-alpha (TNF) as a key inducer of DKK-1 in the mouse inflammatory arthritis model and in human rheumatoid arthritis. These results suggest that the Wnt pathway is a key regulator of joint remodeling.

ABSTRACT: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. Materials and Methods: Gene targeting was used to inactivate SOST and generate a line of SOST KO mice. Radiography, densitometry, CT, 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. Results: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. CT 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. Conclusions: 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.

Sclerostin, the Wnt signaling antagonist encoded by the Sost gene, is secreted by osteocytes and inhibits bone formation by osteoblasts. Mechanical stimulation reduces sclerostin expression, suggesting that osteocytes might coordinate the osteogenic response to mechanical force by locally unleashing Wnt signaling. To investigate whether sclerostin downregulation is a pre-requisite for load-induced bone formation, we conducted experiments in transgenic mice (TG) engineered to maintain high levels of SOST expression during mechanical loading. This was accomplished by introducing a human SOST transgene driven by the 8kb fragment of the DMP1 promoter that also provided osteocyte specificity of the transgene. Right ulnae were subjected to in vivo cyclic axial loading at equivalent strains for 1 min/day at 2Hz; left ulnae served as internal controls. Endogenous murine Sost mRNA expression measured 24h after 1 loading bout was decreased by about 50% in TG and wild type (WT) littermates. In contrast, human SOST, only expressed in TG mice, remained high after loading. Mice were loaded on 3 consecutive days and bone formation was quantified 16 days after initiation of loading. Periosteal bone formation in control ulnae was similar in WT and TG mice. Loading induced the expected strain-dependent increase in bone formation in WT mice, resulting from increases in both mineralizing surface (MS/BS) and mineral apposition rate (MAR). In contrast, load-induced bone formation was reduced by 70–85% in TG mice, due to lower MS/BS and complete inhibition of MAR. Moreover, Wnt target gene expression induced by loading in WT mice was absent in TG mice. Thus, downregulation of Sost/sclerostin in osteocytes is an obligatory step in the mechanotransduction cascade that activates Wnt signaling and directs osteogenesis to where bone is structurally needed.

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.

Therapeutic enhancement of fracture healing would help to prevent the occurrence of orthopedic complications such as nonunion and revision surgery. Sclerostin is a negative regulator of bone formation, and treatment with a sclerostin monoclonal antibody (Scl-Ab) results in increased bone formation and bone mass in animal models. Our objective was to investigate the effects of systemic administration of Scl-Ab in two models of fracture healing. In both a closed femoral fracture model in rats and a fibular osteotomy model in cynomolgus monkeys, Scl-Ab significantly increased bone mass and bone strength at the site of fracture. After 10 weeks of healing in nonhuman primates, the fractures in the Scl-Ab group had less callus cartilage and smaller fracture gaps containing more bone and less fibrovascular tissue. These improvements at the fracture site corresponded with improvements in bone formation, bone mass, and bone strength at nonfractured cortical and trabecular sites in both studies. Thus the potent anabolic activity of Scl-Ab throughout the skeleton also was associated with an anabolic effect at the site of fracture. These results support the potential for systemic Scl-Ab administration to enhance fracture healing in patients. ß

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 (mCT) 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 boneformation 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. ß

Background-The role of osteoprotegerin in vascular disease is unclear. Recent observational studies show that serum osteoprotegerin levels are associated with the severity and progression of coronary artery disease, atherosclerosis, and vascular calcification in patients. However, genetic and treatment studies in mice suggest that osteoprotegerin may protect against vascular calcification. Methods and Results-To test whether osteoprotegerin induces or prevents vascular disease, we treated atherogenic diet-fed ldlr (Ϫ/Ϫ) mice with recombinant osteoprotegerin (Fc-OPG) or vehicle for 5 months. Vehicle-treated mice developed significant, progressive atherosclerosis with increased plasma osteoprotegerin levels, consistent with observational studies, and Ϸ15% of these atherosclerotic lesions developed calcified cartilage-like metaplasia. Treatment with Fc-OPG significantly reduced the calcified lesion area without affecting atherosclerotic lesion size or number, vascular cytokines, or plasma cholesterol levels. Treatment also significantly reduced tissue levels of aortic osteocalcin, a marker of mineralization. Conclusions-These data support a role for osteoprotegerin in the vasculature as an inhibitor of calcification and a marker, rather than a mediator, of atherosclerosis. (Circulation. 2008;117:411-420.)

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.