Marina Stolina

Dickkopf-1 is a master regulator of joint remodeling

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-α (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.

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.

APRIL and TALL-1 and receptors BCMA and TACI: system for regulating humoralimmunity

We report that the tumor neurosis factor homolog APRIL (a proliferation-inducing ligand) stimulates in vitro proliferation of primary B and T cells and increases spleen weight due to accumulation of B cells in vivo. APRIL functions via binding to BCMA (B cell maturation antigen) and TACI (transmembrane activator and CAML-interactor) and competes with TALL-1 (also called BLyS or BAFF) for receptor binding. Soluble BCMA and TACI specifically prevent binding of APRIL and block APRIL-stimulated proliferation of primary B cells. BCMA-Fc also inhibits production of antibodies against keyhole limpet hemocyanin and Pneumovax in mice, indicating that APRIL and/or TALL-1 signaling via BCMA and/or TACI are required for generation of humoral immunity. Thus, APRIL–TALL-1 and BCMA-TACI form a two ligands–two receptors pathway involved in stimulation of B and T cell function.

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.

Sost downregulation and local Wnt signaling are required for the osteogenic response to mechanical loading

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 8 kb 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 2 Hz; left ulnae served as internal controls. Endogenous murine Sost mRNA expression measured 24 h 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.

Inhibition of sclerostin by monoclonal antibody enhances bone healing and improves bone density and strength of nonfractured bones.

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.

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.

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.

Sclerostin antibody inhibits skeletal deterioration due to reduced mechanical loading

Sclerostin, a product of the SOST gene produced mainly by osteocytes, is a potent negative regulator of bone formation that appears to be responsive to mechanical loading, with SOST expression increasing following mechanical unloading. We tested the ability of a murine sclerostin antibody (SclAbII) to prevent bone loss in adult mice subjected to hindlimb unloading (HLU) via tail suspension for 21 days. Mice (n = 11–17/group) were assigned to control (CON, normal weight bearing) or HLU and injected with either SclAbII (subcutaneously, 25 mg/kg) or vehicle (VEH) twice weekly. SclAbII completely inhibited the bone deterioration due to disuse, and induced bone formation such that bone properties in HLU‐SclAbII were at or above values of CON‐VEH mice. For example, hindlimb bone mineral density (BMD) decreased –9.2% ± 1.0% in HLU‐VEH, whereas it increased 4.2% ± 0.7%, 13.1% ± 1.0%, and 30.6% ± 3.0% in CON‐VEH, HLU‐SclAbII, and CON‐SclAbII, respectively (p < 0.0001). Trabecular bone volume, assessed by micro–computed tomography (µCT) imaging of the distal femur, was lower in HLU‐VEH versus CON‐VEH (p < 0.05), and was 2‐ to 3‐fold higher in SclAbII groups versus VEH (p < 0.001). Midshaft femoral strength, assessed by three‐point bending, and distal femoral strength, assessed by micro–finite element analysis (µFEA), were significantly higher in SclAbII versus VEH‐groups in both loading conditions. Serum sclerostin was higher in HLU‐VEH (134 ± 5 pg/mL) compared to CON‐VEH (116 ± 6 pg/mL, p < 0.05). Serum osteocalcin was decreased by hindlimb suspension and increased by SclAbII treatment. Interestingly, the anabolic effects of sclerostin inhibition on some bone outcomes appeared to be enhanced by normal mechanical loading. Altogether, these results confirm the ability of SclAbII to abrogate disuse‐induced bone loss and demonstrate that sclerostin antibody treatment increases bone mass by increasing bone formation in both normally loaded and underloaded environments.

RANKL inhibition with osteoprotegerin increases bone strength by improving cortical and trabecular bone architecture in ovariectomized rats.

Introduction: Ovariectomy (OVX) results in bone loss caused by increased bone resorption. RANKL is an essential mediator of bone resorption. We examined whether the RANKL inhibitor osteoprotegerin (OPG) would preserve bone volume, density, and strength in OVX rats.