Dickkopf-1 regulates bone formation in young growing rodents and upon traumatic injury.

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.

Increased RANK ligand in bone marrow of orchiectomized rats and prevention of their bone loss by the RANK ligand inhibitor osteoprotegerin.

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.

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. MATERIALS AND METHODS: Rats were OVX or sham-operated at 3 mo of age. Sham controls were treated for 6 wk with vehicle (Veh, PBS). OVX rats were treated with Veh or human OPG-Fc (10 mg/kg, 2/wk). Serum RANKL and TRACP5b was measured by ELISA. BMD of lumbar vertebrae (L(1)-L(5)) and distal femur was measured by DXA. Right distal femurs were processed for bone histomorphometry. Left femurs and the fifth lumbar vertebra (L(5)) were analyzed by muCT and biomechanical testing, and L(6) was analyzed for ash weight. RESULTS: OVX was associated with significantly greater serum RANKL and osteoclast surface and with reduced areal and volumetric BMD. OPG markedly reduced osteoclast surface and serum TRACP5b while completely preventing OVX-associated bone loss in the lumbar vertebrae, distal femur, and femur neck. Vertebrae from OPG-treated rats had increased dry and ash weight, with no significant differences in tissue mineralization versus OVX controls. muCT showed that trabecular compartments in OVX-OPG rats had significantly greater bone volume fraction, vBMD, bone area, trabecular thickness, and number, whereas their cortical compartments had significantly greater bone area (p < 0.05 versus OVX-Veh). OPG improved cortical area in L(5) and the femur neck to levels that were significantly greater than OVX or sham controls (p < 0.05). Biomechanical testing of L(5) and femur necks showed significantly greater maximum load values in the OVX-OPG group (p < 0.05 versus OVX-Veh). Bone strength at both sites was linearly correlated with total bone area (r(2) = 0.54-0.74, p < 0.0001), which was also significantly increased by OPG (p < 0.05 versus OVX). CONCLUSIONS: OPG treatment prevented bone loss, preserved trabecular architecture, and increased cortical area and bone strength in OVX rats.

Infrequent delivery of a long-acting PTH-Fc fusion protein has potent anabolic effects on cortical and cancellous bone.

UNLABELLED: Skeletal anabolism with PTH is achieved through daily injections that result in brief exposure to the peptide. We hypothesized that similar anabolic effects could be achieved with less frequent but more sustained exposures to PTH. A PTH-Fc fusion protein with a longer half-life than PTH(1-34) increased cortical and cancellous BMD and bone strength with once- or twice-weekly injections. INTRODUCTION: The anabolic effects of PTH are currently achieved with, and thought to require, daily injections that result in brief exposure to the peptide. We hypothesized that less frequent but more sustained exposures to PTH could also be anabolic for bone, provided that serum levels of PTH were not constant. MATERIALS AND METHODS: PTH(1-34) was fused to the Fc fragment of human IgG1 to increase the half-life of PTH. Skeletal anabolism was examined in mice and rats treated once or twice per week with this PTH-Fc fusion protein. RESULTS: PTH-Fc and PTH(1-34) had similar effects on PTH/PTHrP receptor activation, internalization, and signaling in vitro. However, PTH-Fc had a 33-fold longer mean residence time in the circulation of rats compared with that of PTH(1-34). Subcutaneous injection of PTH-Fc once or twice per week resulted in significant increases in bone volume, density, and strength in osteopenic ovariectomized mice and rats. These anabolic effects occurred in association with hypercalcemia and were significantly greater than those achievable with high concentrations of daily PTH(1-34). PTH-Fc also significantly improved cortical bone volume and density under conditions where daily PTH(1-34) did not. Antiresorptive co-therapy with estrogen further enhanced the ability of PTH-Fc to increase bone mass and strength in ovariectomized rats. CONCLUSIONS: These results challenge the notion that brief daily exposure to PTH is essential for its anabolic effects on cortical and cancellous bone. PTH-derived molecules with a sustained circulating half-life may represent a powerful and previously undefined anabolic regimen for cortical and cancellous bone.

RANKL is a marker and mediator of local and systemic bone loss in two rat models of inflammatory arthritis.

UNLABELLED: RANKL is an essential mediator of bone erosions, but the role of RANKL in systemic bone loss had not been studied in arthritis. RANKL protein was increased in rat joint extracts and serum at the earliest stages of arthritis. Osteoprotegerin (OPG) treatment reversed local and systemic bone loss, suggesting that RANKL is both a marker and mediator of bone loss in arthritis. INTRODUCTION: RANKL is well established as an essential mediator of bone erosions in inflammatory arthritis, but the role of RANKL in systemic bone loss in arthritis had not been studied. We hypothesized that serum RANKL could serve as both a mediator and as a novel biomarker for local and systemic bone loss in arthritis. We challenged this hypothesis in two established rat models of inflammatory arthritis. We sought to determine whether serum RANKL was elevated early in disease progression and whether RANKL suppression could prevent both local and systemic bone loss in these models. MATERIALS AND METHODS: Detailed time-course studies were conducted in animals with collagen-induced (CIA) or adjuvant-induced (AIA) arthritis to evaluate the onset and progression of inflammation (paw swelling), bone erosions, osteoclast numbers, and RANKL protein levels in arthritic joints and in serum. Additional CIA and AIA rats (n=8/group) received placebo (PBS) or recombinant OPG (3 mg/kg three times weekly) for 10 days beginning 4 days after disease onset (first macroscopic evidence of hind paw erythema and edema) to assess the role of RANKL in local and systemic bone loss. RESULTS: RANKL protein was significantly elevated in the joints and serum of CIA and AIA rats within 1-2 days of disease onset. Increased RANKL levels were associated with local (hind paw) and systemic (vertebral) osteopenia in both models. The RANKL inhibitor OPG prevented local and systemic osteopenia in both models of established disease. CONCLUSIONS: RANKL protein is significantly increased both locally and systemically during the earliest stages of inflammatory arthritis in rats, suggesting that serum RANKL might have prognostic value for bone erosions and systemic osteopenia in this condition. RANKL inhibition through OPG prevented local and systemic bone loss in these arthritis models, suggesting that RANKL inhibition is a promising new approach for treating bone loss in arthritis.

The inhibition of RANKL causes greater suppression of bone resorption and hypercalcemia compared with bisphosphonates in two models of humoral hypercalcemia of malignancy.

Humoral hypercalcemia of malignancy (HHM) is mediated primarily by skeletal and renal responses to tumor-derived PTHrP. PTHrP mobilizes calcium from bone by inducing the expression of receptor activator for nuclear factor-kappaB ligand (RANKL), a protein that is essential for osteoclast formation, activation, and survival. RANKL does not influence renal calcium reabsorption, so RANKL inhibition is a rational approach to selectively block, and thereby reveal, the relative contribution of bone calcium to HHM. We used the RANKL inhibitor osteoprotegerin (OPG) to evaluate the role of osteoclast-mediated hypercalcemia in two murine models of HHM. Hypercalcemia was induced either by sc inoculation of syngeneic colon (C-26) adenocarcinoma cells or by sc injection of high-dose recombinant PTHrP (0.5 mg/kg, s.c., twice per day). In both models, OPG (0.2-5 mg/kg) caused rapid reversal of established hypercalcemia, and the speed and duration of hypercalcemia suppression were significantly greater with OPG (5 mg/kg) than with high-dose bisphosphonates (pamidronate or zoledronic acid, 5 mg/kg). OPG also caused greater reductions in osteoclast surface and biochemical markers of bone resorption compared with either bisphosphonate. In both models, hypercalcemia gradually returned despite clear evidence of ongoing suppression of bone resorption by OPG. These data demonstrate that osteoclasts and RANKL are important mediators of HHM, particularly in the early stages of the condition. Aggressive antiresorptive therapy with a RANKL inhibitor therefore might be a rational approach to controlling HHM.

Gene therapy with human recombinant osteoprotegerin reverses established osteopenia in ovariectomized mice.

Osteoporosis is a chronic condition that is typically treated by the long-term repeated administration of antiresorptive agents. Gene therapy has the potential to deliver protein-based antiresorptive agents without the need for repeated administration. Osteoprotegerin (OPG) is a naturally occuring protein that prevents bone resorption by inhibiting osteoclast formation, function and survival. We tested whether adeno-associated virus (AAV) could deliver OPG at levels that are sufficient to reverse established osteopenia in ovariectomized (OVX) mice without causing liver toxicity. Tibial bone mineral density (BMD) was measured by peripheral quantitative computed tomography (pQCT) in 12-week-old CDF1 mice prior to OVX or sham surgery. Six weeks later, BMD was significantly reduced in OVX mice compared to sham controls or pre-surgery values. Sham and OVX mice were then injected once IV with an AAV vector carrying cDNA for recombinant hOPG (AAV-OPG) or beta-galactosidase (AAV-betaGal). BMD and bone histomorphometry were assessed 10 weeks after treatment. A single injection of AAV-OPG led to the appearance of human OPG (hOPG) in the serum of mice within 7 days, and high serum levels of hOPG were maintained for the duration of the 10-week study. At the end of the study, OVX mice given AAV-OPG had significantly greater tibial BMD compared to age-matched OVX animals given AAV-betaGal. In sham-operated mice, AAV-OPG also significantly increased tibial BMD compared to AAV-betaGal. The increased BMD in AAV-OPG animals was accompanied by significantly increased bone volume and significantly reduced osteoclast surfaces in the proximal tibial metaphysis. Liver histology was normal, and circulating activities of hepatocyte cytosolic enzymes were unaffected by AAV exposure. In an accompanying experiment, young (3-4 weeks) C57BL/6 mice treated once IV with AAV-OPG maintained pharmacologically active levels of OPG in serum for at least 16 months. In summary, a single AAV-OPG treatment reversed established osteopenia in OVX mice without evidence of liver toxicity. AAV delivery appears to be a safe and effective method for producing sustained systemic exposure to OPG.