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.

One year of transgenic overexpression of osteoprotegerin in rats suppressed bone resorption and increased vertebral bone volume, density, and strength.

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.

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.