Osteocyte Apoptosis Caused by Hindlimb Unloading is Required to Trigger Osteocyte RANKL Production and Subsequent Resorption of Cortical and Trabecular Bone in Mice Femurs

Abstract

Osteocyte apoptosis is essential to activate bone remodeling in response to fatigue microdamage and estrogen withdrawal, such that apoptosis inhibition in vivo prevents the onset of osteoclastic resorption. Osteocyte apoptosis has also been spatially linked to bone resorption owing to disuse, but whether apoptosis plays a similar controlling role is unclear. We, therefore, 1) evaluated the spatial and temporal effects of disuse from hindlimb unloading (HLU) on osteocyte apoptosis, receptor activator of NF-κB ligand (RANKL) expression, bone resorption, and loss in mouse femora, and 2) tested whether osteocyte apoptosis was required to activate osteoclastic activity in cortical and trabecular bone by treating animals subjected to HLU with the pan-caspase apoptosis inhibitor, QVD (quinolyl-valyl-O-methylaspartyl-[-2,6-difluorophenoxy]-methylketone). Immunohistochemistry was used to identify apoptotic and RANKL-producing osteocytes in femoral diaphysis and distal trabecular bone, and µCT was used to determine the extent of trabecular bone loss owing to HLU. In both cortical and trabecular bone, 5 days of HLU increased osteocyte apoptosis significantly (3- and 4-fold, respectively, p < 0.05 versus Ctrl). At day 14, the apoptotic osteocyte number in femoral cortices declined to near control levels but remained elevated in trabeculae (3-fold versus Ctrl, p < 0.05). The number of osteocytes producing RANKL in both bone compartments was also significantly increased at day 5 of HLU (>1.5-fold versus Ctrl, p < 0.05) and further increased by day 14. Increases in osteocyte apoptosis and RANKL production preceded increases in bone resorption at both endocortical and trabecular surfaces. QVD completely inhibited not only the HLU-triggered increases in osteocyte apoptosis but also RANKL production and activation of bone resorption at both sites. Finally, µCT studies revealed that apoptosis inhibition completely prevented the trabecular bone loss caused by HLU. Together these data indicate that osteocyte apoptosis plays a central and controlling role in triggering osteocyte RANKL production and the activation of new resorption leading to bone loss in disuse. © 2016 American Society for Bone and Mineral Research.

Fig. 1

(A) Femoral mid-diaphyseal section showing the anatomical sampling regions examined. (B) Photomicrographs of mid-diaphyseal cortical bone sections from mice subjected to 5 days of unloading (5d HLU) stained by immunohistochemistry for activated caspase 3 (Casp3, top left), its negative control (Neg Ctrl, bottom left); RANKL (top right), and its negative control (bottom right). Arrows indicate positively stained osteocytes (+Ot); scale bars = 40 μm.

Fig. 2

Circumferential and radial distributions of apoptotic osteocytes (%Casp3+Ot) in femoral mid-diaphyses after HLU (^*p <0.05 versus cage control). (A) Increases in apoptotic osteocytes in principal anatomical regions (posterior cortex p <0.05; anterior, medial, and lateral cortices p >0.1). (B) The radial distribution of apoptotic osteocytes from endocortical to periosteal surface as a percentage of cortical width (endocortical surface = 0%) with this highest concentration of apoptotic osteocytes toward the endocortical region.

Fig. 3

Cortical bone (A) osteocyte apoptosis (%Casp3+Ot), (B) osteocyte RANKL expression (%RANKL+Ot), and (C) endocortical resorption surface (%Ec.Rs.) in femoral mid-diaphyses after HLU. Baseline (day 0) is age-matched cage control value (^*p <0.05 versus baseline [day 0] control).

Fig. 4

Trabecular bone (A) osteocyte apoptosis (%Casp3+Ot), (B) osteocyte RANKL expression (%RANKL+Ot), (C) trabecular bone active resorption surface (%CatK+Pm), and (D) trabecular osteoblastic cell RANKL expression (%RANKL+Pm) after HLU. Baseline (day 0) is cage control value (^*p <0.05 versus control).

Fig. 5

Effects of QVD on osteoclastogenesis and RANKL gene expression in vitro. (A) Number of osteoclasts (TRAP+ multinucleated cells) formed by nonadherent mouse bone marrow mononuclear cells; QVD or carrier (DMSO) was present throughout the differentiation period. (B) RANKL gene expression levels in MLO-Y4 osteocytes with and without QVD.

Fig. 6

Effects of apoptosis inhibition with QVD on femoral diaphyseal cortical bone after HLU versus treatment with vehicle (Veh) alone. (A) Changes in osteocyte apoptosis (%Casp3+Ot) with HLU, (B) changes in osteocytes RANKL expression (%RANKL+Ot) with HLU, and (C) the marked increase in endocortical resorption (%Ec.Rs.) after 14 days of HLU, which was prevented by QVD treatment (^*p <0.05 versus cage controls [Ctrl]).

Fig. 7

Effects of apoptosis inhibition with QVD on trabecular bone after HLU versus treatment with vehicle (Veh) alone. (A) Changes in osteocyte apoptosis (%Casp3+Ot with HLU), (B) changes in osteocytes RANKL expression (%RANKL+Ot) with HLU, and (C) the marked increase in resorption surface (%CatK+Pm) after 14 days of HLU, which was prevented by QVD treatment (^*p <0.05 versus cage controls [Ctrl]).

Fig. 8

μCT-based trabecular bone microarchitectural properties in mouse femora owing to HLU and apoptosis inhibition. Mice subjected to HLU or cage controls (Ctrl) were treated with DMSO vehicle (Veh) or the apoptosis inhibitor QVD (ApInh). (A) μCT images show conspicuous trabecular bone loss in HLU+Veh; HLU+ApInh bones appear similar to cage control (Ctrl) bones. (B–E) Summary data for trabecular bone architecture parameters. Inhibition of osteocyte apoptosis completely prevented HLU-induced bone loss (^*p <0.05 versus Ctrl).