Pannexin-1 and P2X7-Receptor Are Required for Apoptotic Osteocytes in Fatigued Bone to Trigger RANKL Production in Neighboring Bystander Osteocytes

Abstract

Osteocyte apoptosis is required to induce intracortical bone remodeling after microdamage in animal models, but how apoptotic osteocytes signal neighboring "bystander" cells to initiate the remodeling process is unknown. Apoptosis has been shown to open pannexin-1 (Panx1) channels to release adenosine diphosphate (ATP) as a "find-me" signal for phagocytic cells. To address whether apoptotic osteocytes use this signaling mechanism, we adapted the rat ulnar fatigue-loading model to reproducibly introduce microdamage into mouse cortical bone and measured subsequent changes in osteocyte apoptosis, receptor activator of NF-κB ligand (RANKL) expression and osteoclastic bone resorption in wild-type (WT; C57Bl/6) mice and in mice genetically deficient in Panx1 (Panx1KO). Mouse ulnar loading produced linear microcracks comparable in number and location to the rat model. WT mice showed increased osteocyte apoptosis and RANKL expression at microdamage sites at 3 days after loading and increased intracortical remodeling and endocortical tunneling at day 14. With fatigue, Panx1KO mice exhibited levels of microdamage and osteocyte apoptosis identical to WT mice. However, they did not upregulate RANKL in bystander osteocytes or initiate resorption. Panx1 interacts with P2X7 R in ATP release; thus, we examined P2X7 R-deficient mice and WT mice treated with P2X7 R antagonist Brilliant Blue G (BBG) to test the possible role of ATP as a find-me signal. P2X7 RKO mice failed to upregulate RANKL in osteocytes or induce resorption despite normally elevated osteocyte apoptosis after fatigue loading. Similarly, treatment of fatigued C57Bl/6 mice with BBG mimicked behavior of both Panx1KO and P2X7 RKO mice; BBG had no effect on osteocyte apoptosis in fatigued bone but completely prevented increases in bystander osteocyte RANKL expression and attenuated activation of resorption by more than 50%. These results indicate that activation of Panx1 and P2X7 R are required for apoptotic osteocytes in fatigued bone to trigger RANKL production in neighboring bystander osteocytes and implicate ATP as an essential signal mediating this process.

Keywords: BYSTANDER SIGNALING; FATIGUE MICRODAMAGE; OSTEOCYTE APOPTOSIS; P2X7 RECEPTOR; PANNEXIN 1; RANKL.

Figure 1

Summary of mouse ulnar fatigue studies. (A) Basic fuchsin-stained WT mouse ulnar mid diaphysis bone isolated immediately following fatigue loading; arrows show the locations of microdamage in the bone. (B) The enlargement region shows a fluorescence image of the microcracks (arrow) caused by the fatigue loading regime used (bar = 50 µm). (C) Microcrack content (Cr.Dn) in fatigue loaded ulnae vs. non-loaded ulnae and NON-MDX regions within same ulnae (FATIGUE vs No FATIGUE control, p<0.01). (D) Schematic showing principal microdamage locations (MDX) in fatigued ulnae and non-damaged (NON-MDX) regions within the same bone section. (E and F): Osteocyte apoptosis (% Casp3+ Ot) and osteocyte RANKL expression (% RANKL+ Ot) in fatigued ulnae at 3 days after loading. Both osteocyte apoptosis and RANKL expression are dramatically increased, and were highly localized to the MDX regions noted in A and B (above); osteocytes in NON-MDX areas were similar to non-loaded controls (data not shown). (G and H) demonstrate intracortical resorption (thin arrow), endocortical tunneling site (thick arrow) and a microcrack in fatigued ulnae at 14 days post loading; this resorption is not seen in baseline mouse ulnae (bar = 50 µm).

Figure 2

Cortical bone morphology of WT, Panx1KO and P2X₇R KO mice. (A) Micro-CT images of diaphyseal cross-sections. (B) Summary of micro-CT-based architectural parameters for mid-femoral diaphyses. (Tt.Ar = Total Cross-section Area; Ct.Ar = Cortical bone Area; Ma.Ar = Marrow Area; Ct.Th = Cortical Thickness; Ct.Ar./Tt.Ar = Cortical Area / Total Area; CSMI = Cross-Sectional Moment of Inertia)

Figure 3

Osteocyte apoptosis, osteocyte RANKL expression and resorption in fatigued ulnae of WT, Panx1KO and P2X₇RKO mice. (A) Left side photomicrographs show IHC staining for osteocyte apoptosis within MDX regions of WT, Panx1KO and P2X₇RKO mice at 3 days post-fatigue (bar = 25 µm) (c-Caspase 3 staining); arrows illustrate examples of positively-stained cells (brown colored). Right side photomicrographs show IHC staining for osteocyte RANKL expression in MDX regions at 3 days post-fatigue; RANKL staining (arrows) is evident in WT ulnae, but effectively absent from fatigued ulnae from Panx1KO and P2X₇RKO mice. (B) Histomorphometry data for osteocyte apoptosis (%Casp3+ Ot.) and osteocyte RANKL expression (%RANKL+ Ot.) in fatigued ulnae at 3 days after loading: Osteocyte apoptosis at microdamage sites occurred similarly in Panx1KO, P2X₇RKO and WT ulnae, consistent with IHC images shown in (A), with apoptosis increased almost 5-fold vs. NON-MDX bone areas with same bone (p<0.02). In contrast, osteocyte RANKL expression was absent from fatigued Panx1KO and P2X₇RKO bones. (C) Histomorphometry data for resorption space number (Rs.N) and endocortical tunneling foci number (En.Tun.N) in WT, Panx1KO and P2X₇RKO ulnae at 14 days after fatigue (p<0.02 vs WT), showing complete absence of new resorption activity in both KO strains.

Figure 4

(A and B) Osteocyte apoptosis and RANKL expression in fatigued mouse ulnae treated in vivo with the P2X₇R antagonist, Brilliant Blue G (BBG) or Vehicle (VEH). BBG inhibited the normal increase in osteocyte RANKL expression in ulnar cortex following fatigue loading (p<0.05), but did not alter the amount of osteocyte apoptosis. (C and D) BBG treatment dramatically reduced the activation of resorption after fatigue at both intracortical (Rs.N, p<0.06 vs VEH) and endocortical (En.Tun.N, p<0.05 vs VEH) locations.