title: 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.
authors.affiliation: Department of Biomedical Engineering, City College of New York, New York, NY, USA.
authors.forename: Wing Yee
authors.initials: WY
authors.lastname: Cheung
authors.affiliation: Department of Orthopaedic Surgery, New Jersey Medical School, Rutgers University, Newark, NJ, USA.
authors.forename: J Christopher
authors.initials: JC
authors.lastname: Fritton
authors.affiliation: Department of Biomedical Engineering, City College of New York, New York, NY, USA.
authors.forename: Stacy Ann
authors.initials: SA
authors.lastname: Morgan
authors.affiliation: Department of Biomedical Engineering, City College of New York, New York, NY, USA.
authors.forename: Zeynep
authors.initials: Z
authors.lastname: Seref-Ferlengez
authors.affiliation: Department of Biomedical Engineering, City College of New York, New York, NY, USA.
authors.forename: Jelena
authors.initials: J
authors.lastname: Basta-Pljakic
authors.affiliation: Departments of Orthopaedic Surgery, Urology, and Neuroscience, Albert Einstein College of Medicine and Montefiore Medical Center, Bronx, NY, USA.
authors.forename: Mia M
authors.initials: MM
authors.lastname: Thi
authors.affiliation: Departments of Orthopaedic Surgery, Urology, and Neuroscience, Albert Einstein College of Medicine and Montefiore Medical Center, Bronx, NY, USA.
authors.forename: Sylvia O
authors.initials: SO
authors.lastname: Suadicani
authors.affiliation: Departments of Orthopaedic Surgery, Urology, and Neuroscience, Albert Einstein College of Medicine and Montefiore Medical Center, Bronx, NY, USA.
authors.forename: David C
authors.initials: DC
authors.lastname: Spray
authors.affiliation: Department of Biomedical Engineering, City College of New York, New York, NY, USA.
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Majeska
authors.affiliation: Department of Biomedical Engineering, City College of New York, New York, NY, USA.
authors.forename: Mitchell B
authors.initials: MB
authors.lastname: Schaffler
mesh_terms: D000818:Animals; D017209:Apoptosis; D024201:Bystander Effect; D017630:Connexins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D010011:Osteocytes / Q000378:metabolism* / Q000473:pathology; D053245:RANK Ligand / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D058486:Receptors, Purinergic P2X7 / Q000235:genetics / Q000378:metabolism*
chemical_list: D017630:Connexins; D009419:Nerve Tissue Proteins; C503631:Panx1 protein, mouse; D053245:RANK Ligand; D058486:Receptors, Purinergic P2X7; C505689:Tnfsf11 protein, mouse
keywords: BYSTANDER SIGNALING; FATIGUE MICRODAMAGE; OSTEOCYTE APOPTOSIS; P2X7 RECEPTOR; PANNEXIN 1; RANKL