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

Wing Yee Cheung, J. Christopher Fritton, Stacy Ann Morgan, Zeynep Seref-Ferlengez, Jelena Basta-Pljakic, Mia M. Thi, Sylvia O. Suadicani, David C. Spray, Robert J. Majeska, Mitchell B. Schaffler

Research output: Contribution to journalArticle

16 Citations (Scopus)

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 P2X7R in ATP release; thus, we examined P2X7R-deficient mice and WT mice treated with P2X7R antagonist Brilliant Blue G (BBG) to test the possible role of ATP as a find-me signal. P2X7RKO 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 P2X7RKO 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 P2X7R 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.

Original languageEnglish (US)
JournalJournal of Bone and Mineral Research
DOIs
StateAccepted/In press - 2016

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Purinergic P2X7 Receptors
Osteocytes
Bone and Bones
Apoptosis
Adenosine Triphosphate
Fatigue
Up-Regulation
Bone Remodeling
Phagocytes
Bone Resorption

Keywords

  • BYSTANDER SIGNALING
  • FATIGUE MICRODAMAGE
  • OSTEOCYTE APOPTOSIS
  • P2X7 RECEPTOR
  • PANNEXIN 1
  • RANKL

ASJC Scopus subject areas

  • Orthopedics and Sports Medicine
  • Endocrinology, Diabetes and Metabolism

Cite this

Pannexin-1 and P2X7-Receptor Are Required for Apoptotic Osteocytes in Fatigued Bone to Trigger RANKL Production in Neighboring Bystander Osteocytes. / Cheung, Wing Yee; Fritton, J. Christopher; Morgan, Stacy Ann; Seref-Ferlengez, Zeynep; Basta-Pljakic, Jelena; Thi, Mia M.; Suadicani, Sylvia O.; Spray, David C.; Majeska, Robert J.; Schaffler, Mitchell B.

In: Journal of Bone and Mineral Research, 2016.

Research output: Contribution to journalArticle

Cheung, Wing Yee ; Fritton, J. Christopher ; Morgan, Stacy Ann ; Seref-Ferlengez, Zeynep ; Basta-Pljakic, Jelena ; Thi, Mia M. ; Suadicani, Sylvia O. ; Spray, David C. ; Majeska, Robert J. ; Schaffler, Mitchell B. / Pannexin-1 and P2X7-Receptor Are Required for Apoptotic Osteocytes in Fatigued Bone to Trigger RANKL Production in Neighboring Bystander Osteocytes. In: Journal of Bone and Mineral Research. 2016.
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AU - Cheung, Wing Yee

AU - Fritton, J. Christopher

AU - Morgan, Stacy Ann

AU - Seref-Ferlengez, Zeynep

AU - Basta-Pljakic, Jelena

AU - Thi, Mia M.

AU - Suadicani, Sylvia O.

AU - Spray, David C.

AU - Majeska, Robert J.

AU - Schaffler, Mitchell B.

PY - 2016

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N2 - 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 P2X7R in ATP release; thus, we examined P2X7R-deficient mice and WT mice treated with P2X7R antagonist Brilliant Blue G (BBG) to test the possible role of ATP as a find-me signal. P2X7RKO 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 P2X7RKO 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 P2X7R 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.

AB - 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 P2X7R in ATP release; thus, we examined P2X7R-deficient mice and WT mice treated with P2X7R antagonist Brilliant Blue G (BBG) to test the possible role of ATP as a find-me signal. P2X7RKO 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 P2X7RKO 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 P2X7R 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.

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KW - FATIGUE MICRODAMAGE

KW - OSTEOCYTE APOPTOSIS

KW - P2X7 RECEPTOR

KW - PANNEXIN 1

KW - RANKL

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