Apoptotic Osteocytes Induce RANKL Production in Bystanders via Purinergic Signaling and Activation of Pannexin Channels

Abstract

Localized apoptosis of osteocytes, the tissue-resident cells within bone, occurs with fatigue microdamage and activates bone resorption. Osteoclasts appear to target and remove dying osteocytes, resorbing damaged bone matrix as well. Osteocyte apoptosis similarly activates bone resorption with estrogen loss and in disuse. Apoptotic osteocytes trigger viable neighbor (ie, bystander) osteocytes to produce RANKL, the cytokine required for osteoclast activation. Signals from apoptotic osteocytes that trigger this bystander RANKL expression remain obscure. Studying signaling among osteocytes has been hampered by lack of in vitro systems that model the limited communication among osteocytes in vivo (ie, via gap junctions on cell processes and/or paracrine signals through thin pericellular fluid spaces around osteocytes). Here, we used a novel multiscale fluidic device (the Macro-micro-nano, or Mμn) that reproduces these key anatomical features. Osteocytes in discrete compartments of the device communicate only via these limited pathways, which allows assessment of their roles in triggering osteocytes RANKL expression. Apoptosis of MLOY-4 osteocytes in the Mμn device caused increased osteocyte RANKL expression in the neighboring compartment, consistent with in vivo findings. This RANKL upregulation in bystander osteocytes was prevented by blocking Pannexin 1 channels as well as its ATP receptor. ATP alone caused comparable RANKL upregulation in bystander osteocytes. Finally, blocking Connexin 43 gap junctions did not abolish osteocyte RANKL upregulation, but did alter the distribution of RANKL expressing bystander osteocytes. These findings point to extracellular ATP, released from apoptotic osteocytes via Panx1 channels, as a major signal for triggering bystander osteocyte RANKL expression and activating bone remodeling. n 966 MCCUTCHEON ET AL. Journal of Bone and Mineral ResearchAPOPTOTIC OSTEOCYTES INDUCE RANKL IN BYSTANDERS 967 nFig. 2. Osteocyte process ingrowth and gap junction functionality. (A) Confocal photomicrograph showing MLOY-4 osteocyte dendritic processes growing into the nanochannel array at 7 days culture in the Mμn device, visualized by AlexaFluor488-labeled phalloidin staining for F-Actin. (B) Parachute dye transfer assay image, showing Calcein-AM (green) transfer from donor compartment osteocytes to acceptor osteocytes in the "bystander" compartment of Mμn device. (C) Fluorescence and corresponding phase-contrast micrographs showing dye transfer across the channel array in osteocytes cultured under control conditions (top row), while no transfer to the acceptor side of the Mμn occurred when the connexin 43 blocker AGA was added, confirming gap junction communication between compartments. MLO-Y4 cells, labeled with gap junction permeable dye Calcein-AM (green) and gap junction impermeable membrane dye DiI (red), were parachuted onto one side of a previously MLO-Y4-seeded Mμn. Calcein-only labeled cells indicate gap junctional transfer from parachuted cells (donors) to cells on same side of the channe...

Osteocytes and Bone Metastasis

“…As mentioned above, bones inevitably age and undergo numerous changes, including bone loss, osteocyte apoptosis, and increased oxidative stress. Osteocyte apoptosis is a key stimulus that triggers bone resorption ( Figure 1 , step ⑤) ( 73 , 74 ). The slowed production of sex hormones that comes with aging promotes osteoclast activity ( 75 , 76 ), osteocyte apoptosis ( 73 ), elevated oxidative stress ( 76 , 77 ), and a reduction in osteoblast function ( 78 ).…”

A Micro-Optic Stalk (μOS) System to Model the Collective Migration of Retinal Neuroblasts

“…Each reservoir is 150-µm wide, 500-µm long, and 50 µm in height, to represent the large BL and EID regions. These vertical reservoirs were additionally sized to minimize the volume needed for primary cell suspensions and reduce entrance effects via a large volume ratio with the microarray (50:1) [49][50][51].…”

“…represent the large BL and EID regions. These vertical reservoirs were additionally sized to minimize the volume needed for primary cell suspensions and reduce entrance effects via a large volume ratio with the microarray (50:1) [49][50][51]. Each microchannel of the μOS array represents one OS segment and has a length (LOS) of 90 μm, depth of 10 μm, and characteristic width of 37 μm (WOS1) on the BL side and 35 μm (WOS2) on the EID side.…”

“…As per Figure 2D, RNBs exit the EID reservoir and migrate toward the BL reservoir through L OS segments of the µOS that represent the anatomical geometry of the developing visual system of Drosophila. Changes in the anatomical constraints experienced by cells were modeled using two-layer photolithography to produce similar differences in system height, as done previously by our group for biosystems with similar constraints [49,51].…”

“…This extracellular substrate was selected based on our previous work comparing RNB adhesion and viability upon a panel of substrates [37] as well as numerous citations in the Drosophila literature validating ConA as an appropriate substrate [39,48]. The coating was then aspirated, and the device washed with PBS and placed in a flow hood to air dry for 1 h. A 150-µL cell suspension was flushed into the system (NE-1000, New Era Pump Systems Inc, Farmingdale, NY, USA) at volume flow rates of Q L < 5 µL/min to minimize shear [41,51] and enable RNB adhesion. Cells were left to adhere within the µOS for 2 h. Afterwards, the source reservoir (vertical compartment on the right-hand side) was continually flushed (Q R ) with 100 ng/mL of FGF, and the sink reservoir (vertical compartment on the left-hand side) was continually flushed with cell media (Q L ) to generate concentration gradient fields across the horizontal microchannel array.…”