TY - JOUR
T1 - Ex vivo real-time observation of Ca2+ signaling in living bone in response to shear stress applied on the bone surface
AU - Ishihara, Yoshihito
AU - Sugawara, Yasuyo
AU - Kamioka, Hiroshi
AU - Kawanabe, Noriaki
AU - Hayano, Satoru
AU - Balam, Tarek A.
AU - Naruse, Keiji
AU - Yamashiro, Takashi
N1 - Funding Information:
The authors thank Kayo Nagasaka for many helpful comments and support in writing the manuscript. The present work was supported by a Grant-in-Aid for Scientific Research (to Y. Ishihara) from the Ministry of Education, Culture, Sports, Science and Technology, Japan as part of the Research Fellowships for Young Scientists, and was supported in part by Grants-in-Aid for Scientific Research (to T. Yamashiro) from the Japan Society for the Promotion of Science, Japan .
PY - 2013/3
Y1 - 2013/3
N2 - Bone cells respond to mechanical stimuli by producing a variety of biological signals, and one of the earliest events is intracellular calcium ([Ca2+]i) mobilization. Our recently developed ex vivo live [Ca2+]i imaging system revealed that bone cells in intact bone explants showed autonomous [Ca2+]i oscillations, and osteocytes specifically modulated these oscillations through gap junctions. However, the behavior and connectivity of the [Ca2+]i signaling networks in mechanotransduction have not been investigated in intact bone. We herein introduce a novel fluid-flow platform for probing cellular signaling networks in live intact bone, which allows the application of capillary-driven flow just on the bone explant surface while performing real-time fluorogenic monitoring of the [Ca2+]i changes. In response to the flow, the percentage of responsive cells was increased in both osteoblasts and osteocytes, together with upregulation of c-fos expression in the explants. However, enhancement of the peak relative fluorescence intensity was not evident. Treatment with 18 α-GA, a reversible inhibitor of gap junction, significantly blocked the [Ca2+]i responsiveness in osteocytes without exerting any major effect in osteoblasts. On the contrary, such treatment significantly decreased the flow-activated oscillatory response frequency in both osteoblasts and osteocytes. The stretch-activated membrane channel, when blocked by Gd3+, is less affected in the flow-induced [Ca2+]i response. These findings indicated that flow-induced mechanical stimuli accompanied the activation of the autonomous [Ca2+]i oscillations in both osteoblasts and osteocytes via gap junction-mediated cell-cell communication and hemichannel. Although how the bone sense the mechanical stimuli in vivo still needs to be elucidated, the present study suggests that cell-cell signaling via augmented gap junction and hemichannel-mediated [Ca2+]i mobilization could be involved as an early signaling event in mechanotransduction.
AB - Bone cells respond to mechanical stimuli by producing a variety of biological signals, and one of the earliest events is intracellular calcium ([Ca2+]i) mobilization. Our recently developed ex vivo live [Ca2+]i imaging system revealed that bone cells in intact bone explants showed autonomous [Ca2+]i oscillations, and osteocytes specifically modulated these oscillations through gap junctions. However, the behavior and connectivity of the [Ca2+]i signaling networks in mechanotransduction have not been investigated in intact bone. We herein introduce a novel fluid-flow platform for probing cellular signaling networks in live intact bone, which allows the application of capillary-driven flow just on the bone explant surface while performing real-time fluorogenic monitoring of the [Ca2+]i changes. In response to the flow, the percentage of responsive cells was increased in both osteoblasts and osteocytes, together with upregulation of c-fos expression in the explants. However, enhancement of the peak relative fluorescence intensity was not evident. Treatment with 18 α-GA, a reversible inhibitor of gap junction, significantly blocked the [Ca2+]i responsiveness in osteocytes without exerting any major effect in osteoblasts. On the contrary, such treatment significantly decreased the flow-activated oscillatory response frequency in both osteoblasts and osteocytes. The stretch-activated membrane channel, when blocked by Gd3+, is less affected in the flow-induced [Ca2+]i response. These findings indicated that flow-induced mechanical stimuli accompanied the activation of the autonomous [Ca2+]i oscillations in both osteoblasts and osteocytes via gap junction-mediated cell-cell communication and hemichannel. Although how the bone sense the mechanical stimuli in vivo still needs to be elucidated, the present study suggests that cell-cell signaling via augmented gap junction and hemichannel-mediated [Ca2+]i mobilization could be involved as an early signaling event in mechanotransduction.
KW - Ex-vivo calcium imaging
KW - Gap junction
KW - Osteoblast
KW - Osteocyte
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U2 - 10.1016/j.bone.2012.12.002
DO - 10.1016/j.bone.2012.12.002
M3 - Article
C2 - 23246671
AN - SCOPUS:84872179683
VL - 53
SP - 204
EP - 215
JO - Bone
JF - Bone
SN - 8756-3282
IS - 1
ER -