title: Ex vivo real-time observation of Ca(2+) signaling in living bone in response to shear stress applied on the bone surface.
abstract: Bone cells respond to mechanical stimuli by producing a variety of biological signals, and one of the earliest events is intracellular calcium ([Ca(2+)](i)) mobilization. Our recently developed ex vivo live [Ca(2+)](i) imaging system revealed that bone cells in intact bone explants showed autonomous [Ca(2+)](i) oscillations, and osteocytes specifically modulated these oscillations through gap junctions. However, the behavior and connectivity of the [Ca(2+)](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 [Ca(2+)](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 [Ca(2+)](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 Gd(3+), is less affected in the flow-induced [Ca(2+)](i) response. These findings indicated that flow-induced mechanical stimuli accompanied the activation of the autonomous [Ca(2+)](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 [Ca(2+)](i) mobilization could be involved as an early signaling event in mechanotransduction.
authors.affiliation: Department of Orthodontics, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, 700-8525, Japan. ishihara@md.okayama-u.ac.jp
authors.forename: Yoshihito
authors.initials: Y
authors.lastname: Ishihara
authors.forename: Yasuyo
authors.initials: Y
authors.lastname: Sugawara
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kamioka
authors.forename: Noriaki
authors.initials: N
authors.lastname: Kawanabe
authors.forename: Satoru
authors.initials: S
authors.lastname: Hayano
authors.forename: Tarek A
authors.initials: TA
authors.lastname: Balam
authors.forename: Keiji
authors.initials: K
authors.lastname: Naruse
authors.forename: Takashi
authors.initials: T
authors.lastname: Yamashiro
mesh_terms: D000818:Animals; D001483:Base Sequence; D001842:Bone and Bones / Q000378:metabolism*; D020013:Calcium Signaling; D002642:Chick Embryo; D017931:DNA Primers; D016762:Genes, fos; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013315:Stress, Psychological
chemical_list: D017931:DNA Primers; D012333:RNA, Messenger