Ex vivo real-time observation of Ca2+ signaling in living bone in response to shear stress applied on the bone surface

Yoshihito Ishihara, Yasuyo Sugawara, Hiroshi Kamioka, Noriaki Kawanabe, Satoru Hayano, Tarek A. Balam, Keiji Naruse, Takashi Yamashiro

Research output: Contribution to journalArticlepeer-review

36 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)204-215
Number of pages12
JournalBone
Volume53
Issue number1
DOIs
Publication statusPublished - Mar 2013

Keywords

  • Ex-vivo calcium imaging
  • Gap junction
  • Osteoblast
  • Osteocyte

ASJC Scopus subject areas

  • Endocrinology, Diabetes and Metabolism
  • Physiology
  • Histology

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