Fluid Shear Stress Induces Less Calcium Response in a Single Primary Osteocyte Than in a Single Osteoblast: Implication of Different Focal Adhesion Formation

Abstract

The immediate calcium response to fluid shear stress was compared between osteocytes and osteoblasts on glass using real-time calcium imaging. The osteoblasts were responsive to fluid shear stress of up to 2.4 Pa, whereas the osteocytes were not. The difference in flow-induced calcium may be related to differences in focal adhesion formation.Introduction: To explore the immediate response to mechanical stress in a bone cell population, we examined flow-induced calcium transients. In addition, the involvement of focal adhesion-related calcium transients in response to fluid flow in the cells was studied. Materials and Methods: Bone cells were isolated from 16-day-old embryonic chicken calvaria by serial treatment with EDTA and collagenase. Single cells on glass without intercellular connections were subjected to fluid flow, and intracellular calcium concentration was measured using imaging with fluo-3. The identification of cell populations in the same field was performed with a chick osteocyte-specific antibody, OB7.3, and an alkaline phosphatase substrate, ELF-97, for osteoblast identification afterward. Immunofluorescence staining of vinculin was performed to visualize focal adhesions. Results: The percentage of cells responding to fluid shear stress at 1.2 Pa was 5.5% in osteocytes, 32.4% in osteoblasts, and 45.6% in OB7.3/ELF-97-negative cells. Furthermore, osteoblasts and OB7.3/ELF-97-negative cells were more responsive to 2.4 Pa than 1.2 Pa, whereas osteocytes were less responsive. The elevation of calcium transients over baseline did not show any significant differences in the populations. To elucidate the mechanism accounting for the fact that single osteocytes are less sensitive to fluid shear stress of up to 2.4 Pa than osteoblasts, we studied focal adhesion-related calcium transients. First, we compared focal adhesion formation between osteocytes and osteoblasts and found a larger number of focal adhesions in osteoblasts than in osteocytes. Next, when the cells were pretreated with GRGDS (0.5 mM) before flow treatment, a significant reduction of calcium transients in osteoblasts (18%) was observed, whereas calcium transients in osteocytes were not changed by GRGDS. Control peptide GRGES did not reduce the calcium transients in either cell type. Furthermore, we confirmed that osteoblasts in calvaria showed a marked formation of vinculin plaques in the periphery of the cells. However, osteocytes in calvaria showed faint vinculin plaques only at the base of the processes. Conclusions: On glass, single osteocytes are less sensitive to fluid shear stress up to 2.4 Pa than osteoblasts. The difference in calcium transients might be related to differences in focal adhesion formation. Shear stress of a higher magnitude or direct deformation may be responsible for the mechanical response of osteocytes in bone.

Protein Kinase G and Focal Adhesion Kinase Converge on Src/Akt/β-Catenin Signaling Module in Osteoblast Mechanotransduction

“…Osteoblasts and osteocytes differ in their sensitivity to FSS or stretching and demonstrate different kinetics of ␤-catenin nuclear translocation, but both cell types respond to mechanical stimulation with an increase in intracellular calcium and NO production (53)(54)(55)(56)(57). Nuclear ␤-catenin together with its binding partner Lef1 regulates genes in osteoblasts and osteocytes necessary for maintenance of normal bone mass in vivo, including cox-2, and control osteoblast as well as osteoclast differentiation and survival (27-30, 41, 58).…”

Osteocytes, mechanosensing and Wnt signaling

“…Regardless of how difficult or tedious the isolation, several investigators used primary osteoblasts and osteocytes to show significant differences in markers, function, sensitivity and response to mechanical strain (89)(90)(81)(82). For example, it has been shown the primary osteoblasts are less sensitive than primary osteocytes to shear stress (39)(64) that shear stress induced less of a calcium response in a single primary osteocyte than a single primary osteoblast, (60), that shear stress induced prostaglandin production in osteocytes is inhibited by cyoskeletal disruption, but not in osteoblasts (79), different ion channels are involved in osteoblast as compared to osteocytes' response to strain (101), stress fiber organization is delayed in osteocytes compared to osteoblast-like cells in response to fluid flow (98), and osteocytes have a gene expression profile distinct from osteoblasts (45).…”

Functional heterogeneity of osteocytes in FGF23 production: the possible involvement of DMP1 as a direct negative regulator

“…Our in vitro observations in this study are consistent with the findings of this previous report and further suggest that FAK-mediated signaling has a pivotal role in the maintenance of the proper shape of osteocytes and their network, concomitant with the regulation of FGF23 expression mediated by the MAPK pathway. Therefore, DMP1 bridges the structural properties and functional characteristics of osteocytes by stimulating multiple 50 reported that molecules associated with focal adhesion are predominantly found in osteocytic cellular processes but not in cell bodies. Our in vitro findings also suggest that DMP1 is not the only molecule to be involved in the regulation of cellular shape and FGF23 production; other RGD-containing extracellular molecules may be involved.…”