Activation of β-catenin signaling in MLO-Y4 osteocytic cells versus 2T3 osteoblastic cells by fluid flow shear stress and PGE 2: Implications for the study of mechanosensation in bone

Abstract

The osteocyte is hypothesized to be the mechanosensory cell in bone. However, osteoblastic cell models have been most commonly used to investigate mechanisms of mechanosensation in bone. Therefore, we sought to determine if differences might exist between osteocytic and osteoblastic cell models relative to the activation of β-catenin signaling in MLO-Y4 osteocytic, 2T3 osteoblastic and primary neonatal calvarial cells (NCCs) in response to pulsatile fluid flow shear stress (PFFSS). β-catenin nuclear translocation was observed in the MLO-Y4 cells at 2 and 16 dynes/cm 2 PFFSS, but only at 16 dynes/cm 2 in the 2T3 or NCC cultures. The MLO-Y4 cells released high amounts of PGE 2 into the media at all levels of PFFSS (2–24 dynes/cm 2) and we observed a biphasic pattern relative to the level of PFFSS. In contrast PGE 2 release by 2T3 cells was only detected during 16 and 24 dynes/cm 2 PFFSS starting at > 1 h and never reached the levels produced by the MLO-Y4 cells. Exogenously added PGE 2 was able to induce β-catenin nuclear translocation in all cells suggesting that the differences between the cell lines observed for β-catenin nuclear translocation were associated with the differences in PGE 2 production. To investigate a possible mechanism for the differences in PGE 2 release by the MLO-Y4 and 2T3 cells we examined the regulation of Ptgs2 (Cox-2) gene expression by PFFSS. 2T3 cell Ptgs2 mRNA levels at both 0 and 24 h after 2 h of PFFSS showed biphasic increases with peaks at 4 and 24 dynes/cm 2 and 24-hour levels were higher than zero-hour levels. MLO-Y4 cell Ptgs2 expression was similarly biphasic; however at 24-hour post-flow Ptgs2 mRNA levels were lower. Our data suggest significant differences in the sensitivity and kinetics of the response mechanisms of the 2T3 and neonatal calvarial osteoblastic versus MLO-Y4 osteocytic cells to PFFSS. Furthermore our data support a role for PGE 2 in mediating the activation of β-catenin signaling in response to the fluid flow shear stress.