Gap Junctions and Osteoblast-like Cell Gene Expression in Response to Fluid Flow

Abstract

Bone formation occurs in vivo in response to mechanical stimuli, but the signaling pathways involved remain unclear. The ability of bone cells to communicate with each other in the presence of an applied load may influence the overall osteogenic response. The goal of this research was to determine whether inhibiting cell-to-cell gap junctional communication between bone-forming cells would affect the ensemble cell response to an applied mechanical stimulus in vitro. In this study, we investigated the effects of controlled oscillatory fluid flow (OFF) on osteoblastic cells in the presence and the absence of a gap-junction blocker. MC3T3-E1 Clone 14 cells in a monolayer were exposed to 2h of OFF at a rate sufficient to create a shear stress of 20dynes∕cm2 at the cell surface, and changes in steady-state mRNA levels for a number of key proteins known to be involved in osteogenesis were measured. Of the five proteins investigated, mRNA levels for osteopontin (OPN) and osteocalcin were found to be significantly increased 24h postflow. These experiments were repeated in the presence of 18β-glycyrrhetinic acid (BGA), a known gap-junction blocker, to determine whether gap-junction intercellular communication is necessary for this response. We found that the increase in OPN mRNA levels is not observed in the presence of BGA, suggesting that gap junctions are involved in the signaling process. Interestingly, enzyme linked immunosorbent assay data showed that levels of secreted OPN protein increased 48h postflow and that this increase was unaffected by the presence of intact gap junctions.