Oscillating fluid flow regulates gap junction communication in osteocytic MLO-Y4 cells by an ERK1/2 MAP kinase-dependent mechanism

The present work was designed to investigate the effects of oscillating fluid flow on gap junctional intercellular communication (GJIC) and the gap junction protein connexin (Cx) 43 in osteocyte-like MLOY-4 cells. Cells were exposed for 1 h to oscillating fluid flow at a shear stress of ±10 dyn/cm2 and a frequency of 1 Hz in a parallel plate flow chamber. Control cells were incubated in the chamber but were not exposed to oscillating fluid flow. Functional analysis of GJIC indicated that MLOY-4 cells exposed to oscillating fluid flow established more gap junctions with an independent population of dye-labeled cells than did control cells. Phosphorylation of Cx43 was quantified by immunoprecipitation with an anti-Cx43 antibody followed by immunoblot analysis using an anti-phosphoserine antibody. Phosphoserine was normalized to Cx43 in each sample. Compared to control cells, phosphoserine content of Cx43 increased approximately twofold in cells exposed to oscillating fluid flow. The possible role of the extracellular signal regulated kinase (ERK1/2) in the flow-induced upregulation of GJIC was also investigated. The ERK1/2 inhibitor PD-98059 significantly attenuated the effects of oscillating fluid flow on MLOY-4 cells GJIC. These results indicate that oscillating fluid flow regulates GJIC in MLOY-4 cells via the ERK1/2 MAP kinase. In addition, increased serine phosphorylation of Cx43 correlates with the flow-induced increase in GJIC.