Expression of functional gap junctions and regulation by fluid flow in osteocyte-like MLO-Y4 cells

Osteocytes are thought to be mechanosensory cells that respond to mechanica l stress by sending signals to other bone cells to initiate bone remodeling . An osteocyte-like cell line MLO-Y4 provides a model system to examine whe ther gap junctions participate in the regulation of osteocyte function and signaling by mechanical stress. In this study, we show that MLO-Y4 cells ar e coupled and that gap junction channels mediate this coupling. Biochemical analyses show that connexin 43 (Cx43) is a major gap junction protein expr essed in MLO-Y4 cells and approximately 5% of Cx43 protein is phosphorylate d, MLO-Y4 cells were exposed to mechanical stress using a parallel plate fl ow chamber to model bone fluid how shear stress. Fluid how increased signif icantly the length of the dendritic processes, a morphological characterist ic of osteocytes, A redistribution of the gap junction protein, Cx43 also w as observed from a location circling the nucleus to punctate spots in the c ytoplasm and in the dendritic processes. "Scrape-loading" dye transfer anal yses show ed that fluid flow increased intercellular coupling and increased the number of cells coupled immediately after fluid flow treatment, in dir ect proportion to shear stress magnitude. Although intercellular coupling c ontinued to increase, stimulation of Cx43 protein expression during the pos tstress period was found to be biphasic. Cx43 protein was elevated 30 minut es after application of stress but decreased at 24 h poststress. Pulsating fluid flow had a similar stimulatory effect as steady fluid flow on gap jun ctions. However, this stimulatory effect in osteocyte-like cells was not ob served in osteoblast-like 2T3 cells. Together, these results show that flui d flow has stimulatory effects on osteocyte-like MLO-Y4 cells with early ef fects on cellular morphology, opening of gap junctions, and redistribution of Cx43 protein and delayed effects on Cx43 protein expression. The high ex pression of Cx43 and its location in the cytoplasm also suggest that Cx43 m ay have unknown functions in addition to forming gap junctions. These studi es indicate that gap junctions may serve as channels for signals generated by osteocytes in response to mechanical loading.