Fluid shear stress activates c-Src and promotes RANKL localization in the plasma membrane in osteoblast-like MC3T3-E1 cells.

Receptor activator of NF-κB ligand (RANKL) produced by osteoblastic lineage cells is essential for osteoclastogenesis, yet RANKL can be sequestered in intracellular, lysosome-like compartments under basal conditions. How mechanical cues mobilize RANKL toward the cell surface remains poorly defined. Here, we tested whether fluid shear stress alters RANKL subcellular distribution in osteoblast-like MC3T3-E1 cells and examined the involvement of the non-receptor tyrosine kinase c-Src. MC3T3-E1 cells expressing fluorescently tagged RANKL were subjected to fluid shear stress, and RANKL localization was analyzed by microscopy and subcellular fractionation. Fluid shear stress increased c-Src activation (Tyr416 phosphorylation) and promoted redistribution of RANKL toward the cell periphery, accompanied by an increase of RANKL in the membrane fraction. Co-expression experiments showed spatial association of RANKL with c-Src at the cell periphery after shear stimulation. Moreover, constitutively active c-Src (Y527F) enhanced peripheral localization of RANKL even in the absence of shear stress. Together, these data support a model in which shear stress activates c-Src to facilitate RANKL localization from intracellular stores toward membrane-proximal regions, thereby providing a mechanistic link between mechanical cues and osteoblast-derived osteoclastogenic signaling.