Primary Cilia-Mediated Osteogenic Response to Fluid Flow Occurs via Increases in Focal Adhesion and Akt Signaling Pathway in MC3T3-E1 Osteoblastic Cells

摘要

Fluid flow is an important anabolic mechanical signal that results in the upregulation of osteogenic responses in bone cells. Primary cilium may be responsible for the sensing of mechanical signals and converting these into biochemical signals to maintain bone homeostasis through an unknown mechanism. Focal adhesions, sites of mechanical linkage between the cytoskeleton and extracellular matrix, play a role as mechanoreceptors that are involved in fluid flow-induced signaling. In this study, we firstverified whether focal adhesions are required for primary cilia to upregulate fluid flow-induced bone forming activities in osteoblasts. Specifically, mRNA and protein levels of cyclo-oxygenase-2 (COX-2) and release of prostaglandin E2 (PGE2), which areimportant molecules that regulate bone formation, were quantified in MC3T3-E1 osteoblastic cells with or without primary cilia after exposure to 1 Pa of fluid flow-induced shear stress. Then, this experiment was continued on Arg-Gly-Asp-Ser (RGDS) peptide-treated cells after determining the formation of focal adhesions were inhibited. Second, signaling pathways, including Ras, ERK, and Akt, whereby primary cilia transmit fluid flow to cellular responses in osteoblastic cells were studied. Removal of primary cilia resulted in a significant decrease in COX-2 mRNA and protein and PGE2 release with a similar decrease in FAK mRNA and vinculin-positive focal adhesion sites in response to fluid flow. Furthermore, the removal of primary cilia suppressed the activation of p-Akt, but not Ras and p-ERK in response to fluid flow. Our results suggest that primary cilia lead to fluid flow-induced COX-2 induction and PGE2 release via increases in focal adhesions and their responses are activated by Akt phosphorylation in osteoblastic cells.