Fluid shear-induced mechanical signaling in MC3T3-E1 osteoblasts requires cytoskeleton-integrin interactions

TLDR

Mechanical stimulation of bone promotes new bone formation in vivo and boosts metabolic activity and gene expression in cultured osteoblasts. The study aimed to determine how the actin cytoskeleton and actin‑membrane interactions transmit mechanical signals to alter gene expression in MC3T3‑E1 osteoblasts. Inhibition of actin stress‑fiber formation by cytochalasin D, dominant‑negative Rho, or an α‑actinin fragment that blocks actin‑integrin anchorage prevented fluid‑shear‑induced gene expression. Fluid shear reorganizes actin into stress fibers, recruits β1‑integrins and α‑actinin to focal adhesions, and upregulates COX‑2 and c‑Fos, with these responses blocked by disrupting actin stress fibers or α‑actinin anchorage, demonstrating that Rho‑mediated stress‑fiber formation and α‑actinin‑dependent integrin anchorage drive osteoblast mechanotransduction.

Abstract

Mechanical stimulation of bone induces new bone formation in vivo and increases the metabolic activity and gene expression of osteoblasts in culture. We investigated the role of the actin cytoskeleton and actin-membrane interactions in the transmission of mechanical signals leading to altered gene expression in cultured MC3T3-E1 osteoblasts. Application of fluid shear to osteoblasts caused reorganization of actin filaments into contractile stress fibers and involved recruitment of beta1-integrins and alpha-actinin to focal adhesions. Fluid shear also increased expression of two proteins linked to mechanotransduction in vivo, cyclooxygenase-2 (COX-2) and the early response gene product c-fos. Inhibition of actin stress fiber development by treatment of cells with cytochalasin D, by expression of a dominant negative form of the small GTPase Rho, or by microinjection into cells of a proteolytic fragment of alpha-actinin that inhibits alpha-actinin-mediated anchoring of actin filaments to integrins at the plasma membrane each blocked fluid-shear-induced gene expression in osteoblasts. We conclude that fluid shear-induced mechanical signaling in osteoblasts leads to increased expression of COX-2 and c-Fos through a mechanism that involves reorganization of the actin cytoskeleton. Thus Rho-mediated stress fiber formation and the alpha-actinin-dependent anchorage of stress fibers to integrins in focal adhesions may promote fluid shear-induced metabolic changes in bone cells.