Oscillatory Flow-induced Proliferation of Osteoblast-like Cells Is Mediated by αvβ3 and β1 Integrins through Synergistic Interactions of Focal Adhesion Kinase and Shc with Phosphatidylinositol 3-Kinase and the Akt/mTOR/p70S6K Pathway

Ding-Yu Lee,Yi-Shuan J Li,Shun-Fu Chang,Jing Zhou,Hui-Min Ho,Jeng-Jiann Chiu,Shu Chien

doi:10.1074/jbc.m109.010512

Abstract

Interstitial flow in and around bone tissue is oscillatory in nature and affects the mechanical microenvironment for bone cell growth and formation. We investigated the role of oscillatory shear stress (OSS) in modulating the proliferation of human osteoblast-like MG63 cells and its underlying mechanisms. Application of OSS (0.5 +/- 4 dynes/cm(2)) to MG63 cells induced sustained activation of phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR/p70S6K (p70S6 kinase) signaling cascades and hence cell proliferation, which was accompanied by increased expression of cyclins A and D1, cyclin-dependent protein kinases-2, -4, and -6, and bone formation-related genes (c-fos, Egr-1, and Cox-2) and decreased expression of p21(CIP1) and p27(KIP1). OSS-induced activation of PI3K/Akt/mTOR/p70S6K and cell proliferation were inhibited by specific antibodies or small interference RNAs of alpha(v)beta(3) and beta(1) integrins and by dominant-negative mutants of Shc (Shc-SH2) and focal adhesion kinase (FAK) (FAK(F397Y)). Co-immunoprecipitation assay showed that OSS induces sustained increases in association of Shc and FAK with alpha(v)beta(3) and beta(1) integrins and PI3K subunit p85, which were abolished by transfecting the cells with FAK(F397Y) or Shc-SH2. OSS also induced sustained activation of ERK, which was inhibited by the specific PI3K inhibitor LY294002 and was required for OSS-induced activation of mTOR/p70S6K and proliferation in MG63 cells. Our findings provide insights into the mechanisms by which OSS induces osteoblast-like cell proliferation through activation of alpha(v)beta(3) and beta(1) integrins and synergistic interactions of FAK and Shc with PI3K, leading to the modulation of downstream ERK and Akt/mTOR/p70S6K pathways.

Highlights

Mechanical loading is critical for the formation of new bone [1,2,3]
oscillatory shear stress (OSS) Induces MG63 Cell Proliferation via the Activation of phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR/p70S6K Pathway—MG63 cells were kept as controls or subjected to OSS (0.5 Ϯ 4 dynes/cm2) for 5 min, 15 min, 30 min, 1 h, 4 h, and 24 h, and their phosphorylation of Akt, mTOR, and p70S6K was examined by using Western blot analysis
These results indicate that OSS induces MG63 cell proliferation through the activation of the PI3K/ Akt/mTOR/p70S6K pathway

Summary

Introduction

Mechanical loading is critical for the formation of new bone [1,2,3]. During dynamic and periodic loading of intact bone, the reciprocating flow of interstitial fluid through the canaliculi generates oscillatory shear stress (OSS), which is detected by osteocytes in the canaliculi and osteoblasts lining the endosteal and periosteal surfaces of bone [4, 5]. Recent studies using flow channels have demonstrated that application of steady fluid shear stress to osteoblasts induces cell proliferation [10, 11] and the expression of many genes, including c-fos [8, 12], Egr-1 (early growth response-1) [8, 13], and Cox-2 (cyclooxygenase-2) [8, 12], all of which have been shown to play a role in bone formation in vivo (14 –17). Our findings have provided a molecular basis for the mechanisms by which OSS induces proliferation of osteoblast-like cells through integrin-mediated synergistic association of FAK and Shc with PI3K and their modulation of the downstream ERK and Akt/mTOR/p70S6K pathways

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