| Summary: | 博士 === 國防醫學院 === 生命科學研究所 === 98 === Understanding the factors that affect the function of bone cells, such as mechanical stimulus,will be of help for the development of bone tissue engineering. Fluid flow is thought to be an
important mechanical stimulus involved in load-induced bone formation. The aims of this thesis were (1) to investigate the roles of integrins and different extracellular matrix (ECM) in
shear stress-mediated signal transduction and expressions of bone formation-related genes, including egr-1, c-fos, COX-2, and OPN, in human osteoblast-like MG63 cells. (2) to investigate the role of oscillatory fluid, which is more similar to physiological flow pattern, in modulating proliferation of osteoblast-like cells and the mechanisms underlying the shear stress effect. Our results show that shear stress induces sustained associations of αvβ3 and β1
with Shc when seeded on fibronectin (FN), but only sustained associations between β1 and Shc when seeded on collagen I (COL1) and laminin (LM). Shear inductions of MAPKs (MAP Kinase) and bone formation–related genes were sustained (24 h) in cells on FN, but some of these responses were transient in cells on COL1/LM. The shear activations of extracellular signal-regulated kinase [ERK], c-jun-NH2-terminal kinase [JNK], and p38 were mediated by integrins and Shc, and these pathways differentially modulated downstream bone
formation–related gene expression. Our findings show that the β1 integrin plays a predominant role in shear-induced signaling and gene expression in osteoblast-like cells on FN, COL1, and LM and that αvβ3 also plays a significant role in these responses in cells on FN. In addition, we further studied the roles of oscillatory flow in integrin-mediated signaling and proliferation in osteoblasts. Our results show that oscillatory shear stress induces the sustained activation of phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR/p70S6K (p70S6 kinase) signaling cascades and hence cell proliferation, as indicated by the shear-induced increase in the percentage of cells in the synthetic phase. Oscillatory shear stress induced sustained increases in the associations of αvβ3 and β1 integrins with Shc and focal adhesion kinase (FAK), as well as in FAK phosphorylation. Blockage of these integrins with specific antibodies and small interfering RNAs (siRNAs) inhibited oscillatory shear-induced
phosphorylation of FAK, Akt, mTOR, and p70S6K. Dominant-negative mutants of FAK (FAK[F397Y]) and Shc (Shc-SH2) inhibited not only the oscillatory shear-activations of the downstream molecules Akt, mTOR, and p70S6K but also the upstream αvβ3 and β1 integrins. A co-immunoprecipitation assay showed that oscillatory shear stress induces sustained increases in FAK/Shc/PI3K heteromeric complex formation. This shear-induced FAK/Shc/PI3K complex formation was abolished by transfecting the cells with FAK(F397Y) or Shc-SH2. Transfections of MG63 cells with FAK(F397Y), Shc-SH2, and β1- and
β3-specific siRNAs inhibited oscillatory shear-induced increases in the percentage of cells in the synthetic phase. Our findings provide insights into the mechanisms by which oscillatory shear stress induces osteoblast-like cell proliferation through the activation of αvβ3 and β1 integrins and the synergistic interactions of FAK and Shc with PI3K, leading to the modulation of the downstream Akt/mTOR/p70S6K pathway. Taken together, we show that load-induced fluid flow is an important factor that can activate integrin-mediated
mechanotransduction to regulate bone formation-related gene expression and proliferation signaling in osteoblasts. The identification of these mechanotransduction pathways could not
only provide new insights into the mechanisms of load-induced bone formation but also aid in the future development of bone tissue engineering and pharmacologic therapies to increase bone formation.
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