Fluid Shear Stress Synergizes with Insulin-like Growth Factor-I (IGF-I) on Osteoblast Proliferation through Integrin-dependent Activation of IGF-I Mitogenic Signaling Pathway

Abstract

This study tested the hypothesis that shear stress interacts with the insulin-like growth factor-I (IGF-I) pathway to stimulate osteoblast proliferation. Human TE85 osteosarcoma cells were subjected to a steady shear stress of 20 dynes/cm(2) for 30 min followed by 24-h incubation with IGF-I (0-50 ng/ml). IGF-I increased proliferation dose-dependently (1.5-2.5-fold). Shear stress alone increased proliferation by 70%. The combination of shear stress and IGF-I stimulated proliferation (3.5- to 5.5-fold) much greater than the additive effects of each treatment alone, indicating a synergistic interaction. IGF-I dose-dependently increased the phosphorylation level of Erk1/2 by 1.2-5.3-fold and that of IGF-I receptor (IGF-IR) by 2-4-fold. Shear stress alone increased Erk1/2 and IGF-IR phosphorylation by 2-fold each. The combination treatment also resulted in synergistic enhancements in both Erk1/2 and IGF-IR phosphorylation (up to 12- and 8-fold, respectively). Shear stress altered IGF-IR binding only slightly, suggesting that the synergy occurred primarily at the post-ligand binding level. Recent studies have implicated a role for integrin in the regulation of IGF-IR phosphorylation and IGF-I signaling. To test whether the synergy involves integrin-dependent mechanisms, the effect of echistatin (a disintegrin) on proliferation in response to shear stress +/- IGF-I was measured. Echistatin reduced basal proliferation by approximately 60% and the shear stress-induced mitogenic response by approximately 20%. It completely abolished the mitogenic effect of IGF-I and that of the combination treatment. Shear stress also significantly reduced the amounts of co-immunoprecipitated SHP-2 and -1 with IGF-IR, suggesting that the synergy between shear stress and IGF-I in osteoblast proliferation involves integrin-dependent recruitment of SHP-2 and -1 away from IGF-IR.