Bone mechanotransduction is vital for skeletal integrity. Osteocytes are thought to be the cellular structures that sense physical forces and transform these signals into a biological response. The Wnt/beta-catenin signaling pathway has been identified as one of the signaling pathways that is activated in response to mechanical loading, but the molecular events that lead to an activation of this pathway in osteocytes are not Well Understood. We assessed whether nitric oxide, focal adhesion kinase, and/or the phosphatidyl inositol-3 kinase/Akt signaling pathway mediate loading-induced beta-catenin pathway activation in MLO-Y4 osteocytes. We found that mechanical stimulation by pulsating fluid flow (PFF, 0.7 +/- 0.3 Pa, 5 Hz) for 30 min induced beta-catenin stabilization and activation of the Wnt/beta-catenin signaling pathway The PFF-induced stabilization of beta-catenin and activation of the beta-catenin signaling pathway was abolished by adding focal kinase inhibitor FAK inhibitor-14 (50 mu M), or phosphatidyl inositol-3 kinase inhibitor LY-294002 (50 mu M). Addition of nitric oxide synthase inhibitor L-NAME (1.0 mM) also abolished PFF-induced stabilization of beta-catenin This Suggests that mechanical loading activates, the beta-catenin signaling pathway by a mechanism involving nitric oxide, focal adhesion kinase, and the Akt signaling pathway. These data provide a framework for understanding the role of beta-catenin in mechanical adaptation of bone (C) 2009 Elsevier Inc. All rights reserved.