beta-Catenin plays a dual role in cells: one at cell-cell junctions and one regulating gene transcription together with TCF (T-cell Factor) in the nucleus. Recently, a role for beta-catenin in osteoblast differentiation and gene expression has begun to be elucidated. Herein we investigated the effects of fluid shear stress (FSS) on beta-catenin signaling. FSS is a well-characterized anabolic stimulus for osteoblasts; however, the molecular mechanisms for the effects of this stimulation remain largely unknown. We found that 1 hour of laminar FSS (10 dynes/cm(2)) induced translocation of beta-catenin to the nucleus and activated a TCF-reporter gene. Analysis of upstream signals that may regulate beta-catenin signaling activity revealed two potential mechanisms for increased beta-catenin signaling. First, FSS induced a transient, but significant, increase in the phosphorylation of both glycogen synthase kinase 3beta (GSK-3beta) and Akt. Second, FSS reduced the levels of beta-catenin associated with N-cadherin, suggesting that less sequestration of beta-catenin by cadherins occurs in osteoblasts subjected to FSS. Functional analysts of potential genes regulated by beta-catenin signaling in osteoblasts revealed two novel observations. First, endogenous, nuclear beta-catenin purified from osteoblasts formed a complex with a TCF -binding element in the cyclooxygenase-2 promoter, and, second, overexpression of either a constitutively active beta-catenin molecule or inhibition of GSK-3beta activity increased basal cyclooxygenase-2 levels. Together, these data demonstrate for the first time that FSS modulates the activity of both GSK-3beta and beta-catenin and that these signaling molecules regulate cyclooxygenase-2 expression in osteoblasts.