Signaling pathways involved in oxidative stress-induced inhibition of osteoblast differentiation are not known. We showed in this report that H2O2 (0.1-0.2mM)-induced oxidative stress suppressed the ostcoblastic differentiation process of primary rabbit bone marrow stromal cells (BMSC) and calvarial osteoblasts, manifested by a reduction of differentiation markers including alkaline phosphatase (ALP), type I collagen, colony-forming unit-osteoprogenitor (CFU-O) formation, and nuclear phosphorylation of Runx2. H2O2 treatment stimulated phospholipase C-gamma1 (PLC-gamma1), extracellular signal-regulated kinase 1/2 (ERK1/2), and NF-kappaB signaling but inhibited p38 mitogen-activated protein kinase (MAPK) activation. In the presence of 20 muM PD98059 or 50 muM caffeic acid phenethyl ester (CAPE), specific inhibitor for ERKs or NF-kappaB, respectively, could significantly reverse the decrease of above-mentioned osteoblastic differentiation markers elicited by H2O2 (0.1 mM). Furthermore, PD98059 also suppressed H2O2-stimulated NF-kappaB signaling in this process. These data suggest that ERK and ERK-dependent NF-Kbeta activation is required for oxidative stress-induced inhibition of osteoblastic differentiation in rabbit BMSC and calvarial osteoblasts. (C) 2003 Elsevier Inc. All rights reserved.