We aimed at elucidating the molecular basis of c-fos promoter activation in vascular endothelial cells (ECs) in response to shear stress, with emphases on Rho family GTPases (Rho, Cdc42, and Rac) and intracellular calcium. Dominant-negative and constitutively activated mutants of these GTPases were used to block the action of upstream signals and to activate the downstream pathways, respectively. The role of intracellular calcium was assessed with intracellular calcium chelators. Only Rho, but not Cdc42 or Rac, is involved in the shear stress induction of c-fos. This Rho-mediated shear-induction of c-fos is dependent on intracellular calcium, but not on the Rho effector p160ROCK or actin filaments. While the inhibition of p160ROCK and its ensuing disruption of actin filaments decreased the basal c-fos activity in static ECs (no flow), it did not affect the shear-inductive effect. The calcium chelator BAPTA-AM inhibits the shear-induction, as well as the static level, of c-fos activity. (C) 2003 Elsevier Science (USA). All rights reserved.