1 Uniaxial cyclic stretch leads to an upregulation of cyclooxygenase (COX)-2 through increases in the intracellular Ca(2+) concentration via the stretch-activated (SA) channel and following nuclear factor kappa B (NF-kappaB) activation in human fibroblasts. However, the signaling mechanism as to how the elevated Ca(2+) activates NF-kappaB is unknown. In this study, we examined the involvement of reactive oxygen species (ROS) as an intermediate signal, which links the elevated Ca(2+) with NF-kappaB activation. 2 4-Hydroxy-2-nonenal (HNE) was produced and modified IkappaB peaking at 2 min. The phosphorylation of IkappaB peaked at 8 min. HNE modification and IkappaB phosphorylation, NF-kappaB translocation to the nucleus, and following COX-2 production were inhibited by extracellular Ca(2+) removal or Gd(3+) application, as well as by the antioxidants. The stretch-induced Ca(2+) increase was inhibited by extracellular Ca(2+) removal, or Gd(3+) application. 3 IkappaB kinase (IKK) activity peaked at 4 min, which was inhibited by extracellular Ca(2+) removal, Gd(3+) or the antioxidants. IKK was also HNE-modified and, similarly to IkappaB, peaked at 2 min. IKK under static conditions was activated by exogenously applied HNE at a relatively low dose (1 microM), while it was inhibited at higher concentrations, suggesting that HNE could be one of the candidate signals in the stretch-induced NF-kappaB activation. 4 The present study suggests that the NF-kappaB activation by cyclic stretch is mediated by the following signal cascade: SA channel activation --> intracellular Ca(2+) increase --> production of ROS --> activation of IKK --> phosphorylation of IkappaB --> NF-kappaB translocation to the nucleus.