Involvement of reactive oxygen species in cyclic stretch-induced NF-κB activation in human fibroblast cells

1 Uniaxial cyclic stretch leads to an upregulation of cyclooxygenase (COX)-2 through increases in the intracellular Ca2+ concentration via the stretch-activated (SA) channel and following nuclear factor kappa B (NF-kappa B)activation in human fibroblasts. However, the signaling mechanism as to how the elevated Ca2+ activates NF-kappa B is unknown. In this study, weexamined the involvement of reactive oxygen species (ROS) as an intermediate signal, which links the elevated Ca2+ with NF-kappa B activation. 2 4-Hydroxy-2-nonenal (HNE) was produced and modified I kappa B peaking at 2 min. The phosphorylation of I kappa B peaked at 8min. HNE modification and I kappa B phosphorylation, NF-kappa B translocation to the nucleus, and following COX-2 production were inhibited by extracellular Ca2+ removal or Gd3+ application, as well as by the antioxidants. The stretch-inducedCa(2+) increase was inhibited by extracellular Ca2+ removal, or Gd3+ application. 3 I kappa B kinase (IKK) activity peaked at 4 min, which was inhibited by extracellular Ca2+ removal, Gd3+ or the antioxidants. IKK was also HNE-modified and, similarly to I kappa B, peaked at 2 min. IKK under static conditions was activated by exogenously applied HNE at a relatively low dose (1 mu M), while it was inhibited at higher concentrations, suggesting thatHNE could be one of the candidate signals in the stretch-induced NF-kappa B activation. 4 The present study suggests that the NF-kappa B activation by cyclic stretch is mediated by the following signal cascade: SA channel activation -> intracellular Ca2+ increase -> production of ROS -> activation of IKK -> phosphorylation of I kappa B -> NF-kappa B translocation to the nucleus.