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

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-κB) activation in human fibroblasts. However, the signaling mechanism as to how the elevated Ca ²⁺ activates NF-κB 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-κB activation. 4-Hydroxy-2-nonenal (HNE) was produced and modified IκB peaking at 2 min. The phosphorylation of IκB peaked at 8 min. HNE modification and IκB phosphorylation, NF-κB 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. IκB 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 IκB, peaked at 2 min. IKK under static conditions was activated by exogenously applied HNE at a relatively low dose (1 μM), while it was inhibited at higher concentrations, suggesting that HNE could be one of the candidate signals in the stretch-induced NF-κB activation. The present study suggests that the NF-κB 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 IκB → NF-κB translocation to the nucleus.