Involvement of nuclear factor-kB activation through RhoA/Rho-kinase pathway in LPS-induced IL-8 production in human cervical stromal cells

Abstract

Interleukin-8 (IL-8) is a chemokine that recruits and activates neutrophils in stromal tissue and plays an essential role in cervical ripening. Nuclear factor-kB (NF-kB) is known to be important for the up-regulation of IL-8 gene expression. We examined the molecular mechanisms responsible for NF-kB activation in IL-8 production in cervical stromal cells. Lipopolysaccharide (LPS) and IL-1beta stimulated IL-8 production by cervical stromal cells in a dose-dependent manner. Pretreatment of cervical stromal cells with inhibitors of RhoA (C3 transferase exoenzyme), Rho-kinase (Y-27632) or NF-kB (BAY11-7082) effectively blocked LPS-induced IL-8 release. In contrast, IL-1beta-induced IL-8 production was significantly blocked by BAY11-7082, but not by C3 transferase exoenzyme or Y-27632. Pull-down assays showed that LPS activated RhoA, but IL-1beta caused only a lower level of activation. Transfection of the cervical stromal cells with RhoA small interfering RNA (siRNA) inhibited LPS-stimulated IL-8 production, whereas IL-1beta-induced IL-8 production was not significantly inhibited by knockdown of RhoA with siRNA. Using an NF-kB transcription reporter vector, luciferase assays demonstrated that incubation with LPS or IL-1beta induced the activation of NF-kB in cervical stromal cells. Activation of NF-kB by LPS was inhibited by treatment with C3 exoenzyme, Y-27632 or RhoA siRNA. However, inhibition of the RhoA/Rho-kinase pathway did not attenuate the activation of NF-kB by IL-1beta. These results suggest that LPS-induced IL-8 production is accompanied by enhanced NF-kB activation through the RhoA/Rho-kinase pathway in human cervical cells.