LPS-induced nuclear translocation of RhoA is dependent on NF-κB in the human lung cancer cell line A549

Abstract

RhoA, an extensively studied member of the Rho GTPase family, has been identified as a mediator of pro-inflammatory responses and aggressive carcinogenesis. Bacterial lipopolysaccharide (LPS) is known to be a potent stimulator of inflammatory cytokine production. LPS is able to alter the activity of RhoA and the subcellular distribution of RhoA is altered according to its activity. In this study, we investigated a possible link between RhoA and the LPS/nuclear factor (NF)-κB signaling pathway. In the present study, western blotting and pull-down and immunofluorescence assays were performed to investigate the activity of RhoA in A549 cells following LPS stimulation. The results showed that LPS was able to activate RhoA. Furthermore, western blotting and an immunofluorescence assay were carried out to investigate the nuclear expression of RhoA in A549 cells following LPS stimulation. The results indicated that LPS triggers the nuclear translocation of RhoA. Furthermore, western blotting, NF-κB small interfering RNA (siRNA) transfection and an immunofluorescence assay were performed to investigate the role of NF-κB in LPS-induced RhoA nuclear translocation in A549 cells. The results showed that LPS-induced RhoA nuclear translocation was inhibited by NF-κB depletion in A549 cells. RhoA and NF-κB siRNA transfection, western blotting and ELISA were carried out to investigate the role of RhoA in the LPS-induced secretion of interleukin (IL)-6 and IL-8 in A549 cells. The depletion of RhoA using RhoA siRNA decreased the LPS-induced secretion of IL-6 and IL-8, similar to the effect of NF-κB depletion. These results demonstrate that LPS is able to activate RhoA and trigger its nuclear translocation, which is dependent on NF-κB, and that RhoA plays a significant role in the LPS/NF-κB signaling pathway.

Figure 1

Expression of RhoA induced by LPS in the nuclei of A549 cells. A549 cells were treated with or without 10 μg/ml LPS for 4 h. (A) The activity of RhoA was analyzed using the pull-down assay. (B) The cells were stained with antibodies against RhoA (red) and with nuclear dye Hoechst 33342 (blue) to visualize the expression of RhoA in the nuclei. (C) The nuclear extracts were prepared and analyzed by western blotting with antibodies against RhoA and GAPDH. GAPDH expression served as a control. LPS, lipopolysaccharide.

Figure 2

Expression of RhoA induced by LPS following NF-κB depletion in the nuclei. (A) The binding between NF-κB P50 and RhoA was detected using the Co-IP assay. (B) A549 cells were transfected with control siRNA and NF-κB P50 siRNA and the cell extracts were analyzed by western blotting with antibodies against NF-κB P50 and GAPDH at 48 h after transfection. (C) A549 cells were transfected with control siRNA and NF-κB P50 siRNA and stained with antibody against NF-κB P50 (red) and the nuclear dye Hoechst 33342 (blue) to visualize the expression of NF-κB P50. (D) Forty-eight hours after siRNA transfection, the cells were treated with 10 μg/ml LPS for 4 h, then stained with antibody against RhoA (red) and the nuclear dye Hoechst 33342 (blue) to show the expression of RhoA in the nuclei. (E) The cells were treated with 10 μg/ml LPS for 4 h and the nuclear extracts were prepared and analyzed by western blotting with antibodies against RhoA and GAPDH. GAPDH expression served as a control. LPS, lipopolysaccharide; NF-κB, nuclear factor-κB; Co-IP, co-immunoprecipitation; siRNA, small interfering RNA.

Figure 3

Effect of RhoA depletion on LPS-induced IL-6 and IL-8 secretion in A549 cells. (A and B) siRNAs of RhoA and NF-κB P50 were applied to deplete their expression in A549 cells. The effects of the siRNAs on their expression were detected by western blotting with antibodies against RhoA, NF-κB P50 and GAPDH. GAPDH expression served as a control. (C and D) Forty-eight hours after siRNA transfection, the cells were treated with or without 10 μg/ml LPS for 24 h and the concentration of IL-6 and IL-8 in the supernatants was measured by ELISA. Data are shown as the mean + SE (n=6). ^*P<0.05. LPS, lipopolysaccharide; NF-κB, nuclear factor-κB; siRNA, small interfering RNA; IL, interleukin.