IKKβ-I-κBɛ-c-Rel/p50: a new axis of NF-κB activation in lung epithelial cells

Abstract

Cigarette smoke (CS), a major risk factor for developing lung cancer, is known to activate transcriptional activator nuclear factor kappa B (NF-κB). However, the underlying mechanism of this activation remains unclear because of conflicting reports. As NF-κB has a pivotal role in the generation and maintenance of malignancies, efforts were targeted towards understanding its activation mechanism using both ex vivo and in vivo studies. The results show that CS-induced NF-κB activation mechanism is different from that of other pro-inflammatory signals such as lipopolysaccharide (LPS). The NF-κB dimer that translocates to the nucleus upon stimulation with CS is predominantly composed of c-Rel/p50 and this translocation involves degradation of I-κBɛ and not I-κBα. This degradation of I-κBɛ depends on IKKβ activity, which preferentially targets I-κBɛ. Consistently, CS-activated form of IKKβ was found to be different from that involved in LPS activation as neither Ser177 nor Ser181 of IKKβ is crucial for CS-induced NF-κB activation. Thus, unlike other pro-inflammatory stimulations where p65 and I-κBα have a central role, the predominantly active signaling cascade in CS-induced NF-κB activation in the lung epithelial cells comprises of IKKβ-I-κBɛ-c-Rel/p50. Thus, this study uncovers a new axis of NF-κB activation wherein I-κBɛ and c-Rel have the central role.

Figure 1

CSE-induced NF-κB activation in alveolar epithelial A549 cells predominantly involves nuclear translocation of c-Rel and p50. (a) CSE treatment predominantly induces nuclear translocation of c-Rel and p50. A549 cells were treated with either 2% CSE (upper panels) or 1 μg/ml LPS (lower panels) for different time periods as indicated. Nuclear and cytosolic fractions were prepared and separated by SDS–polyacrylamide gel electrophoresis. Western blot analysis was performed with anti-c-Rel, anti-RelA, anti-p50 and anti-tubulin antibodies. C and N indicate cytosolic and nuclear fractions, respectively. (b) Immunolocalization of c-Rel, p65 and p50. A549 cells were treated with 2% CSE for 30 min, fixed and probed with anti-c-Rel, anti-p65 and anti-p50 primary antibodies. (c) c-Rel downregulation inhibits CSE-induced NF-κB activation. A549 cells were transfected with pSuper (empty vector), anti-c-Rel (si-c-Rel) and anti-p65 (si-p65) si-RNA constructs. After 24 h of transfection, cells were harvested and cell extracts were analyzed by western blotting (left panel). Tubulin serves as loading control. These transfectants harboring pSuper, si-c-Rel and si-p65 constructs were treated with 2% CSE for 30 min and harvested. Nuclear extracts were analyzed by EMSA using radiolabeled NF-κB probe (right panel). The first lane of the gel was loaded with the free probe. (d) Chromatin immunoprecipitation (ChIP) analysis of p65, c-Rel and p50 recruitment at IL-8 and cyclin D1 upstream promoter sequences. A549 cells were treated with 2% CSE for 30 min and cross-linked with paraformaldehyde. Immunoprecipitations were carried out using anti- p65, c-Rel and p50 antibodies. Immunoprecipitated DNA was amplified by PCR primers corresponding to the NF-κB-binding site(s) at IL-8 and cyclin D1 upstream promoter sequences as indicated in the upper panel and analyzed by agarose gel electrophoresis. DAPI, 4, 6-diamidino-2-phenyl indole; FITC, fluorescein isothiocyanate.

Figure 2

CS-induced NF-κB activation in guinea pig lung. (a) Exposure of guinea pigs to CS induces alveolar NF-κB activation. Guinea pigs were exposed to CS for 0, 3, 4, 5 and 6 days. Nuclear extracts were prepared from lung tissues, and NF-κB activation was assayed by EMSA using radiolabeled wild-type NF-κB probe. (b) Immunohistochemistry of c-Rel and p65. Lung tissue from guinea pigs that were either exposed to CS for 4 days or left unexposed (0 day) were fixed and paraffin sections were prepared. Thereafter sections were immunostained with anti-c-Rel and anti-p65 antibodies. Nuclei were stained with DAPI. FITC, fluorescein isothiocyanate.

Figure 3

I-κBɛ undergoes degradation upon exposure to CS. (a) Effect of CSE on I-κB in A549 cells. Cells were treated with 2% CSE for different time periods (as indicated) and harvested. Whole-cell extracts were prepared and examined for I-κBɛ and I-κBα by western blotting. Tubulin serves as loading control. (b) Effect of I-κBα super repressor on CS-induced NF-κB activity. A549 cells were transiently transfected with a plasmid construct that expresses I-κBα super repressor (I-κBα^s) along with a NF-κB reporter construct and a lacZ construct. After 24 h of transfection, cells were treated either with 2% CSE or with LPS (1 μg/ml) for 60 min or left untreated (control). Cell extracts were prepared and tested for luciferase activity. Results were normalized for transfection efficiencies with respect to beta galactosidase activity. Result represents the mean ±s.d. of three independent experiments. (c) Interaction of c-Rel with p50 and I-κBɛ in A549 cells. Cells were treated either with 2% CSE for 60 min or left untreated. Cell extracts were prepared and immunoprecipitations were performed using anti-c-Rel antibody. Immunoprecipitates were analyzed for I-κBɛ and p50 by western blotting. The panel marked with star (_^*) sign shows the immunoglobulin heavy-chain band. Bottom panel shows the input. (d) Time-dependent loss of I-κBɛ from c-Rel upon CSE-treatment. A549 cells were treated with 2% CSE for different lengths of time as indicated. Whole-cell extracts were prepared and immunoprecipitations were performed with mouse monoclonal anti-c-Rel antibody. Immunoprecipitates were analyzed by immunoblotting with rabbit polyclonal antibodies against I-κBɛ and c-Rel. (e) CS exposure induces I-κBɛ degradation in guinea pig lung. Guinea pigs were either exposed to CS for 4 days or left unexposed. Lung tissue extracts were analyzed for I-κBɛ and I-κBα by western blotting. Tubulin serves as loading control.

Figure 4

CSE-induced NF-κB activation requires IKK activity. (a) CSE activates IKK. A549 cells were treated with 2% CSE and harvested at different time points as indicated. Cell extracts were prepared and subjected to immunoprecipitation (IP) using anti-IKKγ anibody. Kinase assays were performed with the immunoprecipitates and purified recombinant substrates, either GST-I-κBɛ (1–27 aa) or GST-I-κBα (1–32 aa) in presence [γ-³²P]-ATP. The mixtures were separated by SDS–polyacrylamide gel electrophoresis. The gels were stained with Coomassie blue and autoradiograms were captured. I and III are autoradiograms, whereas II and IV are corresponding Coomassie-stained gels. (b) Downregulation of IKKβ inhibits CSE-induced I-κBɛ degradation. A549 cells were either transfected with pSuper (empty vector) or anti-IKKβ siRNA construct (si-IKKβ). Twenty-four hours after transfection, cells were treated with 2% CSE for different time periods as indicated. Cell extracts were analyzed by western blotting with antibodies against I-κBɛ, IKKβ and tubulin. (c) IKKβ downregulation impairs CSE-induced NF-κB activation. A549 cells transfected with either pSuper or si-IKKβ were treated with 2% CSE for 30 min and harvested. Nuclear extracts were analyzed by EMSA using radiolabeled NF-κB probe.

Figure 5

IKKβ activated by CS is different from LPS-activated IKKβ. A549 cells were transiently transfected with a NF-κB reporter construct, a lacZ construct and any one of the following: (i) wild-type IKKβ, (ii) mutant IKKβ (IKKβ S177A), (iii) mutant IKKβ (IKKβ S181A) and (iv) empty vector. After 24 h of transfection, cells were treated either with 2% CSE or with 1 μg/ml LPS for 60 min or left untreated (control). Cell extracts were prepared and tested for luciferase activity. Results were normalized for transfection efficiencies by beta galactosidase activity assay. Result represents the mean±s.d. of three independent experiments. RLU, relative luminescence unit.