Figure 2

IKKβ-IκBα-dependent activation of NF-κB is required for IL-1β-induced TLR2 up-regulation. A, IL-1β induces IκBα phosphorylation and degradation in HeLa cells as assessed by Western blot analysis. Cells were treated with IL-1β (10 ng/ml) for the indicated time periods. B, MG 132 inhibits IL-1β-induced NF-κB nuclear translocation, IκBα degradation and TLR2 up-regulation in HeLa cells. For Western blotting and immunofluorescent staining, cells were pretreated with MG132 (1 μM) for 2 h prior to treatment with IL-1β (10 ng/ml) for 20 min. For the measurement of TLR2 mRNA, cells were treated with IL-1β (10 ng/ml) for 3 h after MG132 pretreatment. Values are the mean ± SD; n = 3. The symbols indicate results significantly different (p < 0.01) from unstimulated condition (*) and IL-1β-stimulated condition (without MG132)(**). C, Overexpression of a transdominant mutant of IκBα [IκBα [(S32A, S36A)] and a dominant-negative mutant of IKKβ [IKKβ (K44A)] inhibit IL-1β-induced TLR2 up-regulation in HeLa cells. HeLa cells were treated with IL-1β (10 ng/ml) for 3 h. Values are the mean ± SD; n = 3. The symbols indicate results significantly different (p < 0.01) from unstimulated condition (*) and IL-1β-stimulated condition (without dominant-negative construct)(**). D, MG 132 also inhibits IL-1β-induced NF-κB nuclear translocation and TLR2 up-regulation in NHBE cells. For immunofluorescent staining, cells were pretreated with MG132 (1 μM) for 2 h prior to treatment with IL-1β (10 ng/ml) for 20 min. For the measurement of TLR2 mRNA, cells were treated with IL-1β (10 ng/ml) for 3 h after MG132 pretreatment. Values are the mean ± SD; n = 3. The symbols indicate results significantly different (p < 0.01) from unstimulated condition (*) and IL-1β-stimulated condition (without MG132)(**). E, Overexpression of a transdominant mutant of IκBα (S32A, S36A) and a dominant-negative mutant of IKKβ (K44A) reduce IL-1β-induced TLR2 up-regulation in NHBE cells. Cells were treated with IL-1β (10 ng/ml) for 3 h. Values are the mean ± SD; n = 3. The symbols indicate results significantly different (p < 0.01) from unstimulated condition (*) and IL-1β-stimulated condition (without dominant-negative construct)(**).