CS exposure led to recruitment of IKKα and RelA/p65 (total and acetylated) on promoters of IL-6 and MIP-2 genes. Mice were exposed to CS for 3 days and killed 24 hours after the last CS exposure. (A) CS exposure caused recruitment of IKKα, RelA/p65 (total and acetylated) on MIP-2 and IL-6 gene promoters. The nuclear extracts were immunoprecipitated with specific antibodies, and binding to the promoter of pro-inflammatory mediator genes was detected by PCR-primers for IL-6 or MIP-2 (n = 3/group). Binding to the promoters is compared with PCR of the input DNA. (B) The bands were measured by densitometry (fold induction versus control). Data are shown as mean ± SEM; **P < 0.01, and ***P < 0.001, significantly different from respective air-exposed mice. Open bars, air; solid bars, CS.

Nuclear levels of IKKα and RelA/p65 (total and acetylated) were increased in lung tissue of mouse exposed to CS. (A) Mice were exposed to CS for 3 days and 8 weeks, and were killed 24 hours after the last CS exposure. CS increased the nuclear levels of IKKα and RelA/p65, and induced hyper-acetylation of RelA/p65 in mouse lung tissue. The levels of IKKα, RelA/p65, and acetylated RelA/p65 were determined by Western blotting. Histone H3 and β-actin were used for nuclear protein loading controls. β-tubulin was used for cytoplasmic protein loading control, which was not expressed in the nuclear extracts of mouse lung tissue (data not shown). (B) The bands were measured by densitometry (fold induction versus control). Data are shown as mean ± SEM; *P < 0.05, **P < 0.01, and ***P < 0.001, significantly different from respective air-exposed mice (n = 4/group).

Cigarette smoke (CS) exposure increased the levels of RelA/p65 NF-κB–dependent pro-inflammatory mediators in mouse lung. (A) Levels of RelA/p65 NF-κB–dependent pro-inflammatory cytokines were analyzed by Luminex assay in bronchoalveolar lavage fluid (150 μl) of mice exposed to CS for 3 days. The animals were killed 24 hours after the last CS exposure. Open bars indicate air-exposed mice, and solid bars indicate CS-exposed mice. (A) MIP-2 (macrophage-inflammatory protein-2) and IL-6. Acute and sub-chronic CS exposure significantly increased the levels of keratinocyte chemoattractant, MCP-1, IL-6, and granulocyte macrophage colony-stimulating factor in the lungs of mice exposed for (B) 3 days and (C) 8 weeks. The levels of pro-inflammatory mediators in lung homogenates were measured by the Luminex 100 using a beadlyte mouse multi-cytokine beadmaster kit. Data are shown as mean ± SEM (n = 5–6/group). **P < 0.01, ***P < 0.001, significant compared with air-exposed group.

CS exposure increased the expression of IKKα in alveolar macrophages and alveolar/airway epithelial cells in mouse lung. Immunohistochemistry data revealed that IKKα is not only localized, but also increased, in alveolar macrophages, alveolar type II cells, and airway epithelial cells in response to CS both after 3 days and 8 weeks. (A) Representative photographs (×200) from immunostaining for IKKα in lung tissues from air- and CS-exposed mice. The insets in boxes are magnified alveolar macrophages and epithelial cells showing increased IKKα staining in response to CS compared with air-exposure. Appearance of dark brown color represents the presence of IKKα, which was increased in CS exposed mouse lung. E, epithelial cells; M, macrophage; Alv, alveoli; Aw, airway. (B) Immunostaining scores for IKKα per cell type in alveolar and airway regions of the lung. The assessment of immunostaining intensity was performed semi-quantitatively and in a blinded fashion. Black bars, intense staining; grey bars, moderate/weak staining; open bars, no staining. Results are mean of four experiments ± SEM. **P < 0.01 and ***P < 0.001, significant compared with air-exposed mice.

CS exposure caused histone H3 phospho-acetylation and H4 acetylation on pro-inflammatory gene promoters in mouse lung. CS caused acetylation of histone H3 and H4 on promoter sites of MIP-2 and IL-6 genes after 3 days of CS exposure and killed 24 hours after the last exposure (A). Phosphorylation on ser10 and acetylation on lys9 of histone H3, and acetylation on lys12 of histone H4 were increased in response to CS exposure after 3 days. Lung homogenates were immunoprecipitated with anti-acetylated histone H3 and H4 antibodies, and chromatin modification on the promoter region of pro-inflammatory cytokine genes was detected by PCR using primers for IL-6 and MIP-2. The bands were measured by densitometry (fold induction versus control). Chromatin modification on various promoters was compared with input DNA against IL-6 and MIP-2 primers. PCR products were separated on agarose gel and stained with SYBR green. Immunoglobulin G was used as a negative control in ChIP assay. (B) Acid-extracted protein was used for immunoblotting against anti-acetylated and phosphorylated-histone H3 (lys9/ser10) and acetylated histone H4 (lys12). The levels of phosphorylation (histone H3 on ser10) and acetylation (histone H3 on lys9 and histone H4 on lys12) of histones were increased in response to CS exposure after 3 days. Data are shown as mean ± SEM, **P < 0.01, ***P < 0.001, n = 4/group significantly different from respective air-exposed mice. Ac-/P-H3, acetylated- and phosphorylated histone H3; Ac-H3, acetylated histone H3; Ac-H4, acetylated histone H4. Open bars, air; solid bars, CS.

IKKα regulated the release of IL-8–associated with histone H3 phospho-acetylation (lys9/ser10) and phosphorylation/acetylation of RelA/p65 in response to cigarette smoke extract (CSE) in macrophages (MonoMac6 cells). (A) MonoMac6 cells were transfected with IKKα lacking/expressing plasmids, and treated with CSE (0.5%, 1.0%, and 2.5%) for 1 hour; then the level of IL-8 in supernatant was measured by enzyme-linked immunosorbent assay. CSE significantly increased the level of IL-8 release, and cells lacking IKKα showed decreased level of IL-8 in MonoMac6 cells in response to CSE. Data are shown as mean ± SEM; ***P < 0.001, ^###P < 0.001, significant compared with control groups (n = 4). (B) CSE increased acetylated and phosphorylated histone H3, and the cells lacking IKKα showed decreased levels of acetylated and phosphorylated histone H3. However, the cells overexpressing IKKα increased the levels of acetylated and phosphorylated histone H3 in response to CSE treatments. The results are presented as an average of three separate experiments performed in triplicate. For immunocytochemistry, cells were fixed, and the expression of acetylated and phosphorylated histone H3 and IKKα were determined by immunofluorescence. Acetylated and phosphorylated histone H3 is shown in red, IKKα in green, and DNA (Hoechst nuclear staining) in blue. The images were taken by magnification (×400), and selected a representative cellular morphology from three separate experiments. The group without primary antibodies was used for negative control. (C) CSE increased the levels of total IKKα and phosphorylation of IKKα, and overexpression of IKKα augmented the phosphorylation of RelA/p65 on ser276 and acetylation of RelA/p65 on lys310/K310 in MonoMac6 cells. MonoMac6 cells were transfected with overexpression or dominant-negative IKKα plasmid and treated with CSE (0.5%, 1.0%, and 2.5%) for 1 hour; and then whole cell lysate was used to determine the protein levels of phosphorylated (ser176/180 residue) and total IKKα, IKKβ, total RelA/p65, phosphorylation and acetylation of RelA/p65. β-actin was used as a loading control (n = 4).