(A) H292, BEAS-2B and SAEC cells were treated with or without 1% CSE (time course: 0, 15, 30, and 60 minutes). Cells were harvested, nuclear extracts were prepared and immunoblotted for p-MSK1 (Thr581), total MSK1, phosphorylated RelA/p65 (Ser276), acetylated RelA/p65 (Lys310), and total RelA/p65. Lamin B was used as nuclear protein loading controls. Gel pictures shown are representative of at least three separate experiments. (B) The band intensity was measured by densitometry and data shown as fold change relative to Lamin B control. Data are shown as mean ± SEM; *, P<0.05; **, P<0.01 significant compared with control or without treatment.

Acid extracted histone proteins were used for immmunoblotting against anti-phospho-acetylated histone H3 (Ser10/Lys9) and acetylated histone H4 (Lys12). (A) The levels of phospho-acetylated (histone H3 on Ser10/Lys9) and acetylated (histone H4 on Lys12) histones were increased in response to CSE in H292 cells. The band intensity was measured by densitometry and data shown as fold change relative to total histones H3/H4. Data are shown as mean ± SEM (n = 3). ***, P<0.001 significant compared to control group. p-Ac-H3, phospho-acetylated histone H3; Ac-H4, acetylated histone H4. (B) Immunocytochemistry showing histone modifications in H292 and SAEC cells treated with CSE (1%) for 1 h. For immunocytochemistry, cells were fixed, permeabilized, and stained for expression of phospho-acetylated histone H3 (Ser10/Lys9) and acetylated histone H4 (Lys12) by immunofluorescence. Phospho-acetylated histone H3 and acetylated histone H4 are shown in red, and DNA (Hoechst nuclear staining) in blue, and merge represented as dark purple or pink. Gel pictures and immunocytochemistry figures are representative of at least three separate experiments.

MSK1 knock-down by siRNA approach attenuated the phosphorylation of RelA/p65 by CSE in human bronchial epithelial cells. H292 cells were transfected with 100 nM scrambled siRNA (non-targeted siRNA) or siRNA directed against MSK1 (MSK1 siRNA) for 24 h, followed by starvation in serum free medium and treated for 1 h after 72 h of transfection with CSE (1%). Whole cell lysate was prepared after 1 h treatment and assayed for effect of MSK1 knock-down by immunoblotting. The levels of p-MSK1 (Thr581), total MSK1, phosphorylated RelA/p65 (Ser276), acetylated RelA/p65 (Lys310), and total RelA/p65 were determined by immunoblotting. β-actin was used as a loading control. Gel pictures are representative of at least three separate experiments. The band intensity was measured by densitometry and data shown as fold change relative to β-actin control. Data are shown as mean ± SEM; *, P<0.05; **, P<0.01; ***, P<0.001, significant compared with control or without treatment non-targeted siRNA group; #, P<0.05; ##, P<0.01, significant compared to CSE treated non-targeted siRNA group.

(A) MSK1 knock-down by siRNA approach attenuated activation of MSK1 by CSE in H292 cells. CS-induced activation of MSK1 (p-MSK1 Thr581) was observed in control siRNA transfected cells compared to MSK1 siRNA transfected human bronchial epithelial cells. H292 cells were transfected with 100 nM scrambled siRNA (non-targeted siRNA) or siRNA directed against MSK1 (MSK1 siRNA) for 24 h, followed by starvation in serum free medium and treated for 1 h after 72 h of transfection with CSE (1.0%). For immunocytochemistry, cells were fixed, permeabilized, and stained for the expression of p-MSK1 (Thr581), and total MSK1 by immunofluorescence. Phospho-MSK1 is shown in red, and total MSK1 is shown green, and DNA (Hoechst nuclear staining) in blue, and merge represented as orange yellow. (B) MSK1 knock-down by siRNA approach attenuated CSE-induced histone modifications (phospho-acetylated histone H3) in human bronchial epithelial cells. H292 cells were transfected with 100 nM scrambled siRNA (non-targeted siRNA) or siRNA directed against MSK1 (MSK1 siRNA) for 24 h, followed by starvation in serum free medium and treated for 1 h after 72 h of transfection with CSE (1.0%). For immunocytochemistry, cells were fixed, permeabilized, and stained for the expression of p-Ac-H3 (Ser10/Lys9), and total MSK1 by immunofluorescence. Phospho-acetylated histone H3 is shown in red, and total MSK1 is shown green, and DNA (Hoechst nuclear staining) in blue, and merge represented as orange yellow. Results are representative cells from at least three separate experiments. p-Ac-H3, phospho-acetylated histone H3. Quantification of fluorescence intensity in immunofluorescence data was measured using ImageJ and the corrected total cell fluorescence (CTCF) values were converted into fold change values and represented as histograms. Data are shown as mean ± SEM; *, P<0.05; ***, P<0.001, significant compared with respective control or without treatment; ###, P<0.001, significant compared to CSE treated non-targeted siRNA group.

(A) H292 cells were transiently transfected with wild-type MSK1 or MSK1 N-terminal kinase-dead mutant MSK1 flag construct and treated with or without CSE (1.0%) for 1 h. Flag-tagged precipitated complexes were analyzed through immunoblotting using anti-flag and anti-RelA/p65 antibodies. Whole cell lysate from pCMV-Flag-MSK1 wild-type transfected cells were used as lysate control. (B) H292 cells were treated for 1 h with or without CSE (2.0%) and TNFα (10 ng/ml) was used as positive control. Nuclear extract (1000 µg) was used for immunoprecipitation of MSK1 bound complexes and analyzed through immunoblotting using anti-MSK1, anti-RelA/p65 and anti-p300 antibodies. (C) H292 cells were treated for 1 h with or without CSE (2.0%), and TNFα (10 ng/ml) was used as positive control. Nuclear extract (1000 µg) was used for immunoprecipitation of RelA/p65 bound complexes and analyzed through immunoblotting using anti-RelA/p65 and anti-MSK1 antibodies. (D) CS-mediated interaction of MSK1-p300 in acute CS exposed mouse lung. Adult C57BL/6J mice were exposed to CS at 300 mg/m³ total particulate matter (TPM) for 3 days and were sacrificed 24 h after the final exposure. Whole tissue extract (500 µg) was used for immunoprecipitation of MSK1 bound complexes, and analyzed by immunoblotting using anti-MSK1 and anti-p300 antibodies. (E) CS-mediated interaction of RelA/p65 with MSK1 and p300 in acute CS exposed mouse lung. Adult C57BL/6J mice were exposed to CS at 300 mg/m³ total particulate matter (TPM) for 3 days and were sacrificed 24 h after the final exposure. Whole tissue extract (500 µg) was used for immunoprecipitation of RelA/p65 bound complexes, and analyzed by immunoblotting using anti-RelA/p65, anti-MSK1 and anti-p300 antibodies. Results are representative of at least three separate experiments.

Cigarette smoke/aldehyde/oxidative stress via activation of MSK1 results in phosphorylation, and nuclear transclocation of NF-κB RelA/p65. Activated nuclear MSK1 subsequently causes chromatin modifications of histones (phospho-acetylation of histone H3 [Ser10/Lys9] and acetylation of histone H4 [Lys12]). MSK1 N-terminal kinase dead mutant, siRNA-mediated knock-down and stable MSK1 knock-down MEFs represses the nuclear MSK1, phosphorylation of RelA/p65, and phospho-acetylation of histone H3 and acetylation of histone H4. CS activates MSK1 which forms a complex with NF-κB RelA/p65 and coactivator p300 which further mediates transcriptional activation of NF-κB-dependent pro-inflammatory genes (IL-6, IL-8 and COX-2). (↑, Induction; ⊢ inhibition).

(A) Activation of MSK1 and RelA/p65 (Ser276) in H292 cells treated with CSE (1%) for 1 h. For immunocytochemistry, cells were fixed, permeabilized, and stained for the expression of p-MSK1 (Thr581), total MSK1, and phosphorylated RelA/p65 (Ser276) by immunofluorescence. Phosphorylated MSK1, total MSK1, and phosphorylated RelA/p65 is shown in red, and DNA (Hoechst nuclear staining) in blue, and merge represented as dark purple or pink. Results are representative cells from three separate experiments. The panel without primary antibodies was considered as negative control (NC). (B) Activation of MSK1 and RelA/p65 (Ser276) in SAEC cells treated with CSE (1%) for 1 h. For immunocytochemistry, cells were fixed, permeabilized, and stained for the expression of p-MSK1 (Thr581), total MSK1, and phosphorylated RelA/p65 (Ser276) by immunofluorescence. Phosphorylated MSK1, and phosphorylated RelA/p65 is shown in red, total MSK1 in green, and DNA (Hoechst nuclear staining) in blue, and merge represented as dark purple or pink. Results are representative cells from three separate experiments. The panel without primary antibodies was considered as negative control (NC). (C) Nuclear levels of MSK1 (phosphorylated and total MSK1) were increased in lung tissue of mouse exposed to CS determined by immunoblot analysis. Adult C57BL/6J mice were exposed to CS for 3 days and were sacrificed 24 hrs post-final CS exposure. Lamin B was used as protein loading control. The band intensity was measured by densitometry and data shown as fold change relative to Lamin B control. Data are shown as mean ± SEM (n = 4/group); **, P<0.01, significant compared to air-exposed controls. p-MSK1 (Thr581), phosphorylated MSK1.

Cigarette smoke increased the cellular levels of phosphorylated MSK1 as well as phosphorylated RelA/p65 levels in control vector (pCMV-FLAG) and MSK1 WT (pCMV-FLAG-MSK1 wild-type) transfected human bronchial epithelial cells compared to MSK1 N- and C-terminal kinase-dead mutants (pCMV-FLAG-MSK1 ND and pCMV-FLAG-MSK1 CD). H292 cells were transiently transfected with control vector, MSK1 WT, MSK1 N-terminal kinase-dead (ND) mutant, MSK1 C-terminal kinase-dead (CD) mutant and treated with or without CSE (1%) after 24-48 hrs of transfection for 1 h. Cells were harvested (1 h later). Whole cell extracts were prepared and immunoblotted for p-MSK1 (Thr581), total MSK1, phosphorylated RelA/p65 (Ser276), acetylated RelA/p65 (Lys310), and total RelA/p65. β-actin was used as protein loading control. Gel pictures are representative of at least three separate experiments. The band intensity was measured by densitometry and data shown as fold change relative to β-actin control. Data are shown as mean ± SEM; **, P<0.01; ***, P<0.001, significant compared with respective control group; #, P<0.05; ##, P<0.01; significant compared to CSE treated control vector transfected group.

CSE-induced activation of MSK1 and RelA/p65 (Ser276) in non-targeted siRNA transfected human bronchial epithelial cells. MSK1 knock-down by siRNA approach attenuated the activation of MSK1 and phosphorylation of RelA/p65 by CSE in human bronchial epithelial cells. H292 cells were transfected with 100 nM scrambled siRNA (non-targeted siRNA) or MSK1 siRNA for 24 h, followed by starvation in serum free medium and then treated for 1 h (72 h post-transfection) with CSE (1.0%). For immunocytochemistry, cells were fixed, permeabilized, and stained for the expression of p-MSK1 (Thr581), total MSK1, and phosphorylated RelA/p65 (Ser276) by immunofluorescence. Phosphorylated MSK1, total MSK1, and phosphorylated RelA/p65 are shown in red, and DNA (Hoechst nuclear staining) in blue, and merge represented as dark purple or pink. Results are representative cells from at least three separate experiments. The quantification of fluorescence intensity in immunofluorescence data was measured using ImageJ and the corrected total cell fluorescence (CTCF) values were converted into fold change values and represented as histograms. Data are shown as mean ± SEM; **, P<0.01; ***, P<0.001, significant compared with respective control without treatment; #, P<0.05; ##, P<0.01, significant compared to CSE treated non-targeted siRNA group.

CSE induced histone modification (phosphorylated/acetylated histone H3/H4) in 10T1/2 wild-type mouse embryonic fibroblasts (MEFs) compared to stable MSK1 knock-down MEFs. Wild-type MEFs and MSK1 knock-down MEFs were treated for 1 h with CSE (1.0%). For immunocytochemistry, cells were fixed, permeabilized and stained for the expression of p-Ac H3 (Ser10/Lys9), and Ac-H4 (Lys12) by immunofluorescence. Phosphorylated/acetylated histone H3 and acetylated histone H4 are shown in red, and DNA (Hoechst nuclear staining) in blue, and merge represented as purple or pink. Results are representative cells from at least three separate experiments. The panel without primary antibodies was considered as negative control (NC). The quantification of fluorescence intensity in immunofluorescence data was measured using ImageJ and the corrected total cell fluorescence (CTCF) values were converted into fold change values and represented as histograms. Data are shown as mean ± SEM; ***, P<0.001, significant compared with control or without treatment; ###, P<0.001, significant compared to CSE treated wild-type group.

H292 cells were treated with CSE (1%) for 1 h. ChIP analysis was performed against MSK1, phospho-RelA/p65 (Ser276), phospho-histone H3 (Lys9) and acetylated histone H4 (Lys12) antibodies. After reverse cross-linking, co-immunoprecipitated genomic DNA fragments were analyzed by semi-quantitative PCR with IL-6, IL-8, and COX-2 promoter specific primer sets. IgG was used as negative control, and RNA Polymerase II antibody was used as positive control. Input reflects the relative amount of sonicated DNA fragments present before immunoprecipitation and revealed by semi-quantitative PCR with pro-inflammatory gene specific primers. The band intensity was measured by densitometry and data shown as fold change versus input. Data are shown as mean ± SEM (n = 3). *, P<0.05; **, P<0.01, significant compared to respective control group.