Cigarette smoke is the common etiological factor in the pathogenesis of COPD. We and others have demonstrated the role of the NF-κB signaling pathway in pro-inflammatory gene transcription and chromatin modifications 
. Studies in the past have shown that NF-κB is recruited to the promoter of pro-inflammatory genes in CS-exposed rodent lungs, resulting in increased histone acetylation 
MSK1 has been shown to regulate transcription of several pro-inflammatory genes at multiple levels 
. Transcription factor CREB was the first MSK1 substrate to be identified 
. MSK1 also phosphorylates RelA/p65 at Ser276 residue in response to TNFα 
. In addition to transcription factor regulation, MSK1 can contribute to gene activation by phosphorylation of chromatin proteins histone H3 (Ser10 and Ser28) and HMG-N1 (Ser6) 
. However, the underlying molecular signaling mechanism that causes activation of MSK1 and downstream signaling event, including chromatin histone modifications, in response to CS is not known. We tested the hypothesis that CS induces histone modifications via activation of MSK1 and phospho-acetylation of RelA/p65 followed by recruitment of MSK1 and its substrates to the promoters of pro-inflammatory genes, thereby increasing the transcriptional activity of pro-inflammatory mediators in human bronchial and small airway epithelial cells and in mouse lungs.
CS exposure resulted in phosphorylation of MSK1 at Thr581 in transformed human bronchial epithelial cells (H292 and BEAS-2B), human primary small airway epithelial cells (SAEC) and in mouse lung. We observed similar dose-dependent activation of MSK1 and RelA/p65 by CSE (0.1% to 1.0%) in H292, BEAS-2B and SAEC cells (data not shown). Phosphorylation of MSK1 at Thr581 residue is an event of activated MSK1 protein 
. MSKs are activated by several mitogenic and stress stimuli, such as epidermal growth factor (EGF), phorbol 12-myristate 13-acetate/TPA and UV possibly via oxidative stress 
. Our results show that CS activates MSK1, leading to phospho-acetylation of RelA/p65 at Ser276 and Lys310, respectively, in epithelial cells. Similar observations were found when the cells were treated with a component of CS, the aldehyde acrolein (data not shown). Other studies have shown the control of phosphorylation of RelA/p65 at multiple serine residues regulates transcriptional activity of NF-κB RelA/p65 by TNF-α, IL-1β, UV, farnesol, respiratory syncytial virus (RSV) in various cell types 
. Hence, it is possible that MSK1 activation regulates RelA/p65 phosphorylation in lung epithelial cells in response to CS. This is shown by the evidence that CS causes a significant increase in the levels of phosphorylated MSK1 and phospho-acetylation of RelA/p65 in control vector and wild-type MSK1 transfected H292 cells. There was a modest but significant reduction of MSK1 activation and RelA/p65 phosphorylation without any change in acetylation of RelA/p65 in MSK1 N- and C-terminal kinase-dead mutant transfected H292 cells suggest the role of both N- and C- terminal domains of MSK1 are crucial for it activation. Based on previous report 
, we speculated that the kinase activity will be affected in cells transfected with MSK1 N- and C-terminal kinase-dead mutants without any appreciable effects on MSK1 levels. Nevertheless, CS-induced activation of MSK1 and RelA/p65 was modest, but a significant reduction was seen in MSK1 siRNA transfected H292 cells compared to non-targeted control siRNA transfected cells. This suggests that the activation of RelA/p65 (Ser276) is MSK1-dependent in epithelial cells.
MSK1 contains two catalytic active kinase domains (N- and C-terminal), which are required for proper function 
. Earlier reports show that neither of the kinase-dead mutants (N- or C-terminal) of MSK1 possessed detectable activity nor showed any change in the total levels of MSK1 either before or after stimulation of cells with TPA or exposure to UV 
. Therefore, both the N- and C-terminal kinase domains play an essential role in activity of MSKs 
. MSK1 also acts as a regulator of inflammation 
. However, the signaling mechanism by which CS activates MSK1 is not known. It has been shown that IKKα translocated into the nucleus and is required for optimal NF-κB-mediated transcription and phosphorylation of histone H3 at Ser10 of NF-κB target genes 
, as well as EGF-induced transcriptional regulation of immediate early genes (IEGs) 
. Hence, we proposed that CS activates MSK1 via IKKα, leading to chromatin modifications in human lung epithelial cells. In support of this, we found that CS increases the levels of phosphorylated MSK1 and phospho-acetylated RelA/p65 in H292 cells overexpressing IKKα compared both to cells expressing a dominant-negative IKKα and to non-transfected control cells. These data suggest that IKKα plays an important role in CSE-induced activation of MSK1 and histone modifications in epithelial cells. Immunocytochemistry data also suggest that CS-mediated activation of MSK1 occurs via IKKα (unpublished observations). Recently, we have reported that NF-κB inducing kinase (NIK) activation by CS and TNFα induces RelA/p65 and histone H3K9 acetylation in human bronchial epithelial cells. Nuclear accumulation and recruitment of NIK on the proinflammatory gene promoters result in NF-κB-dependent gene activation 
Our data show that CS-mediated activation of MSK1 further leads to phospho-acetylation of histone H3 (Ser10/Lys9) and acetylation of histone H4 (Lys12) in bronchial epithelial cells. These modifications were confirmed by LC-MS/MS analysis of histone H3 and H4 fractions from CS-treated H292 cells. Apart from this, MS analysis also revealed CSE-mediated phosphorylation of histone H3 at Ser28 and acetylation of histone H4 at Lys5, Lys8, Lys12 and Lys16 (unpublished observations). Our in vitro
data are in agreement with other studies on MSK1-mediated histone H3 Ser10 and Ser28 phosphorylation 
. Earlier reports have shown that IKKα phosphorylates histone H3 Ser10, and RelA/p65 phospho-acetylation (Ser276/Lys310) occurs by direct interaction with CBP/p300 
. We further demonstrate that CS-mediated activation of MSK1 leads to phospho-acetylation of histone H3 (Ser10/Lys9), which was significantly reduced when MSK1 was knocked down in human bronchial epithelial cells, as well as in stable MSK1 knock-down mouse embryonic fibroblast (MEF) treated with CSE. Similarly, histone H3 is phosphorylated at Ser10 or Ser28 in response to other mitogenic or stress stimuli associated with induction of IEGs 
. Notably, studies using MSK1 and MSK2 knock-out MEFs or using the MSK1 dominant-negative mutant or MSK1 knock-down approaches have demonstrated that these kinases play a vital role in phosphorylation of histone H3 Ser10 and Ser28 in various cell lines 
. Thus, our data highlight the importance of MSK1-mediated nucleosomal response by CS-mediated oxidative/carbonyl stress potentially leading to inflammatory response.
We have observed CS-induced direct interactions between MSK1 and RelA/p65 in epithelial cells in vitro
and in acute CS-exposed mouse lung in vivo
by coimmunoprecipitation. p300 also coimmunoprecipitated with MSK1 and RelA/p65 suggesting that acetylation of histones H3 and H4 in response to CS is mediated by CBP/p300. This interaction plays a vital role in the regulation of DNA binding by transcription factors, such as NF-κB to the promoters of pro-inflammatory genes 
. Recruitment of MSK1 along with other DNA-binding proteins and coactivators, such as CBP/p300 may result in phosphorylation of chromatin proteins, particularly histone H3 (Ser10 or Ser28), and thus activate gene transcription 
. MSK1 interaction with coactivators (CBP/p300 and ER81) and other chromatin modifying enzymes stimulate the transactivation of target genes 
. Earlier reports have described that phosphorylation of RelA/p65 at Ser276 enhances its assembly with CBP/p300 
, and this interaction is augmented due to the acetylation of a key lysine residues in RelA/p65 (Lys310), which potentiates transcriptional activity 
. Similarly, we have previously reported that CS/aldehyde- or LPS-induced lung inflammation resulted in acetylation of RelA/p65 and histone modifications (phospho-acetylation of histone H3) via the interaction of RelA/p65 with coactivator CBP/p300 in mouse lung 
. Thus, based on our data, we conclude that CS/aldehyde-induced MSK1 activation leads to the formation of a complex that includes MSK1, RelA/p65 and CBP/p300, which localizes to pro-inflammatory gene promoters and modifies histones to promote transcriptional activation.
We also speculated that MSK1-mediated phosphorylation of RelA/p65 at Ser276 is required for the RelA/p65 and p300 in response to CSE. In support of this, we found that a S276A mutant Flag-tagged RelA/p65 protein did not coimmunoprecipitate with p300 either in the presence or absence of CSE (unpublished observations). This suggests a crucial role for RelA/p65 phosphorylation as an important event in CS-mediated MSK1 activation and NF-κB signaling. Our ChIP analysis revealed that MSK1, and its substrates RelA/p65 (Ser276), acetylated histone H3 (Lys9) and histone H4 (Lys12) are recruited to the promoters of pro-inflammatory genes in response to CS in epithelial cells. This is similar to the findings that CS causes recruitment of IKKα and RelA/p65 to the promoters of pro-inflammatory genes, such as MIP-2 and IL-6 in mouse lung 
. Earlier study by Gilmour et al.
demonstrated a role of histone H4 acetylation in regulation of IL-8 gene expression using the ChIP assay showing an increased association of acetylated H4 on IL-8 gene promoter following TSA, PM10, and TNF treatments after 24 h 
. In light of this, we propose a similar phenomenon that CSE treatment in H292 cells for 24 hrs may show a dynamic recruitment of MSK1, phosphorylated RelA/p65, and acetylated histone H3 and H4 on the promoters of pro-inflammatory genes. Thus, MSK1 kinase appears not only to modify and activate the factors involved in transcriptional regulation, but also participate in the complex that mediates chromatin remodeling on pro-inflammatory gene promoters. This is corroborated by the findings of Beck et al.
who demonstrated the presence of MSK1 on inflammatory gene promoters, proximal to or in the κB-site, which was significantly reduced by glucocorticoids in A549 epithelial cells 
. Similarly, other studies have demonstrated that MSK1 and its substrates RelA/p65 (Ser276) and phospho-histone H3 (Ser10) were localized to pro-inflammatory promoters 
. Thus, MSK1 plays an active role in linking the signaling cascade and gene transcription in response to pro-inflammatory environmental stimuli, such as CS and aldehydes.
In summary, we have demonstrated a novel role of MSK1 in CS-induced activation of NF-κB RelA/p65 (Ser276/Lys310) and chromatin modifications in human lung epithelial cells and mouse lung (). MSK1 serves as a specific NF-κB RelA/p65 kinase, promoting transcriptional activation of RelA/p65-dependent pro-inflammatory genes via IKKα-mediated activation of MSK1 and RelA/p65. Knock-down of MSK1 reduces CSE-induced activation of MSK1, RelA/p65 phosphorylation and posttranslational modifications of histones. CS-induced interactions between MSK1, RelA/p65 and p300 play a crucial role in sustained pro-inflammatory gene transcription by causing acetylation of RelA/p65 at Lys310, and modulating chromatin modifications at specific histone residues both in vitro and in vivo. CSE-induced MSK1-mediated phosphorylation of RelA/p65 at Ser276 is required for the interaction of RelA/p65 with p300. The ChIP assay demonstrates that MSK1 and its substrates associate with the promoters of NF-κB-dependent pro-inflammatory genes. These findings provide direct evidence that MSK1 is a kinase that plays a crucial role in CS-induced chromatin modifications. Thus, MSK1 represents a potential target for therapy in controlling CS-mediated chronic inflammatory response seen in several diseases, including COPD and lung cancer.
A schematic model showing the role of MSK1 in cigarette smoke-induced NF-κB signaling and chromatin modifications.