Abstract
Cigarette smoke (CS) causes sustained lung inflammation, which is an important event in the pathogenesis of chronic obstructive pulmonary disease (COPD). We have previously reported that IKKα (I kappaB kinase alpha) plays a key role in CS-induced pro-inflammatory gene transcription by chromatin modifications; however, the underlying role of downstream signaling kinase is not known. Mitogen- and stress-activated kinase 1 (MSK1) serves as a specific downstream NF-κB RelA/p65 kinase, mediating transcriptional activation of NF-κB-dependent pro-inflammatory genes. The role of MSK1 in nuclear signaling and chromatin modifications is not known, particularly in response to environmental stimuli. We hypothesized that MSK1 regulates chromatin modifications of pro-inflammatory gene promoters in response to CS. Here, we report that CS extract activates MSK1 in human lung epithelial (H292 and BEAS-2B) cell lines, human primary small airway epithelial cells (SAEC), and in mouse lung, resulting in phosphorylation of nuclear MSK1 (Thr581), phospho-acetylation of RelA/p65 at Ser276 and Lys310 respectively. This event was associated with phospho-acetylation of histone H3 (Ser10/Lys9) and acetylation of histone H4 (Lys12). MSK1 N- and C-terminal kinase-dead mutants, MSK1 siRNA-mediated knock-down in transiently transfected H292 cells, and MSK1 stable knock-down mouse embryonic fibroblasts significantly reduced CS extract-induced MSK1, NF-κB RelA/p65 activation, and posttranslational modifications of histones. CS extract/CS promotes the direct interaction of MSK1 with RelA/p65 and p300 in epithelial cells and in mouse lung. Furthermore, CS-mediated recruitment of MSK1 and its substrates to the promoters of NF-κB-dependent pro-inflammatory genes leads to transcriptional activation, as determined by chromatin immunoprecipitation. Thus, MSK1 is an important downstream kinase involved in CS-induced NF-κB activation and chromatin modifications, which have implications in pathogenesis of COPD.
Highlights
Cigarette smoke (CS) contains more than 1014–16 free radicals/ oxidants per puff and is composed of,4700 chemical compounds, including a major aldehyde, acrolein
We hypothesized that CS activates Mitogen- and stress-activated kinase 1 (MSK1), leading to phospho-acetylation of the RelA/p65 subunit of NF-kB in human bronchial epithelial cells (H292 and BEAS-2B), and human primary small airway epithelial cells (SAEC)
To further investigate CS-mediated activation of MSK1, immunocytochemistry was performed in H292 and SAEC cells after 1 h CSE (1%) treatment, using antibodies specific for p-MSK1 (Thr581), total MSK1 and p-RelA/p65 (Ser276), and immunohistochemistry for MSK1 in CS-exposed mouse lung sections
Summary
Cigarette smoke (CS) contains more than 1014–16 free radicals/ oxidants per puff and is composed of ,4700 chemical compounds, including a major aldehyde, acrolein. CS mediates pro-inflammatory effects by carbonyl and oxidative stress in the lung via the generation of reactive oxygen species (ROS) and aldehydes, as well as through endogenous generation of ROS from inflammatory/ structural cells [1,2]. Chronic CS exposure to mice leads to increased lung inflammatory response and airspace enlargement, which are characteristics of COPD/ pulmonary emphysema [2,5]. Inflammatory cells release numerous mediators that can cause airway constriction and remodeling. These cells produce proteases (elastase, cathepsins, granzymes, and MMPs) that can cause destruction of lung parenchyma leading to airspace enlargement [2,4,5]. Site-specific posttranslational modifications, such as phosphorylation and acetylation of RelA/p65 (a subunit of NF-kB), play an essential role in NF-kB activation and CS-mediated lung inflammation [7,8]
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