Histone deacetylase 2 is phosphorylated, ubiquitinated, and degraded by cigarette smoke

Cigarette smoke (CS)–induced lung inflammation involves the reduction of histone deacetylase 2 (HDAC2) abundance, which is associated with steroid resistance in patients with chronic obstructive pulmonary disease and in individuals with severe asthma who smoke cigarettes. However, the molecular mechanism of CS-mediated reduction of HDAC2 is not clearly known. We hypothesized that HDAC2 is phosphorylated and subsequently degraded by the proteasome in vitro in macrophages (MonoMac6), human bronchial and primary small airway epithelial cells, and in vivo in mouse lungs in response to chronic CS exposure. Cigarette smoke extract (CSE) exposure in MonoMac6 and in bronchial and airway epithelial cells led to phosphorylation of HDAC2 on serine/threonine residues by a protein kinase CK2-mediated mechanism, decreased HDAC2 activity, and increased ubiquitin-proteasome–dependent HDAC2 degradation. CK2 and proteasome inhibitors reversed CSE-mediated HDAC2 degradation, whereas serine/threonine phosphatase inhibitor, okadaic acid, caused phosphorylation and subsequent ubiquitination of HDAC2. CS-induced HDAC2 phosphorylation was detected in mouse lungs from 2 weeks to 4 months of CS exposure, and mice showed significantly lower lung HDAC2 levels. Thus, CS-mediated down-regulation of HDAC2 in human macrophages and lung epithelial cells in vitro and in mouse lung in vivo involves the induction of serine/threonine phosphorylation and proteasomal degradation, which may have implications for steroid resistance and abnormal inflammation caused by cigarette smoke

Nrf2 deficiency influences susceptibility to steroid resistance via HDAC2 reduction

Abnormal lung inflammation and oxidant burden are associated with a significant reduction in histone deacetylase 2 (HDAC2) abundance and steroid resistance. We hypothesized that Nrf2 regulates steroid sensitivity via HDAC2 in response to inflammation in mouse lung. Furthermore, HDAC2 deficiency leads to steroid resistance in attenuating lung inflammatory response, which may be due to oxidant/antioxidant imbalance. Loss of antioxidant transcription factor Nrf2 resulted in decreased HDAC2 in lung, and increased inflammatory lung response which was not reversed by steroid. Thus, steroid resistance or inability of steroids to control lung inflammatory response is dependent on Nrf2-HDAC2 axis. These findings have implications in steroid resistance, particularly during the conditions of oxidative stress when the lungs are more susceptible to inflammatory response, which is seen in patients with chronic obstructive pulmonary disease, asthma, rheumatoid arthritis, and inflammatory bowel disease

Deacetylases and NF-kappaB in redox regulation of cigarette smoke-induced lung inflammation: epigenetics in pathogenesis of COPD

Oxidative stress has been implicated in the pathogenesis of several inflammatory lung disorders including chronic obstructive pulmonary disease (COPD) due to its effect on pro-inflammatory gene transcription. Cigarette smoke-mediated oxidative stress activates NF-κB-dependent transcription of pro-inflammatory mediators either through activation of inhibitor κB-α kinase (IKK) and/or the enhanced recruitment and activation of transcriptional co-activators. Enhanced NF-κB-co-activator complex formation results in targeted increase in chromatin modifications, such as histone acetylation leading to inflammatory gene transcription. NF-κB-dependent gene expression, at least in part, is regulated by changes in deacetylases such as histone deacetylases (HDACs) and sirtuins. Cigarette smoke and oxidants also alter the levels/activity of HDAC by post-translational modifications and in doing so further induces gene expression of pro-inflammatory mediators. In addition, cigarette smoke/oxidants can reduce glucocorticoid sensitivity by attenuating HDAC2 activity and expression, which may account for the glucocorticoid insensitivity in patients with COPD. Understanding the mechanisms of NF-κB regulation, and the balance between histone acetylation and deacetylation may lead to the development of novel therapies based on the pharmacological manipulation of IKK and deacetylases in lung inflammation and injury

Protein kinase CK2-mediated phosphorylation of HDAC2 regulates co-repressor formation, deacetylase activity and acetylation of HDAC2 by cigarette smoke and aldehydes

Histone deacetylase 2 (HDAC2) mediates the repression of pro-inflammatory genes by deacetylating core histones, RelA/p65 and the glucocorticoid receptor. Reduced level of HDAC2 is associated with steroid resistant inflammation caused by oxidants. However, the molecular mechanisms regulating HDAC2 in response to oxidants and unsaturated aldehydes is not known. Here, we report that oxidative stress imposed by cigarette smoke extract (CSE), and aldehyde acrolein induced phosphorylation of HDAC2 which was abolished by serine-alanine mutations at serine sites S(394), S(411), S(422) and S(424). HDAC2 phosphorylation required direct interaction with serine-phosphorylated protein kinase CK2α and involved reduced HDAC2 deacetylase activity. Furthermore, HDAC2 phosphorylation was required for HDAC2 interaction with transcription factors, co-repressor complex formation, CBP recruitment, acetylation on lysine residues and consequently increased transrepression activity. Thus, phospho-acetylation of HDAC2 negatively regulates its deacetylase activity which has implications in steroid resistance in chronic inflammatory conditions

Cigarette Smoke Regulates Lung Epithelial HDAC2 via Serine Phosphorylation by a Protein Kinase CK2-Dependent Mechanism: Implications for Steroid Resistance

Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Environmental Medicine, 2010.Histone deacetylase 2 (HDAC2) is a critical part of a co-repressor complex recruited by glucocorticoid receptors on binding of steroid ligands to suppress inflammatory gene transcription. Loss of HDAC2 in lungs of chronic obstructive pulmonary disease (COPD) patients is linked to an impaired response to glucocorticoids. Cigarette smoke (CS), the main etiological factor in the pathogenesis of COPD, negatively alters lung HDAC2 activity and protein expression. We recently found that HDAC2, a phospho-protein, is an early target of CS-induced serine phosphorylation associated with increased binding of activated protein kinase CK2α. Cigarette smoke extract (CSE)-induced phosphorylation of HDAC2 required serine sites S394, S411, S422 and S424, is dependent on CK2 catalytic subunits and negatively regulates HDAC2 deacetylase activity. Most importantly however, HDAC2 phosphorylation is critical for CREB binding protein (CBP)-HDAC2 interaction and subsequently lysine residue acetylation of HDAC2. Loss of HDAC2 catalytic activity in vivo either via genetic knock-out or in mice with increased susceptibility to oxidative-stress markedly enhances the lung inflammatory response with subsequent resistance to steroid therapy following LPS exposure. Overall, these data suggests that phosphorylation and acetylation are key factors that control the relative expression and enzyme activity of HDAC2; a critical factor in the cellular response to pro-inflammatory stimuli with corticosteroid therapy. Funding: Supported by the NIH-NHLBI Grant R01-HL-085613, NIEHS Toxicology training grant ES-07026 and NIEHS Environmental Health Sciences Centre Grant ES-01247

Disruption of p21 Attenuates Lung Inflammation Induced by Cigarette Smoke, LPS, and fMLP in Mice

The cyclin-dependent kinase inhibitor p21CIP1/WAF1/SDI1 (p21) is an important inhibitory checkpoint regulator of cell cycle progression in response to oxidative and genotoxic stresses. It is known that p21 potentiates inflammatory response and inhibits apoptosis and proliferation, leading to cellular senescence. However, the role of endogenous p21 in regulation of lung inflammatory and injurious responses by cigarette smoke (CS) or other pro-inflammatory stimuli is not known. We hypothesized that p21 is an important modifier of lung inflammation and injury, and genetic ablation of p21 will confer protection against CS and other pro-inflammatory stimuli (lipopolysacchride [LPS] and N-formyl-methionyl-leucyl-phenylalanine [fMLP])-mediated lung inflammation and injury. To test this hypothesis, p21-deficient (p21−/−) and wild-type mice were exposed to CS, LPS, or fMLP, and the lung oxidative stress and inflammatory responses as well as airspace enlargement were assessed. We found that targeted disruption of p21 attenuated CS-, LPS-, or fMLP-mediated lung inflammatory responses in mice. CS-mediated oxidative stress and fMLP-induced airspace enlargement were also decreased in lungs of p21−/− mice compared with wild-type mice. The mechanism underlying this finding was associated with decreased NF-κB activation, and reactive oxygen species generation by decreased phosphorylation of p47phox and down-modulating the activation of p21-activated kinase. Our data provide insight into the mechanism of pro-inflammatory effect of p21, and the loss of p21 protects against lung oxidative and inflammatory responses, and airspace enlargement in response to multiple pro-inflammatory stimuli. These data may have ramifications in CS-induced senescence in the pathogenesis of chronic obstructive pulmonary disease/emphysema