Glucocorticoid receptor acetylation and corticosteroid resistance in chronic obstructive pulmonary disease

Chronic obstructive pulmonary disease (COPD) or smokers? lung is a growing problem and already one of the commonest causes of death and disability in the UK. It is associated with inflammation in the lung, which gets worse as the disease progresses. Asthma also involves inflammation in the lung but this is usually easy to control with steroid inhalers, which are very effective treatment. By contrast, the inflammation in COPD does not respond to steroid therapy and we have shown that cells from COPD patients also fail to respond, indicating that this problem is likely to be due to a molecular defect. Steroids work by binding to a specific recognition protein called a glucocorticoid receptor (GR) which goes into the nucleus of the cell and switches off genes that code for inflammatory products whereas switching on other genes that lead to side effects. We have found that GR becomes changed by acetylation when it interacts with a steroid, but has to be changed back by enzymes called deacetylases if it is to switch off inflammatory genes and control inflammation. We have also found that these deacetylases are markedly defective in cells from COPD patients so GR is less able to be converted to a form which is needed to switch off inflammatory genes, thus accounting for the lack of response to steroids in these patients. The aim of this project is to study acetylation and deacetylation of GR and how this is altered in COPD cells. We hope that this will provide a means to reverse this abnormality and eventually to the development of treatments that will make steroids more effective in the treatment of COPD. As no anti-inflammatory treatments currently exist for the treatment of COPD and stopping smoking does appear to little or no effect on the inflammation, there is an urgent need to understand the mechanisms of steroid resistance and to find treatments that may reverse this situation. The same mechanisms may also be important in other severe inflammatory diseases, such as inflammatory diseases of joints and the gut, so that the health implications of this research are potentially enormous. We have an excellent Media Relations Department at Imperial College and commonly publicise any research that has public interest when it is published. Any advance in understanding the underlying mechanisms could be presented in a very favourable light if the enormous unmet needs in this disease are emphasised.

Chronic obstructive pulmonary disease (COPD) is a major and increasing global health problem. It is characterised by a specific pattern of chronic inflammation in small airways and lung parenchyma which increases with disease progression. While corticosteroids are highly effective in suppressing airway inflammation in asthmatic patients, they are essentially ineffective in COPD. Corticosteroids fail to inhibit inflammatory gene expression stimulated by enhanced nuclear factor-kappaB (NF-kB) activation in cells from COPD patients. We have shown that glucocorticoid receptors (GR) are acetylated upon ligand binding and that this is essential for DNA binding and gene transactivation, including those that mediate side-effects. However, for GR to inhibit NF-kB and switch off inflammatory genes it is necessary for GR to be deacetylated. Histone deacetylases (HDACs) can also deacetylate non-histone proteins, including transcription factors. We have shown that certain HDACs, particularly HDAC2, are inactivated in COPD cells and that there is an increased proportion of acetylated GR in COPD macrophages. We hypothesise that the reduction in HDAC activity in COPD cells prevents GR from inhibiting NF-kB, thus leading to corticosteroid resistance. This may explain why COPD patients are resistant to the anti-inflammatory effects of corticosteroids but still suffer from detrimental side effects. We aim to explore GR acetylation and deacetylation in cell lines and relevant primary cells (alveolar macrophages and epithelial cells) and how this affects DNA binding and gene activation compared to NF-kB inhibition and suppression of relevant inflammatory genes such as interleukin-8 and matrix metalloproteinase-9 (using chromatin immunoprecipitation assays). We will determine the site(s) of GR acetylation using site-directed mutagenesis, with particular focus on amino acid 492-495 by analogy with other nuclear receptors. We will identify the histone acetyltransferases involved in GR acetylation and the HDACs or sirtuins involved in GR deacetylation using in-gel analysis and/or RNA interference to knock-down specific enzymes. We will then study the effect of oxidative/nitrative stress on GR acetylation/deacetylation in alveolar macrophages and primary airway epithelial cells and how this affects their corticosteroid responsiveness. This will be compared to GR acetylation status in cells from COPD patients. Finally, we will investigate how antioxidants and theophylline may restore GR deacetylation after oxidative stress and in COPD cells. These studies should shed light on the molecular mechanisms of corticosteroid resistance in COPD but may also be relevant to other chronic inflammatory diseases. They may also lead to new therapeutic approaches aimed at reversing this resistance mechanism.