Chronic obstructive pulmonary disease (COPD) is a common chronic inflammatory disease of the lungs with little or no response to glucocorticoids and a high level of oxidative stress. Histone deacetylase (HDAC) activity is reduced in cells of cigarette smokers, and low concentrations of theophylline can increase HDAC activity. We measured the effect of theophylline on HDAC activity and inflammatory gene expression in alveolar macrophages (AM) from patients with COPD. AM from normal smokers showed a decrease in HDAC activity compared with normal control subjects, and this was further reduced in COPD patients (51% decrease, P < 0.01). COPD AMs also showed increased basal release of IL-8 and TNF-α, which was poorly suppressed by dexamethasone. Theophylline induced a sixfold increase in HDAC activity in COPD AM lysates and significantly enhanced dexamethasone suppression of induced IL-8 release, an effect that was blocked by the HDAC inhibitor trichostatin A. Therefore, theophylline might restore steroid responsiveness in COPD patients.

Scientific Knowledge on the Subject: Glucocorticoid unresponsiveness in severe asthma COPD may involve an oxidant mediated impairment of glucocorticoid receptor alpha (GRα) function through reduction of histone deacetylase activity and co-repressor expression.What This Study Adds to the Field: Histone deacetylase 2 activity is reduced in smoke exposed mice lungs correlating with reduced glucocorticoid function which is restored by PI3Kδ but not γ inhibition. GRα expression also is reduced in smoke exposed mouse and in COPD patient lungs. Abstract Rational:There is an increasing prevalence of reduced responsiveness to glucocorticoid therapy in severe asthma and chronic obstructive pulmonary disease, however the molecular mechanism of this remains unknown. Recent studies have shown that histone deacetylase activity, which is critical to glucocorticoid function, is altered by oxidant stress and may be involved in the development of glucocorticoid insensitivity.Objectives: To determine the role of phosphoinositol-3-kinase (PI3K) in the development of cigarette smoke induced glucocorticoid insensitivity. Methods:Wild type, PI3Kγ knock-out and PI3Kδ kinase dead knock-in transgenic mice were used in a model of cigarette smoke induced glucocorticoid insensitivity. Peripheral lung tissue was obtained 6 healthy non-smokers, 9 smokers with normal lung function and 8 patients with chronic obstructive pulmonary disease. Measurements and Main Results:Glucocorticoid receptor expression was significantly reduced in both the lungs of chronic obstructive pulmonary disease patients and in cigarette smoke-exposed mice. Furthermore, cigarette smoke exposure in mice increased tyrosine nitration of histone deacetylase 2 in the lung correlating with both reduced histone deacetylase 2 activity and reduced glucocorticoid function. Oxidative stress activated Akt and induced glucocorticoid insensitivity in vitro, which was restored by inhibition of PI3K. In vivo, histone deacetylase 2 activity and the anti-inflammatory effects of glucocorticoids were restored in PI3Kδ kinase dead knock-in but not PI3Kγ knock-out smoke exposed mice compared to wild types, correlating with reduced histone deacetylase 2 tyrosine nitration.Conclusion: Together these data shows that therapeutic inhibition of PI3Kδ may restore glucocorticoid function in oxidative stress induced glucocorticoid insensitivity. Abstract Word count: 247

Histone acetylation status is a key factor in the regulation of inflammatory gene transcription. We investigated the activity of histone acetylases (HAT) and deacetylases (HDAC), and the effect of glucocorticoids in alveolar macrophages (AM) and peripheral blood mononuclear cells (PBMC) from subjects with asthma. Bronchoalveolar lavage was performed in 10 patients with intermittent asthma, 8 with persistent asthma, and 10 healthy control subjects. PBMCs and granulocytes were isolated from six patients with mild and severe asthma, before and after a 7-day course of prednisolone (30 mg/day). AMs were isolated for HDAC assay or incubated with dexamethasone (1 microM). HAT activity was increased (1.43 +/- 0.1 vs. 1.01 +/- 0.1 standard units/10 microg, p < 0.05), and HDAC activity was reduced (3031 +/- 243 vs. 5004 +/- 164 arbitrary fluorescence units/10 microg, p < 0.001) in AMs of subjects with asthma compared with control subjects. Dexamethasone suppressed LPS-induced granulocyte macrophage-colony stimulating factor, tumor necrosis factor-alpha, and interleukin-8 release by 83 +/- 1%, 51 +/- 7% and 20 +/- 9% (p < 0.001), respectively. Similar effects were seen on nuclear factor-kappaB inhibition, and interleukin-8 release was further reduced by the HDAC enhancer, theophylline (37 +/- 6%). Prednisolone increased HDAC activity in PBMCs from subjects with mild asthma. The increased inflammatory response in asthma may be due to reduced HDAC and enhanced HAT activity. Glucocorticoids and theophylline may downregulate the inflammatory response by modulating HAT and HDAC activity, and nuclear factor-kappaB activation.

Abnormal expression of TGF-β1 is believed to play an important role in the pathogenesis of a number of chronic inflammatory and immune lung diseases, including asthma, chronic obstructive pulmonary disease, and pulmonary fibrosis. Gene activation in eukaryotes requires coordinated use of specific cell signals, chromatin modifications, and chromatin remodeling. We studied the roles of the ubiquitous inflammatory transcription factors, NF-κB and AP-1, in activation of the TGF-β1 gene and histone acetylation at the TGF-β1 promoter. IL-1β-induced TGF-β1 protein secretion and mRNA expression were prevented by actinomycin D and were attenuated by the inhibitor of κB kinase 2 inhibitor AS602868 and the JNK inhibitor SP600125, suggesting a degree of transcriptional regulation mediated by the NF-κB and AP-1 pathways. We demonstrated that IL-1β activated the p65 subunit of NF-κB and the c-Jun subunit of AP-1. Using chromatin immunoprecipitation assays, we observed a sequential recruitment of p65 and c-Jun, accompanying ordered elevation of the levels of histone H4 and H3 acetylation and recruitment of RNA polymerase II at distinct regions in the native TGF-β1 promoter. The specific NF-κB and AP-1 binding sites in the TGF-β1 promoter were confirmed by an ELISA-based binding assay, and evidence for histone hyperacetylation in TGF-β1 induction was supported by the observation that the histone deacetylase inhibitor trichostatin A enhanced basal and IL-1β-induced TGF-β1 mRNA expression. Our results suggest that IL-1β-stimulated transcription of TGF-β1 is temporally regulated by NF-κB and AP-1 and involves histone hyperacetylation at distinct promoter sites.

The CBP and p300 proteins are transcriptional coactivators that are involved in a variety of transcriptional pathways in development and in response to specific signaling pathways. We have previously demonstrated that the ability of both these factors to stimulate transcription is greatly enhanced by treatment of cardiac cells with the hypertrophic agent phenylephrine (PE). Here, we show that inhibition of either CBP or p300 with antisense or dominant negative mutant constructs inhibits PE-induced hypertrophy as assayed by atrial naturetic protein production, cardiac cell protein: DNA ratio and cell size. Furthermore, we show that overexpression of CBP or p300 can induce hypertrophy and that this effect requires their histone acetyltransferase (HAT) activity. Moreover, we show that PE can directly enhance CBP HAT activity and that artificial enhancement of HAT activity is sufficient to induce hypertrophy. Hence, CBP and p300 play an essential role in hypertrophy induced by PE, and this effect is mediated via PE-induced enhancement of their HAT activity. This is the first time a role for these factors, and their HAT activity, in hypertrophy has been directly demonstrated.