Nicolas Mercado

Decreased histone deacetylase 2 impairs Nrf2 activation by oxidative stress.

Research highlights ► Nrf2 anti-oxidant function is impaired when HDAC activity is inhibited. ► HDAC inhibition decreases Nrf2 protein stability. ► HDAC2 is involved in reduced Nrf2 stability and both correlate in COPD samples. ► HDAC inhibition increases Nrf2 acetylation.

Denitrosylation of HDAC2 by targeting Nrf2 restores glucocorticosteroid sensitivity in macrophages from COPD patients

5 5 2 1 jci.org Volume 124 Number 12 December 2014 Retraction Denitrosylation of HDAC2 by targeting Nrf2 restores glucocorticosteroid sensitivity in macrophages from COPD patients Deepti Malhotra, Rajesh K. Thimmulappa, Nicolas Mercado, Kazuhiro Ito, Ponvijay Kombairaju, Sarvesh Kumar, Jinfang Ma, David Feller-Kopman, Robert Wise, Peter Barnes, and Shyam Biswal Original citation: J Clin Invest. 2011;121(11):4289–4302. doi:10.1172/JCI45144. Citation for this retraction: J Clin Invest. 2014;124(12):5521. doi:10.1172/JCI79606. The JCI, with the agreement of the corresponding authors and coauthors, is retracting the article “Denitrosylation of HDAC2 by targeting Nrf2 restores glucocorticosteroid sensitivity in macrophages from COPD patients.” The IP: streptavidin/IB: anti-HDAC2 immunoblot panel in Figure 3B was recently identified as an inverted image of the right four lanes of the anti-H4 acetyl CHIP gel presented in Figure 2A. Additionally, the IP: anti-streptavidin/IB: anti-DDK immunoblot presented in Supplemental Figure 3A was identified as an inverted image of the left four lanes of the anti-H4 acetyl CHIP gel presented in Figure 2A. Further, in Figure 9B, the incorrect anti-HDAC2 immuno blot was presented. The authors sincerely apologize for any misinterpretation of the data as a result of these errors.

Accelerated ageing of the lung in COPD: new concepts

The rise in life expectancy worldwide has been accompanied by an increased incidence of age-related diseases, representing an enormous burden on healthcare services and society. All vital organs lose function with age, and this is well described in the lung, with a progressive decline in pulmonary function after the age of about 25 years. The lung ages, like any other organ, with progressive functional impairment and reduced capacity to respond to environmental stresses and injury. Normal physiological ageing results in enlarged alveolar spaces and loss of lung elasticity in the elderly known as ‘senile emphysema’, whereas in COPD there is destruction of the alveolar walls and fibrosis of peripheral airways. However, COPD shows striking age-associated features, such as an increase in cellular senescence, stem cell exhaustion, increased oxidative stress, alteration in the extracellular matrix and a reduction in endogenous antiageing molecules and protective pathways such as autophagy. In this review we discuss the evidence showing how oxidative stress induces accelerated ageing by upregulating the phosphatidylinositol-4,5-bisphosphate 3-kinase/AKT/mechanistic target of rapamycin signalling pathway resulting in depletion of stem cells, defective autophagy, reduced antioxidant responses and defective mitochondrial function thus generating further oxidative stress. Understanding the mechanisms of accelerated ageing in COPD may identify novel therapeutic approaches.

Denitrosylation of HDAC2 by targeting Nrf2 restores glucocorticosteroid sensitivity in macrophages from COPD patients (Retracted article. See vol. 124, pg. 5521, 2014)

Chronic obstructive pulmonary disease (COPD), which is caused primarily by cigarette smoking, is a major health problem worldwide. The progressive decline in lung function that occurs in COPD is a result of persistent inflammation of the airways and destruction of the lung parenchyma. Despite the key role of inflammation in the pathogenesis of COPD, treatment with corticosteroids - normally highly effective antiinflammatory drugs - has little therapeutic benefit. This corticosteroid resistance is largely caused by inactivation of histone deacetylase 2 (HDAC2), which is critical for the transrepressive activity of the glucocorticoid receptor (GR) that mediates the antiinflammatory effect of corticosteroids. Here, we show that in alveolar macrophages from patients with COPD, S-nitrosylation of HDAC2 is increased and that this abolishes its GR-transrepression activity and promotes corticosteroid insensitivity. Cys-262 and Cys-274 of HDAC2 were found to be the targets of S-nitrosylation, and exogenous glutathione treatment of macrophages from individuals with COPD restored HDAC2 activity. Treatment with sulforaphane, a small-molecule activator of the transcription factor nuclear factor erythroid 2-related factor 2 (NRF2), was also able to denitrosylate HDAC2, restoring dexamethasone sensitivity in alveolar macrophages from patients with COPD. These effects of sulforaphane were glutathione dependent. We conclude that NRF2 is a novel drug target for reversing corticosteroid resistance in COPD and other corticosteroid-resistant inflammatory diseases.

Restoration of Corticosteroid Sensitivity by p38 Mitogen Activated Protein Kinase Inhibition in Peripheral Blood Mononuclear Cells from Severe Asthma

Background Severe asthma accounts for a small number of asthmatics but represents a disproportionate cost to health care systems. The underlying mechanism in severe asthma remains unknown but several mechanisms are likely to be involved because of a very heterogeneous profile. We investigated the effects of a p38MAPK inhibitor in corticosteroid sensitivity in peripheral blood mononuclear cells (PBMCs) from severe asthmatics and the profile of its responders. Methodology/Principal Findings Corticosteroid sensitivity was determined by measuring dexamethasone inhibition of CD3/28 and TNF-α induced IL-8 production in PBMCs by using ELISA. PBMCs from severe asthmatics were relatively less sensitive to dexamethasone (Dex) as compared to those of non-severe asthmatics and healthy volunteers. The IC50 values of Dex negatively correlated with decreased glucocorticoid receptor (GR) nuclear translocation assessed using immunocytochemistry (r = −0.65; p<0.0005) and with decreased FEV1 (% predicted) (r = 0.6; p<0.0005). A p38α/β inhibitor (SB203580) restored Dex-sensitivity in a subpopulation of severe asthma that was characterized by a defective GR nuclear translocation, clinically by lower FEV1 and higher use of oral prednisolone. We also found that SB203580 partially inhibited GR phosphorylation at serine 226, resulting in increased GR nuclear translocation in IL-2/IL-4 treated corticosteroid insensitive U937s. Conclusions/Significance p38MAPKα/β is involved in defective GR nuclear translocation due to phosphorylation at Ser226 and this will be a useful biomarker to identify responders to p38MAPKα/β inhibitor in the future.

Defects of Protein Phosphatase 2A Causes Corticosteroid Insensitivity in Severe Asthma

Background Corticosteroid insensitivity is a major barrier of treatment for some chronic inflammatory diseases, such as severe asthma, but the molecular mechanism of the insensitivity has not been fully elucidated. The object of this study is to investigate the role of protein phosphate 2A (PP2A), a serine/threonine phosphatase, on corticosteroid sensitivity in severe asthma. Methodology/Principal Findings Corticosteroid sensitivity was determined by the dexamethasone ability to inhibit TNFα-induced IL-8 or LPS-induced TNFα production. PP2A expression, glucocorticoid receptor (GR) nuclear translocation defined as the nuclear/cytoplasmic GR ratio and phosphorylation of GR-Ser226, c-Jun N-terminal kinase 1 (JNK1) and PP2A were analysed by Western-blotting. Phosphatase activity was measured by fluorescence-based assay. Okadaic acid (OA), a PP2A inhibitor, reduced corticosteroid sensitivity with reduced GR nuclear translocation and increased GR phosphorylation in U937 monocytic cells. PP2A knockdown by RNA interference showed similar effects. IL-2/IL-4 treatment to U937 reduced corticosteroid sensitivity, and PP2A expression/activity. In peripheral blood mononuclear cells (PBMCs) from severe asthma, the PP2A expression and activity were significantly reduced with concomitant enhancement of PP2AC-Tyr307 phosphorylation compared with those in healthy volunteers. As the results, GR-Ser226 and JNK1 phosphorylation were increased. The expression and activity of PP2A were negatively correlated with phosphorylation levels of GR-Ser226. Furthermore, co-immunoprecipitation assay in U937 cells revealed that PP2A associated with GR and JNK1 and IL-2/IL-4 exposure caused dissociation of each molecule. Lastly, PP2A overexpression increased corticosteroid sensitivity in U937 cells. Conclusions/Significance PP2A regulates GR nuclear translocation and corticosteroid sensitivity possibly by dephosphorylation of GR-Ser226 via dephosphorylation of upstream JNK1. This novel mechanism will provide new insight for the development of new therapy for severe asthma.

Nortriptyline Reverses Corticosteroid Insensitivity by Inhibition of Phosphoinositide-3-Kinase-δ

Corticosteroid insensitivity represents a major barrier to the treatment of chronic obstructive pulmonary disease (COPD) and severe asthma. It is caused by oxidative stress, leading to reduced histone deacetylase-2 (HDAC2) function through activation of phosphoinositide-3-kinase-δ (PI3Kδ). The tricyclic antidepressant nortriptyline has been identified in high-throughput screens as an agent that increases corticosteroid responsiveness. The aim of this study was to identify the molecular mechanism whereby nortriptyline increases corticosteroid sensitivity. Phosphorylation of Akt, a footprint of PI3K activation, and HDAC activity were evaluated by Western blotting and fluorescent activity assay in U937 monocytic cells. Corticosteroid sensitivity was evaluated by the inhibition of tumor necrosis factor α (TNFα)-induced interleukin 8 (IL-8) production by budesonide. Hydrogen peroxide (H2O2) or cigarette smoke extract (CSE) increased the level of phosphorylated Akt (pAkt) and reduced HDAC activity. Pretreatment with nortriptyline inhibited pAkt induced by CSE and H2O2 as well as restored HDAC activity that had been decreased by H2O2 and CSE. In addition, nortriptyline inhibited PI3Kδ activity, but had no effect on the PI3Kα and PI3Kγ isoforms. Although CSE reduced the effects of budesonide on TNFα-induced IL-8 production in U937 cells, nortriptyline reversed CSE-induced corticosteroid insensitivity. Nortriptyline restores corticosteroid sensitivity induced by oxidative stress via direct inhibition of PI3Kδ and is a potential treatment for corticosteroid-insensitive diseases such as COPD and severe asthma.

Geroprotectors as a novel therapeutic strategy for COPD, an accelerating aging disease.

Chronic obstructive pulmonary disease (COPD) progresses very slowly and the majority of patients are therefore elderly. COPD is characterized by an abnormal persistent inflammatory response to noxious environmental stimuli and there are increasing evidences for a close relationship between premature aging and chronic inflammatory diseases. Thus, COPD is considered to be a disease of an accelerating aging. In this review, we collected the evidence for roles of aging on pathogenesis of COPD and considered future therapeutic strategy for COPD based on this senescence hypothesis. Since calorie restriction has been proved to extend lifespan, many efforts were made to clarify the molecular mechanism of aging. Aging is defined as the progressive decline of homeostasis that occurs after the reproductive phase of life is complete, leading to an increasing risk of disease or death due to impaired DNA repair after damage by oxidative stress or telomere shortening as a result of repeated cell division. During aging, pulmonary function progressively deteriorates; innate immunity is impaired and pulmonary inflammation increases, accompanied by structural changes, such as an enlargement of airspaces. Noxious environmental gases, such as cigarette smoke, may worsen these aging-related events in the lung or accelerate aging of the lung due to reduction in anti-aging molecules and/or stimulation of aging molecules. Aging signaling are complex but conserved in divert species, such as worm, fruit fry, rodent and humans. Especially the insulin like growth factor (IGF-1) signaling was well documented. Geroprotectors are therapeutics that affect the root cause of aging and age-related diseases, and thus prolong the life-span of animals. Most of geroprotectors such as melatonin, metformin, rapamycin and resveratrol are anti-oxidant or anti-aging molecule regulators. Therefore, geroprotection for the lung might be an attractive approach for the treatment of COPD by preventing premature aging of lung.

Passive Smoking Impairs Histone Deacetylase-2 in Children With Severe Asthma

Background: Parental smoking is known to worsen asthma symptoms in children and to make them refractory to asthma treatment, but the molecular mechanism is unclear. Oxidative stress from tobacco smoke has been reported to impair histone deacetylase-2 (HDAC2) via phosphoinositide-3-kinase (PI3K)/Akt activation and, thus, to reduce corticosteroid sensitivity. The aim of this study was to investigate passive smoking-dependent molecular abnormalities in alveolar macrophages (AMs) by comparing passive smoke-exposed children and non-passive smoke-exposed children with uncontrolled severe asthma. Methods: BAL fluid (BALF) was obtained from 19 children with uncontrolled severe asthma (10 non-passive smoking-exposed subjects and nine passive smoking-exposed subjects), and HDAC2 expression/activity, Akt/HDAC2 phosphorylation levels, and corticosteroid responsiveness in AMs were evaluated. Results: Parental smoking reduced HDAC2 protein expression by 54% and activity by 47%, with concomitant enhancement of phosphorylation of Akt1 and HDAC2. In addition, phosphorylation levels of Akt1 correlated positively with HDAC2 phosphorylation levels and negatively with HDAC2 activity. Furthermore, passive smoke exposure reduced the inhibitory effects of dexamethasone on tumor necrosis factor-α-induced CXCL8 release in AMs. There were relatively higher neutrophil counts and CXCL8 concentrations in BALF and lower Asthma Control Test scores compared with non-passive smoke-exposed children with uncontrolled severe asthma. Conclusions: Passive smoking impairs HDAC2 function via PI3K signaling activation, which could contribute to corticosteroid-insensitive inflammation in children with severe asthma. This novel mechanism will be a treatment target in children with severe asthma and stresses the need for a smoke-free environment for asthmatic children.

Increased corticosteroid sensitivity by a long acting β2 agonist formoterol via β2 adrenoceptor independent protein phosphatase 2A activation.

Long-acting β2-adrenoceptor agonists (LABAs) are reported to enhance anti-inflammatory effects of corticosteroids in vitro and in vivo, although the molecular mechanisms have not yet been elucidated. We investigated the role of serine/threonine protein phosphatase 2A (PP2A) on regulation of corticosteroid sensitivity via inhibition of glucocorticoid receptor (GR) phosphorylation as the target of formoterol, an LABA. Corticosteroid sensitivity was determined as IC50 to dexamethasone (Dex) on TNFα-induced IL-8 release in a U937 monocytic cell line (Dex-IC50). Phosphorylation levels of GR-Ser226 and c-Jun N-terminal kinase (JNK) were determined by western-blotting. Phosphatase activity of immunopurified PP2A was measured by fluorescence-based assay. Exposure to IL-2/IL-4 for 48 h decreased Dex sensitivity with a concomitant increase of GR phosphorylation at Ser226 with JNK1 activation. Formoterol restored Dex sensitivity by inhibiting phosphorylation of GR-Ser226 and JNK1. PP2A inhibition by okadaic acid, a phosphatase inhibitor, abrogated formoterol-mediated effects. In addition, formoterol enhanced PP2A activity in intact or IL-2/IL-4 treated U937 cells and human peripheral blood mononuclear cells. In addition, PP2A activation by formoterol was not antagonized by ICI-118551, and formoterol could activate PP2A directly in cell free system. Taken together, formoterol increases corticosteroid sensitivity via activation of PP2A in receptor independent manner, explaining its benefits as add-on therapy for the treatment of corticosteroid-insensitive diseases, such as severe asthma.