β2-Adrenoceptor Agonists, Like Glucocorticoids, Repress Eotaxin Gene Transcription by Selective Inhibition of Histone H4 Acetylation

Abstract

Eotaxin is a potent eosinophil chemoattractant implicated in various allergic inflammatory conditions including asthma, but relatively little is known about its regulation. Human airway smooth muscle cells are an important source of eotaxin in the airway. We have previously demonstrated that β2-adrenoceptor agonists (β2-agonists) and glucocorticoids additively inhibit eotaxin production in human airway smooth muscle cells, but the underlying mechanisms are unclear. Here, we studied the molecular mechanisms of their actions and interactions on eotaxin gene transcription. TNF-α-induced eotaxin gene transcription was mediated mainly by the transcription factor NF-κB (p65/p50) as analyzed by luciferase reporter gene assay, Western blotting, EMSA, and electrophoretic mobility supershift assay. Chromatin immunoprecipitation assay demonstrated that TNF-α also induced selective histone H4 acetylation on lysines 5 and 12 at the eotaxin promoter site and p65 binding to the eotaxin promoter, resulting in eotaxin gene transcription. The inhibition of eotaxin production by β2-agonists and glucocorticoids was transcriptional and not due to altered NF-κB nuclear translocation or in vitro promoter binding capability, but due to their inhibition of TNF-α-induced histone H4 acetylation and p65 in vivo binding to the promoter. Additive inhibition was achieved when the two groups of drugs were combined. Our findings reveal a novel mechanism by which β2-agonists, like glucocorticoids, regulate NF-κB-mediated inflammatory gene expression through inhibition of histone acetylation. This provides one explanation for the benefits that result when these agents are combined to treat asthma, and may have important implications in a wide range of inflammatory diseases.

PKCβΙΙ Augments NF-κB-Dependent Transcription at the CCL11 Promoter via p300/CBP-Associated Factor Recruitment and Histone H4 Acetylation

“…ChIP assay showed a time-dependent increase of TNF-␣-induced in vivo p65 association with the CCL11 promoter DNA in HASM cells, as previously reported (9). At 1 h, p65, p300, and p/CAF recruitment to the CCL11 promoter was increased 5.6 Ϯ 2.4-fold, 2 flasks were pretreated with Bis I and LY333531 (10 Ϫ6 M) before TNF-␣ (1 ng/ml) for 1 h. The in vivo p65, p300, and p/CAF binding to the CCL11 promoter and histone H4 acetylation at the CCL11 promoter was analyzed by ChIP assay with specific anti-p65, anti-p300 anti-p/CAF, and anti-histone H4 acetylation Abs.…”

“…We studied NF-B gene regulation with particular emphasis on CCL11 because this is an important chemokine relevant to asthma and other allergic disease (37), which we have shown previously is primarily NF-B dependent under TNF-␣-stimulated conditions (9). We used HASM cells because they are a good model system for studying inflammatory gene transcription in primary human cells, and used embryonic fibroblasts from genetically modified mice to allow us to fully characterize the mechanism of the effect.…”

“…␤-actin was used as the housekeeping gene: sense, 5Ј-AAA TCG TGC GTG ACA TCA AA-3Ј and antisense, 5Ј-AAG GAA GGC TGG AAA AGA GC-3Ј. One nanogram of reverse-transcribed cDNA was subjected to real-time PCR using Excite Real Time Mastermix with SYBR Green (Biogene) and the ABI Prism 7700 detection system (Applied Biosystems), as described (9,28). CCL11 expression was normalized to the housekeeping gene by dividing the mean of the CCL11 triplicate by the mean of the ␤ 2 -microglobulin triplicate value for all human cell experiments and by the ␤-actin triplicate value for murine cell experiments.…”

Inhaled corticosteroids as combination therapy with β-adrenergic agonists in airways disease: present and future

“…The precise mechanism for this repression may include binding and recruiting nuclear receptor corepressors such as NCoR and HDACs, direct repression of coactivator complexes, or effects on RNA polymerase II phosphorylation [7,8]. For example, in primary airway smooth-muscle cells, fluticasone attenuates TNF-α-induced histone H4 acetylation leading to a reduction in p65 association with the native CCL11 promoter and suppression of CCL11 expression [9]. In addition, glucocorticoids might play a role in repressing the action of MAPKs, such as the extracellular signal-regulated kinase (ERK), p38 MAPK, and c-Jun N-terminal kinase (JNK) [2,7,8].…”

Early Prescription of Inhalation Corticosteroid in Bronchial Asthma

“…В отношении применения комбинации ИКС и Б2А важ-ным представляется то, что оба компонента демонстриру-ют синергизм в отношении подавления высвобождения хемокинов (особенно вовлеченных в мобилизацию лим-фоцитов и эозинофилов) из бронхиального эпителия и гладкомышечных клеток [15,16]. Интересно то, что in vitro кортикостероиды ингибируют выведение Б2А гладкомы-шечными клетками бронхов и сосудов посредством быстрого (15-минутной продолжительности) негеномного эффекта, тем самым продлевая местное действие Б2А [17].…”