Elsevier

Cellular Immunology

Volume 334, December 2018, Pages 31-37
Cellular Immunology

Research paper
E4BP4 facilitates glucocorticoid sensitivity of human bronchial epithelial cells via down-regulation of glucocorticoid receptor-beta

https://doi.org/10.1016/j.cellimm.2018.08.015Get rights and content

Highlights

  • This first study showing E4BP4 expression in IL-17A induced in 16HBE cells.

  • The transfection factor is important.

  • The PI3K/Akt/E4BP4 signal pathway is our first found in asthma.

Abstract

It has recently been recognized that a subset of asthma patients suffer from glucocorticoid (GC) insensitivity, and glucocorticoid receptor-β (GR-β) is associated with corticosteroid resistance, but the underlying mechanisms remain unknown. Here we demonstrated that Interleukin-17A induced glucocorticoid sensitivity in human bronchial epithelial cells (16HBE) is enhanced, which is depend on E4 promoter-binding protein 4 (E4BP4) mediated GR-β expression. Our data show that the expression of E4BP4 is significantly up-regulated in 16HBE cells, and the depletion of E4BP4 dramatically decreased glucocorticoid sensitivity in IL-17A induced 16HBE cells. Mechanistic studies revealed that E4BP4 plays a crucial role in Interleukin-17A induced glucocorticoid sensitivity in 16HBE cells via down-regulating GR-β, which is probably mediated by PI3K/Akt activation. Collectively, we can draw the conclusion that E4BP4 contribute to enhance the GCs sensitivity, which may offer a new strategy for therapeutic intervention for GC-insensitive asthma.

Introduction

Asthma is an obstructive lung disorder, characterized by airway hyperreactivity, airway remodeling, and invasion of inflammatory cells and cytokine. Inhaled glucocorticoids (GCs) are currently the most effective anti-inflammatory treatment for asthma. Nevertheless, a characteristic of asthmatics is that they are relatively unresponsive to treatment corticosteroids [1], [2].

Steroid-resistant asthma, was first described in 6 patients who did not respond clinically or have reduced blood eosinophilia with high doses of oral corticosteroids [3]. Several molecular mechanisms contributing to corticosteroid resistant in asthmatic patients, including familial glucocorticoid resistance, GR modification, Increased GR-β expression, increased proinflammatory transcription factors and so on [4]. Previous studies revealed that increased expression of GR-β has been described in patients with steroid-resistant asthma [5], [6]. GR-β is induced by proinflammatory cytokines (IL17) and has the capacity to compete for the binding of GR-α to GREs, thus acting as a dominant negative inhibitor [7], [8], [9]. However, in most cell types, apart from neutrophils, the expression of GR-β is considerably less than GR-α [10], so the precise molecular events underlying the effect of GR-β on corticosteroid insensitivity are remain to be defined.

E4BP4 (or NFIL3) is a basic leucine zipper transcription factor, there is evidence that E4BP4 plays important roles in anti-inflammtory response [3], [4], [5], circadian oscillation [6], apoptosis regulation [7], and immune cell development [8], [9]. Recently, numerous studies have demonstrated a diverse and important role for E4BP4 is a glucocorticoid regulated gene [7], [10], [11]. Furthermore, it was observed that E4BP4 as an up-regulated GR target in primary mouse thymocytes, human CEM lymphoblastic leukemia cells and eosinophils induced cell apoptosis. However, very few studies have investigated expression of E4BP4 and addressed their roles in the process of GC-induced bronchial epithelial cells.

In the current study, we observed the effects of E4BP4 on budesonide (BUD)–induced bronchial epithelial cells. We found that the expression of E4BP4 is significantly up-regulated in IL-17A induced epithelial cells. Furthermore, E4BP4 silencing significantly attenuates the GC sensitivety and increase the expression of GR-β. We demonstrate that E4BP4 significantly strengthen GC responsiveness in IL-17A induced human bronchial epithelial cell line 16HBE, which is probably mediated by activation of phosphoinositide-3-kinase (PI3K) signaling. Taken together, our findings indicate that E4BP4 may be enhances Interleukin-17A induces glucocorticoid sensitivity in human bronchial epithelial cells.

Section snippets

Cell culture

Human bronchial epithelial cells (16HBE) were kindly provided by Central South University Advanced Research Center (Changsha, China). 16HBE were placed into 25 cm2 culture flasks (Corning, NY, USA) and cultured with DMEM growth medium (Hyclone, Logan, UT, USA) containing 10% fetal calf serum (Gibco, Carlsbad, CA, USA) and supplemented with 100U/mL penicillin and 100 μg/mL streptomycin (Gibco, Carlsbad, CA, USA) at 37 °C under 5% CO2 and 90% humidity, as previously described [12].

Stimulation of the cells

Cells were

IL-17A induces the expression of E4BP4 in 16HBE cells

We first sought to determine the impact of IL-17A on E4BP4 expression, 16HBE cells were treated with increasing doses of IL-17A (24 h). Quantitative real-time PCR revealed that IL-17A significantly induced E4BP4 expression in a dose-dependent manner when compared with the control (Fig. 1A). Similarly, Western blot analysis indicated the increased expression of E4BP4, which is also dose-dependently (Fig. 1B). These results support that IL-17A induced E4BP4 production in 16HBE cells.

Effect of BUD on E4BP4 expression in IL-17A induced 16HBE cells

Next, we

Discussion

There is increasing evidence that Th17 cells infiltration and its associated cytokines IL-17 have been detected in inflamed airway tissues from asthmatics, underlining their importance in the pathogenesis of asthma. Their role in promoting steroid resistance has also been suggested, although it is not well characterized [16], [17].

Previous studies suggested that IL-17 cytokines can hamper both anti-inflammatory and immunosuppressant actions of dexamethasone on peripheral lymphocytes, in part

Acknowledgements

This work was supported by grants from the National Natural Science Foundation of China (NO. 81460005 and NO. 81760008) and Natural science foundation of key subjects in Guangxi Zhuang Autonomous Region (NO. 2012GXNSFDA053020 and NO. 2015GXNSFAA139107).

Disclosure of conflict of interest

None.

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