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British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 2015 Jul 21;173(4):778–789. doi: 10.1111/bph.13200

Induction of the inflammatory regulator A20 by gibberellic acid in airway epithelial cells

J A Reihill 1,†, B Malcomson 1,†, A Bertelsen 1, S Cheung 1, A Czerwiec 1, R Barsden 1, J S Elborn 1, H Dürkop 2, B Hirsch 3, M Ennis 1, C Kelly 4,†, B C Schock 1,†,✉
Editors: AG Stewart, PM Beart
PMCID: PMC4742295  PMID: 26013851

Abstract

Background and Purpose

NF‐κB‐driven inflammation is negatively regulated by the zinc finger protein A20. Gibberellic acid (GA 3) is a plant‐derived diterpenoid with documented anti‐inflammatory activity, which is reported to induce A20‐like zinc finger proteins in plants. Here, we sought to investigate the anti‐inflammatory effect of GA 3 in airway epithelial cells and determine if the anti‐inflammatory action relates to A20 induction.

Experimental Approach

Primary nasal epithelial cells and a human bronchial epithelial cell line (16HBE14o‐) were used. Cells were pre‐incubated with GA 3, stimulated with P seudomonas aeruginosa  LPS; IL‐6 and IL‐8 release, A20, NF‐κB and IκBα expression were then evaluated. To determine if any observed anti‐inflammatory effect occurred via an A20‐dependent mechanism, A20 was silenced using siRNA.

Key Results

Cells pre‐incubated with GA 3 had significantly increased levels of A20 mRNA (4 h) and protein (24 h), resulting in a significant reduction in IL‐6 and IL‐8 release. This effect was mediated via reduced IκBα degradation and reduced NF‐κB (p65) expression. Furthermore, the anti‐inflammatory action of GA 3 was abolished in A20‐silenced cells.

Conclusions and Implications

We showed that A20 induction by GA 3 attenuates inflammation in airway epithelial cells, at least in part through its effect on NF‐κB and IκBα. GA 3 or gibberellin‐derived derivatives could potentially be developed into anti‐inflammatory drugs for the treatment of chronic inflammatory diseases associated with A20 dysfunction.

Linked Articles

This article is part of a themed section on Inflammation: maladies, models, mechanisms and molecules. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2016.173.issue-4

Abbreviations

16HBE14o‐

human bronchial epithelial cells

CF

cystic fibrosis

Dex

dexamethasone

GA3

gibberellic acid 3

NEMO

NF‐κB essential modulator

OTU

ovarian tumour domain

PNEC

primary nasal epithelial cell

TLR4

toll‐like receptor 4

TRAF6

TNF receptor‐associated factor 6

Tables of Links

TARGETS
Catalytic receptors a Enzymes b
TLR2 IKK
TLR4
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LIGANDS
Dexamethasone IL‐8
Forskolin LPS
IL‐6
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These Tables list key protein targets and ligands in this article which are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Pawson et al., 2014) and are permanently archived in the Concise Guide to PHARMACOLOGY 2013/14 (a,bAlexander et al., 2013a, 2013b).

Introduction

The airway mucosa is under constant attack from invading pathogens, which are recognized by pattern recognition receptors on the surface of airway epithelial cells and circulating immune cells. Toll‐like receptor 4 (TLR4) recognizes the LPS component of Gram‐negative bacterial cell walls. Activation of TLR4 triggers an innate immune response leading to an acute inflammatory response that is largely mediated by the transcription factor NF‐κB. Following receptor‐ligand binding at the plasma membrane, the intracellular stages of the NF‐κB signalling cascade are tightly regulated to ensure timely termination of the inflammatory response. A20 [TNFAIP3 (TNF‐α‐induced protein 3)] is an endogenous negative regulator of NF‐κB signalling that is rapidly and transiently induced in response to bacterial and viral stimuli. A20 terminates NF‐κB‐driven inflammation in response to LPS by inhibiting the polyubiquitination and activation of the central adaptor protein TNF receptor‐associated factor 6 (TRAF6) (Lin et al., 2008).

In the lung, Gram‐negative Pseudomonas aeruginosa challenge rapidly induces A20 in mice (Tiesset et al., 2009), while A20 is essential for termination of TLR2/4‐mediated IL‐8 release from primary airway epithelial cells (Gon et al., 2004). Furthermore, A20 protects against ovalbumin‐induced ‘asthma’ in mice (Kang et al., 2009). Li et al. (2013) showed that A20 is involved in antigen degradation by facilitating the fusion between the endosome and lysosome, resulting in reduced antigenicity upon absorption of the antigen. We recently demonstrated that A20 is significantly reduced in the chronically inflamed cystic fibrosis (CF) airway epithelium and that basal mRNA expression correlates strongly with lung function (FEV1 % predicted) in people with CF (Kelly et al., 2013a, 2013b). In addition to observations in the airways, A20 is known to be important in the pathogenesis of various inflammatory diseases (rheumatoid arthritis, systemic lupus erythematosus, type 1 diabetes, multiple sclerosis, psoriasis, colitis, chronic inflammatory bowel disease and Crohn's disease), where it may also serve as a susceptibility gene and biomarker of disease development (Barmada et al., 2004; Wellcome Trust Case Control Consortium, 2007; Martin and Dixit, 2011). A20 knockout in epithelial cells promotes severe mucosal inflammation (Vereecke et al., 2011). The therapeutic potential of targeting the A20 gene and resulting protein has been widely recognized but a therapeutic agent is not currently available.

Although a critical regulator of mammalian immune responses, A20‐like zinc finger (ZF) proteins are also important in controlling plant stress responses. Liu et al. (2011) reported that gibberellic acid (GA3), a tetracyclic diterpene, induces A20‐like ZF proteins in rice crops. Interestingly, GA3 has previously been used in the treatment of human lung cancer and in a rodent model of diabetes, and was shown to exert anti‐inflammatory and analgesic effects (Miklussak et al., 1980; Muthuraman and Srikumar, 2009). Furthermore, a recently developed gibberellin derivative possesses potent anti‐tumour and anti‐angiogenic activity in various tumour‐derived cell lines (Zhang et al., 2012). Gibberellins comprise a large family of tetracyclic diterpenoid plant hormones biosynthesized via ent‐kaurene intermediates, which have diverse biological roles in plant growth and development. In plants, gibberellins play signalling roles in response to environmental changes (temperature, stress and light) and are involved in the control of stem and root elongation, leaf expansion, seed germination and flowering (Yamaguchi, 2008). Many terpenes, including diterpenes, triterpenes and sesquiterpenes, possess anti‐inflammatory activity both in vivo and in vitro. Furthermore, most terpenes inhibit NF‐κB activity, although the precise mechanisms of action have not been fully characterized. In particular, forskolin has been used successfully in the treatment of asthma to reduce inflammation and histamine release through activation of cAMP‐dependent mechanisms (Huerta et al., 2010). In the case of kaurenes, Castrillo et al. (2001) have identified relevant targets for this inhibition, showing that the inhibition of NF‐κB inducing kinase (non‐canonical NF‐κB pathway) and p38 and/or ERK1 and ERK2 activation prevents the inflammatory response of genes dependent on NF‐κB activation in particular. The fact that kaurenes are intermediates in the biosynthesis of plant hormones, such as gibberellins, offers the possibility of envisaging further analysis of the interaction of these molecules in several aspects of mammalian cell biology (Castrillo et al., 2001).

Several gibberellin‐based preparations are under patent for the treatment of diabetes (Patent reference US 20050215496 A1), psoriasis and prostatitis (Patent reference WO 1991008751 A1), highlighting the increasing interest in developing gibberellins for preclinical and clinical trials. However, gibberellins have not yet been used clinically as anti‐inflammatory drugs and the mechanism of action has not been reported. Here, we seek to investigate if GA3 can induce A20 and thereby reduce the innate inflammatory response to bacterial LPS in airway epithelial cells [16HBE14o‐ and primary nasal epithelial cells (PNECs)].

Methods

Cell culture

The immortalized bronchial epithelial cell line 16HBE14o‐ (obtained from D. Gruenert UCSF, USA) was grown as described previously (Kelly et al., 2013c). PNECs were obtained from healthy volunteers (n = 7) and grown as previously described (de Courcey et al., 2012). The participants did not have any acute airways disease at the time of sampling, or a history of any chronic airways inflammation. The study was approved by the Research Ethics Committee of Northern Ireland (07/NIR02/23) and all participants provided informed consent (de Courcey et al., 2012).

Cell culture stimulations

All cells were treated with LPS (P. aeruginosa; Sigma‐Aldrich, Gillingham, UK, 10 μg·mL−1) for up to 24 h. Cells were exposed to a range of GA3 concentrations (Sigma‐Aldrich, G7645, lot BCBB9751, 3–300 μM) or dexamethasone (Dex, Sigma‐Aldrich, D4902, lot BCBH2988V, 1 μM) for 1 h before LPS stimulation.

Determination of LPS‐induced cytokine release (IL‐6 and IL‐8)

The concentrations of IL‐6 and IL‐8 in cell‐free culture supernatants were measured by a commercially available elisas (PeproTech EC Ltd., London, UK) according to the manufacturer's instructions.

LDH cytotoxicity assay

The effect of GA3 on membrane integrity was determined by quantification of LDH in cell culture supernatants (LDH‐Cytotoxicity Assay Kit; BioVision Ltd., Milpitas, CA, USA) according to the manufacturer's instructions.

Proliferation assay

Cell proliferation was determined using the CellTiter 96® AQueous One Solution Cell Proliferation Assay (Promega, Southampton, UK) following 72 h exposure to GA3 and according to the manufacturer's recommendations. The assay determines the activity of mitochondrial NAD(P)H‐dependent oxidoreductases, which reduces the tetrazolium dye MTS (3‐(4,5‐di‐methylthiazol‐2‐yl)‐5‐(3‐carboxymethoxyphenyl)‐2‐(4‐sulfophenyl)‐2H‐tetrazolium) to insoluble purple formazan, which is measured at λ = 490 nm.

Transfection studies

Transfections were performed at 70–80% confluency. A20 was silenced in 16HBE14o‐ cells using a commercially available human A20 siRNA (GenomeWide siRNA; Qiagen, Manchester, UK) and RNAiFect™ Transfection Reagent (Qiagen). Experiments included mock (transfection reagent only) and scrambled (Allstars Neg siRNA, Qiagen) controls. A20 knock‐down was confirmed by qPCR (transfection efficiency over 70%) and Western blot.

RNA extraction and real‐time qPCR

Total RNA was extracted using an RNeasy kit (Qiagen) and quantified on a Nanodrop (Thermo Scientific, Loughborough, UK). Equal amounts of RNA were reverse transcribed into cDNA (Sensiscript Reverse Transcription Kit, Qiagen). Primers were designed using gene accession numbers and Primer3 open‐source PCR primer design software and obtained from Invitrogen Ltd. (Paisley, UK). Quantitative PCR was performed using a LightCycler rapid thermal cycler system (Roche Diagnostics Ltd, Burgess Hill, UK). Multiple housekeeping genes were tested and β‐actin was chosen for consistency within cycles and between different samples.

Expression of A20, p65 and β‐actin (housekeeping gene) was assessed by qPCR. All primer sequences are given in Supporting Information Table S1. Relative expression to β‐actin was calculated using the ΔΔCt method. To overcome inter‐patient variability in basal gene expression levels in PNECs, mRNA expression after LPS stimulation (24 h) was compared with mRNA expression at 0 h (standardized to 1) for each individual sample. cDNA obtained from Jurkat cells acted as an internal calibrator for all experiments and was used to determine differences in basal gene expression.

Western blotting

Protein expression was determined by Western blotting after extraction of total proteins in RIPA buffer. Cell lysates were diluted in nuclease free water and Laemmli loading buffer, loaded onto Tris–HCl polyacrylamide gels (Thermo Scientific), separated by SDS‐PAGE and transferred to a PVDF membrane. Membranes were incubated with 1 μg·mL−1 primary antibodies (Ber‐A20; Hirsch et al., 2012), A20 C‐terminal (IMGENEX, San Diego, CA, USA, IMG‐161A), p65 (C‐20; Santa Cruz Biotechnology, Dallas, TX, USA), IkBα (C‐21; Santa Cruz Biotechnology) washed, incubated with appropriate HRP‐conjugated secondary antibodies (Thermo Scientific) and visualized on a BioRadChemi Doc XRS system (BioRad NV, Belgium).

P65 activation elisa

Nuclear protein was extracted from 16HBE14o‐ cells using the Ne‐Per™ assay (Cat. No. 78833; Thermo Scientific). Total protein was determined in the extracts using the BCA protein assay (Cat. No. 23225; Pierce, Life Technologies, Renfrew, UK) and equal amounts of nuclear protein (10 μg) were used in the p65 activation elisa (Active Motif, TransAM®, Carlsbad, CA, USA, NF‐κB Family Transcription Factor Assay Kit, Cat. No. 43296). Briefly, each elisa well contained the immobilized NF‐κB consensus site (5′‐GGGACTTTCC‐3′) to which active NF‐κB binds. The primary antibody (here anti‐p65) can only bind to the epitope on the active p65 that is bound to its target DNA. Then an HRP‐conjugated secondary antibody is used to provide a sensitive colorimetric readout that is spectrophotometrically quantified. All assays were performed according to the manufacturer's instructions.

Statistical analysis

All data are presented as the means ± SEM. Differences between groups were analysed using the Kruskal–Wallis non‐parametric anova, which was considered to be significant if P < 0.05. Dunn's post‐test was performed if the Kruskal–Wallis test was significant. To assess the effect of GA3 pretreatment on LPS‐stimulated PNECs, Kruskal–Wallis test with Dunn's post‐test and Wilcoxon paired signed rank test (cytokine release in response to LPS in the presence and absence of GA3) was applied. Statistical significance levels are indicated as *P < 0.05, **P < 0.01 and ***P < 0.001. GraphPad Prism (La Jolla, CA, USA) was used to plot graphs and to analyse the data.

Results

Changes in gene or protein expression are shown relative to the corresponding untreated control. Given the limited amount of material available from primary cultures, all primary NEC investigations were performed at the mRNA level initially and confirmed at the protein level where possible.

Effect of GA 3 pretreatment on cellular viability and LPS‐stimulated cytokine release

In the first instance, we sought to determine an optimal non‐cytotoxic GA3 dose that would also induce significant anti‐inflammatory effects. Therefore, the effect of various GA3 concentrations (0.03–300 μM) on cellular viability and LPS‐stimulated IL‐6 and IL‐8 release was investigated in the bronchial epithelial cell line 16HBE14o‐.

LDH release into cell culture supernatants was analysed 24 h after GA3 treatment and used as a measure of cytotoxicity. GA3 concentrations of 0.03–30 μM did not increase LDH release above the vehicle control, while 300 μM GA3 caused a significant increase in LDH release (P < 0.001, n = 6; Figure 1A). IL‐8 release was significantly increased compared with untreated cells following 24 h LPS stimulation (Figure 1B). In 16HBE14o‐ cells, GA3 treatment alone did not have any effect on basal IL‐8 release, but GA3 pretreatment significantly reduced the LPS‐induced IL‐8 release in a dose‐dependent manner with a significant reduction at 30 μM (P < 0.01, n = 5–9) (Figure 1B). The maximal effective concentration to reduce IL‐8 was 28.19 μM, while a reduction of 40% was achieved at 2.82 μM (Figure 1C). Similarly, GA3 treatment alone did not have any effect on basal IL‐6 release, but GA3 pretreatment significantly reduced the LPS‐induced IL‐6 release with a significant reduction at 30 μM (P < 0.05, n = 5–8) (Figure 1D).

Figure 1.

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Effect of GA 3 on LDH and cytokine release from the bronchial epithelial cell line 16HBE41o‐. (A) 16HBE14o‐ cells were treated with GA 3 for 24 h and LDH release into the culture medium was determined. At 0.03, 0.3, 3 and 30 μM, GA 3 did not cause any significant LDH release, while 300 μM caused a significantly higher LDH release when compared with vehicle controls (P < 0.001, n = 6). (B) 16HBE14o‐ bronchial epithelial cells were treated with GA 3 (0.03–300 μM, 1 h) or Dex (1 μM, 1 h). LPS (10 μg·mL−1) was subsequently added to the cultures. IL‐8 release was determined by elisa after 24 h. GA 3 (3–300 μM) and Dex (1 μM) pretreatment of LPS‐stimulated 16HBE14o cells caused a significant reduction in IL‐8 release (P < 0.01, n = 5–9). (C) The maximal effective concentration to reduce IL‐8 (by 47%) was 28.19 μM, while a reduction of 40% was achieved at 2.82 μM (all green squares). (D) 16HBE14o‐ cells were treated with GA 3 (0.03–30 μM, 1 h) or Dex (1 μM, 1 h) before LPS exposure (10 μg·mL−1). IL‐6 release was determined by elisa after 24 h. GA 3 (30 μM) and Dex (1 μM) pretreatment of LPS‐stimulated 16HBE14o cells caused a significant reduction in IL‐6 release (P < 0.01, n = 5–8). Statistical significance levels are indicated as *P < 0.05, **P < 0.01 and ***P < 0.001.

Based on these results and previous publications, a concentration of 30 μM GA3 was employed in all further experiments. For comparative purposes, the anti‐inflammatory effect of Dex (1 μM) was also investigated (Figure 1B and D). IL‐8 and IL‐6 release from 16HBE14o‐ cells pretreated with Dex prior to LPS stimulation displayed significantly lower levels of each cytokine than cells treated with LPS alone (P < 0.001 for both cytokines, n = 5) or GA3 and LPS (P < 0.05 for both cytokines, n = 5).

Our initial experiments in 16HBE14o‐ cells were confirmed in PNECs. PNECs were pretreated with GA3 (0.3–300 μM) for 1 h before LPS was added to the cultures for a further 24 h. GA3 concentrations of 0.3–30 μM did not increase LDH release above the vehicle control, but 300 μM GA3 caused a significant increase in LDH release compared with 0.3 and 3 μM (P < 0.01 and P < 0.05, respectively, n = 6; Figure 2A). In PNECs, LPS treatment significantly increased IL‐8 and IL‐6 release (P < 0.05 and P < 0.01, respectively, n = 7) (Figure 2B‐1 and C‐1), while GA3 pretreatment significantly reduced it (P = 0.019 and P = 0.008, Wilcoxon paired signed rank test, n = 7) at 24 h (Figure 2B‐2 and C‐2).

Figure 2.

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Effect of GA 3 on LDH and cytokine release from PNECs. (A) PNECs were pretreated with GA 3 (3–300 μM for 1 h) before LPS (10 μg·mL−1) was added to the cultures for 0–24 h. Analysis of LDH in the supernatants showed no significant increase in LDH release above the vehicle control as a result of treatment with GA 3. (B‐1) LPS treatment significantly increased IL‐8 release (P < 0.05, n = 8). (B‐2) GA 3 pretreatment (30 μM, 1 h) significantly reduced LPS‐induced IL‐8 release in PNECs (P < 0.05, n = 8) at 24 h. Consistently, (C‐1) LPS treatment significantly increased IL‐6 release (P < 0.05, n = 7) while (C‐2) GA 3 (30 μM, 1 h) pretreatment significantly reduced LPS‐induced IL‐6 release in PNECs (P < 0.05, n = 7) after 24 h exposure. Statistical significance levels are indicated as *P < 0.05, **P < 0.01 and ***P < 0.001.

GA 3 promotes an A20‐driven, anti‐inflammatory response in 16HBE14o‐ cells

LPS stimulation of 16HBE14o‐ cells resulted in the rapid and transient induction of A20 mRNA with peak expression observed after 1 h. Thereafter, A20 expression slowly declined and returned to basal levels 4 h after LPS stimulation (Figure 3A ). This is consistent with the original findings of Opipari et al. (1990). GA3 pretreatment (30 μM) alone significantly induced A20 mRNA expression compared with culture medium control (1 h, P < 0.05). The subsequent addition of LPS further enhanced and prolonged the induction of A20 mRNA compared with culture medium control (1 and 4 h, P < 0.001; 24 h, P < 0.05), LPS alone (4 h, P < 0.05) or GA3 alone (1 and 4 h, P < 0.05), and A20 expression remained above basal levels in GA3‐pretreated cells even 24 h after LPS treatment (Figure 3A, n = 5–9). A20 mRNA expression was not induced in 16HBE14o‐ cells treated with Dex, prior to LPS stimulation (Figure 3A).

Figure 3.

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Anti‐inflammatory effect of GA 3 in 16HBE14o‐ cells is A20‐dependent. (A) 16HBE14o‐ bronchial epithelial cells were pretreated with LPS (10 μg·mL−1) alone for 24 h, or with GA 3 (30 μM, 1 h) or Dex (1 μM, 1 h) before the addition of LPS (10 μg·mL−1) for a further 24 h, and A20 mRNA was determined by qPCR. Both LPS and GA 3 treatment significantly induced A20 mRNA in 16HBE14o‐ cells (P < 0.01–0.001, n = 5–9). (B) p65 mRNA was significantly increased 4 h after LPS treatment (10 μg·mL−1, P < 0.001, n = 5–9). This effect was significantly attenuated when cells were pretreated with GA 3 (30 μM, 1 h) (P < 0.001, n = 5–9). For A and B: medium control (grey), GA 3 (blue), LPS (red), GA 3+ LPS (purple), Dex (green). (C) When A20 was silenced with siRNA, the anti‐inflammatory effect of GA 3 [determined by IL‐8 release of LPS‐stimulated cells (24 h)] was abolished (P < 0.001, n = 5, C). Statistical significance levels are indicated as *P < 0.05, **P < 0.01 and ***P < 0.001.

Furthermore, analyses of p65 mRNA expression showed a significant induction of p65 by LPS alone (P < 0.001, 4 h vs. medium control, n = 5–9, Figure 3B) and GA3‐pretreated 16HBE14o‐ showed a significant reduction of LPS‐induced p65 (P < 0.001, 4 h vs. medium control, n = 5–9, Figure 3B), suggesting that GA3 negatively affects the NF‐κB pathway. Pretreatment of 16HBE14o‐ cells with Dex also attenuated LPS‐induced p65 expression; however, this effect was not significant (Figure 3B).

To investigate if the observed induction of A20 and the subsequent reduction in p65 by GA3 is responsible for the anti‐inflammatory effects outlined in Figures 1B and 2B‐1, 16HBE14o‐ cells were treated with siRNA targeting A20. Figure 3C shows that when A20 is silenced in 16HBE14o‐ cells, the anti‐inflammatory effect of GA3 on LPS‐induced IL‐8 release is lost (P < 0.001, n = 5, Figure 3C). These experiments suggest that GA3 acts in an A20‐dependent manner.

GA 3 induces A20 mRNA and protein in PNECs

To investigate the effect of GA3 on A20 in PNECs, cells were pretreated with GA3 (30 μM) for 1 h before the addition of LPS to the cultures and incubated for up to 24 h. In non‐stimulated cells, A20 mRNA levels remained unchanged over the duration of the experiment (data not shown). When cells were stimulated with LPS for up to 24 h, A20 mRNA levels increased significantly over culture medium control (P < 0.01, n = 7) with a peak expression at 4 h (Figure 4A). GA3 exposure alone induced A20 mRNA in PNECs in a time‐dependent manner (P < 0.05, 24 h vs. culture medium control, n = 7, Figure 4A). In GA3‐pretreated cells, LPS (10 μg·mL−1) increased A20 mRNA levels significantly over medium control (n = 7 with P < 0.001 at 4 h and P < 0.01 at 24 h). GA3‐pretreated and LPS‐stimulated PNECs also showed a significant increase in A20 mRNA compared with LPS alone at 4 h (P < 0.05, n = 7, Figure 4A). In Dex‐pretreated and LPS‐stimulated 16HBE14o‐, no significant induction of A20 mRNA was observed, n = 5). A20 protein expression (24 h after LPS challenge) was determined by Western blotting and quantified. In accordance with mRNA levels, A20 protein was induced by GA3 in PNECs challenged with LPS (P < 0.05, compared with GA3, n = 6, Figure 4B).

Figure 4.

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GA 3 induces A20 mRNA and protein in PNECs. PNECs were pretreated with LPS alone (10 μg·mL−1), or pretreated with GA 3 (30 μM, 1 h) or Dex (1 μM, 1 h) before the addition of LPS (10 μg·mL−1) to the cultures for 0–24 h and A20 mRNA and protein expression investigated. GA 3 (blue bars) induced (A) A20 mRNA in PNECs in a time‐dependent manner, with a significant increase at 24 h (n = 7, P < 0.05: 24 h vs. medium control). When cells were stimulated with LPS (10 μg·mL−1) for up to 24 h (red bars), A20 mRNA levels increased significantly over medium control (P < 0.05 and P < 0.01, n = 7). In GA 3‐pretreated cells (30 μM), LPS (10 μg·mL−1) increased A20 mRNA levels significantly compared to LPS alone (P < 0.05, n = 7). Pretreatment with Dex (green bar) did not significantly induce A20 mRNA (n = 5). (B) Western blot analysis confirmed an increase in A20 protein expression when cells were pretreated with GA 3 and then challenged with LPS (P < 0.05, n = 6, compared to GA 3 alone). (C) Representative blot and quantification of Western blots after use of IMG‐161A, an antibody against the C‐terminal end of A20 that can identify several ZFs. Incubation with GA 3 in the presence or absence of LPS leads to an increase in all protein bands in similar proportions with the highest increase in the 50 kDa (ZFs 1–2) band compared with medium control or LPS alone. Only in GA 3‐pretreated and LPS‐treated cells, the 60 kDa band, corresponding to ZFs 1–3, was significantly increased compared with control cells (P < 0.05, n = 4, C). Statistical significance levels are indicated as *P < 0.05, **P < 0.01 and ***P < 0.001.

Using an antibody against the C‐terminal end of A20 that has been previously used to identify A20 cleavage products (http://www.novusbio.com/primary‐antibodies/tnfaip3), we showed that GA3‐dependent induction of A20 in PNECs results in an up‐regulation of the whole A20 protein. The representative blot and the quantification in Figure 4C (n = 4) show C‐terminal A20 protein bands with a molecular weight of the expected 80 kDa (whole protein) and those with approximately 60 and 50 kDa corresponding to C‐terminal A20 fractions containing ZFs 1–2 (∼50 kDa) and ZFs 1–3 (∼60 kDa) (Klinkenberg et al., 2001). The antibody also detects a C‐terminal A20 fraction of around 40 kDa, although bands of this size were not detected in this study. Incubation with GA3 in the presence or absence of LPS induced an increase in all protein bands in similar proportions (Figure 4C). Only in GA3 and LPS‐treated cells, the 60 kDa band, corresponding to ZFs 1–3, was significantly increased compared with control cells (P < 0.05, n = 4, Figure 4C).

GA 3 reduces LPS‐induced cytokine release via reduction of NF‐κB (p65)

We and others have previously shown that IL‐8 production in epithelial cells is largely dependent on p65 activation (Dommisch et al., 2010; Kelly et al., 2013b). To investigate if GA3 affects NF‐κB signalling, p65 mRNA was analysed 1, 4 and 24 h after LPS challenge in GA3‐pretreated PNECs. LPS significantly induced p65 mRNA expression compared with culture medium control at 1 and 4 h (P < 0.05 and <0.001, n = 7). However, p65 mRNA was significantly reduced in GA3‐treated PNECs 4 h after LPS challenge (P < 0.05, n = 7) (Figure 5). Dex pretreatment of PNECs and subsequent LPS stimulation, similar to pretreatment with GA3, resulted in a significant reduction in p65 mRNA 4 h after stimulation (P < 0.05, n = 5) (Figure 5).

Figure 5.

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GA 3 suppresses p65 expression in PNECs. PNECs were treated with LPS alone (10 μg·mL−1), or pretreated with GA 3 (30 μM, 1 h) or Dex (1 μM, 1 h) before the addition of LPS (10 μg·mL−1) for 1, 4 or 24 h. LPS‐induced p65 expression (mRNA) was subsequently measured by qPCR and found to be significantly reduced in GA 3‐treated PNECs 4 h after LPS challenge (P < 0.05, n = 5–7). GA 3 (blue), LPS (red), GA 3+ LPS (purple), Dex (green). Statistical significance levels are indicated as *P < 0.05, **P < 0.01 and ***P < 0.001.

LPS‐induced NF‐κB (p65) activation starts with the posttranslational modification, for example, by phosphorylation of p65 (phosphorylation on serine 536) in the cytosol, and is then translocated into the nucleus (Hall et al., 2006). Having shown a reduction of p65 mRNA by GA3 treatment, we further confirmed that GA3 exerts its anti‐inflammatory action via modulation of NF‐κB activation. Firstly, the level of nuclear‐activated NF‐κB (p65) was significantly induced by stimulation with LPS at 15 min returning to culture medium control levels at 24 h (P < 0.001 and P < 0.01, n = 5, Figure 6A). GA3‐pretreated and LPS‐stimulated 16HBE14o‐ showed significantly reduced nuclear active p65 at 15 min compared with LPS stimulation alone (P < 0.05, n = 5, Figure 6A). Dex pretreatment also reduced p65 activity 15 min after LPS stimulation (P < 0.01, n = 5, Figure 6A).

Figure 6.

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Effect of GA 3 is associated with reduced p65 and increased cytosolic IκBα. (A) LPS‐induced activation of nuclear p65 was significantly reduced in GA 3‐pretreated PNECs 4 h after LPS challenge (P < 0.05, n = 5). (B) Phosphorylation of p65 on serine 536 was determined in whole cell extracts in 16HBE14o cells by Western blotting. Results indicate a reduction in the phosphorylation on serine535 in GA 3‐pretreated cells for up to 8 h after LPS challenge. (C) Representative blot and quantification of IkBα protein expression in GA 3‐pretreated PNECs. LPS treatment (24 h) significantly reduced cytosolic IkBα compared with untreated and GA 3‐treated cells (P < 0.05 and P < 0.001, n = 6). Pretreatment with GA 3 (1 h before addition of LPS for 24 h) restored in part cytosolic IkBα protein levels (P < 0.05, n = 6). Statistical significance levels are indicated as *P < 0.05, **P < 0.01 and ***P < 0.001.

Secondly, phosphorylation of p65 on serine 536 was determined in cytosolic whole cell extracts in 16HBE14o‐ by Western blotting. Our results indicate a reduction in the phosphorylation on serine535 in GA3‐pretreated cells for up to 8 h after LPS challenge (Figure 6B).

Finally, we investigated key events in the pathway upstream of p65 in 16HBE14o‐ cells. LPS‐mediated activation of NF‐κB is reliant on the activation and subsequent degradation of IkBα. We determined cytosolic IκBα protein expression in PNECs and showed that LPS treatment significantly reduces cytosolic IκBα (P < 0.05), while treatment with GA3 restored in part IκBα protein levels (P = 0.05) (all n = 6, Figure 6C).

Overall, these results confirm an anti‐inflammatory effect of GA3 in LPS‐stimulated airway epithelial cells. This appears to be mediated, in part at least, by the induction of A20 (mRNA and protein) and subsequent inhibition of key stages in the NF‐κB pathway.

Discussion

The cytoplasmic zinc finger (ZF) protein A20 is a central negative regulator of NF‐κB that governs multiple intracellular pathways. Here, we demonstrated that pre‐incubation of LPS‐stimulated airway epithelial cells (16HBE14o‐ cells and PNECs) with GA3, significantly induces A20 mRNA and protein. Although GA3 alone also induced A20 mRNA, this did not result in a detectable increase in protein, suggesting that GA3 mainly enhances LPS‐induced A20 expression. Maximal induction of mRNA was slightly delayed in PNECs compared with 16HBE14o‐ cells (1 h vs. 4 h), which could be explained by the differences in the cell types [16HBE14o‐ is a simian virus 40 (SV40) immortalized immortalized cell line (Kelly et al., 2013c) whereas PNECs are primary cells cultured for up to three passages]. Furthermore, the induction of A20 was accompanied by a significant increase in IκBα levels and subsequent reduction in NF‐κB expression, and IL‐6 and IL‐8 release in both cell lines and primary epithelial cells. Silencing of A20 confirmed that the reduction in IL‐8 release was A20 dependent.

El‐Mofty et al. (1994) suggested that GA3 had carcinogenic effects and led to tumour formation in Swiss Albino mice. However, we tested GA3 at a range of concentrations from 0 to 300 μM and found no evidence of cellular toxicity or increased proliferation even at 3 or 30 μM (Supporting Information Fig. S1). The proliferation assay was conducted over 72 h to ensure that at least one complete cell cycle had taken place. In our hands, the doubling time of 16HBE14o‐ cells is approximately 28 h.

Our results are in line with data by Kasamatsu et al. (2012), who showed that incubating adipose‐derived stem cells with GA3 (1 mM) did not alter cell morphology or viability. Early work by Miklussak et al. (1980) suggested that administration significantly improves metabolic functions in patients with lung cancer. More recently, Zhang et al. (2012) showed that the gibberellin derivative 3‐chlorine‐3,15‐dioxy‐gibberellic acid methyl ester (GA‐13315) possessed potent anti‐tumour and anti‐angiogenic activity in vitro and in vivo.

LPS activation of the NF‐κB pathway is dependent on the ubiquitination and subsequent proteasomal degradation of TRAF6 (Mabilleau et al., 2011). This process frees bound signalling molecules for subsequent downstream events including the activation of NEMO, degradation of cytosolic IκB and translocation of NF‐κB subunits to the nucleus. NF‐κB is sequestered in the cytoplasm, bound by members of the IκB family of inhibitor proteins, which includes IκBα. IKK phosphorylates IκBα, resulting in its K48‐linked ubiquitination and subsequent proteasomal degradation. This then allows translocation of the NF‐κB subunits p65/p50 to the nucleus to activate target genes. To address where GA3 acts in the NF‐κB pathway, we showed a significant reduction in cytosolic IκBα protein in LPS‐stimulated cells, suggesting that it had been degraded to release the NF‐κB subunits to the nucleus. GA3 treatment inhibited the LPS‐induced degradation of IκBα, which may explain the anti‐inflammatory effect seen (reduced IL‐8 and IL‐6 release and p65 mRNA). Furthermore, we showed that GA3 treatment leads to reduced p65 nuclear activity (Figure 6A) and this was associated with decreased phosphorylation of p65 on serine 536 (Figure 6B).

Information about the induction of A20 is still controversial. A20 has been described as being NF‐κB dependent, and da Silva et al. (2012a) showed that this activation of the ‘anti‐inflammatory arm of NF‐κB’ is associated with reduced expression of phosphorylated (ser536) p65 compared with the induction of phosphorylated (ser536) p65 by TNF‐α alone. Similarly, we showed here that in bronchial epithelial cells the induction of A20 by GA3 pretreatment resulted in a reduction in the expression of phosphorylated (ser536) p65 compared with LPS‐stimulated cells (Figure 6B), suggesting that the phosphorylation of p65 (e.g. by IKK kinases; Hall et al., 2006) could be a target of A20. Our data on p65 nuclear activity (Figure 6A) may suggest that GA3 itself induces low levels of NF‐κB (although not statistically significant), but further phosphorylation states or DNA binding sites of p65 would need to be investigated.

Finally, the A20 protein consists of an N‐terminal OTU (ovarian tumour) domain and a C‐terminal domain that consists of 7 Cys‐Cys ZFs. Work by Evans et al. (2004) assigned the deubiquitination activity to the OTU domain whereas the anti‐inflammatory activity was in the ZF domain (especially ZFs 4 and 7 have been identified to exhibit the anti‐inflammatory effect of A20; O'Reilly and Moynagh, 2003; Dommisch et al., 2010; Tokunaga et al., 2012). Furthermore, De Valck et al. (1996) showed that A20 ZFs have the ability to self‐assemble to build the whole A20 ZF domain. To investigate if GA3 can induce the whole A20 protein, we used two antibodies against full‐length A20 and the C‐terminal domain specifically (O'Reilly and Moynagh, 2003; Dommisch et al., 2010). Our data suggest that in PNECs, incubation with GA3 in the presence or absence of LPS leads to an increase in all protein bands in similar proportions representing the whole protein and the ZF domain of the A20 protein. However, in cells treated with GA3 and LPS, the 60 kDa fraction appeared to be increased compared with unstimulated cells. The 60 kDa band has been described as ZFs 1–3 (∼60 kDa) (Klinkenberg et al., 2001) and our findings are in accordance with the description for this commercial A20 antibody in THP1 cells (http://www.novusbio.com/primary‐antibodies/tnfaip3). ZF 4 has been shown to facilitate the NF‐κB reducing anti‐inflammatory action of A20 (Wertz et al., 2004; Lu et al., 2013), but mice with a non‐functional ZF 4 showed that this domain might not be needed for the anti‐inflammatory function of A20. In T cells, MALT1 cleaves A20 into a 37 and a 50 kDa fraction (McAllister‐Lucas and Lucas, 2008; Malinverni et al., 2010), but to date this 60 kDa band has not been further identified.

The current study has some limitations. Firstly, the effect of GA3 on inflammatory mediators and the mechanism of its anti‐inflammatory action have been investigated in cell culture, which may also impose several limitations. In most experiments, we used an immortalized cell line (16HBE14o‐). A characteristic of these cells is that they release IL‐8 and IL‐6 throughout proliferation. This may explain why cytokine levels were already relatively high in untreated cells. Furthermore, compared with macrophages or monocytic cell lines, relatively high amounts of LPS are needed to stimulate airway epithelial cells and this is true for cell lines and primary cells alike. Another limitation of our study is that the effects of GA3 on p65 and IκBα – although associated with A20 induction – have not been further validated using siRNA experiments. However, we did show that the anti‐inflammatory effect of GA3 is directly dependent on the induction of A20, as GA3 had no effect on the release of inflammatory mediators in cells lacking A20 (knock‐down of A20 via siRNA, Figure 3C). In PNECs, the number of experiments was limited by the low cell number available from nasal brushings and the fact that PNECs can only be expanded up to passage 3. Due to the lack of PNECs, our investigation of induction of ZF fractions was only performed four times, which may lead to an imperfect statistical estimation. Many products are known to contain trace amounts of LPS and small amounts of LPS may desensitize to a subsequent challenge. The GA3 preparation used in this study passes the tests for plant cell culture applications (Sigma‐Aldrich), but has not been specifically tested for its LPS content. However, determination of TLR4 mRNA in GA3‐treated cells showed no change in the expression of TLR4, while in LPS‐stimulated cells TLR4 showed a time‐dependent up‐regulation (Supporting Information Fig. S2). Overall, this may suggest that the GA3 preparation lacks TLR4 stimulating endotoxins. Finally, future work is required to determine if GA3 reduces pro‐inflammatory cytokine secretion when cells are treated after LPS challenge.

In summary, we demonstrated that pre‐incubation of airway epithelial cell lines and PNECs with GA3 (single bolus of 30 μM) induces the NF‐κB regulator A20 at the mRNA and protein level, thereby exerting an anti‐inflammatory effect upon subsequent LPS stimulation. Our work provides molecular evidence (i) for A20 as a therapeutic target in inflammatory diseases; and (ii) for the plant diterpenoid gibberellin in reducing NF‐κB‐driven inflammatory responses. Gibberellin or gibberellin‐derived derivatives could be developed into anti‐inflammatory drugs for the potential treatment of chronic inflammatory diseases associated with A20 dysfunction such as salivary gland degeneration (Kasamatsu et al., 2012), liver degeneration (da Silva et al., 2012b), bone resorption associated with inflammatory diseases (Shimada et al., 2008; Mabilleau et al., 2011) and psoriasis (Gon et al., 2004 and reviewed in Kelly et al., 2011). Furthermore, the concept of pharmacological induction of A20 presents A20 as a potential drug target to reduce or normalize the inflammatory response in chronic inflammatory airway diseases such as CF, asthma and COPD. Further work will investigate GA3 in primary cells from patients with chronic airways disease.

Author contributions

J. R. started the experiments, supervised the day‐to‐day experiments, performed statistical analyses and provided the first manuscript draft. B. M. performed PNEC experiments and some of the analyses. A. B. performed p65 NF‐κB activity elisa. S. C. performed the p65 Western blot. A. C. and R.B. performed the cell line experiments. S. E. was responsible for ethics, clinical governance, patient selection and clinical sampling. H. D. advised on interpretation of A20 protein results. B. H. supplied A20 antibody and advised on interpretation of results. M. E. helped supervise the students, assisted with the data analysis and manuscript preparation. C. K. identified gibberellic acid as an A20 inducer, overlooked the experiments, analyses and manuscript preparation. B. C. S. identified gibberellic acid as an A20 inducer, supervised students and P.D.R.A., overlooked experiments, analyses and manuscript preparation together with C. K.

Conflict of interest

The authors declare no conflict of interest.

Supporting information

Figure S1 GA3 does not induce cellular proliferation.

Figure S2 GA3 preparation does not induce TLR4 mRNA expression.

Table S1 PCR primer sequences.

Click here for additional data file. (57.3KB, docx)

Acknowledgements

The authors would like to acknowledge the financial support through a Nuffield Science Bursary to B. M. administered by Sentinus Northern Ireland. C. K. was supported by a 3ME grant, funded by the EPSRC. The 16HBE14o‐ cell line was a kind gift of D Gruenert (UCSF). The authors also acknowledge the help of F Corr in qPCR analysis. The authors are particularly grateful to Professor P Hedden (Rothamsted Research, UK) for helpful comments on the manuscript.

Reihill, J. A. , Malcomson, B. , Bertelsen, A. , Cheung, S. , Czerwiec, A. , Barsden, R. , Elborn, J. S. , Dürkop, H. , Hirsch, B. , Ennis, M. , Kelly, C. , and Schock, B. C. (2016) Induction of the inflammatory regulator A20 by gibberellic acid in airway epithelial cells. British Journal of Pharmacology, 173: 778–789. doi: 10.1111/bph.13200.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1 GA3 does not induce cellular proliferation.

Figure S2 GA3 preparation does not induce TLR4 mRNA expression.

Table S1 PCR primer sequences.

Click here for additional data file. (57.3KB, docx)

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