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Published ahead of print on February 28, 2008, doi:10.1165/rcmb.2007-0014OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 39, pp. 208-217, 2008
© 2008 American Thoracic Society
DOI: 10.1165/rcmb.2007-0014OC

Corticosteroids Reduce IL-6 in ASM Cells via Up-Regulation of MKP-1

Timo Quante1, Yee Ching Ng1, Emma E. Ramsay1, Sheridan Henness1, Jodi C. Allen1, Johannes Parmentier1, Qi Ge2 and Alaina J. Ammit1

Respiratory Research Group, 1 Faculty of Pharmacy and 2 Discipline of Pharmacology, University of Sydney, Sydney, New South Wales, Australia

Correspondence and requests for reprints should be addressed to Alaina J. Ammit, Ph.D., Faculty of Pharmacy, University of Sydney, Sydney, NSW 2006, Australia. E-mail: ajammit{at}pharm.usyd.edu.au


   Abstract
Top
 Abstract
CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
The mechanisms by which corticosteroids reduce airway inflammation are not completely understood. Traditionally, corticosteroids were thought to inhibit cytokines exclusively at the transcriptional level. Our recent evidence, obtained in airway smooth muscle (ASM), no longer supports this view. We have found that corticosteroids do not act at the transcriptional level to reduce TNF-{alpha}–induced IL-6 gene expression. Rather, corticosteroids inhibit TNF-{alpha}–induced IL-6 secretion by reducing the stability of the IL-6 mRNA transcript. TNF-{alpha}–induced IL-6 mRNA decays at a significantly faster rate in ASM cells pretreated with the corticosteroid dexamethasone (t1/2 = 2.4 h), compared to vehicle (t1/2 = 9.0 h; P < 0.05) (results are expressed as decay constants [k] [mean ± SEM] and half-life [h]). Interestingly, the underlying mechanism of inhibition by corticosteroids is via the up-regulation of an endogenous mitogen-activated protein kinase (MAPK) inhibitor, MAPK phosphatase-1 (MKP-1). Corticosteroids rapidly up-regulate MKP-1 in a time-dependent manner (44.6 ± 10.5-fold increase after 24 h treatment with dexamethasone; P < 0.05), and MKP-1 up-regulation was temporally related to the inhibition of TNF-{alpha}–induced p38 MAPK phosphorylation. Moreover, TNF-{alpha} acts via a p38 MAPK-dependent pathway to stabilize the IL-6 mRNA transcript (TNF-{alpha}, t1/2 = 9.6 h; SB203580 + TNF-{alpha}, t1/2 = 1.5 h), exogenous expression of MKP-1 significantly inhibits TNF-{alpha}–induced IL-6 secretion and MKP-1 siRNA reverses the inhibition of TNF-{alpha}–induced IL-6 secretion by dexamethasone. Taken together, these results suggest that corticosteroid-induced MKP-1 contributes to the repression of IL-6 secretion in ASM cells.

Key Words: asthma • airway remodeling • chronic obstructive pulmonary disease



   CLINICAL RELEVANCE
Top
 Abstract
 CLINICAL RELEVANCE
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 
In this study we investigate the molecular mechanisms by which corticosteroids suppress inflammation in airway smooth muscle, a pivotal immunomodulatory cell in asthma, in order to exploit these mechanisms in future therapies.

 
Asthma, a clinically and socioeconomically important chronic disease, is characterized, in part, by airway hyperresponsiveness and reversible airflow obstruction. Airway inflammation is considered an underlying cause. Numerous inflammatory mediators have been implicated in the pathogenesis of asthma (1). Of these mediators, cytokines are intrinsically involved in the pathophysiology of asthma. Airway smooth muscle (ASM), the major effector responsible for bronchomotor tone, has emerged as playing an important immunomodulatory role in asthma. During an allergic asthma attack, ASM is bathed in a complex mixture of cytokines and growth factors. It responds to this onslaught of exogenous stimuli by rapidly synthesizing and secreting additional proinflammatory mediators. These inflammatory mediators, predominately cytokines (2), can exert juxtacrine, autocrine, and paracrine effects in the airway (3). Thus, through its synthetic function, ASM plays a critical role in provoking, perpetuating and amplifying airway inflammation in asthma (4, 5).

The most common current anti-asthma medications are the inhaled corticosteroids. However, even after 30 years of use, the precise mechanisms by which they reduce airway inflammation are not completely understood. Furthermore, questions have arisen regarding their significant unwanted effects, including glaucoma and reduced bone mineral density (6). In addition, a small subset of asthma sufferers and patients with chronic obstructive pulmonary disease do not respond to clinically relevant doses of corticosteroids. We urgently require more efficacious novel anti-inflammatory strategies, or corticosteroid-sparing therapies, as these would yield substantial health benefits. A first step to towards achieving these outcomes is to understand molecular mechanisms by which corticosteroids suppress inflammation to allow us to exploit these mechanisms in future therapies.

However, our current understanding of how corticosteroids inhibit airway inflammation is lacking. Traditionally, corticosteroids were thought to inhibit cytokines exclusively during mRNA transcription. Our investigations (7–9), using the secretion of IL-6, a pleiotropic cytokine considered to have pro-inflammatory actions in asthma (10), as a model system, have questioned whether transcriptional regulation is the sole mechanism underlying the anti-inflammatory action of corticosteroids on ASM synthetic function. In unstimulated cells, the secretion of IL-6 protein by ASM cells is negligible (7). Upon stimulation with TNF-{alpha}, a pro-inflammatory cytokine found elevated in bronchoalveolar lavage fluid of patients with asthma (11), IL-6 mRNA expression and protein secretion is rapidly induced (7). This occurs via an NF-{kappa}B–dependent pathway (9). Interestingly, when we examined the anti-inflammatory effects of dexamethasone, we achieved partial inhibition of TNF-{alpha}–induced IL-6 protein secretion (9). However, as NF-{kappa}B–mediated transcription in ASM cells has been shown to be unaffected (12) or only partially inhibited (13) by dexamethasone in ASM cells, the question of how dexamethasone mediates its anti-inflammatory effect remains.

Therefore, in this study we investigate the molecular mechanisms by which corticosteroids suppress inflammation in ASM, a pivotal immunomodulatory cell in asthma. We show that anti-inflammatory corticosteroid action does not occur via transcriptional mechanisms on IL-6, but rather that corticosteroids induce up-regulation of an endogenous mitogen-activated protein kinase (MAPK) inhibitor, MAPK phosphatase-1 (MKP-1), and through an inhibitory effect on TNF-{alpha}–induced p38 MAPK phosphorylation, dexamethasone may inhibit p38 MAPK-mediated mRNA stability to reduce IL-6 protein secretion from ASM cells.


   MATERIALS AND METHODS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 References
 
Cell Culture
Human bronchi were obtained from patients undergoing surgical resection for carcinoma or lung transplant donors in accordance with procedures approved by the Central Sydney Area Health Service and the Human Ethics Committee of the University of Sydney. ASM cells were dissected, purified, and cultured as previously described by Johnson and coworkers (14). A minimum of three different ASM primary cell lines were used for each experiment.

Unless otherwise specified, all chemicals used in this study were purchased from Sigma-Aldrich (St. Louis, MO).

IL-6 mRNA Expression
To examine the time-course of induction of IL-6 mRNA expression by TNF-{alpha} and repression by dexamethasone, or the p38 MAPK inhibitor SB203580, growth-arrested ASM cells were pretreated for 1 hour with 100 nM dexamethasone, or 30 minutes with 1 µM SB203580 (Calbiochem, San Diego, CA) (8), compared to vehicle. Cells were then stimulated with TNF-{alpha} (10 ng/ml; R&D Systems, Minneapolis, MN) for 0, 1, 2, 4, 8, 16, and 24 hours, and IL-6 mRNA expression was quantified by real-time RT-PCR as previously described (15).

IL-6 Protein Secretion
To examine the time-course of induction of IL-6 protein secretion by TNF-{alpha}, and repression by dexamethasone or SB203580, growth-arrested ASM cells were pretreated for 1 hour with 100 nM dexamethasone, or 30 minutes with 1 µM SB203580, compared to vehicle. Cells were then stimulated with TNF-{alpha} (10 ng/ml) for 0, 1, 2, 4, 8, 16, and 24 hours, and secreted IL-6 protein was measured by enzyme-linked immunosorbent assay (ELISA). To compare the effect of MAPK inhibition on TNF-{alpha}–induced IL-6 secretion, growth-arrested ASM cells were pretreated for 30 minutes with vehicle, 10 µM PD98059, 10 µM SP600125, or 1 µM SB203580 (all from Calbiochem), to inhibit extracellular signal–regulated kinase (ERK) (8), c-Jun N-terminal kinase (JNK) (16), or p38 MAPK, respectively, then treated with vehicle or 10 ng/ml TNF-{alpha}. After 24 hours of incubation at 37°C in 5% CO2, cell supernatants were removed and frozen at –20°C for later analysis by ELISA. IL-6 (detection limit, 7.8 pg/ml; BD Biosciences Pharmingen, San Jose, CA) ELISAs were performed according to the manufacturer's instructions.

Transfection
The IL-6 promoter construct, pIL-6-luc 651, a pGL3 luciferase reporter vector (Promega, Madison, WI) containing a 651-bp fragment of the human IL-6 gene promoter containing transcription factor consensus binding elements for the glucocorticoid receptor (GRE), activator protein-1 (AP-1), CCAAT enhancer-binding protein-β (C/EBP-β), NF-{kappa}B, and a cAMP response element (CRE), was kindly provided by Dr. Oliver Eickelberg (Giessen University School of Medicine, Giessen, Germany) (17), with permission from Dr. Shigeru Katamine (Nagasaki University, Nagasaki, Japan) (18). Transient transfection of ASM cells was performed using Lipofectamine 2000 (Invitrogen Corporation, Carlsbad, CA) as previously described (15). Briefly, ASM cells were plated onto 6-well plates at a density of 2.5 x 105 cells/well for 24 hours, then transfected with 2.4 µg of pIL-6-luc 651 as well as 1.4 µg of pSV-β-galactosidase control vector (Promega) to normalize transfection efficiencies.

To measure the effect of dexamethasone on IL-6 promoter activity, ASM transfected with pIL-6-luc 651 were growth-arrested, pretreated for 1 hour with vehicle or 0.01 to 1 µM dexamethasone, then either left without further treatment for 24 hours to examine the effect of dexamethasone on the GRE in the IL-6 promoter, or stimulated with 10 ng/ml TNF-{alpha} for 24 hours to examine the repressive effects of dexamethasone on non-GRE transcriptional elements. To examine the effect of inhibition of p38 MAPK on IL-6 gene expression, transfected ASM cells were incubated for 30 minutes with the p38 MAPK inhibitor SB203580, or the inactive congener, SB202474 (both at 1 and 10 µM) (8), then treated with vehicle or 10 ng/ml TNF-{alpha} for 24 hours. Cells were then harvested and luciferase and β-galactosidase activities assessed according to the manufacturer's instructions (Promega). Data are presented as normalized luciferase activity, relative to vehicle-treated cells (expressed as fold difference).

To overexpress MKP-1 in ASM cells and examine the effect on TNF-{alpha}–induced IL-6 protein secretion, the MKP-1 expression vector pCMV-Flag-MKP-1 (19) was generously provided by Andrew R. Clark (Kennedy Institute of Rheumatology Division, Imperial College London, London, UK). ASM cells (1.5 x 105 cells/well) were transfected with 500 ng of pCMV-Flag-MKP-1, or empty vector control, according to previously established procedures using Lipofectamine 2000 (20). After 24 hours of stimulation with TNF-{alpha} (10 ng/ml), supernatants were removed, IL-6 protein measured by ELISA, and lysates prepared for Western blotting. To determine transfection efficiency under these conditions, we performed in situ staining of ASM cells for β-galactosidase activity, using an established protocol used to quantitate transfection efficiency (Promega). Briefly, ASM cells (1.5 x 105 cells/well) were transfected with 500 ng of MKP-1, and either 500 ng of pSV-β-galactosidase reporter gene or empty vector control. Cells expressing β-galactosidase were visualized by microscopy using the β-galactosidase reporter gene staining kit, according to manufacturer's instructions (Sigma), as the cells appear blue after fixation and incubation with the substrate X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside). In ASM cells (n = 3 primary cell lines) transfected with the pSV-β-galactosidase reporter gene and the MKP-1 expression vector, the transfection efficiency was 19.2 ± 2.3%. The degree of background activity due to endogenous β-galactosidase observed in control cells that had not been transfected with a β-galactosidase vector was 9.2 ± 1.3%.

To reduce protein levels of MKP-1 and examine the effect on dexamethasone-inhibited TNF-{alpha}–induced IL-6 secretion, MKP-1 was inhibited by RNA interference technology, using methods established by Issa and coworkers in 2007 (21). Briefly, ASM cells (0.5 x 106 cells) were transiently transfected with 1 µg MKP-1–specific SMART pool siRNA, consisting of a pool of four individual siRNA from Dharmacon (Lafayette, CO). A pool of scrambled sequences not complementary to any known genes was transfected as a negative control. In order to achieve greater transfection efficiences than previously obtained using Lipofectamine 2000 transfection, ASM cells were transfected with the Nucleofector (Amaxa, Koln, Germany), using the basic kit for primary smooth muscle cells with the manufacturer's optimized protocol of P-024. Transfection efficiency was monitored using pmaxGFP (Amaxa). We achieved 58% transfection, which was in line with the manufacturer's recommendation of 63%. After transfection, ASM cells were plated onto 6-well plates at a density of 4 x 105 cells/well for 16 hours, before being growth-arrested for a further 24 hours. Cells were then pretreated for 1 hour with 100 nM dexamethasone, or vehicle, then stimulated with TNF-{alpha} (10 ng/ml) for 24 hours before supernatants were removed for IL-6 protein measurement by ELISA and lysates utilized for MKP-1 Western blotting.

IL-6 mRNA Stability
To measure IL-6 mRNA stability, growth-arrested ASM cells pretreated for 1 hour with vehicle or 100 nM dexamethasone were stimulated with TNF-{alpha} (10 ng/ml) for 9 hours. In parallel experiments, the role of the p38 MAPK pathway in IL-6 mRNA stability was assessed by pretreating the cells for 30 minutes with vehicle or 1 µM SB203580 (20), respectively, before stimulation for 9 hours with TNF-{alpha} (at 10 ng/ml). Cells were then washed and incubated with actinomycin D (5 µg/ml) to inhibit further transcription. Total RNA was extracted after 0, 0.5, 1, 2, 3, 6, 9, and 12 hours of incubation with actinomycin D, and IL-6 mRNA expression was quantified by real-time RT-PCR as previously described (15). Results are presented as % mRNA remaining (i.e., compared with steady-state levels of mRNA expression after 9 h of cytokine treatment). Decay constants (k) were solved by nonlinear regression (one phase exponential decay: GraphPad Prism version 4.00 for Windows; GraphPad Software, San Diego, CA) of the % mRNA remaining versus time after actinomycin D (22), where the span was constrained to 100%. Results are expressed as decay constants (k) (mean ± SEM) and half-life (h).

Western Blotting
To measure corticosteroid-induced up-regulation of MKP-1, growth-arrested ASM cells were treated with vehicle, 100 nM dexamethasone, or 1 nM fluticasone propionate (AstraZeneca, Basel, Switzerland) for 0, 0.5, 1, 2, 4, 8, and 24 hours. Cells were lysed, then MKP-1 quantified by Western blotting using a rabbit polyclonal antibody against MKP-1 (M18; Santa Cruz Biotechnology, Santa Cruz, CA), compared to {alpha}-tubulin as the loading control (mouse monoclonal IgG1, clone DM 1A; Santa Cruz). Primary antibodies were detected with goat anti-mouse or anti-rabbit horseradish peroxidase–conjugated secondary antibodies (Cell Signaling Technology, Danvers, MA) and visualized by enhanced chemiluminescence (PerkinElmer, Wellesley, MA). Densitometry was performed using ImageJ (23).

To measure the effects of dexamethasone on the kinetics of TNF-{alpha}–induced MAPK phosphorylation, growth-arrested ASM cells pretreated for 1 hour with vehicle or 100 nM dexamethasone were stimulated with TNF-{alpha} (10 ng/ml) for 0, 5, 10, 30, and 60 minutes. Cells were lysed, then analyzed by Western blotting using rabbit polyclonal IgG antibodies against total and phosphorylated p38 MAPK (Thr180/Tyr182), total and phosphorylated ERK (Thr202/Tyr204), and total and phosphorylated JNK (Thr183/Tyr185). All MAPK antibodies were from Cell Signaling Technology. The kinetics of MAPK phosphorylation relative to the temporal up-regulation of MKP-1 was compared, while {alpha}-tubulin was used as the loading control.

To confirm exogenous expression of MKP-1 in ASM cells transfected with the MKP-1 expression vector, as well as reduction of MKP-1 by MKP-1 siRNA, lysates were immunoblotted for MKP-1 and compared with {alpha}-tubulin used as a loading control.

Statistical Analysis
Statistical analysis was performed using one-way ANOVA, then Fisher's post hoc multiple comparison test, or Student's unpaired t test. P values < 0.05 were sufficient to reject the null hypothesis for all analyses. Data represent mean ± SEM.


   RESULTS
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 References
 
Dexamethasone Inhibits TNF-{alpha}–Induced IL-6 mRNA Expression and Protein Secretion by Reducing IL-6 mRNA Stability, Rather than Inhibiting IL-6 Transcriptional Regulation
To examine the time-course of induction of IL-6 by TNF-{alpha}, and repression by dexamethasone, growth-arrested ASM cells pretreated for 1 hour with vehicle or 100 nM dexamethasone were stimulated with TNF-{alpha} for up to 24 hours. As shown in Figure 1A, IL-6 mRNA continued to be expressed up to 24 hours after TNF-{alpha} stimulation. These data are in line with our previously published temporal profile of TNF-{alpha}–induced IL-6 mRNA expression (15), although in contrast to those of McKay and colleagues (24), where a peak of mRNA expression was observed at approximately 1 hour. Furthermore, Figure 1B demonstrates that IL-6 protein secretion from ASM after TNF-{alpha} stimulation increases in a temporally dependent manner; corroborating earlier work (7, 24). When the repressive effect of corticosteroid pretreatment was examined, dexamethasone-dependent repression was lacking at very short time points, while at 8 and 16 hours, TNF-{alpha}–induced IL-6 mRNA was significantly inhibited by 100 nM dexamethasone (Figure 1A; P < 0.05), and a resultant attenuation of TNF-{alpha}–induced IL-6 protein secretion occurred at 24 hours (Figure 1B; P < 0.05).


Figure 1
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  		Figure 1. Dexamethasone inhibits TNF-{alpha}–induced IL-6 mRNA expression and protein secretion by reducing IL-6 mRNA stability, rather than inhibiting IL-6 transcriptional regulation. To examine the time-course of induction of IL-6 by TNF-{alpha} and repression by dexamethasone, growth-arrested airway smooth muscle (ASM) cells pretreated for 1 hour with vehicle (open bars) or 100 nM dexamethasone (solid bars) were stimulated with TNF-{alpha} (10 ng/ml) for indicated times. (A) IL-6 mRNA expression was quantified by real-time RT-PCR (results expressed as % increase over 0 h) and (B) secreted IL-6 protein (pg/ml) measured by enzyme-linked immunosorbent assay (ELISA). Statistical analysis was performed using the Student's unpaired t test (where * denotes significant inhibition [P < 0.05]). Data are mean + SEM values from (A) 4 to 6 replicates or (B) 6 to 10 replicates. To measure the effect of dexamethasone on IL-6 promoter activity, ASM transfected with pIL-6-luc 651 were growth-arrested and then incubated for 1 hour with the indicated concentrations of dexamethasone, then either (C) left without further treatment for 24 hours to examine the effect on the GRE in the IL-6 promoter, or (D) stimulated with 10 ng/ml TNF-{alpha} for 24 hours to examine the repressive effects on non-GRE transcriptional elements. Cells were then harvested and luciferase and β-galactosidase activities assessed. Data represent normalized luciferase activity, relative to vehicle-treated cells (expressed as fold difference). Statistical analysis was performed using the Student's unpaired t test (where * denotes a significant effect of TNF-{alpha} on luciferase activity [P < 0.05]). Data are mean + SEM values from 11 to 18 replicates. To determine whether dexamethasone inhibits TNF-{alpha}–induced IL-6 secretion by reducing IL-6 mRNA stability, growth-arrested ASM cells pretreated for 1 hour with vehicle (open squares) or 100 nM dexamethasone (solid squares) were stimulated with TNF-{alpha} (10 ng/ml) for 9 hours. Cells were then washed and incubated with actinomycin D (5 µg/ml) to inhibit further transcription. Total RNA was extracted after 0, 0.5, 1, 2, 3, 6, 9, and 12 hours, and IL-6 mRNA expression quantified. Results are expressed as % mRNA remaining over time (where the dashed lines represent nonlinear regression of the % mRNA remaining versus time after actinomycin D). Data are mean + SEM (TNF-{alpha}) or mean – SEM (dexamethasone + TNF-{alpha}) values from n = 9 primary ASM cell lines.

 
We have previously shown that TNF-{alpha}–induced IL-6 secretion from ASM cells occurs via an NF-{kappa}B–mediated pathway (9), but that dexamethasone cannot fully prevent cytokine-mediated NF-{kappa}B DNA binding or NF-{kappa}B–mediated reporter activity (12, 13). Despite this, we have observed inhibition of TNF-{alpha}–induced IL-6 secretion by dexamethasone (9). One possible explanation for the NF-{kappa}B–independent anti-inflammatory effects of dexamethasone is via repression of a classic (negative) GRE binding sequence in the IL-6 promoter. In order to examine this possibility we transiently transfected cells with pIL-6-luc 651, a pGL3 luciferase reporter vector containing a 651-bp fragment of the human IL-6 gene promoter, including, among other consensus sequences, two cis-acting binding elements for GRE at positions –551 and –460 relative to the start site (17, 18). As shown in Figure 1C, dexamethasone had no significant effect on the luciferase activity of the IL-6 promoter construct compared with vehicle-treated control cells, suggesting that repression via negative GRE is not the underlying anti-inflammatory mechanism. To confirm earlier results (12, 13), and to specifically exclude repression of the IL-6 promoter by dexamethasone via the NF-{kappa}B–mediated pathway (9), cells transfected with pIL-6-luc 651 were pretreated with dexamethasone, or vehicle, before stimulation with TNF-{alpha}. In agreement with our previous studies (9, 15), TNF-{alpha} significantly increased IL-6 promoter activity by 3.3 ± 0.7-fold (Figure 1D) (P < 0.05). Importantly, dexamethasone did not inhibit TNF-{alpha}–induced IL-6 promoter activity. As shown in Figure 1D, the IL-6 promoter activity induced by TNF-{alpha} pretreated with either 0.01 µM or 1 µM dexamethasone was 3.7 ± 0.7-fold and 4.1 ± 1.2-fold, respectively, and these luciferase activities did not significantly differ from those induced by TNF-{alpha} alone (P > 0.05). Collectively, our results suggest that dexamethasone does not exert a repressive effect on IL-6 gene expression at the transcriptional level; however, it is important to note the possibility that the low reporter activity of the pIL-6-luc 651 construct may not mimic the real promoter and that lack of repression by dexamethasone may in part be due to a lack of overall inducibility of the construct. However, our assertion that dexamethasone does not affect IL-6 gene transcription is further supported by the data showing no effect of the corticosteroid on IL-6 mRNA expression at early time points (1–4 h; Figure 1A).

Because corticosteroids do not appear to act via repressive mechanisms to reduce IL-6 gene transcription, we next examined whether dexamethasone acts at the post-transcriptional level to reduce the stability of TNF-{alpha}–induced IL-6 mRNA transcript. Growth-arrested ASM cells were pretreated (for 1 h) with dexamethasone before stimulation with TNF-{alpha} for 9 hours. Transcription was then halted using actinomycin D, and real-time RT-PCR used to measure IL-6 mRNA degradation over time to determine the kinetics of decay. As shown in Figure 1E, corticosteroids reduce IL-6 mRNA stability. TNF-{alpha}–induced IL-6 mRNA decays at a significantly faster rate in ASM cells pretreated with the corticosteroid dexamethasone (–0.2860 ± 0.0747, t1/2 = 2.4 h), compared with vehicle-treated cells (–0.0773 ± 0.0290 t1/2 = 9.0 h) (P < 0.05). Taken together, these results suggest that dexamethasone inhibits TNF-{alpha}–induced IL-6 mRNA expression and protein secretion from ASM by reducing IL-6 mRNA stability, and suggest that TNF-{alpha}–induced IL-6 gene expression is regulated via a corticosteroid-sensitive post-transcriptional pathway.

Corticosteroids Up-Regulate MKP-1
In cell types apart from ASM it has been known for some time that corticosteroids up-regulate MKP-1, an early response gene and a dual-specificity phosphatase that inhibits MAPK superfamily members in a substrate- and cell type–specific manner (reviewed in Ref. 25). In a recent publication, Issa and colleagues (21) were the first to demonstrate dexamethasone-induced up-regulation of MKP-1 expression in ASM cells, and showed that corticosteroid inhibition of the inflammatory chemokine, growth-related oncogene protein-{alpha} (GRO-{alpha}), was partly dependent on MKP-1 induction. To further support the assertion that the anti-inflammatory action of corticosteroids on ASM synthetic function occurs, in part, via up-regulation of MKP-1, we treated ASM cells for up to 24 hours with dexamethasone, or the clinically relevant corticosteroid, fluticasone propionate. Up-regulation of MKP-1 was determined by Western blotting. As shown in Figure 2, corticosteroids rapidly and robustly induce the endogenous MAPK inhibitor MKP-1. Both dexamethasone and fluticasone propionate produced a significant induction in MKP-1 protein after 2 hours that was sustained for 24 hours (dexamethasone, 44.6 ± 10.5-fold increase at 24 h [Figures 2A and 2B]; fluticasone propionate, 47.9 ± 10.6-fold increase at 24 h [Figures 2C and 2D]) (P < 0.05).


Figure 2
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  		Figure 2. Corticosteroids up-regulate mitogen-activated protein kinase (MAPK) phosphatase-1 (MKP-1). ASM cells were pretreated with either (A, B) 100 nM dexamethasone or (C, D) 1 nM fluticasone propionate for the indicated times, or vehicle for 24 hours. MKP-1 expression was quantified by Western blotting, using {alpha}-tubulin as the loading control. A and C illustrate representative Western blots (top band is MKP-1), while B and D demonstrate densitometric analysis (mean + SEM values from n = 3 primary ASM cell lines). Statistical analysis was performed using one-way ANOVA, followed by Fisher's post hoc multiple comparison test (where * denotes a significant effect of corticosteroids on MKP-1 up-regulation, compared with 0 h [P < 0.05]).

 
Effect of Corticosteroid-Induced MKP-1 on MAPK Phosphorylation
MKP-1 dephosphorylates members of the MAPK superfamily (p38 MAPK, p42/p44 MAPK [ERK], and JNK) in a cell type–specific manner. In order to examine the specificity of phosphatase action of MKP-1 against MAP kinases in ASM cells, we investigated the action of dexamethasone on the kinetics of TNF-{alpha}–induced MAPK phosphorylation.

In corroboration of earlier work (21, 26), we show that TNF-{alpha} induces phosphorylation of MAPK phosphoproteins in a temporally distinct pattern. As demonstrated in Figure 3A, TNF-{alpha} induced a rapid and sustained activation of p38 MAPK; p38 MAPK phosphorylation occurred within 5 minutes, achieved peak phosphorylation by 10 minutes, and had not returned to unstimulated levels by 60 minutes. In contrast, the temporal pattern of ERK and JNK activation was comparatively transient. The peak of ERK and JNK phosphorylation occurred at 10 minutes, but by 60 minutes after stimulation with TNF-{alpha}, levels of phospho-ERK and phospho-JNK had returned to baseline. Total p38 MAPK, ERK, and JNK were included for comparison (Figure 3A), as well as {alpha}-tubulin to demonstrate equal protein loading.


Figure 3
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  		Figure 3. Effect of corticosteroid-induced MKP-1 on MAPK phosphorylation. Growth-arrested ASM pretreated for 1 hour with vehicle or 100 nM dexamethasone were stimulated with TNF-{alpha} (10 ng/ml) for 0, 5, 10, 30, and 60 minutes. Cells were lysed then analyzed by Western blotting using specific antibodies against phosphorylated (Thr180/Tyr182) and total p38 MAPK, phosphorylated (Thr202/Tyr204) and total extracellular signal–regulated kinase (ERK), phosphorylated (Thr183/Tyr185) and total c-Jun N-terminal kinase (JNK), and MKP-1 (top band). {alpha}-tubulin is used as the loading control. A illustrates a representative Western blot, while B demonstrates densitometric analysis of dexamethasone-mediated inhibition of TNF-{alpha}–induced p38 MAPK phosphorylation at 60 minutes (mean + SEM values from n = 15 primary ASM cell lines). Statistical analysis was performed using the Student's unpaired t test (where * denotes a significant inhibition on TNF-{alpha}–induced p38 MAPK phosphorylation [P < 0.05]).

 
When ASM cells were pretreated for 1 hour with dexamethasone before TNF-{alpha} stimulation, up-regulated MKP-1 was evident at 30 and 60 minutes after TNF-{alpha} stimulation (Figure 3A). Importantly, the increase in MKP-1 after corticosteroid treatment appeared to mirror the decrease in TNF-{alpha}–induced p38 MAPK phosphorylation at 30 and 60 minutes, suggesting that dexamethasone-induced MKP-1 shortens the temporal phosphorylation of p38 MAPK in response to TNF-{alpha}. This inhibition was quantified by densitometric analysis, as shown in Figure 3B, where dexamethasone pretreatment significantly inhibited TNF-{alpha}–induced p38 MAPK phosphorylation at 60 minutes in ASM (P < 0.05). Furthermore, JNK phosphorylation after dexamethasone pretreatment at 60 minutes was inhibited, while the kinetics of TNF-{alpha}–induced ERK phosphorylation appeared unaffected by corticosteroid-induced MKP-1 up-regulation. These results support those obtained by Issa and coworkers (21), where pretreatment with dexamethasone inhibited TNF-{alpha}–induced JNK phosphorylation. The effect of dexamethasone on TNF-{alpha}–induced p38 MAPK and ERK phosphorylation was not examined in their study (21).

It is of interest to note that TNF-{alpha} alone can also induce up-regulation of MKP-1, although amount of MKP-1 up-regulation is at a lower level and transient (data not shown) when compared with the sustained and robust up-regulation of MKP-1 achieved by corticosteroid pretreatment. Issa and colleagues demonstrated similar results with IL-1β as well as TNF-{alpha} (21). This is evidence of a negative feedback mechanism, whereby inflammatory stimuli activate phosphoproteins, as well as their own phosphatases, in order to regulate signal transduction.

Effect of MAPK Inhibition on TNF-{alpha}–Induced IL-6 Secretion
As shown in Figure 3A, TNF-{alpha} activated all members of the MAPK superfamily and both p38 MAPK and JNK phosphorylation were inhibited by corticosteroid pretreatment. Therefore, to examine which MAPK signaling pathway mediated TNF-{alpha}–induced IL-6 secretion from ASM cells, we pretreated ASM cells with inhibitors of the ERK, JNK, and p38 MAPK pathways, before stimulation with TNF-{alpha}, using PD98059, SP600125, and SB203580, respectively. Secreted IL-6 was measured by ELISA. As shown in Figure 4, PD98059 and SP600125 did not significantly inhibit TNF-{alpha}–induced IL-6 secretion (P > 0.05), suggesting that although TNF-{alpha} induces ERK and JNK phosphorylation, and that dexamethasone inhibits TNF-{alpha}–induced JNK phosphorylation (as demonstrated in Figure 3A), TNF-{alpha}–induced IL-6 secretion in ASM cells was independent of the ERK and JNK pathways. In contrast, inhibition of the p38 MAPK pathway by SB203580 significantly reduced IL-6 secretion in response to TNF-{alpha} (TNF-{alpha}, 4,996.3 ± 693.3 pg/ml; SB203580 + TNF-{alpha}, 2,439.7 ± 333.2 pg/ml) (P < 0.05), and notably, the level of IL-6 protein secretion after inhibition of the p38 MAPK pathway was not significantly different to that obtained from ASM cells pretreated with the corticosteroid dexamethasone (dexamethasone + TNF-{alpha}, 1,759.9 ± 236.8 pg/ml) (data not shown).


Figure 4
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  		Figure 4. Effect of MAPK inhibition on TNF-{alpha}–induced IL-6 secretion. Growth-arrested ASM were pretreated for 30 minutes with vehicle, or 10 µM PD98059, 10 µM SP600125, or 1 µM SB203580 (to inhibit ERK, JNK, or p38 MAPK, respectively), then treated with vehicle or 10 ng/ml TNF-{alpha} for 24 hours. IL-6 secretion (pg/ml) was measured by ELISA. Statistical analysis was performed using the Student's unpaired t test (where * denotes a significant inhibition of TNF-{alpha}–induced IL-6 secretion [P < 0.05]). Data are mean + SEM values from 14 to 28 replicates.

 
Inhibition of p38 MAPK Does Not Attenuate IL-6 Transcriptional Regulation, but TNF-{alpha} Acts via a p38 MAPK–Dependent Pathway to Stabilize the IL-6 mRNA Transcript
In Figures 5A and 5B we demonstrate the time-course of repression of IL-6 mRNA expression and protein secretion by the p38 MAPK inhibitor SB203580. Both TNF-{alpha}–induced IL-6 mRNA expression and protein secretion are significantly inhibited by SB203580 at 24 hours (P < 0.05). Data presented in Figures 4 and 5B corroborate our earlier findings (8) and confirm that 1 µM SB203580 only partially represses IL-6 protein secretion. Therefore, as inhibition of the p38 MAPK pathway attenuates TNF-{alpha}–induced IL-6 mRNA expression and protein secretion, we now wished to examine whether this inhibition occurred at the transcriptional or post-transcriptional level.


Figure 5
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  		Figure 5. Inhibition of p38 MAPK does not attenuate IL-6 transcriptional regulation, but TNF-{alpha} acts via a p38 MAPK–dependent pathway to stabilize the IL-6 mRNA transcript. To examine the time-course of repression of IL-6 mRNA expression and protein secretion by the p38 MAPK inhibitor SB203580, growth-arrested ASM cells pretreated for 30 minutes with vehicle (open bars) or 1 µM SB203580 (solid bars) were stimulated with TNF-{alpha} (10 ng/ml) for indicated times. (A) IL-6 mRNA expression was quantified by real-time RT-PCR (results expressed as % increase over 0 h) and (B) secreted IL-6 protein (pg/ml) measured by ELISA. Statistical analysis was performed using the Student's unpaired t test (where * denotes significant inhibition [P < 0.05]). Data are mean + SEM values from (A) 4 to 6 replicates or (B) 6 to 10 replicates. (C) ASM cells transfected with pIL-6-luc 651 were incubated for 30 minutes with vehicle, the p38 MAPK inhibitor SB203580, or the inactive congener, SB202474 (both at 1 µM and 10 µM), then treated with vehicle or 10 ng/ml TNF-{alpha} for 24 hours. Cells were then harvested and luciferase and β-galactosidase activities assessed. Data represent normalized luciferase activity, relative to vehicle-treated cells (expressed as fold difference). Statistical analysis was performed using the Student's unpaired t test (where * denotes significant effect of TNF-{alpha} on luciferase activity [P < 0.05]). Data are mean + SEM values from 7 to 18 replicates. (D) Growth-arrested ASM cells pretreated for 30 minutes with vehicle (open squares) or 1 µM SB203580 (solid squares) were stimulated with TNF-{alpha} (both at 10 ng/ml) for 9 hours. Stability of IL-6 mRNA transcripts was measured by an actinomycin D time course using real-time RT-PCR. Results are expressed as % mRNA remaining over time (where the dashed lines represent nonlinear regression of the % mRNA remaining versus time after actinomycin D). Data are mean + SEM (TNF{alpha}) or mean – SEM (dexamethasone + TNF-{alpha}) values from n = 15 primary ASM cell lines.

 
In order to examine the transcriptional regulation of IL-6 gene expression, ASM cells transfected with pIL-6-luc 651 were incubated with the p38 MAPK inhibitor SB203580, or the inactive congener, SB202474, before stimulation with TNF-{alpha}. TNF-{alpha}–induced IL-6 promoter activity was not significantly affected by SB203580 (at either 1 or 10 µM), as shown in Figure 5C. Furthermore, the TNF-{alpha}–induced IL-6 promoter activity in cells pretreated with 1 µM SB203580 (4.7 ± 1.8-fold) was not significantly different from cells pretreated with an equivalent concentration of negative control SB202474 (4.0 ± 1.0-fold) (Figure 5C). The lack of inhibitory effect of SB203580 on the IL-6 luciferase promoter was supported by the demonstration that SB203580 did not reduce TNF-{alpha}–induced IL-6 mRNA at all times before 24 h (Figure 5A).

In order to assess the effect of p38 MAPK inhibition on IL-6 mRNA stability, we measured the decay kinetics in ASM cells from a total of 15 subjects and discovered that the decay of TNF-{alpha}–induced IL-6 mRNA transcripts proceeded at a significantly faster rate when cells were pretreated with the p38 MAPK inhibitor SB203580 (–0.4750 ± 0.0782, t1/2 = 1.5 h), compared with vehicle (–0.0722 ± 0.0199, t1/2 = 9.6 h) (P < 0.05; Figure 5D). Taken together, our data indicate that p38 MAPK signaling does not regulate IL-6 transcription. Rather, TNF-{alpha} acts via a p38 MAPK-dependent pathway to stabilize the IL-6 mRNA transcript.

Exogenous Expression of MKP-1 Inhibits TNF-{alpha}-Induced IL-6 Secretion
In order to confirm that corticosteroids act via MKP-1–mediated inhibition of p38 MAPK signaling, we transfected ASM cells using Lipofectamine 2000 with an MKP-1 expression vector, compared with vector alone, and measured the resultant effect on TNF-{alpha}–induced IL-6 secretion. As shown in Figure 6A, immunoblotting confirmed that MKP-1 protein levels were increased in ASM cells transfected with the MKP-1 expression vector, compared with the empty vector control. As shown in Figure 6B, exogenous expression of MKP-1 significantly inhibited TNF-{alpha}–induced IL-6 secretion from ASM, when compared with cells transfected with vector alone before TNF-{alpha} stimulation (P < 0.05). The percentage inhibition of TNF-{alpha}–induced IL-6 secretion achieved was 15.9 ± 5.3% (Figure 6B), which is line with the transfection efficiencies of approximately 10%, estimated by β-galactosidase reporter gene assay (as detailed in MATERIALS AND METHODS). Furthermore, the amount of IL-6 secreted by nontransfected cells stimulated for 24 hours with TNF-{alpha} (11.8 ± 2.6 ng/ml; Figure 1B) was not statistically different to the levels achieved in cells transfected with vector alone (14.4 ± 2.5 ng/ml) (P < 0.05); excluding the possibility that there was an overt effect of the plasmid on TNF-{alpha}–induced IL-6 secretion.


Figure 6
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  		Figure 6. Exogenous expression of MKP-1 inhibits TNF-{alpha}–induced IL-6 secretion. ASM cells transfected with a MKP-1 expression vector, or empty vector control, were stimulated for 24 hours with 10 ng/ml TNF-{alpha}. (A) To confirm exogenous expression of MKP-1, cells were lysed and immunoblotted for MKP-1 (top band is MKP-1) using {alpha}-tubulin as the loading control. Data are representative results of n = 7 primary ASM cell lines. (B) To measure the effect of exogenous expression of MKP-1 on TNF-{alpha}–induced IL-6, supernatants were removed and IL-6 protein measured by ELISA. Data are expressed as % TNF-{alpha}–induced IL-6 secretion in cell transfected with empty vector alone, and statistical analysis was performed using the Student's unpaired t test (where * denotes a significant inhibition of TNF-{alpha}–induced IL-6 secretion [P < 0.05]). Data are mean + SEM values from 20 replicates.

 
MKP-1 siRNA Reverses the Inhibition of TNF-{alpha}-Induced IL-6 Secretion by Dexamethasone
To further support our assertion that the anti-inflammatory action of corticosteroids on ASM IL-6 secretion occurs via up-regulation of MKP-1, we reduced the levels of MKP-1 protein using MKP-1 siRNA and examined the secretion of IL-6 in response to dexamethasone and TNF-{alpha}. In order to increase transfection efficiency in ASM cells (being a relatively hard-to-transfect primary cell), we used nucleofection to allow transfected DNA to directly enter the nucleus with enhanced efficiency. As detailed in MATERIALS AND METHODS, we achieved a transfection efficiency of 58%, which was in line with the manufacturer's recommendation of 63% for ASM cells (Amaxa).

As shown in Figures 7A and 7B, when ASM cells transiently transfected with scrambled siRNA control were treated with dexamethasone and TNF-{alpha}, we observed a significant enhancement in MKP-1 protein levels to 3.7 ± 0.7-fold (P < 0.05), compared with cells treated with TNF-{alpha} alone. This MKP-1 protein up-regulation was significantly reduced by MKP-1 siRNA; MKP-1 siRNA-transfected cells treated with dexamethasone and TNF-{alpha} showed a significant 61.9 ± 4.2% reduction in MKP-1 protein levels, when compared with cells transfected with scrambled control (Figure 7B; P < 0.05). We then examined the effect of MKP-1 siRNA on the inhibition of IL-6 secretion by corticosteroids. Cell supernatants were assayed for IL-6 by ELISA and results expressed as a percentage of IL-6 secreted by cells transfected with control siRNA and treated with TNF-{alpha} alone (Figure 7C). Notably, dexamethasone-mediated inhibition could be significantly reversed by transfection with MKP-1 siRNA. Supernatants from cells transfected with MKP-1 siRNA and treated with dexamethasone and TNF-{alpha} showed significantly increased IL-6 secretion (60.2 ± 8.4%), compared with IL-6 in ASM cells transfected with scrambled control (39.3 ± 2.8%) (P < 0.05). Although the degree of reversal was partial (due to the transfection efficiency and the contribution of other steroid-inducible genes to IL-6 repression), we show that MKP-1 siRNA reduced protein levels of MKP-1 in ASM cells and reversed the inhibition of TNF-{alpha}–induced IL-6 secretion by dexamethasone. Taken together, these results suggest that corticosteroid-induced MKP-1 contributes to the repression of IL-6 secretion in ASM cells.


Figure 7
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  		Figure 7. MKP-1 siRNA reduces protein levels of MKP-1 in ASM cells and reverses the inhibition of TNF-{alpha}–induced IL-6 secretion by dexamethasone. ASM cells were transiently transfected using nucleofection with scrambled siRNA (scr) or MKP-1 siRNA (siRNA), then pretreated for 1 hour with vehicle or 100 nM dexamethasone, before stimulation for 24 hours with TNF-{alpha} (10 ng/ml). (A) To demonstrate that MKP-1 siRNA reduces protein levels of MKP-1, cells were lysed and immunoblotted for MKP-1, using {alpha}-tubulin as the loading control. A illustrates a representative Western blot, while B demonstrates densitometric analysis of MKP-1 protein levels (mean + SEM values from n = 5 primary ASM cell lines) expressed as fold difference compared with cells transfected with scrambled siRNA treated with TNF-{alpha}. Statistical analysis was performed using the Student's unpaired t test (where * denotes a significant inhibition of MKP-1 protein by DEX + TNF-{alpha} by MKP-1 siRNA [P < 0.05]). (C) To confirm that MKP-1 siRNA reverses the inhibition of TNF-{alpha}–induced IL-6 secretion by dexamethasone, supernatants were removed and IL-6 protein measured by ELISA. Data are expressed as % TNF-{alpha}–induced IL-6 secretion and statistical analysis was performed using the Student's unpaired t test (where * denotes a significant reversal of dexamethasone-inhibited TNF-{alpha}–induced IL-6 secretion by MKP-1 siRNA [P < 0.05]). Data are mean + SEM values from n = 5 primary ASM cell lines.

 

   DISCUSSION
Top
 Abstract
 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
References
 
With this study, we increase our understanding of how corticosteroids reduce inflammation in human ASM, a pivotal cytokine-producing cell in the airway. As corticosteroids do not appear to act at the transcriptional level to reduce IL-6 gene expression, we focused on understanding cytokine message turnover, because after the gene for IL-6 is transcribed, the stability of their mRNA ultimately controls the amount of protein secreted. We discovered that IL-6 mRNA stability is regulated negatively by corticosteroids. Interestingly, the suppressive action of corticosteroids on ASM IL-6 secretion is via inhibition of p38 MAPK, a pro-inflammatory signaling pathway stimulated by TNF-{alpha}. This occurs via a steroid-induced up-regulation of MKP-1, an endogenous inhibitor of MAP kinases.

Corticosteroids are the mainstay of asthma pharmacotherapy. Although the precise mechanisms by which they reduce airway inflammation are not completely understood, insights into the anti-inflammatory signaling pathways have emerged (reviewed in Ref. 27). Corticosteroids can act in a transcriptional manner, initiated by activating the cytosolic glucocorticoid receptor (GR). Activated GR undergoes nuclear translocation where it is thought to either act directly by binding to the GRE of the 5' promoter region, or indirectly by binding to other transcription factors, to inhibit cytokine gene transcription. However, negative GREs (i.e. GREs that act to inhibit inflammatory gene transcription) have not been shown to exist (28), and the complete inhibition of transcription factors such as NF-{kappa}B by corticosteroids does not occur in ASM cells (12, 13). Furthermore, we have shown that corticosteroids do not act predominantly via repressive mechanisms on the IL-6 promoter to reduce IL-6 gene expression in ASM cells.

The anti-inflammatory actions of corticosteroids can also be mediated via the induction of a number of anti-inflammatory proteins (reviewed in Ref. 29), including MKP-1 (reviewed in Refs. 25 and 27). In this study, we show that dexamethasone, and the clinically relevant corticosteroid fluticasone propionate, rapidly up-regulate MKP-1 in a time-dependent manner, supporting the recent publication reporting MKP-1 expression in ASM cells (21). MKP-1 is an early response gene and a phosphatase that inhibits MAPK family members in a substrate- and cell type–specific manner. In this way, corticosteroids can affect the myriad signaling functions controlled by MAP kinases, including post-transcriptional regulation of gene expression.

The expression of many inflammatory genes implicated in asthma and airway remodeling are controlled by post-transcriptional mechanisms (reviewed in Ref. 30). The 3'-untranslated region (3'-UTR) of the IL-6 mRNA transcript contains multiple adenylate + uridylate-rich elements (ARE) (GeneBank accession number BC015511); cis-acting motifs known to be associated with regulation of mRNA stability. After genes are transcribed, the stability of their mRNA ultimately controls the amount of protein produced; thus, post-transcriptional regulation is a critical point in gene expression. To date, however, most studies have focused on the transcriptional regulation of genes, leaving post-transcriptional mechanisms, such as control of mRNA stability, largely unexplored. Our recent work (15, 20), and that of others (31–33), has clearly shown that mRNA stability is an important regulatory mechanism in the control of gene expression in human ASM. In this study we continue our investigations into the regulation of mRNA decay and discover that corticosteroids act to reduce TNF-{alpha}–induced IL-6 mRNA stability at the post-transcriptional level. This work supports earlier studies in ASM cells demonstrating an effect of corticosteroids on the decay of other clinically relevant genes that contain cis-acting ARE motifs in the 3'-UTR of their mRNA transcripts, such as cyclo-oxygenase 2 (COX-2) (31) and granulocyte macrophage-colony stimulating factor (GM-CSF) (32). We confirmed that TNF-{alpha} activates all members of the MAPK superfamily in a temporally specific manner (21, 26); however, pharmacological inhibition revealed that TNF-{alpha}–induced IL-6 secretion is mediated via a p38 MAPK-dependent pathway. Extracellular stimulation by TNF-{alpha}, in addition to stimulating NF-{kappa}B–mediated gene transcription, also antagonized ARE-dependent mRNA decay via activation of p38 MAPK. Activated p38 MAPK signal transduction pathways may modulate ARE-directed mRNA stability by affecting RNA-binding proteins directly or indirectly to stabilize ARE-containing transcripts (reviewed in Ref. 30). Interestingly, the suppressive action of corticosteroids in ASM occurs, in part, via steroid-induced up-regulation of MKP-1, an endogenous inhibitor of MAP kinases, including p38 MAPK. These studies provide further evidence to address the important question of how corticosteroids work in ASM cells (21); and are in line with investigations in cell types apart from ASM, where MKP-1 acts to reduce inflammation by attenuating p38 MAPK-mediated pathways (reviewed in Ref. 34), such as post-transcriptional regulation (19). Moreover, earlier studies in ASM cells demonstrate that the induction of COX-2 (31) and GM-CSF (32) is also p38 MAPK mediated, suggesting the intriguing possibility that the underlying mechanism of corticosteroid action in these studies (31, 32) also occurs via up-regulation of the anti-inflammatory protein MKP-1. Although our study has focused on the transcriptional and post-transcriptional regulation of IL-6 mRNA, it is also important to note that p38 MAPK could also be playing a role in the translation of IL-6 mRNA (in addition to the effect on mRNA destabilization), as p38 MAPK is known to phosphorylate a number of important translation factor proteins responsible for regulating the translational machinery of mammalian cells (reviewed in Ref. 35).

However, although accumulating evidence, including data presented in this publication, implicates the p38 MAPK pathway as critical in modulating the inflammatory response, it is important to note that, in cell types apart from ASM, other MAPK family members such as ERK (36) and JNK (37) have also been shown to be regulated by MKP-1, indicating that this phosphatase has an increasingly important and complex role in regulating MAP kinase activity that is not limited to the p38 MAPK pathway. In corroboration, the recent publication by Issa and colleagues (21) demonstrated that the suppressive action of corticosteroids in ASM cells occur, in part, via inhibition of JNK phosphorylation due to steroid-induced up-regulation of MKP-1. Important stimulus-dependent differences in MAPK signaling pathways exist in ASM cells, and because of this, differences will exist in the extent of regulation of cytokine secretion achieved by dexamethasone-mediated MKP-1. Issa and coworkers have demonstrated that TNF-{alpha}– and IL-1β–mediated GRO-{alpha} is JNK mediated (38), thus corticosteroids repress GRO-{alpha} secretion via MKP-1–mediated inhibition of JNK phosphorylation (21). In our study, we determined that TNF-{alpha}–induced IL-6 secretion was p38 MAPK mediated, and although JNK phosphorylation was inhibited by dexamethasone, IL-6 secretion in ASM cells was independent of the JNK pathway. Collectively, these studies serve to underscore the increasingly important and complex role that this phosphatase has in regulating MAP kinase activity.

The dual-specificity phosphatase MKP-1 (also known as DUSP1) has recently been generating much interest as a novel anti-inflammatory target for the regulation of inflammatory disease (reviewed in Refs. 39 and 40), as many immune responses are critically dependent on MAPK signaling pathways. While corticosteroids continue to provide a widely effective means of controlling inflammation in patients with asthma, clinicians and patients will continue to be challenged by the adverse effects and lack of universal efficacy of these drugs. Targeting molecular control devices, such as MKP-1, or other members of the DUSP family, may provide an alternative basis for the development of drug therapies for debilitating diseases in which innate and adaptive immune functions are perturbed. Drugs targeting MKP-1 levels and activity may not just provide an alternative to corticosteroids in nonresponsive or adverse effect–prone patient groups in asthma; they may also provide a corticosteroid-sparing adjunct to these medications.

Together, our results suggest that the anti-inflammatory action of corticosteroids on ASM synthetic function occurs via up-regulation of MKP-1 and inhibition of p38 MAPK–mediated mRNA stability. Collectively, these results demonstrate that mRNA stability is an important regulatory mechanism in the control of cytokine gene expression in ASM; that the anti-inflammatory effects of corticosteroids occur at the post-transcriptional level; and that phosphatases, such as MKP-1, represent attractive pharmacological targets to control inflammatory diseases such as asthma and airway remodeling.


   Acknowledgments
 
The authors thank Dr. Oliver Eickelberg (Giessen University School of Medicine, Giessen, Germany) for kindly providing the IL-6 promoter constructs; Dr. Shigeru Katamine (Nagasaki University, Nagasaki, Japan) for permission to use the IL-6 promoter constructs; Dr. Andrew R. Clark (Kennedy Institute of Rheumatology Division, Imperial College London, London, UK) for the MKP-1 expression vector; Sandra Chesoni and Prof. Gary Brewer (Robert Wood Johnson Medical School, University of Medicine and Dentistry, NJ) for helpful discussions regarding analysis of mRNA decay kinetics; and their colleagues in the Respiratory Research Group. The authors acknowledge the collaborative effort of the cardiopulmonary transplant team and the pathologists at St Vincent's Hospital, Sydney, and the thoracic physicians and pathologists at Royal Prince Alfred Hospital, Concord Repatriation Hospital and Strathfield Private Hospital and Rhodes Pathology, Sydney.


   Footnotes
 
This work was funded by the Australian Lung Foundation, Asthma Foundation of New South Wales, and the National Health and Medical Research Council.

Originally Published in Press as DOI: 10.1165/rcmb.2007-0014OC on February 28, 2008

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form January 17, 2007

Accepted in final form January 10, 2008


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 CLINICAL RELEVANCE
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 References
 

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Proc. Am. Thorac. Soc. Am. J. Respir. Crit. Care Med.
Copyright © 2008 American Thoracic Society.