Long-acting β2-agonists increase fluticasone propionate-induced mitogen-activated protein kinase phosphatase 1 (MKP-1) in airway smooth muscle cells

Abstract

Mitogen-activated protein kinase phosphatase 1 (MKP-1) represses MAPK-driven signalling and plays an important anti-inflammatory role in asthma and airway remodelling. Although MKP-1 is corticosteroid-responsive and increased by cAMP-mediated signalling, the upregulation of this critical anti-inflammatory protein by long-acting β2-agonists and clinically-used corticosteroids has been incompletely examined to date. To address this, we investigated MKP-1 gene expression and protein upregulation induced by two long-acting β2-agonists (salmeterol and formoterol), alone or in combination with the corticosteroid fluticasone propionate (abbreviated as fluticasone) in primary human airway smooth muscle (ASM) cells in vitro. β2-agonists increased MKP-1 protein in a rapid but transient manner, while fluticasone induced sustained upregulation. Together, long-acting β2-agonists increased fluticasone-induced MKP-1 and modulated ASM synthetic function (measured by interleukin 6 (IL-6) and interleukin 8 (IL-8) secretion). As IL-6 expression (like MKP-1) is cAMP/adenylate cyclase-mediated, the long-acting β2-agonist formoterol increased IL-6 mRNA expression and secretion. Nevertheless, when added in combination with fluticasone, β2-agonists significantly repressed IL-6 secretion induced by tumour necrosis factor α (TNFα). Conversely, as IL-8 is not cAMP-responsive, β2-agonists significantly inhibited TNFα-induced IL-8 in combination with fluticasone, where fluticasone alone was without repressive effect. In summary, long-acting β2-agonists increase fluticasone-induced MKP-1 in ASM cells and repress synthetic function of this immunomodulatory airway cell type.

Figure 1. Salmeterol increases fluticasone-induced MKP-1 protein upregulation.

(A) Growth-arrested ASM cells were treated with vehicle, salmeterol (100 nM), fluticasone (1 nM), salmeterol (100 nM)+fluticasone (1 nM), for 0, 0.5, 1, 2, 4, 8, and 24 h. MKP-1 protein (molecular weight:∼39 kDa) was quantified by Western blotting, using α-tubulin as the loading control (molecular weight: 55∼kDa), where (A) illustrates representative Western blots and (B) demonstrates densitometric analysis where results are expressed as fold increase over 0 h (mean+SEM values from n = 6–8 primary ASM cell cultures). Statistical analysis was performed using two-way ANOVA then Bonferroni's post-test (where * indicates a significant effect of salmeterol or fluticasone on MKP-1 protein upregulation, compared to vehicle-treated cells, and § indicates a significant effect of salmeterol on fluticasone-induced MKP-1 (P<0.05)).

Figure 2. Formoterol increases fluticasone-induced MKP-1 protein upregulation.

(A) Growth-arrested ASM cells were treated with vehicle, formoterol (10 nM), fluticasone (1 nM), formoterol (10 nM)+fluticasone (1 nM), for 0, 0.5, 1, 2, 4, 8, and 24 h. MKP-1 protein (molecular weight:∼39 kDa) was quantified by Western blotting, using α-tubulin as the loading control (molecular weight:∼55 kDa), where (A) illustrates representative Western blots and (B) demonstrates densitometric analysis where results are expressed as fold increase over 0 h and results for fluticasone (from Figure 1B) shown graphically for comparative purposes (mean+SEM values from n = 3–8 primary ASM cell cultures). Statistical analysis was performed using two-way ANOVA then Bonferroni's post-test (where * indicates a significant effect of formoterol or fluticasone on MKP-1 protein upregulation, compared to vehicle-treated cells, and § indicates a significant effect of formoterol on fluticasone-induced MKP-1 (P<0.05)).

Figure 3. Long-acting β₂-agonists increase fluticasone-induced MKP-1 mRNA expression.

Growth-arrested ASM cells were treated for 1 h with vehicle, fluticasone (1 nM), salmeterol (100 nM), formoterol (10 nM), salmeterol (100 nM)+fluticasone (1 nM), formoterol (10 nM)+fluticasone (1 nM). MKP-1 mRNA expression was quantified by real-time RT-PCR and results expressed as fold increase compared to vehicle-treated cells (mean+SEM values from n = 9 primary ASM cell cultures). Statistical analysis was performed using repeated measures ANOVA with Bonferroni’s multiple comparison test (where * denotes a significant increase in MKP-1 mRNA expression and § denotes a significant effect of the β₂-agonists on fluticasone-induced MKP-1 mRNA expression (P<0.05)).

Figure 4. Effect of long-acting β₂-agonists on IL-6 and IL-8 mRNA expression and secretion.

(A, B, C, D) Growth-arrested ASM cells were treated with vehicle, salmeterol (100 nM) or formoterol (10 nM). (A) IL-6 and (B) IL-8 mRNA expression was quantified by real-time RT-PCR at 1 h and results expressed as fold increase compared to vehicle-treated cells (mean+SEM values from n = 9 primary ASM cell cultures). (C) IL-6 and (D) IL-8 secretion was measured by ELISA at 24 h and results are expressed as fold increase compared to vehicle-treated cells (where IL-6 and IL-8 protein levels in cells treated with vehicle were 295.0±34.8 and 26.3±4.6 pg/ml, respectively (mean+SEM values from n = 6 primary ASM cell cultures)). Statistical analysis was performed using repeated measures ANOVA with Bonferroni’s multiple comparison test (where * denotes a significant increase in mRNA expression or secretion (P<0.05)). (E, F) Growth-arrested ASM cells were treated with vehicle, dibutyryl cAMP (1 mM), forskolin (10 µM) and (E) IL-6 and (F) IL-8 mRNA expression was quantified by real-time RT-PCR at 1 h. Results are expressed as fold increase compared to vehicle-treated cells (mean+SEM values from n = 3 primary ASM cell cultures). Statistical analysis was performed using the Student's unpaired t test where * denotes a significant increase in mRNA expression or secretion (P<0.05).

Figure 5. The effect of long-acting β₂-agonists and fluticasone, alone or in combination, on repression of TNFα-induced IL-6 and IL-8 secretion.

Growth-arrested ASM cells were pretreated for 1 h with vehicle, salmeterol (100 nM) or formoterol (10 nM), alone or in combination with fluticasone (1 nM), before stimulation for 24 h with TNFα (10 ng/ml). (A) IL-6 and (B) IL-8 secretion was measured by ELISA and results expressed as (A) % TNFα-induced IL-6 secretion or (B) % TNFα-induced IL-8 secretion (where TNFα-induced IL-6 and IL-8 protein secretion was 5153.9±618.9 and 5913.1±480.0 pg/ml, respectively (mean+SEM values from n = 6 primary ASM cell cultures)). Statistical analysis was performed using repeated measures ANOVA with Bonferroni’s multiple comparison test where * denotes a significant increase in IL-6 secretion and § denotes a significant repression of TNFα-induced cytokine secretion (P<0.05).

Figure 6. Effect of MKP-1 siRNA.

ASM cells were transiently transfected using nucleofection with scrambled control or MKP-1 siRNA, growth-arrested, then pretreated for 1 h with formoterol (10 nM)+fluticasone (1 nM) (along with relevant controls) before stimulation for 24 h with TNFα (10 ng/ml). Supernatants were then removed for IL-6 and IL-8 protein secretion by ELISA and lysates utilized for MKP-1 Western blotting (compared to α-tubulin as a loading control). (A) illustrates a representative Western blot, while (B, C) demonstrates results expressed as % TNFα-induced (B) IL-6 or (C) IL-8 secretion in cells transfected with scrambled control siRNA (mean+SEM values from n = 6 primary ASM cell cultures).