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

<p>(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 (<i>P</i><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 <i>t</i> test where * denotes a significant increase in mRNA expression or secretion (<i>P</i><0.05).</p

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

<p>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 (<i>P</i><0.05)).</p

Formoterol increases fluticasone-induced MKP-1 protein upregulation.

<p>(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 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059635#pone-0059635-g001" target="_blank">Figure 1B</a>) 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 (<i>P</i><0.05)).</p

Salmeterol increases fluticasone-induced MKP-1 protein upregulation.

<p>(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 (<i>P</i><0.05)).</p

Effect of MKP-1 siRNA.

<p>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).</p

Inhibition of Mitogen-Activated Protein Kinase Erk1/2 Promotes Protein Degradation of ATP Binding Cassette Transporters A1 and G1 in CHO and in HuH7 cells.

Signal transduction modulates expression and activity of cholesterol transporters. We recently demonstrated that the Ras/mitogen-activated protein kinase (MAPK) signaling cascade regulates protein stability of Scavenger Receptor BI (SR-BI) through Proliferator Activator Receptor (PPARα) -dependent degradation pathways. In addition, MAPK (Mek/Erk 1/2) inhibition has been shown to influence liver X receptor (LXR) -inducible ATP Binding Cassette (ABC) transporter ABCA1 expression in macrophages. Here we investigated if Ras/MAPK signaling could alter expression and activity of ABCA1 and ABCG1 in steroidogenic and hepatic cell lines. We demonstrate that in Chinese Hamster Ovary (CHO) cells and human hepatic HuH7 cells, extracellular signal-regulated kinase 1/2 (Erk1/2) inhibition reduces PPARα-inducible ABCA1 protein levels, while ectopic expression of constitutively active H-Ras, K-Ras and MAPK/Erk kinase 1 (Mek1) increases ABCA1 protein expression, respectively. Furthermore, Mek1/2 inhibitors reduce ABCG1 protein levels in ABCG1 overexpressing CHO cells (CHO-ABCG1) and human embryonic kidney 293 (HEK293) cells treated with LXR agonist. This correlates with Mek1/2 inhibition reducing ABCG1 cell surface expression and decreasing cholesterol efflux onto High Density Lipoproteins (HDL). Real Time reverse transcriptase polymerase chain reaction (RT-PCR) and protein turnover studies reveal that Mek1/2 inhibitors do not target transcriptional regulation of ABCA1 and ABCG1, but promote ABCA1 and ABCG1 protein degradation in HuH7 and CHO cells, respectively. In line with published data from mouse macrophages, blocking Mek1/2 activity upregulates ABCA1 and ABCG1 protein levels in human THP1 macrophages, indicating opposite roles for the Ras/MAPK pathway in the regulation of ABC transporter activity in macrophages compared to steroidogenic and hepatic cell types. In summary, this study suggests that Ras/MAPK signaling modulates PPARα- and LXR-dependent protein degradation pathways in a cell-specific manner to regulate the expression levels of ABCA1 and ABCG1 transporters

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

<p>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 (<i>P</i><0.05).</p

Inhibition of Mitogen-Activated Protein Kinase Erk1/2 Promotes Protein Degradation of ATP Binding Cassette Transporters A1 and G1 in CHO and in HuH7 cells.

Signal transduction modulates expression and activity of cholesterol transporters. We recently demonstrated that the Ras/mitogen-activated protein kinase (MAPK) signaling cascade regulates protein stability of Scavenger Receptor BI (SR-BI) through Proliferator Activator Receptor (PPARα) -dependent degradation pathways. In addition, MAPK (Mek/Erk 1/2) inhibition has been shown to influence liver X receptor (LXR) -inducible ATP Binding Cassette (ABC) transporter ABCA1 expression in macrophages. Here we investigated if Ras/MAPK signaling could alter expression and activity of ABCA1 and ABCG1 in steroidogenic and hepatic cell lines. We demonstrate that in Chinese Hamster Ovary (CHO) cells and human hepatic HuH7 cells, extracellular signal-regulated kinase 1/2 (Erk1/2) inhibition reduces PPARα-inducible ABCA1 protein levels, while ectopic expression of constitutively active H-Ras, K-Ras and MAPK/Erk kinase 1 (Mek1) increases ABCA1 protein expression, respectively. Furthermore, Mek1/2 inhibitors reduce ABCG1 protein levels in ABCG1 overexpressing CHO cells (CHO-ABCG1) and human embryonic kidney 293 (HEK293) cells treated with LXR agonist. This correlates with Mek1/2 inhibition reducing ABCG1 cell surface expression and decreasing cholesterol efflux onto High Density Lipoproteins (HDL). Real Time reverse transcriptase polymerase chain reaction (RT-PCR) and protein turnover studies reveal that Mek1/2 inhibitors do not target transcriptional regulation of ABCA1 and ABCG1, but promote ABCA1 and ABCG1 protein degradation in HuH7 and CHO cells, respectively. In line with published data from mouse macrophages, blocking Mek1/2 activity upregulates ABCA1 and ABCG1 protein levels in human THP1 macrophages, indicating opposite roles for the Ras/MAPK pathway in the regulation of ABC transporter activity in macrophages compared to steroidogenic and hepatic cell types. In summary, this study suggests that Ras/MAPK signaling modulates PPARα- and LXR-dependent protein degradation pathways in a cell-specific manner to regulate the expression levels of ABCA1 and ABCG1 transporters