Mitogen Activated Protein Kinases and cAMP-dependent Kinases Mediate β 2 -Adrenoceptor Dependent Mucin MUC5AC Expression in Normal Human Bronchial Epithelial Cells in Response to IL-13.

N Al-Sawalha, I Pokkunuri, A Hernandez, RA Bond, BJ Knoll. University of Houston, Houston, TX, USA

Mucus hypersecretion by airway epithelium is a common feature in asthma, resulting in airflow obstruction. We have shown that chronic β2-adrenoceptors (β2ARs) inverse agonist treatment, or genetic ablation of β2ARs in m ice causes a reduction in mucus secretion, an index of inflammatory responses, in response to allergen or IL-13 instillation (Nguyen et al, 2009). The detailed molecular components of these effects are still to be elucidated.

Our objectives were to examine the role of mitogen activated protein kinases (MAPKs), and cAMP-dependent protein kinases in β2AR signaling pathways in enhancing the expression of MUC5AC, the predominant mucin-producing gene in airway epithelium, in response to IL-13 in cultured normal human bronchial epithelial cells (NHBECs).

NHBECs, obtained from Lonza (Walkersville, MD), were cultured on Transwell filter supports in a media composed of 1:1 mixture of bronchial epithelial basal media and Dulbecco′s Modified Eagle Medium, supplemented with growth factors from Lonza (Park et al, 2009) until they reached air-liquid interface (ALI). NHBECs were grown with epinephrine (3μM), with or without IL-13 (20 ng/ml, dissolved in water). Some cells were treated with a selective β2AR antagonist (1μM ICI-118,551), or a selective β1AR antagonist (3μM CGP-20712A, both dissolved in water). We then investigated the effect of inhibitors of the MAPKs MEK1/2, ERK1/2, p38 and JNK using 3μM U0126, FR180204, SB203580, SP600125 respectively (all dissolved in DMSO), and the effect of cAMP-dependent protein kinases using the inhibitor Rp-cAMP (100μM, dissolved in water). All treatments were for 14 days. MUC5AC transcripts were quantified by RT-PCR and Trans-epithelial electric resistance (TEER) was measured to assess the integrity of epithelial monolayer. All experiments were done with NHBECs from 3 donors (n=3).

In the presence of epinephrine, MUC5AC expression was significantly induced by IL-13 compared to control cells (15.18±3.76 fold increase, p<0.05, one-way t-test). (Numbers are mean values ± SD, fold changes are compared to control cells). ICI-118,551 inhibited IL-13 induced MUC5AC expression (0.039±0.038 fold vs 15.18±3.76 fold increase by IL-13. p<0.05, one-way t-test) while CGP-20712A did not alter the increase in MUC5AC by IL-13 (14.75±0.97 fold vs 15.18±3.76 fold increase by IL-13, p>0.05, one-way t-test). Incubation with inhibitors of MAPKs, or cAMP-dependent protein kinases, produced a marked reduction in IL-13 induced MUC5AC expression back to near basal levels (15.18±3.76 fold increase by IL-13 vs 2.51±1.93, 1.82±0.68, 0.77±0.39, 0.80±0.65, 2.77±2.08 fold by U0126, FR180204, SB203580, SP600125 and Rp-cAMP respectively. p<0.05 comparing -/+ inhibitors, one-way t-test). TEER was measured at different time points throughout the culture period and at each time point there was no significant reduction between cells treated with inhibitors or βAR ligands as compared to control cells (p>0.05, one-way ANOVA, Dunnett's post test).

We conclude that, in human bronchial epithelial cells, β2AR signaling is required to induce mucus production in response to IL-13. Moreover, MAPKs and cAMP-dependent protein kinases are required components of the β2AR /IL-13 receptor signaling pathways for producing mucus.

Nguyen LP et al., (2009) Proc Natl Acad Sci U S A. 106, 2435-40.

Park JA et al., (2009). Am J Respir Cell Mol Biol . 41,459-66.