Mucus hypersecretion by airway epithelium is a hallmark of inflammation in allergic asthma and leads to airway narrowing and blockage. epinephrine NHBE cells had been incubated with the preferential β2AR antagonist (1 μM ICI-118 551 or a preferential β1AR antagonist (3 μM CGP-20712A). ICI-118 551 totally abolished (>99%) IL-13 induced MUC5AC manifestation (0.039 ± 0.038 fold 15.99 ± 1.48 fold increase by IL-13. p<0.05). On the other hand CGP-20712A did not affect the MUC5AC expression level (14.75 ± 0.96 fold 15.99 ± 1.48 Roburic acid fold increase by IL-13 p>0.05) (Fig 2A). CGP-20712A did not affect the intracellular mucin levels induced by IL-13 while ICI-118 551 brought the levels back to baseline (Fig Rabbit polyclonal to ZMAT5. 2B and 2C; for representative images see S3A and S3B Fig). Fig 2 β2ARs are required for mucin production in response to IL-13 in NHBE cells. We next asked if the increased MUC5AC expression in response to IL-13 is due to agonist induced or constitutive β2AR signaling. NHBE cells were treated with 10 μM nadolol a non-selective βAR ligand with inverse agonist activity at β2ARs that blocks both constitutive and agonist-induced receptor activity or with 10 μM alprenolol a non-selective βAR antagonist with no inverse agonist activity for 14 days in combination with IL-13 and in Roburic acid the presence of epinephrine. Treatment with nadolol reduced IL-13 induced MUC5AC expression (3.36 ± 4.10 fold 25.37 ± 16.30 fold increase by IL-13 p<0.05) intracellular mucin 5AC protein and mucin content (Fig 3A 3 and 3C; for representative images see S4A and S4B Fig). Treatment with alprenolol reduced IL-13-induced MUC5AC expression to a similar extent (3.19 ± 3.73 fold 25.37 ± 16.30 fold increase by Roburic acid IL-13 p<0.05) and also reduced intracellular mucin 5AC and mucin content (Fig 3A 3 and 3C and S4A and S4B Fig for representative images). Fig 3 Agonist induced β2AR signaling is required for mucin production in response to IL-13 in NHBE cells. To investigate the role of mitogen activated protein kinases (MAPKs) we examined their activation using antibodies specific for phosphorylated (activated) MAPKs. In the absence of epinephrine IL-13 did not affect the phosphorylation of ERK1/2 (Fig 4A) c-Jun (Fig 4B) or p38 (Fig 4C) as compared to their corresponding controls. When epinephrine was included in the medium IL-13 induced an approximately 3-fold increase in the phosphorylation of ERK1/2 and c-Jun when compared to their corresponding controls (Fig 4A and 4B). However phosphorylation of p38 was unaffected by IL-13 even in the presence of epinephrine (Fig 4C). Next we treated NHBE cells with 3 μM "type":"entrez-nucleotide" attrs :"text":"FR180204" term_id :"258307209" term_text :"FR180204"FR180204 SP600125 or SB203580 (inhibitors of ERK1/2 JNK and p38 respectively) in combination with IL-13 and epinephrine for 14 days. All three MAPKs inhibitors significantly reduced MUC5AC gene expression (15.18 ± 3.76 fold increase by IL-13 vs 1.82 ± 0.68 0.77 ± 0.39 and 0.80 ±0.65 fold by "type":"entrez-nucleotide" attrs :"text":"FR180204" term_id :"258307209" term_text :"FR180204"FR180204 SP600125 and SB203580 respectively) (Fig 4D). While all MAPK inhibitors reduced the intracellular mucin 5AC protein (see Fig 4E and S5A Fig for representative images) only "type":"entrez-nucleotide" attrs :"text":"FR180204" term_id :"258307209" term_text :"FR180204"FR180204 and SP600125 reduced intracellular mucin content when compared to IL-13 treated cells (see Fig 4F and S5B Fig for representative images). Fig 4 MAPK signaling is required for mucin production in response to IL-13 in NHBE cells. To explore a possible role for PKA in the induction of MUC5AC we treated NHBE cells with a competitive cAMP analogue Rp-cAMPS for 14 days in combination with IL-13 and epinephrine. Rp-cAMPS did not significantly reduce the levels of MUC5AC expression at 50 μM (5.97 ± 4.29 fold 12.50 ± 5.38 fold increase by IL-13 p>0.05 ) while at 100 μM there is a Roburic acid substantial reduction (2.35 ± 1.63 fold 12.50 ± 5.38 fold increase by IL-13 p<0.05)(Fig 5A). The intracellular mucin 5AC proteins level was considerably decreased when the cells had been treated with 100 μM Rp-cAMPS however not at 50 μM while mucin.