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Epinephrine Activation of the β2-Adrenoceptor Is Required for IL-13-Induced Mucin Production in Human Bronchial Epithelial Cells.

Al-Sawalha N, Pokkunuri I, Omoluabi O, Kim H, Thanawala VJ, Hernandez A, Bond RA, Knoll BJ - PLoS ONE (2015)

Bottom Line: A clinically important long-acting β-agonist, formoterol, was as effective as epinephrine in potentiating IL-13 induced MUC5AC transcription.IL-13 induced mucin production in the presence of epinephrine was significantly reduced by treatment with selective inhibitors of ERK1/2 (FR180204), p38 (SB203580) and JNK (SP600125).Replacement of epinephrine with forskolin + IBMX resulted in a marked increase in mucin production in NHBE cells in response to IL-13, and treatment with the inhibitory cAMP analogue Rp-cAMPS decreased mucin levels induced by epinephrine + IL-13.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacological and Pharmaceutical Sciences, University of Houston, 4800 Calhoun Road, Houston, Texas, 77204, United States of America.

ABSTRACT
Mucus hypersecretion by airway epithelium is a hallmark of inflammation in allergic asthma and results in airway narrowing and obstruction. Others have shown that administration a TH2 cytokine, IL-13 is sufficient to cause mucus hypersecretion in vivo and in vitro. Asthma therapy often utilizes β2-adrenoceptor (β2AR) agonists, which are effective acutely as bronchodilators, however chronic use may lead to a worsening of asthma symptoms. In this study, we asked whether β2AR signaling in normal human airway epithelial (NHBE) cells affected mucin production in response to IL-13. This cytokine markedly increased mucin production, but only in the presence of epinephrine. Mucin production was blocked by ICI-118,551, a preferential β2AR antagonist, but not by CGP-20712A, a preferential β1AR antagonist. Constitutive β2AR activity was not sufficient for IL-13 induced mucin production and β-agonist-induced signaling is required. A clinically important long-acting β-agonist, formoterol, was as effective as epinephrine in potentiating IL-13 induced MUC5AC transcription. IL-13 induced mucin production in the presence of epinephrine was significantly reduced by treatment with selective inhibitors of ERK1/2 (FR180204), p38 (SB203580) and JNK (SP600125). Replacement of epinephrine with forskolin + IBMX resulted in a marked increase in mucin production in NHBE cells in response to IL-13, and treatment with the inhibitory cAMP analogue Rp-cAMPS decreased mucin levels induced by epinephrine + IL-13. Our findings suggest that β2AR signaling is required for mucin production in response to IL-13, and that mitogen activated protein kinases and cAMP are necessary for this effect. These data lend support to the notion that β2AR-agonists may contribute to asthma exacerbations by increasing mucin production via activation of β2ARs on epithelial cells.

No MeSH data available.


Related in: MedlinePlus

cAMP potentiates mucin production in response to IL-13 in NHBE cells.Cells were grown in the absence of epinephrine, then at ALI, they were incubated with or without 20 ng/ml IL-13 with or without 10 μM forskolin and 100 μM IBMX for 14 days. A: MUC5AC transcripts were measured by qRT-PCR, and the data presented as fold change compared to cells grown in the absence of IL-13, IBMX and forskolin (control cells) B: Quantification of intracellular mucin 5AC content. The ratio of mucin 5AC integrated density of each group to the integrated density of the cells grown in the presence of epinephrine alone (control cells) was calculated and expressed as fold change. See the supplement for the representative images (S7A Fig). C: Quantification of intracellular mucin glycoproteins. The ratio of mucin integrated density and nucleic acid/cytoplasm integrated density was calculated and the data presented as fold change compared to control cells. See the supplement for the representative images (S7B Fig). Data are presented as means ± SEM from three donors. † and ¥ indicate p<0.05 significance as compared to−epinephrine and −epinephrine + IL-13 treated cells respectively.
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pone.0132559.g006: cAMP potentiates mucin production in response to IL-13 in NHBE cells.Cells were grown in the absence of epinephrine, then at ALI, they were incubated with or without 20 ng/ml IL-13 with or without 10 μM forskolin and 100 μM IBMX for 14 days. A: MUC5AC transcripts were measured by qRT-PCR, and the data presented as fold change compared to cells grown in the absence of IL-13, IBMX and forskolin (control cells) B: Quantification of intracellular mucin 5AC content. The ratio of mucin 5AC integrated density of each group to the integrated density of the cells grown in the presence of epinephrine alone (control cells) was calculated and expressed as fold change. See the supplement for the representative images (S7A Fig). C: Quantification of intracellular mucin glycoproteins. The ratio of mucin integrated density and nucleic acid/cytoplasm integrated density was calculated and the data presented as fold change compared to control cells. See the supplement for the representative images (S7B Fig). Data are presented as means ± SEM from three donors. † and ¥ indicate p<0.05 significance as compared to−epinephrine and −epinephrine + IL-13 treated cells respectively.

Mentions: To provide more evidence for a role for cAMP in mucin production in response to IL-13, we treated cells with 10 μM forskolin combined with 100 μM 3-isobutyl-l-methylxan-thine (IBMX), in the absence of epinephrine. This treatment caused a dramatic increase in MUC5AC expression (75.73 ± 66.59 fold vs 0.56 ± 0.40 fold increase by IL-13, p<0.05) (Fig 6A) when the cells were treated with IL-13. The same trend was also observed at the level of intracellular mucin 5AC protein accumulation and mucin content of NHBE cells (Fig 6B and 6C; for representative images see S7A and S7B Fig).


Epinephrine Activation of the β2-Adrenoceptor Is Required for IL-13-Induced Mucin Production in Human Bronchial Epithelial Cells.

Al-Sawalha N, Pokkunuri I, Omoluabi O, Kim H, Thanawala VJ, Hernandez A, Bond RA, Knoll BJ - PLoS ONE (2015)

cAMP potentiates mucin production in response to IL-13 in NHBE cells.Cells were grown in the absence of epinephrine, then at ALI, they were incubated with or without 20 ng/ml IL-13 with or without 10 μM forskolin and 100 μM IBMX for 14 days. A: MUC5AC transcripts were measured by qRT-PCR, and the data presented as fold change compared to cells grown in the absence of IL-13, IBMX and forskolin (control cells) B: Quantification of intracellular mucin 5AC content. The ratio of mucin 5AC integrated density of each group to the integrated density of the cells grown in the presence of epinephrine alone (control cells) was calculated and expressed as fold change. See the supplement for the representative images (S7A Fig). C: Quantification of intracellular mucin glycoproteins. The ratio of mucin integrated density and nucleic acid/cytoplasm integrated density was calculated and the data presented as fold change compared to control cells. See the supplement for the representative images (S7B Fig). Data are presented as means ± SEM from three donors. † and ¥ indicate p<0.05 significance as compared to−epinephrine and −epinephrine + IL-13 treated cells respectively.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4498766&req=5

pone.0132559.g006: cAMP potentiates mucin production in response to IL-13 in NHBE cells.Cells were grown in the absence of epinephrine, then at ALI, they were incubated with or without 20 ng/ml IL-13 with or without 10 μM forskolin and 100 μM IBMX for 14 days. A: MUC5AC transcripts were measured by qRT-PCR, and the data presented as fold change compared to cells grown in the absence of IL-13, IBMX and forskolin (control cells) B: Quantification of intracellular mucin 5AC content. The ratio of mucin 5AC integrated density of each group to the integrated density of the cells grown in the presence of epinephrine alone (control cells) was calculated and expressed as fold change. See the supplement for the representative images (S7A Fig). C: Quantification of intracellular mucin glycoproteins. The ratio of mucin integrated density and nucleic acid/cytoplasm integrated density was calculated and the data presented as fold change compared to control cells. See the supplement for the representative images (S7B Fig). Data are presented as means ± SEM from three donors. † and ¥ indicate p<0.05 significance as compared to−epinephrine and −epinephrine + IL-13 treated cells respectively.
Mentions: To provide more evidence for a role for cAMP in mucin production in response to IL-13, we treated cells with 10 μM forskolin combined with 100 μM 3-isobutyl-l-methylxan-thine (IBMX), in the absence of epinephrine. This treatment caused a dramatic increase in MUC5AC expression (75.73 ± 66.59 fold vs 0.56 ± 0.40 fold increase by IL-13, p<0.05) (Fig 6A) when the cells were treated with IL-13. The same trend was also observed at the level of intracellular mucin 5AC protein accumulation and mucin content of NHBE cells (Fig 6B and 6C; for representative images see S7A and S7B Fig).

Bottom Line: A clinically important long-acting β-agonist, formoterol, was as effective as epinephrine in potentiating IL-13 induced MUC5AC transcription.IL-13 induced mucin production in the presence of epinephrine was significantly reduced by treatment with selective inhibitors of ERK1/2 (FR180204), p38 (SB203580) and JNK (SP600125).Replacement of epinephrine with forskolin + IBMX resulted in a marked increase in mucin production in NHBE cells in response to IL-13, and treatment with the inhibitory cAMP analogue Rp-cAMPS decreased mucin levels induced by epinephrine + IL-13.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacological and Pharmaceutical Sciences, University of Houston, 4800 Calhoun Road, Houston, Texas, 77204, United States of America.

ABSTRACT
Mucus hypersecretion by airway epithelium is a hallmark of inflammation in allergic asthma and results in airway narrowing and obstruction. Others have shown that administration a TH2 cytokine, IL-13 is sufficient to cause mucus hypersecretion in vivo and in vitro. Asthma therapy often utilizes β2-adrenoceptor (β2AR) agonists, which are effective acutely as bronchodilators, however chronic use may lead to a worsening of asthma symptoms. In this study, we asked whether β2AR signaling in normal human airway epithelial (NHBE) cells affected mucin production in response to IL-13. This cytokine markedly increased mucin production, but only in the presence of epinephrine. Mucin production was blocked by ICI-118,551, a preferential β2AR antagonist, but not by CGP-20712A, a preferential β1AR antagonist. Constitutive β2AR activity was not sufficient for IL-13 induced mucin production and β-agonist-induced signaling is required. A clinically important long-acting β-agonist, formoterol, was as effective as epinephrine in potentiating IL-13 induced MUC5AC transcription. IL-13 induced mucin production in the presence of epinephrine was significantly reduced by treatment with selective inhibitors of ERK1/2 (FR180204), p38 (SB203580) and JNK (SP600125). Replacement of epinephrine with forskolin + IBMX resulted in a marked increase in mucin production in NHBE cells in response to IL-13, and treatment with the inhibitory cAMP analogue Rp-cAMPS decreased mucin levels induced by epinephrine + IL-13. Our findings suggest that β2AR signaling is required for mucin production in response to IL-13, and that mitogen activated protein kinases and cAMP are necessary for this effect. These data lend support to the notion that β2AR-agonists may contribute to asthma exacerbations by increasing mucin production via activation of β2ARs on epithelial cells.

No MeSH data available.


Related in: MedlinePlus