CREB regulates TNF-α-induced GM-CSF secretion via p38 MAPK in human lung fibroblasts.
BACKGROUND
Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine that mediates eosinophilic differentiation, migration and survival, causing respiratory tract inflammation. GM-CSF is also known to be secreted from respiratory tract structural cells. However, the mechanisms of GM-CSF secretion have not been well established.


METHODS
Human fetal lung fibroblasts and human primary asthmatic lung fibroblasts were used for the study of tumor necrosis factor alpha (TNF-α)-induced GM-CSF secretion. GM-CSF secretion and mRNA expression were measured by enzyme-linked immunosorbent assay and quantitative real-time reverse transcription polymerase chain reaction, respectively. Knockdown of cAMP response element-binding protein (CREB) in fibroblasts was carried out by using specific small interfering RNAs of CREB.


RESULTS
Among respiratory tract structural cells, pulmonary fibroblasts exhibited increased GM-CSF secretion and mRNA expression after stimulation with TNF-α in a concentration-dependent manner. Moreover, a p38 mitogen-activated protein kinase (MAPK) inhibitor controlled TNF-α-induced GM-CSF secretion, and roflumilast and rolipram, inhibitors of phosphodiesterase-4, suppressed TNF-α-induced GM-CSF secretion. Consistent with this, forskolin also completely blocked GM-CSF secretion, and similar results were observed in response to cAMP treatment, suggesting that cAMP signaling suppressed TNF-α-induced GM-CSF secretion in human lung fibroblasts. Furthermore, CREB was phosphorylated through p38 MAPK but not cAMP signaling after TNF-α stimulation, and GM-CSF secretion was inhibited by CREB knockdown. Finally, these effects were also demonstrated in human primary lung fibroblasts in a patient with asthma.


CONCLUSIONS
CREB signaled independent of cAMP signaling and was phosphorylated by p38 MAPK following TNF-α stimulation, playing a critical role in GM-CSF secretion in human lung fibroblasts.
Department of Medicine and Molecular Science, Gunma University Graduate School of Medicine, Gunma, Japan. Electronic address: ykoga@gunma-u.ac.jp.
Yasuhiko
Y
Koga
Department of Medicine and Molecular Science, Gunma University Graduate School of Medicine, Gunma, Japan.
Takeshi
T
Hisada
Third Department of Internal Medicine, Faculty of Medical Sciences, University of Fukui, Fukui, Japan.
Tamotsu
T
Ishizuka
Department of Medicine and Molecular Science, Gunma University Graduate School of Medicine, Gunma, Japan; Department of Respiratory Medicine, Kiryu Kosei General Hospital, Gunma, Japan.
Mitsuyoshi
M
Utsugi
Department of Medicine and Molecular Science, Gunma University Graduate School of Medicine, Gunma, Japan.
Akihiro
A
Ono
Department of Medicine and Molecular Science, Gunma University Graduate School of Medicine, Gunma, Japan.
Masakiyo
M
Yatomi
Department of Medicine and Molecular Science, Gunma University Graduate School of Medicine, Gunma, Japan; Clinical Research Center for Allergy and Rheumatology, Sagamihara National Hospital, Kanagawa, Japan.
Yosuke
Y
Kamide
Department of Medicine and Molecular Science, Gunma University Graduate School of Medicine, Gunma, Japan.
Haruka
H
Aoki-Saito
Department of Medicine and Molecular Science, Gunma University Graduate School of Medicine, Gunma, Japan.
Hiroaki
H
Tsurumaki
Gunma University Graduate School of Health Sciences, Gunma, Japan.
Kunio
K
Dobashi
Department of Medicine and Molecular Science, Gunma University Graduate School of Medicine, Gunma, Japan.
Masanobu
M
Yamada
D002460:Cell Line; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics / Q000378:metabolism*; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism*; D015870:Gene Expression; D055785:Gene Knockdown Techniques; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008168:Lung / Q000378:metabolism*; D010766:Phosphorylation; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism* / Q000494:pharmacology; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism*
D017362:Cyclic AMP Response Element-Binding Protein; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D016178:Granulocyte-Macrophage Colony-Stimulating Factor; D000242:Cyclic AMP; D048051:p38 Mitogen-Activated Protein Kinases
Asthma; CREB; GM-CSF; cAMP; p38 MAPK