Yasuhiko Koga1, Takeshi Hisada2, Tamotsu Ishizuka3, Mitsuyoshi Utsugi4, Akihiro Ono2, Masakiyo Yatomi2, Yosuke Kamide5, Haruka Aoki-Saito2, Hiroaki Tsurumaki2, Kunio Dobashi6, Masanobu Yamada2. 1. Department of Medicine and Molecular Science, Gunma University Graduate School of Medicine, Gunma, Japan. Electronic address: ykoga@gunma-u.ac.jp. 2. Department of Medicine and Molecular Science, Gunma University Graduate School of Medicine, Gunma, Japan. 3. Third Department of Internal Medicine, Faculty of Medical Sciences, University of Fukui, Fukui, Japan. 4. Department of Medicine and Molecular Science, Gunma University Graduate School of Medicine, Gunma, Japan; Department of Respiratory Medicine, Kiryu Kosei General Hospital, Gunma, Japan. 5. 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. 6. Gunma University Graduate School of Health Sciences, Gunma, Japan.
Abstract
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.
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.