title: Nintedanib modulates surfactant protein-D expression in A549 human lung epithelial cells via the c-Jun N-terminal kinase-activator protein-1 pathway.
abstract: Idiopathic pulmonary fibrosis (IPF) is a progressive disease with a high mortality rate. Signalling pathways activated by several tyrosine kinase receptors are known to be involved in lung fibrosis, and this knowledge has led to the development of the triple tyrosine kinase inhibitor nintedanib, an inhibitor of vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), and fibroblast growth factor receptor (FGFR), for the treatment of IPF. Pulmonary surfactant protein D (SP-D), an important biomarker of IPF, reportedly attenuates bleomycin-induced pulmonary fibrosis in mice. In this study, we investigated whether nintedanib modulates SP-D expression in human lung epithelial (A549) cells using quantitative real-time reverse transcriptase polymerase chain reaction and western blotting. To investigate the mechanisms underlying the effects of nintedanib, we evaluated the phosphorylation of c-Jun N-terminal kinase (JNK) and its downstream target c-Jun. The effect of the JNK inhibitor SP600125 on c-Jun phosphorylation was also tested. Activation of activator protein-1 (AP-1) was examined using an enzyme-linked immunosorbent assay-based test, and cell proliferation assays were performed to estimate the effect of nintedanib on cell proliferation. Furthermore, we treated mice with nintedanib to examine its in vivo effect on SP-D levels in lungs. These experiments showed that nintedanib up-regulated SP-D messenger RNA expression in a dose-dependent manner at concentrations up to 5 μM, with significant SP-D induction observed at concentrations of 3 μM and 5 μM, in comparison with that observed in vehicle controls. Nintedanib stimulated a rapid increase in phosphorylated JNK in A549 cells within 30 min of treatment and stimulated c-Jun phosphorylation, which was inhibited by the JNK inhibitor SP600125. Additionally, nintedanib was found to activate AP-1. A549 cell proliferation was not affected by nintedanib at any of the tested concentrations. Moreover, blocking FGFR, PDGFR, and VEGFR function did not affect nintedanib-induced SP-D expression, suggesting that nintedanib mediates its effects through a mechanism that is distinct from its known role as a tyrosine kinase inhibitor. Nintedanib is also reported to inhibit Src kinase although pre-treatment of cells with a Src kinase inhibitor had no effect on nintedanib-induced SP-D expression. Increased expression of SFTPD mRNA and SP-D protein in the lungs of nintedanib-treated mice was also observed. In this work, we demonstrated that nintedanib up-regulated SP-D expression in A549 cells via the JNK-AP-1 pathway and did not affect cell proliferation. This is the first report describing SP-D induction by nintedanib.
authors.affiliation: Department of Pulmonary Medicine and Oncology, Graduate School of Medicine, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8603, Japan. Electronic address: bcway@nms.ac.jp.
authors.forename: Koichiro
authors.initials: K
authors.lastname: Kamio
authors.affiliation: Department of Pulmonary Medicine and Oncology, Graduate School of Medicine, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8603, Japan. Electronic address: usukij@nms.ac.jp.
authors.forename: Jiro
authors.initials: J
authors.lastname: Usuki
authors.affiliation: Department of Pulmonary Medicine and Oncology, Graduate School of Medicine, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8603, Japan. Electronic address: a-azuma@nms.ac.jp.
authors.forename: Arata
authors.initials: A
authors.lastname: Azuma
authors.affiliation: Department of Pulmonary Medicine and Oncology, Graduate School of Medicine, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8603, Japan. Electronic address: kuniko-m@nms.ac.jp.
authors.forename: Kuniko
authors.initials: K
authors.lastname: Matsuda
authors.affiliation: Department of Pulmonary Medicine and Oncology, Graduate School of Medicine, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8603, Japan. Electronic address: tishii@nms.ac.jp.
authors.forename: Takeo
authors.initials: T
authors.lastname: Ishii
authors.affiliation: Department of Pulmonary Medicine and Oncology, Graduate School of Medicine, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8603, Japan. Electronic address: inomataminoru@nms.ac.jp.
authors.forename: Minoru
authors.initials: M
authors.lastname: Inomata
authors.affiliation: Department of Pulmonary Medicine and Oncology, Graduate School of Medicine, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8603, Japan. Electronic address: s5075@nms.ac.jp.
authors.forename: Hiroki
authors.initials: H
authors.lastname: Hayashi
authors.affiliation: Department of Pulmonary Medicine and Oncology, Graduate School of Medicine, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8603, Japan. Electronic address: s7035@nms.ac.jp.
authors.forename: Nariaki
authors.initials: N
authors.lastname: Kokuho
authors.affiliation: Department of Pulmonary Medicine and Oncology, Graduate School of Medicine, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8603, Japan. Electronic address: zufujita@nms.ac.jp.
authors.forename: Kazue
authors.initials: K
authors.lastname: Fujita
authors.affiliation: Department of Pulmonary Medicine and Oncology, Graduate School of Medicine, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8603, Japan. Electronic address: yo-saito@nms.ac.jp.
authors.forename: Yoshinobu
authors.initials: Y
authors.lastname: Saito
authors.affiliation: Department of Pulmonary Medicine and Oncology, Graduate School of Medicine, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8603, Japan. Electronic address: tmiya@nms.ac.jp.
authors.forename: Toshimichi
authors.initials: T
authors.lastname: Miya
authors.affiliation: Department of Pulmonary Medicine and Oncology, Graduate School of Medicine, Nippon Medical School, 1-1-5 Sendagi, Bunkyo-ku, Tokyo 113-8603, Japan. Electronic address: agemma@nms.ac.jp.
authors.forename: Akihiko
authors.initials: A
authors.lastname: Gemma
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism; D005260:Female; D006801:Humans; D054990:Idiopathic Pulmonary Fibrosis / Q000188:drug therapy / Q000503:physiopathology; D007211:Indoles / Q000494:pharmacology*; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D008168:Lung / Q000166:cytology / Q000187:drug effects; D020935:MAP Kinase Signaling System / Q000187:drug effects; D051379:Mice; D008810:Mice, Inbred C57BL; D037663:Pulmonary Surfactant-Associated Protein D / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism; D018808:Transcription Factor AP-1 / Q000378:metabolism; D015854:Up-Regulation / Q000187:drug effects*
chemical_list: D007211:Indoles; D037663:Pulmonary Surfactant-Associated Protein D; D012333:RNA, Messenger; D018808:Transcription Factor AP-1; D048031:JNK Mitogen-Activated Protein Kinases; C530716:nintedanib
keywords: A549; Idiopathic pulmonary fibrosis; Nintedanib; Surfactant protein-D