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.