Role of C/EBPβ/STAT3/AP-1 transcriptional complex formation in thrombin-induced connective tissue growth factor expression in human lung fibroblasts

Abstract

CCAAT/enhancer-binding protein-β (C/EBPβ) may play a key role in idiopathic pulmonary fibrosis progression. Despite this, the mechanism through which the formation of the transcriptional complex comprising C/EBPβ, signal transducer and activator of transcription 3 (STAT3), and activator protein-1 (AP-1) impacts the regulation of connective tissue growth factor (CTGF) production by thrombin in human lung fibroblasts (WI-38) is not well understood. In this study, C/EBPβ knockdown suppressed thrombin-stimulated CTGF production and CTGF-luciferase activity. The phosphorylation and cytosol-to-nucleus translocation of C/EBPβ were enhanced by thrombin. C/EBPβ knockdown attenuated thrombin-induced STAT3-and AP-1-luciferase activity. Upon thrombin stimulation, the C/EBPβ/STAT3/AP-1 complex was bound to the CTGF promoter. Both the phosphatidylinositol 3-kinase (PI3K) inhibitor and the dominant negative mutant of Akt inhibited thrombin-stimulated CTGF production, C/EBPβ phosphorylation, and C/EBPβ-luciferase activity. Furthermore, co-inhibition of the Akt, c-Jun N-terminal kinase (JNK), and Janus kinase 2 (JAK2) did not further reduce thrombin-induced C/EBPβ-luciferase activity compared to inhibition of each pathway alone. In bleomycin-induced pulmonary fibrosis, the phosphorylation of C/EBPβ, STAT3, and c-Jun in lung tissue was higher than the control group. In addition, the protein levels of C/EBPβ and CTGF, but not of c-Jun and STAT3, were higher than those in the control group. In conclusion, thrombin activates the PI3K/Akt pathway to activate C/EBPβ, which in turn induces CTGF expression in human lung fibroblasts. C/EBPβ/STAT3/AP-1 transcriptional complex formation plays an essential role in thrombin-triggered CTGF production. C/EBPβ upregulation and C/EBPβ/STAT3/AP-1 complex formation may play crucial roles in lung fibrosis and have potential for use as therapeutic targets.