The Role of ADAM 17 in Hypoxia-Induced CTGF Expression and Pulmonary Fibrosis (III)

Progressive airway fibrosis and hypoxia remain an intractable problem in chronic obstructive asthma (COA). Several studies demonstrated that hypoxia, altered
expression of A Disintegrin and Metalloproteinase-17 (ADAM17), and connective tissue growth factor (CTGF)overexpression play critical roles in tissue fibrosis. In
this study, we investigate the role of ADAM 17 in hypoxiainduced CTGF expression and pulmonary fibrosis. We found that treatment with TAPI-0 (ADAM 17 inhibitor), CMK (furin inhibitor), small interfering RNA (siRNA) of Notch 1
(Notch1 siRNA), RSK1 siRNA and C/EBPb siRNA, and AP-1 site mutation of the CTGF construct inhibited hypoxia-induced CTGF expression or CTGF-lucifease activity in WI-38 cells. Hypoxia caused increases in ADAM 17 phosphorylation at
Thr735 or ADAM 17 activity was attenuated by CMK. Stimulation of cells with hypoxia resulted in an increase in NICD formation, which was inhibited by TRAP-0. Hypoxia induces increase in c-Jun Ser63 phosphorylation, complex formation of c-Jun and NICD, and recruitment of c-Jun and NICD to the AP-1 binding motif of CTGF promoter. Moreover, hypoxia-induced fibroblast differentiation and a-smooth muscle actin (a-SMA) expression were inhibited by Notch 1 siRNA, TAPI-0, or anti-CTGF antibody. We also found that overexpression of ADAM 17, phosphorylation of ADAM 17, and CTGF in lung fibroblasts from patients with COA compared to normal human lung fibroblasts. RSK1 siRNA and C/EBP siRNA reduced hypoxia-induced ADAM 17 expression. Stimulation of cells with hypoxia resulted in an increase in C/EBPb phosphorylation at Thr217, C/EBP-luciferase activity, and recruits C/EBP to the ADAM 17 promoter region. Hypoxiainduced
C/EBPb-luciferase activity was inhibited by RSK 1 siRNA. When treated with ovalbumin (OVA) or bleomycin, ADAM 17 conditional knockout mice showed attenuation of lung fibrosis, fibronectin, a-SMA, and CTGF expression relative
to wild type mice. Taken together, we conclude that furin/ADAM 17/Notch1 is plays vital role in hypoxia-induced CTGF expression. RSK1/C/EBPb mediates hypoxia-induced ADAM 17 overexpression and its plays an important in airway
fibrosis. Our results provide support for the development of therapeutic strategies to reduce airway fibrosis caused by hypoxia in COA.