Anti-CTGF Single-Chain Variable Fragment Dimers Inhibit Human Airway Smooth Muscle (ASM) Cell Proliferation by Down-Regulating p-Akt and p-mTOR Levels

Abstract

Connective tissue growth factor (CTGF) contributes to airway smooth muscle (ASM) cell hyperplasia in asthma. Humanized single-chain variable fragment antibody (scFv) was well characterized as a CTGF antagonist in the differentiation of fibroblast into myofibroblast and pulmonary fibrosis in our previous studies. To further improve the bioactivity of scFv, we constructed a plasmid to express scFv-linker-matrilin-6×His fusion proteins that could self-assemble into the scFv dimers by disulfide bonds in matrilin under non-reducing conditions. An immunoreactivity assay demonstrated that the scFv dimer could highly bind to CTGF in a concentration-dependent manner. The MTT and EdU assay results revealed that CTGF (≥10 ng/mL) promoted the proliferation of ASM cells, and this effect was inhibited when the cells were treated with anti-CTGF scFv dimer. The western blot analysis results showed that increased phosphorylation of Akt and mTOR induced by CTGF could be suppressed by this scFv dimer. Based on these findings, anti-CTGF scFv dimer may be a potential agent for the prevention of airway remodeling in asthma.

Activation of the mTOR signaling pathway is required for asthma onset

“…Further, reducing the levels of p-Akt and p-mTOR suppressed smooth muscle fibrosis in asthma 28 , and rapamycin suppressed asthma onset by inhibiting mTOR signaling 26 . However, despite studies supporting the involvement of mTOR in asthma, controversy exists over the effects of rapamycin treatment in asthma, especially its ability to suppress airway inflammation and airway hyperreactivity 29 .…”

“…Previous studies have suggested the role of mTOR in asthma: airway inflammation was inhibited by reducing the activation of PKC-δ in the mTOR pathway 26 , and airway remodeling was shown to tightly associated with high levels of mTOR expression in an asthmatic mouse model 27 . Further, reducing the levels of p-Akt and p-mTOR suppressed smooth muscle fibrosis in asthma 28 , and rapamycin suppressed asthma onset by inhibiting mTOR signaling 26 . However, despite studies supporting the involvement of mTOR in asthma, controversy exists over the effects of rapamycin treatment in asthma, especially its ability to suppress airway inflammation and airway hyperreactivity 29 .…”

Novel benzofuran derivative DK-1014 attenuates lung inflammation via blocking of MAPK/AP-1 and AKT/mTOR signaling in vitro and in vivo

“…Previous studies have demonstrated that airway inflammation is suppressed by reducing the activation of the mTOR pathway in asthma 35 and that the AKT/mTOR/p70S6K/GSK pathways play an important role in regulating the cell cycle of lung epithelial cells 36 . Therefore, we explored the effects of DK-1014 on the AKT pathway and found that DK-1014 suppressed the phosphorylation of downstream effectors (i.e., mTOR, p70S6K and GSK) and AKT phosphorylation.…”

TAZ promotes cell growth and inhibits Celastrol-induced cell apoptosis

“…Our results suggested that the anti-apoptotic function of TAZ may be relevant with the up-regulation of CYR61 and CTGF and the decrease in Akt phosphorylation which leads to cell apoptosis [ 25 , 26 , 52 , 53 ] may be a mechanism for Celastrol-induced cell apoptosis. On the other hand, some studies indicated that CYR61 and CTGF could increase the phosphorylation of Akt [ 54 – 56 ]. Hence, TAZ may restore Celastrol-induced cell apoptosis by stimulating Akt signalling pathway activation via up-regulation of CYR61 and CTGF.…”