Rapamycin increases CCN2 expression of lung fibroblasts via phosphoinositide 3-kinase
Authors: Xuan Wan
Huaping Dai
Chen Wang
Dianhua Jiang
Fei Li
Jing Geng
Xuefeng Xu
Xiaoxi Huang
Journal: Laboratory Investigation (2015)
Discipline: CLINICAL MEDICINE
Keywords: connective tissue growth factor; CCN2; idiopathic pulmonary fibrosis; rapamycin; mTOR complex
Affiliations: 1] Department of Respiratory and Critical Care Medicine, Beijing Key Laboratory of Respiratory and Pulmonary Circulation Disorders, Beijing Chao-Yang Hospital, Beijing Institute of Respiratory Medicine, Capital Medical University, Beijing, China [2] National Clinical Research Centre for Respiratory Medicine, Beijing Hospital, Beijing, China., Department of Respiratory and Critical Care Medicine, Beijing Key Laboratory of Respiratory and Pulmonary Circulation Disorders, Beijing Chao-Yang Hospital, Beijing Institute of Respiratory Medicine, Capital Medical University, Beijing, China., Department of Medicine, Cedars Sinai Medical Center, Los Angeles, CA, USA.

Abstract:  Excessive production of connective tissue growth factor (CTGF, CCN2) and increased motor ability of the activated fibroblast phenotype contribute to the pathogenesis of idiopathic pulmonary fibrosis (IPF). However, molecules and signal pathways regulating CCN2 expression and migration of lung fibroblasts are still elusive. We hypothesize that rapamycin, via binding and blocking mammalian target of rapamycin (mTOR) complex (mTORC), affects CCN2 expression and migration of lung fibroblasts in vitro. Primary normal and fibrotic human lung fibroblasts were isolated from lung tissues of three patients with primary spontaneous pneumothorax and three with IPF. Cells were incubated with regular medium, or medium containing rapamycin, human recombinant transforming growth factor (TGF)-β1, or both. CCN2 and tissue inhibitor of metalloproteinase (TIMP)-1 expression in cells or supernatant was detected. Wound healing and migration assay was used to measure the migratory potential. TGF-β type I receptor (TβRI)/Smad inhibitor, SB431542 and phosphoinositide 3-kinase (PI3K) inhibitor, LY294002 were used to determine rapamycin's mechanism of action. We demonstrated that rapamycin amplified basal or TGF-β1-induced CCN2 mRNA and protein expression in normal or fibrotic fibroblasts by Smad-independent but PI3K-dependent pathway. Additionally, rapamycin also enhanced TIMP-1 expression as indicated by ELISA. However, wound healing and migrating assay showed rapamycin did not affect the mobility of fibroblasts. Collectively, this study implies a significant fibrogenic induction activity of rapamycin by activating AKT and inducing CCN2 expression in vitro and provides the possible mechanisms for the in vivo findings which previously showed no antifibrotic effect of rapamycin on lung fibrosis. 

Retracted: No

PubPeer comments:
 Figure 8A and Figure 9A share an identical gel lane for p-AKT, marked by red box. Figure 8A: ![file] Figure 9A: ![file] , 
 We thank the reader noting that the p-AKT band in Figure 8A and Figure 9A contained same images. The authors have indicated that the errors occurred during figure preparation. We have provided the corrected panel below. The authors have contacted the Laboratory Investigation for corrigendum and have provided an updated version of Figure 8. Although this error does not affect the conclusion of the article, the authors deeply regret the error. ![file] , 
 This response is on behalf of corresponding author, Dr. Dai. We thank the reader noting that the p-AKT band in Figure 8A and Figure 9A contained same images. The authors have indicated that the errors occurred during figure preparation. We have provided the corrected panel below. The authors have contacted the Laboratory Investigation for corrigendum and have provided an updated version of Figure 8. Although this error does not affect the conclusion of the article, the authors deeply regret the error. ![file] ,