Elsevier

Laboratory Investigation

Volume 95, Issue 8, August 2015, Pages 846-859
Laboratory Investigation

Research Article
Rapamycin increases CCN2 expression of lung fibroblasts via phosphoinositide 3-kinase

https://doi.org/10.1038/labinvest.2015.68Get rights and content
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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.

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Supplementary Information accompanies the paper on the Laboratory Investigation website

In this paper, the authors show that rapamycin significantly augments basal or transforming growth factor (TGF)-β1-induced connective tissue growth factor expression in normal and fibrotic human lung fibroblasts via activation of the phosphatidyl-inositol-3-kinase (PI3K)-AKT pathway. Rapamcin has no antifibrotic effect and in some cases, even promotes the generation of fibrotic lesions. Combined inhibition of PI3K and mTOR may be a more effective regime for antifibrotic treatment.

Supplementary information The online version of this article (doi:10.1038/labinvest.2015.68) contains supplementary material, which is available to authorized users.

A Correction to this article is available online at https://doi.org/10.1038/s41374-020-00501-5.