Lipopolysaccharide Induces Lung Fibroblast Proliferation through Toll-Like Receptor 4 Signaling and the Phosphoinositide3-Kinase-Akt Pathway

Zhengyu He,Yuan Gao,Yongming Chen,Xiangrui Wang,Yuxiao Deng,Jia Ding,Xianyuan Zhao,Wen Li,Shunpeng Xing,Victor Sanchez-Margalet

doi:10.1371/journal.pone.0035926

Abstract

Pulmonary fibrosis is characterized by lung fibroblast proliferation and collagen secretion. In lipopolysaccharide (LPS)-induced acute lung injury (ALI), aberrant proliferation of lung fibroblasts is initiated in early disease stages, but the underlying mechanism remains unknown. In this study, we knocked down Toll-like receptor 4 (TLR4) expression in cultured mouse lung fibroblasts using TLR4-siRNA-lentivirus in order to investigate the effects of LPS challenge on lung fibroblast proliferation, phosphoinositide3-kinase (PI3K)-Akt pathway activation, and phosphatase and tensin homolog (PTEN) expression. Lung fibroblast proliferation, detected by BrdU assay, was unaffected by 1 mug/mL LPS challenge up to 24 hours, but at 72 hours, cell proliferation increased significantly. This proliferation was inhibited by siRNA-mediated TLR4 knockdown or treatment with the PI3K inhibitor, Ly294002. In addition, siRNA-mediated knockdown of TLR4 inhibited the LPS-induced up-regulation of TLR4, down-regulation of PTEN, and activation of the PI3K-Akt pathway (overexpression of phospho-Akt) at 72 hours, as detected by real-time PCR and Western blot analysis. Treatment with the PTEN inhibitor, bpV(phen), led to activation of the PI3K-Akt pathway. Neither the baseline expression nor LPS-induced down-regulation of PTEN in lung fibroblasts was influenced by PI3K activation state. PTEN inhibition was sufficient to exert the LPS effect on lung fibroblast proliferation, and PI3K-Akt pathway inhibition could reverse this process. Collectively, these results indicate that LPS can promote lung fibroblast proliferation via a TLR4 signaling mechanism that involves PTEN expression down-regulation and PI3K-Akt pathway activation. Moreover, PI3K-Akt pathway activation is a downstream effect of PTEN inhibition and plays a critical role in lung fibroblast proliferation. This mechanism could contribute to, and possibly accelerate, pulmonary fibrosis in the early stages of ALI/ARDS.

Highlights

Pulmonary fibrosis is characterized by aberrant proliferation and activation of lung fibroblasts and collagen secretion that results in excessive extracellular matrix (ECM) deposition [1,2]
Effect of LPS on lung fibroblast proliferation To investigate the effect of LPS on various stages of lung fibroblast proliferation, we employed the BrdU assay to temporally quantify DNA synthesis in lung fibroblasts in response to LPS challenge
Aberrant proliferation of lung fibroblasts has been detected in bleomycin-induced lung fibrosis [15], patients with idiopathic pulmonary fibrosis (IPF) [11], and LPS-induced acute lung injury (ALI) and pulmonary fibrosis [4]

Summary

Introduction

Pulmonary fibrosis is characterized by aberrant proliferation and activation of lung fibroblasts and collagen secretion that results in excessive extracellular matrix (ECM) deposition [1,2]. LPS exerts its biological effects on the host by binding to Toll-like receptor 4 (TLR4), a pattern recognition receptor (PPR) that is widely distributed among lung parenchyma cells, including macrophages, epithelial cells, and fibroblasts. Our previous studies have shown that LPS plays an important role in the development of acute lung injury (ALI), acute respiratory distress syndrome (ARDS), and pulmonary fibrosis, through activation of TLR4 and its downstream intracellular signal transduction pathways [3,4]. Our study and those by others have determined that focal aggregation of lung fibroblasts occurs prior to formation of fibrosis [5], implying that aberrant proliferation of fibroblasts takes place in the early stages of ALI/ARDS.

Methods

Results

Conclusion