Lipopolysaccharide induces lung fibroblast proliferation through Toll-like receptor 4 signaling and the phosphoinositide3-kinase-Akt pathway

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.

Figure 1. Effect of LPS on lung fibroblast proliferation.

DNA synthesis in lung fibroblasts was detected by BrdU assay after LPS challenge at 0, 6, 24, and 72 hours. * p<0.05 for percentage of OD₄₅₀ absorbance compared to the control group at the same time point. Columns represent mean values (n = 3) and error bars represent SD.

Figure 2. Expression of TLR4 in lung fibroblasts and its effect on lung fibroblast proliferation.

TLR4 mRNA (A, real-time PCR) and protein (B, Western blot) expression in lung fibroblasts at 72 hours after 1 µg/mL LPS challenge. Effect of siRNA-mediated knockdown of TLR4 (1×10⁸ TU/mL for 48 hours) on lung fibroblast proliferation (C, BrdU assay). * p<0.05 vs. negative control group (Column 2); ^† p<0.05 vs. positive control group (Column 3). Columns represent mean values and error bars represent SD. Blots are representative of three independent experiments.

Figure 3. Expression of PTEN in lung fibroblasts after LPS challenge or PI3K inhibition.

PTEN mRNA (A and B, real-time PCR) and protein (C and D, Western blot) expression in lung fibroblasts at 72 hours after 1 µg/mL LPS challenge (A, C) or PI3K-Akt pathway inhibition (B, D). siRNA-mediated knockdown of TLR4 (1×10⁸ TU/mL for 48 hours) was used to assess the effect of TLR4 on PTEN expression in lung fibroblasts. Ly294002 (50 µmol/L for one hour) was applied to determine whether PTEN expression is regulated by PI3K activation. * p<0.05 vs. positive control group (A and C, Column 3). * p<0.05 vs. negative control group (B and D, Column 1); ^† p<0.05 vs. positive control group (Column 2). Columns represent mean values and error bars represent SD. Blots are representative of three independent experiments.

Figure 4. Effect of PI3K-Akt pathway activation and PTEN expression on lung fibroblast proliferation.

Activation of PI3K-Akt pathway (A, Western blot) in lung fibroblasts at 72 hours after 1 µg/mL LPS challenge, as detected by expression of Ser473 phospho-Akt. siRNA-mediated TLR4 knockdown (1×10⁸ TU/mL for 48 hours) was used to assess the effect of TLR4 on PI3K-Akt pathway activation. PI3K inhibitor Ly294002 (50 µmol/L for one hour) was used to assess the effect of PI3K-Akt pathway on lung fibroblast proliferation. * p<0.05 vs. negative control group (Column 2); ^† p<0.05 vs. positive control group (Column 3). Activation of PI3K-Akt pathway in lung fibroblasts (B, Western blot), as detected by expression of Ser473 phospho-Akt after treatment with different concentrations of PTEN inhibitor bpV(phen) for 0.5 hour. * p<0.05 vs. negative control group (Column 1). DNA synthesis (C, BrdU assay) in LPS-challenged lung fibroblasts after bpV(phen) (1 µM for 0.5 hour) and/or Ly294002 (50 µmol/L for one hour) treatment. * p<0.05 vs. negative control group (Column 1); ^† p<0.05 vs. positive control group (Column 2). Columns represent mean values and error bars represent SD. Blots are representative of three independent experiments.