Abstract
BackgroundAbnormal and uncontrolled proliferation of lung fibroblasts may contribute to pulmonary fibrosis. Lipopolysaccharide (LPS) can induce fibroblast proliferation and differentiation through activation of phosphoinositide3-Kinase (PI3-K) pathway. However, the detail mechanism by which LPS contributes to the development of lung fibrosis is not clearly understood. To investigate the role of phosphatase and tensin homolog (PTEN), a PI3-K pathway suppressor, on LPS-induced lung fibroblast proliferation, differentiation, collagen secretion and activation of PI3-K, we transfected PTEN overexpression lentivirus into cultured mouse lung fibroblasts with or without LPS treatment to evaluate proliferation by MTT and Flow cytometry assays. Expression of PTEN, alpha-smooth muscle actin (alpha-SMA), glycogen synthase kinase 3 beta (GSK3beta) and phosphorylation of Akt were determined by Western-blot or real-time RT-PCR assays. The PTEN phosphorylation activity was measured by a malachite green-based assay. The content of C-terminal propeptide of type I procollagen (PICP) in cell culture supernatants was examined by ELISA.ResultsWe found that overexpression of PTEN effectively increased expression and phosphatase activity of PTEN, and concomitantly inhibited LPS-induced fibroblast proliferation, differentiation and collagen secretion. Phosphorylation of Akt and GSK3beta protein expression levels in the LPS-induced PTEN overexpression transfected cells were significantly lower than those in the LPS-induced non-transfected cells, which can be reversed by the PTEN inhibitor, bpV(phen).ConclusionsCollectively, our results show that overexpression and induced phosphatase activity of PTEN inhibits LPS-induced lung fibroblast proliferation, differentiation and collagen secretion through inactivation of PI3-K-Akt-GSK3beta signaling pathways, which can be abrogated by a selective PTEN inhibitor. Thus, expression and phosphatase activity of PTEN could be a potential therapeutic target for LPS-induced pulmonary fibrosis. Compared with PTEN expression level, phosphatase activity of PTEN is more crucial in affecting lung fibroblast proliferation, differentiation and collagen secretion.
Highlights
Abnormal and uncontrolled proliferation of lung fibroblasts may contribute to pulmonary fibrosis
phosphatase and tensin homolog (PTEN) expression and dephosphorylation activity in mouse lung fibroblasts transfected with Pten overexpression lentivirus In the Pten-transfected primary cultured mouse lung fibroblasts, overexpression of PTEN and changes in PTEN dephosphorylation activity was detected by measuring
In Pten-transfected cells treated with LPS, treatment with the PTEN inhibitor 1 μM bpV(phen) 72 h after the LPS challenge(PTENLPS/bpV(phen) group) significantly reduced PTEN dephosphorylation activity, but had no effect on Pten mRNA and PTEN protein expression levels, compared to Pten-transfected cells treated with LPS but without the PTEN inhibitor(PTENLPS group, Figure 1B, C)
Summary
Abnormal and uncontrolled proliferation of lung fibroblasts may contribute to pulmonary fibrosis. Lipopolysaccharide (LPS) can induce fibroblast proliferation and differentiation through activation of phosphoinositide3-Kinase (PI3-K) pathway. To investigate the role of phosphatase and tensin homolog (PTEN), a PI3-K pathway suppressor, on LPS-induced lung fibroblast proliferation, differentiation, collagen secretion and activation of PI3-K, we transfected PTEN overexpression lentivirus into cultured mouse lung fibroblasts with or without LPS treatment to evaluate proliferation by MTT and Flow cytometry assays. Our previous studies have shown that LPS was able to directly induce secretion of collagen in primary cultured mouse lung fibroblasts via Toll-like receptor 4 (TLR4)-mediated activation of the phosphoinositide3-kinase-Akt (PI3-K-Akt) pathway [8,9]. LPS was reported to induce fibroblasts proliferation [10], down-regulate phosphatase and tensin homolog (PTEN) expression [11,12]
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