S136 Mtor Signalling Is An Essential Pathway For Tgf- 1 Induced sma And Collagen Gene Expression

Abstract

<h3></h3> Emerging evidence suggests a role for PI3K/mTOR signalling in the pathology of organ fibrosis. The aim of this study was to delineate PI3K/mTOR signalling in response to TGF-β₁stimulation of primary human lung fibroblasts (HLFs), and to investigate the role of this pathway in TGF-β₁ mediated myofibroblast differentiation and collagen synthesis. A time-course of SMAD 2/3 and Akt (Ser473) phosphorylation, the major downstream effector of the PI3K/mTOR pathway, was constructed to assess TGF-β₁ induced signalling kinetics in HLFs. TGF-β₁(1 ng/ml) induced rapid phosphorylation of SMAD2/3, peaking at 1 h, followed by Akt phosphorylation which peaked 12 h after initial stimulation. Maximal expression of <i>ACTA2</i> and <i>COL1A1</i> was observed 36 h after TGF-β₁ stimulation, correlating with the delayed time-course of Akt phosphorylation. To investigate the role of the PI3K/mTOR pathway in TGF-β₁ induced myofibroblast differentiation and collagen gene expression, HLFs were treated with pharmacological titrations of potent pathway inhibitors. Maximal Akt signalling and expression of <i>ACTA2 </i>and <i>COL1A1</i> were significantly inhibited by a dual PI3K/mTOR inhibitor, while SMAD phosphorylation was unaffected. Treatment with an ATP competitive mTOR inhibitor also resulted in significantly reduced Akt phosphorylation and expression of <i>ACTA2</i> and <i>COL1A1</i>, in response to TGF-β₁. In contrast, treatment of HLFs with either an allosteric or ATP competitive Akt inhibitor showed no inhibitory effect on TGF-β₁induced gene expression. These data suggest PI3 kinase/mTOR signalling is an important component in TGF-β₁ induced αSMA and collagen gene expression and that an mTOR dependent, Akt independent pathway mediates this functional response in primary HLFs.