OC-028 Identification of SOX9 as a novel mechanism to explain aspects of liver fibrosis

Abstract

<h3>Introduction</h3> Fibrosis of the liver is characterised by progressive accumulation of extracellular matrix (ECM) proteins and is a major cause of morbidity and mortality in the UK. Several cell-types are responsible for this, but a major role is played by activation of the hepatic stellate cell (HSC). We have identified ectopic expression of the Sry-box transcription factor, SOX9, in activated HSCs as a novel mechanism to explain aspects of liver fibrosis. <h3>Methods</h3> Livers were collected from carbon tetrachloride induced fibrotic and control rats and processed for fixed tissue. Rat hepatic stellate cells (HSCs) were isolated using established perfusion techniques and cultured on tissue culture plastic to activate over 4, 7 and 10 days. The immortalised human LX2 cell line was also used. <h3>Results</h3> In fixed fibrotic rat livers α-smooth muscle actin (α-sma) was detected in activated HSCs with collagen type 1 (Col1) rich fibrotic tracts found disrupting normal tissue architecture. Nuclear Sox9 staining was detected in the same regions as α-sma and Col1. In quiescent HSCs isolated from rat liver, negative for α-sma and col1, Sox9 was absent. However during activation of HSCs, both Sox9 and Col1 were robustly expressed. In these activated Col1-expressing HSCs, Sox9 localised to the nucleus surrounded by α-sma positive cytoplasm. SOX9 knockdown in activated HSCs using RNA interference caused a commensurate reduction in Col1 protein expression (∼60%). The pro-fibrotic cytokine transforming growth factor β (TGF-β) induced expression of SOX9 by ∼3-fold in rat HSCs and ∼2.5-fold in the human stellate cell line LX2s. <h3>Conclusion</h3> We have identified SOX9 as a novel mediator of ECM in liver fibrosis. Our data demonstrate that SOX9 expression occurs during activation of HSCs, a major cell type responsible for liver fibrosis, when, under the influence of TGF-β signalling it causes Col1 production, the predominant collagen deposited in organ fibrosis. Lessening SOX9 levels similarly reduced Col1 production. These data suggest targeted reduction of SOX9 offers potential therapeutic application to ameliorate fibrosis and related conditions of the liver.