RNA-seq from human hepatic stellate cells treated with TGF-ß
Background and Aims: Transforming growth factor (TGF-ß) induced activation of quiescent hepatic stellate cells (HSC) and their transformation to myofibroblasts is a key event in liver fibrosis and portal hypertension. GIPC (also referred to as synectin) is a downstream signal activation molecule of TGF-ß and other receptors. In this study, we sought to identify novel genes targeted by TGF-ß and GIPC and elucidate if and how they may contribute to liver fibrosis. Methods and Results: We performed sequential mRNA sequencing analysis on TGF-ß stimulated HSC and then on TGF-ß-stimulated HSC in presence and absence of GIPC knockdown. IGFBP-3, an insulin growth factor transport protein, emerged as a top activation target of both TGF-ß and GIPC, which was confirmed by qPCR, ELISA and Western blot (WB) analysis.  Targeted chromatin immunoprecipitation (ChIP) revealed that GIPC increases the histone 3 lysine 27 (H3K27) acetylation activating mark and concurrently decreases the H3K27 inhibitory trimethylation (H3K27m3) mark providing an epigenetic correlate to the gene regulation changes. In vivo, global knockout of IGFBP-3 mice resulted in attenuation of HSC activation markers and attenuation of portal pressure in response to chronic liver injury models.  Analysis of serum levels from cirrhotic patients also showed IGFBP-3 increase of more than 2-fold compared to healthy controls. Finally, in vitro mechanism studies revealed that IGFBP-3 promotes HSC migration through integrin dependent phosphorylation of AKT. Conclusion: TGF-ß upregulates IGFBP-3 through GIPC leading to increased HSC migration in vitro and promotes portal hypertension in vivo.  These studies support the role of IGFBP-3 as a potential pathophysiologic target or biomarker in chronic liver disease. Overall design: next generation RNAseq data from human hepatic stellate cells treated with TGFb compared to non-treated hepatic stellate cells reveals IGFBP3 as one of the top target genes downstream of TGFb