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. 2019 Jun 6;68(12):2214–2227. doi: 10.1136/gutjnl-2018-317872

Figure 8.

Figure 8

The glial cell line-derived neurotrophic factor (GDNF)/activin receptor-like kinase 5 (ALK5)/Ret/Smad2/3 circuit promotes hepatic stellate cell (HSC) activation. (A) Real-time PCR Gdnf mRNA detection after transforming growth factor-β (TGFβ) treatment in human and mouse HSCs. (B) Real-time PCR for Tgfβ and Gdnf mRNA in carbon tetrachloride (CCl4)-induced liver fibrosis. (C) GDNF mRNA was not induced in response to TGFβ and GDNF stimulation, when Smad2/3 were depleted using specific small interfering RNAs in LX2 cells. (D) Schematic diagram of the putative Smad2/3 binding site within the human GDNF promoter. LX2 cells were subjected to ChIP assays with anti-Smad2/3 or IgG antibodies. Representative results from three independent experiments are shown. mRNA expression of Smad2 (E) and Smad3 (F) in 165 human liver specimens. The levels of Smad2/3 mRNAs were normalised to that of 18S rRNA (F0=13; F1=35; F2=61; F3=40; F4=16). Correlation analysis between Smad2/3 and GDNF mRNA expression in patients with liver fibrosis. Spearman’s correlation coefficients (r), p values and the number of patients are indicated. Bars indicate the mean±SD of three independent experiments; n=3 per group; the t-test with the non-parametric Mann-Whiney U test was used in part A, one-way analysis of variance with the non-parametric Kruskal-Wallis test was used in parts C and E, and the non-parametric correlation (Spearman’s) two-tailed test was used in parts E and F. (G) Graphical summary of GDNF fibrogenic signalling in HSCs. HSCs express the GDNF receptors GFRα1 and Ret, whereas only Ret is upregulated in patients with liver fibrosis. GDNF induces AKT and Smad2/3 phosphorylation as a short-term response. As downstream events, GDNF induces GDNF transcription as an autocrine feedback stimulation and fibrogenic gene expression, as exemplified by α-SMA and Col1. ALK5 and Ret, but not GFRα1, are required for AKT/Smad2/3 activation and subsequent GDNF and α-SMA/Col1 expression. ALD, alcoholic liver disease; NASH, non-alcoholic steatohepatitis.