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

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© Author(s) (or their employer(s)) 2019. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.

This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/.

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Glial cell line-derived neurotrophic factor (GDNF) induces hepatic stellate cell (HSC) activation via the activin receptor-like kinase 5 (ALK5)/Smad pathway. Western blot analysis for (p-)AKT and (p-)Erk and semiquantification. Human (A) and mouse (B) HSCs were treated with GDNF (10 ng/mL) for the indicated time. (C) Western blot analysis for α-SMA and Col1A1. AKT small interfering RNA (siRNA) was transfected into human HSCs for 12 hours and then the cells were treated with 10 ng/mL GDNF for an additional 48 hours. (D) Western blot analysis for α-SMA and Col1A1. Human HSCs were treated with MK2206 2HCL (0.5 µM) for 1 hour, followed by 10 ng/mL GDNF for an additional 48 hours. (E) Western blot analysis for (p-)AKT and (p-)Smad2. Human HSCs were transfected with GFRα1 siRNA for 48 hours and subsequently treated with 10 ng/mL GDNF for an additional 2 hours. (F) Western blot analysis for (p-)AKT and (p-)Smad2. Human HSCs were treated with RPI-1 (60 µM) for 1 hour, followed by treatment with 10 ng/mL GDNF for additional 2 hours. (G, H) GFRα1 and Ret mRNA expression in 165 human liver specimens. The levels of target mRNAs were normalised to that of 18S rRNA (F0=13; F1=35; F2=61; F3=40; F4=16). (I, J) Western blot analysis for (p-)Smad2 and (p-)Smad3 and semiquantification. Human (I) and mouse (J) HSCs were treated with GDNF (10 ng/mL) as indicated. (K) Western blot analysis for Smad2 and Smad3. Human HSCs were treated with GDNF (10 ng/mL) for 2 hours, and nuclear and cytoplasmic extracts were tested for Smad2/3 levels. (L, M) Western blot analysis for α-SMA and Col1A1. Human HSCs were transfected with siRNA for Smad2 or Smad3 for 12 hours and then treated with GDNF (10 ng/mL) for additional 48 hours. (N) Western blot analysis for α-SMA and Col1A1. Human HSCs were treated with LY2157299 (0.5 µM) for 1 hour and then GDNF (10 ng/mL) for an additional 48 hours. (O) Western blot analysis for (p-)AKT and (p-)Smad2. Human HSCs were treated with SB431542 (10 µM) for 1 hour and then GDNF (10 ng/mL) for an additional 2 hours. (P) Western blot analysis for (p-)Smad2 and (p-)Smad3. Human HSCs were transfected with ALK5 siRNA for 12 hours and then GDNF (10 ng/mL) for an additional 48 hours. (Q, R) mRNA expression of ALK5 in 165 human liver specimens. The level of ALK mRNAs was normalised to that of 18S rRNA (F0=13; F1=35; F2=61; F3=40; F4=16). Correlation analysis between ALK5 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; one-way analysis of variance with the non-parametric Kruskal-Wallis test was used in parts A, B, G–I, J and K, and the non-parametric correlation (Spearman’s) two-tailed test was used in part R.