Antagonistic Roles Of TGF-β/Smad And TGF-β/Non-Smad Signaling In Inducing EMT Of Hepatic Progenitor Cells(HPCs)

Objectives:To investigate the roles of TGF- β /Smad and TGF- β /non-Smad signaling induced Epithelium-to-mesenchymal transition of hepatic progenitor cells(HPCs)Methods: 1.WB-F344 and LE/6 cells were treated with TGF-β1(10ng/ml) for three days,We observed morphologic changes by fluorescent staining of F-actin and analysised expression of epithelial or mesenchymal markers through western blot.2.WB-F344 and LE/6 cells were treated with TGF- β 1(10ng/ml) for the indicated times. Lysates were subjected to Western blot analyses with antibodies against indicated proteins of MAPK and Smad signaling.3.TGF- β /non-Smad signaling include ERK signaling 、 JNK signaling and P38 MAPK signaling.WB-F344 and LE/6 cells were treated with TGF-β1 and kinase inhibitors( u0126, SP600125,SB203580).We observed morphologic changes by fluorescent staining of F-actin and analysised expression of epithelial or mesenchymal markers through western bloting.4.We established the smad4 knock-down WB-F344 and LE-6 cell lines using lentivirus,and detected the expression of smad4 by Western Blot.We further performed Western blotting to measure the phosphorylation of Erk, c-jun and p38,and epithelial or mesenchymal markers between control group.Result: 1.WB-F344 and LE/6 cells were treated with TGF-β1(10ng/ml) for three days,we first carried out fluorescent staining of F-actin and found that the morphologic is not obviously changed.Consistently, TGF- β induced the Slightly changed expression of epithelial or mesenchymal markers.2.We works showed that besides activation of Smad-independent signaling,TGF-beta induced activated Smad signaling such as MAPK signaling.3.WB-F344 and LE/6 cells were treated with TGF-β1 and kinase inhibitors(u0126,sp600125,SB203580).we carried out fluorescent staining of F-actin and found that TGF-β stimulated actin cytoskeleton reorganization, by inducing membrane ruffling,lamellipodia and stress fibres.Consistently, TGF-β induced the decreased expression of epithelial markers(E-cadherin) and increased expression of mesenchymal markers( vimentin),as evidenced by western blot analysis.4.The expression of the phosphorylation of Erk, c-jun and p38 are increased and increased expression of epithelial markers(E-cadherin) and decreased expression of mesenchymal markers( vimentin) after knock down of smad4 between control groupConclusions: TGF- β drives epithelial-mesenchymal transition(EMT) of hepatic progenitor cells(HPCs).Although it is not obvious,interestingly,we found that Antagonistic roles of TGF- β /Smad and TGF- β /non-Smad signaling in hepatic progenitor cells(HPCs).This finding contributes to the stem cell therapy, and it will be argeted therapy of liver fibrosis or tumour.