1.Rapamycin Upregulates Connective Tissue Growth Factor Expression In Hepatic Progenitor Cells Through TGF-?-Smad2 Dependent Signaling 2.Autocrine Transforming Growth Factor-?/activin A-Smad Signaling Induces Hepatic Progenitor Cells Undergoing Partial E

Part 1: Rapamycin upregulates connective tissue growth factor expression in hepatic progenitor cells through TGF-?-Smad2 dependent signalingObjectives: Rapamycin(sirolimus)is a m TOR kinase inhibitor and is widely used as immunosuppressive drug to prevent graft rejection in organ transplantation currently.Whereas recent investigations reported that it had profibrotic effect in the progression of organ fibrosis,and its precise role in the liver fibrosis is still poorly understood.Methods: Western blot analysis was performed to investigate the expression of CTGF and the activation of m TOR signaling,PI3K-Akt signaling and TGF-?-Smad signaling.We used dual-luciferase reporter assay to measure the activation of CTGF promoter and Smad binding element.We used lentivirus to stably knock down the expression of m TOR,Raptor,Rictor,Smad4,Smad2 and Smad3,and used an adenovirus to overexpress Smad3 in HPCs,thus we can study the role of those proteins in rapamycin's upregulation of CTGF.We used various kinase inhibitors to investigate the role of rapamycin activated signaling.Fluorimetric probe DCFH-DA was used to detect the generation of ROS.Results: Here we showed that rapamycin could upregulate connective tissue growth factor(CTGF)expression at the transcriptional level in hepatic progenitor cells(HPCs).Using lentivirus mediated small hairpin RNA(sh RNA)we demonstrated that knockdown of m TOR,Raptor and Rictor mimicked the effect of rapamycin treatment.Mechanistically,inhibition of m TOR activity with rapamycin resulted in a hyperactive PI3K-Akt pathway,whereas this activation inhibited the expression of CTGF in HPCs.Besides,rapamycin activated the TGF-?-Smad signaling,while TGF-? receptor type I(TGF?RI)serine/threonine kinase inhibitors completely blocked the effects of rapamycin on HPCs.Moreover,we found that Smad2 is involved in the induction of CTGF through rapamycin activated TGF-?-Smad signaling.Knockdown of Smad4 expression exhibited a partial effect,whereas knockdown of Smad3 expression had no effect on rapamycin-induced CTGF expression.Furthermore,rapamycin induced ROS generation and latent TGF-? activation which contribute to TGF-?-Smad signaling.Conclusion: This study demonstrates that rapamycin upregulates CTGF in HPCs to aggravate liver fibrosis and suggests it should be handled carefully for clinical uses.Part 2: Autocrine transforming growth factor-?/activin A-Smad signaling induces hepatic progenitor cells undergoing partial epithelial-mesenchymal transition statesHepatic progenitor cells(HPCs)are a subpopulation of cells which was usually expanded in chronic liver injury and are contributed to liver regeneration through differentiating into hepatocytes and cholangiocytes.Epithelial-mesenchymal transition is a dynamic process which is important for the progression of liver fibrosis and cancer initiation.This study demonstrated that LE/6 and WB-F344 cells,both of which were HPC derived cell lines,were undergoing partial epithelial-mesenchymal transition states,which was indicated by the co-expression of epithelial markers(E-cadherin and zona occludin 1),and mesenchymal markers(vimentin,fibronectin,collagen 1and a-SMA).Furthermore,autocrine TGF-? and activin A signaling contributed to the maintenance of partial EMT in HPCs.In addition,Smad signaling,a classic downstream signaling cascade of both TGF-? and activin A,also participated in the partial EMT.These findings revealed the existence of partial EMT states in HPCs and confirmed some partial EMT related autocrine signaling cascades,and may help to further the understanding and explore the functional role of HPCs in the process of hepatic fibrosis and liver cancer initiation.