Effect And Mechanism Of Autophagy In Biliary Differentiation Of Hepatic Progenitor Cells

BackgroundAutophagy, characterized as an important process of cellular homeostasis, takes place in all eukaryotic cells and involves the sequestration of cytoplasmic components in double membrane autophagosomes, which is a basic mechanism involving cell degradation of unnecessary or dysfunctional cellular components by lysosomes. Recent studies have demonstrated autophagy plays important roles in self-renewal and differentiation of normal stem cells. However, how autophagy contributes to self-renewal and differentiation of hepatic progenitor cells is not well understood.Hepatic progenitor cells(HPCs), located in portal area, are very few and in quiescent condition, when the liver is chronically injured, adult liver cells are damaged, proliferation is inhibited, HPCs are activated and differentiate into mature hepatocytes and biliary cells to repair liver damage. Previous reports showed that the process of hepatic progenitor cell differentiation was controlled by many signaling pathways, such as Notch, Wnt, BMP, TGF-B, HGF and FGF signaling pathways.Through the model of biliary differentiation of hepatic progenitor cells, to observe the change of autophagy level in the biliary differentiation process of hepatic progenitor cells. Further applying starvation and mTOR inhibitors rapamycin induced autophagy to study the role of autophagy in the biliary differentiation process of hepatic progenitor cells. Finally, we discuss the mechanism of autophagy in the biliary differentiation process of hepatic progenitor cells.Methods1. Using a well-established rat hepatic progenitor cell lines called WB-F344,which was treated with 3.75 mM sodium butyrate(SB) to promote thedifferentiation of WB-F344 along the biliary phenotype. The morphological change of hepatic progenitor cell WB-F344 was observed by microscope, cell proliferation experiment and the expression of biliary lineage markers of WB-F344 cells were detected by RT-PCR when cultured with 3.75 mM Sodium butyrate(SB). At the same time, we detected the level of autophagy during the whole stage of differentiation in different time points(0 d to 5 d) by Western blot, electron microscopy.2. WB-F344 cells were treated with SB for 4 days, and co-cultured DMSO,rapamycin or starvation at the fifth day. The expression of biliary lineage markers were detected by RT-PCR and Western blot.3. Further to explore the mechanism of autophagy in differentiation of hepatic progenitor cell. Autophagy and Notch1 signaling pathway were detected by Western blot, RT-PCR and Immunofluorescence.ResμLts1. Autophagy was decreased in the early stage of biliary differentiation of WB-F344 cells and maintained a low level at the late stage. Activation of autophagy by rapamycin or starvation suppressed the biliary differentiation of WB-F344 cells.2. Notch1 signaling pathway was up-regulated in the biliary differentiation of WB-F344,The inhibition of Notch signaling pathway suppressed biliary differentiation.3.Activation of autophagy by rapamycin or starvation attenuated Notch1 signaling pathway.ConclusionThese results demonstrate that autophagy regulates biliary differentiation of hepatic progenitor cells through Notch1 signaling pathway.