TGF-? Modulates Hepatic Progenitor Cells Migration Through PI3K/AKT/mTOR/p70S6K Signaling

BackgroundWhen the liver experiences chronic/severe injury,adult hepatic progenitor cells(HPCs)located in the terminal bile duct and Hering tube of the portal vein area are activated,proliferated,differentiate into mature hepatocytes and bile duct cells,and migrate into the hepatic lobules,so as to repair the damaged liver.Studies have shown that HPCs can differentiate into mature hepatocytes in severe viral hepatitis,whereas they can differentiate into cholangiocarcinoma cells in primary biliary cirrhosis and other diseases.Animal experiments have also shown that HPCs can differentiate into HNF-4a-positive hepatocytes and CK19-positive cholangio-cytomas.The activation of HPCs is closely related to the severity of liver injury.At the same time,its activation,proliferation,differentiation,and migration are affected by various factors.The directional migration of HPCs is also an important part of liver regeneration and is regulated by various cytokines and signalings.It has been found that cytokines and inflammatory factors such as HGF,IL-6,and CTGF can regulate the biological function of HPCs.Hepatocyte Growth Factor(HGF)/c-Met are regulators of HPCs migration.C-Met-deficient mice have reduced HPCs activation,impaired migration,and decreased the number of differentiated liver cells.Yeoh et al found that IL-6 can promote the proliferation and migration of HPCs in mice,and ERK-1/2 activation can inhibit this reaction.Pi et al found that connective tissue growth factor(CTGF)can promote the proliferation and migration of HPCs in 2-acetylaminoanthraquinone/partial hepatectomy(2-AAF/PHx)rats.It has been reported that transforming growth factor beta(TGF-?)is a crucial regulator of chronic liver disease.It plays an important role in the process of liver injury,inflammation,regeneration,hepatic fibrosis and hepatocellular carcinoma.After severe hepatic injury,TGF-? has an inhibitory and pro-apoptotic effect on the growth of mature hepatocytes with the increase of TGF-?.However,HPCs are still able to initiate liver regeneration.Robert et al discovered that combination of TGF-?1 and EGF result in changes in cell morphology of HPCs.Yang et al transplanted HPCs pretreated with TGF-?1 into rats,and found that hepatic extracellular matrix increases and hepatic fibrosis rises.Based on the above studies,we believe that TGF-? can regulate the biological behavior of HPCs during liver regeneration.This study was to investigate the effect of TGF-? on the gene expression profile and migration ability of HPCs and its potential molecular mechanism.ObjectiveTo investigate the effect of TGF-? on the migration of HPCs and to explore the mechanism.MethodsPrimary HPCs were isolated from male wild-type C57BL/6J mice by two-step perfusion.HPCs were treated with different concentrations of TGF-?,morphological changes of which were observed under phase contrast microscope.Wound healing and Transwell assays were used to evaluate cell migration of HPCs.Gene expression profiling were used for detecting the gene expression of HPCs exposured to TGF-?.The differentially expressed genes were analyzed by functional enrichment and signal pathway analysis.TGF-? signaling Phospho antibody microarray PTG176 was used to evaluate TGF-? on the regulation of HPCs.We observed the protein level of PI3K/AKT/mTOR/p70S6K signaling,and the localization of various signal molecules in HPCs.The signaling pathway inhibitors LY294002,MK2206,Rapamycin,and LY2584702 were used to further confirm the mechanism of TGF-?on the migration of HPCs.Results1.TGF-? changed HPCs from oval into long shuttle.Wound healing and Transwell assays revealed that TGF-? can promote the migration of HPCs.2.The results of RNA-sequencing results showed that TGF-? regulated the gene expression of HPCs.The differentially expressed genes were involved in the regulation of cell morphology and cell migration.KEGG signaling pathway analysis revealed that differentially expressed genes are related to ECM-receptor interaction and PI3K-AKT signaling.3.Phosphorylation antibody chip showed that TGF-? up upregulated the phosphorylation levels of myc,Abll,AKT1,Shc,PKC zata and Smadl.Among them,AKT1 is in agreement with the PI3K-AKT signaling in our previous RNA-sequencing signal pathway analysis.Western blot showed that the levels of PI3K110,pAKTl,mTOR and p70S6K were significantly increased in HPCs treated with TGF-?.At the same time,immunofluorescence results showed that p70S6K,Akt1 and PI3K co-localized with F-actin.4.The signal pathway inhibitors LY294002,MK2206,Rapamycin and LY2584702 can can inhibit TGF-?-mediated morphological changes and migration of HPCs.ConclusionsTGF-? promotes HPCs migration through the PI3K/AKT/mTOR/p70S6K signaling.The p70S6K,AKT1 and PI3K signaling molecules are involved in the regulation of HPC morphology and migration.