Ethanol negatively regulates hepatic differentiation of hESC by inhibition of the MAPK/ERK signaling pathway in vitro

Abstract

Background: Alcohol insult triggers complex events in the liver, promoting fibrogenic/inflammatory signals and in more advanced cases, aberrant matrix deposition. It is well accepted that the regenerative capacity of the adult liver is impaired during alcohol injury. The liver progenitor/stem cells have been shown to play an important role in liver regeneration -in response to various chronic injuries; however, the effects of alcohol on stem cell differentiation in the liver are not well understood.

Methods: We employed hepatic progenitor cells derived from hESCs to study the impact of ethanol on hepatocyte differentiation by exposure of these progenitor cells to ethanol during hepatocyte differentiation.

Results: We found that ethanol negatively regulated hepatic differentiation of hESC-derived hepatic progenitor cells in a dose-dependent manner. There was also a moderate cell cycle arrest at G1/S checkpoint in the ethanol treated cells, which is associated with a reduced level of cyclin D1 in these cells. Ethanol treatment specifically inhibited the activation of the ERK but not JNK nor the p38 MAP signaling pathway. At the same time, the WNT signaling pathway was also reduced in the cells exposed to ethanol. Upon evaluating the effects of the inhibitors of these two signaling pathways, we determined that the Erk inhibitor replicated the effects of ethanol on the hepatocyte differentiation and attenuated the WNT/β-catenin signaling, however, inhibitors of WNT only partially replicated the effects of ethanol on the hepatocyte differentiation.

Conclusion: Our results demonstrated that ethanol negatively regulated hepatic differentiation of hESC-derived hepatic progenitors through inhibiting the MAPK/ERK signaling pathway, and subsequently attenuating the WNT signaling pathway. Thus, our finding provides a novel insight into the mechanism by which alcohol regulates cell fate selection of hESC-derived hepatic progenitor cells, and the identified pathways may provide therapeutic targets aimed at promoting liver repair and regeneration during alcoholic injury.

Figure 1. Effects of ethanol on the differentiation of hESC towards hepatocytes.

(A) Schematic illustration of the differentiation protocol and the time frame of ethanol treatment. (B) Immunostaining of albumin in the hESC-derived hepatocytes (hEH) at day 24 exposed to the indicated doses of ethanol (x100). (C) Cell viability was assessed at day 24 by MTT assay. (D, E) qPCR was performed to determine the expression of liver genes, ALB and ASGPR1, C/EBPα, and HNF1α at day 24 exposed to the indicated doses of ethanol. (F) Secreted ALB levels in the culture media by hEH were assessed by ELISA at day 24 with or without ethanol treatment. (G) Dynamic expression changes of metabolizing enzymes, CYP3A4, CYP7A1, and UTG1A6 in hEH were evaluated by qPCR at different time points after differentiation in the presence or absence of ethanol at 100 mM. (*p<0.05 vs. no EtOH; #p<0.005 vs. no EtOH).

Figure 2. Effects of ethanol on cell proliferation.

(A, B) Flow cytometry to assess the amount of DNA in the G1, S, and G2 phases during the cell cycle at day 8 after differentiation in the presence or absence of ethanol at 100 mM. (C) qPCR was performed to evaluate the expression of cyclin D1 at days 4 and 8 after differentiation in the presence or absence of ethanol at 100 mM (*p<0.05 vs. no EtOH). (D) Western blot analysis was used to determine protein expression of albumin (ALB), and cyclin D1 at day 8 after differentiation in the presence or absence of ethanol at 100 mM. GAPGH was used as housekeeping gene control.

Figure 3. Expression of ethanol metabolizing genes during hepatocyte differentiation of hESC.

qRT-PCR was employed to evaluate the dynamic expression of alcohol dehydrogenases (ADHs), ADH1A, ADH1B and ADH4, and by aldehyde dehydrogenases (ALDH), ALDH2 during the hepatocyte differentiation from ESC-derived hepatic progenitor cells.

Figure 4. Effects of U0126, Erk inhibitor, on the differentiation of hESC towards hepatocytes.

(A) Western blot analysis was employed to determine the effect of ethanol on the MAPK signaling pathway at day 8 after differentiation in the presence or absence of ethanol at 100 mM. (B) qPCR was used to determine the expression of albumin (ALB), CYP3A4 (3A4), β-catenin (β-cat), and cyclin D1 (D1) at day 8 after differentiation in the presence or absence of U0126 at 10 µM (*p<0.05 vs. no inhibitor; #p<0.005 vs. no inhibitor). (C) qPCR and ELISA were employed to evaluate the relative expression levels of albumin (ALB) and secreted ALB into the medium in the same samples in the presence or absence of U0126 at 10 µM. (p<0.05 vs. no inhibitor). (D–G) Western blot analysis was performed to determine protein expression of albumin (ALB), phosphorylated Erk (p-ERK), phosphorylated GSK-3β at the Ser9 residue (p-GSK3β), and cyclin D1 cytoplasm, and β-catenin in nucleus at day 8 after differentiation in the presence or absence of U0126 at 10 µM. GAPDH and α-Tubulin were control housekeeping genes in cytoplasm and nucleus. (H) Western blot analysis was used to evaluate the expression of albumin (ALB), and cyclin D1 by supplementing high dose FGF-4 (F) or HGF (H) at day 8 after differentiation in the presence or absence of U0126 at 10 µM.

Figure 5. Effects of IWR-1-endo, a WNT1 inhibitor, on the differentiation of hESC towards hepatocytes.

(A) The entire view of the expression changes of 84 genes representing 10 pathways by PCR Array analysis at day 10 after differentiation in the presence or absence of ethanol at 100 mM. The names of the 84 genes and their expression fold changes are listed in Figure S1. (B) qPCR was performed to evaluate the expression of Wnt1 at 8 after differentiation in the presence or absence of ethanol at 100 mM (p<0.05 vs. no inhibitor). (C) Western blot analysis was used to determine protein expression of Wnt1, phosphorylated GSK-3β at the Ser9 residue, and TCF1 in cytoplasm, and β-catenin in nucleus at day 8 after differentiation in the presence or absence of ethanol at 100 mM. GAPGH and α-Tubulin were used to as housekeeping gene controls in the cytoplasm and nucleus respectively. (D) qPCR was used to measure the expression of albumin (ALB), alpha fetoprotein (AFP), CYP3A4 (3A4), and TCF1 at day 8 after differentiation in the presence or absence of IWR-1-endo at 0.2 µM (#p<0.005 vs. no inhibitor). (E–G) Western blot analysis was employed to determine protein expression of albumin (ALB), and TCF1 in cytoplasm, and β-catenin in nucleus at day 8 after differentiation in the presence or absence of IWR-1-endo, at 0.2 µM. GAPDH and α-Tubulin as control housekeeping genes in cytoplasm and nucleus.

Figure 6. Effects of ethanol on promotion of non-hepatocyte lineage and model for effects of ethanol.

Model for effects of ethanol on the differentiation of hESC towards hepatocytes.