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

Gao W, Zhou P, Ma X, Tschudy-Seney B, Chen J, Magner NL, Revzin A, Nolta JA, Zern MA, Duan Y - PLoS ONE (2014)

Bottom Line: 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.At the same time, the WNT signaling pathway was also reduced in the cells exposed to ethanol.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, College of Biology, Hunan University, Changsha, Hunan, China; Department of Internal Medicine, University of California Davis Medical Center, Sacramento, California, United States of America; Institute for Regenerative Cures, University of California Davis Medical Center, Sacramento, California, United States of America.

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.

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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.
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pone-0112698-g005: 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.

Mentions: In order to investigate whether specific signaling pathways are involved in hepatic differentiation in the presence of ethanol, the Signal Transduction PathwayFinder PCR Array containing 84 genes representing 10 pathways was employed to assess the effects of ethanol treatment on the progenitor cells. Four genes including Wnt1 were found to be down-regulated by ethanol (Figure 5A, Figure S1). Since the Wnt signaling pathway has been associated with hepatocyte and cholangiocyte differentiation [29], [30], [31], we sought to further determine whether this pathway was indeed affected by ethanol. The reduced expression of Wnt 1 in the ethanol- treated progenitor cells was confirmed by qPCR and Western blots (Figure 5B and C). Phosphorylation of GSK-3β, β-catenin nuclear translocation, and TCF1 up-regulation have been shown to be involved in the Wnt pathways [32], [33], [34]. The phosphorylation of GSK-3β is essential for de-phosphorylation of β-catenin and release of β-catenin from the destructive complex to translocate into the nucleus [32]. To further assess whether these downstream Wnt signaling components are altered by ethanol, the level of phosphorylated GSK-3β, TCF1 and nuclear β-catenin in the progenitor cells treated with or without ethanol was determined by Western blot analysis. We found that ethanol markedly reduced the level of phosphorylated GSK-3β as well as the level of TCF1 and nuclear β-catenin (Figure 5C). Thus, our data demonstrated that the WNT pathway was attenuated during early hepatic differentiation in the presence of ethanol.


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

Gao W, Zhou P, Ma X, Tschudy-Seney B, Chen J, Magner NL, Revzin A, Nolta JA, Zern MA, Duan Y - PLoS ONE (2014)

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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4231066&req=5

pone-0112698-g005: 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.
Mentions: In order to investigate whether specific signaling pathways are involved in hepatic differentiation in the presence of ethanol, the Signal Transduction PathwayFinder PCR Array containing 84 genes representing 10 pathways was employed to assess the effects of ethanol treatment on the progenitor cells. Four genes including Wnt1 were found to be down-regulated by ethanol (Figure 5A, Figure S1). Since the Wnt signaling pathway has been associated with hepatocyte and cholangiocyte differentiation [29], [30], [31], we sought to further determine whether this pathway was indeed affected by ethanol. The reduced expression of Wnt 1 in the ethanol- treated progenitor cells was confirmed by qPCR and Western blots (Figure 5B and C). Phosphorylation of GSK-3β, β-catenin nuclear translocation, and TCF1 up-regulation have been shown to be involved in the Wnt pathways [32], [33], [34]. The phosphorylation of GSK-3β is essential for de-phosphorylation of β-catenin and release of β-catenin from the destructive complex to translocate into the nucleus [32]. To further assess whether these downstream Wnt signaling components are altered by ethanol, the level of phosphorylated GSK-3β, TCF1 and nuclear β-catenin in the progenitor cells treated with or without ethanol was determined by Western blot analysis. We found that ethanol markedly reduced the level of phosphorylated GSK-3β as well as the level of TCF1 and nuclear β-catenin (Figure 5C). Thus, our data demonstrated that the WNT pathway was attenuated during early hepatic differentiation in the presence of ethanol.

Bottom Line: 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.At the same time, the WNT signaling pathway was also reduced in the cells exposed to ethanol.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, College of Biology, Hunan University, Changsha, Hunan, China; Department of Internal Medicine, University of California Davis Medical Center, Sacramento, California, United States of America; Institute for Regenerative Cures, University of California Davis Medical Center, Sacramento, California, United States of America.

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.

Show MeSH
Related in: MedlinePlus