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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 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.
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pone-0112698-g002: 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.

Mentions: To assess the effect of ethanol on the proliferation of the hESC-derived hepatic progenitor cells, these progenitor cells were exposed to ethanol for 8 days, and their cell cycle distribution was determined by flow cytometry. Ethanol treatment increased the population in G1 phase and reduced the population in S phase (Figure 2B) compared to the control cells (Figure 2A), suggesting that a cell cycle arrest at G1/S phase was induced in ethanol-exposed cells. Since G1/S transition is controlled by CDK4/6-cyclin D complexes, we next assessed the level of cyclin D1 in the progenitor cells with or without ethanol treatment by qPCR and Western blot. Consistent with the G1/S phase arrest, the level of cyclin D in ethanol exposed cells was reduced (Figure 2C and D). Thus, ethanol induced a moderate growth arrest at the G/S phase in hESC-derived hepatic progenitor cells associated with a decreased cyclin D 1 expression.


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 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.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0112698-g002: 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.
Mentions: To assess the effect of ethanol on the proliferation of the hESC-derived hepatic progenitor cells, these progenitor cells were exposed to ethanol for 8 days, and their cell cycle distribution was determined by flow cytometry. Ethanol treatment increased the population in G1 phase and reduced the population in S phase (Figure 2B) compared to the control cells (Figure 2A), suggesting that a cell cycle arrest at G1/S phase was induced in ethanol-exposed cells. Since G1/S transition is controlled by CDK4/6-cyclin D complexes, we next assessed the level of cyclin D1 in the progenitor cells with or without ethanol treatment by qPCR and Western blot. Consistent with the G1/S phase arrest, the level of cyclin D in ethanol exposed cells was reduced (Figure 2C and D). Thus, ethanol induced a moderate growth arrest at the G/S phase in hESC-derived hepatic progenitor cells associated with a decreased cyclin D 1 expression.

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