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Genetic abolishment of hepatocyte proliferation activates hepatic stem cells.

Endo Y, Zhang M, Yamaji S, Cang Y - PLoS ONE (2012)

Bottom Line: Purified OCs express both hepatocyte and cholangiocyte markers, form colonies in vitro, and differentiate to hepatocytes after transplantation.Microarray analysis reveals several previously unrecognized markers, including Reelin, enriched in oval cells.Here we report a genetic model in which irreversible inhibition of hepatocyte duplication results in HSC-driven liver regeneration.

View Article: PubMed Central - PubMed

Affiliation: Signal Transduction Program, Sanford-Burnham Medical Research Institute, La Jolla, California, United States of America.

ABSTRACT
Quiescent hepatic stem cells (HSCs) can be activated when hepatocyte proliferation is compromised. Chemical injury rodent models have been widely used to study the localization, biomarkers, and signaling pathways in HSCs, but these models usually exhibit severe promiscuous toxicity and fail to distinguish damaged and non-damaged cells. Our goal is to establish new animal models to overcome these limitations, thereby providing new insights into HSC biology and application. We generated mutant mice with constitutive or inducible deletion of Damaged DNA Binding protein 1 (DDB1), an E3 ubiquitin ligase, in hepatocytes. We characterized the molecular mechanism underlying the compensatory activation and the properties of oval cells (OCs) by methods of mouse genetics, immuno-staining, cell transplantation and gene expression profiling. We show that deletion of DDB1 abolishes self-renewal capacity of mouse hepatocytes in vivo, leading to compensatory activation and proliferation of DDB1-expressing OCs. Partially restoring proliferation of DDB1-deficient hepatocytes by ablation of p21, a substrate of DDB1 E3 ligase, alleviates OC proliferation. Purified OCs express both hepatocyte and cholangiocyte markers, form colonies in vitro, and differentiate to hepatocytes after transplantation. Importantly, the DDB1 mutant mice exhibit very minor liver damage, compared to a chemical injury model. Microarray analysis reveals several previously unrecognized markers, including Reelin, enriched in oval cells. Here we report a genetic model in which irreversible inhibition of hepatocyte duplication results in HSC-driven liver regeneration. The DDB1 mutant mice can be broadly applied to studies of HSC differentiation, HSC niche and HSCs as origin of liver cancer.

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Deletion of p21 partially restores proliferation of DDB1-deficient hepatocytes and alleviates OC activation.(A) Western blot for some substrates of DDB1-Cul4A ubiquitin ligase using lysates of hepatocytes isolated from DDB1F/F and DDB1F/F;Alb-Cre+/+ mice. (B) Western blot for DDB1 and p21 using lysates of cytoplasmic fraction (C) and nuclear fraction (N) prepared from MEFs. (C) IHC staining for DDB1 and Ki67 on liver sections from DDB1F/F;Alb-Cre+/−, DDB1F/F;p21−/− and DDB1F/F;Alb-Cre+/−;p21−/− mice. (D) Co-IF staining for A6 and DDB1 on DDB1F/F;p21−/− and DDB1F/F;Alb-Cre+/−;p21−/− liver sections. Arrows in DDB1F/F;Alb-Cre+/−;p21−/− mice indicate A6 positive oval cells.
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pone-0031846-g003: Deletion of p21 partially restores proliferation of DDB1-deficient hepatocytes and alleviates OC activation.(A) Western blot for some substrates of DDB1-Cul4A ubiquitin ligase using lysates of hepatocytes isolated from DDB1F/F and DDB1F/F;Alb-Cre+/+ mice. (B) Western blot for DDB1 and p21 using lysates of cytoplasmic fraction (C) and nuclear fraction (N) prepared from MEFs. (C) IHC staining for DDB1 and Ki67 on liver sections from DDB1F/F;Alb-Cre+/−, DDB1F/F;p21−/− and DDB1F/F;Alb-Cre+/−;p21−/− mice. (D) Co-IF staining for A6 and DDB1 on DDB1F/F;p21−/− and DDB1F/F;Alb-Cre+/−;p21−/− liver sections. Arrows in DDB1F/F;Alb-Cre+/−;p21−/− mice indicate A6 positive oval cells.

Mentions: We previously reported that DDB1-deficient hepatocytes accumulate cell cycle inhibitor p21, a substrate of DDB1 ubiquitin ligase [24], [25], and fail to proliferate during liver growth or after partial hepatectomy [17]. Similarly, hepatocytes from DDB1F/F;Alb-Cre+/+ mice express high levels of p21, but not Cdt1 (Figure 3A), another substrate of the E3 ligase [16]. p21 is dramatically enriched in the nuclear fraction of DDB1-depleted MEFs [19] (Figure 3B), consistent with a role of p21 in blocking proliferation of these cells. Indeed, deletion of p21 abrogates the cell cycle arrest of cells with inactivated Cul4A-DDB1 ubiquitin ligase [25], [26]. We therefore generated compound mutant mice by crossing DDB1F/F;Alb-Cre+/− mice and p21−/− mice [27]. Deletion of p21 in DDB1F/F;Alb-Cre+/− mice allowed DDB1-decificent hepatocytes to proliferate (Figure 3C), resulting in significantly less compensatory proliferation of DDB1-expressing OCs (Figure 3D). However, p21 deletion did not fully reverse the proliferative arrest of DDB1-deficient hepatocytes (data not shown). These hepatocytes may still have other defects such as epigenetic deregulation due to the loss of the DDB1Cul4 ubiquitin ligase activity [28], [29], [30]. We conclude that failed proliferation of DDB1-deficient hepatocytes is responsible for activation of DDB1-expressing OCs.


Genetic abolishment of hepatocyte proliferation activates hepatic stem cells.

Endo Y, Zhang M, Yamaji S, Cang Y - PLoS ONE (2012)

Deletion of p21 partially restores proliferation of DDB1-deficient hepatocytes and alleviates OC activation.(A) Western blot for some substrates of DDB1-Cul4A ubiquitin ligase using lysates of hepatocytes isolated from DDB1F/F and DDB1F/F;Alb-Cre+/+ mice. (B) Western blot for DDB1 and p21 using lysates of cytoplasmic fraction (C) and nuclear fraction (N) prepared from MEFs. (C) IHC staining for DDB1 and Ki67 on liver sections from DDB1F/F;Alb-Cre+/−, DDB1F/F;p21−/− and DDB1F/F;Alb-Cre+/−;p21−/− mice. (D) Co-IF staining for A6 and DDB1 on DDB1F/F;p21−/− and DDB1F/F;Alb-Cre+/−;p21−/− liver sections. Arrows in DDB1F/F;Alb-Cre+/−;p21−/− mice indicate A6 positive oval cells.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3285627&req=5

pone-0031846-g003: Deletion of p21 partially restores proliferation of DDB1-deficient hepatocytes and alleviates OC activation.(A) Western blot for some substrates of DDB1-Cul4A ubiquitin ligase using lysates of hepatocytes isolated from DDB1F/F and DDB1F/F;Alb-Cre+/+ mice. (B) Western blot for DDB1 and p21 using lysates of cytoplasmic fraction (C) and nuclear fraction (N) prepared from MEFs. (C) IHC staining for DDB1 and Ki67 on liver sections from DDB1F/F;Alb-Cre+/−, DDB1F/F;p21−/− and DDB1F/F;Alb-Cre+/−;p21−/− mice. (D) Co-IF staining for A6 and DDB1 on DDB1F/F;p21−/− and DDB1F/F;Alb-Cre+/−;p21−/− liver sections. Arrows in DDB1F/F;Alb-Cre+/−;p21−/− mice indicate A6 positive oval cells.
Mentions: We previously reported that DDB1-deficient hepatocytes accumulate cell cycle inhibitor p21, a substrate of DDB1 ubiquitin ligase [24], [25], and fail to proliferate during liver growth or after partial hepatectomy [17]. Similarly, hepatocytes from DDB1F/F;Alb-Cre+/+ mice express high levels of p21, but not Cdt1 (Figure 3A), another substrate of the E3 ligase [16]. p21 is dramatically enriched in the nuclear fraction of DDB1-depleted MEFs [19] (Figure 3B), consistent with a role of p21 in blocking proliferation of these cells. Indeed, deletion of p21 abrogates the cell cycle arrest of cells with inactivated Cul4A-DDB1 ubiquitin ligase [25], [26]. We therefore generated compound mutant mice by crossing DDB1F/F;Alb-Cre+/− mice and p21−/− mice [27]. Deletion of p21 in DDB1F/F;Alb-Cre+/− mice allowed DDB1-decificent hepatocytes to proliferate (Figure 3C), resulting in significantly less compensatory proliferation of DDB1-expressing OCs (Figure 3D). However, p21 deletion did not fully reverse the proliferative arrest of DDB1-deficient hepatocytes (data not shown). These hepatocytes may still have other defects such as epigenetic deregulation due to the loss of the DDB1Cul4 ubiquitin ligase activity [28], [29], [30]. We conclude that failed proliferation of DDB1-deficient hepatocytes is responsible for activation of DDB1-expressing OCs.

Bottom Line: Purified OCs express both hepatocyte and cholangiocyte markers, form colonies in vitro, and differentiate to hepatocytes after transplantation.Microarray analysis reveals several previously unrecognized markers, including Reelin, enriched in oval cells.Here we report a genetic model in which irreversible inhibition of hepatocyte duplication results in HSC-driven liver regeneration.

View Article: PubMed Central - PubMed

Affiliation: Signal Transduction Program, Sanford-Burnham Medical Research Institute, La Jolla, California, United States of America.

ABSTRACT
Quiescent hepatic stem cells (HSCs) can be activated when hepatocyte proliferation is compromised. Chemical injury rodent models have been widely used to study the localization, biomarkers, and signaling pathways in HSCs, but these models usually exhibit severe promiscuous toxicity and fail to distinguish damaged and non-damaged cells. Our goal is to establish new animal models to overcome these limitations, thereby providing new insights into HSC biology and application. We generated mutant mice with constitutive or inducible deletion of Damaged DNA Binding protein 1 (DDB1), an E3 ubiquitin ligase, in hepatocytes. We characterized the molecular mechanism underlying the compensatory activation and the properties of oval cells (OCs) by methods of mouse genetics, immuno-staining, cell transplantation and gene expression profiling. We show that deletion of DDB1 abolishes self-renewal capacity of mouse hepatocytes in vivo, leading to compensatory activation and proliferation of DDB1-expressing OCs. Partially restoring proliferation of DDB1-deficient hepatocytes by ablation of p21, a substrate of DDB1 E3 ligase, alleviates OC proliferation. Purified OCs express both hepatocyte and cholangiocyte markers, form colonies in vitro, and differentiate to hepatocytes after transplantation. Importantly, the DDB1 mutant mice exhibit very minor liver damage, compared to a chemical injury model. Microarray analysis reveals several previously unrecognized markers, including Reelin, enriched in oval cells. Here we report a genetic model in which irreversible inhibition of hepatocyte duplication results in HSC-driven liver regeneration. The DDB1 mutant mice can be broadly applied to studies of HSC differentiation, HSC niche and HSCs as origin of liver cancer.

Show MeSH
Related in: MedlinePlus