Limits...
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.

Show MeSH

Related in: MedlinePlus

DDB1 mutant mice exhibit minor liver damage compared with DDC-treated mice.(A): Co-IF staining for A6 and EpCAM on liver sections from DDC-treated mice. (B): Gross appearance of liver from mice fed with DDC diet for 4 weeks. (C): H&E staining of the liver in (B). (D): IHC staining for CD45 (upper panels) and F4/80 (lower panels) on liver sections from DDB1F/F, DDB1F/F;Alb-Cre+/+ and DDC-treated mice. (E): Percentage of EpCAM+ cells from DDB1F/F;Alb-Cre+/+ and DDC-diet liver, determined by FACS analysis. Data are representative of 4 independent experiments with 3 mice per group in each experiment. Values are expressed as the means±SEM; n = 3. (F): Serum alanine aminotransferase (ALT) levels. Data are representative of 4 independent experiments with 3 mice per group in each experiment. Values are expressed as means±SEM; n = 3 **P<0.01.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3285627&req=5

pone-0031846-g005: DDB1 mutant mice exhibit minor liver damage compared with DDC-treated mice.(A): Co-IF staining for A6 and EpCAM on liver sections from DDC-treated mice. (B): Gross appearance of liver from mice fed with DDC diet for 4 weeks. (C): H&E staining of the liver in (B). (D): IHC staining for CD45 (upper panels) and F4/80 (lower panels) on liver sections from DDB1F/F, DDB1F/F;Alb-Cre+/+ and DDC-treated mice. (E): Percentage of EpCAM+ cells from DDB1F/F;Alb-Cre+/+ and DDC-diet liver, determined by FACS analysis. Data are representative of 4 independent experiments with 3 mice per group in each experiment. Values are expressed as the means±SEM; n = 3. (F): Serum alanine aminotransferase (ALT) levels. Data are representative of 4 independent experiments with 3 mice per group in each experiment. Values are expressed as means±SEM; n = 3 **P<0.01.

Mentions: To determine the severity of liver damage in the DDB1 mutant mice, we compared 4-week old DDB1F/F;Alb-Cre+/+ mice with a conventional chemical injury model for OC proliferation, mice treated with diet containing 3,5-diethoxycarbonyl-1,4-dihydro-collidine (DDC). Consistent with previous reports [13], [32], [33], ductal cells from DDC-treated wild type mice expressed both A6 and EpCAM (Figure 5A). After treating mice with the diet for 4 weeks, their livers turned dark brown (Figure 5B), due to hepatic porphyria resulting from the inhibition of the heme biosynthetic pathway [34]. H&E staining showed some ductal cells around the portal veins with brown metabolite deposits (Figure 5C). To characterize the extent of liver inflammation, we performed CD45 and F4/80 staining for leukocytes and macrophages, respectively. In liver sections from DDC-treated mice, we found greatly enriched CD45+ and F4/80+ cells surrounding OCs (Figure 5D). In contrast, DDB1F/F;Alb-Cre+/+ mouse liver did not show significant increase of these immune cells compared with DDB1F/F liver (Figure 5D), even though there are more OCs in the DDB1 mutant liver than DDC mice (Figure 5E). We further measured the severity of liver damage by serum alanine aminotransferase (ALT) assays. The ALT level in the DDB1 mutant mice was 191±73 (IU/L); however, it was nearly 10 times higher, reaching 1679±475 (IU/L) (normal range: 28–132 U/L) in the DDC-treated mice (Figure 5F), consistent with the overall toxicity of DDC to the liver. With seemingly insignificant damage to hepatocytes, our genetic model provides a better system for studying HSC and OC biomarkers and their extracellular activation signals.


Genetic abolishment of hepatocyte proliferation activates hepatic stem cells.

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

DDB1 mutant mice exhibit minor liver damage compared with DDC-treated mice.(A): Co-IF staining for A6 and EpCAM on liver sections from DDC-treated mice. (B): Gross appearance of liver from mice fed with DDC diet for 4 weeks. (C): H&E staining of the liver in (B). (D): IHC staining for CD45 (upper panels) and F4/80 (lower panels) on liver sections from DDB1F/F, DDB1F/F;Alb-Cre+/+ and DDC-treated mice. (E): Percentage of EpCAM+ cells from DDB1F/F;Alb-Cre+/+ and DDC-diet liver, determined by FACS analysis. Data are representative of 4 independent experiments with 3 mice per group in each experiment. Values are expressed as the means±SEM; n = 3. (F): Serum alanine aminotransferase (ALT) levels. Data are representative of 4 independent experiments with 3 mice per group in each experiment. Values are expressed as means±SEM; n = 3 **P<0.01.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3285627&req=5

pone-0031846-g005: DDB1 mutant mice exhibit minor liver damage compared with DDC-treated mice.(A): Co-IF staining for A6 and EpCAM on liver sections from DDC-treated mice. (B): Gross appearance of liver from mice fed with DDC diet for 4 weeks. (C): H&E staining of the liver in (B). (D): IHC staining for CD45 (upper panels) and F4/80 (lower panels) on liver sections from DDB1F/F, DDB1F/F;Alb-Cre+/+ and DDC-treated mice. (E): Percentage of EpCAM+ cells from DDB1F/F;Alb-Cre+/+ and DDC-diet liver, determined by FACS analysis. Data are representative of 4 independent experiments with 3 mice per group in each experiment. Values are expressed as the means±SEM; n = 3. (F): Serum alanine aminotransferase (ALT) levels. Data are representative of 4 independent experiments with 3 mice per group in each experiment. Values are expressed as means±SEM; n = 3 **P<0.01.
Mentions: To determine the severity of liver damage in the DDB1 mutant mice, we compared 4-week old DDB1F/F;Alb-Cre+/+ mice with a conventional chemical injury model for OC proliferation, mice treated with diet containing 3,5-diethoxycarbonyl-1,4-dihydro-collidine (DDC). Consistent with previous reports [13], [32], [33], ductal cells from DDC-treated wild type mice expressed both A6 and EpCAM (Figure 5A). After treating mice with the diet for 4 weeks, their livers turned dark brown (Figure 5B), due to hepatic porphyria resulting from the inhibition of the heme biosynthetic pathway [34]. H&E staining showed some ductal cells around the portal veins with brown metabolite deposits (Figure 5C). To characterize the extent of liver inflammation, we performed CD45 and F4/80 staining for leukocytes and macrophages, respectively. In liver sections from DDC-treated mice, we found greatly enriched CD45+ and F4/80+ cells surrounding OCs (Figure 5D). In contrast, DDB1F/F;Alb-Cre+/+ mouse liver did not show significant increase of these immune cells compared with DDB1F/F liver (Figure 5D), even though there are more OCs in the DDB1 mutant liver than DDC mice (Figure 5E). We further measured the severity of liver damage by serum alanine aminotransferase (ALT) assays. The ALT level in the DDB1 mutant mice was 191±73 (IU/L); however, it was nearly 10 times higher, reaching 1679±475 (IU/L) (normal range: 28–132 U/L) in the DDC-treated mice (Figure 5F), consistent with the overall toxicity of DDC to the liver. With seemingly insignificant damage to hepatocytes, our genetic model provides a better system for studying HSC and OC biomarkers and their extracellular activation signals.

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