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Notch Signaling and Progenitor/Ductular Reaction in Steatohepatitis

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Notch Signaling and Progenitor/Ductular Reaction in Steatohepatitis

Carola M Morell et al. PLoS One.

Abstract

Background and objective: Persistent hepatic progenitor cells (HPC) activation resulting in ductular reaction (DR) is responsible for pathologic liver repair in cholangiopathies. Also, HPC/DR expansion correlates with fibrosis in several chronic liver diseases, including steatohepatitis. Increasing evidence indicates Notch signaling as a key regulator of HPC/DR response in biliary and more in general liver injuries. Therefore, we aimed to investigate the role of Notch during HPC/DR activation in a mouse model of steatohepatitis.

Methods: Steatohepatitis was generated using methionine-choline deficient (MCD) diet. For hepatocyte lineage tracing, R26R-YFP mice were infected with AAV8-TBG-Cre.

Results: MCD diet promoted a strong HPC/DR response that progressively diffused in the lobule, and correlated with increased fibrosis and TGF-β1 expression. Notch signaling was unchanged in laser-capture microdissected HPC/DR, whereas Notch receptors were down regulated in hepatocytes. However, in-vivo lineage tracing experiments identified discrete hepatocytes showing Notch-1 activation and expressing (the Notch-dependent) Sox9. Stimulation of AML-12 hepatocyte-cell line with immobilized Jag1 induced Sox9 and down-regulated albumin and BSEP expression. TGF-β1 treatment in primary hepatic stellate cells (HSC) induced Jag1 expression. In MCD diet-fed mice, αSMA-positive HSC were localized around Sox9 expressing hepatocytes, suggesting that Notch activation in hepatocytes was promoted by TGF-β1 stimulated HSC. In-vivo Notch inhibition reduced HPC response and fibrosis progression.

Conclusion: Our data suggest that Notch signaling is an important regulator of DR and that in steatohepatitis, hepatocytes exposed to Jag1-positive HSC, contribute to pathologic DR by undergoing Notch-mediated differentiation towards an HPC-like phenotype. Given the roles of Notch in fibrosis and liver cancer, these data suggest mesenchymal expression of Jag1 as an alternative therapeutic target.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. MCD diet induces HPC response and inflammation.
(A) Immunohistochemical analysis on mouse liver specimens shows a progressive expansion into the liver parenchyma of the HPC compartment (CK19+ve cells) in mice treated with MCD diet respect to untreated controls. (B) The progressive spread of HPC from portal areas into the hepatic lobules was further confirmed by morphometric analysis. CK19 positivity in portal areas increased after 4 weeks of MCD diet and persisted throughout the treatment, whereas in the lobule the percentage of CK19+ve cells was significantly higher by the 8th week of diet as compared to CTRL and MCD4wks groups. (n = 10–11 mice per group; *p<0.05, **p<0.01; ***p<0.001; CTRL = control, MCD4wks = MCD diet for 4 weeks; MCD8wks = MCD diet for 8 weeks). Original magnification: 100X. (C) Real time PCR analysis shows that MCD diet stimulated the inflammatory process, as confirmed by the mRNA expression of CD11b and TNFα, which positively correlated with the increase of CK19+ve cells (D) (n = 6–11 mice per group; *p<0.05, **p<0.01; ***p<0.001).
Fig 2
Fig 2. Fibrosis increases in MCD diet-induced steatohepatitis and correlates with HPC activation.
(A) Histological analysis on Sirius Red stained livers revealed a progressive collagen deposition in MCD diet-treated mice from the 4th to the 8th week of treatment, as confirmed by morphometric quantification (B, upper panel). Collagen deposition strongly correlated with CK19 positivity (B, lower panel). MCD diet also stimulated the mRNA expression of profibrogenic mediators TGF-β1 and COL1A1(C, D upper panel, respectively), both of which correlated with increased HPC response (C, D lower panels) (n = 6–11 mice per group; *p<0.05, **p<0.01; ***p<0.001). Original magnification: 100X.
Fig 3
Fig 3. Gene expression analysis on total liver lysates of Notch signaling components.
Real-time PCR analysis of Notch signaling components performed on total liver lysates revealed an overall downregulation of the expression of Notch receptors (Notch-1 and -2) and the ligand Dll4, without reducing the expression of the Notch ligand Jag1 or of the Notch-targets Hes1 and Hey1. Data are expressed as 2-dCt (n = 10 mice per group; *p<0.05, **p<0.01; ***p<0.001).
Fig 4
Fig 4. Notch signaling is not modulated in the CK19+ve population by MCD diet.
The expression of Notch-related factors was evaluated on laser-capture CK19+ve microdissected samples, which comprise HPC/DR and biliary cells. Expression of the Notch pathway components was not significantly influenced by MCD diet in the CK19+ve population overall. Of note, Numb expression was not altered in these cells. Data are expressed as 2-dCt (n = 5 mice per group; *p<0.05, **p<0.01; ***p<0.001).
Fig 5
Fig 5. Notch signaling is modulated in laser-microdissected CK19-ve population.
Real time PCR analysis of Notch components on CK19-ve microdissected samples showed a significant reduction, induced by MCD diet, of Notch receptors (-1 and -2) and Jag1 ligand expression in hepatocytes, respect to controls; on the contrary, the expression of Notch target genes was not altered. The expression of the Notch endogenous inhibitor Numb was strongly downregulated in hepatocytes by MCD diet. Data are expressed as 2^-dCt (n = 5 mice per group; *p<0.05, **p<0.01; ***p<0.001).
Fig 6
Fig 6. Lineage tracing experiments show hepatocytes conversion to HPC-like cells promoted by MCD diet-treatment.
(A) R26R-YFP reporter mice were infected with AAV2/8 viruses containing Cre recombinase under the control of the hepatocyte-specific thyroid binding globulin (TBG) promoter (AAV8-TBG-Cre) to specifically and permanently label the hepatocyte cell lineage. The images depict YFP+ lineage-labelled cells 8 weeks after injection. Efficient and specific marking of hepatocytes with no marking of BECs (CK19+ cells) was observed. PV: portal vein. Fate tracing experiments were performed in R26R-YFP mice infected with AAV8-TBG-Cre. After MCD treatment, approximately 10% of hepatocytes (YFP, green) expressed Sox9 (red) (B), and about 2% of YFP+ve/CK19+ve cells appeared in liver lobule (C). The presence of Sox9 and CK19+ve hepatocytes was negligible in controls, as showed by immunofluorescent staining and histological analysis (n = 4–8 mice per group; *p<0.05, **p<0.01; ***p<0.001). Original magnification: 200X, insets 400X.
Fig 7
Fig 7. Notch is active in Sox9 positive hepatocytes in MCD diet-injured livers.
Nuclei were isolated from paraffin liver sections of R26R-YFP mice infected with AAV8-TBG-Cre (untreated or treated with MCD diet for 8 weeks) to perform FACS analysis. DAPI/YFP+ve nuclei (hepatocytic population) were gated. (A) As shown in the representative FACS plot, YFP+ve nuclei were negative for N2ICD staining both in control and MCD treated mice. (B) On the contrary, a sub-population of YFP+ve nuclei were positive for Sox9 and the majority of them were also co-expressing N1ICD. The bar graphs show the quantification of FACS analysis results. (*p<0.05; ***p<0.001).
Fig 8
Fig 8. Notch induces phenotypic changes in the hepatocytic cell line AML-12.
AML-12 cells were treated with either Jag1 or Shh for 24hours and 48hours to induce respectively Notch and Hedgehog activation. The activation of Notch signaling reduced the expression of markers of hepatocytes (albumin and Bsep), while rapidly increasing the expression of Sox9. The increased expression of Notch target genes Hes1, Hey1 and Hey2 in Jag1 treated cells confirmed that this was a Notch-dependent process. No significant changes in gene expression were detected in the cells treated with Shh at any time point. Data are expressed as fold increase/decrease respect to control (n = 3–4 experiments; *p<0.05, **p<0.01, ***p<0.001).
Fig 9
Fig 9. The fibrotic environment is linked to Notch activation in hepatocytes in MCD diet-treated mice.
The hepatic expression of Jag1 and Hey1 mRNA positively correlated with that of COL1A1 (A), suggesting a link between the fibrotic environment and Notch pathway. Moreover, in mice treated with MCD diet for 8 weeks, Sox9+ve hepatocyte-like cells (green) established close contact with αSMA+ve cells (red) (B); accordingly, αSMA+ve cells (red) localized in proximity of CK19+ve cells (green) (C), indicating HSC/hepatocytes cross-talk thus suggesting HSC involvement in persistent stimulation of Notch receptors on hepatocytes. Original magnification: 200X, insets 400X.
Fig 10
Fig 10. HSC express high levels of Jag1 after TGF-β1-induced activation in vitro.
Jag1 was strongly upregulated in primary mouse HSC (A) as well as in the LX2 cell line (B), when treated with increasing doses of TGF-β1 (1-5-10ng/mL) for respectively 4 days or 72hrs; COL1A1 and αSMA expression confirmed TGF-β1-induced HSC activation (C). (n = 3–15 experiments; *p<0.05, **p<0.01; ***p<0.001).
Fig 11
Fig 11. Notch inhibition reduces HPC expansion and ameliorates steatohepatitis in MCD diet-fed mice.
Immuno-peroxidase staining for CK19 showed a significant reduction in HPC/DR activation after Notch inhibition, as demonstrated by morphometric quantification reported in the bar graph (A). In DBZ-treated mice with steatohepatitis, also the progression of liver fibrosis was attenuated: morphometric quantification confirmed that the percentage of the area positive for Sirius red was comparable to that reported in the group fed with MCD diet for 4 weeks (B). H&E staining shows reduction of steatosis after Notch inhibition, as confirmed by steatotic score quantification (C). (n = 4–5 mice per group; *p<0.05, **p<0.01; ***p<0.001). Original magnification: 100X.

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