Mechanism of NOTCH2 in promoting intrahepatic bile duct development

Abstract

Background: The Notch signaling pathway plays a crucial role in intrahepatic bile duct development. Here, we aimed to investigate the effect of the Notch receptor, NOTCH2, on intrahepatic bile duct development to better understand congenital intrahepatic bile duct dysplasia.

Results: Estradiol increased NOTCH2 and its downstream proteins expression, which promoted the differentiation of hepatoblasts into intrahepatic cholangiocytes and the development of intrahepatic bile ducts by upregulating the Notch signaling pathway. NOTCH2 siRNA inhibited the above processes (P < 0.05). There was no significant difference between estradiol and estradiol + non-targeting siRNA groups (P > 0.1).

Conclusions: In conclusion, the activation of the Notch signaling pathway leads to increased NOTCH2 expression, which promotes the differentiation of hepatoblasts into intrahepatic cholangiocytes and the development of intrahepatic bile ducts during embryonic stages in C57BL/6CrSlc mice. The results of this study may provide a theoretical basis for infantile intrahepatic cholestasis treatment.

Keywords: Hepatoblast; Intrahepatic bile duct; NOTCH2; Notch signaling pathway.

Fig. 1

Differentiation of hepatoblasts into intrahepatic cholangiocytes in different experimental groups in vitro (intrahepatic cholangiocytes are defined as CK19 + cells). (A-D) The proportions of hepatoblasts differentiating into intrahepatic cholangiocytes in the control group, estradiol group, estradiol + non-targeting siRNA group, and estradiol + NOTCH2 siRNA group were 2%, 41%, 41%, and 27%, respectively. P2 in A-D is the gating of CK19⁺ cells. (E) * p < 0.05 vs. Control, # p < 0.05 vs. Estradiol. (A–D: flow cytometry dot plots for each group; E: quantification of CK19⁺cell percentages) (Three independent experiments have been completed. Every time, three pregnant mice were included in each group. Three cultures from embryos of different pregnant mice were analyzed per condition. Therefore, nine cultures of embryos were examined in each group in total.)

Fig. 2

Protein expression levels of NOTCH2 and downstream Hes1 of the Notch signaling pathway in different experimental groups in vitro. (A, C) Hepatoblasts were incubated with anti-Hes1 and anti-NOTCH2 antibodies, and cell nuclei were stained with DAPI (blue). Scale bar, 10 μm. (B, D) Quantitative fluorescence analysis comparing the IOD/pixel values of proteins in hepatoblasts between the estradiol-treated group and the control group, as well as between the estradiol-treated group and the estradiol + NOTCH2 siRNA group (* p < 0.05). (A: Hes1 immunofluorescence; B: quantification of Hes1 staining; C: NOTCH2 immunofluorescence; D: quantification of NOTCH2 staining in vitro) (Three independent experiments have been completed. Every time, three pregnant mice were included in each group. Three cultures from embryos of different pregnant mice were analyzed per condition. Therefore, nine cultures of embryos were examined in each group in total.)

Fig. 3

Gene transcription levels of NOTCH2 and downstream Hes1 of the Notch signaling pathway in different experimental groups in vitro. RT-PCR was used to measure the relative mRNA expression levels of Hes1 and NOTCH2 normalized to β-actin in the control group, estradiol-treated group, estradiol + blank siRNA group, and estradiol + NOTCH2 siRNA group (3 A for Hes1, 3B for Notch2). Gene expression was normalized to β-actin and then to control = 1. The y-axis indicated that fold change in mRNA was relative to control. Comparisons were made between the estradiol-treated group and the control group, as well as between the estradiol-treated group and the estradiol + NOTCH2 siRNA group. * p < 0.05 vs. Control, # p < 0.05 vs. Estradiol. (Three independent experiments have been completed. Every time, three pregnant mice were included in each group. Three cultures from embryos of different pregnant mice were analyzed per condition. Therefore, nine cultures of embryos were examined in each group in total.)

Fig. 4

Protein expression of NOTCH2, downstream Hes1 of the Notch signaling pathway, and biliary lineage marker CK19 in different experimental groups in vivo. (A, C, E) Liver progenitor cells were incubated with anti-CK19, anti-Hes1, and anti-NOTCH2 antibodies, respectively, and nuclei were stained with DAPI (blue). Scale bar: 50 μm. (B, D) Quantitative fluorescence analysis showing the IOD/pixel values of protein expression in liver progenitor cells, comparing the estradiol-treated group with the control group and the estradiol-treated group with the estradiol + NOTCH2 siRNA group. * p < 0.05 vs. Control, # p < 0.05 vs. Estradiol. Fluorescence intensity measured through average area positive percentage per group, n = 3 experiments. (A: CK19 immunofluorescence; B: quantification of CK19 staining; C: Hes1 immunofluorescence; D: quantification of Hes1 staining; E: NOTCH2 immunofluorescence; F: quantification of NOTCH2 staining in vivo) (Three independent experiments have been completed. Every time, three pregnant mice were included in each group. Three litters from different pregnant mice were analyzed per condition. Therefore, nine embryos were examined in each group in total.)

Fig. 5

Gene transcription of NOTCH2, downstream Hes1 of the Notch signaling pathway, and biliary lineage marker CK19 in different experimental groups in vivo. The expression levels of mRNA relative to β-Actin were measured by RT-PCR in the control group, estradiol-treated group, estradiol + blank siRNA group, and estradiol + NOTCH2 siRNA group. (3 A for CK19, 3B for Hes1, 3 C for Notch2). Gene expression was normalized to β-actin and then to control = 1. The y-axis indicated that fold change in mRNA was relative to control. Comparisons were made between the estradiol group and the control group, as well as between the estradiol group and the estradiol + NOTCH2 siRNA group. * p < 0.05 vs. Control, # p < 0.05 vs. Estradiol. (Three independent experiments have been completed. Every time, three pregnant mice were included in each group. Three litters from different pregnant mice were analyzed per condition. Therefore, nine embryos were examined in each group in total.)