Expression of Notch-1 and its ligand Jagged-1 in rat liver during liver regeneration

Abstract

The Notch/Jagged signaling pathway is important for cellular differentiation and proliferation. Its dysfunction is associated with human pathologies in several tissues including liver. Point mutations in Jagged-1 gene are the cause for Alagille syndrome, associated with paucity of intrahepatic bile ducts. To determine the putative role of the trans-membrane receptor Notch and its ligand Jagged-1 in liver regeneration, we investigated the expression of Notch and Jagged-1 in rat liver following 2/3 partial hepatectomy. Immunohistochemical staining of normal rat liver showed that Notch was expressed in hepatocytes, bile duct cells and endothelial cells, whereas Jagged-1 was expressed in bile duct cells and hepatocytes. Both Notch-1 and Jagged-1 proteins were upregulated in hepatocytes after partial hepatectomy up to day 4. After partial hepatectomy, nuclear translocation of the intracellular cytoplasmic domain of Notch (NICD) increased and peaked within 15 minutes, indicating the activation of Notch. Expression of the Notch-dependent target gene (HES-1) expression increased within 30-60 minutes. Addition of recombinant Jagged-1 protein to primary cultures of hepatocytes stimulated hepatocyte DNA synthesis. Furthermore, injection of silencing RNA for Notch and Jagged-1 to livers 2 days before partial hepatectomy significantly suppressed proliferation of hepatocytes at days 2 to 4 of the regenerative response. In conclusion, Notch/Jagged signaling pathway is activated during liver regeneration and is potentially contributing to signals affecting cell growth and differentiation.

Fig. 1

Real-time PCR analysis of Notch (A) and Jagged (B) gene expression in normal, sham operated, and partially hepatectomized (partial hepatectomy) rat liver. Data are normalized to expression in normal liver (actual expression of normal liver samples was Ct_Notch = 26; Ct_Jagged = 27.9). Data represent the mean value ± SEM (n ≥ 3).

Fig. 2

Detection of Notch and Jagged expression by Western blot analysis. Plasma membrane proteins were isolated from normal and partial hepatectomy or sham liver. Monoclonal antibody against Notch (hamster, Upstate) or polyclonal antibody against Jagged were used to analyze Notch (A) and Jagged (B) expression. (C) Jagged detection in whole cell lysate of normal (t = 0), partial hepatectomy, and sham rat livers. PLEASE NOTE: Due to the very low level of expression of both Notch and Jagged in normal and sham operated liver, a longer time exposure was used for the sham samples. The t = 0 sample of both sham and partial hepatectomy is the same tissue sample. It appears over-expressed in the sham samples only due to the longer exposure applied to the sham samples to allow detection of the protein.

Fig. 3

Time course of Notch and Jagged expression in regenerating liver determined by immunohistochemical staining. (A) Normal liver, magnification at 40×. Notch staining is shown on bile ductules (single long arrow), sinusoidal endothelial and small vessel endothelial cells (double arrows), and hepatocyte plasma membranes (short arrows). (B and C) Regenerating liver, 4 days after partial hepatectomy. Figure 3B was taken at a magnification of 10× and Fig. 3C at a magnification of 40×. Both pictures demonstrate staining of Notch in endothelial cells and periportal hepatocytes. (D) Normal liver (magnification: 20×). Jagged staining predominantly in bile ductules with weaker staining seen in hepatocytes. (E and F) Regenerating liver. Magnifications at 10× and 40×, respectively. Strong immunoreactivity for Jagged is seen in bile ductules and periportal hepatocytes. B.D. = bile ductile; P.V. = portal vein.

Fig. 4

Detection of cytoplasmic domain of Notch (NICD) in nuclear protein (NP) extracts by Western blot analysis (rabbit polyclonal antibody, Upstate). (A) Densitometric analysis of Western blots for NICD in nuclear protein (NP). Data are shown as mean ± SEM (n = 3). (B) Representative Western blot of NICD detection in NP of rat liver. Ponceau-S stain of a band at 176 kDa are used as loading control. Numbers indicate time elapsed after operation in minutes, hours, and days.

Fig. 5

Detection of localization of the intra-cytoplasmic domain of Notch (NICD) in normal liver (A-a and A-b), liver at 15 minutes after partial hepatectomy (B-a and B-b), or sham operation (C-a and C-b). In normal liver and in sham-operated animals, NICD is localized only on the cytoplasm or the plasma membrane. There is no green fluorescence in the nuclei. Green fluorescence is seen in the nuclei at 15 minutes after partial hepatectomy. The nuclei were counter-stained with Hoechst dye shown in Fig. A-b, B-b, and C-b, to serve as comparison with the corresponding (a) figures in order to facilitate visual localization of the nuclei. Cytoplasmic and membrane localization of NICD is shown by long arrows. Nuclear localization (seen only in B-a) is shown by short arrows.

Fig. 6

Immunohistochemical stains for Notch and Jagged proteins in animals injected with “scramble” RNA (scra-siRNA), Jagged-1 silencing RNA (J1-siRNA), and Notch1 silencing RNA (N1-siRNA). The animals were injected 2 days before partial hepatectomy. The sections were performed on livers at 4 days after partial hepatectomy (6 days after the RNA injections). There is evident decrease in the expression of both Jagged and Notch in the periportal regions of the hepatic lobules in the animals injected with the silencing RNA for either of the two genes. The portal triads in each section are shown by arrows. PT: portal triad.