Emerging roles of Notch signaling in liver disease

Abstract

This review critically discusses the most recent advances in the role of Notch signaling in liver development, homeostasis, and disease. It is now clear that the significance of Notch in determining mammalian cell fates and functions extends beyond development, and Notch is a major regular of organ homeostasis. Moreover, Notch signaling is reactivated upon injury and regulates the complex interactions between the distinct liver cell types involved in the repair process. Notch is also involved in the regulation of liver metabolism, inflammation, and cancer. The net effects of Notch signaling are highly variable and finely regulated at multiple levels, but also depend on the specific cellular context in which Notch is activated. Persistent activation of Notch signaling is associated with liver malignancies, such as hepatocellular carcinoma with stem cell features and intrahepatic cholangiocarcinoma. The complexity of the pathway provides several possible targets for agents able to inhibit Notch. However, further cell- and context-specific in-depth understanding of Notch signaling in liver homeostasis and disease will be essential to translate these concepts into clinical practice and be able to predict benefits and risks of evolving therapies.

Figure 1. The core ‘canonical’ Notch signaling pathway (A), ‘non-canonical’ Notch signaling, and cross-talk with other signaling pathways (B)

(A) After synthesis Notch receptors are cleaved by a Furin-like convertase in the trans-Golgi (S1 cleavage) to produce heterodimeric receptors containing an intra- and extracellular domain. Notch receptors are exocytosed to the cell surface of the signal-receiving cell where Notch signaling is initiated upon binding to Notch ligands belonging to the Delta or Jagged family expressed on neighboring signal-sending cells. Steady state levels of non-ligand bound Notch receptors are controlled by E3 ligases and several proteins such as Numb and α-Adaptin that control Notch turnover by recycling or lysosomal degradation. Notch ligand-receptor binding enables proteolytic cleavage of the Notch extracellular domain by ADAM10/TACE metalloprotease (S2 cleavage) that remains bound to its ligand and is further subjected to lysosomal degradation in the signal-sending cell. The remnant receptor is then cleaved by the γ-secretase complex within its transmembrane domain at site 3 (S3 cleavage) to allow release and nuclear translocation of the Notch intracellular domain (NICD) where it associates with RBP-Jκ, a DNA-binding adaptor protein mediating the corpus of canonical Notch signals. In the absence of NICD, RBP-Jκ forms a co-repressor complex (red) with many proteins including histone deacetylases (HDACs) and SHARP. NICD binding to RBP-Jκ enables displacement of the co-repressor complex and binding of the adaptor protein Mastermind-like (MAML) that recruits other proteins to form a co-activator complex (green) resulting in the transcriptional activation of Notch target genes. (B) Some effects of Notch signaling were found to be independent from canonical ligands and/or from RBP-Jκ-mediated transcription that may or may not require cleavage of Notch (‘non-canonical’ Notch signaling). Furthermore, at virtually all stages of the Notch signaling cascade, interactions of components of multiple other signaling pathways are found that may influence Notch turnover, non-nuclear signaling, or modulate RBP-Jκ-dependent or –independent transcriptional pathway activity (3, 59). The best-characterized ligand/RBP-Jκ-independent Notch pathway involves the regulation of Wnt/β-catenin signaling. The Notch and Wnt/β-catenin pathway have long been known to interact in multiple and even opposing ways. In a very simplified view, the synergistic interactions between Notch and Wnt/β-catenin depend on RBP-Jκ-mediated canonical signaling, whereas the antagonistic effects are mediated by ligand/RBP-Jκ-independent Notch signaling. Several hypotheses have been proposed to explain this non-canonical mechanism, including binding and degradation of activated β-catenin by plasma membrane-bound Notch involving interaction with proteins such as Numb, Numb-like, and α-Adaptin.

Figure 2. Putative multiple functions of Notch in epithelial lineage control in liver development, homeostasis, and disease

Notch signaling may act at multiple stages in the embryonic and adult liver to control cell fate decisions and cell growth in development, homeostasis, regeneration, and disease. (1.) During embryonic development Notch converts bipotential hepatoblast to the biliary lineage (12, 20, 21) and (2./3.) is required for proper morphogenesis and maturation of the intrahepatic biliary tree (, –12, 14). (4./5.) Notch signaling may also contribute to biliary injury repair by regulating expansion and morphogenesis of ductular reactions and bile ducts (30, 31). (6./7.) Further, Notch has been proposed to function as the key signaling pathway to destine the cellular fate of adult liver progenitor cells either towards the biliary (Notch activation) or hepatocyte lineage (Notch suppression) both, in liver regeneration (30) and carcinogenesis (46). (8.) Notch has also been shown to be capable to reprogram adult hepatocytes to biliary cells (17, 35) that may give rise to intrahepatic cholangiocarcinoma (37, 38). (9.) Further, data support a role for Notch in hepatocyte regeneration after partial hepatectomy (24) and hepatocellular carcinoma development arising from the hepatocyte compartment (47, 48).

Figure 3. Normal embryonic development of the intrahepatic biliary tree and consequences of alterations in Notch signaling

(A) Embryonic development of the intrahepatic biliary tree starts with the differentiation of a subset of Notch2+ periportal hepatoblasts expressing biliary lineage defining proteins such as Sox9, HNF1β, OPN, or CK19. These cells, committed to the biliary fate, encircle the Jag1+ portal mesenchyme in a mono-layered ring, called the ‘ductal plate’, for review see (22, 23). In a second step, lumina arise at distinct sites of the ductal plate that are lined asymmetrically by biliary cells of the first ductal plate layer (portal side) and hepatoblasts (parenchymal side). Later, during development, this asymmetry of immature ducts resolves when the duct-lining hepatoblasts maturate to biliary cells (second ductal plate layer) by loosing HNF4α and gradually expressing Hes1, OPN, SOX9, HNF1β and CK19. This two-step specification process in IHBD tubulogenesis is observed in mice and humans and starts at around E15.5 and W12 respectively. Because IHBD development proceeds from the hilum to the periphery, mature symmetric ducts are first observed at the hilar region when more peripheral IHBDs may still display asymmetric tubules beyond birth before completing their remodeling program by around P2 (12). Thus, differentiation of hepatoblasts to biliary cells represents the main mechanism for intrahepatic biliary morphogenesis in the embryonic and early postnatal period. (B) Bile duct development is disturbed in Notch mutants. At P10, panCytokeratin+ mature bile ducts are well incorporated in the mesenchyme of medium size portal tracts in wildtype mice. In contrast, P10 mice with conditional deletion of Notch2 (N2^F/F;AlbCre) or RBP-Jκ (Rbpj^F/F;AlbCre) lack mature bile ducts due to impaired perinatal IHBD morphogenesis/elongation. Conversely, conditional expression of the intracellular domain of Notch2 in embryonic hepatoblasts (R26^N2IC;AlbCre) directs commitment of embryonic hepatoblasts to the biliary lineage with ectopic formation of biliary-like tubular-cystic structures expressing biliary-specific DBA at P0. In human ALGS portal tracts typically lack mature bile ducts. pv = portal vein; bd = bile duct; ha = hepatic artery.