Group 2 (PI : Neetu GUPTA-ROSSI)

Past results
Our work was originally focused on the mechanisms involved in Notch signalling, and in particular the events associated with trafficking of the receptor following activation (Gupta-Rossi et al., 2001; 2004). In parallel, since the last 5 years, we have introduced an in vitro differentiation model based on hepatic stem cells, which allowed us to characterize the non-redundant role of the four Notch paralogues in liver development.
The Notch pathway is involved in cell-cell signaling during development and adulthood from invertebrates to higher eukaryotes. Activation of the Notch receptor by its ligands relies upon a multi-step processing. A metalloprotease chops off the extracellular domain of the receptor  and the remaining fragment is cleaved within its transmembrane domain by a presenilin-dependent γ-secretase activity (Brou et al., 2000). The intracellular domain of the receptor then migrates to the nucleus where it acts as a transcriptional co-activator and directly activates its target genes.
Notch trafficking and activation
We have shown that Notch activation depends upon monoubiquitination and clathrin-mediated endocytosis (Gupta-Rossi et al., 2004). Ligand-induced activation of the receptor is finely tuned by regulators present throughout the pathway, from the membrane to the nucleus. Genetic screens in worms and flies have revealed many of these modulators. The interplay between two such proteins, Numb and AAK1 (Adaptor-Associated Kinase 1), linked with both Notch and the endocytic control of the pathway, caught our attention. AAK1, the mammalian ortholog of NAK (Numb-Associated Kinase, (Chien et al., 1998)) and SEL-5 (Fares and Greenwald, 1999) is a Ser/Thr kinase involved in receptor internalization and early/sorting clathrin-mediated endocytosis. AAK1 can phosphorylate Numb and lead to its relocalization from the plasma membrane to perinuclear endosomes (Sorensen and Conner, 2008). In invertebrates, its orthologs act as positive regulators upstream of the g-secretase cleavage step. Based on these data, we investigated whether AAK1 could be a regulator of the Notch pathway in mammals.
We showed that AAK1, the Adaptor-Associated kinase 1, directly interacts with the membrane-tethered form of Notch resulting from the ligand-induced metalloprotease cleavage. Active AAK1 acts upstream of γ-secretase processing by stabilizing the membrane-tethered, activated form of Notch and its monoubiquitinated counterpart. We propose that AAK1 acts as an adaptor for Notch interaction with components of the clathrin-mediated pathway such as Eps15b. Moreover, transfected AAK1 favors the localization of activated Notch to Rab5-positive endocytic vesicles, while AAK1 depletion or overexpression of Numb, an inhibitor of the pathway, interferes with this routing (see Figure1). We therefore propose a model suggesting that, after ligand-induced activation of Notch, the membrane-tethered form can be directed to different endocytic pathways leading to distinct fates linked with the AAK1 or Numb factors (Gupta-Rossi et al., 2011).
Figure 1 : AAK1 and Numb differentially regulate NotchDE localization to Rab5-positive endosomes (in collaboration with V. Meas-Yedid, AIQ, Pasteur Institute) Pr
Proposed model
Current project
Notch receptors in the proliferation and the hepatocytic differentiation of liver progenitors
Ortica S, Tarantino N and Gupta-Rossi N
In mammals, four Notch receptors (Notch1-4) and five ligands (Jagged1-2, Deltalike1, 3, 4) have been identified. In liver, Notch cascade dysfunction has been associated with developmental pathologies, like the Alagille syndrome (OMIM#118450 and 610205) and hepatic cancers. Notch signaling is required for the lineage specification of the hepatoblast, the common hepatic progenitor, towards the cholangiocytic fate, and appropriate bile duct morphogenesis.Data get more controversial when proliferation of precursors (the hepatoblasts) or hepatocytic commitment are considered. To get a better insight into the mechanisms of Notch function in liver progenitors we chose to use an in vitro(Strick-Marchand and Weiss, 2002; Strick-Marchand et al., 2004). These non transformed progenitors can be maintained in the undifferentiated state or differentiated into hepatocytes or cholangiocytes. differentiation system based on Bipotential Mouse Embryonic Liver (BMEL) cells
Notch is active in undifferentiated BMEL cells (Ochsner et al., 2007), and we demonstrate that its inhibition decreases BMEL cell proliferation through inhibition of S phase entry. To test the hypothesis that the four mammalian Notch receptors act differentially on liver progenitors, we generated BMEL cells stably expressing the active forms of each Notch paralogue. Our results show that only the active forms of Notch2 and Notch4 can complement the inhibition of hepatoblast proliferation caused by Notch inhibition. The active form of Notch3, on the contrary, impairs hepatoblast proliferation and increases cellular multinucleation. Moreover, we find that this active form is conducive of hepatocytic specification, while the other Notch receptors inhibit this fate.
In conclusion, our results show that, during hepatic development, the four Notch receptors have non-redundant roles, and that the type of the receptor involved can be more important than the overall pathway activation. On one hand, Notch2 and Notch4 are mainly responsible for progenitor proliferation; on the other hand, all receptors, except Notch3, need to be shut down to allow hepatocyte cell fate (Ortica & al., submitted)."
Brou, C., Logeat, F., Gupta, N., Bessia, C., LeBail, O., Doedens, J.R., Cumano, A., Roux, P., Black, R.A., and Israel, A. (2000). A novel proteolytic cleavage involved in Notch signaling: the role of the disintegrin-metalloprotease TACE. Mol Cell 5, 207–216.
Chien, C.T., Wang, S., Rothenberg, M., Jan, L.Y., and Jan, Y.N. (1998). Numb-associated kinase interacts with the phosphotyrosine binding domain of Numb and antagonizes the function of Numb in vivo. Mol Cell Biol 18, 598–607.
Fares, H., and Greenwald, I. (1999). SEL-5, a serine/threonine kinase that facilitates lin-12 activity in Caenorhabditis elegans. Genetics 153, 1641–1654.
Gupta-Rossi, N., Le Bail, O., Gonen, H., Brou, C., Logeat, F., Six, E., Ciechanover, A., and Israel, A. (2001). Functional interaction between SEL-10, an F-box protein, and the nuclear form of activated Notch1 receptor. J Biol Chem 276, 34371–34378.
Gupta-Rossi, N., Six, E., LeBail, O., Logeat, F., Chastagner, P., Olry, A., Israël, A., and Brou, C. (2004). Monoubiquitination and endocytosis direct gamma-secretase cleavage of activated Notch receptor. J Cell Biol 166, 73–83.
Gupta-Rossi, N., Ortica, S., Meas-Yedid, V., Heuss, S., Moretti, J., Olivo-Marin, J.-C., and Israël, A. (2011). The adaptor-associated kinase 1, AAK1, is a positive regulator of the Notch pathway. J Biol Chem 286, 18720–18730.
Ochsner, S.A., Strick-Marchand, H., Qiu, Q., Venable, S., Dean, A., Wilde, M., Weiss, M.C., and Darlington, G.J. (2007). Transcriptional profiling of bipotential embryonic liver cells to identify liver progenitor cell surface markers. Stem Cells 25, 2476–2487.
Sorensen, E.B., and Conner, S.D. (2008). AAK1 regulates Numb function at an early step in clathrin-mediated endocytosis. Traffic 9, 1791–1800.
Strick-Marchand, H., and Weiss, M.C. (2002). Inducible differentiation and morphogenesis of bipotential liver cell lines from wild-type mouse embryos. Hepatology 36, 794–804.
Strick-Marchand, H., Morosan, S., Charneau, P., Kremsdorf, D., and Weiss, M.C. (2004). Bipotential mouse embryonic liver stem cell lines contribute to liver regeneration and differentiate as bile ducts and hepatocytes. Proc Natl Acad Sci USA 101, 8360–8365.
Gupta-Rossi N, Ortica S, Meas-Yedid V, Heuss S, Moretti J, Olivo-Marin J-C & Israël A. 2011.
The Adaptor-associated Kinase 1, AAK1, Is a Positive Regulator of the Notch Pathway. J. Biol. Chem. 286, 18720-18730.
Gupta-Rossi N., Six E., LeBail O., Logeat F., Chastagner P., Olry A., Israël A. & Brou C. 2004. Monoubiquitination and endocytosis direct g-secretase cleavage of activated Notch receptor.
J. Cell Biol. 166, 73-83.
Six E., Ndiaye D., Laâbi Y., Brou C., Gupta-Rossi N., Israël A. & Logeat F. 2003. The Notch ligand Delta1 is sequentially cleaved by an ADAM protease and g-secretase. Proc. Natl. Acad. Sci. USA, 100, 7638-7643.
Gupta-Rossi N., Storck S., Griebel P.J., Reynaud C.-A., Weill J.-C. & Dahan A. 2003. Specific over- expression of Deltex and a new Kelch-like protein in human germinal center B cells. Mol. Immunol., 39, 791-799.
Gupta-Rossi N., LeBail O., Brou C., Logeat F., Six E., & Israël A. 2002. Control of Notch activity by the ubiquitin-proteasome pathway. In : Notch from Neurodevelopment to Neurodegeneration : Keeping the fate, Ed. Springer-Verlag, Berlin, Heidelberg, pages 41-58.
- Gupta-Rossi N., LeBail O., Gönen H., Brou C., Logeat F., Six E., Ciechanover A. & Israël A . 2001. Functional interaction between SEL-10, an F-box protein, and the nuclear form of activated Notch1 receptor. J. Biol. Chem., 276, 34371-34378.
Brou C., Logeat F., Gupta N., Bessia C., LeBail O., Doedens J.R., Cumano A., Roux P., Black R.A. & Israël A. 2000. A novel proteolytic cleavage involved in Notch signaling : the role of the disintegrin-metallopratease TACE. Mol. Cell., 5, 207-216.
Team members

Chargée de Recherche, Institut Pasteur
Phone 01 40 61 30 39
Sara Ortica
Etudiante en Thèse