Projet ANR-07-BLAN-0352 – CASER : Rôle et régulation de l'activité des caspases au cours de l'érythropoïèse normale et pathologique
Programme : Blanc (BLANC) 2007
Année de financement : 2007
Coordinateur du projet : Monsieur Olivier Hermine (CNRS DELEGATION REGIONALE PARIS A)
Partenaires : Centre national de la recherche scientifique; Institut national de la santé et de la recherche médicale; Institut national de la santé et de la recherche médicale
Montant : 550000.0 euros
Date de début : 2007
Durée : 36 mois
Résumé : Objectives of the project Two phases can be identified during the erythroid differentiation process: one first step encompassing a strong cell proliferation with a low degree of progenitor maturation and a second phase of differentiation where the erythroid precursors become red blood cells with a restricted proliferative tendency. We previously showed that a transitory caspase activation that did not lead to apoptosis was necessary for the achievement of the erythroid differentiation process. Some substrates of the caspases such as the transcription factor GATA-1 cleaved during apoptosis are protected during erythroid differentiation. The aim of this project is to understand how the caspases are activated during erythroid differentiation and what is the exact role of this caspase activation. To do that, we will identify the proteins that are cleaved during this process and determine the role of this cleavage. The different caspase targets will be also identified during apoptosis and the mechanism of their protection during erythroid differentiation will be elucidated. We recently showed that GATA-1 protection was due to its association with the HSP70 protein. This protein association is under the control of Epo that determines HSP70 nuclear localization. We aim to determine 1- how the association between HSP70 and GATA-1 proteins is controlled by Epo, 2- if other substrates behaving differently during differentiation and apoptosis are also protected by a similar mechanism. A last question is to determine if this system involving heat chock proteins is modified in two human disorders: the myelodysplastic syndromes (MDS) and the polycythemia vera (PV). Description of the project We will use the I-11 cell line and erythroblastic progenitors derived from murine ES cells for the identification of caspase substrates. In both situations, the cell population is greatly amplified at the stage of proerythroblast progenitor and a synchronous differentiation can be obtained with a change of cytokines in the medium. Permanent clones of these cells expressing the caspase-inhibitor protein p35 will be derived. Both control and p35-expressing cells will be either induced towards differentiation or apoptosis by cytokine deprivation. In both cases, the proteins cleaved by caspases will be identified by 2D electrophoresis. Total cell lysates and nuclear extracts will be analysed. A gel free analysis will be also performed by the COFRADIC technique to overcome the E2D method limitations. The role of the cleavage of the identified proteins will be studied in a model of human erythroid progenitor expansion. Cleaved fragments, non cleavable forms or siRNA against these candidate proteins will be tested. On the other hand, if murine models are available, we plan to derive murine ES cells from mice harbouring genetic modifications for these candidate proteins. Several results suggest that the intrinsic apoptosis pathway could be responsible for caspase activation during the differentiation process. One question is to ask whether pro or anti-apoptotic proteins belonging to the Bcl-2 family play a role in this activation. In addition, it seems that the decrease of the intracellular signalling pathway due to the down regulation of both SCF and Epo receptors contributes to the process of caspase activation. This hypothesis will be tested by using modified Epo receptors, where motifs permitting receptor down regulation are deleted. The pathways that control HSP70 nuclear localization will be identified by means of chemical inhibitors on the one hand, and with mutant EpoR that do not activate selected intracellular pathways on the other hand. The resulted data will be verified by overexpression or inactivation by siRNA of key signalling proteins involved in these pathways. GATA-1 is the target of several post translational modifications (acetylation, sumoylation, phosphorylation); some of these modifications are induced by Epo signalling. By using severa..