Projet ANR-07-BLAN-0102 – CALYTE : Caractérisation de canaux calciques propres aux lymphocytes Th2. Application au traitement de maladies allergiques.
Programme : Blanc (BLANC) 2007
Année de financement : 2007
Coordinateur du projet : Madame Lucette PELLETIER (INSTITUT NATIONALE DE LA SANTE ET DE LA RECHERCHE MEDICALE TOULOUSE ADR4 UNITES 28 326 395 455)
Partenaires : Centre national de la recherche scientifique; Institut national de la santé et de la recherche médicale; Université de Toulouse III - Paul Sabatier
Montant : 368200.0 euros
Date de début : 2007
Durée : 36 mois
Résumé : In collaboration with team 2, we showed that Th2 cells responsible for allergic diseases selectively expressed dihydropyridine (DHP) receptors that were implicated in calcium influx upon T-cell receptor (TCR) stimulation. Calcium signaling is essential for type 2-cytokines (IL-4, IL-5, IL-13) production; all of these cytokines contributing to the pathogenesis asthma. This is likely to explain our results showing that DHP antagonists prevented airway inflammation in experimental models of Th2-induced allergic asthma. In sharp collaboration with team 2, we found voltage-dependent (Cav1.2 and Cav1.3) alpha1 specific PCR products in Th2 but not in Th1 cells. Of note, these channels that characterize excitable cells are defined as DHP receptors in these cells. The project CALYTE aims at deciphering the structure and the functions of Cav1 channels in Th2 cells, in order to understand if Th2 cells express the canonical channels and to establish a model accounting for their function in non-excitable cells. In addition, it has been recently shown that the COOH-terminus of the Cav1.2 channel (CCAT) was a transcription factor. Therefore, we will study the potential role of CCAT in controlling gene expression in Th2-lymphocytes. 1) After completing the sequence of the Cav1.2 and Cav1.3-related channels in Th2 cells, we will clone the full-length cDNA, tag them (in their COOH and NH2 terminus parts) and clone them into a retrovirus in order to transduce T-cells. 2) In sharp collaboration with team 2, we are developing imaging technics (optic patch) allowing to visualize very localized calcium fluxes (TIRFM) and the softwares allowing to determine the biophysical characteristics of the channels. To do this study, we will compare Th2 cells to Th2 cells transfected with Cav1.2 and Cav1.3 specific antisense/siRNA oligonucleotides. The efficacy and specificity of transfection will be evaluated by quantification of cDNAs encoding Cav1 channels and other calcium signaling actors (store-operated calcium channels ...). 3) We will study the subcellular location (cell membrane versus endoplasmic reticulum, immunological synapse) and a putative colocalization with kinases such as protein kinase C in Th2 cells retrovirally transduced with the tagged Cav1 channels. The overexpression of the channels in Xenopus oocytes and in T-cells will allow to detemine whether these channels can actually be voltage-operated. 4) The role of CCAT as a transcription factor in Th2 cells and the putative modifications of nuclear localization in resting and activated Th2 cells will be studied by confocal microscopy and or Western blotting by using an antibody that will be generated. 5) The kinetics of expression of the channels during Th2 cell differentiation show that these channels are induced quite late, after the induction of the Th2-lineage specific transcription factor GATA-3. We will approach a potential role for GATA-3 in controlling Cav1 channel expression by transducing naive CD4+ T and Th1 cells with a retrovirus encoding GATA-3 and we we will follow the expression of Cav1 channels by real time quantitative RT-PCR. 6) We will assess the capacity of Cav1 antisense oligonucleotides to inhibit the Th2-dependent airway inflammation in experimental models of asthma that are available in the lab. 7) We plan to test whether T lymphocytes collected from the bronchoalveolar lavage fluid of asthmatic patients preferentially express Cav1/DHP receptors. This study could allow to validate our model relying on the hypothesis that Th2 cells express a few channels (as compared to excitable cells), the probability of which to cluster and to interact with protein kinase C will be enhanced upon TCR engagement. This work would support the notion that TCR-associated signaling pathways vary depending upon the state of activation, of differentiation and upon the functions exerted by a T-cell subset. Obviously, it can offer opportunity in therapeutics, based on the possibility...