Calcium ions (Ca2+) are extremely important intracellular messengers, activating a plethora of cellular processes. Growing evidence now points to a major role for the local Ca2+ signal in driving specific cellular responses. The simplest and most fundamental local Ca2+ signal is the Ca2+ microdomain, which rapidly forms when Ca2+ permeable ion channels open. In non-excitable cells the dominant Ca2+ entry channels are store-operated Ca2+ channels (SOCCs). The best characterised is the Ca2+ release activated Ca2+ (CRAC) channel. How local Ca2+ entry through CRAC channels impacts on channel function however is unclear. I have investigated the interaction between the Ca2+ binding protein calmodulin and CRAC channel activity and subsequent agonist-induced Ca2+ signals. Furthermore, I have investigated a role for mitofusin 2 (a protein that is known to tether the ER and mitochondria) on these Ca2+ signals. Using three different calmodulin mutant constructs with alterations to their Ca2+ binding sensitivities, I have shown that calmodulin facilitates CRAC channel dependent Ca2+ entry and maintains agonist-induced cytosolic Ca2+ oscillations in a lobe-specific manner. Calmodulin has four Ca2+ binding sites, two on the N-lobe and two on the C-lobe. I found a dominant negative calmodulin mutant (CAM4M, where all four binding sites had been mutated), or one where the C-lobe could not bind Ca2+ (CAM2C), impaired both Ca2+ influx through CRAC channels and maintenance of cytosolic Ca2+ oscillations. In contrast, a Ca2+-insensitive N-lobe mutant had little effect, (CAM2N). Knockdown of the mitochondrial Ca2+ uniporter regulator (MICU1) or mitochondrial membrane depolarization had similar effects to those seen with CAM4M or CAM2C, suggesting that at least in part, the action of calmodulin was through regulation of mitochondrial Ca2+ dynamics. This was confirmed by directly measuring the mitochondrial matrix Ca2+ concentration in intact RBL-1 cells using the mitochondrial targeted, fluorescent protein, pericam. Both CAM4M and disruption of mitochondrial Ca2+ buffering impaired agonist-induced mitochondrial Ca2+ uptake, suggesting that the modulation of CRAC channels occurred through Ca2+-calmodulin facilitation of mitochondrial Ca2+ uptake. Using a mutant Orai1 (A73E) that cannot bind calmodulin, I have shown that calmodulin tethered to the CRAC channel provides a major source of calmodulin for effective mitochondrial Ca2+ uptake. Physiological relevance of my proposed pathway was provided from experiments where I showed knockdown of MICU1 impaired agonist-induced CRAC channel dependent NFAT-1-driven gene expression. In addition, I establish a crucial role for mitochondrial MFN2 and presumably its ability to properly link the mitochondria and ER in the control of CRAC channels and agonist-induced Ca2+ signals.

Les cancers de l’ovaire représentent la première cause de décès par cancer gynécologique. L’échec des traitements, lié à l’apparition d’une chimiorésistance, implique de trouver de nouvelles stratégies thérapeutiques. Dans ce cadre, le laboratoire a mis en évidence la coopération de deux molécules anti-apoptotiques surexprimées dans les cancers de l’ovaire, Mcl-1 et Bcl-xL, qui empêchent alors le déclenchement de la mort cellulaire par apoptose. L’inhibition de ces cibles est alors mise en place. Un BH3-mimétique inhibiteur de Bcl-xL a été développé par la société Abbvie, l’ABT-737, qui possède un dérivé administrable par voie orale l’ABT-263 en essai clinique. En revanche, aucun inhibiteur de Mcl-1 n’est actuellement en clinique. L’inhibition de cet anti-apoptotique est donc l’un des objectifs du laboratoire. Sachant que Mcl-1 est extrêmement régulée, que l’inhibition de la voie de signalisation PI3K/Akt/mTOR conduit à l’inhibition de cet anti-apoptotique, et que le calcium est capable de moduler cette voie, nous nous sommes demandés si la modulation des flux calciques permettait l’inhibition de Mcl-1 dans nos cellules. Ces travaux de thèse ont pu montrer dans un premier temps que la chélation calcique par le BAPTA-AM permettait d’inhiber Mcl-1 via la voie mTORC1 et de sensibiliser les cellules à l’ABT-737. Dans un second temps, l’étude de l’effet d’un inhibiteur des flux calciques en essais cliniques, le carboxyamidotriazole a permis de mettre en évidence que l’inhibition des canaux calciques capacitifs pouvait entraîner l’inhibition de Mcl-1 de nouveau en inhibant mTORC1 et induire la mort cellulaire en combinaison avec l’ABT-737. Enfin des observations morphologiques ont montré que le CAI induisait un changement morphologique aboutissant à la mort des cellules. Ce type de mort semble être lié à une perturbation du métabolisme des cellules cancéreuses IGROV1-R10 et se rapprocherait d’une mort récemmement décrite dans la littérature : l’autosis. / Ovarian cancer is the leading cause of death from gynecological cancer. There is an urgent need to find new therapeutic strategies due to failure of treatments associated to development of chemoresistance. In this context, the laboratory has shown the cooperation of two anti-apoptotic proteins overexpressed in ovarian cancer, Mcl-1 and Bcl-xL, for preventing apoptotic cell death. ABT-737, a Bcl-xL inhibitor BH3-mimetic, was developed by Abbvie and has a clinically derivative named ABT-263. In contrast, no Mcl-1 inhibitor is currently available in clinic. The inhibition of this anti-apoptotic protein is therefore one of the objectives of the laboratory. Since inhibition of PI3K / Akt / mTOR signaling pathway leads to inhibition of Mcl-1 expression and calcium is able to modulate this pathway, we wondered if the modulation of calcium fluxes allowed the inhibition of Mcl-1 in ovarian cancer cells. First we were able to show that calcium chelation by BAPTA-AM allowed to inhibit Mcl-1 expression via mTORC1 pathway and to sensitize the cells to ABT-737. In a second step, we investigated the effect of an inhibitor of calcium fluxes that is evaluated in clinical studies, carboxyamidotriazole. We show that the inhibition of capacitive calcium channels could lead to Mcl-1 down-expression via inhibition of mTORC1 and promote apoptosis in combination with ABT-737. Finally, we observed that CAI induces a morphological change resulting in cell death. This type of death seems to be related to disruption of metabolism in IGROV1-R10 cancer cells and would be close to a cell death recently described in the literature: autosis.

Cholestasis is an important liver pathology. During cholestasis bile acids accumulate in the bile canaliculus affecting hepatocyte viability. The actions of bile acids require changes in the release of Ca2+ from intracellular stores and in Ca2+ entry. The target(s) of the Ca2+ entry pathway affected by bile acids is, however, not known. The overall objective of the work described in this thesis was to elucidate the target(s) and mechanism(s) of bile acids-induced modulation of hepatocytes calcium homeostasis. First, it was shown that a 12 h pre-incubation with cholestatic bile acids (to mimic cholestasis conditions) induced the inhibition of Ca2+ entry through store-operated Ca2+ channels (SOCs), while the addition of choleretic bile acids to the incubation medium caused the reversible activation of Ca2+ entry through SOCs. Moreover, it was shown that incubation of liver cells with choleretic bile acids counteracts the inhibition of Ca2+ entry caused by pre-incubation with cholestatic bile acids. Thus, it was concluded that SOCs are the target of bile acids action in liver cells. Surprisingly, despite the effect of choleretic bile acids in activating SOCs, the Ca2+ dye fura-2 failed to detect choleretic bile acid-induced Ca2+ release from intracellular stores in the absence of extracellular Ca2+. However, under the same conditions, when the sub-plasma membrane Ca2+ levels were measured using FFP-18 Ca2+ dye, choleretic bile acid induced a transient increase in FFP-18 fluorescence. This evidence suggested that choleretic bile acids-induced activation of Ca2+ entry through SOCs, involving the release of Ca2+ from a region of the endoplasmic reticulum (ER) located in the vicinity of the plasma membrane.