The presence of microglia at sites of CNS injury can potentially shift the balance between neuronal survival and death; however, the mechanisms regulating their mobilization to these sites are still poorly understood. Here I report that microglia express podosomes, short-lived, punctate organelles which adhere to and degrade extracellular matrix (ECM). Podosomes and related invadopodia of cancer cells have recently been the focus of much interest for their roles in migration and invasion. Microglial podosomes degraded ECM, providing impetus for further study of their function in microglia. Further, I report the Ca2+-activated SK3 channel as a novel component of the podosome core. While SK3 and SK4 channels are reported to play redundant roles in activated microglia (Kaushal et al., 2007; Schlichter et al., 2010), immunostaining work suggests that they are differentially regulated during microglial activation. Together, these results suggest unique functions for these channel subtypes in microglia.

The presence of microglia at sites of CNS injury can potentially shift the balance between neuronal survival and death; however, the mechanisms regulating their mobilization to these sites are still poorly understood. Here I report that microglia express podosomes, short-lived, punctate organelles which adhere to and degrade extracellular matrix (ECM). Podosomes and related invadopodia of cancer cells have recently been the focus of much interest for their roles in migration and invasion. Microglial podosomes degraded ECM, providing impetus for further study of their function in microglia. Further, I report the Ca2+-activated SK3 channel as a novel component of the podosome core. While SK3 and SK4 channels are reported to play redundant roles in activated microglia (Kaushal et al., 2007; Schlichter et al., 2010), immunostaining work suggests that they are differentially regulated during microglial activation. Together, these results suggest unique functions for these channel subtypes in microglia.

Nous avons montré un rôle d’un canal K+, le canal SK3, dans la migration des cellules cancéreuses de sein MDA-MB-435s et le développement de métastases ostéolytiques du cancer du sein. Lors de l’ostéolyse, la [Ca²+]ext augmente dans le microenvironnement osseux. Nous avons voulu déterminer si cette élévation de [Ca²+]ext, pouvait moduler l’expression et l’activité du canal SK3. Nous avons montré que l’augmentation de la [Ca²+]ext: i) favorise l’expression du canal SK3. Cet effet fait intervenir le récepteur au calcium (CaSR), qui en diminuant la [AMPc]int réduit l’activité de la PKA et lève ainsi son inhibition de la transcription du gène KCNN3 (codant pour SK3) ; ii) favorise la migration cellulaire dépendante du canal SK3, mécanisme impliquant également le CaSR ; iii) active le canal SK3 qui, par ailleurs, voit son activité réduite par l’élévation d’AMPc intracellulaire. De plus, l’augmentation d’AMPc délocalise un canal calcique partenaire de SK3, le canal Orai1, et diminue l’entrée constitutive de Ca²+ et la migration dépendantes du canal SK3. En conclusion, nos résultats montrent que l’expression et l’activité de SK3 sont régulées par l’AMPc et le Ca2+ extracellulaire. Ceci permet d’envisager une nouvelle stratégie thérapeutique ciblant l’AMPc pour le traitement des métastases osseuses du cancer du sein. / We showed that a K+ channel, SK3 channel, is a mediator of MDA-MB-435s breast cancer cells migration and of osteolytic bone metastasis development of breast cancer. Since [Ca²+]out rises during osteolysis, in bone microenvironment, we study if this [Ca²+]out elevation could modulate SK3 expression and activity. We show that [Ca²+]out elevation: i) increases SK3 expression threw CaSR activation which, in turn, decreases [cAMP]int and PKA activation, leading to loss of its inhibitory effect on KCNN3 transcription; ii) increases SK3-dependent migration threw CaSR activation; iii) increases SK3 channel activity that is in addition, decreased by [cAMP]int elevation. Furthermore, cAMP elevation moves the Ca2+ channel Orai1 (SK3 partner) outside of lipid rafts and reduces the SK3 dependent-constitutive Ca²+ entry and cell migration. Our results show that both SK3 expression and activity are regulated by cAMP and extracellular Ca²+. These results underscore an innovative opportunity to use therapeutic approaches targeting cAMP for the treatment of breast cancer bone metastasis.

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SK3 channels contribute to membrane repolarization and hyperpolarization that leads to both relaxation of smooth muscle and vascular branching. These two distinct properties are intensified in the SK3T/T mice possibly influencing pregnancy by dampening uterine contractions and causing dysfunctional placental development. SK3T/T mice have delayed or hindered parturition, suggesting a role for SK3 channels in labor contractions (Chapter 2 & 3). Based on these findings, we hypothesized that SK3 channel expression must be reduced late in normal pregnancy to enable the uterus to produce the forceful contractions required for parturition. The mechanism(s) downregulating this channel in the uterus during pregnancy is unknown. The SK3 gene promoter region contains two Specificity Protein (Sp) binding sites; Sp1, a transcription factor that enhances transcription of genes in response to estrogen, and Sp3, a factor that competes for the same binding motif as Sp1 to reduce gene expression (Chapter 4). SK3 channels may also be involved in the vascular remodeling that occurs during pregnancy. The SK3 channel is present in vascular endothelial cells and overexpression of this channel leads to abnormal vessel branching and an increase in vessel diameter. During pregnancy, the vascular system must adapt to accommodate dramatic increases in blood volume necessary to sustain the developing fetus. Overexpression of SK3 channels could produce abnormalities in the placental vascular network, similar to the abnormal vessel branching and vasodilatation found in the mesenteric circulation, thus leading to poor fetal outcome (Chapter 5). The aim of this research was to determine the function of the SK3 channel in pregnancy by focusing on its role in myometrial contractility in addition to identifying its role in remodeling the maternal vasculature and its impact on placental blood flow and fetal demise.

Il a été mis en évidence que le canal SK3 est un médiateur de la migration de cellules cancéreuses mammaires, une propriété essentielle à la formation de métastases. Par ailleurs, ce canal est inhibé par l’édelfosine, un éther-lipide ayant des propriétés anti-tumorales in vitro mais son usage en clinique a été abandonné en raison d’effets secondaires. Une première partie de ce travail a permis de déterminer les parties de l’édelfosine nécessaires à l’inhibition du canal SK3 et de la migration cellulaire. Ceci nous a permis de sélectionner l’Ohmline (1-O-Hexadécyl-2-O-Méthyl-sn-glycéro-lactose), un analogue non toxique de l’édelfosine qui conserve son activité inhibitrice de SK3 et de la migration. Dans un deuxième temps, nous avons testé ce lipide dans un modèle murin de développement tumoral et nous avons montré qu’il réduisait le développement des métastases sans modifier la tumeur primaire. En conclusion, nous avons décrit l’Ohmline qui est le premier inhibiteur lipidique de SK3 et qui pourrait devenir le premier membre d’une famille de composés lipidiques inhibiteurs de la formation de métastases. / It has been shown that SK3 channel was a mediator of breast cancer cells migration, a fundamental property for metastasis formation. In addition, edelfosine inhibits SK3 channel. This ether-lipid owns a high anti cancerous potential in vitro but its clinical use was hampered by some side effects, Firstly, we showed the structural parts of edelfosine required for SK3 channel inhibition and cell motility inhibition. Moreover, we selected Ohmline (1-O-Hexadécyl-2-O-Méthyl-sn-glycéro-lactose), an edlefosine’s analogue that preserves SK3 channel and motility inhibitory properties. Secondly, we evaluated this lipid on tumor development in nude mice model. We showed that this lipid reduces metastasis formation without effect on primary tumor. To conclude, we described Ohmline, the first lipid inhibitor of SK3. This compound should become the first member of a new family of metastasis lipid inhibitors.

De plus en plus d'études désignent les canaux K+ et Ca2+ comme de nouvelles cibles très prometteuses pour des thérapies anticancéreuses. Parmi ces canaux, les canaux SK3 et CaV1.3 ont été identifiés comme des régulateurs de la migration des cellules cancéreuses. Lors de mes travaux de thèse, nous avons découvert une nouvelle voie de signalisation montrant que la protéine αlD du canal CaVl.3 est impliquée dans la régulation de l'homéostasie Ca2+ ainsi que dans la migration cellulaire, par un mécanisme indépendant de sa fonction canonique. En parallèle nous avons identifié des résidus sérines du canal SK3 qui sont importants dans la régulation négative du canal SK3 par l'activation de la voie AMPc-PKA. Ces résultats suggèrent également que l'Ohmline, un éther-lipide de synthèse, inhibiteur du canal SK3, ne réduit pas l' activité du canal SK3 en favorisant sa phosphorylation suite à l'activation de la voie AMPc-PKA. / Many studies show that K+ and Ca2+ channels are promising new targets for anticancer therapies. Among these channels, SK3 and Ca V 1.3 ion channels have been identified as regulators of cancer cell migration. Here, we have discovered a new signalization pathway showing that the αlD protein of the CaV1 .3 channel is involved in the regulation of Ca2+ homeostasis and in cell migration by a mechanism independent of its canonical function. In parallel, we have identified serine residues of SK3 channel that are important in the negative regulation of the SK3 channel by activation of the cAMP-PKA pathway. These results also suggest that Ohmline, a synthetic ether-lipid, and inhibitor of SK3 channel, does not reduce the activity of SK3 channel by promoting its phosphorylation following the activation of the cAMP-PKA pathway.