L'altération de glycosylation est l'une des caractéristiques du cancer du sein. Ainsi le défaut de glycosylation affecte différentes protéines glycosylées responsables des différents processus cancéreux. Les canaux SOC (Store operated channels) constituent la voie majeure de l'entrée du calcium dans les cellules et sont impliqués dans la prolifération, la migration et la survie des cellules cancéreuses du sein. CO-OCS est un nouvel inhibiteur de la glycosylation avec plus de sélectivité vis-à-vis des α-glucosidases, et montre des activités anticancéreuses des cellules cancéreuses du sein, sans affecter les cellules mammaires normales. L'objectif de ma thèse est d'étudier les mécanismes moléculaires par lesquels CO-OCS induit ses effets anti-tumoraux. CO-OCS inhibe la migration des cellules cancéreuses à fort potentiel métastatique. Cet effet anti-migratoire est dû à une réduction de l'expression de la β1-intégrine, de Stim1, et de l'activation des voies de signalisation FAK et ERK1/2 par CO-OCS. Dans les cellules cancéreuses peu invasives, CO-OCS diminue la prolifération et augmente la mortalité de ces cellules en affectant l'expression de 3 protéines : Stim1 et Orai1 : protéines N-glycosylées au niveau du réticulum endoplasmique (RE), et GRP78, protéine de stress du RE. Ainsi en supprimant complétement l'expression de Stim1, CO-OCS réduit la prolifération en accumulant les cellules dans les phases G1 et G2/M du cycle cellulaire. Alors que la réduction de l'expression de GRP78 et d'Orai1 par le CO-OCS augmente respectivement l'apoptose et la nécrose. Par ailleurs, l'invalidation de Stim1 atténue l'effet apoptotique induit par CO-OCS. CO-OCS réduit aussi le contenu calcique du RE. Cette réduction du calcium réticulaire est due à une fuite de calcium par le Translocon. En effet, l'Anisomycine, inhibiteur du Translocon, restore de contenu calcique réticulaire et antagonise l'apoptose induite par le CO-OCS. En conclusion, CO-OCS induit une accumulation de protéines mal-repliées dans le RE induisant ainsi un stress réticulaire. Trois cibles du CO-OCS ont été identifiées : l'expression de Stim1 favorise la prolifération tandis que celle d'Orai1 et de GRP78 protègent respectivement les cellules de l'apoptose et de la nécrose induites par CO-OCS. De plus, en diminuant l'expression de GRP78, CO-OCS induit une fuite du calcium du RE par le Translocon / Alteration in glycosylation pattern is one of the hallmarks of breast cancer. The levels and the abnormal expressions of glycan were found in breast cancer patients. Glycosylation defect can affect different glycosylated proteins which are implicated in cancerogenesis. Changes in intracellular Ca2+ levels can regulate different cellular processes. SOC channels are implicated in breast cancer proliferation, migration and survival. CO-OCS is a new glycosylation inhibitor with more selectivity toward theα- glucosidases exhibited anti-cancer activities in breast cancer cells without affecting the normal mammary cells. The objective of my thesis is investigating the related molecular mechanisms by which CO-OCS induced its anti-tumour effects.CO-OCS impaired breast cancer migration through decrease β1-integrin expression and the activation of FAK and ERK1/2 signalling pathways. CO-OCS also induced anti-migratory effect via Stim1 protein expression down-regulation leading to inhibition of SOCE. Additionally, CO-OCS affected the expression of both Orai1 and Stim1 proteins leading to anti-proliferative effects and cell cycle arrest in G1 and G2/M phase respectively. Moreover, CO-OCS affected the expression of Stim1 at the protein level without affecting its transcript level. GRP78 implicated in CO-OCS apoptotic death. The expression of Stim1 regulated the apoptosis induced by CO-OCS via modulating GRP78 expression. Orai1 down-regulation promoted CO-OCS necrotic effect. CO-OCS induced ER- calcium depletion due to increase in ER calcium leak via the Translocon; Anisomycin (Translocon inhibitor) decreased the apoptosis induced by CO-OCS. In conclusion, these results show that in breast cancer, by targeting Stim1, Orai1 and GRP78, CO-OCS reduced cell proliferation and induced apoptosis and necrosis cell death. Stim1 favours CO-OCS apoptotic effect while Orai1 protected from necrosis induced by CO-OCS. The inhibition of Translocon decreased CO-OCS apoptotic cell death via restoring the ER calcium homeostasis

Ein durch „Ca2+ Release Activated Ca2+ (CRAC)“-Kanal vermittelter Ca2+-Einstrom ist unverzichtbar für die vollständige Aktivierung von T-Zellen und eine produktive Immunantwort. Im Jahr 2006 führte die Entdeckung des transmembranen Proteins Orai1, einer porenbildenden Untereinheit des CRAC-Kanals, zu einem besseren Verständnis dieses Signalweges. Eine Mutation in Orai1 hat durch die Aufhebung der CRAC-Kanal Funktion eine schwere kombinierte Immundefizienz (SCID) zur Folge (Feske, S. et al. 2006). Die im Rahmen dieser Arbeit präsentierten Experimente hatten die nähere Erforschung der Rolle von Orai1 in Bezug auf die Aktivierung und Entwicklung von Lymphozyten sowie auf die pathogenetische Bedeutung für humane Immundefektsyndrome zum Ziel. So konnte hier durch das Sequenzieren genomischer DNA mehrerer SCID-Patienten eine neue Mutation in Orai1 aufgedeckt werden. Mithilfe intrazellulärer Durchflusszytometrie und Real-Time-PCR gelang es, die Expression von Orai1 auf humanen und murinen Immunzellen, einschließlich T- und B-Lymphozyten, nachzuweisen. Darüber hinaus wurden Orai1 „knock-in“ Mäuse analysiert, welche transgen für eine bei zwei SCID-Patienten gefundene Mutation (R91W) (Feske, S. et al. 2006) sind. Dadurch war es möglich die Funktion von Orai1 und die des CRAC-Kanal vermittelten Ca2+-Einstroms für die Entwicklung und Aktivierung von Lymphozyten zu analysieren. Diese transgenen Mäuse stellen das zu diesem Zeitpunkt erste Tiermodell dar, mit dessen Hilfe die Rolle von CRAC-Kanälen in vivo studiert werden kann.

Ein durch „Ca2+ Release Activated Ca2+ (CRAC)“-Kanal vermittelter Ca2+-Einstrom ist unverzichtbar für die vollständige Aktivierung von T-Zellen und eine produktive Immunantwort. Im Jahr 2006 führte die Entdeckung des transmembranen Proteins Orai1, einer porenbildenden Untereinheit des CRAC-Kanals, zu einem besseren Verständnis dieses Signalweges. Eine Mutation in Orai1 hat durch die Aufhebung der CRAC-Kanal Funktion eine schwere kombinierte Immundefizienz (SCID) zur Folge (Feske, S. et al. 2006). Die im Rahmen dieser Arbeit präsentierten Experimente hatten die nähere Erforschung der Rolle von Orai1 in Bezug auf die Aktivierung und Entwicklung von Lymphozyten sowie auf die pathogenetische Bedeutung für humane Immundefektsyndrome zum Ziel. So konnte hier durch das Sequenzieren genomischer DNA mehrerer SCID-Patienten eine neue Mutation in Orai1 aufgedeckt werden. Mithilfe intrazellulärer Durchflusszytometrie und Real-Time-PCR gelang es, die Expression von Orai1 auf humanen und murinen Immunzellen, einschließlich T- und B-Lymphozyten, nachzuweisen. Darüber hinaus wurden Orai1 „knock-in“ Mäuse analysiert, welche transgen für eine bei zwei SCID-Patienten gefundene Mutation (R91W) (Feske, S. et al. 2006) sind. Dadurch war es möglich die Funktion von Orai1 und die des CRAC-Kanal vermittelten Ca2+-Einstroms für die Entwicklung und Aktivierung von Lymphozyten zu analysieren. Diese transgenen Mäuse stellen das zu diesem Zeitpunkt erste Tiermodell dar, mit dessen Hilfe die Rolle von CRAC-Kanälen in vivo studiert werden kann.

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.

Alors que l’entrée SOC (store-operated Ca2+ entry) portée par les canaux calciques TRPCs (transient receptor potential canonical) et Orai1 est essentielle dans les cellules non-excitables, son rôle physiologique dans les cardiomyocytes adultes reste à élucider. Néanmoins, il est largement admis qu’une entrée SOC exacerbée dépendante des canaux TRPCs et de la protéine régulatrice STIM1 participe à la pathogenèse de l’hypertrophie et de l’insuffisance cardiaque (IC) par induction de voies pro-hypertrophiques telles que la CaMKII (Ca2+/calmoduline-dépendante kinase II ) et la calcineurine (CaN)/NFAT (Nuclear factor of activated T-cells). Au contraire, une inhibition fonctionnelle ou une extinction génique des canaux TRPCs et de la protéine STIM1 serait cardioprotectrice contre le stress hypertrophique. Cependant, le rôle physiopathologique des canaux calciques Orai1 dans le cœur reste, à ce jour, méconnu et débattu puisque son extinction in vitro présente un effet bénéfique contre l’hypertrophie des cardiomyocytes alors que son extinction in vivo présente des effets délétères avec le développement d’une cardiomyopathie dilatée. De plus amples investigations quant au rôle d’Orai1 dans la physiopathologie cardiaque apparaissent donc primordiales. De ce fait, les objectifs de ma thèse sont d’explorer le rôle de la signalisation calcique dépendante d’Orai1 dans le cœur dans des conditions physiologiques et pathologiques grâce à un modèle de souris transgéniques exprimant un mutant non fonctionnel d’Orai1, spécifiquement dans le cœur (dn-Orai1R91W/tTa) et un inhibiteur pharmacologique sélectif, le JPIII. Tout d’abord, nous montrons que les souris dn-Orai1R91W/tTa présentent une fonction cardiaque normale et une homéostasie calcique impliquée dans le couplage excitation-contraction conservée suggérant qu’Orai1 n’a pas de rôle majeur dans le coeur adulte en condition physiologique. Cependant, nous avons démontré une augmentation de l’expression et de l’activité d’Orai1 dans un modèle murin d’hypertrophie cardiaque induite par surcharge de pression, qui serait délétère pour la fonction ventriculaire. Au contraire, l’inhibition fonctionnelle d’Orai1 par manipulation génétique ou par l’outil pharmacologique (JPIII) semble protéger le coeur des dysfonctions ventriculaires au cours de l’hypertrophie. Cet effet bénéfique passerait par une restauration de l’homéostasie calcique et notamment par un maintien de l’expression de la pompe ATPase SERCA2a. Nous avons également mis en évidence que la voie de l’aldostérone/récepteurs aux minéralocorticoïdes modulait l’expression des canaux TRPC1, -C4, -C5 et notamment Orai1 via la protéine SGK1 (Serum and Glucocorticoid-regulated Kinase 1) dans les cardiomyocytes ventriculaires de rat nouveaux-nés. L’activation de cette voie de signalisation pourrait être à l’origine de la surexpression des canaux TRPCs/Orai1 retrouvée au cours de l’hypertrophie cardiaque. Ces travaux décrivent donc Orai1 comme une cible thérapeutique potentielle dans le traitement de l’hypertrophie cardiaque et de l’IC. / While the SOCE (store-operated Ca2+ entry), carried by TRPCs (transient receptor potential canonical) and Orai1 channels, is essential in non-excitable cells, its physiological role in adult cardiomyocytes remains elusive. Nevertheless, it is well established that exacerbated TRPCs/STIM1-dependent Ca2+ entry participates in the pathogenesis of hypertrophy and heart failure (HF) via the induction of pro-hypertrophic signaling pathways, such as CaMKII (Ca2+/calmodulin-kinase II) and calcineurin (CaN)/ NFAT (nuclear factor of activated T-cells). By contrast, functional inhibition or gene silencing of TRPCs and STIM1 is cardioprotective against hypertrophic insults. As for Orai1 Ca2+ channels, their pathophysiological roles in the heart remain unknown and under debate, since in vitro Orai1 silencing has a beneficial effect against cardiomyocyte hypertrophy, whereas in vivo silencing has deleterious effects with the development of dilated cardiomyopathy. Further investigations are necessary to determine the pathophysiological role of Orai1 in the heart. My thesis objectives are to explore the role of Orai1-dependent Ca2+ signaling in the heart under physiological and pathological conditions using a transgenic mouse model expressing a non functional mutant of Orai1, specifically in the heart (dn-Orai1R91W/tTa) and a selective pharmacological inhibitor, JPIII. First, we showed that dn-Orai1R91W/tTa mice have normal cardiac function and conserved Ca2+ homeostasis involved in the excitation-contraction coupling suggesting that Orai1 is not instrumental in regulating cardiac function under physiological conditions. However, we demonstrated an increased Orai1 expression and activity in a mouse model of cardiac hypertrophy induced by pressure overload, which is a maladaptive alteration involved in pathological ventricular dysfunction. By contrast, functional inhibition of Orai1 by genetic manipulation or by the pharmacological tool (JPIII) protects the heart from ventricular dysfunction after pressure overload-induced cardiac hypertrophy. This beneficial effect is related to a restoration of Ca2+ homeostasis and more specifically, is due to preserved ATPase SERCA2a pump expression. We also showed that the aldosterone/mineralocorticoid receptor signaling pathway modulates the expression of TRPC1, -C4, -C5 channels and also the Orai1 channels expression via the SGK1 (Serum and Glucocorticoid-regulated Kinase 1) protein, in neonatal rat ventricular cardiomyocytes. The activation of this signaling pathway could be the cause of the TRPCs/Orai1 channels overexpression found during cardiac hypertrophy. In conclusion, our studies highlighted that Orai1 Ca2+ channels could constitute potential therapeutic target in the treatment of cardiac hypertrophy and HF.

Calcium is the major mineral component of milk and is essential for neonatal development. To enrich the milk, calcium must pass from the maternal bloodstream, through mammary epithelial cells, into the alveolar lumen. While calcium extrusion from the epithelial cells is well characterized, no calcium channel or transporter has been identified as the major conduit for calcium to enter the mammary epithelial cell from the bloodstream. A major aim of this thesis was to identify a calcium channel or channels responsible for calcium influx into mammary epithelial cells during lactation. Real time reverse transcription-polymerase chain reaction was used to investigate in vivo expression of calcium channels in the murine mammary gland at the four main stages of mammary gland development. The store-operated calcium channel Orai1 was upregulated during lactation relative to its expression in the nulliparous gland. The classic ORAI1 regulator Stim1 was not similarly overexpressed during lactation, however, its isoform Stim2 was modestly upregulated. HC11 murine mammary cells were used as a model to further investigate the role of STIM2 on calcium handling during lactation. siRNA knockdown of Stim2 reduced both basal and agonist-induced peak cytosolic calcium levels, indicative of its role in calcium regulation. In addition to investigating the role of calcium channels in normal mammary development, their role in breast cancer was examined. Real time reverse transcription-polymerase chain reaction was used to identify calcium channels upregulated in human breast cancer cell lines, relative to non-tumorigenic mammary cell lines. TRPV1, TRPV6, and ORAI1 were upregulated in the breast cancer cell lines. Pharmacological modulation of ORAI1 resulted in modest changes in proliferation, but as there was no specific ORAI1 inhibitor, this effect could not be conclusively attributed to ORAI1 inhibition. siRNA was used to specifically target ORAI1 in three human breast cancer cell lines: MCF-7, MDA-MB-231, and T-47D. siRNA knockdown of ORAI1 was specific and potent, and reduced cell viability and altered calcium handing in all three cell lines. The alterations caused by ORAI1 knock down were not related to the expression of the genes CDK2 and FOS, as these did not change upon ORAI1 knockdown. Data mining was performed using the National Center for Biotechnology Information’s (NCBI’s) expressed sequence tag (EST) database, dbEST, and the Oncomine database. ORAI1 was elevated in estrogen receptor negative breast cancers and in the basal breast cancer molecular subtype, a subtype that has a poor prognosis. Other data suggested that breast cancer cells with high STIM1 and low STIM2 expression also correlated with the basal breast cancer subtype. These data indicate that ORAI and STIM proteins have a role in the physiological process of lactation as well as in the regulation of tumorigenic pathways in the breast, and particular gene expression profiles may be predictors of disease prognosis.

Calcium is the major mineral component of milk and is essential for neonatal development. To enrich the milk, calcium must pass from the maternal bloodstream, through mammary epithelial cells, into the alveolar lumen. While calcium extrusion from the epithelial cells is well characterized, no calcium channel or transporter has been identified as the major conduit for calcium to enter the mammary epithelial cell from the bloodstream. A major aim of this thesis was to identify a calcium channel or channels responsible for calcium influx into mammary epithelial cells during lactation. Real time reverse transcription-polymerase chain reaction was used to investigate in vivo expression of calcium channels in the murine mammary gland at the four main stages of mammary gland development. The store-operated calcium channel Orai1 was upregulated during lactation relative to its expression in the nulliparous gland. The classic ORAI1 regulator Stim1 was not similarly overexpressed during lactation, however, its isoform Stim2 was modestly upregulated. HC11 murine mammary cells were used as a model to further investigate the role of STIM2 on calcium handling during lactation. siRNA knockdown of Stim2 reduced both basal and agonist-induced peak cytosolic calcium levels, indicative of its role in calcium regulation. In addition to investigating the role of calcium channels in normal mammary development, their role in breast cancer was examined. Real time reverse transcription-polymerase chain reaction was used to identify calcium channels upregulated in human breast cancer cell lines, relative to non-tumorigenic mammary cell lines. TRPV1, TRPV6, and ORAI1 were upregulated in the breast cancer cell lines. Pharmacological modulation of ORAI1 resulted in modest changes in proliferation, but as there was no specific ORAI1 inhibitor, this effect could not be conclusively attributed to ORAI1 inhibition. siRNA was used to specifically target ORAI1 in three human breast cancer cell lines: MCF-7, MDA-MB-231, and T-47D. siRNA knockdown of ORAI1 was specific and potent, and reduced cell viability and altered calcium handing in all three cell lines. The alterations caused by ORAI1 knock down were not related to the expression of the genes CDK2 and FOS, as these did not change upon ORAI1 knockdown. Data mining was performed using the National Center for Biotechnology Information’s (NCBI’s) expressed sequence tag (EST) database, dbEST, and the Oncomine database. ORAI1 was elevated in estrogen receptor negative breast cancers and in the basal breast cancer molecular subtype, a subtype that has a poor prognosis. Other data suggested that breast cancer cells with high STIM1 and low STIM2 expression also correlated with the basal breast cancer subtype. These data indicate that ORAI and STIM proteins have a role in the physiological process of lactation as well as in the regulation of tumorigenic pathways in the breast, and particular gene expression profiles may be predictors of disease prognosis.

Calcium is the major mineral component of milk and is essential for neonatal development. To enrich the milk, calcium must pass from the maternal bloodstream, through mammary epithelial cells, into the alveolar lumen. While calcium extrusion from the epithelial cells is well characterized, no calcium channel or transporter has been identified as the major conduit for calcium to enter the mammary epithelial cell from the bloodstream. A major aim of this thesis was to identify a calcium channel or channels responsible for calcium influx into mammary epithelial cells during lactation. Real time reverse transcription-polymerase chain reaction was used to investigate in vivo expression of calcium channels in the murine mammary gland at the four main stages of mammary gland development. The store-operated calcium channel Orai1 was upregulated during lactation relative to its expression in the nulliparous gland. The classic ORAI1 regulator Stim1 was not similarly overexpressed during lactation, however, its isoform Stim2 was modestly upregulated. HC11 murine mammary cells were used as a model to further investigate the role of STIM2 on calcium handling during lactation. siRNA knockdown of Stim2 reduced both basal and agonist-induced peak cytosolic calcium levels, indicative of its role in calcium regulation. In addition to investigating the role of calcium channels in normal mammary development, their role in breast cancer was examined. Real time reverse transcription-polymerase chain reaction was used to identify calcium channels upregulated in human breast cancer cell lines, relative to non-tumorigenic mammary cell lines. TRPV1, TRPV6, and ORAI1 were upregulated in the breast cancer cell lines. Pharmacological modulation of ORAI1 resulted in modest changes in proliferation, but as there was no specific ORAI1 inhibitor, this effect could not be conclusively attributed to ORAI1 inhibition. siRNA was used to specifically target ORAI1 in three human breast cancer cell lines: MCF-7, MDA-MB-231, and T-47D. siRNA knockdown of ORAI1 was specific and potent, and reduced cell viability and altered calcium handing in all three cell lines. The alterations caused by ORAI1 knock down were not related to the expression of the genes CDK2 and FOS, as these did not change upon ORAI1 knockdown. Data mining was performed using the National Center for Biotechnology Information’s (NCBI’s) expressed sequence tag (EST) database, dbEST, and the Oncomine database. ORAI1 was elevated in estrogen receptor negative breast cancers and in the basal breast cancer molecular subtype, a subtype that has a poor prognosis. Other data suggested that breast cancer cells with high STIM1 and low STIM2 expression also correlated with the basal breast cancer subtype. These data indicate that ORAI and STIM proteins have a role in the physiological process of lactation as well as in the regulation of tumorigenic pathways in the breast, and particular gene expression profiles may be predictors of disease prognosis.

Os distúrbios na regulação da concentração de cálcio (Ca2+) citoplasmático contribuem para a patogênese da hipertensão arterial. Evidências sugerem que as moléculas de interação estromal (STIM) atuam como sensores dos estoques intracelulares de Ca2+, enquanto as proteínas Orai representam as subunidades que formam os canais de Ca2+ ativados pela liberação de Ca2+ (CRAC). Neste estudo avaliamos a participação de STIM1/Orai1 na regulação das concentrações de Ca2+ citoplasmático e na ativação da contração vascular em aortas de ratos hipertensos. Nossos resultados sugerem que a ativação de STIM1/Orai1 pode representar um novo mecanismo que modula alterações vasculares nos níveis de Ca2+ intracelular na hipertensão arterial e que contribui para as diferenças sexuais de reatividade vascular em animais hipertensos. / Disturbance in the regulation of cytoplasmic calcium (Ca2+) concentration contributes to the pathogenesis of hypertension. Evidences suggest that the stromal interaction molecule (STIM) acts as a sensor of intracellular Ca2+ stores, whereas Orai proteins are the subunits that form CRAC channels. In this study, we evaluated the role of STIM1/Orai1 in the regulation of cytoplasmic Ca2+ concentrations and in the activation of contraction in aortas from hypertensive rats. We also studied how the differential activation of this pathway contributes to sex differences observed between hypertensive rats, as well as the protective effects of the female sex hormones in the vasculature. Our results suggest that activation of STIM1/Orai1 may represent a new mechanism that modulates intracellular Ca2+ concentration during hypertension and contributes to sex differences in the vascular reactivity of hypertensive animals.

A O-glicosilação com N-acetil-glucosamina (O-GlcNAc) é uma modificação pós-translacional altamente dinâmica que modula diversas vias de sinalização. O processo de O-GlcNAc é controlado por duas enzimas: a enzima OGT é responsável por catalisar a adição de N-acetil-glucosamina no grupo hidroxila dos resíduos de serina e treonina, enquanto a OGA catalisa a remoção de O-GlcNAc das proteínas modificadas. Proteínas com importante papel na função vascular são alvo de O-GlcNAc e o aumento da expressão de proteínas modificadas por O-GlcNAc promove aumento da reatividade vascular para estímulos contráteis. Um dos mecanismos de extrema importância no controle do tônus vascular está ligado à regulação da concentração de cálcio (Ca2+) intracelular, onde destacamos a participação do sistema STIM1/Orai1. As moléculas de interação estromal (STIM) atuam como sensores dos estoques intracelulares de Ca2+ e as proteínas Orai representam as subunidades que formam os canais de Ca2+ ativados pela liberação de Ca2+ (CRAC). Neste estudo investigamos a hipótese de que o aumento dos níveis vasculares de proteínas glicosiladas aumenta a resposta contrátil em aorta de ratos, por mecanismos relacionados ao controle da concentração intracelular de Ca2+.Em nossos experimentos, utilizamos aortas torácicas de ratos incubadas com PugNAc (inibidor seletivo da OGA, ), por 24h. Utilizando protocolo experimental que permite avaliar contrações induzidas pelo influxo de Ca2+ e liberação de Ca2+ intracelular, demonstramos que a incubação com PugNAc aumentou a resposta contrátil à PE bem como a contração durante o período de influxo de Ca2+, induzida pela reintrodução de solução fisiológica contendo Ca2+ (1,56 mM). O bloqueio dos canais CRAC com 2-APB (100 ) e gadolíneo (Gd3+, 100 ) diminuiu significativamente as contrações induzidas pelo influxo de Ca2+ em aortas incubadas com PugNAc. Além disso, estas aortas apresentaram aumento da expressão protéica de STIM1, o que resultaria em maior influxo de Ca2+. A contração induzida por cafeína (20 mM) e serotonina (10 ), a qual reflete a capacidade funcional do retículo sarcoplasmático (RS) em captar Ca2+, foi maior em aortas incubadas com PugNAc. O papel da Ca2+-ATPase (SERCA) foi avaliado com a utilização de tapsigargina, bloqueador da SERCA. O efeito da tapsigargina foi semelhante em artérias incubadas com PugNAc e veículo, apesar do aumento de expressão proteica da SERCA em aortas incubadas com PugNAc. Como a proteína cinase C (PKC) é ativada por aumentos de Ca2+ intracelular, determinamos se a atividade de proteínas alvo da PKC estavam aumentadas. A incubação com PugNAc aumentou a expressão das formas fosforiladas da CPI-17, MYPT-1 e MLC. Em conjunto, estes resultados sugerem que a ativação de STIM1/Orai1, aumento da liberação de Ca2+ intracelular e ativação da via de sinalização da PKC podem representar mecanismos que modulam as alterações vasculares em resposta ao aumento de proteínas glicosiladas por O-GlcNAc. / Glycosylation with O-linked -N-acetyl-glucosamine (O-GlcNAc) is a highly dynamic post-translational modification. The process of O-GlcNAc is controlled by two enzymes: the OGT enzyme catalyses the addition of N-acetyl-glucosamine to the hydroxyl group of serine and threonine residues of a target protein, while OGA catalyzes the cleavage of O-GlcNAc from post-translationally-modified proteins. Proteins with an important role in vascular function are targets of O-GlcNAc and increased levels of proteins modified by O-GlcNAc increase vascular reactivity to contractile stimuli. The regulation of intracellular calcium (Ca2+) concentration, including the activation of STIM1/Orai1, is key in the control of vascular tone. The stromal interaction molecules (STIM) act as sensors of intracellular Ca2+ stores whereas the Orai proteins represent subunits of the Ca2+ release-activated Ca2+ channels (CRAC). We hypothesized that increased levels of vascular O-GlcNAc proteins augment vascular contractile responses by altering mechanisms that regulate the intracellular Ca2+. Rat thoracic aortas were incubated with PugNAc (OGA selective inhibitor, ) for 24h. Using an experimental protocol that evaluates contractions induced by Ca2+ influx and release, we demonstrated that incubation with PugNAc increases contractile responses to phenylephrine (PE) as well as the contraction induced by Ca2+ influx, after depletion of intracellular Ca2+ stores. The CRAC channel blockers, 2-APB (100 ) and gadolinium (Gd3+, 100 ), significantly reduced the contractions induced by Ca2+ influx in aortas incubated with PugNAc. Furthermore, these aortas showed increased STIM1 protein expression, which could result in increased influx of Ca2+ and, in turn, increase vascular contraction. The contraction induced by the release of intracellular Ca2+ stores, stimulated by caffeine (20 mM) and serotonin (10 ), was increased in aortas incubated with PugNAc. The Ca2+-ATPase (SERCA) inhibitor thapsigargin produced similar effects in arteries incubated with PugNAc or vehicle, despite the increased SERCA protein expression in aortas incubated with PugNAc. Since PKC is activated by increases in intracellular Ca2+ and arteries incubated with PugNAc show activation of PKC, we determined whether the activity of proteins that are targets of PKC was increased in PugNAc-treated aortas. Incubation with PugNAc increased the expression of phosphorylated forms of CPI-17, MYPT-1 and MLC. Together, these results suggest that activation of STIM1/Orai1, increased release of intracellular Ca2+ and PKC activation may represent mechanisms that modulate vascular responses upon increased O-GlcNAc proteins.