Adenylyl Cyclase Type 8 Overexpression Impairs Phosphorylation-Dependent Orai1 Inactivation and Promotes Migration in MDA-MB-231 Breast Cancer Cells.

Orai1 plays a major role in store-operated Ca 2+ entry (SOCE) in triple-negative breast cancer (TNBC) cells. This channel is inactivated via different mechanisms, including protein kinase C (PKC) and protein kinase A (PKA)-dependent phosphorylation at Ser-27 and Ser-30 or Ser-34, respectively, which shapes the Ca 2+ responses to agonists. The Ca 2+ calmodulin-activated adenylyl cyclase type 8 (AC8) was reported to interact directly with Orai1, thus mediating a dynamic interplay between the Ca 2+ - and cyclic adenosine monophosphate (cAMP)-dependent signaling pathways. Here, we show that the breast cancer cell lines MCF7 and MDA-MB-231 exhibit enhanced expression of Orai1 and AC8 as compared to the non-tumoral breast epithelial MCF10A cell line. In these cells, AC8 interacts with the Orai1α variant in a manner that is not regulated by Orai1 phosphorylation. AC8 knockdown in MDA-MB-231 cells, using two different small interfering RNAs (siRNAs), attenuates thapsigargin (TG)-induced Ca 2+ entry and also Ca 2+ influx mediated by co-expression of Orai1 and the Orai1-activating small fragment (OASF) of STIM1 (stromal interaction molecule-1). Conversely, AC8 overexpression enhances SOCE, as well as Ca 2+ entry, in cells co-expressing Orai1 and OASF. In MDA-MB-231 cells, we found that AC8 overexpression reduces the Orai1 phosphoserine content, thus suggesting that AC8 interferes with Orai1 serine phosphorylation, which takes place at residues located in the AC8-binding site. Consistent with this, the subset of Orai1 associated with AC8 in naïve MDA-MB-231 cells is not phosphorylated in serine residues in contrast to the AC8-independent Orai1 subset. AC8 expression knockdown attenuates migration of MCF7 and MDA-MB-231 cells, while this maneuver has no effect in the MCF10A cell line, which is likely attributed to the low expression of AC8 in these cells. We found that AC8 is required for FAK (focal adhesion kinase) phosphorylation in MDA-MB-231 cells, which might explain its role in cell migration. Finally, we found that AC8 is required for TNBC cell proliferation. These findings indicate that overexpression of AC8 in breast cancer MDA-MB-231 cells impairs the phosphorylation-dependent Orai1 inactivation, a mechanism that might support the enhanced ability of these cells to migrate.Ministerio de Asuntos Económicos y de Transformación Digital.Fondo Europeo de Desarrollo Regional Grants.Junta de Extremadura.Juan de la Cierva (Ministro de Industria, Economía y Competitividad)

Orai2 Modulates Store-Operated Ca2+ Entry and Cell Cycle Progression in Breast Cancer Cells

Breast cancer is a heterogeneous disease from the histological and molecular expression point of view, and this heterogeneity determines cancer aggressiveness. Store-operated Ca2+ entry (SOCE), a major mechanism for Ca2+ entry in non-excitable cells, is significantly remodeled in cancer cells and plays an important role in the development and support of different cancer hallmarks. The store-operated CRAC (Ca2+ release-activated Ca2+) channels are predominantly comprised of Orai1 but the participation of Orai2 and Orai3 subunits has been reported to modulate the magnitude of Ca2+ responses. Here we provide evidence for a heterogeneous expression of Orai2 among different breast cancer cell lines. In the HER2 and triple negative breast cancer cell lines SKBR3 and BT20, respectively, where the expression of Orai2 was greater, Orai2 modulates the magnitude of SOCE and sustain Ca2+ oscillations in response to carbachol. Interestingly, in these cells Orai2 modulates the activation of NFAT1 and NFAT4 in response to high and low agonist concentrations. Finally, we have found that, in cells with high Orai2 expression, Orai2 knockdown leads to cell cycle arrest at the G0-G1 phase and decreases apoptosis resistance upon cisplatin treatment. Altogether, these findings indicate that, in breast cancer cells with a high Orai2 expression, Orai2 plays a relevant functional role in agonist-evoked Ca2+ signals, cell proliferation and apoptosis resistance.Ministerio de Ciencia e Innovación: PID2019-104084GB-C21Ministerio de Ciencia e Innovación: PID2019-104084GB-C22Consejería de Educación y Empleo, Junta de Extremadura: IB20007Consejería de Educación y Empleo, Junta de Extremadura: GR18061Consejería de Educación y Empleo, Junta de Extremadura: PD16072Consejería de Educación y Empleo, Junta de Extremadura: GR2100

TRPC Channels in the SOCE Scenario

Transient receptor potential (TRP) proteins form non-selective Ca2+ permeable channels that contribute to the modulation of a number of physiological functions in a variety of cell types. Since the identification of TRP proteins in Drosophila, it is well known that these channels are activated by stimuli that induce PIP2 hydrolysis. The canonical TRP (TRPC) channels have long been suggested to be constituents of the store-operated Ca2+ (SOC) channels; however, none of the TRPC channels generate Ca2+ currents that resemble ICRAC. STIM1 and Orai1 have been identified as the components of the Ca2+ release-activated Ca2+ (CRAC) channels and there is a body of evidence supporting that STIM1 is able to gate Orai1 and TRPC1 in order to mediate non-selective cation currents named ISOC. STIM1 has been found to interact to and activate Orai1 and TRPC1 by different mechanisms and the involvement of TRPC1 in store-operated Ca2+ entry requires both STIM1 and Orai1. In addition to the participation of TRPC1 in the ISOC currents, TRPC1 and other TRPC proteins might play a relevant role modulating Orai1 channel function. This review summarizes the functional role of TRPC channels in the STIM1–Orai1 scenario.Junta de Extremadura Consejería de Economía e Infraestructura-FEDER Grant IB16046 y GR18061Junta de Extremadura TA18011 y TA18054Ministerio de Ciencia, Innovación y Universidade

Innate Immune Receptors, Key Actors in Cardiovascular Diseases

Cardiovascular diseases (CVDs) are the leading cause of death in the industrialized world. Most CVDs are associated with increased inflammation that arises mainly from innate immune system activation related to cardiac damage. Sustained activation of the innate immune system frequently results in maladaptive inflam- matory responses that promote cardiovascular dysfunction and remodeling. Much research has focused on determining whether some mediators of the innate immune system are potential targets for CVD therapy. The innate immune system has specific receptors—termed pattern recognition receptors (PRRs)—that not only recognize pathogen-associated molecular patterns, but also sense danger-associated molecular signals. Acti- vation of PRRs triggers the inflammatory response in different physiological systems, including the cardio- vascular system. The classic PRRs, toll-like receptors (TLRs), and the more recently discovered nucleotide- binding oligomerization domain-like receptors (NLRs), have been recently proposed as key partners in the progression of several CVDs (e.g., atherosclerosis and heart failure). The present review discusses the key findings related to the involvement of TLRs and NLRs in the progression of several vascular and cardiac diseases, with a focus on whether some NLR subtypes (nucleotide-binding oligomerization domain, leucine rich repeat and pyrin domain-containing receptor 3 and nucleotide-binding oligomerization domain-containing protein 1) can be candidates for the development of new therapeutic strategies for several CVDs

miR-7 Modulates hESC Differentiation into Insulin-Producing Beta-like Cells and Contributes to Cell Maturation

Human pluripotent stem cells retain the extraordinary capacity to differentiate into pancreatic beta cells. For this particular lineage, more effort is still required to stress the importance of developing an efficient, reproducible, easy, and cost-effective differentiation protocol to obtain more mature, homogeneous, and functional insulin-secreting cells. In addition, microRNAs (miRNAs) have emerged as a class of small non-coding RNAs that regulate many cellular processes, including pancreatic differentiation. Some miRNAs are known to be preferentially expressed in islets. Of note, miR-375 and miR-7 are two of the most abundant pancreatic miRNAs, and they are necessary for proper pancreatic islet development. Here we provide new insight into specific miRNAs involved in pancreatic differentiation. We found that miR-7 is differentially expressed during the differentiation of human embryonic stem cells (hESCs) into a beta cell-like phenotype and that its modulation plays an important role in generating mature pancreatic beta cells. This strategy may be exploited to optimize the potential for in vitro differentiation of hESCs into insulin-producing beta-like cells for use in preclinical studies and future clinical applications as well as the prospective uses of miRNAs to improve this process.Spanish Ministry of Economy and Competitiveness BFU2016-74932-C2 BFU2013-45564-C2FEDER Funds PI-0272-2017Andalusian Regional Ministry of Health PI-0272-2017European Cooperation in Science and Technology BM1305Spanish Ministry of Economy, Industry and Competitiveness CD16/00118Spanish Institute of Health Carlos III PI16/00259 PI17/02104 RD16/0011/0034 CD16/0011

EFHB is a Novel Cytosolic Ca2+ Sensor That Modulates STIM1-SARAF Interaction

Background/aims: STIM1 and Orai1 are the key components of store-operated Ca2+ entry (SOCE). Among the proteins involved in the regulation of SOCE, SARAF prevents spontaneous activation of SOCE and modulates STIM1 function. Methods: Cytosolic Ca2+ mobilization was estimated in fura-2-loaded cells using an epifluorescence inverted microscope. STIM1 interaction with Orai1, EFHB (EF-hand domain family member B, also known as CFAP21) and SARAF was detected by immunoprecipitation followed by Western blotting using specific antibodies. The involvement of EFHB in the translocation of NFAT to the nucleus was detected by confocal microscopy. Results: Here, we report the identification of EFHB as a new SOCE regulator. EFHB interacts with STIM1 upon store depletion and dissociates through a Ca2+-dependent mechanism. RNAi-mediated silencing as well as overexpression studies revealed that EFHB plays a relevant role in the interaction of STIM1 and Orai1 upon store depletion, the activation of SOCE and NFAT translocation from the cytosol to the nucleus. Silencing EFHB expression abolished the dissociation of SARAF from STIM1, which indicates that EFHB might play an important role in the dynamic interaction between both proteins, which is relevant for the activation of Orai1 channels upon Ca2+ store depletion and their subsequent modulation via slow Ca2+-dependent inactivation. Conclusion: Our results indicate that EFHB is a new SOCE regulator that modulates STIM1-SARAF interaction.MINECO BFU2013-45564-C2-1-P/2-P, BFU2016-74932-C2-1-P/2-P, BFU2016-74932-C2-1-PJunta de Extremadura-FEDER (Fondo Europeo de Desarrollo Regional Grants) IB16046, GR18061Junta de Extremadura-FEDER European Union (EU) IB16046Ministerio Economía y Competitividad, España IJCI-2015-25665MINECO Grant BFU2016-74932-C2-1-

TRPC6 channels are required for proliferation, migration and invasion of breast cancer cell lines by modulation of Orai1 and Orai3 surface exposure

Transient receptor potential channels convey signaling information from a number of stimuli to a wide variety of cellular functions, mainly by inducing changes in cytosolic Ca2+ concentration. Different members of the TRPC, TRPM and TRPV subfamilies have been reported to play a role in tumorigenesis. Here we show that the estrogen receptor positive and triple negative breast cancer cell lines, MCF7 and MDA-MB-231, respectively, exhibit enhanced expression of the TRPC6 channel as compared to the non-tumoral MCF10A cell line. In vitro TRPC6 knockdown using shRNA impaired MCF7 and MDA-MB-231 cell proliferation, migration and invasion detected by BrdU incorporation, wound healing and Boyden chamber assays, respectively. Using RNAi-mediated TRPC6 silencing as well as overexpression of the pore-dead dominant-negative TRPC6 mutant we have found that TRPC6 plays a relevant role in the activation of store-operated Ca2+ entry in the breast cancer cell lines but not in non-tumoral breast cells. Finally, we have found that TRPC6 interacts with Orai1 and Orai3 in MCF7 and MDA-MB-231 cells and is required for the translocation of Orai1 and Orai3 to the plasma membrane in MDA-MB-231 and MCF7 cells, respectively, upon Ca2+ store depletion. These findings introduce a novel mechanism for the modulation of Ca2+ influx and the development of different cancer hallmarks in breast cancer cells

TRP Channels: Current Perspectives in the Adverse Cardiac Remodeling

Calcium is an important second messenger required not only for the excitation-contraction coupling of the heart but also critical for the activation of cell signaling pathways involved in the adverse cardiac remodeling and consequently for the heart failure. Sustained neurohumoral activation, pressure-overload, or myocardial injury can cause pathologic hypertrophic growth of the heart followed by interstitial fibrosis. The consequent heart’s structural and molecular adaptation might elevate the risk of developing heart failure and malignant arrhythmia. Compelling evidences have demonstrated that Ca2+ entry through TRP channels might play pivotal roles in cardiac function and pathology. TRP proteins are classified into six subfamilies: TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPA (ankyrin), TRPML (mucolipin), and TRPP (polycystin), which are activated by numerous physical and/or chemical stimuli. TRP channels participate to the handling of the intracellular Ca2+ concentration in cardiac myocytes and are mediators of different cardiovascular alterations. This review provides an overview of the current knowledge of TRP proteins implication in the pathologic process of some frequent cardiac diseases associated with the adverse cardiac remodeling such as cardiac hypertrophy, fibrosis, and conduction alteration.Spanish Ministry of Economy and Competitiveness BFU2016–74932-C2Institute of Carlos III PI15/00203; PI16/00259; CB16/11/00431Andalusia Government PI-0313-201

Orai1 and TRPC1 Proteins Co-localize with CaV1.2 Channels to Form a Signal Complex in Vascular Smooth Muscle Cells

Voltage-dependent CaV1.2 L-type Ca2 channels (LTCC) are the main route for calcium entry in vascular smooth muscle cells (VSMC). Several studies have also determined the relevant role of store-operated Ca2 channels (SOCC) in vascular tone regulation. Nevertheless, the role of Orai1- and TRPC1-dependent SOCC in vascular tone regulation and their possible interaction with CaV1.2 are still unknown. The current study sought to characterize the co-activation of SOCC and LTCC upon stimulation by agonists, and to determine the possible crosstalk between Orai1, TRPC1, and CaV1.2. Aorta rings and isolated VSMCobtained from wild type or smooth muscle-selective conditional CaV1.2 knock-out (CaV1.2KO) mice were used to study vascular contractility, intracellular Ca2 mobilization, and distribution of ion channels. We found that serotonin (5-HT) or store depletion with thapsigargin (TG) enhanced intracellular free Ca2 concentration ([Ca2 ]i) and stimulated aorta contraction. These responses were sensitive to LTCC and SOCC inhibitors. Also, 5-HT- and TG-induced responses were significantly attenuated in CaV1.2KO mice. Furthermore, hyperpolarization induced with cromakalim or valinomycin significantly reduced both 5-HT and TG responses, whereas these responses were enhanced with LTCC agonist Bay-K-8644. Interestingly, in situ proximity ligation assay revealed that CaV1.2 interacts with Orai1 and TRPC1 in untreated VSMC. These interactions enhanced significantly after stimulation of cells with 5-HT and TG. Therefore, these data indicate for the first time a functional interaction between Orai1, TRPC1, and CaV1.2 channels in VSMC, confirming that upon agonist stimulation, vessel contraction involves Ca2 entry due to co-activation of Orai1- and TRPC1-dependent SOCC and LTCC.Ministerio de Economía y Competitividad BFU2013-45564-C2-1-PMinisterio de Economía y Competitividad BFU2013-45564-C2-2-PInstituto de Salud Carlos III RD12/0042/ 0041Cardiovascular Network “RIC” PI12/00941Junta de Andalucía PI-0108-2012Junta de Andalucía P12- CTS-196

Homeostasis del calcio en miocitos del árbol arterial pulmonar

En el presente trabajo se ha estudiado la homeostasis de la (Ca2+) i en miocitos aislados de la arteria pulmonar y el efecto de la liberación del ión de los almacenes intracelulares sobre las conductancias iónicas presentes en la membrana plasmática. Los resultados indican que tanto la regulación de la (Ca2+)i como los efectos elecrofisiológicos que se derivan de los cambios de los niveles de Ca2+ intracelulares dependen de la localización de los miocitos a lo largo del árbol arterial pulmonar. Aunque los dos tipos de receptores que existen en el RS ( receptor para InsP3 y para rianodina) se encuentran en todos los miocitos, parece observarse una especialización de estos al menos en la forma en que participan en la génesis de las oscilaciones espontáneas de Ca2+ (espigas de Ca2+). El receptor para InsP3 se activa preferentemente durante las oscilaciones de Ca2+ en los miocitos de conducción mientras que en los miocitos de resistencia tiene preporndración funcional el receptor rianodina. En paralelo a estas diferencias en las dos poblaciones de mioctios pulmonares existen especializaciones electrofisiológicas de importancia funcional muy relevantes. En los miocitos de conducción predominan los canales de Kca sobre los de Clca, y en los miocitos de resistencia prevalecen los canales de Clca de la membrana plasmática