Activation Of Store-operated Calcium Entry Research Articles

Tmem178 Negatively Regulates IL-1β Production Through Inhibition of the NLRP3 Inflammasome.

Inflammasomes modulate the release of bioactive interleukin (IL)-1β. Excessive IL-1β levels are detected in patients with systemic juvenile idiopathic arthritis (sJIA) and cytokine storm syndrome (CSS) with mutated and unmutated inflammasome components, raising questions on the mechanisms of IL-1β regulation in these disorders. To investigate how the NLRP3 inflammasome is modulated in sJIA, we focused on Transmembrane protein 178 (Tmem178), a negative regulator of calcium levels in macrophages, and measured IL-1β and caspase-1 activation in wild-type (WT) and Tmem178-/- macrophages after calcium chelators, silencing of Stim1, a component of store-operated calcium entry (SOCE), or by expressing a Tmem178 mutant lacking the Stromal Interaction Molecule 1 (Stim1) binding site. Mitochondrial function in both genotypes was assessed by measuring oxidative respiration, mitochondrial reactive oxygen species (mtROS), and mitochondrial damage. CSS development was analyzed in Perforin-/- /Tmem178-/- mice infected with lymphocytic choriomeningitis virus (LCMV) in which inflammasome or IL-1β signaling was pharmacologically inhibited. Human TMEM178 and IL1B transcripts were analyzed in data sets of whole blood and peripheral blood monocytes from healthy controls and patients with active sJIA. TMEM178 levels are reduced in whole blood and monocytes from patients with sJIA while IL1B levels are increased. Accordingly, Tmem178-/- macrophages produce elevated IL-1β compared with WT cells. The elevated intracellular calcium levels after SOCE activation in Tmem178-/- macrophages induce mitochondrial damage, release mtROS, and ultimately promote NLRP3 inflammasome activation. In vivo, inhibition of inflammasome or IL-1β neutralization prolongs Tmem178-/- mouse survival in LCMV-induced CSS. Down-regulation of TMEM178 levels may represent a marker of disease activity and help identify patients who could benefit from inflammasome targeting.

Homoplantaginin attenuates high glucose-induced vascular endothelial dysfunction via inhibiting store-operated calcium entry channel and endoplasmic reticulum stress.

The activation of store-operated calcium entry (SOCE) channel and endoplasmic reticulum stress (ERS) induced by high glucose (HG) is recognized as a major cause of vascular endothelial dysfunction. This study aims to investigate the protective effect of homoplantaginin (Hom) on HG-induced endothelial dysfunction. HG-induced vascular endothelial dysfunction model in human umbilical vein endothelial cells (HUVECs) and rat-isolated thoracic aortas were established to observe the protective effect of Hom, further evaluated the mechanism of SOCE channel and ERS in the pathogenesis. Hom increased the levels of nitric oxide (NO) and phospho-endothelial nitric oxide synthase (p-eNOS) in HUVECs and isolated rat thoracic aortas in a dose-dependent manner, restored acetylcholine-mediated endothelium-dependent vasodilation. Network pharmacology showed that the pathogenesis of diabetic vascular complications may involve calcium (Ca2+) signal pathway. Hom reduced Ca2+ concentration via blocking SOCE channel in HUVECs, and resisted ERS activation by down-regulating ERS-related proteins expression. Importantly, SKF96365 (SOCE inhibitor) intervention experiment showed that Hom inhibited ERS activation by blocking the SOCE channel, further increased the levels of NO and p-eNOS. Hom could alleviate HG-induced vascular endothelial dysfunction by inhibiting SOCE channel and ERS. This provided a potential pharmacological intervention strategy for the treatment of vascular endothelial dysfunction.

Purinergic Activation of Store-Operated Calcium Entry (SOCE) Regulates Cell Migration in Metastatic Ovarian Cancer Cells.

Store-operated calcium entry (SOCE) is an important process in calcium signaling. Its role in physiological and pathological events is well recognized. However, in cancerous systems, the importance of SOCE in relation to the degree of cancer aggressiveness, as well as its regulation by ligands such as purinergic molecules, are not well documented. This study aimed to characterize a differential effect of the P2Y2 receptor (promoted by UTP of 10 µM and inhibited by ARC118925XX of 1 µM) on intracellular calcium response between metastatic (SKOV-3) and non-metastatic (CAOV-3) ovarian cell lines in conditions of normal (1.5 mM) and zero extracellular calcium concentration. The sustained calcium influx observed exclusively in SKOV-3 cells was associated with the presence of SOCE (promoted by thapsigargin (74.81 ± 0.94 ΔF) and sensitive to 2-APB (20.60 ± 0.85 ΔF)), whereas its absence in CAOV-3 cells (26.2 ± 6.1 ΔF) was correlated with a low expression of ORAI1. The relevance of SOCE in metastatic SKOV-3 cells was further corroborated when 2-APB significantly inhibited (40.4 ± 2.8% of covered area) UTP-induced cell migration (54.6 ± 3.7% of covered area). In conclusion, our data suggest that SOCE activation elicited by the P2Y2 receptor is involved in the aggressiveness of ovarian cancer cells.

Identifying 2PHDO as a SOCE inhibitor from Chinese herbal extracts.

STIM- and Orai-mediated store operated calcium entry (SOCE) is a ubiquitous Ca2+ signaling process, crucial for the proper function of immune, muscle and neuronal systems. To treat SOCE-related disorder or diseases of these systems, and to mechanistically dissect activation and function of SOCE, specific SOCE inhibitors are needed. However, strategies for developing new SOCE modifiers are still limited. <P/> Methodology: In this study, we identified a novel SOCE inhibitor named 2PHDO from a small pool of Chinese herbal extracts used for treating psoriasis. It could block SOCE and SOCE-mediated NFAT translocation in multiple types of cells with a half inhibitory concentration around 1 µM. At this concentration, 2PHDO was specific for SOCE. Mechanistically, 2PHDO didn't affect the activation of STIM1 or its physical coupling with Orai1. Rather, 2PHDO inhibited SOCE via its actions on Orai1. <P/> Results: 2PHDO may serve as a good template for developing new medicines aiming to treat SOCE related diseases. <P/> Conclusion: Overall, we proved the feasibility of screening and identification of novel SOCE inhibitors from active monomers of Chinese herbal medicine.

CAPN1 (Calpain1)-Dependent Cleavage of STIM1 (Stromal Interaction Molecule 1) Results in an Enhanced SOCE (Store-Operated Calcium Entry) in Human Neonatal Platelets.

Altered intracellular Ca2+ homeostasis in neonatal platelets has been previously reported. This study aims to examine the changes in the Ca2+ entry through the store-operated calcium entry (SOCE) mechanism in neonatal platelets. Human platelets from either control women, mothers, and neonates were isolated and, following, were fixed after being treated as required. Platelet samples were analyzed by Western blotting, qRT-PCR, and MALDITOF/TOF. Ca2+ homeostasis was also determined. Culture cells were used as surrogated of platelets to overexpress the proteins of interest to reproduce the alterations observed in platelets. Altered thapsigargin-evoked SOCE, alternative molecular weight form of STIM1 (stromal interaction molecule 1; s-STIM1 [short STIM1 isoform (478 aa)], around 60 kDa) and overexpression of SARAF (SOCE-associated regulatory factor [TMEM66]) were found in neonatal platelet as compared to maternal and control women platelets. s-STIM1 may result due to CAPN1 (calpain1)-dependent processing, as confirmed in platelets and MEG01 cells by using calpeptin and overexpressing CAPN1, respectively. In HEK293 (STIM1 and STIM2 [stromal interaction molecule 2] double knockout) cells transfected either with c-STIM1 (canonical STIM1 [685 aa]), s-STIM1 (478), STIM1B (540), and CAPN1 overexpression plasmids, we found s-STIM1 and c-STIM1, except in cells overexpressing s-STIM1 (478) that lacked CAPN1 target residues. These results and the in silico analysis, lead us to conclude that STIM1 is cleaved at Q496 by CAPN1. Ca2+ imaging analysis and coimmunoprecipitation assay using MEG01 and HEK293 cells overexpressing SARAF together with s-STIM1 (478) reported a reduced slow Ca2+-dependent inactivation, so reproducing the Ca2+-homeostasis pattern observed in neonatal platelets. CAPN1 may cleave STIM1 in neonatal platelets, hence, impairing SARAF coupling after SOCE activation. S-STIM1 may avoid slow Ca2+-dependent inactivation and, subsequently, result in an enhanced thapsigargin-evoked SOCE as observed in neonatal platelets.

ORAI1-Regulated Gene Expression in Breast Cancer Cells: Roles for STIM1 Binding, Calcium Influx and Transcription Factor Translocation.

A remodeling of calcium homeostasis, including calcium influx via store-operated calcium entry (SOCE), is a feature of breast cancers. SOCE is critical to maintain calcium balance in the endoplasmic reticulum calcium store and is an important mechanism for calcium signaling in a variety of cell types, including breast cancer cells. The canonical mechanism of SOCE is stromal interacting molecule 1 (STIM1)-mediated activation of ORAI. Elevated ORAI1 expression is a feature of basal breast cancer cells. However, the role of ORAI1 in the regulation of transcription in breast cancer cells of the basal molecular subtype is still unclear. Using CRISPR-Cas9 gene editing, ORAI1 protein expression was disrupted in MDA-MB-231 and MDA-MB-468 basal breast cancer cells. The ORAI1 wild-type and mutants were reintroduced into ORAI1 knockout cells to study the role of ORAI1 in gene transcriptional regulation. In the absence of calcium store depletion, ORAI1 regulated PTGS2 in MDA-MB-231 cells, and this was dependent on ORAI1 pore function and STIM1 binding. The activation of SOCE by thapsigargin resulted in ORAI1-dependent increases in IL6 transcription in MDA-MB-468 cells; this was also dependent on ORAI1 pore function and STIM1 binding and was associated with the translocation of NFAT1. Given the upregulation of ORAI1 in basal breast cancer cells, our results provide further evidence that ORAI1 may contribute to cancer progression through regulation of gene expression.

Exaggerated Store‐Operated Calcium Entry: A Mechanism for High Glucose‐induced Podocyte Injury and Mitochondria Damage

Purpose and hypothesisPodocyte injury induced by hyperglycemia is the key factor contributing to proteinuria and kidney dysfunction in diabetic nephropathy (DN). Accumulating evidence suggests that mitochondria dysfunction leads to DN‐associated podocyte injury. However, the mechanism of podocyte mitochondria injury by high glucose (HG) is poorly understood. Store‐operated calcium entry (SOCE) is essential for multiple cell functions in both excitable and non‐excitable cells. However, the role of this Ca2+ signaling in podocytopathy remains unknown. The present study was carried out to test the hypothesis that enhanced SOCE mediated HG‐induced podocyte injury and mitochondria damage.MethodsAll experiments were carried out using cultured immortalized human podocytes. The conventional whole‐cell patch‐clamp was performed to measure Orai1 channel currents. Western blot was conducted to evaluate protein abundance of Orai1 (the channel protein mediating SOCE) and STIM1. Calcium imaging was used to evaluate SOCE. Confocal microscopy was used to visualize podocyte actin arrangement. Mitochondrial function was evaluated by measuring: 1) the mitochondria membrane potential (MMP) using TMRE fluorescence; 2) generation of mitochondria reactive oxygen species (ROS) using MitoSox Red Mitochondrial Superoxide Indicator; 3) ATP production using ATP assay kit. In addition, CRISPR‐CAS 9 approach was used to delete Orai1 in podocytes.ResultsThapsigargin (TG, 1 µM), the activator of Orai1 channel induced robust inward currents which were blocked by La3+ (2 µM), an inhibitor of Orai1 channel. Ang II (1 µM) also evoked inward currents, which were significantly blunted by BTP2 (10 µM), an Orai1 channel blocker. Orai1 protein abundance significantly increased by HG (25 mM) treatment for the time periods ranging from 2 to 12 h. This HG induced Orai1 increase was dose‐dependent. Consistently, Ca2+ imaging experiment showed that HG treatment for 12 h significantly increased podocyte SOCE. Thus, the biochemical and functional data suggested an enhancement of SOCE by HG. Furthermore, HG treatment resulted in podocyte cytoskeleton rearrangement by formation of cortical F‐actin, an indication of podocyte injury. This HG response was significantly blunted by BTP2 and Orai1 CRISPR‐CAS 9 lentivirus. Moreover, HG treatment significantly decreased podocyte MMP, ATP production and increased mitochondrial ROS generation, all of which were significantly inhibited by BTP2. Furthermore, activation of SOCE by TG significantly increased mitochondrial ROS generation, which was prevented by pre‐treatment of BTP2.ConclusionEnhanced SOCE contributed to HG induced podocyte injury and mitochondrial dysfunction.

Oscillatory calcium release and sustained store-operated oscillatory calcium signaling prevents differentiation of human oligodendrocyte progenitor cells

Endogenous remyelination in demyelinating diseases such as multiple sclerosis is contingent upon the successful differentiation of oligodendrocyte progenitor cells (OPCs). Signaling via the Gαq-coupled muscarinic receptor (M1/3R) inhibits human OPC differentiation and impairs endogenous remyelination in experimental models. We hypothesized that calcium release following Gαq-coupled receptor (GqR) activation directly regulates human OPC (hOPC) cell fate. In this study, we show that specific GqR agonists activating muscarinic and metabotropic glutamate receptors induce characteristic oscillatory calcium release in hOPCs and that these agonists similarly block hOPC maturation in vitro. Both agonists induce calcium release from endoplasmic reticulum (ER) stores and store operated calcium entry (SOCE) likely via STIM/ORAI-based channels. siRNA mediated knockdown (KD) of obligate calcium sensors STIM1 and STIM2 decreased the magnitude of muscarinic agonist induced oscillatory calcium release and attenuated SOCE in hOPCs. In addition, STIM2 expression was necessary to maintain the frequency of calcium oscillations and STIM2 KD reduced spontaneous OPC differentiation. Furthermore, STIM2 siRNA prevented the effects of muscarinic agonist treatment on OPC differentiation suggesting that SOCE is necessary for the anti-differentiative action of muscarinic receptor-dependent signaling. Finally, using a gain-of-function approach with an optogenetic STIM lentivirus, we demonstrate that independent activation of SOCE was sufficient to significantly block hOPC differentiation and this occurred in a frequency dependent manner while increasing hOPC proliferation. These findings suggest that intracellular calcium oscillations directly regulate hOPC fate and that modulation of calcium oscillation frequency may overcome inhibitory Gαq-coupled signaling that impairs myelin repair.

STIM1-Orai1 interaction mediated calcium influx activation contributes to cardiac contractility of insulin-resistant rats

PurposeMetabolic syndrome (MetS) became a tremendous public health burden in the last decades. Store-operated calcium entry (SOCE) is a unique mechanism that causes a calcium influx, which is triggered by calcium store depletion. MetS-induced alterations in cardiac calcium signaling, especially in SOCE are still unclear. Therefore, we aim to examine the possible role of SOCE and its components (STIM1 and Orai1) in the MetS-induced cardiac remodeling.MethodsWe used male, adult (12 weeks) Wistar albino rats (n = 20). Animals were randomly divided into two groups which were: control (C) and MetS. We gave 33% sucrose solution to animals instead of water for 24 weeks to establish MetS model. In the end, papillary muscle function was evaluated, and various electrophysiological analyses were made in isolated cardiomyocytes. Additionally, STIM1 and Orai1 protein and mRNA expressions were analyzed.ResultsWe observed a deterioration in contractility in MetS animals and demonstrated the contribution of SOCE by applying a SOCE inhibitor (BTP2). Calcium spark frequency was increased while its amplitude was decreasing in MetS hearts, which was reversed after SOCE inhibition. The amplitude of transient calcium changes in the MetS group was decreased, and it decreased further BTP2 application. Both protein and mRNA levels of STIM1 and Orai1 were increased significantly in MetS hearts.ConclusionCurrent data indicate the significant contribution of SOCE to cardiac calcium handling in the MetS model. We think MetS-induced SOCE activation is a compensation mechanism that is required for the continuum of proper cardiac functioning, although the activation can also cause cardiac hypertrophy.

Ablation of collagen VI leads to the release of platelets with altered function

AbstractHemostatic abnormalities and impaired platelet function have been described in patients affected by connective tissue disorders. We observed a moderate bleeding tendency in patients affected by collagen VI–related disorders and investigated the defects in platelet functionality, whose mechanisms are unknown. We demonstrated that megakaryocytes express collagen VI that is involved in the regulation of functional platelet production. By exploiting a collagen VI–null mouse model (Col6a1−/−), we found that collagen VI–null platelets display significantly increased susceptibility to activation and intracellular calcium signaling. Col6a1−/− megakaryocytes and platelets showed increased expression of stromal interaction molecule 1 (STIM1) and ORAI1, the components of store-operated calcium entry (SOCE), and activation of the mammalian target of rapamycin (mTOR) signaling pathway. In vivo mTOR inhibition by rapamycin reduced STIM1 and ORAI1 expression and calcium flows, resulting in a normalization of platelet susceptibility to activation. These defects were cell autonomous, because transplantation of lineage-negative bone marrow cells from Col6a1−/− mice into lethally irradiated wild-type animals showed the same alteration in SOCE and platelet activation seen in Col6a1−/− mice. Peripheral blood platelets of patients affected by collagen VI–related diseases, Bethlem myopathy and Ullrich congenital muscular dystrophy, displayed increased expression of STIM1 and ORAI1 and were more prone to activation. Altogether, these data demonstrate the importance of collagen VI in the production of functional platelets by megakaryocytes in mouse models and in collagen VI–related diseases.

RNA editing of 5-HT2C R impairs insulin secretion of pancreatic beta cells via altered store-operated calcium entry.

Recent studies emphasize the importance of 5-HT2C receptor (5-HT2C R) signaling in the regulation of energy homeostasis. The 5-HT2C R is the only G-protein-coupled receptor known to undergo post-transcriptional adenosine to inosine (A-to-I) editing by adenosine deaminase acting on RNA (ADAR). 5-HT2C R has emerged as an important role in the modulation of pancreatic β cell functions. This study investigated mechanisms behind the effects of palmitic acid (PA) on insulin secretion in different overexpressed 5-HT2C R edited isoforms in pancreatic MIN6 β cells. Results showed that the expressions of 5HT2C R and ADAR2 were upregulated in the pancreatic islets of mice fed with high-fat diet (HFD) compared to control mice. PA treatment significantly induced the expressions of 5-HT2C R and ADAR2 in pancreatic MIN6 β cells. PA treatment significantly induced the editing of 5-HT2C R in pancreatic MIN6 β cells. There was no significant difference in cell viability between naïve cells and three overexpressed 5-HT2C R edited isoforms in pancreatic MIN6 β cells. Overexpressed 5-HT2C R edited isoforms showed reduced glucose-stimulated insulin secretion (GSIS) compared with green fluorescent protein (GFP) expressed cells. Moreover, 5-HT2C R edited isoforms displayed reduced endoplasmic reticulum (ER) calcium release and store-operated calcium entry (SOCE) activation, probably through inhibition of stromal interaction molecule 1 trafficking under PA treatment. Altogether, our results show that PA-mediated editing of 5-HT2C R modulates GSIS through alteration of ER calcium release and SOCE activation in pancreatic MIN6 β cells.

Apoptotic and Non-Apoptotic Modalities of Thymoquinone-Induced Lymphoma Cell Death: Highlight of the Role of Cytosolic Calcium and Necroptosis.

Simple SummaryDiffuse large B cell lymphoma (DLBCL) represents the most common type of non-Hodgkin lymphoma with a high curability rate. However, 40% of patients will relapse or exhibit refractory disease, and compromised apoptotic pathways is among the prognosis-worsening factors. Therefore, drugging non-apoptotic modalities might be therapeutically promising. Thymoquinone (TQ) has been reported to promote apoptosis in cancer cells. Herein, we show that TQ selectively kills DLBCL cells, either cell lines or primary lymphoma cells bearing resistance features to standard treatment. Investigations show that, although TQ induced apoptotic markers, non-apoptotic death was the major mechanism responsible for TQ-induced cellular demise. We demonstrate critical and selective roles of cytosolic calcium and necroptosis in TQ-induced non-apoptotic cell death. Finally, TQ exhibits an improved selectivity profile over conventional chemotherapy. Collectively, this work provides new insights into the mode of action of TQ and points to the therapeutic relevance of non-apoptotic modalities as a fail-safe mechanism for pro-apoptotic DLBCL therapies.Targeting non-apoptotic modalities might be therapeutically promising in diffuse large B cell lymphoma (DLBCL) patients with compromised apoptotic pathways. Thymoquinone (TQ) has been reported to promote apoptosis in cancer cells, but little is known about its effect on non-apoptotic pathways. This work investigates TQ selectivity against DLBCL cell lines and the cell death mechanisms. TQ reduces cell viability and kills cell lines with minimal toxicity on normal hematological cells. Mechanistically, TQ promotes the mitochondrial caspase pathway and increases genotoxicity. However, insensitivity of most cell lines to caspase inhibition by z-VAD-fmk (benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone) pointed to a critical role of non-apoptotic signaling. In cells dying through non-apoptotic death, TQ increases endoplasmic reticulum (ER) stress markers and substantially increases cytosolic calcium ([Ca2+]c) through ER calcium depletion and activation of store-operated calcium entry (SOCE). Chelation of [Ca2+]c, but not SOCE inhibitors, reduces TQ-induced non-apoptotic cell death, highlighting the critical role of calcium in a non-apoptotic effect of TQ. Investigations showed that TQ-induced [Ca2+]c signaling is primarily initiated by necroptosis upstream to SOCE, and inhibition necroptosis by necrostatin-1 alone or with z-VAD-fmk blocks the cell death. Finally, TQ exhibits an improved selectivity profile over standard chemotherapy agents, suggesting a therapeutic relevance of the pro-necroptotic effect of TQ as a fail-safe mechanism for DLBCL therapies targeting apoptosis.

Cyclopiazonic Acid-Induced Ca2+ Store Depletion Initiates Endothelium-Dependent Hyperpolarization-Mediated Vasorelaxation of Mesenteric Arteries in Healthy and Colitis Mice.

Purposes: Since the role of store-operated calcium entry (SOCE) in endothelium-dependent hyperpolarization (EDH)-mediated vasorelaxation of mesenteric arteries in health and colitis is not fully understood, cyclopiazonic acid (CPA), a specific inhibitor of the sarco(endo) plasmic reticulum calcium-ATPases (SERCA), was used as a SOCE activator to investigate its role in normal mice and its alteration in colitis mice.Methods: The changes in Ca2+ signaling in vascular endothelial cells (VEC) were examined by single cell Ca2+ imaging and tension of mesenteric arteries in response to CPA were examined using Danish DMT520A microvascular measuring system.Results: CPA activated the SOCE through depletion of the endoplasmic reticulum (ER) Ca2+ in endothelial cells. CPA had a concentration-dependent vasorelaxing effect in endothelium-intact mesenteric arteries, which was lost after endothelial removal. Both nitric oxide (NO) and prostacyclin (PGI2) inhibitors did not affect CPA-induced vasorelaxation; however, after both NO and PGI2 were inhibited, KCa channel blocker [10 mM tetraethylammonium chloride (TEA)] inhibited CPA-induced vasorelaxation while KCa channel activator (0.3 μM SKA-31) promoted it. Two SOCE blockers [30 μM SKF96365 and 100 μM flufenamic acid (FFA)], and an Orai channel blocker (30 μM GSK-7975A) inhibited this vasorelaxation. The inhibition of both Na+/K+-ATPase (NKA) and Na+/Ca2+-exchange (NCX) also inhibited CPA-induced vasorelaxation. Finally, the CPA involved in EDH-induced vasorelaxation by the depletion of ER Ca2+ of mesenteric arteries was impaired in colitis mice.Conclusion: Depletion of ER Ca2+ by CPA induces a vasorelaxation of mesenteric arteries that is mediated through EDH mechanism and invokes the activation of SOCE. The CPA-induced endothelium-dependent dilation is impaired in colitis which may limit blood perfusion to the intestinal mucosa.

TMEM97 facilitates the activation of SOCE by downregulating the association of cholesterol to Orai1 in MDA-MB-231 cells

The expression of TMEM97, a regulator of cholesterol transport, has been reported to be enhanced in some tumour cells. We have recently shown that TMEM97 is involved in the proliferation of the breast cancer cell line MDA-MB-231, probably through changes in store-operated calcium entry (SOCE). By using silencing and overexpression of TMEM97 in MDA-MB-231 cells (two manoeuvres that either reduce or increase the calcium influx, respectively), we show enhanced cholesterol uptake in these cells as compared to the non-tumoral breast cell line, MCF10A. The enhanced cholesterol uptake in MDA-MB-231 cells was inhibited by silencing TMEM97, while overexpression of this protein increased cholesterol uptake in MCF10A cells and, therefore, indicating that this protein plays a role in the enhanced cholesterol uptake in MDA-MB-231 cancer cell line. TMEM97 silencing and overexpression resulted in an increase and decrease in the association of cholesterol to the SOCE calcium channel Orai1, respectively. Interestingly, silencing of TMEM97 in MDA-MB-231 cells significantly reduced the co-localization of Orai1 with the SOCE regulatory protein STIM1. Finally, neither silencing nor overexpression of TMEM97 altered SOCE in MDA-MB-231 cells transfected with the cholesterol insensible mutant of Orai1(Y80E). Our results reveal a novel regulatory mechanism of SOCE that relies on TMEM97 activity that courses through the reduction of the cholesterol content in the plasma membrane, and subsequently, by impairing its interaction with Orai1.

Phasic Store-Operated Ca2+ Entry During Excitation-Contraction Coupling in Skeletal Muscle Fibers From Exercised Mice.

Store-operated calcium entry (SOCE) plays a pivotal role in skeletal muscle physiology as, when impaired, the muscle is prone to early fatigue and the development of different myopathies. A chronic mode of slow SOCE activation is carried by stromal interaction molecule 1 (STIM1) and calcium-release activated channel 1 (ORAI1) proteins. A phasic mode of fast SOCE (pSOCE) occurs upon single muscle twitches in synchrony with excitation-contraction coupling, presumably activated by a local and transient depletion at the terminal cisternae of the sarcoplasmic reticulum Ca2+-stores. Both SOCE mechanisms are poorly understood. In particular, pSOCE has not been described in detail because the conditions required for its detection in mouse skeletal muscle have not been established to date. Here we report the first measurements of pSOCE in mouse extensor digitorum longus muscle fibers using electrical field stimulation (EFS) in a skinned fiber preparation. We show moderate voluntary wheel running to be a prerequisite to render muscle fibers reasonably susceptible for EFS, and thereby define an experimental paradigm to measure pSOCE in mouse muscle. Continuous monitoring of the physical activity of mice housed in cages equipped with running wheels revealed an optimal training period of 5–6 days, whereby best responsiveness to EFS negatively correlated with running distance and speed. A comparison of pSOCE kinetic data in mouse with those previously derived from rat muscle demonstrated very similar properties and suggests the existence and similar function of pSOCE across mammalian species. The new technique presented herein enables future experiments with genetically modified mouse models to define the molecular entities, presumably STIM1 and ORAI1, and the physiological role of pSOCE in health and under conditions of disease.

Tmem178 negatively regulates store-operated calcium entry in myeloid cells via association with STIM1

Store-operated calcium entry (SOCE) modulates cytosolic calcium in multiple cells. Endoplasmic reticulum (ER)-localized STIM1 and plasma membrane (PM)-localized ORAI1 are two main components of SOCE. STIM1:ORAI1 association requires STIM1 oligomerization, its re-distribution to ER-PM junctions, and puncta formation. However, little is known about the negative regulation of these steps to prevent calcium overload. Here, we identified Tmem178 as a negative modulator of STIM1 puncta formation in myeloid cells. Using site-directed mutagenesis, co-immunoprecipitation assays and FRET imaging, we determined that Tmem178:STIM1 association occurs via their transmembrane motifs. Mutants that increase Tmem178:STIM1 association reduce STIM1 puncta formation, SOCE activation, impair inflammatory cytokine production in macrophages and osteoclastogenesis. Mutants that reduce Tmem178:STIM1 association reverse these effects. Furthermore, exposure to plasma from arthritic patients decreases Tmem178 expression, enhances SOCE activation and cytoplasmic calcium. In conclusion, Tmem178 modulates the rate-limiting step of STIM1 puncta formation and therefore controls SOCE in inflammatory conditions.

Involvement of MURC/Cavin‐4 in store‐operated Ca2+ entry in neonatal cardiomyocytes

STIM1 is a regulatory protein of store‐operated calcium entry (SOCE) and its expressions of in the cardiomyocytes suggests that it may play a role in cardiomyocytes function. To better understand this role, we sought to identify unknown protein partners of STIM1. We fused the ERM domain of STIM1 (amino acids 219–548) to GST and used it for a GST‐pulldown assay combined with analytical liquid chromatography‐mass spectrometry. From the lysate of RIPA‐solubilized proteins from adult rat hearts, we identified the muscle‐related coiled‐coil protein (MURC), also known as Cavin‐4, as a potential candidate. Also, we show that is the HR1 domain of MURC interacts with the ERM domains of STIM1.To understand the role of this interaction, we overexpressed MURC in neonatal rat ventricular myocytes (NRVM) and determined is impact on SOCE. NRVM were loaded with the Ca2+ probe, FURA‐2AM, and incubated with nifedipine to prevent Ca2+ entry at the plasma membrane via L‐type calcium channels. To induce intracellular store depletion and SOCE activation, 10mM caffeine and 2μM thapsigargin were added to the perfusion buffer in the absence of extracellular Ca2+ and Ca2+ entry was determined when Ca2+ was re‐introduced in the extracellular medium. Using this protocol, we demonstrated that the Ca2+ entry in NRVM overexpressing MURC is 1.8‐fold higher that control bGal condition. This effect is reduced by SOCE inhibitors (SKF‐96365, 2‐APB, and YM‐58483). The Ca2+ entry measured in NRVM overexpressing ΔHR1‐MURC was similar to the Ca2+ entry in the bGal control. These results indicate that MURC overexpression relies on its HR1 domain to potentiate SOCE in these cells.The increase in SOCE in MURC overexpressing NRVM may be the result from an enhanced interaction between Orai1 and STIM1. To test this possibility, we assessed the extent of STIM1‐Orai1 complex formation with a co‐immunoprecipitation assay using HA‐Orai1 as the target protein. The STIM1‐Orai1 interaction was evaluated in resting conditions or upon SOCE activation. As expected, SOCE activation enhanced the interaction between STIM1 and Orai1 by 2.80‐fold compared to the control condition. MURC overexpression was also sufficient to increase the STIM1‐Orai1 complex formation in resting (1.78‐fold) and Ca2+ store‐depleted (2.44‐fold) conditions. These results show that MURC overexpression in NRVM favors the interaction of STIM1 with Orai1.Also, we show that R140W‐MURC mutant, a missense variant of the HR1 domain associated with human dilated cardiomyopathy, exacerbates the SOCE increase in NRVM. Our study provides novel evidence that MURC regulates SOCE by interacting with STIM1 in cardiomyocytes. In addition, we identified a first potential mechanism by which the R140W mutation may contribute to Ca2+ mishandling and the development of cardiomyopathies.Support or Funding InformationSupported by the Natural Sciences and Engineering Research Council of Canada (NSERC) [M.A.M, and G.B.], CRMUS graduate student research award [J.M.], FRQS doctoral training award [J.D.], CIHR graduate scholarship [H.G.], André‐Lussier Research Chair [J.‐L.P.], and FRQS and Heart and Stroke Foundation of Canada Investigator awards [M.A‐M.].This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Store‐Operated Calcium Entry in Mesangial Cells and Glomerular Inflammatory Responses

Emerging evidence indicates that immunological and inflammatory mechanisms play a significant role in the development of diabetic nephropathy (DN). The early features of DN include accumulation of extracellular matrix (ECM) in the glomerular mesangium. Inflammatory cell infiltration mediated by locally produced cytokines/chemokines contributes to the histological impairment in DN. Glomerular mesangial cell (MC) is a major cell type in glomerulus to produce cytokines/chemokines in response to diabetes and a major contributor to mesangial expansion in DN. We have previously demonstrated that the Orai‐1 mediated store‐operated calcium entry (SOCE) suppressed ECM protein production by MCs. The aim of this study was to determine whether SOCE in MCs regulated cytokine/chemokine production by MCs and macrophage infiltration into glomerulus.MethodsOrai‐1 channel protein in MCs was knocked down using the targeted nanoparticle‐siRNA delivery system in wild‐type C57BL6 mice at the age of 16 weeks. Immunohistochemistry was performed on the paraffin‐embedded kidney sections to examine macrophages infiltration in glomeruli using F4/80 as a marker. In cultured human MCs, the levels of various inflammatory cytokines (MCP‐1, TNF‐α, IL‐6) were examined using ELISA in the presence of normal glucose (5 mM D‐glucose + 20 mM L‐glucose) or high glucose (25 mM D‐glucose) with/without an activator (thapsigargin at 1 μM) of SOCE.ResultsIn vivo knockdown of Orai1 in MCs induced infiltration of F4/80 stained macrophages into the glomeruli in the mice treated with nanoparticle/Orai1 siRNA for 5 days compared to the control mice. In cultured human MCs, 24‐hour treatment with high glucose significantly increased the expression levels of MCP‐1 and TNF‐α. The high glucose responses were significantly blunted by thapsigargin. However, high glucose treatment for 24 hours significantly decreased and activation of SOCE by thapsigargin significantly increased IL‐6 expression level in MCs.ConclusionOrai‐1 mediated SOCE in MCs inhibits the glomerular macrophage infiltration in mice and has differential effects on the expression of different cytokine/chemokines in MCs.Support or Funding InformationNIH/NIDDK (1RO1 DK115424); AHA Southwest Affiliate (16GRNT27780043); Harry S. Moss Heart Trust Award.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Store-Operated Calcium Entry Contributes to Cisplatin-Induced Cell Death in Non-Small Cell Lung Carcinoma.

Cisplatin (CDDP) is one of the principal chemotherapeutic agents used for the first-line treatment of many malignancies, including non-small cell lung carcinoma (NSCLC). Despite its use for over 40 years, its mechanism of action is not yet fully understood. Store-operated calcium entry (SOCE), the main pathway allowing Ca2+ entry in non-excitable cells, is involved in tumorogenesis, cancer progression and chemoresistance. It has become an attractive target in cancer treatment. In this study, we showed that siRNA-mediated depletion of stromal interaction molecule 1 (STIM1) and transient receptor potential channel 1 (TRPC1), two players of the store-operated calcium entry, dramatically reduced CDDP cytotoxicity in NSCLC cells. This was associated with an inhibition of the DNA damage response (DDR) triggered by CDDP. Moreover, STIM1 depletion also reduced CDDP-dependent oxidative stress. In parallel, SOCE activation induced Ca2+ entry into the mitochondria, a major source of reactive oxygen species (ROS) within the cell. This effect was highly decreased in STIM1-depleted cells. We then conclude that mitochondrial Ca2+ peak associated to the SOCE contributes to CDDP-induced ROS production, DDR and subsequent apoptosis. To the best of our knowledge, this is the first time that it is shown that Ca2+ signalling constitutes an initial step in CDDP-induced apoptosis.

Digitoxin Suppresses Store Operated Calcium Entry by Modulating Phosphorylation and the Pore Region of Orai1.

Store-operated calcium entry (SOCE), primarily mediated by Orai1 and stromal interaction molecule 1 (STIM1), is a major Ca2+ influx pathway that has been linked to human diseases including myopathy, epilepsy, immunodeficiency, and cancer. Despite of the recent rapid progress of dissecting molecular mechanisms underlying SOCE activation, the development of therapies against dysfunctional SOCE significantly lags behind, partly due to the lack of more specific pharmacological tools and poor understanding of currently available SOCE modifiers, including the a newly identified SOCE inhibitor, digitoxin. Capitalizing on Ca2+ imaging and pharmacological tools, we aimed to systemically delineate the mechanism of action of digitoxin by defining how it impinges on Orai1 to exert its suppressive effect on SOCE. The SOCE-suppressive function of digitoxin is dependent on S27-S30 residues of wild-type Orai1. With 8h-incubation of digitoxin with STIM1-prebound Orai1 or a constitutively active mutant Orai1-ANSGA, its inhibition was no longer dependent on S27-S30 residues. Instead, the inhibition may involve the pore region of Orai1 channels, as V102C mutant at the pore region would greatly diminish or abolish the inhibition on pre-activated Orai1. Our study identified two regions that are critical for the inhibition on Orai1 channels, providing valuable hotspots for future design of SOCE inhibitors.