Store-operated Ca2+ Entry Inhibitors Research Articles

Suppression of Ca2+ influx in endotoxin-treated mouse cerebral cortex endothelial bEND.3 cells

Release of nitric oxide (NO) is triggered by a rise in endothelial cell (EC) cytosolic Ca2+ concentration ([Ca2+]i) and is of prime importance in vascular tone regulation as NO relaxes vascular smooth muscle. Agonists could stimulate EC [Ca2+]i elevation by triggering Ca2+ influx via plasma membrane ion channels, one of which is the store-operated Ca2+ channel; the latter opens as a result of agonist-triggered internal Ca2+ release. Endotoxin (lipopolysaccharide, LPS) could cause sepsis, which is often the fatal cause in critically ill patients. One of the LPS-induced damages is EC dysfunction, eventually leading to perturbations in hemodynamics. We obtained data showing that LPS-challenged mouse cerebral cortex endothelial bEND.3 cells did not suffer from apoptotic death, and in fact had intact agonist-triggered intracellular Ca2+ release; however, they had reduced store-operated Ca2+ entry (SOCE) after LPS treatment for 3h or more. Using real-time PCR, we did not find a decrease in gene expression of stromal interaction molecule 1 (STIM1) and Orai1 (two SOCE protein components) in bEND.3 cells treated with LPS for 15h. LPS inhibitory effects could be largely prevented by sodium salicylate (an inhibitor of nuclear factor-κB; NF-κB) or SB203580 (an inhibitor of p38 mitogen-activated protein kinases; p38 MAPK), suggesting that the p38 MAPK–NF-κB pathway is involved in SOCE inhibition.

Mitochondrial Ca2+ uniporter is critical for store-operated Ca2+ entry-dependent breast cancer cell migration

Metastasis of cancer cells is a complicated multistep process requiring extensive and continuous cytosolic calcium modulation. Mitochondrial Ca2+ uniporter (MCU), a regulator of mitochondrial Ca2+ uptake, has been implicated in energy metabolism and various cellular signaling processes. However, whether MCU contributes to cancer cell migration has not been established. Here we examined the expression of MCU mRNA in the Oncomine database and found that MCU is correlated to metastasis and invasive breast cancer. MCU inhibition by ruthenium red (RuR) or MCU silencing by siRNA abolished serum-induced migration in MDA-MB-231 breast cancer cells and reduced serum- or thapsigargin (TG)-induced store-operated Ca2+ entry (SOCE). Serum-induced migrations in MDA-MB-231 cells were blocked by SOCE inhibitors. Our results demonstrate that MCU plays a critical role in breast cancer cell migration by regulating SOCE.

Probing the Functional Coupling Interface between STIM1 and Orai1

Store operated Ca2+ entry (SOCE) plays a crucial physiological roles and involves two key proteins, STIM and Orai. Triggered by store-depletion, ER STIM proteins aggregate at ER-PM junctions and directly activate PM Orai Ca2+ channels. Few SOCE inhibitors have been described that specifically target the STIM/Orai machinery. Recently, we revealed a previously described SOCE modifier, DPB162-AE, potently and specifically blocks STIM1-induced Orai1 channel activation. We narrowed the DPB162-AE site of action to the STIM-Orai activating region (SOAR) of STIM1. DPB162-AE does not affect SOAR-dimerization nor does it prevent the SOAR-Orai1 interaction. However, it potently blocks SOAR-mediated Orai1 channel activation, yet its action is not as an Orai1 channel pore blocker. Using the SOAR-F394H mutant which prevents both physical and functional coupling to Orai1, we reveal DPB162-AE rapidly restores Orai-binding of this SOAR mutant but only slowly restores its activation of Orai1 channel-mediated Ca2+ entry. With the same SOAR mutant, 2-APB induces rapid physical and functional coupling to Orai1, but channel activation is transient. In STIM2, the equivalent residue to Phe-394 is Leu-485 resulting in STIM2's lower efficacy for Orai channel activation. This difference may explain the unusual specificity of DPB162-AE in blocking the action of STIM1 but not STIM2. We infer that the actions of both 2-APB and DPB162-AE are directed toward the STIM1-Orai1 coupling interface. Compared to 2-APB, DPB162-AE is a much more potent and specific STIM1/Orai1 functional uncoupler. DPB162-AE provides an important pharmacological tool and a useful mechanistic probe for the function and coupling between STIM1 and Orai1 channels.

Inhibition of SOCE disrupts cytokinesis in zebrafish embryos via inhibition of cleavage furrow deepening.

During the first few cell division cycles in zebrafish, distinct Ca(2+) transients are localized to the early embryonic cleavage furrows, where they accompany (and are required for) furrow positioning, propagation, deepening and apposition. It has previously been shown that the endoplasmic reticulum (ER) acts as the primary store for generating these Ca(2+) transients via release through inositol 1,4,5-trisphosphate receptors (IP 3Rs). We hypothesised that maintaining the elevated levels of intracellular Ca(2+) required for deepening and apposition of the cleavage furrows in these large eggs might result in the depletion of the available ER Ca(2+) store, thus the role of store-operated Ca(2+) entry (SOCE) was examined. Newly fertilized, dechorionated embryos were incubated with various SOCE inhibitors, starting just prior to the onset of the first cell division cycle. The effect of these inhibitors on mitosis, furrow positioning, propagation, deepening and apposition, and the generation of the cytokinetic Ca(2+) transients was determined. Treatment with 2-APB or SKF 96365 had no major effect on mitosis, furrow positioning or propagation, but inhibited furrow deepening resulting in regression of the cleavage furrow. Both of these inhibitors also blocked the furrowing Ca(2+) transient, with SKF 96365 having a more profound inhibitory effect than 2-APB. In zebrafish, SOCE does not appear to be required for mitosis or the early stages of cytokinesis during the early embryonic cell division cycles, but it does appear to be essential for maintaining the elevated levels of [Ca(2+)]i for the extended periods that are required during furrow deepening and daughter cell apposition.

Store-operated Ca(2+) entry regulates glioma cell migration and invasion via modulation of Pyk2 phosphorylation.

BackgroundThe ubiquitous second messenger Ca2+ has been demonstrated to play an important role in cancer progression. Store-operated Ca2+ entry (SOCE) is the main Ca2+ entry pathway regulating intracellular Ca2+ concentration in a variety of cancer types. The present study aimed to explore the specific mechanisms of SOCE in the processes of glioma migration and invasion.MethodsThe expression of Orai1, a key component of SOCE, was examined in glioma samples and glioma cell lines by immunohistochemistry and western blot analysis. Both pharmacological intervention and RNA interference were employed to investigate the role of SOCE in glioma cell migration and invasion in vitro. The intracellular Ca2+ was certified through Fluo-4/AM based Ca2+ measurement. The effect of SOCE on cell viability, migration, and invasion was explored by methyl thiazolyl tetrazolium (MTT) assay, wound healing assay, transwell invasion assay. Western blot analysis and immunofluorescence assay were used to observe the changes of downstream related protein and cell morpholog.ResultsOrai1 expression was elevated in glioma tissues and several glioma cell lines compared with non-neoplastic brain tissues. Either inhibition of SOCE by a pharmacological inhibitor or Orai1 downregulation suppressed glioma cell migration and invasion. However, re-expression of Orai1 could rescue glioma cell motility. Furthermore, phosphorylation of proline-rich tyrosine kinase 2 (Pyk2) participated in the mechanisms by which SOCE regulated focal adhesion turnover and epithelial-to-mesenchymal (−like) transition in glioma cells, both of which are considered to be critical for tumor progression.ConclusionsThe SOCE-Pyk2 pathway is essential for glioma migration and invasion. The study indicates the potential value of Orai1 as a molecular target for anti-invasion therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s13046-014-0098-1) contains supplementary material, which is available to authorized users.

A functional transient receptor potential vanilloid 4 (TRPV4) channel is expressed in human endothelial progenitor cells.

Endothelial progenitor cells (EPCs) are mobilized into circulation to replace damaged endothelial cells and recapitulate the vascular network of injured tissues. Intracellular Ca(2+) signals are key to EPC activation, but it is yet to be elucidated whether they are endowed with the same blend of Ca(2+) -permeable channels expressed by mature endothelial cells. For instance, endothelial colony forming cells (ECFCs), the only EPC subset truly committed to acquire a mature endothelial phenotype, lack canonical transient receptor potential channels 3, 5 and 6 (TRPC3, 5 and 6), which are widely distributed in vascular endothelium; on the other hand, they express a functional store-operated Ca(2+) entry (SOCE). The present study was undertaken to assess whether human circulating EPCs possess TRP vanilloid channel 4 (TRPV4), which plays a master signalling role in mature endothelium, by controlling both vascular remodelling and arterial pressure. We found that EPCs express both TRPV4 mRNA and protein. Moreover, both GSK1016790A (GSK) and phorbol myristate acetate and, two widely employed TRPV4 agonists, induced intracellular Ca(2+) signals uniquely in presence of extracellular Ca(2+). GSK- and PMA-induced Ca(2+) elevations were inhibited by RN-1734 and ruthenium red, which selectively target TRPV4 in mature endothelium. However, TRPV4 stimulation with GSK did not cause EPC proliferation, while the pharmacological blockade of TRPV4 only modestly affected EPC growth in the presence of a growth factor-enriched culture medium. Conversely, SOCE inhibition with BTP-2, La(3+) and Gd(3+) dramatically decreased cell proliferation. These data indicate that human circulating EPCs possess a functional TRPV4 protein before their engraftment into nascent vessels.

Polyunsaturated fatty acids inhibit stimulated coupling between the ER Ca2 + sensor STIM1 and the Ca2 + channel protein Orai1 in a process that correlates with inhibition of stimulated STIM1 oligomerization

Polyunsaturated fatty acids (PUFAs) have been found to be effective inhibitors of cell signaling in numerous contexts, and we find that acute addition of micromolar PUFAs such as linoleic acid effectively inhibit of Ca2+ responses in mast cells stimulated by antigen-mediated crosslinking of FcεRI or by the SERCA pump inhibitor, thapsigargin. In contrast, the saturated fatty acid, stearic acid, with the same carbon chain length as linoleic acid does not inhibit these responses. Consistent with this inhibition of store-operated Ca2+ entry (SOCE), linoleic acid inhibits antigen-stimulated granule exocytosis to a similar extent. Using the fluorescently labeled plasma membrane Ca2+ channel protein, AcGFP–Orai1, together with the labeled ER Ca2+ sensor protein, STIM1–mRFP, we monitor stimulated coupling of these proteins that is essential for SOCE with a novel spectrofluorimetric resonance energy transfer method. We find effective inhibition of this stimulated coupling by linoleic acid that accounts for the inhibition of SOCE. Moreover, we find that linoleic acid induces some STIM1–STIM1 association, while inhibiting stimulated STIM1 oligomerization that precedes STIM1–Orai1 coupling. We hypothesize that linoleic acid and related PUFAs inhibit STIM1–Orai1 coupling by a mechanism that involves perturbation of ER membrane structure, possibly by disrupting electrostatic interactions important in STIM1 oligomerization. Thisarticle is part of a Special Issue entitled Tools to study lipid functions.

Regulation of Store‐Operated Ca2+ Entry in the Vascular Endothelium by the G Protein‐coupled Estrogen Receptor 1 (LB570)

The novel G protein‐coupled estrogen receptor 1 (GPER/GPR30) has been found to participate in numerous cardiovascular functions. Store‐operated Ca2+ entry (SOCE) is an essential mechanism that is required for many endothelial cell functions. We found that activation of GPER using the GPER specific agonist G1 causes a dose‐dependent inhibition of SOCE in primary vascular endothelial cells. Heterologous of GPER in HEK 293 cells is associated with substantial decreases in the total Ca2+ signal triggered by thapsigargin, and a 40% decrease in the rate of SOCE. Knockdown of GPER in endothelial cells using antisense directed against GPER increases SOCE by approximately 50% compared to cells transfected scrambled oligo. Interestingly, GPER specific antagonist G15 triggers total Ca2+ signals via SOCE by releasing the same internal Ca2+ stores as does thapsigargin. In primary endothelial cells, GPER forms a complex with the stromal interaction molecule 1 (Stim1), an essential molecular switcher of SOCE. In addition, Stim1‐ECFP colocalizes with GPER‐tetracysteine when heterologously expressed in HEK 293 cells. Heterologous expression of GPER‐DsRed2 fusion substantially decreases total Ca2+ signal stimulated by thapsigargin. Interestingly, SOCE measured in cells stably expressing fusions between EYFP and individual sub‐membrane domains (SMDs) 1 ‐ 4 of GPER does not differ from internal controls. These data indicate that GPER may be an important regulatory input of store‐operated Ca2+ entry via its interaction with Stim1. Since the EYFP‐SMD fusions are all cytoplasmically expressed, the data may also suggest that proper membrane docking (PM or ER) of full‐length GPER is required for it to exert its effect on SOCE.Grant Funding Source: National Institutes of Health

Store-Operated Ca2+ Entry (SOCE) Regulates Melanoma Proliferation and Cell Migration

Store-operated Ca2+ entry (SOCE) is a major mechanism of Ca2 + import from extracellular to intracellular space, involving detection of Ca2+ store depletion in endoplasmic reticulum (ER) by stromal interaction molecule (STIM) proteins, which then translocate to plasma membrane and activate Orai Ca2+ channels there. We found that STIM1 and Orai1 isoforms were abundantly expressed in human melanoma tissues and multiple melanoma/melanocyte cell lines. We confirmed that these cell lines exhibited SOCE, which was inhibited by knockdown of STIM1 or Orai1, or by a pharmacological SOCE inhibitor. Inhibition of SOCE suppressed melanoma cell proliferation and migration/metastasis. Induction of SOCE was associated with activation of extracellular-signal-regulated kinase (ERK), and was inhibited by inhibitors of calmodulin kinase II (CaMKII) or Raf-1, suggesting that SOCE-mediated cellular functions are controlled via the CaMKII/Raf-1/ERK signaling pathway. Our findings indicate that SOCE contributes to melanoma progression, and therefore may be a new potential target for treatment of melanoma, irrespective of whether or not Braf mutation is present.

Store-operated Ca 2+ entry regulates glioma cell migration and invasion via modulation of Pyk2 phosphorylation

The ubiquitous second messenger Ca2+ has been demonstrated to play an important role in cancer progression. Store-operated Ca2+ entry (SOCE) is the main Ca2+ entry pathway regulating intracellular Ca2+ concentration in a variety of cancer types. The present study aimed to explore the specific mechanisms of SOCE in the processes of glioma migration and invasion. The expression of Orai1, a key component of SOCE, was examined in glioma samples and glioma cell lines by immunohistochemistry and western blot analysis. Both pharmacological intervention and RNA interference were employed to investigate the role of SOCE in glioma cell migration and invasion in vitro. The intracellular Ca2+ was certified through Fluo-4/AM based Ca2+ measurement. The effect of SOCE on cell viability, migration, and invasion was explored by methyl thiazolyl tetrazolium (MTT) assay, wound healing assay, transwell invasion assay. Western blot analysis and immunofluorescence assay were used to observe the changes of downstream related protein and cell morpholog. Orai1 expression was elevated in glioma tissues and several glioma cell lines compared with non-neoplastic brain tissues. Either inhibition of SOCE by a pharmacological inhibitor or Orai1 downregulation suppressed glioma cell migration and invasion. However, re-expression of Orai1 could rescue glioma cell motility. Furthermore, phosphorylation of proline-rich tyrosine kinase 2 (Pyk2) participated in the mechanisms by which SOCE regulated focal adhesion turnover and epithelial-to-mesenchymal (−like) transition in glioma cells, both of which are considered to be critical for tumor progression. The SOCE-Pyk2 pathway is essential for glioma migration and invasion. The study indicates the potential value of Orai1 as a molecular target for anti-invasion therapy.

Urotensin-II Induces Vascular Smooth Muscle Cell Proliferation and Creb Phosporylation Through Store Operated Calcium Entry and EGFR Transactivation

Urotensin-II (U-II) is a vasoactive peptide with many effects in the cardiovascular system. It has been described to induce vascular smooth muscle cell (SMC) proliferation, and is involved in the pathogenesis of atherosclerosis, restenosis, and vascular remodelling; however, its signalling pathway still remains unclear. The aim of this study was to elucidate the role of calcium (Ca2+)-dependent signalling and alternative signalling pathways in UII-evoked VSMCs proliferation, focussing our attention on store-operated Ca2+ entry (SOCE) pathway and epithelium growth factor receptor (EGFR) transactivation.We used 5-bromo-2-deoxyuridine (BrdU) labeling, molecular knockdown with small interfering RNA (siRNA) and immunofluorescence to study UII-promoted aortic SMC proliferation. Ca2+ mobilization assays were performed to study Ca2+ entry in cultured SMC.We found that UII enhanced intracellular Ca2+ concentration ([Ca2+]i) which was significantly reduced by classical SOCE inhibitors and by knockdown of essential components of the SOCE such as STIM1, Orai1, or TRPC1. Moreover, UII stimulated SMC proliferation and Ca2+/cAMP response element-binding protein (CREB) activation through SOCE pathway that involved STIM1, Orai1, and TRPC1. Co-immunoprecipitation experiments showed that UII promoted the association between Orai1 and STIM1, and between Orai1 and TRPC1. Additionally, we determined that epithelium growth factor receptor (EGFR) transactivation, extracellular signal-regulated kinase (ERK) and Ca2+/calmodulin-dependent kinase (CaMK) signaling pathways were involved in UII-mediated Ca2+ influx, CREB activation and VSMCs proliferation.Our data show for the first time that UII-induced SMC proliferation and CREB activation requires a complex signalling pathway that involves on the one hand SOCE mediated by STIM1, Orai1 and TRPC1, and on the other hand EGFR, ERK, and CaMK activation.Acknowledgements: This study was supported by Spanish Ministry of Science and Innovation [BFU-2010-21043-C02-01; BFU-2010-21043-C02-02]; Instituto Carlos III [RD12/0042/0041, PI12/00941]; and The Andalusian Government [P10-CVI-6095; PI-0108-2012].

Blockade of SOCE protects HT22 cells from hydrogen peroxide-induced apoptosis

Oxidative stress is an established event in the pathology of neurobiological diseases. Previous studies indicated that store-operated Ca2+ entry (SOCE) has been involved in oxidative stress. The present study was carried out to investigate the effects of SOCE inhibition on neuronal oxidative stress injury induced by hydrogen peroxide (H2O2) in HT22 cells, a murine hippocampal neuronal model. H2O2 insult induced significant intracellular Ca2+ overload, mitochondrial dysfunction and cell viability decrease. Inhibition of SOCE by pharmacological inhibitor and STIM1 RNAi significantly alleviated intracellular Ca2+ overload, restored the mitochondrial membrane potential (MMP), decreased cytochrome C release and eventually inhibited H2O2-induced cell apoptosis. These findings suggest that SOCE inhibition exhibited neuroprotection against oxidative stress induced by H2O2 and SOCE might be a useful therapeutic target in neurobiological disorders.

Urotensin-II promotes vascular smooth muscle cell proliferation through store-operated calcium entry and EGFR transactivation

Urotensin-II (UII) is a vasoactive peptide that promotes vascular smooth muscle cells (VSMCs) proliferation and is involved in the pathogenesis of atherosclerosis, restenosis, and vascular remodelling. This study aimed to determine the role of calcium (Ca(2+))-dependent signalling and alternative signalling pathways in UII-evoked VSMCs proliferation focusing on store-operated Ca(2+) entry (SOCE) and epithelium growth factor receptor (EGFR) transactivation. We used primary cultures of VSMCs isolated from Wistar rat aorta to investigate the effects of UII on intracellular Ca(2+) mobilization, and proliferation determined by the 5-bromo-2-deoxyuridine (BrdU) assay. We found that UII enhanced intracellular Ca(2+) concentration ([Ca(2+)]i) which was significantly reduced by classical SOCE inhibitors and by knockdown of essential components of the SOCE such as stromal interaction molecule 1 (STIM1), Orai1, or TRPC1. Moreover, UII activated a Gd(3+)-sensitive current with similar features of the Ca(2+) release-activated Ca(2+) current (ICRAC). Additionally, UII stimulated VSMCs proliferation and Ca(2+)/cAMP response element-binding protein (CREB) activation through the SOCE pathway that involved STIM1, Orai1, and TRPC1. Co-immunoprecipitation experiments showed that UII promoted the association between Orai1 and STIM1, and between Orai1 and TRPC1. Moreover, we determined that EGFR transactivation, extracellular signal-regulated kinase (ERK) and Ca(2+)/calmodulin-dependent kinase (CaMK) signalling pathways were involved in both UII-mediated Ca(2+) influx, CREB activation and VSMCs proliferation. Our data show for the first time that UII-induced VSMCs proliferation and CREB activation requires a complex signalling pathway that involves on the one hand SOCE mediated by STIM1, Orai1, and TRPC1, and on the other hand EGFR, ERK, and CaMK activation.

The apoptosis of non-small cell lung cancer induced by cisplatin through modulation of STIM1

Cis-diamminedichloroplatinum (II) (cisplatin) is one of the most active antitumor agents used in human chemotherapy of non-small cell lung cancer. Cisplatin forms crosslinked DNA adducts and its cytotoxicity has been shown to be mediated by propagation of DNA damage recognition signals to downstream pathways prompting apoptosis. The steps involved in the process include changes in Ca(2+) signaling with dysregulated tumor cell turn-over. Stromal interaction molecules 1 (STIM1), as one of the most potent tumor suppressor genes, are identified as the endoplasmic-reticulum (ER) Ca(2+) sensor controlling store-operated Ca(2+) entry (SOCE) in non-excitable cells, which is main pathway to extracellular Ca(2+) influx. Its role in STIM1 cisplatin-induced apoptosis of non-small cell lung cancer was the focus of study with focus on SOCE inhibitors 2-APB- and SKF96365-cisplatin-induced apoptosis in the non-small cell lung cancer (NSCLC) cell lines A549 and H460. In this experimental model, cisplatin-induced apoptosis and decreased concentration of intracellular Ca(2+) was demonstrated. The expression of STIM1 was significantly higher in carcinoma tissue than in the adjacent non-neoplastic lung tissue. These findings support the conclusion that STIM1 may play an important role in the development of NSCLC which makes drugs that repress the expression of STIM1 to be a potential target for lung cancer therapy.

Store-operated calcium entry and calcium influx via voltage-operated calcium channels regulate intracellular calcium oscillations in chondrogenic cells

Chondrogenesis is known to be regulated by calcium-dependent signalling pathways in which temporal aspects of calcium homeostasis are of key importance. We aimed to better characterise calcium influx and release functions with respect to rapid calcium oscillations in cells of chondrifying chicken high density cultures. We found that differentiating chondrocytes express the α1 subunit of voltage-operated calcium channels (VOCCs) at both mRNA and protein levels, and that these ion channels play important roles in generating Ca2+ influx for oscillations as nifedipine interfered with repetitive calcium transients. Furthermore, VOCC blockade abrogated chondrogenesis and almost completely blocked cell proliferation. The contribution of internal Ca2+ stores via store-operated Ca2+ entry (SOCE) seems to be indispensable to both Ca2+ oscillations and chondrogenesis. Moreover, this is the first study to show the functional expression of STIM1/STIM2 and Orai1, molecules that orchestrate SOCE, in chondrogenic cells. Inhibition of SOCE combined with ER calcium store depletion abolished differentiation and severely diminished proliferation, suggesting the important role of internal pools in calcium homeostasis of differentiating chondrocytes. Finally, we present an integrated model for the regulation of calcium oscillations of differentiating chondrocytes that may have important implications for studies of chondrogenesis induced in various stem cell populations.

Staurosporine maintains the activation of store-operated Ca2+ entry even after the refilling of Ca2+ stores

Store-operated Ca2+ entry (SOCE) from the extracellular space plays a critical role in agonist-mediated Ca2+ signaling in non-excitable cells. Here we show that SOCE is enhanced in COS-7 cells treated with staurosporine (ST), a protein kinase inhibitor. In COS-7 cells, stimulation with ATP induced Ca2+ release from intracellular Ca2+ stores and Ca2+ entry from the extracellular space. Ca2+ release was not affected by treatment with ST, but Ca2+ entry continued in the ST-treated cells even after the removal of ATP. ST did not inhibit Ca2+ sequestration into Ca2+ stores. The Ca2+ entry induced by cyclopiazonic acid (CPA), a reversible ER Ca2+ pump inhibitor, was maintained in ST-treated cells even after the removal of CPA, but was not maintained in the control cells. The sustained Ca2+ entry in ST-treated cells was completely attenuated by the SOCE inhibitors, La3+ and 2-APB. The large increase in Ca2+ entry produced in the cells co-expressing Venus-Orai1 and STIM1-mKO1 was stabilized with ST treatment, and confocal imaging of these cells suggested that the complex between Orai1 and STIM1 did not completely dissociate following the refilling of Ca2+ stores. These results show that SOCE remains activated even after the refilling of Ca2+ stores in ST-treated cells and that the effect of ST on SOCE may result from a stabilization of the Orai1–STIM1 interaction.

Abstract 5244: Store-operated Ca2+ entry (SOCE) regulates melanoma progression.

Abstract Background: Melanoma is the worst poor-prognosis skin cancer in the U.S. Although Ca2+ is well recognized as a major second messenger, the role of Ca2+ remains poorly understood in the cancer research field including melanoma. Store-operated calcium entry (SOCE) is the mechanism of Ca2+ entry from the extracellular space in response to Ca2+ depletion in the endoplasmic reticulum (ER). SOCE is regulated by interaction between STIM1 (stromal interaction molecule 1) in the ER and Orai (ORAI calcium release-activated calcium modulator) in the plasma membrane. Our previous studies have demonstrated the presence of SOCE is observed in melanoma cells, and potentially regulates cell migration and cell cycle. In the present study, we extensively examined the effects of SOCE on migration and proliferation in multiple melanoma and melanocyte cell lines as well as on metastasis in mice. Method: Changes in intracellular Ca2+ level were measured by Fluo4-AM, a Ca2+-sensing fluorescent dye. In order to ablate STIM-1 or Orai-1, shRNA for each protein was induced with lentiviral infection in SK-Mel-2, SK-Mel-24 and C8161 cells. Proliferation was measured by the MTT assay. Migration was examined with the Boyden chambers and the scratch assay. Immunocytochemistry was performed to count the number of lamellipodia with F-actin staining. The experimental metastasis assay was performed as follows. Three weeks after the intravenous injection of SK-Mel-2 cells with knockdown of Orai1 or STIM1 in Balb/c nu/nu mice, the lungs were removed and fixed with picric acid. The number of metastatic colonies in the lung surface was counted. Results: SOCE, as demonstrated by enhancement of Ca2+ entry from extracellular space after Ca2+ depletion in the ER, was observed in 5 primary and 3 metastatic human melanoma cell lines, a mouse melanoma cell line, human and mouse melanocyte cell lines. In addition, metastatic, but not primary, melanoma cell lines showed significantly greater SOCE compared to melanocytes, suggesting that SOCE may positively correlate with malignancy of melanoma. Knockdown of STIM1 inhibited proliferation in metastatic melanoma cells (SK-Mel-2, SK-Mel-24 and C8161) (p<0.01), suggesting that SOCE activates proliferation of melanoma. Inhibition of SOCE with knockdown of Orai1 or STIM1 suppressed migration in cell lines tested (SK-Mel-2 and SK-Mel-24) (p<0.01) and the SOCE inhibitors suppressed it in cell lines (SK-Mel-2 and C8161) (p<0.01 for both cell lines), indicating that SOCE regulates melanoma migration. Indeed, the number of lamellipodia, which reflects the activity of migration, was decreased by deletion of STIM1 or Orai1 (p<0.01). The experimental metastasis assay demonstrated that the number of lung colonies was reduced by knockdown of STIM1 or Orai1 (p<0.01). Conclusion: Our results demonstrated that inhibition of SOCE suppressed proliferation, migration, and metastasis in melanoma, thus SOCE could be a new target for melanoma therapy. Citation Format: Masanari Umemura, Erdene Balijinnyam, Stefan Feske, Mariana S. De Lorenzo, Lai-Hua Xie, Yoshihiro Ishikawa, Kousaku Iwatsubo. Store-operated Ca2+ entry (SOCE) regulates melanoma progression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5244. doi:10.1158/1538-7445.AM2013-5244

Regulation of Store‐Operated Ca2+ Entry in the Vascular Endothelium by the G Protein‐Coupled Estrogen Receptor

The novel G protein‐coupled estrogen receptor (GPER/GPR30) has been found to participate in numerous cardiovascular functions. Store‐operated Ca2+ entry (SOCE) is an essential mechanism that is required for many endothelial cell functions. We found that activation of GPER using the GPER specific agonist G1 is associated with a dose‐dependent inhibition of SOCE in primary vascular endothelial cells. Interestingly, the GPER specific antagonist G15 increases SOCE in cells unstimulated by GPER intrinsic or exogenous ligand. Overexpression of GPER in HEK 293 cells is associated with a 40% decrease in the rate of SOCE, while knockdown of GPER in endothelial cells using shRNA or antisense oligomer directed against GPER increases SOCE by approximately 50%. In addition, coimmunoprecipitation revealed that GPER exists in endothelial cells as a glycosylated protein and forms a complex with the stromal interaction molecule 1 (Stim1). These data suggest that GPER may be an important regulatory input of store‐operated Ca2+ entry via its interaction with Stim1.

Characterization of ryanodine receptor‐gated store‐operated calcium entry in rat pulmonary artery smooth muscle cells

Ca2+ homeostasis in pulmonary arterial smooth muscle cells (PASMCs) involves interactions between Ca2+ influx and release pathways. Depletion of sarcoplasmic reticulum (SR) Ca2+ content activates the Ca2+ sensor, stromal interacting molecule (STIM), which induces store‐operated Ca2+ entry (SOCE). Previous studies on SOCE focused on inositol 1,4,5‐trisphosphate receptor (IP3R)‐ related pathways and how ryanodine receptor (RyR)‐gated store regulates SOCE remained largely unknown. Here we characterized the RyR‐gated SOCE in rat PASMCs. Intracellular Ca2+ was measured in the presence of 1 μM nifedipine to prevent voltage‐dependent Ca2+ entry and extracellular Ca2+ was introduced to induce SOCE after 20‐minute store depletion in Ca2+‐free environment. The RyR agonist, 4‐chloro‐m‐cresol (4‐CmC; 1mM), induced SOCE in rat PASMCs. This Ca2+ response was blocked by a low concentration of the SOCE inhibitor, Gd3+ (1 μM). The RyR1 and RyR3 inhibitor, dantrolene (30 μM), did not inhibit 4‐ CmC‐induced SOCE. In addition, maurocalcine (400 nM), a RyR1 agonist, failed to induce RyR‐gated Ca2+ release in rat PASMCs and hence did not activate SOCE. These data shows that RyR2 is the major RyR subtype that mediates SOCE in rat PASMCs. (This project is supported by NIH grant NL‐071835)

Lysophosphatidic Acid Promotes Cell Migration through STIM1- and Orai1-Mediated Ca2+i Mobilization and NFAT2 Activation

Lysophosphatidic acid (LPA) enhances cell migration and promotes wound healing in vivo, but the intracellular signaling pathways regulating these processes remain incompletely understood. Here we investigated the involvement of agonist-induced Ca2+ entry and STIM1 and Orai1 proteins in regulating nuclear factor of activated T cell (NFAT) signaling and LPA-induced keratinocyte cell motility. As monitored by Fluo-4 imaging, stimulation with 10 μℳ LPA in 60 μℳ Ca2+o evoked Ca2+i transients owing to store release, whereas addition of LPA in physiological 1.2 mℳ Ca2+o triggered store release coupled to extracellular Ca2+ entry. Store-operated Ca2+ entry (SOCE) was blocked by the SOCE inhibitor diethylstilbestrol (DES), STIM1 silencing using RNA interference (RNAi), and expression of dominant/negative Orai1R91W. LPA induced significant NFAT activation as monitored by nuclear translocation of green fluorescent protein-tagged NFAT2 and a luciferase reporter assay, which was impaired by DES, expression of Orai1R91W, and inhibition of calcineurin using cyclosporin A (CsA). By using chemotactic migration assays, LPA-induced cell motility was significantly impaired by STIM1, CsA, and NFAT2 knockdown using RNAi. These data indicate that in conditions relevant to epidermal wound healing, LPA induces SOCE and NFAT activation through Orai1 channels and promotes cell migration through a calcineurin/NFAT2-dependent pathway.