Store-operated Ca2+ Entry In Cells Research Articles

Dynasore modulates store-operated calcium entry and mitochondrial calcium release in corneal epithelial cells

Dysregulation of calcium homeostasis can precipitate a cascade of pathological events that lead to tissue damage and cell death. Dynasore is a small molecule that inhibits endocytosis by targeting classic dynamins. In a previous study, we showed that dynasore can protect human corneal epithelial cells from damage due to tert-butyl hydroperoxide (tBHP) exposure by restoring cellular calcium (Ca2+) homeostasis. Here we report results of a follow-up study aimed at identifying the source of the damaging Ca2+. Store-operated Ca2+ entry (SOCE) is a cellular mechanism to restore intracellular calcium stores from the extracellular milieu. We found that dynasore effectively blocks SOCE in cells treated with thapsigargin (TG), a small molecule that inhibits pumping of Ca2+ into the endoplasmic reticulum (ER). Unlike dynasore however, SOCE inhibitor YM-58483 did not interfere with the cytosolic Ca2+ overload caused by tBHP exposure. We also found that dynasore effectively blocks Ca2+ release from internal sources. The inefficacy of inhibitors of ER Ca2+ channels suggested that this compartment was not the source of the Ca2+ surge caused by tBHP exposure. However, using a Ca2+-measuring organelle-entrapped protein indicator (CEPIA) reporter targeted to mitochondria, we found that dynasore can block mitochondrial Ca2+ release due to tBHP exposure. Our results suggest that dynasore exerts multiple effects on cellular Ca2+ homeostasis, with inhibition of mitochondrial Ca2+ release playing a key role in protection of corneal epithelial cells against oxidative stress due to tBHP exposure.

Extended Synaptotagmins 1 and 2 Are Required for Store-Operated Calcium Entry, Cell Migration and Viability in Breast Cancer Cells.

Extended synaptotagmins (E-Syts) are endoplasmic reticulum (ER)-associated proteins that facilitate the tethering of the ER to the plasma membrane (PM), participating in lipid transfer between the membranes and supporting the Orai1-STIM1 interaction at ER-PM junctions. Orai1 and STIM1 are the core proteins of store-operated Ca2+ entry (SOCE), a major mechanism for Ca2+ influx that regulates a variety of cellular functions. Aberrant modulation of SOCE in cells from different types of cancer has been reported to underlie the development of several tumoral features. Here we show that estrogen receptor-positive (ER+) breast cancer MCF7 and T47D cells and triple-negative breast cancer (TNBC) MDA-MB-231 cells overexpress E-Syt1 and E-Syt2 at the protein level; the latter is also overexpressed in the TNBC BT20 cell line. E-Syt1 and E-Syt2 knockdown was without effect on SOCE in non-tumoral MCF10A breast epithelial cells and ER+ T47D breast cancer cells; however, SOCE was significantly attenuated in ER+ MCF7 cells and TNBC MDA-MB-231 and BT20 cells upon transfection with siRNA E-Syt1 or E-Syt2. Consistent with this, E-Syt1 and E-Syt2 knockdown significantly reduced cell migration and viability in ER+ MCF7 cells and the TNBC cells investigated. To summarize, E-Syt1 and E-Syt2 play a relevant functional role in breast cancer cells.

Ginsenoside Rg-1 prevents elevated cytosolic Ca2+ via store-operated Ca2+ entry in high-glucose–stimulated vascular endothelial and smooth muscle cells

BackgroundGinsenoside Rg-1 (Rg-1), a triterpenoid saponin abundantly present in Panax ginseng, is a type of naturally occurring steroid with known anti-diabetic and anti-inflammatory effects. In this study, we sought to confirm the effects and mechanisms of action of Rg-1 on store-operated Ca2+ entry (SOCE) in human vascular endothelial cell line (EA) and murine aortic vascular smooth muscle cell line (MOVAS) cells exposed to high glucose.MethodsCytosolic Ca2+ concentrations in EA and MOVAS cells were measured by monitoring fluorescence of the ratiometric Ca2+-indicator, Fura-2 AM.ResultsHigh glucose significantly increased Ca2+ influx by abnormally activating SOCE in EA and MOVAS cells. Notably, this high glucose-induced increase in SOCE was restored to normal levels in EA and MOVAS cells by Rg-1. Moreover, Rg-1 induced reductions in SOCE in cells exposed to high glucose were significantly inhibited by the plasma membrane Ca2+ ATPase (PMCA) blocker lanthanum, the Na+/K+-ATPase blocker ouabain, or the Na+/Ca2+ exchanger (NCX) blockers Ni2+ and KB-R7943. These observations suggest that the mechanism of action of Rg-1 inhibition of SOCE involves PMCA and Na+/K+-ATPase, and an increase in Ca2+ efflux via NCXs in both EA and MOVAS cells exposed to high glucose.ConclusionsThese findings indicate that Rg-1 may protect vascular endothelial and smooth muscle cells from Ca2+ increases following exposure to hyperglycemic conditions.

SARAF and EFHB Modulate Store-Operated Ca2+ Entry and Are Required for Cell Proliferation, Migration and Viability in Breast Cancer Cells.

Simple SummaryBreast cancer accounts as the most extended disease among women worldwide. Store-operated calcium entry (SOCE), a major mechanism that allows calcium entry from the extracellular region through the plasma membrane, is required for several physiological processes. In recent years, it has been revealed that several breast cancer types present dysregulated calcium homeostasis, which contribute to their malignancy. Here we show the role of two important regulators of SOCE, SARAF and EFHB, which are necessary for cell viability, proliferation, and migration in breast cancer and pre-neoplastic cells, respectively, thus suggesting that these regulators play a key function in breast cancer development and progression.Breast cancer is among the most common malignancies in women. From the molecular point of view, breast cancer can be grouped into different categories, including the luminal (estrogen receptor positive (ER+)) and triple negative subtypes, which show distinctive features and, thus, are sensitive to different therapies. Breast cancer cells are strongly dependent on Ca2+ influx. Store-operated Ca2+ entry (SOCE) has been found to support a variety of cancer hallmarks including cell viability, proliferation, migration, and metastasis. The Ca2+ channels of the Orai family and the endoplasmic reticulum Ca2+ sensor STIM1 are the essential components of SOCE, but the extent of Ca2+ influx is fine-tuned by several regulatory proteins, such as the STIM1 modulators SARAF and EFHB. Here, we show that the expression and/or function of SARAF and EFHB is altered in breast cancer cells and both proteins are required for cell proliferation, migration, and viability. EFHB expression is upregulated in luminal and triple negative breast cancer (TNBC) cells and is essential for full SOCE in these cells. SARAF expression was found to be similar in breast cancer and pre-neoplastic breast epithelial cells, and SARAF knockdown was found to result in enhanced SOCE in pre-neoplastic and TNBC cells. Interestingly, silencing SARAF expression in ER+ MCF7 cells led to attenuation of SOCE, thus suggesting a distinctive role for SARAF in this cell type. Finally, we used a combination of approaches to show that molecular knockdown of SARAF and EFHB significantly attenuates the ability of breast cancer cells to proliferate and migrate, as well as cell viability. In aggregate, SARAF and EFHB are required for the fine modulation of SOCE in breast cancer cells and play an important role in the maintenance of proliferation, migration, and viability in these cells.

Polychlorinated biphenyl 19 blocks the most common form of store-operated Ca2+ entry through Orai.

PCB19, a 2,2',6-trichlorinated biphenyl, is one of many non-dioxin-like polychlorinated biphenyls (NDL-PCBs), which are ubiquitous pollutants. NDL-PCBs affect cytosolic Ca2+ signaling by promoting Ca2+ release from ryanodine receptor-sensitive Ca2+ pools and inhibiting store-operated Ca2+ entry (SOCE) from the extracellular space. However, NDL-PCB-mediated SOCE inhibition has only been demonstrated in PC12 cells, in which SOCE is thought to be mainly mediated by TRPC family channels. Here, we investigated the effect of PCB19 on SOCE using human embryonic kidney 293 (HEK293) cells, human leukemia T cell line Jurkat-T cells and human promyelocytoma HL-60 cells which are the cell lines that are previously demonstrated to mediate the most common form of SOCE solely by the intrinsic Orai channels. PCB19 reduced thapsigargin-induced Ca2+ influx after Ca2+ pool depletion in HEK293 cells. SOCEs in HEK293, Jurkat T, HL-60 and PC12 cells showed distinct sensitivities to SOCE inhibitors such as Gd3+ and ML-9; however, PCB19 also showed a common effect of inhibiting SOCEs in all cell lines. PCB19-mediated SOCE inhibition was confirmed by demonstrating the ability of PCB19 to inhibit the SOCE current but not the TRPM7 current. These results imply that PCB19 inhibits not only TRPC-mediated SOCE as in PC12 cells but also Orai-mediated SOCE as in many other cells including HEK293, Jurkat T and HL-60 cells.

Hydrogen peroxide is a critical regulator of the hypoxia-induced alterations of store-operated Ca2+ entry into rat pulmonary arterial smooth muscle cells.

To investigate the association between store-operated Ca2+ entry (SOCE) and reactive oxygen species (ROS) during hypoxia, this study determined the changes of transient receptor potential canonical 1 (TRPC1) and Orai1, two candidate proteins for store-operated Ca2+ (SOC) channels and their gate regulator, stromal interaction molecule 1 (STIM1), in a hypoxic environment and their relationship with ROS in pulmonary arterial smooth muscle cells (PASMCs). Exposure to hypoxia caused a transient Ca2+ spike and subsequent Ca2+ plateau of SOCE to be intensified in PASMCs when TRPC1, STIM1, and Orai1 were upregulated. SOCE in cells transfected with specific short hairpin RNA (shRNA) constructs was almost completely eliminated by the knockdown of TRPC1, STIM1, or Orai1 alone and was no longer affected by hypoxia exposure. Hypoxia-induced SOCE enhancement was further strengthened by PEG-SOD but was attenuated by PEG-catalase, with correlated changes to intracellular hydrogen peroxide (H2O2) levels and protein levels of TRPC1, STIM1, and Orai1. Exogenous H2O2 could mimic alterations of the interactions of STIM1 with TRPC1 and Orai1 in hypoxic cells. These findings suggest that TRPC1, STIM1, and Orai1 are essential for the initiation of SOCE in PASMCs. Hypoxia-induced ROS promoted the expression and interaction of the SOC channel molecules and their gate regulator via their converted product, H2O2.

Conformation of ryanodine receptor-2 gates store-operated calcium entry in rat pulmonary arterial myocytes.

Store-operated Ca(2+) entry (SOCE) contributes to a multitude of physiological and pathophysiological functions in pulmonary vasculatures. SOCE attributable to inositol 1,4,5-trisphosphate receptor (InsP3R)-gated Ca(2+) store has been studied extensively, but the role of ryanodine receptor (RyR)-gated store in SOCE remains unclear. The present study aims to delineate the relationship between RyR-gated Ca(2+) stores and SOCE, and characterize the properties of RyR-gated Ca(2+) entry in pulmonary artery smooth muscle cells (PASMCs). PASMCs were isolated from intralobar pulmonary arteries of male Wister rats. Application of the RyR1/2 agonist 4-chloro-m-cresol (4-CmC) activated robust Ca(2+) entry in PASMCs. It was blocked by Gd(3+) and the RyR2 modulator K201 but was unaffected by the RyR1/3 antagonist dantrolene and the InsP3R inhibitor xestospongin C, suggesting RyR2 is mainly involved in the process. siRNA knockdown of STIM1, TRPC1, and Orai1, or interruption of STIM1 translocation with ML-9 significantly attenuated the 4-CmC-induced SOCE, similar to SOCE induced by thapsigargin. However, depletion of RyR-gated store with caffeine failed to activate Ca(2+) entry. Inclusion of ryanodine, which itself did not cause Ca(2+) entry, uncovered caffeine-induced SOCE in a concentration-dependent manner, suggesting binding of ryanodine to RyR is permissive for the process. This Ca(2+) entry had the same molecular and pharmacological properties of 4-CmC-induced SOCE, and it persisted once activated even after caffeine washout. Measurement of Ca(2+) in sarcoplasmic reticulum (SR) showed that 4-CmC and caffeine application with or without ryanodine reduced SR Ca(2+) to similar extent, suggesting store-depletion was not the cause of the discrepancy. Moreover, caffeine/ryanodine and 4-CmC failed to initiate SOCE in cells transfected with the ryanodine-binding deficient mutant RyR2-I4827T. RyR2-gated Ca(2+) store contributes to SOCE in PASMCs; however, store-depletion alone is insufficient but requires a specific RyR conformation modifiable by ryanodine binding to activate Ca(2+) entry.

Zinc Inhibits Orai1-Mediated Calcium Signals in Esophageal Cancer Cells

Intracellular Ca2+ signals, such as oscillations, regulate proliferation, migration and other cellular events in cancer cells. Zinc (Zn), a major dietary micronutrient, plays important roles in chemoprevention. However, the molecular mechanisms underlying Zn-preventive function as well as the cross-talk with Ca2+ signaling are still unclear. We previously demonstrated that Orai1, a store-operated Ca2+ entry (SOCE) channel, is highly expressed in esophageal squamous cell carcinoma (ESCC) compared to adjacent normal tissues from patients, and the elevated expression of Orai1 is strongly associated with poor prognosis. The present study shows that Zn can greatly inhibit cell proliferation and Orai1-mediated SOCE in ESCC cells. We also found that the inhibitory function of Zn on SOCE is redox sensitive; reducing agent DTT could recover the full activity of Orai1-mediated SOCE. Based on the topology information of Orai1, we hypothesized that a histidine residue in the linker region between two transmembranes of Orai1 (H113) as well as three cysteine residues may play critical roles in the Zn-inhibitory effects. The data showed that, in the cells containing H113A-mutated Orai1, the Zn-inhibitory effect is vanished whereas Zn treatment reduces SOCE in the cell expressing wildtype Orai1. Similarly, the ESCC cells expressing any of three mutations in cysteine residues (C126A, C143A and C195A) of Orai1 displayed partial loss of the Zn-inhibitory function. These results suggest that the H113 and three cysteine residues likely bind Zn, which modulate Zn-inhibitory functions on the Orai1-mediated SOCE in ESCC cells.This study reveals how dietary Zn inhibits SOCE and thus Zn and Ca2+ signals may cross-talk, which in turn regulates cancer cell proliferation and migration in esophageal epithelia. Further investigations are required to search for novel and effective prevention strategies as well as therapeutic options targeting on Zn and Ca2+ signaling in ESCC.

Effects of the Inflammatory Cytokines TNF-α and IL-13 on Stromal Interaction Molecule–1 Aggregation in Human Airway Smooth Muscle Intracellular Ca2+ Regulation

Inflammation elevates intracellular Ca(2+) ([Ca(2+)]i) concentrations in airway smooth muscle (ASM). Store-operated Ca(2+) entry (SOCE) is an important source of [Ca(2+)]i mediated by stromal interaction molecule-1 (STIM1), a sarcoplasmic reticulum (SR) protein. In transducing SR Ca(2+) depletion, STIM1 aggregates to form puncta, thereby activating SOCE via interactions with a Ca(2+) release-activated Ca(2+) channel protein (Orai1) in the plasma membrane. We hypothesized that STIM1 aggregation is enhanced by inflammatory cytokines, thereby augmenting SOCE in human ASM cells. We used real-time fluorescence microscopic imaging to assess the dynamics of STIM1 aggregation and SOCE after exposure to TNF-α or IL-13 in ASM cells overexpressing yellow fluorescent protein-tagged wild-type STIM1 (WT-STIM1) and STIM1 mutants lacking the Ca(2+)-sensing EF-hand (STIM1-D76A), or lacking the cytoplasmic membrane binding site (STIM1ΔK). STIM1 aggregation was analyzed by monitoring puncta size during the SR Ca(2+) depletion induced by cyclopiazonic acid (CPA). We found that puncta size was increased in cells expressing WT-STIM1 after CPA. However, STIM1-D76A constitutively formed puncta, whereas STIM1ΔK failed to form puncta. Furthermore, cytokines increased basal WT-STIM1 puncta size, and the SOCE triggered by SR Ca(2+) depletion was increased in cells expressing WT-STIM1 or STIM1-D76A. Meanwhile, SOCE in cells expressing STIM1ΔK and STIM1 short, interfering RNA (siRNA) was decreased. Similarly, in cells overexpressing STIM1, the siRNA knockdown of Orai1 blunted SOCE. However, exposure to cytokines increased SOCE in all cells, increased basal [Ca(2+)]i, and decreased SR Ca(2+) content. These data suggest that cytokines induce a constitutive increase in STIM1 aggregation that contributes to enhanced SOCE in human ASM after inflammation. Such effects of inflammation on STIM1 aggregations may contribute to airway hyperresponsiveness.

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.

Overexpression of Orail Contributes to Abnormal Cell Proliferation in Esophageal Squamous Carcinoma Cancer

Store-operated Ca2+ entry (SOCE), an important Ca2+ signaling pathway, has been shown to involve into a diversity of cellular processes, including cell proliferation, apoptosis, tumor migration and metastasis. While Orai1 was recently identified as the long-sought channel pore-forming unit in SOCE pathway, the pathophysiological roles of Orai1 in various cancers have remained elusive.Here we report that Orai1 was strongly expressed in tumors compared with that in adjacent normal tissues from patients suffering from esophageal squamous cell carcinoma (ESCC), a major form of esophageal cancer with poor prognosis. The cell lines derived from ESCC patients, such as KYSE-150, KYSE-510, KYSE-190, presented upregulated Orai1 at both mRNA and protein levels than that in HET-1A, a non-tumorous esophageal epithelial cell line. Using fluorescence based Mn quenching assay, we examined thapsigargin induced, 2-APB (a SOCE channel blocker) sensitive SOCE in these cells. The activity of SOCE was greatly enhanced in all tested ESCC cell lines than in HET-1A cells. Interestingly, knocking-down of Orai1 by shRNA specifically targeting at the gene not only significantly reduced SOCE in KYSE cells but also inhibited the rampant proliferation of the cancer cells, e.g. the doubling time of KYSE150 was changed from 22.3h to 54.7h. Next, we evaluated the role of Orai1 on tumor growth in xenografted nude mice model. Up to 4 weeks, the sizes of tumors were much smaller or even undetectable in mice injected with KYSE-150 cells containing anti-orai1 shRNA than mice with scramble shRNA, indicating that reduction of Orai1 could inhibit tumor growth in vivo.In brief, out data suggest that overexpression of Orai1 contributes to abnormal proliferation in esophageal cancer cells and inhibition of SOCE may become a novel prevention strategy and a therapeutic intervention for esophageal cancer.

γ-Secretase activity modulates store-operated Ca 2+ entry into rat sensory neurons

Presenilin-1 is required for γ-secretase activity, which participates in Notch receptor processing, the pathogenesis of Alzheimer's disease and the modulation of Ca 2+ signaling. We tested the hypothesis that γ-secretase proteolytic activity modulates store-operated Ca 2+ entry (SOCE) in rat dorsal root ganglion (DRG) neurons. Depletion of intracellular Ca 2+ stores by blocking the endoplasmic reticulum (ER) Ca 2+ pump with cyclopiazonic acid (CPA) evoked a transient increase in [Ca 2+] i but no sustained Ca 2+ influx. However, in cells expressing a dominant negative presenilin-1 mutant (PS1-D257A), γ-secretase activity was inhibited and treatment with CPA evoked sustained Ca 2+ influx. Similarly, pharmacologic inhibition of γ-secretase with DAPT for 48 h enhanced SOCE. SKF96365, an inhibitor of store-operated channels, blocked SOCE in cells expressing PS1-D257A. Thus, γ-secretase proteolytic activity regulates a SOCE pathway in sensory neurons.