Knockdown Of Orai1 Research Articles

Emergence of Orai3 activity during cardiac hypertrophy.

Stromal interaction molecule 1 (STIM1) has been shown to control a calcium (Ca(2+)) influx pathway that emerges during the hypertrophic remodelling of cardiomyocytes. Our aim was to determine the interaction of Orai1 and Orai3 with STIM1 and their role in the constitutive store-independent and the store-operated, STIM1-dependent, Ca(2+) influx in cardiomyocytes. We characterized the expression profile of Orai proteins and their interaction with STIM1 in both normal and hypertrophied adult rat ventricular cardiomyocytes. Orai1 and 3 protein levels were unaltered during the hypertrophic process and both proteins co-immunoprecipitated with STIM1. The level of STIM1 and Orai1 were significantly greater in the macromolecular complex precipitated by the Orai3 antibody in hypertrophied cardiomyocytes. We then used a non-viral method to deliver Cy3-tagged siRNAs in vivo to adult ventricular cardiomyocytes and silence Orai channel candidates. Cardiomyocytes were subsequently isolated then the voltage-independent, i.e. store-independent and store-operated Ca(2+) entries were measured on Fura-2 AM loaded Cy3-labelled and control isolated cardiomyocytes. The whole cell patch-clamp technique was used to measure Orai-mediated currents. Specific Orai1 and Orai3 knockdown established Orai3, but not Orai1, as the critical partner of STIM1 carrying these voltage-independent Ca(2+) entries in the adult hypertrophied cardiomyocytes. Orai3 also drove an arachidonic acid-activated inward current. Cardiac Orai3 is the essential partner of STIM1 and drives voltage-independent Ca(2+) entries in adult cardiomyocytes. Arachidonic acid-activated currents, which are supported by Orai3, are present in adult cardiomyocytes and increased during hypertrophy.

Native store-operated calcium channels are functionally expressed in mouse spinal cord dorsal horn neurons and regulate resting calcium homeostasis.

Store-operated calcium channels (SOCs) are calcium-selective cation channels that mediate calcium entry in many different cell types. Store-operated calcium entry (SOCE) is involved in various cellular functions. Increasing evidence suggests that impairment of SOCE is responsible for numerous disorders. A previous study demonstrated that YM-58483, a potent SOC inhibitor, strongly attenuates chronic pain by systemic or intrathecal injection and completely blocks the second phase of formalin-induced spontaneous nocifensive behaviour, suggesting a potential role of SOCs in central sensitization. However, the expression of SOCs, their molecular identity and function in spinal cord dorsal horn neurons remain elusive. Here, we demonstrate that SOCs are expressed in dorsal horn neurons. Depletion of calcium stores from the endoplasmic reticulum (ER) induced large sustained calcium entry, which was blocked by SOC inhibitors, but not by voltage-gated calcium channel blockers. Depletion of ER calcium stores activated inward calcium-selective currents, which was reduced by replacing Ca(2+) with Ba(2+) and reversed by SOC inhibitors. Using the small inhibitory RNA knockdown approach, we identified both STIM1 and STIM2 as important mediators of SOCE and SOC current, and Orai1 as a key component of the Ca(2+) release-activated Ca(2+) channels in dorsal horn neurons. Knockdown of STIM1, STIM2 or Orai1 decreased resting Ca(2+) levels. We also found that activation of neurokinin 1 receptors led to SOCE and activation of SOCs produced an excitatory action in dorsal horn neurons. Our findings reveal that a novel SOC signal is present in dorsal horn neurons and may play an important role in pain transmission.

Reversal of Murine Epidermal Atrophy by Topical Modulation of Calcium Signaling

Cytosolic Ca(2+) signals are performed by Ca(2+) releases from the endoplasmic reticulum and Ca(2+) influx from the extracellular medium. Releases rely on the refilling of the intracellular Ca(2+) stores by the Ca(2+) influx "Store-Operated Calcium Entry" (SOCE) via the channel Orai1. Here we show that Orai1 expression, SOCE amplitude, and epidermal proliferation are decreased in the epidermis of patients with skin fragility when compared with aged nonatrophic skin. Epidermal atrophy was induced in mice by the inhibition of Orai1 with small interfering RNA and the topical application of a SOCE blocker BTP2. The inhibition of Orai1 impaired the heparin-binding epidermal growth factor (HB-EGF)-induced Ca(2+) influxes and fully prevented the mitogen effect of HB-EGF in primary human keratinocytes. Importantly, epidermal proliferation correlated with Orai1 expression in mice. Conversely, the topical application of an Orai1 activator, the benzohydroquinone (BHQ), increased the epidermal thickness and proliferation, whereas the pro-proliferative effect of BHQ was prevented by the inhibition of Orai1. Finally, the topical application of BHQ reversed the epidermal atrophy induced by corticosteroids in mice. The topical modulation of Ca(2+) signals may thus be a promising therapeutic strategy in dermatology.

Orai1 and STIM1 are critical for cell migration and proliferation of clear cell renal cell carcinoma

The intracellular Ca2+ regulation has been implicated in tumorigenesis and tumor progression. Notably, store-operated Ca2+ entry (SOCE) is a major Ca2+ entry mechanism in non-excitable cells, being involved in cell proliferation and migration in several types of cancer. However, the expression and biological role of SOCE have not been investigated in clear cell renal cell carcinoma (ccRCC). Here, we demonstrate that Orai1 and STIM1, not Orai3, are crucial components of SOCE in the progression of ccRCC. The expression levels of Orai1 in tumor tissues were significantly higher than those in the adjacent normal parenchymal tissues. In addition, native SOCE was blunted by inhibiting SOCE or by silencing Orai1 and STIM1. Pharmacological blockade or knockdown of Orai1 or STIM1 also significantly inhibited RCC cell migration and proliferative capability. Taken together, Orai1 is highly expressed in ccRCC tissues illuminating that Orai1-mediated SOCE may play an important role in ccRCC development. Indeed, Orai1 and STIM1 constitute a native SOCE pathway in ccRCC by promoting cell proliferation and migration.

Orai1 and STIM1 mediate SOCE and contribute to apoptotic resistance of pancreatic adenocarcinoma

The store-operated calcium channels (SOCs) represent one of the major calcium-entry pathways in non-excitable cells. SOCs and in particular their major components ORAI1 and STIM1 have been shown to be implicated in a number of physiological and pathological processes such as apoptosis, proliferation and invasion. Here we demonstrate that ORAI1 and STIM1 mediate store-operated calcium entry (SOCE) in pancreatic adenocarcinoma cell lines. We show that both ORAI1 and STIM1 play pro-survival anti-apoptotic role in pancreatic adenocarcinoma cell lines, as siRNA-mediated knockdown of ORAI1 and/or STIM1 increases apoptosis induced by chemotherapy drugs 5-fluorouracil (5-FU) or gemcitabine. We also demonstrate that both 5-FU and gemcitabine treatments increase SOCE in Panc1 pancreatic adenocarcinoma cell line via upregulation of ORAI1 and STIM1. Altogether our results reveal the novel calcium-dependent mechanism of action of the chemotherapy drugs 5-FU and gemcitabine and emphasize the anti-apoptotic role of ORAI1 and STIM1 in pancreatic adenocarcinoma cells. This article is part of a Special Issue entitled: Calcium signaling in health and disease. Guest Editors: Geert Bultynck, Jacques Haiech, Claus W. Heizmann, Joachim Krebs, and Marc Moreau.

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 Ca2+ Entry Channels Regulate Cell Growth and Migration in Human Cardiac C-Kit+ Progenitor Cells

Background and objective: It is believed that cardiac c-kit+ progenitor cells maintain cardiac homeostasis and have the potential of multipotent differentiation and myocardial repair. However, cellular physiology is not well understood in cardiac c-kit+ progenitor cells. The present study investigated the functional store-operated Ca2+ entry (SOCE) channels and the potential role in regulating cell proliferation and migration in human cardiac c-kit+ progenitor cells.Methods: Multiple techniques were employed in the present study, including confocal scanning microscope, RT-PCR, Western blot analysis, co-immunoprecipitation, and cell proliferation and migration assays.Results: We found that Ca2+ influx through SOCE channels was inhibited by La3+ or 2-aminoethoxydiphenyl borate. Genes and proteins of the SOCE components TRPC1, STIM1 and Orai1 were abundant in human cardiac c-kit+ progenitor cells, and the protein-protein interaction between TRPC1, STIM1 and Orai1 was revealed by co-immunoprecipitation. Silence of TRPC1, STIM1, or Orai1 with the corresponding siRNA significantly reduced the Ca2+ influx through SOCE channels. Interestingly, knockdown of TRPC1, STIM1, or Orai1 decreased cell proliferation by reducing cyclin D1 and cyclin E, while inhibited cell migration via reducing p-Akt kinase.Conclusion: Our results demonstrate the novel information that TRPC1, STIM1 and Orai1 are the major components of SOCE channels in human cardiac c-kit+ cells. They regulate cell proliferation by increasing cyclin D1 and cyclin E, modulate cell migration by activating p-Akt.

Role of STIM1 in survival and neural differentiation of mouse embryonic stem cells independent of Orai1-mediated Ca2 + entry

Store-operated Ca2+ entry (SOCE) is an important Ca2+ influx pathway in non-excitable cells. STIM1, an ER Ca2+ sensor, and Orai1, a plasma membrane Ca2+ selective channel, are the two essential components of the Ca2+ release activated channel (CRAC) responsible for SOCE activity. Here we explored the role of STIM1 and Orai1 in neural differentiation of mouse embryonic stem (ES) cells. We found that STIM1 and Orai1 were expressed and functionally active in ES cells, and expressions of STIM1 and Orai1 were dynamically regulated during neural differentiation of mouse ES cells. STIM1 knockdown inhibited the differentiation of mouse ES cells into neural progenitors, neurons, and astrocytes. In addition, STIM1 knockdown caused severe cell death and markedly suppressed the proliferation of neural progenitors. Surprisingly, Orai1 knockdown had little effect on neural differentiation of mouse ES cells, but the neurons derived from Orai1 knockdown ES cells, like those from STIM1 knockdown cells, had defective SOCE. Taken together, our data indicate that STIM1 is involved in both early neural differentiation of ES cells and survival of early differentiated ES cells independent of Orai1-mediated SOCE.

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.

The Contribution of Orai(CRACM)1 and Orai(CRACM)2 Channels in Store-Operated Ca2+ Entry and Mediator Release in Human Lung Mast Cells

BackgroundThe influx of extracellular Ca2+ into mast cells is critical for the FcεR1-dependent release of preformed granule-derived mediators and newly synthesised autacoids and cytokines. The Orai(CRACM) ion channel family provide the major pathway through which this Ca2+ influx occurs. However the individual role of each of the three members of the Orai channel family in Ca2+ influx and mediator release has not been defined in human mast cells.ObjectiveTo assess whether there might be value in targeting individual Orai family members for the inhibition of FcεRI-dependent human lung mast cells (HLMC) mediator release.MethodsWe used an adenoviral delivery system to transduce HLMCs with shRNAs targeted against Orai1 and Orai2 or with cDNAs directing the expression of dominant-negative mutations of the three known Orai channels.ResultsshRNA-mediated knockdown of Orai1 resulted in a significant reduction of approximately 50% in Ca2+ influx and in the release of β-hexosaminidase (a marker of degranulation) and newly synthesized LTC4 in activated HLMCs. In contrast shRNA knockdown of Orai2 resulted in only marginal reductions of Ca2+ influx, degranulation and LTC4 release. Transduced dominant-negative mutants of Orai1, -2 and -3 markedly reduced Orai currents and completely inhibited HLMC degranulation suggesting that Orai channels form heteromultimers in HLMCs, and that Orai channels comprise the dominant Ca2+ influx pathway following FceRI-dependent HLMC activation. Inhibition of Orai currents did not alter HLMC survival. In addition we observed a significant down-regulation of the level of CRACM3 mRNA transcripts together with a small increase in the level of CRACM1 and CRACM2 transcripts following a period of sustained HLMC activation.Conclusion and Clinical RelevanceOrai1 plays an important role in Ca2+ influx and mediator release from HLMCs. Strategies which target Orai1 will effectively inhibit FcεRI-dependent HLMC activation, but spare off-target inhibition of Orai2 in other cells and body systems.

TRPC3 regulates release of brain-derived neurotrophic factor from human airway smooth muscle

Exogenous brain-derived neurotrophic factor (BDNF) enhances Ca2+ signaling and cell proliferation in human airway smooth muscle (ASM), especially with inflammation. Human ASM also expresses BDNF, raising the potential for autocrine/paracrine effects. The mechanisms by which ASM BDNF secretion occurs are not known. Transient receptor potential channels (TRPCs) regulate a variety of intracellular processes including store-operated Ca2+ entry (SOCE; including in ASM) and secretion of factors such as cytokines. In human ASM, we tested the hypothesis that TRPC3 regulates BDNF secretion. At baseline, intracellular BDNF was present, and BDNF secretion was detectable by enzyme linked immunosorbent assay (ELISA) of cell supernatants or by real-time fluorescence imaging of cells transfected with GFP–BDNF vector. Exposure to the pro-inflammatory cytokine tumor necrosis factor-alpha (TNFα) (20ng/ml, 48h) or a mixture of allergens (ovalbumin, house dust mite, Alternaria, and Aspergillus extracts) significantly enhanced BDNF secretion and increased TRPC3 expression. TRPC3 knockdown (siRNA or inhibitor Pyr3; 10μM) blunted BDNF secretion, and prevented inflammation effects. Chelation of extracellular Ca2+ (EGTA; 1mM) or intracellular Ca2+ (BAPTA; 5μM) significantly reduced secreted BDNF, as did the knockdown of SOCE proteins STIM1 and Orai1 or plasma membrane caveolin-1. Functionally, secreted BDNF had autocrine effects suggested by phosphorylation of high-affinity tropomyosin-related kinase TrkB receptor, prevented by chelating extracellular BDNF with chimeric TrkB-Fc. These data emphasize the role of TRPC3 and Ca2+ influx in the regulation of BDNF secretion by human ASM and the enhancing effects of inflammation. Given the BDNF effects on Ca2+ and cell proliferation, BDNF secretion may contribute to altered airway structure and function in diseases such as asthma.

SPCA2 regulates Orai1 trafficking and store independent Ca2+ entry in a model of lactation.

An unconventional interaction between SPCA2, an isoform of the Golgi secretory pathway Ca2+-ATPase, and the Ca2+ influx channel Orai1, has previously been shown to contribute to elevated Ca2+ influx in breast cancer derived cells. In order to investigate the physiological role of this interaction, we examined expression and localization of SPCA2 and Orai1 in mouse lactating mammary glands. We observed co-induction and co-immunoprecipitation of both proteins, and isoform-specific differences in the localization of SPCA1 and SPCA2. Three-dimensional cultures of normal mouse mammary epithelial cells were established using lactogenic hormones and basement membrane. The mammospheres displayed elevated Ca2+ influx by store independent mechanisms, consistent with upregulation of both SPCA2 and Orai1. Knockdown of either SPCA2 or Orai1 severely depleted Ca2+ influx and interfered with mammosphere differentiation. We show that SPCA2 is required for plasma membrane trafficking of Orai1 in mouse mammary epithelial cells and that this function can be replaced, at least in part, by a membrane-anchored C-terminal domain of SPCA2. These findings clearly show that SPCA2 and Orai1 function together to regulate Store-independent Ca2+ entry (SICE), which mediates the massive basolateral Ca2+ influx into mammary epithelia to support the large calcium transport requirements for milk secretion.

Inhibition of store-operated Ca2+ entry suppresses EGF-induced migration and eliminates extravasation from vasculature in nasopharyngeal carcinoma cell

Store-operated Ca2+ entry (SOCE) mediates Ca2+ responses evoked by extracellular signaling molecules to promote increases in cytosolic Ca2+, thereby triggering downstream signal transduction. Here we demonstrated that either the pharmacological blockage of Ca2+ influx through SOCE or the knockdown of Orai1, a key molecule of SOCE, suppressed the epidermal growth factor-induced migration by disturbing Ca2+ signaling in nasopharyngeal carcinoma (NPC) cell. Furthermore, Orai1 depletion led to a delayed cell attachment to the extracellular matrix surface in vitro and eliminated the extravasation of microinjected cells from vasculature in a zebrafish hematogenous metastasis model. Our findings thus indicate that SOCE acts as a predominant Ca2+ signaling involved in NPC cell metastasis, and may serve as a candidate target for anti-metastasis therapy in NPC.

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

STIM1 and Orai1 mediate CRAC channel activity and are essential for human glioblastoma invasion

The Ca(2+) sensor stromal interacting molecule 1 (STIM1) and the Ca(2+) channel Orai1 mediate the ubiquitous store-operated Ca(2+) entry (SOCE) pathway activated by depletion of internal Ca(2+) stores and mediated through the highly Ca(2+)-selective, Ca(2+) release-activated Ca(2+) (CRAC) current. Furthermore, STIM1 and Orai1, along with Orai3, encode store-independent Ca(2+) currents regulated by either arachidonate or its metabolite, leukotriene C4. Orai channels are emerging as important contributors to numerous cell functions, including proliferation, migration, differentiation, and apoptosis. Recent studies suggest critical involvement of STIM/Orai proteins in controlling the development of several cancers, including malignancies of the breast, prostate, and cervix. Here, we quantitatively compared the magnitude of SOCE and the expression levels of STIM1 and Orai1 in non-malignant human primary astrocytes (HPA) and in primary human cell lines established from surgical samples of the brain tumor glioblastoma multiforme (GBM). Using Ca(2+) imaging, patch-clamp electrophysiology, pharmacological reagents, and gene silencing, we established that in GBM cells, SOCE and CRAC are mediated by STIM1 and Orai1. We further found that GBM cells show upregulation of SOCE and increased Orai1 levels compared to HPA. The functional significance of SOCE was evaluated by studying the effects of STIM1 and Orai1 knockdown on cell proliferation and invasion. Utilizing Matrigel assays, we demonstrated that in GBM, but not in HPA, downregulation of STIM1 and Orai1 caused a dramatic decrease in cell invasion. In contrast, the effects of STIM1 and Orai1 knockdown on GBM cell proliferation were marginal. Overall, these results demonstrate that STIM1 and Orai1 encode SOCE and CRAC currents and control invasion of GBM cells. Our work further supports the potential use of channels contributed by Orai isoforms as therapeutic targets in cancer.

The plasma membrane channel ORAI1 mediates detrimental calcium influx caused by endogenous oxidative stress

The mouse hippocampal cell line HT22 is an excellent model for studying the consequences of endogenous oxidative stress. Addition of extracellular glutamate depletes the cells of glutathione (GSH) by blocking the glutamate−cystine antiporter system xc−. GSH is the main antioxidant in neurons and its depletion induces a well-defined program of cell death called oxytosis, which is probably synonymous with the iron-dependent form of non-apoptotic cell death termed ferroptosis. Oxytosis is characterized by an increase of reactive oxygen species and a strong calcium influx preceding cell death. We found a significant reduction in store-operated calcium entry (SOCE) in glutamate-resistant HT22 cells caused by downregulation of the Ca2+ channel ORAI1, but not the Ca2+ sensors STIM1 or STIM2. Pharmacological inhibition of SOCE mimicked this protection similarly to knockdown of ORAI1 by small interfering RNAs. Long-term calcium live-cell imaging after induction of the cell death program showed a specific reduction in Ca2+-positive cells by ORAI1 knockdown. These results suggest that dysregulated Ca2+ entry through ORAI1 mediates the detrimental Ca2+ entry in programmed cell death induced by GSH depletion. As this detrimental Ca2+ influx occurs late in the course of the cell death program, it might be amenable to therapeutic intervention in diseases caused by oxidative stress.

Orai1 Mediates Exacerbated Ca2+ Entry in Dystrophic Skeletal Muscle

There is substantial evidence indicating that disruption of Ca2+ homeostasis and activation of cytosolic proteases play a key role in the pathogenesis of Duchenne Muscular Dystrophy (DMD). However, the exact nature of the Ca2+ deregulation and Ca2+ signaling pathways that are altered in dystrophic muscles have not yet been resolved. Here we examined the contribution of the store-operated Ca2+ entry (SOCE) for the pathogenesis of DMD. RT-PCR and Western blot found that the expression level of Orai1, the pore-forming unit of SOCE, was significantly elevated in the dystrophic muscles, while parallel increases in SOCE activity and SR Ca2+ storage were detected in adult mdx muscles using Fura-2 fluorescence measurements. High-efficient shRNA probes against Orai1 were delivered into the flexor digitorum brevis muscle in live mice and knockdown of Orai1 eliminated the differences in SOCE activity and SR Ca2+ storage between the mdx and wild type muscle fibers. SOCE activity was repressed by intraperitoneal injection of BTP-2, an Orai1 inhibitor, and cytosolic calpain1 activity in single muscle fibers was measured by a membrane-permeable calpain substrate. We found that BTP-2 injection for 2 weeks significantly reduced the cytosolic calpain1 activity in mdx muscle fibers. Additionally, ultrastructural changes were observed by EM as an increase in the number of triad junctions was identified in dystrophic muscles. Compensatory changes in protein levels of SERCA1, TRP and NCX3 appeared in the mdx muscles, suggesting that comprehensive adaptations occur following altered Ca2+ homeostasis in mdx muscles. Our data indicates that upregulation of the Orai1-mediated SOCE pathway and an overloaded SR Ca2+ store contributes to the disrupted Ca2+ homeostasis in mdx muscles and is linked to elevated proteolytic activity, suggesting that targeting Orai1 activity may be a promising therapeutic approach for the prevention and treatment of muscular dystrophy.

Stromal Interaction Molecule 1 (STIM1) Is Involved in the Regulation of Mitochondrial Shape and Bioenergetics and Plays a Role in Oxidative Stress

Calcium ions are involved in a plethora of cellular functions including cell death and mitochondrial energy metabolism. Store-operated Ca(2+) entry over the plasma membrane is activated by depletion of intracellular Ca(2+) stores and is mediated by the sensor STIM1 and the channel ORAI1. We compared cell death susceptibility to oxidative stress in STIM1 knock-out and ORAI1 knockdown mouse embryonic fibroblasts and in knock-out cells with reconstituted wild type and dominant active STIM1. We show that STIM1 and ORAI1 deficiency renders cells more susceptible to oxidative stress, which can be rescued by STIM1 and ORAI1 overexpression. STIM1 knock-out mitochondria are tubular, have a higher Ca(2+) concentration, and are metabolically more active, resulting in constitutive oxidative stress causing increased nuclear translocation of the antioxidant transcription factor NRF2 triggered by increased phosphorylation of the translation initiation factor eIF2α and the protein kinase-like endoplasmic reticulum kinase PERK. This leads to increased transcription of antioxidant genes and a high basal glutathione in STIM1 knock-out cells, which is, however, more rapidly expended upon additional stress, resulting in increased release and nuclear translocation of apoptosis-inducing factor with subsequent cell death. Our data suggest that store-operated Ca(2+) entry and STIM1 are involved in the regulation of mitochondrial shape and bioenergetics and play a role in oxidative stress.

Orai1 Mediates Exacerbated Ca2+ Entry in Dystrophic Skeletal Muscle

There is substantial evidence indicating that disruption of Ca2+ homeostasis and activation of cytosolic proteases play a key role in the pathogenesis and progression of Duchenne Muscular Dystrophy (DMD). However, the exact nature of the Ca2+ deregulation and the Ca2+ signaling pathways that are altered in dystrophic muscles have not yet been resolved. Here we examined the contribution of the store-operated Ca2+ entry (SOCE) for the pathogenesis of DMD. RT-PCR and Western blot found that the expression level of Orai1, the pore-forming unit of SOCE, was significantly elevated in the dystrophic muscles, while parallel increases in SOCE activity and SR Ca2+ storage were detected in adult mdx muscles using Fura-2 fluorescence measurements. High-efficient shRNA probes against Orai1 were delivered into the flexor digitorum brevis muscle in live mice and knockdown of Orai1 eliminated the differences in SOCE activity and SR Ca2+ storage between the mdx and wild type muscle fibers. SOCE activity was repressed by intraperitoneal injection of BTP-2, an Orai1 inhibitor, and cytosolic calpain1 activity in single muscle fibers was measured by a membrane-permeable calpain substrate. We found that BTP-2 injection for 2 weeks significantly reduced the cytosolic calpain1 activity in mdx muscle fibers. Additionally, ultrastructural changes were observed by EM as an increase in the number of triad junctions was identified in dystrophic muscles. Compensatory changes in protein levels of SERCA1, TRP and NCX3 appeared in the mdx muscles, suggesting that comprehensive adaptations occur following altered Ca2+ homeostasis in mdx muscles. Our data indicates that upregulation of the Orai1-mediated SOCE pathway and an overloaded SR Ca2+ store contributes to the disrupted Ca2+ homeostasis in mdx muscles and is linked to elevated proteolytic activity, suggesting that targeting Orai1 activity may be a promising therapeutic approach for the prevention and treatment of muscular dystrophy.

Role of Molecular Determinants of Store-operated Ca2+ Entry (Orai1, Phospholipase A2 Group 6, and STIM1) in Focal Adhesion Formation and Cell Migration

Store-operated Ca(2+) entry is important for cell migration. This study presents characterization of localization and roles of Orai1, STIM1, and PLA2g6 in adhesion dynamics during cell migration. Orai1 and PLA2g6 are involved in adhesion formation at the front, whereas STIM1 participates in both adhesion formation and disassembly. Results uncovered new parameters of Orai1, STIM1, and PLA2g6 involvement in cell migration. Store-operated Ca(2+) entry and its major determinants are known to be important for cell migration, but the mechanism of their involvement in this complex process is unknown. This study presents a detailed characterization of distinct roles of Orai1, STIM1, and PLA2g6 in focal adhesion (FA) formation and migration. Using HEK293 cells, we discovered that although molecular knockdown of Orai1, STIM1, or PLA2g6 resulted in a similar reduction in migration velocity, there were profound differences in their effects on number, localization, and lifetime of FAs. Knockdown of STIM1 caused an increase in lifetime and number of FAs, their redistribution toward lamellae region, and an increase in cell tail length. In contrast, the number of FAs in Orai1- or PLA2g6-deficient cells was significantly reduced, and FAs accumulated closer to the leading edge. Assembly rate and Vinculin phosphorylation of FAs was similarly reduced in Orai1, PLA2g6, or STIM1-deficient cells. Although Orai1 and PLA2g6 accumulated and co-localized at the leading edge, STIM1 distribution was more complex. We found STIM1 protrusions in lamellipodia, which co-localized with FAs, whereas major accumulation could be seen in central and retracting parts of the cell. Interestingly, knockdown of Orai1 and PLA2g6 produced similar and non-additive effect on migration, whereas knockdown of STIM1 simultaneously with either Orai1 or PLA2g6 produced additional inhibition. Together these data suggest that although Orai1, PLA2g6, and STIM1 play major roles in formation of new FAs at the leading edge, STIM1 may also be involved in Orai1- and PLA2g6-independent disassembly of FAs in the back of cells.