Orai1-mediated Store-operated Ca2+ Entry Research Articles

STIM2 variants regulate Orai1/TRPC1/TRPC4-mediated store-operated Ca2+ entry and mitochondrial Ca2+ homeostasis in cardiomyocytes

The stromal interaction molecules (STIMs) are the sarcoplasmic reticulum (SR) Ca2+ sensors that trigger store-operated Ca2+ entry (SOCE) in a variety of cell types. While STIM1 isoform has been the focus of the research in cardiac pathophysiology, the function of the homolog STIM2 remains unknown. Using Ca2+ imaging and patch-clamp techniques, we showed that knockdown (KD) of STIM2 by siRNAs increased SOCE and the ISOC current in neonatal rat ventricular cardiomyocytes (NRVMs). Within this cardiomyocyte model, we identified the transcript expression of Stim2.1 and Stim2.2 splice variants, with predominance for Stim2.2. Using conventional and super-resolution confocal microscopy (STED), we found that exogenous STIM2.1 and STIM2.2 formed pre-clusters with a reticular organization at rest. Following SR Ca2+ store depletion, some STIM2.1 and STIM2.2 clusters were translocated to SR-plasma membrane (PM) junctions and co-localized with Orai1. The overexpression strategy revealed that STIM2.1 suppressed Orai1-mediated SOCE and the ISOC current while STIM2.2 enhanced SOCE. STIM2.2-enhanced SOCE was also dependent on TRPC1 and TRPC4. Even if STIM2 KD or splice variants overexpression did not affect cytosolic Ca2+ cycling, we observed, using Rhod-2/AM Ca2+ imaging, that Orai1 inhibition or STIM2.1 overexpression abolished the mitochondrial Ca2+ (mCa2+) uptake, as opposed to STIM2 KD. We also found that STIM2 was present in the mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) by interacting with the inositol trisphosphate receptors (IP3Rs), voltage-dependent anion channel (VDAC), mitochondrial Ca2+ uniporter (MCU), and mitofusin-2 (MNF2). Our results suggested that, in NRVMs, STIM2.1 constitutes the predominant functional variant that negatively regulates Orai1-generated SOCE. It participates in the control of mCa2+ uptake capacity possibly via the STIM2-IP3Rs-VDAC-MCU and MNF2 complex.

Parathyroid Hormone Promotes Human Umbilical Vein Endothelial Cell Migration and Proliferation Through Orai1-Mediated Calcium Signaling.

Parathyroid hormone is the main endocrine regulator of extracellular calcium and phosphorus levels. Secondary hyperparathyroidism–induced endothelial dysfunction may be related to calcium homeostasis disorders. Here, we investigated the effects of parathyroid hormone on human umbilical vein endothelial cells (HUVECs) and characterized the involvement of store-operated Ca2+ entry (SOCE) and the nuclear factor of activated T cells (NFAT) signaling pathway. We used immunoblot experiments to find that parathyroid hormone significantly enhanced the expression of the Orai1 channel, a type of channel mediating SOCE, SOCE activity, and Orai1-mediated proliferation of HUVECs but did not increase Orai2 and Orai3. RNA-seq was utilized to identify 1,655 differentially expressed genes (823 upregulated and 832 downregulated) in parathyroid hormone–treated HUVECs as well as enhanced focal adhesion signaling and expression levels of two key genes, namely, COL1A1 and NFATC1. Increased protein and mRNA expression levels of COL1A1 and NFATC1 were confirmed by immunoblotting and quantitative RT-PCR, respectively. Cytosol and nuclei fractionation experiments and immunofluorescence methods were used to show that parathyroid hormone treatment increased NFATC1 nuclear translocation, which was inhibited by a calcineurin inhibitor (CsA), a selective calmodulin antagonist (W7), an Orai channel inhibitor (BTP2), or Orai1 small interfering RNA (siRNA) transfection. Parathyroid hormone also increased COL1A1 expression, cell migration, and proliferation of HUVECs. The PTH-induced increase in HUVEC migration and proliferation were inhibited by CsA, W7, BTP2, or COL1A1 siRNA transfection. These findings indicated that PTH increased Orai1 expression and Orai1-mediated SOCE, causing the nuclear translocation of NFATC1 to increase COL1A1 expression and COL1A1-mediated HUVEC migration and proliferation. These results suggest potential key therapeutic targets of Orai1 and the downstream calmodulin/calcineurin/NFATC1/COL1A1 signaling pathway in parathyroid hormone–induced endothelial dysfunction and shed light on underlying mechanisms that may be altered to prevent or treat secondary hyperparathyroidism–associated cardiovascular disease.

STIM2 protein regulates Orai1-mediated store-operated Ca2+ entry in cardiomyocytes

Stromal interaction molecules, STIM1 and STIM2, are ER Ca 2+ sensors that initiate store-operated Ca 2+ entry (SOCE). It has been recently identified three STIM2 splice variants (STIM2.1, 2.2 and 2.3), STIM2.2 being SOCE activator whereas STIM2.1 will be SOCE inhibitor. The role of STIM1-mediated SOCE has been highlighted in cardiac pathobiology but the one of STIM 2 .1 and STIM2.2 is unknown. To assess whether and how STIM2.1 and STIM2.2 regulate SOCE in neonatal rat ventricular cardiomyocytes (NRVMs). To analyze the function of STIM2.1 and STIM2.2 variants, the NRVMs were transfected with STIM2.2 or STIM2.1 in combination or not with STIM1 or dn-Orai1 106A mutant plasmids or with a mixture of 4 STIM2 siRNAs. At rest, both native STIM2 and exogenous STIM2.2 proteins present a reticular organization. Live cell confocal imaging in STIM2.2 overexpressing cells showed that STIM2.2 is aggregated and translocated towards the sarcolemma following the SR Ca 2+ store depletion. With Ca 2+ imaging, we found that STIM2.2 overexpression significantly increases SOCE, similarly to STIM1 overexpression, but the STIM1 and STIM2.2 co-expression has no additional effect. Importantly, the STIM2.2-enhanced SOCE is suppressed by functional Orai1 inhibition, either by co-expressing STIM2.2 with the dn-Orai1 106A mutant or with the selective Orai1 inhibitor, JPIII. In addition, the JPIII-sensitive Isoc current is also higher in STIM2.2 overexpressing cells. Nonetheless, silencing of endogenous STIM2 also increases SOCE. By co-immunoprecipitation, we found that STIM2, STIM1 and Orai1 interact together suggesting that all these proteins form a complex and constitute functional SOCE machinery. We demonstrated that, in overexpressing system, STIM2.2 functions together with STIM1 as a positive regulator of Orai1-mediated SOCE and that, in endogenous system, the STIM2.1 variant may be the predominant variant that negatively regulates SOCE in NRVMs.

Soluble \u03b1Klotho downregulates Orai1-mediated store-operated Ca2+ entry via PI3K-dependent signaling

αKlotho is a type 1 transmembrane anti-aging protein. αKlotho-deficient mice have premature aging phenotypes and an imbalance of ion homeostasis including Ca2+ and phosphate. Soluble αKlotho is known to regulate multiple ion channels and growth factor-mediated phosphoinositide-3-kinase (PI3K) signaling. Store-operated Ca2+ entry (SOCE) mediated by pore-forming subunit Orai1 and ER Ca2+ sensor STIM1 is a ubiquitous Ca2+ influx mechanism and has been implicated in multiple diseases. However, it is currently unknown whether soluble αKlotho regulates Orai1-mediated SOCE via PI3K-dependent signaling. Among the Klotho family, αKlotho downregulates SOCE while βKlotho or γKlotho does not affect SOCE. Soluble αKlotho suppresses serum-stimulated SOCE and Ca2+ release-activated Ca2+ (CRAC) channel currents. Serum increases the cell-surface abundance of Orai1 via stimulating vesicular exocytosis of the channel. The serum-stimulated SOCE and cell-surface abundance of Orai1 are inhibited by the preincubation of αKlotho protein or PI3K inhibitors. Moreover, the inhibition of SOCE and cell-surface abundance of Orai1 by pretreatment of brefeldin A or tetanus toxin or PI3K inhibitors prevents further inhibition by αKlotho. Functionally, we further show that soluble αKlotho ameliorates serum-stimulated SOCE and cell migration in breast and lung cancer cells. These results demonstrate that soluble αKlotho downregulates SOCE by inhibiting PI3K-driven vesicular exocytosis of the Orai1 channel and contributes to the suppression of SOCE-mediated tumor cell migration.

Oxidative stress facilitates cell death by inhibiting Orai1-mediated Ca2+ entry in brain capillary endothelial cells

Brain capillary endothelial cells (BCECs) form the blood-brain barrier (BBB) and play an essential role in the regulation of its functions. Oxidative stress accumulates excessive reactive oxygen species (ROS) and facilitates the death of BCECs, leading to a dysfunctional BBB. However, the mechanisms underlying the death of BCECs under oxidative stress remain unclear. In the present study, the effects of oxidative stress on cell viability, ROS production, intracellular Ca2+ concentration, and protein expression were examined using a cell line derived from bovine BCECs, t-BBEC117. When t-BBEC117 cells were exposed to oxidative stress induced by hydrogen peroxide (H2O2, 10–100 μM), cell growth was inhibited in a dose-dependent manner. Oxidative stress by 30 μM H2O2 increased the production of ROS and its effects were blocked by the ROS scavenger, 10 mM N-acetyl-l-cysteine (NAC). In addition, oxidative stress reduced store-operated Ca2+ entry (SOCE) and this decrease was recovered by NAC or the Orai channel activator, 5 μM 2-aminoethyl diphenylborinate (2-APB). The siRNA knockdown of Orai1 revealed that Orai1 was mainly responsible for SOCE channels and its activity was decreased by oxidative stress. However, the protein expression of Orai1 and STIM1 was not affected by oxidative stress. Oxidative stress-induced cell death was rescued by 2-APB, NAC, or the STIM-Orai activating region. In conclusion, oxidative stress reduces Orai1-mediated SOCE and, thus, facilitates the death of BCECs.

Targeting Orai1-mediated store-operated calcium entry by RP4010 for anti-tumor activity in esophagus squamous cell carcinoma

Esophageal cancer (EC) is the 6th leading cause of cancer mortality worldwide with poor prognosis, hence more effective chemotherapeutic drugs for this deadly disease are urgently needed. We previously reported that high expression of Orai1, a store-operated Ca2+entry (SOCE) channel, was associated with poor survival rate in EC patients and Orai1-mediated intracellular Ca2+ oscillations regulated cancer cell proliferation. Previous studies suggested that Orai1-mediated SOCE is a promising target for EC chemotherapy. Here, we evaluated the anti-cancer effect of a novel SOCE inhibitor, RP4010, in cultured EC cells and xenograft models. Compared to other previously reported SOCE channel inhibitors, RP4010 is more potent in blocking SOCE and inhibiting cell proliferation in EC and other cancer cells. Treatment with RP4010 resulted in reduction of intracellular Ca2+ oscillations, caused cell cycle arrest at G0/G1 phase in vitro, decreased nuclear translocation of nuclear factor kappa B (NF-κB) in vivo and in vitro, and inhibited tumor growth in vivo. Taken together, data demonstrated the therapeutic potential of RP4010 in EC patients via inhibition of SOCE-mediated intracellular Ca2+ signaling.

Reduced membrane cholesterol after chronic hypoxia limits Orai1-mediated pulmonary endothelial Ca2+ entry.

Endothelial dysfunction in chronic hypoxia (CH)-induced pulmonary hypertension is characterized by reduced store-operated Ca2+ entry (SOCE) and diminished Ca2+-dependent production of endothelium-derived vasodilators. We recently reported that SOCE in pulmonary arterial endothelial cells (PAECs) is tightly regulated by membrane cholesterol and that decreased membrane cholesterol is responsible for impaired SOCE after CH. However, the ion channels involved in cholesterol-sensitive SOCE are unknown. We hypothesized that cholesterol facilitates SOCE in PAECs through the interaction of Orai1 and stromal interaction molecule 1 (STIM1). The role of cholesterol in Orai1-mediated SOCE was initially assessed using CH exposure in rats (4 wk, 380 mmHg) as a physiological stimulus to decrease PAEC cholesterol. The effects of Orai1 inhibition with AnCoA4 on SOCE were examined in isolated PAEC sheets from control and CH rats after cholesterol supplementation, substitution of endogenous cholesterol with epicholesterol (Epichol), or vehicle treatment. Whereas cholesterol restored endothelial SOCE in CH rats, both Epichol and AnCoA4 attenuated SOCE only in normoxic controls. The Orai1 inhibitor had no further effect in cells pretreated with Epichol. Using cultured pulmonary endothelial cells to allow better mechanistic analysis of the molecular components of cholesterol-regulated SOCE, we found that Epichol, AnCoA4, and Orai1 siRNA each inhibited SOCE compared with their respective controls. Epichol had no additional effect after knockdown of Orai1. Furthermore, Epichol substitution significantly reduced STIM1-Orai1 interactions as assessed by a proximity ligation assay. We conclude that membrane cholesterol is required for the STIM1-Orai1 interaction necessary to elicit endothelial SOCE. Furthermore, reduced PAEC membrane cholesterol after CH limits Orai1-mediated SOCE. NEW & NOTEWORTHY This research demonstrates a novel contribution of cholesterol to regulate the interaction of Orai1 and stromal interaction molecule 1 required for pulmonary endothelial store-operated Ca2+ entry. The results provide a mechanistic basis for impaired pulmonary endothelial Ca2+ influx after chronic hypoxia that may contribute to pulmonary hypertension.

Selective inhibitory effects of zinc on cell proliferation in esophageal squamous cell carcinoma through Orai1.

Zinc, an essential micronutrient, has a cancer preventive role. Zinc deficiency has been shown to contribute to the progression of esophageal cancer. Orai1, a store-operated Ca2+ entry (SOCE) channel, was previously reported to be highly expressed in tumor tissues removed from patients with esophageal squamous cell carcinoma (ESCC) with poor prognosis, and elevation of its expression contributes to both hyperactive intracellular Ca2+ oscillations and fast cell proliferation in human ESCC cells. However, the molecular basis of cancer preventive functions of zinc and its association with Orai1-mediated cell proliferation remains unknown. The present study shows that zinc supplementation significantly inhibits proliferation of ESCC cell lines and that the effect of zinc is reversible with N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine, a specific Zn2+ chelator, whereas nontumorigenic esophageal epithelial cells are significantly less sensitive to zinc treatment. Fluorescence live cell imaging revealed that extracellular Zn2+ exerted rapid inhibitory effects on Orai1-mediated SOCE and on intracellular Ca2+ oscillations in the ESCC cells. Knockdown of Orai1 or expression of Orai1 mutants with compromised zinc binding significantly diminished sensitivity of the cancer cells to zinc treatment in both SOCE and cell proliferation analyses. These data suggest that zinc may inhibit cell proliferation of esophageal cancer cells through Orai1-mediated intracellular Ca2+ oscillations and reveal a possible molecular basis for zinc-induced cancer prevention and Orai1-SOCE signaling pathway in cancer cells.-Choi, S., Cui, C., Luo, Y., Kim, S.-H., Ko, J.-K., Huo, X., Ma, J., Fu, L.-W., Souza, R. F., Korichneva, I., Pan, Z. Selective inhibitory effects of zinc on cell proliferation in esophageal squamous cell carcinoma through Orai1.

Abstract 2612: Zinc inhibits Orai1-mediated Ca2+ signals and proliferation in esophageal cancer cells

Abstract Intracellular Ca2+ signals, including oscillations, regulate proliferation, migration, and other cellular events in cancer cells. Zinc (Zn), an essential micronutrient, has been studied for its chemopreventive effects in several cancers, including esophageal cancer. Although there is a growing research interest in the cross-talk between Ca2+ and Zn signaling, it remains elusive as to how Zn prevents tumor growth and whether Ca2+ signaling is involved. Our previous report demonstrated that Orai1, a store-operated Ca2+ entry (SOCE) channel, is highly expressed in esophageal squamous cell carcinoma (ESCC) compared to normal tissues, and that the elevated expression of Orai1 is strongly associated with poor prognosis in patients. In the current study, we show that physiological levels of Zn can significantly suppress cell proliferation in KYSE-150, a human ESCC cell line. We also show that Zn is able to inhibit Orai1-mediated SOCE and intracellular Ca2+ oscillations, both which are known as proliferation signals. Based on the topology information of Orai1, we hypothesized that the histidine residue in linker region between transmembrane 1 and 2 of Orai1 (H113) as well as three cysteine residues may play a critical role in Zn-inhibitory effects. Using a point mutation approach, we exhibit that the Zn-inhibitory effects on both SOCE and cell proliferation are vanished in KYSE-150 cells containing Orai1 H113A mutant. Furthermore, KYSE-150 cells expressing Orai1 with any mutation in cysteine residues (C126A, C143A and C195A) display significant loss of Zn-inhibitory functions. These results indicate that the four amino acid residues are likely to be involved in Zn-inhibitory effects on SOCE and cell proliferation. Taken together, our data suggest that dietary Zn may inhibit Orai1-mediated SOCE and intracellular Ca2+ signaling, which in turn suppresses cell proliferation in ESCC. Further studies are required to search for novel and effective prevention strategies as well as therapeutic options targeting on Zn and Ca2+ signaling in ESCC. Citation Format: Sangyong Choi, Chaochu Cui, Yanhong Luo, Jae-Kyun Ko, Jianjie Ma, Liwu Fu, Irina Korichneva, Zui Pan. Zinc inhibits Orai1-mediated Ca2+ signals and proliferation in esophageal cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2612.

Transient Receptor Potential Canonical (TRPC)/Orai1-dependent Store-operated Ca2+ Channels: NEW TARGETS OF ALDOSTERONE IN CARDIOMYOCYTES

Store-operated Ca(2+) entry (SOCE) has emerged as an important mechanism in cardiac pathology. However, the signals that up-regulate SOCE in the heart remain unexplored. Clinical trials have emphasized the beneficial role of mineralocorticoid receptor (MR) signaling blockade in heart failure and associated arrhythmias. Accumulated evidence suggests that the mineralocorticoid hormone aldosterone, through activation of its receptor, MR, might be a key regulator of Ca(2+) influx in cardiomyocytes. We thus assessed whether and how SOCE involving transient receptor potential canonical (TRPC) and Orai1 channels are regulated by aldosterone/MR in neonatal rat ventricular cardiomyocytes. Molecular screening using qRT-PCR and Western blotting demonstrated that aldosterone treatment for 24 h specifically increased the mRNA and/or protein levels of Orai1, TRPC1, -C4, -C5, and stromal interaction molecule 1 through MR activation. These effects were correlated with a specific enhancement of SOCE activities sensitive to store-operated channel inhibitors (SKF-96365 and BTP2) and to a potent Orai1 blocker (S66) and were prevented by TRPC1, -C4, and Orai1 dominant negative mutants or TRPC5 siRNA. A mechanistic approach showed that up-regulation of serum- and glucocorticoid-regulated kinase 1 mRNA expression by aldosterone is involved in enhanced SOCE. Functionally, 24-h aldosterone-enhanced SOCE is associated with increased diastolic [Ca(2+)]i, which is blunted by store-operated channel inhibitors. Our study provides the first evidence that aldosterone promotes TRPC1-, -C4-, -C5-, and Orai1-mediated SOCE in cardiomyocytes through an MR and serum- and glucocorticoid-regulated kinase 1 pathway.

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.

Store Operated Calcium Channels, New Targets of Aldosterone in Cardiomyocytes

Recently, the Store-Operated Ca2+ Entry (SOCE) has emerged as an important mechanism in cardiac pathology. However, the signals that upregulate SOCE in the heart remain unexplored. Clinical trials have emphasized the beneficial role of mineralocorticoid receptor (MR) signaling blockade in heart failure and associated arrhythmias. Accumulated evidences suggest that the mineralocorticoid hormone, aldosterone, through activation of its receptor (MR), might be a key regulator of Ca2+ influx in cardiomyocytes.We thus assessed whether and how SOCE involving TRPCs (Transient Receptor Potential Canonical) and Orai1 channels are regulated by aldosterone/MR in ventricular cardiomyocytes.Molecular screening using qRT-PCR and western-blot demonstrated that aldosterone treatment for 24 h specifically increased the mRNA and/or protein levels of Orai1, TRPC1, TRPC4, TRPC5 and STIM1 through MR activation. These effects were correlated with a specific enhancement of SOCE sensitive to SOC inhibitors (SKF-96365 and BTP2), to the potent Orai1 blocker, S66 and were prevented by TRPC1, TRPC4 and Orai1 dominant negative-mutants, or TRPC5 siRNA. Mechanistic approach showed that upregulation of Serum and Glucocorticoid-regulated Kinase 1 (SGK1) mRNA expression by aldosterone is involved in enhanced SOCE. Functionally, 24 h aldosterone-enhanced SOCE is associated with increased diastolic [Ca2+]i which is blunted by SOC inhibitors.Our study provides the first evidence that aldosterone promotes TRPC1, TRPC4, TRPC5 and Orai1-mediated SOCE in cardiomyocytes, through MR and SGK1 pathway.

Nitric Oxide-cGMP-PKG Pathway Acts on Orai1 to Inhibit the Hypertrophy of Human Embryonic Stem Cell-Derived Cardiomyocytes.

Cardiac hypertrophy is an abnormal enlargement of heart muscle. It frequently results in congestive heart failure, which is a leading cause of human death. Previous studies demonstrated that the nitric oxide (NO), cyclic GMP (cGMP), and protein kinase G (PKG) signaling pathway can inhibit cardiac hypertrophy and thus improve cardiac function. However, the underlying mechanisms are not fully understood. Here, based on the human embryonic stem cell-derived cardiomyocyte (hESC-CM) model system, we showed that Orai1, the pore-forming subunit of store-operated Ca(2+) entry (SOCE), is the downstream effector of PKG. Treatment of hESC-CMs with an α-adrenoceptor agonist phenylephrine (PE) caused a marked hypertrophy, which was accompanied by an upregulation of Orai1. Moreover, suppression of Orai1 expression/activity using Orai1-siRNAs or a dominant-negative construct Orai1(G98A) inhibited the hypertrophy, suggesting that Orai1-mediated SOCE is indispensable for the PE-induced hypertrophy of hESC-CMs. In addition, the hypertrophy was inhibited by NO and cGMP via activating PKG. Importantly, substitution of Ala for Ser(34) in Orai1 abolished the antihypertrophic effects of NO, cGMP, and PKG. Furthermore, PKG could directly phosphorylate Orai1 at Ser(34) and thus prevent Orai1-mediated SOCE. Together, we conclude that NO, cGMP, and PKG inhibit the hypertrophy of hESC-CMs via PKG-mediated phosphorylation on Orai1-Ser-34. These results provide novel mechanistic insights into the action of cGMP-PKG-related antihypertrophic agents, such as NO donors and sildenafil.

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‐induced store‐operated Ca2+ entry enhances phospholipase activity and modulates canonical transient receptor potential channel 6 function in murine platelets

Orai1, the major store-operated Ca(2+) entry (SOCE) channel in platelets, is not only critical for enhancing diverse signaling pathways, but may also regulate receptor-operated Ca(2+) entry (ROCE). Dynamic coupling of the Orai1 signalosome to canonical transient receptor potential channels (TRPCs) has been suggested as an essential step in the activation of SOCE and ROCE. However, the functional significance of the biochemical interaction between Orai and TRPC isoforms remains controversial. We aimed to elucidate the role of Orai1 in diacylglycerol (DAG)-mediated ROCE. Trpc6(-/-) , Orai1(-/-) and Orai1(-/-) /Trpc6(-/-) mice were generated, and their platelets were analyzed. Thapsigargin (TG)-induced SOCE was further reduced in Orai1(-/-) /Trpc6(-/-) platelets as compared with Orai1(-/-) platelets, thus revealing that TG-induced signaling pathways can activate TRPC6. Thapsigargin-induced SOCE leads to enhanced phospholipaseC and D activity in wild-type platelets. The activity of both enzymes was significantly reduced in Orai1(-/-) platelets upon TG stimulation, whereas receptor-induced phospholipase activity was not affected. Furthermore, TG-induced and glycoproteinVI-mediated thromboxaneA2 release was strongly dependent on Orai1-mediated SOCE. The regulation of TRPC6 activity can occur independently of the physical interaction with Orai1. TRPC6 operates in crosstalk with Orai1 through Orai1-induced DAG production via phospholipase activation. Orai1-induced DAG production and thromboxane release amplify the second phase of Ca(2+) signaling in platelets.

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.

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.

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.

Deletion of Orai1 alters expression of multiple genes during osteoclast and osteoblast maturation

Store-operated Ca2+ entry (SOCE) is a major Ca2+ influx pathway in most non-excitable cell types and Orai1 was recently identified as an essential pore-subunit of SOCE channels. Here we investigate the physiological role of Orai1 in bone homeostasis using Orai1-deficient mice (Orai1−/−). Orai1−/− mice developed osteopenia with decreased bone mineral density and trabecular bone volume. To identify the nature and origin of the bone defect, bone-resorbing osteoclasts and bone-forming osteoblasts from Orai1−/− mice were examined. Orai1-mediated SOCE was completely abolished in Orai1−/− osteoclast precursor cells and osteoclastogenesis in vitro from Orai1−/− mice was impaired due to a defect in cell fusion of pre-osteoclasts. Also, resorption activity in vitro was comparable but the size of pits formed by Orai1−/− osteoclasts was smaller. We next assessed the role of Orai1 in osteoblast differentiation and function by using a pre-osteoblast cell line, as well as primary osteoblasts from wild-type and Orai1−/− mice. SOCE in MC3T3-E1 pre-osteoblastic cells was inactivated by lentiviral overexpression of a pore-dead Orai1 mutant. Lack of SOCE in MC3T3-E1 had no effect on alkaline phosphatase staining and expression but substantially inhibited mineralized nodule formation. Consistent with this finding, Orai1-mediated SOCE was markedly reduced in Orai1−/− osteoblast precursor cells and osteoblastogenesis in vitro from Orai1−/− stromal cells showed impaired mineral deposition but no change in differentiation. This indicates that Orai1 is involved in the function but not in the differentiation of osteoblasts. Together, these results suggest that Orai1 plays a critical role in bone homeostasis by regulating both osteoblasts and osteoclasts.

Orai1 Mediates Store-Operated Ca2+ Entry in Normal Skeletal Muscle and Exacerbated Ca2+ Entry in Dystrophic Muscle

Duchenne muscular dystrophy (DMD) is the most common form of muscular dystrophy, in which loss of dystrophin expression results in compromised sarcolemmal integrity. Although evidence shows that defects in Ca2+ homeostasis is a causal factor for the progressive cell death observed in DMD, the mechanism of Ca2+ deregulation is still under debate. Several laboratories showed that enhanced Ca2+ entry might serve as a pathological factor in dystrophic muscles. In this study, we explored the role of store operated Ca2+ entry (SOCE) in Ca2+ deregulation of dystrophic muscles. We used real-time PCR and Western blotting to detect known isoforms of Orai and STIM1 and determined that Orai1 was the most abundant in skeletal muscle and was significantly upregulated in muscles from mdx mice, while STIM1 levels remained largely unchanged. Furthermore, Mn2+ quenching of fura-2 fluorescence was applied to measure SOCE activity in flexor digitorum brevis (FDB) fibers and a significant increase in SOCE activity was detected in mdx fibers. Similar levels of resting [Ca]i was identified in wt and mdx groups, while peak response to C/R was significantly higher in mdx fibers than wt. Furthermore, we electroporated shRNA probe against mouse Orai1 into FDB muscle of living mice to produce effective knockdown (KD) of Orai1 expression. Two weeks after Orai1 KD, SOCE activity was eliminated in both wt and mdx muscle fibers and peak response to caffeine and ryanodine in mdx fiber sreturned to a level comparable to wt muscle fiber. Therefore, our study established that Orai1 is an essential component of SOCE machinery in adult skeletal muscle and indicates that Orai1-mediated SOCE could be the major pathway for additional Ca2+ entry into mdx muscle fibers, which would eventually lead to progression of DMD.