Expression Of Orai1 Research Articles

Similarities and Differences between the Orai1 Variants: Orai1α and Orai1β

Orai1, the first identified member of the Orai protein family, is ubiquitously expressed in the animal kingdom. Orai1 was initially characterized as the channel responsible for the store-operated calcium entry (SOCE), a major mechanism that allows cytosolic calcium concentration increments upon receptor-mediated IP3 generation, which results in intracellular Ca2+ store depletion. Furthermore, current evidence supports that abnormal Orai1 expression or function underlies several disorders. Orai1 is, together with STIM1, the key element of SOCE, conducting the Ca2+ release-activated Ca2+ (CRAC) current and, in association with TRPC1, the store-operated Ca2+ (SOC) current. Additionally, Orai1 is involved in non-capacitative pathways, as the arachidonate-regulated or LTC4-regulated Ca2+ channel (ARC/LRC), store-independent Ca2+ influx activated by the secretory pathway Ca2+-ATPase (SPCA2) and the small conductance Ca2+-activated K+ channel 3 (SK3). Furthermore, Orai1 possesses two variants, Orai1α and Orai1β, the latter lacking 63 amino acids in the N-terminus as compared to the full-length Orai1α form, which confers distinct features to each variant. Here, we review the current knowledge about the differences between Orai1α and Orai1β, the implications of the Ca2+ signals triggered by each variant, and their downstream modulatory effect within the cell.

Functional Role of STIM-1 and Orai1 in Human Microvascular Aging.

The impact of aging on vascular function is heterogeneous depending on the vascular territories. Calcium regulation plays a key role in vascular function and has been implicated in aging-related hypercontractility of corpus cavernosum. We aimed to evaluate stromal interaction molecule (STIM)/Orai system involvement in aging-related vascular alterations in the human macro and microvasculature. Aortae specimens and mesenteric arteries (MA), obtained from 45 organ donors, were functionally evaluated in organ chambers and wire myographs. Subjects were divided into groups either younger or older than 65-years old. The expressions of STIM-1, Orai1, and Orai3 were determined by immunofluorescence in the aorta and MA, and by Western blot in the aorta homogenates. The inhibition of STIM/Orai with YM-58483 (20 μM) reversed adrenergic hypercontractility in MA from older subjects but did not modify aging-related hypercontractility in the aortic strips. Aging was related to an increased expression of Orai1 in human aorta, while Orai1 and STIM-1 were upregulated in MA. STIM-1 and Orai1 protein expressions were inversely correlated to endothelial function in MA. Circulating levels of Orai1 were correlated with the inflammatory factor TNF-α and with the endothelial dysfunction marker asymmetric dimethylarginine. Aging is associated with an increased expression of the STIM/Orai system in human vessels with functional relevance only in the microvascular territory, suggesting its role in aging-related microvascular dysfunction.

Abstract 9359: Orai1 Inhibitors as Potential Treatments for Pulmonary Arterial Hypertension

Rationale: Pulmonary arterial hypertension (PAH) is characterized by progressive distal pulmonary artery (PA) obstruction, leading to right ventricular hypertrophy and failure. Exacerbated intracellular calcium (Ca 2+ ) signaling contributes to abnormalities in PA smooth muscle cells (PASMCs), including aberrant proliferation, apoptosis resistance, exacerbated migration, and arterial contractility. Store-operated Ca 2+ entry (SOCE) is involved in Ca 2+ homeostasis in PASMCs, but its properties in PAH are unclear. Methods: Using a combination of Ca 2+ imaging, molecular biology, in vitro , ex vivo , and in vivo approaches, we investigated the roles of SOCE channel Orai1 in PA remodeling in PAH, and determined the consequences of pharmacological Orai1 inhibition in vivo using experimental models of pulmonary hypertension (PH). Results: Human PASMCs (hPASMCs) from patients with PAH (PAH-hPASMCs) showed upregulation of SOCE and Orai1 expression, in which ERK1/2, nuclear factor of activated T cells (NFAT), and nuclear factor-kappa B (NFκB) were contributing factors. Using siRNA and two Orai1 inhibitors, we found that Orai1 inhibition reduced SOCE, mitochondrial Ca 2+ entry, aberrant proliferation, apoptosis resistance, migration, and excessive calcineurin activity in PAH-hPASMCs. Orai1 inhibitors reduced agonist-evoked constriction in human PAs. In experimental rat models of PH evoked by chronic-hypoxia or monocrotaline or Sugen/hypoxia, administration of Orai1 inhibitors (BTP2, JPIII, or 5J4) protected against PH. Conclusions: In human PAH and experimental PH, Orai1 expression and activity are increased. Orai1 inhibition normalizes the PAH-hPASMC phenotype and attenuates PH in rat models, suggesting that Orai1 should be considered as a relevant therapeutic target for PAH.

Constitutive assembly of Ca2+ entry units in soleus muscle from calsequestrin knockout mice.

Calcium (Ca2+) entry units (CEUs) are junctions within the I band of the sarcomere between stacks of sarcoplasmic reticulum (SR) cisternae and extensions of the transverse (T)-tubule. CEUs contain STIM1 and Orai1 proteins, the molecular machinery of store-operated Ca2+ entry (SOCE). In extensor digitorum longus (EDL) fibers of wild-type (WT) mice, CEUs transiently assemble during acute exercise and disassemble several hours thereafter. By contrast, calsequestrin-1 (CASQ1) ablation induces a compensatory constitutive assembly of CEUs in EDL fibers, resulting in enhanced constitutive and maximum SOCE that counteracts SR Ca2+ depletion during repetitive activity. However, whether CEUs form in slow-twitch fibers, which express both the skeletal CASQ1 and the cardiac CASQ2 isoforms, is unknown. Herein, we compared the structure and function of soleus muscles from WT and knockout mice that lack either CASQ1 (CASQ1-null) or both CASQs (dCASQ-null). Ultrastructural analyses showed that SR/T-tubule junctions at the I band, virtually identical to CEUs in EDL muscle, were present and more frequent in CASQ1-null than WT mice, with dCASQ-null exhibiting the highest incidence. The greater incidence of CEUs in soleus from dCASQ-null mice correlated with increased specific force production during repetitive, high-frequency stimulation, which depended on Ca2+ entry. Consistent with this, Orai1 expression was significantly increased in soleus of CASQ1-null mice, but even more in dCASQ-null mice, compared with WT. Together, these results strengthen the concept that CEU assembly strongly depends on CASQ expression and provides an alternative source of Ca2+ needed to refill SR Ca2+ stores to maintain specific force production during sustained muscle activity.

Orai1-mediated store-operated Ca2+ entry promotes cervical cancer progression through IL-6 signaling.

Cervical cancer is a major cause of cancer-associated mortality among women in developing countries. Orai1-mediated store-operated Ca2+ entry (SOCE) is the primary mechanism underlying most of the non-excitable calcium influx into cells. There is at present limited evidence showing that Orai1 can function as an oncogene or a tumor suppressor depending on the cancer type. Furthermore, the exact biological functions of Orai1 in cervical cancer and the underlying mechanisms are still poorly understood. In this study, we found that Orai1 was upregulated in cervical cancer tissues, and promoted the growth of human cervical cancer cells both in vitro and in vivo. Gene silencing of Orai1 in cervical cancer cells significantly decreased interleukin (IL)-6 secretion. Interestingly, exogenous IL-6 abrogated the effects of Orai1 silencing and restored the clonogenicity of cervical cancer cells. Furthermore, we also observed a positive correlation between Orai1 and IL-6 expression in human cervical cancer samples. Taken together, our findings indicate that Orai1 functions as an oncogene in cervical cancer and is a promising therapeutic target.

STIM/Orai Inhibition as a Strategy for Alleviating Diabetic Erectile Dysfunction Through Modulation of Rat and Human Penile Tissue Contractility and in vivo Potentiation of Erectile Responses

Stromal interaction molecule (STIM)/Orai calcium entry system appears to have a role in erectile dysfunction (ED) pathophysiology but its specific contribution to diabetic ED was not elucidated. To evaluate STIM/Orai inhibition on functional alterations associated with diabetic ED in rat and human penile tissues and on in vivo erectile responses in diabetic rats. Rat corpus cavernosum (RCC) strips from nondiabetic (No DM) and streptozotocin-induced diabetic (DM) rats and human penile resistance arteries (HPRA) and corpus cavernosum (HCC) from ED patients undergoing penile prosthesis insertion were functionally evaluated in organ chambers and wire myographs. Erectile function in vivo in rats was assessed by intracavernosal pressure (ICP) responses to cavernous nerve electrical stimulation (CNES). Expression of STIM/Orai elements in HCC was determined by immunofluorescence and immunoblot. Functional responses in RCC, HCC and HPRA and STIM/Orai protein expression in HCC. In vivo erectile responses to CNES. Inhibition of Orai channels with YM-58483 (20 µM) significantly reduced adrenergic contractions in RCC but more effectively in DM. Thromboxane-induced and neurogenic contractions were reduced by STIM/Orai inhibition while defective endothelial, neurogenic and PDE5 inhibitor-induced relaxations were enhanced by YM-58483 (10 µM) in RCC from DM rats. In vivo, YM-58483 caused erections and attenuated diabetes-related impairment of erectile responses. YM-58483 potentiated the effects of PDE5 inhibition. In human tissues, STIM/Orai inhibition depressed adrenergic and thromboxane-induced contractions in ED patients more effectively in those with type 2 diabetes. Diabetes was associated with increased expression of Orai1 and Orai3 in ED patients. Targeting STIM/Orai to alleviate diabetes-related functional alterations of penile vascular tissue could improve erectile function and potentiate therapeutic effects of PDE5 inhibitors in diabetic ED. Improving effects of STIM/Orai inhibition on diabetes-related functional impairment was evidenced in vitro and in vivo in an animal model and validated in human tissues from ED patients. Functional findings were complemented with expression results. Main limitation was low numbers of human experiments due to limited human tissue availability. STIM/Orai inhibition alleviated alterations of functional responses in vitro and improved erectile responses in vivo in diabetic rats, potentiating the effects of PDE5 inhibition. STIM/Orai inhibition was validated as a target to modulate functional alterations of human penile vascular tissue in diabetic ED where Orai1 and Orai3 channels were upregulated. STIM/Orai inhibition could be a potential therapeutic strategy to overcome poor response to conventional ED therapy in diabetic patients. Sevilleja-Ortiz A, El Assar M, García-Gómez B, et al. STIM/Orai Inhibition as a Strategy for Alleviating Diabetic Erectile Dysfunction Through Modulation of Rat and Human Penile Tissue Contractility and in vivo Potentiation of Erectile Responses. J Sex Med 2022;19:1733-1749.

Orai1 Inhibitors as Potential Treatments for Pulmonary Arterial Hypertension.

Pulmonary arterial hypertension (PAH) is characterized by progressive distal pulmonary artery (PA) obstruction, leading to right ventricular hypertrophy and failure. Exacerbated intracellular calcium (Ca2+) signaling contributes to abnormalities in PA smooth muscle cells (PASMCs), including aberrant proliferation, apoptosis resistance, exacerbated migration, and arterial contractility. Store-operated Ca2+ entry is involved in Ca2+ homeostasis in PASMCs, but its properties in PAH are unclear. Using a combination of Ca2+ imaging, molecular biology, in vitro, ex vivo, and in vivo approaches, we investigated the roles of the Orai1 SOC channel in PA remodeling in PAH and determined the consequences of pharmacological Orai1 inhibition in vivo using experimental models of pulmonary hypertension (PH). Store-operated Ca2+ entry and Orai1 mRNA and protein were increased in human PASMCs (hPASMCs) from patients with PAH (PAH-hPASMCs). We found that MEK1/2 (mitogen-activated protein kinase kinase 1/2), NFAT (nuclear factor of activated T cells), and NFκB (nuclear factor-kappa B) contribute to the upregulation of Orai1 expression in PAH-hPASMCs. Using small interfering RNA (siRNA) and Orai1 inhibitors, we found that Orai1 inhibition reduced store-operated Ca2+ entry, mitochondrial Ca2+ uptake, aberrant proliferation, apoptosis resistance, migration, and excessive calcineurin activity in PAH-hPASMCs. Orai1 inhibitors reduced agonist-evoked constriction in human PAs. In experimental rat models of PH evoked by chronic hypoxia, monocrotaline, or Sugen/hypoxia, administration of Orai1 inhibitors (N-{4-[3,5-bis(Trifluoromethyl)-1H-pyrazol-1-yl]phenyl}-4-methyl-1,2,3-thiadiazole-5-carboxamide [BTP2], 4-(2,5-dimethoxyphenyl)-N-[(pyridin-4-yl)methyl]aniline [JPIII], or 5J4) protected against PH. In human PAH and experimental PH, Orai1 expression and activity are increased. Orai1 inhibition normalizes the PAH-hPASMCs phenotype and attenuates PH in rat models. These results suggest that Orai1 should be considered as a relevant therapeutic target for PAH.

IL-13 Regulates Orai1 Expression in Human Bronchial Smooth Muscle Cells and Airway Remodeling in Asthma Mice Model via LncRNA H19.

BackgroundIncreased proliferation and hypertrophy of airway smooth muscle cells (ASMCs) contribute substantially to airway remodeling in asthma. Interleukin (IL)-13 regulates ASMC proliferation by increasing Orai1 expression, the pore-forming subunit of store-operated Ca2+ entry (SOCE). The underlying mechanisms of this effect are not fully understood.MethodsBioinformatic analysis identified an interaction between microRNA 93–5p (miR-93-5p) and long non-coding RNA (lncRNA) H19, and between miR-93-5p and Orai1. RNA interference was used to investigate H19 knockdown on IL-13-induced proliferation and migration of in vitro cultured human bronchial smooth muscle cells (hBSMCs). Functional relevance of H19 in airway inflammation and airway remodeling was investigated in murine models of acute and chronic asthma.ResultsIL-13 concentration-dependently increased the expression of H19 and Orai1 and decreased the expression of miR-93-5p in hBSMCs. H19 knockdown partly reversed the effects of IL-13 on the expression of miR-93-5p and Orai1 and attenuated the proliferation and migration of hBSMCs promoted by IL-13. IL-13-promoted expression of Orai1 was attenuated by miR-93-5p mimic and increased by miR-93-5p inhibitor. IL-13-promoted proliferation of hBSMCs was increased by miR-93-5p inhibitor but not affected by miR-93-5p mimic, whereas IL-13-promoted migration of hBSMCs was increased by miR-93-5p inhibitor and attenuated by miR-93-5p mimic. The inhibiting effect of H19 knockdown on IL-13-induced Orai1 expression and the proliferation and migration of hBSMCs was counteracted by miR-93-5p inhibitor but only marginally or not impacted by miR-93-5p mimic. The expression of H19 and Orai1 was higher in the lungs of asthmatic mice than in control mice. In asthmatic mice, H19 siRNA reduced Orai1 expression, inflammatory cell infiltration, goblet cell hyperplasia, collagen deposition and smooth muscle mass in the lungs.ConclusionH19 may mediate the effects of IL-13 on Orai1 expression by inhibition of miR-93-5p in hBSMCs. H19 may be a therapeutic target for airway inflammation and airway remodeling.

Super resolution microscopy analysis reveals increased Orai1 activity in asthma and cystic fibrosis lungs

QuestionIn diseases such as asthma and cystic fibrosis (CF), the immune response is dysregulated and the lung is chronically inflamed. Orai1 activation is required for the initiation and persistence of inflammation. However, Orai1 expression in the lung is poorly understood. We therefore tested the hypothesis that Orai1 expression was upregulated in asthmatic and CF lungs. Materials and methodsWe used LungMAP to analyze single-cell RNAseq data of Orai1 and stromal interaction molecule 1 (STIM1) expression in normal human lungs. We then performed RNAscope analysis and immunostaining on lung sections from normal, asthma, and CF donors. We imaged sections by confocal and super resolution microscopy, and analyzed Orai1 and STIM1 expression in different pulmonary cell types. ResultsOrai1 was broadly-expressed, but expression was greatest in immune cells. At mRNA and protein levels, there were no consistent trends in expression levels between the three phenotypes. Orai1 must interact with STIM1 in order to activate and conduct Ca2+. We therefore used STIM1/Orai1 co-localization as a marker of Orai1 activity. Using this approach, we found significantly increased co-localization between these proteins in epithelia, interstitial and luminal immune cells, but not alveoli, from asthma and CF lungs. Orai1 also aggregates as part of its activation process. Using super resolution microscopy, we also found significantly increased Orai1 aggregation in immune cells from asthmatic and CF lungs. ConclusionWe found evidence that Orai1 was more active in asthma and CF than normal lungs. These data suggest that Orai1 is a relevant target for reducing pulmonary inflammation.

Defective bone repletion in aged Balb/cBy mice was caused by impaired osteoblastic differentiation.

This study was undertaken to gain mechanistic information about bone repair using the bone repletion model in aged Balb/cBy mice. onemonth-old (young) mice were fed a calcium-deficient diet for 2weeks and 8month-old (adult) and 21-25month-old (aged) female mice for 4weeks during depletion, which was followed by feeding a calcium-sufficient diet for 16days during repletion. To determine if prolonged repletion would improve bone repair, an additional group of aged mice were repleted for 4 additional weeks. Control mice were fed calcium-sufficient diet throughout. In vivo bone repletion response was assessed by bone mineral density gain and histomorphometry. In vitro response was monitored by osteoblastic proliferation, differentiation, and senescence. There was no significant bone repletion in aged mice even with an extended repletion period, indicating an impaired bone repletion. This was not due to an increase in bone cell senescence or reduction in osteoblast proliferation, but to dysfunctional osteoblastic differentiation in aged bone cells. Osteoblasts of aged mice had elevated levels of cytosolic and ER calcium, which were associated with increased Cav1.2 and CaSR (extracellular calcium channels) expression but reduced expression of Orai1 and Stim1, key components of Stored Operated Ca2+ Entry (SOCE). Activation of Cav1.2 and CaSR leads to increased osteoblastic proliferation, but activation of SOCE is associated with osteoblastic differentiation. The bone repletion mechanism in aged Balb/cBy mice is defective that is caused by an impaired osteoblast differentiation through reducedactivationof SOCE.

Silencing of the Ca2+ Channel ORAI1 Improves the Multi-Systemic Phenotype of Tubular Aggregate Myopathy (TAM) and Stormorken Syndrome (STRMK) in Mice.

Tubular aggregate myopathy (TAM) and Stormorken syndrome (STRMK) form a clinical continuum associating progressive muscle weakness with additional multi-systemic anomalies of the bones, skin, spleen, and platelets. TAM/STRMK arises from excessive extracellular Ca2+ entry due to gain-of-function mutations in the Ca2+ sensor STIM1 or the Ca2+ channel ORAI1. Currently, no treatment is available. Here we assessed the therapeutic potential of ORAI1 downregulation to anticipate and reverse disease development in a faithful mouse model carrying the most common TAM/STRMK mutation and recapitulating the main signs of the human disorder. To this aim, we crossed Stim1R304W/+ mice with Orai1+/− mice expressing 50% of ORAI1. Systematic phenotyping of the offspring revealed that the Stim1R304W/+Orai1+/− mice were born with a normalized ratio and showed improved postnatal growth, bone architecture, and partly ameliorated muscle function and structure compared with their Stim1R304W/+ littermates. We also produced AAV particles containing Orai1-specific shRNAs, and intramuscular injections of Stim1R304W/+ mice improved the skeletal muscle contraction and relaxation properties, while muscle histology remained unchanged. Altogether, we provide the proof-of-concept that Orai1 silencing partially prevents the development of the multi-systemic TAM/STRMK phenotype in mice, and we also established an approach to target Orai1 expression in postnatal tissues.

Activation of STIM1/Orai1‑mediated SOCE in sepsis‑induced myocardial depression

Unbalanced Ca2+ homeostasis serves an essential role in the occurrence and development of septic myocardial injury. However, the mechanism of Ca2+ homeostasis in septic myocardial depression is poorly understood due to the complexity of Ca2+ transporters in excitable cells. It was therefore hypothesized that cardiac dysfunction, myocardial injury and cardiac apoptosis in septic myocardial depression are associated with elevated intracellular Ca2+ concentrations caused by stromal interaction molecule 1 (STIM1)/Orai calcium release‑activated calcium modulator 1 (Orai1)‑mediated store‑operated Ca2+ entry (SOCE). A septic myocardial depression model was established using the cecal ligation and puncture operation (CLP) in mice and was simulated in H9C2 cells via lipopolysaccharide (LPS) stimulation. Cardiac function, myocardial injury, cardiac apoptosis and the expression levels of Bax, Bcl‑2, STIM1 and Orai1 were quantified invivo at6, 12and24h. Changes in the intracellular Ca2+ concentration, SOCE and the distribution of STIM1 were assessed invitro within 6h. The morphological changes of heart tissue were observed by hematoxylin‑eosin staining. Myocardial cellular apoptosis was determined by TUNEL method. The expression of Bax, Bcl‑2, STIM1 and Orai1 were visualized by western blot. Cytosolic calcium concentration and SOCE were evaluated by confocal microscopy. The results demonstrated that cardiac contractile function was significantly reduced at6h and morphological changes in cardiac tissues, as well as the myocardial apoptosis rate, were markedly increased at6, 12and24h following CLP. mRNA and protein expression levels of Bax/Bcl‑2 were significantly enhanced at6and12h and glycosylation of Orai1 in the myocardium of septic mice was significantly increased at6h following CLP. The intracellular Ca2+ concentration, SOCE, was significantly increased at1‑2h and the clustering and distribution of STIM1 were markedly changed in H9C2 cells at1and2h. These findings suggested that myocardial dysfunction, cardiac injury and myocardial depression may be related to increased intracellular Ca2+ concentration resulting from STIM1/Orai1‑mediated SOCE, which may provide a potential method to alleviate septic myocardial depression.

Sex-dependent effect of aging on calcium signaling and expression of TRPM2 and CRAC channels in human neutrophils

The vulnerability of older adults to bacterial infections has been associated with age-related changes in neutrophils. We analyzed the consequences of aging on calcium (Ca2+) mobilization and TRPM2 and CRAC channels expression in human neutrophils. The percentages of granulocytes, mature neutrophils, and neutrophil precursors were equivalent between young and older adults. However, neutrophil chemotaxis towards IL-8, C5a, or fMLP was lower in older adults of both sexes. Interestingly, a stronger Ca2+ transient followed by an identical Ca2+ influx to IL-8 was observed in older adult females. In addition, the Ca2+ response to LPS was delayed and prolonged in neutrophils of older adult males. There was no significant difference in Ca2+ response to fMLP, C5a, or store-operated Ca2+ entry in the older adults. There were also no differences in the expression of CXCR2, CD88, FPLR1, and TLR4. Interestingly, TRPM2- and ORAI1-mRNA expression was lower in neutrophils of older adults, mainly in females. Both channels were detected intracellularly in the neutrophils. TRPM2 was in late endosomes in young adults and in lysosomes in older adult neutrophils. In summary, defective neutrophil chemotaxis in aging seemed not to stem from alterations in Ca2+ signals; nevertheless, the low TRPM2 and ORAI1 expression may affect other functions.

Propofol pretreatment alleviates mast cell degranulation by inhibiting SOC to protect the myocardium from ischemia-reperfusion injury.

Propofol (PPF) has a protective effect on myocardial ischemia-reperfusion (I/R) injury (MIRI). The purpose of this study was to investigate whether the myocardial protective effect of propofol is related to the inhibition of mast cell degranulation and explore the possible mechanisms involved. Our in vivo results showed that compared with the sham group, cardiac function, infarct size, histopathological damage, apoptosis, and markers of myocardial necrosis were significantly increased in the ischemia-reperfusion group, and propofol pretreatment alleviated these effects. In the coculture system, propofol-treated mast cells reduced their tryptase activity, resulting in cardiomyocyte protective effects, such as decreased apoptosis of cardiomyocytes and decreased expression of myocardial necrosis markers. Finally, experimental results in vitro revealed that thapsigargin (TG) can increase mast cell degranulation, tryptase release, calcium ion concentration, and the expression of STIM1 and Orai1 induced by H/R, but propofol pretreatment can partially reverse the above effects. These results suggested that the cardioprotective effect of propofol is achieved in part by inhibiting calcium influx through store-operated Ca2+ channels (SOCs) and thus alleviating mast cell degranulation.

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

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

Comprehensive Assessment of the STIMs and Orais Expression in Polycystic Ovary Syndrome.

BackgroundPolycystic ovary syndrome (PCOS) is a heterogeneous endocrine disease characterized by irregular menstrual, hyperandrogenism, and polycystic ovaries. The definitive mechanism of the disorder is not fully elucidated. Store-operated Ca2+ entry (SOCE) plays a role in glucose and lipid metabolism, inflammation, hormone secretion, and cell proliferation. STIMs and Orais are the main elements of SOCE. The potential role of SOCE in PCOS pathogenesis remains unclear.MethodsThe expression of STIMs and Orais in granulosa cells (GCs) derived from 83 patients with PCOS and 83 controls were analyzed, respectively, by using quantitative reverse transcription polymerase chain reaction. Binary regression analysis was used to identify the factors affecting PCOS after adjusted by body mass index and age. Pearson correlation analysis was used to determine the association between PCOS phenotypes and SOCE genes expression.ResultsSignificantly increased expression of STIM1, STIM2, Orai1, and Orai2 were observed in patients with PCOS compared with controls (P = 0.037, P = 0.004, P ≤ 0.001, and P = 0.013, respectively), whereas the expression of Orai3 was decreased (P = 0.003). In addition, the expression levels of STIMs and Orais were identified as the factors affecting PCOS (P < 0.05). The expressions of these genes were correlated with hormone level and antral follicle count (P < 0.05).ConclusionsFor the first time, our findings indicated that the elements of SOCE were differently expressed, where STIM1, STIM2, Orai1, and Orai2 significantly increased, whereas Orai3 decreased in PCOS GCs, which might be dominantly involved in dysfunction of ovarian GCs and hormonal changes in PCOS.

Involvement of ORAI1/SOCE in Human AML Cell Lines and Primary Cells According to ABCB1 Activity, LSC Compartment and Potential Resistance to Ara-C Exposure.

Acute myeloid leukemia (AML) is a hematological malignancy with a high risk of relapse. This issue is associated with the development of mechanisms leading to drug resistance that are not yet fully understood. In this context, we previously showed the clinical significance of the ATP binding cassette subfamily B-member 1 (ABCB1) in AML patients, namely its association with stemness markers and an overall worth prognosis. Calcium signaling dysregulations affect numerous cellular functions and are associated with the development of the hallmarks of cancer. However, in AML, calcium-dependent signaling pathways remain poorly investigated. With this study, we show the involvement of the ORAI1 calcium channel in store-operated calcium entry (SOCE), the main calcium entry pathway in non-excitable cells, in two representative human AML cell lines (KG1 and U937) and in primary cells isolated from patients. Moreover, our data suggest that in these models, SOCE varies according to the differentiation status, ABCB1 activity level and leukemic stem cell (LSC) proportion. Finally, we present evidence that ORAI1 expression and SOCE amplitude are modulated during the establishment of an apoptosis resistance phenotype elicited by the chemotherapeutic drug Ara-C. Our results therefore suggest ORAI1/SOCE as potential markers of AML progression and drug resistance apparition.

STIM1-Orai1 Interaction Exacerbates LPS-Induced Inflammation and Endoplasmic Reticulum Stress in Bovine Hepatocytes through Store-Operated Calcium Entry.

(1) Background: The basic mechanism of store-operated Ca2+ entry (SOCE) in bovine hepatocytes (BHEC) is related to the activation of STIM1 and Orai1. The effect of STIM1- and Orai1-dependent calcium ion signaling on the NF-κB signaling pathway is unclear. (2) Methods: In this study, the expression of STIM1 and Orai1 in BHEC was regulated. RT-qPCR, Western blotting, and an immunofluorescence antibody (IFA) assay were performed to elucidate the effect of inflammation and endoplasmic reticulum stress (ERS) in BHEC. (3) Results: First of all, in this study, RT-PCR and Western blotting were used to detect the levels of IκB, NF-κB, and inflammatory factors (IL-6, IL-8, and TNF-α) and the expression of genes and proteins related to ERS (PERK, IRE1, ATF6, GRP78, and CHOP), which reached peak levels simultaneously when BHEC were treated with 16 μg/mL LPS for 1 h. For STIM1, we overexpressed STIM1 in BHEC by using plasmid transfection technology. The results showed that after overexpression of STIM1, the gene and protein expression of STIM1 levels were significantly upregulated, and the expression of Orai1 on the cell membrane was also upregulated, which directly activated the SOCE channel and induced inflammation and ERS in BHEC. The overexpression group was then treated with LPS, and it was found that the overexpression of STIM1 could enhance LPS-induced BHEC inflammation and ERS in BHEC. For Orai1, BHEC were pretreated with 8 μg/mL of the specific inhibitor BTP2 for 6 h. It was found that BTP2 could inhibit the expression of mRNA in Orai1, significantly reduce the gene expression of STIM1, inhibit the activation of the NF-κB signaling pathway, and alleviate inflammation and ERS in BHEC under LPS stimulation. (4) Conclusions: In conclusion, STIM1/Orai1 can intervene and exacerbate LPS-induced inflammation and ERS in bovine hepatocytes through SOCE.

Orai2 channel regulates prostaglandin E2 production in TNFα/IL1α-stimulated astrocytes.

Astrocytes are glial cells that serve homeostatic functions in the central nervous system (CNS). Recent research, however, suggests that under pathological conditions, astrocytes are stimulated by various factors and actively participate in CNS inflammation. In the present study, we found that astrocytes upregulate various inflammatory factors including prostaglandin E2 (PGE2 ) by co-stimulation with tumor necrosis factor-alpha (TNFα) and interleukin-1alpha (IL1α). These TNFα/IL1α-stimulated astrocytes also showed increased Ca2+ release from the endoplasmic reticulum (ER) and increased expression of Orai2, a member of the store-operated calcium channel (SOCC) family. To reveal the role of Orai2, we used astrocytes in which Orai2 was knocked-down (KD) or knocked-out (KO). The expression of the prostaglandin E synthase Ptges and the production of PGE2 were higher in Orai2-KD astrocytes than in WT astrocytes when stimulated with TNFα and IL1α. Orai2-KO astrocytes also showed increased expression of Ptges and increased PGE2 production. The expression of Ptgs2, another PGE2 synthetic enzyme, was also upregulated in Orai2-KO astrocytes. Moreover, Orai2-KO astrocytes showed increased store-operated calcium entry (SOCE) and increased Orai1 expression. These results suggest that Orai2 is upregulated in TNFα/IL1α-stimulated astrocytes and reduces PGE2 production to some extent, modulating CNS inflammation. Our findings may aid in understanding how astrocytes are associated with inflammatory responses, and the identification of new targets that modulate astrocytic reactivity.

Orai1 downregulation causes proliferation reduction and cell cycle arrest via inactivation of the Ras-NF-\u03baB signaling pathway in osteoblasts

BackgroundThe purpose of this study was to determine the role of Orai1 in the regulation of the proliferation and cell cycle of osteoblasts.MethodsThe expression of Orai1 was inhibited by Orai1 small interfering RNA (siRNA) in MC3T3-E1 cells. Following Orai1 downregulation, cell proliferation and cell cycle were examined. Furthermore, the expression of cyclin D1, cyclin E, CDK4, and CDK6 was analyzed. The activity of the Ras-NF-κB signaling pathway was investigated to identify the role of Orai1 in the regulation of osteoblast proliferation.ResultsOrai1 was successfully downregulated in MC3T3-E1 cells by the Orai1 siRNA transfection (p < 0.05). We found that MC3T3-E1 cell proliferation was decreased, and the cell cycle was arrested by Orai1 downregulation (p < 0.05). Additionally, the expression of cyclin D1 was decreased by Orai1 downregulation (p < 0.05), as was the activity of the Ras-NF-κB signaling pathway (p < 0.05). Orai1 siRNA did not further reduce cell proliferation, the proportion of cells in the S phase, and cyclin D1 expression after chemical blockage of the Ras signaling pathway in MC3T3-E1 cells (p > 0.05).ConclusionsThe results reveal that Orai1 downregulation may reduce cyclin D1 expression by inactivating the Ras-NF-κB signaling pathway thus blocking osteoblast proliferation and cell cycle.