Immune Synapse Residency of Orai1 Alters Ca2+ Response of T Cells.

CRAC, which plays important role in Ca2+-dependent T-lymphocyte activation, is composed of the ER-resident STIM1 and the plasma membrane Orai1 pore-forming subunit. Both accumulate at the immunological synapse (IS) between a T cell and an antigen-presenting cell (APC). We hypothesized that adapter/interacting proteins regulate Orai1 residence in the IS. We could show that mGFP-tagged Orai1-Full channels expressed in Jurkat cells had a biphasic IS-accumulation kinetics peaked at 15 min. To understand the background of Orai1 IS-redistribution we knocked down STIM1 and SAP97 (adaptor protein with a short IS-residency (15 min) and ability to bind Orai1 N-terminus): the mGFP-Orai1-Full channels kept on accumulating in the IS up to the 60th minute in the STIM1- and SAP97-lacking Jurkat cells. Deletion of Orai1 N terminus (mGFP-Orai1-Δ72) resulted in the same time course as described for STIM1/SAP97 knock-down cells. Ca2+-imaging of IS-engaged T-cells revealed that of Orai1 residency modifies the Ca2+-response: cells expressing mGFP-Orai1-Δ72 construct or mGFP-Orai1-Full in SAP-97 knock-down cells showed higher number of Ca2+-oscillation up to the 90th minute after IS formation. Overall, these data suggest that SAP97 may contribute to the short-lived IS-residency of Orai1 and binding of STIM1 to Orai1 N-terminus is necessary for SAP97-Orai1 interaction.

Pyridostigmine improves cardiac function and rhythmicity through RyR2 stabilization and inhibition of STIM1-mediated calcium entry in heart failure.

Heart failure (HF) is characterized by asymmetrical autonomic balance. Treatments to restore parasympathetic activity in human heart failure trials have shown beneficial effects. However, mechanisms of parasympathetic-mediated improvement in cardiac function remain unclear. The present study examined the effects and underpinning mechanisms of chronic treatment with the cholinesterase inhibitor, pyridostigmine (PYR), in pressure overload HF induced by transverse aortic constriction (TAC) in mice. TAC mice exhibited characteristic adverse structural (left ventricular hypertrophy) and functional remodelling (reduced ejection fraction, altered myocyte calcium (Ca) handling, increased arrhythmogenesis) with enhanced predisposition to arrhythmogenic aberrant sarcoplasmic reticulum (SR) Ca release, cardiac ryanodine receptor (RyR2) hyper-phosphorylation and up-regulated store-operated Ca entry (SOCE). PYR treatment resulted in improved cardiac contractile performance and rhythmic activity relative to untreated TAC mice. Chronic PYR treatment inhibited altered intracellular Ca handling by alleviating aberrant Ca release and diminishing pathologically enhanced SOCE in TAC myocytes. At the molecular level, these PYR-induced changes in Ca handling were associated with reductions of pathologically enhanced phosphorylation of RyR2 serine-2814 and STIM1 expression in HF myocytes. These results suggest that chronic cholinergic augmentation alleviates HF via normalization of both canonical RyR2-mediated SR Ca release and non-canonical hypertrophic Ca signaling via STIM1-dependent SOCE.

Synthesis and Pharmacological Characterization of 2-Aminoethyl Diphenylborinate (2-APB) Derivatives for Inhibition of Store-Operated Calcium Entry (SOCE) in MDA-MB-231 Breast Cancer Cells.

Calcium ions regulate a wide array of physiological functions including cell differentiation, proliferation, muscle contraction, neurotransmission, and fertilization. The endoplasmic reticulum (ER) is the major intracellular Ca2+ store and cellular events that induce ER store depletion (e.g., activation of inositol 1,4,5-triphosphate (IP3) receptors) trigger a refilling process known as store-operated calcium entry (SOCE). It requires the intricate interaction between the Ca2+ sensing stromal interaction molecules (STIM) located in the ER membrane and the channel forming Orai proteins in the plasma membrane (PM). The resulting active STIM/Orai complexes form highly selective Ca2+ channels that facilitate a measurable Ca2+ influx into the cytosol followed by successive refilling of the ER by the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA). STIM and Orai have attracted significant therapeutic interest, as enhanced SOCE has been associated with several cancers, and mutations in STIM and Orai have been linked to immunodeficiency, autoimmune, and muscular diseases. 2-Aminoethyl diphenylborinate (2-APB) is a known modulator and depending on its concentration can inhibit or enhance SOCE. We have synthesized several novel derivatives of 2-APB, introducing halogen and other small substituents systematically on each position of one of the phenyl rings. Using a fluorometric imaging plate reader (FLIPR) Tetra-based calcium imaging assay we have studied how these structural changes of 2-APB affect the SOCE modulation activity at different compound concentrations in MDA-MB-231 breast cancer cells. We have discovered 2-APB derivatives that block SOCE at low concentrations, at which 2-APB usually enhances SOCE.

Melatonin induces mitochondrial apoptosis in osteoblasts by regulating the STIM1/cytosolic calcium elevation/ERK pathway.

AIMS: Idiopathic scoliosis is a common deformity of the spine that has an especially high incidence rate in adolescents. Some studies have demonstrated a close relationship between idiopathic scoliosis and melatonin deficiency. Our team's previous research showed that melatonin can inhibit the proliferation of osteoblasts, but the mechanism remains unclear. This study aimed to determine the mechanism by which melatonin inhibits the proliferation of osteoblasts. MAIN METHODS: Cell viability experiment, DNA fragment detection and alkaline phosphatase (ALP) activity assays were performed to determine the effects of melatonin on the proliferation, apoptosis and differentiation of osteoblasts. We used immunofluorescence to detect the expression of STIM1 in melatonin-treated osteoblasts. STIM1 interference was achieved using a specific siRNA, and a TRPC inhibitor was used to block the influx of Ca2+. The mRNA expression was determined by RT-qPCR, and protein levels were measured by Western blot. KEY FINDINGS: In this study, we found that melatonin inhibited the proliferation, differentiation and apoptosis of osteoblasts in a concentration-dependent manner. Additional studies showed that melatonin elevated cytosolic calcium levels by upregulation of STIM1, leading to osteoblast apoptosis via the mitochondrial pathway. Finally, we demonstrated that the STIM1-mediated increase in cytosolic calcium levels induced apoptosis through the ERK pathway. SIGNIFICANCE: Melatonin induces mitochondrial apoptosis in osteoblasts by regulating the STIM1/cytosolic calcium elevation/ERK pathway. These basic findings provide a basis for further clinical studies on melatonin as a drug therapeutic for idiopathic scoliosis.

Knockdown of STIM1 expression inhibits non-small-cell lung cancer cell proliferation in vitro and in nude mouse xenografts.

Stromal interaction molecule 1 (STIM1) is a calcium-sensing protein localized in the membrane of the endoplasmic reticulum. The expression of STIM1 has been shown to be closely associated with cell proliferation. The aim of the present study was to investigate the role of STIM1 in the regulation of cancer progression and its clinical relevance. The data demonstrated that the expression of the STIM1 was significantly higher in non-small-cell lung cancer (NSCLC) tissues than in benign lesions and was associated with advanced NSCLC T stage. Knockdown of STIM1 expression in NSCLC cell lines A549 and SK-MES-1 significantly inhibited cell proliferation and induces A549 and SK-MES-1 cell arrest at the G2/M and S phases of the cell cycle. Western blotting showed that the expression of cyclin-dependent kinase (CDK) 1 and CDK2 were reduced while knockdown of STIM1 expression. Furthermore, knockdown of STIM1 in NSCLC cells significantly reduced the levels of xenograft tumor growth in nude mice. These data indicate that aberrant expression of the STIM1 protein may contribute to NSCLC progression. Future studies should focus on targeting STIM1 as a novel strategy for NSCLC therapy.

Chronic dysfunction of Stromal interaction molecule by pulsed RNAi induction in fat tissue impairs organismal energy homeostasis in Drosophila.

Obesity is a progressive, chronic disease, which can be caused by long-term miscommunication between organs. It remains challenging to understand how chronic dysfunction in a particular tissue remotely impairs other organs to eventually imbalance organismal energy homeostasis. Here we introduce RNAi Pulse Induction (RiPI) mediated by short hairpin RNA (shRiPI) or double-stranded RNA (dsRiPI) to generate chronic, organ-specific gene knockdown in the adult Drosophila fat tissue. We show that organ-restricted RiPI targeting Stromal interaction molecule (Stim), an essential factor of store-operated calcium entry (SOCE), results in progressive fat accumulation in fly adipose tissue. Chronic SOCE-dependent adipose tissue dysfunction manifests in considerable changes of the fat cell transcriptome profile, and in resistance to the glucagon-like Adipokinetic hormone (Akh) signaling. Remotely, the adipose tissue dysfunction promotes hyperphagia likely via increased secretion of Akh from the neuroendocrine system. Collectively, our study presents a novel in vivo paradigm in the fly, which is widely applicable to model and functionally analyze inter-organ communication processes in chronic diseases.

STIM1 at the plasma membrane as a new target in progressive chronic lymphocytic leukemia.

BACKGROUND: Dysregulation in calcium (Ca2+) signaling is a hallmark of chronic lymphocytic leukemia (CLL). While the role of the B cell receptor (BCR) Ca2+ pathway has been associated with disease progression, the importance of the newly described constitutive Ca2+ entry (CE) pathway is less clear. In addition, we hypothesized that these differences reflect modifications of the CE pathway and Ca2+ actors such as Orai1, transient receptor potential canonical (TRPC) 1, and stromal interaction molecule 1 (STIM1), the latter being the focus of this study. METHODS: An extensive analysis of the Ca2+ entry (CE) pathway in CLL B cells was performed including constitutive Ca2+ entry, basal Ca2+ levels, and store operated Ca2+ entry (SOCE) activated following B cell receptor engagement or using Thapsigargin. The molecular characterization of the calcium channels Orai1 and TRPC1 and to their partner STIM1 was performed by flow cytometry and/or Western blotting. Specific siRNAs for Orai1, TRPC1 and STIM1 plus the Orai1 channel blocker Synta66 were used. CLL B cell viability was tested in the presence of an anti-STIM1 monoclonal antibody (mAb, clone GOK) coupled or not with an anti-CD20 mAb, rituximab. The Cox regression model was used to determine the optimal threshold and to stratify patients. RESULTS: Seeking to explore the CE pathway, we found in untreated CLL patients that an abnormal CE pathway was (i) highly associated with the disease outcome; (ii) positively correlated with basal Ca2+ concentrations; (iii) independent from the BCR-PLCγ2-InsP3R (SOCE) Ca2+ signaling pathway; (iv) supported by Orai1 and TRPC1 channels; (v) regulated by the pool of STIM1 located in the plasma membrane (STIM1PM); and (vi) blocked when using a mAb targeting STIM1PM. Next, we further established an association between an elevated expression of STIM1PM and clinical outcome. In addition, combining an anti-STIM1 mAb with rituximab significantly reduced in vitro CLL B cell viability within the high STIM1PM CLL subgroup. CONCLUSIONS: These data establish the critical role of a newly discovered BCR independent Ca2+ entry in CLL evolution, provide new insights into CLL pathophysiology, and support innovative therapeutic perspectives such as targeting STIM1 located at the plasma membrane.

FUN14 domain-containing 1 promotes breast cancer proliferation and migration by activating calcium-NFATC1-BMI1 axis.

BACKGROUND: FUN14 domain-containing 1 (FUNDC1), as a novel member of mitochondria-associated endoplasmic reticulum (ER) membranes associates with mitochondrial division and mitophagy. However, the expression profile and functional roles of FUNDC1 remain largely unclear in human cancer biology, including breast cancer (BC). METHODS: Immunohistochemistry and western blot analysis were used to determine the expression of FUNDC1 and BMI1 polycomb ring finger oncogene (BMI1). CCK8, cell counting and transwell assays were used to analyze cell proliferation, migration and invasion, respectively. Luciferase reporter and chromatin immunoprecipitation (ChIP) assays were used to detect the transcriptional regulation of Nuclear factor of activated T-cells, cytoplasmic 1 (NFATC1). The prognostic merit of NFATC1 expression was assessed by Kaplan-Meier assay. FINDINGS: Immunohistochemistry revealed strong immunostaining for FUNDC1 in cytoplasmic and nuclear membrane distribution in BC tissues as compared with normal breast epithelium. Kaplan-Meier survival analysis showed worse outcome for BC patients with high FUNDC1 expression. In vitro assay of gain- and loss-of-function of FUNDC1 suggested that FUNDC1 could stimulate BC cell proliferation, migration and invasion. Furthermore, elevated FUNDC1 level promoted Ca2+ cytosol influx from ER and extracellular, as well as NFATC1 nuclear translocation and activity. Nuclear NFATC1 bound to the BMI1 gene promoter and transcriptionally upregulated its expression. Notably, BMI1 overexpression could rescue the loss of function of FUNDC1. Co-expression of FUNDC1 and BMI1 in BC patients predicted worse prognosis than without either expression. INTERPRETATION: FUNDC1 might promote BC progression by activating the Ca2+-NFATC1-BMI1 axis. This pathway may be promising for developing multiple targets for BC therapy.

The deregulation of STIM1 and store operative calcium entry impaired aortic smooth muscle cells contractility in aortic medial degeneration.

Background: Microarray analysis of clinical aortic samples suggested a potential role for stromal interaction molecule 1 (STIM1) in the modulation of aortic medial degeneration (AMD), despite the uncertainty about STIM1 in normal aortic smooth muscle cells (ASMCs). Here, we aimed to explore changes in STIM1 expression in AMD, and the possible mechanisms. Methods: An AMD model was established using auto-delivery of angiotensin II (Ang II) into ApoE-/- mice. We assessed the effects of SKF96365, a STIM1 inhibitor, in AMD model and in vitro cultured ASMCs. Elastic van Gieson (EVG) staining was used to visualize elastic fiber injury. Mitochondria changes were viewed by TEM. Cytoplasmic calcium was quantified by measuring fluo-4 staining in a flow cytometer. Mechanical stretching device was used to mimic stretching that ASMCs experience in vivo Cell apoptosis was determined by using Annexin V/propidium iodide (PI) staining. The expression of STIM1, contractile related proteins (α-smooth muscle actin (α-SMA), myosin light chain (MLC)), endoplasmic reticulum (ER) stress-related proteins (CHOP, activating transcription factor 6 (ATF-6)) and smad2/3 were assessed by Western blotting, immunohistochemistry (IHC), and immunofluorescence (IF). Results: SKF96365 exacerbated aortic injury in the AMD model. SKF96365 reduced cytoplasmic calcium concentration in ASMCs, caused mitochondrial swelling, and elevated the expression of ATF-6 and CHOP. SKF96365 decreased the expression of MLC and α-SMA in ASMCs, causing them to be vulnerable to mechanical stretch. SKF96365 suppressed smad2/3 activation after treatment with transforming growth factor (TGF) ß1 (TGFß1). Conclusions: STIM1 is indispensable in ASMCs. Interfering with STIM1 exaggerated the AMD process by modulating the expression of contractile proteins, inducing ER stress in ASMCs.

Orai1 and Stim1 Mediate the Majority of Store-Operated Calcium Entry in Multiple Myeloma and Have Strong Implications for Adverse Prognosis.

BACKGROUND/AIMS: Multiple myeloma (MM) is a plasma cell neoplasm which constitutes about 10% of all hematologic malignancies. Despite the development and application of novel agents, MM still undergoes an aggressive and incurable course in the vast majority of patients. Ca2+ is one of the critical regulators of cell migration. Ca2+ influx is essential for the migration of various types of cells including tumor cells. However, the role of store-operated calcium entry (SOC) channels, the only Ca2+ channels of non-excitable cells, has not yet been reported in MM cell survival. METHODS: We evaluated the expression of Stim1 and Orai1 (two key regulators of SOC) in MM tissues and cell lines by immunohistochemical assay, quantitative real-time PCR assay and western blot. MM cell lines were pretreated with pharmacological blockers and siRNAs, and then MM cell proliferation, cell cycle arrest, and apoptosis were examined by FACS (flow cytometry) assay, and Annexin V-FITC/PI staining. The correlation between the expression of Stim1 (or Orai1) level and outcome in MM were assessed by using Progress Free Survival (PFS). RESULTS: Stim1 and Orai1 were both abundantly expressed in MM tissue and MM cell lines. Inhibition of SOCE reduced MM cell viability, and induced cell cycle arrest and apoptosis. Stim1 or Orai1 silencing also reduced cell viability, caused cell apoptosis and cell cycle arrest in MM cell lines. Over-expression of Stim1/Orai1 in MM patients was closely associated with the clinical outcome of MM. CONCLUSION: The Stim1/Orai1-mediated signaling participates in the pathogenesis of MM, which represents an attractive target for future therapeutic intervention.

Bradykinin-mediated Ca2+ signalling regulates cell growth and mobility in human cardiac c-Kit+ progenitor cells.

Our recent study showed that bradykinin increases cell cycling progression and migration of human cardiac c-Kit+ progenitor cells by activating pAkt and pERK1/2 signals. This study investigated whether bradykinin-mediated Ca2+ signalling participates in regulating cellular functions in cultured human cardiac c-Kit+ progenitor cells using laser scanning confocal microscopy and biochemical approaches. It was found that bradykinin increased cytosolic free Ca2+ ( Cai2+ ) by triggering a transient Ca2+ release from ER IP3Rs followed by sustained Ca2+ influx through store-operated Ca2+ entry (SOCE) channel. Blockade of B2 receptor with HOE140 or IP3Rs with araguspongin B or silencing IP3R3 with siRNA abolished both Ca2+ release and Ca2+ influx. It is interesting to note that the bradykinin-induced cell cycle progression and migration were not observed in cells with siRNA-silenced IP3R3 or the SOCE component TRPC1, Orai1 or STIM1. Also the bradykinin-induced increase in pAkt and pERK1/2 as well as cyclin D1 was reduced in these cells. These results demonstrate for the first time that bradykinin-mediated increase in free Cai2+ via ER-IP3R3 Ca2+ release followed by Ca2+ influx through SOCE channel plays a crucial role in regulating cell growth and migration via activating pAkt, pERK1/2 and cyclin D1 in human cardiac c-Kit+ progenitor cells.

Inhibition of stromal-interacting molecule 1-mediated store-operated Ca2+ entry as a novel strategy for the treatment of acquired imatinib-resistant gastrointestinal stromal tumors.

Imatinib has revolutionized the treatment of gastrointestinal stromal tumors (GIST); however, primary and secondary resistance to imatinib is still a major cause of treatment failure. Multiple mechanisms are involved in this progression. In the present study, we reported a novel mechanism for the acquired resistance to imatinib, which was induced by enhanced Ca2+ influx via stromal-interacting molecule 1 (STIM1)-mediated store-operated Ca2+ entry (SOCE). We found that the STIM1 expression level was related to the acquired resistance to imatinib in our studied cohort. The function of STIM1 in imatinib-resistant GIST cells was also confirmed both in vivo and in vitro. The results showed that STIM1 overexpression contributed to SOCE and drug response in imatinib-sensitive GIST cells. Blockage of SOCE by STIM1 knockdown suppressed the proliferation of imatinib-resistant GIST cell lines and xenografts. In addition, STIM1-mediated SOCE exerted an antiapoptotic effect via the MEK/ERK pathway. The results from this study provide a basis for further research into potential novel therapeutic strategies in acquired imatinib-resistant GIST.

STIM2 Induces Activated Conformation of STIM1 to Control Orai1 Function in ER-PM Junctions.

Ca2+ entry mediated by the calcium channel, Orai1, provides critical Ca2+ signals that regulate cell function. The ER-Ca2+ sensor protein, STIM1, recruits and strongly activates Orai1 within ER-PM junctions. STIM2 is a poor activator of Orai1, and its physiological role is not well understood. Herein, we report a crucial function for STIM2 in inducing the activated conformation of STIM1. By using conformational sensors of STIM2 and STIM1, together with protein interaction and functional studies, we show that STIM2 is constitutively localized within ER-PM junctions in ER-Ca2+ store replete cells. Importantly, STIM2 traps STIM1 and triggers remodeling of STIM1 C terminus, causing STIM1/Orai1 coupling and enhancement of Orai1 function in cells with relatively high ER-[Ca2+]. The increase in Ca2+ entry controls Ca2+-dependent transcription factor, NFAT, activation at low [agonist]. Our findings reveal that STIM2 modulates STIM1/Orai1 function to tune the fidelity of receptor-evoked Ca2+ signaling and the physiological response of cells.

Store operated calcium channels are associated with diabetic cystopathy in streptozotocin­induced diabetic rats.

Store operated calcium channels (SOCCs) have been suggested to play a critical role in many diabetic complications. Diabetic cystopathy (DCP) is common in patients with diabetes, but the role of SOCCs in DCP is still unclear. The aim of the present study was to investigate the role of SOCCs in DCP with streptozocin (STZ)­induced diabetic rats. Specifically, the authors investigated whether SOCCs were altered in streptozocin (STZ)­induced diabetic rats and, if so, how this may contribute to the contraction of bladder detrusor strips and the intracellular Ca2+ concentration of bladder smooth muscle cells in diabetic rats. Cyclopiazonic acid (CPA, 10 µM) and SKF­96365 (10 µM) were used to activate and inhibit SOCCs respectively, to research the effects of SOCCs on the contraction of the bladder detrusor strips in normal and STZ­induced diabetic rats at the 4th, 8th and 12th week after the diabetic rat model was established. The changes of intracellular Ca2+ were also evaluated under confocal microscopy with pretreated Fluo­4AM. In addition, the expressions of Orai1 and STIM1 were detected by reverse transcription­quantitative polymerase chain reaction and western blotting at different time points. According to the results, the contractive frequency of diabetic bladder muscle strips was higher than that of controls in the 4th and 8th week. The increased fluorescence intensity was detected after using CPA and SKF­96365 in diabetic groups. The expressions of Orai1 and STIM1 changed in a time­dependent manner.

Wogonin Suppresses the Activity of Matrix Metalloproteinase-9 and Inhibits Migration and Invasion in Human Hepatocellular Carcinoma.

As one of the major active ingredients in Radix Scutellariae, wogonin has been shown to be associated with various pharmacological activities on cancer cell growth, apoptosis, and cell invasion and migration. Here, we demonstrated that wogonin may harbor potential anti-metastatic activities in hepatocarcinoma (HCC). The anti-metastasis potential of wogonin and its underlying mechanisms were evaluated by ligand-protein docking approach, surface plasmon resonance assay, and in vitro gelatin zymography studies. Our results showed that wogonin (100 µM, 50 µM) suppressed MHCC97L and PLC/PRF/5 cells migration and invasion in vitro. The docking approach and surface plasmon resonance assay indicated that the potential binding affinity between wogonin and matrix metalloproteinase-9 (MMP-9) may lead to inhibition of MMP-9 activity and further leads to suppression of tumor metastasis. This conclusion was further verified by Western blot results and gelatin zymography analysis. Wogonin might be a potent treatment option for disrupting the tumor metastasis that favors HCC development. The potential active targets from computational screening integrated with biomedical study may help us to explore the molecular mechanism of herbal medicines.

The STIM1 inhibitor ML9 disrupts basal autophagy in cardiomyocytes by decreasing lysosome content.

Stromal-interaction molecule 1 (STIM1)-mediated store-operated Ca2+ entry (SOCE) plays a key role in mediating cardiomyocyte hypertrophy, both in vitro and in vivo. Moreover, there is growing support for the contribution of SOCE to the Ca2+ overload associated with ischemia/reperfusion injury. Therefore, STIM1 inhibition is proposed as a novel target for controlling both hypertrophy and ischemia/reperfusion-induced Ca2+ overload. Our aim was to evaluate the effect of ML9, a STIM1 inhibitor, on cardiomyocyte viability. ML9 was found to induce cell death in cultured neonatal rat cardiomyocytes. Caspase-3 activation, apoptotic index and release of the necrosis marker lactate dehydrogenase to the extracellular medium were evaluated. ML9-induced cardiomyocyte death was not associated with increased intracellular ROS or decreased ATP levels. Moreover, treatment with ML9 significantly increased levels of the autophagy marker LC3-II, without altering Beclin1 or p62 protein levels. However, treatment with ML9 followed by bafilomycin-A1 did not produce further increases in LC3-II content. Furthermore, treatment with ML9 resulted in decreased LysoTracker® Green staining. Collectively, these data suggest that ML9-induced cardiomyocyte death is triggered by a ML9-dependent disruption of autophagic flux due to lysosomal dysfunction.

Dengue virus induced changes in Ca2+ homeostasis in human hepatic cells that favor the viral replicative cycle.

The role of Ca2+ during dengue virus (DENV) replication is unknown; thus, changes in Ca2+ homeostasis in DENV infected human hepatic HepG2 and Huh-7 cells were analyzed. Infected HepG2 cells, but not Huh-7 cells, showed a significant increase in plasma membrane permeability to Ca2+, while both cell lines showed marked reduced levels of Ca2+ stored in the endoplasmic reticulum. While the expression levels of STIM1 and ORAI1 showed no changes, STIM1 and ORAI1 were shown to co-localized in infected cells, indicating activation of the store-operated Ca2+ entry (SOCE) pathway. Finally, manipulation in the infected cells of the intra and extracellular Ca2+ levels by chelators (BAPTA-AM and EGTA), SOC inhibitor (SKF96365), IP3 Receptor antagonist (2APB) or increase of extracellular [Ca2+], significantly reduced DENV yield, but not vesicular stomatitis virus yield, used as a control. These results show that DENV infection alters cell Ca2+ homeostasis and that such changes favor viral replication.

Suppression of Stim1 reduced intracellular calcium concentration and attenuated hypoxia/reoxygenation induced apoptosis in H9C2 cells.

OBJECTIVE: Previous studies have demonstrated Stromal interaction molecule 1 (STIM1)-mediated store-operated Ca2+ entry (SOCE) contributes to intracellular Ca2+ accumulation. The present study aimed to investigate the expression of STIM1 and its downstream molecules Orai1/TRPC1 in the context of myocardial ischemia/reperfusion injury (MIRI) and the effect of STIM1 inhibition on Ca2+ accumulation and apoptosis in H9c2 cardiomyocytes subjected to hypoxia/reoxygenation (H/R). METHODS: Expression of STIM1/Orai1/TRPC1 was determined by RT-PCR and Western blot in mice subjected to MIRI and H9C2 cardiomyocytes subjected to H/R. To knock-down STIM1, H9C2 cardiomyocytes was transfected with Stealth SiRNA. Apoptosis was analyzed by both flow cytometry and TUNEL assay. Cell viability was measured by MTT assay. Intracellular Ca2+ concentration was detected by laser scanning confocal microscopy using Fluo-3/AM probe. Furthermore, the opening of mitochondrial permeability transition pore (mPTP) was assessed by coloading with calcein AM and CoCl2, while ROS generation was evaluated using the dye DCFH-DA in H9C2 cardiomyocytes. RESULTS: Expression of STIM1/Orai1/TRPC1 significantly increased in transcript and translation level after MIRI in vivo and H/R in vitro In H9C2 cardiomyocytes subjected to H/R, intracellular Ca2+ accumulation significantly increased compared with control group, along with enhanced mPTP opening and elevated ROS generation. However, suppression of STIM1 by SiRNA significantly decreased apoptosis and intracellular Ca2+ accumulation induced by H/R in H9C2 cardiomyocytes, accompanied by attenuated mPTP opening and decreased ROS generation. In addition, suppression of STIM1 increased the Bcl-2/Bax ratio, decreased Orai1/TRPC1, and cleaved caspase-3 expression. CONCLUSION: Suppression of STIM1 reduced intracellular calcium level and attenuated hypoxia/reoxygenation induced apoptosis in H9C2 cardiomyocytes. Our findings provide a new perspective in understanding STIM1-mediated calcium overload in the setting of MIRI.

Suppression of STIM1 inhibits the migration and invasion of human prostate cancer cells and is associated with PI3K/Akt signaling inactivation.

Store-operated calcium entry (SOCE) plays an important role in the invasion and migration of cancer cells. Stromal-interacting molecule 1 (STIM1) is a critical component in the SOCE. STIM1 has been attracting more and more attention due to its oncogenic potential. STIM1 inhibition suppresses cell proliferation, migration and invasion in a variety of cancer models both in vitro and in vivo. However, the role of STIM1 in prostate carcinogenesis, in particular, in tumor migration and invasion is unclear. Herein, we downregulated STIM1 in prostate cancer cells by lentivirus-mediated short hairpin (shRNA), and then studied its impacts on cell migration and invasion. We found that migration and invasion of prostate cancer cells were significantly inhibited after the suppression of STIM1. Furthermore, we demonstrated that the PI3K/Akt signaling pathway was inactivated by STIM1 knockdown. The PI3K inhibitor LY294002 synergized with STIM1 knockdown to inhibit cell motility. Our results revealed that STIM1 may act as a novel regulator to promote migration and invasion of prostate cancer cells and is associated with the activation of the PI3K/Akt signaling pathway.

Suppression of store-operated Ca2+ entry by activation of GPER: contribution to a clamping effect on endothelial Ca2+ signaling.

The G protein-coupled estrogen receptor 1 (GPER, formerly also known as GPR30) modulates many Ca2+-dependent activities in endothelial cells. However, the underlying mechanisms are poorly understood. We recently reported that GPER acts to prolong cytoplasmic Ca2+ signals by interacting with and promoting inhibitory phosphorylation of the plasma membrane Ca2+-ATPase. In the present study, we examined the role of GPER activation in modulating store-operated Ca2+ entry (SOCE) via effects on the stromal interaction molecule 1 (STIM1). GPER activation by agonist G-1 reduces the peak but prolongs the plateau of bradykinin-induced Ca2+ signals in primary endothelial cells. G-1 dose-dependently inhibits thapsigargin-induced SOCE measured by the Mn2+ quenching method. GPER heterologous expression reduces SOCE, which is further pronounced by G-1 treatment. Consistently, GPER gene silencing in endothelial cells is associated with an increase in SOCE. Treatment with G-1 reduces puncta formation by STIM1 triggered by the activation of SOCE. The effect of GPER activation to inhibit SOCE is not affected by combined nonphosphorylatable substitutions at serines 486 and 668 on STIM1, but is substantially reduced by similar substitutions at serines 575, 608 and 621. Taken together with our recently reported inhibitory actions of GPER on Ca2+ efflux, the current data contribute to a model in which GPER acts to clamp agonist-induced cytoplasmic Ca2+ signals. Kinetic modeling based on current and reported data is used to estimate the overall effect of GPER activation on point activity of endothelial nitric oxide synthase during the time course of agonist-induced total Ca2+ signals.