Store-operated Ca2+ Entry Channel Research Articles

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.

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.

A microRNA checkpoint for Ca2+ signaling and overload in acute pancreatitis

Acute pancreatitis (AP) is a common digestive disease without specific treatment, and its pathogenesis features multiple deleterious amplification loops dependent on translation, triggered by cytosolic Ca2+ ([Ca2+]i) overload; however, the underlying mechanisms in Ca2+ overload of AP remains incompletely understood. Here we show that microRNA-26a (miR-26a) inhibits pancreatic acinar cell (PAC) store-operated Ca2+ entry (SOCE) channel expression, Ca2+ overload, and AP. We find that major SOCE channels are post-transcriptionally induced in PACs during AP, whereas miR-26a expression is reduced in experimental and human AP and correlated with AP severity. Mechanistically, miR-26a simultaneously targets Trpc3 and Trpc6 SOCE channels and attenuates physiological oscillations and pathological elevations of [Ca2+]i in PACs. MiR-26a deficiency increases SOCE channel expression and [Ca2+]i overload, and significantly exacerbates AP. Conversely, global or PAC-specific overexpression of miR-26a in mice ameliorates pancreatic edema, neutrophil infiltration, acinar necrosis, and systemic inflammation, accompanied with remarkable improvements on pathological determinants related with [Ca2+]i overload. Moreover, pancreatic or systemic administration of an miR-26a mimic to mice significantly alleviates experimental AP. These findings reveal a previously unknown mechanism underlying AP pathogenesis, establish a critical role for miR-26a in Ca2+ signaling in the exocrine pancreas, and identify a potential target for the treatment of AP.

Orai3 Regulates Pancreatic Cancer Metastasis by Encoding a Functional Store Operated Calcium Entry Channel.

Simple SummaryPancreatic cancer (PC) is one of the most lethal forms of cancers with 5-year mean survival rate of less than 10%. Most of the PC associated deaths are due to metastasis to secondary sites. Calcium (Ca2+) signaling plays a critical role in regulating hallmarks of cancer progression including cell proliferation, migration and apoptotic resistance. Here, we demonstrate that a highly Ca2+ selective plasma membrane channel Orai3 is overexpressed in PC and is associated with poor prognosis in PC patients. Our data demonstrate that Orai3 modulates PC cell proliferation, apoptosis and migration. We further reveal that Orai3 regulates PC metastasis in immune-compromised mice. Collectively, our study establishes Orai3 as an attractive therapeutic target for managing PC metastasis, which may lead to better prognosis.Store operated Ca2+ entry (SOCE) mediated by Orai1/2/3 channels is a highly regulated and ubiquitous Ca2+ influx pathway. Although the role of Orai1 channels is well studied, the significance of Orai2/3 channels is still emerging in nature. In this study, we performed extensive bioinformatic analysis of publicly available datasets and observed that Orai3 expression is inversely associated with the mean survival time of PC patients. Orai3 expression analysis in a battery of PC cell lines corroborated its differential expression profile. We then carried out thorough Ca2+ imaging experiments in six PC cell lines and found that Orai3 forms a functional SOCE channel in PC cells. Our in vitro functional assays show that Orai3 regulates PC cell cycle progression, apoptosis and migration. Most importantly, our in vivo xenograft studies demonstrate a critical role of Orai3 in PC tumor growth and secondary metastasis. Mechanistically, Orai3 controls G1 phase progression, matrix metalloproteinase expression and epithelial-mesenchymal transition in PC cells. Taken together, this study for the first-time reports that Orai3 drives aggressive phenotypes of PC cells, i.e., migration in vitro and metastasis in vivo. Considering that Orai3 overexpression leads to poor prognosis in PC patients, it appears to be a highly attractive therapeutic target.

The carboxy terminal coiled-coil modulates Orai1 internalization during meiosis

Regulation of Ca2+ signaling is critical for the progression of cell division, especially during meiosis to prepare the egg for fertilization. The primary Ca2+ influx pathway in oocytes is Store-Operated Ca2+ Entry (SOCE). SOCE is tightly regulated during meiosis, including internalization of the SOCE channel, Orai1. Orai1 is a four-pass membrane protein with cytosolic N- and C-termini. Orai1 internalization requires a caveolin binding motif (CBM) in the N-terminus as well as the C-terminal cytosolic domain. However, the molecular determinant for Orai1 endocytosis in the C-terminus are not known. Here we show that the Orai1 C-terminus modulates Orai1 endocytosis during meiosis through a structural motif that is based on the strength of the C-terminal intersubunit coiled coil (CC) domains. Deletion mutants show that a minimal C-terminal sequence after transmembrane domain 4 (residues 260–275) supports Orai1 internalization. We refer to this region as the C-terminus Internalization Handle (CIH). Access to CIH however is dependent on the strength of the intersubunit CC. Mutants that increase the stability of the coiled coil prevent internalization independent of specific mutation. We further used human and Xenopus Orai isoforms with different propensity to form C-terminal CC and show a strong correlation between the strength of the CC and Orai internalization. Furthermore, Orai1 internalization does not depend on clathrin, flotillin or PIP2. Collectively these results argue that Orai1 internalization requires both the N-terminal CBM and C-terminal CIH where access to CIH is controlled by the strength of intersubunit C-terminal CC.

The Orai1 inhibitor BTP2 has multiple effects on Ca2+ handling in skeletal muscle.

BTP2 is an inhibitor of the Ca2+ channel Orai1, which mediates store-operated Ca2+ entry (SOCE). Despite having been extensively used in skeletal muscle, the effects of this inhibitor on Ca2+ handling in muscle cells have not been described. To address this question, we used intra- and extracellular application of BTP2 in mechanically skinned fibers and developed a localized modulator application approach, which provided in-preparation reference and test fiber sections to enhance detection of the effect of Ca2+ handling modulators. In addition to blocking Orai1-dependent SOCE, we found a BTP2-dependent inhibition of resting extracellular Ca2+ flux. Increasing concentrations of BTP2 caused a shift from inducing accumulation of Ca2+ in the t-system due to Orai1 blocking to reducing the resting [Ca2+] in the sealed t-system. This effect was not observed in the absence of functional ryanodine receptors (RYRs), suggesting that higher concentrations of BTP2 impair RYR function. Additionally, we found that BTP2 impaired action potential-induced Ca2+ release from the sarcoplasmic reticulum during repetitive stimulation without compromising the fiber Ca2+ content. BTP2 was found to have an effect on RYR-mediated Ca2+ release, suggesting that RYR is the point of BTP2-induced inhibition during cycles of EC coupling. The effects of BTP2 on the RYR Ca2+ leak and release were abolished by pre-exposure to saponin, indicating that the effects of BTP2 on the RYR are not direct and require a functional t-system. Our results demonstrate the presence of a SOCE channels-mediated basal Ca2+ influx in healthy muscle fibers and indicate that BTP2 has multiple effects on Ca2+ handling, including indirect effects on the activity of the RYR.

2,3′,4,4′,5-Pentachlorobiphenyl Induced Thyrocyte Autophagy by Promoting Calcium Influx via Store-Operated Ca2+ Entry

PCB118, a 2,3',4,4',5-pentachlorobiphenyl, has been shown to destroy thyroidal ultrastructure and induce thyrocyte autophagy. Previously, we reported that PCB118 promoted autophagosome formation in vivo and in vitro, but more details remain to be revealed. To explore the underlying mechanism by which PCB118 regulates thyrocyte autophagy, Fischer rat thyroid cell line-5 (FRTL-5) cells were exposed to different doses of PCB118 at 0, 0.25, 2.5, and 25 nM for 0-48 h. Western blot analysis of autophagy-related proteins P62, BECLIN1, and LC3 demonstrated that PCB118 induced autophagy formation in dose- and time-dependent manner. Moreover, laser scanning confocal microscopy and flow cytometry showed PCB118 treatment led to time- and dose-dependent increase in intracellular calcium concentration ([Ca2+]i). Additionally, PCB118 promoted store-operated Ca2+ entry (SOCE) channel followed by significant increase of ORAI1 and STIM1 protein levels. Onthe other hand, PCB118 induced thyroidal autophagy via class III β-tubulin (TUBB3)/death-associated protein kinase 2(DAPK2)/myosin regulatory light chain (MRLC)/autophagy-related 9A (ATG9A) pathway in FRTL-5 cells. Pretreatment with SOCE inhibitor SKF96365 reduced cytosolic Ca2+, ORAI1, STIM1, and BECLIN1 levels as well as LC3 II/LC3 I ratio, while increased P62 expression. SKF96365 also inhibited TUBB3/DAPK2/MRLC/ATG9A pathway in FRTL-5 cells treated by PCB118. Our results provide evidence that PCB118 may induce thyroidal autophagy through TUBB3-related signaling pathway, and these effects are likely to be regulated by calcium influx via SOCE channel.

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.

Integrity of Actin-Network Is Involved in Uridine 5′-Triphosphate Evoked Store-Operated CaM2+ Entry in Bovine Adrenocortical Fasciculata Cells

Store-operated Ca2+ entry channels (SOCs) play an important role in the regulation of diverse non-excitable cell functions. However, the precise mechanism of SOCs activation is still controversial. Uridine 5′-triphosphate (UTP) was shown to induce Ca2+ entry in a dihydropyridines-insensitive manner and accelerated steroidogenesis in bovine adrenocortical fasciculata cells (BAFCs) via the Gq/11 protein-coupled P2Y2 receptor. Therefore we investigated whether UTP is involved in SOCs activation and the mechanism of UTP-induced SOCs activation. Fura 2-loaded BAFCs were used for the measurement of intracellular concentration of Ca2+ ([Ca2+]i) mobilization. Extracellular UTP evoked Ca2+ release from intracellular stores followed by an increase in Ca2+ entry. The Ca2+ influx elicited by UTP was inhibited not by nifedipine, but by Zn2+, Cd2+, and Ni2+ (potency order: Zn2+ > Cd2+ >> Ni2+), and the effect of UTP was also attenuated by a phospholipase C inhibitor (U73122). These results indicate that UTP activates SOCs in BAFCs. The increase in [Ca2+]i by UTP was attenuated by ML-9, a myosin-light chain kinase inhibitor, and calmodulin inhibitors, W-7 and E6 berbamine, in a concentration-dependent manner. These reagents depolymerized actin filaments with rhodamine staining in BAFCs. Cytochalasin D also inhibited UTP-activated SOCs and depolymerized actin filaments. From these results, we proposed that calcium/calmodulin dependent myosin-light chain kinase is involved in the mobilization of actin filaments and the integrity of actin-network plays an important role in UTP-induced SOCs activation in BAFCs.

Experimental and Mathematical Modeling of Intracellular Calcium Dynamics for Anticancer Effects Evaluation in Esophageal Cancer

Targeting dysregulated Ca2+ signaling in cancer cells is an emerging chemotherapy approach. This group previously reported that store‐operated Ca2+ entry (SOCE) blocker, such as RP4010, could effectively inhibit intracellular Ca2+ oscillations and cell proliferation in esophageal cancer, a leading cancer worldwide with low 5‐year survival rate. While many current clinical chemotherapy drugs, such as tyrosine kinase inhibitors (TKIs) can induce intracellular Ca2+ release, it is not known whether combination of SOCE blockers and TKIs could achieve better chemotherapy effects. The present study employed both experimental and mathematical models to evaluate the effect of RP4010 and TKI (Afatinib) on intracellular Ca2+ oscillation periods in KYSE‐150 cells, an esophageal cancer cell line. The intracellular Ca2+ oscillation period in KYSE‐150 cells was measured as ~32.8 sec; both Afatinib and RP4010 could reduce intracellular Ca2+ and prolong the period in a dose‐dependent manner. Using a modified canonical model with variables of SOCE channel and IP3 receptor affected by RP4010 and Afatinib receptively, the intracellular Ca2+ oscillation dynamics were simulated. The theoretical data of the combined implementations of drugs also fitted well with experimental data. Both results demonstrated a synergetic effect of RP4010 and Afatinib on inhibiting intracellular Ca2+ oscillation period, which was further confirmed by cell proliferation analysis. This study suggests that experimental and mathematical modeling of intracellular Ca2+ dynamics could be used as a rapid and cost‐effective tool for evaluation of combined chemotherapy drugs.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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.

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.

Mitochondria control store-operated Ca2+ entry through Na+ and redox signals.

Mitochondria exert important control over plasma membrane (PM) Orai1 channels mediating store-operated Ca2+ entry (SOCE). Although the sensing of endoplasmic reticulum (ER) Ca2+ stores by STIM proteins and coupling to Orai1 channels is well understood, how mitochondria communicate with Orai1 channels to regulate SOCE activation remains elusive. Here, we reveal that SOCE is accompanied by a rise in cytosolic Na+ that is critical in activating the mitochondrial Na+/Ca2+ exchanger (NCLX) causing enhanced mitochondrial Na+ uptake and Ca2+ efflux. Omission of extracellular Na+ prevents the cytosolic Na+ rise, inhibits NCLX activity, and impairs SOCE and Orai1 channel current. We show further that SOCE activates a mitochondrial redox transient which is dependent on NCLX and is required for preventing Orai1 inactivation through oxidation of a critical cysteine (Cys195) in the third transmembrane helix of Orai1. We show that mitochondrial targeting of catalase is sufficient to rescue redox transients, SOCE, and Orai1 currents in NCLX-deficient cells. Our findings identify a hitherto unknown NCLX-mediated pathway that coordinates Na+ and Ca2+ signals to effect mitochondrial redox control over SOCE.

Stromal Interaction Molecule 1 (STIM1) Regulates ATP-sensitive Potassium (KATP) and Store-operated Ca2+ Channels in MIN6 β-Cells

Stromal interaction molecule 1 (STIM1) regulates store-operated Ca2+ entry (SOCE) and other ion channels either as an endoplasmic reticulum Ca2+-sensing protein or when present in the plasma membrane. However, the role of STIM1 in insulin-secreting β-cells is unresolved. We report that lowering expression of STIM1, the gene that encodes STIM1, in insulin-secreting MIN6 β-cells with RNA interference inhibits SOCE and ATP-sensitive K+ (KATP) channel activation. The effects of STIM1 knockdown were reversed by transduction of MIN6 cells with an adenovirus gene shuttle vector that expressed human STIM1 Immunoprecipitation studies revealed that STIM1 binds to nucleotide binding fold-1 (NBF1) of the sulfonylurea receptor 1 (SUR1) subunit of the KATP channel. Binding of STIM1 to SUR1 was enhanced by poly-lysine. Our data indicate that SOCE and KATP channel activity are regulated by STIM1. This suggests that STIM1 is a multifunctional signaling effector that participates in the control of membrane excitability and Ca2+ signaling events in β-cells.

Dental enamel cells express functional SOCE channels

Dental enamel formation requires large quantities of Ca2+ yet the mechanisms mediating Ca2+ dynamics in enamel cells are unclear. Store-operated Ca2+ entry (SOCE) channels are important Ca2+ influx mechanisms in many cells. SOCE involves release of Ca2+ from intracellular pools followed by Ca2+ entry. The best-characterized SOCE channels are the Ca2+ release-activated Ca2+ (CRAC) channels. As patients with mutations in the CRAC channel genes STIM1 and ORAI1 show abnormal enamel mineralization, we hypothesized that CRAC channels might be an important Ca2+ uptake mechanism in enamel cells. Investigating primary murine enamel cells, we found that key components of CRAC channels (ORAI1, ORAI2, ORAI3, STIM1, STIM2) were expressed and most abundant during the maturation stage of enamel development. Furthermore, inositol 1,4,5-trisphosphate receptor (IP3R) but not ryanodine receptor (RyR) expression was high in enamel cells suggesting that IP3Rs are the main ER Ca2+ release mechanism. Passive depletion of ER Ca2+ stores with thapsigargin resulted in a significant raise in [Ca2+]i consistent with SOCE. In cells pre-treated with the CRAC channel blocker Synta-66 Ca2+ entry was significantly inhibited. These data demonstrate that enamel cells have SOCE mediated by CRAC channels and implicate them as a mechanism for Ca2+ uptake in enamel formation.

Roles of store-operated Ca2+ channels in regulating cell cycling and migration of human cardiac c-kit+ progenitor cells.

Cardiac c-kit(+) progenitor cells are important for maintaining cardiac homeostasis and can potentially contribute to myocardial repair. However, cellular physiology of human cardiac c-kit(+) progenitor cells is not well understood. The present study investigates the functional store-operated Ca(2+) entry (SOCE) channels and the potential role in regulating cell cycling and migration using confocal microscopy, RT-PCR, Western blot, coimmunoprecipitation, cell proliferation, and migration assays. We found that SOCE channels mediated Ca(2+) influx, and TRPC1, STIM1, and Orai1 were involved in the formation of SOCE channels in human cardiac c-kit(+) progenitor cells. Silencing TRPC1, STIM1, or Orai1 with the corresponding siRNA significantly reduced the Ca(2+) signaling through SOCE channels, decreased cell proliferation and migration, and reduced expression of cyclin D1, cyclin E, and/or p-Akt. Our results demonstrate the novel information that Ca(2+) signaling through SOCE channels regulates cell cycling and migration via activating cyclin D1, cyclin E, and/or p-Akt in human cardiac c-kit(+) cells.

Differential Redox Regulation of Ca2+ Signaling and Viability in Normal and Malignant Prostate Cells

In prostate cancer, reactive oxygen species (ROS) are elevated and Ca2+ signaling is impaired. Thus, several novel therapeutic strategies have been developed to target altered ROS and Ca2+ signaling pathways in prostate cancer. Here, we investigate alterations of intracellular Ca2+ and inhibition of cell viability caused by ROS in primary human prostate epithelial cells (hPECs) from healthy tissue and prostate cancer cell lines (LNCaP, DU145, and PC3). In hPECs, LNCaP and DU145 H2O2 induces an initial Ca2+ increase, which in prostate cancer cells is blocked at high concentrations of H2O2. Upon depletion of intracellular Ca2+ stores, store-operated Ca2+ entry (SOCE) is activated. SOCE channels can be formed by hexameric Orai1 channels; however, Orai1 can form heteromultimers with its homolog, Orai3. Since the redox sensor of Orai1 (Cys-195) is absent in Orai3, the Orai1/Orai3 ratio in T cells determines the redox sensitivity of SOCE and cell viability. In prostate cancer cells, SOCE is blocked at lower concentrations of H2O2 compared with hPECs. An analysis of data from hPECs, LNCaP, DU145, and PC3, as well as previously published data from naive and effector TH cells, demonstrates a strong correlation between the Orai1/Orai3 ratio and the SOCE redox sensitivity and cell viability. Therefore, our data support the concept that store-operated Ca2+ channels in hPECs and prostate cancer cells are heteromeric Orai1/Orai3 channels with an increased Orai1/Orai3 ratio in cells derived from prostate cancer tumors. In addition, ROS-induced alterations in Ca2+ signaling in prostate cancer cells may contribute to the higher sensitivity of these cells to ROS.

Store-operated Ca2+ Entry Modulates the Expression of Enamel Genes

Dental enamel formation is an intricate process tightly regulated by ameloblast cells. The correct spatiotemporal patterning of enamel matrix protein (EMP) expression is fundamental to orchestrate the formation of enamel crystals, which depend on a robust supply of Ca2+. In the extracellular milieu, Ca2+-EMP interactions occur at different levels. Despite its recognized role in enamel development, the molecular machinery involved in Ca2+ homeostasis in ameloblasts remains poorly understood. A common mechanism for Ca2+ influx is store-operated Ca2+ entry (SOCE). We evaluated the possibility that Ca2+ influx in enamel cells might be mediated by SOCE and the Ca2+ release-activated Ca2+ (CRAC) channel, the prototypical SOCE channel. Using ameloblast-like LS8 cells, we demonstrate that these cells express Ca2+-handling molecules and mediate Ca2+ influx through SOCE. As a rise in the cytosolic Ca2+ concentration is a versatile signal that can modulate gene expression, we assessed whether SOCE in enamel cells had any effect on the expression of EMPs. Our results demonstrate that stimulating LS8 cells or murine primary enamel organ cells with thapsigargin to activate SOCE leads to increased expression of Amelx, Ambn, Enam, Mmp20. This effect is reversed when cells are treated with a CRAC channel inhibitor. These data indicate that Ca2+ influx in LS8 cells and enamel organ cells is mediated by CRAC channels and that Ca2+ signals enhance the expression of EMPs. Ca2+ plays an important role not only in mineralizing dental enamel but also in regulating the expression of EMPs.

PWE-209 Orai inhibition prevents cytosolic ca2+ overload and acute pancreatitis

Introduction Cytosolic calcium overload triggers pancreatic acinar injury induced by pancreatitis toxins. Sustained Ca 2+ elevation depends on Ca 2+ entry through store-operated Ca 2+ entry (SOCE) channel Orai1, but the role of which in experimental acute pancreatitis (EAP) and human pancreatic acinar cell injury has not been determined. Method Confocal and patch clamp technology were used to examine the effects of GSK-7975A and CM_128, inhibitors of SOCE channel Orai1 on bile acid-, hystimulation-, thapsigargin-, or cyclopiazonic acid-induced calcium entry into murine and human pancreatic acinar as well as human Orai1/STIM1-transfected HEK 293 cells. The effects of GSK-7975A and CM_128 on human necrotic pancreatic acinar cell death pathway activation induced by bile acid were monitored. EAP was induced by seven hourly intraperitoneal cerulein injections (50 mg/kg), retrograde pancreatic ductal TLCS infusion (50 mL 3 mM) or two hourly intraperitoneal injections of 150 mg/kg palmitoleic acid and 1.35g/kg ethanol. Different doses of either compound were tested in three diverse clinical representative models of EAP, begun at different time point after disease induction. EAP severity was assessed by standard biochemical parameters and blinded histopathology. Results GSK-7975A and CM_128 inhibited toxin-induced SOCE and/or Ca 2+ release-activated Ca 2+ currents in a concentration-dependent manner up to >90% of control in all cells tested and significantly inhibited murine and human necrotic pancreatic acinar cell death pathway activation (p Conclusion This study confirms the pivotal role of cytosolic calcium overload in the pathogenesis of acute pancreatitis and provides robust preclinical validation for Orai inhibition as a treatment for acute pancreatitis. Disclosure of interest L. Wen: None Declared, S. Voronina: None Declared, M. Javed: None Declared, M. Awais: None Declared, P. Szatmary: None Declared, D. Latawiec: None Declared, M. Chvanov: None Declared, D. Collier: None Declared, J. Barrett Employee of: GlaxoSmithKline, M. Begg Employee of: GlaxoSmithKline, K. Stauderman Employee of: CalciMedica, M. Dunn Employee of: CalciMedica, A. Tepikin: None Declared, D. Criddle: None Declared, R. Sutton: None Declared.

Store-operated Ca2+ entry plays a role in HMGB1-induced vascular endothelial cell hyperpermeability.

AimsEndothelial dysfunction, including increased endothelial permeability, is considered an early marker for atherosclerosis. High-mobility group box 1 protein (HMGB1) and extracellular Ca2+ entry, primarily mediated through store-operated Ca2+ entry (SOCE), are known to be involved in increasing endothelial permeability. The aim of this study was to clarify how HMGB1 could lead to endothelia hyperpermeability.Methods and ResultsWe have shown that human vascular endothelial cell permeability is increased, while transendothelial electrical resistance and VE-cadherin expression were reduced by HMGB1 treatment. Two SOCE inhibitors and knockdown of stromal interaction molecule 1 (STIM1), a Ca2+ sensor mediating SOCE, inhibited the HMGB1-induced influx of Ca2+ and Src activation followed by significant suppression of endothelial permeability. Moreover, knockdown of Orai1, an essential pore-subunit of SOCE channels, decreased HMGB1-induced endothelial hyperpermeability.ConclusionsThese data suggest that SOCE, acting via STIM1, might be the predominant mechanism of Ca2+ entry in the modulation of endothelial cell permeability. STIM1 may thus represent a possible new therapeutic target against atherosclerosis.