Regulation of store-operated Ca entry in pulmonary artery smooth muscle cells

Store-operated Ca²⁺ entry (SOCE) is an important mechanism for Ca²⁺ influx in smooth muscle cells; however the activation and regulation of this influx pathway are incompletely understood. In the present study we have examined the effect of several protein kinases in regulating SOCE in pulmonary artery smooth muscle cells (PASMCs) of the rat. Inhibition of protein kinase C with chelerythrine (3 μM) potentiated SOCE by 47 ± 2%, while the tyrosine kinase inhibitors genistein (100 μM) and tyrphostin 23 (100 μM) caused a significant reduction in SOCE of 55 ± 9% and 43 ± 7%, respectively. It has been proposed that Ca²⁺-insensitive phospholipase A₂ (iPLA₂) is involved in the activation of SOCE in many different cell types. The iPLA₂ inhibitor, bromoenol lactone had no effect on SOCE, suggesting that this mechanism was not involved in the activation of the pathway. The calmodulin antagonists, calmidazolium (CMZ) (10 μM) and W-7 (10 μM) appeared to potentiate SOCE in PASMCs. Further investigation established that CMZ was actually activating a Ca²⁺ influx pathway that was independent of the filling state of the sarcoplasmic reticulum. The CMZ-activated Ca²⁺ influx was blocked by Gd³⁺ (10 μM), but unaffected by 2-APB (75 μM), indicating a pharmacological profile distinct from the classical SOCE pathway.     

Tyrosine kinase modulation of protein kinase C activity regulates G protein-linked Ca2+ signaling in leukemic hematopoietic cells

We have used a recombinant mouse pre-B cell line (TonB210.1, expressing Bcr/Abl under the control of an inducible promoter) and several human leukemia cell lines to study the effect of high tyrosine kinase activity on G protein-coupled receptor (GPCR) agonist-stimulated cellular Ca²⁺ release and store-operated Ca²⁺ entry (SOCE). After induction of Bcr/Abl expression, GPCR-linked SOCE increased. The effect was reverted in the presence of the specific Abl inhibitor imatinib (1 μM) and the Src inhibitor PP2 (10 μM). In leukemic cell lines constitutively expressing high tyrosine kinase activity, Ca²⁺ transients were reduced by imatinib and/or PP2. Ca²⁺ transients were enhanced by specific inhibitors of PKC subtypes and this effect was amplified by tyrosine kinase inhibition in Bcr/Abl expressing TonB210.1 and K562 cells. Under all conditions Ca²⁺ transients were essentially blocked by the PKC activator PMA. In Bcr/Abl expressing (but not in native) TonB210.1 cells, tyrosine kinase inhibitors enhanced PKCα catalytic activity and PKCα co-immunoprecipitated with Bcr/Abl.Unlike native TonB210.1 cells, Bcr/Abl expressing cells showed a high rate of cell death if Ca²⁺ influx was reduced by complexing extracellular Ca²⁺ with BAPTA. Our data suggest that tonic inhibition of PKC represents a mechanism by which high tyrosine kinase activity can enhance cellular Ca²⁺ transients and thus exert profound effects on the proliferation, apoptosis and chemotaxis of leukemic cells.     

Chronic exposure to stress hormones alters the subtype of store‐operated channels expressed in H19‐7 hippocampal neuronal cells

Differentiating H19‐7 hippocampal precursor cells up‐regulate (～4.3‐fold) store‐operated channel (SOC) activity; relatively linear current‐voltage curves indicate an I_SOC subtype of SOC. In differentiated H19‐7 neurons, the majority of agonist (arginine vasopressin, AVP)‐stimulated Ca²⁺ entry occurs via SOCs, based on 2‐aminoethyldiphenylborinate (2‐APB) inhibition data and the observation that transient receptor potential C1 (TRPC1) channel knock down cells show a dramatic reduction of thapsigargin‐stimulated store‐operated Ca²⁺ entry (SOCE) and inhibition of AVP‐stimulated Ca²⁺ entry. Treatment of H19‐7 cells with the rat stress hormone corticosterone during differentiation induces a significant increase in AVP‐stimulated Ca²⁺ entry, which is virtually eliminated by 2‐APB, suggesting a corticosterone‐induced increase of SOCE. Corticosterone also enhances AVP‐stimulated Mn²⁺ entry, confirming an elevated Ca²⁺ entry pathway, rather than a decreased Ca²⁺ extrusion. When corticosterone addition is delayed until after H19‐7 cells have fully differentiated, it still elevates SOCE. In corticosterone‐treated H19‐7 cells, the knock down of TRPC1 no longer blocks thapsigargin‐stimulated Ca²⁺ entry suggesting that the subtype of SOCs expressed in H19‐7 cells is altered by corticosterone treatment. Electrophysiological studies demonstrate that store‐operated currents in corticosterone‐treated H19‐7 cells exhibit a highly inward rectifying current‐voltage curve consistent with an I_CRAC subtype of SOCs. Consistent with this finding is the observation that corticosterone treatment of H19‐7 cells increases the expression of the I_CRAC channel subunit Orai1. Thus, the subtype of SOCs expressed in H19‐7 hippocampal neurons can be altered from I_SOC to I_CRAC by chronic treatment with stress hormones. J. Cell. Physiol. 228: 1332–1343, 2013. © 2012 Wiley Periodicals, Inc.     

Muscarinic Acetylcholine Receptors Potentiate 5′-Adenosine Monophosphate-Activated Protein Kinase Stimulation and Glucose Uptake Triggered by Thapsigargin-Induced Store-Operated Ca<Superscript>2+</Superscript> Entry in Human Neuroblastoma Cells

The 5′-adenosine monophosphate-activated protein kinase (AMPK) is a key regulator of the cellular energy metabolism and may induce either cell survival or death. We previously reported that in SH-SY5Y human neuroblastoma cells stimulation of muscarinic acetylcholine receptors (mAChRs) activate AMPK by triggering store-operated Ca²⁺ entry (SOCE). However, whether mAChRs may control AMPK activity by regulating additional mechanisms beyond SOCE remains to be investigated. In the present study we examined the effects of mAChRs on AMPK when SOCE was induced by the sarco–endoplasmic reticulum Ca²⁺-ATPase inhibitor thapsigargin. We found that in SH-SY5Y cells depleted of Ca²⁺ by thapsigargin, the re-addition Ca²⁺ to the medium stimulated AMPK phosphorylation at Thr172, which is required for full kinase activity. This response occurred through SOCE, as it was blocked by either the SOCE modulator 2-aminoethoxydiphephenyl borate, knockdown of the SOCE molecular component STIM1, or inhibition of Ca²⁺/calmodulin (CaM)-dependent protein kinase kinase β (CaMKKβ). In thapsigargin-pretreated cells, stimulation of pharmacologically defined M₃ mAChRs potentiated SOCE-induced AMPK activation. This potentiation did not involve an increased Ca²⁺ influx, but was associated with CaM mobilization from membrane to cytosol, increased CaM/CaMKKβ interaction, and enhanced CaMKK stimulation by thapsigargin-induced SOCE. In thapsigargin-pretreated cells Ca²⁺ re-addition stimulated glucose uptake and increased the membrane expression of the glucose transporter GLUT1. Both responses were significantly potentiated by mAChRs. These data indicate that in human neuroblastoma cells mAChRs up-regulate AMPK and the downstream glucose uptake by triggering not only SOCE but also CaM translocation and enhanced formation of active CaM/CaMKKβ complexes.     

Staurosporine maintains the activation of store-operated Ca2+ entry even after the refilling of Ca2+ stores

Store-operated Ca²⁺ entry (SOCE) from the extracellular space plays a critical role in agonist-mediated Ca²⁺ signaling in non-excitable cells. Here we show that SOCE is enhanced in COS-7 cells treated with staurosporine (ST), a protein kinase inhibitor. In COS-7 cells, stimulation with ATP induced Ca²⁺ release from intracellular Ca²⁺ stores and Ca²⁺ entry from the extracellular space. Ca²⁺ release was not affected by treatment with ST, but Ca²⁺ entry continued in the ST-treated cells even after the removal of ATP. ST did not inhibit Ca²⁺ sequestration into Ca²⁺ stores. The Ca²⁺ entry induced by cyclopiazonic acid (CPA), a reversible ER Ca²⁺ pump inhibitor, was maintained in ST-treated cells even after the removal of CPA, but was not maintained in the control cells. The sustained Ca²⁺ entry in ST-treated cells was completely attenuated by the SOCE inhibitors, La³⁺ and 2-APB. The large increase in Ca²⁺ entry produced in the cells co-expressing Venus-Orai1 and STIM1-mKO1 was stabilized with ST treatment, and confocal imaging of these cells suggested that the complex between Orai1 and STIM1 did not completely dissociate following the refilling of Ca²⁺ stores. These results show that SOCE remains activated even after the refilling of Ca²⁺ stores in ST-treated cells and that the effect of ST on SOCE may result from a stabilization of the Orai1–STIM1 interaction.     

Mechanism of Catecholamine Synthesis Inhibition by Neuropeptide Y: Role of Ca2+ Channels and Protein Kinases

Abstract: We have previously demonstrated that neuropeptide Y (NPY) inhibits depolarization-stimulated catecholamine synthesis in rat pheochromocytoma (PC12) cells differentiated to a sympathetic neuronal phenotype with nerve growth factor (NGF). The present study uses multiple selective Ca²⁺ channel and protein kinase agonists and antagonists to elucidate the mechanisms by which NPY modulates catecholamine synthesis as determined by in situ measurement of DOPA production in the presence of the decarboxylase inhibitor m-hydroxybenzylhydrazine (NSD-1015). The L-type Ca²⁺ channel blocker nifedipine inhibited the depolarization-induced stimulation of DOPA production by ～90% and attenuated the inhibitory effect of NPY. In contrast, the N-type Ca²⁺ channel blocker ω-conotoxin GVIA inhibited neither the stimulation of DOPA production nor the effect of NPY. Antagonism of Ca²⁺/calmodulin-dependent protein kinase (CaM kinase) greatly inhibited the stimulation of DOPA production by depolarization and prevented the inhibitory effect of NPY, whereas alterations in the cyclic AMP-dependent protein kinase pathway modulated DOPA production but did not prevent the effect of NPY. Stimulation of Ca²⁺/phospholipid-dependent protein kinase (PKC) with phorbol 12-myristate 13-acetate (PMA) did not affect the basal rate of DOPA production in NGF-differentiated PC12 cells but did produce a concentration-dependent inhibition of depolarization-stimulated DOPA production. In addition, NPY did not produce further inhibition of DOPA production in the presence of PMA, and the inhibition by both PMA and NPY was attenuated by the specific PKC inhibitor chelerythrine. These results indicate that NPY inhibits Ca²⁺ influx through L-type voltage-gated Ca²⁺ channels, possibly through a PKC-mediated pathway, resulting in attenuation of the activation of CaM kinase and inhibition of depolarization-stimulated catecholamine synthesis.     

Intracellular Ca2+ stores modulate SOCCs and NMDA receptors via tyrosine kinases in rat hippocampal neurons

The regulation of intracellular Ca²⁺ signalling by phosphorylation processes remains poorly defined, particularly with regards to tyrosine phosphorylation. Evidence from non-excitable cells implicates tyrosine phosphorylation in the activation of so-called store-operated Ca²⁺ channels (SOCCs), but their involvement in neuronal Ca²⁺ signalling is still elusive.In the present study, we determined the role of protein tyrosine kinases (PTKs) and tyrosine phosphatases (PTPs) in the coupling between intracellular Ca²⁺ stores and SOCCs in neonatal rat hippocampal neurons by Fura-2 Ca²⁺ imaging. An early Ca²⁺ response from intracellular stores was triggered with thapsigargin, and followed by a secondary plasma membrane Ca²⁺ response. This phase was blocked by the non-specific Ca²⁺ channel blocker NiCl and the SOCC blocker, 2-aminoethoxydiphenyl borate (2-APB). Interestingly, two structurally distinct PTK inhibitors, genistein and AG126, also inhibited this secondary response.Application of the PTP inhibitor sodium orthovanadate (OV) also activated a sustained and tyrosine kinase dependent Ca²⁺ response, blocked by NiCl and 2-APB. In addition, OV resulted in a Ca²⁺ store dependent enhancement of NMDA responses, corresponding to, and occluding the signalling pathway for group I metabotropic glutamate receptors (mGluRs). This study provides first evidence for tyrosine based phospho-regulation of SOCCs and NMDA signalling in neurons.     

Tyrphostin AG556 increases the activity of large conductance Ca2+‐activated K+ channels by inhibiting epidermal growth factor receptor tyrosine kinase

The present study was designed to investigate whether large conductance Ca²⁺‐activated K⁺ (BK) channels were regulated by epidermal growth factor (EGF) receptor (EGFR) tyrosine kinase. BK current and channel tyrosine phosphorylation level were measured in BK‐HEK 293 cells expressing both functional α‐subunits and the auxiliary β1‐subunits using electrophysiology, immunoprecipitation and Western blotting approaches, respectively, and the function of rat cerebral basilar arteries was determined with a wire myography system. We found that BK current in BK‐HEK 293 cells was increased by the broad spectrum protein tyrosine kinase (PTK) inhibitor genistein and the selective EGFR tyrosine kinase inhibitor AG556, one of the known tyrphostin. The effect of genistein or AG556 was antagonized by the protein tyrosine phosphatase (PTP) inhibitor orthovanadate. On the other hand, orthovanadate or EGF decreased BK current, and the effect was counteracted by AG556. The tyrosine phosphorylation level of BK channels (α‐ and β1‐subunits) was increased by EGF and orthovanadate, while decreased by genistein and AG556, and the reduced tyrosine phosphorylation of BK channels by genistein or AG556 was reversed by orthovanadate. Interestingly, AG556 induced a remarkable enhancement of BK current in rat cerebral artery smooth muscle cells and relaxation of pre‐contracted rat cerebral basilar arteries with denuded endothelium, and these effects were antagonized by the BK channel blocker paxilline or orthovanadate. These results demonstrate that tyrosine phosphorylation of BK channels by EGFR kinase decreases the channel activity, and inhibition of EGFR kinase by AG556 enhances the channel activity and dilates rat cerebral basilar arteries.     

T‐type calcium channel blockers inhibit autophagy and promote apoptosis of malignant melanoma cells

We have recently reported that human melanoma cells express a variety of voltage‐gated calcium (Ca²⁺) channel types, including low‐voltage‐activated T‐type channels that play a significant role in melanoma cell cycle progression. Here, we challenged melanoma metastatic cells with T‐type channel blockers of clinical use and found a dual effect on cell viability: (i) a reduction in the proliferation rate, through a halt in the progression to the G₁‐S phase; and (ii) a promotion of cell death that was partially dependent on the activation of caspases. An in‐depth analysis of the death process showed that the apoptotic pathway is preceded by endoplasmic reticulum stress and the subsequent inhibition of the basal macroautophagy which is active in these cells. The effects of pharmacological blockers on Ca²⁺ homeostasis, autophagy, and cell death were mimicked by T‐type channel gene silencing. These results provide the basis for a new pharmacological and/or gene silencing approach toward tackling melanoma metastasis.     

Store-operated calcium entry and non-capacitative calcium entry have distinct roles in thrombin-induced calcium signalling in human platelets

Phosphatidylserine (PS)-exposing platelets accelerate coagulation at sites of vascular injury. PS exposure requires sustained Ca²⁺ signalling. Two distinct Ca²⁺ entry pathways amplify and sustain platelet Ca²⁺ signalling, but their relative importance in human platelets is not known. Here we examined the relative roles of store-operated Ca²⁺ entry (SOCE) and non-capacitative Ca²⁺ entry (NCCE) in thrombin-induced Ca²⁺ signalling and PS exposure by using two Ca²⁺ channel blockers. BTP-2 showed marked selectivity for SOCE over NCCE. LOE-908 specifically blocked NCCE under our conditions. Using these agents we found that SOCE is important at low thrombin concentrations whereas NCCE became increasingly important as thrombin concentration was increased. PS exposure was reduced by LOE-908, and only activated at thrombin concentrations that also activate NCCE. In contrast, BTP-2 had no effect on PS exposure. We suggest that SOCE amplifies and sustains Ca²⁺ signalling in response to low concentrations of thrombin whereas both NCCE and SOCE are important contributors to Ca²⁺ signalling at higher thrombin concentrations. However, despite being involved in Ca²⁺ signalling at high thrombin concentrations, SOCE is not important for thrombin-induced PS exposure in human platelets. This suggests that the route of Ca²⁺ entry is an important regulator of thrombin-induced PS exposure in platelets.     

Store-operated Ca2+ entry and Ca2+ responses to hypothalamic releasing hormones in anterior pituitary cells from Orai1−/− and heptaTRPC knockout mice

Store operated Ca²⁺ entry (SOCE) is the most important Ca²⁺ entry pathway in non-excitable cells. However, SOCE can also play a pivotal role in excitable cells such as anterior pituitary (AP) cells. The AP gland contains five different cell types that release six major AP hormones controlling most of the entire endocrine system. AP hormone release is modulated by Ca²⁺ signals induced by different hypothalamic releasing hormones (HRHs) acting on specific receptors in AP cells. TRH and LHRH both induce Ca²⁺ release and Ca²⁺ entry in responsive cells while GHRH and CRH only induce Ca²⁺ entry. SOCE has been shown to contribute to Ca²⁺ responses induced by TRH and LHRH but no molecular evidence has been provided. Accordingly, we used AP cells isolated from mice devoid of Orai1 channels (noted as Orai1−/− or Orai1 KO mice) and mice lacking expression of all seven canonical TRP channels (TRPC) from TRPC1 to TRPC7 (noted as heptaTRPC KO mice) to investigate contribution of these putative channel proteins to SOCE and intracellular Ca²⁺ responses induced by HRHs. We found that thapsigargin-evoked SOCE is lost in AP cells from Orai1−/− mice but unaffected in cells from heptaTRPC KO mice. Conversely, while spontaneous intracellular Ca²⁺-oscillations related to electrical activity were not affected in the Orai1−/− mice, these responses were significantly reduced in heptaTRPC KO mice. We also found that Ca²⁺ entry induced by TRH and LHRH is decreased in AP cells isolated from Orai1−/−. In addition, Ca²⁺ responses to several HRHs, particularly TRH and GHRH, are decreased in the heptaTRPC KO mice. These results indicate that expression of Orai1, and not TRPC channel proteins, is necessary for thapsigargin-evoked SOCE and is required to support Ca²⁺ entry induced by TRH and LHRH in mouse AP cells. In contrast, TRPC channel proteins appear to contribute to spontaneous Ca²⁺-oscillations and Ca²⁺ responses induced by TRH and GHRH. We conclude that expression of Orai1 and TRPC channels proteins may play differential and significant roles in AP physiology and endocrine control.     

Store-operated Ca2+ Entry (SOCE) Induced by Protease-activated Receptor-1 Mediates STIM1 Protein Phosphorylation to Inhibit SOCE in Endothelial Cells through AMP-activated Protein Kinase and p38β Mitogen-activated Protein Kinase

The Ca²⁺ sensor STIM1 is crucial for activation of store-operated Ca²⁺ entry (SOCE) through transient receptor potential canonical and Orai channels. STIM1 phosphorylation serves as an “off switch” for SOCE. However, the signaling pathway for STIM1 phosphorylation is unknown. Here, we show that SOCE activates AMP-activated protein kinase (AMPK); its effector p38β mitogen-activated protein kinase (p38β MAPK) phosphorylates STIM1, thus inhibiting SOCE in human lung microvascular endothelial cells. Activation of AMPK using 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) resulted in STIM1 phosphorylation on serine residues and prevented protease-activated receptor-1 (PAR-1)-induced Ca²⁺ entry. Furthermore, AICAR pretreatment blocked PAR-1-induced increase in the permeability of mouse lung microvessels. Activation of SOCE with thrombin caused phosphorylation of isoform α1 but not α2 of the AMPK catalytic subunit. Moreover, knockdown of AMPKα1 augmented SOCE induced by thrombin. Interestingly, SB203580, a selective inhibitor of p38 MAPK, blocked STIM1 phosphorylation and led to sustained STIM1-puncta formation and Ca²⁺ entry. Of the three p38 MAPK isoforms expressed in endothelial cells, p38β knockdown prevented PAR-1-mediated STIM1 phosphorylation and potentiated SOCE. In addition, inhibition of the SOCE downstream target CaM kinase kinase β (CaMKKβ) or knockdown of AMPKα1 suppressed PAR-1-mediated phosphorylation of p38β and hence STIM1. Thus, our findings demonstrate that SOCE activates CaMKKβ-AMPKα1-p38β MAPK signaling to phosphorylate STIM1, thereby suppressing endothelial SOCE and permeability responses.     

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.     

Store-operated Ca2+ entry in muscle physiology and diseases

Ca²⁺ release from intracellular stores and influx from extracellular reservoir regulate a wide range of physiological functions including muscle contraction and rhythmic heartbeat. One of the most ubiquitous pathways involved in controlled Ca²⁺ influx into cells is store-operated Ca²⁺ entry (SOCE), which is activated by the reduction of Ca²⁺ concentration in the lumen of endoplasmic or sarcoplasmic reticulum (ER/SR). Although SOCE is pronounced in non-excitable cells, accumulating evidences highlight its presence and important roles in skeletal muscle and heart. Recent discovery of STIM proteins as ER/SR Ca²⁺ sensors and Orai proteins as Ca²⁺ channel pore forming unit expedited the mechanistic understanding of this pathway. This review focuses on current advances of SOCE components, regulation and physiologic and pathophysiologic roles in muscles. The specific property and the dysfunction of this pathway in muscle diseases, and new directions for future research in this rapidly growing field are discussed. [BMB Reports 2014; 47(2): 69-79]     

Mitochondrial modulation of store-operated Ca2+ entry in model cells of Alzheimer’s disease

Mitochondrial malfunction and calcium dyshomeostasis are early pathological events considered as important features of the Alzheimer’s disease (AD) brain. Recent studies have suggested mitochondrion as an active regulator of Ca²⁺ signaling based on its calcium buffering capacity. Herein, we investigated the mitochondrial involvement in the modulation of store-operated calcium entry (SOCE) in neural 2a (N2a) transgenic AD model cells. Results showed that SOCE was significantly depressed in N2a cells transfected with wild-type human APP695 (N2a APPwt) compared with empty vector control (N2a WT) cells. Pharmacological manipulation with mitochondrial function blockers, such as FCCP, RuR, or antimycin A/oligomycin, could inhibit mitochondrial calcium handling, and then impair SOCE pathway in N2a WT cells. Furthermore, mitochondria of N2a APPwt cells exhibited more severe swelling in response to Ca²⁺, which is an indication of mitochondrial membrane permeability transition (MPT), than the wild-type controls. Additionally, treatment with cyclosporin A, a potent inhibitor of cyclophilin D, which can block MPT, could significantly restore the attenuated SOCE in N2a APPwt cells. Therefore, inhibition of cyclophilin D might be a therapeutic strategy for Alzheimer’s disease.     

Inhibition of Orai1‐mediated Ca2+ entry enhances chemosensitivity of HepG2 hepatocarcinoma cells to 5‐fluorouracil

Increasing evidence supports that activation of store‐operated Ca²⁺ entry (SOCE) is implicated in the chemoresistance of cancer cells subjected to chemotherapy. However, the molecular mechanisms underlying chemoresistance are not well understood. In this study, we aim to investigate whether 5‐FU induces hepatocarcinoma cell death through regulating Ca²⁺‐dependent autophagy. [Ca²⁺]_i was measured using fura2/AM dye. Protein expression was determined by Western blotting and immunohistochemistry. We found that 5‐fluorouracil (5‐FU) induced autophagic cell death in HepG2 hepatocarcinoma cells by inhibiting PI3K/AKT/mTOR pathway. Orai1 expression was obviously elevated in hepatocarcinoma tissues. 5‐FU treatment decreased SOCE and Orai1 expressions, but had no effects on Stim1 and TRPC1 expressions. Knockdown of Orai1 or pharmacological inhibition of SOCE enhanced 5‐FU‐induced inhibition of PI3K/AKT/mTOR pathway and potentiated 5‐FU‐activated autophagic cell death. On the contrary, ectopic overexpression of Orai1 antagonizes 5‐FU‐induced autophagy and cell death. Our findings provide convincing evidence to show that Orai1 expression is increased in hepatocarcinoma tissues. 5‐FU can induce autophagic cell death in HepG2 hepatocarcinoma cells through inhibition of SOCE via decreasing Orai1 expression. These findings suggest that Orai1 expression is a predictor of 5‐FU sensitivity for hepatocarcinoma treatment and blockade of Orai1‐mediated Ca²⁺ entry may be a promising strategy to sensitize hepatocarcinoma cells to 5‐FU treatment.     

The Ca2+ release-activated Ca2+ current (ICRAC) mediates store-operated Ca2+ entry in rat microglia

Ca²⁺ signaling plays a central role in microglial activation, and several studies have demonstrated a store-operated Ca²⁺ entry (SOCE) pathway to supply this ion. Due to the rapid pace of discovery of novel Ca²⁺ permeable channels, and limited electrophysiological analyses of Ca²⁺ currents in microglia, characterization of the SOCE channels remains incomplete. At present, the prime candidates are ‘transient receptor potential’ (TRP) channels and the recently cloned Orai1, which produces a Ca²⁺-release-activated Ca²⁺ (CRAC) current. We used cultured rat microglia and real-time RT-PCR to compare expression levels of Orai1, Orai2, Orai3, TRPM2, TRPM7, TRPC1, TRPC2, TRPC3, TRPC4, TRPC5, TRPC6 and TRPC7 channel genes. Next, we used Fura-2 imaging to identify a store-operated Ca²⁺ entry (SOCE) pathway that was reduced by depolarization and blocked by Gd3+, SKF-96365, diethylstilbestrol (DES), and a high concentration of 2-aminoethoxydiphenyl borate (50 μM 2-APB). The Fura-2 signal was increased by hyperpolarization, and by a low concentration of 2-APB (5 μM), and exhibited Ca²⁺-dependent potentiation. These properties are entirely consistent with Orai1/CRAC, rather than any known TRP channel and this conclusion was supported by patch-clamp electrophysiological analysis. We identified a store-operated Ca²⁺ current with the same properties, including high selectivity for Ca²⁺ over monovalent cations, pronounced inward rectification and a very positive reversal potential, Ca²⁺-dependent current potentiation, and block by SKF-96365, DES and 50 μM 2-APB. Determining the contribution of Orai1/CRAC in different cell types is crucial to future mechanistic and therapeutic studies; this comprehensive multi-strategy analysis demonstrates that Orai1/CRAC channels are responsible for SOCE in primary microglia.     

Protein Kinase C-induced Phosphorylation of Orai1 Regulates the Intracellular Ca2+ Level via the Store-operated Ca2+ Channel

Ca²⁺ signals through store-operated Ca²⁺ (SOC) channels, activated by the depletion of Ca²⁺ from the endoplasmic reticulum, regulate various physiological events. Orai1 is the pore-forming subunit of the Ca²⁺ release-activated Ca²⁺ (CRAC) channel, the best characterized SOC channel. Orai1 is activated by stromal interaction molecule (STIM) 1, a Ca²⁺ sensor located in the endoplasmic reticulum. Orai1 and STIM1 are crucial for SOC channel activation, but the molecular mechanisms regulating Orai1 function are not fully understood. In this study, we demonstrate that protein kinase C (PKC) suppresses store-operated Ca²⁺ entry (SOCE) by phosphorylation of Orai1. PKC inhibitors and knockdown of PKCβ both resulted in increased Ca²⁺ influx. Orai1 is strongly phosphorylated by PKC in vitro and in vivo at N-terminal Ser-27 and Ser-30 residues. Consistent with these results, substitution of endogenous Orai1 with an Orai1 S27A/S30A mutant resulted in increased SOCE and CRAC channel currents. We propose that PKC suppresses SOCE and CRAC channel function by phosphorylation of Orai1 at N-terminal serine residues Ser-27 and Ser-30.