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.     

Ubiquilin 1 interacts with Orai1 to regulate calcium mobilization

Store-operated calcium entry (SOCE) channels composed of Stim and Orai proteins play a critical role in diverse biological processes. Upon endoplasmic reticulum (ER)-mediated calcium (Ca²⁺) depletion, Stim proteins oligomerize with Orai to initiate Ca²⁺ influx across the plasma membrane. The ubiquitin-like (UBL) and ubiquitin-associated (UBA) domains of ubiquilin 1 are involved in the degradation of presenilin and polyglutamine proteins. Through screening of Orai1 interaction partner(s) that might have an effect on SOCE, ubiquilin 1 was identified as a target of Orai1. However, the UBL and UBA domains of ubiquilin 1 were dispensable for this interaction. Additionally, ubiquilin 1 and Orai1 colocalized in the cytosolic compartment. Ubiquilin 1 increased the ubiquitination of Orai1, resulting in the formation of a high-molecular-weight form. MG132, a proteasome inhibitor, failed to block the degradation of Orai1, whereas bafilomycin A, a lysosome inhibitor, prevented Orai1 degradation. Confocal microscopy studies demonstrated that a fraction of Orai1 colocalized with ubiquilin 1 and the autophagosomal marker LC3. Because Orai1 is a constituent of SOCE, we determined the effect of ubiquilin 1 on Orai1-mediated Ca²⁺ influx. As we expected, intracellular Ca²⁺ mobilization, a process normally potentiated by Orai1, was downregulated by ubiquilin 1. Taken together, these findings suggest that ubiquilin 1 downregulates intracellular Ca²⁺ mobilization and its downstream signaling by promoting the ubiquitination and lysosomal degradation of Orai1.     

TRPC1, Orai1, and STIM1 in SOCE: Friends in tight spaces

Store-operated calcium entry (SOCE) is a ubiquitous Ca²⁺ entry pathway that is activated in response to depletion of ER-Ca²⁺ stores and critically controls the regulation of physiological functions in miscellaneous cell types. The transient receptor potential canonical 1 (TRPC1) is the first member of the TRPC channel subfamily to be identified as a molecular component of SOCE. While TRPC1 has been shown to contribute to SOCE and regulate various functions in many cells, none of the reported TRPC1-mediated currents resembled I_CRAC, the highly Ca²⁺-selective store-dependent current first identified in lymphocytes and mast cells. Almost a decade after the cloning of TRPC1 two proteins were identified as the primary components of the CRAC channel. The first, STIM1, is an ER-Ca²⁺ sensor protein involved in activating SOCE. The second, Orai1 is the pore-forming component of the CRAC channel. Co-expression of STIM1 and Orai1 generated robust I_CRAC. Importantly, STIM1 was shown to also activate TRPC1 via its C-terminal polybasic domain, which is distinct from its Orai1-activating domain, SOAR. In addition, TRPC1 function critically depends on Orai1-mediated Ca²⁺ entry which triggers recruitment of TRPC1 into the plasma membrane where it is then activated by STIM1. TRPC1 and Orai1 form discrete STIM1-gated channels that generate distinct Ca²⁺ signals and regulate specific cellular functions. Surface expression of TRPC1 can be modulated by trafficking of the channel to and from the plasma membrane, resulting in changes to the phenotype of TRPC1-mediated current and [Ca²⁺]_i signals. Thus, TRPC1 is activated downstream of Orai1 and modifies the initial [Ca²⁺]_i signal generated by Orai1 following store depletion. This review will summarize the important findings that underlie the current concepts for activation and regulation of TRPC1, as well as its impact on cell function.     

Orai1 Mediates Exacerbated Ca2+ Entry in Dystrophic Skeletal Muscle

There is substantial evidence indicating that disruption of Ca²⁺ homeostasis and activation of cytosolic proteases play a key role in the pathogenesis and progression of Duchenne Muscular Dystrophy (DMD). However, the exact nature of the Ca²⁺ deregulation and the Ca²⁺ signaling pathways that are altered in dystrophic muscles have not yet been resolved. Here we examined the contribution of the store-operated Ca²⁺ entry (SOCE) for the pathogenesis of DMD. RT-PCR and Western blot found that the expression level of Orai1, the pore-forming unit of SOCE, was significantly elevated in the dystrophic muscles, while parallel increases in SOCE activity and SR Ca²⁺ storage were detected in adult mdx muscles using Fura-2 fluorescence measurements. High-efficient shRNA probes against Orai1 were delivered into the flexor digitorum brevis muscle in live mice and knockdown of Orai1 eliminated the differences in SOCE activity and SR Ca²⁺ storage between the mdx and wild type muscle fibers. SOCE activity was repressed by intraperitoneal injection of BTP-2, an Orai1 inhibitor, and cytosolic calpain1 activity in single muscle fibers was measured by a membrane-permeable calpain substrate. We found that BTP-2 injection for 2 weeks significantly reduced the cytosolic calpain1 activity in mdx muscle fibers. Additionally, ultrastructural changes were observed by EM as an increase in the number of triad junctions was identified in dystrophic muscles. Compensatory changes in protein levels of SERCA1, TRP and NCX3 appeared in the mdx muscles, suggesting that comprehensive adaptations occur following altered Ca²⁺ homeostasis in mdx muscles. Our data indicates that upregulation of the Orai1-mediated SOCE pathway and an overloaded SR Ca²⁺ store contributes to the disrupted Ca²⁺ homeostasis in mdx muscles and is linked to elevated proteolytic activity, suggesting that targeting Orai1 activity may be a promising therapeutic approach for the prevention and treatment of muscular dystrophy.     

Local Ca²+ entry via Orai1 regulates plasma membrane recruitment of TRPC1 and controls cytosolic Ca²+ signals required for specific cell functions

Store-operated Ca²⁺ entry (SOCE) has been associated with two types of channels: CRAC channels that require Orai1 and STIM1 and SOC channels that involve TRPC1, Orai1, and STIM1. While TRPC1 significantly contributes to SOCE and SOC channel activity, abrogation of Orai1 function eliminates SOCE and activation of TRPC1. The critical role of Orai1 in activation of TRPC1-SOC channels following Ca²⁺ store depletion has not yet been established. Herein we report that TRPC1 and Orai1 are components of distinct channels. We show that TRPC1/Orai1/STIM1-dependent I_SOC, activated in response to Ca²⁺ store depletion, is composed of TRPC1/STIM1-mediated non-selective cation current and Orai1/STIM1-mediated I_CRAC; the latter is detected when TRPC1 function is suppressed by expression of shTRPC1 or a STIM1 mutant that lacks TRPC1 gating, STIM1(⁶⁸⁴EE⁶⁸⁵). In addition to gating TRPC1 and Orai1, STIM1 mediates the recruitment and association of the channels within ER/PM junctional domains, a critical step in TRPC1 activation. Importantly, we show that Ca²⁺ entry via Orai1 triggers plasma membrane insertion of TRPC1, which is prevented by blocking SOCE with 1 µM Gd³⁺, removal of extracellular Ca²⁺, knockdown of Orai1, or expression of dominant negative mutant Orai1 lacking a functional pore, Orai1-E106Q. In cells expressing another pore mutant of Orai1, Orai1-E106D, TRPC1 trafficking is supported in Ca²⁺-containing, but not Ca²⁺-free, medium. Consistent with this, I_CRAC is activated in cells pretreated with thapsigargin in Ca²⁺-free medium while I_SOC is activated in cells pretreated in Ca²⁺-containing medium. Significantly, TRPC1 function is required for sustained K_Ca activity and contributes to NFκB activation while Orai1 is sufficient for NFAT activation. Together, these findings reveal an as-yet unidentified function for Orai1 that explains the critical requirement of the channel in the activation of TRPC1 following Ca²⁺ store depletion. We suggest that coordinated regulation of the surface expression of TRPC1 by Orai1 and gating by STIM1 provides a mechanism for rapidly modulating and maintaining SOCE-generated Ca²⁺ signals. By recruiting ion channels and other signaling pathways, Orai1 and STIM1 concertedly impact a variety of critical cell functions that are initiated by SOCE.     

Orai1 and STIM1 are critical for cell migration and proliferation of clear cell renal cell carcinoma

The intracellular Ca²⁺ regulation has been implicated in tumorigenesis and tumor progression. Notably, store-operated Ca²⁺ entry (SOCE) is a major Ca²⁺ entry mechanism in non-excitable cells, being involved in cell proliferation and migration in several types of cancer. However, the expression and biological role of SOCE have not been investigated in clear cell renal cell carcinoma (ccRCC). Here, we demonstrate that Orai1 and STIM1, not Orai3, are crucial components of SOCE in the progression of ccRCC. The expression levels of Orai1 in tumor tissues were significantly higher than those in the adjacent normal parenchymal tissues. In addition, native SOCE was blunted by inhibiting SOCE or by silencing Orai1 and STIM1. Pharmacological blockade or knockdown of Orai1 or STIM1 also significantly inhibited RCC cell migration and proliferative capability. Taken together, Orai1 is highly expressed in ccRCC tissues illuminating that Orai1-mediated SOCE may play an important role in ccRCC development. Indeed, Orai1 and STIM1 constitute a native SOCE pathway in ccRCC by promoting cell proliferation and migration.     

STIM1 long and STIM1 gate differently TRPC1 during store-operated calcium entry

During myogenesis, a long splice variant of STIM1, called STIM1L is getting expressed, while the level of STIM1 remains constant. Previous work demonstrated that STIM1L is more efficient in eliciting store-operated Ca²⁺ entry (SOCE), but no current analysis of the channel(s) activated by this new STIM1L isoform was performed until now. In this study, we investigate the ionic channel(s) activated by STIM1L and whether differences exist between the two STIM1 isoforms, using HEK-293 T cells as a model system. Our data show that STIM1 and STIM1L activate Orai1 channel but also the endogenously expressed TRPC1. The channel activation occurs in two steps, with first Orai1 activation followed, in a subset of cells, by TRPC1 opening. Remarkably, STIM1L more frequently activates TRPC1 and preferentially interacts with TRPC1. In low intracellular Ca²⁺ buffering condition, the frequency of TRPC1 opening increases significantly, strongly suggesting a Ca²⁺-dependent channel activation. The ability of STIM1L to open Orai1 appears decreased compared to STIM1, which might be explained by its stronger propensity towards TRPC1. Indeed, increasing the amount of STIM1L results in an enhanced Orai1 current. The role of endogenous TRPC1 in STIM1- and STIM1L-induced SOCE was confirmed by Ca²⁺ imaging experiments. Overall, our findings provide a detailed analysis of the channels activated by both STIM1 isoforms, revealing that STIM1L is more prone to open TRPC1, which might explain the larger SOCE elicited by this isoform.     

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.     

TRPC1 and ORAI1 channels in colon cancer

Colon cancer cells, like other types of cancer cells, undergo the remodeling of the intracellular Ca²⁺ homeostasis that contributes to cancer cell hallmarks including enhanced cell proliferation, migration, and survival. Colon cancer cells display enhanced store-operated Ca²⁺ entry (SOCE) compared with their non-cancer counterparts. Colon cancer cells display an abnormal expression of SOCE molecular players including Orai1 and TRPC1 channels, and the stromal interacting molecule (STIM) 1 and 2. Interestingly, upregulation of Orai1 and TRPC1 channels and their contribution to SOCE are associated with cancer malignancy in colon cancer cells. In a specific cellular model of colon cancer, whereas in non-cancer colon cells SOCE is composed of the Ca²⁺ release activated (CRAC) currents, in colon cancer cells SOCE is composed of CRAC- and cationic, non-selective store operated (SOC) currents. Former SOCs are mediated by TRPC1 channels. Moreover, colon cancer cells also display dysregulation of the expression of 1,4,5-triphosphate receptors (IP₃R) that could contribute to the enhanced SOCE. Another important factor underlying the enhanced SOCE is the differential mitochondrial modulation of the CRAC and SOC currents in non-cancer and colon cancer cells. In colon cancer cells, mitochondria take up more Ca²⁺ that prevent the Ca²⁺-dependent inactivation of the SOCs, leading to sustained Ca²⁺ entry. Notably, the inhibition of SOCE in cancer colon cells abolishes their cancer hallmarks. Robust evidence has shown the efficiency of non-steroidal anti-inflammatory drugs (NSAIDs) and difluoromethylornithine (DFMO) to reverse the enhanced cell proliferation, migration, and apoptosis resistance of cancer cells. In colon cancer cells, both NSAIDs and DFMO decrease SOCE, but they target different molecular components of SOCE. NSAIDs decrease the Ca²⁺ uptake by mitochondria, limiting their ability to prevent the Ca²⁺-dependent inactivation of the SOCs that underlie SOCE. On the other hand, DFMO inhibits the expression of TRPC1 channels in colon cancer cells, eliminating their contribution to SOCE. The identification of players of SOCE in colon cancer cells may help to better understand the remodeling of the Ca²⁺ homeostasis in cancer. Importantly, the use of different pharmacological tools that target different SOCE molecular players in colon cancer cells may play a pivotal role in designing better chemoprevention strategies.     

The dynamic complexity of the TRPC1 channelosome

A rise in cytoplasmic [Ca²⁺] due to store-operated Ca²⁺ entry (SOCE) triggers a plethora of responses, both acute and long term. This leads to the important question of how this initial signal is decoded to regulate specific cellular functions. It is now clearly established that local [Ca²⁺] at the site of SOCE can vary significantly from the global [Ca²⁺] in the cytosol. Such Ca²⁺ microdomains are generated by the assembly of key Ca²⁺ signaling proteins within the domains. For example, GPCR, IP₃ receptors, TRPC3 channels, the plasma membrane Ca²⁺ pump and the endoplasmic reticulum (ER) Ca²⁺ pump have all been found to be assembled in a complex and all of them contribute to the Ca²⁺ signal. Recent studies have revealed that two other critical components of SOCE, STIM1 and Orai1, are also recruited to these regions. Thus, the entire machinery for activation and regulation of SOCE is compartmentalized in specific cellular domains which facilitates the specificity and rate of protein-protein interactions that are required for activation of the channels. In the case of TRPC1-SOC channels, it appears that specific lipid domains, lipid raft domains (LRDs), in the plasma membrane, as well as cholesterol-binding scaffolding proteins such as caveolin-1 (Cav-1), are involved in assembly of the TRPC channel complexes. Thus, plasma membrane proteins and lipid domains as well as ER proteins contribute to the SOCE-Ca²⁺ signaling microdomain and modulation of the Ca²⁺ signals per se. Of further interest is that modulation of Ca²⁺ signals, i.e. amplitude and/or frequency, can result in regulation of specific cellular functions. The emerging data reveal a dynamic Ca²⁺ signaling complex composed of TRPC1/Orai1/STIM1 that is physiologically consistent with the dynamic nature of the Ca²⁺ signal that is generated. This review will focus on the recent studies which demonstrate critical aspects of the TRPC1 channelosome that are involved in the regulation of TRPC1 function and TRPC1-SOC-generated Ca²⁺ signals.     

Roles of Platelet STIM1 and Orai1 in Glycoprotein VI- and Thrombin-dependent Procoagulant Activity and Thrombus Formation

In platelets, STIM1 has been recognized as the key regulatory protein in store-operated Ca²⁺ entry (SOCE) with Orai1 as principal Ca²⁺ entry channel. Both proteins contribute to collagen-dependent arterial thrombosis in mice in vivo. It is unclear whether STIM2 is involved. A key platelet response relying on Ca²⁺ entry is the surface exposure of phosphatidylserine (PS), which accomplishes platelet procoagulant activity. We studied this response in mouse platelets deficient in STIM1, STIM2, or Orai1. Upon high shear flow of blood over collagen, Stim1^−/− and Orai1^−/− platelets had greatly impaired glycoprotein (GP) VI-dependent Ca²⁺ signals, and they were deficient in PS exposure and thrombus formation. In contrast, Stim2^−/− platelets reacted normally. Upon blood flow in the presence of thrombin generation and coagulation, Ca²⁺ signals of Stim1^−/− and Orai1^−/− platelets were partly reduced, whereas the PS exposure and formation of fibrin-rich thrombi were normalized. Washed Stim1^−/− and Orai1^−/− platelets were deficient in GPVI-induced PS exposure and prothrombinase activity, but not when thrombin was present as co-agonist. Markedly, {"type":"entrez-protein","attrs":{"text":"SKF96365","term_id":"1156357400","term_text":"SKF96365"}}SKF96365, a blocker of (receptor-operated) Ca²⁺ entry, inhibited Ca²⁺ and procoagulant responses even in Stim1^−/− and Orai1^−/− platelets. These data show for the first time that: (i) STIM1 and Orai1 jointly contribute to GPVI-induced SOCE, procoagulant activity, and thrombus formation; (ii) a compensating Ca²⁺ entry pathway is effective in the additional presence of thrombin; (iii) platelets contain two mechanisms of Ca²⁺ entry and PS exposure, only one relying on STIM1-Orai1 interaction.     

Role of lipid rafts in the interaction between hTRPC1, Orai1 and STIM1

Store-operated Ca²⁺ entry (SOCE) is a mechanism regulated by the filling state of the intracellular Ca²⁺ stores that requires the participation of the Ca²⁺ sensor STIM1, which communicates the Ca²⁺ content of the stores to the plasma membrane Ca²⁺-permeable channels. We have recently reported that Orai1 mediates the communication between STIM1 and the Ca²⁺ channel hTRPC1. This event is important to confer hTRPC1 store depletion sensitivity, thus supporting the functional role of the STIM1-Orai1-hTRPC1 complex in the activation of SOCE. Here we have explored the relevance of lipid rafts in the formation of the STIM1-Orai1-hTRPC1 complex and the activation of SOCE. Disturbance of lipid raft domains, using methyl-β-cyclodextrin, reduces the interaction between endogenously expressed Orai1 and both STIM1 and hTRPC1 upon depletion of the intracellular Ca²⁺ stores and attenuates thapsigargin-evoked Ca²⁺ entry. These findings suggest that TRPC1, Orai1 and STIM1 form a heteromultimer associated with lipid raft domains and regulated by the intracellular Ca²⁺ stores.     

A Novel Native Store-operated Calcium Channel Encoded by Orai3: SELECTIVE REQUIREMENT OF Orai3 VERSUS Orai1 IN ESTROGEN RECEPTOR-POSITIVE VERSUS ESTROGEN RECEPTOR-NEGATIVE BREAST CANCER CELLS*

Store-operated calcium (Ca²⁺) entry (SOCE) mediated by STIM/Orai proteins is a ubiquitous pathway that controls many important cell functions including proliferation and migration. STIM proteins are Ca²⁺ sensors in the endoplasmic reticulum and Orai proteins are channels expressed at the plasma membrane. The fall in endoplasmic reticulum Ca²⁺ causes translocation of STIM1 to subplasmalemmal puncta where they activate Orai1 channels that mediate the highly Ca²⁺-selective Ca²⁺ release-activated Ca²⁺ current (I_CRAC). Whereas Orai1 has been clearly shown to encode SOCE channels in many cell types, the role of Orai2 and Orai3 in native SOCE pathways remains elusive. Here we analyzed SOCE in ten breast cell lines picked in an unbiased way. We used a combination of Ca²⁺ imaging, pharmacology, patch clamp electrophysiology, and molecular knockdown to show that native SOCE and I_CRAC in estrogen receptor-positive (ER⁺) breast cancer cell lines are mediated by STIM1/2 and Orai3 while estrogen receptor-negative (ER⁻) breast cancer cells use the canonical STIM1/Orai1 pathway. The ER⁺ breast cancer cells represent the first example where the native SOCE pathway and I_CRAC are mediated by Orai3. Future studies implicating Orai3 in ER⁺ breast cancer progression might establish Orai3 as a selective target in therapy of ER⁺ breast tumors.     

Functional interactions among STIM1, Orai1 and TRPC1 on the activation of SOCs in HL-7702 cells

STIM1, Orai1 and TRPC1 are all reported to be important for store-operated Ca²⁺ entry (SOCE) in diverse cells. However, there is no evidence for the functional interaction of the three proteins in SOCE in human liver cells. The objective of this study is to determine whether they are involved in SOCE in normal human liver cells. Liposomal transfection method was used to increase expression levels of the three proteins in HL-7702 cells, a normal human liver cell line. Western blot and single cell RT–PCR were applied to evaluate transfection effectiveness. Changes in store-operated current (I_SOC) and SOCE were investigated using whole-cell patch-clamp recording and calcium imaging. I_SOC is detected in HL-7702 cells and it is inhibited either by 2-Aminoethoxydiphenyl borate (2-APB) or La³⁺. Overexpression of STIM1 or Orai1 alone did not induce any change in I_SOC. TRPC1-transfection, however, caused approximate 2.5-fold increase in I_SOC. A large increase (>10-fold) in I_SOC emerged when both STIM1 and Orai1 were co-transfected into HL-7702 cells. Co-overexpression of STIM1 + TRPC1 also caused >10-fold increase in I_SOC, and addition of Orai1 did not cause any further increase. In HL-7702 cells, TRPC1 and Orai1 take part in SOCE independently of each other. Functional interactions of STIM1 and Orai1 or TRPC1 contribute to I_SOC activation.     

Orai3 mediates Orai channel remodelling to activate fibroblast in pulmonary fibrosis

Orai family are a calcium channel of cell membrane extracellular Ca²⁺ influx which participates in tissue fibrosis. But the roles of Orai3 have less attention on the mechanism of regulating lung fibrosis. In this study, we found that Orai3 expression was increased significantly in BLM‐induced lung fibrosis. The knockdown of Orai3 decreased TGF‐β1‐induced fibroblast proliferation, ECM production, activation of NFAT1 and Calpain/ERK signal pathway and glycolysis levels. Orai3 interacting with Orai1 was increased in BLM‐induced lung fibrosis and TGF‐β1‐induced fibroblast, while the Stim1 interacting with Orai1 and SOCE activity was suppressed, leading in a high and stable extracellular Ca²⁺ influx. Furthermore, the over‐expression of Orai3 did not enhance Orai3 interacting with Orai1 under TGF‐β1 free fibroblast. And then, the deeper mechanism of TGF‐β1‐induced increased SEPTIN4 promoted Orai3 interacting with Orai1. Our results indicated that Orai3 could be one of the therapy targets for PF in which remodels Orai channel, suppresses SOCE activity and activated fibroblast to alleviate fibrosis progress.     

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.     

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 T_H 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 Plays a Role in HMGB1-Induced Vascular Endothelial Cell Hyperpermeability

Aims

Endothelial 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 Results

We 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.

Conclusions

These 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.