Store-operated Calcium Entry Pathway Research Articles

Novel insights into STIM1's role in store-operated calcium entry and its implications for T-cell mediated inflammation in trigeminal neuralgia.

This investigation aims to elucidate the novel role of Stromal Interaction Molecule 1 (STIM1) in modulating store-operated calcium entry (SOCE) and its subsequent impact on inflammatory cytokine release in T lymphocytes, thereby advancing our understanding of trigeminal neuralgia (TN) pathogenesis. Employing the Gene Expression Omnibus (GEO) database, we extracted microarray data pertinent to TN to identify differentially expressed genes (DEGs). A subsequent comparison with SOCE-related genes from the Genecards database helped pinpoint potential target genes. The STRING database facilitated protein-protein interaction (PPI) analysis to spotlight STIM1 as a gene of interest in TN. Through histological staining, transmission electron microscopy (TEM), and behavioral assessments, we probed STIM1's pathological effects on TN in rat models. Additionally, we examined STIM1's influence on the SOCE pathway in trigeminal ganglion cells using techniques like calcium content measurement, patch clamp electrophysiology, and STIM1- ORAI1 co-localization studies. Changes in the expression of inflammatory markers (TNF-α, IL-1β, IL-6) in T cells were quantified using Western blot (WB) and enzyme-linked immunosorbent assay (ELISA) in vitro, while immunohistochemistry and flow cytometry were applied in vivo to assess these cytokines and T cell count alterations. Our bioinformatic approach highlighted STIM1's significant overexpression in TN patients, underscoring its pivotal role in TN's etiology and progression. Experimental findings from both in vitro and in vivo studies corroborated STIM1's regulatory influence on the SOCE pathway. Furthermore, STIM1 was shown to mediate SOCE-induced inflammatory cytokine release in T lymphocytes, a critical factor in TN development. Supportive evidence from histological, ultrastructural, and behavioral analyses reinforced the link between STIM1-mediated SOCE and T lymphocyte-driven inflammation in TN pathogenesis. This study presents novel evidence that STIM1 is a key regulator of SOCE and inflammatory cytokine release in T lymphocytes, contributing significantly to the pathogenesis of trigeminal neuralgia. Our findings not only deepen the understanding of TN's molecular underpinnings but also potentially open new avenues for targeted therapeutic strategies.

Electrophysiological Methods to Measure Ca2+ Current.

To achieve the most accurate assessment of functional Ca2+ channel or modulator properties and their regulation, a patch-clamp technique to record membrane currents is required. This technique has wide applications ranging from recording the activity of native channels in their natural environment to that of recombinant channels expressed in heterologous cells. This chapter introduces the methods that have been used for the detection of calcium release-activated calcium (CRAC) currents, one of the store-operated calcium entry pathways, in human primary T cells. This standard protocol is for laboratories already equipped with a full patch-clamp set-up or for investigators collaborating with laboratories experienced in patch clamp.

Protective effect of total flavonoids of Rhododendron simsii on focal cerebral ischemia-reperfusion injury through SOCE pathway in rats

This paper explored the protective effect of total flavonoids of Rhododendron simsii(TFR) on focal cerebral ischemia-reperfusion injury(CIRI) in rats and its relationship with the store-operated calcium entry(SOCE) pathway regulated by stromal intera-ction molecule(STIM) and calcium release-activated calcium modulator(Orai).Rats were randomly assigned into the sham group, model(middle cerebral artery occlusion, MCAO) group, TFR(60 mg·kg~(-1)) group, TFR(60 mg·kg~(-1))+SOCE pathway inhibitor 2-aminoethoxydiphenyl borate(2-APB, 2.5 mg·kg~(-1)) group, and 2-APB(2.5 mg·kg~(-1)) group.The rats in the sham group and MCAO group were administrated with normal saline, and those in the TFR group and TFR+2-APB group were administrated with TFR(60 mg·kg~(-1)) by gavage for 14 days until sampling.The rats in the 2-APB group and TFR+2-APB group were intraperitoneally injected with 2-APB(2.5 mg·kg~(-1)) after operation.The levels of interleukin-1(IL-1), interleukin-6(IL-6), and tumor necrosis factor-alpha(TNF-α) in serum were measured by ELISA.The cerebral infarction and the pathological status of ischemic brain tissue were detected via TTC staining and HE staining, respectively.The protein and mRNA levels of STIM1, STIM2, Orai1, cysteinyl aspartate specific proteinase 3(caspase-3), and protein kinase B(PKB) in brain tissue were respectively determined by Western blot and RT-qPCR.The growth of brain neurons in each group was observed via immunofluorescence method.The results showed that compared with the MCAO group, TFR lowered the levels of IL-1, IL-6 and TNF-α in serum and the score of neurological function, ameliorated the pathological injury of brain tissue, and decreased the infarct size.Moreover, TFR up-regulated the mRNA and protein levels of STIM1, STIM2, Orai1, and PKB, down-regulated those of caspase-3 in brain tissue, and increased the double-labeled positive cells under fluorescence microscope.However, the above effects were significantly weakened by the addition of 2-APB, a SOCE inhibitor.The results suggested that TFR may play a protective role against focal cerebral ischemia-reperfusion injury by up-regulating the expression of SOCE-related signal molecules, promoting neurogenesis around the ischemic area, improving the survival state of neurons, and redu-cing the activity of inflammatory mediators.

Nanoscale molecular architecture controls calcium diffusion and ER replenishment in dendritic spines.

Dendritic spines are critical components of neuronal synapses as they receive and transform synaptic inputs into a succession of calcium-regulated biochemical events. The spine apparatus (SA), an extension of smooth endoplasmic reticulum, regulates slow and fast calcium dynamics in spines. Calcium release events deplete SA calcium ion reservoir rapidly, yet the next cycle of signaling requires its replenishment. How spines achieve this replenishment without triggering calcium release remains unclear. Using computational modeling, calcium and STED superresolution imaging, we show that the SA replenishment involves the store-operated calcium entry pathway during spontaneous calcium transients. We identified two main conditions for SA replenishment without depletion: a small amplitude and a slow timescale for calcium influx, and a close proximity between SA and plasma membranes. Thereby, spine’s nanoscale organization separates SA replenishment from depletion. We further conclude that spine’s receptor organization also determines the calcium dynamics during the induction of long-term synaptic changes.

3,3'-Diindolylmethane induces gastric cancer cells death via STIM1 mediated store-operated calcium entry.

3,3'-Diindolylmethane (DIM), a natural phytochemicals isolated from cruciferous vegetables, has been reported to inhibit human gastric cancer cells proliferation and induce cells apoptosis as well as autophagy, but its mechanisms are still unclear. Store-operated calcium entry (SOCE) is a main Ca2+ influx pathway in various of cancers, which is activated by the depletion of endoplasmic reticulum (ER) Ca2+ store. Stromal interaction molecular 1 (STIM1) is the necessary component of SOCE. In this study, we focus on to examine the regulatory mechanism of SOCE on DIM-induced death in gastric cancer. After treating the human BGC-823 and SGC-7901 gastric cancer cells with DIM, cellular proliferation was determined by MTT, apoptosis and autophagy were detected by flow cytometry or Hoechst 33342 staining. The expression levels of related proteins were evaluated by Western blotting. Free cytosolilc Ca2+ level was assessed by fluorescence monitoring under a laser scanning confocal microscope. The data have shown that DIM could significantly inhibit proliferation and induce apoptosis as well as autophagy in two gastric cancer cell lines. After DIM treatment, the STIM1-mediated SOCE was activated by upregulating STIM1 and decreasing ER Ca2+ level. Knockdown STIM1 with siRNA or pharmacological inhibition of SOCE attenuated DIM induced apoptosis and autophagy by inhibiting p-AMPK mediated ER stress pathway. Our data highlighted that the potential of SOCE as a promising target for treating cancers. Developing effective and selective activators targeting STIM1-mediated SOCE pathway will facilitate better therapeutic sensitivity of phytochemicals acting on SOCE in gastric cancer. Moreover, more research should be performed to validate the efficacy of combination chemotherapy of anti-cancer drugs targeting SOCE for clinical application.

STIM Proteins: An Ever-Expanding Family.

Stromal interaction molecules (STIM) are a distinct class of ubiquitously expressed single-pass transmembrane proteins in the endoplasmic reticulum (ER) membrane. Together with Orai ion channels in the plasma membrane (PM), they form the molecular basis of the calcium release-activated calcium (CRAC) channel. An intracellular signaling pathway known as store-operated calcium entry (SOCE) is critically dependent on the CRAC channel. The SOCE pathway is activated by the ligand-induced depletion of the ER calcium store. STIM proteins, acting as calcium sensors, subsequently sense this depletion and activate Orai ion channels via direct physical interaction to allow the influx of calcium ions for store refilling and downstream signaling processes. This review article is dedicated to the latest advances in the field of STIM proteins. New results of ongoing investigations based on the recently published functional data as well as structural data from nuclear magnetic resonance (NMR) spectroscopy and molecular dynamics (MD) simulations are reported and complemented with a discussion of the latest developments in the research of STIM protein isoforms and their differential functions in regulating SOCE.

TRPC6 channel and its implications in breast cancer: an overview

TRPC6 channel is widely expressed in most human tissues and participates in a number of physiological processes. TRPC6 belongs to the DAG-activated subfamily of channels, but has also been postulated as a mediator in the store-operated calcium entry pathway. The recent characterization of TRPC6 crystal structure has granted a wonderful tool to finally dissect and understand TRPC6 physiological and biophysical properties. Growing evidences have demonstrated that the pattern of expression of TRPC6 proteins is upregulated in several pathophysiological conditions, including breast cancer. However, the real role of TRPC6 in breast cancer persists still unknown. Here we present the current state of the art concerning the function and significance of TRPC6 in this disease. Future investigations should be focus in the creation and identification of compounds that specifically target the channel to ameliorate TRPC6-related diseases.

P060 The pharmacologic inhibition of store-operated calcium entry pathway in inflammatory bowel disease

Abstract Background Store-operated calcium entry (SOCE) represents the major calcium influx pathway in T cells which not only controls the activation and function of lymphocytes, but which also has been implicated in the metabolic homeostasis and survival of murine CD4+ and CD8+ T cells. Conditional knockout mice, in which SOCE signalling components are deleted in T cells, revealed that SOCE is required for the induction of intestinal inflammation in mouse models of colitis. However, the effects of SOCE inhibition have not been studied in inflammatory bowel disease (IBD) and it remains elusive, which immune cell subset is affected by the pharmaceutical blockade of SOCE. We therefore aim to investigate the effects of SOCE inhibitor BTP-2 on functions and metabolic homeostasis of human lymphocytes isolated from IBD patients. Methods PBMC and/or lamina propria lymphocytes were isolated from colitis patients undergoing colon resection and mass cytometry served in order to evaluate the cytokine production and activation of B, T, NK as well as myeloid cells. Additionally, Ca2+ influx measurement and Seahorse analyses were performed in order to assess the metabolic status of immune cell subsets after SOCE inhibition. Results Data on B, T, NK, myeloid cells and neutrophils isolated from peripheral blood or colon lamina propria revealed that each immune cell subset harbours a distinctive SOCE-dependent Ca2+ influx rate, suggesting that SOCE might differentially regulate the activation and function of each cell subtype. In particular, CD4+ and CD8+ T cells, B and NK cells as well as monocytes were highly susceptible to extracellular Ca2+ influx, followed by granulocytes. Furthermore, inhibition of SOCE in lymphocytes resulted in impaired metabolic fitness, reduced glycolytic capacity and impaired fatty acid oxidation. Finally, BTP-2 was able to decrease the production of key pro-inflammatory cytokines involved in IBD, including TNFα and IL-17 in lamina propria resident T cells. Conclusion Our data revealed for the first time that the cytokine production and the activation of several immune cell subtypes can be modulated by SOCE blockade in human intestinal inflammation, identifying SOCE as a novel therapeutic target in colitis. Moreover, we hope that a wide phenotypical characterisation of immune cells via mass cytometry will provide a better insight into positive as well as negative effects of SOCE inhibitors that might interfere with the clinical applicability of SOCE inhibitors for treating IBD.

SARAF Luminal Domain Structure Reveals a Novel Domain-Swapped β-Sandwich Fold Important for SOCE Modulation

Store-Operated Calcium Entry (SOCE) plays key roles in cell proliferation, muscle contraction, immune responses, and memory formation. The coordinated interactions of a number of proteins from the plasma and endoplasmic reticulum membranes control SOCE to replenish internal Ca2+ stores and generate intracellular Ca2+ signals. SARAF, an endoplasmic reticulum resident component of the SOCE pathway having no homology to any characterized protein, serves as an important brake on SOCE. Here, we describe the X‐ray crystal structure of the SARAF luminal domain, SARAFL. This domain forms a novel 10-stranded β-sandwich fold that includes a set of three conserved disulfide bonds, denoted the “SARAF-fold.” The structure reveals a domain-swapped dimer in which the last two β-strands (β9 and β10) are exchanged forming a region denoted the “SARAF luminal switch” that is essential for dimerization. Sequence comparisons reveal that the SARAF-fold is highly conserved in vertebrates and in a variety of pathologic fungi. Förster resonance energy transfer experiments using full-length SARAF validate the formation of the domain-swapped dimer in cells and demonstrate that dimerization is reversible. A designed variant lacking the SARAF luminal switch shows that the domain swapping is essential to function and indicates that the SARAF dimer accelerates SOCE inactivation.

The Complex Role of Store Operated Calcium Entry Pathways and Related Proteins in the Function of Cardiac, Skeletal and Vascular Smooth Muscle Cells.

Cardiac, skeletal, and smooth muscle cells shared the common feature of contraction in response to different stimuli. Agonist-induced muscle's contraction is triggered by a cytosolic free Ca2+ concentration increase due to a rapid Ca2+ release from intracellular stores and a transmembrane Ca2+ influx, mainly through L-type Ca2+ channels. Compelling evidences have demonstrated that Ca2+ might also enter through other cationic channels such as Store-Operated Ca2+ Channels (SOCCs), involved in several physiological functions and pathological conditions. The opening of SOCCs is regulated by the filling state of the intracellular Ca2+ store, the sarcoplasmic reticulum, which communicates to the plasma membrane channels through the Stromal Interaction Molecule 1/2 (STIM1/2) protein. In muscle cells, SOCCs can be mainly non-selective cation channels formed by Orai1 and other members of the Transient Receptor Potential-Canonical (TRPC) channels family, as well as highly selective Ca2+ Release-Activated Ca2+ (CRAC) channels, formed exclusively by subunits of Orai proteins likely organized in macromolecular complexes. This review summarizes the current knowledge of the complex role of Store Operated Calcium Entry (SOCE) pathways and related proteins in the function of cardiac, skeletal, and vascular smooth muscle cells.

Drug discovery and development for acute pancreatitis

STIM1 and ORAI1 constitute the core machinery of the ubiquitous store-operated calcium entry pathway and loss of function in these proteins is associated with severe immune and muscular disorders. Other isoforms—STIM1L, STIM2, ORAI2 and ORAI3 exhibit varied expression levels in different cell types along with several other interaction partners and thereby play different roles to facilitate, regulate and fine-tune the calcium entry. STIM proteins convey the Ca2+ store-depletion message to the PM and thereby participate in refilling of the ER by physically interacting with the Ca2+-selective ORAI channels at the PM. STIM and ORAI are exposed to oxidative modifications in the ER, the cytosol, and at the cell surface, and redox-mediated alterations in STIM/ORAI coupling might contribute to autoimmune disorders and cancer progression. This review discusses the redox reactivity of cysteine residues in STIM and ORAI isoforms, focusing on the oxidative modifications of STIM and ORAI proteins by which STIM-ORAI signaling can be modulated.

The heterogeneity of store-operated calcium entry in melanoma.

Calcium is a key regulator of many physiological processes that are perturbed in cancer, such as migration, proliferation and apoptosis. The proteins STIM and Orai mediate store-operated calcium entry (SOCE), the main pathway for calcium entry in non-excitable cells. Changes in the expression and function of STIM and Orai have been found in a range of cancer types and thus implicated in disease progression. Here we discuss the role of STIM, Orai and the SOCE pathway in the progression of melanoma and explore how the heterogeneous nature of melanoma may explain the lack of consensus in the field regarding the role of SOCE in the progression of this disease.

STIM-Orai Interaction in Schistosoma Mansoni Indicates the Existence of Functional Store-Operated Calcium Entry in the Parasite

Store-operated calcium entry (SOCE) is a conserved calcium pathway in eukaryotes, although not yet studied in Schistosoma mansoni, the main causative agent of schistosomiasis in America. The proteins STIM1 (calcium sensor) and Orai1 (calcium channel) are the central components of SOCE and the recently published genome of this parasite encodes orthologs of these two proteins, indicating that this Ca2+ pathway is present in S. mansoni. STIM comprises several domains, including three cytosolic coiled coil domains (CC). The first CC (STIM_1CC) is predicted to interact with the other two CC domains in the resting state, and upon activation by calcium depletion, the channel activating domain (CAD), comprised by the second and third CC, is exposed to interact with the C-terminus of Orai, opening the channel. The aim of the present work is to characterize the SOCE pathway in S. mansoni, through the expression of the recombinant proteins from this pathway and interaction assays among STIM and Orai domains, in order to get insights into the mechanism of action of this calcium channel in platyhelminths. S. mansoni STIM_1CC (SmSTIM_1CC), SmSTIM_CAD and SmOrai-C were obtained by recombinant expression in E. coli. Pull down and Isotermic Titration calorimetry experiments showed that SmOrai-C interacts with SmSTIM_CAD in vitro, indicating that the SOCE pathway might be functional in Schistosomes. Additionally, pull down and surface plasmon resonance experiments indicated that SmSTIM_1CC and SmSTIM_CAD also interact with each other, which may consist in a mechanism of autoinhibition of the channel in the resting state, as reported for other organisms. Therefore, the mechanisms by which SmStim activates SmOrai appear highly conserved and the characterization of a new calcium channel for this important human parasite sheds light into possible new targets against schistosomiasis.

Neuronal Store-Operated Calcium Entry and Mushroom Spine Loss in Amyloid Precursor Protein Knock-In Mouse Model of Alzheimer's Disease.

Alzheimer's disease (AD) is the most common reason for elderly dementia in the world. We proposed that memory loss in AD is related to destabilization of mushroom postsynaptic spines involved in long-term memory storage. We demonstrated previously that stromal interaction molecule 2 (STIM2)-regulated neuronal store-operated calcium entry (nSOC) in postsynaptic spines play a key role in stability of mushroom spines by maintaining activity of synaptic Ca(2+)/calmodulin kinase II (CaMKII). Furthermore, we demonstrated previously that the STIM2-nSOC-CaMKII pathway is downregulated in presenilin 1 M146V knock-in (PS1-M146V KI) mouse model of AD, leading to loss of hippocampal mushroom spines in this model. In the present study, we demonstrate that hippocampal mushroom postsynaptic spines are also lost in amyloid precursor protein knock-in (APPKI) mouse model of AD. We demonstrated that loss of mushroom spines occurs as a result of accumulation of extracellular β-amyloid 42 in APPKI culture media. Our results indicate that extracellular Aβ42 acts by overactivating mGluR5 receptor in APPKI neurons, leading to elevated Ca(2+) levels in endoplasmic reticulum, compensatory downregulation of STIM2 expression, impaired synaptic nSOC, and reduced CaMKII activity. Pharmacological inhibition of mGluR5 or overexpression of STIM2 rescued synaptic nSOC and prevented mushroom spine loss in APPKI hippocampal neurons. Our results indicate that downregulation of synaptic STIM2-nSOC-CaMKII pathway causes loss of mushroom synaptic spines in both presenilin and APPKI mouse models of AD. We propose that modulators/activators of this pathway may have a potential therapeutic value for treatment of memory loss in AD. Significance statement: A direct connection between amyloid-induced synaptic mushroom spine loss and neuronal store-operated calcium entry pathway is shown. These results provide strong support for the calcium hypothesis of neurodegeneration and further validate the synaptic store-operated calcium entry pathway as a potential therapeutic target for Alzheimer's disease.

STIM1 and STIM2 Proteins Differently Regulate Endogenous Store-operated Channels in HEK293 Cells

The endoplasmic reticulum calcium sensors stromal interaction molecules 1 and 2 (STIM1 and STIM2) are key modulators of store-operated calcium entry. Both these sensors play a major role in physiological functions in normal tissue and in pathology, but available data on native STIM2-regulated plasma membrane channels are scarce. Only a few studies have recorded STIM2-induced CRAC (calcium release-activated calcium) currents. On the other hand, many cell types display store-operated currents different from CRAC. The STIM1 protein regulates not only CRAC but also transient receptor potential canonical (TRPC) channels, but it has remained unclear whether STIM2 is capable of regulating store-operated non-CRAC channels. Here we present for the first time experimental evidence for the existence of endogenous non-CRAC STIM2-regulated channels. As shown in single-channel patch clamp experiments on HEK293 cells, selective activation of native STIM2 proteins or STIM2 overexpression results in store-operated activation of Imin channels, whereas STIM1 activation blocks this process. Changes in the ratio between active STIM2 and STIM1 proteins can switch the regulation of Imin channels between store-operated and store-independent modes. We have previously characterized electrophysiological properties of different Ca(2+) influx channels coexisting in HEK293 cells. The results of this study show that STIM1 and STIM2 differ in the ability to activate these store-operated channels; Imin channels are regulated by STIM2, TRPC3-containing INS channels are induced by STIM1, and TRPC1-composed Imax channels are activated by both STIM1 and STIM2. These new data about cross-talk between STIM1 and STIM2 and their different roles in store-operated channel activation are indicative of an additional level in the regulation of store-operated calcium entry pathways.

Open Sesame: treasure in store-operated calcium entry pathway for cancer therapy.

Store-operated Ca2+ entry (SOCE) controls intracellular Ca2+ homeostasis and regulates a wide range of cellular events including proliferation, migration and invasion. The discovery of STIM proteins as Ca2+ sensors and Orai proteins as Ca2+ channel pore forming units provided molecular tools to understand the physiological function of SOCE. Many studies have revealed the pathophysiological roles of Orai and STIM in tumor cells. This review focuses on recent advances in SOCE and its contribution to tumorigenesis. Altered Orai and/or STIM functions may serve as biomarkers for cancer prognosis, and targeting the SOCE pathway may provide a novel means for cancer treatment.

Pharmacological blockers of STIM1 inhibit increases in intracellular calcium in horse sweat gland cells (650.2)

Calcium is a key second messenger in stimulus‐secretion coupling in human and Equidae sweat gland secretory cells. Upon stimulation, calcium is released from internal stores, facilitating the opening of membrane ion channels and the subsequent formation of secretion in the lumen of the gland. Sustained secretion requires an influx of calcium from the external milieu. This calcium influx is thought to enter the secretory cells via store operated calcium channels. Little is known about store‐operated calcium entry (SOCE) and so we studied its role in horse sweat gland cells from control animals and those that had developed anhidrosis, an inability to produce sweat.We had previously shown the presence of STIM and Orai proteins in cell lines derived from control and anhidrotic animals. The results also indicated a reduction in STIM1 mRNA and protein expression in anhidrotic sweat gland cells. The functionality of the SOCE pathway in both cell lines was investigated in response to agonist stimulation using FURA‐2AM calcium‐imaging techniques. Increases in [Ca2+]i could be abolished to varying degrees in control cells by a variety of known pharmacological blockers of SOCE. Additionally anhidrotic cells showed a significant decrease in [Ca2+]i FURA‐2 ratio in response to agonists when compared to control cells.The results suggest that a reduction of STIM1 expression in the SOCE pathway may play a role in anhidrosis in horses.Grant Funding Source: Supported by Glasgow Caledonian University.

Positive regulation of STIM1/Orai1‐mediated store operated calcium entry by reactive oxygen species in human mesangial cells (LB731)

Store operated calcium entry (SOCE) plays a pivotal role in physiological responses of glomerular mesangial cells (MCs). STIM1 and Orai1 proteins are the essential components of SOCE pathway. We have previously demonstrated that STIM1and Orai1 were required for SOCE in cultured human MCs. Reactive oxygen species (ROS) can function as intracellular second messengers and as pathogenic factors in a variety of cell types. Whether and how ROS regulate the STIM1/Orai1‐dependent SOCE in MCs is unknown. The aim of the present study was to explore the effect of ROS on SOCE and the mechanism involved in cultured human MCs. Using fura‐2 fluorescence ratiometry approach, we found that the cyclopiazonic acid (CPA, 25 μM)‐stimulated SOCE was significantly enhanced in MCs with H2O2 treatment (100 μM for 5 days). Consistent with the functional data, western blot analysis of whole cell lysates showed significant increases in expressions of STIM1 and Orai1 proteins by H2O2 treatment in the range of 10‐100 μM for the same time period. However, H2O2 treatment did not affect mRNA expression levels of both STIM1 and Orai1. Furthermore, NAC (4 mM), an antioxidant, had tendency to attenuation of high glucose‐stimulated STIM1 protein expression. Taken together, our results indicate that ROS promote SOCE by increasing STIM1/Orai1 protein expression via a post‐transcriptional mechanism.Grant Funding Source: NIH

Role of Orai1 and store‐operated calcium entry in mouse lacrimal gland signalling and function

Lacrimal glands function to produce an aqueous layer, or tear film, that helps to nourish and protect the ocular surface. Lacrimal glands secrete proteins, electrolytes and water, and loss of gland function can result in tear film disorders such as dry eye syndrome, a widely encountered and debilitating disease in ageing populations. To combat these disorders, understanding the underlying molecular signalling processes that control lacrimal gland function will give insight into corrective therapeutic approaches. Previously, in single lacrimal cells isolated from lacrimal glands, we demonstrated that muscarinic receptor activation stimulates a phospholipase C-coupled signalling cascade involving the inositol trisphosphate-dependent mobilization of intracellular calcium and the subsequent activation of store-operated calcium entry (SOCE). Since intracellular calcium stores are finite and readily exhausted, the SOCE pathway is a critical process for sustaining and maintaining receptor-activated signalling. Recent studies have identified the Orai family proteins as critical components of the SOCE channel activity in a wide variety of cell types. In this study we characterize the role of Orai1 in the function of lacrimal glands using a mouse model in which the gene for the calcium entry channel protein, Orai1, has been deleted. Our data demonstrate that lacrimal acinar cells lacking Orai1 do not exhibit SOCE following activation of the muscarinic receptor. In comparison with wild-type and heterozygous littermates, Orai1 knockout mice showed a significant reduction in the stimulated tear production following injection of pilocarpine, a muscarinic receptor agonist. In addition, calcium-dependent, but not calcium-independent exocytotic secretion of peroxidase was eliminated in glands from knockout mice. These studies indicate a critical role for Orai1-mediated SOCE in lacrimal gland signalling and function.

Orai3 Constitutes a Native Store-Operated Calcium Entry That Regulates Non Small Cell Lung Adenocarcinoma Cell Proliferation

Orai channels have been associated with cell proliferation, survival and metastasis in several cancers. The present study investigates the expression and the role of Orai3 in cell proliferation in non-small cell lung cancer (NSCLC). We show that Orai3 is over-expressed in cancer tissues as compared to the non-tumoral ones. Furthermore, Orai3 staining is stronger in high grade tumors. Pharmacological inhibition or knockdown of Orai3 significantly reduced store operated calcium entry (SOCE), inhibited cell proliferation and arrested cells of two NSCLC cell lines in G0/G1 phase. These effects were concomitant with a down-regulation of cyclin D1, cyclin E, CDK4 and CDK2 expression. Moreover, Orai3 silencing decreased Akt phosphorylation levels. In conclusion, Orai3 constitutes a native SOCE pathway in NSCLC that controls cell proliferation and cell cycle progression likely via Akt pathway.