Store-operated Ca2+ Entry Inhibitors Research Articles

Eldecalcitol ameliorates diabetic osteoporosis and glucolipid metabolic disorder by promoting Treg cell differentiation through SOCE

Active vitamin D, known for its role in promoting osteoporosis, has immunomodulatory effects according to the latest evidence. Eldecalcitol (ED-71) is a representative of the third-generation novel active vitamin D analogs, and its specific immunological mechanisms in ameliorating diabetic osteoporosis remain unclear. We herein evaluated the therapeutic effects of ED-71 in the context of type 2 diabetes mellitus (T2DM), delving into its underlying mechanisms. In a T2DM mouse model, ED-71 attenuated bone loss and marrow adiposity. Simultaneously, it rectified imbalanced glucose homeostasis and dyslipidemia, ameliorated pancreatic β-cell damage and hepatic glycolipid metabolism disorder. Subsequently, in mice injected with the Treg cell-depleting agent CD25, we observed that the beneficial effects of ED-71 mentioned earlier were partially contingent on the Treg subsets ratio. Mechanistically, ED-71 promoted the differentiation of CD4+ T cells into Treg subsets, facilitating Ca2+ influx and the expression of ORAI1 and STIM1, pivotal proteins in store-operated Ca2+ entry (SOCE). The SOCE inhibitor, 2-APB, partially attenuated the positive effects of ED-71 observed in the above results. Overall, ED-71 regulates SOCE-mediated Treg cell differentiation, accomplishing the dual purpose of simultaneously ameliorating diabetic osteoporosis and glucolipid metabolic disorders, showcasing its potential in osteoimmunity therapy and interventions for diseases involving SOCE.

TAM-associated CASQ1 mutants diminish intracellular Ca2+ content and interfere with regulation of SOCE.

Tubular aggregate myopathy (TAM) is a rare myopathy characterized by muscle weakness and myalgia. Muscle fibers from TAM patients show characteristic accumulation of membrane tubules that contain proteins from the sarcoplasmic reticulum (SR). Gain-of-function mutations in STIM1 and ORAI1, the key proteins participating in the Store-Operated Ca2+ Entry (SOCE) mechanism, were identified in patients with TAM. Recently, the CASQ1 gene was also found to be mutated in patients with TAM. CASQ1 is the main Ca2+ buffer of the SR and a negative regulator of SOCE. Previous characterization of CASQ1 mutants in non-muscle cells revealed that they display altered Ca2+dependent polymerization, reduced Ca2+storage capacity and alteration in SOCE inhibition. We thus aimed to assess how mutations in CASQ1 affect calcium regulation in skeletal muscles, where CASQ1 is naturally expressed. We thus expressed CASQ1 mutants in muscle fibers from Casq1 knockout mice, which provide a valuable model for studying the Ca2+ storage capacity of TAM-associated mutants. Moreover, since Casq1 knockout mice display a constitutively active SOCE, the effect of CASQ1 mutants on SOCE inhibition can be also properly examined in fibers from these mice. Analysis of intracellular Ca2+ confirmed that CASQ1 mutants have impaired ability to store Ca2+and lose their ability to inhibit skeletal muscle SOCE; this is in agreement with the evidence that alterations in Ca2+entry due to mutations in either STIM1, ORAI1 or CASQ1 represents a hallmark of TAM.

Dynasore modulates store-operated calcium entry and mitochondrial calcium release in corneal epithelial cells

Dysregulation of calcium homeostasis can precipitate a cascade of pathological events that lead to tissue damage and cell death. Dynasore is a small molecule that inhibits endocytosis by targeting classic dynamins. In a previous study, we showed that dynasore can protect human corneal epithelial cells from damage due to tert-butyl hydroperoxide (tBHP) exposure by restoring cellular calcium (Ca2+) homeostasis. Here we report results of a follow-up study aimed at identifying the source of the damaging Ca2+. Store-operated Ca2+ entry (SOCE) is a cellular mechanism to restore intracellular calcium stores from the extracellular milieu. We found that dynasore effectively blocks SOCE in cells treated with thapsigargin (TG), a small molecule that inhibits pumping of Ca2+ into the endoplasmic reticulum (ER). Unlike dynasore however, SOCE inhibitor YM-58483 did not interfere with the cytosolic Ca2+ overload caused by tBHP exposure. We also found that dynasore effectively blocks Ca2+ release from internal sources. The inefficacy of inhibitors of ER Ca2+ channels suggested that this compartment was not the source of the Ca2+ surge caused by tBHP exposure. However, using a Ca2+-measuring organelle-entrapped protein indicator (CEPIA) reporter targeted to mitochondria, we found that dynasore can block mitochondrial Ca2+ release due to tBHP exposure. Our results suggest that dynasore exerts multiple effects on cellular Ca2+ homeostasis, with inhibition of mitochondrial Ca2+ release playing a key role in protection of corneal epithelial cells against oxidative stress due to tBHP exposure.

The Rise in Tubular pH during Hypercalciuria Exacerbates Calcium Stone Formation.

In calcium nephrolithiasis (CaNL), most calcium kidney stones are identified as calcium oxalate (CaOx) with variable amounts of calcium phosphate (CaP), where CaP is found as the core component. The nucleation of CaP could be the first step of CaP+CaOx (mixed) stone formation. High urinary supersaturation of CaP due to hypercalciuria and an elevated urine pH have been described as the two main factors in the nucleation of CaP crystals. Our previous in vivo findings (in mice) show that transient receptor potential canonical type 3 (TRPC3)-mediated Ca2+ entry triggers a transepithelial Ca2+ flux to regulate proximal tubular (PT) luminal [Ca2+], and TRPC3-knockout (KO; -/-) mice exhibited moderate hypercalciuria and microcrystal formation at the loop of Henle (LOH). Therefore, we utilized TRPC3 KO mice and exposed them to both hypercalciuric [2% calcium gluconate (CaG) treatment] and alkalineuric conditions [0.08% acetazolamide (ACZ) treatment] to generate a CaNL phenotype. Our results revealed a significant CaP and mixed crystal formation in those treated KO mice (KOT) compared to their WT counterparts (WTT). Importantly, prolonged exposure to CaG and ACZ resulted in a further increase in crystal size for both treated groups (WTT and KOT), but the KOT mice crystal sizes were markedly larger. Moreover, kidney tissue sections of the KOT mice displayed a greater CaP and mixed microcrystal formation than the kidney sections of the WTT group, specifically in the outer and inner medullary and calyceal region; thus, a higher degree of calcifications and mixed calcium lithiasis in the kidneys of the KOT group was displayed. In our effort to find the Ca2+ signaling pathophysiology of PT cells, we found that PT cells from both treated groups (WTT and KOT) elicited a larger Ca2+ entry compared to the WT counterparts because of significant inhibition by the store-operated Ca2+ entry (SOCE) inhibitor, Pyr6. In the presence of both SOCE (Pyr6) and ROCE (receptor-operated Ca2+ entry) inhibitors (Pyr10), Ca2+ entry by WTT cells was moderately inhibited, suggesting that the Ca2+ and pH levels exerted sensitivity changes in response to ROCE and SOCE. An assessment of the gene expression profiles in the PT cells of WTT and KOT mice revealed a safeguarding effect of TRPC3 against detrimental processes (calcification, fibrosis, inflammation, and apoptosis) in the presence of higher pH and hypercalciuric conditions in mice. Together, these findings show that compromise in both the ROCE and SOCE mechanisms in the absence of TRPC3 under hypercalciuric plus higher tubular pH conditions results in higher CaP and mixed crystal formation and that TRPC3 is protective against those adverse effects.

Side-by-side comparison of published small molecule inhibitors against thapsigargin-induced store-operated Ca2+ entry in HEK293 cells.

Calcium (Ca2+) is a key second messenger in eukaryotes, with store-operated Ca2+ entry (SOCE) being the main source of Ca2+ influx into non-excitable cells. ORAI1 is a highly Ca2+-selective plasma membrane channel that encodes SOCE. It is ubiquitously expressed in mammals and has been implicated in numerous diseases, including cardiovascular disease and cancer. A number of small molecules have been identified as inhibitors of SOCE with a variety of potential therapeutic uses proposed and validated in vitro and in vivo. These encompass both nonselective Ca2+ channel inhibitors and targeted selective inhibitors of SOCE. Inhibition of SOCE can be quantified both directly and indirectly with a variety of assay setups, making an accurate comparison of the activity of different SOCE inhibitors challenging. We have used a fluorescence based Ca2+ addback assay in native HEK293 cells to generate dose-response data for many published SOCE inhibitors. We were able to directly compare potency. Most compounds were validated with only minor and expected variations in potency, but some were not. This could be due to differences in assay setup relating to the mechanism of action of the inhibitors and highlights the value of a singular approach to compare these compounds, as well as the general need for biorthogonal validation of novel bioactive compounds. The compounds observed to be the most potent against SOCE in our study were: 7-azaindole 14d (12), JPIII (17), Synta-66 (6), Pyr 3 (5), GSK5503A (8), CM4620 (14) and RO2959 (7). These represent the most promising candidates for future development of SOCE inhibitors for therapeutic use.

Inhibition of altered Orai1 channels in Müller cells protects photoreceptors in retinal degeneration.

The expressions of ion channels by Müller glial cells (MGCs) may change in response to various retinal pathophysiological conditions. There remains a gap in our understanding of MGCs' responses to photoreceptor degeneration towards finding therapies. The study explores how an inhibition of store-operated Ca2+ entry (SOCE) and its major component, Orai1 channel, in MGCs protects photoreceptors from degeneration. The study revealed increased Orai1 expression in the MGCs of retinal degeneration 10 (rd10) mice. Enhanced expression of oxidative stress markers was confirmed as a crucial pathological mechanism in rd10 retina. Inducing oxidative stress in rat MGCs resulted in increasing SOCE and Ca2+ release-activated Ca2+ (CRAC) currents. SOCE inhibition by 2-Aminoethoxydiphenyl borate (2-APB) protected photoreceptors in degenerated retinas. Finally, molecular simulations proved the structural and dynamical features of 2-APB to the target structure Orai1. Our results provide new insights into the physiology of MGCs regarding retinal degeneration and shed a light on SOCE and Orai1 as new therapeutic targets.

2-Methoxyestradiol-3,17-O,O-bis-sulfamate inhibits store-operated Ca2+ entry in T lymphocytes and prevents experimental autoimmune encephalomyelitis

Ca2+ signaling is one of the essential signaling systems for T lymphocyte activation, the latter being an essential step in the pathogenesis of autoimmune diseases such as multiple sclerosis (MS). Store-operated Ca2+ entry (SOCE) ensures long lasting Ca2+ signaling and is of utmost importance for major downstream T lymphocyte activation steps, e.g. nuclear localization of the transcription factor ‘nuclear factor of activated T cells’ (NFAT). 2-Methoxyestradiol (2ME2), an endogenous metabolite of estradiol (E2), blocks nuclear translocation of NFAT. The likely underlying mechanism is inhibition of SOCE, as shown for its synthetic sulfamate ester analogue 2-ethyl-3-sulfamoyloxy-17β-cyanomethylestra-1,3,5(10)-triene (STX564).Here, we demonstrate that another synthetic bis-sulfamoylated 2ME2 derivative, 2-methoxyestradiol-3,17-O,O-bis-sulfamate (2-MeOE2bisMATE, STX140), an orally bioavailable, multi-targeting anticancer agent and potent steroid sulfatase (STS) inhibitor, antagonized SOCE in T lymphocytes. Downstream events, e.g. secretion of the pro-inflammatory cytokines interferon-γ and interleukin-17, were decreased by STX140 in in vitro experiments.Remarkably, STX140 dosed in vivo completely blocked the clinical disease in both active and transfer experimental autoimmune encephalomyelitis (EAE) in Lewis rats, a T cell-mediated animal model for MS, at a dose of 10 mg/kg/day i.p., whereas neither 2ME2 nor Irosustat, a pure STS inhibitor, showed any effect. The STS inhibitory activity of STX140 is therefore not responsible for its activity in this model.Taken together, inhibition of SOCE by STX140 resulting in full antagonism of clinical symptoms in EAE in the Lewis rat, paired with the known excellent bioavailability and pharmaceutical profile of this drug, open potentially new therapeutic avenues for the treatment of MS.

Lysosomal Ca2+ as a mediator of palmitate-induced lipotoxicity

While the mechanism of lipotoxicity by palmitic acid (PA), an effector of metabolic stress in vitro and in vivo, has been extensively investigated, molecular details of lipotoxicity are still not fully characterized. Since recent studies reported that PA can exert lysosomal stress in addition to well-known ER and mitochondrial stress, we studied the role of lysosomal events in lipotoxicity by PA, focusing on lysosomal Ca2+. We found that PA induced accumulation of mitochondrial ROS and that mitochondrial ROS induced release of lysosomal Ca2+ due to lysosomal Ca2+ exit channel activation. Lysosomal Ca2+ release led to increased cytosolic Ca2+ which induced mitochondrial permeability transition (mPT). Chelation of cytoplasmic Ca2+ or blockade of mPT with olesoxime or decylubiquinone (DUB) suppressed lipotoxicity. Lysosomal Ca2+ release led to reduced lysosomal Ca2+ content which was replenished by ER Ca2+, the largest intracellular Ca2+ reservoir (ER → lysosome Ca2+ refilling), which in turn activated store-operated Ca2+ entry (SOCE). Inhibition of ER → lysosome Ca2+ refilling by blockade of ER Ca2+ exit channel using dantrolene or inhibition of SOCE using BTP2 inhibited lipotoxicity in vitro. Dantrolene or DUB also inhibited lipotoxic death of hepatocytes in vivo induced by administration of ethyl palmitate together with LPS. These results suggest a novel pathway of lipotoxicity characterized by mPT due to lysosomal Ca2+ release which was supplemented by ER → lysosome Ca2+ refilling and subsequent SOCE, and also suggest the potential role of modulation of ER → lysosome Ca2+ refilling by dantrolene or other blockers of ER Ca2+ exit channels in disease conditions characterized by lipotoxicity such as metabolic syndrome, diabetes, cardiomyopathy or nonalcoholic steatohepatitis.

Stromal Interaction Molecule 1-Mediated Store-Operated Calcium Entry Promotes Autophagy Through AKT/Mammalian Target of Rapamycin Pathway in Hippocampal Neurons After Ischemic Stroke

The pathophysiological process of neuronal injury due to cerebral ischemia is complex among which disturbance of calcium homeostasis and autophagy are two major pathogenesis. However, it remains ambiguous whether the two factors are independent. Stromal interaction molecule 1 (STIM1) is the most important Ca2+ sensor mediating the store-operated Ca2+ entry (SOCE) through interacting with Orai1 and has recently been proven to participate in autophagy in multiple cells. In this study, we aimed to investigate the potential role of STIM1-induced SOCE on autophagy and whether its regulator function contributes to neuronal injury under hypoxic conditions using in vivo transient middle cerebral artery occlusion (tMCAO) model and in vitro oxygen and glucose deprivation (OGD) primary cultured neuron model respectively. The present data indicated that STIM1 induces autophagic flux impairment in neurons through promoting SOCE and inhibiting AKT/mTOR signaling pathway. Pharmacological inhibition of SOCE or downregulation of STIM1 with siRNA suppressed the autophagic activity in neurons. Moreover, stim1 knockdown attenuated neurological deficits and brain damage after tMCAO, which could be reversed by AKT/mTOR pathway inhibitor AZD5363. Together, the modulation of STIM1 on autophagic activation indicated the potential link between Ca2+ homeostasis and autophagy which provided evidence that STIM1 could be a promising therapeutic target for ischemic stroke.

Chronic reduction of store operated Ca2+ entry is viable therapeutically but is associated with cardiovascular complications.

Loss of function mutations in store-operated Ca2+ entry (SOCE) are associated with severe paediatric disorders in humans, including combined immunodeficiency, anaemia, thrombocytopenia, anhidrosis and muscle hypotonia. Given its central role in immune cell activation, SOCE has been a therapeutic target for autoimmune and inflammatory diseases. Treatment for such chronic diseases would require prolonged SOCE inhibition. It is, however, unclear whether chronic SOCE inhibition is viable therapeutically. Here we address this issue using a novel genetic mouse model (SOCE hypomorph) with deficient SOCE, nuclear factor of activated T cells activation, and T cell cytokine production. SOCE hypomorph mice develop and reproduce normally and do not display muscle weakness or overt anhidrosis. They do, however, develop cardiovascular complications, including hypertension and tachycardia, which we show are due to increased sympathetic autonomic nervous system activity and not cardiac or vascular smooth muscle autonomous defects. These results assert that chronic SOCE inhibition is viable therapeutically if the cardiovascular complications can be managed effectively clinically. They further establish the SOCE hypomorph line as a genetic model to define the therapeutic window of SOCE inhibition and dissect toxicities associated with chronic SOCE inhibition in a tissue-specific fashion. KEY POINTS: A floxed stromal interaction molecule 1 (STIM1)hypomorph mouse model was generated with significant reduction in Ca2+ influx through store-operated Ca2+ entry (SOCE), resulting in defective nuclear translocation of nuclear factor of activated T cells, cytokine production and inflammatory response. The hypomorph mice are viable and fertile, with no overt defects. Decreased SOCE in the hypomorph mice is due to poor translocation of the mutant STIM1 to endoplasmic reticulum-plasma membrane contact sites resulting in fewer STIM1 puncta. Hypomorph mice have similar susceptibility to controls to develop diabetes but exhibit tachycardia and hypertension. The hypertension is not due to increased vascular smooth muscle contractility or vascular remodelling. The tachycardia is not due to heart-specific defects but rather seems to be due to increased circulating catecholamines in the hypomorph. Therefore, long term SOCE inhibition is viable if the cardiovascular defects can be managed clinically.

Distinct roles of ORAI1 in T cell-mediated allergic airway inflammation and immunity to influenza A virus infection.

T cell activation and function depend on Ca2+ signals mediated by store-operated Ca2+ entry (SOCE) through Ca2+ release–activated Ca2+ (CRAC) channels formed by ORAI1 proteins. We here investigated how SOCE controls T cell function in pulmonary inflammation during a T helper 1 (TH1) cell–mediated response to influenza A virus (IAV) infection and TH2 cell–mediated allergic airway inflammation. T cell–specific deletion of Orai1 did not exacerbate pulmonary inflammation and viral burdens following IAV infection but protected mice from house dust mite–induced allergic airway inflammation. ORAI1 controlled the expression of genes including p53 and E2F transcription factors that regulate the cell cycle in TH2 cells in response to allergen stimulation and the expression of transcription factors and cytokines that regulate TH2 cell function. Systemic application of a CRAC channel blocker suppressed allergic airway inflammation without compromising immunity to IAV infection, suggesting that inhibition of SOCE is a potential treatment for allergic airway disease.

Store‐operated calcium entry controls innate and adaptive immune cell function in inflammatory bowel disease

Inflammatory bowel disease (IBD) is characterized by dysregulated intestinal immune responses. Using mass cytometry (CyTOF) to analyze the immune cell composition in the lamina propria (LP) of patients with ulcerative colitis (UC) and Crohn's disease (CD), we observed an enrichment of CD4+ effector T cells producing IL‐17A and TNF, CD8+ T cells producing IFNγ, T regulatory (Treg) cells, and innate lymphoid cells (ILC). The function of these immune cells is regulated by store‐operated Ca2+ entry (SOCE), which results from the opening of Ca2+ release‐activated Ca2+ (CRAC) channels formed by ORAI and STIM proteins. We observed that the pharmacologic inhibition of SOCE attenuated the production of proinflammatory cytokines including IL‐2, IL‐4, IL‐6, IL‐17A, TNF, and IFNγ by human colonic T cells and ILCs, reduced the production of IL‐6 by B cells and the production of IFNγ by myeloid cells, but had no effect on the viability, differentiation, and function of intestinal epithelial cells. T cell‐specific deletion of CRAC channel genes in mice showed that Orai1, Stim1, and Stim2‐deficient T cells have quantitatively distinct defects in SOCE, which correlate with gradually more pronounced impairment of cytokine production by Th1 and Th17 cells and the severity of IBD. Moreover, the pharmacologic inhibition of SOCE with a selective CRAC channel inhibitor attenuated IBD severity and colitogenic T cell function in mice. Our data indicate that SOCE inhibition may be a suitable new approach for the treatment of IBD.

Ginsenoside Rg-1 prevents elevated cytosolic Ca2+ via store-operated Ca2+ entry in high-glucose–stimulated vascular endothelial and smooth muscle cells

BackgroundGinsenoside Rg-1 (Rg-1), a triterpenoid saponin abundantly present in Panax ginseng, is a type of naturally occurring steroid with known anti-diabetic and anti-inflammatory effects. In this study, we sought to confirm the effects and mechanisms of action of Rg-1 on store-operated Ca2+ entry (SOCE) in human vascular endothelial cell line (EA) and murine aortic vascular smooth muscle cell line (MOVAS) cells exposed to high glucose.MethodsCytosolic Ca2+ concentrations in EA and MOVAS cells were measured by monitoring fluorescence of the ratiometric Ca2+-indicator, Fura-2 AM.ResultsHigh glucose significantly increased Ca2+ influx by abnormally activating SOCE in EA and MOVAS cells. Notably, this high glucose-induced increase in SOCE was restored to normal levels in EA and MOVAS cells by Rg-1. Moreover, Rg-1 induced reductions in SOCE in cells exposed to high glucose were significantly inhibited by the plasma membrane Ca2+ ATPase (PMCA) blocker lanthanum, the Na+/K+-ATPase blocker ouabain, or the Na+/Ca2+ exchanger (NCX) blockers Ni2+ and KB-R7943. These observations suggest that the mechanism of action of Rg-1 inhibition of SOCE involves PMCA and Na+/K+-ATPase, and an increase in Ca2+ efflux via NCXs in both EA and MOVAS cells exposed to high glucose.ConclusionsThese findings indicate that Rg-1 may protect vascular endothelial and smooth muscle cells from Ca2+ increases following exposure to hyperglycemic conditions.

Store-Operated Ca2+ Entry Contributes to Piezo1-Induced Ca2+ Increase in Human Endometrial Stem Cells.

Endometrial mesenchymal stem cells (eMSCs) are a specific class of stromal cells which have the capability to migrate, develop and differentiate into different types of cells such as adipocytes, osteocytes or chondrocytes. It is this unique plasticity that makes the eMSCs significant for cellular therapy and regenerative medicine. Stem cells choose their way of development by analyzing the extracellular and intracellular signals generated by a mechanical force from the microenvironment. Mechanosensitive channels are part of the cellular toolkit that feels the mechanical environment and can transduce mechanical stimuli to intracellular signaling pathways. Here, we identify previously recorded, mechanosensitive (MS), stretch-activated channels as Piezo1 proteins in the plasma membrane of eMSCs. Piezo1 activity triggered by the channel agonist Yoda1 elicits influx of Ca2+, a known modulator of cytoskeleton reorganization and cell motility. We found that store-operated Ca2+ entry (SOCE) formed by Ca2+-selective channel ORAI1 and Ca2+ sensors STIM1/STIM2 contributes to Piezo1-induced Ca2+ influx in eMSCs. Particularly, the Yoda1-induced increase in intracellular Ca2+ ([Ca2+]i) is partially abolished by 2-APB, a well-known inhibitor of SOCE. Flow cytometry analysis and wound healing assay showed that long-term activation of Piezo1 or SOCE does not have a cytotoxic effect on eMSCs but suppresses their migratory capacity and the rate of cell proliferation. We propose that the Piezo1 and SOCE are both important determinants in [Ca2+]i regulation, which critically affects the migratory activity of eMSCs and, therefore, could influence the regenerative potential of these cells.

Extracellular cysteines C226 and C232 mediate hydrogen sulfide-dependent inhibition of Orai3-mediated store-operated calcium entry.

The gaseous signaling molecule hydrogen sulfide (H2S) physiologically regulates store-operated Ca2+ entry (SOCE). The SOCE machinery consists of the plasma membrane-localized Orai channels (Orai1-3) and endoplasmic reticulum-localized stromal interaction molecule (STIM)1 and STIM2 proteins. H2S inhibits Orai3- but not Orai1- or Orai2-mediated SOCE. The current objective was to define the mechanism by which H2S selectively modifies Orai3. We measured SOCE and STIM1/Orai3 dynamics and interactions in HEK293 cells exogenously expressing fluorescently tagged human STIM1 and Orai3 in the presence and absence of the H2S donor GYY4137. Two cysteines (C226 and C232) are present in Orai3 that are absent in the Orai1 and Orai2. When we mutated either of these cysteines to serine, alone or in combination, SOCE inhibition by H2S was abolished. We also established that inhibition was dependent on an interaction with STIM1. To further define the effects of H2S on STIM1/Orai3 interaction, we performed a series of fluorescence recovery after photobleaching (FRAP), colocalization, and fluorescence resonance energy transfer (FRET) experiments. Treatment with H2S did not affect the mobility of Orai3 in the membrane, nor did it influence STIM1/Orai3 puncta formation or STIM1-Orai3 protein-protein interactions. These data support a model in which H2S modification of Orai3 at cysteines 226 and 232 limits SOCE evoked upon store depletion and STIM1 engagement, by a mechanism independent of the interaction between Orai3 and STIM1.

Emergent role of SARAF and store-operated Ca2+ entry in angiogenesis

Angiogenesis is a multistep process that controls endothelial cell (EC) functioning to form new blood vessels from preexisting vascular beds. This process is tightly regulated by pro-angiogenic factors, such as vascular endothelial growth factor (VEGF), which promotes signaling pathways involving the increase in the intracellular Ca2+ concentration ([Ca2+]i). Recent evidence suggests that store-operated Ca2+ entry (SOCE) might play a role in angiogenesis. However, little is known regarding the role of SARAF, SOCE-associated regulatory factor in this process. The aim of this study is to examine the role of SARAF in angiogenesis. In vitro angiogenesis was studied using human umbilical endothelial cells (HUVECs) for tube formation assay and vessel sprouting using rat aortic ring by Matrigel assay supplemented with endothelial cell basal medium enriched with different growth factors (VEGF, FGF, b-EGF, and IGF). HUVECs migration was evaluated by wound healing assay, and HUVECs proliferation using Ki67+ marker. Ex vivo angiogenesis was examined by whole mount mice retina on P6 in neonatal mice injected with increasing concentrations of a SOCE inhibitor, GSK-7975A, on P3, P4, and P5. We observed that SOCE inhibition with GSK-7975A blocks aorta sprouting, as well as HUVEC tube formation and migration. The intraperitoneal injection of GSK-7975A also delays the development of retinal vasculature assessed at postnatal day 6 in mice since it reduces vessel length and the number of junctions while it increases lacunarity. Moreover, we found that knockdown of SARAF using siRNA impairs VEGF-mediated [Ca2+]i increase and HUVEC tube formation, proliferation, and migration. Our data show for the first that SOCE inhibition prevents angiogenesis using different approaches and we provide evidence indicating that SARAF plays a critical role in angiogenesis.

Glial ER and GAP junction mediated Ca2+ waves are crucial to maintain normal brain excitability.

Astrocytes play key roles in regulating multiple aspects of neuronal function from invertebrates to humans and display Ca2+ fluctuations that are heterogeneously distributed throughout different cellular microdomains. Changes in Ca2+ dynamics represent a key mechanism for how astrocytes modulate neuronal activity. An unresolved issue is the origin and contribution of specific glial Ca2+ signaling components at distinct astrocytic domains to neuronal physiology and brain function. The Drosophila model system offers a simple nervous system that is highly amenable to cell-specific genetic manipulations to characterize the role of glial Ca2+ signaling. Here we identify a role for ER store-operated Ca2+ entry (SOCE) pathway in perineurial glia (PG), a glial population that contributes to the Drosophila blood-brain barrier. We show that PG cells display diverse Ca2+ activity that varies based on their locale within the brain. Ca2+ signaling in PG cells does not require extracellular Ca2+ and is blocked by inhibition of SOCE, Ryanodine receptors, or gap junctions. Disruption of these components triggers stimuli-induced seizure-like episodes. These findings indicate that Ca2+ release from internal stores and its propagation between neighboring glial cells via gap junctions are essential for maintaining normal nervous system function.

Development of Store-Operated Calcium Entry-Targeted Compounds in Cancer.

Store-operated Ca2+ entry (SOCE) is the major pathway of Ca2+ entry in mammalian cells, and regulates a variety of cellular functions including proliferation, motility, apoptosis, and death. Accumulating evidence has indicated that augmented SOCE is related to the generation and development of cancer, including tumor formation, proliferation, angiogenesis, metastasis, and antitumor immunity. Therefore, the development of compounds targeting SOCE has been proposed as a potential and effective strategy for use in cancer therapy. In this review, we summarize the current research on SOCE inhibitors and blockers, discuss their effects and possible mechanisms of action in cancer therapy, and induce a new perspective on the treatment of cancer.

Ang II Promotes Cardiac Autophagy and Hypertrophy via Orai1/STIM1.

The pathophysiology of cardiac hypertrophy is complex and multifactorial. Both the store-operated Ca2+ entry (SOCE) and excessive autophagy are the major causative factors for pathological cardiac hypertrophy. However, it is unclear whether these two causative factors are interdependent. In this study, we examined the functional role of SOCE and Orai1 in angiotensin II (Ang II)-induced autophagy and hypertrophy using in vitro neonatal rat cardiomyocytes (NRCMs) and in vivo mouse model, respectively. We show that YM-58483 or SKF-96365 mediated pharmacological inhibition of SOCE, or silencing of Orai1 with Orail-siRNA inhibited Ang II-induced cardiomyocyte autophagy both in vitro and in vivo. Also, the knockdown of Orai1 attenuated Ang II-induced pathological cardiac hypertrophy. Together, these data suggest that Ang II promotes excessive cardiomyocyte autophagy through SOCE/Orai1 which can be the prime contributing factors in cardiac hypertrophy.

STIM2 protein regulates Orai1-mediated store-operated Ca2+ entry in cardiomyocytes

Stromal interaction molecules, STIM1 and STIM2, are ER Ca 2+ sensors that initiate store-operated Ca 2+ entry (SOCE). It has been recently identified three STIM2 splice variants (STIM2.1, 2.2 and 2.3), STIM2.2 being SOCE activator whereas STIM2.1 will be SOCE inhibitor. The role of STIM1-mediated SOCE has been highlighted in cardiac pathobiology but the one of STIM 2 .1 and STIM2.2 is unknown. To assess whether and how STIM2.1 and STIM2.2 regulate SOCE in neonatal rat ventricular cardiomyocytes (NRVMs). To analyze the function of STIM2.1 and STIM2.2 variants, the NRVMs were transfected with STIM2.2 or STIM2.1 in combination or not with STIM1 or dn-Orai1 106A mutant plasmids or with a mixture of 4 STIM2 siRNAs. At rest, both native STIM2 and exogenous STIM2.2 proteins present a reticular organization. Live cell confocal imaging in STIM2.2 overexpressing cells showed that STIM2.2 is aggregated and translocated towards the sarcolemma following the SR Ca 2+ store depletion. With Ca 2+ imaging, we found that STIM2.2 overexpression significantly increases SOCE, similarly to STIM1 overexpression, but the STIM1 and STIM2.2 co-expression has no additional effect. Importantly, the STIM2.2-enhanced SOCE is suppressed by functional Orai1 inhibition, either by co-expressing STIM2.2 with the dn-Orai1 106A mutant or with the selective Orai1 inhibitor, JPIII. In addition, the JPIII-sensitive Isoc current is also higher in STIM2.2 overexpressing cells. Nonetheless, silencing of endogenous STIM2 also increases SOCE. By co-immunoprecipitation, we found that STIM2, STIM1 and Orai1 interact together suggesting that all these proteins form a complex and constitute functional SOCE machinery. We demonstrated that, in overexpressing system, STIM2.2 functions together with STIM1 as a positive regulator of Orai1-mediated SOCE and that, in endogenous system, the STIM2.1 variant may be the predominant variant that negatively regulates SOCE in NRVMs.