Rapamycin Controls Lymphoproliferation and Reverses T-Cell Responses in a Patient with a Novel STIM1 Loss-of-Function Deletion.

PURPOSE: Deficiency of stromal interaction molecule 1 (STIM1) results in combined immunodeficiency accompanied by extra-immunological findings like enamel defects and myopathy. We here studied a patient with a STIM1 loss-of-function mutation who presented with severe lymphoproliferation. We sought to explore the efficacy of the mTOR inhibitor rapamycin in controlling disease manifestations and reversing aberrant T-cell subsets and functions, which has never been used previously in this disorder. METHODS: Clinical findings of the patient were collected over time. We performed immunological evaluations before and after initiation of rapamycin treatment, including detailed lymphocyte subset analyses, alterations in frequencies of circulating T follicular helper (cTFH) and regulatory T (Treg) cells and their subtypes as well as T cell activation and proliferation capacities. RESULTS: A novel homozygous exon 2 deletion in STIM1 was detected in a 3-year-old girl with severe lymphoproliferation, recurrent infections, myopathy, iris hypoplasia, and enamel hypoplasia. Lymphoproliferation was associated with severe T-cell infiltrates. The deletion resulted in a complete loss of protein expression, associated with a lack of store-operated calcium entry response, defective T-cell activation, proliferation, and cytokine production. Interestingly, patient blood contained fewer cTFH and increased circulating follicular regulatory (cTFR) cells. Abnormal skewing towards TH2-like responses in certain T-cell subpopulations like cTFH, non-cTFH memory T-helper, and Treg cells was associated with increased eosinophil numbers and serum IgE levels. Treatment with rapamycin controlled lymphoproliferation, improved T-cell activation and proliferation capacities, reversed T-cell responses, and repressed high IgE levels and eosinophilia. CONCLUSIONS: This study enhances our understanding of STIM1 deficiency by uncovering additional abnormal T-cell responses, and reveals for the first time the potential therapeutic utility of rapamycin for this disorder.

The Role and Mechanism of STIM1/Orai1-Regulated Ca2+ Influx in Myocardial Hypertrophy in Type 2 Diabetes Mellitus.

OBJECTIVE: Diabetic cardiomyopathy (DCM) is the most common cardiovascular complication of type 2 diabetes mellitus (T2DM). Patients affected with DCM face a notably higher risk of progressing to congestive heart failure compared to other populations. Myocardial hypertrophy, a clearly confirmed pathological change in DCM, plays an important role in the development of DCM, with abnormal Ca2+ homeostasis serving as the key signal to induce myocardial hypertrophy. Therefore, investigating the mechanism of Ca2+ transport is of great significance for the prevention and treatment of myocardial hypertrophy in T2DM. METHODS: The rats included in the experiment were divided into wild type (WT) group and T2DM group. The T2DM rat model was established by feeding the rats with high-fat and high-sugar diets for three months combined with low dose of streptozotocin (100mg/kg). Afterwards, primary rat cardiomyocytes were isolated and cultured, and cardiomyocyte hypertrophy was induced through high-glucose treatment. Subsequently, mechanistic investigations were carried out through transfection with si-STIM1 and oe-STIM1. Western blot (WB) was used to detect the expression of the STIM1, Orai1 and p-CaMKII. qRT-PCR was used to detect mRNA levels of myocardial hypertrophy marker proteins. Cell surface area was detected using TRITC-Phalloidin staining, and intracellular Ca2+ concentration in cardiomyocytes was measured using Fluo-4 fluorescence staining. RESULTS: Through animal experiments, an upregulation of Orai1 and STIM1 was revealed in the rat model of myocardial hypertrophy induced by T2DM. Meanwhile, through cell experiments, it was found that in high glucose (HG)-induced hypertrophic cardiomyocytes, the expression of STIM1, Orai1, and p-CaMKII was upregulated, along with increased levels of store-operated Ca2+ entry (SOCE) and abnormal Ca2+ homeostasis. However, when STIM1 was downregulated in HG-induced cardiomyocytes, SOCE levels decreased and p-CaMKII was downregulated, resulting in an improvement in myocardial hypertrophy. To further elucidate the mechanism of action involving SOCE and CaMKII in T2DM-induced myocardial hypertrophy, high-glucose cardiomyocytes were respectively treated with BTP2 (SOCE blocker) and KN-93 (CaMKII inhibitor), and the results showed that STIM1 can mediate SOCE, thereby affecting the phosphorylation level of CaMKII and improving cardiomyocyte hypertrophy. CONCLUSION: STIM1/Orai1-mediated SOCE regulates p-CaMKII levels, thereby inducing myocardial hypertrophy in T2DM.

STIM2 variants regulate Orai1/TRPC1/TRPC4-mediated store-operated Ca2+ entry and mitochondrial Ca2+ homeostasis in cardiomyocytes.

The stromal interaction molecules (STIMs) are the sarcoplasmic reticulum (SR) Ca2+ sensors that trigger store-operated Ca2+ entry (SOCE) in a variety of cell types. While STIM1 isoform has been the focus of the research in cardiac pathophysiology, the function of the homolog STIM2 remains unknown. Using Ca2+ imaging and patch-clamp techniques, we showed that knockdown (KD) of STIM2 by siRNAs increased SOCE and the ISOC current in neonatal rat ventricular cardiomyocytes (NRVMs). Within this cardiomyocyte model, we identified the transcript expression of Stim2.1 and Stim2.2 splice variants, with predominance for Stim2.2. Using conventional and super-resolution confocal microscopy (STED), we found that exogenous STIM2.1 and STIM2.2 formed pre-clusters with a reticular organization at rest. Following SR Ca2+ store depletion, some STIM2.1 and STIM2.2 clusters were translocated to SR-plasma membrane (PM) junctions and co-localized with Orai1. The overexpression strategy revealed that STIM2.1 suppressed Orai1-mediated SOCE and the ISOC current while STIM2.2 enhanced SOCE. STIM2.2-enhanced SOCE was also dependent on TRPC1 and TRPC4. Even if STIM2 KD or splice variants overexpression did not affect cytosolic Ca2+ cycling, we observed, using Rhod-2/AM Ca2+ imaging, that Orai1 inhibition or STIM2.1 overexpression abolished the mitochondrial Ca2+ (mCa2+) uptake, as opposed to STIM2 KD. We also found that STIM2 was present in the mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) by interacting with the inositol trisphosphate receptors (IP3Rs), voltage-dependent anion channel (VDAC), mitochondrial Ca2+ uniporter (MCU), and mitofusin-2 (MNF2). Our results suggested that, in NRVMs, STIM2.1 constitutes the predominant functional variant that negatively regulates Orai1-generated SOCE. It participates in the control of mCa2+ uptake capacity possibly via the STIM2-IP3Rs-VDAC-MCU and MNF2 complex.

The D84G mutation in STIM1 causes nuclear envelope dysfunction and myopathy in mice.

Stromal interaction molecule 1 (STIM1) is a Ca2+ sensor located in the sarcoplasmic reticulum (SR) of skeletal muscle, where it is best known for its role in store-operated Ca2+ entry (SOCE). Genetic syndromes resulting from STIM1 mutations are recognized as a cause of muscle weakness and atrophy. Here, we focused on a gain-of-function mutation that occurs in humans and mice (STIM1+/D84G mice), in which muscles exhibited constitutive SOCE. Unexpectedly, this constitutive SOCE did not affect global Ca2+ transients, SR Ca2+ content, or excitation-contraction coupling (ECC) and was therefore unlikely to underlie the reduced muscle mass and weakness observed in these mice. Instead, we demonstrate that the presence of D84G STIM1 in the nuclear envelope of STIM1+/D84G muscle disrupted nuclear-cytosolic coupling, causing severe derangement in nuclear architecture, DNA damage, and altered lamina A-associated gene expression. Functionally, we found that D84G STIM1 reduced the transfer of Ca2+ from the cytosol to the nucleus in myoblasts, resulting in a reduction of [Ca2+]N. Taken together, we propose a novel role for STIM1 in the nuclear envelope that links Ca2+ signaling to nuclear stability in skeletal muscle.

STIM1 translocation to the nucleus protects cells from DNA damage.

DNA damage represents a challenge for cells, as this damage must be eliminated to preserve cell viability and the transmission of genetic information. To reduce or eliminate unscheduled chemical modifications in genomic DNA, an extensive signaling network, known as the DNA damage response (DDR) pathway, ensures this repair. In this work, and by means of a proteomic analysis aimed at studying the STIM1 protein interactome, we have found that STIM1 is closely related to the protection from endogenous DNA damage, replicative stress, as well as to the response to interstrand crosslinks (ICLs). Here we show that STIM1 has a nuclear localization signal that mediates its translocation to the nucleus, and that this translocation and the association of STIM1 to chromatin increases in response to mitomycin-C (MMC), an ICL-inducing agent. Consequently, STIM1-deficient cell lines show higher levels of basal DNA damage, replicative stress, and increased sensitivity to MMC. We show that STIM1 normalizes FANCD2 protein levels in the nucleus, which explains the increased sensitivity of STIM1-KO cells to MMC. This study not only unveils a previously unknown nuclear function for the endoplasmic reticulum protein STIM1 but also expands our understanding of the genes involved in DNA repair.

Lead inhibits microglial cell migration via suppression of store-operated calcium entry.

Lead (Pb) is a non-biodegradable environmental pollutant that can lead to neurotoxicity by inducing neuroinflammation. Microglial activation plays a key role in neuroinflammation, and microglial migration is one of its main features. However, whether Pb affects microglial migration has not yet been elucidated. Herein, the effect of Pb on microglial migration was investigated using BV-2 microglial cells and primary microglial cells. The results showed that cell activation markers (TNF-α and CD206) in BV-2 cells were increased after Pb treatment. The migration ability of microglia was inhibited by Pb. Both store-operated calcium entry (SOCE) and the Ca2+ release-activated Ca2+ (CRAC) current were downregulated by microglia treatment with Pb in a dose-dependent manner. However, there was no statistical difference in the protein levels of stromal interaction molecule (STIM) 1, STIM2, or Ca2+ release-activated Ca2+ channel protein (Orai) 1 in microglia. The external Ca2+ influx and cell migration ability were restored to a certain extent after overexpression of either STIM1 or its CRAC activation domain in microglia. These results indicated that Pb inhibits microglial migration by downregulation of SOCE and impairment of the function of STIM1.

Genotypes of Stim1 and the proximal region on chromosome 1 exert opposite effects on stroke susceptibility in stroke-prone spontaneously hypertensive rat.

BACKGROUND: The stroke-prone spontaneously hypertensive rat (SHRSP) is a genetic model for cerebral stroke. Although a recent study on a congenic SHRSP suggested that a nonsense mutation in stromal interaction molecule 1 ( Stim1 ) encoding a major component of store-operated Ca 2+ entry was a causal variant for stroke in SHRSP, this was not conclusive because the congenic region including Stim1 in that rat was too wide. On the other hand, we demonstrated that the Wistar-Kyoto (WKY)-derived congenic fragment adjacent to Stim1 exacerbated stroke susceptibility in a congenic SHRSP called SPwch1.71. In the present study, we directly examined the effects of the Stim1 genotype on stroke susceptibility using SHRSP in which wild-type Stim1 was knocked in (called Stim1 -KI SHRSP). The combined effects of Stim1 and the congenic fragment of SPwch1.71 were also investigated. METHODS: Stroke susceptibility was assessed by the stroke symptom-free and survival periods based on observations of behavioral symptoms and reductions in body weight. RESULTS: Stim1 -KI SHRSP was more resistant to, while SPwch1.71 was more susceptible to stroke than the original SHRSP. Introgression of the wild-type Stim1 of Stim1 -KI SHRSP into SPwch1.71 by the generation of F1 rats ameliorated stroke susceptibility in SPwch1.71. Gene expression, whole-genome sequencing, and biochemical analyses identified Art2b , Folr1 , and Pde2a as possible candidate genes accelerating stroke in SPwch1.71. CONCLUSION: The substitution of SHRSP-type Stim1 to wild-type Stim1 ameliorated stroke susceptibility in both SHRSP and SPwch1.71, indicating that the nonsense mutation in Stim1 is causally related to stroke susceptibility in SHRSP.

An apical Phe-His pair defines the Orai1-coupling site and its occlusion within STIM1.

Ca2+ signal-generation through inter-membrane junctional coupling between endoplasmic reticulum (ER) STIM proteins and plasma membrane (PM) Orai channels, remains a vital but undefined mechanism. We identify two unusual overlapping Phe-His aromatic pairs within the STIM1 apical helix, one of which (F394-H398) mediates important control over Orai1-STIM1 coupling. In resting STIM1, this locus is deeply clamped within the folded STIM1-CC1 helices, likely near to the ER surface. The clamped environment in holo-STIM1 is critical-positive charge replacing Phe-394 constitutively unclamps STIM1, mimicking store-depletion, negative charge irreversibly locks the clamped-state. In store-activated, unclamped STIM1, Phe-394 mediates binding to the Orai1 channel, but His-398 is indispensable for transducing STIM1-binding into Orai1 channel-gating, and is spatially aligned with Phe-394 in the exposed Sα2 helical apex. Thus, the Phe-His locus traverses between ER and PM surfaces and is decisive in the two critical STIM1 functions-unclamping to activate STIM1, and conformational-coupling to gate the Orai1 channel.

A multiple-oscillator mechanism underlies antigen-induced Ca2+ oscillations in Jurkat T-cells.

T-cell receptor stimulation triggers cytosolic Ca2+ signaling by inositol-1,4,5-trisphosphate (IP3)-mediated Ca2+ release from the endoplasmic reticulum (ER) and Ca2+ entry through Ca2+ release-activated Ca2+ (CRAC) channels gated by ER-located stromal-interacting molecules (STIM1/2). Physiologically, cytosolic Ca2+ signaling manifests as regenerative Ca2+ oscillations, which are critical for nuclear factor of activated T-cells-mediated transcription. In most cells, Ca2+ oscillations are thought to originate from IP3 receptor-mediated Ca2+ release, with CRAC channels indirectly sustaining them through ER refilling. Here, experimental and computational evidence support a multiple-oscillator mechanism in Jurkat T-cells whereby both IP3 receptor and CRAC channel activities oscillate and directly fuel antigen-evoked Ca2+ oscillations, with the CRAC channel being the major contributor. KO of either STIM1 or STIM2 significantly reduces CRAC channel activity. As such, STIM1 and STIM2 synergize for optimal Ca2+ oscillations and activation of nuclear factor of activated T-cells 1 and are essential for ER refilling. The loss of both STIM proteins abrogates CRAC channel activity, drastically reduces ER Ca2+ content, severely hampers cell proliferation and enhances cell death. These results clarify the mechanism and the contribution of STIM proteins to Ca2+ oscillations in T-cells.

TSPAN18 facilitates bone metastasis of prostate cancer by protecting STIM1 from TRIM32-mediated ubiquitination.

BACKGROUND: Bone metastasis is a principal cause of mortality in patients with prostate cancer (PCa). Increasing evidence indicates that high expression of stromal interaction molecule 1 (STIM1)-mediated store-operated calcium entry (SOCE) significantly activates the calcium (Ca2+) signaling pathway and is involved in multiple steps of bone metastasis in PCa. However, the regulatory mechanism and target therapy of STIM1 is poorly defined. METHODS: Liquid chromatography-mass spectrometry analysis was performed to identify tetraspanin 18 (TSPAN18) as a binding protein of STIM1. Co-IP assay was carried out to explore the mechanism by which TSPAN18 inhibits STIM1 degradation. The biological function of TSPAN18 in bone metastasis of PCa was further investigated in vitro and in vivo models. RESULT: We identified that STIM1 directly interacted with TSPAN18, and TSPAN18 competitively inhibited E3 ligase tripartite motif containing 32 (TRIM32)-mediated STIM1 ubiquitination and degradation, leading to increasing STIM1 protein stability. Furthermore, TSPAN18 significantly stimulated Ca2+ influx in an STIM1-dependent manner, and then markedly accelerated PCa cells migration and invasion in vitro and bone metastasis in vivo. Clinically, overexpression of TSPAN18 was positively associated with STIM1 protein expression, bone metastasis and poor prognosis in PCa. CONCLUSION: Taken together, this work discovers a novel STIM1 regulative mechanism that TSPAN18 protects STIM1 from TRIM32-mediated ubiquitination, and enhances bone metastasis of PCa by activating the STIM1-Ca2+ signaling axis, suggesting that TSPAN18 may be an attractive therapeutic target for blocking bone metastasis in PCa.

Stromal Interaction Molecule 1 Maintains ß-Cell Identity and Function in Female Mice Through Preservation of G-Protein-Coupled Estrogen Receptor 1 Signaling.

Altered endoplasmic reticulum (ER) Ca2+ signaling has been linked with ß-cell dysfunction and diabetes development. Store-operated Ca2+ entry replenishes ER Ca2+ through reversible gating of plasma membrane Ca2+ channels by the ER Ca2+ sensor, stromal interaction molecule 1 (STIM1). For characterization of the in vivo impact of STIM1 loss, mice with ß-cell-specific STIM1 deletion (STIM1Δß mice) were generated and challenged with high-fat diet. Interestingly, ß-cell dysfunction was observed in female, but not male, mice. Female STIM1Δß mice displayed reductions in ß-cell mass, a concomitant increase in α-cell mass, and reduced expression of markers of ß-cell maturity, including MafA and UCN3. Consistent with these findings, STIM1 expression was inversely correlated with HbA1c levels in islets from female, but not male, human organ donors. Mechanistic assays demonstrated that the sexually dimorphic phenotype observed in STIM1Δß mice was due, in part, to loss of signaling through the noncanonical 17-ß estradiol receptor (GPER1), as GPER1 knockdown and inhibition led to a similar loss of expression of ß-cell maturity genes in INS-1 cells. Together, these data suggest that STIM1 orchestrates pancreatic ß-cell function and identity through GPER1-mediated estradiol signaling. ARTICLE HIGHLIGHTS: Store-operated Ca2+ entry replenishes endoplasmic reticulum (ER) Ca2+ through reversible gating of plasma membrane Ca2+ channels by the ER Ca2+ sensor, stromal interaction molecule 1 (STIM1). ß-Cell-specific deletion of STIM1 results in a sexually dimorphic phenotype, with ß-cell dysfunction and loss of identity in female but not male mice. Expression of the noncanonical 17-ß estradiol receptor (GPER1) is decreased in islets of female STIM1Δß mice, and modulation of GPER1 levels leads to alterations in expression of ß-cell maturity genes in INS-1 cells.

The Mechanism of the Anti-Cardiac Hypertrophy Effect of Glycyrrhizic Acid Is Related to Reducing STIM1-Dependent Store-Operated Calcium Entry.

We explored the anti-cardiac hypertrophy mechanism of glycyrrhizic acid from the perspective of calcium regulation under pathological conditions. For this purpose, we used a rat model of myocardial hypertrophy induced by pressure overload. The effect of glycyrrhizic acid on BP was measured non-invasively with a sphygmomanometer and recorded in PC. In rats with modeled cardiac hypertrophy, the effect of GA on expression of type 1 matrix interaction molecules was determined in horizontal tissues and cultured cardiomyocytes of the left ventricle. The laser confocal microscopy and calcium ion probe Fluo-4 AM were used to assess the effect of glycyrrhizic acid on stromal interaction molecule 1 (STIM1)-dependent store-operated calcium entry in cultured cardiomyocytes derived from the hypertrophic myocardium. Glycyrrhizic acid exerted the anti-hypertrophic effect in rats with hypertrophic myocardium by down-regulating STIM1 protein expression and reducing the intensity of STIM1-dependent store-operated calcium entry.

Muscle magnetic resonance characterization of STIM1 tubular aggregate myopathy using unsupervised learning.

PURPOSE: Congenital myopathies are a heterogeneous group of diseases affecting the skeletal muscles and characterized by high clinical, genetic, and histological variability. Magnetic Resonance (MR) is a valuable tool for the assessment of involved muscles (i.e., fatty replacement and oedema) and disease progression. Machine Learning is becoming increasingly applied for diagnostic purposes, but to our knowledge, Self-Organizing Maps (SOMs) have never been used for the identification of the patterns in these diseases. The aim of this study is to evaluate if SOMs may discriminate between muscles with fatty replacement (S), oedema (E) or neither (N). METHODS: MR studies of a family affected by tubular aggregates myopathy (TAM) with the histologically proven autosomal dominant mutation of the STIM1 gene, were examined: for each patient, in two MR assessments (i.e., t0 and t1, the latter after 5 years), fifty-three muscles were evaluated for muscular fatty replacement on the T1w images, and for oedema on the STIR images, for reference. Sixty radiomic features were collected from each muscle at t0 and t1 MR assessment using 3DSlicer software, in order to obtain data from images. A SOM was created to analyze all datasets using three clusters (i.e., 0, 1 and 2) and results were compared with radiological evaluation. RESULTS: Six patients with TAM STIM1-mutation were included. At t0 MR assessments, all patients showed widespread fatty replacement that intensifies at t1, while oedema mainly affected the muscles of the legs and appears stable at follow-up. All muscles with oedema showed fatty replacement, too. At t0 SOM grid clustering shows almost all N muscles in Cluster 0 and most of the E muscles in Cluster 1; at t1 almost all E muscles appear in Cluster 1. CONCLUSION: Our unsupervised learning model appears to be able to recognize muscles altered by the presence of edema and fatty replacement.

The roles and mechanism of STIM1 in tumorigenesis and metastasis.

STIM1 (stromal interaction molecule 1) is one of the key components of the store operated Ca2+ entry channel (SOCE), which is located on the endoplasmic reticulum membrane and highly expressed in most kinds of tumors. STIM1 promotes tumorigenesis and metastasis by modulating the formation of invadopodia, promoting angiogenesis, mediating inflammatory response, altering the cytoskeleton and cell dynamics. However, the roles and mechanism of STIM1 in different tumors have not been fully elucidated. In this review, we summarize the latest progress and mechanisms of STIM1 in tumorigenesis and metastasis, thereby providing insights and references for the study on STIM1 in the field of cancer biology in the future.

Reduction of TRPC1/TRPC3 mediated Ca2+-signaling protects oxidative stress-induced COPD.

Oxidative stress is a predisposing factor in Chronic Obstructive Pulmonary Disease (COPD). Specifically, pulmonary epithelial (PE) cells reduce antioxidant capacity during COPD because of the continuous production of reactive oxygen species (ROS). However, the molecular pathogenesis that governs such ROS activity is unclear. Here we show that the dysregulation of intracellular calcium concentration ([Ca2+]i) in PE cells from COPD patients, compared to the healthy PE cells, is associated with the robust functional expressions of Transient Receptor Potential Canonical (TRPC)1 and TRPC3 channels, and Ca2+ entry (SOCE) components, Stromal Interaction Molecule 1 (STIM1) and ORAI1 channels. Additionally, the elevated expression levels of fibrotic, inflammatory, oxidative, and apoptotic markers in cells from COPD patients suggest detrimental pathway activation, thereby reducing the ability of lung remodeling. To further delineate the mechanism, we used human lung epithelial cell line, A549, since the behavior of SOCE and the expression patterns of TRPC1/C3, STIM1, and ORAI1 were much like PE cells. Notably, the knockdown of TRPC1/C3 in A549 cells substantially reduced the SOCE-induced [Ca2+]i rise, and reversed the ROS-mediated oxidative, fibrotic, inflammatory, and apoptotic responses, thus confirming the role of TRPC1/C3 in SOCE driven COPD-like condition. Higher TRPC1/C3, STIM1, and ORAI1 expressions, along with a greater Ca2+ entry, via SOCE in ROS-induced A549 cells, led to the rise in oxidative, fibrotic, inflammatory, and apoptotic gene expression, specifically through the extracellular signal-regulated kinase (ERK) pathway. Abatement of TRPC1 and/or TRPC3 reduced the mobilization of [Ca2+]i and reversed apoptotic gene expression and ERK activation, signifying the involvement of TRPC1/C3. Together these data suggest that TRPC1/C3 and SOCE facilitate the COPD condition through ROS-mediated cell death, thus implicating their likely roles as potential therapeutic targets for COPD. SUMMARY: Alterations in Ca2+ signaling modalities in normal pulmonary epithelial cells exhibit COPD through oxidative stress and cellular injury, compromising repair, which was alleviated through inhibition of store-operated calcium entry. SUBJECT AREA: Calcium, ROS, Cellular signaling, lung disease.

p38 MAPK activation and STIM1-Orai3 association mediate TRPC6 externalization.

Endothelial cell (EC) migration is critical for the repair of monolayer disruption following angioplasties, but migration is inhibited by lipid oxidation products, including lysophosphatidylcholine (lysoPC), which open canonical transient receptor potential 6 (TRPC6) channels. TRPC6 activation requires an increase in intracellular Ca2+ concentration ([Ca2+]i), the source of which is unknown. LysoPC can activate phospholipase A2 to release arachidonic acid (ArA). ArA can activate arachidonic acid-regulated calcium (ARC) channels that are formed by stromal interaction molecule 1 (STIM1) and Orai1 and Orai3 proteins. Both lysoPC and ArA can activate p38 mitogen-activated protein kinase (MAPK) that induces the phosphorylation required for STIM1-Orai3 association. This is accompanied by an increase in [Ca2+]i and TRPC6 externalization. The effect of lysoPC and ArA is not additive, suggesting activation of the same pathway. The increase in [Ca2+]i activates an Src kinase that leads to TRPC6 activation. Downregulation of Orai3 using siRNA blocks the lysoPC- or ArA-induced increase in [Ca2+]i and TRPC6 externalization and preserves EC migration. These data show that lysoPC induces activation of p38 MAPK, which leads to STIM1-Orai3 association and increased [Ca2+]i. This increase in [Ca2+]i activates an Src kinase leading to TRPC6 externalization, which initiates a cascade of events ending in cytoskeletal changes that disrupt EC migration. Blocking this pathway preserves EC migration in the presence of lipid oxidation products.NEW & NOTEWORTHY The major lysophospholipid component in oxidized LDL, lysophosphatidylcholine (lysoPC), can activate p38 MAP kinase, which in turn promotes externalization of Orai3 and STIM1-Orai3 association, suggesting involvement of arachidonic acid-regulated calcium (ARC) channels. The subsequent increase in intracellular calcium activates an Src kinase required for TRPC6 externalization. TRPC6 activation, which has been shown to inhibit endothelial cell migration, is blocked by p38 MAP kinase or Orai3 downregulation, and this partially preserves endothelial migration in lysoPC.

Chronic suppression of STIM1-mediated calcium signaling in Purkinje cells rescues the cerebellar pathology in spinocerebellar ataxia type 2.

Distorted neuronal calcium signaling has been reported in many neurodegenerative disorders, including different types of spinocerebellar ataxias (SCAs). Cerebellar Purkinje cells (PCs) are primarily affected in SCAs and the disturbances in the calcium homeostasis were observed in SCA PCs. Our previous results have revealed that 3,5-dihydroxyphenylglycine (DHPG) induced greater calcium responses in SCA2-58Q PC cultures than in wild type (WT) PC cultures. Here we observed that glutamate-induced calcium release in PCs cells bodies is significantly higher in SCA2-58Q PCs from acute cerebellar slices compared to WT PCs of the same age. Recent studies have demonstrated that the stromal interaction molecule 1 (STIM1) plays an important role in the regulation of the neuronal calcium signaling in cerebellar PCs in mice. The main function of STIM1 is to regulate store-operated calcium entry through the TRPC/Orai channels formation to refill the calcium stores in the ER when it is empty. Here we demonstrated that the chronic viral-mediated expression of the small interfering RNA (siRNA) targeting STIM1 specifically in cerebellar PCs alleviates the deranged calcium signaling in SCA2-58Q PCs, rescues the spine loss in these cerebellar neurons, and also improves the motor decline in SCA2-58Q mice. Thus, our preliminary results support the important role of the altered neuronal calcium signaling in SCA2 pathology and also suggest the STIM1-mediated signaling pathway as a potential therapeutic target for treatment of SCA2 patients.

Rescue of astrocyte activity by the calcium sensor STIM1 restores long-term synaptic plasticity in female mice modelling Alzheimer's disease.

Calcium dynamics in astrocytes represent a fundamental signal that through gliotransmitter release regulates synaptic plasticity and behaviour. Here we present a longitudinal study in the PS2APP mouse model of Alzheimer's disease (AD) linking astrocyte Ca2+ hypoactivity to memory loss. At the onset of plaque deposition, somatosensory cortical astrocytes of AD female mice exhibit a drastic reduction of Ca2+ signaling, closely associated with decreased endoplasmic reticulum Ca2+ concentration and reduced expression of the Ca2+ sensor STIM1. In parallel, astrocyte-dependent long-term synaptic plasticity declines in the somatosensory circuitry, anticipating specific tactile memory loss. Notably, we show that both astrocyte Ca2+ signaling and long-term synaptic plasticity are fully recovered by selective STIM1 overexpression in astrocytes. Our data unveil astrocyte Ca2+ hypoactivity in neocortical astrocytes as a functional hallmark of early AD stages and indicate astrocytic STIM1 as a target to rescue memory deficits.

CAPN1 (Calpain1)-Dependent Cleavage of STIM1 (Stromal Interaction Molecule 1) Results in an Enhanced SOCE (Store-Operated Calcium Entry) in Human Neonatal Platelets.

BACKGROUND: Altered intracellular Ca2+ homeostasis in neonatal platelets has been previously reported. This study aims to examine the changes in the Ca2+ entry through the store-operated calcium entry (SOCE) mechanism in neonatal platelets. METHODS: Human platelets from either control women, mothers, and neonates were isolated and, following, were fixed after being treated as required. Platelet samples were analyzed by Western blotting, qRT-PCR, and MALDITOF/TOF. Ca2+ homeostasis was also determined. Culture cells were used as surrogated of platelets to overexpress the proteins of interest to reproduce the alterations observed in platelets. RESULTS: Altered TG (thapsigargin)-evoked SOCE, alternative molecular weight form of STIM1 (stromal interaction molecule 1; s-STIM1 [short STIM1 isoform (478 aa)], around 60 kDa) and overexpression of SARAF (SOCE-associated regulatory factor) were found in neonatal platelets as compared to maternal and control women platelets. s-STIM1 may result due to CAPN1 (calpain1)-dependent processing, as confirmed in platelets and MEG01 cells by using calpeptin and overexpressing CAPN1, respectively. In HEK293 (STIM1 and STIM2 [stromal interaction molecule 2] double knockout) cells transfected either with c-STIM1 (canonical STIM1 [685 aa]), s-STIM1 (478), STIM1B (540), and CAPN1 overexpression plasmids, we found s-STIM1 and c-STIM1, except in cells overexpressing s-STIM1 (478) that lacked CAPN1 target residues. These results and the in silico analysis, lead us to conclude that STIM1 is cleaved at Q496 by CAPN1. Ca2+ imaging analysis and coimmunoprecipitation assay using MEG01 and HEK293 cells overexpressing SARAF together with s-STIM1 (478) reported a reduced slow Ca2+-dependent inactivation, so reproducing the Ca2+-homeostasis pattern observed in neonatal platelets. CONCLUSIONS: CAPN1 may cleave STIM1 in neonatal platelets, hence, impairing SARAF coupling after SOCE activation. s-STIM1 may avoid slow Ca2+-dependent inactivation and, subsequently, results in an enhanced TG-evoked SOCE as observed in neonatal platelets.