The Ca2+ sensor STIM1 regulates the type I interferon response by retaining the signaling adaptor STING at the endoplasmic reticulum.

Stimulator of interferon genes (STING) is an endoplasmic reticulum (ER) signaling adaptor that is essential for the type I interferon response to DNA pathogens. Aberrant activation of STING is linked to the pathology of autoimmune and autoinflammatory diseases. The rate-limiting step for the activation of STING is its translocation from the ER to the ER-Golgi intermediate compartment. Here, we found that deficiency in the Ca2+ sensor stromal interaction molecule 1 (STIM1) caused spontaneous activation of STING and enhanced expression of type I interferons under resting conditions in mice and a patient with combined immunodeficiency. Mechanistically, STIM1 associated with STING to retain it in the ER membrane, and coexpression of full-length STIM1 or a STING-interacting fragment of STIM1 suppressed the function of dominant STING mutants that cause autoinflammatory diseases. Furthermore, deficiency in STIM1 strongly enhanced the expression of type I interferons after viral infection and prevented the lethality of infection with a DNA virus in vivo. This work delineates a STIM1-STING circuit that maintains the resting state of the STING pathway.

Store-Operated Ca2+ Entry Controls Clonal Expansion of T Cells through Metabolic Reprogramming.

Store-operated Ca2+ entry (SOCE) is the main Ca2+ influx pathway in lymphocytes and is essential for T cell function and adaptive immunity. SOCE is mediated by Ca2+ release-activated Ca2+ (CRAC) channels that are activated by stromal interaction molecule (STIM) 1 and STIM2. SOCE regulates many Ca2+-dependent signaling molecules, including calcineurin, and inhibition of SOCE or calcineurin impairs antigen-dependent T cell proliferation. We here report that SOCE and calcineurin regulate cell cycle entry of quiescent T cells by controlling glycolysis and oxidative phosphorylation. SOCE directs the metabolic reprogramming of naive T cells by regulating the expression of glucose transporters, glycolytic enzymes, and metabolic regulators through the activation of nuclear factor of activated T cells (NFAT) and the PI3K-AKT kinase-mTOR nutrient-sensing pathway. We propose that SOCE controls a critical "metabolic checkpoint" at which T cells assess adequate nutrient supply to support clonal expansion and adaptive immune responses.

CRAC Channels and Calcium Signaling in T Cell-Mediated Immunity.

Calcium (Ca2+) signals play fundamental roles in immune cell function. The main sources of Ca2+ influx in mammalian lymphocytes following antigen receptor stimulation are Ca2+ release-activated Ca2+ (CRAC) channels. These are formed by ORAI proteins in the plasma membrane and are activated by stromal interaction molecules (STIM) located in the endoplasmic reticulum (ER). Human loss-of-function (LOF) mutations in ORAI1 and STIM1 that abolish Ca2+ influx cause a unique disease syndrome called CRAC channelopathy that is characterized by immunodeficiency autoimmunity and non-immunological symptoms. Studies in mice lacking Stim and Orai genes have illuminated many cellular and molecular mechanisms by which these molecules control lymphocyte function. CRAC channels are required for the differentiation and function of several T lymphocyte subsets that provide immunity to infection, mediate inflammation and prevent autoimmunity. This review examines new insights into how CRAC channels control T cell-mediated immunity.

Stromal interaction molecule 1 promotes tumor growth in Esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is a disease with poor prognosis which urgently is in need of effective prognostic marker. To discover novel prognostic protein marker for ESCC, we applied a high-throughput monoclonal antibody microarray to compare tumor and adjacent non-tumor tissues from ESCC patients. Antibody #ESmAb270 was consistent higher expressed in tumors and it was identified via mass spectrometry to be stromal interaction molecule 1 (STIM1). STIM1 H scores in tumor tissues were significantly up-regulated in esophageal tumor tissues compared to non-tumor tissues in 105 ESCC patients. We also observed that high STIM1 expression was correlated with advanced tumor grade and poor prognosis of ESCC. In addition, attenuation of STIM1 by siRNA or chemical inhibitors significantly inhibited cell viability and migration of ESCC cells. Evidence from high-throughput monoclonal antibody microarray, IHC microarray with associated survival data and functional analysis show that STIM1 is an unfavorable prognostic biomarker in ESCC.

Natural genetic variation in Stim1 creates stroke in the spontaneously hypertensive rat.

Similar to humans, the risk of cerebrovascular disease in stroke-prone spontaneously hypertensive rats (SHR-A3/SHRSP) arises from naturally occurring genetic variation. In the present study, we show the involvement of genetic variation affecting the store-operated calcium signaling gene, Stim1, in the pathogenesis of stroke in SHR. Stim1 is a key lymphocyte activation signaling molecule and contains functional variation in SHR-A3 that diverges from stroke-resistant SHR-B2. We created a SHR-A3 congenic line in which Stim1 was substituted with the corresponding genomic segment from SHR-B2. Compared with SHR-A3 rats, Stim1 congenic SHR-A3 (SHR-A3(Stim1-B2)) have reduced cerebrovascular disease in response to salt loading including lower neurological deficit scores and cerebral edema. Microbleeds and major hemorrhages occurred in over half of SHR-A3 rats. These lesions were absent in SHR-A3(Stim1-B2) rats. Loss of Stim1 function in mice and humans is associated with antibody-mediated autoimmunity due to defects in T lymphocyte helper function to B cells. We investigated autoantibody formation using a high-density protein array to detect the presence of IgG and IgM autoantibodies in SHR-A3. Autoantibodies to key cerebrovascular stress proteins were detected that were reduced in the congenic line.

STIM1 knock-down decreases the affinity of obinutuzumab for CD20 by altering CD20 localization to Triton-soluble membrane.

Obinutuzumab is thought to exert its effects through its high antibody-dependent cellular cytotoxicity (ADCC) via glyco-engineering of the Fc region. In addition, obinutuzumab causes direct binding-induced cell death (DCD) only by specifically binding to its target CD20, a Ca2+ channel. However, the specific features of CD20 related to obinutuzumab binding-induction of cell death are not clearly understood. In this study, we evaluated the relationship between the Ca2+ channel features of CD20 as a store-operated Ca2+ channel (SOC) and obinutuzumab binding-induced cell death. Ca2+ channel function and biochemical analysis revealed that CD20 is an Orai1- and stromal interaction molecule (STIM1)-dependent Ca2+ pore. However, binding of obinutuzumab on CD20 did not have any effect on Ca2+ influx activity of CD20; the direct cell death rate mediated by obinutuzumab binding was almost equivalent with or without the extracellular Ca2+ condition. Given the apparent interaction between STIM1 and CD20, we observed Triton-X solubilized obinutuzumab-bound CD20 accompanied by STIM1. Subsequently, obinutuzumab binding and cell death were decreased by STIM1 knock-down in Ramos B cells. Thus, STIM1 directly contributes to cell death by increasing the affinity of cells for obinutuzumab by transferring CD20 to the Triton-soluble membrane region.

STIM1 and STIM2 cooperatively regulate mouse neutrophil store-operated calcium entry and cytokine production.

Neutrophils are key effector cells of the innate immune system. Calcium-dependent signaling pathways initiated by store-operated calcium entry (SOCE) are known to regulate neutrophil activation; however, the precise mechanism of this process remains unclear. STIM1 and STIM2 are calcium-sensing molecules that link calcium depletion of the endoplasmic reticulum with opening of plasma membrane calcium channels. Although a role for STIM1 in neutrophil SOCE and activation has been established, the function of STIM2 is unknown. Here we use mice with conditional ablation of Stim1 and/or Stim2 to investigate the role of STIM2 in neutrophil activation. We demonstrate that loss of STIM2 results in decreased SOCE, particularly at lower doses of agonists. Reactive oxygen species (ROS) production, degranulation, and phagocytosis are normal in the absence of STIM2, suggesting STIM1 is the dominant calcium sensor required for classical short-term neutrophil responses. However, neutrophil cytokine production required STIM2, but not STIM1, at least in part as a result of redox regulation of cytokine gene expression. In vivo loss of STIM2 results in lower cytokine levels and protection from mortality in a mouse model of systemic inflammatory response syndrome. These data, combined with previous studies focusing on STIM1, define distinct but cooperative functions for STIM1 and STIM2 in modulating neutrophil bactericidal and cytokine responses.

Single Nucleotide Polymorphism Analysis in HIV and Kaposi's Sarcoma Disease by Microarray Technique.

BACKGROUND: Emergence of Kaposi's Sarcoma in the cases other than HIV, following the use of immunosuppressant drugs, demonstrates that it is related to weak immunity. The fact that this malignancy does not occur in every HIV-positive patient suggests that genetic predisposition may also be effective. Replacement of one of the base pairs of adenine, guanine, cytosine, and thymine that constitute the DNA sequence in the human genome with another base pair can affect susceptibility to disease, response to treatment, and immunity. OBJECTIVE: The purpose of this study is to analyze the Single Nucleotide Polymorphism that could predispose to Kaposi's sarcoma of an HIV-infected patient and to identify which nucleotides such SNPs correspond to, using the microarray technology. MATERIALS AND METHODS: The blood samples of individuals, one of whom was diagnosed with Kaposi's Sarcoma HIV (+) visiting the outpatient clinic of infectious diseases polyclinic of Harran University Research and Practice Hospital and of a healthy individual with no Kaposi's Sarcoma, were used in the study. Following the DNA isolation of the blood samples taken from the respective individuals, a SNP analysis was conducted on the microarray device. 204,000 SNPs obtained were scanned later on in the databases in an attempt to identify the SNPs related to Kaposi's Sarcoma. RESULTS: In the 204,000 SNP screenings, we scrutinized the SNPs that differ in the case of Kaposi's Sarcoma [KS (+) and HIV (+)] on the basis of Control [KS(-) and HIV(-)] and HIV+ [KS(-)], and two SNPs of the ENDRA gene, three SNPs of the ADRA1A gene, six SNPs of the STIM1 gene, four SNPs of the EFNB2 gene, and one SNP of the CD209 gene were found to be different. However, when it comes to all SNPs (all the 204.000 SNPs) screened in terms of allele, it was observed that the AA and BB alleles were lower in the patient with Kaposi's Sarcoma [KS (+) and HIV (+)] compared to other groups and AB alleles were found to be higher than others in the patient with Kaposi's sarcoma [KS] (+) and HIV (+)]. CONCLUSION: In the microarray study we have conducted, 204,000 SNPs were screened for Control (HIV-) HIV (+) and HIV (+) patient with Kaposi's Sarcoma. It was found that 32,362 of those SNPs had different alleles in the Kaposi's Sarcoma [KS + HIV (+)] patient, while they had the same ones in the control [KS (-) and HIV (-)] and HIV + [KS (-)] group. 16 of the 32,362 SNPs took place among the genes related to Kaposi's Sarcoma. In the cases of Kaposi's Sarcoma with suspected diagnosis, it can be used as a beneficial laboratory test.

Stepping out of the shadow: STIM2 promotes IL-3-induced cytokine release.

Basophils are a small population of innate immune cells, but their release of the cytokine interleukin-4 (IL-4) is important for mounting an efficient immune response against distinct parasites. Yoshikawa et al (in the 9 April 2019 issue) showed that whereas STIM1 is essential for IL-4 release after stimulation of FcεRI, STIM2 mediates a delayed IL-3/IL-33-induced IL-4 release independent of STIM1.

Store-Operated Calcium Entry via STIM1 Contributes to MRGPRX2 Induced Mast Cell Functions.

Mast cells are inflammatory immune cells that play an essential role in mediating allergic reactions in humans. It is well-known that mast cell activation is critically regulated by intracellular calcium ion (Ca2+) concentrations. MAS-related G-protein coupled receptor-X2 (MRGPRX2) is a G-protein coupled receptor (GPCR) expressed on mast cells that is activated by various ligands, including several FDA approved drugs; consequently, this receptor has been implicated in causing pseudo-allergic reactions in humans. MRGPRX2 activation leads to an increase in intracellular Ca2+ levels; however, the Ca2+ mobilizing mechanisms utilized by this receptor are largely unknown. Previous reports showed that store-operated Ca2+ entry (SOCE) via the calcium sensor, stromal interaction molecule 1 (STIM1), regulates mast cell response induced by the high-affinity IgE receptor (FcεRI). In this study, using complementary pharmacologic and genetic ablation approaches we demonstrate that SOCE through STIM1 promotes MRGPRX2-induced human mast cell response in vitro. Importantly, SOCE also critically modulates MrgprB2 (mouse ortholog of human MRGPRX2) dependent inflammation in in vivo mouse models of pseudo-allergy. Collectively, our data suggests that MRGPRX2/MrgprB2 activation of mast cells is dependent on SOCE via STIM1, and further characterization of the MRGPRX2-SOCE-STIM1 pathway will lead to the identification of novel targets for the treatment of pseudo-allergic reactions in humans.

Gut Bacterial Metabolite Urolithin A (UA) Mitigates Ca2+ Entry in T Cells by Regulating miR-10a-5p.

The gut microbiota influences several biological functions including immune responses. Inflammatory bowel disease is favorably influenced by consumption of several dietary natural plant products such as pomegranate, walnuts, and berries containing polyphenolic compounds such as ellagitannins and ellagic acid. The gut microbiota metabolizes ellagic acid resulting in the formation of bioactive urolithins A, B, C, and D. Urolithin A (UA) is the most active and effective gut metabolite and acts as a potent anti-inflammatory and anti-oxidant agent. However, whether gut metabolite UA affects the function of immune cells remains incompletely understood. T cell proliferation is stimulated by store operated Ca2+ entry (SOCE) resulting from stimulation of Orai1 by STIM1/STIM2. We show here that treatment of murine CD4+ T cells with UA (10 µM, 3 days) significantly blunted SOCE in CD4+ T cells, an effect paralleled by significant downregulation of Orai1 and STIM1/2 transcript levels and protein abundance. UA treatment further increased miR-10a-5p abundance in CD4+ T cells in a dose dependent fashion. Overexpression of miR-10a-5p significantly decreased STIM1/2 and Orai1 mRNA and protein levels as well as SOCE in CD4+ T cells. UA further decreased CD4+ T cell proliferation. Thus, the gut bacterial metabolite UA increases miR-10a-5p levels thereby downregulating Orai1/STIM1/STIM2 expression, store operated Ca2+ entry, and proliferation of murine CD4+ T cells.

Pivotal role of STIM2, but not STIM1, in IL-4 production by IL-3-stimulated murine basophils.

Basophils have nonredundant roles in various immune responses that require Ca2+ influx. Here, we examined the role of two Ca2+ sensors, stromal interaction molecule 1 and 2 (STIM1 and STIM2), in basophil activation. We found that loss of STIM1, but not STIM2, impaired basophil IL-4 production after stimulation with immunoglobulin E (IgE)-containing immune complexes. In contrast, when basophils were stimulated with IL-3, loss of STIM2, but not STIM1, reduced basophil IL-4 production. This difference in STIM proteins was associated with distinct time courses of Ca2+ influx and transcription of the Il4 gene that were elicited by each stimulus. Similarly, basophil-specific STIM1 expression was required for IgE-driven chronic allergic inflammation in vivo, whereas STIM2 was required for IL-4 production after combined IL-3 and IL-33 treatment in mice. These data indicate that STIM1 and STIM2 have differential roles in the production of IL-4, which are stimulus dependent. Furthermore, these results illustrate the vital role of STIM2 in basophils, which is often considered to be less important than STIM1.

Pro-inflammatory role of transient receptor potential canonical channel 6 in the pathogenesis of chronic rhinosinusitis with nasal polyps.

BACKGROUND: The pathogenesis of chronic rhinosinusitis with nasal polyps (CRSwNP) has not been fully elucidated. This study sought to explore the role and mechanism of transient receptor potential canonical channel 6 (TRPC6) in the pathogenesis of CRSwNP. METHODS: Immunohistochemistry (IHC) was employed to evaluate TRPC6 immunolabeling. Real-time polymerase chain reaction (PCR) was conducted to assay TRPC6, stromal interaction molecule 1 (STIM1), and calcium release-activated calcium channel protein 1 (Orai1) messenger RNA (mRNA) levels in 70 patients with CRSwNP, including eosinophilic CRSwNP (ECRSwNP) or non-eosinophilic CRSwNP (nECRSwNP), and 28 control subjects. The concentrations of inflammatory mediators, including interleukin (IL)-1ß, IL-5, and IL-25, were assayed by enzyme-linked immunosorbent assay (ELISA). In experiments on human nasal epithelial cell (HNEC) culture and stimulation, the mean fluorescence intensity (MFI) of intracellular Ca2+ was assayed by flow cytometry. Western blotting, real-time PCR, and ELISA were also conducted to assess the effects and mechanisms of TRPC6 activator 1-oleoyl-2-acetyl-glycerol (OAG) and TRPC6 inhibitor 1-[2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl) propoxy]ethyl-1H-imidazole (SKF-96365) on HNECs. RESULTS: Upregulation of TRPC6, STIM1, and Orai1 levels was found in CRSwNP patients, particularly in those with ECRSwNP. TRPC6-positive cells correlated positively with the numbers of eosinophils and neutrophils, respectively. Moreover, TRPC6 mRNA was positively correlated with STIM1 and Orai1 mRNA levels. The concentrations of inflammatory mediators, including IL-1ß, IL-5, and IL-25, were elevated in CRSwNP, especially in ECRSwNP. In cultured HNECs, TRPC6, STIM1, Orai1, Ca2+ MFI levels, and inflammatory mediators were upregulated by lipopolysaccharide (LPS) and OAG but were inhibited by SKF-96365. CONCLUSION: TRPC6 plays a pro-inflammatory role in the pathogenesis of CRSwNP via regulating Ca2+ flow.

UNC93B1 interacts with the calcium sensor STIM1 for efficient antigen cross-presentation in dendritic cells.

Dendritic cells (DC) have the unique ability to present exogenous antigens via the major histocompatibility complex class I pathway to stimulate naive CD8+ T cells. In DCs with a non-functional mutation in Unc93b1 (3d mutation), endosomal acidification, phagosomal maturation, antigen degradation, antigen export to the cytosol and the function of the store-operated-Ca2+-entry regulator STIM1 are impaired. These defects result in compromised antigen cross-presentation and anti-tumor responses in 3d-mutated mice. Here, we show that UNC93B1 interacts with the calcium sensor STIM1 in the endoplasmic reticulum, a critical step for STIM1 oligomerization and activation. Expression of a constitutively active STIM1 mutant, which no longer binds UNC93B1, restores antigen degradation and cross-presentation in 3d-mutated DCs. Furthermore, ablation of STIM1 in mouse and human cells leads to a decrease in cross-presentation. Our data indicate that the UNC93B1 and STIM1 cooperation is important for calcium flux and antigen cross-presentation in DCs.