RESUMO
The single-pass transmembrane protein Stromal Interaction Molecule 1 (STIM1), located in the endoplasmic reticulum (ER) membrane, possesses two main functions: It senses the ER-Ca2+ concentration and directly binds to the store-operated Ca2+ channel Orai1 for its activation when Ca2+ recedes. At high resting ER-Ca2+ concentration, the ER-luminal STIM1 domain is kept monomeric but undergoes di/multimerization once stores are depleted. Luminal STIM1 multimerization is essential to unleash the STIM C-terminal binding site for Orai1 channels. However, structural basis of the luminal association sites has so far been elusive. Here, we employed molecular dynamics (MD) simulations and identified two essential di/multimerization segments, the α7 and the adjacent region near the α9-helix in the sterile alpha motif (SAM) domain. Based on MD results, we targeted the two STIM1 SAM domains by engineering point mutations. These mutations interfered with higher-order multimerization of ER-luminal fragments in biochemical assays and puncta formation in live-cell experiments upon Ca2+ store depletion. The STIM1 multimerization impeded mutants significantly reduced Ca2+ entry via Orai1, decreasing the Ca2+ oscillation frequency as well as store-operated Ca2+ entry. Combination of the ER-luminal STIM1 multimerization mutations with gain of function mutations and coexpression of Orai1 partially ameliorated functional defects. Our data point to a hydrophobicity-driven binding within the ER-luminal STIM1 multimer that needs to switch between resting monomeric and activated multimeric state. Altogether, these data reveal that interactions between SAM domains of STIM1 monomers are critical for multimerization and activation of the protein.
Assuntos
Proteínas de Neoplasias , Multimerização Proteica , Molécula 1 de Interação Estromal , Humanos , Sítios de Ligação , Cálcio/metabolismo , Retículo Endoplasmático/metabolismo , Células HEK293 , Simulação de Dinâmica Molecular , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/química , Proteína ORAI1/metabolismo , Proteína ORAI1/genética , Proteína ORAI1/química , Ligação Proteica , Domínios Proteicos , Molécula 1 de Interação Estromal/metabolismo , Molécula 1 de Interação Estromal/genética , Molécula 1 de Interação Estromal/químicaRESUMO
The upregulation of Orai1 and subsequent store-operated Ca2+ entry (SOCE) has been associated with adverse cardiac remodeling and heart failure (HF). However, the mechanism underlying Orai1 upregulation and its role in myocardial infarction remains unclear. Our study investigated the role of Orai1 in activating adenylyl cyclase 8 (AC8) and cyclic AMP (cAMP) response element-binding protein (CREB), as well as its contribution to cardiac dysfunction induced by ischemia and reperfusion (I/R). We found that I/R evoked an increase in the expression of Orai1 and AC8 in rats' hearts, resulting in a substantial rise in diastolic Ca2+ concentration ([Ca2+]i), and reduced ventricular contractions. The expression of Orai1 and AC8 was also increased in ventricular biopsies of post-ischemic HF patients. Mechanistically, we demonstrate that I/R activation of Orai1 stimulated AC8, which produced cAMP and phosphorylated CREB. Subsequently, p-CREB activated the ORAI1 promoter, resulting in Orai1 upregulation and SOCE exacerbation. Intramyocardial administration of AAV9 carrying AC8 short hairpin RNA decreased the expression of AC8, Orai1 and CREB, which restored diastolic [Ca2+]i and improved cardiac contraction. Therefore, our data suggests that the axis composed by Orai1/AC8/CREB plays a critical role in I/R-induced cardiac dysfunction, representing a potential new therapeutic target to limit the progression of the disease toward HF.
Assuntos
Adenilil Ciclases , Infarto do Miocárdio , Humanos , Ratos , Animais , Regulação para Cima , Adenilil Ciclases/genética , Adenilil Ciclases/metabolismo , AMP Cíclico/metabolismo , Sinalização do Cálcio , Infarto do Miocárdio/genética , Cálcio/metabolismo , Proteína ORAI1/genética , Proteína ORAI1/metabolismoRESUMO
Alteration in the normal mechanical forces of breathing can contribute to changes in contractility and remodeling characteristic of airway diseases, but the mechanisms that mediate these effects in airway cells are still under investigation. Airway smooth muscle (ASM) cells contribute to both contractility and extracellular matrix (ECM) remodeling. In this study, we explored ASM mechanisms activated by mechanical stretch, focusing on mechanosensitive piezo channels and the key Ca2+ regulatory protein stromal interaction molecule 1 (STIM1). Expression of Ca2+ regulatory proteins, including STIM1, Orai1, and caveolin-1, mechanosensitive ion channels Piezo-1 and Piezo-2, and NLRP3 inflammasomes were upregulated by 10% static stretch superimposed on 5% cyclic stretch. These effects were blunted by STIM1 siRNA. Histamine-induced [Ca2+]i responses and inflammasome activation were similarly blunted by STIM1 knockdown. These data show that the effects of mechanical stretch in human ASM cells are mediated through STIM1, which activates multiple pathways, including Piezo channels and the inflammasome, leading to potential downstream changes in contractility and ECM remodeling.NEW & NOTEWORTHY Mechanical forces on the airway can contribute to altered contractility and remodeling in airway diseases, but the mechanisms are not clearly understood. Using human airway smooth muscle cells exposed to cyclic forces with static stretch to mimic breathing and static pressure, we found that the effects of stretch are mediated through STIM1, resulting in the activation of multiple pathways, including Piezo channels and the inflammasome, with potential downstream influences on contractility and remodeling.
Assuntos
Miócitos de Músculo Liso , Molécula 1 de Interação Estromal , Humanos , Molécula 1 de Interação Estromal/metabolismo , Molécula 1 de Interação Estromal/genética , Miócitos de Músculo Liso/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/genética , Inflamassomos/metabolismo , Estresse Mecânico , Mecanotransdução Celular , Músculo Liso/metabolismo , Canais Iônicos/metabolismo , Caveolina 1/metabolismo , Caveolina 1/genética , Transdução de Sinais , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Cálcio/metabolismo , Células Cultivadas , Contração Muscular/fisiologia , Remodelação das Vias Aéreas/fisiologia , Proteína ORAI1/metabolismo , Proteína ORAI1/genéticaRESUMO
Store-operated calcium entry (SOCE) plays a crucial role in maintaining cellular calcium homeostasis. This mechanism involves proteins, such as stromal interaction molecule 1 (STIM1) and ORAI1. Mutations in the genes encoding these proteins, especially STIM1, can lead to various diseases, including CRAC channelopathies associated with severe combined immunodeficiency. Herein, we describe a novel homozygous mutation, NM_003156 c.792-3C > G, in STIM1 in a patient with a clinical profile of CRAC channelopathy, including immune system deficiencies and muscle weakness. Functional analyses revealed three distinct spliced forms in the patient cells: wild-type, exon 7 skipping, and intronic retention. Calcium influx analysis revealed impaired SOCE in the patient cells, indicating a loss of STIM1 function. We developed an antisense oligonucleotide treatment that improves STIM1 splicing and highlighted its potential as a therapeutic approach. Our findings provide insights into the complex effects of STIM1 mutations and shed light on the multifaceted clinical presentation of the patient.
Assuntos
Cálcio , Mutação , Proteínas de Neoplasias , Molécula 1 de Interação Estromal , Humanos , Molécula 1 de Interação Estromal/genética , Molécula 1 de Interação Estromal/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Cálcio/metabolismo , Canalopatias/genética , Masculino , Canais de Cálcio Ativados pela Liberação de Cálcio/genética , Canais de Cálcio Ativados pela Liberação de Cálcio/metabolismo , Feminino , Imunodeficiência Combinada Severa/genética , Proteína ORAI1/genética , Proteína ORAI1/metabolismoRESUMO
An important calcium (Ca2+) entry pathway into the cell is the Ca2+ release-activated Ca2+ (CRAC) channel, which controls a series of downstream signaling events such as gene transcription, secretion and proliferation. It is composed of a Ca2+ sensor in the endoplasmic reticulum (ER), the stromal interaction molecule (STIM), and the Ca2+ ion channel Orai in the plasma membrane (PM). Their activation is initiated by receptor-ligand binding at the PM, which triggers a signaling cascade within the cell that ultimately causes store depletion. The decrease in ER-luminal Ca2+ is sensed by STIM1, which undergoes structural rearrangements that lead to coupling with Orai1 and its activation. In this review, we highlight the current understanding of the Orai1 pore opening mechanism. In this context, we also point out the questions that remain unanswered and how these can be addressed by the currently emerging genetic code expansion (GCE) technology. GCE enables the incorporation of non-canonical amino acids with novel properties, such as light-sensitivity, and has the potential to provide novel insights into the structure/function relationship of CRAC channels at a single amino acid level in the living cell.
Assuntos
Canais de Cálcio Ativados pela Liberação de Cálcio , Cálcio , Retículo Endoplasmático , Proteína ORAI1 , Molécula 1 de Interação Estromal , Animais , Humanos , Cálcio/metabolismo , Canais de Cálcio Ativados pela Liberação de Cálcio/metabolismo , Sinalização do Cálcio , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Proteína ORAI1/metabolismo , Molécula 1 de Interação Estromal/metabolismoRESUMO
BACKGROUND: Store-operated Ca2+ entry (SOCE) mediated by ORAI1 channel plays a crucial role in acute pancreatitis (AP). Macrophage is an important regulator in amplifying pancreatic tissue damage, but little is known about the role of ORAI1 in macrophages. In this study, we examined the effects of macrophage-specific ORAI1 on pancreatic tissue damage in AP. METHOD: Myeloid-specific Orai1 deficient mice was generated by crossing a LysM-Cre mouse line with Orai1f/f mice. Bone marrow-derived macrophages (BMDMs) were isolated, cultured, and stimulated to induce M1 or M2 macrophage polarization. Intracellular Ca2+ signals were measured by time-lapse confocal microscope imaging, with a Ca2+ indicator (Fluo 4). Experimental AP was induced by hourly intraperitoneal injections of caerulein or retrograde biliopancreatic infusion of sodium taurocholate. Pancreatic tissue damage was assessed by histopathological scoring and immunostaining. Sepsis was induced by intraperitoneal injection of lipopolysaccharide; organ damage and serum pro-inflammatory cytokines were measured. RESULT: Myeloid-specific Orai1 deletion exhibited minimal effect on SOCE in M0 macrophages and promoted M2 macrophage polarization ex vivo. Myeloid-specific Orai1 deletion did not affect pancreatic tissue damage, nor neutrophil or macrophage infiltration in two models of AP. Similarly, myeloid-specific Orai1 deletion did not influence overall survival rate in a model of sepsis, nor lung, kidney, and liver damage; while serum pro-inflammatory cytokines, including IL-6, TNF-α, and IL-1ß were higher in Orai1ΔLysM mice, but were largely reduced in mice with Orai1 inhibitor. CONCLUSION: Our data suggest that ORAI1 may not be a predominant SOCE channel in macrophages and play a limited role in mediating pancreatic tissue damage in AP.
Assuntos
Macrófagos , Proteína ORAI1 , Pâncreas , Pancreatite , Animais , Proteína ORAI1/metabolismo , Proteína ORAI1/genética , Pancreatite/patologia , Pancreatite/metabolismo , Pancreatite/induzido quimicamente , Pancreatite/genética , Camundongos , Macrófagos/metabolismo , Pâncreas/patologia , Pâncreas/metabolismo , Camundongos Endogâmicos C57BL , Células Mieloides/metabolismo , Camundongos Knockout , Modelos Animais de Doenças , Deleção de GenesRESUMO
Endothelium-dependent contraction (EDC) exists in blood vessels of normotensive animals, but is exaggerated in hypertension. An early signal in EDC is cytosolic Ca2+ rise in endothelial cells. In this study we investigated the functional role of Orai1, a major endothelial cell Ca2+ entry channel, in EDC. Hypertension model was established in WT mice by intake of L-NNA in the drinking water (0.5 g/L) for 4 weeks or osmotic pump delivery of Ang II (1.5 mg·kg-1·d-1) for 2 weeks. In TRPC5 KO mice, the concentration of L-NNA and Ang II were increased to 1 g/L or 2 mg·kg-1·d-1, respectively. Arterial segments were prepared from carotid arteries and aortas, and EDC was elicited by acetylcholine in the presence of Nω-nitro-L-arginine methyl ester. We showed that low concentration of acetylcholine (3-30 nM) initiated relaxation in phenylephrine-precontracted carotid arteries of both normotensive and hypertensive mice, while high concentration of acetylcholine (0.1-2 µM) induced contraction. Application of selective Orai1 inhibitors AnCoA4 (100 µM) or YM58483 (400 nM) had no effect on ACh-induced relaxation but markedly reduced acetylcholine-induced EDC. We found that EDC was increased in hypertensive mice compared with that of normotensive mice, which was associated with increased Orai1 expression in endothelial cells of hypertensive mice. Compared to TRPC5 and TRPV4, which were also involved in EDC, endothelial cell Orai1 had relatively greater contribution to EDC than either TRPC5 or TRPV4 alone. We identified COX-2, followed by PGF2α, PGD2 and PGE2 as the downstream signals of Orai1/TRPC5/TRPV4. In conclusion, Orai1 coordinates together with TRPC5 and TRPV4 in endothelial cells to regulate EDC responses. This study demonstrates a novel function of Orai1 in EDC in both normotensive and hypertensive mice, thus providing a general scheme about the control of EDC by Ca2+-permeable channels.
Assuntos
Artérias Carótidas , Células Endoteliais , Endotélio Vascular , Hipertensão , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína ORAI1 , Canais de Cátion TRPC , Animais , Proteína ORAI1/metabolismo , Hipertensão/metabolismo , Hipertensão/fisiopatologia , Masculino , Camundongos , Células Endoteliais/metabolismo , Células Endoteliais/efeitos dos fármacos , Artérias Carótidas/efeitos dos fármacos , Artérias Carótidas/metabolismo , Canais de Cátion TRPC/metabolismo , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/metabolismo , Acetilcolina/farmacologia , Angiotensina II/farmacologia , Vasoconstrição/efeitos dos fármacos , Canais de Cátion TRPV/metabolismoRESUMO
Colon cancer is a complex malignancy characterized by intricate molecular interactions that influence its progression. This study investigates the role of calcium channel gene expression (ORAI1 and Piezo1) and their interplay with angiogenesis-related genes (VEGFA, CCL3, and NF-KB1) in colon cancer tissue biopsies. Additionally, we explore the mutation profiles of pivotal oncogenes (KRAS, PI3KCA, and BRAF) and their potential correlations with calcium channel and angiogenesis-related gene expression. The results indicate significant upregulation of ORAI1 and Piezo1, suggesting their involvement in colon cancer pathogenesis. Correlations between ORAI1 and VEGFA/CCL3 highlight potential crosstalk between calcium signaling and angiogenesis. The mutation analysis identifies prevalent oncogenic mutations, while intriguing connections between gene expression and oncogenic mutations emerge. Notably, mutant KRAS exon 2 samples exhibit elevated CCL3 and VEGFA expression, suggesting a nuanced link between specific KRAS mutations and the tumor microenvironment. These findings illuminate the intricate molecular landscape of colon cancer and emphasize the potential roles of calcium channels, angiogenesis-related genes, and oncogenic mutations as prognostic markers and therapeutic targets.
Assuntos
Neoplasias do Colo , Neoplasias Colorretais , Canais Iônicos , Proteína ORAI1 , Humanos , Angiogênese , Biomarcadores Tumorais/genética , Biópsia , Canais de Cálcio/genética , Proliferação de Células , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Neoplasias Colorretais/patologia , Mutação/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Microambiente Tumoral/genética , Canais Iônicos/metabolismo , Proteína ORAI1/metabolismoRESUMO
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.
Assuntos
Sinalização do Cálcio , Microglia , Humanos , Cálcio/metabolismo , Chumbo/toxicidade , Chumbo/metabolismo , Doenças Neuroinflamatórias , Proteína ORAI1/genética , Proteína ORAI1/metabolismo , Proteína ORAI1/farmacologia , Molécula 1 de Interação Estromal/genética , Molécula 1 de Interação Estromal/metabolismo , Movimento CelularRESUMO
Calcium (Ca2+) is a secondary messenger that regulates various cellular processes. However, Ca2+ mishandling could lead to pathological conditions. Orai1 is a Ca2+channel contributing to the store-operated calcium entry (SOCE) and plays a critical role in Ca2+ homeostasis in several cell types. Dysregulation of Orai1 contributed to severe combined immune deficiency syndrome, some cancers, pulmonary arterial hypertension (PAH), and other cardiorespiratory diseases. During its activation process, Orai1 is mainly regulated by stromal interacting molecule (STIM) proteins, especially STIM1; however, many other regulatory partners have also been recently described. Increasing knowledge about these regulatory partners provides a better view of the downstream signalling pathways of SOCE and offers an excellent opportunity to decipher Orai1 dysregulation in these diseases. These proteins participate in other cellular functions, making them attractive therapeutic targets. This review mainly focuses on Orai1 regulatory partners in the physiological and pathological conditions of the pulmonary circulation and inflammation.
Assuntos
Proteína ORAI1 , Humanos , Proteína ORAI1/metabolismo , Animais , Moléculas de Interação Estromal/metabolismo , Sinalização do Cálcio , Cálcio/metabolismo , Doenças Vasculares/metabolismo , Doenças Vasculares/patologiaRESUMO
INTRODUCTION: This study explores the immunogenetic associations of human leukocyte antigens (HLA) and the calcium release-activated calcium modulator 1 (ORAI1) and stromal interaction molecule 1 (STIM1) genes in HIV-1âpositive patients with HIV-related skin disorders. METHODS: This study assessed the distribution of variants of HLA class II alleles and expression levels of ORAI1 and STIM1 genes in the blood between HIV-1âpositive patients with HIV-related skin disorders and the control group with no HIV within the Latvian population. RESULTS: The research group comprised 115 HIV-1âpositive patients with HIV-related skin disorders, and the control group included 80 healthy individuals. Risk alleles (HLA- DQB1*02:01-0301 and HLA-DQA1*01:01-0501) and protective alleles (HLA-DRB1*07-13, DRB1*01-13, DRB1*04-11, and HLA-DQA1*05:01-0501) showed statistical significance in the groups. In 38 out of 115 patients, higher expression levels of ORAI1 and STIM1 genes were detected in the blood at the beginning of treatment. A significantly higher level of the microribonucleic acid (mRNA) ORAI1 gene was also found in the control group. CONCLUSIONS: The results demonstrate that HLA class II alleles are associated with a trend toward risk/protection concerning HIV-related skin disorders in HIV-1âpositive patients. It was also shown that a low level of ORAI1 mRNA and the risk allele HLA-DQB1*0201-0301 were simultaneously present in the research group.
Assuntos
Infecções por HIV , Proteína ORAI1 , Polimorfismo Genético , Molécula 1 de Interação Estromal , Humanos , Proteína ORAI1/genética , Masculino , Letônia , Feminino , Molécula 1 de Interação Estromal/genética , Adulto , Infecções por HIV/genética , Pessoa de Meia-Idade , Dermatopatias/genética , Estudos de Casos e Controles , Proteínas de Neoplasias/genética , Predisposição Genética para DoençaRESUMO
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.
Assuntos
Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina , Cálcio , Cardiomegalia , Diabetes Mellitus Tipo 2 , Cardiomiopatias Diabéticas , Glucose , Proteína ORAI1 , Molécula 1 de Interação Estromal , Animais , Ratos , Cálcio/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Cardiomegalia/etiologia , Cardiomegalia/metabolismo , Diabetes Mellitus Tipo 2/complicações , Glucose/metabolismo , Glucose/farmacologia , Proteína ORAI1/genética , Proteína ORAI1/metabolismo , Molécula 1 de Interação Estromal/genética , Molécula 1 de Interação Estromal/metabolismo , Regulação para Cima , Cardiomiopatias Diabéticas/complicações , Ratos Sprague-Dawley , MasculinoRESUMO
Tubular aggregate myopathy (TAM) and Stormorken syndrome (STRMK) are clinically overlapping disorders characterized by childhood-onset muscle weakness and a variable occurrence of multisystemic signs, including short stature, thrombocytopenia, and hyposplenism. TAM/STRMK is caused by gain-of-function mutations in the Ca2+ sensor STIM1 or the Ca2+ channel ORAI1, both of which regulate Ca2+ homeostasis through the ubiquitous store-operated Ca2+ entry (SOCE) mechanism. Functional experiments in cells have demonstrated that the TAM/STRMK mutations induce SOCE overactivation, resulting in excessive influx of extracellular Ca2+. There is currently no treatment for TAM/STRMK, but SOCE is amenable to manipulation. Here, we crossed Stim1R304W/+ mice harboring the most common TAM/STRMK mutation with Orai1R93W/+ mice carrying an ORAI1 mutation partially obstructing Ca2+ influx. Compared with Stim1R304W/+ littermates, Stim1R304W/+Orai1R93W/+ offspring showed a normalization of bone architecture, spleen histology, and muscle morphology; an increase of thrombocytes; and improved muscle contraction and relaxation kinetics. Accordingly, comparative RNA-Seq detected more than 1,200 dysregulated genes in Stim1R304W/+ muscle and revealed a major restoration of gene expression in Stim1R304W/+Orai1R93W/+ mice. Altogether, we provide physiological, morphological, functional, and molecular data highlighting the therapeutic potential of ORAI1 inhibition to rescue the multisystemic TAM/STRMK signs, and we identified myostatin as a promising biomarker for TAM/STRMK in humans and mice.
Assuntos
Transtornos Plaquetários , Dislexia , Ictiose , Transtornos de Enxaqueca , Miopatias Congênitas Estruturais , Proteína ORAI1 , Baço , Animais , Camundongos , Cálcio/metabolismo , Eritrócitos Anormais , Transtornos de Enxaqueca/tratamento farmacológico , Miose/tratamento farmacológico , Miose/genética , Miose/metabolismo , Fadiga Muscular , Miopatias Congênitas Estruturais/tratamento farmacológico , Miopatias Congênitas Estruturais/genética , Miopatias Congênitas Estruturais/metabolismo , Proteína ORAI1/genética , Proteína ORAI1/metabolismo , Baço/metabolismo , Baço/anormalidadesRESUMO
BACKGROUND: One of major challenges in breast tumor therapy is the existence of breast cancer stem cells (BCSCs). BCSCs are a small subpopulation of tumor cells that exhibit characteristics of stem cells. BCSCs are responsible for progression, recurrence, chemoresistance and metastasis of breast cancer. Ca2+ signalling plays an important role in diverse processes in cancer development. However, the role of Ca2+ signalling in BCSCs is still poorly understood. METHODS: A highly effective 3D soft fibrin gel system was used to enrich BCSC-like cells from ER+ breast cancer lines MCF7 and MDA-MB-415. We then investigated the role of two Ca2+-permeable ion channels Orai1 and Orai3 in the growth and stemness of BCSC-like cells in vitro, and tumorigenicity in female NOD/SCID mice in vivo. RESULTS: Orai1 RNA silencing and pharmacological inhibition reduced the growth of BCSC-like cells in tumor spheroids, decreased the expression levels of BCSC markers, and reduced the growth of tumor xenografts in NOD/SCID mice. Orai3 RNA silencing also had similar inhibitory effect on the growth and stemness of BCSC-like cells in vitro, and tumor xenograft growth in vivo. Mechanistically, Orai1 and SPCA2 mediate store-operated Ca2+ entry. Knockdown of Orai1 or SPCA2 inhibited glycolysis pathway, whereas knockdown of Orai3 or STIM1 had no effect on glycolysis. CONCLUSION: We found that Orai1 interacts with SPCA2 to mediate store-independent Ca2+ entry, subsequently promoting the growth and tumorigenicity of BCSC-like cells via glycolysis pathway. In contrast, Orai3 and STIM1 mediate store-operated Ca2+ entry, promoting the growth and tumorigenicity of BCSC-like cells via a glycolysis-independent pathway. Together, our study uncovered a well-orchestrated mechanism through which two Ca2+ entry pathways act through distinct signalling axes to finely control the growth and tumorigenicity of BCSCs.
Assuntos
Neoplasias da Mama , Canais de Cálcio , Camundongos Endogâmicos NOD , Camundongos SCID , Células-Tronco Neoplásicas , Proteína ORAI1 , Proteína ORAI1/metabolismo , Proteína ORAI1/genética , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Humanos , Animais , Feminino , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Neoplasias da Mama/genética , Camundongos , Canais de Cálcio/metabolismo , Canais de Cálcio/genética , Carcinogênese/metabolismo , Carcinogênese/patologia , Linhagem Celular Tumoral , Transdução de Sinais , Sinalização do Cálcio , Células MCF-7RESUMO
BACKGROUND: Airway epithelial barrier dysfunction has been proved to contribute to the development of type 2 inflammation of asthma. Interleukin (IL)-37 is a negative regulator of immune responses and allergic airway inflammation. However, whether IL-37 has any effect on airway epithelial barrier has been unknown. METHODS: We evaluated the role of IL-37 in both mouse model and cultured 16HBE cells. Histology and ELISA assays were used to evaluate airway inflammation. FITC-dextran permeability assay was used to evaluate the airway epithelial barrier function. Immunofluorescence, western blot and quantitative Real-Time PCR (RT-PCR) were used to evaluate the distribution and expression of tight junction proteins. RT-PCR and Ca2+ fluorescence measurement were used to evaluate the mRNA expression and activity of store-operated calcium entry (SOCE). RESULTS: IL-37 inhibited house dust mite (HDM)-induced airway inflammation and decreased the levels of IgE in serum and type 2 cytokines in bronchoalveolar lavage fluid (BALF) compared to asthmatic mice. IL-37 protected against HDM-induced airway epithelial barrier dysfunction, including reduced leakage of FITC-dextran, enhanced expression of TJ proteins, and restored the membrane distribution of TJ proteins. Moreover, IL-37 decreased the level of IL-33 in the BALF of asthmatic mice and the supernatants of HDM-treated 16HBE cells. IL-37 decreased the peak level of Ca2+ fluorescence induced by thapsigargin and HDM, and inhibited the mRNA expression of Orai1, suggesting an inhibiting effect of IL-37 on SOCE in airway epithelial cells. CONCLUSION: IL-37 plays a protective role in airway inflammation and HDM-induced airway epithelial barrier dysfunction by inhibiting SOCE.
Assuntos
Asma , Cálcio , Interleucina-1 , Pyroglyphidae , Animais , Feminino , Humanos , Camundongos , Asma/imunologia , Asma/metabolismo , Líquido da Lavagem Broncoalveolar/imunologia , Cálcio/metabolismo , Linhagem Celular , Citocinas/metabolismo , Modelos Animais de Doenças , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Imunoglobulina E/sangue , Imunoglobulina E/imunologia , Interleucina-1/metabolismo , Proteína ORAI1/metabolismo , Proteína ORAI1/genética , Pyroglyphidae/imunologia , Mucosa Respiratória/imunologia , Mucosa Respiratória/metabolismoRESUMO
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.
Assuntos
Debilidade Muscular , Membrana Nuclear , Molécula 1 de Interação Estromal , Animais , Humanos , Camundongos , Cálcio/metabolismo , Sinalização do Cálcio , Debilidade Muscular/genética , Debilidade Muscular/metabolismo , Músculo Esquelético/metabolismo , Mutação , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Membrana Nuclear/genética , Membrana Nuclear/metabolismo , Proteína ORAI1/genética , Molécula 1 de Interação Estromal/genética , Molécula 1 de Interação Estromal/metabolismoRESUMO
Orai1 channel capacity to control store-operated Ca2+ entry (SOCE) and B-cell functions is poorly understood and more specifically in B-cell cancers, including human lymphoma and leukemia. As compared to normal B-cells, Orai1 is overexpressed in B-chronic lymphocytic leukemia (B-CLL) and contributes in resting B-CLL to mediate an elevated basal Ca2+ level through a constitutive Ca2+ entry, and in BCR-activated B-cell to regulate the Ca2+ signaling response. Such observations were confirmed in human B-cell lymphoma and leukemia lines, including RAMOS, JOK-1, MEC-1 and JVM-3 cells. Next, the use of pharmacological Orai1 inhibitors (GSK-7975 A and Synta66) blocks constitutive Ca2+ entry and in turn affects B-cell cancer (primary and cell lines) survival and migration, controls cell cycle, and induces apoptosis through a mitochondrial and caspase-3 independent pathway. Finally, the added value of Orai1 inhibitors in combination with B-CLL drugs (ibrutinib, idelalisib, rituximab, and venetoclax) on B-CLL survival was tested, showing an additive/synergistic effect including in the B-cell cancer lines. To conclude, this study highlights the pathophysiological role of the Ca2+ channel Orai1 in B-cell cancers, and pave the way for the use of ORAI1 modulators as a plausible therapeutic strategy.
Assuntos
Leucemia Linfocítica Crônica de Células B , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Sinalização do Cálcio , Sobrevivência Celular , Linfócitos B/metabolismo , Linhagem Celular , Proteína ORAI1/metabolismo , Cálcio/metabolismo , Molécula 1 de Interação Estromal/metabolismoRESUMO
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.