RESUMO
BACKGROUND: Remodeling of vascular smooth muscle cells (VSMCs), as a pathological hallmark of cardiovascular diseases, is related to the molecular rewiring of Calcium signaling, which induces upregulation of stromal interaction molecule (STIM) proteins. This study analyzed the influence of STIM1 proteins on the remodeling of VSMCs in atherosclerosis (AS). METHODS: After oxidized low-density lipoprotein (ox-LDL) treatment and transfection, VSMC viability, migration, and invasion were separately measured using Cell Counting Kit-8, Scratch assay, and Transwell assay. An animal AS model was constructed, and histological analysis via hematoxylin-eosin staining was conducted on the aorta. RESULTS: Ox-LDL promoted expression of STIM1 and Orai calcium release-activated calcium modulator 1 (Orai1). STIM1 or Orai1 downregulation suppressed viability, migration, invasion, and phenotypic switching of ox-LDL-treated VSMCs, whereas STIM1 or Orai1 upregulation had opposite effects. Orai1 level was upregulated by STIM1 overexpression. Orai1 silencing reversed the effects of STIM1 overexpression in VSMCs. STIM1 deficiency alleviated AS and regulated expression of Orai1 and phenotypic switch-related factors in vivo. CONCLUSION: STIM1 deficiency suppresses viability, migration, invasion, and phenotypic switching of ox-LDL-induced VSMCs and alleviates AS by inhibiting Orai1.
Assuntos
Aterosclerose , Movimento Celular , Lipoproteínas LDL , Músculo Liso Vascular , Miócitos de Músculo Liso , Proteína ORAI1 , Molécula 1 de Interação Estromal , Molécula 1 de Interação Estromal/metabolismo , Molécula 1 de Interação Estromal/genética , Aterosclerose/patologia , Aterosclerose/metabolismo , Músculo Liso Vascular/patologia , Músculo Liso Vascular/metabolismo , Animais , Proteína ORAI1/metabolismo , Proteína ORAI1/genética , Proteína ORAI1/antagonistas & inibidores , Miócitos de Músculo Liso/metabolismo , Miócitos de Músculo Liso/patologia , Lipoproteínas LDL/metabolismo , Movimento Celular/efeitos dos fármacos , Humanos , Masculino , Camundongos , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Remodelação Vascular/efeitos dos fármacos , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/genéticaRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Flos Magnoliae (the dried flower buds of Magnolia biondii Pamp, FM) is a known herbal traditional medicine used for the symptomatic relief of nasal congestion and rhinorrhea caused by rhinitis and sinusitis. Magnolol, a neolignan from the magnolia family, is a secondary metabolite known to have anti-allergic and anti-inflammatory effects. However, the underlying mechanisms and therapeutic effect of magnolol in the treatment of allergic rhinitis (AR) remain elusive. AIMS OF THE STUDY: Anoctamin 1 (ANO1), a calcium-activated anion channel, mediates mucus and electrolyte secretion in nasal airway epithelial cells, whereas calcium release-activated calcium channel protein 1 (ORAI1) participates in the activation of T-lymphocytes and mast cells. The aim of our study is to understand the mechanisms of action of magnolol against AR, i.e., whether it acts through the modulation of ANO1 and ORAI1 channels that are expressed in nasal epithelial cells and T-lymphocytes, respectively. MATERIALS AND METHODS: Whole-cell patch clamp was used to record the activity of ORAI1 and ANO1 ion channels in ORAI1 or ANO1 overexpressed HEK293T cells, while the Ussing chamber apparatus was used to measure electrolyte transport via the epithelium, in Calu-3 cells cultured in an air-liquid interface. Additionally, calcium imaging of Jurkat T-lymphocytes was used to assess changes in the intracellular calcium concentration. Magnolol toxicity was assessed using the CCK-8 assay, and its effect on T-lymphocyte proliferation was measured by labeling human primary T-lymphocytes with carboxyfluorescein succinimidyl ester. Finally, OVA-induced Balb/c mice were employed to evaluate the effect of magnolol on nasal symptoms, as well as cytokine and eosinophil infiltration in AR. RESULTS: Magnolol inhibits ORAI1 and ANO1 channels in a concentration-dependent manner. Magnolol (30 µM) inhibits anti-CD3 induced cellular proliferation and production of IL-2 via ORAI1 channels in T-lymphocytes. Further, ATP-induced electrolyte transport mediated by ANO1 channels is significantly inhibited by magnolol in IL-4 sensitized Calu-3 cells. Notably, 300 µM magnolol significantly attenuates cytokine and eosinophil infiltration, thus alleviating AR symptoms in mice OVA-induced AR. CONCLUSION: Magnolol may be a promising therapeutic agent for the treatment and prevention of AR.
Assuntos
Antialérgicos/farmacologia , Compostos de Bifenilo/farmacologia , Lignanas/farmacologia , Magnolia/química , Rinite Alérgica/tratamento farmacológico , Animais , Anoctamina-1/antagonistas & inibidores , Antialérgicos/administração & dosagem , Antialérgicos/isolamento & purificação , Compostos de Bifenilo/administração & dosagem , Compostos de Bifenilo/isolamento & purificação , Linhagem Celular Tumoral , Citocinas/metabolismo , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Feminino , Flores , Células HEK293 , Humanos , Lignanas/administração & dosagem , Lignanas/isolamento & purificação , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Neoplasias/antagonistas & inibidores , Proteína ORAI1/antagonistas & inibidores , Ovalbumina , Técnicas de Patch-ClampRESUMO
Calcium signaling plays a vital role in the regulation of various cellular processes, including activation, proliferation, and differentiation of T-lymphocytes, which is mediated by ORAI1 and potassium (K+) channels. These channels have also been identified as highly attractive therapeutic targets for immune-related diseases. Licochalcone A is a licorice-derived chalconoid known for its multifaceted beneficial effects in pharmacological treatments, including its anti-inflammatory, anti-asthmatic, antioxidant, antimicrobial, and antitumorigenic properties. However, its anti-inflammatory effects involving ion channels in lymphocytes remain unclear. Thus, the present study aimed to investigate whether licochalcone A inhibits ORAI1 and K+ channels in T-lymphocytes. Our results indicated that licochalcone A suppressed all three channels (ORAI1, Kv1.3, and KCa3.1) in a concentration-dependent matter, with IC50 values of 2.97 ± 1.217 µM, 0.83 ± 1.222 µM, and 11.21 ± 1.07 µM, respectively. Of note, licochalcone A exerted its suppressive effects on the IL-2 secretion and proliferation in CD3 and CD28 antibody-induced T-cells. These results indicate that the use of licochalcone A may provide an effective treatment strategy for inflammation-related immune diseases.
Assuntos
Anti-Inflamatórios/farmacologia , Chalconas/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/antagonistas & inibidores , Canal de Potássio Kv1.3/antagonistas & inibidores , Proteína ORAI1/antagonistas & inibidores , Linfócitos T/efeitos dos fármacos , Cálcio/metabolismo , Sinalização do Cálcio , Células HEK293 , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/genética , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Células Jurkat , Canal de Potássio Kv1.3/genética , Canal de Potássio Kv1.3/metabolismo , Proteína ORAI1/genética , Proteína ORAI1/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismoRESUMO
Vascular calcification may result from stimulation of osteogenic signalling with upregulation of the transcription factors CBFA1, MSX2 and SOX9, as well as alkaline phosphatase (ALPL), which degrades and thus inactivates the calcification inhibitor pyrophosphate. Osteogenic signalling further involves upregulation of the Ca2+-channel ORAI1. The channel is activated by STIM1 and then accomplishes store-operated Ca2+ entry. ORAI1 and STIM1 are upregulated by the serum & glucocorticoid inducible kinase 1 (SGK1) which is critically important for osteogenic signalling. Stimulators of vascular calcification include vasopressin. The present study explored whether exposure of human aortic smooth muscle cells (HAoSMCs) to vasopressin upregulates ORAI1 and/or STIM1 expression, store-operated Ca2+ entry and osteogenic signalling. To this end, HAoSMCs were exposed to vasopressin (100 nM, 24 h) without or with additional exposure to ORAI1 blocker MRS1845 (10 µM) or SGK1 inhibitor GSK-650394 (1 µM). Transcript levels were measured using q-RT-PCR, cytosolic Ca2+-concentration ([Ca2+]i) by Fura-2-fluorescence, and store-operated Ca2+ entry from increase of [Ca2+]i following re-addition of extracellular Ca2+ after store depletion with thapsigargin (1 µM). As a result, vasopressin enhanced the transcript levels of ORAI1 and STIM1, store-operated Ca2+ entry, as well as the transcript levels of CBFA1, MSX2, SOX9 and ALPL. The effect of vasopressin on store-operated Ca2+ entry as well as on transcript levels of CBFA1, MSX2, SOX9 and ALPL was virtually abrogated by MRS1845 and GSK-650394. In conclusion, vasopressin stimulates expression of ORAI1/STIM1, thus augmenting store-operated Ca2+ entry and osteogenic signalling. In HAoSMCs, vasopressin (VP) upregulates Ca2+ channel ORAI1 and its activator STIM1. VP upregulates store-operated Ca2+ entry (SOCE) and osteogenic signalling (OS). VP-induced SOCE, OS and Ca2+-deposition are disrupted by ORAI1 inhibitor MRS1845. VP-induced SOCE, OS and Ca2+-deposition are disrupted by SGK1 blocker GSK-650394. KEY MESSAGES: ⢠In HAoSMCs, vasopressin (VP) upregulates Ca2+ channel ORAI1 and its activator STIM1. ⢠VP upregulates store-operated Ca2+ entry (SOCE) and osteogenic signalling (OS). ⢠VP-induced SOCE, OS and Ca2+-deposition are disrupted by ORAI1 inhibitor MRS1845. ⢠VP-induced SOCE, OS and Ca2+-deposition are disrupted by SGK1 blocker GSK-650394.
Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/efeitos dos fármacos , Proteína ORAI1/biossíntese , Calcificação Vascular/metabolismo , Vasopressinas/farmacologia , Aorta/citologia , Benzoatos/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Sinalização do Cálcio/fisiologia , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos , Humanos , Proteínas Imediatamente Precoces/antagonistas & inibidores , Proteínas Imediatamente Precoces/fisiologia , Miócitos de Músculo Liso/metabolismo , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiologia , Nitrendipino/análogos & derivados , Nitrendipino/farmacologia , Proteína ORAI1/antagonistas & inibidores , Proteína ORAI1/genética , Osteogênese/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/fisiologia , Molécula 1 de Interação Estromal/biossíntese , Molécula 1 de Interação Estromal/genética , Molécula 1 de Interação Estromal/fisiologia , Transcrição Gênica/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Calcificação Vascular/prevenção & controleRESUMO
Brain capillary endothelial cells (BCECs) form the blood-brain barrier (BBB) and play an essential role in the regulation of its functions. Oxidative stress accumulates excessive reactive oxygen species (ROS) and facilitates the death of BCECs, leading to a dysfunctional BBB. However, the mechanisms underlying the death of BCECs under oxidative stress remain unclear. In the present study, the effects of oxidative stress on cell viability, ROS production, intracellular Ca2+ concentration, and protein expression were examined using a cell line derived from bovine BCECs, t-BBEC117. When t-BBEC117 cells were exposed to oxidative stress induced by hydrogen peroxide (H2O2, 10-100 µM), cell growth was inhibited in a dose-dependent manner. Oxidative stress by 30 µM H2O2 increased the production of ROS and its effects were blocked by the ROS scavenger, 10 mM N-acetyl-l-cysteine (NAC). In addition, oxidative stress reduced store-operated Ca2+ entry (SOCE) and this decrease was recovered by NAC or the Orai channel activator, 5 µM 2-aminoethyl diphenylborinate (2-APB). The siRNA knockdown of Orai1 revealed that Orai1 was mainly responsible for SOCE channels and its activity was decreased by oxidative stress. However, the protein expression of Orai1 and STIM1 was not affected by oxidative stress. Oxidative stress-induced cell death was rescued by 2-APB, NAC, or the STIM-Orai activating region. In conclusion, oxidative stress reduces Orai1-mediated SOCE and, thus, facilitates the death of BCECs.
Assuntos
Acetilcisteína/farmacologia , Cálcio/metabolismo , Células Endoteliais/efeitos dos fármacos , Proteína ORAI1/antagonistas & inibidores , Estresse Oxidativo , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Barreira Hematoencefálica/metabolismo , Bovinos , Morte Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Células Endoteliais/metabolismo , Peróxido de Hidrogênio/antagonistas & inibidores , Peróxido de Hidrogênio/farmacologia , Proteína ORAI1/metabolismo , Estresse Oxidativo/efeitos dos fármacosRESUMO
Calcium (Ca2+) and its relevant transmembrane and intracellular calcium channels were previously thought to be chiefly associated with the regulation of cardiovascular and neuronal systems. Nowadays, an increasing evidence shows those calcium channels are also responsible for tumorigenesis and progression. However, the general underlying mechanisms and the involving signaling transduction pathways still remain unclear. Therefore, in this mini-review, we are mainly focusing on the linkage between calcium channels and major characteristics of tumors such as multi-drug resistance (MDR), metastasis, apoptosis, proliferation, evasion of immune surveillance, and the alterations of tumor microenvironment. We will shed light on the possible therapeutic approaches to counteract tumors regarding the intervention of calcium channel.
Assuntos
Canais de Cálcio Ativados pela Liberação de Cálcio/metabolismo , Sinalização do Cálcio , Cálcio/metabolismo , Neoplasias/metabolismo , Proteína ORAI1/metabolismo , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Bloqueadores dos Canais de Cálcio/uso terapêutico , Canais de Cálcio Ativados pela Liberação de Cálcio/antagonistas & inibidores , ATPases Transportadoras de Cálcio/antagonistas & inibidores , Carcinogênese/metabolismo , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Humanos , Neoplasias/tratamento farmacológico , Proteína ORAI1/antagonistas & inibidores , Canais de Potencial de Receptor Transitório/antagonistas & inibidores , Canais de Potencial de Receptor Transitório/metabolismo , Microambiente TumoralRESUMO
BACKGROUND AND PURPOSE: Mibefradil, a T-type Ca2+ channel blocker, has been investigated for treating solid tumours. However, its underlying mechanisms are still unclear. Here, we have investigated the pharmacological actions of mibefradil on Orai store-operated Ca2+ channels. EXPERIMENTAL APPROACH: Human Orai1-3 cDNAs in tetracycline-regulated pcDNA4/TO vectors were transfected into HEK293 T-REx cells with stromal interaction molecule 1 (STIM1) stable expression. The Orai currents were recorded by whole-cell and excised-membrane patch clamp. Ca2+ influx or release was measured by Fura-PE3/AM. Cell growth and death were monitored by WST-1, LDH assays and flow cytometry. KEY RESULTS: Mibefradil inhibited Orai1, Orai2, and Orai3 currents dose-dependently. The IC50 for Orai1, Orai2, and Orai3 channels was 52.6, 14.1, and 3.8 µM respectively. Outside-out patch demonstrated that perfusion of 10-µM mibefradil to the extracellular surface completely blocked Orai3 currents and single channel activity evoked by 2-APB. Intracellular application of mibefradil did not alter Orai3 channel activity. Mibefradil at higher concentrations (>50 µM) inhibited Ca2+ release but had no effect on cytosolic STIM1 translocation evoked by thapsigargin. Inhibition on Orai channels by mibefradil was structure-related, as other T-type Ca2+ channel blockers with different structures, such as ethosuximide and ML218, had no or minimal effects on Orai channels. Moreover, mibefradil inhibited cell proliferation, induced apoptosis, and arrested cell cycle progression. CONCLUSIONS AND IMPLICATIONS: Mibefradil is a potent cell surface blocker of Orai channels, demonstrating a new pharmacological action of this compound in regulating cell growth and death, which could be relevant to its anti-cancer activity.
Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/metabolismo , Mibefradil/farmacologia , Proteína ORAI1/antagonistas & inibidores , Proteína ORAI2/antagonistas & inibidores , Cálcio/análise , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/química , Morte Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Células HEK293 , Humanos , Mibefradil/química , Proteína ORAI1/metabolismo , Proteína ORAI2/metabolismo , Imagem ÓpticaRESUMO
BACKGROUND: Dysregulation in calcium (Ca2+) signaling is a hallmark of chronic lymphocytic leukemia (CLL). While the role of the B cell receptor (BCR) Ca2+ pathway has been associated with disease progression, the importance of the newly described constitutive Ca2+ entry (CE) pathway is less clear. In addition, we hypothesized that these differences reflect modifications of the CE pathway and Ca2+ actors such as Orai1, transient receptor potential canonical (TRPC) 1, and stromal interaction molecule 1 (STIM1), the latter being the focus of this study. METHODS: An extensive analysis of the Ca2+ entry (CE) pathway in CLL B cells was performed including constitutive Ca2+ entry, basal Ca2+ levels, and store operated Ca2+ entry (SOCE) activated following B cell receptor engagement or using Thapsigargin. The molecular characterization of the calcium channels Orai1 and TRPC1 and to their partner STIM1 was performed by flow cytometry and/or Western blotting. Specific siRNAs for Orai1, TRPC1 and STIM1 plus the Orai1 channel blocker Synta66 were used. CLL B cell viability was tested in the presence of an anti-STIM1 monoclonal antibody (mAb, clone GOK) coupled or not with an anti-CD20 mAb, rituximab. The Cox regression model was used to determine the optimal threshold and to stratify patients. RESULTS: Seeking to explore the CE pathway, we found in untreated CLL patients that an abnormal CE pathway was (i) highly associated with the disease outcome; (ii) positively correlated with basal Ca2+ concentrations; (iii) independent from the BCR-PLCγ2-InsP3R (SOCE) Ca2+ signaling pathway; (iv) supported by Orai1 and TRPC1 channels; (v) regulated by the pool of STIM1 located in the plasma membrane (STIM1PM); and (vi) blocked when using a mAb targeting STIM1PM. Next, we further established an association between an elevated expression of STIM1PM and clinical outcome. In addition, combining an anti-STIM1 mAb with rituximab significantly reduced in vitro CLL B cell viability within the high STIM1PM CLL subgroup. CONCLUSIONS: These data establish the critical role of a newly discovered BCR independent Ca2+ entry in CLL evolution, provide new insights into CLL pathophysiology, and support innovative therapeutic perspectives such as targeting STIM1 located at the plasma membrane.
Assuntos
Antineoplásicos Imunológicos/farmacologia , Linfócitos B/efeitos dos fármacos , Sinalização do Cálcio/genética , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Proteínas de Neoplasias/antagonistas & inibidores , Molécula 1 de Interação Estromal/antagonistas & inibidores , Idoso , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Antineoplásicos Imunológicos/uso terapêutico , Linfócitos B/citologia , Linfócitos B/imunologia , Linfócitos B/metabolismo , Cálcio/imunologia , Cálcio/metabolismo , Sinalização do Cálcio/imunologia , Membrana Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/imunologia , Progressão da Doença , Feminino , Técnicas de Silenciamento de Genes , Humanos , Leucemia Linfocítica Crônica de Células B/sangue , Leucemia Linfocítica Crônica de Células B/imunologia , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/antagonistas & inibidores , Proteína ORAI1/genética , Proteína ORAI1/imunologia , Proteína ORAI1/metabolismo , Cultura Primária de Células , Estudos Prospectivos , RNA Interferente Pequeno/metabolismo , Molécula 1 de Interação Estromal/genética , Molécula 1 de Interação Estromal/metabolismo , Canais de Cátion TRPC/genética , Canais de Cátion TRPC/imunologia , Canais de Cátion TRPC/metabolismo , Resultado do Tratamento , Células Tumorais CultivadasRESUMO
Glioblastomas (GBM) are the most aggressive brain cancers without effective therapeutics. The Hippo pathway transcriptional coactivators YAP/TAZ were implicated as drivers in GBM progression and could be therapeutic targets. Here we found in an unbiased screen of 1650 compounds that amlodipine is able to inhibit survival of GBM cells by suppressing YAP/TAZ activities. Instead of its known function as an L-type calcium channel blocker, we found that amlodipine is able to activate Ca2+ entry by enhancing store-operated Ca2+ entry (SOCE). Amlodipine as well as approaches that cause store depletion and activate SOCE trigger phosphorylation and activation of Lats1/2, which in turn phosphorylate YAP/TAZ and prevent their accumulation in the cell nucleus. Furthermore, we identified that protein kinase C (PKC) beta II is a major mediator of Ca2+-induced Lats1/2 activation. Ca2+ induces accumulation of PKC beta II in an actin cytoskeletal compartment. Such translocation depends on inverted formin-2 (INF2). Depletion of INF2 disrupts both PKC beta II translocation and Lats1/2 activation. Functionally, we found that elevation of cytosolic Ca2+ or PKC beta II expression inhibits YAP/TAZ-mediated gene transcription. In vivo PKC beta II expression inhibits GBM tumor growth and prolongs mouse survival through inhibition of YAP/TAZ in an orthotopic mouse xenograft model. Our studies indicate that Ca2+ is a crucial intracellular cue that regulates the Hippo pathway and that triggering SOCE could be a strategy to target YAP/TAZ in GBM.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Anlodipino/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Glioblastoma/patologia , Proteínas de Neoplasias/antagonistas & inibidores , Fosfoproteínas/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/fisiologia , Transdução de Sinais/efeitos dos fármacos , Fatores de Transcrição/antagonistas & inibidores , Aciltransferases , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Linhagem Celular Tumoral , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Ativação Enzimática/efeitos dos fármacos , Feminino , Técnicas de Silenciamento de Genes , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Via de Sinalização Hippo , Humanos , Ionomicina/farmacologia , Camundongos , Camundongos Nus , Proteínas de Neoplasias/fisiologia , Proteína ORAI1/antagonistas & inibidores , Proteína ORAI1/genética , Proteína ORAI1/fisiologia , Fosfoproteínas/genética , Fosforilação/efeitos dos fármacos , Proteína Quinase C beta/fisiologia , Proteínas Quinases/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , RNA Interferente Pequeno/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais/fisiologia , Tapsigargina/farmacologia , Fatores de Transcrição/genética , Proteínas de Sinalização YAPRESUMO
OBJECTIVE: To investigate mechanisms underlying the capability of ADAM15 to transform FasL-mediated death-inducing signals into prosurvival activation of Src and focal adhesion kinase (FAK) in rheumatoid arthritis synovial fibroblasts (RASFs). METHODS: Caspase 3/7 activity and apoptosis rate were determined in RASFs and ADAM15-transfected T/C28a4 cells upon Fas/CD95 triggering using enzyme assays and annexin V staining. Phosphorylated Src and FAK were analyzed by immunoblotting. Interactions of ADAM15 and CD95 with calmodulin (CaM), Src, or FAK were analyzed by pull-downs using CaM-Sepharose and coimmunoprecipitations with specific antibodies. Protein binding assays were performed using recombinant CaM and ADAM15. Immunofluorescence was performed to investigate subcellular colocalization of ADAM15, Fas/CD95, and CaM. RESULTS: The antiapoptotic effect of ADAM15 in FasL-stimulated cells was demonstrated either by increased apoptosis of cells transfected with an ADAM15 construct lacking the cytoplasmic domain compared to cells transfected with full-length ADAM15 or by reduced apoptosis resistance of RASFs upon RNA interference silencing of ADAM15. Fas ligation triggered a Ca2+ release-activated Ca2+ /calcium release-activated calcium channel protein 1 (CRAC/Orai1) channel-dependent CaM recruitment to Fas/CD95 and ADAM15 in the cell membrane. Simultaneously, Src associated with CaM was shown to become engaged in the ADAM15 complex also containing cytoplasmic-bound FAK. Accordingly, Fas ligation in RASFs led to ADAM15-dependent phosphorylation of Src and FAK, which was associated with increased survival. Pharmacologic interference with either the CaM inhibitor trifluoperazine or the CRAC/Orai inhibitor BTP-2 simultaneously applied with FasL synergistically enhanced Fas-mediated apoptosis in RASFs. CONCLUSION: ADAM15 provides a scaffold for formation of CaM-dependent prosurvival signaling complexes upon CRAC/Orai coactivation by FasL-induced death signals and a potential therapeutic target to break apoptosis resistance in RASFs.
Assuntos
Proteínas ADAM/genética , Apoptose/genética , Artrite Reumatoide/metabolismo , Calmodulina/metabolismo , Condrócitos/metabolismo , Proteína Ligante Fas/metabolismo , Fibroblastos/metabolismo , Proteínas de Membrana/genética , Proteínas ADAM/metabolismo , Anilidas/farmacologia , Apoptose/efeitos dos fármacos , Calmodulina/antagonistas & inibidores , Linhagem Celular , Quinase 1 de Adesão Focal/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Proteína ORAI1/antagonistas & inibidores , Proteína ORAI1/metabolismo , Fosforilação , Interferência de RNA , Membrana Sinovial/citologia , Tiadiazóis/farmacologia , Trifluoperazina/farmacologia , Receptor fas/metabolismo , Quinases da Família src/metabolismoRESUMO
BACKGROUND: Store-operated calcium entry (SOCE), primarily mediated by Orai1 and stromal interaction molecule 1 (STIM1), is a major Ca2+ influx pathway that has been linked to human diseases including myopathy, epilepsy, immunodeficiency, and cancer. Despite of the recent rapid progress of dissecting molecular mechanisms underlying SOCE activation, the development of therapies against dysfunctional SOCE significantly lags behind, partly due to the lack of more specific pharmacological tools and poor understanding of currently available SOCE modifiers, including the a newly identified SOCE inhibitor, digitoxin. OBJECTIVE AND METHODS: Capitalizing on Ca2+ imaging and pharmacological tools, we aimed to systemically delineate the mechanism of action of digitoxin by defining how it impinges on Orai1 to exert its suppressive effect on SOCE. RESULTS: The SOCE-suppressive function of digitoxin is dependent on S27-S30 residues of wild-type Orai1. With 8h-incubation of digitoxin with STIM1-prebound Orai1 or a constitutively active mutant Orai1-ANSGA, its inhibition was no longer dependent on S27-S30 residues. Instead, the inhibition may involve the pore region of Orai1 channels, as V102C mutant at the pore region would greatly diminish or abolish the inhibition on pre-activated Orai1. CONCLUSIONS: Our study identified two regions that are critical for the inhibition on Orai1 channels, providing valuable hotspots for future design of SOCE inhibitors.
Assuntos
Sinalização do Cálcio , Cálcio , Digitoxina , Proteína ORAI1 , Substituição de Aminoácidos , Cálcio/química , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/genética , Digitoxina/química , Digitoxina/farmacologia , Células HEK293 , Humanos , Mutação de Sentido Incorreto , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/antagonistas & inibidores , Proteína ORAI1/química , Proteína ORAI1/genética , Proteína ORAI1/metabolismo , Fosforilação/efeitos dos fármacos , Domínios Proteicos , Molécula 1 de Interação Estromal/química , Molécula 1 de Interação Estromal/genética , Molécula 1 de Interação Estromal/metabolismoRESUMO
BACKGROUND/AIMS: Multiple myeloma (MM) is a plasma cell neoplasm which constitutes about 10% of all hematologic malignancies. Despite the development and application of novel agents, MM still undergoes an aggressive and incurable course in the vast majority of patients. Ca2+ is one of the critical regulators of cell migration. Ca2+ influx is essential for the migration of various types of cells including tumor cells. However, the role of store-operated calcium entry (SOC) channels, the only Ca2+ channels of non-excitable cells, has not yet been reported in MM cell survival. METHODS: We evaluated the expression of Stim1 and Orai1 (two key regulators of SOC) in MM tissues and cell lines by immunohistochemical assay, quantitative real-time PCR assay and western blot. MM cell lines were pretreated with pharmacological blockers and siRNAs, and then MM cell proliferation, cell cycle arrest, and apoptosis were examined by FACS (flow cytometry) assay, and Annexin V-FITC/PI staining. The correlation between the expression of Stim1 (or Orai1) level and outcome in MM were assessed by using Progress Free Survival (PFS). RESULTS: Stim1 and Orai1 were both abundantly expressed in MM tissue and MM cell lines. Inhibition of SOCE reduced MM cell viability, and induced cell cycle arrest and apoptosis. Stim1 or Orai1 silencing also reduced cell viability, caused cell apoptosis and cell cycle arrest in MM cell lines. Over-expression of Stim1/Orai1 in MM patients was closely associated with the clinical outcome of MM. CONCLUSION: The Stim1/Orai1-mediated signaling participates in the pathogenesis of MM, which represents an attractive target for future therapeutic intervention.
Assuntos
Mieloma Múltiplo/patologia , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/metabolismo , Molécula 1 de Interação Estromal/metabolismo , Adulto , Idoso , Apoptose/efeitos dos fármacos , Medula Óssea/metabolismo , Medula Óssea/patologia , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Feminino , Humanos , Imidazóis/farmacologia , Masculino , Pessoa de Meia-Idade , Mieloma Múltiplo/metabolismo , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteína ORAI1/antagonistas & inibidores , Proteína ORAI1/genética , Prognóstico , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Molécula 1 de Interação Estromal/antagonistas & inibidores , Molécula 1 de Interação Estromal/genéticaRESUMO
Our recent study showed that bradykinin increases cell cycling progression and migration of human cardiac c-Kit+ progenitor cells by activating pAkt and pERK1/2 signals. This study investigated whether bradykinin-mediated Ca2+ signalling participates in regulating cellular functions in cultured human cardiac c-Kit+ progenitor cells using laser scanning confocal microscopy and biochemical approaches. It was found that bradykinin increased cytosolic free Ca2+ ( Cai2+ ) by triggering a transient Ca2+ release from ER IP3Rs followed by sustained Ca2+ influx through store-operated Ca2+ entry (SOCE) channel. Blockade of B2 receptor with HOE140 or IP3Rs with araguspongin B or silencing IP3R3 with siRNA abolished both Ca2+ release and Ca2+ influx. It is interesting to note that the bradykinin-induced cell cycle progression and migration were not observed in cells with siRNA-silenced IP3R3 or the SOCE component TRPC1, Orai1 or STIM1. Also the bradykinin-induced increase in pAkt and pERK1/2 as well as cyclin D1 was reduced in these cells. These results demonstrate for the first time that bradykinin-mediated increase in free Cai2+ via ER-IP3R3 Ca2+ release followed by Ca2+ influx through SOCE channel plays a crucial role in regulating cell growth and migration via activating pAkt, pERK1/2 and cyclin D1 in human cardiac c-Kit+ progenitor cells.
Assuntos
Bradicinina/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Cálcio/metabolismo , Proteínas Proto-Oncogênicas c-kit/genética , Células-Tronco/efeitos dos fármacos , Cátions Bivalentes , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ciclina D1/genética , Ciclina D1/metabolismo , Feminino , Regulação da Expressão Gênica , Humanos , Receptores de Inositol 1,4,5-Trifosfato/genética , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Transporte de Íons/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Miocárdio/citologia , Miocárdio/metabolismo , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/antagonistas & inibidores , Proteína ORAI1/genética , Proteína ORAI1/metabolismo , Cultura Primária de Células , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-kit/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-kit/metabolismo , Quinolizinas/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Molécula 1 de Interação Estromal/antagonistas & inibidores , Molécula 1 de Interação Estromal/genética , Molécula 1 de Interação Estromal/metabolismo , Canais de Cátion TRPC/antagonistas & inibidores , Canais de Cátion TRPC/genética , Canais de Cátion TRPC/metabolismoRESUMO
The proteins Orai1 and STIM1 control store-operated Ca2+ entry (SOCE) into cells. SOCE is important for migration, invasion and metastasis of MDA-MB-231 human triple negative breast cancer (TNBC) cells and has been proposed as a target for cancer drug discovery. Two hit compounds from a medium throughput screen, displayed encouraging inhibition of SOCE in MDA-MB-231 cells, as measured by a Fluorescence Imaging Plate Reader (FLIPR) Ca2+ assay. Following NMR spectroscopic analysis of these hits and reassignment of their structures as 5-hydroxy-5-trifluoromethylpyrazolines, a series of analogues was prepared via thermal condensation reactions between substituted acylhydrazones and trifluoromethyl 1,3-dicarbonyl arenes. Structure-activity relationship (SAR) studies showed that small lipophilic substituents at the 2- and 3-positions of the RHS and 2-, 3- and 4-postions of the LHS terminal benzene rings improved activity, resulting in a novel class of potent and selective inhibitors of SOCE.
Assuntos
Bloqueadores dos Canais de Cálcio/química , Proteína ORAI1/antagonistas & inibidores , Pirazóis/química , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Linhagem Celular Tumoral , Feminino , Humanos , Proteína ORAI1/metabolismo , Análise Serial de Proteínas , Pirazóis/metabolismo , Pirazóis/farmacologia , Espectrometria de Fluorescência , Relação Estrutura-AtividadeRESUMO
Ca2+ entry mediated by the calcium channel, Orai1, provides critical Ca2+ signals that regulate cell function. The ER-Ca2+ sensor protein, STIM1, recruits and strongly activates Orai1 within ER-PM junctions. STIM2 is a poor activator of Orai1, and its physiological role is not well understood. Herein, we report a crucial function for STIM2 in inducing the activated conformation of STIM1. By using conformational sensors of STIM2 and STIM1, together with protein interaction and functional studies, we show that STIM2 is constitutively localized within ER-PM junctions in ER-Ca2+ store replete cells. Importantly, STIM2 traps STIM1 and triggers remodeling of STIM1 C terminus, causing STIM1/Orai1 coupling and enhancement of Orai1 function in cells with relatively high ER-[Ca2+]. The increase in Ca2+ entry controls Ca2+-dependent transcription factor, NFAT, activation at low [agonist]. Our findings reveal that STIM2 modulates STIM1/Orai1 function to tune the fidelity of receptor-evoked Ca2+ signaling and the physiological response of cells.
Assuntos
Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/metabolismo , Molécula 1 de Interação Estromal/metabolismo , Molécula 2 de Interação Estromal/metabolismo , Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Células HEK293 , Humanos , Indóis/farmacologia , Microscopia Confocal , Fatores de Transcrição NFATC/metabolismo , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteína ORAI1/antagonistas & inibidores , Proteína ORAI1/genética , Técnicas de Patch-Clamp , Conformação Proteica , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Molécula 1 de Interação Estromal/antagonistas & inibidores , Molécula 1 de Interação Estromal/genética , Molécula 2 de Interação Estromal/genéticaRESUMO
Store operated calcium channels (SOCCs) have been suggested to play a critical role in many diabetic complications. Diabetic cystopathy (DCP) is common in patients with diabetes, but the role of SOCCs in DCP is still unclear. The aim of the present study was to investigate the role of SOCCs in DCP with streptozocin (STZ)induced diabetic rats. Specifically, the authors investigated whether SOCCs were altered in streptozocin (STZ)induced diabetic rats and, if so, how this may contribute to the contraction of bladder detrusor strips and the intracellular Ca2+ concentration of bladder smooth muscle cells in diabetic rats. Cyclopiazonic acid (CPA, 10 µM) and SKF96365 (10 µM) were used to activate and inhibit SOCCs respectively, to research the effects of SOCCs on the contraction of the bladder detrusor strips in normal and STZinduced diabetic rats at the 4th, 8th and 12th week after the diabetic rat model was established. The changes of intracellular Ca2+ were also evaluated under confocal microscopy with pretreated Fluo4AM. In addition, the expressions of Orai1 and STIM1 were detected by reverse transcriptionquantitative polymerase chain reaction and western blotting at different time points. According to the results, the contractive frequency of diabetic bladder muscle strips was higher than that of controls in the 4th and 8th week. The increased fluorescence intensity was detected after using CPA and SKF96365 in diabetic groups. The expressions of Orai1 and STIM1 changed in a timedependent manner.
Assuntos
Complicações do Diabetes/metabolismo , Diabetes Mellitus Experimental/metabolismo , Regulação da Expressão Gênica , Proteína ORAI1/metabolismo , Molécula 1 de Interação Estromal/metabolismo , Doenças da Bexiga Urinária/metabolismo , Animais , Complicações do Diabetes/patologia , Diabetes Mellitus Experimental/patologia , Feminino , Imidazóis/farmacologia , Indóis/farmacologia , Proteína ORAI1/antagonistas & inibidores , Ratos , Ratos Sprague-Dawley , Molécula 1 de Interação Estromal/antagonistas & inibidores , Doenças da Bexiga Urinária/patologiaRESUMO
Zinc, an essential micronutrient, has a cancer preventive role. Zinc deficiency has been shown to contribute to the progression of esophageal cancer. Orai1, a store-operated Ca2+ entry (SOCE) channel, was previously reported to be highly expressed in tumor tissues removed from patients with esophageal squamous cell carcinoma (ESCC) with poor prognosis, and elevation of its expression contributes to both hyperactive intracellular Ca2+ oscillations and fast cell proliferation in human ESCC cells. However, the molecular basis of cancer preventive functions of zinc and its association with Orai1-mediated cell proliferation remains unknown. The present study shows that zinc supplementation significantly inhibits proliferation of ESCC cell lines and that the effect of zinc is reversible with N,N,N',N'-tetrakis (2-pyridylmethyl) ethylenediamine, a specific Zn2+ chelator, whereas nontumorigenic esophageal epithelial cells are significantly less sensitive to zinc treatment. Fluorescence live cell imaging revealed that extracellular Zn2+ exerted rapid inhibitory effects on Orai1-mediated SOCE and on intracellular Ca2+ oscillations in the ESCC cells. Knockdown of Orai1 or expression of Orai1 mutants with compromised zinc binding significantly diminished sensitivity of the cancer cells to zinc treatment in both SOCE and cell proliferation analyses. These data suggest that zinc may inhibit cell proliferation of esophageal cancer cells through Orai1-mediated intracellular Ca2+ oscillations and reveal a possible molecular basis for zinc-induced cancer prevention and Orai1-SOCE signaling pathway in cancer cells.-Choi, S., Cui, C., Luo, Y., Kim, S.-H., Ko, J.-K., Huo, X., Ma, J., Fu, L.-W., Souza, R. F., Korichneva, I., Pan, Z. Selective inhibitory effects of zinc on cell proliferation in esophageal squamous cell carcinoma through Orai1.
Assuntos
Carcinoma de Células Escamosas/tratamento farmacológico , Carcinoma de Células Escamosas/metabolismo , Neoplasias Esofágicas/tratamento farmacológico , Neoplasias Esofágicas/metabolismo , Proteína ORAI1/metabolismo , Zinco/farmacologia , Substituição de Aminoácidos , Sinalização do Cálcio/efeitos dos fármacos , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Quelantes/farmacologia , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago , Etilenodiaminas/farmacologia , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Modelos Biológicos , Mutagênese Sítio-Dirigida , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteína ORAI1/antagonistas & inibidores , Proteína ORAI1/genéticaRESUMO
The role of Ca2+ during dengue virus (DENV) replication is unknown; thus, changes in Ca2+ homeostasis in DENV infected human hepatic HepG2 and Huh-7 cells were analyzed. Infected HepG2 cells, but not Huh-7 cells, showed a significant increase in plasma membrane permeability to Ca2+, while both cell lines showed marked reduced levels of Ca2+ stored in the endoplasmic reticulum. While the expression levels of STIM1 and ORAI1 showed no changes, STIM1 and ORAI1 were shown to co-localized in infected cells, indicating activation of the store-operated Ca2+ entry (SOCE) pathway. Finally, manipulation in the infected cells of the intra and extracellular Ca2+ levels by chelators (BAPTA-AM and EGTA), SOC inhibitor (SKF96365), IP3 Receptor antagonist (2APB) or increase of extracellular [Ca2+], significantly reduced DENV yield, but not vesicular stomatitis virus yield, used as a control. These results show that DENV infection alters cell Ca2+ homeostasis and that such changes favor viral replication.
Assuntos
Quelantes de Cálcio/farmacologia , Cálcio/metabolismo , Vírus da Dengue/efeitos dos fármacos , Homeostase/efeitos dos fármacos , Interações Hospedeiro-Patógeno , Replicação Viral/efeitos dos fármacos , Animais , Compostos de Boro/farmacologia , Bloqueadores dos Canais de Cálcio/farmacologia , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Membrana Celular/virologia , Permeabilidade da Membrana Celular , Chlorocebus aethiops , Vírus da Dengue/fisiologia , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Retículo Endoplasmático/virologia , Expressão Gênica , Células Hep G2 , Humanos , Imidazóis/farmacologia , Receptores de Inositol 1,4,5-Trifosfato/antagonistas & inibidores , Receptores de Inositol 1,4,5-Trifosfato/genética , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Transporte de Íons , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/antagonistas & inibidores , Proteína ORAI1/genética , Proteína ORAI1/metabolismo , Molécula 1 de Interação Estromal/antagonistas & inibidores , Molécula 1 de Interação Estromal/genética , Molécula 1 de Interação Estromal/metabolismo , Células Vero , Vírus da Estomatite Vesicular Indiana/efeitos dos fármacos , Vírus da Estomatite Vesicular Indiana/fisiologia , Replicação Viral/genéticaRESUMO
Impaired albumin reabsorption by proximal tubular epithelial cells (PTECs) has been highlighted in diabetic nephropathy (DN), but little is known about the underlying molecular mechanisms. Here we find that ORAI1-3, are preferentially expressed in PTECs and downregulated in patients with DN. Hyperglycemia or blockade of insulin signaling reduces the expression of ORAI1-3. Inhibition of ORAI channels by BTP2 and diethylstilbestrol or silencing of ORAI expression impairs albumin uptake. Transgenic mice expressing a dominant-negative Orai1 mutant (E108Q) increases albuminuria, and in vivo injection of BTP2 exacerbates albuminuria in streptozotocin-induced and Akita diabetic mice. The albumin endocytosis is Ca2+-dependent and accompanied by ORAI1 internalization. Amnionless (AMN) associates with ORAIs and forms STIM/ORAI/AMN complexes after Ca2+ store depletion. STIM1/ORAI1 colocalizes with clathrin, but not with caveolin, at the apical membrane of PTECs, which determines clathrin-mediated endocytosis. These findings provide insights into the mechanisms of protein reabsorption and potential targets for treating diabetic proteinuria.
Assuntos
Albuminas/metabolismo , Albuminúria/genética , Canais de Cálcio/genética , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Nefropatias Diabéticas/metabolismo , Células Epiteliais/metabolismo , Túbulos Renais Proximais/metabolismo , Proteína ORAI1/genética , Proteína ORAI2/genética , Albuminas/efeitos dos fármacos , Albuminúria/metabolismo , Anilidas/farmacologia , Animais , Cálcio/metabolismo , Bloqueadores dos Canais de Cálcio , Canais de Cálcio/metabolismo , Estudos de Casos e Controles , Caveolinas/metabolismo , Linhagem Celular , Clatrina/metabolismo , Dietilestilbestrol/farmacologia , Regulação para Baixo , Endocitose , Células Epiteliais/efeitos dos fármacos , Estrogênios não Esteroides/farmacologia , Feminino , Humanos , Hiperglicemia/genética , Hiperglicemia/metabolismo , Rim/metabolismo , Túbulos Renais Proximais/citologia , Túbulos Renais Proximais/efeitos dos fármacos , Masculino , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Mutação , Proteína ORAI1/antagonistas & inibidores , Proteína ORAI1/metabolismo , Proteína ORAI2/antagonistas & inibidores , Proteína ORAI2/metabolismo , Reabsorção Renal/efeitos dos fármacos , Reabsorção Renal/genética , Molécula 1 de Interação Estromal/metabolismo , Tiadiazóis/farmacologiaRESUMO
Mast cells play a primary role in allergic diseases. During an allergic reaction, mast cell activation is initiated by cross-linking IgE-FcεRI complex by multivalent antigen resulting in degranulation. Additionally, G protein-coupled receptors also induce degranulation upon activation. However, the spatio-temporal relationship between Ca2+ mobilization and mast cell degranulation is not well understood. We investigated the relationship between oscillations in Ca2+ level and mast cell degranulation upon stimulation in rat RBL-2H3 cells. Nile red and Fluo-4 were used as probes for monitoring histamine and intracellular Ca2+ levels, respectively. Histamine release and Ca2+ oscillations in real-time were monitored using total internal reflection fluorescence microscopy (TIRFM). Mast cell degranulation followed immediately after FcεRI and GPCR-mediated Ca2+ increase. FcεRI-induced Ca2+ increase was higher and more sustained than that induced by GPCRs. However, no significant difference in mast cell degranulation rates was observed. Although intracellular Ca2+ release was both necessary and sufficient for mast cell degranulation, extracellular Ca2+ influx enhanced the process. Furthermore, cytosolic Ca2+ levels and mast cell degranulation were significantly decreased by downregulation of store-operated Ca2+ entry (SOCE) via Orai1 knockdown, 2-aminoethyl diphenylborinate (2-APB) or tubastatin A (TSA) treatment. Collectively, this study has demonstrated the role of Ca2+ signaling in regulating histamine degranulation.