RESUMO
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.
Assuntos
Interferon Tipo I/imunologia , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Molécula 1 de Interação Estromal/metabolismo , Animais , Pré-Escolar , Chlorocebus aethiops , DNA Viral/imunologia , Modelos Animais de Doenças , Retículo Endoplasmático/metabolismo , Fibroblastos , Técnicas de Inativação de Genes , Células HEK293 , Herpes Simples/imunologia , Herpes Simples/virologia , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/imunologia , Humanos , Imunidade Inata , Células Jurkat , Macrófagos , Masculino , Proteínas de Membrana/imunologia , Camundongos , Camundongos Knockout , Células NIH 3T3 , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/imunologia , Cultura Primária de Células , Imunodeficiência Combinada Severa/sangue , Imunodeficiência Combinada Severa/genética , Imunodeficiência Combinada Severa/imunologia , Molécula 1 de Interação Estromal/genética , Molécula 1 de Interação Estromal/imunologia , Células VeroRESUMO
TRPV1 is a Ca(2+)-permeable channel studied mostly as a pain receptor in sensory neurons. However, its role in other cell types is poorly understood. Here we found that TRPV1 was functionally expressed in CD4(+) T cells, where it acted as a non-store-operated Ca(2+) channel and contributed to T cell antigen receptor (TCR)-induced Ca(2+) influx, TCR signaling and T cell activation. In models of T cell-mediated colitis, TRPV1 promoted colitogenic T cell responses and intestinal inflammation. Furthermore, genetic and pharmacological inhibition of TRPV1 in human CD4(+) T cells recapitulated the phenotype of mouse Trpv1(-/-) CD4(+) T cells. Our findings suggest that inhibition of TRPV1 could represent a new therapeutic strategy for restraining proinflammatory T cell responses.
Assuntos
Linfócitos T CD4-Positivos/imunologia , Inflamação/imunologia , Ativação Linfocitária/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Canais de Cátion TRPV/genética , Anilidas/farmacologia , Animais , Linfócitos T CD4-Positivos/citologia , Cálcio/metabolismo , Canais de Cálcio/imunologia , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/imunologia , Capsaicina/farmacologia , Células Cultivadas , Cinamatos/farmacologia , Colite/imunologia , Humanos , Interleucina-10/genética , Intestinos/imunologia , Intestinos/patologia , Ativação Linfocitária/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fármacos do Sistema Sensorial/farmacologia , Canais de Cátion TRPV/antagonistas & inibidores , Canais de Cátion TRPV/biossínteseRESUMO
ORAI1 and stromal interaction molecule 1 (STIM1) are the critical mediators of store-operated Ca2+ entry by acting as the pore subunit and an endoplasmic reticulum-resident signaling molecule, respectively. In addition to Ca2+ signaling, STIM1 is also involved in regulation of the type I IFN (IFN-I) response. To examine their potential role in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, we generated ORAI1 and STIM1 knockout human HEK293-angiotensin-converting enzyme 2 cells and checked their responses. STIM1 knockout cells showed strong resistance to SARS-CoV-2 infection as a result of enhanced IFN-I response. On the contrary, ORAI1 deletion induced high susceptibility to SARS-CoV-2 infection. Mechanistically, ORAI1 knockout cells showed reduced homeostatic cytoplasmic Ca2+ concentration and severe impairment in tonic IFN-I signaling. Transcriptome analysis showed downregulation of multiple antiviral signaling pathways in ORAI1 knockout cells, likely because of reduced expression of the Ca2+-dependent transcription factors of the AP-1 family and MEF2C Accordingly, modulation of homeostatic Ca2+ concentration by pretreatment with ORAI1 blocker or agonist could influence baseline IFNB expression and resistance to SARS-CoV-2 infection in a human lung epithelial cell line. Our results identify a novel role of ORAI1-mediated Ca2+ signaling in regulating the tonic IFN-I levels, which determine host resistance to SARS-CoV-2 infection.
Assuntos
COVID-19/metabolismo , Interferon Tipo I/metabolismo , Pulmão/imunologia , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/metabolismo , Mucosa Respiratória/metabolismo , SARS-CoV-2/fisiologia , Molécula 1 de Interação Estromal/metabolismo , Células A549 , Enzima de Conversão de Angiotensina 2/genética , Enzima de Conversão de Angiotensina 2/metabolismo , COVID-19/imunologia , Sinalização do Cálcio , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Resistência à Doença , Suscetibilidade a Doenças , Perfilação da Expressão Gênica , Células HEK293 , Humanos , Pulmão/virologia , Fatores de Transcrição MEF2/genética , Proteínas de Neoplasias/genética , Proteína ORAI1/genética , Molécula 1 de Interação Estromal/genética , Fator de Transcrição AP-1/genéticaRESUMO
Activation of the Ca2+ release-activated Ca2+ (CRAC) channel is crucial for T cell functions. It was recently shown that naked cuticle homolog 2 (NKD2), a signaling adaptor molecule, orchestrates trafficking of ORAI1, a pore subunit of the CRAC channels, to the plasma membrane for sustained activation of the CRAC channels. However, the physiological role of sustained Ca2+ entry via ORAI1 trafficking remains poorly understood. Using NKD2 as a molecular handle, we show that ORAI1 trafficking is crucial for sustained Ca2+ entry and cytokine production, especially in inflammatory Th1 and Th17 cells. We find that murine T cells cultured under pathogenic Th17-polarizing conditions have higher Ca2+ levels that are NKD2-dependent than those under nonpathogenic conditions. In vivo, deletion of Nkd2 alleviated clinical symptoms of experimental autoimmune encephalomyelitis in mice by selectively decreasing effector T cell responses in the CNS. Furthermore, we observed a strong correlation between NKD2 expression and proinflammatory cytokine production in effector T cells. Taken together, our findings suggest that the pathogenic effector T cell response demands sustained Ca2+ entry supported by ORAI1 trafficking.
Assuntos
Canais de Cálcio , Canais de Cálcio Ativados pela Liberação de Cálcio , Animais , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Canais de Cálcio Ativados pela Liberação de Cálcio/metabolismo , Sinalização do Cálcio , Citocinas/metabolismo , Camundongos , Proteína ORAI1/genética , Proteína ORAI1/metabolismo , Molécula 1 de Interação EstromalRESUMO
Ca2+ release-activated Ca2+ channel regulator 2A (CRACR2A) is expressed abundantly in T cells and acts as a signal transmitter between TCR stimulation and activation of the Ca2+/NFAT and JNK/AP1 pathways. CRACR2A has been linked to human diseases in numerous genome-wide association studies and was shown to be one of the most sensitive targets of the widely used statin drugs. However, the physiological role of CRACR2A in T cell functions remains unknown. In this study, using transgenic mice for tissue-specific deletion, we show that CRACR2A promotes Th1 responses and effector function of Th17 cells. CRACR2A was abundantly expressed in Th1 and Th17 cells. In vitro, deficiency of CRACR2A decreased Th1 differentiation under nonpolarizing conditions, whereas the presence of polarizing cytokines compensated this defect. Transcript analysis showed that weakened TCR signaling by deficiency of CRACR2A failed to promote Th1 transcriptional program. In vivo, conditional deletion of CRACR2A in T cells alleviated Th1 responses to acute lymphocytic choriomeningitis virus infection and imparted resistance to experimental autoimmune encephalomyelitis. Analysis of CNS from experimental autoimmune encephalomyelitis-induced mice showed impaired effector functions of both Th1 and Th17 cell types, which correlated with decreased pathogenicity. Collectively, our findings demonstrate the requirement of CRACR2A-mediated TCR signaling in Th1 responses as well as pathogenic conversion of Th17 cells, which occurs at the site of inflammation.
Assuntos
Infecções por Arenaviridae/imunologia , Proteínas de Ligação ao Cálcio/metabolismo , Encefalomielite Autoimune Experimental/imunologia , Vírus da Coriomeningite Linfocítica/fisiologia , Células Th1/imunologia , Células Th17/imunologia , Animais , Proteínas de Ligação ao Cálcio/genética , Diferenciação Celular , Células Cultivadas , Citocinas , Resistência à Doença , Humanos , Camundongos , Camundongos Knockout , Transdução de SinaisRESUMO
RATIONALE: Lymphatic vessels function to drain interstitial fluid from a variety of tissues. Although shear stress generated by fluid flow is known to trigger lymphatic expansion and remodeling, the molecular basis underlying flow-induced lymphatic growth is unknown. OBJECTIVE: We aimed to gain a better understanding of the mechanism by which laminar shear stress activates lymphatic proliferation. METHODS AND RESULTS: Primary endothelial cells from dermal blood and lymphatic vessels (blood vascular endothelial cells and lymphatic endothelial cells [LECs]) were exposed to low-rate steady laminar flow. Shear stress-induced molecular and cellular responses were defined and verified using various mutant mouse models. Steady laminar flow induced the classic shear stress responses commonly in blood vascular endothelial cells and LECs. Surprisingly, however, only LECs showed enhanced cell proliferation by regulating the vascular endothelial growth factor (VEGF)-A, VEGF-C, FGFR3, and p57/CDKN1C genes. As an early signal mediator, ORAI1, a pore subunit of the calcium release-activated calcium channel, was identified to induce the shear stress phenotypes and cell proliferation in LECs responding to the fluid flow. Mechanistically, ORAI1 induced upregulation of Krüppel-like factor (KLF)-2 and KLF4 in the flow-activated LECs, and the 2 KLF proteins cooperate to regulate VEGF-A, VEGF-C, FGFR3, and p57 by binding to the regulatory regions of the genes. Consistently, freshly isolated LECs from Orai1 knockout embryos displayed reduced expression of KLF2, KLF4, VEGF-A, VEGF-C, and FGFR3 and elevated expression of p57. Accordingly, mouse embryos deficient in Orai1, Klf2, or Klf4 showed a significantly reduced lymphatic density and impaired lymphatic development. CONCLUSIONS: Our study identified a molecular mechanism for laminar flow-activated LEC proliferation.
Assuntos
Proliferação de Células , Células Endoteliais/metabolismo , Endotélio Linfático/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Linfangiogênese , Mecanotransdução Celular , Proteína ORAI1/metabolismo , Animais , Inibidor de Quinase Dependente de Ciclina p57/genética , Inibidor de Quinase Dependente de Ciclina p57/metabolismo , Endotélio Linfático/patologia , Endotélio Linfático/fisiopatologia , Endotélio Vascular/metabolismo , Regulação da Expressão Gênica , Genótipo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/deficiência , Fatores de Transcrição Kruppel-Like/genética , Camundongos Knockout , Proteína ORAI1/deficiência , Proteína ORAI1/genética , Fenótipo , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/metabolismo , Estresse Mecânico , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/metabolismo , Fator C de Crescimento do Endotélio Vascular/genética , Fator C de Crescimento do Endotélio Vascular/metabolismoRESUMO
Orai1 and stromal interaction molecule 1 (STIM1) mediate store-operated Ca(2+) entry (SOCE) in immune cells. STIM1, an endoplasmic reticulum (ER) Ca(2+) sensor, detects store depletion and interacts with plasma membrane (PM)-resident Orai1 channels at the ER-PM junctions. However, the molecular composition of these junctions in T cells remains poorly understood. Here, we show that junctophilin-4 (JP4), a member of junctional proteins in excitable cells, is expressed in T cells and localized at the ER-PM junctions to regulate Ca(2+) signaling. Silencing or genetic manipulation of JP4 decreased ER Ca(2+) content and SOCE in T cells, impaired activation of the nuclear factor of activated T cells (NFAT) and extracellular signaling-related kinase (ERK) signaling pathways, and diminished expression of activation markers and cytokines. Mechanistically, JP4 directly interacted with STIM1 via its cytoplasmic domain and facilitated its recruitment into the junctions. Accordingly, expression of this cytoplasmic fragment of JP4 inhibited SOCE. Furthermore, JP4 also formed a complex with junctate, a Ca(2+)-sensing ER-resident protein, previously shown to mediate STIM1 recruitment into the junctions. We propose that the junctate-JP4 complex located at the junctions cooperatively interacts with STIM1 to maintain ER Ca(2+) homeostasis and mediate SOCE in T cells.
Assuntos
Sinalização do Cálcio , Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Linfócitos T/metabolismo , Animais , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Células Cultivadas , Células HEK293 , Células HeLa , Humanos , Immunoblotting , Junções Intercelulares/metabolismo , Células Jurkat , Proteínas de Membrana/genética , Camundongos , Microscopia Confocal , Microscopia Eletrônica , Proteínas do Tecido Nervoso/genética , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Molécula 1 de Interação Estromal , Linfócitos T/ultraestruturaRESUMO
Engagement of T cell receptors (TCRs) with cognate antigens triggers cascades of signaling pathways in helper T cells. TCR signaling is essential for the effector function of helper T cells including proliferation, differentiation, and cytokine production. It also modulates effector T cell fate by inducing cell death, anergy (nonresponsiveness), exhaustion, and generation of regulatory T cells. One of the main axes of TCR signaling is the Ca2+-calcineurin-nuclear factor of activated T cells (NFAT) signaling pathway. Stimulation of TCRs triggers depletion of intracellular Ca2+ store and, in turn, activates store-operated Ca2+ entry (SOCE) to raise the intracellular Ca2+ concentration. SOCE in T cells is mediated by the Ca2+ release-activated Ca2+ (CRAC) channels, which have been very well characterized in terms of their electrophysiological properties. Identification of STIM1 as a sensor to detect depletion of the endoplasmic reticulum (ER) Ca2+ store and Orai1 as the pore subunit of CRAC channels has dramatically advanced our understanding of the regulatory mechanism of Ca2+ signaling in T cells. In this review, we discuss our current understanding of Ca2+ signaling in T cells with specific focus on the mechanism of CRAC channel activation and regulation via protein interactions. In addition, we will discuss the role of CRAC channels in effector T cells, based on the analyses of genetically modified animal models.
Assuntos
Canais de Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Doenças do Sistema Imunitário/metabolismo , Linfócitos T/metabolismo , Animais , Humanos , Receptores de Antígenos de Linfócitos T/metabolismo , Moléculas de Interação Estromal/metabolismoRESUMO
Orai1 is a pore-subunit of store-operated Ca(2+) release-activated Ca(2+) (CRAC) channel that mediates Ca(2+) influx in most non-excitable cells via store-operated Ca(2+) entry (SOCE) mechanism. We previously demonstrated that Orai1 is involved in mediating osteogenic potential of mesenchymal stem cells (MSCs), but the underlying mechanism of this function remains unknown. Here, we report that Orai1 mediates osteogenic differentiation via bone morphogenic protein (BMP) signaling pathway in bone marrow MSCs (BMSCs). In osteogenic conditions, BMSCs derived from wild-type mice underwent osteoblastic differentiation and induced mineralization as demonstrated by increased alkaline phosphatase activity and alizarin red S staining, respectively. The expression of Runx2, a master regulator of osteoblast differentiation, and osteogenic differentiation markers were markedly increased in wild-type BMSCs under osteogenic conditions. In contrast, osteogenic conditions failed to induce such effects in BMSCs derived from Orai1-deficient (Orai1(-/-)) mice, indicating that Orai1 is, in part, necessary for osteogenic differentiation of MSCs. We also found that BMP2 successfully induced phosphorylation of Smad1/5/8, the immediate effector molecules of BMP signaling, in wild-type BMSCs, but failed to do so in Orai1(-/-) BMSCs. Downstream target genes of BMP signaling pathway were consistently increased by osteogenic conditions in wild-type BMSCs, but not in Orai1(-/-) BMSCs, suggesting a novel molecular link between Orai1 and BMP signaling pathway in the osteogenic differentiation process. Further functional studies demonstrated that activation of BMP signaling rescues osteogenic differentiation capacity of Orai1(-/-) BMSCs. In conclusion, Orai1 regulates osteogenic differentiation through BMP signaling, and the Orai1-BMP signaling may be a possible therapeutic target for treating bone-related diseases.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Calcificação Fisiológica/fisiologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/fisiologia , Osteogênese/fisiologia , Transdução de Sinais/fisiologia , Animais , Células da Medula Óssea/citologia , Células da Medula Óssea/fisiologia , Sinalização do Cálcio , Diferenciação Celular/fisiologia , Células Cultivadas , CamundongosRESUMO
Orai1 is the pore subunit of Ca(2+) release-activated Ca(2+) (CRAC) channels that stimulate downstream signaling pathways crucial for T cell activation. CRAC channels are an attractive therapeutic target for alleviation of autoimmune diseases. Using high-throughput chemical library screening targeting Orai1, we identified a novel class of small molecules that inhibit CRAC channel activity. One of these molecules, compound 5D, inhibited CRAC channel activity by blocking ion permeation. When included during differentiation, Th17 cells showed higher sensitivity to compound 5D than Th1 and Th2 cells. The selectivity was attributable to high dependence of promoters of retinoic-acid-receptor-related orphan receptors on the Ca(2+)-NFAT pathway. Blocking of CRAC channels drastically decreased recruitment of NFAT and histone modifications within key gene loci involved in Th17 differentiation. The impairment in Th17 differentiation by treatment with CRAC channel blocker was recapitulated in Orai1-deficient T cells, which could be rescued by exogenous expression of retinoic-acid-receptor-related orphan receptors or a constitutive active mutant of NFAT. In vivo administration of CRAC channel blockers effectively reduced the severity of experimental autoimmune encephalomyelitis by suppression of differentiation of inflammatory T cells. These results suggest that CRAC channel blockers can be considered as chemical templates for the development of therapeutic agents to suppress inflammatory responses.
Assuntos
Canais de Cálcio/metabolismo , Sinalização do Cálcio , Receptores Nucleares Órfãos/metabolismo , Células Th17/citologia , Células Th17/metabolismo , Animais , Bloqueadores dos Canais de Cálcio/química , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/genética , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/genética , Encefalomielite Autoimune Experimental/imunologia , Encefalomielite Autoimune Experimental/metabolismo , Humanos , Íons/metabolismo , Camundongos , Fatores de Transcrição NFATC/metabolismo , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Proteína ORAI1 , Receptores Nucleares Órfãos/deficiência , Receptores Nucleares Órfãos/genética , Regiões Promotoras Genéticas , Ligação Proteica , Elementos de Resposta , Bibliotecas de Moléculas Pequenas , Células Th1/citologia , Células Th1/imunologia , Células Th1/metabolismo , Células Th17/imunologia , Células Th2/citologia , Células Th2/imunologia , Células Th2/metabolismoRESUMO
Orai1 and stromal interaction molecule (STIM)1 are critical components of Ca(2+) release-activated Ca(2+) (CRAC) channels. Orai1 is a pore subunit of CRAC channels, and STIM1 acts as an endoplasmic reticulum (ER) Ca(2+) sensor that detects store depletion. Upon store depletion after T-cell receptor stimulation, STIM1 translocates and coclusters with Orai1 at sites of close apposition of the plasma membrane (PM) and the ER membrane. However, the molecular components of these ER-PM junctions remain poorly understood. Using affinity protein purification, we uncovered junctate as an interacting partner of Orai1-STIM1 complex. Furthermore, we identified a Ca(2+)-binding EF-hand motif in the ER-luminal region of junctate. Mutation of this EF-hand domain of junctate impaired its Ca(2+) binding and resulted in partial activation of CRAC channels and clustering of STIM1 independently of store depletion. In addition to the known mechanisms of STIM1 clustering (i.e., phosphoinositide and Orai1 binding), our study identifies an alternate mechanism to recruit STIM1 into the ER-PM junctions via binding to junctate. We propose that junctate, a Ca(2+)-sensing ER protein, is a structural component of the ER-PM junctions where Orai1 and STIM1 cluster and interact in T cells.
Assuntos
Canais de Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Membrana/metabolismo , Oxigenases de Função Mista/metabolismo , Proteínas Musculares/metabolismo , Proteínas de Neoplasias/metabolismo , Cálcio/metabolismo , Canais de Cálcio/genética , Proteínas de Ligação ao Cálcio/genética , Membrana Celular/metabolismo , Motivos EF Hand/genética , Retículo Endoplasmático/metabolismo , Células HEK293 , Células HeLa , Humanos , Immunoblotting , Células Jurkat , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas de Membrana/genética , Microscopia de Fluorescência , Oxigenases de Função Mista/genética , Proteínas Musculares/genética , Mutação , Proteínas de Neoplasias/genética , Proteína ORAI1 , Ligação Proteica , Transporte Proteico , Molécula 1 de Interação EstromalAssuntos
Síndromes de Imunodeficiência/diagnóstico , Linfócitos/fisiologia , Proteínas de Neoplasias/genética , Deleção de Sequência/genética , Molécula 1 de Interação Estromal/genética , Células Cultivadas , Pré-Escolar , Transplante de Células-Tronco de Sangue do Cordão Umbilical , Eczema , Humanos , Ictiose , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/terapia , Lactente , Infecções , Masculino , Linhagem , FenótipoRESUMO
DRAK2 is a serine/threonine kinase highly enriched in lymphocytes that raises the threshold for T cell activation and maintains T cell survival following productive activation. T cells lacking DRAK2 are prone to activation under suboptimal conditions and exhibit enhanced calcium responses to AgR stimulation. Despite this, mice lacking DRAK2 are resistant to organ-specific autoimmune diseases due to defective autoreactive T cell survival. DRAK2 kinase activity is induced by AgR signaling, and in this study we show that the induction of DRAK2 activity requires Ca(2+) influx through the Ca(2+) release-activated Ca(2+) channel formed from Orai1 subunits. Blockade of DRAK2 activity with the protein kinase D (PKD) inhibitor Gö6976 or expression of a kinase-dead PKD mutant prevented activation of DRAK2, whereas a constitutively active PKD mutant promoted DRAK2 function. Knockdown of PKD in T cells strongly blocked endogenous DRAK2 activation following TCR ligation, implicating PKD as an essential intermediate in the activation of DRAK2 by Ca(2+) influx. Furthermore, we identify DRAK2 as a novel substrate of PKD, and demonstrate that DRAK2 and PKD physically interact under conditions that activate PKD. Mitochondrial generation of reactive oxygen intermediates was necessary and sufficient for DRAK2 activation in response to Ca(2+) influx. Taken together, DRAK2 and PKD form a novel signaling module that controls calcium homeostasis following T cell activation.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Sinalização do Cálcio/imunologia , Mitocôndrias/imunologia , Mitocôndrias/metabolismo , Proteína Quinase C/fisiologia , Proteínas Serina-Treonina Quinases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Receptores de Antígenos de Linfócitos T/fisiologia , Animais , Proteínas Reguladoras de Apoptose/deficiência , Proteínas Reguladoras de Apoptose/genética , Sinalização do Cálcio/genética , Células Clonais , Ativação Enzimática/genética , Ativação Enzimática/imunologia , Homeostase/genética , Homeostase/imunologia , Humanos , Células Jurkat , Ativação Linfocitária/genética , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Linfócitos T/enzimologia , Linfócitos T/imunologiaRESUMO
ORAI1 is a pore subunit of Ca(2+) release-activated Ca(2+) channels that mediate TCR stimulation-induced Ca(2+) entry. A point mutation in ORAI1 (ORAI1(R91W)) causes SCID in human patients that is recapitulated in Orai1(-/-) mice, emphasizing its important role in the immune cells. In this study, we have characterized a novel function of ORAI1 in T cell death. CD4(+) T cells from Orai1(-/-) mice showed robust proliferation with repetitive stimulations and strong resistance to stimulation-induced cell death due to reduced mitochondrial Ca(2+) uptake and altered gene expression of proapoptotic and antiapoptotic molecules (e.g., Fas ligand, Noxa, and Mcl-1). Nuclear accumulation of NFAT was severely reduced in ORAI1-deficient T cells, and expression of ORAI1 and a constitutively active mutant of NFAT recovered cell death. These results indicate NFAT-mediated cell death pathway as one of the major downstream targets of ORAI1-induced Ca(2+) entry. By expressing various mutants of ORAI1 in wild-type and Orai1(-/-) T cells to generate different levels of intracellular Ca(2+), we have shown that activation-induced cell death is directly proportional to the intracellular Ca(2+) concentration levels. Consistent with the in vitro results, Orai1(-/-) mice showed strong resistance to T cell depletion induced by injection of anti-CD3 Ab. Furthermore, ORAI1-deficient T cells showed enhanced survival after adoptive transfer into immunocompromised hosts. Thus, our results demonstrate a crucial role of the ORAI1-NFAT pathway in T cell death and highlight the important role of ORAI1 as a major route of Ca(2+) entry during activated T cell death.
Assuntos
Apoptose/imunologia , Linfócitos T CD4-Positivos/imunologia , Canais de Cálcio/imunologia , Sinalização do Cálcio/imunologia , Animais , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Canais de Cálcio/metabolismo , Separação Celular , Sobrevivência Celular , Citometria de Fluxo , Humanos , Immunoblotting , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Fatores de Transcrição NFATC/imunologia , Fatores de Transcrição NFATC/metabolismo , Proteína ORAI1 , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução GenéticaRESUMO
Store-operated Ca(2+) entry (SOCE) is a fundamental mechanism ubiquitously employed by cells to elevate intracellular Ca(2+) concentrations ([Ca(2+)]i). Increased intracellular Ca(2+) ions act as a second messenger that can stimulate a variety of downstream signaling pathways affecting proliferation, secretion, differentiation, and death of cells. In immune cells, immune receptor stimulation induces endoplasmic reticulum Ca(2+) store depletion that subsequently activates Ca(2+)-release-activated-Ca(2+) (CRAC) channels, a prototype of store-operated Ca(2+) (SOC) channels. Identification of Orai1 as the pore subunit of CRAC channels has provided the much-needed molecular tool to dissect the mechanism of activation and regulation of these channels. In this review, we discuss the recent advances in understanding the regulatory mechanisms and posttranslational modifications that regulate diverse aspects of CRAC channel function.
Assuntos
Canais de Cálcio/metabolismo , Sinalização do Cálcio , Processamento de Proteína Pós-Traducional , Adenilil Ciclases/metabolismo , Animais , Proteínas de Ligação ao Cálcio/metabolismo , Calmodulina/metabolismo , Retículo Endoplasmático/metabolismo , Humanos , Ativação do Canal Iônico , Mitocôndrias/metabolismo , Proteína Básica da Mielina/metabolismo , Proteína ORAI1 , Transporte ProteicoRESUMO
Ca2+ entry via Ca2+ release-activated Ca2+ (CRAC) channels is a predominant mechanism of intracellular Ca2+ elevation in immune cells. Here we show the immunoregulatory role of CRAC channel components Orai1 and Orai2 in Group 2 innate lymphoid cells (ILC2s), that play crucial roles in the induction of type 2 inflammation. We find that blocking or genetic ablation of Orai1 and Orai2 downregulates ILC2 effector function and cytokine production, consequently ameliorating the development of ILC2-mediated airway inflammation in multiple murine models. Mechanistically, ILC2 metabolic and mitochondrial homeostasis are inhibited and lead to the upregulation of reactive oxygen species production. We confirm our findings in human ILC2s, as blocking Orai1 and Orai2 prevents the development of airway hyperreactivity in humanized mice. Our findings have a broad impact on the basic understanding of Ca2+ signaling in ILC2 biology, providing potential insights into the development of therapies for the treatment of allergic and atopic inflammatory diseases.
Assuntos
Asma , Imunidade Inata , Camundongos , Humanos , Animais , Linfócitos , Homeostase , Inflamação , Proteína ORAI1/genéticaRESUMO
Emerging evidence indicates that intracellular calcium (Ca2+) levels and their regulatory proteins play essential roles in normal stem cell proliferation and differentiation. Cancer stem-like cells (CSCs) are subpopulations of cancer cells that retain characteristics similar to stem cells and play an essential role in cancer progression. Recent studies have reported that the Orai3 calcium channel plays an oncogenic role in human cancer. However, its role in CSCs remains underexplored. In this study, we explored the effects of Orai3 in the progression and stemness of oral/oropharyngeal squamous cell carcinoma (OSCC). During the course of OSCC progression, the expression of Orai3 exhibited a stepwise augmentation. Notably, Orai3 was highly enriched in CSC populations of OSCC. Ectopic Orai3 expression in non-tumorigenic immortalized oral epithelial cells increased the intracellular Ca2+ levels, acquiring malignant growth and CSC properties. Conversely, silencing of the endogenous Orai3 in OSCC cells suppressed the CSC phenotype, indicating a pivotal role of Orai3 in CSC regulation. Moreover, Orai3 markedly increased the expression of inhibitor of DNA binding 1 (ID1), a stemness transcription factor. Orai3 and ID1 exhibited elevated expression within CSCs compared to their non-CSC counterparts, implying the functional importance of the Orai3/ID1 axis in CSC regulation. Furthermore, suppression of ID1 abrogated the CSC phenotype in the cell with ectopic Orai3 overexpression and OSCC. Our study reveals that Orai3 is a novel functional CSC regulator in OSCC and further suggests that Orai3 plays an oncogenic role in OSCC by promoting cancer stemness via ID1 upregulation.
Assuntos
Carcinoma de Células Escamosas , Neoplasias de Cabeça e Pescoço , Neoplasias Bucais , Neoplasias Orofaríngeas , Humanos , Neoplasias Bucais/genética , Carcinoma de Células Escamosas de Cabeça e Pescoço , Canais de Cálcio , Hiperplasia , Proteína 1 Inibidora de DiferenciaçãoRESUMO
Background: Hantaviruses - dichotomized into New World (i.e. Andes virus, ANDV; Sin Nombre virus, SNV) and Old-World viruses (i.e. Hantaan virus, HTNV) - are zoonotic viruses transmitted from rodents to humans. Currently, no FDA-approved vaccines against hantaviruses exist. Given the recent breakthrough to human-human transmission by the ANDV, an essential step is to establish an effective pandemic preparedness infrastructure to rapidly identify cell tropism, infective potential, and effective therapeutic agents through systematic investigation. Methods: We established human cell model systems in lung (airway and distal lung epithelial cells), heart (pluripotent stem cell-derived (PSC-) cardiomyocytes), and brain (PSC-astrocytes) cell types and subsequently evaluated ANDV, HTNV and SNV tropisms. Transcriptomic, lipidomic and bioinformatic data analyses were performed to identify the molecular pathogenic mechanisms of viruses in different cell types. This cell-based infection system was utilized to establish a drug testing platform and pharmacogenomic comparisons. Results: ANDV showed broad tropism for all cell types assessed. HTNV replication was predominantly observed in heart and brain cells. ANDV efficiently replicated in human and mouse 3D distal lung organoids. Transcriptomic analysis showed that ANDV infection resulted in pronounced inflammatory response and downregulation of cholesterol biosynthesis pathway in lung cells. Lipidomic profiling revealed that ANDV-infected cells showed reduced level of cholesterol esters and triglycerides. Further analysis of pathway-based molecular signatures showed that, compared to SNV and HTNV, ANDV infection caused drastic lung cell injury responses. A selective drug screening identified STING agonists, nucleoside analogues and plant-derived compounds that inhibited ANDV viral infection and rescued cellular metabolism. In line with experimental results, transcriptome data shows that the least number of total and unique differentially expressed genes were identified in urolithin B- and favipiravir-treated cells, confirming the higher efficiency of these two drugs in inhibiting ANDV, resulting in host cell ability to balance gene expression to establish proper cell functioning. Conclusions: Overall, our study describes advanced human PSC-derived model systems and systems-level transcriptomics and lipidomic data to better understand Old and New World hantaviral tropism, as well as drug candidates that can be further assessed for potential rapid deployment in the event of a pandemic.
RESUMO
Store-operated Ca(2+) (SOC) entry is one of the major mechanisms to raise intracellular Ca(2+) concentration in non-excitable cells. Ca(2+)-release-activated Ca(2+) (CRAC) channels are a subtype of SOC channels that are extensively characterized in immune cells. Identification of STIM1 as an endoplasmic reticulum Ca(2+) sensor and Orai1 as the pore subunit has dramatically advanced the molecular understanding of CRAC channels. Recent efforts have focused on understanding the physiological aspects of CRAC channels at an organism level using transgenic animal models and at a molecular level using electrophysiological and biochemical tools. In this review, we summarize our current understanding of the interacting partners of Orai and STIM proteins in the regulation of CRAC channel activity and other non-CRAC channel-related functions.