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1.
Nat Commun ; 15(1): 1530, 2024 Feb 27.
Artigo em Inglês | MEDLINE | ID: mdl-38413581

RESUMO

Homoeostatic regulation of the acid-base balance is essential for cellular functional integrity. However, little is known about the molecular mechanism through which the acid-base balance regulates cellular responses. Here, we report that bicarbonate ions activate a G protein-coupled receptor (GPCR), i.e., GPR30, which leads to Gq-coupled calcium responses. Gpr30-Venus knock-in mice reveal predominant expression of GPR30 in brain mural cells. Primary culture and fresh isolation of brain mural cells demonstrate bicarbonate-induced, GPR30-dependent calcium responses. GPR30-deficient male mice are protected against ischemia-reperfusion injury by a rapid blood flow recovery. Collectively, we identify a bicarbonate-sensing GPCR in brain mural cells that regulates blood flow and ischemia-reperfusion injury. Our results provide a perspective on the modulation of GPR30 signalling in the development of innovative therapies for ischaemic stroke. Moreover, our findings provide perspectives on acid/base sensing GPCRs, concomitantly modulating cellular responses depending on fluctuating ion concentrations under the acid-base homoeostasis.


Assuntos
Isquemia Encefálica , Traumatismo por Reperfusão , Acidente Vascular Cerebral , Masculino , Camundongos , Animais , Bicarbonatos , Cálcio/metabolismo , Receptores de Estrogênio/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo
2.
Glia ; 70(9): 1666-1680, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35506586

RESUMO

Astrocytes are glial cells that serve homeostatic functions in the central nervous system (CNS). Recent research, however, suggests that under pathological conditions, astrocytes are stimulated by various factors and actively participate in CNS inflammation. In the present study, we found that astrocytes upregulate various inflammatory factors including prostaglandin E2 (PGE2 ) by co-stimulation with tumor necrosis factor-alpha (TNFα) and interleukin-1alpha (IL1α). These TNFα/IL1α-stimulated astrocytes also showed increased Ca2+ release from the endoplasmic reticulum (ER) and increased expression of Orai2, a member of the store-operated calcium channel (SOCC) family. To reveal the role of Orai2, we used astrocytes in which Orai2 was knocked-down (KD) or knocked-out (KO). The expression of the prostaglandin E synthase Ptges and the production of PGE2 were higher in Orai2-KD astrocytes than in WT astrocytes when stimulated with TNFα and IL1α. Orai2-KO astrocytes also showed increased expression of Ptges and increased PGE2 production. The expression of Ptgs2, another PGE2 synthetic enzyme, was also upregulated in Orai2-KO astrocytes. Moreover, Orai2-KO astrocytes showed increased store-operated calcium entry (SOCE) and increased Orai1 expression. These results suggest that Orai2 is upregulated in TNFα/IL1α-stimulated astrocytes and reduces PGE2 production to some extent, modulating CNS inflammation. Our findings may aid in understanding how astrocytes are associated with inflammatory responses, and the identification of new targets that modulate astrocytic reactivity.


Assuntos
Astrócitos , Interleucina-1alfa , Proteína ORAI2 , Prostaglandinas E , Fator de Necrose Tumoral alfa , Animais , Astrócitos/metabolismo , Cálcio/metabolismo , Sinalização do Cálcio , Inflamação , Interleucina-1alfa/metabolismo , Interleucina-1alfa/farmacologia , Camundongos , Proteína ORAI2/metabolismo , Prostaglandinas E/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologia
3.
Int Immunol ; 34(4): 197-206, 2022 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-34953165

RESUMO

Myeloid cells play a pivotal role in immune responses against bacterial and fungal infection. Among innate immune receptors, C-type lectin receptors (CLRs) can induce a wide spectrum of cytokines through immunoreceptor tyrosine-based activation motifs (ITAMs)-mediated signaling pathways. Dendritic cells (DCs) produce IL-10 through CLR stimulation; however, the regulatory mechanism of IL-10 expression has not been elucidated. In the current study, we report that calcium (Ca2+) signaling-deficient DCs produced more IL-10 than wild-type DCs. Mechanistically, Ca2+-dependent phosphatase calcineurin directly inactivates cAMP response element-binding protein (CREB), a transcription factor of Il10 in DCs, through dephosphorylating CREB at serine 133. In calcineurin-deficient DCs, CREB was highly phosphorylated and increased its binding to the Il10 promoter. Elimination of mitogen-activated protein kinase (MAPK) signaling that phosphorylates CREB, deficiency of CREB, as well as deletion of a CREB-binding site in the Il10 promoter could diminish IL-10 production in DCs. Our findings identified a novel substrate of calcineurin as well as a mechanism through which Ca2+ signaling regulates IL-10 expression downstream of CLRs. As IL-10 is a crucial immunosuppressive cytokine, this mechanism may counteract the over-activated IL-10-producing signals induced by CARD9 and MAPK pathways, preventing the ineffectiveness of the immune system during bacterial and fungal infection.


Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico , Interleucina-10 , Calcineurina/metabolismo , Cálcio/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Citocinas/metabolismo , Células Dendríticas , Lectinas Tipo C , Fosforilação
4.
Sci Signal ; 12(576)2019 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-30967512

RESUMO

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


Assuntos
Basófilos/imunologia , Sinalização do Cálcio/imunologia , Interleucina-3/imunologia , Interleucina-4/imunologia , Molécula 1 de Interação Estromal/imunologia , Molécula 2 de Interação Estromal/imunologia , Animais , Basófilos/citologia , Sinalização do Cálcio/genética , Imunoglobulina E/imunologia , Interleucina-3/genética , Interleucina-4/genética , Camundongos , Camundongos Knockout , Molécula 1 de Interação Estromal/genética , Molécula 2 de Interação Estromal/genética
5.
J Immunol ; 202(9): 2616-2627, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-30910863

RESUMO

Appropriate T cell responses are controlled by strict balance between activatory and inhibitory pathways downstream of TCR. Although mice or humans with impaired TCR signaling develop autoimmunity, the precise molecular mechanisms linking reduced TCR signaling to autoimmunity are not fully understood. Engagement of TCR activates Ca2+ signaling mainly through store-operated Ca2+ entry activated by stromal interaction molecule (Stim) 1 and Stim2. Despite defective T cell activation, mice deficient in both Stim1 and Stim2 in T cells (conditional double knockout [cDKO]) developed lymphoproliferative disorders and skin inflammation with a concomitant increase in serum IgG1 and IgE levels. In cDKO mice, follicular helper T (Tfh) cells were dramatically increased in number, and they produced IL-4 spontaneously. These inflammatory symptoms were abolished by the deletion of IL-4 in cDKO mice. Tfh development and inflammatory symptoms in cDKO mice were abrogated by further deletion of NFAT2 in T cells. These findings suggest that Tfh cells spontaneously developed in the absence of Ca2+ signaling and caused unregulated type 2 responses.


Assuntos
Doenças do Sistema Imunitário/imunologia , Molécula 1 de Interação Estromal/deficiência , Molécula 2 de Interação Estromal/deficiência , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Sinalização do Cálcio/genética , Sinalização do Cálcio/imunologia , Doenças do Sistema Imunitário/genética , Doenças do Sistema Imunitário/patologia , Imunoglobulina E/genética , Imunoglobulina E/imunologia , Imunoglobulina G/genética , Imunoglobulina G/imunologia , Interleucina-4/genética , Interleucina-4/imunologia , Transtornos Linfoproliferativos/genética , Transtornos Linfoproliferativos/imunologia , Transtornos Linfoproliferativos/patologia , Camundongos , Camundongos Knockout , Fatores de Transcrição NFATC/genética , Fatores de Transcrição NFATC/imunologia , Dermatopatias/genética , Dermatopatias/imunologia , Dermatopatias/patologia , Linfócitos T Auxiliares-Indutores/patologia
6.
Front Immunol ; 8: 1348, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29085376

RESUMO

Ticks, blood-sucking arthropods, serve as vectors for transmission of infectious diseases including Lyme borreliosis. After tick infestation, several animal species can develop resistance to subsequent infestations, reducing the risk of transmission. In a mouse model, basophils reportedly infiltrate tick-feeding sites during the second but not first infestation and play a crucial role in the expression of acquired tick resistance. However, the mechanism underlying basophil recruitment to the second tick-feeding site remains ill-defined. Here, we investigated cells and their products responsible for the basophil recruitment. Little or no basophil infiltration was detected in T-cell-deficient mice, and adoptive transfer of CD4+ but not CD8+ T cells reconstituted it. Il3 gene expression was highly upregulated at the second tick-feeding site, and adoptive transfer of interleukin-3 (IL-3)-sufficient but not IL-3-deficient CD4+ T cells conferred the basophil infiltration on T-cell-deficient mice, indicating that the CD4+ T-cell-derived IL-3 is essential for the basophil recruitment. Notably, IL-3+ resident CD4+ memory T cells were detected even before the second infestation in previously uninfested skin distant from the first tick-feeding site. Taken together, IL-3 produced locally by skin CD4+ memory T cells appears to play a crucial role in basophil recruitment to the second tick-feeding site.

7.
Neurobiol Learn Mem ; 138: 54-61, 2017 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27544849

RESUMO

STIM2 is an integral membrane protein of the endoplasmic reticulum (ER) that regulates the activity of plasma membrane (PM) channels at ER-PM contact sites. Recent studies show that STIM2 promotes spine maturation and surface expression of the AMPA receptor (AMPAR) subunit GluA1, hinting at a probable role in synaptic plasticity. Here, we used a Stim2 cKO mouse to explore the function of STIM2 in Long-Term Potentiation (LTP) and Depression (LTD), two widely-studied models of synaptic plasticity implicated in information storage. We found that STIM2 is required for the stable expression of both LTP and LTD at CA3-CA1 hippocampal synapses. Altered plasticity in Stim2 cKO mice is associated with subtle alterations in the shape and density of dendritic spines in CA1 neurons. Further, surface delivery of GluA1 in response to LTP-inducing chemical manipulations was markedly reduced in excitatory neurons derived from Stim2 cKO mice. GluA1 endocytosis following chemically-induced LTD was also impaired in Stim2 cKO neurons. We conclude that STIM2 facilitates synaptic delivery and removal of AMPARs and regulates activity-dependent changes in synaptic strength through a unique mode of communication between the ER and the synapse.


Assuntos
Hipocampo/metabolismo , Plasticidade Neuronal/fisiologia , Transporte Proteico/fisiologia , Receptores de AMPA/metabolismo , Molécula 2 de Interação Estromal/metabolismo , Sinapses/metabolismo , Animais , Forma Celular/fisiologia , Espinhas Dendríticas/genética , Espinhas Dendríticas/metabolismo , Retículo Endoplasmático/metabolismo , Masculino , Camundongos , Camundongos Knockout , Neurônios/citologia , Neurônios/metabolismo , Molécula 2 de Interação Estromal/genética , Sinapses/genética
8.
Immunity ; 43(5): 859-69, 2015 Nov 17.
Artigo em Inglês | MEDLINE | ID: mdl-26522985

RESUMO

The precise impact of thymic positive and negative selection on the T cell receptor (TCR) repertoire remains controversial. Here, we used unbiased, high-throughput cloning and retroviral expression of individual pre-selection TCRs to provide a direct assessment of these processes at the clonal level in vivo. We found that 15% of random TCRs induced signaling and directed positive (7.5%) or negative (7.5%) selection, depending on strength of signal, whereas the remaining 85% failed to induce signaling or selection. Most negatively selected TCRs exhibited promiscuous crossreactivity toward multiple other major histocompatibility complex (MHC) haplotypes. In contrast, TCRs that were positively selected or non-selected were minimally crossreactive. Negative selection of crossreactive TCRs led to clonal deletion but also recycling into intestinal CD4(-)CD8ß(-) intraepithelial lymphocytes (iIELs). Thus, broadly crossreactive TCRs arise at low frequency in the pre-selection repertoire but constitute the primary drivers of thymic negative selection and iIEL lineage differentiation.


Assuntos
Reações Cruzadas/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Timócitos/imunologia , Animais , Ativação Linfocitária/imunologia , Complexo Principal de Histocompatibilidade/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Transdução de Sinais/imunologia , Subpopulações de Linfócitos T/imunologia
9.
J Biol Chem ; 290(42): 25322-32, 2015 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-26296894

RESUMO

C-type lectin receptors (CLRs) are an emerging family of pattern recognition receptors that recognizes pathogens or damaged tissue to trigger innate immune responses. However, endogenous ligands for CLRs are not fully understood. In this study, we sought to identify an endogenous ligand(s) for human macrophage-inducible C-type lectin (hMincle). A particular fraction of lipid extracts from liver selectively activated reporter cells expressing hMincle. MS analysis determined the chemical structure of the active component as cholesterol. Purified cholesterol in plate-coated and crystalized forms activates reporter cells expressing hMincle but not murine Mincle (mMincle). Cholesterol crystals are known to activate immune cells and induce inflammatory responses through lysosomal damage. However, direct innate immune receptors for cholesterol crystals have not been identified. Murine macrophages transfected with hMincle responded to cholesterol crystals by producing pro-inflammatory cytokines. Human dendritic cells expressed a set of inflammatory genes in response to cholesterol crystals, and this was inhibited by anti-human Mincle. Importantly, other related CLRs did not bind cholesterol crystals, whereas other steroids were not recognized by hMincle. These results suggest that cholesterol crystals are an endogenous ligand for hMincle and that they activate innate immune responses.


Assuntos
Colesterol/metabolismo , Imunidade Inata , Mediadores da Inflamação/metabolismo , Lectinas Tipo C/metabolismo , Receptores Imunológicos/metabolismo , Animais , Linhagem Celular , Colesterol/química , Cristalização , Humanos , Camundongos , Ligação Proteica , Transcrição Gênica
10.
Front Behav Neurosci ; 9: 180, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26236206

RESUMO

Recent findings point to a central role of the endoplasmic reticulum-resident STIM (Stromal Interaction Molecule) proteins in shaping the structure and function of excitatory synapses in the mammalian brain. The impact of the Stim genes on cognitive functions remains, however, poorly understood. To explore the function of the Stim genes in learning and memory, we generated three mouse strains with conditional deletion (cKO) of Stim1 and/or Stim2 in the forebrain. Stim1, Stim2, and double Stim1/Stim2 cKO mice show no obvious brain structural defects or locomotor impairment. Analysis of spatial reference memory in the Morris water maze revealed a mild learning delay in Stim1 cKO mice, while learning and memory in Stim2 cKO mice was indistinguishable from their control littermates. Deletion of both Stim genes in the forebrain resulted, however, in a pronounced impairment in spatial learning and memory reflecting a synergistic effect of the Stim genes on the underlying neural circuits. Notably, long-term potentiation (LTP) at CA3-CA1 hippocampal synapses was markedly enhanced in Stim1/Stim2 cKO mice and was associated with increased phosphorylation of the AMPA receptor subunit GluA1, the transcriptional regulator CREB and the L-type Voltage-dependent Ca(2+) channel Cav1.2 on protein kinase A (PKA) sites. We conclude that STIM1 and STIM2 are key regulators of PKA signaling and synaptic plasticity in neural circuits encoding spatial memory. Our findings also reveal an inverse correlation between LTP and spatial learning/memory and suggest that abnormal enhancement of cAMP/PKA signaling and synaptic efficacy disrupts the formation of new memories.

11.
Immunity ; 41(3): 402-413, 2014 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-25176311

RESUMO

Mycobacteria possess various immunomodulatory molecules on the cell wall. Mannose-capped lipoarabinomannan (Man-LAM), a major lipoglycan of Mycobacterium tuberculosis, has long been known to have both inhibitory and stimulatory effects on host immunity. However, the direct Man-LAM receptor that explains its pleiotropic activities has not been clearly identified. Here, we report that a C-type lectin receptor Dectin-2 (gene symbol Clec4n) is a direct receptor for Man-LAM. Man-LAM activated bone-marrow-derived dendritic cells (BMDCs) to produce pro- and anti-inflammatory cytokines, whereas it was completely abrogated in Clec4n(-/-) BMDCs. Man-LAM promoted antigen-specific T cell responses through Dectin-2 on DCs. Furthermore, Man-LAM induced experimental autoimmune encephalitis (EAE) as an adjuvant in mice, whereas Clec4n(-/-) mice were resistant. Upon mycobacterial infection, Clec4n(-/-) mice showed augmented lung pathology. These results demonstrate that Dectin-2 contributes to host immunity against mycobacterial infection through the recognition of Man-LAM.


Assuntos
Encefalomielite Autoimune Experimental/imunologia , Lectinas Tipo C/imunologia , Lipopolissacarídeos/imunologia , Infecções por Mycobacterium/imunologia , Animais , Antígenos CD/genética , Moléculas de Adesão Celular/genética , Citocinas/biossíntese , Células Dendríticas/imunologia , Encefalomielite Autoimune Experimental/genética , Inflamação/imunologia , Interferon gama/biossíntese , Interleucina-10/biossíntese , Lectinas Tipo C/genética , Lipopolissacarídeos/química , Manose/química , Receptor de Manose , Lectinas de Ligação a Manose/imunologia , Camundongos , Camundongos Knockout , Infecções por Mycobacterium/genética , Mycobacterium bovis/imunologia , Mycobacterium tuberculosis/imunologia , Fator 88 de Diferenciação Mieloide/genética , Ligação Proteica/imunologia , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/imunologia , Receptores Imunológicos/genética , Linfócitos T/imunologia
12.
Nat Med ; 20(1): 62-8, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24362934

RESUMO

Autoimmune diseases often result from an imbalance between regulatory T (Treg) cells and interleukin-17 (IL-17)-producing T helper (TH17) cells; the origin of the latter cells remains largely unknown. Foxp3 is indispensable for the suppressive function of Treg cells, but the stability of Foxp3 has been under debate. Here we show that TH17 cells originating from Foxp3(+) T cells have a key role in the pathogenesis of autoimmune arthritis. Under arthritic conditions, CD25(lo)Foxp3(+)CD4(+) T cells lose Foxp3 expression (herein called exFoxp3 cells) and undergo transdifferentiation into TH17 cells. Fate mapping analysis showed that IL-17-expressing exFoxp3 T (exFoxp3 TH17) cells accumulated in inflamed joints. The conversion of Foxp3(+)CD4(+) T cells to TH17 cells was mediated by synovial fibroblast-derived IL-6. These exFoxp3 TH17 cells were more potent osteoclastogenic T cells than were naive CD4(+) T cell-derived TH17 cells. Notably, exFoxp3 TH17 cells were characterized by the expression of Sox4, chemokine (C-C motif) receptor 6 (CCR6), chemokine (C-C motif) ligand 20 (CCL20), IL-23 receptor (IL-23R) and receptor activator of NF-κB ligand (RANKL, also called TNFSF11). Adoptive transfer of autoreactive, antigen-experienced CD25(lo)Foxp3(+)CD4(+) T cells into mice followed by secondary immunization with collagen accelerated the onset and increased the severity of arthritis and was associated with the loss of Foxp3 expression in the majority of transferred T cells. We observed IL-17(+)Foxp3(+) T cells in the synovium of subjects with active rheumatoid arthritis (RA), which suggests that plastic Foxp3(+) T cells contribute to the pathogenesis of RA. These findings establish the pathological importance of Foxp3 instability in the generation of pathogenic TH17 cells in autoimmunity.


Assuntos
Artrite/imunologia , Doenças Autoimunes/imunologia , Linfócitos T CD4-Positivos/citologia , Diferenciação Celular/imunologia , Fatores de Transcrição Forkhead/metabolismo , Células Th17/citologia , Transferência Adotiva , Animais , Linfócitos T CD4-Positivos/metabolismo , Citometria de Fluxo , Fatores de Transcrição Forkhead/imunologia , Técnicas de Introdução de Genes , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Análise de Sequência com Séries de Oligonucleotídeos , Membrana Sinovial/citologia , Membrana Sinovial/imunologia , Células Th17/imunologia
13.
Immunity ; 38(5): 881-95, 2013 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-23499491

RESUMO

T cell receptor (TCR) signaling driven by interaction of the TCR with specific complexes of self-peptide and the major histocompatibility complex determines T cell fate in thymic development. However, the signaling pathway through which TCR signal strength regulates distinct T cell lineages remains unknown. Here we have used mice lacking the endoplasmic reticulum Ca2+ sensors stromal interaction molecule 1 (STIM1) and STIM2 to show that STIM-induced store-operated Ca2+ entry is not essential for thymic development of conventional TCRαß+ T cells but is specifically required for the development of agonist-selected T cells (regulatory T cells, invariant natural killer T cells, and TCRαß+ CD8αα+ intestinal intraepithelial lymphocytes). The severe impairment of agonist-selected T cell development is mainly due to a defect in interleukin-2 (IL-2) or IL-15 signaling. Thus, STIM1 and STIM2-mediated store-operated Ca2+ influx, leading to efficient activation of NFAT (nuclear factor of activated T cells), is critical for the postselection maturation of agonist-selected T cells.


Assuntos
Cálcio/metabolismo , Glicoproteínas de Membrana/metabolismo , Fatores de Transcrição NFATC/metabolismo , Linfócitos T Reguladores/imunologia , Animais , Canais de Cálcio/imunologia , Canais de Cálcio/metabolismo , Sinalização do Cálcio/imunologia , Retículo Endoplasmático/imunologia , Retículo Endoplasmático/metabolismo , Interleucina-15/metabolismo , Interleucina-2/metabolismo , Transporte de Íons/imunologia , Ativação Linfocitária/imunologia , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Transgênicos , Células T Matadoras Naturais/imunologia , Células T Matadoras Naturais/metabolismo , Receptores de Antígenos de Linfócitos T alfa-beta/metabolismo , Molécula 1 de Interação Estromal , Molécula 2 de Interação Estromal , Linfócitos T Reguladores/metabolismo
14.
Curr Biol ; 22(21): 1990-7, 2012 Nov 06.
Artigo em Inglês | MEDLINE | ID: mdl-23041196

RESUMO

BACKGROUND: Endoplasmic reticulum (ER) membranes are recruited to phagosomes, but the mechanism and functional significance of this ER recruitment is not known. Here, we show that the ER Ca(2+) sensor stromal interaction molecule 1 (STIM1) sustains high-efficiency phagocytosis by recruiting thin ER cisternae that interact productively but do not fuse with phagosomes. RESULTS: Endogenous STIM1 was recruited to phagosomes upon ER Ca(2+) depletion in mouse neutrophils, and exogenous YFP-STIM1 puncta coincided with localized Ca(2+) elevations around phagosomes in fibroblasts expressing phagocytic receptors. STIM1 ablation decreased phagocytosis, ER-phagosome contacts, and periphagosomal Ca(2+) elevations in both neutrophils and fibroblasts, whereas STIM1 re-expression in Stim1(-/-) fibroblasts rescued these defects, promoted the formation and elongation of tight ER-phagosome contacts upon ER Ca(2+) depletion and increased the shedding of periphagosomal actin rings. Re-expression of a signaling-deficient STIM1 mutant unable to open Ca(2+) channels recruited ER cisternae to the vicinity of phagosomes but failed to rescue phagocytosis, actin shedding, and periphagosomal Ca(2+) elevations. The periphagosomal Ca(2+) hotspots were decreased by extracellular Ca(2+) chelation and by Ca(2+) channels inhibitors, revealing that the Ca(2+) ions originate at least in part from phagosomes. CONCLUSIONS: Our findings indicate that STIM1 recruits ER cisternae near phagosomes for signaling purposes and that the opening of phagosomal Ca(2+) channels generates localized Ca(2+) elevations that promote high-efficiency phagocytosis.


Assuntos
Cálcio/análise , Retículo Endoplasmático/metabolismo , Glicoproteínas de Membrana/metabolismo , Fagocitose , Fagossomos/metabolismo , Animais , Bloqueadores dos Canais de Cálcio , Canais de Cálcio/metabolismo , Sinalização do Cálcio , Linhagem Celular , Células HL-60 , Humanos , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neutrófilos/metabolismo , Transdução de Sinais , Molécula 1 de Interação Estromal , Junções Íntimas
15.
Proc Natl Acad Sci U S A ; 109(36): 14544-9, 2012 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-22904194

RESUMO

Primary Sjögren's Syndrome (pSS) is an autoimmune disease involving salivary and other exocrine glands that leads to progressive lymphocytic infiltration into the gland, tissue damage, and secretory defects. The mechanism underlying this disease remains poorly understood. Here we report that mice with T-cell-targeted deletion of Stromal Interaction Molecule (STIM) 1 and STIM2 [double-knockout (DKO)] mice develop spontaneous and severe pSS-like autoimmune disease, displaying major hallmarks of the disease. In DKO mice, diffuse lymphocytic infiltration was seen in submandibular glands, a major target of pSS, by age 6 wk, progressing to severe inflammation by age 12 wk. Sjögren's syndrome-specific autoantibodies (SSA/Ro and SSB/La) were detected in the serum, and progressive salivary gland destruction and loss of fluid secretion were also seen. Importantly, we report that peripheral blood mononuclear cells as well as lymphocytic infiltrates in submandibular glands from patients with pSS demonstrated significant reductions in STIM1 and STIM2 proteins. Store-operated calcium entry was also reduced in peripheral blood mononuclear cells from pSS patients compared with those from healthy controls. Thus, deficiency of STIM1 and STIM2 proteins in T cells, and consequent defects in Ca(2+) signaling, are associated with salivary gland autoimmunopathy in DKO mice and pSS patients. These data reveal a previously unreported link between STIM1 and STIM2 proteins and pSS.


Assuntos
Glicoproteínas de Membrana/deficiência , Síndrome de Sjogren/genética , Glândula Submandibular/patologia , Linfócitos T/metabolismo , Animais , Autoanticorpos/sangue , Western Blotting , Cálcio/metabolismo , Canais de Cálcio , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Knockout , Microscopia de Fluorescência , Molécula 1 de Interação Estromal , Molécula 2 de Interação Estromal , Glândula Submandibular/imunologia
16.
Mol Cell Biol ; 32(15): 3009-17, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22645307

RESUMO

Immediately after birth, skeletal muscle must undergo an enormous period of growth and differentiation that is coordinated by several intertwined growth signaling pathways. How these pathways are integrated remains unclear but is likely to involve skeletal muscle contractile activity and calcium (Ca(2+)) signaling. Here, we show that Ca(2+) signaling governed by stromal interaction molecule 1 (STIM1) plays a central role in the integration of signaling and, therefore, muscle growth and differentiation. Conditional deletion of STIM1 from the skeletal muscle of mice (mSTIM1(-/-) mice) leads to profound growth delay, reduced myonuclear proliferation, and perinatal lethality. We show that muscle fibers of neonatal mSTIM1(-/-) mice cannot support the activity-dependent Ca(2+) transients evoked by tonic neurostimulation, even though excitation contraction coupling (ECC) remains unperturbed. In addition, disruption of tonic Ca(2+) signaling in muscle fibers attenuates downstream muscle growth signaling, such as that of calcineurin, mitogen-activated protein (MAP) kinases, extracellular signal-regulated kinase 1 and 2 (ERK1/2), and AKT. Based on our findings, we propose a model wherein STIM1-mediated store-operated calcium entry (SOCE) governs the Ca(2+) signaling required for cellular processes that are necessary for neonatal muscle growth and differentiation.


Assuntos
Canais de Cálcio/metabolismo , Sinalização do Cálcio/genética , Glicoproteínas de Membrana/metabolismo , Desenvolvimento Muscular , Músculo Esquelético/metabolismo , Animais , Transporte Biológico Ativo/genética , Transporte Biológico Ativo/fisiologia , Cálcio/metabolismo , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Sistema de Sinalização das MAP Quinases , Glicoproteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Esquelético/crescimento & desenvolvimento , Técnicas de Patch-Clamp , Proteínas Proto-Oncogênicas c-akt/biossíntese , Proteínas Proto-Oncogênicas c-akt/metabolismo , Molécula 1 de Interação Estromal , Proteínas Quinases p38 Ativadas por Mitógeno/biossíntese , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo
17.
Nat Med ; 17(10): 1231-4, 2011 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-21909105

RESUMO

Osteocytes embedded in bone have been postulated to orchestrate bone homeostasis by regulating both bone-forming osteoblasts and bone-resorbing osteoclasts. We find here that purified osteocytes express a much higher amount of receptor activator of nuclear factor-κB ligand (RANKL) and have a greater capacity to support osteoclastogenesis in vitro than osteoblasts and bone marrow stromal cells. Furthermore, the severe osteopetrotic phenotype that we observe in mice lacking RANKL specifically in osteocytes indicates that osteocytes are the major source of RANKL in bone remodeling in vivo.


Assuntos
Osso e Ossos/fisiologia , Homeostase/fisiologia , Osteócitos/metabolismo , Osteopetrose/metabolismo , Ligante RANK/metabolismo , Animais , Diferenciação Celular/fisiologia , Fêmur/citologia , Fêmur/diagnóstico por imagem , Citometria de Fluxo , Perfilação da Expressão Gênica , Proteínas de Fluorescência Verde/metabolismo , Imuno-Histoquímica , Camundongos , Camundongos Transgênicos , Osteoclastos/metabolismo , Osteoclastos/fisiologia , Osteócitos/fisiologia , Ligante RANK/deficiência , Reação em Cadeia da Polimerase em Tempo Real , Microtomografia por Raio-X
18.
Mol Cell Biol ; 31(18): 3710-22, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21746875

RESUMO

The regulation of cellular Ca(2+) homeostasis is essential for innumerable physiological and pathological processes. Stanniocalcin 1, a secreted glycoprotein hormone originally described in fish, is a well-established endocrine regulator of gill Ca(2+) uptake during hypercalcemia. While there are two mammalian Stanniocalcin homologs (STC1 and STC2), their precise molecular functions remain unknown. Notably, STC2 is a prosurvival component of the unfolded protein response. Here, we demonstrate a cell-intrinsic role for STC2 in the regulation of store-operated Ca(2+) entry (SOCE). Fibroblasts cultured from Stc2 knockout mice accumulate higher levels of cytosolic Ca(2+) following endoplasmic reticulum (ER) Ca(2+) store depletion, specifically due to an increase in extracellular Ca(2+) influx through store-operated Ca(2+) channels (SOC). The knockdown of STC2 expression in a hippocampal cell line also potentiates SOCE, and the overexpression of STC2 attenuates SOCE. Moreover, STC2 interacts with the ER Ca(2+) sensor STIM1, which activates SOCs following ER store depletion. These results define a novel molecular function for STC2 as a negative modulator of SOCE and provide the first direct evidence for the regulation of Ca(2+) homeostasis by mammalian STC2. Furthermore, our findings implicate the modulation of SOCE through STC2 expression as one of the prosurvival measures of the unfolded protein response.


Assuntos
Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Glicoproteínas/metabolismo , Animais , Canais de Cálcio/metabolismo , Proliferação de Células , Células Cultivadas , Retículo Endoplasmático/metabolismo , Fibroblastos/metabolismo , Glicoproteínas/biossíntese , Glicoproteínas/genética , Homeostase , Peptídeos e Proteínas de Sinalização Intercelular , Peptídeos e Proteínas de Sinalização Intracelular , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Molécula 1 de Interação Estromal
19.
J Immunol ; 185(10): 5845-58, 2010 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-20956344

RESUMO

ORAI1 is the pore-forming subunit of the Ca(2+) release-activated Ca(2+) (CRAC) channel, which is responsible for store-operated Ca(2+) entry in lymphocytes. A role for ORAI1 in T cell function in vivo has been inferred from in vitro studies of T cells from human immunodeficient patients with mutations in ORAI1 and Orai1(-/-) mice, but a detailed analysis of T cell-mediated immune responses in vivo in mice lacking functional ORAI1 has been missing. We therefore generated Orai1 knock-in mice (Orai1(KI/KI)) expressing a nonfunctional ORAI1-R93W protein. Homozygosity for the equivalent ORAI1-R91W mutation abolishes CRAC channel function in human T cells resulting in severe immunodeficiency. Homozygous Orai1(KI/KI) mice die neonatally, but Orai1(KI/KI) fetal liver chimeric mice are viable and show normal lymphocyte development. T and B cells from Orai1(KI/KI) mice display severely impaired store-operated Ca(2+) entry and CRAC channel function resulting in a strongly reduced expression of several key cytokines including IL-2, IL-4, IL-17, IFN-γ, and TNF-α in CD4(+) and CD8(+) T cells. Cell-mediated immune responses in vivo that depend on Th1, Th2, and Th17 cell function were severely attenuated in ORAI1-deficient mice. Orai1(KI/KI) mice lacked detectable contact hypersensitivity responses and tolerated skin allografts significantly longer than wild-type mice. In addition, T cells from Orai1(KI/KI) mice failed to induce colitis in an adoptive transfer model of inflammatory bowel disease. These findings reaffirm the critical role of ORAI1 for T cell function and provide important insights into the in vivo functions of CRAC channels for T cell-mediated immunity.


Assuntos
Autoimunidade/imunologia , Canais de Cálcio/metabolismo , Cálcio/metabolismo , Rejeição de Enxerto/metabolismo , Linfócitos T/imunologia , Animais , Canais de Cálcio/genética , Canais de Cálcio/imunologia , Sinalização do Cálcio/genética , Sinalização do Cálcio/imunologia , Separação Celular , Citocinas/biossíntese , Citocinas/imunologia , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Transferência Ressonante de Energia de Fluorescência , Técnicas de Introdução de Genes , Rejeição de Enxerto/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Mutação , Proteína ORAI1 , Técnicas de Patch-Clamp , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Linfócitos T/metabolismo , Quimeras de Transplante , Transplante Homólogo
20.
Cell Calcium ; 48(2-3): 161-7, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20801505

RESUMO

The stromal interaction molecules STIM1 and STIM2 sense a decreasing Ca(2+) concentration in the lumen of the endoplasmic reticulum and activate Ca(2+) channels in the plasma membrane. In addition, at least 2 reports suggested that STIM1 may also interact with the inositol 1,4,5-trisphosphate (IP(3)) receptor. Using embryonic fibroblasts from Stim1(-/-), Stim2(-/-) and wild-type mice, we now tested the hypothesis that STIM1 and STIM2 would also regulate the IP(3) receptor. We investigated whether STIM1 or STIM2 would be the luminal Ca(2+) sensor that controls the loading dependence of the IP(3)-induced Ca(2+) release. Partial emptying of the stores in plasma-membrane permeabilized cells resulted in an increased EC(50) and a decreased Hill coefficient for IP(3)-induced Ca(2+) release. This effect occurred both in the presence and absence of STIM proteins, indicating that these proteins were not the luminal Ca(2+) sensor for the IP(3) receptor. Although Stim1(-/-) cells displayed a normal IP(3)-receptor function, agonist-induced Ca(2+) release was reduced. This finding suggests that the presence of STIM1 is required for proper agonist-induced Ca(2+) signaling. Our data do not provide experimental evidence for the suggestion that STIM proteins would directly control the function of the IP(3) receptor.


Assuntos
Sinalização do Cálcio/fisiologia , Cálcio/fisiologia , Fibroblastos/metabolismo , Glicoproteínas de Membrana/fisiologia , Animais , Cálcio/antagonistas & inibidores , Canais de Cálcio , Sinalização do Cálcio/genética , Células Cultivadas , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Inositol 1,4,5-Trifosfato/agonistas , Inositol 1,4,5-Trifosfato/metabolismo , Receptores de Inositol 1,4,5-Trifosfato/biossíntese , Receptores de Inositol 1,4,5-Trifosfato/genética , Receptores de Inositol 1,4,5-Trifosfato/fisiologia , Glicoproteínas de Membrana/agonistas , Glicoproteínas de Membrana/deficiência , Camundongos , Camundongos Knockout , Isoformas de Proteínas/agonistas , Isoformas de Proteínas/deficiência , Isoformas de Proteínas/fisiologia , Molécula 1 de Interação Estromal , Molécula 2 de Interação Estromal
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