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1.
Annu Rev Immunol ; 33: 291-353, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25861976

RESUMO

Ion channels and transporters mediate the transport of charged ions across hydrophobic lipid membranes. In immune cells, divalent cations such as calcium, magnesium, and zinc have important roles as second messengers to regulate intracellular signaling pathways. By contrast, monovalent cations such as sodium and potassium mainly regulate the membrane potential, which indirectly controls the influx of calcium and immune cell signaling. Studies investigating human patients with mutations in ion channels and transporters, analysis of gene-targeted mice, or pharmacological experiments with ion channel inhibitors have revealed important roles of ionic signals in lymphocyte development and in innate and adaptive immune responses. We here review the mechanisms underlying the function of ion channels and transporters in lymphocytes and innate immune cells and discuss their roles in lymphocyte development, adaptive and innate immune responses, and autoimmunity, as well as recent efforts to develop pharmacological inhibitors of ion channels for immunomodulatory therapy.


Assuntos
Imunidade Adaptativa/fisiologia , Imunidade Inata/fisiologia , Canais Iônicos/metabolismo , Animais , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Humanos , Hipersensibilidade/genética , Hipersensibilidade/imunologia , Hipersensibilidade/metabolismo , Síndromes de Imunodeficiência/tratamento farmacológico , Síndromes de Imunodeficiência/genética , Síndromes de Imunodeficiência/imunologia , Síndromes de Imunodeficiência/metabolismo , Imunoterapia/métodos , Canais Iônicos/genética , Linfócitos/citologia , Linfócitos/imunologia , Linfócitos/metabolismo , Mastócitos/imunologia , Mastócitos/metabolismo , Terapia de Alvo Molecular , Mutação , Transdução de Sinais
2.
Mol Cell ; 63(3): 457-69, 2016 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-27453048

RESUMO

Whereas phosphorylation of serine, threonine, and tyrosine is exceedingly well characterized, the role of histidine phosphorylation in mammalian signaling is largely unexplored. Here we show that phosphoglycerate mutase family 5 (PGAM5) functions as a phosphohistidine phosphatase that specifically associates with and dephosphorylates the catalytic histidine on nucleoside diphosphate kinase B (NDPK-B). By dephosphorylating NDPK-B, PGAM5 negatively regulates CD4(+) T cells by inhibiting NDPK-B-mediated histidine phosphorylation and activation of the K(+) channel KCa3.1, which is required for TCR-stimulated Ca(2+) influx and cytokine production. Using recently developed monoclonal antibodies that specifically recognize phosphorylation of nitrogens at the N1 (1-pHis) or N3 (3-pHis) positions of the imidazole ring, we detect for the first time phosphoisoform-specific regulation of histidine-phosphorylated proteins in vivo, and we link these modifications to TCR signaling. These results represent an important step forward in studying the role of histidine phosphorylation in mammalian biology and disease.


Assuntos
Linfócitos T CD4-Positivos/enzimologia , Ativação Linfocitária , Proteínas Mitocondriais/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Animais , Linfócitos T CD4-Positivos/imunologia , Sinalização do Cálcio , Citocinas/metabolismo , Predisposição Genética para Doença , Doença Enxerto-Hospedeiro/enzimologia , Doença Enxerto-Hospedeiro/genética , Doença Enxerto-Hospedeiro/imunologia , Células HEK293 , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Histidina , Humanos , Mediadores da Inflamação/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Células Jurkat , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Mitocondriais/genética , Núcleosídeo-Difosfato Quinase/metabolismo , Fenótipo , Fosfoproteínas Fosfatases/deficiência , Fosfoproteínas Fosfatases/genética , Fosforilação , Interferência de RNA , Receptores de Antígenos de Linfócitos T/metabolismo , Fatores de Tempo , Transfecção
3.
Proc Natl Acad Sci U S A ; 114(10): 2693-2698, 2017 03 07.
Artigo em Inglês | MEDLINE | ID: mdl-28213494

RESUMO

Regulation of integrins is critical for lymphocyte adhesion to endothelium and migration throughout the body. Inside-out signaling to integrins is mediated by the small GTPase Ras-proximate-1 (Rap1). Using an RNA-mediated interference screen, we identified phospholipase Cε 1 (PLCε1) as a crucial regulator of stromal cell-derived factor 1 alpha (SDF-1α)-induced Rap1 activation. We have shown that SDF-1α-induced activation of Rap1 is transient in comparison with the sustained level following cross-linking of the antigen receptor. We identified that PLCε1 was necessary for SDF-1α-induced adhesion using shear stress, cell morphology alterations, and crawling on intercellular adhesion molecule 1 (ICAM-1)-expressing cells. Structure-function experiments to separate the dual-enzymatic function of PLCε1 uncover necessary contributions of the CDC25, Pleckstrin homology, and Ras-associating domains, but not phospholipase activity, to this pathway. In the mouse model of delayed type hypersensitivity, we have shown an essential role for PLCε1 in T-cell migration to inflamed skin, but not for cytokine secretion and proliferation in regional lymph nodes. Our results reveal a signaling pathway where SDF-1α induces T-cell adhesion through activation of PLCε1, suggesting that PLCε1 is a specific potential target in treating conditions involving migration of T cells to inflamed organs.


Assuntos
Quimiocina CXCL12/genética , Inflamação/genética , Fosfoinositídeo Fosfolipase C/genética , Proteínas de Ligação a Telômeros/genética , Animais , Adesão Celular/genética , Adesão Celular/imunologia , Movimento Celular/genética , Movimento Celular/imunologia , Quimiocina CXCL12/imunologia , Humanos , Inflamação/imunologia , Inflamação/patologia , Molécula 1 de Adesão Intercelular/imunologia , Linfócitos/imunologia , Linfócitos/patologia , Camundongos , Fosfoinositídeo Fosfolipase C/imunologia , Receptores de Antígenos/genética , Receptores de Antígenos/imunologia , Complexo Shelterina , Transdução de Sinais , Linfócitos T/imunologia , Linfócitos T/patologia , Proteínas de Ligação a Telômeros/imunologia , ras-GRF1/imunologia
4.
Circulation ; 135(9): 881-897, 2017 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-27927712

RESUMO

BACKGROUND: Chronic heart failure (HF) is associated with altered signal transduction via ß-adrenoceptors and G proteins and with reduced cAMP formation. Nucleoside diphosphate kinases (NDPKs) are enriched at the plasma membrane of patients with end-stage HF, but the functional consequences of this are largely unknown, particularly for NDPK-C. Here, we investigated the potential role of NDPK-C in cardiac cAMP formation and contractility. METHODS: Real-time polymerase chain reaction, (far) Western blot, immunoprecipitation, and immunocytochemistry were used to study the expression, interaction with G proteins, and localization of NDPKs. cAMP levels were determined with immunoassays or fluorescent resonance energy transfer, and contractility was determined in cardiomyocytes (cell shortening) and in vivo (fractional shortening). RESULTS: NDPK-C was essential for the formation of an NDPK-B/G protein complex. Protein and mRNA levels of NDPK-C were upregulated in end-stage human HF, in rats after long-term isoprenaline stimulation through osmotic minipumps, and after incubation of rat neonatal cardiomyocytes with isoprenaline. Isoprenaline also promoted translocation of NDPK-C to the plasma membrane. Overexpression of NDPK-C in cardiomyocytes increased cAMP levels and sensitized cardiomyocytes to isoprenaline-induced augmentation of contractility, whereas NDPK-C knockdown decreased cAMP levels. In vivo, depletion of NDPK-C in zebrafish embryos caused cardiac edema and ventricular dysfunction. NDPK-B knockout mice had unaltered NDPK-C expression but showed contractile dysfunction and exacerbated cardiac remodeling during long-term isoprenaline stimulation. In human end-stage HF, the complex formation between NDPK-C and Gαi2 was increased whereas the NDPK-C/Gαs interaction was decreased, producing a switch that may contribute to an NDPK-C-dependent cAMP reduction in HF. CONCLUSIONS: Our findings identify NDPK-C as an essential requirement for both the interaction between NDPK isoforms and between NDPK isoforms and G proteins. NDPK-C is a novel critical regulator of ß-adrenoceptor/cAMP signaling and cardiac contractility. By switching from Gαs to Gαi2 activation, NDPK-C may contribute to lower cAMP levels and the related contractile dysfunction in HF.


Assuntos
AMP Cíclico/análise , Insuficiência Cardíaca/patologia , Nucleosídeo NM23 Difosfato Quinases/análise , Animais , Linhagem Celular , Membrana Celular/metabolismo , AMP Cíclico/metabolismo , Modelos Animais de Doenças , Embrião não Mamífero/metabolismo , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/metabolismo , Insuficiência Cardíaca/metabolismo , Humanos , Isoproterenol/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Nucleosídeo NM23 Difosfato Quinases/antagonistas & inibidores , Nucleosídeo NM23 Difosfato Quinases/genética , Nucleosídeo NM23 Difosfato Quinases/metabolismo , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Ratos , Ratos Wistar , Peixe-Zebra/crescimento & desenvolvimento
5.
Nephrol Dial Transplant ; 33(8): 1343-1353, 2018 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-29420817

RESUMO

Background: Metabolism of glutamine by glutaminase 1 (GLS1) plays a key role in tumor cell proliferation via the generation of ATP and intermediates required for macromolecular synthesis. We hypothesized that glutamine metabolism also plays a role in proliferation of autosomal-dominant polycystic kidney disease (ADPKD) cells and that inhibiting GLS1 could slow cyst growth in animal models of ADPKD. Methods: Primary normal human kidney and ADPKD human cyst-lining epithelial cells were cultured in the presence or absence of two pharmacologic inhibitors of GLS1, bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide 3 (BPTES) and CB-839, and the effect on proliferation, cyst growth in collagen and activation of downstream signaling pathways were assessed. We then determined if inhibiting GLS1 in vivo with CB-839 in the Aqp2-Cre; Pkd1fl/fl and Pkhd1-Cre; Pkd1fl/fl mouse models of ADPKD slowed cyst growth. Results: We found that an isoform of GLS1 (GLS1-GAC) is upregulated in cyst-lining epithelia in human ADPKD kidneys and in mouse models of ADPKD. Both BPTES and CB-839 blocked forskolin-induced cyst formation in vitro. Inhibiting GLS1 in vivo with CB-839 led to variable outcomes in two mouse models of ADPKD. CB-839 slowed cyst growth in Aqp2-Cre; Pkd1fl/fl mice, but not in Pkhd1-Cre; Pkd1fl/fl mice. While CB-839 inhibited mammalian target of rapamycin (mTOR) and MEK activation in Aqp2-Cre; Pkd1fl/fl, it did not in Pkhd1-Cre; Pkd1fl/fl mice. Conclusion: These findings provide support that alteration in glutamine metabolism may play a role in cyst growth. However, testing in other models of PKD and identification of the compensatory metabolic changes that bypass GLS1 inhibition will be critical to validate GLS1 as a drug target either alone or when combined with inhibitors of other metabolic pathways.


Assuntos
Proliferação de Células/efeitos dos fármacos , Glutaminase/metabolismo , Glutamina/metabolismo , Rim Policístico Autossômico Dominante/metabolismo , Rim Policístico Autossômico Dominante/patologia , Animais , Aquaporina 2/fisiologia , Benzenoacetamidas/farmacologia , Células Cultivadas , Feminino , Glutaminase/antagonistas & inibidores , Humanos , Masculino , Camundongos , Camundongos Knockout , Receptores de Superfície Celular/fisiologia , Transdução de Sinais , Tiadiazóis/farmacologia
6.
Proc Natl Acad Sci U S A ; 111(11): E978-87, 2014 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-24591580

RESUMO

Macropinocytosis is a highly conserved endocytic process by which extracellular fluid and solutes are internalized into cells. Macropinocytosis starts with the formation of membrane ruffles at the plasma membrane and ends with their closure. The transient and sequential emergence of phosphoinositides PI(3,4,5)P3 and PI(3,4)P2 in the membrane ruffles is essential for macropinocytosis. By making use of information in the Caenorhabditis elegans mutants defective in fluid-phase endocytosis, we found that mammalian phosphoinositide phosphatase MTMR6 that dephosphorylates PI(3)P to PI, and its binding partner MTMR9, are required for macropinocytosis. INPP4B, which dephosphorylates PI(3,4)P2 to PI(3)P, was also found to be essential for macropinocytosis. These phosphatases operate after the formation of membrane ruffles to complete macropinocytosis. Finally, we showed that KCa3.1, a Ca(2+)-activated K(+) channel that is activated by PI(3)P, is required for macropinocytosis. We propose that the sequential breakdown of PI(3,4,5)P3 → PI(3,4)P2 → PI(3)P → PI controls macropinocytosis through specific effectors of the intermediate phosphoinositides.


Assuntos
Caenorhabditis elegans/fisiologia , Fosfatos de Fosfatidilinositol/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Pinocitose/fisiologia , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Animais , Caenorhabditis elegans/metabolismo , Linhagem Celular , Primers do DNA/genética , Humanos , Microscopia Eletrônica de Varredura , Microscopia de Fluorescência , Fosforilação , Interferência de RNA , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
7.
Arterioscler Thromb Vasc Biol ; 35(8): 1852-61, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26088577

RESUMO

OBJECTIVE: Vascular smooth muscle cells (VSMC) proliferation is a hallmark of atherosclerosis and vascular restenosis. The intermediate conductance Ca(2+)-activated K(+) (SK4) channel is required for pathological VSMC proliferation. In T lymphocytes, nucleoside diphosphate kinase B (NDPKB) has been implicated in SK4 channel activation. We thus investigated the role of NDPKB in the regulation of SK4 currents (ISK4) in proliferating VSMC and neointima formation. APPROACH AND RESULTS: Function and expression of SK4 channels in VSMC from injured mouse carotid arteries were assessed by patch-clamping and real-time polymerase chain reaction. ISK4 was detectable in VSMC from injured but not from uninjured arteries correlating with the occurrence of the proliferative phenotype. Direct application of NDPKB to the membrane of inside-out patches increased ISK4, whereas NDPKB did not alter currents in VSMC obtained from injured vessels of SK4-deficient mice. The NDPKB-induced increase in ISK4 was prevented by protein histidine phosphatase 1, but not an inactive protein histidine phosphatase 1 mutant indicating that ISK4 is regulated via histidine phosphorylation in proliferating VSMC; moreover, genetic NDPKB ablation reduced ISK4 by 50% suggesting a constitutive activation of ISK4 in proliferating VSMC. In line, neointima formation after wire injury of the carotid artery was substantially reduced in mice deficient in SK4 channels or NDPKB. CONCLUSIONS: NDPKB to SK4 signaling is required for neointima formation. Constitutive activation of SK4 by NDPKB in proliferating VSMC suggests that targeting this interaction via, for example, activation of protein histidine phosphatase 1 may provide clinically meaningful effects in vasculoproliferative diseases such as atherosclerosis and post angioplasty restenosis.


Assuntos
Lesões das Artérias Carótidas/enzimologia , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Músculo Liso Vascular/enzimologia , Miócitos de Músculo Liso/enzimologia , Nucleosídeo NM23 Difosfato Quinases/metabolismo , Neointima , Animais , Artérias Carótidas/enzimologia , Artérias Carótidas/patologia , Lesões das Artérias Carótidas/genética , Lesões das Artérias Carótidas/patologia , Proliferação de Células , Células Cultivadas , Modelos Animais de Doenças , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/deficiência , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/genética , Potenciais da Membrana , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Músculo Liso Vascular/patologia , Miócitos de Músculo Liso/patologia , Nucleosídeo NM23 Difosfato Quinases/deficiência , Nucleosídeo NM23 Difosfato Quinases/genética , Transdução de Sinais
8.
AJR Am J Roentgenol ; 207(2): 344-53, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27305103

RESUMO

OBJECTIVE: The purpose of this article is to compare the effectiveness of a treatment algorithm for small renal tumors incorporating the nephrometry score, a renal tumor anatomy scoring system developed by urologists, with the current standard of uniformly recommended partial nephrectomy in patients with mild-to-moderate chronic kidney disease (CKD). MATERIALS AND METHODS: We developed a state-transition microsimulation model to project life expectancy (LE) in hypothetic patients with baseline mild or moderate CKD undergoing treatment of small renal masses. Our model incorporated the nephrometry score, which is predictive of postsurgical renal function loss. The two tested strategies were uniform treatment with partial nephrectomy and selective treatment based on nephrometry score and CKD stage, including percutaneous ablation for CKD stages 2 or 3a and intermediate-to-high nephrometry score or stage 3b CKD and any nephrometry score; otherwise, partial nephrectomy was assumed for other CKD stages and nephrometry scores. The model accounted for benign and malignant lesions, renal function decline, recurrence, and metastatic disease rates specific to each treatment, mortality by CKD stage, and comorbidities. Sensitivity analysis tested the stability of results when varying key parameters. RESULTS: Selective treatment with partial nephrectomy resulted in an average LE benefit of 0.48 year (95% interpercentile range, 0.42-0.54 year) in 65-year-old men and 0.37 year (95% interpercentile range, 0.30-0.43 year) in 65-year-old women relative to nondiscriminatory surgery, due to worsening CKD and cardiovascular mortality associated with partial nephrectomy. Model results were most sensitive to the rate of renal function decline and CKD-related mortality. CONCLUSION: Nephron-sparing treatment selection for small renal masses based on nephrometry score may improve LE in patients with mild or moderate CKD.


Assuntos
Técnicas de Apoio para a Decisão , Neoplasias Renais/cirurgia , Nefrectomia/métodos , Idoso , Algoritmos , Comorbidade , Feminino , Humanos , Testes de Função Renal , Neoplasias Renais/mortalidade , Neoplasias Renais/patologia , Expectativa de Vida , Masculino , Seleção de Pacientes , Valor Preditivo dos Testes , Taxa de Sobrevida , Resultado do Tratamento
10.
Arterioscler Thromb Vasc Biol ; 34(10): 2292-300, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25147336

RESUMO

OBJECTIVE: Nucleoside diphosphate kinase B (NDPKB) participates in the activation of heterotrimeric and monomeric G proteins, which are pivotal mediators in angiogenic signaling. The role of NDPKB in angiogenesis has to date not been defined. Therefore, we analyzed the contribution of NDPKB to angiogenesis and its underlying mechanisms in well-characterized in vivo and in vitro models. APPROACH AND RESULTS: Zebrafish embryos were depleted of NDPKB by morpholino-mediated knockdown. These larvae displayed severe malformations specifically in vessels formed by angiogenesis. NDPKB-deficient (NDPKB(-/-)) mice were subjected to oxygen-induced retinopathy. In this model, the number of preretinal neovascularizations in NDPKB(-/-) mice was strongly reduced in comparison with wild-type littermates. In accordance, a delayed blood flow recovery was detected in the NDPKB(-/-) mice after hindlimb ligation. In in vitro studies, a small interfering RNA-mediated knockdown of NDPKB was performed in human umbilical endothelial cells. NDPKB depletion impaired vascular endothelial growth factor (VEGF)-induced sprouting and hampered the VEGF-induced spatial redistributions of the VEGF receptor type 2 and VE-cadherin at the plasma membrane. Concomitantly, NDPKB depletion increased the permeability of the human umbilical endothelial cell monolayer. CONCLUSIONS: This is the first report to show that NDPKB is required for VEGF-induced angiogenesis and contributes to the correct localization of VEGF receptor type 2 and VE-cadherin at the endothelial adherens junctions. Therefore, our data identify NDPKB as a novel molecular target to modulate VEGF-dependent angiogenesis.


Assuntos
Antígenos CD/metabolismo , Caderinas/metabolismo , Células Endoteliais/enzimologia , Músculo Esquelético/irrigação sanguínea , Nucleosídeo NM23 Difosfato Quinases/metabolismo , Neovascularização Fisiológica , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/metabolismo , Animais , Células Cultivadas , Modelos Animais de Doenças , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Membro Posterior , Células Endoteliais da Veia Umbilical Humana/enzimologia , Humanos , Isquemia/enzimologia , Isquemia/genética , Isquemia/fisiopatologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nucleosídeo NM23 Difosfato Quinases/deficiência , Nucleosídeo NM23 Difosfato Quinases/genética , Interferência de RNA , Recuperação de Função Fisiológica , Fluxo Sanguíneo Regional , Neovascularização Retiniana/enzimologia , Neovascularização Retiniana/genética , Neovascularização Retiniana/fisiopatologia , Transdução de Sinais , Fatores de Tempo , Transfecção , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/genética
11.
Proc Natl Acad Sci U S A ; 108(50): 20072-7, 2011 Dec 13.
Artigo em Inglês | MEDLINE | ID: mdl-22128329

RESUMO

The K(+) channel KCa3.1 is required for Ca(2+) influx and the subsequent activation of CD4 T cells. The class II phosphatidylinositol 3 kinase C2ß (PI3KC2ß) is activated by the T-cell receptor (TCR) and is critical for KCa3.1 channel activation. Tripartite motif containing protein 27 (TRIM27) is a member of a large family of proteins that function as Really Interesting New Gene (RING) E3 ubiquitin ligases. We now show that TRIM27 functions as an E3 ligase and mediates lysine 48 polyubiquitination of PI3KC2ß, leading to a decrease in PI3K enzyme activity. By inhibiting PI3KC2ß, TRIM27 also functions to negatively regulate CD4 T cells by inhibiting KCa3.1 channel activity and TCR-stimulated Ca(2+) influx and cytokine production in Jurkat, primary human CD4 T cells, and Th0, Th1, and Th2 CD4 T cells generated from TRIM27(-/-) mice. These findings provide a unique mechanism for regulating class II PI3Ks, and identify TRIM27 as a previously undescribed negative regulator of CD4 T cells.


Assuntos
Linfócitos T CD4-Positivos/enzimologia , Linfócitos T CD4-Positivos/imunologia , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Ubiquitinação , Animais , Cálcio/metabolismo , Citocinas/biossíntese , Proteínas de Ligação a DNA/deficiência , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Ativação do Canal Iônico , Células Jurkat , Camundongos , Mucoproteínas/metabolismo , Proteínas Nucleares/deficiência , Fosfatidilinositol 3-Quinases/metabolismo , Poliubiquitina/metabolismo , Ligação Proteica , Proteólise , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais/imunologia , Células Th1/imunologia , Células Th2/imunologia , Técnicas do Sistema de Duplo-Híbrido , Ubiquitina-Proteína Ligases
12.
Proc Natl Acad Sci U S A ; 107(4): 1541-6, 2010 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-20080610

RESUMO

The calcium-activated K(+) channel KCa3.1 plays an important role in T lymphocyte Ca(2+) signaling by helping to maintain a negative membrane potential, which provides an electrochemical gradient to drive Ca(2+) influx. To assess the role of KCa3.1 channels in lymphocyte activation in vivo, we studied T cell function in KCa3.1(-/-) mice. CD4 T helper (i.e., Th0) cells isolated from KCa3.1(-/-) mice lacked KCa3.1 channel activity, which resulted in decreased T cell receptor-stimulated Ca(2+) influx and IL-2 production. Although loss of KCa3.1 did not interfere with CD4 T cell differentiation, both Ca(2+) influx and cytokine production were impaired in KCa3.1(-/-) Th1 and Th2 CD4 T cells, whereas T-regulatory and Th17 function were normal. We found that inhibition of KCa3.1(-/-) protected mice from developing severe colitis in two mouse models of inflammatory bowel disease, which were induced by (i) the adoptive transfer of mouse naïve CD4 T cells into rag2(-/-) recipients and (ii) trinitrobenzene sulfonic acid. Pharmacologic inhibitors of KCa3.1 have already been shown to be safe in humans. Thus, if these preclinical studies continue to show efficacy, it may be possible to rapidly test whether KCa3.1 inhibitors are efficacious in patients with inflammatory bowel diseases such as Crohn's disease and ulcerative colitis.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Colite/imunologia , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/imunologia , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Animais , Linfócitos B/citologia , Linfócitos B/imunologia , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Diferenciação Celular , Células Cultivadas , Colite/genética , Colite/metabolismo , Colite/patologia , Citocinas/biossíntese , Citocinas/imunologia , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/imunologia , Modelos Animais de Doenças , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T Auxiliares-Indutores/citologia , Linfócitos T Auxiliares-Indutores/imunologia , Linfócitos T Auxiliares-Indutores/metabolismo
13.
J Biol Chem ; 285(50): 38765-71, 2010 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-20884616

RESUMO

Nucleoside diphosphate kinases (NDPKs) are encoded by the Nme (non-metastatic cell) gene family. Although they comprise a family of 10 genes, NDPK-A and -B are ubiquitously expressed and account for most of the NDPK activity. We previously showed that NDPK-B activates the K(+) channel KCa3.1 via histidine phosphorylation of the C terminus of KCa3.1, which is required for T cell receptor-stimulated Ca(2+) flux and proliferation of activated naive human CD4 T cells. We now report the phenotype of NDPK-B(-/-) mice. NDPK-B(-/-) mice are phenotypically normal at birth with a normal life span. Although T and B cell development is normal in NDPK-B(-/-) mice, KCa3.1 channel activity and cytokine production are markedly defective in T helper 1 (Th1) and Th2 cells, whereas Th17 function is normal. These findings phenocopy studies in the same cells isolated from KCa3.1(-/-) mice and thereby support genetically that NDPK-B functions upstream of KCa3.1. NDPK-A and -B have been linked to an astonishing array of disparate cellular and biochemical functions, few of which have been confirmed in vivo in physiological relevant systems. NDPK-B(-/-) mice will be an essential tool with which to definitively address the biological functions of NDPK-B. Our finding that NDPK-B is required for activation of Th1 and Th2 CD4 T cells, together with the normal overall phenotype of NDPK-B(-/-) mice, suggests that specific pharmacological inhibitors of NDPK-B may provide new opportunities to treat Th1- and Th2-mediated autoimmune diseases.


Assuntos
Canais de Potássio Ativados por Cálcio de Condutância Intermediária/genética , Nucleosídeo NM23 Difosfato Quinases/genética , Potássio/química , Linfócitos T/metabolismo , Animais , Linfócitos T CD4-Positivos/metabolismo , Citometria de Fluxo , Histidina Quinase , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Técnicas de Patch-Clamp , Proteínas Quinases/metabolismo , Transdução de Sinais , Linfócitos T/citologia
14.
Kidney Int ; 80(7): 719-30, 2011 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-21544061

RESUMO

Micro-RNAs (miRNAs) are short (average 22 nucleotides) noncoding regulatory RNAs that inhibit gene expression by targeting complementary 3'-untranslated regions of protein-encoding mRNAs for translational repression or degradation. miRNAs play key roles in both the function and differentiation of many cell types. Drosha and Dicer, two RNAase III enzymes, function in a stepwise manner to generate a mature miRNA. Previous studies have shown that podocyte-specific deletion of Dicer during development results in proteinuric renal disease and collapsing glomerulopathy (CG); however, Dicer has functions other than the generation of miRNAs. Here we found that the podocyte-specific deletion of Drosha results in a similar phenotype to Dicer mutants, confirming that the Dicer mutant phenotype is due to the loss of miRNAs. Moreover, the inducible deletion of Drosha in 2- to 3-month-old mice (Tet-On system) resulted in CG. Thus, continuous generation of miRNAs are required for the normal function of mature podocytes and their loss leads to CG. Identifying these miRNAs may provide new insight into disease pathogenesis and novel therapeutic targets in various podocytopathies.


Assuntos
Nefropatias/genética , MicroRNAs/genética , Podócitos/metabolismo , Ribonuclease III/genética , Animais , Apoptose , Biomarcadores/metabolismo , Desdiferenciação Celular , Diferenciação Celular , Proliferação de Células , RNA Helicases DEAD-box/deficiência , RNA Helicases DEAD-box/genética , Nefropatias/patologia , Camundongos , Camundongos Knockout , Camundongos Mutantes , MicroRNAs/metabolismo , Podócitos/patologia , Ribonuclease III/deficiência
15.
Radiology ; 259(2): 462-70, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21386050

RESUMO

PURPOSE: To assess the accuracy of glomerular filtration rate (GFR) measurements obtained with low-contrast agent dose dynamic contrast material-enhanced magnetic resonance (MR) renography in patients with liver cirrhosis who underwent routine liver MR imaging, with urinary clearance of technetium 99m ((99m)Tc) pentetic acid (DTPA) as the reference standard. MATERIALS AND METHODS: This HIPAA-compliant study was institutional review board approved. Written informed patient consent was obtained. Twenty patients with cirrhosis (14 men, six women; age range, 41-70 years; mean age, 54.6 years) who were scheduled for routine 1.5-T liver MR examinations to screen for hepatocellular carcinoma during a 6-month period were prospectively included. Five-minute MR renography with a 3-mL dose of gadoteridol was performed instead of a routine test-dose timing examination. The GFR was estimated at MR imaging with use of two kinetic models. In one model, only the signal intensities in the aorta and kidney parenchyma were considered, and in the other, renal cortical and medullary signal intensities were treated separately. The GFR was also calculated by using serum creatinine levels according to the Cockcroft-Gault and modification of diet in renal disease (MDRD) formulas. All patients underwent a (99m)Tc-DTPA urinary clearance examination on the same day to obtain a reference GFR measurement. The accuracies of all MR- and creatinine-based GFR estimations were compared by using Wilcoxon signed rank tests. RESULTS: The mean reference GFR, based on (99m)Tc-DTPA clearance, was 74.9 mL/min/1.73 m(2) ± 27.7 (standard deviation) (range, 10.3-120.7 mL/min/1.73 m(2)). With both kinetic models, 95% of MR-based GFRs were within 30% of the reference values, whereas only 40% and 60% of Cockcroft-Gault- and MDRD-based GFRs, respectively, were within this range. MR-based GFR estimates were significantly more accurate than creatinine level-based estimates (P < .001). CONCLUSION: GFR assessment with MR imaging, which outperformed the Cockcroft-Gault and MDRD formulas, adds less than 10 minutes of table time to a clinically indicated liver MR examination without ionizing radiation. SUPPLEMENTAL MATERIAL: http://radiology.rsna.org/lookup/suppl/doi:10.1148/radiol.11101338/-/DC1.


Assuntos
Taxa de Filtração Glomerular/fisiologia , Cirrose Hepática/fisiopatologia , Imageamento por Ressonância Magnética/métodos , Adulto , Idoso , Análise de Variância , Meios de Contraste/farmacocinética , Feminino , Humanos , Interpretação de Imagem Assistida por Computador , Testes de Função Renal , Cinética , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Compostos Radiofarmacêuticos/farmacocinética , Estatísticas não Paramétricas , Pentetato de Tecnécio Tc 99m/farmacocinética
16.
Proc Natl Acad Sci U S A ; 105(38): 14442-6, 2008 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-18796614

RESUMO

The calcium activated K(+) channel KCa3.1 plays an important role in T lymphocyte Ca(2+) signaling by helping to maintain a negative membrane potential, which provides an electrochemical gradient to drive Ca(2+) influx. We previously showed that nucleoside diphosphate kinase beta (NDPK-B), a mammalian histidine kinase, is required for KCa3.1 channel activation in human CD4 T lymphocytes. We now show that the mammalian protein histidine phosphatase (PHPT-1) directly binds and inhibits KCa3.1 by dephosphorylating histidine 358 on KCa3.1. Overexpression of wild-type, but not a phosphatase dead, PHPT-1 inhibited KCa3.1 channel activity. Decreased expression of PHPT-1 by siRNA in human CD4 T cells resulted in an increase in KCa3.1 channel activity and increased Ca(2+) influx and proliferation after T cell receptor (TCR) activation, indicating that endogenous PHPT-1 functions to negatively regulate CD4 T cells. Our findings provide a previously unrecognized example of a mammalian histidine phosphatase negatively regulating TCR signaling and are one of the few examples of histidine phosphorylation/dephosphorylation influencing a biological process in mammals.


Assuntos
Linfócitos T CD4-Positivos/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Adulto , Animais , Linfócitos T CD4-Positivos/citologia , Células CHO , Cálcio/metabolismo , Sinalização do Cálcio/fisiologia , Proliferação de Células , Cricetinae , Cricetulus , Expressão Gênica , Inativação Gênica , Histidina/metabolismo , Humanos , Imunoprecipitação , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/antagonistas & inibidores , Técnicas de Patch-Clamp , Monoéster Fosfórico Hidrolases/genética , RNA Interferente Pequeno/metabolismo
17.
Mol Cell Biol ; 26(15): 5595-602, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16847315

RESUMO

Intracellular Ca2+ levels rapidly rise following cross-linking of the T-cell receptor (TCR) and function as a critical intracellular second messenger in T-cell activation. It has been relatively under appreciated that K+ channels play an important role in Ca2+ influx into T lymphocytes by helping to maintain a negative membrane potential which provides an electrochemical gradient to drive Ca2+ influx. Here we show that the Ca2+-activated K+ channel, KCa3.1, which is critical for Ca2+ influx in reactivated naive T cells and central memory T cells, requires phosphatidylinositol-3 phosphatase [PI(3)P] for activation and is inhibited by the PI(3)P phosphatase myotubularin-related protein 6 (MTMR6). Moreover, by inhibiting KCa3.1, MTMR6 functions as a negative regulator of Ca2+ influx and proliferation of reactivated human CD4 T cells. These findings point to a new and unexpected role for PI(3)P and the PI(3)P phosphatase MTMR6 in the regulation of Ca2+ influx in activated CD4 T cells and suggest that MTMR6 plays a critical role in setting a minimum threshold for a stimulus to activate a T cell.


Assuntos
Linfócitos T CD4-Positivos/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Linfócitos T CD4-Positivos/citologia , Cálcio/metabolismo , Proliferação de Células , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/genética , Ativação Linfocitária , Técnicas de Patch-Clamp , Monoéster Fosfórico Hidrolases/genética , Proteínas Tirosina Fosfatases não Receptoras , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo
18.
Mol Biol Cell ; 17(1): 146-54, 2006 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-16251351

RESUMO

KCa3.1 is an intermediate conductance Ca2+-activated K+ channel that is expressed predominantly in hematopoietic cells, smooth muscle cells, and epithelia where it functions to regulate membrane potential, Ca2+ influx, cell volume, and chloride secretion. We recently found that the KCa3.1 channel also specifically requires phosphatidylinositol-3 phosphate [PI(3)P] for channel activity and is inhibited by myotubularin-related protein 6 (MTMR6), a PI(3)P phosphatase. We now show that PI(3)P indirectly activates KCa3.1. Unlike KCa3.1 channels, the related KCa2.1, KCa2.2, or KCa2.3 channels do not require PI(3)P for activity, suggesting that the KCa3.1 channel has evolved a unique means of regulation that is critical for its biological function. By making chimeric channels between KCa3.1 and KCa2.3, we identified a stretch of 14 amino acids in the carboxy-terminal calmodulin binding domain of KCa3.1 that is sufficient to confer regulation of KCa2.3 by PI(3)P. However, mutation of a single potential phosphorylation site in these 14 amino acids did not affect channel activity. These data together suggest that PI(3)P and these 14 amino acids regulate KCa3.1 channel activity by recruiting an as yet to be defined regulatory subunit that is required for Ca2+ gating of KCa3.1.


Assuntos
Canais de Potássio Ativados por Cálcio de Condutância Intermediária/química , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Sequência de Aminoácidos , Animais , Células CHO , Membrana Celular/metabolismo , Sequência Conservada , Cricetinae , Citosol , Eletrofisiologia , Ativação Enzimática , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/genética , Dados de Sequência Molecular , Mutação/genética , Técnicas de Patch-Clamp , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Monoéster Fosfórico Hidrolases , Fosforilação , Proteínas Tirosina Fosfatases não Receptoras , Ratos , Alinhamento de Sequência
19.
PLoS One ; 14(7): e0211670, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31260458

RESUMO

Tolvaptan is the only drug approved to slow cyst growth and preserve kidney function in patients with autosomal dominant polycystic kidney disease (ADPKD). However, its limited efficacy combined with significant side effects underscores the need to identify new and safe therapeutic drug targets to slow progression to end stage kidney disease. We identified Discoidin Domain Receptor 1 (DDR1) as receptor tyrosine kinase upregulated in vivo in 3 mouse models of ADPKD using a novel mass spectrometry approach to identify kinases upregulated in ADPKD. Previous studies demonstrating critical roles for DDR1 to cancer progression, its potential role in the pathogenesis of a variety of other kidney disease, along with the possibility that DDR1 could provide new insight into how extracellular matrix impacts cyst growth led us to study the role of DDR1 in ADPKD pathogenesis. However, genetic deletion of DDR1 using CRISPR/Cas9 failed to slow cyst growth or preserve kidney function in both a rapid and slow mouse model of ADPKD demonstrating that DDR1 does not play a role in PKD pathogenesis and is thus a not viable drug target. In spite of the negative results, our studies will be of interest to the nephrology community as it will prevent others from potentially conducting similar experiments on DDR1 and reinforces the potential of performing unbiased screens coupled with in vivo gene editing using CRISPR/Cas9 to rapidly identify and confirm new potential drug targets for ADPKD.


Assuntos
Receptor com Domínio Discoidina 1/biossíntese , Regulação Enzimológica da Expressão Gênica , Rim/enzimologia , Doenças Renais Policísticas/enzimologia , Regulação para Cima , Animais , Receptor com Domínio Discoidina 1/genética , Modelos Animais de Doenças , Rim/patologia , Camundongos , Camundongos Transgênicos , Doenças Renais Policísticas/genética , Doenças Renais Policísticas/patologia
20.
Kidney Int ; 74(6): 740-9, 2008 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-18547995

RESUMO

Autosomal-dominant polycystic kidney disease (ADPKD) is characterized by numerous fluid-filled kidney cysts. Net fluid secretion into renal cysts is caused by transepithelial transport mediated by the apical cystic fibrosis transmembrane conductance regulator chloride channel, which leads to cyst enlargement. Here we found that forskolin, a potent adenylyl cyclase agonist, stimulated anion secretion by monolayers of kidney cells derived from patients with ADPKD. TRAM-34, a specific KCa3.1 potassium channel blocker, inhibited this current, and in vitro cyst formation and enlargement by the cells cultured within a collagen gel. Net chloride secretion was enhanced by the KCa3.1 activator DCEBIO and both chloride secretion and in vitro cyst growth were inhibited by overexpression of myotubularin-related protein-6, a phosphatase that specifically inhibits KCa3.1 channel activity. Our study suggests that KCa3.1 channels play a critical role in transcellular chloride secretion and net fluid transport into the kidney cysts of patients with ADPKD by maintaining the electrochemical driving force for chloride efflux through apical chloride channels. Pharmacological inhibitors of KCa3.1 channels may provide a novel and effective therapy to delay progression to kidney failure in patients with ADPKD.


Assuntos
Cloretos/metabolismo , AMP Cíclico , Cistos/patologia , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/fisiologia , Rim Policístico Autossômico Dominante/patologia , Animais , Transporte Biológico , Células Cultivadas , AMP Cíclico/agonistas , Líquido Cístico/metabolismo , Cães , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/antagonistas & inibidores , Rim Policístico Autossômico Dominante/metabolismo
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