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1.
Proc Natl Acad Sci U S A ; 116(33): 16463-16472, 2019 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-31346092

RESUMO

Heterozygous in-frame mutations in coding regions of human STAT3 underlie the only known autosomal dominant form of hyper IgE syndrome (AD HIES). About 5% of familial cases remain unexplained. The mutant proteins are loss-of-function and dominant-negative when tested following overproduction in recipient cells. However, the production of mutant proteins has not been detected and quantified in the cells of heterozygous patients. We report a deep intronic heterozygous STAT3 mutation, c.1282-89C>T, in 7 relatives with AD HIES. This mutation creates a new exon in the STAT3 complementary DNA, which, when overexpressed, generates a mutant STAT3 protein (D427ins17) that is loss-of-function and dominant-negative in terms of tyrosine phosphorylation, DNA binding, and transcriptional activity. In immortalized B cells from these patients, the D427ins17 protein was 2 kDa larger and 4-fold less abundant than wild-type STAT3, on mass spectrometry. The patients' primary B and T lymphocytes responded poorly to STAT3-dependent cytokines. These findings are reminiscent of the impaired responses of leukocytes from other patients with AD HIES due to typical STAT3 coding mutations, providing further evidence for the dominance of the mutant intronic allele. These findings highlight the importance of sequencing STAT3 introns in patients with HIES without candidate variants in coding regions and essential splice sites. They also show that AD HIES-causing STAT3 mutant alleles can be dominant-negative even if the encoded protein is produced in significantly smaller amounts than wild-type STAT3.

2.
J Allergy Clin Immunol ; 144(1): 236-253, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30738173

RESUMO

BACKGROUND: Gain-of-function (GOF) mutations in PIK3CD cause a primary immunodeficiency characterized by recurrent respiratory tract infections, susceptibility to herpesvirus infections, and impaired antibody responses. Previous work revealed defects in CD8+ T and B cells that contribute to this clinical phenotype, but less is understood about the role of CD4+ T cells in disease pathogenesis. OBJECTIVE: We sought to dissect the effects of increased phosphoinositide 3-kinase (PI3K) signaling on CD4+ T-cell function. METHODS: We performed detailed ex vivo, in vivo, and in vitro phenotypic and functional analyses of patients' CD4+ T cells and a novel murine disease model caused by overactive PI3K signaling. RESULTS: PI3K overactivation caused substantial increases in numbers of memory and follicular helper T (TFH) cells and dramatic changes in cytokine production in both patients and mice. Furthermore, PIK3CD GOF human TFH cells had dysregulated phenotype and function characterized by increased programmed cell death protein 1, CXCR3, and IFN-γ expression, the phenotype of a TFH cell subset with impaired B-helper function. This was confirmed in vivo in which Pik3cd GOF CD4+ T cells also acquired an aberrant TFH phenotype and provided poor help to support germinal center reactions and humoral immune responses by antigen-specific wild-type B cells. The increase in numbers of both memory and TFH cells was largely CD4+ T-cell extrinsic, whereas changes in cytokine production and TFH cell function were cell intrinsic. CONCLUSION: Our studies reveal that CD4+ T cells with overactive PI3K have aberrant activation and differentiation, thereby providing mechanistic insight into dysfunctional antibody responses in patients with PIK3CD GOF mutations.

3.
Sci Immunol ; 3(30)2018 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-30578351

RESUMO

Hundreds of patients with autosomal recessive, complete IL-12p40 or IL-12Rß1 deficiency have been diagnosed over the last 20 years. They typically suffer from invasive mycobacteriosis and, occasionally, from mucocutaneous candidiasis. Susceptibility to these infections is thought to be due to impairments of IL-12-dependent IFN-γ immunity and IL-23-dependent IL-17A/IL-17F immunity, respectively. We report here patients with autosomal recessive, complete IL-12Rß2 or IL-23R deficiency, lacking responses to IL-12 or IL-23 only, all of whom, unexpectedly, display mycobacteriosis without candidiasis. We show that αß T, γδ T, B, NK, ILC1, and ILC2 cells from healthy donors preferentially produce IFN-γ in response to IL-12, whereas NKT cells and MAIT cells preferentially produce IFN-γ in response to IL-23. We also show that the development of IFN-γ-producing CD4+ T cells, including, in particular, mycobacterium-specific TH1* cells (CD45RA-CCR6+), is dependent on both IL-12 and IL-23. Last, we show that IL12RB1, IL12RB2, and IL23R have similar frequencies of deleterious variants in the general population. The comparative rarity of symptomatic patients with IL-12Rß2 or IL-23R deficiency, relative to IL-12Rß1 deficiency, is, therefore, due to lower clinical penetrance. There are fewer symptomatic IL-23R- and IL-12Rß2-deficient than IL-12Rß1-deficient patients, not because these genetic disorders are rarer, but because the isolated absence of IL-12 or IL-23 is, in part, compensated by the other cytokine for the production of IFN-γ, thereby providing some protection against mycobacteria. These experiments of nature show that human IL-12 and IL-23 are both required for optimal IFN-γ-dependent immunity to mycobacteria, both individually and much more so cooperatively.


Assuntos
Imunidade Inata/imunologia , Interferon gama/imunologia , Interleucina-12/imunologia , Interleucina-23/imunologia , Infecções por Micobactéria não Tuberculosa/imunologia , Mycobacterium/imunologia , Humanos , Interleucina-12/deficiência , Interleucina-12/genética , Interleucina-23/deficiência , Interleucina-23/genética , Linhagem
4.
Sci Immunol ; 3(30)2018 12 21.
Artigo em Inglês | MEDLINE | ID: mdl-30578352

RESUMO

Inherited IL-12Rß1 and TYK2 deficiencies impair both IL-12- and IL-23-dependent IFN-γ immunity and are rare monogenic causes of tuberculosis, each found in less than 1/600,000 individuals. We show that homozygosity for the common TYK2 P1104A allele, which is found in about 1/600 Europeans and between 1/1000 and 1/10,000 individuals in regions other than East Asia, is more frequent in a cohort of patients with tuberculosis from endemic areas than in ethnicity-adjusted controls (P = 8.37 × 10-8; odds ratio, 89.31; 95% CI, 14.7 to 1725). Moreover, the frequency of P1104A in Europeans has decreased, from about 9% to 4.2%, over the past 4000 years, consistent with purging of this variant by endemic tuberculosis. Surprisingly, we also show that TYK2 P1104A impairs cellular responses to IL-23, but not to IFN-α, IL-10, or even IL-12, which, like IL-23, induces IFN-γ via activation of TYK2 and JAK2. Moreover, TYK2 P1104A is properly docked on cytokine receptors and can be phosphorylated by the proximal JAK, but lacks catalytic activity. Last, we show that the catalytic activity of TYK2 is essential for IL-23, but not IL-12, responses in cells expressing wild-type JAK2. In contrast, the catalytic activity of JAK2 is redundant for both IL-12 and IL-23 responses, because the catalytically inactive P1057A JAK2, which is also docked and phosphorylated, rescues signaling in cells expressing wild-type TYK2. In conclusion, homozygosity for the catalytically inactive P1104A missense variant of TYK2 selectively disrupts the induction of IFN-γ by IL-23 and is a common monogenic etiology of tuberculosis.


Assuntos
Interferon gama/imunologia , Interleucina-23/imunologia , Mutação de Sentido Incorreto/genética , TYK2 Quinase/genética , Tuberculose/imunologia , Células Cultivadas , Homozigoto , Humanos , Interleucina-23/deficiência , TYK2 Quinase/imunologia
5.
Nat Immunol ; 19(9): 973-985, 2018 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30127434

RESUMO

Human inborn errors of IFN-γ immunity underlie mycobacterial diseases. We describe patients with Mycobacterium bovis (BCG) disease who are homozygous for loss-of-function mutations of SPPL2A. This gene encodes a transmembrane protease that degrades the N-terminal fragment (NTF) of CD74 (HLA invariant chain) in antigen-presenting cells. The CD74 NTF therefore accumulates in the HLA class II+ myeloid and lymphoid cells of SPPL2a-deficient patients. This toxic fragment selectively depletes IL-12- and IL-23-producing CD1c+ conventional dendritic cells (cDC2s) and their circulating progenitors. Moreover, SPPL2a-deficient memory TH1* cells selectively fail to produce IFN-γ when stimulated with mycobacterial antigens in vitro. Finally, Sppl2a-/- mice lack cDC2s, have CD4+ T cells that produce small amounts of IFN-γ after BCG infection, and are highly susceptible to infection with BCG or Mycobacterium tuberculosis. These findings suggest that inherited SPPL2a deficiency in humans underlies mycobacterial disease by decreasing the numbers of cDC2s and impairing IFN-γ production by mycobacterium-specific memory TH1* cells.

6.
Sci Immunol ; 3(24)2018 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-29907691

RESUMO

Heterozygosity for human signal transducer and activator of transcription 3 (STAT3) dominant-negative (DN) mutations underlies an autosomal dominant form of hyper-immunoglobulin E syndrome (HIES). We describe patients with an autosomal recessive form of HIES due to loss-of-function mutations of a previously uncharacterized gene, ZNF341 ZNF341 is a transcription factor that resides in the nucleus, where it binds a specific DNA motif present in various genes, including the STAT3 promoter. The patients' cells have low basal levels of STAT3 mRNA and protein. The autoinduction of STAT3 production, activation, and function by STAT3-activating cytokines is strongly impaired. Like patients with STAT3 DN mutations, ZNF341-deficient patients lack T helper 17 (TH17) cells, have an excess of TH2 cells, and have low memory B cells due to the tight dependence of STAT3 activity on ZNF341 in lymphocytes. Their milder extra-hematopoietic manifestations and stronger inflammatory responses reflect the lower ZNF341 dependence of STAT3 activity in other cell types. Human ZNF341 is essential for the STAT3 transcription-dependent autoinduction and sustained activity of STAT3.

7.
Biochim Biophys Acta Mol Cell Biol Lipids ; 1863(4): 359-368, 2018 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-29306077

RESUMO

The ABC lipid transporters, ABCA1 and ABCG1, are essential for maintaining lipid homeostasis in cells such as macrophages by exporting excess cholesterol to extracellular acceptors. These transporters are highly regulated at the post-translational level, including protein ubiquitination. Our aim was to investigate the role of the E3 ubiquitin ligase HECTD1, recently identified as associated with ABCG1, on ABCG1 and ABCA1 protein levels and cholesterol export function. Here, we show that HECTD1 protein is widely expressed in a range of human and murine primary cells and cell lines, including macrophages, neuronal cells and insulin secreting ß-cells. siRNA knockdown of HECTD1 unexpectedly decreased overexpressed ABCG1 protein levels and cell growth, but increased native ABCA1 protein in CHO-K1 cells. Knockdown of HECTD1 in unloaded THP-1 macrophages did not affect ABCG1 but significantly increased ABCA1 protein levels, in wild-type as well as THP-1 cells that do not express ABCG1. Cholesterol export from macrophages to apoA-I over time was increased after knockdown of HECTD1, however these effects were not sustained in cholesterol-loaded cells. In conclusion, we have identified a new candidate, the E3 ubiquitin ligase HECTD1, that may be involved in the regulation of ABCA1-mediated cholesterol export from unloaded macrophages to apoA-I. The exact mechanism by which this ligase affects this pathway remains to be elucidated.


Assuntos
Transportador 1 de Cassete de Ligação de ATP/metabolismo , Colesterol/metabolismo , Macrófagos/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Transportador 1 de Cassete de Ligação de ATP/genética , Animais , Apolipoproteína A-I/metabolismo , Transporte Biológico , Células CHO , Proliferação de Células , Cricetinae , Cricetulus , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Imunoprecipitação , Receptores X do Fígado/metabolismo , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Ubiquitina-Proteína Ligases/genética
8.
Oncotarget ; 7(37): 59220-59235, 2016 Sep 13.
Artigo em Inglês | MEDLINE | ID: mdl-27494876

RESUMO

MICA/B (the major histocompatibility antigen-related chain A and B) and Rae I are stress-inducible ligands for the immune-receptor NKG2D. Mechanisms by which genotoxic stress and DNA damage induce the expression of NKG2D ligands remain incompletely understood. Here, we report that inhibition of xanthine oxidoreductase (XOR) activity by allopurinol or inhibition of XOR expression by gene knockdown abrogated genotoxic stress-induced expression of MICA/B and Rae I in three tumor cell lines. XOR knockdown also blocked gemcitabine-mediated antitumor activity in an orthotopic syngeneic mouse model of breast cancer. As a rate-limiting enzyme in the purine catabolic pathway, XOR generates two end-products, uric acid and reactive oxygen species (ROS). ROS scavenging had an insignificant effect on genotoxic drug-induced MICA/B expression but modestly inhibited radiation-induced MICA/B expression. Exogenous uric acid (in the form of monosodium urate) induced MICA/B expression by activating the MAP kinase pathway. Allopurinol blocked genotoxic stress-induced MAP kinase activation. Our study provides mechanistic insights into genotoxic stress-induced activation of the MAP kinase pathway and suggests that XOR is required for genotoxic stress-induced NKG2D ligand expression and gemcitabine-mediated antitumor activity.


Assuntos
Neoplasias da Mama/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Proteínas Associadas à Matriz Nuclear/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Xantina Desidrogenase/metabolismo , Alopurinol/farmacologia , Animais , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Dano ao DNA , Feminino , Regulação Neoplásica da Expressão Gênica , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Células Matadoras Naturais/imunologia , Sistema de Sinalização das MAP Quinases , Camundongos , Subfamília K de Receptores Semelhantes a Lectina de Células NK/metabolismo , Proteínas Associadas à Matriz Nuclear/genética , Proteínas de Transporte Nucleocitoplasmático/genética , RNA Interferente Pequeno/genética , Xantina Desidrogenase/antagonistas & inibidores , Xantina Desidrogenase/genética , Ensaios Antitumorais Modelo de Xenoenxerto
9.
J Exp Med ; 213(8): 1589-608, 2016 07 25.
Artigo em Inglês | MEDLINE | ID: mdl-27401342

RESUMO

Naive CD4(+) T cells differentiate into specific effector subsets-Th1, Th2, Th17, and T follicular helper (Tfh)-that provide immunity against pathogen infection. The signaling pathways involved in generating these effector cells are partially known. However, the effects of mutations underlying human primary immunodeficiencies on these processes, and how they compromise specific immune responses, remain unresolved. By studying individuals with mutations in key signaling pathways, we identified nonredundant pathways regulating human CD4(+) T cell differentiation in vitro. IL12Rß1/TYK2 and IFN-γR/STAT1 function in a feed-forward loop to induce Th1 cells, whereas IL-21/IL-21R/STAT3 signaling is required for Th17, Tfh, and IL-10-secreting cells. IL12Rß1/TYK2 and NEMO are also required for Th17 induction. Strikingly, gain-of-function STAT1 mutations recapitulated the impact of dominant-negative STAT3 mutations on Tfh and Th17 cells, revealing a putative inhibitory effect of hypermorphic STAT1 over STAT3. These findings provide mechanistic insight into the requirements for human T cell effector function, and explain clinical manifestations of these immunodeficient conditions. Furthermore, they identify molecules that could be targeted to modulate CD4(+) T cell effector function in the settings of infection, vaccination, or immune dysregulation.


Assuntos
Diferenciação Celular/imunologia , Células Th1/imunologia , Células Th17/imunologia , Células Th2/imunologia , Antígenos de Diferenciação/genética , Antígenos de Diferenciação/imunologia , Diferenciação Celular/genética , Feminino , Humanos , Interleucina-10/genética , Interleucina-10/imunologia , Masculino , Mutação , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT1/imunologia , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/imunologia , Células Th1/citologia , Células Th17/citologia
10.
J Exp Med ; 212(10): 1641-62, 2015 Sep 21.
Artigo em Inglês | MEDLINE | ID: mdl-26304966

RESUMO

Autosomal recessive, complete TYK2 deficiency was previously described in a patient (P1) with intracellular bacterial and viral infections and features of hyper-IgE syndrome (HIES), including atopic dermatitis, high serum IgE levels, and staphylococcal abscesses. We identified seven other TYK2-deficient patients from five families and four different ethnic groups. These patients were homozygous for one of five null mutations, different from that seen in P1. They displayed mycobacterial and/or viral infections, but no HIES. All eight TYK2-deficient patients displayed impaired but not abolished cellular responses to (a) IL-12 and IFN-α/ß, accounting for mycobacterial and viral infections, respectively; (b) IL-23, with normal proportions of circulating IL-17(+) T cells, accounting for their apparent lack of mucocutaneous candidiasis; and (c) IL-10, with no overt clinical consequences, including a lack of inflammatory bowel disease. Cellular responses to IL-21, IL-27, IFN-γ, IL-28/29 (IFN-λ), and leukemia inhibitory factor (LIF) were normal. The leukocytes and fibroblasts of all seven newly identified TYK2-deficient patients, unlike those of P1, responded normally to IL-6, possibly accounting for the lack of HIES in these patients. The expression of exogenous wild-type TYK2 or the silencing of endogenous TYK2 did not rescue IL-6 hyporesponsiveness, suggesting that this phenotype was not a consequence of the TYK2 genotype. The core clinical phenotype of TYK2 deficiency is mycobacterial and/or viral infections, caused by impaired responses to IL-12 and IFN-α/ß. Moreover, impaired IL-6 responses and HIES do not appear to be intrinsic features of TYK2 deficiency in humans.


Assuntos
Síndrome de Job/etiologia , TYK2 Quinase/deficiência , Adolescente , Estudos de Casos e Controles , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Interferon gama/metabolismo , Interleucina-10/farmacologia , Interleucina-12/metabolismo , Interleucina-12/farmacologia , Interleucina-23/farmacologia , Interleucina-6/farmacologia , Síndrome de Job/complicações , Síndrome de Job/genética , Leucócitos/efeitos dos fármacos , Leucócitos/metabolismo , Masculino , Mutação , Infecções por Mycobacterium/etiologia , Linfócitos T/metabolismo , Linfócitos T/patologia , TYK2 Quinase/genética , TYK2 Quinase/metabolismo , Viroses/etiologia , Adulto Jovem
11.
J Biol Chem ; 290(40): 24604-13, 2015 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-26296893

RESUMO

The ATP-binding cassette transporter ABCG1 has an essential role in cellular cholesterol homeostasis, and dysregulation has been associated with a number of high burden diseases. Previous studies reported that ABCG1 is ubiquitinated and degraded via the ubiquitin proteasome system. However, so far the molecular mechanism, including the identity of any of the rate-limiting ubiquitination enzymes, or E3 ligases, is unknown. Using liquid chromatography mass spectrometry, we identified two HECT domain E3 ligases associated with ABCG1, named HUWE1 (HECT, UBA, and WWE domain containing 1, E3 ubiquitin protein ligase) and NEDD4-1 (Neural precursor cell-expressed developmentally down regulated gene 4), of which the latter is the founding member of the NEDD4 family of ubiquitin ligases. Silencing both HUWE1 and NEDD4-1 in cells overexpressing human ABCG1 significantly increased levels of the ABCG1 monomeric and dimeric protein forms, however ABCA1 protein expression was unaffected. In addition, ligase silencing increased ABCG1-mediated cholesterol export to HDL in cells overexpressing the transporter as well as in THP-1 macrophages. Reciprocally, overexpression of both ligases resulted in a significant reduction in protein levels of both the ABCG1 monomeric and dimeric forms. Like ABCG1, ABCG4 protein levels and cholesterol export activity were significantly increased after silencing both HUWE1 and NEDD4-1 in cells overexpressing this closely related ABC half-transporter. In summary, we have identified for the first time two E3 ligases that are fundamental enzymes in the post-translational regulation of ABCG1 and ABCG4 protein levels and cellular cholesterol export activity.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Lipídeos/química , Ubiquitina-Proteína Ligases/metabolismo , Subfamília G de Transportadores de Cassetes de Ligação de ATP , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Animais , Transporte Biológico , Células CHO , Linhagem Celular , Colesterol/química , Cromatografia Líquida , Cricetulus , Regulação da Expressão Gênica , Inativação Gênica , Humanos , Macrófagos/metabolismo , Espectrometria de Massas , Ubiquitina-Proteína Ligases Nedd4 , Ligação Proteica , Multimerização Proteica , Estrutura Terciária de Proteína , Transporte Proteico , RNA Interferente Pequeno/metabolismo , Proteínas Supressoras de Tumor
12.
J Allergy Clin Immunol ; 136(4): 993-1006.e1, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26162572

RESUMO

BACKGROUND: Follicular helper T (TFH) cells underpin T cell-dependent humoral immunity and the success of most vaccines. TFH cells also contribute to human immune disorders, such as autoimmunity, immunodeficiency, and malignancy. Understanding the molecular requirements for the generation and function of TFH cells will provide strategies for targeting these cells to modulate their behavior in the setting of these immunologic abnormalities. OBJECTIVE: We sought to determine the signaling pathways and cellular interactions required for the development and function of TFH cells in human subjects. METHODS: Human primary immunodeficiencies (PIDs) resulting from monogenic mutations provide a unique opportunity to assess the requirement for particular molecules in regulating human lymphocyte function. Circulating follicular helper T (cTFH) cell subsets, memory B cells, and serum immunoglobulin levels were quantified and functionally assessed in healthy control subjects, as well as in patients with PIDs resulting from mutations in STAT3, STAT1, TYK2, IL21, IL21R, IL10R, IFNGR1/2, IL12RB1, CD40LG, NEMO, ICOS, or BTK. RESULTS: Loss-of-function (LOF) mutations in STAT3, IL10R, CD40LG, NEMO, ICOS, or BTK reduced cTFH cell frequencies. STAT3 and IL21/R LOF and STAT1 gain-of-function mutations skewed cTFH cell differentiation toward a phenotype characterized by overexpression of IFN-γ and programmed death 1. IFN-γ inhibited cTFH cell function in vitro and in vivo, as corroborated by hypergammaglobulinemia in patients with IFNGR1/2, STAT1, and IL12RB1 LOF mutations. CONCLUSION: Specific mutations affect the quantity and quality of cTFH cells, highlighting the need to assess TFH cells in patients by using multiple criteria, including phenotype and function. Furthermore, IFN-γ functions in vivo to restrain TFH cell-induced B-cell differentiation. These findings shed new light on TFH cell biology and the integrated signaling pathways required for their generation, maintenance, and effector function and explain the compromised humoral immunity seen in patients with some PIDs.


Assuntos
Síndromes de Imunodeficiência/imunologia , Subpopulações de Linfócitos T/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Tirosina Quinase da Agamaglobulinemia , Linfócitos B/imunologia , Ligante de CD40/genética , Diferenciação Celular/genética , Proliferação de Células/genética , Células Cultivadas , Humanos , Quinase I-kappa B/genética , Imunidade Humoral/genética , Síndromes de Imunodeficiência/genética , Memória Imunológica , Proteína Coestimuladora de Linfócitos T Induzíveis/genética , Interferon gama/genética , Interferon gama/metabolismo , Ativação Linfocitária , Mutação/genética , Proteínas Tirosina Quinases/genética , Receptores de Citocinas/genética , Fator de Transcrição STAT1/genética , Fator de Transcrição STAT3/genética , Transdução de Sinais/genética , Transdução de Sinais/imunologia
13.
J Exp Med ; 212(6): 855-64, 2015 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-25941256

RESUMO

Unconventional T cells such as γδ T cells, natural killer T cells (NKT cells) and mucosal-associated invariant T cells (MAIT cells) are a major component of the immune system; however, the cytokine signaling pathways that control their development and function in humans are unknown. Primary immunodeficiencies caused by single gene mutations provide a unique opportunity to investigate the role of specific molecules in regulating human lymphocyte development and function. We found that individuals with loss-of-function mutations in STAT3 had reduced numbers of peripheral blood MAIT and NKT but not γδ T cells. Analysis of STAT3 mosaic individuals revealed that this effect was cell intrinsic. Surprisingly, the residual STAT3-deficient MAIT cells expressed normal levels of the transcription factor RORγt. Despite this, they displayed a deficiency in secretion of IL-17A and IL-17F, but were able to secrete normal levels of cytokines such as IFNγ and TNF. The deficiency in MAIT and NKT cells in STAT3-deficient patients was mirrored by loss-of-function mutations in IL12RB1 and IL21R, respectively. Thus, these results reveal for the first time the essential role of STAT3 signaling downstream of IL-23R and IL-21R in controlling human MAIT and NKT cell numbers.


Assuntos
Fator de Transcrição STAT3/metabolismo , Linfócitos T/citologia , Separação Celular , Citocinas/metabolismo , Citometria de Fluxo , Humanos , Interferon gama/metabolismo , Interleucina-17/metabolismo , Interleucina-23/metabolismo , Interleucinas/metabolismo , Células Matadoras Naturais/citologia , Leucócitos Mononucleares/citologia , Linfócitos/citologia , Mutação , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Polimorfismo de Nucleotídeo Único , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Receptores de Interleucina/metabolismo , Receptores de Interleucina-12/metabolismo , Receptores de Interleucina-21/metabolismo , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismo
14.
Biochim Biophys Acta ; 1851(7): 92017 Apr, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25732853

RESUMO

The ATP-binding cassette (ABC) transporter, ABCG1, is a lipid exporter involved in removal of cholesterol from cells that has been investigated for its role in foam cells formation and atherosclerosis. The mechanism by which ABC lipid transporters bind and recognise their substrates is currently unknown. In this study, we identify a critical region in the final transmembrane domain of ABCG1, which is essential for its export function and stabilisation by cholesterol, a post-translational regulatory mechanism that we have recently identified as dependent on protein ubiquitination. This transmembrane region contains several Cholesterol Recognition/interaction Amino acid Consensus (CRAC) motifs, and its inverse CARC motifs. Mutational analyses identify one CRAC motif in particular with Y667 at its core, that is especially important for transport activity to HDL as well as stability of the protein in the presence of cholesterol. In addition, we present a model of how cholesterol docks to this CRAC motif in an energetically favourable manner. This study identifies for the first time how ABCG1 can interact with cholesterol via a functional CRAC domain, which provides the first insight into the substrate-transporter interaction of an ABC lipid exporter.


Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Transportadores de Cassetes de Ligação de ATP/fisiologia , Colesterol/metabolismo , Domínios e Motivos de Interação entre Proteínas , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Sequência de Aminoácidos , Animais , Transporte Biológico/genética , Células CHO , Membrana Celular/metabolismo , Cricetinae , Cricetulus , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica/genética , Domínios e Motivos de Interação entre Proteínas/genética , Estrutura Secundária de Proteína
15.
Neoplasia ; 16(10): 824-34, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25379019

RESUMO

Leflunomide is a novel immunomodulatory drug prescribed for treating rheumatoid arthritis. It inhibits the activity of protein tyrosine kinases and dihydroorotate dehydrogenase, a rate-limiting enzyme in the pyrimidine nucleotide synthesis pathway. Here, we report that A77 1726, the active metabolite of leflunomide, inhibited the phosphorylation of ribosomal protein S6 and two other substrates of S6K1, insulin receptor substrate-1 and carbamoyl phosphate synthetase 2, in an A375 melanoma cell line. A77 1726 increased the phosphorylation of AKT, p70 S6 (S6K1), ERK1/2, and MEK through the feedback activation of the IGF-1 receptor-mediated signaling pathway. In vitro kinase assay revealed that leflunomide and A77 1726 inhibited S6K1 activity with IC50 values of approximately 55 and 80 µM, respectively. Exogenous uridine partially blocked A77 1726-induced inhibition of A375 cell proliferation. S6K1 knockdown led to the inhibition of A375 cell proliferation but did not potentiate the antiproliferative effect of A77 1726. A77 1726 stimulated bromodeoxyuridine incorporation in A375 cells but arrested the cell cycle in the S phase, which was reversed by addition of exogenous uridine or by MAP kinase pathway inhibitors but not by rapamycin and LY294002 (a phosphoinositide 3-kinase inhibitor). These observations suggest that A77 1726 accelerates cell cycle entry into the S phase through MAP kinase activation and that pyrimidine nucleotide depletion halts the completion of the cell cycle. Our study identified a novel molecular target of A77 1726 and showed that the inhibition of S6K1 activity was in part responsible for its antiproliferative activity. Our study also provides a novel mechanistic insight into A77 1726-induced cell cycle arrest in the S phase.


Assuntos
Compostos de Anilina/farmacologia , Ciclo Celular/efeitos dos fármacos , Hidroxibutiratos/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases S6 Ribossômicas 70-kDa/antagonistas & inibidores , Butadienos/farmacologia , Linhagem Celular Tumoral/efeitos dos fármacos , Linhagem Celular Tumoral/metabolismo , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Cromonas/farmacologia , Retroalimentação Fisiológica , Técnicas de Silenciamento de Genes , Humanos , Imidazóis/farmacologia , Isoxazóis/farmacologia , Leflunomida , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Morfolinas/farmacologia , Nitrilos/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Pirazinas/farmacologia , Receptor IGF Tipo 1/antagonistas & inibidores , Receptor IGF Tipo 1/metabolismo , Proteínas Quinases S6 Ribossômicas 70-kDa/genética , Proteínas Quinases S6 Ribossômicas 70-kDa/metabolismo
16.
PLoS One ; 8(9): e67895, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24069135

RESUMO

The role of mesenchymal stem cells (MSCs) on breast cancer progression, growth and tumorigenesis remains controversial or unknown. In the present study, we investigated the role of MSCs on breast tumor induction and growth in a clinically relevant somatic breast cancer model. We first conducted in vitro studies and found that conditioned media (CM) of RCAS-Neu and RCAS-PyMT breast cancer cell lines and tumor cells themselves dramatically increased the proliferation and motility of MSCs and induced morphological changes of MSCs and differentiation into fibroblast-like cells. In contrast, the CM of MSCs inhibited the proliferation of two breast cancer cell lines by arresting the cell cycle at the G0/G1 phase. In vivo studies revealed that fluorescence dye-labeled MSCs migrated into tumor tissues. Unexpectedly, single or multiple intravenous injections of MSCs did not affect the latency of breast cancer in TVA- transgenic mice induced by intraductal injection of the RCAS vector encoding polyoma middle-T antigen (PyMT) or Neu oncogenes. Moreover, MSCs had no effect on RCAS-Neu tumor growth in a syngeneic ectopic breast cancer model. While our studies consistently demonstrated the ability of breast cancer cells to profoundly induce MSCs migration, differentiation, and proliferation, the anti-proliferative effect of MSCs on breast tumor cells observed in vitro could not be translated into an antitumor activity in vivo, probably reflecting the antagonizing or complex effects of MSCs on tumor environment and tumor cells themselves.


Assuntos
Neoplasias da Mama/patologia , Células-Tronco Mesenquimais/patologia , Animais , Western Blotting , Neoplasias da Mama/metabolismo , Diferenciação Celular/fisiologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células , Células Cultivadas , Feminino , Imunofluorescência , Camundongos , Camundongos Transgênicos
17.
J Lipid Res ; 53(10): 2133-40, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-22872754

RESUMO

ABCG1 is an ABC half-transporter that exports cholesterol from cells to HDL. This study set out to investigate differences in posttranslational processing of two human ABCG1 protein isoforms, termed ABCG1(+12) and ABCG1(-12), that differ by the presence or absence of a 12 amino acid peptide. ABCG1(+12) is expressed in human cells and tissues, but not in mice. We identified two protein kinase A (PKA) consensus sites in ABCG1(+12), absent from ABCG1(-12). Inhibition of PKA with either of two structurally unrelated inhibitors resulted in a dose-dependent increase in cholesterol export from cells expressing ABCG1(+12), whereas ABCG1(-12)-expressing cells were unaffected. This was associated with stabilization of the ABCG1(+12) protein, and ABCG1(+12)-S389 was necessary to mediate these effects. Mutation of this serine to aspartic acid, simulating a constitutively phosphorylated state, resulted in accelerated degradation of ABCG1(+12) and reduced cholesterol export. Engineering an equivalent PKA site into ABCG1(-12) rendered this isoform responsive to PKA inhibition, confirming the relevance of this sequence. Together, these results demonstrate an additional level of complexity to the posttranslational control of this human ABCG1 isoform that is absent from ABCG1(-12) and the murine ABCG1 homolog.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Células CHO , Sobrevivência Celular , Colesterol/metabolismo , Cricetinae , Humanos , Macrófagos , Camundongos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RNA Mensageiro/metabolismo
18.
Endocr Relat Cancer ; 19(2): 167-79, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22241722

RESUMO

The sonic hedgehog (SHH) pathway is activated in several types of malignancy and plays an important role in tumor cell proliferation and tumorigenesis. SHH binding to a 12-pass transmembrane receptor, Patched (PTCH), leads to freeing of Smoothened (SMO) and subsequent activation of GLI transcription factors. In the present study, we analyzed the expression of SHH, PTCH, SMO, and GLI1 in 31 follicular thyroid adenomas (FTA), 8 anaplastic thyroid carcinomas (ATC), and 51 papillary thyroid carcinomas (PTC) by immunohistochemical staining. More than 65% of FTA, PTC, and ATC specimens stained positive for SHH, PTCH, SMO, and GLI. However, the expression of the genes encoding these four molecules did not correlate with any clinicopathologic parameters, including the age, gender, the status of BRAF gene mutation, tumor stage, local invasion, and metastasis. Three thyroid tumor cell lines (KAT-18, WRO82, and SW1736) all expressed the genes encoding these four molecules. 5-Bromo-2-deoxyuridine labeling and MTT cell proliferation assays revealed that cyclopamine (CP), an inhibitor of the SHH pathway, was able to inhibit the proliferation of KAT-18 and WRO82 cells more effectively than SW1736 cells. CP led to the arrest of cell cycle or apoptosis. Knockdown of SHH and GLI expression by miRNA constructs that target SHH or GLI mRNA in KAT-18 and SW1736 cells led to the inhibition of cell proliferation. Our results suggest that the SHH pathway is widely activated in thyroid neoplasms and may have potential as an early marker of thyroid cancer or as a potential therapeutic target for thyroid cancer treatment.


Assuntos
Proteínas Hedgehog/metabolismo , Neoplasias da Glândula Tireoide/metabolismo , Adenoma/genética , Adenoma/metabolismo , Adenoma/patologia , Western Blotting , Carcinoma/metabolismo , Carcinoma/patologia , Carcinoma Papilar/genética , Carcinoma Papilar/metabolismo , Carcinoma Papilar/patologia , Processos de Crescimento Celular/fisiologia , Linhagem Celular Tumoral , Distribuição de Qui-Quadrado , Feminino , Citometria de Fluxo , Proteínas Hedgehog/genética , Humanos , Masculino , MicroRNAs/farmacologia , Receptores Patched , Receptor Patched-1 , RNA Neoplásico/química , RNA Neoplásico/genética , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Receptores Acoplados a Proteínas-G/genética , Receptores Acoplados a Proteínas-G/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Receptor Smoothened , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/patologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Alcaloides de Veratrum/farmacologia , Proteína GLI1 em Dedos de Zinco
19.
BMC Cancer ; 11: 194, 2011 May 23.
Artigo em Inglês | MEDLINE | ID: mdl-21605422

RESUMO

BACKGROUND: Major histocompatibility complex class I-related chain A and B (MICA/B) are two stress-inducible ligands that bind the immunoreceptor NKG2D and play an important role in mediating the cyotoxicity of NK and T cells. In this study, we sought to study MICA/B expression in pancreatic cancer and to determine whether and how genotoxic drugs such as gemcitabine can affect MICA/B expression and natural killer cytotoxity. METHODS: Seven pancreatic cancer cell lines were analyzed for MICA/B expression by flow cytometry and for their sensitivity to NK-92 cell killing by a 51Cr release assay. MICA/B expression in tumor tissues and sera of pancreatic cancer was analyzed by immunohistochemical staining (IHC) and ELISA, respectively. RESULTS: Two MICA/B-positive cell lines were sensitive to the cytotoxic activity of NK-92 cells. Other two MICA/B-positive cell lines and three MICA/B-negative cell lines were resistant to NK-92 cell killing. MICA/B expression was positive in 17 of 25 (68%) pancreatic ductal adenocarcinomas but not in normal pancreatic ductal epithelial cells. Serum MICA/B levels were significantly elevated in patients with pancreatic adenocarcinomas but did not correlate with the stage of pancreatic cancer and patient survival. Gemcitabine therapy led to increased serum MICA levels in 6 of 10 patients with detectable serum MICA. Allopurinol, an inhibitor of xanthine oxidoreductase that converts xanthine to uric acid, blocked uric acid production, MICA/B expression, and sensitivity to NK-92 cell killing toward a PANC-1 cancer cell line exposed to radiation and two genotoxic drugs, gemcitabine and 5-fluorouracil. CONCLUSIONS: The levels of MICA/B expression in serum and tissue of pancreatic cancer are elevated. DNA damage-induced MICA/B expression is mediated through increased uric acid production.


Assuntos
Antimetabólitos Antineoplásicos/uso terapêutico , Carcinoma Ductal Pancreático/metabolismo , Desoxicitidina/análogos & derivados , Antígenos de Histocompatibilidade Classe I/metabolismo , Neoplasias Pancreáticas/metabolismo , Ácido Úrico/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Alopurinol/farmacologia , Antimetabólitos Antineoplásicos/farmacologia , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/patologia , Morte Celular , Linhagem Celular Tumoral , Citotoxicidade Imunológica , Dano ao DNA , Desoxicitidina/farmacologia , Desoxicitidina/uso terapêutico , Feminino , Neoplasias da Vesícula Biliar/metabolismo , Neoplasias da Vesícula Biliar/secundário , Expressão Gênica/efeitos dos fármacos , Antígenos de Histocompatibilidade Classe I/sangue , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Estimativa de Kaplan-Meier , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/secundário , Masculino , Pessoa de Meia-Idade , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/patologia , Neoplasias Peritoneais/metabolismo , Neoplasias Peritoneais/secundário , Xantina Desidrogenase/antagonistas & inibidores
20.
Neoplasia ; 12(11): 946-56, 2010 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21076620

RESUMO

Heparanase-1 (HPR1), an endoglycosidase that specifically degrades heparan sulfate (HS) proteoglycans, is overexpressed in a variety of malignancies. Our present study sought to determine whether oncogene BRAF and RAS mutations lead to increased HPR1 expression. Reverse transcription-polymerase chain reaction analysis revealed that HPR1 gene expression was increased in HEK293 cells transiently transfected with a mutant BRAF or RAS gene. Flow cytometric analysis revealed that B-Raf activation led to loss of the cell surface HS, which could be blocked by two HPR1 inhibitors: heparin and PI-88. Cotransfection of a BRAF or RAS mutant gene with HPR1 promoter-driven luciferase reporters increased luciferase reporter gene expression in HEK293 cells. Knockdown of BRAF expression in a BRAF-mutated KAT-10 tumor cell line led to the suppression of HPR1 gene expression, subsequently leading to increased cell surface HS levels. Truncational and mutational analyses of the HPR1 promoter revealed that the Ets-relevant elements in the HPR1 promoter were critical for BRAF activation-induced HPR1 expression. Luciferase reporter gene expression driven by a four-copy GA binding protein (GABP) binding site was significantly lower in BRAF siRNA-transfected KAT-10 cells than in the control siRNA-transfected cells. We further showed that BRAF knockdown led to suppression of the expression of the GABPß, an Ets family transcription factor involved in regulating HPR1 promoter activity. Taken together, our study suggests that B-Raf kinase activation plays an important role in regulating HPR1 expression. Increased HPR1 expression may contribute to the aggressive behavior of BRAF-mutated cancer.


Assuntos
Glucuronidase/genética , Mutação , Proteínas Proto-Oncogênicas B-raf/genética , Sítios de Ligação/genética , Western Blotting , Linhagem Celular Tumoral , Ativação Enzimática , Fator de Transcrição de Proteínas de Ligação GA/genética , Fator de Transcrição de Proteínas de Ligação GA/metabolismo , Regulação Enzimológica da Expressão Gênica , Glucuronidase/antagonistas & inibidores , Glucuronidase/metabolismo , Células HEK293 , Heparina/farmacologia , Heparitina Sulfato/metabolismo , Humanos , Luciferases/genética , Luciferases/metabolismo , Microscopia de Fluorescência , Oligossacarídeos/farmacologia , Regiões Promotoras Genéticas/genética , Proteínas Proto-Oncogênicas B-raf/metabolismo , Interferência de RNA , Elementos de Resposta/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas ras/genética , Proteínas ras/metabolismo
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