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1.
Emerg Microbes Infect ; 8(1): 734-748, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31130074

RESUMO

Many pathogens infect hosts through various immune evasion strategies. However, the molecular mechanisms by which pathogen proteins modulate and evade the host immune response remain unclear. Enterohemorrhagic Escherichia coli (EHEC) is a pathological strain that can induce mitogen-activated protein (MAP) kinase (Erk, Jnk and p38 MAPK) and NF-κB pathway activation and proinflammatory cytokine production, which then causes diarrheal diseases such as hemorrhagic colitis and hemolytic uremic syndrome. Transforming growth factor ß-activated kinase-1 (TAK1) is a key regulator involved in distinct innate immune signalling pathways. Here we report that EHEC translocated intimin receptor (Tir) protein inhibits the expression of EHEC-induced proinflammatory cytokines by interacting with the host tyrosine phosphatase SHP-1, which is dependent on the phosphorylation of immunoreceptor tyrosine-based inhibition motifs (ITIMs). Mechanistically, the association of EHEC Tir with SHP-1 facilitated the recruitment of SHP-1 to TAK1 and inhibited TAK1 phosphorylation, which then negatively regulated K63-linked polyubiquitination of TAK1 and downstream signal transduction. Taken together, these results suggest that EHEC Tir negatively regulates proinflammatory responses by inhibiting the activation of TAK1, which is essential for immune evasion and could be a potential target for the treatment of bacterial infection.


Assuntos
Escherichia coli Êntero-Hemorrágica/patogenicidade , Infecções por Escherichia coli/fisiopatologia , Proteínas de Escherichia coli/metabolismo , Interações Hospedeiro-Patógeno , Evasão da Resposta Imune , MAP Quinase Quinase Quinases/antagonistas & inibidores , Receptores de Superfície Celular/metabolismo , Fatores de Virulência/metabolismo , Animais , Infecções por Escherichia coli/microbiologia , Células HEK293 , Humanos , Macrófagos Peritoneais , Camundongos , Camundongos Endogâmicos C57BL , Ligação Proteica , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Células RAW 264.7
2.
Cell Commun Signal ; 17(1): 20, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30823936

RESUMO

BACKGROUND: Shp1, a tyrosine-phosphatase-1 containing the Src-homology 2 (SH2) domain, is involved in inflammatory and immune reactions, where it regulates diverse signalling pathways, usually by limiting cell responses through dephosphorylation of target molecules. Moreover, Shp1 regulates actin dynamics. One Shp1 target is Src, which controls many cellular functions including actin dynamics. Src has been previously shown to be activated by a signalling cascade initiated by the cytosolic-phospholipase A2 (cPLA2) metabolite glycerophosphoinositol 4-phosphate (GroPIns4P), which enhances actin polymerisation and motility. While the signalling cascade downstream Src has been fully defined, the mechanism by which GroPIns4P activates Src remains unknown. METHODS: Affinity chromatography, mass spectrometry and co-immunoprecipitation studies were employed to identify the GroPIns4P-interactors; among these Shp1 was selected for further analysis. The specific Shp1 residues interacting with GroPIns4P were revealed by NMR and validated by site-directed mutagenesis and biophysical methods such as circular dichroism, isothermal calorimetry, fluorescence spectroscopy, surface plasmon resonance and computational modelling. Morphological and motility assays were performed in NIH3T3 fibroblasts. RESULTS: We find that Shp1 is the direct cellular target of GroPIns4P. GroPIns4P directly binds to the Shp1-SH2 domain region (with the crucial residues being Ser 118, Arg 138 and Ser 140) and thereby promotes the association between Shp1 and Src, and the dephosphorylation of the Src-inhibitory phosphotyrosine in position 530, resulting in Src activation. As a consequence, fibroblast cells exposed to GroPIns4P show significantly enhanced wound healing capability, indicating that GroPIns4P has a stimulatory role to activate fibroblast migration. GroPIns4P is produced by cPLA2 upon stimulation by diverse receptors, including the EGF receptor. Indeed, endogenously-produced GroPIns4P was shown to mediate the EGF-induced cell motility. CONCLUSIONS: This study identifies a so-far undescribed mechanism of Shp1/Src modulation that promotes cell motility and that is dependent on the cPLA2 metabolite GroPIns4P. We show that GroPIns4P is required for EGF-induced fibroblast migration and that it is part of a cPLA2/GroPIns4P/Shp1/Src cascade that might have broad implications for studies of immune-inflammatory response and cancer.


Assuntos
Movimento Celular , Receptores ErbB/metabolismo , Fosfatos de Inositol/metabolismo , Fosfolipases A2/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Transdução de Sinais , Quinases da Família src/metabolismo , Animais , Sítios de Ligação , Fator de Crescimento Epidérmico/farmacologia , Camundongos , Células NIH 3T3 , Fosforilação , Ligação Proteica , Proteína Tirosina Fosfatase não Receptora Tipo 6/química , Células RAW 264.7 , Cicatrização , Domínios de Homologia de src
3.
Nat Immunol ; 20(4): 447-457, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30833791

RESUMO

Invariant natural killer T cells (iNKT cells) develop through an incompletely understood process that requires positive selection by CD4+CD8+ double-positive thymocytes and SLAM family receptors (SFRs). Here we found that SFRs promoted the development of iNKT cells by reducing the strength of the T cell antigen receptor (TCR) signal after positive selection. This effect improved the survival of iNKT cells and their responses to antigen. Loss of SFRs upregulated the expression of inhibitory receptors, including PD-1, on iNKT cells to mitigate the deleterious effect of SFR deficiency. The role of SFRs could be mimicked by expression of SLAMF6 alone in SFR-deficient mice, and this involved the adaptor SAP-kinase Fyn complex and the phosphatase SHP-1. Thus, SFRs foster iNKT cell development by attenuating TCR signal strength after positive selection.


Assuntos
Células T Matadoras Naturais/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Família de Moléculas de Sinalização da Ativação Linfocitária/fisiologia , Animais , Proliferação de Células , Sobrevivência Celular , Receptores Coestimuladores e Inibidores de Linfócitos T/metabolismo , Humanos , Camundongos , Camundongos Knockout , Células T Matadoras Naturais/enzimologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais , Proteína Associada à Molécula de Sinalização da Ativação Linfocitária/genética , Família de Moléculas de Sinalização da Ativação Linfocitária/genética , Família de Moléculas de Sinalização da Ativação Linfocitária/metabolismo
4.
Mol Med Rep ; 19(4): 3273-3282, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30816454

RESUMO

The human protein tyrosine phosphatase, non­receptor type 6 (PTPN6) gene is located on chromosome 12p13 and encodes an Mr 68,000 non­receptor type protein­tyrosine phosphatase. The PTPN6 gene has been considered as a candidate tumor suppressor in hematological and solid malignancies, and promoter methylation may be an epigenetic modification silencing its expression. However, the detailed role of PTPN6 and its promoter methylation status in the pathogenesis of esophageal squamous cell carcinoma (ESCC) has not been fully elucidated. The aim of the present study was to investigate PTPN6 expression in ESCC tissues and esophageal cancer cell lines, detect the effect of CpG hypermethylation on the activity of PTPN6, and additionally elucidate the role and prognostic significance of PTPN6 in ESCC tumorigenesis and progression. The expression of PTPN6 was identified to be significantly downregulated in esophageal cancer cell lines and ESCC tissues. Marked upregulation of PTPN6 was detected in 5­aza­2'­deoxycytidine­treated esophageal cancer cells, and frequent hypermethylation of the CpG sites within the P2 promoter (P2) was detected in ESCC tissues and esophageal cancer cell lines. The expression and methylation status of PTPN6 was associated with tumor node metastasis stage, pathological differentiation and lymph node metastasis in patients with ESCC. Aberrant hypermethylation of the P2 exhibited marked tumor specificity and was identified to be associated with the expression level of PTPN6. Downregulation and hypermethylation of PTPN6 were identified to be associated with poor ESCC patient survival. Furthermore, upregulation of PTPN6 inhibited the proliferation and invasion of esophageal cancer cells in vitro. The results of the present study suggest that PTPN6 may serve as a tumor suppressor in ESCC, and it may serve as a potential target for antitumor therapy.


Assuntos
Metilação de DNA , Carcinoma de Células Escamosas do Esôfago/genética , Carcinoma de Células Escamosas do Esôfago/patologia , Regulação Neoplásica da Expressão Gênica , Regiões Promotoras Genéticas , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Adulto , Idoso , Azacitidina/farmacologia , Biomarcadores Tumorais , Linhagem Celular Tumoral , Ilhas de CpG , Progressão da Doença , Epigênese Genética , Carcinoma de Células Escamosas do Esôfago/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Estudos de Associação Genética , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Metástase Neoplásica , Estadiamento de Neoplasias , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo
5.
J Exp Clin Cancer Res ; 38(1): 80, 2019 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-30764849

RESUMO

BACKGROUND: The differentiation-based therapy for acute promyelocytic leukemia (APL) is an inspiring example for the search of novel strategies aimed at treatment of other subtypes of acute myeloid leukemia (AML). Thus, the discovery of new molecular players in cell differentiation becomes a paramount research area to achieve this goal. Here, the involvement of the protein tyrosine phosphatases SHP1 and SHP2 on leukemic cells differentiation is shown, along with the therapeutic possibilities of their targeting to enhance the differentiation induction effect of phorbol esters. METHODS: The oxidation status and enzymatic activity of SHP1 and SHP2 during PMA-induced differentiation of HEL cells was evaluated. Additionally, the effects of RNAi-mediated downregulation of these phosphatases on cell differentiation was studied. Afterwards, the impact of chemical inhibition of SHP1 and SHP2 on differentiation both in the presence and absence of phorbol esters was tested. Finally, the anti-leukemic potential of phorbol esters and chemical inhibitors of SHP1 and SHP2 was addressed in several AML model cell lines, a xenograft mouse model and AML primary cells in vitro. RESULTS: An increase of oxidation with a concomitant decrease of activity was observed for both phosphatases at the onset of PMA-induced differentiation. Consistently, silencing of these proteins favored the process, with an enhanced effect upon their simultaneous downregulation. Moreover, the proteins SRC and ß-catenin were identified as downstream targets of SHP1 and SHP2 in this context. In agreement with these findings, chemical inhibition of the phosphatases promoted cell differentiation itself and enhanced the effect of phorbol esters. Interestingly, treatment with the phorbol ester prostratin and the dual inhibitor of SHP1 and SHP2 NSC87877 synergistically hampered the proliferation of AML cell lines, prolonged the survival of xenografted mice and reduced the clonogenic potential of AML primary cells. CONCLUSIONS: SHP1 and SHP2 are relevant mediators of differentiation in AML cells and their inhibition either alone or in combination with prostratin seems a promising differentiation-based therapeutic strategy against different subtypes of AML beyond APL.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Leucemia Mieloide Aguda/patologia , Ésteres de Forbol/farmacologia , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Animais , Feminino , Humanos , Leucemia Mieloide Aguda/metabolismo , Masculino , Camundongos Endogâmicos NOD , Camundongos SCID , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Cell Physiol Biochem ; 49(6): 2111-2123, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30273928

RESUMO

BACKGROUND/AIMS: Adult T-cell leukemia-lymphoma (ATL) is an aggressive disease which is highly resistant to chemotherapy. Studies show that enhanced ability of DNA damage repair (DDR) in cancer cells plays a key role in chemotherapy resistance. Here, we suggest that defect in DDR related genes might be a promising target to destroy the genome stability of tumor cells. METHODS: Since KU70 is highly expressed in Jurkat cells, one of the most representative cell lines of ATL, we knocked down KU70 by shRNA and analyzed the impact of KU70 deficiency in Jurkat cells as well as in NOD-SCID animal models by western blot, immunofluorescence, flow cytometry and measuring DNA repair efficiency. RESULTS: It is observed that silencing of KU70 resulted in accumulated DNA damage and impaired DDR in Jurkat cells, resulting in more apoptosis, decreased cell proliferation and cell cycle arrest. DNA damage leads to DNA double-strand breaks (DSBs), which are processed by either non-homologous end joining(NHEJ) or homologous recombination(HR). In our study, both NHEJ and HR are impaired because of KU70 defect, accompanied with increased protein level of SHP-1, a dephosphorylation enzyme. In turn, SHP-1 led to dephosphorylation of SIRT1, which further impaired HR repair efficiency. Moreover, KU70 deficiency prolonged survival of Jurkat-xenografted mice. CONCLUSION: These findings suggest that targeting KU70 is a promising target for ATL and might overcome the existing difficulties in chemotherapy.


Assuntos
Reparo do DNA por Junção de Extremidades , Autoantígeno Ku/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Reparo de DNA por Recombinação , Sirtuína 1/metabolismo , Animais , Apoptose , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Pontos de Checagem do Ciclo Celular , Quebras de DNA de Cadeia Dupla , Humanos , Células Jurkat , Autoantígeno Ku/antagonistas & inibidores , Autoantígeno Ku/genética , Leucemia-Linfoma de Células T do Adulto/tratamento farmacológico , Leucemia-Linfoma de Células T do Adulto/metabolismo , Leucemia-Linfoma de Células T do Adulto/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Fosforilação , Proteína Tirosina Fosfatase não Receptora Tipo 6/antagonistas & inibidores , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismo , RNA Interferente Pequeno/uso terapêutico , Rad51 Recombinase/metabolismo
7.
J Enzyme Inhib Med Chem ; 33(1): 1248-1255, 2018 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30261753

RESUMO

The unregulated activation of STAT3 has been demonstrated to occur in many cancers and enhances tumour growth, migration, and invasion. Stimulation by cytokines, growth factors, and hormones triggers this activation by phosphorylating STAT3 at tyrosine 705. Novel imidazopyridine compounds were synthesized to evaluate the inhibition of STAT3 at Y705. Among the tested compounds, 16 reduced the level of phospho-STAT3, inhibited the downstream signalling cascade and subsequently attenuated the survival of hepatocellular carcinoma (HCC) cells. Further assays showed that the reduction effects of compound 16 on tyrosine 705 of STAT3 were attributed to up-regulation of protein tyrosine phosphatase SHP-1.


Assuntos
Antineoplásicos/farmacologia , Imidazóis/farmacologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Piridinas/farmacologia , Fator de Transcrição STAT3/antagonistas & inibidores , Antineoplásicos/síntese química , Antineoplásicos/química , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Imidazóis/síntese química , Imidazóis/química , Estrutura Molecular , Piridinas/síntese química , Piridinas/química , RNA Interferente Pequeno/antagonistas & inibidores , RNA Interferente Pequeno/genética , Fator de Transcrição STAT3/metabolismo , Relação Estrutura-Atividade , Células Tumorais Cultivadas
8.
Oncology ; 95(5): 257-269, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29925063

RESUMO

Well-balanced levels of tyrosine phosphorylation, maintained by the reversible and coordinated actions of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs), are critical for a wide range of cellular processes including growth, differentiation, metabolism, migration, and survival. Aberrant tyrosine phosphorylation, as a result of a perturbed balance between the activities of PTKs and PTPs, is linked to the pathogenesis of numerous human diseases, including cancer, suggesting that PTPs may be innovative molecular targets for cancer treatment. Two PTPs that have an important inhibitory role in haematopoietic cells are SHP-1 and SHP-2. SHP-1, 2 promote cell growth and act by both upregulating positive signaling pathways and by downregulating negative signaling pathways. SHIP is another inhibitory phosphatase that is specific for the inositol phospholipid phosphatidylinositol-3,4,5-trisphosphate (PIP3). SHIP acts as a negative regulator of immune response by hydrolysing PIP3, and SHIP deficiency results in myeloproliferation and B-cell lymphoma in mice. The validation of SHP-1, 2 and SHIP as oncology targets has generated interest in the development of inhibitors as potential therapeutic agents for cancers; however, SHP-1, 2 and SHIP have proven to be an extremely difficult target for drug discovery, primarily due to the highly conserved and positively charged nature of their PTP active site, and many PTP inhibitors lack either appro-priate selectivity or membrane permeability. To overcome these caveats, novel techniques have been employed to synthesise new inhibitors that specifically attenuate the PTP-dependent signaling inside the cell and amongst them; some are already in clinical development which are discussed in this review.


Assuntos
Antineoplásicos/uso terapêutico , Inibidores Enzimáticos/uso terapêutico , Neoplasias/tratamento farmacológico , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/antagonistas & inibidores , Proteína Tirosina Fosfatase não Receptora Tipo 11/antagonistas & inibidores , Proteína Tirosina Fosfatase não Receptora Tipo 6/antagonistas & inibidores , Animais , Antineoplásicos/química , Desenho de Drogas , Inibidores Enzimáticos/química , Humanos , Terapia de Alvo Molecular , Neoplasias/enzimologia , Neoplasias/patologia , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/metabolismo , Fosforilação , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-Atividade , Resultado do Tratamento , Tirosina/metabolismo
9.
Cell Physiol Biochem ; 47(2): 641-653, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29794468

RESUMO

BACKGROUND/AIMS: Cholangiocarcinoma (CCA) is a malignant tumor that is resistant to chemotherapy, so new therapeutic agents are needed. Allicin which is rapidly converted from allin by allinase, is one of the most biologically active compounds in freshly crushed garlic and has been shown to have strong anti-tumor effects. Our aim was to explore the molecular mechanism by which allicin affects the cell proliferation and invasion of CCA. METHODS: Cell viability and apoptosis were measured using the CCK-8 assay, colony formation assay, and flow cytometry. Cell migration and invasion were evaluated by wound healing and Transwell assays, respectively. The expression of several proteins involved in cell apoptosis and invasion were assessed by Western blot. The activation of STAT3 signaling was detected by Western blot and immunofluorescence staining. The involvement of SHP-1 was determined using small interfering RNA (siRNA). Moreover, a nude mouse model of human CCA was established to assess the anti-tumor effects of allicin in vivo. RESULTS: Allicin significantly suppressed CCA cell proliferation by activating the caspase cascade, inducing apoptosis, and reducing the expression of proteins downstream of STAT3, such as B-cell lymphoma 2 (Bcl-2), while upregulating Bcl-2-associated X (Bax) protein. In addition, allicin inhibited the migration, invasion, and epithelial-mesenchymal transition (EMT) of CCA cells. Moreover, the protein expression of MMP-2 and MMP-9 was significantly downregulated in CCA cells treated with allicin compared with CCA cells treated with control. Mechanistic investigations indicated that allicin upregulated SHP-1 expression in CCA, and pervanadate treatment reversed the allicin-induced downregulation of STAT3. Moreover, suppression of SHP-1 by siRNA overturned the effect of allicin on the induction of SHP-1 and inhibition of STAT3 activation. Additionally, treatment with allicin attenuated tumor growth in the nude mouse model of CCA. CONCLUSIONS: Our findings suggest that allicin suppresses cell proliferation and invasion via STAT3 signaling and may be a potential therapeutic agent for CCA.


Assuntos
Antineoplásicos/farmacologia , Proliferação de Células/efeitos dos fármacos , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ácidos Sulfínicos/farmacologia , Animais , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Neoplasias dos Ductos Biliares/tratamento farmacológico , Neoplasias dos Ductos Biliares/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Colangiocarcinoma/tratamento farmacológico , Colangiocarcinoma/patologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Humanos , Metaloproteinases da Matriz/metabolismo , Camundongos , Camundongos Nus , Proteína Tirosina Fosfatase não Receptora Tipo 6/antagonistas & inibidores , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Fator de Transcrição STAT3/genética , Ácidos Sulfínicos/química , Ácidos Sulfínicos/metabolismo , Ácidos Sulfínicos/uso terapêutico , Transplante Heterólogo
10.
Dev Comp Immunol ; 84: 396-407, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29555550

RESUMO

Diverse immunoglobulin (Ig) domain-containing protein (DICP) family is a novel bony fish-specific multi-gene family encoding diversified immune receptors. However, their function and the implication of binding partners remain unknown. In this study, we first identified 28 DICPs from three gibel carp gynogenetic clones and revealed their high variability and clone-specific feature. After crucian carp herpesvirus (CaHV) infection, these DICPs were significantly upregulated in head kidney, kidney and spleen. The up-regulation folds in clone A+, F and H were related to the susceptibility to CaHV, progressively increasing from resistant clone to susceptible clone. Overexpression of gibel carp DICPs inhibited interferon (IFN) and viperin promoter-driven luciferase activity. The additions of E. coli extracts and lipid A significantly enhanced the inhibition effect. In addition, gibel carp DICPs can interact with SHP-1 and SHP-2. These findings suggest that gible carp DICPs, as inhibitory receptors, might specifically recognize lipid A, and then interact with SHP-1 and SHP-2 to inhibit the induction of IFN and ISGs.


Assuntos
Carpas/imunologia , Doenças dos Peixes/imunologia , Proteínas de Peixes/genética , Rim Cefálico/fisiologia , Infecções por Herpesviridae/imunologia , Herpesviridae/imunologia , Receptores Imunológicos/genética , Animais , Carpas/genética , Carpas/virologia , Suscetibilidade a Doenças , Evolução Molecular , Doenças dos Peixes/genética , Proteínas de Peixes/metabolismo , Especiação Genética , Rim Cefálico/virologia , Infecções por Herpesviridae/genética , Interferons/genética , Lipídeo A/metabolismo , Partenogênese , Polimorfismo Genético , Ligação Proteica , Proteína Tirosina Fosfatase não Receptora Tipo 11/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Receptores Imunológicos/metabolismo , Especificidade da Espécie , Regulação para Cima
11.
BMC Cancer ; 18(1): 150, 2018 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-29409467

RESUMO

BACKGROUND: We investigated the effect of arsenic trioxide (ATO) for inhibition of signal transducer and activator of transcription 3 (STAT3) and epithelial-mesenchymal transition (EMT) in gastric cancer cells, and the role of SH2 domain-containing phosphatase-1 (SHP-1) during this process. METHODS: We used AGS cells, which showed minimal SHP-1 expression and constitutive STAT3 expression. After treatment of ATO, cellular migration and invasion were assessed by using wound closure assay, Matrigel invasion assay and 3-D culture invasion assay. To validate the role of SHP-1, pervanadate, a pharmacologic phosphatase inhibitor, and SHP-1 siRNA were used. Xenograft tumors were produced, and ATO or pervanadate were administered via intraperitoneal (IP) route. RESULTS: Treatment of ATO 5 and 10 µM significantly decreased cellular migration and invasion in a dose-dependent manner. Western blot showed that ATO upregulated SHP-1 expression and downregulated STAT3 expression, and immunofluorescence showed upregulation with E-cadherin (epithelial marker) and downregulation of Snail1 (mesenchymal marker) expression by ATO treatment. Anti-migration and invasion effect and modulation of SHP-1/STAT3 axis by ATO were attenuated by pervanadate or SHP-1 siRNA. IP injection of ATO significantly decreased the xenograft tumor volume and upregulated SHP-1 expression, which were attenuated by co-IP injection of pervanadate. CONCLUSION: Our data suggest that ATO inhibits STAT3 activity and EMT process by upregulation of SHP-1 in gastric cancer cells.


Assuntos
Arsenicais/farmacologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Óxidos/farmacologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Fator de Transcrição STAT3/metabolismo , Neoplasias Gástricas/tratamento farmacológico , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Antineoplásicos/farmacologia , Trióxido de Arsênio , Caderinas/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Humanos , Camundongos Nus , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Interferência de RNA , Fatores de Transcrição da Família Snail/metabolismo , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , Carga Tumoral/efeitos dos fármacos
12.
EMBO J ; 37(5)2018 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-29449322

RESUMO

Natural killer (NK) cells are a powerful weapon against viral infections and tumor growth. Although the actin-myosin (actomyosin) cytoskeleton is crucial for a variety of cellular processes, the role of mechanotransduction, the conversion of actomyosin mechanical forces into signaling cascades, was never explored in NK cells. Here, we demonstrate that actomyosin retrograde flow (ARF) controls the immune response of primary human NK cells through a novel interaction between ß-actin and the SH2-domain-containing protein tyrosine phosphatase-1 (SHP-1), converting its conformation state, and thereby regulating NK cell cytotoxicity. Our results identify ARF as a master regulator of the NK cell immune response. Since actin dynamics occur in multiple cellular processes, this mechanism might also regulate the activity of SHP-1 in additional cellular systems.


Assuntos
Citoesqueleto de Actina/fisiologia , Actinas/metabolismo , Células Matadoras Naturais/imunologia , Mecanotransdução Celular/fisiologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Actomiosina/metabolismo , Células Cultivadas , Humanos , Conformação Proteica , Transdução de Sinais/imunologia
13.
Artigo em Inglês | MEDLINE | ID: mdl-29321445

RESUMO

The antibody response to RNA-related antigens such as Sm/RNP requires the endosomal RNA sensor TLR7, and this process is crucial in the development of systemic lupus erythematosus at least in animal models. The inhibitory B cell receptor CD72 is unique because it recognizes Sm/RNP and specifically inhibits the activation of Sm/RNP-reactive B cells by activating SH2-containing protein tyrosine phosphatase 1 (SHP-1). In the normal immune system, Sm/RNP-reactive B cells are tolerized by a unique mechanism that probably involves SHP-1. These self-reactive B cells appear in the peripheral lymphoid organs, differentiate into marginal zone B cells, and then undergo apoptosis without further maturation into plasma cells. Thus, CD72 is involved in the suppression of TLR7-mediated response to RNA in complexes with nuclear proteins that are resistant to nucleases, whereas free RNAs are degraded by nucleases before they encounter the endosomal RNA sensor.


Assuntos
Autoantígenos/metabolismo , Linfócitos B/imunologia , Lúpus Eritematoso Sistêmico/metabolismo , RNA/metabolismo , Ribonucleoproteínas Nucleares Pequenas/metabolismo , Animais , Antígenos CD/imunologia , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos B/imunologia , Antígenos de Diferenciação de Linfócitos B/metabolismo , Linfócitos B/metabolismo , Regulação para Baixo , Humanos , Tolerância Imunológica , Lúpus Eritematoso Sistêmico/imunologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , RNA/imunologia , Ribonucleoproteínas Nucleares Pequenas/imunologia , Transdução de Sinais , Receptor 7 Toll-Like/metabolismo
14.
Hepatology ; 67(4): 1303-1319, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29091299

RESUMO

Nonalcoholic fatty liver disease (NAFLD) is an increasingly prevalent liver pathology characterized by hepatic steatosis and commonly accompanied by systematic inflammation and metabolic disorder. Despite an accumulating number of studies, no pharmacological strategy is available to treat this condition in the clinic. In this study, we applied extensive gain- and loss-of-function approaches to identify the key immune factor leukocyte immunoglobulin-like receptor B4 (LILRB4) as a negative regulator of NAFLD. The hepatocyte-specific knockout of LILRB4 (LILRB4-HKO) exacerbated high-fat diet-induced insulin resistance, glucose metabolic imbalance, hepatic lipid accumulation, and systematic inflammation in mice, whereas LILRB4 overexpression in hepatocytes showed a completely opposite phenotype relative to that of LILRB4-HKO mice when compared with their corresponding controls. Further investigations of molecular mechanisms demonstrated that LILRB4 recruits SHP1 to inhibit TRAF6 ubiquitination and subsequent inactivation of nuclear factor kappa B and mitogen-activated protein kinase cascades. From a therapeutic perspective, the overexpression of LILRB4 in a genetic model of NAFLD, ob/ob mice, largely reversed the inherent hepatic steatosis, inflammation, and metabolic disorder. CONCLUSION: Targeting hepatic LILRB4 to improve its expression or activation represents a promising strategy for the treatment of NAFLD as well as related liver and metabolic diseases. (Hepatology 2018;67:1303-1319).


Assuntos
Hepatócitos/metabolismo , Glicoproteínas de Membrana/metabolismo , Hepatopatia Gordurosa não Alcoólica/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Receptores Imunológicos/metabolismo , Fator 6 Associado a Receptor de TNF/metabolismo , Animais , Western Blotting , Técnicas de Cultura de Células , Imunofluorescência , Regulação da Expressão Gênica , Teste de Tolerância a Glucose/métodos , Hepatócitos/patologia , Humanos , Resistência à Insulina , Fígado/patologia , Camundongos , Camundongos Knockout , Hepatopatia Gordurosa não Alcoólica/patologia , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais
15.
J Immunol ; 200(3): 1027-1038, 2018 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-29288206

RESUMO

Emerging data highlight the crucial role of enzymes involved in amino acid metabolism in immune cell biology. IL-4-induced gene-1 (IL4I1), a secreted l-phenylalanine oxidase expressed by APCs, has been detected in B cells, yet its immunoregulatory role has only been explored on T cells. In this study, we show that IL4I1 regulates multiple steps in B cell physiology. Indeed, IL4I1 knockout mice exhibit an accelerated B cell egress from the bone marrow, resulting in the accumulation of peripheral follicular B cells. They also present a higher serum level of natural Igs and self-reactive Abs. We also demonstrate that IL4I1 produced by B cells themselves controls the germinal center reaction, plasma cell differentiation, and specific Ab production in response to T dependent Ags, SRBC, and NP-KLH. In vitro, IL4I1-deficient B cells proliferate more efficiently than their wild-type counterparts in response to BCR cross-linking. Moreover, the absence of IL4I1 increases activation of the Syk-Akt-S6kinase signaling pathway and calcium mobilization, and inhibits SHP-1 activity upon BCR engagement, thus supporting that IL4I1 negatively controls BCR-dependent activation. Overall, our study reveals a new perspective on IL4I1 as a key regulator of B cell biology.


Assuntos
Aminoácido Oxirredutases/genética , Linfócitos B/citologia , Flavoproteínas/genética , Ativação Linfocitária/imunologia , Receptores de Antígenos de Linfócitos B/imunologia , Aminoácido Oxirredutases/metabolismo , Animais , Linfócitos B/imunologia , Diferenciação Celular/imunologia , Flavoproteínas/metabolismo , Imunoglobulinas/sangue , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Quinases S6 Ribossômicas/metabolismo , Transdução de Sinais/fisiologia , Quinase Syk/metabolismo
16.
J Immunotoxicol ; 15(1): 1-11, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-29228829

RESUMO

Vanadium is an air pollutant that imparts immunosuppressive effects on NK cell immune responses, in part, by dysregulating interleukin (IL)-2/IL-2R-mediated JAK signaling pathways and inducing apoptosis. The aim of the present study was to evaluate effects of vanadium pentoxide (V2O5) on other IL-2 receptor-mediated signaling pathways, i.e. PI3K-AKT-mTOR and Ras-MAPK. Here, IL-2-independent NK-92MI cells were exposed to different V2O5 doses for 24 h periods. Expression of PI3K, Akt, mTOR, ERK1/2, MEK1, PTEN, SHP1, BAD and phosphorylated forms, as well as caspases-3, -8, -9, BAX and BAK in/on the cells were then determined by flow cytometry. The results show that V2O5 was cytotoxic to NK cells in a dose-related manner. Exposure increased BAD and pBAD expression and decreased that of BAK and BAX, but cell death was not related to caspase activation. At 400 µM V2O5, expression of PI3K-p85 regulatory subunit increased 20% and pPI3K 50%, while that of the non-pPI3K 110α catalytic subunit decreased by 20%. At 200 µM, V2O5 showed significant decrease in non-pAkt expression (p < 0.05); the decrease in pAkt expression was significant at 100 µM. Non-pmTOR expression displayed a significant downward trend beginning at 100 µM. Expressions of pMEK-1/2 and pERK-1/2 increased substantially at 200 µM V2O5. No differences were found with non-phosphorylated ERK-1/2. PTEN expression increased significantly at 100 µM V2O5 exposure whereas pPTEN decreased by 18% at 25 µM V2O5 concentrations, but remained unchanged thereafter. Lastly, V2O5 at all doses decreased SHP1 expression and increased expression of its phosphorylated form. These results indicated a toxic effect of V2O5 on NK cells that was due in part to dysregulation of signaling pathways mediated by IL-2 via increased PTEN and decreased SHP1 expression. These results can help to explain some of the known deleterious effects of this particular form of vanadium on innate immune responses.


Assuntos
Células Matadoras Naturais/fisiologia , PTEN Fosfo-Hidrolase/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Compostos de Vanádio/toxicidade , Poluição do Ar/efeitos adversos , Apoptose , Linhagem Celular , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação da Expressão Gênica , Humanos , Proteína Oncogênica v-akt/metabolismo , PTEN Fosfo-Hidrolase/genética , Fosfatidilinositol 3-Quinases/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Proteínas ras/metabolismo
17.
Arch Virol ; 163(1): 243-248, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-29058147

RESUMO

Chikungunya virus (CHIKV)-induced myositis is an emerging affliction with high incidence globally. Given the essential regulatory role of protein tyrosine phosphatase non-receptor 6 (PTPN6) in virus-induced myositis, the expression of the PTPN6 and TNF-α genes in a CHIKV-infected muscle cell line was examined by quantitative PCR, and the expression of PTPN6 and STAT 3 was examined by immunoblotting. In addition, the effect of PTPN6 siRNA treatment on TNF-α gene expression was assessed. Increased higher expression of PTPN6 and TNF-α, and significant upregulation of TNF-α upon PTPN6 siRNA treatment were observed, suggesting that CHIKV has the ability to induce host PTPN6 gene expression, which may lead to a decreased pro-inflammatory immune response in the host.


Assuntos
Inflamação/metabolismo , Músculo Esquelético/citologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Animais , Linhagem Celular Tumoral , Vírus Chikungunya , Regulação da Expressão Gênica , Humanos , Imunidade Inata , Inflamação/genética , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Rabdomiossarcoma , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo , Regulação para Cima
18.
Part Fibre Toxicol ; 14(1): 53, 2017 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-29233151

RESUMO

BACKGROUND: Inhaled nanoparticles can deposit in the deep lung where they interact with pulmonary cells. Despite numerous studies on pulmonary nanotoxicity, detailed molecular mechanisms of specific nanomaterial-induced lung injury have yet to be identified. RESULTS: Using whole-body dynamic inhalation model, we studied the interactions between aluminum oxide nanoparticles (Al2O3 NPs) and the pulmonary system in vivo. We found that seven-day-exposure to Al2O3 NPs resulted in emphysema and small airway remodeling in murine lungs, accompanied by enhanced inflammation and apoptosis. Al2O3 NPs exposure led to suppression of PTPN6 and phosphorylation of STAT3, culminating in increased expression of the apoptotic marker PDCD4. Rescue of PTPN6 expression or application of a STAT3 inhibitor, effectively protected murine lungs from inflammation and apoptosis, as well as, in part, from the induction of chronic obstructive pulmonary disease (COPD)-like effects. CONCLUSION: In summary, our studies show that inhibition of PTPN6 plays a critical role in Al2O3 NPs-induced COPD-like lesions.


Assuntos
Óxido de Alumínio/toxicidade , Pulmão/efeitos dos fármacos , Nanopartículas Metálicas/toxicidade , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Doença Pulmonar Obstrutiva Crônica/induzido quimicamente , Fator de Transcrição STAT3/metabolismo , Células A549 , Animais , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose/metabolismo , Progressão da Doença , Relação Dose-Resposta a Droga , Humanos , Mediadores da Inflamação/metabolismo , Exposição por Inalação/efeitos adversos , Pulmão/enzimologia , Pulmão/fisiologia , Masculino , Camundongos Endogâmicos C57BL , Fosforilação , Pneumonia/induzido quimicamente , Pneumonia/enzimologia , Pneumonia/patologia , Doença Pulmonar Obstrutiva Crônica/enzimologia , Doença Pulmonar Obstrutiva Crônica/patologia , Doença Pulmonar Obstrutiva Crônica/prevenção & controle , Enfisema Pulmonar/induzido quimicamente , Enfisema Pulmonar/enzimologia , Enfisema Pulmonar/patologia , Proteínas de Ligação a RNA/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo
19.
Nat Commun ; 8(1): 1286, 2017 11 03.
Artigo em Inglês | MEDLINE | ID: mdl-29097680

RESUMO

Chronic kidney disease (CKD) is defined by reduced estimated glomerular filtration rate (eGFR). Previous genetic studies have implicated regulatory mechanisms contributing to CKD. Here we present epigenome-wide association studies of eGFR and CKD using whole-blood DNA methylation of 2264 ARIC Study and 2595 Framingham Heart Study participants to identify epigenetic signatures of kidney function. Of 19 CpG sites significantly associated (P < 1e-07) with eGFR/CKD and replicated, five also associate with renal fibrosis in biopsies from CKD patients and show concordant DNA methylation changes in kidney cortex. Lead CpGs at PTPN6/PHB2, ANKRD11, and TNRC18 map to active enhancers in kidney cortex. At PTPN6/PHB2 cg19942083, methylation in kidney cortex associates with lower renal PTPN6 expression, higher eGFR, and less renal fibrosis. The regions containing the 243 eGFR-associated (P < 1e-05) CpGs are significantly enriched for transcription factor binding sites of EBF1, EP300, and CEBPB (P < 5e-6). Our findings highlight kidney function associated epigenetic variation.


Assuntos
Metilação de DNA/genética , Insuficiência Renal Crônica/genética , Insuficiência Renal Crônica/fisiopatologia , Idoso , Sítios de Ligação/genética , Proteína beta Intensificadora de Ligação a CCAAT/genética , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Ilhas de CpG , Progressão da Doença , Proteína p300 Associada a E1A/genética , Proteína p300 Associada a E1A/metabolismo , Epigênese Genética , Feminino , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Taxa de Filtração Glomerular/genética , Humanos , Rim/metabolismo , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Transativadores/genética , Transativadores/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
20.
Arterioscler Thromb Vasc Biol ; 37(12): 2291-2300, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-29074590

RESUMO

OBJECTIVE: Ischemia caused by narrowing of femoral artery is a major cause of peripheral arterial disease and morbidity affecting patients with diabetes mellitus. We have previously reported that the inhibition of the angiogenic response to VEGF (vascular endothelial growth factor) in diabetic mice was associated with the increased expression of SHP-1 (SH2 domain-containing phosphatase 1), a protein that can be activated by the AT2 (angiotensin II type 2) receptor. Deletion of AT2 receptor has been shown to promote angiogenesis within the ischemic muscle. However, the relative impact of AT2 receptor in diabetic condition remains unknown. APPROACH AND RESULTS: Nondiabetic and diabetic AT2 null (Atgr2-/Y) mice underwent femoral artery ligation after 2 months of diabetes mellitus. Blood perfusion was measured every week ≤4 weeks post-surgery. Expression of the VEGF, SHP-1, and renin-angiotensin pathways was evaluated. Blood flow in the ischemic muscle of diabetic Atgr2-/Y mice recovered faster and ≤80% after 4 weeks compared with 51% recovery in diabetic control littermates. Diabetic Atgr2-/Y had reduced apoptotic endothelial cells and elevated small vessel formation compared with diabetic Atgr2+/Y mice, as well as increased SHP-1 expression and reduced VEGF receptor activity. In endothelial cells, high glucose levels and AT2 agonist treatment did not change SHP-1, VEGF, and VEGF receptor expression. However, the activity of SHP-1 and its association with the VEGF receptors were increased, causing inhibition of the VEGF action in endothelial cell proliferation and migration. CONCLUSIONS: Our results suggest that the deletion of AT2 receptor reduced SHP-1 activity and restored VEGF actions, leading to an increased blood flow reperfusion after ischemia in diabetes mellitus.


Assuntos
Diabetes Mellitus/metabolismo , Angiopatias Diabéticas/metabolismo , Isquemia/metabolismo , Músculo Esquelético/irrigação sanguínea , Neovascularização Fisiológica , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Receptor Tipo 2 de Angiotensina/deficiência , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Glicemia/metabolismo , Bovinos , Movimento Celular , Proliferação de Células , Células Cultivadas , Diabetes Mellitus/genética , Diabetes Mellitus/fisiopatologia , Angiopatias Diabéticas/genética , Angiopatias Diabéticas/fisiopatologia , Modelos Animais de Doenças , Células Endoteliais/metabolismo , Deleção de Genes , Genótipo , Membro Posterior , Isquemia/genética , Isquemia/fisiopatologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Receptor Tipo 2 de Angiotensina/genética , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Recuperação de Função Fisiológica , Fluxo Sanguíneo Regional , Sistema Renina-Angiotensina , Transdução de Sinais , Fatores de Tempo
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