RESUMO
Myeloid-derived suppressor cells (MDSCs) are key determinants of the immunosuppressive microenvironment in tumors. As ion channels play key roles in the physiology/pathophysiology of immune cells, we aimed at studying the ion channel repertoire in tumor-derived polymorphonuclear (PMN-MDSC) and monocytic (Mo-MDSC) MDSCs. Subcutaneous tumors in mice were induced by the Lewis lung carcinoma cell line (LLC). The presence of PMN-MDSC (CD11b+/Ly6G+) and Mo-MDSCs (CD11b+/Ly6C+) in the tumor tissue was confirmed using immunofluorescence microscopy and cells were identified as CD11b+/Ly6G+ PMN-MDSCs and CD11b+/Ly6C+/F4/80-/MHCII- Mo-MDSCs using flow cytometry and sorting. The majority of the myeloid cells infiltrating the LLC tumors were PMN-MDSC (~60%) as compared to ~10% being Mo-MDSCs. We showed that PMN- and Mo-MDSCs express the Hv1 H+ channel both at the mRNA and at the protein level and that the biophysical and pharmacological properties of the whole-cell currents recapitulate the hallmarks of Hv1 currents: ~40 mV shift in the activation threshold of the current per unit change in the extracellular pH, high H+ selectivity, and sensitivity to the Hv1 inhibitor ClGBI. As MDSCs exert immunosuppression mainly by producing reactive oxygen species which is coupled to Hv1-mediated H+ currents, Hv1 might be an attractive target for inhibition of MDSCs in tumors.
Assuntos
Células Supressoras Mieloides , Neoplasias , Animais , Camundongos , Linhagem Celular , Monócitos , Células Mieloides , Células Supressoras Mieloides/metabolismo , Neoplasias/metabolismo , Microambiente TumoralRESUMO
Signal transducer and activator of transcription 3 (STAT-3) gain-of-function (GOF) syndrome is an early-onset monogenic inborn error of immunity characterized by multi-organ autoimmune disorders, growth failure and lymphoproliferation. We describe that STAT-3 GOF syndrome may be presented with hypogammaglobulinemia and recurrent severe upper and lower respiratory tract infections. In addition, the patient had lymphoproliferation, short stature and interstitial lung disease. Chest computerized tomography examinations showed mild bronchiectasis with areas of non-fibrosing alveolar-interstitial disease and maldevelopment of bilateral first ribs. Using Sanger sequencing, we revealed a novel c.508G>C, p.D170H STAT-3 variant affecting the coiled coil domain of STAT-3. Functional studies confirmed that p.D170H was a GOF variant, as shown by increased phosphorylated STAT-3 (pSTAT-3) and STAT-3 transcriptional activity. Our observation suggests that STAT-3 GOF syndrome can manifest in early childhood with hypogammaglobulinemia and recurrent severe respiratory tract infections. We suggest that patients with lymphoproliferation, hypogammaglobulinemia and severe recurrent infections should be screened for STAT-3 variants, even if autoimmune manifestations are missing.
Assuntos
Agamaglobulinemia/genética , Mutação com Ganho de Função/genética , Transtornos Linfoproliferativos/genética , Infecções Respiratórias/genética , Fator de Transcrição STAT3/genética , Agamaglobulinemia/imunologia , Desenvolvimento Ósseo/genética , Bronquiectasia/genética , Humanos , Masculino , Infecções Respiratórias/imunologia , Infecções Respiratórias/mortalidade , Fator de Transcrição STAT3/metabolismo , Adulto JovemRESUMO
CD84 (SLAMF5) is a member of the SLAM family of cell-surface immunoreceptors. Broadly expressed on most immune cell subsets, CD84 functions as a homophilic adhesion molecule, whose signaling can activate or inhibit leukocyte function depending on the cell type and its stage of activation or differentiation. CD84-mediated signaling regulates diverse immunological processes, including T cell cytokine secretion, natural killer cell cytotoxicity, monocyte activation, autophagy, cognate T:B interactions, and B cell tolerance at the germinal center checkpoint. Recently, alterations in CD84 have been related to autoimmune and lymphoproliferative disorders. Specific allelic variations in CD84 are associated with autoimmune diseases such as systemic lupus erythematosus and rheumatoid arthritis. In chronic lymphocytic leukemia, CD84 mediates intrinsic and stroma-induced survival of malignant cells. In this review, we describe our current understanding of the structure and function of CD84 and its potential role as a therapeutic target and biomarker in inflammatory autoimmune disorders and cancer.
Assuntos
Doenças Autoimunes/imunologia , Biomarcadores/metabolismo , Neoplasias/imunologia , Família de Moléculas de Sinalização da Ativação Linfocitária/imunologia , Animais , Humanos , Polimorfismo GenéticoAssuntos
DNA Topoisomerases Tipo II/genética , Estudos de Associação Genética , Predisposição Genética para Doença , Mutação , Fenótipo , Proteínas de Ligação a Poli-ADP-Ribose/genética , Adolescente , Alelos , Substituição de Aminoácidos , Biomarcadores , DNA Topoisomerases Tipo II/química , Estudos de Associação Genética/métodos , Genótipo , Humanos , Masculino , Proteínas de Ligação a Poli-ADP-Ribose/química , Análise de Sequência de DNA , Avaliação de Sintomas , Sequenciamento do ExomaRESUMO
Mutations in the SH3PXD2B gene coding for the Tks4 protein are responsible for the autosomal recessive Frank-ter Haar syndrome. Tks4, a substrate of Src tyrosine kinase, is implicated in the regulation of podosome formation. Here, we report a novel role for Tks4 in the EGF signaling pathway. In EGF-treated cells, Tks4 is tyrosine-phosphorylated and associated with the activated EGF receptor. This association is not direct but requires the presence of Src tyrosine kinase. In addition, treatment of cells with LY294002, an inhibitor of PI 3-kinase, or mutations of the PX domain reduces tyrosine phosphorylation and membrane translocation of Tks4. Furthermore, a PX domain mutant (R43W) Tks4 carrying a reported point mutation in a Frank-ter Haar syndrome patient showed aberrant intracellular expression and reduced phosphoinositide binding. Finally, silencing of Tks4 was shown to markedly inhibit HeLa cell migration in a Boyden chamber assay in response to EGF or serum. Our results therefore reveal a new function for Tks4 in the regulation of growth factor-dependent cell migration.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Movimento Celular , Anormalidades Craniofaciais/metabolismo , Fator de Crescimento Epidérmico/metabolismo , Cardiopatias Congênitas/metabolismo , Osteocondrodisplasias/congênito , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Células COS , Chlorocebus aethiops , Cromonas/farmacologia , Anormalidades Craniofaciais/genética , Anormalidades Craniofaciais/mortalidade , Deficiências do Desenvolvimento/genética , Deficiências do Desenvolvimento/metabolismo , Deficiências do Desenvolvimento/mortalidade , Inibidores Enzimáticos/farmacologia , Fator de Crescimento Epidérmico/genética , Receptores ErbB/genética , Receptores ErbB/metabolismo , Inativação Gênica , Células HeLa , Cardiopatias Congênitas/genética , Cardiopatias Congênitas/mortalidade , Humanos , Morfolinas/farmacologia , Mutação , Osteocondrodisplasias/genética , Osteocondrodisplasias/metabolismo , Osteocondrodisplasias/mortalidade , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Estrutura Terciária de Proteína , Quinases da Família src/genética , Quinases da Família src/metabolismoRESUMO
The activation of TLRs expressed by macrophages or DCs, in the long run, leads to persistently impaired functionality. TLR signals activate a wide range of negative feedback mechanisms; it is not known, however, which of these can lead to long-lasting tolerance for further stimulatory signals. In addition, it is not yet understood how the functionality of monocyte-derived DCs (MoDCs) is influenced in inflamed tissues by the continuous presence of stimulatory signals during their differentiation. Here we studied the role of a wide range of DC-inhibitory mechanisms in a simple and robust model of MoDC inactivation induced by early TLR signals during differentiation. We show that the activation-induced suppressor of cytokine signaling 1 (SOCS1), IL-10, STAT3, miR146a and CD150 (SLAM) molecules possessed short-term inhibitory effects on cytokine production but did not induce persistent DC inactivation. On the contrary, the LPS-induced IRAK-1 downregulation could alone lead to persistent MoDC inactivation. Studying cellular functions in line with the activation-induced negative feedback mechanisms, we show that early activation of developing MoDCs allowed only a transient cytokine production that was followed by the downregulation of effector functions and the preservation of a tissue-resident non-migratory phenotype.
Assuntos
Citocinas/metabolismo , Células Dendríticas/metabolismo , Regulação da Expressão Gênica , Quinases Associadas a Receptores de Interleucina-1/metabolismo , Antígenos CD/genética , Antígenos CD/metabolismo , Diferenciação Celular , Células Cultivadas , Citocinas/genética , Células Dendríticas/imunologia , Células Dendríticas/patologia , Retroalimentação Fisiológica , Regulação da Expressão Gênica/imunologia , Humanos , Tolerância Imunológica , Inflamação , Quinases Associadas a Receptores de Interleucina-1/genética , Quinases Associadas a Receptores de Interleucina-1/imunologia , Interleucina-10/genética , Interleucina-10/metabolismo , Lipopolissacarídeos/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Monócitos/patologia , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Membro 1 da Família de Moléculas de Sinalização da Ativação Linfocitária , Proteína 1 Supressora da Sinalização de Citocina , Proteínas Supressoras da Sinalização de Citocina/genética , Proteínas Supressoras da Sinalização de Citocina/metabolismo , Receptores Toll-Like/metabolismoRESUMO
5-chloro-2-guanidinobenzimidazole (ClGBI), a small-molecule guanidine derivative, is a known effective inhibitor of the voltage-gated proton (H+) channel (HV1, Kd ≈ 26 µM) and is widely used both in ion channel research and functional biological assays. However, a comprehensive study of its ion channel selectivity determined by electrophysiological methods has not been published yet. The lack of selectivity may lead to incorrect conclusions regarding the role of hHv1 in physiological or pathophysiological responses in vitro and in vivo. We have found that ClGBI inhibits the proliferation of lymphocytes, which absolutely requires the functioning of the KV1.3 channel. We, therefore, tested ClGBI directly on hKV1.3 using a whole-cell patch clamp and found an inhibitory effect similar in magnitude to that seen on hHV1 (Kd ≈ 72 µM). We then further investigated ClGBI selectivity on the hKV1.1, hKV1.4-IR, hKV1.5, hKV10.1, hKV11.1, hKCa3.1, hNaV1.4, and hNaV1.5 channels. Our results show that, besides HV1 and KV1.3, all other off-target channels were inhibited by ClGBI, with Kd values ranging from 12 to 894 µM. Based on our comprehensive data, ClGBI has to be considered a non-selective hHV1 inhibitor; thus, experiments aiming at elucidating the significance of these channels in physiological responses have to be carefully evaluated.
RESUMO
Dendritic cells (DCs) respond to changes in their lipid environment by altering gene expression and immunophenotype. Some of these alterations are mediated via the nuclear receptor superfamily. However, little is known about the contribution of liver X receptor (LXR) to DC biology. In this study, we present a systematic analysis of LXR, activated by synthetic ligands or naturally occurring oxysterols in developing human monocyte-derived DCs. We found that LXRs are present and can be activated throughout DC differentiation in monocyte- and blood-derived DCs. Administration of LXR-specific natural or synthetic activators induced target gene expression accompanied by increased expression of DC maturation markers, such as CD80 and CD86. In mature DCs, LXR activation augmented the production of inflammatory cytokines IL-12, TNF-alpha, IL-6, and IL-8 and resulted in an increased capacity to activate CD4+ T cell proliferation upon ligation with TLR4 or TLR3 ligands. These effects appear to be underpinned by prolonged NF-kappaB signaling. Supporting such an inflammatory role, we found that LXR positive DCs are present in reactive lymph nodes in vivo. We propose that activation of LXR represents a novel lipid-signaling paradigm that alters the inflammatory response of human DCs.
Assuntos
Diferenciação Celular/imunologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Mediadores da Inflamação/fisiologia , Receptores Nucleares Órfãos/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/patologia , Proliferação de Células , Células Cultivadas , Citocinas/biossíntese , Células Dendríticas/patologia , Humanos , Mediadores da Inflamação/metabolismo , Metabolismo dos Lipídeos/imunologia , Receptores X do Fígado , Linfonodos/citologia , Linfonodos/imunologia , Linfonodos/metabolismo , Linfonodos/patologia , Monócitos/citologia , Monócitos/imunologia , Monócitos/metabolismo , NF-kappa B/fisiologia , Receptores Nucleares Órfãos/fisiologia , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/fisiologia , Transdução de Sinais/imunologia , Regulação para Cima/imunologiaRESUMO
SAP (SLAM-associated protein) is a small lymphocyte-specific signalling molecule that is defective or absent in patients with X-linked lymphoproliferative syndrome (XLP). Consistent with its single src homology 2 (SH2) domain architecture and unusually high affinity for SLAM (also called CD150), SAP has been suggested to function by blocking binding of SHP-2 or other SH2-containing signalling proteins to SLAM receptors. Additionally, SAP has recently been shown to be required for recruitment and activation of the Src-family kinase FynT after SLAM ligation. This signalling 'adaptor' function has been difficult to conceptualize, because unlike typical SH2-adaptor proteins, SAP contains only a single SH2 domain and lacks other recognized protein interaction domains or motifs. Here, we show that the SAP SH2 domain binds to the SH3 domain of FynT and directly couples FynT to SLAM. The crystal structure of a ternary SLAM-SAP-Fyn-SH3 complex reveals that SAP binds the FynT SH3 domain through a surface-surface interaction that does not involve canonical SH3 or SH2 binding interactions. The observed mode of binding to the Fyn-SH3 domain is expected to preclude the auto-inhibited conformation of Fyn, thereby promoting activation of the kinase after recruitment. These findings broaden our understanding of the functional repertoire of SH3 and SH2 domains.
Assuntos
Proteínas de Transporte/metabolismo , Glicoproteínas/metabolismo , Imunoglobulinas/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Proto-Oncogênicas/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais/fisiologia , Sequência de Aminoácidos , Animais , Antígenos CD/metabolismo , Sítios de Ligação , Proteínas de Transporte/química , Proteínas de Transporte/genética , Células Cultivadas , Cristalografia por Raios X , Genes Reporter , Glicoproteínas/química , Glicoproteínas/genética , Humanos , Imunoglobulinas/química , Imunoglobulinas/genética , Transtornos Linfoproliferativos/imunologia , Transtornos Linfoproliferativos/metabolismo , Substâncias Macromoleculares , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Estrutura Quaternária de Proteína , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-fyn , Receptores de Superfície Celular , Receptores Virais/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Proteína Associada à Molécula de Sinalização da Ativação Linfocitária , Membro 1 da Família de Moléculas de Sinalização da Ativação Linfocitária , Timo/citologia , Timo/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Domínios de Homologia de srcRESUMO
The nine SLAM-family genes, SLAMF1-9, a subfamily of the immunoglobulin superfamily, encode differentially expressed cell-surface receptors of hematopoietic cells. Engagement with their ligands, which are predominantly homotypic, leads to distinct signal transduction events, for instance those that occur in the T or NK cell immune synapse. Upon phosphorylation of one or more copies of a unique tyrosine-based signaling motif in their cytoplasmic tails, six of the SLAM receptors recruit the highly specific single SH2-domain adapters SLAM-associated protein (SAP), EAT-2A, and/or EAT-2B. These adapters in turn bind to the tyrosine kinase Fyn and/or other protein tyrosine kinases connecting the receptors to signal transduction networks. Individuals deficient in the SAP gene, SH2D1A, develop an immunodeficiency syndrome: X-linked lympho-proliferative disease. In addition to operating in the immune synapse, SLAM receptors initiate or partake in multiple effector functions of hematopoietic cells, for example, neutrophil and macrophage killing and platelet aggregation. Here we discuss the current understanding of the structure and function of these recently discovered receptors and adapter molecules in the regulation of adaptive and innate immune responses.
Assuntos
Imunidade/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Receptores Imunológicos/fisiologia , Animais , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Lúpus Eritematoso Sistêmico/genética , Lúpus Eritematoso Sistêmico/fisiopatologia , Transtornos Linfoproliferativos/genética , Transtornos Linfoproliferativos/fisiopatologia , Modelos Biológicos , Receptores Imunológicos/genética , Proteína Associada à Molécula de Sinalização da Ativação LinfocitáriaRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Tumor cells exposed to a physiological matrix of type I collagen fibers form elongated collagenolytic invadopodia, which differ from dotty-like invadopodia forming on the gelatin substratum model. The related scaffold proteins, TKS5 and TKS4, are key components of the mechanism of invadopodia assembly. The molecular events through which TKS proteins direct collagenolytic invadopodia formation are poorly defined. Using coimmunoprecipitation experiments, identification of bound proteins by mass spectrometry, and in vitro pull-down experiments, we found an interaction between TKS5 and FGD1, a guanine nucleotide exchange factor for the Rho-GTPase CDC42, which is known for its role in the assembly of invadopodial actin core structure. A novel cell polarity network is uncovered comprising TKS5, FGD1, and CDC42, directing invadopodia formation and the polarization of MT1-MMP recycling compartments, required for invadopodia activity and invasion in a 3D collagen matrix. Additionally, our data unveil distinct signaling pathways involved in collagenolytic invadopodia formation downstream of TKS4 or TKS5 in breast cancer cells.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Podossomos/metabolismo , Transdução de Sinais/fisiologia , Proteína cdc42 de Ligação ao GTP/metabolismo , Actinas/metabolismo , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Polaridade Celular/fisiologia , Colágeno/metabolismo , Feminino , Humanos , Transfecção/métodos , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
Endothelial cell motility has fundamental role in vasculogenesis and angiogenesis during developmental or pathological processes. Tks4 is a scaffold protein known to organize the cytoskeleton of lamellipodia and podosomes, and thus modulating cell motility and invasion. In particular, Tks4 is required for the localization and activity of membrane type 1-matrix metalloproteinase, a key factor for extracellular matrix (ECM) cleavage during cell migration. While its role in transformed cells is well established, little is known about the function of Tks4 under physiological conditions. In this study we examined the impact of Tks4 gene silencing on the functional activity of primary human umbilical vein endothelial cells (HUVEC) and used time-lapse videomicrosopy and quantitative image analysis to characterize cell motility phenotypes in culture. We demonstrate that the absence of Tks4 in endothelial cells leads to impaired ECM cleavage and decreased motility within a 3-dimensional ECM environment. Furthermore, absence of Tks4 also decreases the ability of HUVEC cells to form multicellular sprouts, a key requirement for angiogenesis. To establish the involvement of Tks4 in vascular development in vivo, we show that loss of Tks4 leads sparser vasculature in the fetal chorion in the Tks4-deficient 'nee' mouse strain.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Citoesqueleto/genética , Matriz Extracelular/genética , Proteínas Adaptadoras de Transdução de Sinal/antagonistas & inibidores , Animais , Movimento Celular/genética , Células Endoteliais/metabolismo , Inativação Gênica , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Neovascularização Patológica/genética , Neovascularização Patológica/patologia , Neovascularização Fisiológica/genética , Podossomos/genética , Pseudópodes/genética , Transdução de Sinais/genéticaRESUMO
Signaling lymphocyte activation molecule family (SLAMF) receptors are essential regulators of innate and adaptive immune responses. The function of SLAMF5/CD84, a family member with almost ubiquitous expression within the hematopoietic lineage is poorly defined. In this article, we provide evidence that in human monocyte-derived dendritic cells (moDCs) SLAMF5 increases autophagy, a degradative pathway, which is highly active in dendritic cells (DCs) and plays a critical role in orchestration of the immune response. While investigating the underlying mechanism, we found that SLAMF5 inhibited proteolytic degradation of interferon regulatory factor 8 (IRF8) a master regulator of the autophagy process by a mechanism dependent on the E3-ubiquitin ligase tripartite motif-containing protein 21 (TRIM21). Furthermore, we demonstrate that SLAMF5 influences the ratio of CD1a+ cells in differentiating DCs and partakes in the regulation of IL-1ß, IL-23, and IL-12 production in LPS/IFNγ-activated moDCs in a manner that is consistent with its effect on IRF8 stability. In summary, our experiments identified SLAMF5 as a novel cell surface receptor modulator of autophagy and revealed an unexpected link between the SLAMF and IRF8 signaling pathways, both implicated in multiple human pathologies.
Assuntos
Autofagia , Citocinas/metabolismo , Células Dendríticas/metabolismo , Fatores Reguladores de Interferon/metabolismo , Transdução de Sinais , Família de Moléculas de Sinalização da Ativação Linfocitária/metabolismo , Autofagia/efeitos dos fármacos , Diferenciação Celular , Células Dendríticas/imunologia , Regulação da Expressão Gênica , Inativação Gênica , Humanos , Modelos Biológicos , Complexo de Endopeptidases do Proteassoma/metabolismo , Família de Moléculas de Sinalização da Ativação Linfocitária/genética , Sirolimo/farmacologiaRESUMO
Mitochondrial reactive oxygen species (mtROS) generated continuously under physiological conditions have recently emerged as critical players in the regulation of immune signaling pathways. In this study we have investigated the regulation of antiviral signaling by increased mtROS production in plasmacytoid dendritic cells (pDCs), which, as major producers of type I interferons (IFN), are the key coordinators of antiviral immunity. The early phase of type I IFN production in pDCs is mediated by endosomal Toll-like receptors (TLRs), whereas the late phase of IFN response can also be triggered by cytosolic retinoic acid-inducible gene-I (RIG-I), expression of which is induced upon TLR stimulation. Therefore, pDCs provide an ideal model to study the impact of elevated mtROS on the antiviral signaling pathways initiated by receptors with distinct subcellular localization. We found that elevated level of mtROS alone did not change the phenotype and the baseline cytokine profile of resting pDCs. Nevertheless increased mtROS levels in pDCs lowered the TLR9-induced secretion of pro-inflammatory mediators slightly, whereas reduced type I IFN production markedly via blocking phosphorylation of interferon regulatory factor 7 (IRF7), the key transcription factor of the TLR9 signaling pathway. The TLR9-induced expression of RIG-I in pDCs was also negatively regulated by enhanced mtROS production. On the contrary, elevated mtROS significantly augmented the RIG-I-stimulated expression of type I IFNs, as well as the expression of mitochondrial antiviral-signaling (MAVS) protein and the phosphorylation of Akt and IRF3 that are essential components of RIG-I signaling. Collectively, our data suggest that increased mtROS exert diverse immunoregulatory functions in pDCs both in the early and late phase of type I IFN responses depending on which type of viral sensing pathway is stimulated.
Assuntos
Células Dendríticas/metabolismo , Interferon Tipo I/metabolismo , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Linhagem Celular , Células Cultivadas , Proteína DEAD-box 58/metabolismo , Humanos , Fator Regulador 7 de Interferon/metabolismo , Interferon Tipo I/genética , Receptores Imunológicos , Transdução de Sinais , Receptor Toll-Like 9/metabolismoRESUMO
BACKGROUND: BRAF-mutant melanoma is characterized by aggressive metastatic potential and therapeutic resistance. The innate immune receptor RIG-I has emerged as a potential target in melanoma therapies but the contributing pathways involved in anti-cancer activity are poorly characterized. METHODS: Baseline and ATRA-induced expression of RIG-I in nine (3 wild type and 6 BRAF-mutant) melanoma cell lines was measured with Q-PCR and Western blot. Ligand-specific stimulation of RIG-I was detected by Q-PCR and ELISA. Activation of the RIG-I-coupled IRF3, NF-κB and MAPK pathways was tested with protein array and Western blot. Cell proliferation and apoptosis was monitored by flow cytometry and cell counting. Down modulation of MKP-1 expression in melanoma cells was performed by specific siRNA. RESULTS: Short-term ATRA pre-treatment increases the expression of RIG-I in BRAF-mutant melanoma cells. Specific activation of RIG-I by 5'ppp-dsRNA leads to increased activity of the IRF3-IFNß pathway but does not influence NF-κB signaling. RIG-I mediates the targeted dephosphorylation of several MAPKs (p38, RSK1, GSK-3α/ß, HSP27) via the endogenous regulator MKP-1 resulting in decreased melanoma cell proliferation. CONCLUSION: RIG-I has the potential to exert anticancer activity in BRAF-mutant melanoma via controlling IFNß production and MAPK signaling. This is the first study showing that RIG-I activation results in MKP-1-mediated inhibition of cell proliferation via controlling the p38-HSP27, c-Jun and rpS6 pathways thus identifying RIG-I and MKP-1 as novel and promising therapeutical targets.
Assuntos
Proteína DEAD-box 58/metabolismo , Fosfatase 1 de Especificidade Dupla/metabolismo , Sistema de Sinalização das MAP Quinases , Melanoma/enzimologia , Proteínas Proto-Oncogênicas B-raf/genética , Linhagem Celular Tumoral , Proliferação de Células , Citocinas/metabolismo , Humanos , Fator Regulador 3 de Interferon/metabolismo , Melanoma/genética , Melanoma/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Receptores Imunológicos , Tretinoína/farmacologiaRESUMO
The commitment steps of mesenchymal stromal cells (MSCs) to adipogenic and other lineages have been widely studied but not fully understood. Therefore, it is critical to understand which molecules contribute to the conversion of stem cells into differentiated cells. The scaffold protein Tks4 plays a role in podosome formation, EGFR signaling and ROS production. Dysfunction of Tks4 causes a hereditary disease called Frank-ter Haar syndrome with a variety of defects concerning certain mesenchymal tissues (bone, fat and cartilage) throughout embryogenic and postnatal development. In this study, we aimed to analyze how the mutation of Tks4 affects the differentiation potential of multipotent bone marrow MSCs (BM-MSCs). We generated a Tks4 knock-out mouse strain on C57Bl/6 background, and characterized BM-MSCs isolated from wild type and Tks4-/- mice to evaluate their differentiation. Tks4-/- BM-MSCs had reduced ability to differentiate into osteogenic and adipogenic lineages compared to wild type. Studying the expression profile of a panel of lipid-regulated genes during adipogenic induction revealed that the expression of adipogenic transcription factors, genes responsible for lipid droplet formation, sterol and fatty acid metabolism was delayed or reduced in Tks4-/- BM-MSCs. Taken together, these results establish a novel function for Tks4 in the regulation of MSC differentiation.
Assuntos
Adipogenia , Diferenciação Celular , Células-Tronco Mesenquimais/metabolismo , Osteogênese , Fosfoproteínas/metabolismo , Transdução de Sinais , Proteínas Adaptadoras de Transdução de Sinal , Animais , Anormalidades Craniofaciais/genética , Anormalidades Craniofaciais/metabolismo , Deficiências do Desenvolvimento/genética , Deficiências do Desenvolvimento/metabolismo , Cardiopatias Congênitas/genética , Cardiopatias Congênitas/metabolismo , Camundongos , Camundongos Knockout , Osteocondrodisplasias/congênito , Osteocondrodisplasias/genética , Osteocondrodisplasias/metabolismo , Fosfoproteínas/genéticaRESUMO
Regulated production of ROS is mainly attributed to Nox family enzymes. In neutrophil granulocytes and macrophages, Nox2 has a crucial role in bacterial killing, and the absence of phagocytic ROS production leads to the development of CGD. Expression of Nox2 was also described in B lymphocytes, where the role of the enzyme is still poorly understood. Here, we show that peritoneal B cells, which were shown recently to possess phagocytic activity, have a high capacity to produce ROS in a Nox2-dependent manner. In phagocytosing B cells, intense intraphagosomal ROS production is detected. Finally, by studying 2 animal models of CGD, we demonstrate that phagocyte oxidase-deficient B cells have a reduced capacity to kill bacteria. Our observations extend the number of immune cell types that produce ROS to kill pathogens.