RESUMEN
A role for microglia in neuropsychiatric diseases, including major depressive disorder (MDD), has been postulated. Regulation of microglial phenotype by immune receptors has become a central topic in many neurological conditions. We explored preclinical and clinical evidence for the role of the CD300f immune receptor in the fine regulation of microglial phenotype and its contribution to MDD. We found that a prevalent nonsynonymous single-nucleotide polymorphism (C/T, rs2034310) of the human CD300f receptor cytoplasmic tail inhibits the protein kinase C phosphorylation of a threonine and is associated with protection against MDD, mainly in women. Interestingly, CD300f-/- mice displayed several characteristic MDD traits such as augmented microglial numbers, increased interleukin 6 and interleukin 1 receptor antagonist messenger RNA, alterations in synaptic strength, and noradrenaline-dependent and persistent depressive-like and anhedonic behaviors in females. This behavioral phenotype could be potentiated inducing the lipopolysaccharide depression model. RNA sequencing and biochemical studies revealed an association with impaired microglial metabolic fitness. In conclusion, we report a clear association that links the function of the CD300f immune receptor with MDD in humans, depressive-like and anhedonic behaviors in female mice, and altered microglial metabolic reprogramming.
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Anhedonia , Trastorno Depresivo Mayor/patología , Inflamación/etiología , Microglía/patología , Polimorfismo de Nucleótido Simple , Receptores Inmunológicos/genética , Receptores Inmunológicos/fisiología , Animales , Conducta Animal , Estudios de Cohortes , Trastorno Depresivo Mayor/genética , Trastorno Depresivo Mayor/metabolismo , Trastorno Depresivo Mayor/psicología , Femenino , Perfilación de la Expresión Génica , Humanos , Inflamación/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microglía/metabolismo , SinapsisRESUMEN
Tumor recurrence, metastatic spread and progressive gain of chemo-resistance of advanced cancers are sustained by the presence of cancer stem cells (CSCs) within the tumor. Targeted therapies with the aim to eradicate these cells are thus highly regarded. However, often the use of new anti-cancer therapies is hampered by pharmacokinetic demands. Drug delivery through nanoparticles has great potential to increase efficacy and reduce toxicity and adverse effects. However, its production has to be based on intelligent design. Likewise, we developed polymeric nanoparticles loaded with Zileuton™, a potent inhibitor of cancer stem cells (CSCs), which was chosen based on high throughput screening. Its great potential for CSCs treatment was subsequently demonstrated in in vitro and in in vivo CSC fluorescent models. Encapsulated Zileuton™ reduces amount of CSCs within the tumor and effectively blocks the circulating tumor cells (CTCs) in the blood stream and metastatic spread.
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Neoplasias de la Mama , Hidroxiurea/análogos & derivados , Micelas , Células Neoplásicas Circulantes , Células Madre Neoplásicas , Animales , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Femenino , Humanos , Hidroxiurea/química , Hidroxiurea/farmacología , Células MCF-7 , Ratones , Ratones Endogámicos NOD , Ratones SCID , Células Neoplásicas Circulantes/metabolismo , Células Neoplásicas Circulantes/patología , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
IL-4 receptor (R) α, the common receptor chain for IL-4 and IL-13, is a critical component in IL-4- and IL-13-mediated signaling and subsequent effector functions such as those observed in type 2 inflammatory responses. Nonetheless, the existence of intrinsic pathways capable of amplifying IL-4Rα-induced responses remains unknown. In this study, we identified the myeloid-associated Ig receptor CD300f as an IL-4-induced molecule in macrophages. Subsequent analyses demonstrated that CD300f was colocalized and physically associated with IL-4Rα. Using Cd300f(-/-) cells and receptor cross-linking experiments, we established that CD300f amplified IL-4Rα-induced responses by augmenting IL-4/IL-13-induced signaling, mediator release, and priming. Consistently, IL-4- and aeroallergen-treated Cd300f(-/-) mice displayed decreased IgE production, chemokine expression, and inflammatory cell recruitment. Impaired responses in Cd300f(-/-) mice were not due to the inability to generate a proper Th2 response, because IL-4/IL-13 levels were markedly increased in allergen-challenged Cd300f(-/-) mice, a finding that is consistent with decreased cytokine consumption. Finally, CD300f expression was increased in monocytes and eosinophils obtained from allergic rhinitis patients. Collectively, our data highlight a previously unidentified role for CD300f in IL-4Rα-induced immune cell responses. These data provide new insights into the molecular mechanisms governing IL-4Rα-induced responses, and may provide new therapeutic tools to target IL-4 in allergy and asthma.
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Sistema Inmunológico/inmunología , Subunidad alfa del Receptor de Interleucina-4/metabolismo , Interleucina-4/fisiología , Receptores Inmunológicos/metabolismo , Alérgenos/inmunología , Animales , Sistema Inmunológico/citología , Inmunoglobulina E/biosíntesis , Activación de Macrófagos/fisiología , Ratones , Ratones Noqueados , Unión Proteica , Receptores Inmunológicos/genética , Receptores Inmunológicos/fisiología , Regulación hacia Arriba/fisiologíaRESUMEN
SH3-binding protein 2 (3BP2) is a cytoplasmic adaptor protein that acts as a positive regulator in mast cell FcεRI-dependent signaling. The KIT receptor whose ligand is the stem cell factor is necessary for mast cell development, proliferation, and survival as well as for optimal IgE-dependent signal. Activating mutations in KIT have been associated with several diseases including mastocytosis. In the present work, we found that 3BP2 silencing impairs KIT signaling pathways, thus affecting phosphoinositide 3-kinase and MAPK pathways in human mast cells (huMCs) from HMC-1, LAD2 (huMC lines), and CD34(+)-derived mast cells. Unexpectedly, silencing of 3BP2 reduces KIT expression in normal huMCs as well as in HMC-1 cells where KIT is mutated, thus increasing cellular apoptosis and caspase-3/7 activity. 3BP2 silencing reduces KIT transcription expression levels. Interestingly, 3BP2 silencing decreased microphthalmia-associated transcription factor (MITF) expression, a transcription factor involved in KIT expression. Reconstitution of 3BP2 in knockdown cells leads to reversal of KIT expression as well as survival phenotype. Accordingly MITF reconstitution enhances KIT expression levels in 3BP2-silenced cells. Moreover, downregulation of KIT expression by miRNA-221 overexpression or the proteasome inhibitor bortezomib also reduced 3BP2 and MITF expression. Furthermore, KIT tyrosine activity inhibition reduced 3BP2 and MITF expression, demonstrating again a tight and reciprocal relationship between these molecules. Taken together, our results show that 3BP2 regulates huMC survival and participates in KIT-mediated signal transduction by directly controlling KIT receptor expression, suggesting its potential as a therapeutic target in mast cell-mediated inflammatory diseases and deregulated KIT disorders.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Regulación de la Expresión Génica , Mastocitos/metabolismo , Proteínas Proto-Oncogénicas c-kit/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Apoptosis/genética , Caspasa 3/metabolismo , Caspasa 7/metabolismo , Línea Celular , Supervivencia Celular/genética , Silenciador del Gen , Humanos , Mastocitos/inmunología , Factor de Transcripción Asociado a Microftalmía/metabolismo , Mutación , Proteínas Proto-Oncogénicas c-kit/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transducción de Señal , Transcripción GenéticaRESUMEN
BACKGROUND: It has recently become evident that activating/inhibitory cell surface immune receptors play a critical role in regulating immune and inflammatory processes in the central nervous system (CNS). The immunoreceptor CD300f expressed on monocytes, neutrophils, and mast cells modulates inflammation, phagocytosis, and outcome in models of autoimmune demyelination, allergy, and systemic lupus erythematosus. On the other hand, a finely regulated inflammatory response is essential to induce regeneration after injury to peripheral nerves since hematogenous macrophages, together with resident macrophages and de-differentiated Schwann cells, phagocyte distal axonal and myelin debris in a well-orchestrated inflammatory response. The possible roles and expression of CD300f and its ligands have not been reported under these conditions. METHODS: By using quantitative PCR (QPCR) and CD300f-IgG2a fusion protein, we show the expression of CD300f and its ligands in the normal and crush injured sciatic nerve. The putative role of CD300f in peripheral nerve regeneration was analyzed by blocking receptor-ligand interaction with the same CD300f-IgG2a soluble receptor fusion protein in sciatic nerves of Thy1-YFP-H mice injected at the time of injury. Macrophage M1/M2 polarization phenotype was also analyzed by CD206 and iNOS expression. RESULTS: We found an upregulation of CD300f mRNA and protein expression after injury. Moreover, the ligands are present in restricted membrane patches of Schwann cells, which remain stable after the lesion. The lesioned sciatic nerves of Thy1-YFP-H mice injected with a single dose of CD300f-IgG2a show long lasting effects on nerve regeneration characterized by a lower number of YFP-positive fibres growing into the tibial nerve after 10 days post lesion (dpl) and a delayed functional recovery when compared to PBS- or IgG2a-administered control groups. Animals treated with CD300f-IgG2a show at 10 dpl higher numbers of macrophages and CD206-positive cells and lower levels of iNOS expression than both control groups. At later time points (28 dpl), increased numbers of macrophages and iNOS expression occur. CONCLUSIONS: Taken together, these results show that the pair CD300f ligand is implicated in Wallerian degeneration and nerve regeneration by modulating both the influx and phenotype of macrophages.
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Inflamación/patología , Macrófagos/patología , Regeneración Nerviosa , Receptores Inmunológicos/genética , Animales , Axones/patología , Células CHO , Cricetinae , Cricetulus , Femenino , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/genética , Lectinas Tipo C/metabolismo , Ligandos , Masculino , Receptor de Manosa , Lectinas de Unión a Manosa/metabolismo , Ratones , Compresión Nerviosa , Óxido Nítrico Sintasa de Tipo II/biosíntesis , Nervios Periféricos/patología , Fagocitosis , Fenotipo , Receptores de Superficie Celular/metabolismo , Células de Schwann/patología , Neuropatía Ciática/patología , Degeneración Walleriana/patologíaRESUMEN
Adaptor molecules are essential in organizing signaling molecules and in coordinating and compartmentalizing their activity. SH3-binding protein 2 (3BP2) is a cytoplasmic adaptor protein mainly expressed by hematopoietic cells that has been shown to act as a positive regulator in T, B, and NK cell signal transduction. 3BP2 is an important regulator of cytotoxic granule release in NK cells. Mast cells (MCs) similarly degranulate following Ag-dependent aggregation of the FcεRI on the cell surface. Activation of these cells induces the release of preformed inflammatory mediators and the de novo synthesis and secretion of cytokines and chemokines. Thus, MCs participate in both innate and acquired responses. We observed that 3BP2 is expressed in human MCs (huMCs) from diverse origins. Moreover, 3BP2 coimmunoprecipitates with essential MC signaling mediators such as Lyn, Syk, and phospholipase C γ; thus, a role for this adaptor in MC function was postulated. In the present work, we used the short hairpin RNA lentiviral targeting approach to silence 3BP2 expression in huMCs. Our findings point to a requirement for 3BP2 in optimal immediate and late MCs responses such as degranulation and IL-8 or GM-CSF secretion. 3BP2 was determined to be necessary for optimal phosphorylation of Syk, linker for activation of T cells, and phospholipase C γ(1), critical signals for calcium release from intracellular stores. Taken together, our results show that by participating in FcεRI- mediated signal transduction 3BP2 is an important regulator of huMC activation. Thus, 3BP2 could be a potential therapeutic target for IgE-dependent MC-mediated inflammatory disease.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/fisiología , Mastocitos/inmunología , Mastocitos/metabolismo , Receptores de IgE/fisiología , Transducción de Señal/inmunología , Degranulación de la Célula/inmunología , Línea Celular , Humanos , Factores de TiempoRESUMEN
Herein we present the cloning and molecular characterization of CD300d, a member of the human CD300 family of immune receptors. CD300d cDNA was cloned from RNA obtained from human peripheral blood mononuclear cells, and RT-PCR revealed the gene to be expressed in cells of myeloid lineage. The cloned cDNA encoded for a type I protein with a single extracellular Ig V-type domain and a predicted molecular mass of 21.5 kDa. The short cytoplasmic tail is lacking in any known signaling motif, but there is a negatively charged residue (glutamic acid) within the transmembrane domain. CD300d forms complexes with the CD300 family members, with the exception of CD300c. Contrary to other activating members of the CD300 family of receptors, surface expression of CD300d in COS-7-transfected cells required the presence of an immunoreceptor tyrosine-based activating motif-bearing adaptor (FcεRγ). Accordingly, we found that CD300d was able to recruit FcεRγ. Unexpectedly, we could not detect CD300d on the surface of cells expressing FcεRγ, suggesting the existence of unknown mechanisms regulating the trafficking of this molecule. The presence of other CD300 molecules also did not modify the intracellular expression of CD300d. In fact, the presence of CD300d decreased the levels of surface expression of CD300f but not CD300c. Our data suggest that the function of CD300d would be related to the regulation of the expression of other CD300 molecules and the composition of CD300 complexes on the cell surface.
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Regulación de la Expresión Génica/fisiología , Leucocitos Mononucleares/metabolismo , Complejos Multiproteicos/metabolismo , Receptores Inmunológicos/biosíntesis , Receptores Inmunológicos/genética , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Células COS , Chlorocebus aethiops , Clonación Molecular , ADN Complementario/genética , Células HeLa , Humanos , Leucocitos Mononucleares/citología , Datos de Secuencia Molecular , Complejos Multiproteicos/genética , Estructura Terciaria de Proteína , Transporte de Proteínas , Receptores de IgE/genética , Receptores de IgE/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Human SMC2 is part of the condensin complex, which is responsible for tightly packaging replicated genomic DNA prior to segregation into daughter cells. Engagement of the WNT signaling pathway is known to have a mitogenic effect on cells, but relatively little is known about WNT interaction with mitotic structural organizer proteins. In this work, we described the novel transcriptional regulation of SMC2 protein by direct binding of the ß-catenin·TCF4 transcription factor to the SMC2 promoter. Furthermore, we identified the precise region in the SMC2 promoter that is required for ß-catenin-mediated promoter activation. Finally, we explored the functional significance of down-regulating SMC2 protein in vivo. Treatment of WNT-activated intestinal tumor cells with SMC2 siRNA significantly reduced cell proliferation in nude mice, compared with untreated controls (p = 0.02). Therefore, we propose that WNT signaling can directly activate SMC2 transcription as a key player in the mitotic cell division machinery. Furthermore, SMC2 represents a new target for oncological therapeutic intervention.
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Adenosina Trifosfatasas/metabolismo , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Unión al ADN/metabolismo , Complejos Multiproteicos/metabolismo , Proteínas de Neoplasias/metabolismo , Neoplasias/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Vía de Señalización Wnt , Adenosina Trifosfatasas/genética , Animales , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Proteínas Portadoras/genética , Proteínas de Ciclo Celular , Línea Celular Tumoral , Proteínas de Unión al ADN/genética , Humanos , Macaca , Ratones , Ratones Desnudos , Mitosis/genética , Complejos Multiproteicos/genética , Proteínas de Neoplasias/genética , Trasplante de Neoplasias , Neoplasias/genética , Neoplasias/terapia , Proteínas Nucleares/genética , Pan troglodytes , Regiones Promotoras Genéticas , Unión Proteica , Ratas , Factor de Transcripción 4 , Factores de Transcripción/genética , Transcripción Genética/genética , Trasplante Heterólogo , beta Catenina/genética , beta Catenina/metabolismoRESUMEN
CD84 is a self-binding receptor from the CD150 (or signaling lymphocyte activation molecule [SLAM]) family that is broadly expressed in hematopoietic cells. It has been described that the adaptors SLAM-associated protein (SAP) and EWS-FLI1-activated transcript 2 (EAT-2) are critical for CD150 family members' signaling and function. We observed that human mast cells express CD84 but lack SAP or EAT-2, that CD84 is tyrosine phosphorylated upon FcεRI engagement, and that the release of granule contents is reduced when FcεRI is coengaged with CD84 in LAD2 and human CD34(+)-derived mast cells. In addition, we observed that the release of IL-8 and GM-CSF was also reduced in FcεRI/CD84-costimulated cells as compared with FcεRI/Ig control. To understand how CD84 downregulates FcεRI-mediated function, we analyzed signaling pathways affected by CD84 in human mast cells. Our results showed that CD84 dampens FcεRI-mediated calcium mobilization after its co-cross-linking with the receptor. Furthermore, FcεRI-mediated Syk-linker for activation of T cells-phospholipase C-γ1 axis activity is downregulated after CD84 stimulation, compared with FcεRI/Ig control. The inhibitory kinase Fes phosphorylates mainly the inhibitory motif for CD84. Moreover, Fes, which has been described to become phosphorylated after substrate binding, also gets phosphorylated when coexpressed with CD84. Consistently, Fes was observed to be more phosphorylated after CD84 and FcεRI co-cross-linking. The phosphorylation of the protein phosphatase Src homology region 2 domain-containing phosphatase-1 also increases after CD84 and FcεRI coengagement. Taken together, our results show that CD84 is highly expressed in mast cells and that it contributes to the regulation of FcεRI signaling in SAP- and EAT-2-independent and Fes- and Src homology region 2 domain-containing phosphatase-1-dependent mechanisms.
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Antígenos CD/inmunología , Mastocitos/inmunología , Receptores de IgE/inmunología , Transducción de Señal/inmunología , Antígenos CD/metabolismo , Degranulación de la Célula/inmunología , Línea Celular , Separación Celular , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Humanos , Immunoblotting , Inmunoprecipitación , Mastocitos/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Familia de Moléculas Señalizadoras de la Activación Linfocitaria , Transfección , Técnicas del Sistema de Dos HíbridosRESUMEN
The CD300 family of myeloid immunoglobulin receptors includes activating (CD300b, CD300e) and inhibitory members (CD300a, CD300f), as well as molecules of uncertain function presenting a negative charge within their transmembrane domain (CD300c, CD300d). In this paper, we establish that CD300c is a functional immune receptor able to deliver activating signals upon ligation in RBL-2H3 mast cells. CD300c signaling is partially mediated by a direct association with the immune receptor tyrosine-based activation motif-bearing adaptor FcεRγ. The existence of complementary transmembrane-charged residues in certain CD300 receptors suggested the formation of heterodimers within this family. Indeed, we proved the interaction between CD300b and CD300c in transfected COS-7 cells and demonstrated that it has important functional consequences. Unexpectedly, dimmer formation was dependent on the immunoglobulin domains rather than the charged transmembrane residues. Concordantly, all CD300 members were found to interact with each other, even with themselves, forming both homo- and heterodimers. We found that the combination of CD300 receptors in a complex differentially modulates the signaling outcome, strongly suggesting a new mechanism by which CD300 complexes could regulate the activation of myeloid cells upon interaction with their natural ligands.
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Antígenos de Superficie/metabolismo , Glicoproteínas de Membrana/metabolismo , Células Mieloides/citología , Receptores Inmunológicos/metabolismo , Transducción de Señal , Secuencias de Aminoácidos , Animales , Células COS , Membrana Celular/metabolismo , Chlorocebus aethiops , Dimerización , Citometría de Flujo/métodos , Sistema Inmunológico , Inmunidad Innata , ARN Interferente Pequeño/metabolismo , Tirosina/químicaRESUMEN
Hemophagocytic lymphohistiocytosis (HLH) is a life-threatening hyperinflammatory disorder. HLH can be considered as a threshold disease depending on the trigger and the residual NK-cell cytotoxicity. In this study, we analyzed the molecular and functional impact of a novel monoallelic mutation found in a patient with two episodes of HLH. A 9-month-old child was diagnosed at 2 months of age with cutaneous Langerhans cell histiocytosis (LCH). After successful treatment, the patient developed an HLH episode. At 16 month of age, the patient went through an HSCT losing the engraftment 5 months later concomitant with an HLH relapse. The genetic study revealed a monoallelic mutation in the STXBP2 gene (.pArg190Cys). We transfected COS7 cells to analyze the STXBP2-R190C expression and to test the interaction with STX11. We used the RBL-2H3 cell line expressing STXBP2-WT-EGFP or R190C-EGFP for degranulation assays. Mutation STXBP2-R190C did not affect protein expression or interaction with syntaxin-11. However, we have demonstrated that STXBP2-R190C mutation diminishes degranulation in the RBL-2H3 cell line compared with the RBL-2H3 cell line transfected with STXBP2-WT or nontransfected. These results suggest that STXBP2-R190C mutation acts as a modifier of the degranulation process producing a decrease in degranulation. Therefore, under homeostatic conditions, the presence of one copy of STXBP2-R190 could generate sufficient degranulation capacity. However, it is likely that early in life when adaptive immune system functions are not sufficiently developed, an infection may not be resolved with this genetic background, leading to a hyperinflammation syndrome and eventually develop HLH. This analysis highlights the need for functional testing of new mutations to validate their role in genetic susceptibility and to establish the best possible treatment for these patients.
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Histiocitosis de Células de Langerhans/diagnóstico , Histiocitosis de Células de Langerhans/genética , Linfohistiocitosis Hemofagocítica/diagnóstico , Linfohistiocitosis Hemofagocítica/genética , Proteínas Munc18/genética , Citotoxicidad Inmunológica , Predisposición Genética a la Enfermedad , Histiocitosis de Células de Langerhans/complicaciones , Humanos , Lactante , Linfohistiocitosis Hemofagocítica/complicaciones , Masculino , MutaciónRESUMEN
The presence of mutations in PRF1, UNC13D, STX11 and STXBP2 genes in homozygosis or compound heterozygosis results in immune deregulation. Most such cases lead to clinical manifestations of haemophagocytic lymphohistiocytosis (HLH). In the present study, we analyzed degranulation and cytotoxicity in a pediatric patient with a late presentation of HLH associated with Epstein-Barr virus infection. Remarkably, the results of the degranulation assay showed reduction of CD107a median fluorescence intensity (MFI) and absent cytotoxicity. Genetic analysis identified compound heterozygous mutations in STXBP2 gene: a previously reported splicing defect in exon 15 (c.1247-1G>C, p.V417LfsX126) and a novel missense mutation in exon 9 (c.728T>G, p.L243R). Transfection experiments of STXBP2-L243R or STXBP2-WT constructs showed an undetectable protein expression of the STXBP2-L243R mutation. The residue L243 is highly preserved evolutionarily; moreover, computational analysis of its structure revealed its participation in the rich network of interactions that stabilizes domains 2 and 3 of the protein. Altogether, we demonstrated by molecular and in silico analysis that the new L243R mutation in STXBP2 plays a pathogenic role that, together with the p.Val417Leufsc mutation, shows the synergistic negative effect of these two mutations on STXBP2 function, leading to a decrease of degranulatory activity in vivo.
Asunto(s)
Degranulación de la Célula , Linfohistiocitosis Hemofagocítica/genética , Linfohistiocitosis Hemofagocítica/patología , Proteínas Munc18/genética , Mutación , Animales , Células COS , Preescolar , Chlorocebus aethiops , Infecciones por Virus de Epstein-Barr/complicaciones , Humanos , Linfohistiocitosis Hemofagocítica/complicaciones , MasculinoRESUMEN
Natural killer (NK) cell cytotoxicity requires triggering of activation receptors over inhibitory receptors. CD244, a member of CD150 receptor family, positively regulates NK-mediated lyses by activating an intracellular multiproteic signaling network that involves the adaptors X-linked lymphoproliferative gene product SAP and 3BP2. However, the exact mechanisms used by 3BP2 to enhance CD244-mediated cytotoxicity are still not fully understood. Here using the human NK cell line YT-overexpressing 3BP2, we found that the adaptor increases CD244, PI3K, and Vav phosphorylation upon CD244 engagement. The use of enzymatic inhibitors revealed that 3BP2-dependent cytolysis enhancement was PKC-dependent and PI3K-ERK independent. Furthermore, 3BP2 overexpression enhanced PKC delta phosphorylation. SAP knockdown expression inhibited PKC delta activation, indicating that the activating role played by 3BP2 depends upon the presence of SAP. In conclusion, our data show that 3BP2 acts downstream of SAP, increases CD244 phosphorylation and links the receptor with PI3K, Vav, PLC gamma, and PKC downstream events in order to achieve maximum NK killing function.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , Antígenos CD/metabolismo , Citotoxicidad Inmunológica , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteína Quinasa C-delta/metabolismo , Receptores Inmunológicos/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Androstadienos/farmacología , Animales , Línea Celular , Citotoxicidad Inmunológica/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Inmunoprecipitación , Ratones , Modelos Inmunológicos , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfolipasa C gamma/metabolismo , Fosforilación/efectos de los fármacos , Fosfotirosina/metabolismo , Unión Proteica/efectos de los fármacos , Proteínas Proto-Oncogénicas c-vav/metabolismo , Transducción de Señal/efectos de los fármacos , Proteína Asociada a la Molécula de Señalización de la Activación Linfocitaria , Familia de Moléculas Señalizadoras de la Activación Linfocitaria , WortmaninaRESUMEN
Mast cell chemotaxis is essential for cell recruitment to target tissues, where these cells play an important role in adaptive and innate immunity. Stem cell factor (SCF) is a major chemoattractant for mast cells. SCF binds to the KIT receptor, thereby triggering tyrosine phosphorylation in the cytoplasmic domain and resulting in docking sites for SH2 domain-containing molecules, such as Lyn and Fyn, and the subsequent activation of the small GTPases Rac that are responsible for cytoskeletal reorganization and mast cell migration. In previous works we have reported the role of 3BP2, an adaptor molecule, in mast cells. 3BP2 silencing reduces FcεRI-dependent degranulation, by targeting Lyn and Syk phosphorylation, as well as SCF-dependent cell survival. This study examines its role in SCF-dependent migration and reveals that 3BP2 silencing in human mast cell line (LAD2) impairs cell migration due to SCF and IgE. In that context we found that 3BP2 silencing decreases Rac-2 and Cdc42 GTPase activity. Furthermore, we identified Myo1f, an unconventional type-I myosin, as a new partner for 3BP2. This protein, whose functions have been described as critical for neutrophil migration, remained elusive in mast cells. Myo1f is expressed in mast cells and colocalizes with cortical actin ring. Interestingly, Myo1f-3BP2 interaction is modulated by KIT signaling. Moreover, SCF dependent adhesion and migration through fibronectin is decreased after Myo1f silencing. Furthermore, Myo1f silencing leads to downregulation of ß1 and ß7 integrins on the mast cell membrane. Overall, Myo1f is a new 3BP2 ligand that connects the adaptor to actin cytoskeleton and both molecules are involved in SCF dependent mast cell migration.
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Citoesqueleto de Actina/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Mastocitos/fisiología , Miosina Tipo I/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Línea Celular , Movimiento Celular , Quimiotaxis , Humanos , Inmunoglobulina E/metabolismo , Miosina Tipo I/genética , Proteínas Proto-Oncogénicas c-kit/genética , Proteínas Proto-Oncogénicas c-kit/metabolismo , ARN Interferente Pequeño/genética , Transducción de Señal , Factor de Células Madre/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Proteínas de Unión al GTP rac/metabolismo , Proteína RCA2 de Unión a GTPRESUMEN
Development of RNA interference-based therapies with appropriate therapeutic window remains a challenge for advanced cancers. Because cancer stem cells (CSC) are responsible of sustaining the metastatic spread of the disease to distal organs and the progressive gain of resistance of advanced cancers, new anticancer therapies should be validated specifically for this subpopulation of cells. A new amphihilic-based gene delivery system that combines Pluronic® F127 micelles with polyplexes spontaneously formed by electrostatic interaction between anionic siRNA and cationic polyethylenimine (PEI) 10K, was designed (PM). Resultant PM gather the requirements for an efficient and safe transport of siRNA in terms of its physicochemical characteristics, internalization capacity, toxicity profile and silencing efficacy. PM were loaded with a siRNA against AKT2, an important oncogene involved in breast cancer tumorigenesis, with a special role in CSC malignancy. Efficacy of siAKT2-PM was validated in CSC isolated from two breast cancer cell lines: MCF-7 and Triple Negative MDA-MB-231 corresponding to an aggressive subtype of breast cancer. In both cases, we observed significant reduction on cell invasion capacity and strong inhibition of mammosphere formation after treatment. These results prompt AKT2 inhibition as a powerful therapeutic target against CSC and pave the way to the appearance of more effective nanomedicine-based gene therapies aimed to prevent CSC-related tumor recurrence.
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Antineoplásicos/farmacología , Células Madre Neoplásicas/efectos de los fármacos , Polímeros/química , Proteínas Proto-Oncogénicas c-akt/genética , ARN Interferente Pequeño/genética , Antineoplásicos/química , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Línea Celular Tumoral , Sistemas de Liberación de Medicamentos/métodos , Femenino , Técnicas de Transferencia de Gen , Humanos , Células MCF-7 , Micelas , Nanomedicina/métodos , Poloxámero/química , Polietileneimina/química , Interferencia de ARN/efectos de los fármacosRESUMEN
Gastrointestinal stromal tumors (GISTs) represent about 80% of the mesenchymal neoplasms of the gastrointestinal tract. Most GISTs contain oncogenic KIT (85%) or PDGFRA (5%) receptors. The kinase inhibitor imatinib mesylate is the preferential treatment for these tumors; however, the development of drug resistance has highlighted the need for novel therapeutic strategies. Recently, we reported that the adaptor molecule SH3 Binding Protein 2 (SH3BP2) regulates KIT expression and signaling in human mast cells. Our current study shows that SH3BP2 is expressed in primary tumors and cell lines from GIST patients and that SH3BP2 silencing leads to a downregulation of oncogenic KIT and PDGFRA expression and an increase in apoptosis in imatinib-sensitive and imatinib-resistant GIST cells. The microphthalmia-associated transcription factor (MITF), involved in KIT expression in mast cells and melanocytes, is expressed in GISTs. Interestingly, MITF is reduced after SH3BP2 silencing. Importantly, reconstitution of both SH3BP2 and MITF restores cell viability. Furthermore, SH3BP2 silencing significantly reduces cell migration and tumor growth of imatinib-sensitive and imatinib-resistant cells in vivo. Altogether, SH3BP2 regulates KIT and PDGFRA expression and cell viability, indicating a role as a potential target in imatinib-sensitive and imatinib-resistant GISTs.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Neoplasias Gastrointestinales/genética , Tumores del Estroma Gastrointestinal/genética , Silenciador del Gen , Proteínas Proto-Oncogénicas c-kit/genética , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/genética , Animales , Antineoplásicos/farmacología , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Resistencia a Antineoplásicos , Femenino , Neoplasias Gastrointestinales/tratamiento farmacológico , Neoplasias Gastrointestinales/patología , Tumores del Estroma Gastrointestinal/tratamiento farmacológico , Tumores del Estroma Gastrointestinal/patología , Regulación Neoplásica de la Expresión Génica , Humanos , Mesilato de Imatinib/farmacología , Ratones DesnudosRESUMEN
There are remarkable similarities in the description of cancer stem cells (CSCs) and cancer cells with mesenchymal phenotype. Both cell types are highly tumorigenic, resistant against common anticancer treatment, and thought to cause metastatic growth. Moreover, cancer cells are able to switch between CSC and non-CSC phenotypes and vice versa, to ensure the necessary balance within the tumor. Likewise, cancer cells can switch between epithelial and mesenchymal phenotypes via well-described transition (EMT/MET) that is thought to be crucial for tumor propagation. In this review, we discuss whether, and to which extend, the CSCs and mesenchymal cancer cells are overlapping phenomena in terms of mechanisms, origin, and implication for cancer treatment. As well, we describe the dynamism of both phenotypes and involvement of the tumor microenvironment in CSC reversion and in EMT.
RESUMEN
BACKGROUND: Astrocytes contribute to neuroinflammation that accompanies neurodegenerative disorders such as Alzheimer's disease (AD). In this sense, the toxicity of these diseases might be attenuated through the modulation of astrocytic inflammatory responses. Recently, the CD300f immunoreceptor was described as a new member of the CD300 immunoreceptor family, showing promising modulatory properties. OBJECTIVE: Here, we investigated whether overexpression of hCD300f (the human isoform of CD300f) in astrocytes protects hippocampal neurons against the degeneration induced by amyloid-beta (Aß) oligomer. METHOD: Astrocyte monolayers were transfected with hCD300f before seeding the hippocampal neurons, and then the co-culture was exposed to Aß1-42 oligomers (5 µM, 48h). RESULTS: hCD300f expression significantly abrogated the neuronal loss elicited by Aß. This effect was dependent on neuron-astrocyte cell-cell interactions since no protection was observed using conditioned media from transfected astrocytes. Astrocyte modulation was dependent on the cytoplasmic signaling tail of hCD300f. Furthermore hCD300f expression did not affect the ability of astrocytes to uptake Aß1- 42 oligomers by endocytosis, which discards the possibility that increased Aß1-42 clearance could mediate neuroprotection by hCD300f. CONCLUSION: These results suggest that the astrocyte-directed expression of the hCD300f immune receptor can be a neuroprotective strategy in AD disease.
Asunto(s)
Péptidos beta-Amiloides/toxicidad , Astrocitos/metabolismo , Hipocampo/citología , Neuronas/efectos de los fármacos , Fragmentos de Péptidos/toxicidad , Receptores Inmunológicos/metabolismo , Péptidos beta-Amiloides/metabolismo , Análisis de Varianza , Animales , Animales Recién Nacidos , Células Cultivadas , Endocitosis/efectos de los fármacos , Proteína Ácida Fibrilar de la Glía/metabolismo , Masculino , Ratas , Ratas WistarRESUMEN
Herein, we have used bioinformatics tools to predict five clusters defining ligand-binding sites on the extracellular domain of human CD300b receptor, presumably involved in the formation of both homodimers and heterodimers with other CD300 family members. Site-directed mutagenesis revealed residues glutamic acid 28 and glutamine 29 in cluster 5 to be necessary for the formation of CD300b complexes. Surprisingly, the disruption of cluster 2 and 4 reconstituted the binding capability lost by the mutation of residues glutamic acid 28 to alanine, glutamine 29 to alanine (E28A-Q29G). We identified a missense mutation arginine 33 to glutamine (R33Q) in CD300f by direct sequencing of exon 2 in peripheral blood samples from 50 patients with multiple sclerosis (MS). Levels of expression of CD300f were almost undetectable on monocytes from the patient bearing the R33Q mutation compared with healthy individuals. Whereas R33Q mutation had no effect in the formation of CD300f complexes, the inhibition of protein synthesis with cycloheximide indicated that CD300f R33Q is less stable than native CD300f. Finally, we report that the levels of expression of CD300f on the surface of classical and intermediate monocytes from MS patients are significantly lower when compared to the same cell populations in healthy individuals.