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1.
Immunol Rev ; 317(1): 203-222, 2023 08.
Artículo en Inglés | MEDLINE | ID: mdl-37096808

RESUMEN

Lysophosphatidic acid (LPA) is an endogenous bioactive lipid that is produced extracellularly and signals to cells via cognate LPA receptors, which are G-protein coupled receptors (GPCRs). Mature lymphocytes in mice and humans express three LPA receptors, LPA2 , LPA5, and LPA6 , and work from our group has determined that LPA5 signaling by T lymphocytes inhibits specific antigen-receptor signaling pathways that ultimately impair lymphocyte activation, proliferation, and function. In this review, we discuss previous and ongoing work characterizing the ability of an LPA-LPA5 axis to serve as a peripheral immunological tolerance mechanism that restrains adaptive immunity but is subverted during settings of chronic inflammation. Specifically, LPA-LPA5 signaling is found to regulate effector cytotoxic CD8 T cells by (at least) two mechanisms: (i) regulating the actin-microtubule cytoskeleton in a manner that impairs immunological synapse formation between an effector CD8 T cell and antigen-specific target cell, thus directly impairing cytotoxic activity, and (ii) shifting T-cell metabolism to depend on fatty-acid oxidation for mitochondrial respiration and reducing metabolic efficiency. The in vivo outcome of LPA5 inhibitory activity impairs CD8 T-cell killing and tumor immunity in mouse models providing impetus to consider LPA5 antagonism for the treatment of malignancies and chronic infections.


Asunto(s)
Antineoplásicos , Linfocitos T CD8-positivos , Humanos , Ratones , Animales , Lisofosfolípidos/metabolismo , Transducción de Señal , Receptores del Ácido Lisofosfatídico/metabolismo
2.
Proc Natl Acad Sci U S A ; 119(15): e2118816119, 2022 04 12.
Artículo en Inglés | MEDLINE | ID: mdl-35394866

RESUMEN

Cancer and chronic infections often increase levels of the bioactive lipid, lysophosphatidic acid (LPA), that we have demonstrated acts as an inhibitory ligand upon binding LPAR5 on CD8 T cells, suppressing cytotoxic activity and tumor control. This study, using human and mouse primary T lymphocytes, reveals how LPA disrupts antigen-specific CD8 T cell:target cell immune synapse (IS) formation and T cell function via competing for cytoskeletal regulation. Specifically, we find upon antigen-specific T cell:target cell formation, IP3R1 localizes to the IS by a process dependent on mDia1 and actin and microtubule polymerization. LPA not only inhibited IP3R1 from reaching the IS but also altered T cell receptor (TCR)­induced localization of RhoA and mDia1 impairing F-actin accumulation and altering the tubulin code. Consequently, LPA impeded calcium store release and IS-directed cytokine secretion. Thus, targeting LPA signaling in chronic inflammatory conditions may rescue T cell function and promote antiviral and antitumor immunity.


Asunto(s)
Linfocitos T CD8-positivos , Sinapsis Inmunológicas , Infecciones , Lisofosfolípidos , Neoplasias , Animales , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/inmunología , Citoesqueleto/efectos de los fármacos , Citoesqueleto/inmunología , Humanos , Sinapsis Inmunológicas/efectos de los fármacos , Sinapsis Inmunológicas/inmunología , Infecciones/inmunología , Receptores de Inositol 1,4,5-Trifosfato/metabolismo , Lisofosfolípidos/metabolismo , Lisofosfolípidos/farmacología , Ratones , Neoplasias/inmunología , Receptores del Ácido Lisofosfatídico/metabolismo
3.
J Immunol ; 201(3): 1086-1096, 2018 08 01.
Artículo en Inglés | MEDLINE | ID: mdl-29914885

RESUMEN

The bone marrow microenvironment harbors and protects leukemic cells from apoptosis-inducing agents via mechanisms that are incompletely understood. We previously showed SDF-1 (CXCL-12), a chemokine readily abundant within the bone marrow microenvironment, induces apoptosis in acute myeloid leukemia (AML) cells that express high levels of the SDF-1 receptor CXCR4. However, differentiating osteoblasts found within this niche protect cocultured AML cells from apoptosis. Additionally, this protection was abrogated upon treatment of the differentiating osteoblasts with histone deacetylase inhibitors (HDACi). In this study, we begin to characterize and target the molecular mechanisms that mediate this osteoblast protection. Quantitative RT-PCR revealed that HDACi treatment of differentiating osteoblasts (mouse MC3T3 osteoblast cell line) reduced expression of multiple genes required for osteoblast differentiation, including genes important for producing mineralized bone matrix. Interestingly, pretreating differentiating osteoblasts with cyclosporine A, a drug known to inhibit osteoblast differentiation, similarly impaired osteoblast-mediated protection of cocultured AML cells (KG1a and U937 human AML cell lines). Both HDACi and cyclosporine A reduced osteoblast expression of the key mineralization enzyme tissue-nonspecific alkaline phosphatase (TNAP; encoded by Alpl). Moreover, specifically reducing TNAP expression or activity in differentiating osteoblasts significantly impaired the ability of the osteoblasts to protect cocultured AML cells. Together, our results indicate that inhibiting osteoblast matrix mineralization by specifically targeting TNAP is sufficient to significantly impair osteoblast-mediated protection of AML cells. Therefore, designing combination therapies that additionally target the osteoblast-produced mineralized bone matrix may improve treatment of AML by reducing the protection of leukemic cells within the bone marrow microenvironment.


Asunto(s)
Fosfatasa Alcalina/metabolismo , Apoptosis/fisiología , Leucemia Mieloide Aguda/metabolismo , Osteoblastos/metabolismo , Células 3T3 , Animales , Apoptosis/efectos de los fármacos , Médula Ósea/efectos de los fármacos , Médula Ósea/metabolismo , Calcificación Fisiológica/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Línea Celular , Línea Celular Tumoral , Microambiente Celular/efectos de los fármacos , Quimiocina CXCL12/metabolismo , Técnicas de Cocultivo/métodos , Inhibidores de Histona Desacetilasas/farmacología , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Ratones , Receptores CXCR4/metabolismo , Células U937
4.
J Biol Chem ; 293(36): 14022-14039, 2018 09 07.
Artículo en Inglés | MEDLINE | ID: mdl-30018141

RESUMEN

The immune system includes abundant examples of biologically-relevant cross-regulation of signaling pathways by the T cell antigen receptor (TCR) and the G protein-coupled chemokine receptor, CXCR4. TCR ligation induces transactivation of CXCR4 and TCR-CXCR4 complex formation, permitting the TCR to signal via CXCR4 to activate a phosphatidylinositol 3,4,5-trisphosphate-dependent Rac exchanger 1 protein (PREX1)-dependent signaling pathway that drives robust cytokine secretion by T cells. To understand this receptor heterodimer and its regulation, we characterized the molecular mechanisms required for TCR-mediated TCR-CXCR4 complex formation. We found that the cytoplasmic C-terminal domain of CXCR4 and specifically phosphorylation of Ser-339 within this region were required for TCR-CXCR4 complex formation. Interestingly, siRNA-mediated depletion of G protein-coupled receptor kinase-2 (GRK2) or inhibition by the GRK2-specific inhibitor, paroxetine, inhibited TCR-induced phosphorylation of CXCR4-Ser-339 and TCR-CXCR4 complex formation. Either GRK2 siRNA or paroxetine treatment of human T cells significantly reduced T cell cytokine production. Upstream, TCR-activated tyrosine kinases caused inducible tyrosine phosphorylation of GRK2 and were required for the GRK2-dependent events of CXCR4-Ser-339 phosphorylation and TCR-CXCR4 complex formation. Downstream of TCR-CXCR4 complex formation, we found that GRK2 and phosphatidylinositol 3-kinase γ (PI3Kγ) were required for TCR-stimulated membrane recruitment of PREX1 and for stabilization of cytokine mRNAs and robust cytokine secretion. Together, our results identify a novel role for GRK2 as a target of TCR signaling that is responsible for TCR-induced transactivation of CXCR4 and TCR-CXCR4 complex formation that signals via PI3Kγ/PREX1 to mediate cytokine production. Therapeutic regulation of GRK2 or PI3Kγ may therefore be useful for limiting cytokines produced by T cell malignancies or autoimmune diseases.


Asunto(s)
Fosfatidilinositol 3-Quinasa Clase Ib/metabolismo , Citocinas/metabolismo , Quinasa 2 del Receptor Acoplado a Proteína-G/fisiología , Factores de Intercambio de Guanina Nucleótido/metabolismo , Receptores de Antígenos de Linfocitos T/fisiología , Receptores CXCR4/metabolismo , Sitios de Unión , Humanos , Fosforilación , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal , Linfocitos T/metabolismo , Activación Transcripcional
5.
Blood ; 130(8): 982-994, 2017 08 24.
Artículo en Inglés | MEDLINE | ID: mdl-28694325

RESUMEN

As with many immunopathologically driven diseases, the malignant T cells of cutaneous T-cell lymphomas (CTCLs), such as Sézary syndrome, display aberrant cytokine secretion patterns that contribute to pathology and disease progression. Targeting this disordered release of cytokines is complicated by the changing cytokine milieu that drives the phenotypic changes of CTCLs. Here, we characterize a novel signaling pathway that can be targeted to inhibit the secretion of cytokines by modulating either CXCR4 or CXCR4-mediated signaling. We demonstrate that upon ligation of the T-cell antigen receptor (TCR), the TCR associates with and transactivates CXCR4 via phosphorylation of S339-CXCR4 in order to activate a PREX1-Rac1-signaling pathway that stabilizes interleukin-2(IL-2), IL-4, and IL-10 messenger RNA (mRNA) transcripts. Pharmacologic inhibition of either TCR-CXCR4 complex formation or PREX1-Rac1 signaling in primary human T cells decreased mRNA stability and inhibited secretion of IL-2, IL-4, and IL-10. Applying this knowledge to Sézary syndrome, we demonstrate that targeting various aspects of this signaling pathway blocks both TCR-dependent and TCR-independent cytokine secretion from a Sézary syndrome-derived cell line and patient isolates. Together, these results identify multiple aspects of a novel TCR-CXCR4-signaling pathway that could be targeted to inhibit the aberrant cytokine secretion that drives the immunopathogenesis of Sézary syndrome and other immunopathological diseases.


Asunto(s)
Citocinas/genética , Factores de Intercambio de Guanina Nucleótido/metabolismo , Linfoma Cutáneo de Células T/metabolismo , Estabilidad del ARN , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores CXCR4/metabolismo , Transducción de Señal , Proteína de Unión al GTP rac1/metabolismo , Bencilaminas , Ciclamas , Citocinas/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Compuestos Heterocíclicos/farmacología , Humanos , Células Jurkat , Subgrupos Linfocitarios/efectos de los fármacos , Subgrupos Linfocitarios/metabolismo , Linfoma Cutáneo de Células T/patología , Modelos Biológicos , Estabilidad del ARN/efectos de los fármacos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Síndrome de Sézary/patología , Transducción de Señal/efectos de los fármacos , Activación Transcripcional/efectos de los fármacos , Activación Transcripcional/genética
6.
J Biol Chem ; 290(49): 29478-92, 2015 Dec 04.
Artículo en Inglés | MEDLINE | ID: mdl-26491017

RESUMEN

Disrupting the protective signals provided by the bone marrow microenvironment will be critical for more effective combination drug therapies for acute myeloid leukemia (AML). Cells of the osteoblast lineage that reside in the endosteal niche have been implicated in promoting survival of AML cells. Here, we investigated how to prevent this protective interaction. We previously showed that SDF-1, a chemokine abundant in the bone marrow, induces apoptosis of AML cells, unless the leukemic cells receive protective signals provided by differentiating osteoblasts (8, 10). We now identify a novel signaling pathway in differentiating osteoblasts that can be manipulated to disrupt the osteoblast-mediated protection of AML cells. Treating differentiating osteoblasts with histone deacetylase inhibitors (HDACi) abrogated their ability to protect co-cultured AML cells from SDF-1-induced apoptosis. HDACi prominently up-regulated expression of the Nherf1 scaffold protein, which played a major role in preventing osteoblast-mediated protection of AML cells. Protein phosphatase-1α (PP1α) was identified as a novel Nherf1 interacting protein that acts as the downstream mediator of this response by promoting nuclear localization of the TAZ transcriptional modulator. Moreover, independent activation of either PP1α or TAZ was sufficient to prevent osteoblast-mediated protection of AML cells even in the absence of HDACi. Together, these results indicate that HDACi target the AML microenvironment by enhancing activation of the Nherf1-PP1α-TAZ pathway in osteoblasts. Selective drug targeting of this osteoblast signaling pathway may improve treatments of AML by rendering leukemic cells in the bone marrow more susceptible to apoptosis.


Asunto(s)
Inhibidores de Histona Desacetilasas/química , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Leucemia Mieloide Aguda/metabolismo , Fosfoproteínas/metabolismo , Proteína Fosfatasa 1/metabolismo , Intercambiadores de Sodio-Hidrógeno/metabolismo , Microambiente Tumoral , Células 3T3 , Animales , Apoptosis , Médula Ósea/metabolismo , Diferenciación Celular , Núcleo Celular/metabolismo , Quimiocina CXCL12/metabolismo , Técnicas de Cocultivo , Humanos , Ratones , Osteoblastos/citología , Osteoblastos/metabolismo , ARN Interferente Pequeño/metabolismo , Receptores CXCR4/metabolismo , Transducción de Señal , Fracciones Subcelulares/metabolismo , Transactivadores , Factores de Transcripción , Proteínas Coactivadoras Transcripcionales con Motivo de Unión a PDZ
7.
Mol Pharmacol ; 85(4): 542-52, 2014 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-24452472

RESUMEN

CXC chemokine receptor 4 (CXCR4) is a G protein-coupled receptor (GPCR) located on the cell surface that signals upon binding the chemokine stromal derived factor-1 (SDF-1; also called CXCL 12). CXCR4 promotes neuroblastoma proliferation and chemotaxis. CXCR4 expression negatively correlates with prognosis and drives neuroblastoma growth and metastasis in mouse models. All functions of CXCR4 require its expression on the cell surface, yet the molecular mechanisms that regulate CXCR4 cell-surface levels in neuroblastoma are poorly understood. We characterized CXCR4 cell-surface regulation in the related SH-SY5Y and SK-N-SH human neuroblastoma cell lines. SDF-1 treatment caused rapid down-modulation of CXCR4 in SH-SY5Y cells. Pharmacologic activation of protein kinase C similarly reduced CXCR4, but via a distinct mechanism. Analysis of CXCR4 mutants delineated two CXCR4 regions required for SDF-1 treatment to decrease cell-surface CXCR4 in neuroblastoma cells: the isoleucine-leucine motif at residues 328 and 329 and residues 343-352. In contrast, and unlike CXCR4 regulation in other cell types, serines 324, 325, 338, and 339 were not required. Arrestin proteins can bind and regulate GPCR cell-surface expression, often functioning together with kinases such as G protein-coupled receptor kinase 2 (GRK2). Using SK-N-SH cells which are naturally deficient in ß-arrestin1, we showed that ß-arrestin1 is required for the CXCR4 343-352 region to modulate CXCR4 cell-surface expression following treatment with SDF-1. Moreover, GRK2 overexpression enhanced CXCR4 internalization, via a mechanism requiring both ß-arrestin1 expression and the 343-352 region. Together, these results characterize CXCR4 structural domains and ß-arrestin1 as critical regulators of CXCR4 cell-surface expression in neuroblastoma. ß-Arrestin1 levels may therefore influence the CXCR4-driven metastasis of neuroblastoma as well as prognosis.


Asunto(s)
Arrestinas/metabolismo , Quimiocina CXCL12/metabolismo , Neuroblastoma/metabolismo , Receptores CXCR4/metabolismo , Línea Celular Tumoral , Membrana Celular/metabolismo , Quimiocina CXCL12/farmacología , Endocitosis , Quinasa 2 del Receptor Acoplado a Proteína-G/metabolismo , Proteínas de Unión al GTP/metabolismo , Humanos , Metástasis de la Neoplasia , Toxina del Pertussis/farmacología , Fosforilación , Proteína Quinasa C/metabolismo , Acetato de Tetradecanoilforbol/farmacología , Fosfolipasas de Tipo C/metabolismo , beta-Arrestinas
8.
J Biol Chem ; 288(32): 22899-914, 2013 Aug 09.
Artículo en Inglés | MEDLINE | ID: mdl-23798675

RESUMEN

The CXCR4 chemokine receptor promotes survival of many different cell types. Here, we describe a previously unsuspected role for CXCR4 as a potent inducer of apoptosis in acute myeloid leukemia (AML) cell lines and a subset of clinical AML samples. We show that SDF-1, the sole ligand for CXCR4, induces the expected migration and ERK activation in the KG1a AML cell line transiently overexpressing CXCR4, but ERK activation did not lead to survival. Instead, SDF-1 treatment led via a CXCR4-dependent mechanism to apoptosis, as evidenced by increased annexin V staining, condensation of chromatin, and cleavage of both procaspase-3 and PARP. This SDF-1-induced death pathway was partially inhibited by hypoxia, which is often found in the bone marrow of AML patients. SDF-1-induced apoptosis was inhibited by dominant negative procaspase-9 but not by inhibition of caspase-8 activation, implicating the intrinsic apoptotic pathway. Further analysis showed that this pathway was activated by multiple mechanisms, including up-regulation of Bak at the level of mRNA and protein, stabilization of the Bak activator Noxa, and down-regulation of antiapoptotic Bcl-XL. Furthermore, adjusting expression levels of Bak, Bcl-XL, or Noxa individually altered the level of apoptosis in AML cells, suggesting that the combined modulation of these family members by SDF-1 coordinates their interplay to produce apoptosis. Thus, rather than mediating survival, SDF-1 may be a means to induce apoptosis of CXCR4-expressing AML cells directly in the SDF-1-rich bone marrow microenvironment if the survival cues of the bone marrow are disrupted.


Asunto(s)
Apoptosis , Regulación Leucémica de la Expresión Génica , Leucemia Mieloide Aguda/metabolismo , Sistema de Señalización de MAP Quinasas , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Receptores CXCR4/metabolismo , Proteína Destructora del Antagonista Homólogo bcl-2/biosíntesis , Proteína bcl-X/biosíntesis , Anexina A5/genética , Anexina A5/metabolismo , Supervivencia Celular/genética , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Regulación hacia Abajo/genética , Femenino , Células HEK293 , Humanos , Células Jurkat , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patología , Masculino , Estabilidad Proteica , Proteínas Proto-Oncogénicas c-bcl-2/genética , Receptores CXCR4/genética , Células U937 , Regulación hacia Arriba/genética , Proteína Destructora del Antagonista Homólogo bcl-2/genética , Proteína bcl-X/genética
9.
J Cell Biochem ; 115(6): 1128-37, 2014 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-24851270

RESUMEN

The bone marrow provides a protective environment for acute myeloid leukemia (AML) cells that often allows leukemic stem cells to survive standard chemotherapeutic regimens. Targeting these leukemic stem cells within the bone marrow is critical for preventing relapse. We recently demonstrated that SDF-1, a chemokine abundant in the bone marrow, induces apoptosis in AML cell lines and in patient samples expressing high levels of its receptor, CXCR4. Here we show that a subset of osteoblast lineage cells within the bone marrow can protect AML cells from undergoing apoptosis in response to the SDF-1 naturally present in that location. In co-culture systems, osteoblasts at various stages of differentiation protected AML cell lines and patient isolates from SDF-1-induced apoptosis. The differentiation of the osteoblast cell lines, MC3T3 and W-20-17, mediated this protection via a cell contact-independent mechanism. In contrast, bone marrow-derived mesenchymal cells, the precursors of osteoblasts, induced apoptosis in AML cells via a CXCR4-dependent mechanism and failed to protect AML cells from exogenously added SDF-1. These results indicate that osteoblasts in the process of differentiation potently inhibit the SDF-1-driven apoptotic pathway of CXCR4-expressing AML cells residing in the bone marrow. Drugs targeting this protective mechanism could potentially provide a new approach to treating AML by enhancing the SDF-1-induced apoptosis of AML cells residing within the bone marrow microenvironment.


Asunto(s)
Apoptosis/efectos de los fármacos , Quimiocina CXCL12/farmacología , Leucemia Mieloide/patología , Osteoblastos/citología , Enfermedad Aguda , Fosfatasa Alcalina/genética , Animales , Diferenciación Celular/genética , Línea Celular , Línea Celular Tumoral , Células Cultivadas , Técnicas de Cocultivo , Citometría de Flujo , Expresión Génica , Humanos , Leucemia Mieloide/genética , Leucemia Mieloide/metabolismo , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Ratones Endogámicos C57BL , Osteoblastos/metabolismo , Osteocalcina/genética , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células U937
10.
J Immunol ; 187(6): 3177-85, 2011 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-21856938

RESUMEN

RasGRP1, a Ras guanine-nucleotide exchange factor, critically mediates T cell development and function and controls immunodeficiency and autoimmunity. In this study, we describe a unique mechanism of mobilization and activation of RasGRP1 in response to SDF-1, a chemokine that signals via the G protein-coupled receptor CXCR4. Depletion of RasGRP1 impaired SDF-1-stimulated human T cell migration, expression of the activation marker CD69, and activation of the ERK MAPK pathway, indicating that RasGRP1 mediates SDF-1 functions. SDF-1 treatment caused RasGRP1 to localize to the plasma membrane to activate K-Ras and to the Golgi to activate N-Ras. These events were required for cellular migration and for ERK activation that mediates downstream transcriptional events in response to SDF-1. SDF-1-dependent localization of RasGRP1 did not require its diacylglycerol-binding domain, even though diacyglycerol was previously shown to mediate localization of RasGRP1 in response to Ag stimulation. This domain was, however, required for activity of RasGRP1 after its localization. Intriguingly, SDF-1 treatment of T cells induced the formation of a novel molecular signaling complex containing RasGRP1, Gαi2, and ZAP-70. Moreover, SDF-1-mediated signaling by both Gi proteins and ZAP-70 was required for RasGRP1 mobilization. In addition, RasGRP1 mobilization and activation in response to SDF-1 was dependent on TCR expression, suggesting that CXCR4 heterodimerizes with the TCR to couple to ZAP-70 and mobilize RasGRP1. These results increase understanding of the molecular mechanisms that mediate SDF-1 effects on T cells and reveal a novel mechanism of RasGRP1 regulation. Other G protein-coupled receptors may similarly contribute to regulation of RasGRP1.


Asunto(s)
Quimiocina CXCL12/inmunología , Proteínas de Unión al ADN/inmunología , Subunidad alfa de la Proteína de Unión al GTP Gi2/inmunología , Factores de Intercambio de Guanina Nucleótido/inmunología , Transducción de Señal/inmunología , Linfocitos T/inmunología , Proteína Tirosina Quinasa ZAP-70/inmunología , Western Blotting , Membrana Celular/metabolismo , Quimiocina CXCL12/metabolismo , Quimiotaxis de Leucocito/inmunología , Proteínas de Unión al ADN/metabolismo , Activación Enzimática/inmunología , Subunidad alfa de la Proteína de Unión al GTP Gi2/metabolismo , Factores de Intercambio de Guanina Nucleótido/metabolismo , Humanos , Inmunoprecipitación , Activación de Linfocitos/inmunología , Transporte de Proteínas/inmunología , Linfocitos T/metabolismo , Proteína Tirosina Quinasa ZAP-70/metabolismo
11.
J Immunol ; 186(2): 951-8, 2011 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-21148034

RESUMEN

CXCR4, like other G protein-coupled receptors, signals via heterotrimeric guanine nucleotide-binding proteins (G proteins) to regulate gene transcription, migration, development, growth, and transformation. We describe a formerly uncharacterized function of a G protein: a role in receptor trafficking. We previously showed that CXCR4 and the TCR physically associate and form a heterodimer upon stromal cell-derived factor-1 or CXCL12 (SDF-1) stimulation in human T cells to prolong ERK activation and, thereby, lead to gene upregulation and cytokine secretion. The CXCR4-TCR heterodimers occur on the cell surface and in an intracellular compartment in response to SDF-1. Neither the intracellular compartment to which the CXCR4-TCR heterodimers localize nor the mechanism for localization has been elucidated. In this article, we characterize molecular mechanisms required for postendocytic trafficking of CXCR4. Upon SDF-1 stimulation, CXCR4 localizes to Rab11(+) vesicles, a recycling compartment near the microtubule organizing center and Golgi apparatus. This trafficking requires the CXCR4 C-terminal tail domain but not the CXCR4 ubiquitination sites. The TCR also constitutively localizes to this Rab11(+) compartment. Trafficking of CXCR4 into the Rab11(+), TCR-containing endosomes requires actin polymerization. Furthermore, inhibiting Rho activation or depleting Gα13 prevented trafficking of CXCR4 into the Rab11(+) endosomes without hindering the ability of CXCR4 to endocytose. These results indicated that, upon SDF-1 treatment, Gα13 and Rho mediate the actin polymerization necessary for trafficking CXCR4 into the Rab11(+), recycling endosomal compartment, which also contains constitutively recycling TCR and, thus, CXCR4-TCR heterodimers. To our knowledge, this is the first report of Gα13 as a mediator of receptor trafficking.


Asunto(s)
Quimiocina CXCL12/fisiología , Vesículas Citoplasmáticas/metabolismo , Endosomas/metabolismo , Subunidades alfa de la Proteína de Unión al GTP G12-G13/fisiología , Receptores CXCR4/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rho/fisiología , Vesículas Citoplasmáticas/inmunología , Endocitosis/inmunología , Endosomas/enzimología , Endosomas/inmunología , Humanos , Células Jurkat , Estructura Terciaria de Proteína , Transporte de Proteínas/inmunología , Receptores CXCR4/química , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Ubiquitinación/inmunología , Proteínas de Unión al GTP rab/biosíntesis
12.
J Immunol ; 187(3): 1440-7, 2011 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-21705626

RESUMEN

The CXCR4 chemokine receptor is a G protein-coupled receptor that signals in T lymphocytes by forming a heterodimer with the TCR. CXCR4 and TCR functions are consequently highly cross regulated, affecting T cell immune activation, cytokine secretion, and T cell migration. The CXCR4-TCR heterodimer stimulates T cell migration and activation of the ERK MAPK and downstream AP-1-dependent cytokine transcription in response to stromal cell-derived factor-1 (SDF-1), the sole chemokine ligand of CXCR4. These responses require Gi-type G proteins as well as TCR ITAM domains and the ZAP70 tyrosine kinase, thus indicating that the CXCR4-TCR heterodimer signals to integrate G protein-coupled receptor-associated and TCR-associated signaling molecules in response to SDF-1. Yet, the phospholipase C (PLC) isozymes responsible for coupling the CXCR4-TCR heterodimer to distinct downstream cellular responses are incompletely characterized. In this study, we demonstrate that PLC activity is required for SDF-1 to induce ERK activation, migration, and CXCR4 endocytosis in human T cells. SDF-1 signaling via the CXCR4-TCR heterodimer uses PLC-ß3 to activate the Ras-ERK pathway and increase intracellular calcium ion concentrations, whereas PLC-γ1 is dispensable for these outcomes. In contrast, PLC-γ1, but not PLC-ß3, is required for SDF-1-mediated migration via a mechanism independent of LAT. These results increase understanding of the signaling mechanisms employed by the CXCR4-TCR heterodimer, characterize new roles for PLC-ß3 and PLC-γ1 in T cells, and suggest that multiple PLCs may also be activated downstream of other chemokine receptors to distinctly regulate migration versus other signaling functions.


Asunto(s)
Quimiocina CXCL12/fisiología , Fosfolipasa C beta/fisiología , Fosfolipasa C gamma/fisiología , Multimerización de Proteína/inmunología , Receptores de Antígenos de Linfocitos T/fisiología , Receptores CXCR4/fisiología , Transducción de Señal/inmunología , Subgrupos de Linfocitos T/inmunología , Señalización del Calcio/inmunología , Movimiento Celular/inmunología , Endocitosis/inmunología , Humanos , Líquido Intracelular/enzimología , Líquido Intracelular/inmunología , Isoenzimas/fisiología , Células Jurkat , Sistema de Señalización de MAP Quinasas/inmunología , Receptores CXCR4/metabolismo , Subgrupos de Linfocitos T/enzimología
13.
J Clin Invest ; 129(3): 965-968, 2019 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-30714991

RESUMEN

Primary antibody deficiencies are the most common immunodeficiencies in humans; however, identification of the underlying genetic and biochemical basis for these diseases is often difficult, given that these deficiencies typically involve complex genetic etiologies. In this issue of the JCI, Bouafia et al. performed whole-exome sequencing on a pair of siblings with primary antibody deficiencies and identified genetic mutations that result in a deficiency of ARHGEF1, a hematopoietic intracellular signaling molecule that transmits signals from GPCRs. ARHGEF1-deficient lymphocytes from the affected siblings exhibited important functional deficits that indicate that loss of ARHGEF1 accounts for the observed primary antibody deficiency, which manifests in an inability to mount antibody responses to vaccines and pathogens. Thus, this report demonstrates an important role for ARHGEF1 in GPCR signal transduction required for appropriate adaptive immune responses in humans.


Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP G12-G13 , Enfermedades de Inmunodeficiencia Primaria , Humanos , Linfocitos , Factores de Intercambio de Guanina Nucleótido Rho , Transducción de Señal
14.
Front Immunol ; 10: 1159, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31231367

RESUMEN

Persistent T cell antigen receptor (TCR) signaling by CD8 T cells is a feature of cancer and chronic infections and results in the sustained expression of, and signaling by, inhibitory receptors, which ultimately impair cytotoxic activity via poorly characterized mechanisms. We have previously determined that the LPA5 GPCR expressed by CD8 T cells, upon engaging the lysophosphatidic acid (LPA) bioactive serum lipid, functions as an inhibitory receptor able to negatively regulate TCR signaling. Notably, the levels of LPA and autotaxin (ATX), the phospholipase D enzyme that produces LPA, are often increased in chronic inflammatory disorders such as chronic infections, autoimmune diseases, obesity, and cancer. In this report, we demonstrate that LPA engagement selectively by LPA5 on human and mouse CD8 T cells leads to the inhibition of several early TCR signaling events including intracellular calcium mobilization and ERK activation. We further show that, as a consequence of LPA5 suppression of TCR signaling, the exocytosis of perforin-containing granules is significantly impaired and reflected by repressed in vitro and in vivo CD8 T cell cytolytic activity. Thus, these data not only document LPA5 as a novel inhibitory receptor but also determine the molecular and biochemical mechanisms by which a naturally occurring serum lipid that is elevated under settings of chronic inflammation signals to suppress CD8 T cell killing activity in both human and murine cells. As diverse tumors have repeatedly been shown to aberrantly produce LPA that acts in an autocrine manner to promote tumorigenesis, our findings further implicate LPA in activating a novel inhibitory receptor whose signaling may be therapeutically silenced to promote CD8 T cell immunity.


Asunto(s)
Linfocitos T CD8-positivos/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Receptores del Ácido Lisofosfatídico/inmunología , Transducción de Señal/inmunología , Linfocitos T Citotóxicos/inmunología , Animales , Linfocitos T CD8-positivos/metabolismo , Calcio/inmunología , Calcio/metabolismo , Carcinogénesis/genética , Carcinogénesis/inmunología , Carcinogénesis/metabolismo , Línea Celular Tumoral , Células Cultivadas , Exocitosis/inmunología , Humanos , Ratones Endogámicos C57BL , Ratones Noqueados , Perforina/inmunología , Perforina/metabolismo , Hidrolasas Diéster Fosfóricas/inmunología , Hidrolasas Diéster Fosfóricas/metabolismo , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores del Ácido Lisofosfatídico/genética , Receptores del Ácido Lisofosfatídico/metabolismo , Transducción de Señal/genética , Linfocitos T Citotóxicos/metabolismo
15.
Oncotarget ; 8(55): 94569-94579, 2017 Nov 07.
Artículo en Inglés | MEDLINE | ID: mdl-29212250

RESUMEN

The bone marrow microenvironment protects acute myeloid leukemia (AML) cells during chemotherapy and is a major factor in relapse. Here, we examined which type(s) of bone marrow cells are responsible for the relapse of AML following treatment with cytarabine (Ara-C), and we identified a means to inhibit this protection. To determine the protective cell type(s), AML cells were treated with Ara-C, and AML cell survival in the presence or absence of osteoblast lineage cells was assessed. Cultured AML cells and patient bone marrow isolates were each significantly protected from Ara-C-induced apoptosis by co-culture with differentiating osteoblasts. Moreover, pretreating differentiating osteoblasts with the histone deacetylase inhibitors (HDACi) vorinostat and panobinostat abrogated the ability of the differentiating osteoblasts to protect AML cells. Together, our results indicate that differentiating osteoblasts have the potential to promote residual AML in the bone marrow following standard chemotherapy and act via a mechanism requiring HDACi-sensitive gene expression. Using HDACi to target the leukemic microenvironment in combination with Ara-C could potentially improve treatment of AML. Moreover, other strategies for manipulating bone marrow osteoblasts may also help eradicate AML cells and reduce relapse.

16.
J Cell Biol ; 210(2): 257-72, 2015 Jul 20.
Artículo en Inglés | MEDLINE | ID: mdl-26195666

RESUMEN

IQ motif-containing GTPase-activating protein 1 (IQGAP1) is a cytoskeleton-interacting scaffold protein. CXCR4 is a chemokine receptor that binds stromal cell-derived factor-1 (SDF-1; also known as CXCL12). Both IQGAP1 and CXCR4 are overexpressed in cancer cell types, yet it was unclear whether these molecules functionally interact. Here, we show that depleting IQGAP1 in Jurkat T leukemic cells reduced CXCR4 expression, disrupted trafficking of endocytosed CXCR4 via EEA-1(+) endosomes, and decreased efficiency of CXCR4 recycling. SDF-1-induced cell migration and activation of extracellular signal-regulated kinases 1 and 2 (ERK) MAPK were strongly inhibited, even when forced overexpression restored CXCR4 levels. Similar results were seen in KMBC and HEK293 cells. Exploring the mechanism, we found that SDF-1 treatment induced IQGAP1 binding to α-tubulin and localization to CXCR4-containing endosomes and that CXCR4-containing EEA-1(+) endosomes were abnormally located distal from the microtubule (MT)-organizing center (MTOC) in IQGAP1-deficient cells. Thus, IQGAP1 critically mediates CXCR4 cell surface expression and signaling, evidently by regulating EEA-1(+) endosome interactions with MTs during CXCR4 trafficking and recycling. IQGAP1 may similarly promote CXCR4 functions in other cancer cell types.


Asunto(s)
Endosomas/metabolismo , Receptores CXCR4/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Proteínas Activadoras de ras GTPasa/fisiología , Movimiento Celular , Quimiocina CXCL12/metabolismo , Endocitosis , Células HEK293 , Humanos , Células Jurkat , Sistema de Señalización de MAP Quinasas , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Transporte de Proteínas , Receptores Opioides delta/metabolismo
17.
Methods Enzymol ; 460: 379-97, 2009.
Artículo en Inglés | MEDLINE | ID: mdl-19446736

RESUMEN

Multiprotein complexes play an important role in nearly all cell functions; therefore, the characterization of protein-protein interactions in living cells constitutes an important step in the analysis of cellular signaling pathways. Using fluorescence resonance energy transfer (FRET) as a "molecular ruler" is a powerful approach for identifying biologically relevant molecular interactions with high spatiotemporal resolution. Here, we describe two methods that use FRET to detect a physical interaction between the T-cell antigen receptor (TCR) and the CXCR4 chemokine receptor in living T lymphocytes. These FRET approaches use two different sets of chromophores. We discuss the design strategies, control experiments, and pitfalls involved in using these FRET approaches. Although there is no perfect pair of chromophores for FRET, the two FRET methods described here provide complementary and reliable insight into the molecular interactions between these receptor molecules.


Asunto(s)
Transferencia Resonante de Energía de Fluorescencia/métodos , Receptores CXCR4/metabolismo , Linfocitos T/metabolismo , Citometría de Flujo , Humanos , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores CXCR4/genética
18.
J Immunol ; 178(3): 1581-8, 2007 Feb 01.
Artículo en Inglés | MEDLINE | ID: mdl-17237407

RESUMEN

Costimulation by the chemokine, stromal cell-derived factor-1 (SDF-1)/CXCL12, has been shown to increase the amount of IL-10 secreted by TCR-stimulated human T cells; however, the molecular mechanisms of this response are unknown. Knowledge of this signaling pathway may be useful because extensive evidence indicates that deficient IL-10 secretion promotes autoimmunity. The human IL-10 locus is highly polymorphic. We report in this study that SDF-1 costimulates IL-10 secretion from T cells containing all three of the most common human IL-10 promoter haplotypes that are identified by single-nucleotide polymorphisms at -1082, -819, and -592 bp (numbering is relative to the transcription start site). We further show that SDF-1 primarily costimulates IL-10 secretion by a diverse population of CD45RA(-) ("memory") phenotype T cells that includes cells expressing the presumed regulatory T cell marker, Foxp3. To address the molecular mechanisms of this response, we showed that SDF-1 costimulates the transcriptional activities in normal human T cells of reporter plasmids containing 1.1 kb of all three of the common IL-10 promoter haplotypes. IL-10 promoter activity was ablated by mutating two nonpolymorphic binding sites for the AP-1 transcription factor, and chromatin immunoprecipitation assays of primary human T cells revealed that SDF-1 costimulation enhances AP-1 binding to both of these sites. Together, these results delineate the molecular mechanisms responsible for SDF-1 costimulation of T cell IL-10 secretion. Because it is preserved among several human haplotypes and in diverse T cell populations including Foxp3(+) T cells, this pathway of IL-10 regulation may represent a key mechanism for modulating expression of this important immunoregulatory cytokine.


Asunto(s)
Quimiocinas CXC/fisiología , Interleucina-10/genética , Interleucina-10/metabolismo , Regiones Promotoras Genéticas , Factor de Transcripción AP-1/metabolismo , Sitios de Unión/genética , Células Cultivadas , Quimiocina CXCL12 , Haplotipos , Humanos , Unión Proteica , Linfocitos T/química , Transcripción Genética
19.
Immunity ; 25(2): 213-24, 2006 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-16919488

RESUMEN

SDF-1alpha (CXCL12) signaling via its receptor, CXCR4, stimulates T cell chemotaxis and gene expression. The ZAP-70 tyrosine kinase critically mediates SDF-1alpha-dependent migration and prolonged ERK mitogen-activated protein (MAP) kinase activation in T cells. However, the molecular mechanism by which CXCR4 or other G protein-coupled receptors activate ZAP-70 has not been characterized. Here we show that SDF-1alpha stimulates the physical association of CXCR4 and the T cell receptor (TCR) and utilizes the ZAP-70 binding ITAM domains of the TCR for signal transduction. This pathway is responsible for several of the effects of SDF-1alpha on T cells, including prolonged ERK MAP kinase activity, increased intracellular calcium ion concentrations, robust AP-1 transcriptional activity, and SDF-1alpha costimulation of cytokine secretion. These results suggest new paradigms for understanding the effects of SDF-1alpha and other chemokines on immunity.


Asunto(s)
Receptores de Antígenos de Linfocitos T/inmunología , Receptores CXCR4/inmunología , Transducción de Señal/inmunología , Linfocitos T/inmunología , Antígenos CD/metabolismo , Antígenos de Diferenciación de Linfocitos T/metabolismo , Calcio/metabolismo , Células Cultivadas , Quimiocina CXCL12 , Quimiocinas CXC/farmacología , Activación Enzimática/efectos de los fármacos , Humanos , Interleucina-10/metabolismo , Interleucina-2/metabolismo , Lectinas Tipo C , Modelos Inmunológicos , Fosfotirosina/metabolismo , Unión Proteica , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T/efectos de los fármacos , Linfocitos T/metabolismo , Factor de Transcripción AP-1/metabolismo , Activación Transcripcional/genética , Proteína Tirosina Quinasa ZAP-70/metabolismo , Proteínas ras/metabolismo
20.
J Immunol ; 171(1): 360-7, 2003 Jul 01.
Artículo en Inglés | MEDLINE | ID: mdl-12817019

RESUMEN

Stimulation of T lymphocytes with the ligand for the CXCR4 chemokine receptor stromal cell-derived factor-1alpha (SDF-1alpha/CXCL12), results in prolonged activation of the extracellular signal-regulated kinases (ERK) ERK1 and ERK2. Because SDF-1alpha is unique among several chemokines in its ability to stimulate prolonged ERK activation, this pathway is thought to mediate special functions of SDF-1alpha that are not shared with other chemokines. However, the molecular mechanisms of this response are poorly understood. In this study we show that SDF-1alpha stimulation of prolonged ERK activation in Jurkat T cells requires both the ZAP-70 tyrosine kinase and the Src homology 2 domain-containing leukocyte protein of 76 kDa (SLP-76) scaffold protein. This pathway involves ZAP-70-dependent tyrosine phosphorylation of SLP-76 at one or more of its tyrosines, 113, 128, and 145. Because TCR activates ERK via SLP-76-mediated activation of the linker of activated T cells (LAT) scaffold protein, we examined the role of LAT in SDF-1alpha-mediated ERK activation. However, neither the SLP-76 proline-rich domain that links to GADS and LAT, nor LAT, itself are required for SDF-1alpha to stimulate SLP-76 tyrosine phosphorylation or to activate ERK. Together, our results describe the distinct mechanism by which SDF-1alpha stimulates prolonged ERK activation in T cells and indicate that this pathway is specific for cells expressing both ZAP-70 and SLP-76.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Quimiocinas CXC/fisiología , Proteínas de la Membrana , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosfoproteínas/fisiología , Proteínas Tirosina Quinasas/fisiología , Linfocitos T/enzimología , Sitios de Unión/genética , Sitios de Unión/inmunología , Proteínas Portadoras/metabolismo , Quimiocina CXCL12 , Activación Enzimática/genética , Activación Enzimática/inmunología , Humanos , Células Jurkat , Activación de Linfocitos/genética , Fosfoproteínas/deficiencia , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fosforilación , Unión Proteica/genética , Unión Proteica/inmunología , Proteínas Tirosina Quinasas/deficiencia , Proteínas Tirosina Quinasas/genética , Transducción de Señal/genética , Transducción de Señal/inmunología , Especificidad por Sustrato/genética , Especificidad por Sustrato/inmunología , Linfocitos T/inmunología , Tirosina/metabolismo , Proteína Tirosina Quinasa ZAP-70 , Dominios Homologos src/genética , Dominios Homologos src/inmunología
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