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1.
Immunity ; 45(6): 1232-1244, 2016 12 20.
Artículo en Inglés | MEDLINE | ID: mdl-27889108

RESUMEN

B1 and B2 B cells differ in their ability to respond to T-cell-independent (TI) antigens. Here we report that the Src-family kinase (SFK) regulator CD148 has a unique and critical role in the initiation of B1 but not B2 cell antigen receptor signaling. CD148 loss-of-function mice were found to have defective B1 B-cell-mediated antibody responses against the T-cell-independent antigens NP-ficoll and Pneumovax 23 and had impaired selection of the B1 B cell receptor (BCR) repertoire. These deficiencies were associated with a decreased ability of B1 B cells to induce BCR signaling downstream of the SFK Lyn. Notably, Lyn appeared to be selectively regulated by CD148 and loss of this SFK resulted in opposite signaling phenotypes in B1 and B2 B cells. These findings reveal that the function and regulation of Lyn during B1 cell BCR signaling is distinct from other B cell subsets.


Asunto(s)
Subgrupos de Linfocitos B/inmunología , Activación de Linfocitos/inmunología , Receptores de Antígenos de Linfocitos B/inmunología , Familia-src Quinasas/inmunología , Animales , Ensayo de Inmunoadsorción Enzimática , Ensayo de Immunospot Ligado a Enzimas , Citometría de Flujo , Ratones , Ratones Noqueados , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores/inmunología , Transducción de Señal/inmunología
2.
Immunity ; 35(5): 757-69, 2011 Nov 23.
Artículo en Inglés | MEDLINE | ID: mdl-22078799

RESUMEN

Neutrophils, critical innate immune effectors, use bacterial-derived chemoattractant-induced G protein-coupled receptor (GPCR) signaling for their pursuit of bacteria. Tyrosine phosphorylation pathways and receptor-like tyrosine phosphatases (RPTPs) are rarely considered in chemoattractant-mediated GPCR signaling. Here, we report that two RPTPs, CD45 and CD148, previously shown to share redundant roles in positively regulating Src family kinases (SFKs) in immunoreceptor signaling pathways in B cells and macrophages, are critical in the neutrophil response to S. aureus infection and, surprisingly, in chemoattractant-mediated chemotaxis. Remarkably, deficiency in either of these RPTPs influenced neutrophil GPCR responses in unique ways. Our results reveal that CD45 positively while CD148 positively and negatively regulate GPCR function and proximal signals including Ca(2+), phosphatidylinositol 3'OH kinase (PI3K), and phospho-extracellular regulated kinase (pERK) activity. Moreover, our results suggest that CD45 and CD148 preferentially target different SFK members (Hck and Fgr versus Lyn, respectively) to positively and negatively regulate GPCR pathways.


Asunto(s)
Quimiotaxis/inmunología , Antígenos Comunes de Leucocito/metabolismo , Neutrófilos/inmunología , Staphylococcus aureus/inmunología , Animales , Adhesión Celular/inmunología , Células Cultivadas , Quimiotaxis/genética , Antígenos Comunes de Leucocito/genética , Ratones , Ratones Noqueados , Neutrófilos/metabolismo , Fagocitosis/inmunología , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores/genética , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , Superóxidos/metabolismo , Familia-src Quinasas/genética , Familia-src Quinasas/metabolismo
3.
Immunol Rev ; 228(1): 288-311, 2009 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-19290935

RESUMEN

Reciprocal regulation of tyrosine phosphorylation by protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs) is central to normal immune cell function. Disruption of the equilibrium between PTK and PTP activity can result in immunodeficiency, autoimmunity, or malignancy. Src family kinases (SFKs) play a central role in both immune cell function and disease due to their proximal position in numerous signal transduction cascades including those emanating from integrin, T and B-cell antigen receptors, Fc, growth factor, and cytokine receptors. Given that tight regulation of SFKs activity is critical for appropriate responses to stimulation of these various signaling pathways, it is perhaps not surprising that multiple PTPs are involved in their regulation. Here, we focus on the role of three phosphatases, CD45, CD148, and LYP/PEP, which are critical regulators of SFKs in hematopoietic cells. We review our current understanding of their structures, expression, functions in different hematopoietic cell subsets, regulation, and putative roles in disease. Finally, we discuss remaining questions that must be addressed if we are to have a clearer understanding of the coordinated regulation of tyrosine phosphorylation and signaling networks in hematopoietic cells and how they could potentially be manipulated therapeutically in disease.


Asunto(s)
Antígenos Comunes de Leucocito/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 12/metabolismo , Transducción de Señal , Animales , Células Sanguíneas/enzimología , Células Sanguíneas/metabolismo , Humanos , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores/metabolismo
4.
PLoS Pathog ; 5(7): e1000501, 2009 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-19578436

RESUMEN

Delivery of effector proteins is a process widely used by bacterial pathogens to subvert host cell functions and cause disease. Effector delivery is achieved by elaborate injection devices and can often be triggered by environmental stimuli. However, effector export by the L. pneumophila Icm/Dot Type IVB secretion system cannot be detected until the bacterium encounters a target host cell. We used chemical genetics, a perturbation strategy that utilizes small molecule inhibitors, to determine the mechanisms critical for L. pneumophila Icm/Dot activity. From a collection of more than 2,500 annotated molecules we identified specific inhibitors of effector translocation. We found that L. pneumophila effector translocation in macrophages requires host cell factors known to be involved in phagocytosis such as phosphoinositide 3-kinases, actin and tubulin. Moreover, we found that L. pneumophila phagocytosis and effector translocation also specifically require the receptor protein tyrosine phosphate phosphatases CD45 and CD148. We further show that phagocytosis is required to trigger effector delivery unless intimate contact between the bacteria and the host is artificially generated. In addition, real-time analysis of effector translocation suggests that effector export is rate-limited by phagocytosis. We propose a model in which L. pneumophila utilizes phagocytosis to initiate an intimate contact event required for the translocation of pre-synthesized effector molecules. We discuss the need for host cell participation in the initial step of the infection and its implications in the L. pneumophila lifestyle. Chemical genetic screening provides a novel approach to probe the host cell functions and factors involved in host-pathogen interactions.


Asunto(s)
Legionella pneumophila/fisiología , Enfermedad de los Legionarios/microbiología , Animales , Proteínas Bacterianas/metabolismo , Carbonil Cianuro m-Clorofenil Hidrazona/farmacología , Carbonil Cianuro p-Trifluorometoxifenil Hidrazona/farmacología , Proteínas Portadoras/metabolismo , Línea Celular , Citoesqueleto/fisiología , Genes Reporteros , Interacciones Huésped-Patógeno/fisiología , Humanos , Ionóforos/farmacología , Legionella pneumophila/genética , Enfermedad de los Legionarios/genética , Antígenos Comunes de Leucocito/genética , Antígenos Comunes de Leucocito/metabolismo , Macrófagos/metabolismo , Macrófagos/microbiología , Proteínas de la Membrana/metabolismo , Ratones , Proteínas Opsoninas , Fagocitosis/fisiología , Transporte de Proteínas , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores/metabolismo , Bibliotecas de Moléculas Pequeñas , beta-Lactamasas/genética , beta-Lactamasas/metabolismo
5.
Blood ; 113(20): 4942-54, 2009 May 14.
Artículo en Inglés | MEDLINE | ID: mdl-19246339

RESUMEN

Platelets play a fundamental role in hemostasis and thrombosis. They are also involved in pathologic conditions resulting from blocked blood vessels, including myocardial infarction and ischemic stroke. Platelet adhesion, activation, and aggregation at sites of vascular injury are regulated by a diverse repertoire of tyrosine kinase-linked and G protein-coupled receptors. Src family kinases (SFKs) play a central role in initiating and propagating signaling from several platelet surface receptors; however, the underlying mechanism of how SFK activity is regulated in platelets remains unclear. CD148 is the only receptor-like protein tyrosine phosphatase identified in platelets to date. In the present study, we show that mutant mice lacking CD148 exhibited a bleeding tendency and defective arterial thrombosis. Basal SFK activity was found to be markedly reduced in CD148-deficient platelets, resulting in a global hyporesponsiveness to agonists that signal through SFKs, including collagen and fibrinogen. G protein-coupled receptor responses to thrombin and other agonists were also marginally reduced. These results highlight CD148 as a global regulator of platelet activation and a novel antithrombotic drug target.


Asunto(s)
Activación Plaquetaria/genética , Trombosis/genética , Animales , Antígenos de Superficie/metabolismo , Plaquetas/metabolismo , Adhesión Celular/efectos de los fármacos , Células Cultivadas , Fibrinógeno/farmacología , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Agregación Plaquetaria/efectos de los fármacos , Glicoproteínas de Membrana Plaquetaria/metabolismo , Glicoproteínas de Membrana Plaquetaria/fisiología , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Fosfatasas/fisiología , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores/genética , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores/metabolismo , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores/fisiología , Receptores de IgG/genética , Transducción de Señal/inmunología
6.
MAbs ; 11(6): 996-1011, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-31156033

RESUMEN

Agonism of members of the tumor necrosis factor receptor superfamily (TNFRSF) with monoclonal antibodies is of high therapeutic interest due to their role in immune regulation and cell proliferation. A major hurdle for pharmacologic activation of this receptor class is the requirement for high-order clustering, a mechanism that imposes a reliance in vivo on Fc receptor-mediated crosslinking. This extrinsic dependence represents a potential limitation of virtually the entire pipeline of agonist TNFRSF antibody drugs, of which none have thus far been approved or reached late-stage clinical trials. We show that tetravalent biepitopic targeting enables robust intrinsic antibody agonism for two members of this family, OX40 and DR5, that is superior to extrinsically crosslinked native parental antibodies. Tetravalent biepitopic anti-OX40 engagement co-stimulated OX40low cells, obviated the requirement for CD28 co-signal for T cell activation, and enabled superior pharmacodynamic activity relative to native IgG in a murine vaccination model. This work establishes a proof of concept for an engineering approach that addresses a major gap for the therapeutic activation of this important receptor class.


Asunto(s)
Anticuerpos Monoclonales/inmunología , Recubrimiento Inmunológico , Ligando OX40/agonistas , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/agonistas , Transducción de Señal/inmunología , Linfocitos T/inmunología , Animales , Antígenos CD28/inmunología , Células CHO , Cricetulus , Humanos , Células Jurkat , Ratones , Ratones SCID , Ratones Transgénicos , Ligando OX40/inmunología , Receptores Fc/inmunología , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/inmunología , Linfocitos T/citología
7.
Mol Cell Biol ; 23(10): 3607-22, 2003 May.
Artículo en Inglés | MEDLINE | ID: mdl-12724419

RESUMEN

E2F plays critical roles in cell cycle progression by regulating the expression of genes involved in nucleotide synthesis, DNA replication, and cell cycle control. We show that the combined loss of E2F1 and E2F2 in mice leads to profound cell-autonomous defects in the hematopoietic development of multiple cell lineages. E2F2 mutant mice show erythroid maturation defects that are comparable with those observed in patients with megaloblastic anemia. Importantly, hematopoietic defects observed in E2F1/E2F2 double-knockout (DKO) mice appear to result from impeded S phase progression in hematopoietic progenitor cells. During DKO B-cell maturation, differentiation beyond the large pre-BII-cell stage is defective, presumably due to failed cell cycle exit, and the cells undergo apoptosis. However, apoptosis appears to be the consequence of failed maturation, not the cause. Despite the accumulation of hematopoietic progenitor cells in S phase, the combined loss of E2F1 and E2F2 results in significantly decreased expression and activities of several E2F target genes including cyclin A2. Our results indicate specific roles for E2F1 and E2F2 in the induction of E2F target genes, which contribute to efficient expansion and maturation of hematopoietic progenitor cells. Thus, E2F1 and E2F2 play essential and redundant roles in the proper coordination of cell cycle progression with differentiation which is necessary for efficient hematopoiesis.


Asunto(s)
Proteínas de Ciclo Celular , Proteínas de Unión al ADN , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/metabolismo , Factores de Transcripción/metabolismo , Animales , Apoptosis , Western Blotting , Células de la Médula Ósea/citología , Trasplante de Médula Ósea , Bromodesoxiuridina/farmacología , Ciclo Celular , Diferenciación Celular , Linaje de la Célula , Factores de Transcripción E2F , Factor de Transcripción E2F1 , Eritropoyesis , Citometría de Flujo , Genotipo , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Ratones Mutantes , Ratones Transgénicos , Mutación , Unión Proteica , Fase S , Factores de Tiempo
8.
J Clin Invest ; 123(5): 2037-48, 2013 May.
Artículo en Inglés | MEDLINE | ID: mdl-23543053

RESUMEN

Increased airway smooth muscle (ASM) contractility and the development of airway hyperresponsiveness (AHR) are cardinal features of asthma, but the signaling pathways that promote these changes are poorly understood. Tyrosine phosphorylation is tightly regulated by the opposing actions of protein tyrosine kinases and phosphatases, but little is known about whether tyrosine phosphatases influence AHR. Here, we demonstrate that genetic inactivation of receptor-like protein tyrosine phosphatase J (Ptprj), which encodes CD148, protected mice from the development of increased AHR in two different asthma models. Surprisingly, CD148 deficiency minimally affected the inflammatory response to allergen, but significantly altered baseline pulmonary resistance. Mice specifically lacking CD148 in smooth muscle had decreased AHR, and the frequency of calcium oscillations in CD148-deficient ASM was substantially attenuated, suggesting that signaling pathway alterations may underlie ASM contractility. Biochemical analysis of CD148-deficient ASM revealed hyperphosphorylation of the C-terminal inhibitory tyrosine of SRC family kinases (SFKs), implicating CD148 as a critical positive regulator of SFK signaling in ASM. The effect of CD148 deficiency on ASM contractility could be mimicked by treatment of both mouse trachea and human bronchi with specific SFK inhibitors. Our studies identify CD148 and the SFKs it regulates in ASM as potential targets for the treatment of AHR.


Asunto(s)
Asma/patología , Pulmón/patología , Familia-src Quinasas/metabolismo , Animales , Asma/metabolismo , Bronquios/patología , Linaje de la Célula , Femenino , Eliminación de Gen , Inflamación/metabolismo , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Miocitos del Músculo Liso/citología , Ovalbúmina/metabolismo , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores/metabolismo , Transducción de Señal , Tráquea/patología
9.
Immunity ; 28(2): 183-96, 2008 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-18249142

RESUMEN

The receptor-type protein tyrosine phosphatase (RPTP) CD148 is thought to have an inhibitory function in signaling and proliferation in nonhematopoietic cells. However, its role in the immune system has not been thoroughly studied. Our analysis of CD148 loss-of-function mice showed that CD148 has a positive regulatory function in B cells and macrophages, similar to the role of CD45 as a positive regulator of Src family kinases (SFKs). Analysis of CD148 and CD45 doubly deficient B cells and macrophages revealed hyperphosphorylation of the C-terminal inhibitory tyrosine of SFKs accompanied by substantial alterations in B and myeloid lineage development and defective immunoreceptor signaling. Because these findings suggest the C-terminal tyrosine of SFKs is a common substrate for both CD148 and CD45 phosphatases and imply a level of redundancy not previously appreciated, a reassessment of the function of CD45 in the B and myeloid lineages based on prior data from the CD45-deficient mouse is warranted.


Asunto(s)
Linfocitos B/inmunología , Antígenos Comunes de Leucocito/metabolismo , Macrófagos/inmunología , Receptores de Antígenos de Linfocitos B/metabolismo , Familia-src Quinasas/metabolismo , Animales , Linfocitos B/metabolismo , Cruzamientos Genéticos , Citocinas/metabolismo , Antígenos Comunes de Leucocito/deficiencia , Antígenos Comunes de Leucocito/genética , Antígenos Comunes de Leucocito/inmunología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Fagocitosis , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores/deficiencia , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores/genética , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores/inmunología , Proteínas Tirosina Fosfatasas Clase 3 Similares a Receptores/metabolismo , Receptores de Antígenos de Linfocitos B/inmunología , Transducción de Señal
10.
J Immunol ; 173(4): 2324-30, 2004 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-15294945

RESUMEN

CD148 is a receptor-like protein tyrosine phosphatase expressed on a wide variety of cell types. Through the use flow cytometry and immunofluorescence microscopy on tissue sections, we examined the expression of CD148 on multiple murine hemopoietic cell lineages. We found that CD148 is moderately expressed during all stages of B cell development in the bone marrow, as well as peripheral mature B cells. In contrast, CD148 expression on thymocytes and mature T cells is substantially lower. However, stimulation of peripheral T cells through the TCR leads to an increase of CD148 expression. This up-regulation on T cells can be partially inhibited by reagents that block the activity of src family kinases, calcineurin, MEK, or PI3K. Interestingly, CD148 levels are elevated on freshly isolated T cells from MRL lpr/lpr and CTLA-4-deficient mice, two murine models of autoimmunity. Together, these expression data along with previous biochemical data suggest that CD148 may play an important regulatory role to control an immune response.


Asunto(s)
Inmunidad Celular , Leucocitos/metabolismo , Proteínas Tirosina Fosfatasas/biosíntesis , Animales , Linfocitos B/citología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Diferenciación Celular/inmunología , Linaje de la Célula/inmunología , Cricetinae , Inhibidores Enzimáticos/farmacología , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/inmunología , Células Madre Hematopoyéticas/metabolismo , Leucocitos/inmunología , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/inmunología , Proteínas Tirosina Fosfatasas/inmunología , Linfocitos T/citología , Linfocitos T/inmunología , Linfocitos T/metabolismo
11.
Proc Natl Acad Sci U S A ; 100(22): 12935-40, 2003 Oct 28.
Artículo en Inglés | MEDLINE | ID: mdl-14566047

RESUMEN

Our studies of mice deficient for the E2F1 and E2F2 transcription factors have revealed essential roles for these proteins in the cell cycle control of pancreatic exocrine cells and the regulation of pancreatic beta cell maintenance. Pancreatic exocrine cells in E2f1-/-E2f2 mutant mice become increasingly polyploid with age, coinciding with severe exocrine atrophy. Furthermore, mice deficient for both E2F1 and E2F2 develop nonautoimmune, insulin-dependent diabetes with high penetrance. Surprisingly, transplantation of wild-type bone marrow can prevent or rescue diabetes in E2f1-/-E2f2-/-mice. We hypothesize that exocrine degeneration results in a destructive environment for beta cells, which can be alleviated by restoration of the hematopoietic system that is also defective in E2f1-/-E2f2-/-mice The demonstration that beta cell maintenance under conditions of stress is influenced by bone marrow-derived cells may provide important insight into the design of therapies to boost islet mass and function in diabetic patients.


Asunto(s)
Células de la Médula Ósea/fisiología , Trasplante de Médula Ósea , Proteínas de Ciclo Celular , Proteínas de Unión al ADN , Diabetes Mellitus/genética , Islotes Pancreáticos/patología , Factores de Transcripción/genética , Animales , Glucemia/efectos de los fármacos , Glucemia/metabolismo , Diabetes Mellitus/patología , Diabetes Mellitus/terapia , Factores de Transcripción E2F , Factor de Transcripción E2F1 , Femenino , Insulina/uso terapéutico , Masculino , Ratones , Ratones Noqueados , Poliploidía , Caracteres Sexuales , Factores de Transcripción/deficiencia , Trasplante Homólogo
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