RESUMEN
Both conventional T (Tconv) cells and regulatory T (Treg) cells are activated through ligation of the T cell receptor (TCR) complex, leading to the induction of the transcription factor NF-κB. In Tconv cells, NF-κB regulates expression of genes essential for T cell activation, proliferation, and function. However the role of NF-κB in Treg function remains unclear. We conditionally deleted canonical NF-κB members p65 and c-Rel in developing and mature Treg cells and found they have unique but partially redundant roles. c-Rel was critical for thymic Treg development while p65 was essential for mature Treg identity and maintenance of immune tolerance. Transcriptome and NF-κB p65 binding analyses demonstrated a lineage specific, NF-κB-dependent transcriptional program, enabled by enhanced chromatin accessibility. These dual roles of canonical NF-κB in Tconv and Treg cells highlight the functional plasticity of the NF-κB signaling pathway and underscores the need for more selective strategies to therapeutically target NF-κB.
Asunto(s)
Linaje de la Célula/genética , FN-kappa B/metabolismo , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Transcripción Genética , Animales , Autoinmunidad/genética , Autoinmunidad/inmunología , Sitios de Unión , Biomarcadores , Diferenciación Celular , Supervivencia Celular/genética , Supervivencia Celular/inmunología , Análisis por Conglomerados , Citocinas/metabolismo , Eliminación de Gen , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Homeostasis/genética , Homeostasis/inmunología , Tolerancia Inmunológica , Inmunofenotipificación , Inflamación/genética , Inflamación/inmunología , Inflamación/metabolismo , Activación de Linfocitos , Ratones , Ratones Transgénicos , FN-kappa B/genética , Motivos de Nucleótidos , Fenotipo , Unión Proteica , Transducción de Señal , Linfocitos T Reguladores/citología , Factor de Transcripción ReIA/genética , Factor de Transcripción ReIA/metabolismo , TranscriptomaRESUMEN
The NF-κB signaling cascade relays external signals essential for B-cell growth and survival. This cascade is frequently hijacked by cancers that arise from the malignant transformation of germinal center (GC) B cells, underscoring the importance of deciphering the function of NF-κB in these cells. The NF-κB signaling cascade is comprised of two branches, the canonical and alternative NF-κB pathways, mediated by distinct transcription factors. The expression and function of the transcription factors of the alternative pathway, RELB and NF-κB2, in late B-cell development is incompletely understood. Using conditional deletion of relb and nfkb2 in GC B cells, we here report that ablation of both RELB and NF-κB2, but not of the single transcription factors, resulted in the collapse of established GCs. RELB/NF-κB2 deficiency in GC B cells was associated with impaired cell-cycle entry and reduced expression of the cell-surface receptor inducible T-cell costimulator ligand that promotes optimal interactions between B and T cells. Analysis of human tonsillar tissue revealed that plasma cells and their precursors in the GC expressed high levels of NF-κB2 relative to surrounding lymphocytes. Accordingly, deletion of nfkb2 in murine GC B cells resulted in a dramatic reduction of antigen-specific antibody-secreting cells, whereas deletion of relb had no effect. These results demonstrate that the transcription factors of the alternative NF-κB pathway control distinct stages of late B-cell development, which may have implications for B-cell malignancies that aberrantly activate this pathway.
Asunto(s)
Linfocitos B/fisiología , Centro Germinal/fisiología , FN-kappa B/fisiología , Factores de Transcripción/fisiología , Animales , Antígenos CD40/fisiología , Células Cultivadas , Humanos , Ratones , Transducción de Señal/fisiología , Factor de Transcripción ReIB/fisiologíaRESUMEN
Signaling through the canonical nuclear factor-κB (NF-κB) pathway is critical for the generation and maintenance of mature B cells and for antigen-dependent B-cell activation. c-REL (rel) and RELA (rela) are the downstream transcriptional activators of the canonical NF-κB pathway. Studies of B cells derived from constitutional rel knockout mice and chimeric mice repopulated with rela-/- fetal liver cells provided evidence that the subunits can have distinct roles during B-cell development. However, the B cell-intrinsic functions of c-REL and RELA during B-cell generation and antigen-dependent B-cell activation have not been determined in vivo. To clarify this issue, we crossed mice with conditional rel and rela alleles individually or in combination to mice that express Cre-recombinase in B cells. We here report that, whereas single deletion of rel or rela did not impair mature B-cell generation and maintenance, their simultaneous deletion led to a dramatic reduction of follicular and marginal zone B cells. Upon T cell-dependent immunization, B cell-specific deletion of the c-REL subunit alone abrogated the formation of germinal centers (GCs), whereas rela deletion did not affect GC formation. T-independent responses were strongly impaired in mice with B cell-specific deletion of rel, and only modestly in mice with RELA-deficient B cells. Our findings identify differential requirements for the canonical NF-κB subunits c-REL and RELA at distinct stages of mature B-cell development. The subunits are jointly required for the generation of mature B cells. During antigen-dependent B-cell activation, c-REL is the critical subunit required for the initiation of the GC reaction and for optimal T-independent antibody responses, with RELA being largely dispensable at this stage.
Asunto(s)
Linfocitos B/citología , Linfocitos B/metabolismo , Activación de Linfocitos/inmunología , Proteínas Proto-Oncogénicas c-rel/metabolismo , Factor de Transcripción ReIA/metabolismo , Animales , Formación de Anticuerpos/inmunología , Factor Activador de Células B/metabolismo , Células de la Médula Ósea/citología , Diferenciación Celular , Supervivencia Celular , Eliminación de Gen , Centro Germinal/citología , Integrasas/metabolismo , Ratones Endogámicos C57BL , Bazo/citologíaRESUMEN
Psoriasis is an inflammatory skin disease in which activated immune cells and the proinflammatory cytokine TNF are well-known mediators of pathogenesis. The transcription factor NF-κB is a key regulator of TNF production and TNF-induced proinflammatory gene expression, and both the psoriatic transcriptome and genetic susceptibility further implicate NF-κB in psoriasis etiopathology. However, the role of NF-κB in psoriasis remains controversial. We analyzed the function of canonical NF-κB in the epidermis using CRE-mediated deletion of p65 and c-Rel in keratinocytes. In contrast to animals lacking p65 or c-Rel alone, mice lacking both subunits developed severe dermatitis after birth. Consistent with its partial histological similarity to human psoriasis, this condition could be prevented by anti-TNF treatment. Moreover, regulatory T cells in lesional skin played an important role in disease remission. Our results demonstrate that canonical NF-κB in keratinocytes is essential for the maintenance of skin immune homeostasis and is protective against spontaneous dermatitis.
Asunto(s)
Epidermis/inmunología , Homeostasis/inmunología , Proteínas Proto-Oncogénicas c-rel/inmunología , Piel/inmunología , Factor de Transcripción ReIA/inmunología , Animales , Animales Recién Nacidos , Anticuerpos Bloqueadores/inmunología , Anticuerpos Bloqueadores/farmacología , Células Cultivadas , Dermatitis/genética , Dermatitis/inmunología , Dermatitis/metabolismo , Epidermis/metabolismo , Epidermis/patología , Femenino , Citometría de Flujo , Expresión Génica/inmunología , Homeostasis/efectos de los fármacos , Homeostasis/genética , Queratinocitos/inmunología , Queratinocitos/metabolismo , Masculino , Ratones Noqueados , Ratones Transgénicos , Proteínas Proto-Oncogénicas c-rel/genética , Proteínas Proto-Oncogénicas c-rel/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Piel/metabolismo , Piel/patología , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Factor de Transcripción ReIA/genética , Factor de Transcripción ReIA/metabolismo , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunología , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Interferon regulatory factor 4 (IRF4) is a member of the IRF family of transcription factors and is expressed in most cell types of the immune system. Within the B-cell lineage, IRF4 is expressed in all developmental stages except during the germinal center (GC) reaction. IRF4 expression, however, is upregulated during exit from the GC reaction and has been demonstrated to have critical functions in at least three key developmental processes: the termination of the GC B-cell transcriptional program, immunoglobulin (Ig) class switch recombination (CSR), and plasma cell development. Herein, we attempt to reconcile the often contradictory findings regarding IRF4 into a model to explain the role of IRF4 in the transcription factor networks that operate within exiting GC B cells. In addition, a deregulation of the biological programs controlled by IRF4 has recently been implicated in the pathogenesis of various B-cell-derived malignancies. Determining the specific functions of IRF4 in the markedly diverse developmental processes that coordinate B-cell development is therefore likely to have important implications for understanding these malignancies and devising therapeutic interventions.
Asunto(s)
Linfocitos B/citología , Diferenciación Celular , Centro Germinal , Factores Reguladores del Interferón/metabolismo , Animales , Centro Germinal/fisiología , Humanos , Factores Reguladores del Interferón/química , Modelos BiológicosRESUMEN
PURPOSE OF REVIEW: B-cell tumors originating from the transformation of germinal center B cells frequently harbor genetic mutations, leading to constitutive activation of the nuclear factor-κB (NF-κB) signaling pathway. The present review highlights recent insights into the roles of separate NF-κB transcription factors in germinal center B-cell development and discusses implications of the results for germinal center lymphomagenesis. RECENT FINDINGS: Understanding how aberrant NF-κB activation promotes tumorigenesis requires the understanding of the role of NF-κB in the tumor-precursor cells. Despite extensive knowledge on NF-κB biology, the function of this complex signaling pathway in the differentiation of germinal center B cells is largely unknown. The present review will discuss recent findings that revealed distinct roles of separate NF-κB transcription factors during the germinal center reaction in the context of germinal center lymphomagenesis. Most notably, a single NF-κB subunit, c-REL, was found to be required for the maintenance of the germinal center reaction and was associated with the activation of a metabolic program that promotes cell growth. SUMMARY: Identifying the biological roles of the separate NF-κB transcription factor subunits in germinal center biology will help to better understand the pathogenic consequences of their constitutive activation in B-cell tumors. This knowledge may be exploited for the development of targeted antitumor therapies aimed at inhibiting selectively those components of aberrant NF-κB activity which contribute to pathogenesis.
Asunto(s)
Carcinogénesis/metabolismo , Regulación Neoplásica de la Expresión Génica , Linfoma/metabolismo , FN-kappa B/genética , Subunidades de Proteína/genética , Linfocitos B/metabolismo , Linfocitos B/patología , Carcinogénesis/genética , Carcinogénesis/patología , Diferenciación Celular , Centro Germinal/metabolismo , Centro Germinal/patología , Humanos , Linfoma/genética , Linfoma/patología , FN-kappa B/metabolismo , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Subunidades de Proteína/metabolismo , Transducción de SeñalRESUMEN
The function of dendritic cells (DCs), antigen-presenting cells regulating naïve T-cells, is regulated by cytosolic Ca²âº concentration ([Ca²âº]i). [Ca²âº]i is increased by store-operated Ca²âº entry and decreased by Kâº-independent (NCX) and Kâº-dependent (NCKX) Naâº/Ca²âº exchangers. NCKX exchangers are stimulated by immunosuppressive 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], the biologically active form of vitamin D. Formation of 1,25(OH)2D3 is inhibited by the antiaging protein Klotho. Thus 1,25(OH)2D3 plasma levels are excessive in Klotho-deficient mice (klothohm). The present study explored whether Klotho deficiency modifies [Ca²âº]i regulation in DCs. DCs were isolated from the bone marrow of klothohm mice and wild-type mice (klotho+/+) and cultured for 7-9 days with granulocyte-macrophage colony-stimulating factor. According to major histocompatibility complex II (MHC II) and CD86 expression, differentiation and lipopolysaccharide (LPS)-induced maturation were similar in klothohm DCs and klotho+/+ DCs. However, NCKX1 membrane abundance and NCX/NCKX-activity were significantly enhanced in klothohm DCs. The [Ca²âº]i increase upon acute application of LPS (1 µg/ml) was significantly lower in klothohm DCs than in klotho+/+ DCs, a difference reversed by the NCKX blocker 3',4'-dichlorobenzamyl (DBZ; 10 µM). CCL21-dependent migration was significantly less in klothohm DCs than in klotho+/+ DCs but could be restored by DBZ. NCKX activity was enhanced by pretreatment of klotho+/+ DC precursors with 1,25(OH)2D3 the first 2 days after isolation from bone marrow. Feeding klothohm mice a vitamin D-deficient diet decreased NCKX activity, augmented LPS-induced increase of [Ca²âº]i, and enhanced migration of klothohm DCs, thus dissipating the differences between klothohm DCs and klotho+/+ DCs. In conclusion, Klotho deficiency upregulates NCKX1 membrane abundance and Naâº/Ca²âº-exchange activity, thus blunting the increase of [Ca²âº]i following LPS exposure and CCL21-mediated migration. The effects are in large part due to excessive 1,25(OH)2D3 formation.
Asunto(s)
Calcio/metabolismo , Citosol/metabolismo , Células Dendríticas/metabolismo , Glucuronidasa/metabolismo , Animales , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Movimiento Celular/fisiología , Células Cultivadas , Quimiocina CCL21 , Citosol/química , Regulación de la Expresión Génica/fisiología , Glucuronidasa/genética , Proteínas Klotho , Lipopolisacáridos , Macrófagos/citología , Macrófagos/metabolismo , Ratones , Ratones NoqueadosRESUMEN
Ca(2+) signaling includes store-operated Ca(2+) entry (SOCE) following depletion of endoplasmic reticulum (ER) Ca(2+) stores. On store depletion, the ER Ca(2+) sensor STIM1 activates Orai1, the pore-forming unit of Ca(2+)-release-activated Ca(2+) (CRAC) channels. Here, we show that Orai1 is regulated by serum- and glucocorticoid-inducible kinase 1 (SGK1), a growth factor-regulated kinase. Membrane Orai1 protein abundance, I(CRAC), and SOCE in human embryonic kidney (HEK293) cells stably expressing Orai1 and transfected with STIM1 were each significantly enhanced by coexpression of constitutively active (S422D)SGK1 (by+81, +378, and+136%, respectively) but not by inactive (K127N)SGK1. Coexpression of the ubiquitin ligase Nedd4-2, an established negatively regulated SGK1 target, down-regulated SOCE (by -48%) and I(CRAC) (by -60%), an effect reversed by expression of (S422D)SGK1 (by +175 and +173%, respectively). Orai1 protein abundance and SOCE were significantly lower in mast cells from SGK1-knockout (sgk1(-/-)) mice (by -37% and -52%, respectively) than in mast cells from wild-type (sgk1(+/+)) littermates. Activation of SOCE by sarcoplasmic/endoplasmic reticulum Ca(2+)-ATPase-inhibitor thapsigargin (2 µM) stimulated migration, an effect significantly higher (by +306%) in (S422D)SGK1-expressing than in (K127N)SGK1-expressing HEK293 cells, and also significantly higher (by +108%) in sgk1(+/+) than in sgk1(-/-) mast cells. SGK1 is thus a novel key player in the regulation of SOCE.
Asunto(s)
Canales de Calcio/metabolismo , Señalización del Calcio/fisiología , Proteínas Inmediatas-Precoces/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Animales , Canales de Calcio/genética , Línea Celular , Movimiento Celular , Femenino , Regulación de la Expresión Génica/fisiología , Humanos , Proteínas Inmediatas-Precoces/genética , Masculino , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Proteínas de Neoplasias/genética , Proteína ORAI1 , Proteínas Serina-Treonina Quinasas/genética , Molécula de Interacción Estromal 1RESUMEN
Bone-marrow-derived mast cells are matured from bone marrow cells in medium containing 20% fetal calf serum (FCS), interleukin (IL)-3 and stem-cell factor (SCF) and are used as in vitro models to study mast cells (MC) and their role in health and disease. In vivo, however, BM-derived hematopoietic stem cells account for only a fraction of MC; the majority of MC in vivo are and remain tissue resident. In this study we established a side-by-side culture with BMMC, fetal skin MC (FSMC) or fetal liver MC (FLMC) for comparative studies to identify the best surrogates for mature connective tissue MC (CTMC). All three MC types showed comparable morphology by histology and MC phenotype by flow cytometry. Heterogeneity was detected in the transcriptome with the most differentially expressed genes in FSMC compared to BMMC being Hdc and Tpsb2. Expression of ST2 was highly expressed in BMMC and FSMC and reduced in FLMC, diminishing their secretion of type 2 cytokines. Higher granule content, stronger response to FcεRI activation and significantly higher release of histamine from FSMC compared to FLMC and BMMC indicated differences in MC development in vitro dependent on the tissue of origin. Thus, tissues of origin imprint MC precursor cells to acquire distinct phenotypes and signatures despite identical culture conditions. Fetal-derived MC resemble mature CTMC, with FSMC being the most developed.
Asunto(s)
Células del Tejido Conectivo , Mastocitos , Células Cultivadas , Tejido Conectivo , Feto , Mastocitos/metabolismoRESUMEN
Ca(+)-dependent signaling regulates the function of dendritic cells (DCs), antigen-presenting cells linking innate and adaptive immunity. The activity of DCs is suppressed by glucocorticoids, potent immunosuppressive hormones. The present study explored whether the glucocorticoid dexamethasone influences the cytosolic Ca(2+) concentration ([Ca(2+)](i)) in DCs. To this end, DCs were isolated from mouse bone marrow. According to fura-2 fluorescence, exposure of DCs to lipopolysaccharide (LPS, 100 ng/ml) increased [Ca(2+)](i), an effect significantly blunted by overnight incubation with 10 nM dexamethasone before LPS treatment. Dexamethasone did not affect the Ca(2+) content of intracellular stores, sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA)2 and SERCA3 expression, ryanodine receptor (RyR)1 expression, or Ca(2+) entry through store-operated Ca(2+) channels. In contrast, dexamethasone increased the transcript level and the membrane protein abundance of the Na(+)/Ca(2+) exchanger NCX3. The activity of Na(+)/Ca(2+) exchangers was assessed by removal of extracellular Na(+) in the presence of external Ca(2+), a maneuver triggering the Ca(2+) influx mode. Indeed, Na(+) removal resulted in a rapid transient increase of [Ca(2+)](i) and induced an outwardly directed current as measured in whole cell patch-clamp experiments. Dexamethasone significantly augmented the increase of [Ca(2+)](i) and the outward current following removal of extracellular Na(+). The NCX blocker KB-R7943 reversed the inhibitory effect of dexamethasone on LPS-induced increase in [Ca(2+)](i). Dexamethasone blunted LPS-induced stimulation of CD86 expression and TNF-α production, an effect significantly less pronounced in the presence of NCX blocker KB-R7943. In conclusion, our results show that glucocorticoid treatment blunts LPS-induced increase in [Ca(2+)](i) in DCs by increasing expression and activity of Na(+)/Ca(2+) exchanger NCX3. The effect contributes to the inhibitory effect of the glucocorticoid on DC maturation.
Asunto(s)
Células Dendríticas/efectos de los fármacos , Células Dendríticas/metabolismo , Dexametasona/farmacología , Glucocorticoides/farmacología , Intercambiador de Sodio-Calcio/metabolismo , Animales , Antígeno B7-2/metabolismo , Calcio/metabolismo , Células Dendríticas/citología , Femenino , Humanos , Lipopolisacáridos/farmacología , Ratones , Técnicas de Placa-Clamp , Intercambiador de Sodio-Calcio/antagonistas & inhibidores , Tiourea/análogos & derivados , Tiourea/metabolismo , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Previous studies have shown that pharmacological inhibition of the phosphoinositol-3 (PI3) kinase disrupts the activation of mast cells. Through phosphoinositide-dependent kinase PDK1, PI3 kinase activates the serum- and glucocorticoid-inducible kinase 3 (SGK3). The present study explored the role of SGK3 in mast cell function. Mast cells were isolated and cultured from bone marrow (BMMCs) of gene-targeted mice lacking SGK3 (sgk3(-/-)) and their wild-type littermates (sgk3(+/+)). BMMC numbers in the ear conch were similar in both genotypes. Stimulation with IgE and cognate antigen triggered the release of intracellular Ca(2+) and entry of extracellular Ca(2+). Influx of extracellular Ca(2+) but not Ca(2+) release from intracellular stores was significantly blunted in sgk3(-/-) BMMCs compared with sgk3(+/+) BMMCs. Antigen stimulation further led to a rapid increase of a K(+)-selective conductance in sgk3(+/+) BMMCs, an effect again blunted in sgk3(-/-) BMMCs. In contrast, the Ca(2+) ionophore ionomycin activated K(+) currents to a similar extent in sgk3(-/-) and in sgk3(+/+) BMMCs. ß-Hexosaminidase release, triggered by antigen stimulation, was also significantly decreased in sgk3(-/-) BMMCs. IgE-dependent anaphylaxis measured as a sharp decrease in body temperature upon injection of DNP-HSA antigen was again significantly blunted in sgk3(-/-) compared with sgk3(+/+) mice. Serum histamine levels measured 30 min after induction of an anaphylactic reaction were significantly lower in sgk3(-/-) than in sgk3(+/+) mice. In conclusion, both in vitro and in vivo function of BMMCs are impaired in gene targeted mice lacking SGK3. Thus SGK3 is critical for proper mast cell function.
Asunto(s)
Inmunoglobulina E/inmunología , Mastocitos/inmunología , Proteínas Serina-Treonina Quinasas/inmunología , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/inmunología , Calcio/metabolismo , Degranulación de la Célula , Oído/anatomía & histología , Femenino , Masculino , Mastocitos/citología , Ratones , Ratones Noqueados , Técnicas de Placa-Clamp , Proteínas Serina-Treonina Quinasas/genética , beta-N-Acetilhexosaminidasas/metabolismoRESUMEN
Genotoxic stress induces cell cycle arrest and DNA repair which may enable tumor cells to survive radiation therapy. Here, we defined the role of Ca(2+) signaling in the cell cycle control and survival of chronic myeloid leukemia (CML) cells subjected to ionizing radiation (IR). To this end, K562 erythroid leukemia cells were irradiated (0-10 Gy). Tumor survival was analyzed by clonogenic survival assay and cell cycle progression via flow cytometry. Plasma membrane cation conductance was assessed by patch-clamp whole-cell recording and the cytosolic free Ca(2+) concentration ([Ca(2+)](i)) was measured by fura-2 Ca(2+) imaging. Nuclear activity of Ca(2+)/calmodulin-dependent kinase II (CaMKII) was defined by Western blotting. In addition, the effect of IR (5 Gy) on the cation conductance of primary CML cells was determined. The results indicated that IR (10 Gy) induced a G(2)/M cell cycle arrest of K562 cells within 24 h post-irradiation (p.i.) and decreased the clonogenic survival to 0.5 % of that of the control cells. In K562 cells, G(2)/M cell cycle arrest was preceded by activation of TRPV5/6-like nonselective cation channels in the plasma membrane 1-5 h p.i., resulting in an elevated Ca(2+) entry as evident from fura-2 Ca(2+) imaging. Similarly, IR stimulated a Ca(2+)-permeable nonselective cation conductance in primary CML cells within 2-4 h p.i.. Ca(2+) entry, into K562 cells was paralleled by an IR-induced activation of nuclear CaMKII. The IR-stimulated accumulation in G(2) phase was delayed upon buffering [Ca(2+)](i) with the Ca(2+) chelator BAPTA-AM or inhibiting CaMKII with KN93 (1 nM). In addition, KN93 decreased the clonogenic survival of irradiated cells but not of control cells. In conclusion, the data suggest that IR-stimulated cation channel activation, Ca(2+) entry and CaMKII activity participate in control of cell cycle progression and survival of irradiated CML cells.
Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Calcio/metabolismo , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismo , Tolerancia a Radiación , Canales Catiónicos TRPV/metabolismo , Canales de Calcio/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/antagonistas & inhibidores , División Celular , Quelantes/química , Quelantes/farmacología , Ácido Egtácico/análogos & derivados , Ácido Egtácico/química , Ácido Egtácico/farmacología , Fura-2/química , Fura-2/farmacología , Fase G2 , Humanos , Células K562 , Leucemia Mielógena Crónica BCR-ABL Positiva/enzimología , Leucemia Mielógena Crónica BCR-ABL Positiva/radioterapia , Técnicas de Placa-Clamp , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Radiación IonizanteRESUMEN
The function of mast cells is modified by the phosphoinositol-3 (PI3)-kinase pathway. The kinase signals partially through the phosphoinositide-dependent kinase PDK1, which on the one hand activates the serum- and glucocorticoid- inducible kinase SGK1 and on the other hand activates protein kinase PKCδ. SGK1 participates in the stimulation of Ca(2+) entry and degranulation, PKCδ inhibits degranulation. The present experiments explored the role of PDK1 in mast cell function. As mice completely lacking PDK1 are not viable, experiments have been performed in mast cells isolated from bone marrow (BMMCs) of PDK1 hypomorphic mice (pdk1(hm)) and their wild-type littermates (pdk1(wt)). Antigen stimulation via the FceRI receptor was followed by Ca(2+) entry leading to increase of cytosolic Ca(2+) activity in pdk1(wt) BMMCs, an effect significantly blunted in pdk1(hm) BMMCs. In contrast, Ca(2+) release from intracellular stores was not different between BMMCs of the two genotypes. The currents through Ca(2+)-activated K(+) channels following antigen exposure were again significantly larger in pdk1(wt) than in pdk1(hm) cells. The Ca(2+) ionophore ionomycin (1 µM) increased the K(+) channel conductance to similar values in both genotypes. ß-hexosaminidase and histamine release were similar in pdk1(wt) BMMCs and pdk1(hm) BMMCs. PKCδ inhibitor rottlerin increased ß-hexosaminidase release in pdk1(wt) BMMCs but not in pdk1(hm) BMMCs. Phosphorylation of PKCδ and of the SGK1 target NDRG1, was stimulated by the antigen in pdk1(wt) but not in pdk1(hm) cells. The observations reveal a role for PDK1 in the regulation of Ca(2+) entry into and degranulation of murine mast cells.
Asunto(s)
Calcio/metabolismo , Mastocitos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Quinasas Dependientes de 3-Fosfoinosítido , Acetofenonas/farmacología , Animales , Benzopiranos/farmacología , Proteínas de Ciclo Celular/metabolismo , Inhibidores Enzimáticos/farmacología , Histamina/metabolismo , Proteínas Inmediatas-Precoces , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ionomicina/farmacología , Ionóforos/farmacología , Mastocitos/efectos de los fármacos , Mastocitos/enzimología , Ratones , Técnicas de Placa-Clamp , Fosforilación , Canales de Potasio/metabolismo , Proteína Quinasa C-delta/antagonistas & inhibidores , Proteína Quinasa C-delta/metabolismo , Proteínas Serina-Treonina Quinasas/genética , beta-N-Acetilhexosaminidasas/metabolismoRESUMEN
Somatic hypermutation of immunoglobulin variable region (IgV) genes and affinity maturation of the antibody response are the hallmarks of the germinal center (GC) reaction in T cell-dependent immune responses. Determining the consequences of the experimental manipulation of the GC response on somatic hypermutation and affinity maturation requires the availability of a system that allows measuring these parameters. Immunization of mice of the C57/Bl6 genetic background with the hapten 4-hydroxy-3-nitrophenyl-acetyl (NP) coupled to a carrier protein leads to the predominant usage of one particular IgV heavy chain gene segment, V186.2, among the responding B cells. Moreover, a specific somatic mutation in codon 33 of V186.2 that leads to a tryptophan to leucine amino acid exchange increases the affinity of the corresponding antibody by ~10-fold, thus representing a molecular marker for affinity maturation. In addition, due to the simplicity of the antigen and the virtual absence of NP-specific plasma cells prior to immunization, NP-based immunizations represent ideal tools to quantify the plasma cell response by measuring NP-specific antisera by ELISA and the generation of NP-specific plasma cells by ELISPOT analysis. We here describe approaches to (1) measure the anti-NP plasma cell response by ELISA and ELISPOT analysis, and to (2) amplify and sequence V186.2 rearrangements from GC B cells and plasma cells to determine the level of somatic hypermutation and the extent of affinity maturation in the anti-NP response.
Asunto(s)
Nitrofenoles , Fenilacetatos , Hipermutación Somática de Inmunoglobulina , Animales , Formación de Anticuerpos , Linfocitos B/inmunología , Linfocitos B/metabolismo , Biomarcadores , Clonación Molecular , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Reordenamiento Génico de Linfocito B , Centro Germinal/citología , Centro Germinal/inmunología , Centro Germinal/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento , Inmunización , Región Variable de Inmunoglobulina/genética , Memoria Inmunológica , Inmunofenotipificación , Ratones , Nitrofenoles/inmunología , Fenilacetatos/inmunología , Linfocitos T/inmunología , Linfocitos T/metabolismoRESUMEN
IκB kinase/nuclear [corrected] factor κB (IKK/NF-κB) signaling exhibits important yet opposing functions in hepatocarcinogenesis. Mice lacking NEMO in liver parenchymal cells (LPC) spontaneously develop steatohepatitis and hepatocellular carcinoma (HCC) suggesting that NF-κB prevents liver disease and cancer. Here, we show that complete NF-κB inhibition by combined LPC-specific ablation of RelA, c-Rel, and RelB did not phenocopy NEMO deficiency, but constitutively active IKK2-mediated NF-κB activation prevented hepatocellular damage and HCC in NEMO(LPC-KO) mice. Knock-in expression of kinase inactive receptor-interacting protein kinase 1 (RIPK1) prevented hepatocyte apoptosis and HCC, while RIPK1 ablation induced TNFR1-associated death domain protein (TRADD)-dependent hepatocyte apoptosis and liver tumors in NEMO(LPC-KO) mice, revealing distinct kinase-dependent and scaffolding functions of RIPK1. Collectively, these results show that NEMO prevents hepatocarcinogenesis by inhibiting RIPK1 kinase activity-driven hepatocyte apoptosis through NF-κB-dependent and -independent functions.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Hígado Graso/metabolismo , Hepatocitos/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Neoplasias Hepáticas/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Animales , Apoptosis/genética , Carcinoma Hepatocelular/genética , Células Cultivadas , Hígado Graso/genética , Expresión Génica , Quinasa I-kappa B/genética , Quinasa I-kappa B/metabolismo , Immunoblotting , Inmunohistoquímica , Péptidos y Proteínas de Señalización Intracelular/genética , Hígado/metabolismo , Hígado/patología , Neoplasias Hepáticas/genética , Ratones Endogámicos C57BL , Ratones Noqueados , FN-kappa B/genética , FN-kappa B/metabolismo , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/genéticaRESUMEN
Germinal centers (GCs) are the sites where memory B cells and plasma cells producing high-affinity antibodies are generated during T cell-dependent immune responses. The molecular control of GC B cell maintenance and differentiation remains incompletely understood. Activation of the NF-κB signaling pathway has been implicated; however, the distinct roles of the individual NF-κB transcription factor subunits are unknown. We report that GC B cell-specific deletion of the NF-κB subunits c-REL or RELA, which are both activated by the canonical NF-κB pathway, abolished the generation of high-affinity B cells via different mechanisms acting at distinct stages during the GC reaction. c-REL deficiency led to the collapse of established GCs immediately after the formation of dark and light zones at day 7 of the GC reaction and was associated with the failure to activate a metabolic program that promotes cell growth. Conversely, RELA was dispensable for GC maintenance but essential for the development of GC-derived plasma cells due to impaired up-regulation of BLIMP1. These results indicate that activation of the canonical NF-κB pathway in GC B cells controls GC maintenance and differentiation through distinct transcription factor subunits. Our findings have implications for the role of NF-κB in GC lymphomagenesis.
Asunto(s)
Linfocitos B/inmunología , Diferenciación Celular/inmunología , Centro Germinal/inmunología , FN-kappa B/metabolismo , Transducción de Señal/inmunología , Animales , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Fluoresceínas , Eliminación de Gen , Immunoblotting , Inmunohistoquímica , Ratones , Ratones Transgénicos , Factor 1 de Unión al Dominio 1 de Regulación Positiva , Proteínas Proto-Oncogénicas c-rel/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Análisis de Secuencia de ARN , Succinimidas , Factor de Transcripción ReIA/genética , Factores de Transcripción/metabolismoRESUMEN
The transcription factor interferon regulatory factor-4 (IRF4) is expressed in B cells at most developmental stages. In antigen-activated B cells, IRF4 controls germinal center formation, class-switch recombination, and the generation of plasma cells. Here we describe a novel function for IRF4 in the homeostasis of mature B cells. Inducible deletion of irf4 specifically in B cells in vivo led to the aberrant accumulation of irf4-deleted follicular B cells in the marginal zone (MZ) area. IRF4-deficient B cells showed elevated protein expression and activation of NOTCH2, a transmembrane receptor and transcriptional regulator known to be required for MZ B cell development. Administration of a NOTCH2-inhibitory antibody abolished nuclear translocation of NOTCH2 in B cells within 12 h and caused a rapid and progressive disintegration of the MZ that was virtually complete 48 h after injection. The disappearance of the MZ was accompanied by a transient increase of MZ-like B cells in the blood rather than increased B cell apoptosis, demonstrating that continued NOTCH2 activation is critical for the retention of B cells in the MZ. Our results suggest that IRF4 controls the positioning of mature B cells in the lymphoid microenvironments by regulating NOTCH2 expression. These findings may have implications for the understanding of B cell malignancies with dysregulated IRF4 and NOTCH2 activity.