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1.
Int J Mol Sci ; 23(24)2022 Dec 10.
Artículo en Inglés | MEDLINE | ID: mdl-36555322

RESUMEN

Understanding the signaling cascades that govern adipocyte metabolism and differentiation is necessary for the development of therapies for obesity. Toll-like receptors (TLRs) are key mediators in adipogenesis, but their specific role is not completely understood. In this study, siRNA knockdown of Tlr2 in 3T3-L1 cells allowed them to differentiate more efficiently into adipocytes, whereas the opposite was observed for the knockdown of Tlr4. At the same time, we show that TLR2 knock-out mice spontaneously developed mature-onset obesity and insulin resistance. Besides a higher incidence of hyperplasia and hypertrophy in white adipose tissue (WAT), we found a significantly increased number of adipocyte precursor cells in TLR2-/- mice compared to TLR4-/- mice. Interestingly, genetic inactivation of Tlr4 in TLR2-/- mice reverted their increased adiposity, insulin resistance, and restored normal levels of adipocyte precursor cells. These findings provide evidence that TLR2 and TLR4 play opposing roles in WAT homeostasis and point to the existence of cross-regulation among TLR2 and TLR4 during adipocyte differentiation both in vitro and in vivo.


Asunto(s)
Resistencia a la Insulina , Receptor Toll-Like 4 , Ratones , Animales , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismo , Receptor Toll-Like 2/genética , Receptor Toll-Like 2/metabolismo , Resistencia a la Insulina/genética , Obesidad/metabolismo , Diferenciación Celular/genética , Adipocitos/metabolismo , Adipogénesis/genética , Ratones Noqueados , Células 3T3-L1
3.
Biochim Biophys Acta ; 1863(1): 115-27, 2016 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-26516054

RESUMEN

The expression and function of TRPV1 are influenced by its interaction with cellular proteins. Here, we identify Whirlin, a cytoskeletal PDZ-scaffold protein implicated in hearing, vision and mechanosensory transduction, as an interacting partner of TRPV1. Whirlin associates with TRPV1 in cell lines and in primary cultures of rat nociceptors. Whirlin is expressed in 55% of mouse sensory C-fibers, including peptidergic and non-peptidergic nociceptors, and co-localizes with TRPV1 in 70% of them. Heterologous expression of Whirlin increased TRPV1 protein expression and trafficking to the plasma membrane, and promoted receptor clustering. Silencing Whirlin expression with siRNA or blocking protein translation resulted in a concomitant degradation of TRPV1 that could be prevented by inhibiting the proteasome. The degradation kinetics of TRPV1 upon arresting protein translation mirrored that of Whirlin in cells co-expressing both proteins, suggesting a parallel degradation mechanism. Noteworthy, Whirlin expression significantly reduced TRPV1 degradation induced by prolonged exposure to capsaicin. Thus, our findings indicate that Whirlin and TRPV1 are associated in a subset of nociceptors and that TRPV1 protein stability is increased through the interaction with the cytoskeletal scaffold protein. Our results suggest that the Whirlin­TRPV1 complex may represent a novel molecular target and its pharmacological disruption might be a therapeutic strategy for the treatment of peripheral TRPV1-mediated disorders.


Asunto(s)
Regulación de la Expresión Génica/fisiología , Proteínas de la Membrana/metabolismo , Complejos Multiproteicos/metabolismo , Nociceptores/metabolismo , Canales Catiónicos TRPV/biosíntesis , Animales , Células Cultivadas , Proteínas de la Membrana/genética , Ratones , Complejos Multiproteicos/genética , Nociceptores/citología , Complejo de la Endopetidasa Proteasomal/genética , Complejo de la Endopetidasa Proteasomal/metabolismo , Estabilidad Proteica , Proteolisis , ARN Interferente Pequeño , Ratas , Ratas Wistar , Canales Catiónicos TRPV/genética
4.
Immunol Cell Biol ; 94(1): 39-51, 2016 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-26051593

RESUMEN

Understanding the regulation of T-cell responses during inflammation and auto-immunity is fundamental for designing efficient therapeutic strategies against immune diseases. In this regard, prostaglandin E2 (PGE2) is mostly considered a myeloid-derived immunosuppressive molecule. We describe for the first time that T cells secrete PGE2 during T-cell receptor stimulation. In addition, we show that autocrine PGE2 signaling through EP receptors is essential for optimal CD4(+) T-cell activation in vitro and in vivo, and for T helper 1 (Th1) and regulatory T cell differentiation. PGE2 was found to provide additive co-stimulatory signaling through AKT activation. Intravital multiphoton microscopy showed that triggering EP receptors in T cells is also essential for the stability of T cell-dendritic cell (DC) interactions and Th-cell accumulation in draining lymph nodes (LNs) during inflammation. We further demonstrated that blocking EP receptors in T cells during the initial phase of collagen-induced arthritis in mice resulted in a reduction of clinical arthritis. This could be attributable to defective T-cell activation, accompanied by a decline in activated and interferon-γ-producing CD4(+) Th1 cells in draining LNs. In conclusion, we prove that T lymphocytes secret picomolar concentrations of PGE2, which in turn provide additive co-stimulatory signaling, enabling T cells to attain a favorable activation threshold. PGE2 signaling in T cells is also required for maintaining long and stable interactions with DCs within LNs. Blockade of EP receptors in vivo impairs T-cell activation and development of T cell-mediated inflammatory responses. This may have implications in various pathophysiological settings.


Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Reactividad Cruzada/inmunología , Subtipo EP2 de Receptores de Prostaglandina E/metabolismo , Subtipo EP4 de Receptores de Prostaglandina E/metabolismo , Transducción de Señal , Animales , Artritis/inmunología , Artritis/patología , Comunicación Celular/inmunología , Diferenciación Celular , Proliferación Celular , Células Dendríticas/inmunología , Dinoprostona/metabolismo , Modelos Animales de Enfermedad , Humanos , Inflamación/patología , Mediadores de Inflamación/metabolismo , Interleucina-2/biosíntesis , Ganglios Linfáticos/metabolismo , Ratones Noqueados , Subtipo EP2 de Receptores de Prostaglandina E/antagonistas & inhibidores , Subtipo EP4 de Receptores de Prostaglandina E/antagonistas & inhibidores , Linfocitos T Reguladores/inmunología , Células TH1/inmunología , Regulación hacia Arriba
5.
Immunol Cell Biol ; 90(6): 579-86, 2012 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-21946663

RESUMEN

Our understanding of the key players involved in the differential regulation of T-cell responses during inflammation, infection and auto-immunity is fundamental for designing efficient therapeutic strategies against immune diseases. With respect to this, the inhibitory role of the lipid mediator prostaglandin E(2) (PGE(2)) in T-cell immunity has been documented since the 1970s. Studies that ensued investigating the underlying mechanisms substantiated the suppressive function of micromolar concentrations of PGE(2) in T-cell activation, proliferation, differentiation and migration. However, the past decade has seen a revolution in this perspective, since nanomolar concentrations of PGE(2) have been shown to potentiate Th1 and Th17 responses and aid in T-cell proliferation. The understanding of concentration-specific effects of PGE(2) in other cell types, the development of mice deficient in each subtype of the PGE(2) receptors (EP receptors) and the delineation of signalling pathways mediated by the EP receptors have enhanced our understanding of PGE(2) as an immune-stimulator. PGE(2) regulates a multitude of functions in T-cell activation and differentiation and these effects vary depending on the micro-environment of the cell, maturation and activation state of the cell, type of EP receptor involved, local concentration of PGE(2) and whether it is a homeostatic or inflammatory scenario. In this review, we compartmentalize the various aspects of this complex relationship of PGE(2) with T lymphocytes. Given the importance of this molecule in T-cell activation, we also address the possibility of using EP receptor antagonism as a potential therapeutic approach for some immune disorders.


Asunto(s)
Dinoprostona/inmunología , Dinoprostona/metabolismo , Activación de Linfocitos , Receptores de Prostaglandina E/metabolismo , Linfocitos T/inmunología , Animales , Diferenciación Celular , Movimiento Celular , Proliferación Celular , Tolerancia Inmunológica , Inflamación/inmunología , Ratones , Receptores de Prostaglandina E/genética , Transducción de Señal
6.
J Clin Invest ; 118(2): 640-50, 2008 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-18172554

RESUMEN

The aryl hydrocarbon receptor repressor (AHRR) is a bHLH/Per-ARNT-Sim transcription factor located in a region of chromosome 5 (5p15.3) that has been proposed to contain one or more tumor suppressor genes. We report here consistent downregulation of AHRR mRNA in human malignant tissue from different anatomical origins, including colon, breast, lung, stomach, cervix, and ovary, and demonstrate DNA hypermethylation as the regulatory mechanism of AHRR gene silencing. Knockdown of AHRR gene expression in a human lung cancer cell line using siRNA significantly enhanced in vitro anchorage-dependent and -independent cell growth as well as cell growth after transplantation into immunocompromised mice. In addition, knockdown of AHRR in non-clonable normal human mammary epithelial cells enabled them to grow in an anchorage-independent manner. Further, downregulation of AHRR expression in the human lung cancer cell line conferred resistance to apoptotic signals and enhanced motility and invasion in vitro and angiogenic potential in vivo. Ectopic expression of AHRR in tumor cells resulted in diminished anchorage-dependent and -independent cell growth and reduced angiogenic potential. These results therefore demonstrate that AHRR is a putative new tumor suppressor gene in multiple types of human cancers.


Asunto(s)
Genes Supresores de Tumor/fisiología , Neoplasias/patología , Receptores de Hidrocarburo de Aril/fisiología , Animales , Apoptosis/genética , Ciclo Celular/genética , Línea Celular Tumoral , Movimiento Celular/genética , Genes Supresores de Tumor/efectos de los fármacos , Humanos , Ratones , Trasplante de Neoplasias , Neoplasias/genética , Neoplasias/metabolismo , ARN Interferente Pequeño/farmacología , Receptores de Hidrocarburo de Aril/antagonistas & inhibidores , Receptores de Hidrocarburo de Aril/genética
7.
Arch Physiol Biochem ; 114(3): 201-9, 2008 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-18629685

RESUMEN

Prostanoids, including prostaglandins (PGs) and thromboxanes (TXs) are synthesized from arachidonic acid by the combined action of cyclooxygenases (COXs) and PG and TX synthases. Finally after their synthesis, prostanoids are quickly released to the extracellular medium exerting their effects upon interaction with prostanoid receptors present in the neighbouring cells. These agents exert important actions in the cardiovascular system, modulating vascular homeostasis and participating in the pathogenesis of vascular diseases as thrombosis and atherosclerosis. Among prostanoids, Tromboxane (TX)A(2), a potent platelet activator and vasoconstrictor and prostacyclin (PGI2), a platelet inhibitor and vasodilator, are the most important in controlling vascular homeostasis. Although multiple studies using pharmacological inhibitors and genetically deficient mice have demonstrated the importance of prostanoid-mediated actions on cardiovascular physiology, further analysis on the prostanoid mediated actions in the vascular system are required to better understand the benefits and risks for the use of COX inhibitors in cardiovascular diseases.


Asunto(s)
Enfermedades Cardiovasculares/fisiopatología , Prostaglandinas/fisiología , Homeostasis , Humanos
8.
J Endotoxin Res ; 12(5): 285-95, 2006.
Artículo en Inglés | MEDLINE | ID: mdl-17059692

RESUMEN

Activation of TLRs is most closely associated with induction of pro-inflammatory gene expression; however, expression of many other genes, including the TLR genes themselves, has also been shown to be modulated following TLR engagement. A large family of nuclear transcription factors, the interferon regulatory factors (IRFs), have been implicated in TLR signaling leading to pro-inflammatory gene expression. Given that IRF-1 and IRF-2 counter-regulate the transcriptional activity of many genes, we hypothesized that IRF-1 and IRF-2 might also regulate TLR gene expression following LPS stimulation of murine macrophages. mRNA derived from medium- or LPS-treated primary peritoneal macrophages was analyzed for TLR gene expression using quantitative real-time PCR. In wild-type macrophages, LPS up-regulated expression of TLRs 1-3 and 6-9 steady-state mRNA, while TLR4 mRNA was modestly down-regulated. IRF-2(-/-) macrophages responded to LPS with dysregulated expression of TLR3, TLR4, and TLR5 mRNA, whereas IRF-1 deficiency dampened LPS-induced mRNA expression for TLR3, TLR6, and TLR9. Functional studies revealed aberrant TLR3 signaling in IRF-2(-/-) macrophages. Collectively, these findings reveal an additional level of complexity associated with TLR transcriptional regulation and suggest that the trans-acting factors, IRF-1 and IRF-2, contribute to the innate immune response to infections by regulating TLR gene expression.


Asunto(s)
Regulación de la Expresión Génica/efectos de los fármacos , Factor 1 Regulador del Interferón/metabolismo , Factor 2 Regulador del Interferón/metabolismo , Macrófagos Peritoneales/metabolismo , Receptores Toll-Like/metabolismo , Transcripción Genética , Animales , Células Cultivadas , Factor 1 Regulador del Interferón/genética , Factor 2 Regulador del Interferón/genética , Lipopolisacáridos/farmacología , Macrófagos Peritoneales/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , ARN Mensajero/análisis , Transcripción Genética/efectos de los fármacos
9.
J Histochem Cytochem ; 53(6): 773-80, 2005 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-15928326

RESUMEN

Adrenomedullin (AM) is a potent vasodilator peptide present in the lung of mammals where it is expressed mainly in the columnar epithelium and alveolar macrophages. AM increases the secretion of phosphatidylcholine by type II pneumocytes, which suggests a role as an autocrine modulator of surfactant secretion. In this study we show the expression of an AM-like protein in the lung of the pigeon, Columba livia. Using an antibody against its human ortholog, AM-like immunoreactivity was found to be associated with membranous structures of the multivesicular bodies of type II pneumocytes. We also studied the differential expression of AM-like peptide in the lung of pigeons exposed to polluted city air vs cleaner countryside conditions and found that AM-like expression was higher in city animals. Similar results were obtained in an experimental study in which pigeons were exposed to increasing concentrations of a single pollutant, ozone. Taken together, our findings support the implication of AM in the response of type II pneumocytes to air pollutants.


Asunto(s)
Contaminantes Atmosféricos/toxicidad , Pulmón/metabolismo , Péptidos/agonistas , Adrenomedulina , Animales , Columbidae , Humanos , Inmunohistoquímica , Pulmón/citología , Pulmón/ultraestructura , Ozono/toxicidad , Péptidos/metabolismo
10.
J Leukoc Biol ; 74(2): 277-86, 2003 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-12885945

RESUMEN

Toll-like receptor (TLR) proteins mediate cellular activation by microbes and microbial products. To delineate the role of TLR proteins in the development of host immune responses against mycobacteria, wild-type and TLR-deficient mice were infected with nonpathogenic Mycobacterium bovis bacillus Calmette-Guerin (BCG). Two weeks after intraperitoneal challenge with BCG, few bacilli were present in the lungs of wild-type and TLR4(-/-) mice, whereas bacterial loads were tenfold higher in the lungs of infected TLR2(-/-) mice. BCG challenge in vitro strongly induced proinflammatory cytokine secretion by macrophages from wild-type and TLR4(-/-) mice but not by TLR2(-/-) macrophages. In contrast, intracellular uptake, intracellular bacterial growth, and suppression of intracellular bacterial growth in vitro by interferon-gamma (IFN-gamma) were similar in macrophages from all three mouse strains, suggesting that BCG growth in the lungs of TLR2(-/-) mice was a consequence of defective adaptive immunity. Antigenic stimulation of splenocytes from infected wild-type and TLR4(-/-) mice induced T cell proliferation in vitro, whereas T cells from TLR2(-/-) mice failed to proliferate. Unexpectedly, activated CD4(+) T cells from both TLR-deficient mouse strains secreted little IFN-gamma in vitro compared with control T cells. A role for TLR4 in the control of bacterial growth and IFN-gamma production in vivo was observed only when mice were infected with higher numbers of BCG. Thus, TLR2 and TLR4 appear to regulate distinct aspects of the host immune response against BCG.


Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Pulmón/microbiología , Macrófagos/inmunología , Glicoproteínas de Membrana/fisiología , Mycobacterium bovis/fisiología , Receptores de Superficie Celular/fisiología , Tuberculosis/inmunología , Animales , División Celular , Homocigoto , Inmunidad Celular , Técnicas para Inmunoenzimas , Interferón gamma/metabolismo , Activación de Macrófagos/inmunología , Glicoproteínas de Membrana/deficiencia , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Receptores de Superficie Celular/deficiencia , Bazo/metabolismo , Receptor Toll-Like 2 , Receptor Toll-Like 4 , Receptores Toll-Like , Transfección , Tuberculosis/microbiología
11.
Microsc Res Tech ; 57(2): 61-75, 2002 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-11921357

RESUMEN

Adrenomedullin (AM) is a multiregulatory peptide which is expressed in a wide range of tissues. In the pancreas, AM was first found in mammals, including man, and its colocalization with the pancreatic polypeptide (PP) was established in islet F cells. In addition, three different AM receptors have been characterized in B-cells. AM has been also located in the pancreatic cells of other vertebrate classes. The frequency and distribution of AM cells vary between different animals; they can be found scattered among the exocrine tissue, in the islets, or in ductal epithelia. The colocalization of AM with other hormones presents different patterns, although in birds, as in mammals, it seems to colocalize only with PP. The best-determined pancreatic AM function is the inhibition of insulin secretion in B-cells, which seems to be linked to a recently discovered binding protein, factor H. In relation to this physiological role, clinical data show that AM is raised in some groups of both types I and II diabetic patients and AM might have triggered the disease in a subset of them. On the other hand, AM pancreatic cells are also involved in the response to septic shock by increasing AM circulating levels. A third putative function is the inhibition of amylase secretion by the exocrine pancreatic cells. AM has been found in embryonic mammalian pancreas from the earliest stages of the development, colocalizing with all pancreatic hormones, although in adults only coexpression with PP is kept. AM may play a role in the growth and morphogenesis of the pancreas.


Asunto(s)
Páncreas/metabolismo , Hormonas Pancreáticas/metabolismo , Péptidos/metabolismo , Adrenomedulina , Animales , Humanos , Páncreas/citología , Páncreas/embriología , Ratas , Receptores de Adrenomedulina , Receptores de Péptidos/metabolismo
12.
Arch Physiol Biochem ; 117(3): 151-64, 2011 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-21599616

RESUMEN

Obesity is a highly prevalent health problem in Western countries that leads to many important diseases such as type 2 diabetes and metabolic syndrome being now considered an inflammatory chronic disease. Adipocytes are no longer considered passive cells storing fat since they are major producers of inflammatory cytokines during obesity. Adipocytes and macrophages share many biological properties including the synthesis of similar molecules regulating inflammation. Fatty acid levels are elevated in obesity and induce inflammatory pathways by yet a mostly unknown mechanism, leading to the development of insulin and leptin resistance. Recent studies suggest that these effects could be mediated through the activation of toll-like receptors (TLR). TLR signalling pathways might contribute to the development of obesity-associated insulin resistance, thus representing a connection between innate immunity and metabolism. Here, we summarize the recent evidence for the important role that TLRs play in adipose tissue, obesity and insulin resistance.


Asunto(s)
Inflamación/inmunología , Obesidad/inmunología , Receptores Toll-Like/inmunología , Adipocitos/inmunología , Tejido Adiposo/inmunología , Animales , Citocinas/inmunología , Diabetes Mellitus Tipo 2/inmunología , Humanos , Resistencia a la Insulina/inmunología , Leptina/metabolismo , Macrófagos/inmunología , Síndrome Metabólico/inmunología , Transducción de Señal/inmunología
13.
Sci Signal ; 4(161): mr3, 2011 Feb 22.
Artículo en Inglés | MEDLINE | ID: mdl-21343616

RESUMEN

Over the past years, a holistic approach has been applied to the study of the field of receptor signaling, permitting the analysis of how the interaction between receptors and their cellular environment determines receptor function and the study of the role of these receptors, under both normal and pathophysiological conditions, in whole organisms. This has been facilitated by the development of high-resolution microscopy techniques, which allow single-molecule or spatiotemporal resolution, or both, of signaling processes at the cellular and organismal levels. Concurrently, the role of these signaling pathways can be tested in increasingly sophisticated murine disease models. Finally, computational approaches aid in predicting and understanding receptor behavior. The program of the Madrid meeting reflected this integrated approach, highlighting signaling by and dynamics and regulation of immune cell receptors, the T cell receptor and B cell receptor, and signaling by and regulation of G protein-coupled receptors.


Asunto(s)
Enfermedades Metabólicas/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal , Animales , Humanos , Ratones
14.
J Immunol ; 178(6): 3602-11, 2007 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-17339457

RESUMEN

IFN regulatory factor (IRF)-2(-/-) mice are significantly more resistant to LPS challenge than wild-type littermates, and this was correlated with increased numbers of apoptotic Kupffer cells. To assess the generality of this observation, and to understand the role of IRF-2 in apoptosis, responses of peritoneal macrophages from IRF-2(+/+) and IRF-2(-/-) mice to apoptotic stimuli, including the fungal metabolite, gliotoxin, were compared. IRF-2(-/-) macrophages exhibited a consistently higher incidence of apoptosis that failed to correlate with caspase-3/7 activity. Using microarray gene expression profiling of liver RNA samples derived from IRF-2(+/+) and IRF-2(-/-) mice treated with saline or LPS, we identified >40 genes that were significantly down-regulated in IRF-2(-/-) mice, including Stat3, which has been reported to regulate apoptosis. Compared with IRF-2(+/+) macrophages, STAT3alpha mRNA was up-regulated constitutively or after gliotoxin treatment of IRF-2(-/-) macrophages, whereas STAT3beta mRNA was down-regulated. Phospho-Y705-STAT3, phospho-S727-STAT1, and phospho-p38 protein levels were also significantly higher in IRF-2(-/-) than control macrophages. Activation of the STAT signaling pathway has been shown to elicit expression of CASP1 and apoptosis. IRF-2(-/-) macrophages exhibited increased basal and gliotoxin-induced caspase-1 mRNA expression and enhanced caspase-1 activity. Pharmacologic inhibition of STAT3 and caspase-1 abolished gliotoxin-induced apoptosis in IRF-2(-/-) macrophages. A novel IFN-stimulated response element, identified within the murine promoter of Casp1, was determined to be functional by EMSA and supershift analysis. Collectively, these data support the hypothesis that IRF-2 acts as a transcriptional repressor of Casp1, and that the absence of IRF-2 renders macrophages more sensitive to apoptotic stimuli in a caspase-1-dependent process.


Asunto(s)
Apoptosis/inmunología , Caspasa 3/inmunología , Caspasa 7/inmunología , Factor 2 Regulador del Interferón/inmunología , Macrófagos del Hígado/inmunología , Macrófagos Peritoneales/inmunología , Proteínas Represoras/inmunología , Factor de Transcripción STAT1/inmunología , Factor de Transcripción STAT3/inmunología , Animales , Apoptosis/efectos de los fármacos , Apoptosis/genética , Caspasa 3/biosíntesis , Caspasa 7/biosíntesis , Células Cultivadas , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Regulación hacia Abajo/inmunología , Gliotoxina/farmacología , Inmunosupresores/farmacología , Factor 2 Regulador del Interferón/biosíntesis , Factor 2 Regulador del Interferón/deficiencia , Macrófagos del Hígado/metabolismo , Lipopolisacáridos/farmacología , Macrófagos Peritoneales/metabolismo , Ratones , Ratones Noqueados , Regiones Promotoras Genéticas/inmunología , Proteínas Represoras/biosíntesis , Factor de Transcripción STAT1/biosíntesis , Factor de Transcripción STAT3/biosíntesis
15.
J Immunol ; 176(11): 6888-99, 2006 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-16709849

RESUMEN

Francisella tularensis (Ft), a Gram-negative intracellular bacterium, is the etiologic agent of tularemia. Although attenuated for humans, i.p. infection of mice with <10 Ft live vaccine strain (LVS) organisms causes lethal infection that resembles human tularemia, whereas the LD50 for an intradermal infection is >10(6) organisms. To examine the immunological consequences of Ft LVS infection on the innate immune response, the inflammatory responses of mice infected i.p. or intradermally were compared. Mice infected i.p. displayed greater bacterial burden and increased expression of proinflammatory genes, particularly in the liver. In contrast to most LPS, highly purified Ft LVS LPS (10 microg/ml) was found to be only minimally stimulatory in primary murine macrophages and in HEK293T cells transiently transfected with TLR4/MD-2/CD14, whereas live Ft LVS bacteria were highly stimulatory for macrophages and TLR2-expressing HEK293T cells. Despite the poor stimulatory activity of Ft LVS LPS in vitro, administration of 100 ng of Ft LVS LPS 2 days before Ft LVS challenge severely limited both bacterial burden and cytokine mRNA and protein expression in the absence of detectable Ab at the time of bacterial challenge, yet these mice developed a robust IgM Ab response within 2 days of infection and survived. These data suggest that prior administration of Ft LVS LPS protects the host by diminishing bacterial burden and blunting an otherwise overwhelming inflammatory response, while priming the adaptive immune response for development of a strong Ab response.


Asunto(s)
Vacunas Bacterianas/inmunología , Francisella tularensis/inmunología , Tularemia/inmunología , Animales , Vacunas Bacterianas/administración & dosificación , Línea Celular , Regulación de la Expresión Génica/inmunología , Humanos , Inmunidad Innata/genética , Inflamación/genética , Inflamación/inmunología , Inflamación/prevención & control , Lipopolisacáridos/inmunología , Lipopolisacáridos/metabolismo , Hígado/inmunología , Hígado/metabolismo , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/microbiología , Ratones , Ratones Endogámicos C57BL , Transducción de Señal/inmunología , Receptor Toll-Like 2/genética , Receptor Toll-Like 2/fisiología , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/fisiología , Tularemia/microbiología , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/inmunología
16.
Gen Comp Endocrinol ; 143(1): 10-20, 2005 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-15993100

RESUMEN

Adrenomedullin (AM) is a multifunctional evolutionarily highly conserved peptide. Although its genomic and amino acid (aa) sequences are known in several mammalian species and in fish, the structure of the AM gene remains unknown in intermediate phyla, including birds. Here, we report the structure and aa sequence of the chicken (c) AM ortholog. The cAM gene is located at the short arm of chromosome 5, which shows high synteny with the short arm of human (h) chromosome 11, where hAM is located. Key sequences in the third intron have been conserved which allow for an alternative splicing mechanism, similar to the one found in mammals. The preprohormone contains two peptides with high homology to human proadrenomedullin N-terminal 20 peptide (PAMP) and hAM. We found through real-time PCR and immunocytochemistry cAM mRNA and peptide expression in a variety of chicken tissues, which parallel patterns observed for mammals, with the exception that cAM levels are almost non-detectable in brain. Similarly to mammals, cAM expression is upregulated under hypoxic conditions and following dexamethasone treatment. These data demonstrate a high degree of homology between the cAM gene and its mammalian ortholog and evolutionary conservation of the regulatory mechanisms controlling its expression.


Asunto(s)
Dexametasona/farmacología , Hipoxia/complicaciones , Péptidos/genética , Péptidos/metabolismo , Adrenomedulina , Empalme Alternativo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Pollos , Humanos , Inmunohistoquímica , Péptidos/efectos de los fármacos , Reacción en Cadena de la Polimerasa , Regulación hacia Arriba/efectos de los fármacos
17.
J Biol Chem ; 280(48): 39786-94, 2005 Dec 02.
Artículo en Inglés | MEDLINE | ID: mdl-16204251

RESUMEN

The ubiquitously expressed canonical transient receptor potential (TRPC) ion channels are considered important in Ca2+ signal generation, but their mechanisms of activation and roles remain elusive. Whereas most studies have examined overexpressed TRPC channels, we used molecular, biochemical, and electrophysiological approaches to assess the expression and function of endogenous TRPC channels in A7r5 smooth muscle cells. Real time PCR and Western analyses reveal TRPC6 as the only member of the diacylglycerol-responsive TRPC3/6/7 subfamily of channels expressed at significant levels in A7r5 cells. TRPC1, TRPC4, and TRPC5 were also abundant. An outwardly rectifying, nonselective cation current was activated by phospholipase C-coupled vasopressin receptor activation or by the diacylglycerol analogue, oleoyl-2-acetyl-sn-glycerol (OAG). Introduction of TRPC6 small interfering RNA sequences into A7r5 cells by electroporation led to 90% reduction of TRPC6 transcript and 80% reduction of TRPC6 protein without any detectable compensatory changes in the expression of other TRPC channels. The OAG-activated nonselective cation current was similarly reduced by TRPC6 RNA interference. Intracellular Ca2+ measurements using fura-2 revealed that thapsigargin-induced store-operated Ca2+ entry was unaffected by TRPC6 knockdown, whereas vasopressin-induced Ca2+ entry was suppressed by more than 50%. In contrast, OAG-induced Ca2+ transients were unaffected by TRPC6 knockdown. Nevertheless, OAG-induced Ca2+ entry bore the hallmarks of TRPC6 function; it was inhibited by protein kinase C and blocked by the Src-kinase inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2). Importantly, OAG-induced Ca2+ entry was blocked by the potent L-type Ca2+ channel inhibitor, *nimodipine. Thus, TRPC6 activation probably results primarily in Na ion entry and depolarization, leading to activation of L-type channels as the mediators of Ca2+ entry. Calculations reveal that even 90% reduction of TRPC6 channels would allow depolarization sufficient to activate L-type channels. This tight coupling between TRPC6 and L-type channels is probably important in mediating smooth muscle cell membrane potential and muscle contraction.


Asunto(s)
Calcio/metabolismo , Miocitos del Músculo Liso/citología , Canales Catiónicos TRPC/química , Animales , Western Blotting , Calcio/química , Canales de Calcio/química , Cationes , Cartilla de ADN/química , Diglicéridos/farmacología , Electrofisiología , Electroporación , Fura-2/farmacología , Iones , Potenciales de la Membrana , Modelos Biológicos , Nimodipina/farmacología , Oligonucleótidos/química , Técnicas de Placa-Clamp , Pirimidinas/farmacología , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Ratas , Receptores de Vasopresinas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Sodio/química , Canales Catiónicos TRPC/metabolismo , Canal Catiónico TRPC6 , Tapsigargina/farmacología , Factores de Tiempo , Fosfolipasas de Tipo C/química , Vasopresinas/farmacología
18.
J Immunol ; 170(11): 5739-47, 2003 Jun 01.
Artículo en Inglés | MEDLINE | ID: mdl-12759457

RESUMEN

IFN regulatory factors (IRFs) are a family of transcription factors and include several members that regulate expression of pro- and anti-inflammatory genes. Mice with a targeted mutation in IRF-2 (IRF-2(-/-)) were studied after injection of LPS to evaluate the importance of IRF-2 in the regulation of endotoxicity. IRF-2(-/-) mice were highly refractory to LPS-induced lethality. Although hepatic TNF-alpha mRNA and circulating TNF-alpha were significantly elevated in LPS-challenged IRF-2(-/-) mice, levels of IL-1, IL-12, and IFN-gamma mRNA and protein, as well as IL-6 protein, were significantly lower than levels seen in LPS-challenged IRF-2(+/+) mice. IRF-2(-/-) mice were also more refractory to TNF-alpha challenge than were control mice, which was consistent with their diminished sensitivity to LPS, yet no significant difference in the mRNA expression of TNFRs was observed. IL-12R beta 2 mRNA levels from LPS-challenged IRF-2(-/-) mice were significantly different after 1, 6, and 8 h, suggesting that both diminished IL-12 and altered IL-12R expression contribute to the paucity of IFN-gamma produced. IRF-2 knockout mice also failed to sustain LPS-inducible levels of IRF-1 and IFN consensus sequence binding protein mRNA expression, two transacting factors required for IL-12 transcription, perhaps as a result of diminished IL-1 beta, IL-6, and IFN-gamma levels. Liver sections from IRF-2(+/+) and IRF-2(-/-) mice were analyzed 6 h after a typically lethal injection of LPS. IRF-2(-/-) mice exhibited greater numbers of apoptotic Kupffer cells than did wild-type mice, suggesting a novel anti-apoptotic role for IRF-2. Collectively, these findings reveal a critical role for IRF-2 in endotoxicity, and point to a previously unappreciated role for IRF-2 in the regulation of apoptosis.


Asunto(s)
Proteínas de Unión al ADN/fisiología , Lipopolisacáridos/toxicidad , Factores de Transcripción , Animales , Antígenos CD/biosíntesis , Antígenos CD/genética , Apoptosis/inmunología , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Endotoxemia/genética , Endotoxemia/inmunología , Endotoxemia/mortalidad , Regulación de la Expresión Génica/inmunología , Inmunidad Innata/genética , Inyecciones Intraperitoneales , Factor 2 Regulador del Interferón , Factores Reguladores del Interferón , Interferón gamma/antagonistas & inhibidores , Interferón gamma/biosíntesis , Interleucina-1/antagonistas & inhibidores , Interleucina-1/biosíntesis , Interleucina-10/biosíntesis , Interleucina-10/genética , Interleucina-12/antagonistas & inhibidores , Interleucina-12/biosíntesis , Interleucina-12/metabolismo , Interleucina-6/antagonistas & inhibidores , Interleucina-6/biosíntesis , Macrófagos del Hígado/citología , Macrófagos del Hígado/inmunología , Lipopolisacáridos/administración & dosificación , Hígado/inmunología , Hígado/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , ARN Mensajero/biosíntesis , Receptores de Interleucina/biosíntesis , Receptores de Interleucina/genética , Receptores de Interleucina-12 , Receptores del Factor de Necrosis Tumoral/biosíntesis , Receptores del Factor de Necrosis Tumoral/genética , Receptores Tipo I de Factores de Necrosis Tumoral , Receptores Tipo II del Factor de Necrosis Tumoral , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/toxicidad , Proteínas Represoras/biosíntesis , Proteínas Represoras/genética , Factor de Necrosis Tumoral alfa/administración & dosificación , Factor de Necrosis Tumoral alfa/toxicidad
19.
J Immunol ; 171(2): 717-25, 2003 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-12847238

RESUMEN

Stimulation of the APC by Porphyromonas gingivalis LPS has been shown to result in the production of certain pro- and anti-inflammatory cytokines. However, the signaling pathways that regulate these processes are currently unknown. In the present study, the role of the phosphatidylinositol 3 kinase (PI3K)-Akt pathway in regulating P. gingivalis LPS-induced production of IL-10, IL-12 p40, and IL-12 p70 by human monocytes was investigated. P. gingivalis LPS selectively activates the PI3K-Akt pathway via Toll-like receptor 2, and inhibition of this pathway results in an abrogation of extracellular signal-regulated kinase 1/2 phosphorylation, whereas the activation of p38 and c-Jun N-terminal kinase 1/2 kinases were unaffected. Analysis of cytokine production following stimulation of monocytes with P. gingivalis LPS revealed that inhibition of the PI3K pathway differentially regulated IL-10 and IL-12 synthesis. IL-10 production was suppressed, whereas IL-12 levels were enhanced. Inhibition of P. gingivalis LPS-mediated activation of the PI3K-Akt pathway resulted in a pronounced augmentation of NF-kappaB p65 that was independent of IkappaB-alpha degradation. Furthermore, the ability of the PI3K-Akt pathway to modulate IL-10 and IL-12 production appears to be mediated by the selective suppression of extracellular signal-regulated kinase 1/2 activity, as the MEK1 inhibitor PD98059 closely mimicked the effects of wortmannin and LY294002 to differentially regulate IL-10 and IL-12 production by P. gingivalis LPS-stimulated monocytes. These studies provide new insight into how engagement of the PI3K-Akt pathway by P. gingivalis LPS affects the induction of key immunoregulatory cytokines that control both qualitative and quantitative aspects of innate and adaptive immunity.


Asunto(s)
Interleucina-10/biosíntesis , Interleucina-12/biosíntesis , Lipopolisacáridos/farmacología , Sistema de Señalización de MAP Quinasas/inmunología , Fosfatidilinositol 3-Quinasas/fisiología , Porphyromonas gingivalis/inmunología , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Proto-Oncogénicas/fisiología , Células Cultivadas , Activación Enzimática/efectos de los fármacos , Activación Enzimática/inmunología , Inhibidores Enzimáticos/farmacología , Humanos , Interleucina-10/antagonistas & inhibidores , Interleucina-10/fisiología , Interleucina-12/antagonistas & inhibidores , Subunidad p40 de la Interleucina-12 , Glicoproteínas de Membrana/fisiología , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Proteína Quinasa 8 Activada por Mitógenos , Proteína Quinasa 9 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Monocitos/enzimología , Monocitos/inmunología , Monocitos/metabolismo , FN-kappa B/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación , Subunidades de Proteína/antagonistas & inhibidores , Subunidades de Proteína/biosíntesis , Proteínas Proto-Oncogénicas c-akt , Receptores de Superficie Celular/fisiología , Receptor Toll-Like 2 , Receptores Toll-Like , Activación Transcripcional/inmunología , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/inmunología , Proteínas Quinasas p38 Activadas por Mitógenos
20.
J Immunol ; 170(1): 508-19, 2003 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-12496438

RESUMEN

In this study, tolerance induction by preexposure of murine macrophages to Toll-like receptor (TLR)2 and TLR4 agonists was revisited, focusing on the major signaling components associated with NF-kappaB activation. Pretreatment of macrophages with a pure TLR4 agonist (protein-free Escherichia coli (Ec) LPS) or with TLR2 agonists (Porphyromonas gingivalis LPS or synthetic lipoprotein Pam3Cys) led to suppression of TNF-alpha secretion, IL-1R-associated kinase-1, and IkappaB kinase (IKK) kinase activities, c-jun N-terminal kinase, and extracellular signal-regulated kinase phosphorylation, and to suppression of NF-kappaB DNA binding and transactivation upon challenge with the same agonist (TLR4 or TLR2 "homotolerance," respectively). Despite inhibited NF-kappaB DNA binding, increased levels of nuclear NF-kappaB were detected in agonist-pretreated macrophages. For all the intermediate signaling elements, heterotolerance was weaker than TLR4 or TLR2 homotolerance with the exception of IKK kinase activity. IKK kinase activity was unperturbed in heterotolerance. TNF-alpha secretion was also suppressed in P. gingivalis LPS-pretreated, Ec LPS-challenged cells, but not vice versa, while Pam3Cys and Ec LPS did not induce a state of cross-tolerance at the level of TNF-alpha. Experiments designed to elucidate novel mechanisms of NF-kappaB inhibition in tolerized cells revealed the potential contribution of IkappaBepsilon and IkappaBxi inhibitory proteins and the necessity of TLR4 engagement for induction of tolerance to Toll receptor-IL-1R domain-containing adapter protein/MyD88-adapter-like-dependent gene expression. Collectively, these data demonstrate that induction of homotolerance affects a broader spectrum of signaling components than in heterotolerance, with selective modulation of specific elements within the NF-kappaB signaling pathway.


Asunto(s)
Proteínas de Drosophila , Tolerancia Inmunológica , Macrófagos Peritoneales/inmunología , Macrófagos Peritoneales/metabolismo , Glicoproteínas de Membrana/agonistas , Glicoproteínas de Membrana/fisiología , FN-kappa B/metabolismo , Receptores de Superficie Celular/agonistas , Receptores de Superficie Celular/fisiología , Transducción de Señal/inmunología , Animales , Línea Celular , Células Cultivadas , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/metabolismo , Regulación hacia Abajo/genética , Regulación hacia Abajo/inmunología , Activación Enzimática/inmunología , Femenino , Factor C1 de la Célula Huésped , Humanos , Quinasa I-kappa B , Tolerancia Inmunológica/genética , Interferón beta/antagonistas & inhibidores , Interferón beta/biosíntesis , Interferón beta/genética , Quinasas Asociadas a Receptores de Interleucina-1 , Proteínas Quinasas JNK Activadas por Mitógenos , Lipopolisacáridos/farmacología , Macrófagos Peritoneales/enzimología , Ratones , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Proteínas Quinasas Activadas por Mitógenos/metabolismo , FN-kappa B/antagonistas & inhibidores , FN-kappa B/genética , FN-kappa B/fisiología , Subunidad p50 de NF-kappa B , Factor 1 de Transcripción de Unión a Octámeros , Inhibidores de Proteínas Quinasas , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/biosíntesis , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Mensajero/antagonistas & inhibidores , ARN Mensajero/biosíntesis , Eliminación de Secuencia , Transducción de Señal/genética , Receptor Toll-Like 2 , Receptor Toll-Like 4 , Receptores Toll-Like , Transactivadores/antagonistas & inhibidores , Transactivadores/metabolismo , Factor de Transcripción AP-1/metabolismo , Factor de Transcripción ReIA , Factores de Transcripción/metabolismo , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/metabolismo
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