Distinct Responses to IL4 in Macrophages Mediated by JNK

IL(Interleukin)-4 is the main macrophage M2-type activator and induces an anti-inflammatory phenotype called alternative activation. The IL-4 signaling pathway involves the activation of STAT (Signal Transducer and Activator of Transcription)-6 and members of the MAPK (Mitogen-activated protein kinase) family. In primary-bone-marrow-derived macrophages, we observed a strong activation of JNK (Jun N-terminal kinase)-1 at early time points of IL-4 stimulation. Using selective inhibitors and a knockout model, we explored the contribution of JNK-1 activation to macrophages' response to IL-4. Our findings indicate that JNK-1 regulates the IL-4-mediated expression of genes typically involved in alternative activation, such as Arginase 1 or Mannose receptor, but not others, such as SOCS (suppressor of cytokine signaling) 1 or p21Waf−1 (cyclin dependent kinase inhibitor 1A). Interestingly, we have observed that after macrophages are stimulated with IL-4, JNK-1 has the capacity to phosphorylate STAT-6 on serine but not on tyrosine. Chromatin immunoprecipitation assays revealed that functional JNK-1 is required for the recruitment of co-activators such as CBP (CREB-binding protein)/p300 on the promoter of Arginase 1 but not on p21Waf−1. Taken together, these data demonstrate the critical role of STAT-6 serine phosphorylation by JNK-1 in distinct macrophage responses to IL-4

Involvement of the Cellular Phosphatase DUSP1 in Vaccinia Virus Infection

Poxviruses encode a large variety of proteins that mimic, block or enhance host cell signaling pathways on their own benefit. It has been reported that mitogen-activated protein kinases (MAPKs) are specifically upregulated during vaccinia virus (VACV) infection. Here, we have evaluated the role of the MAPK negative regulator dual specificity phosphatase 1 (DUSP1) in the infection of VACV. We demonstrated that DUSP1 expression is enhanced upon infection with the replicative WR virus and with the attenuated VACV viruses MVA and NYVAC. This upregulation is dependent on early viral gene expression. In the absence of DUSP1 in cultured cells, there is an increased activation of its molecular targets JNK and ERK and an enhanced WR replication. Moreover, DUSP1 knock-out (KO) mice are more susceptible to WR infection as a result of enhanced virus replication in the lungs. Significantly, MVA, which is known to produce non-permissive infections in most mammalian cell lines, is able to grow in DUSP1 KO immortalized murine embryo fibroblasts (MEFs). By confocal and electron microscopy assays, we showed that in the absence of DUSP1 MVA morphogenesis is similar as in permissive cell lines and demonstrated that DUSP1 is involved at the stage of transition between IVN and MV in VACV morphogenesis. In addition, we have observed that the secretion of pro-inflammatory cytokines at early times post-infection in KO mice infected with MVA and NYVAC is increased and that the adaptive immune response is enhanced in comparison with WT-infected mice. Altogether, these findings reveal that DUSP1 is involved in the replication and host range of VACV and in the regulation of host immune responses through the modulation of MAPKs. Thus, in this study we demonstrate that DUSP1 is actively involved in the antiviral host defense mechanism against a poxvirus infection

Glucocorticoid Resistance: Interference between the Glucocorticoid Receptor and the MAPK Signalling Pathways

Endogenous glucocorticoids (GCs) are steroid hormones that signal in virtually all cell types to modulate tissue homeostasis throughout life. Also, synthetic GC derivatives (pharmacological GCs) constitute the first-line treatment in many chronic inflammatory conditions with unquestionable therapeutic benefits despite the associated adverse effects. GC actions are principally mediated through the GC receptor (GR), a ligand-dependent transcription factor. Despite the ubiquitous expression of GR, imbalances in GC signalling affect tissues differently, and with variable degrees of severity through mechanisms that are not completely deciphered. Congenital or acquired GC hypersensitivity or resistance syndromes can impact responsiveness to endogenous or pharmacological GCs, causing disease or inadequate therapeutic outcomes, respectively. Acquired GC resistance is defined as loss of efficacy or desensitization over time, and arises as a consequence of chronic inflammation, affecting around 30% of GC-treated patients. It represents an important limitation in the management of chronic inflammatory diseases and cancer, and can be due to impairment of multiple mechanisms along the GC signalling pathway. Among them, activation of the mitogen-activated protein kinases (MAPKs) and/or alterations in expression of their regulators, the dual-specific phosphatases (DUSPs), have been identified as common mechanisms of GC resistance. While many of the anti-inflammatory actions of GCs rely on GR-mediated inhibition of MAPKs and/or induction of DUSPs, the GC anti-inflammatory capacity is decreased or lost in conditions of excessive MAPK activation, contributing to disease susceptibility in tissue- and disease- specific manners. Here, we discuss potential strategies to modulate GC responsiveness, with the dual goal of overcoming GC resistance and minimizing the onset and severity of unwanted adverse effects while maintaining therapeutic potential

The role of the c-Jun N-terminal kinase (JNK) pathway in insulin resistance

Podeu consultar el llibre complet a: http://hdl.handle.net/2445/103042Obesity is usually associated with a decreased response to insulin, a major metabolic defect known as insulin resistance and an early trait in the development of type 2 diabetes. The c-Jun N-terminal kinase (JNK) pathway has emerged as a central regulator of insulin sensitivity, locally and systemically, thereby, of body’s metabolic homeostasis. As the incidence of obesity and type 2 diabetes has alarmingly increased in the last few decades, there is a tremendous necessity to identify novel pharmacological targets to efficiently improve the therapeutic outcome. In this regard, the JNK pathway seems to meet most of the requirements for being an adequate candidate to direct pharmacological intervention

Nuclear receptors: Lipid and hormone sensors with essential roles in the control of cancer development

Nuclear receptors (NRs) are a superfamily of ligand-activated transcription factors that act as biological sensors and use a combination of mechanisms to modulate positively and negatively gene expression in a spatial and temporal manner. The highly orchestrated biological actions of several NRs influence the proliferation, differentiation, and apoptosis of many different cell types. Synthetic ligands for several NRs have been the focus of extensive drug discovery efforts for cancer intervention. This review summarizes the roles in tumour growth and metastasis of several relevant NR family members, namely androgen receptor (AR), estrogen receptor (ER), glucocorticoid receptor (GR), thyroid hormone receptor (TR), retinoic acid receptors (RARs), retinoid X receptors (RXRs), peroxisome proliferator-activated receptors (PPARs), and liver X receptors (LXRs). These studies are key to develop improved therapeutic agents based on novel modes of action with reduced side effects and overcoming resistance

Pharmacological activation of LXR alters the expression profile of tumor-associated macrophages and the abundance of regulatory T cells in the tumor microenvironment

Liver X receptors (LXR) are transcription factors from the nuclear receptor family that are activated by oxysterols and synthetic high-affinity agonists. In this study, we assessed the anti-tumor effects of synthetic LXR agonist TO901317 in a murine model of syngeneic Lewis Lung carcinoma. Treatment with TO901317 inhibited tumor growth in wild-type but not in LXR-deficient mice, indicating that the anti-tumor effects of the agonist depends on functional LXR activity in host cells. Pharmacological activation of the LXR pathway reduced the intratumoral abundance of regulatory T cells (Treg) and the expression of the Treg-attracting chemokine Ccl17 by MHCIIhigh tumor-associated macrophages (TAM). Moreover, gene expression profiling indicated a broad negative impact of the LXR agonist on other mechanisms used by TAM for the maintenance of an immunosuppressive environment. In studies exploring the macrophage response to GM-CSF or IL-4, activated LXR repressed IRF4 expression, resulting in subsequent downregulation of IRF4-dependent genes including Ccl17. Taken together, this work reveals the combined actions of the LXR pathway in the control of TAM responses that contribute to the anti-tumoral effects of pharmacological LXR activation. Moreover, these data provide new insights for the development of novel therapeutic options for the treatment of cancer

The Multivalency of the glucocorticoid receptor ligand-binding domain explains its manifold physiological activities

The glucocorticoid receptor (GR) is a ubiquitously expressed transcription factor that controls metabolic and homeostatic processes essential for life. Although numerous crystal structures of the GR ligand-binding domain (GR-LBD) have been reported, the functional oligomeric state of the full-length receptor, which is essential for its transcriptional activity, remains disputed. Here we present five new crystal structures of agonist-bound GR-LBD, along with a thorough analysis of previous structural work. We identify four distinct homodimerization interfaces on the GR-LBD surface, which can associate into 20 topologically different homodimers. Biologically relevant homodimers were identified by studying a battery of GR point mutants including crosslinking assays in solution, quantitative fluorescence microscopy in living cells, and transcriptomic analyses. Our results highlight the relevance of non-canonical dimerization modes for GR, especially of contacts made by loop L1-3 residues such as Tyr545. Our work illustrates the unique flexibility of GR's LBD and suggests different dimeric conformations within cells. In addition, we unveil pathophysiologically relevant quaternary assemblies of the receptor with important implications for glucocorticoid action and drug design

The nuclear receptor LXR limits bacterial infection of host macrophages through a mechanism that impacts cellular NAD metabolism

Macrophages exert potent effector functions against invading microorganisms but constitute, paradoxically, a preferential niche for many bacterial strains to replicate. Using a model of infection by Salmonella Typhimurium, we have identified a molecular mechanism regulated by the nuclear receptor LXR that limits infection of host macrophages through transcriptional activation of the multifunctional enzyme CD38. LXR agonists reduced the intracellular levels of NAD+ in a CD38-dependent manner, counteracting pathogen-induced changes in macrophage morphology and the distribution of the F-actin cytoskeleton and reducing the capability of nonopsonized Salmonella to infect macrophages. Remarkably, pharmacological treatment with an LXR agonist ameliorated clinical signs associated with Salmonella infection in vivo, and these effects were dependent on CD38 expression in bonemarrow- derived cells. Altogether, this work reveals an unappreciated role for CD38 in bacterial-host cell interaction that can be pharmacologically exploited by activation of the LXR pathway

Caracterización del gen correspondiente a la HmG-CoA reductasa de Arabidopsis thaliana

[spa] Las plantas superiores sintetizan una gran variedad de compuestos de naturaleza isoprenoide, entre los que se encuentran moléculas vitales para el crecimiento y desarrollo celular y compuestos de importancia económica. El enzima HmG-CoA reductasa esta implicado en la ruta de biosíntesis de estos compuestos y es uno de los candidatos mas probables a participar en su regulación. Para el estudio de la HmG-CoA reductasa en un sistema vegetal se planteo el clonaje del gen correspondiente en Arabidopsis thaliana, empleando una sonda heteróloga procedente de un CDNA que codificaba para dicho enzima en hámster. En el trabajo presentado, han sido detectados dos genes de HmG-CoA reductasa en A. thaliana. La caracterización estructural de uno de ellos, denominado HmG1, ha sido llevada a cabo tanto a nivel de la proteína como del gen y del MRNA. El segundo de ellos, denominado HmG2, ha sido aislado y caracterizado parcialmente a nivel genómico. El gen HmG1 se extiende a lo largo de aproximadamente 4 KB del genoma y presenta 3 intrones y 4 exones. EL MRNA correspondiente presenta una secuencia 5' no traducida de 70 nucleótidos a partir de donde empieza un marco de lectura abierto de 1776 nucleótidos. La región 3' no traducida presenta dos sitios de poliadenilación alternativos que distan 96 nucleótidos. La HmG-CoA reductasa, codificada por el gen HmG1, es un polipéptido de 592 aminoácidos y tiene un peso molecular de 63.605 DA. La principal diferencia entre la HmG-CoA reductasa de A. thaliana y las caracterizadas en otros organismos eucariotas (hámster, Drosophila, levadura y erizo de mar) reside en la estructura de su dominio amino-terminal. El dominio carboxi-terminal de la HmG-CoA reductasa de distintos organismos eucariotas, incluida la de A. thaliana, presenta un 38% de aminoácidos idénticos entre todas ellas. Este hecho indica que existe una presión evolutiva para mantener determinados aminoácidos en posiciones específicas

Purificación a homogeneidad y propiedades del enzima 3-hidroxi 3-metil glutaril coenzima A reductasa quinasa de citosol de hígado de rata

Tesi de Llicenciatura per a la obtenció del Grau de Farmàcia. Facultat de Farmàcia. Universitat de Barcelona. Director: Fausto García Hegardt. 1985.En los trabajos publicados en la bibliografía internacional durante los últimos años, se ha comunicado la purificación hasta homogeneidad aparente de una actividad Reductasa quinasa procedente de la fracción microsomal de hígado de rata. Se han comunicado también purificaciones parciales de actividad Reductasa quinasa procedente de la fracción citosóli-' ca. En nuestro propio laboratorio se ha purificado parcialmente Reductasa quinasa citosólica que ya manifestó capacidad para fosforilar e inactivar a la HMG-CoA Reductasa homogénea, aunque se precisaba de* una etapa final de inmunoprecipitación para aislar a la Reductasa de otras proteínas fosforiladas. Con objeto de poder estudiar el proceso de fosfo rilación en condiciones óptimas se consideró necesario purificar la Reductasa quinasa a homogeneidad. Esto permite omitir la etapa de inmunoprecipitación y obtener evidencias más directas del mecanismo de fosforilación de la Reductasa. Disponer de una preparación homogénea permitirá en un futuro emprender estudios estructurales de este enzima y obtener anticuerpos contra el mismo que harán posible estudios "in vivo" de la actividad reguladora de la Reductasa quinasa