Caractérisation de l’activité transcriptionnelle antivirale et immunorégulatrice dépendante de STAT2 et IRF9, mais indépendante de STAT1, induite par la costimulation par TNF

Les cellules épithéliales pulmonaires constituent la première ligne de défense face aux virus respiratoires via la sécrétion de mucus, de peptides, de cytokines et chimiokines qui déterminent l'élimination ou la progression de l’infection. Les principales cytokines antivirales produites par les cellules épithéliales alvéolaires (AEC) sont les interférons (IFN) type I (α/β) et III (λ). La liaison d’IFNβ à son récepteur induit une voie antivirale bien caractérisée qui aboutit à l’activation du complexe ISGF3 (STAT1, STAT2 et IRF9) qui permet la transcription de multiples gènes codant pour des protéines à activité antivirale et immunorégulatrice. Il a récemment été démontré que la costimulation des cellules épithéliales pulmonaires par l’IFNβ et le Tumor Necrosis Factor α (TNFα), également produit lors d’une infection, synergisent pour induire un état antiviral tardif distinct. D’autre part, il a été montré que la synergie entre le TNFα et l'IFNβ induit une voie de signalisation impliquant STAT2 et IRF9, mais indépendante de STAT1 permettant l’expression du gène DUOX2. Notre but est de déterminer l’importance de cette nouvelle voie de signalisation induite par la costimulation du TNFα+IFNβ, impliquant STAT2 et IRF9 indépendamment de STAT1 dans la régulation d’un programme transcriptionnel antiviral et immunorégulateur tardif. Notre premier objectif est de déterminer si des gènes antiviraux et immunorégulateurs qui sont induits par la costimulation par TNFα+IFNβ sont dépendants de la voie STAT2/IRF9, indépendamment de STAT1. En utilisant la technique de qRT-PCR, nous avons identifié 3 gènes immunorégulateurs, CXCL10, IDO et APOBEC3G, induits de manière synergique en réponse à TNFα+IFNβ dans les cellules A549, un modèle de cellules épithéliales pulmonaires. Afin de confirmer que ces gènes sont induits indépendamment de STAT1, nous avons validé leur expression dans la lignée cellulaire U3A déficiente en STAT1. Par l'utilisation d'ARN interférants (ARNi) dirigés contre STAT2 et IRF9, nous avons confirmé que l’induction de ces gènes est dépendante de STAT2 et IRF9. Finalement, l’analyse de l’activité du promoteur de CXCL10 en réponse à TNFα+IFNβ par des essais rapporteurs luciférases a permis de montrer que la régulation se fait au niveau transcriptionnel. Notre deuxième objectif, est de déterminer si STAT6 pourrait remplacer STAT1 dans la voie de signalisation induite par TNFα+IFNβ. En effet, STAT6 est un inducteur connu de l’expression de DUOX2 en réponse à IL4+IL13. Contrairement à notre hypothèse, l’inhibition de STAT6 par ARNi augmente l’expression de DUOX2 en réponse à TNFα+IFNβ suggérant que STAT6 est un régulateur négatif. Nos résultats ont permis de comprendre de manière plus détaillée les mécanismes mis en place dans le développement d’une réponse antivirale. D’autre part, l’étude de l’effet de l’IFNβ et du TNFα est également pertinente pour les maladies chroniques inflammatoires et autoimmunes. De plus, nos résultats illustrent un nouveau paradigme concernant les mécanismes de signalisation cellulaire impliqués dans la synergie entre deux cytokines qui pourrait être applicable à des combinaisons de cytokines autres que TNFα+IFNβ.

Erythropoietin does not enhance skeletal muscle protein synthesis following exercise in young and older adults

PURPOSE: Erythropoietin (EPO) is a renal cytokine that is primarily involved in hematopoiesis while also playing a role in non-hematopoietic tissues expressing the EPO-receptor (EPOR). The EPOR is present in human skeletal muscle. In mouse skeletal muscle, EPO stimulation can activate the AKT serine/threonine kinase 1 (AKT) signaling pathway, the main positive regulator of muscle protein synthesis. We hypothesized that a single intravenous EPO injection combined with acute resistance exercise would have a synergistic effect on skeletal muscle protein synthesis via activation of the AKT pathway.

METHODS: Ten young (24.2 ± 0.9 years) and 10 older (66.6 ± 1.1 years) healthy subjects received a primed, constant infusion of [ring-13C(6)] L-phenylalanine and a single injection of 10,000 IU epoetin-beta or placebo in a double-blind randomized, cross-over design. 2 h after the injection, the subjects completed an acute bout of leg extension resistance exercise to stimulate skeletal muscle protein synthesis.

RESULTS: Significant interaction effects in the phosphorylation levels of the members of the AKT signaling pathway indicated a differential activation of protein synthesis signaling in older subjects when compared to young subjects. However, EPO offered no synergistic effect on vastus lateralis mixed muscle protein synthesis rate in young or older subjects.

CONCLUSIONS: Despite its ability to activate the AKT pathway in skeletal muscle, an acute EPO injection had no additive or synergistic effect on the exercise-induced activation of muscle protein synthesis or muscle protein synthesis signaling pathways.

Exploring the effects of omega-3 and omega-6 fatty Acids on allergy using a HEK-blue cell line.

BACKGROUND: Allergic reactions can result in life-threatening situations resulting in high economic costs and morbidity. Therefore, more effective reagents are needed for allergy treatment. A causal relationship has been suggested to exist between the intake of omega-3/6 fatty acids, such as docosahexanoic acid (DHA), eicosapentanoic acid (EPA), docosapentanoic acid (DPA) and arachidonic acid (AA), and atopic individuals suffering from allergies. In allergic cascades, the hallmark cytokine IL-4 bind to IL-4 receptor (IL-4R) and IL-13 binds to IL-13 receptor (IL-13R), this activates the STAT6 phosphorylation pathway leading to gene activation of allergen-specific IgE antibody production by B cells. The overall aim of this study was to characterize omega-3/6 fatty acids and their effects on STAT6 signaling pathway that results in IgE production in allergic individuals. METHODS: The fatty acids were tested in vitro with a HEK-Blue IL-4/IL-13 reporter cell line model, transfected with a reporter gene that produces an enzyme, secreted embryonic alkaline phosphatase (SEAP). SEAP acts as a substitute to IgE when cells are stimulated with bioactive cytokines IL-4 and/or IL-13. RESULTS: We have successfully used DHA, EPA and DPA in our studies that demonstrated a decrease in SEAP secretion, as opposed to an increase in SEAP secretion with AA treatment. A statistical Student's t-test revealed the significance of the results, confirming our initial hypothesis. CONCLUSION: We have successfully identified and characterised DHA, EPA, DPA and AA in our allergy model. While AA was a potent stimulator, DHA, EPA and DPA were potential inhibitors of IL-4R/IL-13R signalling, which regulates the STAT6 induced pathway in allergic cascades. Such findings are significant in the future design of dietary therapeutics for the treatment of allergies.

Genome-wide detection of CNVs and their association with meat tenderness in Nelore cattle.

Brazil is one of the largest beef producers and exporters in the world with the Nelore breed representing the vast majority of Brazilian cattle (Bos taurus indicus). Despite the great adaptability of the Nelore breed to tropical climate, meat tenderness (MT) remains to be improved. Several factors including genetic composition can influence MT. In this article, we report a genome-wide analysis of copy number variation (CNV) inferred from Illumina1 High Density SNP-chip data for a Nelore population of 723 males. We detected >2,600 CNV regions (CNVRs) representing 6.5% of the genome. Comparing our results with previous studies revealed an overlap in 1400 CNVRs (>50%). A total of 1,155 CNVRs (43.6%) overlapped 2,750 genes. They were enriched for processes involving guanosine triphosphate (GTP), previously reported to influence skeletal muscle physiology and morphology. Nelore CNVRs also overlapped QTLs for MT reported in other breeds (8.9%, 236 CNVRs) and from a previous study with this population (4.1%, 109 CNVRs). Two CNVRs were also proximal to glutathione metabolism genes that were previously associated with MT. Genome-wide association study of CN state with estimated breeding values derived from meat shear force identified 6 regions, including a region on BTA3 that contains genes of the cAMP and cGMP pathway. Ten CNVRs that overlapped regions associated with MT were successfully validated by qPCR. Our results represent the first comprehensive CNV study in Bos taurus indicus cattle and identify regions in which copy number changes are potentially of importance for the MT phenotype.

cAMP regulates IL-2 receptor signaling in human T cells

Proper immune system function is dependent on positive and negative regulation of T cell signaling pathways. Full T cell activation requires sequential signaling through the T cell receptor (TCR), costimulatory molecules and the IL-2 receptor (IL-2R). The IL-2R associated Janus tyrosine kinase 3 (Jak3), as well as Signal transducer and activator of transcription 5 (Stat5), are required for normal T cell function and survival. Constitutive activation of Jak3 and Stat5 have been linked to cancers of hematopoietic origin, including certain lymphomas and leukemias. ^ The production of cAMP by adenylate cyclase has been shown to negatively regulate human TCR mediated cell proliferation. Since cAMP has been shown to negatively regulate T cell activation, we sought to investigate whether crosstalk exists between cAMP and IL-2R signaling. The first objective of this study was to determine the effect of cAMP on the activation of IL-2R signaling molecules Jak3 and Stat5. We found that the potent adenylate cyclase activator, forskolin, inhibited IL-2 activation of Jak3 and Stat5. Indeed, in vitro kinase assays and electrophoretic mobility shift assays verified a loss of Jak3 enzymatic activity and Stat5 DNA binding ability, respectively. Further analysis of IL-2R signaling showed that forskolin treatment reduced IL-2 induced association of the IL-2Rβ and γc chain. ^ Because cAMP activates protein kinase A (PKA), the second objective was to determine the role for PKA in the cAMP directed regulation of IL-2R signaling intermediates. Interestingly, forskolin induced serine phosphorylation of Jak3, suggesting that cAMP can directly regulate Jak3 via activation of a serine/threonine kinase. Indeed, phosphoamino acid analysis revealed that PKA was able to induce Jak3 serine phosphorylation in the human leukemia cell line MT-2. In addition, in vitro kinase assays established that PKA can directly inhibit Jak3 enzymatic activity. Collectively, these data indicate that cAMP negatively regulates IL-2R signaling via various effector molecules by a previously unrecognized mechanism. This new data suggests that the Jak3/Stat5 pathway may be regulated by various pharmacological agents that stimulate cAMP production and thus can be used to uncouple some types of T cell mediated diseases. ^

Molecular mechanism of the relaxin signaling pathways: Activation of cAMP, PI3K, and PKCzeta

Relaxin is a polypeptide hormone that has diverse effects on reproductive and non-reproductive tissues. Relaxin activates the G-protein coupled receptors, LGR7 and LRG8. Early studies described increased cAMP and protein kinase A activity upon relaxin treatment, but cAMP accumulation alone could not account for all of the relaxin-mediated effects. We utilized the human monocyte cell line THP-1 to study the mechanism of relaxin-stimulated CAMP production. ^ Relaxin treatment in THP-1 cells produces a biphasic time course in cAMP accumulation, where the first peak appears as early as 1–2 minutes with a second peak at 10–20 minutes. Selective inhibitors for phosphoinositide 3-kinase (P13K), such as wortmannin and LY294002, show a dose-dependent inhibition of relaxin-stimulated cAMP accumulation, specific for the second peak of the relaxin time course. Neither the effects of relaxin nor the inhibition of relaxin by LY294002 is mediated by the activity of phosphodiesterases. Furthermore, LY294002 blocks upregulation of vascular endothelial growth factor transcript levels by relaxin. ^ To further delineate relaxin signaling pathways, we searched for downstream targets of PI3K that could activate adenylyl cyclase (AC). Protein kinase C ζ (PKCζ) was a prime candidate because it activates types II and V AC. Chelerythrine chloride (a general PKC inhibitor) inhibits relaxin-induced cAMP production to the same degree as LY294002 (∼40%). Relaxin stimulates PKCζ translocation to the plasma membrane in THP-1, MCF-7, PHM1-31, and MMC cells, as shown by immunocytochemistry. PKCζ translocation is P13K-dependent and independent of cAMP production. Antisense PKCζ oligodeoxynucleotides (PKCζ-ODNs) deplete both PKCζ transcript and protein levels in THP-1 cells. PKCζ-ODNs abolish relaxin-mediated PKCζ translocation and inhibit relaxin stimulation of cAMP by 40%, as compared to mock and random ODN controls. Treatment with LY294002 in the presence of PKCζ-ODNs results in little further inhibition. Taken together, we present a novel role for PI3K and PKCζ in relaxin stimulation of cAMP and provide the first example of the PKCζ regulation of AC in an endogenous system. Furthermore, we have identified higher order complexes of AC isoforms and PKA anchoring proteins in attempts to explain the differential coupling of relaxin to cAMP and PI3K-signaling pathways in various cell types. ^

The phosphoprotein DARPP-32 mediates cAMP-dependent potentiation of striatal N-methyl-d-aspartate responses

The signal transduction pathway underlying the cAMP-dependent modulation of rat striatal N-methyl-d-aspartate (NMDA) responses was investigated by using the two-electrode voltage-clamp technique. In oocytes injected with rat striatal poly(A)+ mRNA, activation of cAMP-dependent protein kinase (PKA) by forskolin potentiated NMDA responses. Inhibition of protein phosphatase 1 (PP1) and/or protein phosphatase 2A (PP2A) by the specific inhibitor calyculin A occluded the PKA-mediated potentiation of striatal NMDA responses, suggesting that the PKA effect was mediated by inhibition of a protein phosphatase. Coinjection of oocytes with striatal mRNA and antisense oligodeoxynucleotides directed against the protein phosphatase inhibitor DARPP-32 dramatically reduced the PKA enhancement of NMDA responses. NMDA responses recorded from oocytes injected with rat hippocampal poly(A)+ mRNA were not affected by stimulation of PKA. When oocytes were coinjected with rat hippocampal poly(A)+ mRNA plus complementary RNA coding for DARPP-32, NMDA responses were potentiated after stimulation of PKA. The results provide evidence that DARPP-32, which is enriched in the striatum, may participate in the signaling between the two major afferent striatal pathways, the glutamatergic and the dopaminergic projections, by the cAMP-dependent regulation of striatal NMDA currents.

The PDE1-encoded Low-Affinity Phosphodiesterase in the Yeast Saccharomyces cerevisiae Has a Specific Function in Controlling Agonist-induced cAMP Signaling

The yeast Saccharomyces cerevisiae contains two genes, PDE1 and PDE2, which respectively encode a low-affinity and a high-affinity cAMP phosphodiesterase. The physiological function of the low-affinity enzyme Pde1 is unclear. We show that deletion of PDE1, but not PDE2, results in a much higher cAMP accumulation upon addition of glucose or upon intracellular acidification. Overexpression of PDE1, but not PDE2, abolished the agonist-induced cAMP increases. These results indicate a specific role for Pde1 in controlling glucose and intracellular acidification-induced cAMP signaling. Elimination of a putative protein kinase A (PKA) phosphorylation site by mutagenesis of serine252 into alanine resulted in a Pde1ala252 allele that apparently had reduced activity in vivo. Its presence in a wild-type strain partially enhanced the agonist-induced cAMP increases compared with pde1Δ. The difference between the Pde1ala252 allele and wild-type Pde1 was strongly dependent on PKA activity. In a RAS2val19 pde2Δ background, the Pde1ala252 allele caused nearly the same hyperaccumulation of cAMP as pde1Δ, while its expression in a PKA-attenuated strain caused the same reduction in cAMP hyperaccumulation as wild-type Pde1. These results suggest that serine252 might be the first target site for feedback inhibition of cAMP accumulation by PKA. We show that Pde1 is rapidly phosphorylated in vivo upon addition of glucose to glycerol-grown cells, and this activation is absent in the Pde1ala252 mutant. Pde1 belongs to a separate class of phosphodiesterases and is the first member shown to be phosphorylated. However, in vitro the Pde1ala252 enzyme had the same catalytic activity as wild-type Pde1, both in crude extracts and after extensive purification. This indicates that the effects of the S252A mutation are not caused by simple inactivation of the enzyme. In vitro phosphorylation of Pde1 resulted in a modest and variable increase in activity, but only in crude extracts. This was absent in Pde1ala252, and phosphate incorporation was strongly reduced. Apparently, phosphorylation of Pde1 does not change its intrinsic activity or affinity for cAMP but appears to be important in vivo for protein-protein interaction or for targeting Pde1 to a specific subcellular location. The PKA recognition site is conserved in the corresponding region of the Schizosaccharomyces pombe and Candida albicans Pde1 homologues, possibly indicating a similar control by phosphorylation.

Impaired hippocampal plasticity in mice lacking the Cbeta1 catalytic subunit of cAMP-dependent protein kinase.

Neural pathways within the hippocampus undergo use-dependent changes in synaptic efficacy, and these changes are mediated by a number of signaling mechanisms, including cAMP-dependent protein kinase (PKA). The PKA holoenzyme is composed of regulatory and catalytic (C) subunits, both of which exist as multiple isoforms. There are two C subunit genes in mice, Calpha and Cbeta, and the Cbeta gene gives rise to several splice variants that are specifically expressed in discrete regions of the brain. We have used homologous recombination in embryonic stem cells to introduce an inactivating mutation into the mouse Cbeta gene, specifically targeting the Cbeta1-subunit isoform. Homozygous mutants showed normal viability and no obvious pathological defects, despite a complete lack of Cbeta1. The mice were analyzed in electrophysiological paradigms to test the role of this isoform in long-term modulation of synaptic transmission in the Schaffer collateral-CA1 pathway of the hippocampus. A high-frequency stimulus produced potentiation in both wild-type and Cbeta1-/- mice, but the mutants were unable to maintain the potentiated response, resulting in a late phase of long-term potentiation that was only 30% of controls. Paired pulse facilitation was unaffected in the mutant mice. Low-frequency stimulation produced long-term depression and depotentiation in wild-type mice but failed to produce lasting synaptic depression in the Cbeta1 -/- mutants. These data provide direct genetic evidence that PKA, and more specifically the Cbeta1 isoform, is required for long-term depression and depotentiation, as well as the late phase of long-term potentiation in the Schaffer collateral-CA1 pathway.

Activation of the cAMP pathway by variant human MC1R alleles expressed in HEK and in melanoma cells

alpha-Melanocyte-stimulating hormone (alpha-MSH) activates the melanocortin-1 receptor (MC1R) on melanocytes to promote a switch from red/yellow pheomelanin synthesis to darker eumelanins via positive coupling to adenylate cyclase. The human MC1R locus is highly polymorphic with the specific variants associated with red hair and fair skin (RHC phenotype) postulated to be loss-of-function receptors. We have examined the ability of MC1R variants to activate the cAMP pathway in stably transfected REK293 cells. The RHC associated variants, Arg151Cys, Arg160Trp and Asp294His, demonstrated agonist-mediated increases in cAMP and phosphorylation of cAMP-responsive element-binding protein (CREB). Whereas the Asp294His variant showed severely impaired functional responses, the Arg151Cys and Arg160Trp variants retained considerable signaling capacity. Melanoma cells homozygous for either the Arg151Cys variant or consensus sequence both elicited CREB phosphorylation in response to alpha-MSH in the presence of IBMX. The common RHC alleles, Arg151Cys, Arg160Trp and Asp294His, are neither complete loss-of-function receptors nor are they functionally equivalent. (c) 2005 Elsevier Inc. All rights reserved.

Rod and cone pathway signaling and interaction under mesopic illumination

This study investigates the time-course and post-receptoral pathway signaling of photoreceptor interactions when the rod (R) and three cone (L, M, S) photoreceptor classes contribute to mesopic vision. A four-primary photostimulator independently controls photoreceptor activity in human observers. The first experiment defines the temporal adaptation response of receptoral (L-, S-cone, rod) and post-receptoral (LMS, LMSR,+L-M) signaling and interactions. Here we show that nonopponent cone-cone interactions (L-cone, LMS, LMSR) have monophasic temporal response patterns whereas opponent signals (+L-M, S-cone) show biphasic response patterns with slower recovery. By comparison, rod-cone interactions with nonopponent signals have faster adaptation responses and reduced sensitivity loss whereas opponent rod-cone interactions are small or absent. Additionally, the rod-rod interaction differs from these interaction types and acts to increase rod sensitivity due to temporal summation but with a slower time course. The second experiment shows that the temporal profile of the rod signal alters the relative rod contributions to the three primary post-receptoral pathways. We demonstrate that rod signals generate luminance (þLþM) signals mediated via the MC pathway with all rod temporal profiles and chromatic signals (L/LþM, S/LþM) in both the PC and KC pathways with durations .75 ms. Thus, we propose that the change in relative weighting of rod signals within the post-receptoral pathways contributes to the sensitivity and temporal response of rod and cone pathway signaling and interactions.

Involvement of Src family of kinases and cAMP phosphodiesterase in the luteinizing hormone/chorionic gonadotropin receptor-mediated signaling in the corpus luteum of monkey

Background: In higher primates, during non-pregnant cycles, it is indisputable that circulating LH is essential for maintenance of corpus luteum (CL) function. On the other hand, during pregnancy, CL function gets rescued by the LH analogue, chorionic gonadotropin (CG). The molecular mechanisms involved in the control of luteal function during spontaneous luteolysis and rescue processes are not completely understood. Emerging evidence suggests that LH/CGR activation triggers proliferation and transformation of target cells by various signaling molecules as evident from studies demonstrating participation of Src family of tyrosine kinases (SFKs) and MAP kinases in hCG-mediated actions in Leydig cells. Since circulating LH concentration does not vary during luteal regression, it was hypothesized that decreased responsiveness of luteal cells to LH might occur due to changes in LH/CGR expression dynamics, modulation of SFKs or interference with steroid biosynthesis. Methods: Since, maintenance of structure and function of CL is dependent on the presence of functional LH/CGR its expression dynamics as well as mRNA and protein expressions of SFKs were determined throughout the luteal phase. Employing well characterized luteolysis and CL rescue animal models, activities of SFKs, cAMP phosphodiesterase (cAMP-PDE) and expression of SR-B1 (a membrane receptor associated with trafficking of cholesterol ester) were examined. Also, studies were carried out to investigate the mechanisms responsible for decline in progesterone biosynthesis in CL during the latter part of the non-pregnant cycle. Results and discussion: The decreased responsiveness of CL to LH during late luteal phase could not be accounted for by changes in LH/CGR mRNA levels, its transcript variants or protein. Results obtained employing model systems depicting different functional states of CL revealed increased activity of SFKs pSrc (Y-416)] and PDE as well as decreased expression of SR-B1correlating with initiation of spontaneous luteolysis. However, CG, by virtue of its heroic efforts, perhaps by inhibition of SFKs and PDE activation, prevents CL from undergoing regression during pregnancy. Conclusions: The results indicated participation of activated Src and increased activity of cAMP-PDE in the control of luteal function in vivo. That the exogenous hCG treatment caused decreased activation of Src and cAMP-PDE activity with increased circulating progesterone might explain the transient CL rescue that occurs during early pregnancy.

GLP-1 and related peptides cause concentration-dependent relaxation of rat aorta through a pathway involving KATP and cAMP

increasing evidence from both clinical and experimental studies indicates that the insulin-releasing hormone, glucagon-like peptide-1 (GLP-1) may exert additional protective/reparative effects on the cardiovascular system. The aim of this study was to examine vasorelaxant effects of GLP-1(7-36)amide, three structurally-related peptides and a non-peptide GLP-1 agonist in rat aorta. Interestingly, all GLP-1 compounds, including the established GLP-1 receptor antagonist, exendin (9-39) caused concentration-dependent relaxation. Mechanistic studies employing hyperpolarising concentrations of potassium or glybenclamide revealed that these relaxant effects are mediated via specific activation of ATP-sensitive potassium channels. Further experiments using a specific membrane-permeable cyclic AMP (cAMP) antagonist, and demonstration of increased cAMP production in response to GLP-1 illustrated the critical importance of this pathway. These data significantly extend previous observations suggesting that GLP-1 may modulate vascular function, and indicate that this effect may be mediated by the GLP-1 receptor. However, further studies are required in order to establish whether GLP-1 related agents may confer additional cardiovascular benefits to diabetic patients. (c) 2008 Elsevier Inc. All rights reserved.