Ikkε Is Key To Induction Of Insulin Resistance In The Hypothalamus, And Its Inhibition Reverses Obesity.

IKK epsilon (IKKε) is induced by the activation of nuclear factor-κB (NF-κB). Whole-body IKKε knockout mice on a high-fat diet (HFD) were protected from insulin resistance and showed altered energy balance. We demonstrate that IKKε is expressed in neurons and is upregulated in the hypothalamus of obese mice, contributing to insulin and leptin resistance. Blocking IKKε in the hypothalamus of obese mice with CAYMAN10576 or small interfering RNA decreased NF-κB activation in this tissue, relieving the inflammatory environment. Inhibition of IKKε activity, but not TBK1, reduced IRS-1(Ser307) phosphorylation and insulin and leptin resistance by an improvement of the IR/IRS-1/Akt and JAK2/STAT3 pathways in the hypothalamus. These improvements were independent of body weight and food intake. Increased insulin and leptin action/signaling in the hypothalamus may contribute to a decrease in adiposity and hypophagia and an enhancement of energy expenditure accompanied by lower NPY and increased POMC mRNA levels. Improvement of hypothalamic insulin action decreases fasting glycemia, glycemia after pyruvate injection, and PEPCK protein expression in the liver of HFD-fed and db/db mice, suggesting a reduction in hepatic glucose production. We suggest that IKKε may be a key inflammatory mediator in the hypothalamus of obese mice, and its hypothalamic inhibition improves energy and glucose metabolism.

Jnk And Ikkβ Phosphorylation Is Reduced By Glucocorticoids In Adipose Tissue From Insulin-resistant Rats.

Peripheral insulin resistance (IR) is one of the main side effects caused by glucocorticoid (GC)-based therapies, and the molecular mechanisms of GC-induced IR are not yet fully elucidated. Thus, we aimed to investigate the effects of dexamethasone treatment on the main components of insulin and inflammatory signaling in the adipose tissue of rats. Male Wistar rats received daily injections of dexamethasone (1mg/kg body weight (b.w.), intraperitoneally (i.p.)) for 5 days (DEX), whereas control rats received saline (CTL). The metabolic status was investigated, and the epididymal fat fragments were collected for lipolysis and western blot analyses. The DEX rats became hyperglycemic, hyperinsulinemic, insulin resistant and glucose intolerant, compared with the CTL rats (P<0.05). The basal glycerol release in the fat fragments was 1.5-fold higher in the DEX rats (P<0.05). The phosphorylation of protein kinase B (PKB) at ser(473) decreased by 44%, whereas, the phosphorylation of insulin receptor substrate (IRS)-1 at ser(307) increased by 93% in the adipose tissue of the DEX rats after an oral bolus of glucose (P<0.05). The basal phosphorylation of c-jun-N-terminal kinase (JNK) and inhibitor of nuclear factor kappa-B (IKKβ) proteins was reduced by 46% and 58%, respectively, in the adipose tissue of the DEX rats (P<0.05). This was paralleled with a significant reduction (47%) in the glucocorticoid receptor (GR) protein content in the adipose tissue of the DEX rats (P<0.05). The insulin-resistant status of rats induced by dexamethasone administration have PKB and IRS-1 activity attenuated in epididymal fat without increases in the phosphorylation of the proinflammatory signals JNK and IKKβ.

Die Rezeption von Kundenzeitschriften gesetzlicher Krankenkassen : Eine qualitative Untersuchung am Beispiel des „Gesundheitstelegramms“ der IKK gesund plus

Kundenmagazine sind ein wichtiges Instrument des Marketing und der Öffentlichkeitsarbeit. Dies haben auch die gesetzlichen Krankenkassen erkannt. Im Rahmen des Wettbewerbs nutzen sie ihre Kundenzeitschriften zur Mitgliedererhaltung. Wie aber werden diese Zeitschriften von ihrer Leserschaft rezipiert und welche Handlungsmotive liegen dem zugrunde? Im Rahmen einer qualitativen Untersuchung wurde dies anhand von Befragungen der Rezipienten des "Gesundheitstelegramms" der IKK gesund plus analysiert.

Arenavirus Nucleoprotein Targets Interferon Regulatory Factor-Activating Kinase IKKε.

Arenaviruses perturb innate antiviral defense by blocking induction of type I interferon (IFN) production. Accordingly, the arenavirus nucleoprotein (NP) was shown to block activation and nuclear translocation of interferon regulatory factor 3 (IRF3) in response to virus infection. Here, we sought to identify cellular factors involved in innate antiviral signaling targeted by arenavirus NP. Consistent with previous studies, infection with the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) prevented phosphorylation of IRF3 in response to infection with Sendai virus, a strong inducer of the retinoic acid-inducible gene I (RIG-I)/mitochondrial antiviral signaling (MAVS) pathway of innate antiviral signaling. Using a combination of coimmunoprecipitation and confocal microscopy, we found that LCMV NP associates with the IκB kinase (IKK)-related kinase IKKε but that, rather unexpectedly, LCMV NP did not bind to the closely related TANK-binding kinase 1 (TBK-1). The NP-IKKε interaction was highly conserved among arenaviruses from different clades. In LCMV-infected cells, IKKε colocalized with NP but not with MAVS located on the outer membrane of mitochondria. LCMV NP bound the kinase domain (KD) of IKKε (IKBKE) and blocked its autocatalytic activity and its ability to phosphorylate IRF3, without undergoing phosphorylation. Together, our data identify IKKε as a novel target of arenavirus NP. Engagement of NP seems to sequester IKKε in an inactive complex. Considering the important functions of IKKε in innate antiviral immunity and other cellular processes, the NP-IKKε interaction likely plays a crucial role in arenavirus-host interaction.

Identification of human IKK-2 inhibitors of natural origin (Part II): in Silico prediction of IKK-2 inhibitors in natural extracts with known anti-inflammatory activity.

Human inhibitor NF-κB kinase 2 (hIKK-2) is the primary component responsible for activating NF-κB in response to various inflammatory stimuli. Thus, synthetic ATP-competitive inhibitors for hIKK-2 have been developed as anti-inflammatory compounds. We recently reported a virtual screening protocol (doi:10.1371/journal.pone.0016903) that is able to identify hIKK-2 inhibitors that are not structurally related to any known molecule that inhibits hIKK-2 and that have never been reported to have anti-inflammatory activity. In this study, a stricter version of this protocol was applied to an in-house database of 29,779 natural products annotated with their natural source. The search identified 274 molecules (isolated from 453 different natural extracts) predicted to inhibit hIKK-2. An exhaustive bibliographic search revealed that anti-inflammatory activity has been previously described for: (a) 36 out of these 453 extracts; and (b) 17 out of 30 virtual screening hits present in these 36 extracts. Only one of the remaining 13 hit molecules in these extracts shows chemical similarity with known synthetic hIKK-2 inhibitors. Therefore, it is plausible that a significant portion of the remaining 12 hit molecules are lead-hopping candidates for the development of new hIKK-2 inhibitors.

PPM1B and P-IKKβ expression levels correlated inversely with rat gastrocnemius atrophy after denervation

Activated inhibitor of nuclear factor-κB kinase β (IKKβ) is necessary and sufficient for denervated skeletal muscle atrophy. Although several studies have shown that Mg2+/Mn2+-dependent protein phosphatase 1B (PPM1B) inactivated IKKβ, few studies have investigated the role of PPM1B in denervated skeletal muscle. In this study, we aim to explore the expression and significance of PPM1B and phosphorylated IKKβ (P-IKKβ) during atrophy of the denervated gastrocnemius. Thirty young adult female Wistar rats were subjected to right sciatic nerve transection and were sacrificed at 0 (control), 2, 7, 14, and 28 days after denervation surgery. The gastrocnemius was removed from both the denervated and the contralateral limb. The muscle wet weight ratio was calculated as the ratio of the wet weight of the denervated gastrocnemius to that of the contralateral gastrocnemius. RT-PCR and Western blot analysis showed that mRNA and protein levels of PPM1B were significantly lower than those of the control group at different times after the initiation of denervation, while P-IKKβ showed the opposite trends. PPM1B protein expression persistently decreased while P-IKKβ expression persistently increased for 28 days after denervation. PPM1B expression correlated negatively with P-IKKβ expression by the Spearman test, whereas decreasing PPM1B expression correlated positively with the muscle wet weight ratio. The expression levels of PPM1B and P-IKKβ were closely associated with atrophy in skeletal denervated muscle. These results suggest that PPM1B and P-IKKβ could be markers in skeletal muscle atrophy.

Rôles des kinases IKK et IKK-related dans les maladies inflammatoires chroniques : implications dans l’athérosclérose et la réponse hypoxique

L’inflammation est un procédé complexe qui vise l’élimination de l’agent causal de dommages tissulaires en vue de faciliter la réparation du tissu affecté. La persistance de l’agent causal ou l’incapacité à résoudre l’inflammation mène à un dérèglement homéostatique chronique qui peut avoir une incidence sur la morbidité et la mortalité. L’athérosclérose est une condition inflammatoire chronique des vaisseaux sanguins dont l’origine est multifactorielle. L’hypertension et l’état infectieux représentent respectivement des facteurs de risque classiques et émergents du développement de cette maladie. Les fondements initiaux de l’inflammation font intervenir l’immunité innée, la première ligne de défense dont disposent les cellules pour répondre à un signal de danger. Le but de cette thèse est d’examiner le rôle pro-inflammatoire d’une famille de kinases essentielles à l’immunité innée, soit celle des kinases de IkappaB (IKK) et des kinases IKK-related. Les kinases IKKalpha et IKKbeta forment le complexe IKK avec la molécule adaptatrice NEMO/IKKgamma. Ce complexe est chargé d’effectuer la phosphorylation de l’inhibiteur de NF-kappaB, IkappaBalpha, ce qui mène à sa dégradation et à la libération du facteur de transcription NF-kappaB. Nous montrons que le peptide vasoactif angiotensine II (AngII) induit l’activité phosphotransférase d’IKKbeta dans les VSMC par immunoprécipitation de NEMO puis essai kinase in vitro. Grâce à une approche ARN interférence (ARNi) dirigée contre IKK, nous montrons que cette kinase est responsable de la phosphorylation de p65/RelA. Nous montrons que le mécanisme d’induction de NF-kappaB par l’AngII est atypique, puisqu’il ne module pas IkappaBalpha, et montrons à l’aide d’inhibiteurs pharmacologiques que l’activation de p65 est indépendante des voies MEK-ERK-RSK, PI3K et de la transactivation du récepteur de l’EGF. Les kinases IKK-related Tank-binding kinase 1 (TBK1) et IKK-i sont quant à elles principalement activées suite à une infection bactérienne ou virale. Ces kinases phosphorylent directement le facteur de transcription interferon regulatory factor (IRF)-3. Nous montrons que le cytomégalovirus humain, un pathogène associé à l’athérosclérose, a la capacité d’induire l’activation de TBK1 dans les VSMC. L’usage d’ARNi dirigé contre TBK1 et IKKi montre que les 2 kinases sont impliquées dans l’activation d’IRF-3. De plus, nous montrons à l’aide d’une lignée de VSMC exprimant une version dominante négative d’IRF-3 que ce dernier est essentiel à la synthèse des chimiokines RANTES et IP-10, tel qu’analysé par RT-PCR. Par ailleurs, il a récemment été montré que les kinases IKK-related étaient étroitement liées à la transformation oncogénique, et que TBK1 était pro-angiogénique. Or, l’angiogenèse est le plus souvent modulée par la réponse hypoxique qui est d’ailleurs commune à la majorité des processus inflammatoires. Le facteur de transcription hypoxia inducible factor (HIF)-1 module l’angiogenèse, l’inflammation et la survie cellulaire. Nous montrons à l’aide de cellules Tbk1 et Ikbke -/- et d’une approche lentivirale que TBK1 est spécifiquement impliquée dans l’induction traductionnelle de HIF-1alpha en condition de stress hypoxique. L’expression de TBK1 est induite sous ces conditions, et cette kinase module la phosphorylation de ERK, RSK, Akt et TSC1. Les résultats originaux présentés dans cette thèse montrent donc que les kinases IKK et IKK-related exercent leurs actions pro-inflammatoires par des mécanismes distincts.

Tumor necrosis factor alpha triggers proliferation of adult neural stem cells via IKK/NF-kappaB signaling

BACKGROUND: Brain inflammation has been recognized as a complex phenomenon with numerous related aspects. In addition to the very well-described neurodegenerative effect of inflammation, several studies suggest that inflammatory signals exert a potentially positive influence on neural stem cell proliferation, migration and differentiation. Tumor necrosis factor alpha (TNF-alpha) is one of the best-characterized mediators of inflammation. To date, conclusions about the action of TNF on neural stem or progenitor cells (NSCs, NPCs) have been conflicting. TNF seems to activate NSC proliferation and to inhibit their differentiation into NPCs. The purpose of the present study was to analyze the molecular signal transduction mechanisms induced by TNF and resulting in NSC proliferation. RESULTS: Here we describe for the first time the TNF-mediated signal transduction cascade in neural stem cells (NSCs) that results in increased proliferation. Moreover, we demonstrate IKK-alpha/beta-dependent proliferation and markedly up-regulated cyclin D1 expression after TNF treatment. The significant increase in proliferation in TNF-treated cells was indicated by increased neurosphere volume, increased bromodeoxyuridin (BrdU) incorporation and a higher total cell number. Furthermore, TNF strongly activated nuclear factor-kappa B (NF-kappaB) as measured by reporter gene assays and by an activity-specific antibody. Proliferation of control and TNF-treated NSCs was strongly inhibited by expression of the NF-kappaB super-repressor IkappaB-AA1. Pharmacological blockade of IkappaB ubiquitin ligase activity led to comparable decreases in NF-kappaB activity and proliferation. In addition, IKK-beta gene product knock-down via siRNA led to diminished NF-kappaB activity, attenuated cyclin D1 expression and finally decreased proliferation. In contrast, TGFbeta-activated kinase 1 (TAK-1) is partially dispensable for TNF-mediated and endogenous proliferation. Understanding stem cell proliferation is crucial for future regenerative and anti-tumor medicine. CONCLUSION: TNF-mediated activation of IKK-beta resulted in activation of NF-kappaB and was followed by up-regulation of the bona-fide target gene cyclin D1. Activation of the canonical NF-kappaB pathway resulted in strongly increased proliferation of NSCs.

Effects of P-MAPA immunomodulator on Toll-like receptor 2, ROS, nitric oxide, MAPKp38 and IKK in PBMC and macrophages from dogs with visceral leishmaniasis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

TLR 2, TLR 4, ROS, óxido nítrico, P38 e IKK em células mononucleares de cães com leishmaniose Visceral tratadas com o imunomodulador P-MAPA: Larissa Martins Melo. -

Pós-graduação em Ciência Animal - FMVA

Modulation of NF-kappaB activation in Theileria annulata-infected cloned cell lines is associated with detection of parasite-dependent IKK signalosomes and disruption of the actin cytoskeleton

Summary Apicomplexan parasites within the genus Theileria have the ability to induce continuous proliferation and prevent apoptosis of the infected bovine leukocyte. Protection against apoptosis involves constitutive activation of the bovine transcription factor NF-kappaB in a parasite-dependent manner. Activation of NF-kappaB is thought to involve recruitment of IKK signalosomes at the surface of the macroschizont stage of the parasite, and it has been postulated that additional host proteins with adaptor or scaffolding function may be involved in signalosome formation. In this study two clonal cell lines were identified that show marked differences in the level of activated NF-kappaB. Further characterization of these lines demonstrated that elevated levels of activated NF-kappaB correlated with increased resistance to cell death and detection of parasite-associated IKK signalosomes, supporting results of our previous studies. Evidence was also provided for the existence of host- and parasite-dependent NF-kappaB activation pathways that are influenced by the architecture of the actin cytoskeleton. Despite this influence, it appears that the primary event required for formation of the parasite-dependent IKK signalosome is likely to be an interaction between a signalosome component and a parasite-encoded surface ligand.

Theileria-induced constitutive IKK activation is independent of functional Hsp90

The intracellular parasite Theileria induces uncontrolled proliferation and host cell transformation. Parasite-induced transformation is accompanied by constitutive activation of IkappaB kinase (IKK), resulting in permanently high levels of activated nuclear factor (NF)-kappaB. IKK activation pathways normally require heat shock protein 90 (Hsp90), a chaperone that regulates the stability and activity of signalling molecules and can be blocked by the benzoquinone ansamycin compound geldanamycin (GA). In Theileria-transformed cells, IkappaBalpha and p65 phosphorylation, NF-kappaB nuclear translocation and DNA binding activity are largely resistant to GA and also NF-kappaB-dependent reporter gene expression is only partly affected. Our findings indicate that parasite-induced IKK activity does not require functional Hsp90.

Hijacking of host cell IKK signalosomes by the transforming parasite Theileria

Parasites have evolved a plethora of mechanisms to ensure their propagation and evade antagonistic host responses. The intracellular protozoan parasite Theileria is the only eukaryote known to induce uncontrolled host cell proliferation. Survival of Theileria-transformed leukocytes depends strictly on constitutive nuclear factor kappa B (NF-kappaB) activity. We found that this was mediated by recruitment of the multisubunit IkappaB kinase (IKK) into large, activated foci on the parasite surface. IKK signalosome assembly was specific for the transforming schizont stage of the parasite and was down-regulated upon differentiation into the nontransforming merozoite stage. Our findings provide insights into IKK activation and how pathogens subvert host-cell signaling pathways.