Janus kinases 1 and 2 regulate chemokine-mediated integrin activation and naïve T-cell homing.

Janus kinases (JAKs) are central signaling molecules in cytokine receptor cascades. Although they have also been implicated in chemokine receptor signaling, this function continues to be debated. To address this issue, we established a nucleofection model in primary, nonactivated mouse T lymphocytes to silence JAK expression and to evaluate the ability of these cells to home to lymph nodes. Reduced JAK1 and JAK2 expression impaired naïve T-cell migration in response to gradients of the chemokines CXCL12 and CCL21. In vivo homing of JAK1/JAK2-deficient cells to lymph nodes decreased, whereas intranodal localization and motility were unaffected. JAK1 and JAK2 defects altered CXCL12- and CCL21-triggered ezrin/radixin/moesin (ERM) dephosphorylation and F-actin polymerization, as well as activation of lymphocyte function-associated Ag-1 and very late Ag-4 integrins. As a result, the cells did not adhere firmly to integrin substrates in response to these chemokines. The results demonstrate that JAK1/JAK2 participate in chemokine-induced integrin activation and might be considered a target for modulation of immune cell extravasation and therefore, control of inflammatory reactions.

Tyrosine phosphorylation and activation of STAT5, STAT3, and Janus kinases by interleukins 2 and 15.

The cytokines interleukin 2 (IL-2) and IL-15 have similar biological effects on T cells and bind common hematopoietin receptor subunits. Pathways that involve Janus kinases (JAKs) and signal transducers and activators of transcription (STATs) have been shown to be important for hematopoietin receptor signaling. In this study we identify the STAT proteins activated by IL-2 and IL-15 in human T cells. IL-2 and IL-15 rapidly induced the tyrosine phosphorylation of STAT3 and STAT5, and DNA-binding complexes containing STAT3 and STAT5 were rapidly activated by these cytokines in T cells. IL-4 induced tyrosine phosphorylation and activation of STAT3 but not STAT5. JAK1 and JAK3 were tyrosine-phosphorylated in response to IL-2 and IL-15. Hence, the JAK and STAT molecules that are activated in response to IL-2 and IL-15 are similar but differ from those induced by IL-4. These observations identify the STAT proteins activated by IL-2 and IL-15 and therefore define signaling pathways by which these T-cell growth factors may regulate gene transcription.

Régulation de protéine C-réactive vasculaire dans le diabète de type 2

Les maladies cardiovasculaires sont la principale cause de mortalité dans les pays occidentaux et représentent une complication majeure du syndrome métabolique. Il est maintenant largement admis que l’athérosclérose est une maladie inflammatoire chronique et que l’inflammation joue un rôle pathogénique majeur dans l’initiation et la progression de la maladie athéromateuse. Il a été démontré qu’une augmentation des niveaux sériques de la protéine c-réactive (CRP), une protéine de la phase aigüe et un important constituant de la réponse immunitaire de type inné, est associée à un risque cardiovasculaire accru. Ainsi, il a été documenté qu’une augmentation de CRP, tant chez les sujets sains que chez les sujets diabétiques, était associée à une augmentation du risque de morbidité et de mortalité cardiovasculaires. De multiples évidences suggèrent que la CRP puisse non seulement constituer un marqueur de risque des maladies cardiovasculaires mais aussi représenter un facteur pro-athérogénique direct. La dysfonction endothéliale représente un des stades les plus précoces du processus athérosclérotique et un rôle de la CRP dans la pathogenèse de la dysfonction endothéliale est postulé. Outre son origine systémique, la CRP est produite dans la lésion athérosclérotique et par diverses cellules vasculaires, dont les cellules endothéliales. Afin d’élucider le rôle de la CRP vasculaire dans l’altération de la fonction endothéliale associée au syndrome métabolique, nous avons étudié la régulation de l’expression endothéliale de la CRP par les acides gras libres (AGL) et le rôle de la CRP endothéliale dans l’inhibition de la synthèse d’oxyde nitrique (NO) par les AGL. Nos résultats démontrent que :1) l’acide palmitique (PA) induit l’expression génique de CRP au niveau de cellules endothéliales aortiques humaines (HAECs) en culture et, augmente, de manière dose-dépendante, l’expression protéique de la CRP; 2) La pré-incubation des HAECs avec des antioxydants et des inhibiteurs de la i) protéine kinase C (PKC), ii) du facteur nucléaire-kappa B, iii) des Janus kinases et des protéines de transduction et de régulation de la transcription et iv) des protéines kinases activées par les mitogènes prévient l’effet stimulant du PA sur l’expression protéique et génique de la CRP; 3) Le traitement des HAECs par le PA induit une augmentation de la production des espèces réactives oxygénées, un effet prévenu par les inhibiteurs de la PKC et par l’AICAR(5-amino-4-imidazole carboxamide 1-β-D-ribofuranoside), un activateur de la protéine kinase activée par l’AMP; 4) L’incubation des HAECs en présence de PA résulte enfin en une diminution de la production basale endothéliale de NO, un effet abrogé par la préincubation de ces cellules avec un anticorps anti-CRP. Dans l’ensemble, ces données démontrent un effet stimulant du PA sur l’expression de la CRP endothéliale via l’activation de kinases et de facteurs de transcription sensibles au stress oxydatif. Ils suggèrent en outre un rôle de la CRP dans la dysfonction endothéliale induite par les AGL.

Expression of suppressor of cytokine signaling 1 and 3 in ligature-induced periodontitis in rats

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

Modulation of host cell signaling pathways as a therapeutic approach in periodontal disease

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

Estudo da participação de reguladores negativos endógenos da atividade de STAT1 e STAT3 (SOCS1 e SOCS3) na doença periodontal experimental

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Modulation of host cell signaling pathways as a therapeutic approach in periodontal disease

Recently, new treatment approaches have been developed to target the host component of periodontal disease. This review aims at providing updated information on host-modulating therapies, focusing on treatment strategies for inhibiting signal transduction pathways involved in inflammation. Pharmacological inhibitors of MAPK, NFκB and JAK/STAT pathways are being developed to manage rheumatoid arthritis, periodontal disease and other inflammatory diseases. Through these agents, inflammatory mediators can be inhibited at cell signaling level, interfering on transcription factors activation and inflammatory gene expression. Although these drugs offer great potential to modulate host response, their main limitations are lack of specificity and developments of side effects. After overcoming these limitations, adjunctive host modulating drugs will provide new therapeutic strategies for periodontal treatment.

Molekulare Reaktionsmuster in dendritischen Zellen nach Allergenexposition

Allergische Erkrankungen, wie zum Beispiel die allergische Rhinitis oder das allergische Asthma haben im Verlauf der letzten vier Jahrzehnte stark zugenommen. So leidet heute jeder vierte bis fünfte Mensch an einer Allergie. Ausgelöst wird diese IgE-vermittelte Hypersensibilitätsreaktion des Typs I (Allergie vom Soforttyp) von Allergenen und beruht auf der Aktivierung von Mastzellen durch die Interaktion eines Antigens mit dem an eine Mastzelle über die Fc-Rezeptoren gebundenen IgE-Moleküls. Die degranulierende Mastzelle sezerniert Mediatoren, was zu einem Auftreten von allergischen Symptomen führt. Die Bildung von IgE wird durch das von TH2-Zellen produzierte Zytokin IL-4 induziert. Das von TH1-Zellen produzierte Zytokin IFN- ist in der Lage die Sekretion von IL-4 zu inhibieren, wie auch IL-4 hemmend auf die Produktion von IFN- wirkt. Dieses TH1-/ TH2-Gleichgewicht ist bei allergischen Erkrankungen in Richtung TH2 verschoben. Allergene werden von antigenpräsentierenden Zellen aufgenommen, prozessiert und auf der Zelloberfläche präsentiert. Die potentesten antigenpräsentierenden Zellen sind die dendritischen Zellen, die nach Kontakt mit einem Allergen in die benachbarten Lymphknoten wandern, ausreifen und kostimulatorische Moleküle exprimieren. Sie sind so in der Lage T-Zellen zu aktivieren und entweder in TH1- oder in TH2-Zellen differenzieren zu lassen. Die zytokinabhängige TH1- beziehungsweise TH2-Differenzierung führt zur Aktivierung der Januskinasen. Im aktiven Zustand phosphorylieren sie STAT-Moleküle, die dimerisieren und in den Zellkern translozieren, wo sie unter anderem als Transkriptionsfaktoren für Zytokingene dienen. Unreife humane dendritische Zellen von Allergikern zeigen nach Stimulation mit Proteinallergenen eine schnelle Phosphorylierung des mit der TH2-Entwicklung assoziierten STAT6. Dahingegen sind TH1-Antwort hervorrufende Kontaktallergene nicht in der Lage STAT6 oder andere STAT-Moleküle in dendritischen Zellen zu induzieren. Die Transkriptionsfaktoren T-bet und GATA3 sind ebenfalls von Bedeutung für die TH1-/TH2-Entwicklung, da T-bet ausschließlich in TH1-Zellen, GATA3 nur in TH2-Zellen exprimiert wird. Die Regulation des JAK/STAT-Weg unterliegt den Molekülen der intrazellulär vorkommenden Familie der SOCS-Proteine. SOCS3 ist in TH2-Zellen höher exprimiert als SOCS1, wohingegen SOCS1 in TH1-Zellen eine erhöhte Expression gegenüber SOCS3 aufweist. In der vorliegenden Arbeit wurde der Einfluss von Proteinallergenen auf humane dendritische Zellen untersucht. Zunächst konnte eine morphologische Veränderung der unreifen dendritischen Zellen nach Kontakt mit dem Allergenextrakt beobachtet werden. Die beginnende Ausreifung der Zellen konnte mittels Durchflußzytometrie anhand der kostimulatorischen Moleküle CD80 und CD86, insbesondere aber über den Marker für reife dendritische Zellen CD83, nachgewiesen werden. Die zu beobachtende beginnende Ausreifung scheint ein Effekt des bakteriellen Lipopolysaccharids (LPS) zu sein, das in dem Allergenextrakt vorkommt, da sich durch Zugabe des kationischen Antibiotikums Polymyxin B die beginnende Reifung verhindern ließ. Auf RNA-Ebene war es im Rahmen dieser Arbeit möglich, den Einfluss verschiedener Allergene auf unreifen humanen dendritischen Zellen näher zu charakterisieren. So weisen unreife humane dendritische Zellen nach Kontakt mit Proteinallergenextrakt ein TH2-assoziiertes Genexpressionprofil auf, was sich durch eine erhöhte relative Expression der Gene SOCS3 und GATA3 auszeichnet. Im Gegensatz hierzu zeigen unreife humane dendritische Zellen nach Inkubation mit dem Kontaktallergen MCI/MI eine erhöhte relative Expression des Gens T-bet, was mit einer TH1-Antwort assoziiert ist. Nach Zugabe des „TH1-/ TH2-neutralen“ Tetanustoxoids konnten erhöhte relative Expressionen der Gene GATA3, T-bet und SOCS3 gemessen werden. Die Ergebnisse in dem in dieser Arbeit benutzten humanen in vitro System geben Anlass zur Hypothese, dass die Art der Immunantwort (TH1 versus TH2) sich bereits auf Ebene der dendritischen Zellen anbahnt. GeneChip-Analysen mittels High Density Micro Arrays von unreifen humanen dendritischen Zellen, die entweder mit Proteinallergenextrakt oder mit LPS in Berührung kamen, zeigten statistisch signifikant regulierte Gene, die allerdings keine Gemeinsamkeiten aufwiesen. Es konnten für die mit Alllergenextrakt gepulsten dendritischen Zellen insgesamt 10 Gene identifiziert werden, jedoch gelang es nicht, diese näher zu deuten oder in einen Zusammenhang mit der allergischen Erkrankung oder der dendritischen Zelle zu bringen. Für die mit LPS, dem stärkeren Stimulus, gepulsten dendritischen Zellen konnten 40 Gene identifiziert werden, die unter anderem für die Maturierung der dendritischen Zelle verantwortlich sind. Zudem war es möglich, die Daten der Arrays auf Proteinebene exemplarisch anhand des Chemokins CXCL2 (Gro-β) zu verifizieren.

Identification and functional characterization of novel phosphotyrosine and phosphoserine residues in the interleukin-2 receptor complex

Interleukin-2 (IL-2) is a major T cell growth factor and plays an essential role in the development of normal immune responses. The Janus kinases (Jaks) and Signal transducers and activators of transcription (Stats) are critical for transducing signals from the IL-2 receptors (IL2Rs) to the nucleus to control cell growth and differentiation. In recent years there has been increasing evidence to indicate that the IL-2 activated Jak3/Stat5 pathway provides a new molecular target for immune suppression. Thus, understanding the regulation of this effector cascade has important therapeutic potential.^ One objective of this work was to identify and define the role and molecular mechanism of novel phosphorylation sites in Jak3. Using functional proteomics, three novel Jak3 phosphorylation sites, Y904, Y939 and S574 were identified. Phosphospecific antibodies confirmed that phosphorylation of Y904 and Y939 were mediated by IL-2 and other IL-2 family cytokines in distinct cell types. Biochemical analysis demonstrated that phosphorylation of both Y904 and Y939 positively regulated Jak3 enzymatic activity, while phosphorylation of S574 did not affect Jak3 in vitro kinase activity. However, a gain-of-function mutation of S574 in Jak3 abrogated IL-2 mediated Stat5 activation, suggesting that phosphorylation of this residue might serve a negative role to attenuate IL-2 signaling. Furthermore, mechanistic analysis suggested that phosphorylation of Y904 in Jak3 affects the KmATP of Jak3, while phosphorylation of Y939 in Jak3 was required to bind one of its substrates, Stat5.^ The second objective was to determine the role of serine/threonine phosphatases in the regulation of the IL2R complex. Activation of Jak3 and Stat5 by IL-2 is a transient event mediated by phosphorylation. Using a specific PP1/PP2A inhibitor, we observed that inhibition of PP1/PP2A negatively regulated the IL-2 activated Jak3/Stat5 signaling pathway in a human NK cell line (YT) and primary human T cells. More importantly, coimmunoprecipitation assays indicated that inhibition of PP1/PP2A blocked the formation of an active IL2R complex. Pretreatment of cells with the inhibitor also reduced the electrophoretic mobility of the IL2Rβ and IL2Rγ subunits in YT cells, suggesting that inhibition of PP1/PP2A directly or indirectly regulates undefined serine/threonine kinases which phosphorylate these proteins. Based on these observations, a model has emerged that serine/threonine phosphorylation of the IL2Rβ and IL2Rγ subunits causes a conformational change of these proteins, which disrupts IL2R dimerization and association of Jak3 and Stat5 to these receptors.^

Down-regulation of interferon γ-activated STAT1 by UV light

STAT1 is a cytoplasmic transcription factor that is phosphorylated by Janus kinases (Jak) in response to interferon-γ (IFNγ). Phosphorylated STAT1 translocates to the nucleus, where it turns on specific sets of IFNγ-inducible genes. Here, we show that UV light interferes with tyrosine phosphorylation of STAT1, thereby hindering IFNγ from exerting its biological effects. This effect is not due to a down-regulation of the IFNγ receptor because phosphorylation of upstream-located Jak1 and Jak2 was not suppressed by UV light. In contrast, UV light had no effect on the phosphorylation of STAT3, which is activated by the proinflammatory cytokine interleukin 6. The UV light effect on STAT1 phosphorylation could be antagonized by vanadate, indicating at least partial involvement of a protein tyrosine phosphatase. Therefore, this study indicates a mechanism by which UV light can inhibit gene activation and suggests STAT1 as a new extranuclear UV target closely located to the membrane.

Liver degeneration and lymphoid deficiencies in mice lacking suppressor of cytokine signaling-1

SOCS-1, a member of the suppressor of cytokine signaling (SOCS) family, was identified in a genetic screen for inhibitors of interleukin 6 signal transduction. SOCS-1 transcription is induced by cytokines, and the protein binds and inhibits Janus kinases and reduces cytokine-stimulated tyrosine phosphorylation of signal transducers and activators of transcription 3 and the gp130 component of the interleukin 6 receptor. Thus, SOCS-1 forms part of a feedback loop that modulates signal transduction from cytokine receptors. To examine the role of SOCS-1 in vivo, we have used gene targeting to generate mice lacking this protein. SOCS-1−/− mice exhibited stunted growth and died before weaning with fatty degeneration of the liver and monocytic infiltration of several organs. In addition, the thymus of SOCS-1−/− mice was reduced markedly in size, and there was a progressive loss of maturing B lymphocytes in the bone marrow, spleen, and peripheral blood. Thus, SOCS-1 is required for in vivo regulation of multiple cell types and is indispensable for normal postnatal growth and survival.

A Stat3-interacting protein (StIP1) regulates cytokine signal transduction

Genetic and biochemical studies have led to the identification of the Stat3-Interacting Protein StIP1. The preferential association of StIP1 with inactive (i.e., unphosphorylated) Stat3 suggests that it may contribute to the regulation of Stat3 activation. Consistent with this possibility, StIP1 also exhibits an affinity for members of the Janus kinase family. Overexpression of the Stat3-binding domain of StIP1 blocks Stat3 activation, nuclear translocation, and Stat3-dependent induction of a reporter gene. These studies indicate that StIP1 regulates the ligand-dependent activation of Stat3, potentially by serving as a scaffold protein that promotes the interaction between Janus kinases and their Stat3 substrate. The ability of StIP1 to associate with several additional members of the signal transducer and activator of transcription family suggests that StIP1 may serve a broader role in cytokine-signaling events.

Distinct tyrosine phosphorylation sites in JAK3 kinase domain positively and negatively regulate its enzymatic activity

Cytokines are critically important for the growth and development of a variety of cells. Janus kinases (JAKs) associate with cytokine receptors and are essential for transmitting downstream cytokine signals. However, the regulation of the enzymatic activity of the JAKs is not well understood. Here, we investigated the role of tyrosine phosphorylation of JAK3 in regulating its kinase activity by analyzing mutations of tyrosine residues within the putative activation loop of the kinase domain. Specifically, tyrosine residues 980 and 981 of JAK3 were mutated to phenylalanine individually or doubly. We found that JAK3 is autophosphorylated on multiple sites including Y980 and Y981. Compared with the activity of wild-type (WT) JAK3, mutant Y980F demonstrated markedly decreased kinase activity, and optimal phosphorylation of JAK3 on other sites was dependent on Y980 phosphorylation. The mutant Y980F also exhibited reduced phosphorylation of its substrates, γc and STAT5A. In contrast, mutant Y981F had greatly increased kinase activity, whereas the double mutant, YY980/981FF, had intermediate activity. These results indicate that Y980 positively regulates JAK3 kinase activity whereas Y981 negatively regulates JAK3 kinase activity. These observations in JAK3 are similar to the findings in the kinase that is closely related to the JAK family, ZAP-70; mutations of tyrosine residues within the putative activation loop of ZAP-70 also have opposing actions. Thus, it will be important to determine whether this feature of regulation is unique to JAK3 or if it is also a feature of other JAKs. Given the importance of JAKs and particularly JAK3, it will be critical to fully dissect the positive and negative regulatory function of these and other tyrosine residues in the control of kinase activity and hence cytokine signaling.

Proliferation of adult T cell leukemia/lymphoma cells is associated with the constitutive activation of JAK/STAT proteins

Human T cell leukemia/lymphotropic virus type I (HTLV-I) induces adult T cell leukemia/lymphoma (ATLL). The mechanism of HTLV-I oncogenesis in T cells remains partly elusive. In vitro, HTLV-I induces ligand-independent transformation of human CD4+ T cells, an event that correlates with acquisition of constitutive phosphorylation of Janus kinases (JAK) and signal transducers and activators of transcription (STAT) proteins. However, it is unclear whether the in vitro model of HTLV-I transformation has relevance to viral leukemogenesis in vivo. Here we tested the status of JAK/STAT phosphorylation and DNA-binding activity of STAT proteins in cell extracts of uncultured leukemic cells from 12 patients with ATLL by either DNA-binding assays, using DNA oligonucleotides specific for STAT-1 and STAT-3, STAT-5 and STAT-6 or, more directly, by immunoprecipitation and immunoblotting with anti-phosphotyrosine antibody for JAK and STAT proteins. Leukemic cells from 8 of 12 patients studied displayed constitutive DNA-binding activity of one or more STAT proteins, and the constitutive activation of the JAK/STAT pathway was found to persist over time in the 2 patients followed longitudinally. Furthermore, an association between JAK3 and STAT-1, STAT-3, and STAT-5 activation and cell-cycle progression was demonstrated by both propidium iodide staining and bromodeoxyuridine incorporation in cells of four patients tested. These results imply that JAK/STAT activation is associated with replication of leukemic cells and that therapeutic approaches aimed at JAK/STAT inhibition may be considered to halt neoplastic growth.

Régulation de la voie Jak/STAT par les récepteurs couplés aux protéines G : rôle des petites protéines G de la famille Rho

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.