Tyk2 tyrosine kinase expression is required for the maintenance of mitochondrial respiration in primary pro-B lymphocytes

Tyk2, a member of the Jak family of protein tyrosine kinases, is critical for the biological actions of alpha/beta interferon (IFN-alpha/beta). Although Tyk2(-/-) mice are phenotypically normal, they exhibit abnormal responses to inflammatory challenges in a variety of cells isolated from Tyk2(-/-) mice. The reported phenotypic alterations in both Tyk2-null cells and mice are consistent with the possibility that the expression of this tyrosine kinase may regulate mitochondrial function. We report here that Tyk2-null pro-B cells are markedly deficient in basal oxygen consumption and exhibit a significant decrease in steady-state cellular ATP levels compared to wild-type cells. Tyk2-null cells also exhibit impaired complex I, III, and IV function of the mitochondrial electron transport chain. Reconstitution of Tyk2-null pro-B cells with either the wild type or a kinase-inactive mutant of Tyk2 restores basal mitochondrial respiration. By contrast, the kinase activity of Tyk2 is required for maintenance of both complex I-dependent mitochondrial respiration as well as induction of apoptosis in cells incubated with IFN-beta. Consistent with the role of Tyk2 in the regulation of tyrosine phosphorylation of Stat3, expression of a constitutively active Stat3 can restore the mitochondrial respiration in Tyk2-null cells treated with IFN-beta. Finally, Tyk2(-/-) mice show decreased exercise tolerance compared to wild-type littermates. Our results implicate a novel role for Tyk2 kinase and Stat3 phosphorylation in mitochondrial respiration.     

Tyk2 mediates effects of urokinase on human vascular smooth muscle cell growth

The urokinase (uPA)/uPA receptor (uPAR) system plays a role in the response of the vessel wall to injury, presumably by modulating vascular smooth muscle cell (VSMC) functional behaviour. The Jak/Stat signaling pathway has been implicated to mediate the uPA/uPAR-directed cell migration and proliferation in VSMC. We have therefore investigated the underlying molecular mechanisms, which remained not completely understood. In particular, we aimed at identification of the kinase involved in the signaling cascade leading to Stat1 phosphorylation by uPA and its impact on VSMC growth. We performed expression in VSMC of kinase-deficient mutant forms of the Janus kinases Jak1 and Tyk2 and used different cell culture models imitating the response to vascular injury. We provide evidence that Tyk2, but not Jak1, mediates uPA-induced Stat1 phosphorylation and VSMC growth inhibition and suggest a novel function for Tyk2 as an important modulator of the uPA-directed VSMC functional behaviour at the place of injury.     

Forskolin-inducible cAMP Pathway Negatively Regulates T-cell Proliferation by Uncoupling the Interleukin-2 Receptor Complex

Cytokine-mediated regulation of T-cell activity involves a complex interplay between key signal transduction pathways. Determining how these signaling pathways cross-talk is essential to understanding T-cell function and dysfunction. In this work, we provide evidence that cross-talk exists between at least two signaling pathways: the Jak3/Stat5 and cAMP-mediated cascades. The adenylate cyclase activator forskolin (Fsk) significantly increased intracellular cAMP levels and reduced proliferation of the human T-cells via inhibition of cell cycle regulatory genes but did not induce apoptosis. To determine this inhibitory mechanism, effects of Fsk on IL-2 signaling was investigated. Fsk treatment of MT-2 and Kit 225 T-cells inhibited IL-2-induced Stat5a/b tyrosine and serine phosphorylation, nuclear translocation, and DNA binding activity. Fsk treatment also uncoupled IL-2 induced association of the IL-2Rβ and γ_c chain, consequently blocking Jak3 activation. Interestingly, phosphoamino acid analysis revealed that Fsk-treated cells resulted in elevated serine phosphorylation of Jak3 but not Stat5, suggesting that Fsk can negatively regulate Jak3 activity possibly mediated through PKA. Indeed, in vitro kinase assays and small molecule inhibition studies indicated that PKA can directly serine phosphorylate and functionally inactivate Jak3. Taken together, these findings suggest that Fsk activation of adenylate cyclase and PKA can negatively regulate IL-2 signaling at multiple levels that include IL-2R complex formation and Jak3/Stat5 activation.     

H(2)O(2)-induced tyrosine phosphorylation of protein kinase cdelta by a mechanism independent of inhibition of protein-tyrosine phosphatase in CHO and COS-7 cells

It has been proposed that H(2)O(2) increases tyrosine phosphorylation of cellular proteins by inhibiting protein-tyrosine phosphatase through oxidation of the cysteine residue of the enzyme essential for its catalytic activity. Tyrosine phosphorylation of the delta isoform of protein kinase C (PKC) was induced by H(2)O(2) in CHO and COS-7 cells. H(2)O(2) also induced activation of mitogen-activated protein kinase. Vanadate and molybdate, which inhibit protein-tyrosine phosphatase by binding to its active site, did not induce tyrosine phosphorylation of PKCdelta, but enhanced H(2)O(2)-induced tyrosine phosphorylation of PKCdelta in the cell. The oxoanions, however, generated the active form of mitogen-activated protein kinase. Another protein-tyrosine phosphatase inhibitor, phenylarsine oxide, which bridges the thiol residues of the enzyme, induced tyrosine phosphorylation of PKCdelta, and the reaction was enhanced by vanadate. These results suggest that inhibition of protein-tyrosine phosphatase is insufficient for induction of tyrosine phosphorylation of PKCdelta in the cells, and that presumably activation of protein-tyrosine kinase may be essential for tyrosine phosphorylation of the PKC isoform.     

Activation of Tyk2 and Stat3 is required for the apoptotic actions of interferon-beta in primary pro-B cells

The growth-inhibitory effects of type 1 interferons (IFNs) (IFNalpha/beta) are complex, and the role of apoptosis in their antigrowth effects is variable and not well understood. We have examined primary murine interleukin-7-dependent bone marrow-derived pro-B cells, where IFNbeta, but not IFNalpha, induces programmed cell death (PCD). IFNbeta-stimulated apoptosis is the same in pro-B cells derived from wild type and Stat1(-/-) mice. However, in pro-B cells from Tyk2(-/-) mice, where there is normal activation of Stat1 and Stat2, IFNbeta-stimulated PCD is not observed. Loss of B cells in lymphocytic choriomeningitis virus-infected mice has been shown to be mediated through the expression of IFNalpha/beta (1). In wild type mice infected with lymphocytic choriomeningitis virus, there is a greater loss of B cells in the bone marrow and spleen than in Tyk2(-/-) mice infected with the virus, suggesting that the expression of this kinase plays an in vivo role in IFNalpha/beta-mediated PCD. In contrast to IFNbeta-stimulated tyrosine phosphorylation of Stat1 and Stat2, Stat3 tyrosine phosphorylation is defective in Tyk2(-/-) pro-B cells, suggesting that this Stat family member is required for apoptosis. In support of this hypothesis, inhibition of Stat3 activation in wild type B cells reverses the apoptotic effects of IFNbeta. Furthermore, expression of a constitutively active form of Stat3 in Tyk2(-/-) B cells partially restores IFNbeta-stimulated PCD. These results demonstrate an important role of Tyk2-mediated tyrosine phosphorylation of Stat3 in the ability of IFNbeta to stimulate apoptosis of primary pro-B cells.     

Cells previously desensitized to type 1 interferons display different mechanisms of activation of stat-dependent gene expression from naïve cells

Over the past decade, a wealth of knowledge has been obtained concerning the mechanisms by which interferons (IFNs) and other cytokines activate or down-regulate immediate early genes via the Jak/Stat pathway. In contrast, little information is available on interferon-activated gene expression in na?ve cells compared with cells that have been desensitized and subsequently resensitized to the actions of these cytokines. In na?ve cells, the ISG54 gene is activated via IFN beta-stimulated formation of ISGF3, a heterotrimeric DNA binding complex consisting of p48 (IRF9) and tyrosine-phosphorylated Stat1 and Stat2. In contrast, in previously desensitized cells IFN beta weakly stimulates the assembly of an ISGF3-like complex that lacks Stat1, even though ISG54 mRNA induction is the same as in na?ve cells. The lack of Stat1 tyrosine phosphorylation and DNA binding is due to increased activity of a protein-tyrosine phosphatase. In cells that do not express the tyrosine phosphatase Tc-PTP, the rate of Stat1 dephosphorylation is the same in na?ve and previously desensitized cells. These results implicate Tc-PTP in a novel role in the regulation of type 1 interferon-stimulated gene expression.     

The Stat3-activating Tyk2 V678F mutant does not up-regulate signaling through the type I interferon receptor but confers ligand hypersensitivity to a homodimeric receptor

Tyk2 is a Jak family member involved in cytokine signaling through heterodimeric-type receptors. Here, we analyzed the impact of the Val(678)-to-Phe substitution on Tyk2 functioning. This mutation is homologous to the Jak2 Val(617)-to-Phe mutation, implicated in myeloproliferative disorders. We studied ligand-independent and ligand-dependent Jak/Stat signaling in cells expressing Tyk2 V678F. Moreover, the effect of Tyk2 V678F was monitored in the context of the native heterodimeric interferon alpha receptor and in the context of a homodimeric receptor chimera, EpoR/R1, containing the ectodomain of the erythropoietin receptor. We show that Tyk2 V678F has increased catalytic potential in vivo and in vitro and more so when it is anchored to the homodimeric receptor. Tyk2 V678F leads to constitutive Stat3 phosphorylation but has no notable effect on the canonical interferon alpha-induced signaling. However, if anchored to the homodimeric EpoR/R1, the mutant confers to the cell increased sensitivity to erythropoietin. Thus, despite the catalytic gain of function of Tyk2 V678F, the effect on ligand-induced signaling is manifest only when two mutant enzymes are juxtaposed via the homodimeric receptor.     

Stat4 regulates multiple components of IFN-gamma-inducing signaling pathways

Stat4 is activated in response to IL-12. Most functions of IL-12, including the induction of IFN-gamma, are compromised in the absence of Stat4. Since the precise role of Stat4 in IFN-gamma induction has not been established, experiments were conducted to examine Stat4 activation of IFN-gamma and other genes required for cytokine-induced expression of IFN-gamma. We first examined IL-12 signaling components. Basal expression of IL-12Rss1 and IL-12Rss2 is decreased in Stat4-deficient cells compared with that in control cells. However, IL-12 was still capable of inducing equivalent phosphorylation of Jak2 and Tyk2 in wild-type and Stat4-deficient activated T cells. We have further determined that other cytokine signaling pathways that induce IFN-gamma production are defective in the absence of Stat4. IL-18 induces minimal IFN-gamma production from Stat4-deficient activated T cells compared with control cells. This is due to defective IL-18 signaling, which results from the lack of IL-12-induced, and Stat4-dependent, expression of the IL-18R. Following IL-12 pretreatment to induce IL-18R, wild-type, but not Stat4-deficient, activated T cells demonstrated IL-18-induced NF-kappaB DNA-binding activity. In addition, IL-12-pretreated Stat4-deficient activated T cells have minimal IFN-gamma production followed by stimulation with IL-18 alone or in combination with IL-12 compared with control cells. Thus, Stat4 activation by IL-12 is required for the function of multiple cytokine pathways that result in induction of IFN-gamma.     

Role of IL-6 and CD23 in the resistance to growth arrest and apoptosis in LCL41 B lymphoma cells

Interleukin-6 (IL-6) is a growth and survival factor in Epstein-Barr virus (EBV)-infected B lymphoma cells and IL-6 antagonists have been used in clinical practice for this pathology. We thus wanted to investigate the effect of the IL-6 receptor antagonist Sant7 on proliferative and anti-apoptotic signals in the IL-6-secreting LCL41 B lymphoid cells, taken from a patient with EBV-induced lymphoproliferative disorder. Results show efficient inhibition of constitutive Stat3 activation by Sant7. However, this inhibition is associated with marginal induction of apoptosis and with minor decrease of cell proliferation, contrary to the effect of the Jak kinase inhibitor AG490, which down-regulates both proliferation and Stat3 activation. Anti-apoptotic markers such as Bcl-xL or Mcl-1 are constitutively expressed in these cells, and their expression is not affected by Sant7 treatment. Inhibition of Stat3 activation is therefore not sufficient to prevent proliferation and to induce apoptosis in these cells. In addition, low cell density is a condition favouring inhibition of cell clustering and anti-proliferative Sant7 activity. A marked inhibition of cell cluster formation and proliferation is achieved by antibody treatment against the CD23 mature B cell surface marker expressed in LCL41 cells. These findings may thus contribute to the identification of possible resistance mechanisms to growth arrest in B cell lymphoproliferative conditions.     

Longer and Shorter Forms of Sendai Virus C Proteins Play Different Roles in Modulating the Cellular Antiviral Response

The Sendai virus (SeV) C gene codes for a nested set of four C proteins that carry out several functions, including the modulation of viral RNA synthesis and countering of the cellular antiviral response. Using mutant C genes (and in particular a C gene with a deletion of six amino acids present only in the larger pair of C proteins) and recombinant SeV carrying these mutant C genes, we find that the nested set of C proteins carry out a nested set of functions. All of the C proteins interdict interferon (IFN) signaling to IFN-stimulated genes (ISGs) and prevent pY701-Stat1 formation. However, only the larger C proteins can induce STAT1 instability, prevent IFN from inducing an antiviral state, or prevent programmed cell death. Remarkably, interdiction of IFN signaling to ISGs and the absence of pY701-Stat1 formation did not prevent IFN-alpha from inducing an anti-Vesicular stomatitis virus (VSV) state. It is possible that IFN-alpha signaling to induce an anti-VSV state can occur independently of the well-established Jak/Stat/ISGF3 pathway and that it is this parallel pathway that is targeted by the longer C proteins.