Cloning of a type I cytokine receptor most related to the IL-2 receptor beta chain

We have identified a type I cytokine receptor, which we have termed novel interleukin receptor (NILR), that is most related to the IL-2 receptor beta chain (IL-2Rbeta) and physically adjacent to the IL-4 receptor alpha chain gene on chromosome 16. NILR mRNA is most highly expressed in thymus and spleen, and is induced by phytohemagglutinin in human peripheral blood mononuclear cells. NILR protein was detected on human T cell lymphotropic virus type I-transformed T cell lines, Raji B cells, and YT natural killer-like cells. Artificial homodimerization of the NILR cytoplasmic domain confers proliferation to Ba/F3 murine pro-B cells but not to 32D myeloid progenitor cells or CTLL-2 murine helper T cells. In these latter cells, heterodimerization of IL-2Rbeta and the common cytokine receptor gamma chain (gamma(c)) cytoplasmic domains allows potent proliferation, whereas such heterodimerization of NILR with gamma(c) does not. This finding suggests that NILR has signaling potential but that a full understanding of its signaling partner(s) is not yet clear. Like IL-2Rbeta, NILR associates with Jak1 and mediates Stat5 activation.     

Down-regulation of interleukin-3/granulocyte-macrophage colony-stimulating factor receptor beta-chain in BCR-ABL(+) human leukemic cells: association with loss of cytokine-mediated Stat-5 activation and protection from apoptosis after BCR-ABL inhibition

Several signaling cascades are engaged by expression of the p210 bcr-abl tyrosine kinase, and evidence suggests that these signals drive leukemogenesis. In this report, signaling pathways were examined and compared between cells derived from leukemic patients and cells expressing a bcr-abl construct (MBA). The effects of acute inhibition of bcr-abl with STI-571 on these signals and the survival of bcr-abl-expressing cells were also evaluated. Expression of bcr-abl in interleukin-3 (IL-3)/granulocyte-macrophage colony-stimulating factor (GM-CSF)-dependent Mo7e cells (MBA) resulted in growth factor independence, constitutive activation of Stat-5 phosphorylation, engagement of mitogen-activated protein (MAP) kinase signals, and increased expression of PTP1B and bcl-x(L). STI-571 inhibited cell growth and induced apoptosis in bcr-abl-expressing cells (MBA, K562, BV-173, KBM5) but not in bcr-abl(-) tumor cells (Mo7e, KG-1, ME-180, Daudi). STI-571-mediated apoptosis correlated with the inhibition of Stat-5 and MAP kinase activation and a reduction in overexpressed bcl-x(L) but not in PTP1B. Inhibitor had no effect on IL-3/GM-CSF-dependent Mo7e cell signaling and did not prevent activation of the other Jak/Stat pathways (interferon alpha, IL-3/GM-CSF). However, neither IL-3 nor GM-CSF could reactivate Stat-5 after the STI-571-mediated inhibition of bcr-abl. Expression of the common beta-chain of the IL-3/GM-CSF receptor was down-regulated in Stat-5-activated myeloid leukemic cells, suppressing IL-3/GM-CSF signal transduction and the ability of these cytokines to provide apoptotic protection. These studies suggest that bcr-abl activates cytokine-independent mechanisms of survival while inactivating intrinsic cytokine signaling cascades, making bcr-abl(+) myeloid cells vulnerable to apoptosis after bcr-abl inactivation.     

Cis desensitizes GH induced Stat5 signaling in rat liver cells.

Recently a novel family of proteins, the Cis/Socs family, has been shown to constitute negative regulators of cytokine-induced Jak/Stat signaling. Here we demonstrate that Socs-2 and Cis mRNA expression in rat liver is dependent on the presence of growth hormone (GH), and that GH induce Cis mRNA expression in cultures of primary rat hepatocytes. Furthermore, cotransfection studies in the rat liver cell line, BRL-4, revealed that constitutive expression of Cis, but not Socs-2, inhibited the GH-induced transactivation of a Stat5-responsive reporter gene construct. This indicates a functional role for Cis in the desensitization of GH activated Jak/Stat5 signaling in rat liver cells. In response to the intermittent pattern of GH secretion in male rats, GH activates Stat5b signaling whereas this activation is blunted in female rats having a continuous pattern of GH secretion. We hypothesize that GH induction of Cis could be one mechanism by which sexually dimorphic GH signaling via Stat5b is achieved in the rat liver.     

Regulation of Jak2 tyrosine kinase by protein kinase C during macrophage differentiation of IL-3-dependent myeloid progenitor cells

Differentiation of macrophages from myeloid progenitor cells depends on a discrete balance between cell growth, survival, and differentiation signals. Interleukin-3 (IL-3) supports the growth and survival of myeloid progenitor cells through the activation of Jak2 tyrosine kinase, and macrophage differentiation has been shown to be regulated by protein kinase C (PKC). During terminal differentiation of macrophages, the cells lose their mitogenic response to IL-3 and undergo growth arrest, but the underlying signaling mechanisms have remained elusive. Here we show that in IL-3-dependent 32D myeloid progenitor cells, the differentiation-inducing PKC isoforms PKC-alpha and PKC-delta specifically caused rapid inhibition of IL-3-induced tyrosine phosphorylation. The target for this inhibition was Jak2, and the activation of PKC by 12-O-tetradecanoyl-phorbol-13-acetate treatment also abrogated IL-3-induced tyrosine phosphorylation of Jak2 in Ba/F3 cells. The mechanism of this regulation was investigated in 32D and COS7 cells, and the inhibition of Jak2 required catalytic activity of PKC-delta and involved the phosphorylation of Jak2 on serine and threonine residues by the associated PKC-delta. Furthermore, PKC-delta inhibited the in vitro catalytic activity of Jak2, indicating that Jak2 was a direct target for PKC-delta. In 32D cells, the inhibition of Jak2 either by PKC-delta, tyrosine kinase inhibitor AG490, or IL-3 deprivation caused a similar growth arrest. Reversal of PKC-delta-mediated inhibition by the overexpression of Jak2 promoted apoptosis in differentiating 32D cells. These results demonstrate a PKC-mediated negative regulatory mechanism of cytokine signaling and Jak2, and they suggest that it serves to integrate growth-promoting and differentiation signals during macrophage differentiation. (Blood. 2000;95:1626-1632)     

The molecular basis of IL-21-mediated proliferation

Interleukin-21 (IL-21) is a type I cytokine that modulates functions of T, B, natural killer (NK), and myeloid cells. The IL-21 receptor (IL-21R) is closely related to the IL-2 receptor beta chain and is capable of transducing signals through its dimerization with the common cytokine receptor gamma chain (gamma(c)), the protein whose expression is defective in humans with X-linked severe combined immunodeficiency. To clarify the molecular basis of IL-21 actions, we investigated the role of tyrosine residues in the IL-21R cytoplasmic domain. Simultaneous mutation of all 6 tyrosines greatly diminished IL-21-mediated proliferation, whereas retention of tyrosine 510 (Y510) allowed full proliferation. Y510 efficiently mediated IL-21-induced phosphorylation of Stat1 and Stat3, but not of Stat5, and CD8(+) T cells from Stat1/Stat3 double knock-out mice exhibited decreased proliferation in response to IL-21 + IL-15. In addition, IL-21 weakly induced phosphorylation of Shc and Akt, and consistent with this, specific inhibitors of the MAPK and PI3K pathways inhibited IL-21-mediated proliferation. Collectively, these data indicate the involvement of the Jak-STAT, MAPK, and PI3K pathways in IL-21 signaling.     

Novel in vitro effects of bucillamine: inhibitory effects on proinflammatory cytokine production and transendothelial migration of T cells

OBJECTIVE: To investigate the novel antiinflammatory mechanism of a disease-modifying antirheumatic drug, bucillamine, on activated T cells, specifically its effect on T cell proliferation, cytokine production, and migration of T cells. METHODS: T cells were cultured in wells coated with anti-CD3 monoclonal antibodies (mAb) plus anti-CD26 mAb or anti-CD3 plus anti-CD28 mAb, with or without bucillamine. Proliferative responses and the production of interleukin-2 (IL-2), interferon-gamma (IFNgamma), tumor necrosis factor alpha (TNFalpha), IL-6, IL-4, and IL-5 were measured under these costimulatory conditions. Phytohemagglutinin (PHA)-activated T cells were cultured on human umbilical vein endothelial cell-coated transwells in the presence or absence of bucillamine, and T cells migrating through the endothelial cell layer were counted. Immunofluorescence analysis was also performed to analyze the effect of bucillamine on the surface expression of adhesion molecules on T cells. RESULTS: Bucillamine (64 microM) significantly inhibited T cell proliferation and the production of IL-2, IFNgamma, TNFalpha, and IL-6, whereas it had no inhibitory effects on the production of IL-4 and IL-5 in the cultures with anti-CD3 plus anti-CD26 mAb. In contrast, bucillamine had no effects on T cell proliferation or any cytokine production in the cultures with anti-CD3 plus anti-CD28 mAb. Furthermore, the same concentration of bucillamine inhibited transendothelial migration of PHA-activated T cells, and reduced the expression level of CD44 on T cells. CONCLUSION: This study demonstrated the novel effects of bucillamine in vitro, showing inhibition of type 1 T helper-type cytokine production and proinflammatory cytokine production induced by certain costimulatory conditions, and inhibition of transendothelial migration of T cells. The inhibition of T cell migration appeared to be mediated partly through the reduced expression of CD44, an adhesion molecule on the T cell surface.     

Limitin, an interferon-like cytokine, transduces inhibitory signals on B-cell growth through activation of Tyk2, but not Stat1, followed by induction and nuclear translocation of Daxx

OBJECTIVE: Limitin, an interferon-like cytokine, suppresses B lymphopoiesis through ligation of the interferon-alpha/beta (IFN-alpha/beta) receptor. The aim of this study was to examine the intracellular signal transduction pathways activated by limitin. MATERIALS AND METHODS: The effects of limitin on cell growth, the activation of Jak kinase and Stat proteins, and the induction of interferon regulatory factor-1 (IRF-1) and Daxx were examined using the mouse pre-B-cell line 18.81, wild-type, and Tyk2-deficient mouse bone marrow cells. In addition, the change of localization of the Daxx protein after limitin treatment in wild-type and Tyk2-deficient mice was examined. RESULTS: Limitin phosphorylates Tyk2, Jak1, Stat1, and Stat2 and rapidly induces IRF-1 mRNA production. Phosphorylation of Stat1 by limitin is partially dependent on Tyk2. Suppression of B-cell growth by limitin, however, is severely impaired in the absence of Tyk2, whereas it is unaffected by the absence of Stat1. Limitin also induces the expression and nuclear translocation of Daxx, which is essential for IFN-alpha-induced inhibition of B-lymphocyte development. The absence of Tyk2 abrogates this induction of Daxx expression and nuclear translocation. CONCLUSIONS: Limitin suppresses B-cell growth through activation of Tyk2, resulting in the up-regulation and nuclear translocation of Daxx. This limitin-mediated signaling pathway does not require Stat1.     

Physical and functional interactions between Stat5 and the tyrosine- phosphorylated receptors for erythropoietin and interleukin-3

Erythropoietin (Epo) and interleukin-3 (IL-3) stimulate activation of the Jak2 tyrosine kinase and induce tyrosine phosphorylation and activation of Stat5. In the present study, we have shown that Epo or IL- 3 stimulation induces binding of Stat5 to the tyrosine-phosphorylated Epo receptor (EpoR) or IL-3 receptor beta subunit (betaIL3), respectively, in IL-3-dependent 32D cells expressing the EpoR. The binding of Stat5 to these cytokine receptors was shown to be rapid and transient, occurring within 1 minute of stimulation of cells and significantly decreasing after 5 minutes of cell treatment. In vivo binding experiments in COS cells showed that binding of Stat5 to the EpoR was mediated through the Stat5 Src homology 2 (SH2) domain. In vitro binding studies further showed that Stat5, but not other Stats examined, bound specifically to tyrosine-phosphorylated recombinant EpoR fusion proteins. In these in vivo and in vitro binding studies, Stat5 bound, albeit to a lesser degree, to truncated EpoR mutants in which all the intracellular tyrosines except Y-343 were removed. Furthermore, EpoR-derived synthetic phosphotyrosine peptides corresponding to Y-343, Y-401, Y-431, and Y-479 inhibited the in vitro binding of Stat5. When expressed in 32D cells, a mutant EpoR in which all the intracellular tyrosines were removed by carboxy-terminal truncation showed a significantly impaired ability to induce tyrosine phosphorylation of Stat5, particularly at low concentrations of Epo, but exhibited an increased sensitivity to Epo for growth signaling as compared with the wild-type EpoR. These results indicate that Stat5 specifically and transiently binds to the EpoR through the interaction between the Stat5 SH2 domain and specific phosphorylated tyrosines, including Y-343, in the EpoR cytoplasmic domain. It was implied that betaIL3 may also have similar Stat5 docking sites. The Stat5 docking sites in the EpoR were shown to facilitate specific activation of Stat5, which, however, may not be required for the EpoR-mediated growth signaling.