Cell type-specific roles of Jak3 in IL-2-induced proliferative signal transduction

Binding of interleukin-2 (IL-2) to its specific receptor induces activation of two members of Jak family protein tyrosine kinases, Jak1 and Jak3. An IL-2 receptor (IL-2R)-reconstituted NIH 3T3 fibroblast cell line proliferates in response to IL-2 only when hematopoietic lineage-specific Jak3 is ectopically expressed. However, the mechanism of Jak3-dependent proliferation in the fibroblast cell line is not known. Here, I showed that Jak3 expression is dispensable for IL-2-induced activation of Jak1 and Stat proteins and expression of nuclear proto-oncogenes in the IL-2R-reconstituted fibroblast cell line. Jak3 expression markedly enhanced these IL-2-induced signaling events. In contrast, Jak3 expression was essential for induction of cyclin genes involved in the G1-S transition. These data suggest a critical role of Jak3 in IL-2 signaling in the fibroblast cell line and may provide further insight into the cell type-specific mechanism of cytokine signaling.     

A membrane-proximal region of the interleukin-2 receptor gamma c chain sufficient for Jak kinase activation and induction of proliferation in T cells.

The interleukin-2 (IL-2) receptor (IL-2R) consists of three distinct subunits (alpha, beta, and gamma c) and regulates proliferation of T lymphocytes. Intracellular signalling results from ligand-mediated heterodimerization of the cytoplasmic domains of the beta and gamma c chains. To identify the residues of gamma c critical to this process, mutations were introduced into the cytoplasmic domain, and the effects on signalling were analyzed in the IL-2-dependent T-cell line CTLL2 and T-helper clone D10, using chimeric IL-2R chains that bind and are activated by granulocyte-macrophage colony-stimulating factor. Whereas previous studies of fibroblasts and transformed T cells have suggested that signalling by gamma c requires both membrane-proximal and C-terminal subdomains, our results for IL-2-dependent T cells demonstrate that the membrane-proximal 52 amino acids are sufficient to mediate a normal proliferative response, including induction of the proto-oncogenes c-myc and c-fos. Although gamma c is phosphorylated on tyrosine upon receptor activation and could potentially interact with downstream molecules containing SH2 domains, cytoplasmic tyrosine residues were dispensable for mitogenic signalling. However, deletion of a membrane-proximal region conserved among other cytokine receptors (cytoplasmic residues 5 to 37) or an adjacent region unique to gamma c (residues 40 to 52) abrogated functional interaction of the receptor chain with the tyrosine kinase Jak3. This correlated with a loss of all signalling events analyzed, including phosphorylation of the IL-2R beta-associated kinase Jak1, expression of c-myc and c-fos, and induction of the proliferative response. Thus, it appears in T cells that Jak3 is a critical mediator of mitogenic signaling by the gamma c chain.     

Fes mediates the IL-4 activation of insulin receptor substrate-2 and cellular proliferation

Although Jak kinases are essential for initiating cytokine signaling, the role of other nonreceptor tyrosine kinases in this process remains unclear. We have examined the role of Fes in IL-4 signaling. Examination of Jak1-deficient cell lines demonstrates that Jak1 is required for the activation of Fes by IL-4. Experiments studying signaling molecules activated by IL-4 receptor suggest that IL-4 signaling can be subdivided into Fes-dependent and Fes-independent pathways. Overexpression of kinase-inactive Fes blocks the IL-4 activation of insulin receptor substrate-2, but not STAT6. Fes appears to be a downstream kinase from Jak1/Jak3 in this process. Further examination of downstream signaling demonstrates that kinase-inactive Fes inhibits the recruitment of phosphoinositide 3-kinase to the activated IL-4 receptor complex and decreases the activation of p70(S6k) kinase in response to IL-4. This inhibition correlates with a decrease in IL-4-induced proliferation. In contrast, mutant Fes does not inhibit the activation of Akt by IL-4. These data demonstrate that signaling pathways activated by IL-4 require different tyrosine kinases. This differential requirement predicts that specific kinase inhibitors may permit the disruption of specific IL-4-induced functions.     

Signaling from the IL-2 receptor to the nucleus

Interleukin-2 has pleiotropic actions on the immune system and plays a vital role in the modulation of immune responses. Our current understanding of IL-2 signaling has resulted from in vitro studies that have identified the signaling pathways activated by IL-2, including the Jak-STAT pathways, and from in vivo studies that have analyzed mice in which IL-2, each chain of the receptor, as well a number of signaling molecules have been individually targeted by homologous recombination. Moreover, mutations in IL-2R, γ_c and Jak3 have been found in patients with severe combined immunodeficiency. In addition, with the discovery that two components of the receptor, IL-2Rβ and γ_c, are shared by other cytokine receptors, we have an enhanced appreciation of the contributions of these molecules towards cytokine specificity, pleiotropy and redundancy.     

Activation of the interleukin 2 receptor: a possible role for tyrosine phosphatases

Engagement of interleukin-2 (IL-2) mediates the heterodimeridation of the common beta chain (beta(c)) and common gamma chain (gamma(c)) of the IL-2 receptor (IL-2R). This is sufficient and necessary for receptor activation and signal transduction. It is generally held that the IL-2R is activated by the trans-activity of the protein tyrosine kinases (PTKs) Jak1 and Jak3 associated with beta(c) and gamma(c) respectively. Transduction of proliferative signals requires Jak3 activity. A Jak3 independent signalling pathway involving p56(lck), generating anti-apoptotic signals, can be observed and requires the PROX domain of gamma(c). p56(lck) can be activated by dephosphorylation of an inhibitory carboxyl terminal phosphorylated tyrosine residue (Y505). We propose that this is mediated by a PROX domain associated protein tyrosine phosphatase (PTP). Activation of p56(lck) alone is insufficient for transduction of proliferative signals and thus works in concert with Jak3 mediated receptor activation. This indicates that both gamma(c) domains are vital for signal transduction.     

Reconstitution of ST2 (IL-1R4) specific for IL-33 activity; no suppression by IL-1Ra though a common chain IL-1R3 (IL-1RAcP) shared with IL-1

Interleukin-33 (IL-33) receptors are composed of ST2 (also known as IL-1R4), a ligand binding chain, and IL-1 receptor accessory protein (IL-1RAcP, also known as IL-1R3), a signal transducing chain. IL-1R3 is a common receptor for IL-1α, and IL-1β, IL-33, and three IL-36 isoforms. A549 human lung epithelial cells are highly sensitive to IL-1α and IL-1β but not respond to IL-33. The lack of responsiveness to IL-33 is due to ST2 expression. ST2 was stably transfected into A549 cells to reconstitute its activity. RT-PCR and FACS analysis confirmed ST2 expression on the cell surface of A549/ST2 cells. Upon IL-33 stimulation, A549/ST2 cells induced IL-8 and IL-6 production in a dose dependent manner while A549/mock cells remained unresponsive. There was no difference in IL-1α and IL-1β activity in A549/ST2 cells compared to A549/mock cells despite the fact that IL-33 shares IL-1R3 with IL-1α/β. IL-33 activated inflammatory signaling molecules in a time- and dose-dependent manner. Anti-ST2 antibody and soluble recombinant ST2-Fc abolished IL-33-induced IL-6 and IL-8 production in A549/ST2 cells but the IL-1 receptor antagonist failed to block IL-33-induced cytokines. This result demonstrates for the first time the reconstitution of ST2 in A549 human lung epithelial cell line and verified its function in IL-33-mediated cytokine production and signal transduction.     

Interleukin (IL)-7 induces rapid activation of Pyk2, which is bound to Janus kinase 1 and IL-7Ralpha

Interleukin-7 (IL-7) receptor signaling begins with activation of the Janus tyrosine kinases Jak1 and Jak3, which are associated with the receptor complex. To identify potential targets of these kinases, we examined Pyk2 (a member of the focal adhesion kinase family) using an IL-7-dependent murine thymocyte line, D1. We demonstrate that stimulation of D1 (or normal pro-T) cells by IL-7 rapidly increased tyrosine phosphorylation and enzymatic activity of Pyk2, with kinetics slightly lagging that of Jak1 and Jak3 phosphorylation. Conversely, IL-7 withdrawal resulted in a marked decrease of Pyk2 phosphorylation. Pyk2 was found to be physically associated with Jak1 prior to IL-7 stimulation and to increase its association with IL-7Ralpha chain following IL-7 stimulation. Pyk2 appeared to be involved in cell survival, because antisense Pyk2 accelerated the cell death process. Activation of Pyk2 via the muscarinic and nicotinic receptors using carbachol or via intracellular Ca(2+) rise using ionomycin/phorbol myristate acetate promoted survival in the absence of IL-7. These data support a role for Pyk2 in coupling Jak signaling to the trophic response.     

IL-22-mediated tumor growth reduction correlates with inhibition of ERK1/2 and AKT phosphorylation and induction of cell cycle arrest in the G2-M phase

IL-22 is a recently discovered cytokine of the IL-10 family that binds to a class II cytokine receptor composed of IL-22R1 and IL-10R2(c) and influences a variety of immune reactions. As IL-22 has also been shown to modulate cell cycle and proliferation mediators such as ERK1/2 and JNK, we studied the role of IL-22 in proliferation, apoptosis, and cell cycle regulation in EMT6 murine breast cancer cells in vitro and in vivo. In this study, we report that murine breast cancer cells express functional IL-22R as indicated by RT-PCR studies, immunoblotting, and STAT3 activation assays. Importantly, IL-22 exposure of EMT6 cells resulted in decreased levels of phosphorylated ERK1/2 and AKT protein kinases, indicating an inhibitory effect of IL-22 on signaling pathways promoting cell proliferation. Furthermore, IL-22 induced a cell cycle arrest of EMT6 cells in the G(2)-M phase. IL-22 reduced EMT6 cell numbers and the proliferation rate by approximately 50% as measured by [(3)H]thymidine incorporation. IL-22 treatment of EMT6 tumor-bearing mice lead to a decreased tumor size and a reduced tumor cell proliferation in vivo, as determined by 3'-deoxy-3'-fluorothymidine-positron emission tomography scans. Interestingly, IL-22 did not induce apoptosis, as determined in annexin V binding assay and caspase-3 activation assay and had no effect on angiogenesis in vivo. In conclusion, our results indicate that IL-22 reduced tumor growth by inhibiting signaling pathways such as ERK1/2 and AKT phosphorylation that promote tumor cell proliferation in EMT6 cells. Therefore, IL-22 may play a role in the control of tumor growth and tumor progression.     

Alternate signalling pathways from the interleukin-2 receptor.

Interleukin-2 (IL-2) plays a major role in the proliferation of cell populations during an immune reaction. The beta(c) and gamma(c) subunits of the IL-2 receptor (IL-2R) are sufficient and necessary for signal transduction. Despite lacking known catalytic domains, receptor engagement leads to the activation of a diverse array protein tyrosine kinases (PTKs). In resting or anergised T cells, Jak3 is not activated. Signals arising from the PROX domain of the gamma(c) subunit activate p56(lck) (lck) leading to the induction of anti-apoptotic mechanisms. When Jak3 is activated, in primed T cells, other PTKs predominantly mediate the induction of anti-apoptotic mechanisms and drive cellular proliferation. This review intends to suggest a role for these differences within the context of the immune system.     

Epstein-Barr Virus IL-10 Engages IL-10R1 by a Two-step Mechanism Leading to Altered Signaling Properties

Human interleukin-10 (hIL-10) is a pleiotropic cytokine that is able to suppress or activate cellular immune responses to protect the host from invading pathogens. Epstein-Barr virus (EBV) encodes a viral IL-10 (ebvIL-10) in its genome that has retained the immunosuppressive activities of hIL-10 but lost the ability to induce immunostimulatory activities on some cells. These functional differences are at least partially due to the ～1000-fold difference in hIL-10 and ebvIL-10 binding affinity for the IL-10R1·IL-10R2 cell surface receptors. Despite weaker binding to IL-10R1, ebvIL-10 is more active than hIL-10 in inducing B-cell proliferation. To explore this counterintuitive observation further, a series of monomeric and dimeric ebvIL-10·hIL-10 chimeric proteins were produced and characterized for receptor binding and cellular proliferation on TF-1/hIL-10R1 cells that express high levels of the IL-10R1 chain. On this cell line, monomeric chimeras elicited cell proliferation in accordance with how tightly they bound to the IL-10R1 chain. In contrast, dimeric chimeras exhibiting the highest affinity for IL-10R1 exhibited reduced proliferative activity. These distinct activity profiles are correlated with kinetic analyses that reveal that the ebvIL-10 dimer is impaired in its ability to form a 1:2 ebvIL-10·IL-10R1 complex. As a result, the ebvIL-10 dimer functions like a monomer at low IL-10R1 levels, which prevents efficient signaling. At high IL-10R1 levels, the ebvIL-10 dimer is able to induce signaling responses greater than hIL-10. Thus, the ebvIL-10 dimer scaffold is essential to prevent activation of cells with low IL-10R1 levels but to maintain or enhance activity on cells with high IL-10R1 levels.     

Murine pro-B cells require IL-7 and its receptor complex to up-regulate IL-7R alpha, terminal deoxynucleotidyltransferase, and c mu expression

Phenotypic analysis of bone marrow cells from IL-7 knockout (KO) mice revealed that B cell development is blocked precisely at the transition between pro-B cells and pre-B cells. In contrast, the generation of pre-pro-B cells and pro-B cells appeared to be normal, as judged by total cell numbers, proliferative indexes, D-JH and V-DJH gene rearrangements, and mRNA for recombinase-activating gene-1 (RAG-1), RAG-2, TdT, Ig mu, lambda 5, and VpreB. However, upon closer inspection, several abnormalities in pro-B cell development were identified that could be corrected by injection of rIL-7 in vivo. These included the absence of the subset of late pro-B cells that initiates cmu expression for pre-B cell Ag receptor (BCR) formation, and the failure of pro-B cells to up-regulate TdT and the IL-7R alpha (but not the common gamma-chain) chain. Similar defects were present in common gamma-chain and Jak3 KO mice, but not in lambda 5 or (excluding cytoplasmic Ig mu heavy chain (c mu)) RAG-1 KO mice, all of which also arrest at the late pro-B cell stage. Consequently, up-regulation of TdT and IL-7R alpha expression requires signaling through the high affinity IL-7R, but does not require cmu expression or a functional pre-BCR. Taken together, these results suggest that IL-7 and its receptor complex are essential for 1) up-regulating the expression of TdT and IL-7R alpha, 2) initiating the production of cmu and 3) promoting the formation of a functional pre-BCR in/on pro-B cells. These key events, in turn, appear to be prerequisite both for differentiation of pro-B cells to pre-B cells and for proliferation of these cell subsets upon continued stimulation with IL-7.     

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.     

Transforming growth factor-β activates c-Myc to promote palatal growth

During palatogenesis, the palatal mesenchyme undergoes increased cell proliferation resulting in palatal growth, elevation and fusion of the two palatal shelves. Interestingly, the palatal mesenchyme expresses all three transforming growth factor (TGF) β isoforms (1, 2, and 3) throughout these steps of palatogenesis. However, the role of TGFβ in promoting proliferation of palatal mesenchymal cells has never been explored. The purpose of this study was to identify the effect of TGFβ on human embryonic palatal mesenchymal (HEPM) cell proliferation. Our results showed that all isoforms of TGFβ, especially TGFβ3, increased HEPM cell proliferation by up‐regulating the expression of cyclins and cyclin‐dependent kinases as well as c‐Myc oncogene. TGFβ activated both Smad‐dependent and Smad‐independent pathways to induce c‐Myc gene expression. Furthermore, TBE1 is the only functional Smad binding element (SBE) in the c‐Myc promoter and Smad4, activated by TGFβ, binds to the TBE1 to induce c‐Myc gene activity. We conclude that HEPM proliferation is manifested by the induction of c‐Myc in response to TGFβ signaling, which is essential for complete palatal confluency. Our data highlights the potential role of TGFβ as a therapeutic molecule to correct cleft palate by promoting growth. J. Cell. Biochem. 113: 3069–3085, 2012. © 2012 Wiley Periodicals, Inc.     

Hierarchy of protein tyrosine kinases in interleukin-2 (IL-2) signaling: activation of syk depends on Jak3; however, neither Syk nor Lck is required for IL-2-mediated STAT activation

Interleukin-2 (IL-2) activates several different families of tyrosine kinases, but precisely how these kinases interact is not completely understood. We therefore investigated the functional relationships among Jak3, Lck, and Syk in IL-2 signaling. We first observed that in the absence of Jak3, both Lck and Syk had the capacity to phosphorylate Stat3 and Stat5a. However, neither supported IL-2-induced STAT activation, nor did dominant negative alleles of these kinases inhibit. Moreover, pharmacological abrogation of Lck activity did not inhibit IL-2-mediated phosphorylation of Jak3 and Stat5a. Importantly, ligand-dependent Syk activation was dependent on the presence of catalytically active Jak3, whereas Lck activation was not. Interestingly, Syk functioned as a direct substrate of Jak1 but not Jak3. Additionally, Jak3 phosphorylated Jak1, whereas the reverse was not the case. Taken together, our data support a model in which Lck functions in parallel with Jak3, while Syk functions as a downstream element of Jaks in IL-2 signaling. Jak3 may regulate Syk catalytic activity indirectly via Jak1. However, IL-2-mediated Jak3/Stat activation is not dependent on Lck or Syk. While the essential roles of Jak1 and Jak3 in signaling by gammac-utilizing cytokines are clear, it will be important to dissect the exact contributions of Lck and Syk in mediating the effects of IL-2 and related cytokines.