Interleukin-17 induces rapid tyrosine phosphorylation and activation of raf-1 kinase in human monocytic progenitor cell line U937.

Interleukin-17 is a T cell derived pro-inflammatory cytokine exhibiting multiple biological activities in a variety of cells and believed to fine tune all general phases of hematopoietic response. However, the signaling mechanism of this novel cytokine remains unknown. The purpose of this study was to determine whether Interleukin-17 induces tyrosine phosphorylation of proteins and to find out whether the raf-1 kinase signaling pathway is involved in mediating its signaling. Using immunoblotting and immunocomplex kinase assays, we report that the early signaling events triggered by rhIL-17 involves rapid tyrosine phosphorylation of several cellular proteins including raf-1 within 0.5 to 30 min. Optimal stimulation of tyrosine phosphorylation was observed with 0.5 to 1.0 ng/ml of Interleukin-17. Further, Interleukin-17 stimulates rapid activation of raf-1 kinase. These findings provide the first evidence that the mechanism of IL-17 signaling involves rapid tyrosine phosphorylation and activation of raf-1 serine/threonine kinase.     

Mechanism of STAT3 activation by insulin-like growth factor I receptor

Recent evidence indicates that STAT proteins can be activated by a variety of receptor and non-receptor protein-tyrosine kinases. Unlike cytokine-induced activation of STATs, where JAKs are known to play a pivotal role in phosphorylating STATs, the mechanism for receptor protein-tyrosine kinase-mediated activation of STATs remains elusive. In this study, we investigated the activation of STAT proteins by the insulin-like growth factor I receptor (IGF-IR) in vitro and in vivo and assessed the role of JAKs in the process of activation. We found that STAT3, but not STAT5, was activated in response to IGF-I in 293T cells cotransfected with IGF-IR and STAT expression vectors. Moreover, tyrosine phosphorylation of STAT3, JAK1, and JAK2 was increased upon IGF-I stimulation of endogenous IGF-IR in 293T cells transfected with the respective STAT or JAK expression vector. Supporting the observation in 293T cells, endogenous STAT3 was tyrosine-phosphorylated upon IGF-I stimulation in the muscle cell line C2C12 as well as in various embryonic and adult mouse organs during different stages of development. Dominant-negative JAK1 or JAK2 was able to block the IGF-IR-mediated tyrosine phosphorylation of STAT3 in 293T cells. A newly identified family of proteins called SOCS (suppressor of cytokine signaling), including SOCS1, SOCS2, SOCS3 and CIS, was able to inhibit the IGF-I-induced STAT3 activation as well with varying degrees of potency, in which SOCS1 and SOCS3 appeared to have the higher inhibitory ability. Inhibition of STAT3 activation by SOCS could be overcome by overexpression of native JAK1 and JAK2. We conclude that IGF-I/IGF-IR is able to mediate activation of STAT3 in vitro and in vivo and that JAKs are essential for the process of activation.     

Thrombopoietin signal transduction requires functional JAK2, not TYK2

The Janus family of tyrosine kinases (JAKs) plays a critical role in signal transduction by members of the cytokine receptor superfamily. In response to ligand-receptor interaction, these nonreceptor tyrosine kinases are rapidly phosphorylated and activated, triggering tyrosine phosphorylation and activation of downstream signaling intermediates. Upon binding to its receptor, the product of the proto-oncogene c-mpl, thrombopoietin (TPO) activates both JAK2 and TYK2 in multiple cell lines as well as megakaryocytes and platelets. To study whether one or both of these kinases are essential for TPO signal transduction, we engineered a parental human sarcoma cell line (2C4) as well as sarcoma cell lines that are deficient in JAK2 expression (gamma2A) or TYK2 expression (U1A) to express the wild-type Mpl receptor. The ability of TPO to induce tyrosine phosphorylation of Mpl and multiple intracellular substrates in each cell line was then examined. Our results demonstrate that JAK2-deficient cells (gamma2A-Mpl) are unable to initiate TPO-mediated signaling. In contrast, cells that are TYK2-deficient (U1A-Mpl) are able to induce tyrosine phosphorylation of Mpl, JAK2, STAT3, and Shc as efficiently as parental cells (2C4-Mpl). These data indicate that JAK2 is an essential component of Mpl signaling and that, in the absence of JAK2, TYK2 is incapable of initiating TPO-induced tyrosine phosphorylation.     

Icariside II induces apoptosis in U937 acute myeloid leukemia cells: role of inactivation of STAT3-related signaling

Background

The aim of this study is to determine anti-cancer effect of Icariside II purified from the root of Epimedium koreanum Nakai on human acute myeloid leukemia (AML) cell line U937.

Methodology/Principal Findings

Icariside II blocked the growth U937 cells in a dose- and time-dependent manner. In this anti-proliferation process, this herb compound rendered the cells susceptible to apoptosis, manifested by enhanced accumulation of sub-G1 cell population and increased the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells. Icariside II was able to activate caspase-3 and cleaved poly (ADP-ribose) polymerase (PARP) in a time-dependent manner. Concurrently, the anti-apoptotic proteins, such as bcl-x_L and survivin in U937 cells, were downregulated by Icariside II. In addition, Icariside II could inhibit STAT3 phosphorylation and function and subsequently suppress the activation of Janus activated kinase 2 (JAK2), the upstream activators of STAT3, in a dose- and time-dependent manner. Icariside II also enhanced the expression of protein tyrosine phosphatase (PTP) SH2 domain-containing phosphatase (SHP)-1, and the addition of sodium pervanadate (a PTP inhibitor) prevented Icariside II-induced apoptosis as well as STAT3 inactivation in STAT3 positive U937 cells. Furthermore, silencing SHP-1 using its specific siRNA significantly blocked STAT3 inactivation and apoptosis induced by Icariside II in U937 cells.

Conclusions/Significance

Our results demonstrated that via targeting STAT3-related signaling, Icariside II sensitizes U937 cells to apoptosis and perhaps serves as a potent chemotherapeutic agent for AML.     

Leptin stimulates both JAK2-dependent and JAK2-independent signaling pathways

Leptin controls body weight by activating the long form of the leptin receptor (LEPRb). Janus kinase 2 (JAK2) is associated with LEPRb and autophosphorylates in response to leptin. JAK2 also phosphorylates LEPRb, STAT3, and multiple other downstream molecules. Surprisingly, here we show that JAK2 is not required for leptin stimulation of STAT3 phosphorylation. Leptin time- and dose-dependently stimulated tyrosine phosphorylation of STAT3 in both human and mouse JAK2-null cells. Leptin also increased the viability of JAK2-null cells. Overexpression of c-Src or Fyn, two Src family members, promoted STAT3 phosphorylation, whereas inhibition of the endogenous Src family members by either pharmacological inhibitors or dominant negative Src(K298M) decreased the ability of leptin to stimulate the phosphorylation of STAT3 and ERK1/2. Leptin also stimulated tyrosine phosphorylation of kinase-inactive JAK2(K882E) in JAK2-null cells. Overexpression of JAK2(K882E) enhanced the ability of leptin to stimulate STAT3 phosphorylation in JAK2-null cells. Tyr1138 in LEPRb was required for leptin-stimulated phosphorylation of STAT3 but not JAK2(K882E). These data suggest that leptin stimulates non-JAK2 tyrosine kinase(s), including the Src family members, which phosphorylate JAK2, STAT3, and other molecules downstream of LEPRb. JAK2 mediates leptin signaling by both phosphorylating its substrates and forming a signaling complex as a scaffolding/adaptor protein. The non-JAK2 kinase(s) and JAK2 may act coordinately and synergistically to mediate leptin response.     

Negative regulation of growth hormone receptor/JAK2 signaling by signal regulatory protein alpha

Signal regulatory proteins (SIRPs) are receptor-like transmembrane proteins, the majority of which contain a cytoplasmic proline-rich region and four cytoplasmic tyrosines that, when phosphorylated, bind SH2 domain-containing protein tyrosine phosphatases (SHP). We demonstrated previously that growth hormone (GH) induces tyrosyl phosphorylation of SIRPalpha and association of SIRPalpha with SHP-2. The GH-activated tyrosine kinase JAK2 associates with and tyrosyl-phosphorylates SIRPalpha1. Here we show that JAK2-SIRPalpha1 association does not require phosphotyrosines in SIRPalpha1 or JAK2 or the proline-rich region of SIRPalpha1. However, when the C-terminal 30 amino acids of SIRPalpha1 containing the proline-rich region and tyrosine 495 are deleted, tyrosyl phosphorylation of SIRPalpha1 by JAK2 and association of SHP-2 with SIRPalpha1 are reduced. GH-dependent tyrosyl phosphorylation of JAK2 is reduced when wild-type SIRPalpha1 compared with SIRPalpha1 lacking the four cytoplasmic tyrosines (SIRP 4YF) is expressed in cells, suggesting that SIRPalpha1 negatively regulates GHR/JAK2 signaling. Consistent with reduced JAK2 activity, overexpression of wild-type SIRPalpha1 but not SIRP 4YF reduces GH-induced phosphorylation of ERKs 1 and 2, STAT3, and STAT5B. These results suggest that SIRPalpha1 is a negative regulator of GH signaling and that the ability of SIRPalpha1 mutants to negatively regulate GHR-JAK2 signaling correlates with their ability to bind SHP-2.     

Oncostatin M induces proliferation of human adipose tissue-derived mesenchymal stem cells

Interleukin-6 (IL-6) subfamily of cytokines, including oncostatin M (OSM), leukemia inhibitory factor (LIF), and IL-6, has been implicated in a variety of physiological responses, such as cell growth, differentiation, and inflammation. In the present study, we demonstrated that both OSM and LIF stimulated the proliferation of human adipose tissue-derived mesenchymal stem cells (hATSCs), however, IL-6 had no effect on cell proliferation. OSM treatment induced phosphorylation of ERK, and pretreatment with U0126, a MEK inhibitor, prevented the OSM-stimulated proliferation of hATSCs, suggesting that the MEK/ERK pathway is involved in the OSM-induced proliferation. Treatment with OSM also induced phosphorylation of JAK2 and JAK3, and pretreatment of the cells with WHI-P131, a JAK3 inhibitor, but not with AG490, a JAK2 inhibitor, attenuated the OSM-induced proliferation of hATSCs. Furthermore, OSM treatment elicited phosphorylation of STAT1 and STAT3, and pretreatment with WHI-P131 specifically prevented the OSM-induced phosphorylation of STAT1, without affecting the OSM-induced phosphorylation of ERK and STAT3. These results suggest that two separate signaling pathways, such as MEK/ERK and JAK3/STAT1, are independently involved in the OSM-stimulated proliferation of hATSCs.     

H2O2-induced activation of transcription factors STAT1 and STAT3: the role of EGF receptor and tyrosine kinase JAK2

Reactive oxygen species initiate multiple signal transduction pathways including tyrosine kinase signaling. Here, we demonstrate tyrosine phosphorylation of EGF receptor, STAT3, and, to a lesser extent, STAT1 upon H2O2 treatment of HER14 cells (NIH3T3 fibroblasts transfected with full-length EGF receptor). Maximum phosphorylation levels were observed in 5 min of stimulation at 1-2 mM H2O2. It has been shown that the intrinsic EGF-receptor tyrosine kinase is responsible for the receptor phosphorylation upon H2O2 stimulation. STAT3 and STAT1 activation in HER14 cells was demonstrated to depend on EGF receptor kinase activity, rather than JAK2 activity, while in both K721A and CD126 cells (NIH3T3 transfected with kinase-dead EGF receptor, and EGF receptor lacking major autophosphorylation sites, respectively) STAT1 and STAT3 tyrosine phosphorylation requires JAK2 kinase activity. Furthermore, STAT3 is constitutively phosphorylated in K721A and CD126 cells, and STAT1 H2O2-stimulated activation in these cells is much more prominent than in HER14. In all the cell lines used, Src-kinase activity was demonstrated to be unnecessary for ROS-initiated phosphorylation of STATs. Herein, we postulate that EGF receptor plays a role in H2O2-induced STAT activation in HER14 cells. Our data also prompted a hypothesis of constitutive inhibition of JAK2-dependent STAT activation in this cell line.     

Src family kinase-independent signal transduction and gene induction by leukemia inhibitory factor

Members of the interleukin-6 (IL-6) family of cytokines exert their biological effects via binding to their cognate ligand-binding receptor subunit on a target cell. The subsequent recruitment of the common signal transducer glycoprotein 130 and activation of the JAK/STAT and SHP-2/Ras/mitogen-activated protein kinase (MAPK) pathways are responsible for the majority of cellular responses elicited by IL-6 cytokines. Several types of experiments suggest that the Src family of kinases (SFK) also participates in IL-6 family cytokine-mediated signaling events. SYF cells, which lack expression of SFKs Src, Yes, and Fyn, were used to determine the role of SFKs in IL-6 family cytokine signaling and gene induction. SYF and wild type (WT) control fibroblasts displayed similar activation of signaling intermediates following stimulation with leukemia inhibitory factor (LIF). LIF-stimulated tyrosine phosphorylation of SHP-2 and subsequent activation of MAPK in SYF cells were identical to that seen in LIF-stimulated WT cells. Both LIF-stimulated tyrosine phosphorylation of STAT1 and STAT3, as well as LIF-stimulated DNA binding activity of STAT-containing nuclear complexes were indistinguishable when compared in SYF and WT cells. In addition, the phosphatidylinositol 3-kinase-sensitive Akt kinase and p38 MAPK were activated by LIF in both SYF and WT cells. Furthermore, LIF-stimulated expression of c-fos, egr-1, and suppressor of cytokine signaling-3 was retained in SYF cells. The IL-6 family cytokine oncostatin M was also capable of activating MAPK, STAT3, STAT1, Akt, and p38 in both WT and SYF cells. These results demonstrate that IL-6 family cytokines can activate a full repertoire of signaling pathways and induce gene expression independent of SFKs.     

Simultaneous activation of JAK1 and JAK2 confers IL-3 independent growth on Ba/F3 pro-B cells

JAK1 and JAK2 are tyrosine kinases involved in the regulation of cell proliferation, differentiation, and survival. These proteins may play a key role in mediating the effects of the cytokine IL-3 on hematopoietic cells. IL-3 induces tyrosine phosphorylation of both JAK1 and JAK2. However, it is not clear whether the activation of JAK1, JAK2, or both is sufficient to confer factor-independent growth in IL-3 dependent cells. To address this issue, fusion proteins CD16/CD7/JAK (CDJAK), comprised of a CD16 extracellular domain, a CD7 transmembrane domain, and a JAK cytoplasmic region (either a wild-type JAK or a dominant negative mutant of JAK) were constructed. We established several Ba/F3 derivatives that stably overexpress the conditionally active forms of either CDJAK1, CDJAK2, or both these fusion proteins. In this study, the autophosphorylation of CDJAK1 or CDJAK2 was induced by crosslinking with anti-CD16 antibody. We demonstrated that, like their wild-type counterparts, CDJAK1 and CDJAK2 were preassociated with the IL-3 receptor beta and alpha subunits, respectively. Furthermore, the simultaneous activation of both CDJAK1 and CDJAK2 fusion proteins, but not either one alone, led to the tyrosine phosphorylation of the IL-3 receptor beta subunit, the activation of downstream signaling molecules, including STAT5, Akt, and MAPK, and the conferring of factor-independent growth to IL-3-dependent Ba/F3 cells. Coexpression of dominant negative mutants CDJAK1KE or CDJAK2KE with wild type CDJAK2 or CDJAK1, respectively, inhibited these activation activities. These results suggest that JAK1 and JAK2 must work cooperatively and not independently and that their actions are dependent on having normal kinase activity to trigger downstream signals leading to IL-3 independent proliferation and survival of Ba/F3 cells.     

The T cell protein tyrosine phosphatase is a negative regulator of janus family kinases 1 and 3

BACKGROUND: The immune response is regulated through a tightly controlled cytokine network. The counteracting balance between protein tyrosine kinase (PTK) and protein tyrosine phosphatase (PTP) activity regulates intracellular signaling in the immune system initiated by these extracellular polypeptides. Mice deficient for the T cell protein tyrosine phosphatase (TCPTP) display gross defects in the hematopoietic compartment, indicating a critical role for TCPTP in the regulation of immune homeostasis. To date, the molecular basis underlying this phenotype has not been reported. RESULTS: We have identified two members of the Janus family of tyrosine kinases (JAKs), JAK1 and JAK3, as bona fide substrates of TCPTP. Inherent substrate specificity in the TCPTP-JAK interaction is demonstrated by the inability of other closely related PTP family members to form an in vivo interaction with the JAKs in hematopoietic cells. In keeping with a negative regulatory role for TCPTP in cytokine signaling, expression of TCPTP in T cells abrogated phosphorylation of STAT5 following interleukin (IL)-2 stimulation. TCPTP-deficient lymphocytes treated with IL-2 had increased levels of tyrosine-phosphorylated STAT5, and thymocytes treated with interferon (IFN)-alpha or IFN-gamma had increased tyrosine-phosphorylated STAT1. Hyperphosphorylation of JAK1 and elevated expression of iNOS was observed in IFN-gamma-treated, TCPTP-deficient, bone marrow-derived macrophages. CONCLUSIONS: We have identified JAK1 and JAK3 as physiological substrates of TCPTP. These results indicate a negative regulatory role for TCPTP in cytokine signaling and provide insight into the molecular defect underlying the phenotype of TCPTP-deficient animals.