Interleukin-7 inactivates the pro-apoptotic protein Bad promoting T cell survival

Interleukin-7 (IL-7) is a cytokine that is required for T cell development and survival. The anti-apoptotic function of IL-7 is partly through induction of Bcl-2 synthesis and cytosolic retention of Bax. Here we show that the Bcl-2 homology 3 domain-only protein, Bad, is involved in cell death following IL-7 withdrawal from D1 cells, an IL-7-dependent murine thymocyte cell line. IL-7 stimulation resulted in the inactivation of Bad by phosphorylation at Ser-112, -136, and -155. The phosphoinositide 3-kinase (PI3K)/Akt pathway has been implicated previously in Bad phosphorylation. In response to IL-7, the PI3K/Akt pathway induced phosphorylation at Ser-136 and -155, but Ser-112 was partly independent of the PI3K/Akt pathway, indicating an as yet unknown pathway in this response. Following IL-7 withdrawal, dephosphorylated Bad translocated from cytosol to mitochondria, bound to Bcl-2, and accelerated cell death. Thus, the inactivation of Bad contributes to the survival function of IL-7.     

Interleukin-6 inhibits transforming growth factor-beta-induced apoptosis through the phosphatidylinositol 3-kinase/Akt and signal transducers and activators of transcription 3 pathways.

The multifunctional cytokine interleukin-6 (IL-6) regulates growth and differentiation of many cell types and induces production of acute-phase proteins in hepatocytes. Here we report that IL-6 protects hepatoma cells from apoptosis induced by transforming growth factor-beta (TGF-beta), a well known apoptotic inducer in liver cells. Addition of IL-6 blocked TGF-beta-induced activation of caspase-3 while showing no effect on the induction of plasminogen activator inhibitor-1 and p15(INK4B) genes, indicating that IL-6 interferes with only a subset of TGF-beta activities. To further elucidate the mechanism of this anti-apoptotic effect of IL-6, we investigated which signaling pathway transduced by IL-6 is responsible for this effect. IL-6 stimulation of hepatoma cells induced a rapid tyrosine phosphorylation of the p85 subunit of phosphatidylinositol 3-kinase (PI 3-kinase) and its kinase activity followed by the activation of Akt. Inhibition of PI 3-kinase by wortmannin or LY294002 abolished the protection of IL-6 against TGF-beta-induced apoptosis. A dominant-negative Akt also abrogated this anti-apoptotic effect. Dominant-negative inhibition of STAT3, however, only weakly attenuated the IL-6-induced protection. Finally, inhibition of both STAT3 and PI 3-kinase by treating cells overexpressing the dominant-negative STAT3 with LY294002 completely blocked IL-6-induced survival signal. Thus, concomitant activation of the PI 3-kinase/Akt and the STAT3 pathways mediates the anti-apoptotic effect of IL-6 against TGF-beta, with the former likely playing a major role in this anti-apoptosis.     

IL-12 provides proliferation and survival signals to murine CD4+ T cells through phosphatidylinositol 3-kinase/Akt signaling pathway

IL-12 is a pleiotropic cytokine that plays an important role in innate and adaptive immunity. IL-12 induces T cell proliferation and IFN-gamma secretion from activated T cells. It was also reported that IL-12 prevents apoptosis of CD4(+) T cells. However, the signaling mechanism that regulates these IL-12-induced responses is poorly understood yet. In this study, we demonstrated that IL-12 activates phosphatidylinositol 3-kinase (PI3K)/Akt pathway in murine CD4(+) T cells, and that this signaling pathway is required for IL-12-induced T cell proliferation and antiapoptotic function, but not for IFN-gamma induction. Through PI3K/Akt pathway, IL-12 up-regulates the expression of cell cycle-related molecule such as cyclin D3, and antiapoptotic molecules such as Bcl-2 and cellular inhibitors of apoptosis proteins-2, followed by down-regulation of active caspase-3. These results suggest that PI3K/Akt pathway is critical for mediating IL-12-induced CD4(+) T cell responses such as T cell proliferation and survival.     

Thrombopoietin regulates Bcl-xL gene expression through Stat5 and phosphatidylinositol 3-kinase activation pathways.

Thrombopoietin (TPO), an essential factor for megakaryopoiesis and thrombopoiesis, works as a survival factor for megakaryocytic lineage cells. However, little is known about the molecular mechanism in detail. We show here that TPO supports the survival of TPO-dependent leukemia cell line UT-7/TPO and normal megakaryocytic progenitors via the induction of Bcl-xL, an anti-apoptotic member of the Bcl-2 family. We further analyzed the signal transduction pathways required for TPO-induced Bcl-xL gene expression. A reporter assay with various lengths of Bcl-x gene promoter revealed that both Stat- and nuclear factor kappa B-binding sites are prerequisites for TPO-induced promoter activity. Consistent with these results, TPO induced the binding of Stat5 and subunits of nuclear factor kappa B, p50, and c-Rel to the Bcl-x gene promoter. AG490, a specific inhibitor for Jak2, and LY294002, a specific inhibitor for phosphatidylinositol (PI) 3-kinase, reduced the protein level of Bcl-xL in UT-7/TPO cells, accompanied by an increase in the ratio of apoptotic cells. Interestingly, LY294002 enhanced the TPO-induced DNA binding activity of Stat5 without affecting the Jak2 activation and tyrosine phosphorylation of Stat5. Concomitantly, confocal microscopy revealed that LY294002 clearly inhibited the nuclear export of Stat5, suggesting that PI 3-kinase regulates the subcellular localization of Stat5. Taken together, our results suggest that both Jak-Stat and PI 3-kinase activation pathways regulate the TPO-induced survival of megakaryocytic cells via Bcl-xL gene expression. In addition, our data suggest possible cross-talk between these two signaling pathways.     

Phosphatidylinositol 3'-kinase, but not S6-kinase, is required for insulin-like growth factor-I and IL-4 to maintain expression of Bcl-2 and promote survival of myeloid progenitors

Phosphatidylinositol 3'-kinase (PI 3-kinase) catalyzes the formation of 3' phosphoinositides and has been implicated in an intracellular signaling pathway that inhibits apoptosis in both neuronal and hemopoietic cells. Here, we investigated two potential downstream mediators of PI 3-kinase, the serine/threonine p70 S6-kinase (S6-kinase) and the antiapoptotic protein B cell lymphoma-2 (Bcl-2). Stimulation of factor-dependent cell progenitor (FDCP) cells with either IL-4 or insulin-like growth factor (IGF)-I induced a 10-fold increase in the activity of both PI 3-kinase and S6-kinase. Rapamycin blocked 90% of the S6-kinase activity but did not affect PI 3-kinase, whereas wortmannin and LY294002 inhibited the activity of both S6-kinase and PI 3-kinase. However, wortmannin and LY294002, but not rapamycin, blocked the ability of IL-4 and IGF-I to promote cell survival. We next established that IL-3, IL-4, and IGF-I increase expression of Bcl-2 by >3-fold. Pretreatment with inhibitors of PI 3-kinase, but not rapamycin, abrogated expression of Bcl-2 caused by IL-4 and IGF-I, but not by IL-3. None of the cytokines affected expression of the proapoptotic protein Bax, suggesting that all three cytokines were specific for Bcl-2. These data establish that inhibition of PI 3-kinase, but not S6-kinase, blocks the ability of IL-4 and IGF-I to increase expression of Bcl-2 and protect promyeloid cells from apoptosis. The requirement for PI 3-kinase to maintain Bcl-2 expression depends upon the ligand that activates the cell survival pathway.     

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.     

The anti-apoptotic effect of Notch-1 requires p56lck-dependent, Akt/PKB-mediated signaling in T cells

The Notch family of transmembrane receptors have been implicated in a variety of cellular decisions in different cell types. Here we investigate the mechanism underlying Notch-1-mediated anti-apoptotic function in T cells using model cell lines as the experimental system. Ectopic expression of the intracellular domain of Notch-1/activated Notch (AcN1) increases expression of anti-apoptotic proteins of the inhibitors of apoptosis (IAP) family, the Bcl-2 family, and the FLICE-like inhibitor protein (FLIP) and inhibits death triggered by multiple stimuli that activate intrinsic or extrinsic pathways of apoptosis in human and murine T cell lines. Numb inhibited the AcN1-dependent induction of anti-apoptotic proteins and anti-apoptotic function. Using pharmacological inhibitors and dominant-negative approaches, we describe a functional role for phosphatidylinositol 3-kinase (PI3K)-dependent activation of the serine-threonine kinase Akt/PKB in the regulation of AcN1-mediated anti-apoptotic function and the expression of FLIP and IAP family proteins. Using a cell line deficient for the T cell-specific, Src family protein, the tyrosine kinase p56(lck) and by reconstitution approaches we demonstrate that p56(lck) is required for the Notch-1-mediated activation of Akt/PKB function. Furthermore, the Src tyrosine kinase inhibitor, PP2, abrogated ectopically expressed AcN1-mediated anti-apoptotic function and phosphorylation of p56(lck). We present evidence that endogenous Notch-1 associates with p56(lck) and PI3K but that Akt/PKB does not co-immunoprecipitate with the Notch1.p56(lck).PI3K complex. Finally, we demonstrate that the Notch1.p56(lck).PI3K complex is present in primary T cells that have been activated in vitro and sustained in culture with the cytokine interleukin-2.     

Anti-apoptotic signaling by the interleukin-2 receptor reveals a function for cytoplasmic tyrosine residues within the common gamma (gamma c) receptor subunit

The interleukin-2 receptor (IL-2R) is composed of one affinity-modulating subunit (IL-2Ralpha) and two essential signaling subunits (IL-2Rbeta and gammac). Although most known signaling events are mediated through tyrosine residues located within IL-2Rbeta, no functions have yet been ascribed to gammac tyrosine residues. In this study, we describe a role for gammac tyrosines in anti-apoptotic signal transduction. We have shown previously that a tyrosine-deficient IL-2Rbeta chain paired with wild type gammac stimulated enhancement of bcl-2 mRNA in IL-2-dependent T cells, but it was not determined which region of the IL-2R or which pathway was activated to direct this signaling response. Here we show that up-regulation of Bcl-2 by an IL-2R lacking IL-2Rbeta tyrosine residues leads to increased cell survival after cytokine deprivation; strikingly, this survival signal does not occur in the absence of gammac tyrosine residues. These gammac-dependent signals are revealed only in the absence of IL-2Rbeta tyrosines, indicating that the IL-2R engages at least two distinct signaling pathways to regulate apoptosis and Bcl-2 expression. Mechanistically, the gammac-dependent signal requires activation of Janus kinases 1 and 3 and is sensitive to wortmannin, implicating phosphatidylinositol 3-kinase. Consistent with involvement of phosphatidylinositol 3-kinase, Akt can be activated via tyrosine residues on gammac. Thus, gammac mediates an anti-apoptotic signaling pathway through Akt which cooperates with signals from its partner chain, IL-2Rbeta.     

Cytokine-stimulated T cells induce macrophage IL-10 production dependent on phosphatidylinositol 3-kinase and p70S6K: implications for rheumatoid arthritis

IL-10 is an anti-inflammatory cytokine produced in the joint in rheumatoid arthritis by macrophages and infiltrating blood lymphocytes. Regulation of its expression is poorly understood, but previous findings have suggested that physical interactions with T cells may play a role. This report investigates signalling mechanisms involved in the production of macrophage IL-10 upon interaction with fixed, cytokine-stimulated T cells (Tck). Elutriated monocytes were differentiated to macrophages by macrophage-colony-stimulating factor (M-CSF) and co-cultured with fixed T cells chronically stimulated in a cytokine cocktail of IL-2/IL-6/tumour necrosis factor (TNF)-α in the presence or absence of wortmannin and LY294002, inhibitors of phosphatidylinositol 3-kinase (PI3K), or of rapamycin, an inhibitor of p70 S6-kinase (p70S6K). Spontaneous IL-10 production by rheumatoid arthritis synovial-membrane mononuclear cells (RA-SMCs) and co-cultures of rheumatoid arthritis T cells (RA-Ts) and macrophages was also assessed. RA-T and Tck induction of macrophage IL-10 production was suppressed by cell separation and inhibition of PI3K and p70S6K. PI3K involvement was also shown by phosphorylation of the downstream effector protein kinase B. Spontaneous IL-10 production by RA-SMCs was also inhibited by LY294002 and depletion of the nonadherent (T-cell-enriched) fraction of the cell population. IL-10 production in RA-SMCs and M-CSF-primed macrophages, activated by interaction with Tck, is PI3K- and p70S6K-dependent.     

Cytokine-stimulated T cells induce macrophage IL-10 production dependent on phosphatidylinositol 3-kinase and p70S6K: implications for rheumatoid arthritis

IL-10 is an anti-inflammatory cytokine produced in the joint in rheumatoid arthritis by macrophages and infiltrating blood lymphocytes. Regulation of its expression is poorly understood, but previous findings have suggested that physical interactions with T cells may play a role. This report investigates signalling mechanisms involved in the production of macrophage IL-10 upon interaction with fixed, cytokine-stimulated T cells (Tck). Elutriated monocytes were differentiated to macrophages by macrophage-colony-stimulating factor (M-CSF) and co-cultured with fixed T cells chronically stimulated in a cytokine cocktail of IL-2/IL-6/tumour necrosis factor (TNF)-alpha in the presence or absence of wortmannin and LY294002, inhibitors of phosphatidylinositol 3-kinase (PI3K), or of rapamycin, an inhibitor of p70 S6-kinase (p70S6K). Spontaneous IL-10 production by rheumatoid arthritis synovial-membrane mononuclear cells (RA-SMCs) and co-cultures of rheumatoid arthritis T cells (RA-Ts) and macrophages was also assessed. RA-T and Tck induction of macrophage IL-10 production was suppressed by cell separation and inhibition of PI3K and p70S6K. PI3K involvement was also shown by phosphorylation of the downstream effector protein kinase B. Spontaneous IL-10 production by RA-SMCs was also inhibited by LY294002 and depletion of the nonadherent (T-cell-enriched) fraction of the cell population. IL-10 production in RA-SMCs and M-CSF-primed macrophages, activated by interaction with Tck, is PI3K- and p70S6K-dependent.     

Dok-1 is a positive regulator of IL-4 signalling and IgE response

Interleukin-4 (IL-4) plays an essential role in the control of humoral immunity by regulating lymphocyte proliferation and differentiation, including the T helper type 2 lineage commitment of CD4(+) T cells as well as the isotype switching to IgE in B cells. The adaptor protein Dok-1 is known to have an essential role in the negative regulation of a variety of cytokine signalling events. However, here we have found that the loss of Dok-1 impaired the proliferative response of CD4(+) T cells and B cells to IL-4. Conversely, the forced expression of Dok-1 in the myeloid cell line 32D augmented the IL-4-induced proliferation, indicating a positive role for Dok-1. Tyrosine phosphorylation, and thereby the activation of Stat6 and IRS-2, is critical for IL-4 signalling; however, only the activation of Stat6, not the IRS-2-dependent phosphorylation of Akt, was perturbed in Dok-1-deficient cells stimulated with IL-4. Furthermore, mice lacking Dok-1 showed an impaired IgE response to thymus-dependent antigen. Thus, Dok-1 is a positive regulator of IL-4 signalling and IgE response.     

Interleukin 10 modulation of tumour necrosis factor receptors requires tyrosine kinases but not the PI 3-kinase/p70 S6 kinase pathway

We have previously shown that interleukin (IL-)10-induced proliferation of the murine mast cell line D36, was dependent upon the activation of PI 3-kinase and p70 S6 kinase. Conversely, we were able to show that this pathway was not involved in the signal transduction pathway mediating IL-10 inhibition of pro-inflammatory cytokine release from monocytes. We have extended these studies to investigate the induction of p75 tumour necrosis factor receptor (TNF-R) shedding, another anti-inflammatory property of IL-10. Using the inhibitors of PI 3-kinase (LY294002 and wortmannin) and an inhibitor of p70 S6 kinase activation (rapamycin), we were able to show that this anti-inflammatory effect of IL-10 was not mediated by the PI 3-kinase/p70 S6 kinase pathway, indicating that another signalling cascade(s) was involved. Further studies also investigated the role of tyrosine kinases in the response to IL-10. Two distinct tyrosine kinase inhibitors, herbimycin and genistein affected the expression of TNF-R in response to IL-10 but, surprisingly, with opposite effects. However, both compounds inhibited the activation of both PI 3-kinase and p70 S6 kinase, with a concomitant inhibition of IL-10-induced proliferation. We observed that whilst tyrosine kinase activity was involved in the regulation of TNF-R expression, IL-10-induced activation of JAK kinases was not sensitive to inhibition by the tyrosine kinase inhibitors. These data suggest that multiple unknown tyrosine kinases are mediating the IL-10-induced signal transduction pathways leading to the regulation of TNF-R expression and IL-10-induced proliferation.     

Ubiquitous calpains promote both apoptosis and survival signals in response to different cell death stimuli

The mu- and m-calpain proteases have been implicated in both pro- or anti-apoptotic functions. Here we compared cell death responses and apoptotic or survival signaling pathways in primary mouse embryonic fibroblasts (MEFs) derived from wild type or capn4 knock-out mice which lack both mu- and m-calpain activities. Capn4(-/-) MEFs displayed resistance to puromycin, camptothecin, etoposide, hydrogen peroxide, ultraviolet light, and serum starvation, which was consistent with pro-apoptotic roles for calpain. In contrast, capn4(-/-) MEFs were more susceptible to staurosporine (STS) and tumor necrosis factor alpha-induced cell death, which provided evidence for anti-apoptotic signaling roles for calpain. Bax activation, release of cytochrome c, and activation of caspase-9 and caspase-3 all correlated with the observed cell death responses of wild type or capn4(-/-) MEFs to the various challenges, suggesting that calpain might play distinct roles in transducing different death signals to the mitochondria. There was no evidence that calpain cleaved Bcl-2 family member proteins that regulate mitochondrial membrane permeability including Bcl-2, Bcl-xl, Bad, Bak, Bid, or Bim. However, activation of the phosphatidylinositol 3 (PI3)-kinase/Akt survival signaling pathway was compromised in capn4(-/-) MEFs under all challenges regardless of the cell death outcome, and blocking Akt activation using the PI3-kinase inhibitor LY294002 abolished the protective effect of calpain to STS challenge. We conclude that the anti-apoptotic function of calpain in tumor necrosis factor alpha- and STS-challenged cells relates to a novel role in activating the PI3-kinase/Akt survival pathway.