IL-16 signaling specifically induces STAT6 activation through CD4

Biologic activities of IL-16 have been well described (e.g., chemotaxis of CD4+ cells, CD25 upregulation, secretion of IL-1b, IL-4 and TNF-a secretion) but very few signaling events have been described. To gain a better understanding of how the biologic activities of IL-16 are regulated following receptor engagement (CD4) we have analyzed the activation state of numerous STAT proteins in primary human peripheral blood mononuclear cells (PBMCs) and the human monocytic cell line THP-1 following IL-16 stimulation. Of the four STAT proteins tested, only STAT6 was activated (phosphorylated) in a dose-dependant manner by IL-16. The activation of STAT6 was completely abolished when IL-16 was pre-incubated with soluble CD4 (the IL-16 cell surface receptor), demonstrating the need for CD4 engagement in STAT6 activation. These results are the first to demonstrate a link between IL-16 and STAT6 activation.     

Aspirin and salicylates inhibit the IL-4- and IL-13-induced activation of STAT6.

Allergic diseases, including asthma, represent a major threat to human health. Over the three last decades, their incidence has risen in western countries. Aspirin treatment has been shown to improve allergic diseases, especially asthma, and the decreased use of aspirin has been hypothesized to contribute to the increase in childhood asthma. Because salicylate compounds suppress a number of enzymatic activities, and signaling through IL-4R participates in the development of allergic responses, we tested the effect of salicylates on IL-4 signal transduction. We found that treatment of cell lines and primary cells with aspirin and salicylates, but not acetaminophen, inhibited the activation of STAT6 by IL-4 and IL-13. This effect correlated with the inhibition of IL-4-induced CD23 expression. Although salicylates inhibited the in vivo activation of Janus kinases, their kinase activity was not affected in vitro by salicylates, suggesting that other kinases were involved in IL-4-induced STAT6 activation. Furthermore, we found that an Src kinase was involved in STAT6 activation because 1) Src kinase activity was induced by IL-4, 2) Src kinase activity, but not Janus kinase, was inhibited by salicylates in vitro, 3) cells expressing viral Src had constitutive STAT6 phosphorylation, and 4) cells lacking Src showed low STAT6 phosphorylation in response to IL-4. Because STAT6 activation by IL-4 and IL-13 participates in the development of allergic diseases, our results provide a mechanism to explain the beneficial effects of aspirin and salicylate treatment of these diseases.     

IL-4-dependent CD86 expression requires JAK/STAT6 activation and is negatively regulated by PKCdelta

CD86 expression is up-regulated in activated monocytes and macrophages by a mechanism that is not clearly defined. Here, we report that IL-4-dependent CD86 expression requires activation of ERK1/2 and JAK/STAT6 but is negatively regulated by PKCdelta. PMA differentiated U937 monocytic cells when stimulated with IL-4 increased CD11b and CD86 expression by 52- and 98-fold, respectively. PMA+IL-4 treatment also induced a synergistic enhancement of ERK1/2 activation when compared to the effects of PMA and IL-4 alone. Use of the mitogen or extracellular kinase (MEK) inhibitor, PD98059, completely blocked up-regulation of CD11b and CD86 demonstrating the importance of MEK-activated ERK1/2. JAK inhibition with WHI-P154-abrogated IL-4-dependent CD11b and CD86 up-regulation and inhibited STAT6 tyrosine phosphorylation. Importantly, CD11b and CD86 expression were not reliant on IL-4-dependent activation of phosphatidylinositol 3'-kinase (PI 3-kinase). Blockade of PKCdelta activation with rottlerin prevented CD11b expression but lead to a 75- and 213-fold increase in PMA and PMA+IL-4-dependent CD86 expression, respectively. As anticipated, increasing PKCdelta activity with anti-sense reduction of CD45 increased CD11b expression and reduced CD86 expression. Likewise, rottlerin prevented nuclear localization of activated PKCdelta. We conclude from these data that IL-4-dependent CD11b expression relies predominantly on enhanced activation of ERK1/2, while IL-4-dependent CD86 expression utilizes the JAK/STAT6 pathway.     

IL-1-mediated inhibition of IL-6-induced STAT3 activation is modulated by IL-4, MAP kinase inhibitors and redox state of HepG2 cells

It is known that during acute phase reaction IL-6 activates STAT3 in hepatoma cells and IL-1 downregulates this response. We found that the inhibitory properties of IL-1 on STAT signalling cascade in human hepatoma HepG2 cells are considerably decreased not only in the presence of MAP kinase inhibitors SB203580 and PD98059 but also by some antioxidants (N-acetyl cysteine and pyrrolidine dithiocarbamate) and by anti-inflammatory cytokine IL-4. It appears that cytokine crosstalk between IL-6 and IL-1 includes a direct pathway sensitive to antioxidants and MAP kinase inhibitors, whereas the indirect prolonged response depends probably on synthesis of suppressors of cytokine signalling (SOCS).     

In vitro inhibitory effects of cirsiliol on IL-6-induced STAT3 activation through anti-inflammatory activity

Many studies have identified and described various medicinal effects of cirsiliol. Here, we investigated the signaling pathway involved in the anti-inflammatory effects of cirsiliol on IL-6-induced activity. Cirsiliol showed no cytotoxicity and inhibited pSTAT3-induced luciferase activity. At the molecular level, cirsiliol suppressed the expression of IL-6-induced inflammatory marker genes such as CRP, IL-1β, ICAM-1 and SOCS3, IL-6-induced activation of Jak2, gp130, STAT3 and ERK and nuclear translocation of STAT3, as measured by PCR, immunofluorescence staining and western blot analysis. However, the interaction between IL-6 and its receptor was not affected by cirsiliol treatment. These results indicate that cirsiliol attenuates IL-6-induced cellular signaling by regulating Jak2 phosphorylation. Therefore, cirsiliol could be a therapeutic agent for IL-6-related inflammatory diseases.     

LPS and TNFalpha induce SOCS3 mRNA and inhibit IL-6-induced activation of STAT3 in macrophages

The human N-acetylglucosaminyltransferase I gene was introduced in the genome of Trichoderma reesei strain VTT-D-80133. Expression was studied after induction from the cellobiohydrolase I promoter. Successful in vivo transfer of GlcNAc was demonstrated by analyzing the neutral N-glycans which were synthesized on cellobiohydrolase I. Final proof of the formation of GlcNAcMan5GlcNAc2 was obtained by NMR analysis.     

IL-6-induced Bcl6 variant 2 supports IL-6-dependent myeloma cell proliferation and survival through STAT3

IL-6 is a growth and survival factor for myeloma cells, although the mechanism by which it induces myeloma cell proliferation through gene expression is largely unknown. Microarray analysis showed that some B-cell lymphoma-associated oncogenes such as Bcl6, which is absent in normal plasma cells, were upregulated by IL-6 in IL-6-dependent myeloma cell lines. We found that Bcl6 variant 2 was upregulated by STAT3. ChIP assay and EMSA showed that STAT3 bound to the upstream region of variant 2 DNA. Expression of p53, a direct target gene of Bcl6, was downregulated in the IL-6-stimulated cells, and this process was impaired by an HDAC inhibitor. Bcl6 was knocked down by introducing small hairpin RNA, resulting in decreased proliferation and increased sensitivity to a DNA damaging agent. Thus, STAT3-inducible Bcl6 variant 2 appears to generate an important IL-6 signal that supports proliferation and survival of IL-6-dependent myeloma cells.     

Interleukin-6-induced JAK2/STAT3 signaling pathway in endothelial cells is suppressed by hemodynamic flow

Endothelial cells (ECs) are constantly exposed to shear stress, the action of which triggers signaling pathways and cellular responses. During inflammation, cytokines such as IL-6 increase in plasma. In this study, we examined the effects of steady flow on IL-6-induced endothelial responses. ECs exposed to IL-6 exhibited STAT3 activation via phosphorylation of Tyr705. However, when ECs were subjected to shear stress, shear force-dependent suppression of IL-6-induced STAT3 phosphorylation was observed. IL-6 treatment increased the phosphorylation of JAK2, an upstream activator of STAT3. Consistently, shear stress significantly reduced IL-6-induced JAK2 activation. Pretreatment of ECs with an inhibitor of MEK1 did not alter this suppression by shear stress, indicating that extracellular signal-regulated kinase (ERK1/2) was not involved. However, pretreatment of ECs with an endothelial nitric oxide synthase inhibitor (nitro-L-arginine methyl ester) attenuated this inhibitory effect of shear stress on STAT3 phosphorylation. Shear stress-treated ECs displayed decreased nuclear transmigration of STAT3 and reduced STAT3 binding to DNA. Intriguingly, ECs exposed to IL-6 entered the cell cycle, as evidenced by increasing G₂/M phase, and shear stress to these ECs significantly reduced IL-6-induced cell cycle progression. STAT3-mediated IL-6-induced cell cycle was confirmed by the inhibition of the cell cycle in ECs infected with adenovirus carrying the inactive mutant of STAT3. Our study clearly shows that shear stress exerts its inhibitory regulation by suppressing the IL-6-induced JAK2/STAT3 signaling pathway and thus inhibits IL-6-induced EC proliferation. This shear force-dependent inhibition of IL-6-induced JAK2/STAT3 activation provides new insights into the vasoprotective effects of steady flow on ECs against cytokine-induced responses. shear stress; nitric oxide; cell cycle     

Novel imidazopyridine suppresses STAT3 activation by targeting SHP-1

The unregulated activation of STAT3 has been demonstrated to occur in many cancers and enhances tumour growth, migration, and invasion. Stimulation by cytokines, growth factors, and hormones triggers this activation by phosphorylating STAT3 at tyrosine 705. Novel imidazopyridine compounds were synthesized to evaluate the inhibition of STAT3 at Y705. Among the tested compounds, 16 reduced the level of phospho-STAT3, inhibited the downstream signalling cascade and subsequently attenuated the survival of hepatocellular carcinoma (HCC) cells. Further assays showed that the reduction effects of compound 16 on tyrosine 705 of STAT3 were attributed to up-regulation of protein tyrosine phosphatase SHP-1.     

IL-4 and IL-13 upregulate arginase I expression by cAMP and JAK/STAT6 pathways in vascular smooth muscle cells

The objectives ofthis study were to determine whether rat aortic smooth muscle cells(RASMC) express arginase and to elucidate the possible mechanismsinvolved in the regulation of arginase expression. The results showthat RASMC contain basal arginase I (AI) activity, which issignificantly enhanced by stimulating the cells with either interleukin(IL)-4 or IL-13, but arginase II (AII) expression was not detectedunder any condition studied here. We further investigated the signaltransduction pathways responsible for AI induction. AI mRNA and proteinlevels were enhanced by addition of forskolin (1 µM) and inhibited byH-89 (30 µM), suggesting positive regulation of AI by aprotein kinase A pathway. Genistein (10 µg/ml) and sodiumorthovanadate (Na₃VO₄; 10 µM) were used toinvestigate the role of tyrosine phosphorylation in the control of AIexpression. Genistein inhibited, whereas Na₃VO₄enhanced the induction of AI by IL-4 or IL-13. Along with immunoprecipitation and immunoblot analyses, these data implicate theJAK/STAT6 pathway in AI regulation. Dexamethasone (Dex) and interferon(IFN)- were investigated for their effects on AI induction. Dex (1 µM) and IFN- (100 U/ml) alone had no effect on basal AI expressionin RASMC, but both reduced AI induction by IL-4 and IL-13. Incombination, Dex and IFN- abolished AI induction by IL-4 and IL-13.Finally, both IL-4 and IL-13 significantly increased RASMC DNAsynthesis as monitored by [³H]thymidine incorporation,demonstrating that upregulation of AI is correlated with an increase incell proliferation. Blockade of AI induction by IFN-, H-89, orgenistein also blocked the increase in cell proliferation. Theseobservations are consistent with the possibility that upregulation ofAI might play an important role in the pathophysiology of vasculardisorders characterized by excessive smooth muscle growth.

     

IL-6 regulates in vivo dendritic cell differentiation through STAT3 activation

Dendritic cells (DCs) orchestrate immune responses according to their state of maturation. In response to infection, DCs differentiate into mature cells that initiate immune responses, while in the absence of infection, most of them remain in an immature form that induces tolerance to self Ags. Understanding what controls these opposing effects is an important goal for vaccine development and prevention of unwanted immune responses. A crucial question is what cytokine(s) regulates DC maturation in the absence of infection. In this study, we show that IL-6 plays a major role in maintaining immature DCs. IL-6 knockout (KO) mice had increased numbers of mature DCs, indicating that IL-6 blocks DC maturation in vivo. We examined this effect further in knockin mice expressing mutant versions of the IL-6 signal transducer gp130, with defective signaling through either Src homology region 2 domain-containing phosphatase 2/Gab/MAPK (gp130(F759/F759)) or STAT3 (gp130(FxxQ/FxxQ)), and combined gp130 and IL-6 defects (gp130(F759/F759)/IL-6 KO mice). Importantly, we found STAT3 activation by IL-6 was required for the suppression of LPS-induced DC maturation. In addition, STAT3 phosphorylation in DCs was regulated by IL-6 in vivo, and STAT3 was necessary for the IL-6 suppression of bone marrow-derived DC activation/maturation. DC-mediated T cell activation was enhanced in IL-6 KO mice and suppressed in gp130(F759/F759) mice. IL-6 is thus a potent regulator of DC differentiation in vivo, and IL-6-gp130-STAT3 signaling in DCs may represent a critical target for controlling T cell-mediated immune responses in vivo.