STAT5 induces macrophage differentiation of M1 leukemia cells through activation of IL-6 production mediated by NF-kappaB p65

We recently demonstrated that STAT5 can induce a variety of biological functions in mouse IL-3-dependent Ba/F3 cells; STAT5-induced expression of pim-1, p21(WAF/Cip1), and suppressor of cytokine signaling-1/STAT-induced STAT inhibitor-1/Janus kinase binding protein is responsible for induction of proliferation, differentiation, and apoptosis, respectively. In the present study, using a constitutively active STAT5A (STAT5A1*6), we show that STAT5 induces macrophage differentiation of mouse leukemic M1 cells through a distinct mechanism, autocrine production of IL-6. The supernatant of STAT5A1*6-transduced cells contained sufficient concentrations of IL-6 to induce macrophage differentiation of parental M1 cells, and STAT3 was phosphorylated on their tyrosine residues in these cells. Treatment of the cells with anti-IL-6 blocking Abs profoundly inhibited the differentiation. We also found that the STAT5A1*6 transactivated the IL-6 promoter, which was mediated by the enhanced binding of NF-kappaB p65 (RelA) to the promoter region of IL-6. These findings indicate that STAT5A cooperates with Rel/NF-kappaB to induce production of IL-6, thereby inducing macrophage differentiation of M1 cells in an autocrine manner. In summary, we have shown a novel mechanism by which STAT5 induces its pleiotropic functions. Cytokines     

Interferon-alpha inhibits interleukin-3-induced proliferation of Ba/F3 cells in a protein kinase R-dependent manner

We have previously shown that interferon-alpha (IFN-alpha) inhibits proliferation of Ba/F3 cells by interfering with the action of the mitogen interleukin-3 (IL-3) [Cell Signal 11 (1999) 769]. Here, we have characterised the role of protein kinase R (PKR), an IFN-alpha-inducible enzyme, in the mediation of IL-3-antagonistic IFN-alpha effects. Downregulation of PKR expression by antisense oligonucleotide treatment blocked IFN-alpha-induced growth inhibition. Reduction of PKR levels and overexpression of a dominant-negative PKR mutant correlated with diminished inhibitory IFN-alpha effects on the IL-3-dependent expression of a luciferase reporter construct, GAS-luc. Furthermore, increased nuclear levels of STAT1 (bound in ISGF3 complexes) were observed in PKR-depleted cells cultured with or without IFN-alpha. Together, our data indicate an essential role of PKR in the mediation of IL-3-antagonistic IFN-alpha effects on Ba/F3 cells. They also suggests that activation of STAT1, an essential mediator of IFN effects, is insufficient for growth inhibition if PKR is not expressed.     

Type I IFN induced IL1-Ra expression in hepatocytes is mediated by activating STAT6 through the formation of STAT2: STAT6 heterodimer

The biological activities of type I interferons (IFNs) are mediated by their binding to a heterodimer receptor complex (IFNAR1 and IFNAR2), resulting in the activation of the JAK (JAK1 and TYK2)-STAT (1, 2, 3, 5 isotypes) signalling pathway. Although several studies have indicated that IFN-alpha and IFN-beta can activate complexes containing STAT6, the biological role of this activation is still unknown. We found that exposure of hepatoma cells (HuH7 and Hep3B) to IFN-alpha or IFN-beta led to the activation of STAT6. Activated STAT6 in turn induced the formation of STAT2: STAT6 complexes, which led to the secretion of IL-1Ra. The activation of STAT6 by type I IFN in hepatocytes was mediated by JAK1 and Tyk2. In addition, IFN-alpha or IFN-beta significantly enhanced the stimulatory effect of IL-1beta on production of IL-1Ra. The present study suggests a novel function of IFN-alpha and IFN-beta signalling in human hepatocytes. Our results provide evidence for the mechanism how IFN-alpha and IFN-beta modulate inflammatory responses through activation of STAT6 and production of secreted IL-1Ra.     

Distinct roles for STAT1, STAT3, and STAT5 in differentiation gene induction and apoptosis inhibition by interleukin-9.

Interleukin-9 (IL-9) activates three distinct STAT proteins: STAT1, STAT3, and STAT5. This process depends on one tyrosine of the IL-9 receptor, which is necessary for proliferation, gene induction, and inhibition of apoptosis induced by glucocorticoids. By introduction of point mutations in amino acids surrounding this tyrosine, we obtained receptors that activated either STAT5 alone or both STAT1 and STAT3, thus providing us with the possibility to study the respective roles of these factors in the biological activities of IL-9. Both mutant receptors were able to prevent apoptosis, but only the mutant that activated STAT1 and STAT3 was able to support induction of granzyme A and L-selectin. In line with these results, constitutively activated STAT5 blocked glucocorticoid-induced apoptosis. In Ba/F3 cells, significant proliferation and pim-1 induction were observed with both STAT-restricted mutants, though proliferation was lower than with the wild-type receptor. These results suggest that survival and cell growth are redundantly controlled by multiple STAT factors, whereas differentiation gene induction is more specifically correlated with individual STAT activation by IL-9.     

IL-9 Induces CCL11 Expression via STAT3 Signalling in Human Airway Smooth Muscle Cells

Background

Previous findings support the concept that IL-9 may play a significant role in mediating both pro-inflammatory and changes in airway responsiveness that characterizes the atopic asthmatic state. We previously demonstrated that human airway smooth muscle (ASM) cells express a functional IL-9R that mediate CCL11 expression. However, the signaling pathway governing this effect is not well understood.

Methodology/Principal Findings

In this study, we showed that IL-9 mediated CCL11 expression in ASM cells does not rely on STAT6 or STAT5 but on STAT3 pathway. IL-9 induced rapid STAT3 activation in primary ASM cells that was not observed in case of STAT6 or STAT5. STAT3 binding to CCL11 promoter was also observed in vivo upon IL-9 stimulation of ASM cells. Disruption of STAT3 activity with SH2 domain binding inhibitory peptide results in significant reduction of IL-9 mediated CCL11 promoter activity. DN STAT3β over-expression in ASM cells, but not Ser 727 STAT3 or STAT6 DN, abolishes IL-9 mediated CCL11 promoter activity. Finally, STAT3 but not STAT6 silenced ASM cells showed significant reduction in IL-9 mediated CCL11 promoter activity and mRNA expression.

Conclusion/Significance

Taken together, our results indicate that IL-9 mediated CCL11 via STAT3 signalling pathway may play a crucial role in airway inflammatory responses.     

Interferon-alpha inhibits Stat5 DNA-binding in IL-2 stimulated primary T-lymphocytes.

It has previously been shown that IFN-alpha is a potent inhibitor of IL-2 induced proliferation in primary T-lymphocytes, by selectively abrogating the downstream effects of IL-2 on the core cell cycle machinery regulating the G1/S transition. Theoretically this could be mediated through cross-talk between the signalling cascades activated by these cytokines, as several signalling components are known to be shared. IL-2 activates multiple signalling pathways that are important for T-cell proliferation and differentiation. In the present study, the effects of IFN-alpha on IL-2 signal transduction was investigated. The IFN-alpha induced inhibition of IL-2 induced proliferation in activated T-lymphocytes, was associated with a suppressed Jak3 protein expression as well as an inhibited prolonged Stat5 DNA binding, and a partially reduced expression of the Stat5 inducible gene IL-2R alpha. Our results provide a possible molecular link between the prominent antiproliferative effects of IFN-alpha on IL-2 induced T-cell proliferation and the signal transduction pathways emerging from the IL-2 receptor.