Activation of the STAT3/Acute Phase Response Factor Transcription Factor by Interleukin-5

The receptor for interleukin-5 (IL-5R) is composed of a unique a chain (IL-5Ra) expressed on eosinophils and basophils, associated with a bc subunit, which is shared by the receptors for IL-3 and granulocyte macrophagecolony stimulating factor. One of the molecular events activated via the IL-5R is the JAK/STAT signaling pathway. Recent reports have shown that IL-5 induces tyrosine phosphorylation of JAK2 followed by the subsequent cell type-specific activation of either STAT1a or STAT5. To identify additional STAT proteins activated by IL-5, we co-transfected the IL-5R with STAT cDNAs in COS cells. We found that IL-5 induces binding of STAT3 to the intercellular adhesion molecule-1 pIRE, and activates STAT3-dependent transcription. Moreover, endogenous STAT3 was tyrosine phosphorylated and activated in human IL-5-stimulated BaF3 cells ectopically expressing the human IL-5R (BaF3/IL5R). These data imply that multiple STAT proteins are involved in gene regulation by IL-5 in a cell type-specific manner. We further demonstrate using C-terminal truncations of the aand bc subunits of the IL-5R that the membrane-proximal regions of both subunits are required for STAT activation. Interestingly, a bc receptor mutant lacking intracellular tyrosine residues is able to mediate STAT3 activation, suggesting that tyrosine phosphorylation of the bc receptor is not essential for STAT3 activation

A Composite C/EBP Binding Site Is Essential for the Activity of the Promoter of the IL-3/IL-5/Granulocyte-Macrophage Colony-Stimulating Factor Receptor ßc Gene

The common ß-chain (ßc) is the main signaling component of the heterodimeric receptors for IL-3, IL-5, and GM-CSF and is primarily expressed on myeloid cells. The proximal ßc promoter is regulated by GGAA binding proteins, including PU.1, a hemopoietic specific member of the Ets family. However, it is not likely that PU.1 alone accounts for the myeloid-restricted expression of the ßc subunit. Here we describe the identification of a C/EBP binding enhancer that is located 2 kb upstream of the transcription start site. The enhancer contains two elements that bind C/EBPa and -ß in U937 cells, while C/EBPe is also bound in extracts of HL-60 cells. Importantly, deletion of the enhancer or mutation of either of one of the C/EBP sites results in a complete loss of promoter activity in cell lines as well as in primary cells, showing the importance of C/EBP members inßc gene activation. We further show that PU.1 has to cooperate with C/EBP proteins to induce bc transcription. Since the ßc is already expressed on CD341 cells, these results demonstrate that both C/EBP and PU.1 are not only important for the myeloid-specific gene regulation at later stages of myeloid differentiation

Multiple tyrosine residues in the intracellular domain of the common ß subunit of the interleukin 5 receptor are involved in activation of STAT5

Abstract In contrast to the general model of cytokine-induced JAK/STAT signaling, tyrosine phosphorylation of the IL-5R ß chain seems to be dispensable for STAT activation in cells overexpressing exogenous STAT proteins. In this study we expressed IL-5 receptor mutants in 293 cells and studied IL-5- induced endogenous STAT-dependent transcription. Our results indicate that: (a) tyrosine phosphorylation of the IL-5R ß chain is required for endogenous STAT5 activation, (b) multiple tyrosine residues are phosphorylated upon IL-5 stimulation, including Tyr^(577) , Tyr^(612) , Tyr^(695) , and Tyr^(750) , and (c) Tyr^(612) , Tyr^(695) , and Tyr^(750) are all capable of inducing activation of STAT5, demonstrating a high level of functional redundancy within the IL-5R ß chain

Differential Activation of Functionally Distinct STAT5 Proteins by IL-5 and GM-CSF During Eosinophil and Neutrophil Differentiation from Human CD34^+ Hematopoietic Stem Cells

Interleukin-5 (IL-5) and granulocyte macrophage-colony stimulating factor (GM-CSF) are important cytokines for the proliferation, differentiation, and acti-vation of myeloid lineages. The JAK/STAT pathway is one of the signaling pathways implicated in mediating biological responses induced by these cytokines. Previous studies have demonstrated that these cytokines predomi-nantly activate an 80 kDa STAT5 isoform in mature granulocytes. To better understand the role of STAT pro-teins during growth and differentiation of granulocytes, we evaluated differentiation of human CD34^+ hematopoi-etic stem cells ex vivo toward eosinophils and neutrophils. Bandshift experiments showed that in an early stage of both differentiation pathways (14 days), the 94 kDa STAT5B protein was activated by both IL-5 (eosino-phil lineage) and GM-CSF (neutrophil lineage). How-ever, during maturation of both lineages (days 21 and 28), increased expression of a functionally distinct 80 kDa STAT5 isoform was observed, resulting in het-erodimer DNA-binding complexes containing both the 94 and 80 kDa STAT5 proteins. The finding that functionally distinct isoforms of STAT5 are activated during the early and late differentiation stages of granulocytes suggests that they might be involved in regulating different biological functions in these cells

Activation of 12-O-Tetradecanoylphorbol-13-acetate Response Element- and Dyad Symmetry Element-dependent Transcription by Interleukin-5 Is Mediated by Jun N-terminal Kinase/Stress-activated Protein Kinase Kinases

Interleukin-5 (IL-5) is one of the major regulators of eosinophilic granulocytes in vivo. IL-5 exerts its pleiotropic effects by binding to the IL-5 receptor, which is composed of an IL-5-specific a chain and a common bc chain shared with the receptors for IL-3 and granulocyte- macrophage colony-stimulating factor. Previous studies have shown that binding of IL-5 to its receptor triggers the activation of multiple signaling cascades, including the Ras/mitogen-activated protein kinase, the phosphatidyl -3`-kinase, and the Janus kinase/signal transducer and activator of transcription pathways. Here we describe that IL-5 activates the serine/threonine protein kinase Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) pathway. We show that IL-5 activates TPA response element (TRE)-dependent transcription in transfection experiments. TRE activation by IL-5 is mediated by a region of the bc (577- 581) that is also responsible for activation of JNK/SAPK and for activation of dyad symmetry element (DSE)-dependent transcription. Dominant-negative SAPK or ERK kinase-1 was used to demonstrate that JNK/SAPK activation is necessary for induction of DSE- and TREdependent transcription by IL-5, whereas extracellular signal-regulated kinase 2 was not essential for TRE- and DSE-dependent transcription. By contrast, IL-5-induced activation of the tyrosine kinase Janus kinase 2 seems to be a prerequisite for TRE- and DSE-dependent transcription. Taken together, we show for the first time that IL-5 activates kinases of the JNK/SAPK family, and that this activation is linked to IL-5-induced TRE- and DSE-dependent transcription

Stem cell factor induces phosphatidylinositol 3'-kinase-dependent Lyn/Tec/Dok-1 complex formation in hematopoietic cells

Stem cell factor (SCF) has an important role in the proliferation, differentiation, survival, and migration of hematopoietic cells. SCF exerts its effects by binding to cKit, a receptor with intrinsic tyrosine kinase activity. Activation of phosphatidylinositol 3'-kinase (PI3-K) by cKit was previously shown to contribute to many SCF-induced cellular responses. Therefore, PI3-K-dependent signaling pathways activated by SCF were investigated. The PI3-K-dependent activation and phosphorylation of the tyrosine kinase Tec and the adapter molecule p62Dok-1 are reported. The study shows that Tec and Dok-1 form a stable complex with Lyn and 2 unidentified phosphoproteins of 56 and 140 kd. Both the Tec homology and the SH2 domain of Tec were identified as being required for the interaction with Dok-1, whereas 2 domains in Dok-1 appeared to mediate the association with Tec. In addition, Tec and Lyn were shown to phosphorylate Dok-1, whereas phosphorylated Dok-1 was demonstrated to bind to the SH2 domains of several signaling molecules activated by SCF, including Abl, CrkL, SHIP, and PLCgamma-1, but not those of Vav and Shc. These findings suggest that p62Dok-1 may function as an important scaffold molecule in cKit-mediated signaling

An AP-1 site in the promoter of the human IL-5RK gene is necessary for promoter activity in eosinophilic HL60 cells

Interleukin-5 (IL-5) plays a crucial role in the proliferation, differentiation and activation of eosinophils. The IL-5 receptor is composed of an IL-5-specific a subunit, which is expressed by eosinophils and basophils, and a ßc-subunit shared with the receptors for IL-3 and GM-CSF. We identified an AP-1 element which is important for IL-5Ra promoter activity in eosinophilic HL60 cells. The AP-1 site and the previously identified EOS1 site cooperate, since single mutation of either of the sites decreased promoter activity. We show that the AP-1 site of the IL-5Ra promoter binds multiple proteins, including cJun, CREB, and CREM

STAT3ß, a Splice Variant of Transcription Factor STAT3, Is a Dominant Negative Regulator of Transcription

The 89-kDa STAT3 protein is a latent transcription factor which is activated in response to cytokines (interleukin (IL)-5 and -6) and growth factors (epidermal growth factor). Binding of IL-5 to its specific receptor activates JAK2 which leads to the tyrosine phosphorylation of STAT3 proteins. Here we report the cloning of a cDNA encoding a variant of the transcription factor STAT3 (named STAT3b) which was isolated by screening an eosinophil cDNA library. Compared to wild-type STAT3, STAT3b lacks an internal domain of 50 base pairs located near the C terminus. This splice product is a naturally occurring isoform of STAT3 and encodes a 80-kDa protein. We found by reconstitution of the human IL-5R in COS cells that like STAT3, STAT3bis phosphorylated on tyrosine and binds to the pIRE from the ICAM-1 promoter after IL-5 stimulation. However, STAT3b fails to activate a pIRE containing promoter in transient transfection assays. Instead, co-expression of STAT3binhibits the transactivation potential of STAT3. These results suggests that STAT3b functions as a negative regulator of transcription

Tyrosine kinase receptor RON functions downstream of the erythropoietin

Erythropoietin (EPO) is required for cell survival during differentiation and for progenitor expansion during stress erythropoiesis. Although signaling pathways may couple directly to docking sites on the EPO receptor (EpoR), additional docking molecules expand the signaling platform of the receptor. We studied the roles of the docking molecules Grb2-associated binder-1 (Gab1) and Gab2 in EPO-induced signal transduction and erythropoiesis. Inhibitors of phosphatidylinositide 3-kinase and Src kinases suppressed EPO-dependent phosphorylation of Gab2. In contrast, Gab1 activation depends on recruitment and phosphorylation by the tyrosine kinase receptor RON, with which it is constitutively associated. RON activation induces the phosphorylation of Gab1, mitogen-activated protein kinase (MAPK), and protein kinase B (PKB) but not of signal transducer and activator of transcription 5 (Stat5). RON activation was sufficient to replace EPO in progenitor expansion but not in differentiation. In conclusion, we elucidated a novel mechanism specifically involved in the expansion of erythroblasts involving RON as a downstream target of the Epo

FoxO3a regulates erythroid differentiation and induces BTG1, an activator of protein arginine methyl transferase 1

Erythropoiesis requires tight control of expansion, maturation, and survival of erythroid progenitors. Because activation of phosphatidylinositol-3-kinase (PI3K) is required for erythropoietin/stem cell factor–induced expansion of erythroid progenitors, we examined the role of the PI3K-controlled Forkhead box, class O (FoxO) subfamily of Forkhead transcription factors. FoxO3a expression and nuclear accumulation increased during erythroid differentiation, whereas untimely induction of FoxO3a activity accelerated differentiation of erythroid progenitors to erythrocytes. We identified B cell translocation gene 1 (BTG1)/antiproliferative protein 2 as a FoxO3a target gene in erythroid progenitors. Promoter studies indicated BTG1 as a direct target of FoxO3a. Expression of BTG1 in primary mouse bone marrow cells blocked the outgrowth of erythroid colonies, which required a domain of BTG1 that binds protein arginine methyl transferase 1. During erythroid differentiation, increased arginine methylation coincided with BTG1 expression. Concordantly, inhibition of methyl transferase activity blocked erythroid maturation without affecting expansion of progenitor cells. We propose FoxO3a-controlled expression of BTG1 and subsequent regulation of protein arginine methyl transferase activity as a novel mechanism controlling erythroid expansion and differentiation