Biomedical subjects

R P de Groot

Publications and source records attributed to R P de Groot.

At least 19 recordsLinked to original sources

PubMed2000

The role of STATs in myeloid differentiation and leukemia.

Myeloid differentiation is a highly regulated process governed by various cytokines, such as EPO, TPO, G-CSF, IL-3, IL-5 and GM-CSF. These cytokines act in part through activation of the STAT transcription factor family. In particular, various isoforms of STAT3 and STAT5 are activated during myeloid differentiation in a cell-type and maturation-state dependent fashion. In vitro studies have shown that STAT proteins are essential for cytokine-regulated processes such as cellular proliferation, differentiation as well as survival. Similarly, various STAT knock-outs have highlighted the role of STATs in myeloid differentiation in vivo. STATs also appear to play an important role in various myeloid malignancies, which are characterized by arrested maturation and cytokine-independent proliferation of myeloid progenitors. Constitutive activation of STAT3 and/or STAT5 resulting in enhanced transcription of anti-apoptotic- cell-cycle progression genes is likely to contribute to the pathogenesis of various myeloid leukemia's. Oncogene (2000).

Animals↗

PubMed2000

STAT5-Dependent CyclinD1 and Bcl-xL expression in Bcr-Abl-transformed cells.

Signal transducers and activators of transcription (STATs) are a family of transcription factors that were originally identified as mediators of cytokine-induced gene expression. We and others have recently shown that STAT5 also plays a major role in cellular transformation by the Bcr-Abl oncogene. Here we show that the antiapoptotic bcl-xL gene product and the cell cycle regulator cyclin D1 are targets of STAT5 in Bcr-Abl-transformed cells. In the CML cell line K562 and in BaF3 cells ectopically expressing Bcr-Abl, both the cyclin D1 and bcl-x promoters are highly active. The activity of these promoters can be strongly repressed by cotransfection of a dominant negative (DN) mutant of STAT5. Moreover, the cyclin D1 and bcl-x promoters contain STAT binding sites to which STAT5 constitutively binds in Bcr-Abl transformed cells. These results suggest that STAT5 contributes to transformation by Bcr-Abl by induction of cyclin D1 and bcl-xL expression.

Apoptosis↗

PubMed2000

STAT3beta does not interfere with granulocyte colony-stimulating factor-induced neutrophilic differentiation.

INTRODUCTION: Activation of the signal transducer and activator of transcription protein STAT3 is a crucial step in granulocyte colony-stimulating factor (G-CSF)-mediated cell cycle exit and subsequent neutrophilic differentiation of myeloid precursor cells. We have recently demonstrated that this is mediated, at least in part, by upregulation of the cyclin-dependent kinase inhibitor p27(Kip1). The splice variant STAT3beta, that lacks a C-terminal serine residue implicated in the transcriptional activity of STAT3, has been shown to inhibit STAT3-mediated transcription in certain situations. STAT3beta is known to be expressed in hematopoietic cells, but its role in controlling the balance between proliferation and differentiation has not been established. MATERIALS AND METHODS: We ectopically introduced STAT3beta in differentiation-competent 32D cell transfectants expressing human wild type (WT) G-CSF receptors and studied the consequences for G-CSF-mediated responses. RESULTS: Overexpression of STAT3beta did not alter the kinetics of G-CSF-mediated neutrophilic differentiation or p27 induction in 32D/G-CSF-R WT cells. In addition, we found that p27(Kip1) promoter activity was not inhibited by STAT3beta, while inhibition of p27 transactivation by a dominant-negative STAT3 mutant could in fact be alleviated by coexpression of the beta form. CONCLUSION: These findings argue against a role of STAT3beta as a negative regulator of G-CSF-induced expression of p27 and myeloid differentiation.

Alternative Splicing↗

PubMed1999

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 beta c gene.

The common beta-chain (beta c) 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 beta c 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 beta c 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/EBP alpha and -beta in U937 cells, while C/EBP epsilon 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 in beta c gene activation. We further show that PU.1 has to cooperate with C/EBP proteins to induce beta c transcription. Since the beta c is already expressed on CD34+ 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.

Animals↗

PubMed1999

STAT5 activation by BCR-Abl contributes to transformation of K562 leukemia cells.

Signal transducers and activators of transcription (STATs) belong to a family of transcription factors that were originally identified as mediators of cytokine-induced gene expression. Recent evidence, however, has shown that certain members of the STAT family, including STAT3, are also involved in cellular transformation. Here we show that STAT5 also plays a role in cellular transformation by the BCR-Abl oncogene. In BCR-Abl transformed K562 cells, STAT5A and 5B are constitutively phosphorylated on tyrosine and are transcriptionally active. Moreover, expression of a dominant negative form of STAT5 shows that active STAT5 is necessary for the growth in soft agar of these cells. These results show that besides STAT3, STAT5 can also be involved in cellular transformation.

Cell Transformation, Neoplastic↗

PubMed1999

Leptin treatment increases suppressors of cytokine signaling in central and peripheral tissues.

Leptin concentrations are elevated in the majority of obese individuals raising the possibility that leptin resistance contributes to their obesity. Peripheral leptin administration for 48 h caused a several-fold increase in mRNA encoding the suppressors of cytokine signaling SOCS-3 and CIS in hypothalamus and peripheral tissues. Paradoxically, CIS and SOCS-3 mRNAs are also elevated in the leptin-deficient ob/ob mouse. Forced expression of CIS in insulinoma cells prevented transactivation mediated by leptin. Thus tissues continuously exposed to leptin and/or other factors associated with obesity accumulate excessive amounts of SOCS-3 and CIS which could provide a potential mechanism for leptin resistance.

Animals↗

PubMed1999

Regulation and function of protein kinase B and MAP kinase activation by the IL-5/GM-CSF/IL-3 receptor.

Interleukin (IL)-3, IL-5 and granulocyte-macrophage colony-stimulating factor (GM-CSF) regulate proliferation, differentiation and apoptosis of target cells. Receptors for these cytokines consist of a cytokine-specific alpha subunit and a common shared beta c subunit. Tyrosine phosphorylation of the beta c is thought to play a critical role in mediating signal transduction events. We have examined the effect of mutation of beta c tyrosines on the activation of multiple signal transduction pathways. Activation of protein kinase B (PKB) required JAK2 and was inhibited by dominant-negative phosphatidylinositol 3-kinase (P13K). Overexpression of JAK2 was sufficient to activate both protein kinase B (PKB) and extracellular regulated kinase-1 (ERK1). Tyrosine 577 and 612 were found to be critical for the activation of PKB and ERK1, but not activation of STAT transcription factors. Activation of both PKB and ERK have been implicated in the regulation of proliferation and apoptosis. We generated GM-CSFR stable cell lines expressing receptor mutants to evaluate their effect on these processes. Activation of both PKB and ERK was perturbed, while STAT activation remained unaffected. Tyrosines 577 and 612 were necessary for optimal proliferation, however, mutation of these tyrosine residues did not affect GM-CSF mediated rescue from apoptosis. These data demonstrate that while phosphorylation of beta c tyrosine residues 577 and 612 are important for optimal cell proliferation, rescue from apoptosis can be mediated by alternative signalling routes apparently independent of PKB or ERK activation.

Animals↗

PubMed1999

Interleukin-1beta activates a short STAT-3 isoform in clonal insulin-secreting cells.

Interleukin-1beta (IL-1beta) is a potent inflammatory cytokine involved in type 1 diabetes and acts through defined IL-1beta signaling pathways. In the present work we describe induction of DNA binding activity to signal transducer and activator of transcription (STAT) in response to IL-1beta in clonal insulin-secreting cells. Moreover, IL-1beta activates a short isoform of STAT-3 that potently stimulates transcription. Immunoprecipitation studies reveal an interaction between the activated STAT-3 and the IL-1 receptor accessory protein indicating an association between the two signaling pathways. This may be a novel point of transduction cross talk and an additional mechanism utilised by IL-1beta in the pancreatic beta-cell during the process of type 1 diabetes.

Base Sequence↗

PubMed1999

Lineage-specific activation of STAT3 by interferon-gamma in human neutrophils.

Binding of interferon-gamma (IFN-gamma) to its heterodimeric receptor induces activation of the tyrosine kinases JAK1 and JAK2 followed by tyrosine phosphorylation of STAT1alpha. Selective activation of STAT1alpha at the IFN-gamma receptor is achieved by specific interaction between a cytosolic tyrosine motif including Y440 in the IFN-gamma receptor alpha-chain and the SH2 domain of STAT1alpha. We demonstrate that, in addition to STAT1alpha, STAT3 is also activated by IFN-gamma in human neutrophils. The activation of STAT3 was not found in human eosinophils, monocytes, and HL-60 cells, although the STAT3 protein was expressed in these cells. The cell type-specific activation of STAT3 by IFN-gamma was also observed in neutrophils that are differentiated in vitro from human CD34+ hematopoietic stem cells. These results indicate that a single cytokine receptor can activate different STAT family members in a cell-specific manner, which might result in cell-specific gene transcription.

Antiviral Agents↗

PubMed1999

C/EBP regulates the promoter of the eosinophil-derived neurotoxin/RNS2 gene in human eosinophilic cells.

The eosinophil-derived neurotoxin (EDN), a member of the mammalian ribonuclease family, is found in the large specific granules of human eosinophilic leukocytes. We have investigated the role of the C/EBP transcription factor family in the regulation of EDN promoter activity. Here we show that the C/EBP family is involved in intrinsic regulation of EDN promoter activity. We have identified a C/EBP binding site located at -124 in the proximal promoter of the EDN gene. Mutation of this C/EBP site results in a decrease of promoter activity in HL-60-eos cells as well as in eosinophils differentiated in vitro from CD34+ cells. Different C/EBP proteins are able to bind to the C/EBP site as shown by gel shift assay. Our results indicate the importance of the C/EBP family in the regulation of the EDN gene in eosinophils.

Animals↗

PubMed1999

Activation of a functionally distinct 80-kDa STAT5 isoform by IL-5 and GM-CSF in human eosinophils and neutrophils.

Interleukin-5 (IL-5), IL-3, and granulocyte macrophage colony-stimulating factor (GM-CSF) are hematopoietic cytokines which signal through a common beta subunit (betac) of a heterodimeric receptor. Among the intracellular signaling pathways activated via betac is the JAK/STAT pathway. We show that different STAT5 isoforms are activated by IL-5 and GM-CSF in eosinophils, neutrophils, and differentiated eosinophilic HL-60 cells. Whereas IL-5 activated the wild-type STAT5A and STAT5B proteins in HL60-eos cells, a carboxyl-terminally truncated 80-kDa STAT5 isoform was activated in mature eosinophils and neutrophils. Surprisingly, while both isoforms bind strongly to an element from the beta-casein promoter, only p80 STAT5 binds to the ICAM1-IRE. Consequently, a carboxyl-terminal truncated STAT5 is capable of blocking STAT3-mediated transcription of an IREtkCAT reporter construct. The cell type-specific expression of these functionally distinct STAT5 isoforms might contribute to the pleiotropic effects of IL-5 and GM-CSF on different target cells.

Cell Extracts↗

PubMed1998

Cloning and characterization of the human interleukin-3 (IL-3)/IL-5/ granulocyte-macrophage colony-stimulating factor receptor betac gene: regulation by Ets family members.

High-affinity receptors for interleukin-3 (IL-3), IL-5, and granulocyte-macrophage colony-stimulating factor (GM-CSF) are composed of two distinct subunits, a ligand-specific chain and a common beta chain (betac). Whereas the mouse has two homologous beta subunits (betac and betaIL-3), in humans, only a single beta chain is identified. We describe here the isolation and characterization of the gene encoding the human IL-3/IL-5/GM-CSF receptor beta subunit. The gene spans about 25 kb and is divided into 14 exons, a structure very similar to that of the murine betac/betaIL-3 genes. Surprisingly, we also found the remnants of a second betac chain gene directly downstream of betac. We identified a functional promoter that is active in the myeloid cell lines U937 and HL-60, but not in HeLa cells. The proximal promoter region, located from -103 to +33 bp, contains two GGAA consensus binding sites for members of the Ets family. Single mutation of those sites reduces promoter activity by 70% to 90%. The 5' element specifically binds PU.1, whereas the 3' element binds a yet-unidentified protein. These findings, together with the observation that cotransfection of PU.1 and other Ets family members enhances betac promoter activity in fibroblasts, reinforce the notion that GGAA elements play an important role in myeloid-specific gene regulation.

Animals↗

PubMed1998

An AP-1 site in the promoter of the human IL-5R alpha 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 alpha subunit, which is expressed by eosinophils and basophils, and a beta c-subunit shared with the receptors for IL-3 and GM-CSF. We identified an AP-1 element which is important for IL-5R alpha 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-5R alpha promoter binds multiple proteins, including cJun, CREB, and CREM.

Base Sequence↗

PubMed1998

The role of transcription factor PU.1 in the activity of the intronic enhancer of the eosinophil-derived neurotoxin (RNS2) gene.

Eosinophil-derived neurotoxin (EDN) found in the granules of human eosinophils is a cationic ribonuclease toxin. Expression of the EDN gene (RNS2) in eosinophils is dependent on proximal promoter sequences in combination with an enhancer located in the first intron. We further define here the active region of the intron using transfections in differentiated eosinophilic HL60 cells. We show that a region containing a tandem PU.I binding site is important for intronic enhancer activity. This region binds multiple forms of transcription factor PU.I as judged by gel-shift analysis and DNA affinity precipitation. Importantly, introducing point mutations in the PU.I site drastically reduces the intronic enhancer activity, showing the importance of PU.I for expression of EDN in cells of the eosinophilic lineage.

Binding Sites↗

PubMed1998

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 activation 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 predominantly activate an 80 kDa STAT5 isoform in mature granulocytes. To better understand the role of STAT proteins during growth and differentiation of granulocytes, we evaluated differentiation of human CD34+ hematopoietic 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 (eosinophil lineage) and GM-CSF (neutrophil lineage). However, during maturation of both lineages (days 21 and 28), increased expression of a functionally distinct 80 kDa STAT5 isoform was observed, resulting in heterodimer 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.

Antigens, CD34↗

PubMed1998

Regulation of proliferation, differentiation and survival by the IL-3/IL-5/GM-CSF receptor family.

The receptors for the I1-3/IL-5/GM-CSF cytokine family are composed of a heterodimeric complex of a cytokine-specific alpha chain and a common beta chain (betac). Binding of IL-3/IL-5/GM-CSF to their respective receptors rapidly induces activation of multiple intracellular signalling pathways, including the Ras-Raf-ERK, the JAK/STAT, the phosphatidylinositol 3-kinase PKB, and the JNK/SAPK and p38 signalling pathways. This review focuses on recent advancements in understanding how these different signalling pathways are activated by IL-3/IL-5/GM-CSF receptors, and how the individual pathways contribute to the pleiotropic effects of IL-3/IL-5/GM-CSF on their target cells, including proliferation, differentiation, survival, and effector functions.

Animals↗

PubMed1997

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

In contrast to the general model of cytokine-induced JAK/STAT signaling, tyrosine phosphorylation of the IL-5R beta 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 beta chain is required for endogenous STAT5 activation, (b) multiple tyrosine residues are phosphorylated upon IL-5 stimulation, including Tyr577, Tyr612, Tyr695, and Tyr750, and (c) Tyr612, Tyr695, and Tyr750 are all capable of inducing activation of STAT5, demonstrating a high level of functional redundancy within the IL-5R beta chain.

3T3 Cells↗

PubMed1997

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 alpha chain and a common betac 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 betac (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 TRE-dependent 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.

Animals↗