Biomedical subjects

E Caldenhoven

Publications and source records attributed to E Caldenhoven.

At least 19 recordsLinked to original sources

PubMed2005

Expression profiling of colon cancer cell lines and colon biopsies: towards a screening system for potential cancer-preventive compounds.

Interest in mechanisms of colon cancer prevention by food compounds is strong and research in this area is often performed with cultured colon cancer cells. In order to assess utility for screening of potential cancer-preventive (food) compounds, expression profiles of 14 human cell lines derived from colonic tissue were measured using cDNA microarrays with 4000 genes and compared with expression profiles in biopsies of human colon tumours and normal tissue. Differences and similarities in the gene expression profiles of the cell lines were analysed by clustering and principal component analysis (PCA). Cytoskeleton genes and immune response genes are two functional classes of genes that contributed to the differences between the cell lines. A subset of 72 colon cancer-specific genes was identified by comparing expression profiles in human colon biopsies of tumour tissue and normal tissue. A separation of the cell lines based on the tumour stage of the original adenocarcinoma was observed after PCA of expression data of the subset of colon cancer-specific genes in the cell lines. The results of this study may be useful in the ongoing research into mechanisms of cancer prevention by dietary components.

Adenocarcinoma↗

PubMed2001

Avian erythroleukemia: a model for corepressor function in cancer.

Transcriptional regulation at the level of chromatin plays crucial roles during eukaryotic development and differentiation. A plethora of studies revealed that the acetylation status of histones is controlled by multi-protein complexes containing (de)acetylase activities. In the current model, histone deacetylases and acetyltransferases are recruited to chromatin by DNA-bound repressors and activators, respectively. Shifting the balance between deacetylation, i.e. repressive chromatin and acetylation, i.e. active chromatin can lead to aberrant gene transcription and cancer. In human acute promyelocytic leukemia (APL) and avian erythroleukemia (AEL), chromosomal translocations and/or mutations in nuclear hormone receptors, RARalpha [NR1B1] and TRalpha [NR1A1], yielded oncoproteins that deregulate transcription and alter chromatin structure. The oncogenic receptors are locked in their 'off' mode thereby constitutively repressing transcription of genes that are critical for differentiation of hematopoietic cells. AEL involves an oncogenic version of the chicken TRalpha, v-ErbA. Apart from repression by v-ErbA via recruitment of corepressor complexes, other repressors and corepressors appear to be involved in repression of v-ErbA target genes, such as carbonic anhydrase II (CAII). Reactivation of repressed genes in APL and AEL by chromatin modifying agents such as inhibitors of histone deacetylase or of methylation provides new therapeutic strategies in the treatment of acute myeloid leukemia.

Alpharetrovirus↗

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

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

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

Stat3 activation by Src induces specific gene regulation and is required for cell transformation.

While signal transducers and activators of transcription (STATs) were originally discovered as intracellular effectors of normal signaling by cytokines, increasing evidence also points to a role for STAT transcription factors in oncogenesis. Previous studies have demonstrated that one STAT family member, Stat3, possesses constitutively elevated tyrosine phosphorylation and DNA-binding activity in fibroblasts stably transformed by the Src oncoprotein. To determine if this Stat3 activation by Src could induce Stat3-mediated gene expression, luciferase reporter constructs based on synthetic and authentic promoters were transfected into NIH 3T3 cells. Activation of endogenous cellular Stat3 by the Src oncoprotein induced gene expression through a Stat3-specific binding element (TTCCCGAA) of the C-reactive protein gene promoter. A naturally occurring splice variant of human Stat3 protein, Stat3beta, with a deletion in the C-terminal transactivation domain abolished this gene induction in a dominant negative manner. Expression of Stat3beta did not have any effect on a reporter construct based on the c-fos serum response element, which is not dependent on Stat3 signaling, indicating that Stat3beta does not nonspecifically inhibit other signaling pathways or Src function. Transfection of vectors expressing Stat3beta together with Src blocked cell transformation by Src as measured in a quantitative focus formation assay using NIH 3T3 cells. By contrast, Stat3beta had a much less pronounced effect on focus formation induced by the Ras oncoprotein, which does not activate Stat3 signaling. In addition, three independent clones of NIH 3T3 cells stably overexpressing Stat3beta were generated and characterized, demonstrating that Stat3beta overexpression does not have a toxic effect on cell viability. These Stat3beta-overexpressing clones were shown to be deficient in Stat3-mediated signaling and refractory to Src-induced cell transformation. We conclude that Stat3 activation by the Src oncoprotein leads to specific gene regulation and that Stat3 is one of the critical signaling pathways involved in Src oncogenesis. Our findings provide evidence that oncogenesis-associated activation of Stat3 signaling is part of the process of malignant transformation.

3T3 Cells↗

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↗

PubMed1997

NF-kappa B/Rel family members regulating the ICAM-1 promoter in monocytic THP-1 cells.

A kappa B-site was identified in the promoter of the intercellular adhesion molecule-1 (ICAM-1) gene, which is involved in regulation of ICAM-1 expression by tumor necrosis factor alpha (TNF-alpha) and glucocorticoids. We now report on the transcription factors which bind and transactivate this enhancer sequence. In vitro, the ICAM-1 kappa B site appeared to bind RelA and c-Rel homodimers as well as heterodimers with NF-kappa B1, but weakly NF-kappa B1 homodimers. In addition, both RelA and c-Rel, but not NF-kappa B1, were shown to transactivate an ICAM-1 kappa B-reporter construct. In monocytic THP-1 cells TNF-alpha induced two nuclear complexes which in vitro bound to the ICAM-1 kappa B site. Using antibodies in an electrophoretic mobility supershift assay, one of these complexes was shown to contain NF-kappa B1 and RelA, and to bind with higher affinity to the consensus kappa B site in the HIV long terminal repeat. The second complex contained RelA, and exhibited higher affinity towards the ICAM-1 kappa B than to the HIV kappa B site. The glucocorticoid receptor was shown to repress activity of both the RelA homodimer and the NF-kappa B1/RelA heterodimer. We argue that in vivo RelA homodimers are likely to play a dominant role in TNF-alpha-induced ICAM-1 transcription in monocytic cells.

Animals↗

PubMed1996

Cloning and characterization of Fc alpha Rb, a novel Fc alpha receptor (CD89) isoform expressed in eosinophils and neutrophils.

The Fc receptor for IgA (Fc alpha R, CD89) is a transmembrane glycoprotein found on monocytes, macrophages, neutrophils, and eosinophils. Here we describe the characterization of a novel isoform of the Fc alpha R cloned from a human eosinophil cDNA library. This clone, Fc alpha Rb, lacks the exon encoding the transmembrane/intracellular region of wild type Fc alpha R, which is replaced by 23 new amino acids. Expression of Fc alpha Rb mRNA could be detected in eosinophils and neutrophils. IIA1.6 murine pro-B cells transfected with Fc alpha Rb cDNA secrete high levels of the protein, but also a substantial amount of Fc alpha Rb is expressed at the cell membrane. Membrane-bound Fc alpha Rb binds IgA-coated beads equally well as wild type Fc alpha R. Surface expression is not affected by phosphatidyl inositol phospholipase C, indicating that glycosyl phosphatidyl inositol-linkage of Fc alpha Rb is not likely. In IIA1.6 cells expressing Fc alpha Rb and FcR gamma, which is necessary for signal transduction by wild type Fc alpha R, no tyrosine phosphorylation or Ca(2+)-mobilization could be observed after receptor cross-linking. These results indicate that Fc alpha Rb is likely to have a different function than wild-type Fc alpha R receptor.

Amino Acid Sequence↗

PubMed1996

STAT3beta, 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 STAT3beta) which was isolated by screening an eosinophil cDNA library. Compared to wild-type STAT3, STAT3beta 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, STAT3beta is phosphorylated on tyrosine and binds to the pIRE from the ICAM-1 promoter after IL-5 stimulation. However, STAT3beta fails to activate a pIRE containing promoter in transient transfection assays. Instead, co-expression of STAT3beta inhibits the transactivation potential of STAT3. These results suggests that STAT3beta functions as a negative regulator of transcription.

Amino Acid Sequence↗

PubMed1996

Eosinophil priming by cytokines: from cellular signal to in vivo modulation.

Eosinophils play an important role in the effector phase of allergic inflammation. This review will focus on the conversion of the unprimed eosinophil phenotype in the peripheral blood of normal individuals to the primed phenotype found in the peripheral blood and tissues of allergic patients, a phenomenon called priming. Recent data on the signals initiated after cytokine receptor activation on eosinophils will be reviewed.

Calcium↗

PubMed1996

Molecular mechanisms of steroid action: a novel type of cross-talk between glucocorticoids and NF-kappa B transcription factors.

Despite the widespread use of glucocorticoids in the treatment of diseases characterized by inflammation, the molecular mechanism(s) by which these hormones exert this beneficial effect in patients with asthma remains to be elucidated. Therefore, we have studied the transcriptional regulation of intercellular adhesion molecule-1 (ICAM-1) as adhesion molecules are likely to play a causal role in inflammation in promoting cell-cell and cell-matrix interactions. We observed that in a monocytic (U937) and a bronchial epithelial (H292) cell-line dexamethasone strongly suppressed basal and induced ICAM-1 expression. Subsequent analysis of the human ICAM-1 promoter has revealed that both 12-O-tetradecanoylphorbol 13-acetate (TPA) and tumour necrosis factor-alpha (TNF-alpha) upregulate ICAM-1 expression through the presence of a nuclear factor (NF-kappa B) target sequence (TGGAAATTCC). No glucocorticoid recognition sequences are present in this promoter region and dexamethasone is still able to repress transcription when the multimerized NF-kappa B sequence is transactivated by TNF-alpha upon transfection in 293 cells. We propose that direct interaction between the glucocorticoid receptor and nuclear factor-kappa B factors is at least a partial explanation for the effects of this hormone in inflammatory diseases.

Animals↗

PubMed1995

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 alpha chain (IL-5R alpha) expressed on eosinophils and basophils, associated with a beta c subunit, which is shared by the receptors for IL-3 and granulocyte macrophage-colony 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 STAT1 alpha 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 alpha and beta c subunits of the IL-5R that the membrane-proximal STAT activation. Interestingly, a beta c receptor mutant lacking intracellular tyrosine residues is able to mediate STAT3 activation, suggesting that tyrosine phosphorylation of the beta c receptor is not essential for STAT3 activation.

Acute-Phase Reaction↗