Biomedical subjects

Isabelle Dusanter-Fourt

Publications and source records attributed to Isabelle Dusanter-Fourt.

10 recordsLinked to original sources

PubMed2005

IFN regulatory factor-2 cooperates with STAT1 to regulate transporter associated with antigen processing-1 promoter activity.

Class I MHC complexes (MHC(I)) are essential in mediating immune response. The transport of antigenic peptides (TAP) to MHC(I) and the stable expression of MHC(I) on the cell surface require the presence of a dedicated TAP. In this study we report that IFN-gamma and thrombopoietin (TPO) strongly increase TAP1 protein expression in megakaryocytes, followed by an enhanced expression of MHC(I) on the cell surface. This expression parallels the enhanced TAP1 promoter activity and TAP1 mRNA expression, which are independent of protein synthesis. We also show that this cytokine-dependent expression of TAP1 transcripts depends on STAT1 and IFN regulatory factor-2 (IRF-2), but not on IRF-1, and provide evidence that IRF-2 constitutively binds to the TAP1 gene promoter and enhances TAP1 promoter activity. We show that IRF-2 forms a complex with STAT1 and the cytokine-responsive region of the TAP1 promoter in any TPO or IFN-gamma target cells tested. Interaction of IRF-2 and STAT1 on the promoter depends on the DNA-binding domain of IRF-2. Overall, our data indicate that TPO and IFN-gamma activate the expression of TAP1 via a new mechanism that involves functional cooperation between STAT1 and IRF-2 on the TAP1 promoter.

ATP-Binding Cassette Transporters↗

PubMed2005

[Negative regulation of the JAK/STAT: pathway implication in tumorigenesis].

Cytokines, hormones or growth factors induce a variety of biological responses including proliferation, differentiation and apoptosis. After binding to their specific cell surface receptors, these stimuli induce the activation of a number of signaling pathways including the activation of JAK (JAnus Kinase) proteins by auto and transphosphorylation. Activated JAK phosphorylate the receptor chains on tyrosines, creating docking sites for cytoplasmic transcription factors named STAT (Signal Transducers and Activators of Transcription). Furthermore, the JAK phosphorylate the STAT which form dimers and migrate to the nucleus where they bind to specific DNA sequences leading to the activation of transcription. The multiplicity of JAK (4 members) and STAT (7 members) and their associations with multiple possible partners allow the formation of various STAT homo and heterodimers and STAT-containing transcriptional complexes. Each of these complexes lead to the specific regulation of gene transcription. Negative regulation of the JAK/STAT signaling pathway is crucial to switch off the cytokine/growth factors' signal. Three families of proteins : the phosphotyrosine phosphatases (SHPs, CD45, PTP1B/TC-PTP), the SOCS proteins (Suppressors Of Cytokine Signaling) and the PIAS (Protein Inhibitor of Activated STAT) are involved in this process. These proteins act at various levels of the JAK/STAT pathway. Thus, tyrosine-phosphatases dephosphorylate activated JAK, STAT or cytokine receptors. PIAS interact with activated STAT and inhibit their DNA binding or their transactivating capacity, probably in relation with their intrinsic SUMO E3-ligase activity. The tyrosine phosphatases and the PIAS are constitutively present in the cell and represent a first level of regulation. The SOCS, which represent a second level of JAK/STAT negative control, are induced by cytokines and exert a negative feed-back loop. Indeed, they interact with activated JAK or with phosphorylated receptors, inhibiting the recruitment of STAT, the activation of the JAK enzymatic activity, or inducing the proteasome-dependant degradation of activated JAK or receptors.

Apoptosis↗

PubMed2004

Differential roles of STAT1alpha and STAT1beta in fludarabine-induced cell cycle arrest and apoptosis in human B cells.

Signal transducer and activator of transcription 1 (STAT1), a transcription factor known to participate in antiviral responses, acts as a tumor suppressor inhibiting cell growth and promoting apoptosis. To study the role of STAT1 in DNA damage-induced apoptosis in B lymphocytes, its active form, STAT1alpha, was specifically inhibited by the overexpression of STAT1beta, the STAT1alpha truncated inhibitory isoform. An episomal vector with a tetracycline-inducible bidirectional promoter was created to induce the expression of 2 proteins, STAT1beta and enhanced green fluorescence protein (EGFP). The same vector was used to overexpress STAT1alpha as a control. Expression of STAT1beta inhibited the phosphorylation, the DNA-binding activity, and the transcriptional activity of STAT1alpha, as well as the expression of STAT1alpha target genes such as p21WAF1/CIP1, TAP1, IRF1, and PKR. Inhibiting STAT1alpha by STAT1beta increased the growth rate of transfected cells and their resistance to fludarabine-induced apoptosis and cell cycle arrest. Overexpressing STAT1beta reversed the negative regulation of Mdm2 expression observed after treatment with interferon-gamma (IFN-gamma), which activates STAT1, or with fludarabine. Nuclear translocation of p53 after fludarabine treatment was decreased when STAT1beta was overexpressed, and it was increased when STAT1alpha was induced. Oligonucleotide pull-down experiments showed a physical STAT1/p53 interaction. Our results show that imbalance between the antiproliferative/proapoptotic isoform STAT1alpha and the proliferative isoform STAT1beta is likely to play a crucial role in the regulation of proliferation and apoptosis and that STAT1alpha may regulate p53 activity and sensitize B cells to fludarabine-induced apoptosis.

Apoptosis↗

PubMed2004

Constitutive STAT6 activation in primary mediastinal large B-cell lymphoma.

Primary mediastinal large B-cell lymphoma (PMBL), currently recognized as a diffuse large B-cell lymphoma (DLBCL) subtype, shows increased expression of interleukin 4 (IL-4)/IL-13 signaling effectors and targets, suggesting constitutive activation of these pathways. We therefore investigated the functional state of the signal transducer and activator of transcription 6 (STAT6), mediating IL-4/IL-13 transcriptional effects. Constitutive STAT6 phosphorylation and DNA-binding activity were detected in PMBL cell lines but not DLBCL cell lines. Moreover, immunohistochemical analysis revealed nuclear phosphorylated STAT6 (P-STAT6) in 8 of 11 PMBL, compared with 1 of 10 DLBCL primary tumors (P =.01). IL-4 and IL-13 transcripts were absent in PMBL cell lines and expressed at low levels in tumors, indicating that, contrary to classical Hodgkin lymphoma (cHL), STAT6 activation is not due to an autocrine IL-4/IL-13 secretion. We demonstrated an amplification of the JAK2 gene in 2 of 6 PMBL cases, and showed higher JAK2 mRNA levels in PMBL compared with DLBCL (P =.005). The Janus kinase 2 (JAK2) was constitutively phosphorylated in the PMBL MedB1 cell line. MedB1 treatment with JAK2 inhibitor AG490 partially decreased STAT6 phosphorylation, suggesting that JAK2 is partially involved in STAT6 activation in these cells. Our findings highlight phosphorylated STAT6 as a characteristic distinguishing PMBL from DLBCL, but a common feature to PMBL and cHL, supporting the hypothesis of common pathogenic events in these 2 lymphomas.

Cell Line, Tumor↗

PubMed2004

Selective modification of eukaryotic initiation factor 4F (eIF4F) at the onset of cell differentiation: recruitment of eIF4GII and long-lasting phosphorylation of eIF4E.

mRNA translation is mainly regulated at the level of initiation, a process that involves the synergistic action of the 5' cap structure and the 3' poly(A) tail at the ends of eukaryotic mRNA. The eukaryote initiation factor 4G(eIF4G) is a pivotal scaffold protein that forms a critical link between mRNA cap structure, poly(A) tail, and the small ribosomal subunit. There are two functional homologs of eIF4G in mammals, the original eIF4G, renamed eIF4GI, and eIF4GII that functionally complements eIF4GI. To date, biochemical and functional analysis have not identified differential activities for eIF4GI and eIF4GII. In this report, we demonstrate that eIF4GII, but not eIF4GI, is selectively recruited to capped mRNA at the onset of cell differentiation. This recruitment is coincident with a strong and long-lasting phosphorylation of eIF4E and the release of 4E-BP1, a suppressor of eIF4E function, from the cap structure, without a concomitant change in 4E-BP1's phosphorylation. Our data further indicate that cytokines such as thrombopoietin can differentially regulate eIF4GI/II activities. These results provide the first evidence that eIF4GI/II does fulfill selective roles in mammalian cells.

Cell Differentiation↗

PubMed2003

Constitutive and specific activation of STAT3 by BCR-ABL in embryonic stem cells.

BCR-ABL oncogene, the molecular hallmark of chronic myelogenous leukemia (CML) arises in a primitive hematopoietic stem cell with both differentiation and self-renewal ability. To study the phenotypic effects of BCR-ABL in a clonal in vitro self-renewal and differentiation model, we have introduced BCR-ABL in the ES cell line CCE. The major effect of BCR-ABL expression was the persistence of primitive morphology of ES cells despite LIF deprivation, correlated with a constitutive activation of STAT3, the major self-renewal factor of ES cells, but no evidence of activation of STAT5. The enforced expression of BCR-ABL in an ES cell line, engineered to express a tetracycline-inducible dominant-negative form of a STAT3, triggered ES cell differentiation with an increased generation of hematopoietic cells expressing erythroid and megakaryocytic phenotypes. RT-PCR analysis for Oct4, Brachyury and beta-globin expression confirmed a delay of differentiation in BCR-ABL expressing clones, which could be entirely reversed upon activation of the dominant-negative form of STAT3. To study the possible relevance of STAT3 activation by BCR-ABL in human CML, Western blot analyses performed on the CD34+ cells, purified from CML patients at different stages of their disease, also demonstrated increased levels of STAT3 proteins phosphorylated both on tyrosine and serine residues. These results represent to our knowledge the first functional link between BCR-ABL oncogene and a self-renewal in the context of ES cells through constitutive activation of STAT3. Thus, the BCR-ABL embryonic stem cell model that we developed as well as the results obtained in human CML samples suggests a role for STAT3 in the pathogenesis of human CML.

Antigens, CD34↗

PubMed2003

STAT5 and Oct-1 form a stable complex that modulates cyclin D1 expression.

Signal transducer and activator of transcription 5 (STAT5) is activated by numerous cytokines that control blood cell development. STAT5 was also shown to actively participate in leukemogenesis. Among the target genes involved in cell growth, STAT5 had been shown to activate cyclin D1 gene expression. We now show that thrombopoietin-dependent activation of the cyclin D1 promoter depends on the integrity of a new bipartite proximal element that specifically binds STAT5A and -B transcription factors. We demonstrate that the stable recruitment of STAT5 to this element in vitro requires the integrity of an adjacent octamer element that constitutively binds the ubiquitous POU homeodomain protein Oct-1. We observe that cytokine-activated STAT5 and Oct-1 form a unique complex with the cyclin D1 promoter sequence. We find that STAT5 interacts with Oct-1 in vivo, following activation by different cytokines in various cellular contexts. This interaction involves a small motif in the carboxy-terminal region of STAT5 which, remarkably, is similar to an Oct-1 POU-interacting motif present in two well-known partners of Oct-1, namely, OBF-1/Bob and SNAP190. Our data offer new insights into the transcriptional regulation of the key cell cycle regulator cyclin D1 and emphasize the active roles of both STAT5 and Oct-1 in this process.

Animals↗

PubMed2002

The cytoplasmic domain of Mpl receptor transduces exclusive signals in embryonic and fetal hematopoietic cells.

The Mpl receptor plays an important role at the level of adult hematopoietic stem cells, but little is known of its function in embryonic and fetal hematopoiesis. We investigated the signals sent by the MPL cytoplasmic domain in fetal liver hematopoietic progenitors and during embryonic stem (ES) cell hematopoietic commitment. Mpl was found to be expressed only from day 6 of ES cell differentiation into embryoid bodies. Therefore, we expressed Mpl in undifferentiated ES cells or in fetal progenitors and studied the effects on hematopoietic differentiation. To avoid the inadvertent effect of thrombopoietin, we used a chimeric receptor, PM-R, composed of the extracellular domain of the prolactin receptor (PRL-R) and the transmembrane and cytoplasmic domains of Mpl. This allowed activation of the receptor with a hormone that is not involved in hematopoietic differentiation and assessment of the specificity of responses to Mpl by comparing PM-R with another PRL-R chimeric receptor that includes the cytoplasmic domain of the erythropoietin receptor (EPO-R) ([PE-R]). We have shown that the cytoplasmic domain of the Mpl receptor transduces exclusive signals in fetal liver hematopoietic progenitors as compared with that of EPO-R and that it promotes hematopoietic commitment of ES cells. Our findings demonstrate for the first time the specific role of Mpl in early embryonic or fetal hematopoietic progenitors and stem cells.

Animals↗

PubMed2002

Resistance to fludarabine-induced apoptosis in Epstein-Barr virus infected B cells.

The Epstein-Barr virus (EBV) transforms B cells in part by inhibiting the cellular apoptotic programme. This is also observed when Burkitt lymphoma cell lines are infected with EBV. Induction of apoptosis is one of the mechanisms by which fludarabine inhibits the growth of cells with low proliferative capacity. This compound can also inhibit several other mechanisms in the cell, including inhibition of the synthesis of factors such as STAT1. To analyse the relationship between EBV status, fludarabine-induced apoptosis, and transcription factors we studied the EBV-negative Burkitt lymphoma cell line BL2, its EBV-infected counterpart BL2.B95.8 and the EBV-transformed cell line PRI. The BL2 cell line was found to be very sensitive to fludarabine. The BL2.B95.8 and PRI cells were both resistant but the latter to a lesser extent. In the PRI cells fludarabine activated p53, but not in the BL2.B95.8 cells in which the p53 pathway is inactivated. We observed that this inactivation results in part from the lack of expression of the MDM2 inhibitor p14ARF. Conversely, there was a substantial constitutive activation of STAT1, and not of the other STATs, in the BL2.B95.8 cells and a modest one in the PRI cells. Furthermore, expression of STAT1 was significantly reduced by fludarabine treatment in the PRI cells, but not in the BL2.BL95.8 cells. Finally, the expression of p21WAF1/CIP1 was detected only in the BL2.B95.8 and PRI cells. This protein, known to play a role in cell survival, may therefore be involved in the resistance of the BL2.B95.8 cells to fludarabine.

Antineoplastic Agents↗

PubMed2002

Forced expression of p21 in GPIIb-p21 transgenic mice induces abnormalities in the proliferation of erythroid and megakaryocyte progenitors and primitive hematopoietic cells.

OBJECTIVE: p21(WAF1/Cip/kip) and p27(Kip1) are cyclin-dependant kinase inhibitors controlling cell-cycle exit and differentiation of numerous cell types. Among hematopoietic cells, megakaryocytes express high levels of p21, while in erythroid cells, p27(Kip1) is predominant. As p21 and p27 could display overlapping functions and as megakaryocytes and erythroid cells derive from a bipotent progenitor, we developed an in vivo model to determine the specific role of p21 in controlling the proliferation/differentiation balance of erythroid and megakaryocytic progenitors. METHODS: Transgenic mice that overexpressed p21 under the control of the human GPIIb promoter in early progenitors and along megakaryocytic differentiation were generated. Different subsets of hematopoietic progenitors (BFU and CFU) and primitive cells (CAFC, LTC-IC) were analyzed by methylcellulose assay. Phenotypic evolution and clonogenic properties of the lin(-) population were analyzed along erythroid and megakaryocytic differentiation. RESULTS: We observed p21 ectopic expression in early hematopoietic progenitors (lin(-)Sca(+)), megakaryocytes, and, to a lesser extent, erythroid cells. This expression induced an important decrease in the number of CFU-MK, BFU-E, CFU-E, primitive progenitors (CAFC day 35), and LTC-IC, but did not affect the maturation process of these cells and the blood cell count. CONCLUSIONS: We show that variation of p21 expression level changes the fate of hematopoietic cells by favoring either proliferation or differentiation pathways. This effect of p21 is exerted not only at the level of primitive progenitors but also in more mature progenitors. However, in vivo, a systemic compensation mechanism is most likely activated in response to variations of the flow of progenitor production.

Animals↗