Publications by authors named "Frédérique Verdier"

26 Publications

  • Page 1 of 1

Myotonic dystrophy kinase-related CDC42-binding kinase α, a new transferrin receptor type 2-binding partner, is a regulator of erythropoiesis.

Am J Hematol 2021 04 4;96(4):480-492. Epub 2021 Feb 4.

Université de Paris, Institut Cochin, INSERM U1016-CNRS UMR8104, Paris, France.

Efficient erythropoiesis relies on the expression of the transferrin receptor type 2 (TFR2). In erythroid precursors, TFR2 facilitates the export of the erythropoietin receptor (EPOR) to cell surface, which ensures the survival and proliferation of erythroblasts. Although TFR2 has a crucial role in erythropoiesis regulation, its mechanism of action remains to be clarified. To understand its role better, we aimed at identifying its protein partners by mass-spectrometry after immunoprecipitation in erythroid cells. Here we report the kinase MRCKα (myotonic dystrophy kinase-related CDC42-binding kinase α) as a new partner of both TFR2 and EPOR in erythroblasts. We show that MRCKα is co-expressed with TFR2, and TFR1 during terminal differentiation and regulates the internalization of the two types of transferrin receptors. The knockdown of MRCKα by shRNA in human primary erythroblasts leads to a decreased cell surface expression of both TFR1 and TFR2, an increased cell-surface expression of EPOR, and a delayed differentiation. Additionally, knockout of Mrckα in the murine MEDEP cells also leads to a striking delay in erythropoiesis, showcasing the importance of this kinase in both species. Our data highlight the importance of MRCKα in the regulation of erythropoiesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/ajh.26104DOI Listing
April 2021

Transferrin Receptors in Erythropoiesis.

Int J Mol Sci 2020 Dec 19;21(24). Epub 2020 Dec 19.

Inserm U1016, CNRS UMR8104, Institut Cochin, Université de Paris, 75014 Paris, France.

Erythropoiesis is a highly dynamic process giving rise to red blood cells from hematopoietic stem cells present in the bone marrow. Red blood cells transport oxygen to tissues thanks to the hemoglobin comprised of α- and β-globin chains and of iron-containing hemes. Erythropoiesis is the most iron-consuming process to support hemoglobin production. Iron delivery is mediated via transferrin internalization by the endocytosis of transferrin receptor type 1 (TFR1), one of the most abundant membrane proteins of erythroblasts. A second transferrin receptor-TFR2-associates with the erythropoietin receptor and has been implicated in the regulation of erythropoiesis. In erythroblasts, both transferrin receptors adopt peculiarities such as an erythroid-specific regulation of TFR1 and a trafficking pathway reliant on TFR2 for iron. This review reports both trafficking and signaling functions of these receptors and reassesses the debated role of TFR2 in erythropoiesis in the light of recent findings. Potential therapeutic uses targeting the transferrin-TFR1 axis or TFR2 in hematological disorders are also discussed.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/ijms21249713DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7766611PMC
December 2020

The epigenetic regulator RINF (CXXC5) maintains SMAD7 expression in human immature erythroid cells and sustains red blood cells expansion.

Haematologica 2020 11 26;Online ahead of print. Epub 2020 Nov 26.

Université de Paris, Institut Cochin, INSERM, CNRS, F-75014 Paris, France; Laboratory of Excellence GR-ex, Paris, France; Equipe Labellisée Ligue Nationale Contre le Cancer (LNCC), Paris.

The gene CXXC5, encoding a Retinoid-Inducible Nuclear Factor (RINF), is located within a region at 5q31.2 commonly deleted in myelodysplastic syndrome (MDS) and adult acute myeloid leukemia (AML). RINF may act as an epigenetic regulator and has been proposed as a tumor suppressor in hematopoietic malignancies. However, functional studies in normal hematopoiesis are lacking, and its mechanism of action is unknow. Here, we evaluated the consequences of RINF silencing on cytokineinduced erythroid differentiation of human primary CD34+ progenitors. We found that RINF is expressed in immature erythroid cells and that RINF-knockdown accelerated erythropoietin-driven maturation, leading to a significant reduction (~45%) in the number of red blood cells (RBCs), without affecting cell viability. The phenotype induced by RINF-silencing was TGFβ-dependent and mediated by SMAD7, a TGFβ- signaling inhibitor. RINF upregulates SMAD7 expression by direct binding to its promoter and we found a close correlation between RINF and SMAD7 mRNA levels both in CD34+ cells isolated from bone marrow of healthy donors and MDS patients with del(5q). Importantly, RINF knockdown attenuated SMAD7 expression in primary cells and ectopic SMAD7 expression was sufficient to prevent the RINF knockdowndependent erythroid phenotype. Finally, RINF silencing affects 5’-hydroxymethylation of human erythroblasts, in agreement with its recently described role as a Tet2- anchoring platform in mouse. Altogether, our data bring insight into how the epigenetic factor RINF, as a transcriptional regulator of SMAD7, may fine-tune cell sensitivity to TGFβ superfamily cytokines and thus play an important role in both normal and pathological erythropoiesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3324/haematol.2020.263558DOI Listing
November 2020

ARID1A loss in adult hepatocytes activates β-catenin-mediated erythropoietin transcription.

Elife 2020 10 21;9. Epub 2020 Oct 21.

INSERM, Sorbonne Université, Université de Paris, Centre de Recherche des Cordeliers (CRC), Paris, France.

Erythropoietin (EPO) is a key regulator of erythropoiesis. The embryonic liver is the main site of erythropoietin synthesis, after which the kidney takes over. The adult liver retains the ability to express EPO, and we discovered here new players of this transcription, distinct from the classical hypoxia-inducible factor pathway. In mice, genetically invalidated in hepatocytes for the chromatin remodeler , and for , the major silencer of Wnt pathway, chromatin was more accessible and histone marks turned into active ones at the downstream enhancer. Activating β-catenin signaling increased binding of Tcf4/β-catenin complex and upregulated its enhancer function. The loss of together with β-catenin signaling, resulted in cell-autonomous transcription in mouse and human hepatocytes. In mice with gene invalidations in single hepatocytes, Epo de novo synthesis led to its secretion, to splenic erythropoiesis and to dramatic erythrocytosis. Thus, we identified new hepatic regulation mechanism stimulating erythropoiesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7554/eLife.53550DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7641585PMC
October 2020

p53 activation during ribosome biogenesis regulates normal erythroid differentiation.

Blood 2021 01;137(1):89-102

Université de Paris, Institut Cochin, Unité Mixte de Recherche (UMR) 8104, Centre National de la Recherche Scientifique (CNRS), INSERM U1016, Paris, France.

The role of ribosome biogenesis in erythroid development is supported by the recognition of erythroid defects in ribosomopathies in both Diamond-Blackfan anemia and 5q- syndrome. Whether ribosome biogenesis exerts a regulatory function on normal erythroid development is still unknown. In the present study, a detailed characterization of ribosome biogenesis dynamics during human and murine erythropoiesis showed that ribosome biogenesis is abruptly interrupted by the decline in ribosomal DNA transcription and the collapse of ribosomal protein neosynthesis. Its premature arrest by the RNA Pol I inhibitor CX-5461 targeted the proliferation of immature erythroblasts. p53 was activated spontaneously or in response to CX-5461, concomitant to ribosome biogenesis arrest, and drove a transcriptional program in which genes involved in cell cycle-arrested, negative regulation of apoptosis, and DNA damage response were upregulated. RNA Pol I transcriptional stress resulted in nucleolar disruption and activation of the ATR-CHK1-p53 pathway. Our results imply that the timing of ribosome biogenesis extinction and p53 activation is crucial for erythroid development. In ribosomopathies in which ribosome availability is altered by unbalanced production of ribosomal proteins, the threshold downregulation of ribosome biogenesis could be prematurely reached and, together with pathological p53 activation, prevents a normal expansion of erythroid progenitors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/blood.2019003439DOI Listing
January 2021

Plasmodium falciparum sexual parasites develop in human erythroblasts and affect erythropoiesis.

Blood 2020 09;136(12):1381-1393

INSERM U1016, Centre National de la Recherche Scientifique (CNRS) Unité Mixte de Recherche (UMR) 8104, Université de Paris, Institut Cochin, Paris, France.

Plasmodium falciparum gametocytes, the sexual stage responsible for malaria parasite transmission from humans to mosquitoes, are key targets for malaria elimination. Immature gametocytes develop in the human bone marrow parenchyma, where they accumulate around erythroblastic islands. Notably though, the interactions between gametocytes and this hematopoietic niche have not been investigated. Here, we identify late erythroblasts as a new host cell for P falciparum sexual stages and show that gametocytes can fully develop inside these nucleated cells in vitro and in vivo, leading to infectious mature gametocytes within reticulocytes. Strikingly, we found that infection of erythroblasts by gametocytes and parasite-derived extracellular vesicles delay erythroid differentiation, thereby allowing gametocyte maturation to coincide with the release of their host cell from the bone marrow. Taken together, our findings highlight new mechanisms that are pivotal for the maintenance of immature gametocytes in the bone marrow and provide further insights on how Plasmodium parasites interfere with erythropoiesis and contribute to anemia in malaria patients.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/blood.2019004746DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7498361PMC
September 2020

Comprehensive proteomic analysis of murine terminal erythroid differentiation.

Blood Adv 2020 04;4(7):1464-1477

INSERM U1016, Centre National Recherche Scientifique (CNRS) UMR8104, Institut Cochin, Université de Paris, Paris, France.

Murine-based cellular models have provided and continue to provide many useful insights into the fundamental mechanisms of erythropoiesis, as well as insights into the pathophysiology of inherited and acquired red cell disorders. Although detailed information on many aspects of these cell models is available, comprehensive proteomic data are lacking. This is a critical knowledge gap, as proteins are effectors of most biologic processes. To address this critical unmet need, proteomes of the murine cell lines Friend erythroleukemia (MEL), GATA1 erythroid (G1ER), and embryonic stem cell-derived erythroid progenitor (MEDEP) and proteomes of cultured murine marrow-derived erythroblasts at different stages of terminal erythroid differentiation were analyzed. The proteomes of MEDEP cells and primary murine erythroid cells were most similar, whereas those of MEL and G1ER cells were more distantly related. We demonstrated that the overall cellular content of histones does not decrease during terminal differentiation, despite strong chromatin condensation. Comparison of murine and human proteomes throughout terminal erythroid differentiation revealed that many noted transcriptomic changes were significantly dampened at the proteome level, especially at the end of the terminal differentiation process. Analysis of the early events associated with induction of terminal differentiation in MEDEP cells revealed divergent alterations in associated transcriptomes and proteomes. These proteomic data are powerful and valuable tools for the study of fundamental mechanisms of normal and disordered erythropoiesis and will be of broad interest to a wide range of investigators for making the appropriate choice of various cell lines to study inherited and acquired diseases of the erythrocyte.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/bloodadvances.2020001652DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7160260PMC
April 2020

C3P3-G1: first generation of a eukaryotic artificial cytoplasmic expression system.

Nucleic Acids Res 2019 03;47(5):2681-2698

Medical Microbiology and Immunology, University of Alberta, 6-142J Katz Group Centre for Pharmacy and Health Research, 114 Street NW, Edmonton, Alberta T6G 2E1, Canada.

Most eukaryotic expression systems make use of host-cell nuclear transcriptional and post-transcriptional machineries. Here, we present the first generation of the chimeric cytoplasmic capping-prone phage polymerase (C3P3-G1) expression system developed by biological engineering, which generates capped and polyadenylated transcripts in host-cell cytoplasm by means of two components. First, an artificial single-unit chimeric enzyme made by fusing an mRNA capping enzyme and a DNA-dependent RNA polymerase. Second, specific DNA templates designed to operate with the C3P3-G1 enzyme, which encode for the transcripts and their artificial polyadenylation. This system, which can potentially be adapted to any in cellulo or in vivo eukaryotic expression applications, was optimized for transient expression in mammalian cells. C3P3-G1 shows promising results for protein production in Chinese Hamster Ovary (CHO-K1) cells. This work also provides avenues for enhancing the performances for next generation C3P3 systems.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkz069DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6412113PMC
March 2019

Plasmodium falciparum gametocyte-infected erythrocytes do not adhere to human primary erythroblasts.

Sci Rep 2018 12 14;8(1):17886. Epub 2018 Dec 14.

Inserm U1016, Institut Cochin, Paris, France.

Plasmodium falciparum gametocytes, the sexual stages responsible for malaria parasite transmission, develop in the human bone marrow parenchyma in proximity to the erythroblastic islands. Yet, mechanisms underlying gametocytes interactions with these islands are unknown. Here, we have investigated whether gametocyte-infected erythrocytes (GIE) adhere to erythroid precursors, and whether a putative adhesion may be mediated by a mechanism similar to the adhesion of erythrocytes infected with P. falciparum asexual stages to uninfected erythrocytes. Cell-cell adhesion assays with human primary erythroblasts or erythroid cell lines revealed that immature GIE do not specifically adhere to erythroid precursors. To determine whether adhesion may be dependent on binding of STEVOR proteins to Glycophorin C on the surface of erythroid cells, we used clonal lines and transgenic parasites that overexpress specific STEVOR proteins known to bind to Glycophorin C in asexual stages. Our results indicate that GIE overexpressing STEVOR do not specifically adhere to erythroblasts, in agreement with our observation that the STEVOR adhesive domain is not exposed at the surface of GIE.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-018-36148-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6294825PMC
December 2018

Finely-tuned regulation of AMP-activated protein kinase is crucial for human adult erythropoiesis.

Haematologica 2019 05 11;104(5):907-918. Epub 2018 Oct 11.

Institut Cochin, INSERM U1016

AMP-activated protein kinase (AMPK) is a heterotrimeric complex containing α, β, and γ subunits involved in maintaining integrity and survival of murine red blood cells. Indeed, α , and mice develop hemolytic anemia and the plasma membrane of their red blood cells shows elasticity defects. The membrane composition evolves continuously along erythropoiesis and during red blood cell maturation; defects due to the absence of Ampk could be initiated during erythropoiesis. We, therefore, studied the role of AMPK during human erythropoiesis. Our data show that AMPK activation had two distinct phases in primary erythroblasts. The phosphorylation of AMPK (Thr172) and its target acetyl CoA carboxylase (Ser79) was elevated in immature erythroblasts (glycophorin A), then decreased conjointly with erythroid differentiation. In erythroblasts, knockdown of the α1 catalytic subunit by short hairpin RNA led to a decrease in cell proliferation and alterations in the expression of membrane proteins (band 3 and glycophorin A) associated with an increase in phosphorylation of adducin (Ser726). AMPK activation in mature erythroblasts (glycophorin A), achieved through the use of direct activators (GSK621 and compound 991), induced cell cycle arrest in the S phase, the induction of autophagy and caspase-dependent apoptosis, whereas no such effects were observed in similarly treated immature erythroblasts. Thus, our work suggests that AMPK activation during the final stages of erythropoiesis is deleterious. As the use of direct AMPK activators is being considered as a treatment in several pathologies (diabetes, acute myeloid leukemia), this observation is pivotal. Our data highlighted the importance of the finely-tuned regulation of AMPK during human erythropoiesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3324/haematol.2018.191403DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6518903PMC
May 2019

Dyserythropoiesis evaluated by the RED score and hepcidin:ferritin ratio predicts response to erythropoietin in lower-risk myelodysplastic syndromes.

Haematologica 2019 03 4;104(3):497-504. Epub 2018 Oct 4.

Department of Hematology, CHU Grenoble-Alpes, Grenoble.

Erythropoiesis-stimulating agents are generally the first line of treatment of anemia in patients with lower-risk myelodysplastic syndrome. We prospectively investigated the predictive value of somatic mutations, and biomarkers of ineffective erythropoiesis including the flow cytometry RED score, serum growth-differentiation factor-15, and hepcidin levels. Inclusion criteria were no prior treatment with erythropoiesis-stimulating agents, low- or intermediate-1-risk myelodysplastic syndrome according to the International Prognostic Scoring System, and a hemoglobin level <10 g/dL. Patients could be red blood cell transfusion-dependent or not and were given epoetin zeta 40 000 IU/week. Serum erythropoietin level, iron parameters, hepcidin, flow cytometry Ogata and RED scores, and growth-differentiation factor-15 levels were determined at baseline, and molecular analysis by next-generation sequencing was also conducted. Erythroid response (defined according to the International Working Group 2006 criteria) was assessed at week 12. Seventy patients, with a median age of 78 years, were included in the study. There were 22 patients with refractory cytopenia with multilineage dysplasia, 19 with refractory cytopenia with unilineage dysplasia, 14 with refractory anemia with ring sideroblasts, four with refractory anemia with excess blasts-1, six with chronic myelomonocytic leukemia, two with del5q-and three with unclassifiable myelodysplastic syndrome. According to the revised International Prognostic Scoring System, 13 had very low risk, 47 had low risk, nine intermediate risk and one had high-risk disease. Twenty patients were transfusion dependent. Forty-eight percent had an erythroid response and the median duration of the response was 26 months. At baseline, non-responders had significantly higher RED scores and lower hepcidin:ferritin ratios. In multivariate analysis, only a RED score >4 (=0.05) and a hepcidin:ferritin ratio <9 (=0.02) were statistically significantly associated with worse erythroid response. The median response duration was shorter in patients with growth-differentiation factor-15 >2000 pg/mL and a hepcidin:ferritin ratio <9 (=0.0008 and =0.01, respectively). In multivariate analysis, both variables were associated with shorter response duration. Erythroid response to epoetin zeta was similar to that obtained with other erythropoiesis-stimulating agents and was correlated with higher baseline hepcidin:ferritin ratio and lower RED score. .
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3324/haematol.2018.203158DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6395339PMC
March 2019

New pathogenic mechanisms induced by germline erythropoietin receptor mutations in primary erythrocytosis.

Haematologica 2018 04 21;103(4):575-586. Epub 2017 Dec 21.

INSERM, UMR 1170, Gustave Roussy, Laboratoire d'Excellence GR-Ex, Villejuif, France

Primary familial and congenital polycythemia is characterized by erythropoietin hypersensitivity of erythroid progenitors due to germline nonsense or frameshift mutations in the erythropoietin receptor gene. All mutations so far described lead to the truncation of the C-terminal receptor sequence that contains negative regulatory domains. Their removal is presented as sufficient to cause the erythropoietin hypersensitivity phenotype. Here we provide evidence for a new mechanism whereby the presence of novel sequences generated by frameshift mutations is required for the phenotype rather than just extensive truncation resulting from nonsense mutations. We show that the erythropoietin hypersensitivity induced by a new erythropoietin receptor mutant, p.Gln434Profs*11, could not be explained by the loss of negative signaling and of the internalization domains, but rather by the appearance of a new C-terminal tail. The latter, by increasing erythropoietin receptor dimerization, stability and cell-surface localization, causes pre-activation of erythropoietin receptor and JAK2, constitutive signaling and hypersensitivity to erythropoietin. Similar results were obtained with another mutant, p.Pro438Metfs*6, which shares the same last five amino acid residues (MDTVP) with erythropoietin receptor p.Gln434Profs*11, confirming the involvement of the new peptide sequence in the erythropoietin hypersensitivity phenotype. These results suggest a new mechanism that might be common to erythropoietin receptor frameshift mutations. In summary, we show that primary familial and congenital polycythemia is more complex than expected since distinct mechanisms are involved in the erythropoietin hypersensitivity phenotype, according to the type of erythropoietin receptor mutation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3324/haematol.2017.176370DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5865417PMC
April 2018

Comprehensive Proteomic Analysis of Human Erythropoiesis.

Cell Rep 2016 08 21;16(5):1470-1484. Epub 2016 Jul 21.

INSERM U1016, Institut Cochin, 75014 Paris, France; Centre National de la Recherche Scientifique (CNRS), UMR8104, 75014 Paris, France; Université Paris Descartes, Sorbonne Paris Cité, 75014 Paris, France; Laboratory of Excellence GReX, 75015 Paris, France; Plateforme de Protéomique de l'Université Paris Descartes (3P5), 75014 Paris, France; Ligue Nationale Contre le Cancer, Equipe Labellisée, 75014 Paris, France. Electronic address:

Mass spectrometry-based proteomics now enables the absolute quantification of thousands of proteins in individual cell types. We used this technology to analyze the dynamic proteome changes occurring during human erythropoiesis. We quantified the absolute expression of 6,130 proteins during erythroid differentiation from late burst-forming units-erythroid (BFU-Es) to orthochromatic erythroblasts. A modest correlation between mRNA and protein expression was observed. We identified several proteins with unexpected expression patterns in erythroid cells, highlighting a breakpoint in the erythroid differentiation process at the basophilic stage. We also quantified the distribution of proteins between reticulocytes and pyrenocytes after enucleation. These analyses identified proteins that are actively sorted either with the reticulocyte or the pyrenocyte. Our study provides the absolute quantification of protein expression during a complex cellular differentiation process in humans, and it establishes a framework for future studies of disordered erythropoiesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2016.06.085DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5274717PMC
August 2016

Regulation of cell surface transferrin receptor-2 by iron-dependent cleavage and release of a soluble form.

Haematologica 2015 Apr 30;100(4):458-65. Epub 2015 Jan 30.

Vita Salute San Raffaele University, Milan, Italy Division of Genetics and Cell Biology, IRCCS San Raffaele Scientific Institute, Milan, Italy

Transferrin receptor-2 is a transmembrane protein whose expression is restricted to hepatocytes and erythroid cells. Transferrin receptor-2 has a regulatory function in iron homeostasis, since its inactivation causes systemic iron overload. Hepatic transferrin receptor-2 participates in iron sensing and is involved in hepcidin activation, although the mechanism remains unclear. Erythroid transferrin receptor-2 associates with and stabilizes erythropoietin receptors on the erythroblast surface and is essential to control erythrocyte production in iron deficiency. We identified a soluble form of transferrin receptor-2 in the media of transfected cells and showed that cultured human erythroid cells release an endogenous soluble form. Soluble transferrin receptor-2 originates from a cleavage of the cell surface protein, which is inhibited by diferric transferrin in a dose-dependent manner. Accordingly, the shedding of the transferrin receptor-2 variant G679A, mutated in the Arginine-Glycine-Aspartic acid motif and unable to bind diferric transferrin, is not modulated by the ligand. This observation links the process of transferrin receptor-2 removal from the plasma membrane to iron homeostasis. Soluble transferrin receptor-2 does not affect the binding of erythropoietin to erythropoietin receptor or the consequent signaling and partially inhibits hepcidin promoter activation only in vitro. Whether it is a component of the signals released by erythropoiesis in iron deficiency remains to be investigated. Our results indicate that membrane transferrin receptor-2, a sensor of circulating iron, is released from the cell membrane in iron deficiency.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3324/haematol.2014.118521DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4380718PMC
April 2015

Tyrosine kinase inhibitors induce down-regulation of c-Kit by targeting the ATP pocket.

PLoS One 2013 23;8(4):e60961. Epub 2013 Apr 23.

Institut Cochin, Département d'Immunologie-Hématologie, Paris, France.

The stem cell factor receptor (SCF) c-Kit plays a pivotal role in regulating cell proliferation and survival in many cell types. In particular, c-Kit is required for early amplification of erythroid progenitors, while it must disappear from cell surface for the cell entering the final steps of maturation in an erythropoietin-dependent manner. We initially observed that imatinib (IM), an inhibitor targeting the tyrosine kinase activity of c-Kit concomitantly down-regulated the expression of c-Kit and accelerated the Epo-driven differentiation of erythroblasts in the absence of SCF. We investigated the mechanism by which IM or related masitinib (MA) induce c-Kit down-regulation in the human UT-7/Epo cell line. We found that the down-regulation of c-Kit in the presence of IM or MA was inhibited by a pre-incubation with methyl-β-cyclodextrin suggesting that c-Kit was internalized in the absence of ligand. By contrast to SCF, the internalization induced by TKI was independent of the E3 ubiquitin ligase c-Cbl. Furthermore, c-Kit was degraded through lysosomal, but not proteasomal pathway. In pulse-chase experiments, IM did not modulate c-Kit synthesis or maturation. Analysis of phosphotyrosine peptides in UT-7/Epo cells treated or not with IM show that IM did not modify overall tyrosine phosphorylation in these cells. Furthermore, we showed that a T670I mutation preventing the full access of IM to the ATP binding pocket, did not allow the internalization process in the presence of IM. Altogether these data show that TKI-induced internalization of c-Kit is linked to a modification of the integrity of ATP binding pocket.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0060961PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3634048PMC
November 2013

Transferrin receptor 2 is a component of the erythropoietin receptor complex and is required for efficient erythropoiesis.

Blood 2010 Dec 8;116(24):5357-67. Epub 2010 Sep 8.

Institut Cochin, Université Paris Descartes, Centre National de la recherche Scientifique (Unité Mixte de Recherche 8104), Paris, France.

Erythropoietin (Epo) is required for erythroid progenitor differentiation. Although Epo crosslinking experiments have revealed the presence of Epo receptor (EpoR)-associated proteins that could never be identified, EpoR is considered to be a paradigm for homodimeric cytokine receptors. We purified EpoR-binding partners and identified the type 2 transferrin receptor (TfR2) as a component of the EpoR complex corresponding to proteins previously detected in cross-linking experiments. TfR2 is involved in iron metabolism by regulating hepcidin production in liver cells. We show that TfR2 and EpoR are synchronously coexpressed during the differentiation of erythroid progenitors. TfR2 associates with EpoR in the endoplasmic reticulum and is required for the efficient transport of this receptor to the cell surface. Erythroid progenitors from TfR2(-/-)mice show a decreased sensitivity to Epo and increased circulating Epo levels. In human erythroid progenitors, TfR2 knockdown delays the terminal differentiation. Erythroid cells produce growth differentiation factor-15, a cytokine that suppresses hepatic hepcidin production in certain erythroid diseases such as thalassemia. We show that the production of growth differentiation factor-15 by erythroid cells is dependent on both Epo and TfR2. Taken together, our results show that TfR2 exhibits a non hepatic function as a component of the EpoR complex and is required for efficient erythropoiesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/blood-2010-04-281360DOI Listing
December 2010

IκB kinase overcomes PI3K/Akt and ERK/MAPK to control FOXO3a activity in acute myeloid leukemia.

Blood 2010 Nov 29;116(20):4240-50. Epub 2010 Jul 29.

Institut Cochin, Université Paris Descartes, CNRS (UMR8104), Paris, France.

The FOXO transcription factors are involved in multiple signaling pathways and have tumor-suppressor functions. In acute myeloid leukemia (AML), deregulation of oncogenic kinases, including Akt, extra-signal-regulated kinase, or IκB kinase, is frequently observed, which may potentially inactivate FOXO activity. We therefore investigated the mechanism underlying the regulation of FOXO3a, the only FOXO protein constantly expressed in AML blast cells. We show that in both primary AML samples and in a MV4-11/FOXO3a-GFP cell line, FOXO3a is in a constant inactive state due to its cytoplasmic localization, and that neither PI3K/Akt nor extra-signal-regulated kinase-specific inhibition resulted in its nuclear translocation. In contrast, the anti-Nemo peptide that specifically inhibits IKK activity was found to induce FOXO3a nuclear localization in leukemic cells. Furthermore, an IKK-insensitive FOXO3a protein mutated at S⁶⁴⁴ translocated into the nucleus and activated the transcription of the Fas-L and p21(Cip1) genes. This, in turn, inhibited leukemic cell proliferation and induced apoptosis. These results thus indicate that IKK activity maintains FOXO3a in the cytoplasm and establishes an important role of FOXO3a inactivation in the proliferation and survival of AML cells. The restoration of FOXO3a activity by interacting with its subcellular distribution may thus represent a new attractive therapeutic strategy for AML.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/blood-2009-12-260711DOI Listing
November 2010

Oncostatin M is a potent inducer of hepcidin, the iron regulatory hormone.

FASEB J 2010 Jun 2;24(6):2093-103. Epub 2010 Feb 2.

Université Paris Descartes, Centre National de la Recherche Scientifique (UMR 8104), Paris, France.

Erythropoietic activity is known to affect iron homeostasis through regulation of the liver iron regulatory hormone hepcidin. To identify new factors secreted by the erythroblasts that could influence hepcidin synthesis, we set up a coculture model. HuH7 hepatoma cells cocultured with primary human erythroblasts or erythroleukemic UT7 cells presented a 20- to 35-fold increase of hepcidin gene expression. This induction was fully blunted in the presence of a neutralizing oncostatin M antibody, demonstrating that this cytokine, belonging to the IL-6 family of cytokines, was responsible for increased levels of hepcidin expression. We further demonstrated that recombinant oncostatin M induced a dramatic transcriptional increase of hepcidin in HuH7 cells through specific activation of the STAT pathway. Hepcidin induction by oncostatin M was also observed in hepatocytes in primary culture and is believed to be cell specific since no induction was found in isolated bone marrow cells, macrophagic, stromal, and lymphoma-derived cell lines, nor in erythroblasts. Finally, we show that oncostatin M administration in vivo increases hepcidin expression and leads to significantly decreased serum iron levels. This work identifies a new potent inducer of hepcidin expression in the liver and supports a role for modulators of oncostatin M signaling pathway in treating iron disorders.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1096/fj.09-152561DOI Listing
June 2010

beta-Trcp mediates ubiquitination and degradation of the erythropoietin receptor and controls cell proliferation.

Blood 2007 Jun 27;109(12):5215-22. Epub 2007 Feb 27.

Institut Cochin, Département d'Hématologie, Paris, France.

Control of intensity and duration of erythropoietin (Epo) signaling is necessary to tightly regulate red blood cell production. We have recently shown that the ubiquitin/proteasome system plays a major role in the control of Epo-R signaling. Indeed, after Epo stimulation, Epo-R is ubiquitinated and its intracellular part is degraded by the proteasome, preventing further signal transduction. The remaining part of the receptor and associated Epo are internalized and degraded by the lysosomes. We show that beta-Trcp is responsible for Epo-R ubiquitination and degradation. After Epo stimulation, beta-Trcp binds to the Epo-R. This binding, like Epo-R ubiquitination, requires Jak2 activation. The Epo-R contains a typical DSG binding sequence for beta-Trcp that is highly conserved among species. Interestingly, this sequence is located in a region of the Epo-R that is deleted in patients with familial polycythemia. Mutation of the serine residue of this motif to alanine (Epo-RS462A) abolished beta-Trcp binding, Epo-R ubiquitination, and degradation. Epo-RS462A activation was prolonged and BaF3 cells expressing this receptor are hypersensitive to Epo, suggesting that part of the hypersensitivity to Epo in familial polycythemia could be the result of the lack of beta-Trcp recruitment to the Epo-R.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/blood-2006-10-055350DOI Listing
June 2007

Identification of proteins cleaved downstream of caspase activation in monocytes undergoing macrophage differentiation.

J Biol Chem 2006 Jun 24;281(26):17779-88. Epub 2006 Apr 24.

INSERM UMR 517, IFR 100, Faculty of Medicine, 7 Boulevard Jeanne d'Arc, F-21079 Dijon Cedex, France.

We have shown previously that caspases were specifically involved in the differentiation of peripheral blood monocytes into macrophages while not required for monocyte differentiation into dendritic cells. To identify caspase targets in monocytes undergoing macrophagic differentiation, we used the human monocytic leukemic cell line U937, whose macrophagic differentiation induced by exposure to 12-O-tetradecanoylphorbol 13-acetate (TPA) can be prevented by expression of the baculovirus caspase-inhibitory protein p35. A comparative two-dimensional gel proteomic analysis of empty vector- and p35-transfected cells after 12 h of exposure to 20 nm TPA, followed by mass spectrometry analysis, identified 38 differentially expressed proteins. Those overexpressed in p35-expressing cells (n = 16) were all full-length, whereas half of those overexpressed in control cells (n = 22) were N- or C-terminal cleavage fragments. The cleavage or degradation of seven of these proteins was confirmed in peripheral blood monocytes undergoing macrophage colony-stimulating factor-induced macrophagic differentiation. In U937 cells exposed to TPA, these proteolytic events can be inhibited by expression of a caspase-8 dominant negative mutant or the cowpox virus CrmA caspase inhibitor. These cleavages provide new insights to analyze the role of caspases in this specific differentiation program.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M600537200DOI Listing
June 2006

Both proteasomes and lysosomes degrade the activated erythropoietin receptor.

Blood 2005 Jan 9;105(2):600-8. Epub 2004 Sep 9.

Département d'Hématologie, Institut Cochin, Institut National de la Santé et de la Recherche Médicale U567, Paris, France.

Activation of the erythropoietin receptor (EpoR) after Epo binding is very transient because of the rapid activation of strong down-regulation mechanisms that quickly decrease Epo sensitivity of the cells. Among these down-regulation mechanisms, receptor internalization and degradation are probably the most efficient. Here, we show that the Epo receptor was rapidly ubiquitinated after ligand stimulation and that the C-terminal part of the Epo receptor was degraded by the proteasomes. Both ubiquitination and receptor degradation by the proteasomes occurred at the cell surface and required Janus kinase 2 (Jak2) activation. Moreover, Epo-EpoR complexes were rapidly internalized and targeted to the lysosomes for degradation. Neither Jak2 nor proteasome activities were required for internalization. In contrast, Jak2 activation was necessary for lysosome targeting of the Epo-EpoR complexes. Blocking Jak2 with the tyrphostin AG490 led to some recycling of internalized Epo-Epo receptor complexes to the cell surface. Thus, activated Epo receptors appear to be quickly degraded after ubiquitination by 2 proteolytic systems that proceed successively: the proteasomes remove part of the intracellular domain at the cell surface, and the lysosomes degrade the remaining part of the receptor-hormone complex. The efficiency of these processes probably explains the short duration of intracellular signaling activated by Epo.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/blood-2004-03-1216DOI Listing
January 2005

Protein kinase C phosphorylates ribosomal protein S6 kinase betaII and regulates its subcellular localization.

Mol Cell Biol 2003 Feb;23(3):852-63

Ludwig Institute for Cancer Research, London W1W 7BS, United Kingdom.

The ribosomal protein S6 kinase (S6K) belongs to the AGC family of Ser/Thr kinases and is known to be involved in the regulation of protein synthesis and the G(1)/S transition of the cell cycle. There are two forms of S6K, termed S6Kalpha and S6Kbeta, which have cytoplasmic and nuclear splice variants. Nucleocytoplasmic shuttling has been recently proposed for S6Kalpha, based on the use of the nuclear export inhibitor, leptomycin B. However, the molecular mechanisms regulating subcellular localization of S6Ks in response to mitogenic stimuli remain to be elucidated. Here we present data on the in vitro and in vivo phosphorylation of S6Kbeta, but not S6Kalpha, by protein kinase C (PKC). The site of phosphorylation was identified as S486, which is located within the C-terminal nuclear localization signal. Mutational analysis and the use of phosphospecific antibodies provided evidence that PKC-mediated phosphorylation at S486 does not affect S6K activity but eliminates the function of its nuclear localization signal and causes retention of an activated form of the kinase in the cytoplasm. Taken together, this study uncovers a novel mechanism for the regulation of nucleocytoplasmic shuttling of S6KbetaII by PKC-mediated phosphorylation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC140705PMC
http://dx.doi.org/10.1128/MCB.23.3.852-863.2003DOI Listing
February 2003

Ruk is ubiquitinated but not degraded by the proteasome.

Eur J Biochem 2002 Jul;269(14):3402-8

Ludwig Institute for Cancer Research, University College of London Medical School, London, UK.

The regulator of ubiquitous kinase (Ruk) protein, also known as CIN85 or SETA, is an adaptor-type protein belonging to the CD2AP/CMS family. It was found in complexes with many signaling proteins, including phosphoinositol (PtdIns) 3-kinase (EC 2.7.1.137), Cbl, GRB2, p130Cas and Crk. Functional analysis of these interactions, implicated Ruk in the regulation of apoptosis, receptor endocytosis and cytoskeletal rearrangements. We have recently demonstrated that overexpression of Ruk induces apoptotic death in neurons, which could be reversed by activated forms of PtdIns 3-kinase and PKB/Akt. Furthermore, Ruk was shown to be a negative regulator of PtdIns 3-kinase activity through binding to its P85 regulatory subunit [Gout, I., Middleton, G., Adu, J., Ninkina, N. N., Drobot, L. B., Filonenko, V., Matsuka, G., Davies, A.M., Waterfield, M. & Buchman, V. L. (2000) Embo J.19, 4015-4025]. Here, we report for the first time, that all three isoforms of Ruk (L, M and S) are ubiquitinated. Specific interaction between the E3 ubiquitin ligase Cbl and all three Ruk isoforms was demonstrated by coexpression studies in Hek293 cells. The interaction of Ruk M and S isoforms with Cbl was found to be mediated via heterodimerization with Ruk L. The use of proteosomal and lysosomal inhibitors clearly indicated that ubiquitination of Ruk L does not lead to its degradation. Based on this study, we propose a possible mechanism for the regulation of Ruk function by ubiquitination.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1046/j.1432-1033.2002.03031.xDOI Listing
July 2002

Molecular cloning of CoA Synthase. The missing link in CoA biosynthesis.

J Biol Chem 2002 Jun 29;277(25):22107-10. Epub 2002 Apr 29.

Department of Structure and Function of Nucleic Acid, The Institute of Molecular Biology and Genetics, 150 Zabolotnogo Street, Kyiv 143, Ukraine.

Coenzyme A functions as a carrier of acetyl and acyl groups in living cells and is essential for numerous biosynthetic, energy-yielding, and degradative metabolic pathways. There are five enzymatic steps in CoA biosynthesis. To date, molecular cloning of enzymes involved in the CoA biosynthetic pathway in mammals has been only reported for pantothenate kinase. In this study, we present cDNA cloning and functional characterization of CoA synthase. It has an open reading frame of 563 aa and encodes a protein of approximately 60 kDa. Sequence alignments suggested that the protein possesses both phosphopantetheine adenylyltransferase and dephospho-CoA kinase domains. Biochemical assays using wild type recombinant protein confirmed the gene product indeed contained both these enzymatic activities. The presence of intrinsic phosphopantetheine adenylyltransferase activity was further confirmed by site-directed mutagenesis. Therefore, this study describes the first cloning and characterization of a mammalian CoA synthase and confirms this is a bifunctional enzyme containing the last two components of CoA biosynthesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.C200195200DOI Listing
June 2002
-->