MKL1 Enhances Megakaryocytic Differentiation of Primary CD34+ Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2218-2218
Author(s):  
Matthew J. Renda ◽  
James A. Troy ◽  
Ee-Chun Cheng ◽  
Lin Wang ◽  
Diane S. Krause
Keyword(s):  
Lentiviral Vectors ◽  
Absolute Number ◽  
High Concentration ◽  
Megakaryoblastic Leukemia ◽  
Human Cd34 ◽  
Healthy Donors ◽  
Cd34 Cells ◽  
Megakaryocyte Differentiation ◽  
In Cells

Abstract Acute Megakaryoblastic Leukemia (AMKL or AML variant M7), which occurs most often in infants and young children, is characterized by a failure of megakaryocyte (MK) differentiation, bone marrow fibrosis, cytogenetic abnormalities, and a poor prognosis. We are particularly interested in AMKL that is associated with the translocation t(1;22)(p13;q13), which yields an in-frame fusion of RBM15 (OTT) and MKL1 (MAL) on chromosomes 1 and 22, respectively. The resultant fusion, RBM15-MKL1 is believed to include all of the functional domains of each component. In order to better understand the role of RBM15-MKL1 in AMKL, it is necessary to understand the roles of the constituent genes, RBM15 and MKL1, in hematopoiesis. We have studied the role of human MKL1 in megakaryopoiesis using primary human CD34+ cells purified from G-CSF mobilized PBMC from healthy donors (n=4). To optimize the CD34+ model, we tested the ability of TPO vs. TPO+SCF vs. TPO+SCF+IL–3 to induce megakaryocytopoiesis. TPO and TPO+SCF gave the highest percentages of MK (12% and 7%, respectively) on day 9. However, due to enhanced cell proliferation with TPO+SCF, the absolute number of MK was highest using this cytokine combination. To test the effect of MKL1 overexpression on megakaryopoiesis, we generated VSVG-pseudotyped lentiviral vectors containing human MKL1 and tested the effect of retronectin on viral transduction of CD34 cells. Surprisingly, retronectin decreased the level of transduction when compared to no retronectin (12% vs. 15% transduction respectively). We also found that polybrene enhanced transduction compared to lipofectamine 2000 (20% vs. 6% transduction, respectively). Using our optimized protocols, we examined the effect of MKL1 overexpression on megakaryocytopoiesis. One million CD34+ cells were thawed, infected the following two days with either empty lentivirus (pCCL) or lentivirus containing human MKL1 (pCCL-MKL), and cultured in TPO+SCF for 9 days. Since both lentiviral vectors included GFP driven by the PGK promoter, we measured the levels of CD41a, CD42d, and CD61 in GFP+ cells at day 9. In a representative experiment (of 4), CD41a levels increased in cells containing pCCL-MKL1 vs. pCCL (50% vs. 40%). Moreover, CD42d levels (22% vs. 7%) and CD61 levels (53% vs. 44%) were increased in cells containing pCCL-MKL1 virus when compared to cells containing pCCL virus. We also tested the ability of MKL1 to increase megakaryocyte differentiation using a semisolid Megacult assay from Stem Cell Technologies. CD34+ cells were cultured and infected as described above with either pCCL or pCCL-MKL1 virus. Two days post infection, GFP+ cells were FACS sorted and plated at two different concentrations in semisolid Megacult medium containing collagen, TPO, IL-6, and IL-3. Eleven days post plating, cells were stained for CD41/CD61. Cells infected with pCCL-MKL1 cells gave approximately 2 fold more MK colonies than pCCL infected cells at both low cell concentration plating (395 vs. 182 colonies, respectively) and high concentration plating (900 vs. 389 colonies, respectively). These data suggest that overexpression of human MKL1 enhances megakaryocyte differentiation of primary human CD34+ cells. A further understanding of the normal roles of RBM15 and MKL1 in megakaryopoiesis will allow us to better understand the role of the RBM15-MKL1 fusion in AMKL, and aid in the development of treatments for this disease.

Minimal Engraftment of Human CD34+Cells Mobilized From Healthy Donors in the Infarcted Heart of Athymic Nude Rats

2009 ◽  
Vol 18 (6) ◽  
pp. 845-856 ◽  
Author(s):  
Claus S. Sondergaard ◽  
Jesper Bonde ◽  
Frederik Dagnæs-Hansen ◽  
Jan M. Nielsen ◽  
Vladimir Zachar ◽  
...  
Keyword(s):  
Human Cd34 ◽  
Infarcted Heart ◽  
Nude Rats ◽  
Healthy Donors ◽  
Cd34 Cells

Abnormalities in the myeloid progenitor compartment in Down syndrome fetal liver precede acquisition of GATA1 mutations

Blood ◽  
2008 ◽  
Vol 112 (12) ◽  
pp. 4507-4511 ◽  
Author(s):  
Oliver Tunstall-Pedoe ◽  
Anindita Roy ◽  
Anastasios Karadimitris ◽  
Josu de la Fuente ◽  
Nicholas M. Fisk ◽  
...  
Keyword(s):  
Down Syndrome ◽  
Fetal Liver ◽  
Testable Hypothesis ◽  
Erythroid Progenitor ◽  
Megakaryoblastic Leukemia ◽  
Myeloid Progenitor ◽  
Cd34 Cells ◽  
Progenitor Compartment ◽  
Normal Controls

Abstract Down syndrome (DS) children have a high frequency of acute megakaryoblastic leukemia (AMKL) in early childhood. At least 2 in utero genetic events are required, although not sufficient, for DS-AMKL: trisomy 21 (T21) and N-terminal–truncating GATA1 mutations. To investigate the role of T21 in DS-AMKL, we compared second trimester hemopoiesis in DS without GATA1 mutations to gestation-matched normal controls. In all DS fetal livers (FLs), but not marrows, megakaryocyte-erythroid progenitor frequency was increased (55.9% ± 4% vs 17.1% ± 3%, CD34+CD38+ cells; P < .001) with common myeloid progenitors (19.6% ± 2% vs 44.0% ± 7%) and granulocyte-monocyte (GM) progenitors (15.8% ± 4% vs 34.5% ± 9%) commensurately reduced. Clonogenicity of DS-FL versus normal FL CD34+ cells was markedly increased (78% ± 7% vs 15% ± 3%) affecting megakaryocyte-erythroid (∼ 7-fold higher) and GM and colony-forming unit–granulocyte, erythrocyte macrophage, megakaryocyte (CFU-GEMM) progenitors. Replating efficiency of CFU-GEMM was also markedly increased. These data indicate that T21 itself profoundly disturbs FL hemopoiesis and they provide a testable hypothesis to explain the increased susceptibility to GATA1 mutations in DS-AMKL and DS-associated transient myeloproliferative disorder.

MT1-MMP and RECK Inversely Regulate Hematopoietic Progenitor Cell Egress.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1259-1259
Author(s):  
Abraham Avigdor ◽  
Yaron Vagima ◽  
Polina Goichberg ◽  
Shoham Shivtiel ◽  
Melania Tesio ◽  
...  
Keyword(s):  
Steady State ◽  
Progenitor Cell ◽  
Scid Mice ◽  
Hematopoietic Progenitor Cell ◽  
Chimeric Mice ◽  
Hematopoietic Progenitor ◽  
Human Cd34 ◽  
Healthy Donors ◽  
Cd34 Cells

Abstract Hematopoietic progenitor cell release to the circulation is the outcome of signals provided by cytokines, chemokines, adhesion molecules, and proteolytic enzymes. Clinical recruitment of immature CD34+ cells to the peripheral blood (PB) is achieved by repeated G-CSF stimulations. Yet, the mechanisms governing progenitor cell egress during steady state homeostasis and clinical mobilization are not fully understood. Membrane type-1 metalloproteinase (MT1-MMP) and its endogenous inhibitor, RECK, are established key regulators of tumor and endothelial cell motility. We detected higher MT1-MMP and lower RECK expression on circulating human CD34+ progenitors and maturing leukocytes as compared to immature bone-marrow (BM) cells. MT1-MMP expression was even more prominent on CD34+ cells obtained from PB of G-CSF-treated healthy donors whereas RECK labeling was barely detected. In addition, five daily injections of G-CSF to NOD/SCID mice, previously engrafted with human cells, increased MT1-MMP and decreased RECK expression on human CD45+ leukocytes, immature CD34+ and primitive CD34+/CD38−/low cells, in a PI3K/Akt1-dependent manner, resulting in elevated MT1-MMP activity. Inverse regulation of MT1-MMP and RECK by G-CSF mobilization was confirmed by in situ immuno-labeling of BM sections, as well as by human MT1-MMP and RECK mRNA expression analysis of leukocytes repopulating the BM of chimeric mice. Blocking MT1-MMP function impaired mobilization, while RECK neutralization promoted egress of human CD34+ progenitors in the functional pre-clinical model of NOD/SCID chimeric mice. Targeting MT1-MMP expression by SiRNA or blocking its function reduced the in-vitro chemotactic response to SDF-1 of human CD34+ progenitors via matrigel and impaired to a similar extent the BM homing capacity of transplanted human CD34+ cells in NOD/SCID mice. In accordance, neutralization of RECK function, thus abrogating RECK-mediated inhibition of MT1-MMP, facilitated SDF-1-induced migration of steady state human BM CD34+ cells in vitro. Furthermore, following G-CSF mobilization, we also observed a reduction in CD44 expression on human leukocytes and, specifically, on immature CD34+ progenitor cells in the BM of chimeric mice. This was accompanied by accumulation of CD44 cleaved products of molecular weights, expected for MT1-MMP activity, in the BM supernatants. In chimeric mice co-injected with MT1-MMP-neutralizing Ab, less cleavage of CD44 was detected upon G-CSF mobilization, whereas in the absence of a mobilizing signal, increasing MT1-MMP activity by anti RECK Ab injection facilitated CD44 proteolysis on the BM cells. Finally, MT1-MMP expression correlated with the number of CD34+ cells, collected on the first apheresis day in 29 consecutive patients with lymphoid malignancies and in 21 healthy donors treated with G-CSF. In conclusion, our results indicate that G-CSF inversely regulates MT1-MMP and RECK expression on CD34+ progenitors, resulting in net increase in MT1-MMP activity. MT1-MMP proteolysis of CD44 diminishes progenitor adhesion to BM components, leading to cell egress. These cell autonomous changes provide a previously undefined mechanism for G-CSF recruitment of CD34+ progenitors and might serve as target for new approaches to improve clinical stem cell mobilization.

In Megakaryocytic Differentiation, the Aryl Hydrocarbon Receptor (AhR) Transcription Factor Regulates Hes1 Expression and Megakaryocytic Polyploidization.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2519-2519
Author(s):  
Stephan Lindsey ◽  
Eleftherios Papoutsakis
Keyword(s):  
Cell Cycle ◽  
Transcription Factor ◽  
Notch Signaling ◽  
Cell Cycle Regulation ◽  
Ex Vivo ◽  
Megakaryoblastic Leukemia ◽  
Aryl Hydrocarbon ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Cycle Regulation

Abstract Abstract 2519 Poster Board II-496 Understanding the mechanisms underlying megakaryocytic (Mk) differentiation and maturation is vital to the discovery of novel approaches to treating Mk and platelet disorders such as thrombocytopenia, megakaryoblastic leukemia, and thrombocythemia. The number of platelets released is proportional to the amount of DNA present in a given Mk, so insights into the molecular basis of Mk polyploidization could inspire improved ex vivo culturing methods to promote Mk commitment, expansion, and differentiation, leading to improved autologous transfusion protocols to offset thrombocytopenia associated with HSC transplants following high-dose chemotherapy or MDS progression. Microarray analyses on ex vivo Mk-differentiated primary human CD34+ cells showed that mRNA levels of the Aryl Hydrocarbon Receptor (AhR) increased during Mk differentiation and was elevated 4–6 fold in Mks compared to isogenic granulocytic cultures. These data were further confirmed by quantitative(Q)-RT-PCR analysis of differentiating Mks derived from primary human CD34+ cells as well as from CHRF cells (human megakaryoblastic leukemia). We have shown that CHRF cells are a valid model of human Mk differentiation (Fuhrken PG et al. Exp Hematol, 2007; 35:476–489). Thus, we hypothesized that AhR may act as a novel Mk transcription factor, possibly by influencing or regulating Mk polyploidization. Known as a “toxin sensor”, AhR is involved in the mechanism of action of environmental toxins, likely by altering cell cycle regulation. Epidemiological studies of toxic waste spills and Vietnam veterans suggest that exposure to known AhR ligands may result in increased platelet counts proportional to dioxin exposure level (Webb K et al. Am J Ind Med, 1987;11:685–691, Michalek JE Arch Environ Health, 2001; 56:396–405). These studies offer the intriguing possibility that AhR activation modulates megakaryocyte differentiation and/or platelet production. Interestingly, AhR influences the differentiation of other myeloid lineages including monocytes (Hayashi S et al. Carcinogenesis, 1995; 16:1403–1409) and is upregulated after leukocyte activation (Crawford RB et al. Mol Pharmacol, 1997; 52:921–927). Western blot analyses determined that although initially expressed in both the cytoplasm and nucleus, AhR became solely nuclear in differentiating CHRF cells. EMSA analysis using CHRF nuclear extracts demonstrated that AhR binding to a consensus binding sequence increased as megakaryopoiesis progressed (n=3). Increased AhR-DNA binding during CHRF Mk differentiation correlated with 4.6-fold increased mRNA expression of the AhR transcriptional target Hes1 (n=3, p<0.005), a known cell-cycle regulator and mediator of notch signaling. In order to examine the functional role of AhR in megakaryopoiesis, we generated 3 independent AhR knockdown (KD) CHRF cell lines. Depending on the day of culture, AhR-KD CHRF cell lines differentiated into Mk cells expressed 2-3 fold less AhR mRNA (n=3; p<0.02), 40–60% less AhR protein (n=3), 2.7 times less Hes1 mRNA (n=3; p=0.018), displayed Mk-ploidy distributions shifted towards lower ploidy classes, and were incapable of reaching higher ploidy classes (i.e., ≥32n) seen in control cells. Ploidy levels on day 7 (maximal ploidy in control cells) were 3-fold lower in AhR-KD CHRF cells (n=3; p=0.012 or p=0.005 depending on KD cell line). AhR KD resulted in increased DNA synthesis of low ploidy (<8n; n=3; p<0.05) without influencing apoptosis (n=3, p=0.37). These data suggest that AhR may regulate the cell cycle differently in Mks compared to other cell types, where loss of AhR results in cell cycle blockage and increased apoptosis. As such, AhR deregulation provides a mechanistic explanation for chemical-induced thrombocytopenia, including chemotherapy, and suggests that AhR agonists may provide novel therapies for megakaryoblastic leukemia. AhR-mediated expression of Hes1, an established regulator of the Notch signaling pathway, provides a novel molecular model of endomitotic entry and Mk polyploidization; in drosophila, Notch cell-cycle regulation controls the initial switch toward endomitosis. Disclosures: No relevant conflicts of interest to declare.

PACAP and its receptor VPAC1 regulate megakaryocyte maturation: therapeutic implications

Blood ◽  
2008 ◽  
Vol 111 (4) ◽  
pp. 1885-1893 ◽  
Author(s):  
Kathleen Freson ◽  
Karen Peeters ◽  
Rita De Vos ◽  
Christine Wittevrongel ◽  
Chantal Thys ◽  
...  
Keyword(s):  
Transgenic Mice ◽  
Adenylyl Cyclase ◽  
Independent Manner ◽  
Hematopoietic Stem ◽  
Platelet Recovery ◽  
Therapeutic Implications ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Megakaryocyte Differentiation

Megakaryocytes and platelets express the Gs-coupled VPAC1 receptor, for which the pituitary adenylyl cyclase–activating peptide (PACAP) and the vasointestinal peptide (VIP) are agonists. We here demonstrate a regulatory role for VPAC1 signaling during megakaryopoiesis. A total of 2 patients with trisomy 18p with PACAP overexpression and transgenic mice overexpressing PACAP in megakaryocytes have thrombopathy, a mild thrombocytopenia, and a reduced number of mature megakaryocytes in their bone marrow. In vitro differentiation of hematopoietic stem cells from the patient and transgenic mice shows a reduced number of megakaryocyte colonies compared with controls. The addition of PACAP, VIP, or the adenylyl cyclase activator forskolin to CD34+ cells inhibits megakaryocyte differentiation. In contrast, neutralizing monoclonal anti-PACAP (PP1A4) or anti-VPAC1 (23A11) antibodies inhibit cAMP formation and stimulate megakaryopoiesis in a thrombopoietin-independent manner. Moreover, wild-type mice obtain an increased platelet count after subcutaneous injection of PP1A4 or 23A11. These antibodies also elevate platelet numbers in animal models of myelosuppressive therapy and in GATA1-deficient mice with congenital thrombocytopenia. Furthermore, 23A11 stimulates the in vitro megakaryocyte differentiation of both normal and GATA1-deficient human CD34+ cells. Together, our data strongly suggest that VPAC1 signaling tempers normal megakaryopoiesis, and that inhibition of this pathway stimulates megakaryocyte differentiation, enhancing platelet recovery after myelosuppressive therapy and in GATA1 deficiency.

Differential Effects of a Novel Non-Peptidic Thrombopoietin Mimetic on Proliferation and Differentiation of Human CD34+ Progenitor Cells

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2888-2888 ◽  
Author(s):  
Juha Punnonen ◽  
Marcus O. Muench ◽  
Jeffrey R. Spencer
Keyword(s):  
Molecular Weight ◽  
Progenitor Cells ◽  
Cultured Cells ◽  
Aqueous Solubility ◽  
Similar Proportion ◽  
Low Molecular Weight ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Proliferation And Differentiation ◽  
Megakaryocyte Differentiation

Abstract Thrombopoietin (TPO) and TPO mimetics have been shown to be beneficial in the treatment of thrombocytopenia. Low molecular weight, orally available compounds offer the advantage of convenience and lack of immunogenicity when compared to protein-based drugs. STS-T4 is a novel low-molecular weight (&lt;500 Da), non-peptidic TPO mimetic compound that is a 2nd generation agent in preclinical development with promising in vitro potency and aqueous solubility. We have studied the effects of STS-T4 on the growth and differentiation of primary human CD34+ progenitor cells. A dose-dependent proliferation of highly purified human CD34+ cells was observed with EC50 value of less than 1 μM. A bell-shaped dose-response curve was observed, which is consistent with previous reports on compounds stimulating homodimeric cytokine receptors. In addition, the phenotype of the cultured cells was analyzed by flow cytometry and monoclonal antibodies specific for markers of megakaryocyte differentiation. During a culture period of 10 days, 85% of the cells cultured in the presence of STS-T4 differentiated into CD41+ megakaryocytes in the absence of any other cytokines or mimetics. In addition, 30% of these CD41+ cells coexpressed CD42b as a marker of more mature cells. When compared to recombinant human TPO (rhTPO), a similar proportion of the cultured cells expressed CD41 and CD42b in response to STS-T4 and the levels of expression of these antigens on the surface of the megakaryocytes were similar, suggesting that the effects of STS-T4 and rhTPO on human megakaryocyte differentiation are comparable. However, the level of cell proliferation induced by STS-T4 in cultures of CD34+ cells was 50–60% of that induced by rhTPO, supporting the conclusion that the signals mediating proliferation and differentiation of CD34+ progenitor cells are differentially regulated. In addition to the activity profile, the physical properties of STS-T4 are desirable for further development. Aqueous solubility and gastrointestinal permeability are major contributors to oral absorption, and high solubility and permeability generally also reduce the risk of food effects. The aqueous solubility of STS-T4 was measured by a kinetic method with HPLC and visual detection and was determined to be &gt;1 mM. These data suggest that further evaluation of safety and efficacy of STS-T4 for the treatment of thrombocytopenia is warranted. In addition, the results imply that CD34+ progenitor cells can differentiate into megakaryocytes expressing high levels of CD41 and CD42b in the absence of a full rhTPO-like proliferative response, which may support further investigation of new therapies for the treatment of thrombocytopenia that provide the benefit of megakaryocyte maturation while avoiding excessive expansion of hematopoietic stem cells.

Regulation of AML1/RUNX1 Function by Protein Arginine Methyltransferase 4 (PRMT4) in Myeloid Differentiation

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 549-549
Author(s):  
Ly P. Vu ◽  
Xinyang Zhao ◽  
Fabiana Perna ◽  
Stephen D. Nimer
Keyword(s):  
Arginine Methylation ◽  
Mass Spectrometry Analysis ◽  
Myeloid Differentiation ◽  
Type I ◽  
Arginine Methyltransferase ◽  
Human Cd34 ◽  
Transcriptional Regulatory ◽  
Cd34 Cells ◽  
Functional Complex

Abstract Abstract 549 RUNX1 (also known as AML1) is the DNA binding component of the Core Binding Factor (CBF)-transcriptional regulatory complex, which plays an important role in hematopoiesis. Upon binding to the common binding sequence -PyGpyGGTPy (Py = pyrimidine) in the regulatory regions of promoters and enhancers of its target genes, RUNX1 acts either as an activator or a repressor, depending on promoter context and its interacting partners. Thus, modulation of the network of RUNX1 interactions can influence hematopoiesis. However, how RUNX1 selects one set of partners over another to assemble a functional complex is largely unknown. Posttranslational modifications, including ubiquitination, phosphorylation, acetylation and methylation, present a viable mean to fine-tune its functions. Here we shown that RUNX1 is arginine methylated at a specific residue, R223, by PRMT4, a type I arginine methyltransferase generally thought of as a co-activator molecule. We hypothesized that arginine methylation of RUNX1 by PRMT4 affects its protein-protein interactions, therefore, to identify proteins that specifically interact with unmethylated and/or methylated-R223 RUNX1, in an unbiased manner, we performed a peptide pull-down experiment, using a methyl-R223 RUNX1 peptide and an unmodified RUNX1 peptide as bait, following by mass spectrometry analysis. We identified several proteins that preferentially interacted with the R223 methyl peptide, but focused on a novel interacting protein, DPF2 (double PhD Finger 2), which is a widely expressed member of the d4 protein family, characterized by the presence of a tandem plant-homodomain (PHD domain). We confirmed the specific interaction between methylated-RUNX1 with DPF2 in vivo by immunoprecipitation. We generated an antibody specific for the R223 methylated-RUNX1 protein, and found that RUNX1 methylation decreases during the myeloid differentiation of human CD34+ haematopoietic stem/progenitor cells (HSPCs), without a change in the total level of RUNX1 protein, and this occurred co-incident with a downregulation of PRMT4 protein expression. Having determined that PRMT4 expression declines during myeloid differentiation, we examined the role of PRMT4 in this process, using short hairpin RNAs to knockdown PRMT4 expression in CD34+ cells. Knockdown of PRMT4 accelerates the myeloid differentiation of the cells, whereas overexpression of PRMT4 in human CD34+ cells blocked their myeloid differentiation. When analyzing the expression of several “master” regulators of myeloid differentiation, we identified microRNA-223, a myeloid specific microRNA, as a common target gene of PRMT4 and RUNX1. Furthermore, we have found that by promoting the assembly of a functional complex containing R223 methylRUNX1 and DPF2 at the transcriptional regulatory region of the microRNA-223 promoter, PRMT4 can control miR-223 expression and myeloid differentiation. We have verified the role of DPF2 in this process, as DPF2 represses miR-223 expression and loss of DPF2 promotes myeloid differentiation. Thus, DPF2 acts in a common pathway with PRMT4 to regulate myeloid differentiation. In conclusion, our study elucidates a novel mechanism, where the arginine methylation of RUNX1 regulates its recruitment of interacting partner(s). In addition to demonstrating that PRMT4 can trigger repression of gene expression, we have identified a novel role for PRMT4 (aka CARM1) in myeloid differentiation. Disclosures: No relevant conflicts of interest to declare.

CXCR7 Functions Together with CXCR4 in SDF-1/CXCL12 Induced CD34+ Hematopoietic Progenitor Cell Cycling Through β-Arrestin 2-Dependent Akt Activation

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3448-3448
Author(s):  
Frederic Torossian ◽  
Aurélie Chabanon ◽  
Denis Clay ◽  
Bernadette Guerton ◽  
Adrienne Anginot ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Plasma Membrane ◽  
Hematopoietic Progenitor Cell ◽  
Hematopoietic Stem ◽  
Akt Activation ◽  
Respective Role ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Cell Cycling

Abstract Abstract 3448 Introduction SDF-1/CXCL12 chemokine exhibits a well-known effect on retention, migration and homing of hematopoietic stem/progenitor cells (HSC/HP). We have previously demonstrated that it is also a key regulator of hematopoiesis homeostasis, acting, at low concentrations, as a survival and cell cycle promoting factor for human CD34+ HP. It has long been considered that CXCR4 was responsible for SDF-1 induced biological effects until the recent discovery of its second receptor, CXCR7. In the present study, we explored the respective role of CXCR4 and CXCR7 in the cell cycling and survival promoting effect of SDF-1/CXCL12 on human CD34+HP. Material and Methods We used CD34+ HP purified from the peripheral blood (PB) of healthy un-mobilized donors since they are mainly in G0. This allows to study the role of CXCR4 and CXCR7 receptors in 0.5ng/ml SDF-1/CXCL12 induced G0-G1 transition in synchronized quiescent cells. Gene expression was detected by RT-QPCR. Protein expression was detected and quantified using confocal microscopy, flow cytometry, immunoblotting and immunoprecipitation. Cell cycling experiments were performed using a Ki67 antibody and CXCR7 binding assay was performed using SDF-1/CXCL12AF647. Neutralization experiments were performed using a specific CXCR4 antibody or CXCR7 chemical inhibitors, a kind gift from ChemoCentryx, Inc (CCX771 and CCX733) and their respective controls (IgG and CCX704). Results Flow cytometry and confocal analysis showed that CXCR7 and CXCR4 are differentially distributed in PB CD34+ cells. In contrast to CXCR4 that is present at both the plasma membrane and intracellular level, CXCR7 expression is mainly restricted to the intracellular compartment. Confocal analysis suggested the presence of CXCR4/CXCR7 heterodimers on these cells the presence of which were confirmed by immunoprecipitation in a HP cell line. Despite its very low expression at the surface of CD34+ cells, we found that CXCR7 is capable of binding to exogenous SDF-1/CXCL12. Indeed, pretreatment with CXCR7 antibody or a chemical inhibitor reduces the mean fluorescence of bound fluorescent SDF-1/CXCL12AF647, a fully functional and specific chemokine with similar effects compared to unlabeled SDF-1/CXCL12. Neutralizing either CXCR4 or CXCR7 in PB CD34+ cells strongly reduced Akt activation induced by SDF-1/CXCL12 (0.5 ng/ml) as well as the percentage of cells in cycle (G1 and S + G2/M), colony formation and cell survival. This demonstrates that both receptors cooperate in SDF-1/CXCL12 induced functional effects. We further analyzed the respective role of CXCR4 and CXCR7 in SDF-1/CXCL12 signalization. In contrast to CXCR4, CXCR7 is reported not to activate G protein signaling pathways in response to SDF-1/CXCL12. However, it can transduce cell signaling through the β-arrestin pathway. In the present study, we showed that CXCR7 and β-arrestin 2 colocalize near the plasma membrane in freshly purified PB CD34+ cells, suggesting that CXCR7 is constitutively activated. After SDF-1/CXCL12 treatment, the majority of β-arrestin 2 was translocated to the nucleus and only a partial colocalization persisted in the cytoplasm. Using neutralizing antibodies and specific inhibitors, we showed that β-arrestin 2 nuclear translocation was dependent on both CXCR7 and CXCR4 receptors. Reducing β-arrestin 2 expression using siRNA decreased SDF-1/CXCL12 induced Akt activation in PB CD34+cells indicating the involvement of β-arrestin 2 in this process. Conclusion Altogether, our results demonstrate for the first time the role of CXCR7 together with CXCR4 in SDF-1/CXCL12-induced CD34+ cell cycling/proliferation. They also suggest the involvement of β-arrestin 2 as signalling hubs, downstream of both receptors. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Effects of the NUP98–DDX10 oncogene on primary human CD34+ cells: role of a conserved helicase motif

Leukemia ◽  
2010 ◽  
Vol 24 (5) ◽  
pp. 1001-1011 ◽  
Author(s):  
E R Yassin ◽  
A M Abdul-Nabi ◽  
A Takeda ◽  
N R Yaseen
Keyword(s):  
Human Cd34 ◽  
Cd34 Cells

Selected Contribution: Effect of the aldehyde acrolein on acetylcholine-induced membrane current in airway smooth muscle cells

2001 ◽  
Vol 90 (2) ◽  
pp. 750-754 ◽  
Author(s):  
Jean-Marc Hyvelin ◽  
Jean-Pierre Savineau ◽  
Roger Marthan
Keyword(s):  
Protective Role ◽  
Air Pollutant ◽  
Membrane Currents ◽  
Airway Smooth Muscle Cells ◽  
Induced Current ◽  
High Concentration ◽  
Induced Membrane ◽  
Rat Trachea ◽  
In Cells

Acrolein administered to isolated airways has been shown to alter airway responsiveness as a consequence of its effect on Ca2+ signaling. To examine the mechanisms involved, we studied the effect of acrolein on ACh- and caffeine-induced membrane currents (patch-clamp) in myocytes freshly isolated from rat trachea. In cells clamped at −60 mV, ACh (0.1–10 μM) induced a concentration-dependent inward current, which, in ∼50% of the cells, was followed by current oscillations in response to high concentration of ACh (10 μM). Exposure to acrolein (0.2 μM) for 10 min significantly enhanced the amplitude of the low-ACh (0.1 μM) concentration-induced initial peak of current (318.8 ± 28.3 vs. 251.2 ± 40.3 pA; n = 25, P < 0.05). At a high-ACh concentration (10 μM), the frequency at which subsequent peaks occurred was significantly increased (13.2 ± 1.1 vs. 8.7 ± 2 min−1; n = 20, P < 0.05). ACh-induced current was identified as a Ca2+-activated Cl− current. In contrast, similar exposure to acrolein, which does not alter caffeine-induced Ca2+ release, did not alter caffeine-induced transient membrane currents (595 ± 45 and 640 ± 45 pA in control cells and in cells exposed to acrolein, respectively; n = 15). It is concluded that acrolein alters ACh-induced current as a consequence of its effect on the cytosolic Ca2+ concentration response and that the protective role of inhibitors of Cl− channels in air pollutant-induced airway hyperresponsiveness should be examined.

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