scholarly journals Extracellular DEK Is a Novel Chemotactic Agent of Lineage Negative, Sca-1 Positive, c-Kit Positive Bone Marrow Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1583-1583
Author(s):  
Maegan L. Capitano ◽  
Nirit Mor-Vaknin ◽  
Maureen Legendre ◽  
David Markovitz ◽  
Hal E. Broxmeyer
Keyword(s):  
Bone Marrow ◽  
G Protein ◽  
Pertussis Toxin ◽  
Neutralizing Antibodies ◽  
Conflicts Of Interest ◽  
Regulation Of Transcription ◽  
Mouse Bone Marrow ◽  
Dependent Manner ◽  
Hematopoietic Stem ◽  
G Protein Coupled

Abstract DEK, a nuclear DNA-binding protein that has been implicated in the regulation of transcription, chromatin architecture, and mRNA processing, is known to be secreted by macrophages and act as a proinflammatory molecule (Mor-Vanknin et al., 2006, Mol. Cell. Bio., 26: 9484). Recombinant (r)DEK is known to function as a chemotactic factor that attracts neutrophils, CD8+ T lymphocytes and natural killer cells. Few cytokines are known to be chemoattractants for hematopoietic stem (HSC) and progenitor (HPC) cells, SDF-1 being the most potent of proteins with this capability. To test whether rDEK can serve as a chemotactic agent, transwell assays were performed utilizing lineage negative, sca-1 positive, c-kit positive (LSK) mouse bone marrow and neutrophils (Ly6G+ cells) as a positive control. Both SDF-1 and DEK induced migration of LSK cells at a dose of 100ng/mL, with no significant migration occurring towards 100ng/mL of IL-8 or MIP-2. All four cytokines induced migration of Ly6G+ cells. After examining the ability of LSK cells to migrate towards various doses of rDEK (0-200ng/mL), it was determined that LSK cells can migrate towards rDEK in a dose dependent manner with the maximum chemoattraction potential (~20%) occurring at a dose of DEK equal to or greater than 50ng/mL. A checkerboard assay using LSK cells was performed to determine whether rDEK acted more as a chemotactic (directed cell movement) or a chemokinetic (random migration) agent. Checkerboard analysis demonstrated that DEK acted as a chemotactic molecule. Upon our discovery that the DEK protein has a Glu-Leu-Arg (ELR) motif, similar to that of CXC chemokines such as IL-8, we hypothesized that DEK may manifest at least some of its actions through CXCR2, known to bind and mediate the actions of IL-8 and MIP-2. In order to examine if this is indeed the case we first confirmed expression of CXCR2 on the surface of HSC and HPC. Next, to determine if LSK migration towards DEK is dependent upon its ability to signal through CXCR2, LSK cells were pretreated with a neutralizing monoclonal antibody for CXCR2 immediately prior to being placed in a transwell chemotaxis assay utilizing 100ng/mL of rDEK in the bottom chamber. Neutralizing anti-CXCR2 antibodies inhibited migration of LSK and Ly6G+ cells toward DEK; however, if LSK cells were pretreated with an isotype control or a neutralizing antibody towards CXCR4, migration towards DEK still occurred. To confirm that the neutralizing CXCR2 antibody did not inhibit migration in a non-specific manner, transwell assays were performed examining LSK migration towards SDF-1, IL-8, and MIP-2. LSK cells were still able to migrate towards SDF-1 except when CXCR4 was neutralized. No migration was observed when IL-8 or MIP-2 was utilized. When Ly6G+ cells were used CXCR2 neutralizing antibodies blocked the migration of Ly6G+ cells towards DEK, IL-8 and MIP-2. Neutralizing CXCR4 only blocked Ly6G+ migration towards SDF-1. CXCR2 is known to be a G protein-coupled receptor and this interaction can be blocked through the use of pertussis toxin which prevents G proteins from interacting with G protein-coupled receptors thus interfering with receptor signaling. Pretreatment of LSK cells with pertussis toxin significantly inhibited the migration of LSK cells towards DEK and SDF-1. These data suggest that DEK acts as a chemotactic agent for HSC and HPC in vitro. Thus, DEK may be involved in migration and homing of HSCs and HPCs. Disclosures No relevant conflicts of interest to declare.

scholarly journals Hematopoietic Stem and Progenitor Cells Manifest Direct Migration Towards Secreted DEK in a CXCR2- and G Protein Signaling-Dependent Manner

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1196-1196
Author(s):  
Maegan L. Capitano ◽  
Yasser Sammour ◽  
Maureen Legendre ◽  
Scott Cooper ◽  
David Markovitz ◽  
...  
Keyword(s):  
Cell Migration ◽  
G Protein ◽  
Pertussis Toxin ◽  
Conflicts Of Interest ◽  
Neutrophil Migration ◽  
Regulation Of Transcription ◽  
Mouse Bone Marrow ◽  
Dependent Manner ◽  
Hematopoietic Stem ◽  
G Protein Coupled

DEK, a nuclear DNA-binding protein implicated in the regulation of transcription, chromatin architecture, and mRNA processing, is secreted by macrophages and acts as a proinflammatory molecule (Mor-Vanknin et al., 2006, Mol. Cell. Bio., 26: 9484). Recombinant (r)DEK functions as a chemotactic factor attracting neutrophils, CD8+ T lymphocytes and natural killer cells. Few cytokines/growth modulating proteins are known to be chemoattractants for hematopoietic stem (HSC) and progenitor (HPC) cells; stromal cell-derived factor-1 (SDF-1/CXCL12) being the most potent known protein with this capability. To test whether rDEK can serve as a chemotactic agent, transwell assays were performed utilizing lineage negative mouse bone marrow (BM) cells with neutrophils (Ly6G+ cells) as a positive control. Both SDF-1 and DEK induced directed migration of Lin-Sca1+cKit+ (LSK) BM cells at a dose of 100ng/mL, as determined by flow cytometry of input and migrated cells, with no significant migration occurring towards 100ng/mL of IL-8 or MIP-2. All four cytokines induced migration of Ly6G+ neutrophils. After examining the ability of LSK cells to migrate towards various doses of rDEK (0-200ng/mL), it was determined that LSK cells can migrate towards rDEK in a dose dependent manner with maximum chemoattraction potential (~20%) occurring at a dose of DEK equal to or greater than 50ng/mL. A checkerboard assay using LSK cells was performed to determine whether rDEK acted more as a chemotactic (directed cell movement) or a chemokinetic (random migration) agent. Checkerboard analysis demonstrated that DEK acted as a chemotactic molecule. Upon our recent discovery and report that the DEK protein has a Glu-Leu-Arg (ELR) motif, similar to that of CXC chemokines such as IL-8 and binds to the chemokine receptor CXCR2 to regulate hematopoiesis (Capitano et al., 2019, J.C.I. 130: 2555-2570), we hypothesized that DEK may manifest its chemotactic actions through CXCR2, known previously to only bind and mediate the actions of the chemokines IL-8 and MIP-2. To examine this, we first confirmed expression of CXCR2 on the surface of HSC and HPC. Next, to determine if LSK migration towards DEK is dependent upon its ability to signal through CXCR2, LSK cells were pretreated with a neutralizing monoclonal antibody for CXCR2 immediately prior to being placed in a transwell chemotaxis assay utilizing 100ng/mL of rDEK in the bottom chamber. Neutralizing anti-CXCR2 antibodies inhibited migration of both LSK and Ly6G+ cells toward DEK; however, if LSK cells were pretreated with an isotype control or a neutralizing antibody towards CXCR4, migration towards DEK still occurred. To confirm that the neutralizing CXCR2 antibody did not inhibit migration in a non-specific manner, transwell assays were performed examining LSK cell migration towards SDF-1, IL-8, and MIP-2. LSK cells were still able to migrate towards SDF-1 except when CXCR4 was neutralized. No migration of LSK cells was observed when IL-8 or MIP-2 was utilized. When Ly6G+ neutrophils were used, CXCR2 neutralizing antibodies blocked migration of the Ly6G+ neutrophils towards DEK, IL-8 and MIP-2. Neutralizing CXCR4 only blocked Ly6G+ neutrophil migration towards SDF-1. CXCR2 is a G protein-coupled receptor and this interaction can be blocked using pertussis toxin which prevents G proteins from interacting with G protein-coupled receptors thus interfering with receptor signaling. Pretreatment of LSK cells with pertussis toxin significantly inhibited the migration of LSK cells towards DEK and SDF-1. To determine if DEK and SDF-1 could inhibit one another, checkboard assays were performed where either different concentrations of DEK was used in the top well and different concentrations of SDF-1 was used on the bottom. Starting at 100ng/mL, DEK in the top well inhibited LSK cell migration towards SDF-1. However, when SDF-1 was in the top well, regardless of the SDF-1 dose, SDF-1 always inhibited LSK cell migration towards DEK. These data suggest that DEK acts as a chemotactic agent for HSC and HPC in vitro but is not as strong of a signaling protein for migration when competing against SDF-1. Thus, DEK may be involved as a compensatory chemotactic agent for HSCs and HPCs, especially under certain inflammatory conditions and when SDF-1 signaling is reduced. Disclosures No relevant conflicts of interest to declare.

Potent Strategy for Treatmet of Gvhd and Autoimmune-Diseased by Infusion of Donor-Typed or Even Third-Party Tolerogenic Dendritic Cells by Psoralen Plus UVA Treatment

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2777-2777
Author(s):  
Hideaki Maeba ◽  
Ryosei Nishimura ◽  
Rie Kuroda ◽  
Raita Araki ◽  
Shintaro Mase ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Neutralizing Antibodies ◽  
Conflicts Of Interest ◽  
Third Party ◽  
Cell Contact ◽  
Dependent Manner ◽  
Independent Manner ◽  
Hematopoietic Stem ◽  
Tolerogenic Dendritic Cells ◽  
Inhibition Of Alloreaction

Abstract Abstract 2777 We have reported that bone marrow derived dendritic cells with psoralen and UVA (PUVA-DCs) treatment acquired tolerogenicity in mice. With the purpose of potential application of PUVA-DCs in a clinical hematopoietic stem cell transplantations (HSCT) for graft-versus-host disease (GVHD), we showed that mixed lymphocyte reaction (MLR) was strongly inhibited when PUVA-DCs from the stimulator strain were added to the coculture (Stimulator (S): conventional DCs obtained from C57BL/6, Responder (R): splenocytes obtained from Balb/c, PUVA-DCs: C57BL/6). This suggests that infusion of host-typed PUVA-DCs would become a novel therapeutic approach for GVHD. However utilizing host-typed DCs has problems because of leukemic cell contaminations or low efficiency of cell culture from the patients receiving repetitive chemotherapy. Therefore next concern is whether PUVA-DCs generated from BM donor or even strangers would have same tolerogenicity as host-typed PUVA-DCs do. To test this, we performed MLR by adding PUVA-DC generated from the same strain of responder or third party strain (S: conventional DCs obtained from C57BL/6, R: splenocytes obtained from Balb/c, PUVA-DC: C57BL/6 or C3H). Proliferation was significantly inhibited when PUVA-DC generated from the stimulator strain were added to the coculture (p<0.05). Also significant inhibition was observed (p<0.05) when adding PUVA-DCs generated from third party, suggesting that PUVA-DCs have tolerogenicity in a MHC-independent manner. To clarify the mechanisms of how PUVA-DCs induce tolerogenicity, we performed MLR as mentioned above with the addition of neutralizing antibodies against IL-10 or TGF-beta1 or both, which have immunosuppressive effects. Neutralization of immunosuppressive cytokines had no effects on MLR. We then hypothesized that cell-to-cell contact between PUVA-DCs and alloreactive T-cells was needed to mediate the regulatory effect. To this end, we performed MLR using transwell to prevent cell-to cell contact. MLR was not suppressed when transwell was used, suggesting that PUVA-DCs dominantly regulates the alloreaction in a cell contact-dependent manner. This is the first report that PUVA-DCs prepared not only from host-typed but from donor-typed or even third-party could induce strong inhibition of alloreaction. Tolerogenic DCs prepared previously by several ways could not induce inhibition of alloreaction in vitro when these cells were prepared from donor-typed or third-party strains in mice. To apply tolerogenic dendritic cells for GVHD in clinical settings, it is necessary to obtain sufficient doses of PUVA-DCs with ease and safety guaranteed. Therefore in the future PUVA-DCs generated even from HLA mismatched iPS cells would be a promising approach. In conclusion, infusion of PUVA-DCs from donor-typed or even third party strain could have a potent strategy for treatment of lethal GVHD and autoimmune diseases. Disclosure: No relevant conflicts of interest to declare.

scholarly journals Activation of Non-Canonical Immune Signalling Pathways Drives Marrow Failure in a Murine Model of MDS

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 3246-3246
Author(s):  
Rawa Ibrahim ◽  
Joanna Wegrzyn ◽  
Linda Ya-Ting Chang ◽  
Patricia Umlandt ◽  
Jeff Lam ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Cell Line ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Signalling Pathway ◽  
Gene Set Enrichment Analysis ◽  
Mouse Bone Marrow ◽  
Bone Marrow Niche ◽  
Hematopoietic Stem

Abstract The Myelodysplastic Syndromes (MDS) are the most common hematological malignancies arising from stem/progenitor cells. MDS is characterized by ineffective hematopoiesis in one or more lineages of the bone marrow, resulting in peripheral cytopenias and the propensity to progress to either acute myeloid leukemia (AML) or bone marrow failure (BMF). The most common cytogenetic aberration associated with MDS is deletion of the long arm of chromosome 5. Many of the molecular events involved in the development of del(5q) MDS have been elucidated including haploinsufficiency of the gene encoding the ribosomal protein RPS14, responsible for the anemia observed, and haploinsufficency of the miRNAs miR-145 and miR-146a, which together target the innate immune signaling pathway, specifically, the Toll-like receptor-4 (TLR-4)signalling pathway. It has been demonstrated that overexpression of a target of miR-146a,TRAF6, in mouse bone marrow can recapitulate the phenotype of del(5q) MDS including the cytopenias and progression to BMF or AML. However, enforced expression of TIRAP, a miR-145 target gene, results in rapid BMF independent of TRAF6. The molecular and cellular mechanisms responsible for the differential outcome of overexpression of two genes that act within the same signalling pathway remain to be fully understood. We have identified several differentially expressed cytokines, including interferon gamma (IFNγ) and interleukin-10 (IL-10), following TIRAP overexpression compared with TRAF6 overexpression. Promoter methylation analysis has shown hypermethylation of key adaptors and signal transducers that lie between TIRAP and TRAF6 in the TLR-4 signalling pathway, suggesting activation of different pathways by TIRAP and TRAF6 overexpression. Indeed, blockade of TRAF6 and MyD88 did not inhibit TIRAP induced expression of these cytokines, suggesting that IFNγ and IL-10 production occurs in a TRAF6 and MyD88 independent manner. We identified IFNγ as the critical effector cytokine responsible for TIRAP mediated marrow failure. Gene set enrichment analysis has shown an enrichment of an IFNγ signature in MDS patients with a low risk of transformation to AML compared to healthy controls. Furthermore, interferon signatures were highly enriched in MDS patients compared to patients with AML, suggesting an important role for IFNγ signaling in driving MDS progression toward marrow failure as opposed to leukemic progression. IFNγ has been shown to inhibit components of the bone marrow niche by blocking RANK signalling in stromal cells such as osteoclast progenitors. Using coculture of TIRAP expressing bone marrow cells with the RAW264.7 monocyte cell line, a cell line that is capable of differentiation into osteoclasts, we found an inhibition in the ability of these cells to form osteoclasts compared to control. This provides the first line of evidence suggesting that immune signalling defects arising from genetic perturbations in the hematopoietic stem cell compartment can result in stem cell niche dysfunction leading to marrow failure. Disclosures No relevant conflicts of interest to declare.

Osteocytes Support Hematopoiesis by Altering the Bone Marrow Microenvironment Through Gsα Signaling

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 219-219
Author(s):  
Daniela S. Krause ◽  
Keertik Fulzele ◽  
Kevin Barry ◽  
Sutada Lotinun ◽  
Roland Baron ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Remodeling ◽  
Conflicts Of Interest ◽  
Bone Matrix ◽  
Myeloid Cells ◽  
Hematopoietic Stem ◽  
Inflammatory Protein ◽  
G Protein Coupled

Abstract Abstract 219 Osteocytes, the most abundant and long living cells of bone embedded in the bone matrix, coordinate bone remodeling by regulating osteoblast and osteoclast activity, at least in part, via G-protein coupled receptor signaling. Osteoblasts and osteoclasts control hematopoiesis primarily by influencing self-renewal, differentiation, and mobilization of hematopoietic stem cells in their endosteal bone niche. A role for osteocytes in hematopoiesis has previously not been demonstrated. We engineered mice lacking Gsα in osteocytes (DMP1-GsαKO) using the Cre-loxP recombination technique. Consistent with the previously established role of osteocytes in regulation of bone remodeling, DMP1-GsαKO mice showed severe osteopenia and a decrease in cortical thickness. The osteopenia in the KO mice was due to a dramatic decrease in osteoblast numbers whereas the number and activity of osteoclasts was unaffected. In addition, DMP1-GsαKO mice displayed hematopoietic abnormalities that resembled a myeloproliferative syndrome (MPS) characterized by leukocytosis and neutrophilia. Myeloid cells were increased in the peripheral blood, bone marrow (BM), and spleen in DMP1-GsαKO mice compared to controls (p<0.01 in blood, BM and spleen, N≥6) as assessed by CBC and immunophenotypical flow cytometry analysis. Lineage- negative c-kit-positive and Sca-1+ (LKS) cells and LKS CD150-positive CD48-negative (LKS SLAM) cells were significantly increased in DMP1-GsαKO spleen compared to controls whereas there was no change in the bone marrow suggesting mobilization from the bone marrow in mutant mice. Surprisingly, the number of colonies formed in in-vitro methylcellulose assays from BM cells from DMP1-GsαKO mice were not changed indicating the requirement of the bone microenvironment to induce MPS. Co-culture of osteocyte-enriched bone explants from DMP1-GsαKO mice with control BM cells significantly increased the number of colonies compared to control explants. Transplantation of BM from control to DMP1-GsαKO mice rapidly recapitulated the MPS whereas converse transplantation completely normalized the hematopoietic abnormality. Protein expression of CXCL2 (macrophage inflammatory protein 2 alpha; MIP2-alpha), a chemotactic cytokine known to mobilize hematopoietic stem and myeloid cells, was markedly increased in Gsa deficient osteocytes as assessed by immunohistochemistry. Furthermore, CXCL2 secretion in conditioned media from osteocyte explants cultures was also increased 3-fold in Gsa deficient osteocytes as compared to controls. In summary, our results represent the first evidence for osteocyte-mediated regulation of hematopoiesis via Gsα-signaling-induced alteration of the BM microenvironment, possibly through CXCL2 signaling. Disclosures: No relevant conflicts of interest to declare.

Essential Role for PU.1 in MEIS1 Activation and MLL Fusion Leukemia,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3466-3466
Author(s):  
Jing Zhou ◽  
Bo Li ◽  
Jun Wu ◽  
Fuhong He ◽  
Qiang Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Myeloid Leukemia ◽  
Essential Role ◽  
Conflicts Of Interest ◽  
Leukemia Cell ◽  
Genetic Alterations ◽  
Regulation Of Transcription ◽  
Mouse Bone Marrow ◽  
Down Regulation ◽  
Hematopoietic Stem

Abstract Abstract 3466 Down-regulation of transcription factor PU.1, a key regulator of hematopoiesis, induces myeloid leukemia in mice, demonstrating a role of PU.1 as tumor suppressor. Recent studies, however, have also suggested that PU.1 is required for repopulation/self-renewal capacity of normal hematopoietic stem cells (HSCs), and presence of PU.1 activity may be necessary to favor growth of myeloid leukemia stem cells. To explore whether PU.1 could possibly act as an oncogene in the development of certain type of myeloid leukemia, we set to look for differential up-regulation of PU.1 among AML patients with distinct cytogenetic and genetic alterations in public databases. Consistent with recent molecular studies showing suppression of PU.1 expression by AML1-ETO and PML-RARa fusion proteins, PU.1 expresses at a significant lower level in AML patients with t(8;21) and t(15;17) translocations. In contrast, PU.1 expression level in MLL leukemia patients is significantly higher than that of other subgroups of AML. In addition, we found that a set of PU.1 direct target genes, as defined by genome wide location analysis of this factor, expresses at higher level in MLL leukemia patients comparing with those with t(8;21) and t(15;17) translocations, supporting an increased PU.1 activity in this subgroup of leukemia. In our effort to characterize the functional consequence of high expression of PU.1 in AML, we found that PU.1 plays an essential role in activation of MEIS1, an oncogene essential for MLL leukemia stem cell potential, and in development of MLL fusion leukemia. MEIS1, as PU.1, is differentially up-regulated in MLL leukemia patients, and expresses at a significant lower level in AML patients with t(8;21) and t(15;17) translocations. Among AML patients with higher level MEIS1 expression, a positive correlation was observed between expression of PU.1 and that of MEIS1. Using promoter reporter assay, electro mobility shift assay (EMSA) and chromatin immunoprecipiation (ChIP) analysis, we found that PU.1 directly binds to and activates MEIS1 promoter in vitro and in vivo. Analysis of a hypomorphic PU.1 mouse model indicated that PU.1 is required to maintain Meis1 expression in murine HSCs and progenitors, and knockdown of PU.1 in patient-derived MLL leukemia cell lines resulted in lower enrichment of PU.1 protein at MEIS1 promoter, accompanied by down-regulation of MEIS1 expression and decreased proliferation and survival of these cells. We are now examining whether the ability of MLL-AF9 fusion protein to drive leukemia is compromised in PU.1-deficient mouse HSC/HPCs, and whether introduction of exogenous Meis1 can compensate for the loss of PU.1 in the development of MLL-AF9 leukemia in mouse bone marrow transplantation model. Finally, we are also testing knock-down of PU.1 as a therapeutic approach to primary AMLs isolated from MLL leukemia patients. Collectively, our data indicate that PU.1 is required for the pathogenesis of MLL associated leukemia, at least partially, through direct activation of MEIS1. In veiw of the dependency of MEIS1 in MLL leukemic transformation, targeting PU.1 mediated MEIS1 gene activation could be an alternative or synergistic approach for MLL leukemia therapies aimed at inhibition of DOT1L and HOXA9. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Hematological Disorders in Human Patients with Son Mutations

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3709-3709
Author(s):  
Lana Vukadin ◽  
Jung-Hyun Kim ◽  
Tara Bly Hackwelder ◽  
Nathan Ungerleider ◽  
Erik Flemington ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Rna Splicing ◽  
Conflicts Of Interest ◽  
Bone Marrow Failure ◽  
Regulation Of Transcription ◽  
Heterozygous Deletion ◽  
Hematopoietic Stem ◽  
Hematological Disorders ◽  
Hematopoietic Lineage

Precise regulation of transcription and RNA splicing is critical for controlling hematopoietic cell fate determination and lineage differentiation. Alteration of expression of lineage-specific transcription factors and several core spliceosome components in hematopoietic malignancies highlight the significance of abnormal transcription and RNA splicing as disease-causing factors. Our group previously demonstrated that SON, a large nuclear speckle protein possessing dual abilities to bind both DNA and RNA, functions as a splicing factor as well as a transcriptional repressor. We recently identified heterozygous loss-of-function mutations in the SON gene from children with intellectual disability and developmental delay often with a broad spectrum of other congenital anomalies. The disorder caused by SON haploinsufficiency has been designated as ZTTK syndrome (Zhu-Tokita-Takenouchi-Kim syndrome; OMIM #617140). The majority of the mutations found in these patients are frameshift or nonsense mutations which cause degradation of the mutation-bearing transcript. While the most prominent features of these patients are brain malformations and musculoskeletal abnormalities, we identified various hematological disorders from children with ZTTK syndrome. Notable symptoms include bone marrow failure, severe anemia, immunoglobulin deficiency, thalassemia, polycythemia, polycythemia vera, stroke due to blood clots, and leukocytopenia. Apart from the ZTTK syndrome, SON is known to be upregulated in acute myeloid leukemia (AML) patients and is correlated to altered hematopoietic differentiation. To investigate how altered SON expression affects hematopoiesis, we generated a mouse line with the Son gene deleted specifically in the hematopoietic lineage. Homozygous deletion of Son in hematopoietic lineage led to embryonic lethality, indicating that SON expression in blood cells is indispensable during development. Mice with heterozygous deletion of Son in the hematopoietic lineage were viable and born without notable defects or sign of diseases. However, there is a significant decrease in bone marrow cellularity in the mice with heterozygous deletion of Son. Furthermore, Son haploinsufficiency decreased the size of the lineage negative (Lin-) cell population and short-term hematopoietic stem cell (ST-HSC) population with a concurrent increase in megakaryocyte/erythrocyte lineage-biased multipotent progenitors (MPP2) within hematopoietic stem/progenitor cells. These findings suggest that the level of Son expression potentially affects stem cell maintenance and MPP lineage bias, and the distortion of the subpopulation balance within hematopoietic stem/progenitors is possibly linked to multiple hematological disorders. Our ongoing analyses of hematopoiesis and gene expression changes using this mouse model will expand our knowledge about the role of SON in several hematological disorders and benefit clinical practice for ZTTK syndrome patients. Disclosures No relevant conflicts of interest to declare.

scholarly journals A Novel Mechanism of Megakaryopoiesis from Pre-Adipocytes: Involvement of Transferrin/ CD71/ TPO Pathways

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4306-4306
Author(s):  
Yukako Ono-Uruga ◽  
Keiichi Tozawa ◽  
Sahoko Matsuoka ◽  
Tadashi Horiuchi ◽  
Shinichiro Okamoto ◽  
...  
Keyword(s):  
Bone Marrow ◽  
High Efficiency ◽  
Nuclear Translocation ◽  
Conflicts Of Interest ◽  
Thrombus Formation ◽  
Lineage Commitment ◽  
Mouse Bone Marrow ◽  
Hematopoietic Stem ◽  
Blocking Antibody ◽  
Unique Mechanism

Abstract Platelets are released from terminally differentiated megakaryocytes (MKs) through lineage commitment, although the underlying mechanisms of megakaryopoiesis and subsequent thrombopoiesis are incompletely understood. MKs were reportedly generated from not only hematopoietic stem cells (HSCs), but also pre-adipocytes, without any gene transfer in an in vitro culture system (Matsubara et al, 2012 Methods Mol Biol). Because of high efficiency of platelet production from pre-adipocytes, much interest has been attracted to clarify the mechanisms of megakaryopoiesis from pre-adipocytes. Here we demonstrate a novel mechanism that megakaryopoiesis from pre-adipocytes is regulated by the inducible production of endogenous TPO via transferrin receptor CD71. NF-kB pathway in pre-adipocytes might modulate the TPO production through the stimulation of transferrin. Previously, we reported that pre-adipocyte cell line OP9 has the gene expressions of thrombopoietin (TPO) and its receptor c-MPL. This study first examined the TPO protein levels in supernatant from human (Cell Applications) and mouse pre-adipocytes cultured with MK lineage induction (MKLI) media (Ono et al, 2012 Blood, Matsubara et al, 2012 Methods Mol Biol) in the absence of recombinant TPO (rTPO). The TPO levels were 36.6 ± 9.2 pg/mL in mouse pre-adipocytes on Day 8 and 36.1 ± 5.8 pg/mL in human pre-adipocytes on Day 8, and undetectable levels of TPO were observed in both cells before MK induction. Differentiation efficiency (approximately 20%) of MKs from pre-adipocytes cultured with rTPO was similar to that without rTPO. The function of pre-adipocyte-derived platelets cultured with rTPO was similar to that without rTPO when assessed by incorporation of platelets into thrombus formation on the collagen-coated surface under flow conditions. The number of MKs produced from pre-adipocytes was markedly decreased in the presence of anti-c-MPL blocking antibody, AMM2. Regarding the MK differentiation from HSCs, it is well known that HSCs do not differentiate into MKs in the absence of rTPO. We did not observe the TPO secretion from human bone marrow CD34(+) cells and mouse bone marrow Lin(-)Sca1(+)c-kit(+) cells in a culture condition using MKLI media in the absence of rTPO. The observations suggest that the MK differentiation from pre-adipocytes has a distinct mechanism of megakaryopoiesis from HSCs. These findings indicate the unique mechanism of megakaryopoiesis from pre-adipocytes; the endogenous TPO stimulation via c-MPL in pre-adipocytes has activity for promoting MK differentiation and subsequent production of functional platelets. To elucidate the molecular mechanism of TPO production during MK differentiation from pre-adipocytes, we examined which components of MKLI media were responsible for TPO production. We found iron-saturated transferrin, but not apo-transferrin, included in MKLI media is the critical factor to induce TPO production and MK differentiation from pre-adipocytes. The effects of transferrin were not observed when pre-adipocytes were cultured in the presence of anti-CD71 blocking antibody, 8D3, or deferoxamine mesilate, an iron-chelating agent. The effects of transferrin on TPO production and MK differentiation were also abolished in pre-adipocytes transfected with siRNA-CD71, and the TPO levels were 44.8 ± 15.9 pg/mL for siRNA-control and undetectable levels for siRNA-CD71 on Day 6. We next investigated a mechanism of inducing TPO production via CD71 in pre-adipocytes. The 5’ region of human TPO gene contains transcription factor-binding sequences, such as GATA1, GATA2 and NF-kB. Since NF-kB is a downstream target of CD71, we then examined whether NF-kB involved in MK differentiation derived from pre-adipocytes. Nuclear translocation of NF-kB in pre-adipocytes was observed when cells were stimulated with transferrin. Both TPO secretion and MK production were decreased in the presence of a peptide to inhibit nuclear translocation of NF-kB. Taken together, it is concluded that NF-kB pathway in pre-adipocytes modulates megakaryopoiesis through endogenous TPO production via CD71. The present findings suggest that pre-adipocytes have a unique mechanism of megakaryopoiesis through lineage commitment. Disclosures No relevant conflicts of interest to declare.

Growth Factor Independence 1b (Gfi1b) Regulates The Commitment, Differentiation and Expansion Of Hematopoietic Stem Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2433-2433
Author(s):  
Tarik Moroy ◽  
Cyrus Khandanpour ◽  
Joseph Krongold
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Mouse Bone Marrow ◽  
Paracrine Factors ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract The efficacy of bone marrow stem cell transplantation is the therapy of choice for many hematopoietic diseases, in particular leukemia and lymphoma. This therapy is critically dependent on the transfer of sufficient numbers of hematopoietic stem cells (HSCs), which possess the capacity for self-renewal and can fully reconstitute the hematopoietic system. As such, the development of techniques for the expansion of fully functional HSCs is of significant clinical interest. By transiently manipulating the factors that govern HSC homeostasis it has been proposed that HSCs can be expanded without the loss of essential stem cell characteristics. Previously we have observed that ablation of the gene encoding the transcription factor Gfi1b in-vivo results in a dramatic expansion and mobilization of hematopoietic stem cells in the bone marrow and periphery. More recent data suggest that the blood mobilization of Gfi1b deficient HSCs is very likely mediated by a deregulation of the integrin expression. These data led us to hypothesize that Gfi1b could be a potential target for ex-vivo treatment and expansion of HSCs. Indeed, when deletion of Gfi1b was induced in whole bone marrow ex-vivo, HSCs showed a significant expansion in both in absolute number and in terms of proportion of bone marrow. We followed HSCs in ex-vivo expansion cultures from mouse bone marrow by tracking expression of the surface marker CD48, which indicates whether an HSC has transitioned to a differentiation committed multi-potent progenitor. We observed that Gfi1b null HSCs expanded without up-regulating CD48 in contrast to wt HSCs. This suggests that Gf11b deficient HSCs underwent symmetric self-renewal type cell divisions at a significantly increased frequency, when compared to wt HSCs. We had previously shown that HSCs lacking Gfi1b cycle at a faster rate than control HSCs. The combination of increased cell division and preferential self-renewal of Gfi1b-/- HSCs indicates that inhibition of Gfi1b may be the ideal strategy for ex-vivo HSC expansion. As well, in accordance with this preference for self-renewal, Gfi1b null HSCs that were cultured under myeloid differentiation conditions remained primarily in an undifferentiated state as defined by a lack of the myeloid surface markers Gr1 and Mac1. These cultures also demonstrated increased long term colony forming capacity versus controls, further supporting an undifferentiated phenotype in Gfi1b-/- cells. Because the stem cell niche is a highly complex and heterogeneous environment we also investigated whether bone marrow in which Gfi1b has been deleted exerts paracrine effects that contributed to HSC expansion. Co-Culture assays demonstrated that Gfi1b-/- bone marrow was able to induce an expansion of progenitors in wild-type bone marrow of more than 10 fold compared to Gfi1b-/+ bone marrow. Interestingly cells co-cultured with Gfi1b null bone marrow also exhibited an overall proliferation advantage after short-term cultures. This suggests that not only does Gfi1b deletion induce HSC expansion via cell intrinsic mechanisms, but also points to the possibility that this occurs through paracrine factors that alter bone marrow homeostasis. Disclosures: No relevant conflicts of interest to declare.

The G Protein-Coupled Receptor Associated Sorting Proteins, Gprasp2 and Armcx1 Are Putative Negative Regulators of HSC Engraftment and Repopulation

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2386-2386 ◽  
Author(s):  
Maheen Ferdous ◽  
Miguel Ganuza ◽  
Per Holmfeldt ◽  
Trent Hall ◽  
Megan Walker ◽  
...  
Keyword(s):  
G Protein ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Pcr Analysis ◽  
G Protein Coupled Receptor ◽  
Negative Regulators ◽  
Hematopoietic Stem ◽  
Post Transplant ◽  
Qrt Pcr ◽  
G Protein Coupled

Abstract Although hematopoietic stem cell transplantation (HSCT) is used routinely to cure hematologic disease, the efficacy of transplantation is limited by the paucity of HSC. One way to overcome this is to increase the efficiency of HSC engraftment. Thus, we executed a functional screen for novel regulators of HSCT. Murine HSC were lentivirally transduced with shRNAs targeting prioritized gene candidates prior to transplantation into cohorts of lethally irradiated recipient mice. In total, around 1300 mice were transplanted to assess a putative role for about 50 genes in HSCT. We thereby identified Gprasp2 and Armcx1 as putative negative regulators of HSCT. When transplanted at a 1:4 disadvantage relative to control, recipients of either Gprasp2 or Armcx1 shRNA-treated CD45.2 Lineage- Sca-1+ c-Kit+ (LSK) cells displayed 3.12 (p=0.024) and 2.8 (p=0.04) fold enhanced CD45.2 chimerism in peripheral blood (PB) at 16 weeks post-transplant, respectively, relative to mice transplanted with CD45.2 LSK cells treated with control shRNAs. Although loss of each gene did not favor a particular PB lineage, CD45.2 chimerism was enhanced in all bone marrow (BM) HSC and progenitor (HSPC) compartments in these recipients, correlating with their enhanced PB chimerism. qRT-PCR reveals that both murine Armcx1 and Gprasp2 are highly enriched for expression in LSK CD150+CD48- cells relative to all downstream hematopoietic progeny. Further, HemaExplorer, a bioinformatics database of human hematopoietic gene expression, suggests that GPRASP2 and ARMCX1 are also highly expressed in human HSC. This prediction is currently being validated by qRT-PCR. Interestingly, Gprasp2 and Armcx1 both belong to the G protein-coupled receptor associated sorting protein (GASP) gene family, which has never before been implicated in HSC function. The closely related GASP family member, Gprasp1, sorts G protein-coupled receptors (GPCR) to lysosomes for degradation. As Gprasp1 and Gprasp2 both contain GPCR-binding domains and ~70% amino acid sequence conservation in their C-termini, Gprasp2 may also regulate GPCR trafficking and degradation in HSC. Although Gprasp1 was not tested in our screen, qRT-PCR analysis reveals that it is also highly expressed by murine HSC relative to downstream progeny, suggesting that it too may play a role in HSC function. We are currently assessing this using Gprasp1-shRNAs and competitive transplantation. In contrast, Armcx1 lacks the GPCR binding domain and contains both a nuclear and mitochondrial localization signal and has been shown to localize to mitochondrial networks when expressed in HEK-293 cells, suggesting a role in mitochondrial/nuclear communication. To determine how loss of Gprasp2 and Armcx1 promotes HSC engraftment, we are currently employing transplantation and ex vivo culture assays to analyze the effect of their loss on cell cycle, apoptosis, migration, and adhesion of HSPC post-transplant. Our work may help elucidate the mechanisms underlying efficient engraftment, adhesion, and retention of HSPC in the BM niche, which in turn may shed light on novel pathways that could be targeted to promote the efficiency of HSCT in the clinic. Disclosures No relevant conflicts of interest to declare.

Sign in / Sign up

Export Citation Format

Share Document