Endothelial Cells Are Essential to Sense Lipopolysaccharide in a MYD88-Dependent Manner and to Subsequently Induce Emergency Myelopoiesis

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 641-641
Author(s):  
Steffen Boettcher ◽  
Rahel Gerosa ◽  
Ramin Radpour ◽  
Markus G. Manz
Keyword(s):  
Endothelial Cells ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Hematopoietic Cells ◽  
Clinical Signs ◽  
Dependent Manner ◽  
Perivascular Cells ◽  
Hematopoietic Stem ◽  
Emergency Myelopoiesis

Abstract Abstract 641 Severe systemic infections evoke a number of characteristic clinical signs such as fever, neutrophilia and the appearance of immature myeloid precursors in the circulation (left-shift). This reflects a well-regulated hematopoietic response program to enhance myeloid cell output during times of increased hematopoietic demand, a condition which is referred to as 'emergency myelopoiesis'. Important molecular components of the emergency myelopoiesis cascade, such as cytokines and transcription factors involved, have been elucidated. However, the initial steps of emergency myelopoiesis involving pathogen recognition and translation into accelerated bone marrow (BM) myelopoiesis have only been inferred from findings on Toll-like receptor (TLR) expression on immature hematopoietic stem and progenitor cells (HSPCs) as well as on mature hematopoietic cells (e.g. macrophages). Accordingly, it has been assumed that both immature as well as mature hematopoietic cells are involved in sensing infection and inducing emergency myelopoiesis directly and indirectly, respectively. Surprisingly, by generating reciprocal BM chimeric animals mice with TLR4−/− hematopoiesis on a wild-type (WT) nonhematopoietic background (TLR4−/−→WT mice) and WT hematopoiesis on a TLR4−/− nonhematopoietic background (WT→TLR4−/−mice), we demonstrated that LPS-Induced emergency myelopoiesis depends on TLR4-expressing nonhematopoietic cells (Boettcher et al., J Immunol. 2012 Jun 15;188(12):5824–8.). However, the precise identity and localization of the nonhematopoietic cell type crucial for sensing gramnegative infection-derived lipopolysaccharide (LPS) has remained elusive to date. We now have addressed this fundamental question using BM transplantation experiments and Cre-loxP recombination technology. BM chimeric mice with a myeloid differentiation primary response gene 88 (Myd88)-deficiency in the hematopoietic lineage (MYD88−/−→WT mice) showed a normal LPS response indistinguishable to control (WT→WT) mice, while knocked out Myd88 within the nonhematopoietic compartment (WT→MYD88−/− mice) led to a non-responsiveness towards LPS similar to controls (Myd88−/−→Myd88−/− mice). These results are in line with our earlier data, thus confirming the critical role of the TLR4/MYD88 pathway in nonhematopoietic cells for the induction of emergency myelopoiesis. In order to specifically delete TLR-MyYD88-downstream signaling in various nonhematopoietic cells including BM Nestin+ mesenchymal stem cells (MSCs) and their progeny, perivascular cells, endothelial cells, and hepatocytes, we generated Nes-Cre;Myd88fl/fl, Pdgfrb-Cre;Myd88fl/fl, Tek-Cre;Myd88fl/fl, and Alb-Cre;Myd88fl/fl mice, respectively. We observed a normal increase in the frequency of BM CD11b+Gr-1low immature myeloid precursors accompanied by a decrease of BM CD11b+Gr-1high mature myeloid cells upon LPS stimulation characteristic for efficient emergency myelopoiesis in Nes-Cre;Myd88fl/fl, Pdgfrb-Cre;Myd88fl/fl, and Alb-Cre;Myd88fl/fl mice as compared to control mice. Furthermore, we measured highly-elevated plasma G-CSF levels in these mouse strains upon LPS injection. Hence, intact TLR signaling in mesenchymal stromal cells incl. Nestin+ MSCs, perivascular cells as well as hepatocytes is dispensable for induction of emergency myelopoiesis. Strikingly, Tek-Cre;Myd88fl/fl mice were completely non-responsive towards LPS stimulation as assessed by the above-mentioned parameters. Our results thus demonstrate a fundamental and unanticipated role of the endothelium for sensing of systemically spread pathogens and subsequent stimulation of BM emergency myelopoiesis. Disclosures: No relevant conflicts of interest to declare.

A Critical Role of Mir-9 in Myelopoesis and EVI1-Induced Leukemogenesis.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2332-2332
Author(s):  
Vitalyi Senyuk ◽  
Yunyuan Zhang ◽  
Yang Liu ◽  
Ming Ming ◽  
Jianjun Chen ◽  
...  
Keyword(s):  
Cell Transformation ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Ectopic Expression ◽  
Myeloid Differentiation ◽  
Dna Hypermethylation ◽  
Hematopoietic Stem

Abstract Abstract 2332 MicroRNA-9 (miR-9) is required for normal neurogenesis and organ development. The expression of miR-9 is altered in several types of solid tumors suggesting that it may have a function in cell transformation. However the role of this miR in normal hematopoiesis and leukemogenesis is unknown. Here we show that miR-9 is expressed at low levels in hematopoietic stem/progenitor cells (HSCs/HPCs), and that it is upregulated during hematopoietic differentiation. Ectopic expression of miR-9 strongly accelerates terminal myelopoiesis, while promoting apoptosis in vitro and in vivo. In addition, the inhibition of miR-9 in HPC with a miRNA sponge blocks myelopoiesis. EVI1, required for normal embryogenesis, and is considered an oncogene because inappropriate upregulation induces malignant transformation in solid and hematopoietic cancers. In vitro, EVI1 severely affects myeloid differentiation. Here we show that EVI1 binds to the promoter of miR-9–3 leading to DNA hypermethylation of the promoter as well as repression of miR-9. We also show that ectopic miR-9 reverses the myeloid differentiation block that is induced by EVI1. Our findings suggest that inappropriately expressed EVI1 delays or blocks myeloid differentiation, at least in part by DNA hypermethylation and downregulation of miR-9. It was previously reported that FoxOs genes inhibit myeloid differentiation and prevent differentiation of leukemia initiating cells. Here we identify FoxO3 and FoxO1 as new direct targets of miR-9 in hematopoietic cells, and we find that upregulation of FoxO3 in miR-9-positive cells reduces the acceleration of myelopoiesis. These results reveal a novel role of miR-9 in myelopoiesis and in the pathogenesis of EVI1-induced myeloid neoplasms. They also provide new insights on the potential chromatin-modifying role of oncogenes in epigenetic changes in cancer cells. Disclosures: No relevant conflicts of interest to declare.

Investigating The Role Of ARAP3 In The Regulation Of Hematopoietic Stem and Progenitor Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2409-2409
Author(s):  
Yiwen Song ◽  
Sonja Vermeren ◽  
Wei Tong
Keyword(s):  
Endothelial Cells ◽  
Plasma Membrane ◽  
Progenitor Cells ◽  
Hematopoietic Cells ◽  
Conditional Knockout ◽  
Ph Domain ◽  
Hematopoietic Stem ◽  
Normal Peripheral Blood ◽  
Stem And Progenitor Cells

Abstract ARAP3 is a member of the dual Arf-and-Rho GTPase-activating proteins (GAP) family, functioning specifically to inactivate its substrates Arf6 and RhoA GTPases. ARAP3 is translocated to the plasma membrane after PIP3 binding to the first two of its five PH domains, facilitating its GAP activity in a PI3K-mediated manner. Rho family GTPases are found to play critical roles in many aspects of hematopoietic stem and progenitor cells (HSPCs), such as engraftment and migration, while a role for Arf family GTPases in hematopoiesis is less defined. Previous studies found that either exogenous ARAP3 expression in epithelial cells or RNAi-mediated ARAP3 depletion in endothelial cells disrupts F-actin or lamellipodia formation, respectively, resulting in a cell rounding phenotype and failure to spread. This implies that ARAP3 control of Arf6 and RhoA is tightly regulated, and maintaining precise regulation of ARAP3 levels is crucial to actin organization in the cell. Although ARAP3 was first identified in porcine leukocytes, its function in the hematopoietic system is incompletely understood. Germline deletion of Arap3 results in embryonic lethality due to angiogenic defects. Since endothelial cells are important for the emergence of HSCs during embryonic development, early lethality precludes further studying the role of ARAP3 in definitive hematopoiesis. Therefore, we generated several transgenic mouse models to manipulate ARAP3 in the hematopoietic compartment: (1) Arap3fl/fl;Vav-Cretg conditional knockout mice (CKO) deletes ARAP3 specifically in hematopoietic cells, (2) Arap3fl/fl;VE-Cadherin -Cretg CKO mice selectively deletes ARAP3 in embryonic endothelial cells and thereby hematopoietic cells, and (3) Arap3R302,3A/R302,3A germline knock-in mice (KI/KI) mutates the first PH domain to ablate PI3K-mediated ARAP3 activity in all tissues. We found an almost 100% and 90% excision efficiency in the Vav-Cretg- and VEC-Cretg- mediated deletion of ARAP3 in the bone marrow (BM), respectively. However, the CKO mice appear normal in steady-state hematopoiesis, showing normal peripheral blood (PB) counts and normal distributions of all lineages in the BM. Interestingly, we observed an expansion of the Lin-Scal+cKit+ (LSK) stem and progenitor compartment in the CKO mice. This is due to an increase in the multi-potent progenitor (MPP) fraction, but not the long-term or short-term HSC (LT- or ST-HSC) fractions. Although loss of ARAP3 does not alter the frequency of phenotypically-characterized HSCs, we performed competitive BM transplantation (BMT) studies to investigate the functional impact of ARAP3 deficiency. 500 LSK cells from Arap3 CKO (Arap3fl/fl;Vav-Cretg and Arap3fl/fl;VEC-Cretg) or Arap3fl/fl control littermate donors were transplanted with competitor BM cells into irradiated recipients. We observed similar donor-derived reconstitution and lineage repopulation in the mice transplanted with Arap3fl/fl and Arap3 CKO HSCs. Moreover, Arap3 CKO HSCs show normal reconstitution in secondary transplants. Arap3 KI/KI mice are also grossly normal and exhibit an expanded MPP compartment. Importantly, Arap3KI/KI LSKs show impaired reconstitution compared to controls in the competitive BMT assays. Upon secondary and tertiary transplantation, reconstitution in both PB and BM diminished in the Arap3KI/KI groups, in contrast to sustained reconstitution in the control group. Additionally, we observed a marked skewing towards the myeloid lineage in Arap3KI/KI transplanted secondary and tertiary recipients. These data suggest a defect in HSC function in Arap3KI/KI mice. Myeloid-skewed reconstitution also points to the possibility of selection for “myeloid-primed” HSCs and against “balanced” HSCs, as HSCs exhaust during aging or upon serial transplantation. Taken together, our data suggest that ARAP3 plays a non-cell-autonomous role in HSCs by regulating HSC niche cells. Alternatively, the ARAP3 PH domain mutant that is incapable of locating to the plasma membrane in response to PI3K may exert a novel dominant negative function in HSCs. We are investigating mechanistically how ARAP3 controls HSC engraftment and self-renewal to elucidate the potential cell-autonomous and non-cell-autonomous roles of ARAP3 in HSCs. In summary, our studies identify a previously unappreciated role of ARAP3 as a regulator of hematopoiesis and hematopoietic stem and progenitor cell function. Disclosures: No relevant conflicts of interest to declare.

PRMT5 Methyltransferase Activity Is Required to Sustain Adult Hematopoiesis

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 4335-4335
Author(s):  
Fan LIU ◽  
Guoyan Cheng ◽  
Fabiana Perna ◽  
Xu Haiming ◽  
Pierre-Jacques Hamard ◽  
...  
Keyword(s):  
Cell Biology ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Conditional Knockout ◽  
Major Type ◽  
Cytokine Signaling ◽  
Methyltransferase Activity ◽  
Hematopoietic Stem ◽  
Arginine Residues

Abstract Epigenetic regulators have been shown to play critical roles in normal hematopoiesis, and their activity is frequently altered in hematopoietic cancers. Protein arginine methyltransferase 5 (PRMT5) is the major type II PRMTs, catalyzing the symmetric di-methylation of arginine residues in histones (H2A, H3 and H4) and non-histone proteins. PRMT5 is over-expressed in several cancers, including acute leukemia and non-Hodgkin’s lymphoma. To define the role of PRMT5 in normal adult hematopoiesis, we generated PRMT5 conditional knockout mice using Mx1-cre. The induced deletion of both alleles of PRMT5 leads to severe pancytopenia and bone marrow aplasia with subsequent lethality in two weeks. First, loss of PRMT5 triggers the impaired proliferation and rapid disappearance of progenitor cells. At the same time, PRMT5 deficient HSCs show increased cell cycling and a transient HSC accumulation, which is rapidly followed by stem cell exhaustion. Mechanistically, we show that deletion of PRMT5 severely impairs cytokine signaling. It also up-regulates p53 protein level and the expression of p53 target genes. These effects likely account for the critical role of PRMT5 in HSPCs. We have conducted many additional experiments to show that these effects of PRMT5 deletion on hematopoiesis are cell autonomous; and also that the methyltransferase activity of PRMT5 is required to sustain normal hematopoiesis. Thus, we identify PRMT5 as a critical regulator of normal hematopoietic stem and progenitor cell biology. Disclosures No relevant conflicts of interest to declare.

Isolation and Functional Analysis of CXCR7 Promoter - a Novel Receptor for Stromal Derived Factor-1 (SDF-1): Different Regulation of Expression in Human Hematopoietic Cells Versus Pediatric Sarcomas.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4583-4583 ◽  
Author(s):  
Maciek Tarnowski ◽  
Magdalena Kucia ◽  
Mariusz Z Ratajczak
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Binding Site ◽  
Promoter Activity ◽  
Conflicts Of Interest ◽  
Hematopoietic Cells ◽  
Leukemic Cells ◽  
Dependent Manner ◽  
Hematopoietic Stem ◽  
Pediatric Sarcomas

Abstract Abstract 4583 Stromal derived factor-1 (SDF-1) binds to seven transmembrane-span G-protein coupled receptor CXCR4 and directs homing of CXCR4+ hematopoietic stem cells to bone marrow (BM) as well as metastasis of CXCR4+ cancer cells. Recently, a new SDF-1 binding receptor has been identified and named CXCR7. With identification of this receptor a role of SDF-1 in directing chemotaxis of CXCR7+ hematopoietic cells as well as metastasis of CXCR7+ tumors become more complex. While CXCR7 is expressed at very low level on normal hematopoietic stem cells, its expression becomes high on leukemic cells. Similarly we noticed high expression of CXCR7 on several pediatric sarcomas (e.g., rhabdomyosarcomas and neuroblastomas) that very often metastasize/infiltrate BM. The aim of our study was to evaluate 5′ fragment of CXCR7 gene for promoter activity and analyze how its expression is regulated in CXCR7+ human hematopoietic cells as well as in CXCR7+ human rhabdomyosarcoma cells (RMS). The putative CXCR7 promoter was cloned by employing specific primers for 5′ fragment of CXCR7 gene. We found that this 2.5 kb 5′ DNA fragment adjacent to CXCR7 gene contains three potential hypoxia responsive element (HRE)- (-100-104, -965-969, -1306-1310), five NF-kB- (-32-42, -308-318, -1019-1029, -1375-1379, -2145-2155), four NRF-1 binding sites (-1030-1040, -1468-1478, -1980-1990, -2085-2095), one c-myb binding site at -15-19 and at -702-706 a binding site for negative transcription regulatory factor YY1. We generated 8 constructs containing smaller CXCR7 promoter fragments and three constructs containing mutated distal NF-kB and HREs as well as c-myb that were subcloned into a pGL4.10 vector. The promoter activity of these fragments was tested in transfected human hematopoietic cells (THP-1) and RMS cell line (RD) by measuring luciferase activity. While the minimal promoter activity in human hematopoietic cells was retained in 80 bp short fragment containing c-myb binding site, similar activity in human rhabdomyosarcoma cells required longer 150 bp fragment containing proximal NF-kB binding element. We noticed that while mutation of c-myb binding site in CXCR7 promoter in THP-1 cells reduces promoter activity by ∼50%, mutation of proximal NF-kB-binding site in CXCR7 promoter completely inhibits promoter activity in RD cells. This was confirmed by knock-down of c-myb by shRNA and chemical inhibition of NF-kB respectively. Furthermore, we noticed that during hypoxia in contrast to CXCR4, CXCR7 expression does not change in hematopoietic cells, however is significantly downregulated in rhabdomyosarcoma cells. This could be explained by upregulation of negative transcripton factor YY1 during hypoxia, as evidenced by RQ-PCR and confirmed by CHIP assay. In conclusion we have demonstrated that 5′ fragment of CXCR7 possesses promoter activity and is differently regulated in hematopoietic versus sarcoma cells - in c-myb or NF-kB dependent manner respectively. Furthermore, we also found that in contrast to hematopoietic cells hypoxia inhibits CXCR7 promoter activity in RMS cells in YY1-dependent manner. Disclosures: No relevant conflicts of interest to declare.

Deletion of Pleiotrophin in LepR+ Perivascular Cells Depletes Hematopoietic Stem Cells

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1491-1491
Author(s):  
Heather A Himburg ◽  
Vivian Y. Chang ◽  
Joshua Sasine ◽  
Jenny Kan ◽  
Liman Zhao ◽  
...  
Keyword(s):  
Steady State ◽  
Stromal Cells ◽  
Leptin Receptor ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Hematopoietic Cells ◽  
Heparin Binding ◽  
Perivascular Cells ◽  
Hematopoietic Stem

Abstract Pleiotrophin (PTN) is a heparin binding growth factor which is expressed by bone marrow vascular endothelial cells (BM ECs) and perivascular stromal cells. Treatment of murine or human HSCs ex vivo promotes HSC expansion (Nat Med. 2010 Apr;16(4):475-82) and constitutive deletion of PTN depletes LT-HSCs in steady state and markedly impairs HSC regeneration following myeloablation (Cell Rep. 2012 Oct 25;2(4):964-75; JCI. 2014;18(7):1123-1129). Here, we sought to determine which BM microenvironment cell is responsible for PTN-mediated maintenance of the HSC pool. Utilizing the Cre-loxP system, we deleted PTN from VE-cadherin+ ECs, leptin receptor+ (lepR+) perivascular stromal cells, osteocalcin+ osteoblasts, and vav1+ hematopoietic cells and examined the effects on hematopoiesis. We observed no differences in steady state hematopoiesis or HSC content as measured by long-term competitive repopulation assays in mice lacking PTN expression in osteocalcin+ cells, vav1+ hematopoietic cells or VE-cadherin+ BM ECs. However, deletion of PTN from lepR+ BM perivascular cells caused a significant decrease in BM c-kit+sca-1+lin- cells (KSL cells) and BM SLAM+KSL HSCs, and colony forming cell (CFC) content compared to PTN+/+ controls (*p = 0.04, 0.04, and 0.001, respectively). Importantly, deletion of PTN in lepR+ cells, caused a significant reduction in long-term HSC content as measured by primary and secondary competitive repopulation assays (*P<0.01 for all time points through 20 weeks). These data suggest that LepR+ BM perivascular cells, rather than VE-cadherin+ ECs are the primary source of PTN in the BM niche which contributes to the maintenance of the HSC pool. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures No relevant conflicts of interest to declare.

scholarly journals TRAF6 Is Essential for Maintaining Hematopoietic Stem Cell Homeostasis

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 568-568
Author(s):  
Jing Fang ◽  
Lyndsey Bolanos ◽  
Juana Serrano-Lopez ◽  
Susanne Christie ◽  
Jose A Cancelas ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Conflicts Of Interest ◽  
Gene Dosage ◽  
Critical Role ◽  
Myeloid Cells ◽  
Physiological Role ◽  
Lymphoid Cells ◽  
Post Transplantation ◽  
Hematopoietic Stem

Abstract Tumor necrosis factor (TNF) receptor associated factor 6 (TRAF6), an E3 ubiquitin ligase downstream of Toll-like receptors (TLR), is required for mediating signals in response to foreign pathogens and stress molecules, and is implicated in the pathogenesis of MDS and AML. Although TLRs are expressed on normal HSC and TRAF6 is implicated in malignant HSC function, the normal physiological role of TRAF6 in HSC homeostasis and during hematopoiesis remains unknown. We find that TRAF6 is expressed in human and mouse HSPC (LT-HSC, ST-HSC, and MPP) at comparable or elevated levels relative to mature myeloid and lymphoid cells. To understand the role of TRAF6 in HSPC homeostasis, we generated hematopoietic-specific and inducible TRAF6 deleted mice by crossing Traf6-floxed with Vav-Cre (Traf6-HscKO) or Mx1-Cre (Traf6-iKO after PolyIC treatment) mice, respectively. Traf6-HscKO mice are born smaller and become moribund shortly after birth. Examination of peripheral blood (PB) and bone marrow (BM) revealed a significant expansion of myeloid cells and reduction of lymphoid cells. Moreover, moribund mice developed splenomegaly and extramedullary hematopoiesis. To determine whether the observed phenotype could be driven by loss of TRAF6 in mature myeloid cells, we generated mice in which TRAF6 is only deleted in myeloid cells by crossing Traf6-floxed with LysM-Cre mice (Traf6-MyKO). Interestingly, Traf6-MyKO mice did not develop myeloid expansion in the PB, BM, or spleen, indicating that TRAF6 plays a role in normal HSPC function. To determine the cell-intrinsic role of TRAF6 in hematopoiesis, we transplanted BM cells from Traf6-HscKO mice into lethally-irradiated recipient mice. The recipient mice with Traf6-HscKO BM cells similarly displayed myeloid-biased hematopoiesis in PB, BM, and spleens. Strikingly, LT-HSCs from Traf6-HscKO mice were significantly reduced in the BM of recipient mice. To exclude a possible effect of myeloid cells on the reduction in LT-HSC, we examined BM HSPC from Traf6-MyKO mice. Consistent with a role of TRAF6 in normal HSC function, the LT-HSC proportions and numbers were not affected in Traf6-MyKO mice. We next examined the functional consequences of deleting TRAF6 in HSC by performing competitive BM transplantation assays. Although initial homing to the BM was comparable between WT and Traf6-HscKO cells, the donor-derived chimerism of Traf6-HscKO cells was significantly reduced for myeloid and lymphoid populations 1 month post transplantation, and declined to below 5% after 4 months as compared with control mice. In addition, donor-derived HSC, HPC, and total BM cell chimerism of Traf6-HscKO cells was dramatically reduced. To examine the effects of TRAF6 deletion on HSC function after BM engraftment has been achieved, competitive BMT were performed with BM cells from Traf6-iKO mice. Upon deletion of Traf6 (PolyIC treatment 2 months post transplantation), total PB and BM chimerism, and chimerism of Traf6-deleted LT-HSC and HPC dramatically declined. Collectively, these findings indicate that TRAF6 is essential for normal HSPC function and homeostasis. To understand the function of TRAF6 in HSPC, HSC-enriched Lin-Sca1+Kit+(LSK) BM cells were isolated and examined for gene expression changes by RNA-sequencing. Genes directly implicated in cell cycle control were among the most differentially expressed in Traf6-deficient HSPC. Particularly, the cyclin-dependent kinase inhibitors (CDKIs) p21, p27 and p57 were significantly down-regulated in Traf6-deficient LSK cells as compared to normal LSK cells. CDKIs are negative regulators of cell cycle progression and involved in maintaining HSC quiescence. Consistent with the observed reduction in CDKI genes, LT-HSC and HPC (LSK) from Traf6-HscKO mice were less quiescent (lower proportion of G0 cells) and more actively cycling (higher proportion of G1/S/G2/M cells). Despite the established requirement of TRAF6 in myeloid and lymphoid cells during infection, our study uncovers a critical role of TRAF6 during normal HSC function and homeostasis. Our findings suggest that TRAF6 is a novel hematopoietic-requisite factor for maintaining HSC quiescence and controlling myeloid-biased differentiation. These findings reinforce the importance of innate immune pathway gene dosage and signaling requirements in normal and malignant HSPC. Disclosures No relevant conflicts of interest to declare.

scholarly journals Sumoylation of Pyruvate Kinase M2 Inhibits Myeloid Differentiation in Hematopoietic Cells

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3919-3919
Author(s):  
Li Xia ◽  
Xin-Ran Wang ◽  
Ran Wei ◽  
Jin-Song Yan ◽  
Guo-Qiang Chen ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Pyruvate Kinase ◽  
Nuclear Localization ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Hematopoietic Cells ◽  
Leukemic Cells ◽  
Myeloid Differentiation ◽  
Significant Shift

Abstract The pyruvate kinase (PK) is a rate-limiting glycolytic enzyme catalyzing the dephosphorylation of phosphoenolpyruvate to pyruvate. M2 form of PK (PKM2) is expressed during embryogenesis and is the predominant form in tumors of different types. In contrast to the essential role of PKM2 in solid tumors, much less is known about the effects of PKM2 in hematopoietic cells and the development of leukemia. Here we found that PKM2 is modified by small ubiquitin-like modifier 1(SUMO1), which can be reduced by a SUMO1-specific protease SENP1 in hematopoietic cells. SUMOylation induced nuclear localization and conformation change from tetramer to dimer of PKM2. Importantly, SUMOylation of PKM2 is prevalent in a variety of leukemic cell lines as well as primary samples from patients with hematologic malignancies. In consistency, predominant nuclear localization and dimeric forms of PKM2 in leukemic cells were observed. Using in vitro SUMOylation reaction-coupled liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS), we identified K270 lysine residue of PKM2 as the SUMOylation target. Replacement of endogenous PKM2 with mutant PKM2K270 showed a significant shift of PKM2 from tetramer to dimer. To investigate the potential leukemogenic effect of PKM2 SUMOylation, murine hematopoietic progenitor 32D clone 3 (32Dcl3) transfectants expressing wild type(WT) or mutant PKM2K270 were generated and G-CSF-induced differentiation was evaluated by morphology appearance and expression of myeloid associated surface markers CD11b and Gr-1. The results showed that expression of WT PKM2 but not mutant PKM2K270 significantly blocked myeloid differentiation. Further investigations revealed that SUMO1 modification of PKM2 at K270 is essential in mediating the interaction between PKM2 and Runt-related transcription factor 1(RUNX1), a master transcriptional factor implicated in the differentiation of hematopoietic cells. This interaction led to a downregulation of RUNX1 during G-CSF-induced myeloid differentiation of 32D cells, which could be abrogated by expression of mutant PKM2K270. Collectively, these data indicated that SUMOylated PKM2 blocks myeloid differentiation through suppressing RUNX1. These findings reveal a novel nonmetabolic function of PKM2 in modulating myeloid differentiation and highlight the critical role of SUMOylation in leukemogenesis. Disclosures No relevant conflicts of interest to declare.

Role of Cohesin-Resolving Protease, Separase, In Hematopoiesis.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1593-1593
Author(s):  
Lanelle V. Nakamura ◽  
Malini Mukherjee ◽  
Margaret A. Goodell ◽  
Debananda Pati
Keyword(s):  
Stem Cells ◽  
Hematopoietic Stem Cells ◽  
Protein Complex ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Hematopoietic Stem ◽  
Sister Chromatids

Abstract Abstract 1593 Introduction: Cohesin is an evolutionarily conserved protein complex that forms during the replication of sister chromatids. It is a multi-protein complex that consists of four proteins, Smc1, Smc3, Rad21, and Scc3. Resolution of sister chromatid cohesion at the onset of anaphase depends on Separase, an endopeptidase that separates sister chromatids by cleaving cohesion Rad21. A recent study suggests a new role of Cohesin proteins in gene expression and development with implications in hematopoiesis. Our data indicates that cohesin-resolving protease Separase may play a critical role in hematopoiesis. HYPOTHESIS: We hypothesize that Separase plays a role in hematopoiesis by increasing the quantity of hematopoietic stem cells (HSC). METHODS: Our experimental approach was to isolate murine long-term HSC from WT mice and mice with one mutated copy of Separase (i.e. Separase heterozygotes). In addition, in vivo competitive long term repopulation assays were used assess the function of HSC in Separase heterozyotes. RESULTS: Separase heterozygote have increased HSC numbers (p<0.05) as compared to WT mice. In addition, an improved engraftment in a competitive repopulation assay (p < 0.001) was seen in the Separase heterozyotes. Analysis of the engrafted cells demonstrated no difference between the wild type and Separase heterozygote animals, indicating the increased engraftment may be due to unique features in the primitive hematopoietic stem cells. CONCLUSION: Investigation of the mechanism for improved HSC engraftment in Separase heterozygote mice will significantly contribute to our understanding of marrow engraftment and function. Elucidating the mechanisms of hematopoietic dysregulation will provide insights into the development of life-threatening disorders such as leukemia and, in the setting of bone marrow transplant, engraftment failure. Disclosures: No relevant conflicts of interest to declare.

PRL2 Maintains Hematopoietic Stem and Progenitor Cells Through Regulating SCF/KIT Signaling

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3674-3674
Author(s):  
Michihiro Kobayashi ◽  
Yuanshu Dong ◽  
Hao Yu ◽  
Yunpeng Bai ◽  
Sisi Chen ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Progenitor Cell ◽  
Conflicts Of Interest ◽  
Critical Role ◽  
Hematopoietic Progenitor Cell ◽  
Hematopoietic Stem ◽  
Acute Myeloid

Abstract The phosphatase of regenerating liver family of phosphatases, consisting of PRL1, PRL2 and PRL3, represents an intriguing group of proteins implicated in cell proliferation and tumorigenesis. However, the role of PRLs in normal and malignant hematopoiesis is largely unknown. While SCF/KIT signaling plays an important role in hematopoietic stem and progenitor cell (HSPC) maintenance, how SCF/KIT signaling is regulated in HSPCs remains poorly understood. We here report that PRL2 regulates HSPC maintenance through regulating SCF/KIT signaling. To define the role of PRL2 in hematopoiesis, we analyzed the hematopoietic stem cell (HSC) behavior in Prl2 deficient mice generated by our group. Prl2 deficiency results in ineffective hematopoiesis and impairs the long-term repopulating ability of HSCs. In addition, Prl2 null HSPCs are less proliferative and show decreased colony formation in response to SCF stimulation. Furthermore, Prl2 null HSPCs show reduced activation of the PI3K/AKT and ERK signaling in steady state and following SCF stimulation. Importantly, we found that PRL2 associates with KIT and the ability of PRL2 to enhance SCF signaling depends on its enzymatic activity, demonstrating that PRL2 mediates SCF/KIT signaling in HSPCs. Thus, PRL2 plays a critical role in hematopoietic stem and progenitor cell maintenance through regulating SCF/KIT signaling. Furthermore, loss of Prl2 decreased the ability of oncogenic KITD814V mutant in promoting hematopoietic progenitor cell proliferation and in activation of signaling pathways. We also checked the expression of PRL2 proteins in human AML cell lines and found increased level of PRL2 proteins in some acute myeloid leukemia (AML) cells compared with normal human bone marrow cells, indicating that PRL2 may play a pathological role in AML. Our results suggest that the PRL2 phosphatase may be a druggable target in myeloproliferative disease (MPD) and acute myeloid leukemia (AML) with oncogenic KIT mutations. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Transforming Growth Factor- β1 Signaling in Vascular Endothelial Cells Inhibits Hematopoietic Stem Cell Regeneration

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1493-1493
Author(s):  
Derek Zachman ◽  
Devorah C Goldman ◽  
Chandan Guha ◽  
Beth Wilmot ◽  
William H. Fleming
Keyword(s):  
Endothelial Cells ◽  
Growth Factor ◽  
Transforming Growth Factor ◽  
Conflicts Of Interest ◽  
Umbilical Vein ◽  
Transforming Growth Factor Β1 ◽  
Dependent Manner ◽  
Hematopoietic Stem ◽  
Tgf Β1

Abstract Endothelial cells (EC) are known to be essential for hematopoietic regeneration; however, little is known about the pathways that regulate this activity. By modeling endothelial-dependent HSC interactions in vitro, we found that human umbilical vein endothelial cells (HUVEC) had a markedly reduced capacity to regenerate functional CD150+LSK cells (HSC) compared to other sources of arterial and venous EC. Transcriptional profiling revealed the overexpression of transforming growth factor- β1 (TGF-β1) in HUVEC and indicated that TGF-β1 driven transcriptional programs are highly active in these cells, a finding consistent with autocrine TGF-β1 signaling. Functional studies demonstrated that HSC regeneration by EC was potently inhibited by TGF-β1 and augmented by the ALK5 inhibitor SB431542, in a dose-dependent manner. Importantly, exposure of EC alone to TGF- β1 was sufficient to attenuate subsequent HSC self-renewal. Transcriptome analysis also identified hepatocyte growth factor (HGF) as a candidate EC-derived factor with the potential to enhance hematopoietic regeneration. HGF treatment of HUVEC activated endothelial Akt signaling and led to a >10-fold increase in HSC regeneration that could be blocked by the c-Met inhibitor PF04217903. HGF treatment also dramatically increased long-term multi-lineage hematopoiesis from HUVEC regenerated HSC. Our findings reveal a novel suppressive role for TGF-β1 in the vascular niche and demonstrate that EC-derived growth factors such as HGF have the potential to attenuate this suppression and significantly enhance HSC regeneration. Disclosures No relevant conflicts of interest to declare.

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