A Novel Role for Thrombin in Hematopoietic Stem/Progenitor Cell Homing.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3374-3374
Author(s):  
Neeta Shirvaikar ◽  
Ali Jalili ◽  
Mariusz Z. Ratajczak ◽  
Anna Janowska-Wieczorek
Keyword(s):  
Protein Expression ◽  
Lipid Rafts ◽  
Leading Edge ◽  
Membrane Lipid ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
And Migration ◽  
Stimulation Of

Abstract Thrombin, an important serine protease, not only plays a pivotal role in platelet aggregation and coagulation, but also through activation of its receptor, seven transmembrane, G-protein-coupled receptor PAR-1, elicits numerous cellular responses in platelets and endothelial cells such as induction of adhesion molecules, production of chemokines, activation of matrix metalloproteinase (MMP)-2, cytoskeletal reorganization and migration. Thrombin is also one of the inflammatory molecules elevated during G-CSF mobilization of hematopoietic stem/progenitor cells (HSPC) and their collection by leukapheresis. We recently reported that components of leukapheresis products including thrombin enhance in vitro chemotaxis of CD34+ cells towards an SDF-1 gradient and in vivo homing to bone marrow (BM) niches in a murine model (Blood2005; 105:40). In this study we investigated whether thrombin enhances the homing-related responses of human HSPC (CD34+ cells) through MMPs, especially membrane-type (MT)1-MMP which is known to be localized on the leading edge of migrating cells and both activates latent proMMPs (MMP-2, -9) and itself has strong pericellular proteolytic activity. We found that stimulation of CD34+ cells with thrombin upregulates mRNA for MT1-MMP and MMP-9 as well as MT1-MMP protein expression (Western blot, flow cytometry) and proMMP-2 and proMMP-9 secretion (zymography). Thrombin was also found to (i) prime trans-Matrigel chemoinvasion of CD34+ cells towards a low SDF-1 gradient (20 ng/mL), which was inhibited by epigallocatechin-3-gallate, a potent inhibitor of MT1-MMP, and (ii) activate MMP-2 in of co-cultures of CD34+ cells with stromal cells (BM fibroblasts and HUVEC) which secrete proMMP-2. We also found that SDF-1 upregulates mRNA and protein expression of MT1-MMP. Moreover, using confocal microscopy we demonstrate for the first time that in CD34+ cells, PAR-1, like CXCR4, is localized in the GM1 fraction of lipid rafts and stimulation of these cells with thrombin as well as SDF-1 increases incorporation of MT1-MMP into membrane lipid rafts. Furthermore, disruption of lipid raft formation by the cholesterol-depleting agent methyl-b-cyclodextrin inhibits MT1-MMP incorporation into membrane lipid rafts and also trans-Matrigel chemoinvasion of CD34+ cells towards SDF-1. Thus we conclude that thrombin, through PAR-1 signalling and the SDF-1-CXCR4 axis, upregulates the incorporation of MT1-MMP into membrane lipid rafts and the interaction of these axes enhances the homing-related responses of HSPC towards SDF-1.

Upregulation of MT1-MMP by Molecules Present in Leukaphereis Products Primes CD34+ Cell Homing.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5273-5273
Author(s):  
Neeta Shirvaikar ◽  
Jencet Montano ◽  
A.Robert Turner ◽  
Mariusz Z. Ratajczak ◽  
Anna Janowska-Wieczorek
Keyword(s):  
Lipid Rafts ◽  
Leading Edge ◽  
Membrane Lipid ◽  
Rt Pcr ◽  
Hematopoietic Stem ◽  
Cell Homing ◽  
Mmp Inhibitor ◽  
Cd34 Cells ◽  
Mobilized Peripheral Blood ◽  
Stimulation Of

Abstract Hematopoietic stem/progenitor cells (HSPC) derived from mobilized peripheral blood engraft faster than those from bone marrow (BM) or cord blood (CB). We recently postulated that leukapheresis product supernatants (collected from G-CSF-mobilized patients) and their components (fibrinogen, fibronectin, thrombin, hyaluronic acid (HA) and C3 cleavage fragments) have a priming effect on HSPC homing by enhancing chemotactic responses to SDF-1 through increased incorporation of CXCR4 into membrane lipid rafts and stimulation of matrix metalloproteinase (proMMP-2 and proMMP-9) secretion in these cells (Blood2005; 105:40). As MT1-MMP has been shown to activate latent forms of MMP-2 and (indirectly) MMP-9 and is known to localize proteolytic activity at the leading edge of migrating cells, we decided to investigate MT1-MMPs’ role in the priming of HSPC homing. We observed that stimulation of BM and CB CD34+ cells with priming agents (HA, fibrinogen or thrombin) not only increased secretion of proMMP-2 and proMMP-9 (zymography) but also highly upregulated levels of MT1-MMP transcript (RT-PCR) and protein (Western blotting) in these cells. Moreover, trans-Matrigel chemoinvasion of CD34+ cells towards a low SDF-1 gradient (20 ng/mL), enhanced by priming agents, was inhibited by the potent MT1-MMP inhibitor epigallocatechin-3-gallate. Furthermore, priming agents (HA, fibrinogen and thrombin) increased levels of active MMP-2 in co-cultures of stromal cells (endothelial cells and BM fibroblasts) with CD34+ cells. To elucidate the mechanism of MT1-MMP upregulation by priming agents, we evaluated whether they affected MT1-MMP incorporation in ganglioside M1-enriched membrane lipid rafts in the same way as for CXCR4. We found that stimulation of hematopoietic and THP-1 cells with HA, fibrinogen and thrombin increased incorporation of MT1-MMP into membrane lipid rafts and these observations were confirmed using confocal microscopy. Hence it appears that MT1-MMP, like CXCR4, localizes in membrane lipid rafts, and this is enhanced by priming agents, leading to better homing of HSPC.

MicroRNA Expression and Regulation of Hematopoiesis in CD34+ Cells: A Bioinformatic Circuit Diagram of the Hematopoietic Differentiation Control.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1334-1334
Author(s):  
Robert W. Georgantas ◽  
Richard Hildreth ◽  
Jonathan Alder ◽  
Carlo M. Croce ◽  
George A. Calin ◽  
...  
Keyword(s):  
Protein Expression ◽  
Expression Profiles ◽  
In Vivo Studies ◽  
Luciferase Reporter ◽  
Hematopoietic Differentiation ◽  
Aberrant Expression ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract MicroRNAs (miRs) are a recently realized class of epigenetic elements which block translation of mRNA to protein. MicroRNAs have been shown to control cellular metabolism, apoptosis, differentiation and development in numerous organisms including drosophila, rat, mouse, and humans. Recently, miRs have been implicated in the control of hematopoiesis. Importantly, both aberrant expression and deletion of miRs are have been associated with the development of various cancers. In a previous study, we determined the gene expression profiles of HSC-enriched, HPC-enriched, and total CD34+ cells from human PBSC, BM, and CB. One rather surprising finding from this study was that virtually all of “hematopoietic important” genes were expressed at virtually identical levels within all populations examined. One of our hypotheses to explain this phenomena was that miRs may control differentiation by controlling protein expression from these “hematopoietic” RNAs. To examine the possible role of miRs in normal hematopoiesis and their relation to the HSPC transcriptome, we used mir-miroarrays to determine the miR expression profile of primary normal human mobilized blood and bone marrow CD34+ hematopoietic stem-progenitor cells (HSPCs). We have combined this miR data with (1) our extensive mRNA expression data obtained previously for CD34+ HSPCs, CD34+/CD38−/Lin- stem cell-enriched, CD34+/CD38+/Lin+ progenitor-enriched populations, and total CD34+ HSPC (Georgantas, Cancer Research 64:4434) and (2) miR target predictions from various published algorithms. Combining these datasets into one integrated database allowed us to bioinformaticly examine the global interaction of HSPC mRNAs and miRs during hematopoiesis. The 3′UTR sequences from many of these “hematopoietic” mRNA were cloned behind a luciferase reporter. K562 cells were transfected with these luc-3′UTR constructs, confirmating that expression of many important hematopoietic proteins are controlled by miRs. Based on our bioinformatic and protein expression studies, we present a global in silico model by which microRNAs control and direct hematopoietic differentiation. Actual in vitro and in vivo studies addressing the action of specific miRs in hematopoietic differentiation are presented in separate abstracts.

Fibrinogen Enhances Homing-Related Responses of CD34+ by Incorporating Membrane Type1-Matrix Metalloproteinase into Membrane Lipid Rafts.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3188-3188
Author(s):  
Neeta Shirvaikar ◽  
A. Robert Turner ◽  
Mariusz Z. Ratajczak ◽  
Anna Janowska-Wieczorek
Keyword(s):  
Lipid Rafts ◽  
Stromal Cells ◽  
Leading Edge ◽  
Membrane Lipid ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Chemokine Gradient ◽  
Cord Blood Cells ◽  
First Time ◽  
Mobilized Peripheral Blood

Abstract Clinical studies have shown that transplanted mobilized peripheral blood (mPB) hematopoietic stem/progenitor cells (HSPC) home faster to the bone marrow (BM) than harvested BM or cord blood cells. We previously showed that fibrinogen has a priming effect on HSPC homing by enhancing the chemotactic responses of CD34+ cells towards a low SDF-1 gradient by incorporating CXCR4 into membrane lipid rafts (Blood2005; 105:40). In this study we further investigated the mechanism of priming by determining the effect of fibrinogen on matrix metalloproteinases (MMPs) expressed by CD34+ cells, particularly MT1-MMP which is localized on the leading edge of migrating cells and not only activates latent MMPs but by itself degrades several extracellular matrix (ECM) components such as collagens, laminin, fibrin and fibronectin. We found that fibrinogen highly upregulated MT1-MMP protein expression (Western blot) in CD34+ cells as well as proMMP-2 and proMMP-9 secretion (zymography); primed trans-Matrigel chemoinvasion of CD34+ cells towards a low SDF-1 gradient (20 ng/mL), which was inhibited by epigallocatechin-3-gallate, a potent inhibitor of MT1-MMP; and stimulated MMP-2 activation in co-cultures of stromal cells (BM fibroblasts and HUVEC) with CD34+ cells. Moreover, we demonstrate by confocal microscopy, for the first time, that in CD34+ cells MT1-MMP is localized in the GM1-fraction of lipid rafts where it co-localizes with CXCR4; this co-localization is enhanced when CD34+ cells are stimulated with fibrinogen. Furthermore, disruption of lipid raft formation by the cholesterol-depleting agent methyl-b-cyclodextrin inhibited MT1-MMP incorporation into membrane lipid rafts and also trans-Matrigel chemoinvasion of CD34+ cells towards an SDF-1 gradient. Thus we conclude that fibrinogen enhances homing-related responses of CD34+ cells towards SDF-1 by increased incorporation and co-localization of CXCR4 and MT1-MMP in membrane lipid rafts. Further, we postulate that while the presence of CXCR4 in lipid rafts allows the cells to better sense the SDF-1 chemokine gradient, the upregulated MT1-MMP in the lipid rafts facilitates their migration through the ECM and possibly towards the BM niches.

scholarly journals In Vitro and In Vivo Evidence That Ex Vivo Cytokine Priming of Donor Marrow Cells May Ameliorate Posttransplant Thrombocytopenia

Blood ◽  
1998 ◽  
Vol 91 (1) ◽  
pp. 353-359 ◽  
Author(s):  
Mariusz Z. Ratajczak ◽  
Janina Ratajczak ◽  
Boguslaw Machalinski ◽  
Rosemarie Mick ◽  
Alan M. Gewirtz
Keyword(s):  
Mononuclear Cells ◽  
Recovery Period ◽  
Hematopoietic Stem ◽  
Platelet Recovery ◽  
Serum Free ◽  
Cd34 Cells ◽  
In Vivo Animal Model ◽  
Marrow Cells

AbstractThrombocytopenia is typically observed in patients undergoing hematopoietic stem cell transplantation. We hypothesized that delayed platelet count recovery might be ameliorated by increasing the number of megakaryocyte colony- forming units (CFU-Meg) in the hematopoietic cell graft. To test this hypothesis, we evaluated cytokine combinations and culture medium potentially useful for expanding CFU-Meg in vitro. We then examined the ability of expanded cells to accelerate platelet recovery in an animal transplant model. Depending on the cytokine combination used, we found that culturing marrow CD34+cells for 7 to 10 days in serum-free cultures was able to expand CFU-Meg ∼40 to 80 times over input number. Shorter incubation periods were also found to be effective and when CD34+ cells were exposed to thrombopoietin (TPO), kit ligand (KL), interleukin-1α (IL-1α), and IL-3 in serum-free cultures for as few as 48 hours, the number of assayable CFU-Meg was still increased ∼threefold over input number. Of interest, cytokine primed marrow cells were also found to form colonies in vitro more quickly than unprimed cells. The potential clinical utility of this short-term expansion strategy was subsequently tested in an in vivo animal model. Lethally irradiated Balb-C mice were transplanted with previously frozen syngeneic marrow mononuclear cells (106/mouse), one tenth of which (105) had been primed with [TPO, KL, IL-1a, and IL-3] under serum-free conditions for 36 hours before cryopreservation. Mice receiving the primed frozen marrow cells recovered their platelet and neutrophil counts 3 to 5 days earlier than mice transplanted with unprimed cells. Mice which received marrow cells that had been primed after thawing but before transplantation had similar recovery kinetics. We conclude that pretransplant priming of hematopoietic cells leads to faster recovery of all hematopoietic lineages. Equally important, donor cell priming before transplant may represent a highly cost-effective alternative to constant administration of cytokines during the posttransplant recovery period.

Isolation and characterization of human CD34−Lin− and CD34+Lin− hematopoietic stem cells using cell surface markers AC133 and CD7

Blood ◽  
2000 ◽  
Vol 95 (9) ◽  
pp. 2813-2820 ◽  
Author(s):  
Lisa Gallacher ◽  
Barbara Murdoch ◽  
Dongmei M. Wu ◽  
Francis N. Karanu ◽  
Mike Keeney ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Surface Markers ◽  
Cell Surface Markers ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Cd34 Cells

Recent evidence indicates that human hematopoietic stem cell properties can be found among cells lacking CD34 and lineage commitment markers (CD34−Lin−). A major barrier in the further characterization of human CD34− stem cells is the inability to detect this population using in vitro assays because these cells only demonstrate hematopoietic activity in vivo. Using cell surface markers AC133 and CD7, subfractions were isolated within CD34−CD38−Lin− and CD34+CD38−Lin− cells derived from human cord blood. Although the majority of CD34−CD38−Lin− cells lack AC133 and express CD7, an extremely rare population of AC133+CD7− cells was identified at a frequency of 0.2%. Surprisingly, these AC133+CD7− cells were highly enriched for progenitor activity at a frequency equivalent to purified fractions of CD34+ stem cells, and they were the only subset among the CD34−CD38−Lin− population capable of giving rise to CD34+ cells in defined liquid cultures. Human cells were detected in the bone marrow of non-obese/severe combined immunodeficiency (NOD/SCID) mice 8 weeks after transplantation of ex vivo–cultured AC133+CD7− cells isolated from the CD34−CD38−Lin− population, whereas 400-fold greater numbers of the AC133−CD7− subset had no engraftment ability. These studies provide novel insights into the hierarchical relationship of the human stem cell compartment by identifying a rare population of primitive human CD34− cells that are detectable after transplantation in vivo, enriched for in vitro clonogenic capacity, and capable of differentiation into CD34+ cells.

High-level ectopic HOXB4 expression confers a profound in vivo competitive growth advantage on human cord blood CD34+ cells, but impairs lymphomyeloid differentiation

Blood ◽  
2003 ◽  
Vol 101 (5) ◽  
pp. 1759-1768 ◽  
Author(s):  
Bernhard Schiedlmeier ◽  
Hannes Klump ◽  
Elke Will ◽  
Gökhan Arman-Kalcek ◽  
Zhixiong Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cord Blood ◽  
Therapeutic Window ◽  
Hematopoietic Stem ◽  
Expression Levels ◽  
Cd34 Cells ◽  
Cord Blood Cd34 ◽  
The Impact

Ectopic retroviral expression of homeobox B4 (HOXB4) causes an accelerated and enhanced regeneration of murine hematopoietic stem cells (HSCs) and is not known to compromise any program of lineage differentiation. However, HOXB4 expression levels for expansion of human stem cells have still to be established. To test the proposed hypothesis that HOXB4 could become a prime tool for in vivo expansion of genetically modified human HSCs, we retrovirally overexpressed HOXB4 in purified cord blood (CB) CD34+ cells together with green fluorescent protein (GFP) as a reporter protein, and evaluated the impact of ectopic HOXB4 expression on proliferation and differentiation in vitro and in vivo. When injected separately into nonobese diabetic–severe combined immunodeficient (NOD/SCID) mice or in competition with control vector–transduced cells, HOXB4-overexpressing cord blood CD34+ cells had a selective growth advantage in vivo, which resulted in a marked enhancement of the primitive CD34+ subpopulation (P = .01). However, high HOXB4 expression substantially impaired the myeloerythroid differentiation program, and this was reflected in a severe reduction of erythroid and myeloid progenitors in vitro (P < .03) and in vivo (P = .01). Furthermore, HOXB4 overexpression also significantly reduced B-cell output (P < .01). These results show for the first time unwanted side effects of ectopic HOXB4 expression and therefore underscore the need to carefully determine the therapeutic window of HOXB4 expression levels before initializing clinical trials.

scholarly journals Erythropoietin-independent erythrocyte production: signals through gp130 and c-kit dramatically promote erythropoiesis from human CD34+ cells.

1996 ◽  
Vol 183 (3) ◽  
pp. 837-845 ◽  
Author(s):  
X Sui ◽  
K Tsuji ◽  
S Tajima ◽  
R Tanaka ◽  
K Muraoka ◽  
...  
Keyword(s):  
Stem Cell Factor ◽  
Current Data ◽  
Erythroid Cells ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Interleukin 6 Receptor ◽  
Human Sera ◽  
Stimulation Of

Erythropoietin (EPO) is the primary humoral regulator of erythropoiesis and no other factor has previously been reported to support proliferation and terminal maturation of erythroid cells from hemopoietic stem cells. Here we show that stimulation of glycoprotein (gp130) by a combination of recombinant human soluble interleukin 6 receptor (sIL-6R) and IL-6 but not sIL-6R or IL-6 alone can support proliferation, differentiation, and terminal maturation of erythroid cells in the absence of EPO from purified human CD34+ cells in suspension culture containing stem cell factor (SCF). A number of erythroid bursts and mixed erythroid colonies also developed in methylcellulose culture under the same combination. The addition of anti-gp130 monoclonal antibodies but not anti-EPO antibody to the same culture completely abrogated the generation of erythroid cells. These results clearly demonstrate that mature erythroid cells can be emerged from hemopoietic progenitors without EPO in vitro. Together with the previous reports that human sera contain detectable levels of sIL-6R, IL-6, and SCF, current data suggest that gp130 signaling in association with c-kit activation may play a role in human erythropoiesis in vivo.

Upregulation of MT1-MMP Expression by Hyaluronic Acid Enhances Homing-Related Responses of Hematopoietic CD34+ Cells to an SDF-1 Gradient.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2889-2889 ◽  
Author(s):  
Neeta Shirvaikar ◽  
Jencet Montano ◽  
Andrew R. Turner ◽  
Mariusz Z. Ratajczak ◽  
Anna Janowska-Wieczorek
Keyword(s):  
Hyaluronic Acid ◽  
Leading Edge ◽  
Cell Surface Receptor ◽  
Migration And Invasion ◽  
Hematopoietic Stem ◽  
Tumor Cell Migration ◽  
Cell Engraftment ◽  
Cd34 Cells ◽  
Surface Receptor ◽  
And Migration

Abstract Hyaluronic acid (HA), the bone marrow (BM) extracellular matrix microenvironment (ECM) component, not only supports cell adhesion but also promotes migration and homing of hematopoietic stem/progenitor cells (HSPC) by interacting with its cell surface receptor CD44. CD44 has been shown to co-localize with matrix metalloproteinases (MMPs), particularly membrane-type (MT)-1 MMP and MMP-9, at the leading edge of migrating tumor cells, and the cleavage of CD44 by MT1-MMP is critical for tumor cell migration and invasion. MT1-MMP has strong pericellular proteolytic activity and also activates latent forms of MMP-2 and MMP-9. In this study we examined the effect of HA on MT1-MMP expression and migration of BM, peripheral blood and cord blood CD34+ cells. We found that HA upregulates mRNA for MT1-MMP and MMP-9, increases MT1-MMP protein (as evaluated by Western blotting) and stimulates MMP-9 and MMP-2 activity (as determined by zymography) in CD34+ cells. In chemotaxis assays HA alone did not show any chemotactic activity but primed the chemotaxis of CD34+ cells to a low SDF-1 gradient (10 ng/mL) and their trans-Matrigel chemoinvasion to a low SDF-1 gradient. Similarly, SDF-1 besides stimulating MMP-2 and MMP-9 (as we previously described in Exp Hematol2000; 28:1274), also upregulated MT1-MMP in CD34+ cells. In addition, we found that the HA enhanced activation of latent MMP-2 in co-cultures of CD34+ cells with HUVEC. In conclusion, we demonstrate for the first time that (i) HA primes CD34+ cell chemotaxis and chemoinvasion to a low SDF-1 gradient; (ii) that both HA and SDF-1 stimulate MT1-MMP, MMP-2 and MMP-9 in CD34+ cells, and (iii) HA enhances activation of proMMP-2 in the ECM. Hence the interaction of HA and SDF-1 with MT1-MMP could play an important role in HSPC migration and homing and we postulate that pretreatment of HSPC with HA before transplantation could enhance their homing by inducing expression of MMPs that is optimal for stem cell engraftment.

Enforced Expression of GATA-2 Confers Quiescence on Human Hematopoietic Stem and Progenitor Cells In Vitro and In Vivo.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 83-83
Author(s):  
Alex J. Tipping ◽  
Cristina Pina ◽  
Anders Castor ◽  
Ann Atzberger ◽  
Dengli Hong ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Progenitor Cells ◽  
Zinc Finger ◽  
Human Cord Blood ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Stem And Progenitor Cells ◽  
Colony Number

Abstract Hematopoietic stem cells (HSCs) in adults are largely quiescent, periodically entering and exiting cell cycle to replenish the progenitor pool or to self-renew, without exhausting their number. Expression profiling of quiescent HSCs in our and other laboratories suggests that high expression of the zinc finger transcription factor GATA-2 correlates with quiescence. We show here that TGFβ1-induced quiescence of wild-type human cord blood CD34+ cells in vitro correlated with induction of endogenous GATA-2 expression. To directly test if GATA-2 has a causative role in HSC quiescence we constitutively expressed GATA-2 in human cord blood stem and progenitor cells using lentiviral vectors, and assessed the functional output from these cells. In both CD34+ and CD34+ CD38− populations, enforced GATA-2 expression conferred increased quiescence as assessed by Hoechst/Pyronin Y staining. CD34+ cells with enforced GATA-2 expression showed reductions in both colony number and size when assessed in multipotential CFC assays. In CFC assays conducted with more primitive CD34+ CD38− cells, colony number and size were also reduced, with myeloid and mixed colony number more reduced than erythroid colonies. Reduced CFC activity was not due to increased apoptosis, as judged by Annexin V staining of GATA-2-transduced CD34+ or CD34+ CD38− cells. To the contrary, in vitro cultures from GATA-2-transduced CD34+ CD38− cells showed increased protection from apoptosis. In vitro, proliferation of CD34+ CD38− cells was severely impaired by constitutive expression of GATA-2. Real-time PCR analysis showed no upregulation of classic cell cycle inhibitors such as p21, p57 or p16INK4A. However GATA-2 expression did cause repression of cyclin D3, EGR2, E2F4, ANGPT1 and C/EBPα. In stem cell assays, CD34+ CD38− cells constitutively expressing GATA-2 showed little or no LTC-IC activity. In xenografted NOD/SCID mice, transduced CD34+ CD38−cells expressing high levels of GATA-2 did not contribute to hematopoiesis, although cells expressing lower levels of GATA-2 did. This threshold effect is presumably due to DNA binding by GATA-2, as a zinc-finger deletion variant of GATA-2 shows contribution to hematopoiesis from cells irrespective of expression level. These NOD/SCID data suggest that levels of GATA-2 may play a part in the in vivo control of stem and progenitor cell proliferation. Taken together, our data demonstrate that GATA-2 enforces a transcriptional program on stem and progenitor cells which suppresses their responses to proliferative stimuli with the result that they remain quiescent in vitro and in vivo.

MT1-MMP Associates with Membrane Lipid Rafts and Is Required for G-CSF-Induced Hematopoietic Stem/Progenitor Cell Mobilization.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3536-3536
Author(s):  
Neeta Shirvaikar ◽  
Leah A. Marquez-Curtis ◽  
Andrew Shaw ◽  
A. Robert Turner ◽  
Anna Janowska-Wieczorek
Keyword(s):  
Flow Cytometry ◽  
Steady State ◽  
Western Blot ◽  
Lipid Rafts ◽  
Stromal Cells ◽  
Hematopoietic Cells ◽  
Membrane Lipid ◽  
Rt Pcr ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract Abstract 3536 Poster Board III-473 Hematopoietic stem/progenitor cells (HSPC) that have been mobilized from bone marrow (BM) to peripheral blood (PB) by granulocyte-colony stimulating factor (G-CSF) are being used for autologous and allogeneic transplantation. However, the molecular mechanisms of HSPC mobilization are not completely understood. The key molecules and interactions that regulate HSPC mobilization include various adhesion molecules, chemokine stromal cell-derived factor (SDF)-1 and its receptor CXCR4, and proteases including the soluble matrix metalloproteinase (MMP)-9. Membrane type (MT)-1 MMP, which is localized on the leading edge of migrating cells, has strong pericellular proteolytic activity, activates the latent MMPs especially proMMP-2, and has been implicated in mediating migration of tumor cells, monocytes, endothelial as well as CD34+ HSPC. MT1-MMP not only degrades several extracellular matrix molecules in the pericellular space, but also cleaves cell surface molecules such as CXCR4 and CD44, cytokines, and chemokines including SDF-1. In this study we focused on characterizing the role of MT1-MMP during G-CSF-induced migration, its regulation and subcellular localization in HSPC and mature cells. We found that MT1-MMP mRNA and protein expression (as determined by RT-PCR and flow cytometry) in G-CSF-mobilized mature hematopoietic cells (monocytes and neutrophils) as well as immature CD34+ cells was significantly higher than in their steady-state BM counterparts. Moreover, G-CSF stimulation (i) upregulated MT1-MMP transcription (RT-PCR) and protein synthesis (flow cytometry, Western blot, and confocal microscopy) in BM MNC and CD34+ cells but not in BM stromal cells; and (ii) increased their trans-Matrigel chemoinvasion towards an SDF-1 gradient which was inhibited by the MT1-MMP inhibitor epigallocatechin 3-gallate, by anti-MT1-MMP mAb, and by siRNA silencing of MT1-MMP. To determine the effect of high MT1-MMP expression in hematopoietic cells on the BM microenvironment we co-cultured steady-state BM CD34+ cells with BM fibroblasts. Zymographic analysis of the cell-conditioned media revealed that activation of proMMP-2 occurs only when the co-cultures were stimulated with G-CSF indicating that upregulation of MT1-MMP in CD34+ cells is necessary for proMMP-2 activation as media conditioned by CD34+ cells (silenced with MT1-MMP siRNA) co-cultured with stromal cells did not show proMMP-2 activation. We next focused on determining the signaling pathways that regulate MT1-MMP expression and localization in hematopoietic cells including HSPC during G-CSF-induced migration. We found that although G-CSF activated both phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling pathways (Western blot), upregulation of MT1-MMP by G-CSF, and proMMP-2 activation were PI3K-dependent. Moreover, we demonstrated for the first time that G-CSF incorporated MT1-MMP to membrane lipid rafts of hematopoietic cells in a PI3K-dependent manner since inhibition of this axis by PI3K inhibitor LY290042 reduced MT1-MMP incorporation, an effect not observed with the MAPK inhibitor PD98059. We further demonstrated that by disrupting raft formation using the cholesterol sequestering agent methyl-beta-cyclodextrin, PI3K phosphorylation was inhibited. Subsequently MT1-MMP incorporation into lipid rafts was abrogated resulting in reduced both proMMP-2 activation and HSPC trans-Matrigel migration. We conclude that G-CSF-induced upregulation of MT1-MMP and its incorporation into membrane lipid rafts of hematopoietic cells contributes to the activation of proMMP-2 and to the generation of a highly proteolytic microenvironment in BM, which facilitates egress of HSPC into circulation. Our results suggest that manipulating MT1-MMP expression could become a new strategy to enhance mobilization of HSPC and improve the outcome of transplantation. Disclosures: No relevant conflicts of interest to declare.

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