|
|||||||||
|
|
|
|
|
|
|
|
|
|
|
HEMATOPOIESIS
From the Department of Pathology and Laboratory
Medicine and the Department of Pediatrics, University of Pennsylvania
School of Medicine, Philadelphia, PA; the Department of Medicine,
University of Alberta, and Canadian Blood Services, Edmonton, Alberta,
Canada; and the Department of Molecular and Experimental Medicine, The
Scripps Research Institute, La Jolla, CA.
The role of the chemokine binding stromal-derived factor 1 (SDF-1)
in normal human megakaryopoiesis at the cellular and molecular levels
and its comparison with that of thrombopoietin (TPO) have not been
determined. In this study it was found that SDF-1, unlike TPO, does not
stimulate The immunoglobulin superfamily cytokine receptor
c-mpl binds thrombopoietin (TPO), and the 7-transmembrane
G-protein-coupled chemokine receptor CXCR4 binds stromal-derived
factor 1 (SDF-1).1-8 Targeted disruption of the
genes for TPO or c-mpl results in thrombocytopenia, and animals
defective in either SDF-1 or CXCR4 have a deficit in colony-forming
units-megakaryocyte (CFU-MK) in the adult marrow.1-4 Both
receptors are present in high concentrations on the surfaces of
developing megakaryocytes,1,2,9-11 but though the role of
c-mpl during megakaryopoiesis has been established, that of CXCR4 is
less clear. SDF-1 stimulates chemotaxis of early megakaryocytic progenitors and plays a role in the migration of megakaryocytic ( The aim of this study was to extend our understanding of the molecular
basis of megakaryopoiesis by investigating the biologic responses of
human megakaryocytic cells to stimulation by SDF-1 or TPO and the
intracellular signaling pathways involved (mitogen-activated protein
kinase [MAPK] p42/44, p38, JNK, AKT [protein kinase B], NF- Our results suggest that SDF-1 and TPO activate human megakaryoblastic
Human CD34+ cells, megakaryoblasts, and
platelets
BM CD34+ cells were expanded in a serum-free liquid system,
and growth of CFU-MK was stimulated with recombinant human (rh) TPO (50 ng/mL) and rhIL-3 (10 ng/mL) (both from R&D Systems, Minneapolis, MN)
as described.19,20,31 After incubation for 11 days at 37°C, approximately 85% of the expanded cells were positive for the
megakaryocytic-specific marker
Gel-filtered platelets were prepared from 4 persons as previously
described10,20 and used within 2 hours of preparation. Marrow aspiration from and blood donation by healthy volunteers was
carried out with donors' informed consent obtained through the
Institutional Review Board.
Cell cycle analysis and detection of apoptosis by Annexin V binding
assay, caspase-3 activation, and poly(ADP-ribose) polymerase
cleavage
Apoptosis was assessed by staining with FITC-Annexin V and flow cytometric analysis (FACScan; Becton Dickinson, Mountain View, CA) and by using the apoptosis detection kit (R&D Systems) according to the manufacturer's protocol. Activation of caspase-3 and poly(ADP-ribose) polymerase (PARP) cleavage was determined by FACS and Western blot analysis, respectively, according to the manufacturers' protocols (BD Pharmingen, San Diego, CA). Cellular extracts were assayed for telomerase activity using the PCR-based telomeric repeat amplification protocol assay as described.33 Chemotaxis, trans-Matrigel migration, Ca 2+ fluxes, and MMP and VEGF production Chemotaxis assays to SDF-1 (PeproTech, Rocky Hill, NJ; R&D Systems) or TPO (R&D) through an 8-µm pore filter were performed in Costar-Transwell 24-well plate (Costar Corning, Cambridge, MA) as described before.34 Results were calculated as a percentage of the input number of cells. All experiments were performed in triplicate.Chemoattraction of megakaryoblasts across Matrigel was evaluated
in a trans-Matrigel migration assay according to a method previously
described by us.35,36 The lower chambers were filled with
migration media containing 100 ng/mL SDF-1 Ca++ flux studies on ex vivo-expanded megakaryoblasts were performed using a spectrophotofluorimeter, as previously described.10,34 Secretion of VEGF by normal human megakaryoblastic cells was evaluated by the Quantikine human VEGF immunoassay (R&D) according to the manufacturer's protocol, as described.20
 IIb 3 receptors was
measured using the MoAb PAC-1 (Becton Dickinson) as previously
described.38 The
IIb3+ cells
(1 × 106) were washed twice with PBS, resuspended in 50 µL PBS plus 2% fetal bovine serum, and treated with the appropriate
ligands: thrombin (2 U/mL), SDF-1 (500 ng/mL), or TPO (100 ng/mL)
for 5 minutes. Subsequently, 20 µL FITC-conjugated PAC-1 was added, and the cells were incubated for 15 to 20 minutes at room temperature in the dark. RGDS peptide38 was added to confirm specific
binding of PAC-1 antibody. After staining, cells were analyzed by
FACstar and the Cell Quest program.
Adherence assays of Phosphorylation of intracellular pathway proteins Western blot analysis was performed on extracts prepared from humanIIb3+ cells
(1 × 107), which were kept in RPMI medium containing low
levels of BSA (0.5%) to render the cells quiescent. The cells were
then divided and stimulated with optimal doses of SDF-1 or SDF-1
(500 ng/mL) or TPO (100 ng/mL) for 1 minute to 2 hours at 37°C, and
cells were then lysed for 10 minutes on ice in M-Per lysing buffer
(Pierce, Rockford, IL) containing protease and phosphatase inhibitors
(Sigma). Subsequently, the extracted proteins were separated on either a 12% or 15% SDS-PAGE gel, and the fractionated proteins were transferred to a nitrocellulose membrane (Schleicher & Schuell, Keene,
NH) as previously described.20,34 Phosphorylation of each
of the intracellular kinases MAPK p44/42, MAPK p38, JNK MAPK, p90 RSK,
AKT, ELK-1 and STAT-1, -3, -5, and -6was detected using commercial
mouse phosphospecific monoclonal antibody (p44/42) or rabbit
phosphospecific polyclonal antibodies for each of the remainders (all
from New England Biolabs, Beverly, MA) with horseradish peroxidase-conjugated goat antimouse IgG or goat antirabbit IgG as a
secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA), as
described.13,26 Equal loading in the lanes was evaluated by stripping the blots and reprobing them with the appropriate monoclonal antibodies: p42/44 anti-MAPK antibody clone 9102, anti p38
MAPK antibody clone 9212, an anti-JNK antibody clone 9252, an anti-AKT
antibody clone 9272, an anti-ELK-1 antibody clone 9182, an
anti-STAT-3 9132 (New England Biolabs), an anti-STAT-1 sc-464 and
STAT-6 sc-1689 (Santa Cruz Biotechnology), an anti-STAT-5 89 or p90
RSK 78 (Transduction Laboratories, Lexington, KY). The membranes were
developed with an ECL reagent (Amersham Life Sciences, Little Chalfont,
UK) and subsequently dried and exposed to film (HyperFilm; Amersham
Life Sciences). Densitometric analysis was performed using exposures
that were within the linear range of the densitometer (Personal
Densitometer SI; Molecular Dynamics, Sunnyvale, CA) and ImageQuant
software (Molecular Dynamics).
Blocking of PI-3K and MEK PI-3K and MAPK p42/244 activities were blocked by selective inhibitors. To assess the effects of the PI-3K inhibitor LY294002 (Sigma) or the MEK inhibitor U0126 (Calbiochem, La Jolla, CA), cells were preincubated with each of these compounds for 30 minutes before SDF-1 or TPO stimulation.
Electrophoretic mobility shift assay To evaluate NF- B pathways, nuclear extracts were prepared
from normal human megakaryoblasts using a modified method of Pan et
al,41 and electrophoretic mobility shift assay (EMSA) was performed using 2.5 µg nuclear extract as described
previously.41 Oligonucleotides and their complementary
strands for EMSA were obtained from Promega (Madison, WI) and Santa
Cruz Biotechnology. The sequences were a consensus B site
(underlined), 5'-AGTTGAGGGGACTTTCC CAGGC-3'
(NF-B).41,42 [ -32 P]ATP (greater than
500 Ci/mmol) was from Amersham Pharmacia Biotech.
Statistical analysis Arithmetic means and standard deviations of our FACS and chemotaxis data were calculated on a Macintosh computer PowerBase 180 (Apple, Cupertino, CA), using Instat 1.14 (GraphPad, San Diego, CA) software.
SDF-1 does not affect proliferation of human CFU-MK and megakaryoblasts It has been unknown whether SDF-1 stimulates human CFU-MK colony formation. In our previous studies, we used a plasma clot system10 that might have contained confounding growth factors. Hence, to remove the unwanted influence of growth factors and cytokines in a serum, we now evaluated the growth of CFU-MK orIIb3+ cells stimulated with
SDF-1 or TPO in cultures supplemented with chemically defined
artificial serum.18-20,30 SDF-1 was found not to have any
effect, either on its own or acting as a costimulator with TPO, on the
formation of human CFU-MK colonies from CD34+ cells (Table
1). In addition, when human
megakaryoblastic IIb3+ cells
were expanded ex vivo from CD34+ cells (in the presence of
a suboptimal or an optimal dose of TPO + IL-3) and SDF-1 was added as
a costimulator, we did not observe SDF-1 to affect proliferation or
maturation of these cells at days 6 and 11 of culture (Table
2). Neither the number of IIb3+ cells in the cultures
nor the intensity of expression of
IIb3+ on the cell surface
changed (data not shown). Because it has been recently suggested that
SDF-1 may stimulate proliferation of CD34+ cells if used at
very low concentrations,43 we repeated these experiments
using low doses of SDF-1 (10 pg to 1 ng/mL) and still did not observe
any effect on cell proliferation (data not shown).
Cell cycle analysis of megakaryoblasts growing in the presence of SDF-1
or TPO was also undertaken. The
SDF-1 does not affect survival of human
IIb3+ cells is unknown. To
address this issue, human
IIb3+ cells were cultured for
12 hours in the absence or presence of TPO or SDF-1. We found that
cells that were withdrawn from TPO + IL-3 underwent apoptosis after
12 hours, as evidenced by an Annexin V binding assay (Figure
1Ai).46 The presence
of TPO in the culture media prevented these cells from undergoing
apoptosis (Figure 1Aii), but the presence of SDF-1 did not (Figure
1Aiii). Megakaryoblastic cells cultured in the presence of SDF-1 bound Annexin V to the same extent as cells cultured without TPO + IL-3 (Figure 1Aiii). These data clearly indicate that SDF-1 does not maintain or enhance megakaryopoiesis.
Because PI-3K (a potential target for TPO signaling) plays an important role in inhibiting apoptosis in human hematopoietic cells,47-50 normal human megakaryoblasts were exposed to the PI-3K inhibitor LY294002, and we found that the inhibition of PI-3K activity resulted in increases in Annexin V binding of cells (not shown), activation of caspase-3 (Figure 1B) and PARP cleavage (Figure 1C). Of note, inhibition of the MAPK p42/44 pathway by the MEK inhibitor (UO126) did not affect the survival of human megakaryoblasts (not shown). SDF-1 but not TPO induces Ca++ flux, chemotaxis, trans-Matrigel chemoattraction, MMP-9, and VEGF production Next we extended our studies to define the roles of SDF-1 and TPO in the homing of human megakaryoblastic cells by examining their effects on chemotaxis, Ca++ fluxes, trans-Matrigel chemoattraction, and production of MMP and VEGF.We have reported that SDF-1 induces Ca++ flux in human
megakaryoblasts,10 and TPO has been shown to enhance the
platelet reactivity of other agonists.51 Because the
chemotactic ability of TPO had not yet been studied in human
megakaryoblasts, we investigated whether TPO could induce
Ca++ flux in these cells. We found that in comparison with
SDF-1, which stimulated a measurable Ca++ flux in these
cells, TPO added at various concentrations (physiological and high) did
not (Figure 2A). Moreover, having
previously shown that SDF-1 is a strong chemoattractant for human
megakaryocytes,10 we examined whether TPO could attract
these cells as well. We found that TPO, in contrast to SDF-1, did not
attract human
Because normal human megakaryoblasts have been shown to secrete
VEGF15,16 and because endogenously secreted VEGF plays an
important role in the transendothelial migration of
megakaryocytes,15 we next evaluated whether SDF-1 or TPO
has any effect on the secretion of VEGF by normal human
SDF-1 and TPO activate IIb3 integrin on human megakaryoblasts (data not shown), which is consistent with previous findings by others
that TPO enhances platelet reactivity51 and with our recent
observations that SDF-1 also stimulates it.53 Consistent with these observations, we found that both SDF-1 and TPO increased the
adherence of IIb3 cells to fibrinogen
(Figure 3). Both cytokines, if added
together, also induced adhesiveness to vitronectin (Figures 3,
4). Of note, adhesion of human
megakaryoblasts was inhibited when the cells were pretreated with the
PI-3K inhibitor, suggesting again the involvement of PI-3K in this
process. Interestingly, the adhesion of human differentiating
IIb3+ cells to fibronectin
and VCAM-1 was weak and not affected by SDF-1 or TPO (data not shown).
Thus, it appears that both SDF-1 and TPO increase the adhesion of
megakaryoblasts to their microenvironment.
Phosphorylation of MAPK (p42/44 and p38) and AKT in normal human megakaryoblasts is induced by SDF-1 and TPO To explain the molecular basis of the different biologic effects of SDF-1 and TPO, we examined the intracellular signaling pathways induced by these cytokines in human megakaryocytic cells. It has been reported that the intracellular kinase MAPK p42/44 is phosphorylated in human cell lines, platelets, and murine megakaryoblasts after stimulation by both TPO or SDF-1.23-26 In this study, we examined the MAP kinases (p42/44, p38, and JNK) that have been reported to play an important role in regulating cell proliferation,54 including the intensity and kinetics of their activation in normal human megakaryoblasts. We found that both SDF-1 and TPO induced strong phosphorylation of MAPK p42/44 (Figure 5A); however, after stimulation with SDF-1, it was phosphorylated faster than with TPO (peak at 1 minute for SDF-1 vs 10 minutes for TPO) and more intensely (21- ± 6- vs 11- ± 3-fold increases, respectively) (Figure 5C). We correctly predicted that the activation of MAPK p42/44 should lead to phosphorylation of several MAPK substrates (p90 RSK and ELK-1), and this was confirmed for p90 RSK (Figure 5B) and ELK-1 (not shown). Again, though both SDF-1 and TPO stimulated strong phosphorylation of both substrates, SDF-1 induced an earlier and more intense response.
We next tested whether other members of the MAPK family (p38, JNK) are
activated in AKT is a serine-threonine kinase that plays an important role in the
phosphorylation of several antiapoptotic proteins that may be key to
normal hematopoiesis.47-50 It has been reported that integrin stimulation of human
AKT is also involved in the activation of NF-
SDF-1 induces phosphorylation of MAPK p42/44 and AKT in human CD34+ cells but not in platelets We also investigated the responsiveness of CD34+ cells and circulating platelets to stimulation by SDF-1 or TPO. Examining CD34+ cells, we found that though both factors induced the phosphorylation of MAPK p42/44 and AKT (Figure 8A,B), only TPO stimulated the proliferation of these cells (Table 1) and, as reported previously, protected them from undergoing apoptosis.1,44 This observation again suggests that SDF-1 is not primarily directed toward maintaining or enhancing cell proliferation. In contrast to CD34+ andIIb3+
cells, the stimulation of human platelets by SDF-1 under similar conditions did not lead to phosphorylation of MAPK p42/44 and AKT
(Figure 9A,B). This observation supports
our hypothesis that the responsiveness of CXCR4 to stimulation by SDF-1
(phosphorylation of MAPK p42/44, and AKT) decreases in the final stages
of megakaryocytopoiesis-thrombocytopoiesis. In contrast, TPO
stimulation of human platelets resulted in the phosphorylation of MAPK
p42 and AKT (Figure 9).
SDF-1, in contrast to TPO, does not induce tyrosine phosphorylation of STAT family proteins in human megakaryoblasts Despite the fact that the stimulation of humanIIb3+ cells with SDF-1 led to
the phosphorylation of MAPK p42/44, p38 and the nuclear protein ELK-1,
SDF-1 (as shown above) had no effect on the proliferation or maturation
of normal human megakaryoblasts. To understand the molecular basis of
these findings, we looked at the activation of the JAK-STAT pathways in
normal human IIb3+ cells.
STAT proteins have been shown to play an important role in regulating
cell proliferation5,21,27,54 and in signaling from the
activated c-mpl receptor in various hematopoietic cell lines1,27 and normal human platelets.22,23 In
particular, the stimulation of human platelets by TPO led to the
phosphorylation of STAT-1, STAT-2, STAT-3, and STAT-5
proteins28 and of STAT-3 and STAT-5 in FDCP-2 cells
genetically engineered to constitutively express human
c-mpl.27 However, the effects of SDF-1 on the phosphorylation of STAT proteins in human megakaryocytic cells have not
been studied.
We focused our studies on the phosphorylation of STAT-1 and STAT-3 at
both Tyr705 and Ser727 and of STAT-5 and STAT-6 (Figures 10, 11)
in human megakaryocytes, and we found that among these proteins, only
STAT-3 at Ser727 was phosphorylated after stimulation by SDF-1 (Figure
11B). TPO, however, caused phosphorylation of all the STAT proteins
tested (Figures 10, 11), consistent with previous studies of human
blood platelets.28,29 The phosphorylation of STAT proteins
was maximal 10 minutes after stimulation by TPO. The fact that multiple
STAT proteins were not phosphorylated in
SDF-1, in contrast to TPO, does not induce tyrosine phosphorylation of STAT proteins in human CD34+ cells and platelets Finally, we investigated the responsiveness of both human CD34+ cells and circulating platelets to stimulation by SDF-1 or TPO. We found that only TPO stimulated the phosphorylation of STAT-5 (Figures 8C, 9D) and STAT-3 at Tyr705 (Figure 9C) in these cells. This finding for human CD34+ cells (Figure 8C) may also explain why TPO, but not SDF-1, stimulates the proliferation of CD34+ cells.
Data on CXCR4 or SDF-1 knockout mice1,3,4 and recent findings suggesting that SDF-1 enhances the effect of TPO9,17 on megakaryocyte formation indicate a potential role for SDF-1 in megakaryopoiesis. However, our previous work10 and that of others11 provided evidence that though SDF-1 stimulated homing features in human megakaryocytic progenitors, it did not influence CFU-MK formation and growth. These observations prompted us to compare the effects of SDF-1 with those of TPO on development and activation of human megakaryocytic cells and to study the intracellular signaling pathways activated by these cytokines to better define the molecular basis for the observed differences. We found that the stimulation of human CD34+ cells and megakaryoblasts by SDF-1 does not influence proliferation, apoptosis, or telomerase activity, even when the SDF-1 is added with TPO. Hence, our data collectively suggest that, unlike TPO, SDF-1 is not a megakaryopoietic growth factor. This agrees with a recent report demonstrating lack of a proliferative effect by SDF-1 on CFU-MK formation from human CD34+ cells.11 However, it does not support recent observations on a murine model suggesting that SDF-1 acts together with TPO to enhance the development of CFU-MK17 and that SDF-1 at low doses enhances the proliferation of peripheral blood CD34+ cells.43 We suggest that this difference in results may be due to the different culture systems used (serum-free medium vs serum-supplemented medium) or to the different target cells (human vs murine marrow cells). We next evaluated the role of SDF-1 and TPO in the homing of human
megakaryoblastic cells. We found that TPO, in contrast to SDF-1, does
not induce Ca++ flux and is not a chemoattractant for human
To find an explanation at the molecular level for the differences in
the biologic effects of SDF-1 and TPO, we investigated signal
transduction pathways activated in normal human megakaryoblasts by
these cytokines. First, we found that the stimulation of human AKT has been reported to be important in preventing apoptosis during
hematopoiesis.47-50 In agreement with this observation, we
have now demonstrated that AKT is phosphorylated in normal human
megakaryoblasts after stimulation by both SDF-1 and TPO in a
PI-3K-dependent manner and that the inhibition of PI-3K activity by
LY294002 induced apoptosis in these cells. However, though both factors
activate the PI-3K-AKT axis, our in vitro data showed that only TPO
protected Because TPO has been shown to be crucial for the proliferation and differentiation of developing murine megakaryocytes through JAK-STAT pathways,24,27 we investigated whether similar pathways are activated after SDF-1 stimulation. Identification of these pathways could shed more light on the regulation of proliferation of normal human megakaryocytic cells and explain at a molecular level why TPO and not SDF-1, as demonstrated in this study, stimulated the proliferation of these cells. We found that only STAT-3 was phosphorylated at the serine residue (Ser727) after SDF-1 stimulation, in contrast to much of the STAT family of proteins, which are phosphorylated by TPO at tyrosine residues in megakaryocytic cells.27-29 Because it has been suggested that the phosphorylation of STAT-3 at Ser727 may play a role in the down-regulation of STAT-3 protein activation,61 the phosphorylation of STAT-3 at Ser727 by SDF-1 suggests that it may down-regulate STAT-3 in normal human megakaryoblasts. Hence, we suggest that the tyrosine phosphorylation of JAK-STAT proteins probably plays a crucial role in the proliferation of megakaryocytic cells after TPO stimulation but that activation of the MAPK p42/44 and p38 pathways is not an important intermediate step in the proliferation of these cells given that SDF-1 also activates them. Of note, we found that the inhibition of MEK by UO126 affected neither survival nor proliferation of human megakaryoblasts. These data are consistent with recent studies showing that the MAPK p42/44 pathway is not required for megakaryoblast formation, though it may regulate the transition from proliferation to maturation in this lineage.21,24 In contrast, the phosphorylation of MAPK p42/44, p38, and AKT after stimulation with SDF-1 does not occur in human platelets, and we find this intriguing. We suggest that the differences between human megakaryoblasts and platelets in the composition of G and RGS proteins, coupled to the particular chemokine receptor, could explain these differences.62 In summary, we demonstrated that though both TPO and SDF-1 are important in megakaryopoiesis and stimulate some of the same intracellular pathways, they have distinct biologic effects on human megakaryocytic cells. SDF-1, but not TPO, regulates some steps in the migration of these cells in the hematopoietic microenvironment (eg, chemotaxis and secretion of MMP-9 and VEGF). In contrast, TPO, but not SDF-1, permits growth of megakaryocyte precursors (eg, by enhancing proliferation and by inhibiting apoptosis); both factors regulate their adhesion. Hence, this study sheds light on the relation between 2 distinct cytokine axes critical in human megakaryopoiesis and the molecular basis of the observed differences in cellular responses.
Submitted March 10, 2000; accepted August 21, 2000.
Supported by National Institutes of Health grant R01 HL61796-01 (M.Z.R., M.A.K., and M.P.), Leukemia and Lymphoma Society grant 64907-00 (M.Z.R.), and Canadian Blood Services Research and Development grant XE 0004 (A.J.-W.).
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Mariusz Z. Ratajczak, Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, University of Pennsylvania, 405A Stellar Chance Labs, 422 Curie Blvd, Philadelphia PA 19104; e-mail: mariusz{at}mail.med.upenn.edu.
1.
Kaushansky K.
Thrombopoietin: the primary regulator of platelet production.
Blood.
1995;86:419-431
2.
Debili N, Wendling F, Cosman D, et al.
The mpl receptor is expressed in the megakaryocytic lineage from late progenitors to platelets.
Blood.
1995;85:391-401 3. Nagasawa T, Hirota S, Tachibana K, et al. Defects of B-cell lymphopoiesis and bone-marrow myelopoiesis in mice lacking the CXC chemokine PBSF/SDF-1. Nature. 1996;382:635-638[Medline] [Order article via Infotrieve]. 4. Zou Y, Kottman AH, Kuroda M, Taniuchi I, Littman DR. Function of the chemokine receptor CXCR4 in haematopoiesis and in cerebellar development. Nature. 1998;393:595-599[Medline] [Order article via Infotrieve]. 5. Blalock WL, Weinstein-Oppenheimer C, Chang F, et al. Signal transduction, cell cycle regulatory, and anti-apoptotic pathways regulated by IL-3 in hematopoietic cells: possible sites for intervention with anti-neoplastic drugs. Leukemia. 1999;13:1109-1166[Medline] [Order article via Infotrieve].
6.
Rollins BJ.
Chemokines.
Blood.
1997;90:909-928
7.
Lefkowitz PJ.
G protein-coupled receptors, III: new roles for receptor kinase and
8.
Ji TH, Grossmann M, Ji J.
G protein-coupled receptors.
J Biol Chem.
1998;273:17299-17302
9.
Wang JF, Liu ZY, Groopman JE.
The 10. Kowalska MA, Ratajczak J, Hoxie J, et al. Platelet and megakaryocytes express the HIV co-receptor CXCR4 on their surface but do not respond to stromal derived factor (SDF)-1. Br J Haematol. 1999;104:220-229[Medline] [Order article via Infotrieve].
11.
Riviere C, Subara F, Cohen-Solal K, et al.
Phenotypic and functional evidence for the expression of CXCR4 receptor during megakaryocytopoiesis.
Blood.
1999;93:1511-1523
12.
Aiuti A, Webb IJ, Bleul C, Springer T, Gutierrez-Ramos JC.
The chemokine SDF-1 is a chemoattractant for human CD34+ hematopoietic progenitor cells and provides a new mechanism to explain the mobilization of CD34+ progenitors to peripheral blood.
J Exp Med.
1997;185:111-120
13.
Dutt P, Wang JF, Groopman JE.
Stromal cell-derived factor-1a and stem cell factor/kit ligand share signaling pathways in hemopoietic progenitors: a potential mechanism for cooperative induction of chemotaxis.
J Immunol.
1998;161:3652-3658
14.
Mohle R, Bautz F, Rafii S, Moore MA, Brugger W, Kanz L.
The chemokine receptor CXCR4 is expressed on CD34+ hematopoietic progenitors and leukemic cells and mediates transendothelial migration induced by stromal cell-derived factor-1.
Blood.
1998;91:4523-4530
15.
Naiyer AJ, Jo DY, Ahn J, et al.
Stromal derived factor-1 induced chemokinesis of cord blood CD34+ cells (long-term culture-initiating cells) through endothelial cells is mediated by E-selectin.
Blood.
1999;94:4011-4019
16.
Hamada T, Mohle R, Hesselgesser J, et al.
Transendothelial migration of megakaryocytes in response to stromal cell-derived factor-1 (SDF-1) enhances platelet formation.
J Exp Med.
1998;188:539-548
17.
Hodohara K, Fujii N, Yamamoto N, Kaushansky K.
Stromal cell-derived factor-1 (SDF-1) acts together with thrombopoietin to enhance the development of megakaryocytic progenitor cells (CFU-MK).
Blood.
2000;95:769-775
18.
Ratajczak MZ, Ratajczak J, Machalanski B, Mick R, Gewirtz AM.
In vitro and in vivo evidence that ex vivo cytokine priming of donor marrow cells may ameliorate post-transplant thrombocytopenia.
Blood.
1998;91:353-359 19. Ratajczak J, Machalinski B, Samuel A, et al. A novel serum-free system for cloning human megakaryocytic progenitors (CFU-M): the role of thrombopoietin and other cytokines on bone marrow and cord blood CFU-M growth under serum free conditions. Folia Histochem Cytobiol. 1998;36:55-60[Medline] [Order article via Infotrieve].
20.
Majka M, Rozmyslowicz T, Lee B, et al.
Bone marrow CD34+ cells and megakaryoblasts secrete
21.
Fichelson S, Freyssinier JM, Picard F, et al.
Megakaryocyte growth and developmental factor-induced proliferation and differentiation are regulated by the mitogen-activated protein kinase pathway in primitive cord blood hematopoietic progenitors.
Blood.
1999;94:1601-1613
22.
Faraday N, Rade JJ, Johns DC, et al.
Ex vivo cultured megakaryocytes express functional glycoprotein IIb-IIIa receptors and are capable of adenovirus-mediated transgene expression.
Blood.
1999;94:4084-4092
23.
Rojnuckarin P, Drachman JG, Kaushansky K.
Thrombopoietin-induced activation of the mitogen-activated protein kinase (MAPK) pathway in normal megakaryocytes: role in endomitosis.
Blood.
1999;94:1273-1282
24.
Drachman JG, Millett KM, Kaushansky K.
Thrombopoietin signal transduction requires functional JAK2 not TYK2.
J Biol Chem.
1999;274:13480-13484
25.
Ganju RK, Brubaker SA, Meyer J, et al.
The 26. Banfic H, Downes CP, Rittenhouse SE. Biphasic activation of PKBa/AKT in platelets: evidence for stimulation both by phosphatidylinositol 3,4-biphosphate, produced via a novel pathway, and by phosphatidylinositol 3,4,5-triphosphate. J Biol Chem. 1998;271:11630-11637.
27.
Liu RY, Fan C, Garcia R, Jove R, Zuckerman KS.
Constitutive activation of JAK2/STAT5 signal transduction pathway correlates with growth factor independence of megakaryocytic leukemic cell lines.
Blood.
1999;93:2369-2379
28.
Miyakawa Y, Oda A, Druker BJ, et al.
Thrombopoietin and thrombin induce tyrosine phosphorylation of vav in human blood platelets.
Blood.
1997;89:2789-2798
29.
Miyakawa Y, Oda A, Druker BJ, et al.
Thrombopoietin induces tyrosine phosphorylation of Stat3 and Stat5 in human blood platelets.
Blood.
1996;87:439-446
30.
Ratajczak J, Zhang Q, Pertusini E, Wojczyk B, Wasik M, Ratajczak MZ.
The role of insulin (INS) and insulin like growth factor-I (IGF-I) in regulating human erythropoiesis: studies in vitro under serum free conditions
31.
Lee B, Ratajczak J, Doms RW, Gewirtz AM, Ratajczak MZ.
Coreceptor/chemokine receptor expression on human hematopoietic cells: biological implications for HIV-1 infection.
Blood.
1999;93:1145-1156 32. Mariallo A, Diaz P, Blanco A, et al. Properties of S-phase tumor cells measured by flow cytometry is an independent prognostic factor in meningioma tumors. Cytometry. 1999;38:118-123[Medline] [Order article via Infotrieve].
33.
Kim NW, Wu F.
Advances in quantitation and characterization of telomerase activity by the telomeric repeat amplification protocol (TRAP).
Nucleic Acids Res.
1997;25:2595-2597 34. Majka M, Ratajczak J, Lee B, et al. The role of HIV-related chemokine receptors and chemokines in human erythropoiesis in vitro. Stem Cells. 2000;18:128-138[Medline] [Order article via Infotrieve]. 35. Janiak M, Hashmi HR, Janowska-Wieczorek A. Use of the Matrigel-based assay to measure the invasiveness of leukemic cells. Exp Hematol. 1994;22:559-565[Medline] [Order article via Infotrieve].
36.
Janowska-Wieczorek A, Marquez LA, Nabholtz J-M, et al.
Growth factors and cytokines up-regulate gelatinase expression in bone marrow CD34+ cells and their transmigration through reconstituted basement membrane.
Blood.
1999;93:3379-3390 37. Janowska-Wieczorek A, Marquez LA, Matsuzaki A, et al. Expression of matrix metalloproteinases (MMP-2 and -9) and tissue inhibitors of metalloproteinases (TIMP-1 and -2) in acute myelogenous leukaemia blasts: comparison with normal bone marrow cells. Br J Haematol. 1999;105:402-411[Medline] [Order article via Infotrieve].
38.
Shattil SJ, Hoxie JA, Cunningham M, Brass LF.
Changes in the platelet membrane glycoprotein IIb/IIIa complex during platelet activation.
J Biol Chem.
1985;260:11107-11114
39.
Loh E, Beaverson K, Vilaire G, Qi W, Poncz M, Bennett JS.
Agonist-stimulated ligand binding by the platelet integrin 40. Bellavite P, Andrioli G, Guzzo P, et al. A colorimetric method for the measurements of platelet adhesion in microtiter plates. Anal Biochem. 1994;216:444-450[Medline] [Order article via Infotrieve].
41.
Pan ZK, Christiansen SC, Ptasznik A, Zuraw BL.
Requirement of phosphatidylinositol 3-kinase activity for bradykinin stimulation of NF-
42.
Ghosh S, May MJ, Kopp EB.
NF-
43.
Lataillade J-J, Clay D, Dupuy C, et al.
Chemokine SDF-1 enhances circulating CD34+ cell proliferation in synergy with cytokines: possible role in progenitor survival.
Blood.
2000;95:756-768 44. Ratajczak MZ, Ratajczak J, Marlicz W, et al. Recombinant human thrombopoietin (Tpo) stimulates erythropoiesis by inhibiting erythroid progenitor cell apoptosis. Br J Haematol. 1997;98:8-17[Medline] [Order article via Infotrieve]. 45. Broudy VC, Kaushansky K. Biology of thrombopoietin. Curr Opin Pediatr. 1998;10:60-64[Medline] [Order article via Infotrieve]. 46. van Engeland M, Nielwand LJ, Ramaekens FC, Schutte B, Reutelingsperger A. Annexin V affinity assay: a review on an apoptosis detection system based on phosphatidylserine exposure. Cytometry. 1998;31:1-9[Medline] [Order article via Infotrieve]. 47. Datta SR, Dudek H, Tao X, et al. Akt phosphorylation of BAD couples survival signals to the cell intrinsic death machinery. Cell. 1997;91:231-241[Medline] [Order article via Infotrieve].
48.
Songyang Z, Baltimore D, Cantley LC, Kaplan DR, Franke TF.
Interleukin-3 dependent survival by the Akt protein kinase.
Proc Natl Acad Sci U S A.
1997;94:11345-11350 49. Khwaja A. Akt is more than just Bad kinase. Science. 1999;401:33-34.
50.
Kelly TW, Graham MM, Doseff AI, et al.
Macrophage colony stimulating factor promotes cell survival through Akt/protein kinase.
J Biol Chem.
1999;274:26393-26398 51. Kojima H, Hamazaki Y, Nagata Y, Todokoro K, Nagasawa T, Abe T. Modulation of platelet activation in vitro by thrombopoietin. Thromb Haemost. 1995;74:1541-1545[Medline] [Order article via Infotrieve].
52.
Wang J-F, Park I-W, Groopman JE.
Stromal cell-derived factor-1
53.
Kowalska MA, Ratajczak MZ, Majka M, Brass LW, Poncz M.
SDF-1 and MDC: complementary chemokines at the crossroads between inflammation and thrombosis.
Blood.
2000;96:50-57
54.
Gutkind JS.
The pathways connecting G protein-coupled receptors to the nucleus through divergent mitogen-activated protein kinase cascades.
J Biol Chem.
1998;273:1839-1842
55.
Mohle R, Green D, Moore MAS, Nachman RL, Rafii S.
Constitutive production and thrombin-induced release of vascular endothelial growth factor by human megakaryocytes and platelets.
Proc Natl Acad Sci U S A.
1997;94:663-668 56. Ratajczak MZ, Ratajczak J, Machalinski B, et al. Role of vascular endothelial growth factor (VEGF) and placenta-derived growth factor (PlGF) in regulating human haematopoietic growth. Br J Haematol. 1998;103:969-979[Medline] [Order article via Infotrieve]. 57. Peled A, Grabovsky V, Habler L, et al. The chemokine SDF-1 stimulates integrin-mediated arrest of CD34+ cells on vascular endothelium under shear flow. J Clin Invest. 1999;104:1199-1211[Medline] [Order article via Infotrieve].
58.
Hinton HJ, Welham MJ.
Cytokine-induced protein kinase B activation and Bad phosphorylation do not correlate with cell survival of hemopoietic cells.
J Immunol.
1999;162:7002-7009 59. Bond M, Fabunmi RP, Baker AH, Newby AC. Synergistic up-regulation of metalloproteinase-9 by growth factors and inflammatory cytokines: an absolute requirement for transcriptional factor NF-kappa B. FEBS Lett. 1998;435:29-34[Medline] [Order article via Infotrieve]. 60. Yoshida S, Ono M, Shono T, et al. Involvement of interleukin-8, vascular endothelial growth factor, and basic fibroblast growth factor in tumor necrosis factor alpha-dependent angiogenesis. Mol Cell Biol. 1997;17:4015-4023[Abstract].
61.
Lim CP, Cao X.
Serine phosphorylation and negative regulation of STAT3 by JNK.
J Biol Chem.
1999;274:31055-31061
62.
Dohlman HG, Thorner J.
RGS proteins and signaling by heterotrimeric G proteins.
J Biol Chem.
1997;272:3871-3874
© 2000 by The American Society of Hematology.
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
|
 |
|
  M. P. Lambert, Y. Wang, K. H. Bdeir, Y. Nguyen, M. A. Kowalska, and M. Poncz Platelet factor 4 regulates megakaryopoiesis through low-density lipoprotein receptor-related protein 1 (LRP1) on megakaryocytes Blood, September 10, 2009; 114(11): 2290 - 2298. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. T. Scupoli, M. Donadelli, F. Cioffi, M. Rossi, O. Perbellini, G. Malpeli, S. Corbioli, F. Vinante, M. Krampera, M. Palmieri, et al. Bone marrow stromal cells and the upregulation of interleukin-8 production in human T-cell acute lymphoblastic leukemia through the CXCL12/CXCR4 axis and the NF-{kappa}B and JNK/AP-1 pathways Haematologica, April 1, 2008; 93(4): 524 - 532. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Freson, K. Peeters, R. De Vos, C. Wittevrongel, C. Thys, M. F. Hoylaerts, J. Vermylen, and C. Van Geet PACAP and its receptor VPAC1 regulate megakaryocyte maturation: therapeutic implications Blood, February 15, 2008; 111(4): 1885 - 1893. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-Q. Dai, X.-J. Zhu, F.-Q. Liu, J.-H. Xiang, H. Nagasawa, and W.-J. Yang Involvement of p90 Ribosomal S6 Kinase in Termination of Cell Cycle Arrest during Development of Artemia-encysted Embryos J. Biol. Chem., January 18, 2008; 283(3): 1705 - 1712. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Z. Pasha, Y. Wang, R. Sheikh, D. Zhang, T. Zhao, and M. Ashraf Preconditioning enhances cell survival and differentiation of stem cells during transplantation in infarcted myocardium Cardiovasc Res, January 1, 2008; 77(1): 134 - 142. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. P. Lambert, L. Rauova, M. Bailey, M. C. Sola-Visner, M. A. Kowalska, and M. Poncz Platelet factor 4 is a negative autocrine in vivo regulator of megakaryopoiesis: clinical and therapeutic implications Blood, August 15, 2007; 110(4): 1153 - 1160. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Y. Yoon, Z. Liang, X. Zhang, M. Choe, A. Zhu, H. T. Cho, D. M. Shin, M. M. Goodman, Z. Chen, and H. Shim CXC Chemokine Receptor-4 Antagonist Blocks Both Growth of Primary Tumor and Metastasis of Head and Neck Cancer in Xenograft Mouse Models Cancer Res., August 1, 2007; 67(15): 7518 - 7524. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Raslova, V. Baccini, L. Loussaief, B. Comba, J. Larghero, N. Debili, and W. Vainchenker Mammalian target of rapamycin (mTOR) regulates both proliferation of megakaryocyte progenitors and late stages of megakaryocyte differentiation Blood, March 15, 2006; 107(6): 2303 - 2310. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. D. Gallego, M. A. de la Fuente, I. M. Anton, S. Snapper, R. Fuhlbrigge, and R. S. Geha WIP and WASP play complementary roles in T cell homing and chemotaxis to SDF-1{alpha} Int. Immunol., February 1, 2006; 18(2): 221 - 232. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Z. Ratajczak RGS16 "tightens the reins" on CXCR4 Blood, November 1, 2005; 106(9): 2928 - 2929. [Full Text] [PDF]
|
|
|
|
|
|
M. Nishio, T. Endo, N. Tsukada, J. Ohata, S. Kitada, J. C. Reed, N. J. Zvaifler, and T. J. Kipps Nurselike cells express BAFF and APRIL, which can promote survival of chronic lymphocytic leukemia cells via a paracrine pathway distinct from that of SDF-1{alpha} Blood, August 1, 2005; 106(3): 1012 - 1020. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Moriguchi, B. D. Hissong, M. Gadina, K. Yamaoka, H. L. Tiffany, P. M. Murphy, F. Candotti, and J. J. O'Shea CXCL12 Signaling Is Independent of Jak2 and Jak3 J. Biol. Chem., April 29, 2005; 280(17): 17408 - 17414. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Wysoczynski, R. Reca, J. Ratajczak, M. Kucia, N. Shirvaikar, M. Honczarenko, M. Mills, J. Wanzeck, A. Janowska-Wieczorek, and M. Z. Ratajczak Incorporation of CXCR4 into membrane lipid rafts primes homing-related responses of hematopoietic stem/progenitor cells to an SDF-1 gradient Blood, January 1, 2005; 105(1): 40 - 48. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. A. Jaleel, A. C. Tsai, S. Sarkar, P. V. Freedman, and L. P. Rubin Stromal cell-derived factor-1 (SDF-1) signalling regulates human placental trophoblast cell survival Mol. Hum. Reprod., December 1, 2004; 10(12): 901 - 909. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-M. Paulus, N. Debili, F. Larbret, J. Levin, and W. Vainchenker Thrombopoietin responsiveness reflects the number of doublings undergone by megakaryocyte progenitors Blood, October 15, 2004; 104(8): 2291 - 2298. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Ratajczak, R. Reca, M. Kucia, M. Majka, D. J. Allendorf, J. T. Baran, A. Janowska-Wieczorek, R. A. Wetsel, G. D. Ross, and M. Z. Ratajczak Mobilization studies in mice deficient in either C3 or C3a receptor (C3aR) reveal a novel role for complement in retention of hematopoietic stem/progenitor cells in bone marrow Blood, March 15, 2004; 103(6): 2071 - 2078. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Jankowski, M. Kucia, M. Wysoczynski, R. Reca, D. Zhao, E. Trzyna, J. Trent, S. Peiper, M. Zembala, J. Ratajczak, et al. Both Hepatocyte Growth Factor (HGF) and Stromal-Derived Factor-1 Regulate the Metastatic Behavior of Human Rhabdomyosarcoma Cells, But Only HGF Enhances Their Resistance to Radiochemotherapy Cancer Res., November 15, 2003; 63(22): 7926 - 7935. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Reca, D. Mastellos, M. Majka, L. Marquez, J. Ratajczak, S. Franchini, A. Glodek, M. Honczarenko, L. A. Spruce, A. Janowska-Wieczorek, et al. Functional receptor for C3a anaphylatoxin is expressed by normal hematopoietic stem/progenitor cells, and C3a enhances their homing-related responses to SDF-1 Blood, May 15, 2003; 101(10): 3784 - 3793. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Libura, J. Drukala, M. Majka, O. Tomescu, J. M. Navenot, M. Kucia, L. Marquez, S. C. Peiper, F. G. Barr, A. Janowska-Wieczorek, et al. CXCR4-SDF-1 signaling is active in rhabdomyosarcoma cells and regulates locomotion, chemotaxis, and adhesion Blood, September 18, 2002; 100(7): 2597 - 2606. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Lee, A. Gotoh, H.-J. Kwon, M. You, L. Kohli, C. Mantel, S. Cooper, G. Hangoc, K. Miyazawa, K. Ohyashiki, et al. Enhancement of intracellular signaling associated with hematopoietic progenitor cell survival in response to SDF-1/CXCL12 in synergy with other cytokines Blood, May 29, 2002; 99(12): 4307 - 4317. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-J. Lataillade, D. Clay, P. Bourin, F. Herodin, C. Dupuy, C. Jasmin, and M.-C. Le Bousse-Kerdiles Stromal cell-derived factor 1 regulates primitive hematopoiesis by suppressing apoptosis and by promoting G0/G1 transition in CD34+ cells: evidence for an autocrine/paracrine mechanism Blood, February 15, 2002; 99(4): 1117 - 1129. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. D. Cristillo and B. E. Bierer Identification of Novel Targets of Immunosuppressive Agents by cDNA-based Microarray Analysis J. Biol. Chem., February 1, 2002; 277(6): 4465 - 4476. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. A. Millot, W. Vainchenker, D. Dumenil, and F. Svinarchuk Distinct effects of thrombopoietin depending on a threshold level of activated Mpl in BaF-3 cells J. Cell Sci., January 6, 2002; 115(11): 2329 - 2337. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. Janowska-Wieczorek, M. Majka, J. Kijowski, M. Baj-Krzyworzeka, R. Reca, A. R. Turner, J. Ratajczak, S. G. Emerson, M. A. Kowalska, and M. Z. Ratajczak Platelet-derived microparticles bind to hematopoietic stem/progenitor cells and enhance their engraftment Blood, November 15, 2001; 98(10): 3143 - 3149. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Thelen and M. Baggiolini Is Dimerization Of Chemokine Receptors Functionally Relevant? Sci. Signal., October 16, 2001; 2001(104): pe34 - pe34. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. KIM, S. KIM, H. KOH, S.-O. YOON, A.-S. CHUNG, K. S. CHO, and J. CHUNG Akt/PKB promotes cancer cell invasion via increased motility and metalloproteinase production FASEB J, September 1, 2001; 15(11): 1953 - 1962. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B.-S. Youn, Y. J. Kim, C. Mantel, K.-Y. Yu, and H. E. Broxmeyer Blocking of c-FLIPL-independent cycloheximide-induced apoptosis or Fas-mediated apoptosis by the CC chemokine receptor 9/TECK interaction Blood, August 15, 2001; 98(4): 925 - 933. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. K. Sen, S. Khanna, M. Venojarvi, P. Trikha, E. C. Ellison, T. K. Hunt, and S. Roy Copper-induced vascular endothelial growth factor expression and wound healing Am J Physiol Heart Circ Physiol, May 1, 2002; 282(5): H1821 - H1827. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
 |
 |
 |
|||||||
 |
 |
 |
 |
 |
 |
 |
 |
||
| Copyright © 2000 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
Top
Abstract
Introduction
T
) or 5 to 100 ng/mL TPO or 300 ng/mL SDF-1. (C) Inhibition of SDF-1-dependent
chemotaxis after preincubation of megakaryoblasts with increasing
concentration of LY294002 (10 µmol/L and 30 µmol/L). The experiment
was repeated 3 times in duplicate. (D) Trans-Matrigel migration of
megakaryoblasts toward SDF-1 and TPO gradients and the effect of MMP
inhibitors. The bottom chambers contained media alone (control, c) or
100 ng/mL each of SDF-1 or TPO. The assay was carried out twice using 4 to 5 chambers. Inhibition of trans-Matrigel migration toward SDF-1 was
carried out by preincubating the megakaryoblasts for 2 hours in the
absence or presence of 10 µg/mL rhTIMP-1 or rhTIMP-2. Percentages of
cells that migrated after 3 hours of incubation at 37°C are shown as
mean ± 1 SD. (E) MMP expression in megakaryoblasts in the presence
of SDF-1 and TPO. Media conditioned by megakaryoblasts after incubation
for 3 hours in the absence (control) or presence of 100 ng/mL SDF-1 or
TPO were analyzed by zymography. Media conditioned by KG-1 cells were
used as the standard to show the positions of the MMP-9 and MMP-2
activities in the gel. The experiment was repeated 3 times. (F)
Inhibition of trans-Matrigel migration to SDF-1 after preincubation of
megakaryoblasts with increasing concentration of LY294002 (10 µmol/L
and 30 µmol/L). The experiment was repeated twice, using 6 chambers
each. (G) MMP expression in megakaryoblasts stimulated with SDF-1
without (lane 1) or with (lane 2) LY294002. The experiment was repeated
2 times.
