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PLATELETS AND THROMBOPOIESIS
MAL/SRF complex is involved in platelet formation and megakaryocyte
migration by regulating MYL9 (MLC2) and MMP9
Laure Gilles,1-3 Dominique Bluteau,1-3 Siham Boukour,1-3 Yunhua Chang,1-3 Yanyan Zhang,1-3 Thomas Robert,2,3
Philippe Dessen,2-4 Najet Debili,1-3 Olivier A. Bernard,5 William Vainchenker,1-3 and Hana Raslova1-3
1Inserm,
U790, Villejuif; 2Université Paris XI, Villejuif; 3Institut Gustave Roussy, IFR54, Villejuif; 4Centre National de la Recherche Scientifique, FRE2939,
Villejuif; and 5Inserm, Hôpital Necker, Paris, France
Megakaryoblastic leukemia 1 (MAL) is a
transcriptional coactivator of serum response factor (SRF). In acute megakaryoblastic leukemia, the MAL gene is translocated and fused with the gene encoding
one twenty-two (OTT). Herein, we show
that MAL expression increases during the
late differentiation steps of neonate and
adult human megakaryopoiesis and localized into the nucleus after Rho GTPase activation by adhesion on collagen I or convulxin.
MAL knockdown in megakaryocyte progenitors reduced the percentage of cells
forming filopodia, lamellipodia, and stress
fibers after adhesion on the same substrates, and reduced proplatelet formation. MAL repression led to dysmorphic
megakaryocytes with disorganized demarcation membranes and ␣ granules
heterogeneously scattered in the cytoplasm. Gene expression profiling revealed a marked decrease in metalloproteinase 9 (MMP-9) and MYL9 expression
after MAL inhibition. Luciferase assays in
HEK293T cells and chromatin immunoprecipitation in primary megakaryocytes
showed that the MAL/SRF complex directly regulates MYL9 and MMP9 in vitro.
Megakaryocyte migration in response to
stromal cell–derived factor 1, through Matrigel was considerably decreased after
MAL knockdown, implicating MMP9 in
migration. Finally, the use of a shRNA to
decrease MYL9 expression showed that
MYL9 was involved in proplatelet formation. MAL/SRF complex is thus involved
in platelet formation and megakaryocyte
migration by regulating MYL9 and MMP9.
(Blood. 2009;114:4221-4232)
Introduction
Serum response factor (SRF) is a widely expressed transcription
factor required for the expression of immediate early, musclespecific, and cytoskeletal genes.1-4 SRF contains a MADS domain
that mediates homodimerization and DNA binding, and that allows
recruitment of transcriptional cofactors. SRF binds to a CArG box
present in promoter/enhancer regions of SRF-regulated genes.5
Depending on cell lines, different extracellular stimuli activate SRF
through 2 main signaling pathways: the MAP-kinase pathway
through members of the ternary complex factor (TCF)6,7 and the
small GTPases pathway through the Rho family8 members regulating the myocardin-related transcription factors (MRTFs). The
Rho-actin signaling pathway9-12 stimulates SRF by 2 ubiquitous
MRTFs, megakaryoblastic leukemia 1 (MAL; MKL1, MRTF-A,
BSAC) and MAL16 (MKL2, MRTF-B).
MAL was initially identified in acute megakaryoblastic leukemia (AMKL, M7) as a chromosome 22 encoded protein fused in 3⬘
with RNA-binding motif protein 15 (RBM15; OTT) located on
chromosome 1.13-15 The translocation t(1,22)(p13;q13) leads to the
in-frame fusion of the quasi-totality of OTT/RBM15 to the MAL
gene. The OTT-MAL fusion protein is restricted to AMKL
occurring de novo in infancy,16,17 in children older than 1 year or,
occasionally, in Down syndrome patients.18,19
The subcellular localization of MAL is regulated through its
association with globular actin by its RPEL motifs in the Nterminal region. The modification of the actin treadmilling by the
Rho pathway results in the nuclear accumulation of MAL, and
actin binding may also control MAL transcriptional activity.12,20
The 3 best-characterized Rho GTPase family members are Rho,
Rac, and Cdc42. They regulate assembly and disassembly of actin
cytoskeleton in response to a variety of extracellular stimuli. Rho
regulates stress fibers and focal adhesion assembly, Cdc42 promotes filopodia, and Rac promotes lamellipodia formation. Different effectors of these 3 signaling pathways are involved in
promoting actin polymerization. Except their direct role in actin
cytoskeleton remodeling, Rho, Rac, and Cdc42 also control cell
cycle, including G1 progression and cytokinesis, cell contraction,
polarity and migration, vesicle transport (endocytosis, exocytosis),
enzyme activation, and gene transcription via JNK, p38, nuclear
factor-␬B, and SRF.21,22 Hill et al reported that Rac1 and Cdc42
activate SRF in a RhoA-independent manner8; however, several
studies showed a crosslink between these pathways in SRF
regulation.23,24 In megakaryocytes (MKs), the platelet precursors,
reorganization of actin cytoskeleton plays a crucial role not only in
MK adhesion and migration25 but also at different stages during
maturation, ie, during endomitosis,26 demarcation membrane formation, proplatelet formation, and platelet shedding.27-32 Recently, it
has been shown that thrombopoietin (TPO) better stimulates Rho
activity in immature than in mature MKs and that the Rho/ROCK
pathway is a negative regulator of proplatelet formation. On the
other hand, the use of constitutively active Cdc42 led to increased
proplatelet formation33,34 (Y.C., unpublished data, February 2005).
Interestingly, a recent study strongly supports the hypothesis that a
Submitted March 10, 2009; accepted July 15, 2009. Prepublished online as
Blood First Edition paper, September 1, 2009; DOI 10.1182/blood2009-03-209932.
The online version of this article contains a data supplement.
An Inside Blood analysis of this article appears at the front of this issue.
© 2009 by The American Society of Hematology
BLOOD, 5 NOVEMBER 2009 䡠 VOLUME 114, NUMBER 19
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 USC section 1734.
4221
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BLOOD, 5 NOVEMBER 2009 䡠 VOLUME 114, NUMBER 19
GILLES et al
local inhibition of the Rho/ROCK pathway is at the origin of MK
ploidization by decreasing F-actin and myosin accumulation at
midzone during cytokinesis.26
The involvement of MAL in the transformation of megakaryoblasts and recent experimental evidence35 strongly suggested a role
in MK maturation. We therefore investigated its role in maturation,
adhesion, migration, and proplatelet formation of MKs derived
from neonate and adult CD34⫹ cells. The functional studies
coupled to gene profiling after MAL depletion in MK revealed that
the MAL/SRF complex regulates positively the known cytoskeletal
genes and 2 new targets: metalloproteinase 9 (MMP-9) important for
MK migration and MLC2 (MYL9) involved in platelet formation.
carried out in the ABI Prism GeneAmp 7500 Sequence Detection System
(Applied Biosystems) using TaqMan Universal PCR Master Mix (ABI)
containing the specific primers (1.2 mM) and the specific probe (0.1 mM).
Expression levels of all genes were expressed relatively to hypoxanthine
phosphoribosyl transferase (HPRT).
Western blot analysis
Western blot analyses were performed as previously reported.41 The
following antibodies were used: anti-MRTF-A (C-19), anti-HDAC-1 (H-51;
Santa Cruz Biotechnology), and anti-HSC70 (Stressgen). In addition of
anti-HDAC and anti-HSC70 staining, coloration with Ponceau S solution
(Sigma-Aldrich) after transfer was used to control the quantity of proteins.
Proplatelet formation assay
Methods
In vitro growth of MKs from CD34ⴙ cells
Cord blood and cytapheresis samples were obtained after informed consent
in accordance with the Declaration of Helsinki. Approval was obtained
from the Assistance Publique des Hôpitaux de Paris. CD34⫹ cells were
isolated using an immunomagnetic beads technique36 and grown in
serum-free medium as previously reported.37 The culture medium was
supplemented with recombinant human thrombopoietin (TPO; 10 ng/mL)
or with a cytokine cocktail containing TPO (10 ng/mL), interleukin-3 (IL-3;
100 U/mL; Immunex), interleukin-6 (IL-6; 10 ng/mL; Tebu), stem cell
factor (SCF; 25 ng/mL), and fetal liver tyrosine kinase 3 ligand (FLT3-L;
1 ng/mL; Amgen).
Lentiviral vector construction and production
For MAL, the published small interfering RNA (siRNA) sequence was
used.9 For MYL9, the siRNA sequence was designed using siSearch
software (http://www.dharmacon.com/DesignCenter/DesignCenterPage.aspx).
Small hairpin sequences were created as previously described.38,39 Oligonucleotide MAL and MYL9 short hairpins (sh; supplemental Table 1A,
available on the Blood website; see the Supplemental Materials link at the
top of the online article) were synthesized (Eurogentec) and inserted into a
pBlue Script vector containing the human H1 promoter. H1-shRNA specific
for MAL (shMAL), H1-shMYL9, or H1-SCR (scramble control sequence)
cassettes were inserted into a lentiviral vector (pRRLsin-PGK-eGFPWPRE, Genethon). Lentiviral stocks were prepared and stored, and
concentration was determined, as previously described.40
Cell transduction
CD34⫹ cells (105/mL) were prestimulated for 24 hours with TPO, IL-3,
SCF, and FLT3-L. Lentiviral particles were added at a concentration
corresponding to 125 ng viral p24/100 ␮L for 12 hours, followed by a
second transduction. Cells were then cultured in the presence of TPO alone.
Cell sorting, flow cytometric, and cytologic analysis
Cells were incubated for 30 minutes at 4°C with an allophycocyaninconjugated anti-CD41a monoclonal antibody and a phycoerythrinconjugated anti-CD42a monoclonal antibody (BD Biosciences PharMingen). After washing, cells were incubated for 2 hours in 0.01 mM Hoechst
33342 (Sigma-Aldrich) at 37°C. To study endogenous MAL expression in
the different differentiation classes, cells were sorted into CD34⫹41⫺;
CD34⫺41⫹CD42⫺; CD34⫺41⫹CD42low; CD34⫺41⫹CD42high cell fractions
for cord blood cultures, and into CD34⫹41⫺; CD34⫹41⫹CD42⫺;
CD34⫹41⫹CD42 ⫹; CD34⫺41⫹CD42 ⫹cell fractions for cytapheresis
cultures. To study the ploidy level, CD41⫹CD42⫹ cells stained with
Hoechst were analyzed on a LSRII (BD Biosciences) with the CellQuest
software package (BD Biosciences).
Real-time quantitative RT-PCR
Primers and internal probes for quantitative reverse-transcribed polymerase
chain reaction (RT-PCR) are listed in supplemental Table 1B. PCR was
CD41⫹CD42⫹GFP⫹ MK were sorted at day 8 of culture and plated in
96-well plates at a concentration of 2000 cells/well in serum-free medium
containing TPO, and quantification of proplatelet MK was performed as
previously described.42 Images were obtained using a Zeiss inverted
microscope at a magnification of 40⫻, and the proplatelet network area was
measured using the Axio Vision 4.6 software.
Luciferase assays
The 440-bp and 550-bp promoter regions upper transcription initiation of
MMP9 and MYL9, respectively, were amplified (primer listed in supplemental Table 1C) and subcloned into the pLuc-MCS vector (Stratagene). MAL
cDNA was cloned into the vector pMegix (gift of Dr Morgenstern from
Millennium Pharmaceuticals). HEK293T cells were transfected with pLuc
constructs (pLuc_MCS, pLuc_MMP9, pLuc_MYL9, pLuc_SRE, or pLuc_
FLT3), a Renilla luciferase vector (for normalization) and MAL expression
vector using Lipofectamine 2000 (Invitrogen). Cells were cultured in
12-well plates and transfected with 50 to 200 ng DNA. The total amount of
DNA was kept constant by the inclusion of appropriate amounts of empty
vectors. Cells were lysed with Passive Lysis Buffer 48 hours after
transfection, and the luciferase activity was evaluated using the Dual
Luciferase Reporter Assay System (Promega).
ChIP
Chromatin immunoprecipitation (ChIP) assays were performed with a ChIP
assay kit (Upstate Biotechnology) using the anti-SRF antibody (clone H-65;
Santa Cruz Biotechnology). Assays were performed using chromatin
prepared from human MKs. Quantification of precipitated DNA fragments
was carried out on an ABI PRISM 7500 sequence detection system using
SYBR green (Applied Biosystems) in duplicate. Primers for MYL9,
MMP9, and THSB1 are described in supplemental Table 1D. Fold
enrichment is expressed as the ratio of target antibody signal to IgG signal
calculated by extrapolation from a standard curve of DNA dilutions.
Migration assays
Cell migration was assessed with BD Bio-Coat Matrigel Invasion Chamber
(BD Biosciences) according to the protocol provided by the manufacturer.
Briefly, MKs were washed and prepared to a final concentration of
106 cells/mL. MK aliquots (100 ␮L) were seeded into each 8-␮m pore
transwell inserts, which were placed into the wells of a 24-well plate and
incubated for 22 hours at 37°C. The lower compartment contained 600 ␮L
serum-free media with or without 100 ng to stromal cell-derived factor 1␣
(SDF-1␣; Abcys). The percentage of migrated CD41⫹CD42⫹GFP⫹ MK
was determined by flow cytometry.
Cell adhesion and immunohistochemistry
Slides were coated overnight at 4°C with convulxin (kindly provided by M.
Jandrot-Perrus, Hôpital Bichat, France) at 15 ␮g/mL in 1⫻ PBS or with
collagen type I (Nycomed) at 50 ␮g/mL. pRRL-SCR or pRRL-shMAL
GFP⫹-sorted cells were seeded on coated slides for 2 hours (convulxin) or
1 hour (collagen type I) at 37°C. After a gentle wash, adherent cells were
fixed in 2% paraformaldehyde for 10 minutes, permeabilized with 0.1%
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Triton X-100 for 5 minutes, and washed with 1⫻ PBS for 5 minutes.
Primary antibodies were subsequently applied to the slides at a concentration of 1 ␮g/mL and incubated for 1 hour at room temperature. Slides were
then washed 3 times with 1⫻ PBS for 5 minutes before and after
application of the secondary antibody (20 ␮g/mL) for 30 minutes at room
temperature. Slides were mounted using either Vectashield with 4,6diamidino-2-phenylindole (Invitrogen) and observed under an epifluorescence microscope (Nikon Eclipse 600) equipped with a 60⫻ objective
(Nikon) or TOTO-3 iodide (Molecular Probes) and observed under a Zeiss
LSM 510 laser scanning microscope (Carl Zeiss) with a 63⫻/1.4 numeric
aperture oil objective. Images were processed using the Adobe Photoshop
6.0 and LSM 5 Image Browser software, respectively.
Primary antibodies were: anti-MRTF-A (clone C-19; Santa Cruz
Biotechnology), anti-phalloidin–tetramethylrhodamine isothiocyanate
(TRITC) (P-1951; Sigma-Aldrich). The secondary antibody was anti-goat
TRITC (Jackson ImmunoResearch).
Electron microscopy
Samples were washed in 1⫻ PBS, then fixed in 1.5% glutaraldehyde for
1 hour, and washed 3 times in 0.1 M phosphate buffer, pH 7.4. For
morphologic examination, samples were postfixed in 1% osmic acid,
dehydrated in ethanol, and embedded in Epon by standard methods.
Samples were counterstained and were observed on a Philips Tecnai
electron microscope.
Microarray analysis
We used Agilent long (60-bp) oligonucleotide microarrays and the dualcolor analysis method in which probes from specimens and from the
reference are differentially labeled with Cyanine 5 and Cyanine 3, as
described previously.43 We performed a set of 4 dye swap experiments to
compare RNAs obtained from MKs derived either from cytapheresis or
cord blood CD34⫹ cells after transduction by shMAL. GFP⫹ cells were
sorted at day 9 of culture. The purity of MKs derived from cytapheresis was
approximately 97% (supplemental Figure 1). RNAs from scramblelentivirus–transduced cells were used as the reference. From each of the
4 combined experiments, signatures (supplemental Table 2) were obtained
by an analysis of variance test with P value of 10⫺3, and annotated with
updated databases as Entrez-Gene. Data were analyzed using Resolver
software, and gene selection was obtained by an analysis of variance test
with P ⬍ 10⫺3 value.
Data availability
All data obtained by microarray analyses have been submitted to ArrayExpress44 with the accession number E-TABM-640.
Results
Expression of MAL and MAL16 in MKs
Different studies suggest a redundant role for 2 ubiquitous MRTFs,
MAL and MAL16. Therefore, we first studied their relative
expression in culture of MKs derived from CD34⫹ cells isolated
from cord blood or cytapheresis and grown for 9 days in the
presence of TPO. MAL mRNA level was approximately 7-fold
higher than MAL16 mRNA in both types of MKs, taking HPRT as
an internal control (Figure 1A). Because of the high expression of
MAL and its direct involvement in megakaryoblastic transformation, we focused our study only on the role of MAL in megakaryopoiesis. To determine whether MAL expression varies through MK
maturation during ontogenesis, CD34⫹ cells isolated from cord
Figure 1. MAL expression in MKs. CD34⫹ cells from cord blood or cytapheresis
were cultured with TPO. (A) At day 9 of culture, mRNA levels of MAL and MAL16 were
evaluated by quantitative RT-PCR. Their expression was normalized with respect to
HPRT mRNA. (B) To investigate MAL transcription level during MK differentiation,
cells were sorted at day 6 of culture in 4 populations: for cord blood, CD34⫹CD41⫺,
CD34⫺CD41⫹CD42⫺, CD34⫺CD41⫹CD42low, and CD34⫺CD41⫹CD42high; and for
cytapheresis, CD34⫹CD41⫺, CD34⫹CD41⫹CD42⫺, CD34⫹CD41⫹CD42⫹, and
CD34⫺CD41⫹CD42⫹. MAL mRNA level was evaluated as in panel A. Error bars in
panels A and B represent the SD of the mean of 3 repeated experiments each
performed in triplicate wells. (C) To investigate protein levels by Western blot analysis
during MK differentiation, total cell populations were harvested on day 3 (D3), day 6
(D6), day 9 (D9), and day 12 (D12) for cord blood-derived MK and at day 0 (D0), day 3
(D3), day 6 (D6), and day 9 (D9) for cytapheresis-derived MKs. HDAC-1 and HSC70
were used as internal control. The figure illustrates representative data of 2 experiments with similar results.
blood and cytapheresis were cultured in the presence of TPO and
sorted at day 6 according to the expression of 3 differentiation
markers (“Cell sorting, flow cytometric, and cytologic analysis”).
No difference in MAL expression was found during ontogenesis.
MAL transcripts remained stable during the first stages of differentiation in both neonate and adult-derived MKs but clearly increased
in the most mature fraction (CD34⫺CD41⫹CD42⫹; Figure 1B). We
next investigated the protein expression at different days of culture
in the presence of TPO (until day 12 for cord blood and until day 9
for cytapheresis), as well as in platelets isolated either from cord
blood or cytapheresis samples. Consistent with MAL mRNA level,
the protein level increased continuously until day 12 of culture for
cord blood-derived MKs and until day 9 of culture for cytapheresisderived MKs (⬃ 65% of CD41⫹CD42⫹ MKs; Figure 1C). In our
culture conditions, proplatelet-forming MKs first appeared at day
12 for adult MKs and at day 15 for neonate MKs. Thereafter, MAL
protein level decreased with the appearance of proplatelet-forming
MKs and was undetectable in platelets (data not shown).
MAL localizes into the nucleus of MKs after Rho GTPase
activation
As previously described,8,12 we observed that MAL localized in the
cytoplasm of serum-starved NIH3T3 cells and accumulated in the
nucleus after serum stimulation (Figure 2A). This is mediated
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BLOOD, 5 NOVEMBER 2009 䡠 VOLUME 114, NUMBER 19
Figure 2. MAL nuclear localization after Rho GTPase activation in MKs. (A) MAL cytoplasmic localization in serum-starved NIH3T3 cells and its nuclear localization after
serum stimulation. (B) Cord blood-derived MKs (upper panels) and cytapheresis-derived MKs (lower panels). After adhesion to polylysine for 10 minutes (PLL, 10⬘), MAL is
localized in the cytoplasm. After adhesion to collagen I or convulxin for 2 hours, MAL is localized in the nucleus. Both substrates induce Rho GTPase activation. MAL (red) and
4,6-diamidino-2-phenylindole (blue) staining was detected by immunofluorescence and visualized under a fluorescent light microscope at an original magnification ⫻60.
through small GTPase activation.8,12 To examine the effect of
RhoA and Cdc42 activation on MAL localization in cord blood and
cytapheresis-derived MKs, MKs were cultured for 9 days in
serum-free medium supplemented by TPO and then allowed to
adhere onto different extracellular matrix. After 10 minutes on
polylysine, a substrate that does not allow Rho or Cdc42 activation,33 MAL remained cytoplasmic (Figure 2B). After 2 hours on
collagen type I, a substrate that activates principally Rho/ROCK
leading to fiber stress formation,33 MAL accumulated in the MK
nucleus. After 2 hours onto convulxin, a substrate inducing mainly
filopodia formation through activation of the Cdc42 pathway,33
MAL also accumulated in the MK nucleus (Figure 2B). The
nuclear accumulation of MAL after Rho and Cdc42 pathway
activation is observed in MKs derived either from cord blood or
cytapheresis CD34⫹ cells.
MAL knockdown does not significantly impair the first stages
of MK differentiation
To investigate further the role of MAL in MKs, we developed a
lentivirus encoding an shMAL under the H1 promoter. The vector
efficiency was first tested in the MO7e leukemic cell line. Two days
after transduction, MAL level was determined by RT-PCR and
Western blot in cells sorted on the expression of GFP. The shMAL
reduced MAL transcripts approximately 13-fold compared with the
control shRNA (Figure 3A left panel), and the protein expression
was similarly decreased (Figure 3A right panel). Then, CD34⫹ cells
obtained from cord blood or cytapheresis were prestimulated in
culture by IL-3, FLT3-L, SCF, and TPO and transduced at days 1
and 2 with the control shRNA or shMAL lentiviruses. Thereafter,
cells were cultured in TPO alone. At day 9 of culture, GFP⫹ cells
were sorted. The MK purity was approximately 90% in both cases
(data not shown). As shown in Figure 3B, shMAL led to a major
decrease of MAL protein level compared with the control shRNA.
Recently, it has been reported that inhibition of the Rho/Rock
pathway leads to an increased MK ploidy level.26 Therefore, we
examined whether the Rho effector MAL could be involved in the
endomitotic process. To study the effect of MAL repression on MK
ploidization, CD34⫹ cells isolated from cytapheresis were transduced at days 1 and 2 of culture and analyzed at day 9. The ploidy
level of transduced (GFP⫹) cells was analyzed in the gate of mature
CD41⫹CD42⫹ MKs. As illustrated in Figure 3C, MAL knockdown
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Figure 3. Effect of MAL knockdown on ploidy level and MK differentiation. (A) MO7e cells were transduced with a control lentivirus or the lentivirus-encoding shRNA of
MAL (shMAL). GFP⫹ cells were sorted and analyzed. MAL mRNA level (left panel) was measured as in Figure 1. MAL protein level (right panel) was analyzed at day 9 of culture
by Western blot. HDAC-1 was used as internal control of a quantity. The figure illustrates representative data of 2 experiments with similar results. (B-D) Cord blood and
cytapheresis-isolated CD34⫹ cells were transduced with the control lentivirus or the lentivirus-encoding shRNA of MAL (shMAL). (B) MAL protein level was analyzed at day 9 of
culture by Western blot in GFP⫹ cells. HDAC-1 and HSC70 were used as internal controls. The figure illustrates representative data of 2 experiments with similar results.
(C) The CD41⫹CD42⫹ cell population (left panel), corresponding to mature cytapheresis-derived MKs, was analyzed for ploidy level by Hoechst staining (right panel). The
mean ploidy (N) was calculated from the number of cells at each ploidy level. The figure illustrates representative data of 3 experiments with similar results. (D) The percentage
of mature MKs was evaluated as the percentage of cells coexpressing both CD41 and CD42 markers. Data illustrate analysis of 4 repeated experiments for cytapheresis and
3 for cord blood. Similar results were obtained in all experiments.
resulted in a moderate (21.3% ⫾ 9%) increase in mean ploidy level
that did not reach significance (n ⫽ 3, P ⱕ .09), compared with the
untransduced population.
Then, we analyzed the effect of MAL knockdown on maturation
of MKs issued from cord blood and cytapheresis samples. Cells
were transduced as at days 1 and 2, and GFP⫹ cells were analyzed
at day 7 of culture for the expression of 2 MK markers, CD41 and
CD42. As shown in Figure 3D, the percentage of CD41⫹CD42⫹
mature MKs slightly increased in cytapheresis samples (top panel,
26% ⫾ 13.6%, n ⫽ 4, P ⱕ .004) but decreased in cord blood
samples (bottom panel, 16.8% ⫾ 1.5%, n ⫽ 3, P ⱕ .08) after MAL
repression. The minor difference in the percentage of CD41⫹CD42⫹
MK in both cases after MAL repression and the slight increase in
ploidy level suggest that MAL does not play an essential role in
MK ploidization and during the early differentiation steps.
MAL plays an important role in adhesion, terminal maturation,
and proplatelet formation of MKs
MAL and MAL16 are necessary for the formation of stress fibers
and focal adhesion in NIH3T3 cells.4 Here, we investigated
whether MAL had a similar role in MK. Control shRNA- or
shMAL-transduced MKs were sorted at day 8 of culture on GFP⫹
and allowed to adhere for 1 hour on collagen type I or for 2 hours
on convulxin. As illustrated in Figure 4A, the percentage of
filopodia and stress fibers forming MKs was markedly decreased
after MAL repression (n ⫽ 2, P ⱕ .016; n ⫽ 2, P ⱕ .05, respec-
tively). These results suggested that, like in NIH3T3 cells, MAL
acts as a mediator of actin-polymerized structures by regulating
transcription of several cytoskeletal genes.
Electron microscopy studies revealed structural abnormalities
of maturing MK after MAL knockdown (Figure 4B). Compared
with the control shRNA (Figure 4Bi), the cytoplasm was disorganized with numerous vacuoles (Figure 4Bii-iii); the demarcation
membrane system was heterogeneously dilated and abnormally
distributed with its presence at the periphery of MKs (Figure
4Bii-iii); moreover, the ␣ granules were scattered heterogeneously
in the cytoplasm (Figure 4Bii).
To test whether MAL was involved in the process of proplatelet
formation, control shRNA- and shMAL-transduced MKs were
cultured in a liquid serum-free medium containing TPO.
CD41⫹CD42⫹GFP⫹ MKs were sorted at day 9 and seeded in
96-well plates at 2 ⫻ 103 cells per well. At day 13 of culture, the
percentage of proplatelet-forming MKs was 68% reduced in MAL
knockdown MKs derived both from cytapheresis (n ⫽ 3, P ⱕ .003)
and cord blood (n ⫽ 2, P ⱕ .1; Figure 4C). Moreover, we measured the total surface area of the proplatelet network in cells
transduced with control or shMAL and found that it was decreased
3-fold after MAL inhibition (Figure 4D). Ten cells were analyzed in
each group (P ⱕ .008). These results suggest that the MAL/SRF
complex plays a major role in terminal maturation, proplatelet
formation, and adhesion of MK by transcriptionally regulating
genes implicated in these processes.
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BLOOD, 5 NOVEMBER 2009 䡠 VOLUME 114, NUMBER 19
Figure 4. MAL knockdown alters actin polymerization, terminal maturation, and proplatelet formation of MKs. CD34⫹ cells isolated from cord blood or cytapheresis
were transduced with the control lentivirus or the lentivirus encoding shRNA of MAL (shMAL). (A) GFP⫹ cells (day 8) were allowed to adhere on collagen I or convulxin for
2 hours. Stress fibers, filopodia, and lamellipodia were stained with TRITC-conjugated phalloidin (red) and nucleus with 4,6-diamidino-2-phenylindole (blue). The percentage of
MK-forming stress fibers, filopodia, or lamellipodia was evaluated on a total of 500 cells using fluorescent light microscopy (original magnification ⫻40). (B) Ultrastructural
aspect of control (i) and shMAL (ii-iii) transduced MKs. MKs were sorted at day 10 of culture on the expression of GFP and fixed. N indicates nucleus; DM, demarcation
membranes. Arrowhead represents ␣ granules. (i) Morphology of a typical normal MK. Bar represents 5 ␮m. (ii-iii) Morphology of shMAL-transduced MK. (ii) Bar represents
2 ␮m. (iii) Bar represents 5 ␮m. (C) GFP⫹ cells were sorted at day 9 on the coexpression of CD41 and CD42. Cells were seeded at 2 ⫻ 103 cells/well in a 96-well plate. The
percentage of proplatelet-forming MKs was estimated by counting MKs exhibiting one or more cytoplasmic processes with areas of constriction. A total of 500 cells per well
were counted at day 13 for cytapheresis-derived MKs and at day 15 for cord blood–derived MKs. Error bars in histograms represent the SD of one representative experiment
performed in triplicate wells. Similar results were obtained in 3 repeated experiments with cytapheresis and 2 experiments with cord blood. (D) MKs derived from cytapheresis
were plated as in panel C, and the area of proplatelet network estimated in microns squared was measured using Axio Vision 4.6 software. One representative control- and
shMAL-transduced proplatelet-forming MKs is shown on left, and the area mean of 10 cells from each group is shown in the histogram on right. Error bars in histograms
represent the SD obtained for 10 cells in one representative experiment (n ⫽ 2).
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BLOOD, 5 NOVEMBER 2009 䡠 VOLUME 114, NUMBER 19
Microarray analysis of genes deregulated after MAL
knockdown in MKs
MAL is thought to regulate transcription through its interaction
with SRF. To characterize MAL/SRF target genes in MKs derived
from cord blood or cytapheresis samples, we compared control
shRNA- and shMAL-transduced MKs by microarray analyses.
CD34⫹ cells from cord blood or cytapheresis were transduced as
described in the previous paragraph, cultured in the presence of
TPO alone after transduction and GFP⫹ cells were sorted at day 9
of culture. The purity of MKs was approximately 97%, as illustrated
for cytapheresis-derived MKs in supplemental Figure 1. Gene
expression profiles were obtained using Agilent Human Whole
Genome 44K oligonucleotide arrays allowing a direct comparison
of 2 populations labeled by 2 different dyes (Cy3 and Cy5). Cell
populations transduced by the control lentivirus were used as
reference for transduction-induced changes. The signal from 1929 and
1436 probe sets (corresponding to 1037 and 773 genes, respectively) was significantly deregulated by the expression of the
shMAL in MKs derived from cytapheresis and cord blood,
respectively (Figure 5A). A total of 613 probe sets were common
between cytapheresis- and cord blood–derived MKs (supplemental
Table 2). The signal of 589 probe sets was found to be modified
similarly in both cell types, and the expression of 24 probe set
genes showed an opposite variation in cord blood and cytapheresis
MKs (supplemental Table 3). Figure 5A shows the 20 most
down-regulated genes found in MKs derived from cytapheresis and
MAL REGULATES MMP9 AND MYL9 IN MEGAKARYOCYTES
4227
cord blood. They are listed according to the fold change observed
in the experiment using cytapheresis MKs.
To confirm the microarray data, 12 genes were selected, most
with a key role in cytoskeleton organization. Three of them (zyxin,
profilin 1, and N-wasp) play a major role in actin polymerization
and 3 others (␤-actin, vinculin, and tropomyosin 2) are major
structural proteins of the cytoskeleton and thus could be implicated
in the defects in fiber stress and filopodia formation seen in MAL
knockdown MKs. Gene expression was analyzed by quantitative
RT-PCR (Figure 5B). For some genes (zyxin, thrombospondin,
␤-actin, and vinculin), a discrepancy was found between
microarray (Figure 5Bi) and RT-PCR analyses (Figure 5Bii) that
could be explained by methods sensitivity. Indeed, saturation of
highly expressed genes may lead to their exclusion in microarray analyses.
We next focused our efforts on MMP9 and MYL9 (the myosin
light chain associated with MYH9), 2 genes with a specific role in
megakaryopoiesis.
MMP9 and MYL9 (MLC2) are direct targets of MAL/SRF complex
in MKs
The MAL/SRF complex binds DNA at specific sites through the
SRF DNA-binding domain. Using the ChipMapper software, we
identified putative SRF-binding sites in both MMP9 and MYL9
promoters. To determine whether MMP9 and MYL9 are targets of
the MAL/SRF complex, we cloned 420 bp of the MMP9 promoter
Figure 5. Gene profiling of MKs after MAL knockdown. CD34⫹ cells isolated from cord blood and cytapheresis were transduced with the control lentivirus or the
lentivirus-expressing shMAL. At day 9 of culture, cells expressing GFP were sorted and mRNA was subjected to microarray analyses using Agilent Human Whole Genome 44K
oligonucleotide arrays (A) and to quantitative RT-PCR (B). (A) A total of 1929 and 1436 probe sets were found significantly deregulated in MKs derived from cytapheresis and
cord blood, respectively, with a 613 probe set common to both. The table shows primary sequence name, accession number, and fold change of the 20 most down-regulated
genes after MAL knockdown in cytapheresis- and cord blood-derived MKs. (B) Validation of microarray data (i) by quantitative RT-PCR (ii) for 12 selected genes. The error bars
represent the SD of the mean of 3 repeated experiments each performed in triplicate wells.
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4228
GILLES et al
BLOOD, 5 NOVEMBER 2009 䡠 VOLUME 114, NUMBER 19
Figure 6. MMP9 and MYL9 are directly regulated by MAL/SRF
complex. (A) Schematic representation of MMP9 and MYL9 human
promoter regions cloned into the pLuc-MCS reporter. Arrows represent
the translation start site. (B) Luciferase assay performed by transient
transfection of HEK293 cells with the 50 ng of Megix vector containing
MAL. Luciferase levels are shown as fold change relative to cells
transfected with the reporter construct alone. The total amount of
transfected DNA was kept constant by addition of empty Megix vector.
The histogram shows one representative experiment of 3, each in
triplicate. Error bars represent the SD of triplicate. (C) ChIP assay
performed in cytapheresis-derived MKs (day 10 in culture) with primer
sets directed toward in silico predicted SRF-binding sites: MMP9_C_F
and R, and MMP9_D_F and R for MMP9, and primer sets MYL9_A_F
and MYL9_E_R for MYL9. Localization of primers MMP9_C_F and R for
MMP9 and MYL9_A_F and MYL9_E_R for MYL9 are depicted in panel
A. Primers MMP9_D_F and R are not localized in the cloned promoter
region designed in panel A. Control primer sets allowing amplification of
known SRF-binding sites (THSB1) or DNA region devoid of SRF sites
were also used. Immunoprecipitation was performed with control rabbit
IgG and anti-SRF antibodies. Histograms indicate relative occupancy of
SRF-binding sites by SRF in the MMP9, MYL9, and THSB1 promoters.
Error bars represent the SD of experiments performed in duplicate. The
figure illustrates representative data of 2 independent experiments with
similar results.
(including one SRF-binding site) and 650 bp of the MYL9
promoter (including 3 SRF-binding sites) upstream of the firefly
luciferase gene (Figure 6A) and tested the ability of MAL to
modulate expression in transient reporter assays using HEK293T
cells. Transcription from both MMP9 and MYL9 promoters was
highly stimulated by MAL coexpression (Figure 6B). The
baseline activity of MMP9 and MYL9 promoters compared with
minimal promoter with or without SRF consensus-binding sites
(pLUC_SRE and pLuc_MCS, respectively) or to a control
promoter region without SRF consensus binding site cloned into
the same luciferase vector (pLuc_FLT3) is shown in supplemental Figure 2.
To demonstrate that SRF binds to the MMP9 and MYL9
promoters in vivo, ChIP assays were performed using human MKs
derived in culture from CD34⫹ cells. Thrombospondin, a previously described target of SRF,45 was used as a positive control for a
ChIP experiment. Quantitative RT-PCR with primers amplifying
the regions encompassing the predicted C (Figure 6A) and D
(supplemental primer Table 1d) SRF-binding sites in MMP9
promoter and 3 SRF-binding sites in MYL9 promoter (Figure 6A)
were performed. ChIP experiments revealed a high MYL9 promoter
region enrichment and a slight but significant MMP9 promoter
region enrichment using SRF antibody (Figure 6C). Our results
confirmed that MMP9 and MYL9 are direct targets for MAL/SRF
complex in MK.
MAL plays a key role in MK migration and platelet formation via
MMP9 and MYL9 regulation
To confirm that the drop in MYL9 expression could be responsible
for the defect in proplatelet formation observed on the knockdown
of MAL, we constructed a lentivirus expressing an shRNA specific
for MYL9 (shMYL9). The shMYL9 was expressed in primary
MKs derived from cytapheresis, as described for MAL knockdown.
Two independent experiments showed a 50% decrease in the
MYL9 transcript level (n ⫽ 2, P ⱕ .08; Figure 7A). At day 13 of
culture, knockdown of MYL9 elicited an approximately 35%
decrease in the percentage of proplatelet-forming MKs (n ⫽ 4,
P ⱕ .003; Figure 7B). Moreover, the total surface area of the
proplatelet network in cells transduced with shMYL9 was decreased approximately 2-fold after shMYL9 transduction compared with control-transduced cells. Ten cells were analyzed in
each group (P ⱕ .009; Figure 7C).
MYL9 knockdown was less efficient in affecting the proplatelet
formation than MAL knockdown. This is probably the result of the
lower efficiency of the shMYL9 leading only to 50% reduction of
MYL9 transcript, whereas shMAL resulted in a nearly complete
abolition of MAL expression. Nevertheless, the data confirm that
MAL/SRF complex is involved in proplatelet formation via a
transcriptional regulation of MYL9.
MKs migrate through bone marrow endothelial cells to release
platelets within the sinusoidal space in response to SDF-1.46,47
Because SRF was reported to be involved in cell migration,3 we
investigated the effect of MAL knockdown on MK migration. MKs
were transduced and grown as described in “Cell transduction,”
and chemotactic assays in response to SDF-1 were performed on
day 8 of culture. No difference was detected between control
shRNA- or shMAL-transduced MKs derived from cytapheresis
after migration through noncoated or collagen I–coated transwells
(data not shown). However, when Matrigel-coated transwells were
used, a 2-fold decrease in MK migration in response to SDF-1 was
seen in shMAL-expressing cells (n ⫽ 2, P ⱕ .08; Figure 7D). This
defective MK migration through an extracellular matrix similar to
the endothelial basement membrane suggests a deficiency in MMP
activity.48 It could be explained by the marked decrease of MMP9
expression in shMAL-expressing cells (Figure 5A), which encodes
for an important proteinase involved both in MK migration through
bone marrow endothelial cells in response to SDF-1 and in the
release of platelets into the blood flow.48
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BLOOD, 5 NOVEMBER 2009 䡠 VOLUME 114, NUMBER 19
MAL REGULATES MMP9 AND MYL9 IN MEGAKARYOCYTES
4229
Figure 7. MAL contribution to proplatelet formation and migration of MKs by targeting MYL9 and MMP9, respectively. (A-B) CD34⫹ cells isolated from cytapheresis
were transduced at days 1 and 2 of culture with the lentivirus-encoding control shRNA (control) or MYL9 shRNA (shMYL9). (A) GFP⫹ cells were analyzed at day 9 of culture.
MYL9 mRNAs were measured by quantitative RT-PCR (left panel). The histogram shows one of 2 representative experiments, each in triplicate. Error bars represent the SD of
triplicate wells. (B) The percentage of proplatelet-forming MKs derived from cytapheresis samples was evaluated as described in Figure 4C at day 13 of culture. Error bars
represent the SD of 1 representative experiment performed in triplicate wells. Similar results were obtained in 4 independent experiments. (C) MKs derived from cytapheresis
CD34⫹ cells were plated as in panel C, and the area of proplatelet network estimated in microns squared was measured using Axio Vision 4.6 software. One representative
control- and shMYL9-transduced proplatelet-forming MKs is shown on the left, and the mean area of 10 cells from each group is shown in the histogram on the right. Error bars
in histograms represent the SD obtained for 10 cells in 1 representative experiment (n ⫽ 2). (D) MK migration through Matrigel-coated transwells in response to SDF-1.
Cytapheresis isolated CD34⫹ cells were transduced at days 1 and 2 of culture with a control or shMAL-encoding lentivirus. The experiment was done on day 8 of culture. Data
represent the percentage of migrated CD41⫹CD42⫹GFP⫹ cells compared with total CD41⫹CD42⫹GFP⫹ input. Error bars represent the SD of 1 representative experiment
performed in triplicate. The figure illustrates representative data of 2 independent experiments with similar results.
Discussion
MK differentiation is characterized by DNA endoreduplication,
cytoplasmic maturation, and expansion. In addition, MKs must
migrate in the bone marrow from the endosteal to the vascular
niches, and then form long cytoplasmic extension that crosses
the endothelial barrier to form platelets under the blood flow.
During differentiation, MKs must progressively accumulate the
main platelet proteins localized in the membrane or in secretory
granules but also develop a complex cytoskeleton network to
allow polyploidization, proplatelet formation, and adhesion and
aggregation of future platelets. MK development is controlled
by the concerted action of transcription factors, which form
complexes to specifically activate or repress gene expression.
The transcriptional regulation of platelet-specific genes is now
well understood. It essentially depends on GATA-1/FOG-1
transcription complexes and on members of the Ets family,
especially FLI-1.49 In contrast, the transcriptional regulation of
cytoskeleton components as well as its relationship with the
microenvironment-mediated signal transduction are still poorly
understood. The role of this transcriptional regulation has been
underscored by the knockout of p45NF-E2, which leads to a lethal
thrombocytopenia with the absence of proplatelet formation.
This profound defect is in large part the result of a defect in
␤1-tubulin, a direct target of p45NF-E2.49 These data have
emphasized the major role of microtubules in the process of
proplatelet formation.29 However, there is increasing evidence
that the actomyosin cytoskeleton plays also an important
function in efficient platelet production, not only by regulating
proplatelet branching and amplification of platelet production
but also by regulating MK polyploidization,26 demarcation
membrane distribution, MK migration, and probably also platelet release.32,50
SRF is a major factor that transcriptionally regulates many
cytoskeleton components. Transcriptional regulation of this set of
genes by SRF depends on the myocardin family coactivators. We
have used an shRNA strategy to knockdown MAL in human MKs
derived from CD34⫹ cells and to show that MAL plays a major role
in regulating target genes involved in proplatelet formation and
MK migration.
In fibroblasts, MAL/SRF activation is dependent on the Rho
pathway and controls many Rho-dependent cytoskeleton processes.51 Our data indicate that it might be the same in MK
differentiation. We showed that, in suspension cultures, in the
presence of TPO, a condition in which the Rho and Cdc42
pathways are only slightly activated, MAL is mainly distributed in
the MK cytoplasm. In contrast, when the RhoA or Cdc42 pathways
are activated after adherence on collagen 1 and convulxin, respectively, MAL accumulates in the nucleus. This result strongly
suggests that MAL in MK, as in fibroblasts, is regulated by the
actin treadmilling cycle through RhoA and Cdc42 effectors.
Consequently, knockdown of MAL impairs adhesion, stress fiber,
and filopodia formation in MK. This could be explained by
down-regulation of ␤-actin, thrombospondin 1 (THSB1), and some
proteins involved in a multicomponent focal adhesion complex
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4230
GILLES et al
(zyxin and vinculin). These cytoskeleton components are regulated
by the MAL/SRF complex in other cell types, such as fibroblasts4
and vascular smooth muscle cells.52 THSB1 is an angiogenetic
factor, which may negatively regulate MK progenitors,53 although
THSB1 knockout mice have no MK/platelet defect.54 The expression of other focal adhesion proteins, such as talin, involved in
GPIIb-IIIa–dependent platelet activation55 and focal adhesion
kinase, a negative regulator of megakaryopoiesis,56 were not
modified after MAL inhibition.
Recent reports performed in cell lines and CD34⫹ cells showed
that MAL overexpression promotes MK differentiation and
polyploidization.35 These results differ from ours in which knockdown of MAL has no significant effect on human MK differentiation, as judged by the expression of CD41 and CD42 and
polyploidization. Furthermore, a tendency to increase ploidy was
observed in MAL knockdown MKs, an expected result because
inhibition of RhoA activity also leads to a slight increase in MK
ploidy.26 It is possible that overexpression of MAL in MKs
performed by Cheng et al35 modifies the SRF complexes and
interferes with its association with TCFs. TCFs are important
mediators of the MAP kinase pathway,6,7 which plays a major role
in MK differentiation.57,58 Thus, a part of the effect of MAL
overexpression might be related to an alteration in this pathway.
However, Cheng et al reported also a decrease in ploidy level in
bone marrow MKs of MKL1⫺/⫺ mice.35 We cannot exclude that the
regulation of MK polyploidization between mouse and human is
different. However, it will be interesting to confirm these data in
bone marrow MK from another MKL1⫺/⫺ model developed
by Li et al.59
In contrast, our study demonstrates a major effect of MAL
knockdown on proplatelet formation and MK migration. This result
is in agreement with the phenotype of MAL knockout mice,35
which have a decreased platelet count suggesting a defect in
platelet biogenesis.
Gene expression profiling indicates that MAL knockdown
affects the expression of 2.5% of the genes common between
adult and neonate MK. To further work, we selected 2 of the 20
most down-regulated genes, MMP9 and MYL9. We have demonstrated by luciferase and ChIP experiments that the MAL/SRF
complex directly regulates both MMP9 and MYL9. Very
recently, Medjkane et al reported the decrease of MYL9 and
MYH9 expression in breast carcinoma and melanoma cell lines
as a result of siRNA-mediated MAL knockdown.60 Our study
extends this report in another cell model and further supports the
hypothesis that MAL/SRF regulates genes involved in actomyosin contractility.
MYL9 is a myosin light chain that is associated with MYH9, the
only isoform of nonmuscle myosin II present in platelets.61
Phosphorylation of MYL9 activates MYH9 and negatively regulates proplatelet formation in normal MKs.62 In contrast, deficiency
in myosin IIA leads to a defect in proplatelet formation in the
MYH9 syndrome, and use of a shMYH9 induces a decrease in the
percentage of proplatelet-forming MKs (Y.C. and N.D., unpublished results, February 2008). Again, a difference between mouse
and human model should be noted because, although MYH9deficient mice exhibited a marked thrombocytopenia, an increase in
proplatelet-forming MKs was observed. The cytoplasm of murine
MYH9 knockout MK is totally disorganized compared with WT
MKs. The demarcation membrane system is abnormally dilated
with a vacuole-like appearance.63 Interestingly, very similar ultrastructural abnormalities were observed in human MKs treated with
the shMAL, strongly suggesting that basal regulation of MYL9 by
BLOOD, 5 NOVEMBER 2009 䡠 VOLUME 114, NUMBER 19
MAL/SRF is an important determinant in the regulation of
megakaryopoiesis by this transcriptional complex. This hypothesis
was supported by MYL9 depletion in cultured MKs leading to
inhibition of proplatelet formation.
In addition, we show that MAL is required for MK migration in
response to SDF-1 through Matrigel-coated transwell but not
through noncoated transwell (data not shown). This phenotype may
be related to the MYL9 deficiency, which is implicated in cell
motility, but even more by the marked decrease in MMP9
expression observed in MAL-deficient MKs. Indeed, MMP9 has
been demonstrated to be necessary for in vitro MK migration
through the basement membrane in response to SDF-1.48 In this
study, the authors suggested that MMP9 may be also required for
platelet release. MMP9 plays an important role in platelet functions, and its antiaggregant effect may be at the origin of
hemorrhagic transformation of acute stroke. However, the large
redundancy between various MMPs renders the study of their
respective role in platelet functions difficult.64 Altogether, the
defect in MYL9 and MMP9 induced by MAL knockout could
explain the MK/platelet phenotype of MAL knockout mice recently
described.35
The oncogenic OTT-MAL fusion protein has been shown
to interfere with the activity of SRF in model cell lines.65 Acting as a dominant negative form of MAL, it could block the
terminal stages of MK maturation affecting the expression of
MMP9 and MYL9. Thus, it will be interesting to assess the
role of these 2 new target genes of MAL in the phenotype of
the t(1;22) acute megakaryoblastic leukemia, for example, in
the context of recently developed knock-in OTT-MAL mouse model
of AMKL.66
Acknowledgments
The authors thank F. Wendling for helpful suggestions on the
manuscript, Kirin Brewery (Tokyo, Japan) for human recombinant
thrombopoietin, Dr Morgenstern for Megix vector (Millennium
Pharmaceuticals, Inc, Cambridge, MA), M. Jandrot-Perrus (Hôpital Bichat, France) for the gift of convulxin, and T. Mercher for
MAL cDNA (Hôpital Necker, France) and genomic platform of
IGR for microarrays.
This work was supported by grants from Fondation de France
and the Agence Nationale de la Recherche (ANR contrat blanc).
L.G. was supported in part by the Association pour la Recherche
sur le Cancer (ARC). D.B. was supported by a postdoctoral
fellowship from ANR.
Authorship
Contribution: L.G. performed experiments, analyzed data, and
wrote the paper; D.B. performed experiments and analyzed data;
S.B., Y.Z., T.R., and N.D. performed experiments; Y.C. and O.A.B.
discussed the results; P.D. analyzed data; W.V. designed the work
and wrote the paper; and H.R. designed and supervised the
experiments and wrote the paper.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Hana Raslova, Inserm, U790, 39 rue Camille
Desmoulins, Villejuif, France; e-mail: [email protected].
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BLOOD, 5 NOVEMBER 2009 䡠 VOLUME 114, NUMBER 19
MAL REGULATES MMP9 AND MYL9 IN MEGAKARYOCYTES
4231
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2009 114: 4221-4232
doi:10.1182/blood-2009-03-209932 originally published
online September 1, 2009
MAL/SRF complex is involved in platelet formation and megakaryocyte
migration by regulating MYL9 (MLC2) and MMP9
Laure Gilles, Dominique Bluteau, Siham Boukour, Yunhua Chang, Yanyan Zhang, Thomas Robert,
Philippe Dessen, Najet Debili, Olivier A. Bernard, William Vainchenker and Hana Raslova
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