doi:10.1182/blood-2012-07-446443
Prepublished online December 13, 2012;
2013 121: 781-790
An Zwijsen and Catherine M. Verfaillie
Satish Khurana, Shannon Buckley, Sarah Schouteden, Stephen Ekker, Anna Petryk, Michel Delforge,
4 expressionαhoming via Smad independent regulation of integrinA
novel role of BMP4 in adult hematopoietic stem and progenitor cell
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Regular Article
HEMATOPOIESIS AND STEM CELLS
Anovel role of BMP4 in adult hematopoietic stem and progenitor cell
homing via Smad independent regulation of integrin-␣4 expression
Satish Khurana,1 Shannon Buckley,1 Sarah Schouteden,1 Stephen Ekker,2 Anna Petryk,3,4 Michel Delforge,5 An Zwijsen,6
and Catherine M. Verfaillie1
1Interdepartmental Stem Cell Institute, KU Leuven, Leuven, Belgium; 2Department of Biochemistry and Molecular Biology, Mayo Clinic Cancer Center,
Rochester, MN; Departments of 3Pediatrics, and 4Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN; 5Faculty of Medicine,
UZ Leuven, Leuven, Belgium; and 6Department of Developmental and Molecular Genetics, VIB, KU Leuven, Leuven, Belgium
Key Points
• Cytokine-induced loss of murine
as well as human HSPC
homing during ex vivo culture
can be prevented by addition
of BMP4.
• In HSPCs, BMP4 directly
regulates Integrin-␣4 expression
through SMADindependent
p38 MAPKmediated
signaling.
Although it is well established that BMP4 plays an important role in the development of
hematopoietic system, it is less well understood whether BMP4 affects adult hematopoiesis
and how. Here, we describe a novel mechanism by which BMP4 regulates homing of
murine as well as human hematopoietic stem/progenitor cells (HSPCs). BMP4 treatment
of murine BM derived c-kit؉Lin؊Sca-1؉ (KLS) and CD150؉CD48؊KLS cells for up to
5 days in vitro prevented the culture-induced loss of Integrin-␣4 (ITGA4) expression as
well as homing. The effect on ITGA4 expression in response to BMP4 is mediated via
SMAD-independent phosphorylation of p38 MAPK, which activates microphthalmiaassociated
transcription factor (MITF), known to induce ITGA4 expression. Elevated
ITGA4 expression signiﬁcantly enhanced HSPC attachment to bone marrow stromal
cells, homing and long-term engraftment of the BMP4 treated cells compared with the
cells cultured without BMP4. BMP4 also induced expression of ITGA4 on human BM
derived Lin؊CD34؉ cells in culture, which was associated with improved homingpotential.
Thus, BMP4 prevents culture-induced loss of ITGA4 expression on HSPCs in a
SMAD-independent manner, resulting in improved homing of cultured HSPCs and subsequent hematopoietic reconstitution.
(Blood. 2013;121(5):781-790)
Introduction
BMP4 is a member of the transforming growth factor (TGF)
family. After binding of BMP4 to its receptors, the receptorregulated
R-SMADs, SMAD1/5/8, get activated and bind to
SMAD4. This complex is translocated into the nucleus to regulate
transcription of various target genes.1 In addition to the SMADmediated
canonical pathway, BMPs can activate several mitogenactivated
protein kinases (MAPKs), including extracellular signalregulated
kinases (ERKs), c-Jun N-terminal kinases (JNKs), and
p38 mitogen activated protein kinase (MAPK).2
The role of BMP4 in mesoderm formation and early hematopoietic
development has been well established.3-5 However, studies on
the role of BMP4 in adult hematopoiesis have not been conclusive.
Bhatia et al, demonstrated that BMP-4 improves maintenance of
human umbilical cord blood HSPCs in vitro by acting as a survival
factor for stem cell function.6 BMP4 induces development of stress
erythroid progenitors by stimulating erythropoietin in spleen of
adult mice.7,8 Culture of murine CD34Ϫ KLS cells with BMP4 did
not affect their proliferation, even though it resulted in phosphorylation
of SMAD1/5 in the HSPC-like Lhx2-HPC cell line as well as
in LinϪ BM cells.9 However, normal hematopoiesis was observed
in mice where SMAD5 was conditionally eliminated from HSCs.10
Although SMAD-dependent signaling was shown to be dispensable
in adult hematopoiesis, BMP4 hypomorhic (Bmp4S2G/S2G)
mice, wherein decreased levels of mature BMP4 ligand are present
in the BM niche, showed a signiﬁcant reduction in HSC number in
the BM.11 Thus, although there is evidence that BMP4 plays a role
in postnatal hematopoiesis, the mechanisms whereby BMP4 affects
the fate of HSPCs in vitro or in vivo remains unknown.
The BM niche provides necessary signals to maintain the stem
cell pool,12 as well as signals important for homing and retention of
transplanted HSCs.13 Among others, receptor-ligand pairs, such as
integrin-␣4 (ITGA4)/vascular cell adhesion molecule (VCAM)–1,
integrin-␤1/osteopontin, and N-cadherin/␤-catenin, are involved in
anchoring HSPCs to the BM niche.14 It has been well established
that ITGA4 plays a major role in HSC homing and retention into
the BM.15,16 In addition, there is evidence that ITGA4 is required
for HSC maintenance in vivo.17
Regulation of integrin-mediated cell adhesion largely depends
on changes in the activation state of integrins, induced by
inside-out and later by outside-in signaling events by integrinligand
interactions.18 However, changes in integrin expression
levels have also been implicated in changes in cell adhesion or
Submitted July 31, 2012; accepted November 25, 2012. Prepublished online as
Blood First Edition paper, December 13, 2012; DOI 10.1182/blood-2012-07-446443.
The online version of this article contains a data supplement.
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.
© 2013 by The American Society of Hematology
781BLOOD, 31 JANUARY 2013 ⅐ VOLUME 121, NUMBER 5
motility, even though mechanisms underlying such changes in
expression levels have been less extensively studied. The murine
Itga4 promoter is known to contain binding motifs for Ap1, Ap2,
Spi1, and Pu.119 and is directly activated by Runx3 in monocytes.20
There is extensive evidence that TGF-␤ can enhance expression of
integrins.21
As both the loss of BMP4 in the niche11 as well as loss of
ITGA4 on HSCs17 create a similar hematopoietic phenotype, with
decreased HSC maintenance, we hypothesized that the mechanism
via which BMP4 may affect HSCs might be through BMP4mediated
effects on the ITGA4-mediated anchoring of HSPCs to
the BM niche. We here demonstrate that BMP4 can act as an
external growth factor that regulates ITGA4 expression. This
occurs via SMAD independent, p38-MAPK–mediated activation
of the transcription factor MITF. As a result, adhesion of HSPCs to
stromal cells, in vivo homing, and long-term repopulation is
signiﬁcantly enhanced by BMP4 pretreatment.
Methods
Animals
Six- to 8-week-old C57BL/6J-CD45.2 (Center d’Elevage R. Janvier, Le
Genest-St Isle, France), B6.SJL-PTPRCA-CD45.1 (Charles River Laboratories),
and Rag2Ϫ/ϪIl2R␥cϪ/Ϫ mice (gift from Prof Chantal Mathieu,
Clinical and Experimental Endocrinology, UZ Leuven, Leuven, Belgium)
were bred and maintained in the animal facility at KU Leuven. During the
experiments, mice were maintained in isolator cages, fed with autoclaved
acidiﬁed water, and irradiated food ad libitum. All experiments were
approved by the university institutional ethics committee.
Human bone marrow samples
Human BM aspirates were obtained from healthy donors by aspiration from
the posterior iliac crest. All studies, conducted in accordance with the
Declaration of Helsinki, were approved by the Medical Ethics Committee,
UZ Leuven, Gasthuisberg.
Hematopoietic progenitor cell isolation
Details of the procedures for murine and human HSPC isolation are
provided in supplemental Methods (available on the Blood Web site; see the
Supplemental Materials link at the top of the online article).
Cell culture
The in vitro HSPC culture system was adapted from Zhang et al.22 Sorted
murine KLS cells (50 cells/well), were cultured in U-bottom 96-well plates
(BD Bioscience) in 100 ␮L of Stemspan (StemCell Technologies) supplemented
with 100 ng/mL mTPO and 50 ng/mL mSCF, with or without
100 ng/mL rmBMP-4, 1 ␮g/mL rmTSG, or 1 ␮g/mL rmCHD (all from
R&D Systems), dorsomorphin (2␮M, Sigma-Aldrich) or SB203580 (5␮M,
Sigma-Aldrich). Recombinant human SCF, TPO, and BMP4 (all from R&D
systems) were used to culture hBM-derived LinϪCD34ϩ (50 000 cells per
well in ultra-low attachment 24-well plates). Cells were cultured for 5 days
at 37°C with 5% CO2.
Serial transplantation
Freshly isolated or culture progeny of 200 KLS cells from CD45.1 mice
were transplanted along with 1 ϫ 106 competitor CD45.2 BM cells into
lethally irradiated (10Gy) C57BL/6J-CD45.2 mice. Peripheral blood chimerism
analysis was performed every 4 weeks. After 12 weeks, primary
recipients were killed, BM harvested, and 1 ϫ 106 cells grafted in
2 secondary lethally irradiated CD45.2 mice. After 3 months, chimerism in
secondary recipients was evaluated. Mice with more than 1% multilineage
chimerism were considered engrafted.
Flow cytometry
Chimerism and lineage analysis was performed by ﬂow cytometry.
Lineage-speciﬁc antibodies used were PE-conjugated anti–Mac-1/Gr-1 for
myeloid cells, APC-conjugated anti-B220 for B cells, PE-conjugated
anti-CD4/CD8 for T cells were used. For chimerism analysis FITC
conjugated anti-CD45.1 and PerCPCy5.5-conjugated anti-CD45.2 antibodies
were used. All antibodies were procured from BD Pharmingen. ITGA4
expression and phosphorylation status of MAPK-p38 was assayed using
anti-h/mITGA4 FITC (BD Pharmingen) and anti-phospho-p38 antibody
(Cell Signaling).
In vitro adhesion assays
OP9 cells (5 ϫ 104) were plated per well in 24-well plates. Two
ϫ 104 PKH-26 labeled (details in supplemental Methods) KLS progeny
were added per well and incubated for 3 hours at 37°C and 5% CO2.
Nonadherent cells were removed and adherent cells were harvested along
with the feeder layer. Flow cytometry was used to quantify the labeled cells
to compare cell attachment.23 Results are represented as percentage of cells
adhered. For ITGA4 neutralization experiments, cells were pre-incubated
with a blocking anti-ITGA4 antibody (PS/2 against CD49d, Novus
Biologicals).24
Quantitative RT-PCR analysis
Quantitative RT-PCR was performed using standard protocols. The details
of the procedures, reagent,s and the list of primers used are provided in
supplemental Methods.
Immunoblotting
Immunoblotting was performed using standard protocols and reagents.
Details of the procedures and antibodies used are provided in supplemental
Methods. Band intensities were quantiﬁed using ImageJ 1.32 software
(National Institutes of Health) after densitometric scanning of the ﬁlms, and
compared with ␤-actin or histone-H3.
Immunostaining
A protocol adapted from earlier published method25 was used to stain the
cultured murine BM derived KLS or CD48ϪCD150ϩKLS cells for
phospho-SMAD1/5/8, phospho-p38 MAPK and MITF. Details of the
procedure and antibodies used are provided in supplemental Methods.
Images were captured on a Zeiss AxioImager microscope (Zeiss) ﬁtted with
an AxioCam MRc5 digital camera. Images were captured using AxioVision
AC Version 4.8 software (Zeiss) and assembled in Adobe Photoshop CS2.
Homing assay
A protocol adapted from previously published results26,27 was used to
examine the homing potential of KLS progeny or human LinϪCD34ϩ
progeny. Details of the procedures are provided in supplemental Methods.
Transduction of Lin؊ murine BM cells
Scrambled or Mitf speciﬁc shRNAs in the miR-30 backbone of the pTRIPZ
lentiviral vector were procured from Open Biosystems. Details of the
procedures for producing viruses and transduction of the HSPCs are
provided in supplemental Methods.
Statistical analysis
Data are shown as mean Ϯ SE. Statistical analysis was performed using a
2-tailed student t test. P values less than .05 were considered statistically
signiﬁcant. For radiation rescue experiments, Kaplan-Meier survival curves
were plotted using GraphPad Prism Version 5.0a software.
782 KHURANA et al BLOOD, 31 JANUARY 2013 ⅐ VOLUME 121, NUMBER 5
Results
Culture with BMP4 affects engraftment but not expansion of
murine KLS cells
We assessed the effect of BMP4, or its inhibitors chordin (CHD)
and twisted gastrulation (TSG) on murine KLS cells cultured in
vitro in serum-free medium with stem cell factor (SCF) and
thrombopoietin (TPO). Total cell expansion was similar for all
culture conditions (supplemental Figure 1A). Likewise the frequency
of KLS cells (Figure 1A) as well as colony forming cells
(CFCs; supplemental Figure 1B) was similar in all culture conditions,
as was the percentage KLS progeny in G0 (supplemental
Figure 1C) or G2/M (Figure 1B) phase of the cell cycle. These
studies suggested that BMP4 or its inhibitors do not affect
expansion of HSPCs cultured in vitro.
Next, we assessed the effect of BMP4 and its antagonists on the
ability of KLS cells to provide radioprotection (Figure 1C).
Lethally irradiated CD45.2 mice received progeny of 200 KLS
cells cultured with SCFϩTPO (ST) alone or combined with BMP4
(STB), or the BMP4 inhibitors CHD (STC) or TSG (STT).
Irradiated mice without graft died within 2 weeks (median 9 days),
whereas the group transplanted with the progeny of ST supplemented
cultures survived for a median of 16 days and Ͼ 10% of
mice survived long-term (Ͼ 8 months; Figure 1C). Addition of
BMP4 during culture enhanced median (27 days, P ϭ .0001) as
well as overall survival (27.78% of mice survived long-term). This
effect was partially abolished by addition of BMP4 inhibitor TSG
(STBT). The ability of KLS cell-progeny from STC or STT
cultures to rescue mice from irradiation was signiﬁcantly decreased
compared with cells from ST cultures (median 14 and 13.5 days,
respectively; log rank test method for trend, P Ͻ .01, n ϭ 10-18).
We also performed competitive repopulation experiments to
examine the effect of BMP4 on the long-term repopulation
(LTR)–potential of HSPCs. Freshly isolated or progeny of
200 cultured CD45.1 KLS cells were transplanted along with
1 ϫ 106 competitor CD45.2 BM cells intravenously into lethally
irradiated CD45.2 mice.After 3 months, 1 ϫ 106 BM cells from the
primary recipients were injected in lethally irradiated secondary
recipients. Chimerism in primary recipients that received cells
cultured with STB was signiﬁcantly better than from cells cultured
with ST (n ϭ 8, P Ͻ .02; Figure 1D). KLS cells cultured in the
presence of SCF/TPO showed signiﬁcantly lower engraftment than
freshly isolated cells. BMP4 treatment resulted in chimerism levels
in primary as well as secondary recipients comparable with those of
freshly isolated cells. By contrast, chimerism from cells treated
Figure 1. Improved radioprotection and chimerism because of culture of KLS cells with BMP4. Murine KLS cells were cultured for 5 days in serum-free medium with
SCFϩTPO with or without BMP4 and/or TSG or CHD (ST ϭ SCF/TPO; STT ϭ SCF/TPO/TSG; STB ϭ SCF/TPO/BMP4; STBT ϭ SCF/TPO/BMP4 /TSG; STC ϭ SCF/TPO/
CHD). (A) Flow cytometry was performed to quantify cells maintaining KLS phenotype after 5 days of culture under different conditions (n ϭ 3). (B) Cell-cycle status was
analyzed by propidium iodide staining. The percentage cells in G2/M phase of cell cycle were plotted for the different conditions (n ϭ 3). (C) Survival of mice grafted with the
progeny of 200 KLS cells from the different cultures after lethal irradiation. Control denotes mice that did not receive KLS cell progeny. Results were analyzed by log-rank test
method for trend and expressed as Kaplan-Meier survival curves (n ϭ 10-18, *P Ͻ .01). (D) Donor derived chimerism was determined by FACS in primary recipients, 3 months
after the transplantation of either 200 uncultured KLS cells, or of progeny from 200 KLS cells from different culture conditions along with 1 ϫ 106 CD45.2 BM cells. One million
total BM cells from primary recipients were transplanted in secondary hosts. Chimerism in secondary recipients was determined after 3 months (n ϭ 8-12; *P Ͻ .05). (E) In vitro
transwell migration assays was performed to compare the migration potential of the KLS cell progeny (n ϭ 4). (F) PKH-26 labeled KLS cell progeny were allowed to adhere to
OP9 cells for 3h. Nonadherent cells were washed and the percentage adherent cells was enumerated and compared for different conditions. In some conditions, a blocking
anti-ITGA4 antibody was added in addition to BMP4 (n ϭ 3-4; *P Ͻ .05). (G) KLS cell progeny (0.1 ϫ 106) from the different cultures were transplanted intravenously in lethally
irradiated mice, and BM harvested after 16 hours. Total BM cells (0.1 ϫ 106) were assayed in methylcellulose colony forming assays and CFU-Cs were enumerated.
Percentage homing was determined by comparing total CFU-Cs homed with CFU-Cs injected (n ϭ 6, *P Ͻ .05, **P Ͻ .01).
BMP4 IN ADULT HSC HOMING 783BLOOD, 31 JANUARY 2013 ⅐ VOLUME 121, NUMBER 5
with STT or STC was signiﬁcantly lower than from ST treated cells
(P Ͻ .05). Chimerism in secondary recipients after 3 months was
signiﬁcantly higher for STB compared with ST-treated cells
(n ϭ 12, P Ͻ .05; Figure 1D). We did not ﬁnd any differences in
the multilineage potential of the transplanted cells in any of the
groups (supplemental Figure 2).
BMP4 enhances adhesion of KLS progeny to stroma and their
homing in vivo
Initial studies demonstrated thus that although BMP4 or its
inhibitors signiﬁcantly affect the engraftment of short and longterm
repopulating HSPCs, this is not associated with effects on
KLS cell proliferation in vitro. We therefore examined whether the
improved engraftment could be because of changes in HSPC
homing. As homing requires attachment of HSPCs to the endothelial
lining of BM sinusoids and migration through the endothelium
into the BM niche, we performed both adhesion and migration
assays with KLS progeny treated with or without BMP4 or its
inhibitors. Addition of BMP4 did not affect the migration potential
of KLS cell progeny toward OP9 stromal feeders (Figure 1E).
However, adhesion of KLS progeny to OP9 stromal cells was
signiﬁcantly increased by BMP4 (n ϭ 3; P ϭ .02), whereas KLS
progeny from CHD or TSG containing cultures adhered signiﬁcantly
less to the stromal cells (P Ͻ .05; Figure 1F). The effect of
BMP4 inhibitors on the adhesion potential of HSPCs was found to
be reversible as removal of the inhibitor and culturing the cells in
the presence of BMP4 for additional 2 days led to increase in their
adhesion potential (supplemental Figure 3A) further indicating an
effect on HSPC homing.
To further substantiate this, direct in vivo homing experiments
were performed with KLS cell progeny from ST, STB, and STC
cultures (Figure 1G). CD45.1 mice derived KLS cells cultured
under different conditions were transplanted IV into lethally
irradiated animals. After 16 hours, mice were killed and the
proportion of transplanted CFCs that homed into the recipient BM
was measured by methylcellulose assay. Lethal irradiation depletes
the colony forming activity of BM cells; hence, colonies formed
from the BM cells after irradiation are derived from the grafted
donor cells. The number of homed CFCs compared with the
number of CFCs in the graft and homing efﬁciency was determined.27
Details of the calculations are provided in supplemental
Methods. The percentage of homed CFCs was signiﬁcantly higher
for KLS cell progeny from STB cultures than ST control cultures
(n ϭ 6; P ϭ .033), whereas progeny from STC cultured cells
homed signiﬁcantly less (Figures 1G; P ϭ .04). These results were
also compared with ﬂow cytometric detection of homed donor
derived (CD45.1) cells as shown in Figure 3C and supplemental
Figure 6A-B.
BMP4 improves cell adhesion by SMAD independent induction
of ITGA4 expression
We examined transcript levels of various adhesion molecules
commonly expressed by HSPCs. No signiﬁcant change in expression
of most of these genes was found (data not shown), except for
a signiﬁcant increase in Itga4 expression (n ϭ 6, P ϭ .045; Figure
2A). Itga4 expression decreased after culture of KLS cells in the
presence of SCF and TPO without BMP4, whereas addition of
BMP4 prevented this effect. Exposure to CHD/TSG caused further
Figure 2. BMP4-induced activation of ITGA4 expression is mediated via SMAD independent p38 MAPK phosphorylation. KLS cells were cultured with SCFϩTPO in
addition to BMP4 alone or combined with various inhibitors, for 5 days (ST ϭ SCF/TPO; STC ϭ SCF/TPO/CHD; STT ϭ SCF/TPO/TSG; STB ϭ SCF/TPO/BMP4;
STBD ϭ SCF/TPO/BMP4/Dorsomorphin; STBS ϭ SCF/TPO/BMP4/SB203580), and analyzed by qRT-PCR, Western blot and ﬂow cytometry. (A) Fold change in
Itga4 transcript levels in KLS progeny from cultures stimulated with SCFϩTPO and either BMP4, CHD, or TSG compared with KLS progeny from SCFϩTPO cultures
(n ϭ 6; *P Ͻ .05). (B) Immunoblotting with antibodies against ITGA4 and ␤-actin (n ϭ 3). (C) qRT-PCR analysis for Itga4 using KLS progeny cultured with ST, STC, STB,
STBD, and STBS. (n ϭ 3; *P Ͻ .05). (D). FACS analysis of KLS progeny cultured with ST, STB, STBD, and STBS using antibodies against ITGA4 (n ϭ 4). (A) Western blot
analysis using antibodies against ITGA4 and ␤-actin in KLS progeny cultured with ST, STC, STB, STBD, and STBS. (E) Western blot analysis of KLS progeny cultured with
ST or STB using phospho-speciﬁc antibodies against JNK/SAPK, Erk1/2, and S6 kinase. (F) Western blot analysis of KLS progeny cultured with ST, STB using antibodies
against phospho-p38 MAPK and total p38 MAPK. (G) Western blot analysis of KLS progeny cultured with ST or STB using antibodies against phospho-TAK1 and
phospho-MKK3/6. ␤-actin was used as internal control (representative for panels D through F). All western blots are representative examples of 3 independent experiments.
784 KHURANA et al BLOOD, 31 JANUARY 2013 ⅐ VOLUME 121, NUMBER 5
decrease in Itga4 expression (n ϭ 6; P Ͻ .02). Consistent with the
gene expression data, Western blotting (n ϭ 3; P Ͻ .03; Figure
2B, quantitation in supplemental Figure 3B) and ﬂow cytometric
analysis (Figure 2D) demonstrated elevated levels of ITGA4
expression in STB treated KLS cell progeny, and decreased levels
in STC and STT treated cells (n ϭ 3; P Ͻ .05). That the changes in
ITGA4 expression levels were responsible for the BMP4–induced
increase in cell adhesion was conﬁrmed by incubation of KLS
cell progeny with BMP4 and a blocking anti-ITGA4 antibody
(P ϭ .01; Figure 1F).
To determine the signaling pathway involved in regulation of
ITGA4 expression by BMP4, immunostaining (supplemental Figure
4A) and immunoblotting (supplemental Figure 4B) was
performed to detect SMAD1/5/8 phosphorylation in KLS cells
treated with different combinations of growth factors for 5 days.
We detected signiﬁcantly increased SMAD1/5/8 phosphorylation
after incubation with BMP4 (n ϭ 3; P ϭ .006), consistent with
previously published studies.9 By contrast, the addition of CHD
decreased SMAD1/5/8 phosphorylation (supplemental Figure 4A).
However, no change in transcript levels of SMAD target genes,
such as Id1, Id2, Id3, Runx2, and Gata2 was detected (supplemental
Figure 4C).
To further examine whether SMAD1/5/8 was involved in
regulating ITGA4 expression, we cultured KLS cells with dorsomorphin,
an inhibitor of SMAD1/5/8 phosphorylation (referred to as
D)28 along with BMP4, and evaluated transcript (Figure 2C) as well
as protein levels of ITGA4 by ﬂow cytometry (Figure 2D) and
immunoblotting (Figure 2E, quantitation in supplemental Figure
5A). Although dorsomorphin inhibited SMAD1/5/8 phosphorylation
(supplemental Figure 4B), it did not reverse the effects of
BMP4 on the levels of ITGA4 expression (n ϭ 3; P Ͻ .02; Figure
2C-E). Hence, although BMP4 activated SMAD1/5/8, this signaling
pathway was not involved in BMP4-mediated regulation of
ITGA4 expression.
BMP4-induced p38 MAPK activation is responsible for the
regulation of ITGA4 expression
To further delineate the BMP4 signaling pathway involved in the
process, we performed immunoblotting experiments using phosphospeciﬁc
antibodies against various signal transducers that mediate
SMAD-independent signaling.2 No changes were seen in the
phosphorylation status of JNK, ERK-MAPK, and S6K on culture
with BMP4 (Figure 2F, quantitation in supplemental Figure 5B).
However, a signiﬁcant increase in phosphorylation of p38 MAPK
was detected (Figure 2G, quantitation in supplemental Figure 5B).
In addition, the upstream regulators of p38 MAPK, TAK1, and
MKK3/6, were phosphorylated in response to BMP4 treatment
(Figure 2H, quantitation in supplemental Figure 5B).
To test whether p38 MAPK phosphorylation was involved in
the BMP4-mediated regulation of ITGA4 expression, the
p38 MAPK inhibitor, SB203580 (referred to as S), was added to
cultures stimulated with BMP4. As expected, SB203580 did not
affect BMP4-induced phosphorylation of SMAD1/5/8 although it
inhibited phosphorylation of p38 MAPK (supplemental Figure
4B). In contrast to dorsomorphin, addition of SB203580 together
with BMP4 reversed the effect of BMP4 on Itga4 transcript and
protein expression (n ϭ 3, P Ͻ .03; Figure 2C-E). These studies
strongly suggest that BMP4 inﬂuences ITGA4 expression via
SMAD-independent signaling pathways. To extend these ﬁndings
in vivo, radiation rescue experiments and 16-hour homing assays
were performed. As previously shown (Figure 1C), KLS cells
cultured in the presence of BMP4 rescued the lethally irradiated
mice better than the cells cultured without BMP4 (Figure 3A). We
found that this effect was abolished after addition of SB203580 but
not by dorsomorphin (P Ͻ .0002), suggesting that SMADindependent
p38 MAPK-mediated BMP4 signaling was involved
in this effect. To assess homing, we evaluated the number of CFCs,
CD45.1, as well as CD45.1-KLS cells in recipient mice 16 hours
after infusion of the graft (Figure 3B-C, supplemental Figure
6A-C). In coherence with the previous results, we found a
signiﬁcant increase in total number of CD45.1 KLS cells as well as
CD45.1 KLS cells (Figure 3C). SB203580 but not dorsomorphin
reversed the BMP4-mediated enhanced in vivo homing (Figure
3B-C; P ϭ .003). The percentage of homed CFCs was signiﬁcantly
higher for KLS cell progeny from STB cultures than ST control
cultures (n ϭ 6; P ϭ .033), whereas progeny from STC cultured
cells homed signiﬁcantly less (Figure 1G, P ϭ .04).
BMP4 results in p38 MAPK-mediated activation of MITF
Although little is known regarding the transcription factor(s) that
regulate ITGA4 expression in HSPCs, MITF has been shown to
bind to a CACTTG motif in the ITGA4 promoter resulting in
increased ITGA4 expression in murine mast cells, hence increasing
their adhesion potential.29 One of the signaling pathways that
regulates the expression and activation of MITF is p38 MAPK.30,31
As there was phosphorylation of p38 MAPK in KLS cells after
BMP4 stimulation, we examined whether MITF was activated in
response to BMP4 treatment. qRT-PCR (Figure 4A) analysis
demonstrated that CD45ϩ BMCs expressed signiﬁcantly higher
levels of Mitf than CD45Ϫ cells (n ϭ 3; P ϭ .034). Mitf was
Figure 3. Activation of SMAD independent BMP4 signaling leads to better homing potential and radioprotection. KLS cells were cultured for 5 days in SCFϩTPO
with BMP4 alone or combined with dorsomorphin or SB203580 and radioprotection and homing ability was evaluated. (A) Survival of mice grafted with 0.1 ϫ 106 KLS cell
progeny from different culture conditions after lethal irradiation. Results were analyzed by log-rank test method for trend and expressed as Kaplan-Meier survival curves
(n ϭ 10-18, P Ͻ .002). (B) KLS cell progeny (1 ϫ 105) from the different cultures were transplanted intravenously in lethally irradiated mice, and BM harvested after 16 hours.
Total BM cells (0.1 ϫ 106) were assayed in methylcellulose colony forming assays and CFU-Cs were enumerated. Percentage homing was determined by comparing total
CFU-Cs homed with CFU-Cs injected (n ϭ 9, P Ͻ .01). (C) Aside from analyzing homed CFCs, in homing assays BM of the recipient mice was analyzed by ﬂow cytometry for
donor derived CD45.1 cells. Flow cytometric analysis was extended to quantify the KLS cells homed in the 16 hours after transplantation. Total KLS cells homed in each mice
was calculated for different conditions (n ϭ 8, P Ͻ .02).
BMP4 IN ADULT HSC HOMING 785BLOOD, 31 JANUARY 2013 ⅐ VOLUME 121, NUMBER 5
expressed signiﬁcantly higher in LinϪ BM cells compared with
Linϩ cells (P ϭ .04), and the highest expression levels were found
in KLS cells.
As no phospho-speciﬁc antibodies against MITF are available,
we examined its nuclear translocation after culture of KLS cells in
different growth factor combinations as a measure of its activation.
Immunostaining for MITF demonstrated its nuclear translocation
after BMP4 treatment (Figure 4B). We quantiﬁed this effect by
immunoblotting nuclear extracts isolated from KLS cells cultured
under different conditions (Figure 4C-D). BMP4 treatment signiﬁcantly
increased the nuclear concentration of MITF (n ϭ 3; P Ͻ .01)
whereas CHD led to a decrease in nuclear localization of MITF
(P ϭ .04). In addition, SB203580 but not dorsomorphin inhibited
the increase in nuclear levels, and hence the activation of MITF
induced by BMP4.
BMP4-dependent activation of MITF is responsible for
induction of ITGA4 expression
To prove that MITF is the transcription factor responsible for
BMP4-mediated regulation of ITGA4 expression, we transduced
mouse BM derived LinϪ cells with an inducible lentiviral vector
encoding either a doxycycline inducible nonsilencing construct
(NSC) or a short hairpin RNA (shRNA) targeting Mitf (Mitf-KD;
Figure 5A). Transduced cells were selected using hygromycin, and
on induction with doxycycline, were identiﬁable by red ﬂuorescent
protein (RFP) expression. Mitf-KD and NSC cells were then
cultured with or without BMP4. Before further analyses, downregulation
of Mitf expression at the transcript and protein level was
conﬁrmed by qRT-PCR (Figure 5B) and immunoblotting (Figure
5C), respectively. We obtained 65% to 85% knockdown of Mitf in
the 3 independent experiments, which was accompanied with a
Ͼ 75% decrease in Itga4 expression (Figure 5B). As expected, in
NSC cells cultured with BMP4, the expression of Itga4 increased
signiﬁcantly. However, knockdown of Mitf inhibited BMP4mediated
induction of Itga4 expression (Figure 5B). Consistent
with the gene expression data, MITF protein levels were decreased
by 76% Ϯ 3.6% in Mitf-KD cells and this persisted when the cells
Figure 4. SMAD independent BMP4 signaling leads to activation of MITF.
(A) Quantitative RT-PCR for Mitf expression in different subpopulations of mouse BM
(n ϭ 3, P Ͻ .04). (B-D) KLS cells were cultured with SCFϩTPO in addition to BMP4
alone or combined with dorsomorphin or SB203580, for 5 days (ST ϭ SCF/TPO;
STC ϭ SCF/TPO/Chd; STB ϭ SCF/TPO/BMP4; STBD ϭ SCF/TPO/BMP4/Dorsomorphin;
STBS ϭ SCF/TPO/BMP4/SB203580), and analyzed by Western blot or
immunostaining. (B) Immunostaining with an antibody against MITF and
Hoechst33342. Representative example of 3 experiments. (C) Western blotting on
nuclear extracts of KLS cell progeny with antibodies against MITF and Histone H3 as
control. Representative example of 3 experiments. (D) Quantiﬁcation of nuclear MITF
protein levels in KLS cell progeny cultured for 5 days by densitometric analysis of
MITF and histone H3 intensities (n ϭ 3; *P Ͻ .04).
Figure 5. MITF is essential for BMP4-mediated increase in ITGA4 expression, and HSPC homing. (A) Schematic representation of Mitf knockdown experiments.
LinϪ cells were transduced with a lentiviral vector containing doxycycline inducible anti–Mitf-shRNA or nonsilencing construct (NSC). Expression of the shRNA was induced
with doxycycline and the transduced cells were selected using hygromycin. Expression of the anti–Mitf-shRNA or NSC was detected by RFP expression. Mitf-KD and NSC
LinϪ progeny were then cultured with or without BMP4 and evaluated by qRT-PCR, Western blotting, FACS and in vivo homing assays. (B) qRT-PCR for Mitf and Itga4
expression (n ϭ 3; P Ͻ .02). (C) Western blotting using antibodies against MITF and ␤-actin. Representative example of 3 independent experiments. (D) FACS analysis with
antibodies against ITGA4. Representative example of 3 experiments. (E) KLS cell progeny (0.1 ϫ 106) from the different cultures were transplanted intravenously in lethally
irradiated mice, and the BM harvested after 16 hours. Total BM cells (0.1 ϫ 106) were assayed in methylcellulose colony forming assay, Percentage homing was determined by
comparing total CFU-Cs homed with CFU-Cs injected (n ϭ 3, P Ͻ .02).
786 KHURANA et al BLOOD, 31 JANUARY 2013 ⅐ VOLUME 121, NUMBER 5
were cultured with BMP4 (Figure 5C; quantiﬁcation in supplemental
Figure 5C). Cell surface expression of ITGA4 was signiﬁcantly
decreased in cells with decreased expression of MITF (Figure 5D).
These results, together with the qRT-PCR results, suggest that
MITF might be regulating the basal level of ITGA4 expression in
HSPCs. Autocrine regulation of the BMP4 signaling could be one
of the determining factors. Further, unlike in NSC cells, BMP4 did
not induce increased cell-surface levels of ITGA4 in Mitf-KD cells
(Figure 5D).
We also performed homing assays using Mitf-KD and NSC cells
cultured with or without BMP4. The number of CFCs recovered
from recipient BM 16 hours after transplantation of NSC and
Mitf-KD cells was compared with the number of CFCs in the graft.
Homing of the cells from NSC group was signiﬁcantly increased
after incubation with BMP4 (P ϭ .02, Figure 5E), whereas homing
of cells from the Mitf-KD group cells was signiﬁcantly lower
compared with the NSC group. Further, culture of Mitf-KD cells
with BMP4 did not result in improved homing potential, conﬁrming
that the effect of BMP4 is MITF-mediated (Figure 5E).
BMP4 induces ITGA4 expression in primitive HSCs via p38
MAPK phosphorylation
In line with the LTR-engraftment data, we also demonstrated by
FACS (Figure 6A) that BMP4 activated the p38 MAPK pathway,
which was associated with increased ITGA4 expression in primitive
CD48ϪCD150ϩKLS cells. Clearly, inhibition of p38 MAPK
phosphorylation by SB203580 inhibited ITGA4 expression. In
addition, we sorted CD48ϪCD150ϩKLS cells and performed
immunostaining to analyze activation of p38 MAPK as well as
nuclear localization of MITF in response to BMP4 treatment
(Figure 6B). We observed that addition of BMP4 resulted in
phosphorylation of p38 MAPK, which also resulted in activation of
MITF as conﬁrmed by its nuclear localization (middle panel). We
also conﬁrmed inhibition of phospho-p38 MAPK by SB203580
(bottom panel), which resulted in reversal of the effect of BMP4 on
activation of MITF.
To further deﬁne the kinetics with which BMP4 affected ITGA4
expression, we cultured freshly isolated KLS cells with ST or STB
for 3 hours (top panel) and 6 hours (bottom panel) and evaluated
activation of p38 MAPK phosphorylation and subsequent effects
on ITGA4 expression (Figure 6C) and in vivo homing into the BM
(Figure 6D). BMP4 increased phosphorylation of p38 MAPK
within 3 hours (Figure 6C top left) and induced increased
expression of ITGA4 after 6 hours of BMP4 treatment (Figure
6C bottom right). Compared with ST treated cells, cells cultured for
6 hours with STB homed signiﬁcantly better (Figure 6D). After 6
hours of culture with SCFϩTPO homing potential of KLS progeny
was already decreased compared with uncultured cells, whereas
Figure 6. BMP4 enhances ITGA4 expression via p38 phosphorylation in KLS/CD150؉CD48؊ cells within 6 hours. (A-B) LinϪ BM cells were cultured in serum free
medium in the presence of SCFϩTPO in the presence or absence of BMP4 alone or with SB203580 for 5 days. ST ϭ SCF/TPO; STB ϭ SCF/TPO/BMP4;
STBS ϭ SCF/TPO/BMP4/SB203580. (A) After 5 days, ﬂow cytometry was performed to analyze p38 MAPK phosphorylation as well as ITGA4 expression. The cells were
harvested and stained with antibodies against Lineage positive cells, CD48, CD150, Sca-1, and c-Kit combined with anti–phospho-p38 MAPK or ITGA4 antibodies.
CD48ϪCD150ϩKLS cells were gated (top panel) and the levels of phosphorylated p38 MAPK (bottom left) and ITGA4 expression (bottom right) were compared among different
conditions. Representative example of 5 experiments. (B)The CD48ϪCD150ϩKLS subpopulation was sorted from LinϪ BM cell progeny cultured under the different conditions.
Immunostaining was performed to evaluate p38 MAPK and MITF activation using antibodies against phospho-p38 MAPK and MITF. Representative example of 3 experiments.
(C) Fresh KLS cells were cultured in the presence of SCFϩTPO with or without BMP4 for 3 hours (top panel) and 6 hours (bottom panel). Flow cytometry was performed to
analyze phosphorylation status of MAPK p38 (left) and ITGA4 (right) expression. Representative example of 4 independent experiments. (D) Homing potential of KLS cells
cultured for 6 hours in SCFϩTPO with or without BMP4 was compared with freshly isolated KLS cells (n ϭ 8; *P ϭ .002).
BMP4 IN ADULT HSC HOMING 787BLOOD, 31 JANUARY 2013 ⅐ VOLUME 121, NUMBER 5
BMP4 prevented this loss. Although not statistically signiﬁcant, we
consistently observed a minor increase in the homing potential of
the STB treated cells than the freshly isolated KLS cells (Figure
6D), consistent with the ﬁnding that secondary engraftment from
SCFϩTPOϩBMP4 treated cells appears higher than that of the
uncultured cells (Figure 1D).
BMP4 also activates p38 MAPK and induces ITGA4 in human
BM derived CD34؉Lin؊ cells
Finally, we tested whether BMP4 has similar effects on human BM
derived HSPCs. LinϪCD34ϩ cells were magnetically sorted from
BM mononuclear cells and cultured in the presence of SCFϩTPO
with or without BMP4. After 5 days, cells were harvested and gene
and protein expression analysis was performed by qRT-PCR and
ﬂow cytometry, respectively. As in murine BM HSPCs, the
expression of ITGA4 mRNA in human LinϪCD34ϩ cells was
signiﬁcantly increased in response to BMP4 (3.76- Ϯ 0.58-fold,
P ϭ .009, n ϭ 5; Figure 7A), which was conﬁrmed by FACS
(Figurs 7B-C). We also demonstrated by FACS using a phosphop38
MAPK speciﬁc antibody, that BMP4 activates p38 MAPK
(Figure 7C left) in the LinϪCD34ϩ subpopulation (Figure 7B) of
cultured human BM cells. Moreover, BMP4 affected ITGA4
expression in human HSPCs via the p38 MAPK signaling pathway,
as addition of SB203580 prevented BMP4-mediated ITGA4 expression
(Figure 7C right). To extend these ﬁndings in vivo, we
performed homing experiments (Figure 7D). Human BM-derived
linϪCD34ϩ cells were cultured in the presence of SCFϩTPO with
or without BMP4 and/or SB203580. After 5 days, the freshly
isolated cells as well as the cultured cells were transplanted in
lethally irradiated Rag2Ϫ/Ϫ␥CϪ/Ϫ mice. The number of homed
progenitors was calculated by performing colony assay on the
injected cells as well as on BM cells harvested 16 hours after
infusion of the LinϪCD34ϩ progeny. As observed in the murine
system, LinϪCD34ϩ progeny from cultures with BMP4 homed
signiﬁcantly better than cells cultured without BMP4 (P ϭ .002,
n ϭ 8; Figure 7D). Although we did not ﬁnd a signiﬁcant increase
in homing potential of the cells cultured with BMP4 over the
uncultured cells, BMP4 completely overcame the culture-induced
loss of homing potential of human HSPCs. Addition of SB203580
abrogated the effect of BMP4. Hence, BMP4 increases homing and
engraftment of both human and murine HSPCs via p38 MAPKmediated
increased ITGA4 expression.
Discussion
Apart from cell-intrinsic molecular interactions that control HSC
function,32 multiple extra-cellular signals play important roles in
the regulation of HSPC function.33 BMP4, a member of the TGF-␤
super-family, is important in the development of the hematopoietic
Figure 7. BMP4 increases ITGA4 expression in human BM-derived
HSPCs resulting in better homing after transplantation in mice.
Human BM derived LinϪCD34ϩ cells were cultured with SCFϩTPO with or
without BMP4 alone or with SB203580 for 5 days (ST ϭ SCF/TPO;
STB ϭ SCF/TPO/BMP4; STBS ϭ SCF/TPO/BMP4/ SB203580). Levels
of ITGA4 mRNA were assessed by qRT-PCR, expression of phospho-p38
MAPK and ITGA4 by FACS, or cells were used for in vivo homing assays.
(A) qRT-PCR analysis for ITGA4 in hBM derived LinϪCD34ϩ progeny from
ST, STB and STBS cultures. (n ϭ 5; * P Ͻ .01). (B-C) LinϪCD34ϩ
BM progeny were harvested and stained with antibodies against Lineage
markers, CD34, phospho-p38 MAPK or ITGA4. LinϪCD34ϩ cells were
gated (B), and phosphorylated p38 MAPK levels (C, left) and ITGA4 levels
(C, right) were compared among different conditions by FACS. Representative
example of 5 experiments. (D) Freshly isolated or cultured
hBM LinϪCD34ϩ cells (0.1 ϫ 106) were transplanted intravenously in
lethally irradiated Rag2Ϫ/ϪIl2R␥cϪ/Ϫ mice, BM was harvested after
16 hours and CFU-Cs enumerated in the recipient BM. Percentage
homing was determined by comparing total CFU-Cs homed with CFU-Cs
injected (n ϭ 8, P Ͻ .005).
788 KHURANA et al BLOOD, 31 JANUARY 2013 ⅐ VOLUME 121, NUMBER 5
system. Whether it is also important in postnatal hematopoiesis is
less clear. Elimination of SMAD5 does not affect adult hematopoiesis
in mice,10 suggesting that canonical SMAD-dependent BMP4
signaling might not be important. However, a recent study demonstrated
that hypomorphic BMP4 mice have decreased numbers of
HSCs,11 suggesting a role of BMP4 in postnatal hematopoiesis.
Moreover, Bhatia et al demonstrated that BMP4 maintains, although
does not expand, the engraftment ability of human SCIDrepopulating
cells in culture.6
Here, we describe a novel mechanism by which BMP4 regulates
murine and human HSPCs, which provides an explanation for
the somewhat contradictory ﬁndings described in this paper.
Consistent with Utsugisawa et al,9 BMP4 did not affect murine
BM-HSPC proliferation. Likewise, consistent with Bhatia et al, we
found signiﬁcantly improved short-term and long-term repopulation
from cells cultured with high concentrations of BMP4
compared with cells cultured under the same deﬁned conditions
with only SCFϩTPO. We demonstrate that the mechanism whereby
BMP4 affects repopulation of cultured cells is via increased
homing, because of maintained ITGA4 expression during the in
vitro culture step, and this via BMP4–mediated SMAD-independent
activation of the transcription factor MITF (supplemental Figure 7).
Homing depends both on migration and attachment of HSPCs
to the BM niche. Migration of HSPCs is predominantly governed
by the CXCL12-CXCR4 axis.34,35 However, we did not observe
any change in migration of HSPCs cultured with BMP4 to stromal
feeders, strongly suggesting that BMP4 does not affect homing and
engraftment of HSPCs by inﬂuencing this pathway. Attachment of
HSPCs to the BM niche is regulated in large part by ITGA4, and
ITGA4 is therefore important for both homing and maintenance of
HSCs.16,17,36,37 Here, we demonstrate that cells cultured for as long
as 5 days with SCFϩTPO in combination with BMP4 home and
establish long-term engraftment at least as well as freshly isolated
KLS cells. Of note, when KLS cells were treated with either CHD
or TSG, 2 extracellular inhibitors of BMP4, an opposite effect was
seen, that is, decreased radioprotection and long-term repopulation,
along with decreased adhesion and homing, as well as decreased
expression of ITGA4. This can be explained by autocrine regulation
by BMP4 signaling, as BMP4 expression in HSPCs has been
reported.9,38
Although BMP4 caused phosphorylation of SMAD1/5/8, inhibition
of this SMAD-dependent pathway by dorsomorphin did not
affect ITGA4 expression, the ability of KLS progeny to home into
the BM or impart radioprotection. In contrast, when KLS cells were
cultured in the presence of the p38 MAPK inhibitor SB203580
along with BMP4, ITGA4 up-regulation was inhibited. This was
associated with reversal of BMP4-enhanced homing and radioprotection.
Other signaling molecules that can partake in SMADindependent
signaling, such as ERK1/2, JNK, and S6K,2 already
active in KLS progeny cultured with SCFϩTPO, were not further
activated by BMP4 stimulation. The SMAD-independent signaling
pathway is initiated by TAK1 phosphorylation,39 which then
phosphorylates MKK3/6 or MKK4 to activate p38 MAPK40 or
JNK,41 respectively. In KLS cell progeny, BMP4 induced the
phosphorylation of TAK1 as well as MKK3/6. Consistent with the
ﬁnding that BMP4 treatment increased competitive long-term
repopulation of the KLS cells we also demonstrated that BMP4
activates p38 MAPK, which causes increased expression of ITGA4
in the primitive CD48ϪCD150ϩKLS subpopulation.
Although integrin-mediated adhesion is known to be regulated
by changes in the activation state of the receptor,18 changes in
integrin transcription also play a role. For instance, it is well
established that TGF-␤ up-regulates expression of ␣v␤3, ␣v␤5,
␣v␤6, and several ␤1 integrins in several cell types as well as in
cancer.21 In general, TGF-␤ inﬂuences integrin expression via
SMAD2/3 activation, even though noncanonical pathways also
play a role. Among the TFs that regulate ITGA4 expression is
MITF.42 MITF binds to a CACTTG motif in the Itga4 promoter.29 It
is a member of the basic helix-loop-helix family of TFs and plays
an important role in melanoblast biology.42 Mitf expression in
melanoblasts is inhibited by TGF-␤43 and Notch,44 and positively
regulated by Wnt3a43 and p38 MAPK.45 The latter also phosphorylates
MITF on Ser307, increasing its ability to activate transcription.31
In the mi/mi mouse, mast cells do not express ITGA4, which
is associated with their impaired adhesion to ﬁbroblasts, but is
restored on reintroduction of MITF.29,46
These studies led us to test whether MITF is the transcription
factor that mediates BMP4-induced increase in ITGA4 expression.
On stimulation with BMP4, MITF translocated into the nucleus of
KLS cell progeny, consistent with its activation. The same effect
was observed in immunocytochemistry experiments in which the
primitive CD48ϪCD150ϩKLS fraction was sorted from cultured
KLS progeny. This effect could be prevented by SB203580 but not
by dorsomorphin. When Mitf was knocked down, ITGA4 levels
were signiﬁcantly reduced in HSPCs, and BMP4–mediated induction
of ITGA4 was abrogated. Knockdown of Mitf signiﬁcantly
impeded homing and radioprotection of KLS cell progeny, which
could no longer be induced by addition of BMP4.
In conclusion, BMP4 treatment improves homing and engraftment
of HSPCs, which is mediated by p38 MAPK-induced
activation of MITF that up-regulates ITGA4 expression. We also
demonstrate that BMP4 increases ITGA4 expression and in vivo
homing of human BM linϪCD34ϩ cells via a similar signaling
pathway. As loss of ITGA4 is associated with decreased maintenance
of adult HSCs in the BM, our results help to explain the
hematopoietic defects in hypomorphic BMP4 mice, which might
be because of loss of retention in the BM niche.
Acknowledgments
The authors thank Vik Vanduppen for excellent assistance with
FACS sorting and analysis, and Rangarajan Sambathkumar and Drs
Valerie Roobrouck and Anujith Kumar for critical review for
accurateness of the paper.
This work was supported by an FWO grant (1.2.665.11.N.00) to
S.K.; FWO funding (G085111N), NIH-PO1-CA-65 493-06, Odysseus
funding, CoE and GOA/11/012 funding from KU Leuven, and
the Vanwayenberghe fund to C.M.V.
Authorship
Contribution: S.K. designed and performed the experiments,
analyzed the data, and wrote the paper; S.B. performed chimerism
experiments; S.S. performed FACS analyses and some of the
murine homing and engraftment studies; S.E. discussed the experiments
and conclusions and reviewed the paper; A.P. discussed the
experiments and conclusions and reviewed the paper; M.D. discussed
the experiments and conclusions and collected human BM
BMP4 IN ADULT HSC HOMING 789BLOOD, 31 JANUARY 2013 ⅐ VOLUME 121, NUMBER 5
samples; A.Z. discussed the experiments and conclusions and
reviewed the paper; and C.V. supervised all the research and edited
the paper.
Conﬂict-of-interest disclosure: The authors declare no competing
ﬁnancial interests.
Correspondence: Satish Khurana, Stem Cell Institute, KU
Leuven, O&N4, Bus 804, Herestraat 49, 3000 Leuven, Belgium;
e-mail: satish.khurana@med.kuleuven.be; or Catherine Verfaillie,
Stem Cell Institute, KU Leuven, O&N4, Bus 804, Herestraat 49,
3000 Leuven, Belgium; e-mail: catherine.verfaillie@med.kuleuven.be.
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