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Blood, 15 November 2002, Vol. 100, No. 10, pp. 3578-3587
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
Furin gene (fur) regulation in differentiating human
megakaryoblastic Dami cells: involvement of the proximal GATA
recognition motif in the P1 promoter and impact on the maturation
of furin substrates
Marie-Hélène Laprise,
Francine Grondin,
Pauline Cayer,
Patrick P. McDonald, and
Claire M. Dubois
From the Immunology Division, Department of Pediatrics,
Faculty of Medicine, University of Sherbrooke, Québec, PQ,
Canada.
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Abstract |
The convertase furin is involved in the maturation of key
growth/aggregation mediators synthesized by the platelet producers, megakaryocytes, but the regulation of furin in these cells remains unknown. Computer-assisted search of the furin promoter sequence revealed multiple potential binding motifs for GATA-1, suggesting that
furin is expressed and regulated in these cells. Using megakaryoblastic Dami cells, we observed that fur mRNA expression increased
gradually on phorbol 12-myristate 13-acetate-induced differentiation,
reaching maximum levels (8.3-fold increase) at 10 days. Transient
transfections with P1, P1A, or P1B fur-LUC-promoter
constructs revealed that in Dami cells, the P1 promoter is the
strongest and the most sensitive to forced expression of GATA-1.
Coexpression of GATA-1 and its comodulator, Friend of GATA-1 (FOG-1),
resulted in a cooperative increase in P1 activity. Deletion analysis
indicated that important GATA-1-regulated sequences are located in the
most proximal region of the P1 promoter. Further analysis revealed 2 potential GATA-binding motifs at positions  66 and +62. Point mutation
of each of the 2 motifs indicated that the intactness of the first GATA
site is required for full basal and GATA-1-stimulated promoter
activity. Finally, the inhibition of furin activity through gene
transfer of the inhibitor  1-AT-PDX led to a block in maturation of
the furin substrates transforming growth factor- 1 and
platelet-derived growth factor. Taken together, these results indicate
that the most proximal GATA element in the P1 promoter is needed for
fur gene expression in megakaryoblastic cells. They also
suggest that proper regulation of the fur gene in
megakaryocytes has an impact on the activation of furin substrates
involved in megakaryocyte maturation and platelet functions.
(Blood. 2002;100:3578-3587)
© 2002 by The American Society of Hematology.
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Introduction |
Platelet generation implies a set of
well-regulated processes that take place during megakaryocyte
differentiation. These processes are mediated by bioactive proteins;
among them are the growth/differentiation factors transforming growth
factor-1 (TGF-1)1,2 and platelet-derived growth
factor (PDGF),3 the key platelet adhesion molecules
IIb4,5 and von Willebrand factor
(VWF),6 and the cell-surface receptor
Notch-1,7 which are first synthesized as larger, inactive
precursor molecules. Their activation occurs through limited
endoproteolytic cleavage after a sequence of 2 or more basic residues
(K or R). In the 1990s, 7 closely related mammalian
subtilisin/kexin-like serine proteases with this cleavage specificity
were discovered. They are grouped under the generic name proprotein
convertases (PCs) and include PC1/PC3, PC2, PC4, PC5/PC6, PC7, PACE 4, and furin.8 Among the convertases, furin represents the
first and so far the best-characterized enzyme. The biologic importance
of this convertase stems from the large number and variety of bioactive
proteins and peptides that can be generated through its activity. These
include key elements involved in normal and physiopathologic
conditions, as exemplified by growth/differentiation processes. In
contrast to PC1/PC3, PC2, and PC4, which display tissue-specific
expression pattern, the fur gene is ubiquitously
expressed,9-11 but its levels of expression vary depending
on cell type and degree of cell differentiation.
For instance, high levels of furin mRNA were observed in liver and
kidney, whereas lower levels were detected in brain, spleen, and thymus
and even lower levels in heart muscle, lung, and
testis.2,12,13 In addition, the amounts of furin mRNA
varied throughout development; they are found coordinately expressed
with known furin substrates such as TGF-1, TGF-2, bone
morphogenic protein-4 (BMP4), BMP7,14-17 and
insulinlike growth factor.18 In particular, by
embryonic day 12 (E12) and the midgestational stage, there is
remarkable temporal correlation between the expression of furin and
TGF-1 in developing liver.19 This corresponds to the
establishment of hematopoiesis in this tissue, in particular for the
megakaryocyte lineage.20 It is well known that
megakaryocytes and platelets are a predominant source of the TGF-1
isoform involved in a variety of processes associated with
megakaryocyte differentiation and platelet reactivity.21
The mechanisms by which the fur gene is differentially
expressed and regulated are still poorly defined. It is known that at
least 3 distinct promoters mediate transcription of the fur gene. The fur mRNAs generated differ in their 5' end but are
all translated from the same AUG in exon 2, giving rise to identical furin proteins.22 The P1A and P1B promoters
resemble housekeeping genes with multiple Sp1-binding sites. On the
other hand, the P1 promoter has TATA and CCAAT elements in the proximal
region and has been shown to be transactivated by
C/EBP-.22 Differential expression of the P1 and
P1A promoters has been observed in hepatocytes, where the P1 promoter
was found to be the strongest; in endocrine cells, the P1A promoter
showed the strongest activity.22 Recent observations
indicated that in HepG2 hepatic cells, the P1 promoter is the strongest
and the most sensitive to TGF-.23 Studies of the
transcriptional activity involved indicate that the Smad signal
transducers are central participants of TGF--induced fur regulation in these cells. However, the transcriptional machinery involved in the regulation of furin in other cell types, including megakaryocytes, remains unknown. Interestingly, analysis of the fur P1, P1A, and P1B promoter sequences reveals the presence
of putative recognition elements for the transcription factor GATA-1 (A/T)GATA(A/G), including several closely spaced sites characteristic of GATA-1-regulated genes. This suggests that GATA-1 may regulate fur promoter activity in megakaryocytes.
Megakaryocyte differentiation leading to platelet generation is
characterized by successive and highly controlled stages coordinated by
the expression of transcription factors.24 Among the
transcription factors, GATA-1 is known to be essential for the
expression of several characterized megakaryocyte genes. For example,
the cis-regulatory promoter regions of VWF,25
IIb,26 and platelet factor 4,27 which are expressed in megakaryocytes, have critical GATA-1 recognition motifs. Gene disruption study involving a loss of GATA-1 in the megakaryocyte lineage resulted in developmental arrest at a late differentiation stage, firmly establishing the role of GATA-1 in the
differentiation process.28
GATA-1 is the first recognized member of the GATA family, which now
comprises 6 members named GATA-1 to -6 (for reviews, see Orkin et
al29 and Charron and Nemer30). In
hematopoietic tissues, the transcription factor GATA-1 is expressed in
multipotent progenitors, erythrocytes,31 megakaryocytes,
mast cells,32 and eosinophils,33 whereas
GATA-2 and GATA-3 are mostly expressed in early hematopoietic progenitors and T lymphocytes, respectively.34 Even if
their pattern of expression differs, these transcription factors can recognize the same consensus target sequence (T/A)GATA(A/G) through their highly conserved zinc fingers. A characteristic feature of many
GATA-1-sensitive promoter regions is the presence of recognition motifs in repeats or in a palindromic configuration that may stabilize GATA-1 anchoring.35 GATA-1 activity can also be modulated
by combinatorial association with other zinc finger proteins. Recently, a GATA-1 cofactor, named Friend of GATA-1 (FOG-1), was identified using
a yeast 2-hybrid screen for GATA-1-interacting
proteins.36 FOG is a large zinc finger protein that
interacts with GATA-1 through binding to its n-terminal zinc
finger.37 FOG-1 and GATA-1 cooperate to drive
megakaryocyte differentiation, and they have been shown to
synergistically activate the p45 NF-E2 and IIb promoters.36,38 FOG-1 is largely expressed in
hematopoietic tissue and in the liver.36 Like GATA-1,
FOG-1 is essential for megakaryopoiesis, but unlike GATA-1, which acts
late in megakaryocyte differentiation, the disruption of FOG leads to
the ablation of megakaryocyte lineage at an early stage.39
The finding that furin is involved in the maturation/activation of
several growth factors and integrins synthesized by megakaryocytes, coupled with the observation that the fur promoters contain
multiple GATA-1 motifs, prompted us to investigate the expression of
the fur gene in these cells. In this report, we demonstrate
that the expression of the fur gene is rapidly induced on
differentiation of the human megakaryocytic Dami cells, and we
identified a GATA-binding sequence within the proximal region of the
fur P1 promoter that is required for full basal and
GATA-1-induced activation. Blockage of furin activity in
differentiated cells has an impact on the maturation of furin
substrates TGF-1 and PDGF. These findings provide insight into the
mechanisms by which furin is expressed in differentiating
megakaryocytes and suggest that furin is involved in megakaryocyte
differentiation and platelet functions.
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Materials and methods |
Cell lines
Megakaryoblastic Dami cells were routinely grown in Iscove
medium (Gibco BRL, Burlington, ON, Canada) supplemented with 10% fetal
bovine serum (FBS; Biomedia Canada, Drummondville, PQ, Canada). For
differentiation experiments, 5 × 106 cells were cultured
in 100-mm plates (Costar, Cambridge, MA) containing 5 mL complete
culture medium and were treated with 100 nM on phorbol 12-myristate
13-acetate (PMA; Sigma, Oakville, ON, Canada) for various time periods,
as indicated in the figure legends. In Petri dishes, PMA treatment
increased the main ploidy of the culture, a unique developmental
feature of megakaryocytes.40 The other human cell lines
used in this study are the K562 cells (American Type Culture Collection
[ATCC], Manassas, VA), Meg-01 megakaryoblastic cells (ATCC),
HL-60 promyelocytic cells (ATCC), and Jurkat T cells (ATCC). The murine
myeloid leukemia clonal lines M1/pMGS (control vector) and M1GATA-Y22
(enforced GATA-1 expressing), generously provided by Dr Y. Yamaguchi
(Kumamoto University, Japan), have been characterized
elsewhere.41 The murine thymoma cell line EL-4 and the
pre-B cell line 70Z/3 were provided by Dr H. R. MacDonald (Ludwig
Institute for Cancer Research, Basel, Switzerland), and the pre-B cell
line 70Z/3 was from ATCC.
Northern blot analysis
Total cellular RNA was extracted from Dami cells with guanidine
isothiocyanate according to the previously described
TRI-reagent protocol.42 Northern blot analysis was
performed as previously described using a rat cDNA probe (generously
provided by Dr Robert Day, University of Sherbrooke, Canada).
2,23
Immunocytochemistry
Dami cells were grown on Goldline microscope slides (VWR Canlab,
ville Mont-Royal, QC, Canada) in the presence or absence of 100 nM PMA
for 3 days. Cells were fixed in cold methanol and permeabilized in cold
phosphate-buffered saline-0.1% Triton. For immunolabeling, cells were
incubated overnight with antihuman furin (1/1000; Chiron, Emeryville,
CA), antihuman GATA-1 (1/100; Santa Cruz Biotechnology, Santa
Cruz, CA), or monoclonal anti-Golgi 58K protein (1/100;
Sigma). Cells were washed and incubated for 30 minutes with
fluorescein-conjugated antibodies (antirabbit or antimouse, 1/200) or
rhodamine-conjugated antibody (antimouse, 1/800). Negative controls
were incubated with either preimmune rabbit serum or mouse
immunoglobulin G (IgG; Sigma).
Western blot analysis
Western blotting was performed on total cell lysates transferred
to nitrocellulose membranes (Roche Molecular Biochemicals, Québec, PQ, Canada) using antibodies directed against
IIb (1/1000; Immunotech, Marseilles, France), FOG-1
(1/1000; a generous gift from Dr Stuart H. Orkin), GATA-1 (1/500; Santa
Cruz Biotechnology), or actin (1/200; Sigma). Secondary antibodies were
peroxidase-conjugated antimouse IgG (1/2500; Amersham Pharmacia
Biotech), antigoat IgG (1/8000; Sigma), or antirabbit IgG (1/5000;
Amersham Pharmacia Biotech). Blots were developed using ECL Western
blotting detection reagent (Amersham Pharmacia Biotech).
Plasmids for transient transfections
The human fur promoter luciferase constructs pGL2-P1,
pGL2-P1-SacI, pGL2-P1-NheI,
pGL2-P1-KpnI, pGL2-P1A, and pGL2-P1B were generously
provided by Dr Torik A. Y. Ayoubi (University of Leuven and
Flanders Interuniversity, Belgium). The plasmid pMGS-GATA-1 was kindly
given by Dr Toshio Suda (University School of Medicine, Japan). As a
control vector, we used the pMGS plasmid from which GATA-1 cDNA was
previously excised with the restriction enzyme XhoI. FOG
cDNA, kindly provided by Dr Stuart H. Orkin, was inserted in pCDNA3.
Luciferase assays
Cells were transiently transfected by CaPO4
precipitation technique using a Mammalian Cell Transfection Kit
(Specialty Media, Lavallette, NJ) as previously
described.43 Briefly, 24 hours before transfection, Dami
cells were plated at a density of 500 000 cells/well in 6-well plates
(Falcon Labware, Missassauga, ON, Canada) and were differentiated with
100 nM PMA in 2 mL Iscove medium supplemented with 10% FBS. Cells were
fed with fresh complete media 3 to 4 hours before transfection. Dami
cells were transfected with 1 to 5 µg total plasmid/well, as
indicated in the figure legends. Plates were incubated overnight, and
cell lysates were assayed for luciferase activity as previously
described.23 Data were from at least 3 independent
experiments performed in duplicate. Values were normalized for
transfection efficiency with either green fluorescence protein (GFP)
mean fluorescence or -galactosidase activity.
Elimination of the GATA-1 recognition motifs on the
fur P1-KpnI promoter
The following oligonucleotides were used to engineer the
different fur P1-KpnI mutants:
5'-GCATTCTAGTTGTGGTTTGTCC-3' (sense), 5'-GTGCGACCATCTATGTCACCACCAC-3'
(sense), 5'-CCTGTGAAGGTCTCTGAGCCTGACTG-3' (sense),
5'-GTGGTGGTGACATAGATGGTCGCA-3' (antisense),
5'-CAGTCAGGCTCAGAGACCTTCACAGG-3' (antisense), and
5'-CATAGCCTTATGCAGTTGCTCTCCA-5' (antisense). First, the fur
P1-KpnI-Mut1 was obtained by polymerase chain reaction (PCR)
using the pGL2-P1-KpnI plasmid with primers 1 and 4 or 2 and
6. After amplification, the 2 PCR products were mixed together, and a
subsequent PCR was performed with primers 1 and 6. The obtained fragment was excised KpnI and HindIII and
subcloned into pGL2. Fur P1-KpnI-Mut2 was
performed using primers 1 and 5 or 3 and 6 in separate reactions. The 2 distinct products were pooled together and subjected to another PCR
with primers 1 and 6. The resultant sequence was excised with
KpnI and HindIII and subcloned into pGL2.
Fur P1-KpnI-Mut1/2 was obtained by replacement of
the fur P1-KpnI-Mut1
ApaI/HindIII region by the fur
P1-KpnI-Mut2 ApaI/HindIII portion. All
mutants were sequenced before transfection.
Electrophoretic mobility shift assays
Gel mobility retardation assays were performed as
described.44,45 Synthetic nucleotides used in
electrophoretic mobility shift assay (EMSA) experiments were designed
to include the 66 GATA sites of the P1 promoter with the respective
sequences (74) 5'-GTGCGACCAGATATGTCACCACCACATCACTTTTAG-3'. Other
nucleotides used for cold-competition corresponded to 66 GATA
oligonucleotide with the following mutations (bold italic
letters) within the GATA site sequence (66)
5'-GTGCGACCATCTATGTCACCACCACATCACTTTTAG-3' or a GATA
consensus oligonucleotide featuring 2 GATA sites in tandem
5'-CACTTGATAACAGAAAGTGATAACTCT-3' (Santa Cruz Biotechnology).
Adenoviral vector construction
The gene encoding full-length  1
antitrypsin-Portland (PDX; kindly provided by Jeff Lipps, Hedral
Therapeutics, Portland, OR), human TGF-1 (ATCC), or human
fur (from Dr Gary Thomas, Vollum Institute) was inserted
into the multiple cloning site of the transfer vector pAd-TR5F-DC-GFP
and was placed under the control of a modified cytomegalovirus (CMV)
promoter containing a tetracycline (tet)-regulated expression
cassette46,47 and expressed together with the GFP tracer.
Adenoviral vectors were produced as described47 and were
titered by flow cytometry using GFP as a marker of infection. AdCMVtTA,
expressing the transactivator tTA under the control of a constitutive
CMV promoter, was obtained from Dr Bernard Massie (Biotechnology
Research Institute of Montreal, QC, Canada).
Measure of mature TGF-1 and PDGF-AB
Supernatants were assayed for TGF-1 and PDGF-AB using a
commercially available enzyme-linked immunosorbent assay specific for
bioactive TGF-1 or mature PDGF-AB (R&D Systems, Minneapolis, MN).
Supernatants were activated for 10 minutes at 80°C before TGF-1
detection. The detection limit for TGF-1 is 30 pg/mL and 9 pg/mL
for PDGF-AB.
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Results |
Increased expression of endogenous furin in differentiating
Dami cells
To define the pattern of furin expression during megakaryocyte
differentiation, we used the megakaryoblastic Dami cell line. These
cells exhibit many morphologic and biochemical characteristics of human
megakaryocytes and can be induced to further differentiation along
megakaryocyte/platelet lineage on PMA treatment.48 To investigate the expression of furin in megakaryocytes, Dami cells were
differentiated for different time periods with 100 nM PMA. Northern
blot analysis revealed a rapid increase in furin mRNA levels, with a
7.9-fold increase observed at 3 days and maximum levels (8.3-fold)
reached at 10 days (Figure 1A). As
previously demonstrated, differentiation of these cells with PMA
results in cell adhesion, DNA ploidy, and augmentation in structures
characteristic of proplatelet formation48 (data not
shown). This was correlated with an increase in the transcription
factor GATA-1 (Figure 1B), a molecule known to be up-regulated on
megakaryocyte differentiation.45,31 In contrast, the
expression levels of FOG-1, a GATA-1 cofactor known to act early in
megakaryopoiesis,39 remained essentially unchanged.
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| Figure 1.
Expression of furin in differentiating megakaryocytic
cells.
Megakaryoblastic Dami cells were cultured with 100 nM PMA for various
time periods as indicated. (A) Kinetics of fur mRNA
accumulation. Total mRNA (5 µg/lane) was probed with rat riboprobe
specific for furin or 18S. The autoradiogram and the densitometry ratio
of furin/18S (control set at 1) is represented. (B) Western blot
analysis of GATA-1 and FOG-1 in differentiated Dami cells.
Cell lysates were separated on 10% sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and
transferred to nitrocellulose membranes. Immunoblotting of GATA-1 and
FOG-1 were performed using GATA-1(1/500) or FOG-1 (1/1000)-specific
antibodies. Equal loading was confirmed using an antibody against actin
(1/200). (C) Fur expression in megakaryocytic cells. Total
mRNA was extracted from unstimulated murine and human hematopoietic
cell lines and from HL-60 and K562 cells cultured with 100 nM PMA for 3 days; mRNA was probed with rat riboprobe specific for furin or
18S.
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We next examined the regulated expression of the fur gene in
other megakaryocytic cell differentiation systems. For this, Northern
blot analysis was performed using mRNA from the human K562 cells
differentiated along megakaryocyte/erythroid lineage with PMA, the
murine myeloid cell line M1, a murine myeloid cell that undergoes
megakaryocytic differentiation on enforced GATA-1 expression,41 and MEG-01 cells, a human megakaryoblastic
cell line with features characteristic of differentiated
megakaryocytes.49,50 As a control, we used human
promyelocytic leukemia HL-60 cells, which are known to differentiate
along the monocytic lineage in the presence of PMA.51 As
observed with the Dami cell differentiation system, treatment of K562
cells with PMA for 3 days resulted in a marked increase in fur
expression (Figure 1C). In contrast, similar treatment did not
significantly affect furin mRNA levels in myeloid HL-60 cells. These
results suggest that fur regulation is associated with the
cell differentiation process toward the erythroid/megakaryocyte (K562)
and megakaryocyte (Dami) lineages rather than direct PMA stimulation.
Further supporting this possibility, fur expression levels
were also elevated in myeloid M1 cells transfected with GATA-1, a
process known to induce a change in the phenotype of these cells from
myeloid to erythroid/megakaryocyte lineage.41
To ensure that the augmentation in furin mRNA levels resulted in an
increase in the furin protein, we assessed the relative levels of
immunoreactive furin in Dami cells that were or were not differentiated
with 100 pM PMA for 3 days. Results expressed in Figure
2A indicated that PMA treatment resulted
in a strong increase in furin immunoreactivity compared with untreated
cells. Furin immunostaining was found to overlap with the staining of the 58K Golgi marker (Figure 2B), a feature consistent with the main
localization of furin within the trans-Golgi
compartment.52,53
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| Figure 2.
Localized expression of furin convertase in
differentiated Dami cells.
Immunofluorescence was performed using Dami cells undifferentiated or
differentiated for 3 days with 107 M PMA. Cells were
fixed, permeabilized, and stained with (A) mouse anti-GATA-1 (1/100)
or rabbit anti-furin (1/1000) and then visualized by fluorescence
microscopy, using a secondary antibody coupled to fluorescein
isothiocyanate. (B) Three-day differentiated cells were costained with
rabbit anti-furin (FITC) and mouse anti-58K (1/100; rhodamine).
Fluorescence colocalization corresponds to Golgi structures around the
nuclei. Preimmune serum was used as negative control.
Magnification, × 400.
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Differential sensitivity of the fur promoters to the
transcription factor GATA-1
Furin transcription is mediated by at least 3 distinct
promoters P1, P1A, and P1B. As mentioned, the P1 promoter has features of regulated promoters, whereas P1A and P1B have the characteristics of
promoters for constitutive/housekeeping genes. Computer-assisted search
for the presence of the consensus GATA-binding motif 5'-(A/T) GATA
(A/G)-3'54 within these promoters identified 10, 8, and 4 potential GATA sites for P1, P1A, and P1B promoters, respectively (illustrated in Figure 3A). To determine
the capacity of each promoter to be transactivated by GATA-1, Dami
cells were transiently transfected with P1, P1A, or P1B promoter-Luc
construct with or without construct-encoding GATA-1. As indicated in
Figure 3B, enforced expression of GATA-1 increased fur P1
and P1B promoter activity by 9.54 ± 0.68-fold and
6.36 ± 0.34-fold, respectively. In the same set of experiments, the
P1A promoter did not express any significant sensitivity to exogenous
GATA-1. Further studies, therefore, were performed with the most
sensitive P1 promoter of GATA-1.
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| Figure 3.
Basal and GATA-1-induced transactivation of the fur P1, P1A, and P1B
promoters.
(A) Schematic representation of localization and sequence of the GATA-1
recognition motifs present in the 5' noncoding exons 1, 1A, and 1B of
the human fur gene. (B) Transient cotransfection of Dami
cells, with fur P1, P1A, or P1B promoters and either pMGS
control vector or pMGS-GATA-1 vector. Before transfection, Dami cells
were differentiated overnight with 100 nM PMA. Luciferase activity is
expressed as fold-increase relative to P1 promoter activity (set at 1)
cotransfected with pMGS control vector. Data are expressed as the
mean ± SEM; n = 3. **P < .001, compared with
promoter constructs without GATA-1.
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FOG acts in cooperation with GATA-1 to amplify fur
P1 promoter transactivation
In addition to contacting DNA, GATA-1 was reported to interact
with transcriptional cofactors such as the multitype zinc finger FOG-1.37 FOG-1 and GATA-1 can cooperate to
activate or to repress promoter activities, depending on the cell type
or the promoters studied.36,38,55 To define the impact of
FOG on fur P1 promoter transactivation, Dami cells were
transfected with FOG-1 in the presence or absence of GATA-1. As
demonstrated in Figure 4A, GATA-1 alone
increased by 8.1 ± 2.3-fold the levels of fur P1 promoter transactivation compared to control P1 vector, whereas FOG-1 expression resulted in a milder 2.8 ± 0.2-fold increase in activity.
Coexpression of FOG-1 with GATA-1 induced an additional amplification
(2.4-fold) of fur P1 promoter activity. Therefore, FOG-1,
used in the fur P1 promoter context, acts as a potent
coactivator of fur expression in Dami cells. Coactivation by
FOG-1 and GATA-1 was not dependent on other megakaryocyte-specific
transcription factors because similar results were observed using the
nonhematopoietic HepG2 cell line (Figure 4B).
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| Figure 4.
Transactivation of the fur P1 promoter by GATA-1 and
FOG-1.
(A) Dami cells were differentiated overnight with 100 nM PMA, or (B)
HepG2 cells were transiently cotransfected with fur P1
promoter and either pMGS control vector or pMGS-GATA-1, in combination
with pCDNA3 control vector or pCDNA3-FOG-1. Luciferase activity is
expressed as fold-increase relative to the activity of P1 promoter
cotransfected with pMGS control vector (value set at 1). Data are
expressed as the mean ± SEM; n = 3. **P < .001,
compared with P1 promoter activity.
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Analysis by 5' deletion of P1 promoter activity in Dami
cells
To delineate the promoter regions implicated in the constitutive
and GATA-1-induced regulation of the fur P1 promoter, 5' deletion constructs were tested in transient transfection assays. As
the promoter sequence was progressively deleted in 5', constitutive promoter activity gradually increased in Dami cells, reaching 9.5 ± 1.7-fold more luciferase activity for the shorter 502-base pair (bp) KpnI fragment (Figure
5). In contrast, the sensitivity of the
P1 promoter fragment to overexpressed GATA-1 gradually decreased with
truncation of the distal promoter region with 3.6 ± 0.6-fold
stimulation for the KpnI fragment compared to
13.3 ± 1.3-fold for the intact P1 promoter. These results indicate that even though several of the putative GATA sites dispersed throughout the P1 promoter are presumably functional, 27% of the P1
sensitivity to GATA-1 remains within the shorter 502-bp region between
position 413 to +89 of the fur P1 promoter. They also suggest the existence of repressor region(s) upstream of the most proximal 413 promoter region.
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| Figure 5.
Basal and GATA-1-induced transactivation of P1 5' deletion constructs.
(A) Schematic representation of GATA recognition sequences present in
fur P1 promoter fragments shortened in 5' accordingly to
endogenous SacI, NheI, or KpnI
restriction sites. Base positions are numbered relative to the TATA
box. (B) Dami cells were incubated overnight with 100 nM PMA and were
cotransfected with fur P1, P1-SacI,
P1-NheI, or P1-KpnI constructs and either pMGS
control vector or pMGS-GATA-1 vector. Luciferase activity is expressed
as fold-increase relative to the P1 promoter cotransfected with the
pMGS control vector. Data are expressed as the mean ± SEM;
n = 3. **P < .001, compared with promoter constructs
without GATA-1.
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Functional analysis of GATA-binding sites within the
KpnI- P1 promoter fragment
Analysis of the nucleotide sequence of the 502-bp DNA fragment
extending upstream of the transcription start site of the
fur gene reveals the presence of 2 potential GATA-binding
sites (Figure 6A). One site, at position
66, exhibited the sequence 5'-AGATAT- 3', with one
mismatch with the consensus GATA-binding motif
(5'-(T/A)(GATA)(A/G)-3'54 at its 3'end (T instead of A/G).
The second site, 5'-GGATAG-3' located +62 bp downstream of
the first site, also diverges slightly from the consensus sequence,
with one mismatch at the 5' end (G instead of T/A).
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| Figure 6.
Functional analysis of GATA-binding sites within
fur P1-Kpn1 promoter
region. Dami cells were incubated overnight with 100 nM PMA and
transiently transfected with the fur P1-KpnI
mutant promoter constructs illustrated on the left side of the figure.
(A) Measure of basal promoter activity (cotransfection with pMGS
control vector); data are expressed as percentage of nonmutated
P1-KpnI construct. (B) GATA-1-induced activity
(cotransfection with pMGS or pMGS-GATA-1 vectors). Data in panels A
and B are expressed as percentage of the nonmutated P1-KpnI
construct. Results are expressed as the mean ± SEM; n = 4.
**P < .001, compared with nonmutated P1-KpnI
promoter construct. (C) Binding of GATA-1 to the 66 P1 GATA site.
Nuclear extracts (1.5 µg) from PMA-differentiated Dami cells were
incubated (1 hour at 4°C) in binding buffer containing 0.8 µg poly
(dI-dC) before further incubation (10 minutes at room temperature) with
a 32P-labeled oligonucleotide spanning the 66 GATA motif
(bp 74 to 39) of the fur P1 promoter. The specificity of
complex formation was tested by the inclusion of unlabeled competitors
in the binding buffer (cold probe, lanes 6, 7; cold P1 probe with
mutated 66 GATA sites, lanes 8, 9; or unlabeled consensus GATA probe,
lanes 10, 11), or by including antibodies to GATA-1 (antibodies N6,
sc-265 and C-20, sc-1233; Santa Cruz Biotechnology), lanes 2 and 4, respectively) or isotype-matched controls (rat IgG or goat IgG, lanes 3 and 5, respectively). SS indicates supershift complex; ns, nonspecific
band; I, cluster of slowly migrating bands; II and III, faster
migrating complexes.
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Site-directed mutagenesis was performed with the AGATAT site changed
for ATCTAT and the GGATAG site changed for a
GTCTCT sequence.27 This resulted in
3 distinct mutantsP1-KpnI-Mut1, P1-KpnI-Mut2, and P1-KpnI-Mut1/2that correspond to the elimination of
the first, the second, or both GATA recognition sites. Expression of
the P1-KpnI-Mut1 in Dami cells, which constitutively express
endogenous GATA-1, reduced the activity to 39% ± 3% of the intact
fragment, indicating that this site is critical for promoter activity.
In contrast, expression of P1-KpnI-Mut2 resulted in a more
modest decrease in luciferase activity (to 70% ± 9%), suggesting
that this site has a minor contribution to P1 promoter transactivation. Finally, mutation of the 2 GATA sites (P1-KpnI-Mut1/2)
decreased Luc activity to a level (33% ± 2%) similar to the one
observed with P1-KpnI-Mut1 (Figure 6A) but resulted in an
additive reduction in GATA-induced transactivation (Figure 6B). In
parallel, results from coexpression of the transcription factor GATA-1
indicated that the first AGATAT motif of the KpnI P1
fragment is indeed the most responsive to GATA-1 (Figure 6B). From
these results, we conclude that the intactness of the 66 AGATAT site
is critical for high-level expression of the fur gene in
Dami cells and that the second +62 site can act in cooperation,
especially when GATA-1 is highly expressed.
We also tested whether the 66 GATA site of the fur P1
promoter was likely to be occupied by its cognate transcription factors in PMA-differentiated Dami cells. To this end, nuclear extracts were
analyzed in EMSA using an oligonucleotide probe spanning the 66 GATA
site. This revealed the existence of 3 groups of DNA-binding activities
(Figure 6C, lane 1): a cluster of slowly migrating bands (complex 1)
and 2 faster-migrating complexes (2 and 3). All complexes showed
specificity in their binding to the P1 region used as a probe because
they were effectively competed by an excess of cold probe (lanes 6, 7)
but mostly were unaffected by equivalent quantities of a similar
oligonucleotide featuring mutated GATA sites (lanes 8, 9). By contrast,
only the middle complex (complex 2) was competed using a commercially
available oligonucleotide featuring a consensus GATA sequence (lanes
10, 11). More direct evidence that complex 2 contains GATA proteins was
obtained using an antibody that interferes with the DNA-binding activity of GATA-1, which displaced complex 2 (lane 2). Similarly, an
antibody recognizing the C-terminal domain of GATA-1 supershifted complex 2 (lane 4). Because neither antibody displayed cross-reactivity with other GATA family members, we conclude that endogenous GATA-1 is a
component of the DNA-binding complex interacting with the fur P1 promoter in PMA-differentiated Dami cells.
Impact of furin regulation in megakaryocytes on the production of
mature furin substrates
Megakaryocyte differentiation is accompanied by sequential
expressions of growth factors/receptors and adhesion molecules characterized by the presence of a consensus furin recognition motif at
the maturation site.1-7 Among them are the key growth factors TGF- and PDGF and the IIb chain component of
the integrin IIb3 complex. As a first step to define
the biologic relevance of regulated furin expression in megakaryocytes,
we investigated whether this convertase is coordinately regulated with
furin substrates in Dami cells. Results expressed in Figure
7A indicate that PMA-induced Dami cell
differentiation is associated with a gradual increase in the
accumulation of TGF-1 and PDGFAB in the culture medium. Interestingly, the production and the maturation of the IIb chain of
the integrin IIb3 are also up-regulated, as evidenced
by the increased abundance of the precursor and the mature forms, combined with a gradual augmentation in conversion ratios.
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| Figure 7.
Role of furin in the production of mature integrin and growth factors.
(A) Megakaryoblastic Dami cells were cultured with 100 nM PMA for
various time periods, as indicated, supernatants were collected, and
TGF-1 and PDGF AB were measured by ELISA. Corresponding cell lysates
were separated by SDS-PAGE and immunoblotted using IIb-specific
antibodies (1:1000). Western blots were scanned, and differences in
staining intensity were measured using NIH image software. Western blot
results are expressed as a ratio of mature to precursor forms, which is
an estimation of conversion efficiency. (B) Dami cells were incubated
overnight in the presence of 100 nM PMA. Cells were infected with the
indicated recombinant adenovirus. Forty-eight-hour supernatants were
collected for TGF-1 and PDGFAB determination. Data are
expressed as the mean ± SEM; n = 3. **P < .001,
compared with control adenovirus. (C) Cells were coinfected with the
indicated recombinant adenovirus. 48 hours after infection,
supernatants were concentrated, separated on SDS-PAGE gels, and
immunoblotted using an anti-TGF--specific antibody (1:1000;
R&D Systems).
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In parallel experiments, we tested whether blockage of furin activity
impacts the production of mature forms of furin substrates. For this,
we used a modified adenovirus vector (AdTR5) for cell delivery of
1-AT-PDX, a potent furin inhibitor.56 In this system, the 1-AT PDX gene is placed under the negative control of
a tet/doxycycline-regulated promoter sensitive to low concentrations of
doxycycline in cell cultures.46 As observed in Figure 7B,
the infection of differentiated Dami cells with AdTR5PDX significantly
reduced the amounts of mature and active TGF-1 and
PDGFAB released in the cell supernatants. Similarly,
complete blockage in the processing IIb chain of integrin IIb3 was observed on adenovirus-induced transduction
of 1-ATPDX in Dami cells (data not shown). The addition of 1 µg/mL
Dox to the cultures abolished AT-PDX production and its effect on the production of mature growth factors. To confirm that, in our system, PDX overexpression indeed resulted in an inhibition of furin-dependent processing, coinfection experiments were performed using recombinant adenovirus encoding for the TGF-1 substrate or for 1-ATPDX, and
concentrated supernatants were analyzed for TGF-1 processing by
immunoblotting. As illustrated in Figure 7C, supernatants from TGF-1-infected cells produced approximately 60% of processed TGF-1 as seen by the relative intensity of the proregion and the
pro-TGF-1 bands. Coinfection of cells with 100 multiplicity of
infection (MOI) of adenovirus encoding 1-ATPDX abrogated
pro-TGF-1 proteolytic processing mediated either by
endogenous furin activity or by overexpressed furin. Taken
together, these results indicate that the TGF-1, PDGFAB,
and IIb substrates that are associated (integrin) or
involved (growth factors) in megakaryocyte differentiation undergo furin-dependent cleavage.
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Discussion |
The mammalian convertase furin is responsible for the maturation
of key platelet aggregation/coagulation mediators synthesized by the
platelet producers, megakaryocytes.1-6 To date, however, there has been no study on the expression/regulation of the
fur gene in these cells. In this report, we demonstrate that
the fur gene and furin-converting activities are expressed
at low levels in human megakaryocytic cells, and their expression is
rapidly induced on cell differentiation with PMA. By promoter deletion and mutation analysis, we identified a region within the human fur gene that regulates fur transcription in
megakaryocytes. This site contains the core-binding
(A/T)GATA(A/G)55 sequence for GATA zinc finger
transcription factors and is required for constitutive and
GATA-1-induced transactivation of the proximal P1 promoter region.
Three alternative promoters can drive transcription of the
fur gene. The P1A and P1B promoters resemble housekeeping
genes with multiple Sp1-binding sites. On the other hand, the P1
promoter has TATA and CCAAT elements in the proximal region and has
been shown to be transactivated by C/EBP-22 and members
of the Smad family.23 Herein, we show that even though
each of the 3 fur promoters has several potential GATA
recognition sequences, in Dami cells, the P1 promoter is the strongest
and the most sensitive to forced expression of GATA-1, whereas little
or no induction was observed for P1B or P1A. The exact reason for this
discrepancy is unclear, but it has been reported for several
GATA-1-controlled genes (including the maj-globin57
and the mpl receptor58) that GATA motifs located proximal
to the translation initiation site are critical for promoter
transactivation. As demonstrated in Figure 3A, proximal GATA elements
are found within the P1 and P1B promoters only, and this correlates
with their capacity to be transactivated by GATA-1. In addition, for
the P1 promoter, the GATA sites (66 and +62) were found proximal to
CCAAT (129) and CACCC (28) elements, a promoter context that might
contribute to its robust transcriptional activation by GATA-1a
situation that has been described for the PECAM-1
promoter.59 In contrast to other
megakaryocyte-restricted genes, such as the integrin IIb3, the fur P1 promoter more likely
qualifies as a megakaryocyte-sensitive promoter because it
is expressed in other cell types, such as HepG2 and COS cells.
In the nonhematopoietic HepG2 cell, for example, the transcriptional
activity of the P1 promoter is in part under the control of endogenous
C/EBP- and of members of the Smad transducers.22,23 The
known capacity and yet to be uncovered capacity of the
fur promoters to respond to various transcriptional contexts
would likely help explain the temporal and spatial expression of furin in megakaryocytes and in other tissues and organs.
The proximal region of the fur P1 promoter contains 2 potential GATA-binding sites at positions 66 and +62. The relative contribution of each GATA-1-binding site to the transcriptional activity of the promoter was examined. Ablation by mutation of the
first AGATAT site resulted in a strong (61%) reduction in constitutive promoter activity and a 52% inhibition in overexpressed GATA-1-induced P1-Mut1 promoter construct. The importance of its integrity for fur promoter activity in Dami cells clearly
indicates that this GATA site represents a major cis-acting
element for the human fur gene in these cells. Even though
the AGATAT site diverges slightly from the well-known
optimal (A/T)GATA(A/G) consensus sequence, EMSA indicated that it does
interact with GATA-1. In contrast to the 66 site, destruction by
mutation of the second GATA site resulted in milder 30% reduction in
constitutive promoter activity and 46% inhibition in GATA-1-induced
promoter activation. This site, GGATAG, slightly differs
from the consensus GATA recognition site and is located downstream of
the TATA box; neither situation may be favored within the
fur promoter context. Finally, mutation of the 2 GATA sites
(P1-KpnI-Mut1/2) decreased basal promoter activity to a
level similar to that observed with mutation of the first GATA site
only, but it resulted in an additive reduction in GATA-induced
transactivation. From these results, we conclude that the intactness of
the 66 AGATAT site is critical for high-level expression of the
fur gene in Dami cells and that the second +62 site may have
lower affinity for GATA-1 because it is involved in conditions in which
GATA-1 is overexpressed.
Several studies have shown that GATA-1 site repeats found in 5' or 3'
orientation or high-affinity palindromic sites, composed of one
complete (A/T)GATA(A/G) and one partial (GAT) canonical motif found on
the opposite DNA strand, are the hallmark of erythroid/megakaryocyte cell-expressed genes, including GATA-1 and the human  -globin promoter.35 It has been demonstrated that the palindromic
sites can bind one molecule of GATA-1 with high affinity because they are able to engage the N-terminal and C-terminal zinc fingers of the protein.35,60 Interestingly, the prominent P1 GATA
site is flanked by a partial GATA motif, TGATGT, found in a
3' position, 9 bp on the opposite strand of the AGATAT site.
Such positioning evokes the reported high-affinity palindromic GATA
site, with more distance between the minor and the major GATA sites (9 bp instead of 3 bp).
Deletion studies of the P1 promoter indicated that as the promoter
sequence was progressively truncated in 5', constitutive promoter
activity gradually increased in Dami cells, reaching 9.5 ± 1.7-fold
more luciferase activity for the more proximal 502-bp KpnI
fragment. This could indicate the existence of a repressor region
upstream of the most proximal 2554 to 413 region. The exact nature
of this repressor region is unknown, but analysis of the P1 sequence
indicates the presence of a series of CATGAG sequences upstream of the
more responsive GATA-1 region. One site is located between the
NheI-KpnI restriction sites and 3 sites between
SacI and NheI. Because CATGAG sites have been
shown to repress cis-acting elements of the
IIb promoter,61 it is conceivable that such
sites act in a similar fashion for fur P1 promoter
regulation. In this context, temporal expression of furin during
megakaryocyte differentiation would involve a block in the activity of
these repressor regions.
In addition, we provide evidence that FOG-1, a cofactor for
GATA-binding proteins, acts as a positive cofactor for fur
P1 transactivation. FOG, in association with GATA-1, consistently resulted in more than a 2-fold increase in P1 transactivation than
GATA-1. Similar cooperation between FOG and GATA-1 had been observed
for the transactivation of the megakaryocytic gene
IIb38 and the erythromegakaryocytic gene
p45 NF-E2,36 among others. This is consistent
with its essential participation in the maturation of erythroid cells
and the early development of thrombocytic lineage.39 On
the other hand, FOG recruited by GATA-1 acts as a repressor of the
eosinophil lineage and down-regulates the eosinophil-specific markers
EOS47, C/EBP, and Mim-1.55 In contrast to GATA-1, which is expressed at low levels in Dami cells and is up-regulated on cell
differentiation, the FOG-1 protein remained at a relatively high level
of expression throughout the differentiation process (Figure 1B). In
this context, cooperative interaction between GATA-1 and FOG-1 might be
important for the rapid elevation of furin observed at an early stage
of megakaryocyte cell differentiation, a situation in which
others62 and we have detected only threshold levels of
endogenous GATA-1.
The observed increase of furin expression in megakaryocytes suggests
the implication of this convertase in the bioavailability of selected
mediators. Among the known furin substrates, the potent regulator of
cell growth/differentiation PDGF and TGF-, and the platelet adhesion
molecules to VWF, integrin subunits 6 and
IIb were observed to be increased along megakaryocyte
differentiation.48,63-66 A body of literature has firmly
established that VWF and IIb are essential for thrombus
formation at the vascular injury site, whereas platelet
-granule-derived TGF- and PDGF are major players of endothelial
cell proliferation and vessel repair.67-70 In addition, TGF-1 has been shown to enhance platelet aggregation through a
nontranscriptional effect on the fibrinogen receptor.71
Furin has also been identified as the convertase responsible for the maturation of Notch-1, a cell-surface receptor involved in multiple developmental processes.7 Interestingly, a recent study
using a constitutively active intracellular domain of Notch-1 in
bipotent hematopoietic cells indicated that Notch-1 is permissive for
the differentiation of megakaryocytes while it suppresses erythroid maturation.72 Therefore, the proper expression/regulation
of furin in megakaryocytes would likely be important for correct processing of mediators involved in megakaryocyte differentiation and
platelet function (Figure 8).
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| Figure 8.
Proposed impacts of fur expression/regulation in
megakaryocytes.
The events depicted here are initiated with hematopoietic cell
differentiation into megakaryocytes. In this scheme, we extrapolate the
results observed with various megakaryocytic cell lines to the events
proposed to occur in primary cells. Fur regulation in
megakaryocytes is, in part, under the control of the transcription
factor GATA-1, and the relevance of other transcription factors in this
regulation is not excluded. The role of furin in the
maturation/activation of the integrin IIb will be
published elsewhere (M.-H. L. et al, mansucript in prepration).
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Acknowledgments |
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Abstract
Introduction