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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Immunology Division, Department of Pediatrics,
Faculty of Medicine, University of Sherbrooke, Québec, PQ,
Canada.
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 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- 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- 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- 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
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 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- Cell lines
Northern blot analysis
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 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.
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.
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
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.
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).
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 position413 to +89 of the fur P1 promoter. They also suggest the existence of repressor region(s) upstream of the most proximal 413 promoter region.
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 position66, 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).
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 mutants We also tested whether the 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.
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
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- The proximal region of the fur P1 promoter contains 2 potential GATA-binding sites at positions 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 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 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
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-
We thank David Bouchard for performing the site-directed mutagenesis, Dr Jean Luc-Parent for his help in mutagenesis design, Penny Rudd for her assistance in luciferase assays, and Tayna Bardati for performing the gel shift assays.
Submitted February 15, 2002; accepted June 25, 2002.
Supported by the Canadian Institutes of Health Research grant MT-13222.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Claire M. Dubois, Department of Pediatrics, Immunology Division, Faculty of Medicine, University of Sherbrooke, Québec, QC, Canada J1H 5N4; e-mail: claire.m.dubois{at}usherbrooke.ca.
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© 2002 by The American Society of Hematology.
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Top
Abstract
Introduction
-globin promoter.