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Blood, Vol. 93 No. 5 (March 1), 1999:
pp. 1600-1611
The Megakaryocyte/Platelet-Specific Enhancer of the
 2 1 Integrin Gene: Two Tandem AP1 Sites
and the Mitogen-Activated Protein Kinase Signaling Cascade
By
Mary M. Zutter,
Audrey D. Painter, and
Xun Yang
From the Department of Pathology, Washington University School of
Medicine, St Louis, MO.
 |
ABSTRACT |
The  2 1 integrin, a collagen receptor
on platelets and megakaryocytes, is required for normal platelet
function. Transcriptional regulation of the 2 integrin
gene in cells undergoing megakaryocytic differentiation requires a core
promoter between bp  30 and 92, a silencer between bp 92 and
351, and megakaryocytic enhancers in the distal 5' flank. We
have now identified a 229-bp region of the distal 5' flank of the
2 integrin gene required for high-level enhancer
activity in cells with megakaryocytic features. Two tandem AP1 binding
sites with dyad symmetry are required for enhancer activity and for
DNA-protein complex formation with members of the c-fos/c-jun family.
The requirement for AP1 activation suggested a role for the
mitogen-activated protein kinase (MAPK) signaling pathway in regulating
2 integrin gene expression. Inhibition of the MAP kinase
cascade with PD98059, a specific inhibitor of MAPK kinase 1, prevented
the expression of the 2 integrin subunit in cells
induced to become megakaryocytic. We provide a model of megakaryocytic
differentiation in which expression of the 2 integrin
gene requires signaling via the MAP kinase pathway to activate two
tandem AP1 binding sites in the 2 integrin enhancer.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
DISTINCT MEMBERS OF the integrin family
of cell adhesion receptors are expressed at different times during
hematopoietic differentiation. The expression of some integrin
receptors, such as the  51 integrin on
erythroid cells, is required for differentiation; the expression of
other receptors, such as 21 and
IIb3 on megakaryocytes and platelets, is
required for normal function.1-3 The
21 integrin mediates platelet adhesion to
collagen in vessel walls and is essential for the formation of a normal
platelet plug.3 Lack of 21
integrin expression by platelets, either due to congenital
abnormalities or the development of myelodysplastic/myeloproliferative disorders, is associated with bleeding.4-9 The regulation
of integrin gene expression during hematopoietic differentiation, specifically megakaryocytic differentiation, has been a major focus of
our laboratory.
Expression of the 21 integrin is
exquisitely regulated by distinctly different transcriptional
regulatory elements in epithelial cells, fibroblasts, megakaryocytes,
and platelets.10-15 To evaluate the molecular mechanisms by
which differentiation-dependent expression of the
21 integrin is regulated during
megakaryocytic differentiation, we have used several models of
megakaryocytic differentiation including K562 cells, a pluripotent
hematopoietic cell line, and DAMI cells, a megakaryocytic cell
line.10,14 K562 cells cultured in the presence of 40 nmol/L
phorbol dibutyrate (PDB) acquired megakaryocytic features, including
the expression of the platelet/megakaryocyte integrins
IIb3 and
21. The increased
21 integrin expression was a result of
increased steady-state levels of 2 mRNA due to transcriptional activation of the 2 integrin
gene.11 Characterization of the 5' flanking region of
the 2 integrin gene showed three distinct regions of the
5' flank that are responsible for the expression of the
2 integrin gene.13,14,16 Regulation of the
2 integrin gene in both the K562 and DAMI cell models
undergoing megakaryocyte differentiation requires a core promoter
between bp 30 and 92, a silencer between bp 92 and
351, and additional megakaryocytic enhancers within the distal
5' flank.14,16
Our earlier studies localized the strong megakaryocytic enhancer to the
region between bp 1426 and 2592.14 We now
verify that the 1,166-bp region serves as an enhancer in the irrelevant promoter construct SV40pCAT in both the K562 and DAMI models of megakaryocytic differentiation. Enhancer activity requires two tandem
AP1 binding sites with dyad symmetry. The short enhancer fragment
containing the tandem AP1 consensus sites binds members of the c-fos
and c-jun family. Inhibition of the mitogen activation protein kinase
(MAPK) cascade prevents the upregulation of
21 integrin expression induced by PDB. We
present a model in which activation of the MAPK cascade signals via
c-fos/c-jun family members to activate 2 integrin gene expression.
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MATERIALS AND METHODS |
Cell cultures and transfection assays.
The K562 cell line obtained from the American Type Culture Collection
(Rockville, MD) and the DAMI cell line obtained from Dr Robert I. Handin (Harvard Medical School, Boston, MA) were propagated in RPMI 1640 medium. Megakaryocytic differentiation was
induced by the addition of 40 nmol/L PDB in dimethyl sulfoxide, as
previously described.10 In experiments using the MAPK
kinase 1 inhibitor, PD98059, cells were treated with PD98059 (50 µmol/L) in dimethyl sulfoxide for 15 minutes before the addition of PDB.
The K562 cell line was transfected by electroporation using a BTX
Electro Cell Manipulator 600 (BTX Inc, San Diego, CA).17 Approximately 1.0 × 107 cells were transfected in
RPMI medium containing 100 µg/mL salmon sperm DNA, 30 µg of plasmid
DNA, and 3 µg of RSV-luciferase DNA by electroporation at 275 V and
600 µF. Cell extracts were harvested after 48 hours. Luciferase
activity was analyzed using a Monolight 2010 luminometer (Analytical
Luminescence Laboratory, San Diego, CA), as described
previously,18 and was used to normalize for transfection
efficiency. Cell extracts containing identical amounts of luciferase
activity were then assayed for chloramphenicol acetyl transferase (CAT)
activity using the standard method of Gorman et al.19
Acetylation of chloramphenicol was quantified by thin-layer chromatography.
Gel mobility shift analysis.
Nuclear extracts were obtained from uninduced and phorbol-induced K562
cells by isolating nuclei, as previously described.20 The
cells were washed with phosphate-buffered saline (PBS) and lysed in
0.5% Triton X-100 in 10 mL of STKM buffer (30% sucrose, 40 mmol/L
Tris, pH 7.5, 37 mmol/L KCI, and 12 mmol/L MgC12). After pelleting, the nuclei were washed in TNM buffer (100 mmol/L Tris, pH
7.5, 10 mmol/L NaCl, and 30 mmol/L MgCl2). The proteins
were extracted from the nuclei using a buffer containing 20 mmol/L HEPES, 20% glycerol, 0.35 mol/L KCl, 0.1 mmol/L EGTA, 0.5 mmol/L EDTA,
10 µg/mL aprotinin, and 10 µg/mL leupeptin. After incubation with
gentle rotation for 1 hour at 4°C, the sample was centrifuged at
100,000g for 2 hours. The supernatant was collected and the concentration of nuclear proteins in the extracts was determined by
Bradford assay (Bio-Rad Laboratory, Hercules, CA). The gel-purified double-stranded 75-bp DNA fragment extending from bp 1503 to 1578 or the double-stranded oligonucleotides were end-labeled with 32P-dCTP using Klenow DNA polymerase. Oligonucleotides
containing a single AP1 consensus element
(5'-CGCTTGATGACTCAGCCGGAA-3'), a consensus element with a
2-bp mutation of the AP1 site (AP1m; 5'-CGCTTGATGACTTGGCCGGAA-3'), a single Sp1 consensus
element (5'-ATT CGATCGGGGCGGGGCGAGC-3'), or an early growth
response (Egr) consensus element
(5'-GGATCCAGCGGGGGCGAGCGGGGGCGA-3') were obtained from Santa Cruz Biotech, Inc (Santa Cruz, CA). Approximately 1 to 2 µg of
nuclear protein extract was added to 20,000 counts per minute of
labeled DNA in 15 µL of binding buffer (10% glycerol, 25 mmol/L Tris
HCl, pH 7.5, 100 mmol/L KCI, 5 mmol/L spermidine, 5 mmol/L EDTA, 1 mmol/L dithiothreitol, and 2 mmol/L phenylmethylsulfonyl fluoride) and
incubated on ice for 15 minutes. Some incubations contained an excess
of unlabeled competitor DNA fragment, or oligonucleotide, as indicated.
In antibody inhibition experiments, the indicated amount of anti-c-fos
or anti-c-jun antibody (Santa Cruz Biotech, Inc) was preincubated with
the nuclear extract for 18 hours at 4°C before the addition of
32P-labeled probe. The reactions were then analyzed by
polyacrylamide gel electrophoresis using 6% acrylamide/Bis (19:1) in
Tris, pH 8.8.
2-CAT fusion constructs.
The p2 2592-CAT and p2 1426-CAT
constructs were generated by restriction enzyme digestion of the
original p2 5000-CAT construct at the restriction enzyme
sites Stu I (2592) and Sac I (1426), respectively, as described earlier.14 The Stu
I/Sac I fragment was then inserted into the blunted Sal
I site within the multiple cloning site of the SV40pCAT plasmid
(Promega, Madison, WI). Two SV40pCAT constructs containing the bp
1426 to 2592 region in opposite orientations were
selected by restriction enzyme and sequence analyses. A series of
deletion mutants of the 1166 bp (bp 1426 to 2592) were
generated by restriction enzyme digestion of the
1426/2592 SV40pCAT plasmid at the restriction sites
Xmn (1842), Nsi (1655), Mnl
I(1578) and Mva I (1503). The
1426/1655 SV40pCAT plasmid was used as a template for
polymerase chain reaction (PCR) to generate the site-specific mutations
that disrupt the AP2, GATA, or both binding sites in the construct. The
1503/1578 SV40pCAT construct was used as a template for
PCR-directed mutagenesis to generate the constructs containing
site-specific mutations that disrupt the two AP1 binding sites,
including 5' AP1m 1503/1578 SV40pCAT, 3' AP1m
1503/1578 SV40pCAT, and Dbl AP1m 1503/1578 SV40pCAT. The following 5' primers were used for the GATA and AP2
mutant constructs: GATAm (5'-GTT ATT TCC CCC CAC CCC CAG ATT TAA
AAC AC-3'), AP2m (5'-GTT ATT TCC CCC TTT CCC CAG AGA TAA
AAC AC-3'), or the AP2-GATAm (5'-GTT ATT TCC CCC TTT CCC
CAG ATT TAA AAC AC-3'). The following primers were used for the
AP1 mutant constructs: 5'AP1m 1503/1578 SV40pCAT
(5'-GAA ATG TAT GGG AGT GTT TGG-3'), 3' AP1m
1503/1578 SV40pCAT (5'-CCT TC TAT CGG ACT GAA
ATG-3'), and Dbl AP1m 1503/1578 SV40pCAT
(5'-CCA AAC ACT CCC ATA CAT TTC AGT CCG ATA GAA-3'). A
common 3' primer from the M13 vector sequence was used in all
reactions. All constructs were completely sequenced by the
dideoxynucleotide chain termination method of Sanger et
al21 to insure that the site-specific mutations were
incorporated with no error. Mutations in the full-length CAT construct
p2 2592-CAT were produced by site-directed
mutagenesis.22
Flow cytometric analysis.
Flow cytometric analysis was performed on uninduced K562 cells, K562
cells induced for 6 days with PDB, or K562 cells incubated with PD98059
for 15 minutes before induction with PDB. Single cells (1 × 106) in PBS with 1.5% horse serum were incubated with the
monoclonal anti-2 antibody (P1E6) at a concentration of
2 µg/mL for 45 minutes at 4°C. Cells were washed three times and
incubated with 2.5 µg/mL of a secondary goat antimouse coupled to
fluorescein (Tago, Inc, Burlingame, CA) for 45 minutes at 4°C,
washed twice, and resuspended in PBS. Fluorescein-labeled cells were
analyzed using a FACScan instrument (Becton Dickinson, Mountain View, CA).
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RESULTS |
A 229-bp region of the distal 5' flank functions as an
2 integrin subunit enhancer.
Three distinct regions of the 5' flank of the 2
integrin gene were required for high-level gene expression in cells
with megakaryocytic features (Fig
1).14 To verify that the region between bp 1426 and
2592 served enhancer function in cells with megakaryocytic
features, the 1,166-bp fragment was placed in the irrelevant promoter
construct SV40pCAT in both 5' to 3' and 3' to
5' orientations (Fig 2A). Both the
5' to 3' and 3' to 5' 1426/2592 SV40pCAT constructs, independent of orientation, directed high-level, reporter gene activity in K562 cells induced with phorbol dibutyrate to
undergo megakaryocytic differentiation (Fig 2B and data not shown). The
1426/2592 SV40pCAT constructs directed only low-level CAT
activity in uninduced K562 cells. The enhancer activity of the 1,166-bp
region in the irrelevant SV40pCAT construct responded to induction in a
manner similar to the activity of the original intact promoter/enhancer
construct containing the entire 5' flank of the 2
integrin gene, as shown in Fig 2A and B. The orientation-independent activity of the region between bp 1426 and 2592 in an
irrelevant promoter construct established its role as an enhancer.
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| Fig 1.
A schematic diagram of the distal 5' flank of the
2 integrin gene. Identification of numerous potential
binding sites for ubiquitous as well as megakaryocyte-specific
transcription factors are shown. Three distinct regions are responsible
for 2 integrin gene expression in cells with
megakaryocytic origin. These include a core promoter between bp 30
and 92, a silencer/repressor between bp 92 and 351, and
megakaryocyte-specific enhancers in the distal 5' flank between
bp 1426 and 1655. The megakaryocyte-specific enhancer is
underlined and bracketed. Data from Zutter et
al.14
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| Fig 2.
A schematic diagram of the 2 promoter-CAT
construct and the megakaryocytic enhancer in the irrelevant promoter
construct SV40pCAT. (A) The construct p22592-CAT
contains the entire 5' flanking region from bp 2592 to +109
of the 5' untranslated region of the 2 integrin
gene upstream to the CAT structural gene. The construct 1426/2592
SV40pCAT consists of the megakaryocyte enhancer region extending from
bp 1426 to 2592 in the irrelevant promoter construct SV40pCAT.
Constructs 1842/2592 SV40pCAT, 1426/1842 SV40pCAT,
1426/1655 SV40pCAT, and 1426/1542 SV40pCAT were derived
from the construct 1426/2592 SV40pCAT. (B) The 1,166-bp region of
the distal 5' flank serves as a megakaryocytic enhancer. The
constructs p22592-CAT, 1426/2592 SV40pCAT,
1842/2592 SV40pCAT, 1426/1842 SV40pCAT, 1426/1655
SV40pCAT, and 1655/1842 SV40pCAT were transfected in parallel
with the SV40pCAT plasmid that contains the strong viral promoter SV40
without an enhancer into either uninduced (⊠) or induced ( ) K562
cells. Cotransfection with RSV-luciferase was used to control for
transfection efficiency. After 48 hours of incubation, cell extracts
were assayed. After normalization for transfection efficiency, CAT
activity of the constructs was determined using thin-layer
chromatography and differential extraction. The mean and standard
deviation of CAT activity of the mutated constructs in uninduced or
induced K562 cells from three separate experiments was determined
relative to the activity of SV40pCAT in uninduced K562 cells, which was
assigned a value of 1.0.
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To determine which elements within the 1,166-bp region were required
for enhancer activity in cells undergoing megakaryocytic differentiation, a series of deletion mutants of the
1426/2592 SV40pCAT construct were constructed as shown in
Fig 2A. This series of deletion mutants localized strong enhancer
activity to the region between bp 1426 and 1655 relative
to the activity of SV40pCAT in uninduced K562 cells (Fig 2B). The
construct 1426/1655 SV40pCAT exhibited enhancer activity
comparable to that of the longer enhancer constructs
1426/1842 SV40pCAT and 1426/2592 SV40pCAT
(Fig 2A and B). These findings suggested that the enhancer was located
in the 229-bp region between bp 1426 and 1655.
AP2 and GATA elements do not mediate enhancer function.
Sequence analysis of this 229-bp region showed a single GATA box
between bp 1628 to 1684 adjacent to an AP2 site between bp 1637 to 1644 and two tandem, but inversely oriented
AP1 binding sites at bp 1543 and 1557 (Fig 1). Based on
data from other laboratories suggesting that members of the GATA family
are involved in erythro-megakaryocytic differentiation, we initially
focused on the role of the GATA and AP2 sites in megakaryocytic
enhancer activity.23-29 To evaluate the role of these two
potential binding sites in enhancer activity, mutations in the AP2
site, the GATA site, or both sites that are known to eliminate protein
binding were constructed by PCR-directed mutagenesis of the
1426/1655 SV40pCAT construct
(Fig 3A).30-35 Activity of the
mutant constructs in uninduced and induced K562 cells was compared with
the activity of the intact 1426/1655 SV40pCAT construct
in uninduced K562 cells (Fig 3B). The activity of the intact
1426/1655 SV40pCAT construct in uninduced K562 cells was
assigned a value of 1.0. Results are represented as the mean and
standard deviation of at least three separate experiments. The
1426/1655 SV40pCAT construct directed at least 10-fold
greater enhancer activity in K562 cells induced to become
megakaryocytic than in uninduced K562 cells. In contrast to the loss of
enhancer activity we expected upon mutation of the AP2 or GATA sites,
mutation of either the AP2 site, the GATA site, or both sites increased
enhancer activity twofold to threefold in both uninduced and induced
K562 cells. Clearly, the AP2 and GATA sites do not mediate enhancer
activity for the 2 integrin gene and may serve to
diminish enhancer activity in hematopoietic cells.
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| Fig 3.
Site-directed mutagenesis of the AP2 and GATA consensus
sites. (A) A diagram demonstrates the sequence of a 32-bp
region within the megakaryocytic enhancer extending between bp 1623
and 1655 containing the intact AP2 and GATA binding sites and the
sequence of 3 mutant constructs prepared by PCR to produce mutations of
either the AP2 site, the GATA site, or both AP2 and GATA recognition
sites. (B) Mutations in either the AP2 site, the GATA site, or both AP2
and GATA sites were incorporated into the construct 1426/1655
SV40pCAT. The enhancer activity of the mutant constructs containing
either a single mutation of the AP2 site, GATA site, or the AP2 and
GATA sites was compared with the activity of the intact 1426/1655
SV40pCAT in uninduced and induced K562 cells. Cotransfection with
RSV-luciferase was used to control for transfection efficiency. After
48 hours of incubation, cell extracts were assayed. After normalization
for transfection efficiency, CAT activity of the constructs was
determined by thin-layer chromatography and differential extraction.
The mean and standard deviation of CAT activity of the mutant
constructs from at least three separate electroporations was determined
relative to 1426/1655 SV40pCAT in uninduced K562 cells, which was
assigned the value of 1.0.
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Two tandem, inversely oriented AP1 sites demonstrate full enhancer
activity.
We then turned our attention to the potential role of the two tandem
AP1 sites with dyad symmetry located between bp 1543 and
1557. To focus only on the two tandem AP1 sites, a shorter double-stranded DNA fragment extending from bp 1503 to
1578 that included both AP1 binding sites but eliminated 77 bp
of the 5' region including the GATA and AP2 sites and 77 bp of
3' sequence was prepared by PCR and placed in the enhancer
location of the SV40pCAT vector (Fig 4A and
B). As shown in Fig 4B, the short construct 1530/1578
SV40pCAT that lacked 5' and 3' elements including the GATA
and AP2 sites contained all of the enhancer activity of the longer
construct 1426/1655 SV40pCAT in induced K562 cells. To
determine the role of one or both AP1 sites, mutations of either the
5' AP1 site, the 3' AP1 site, or both sites were prepared
in the shorter construct 1503/1578 SV40pCAT, as shown in
Fig 4A. The ability of the mutant constructs to direct reporter gene
activity in the irrelevant promoter construct SV40pCAT was compared
with the activity of the nonmutated construct 1503/1578 SV40pCAT and the longer 1426/1655 SV40pCAT construct in
both induced and uninduced K562 cells. In contrast to the high level of
reporter gene activity generated by the intact 1503/1578 SV40pCAT construct, the activity of the constructs with mutations of
either the 5' AP1 site, the 3' AP1 site, or both AP1 sites was markedly reduced to levels only slightly above background in both
uninduced and induced K562 cells (Fig 4B). These results indicate that
the two adjacent but inversely oriented AP1 binding sites play an
important role in enhancer activity of the 2 integrin gene in K562 cells induced to become megakaryocytic. The tandem AP1
binding sites appear responsible for both enhancer gene activity in
K562 cells induced to differentiate along the megakaryocyte lineage and
for the low level of reporter gene activity in uninduced K562 cells.
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| Fig 4.
Site-directed mutagenesis of tandem AP1 consensus binding
sites. (A) The diagram demonstrates the sequence of the 40-bp region of
the 2 enhancer extending from bp 1530 bp to 1570
containing the two intact but inversely oriented AP1 binding sites. The
sequence of the three mutant constructs prepared by PCR containing
mutations of the 5' AP1 site, the 3' AP1 site, or both AP1
binding sites is demonstrated. (B) To determine the role of one or both
AP1 sites in enhancer activity, mutations of either the 5' AP1
site, the 3' AP1 site, or both sites were introduced into the
shorter construct 1503/1578 SV40pCAT. This shorter construct
includes both AP1 binding sites but eliminates 77 bp of the 5'
region including the GATA and AP2 site and 77 bp of 3' sequence.
The enhancer activity of the original 1426/1655 SV40pCAT
construct was compared with the shorter intact construct
1503/1578 SV40pCAT and the three mutant constructs, 5' AP1m
1503/1578 SV40pCAT, 3' AP1m 1503/1578 SV40pCAT,
and Dbl AP1m 1503/1578 SV40pCAT in uninduced and induced K562
cells after transient transfection. Cotransfection with RSV-luciferase
was used to control for transfection efficiency. After 48 hours of
incubation, cell extracts were assayed after normalization for
transfection efficiency. CAT activity of the constructs was determined
by thin-layer chromatography and differential extraction. The mean and
standard deviation of CAT activity of the mutant constructs from at
least three separate electroporations was determined relative to the
1426/1655 SV40pCAT construct in uninduced K562 cells, which was
assigned a value of 1.0.
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To further demonstrate that the two AP1 sites within the distal
5' flanking region are required for expression of the
2 integrin gene in cells with megakaryocytic features,
either one or both of the AP1 binding sites were mutated in the context
of the original intact 2 integrin gene promoter/enhancer
construct, p22592-CAT. Identical mutations of either the
5' AP1 site, the 3' AP1 site, or both AP1 sites, as
outlined above, were introduced into the original
p22592-CAT construct by site-directed mutagenesis.
Mutation of either the 5' AP1 site, the 3' AP1 site, or
both AP1 sites completely eliminated reporter gene activity mediated by
the full-length construct containing the entire 5' flank of the
2 integrin gene in both induced and uninduced K562 cells
(Fig 5), just as it did for the simpler
reporter construct (Fig 4B).
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| Fig 5.
Site-directed mutagenesis of the tandem AP1 binding sites
markedly reduced the promoter/enhancer activity of the intact
p22592-CAT construct. Mutation of the 5' AP1 site,
the 3' AP1 site, or both AP1 sites was introduced into the
original p22592-CAT construct by site-directed
mutagenesis. The promoter/enhancer activity of the original
p22592-CAT construct was compared with the activity of
the construct with mutations in the 5'AP1 site, the 3' AP1
site, or both AP1 sites after transient transfection into uninduced and
induced K562 cells and uninduced and induced DAMI cells. Cotransfection
with RSV-luciferase was used to control for transfection efficiency.
After 48 hours of incubation, cell extracts were assayed. After
normalization for transfection efficiency, CAT activity of the
constructs was determined by thin-layer chromatography and differential
extraction. The mean and standard deviation of CAT activity of the
mutant constructs in either K562 cells or DAMI cells from at least
three separate electroporations was determined relative to the
pCAT-Basic construct in uninduced K562 or uninduced DAMI cells,
respectively, which was assigned a value of 1.0.
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The two AP1 sites serve enhancer function in DAMI cells.
The enhancer activity of the two AP1 sites in DAMI cells, another model
of megakaryocytic differentiation, was established. Uninduced DAMI
cells express low levels of the 21
integrin. In DAMI cells, 21 integrin
expression is markedly increased by the addition of PDB.14
Reporter gene activity of the p22592-CAT construct with
mutations of the 5' AP1 site, the 3' AP1 site, and both AP1
sites was compared with the original p22592-CAT construct in uninduced and induced DAMI cells. The
p22592-CAT construct exhibited low levels of reporter
gene activity in uninduced DAMI cells and high levels of reporter
activity in induced DAMI cells. Mutations of either the 5' AP1
site, the 3' AP1 site, or both AP1 sites completely eliminated
reporter gene activity of the p22592-CAT construct in
uninduced and induced DAMI cells as well (Fig 5). These findings
provide additional evidence that the tandem AP1 sites located in the
distal 5' flank of the 2 integrin gene are
required for 2 integrin gene activity in hematopoietic cells either poised to become megakaryocytic or induced with phorbol dibutyrate to acquire megakaryocytic features.
DNA-protein complex formation with the tandem AP1 binding sites.
To identify transcription factors that specifically interact with the
2 integrin enhancer, binding of nuclear proteins from uninduced and induced K562 cells to the 75-bp double-stranded DNA
fragment (bp 1503 to 1578) containing the two tandem AP1 sites was evaluated by gel mobility shift assays. Nuclear proteins from
both uninduced K562 cells and K562 cells induced to differentiate for 4 days with phorbol dibutyrate bound the 75-bp enhancer element (Fig 6A). Nuclear extracts from both
uninduced K562 cells and K562 cells induced with phorbol dibutyrate
formed a complex of intermediate migration (B). Nuclear extracts from
uninduced K562 cells also formed a faster migrating complex (C) that
was not as prominent with extracts from induced cells. Furthermore,
nuclear extracts from K562 cells induced to develop megakaryocytic
features bound the 75-bp DNA fragment to form an additional, slower
migrating complex (A) that was not as abundant with extracts from
uninduced cells. Formation of all three DNA-protein complexes could be
specifically inhibited by the unlabeled 75-bp DNA fragment but not by
an irrelevant 119-bp double-stranded DNA fragment. The unlabeled 75-bp
fragment containing a mutation of the 5' AP1 site, the 3'
AP1 site, or both AP1 sites (Dbl AP1m) failed to inhibit complex
formation (Fig 6B, data not shown). More rapidly migrating, nonspecific complexes, as judged by the inability of the unlabeled DNA fragment to
compete, were present in some experiments.
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| Fig 6.
Gel mobility shift analysis of the megakaryocyte
enhancer. (A and B) Gel mobility shift analysis was performed with
32P end-labeled double-stranded DNA fragment containing the
75-bp enhancer region (bp 1503/1578) with two tandem AP1 binding
sites and 1 µg of nuclear protein from either uninduced K562 cells
(K562) or K562 cells induced for 4 days (4d Ind) with phorbol. Three
major DNA protein complexes are indicated as A, B, and C. DNA-protein
complexes were formed in the absence of competitive inhibitor (ø) (A
and B), in the presence of a non-specific (NS) inhibitor consisting of
119-bp double-stranded DNA fragment from the coding region of the
2 cDNA (A), or in the presence of 75-bp DNA fragment
containing mutations to both AP1 binding sites (Dbl AP1m) (B). The
presence of an unlabeled specific inhibitor (75 bp) inhibited
DNA-protein complex formation (A and B). Gel mobility shift analysis
was performed as described in Materials and Methods.
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The involvement of two tandem AP1 consensus elements in the enhancer
suggests a role for the AP1 family of transcription factors in
2 integrin gene regulation. A double-stranded
oligonucleotide containing only a single consensus AP1 site
specifically bound nuclear extracts from both uninduced and induced
K562 cells (Fig 7A, B, and C). The complex
formed between the consensus AP1 oligonucleotide and nuclear extracts
from induced K562 cells was more abundant than the complex formed with
nuclear extracts from uninduced K562 cells. Complex formation between
the consensus AP1 site and nuclear extracts from both uninduced and
induced K562 cells was partially inhibited by 10-fold and almost
completely inhibited by 20-fold addition of the identical unlabeled
oligonucleotide and completely inhibited by 10-fold or 20-fold addition
of the 75-bp enhancer fragment but was not inhibited by the AP1
consensus oligonucleotide with a 2-bp mutation (AP1m; Fig 7A and C). In
addition, an oligonucleotide containing the consensus binding site for
the Egr family of transcription factors failed to inhibit complex
formation (Fig 7B).
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| Fig 7.
A consensus AP1 site forms DNA-protein complexes. (A, B,
and C) Gel mobility shift experiments were performed with
32P end-labeled double-stranded oligonucleotide containing
a single AP1 consensus binding site and 1 µg of nuclear extract from
uninduced K562 cells (K562) or phorbol ester-induced K562 cells (4d
Ind) (A, B, and C). A DNA-protein complex formed in the absence of
competitive inhibitor (ø) (A, B, and C), in the presence of an
identical oligonucleotide with a 2-bp mutation in the consensus binding
site (AP1m) (A), in the presence of the unlabeled specific 75-bp
enhancer region (bp 1503/1578) with two tandem AP1 binding sites
(C), or in the presence of an irrelevant double-stranded
oligonucleotide containing the consensus binding site for transcription
factors of the early growth response family (Egr) (B). The unlabeled
AP1 oligonucleotide inhibited DNA-protein complex formation (A, B, and
C). Monoclonal or polyclonal antibodies against either c-fos or c-jun
(at the indicated concentration) were preincubated with nuclear
extracts for 18 hours at 4°C before the addition of 32P
end-labeled probe, as described in Materials and Methods. Antisera
against both c-fos and anti-c-jun inhibited DNA-protein complex
formation in a concentration-dependent manner. The molar excess of
unlabeled competitor or the concentration of antibody is indicated. Gel
mobility shift analyses were performed as described in Materials and
Methods.
|
|
Antibodies against c-fos/c-jun heterodimers prevent DNA-protein
complex formation.
The AP1 consensus element of the 2 integrin enhancer is
separated by one nucleotide (TGANTCA).36 c-fos/c-jun
heterodimers prefer such DNA targets. In contrast, ATF/CREB-containing
dimers prefer AP1 consensus elements separated by two
nucleotides (TGANNTCA).37 Antibodies against c-fos or c-jun
family members inhibited complex formation between K562 nuclear
extracts and the oligonucleotide containing the AP1 consensus site, but
not oligonucleotides containing the SP1 or NF B consensus sites in a
concentration-dependent manner under our experiment conditions (Fig 7A
and data not shown).
Under similar experimental conditions, antibodies against c-fos also
inhibited formation of complexes B and C between the 75-bp enhancer
fragment and uninduced K562 cell extracts, as shown in
Fig 8. Anti-c-fos antibodies inhibited
complexes A and B formed between the 75-bp enhancer and nuclear
extracts from induced K562 cells. An antibody against c-jun family
members diminished formation of complex B formed by the 75-bp enhancer
and uninduced nuclear extracts and formation of complexes A and B
between the 75-bp fragment and extracts from induced K562 cells. These
findings suggest that members of the c-fos and c-jun family bind to the 75-bp enhancer fragment of the 2 integrin gene.
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| Fig 8.
Anti-c-fos and anti-c-jun antibodies inhibit
DNA-protein complex formation. Gel mobility shift experiments were
performed with 32P end-labeled 75-bp enhancer fragment (bp
1578 to bp 1503) and 1 µg of nuclear extract from either
uninduced K562 cells (K562) or K562 cells induced for 4 days with
phorbol dibutyrate (4d Ind). Monoclonal or polyclonal antibodies
against either c-fos or c-jun (at the indicated concentration) were
preincubated with nuclear extracts for 18 hours at 4°C before the
addition of 32P end-labeled probe, as described in
Materials and Methods. Anti-c-fos and anti-c-jun inhibited
DNA-protein complex formation.
|
|
A role for the MAPK cascade in 2 integrin
gene regulation.
The signaling cascades that lead to AP1 activation include numerous
mitogen activated protein kinase intermediates and transcriptional induction of c-fos and c-jun proteins.38,39 Recent
observations showed that inhibition of MAPK kinase 1 (MAPKK1) prevented
some features of phorbol-induced megakaryocytic differentiation,
including expression of the IIb and 3
integrin genes.40 The role of MAPKK1 in regulating
2 integrin gene expression was therefore evaluated. As
shown previously, uninduced K562 cells fail to express detectable
levels of the 21 integrin
(Fig 9). After 6 days of PDB induction,
expression of the 21 integrin is greatly
increased. The addition of PD98059, the MAPKK1 specific inhibitor, to
K562 cells 15 minutes before the addition of PDB completely eliminated 21 integrin subunit expression, as shown
in Fig 9.
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| Fig 9.
Inhibition of 21 integrin
expression by PD98059. Flow cytometric analysis of the
21 integrin expression by uninduced K562
cells, K562 cells induced for 6 days with PDB (40 nmol/L) (6d Ind), or
K562 cells treated with PD98059 (50 µmol/L) for 15 minutes before the
addition of PDB (40 nmol/L) (PD98059 + 6d Ind).
|
|
The role of the MAPK cascade in activating the 2
promoter/enhancer was assessed. As shown in Figs 2B and
10, the reporter gene activity of the
original intact 2 integrin promoter/enhancer construct
p22592-CAT was high in induced K562 cells and at
background levels in uninduced K562. Reporter gene activity of the
p22592-CAT in K562 cells treated with both PDB and
PD98059 was markedly reduced compared with the reporter activity in PDB
induced K562 cells (Fig 10). Inhibition of the MAPK kinase cascade
diminished the function of the 2 integrin
promoter/enhancer. Based on these findings, we propose a model by which
activation of the MAPK cascade leads to 2 integrin gene
expression in cells induced to become megakaryocytic
(Fig 11).
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| Fig 10.
Diminution of 2 promoter/enhancer
function by PD98059. The constructs pCAT-Basic and
p22592-CAT were transfected into uninduced K562 cells
( ), K562 cells treated with PDB (40 nmol/L; ), and K562 cells
treated for 15 minutes with PD98059 (50 µmol/L) before the addition
of PDB (40 nmol/L; ⊠). Cotransfection with RSV-luciferase was used to
control for transfection efficiency. After 48 hours of incubation, cell
extracts were assayed. After normalization for transfection efficiency,
CAT activity of the constructs was determined using thin-layer
chromatography. The mean and standard deviation of CAT activity in
uninduced K562 cells, PDB-induced K562 cells, or K562 cells treated
with PD98059 and PDB was determined. CAT activity relative to the
pCAT-Basic construct in uninduced K562, which was assigned a value of
1.0, is shown.
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|
View larger version (38K):
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| Fig 11.
A proposed pathway for increased expression of the
2 integrin subunit in cells with megakaryocytic
features. Both growth factors and PDB indirectly activate the MAPK
cascade, including the mitogen-activated protein kinase kinase (MAPKK),
extracellular receptor activated kinase (Erk), and Jun kinase via
intermediate signaling molecules, including protein kinase C (PKC).
Activated Erk and Jun kinase increase c-fos and c-jun synthesis by
phosphorylation and activation of transcription factors, including
complex factors (TCF), serum response factors (SRF), c-jun, and
activating transcription factors (ATF).38 In addition, Erk
and Jun kinase phosphorylation augment the activity of both c-fos and
c-jun. Newly synthesized ( ) and phosphorylated (- - ) c-fos and c-jun family members heterodimerize to bind the
tandem and inversely oriented AP1 binding sites of the
2 integrin enhancer and upregulate 2
integrin gene expression.
|
|
| |
DISCUSSION |
In earlier studies we demonstrated that expression of the
2 integrin gene in cells with megakaryocytic features,
both induced K562 cells and DAMI cells, is regulated by three distinct
regions of the 5' flank of the 2 integrin gene
containing both positive and negative elements.14 The three
regions include a core promoter between bp 30 and 92, a
silencer between bp 92 and 351, and megakaryocytic
enhancers between bp 1426 and 2592. In this study, using
a combination of deletional and mutational analyses, we identified a
megakaryocytic enhancer of the 2 integrin gene. The
enhancer requires two tandem inversely oriented AP1 consensus binding
sites for interaction with c-fos/c-jun family members. These data
suggest a model in which signaling via the MAPK cascade leads to
activation of the megakaryocytic enhancer of the 2
integrin gene (Fig 11). Extracellular stimuli such as phorbol
dibutyrate under our experimental conditions or growth factors during
thrombopoiesis directly or indirectly activate the MAPK signaling cascade.
The MAP kinase cascade involves at least three distinct signaling
pathways, including the extracellular regulated kinases (ERK), jun
kinase, and p38 MAPK. The best delineated pathway initiated via growth
factor receptors or phorbol esters initiates a cascade from ras, raf,
MAPKK1 (MEK1), or MAPKK2 (MEK2) to ERK1 or ERK2.39,41 The
activated ERK proteins then translocate to the nucleus to phosphorylate
and activate nuclear transcription factors, such as ternary complex
factors (TCF).42 ERK activation directly increases AP1
activity via c-jun activation and increased c-fos synthesis, leading to
an increase in c-fos/c-jun heterodimerization and DNA
binding.43
A role for the MAP kinase pathway in megakaryocytic differentiation was
originally suggested by the ability of phorbol esters to initiate
megakaryocytic differentiation and induce the expression of
megakaryocyte-specific genes.10,11 A direct role for the MAP kinase pathway in megakaryocytic differentiation has been demonstrated by a number of investigators.39-41,44,45
Megakaryocytic differentiation of K562 cells induced with phorbol
dibutyrate, including the morphologic aspects of cell adhesion and cell
spreading and the expression of the megakaryocytic genes encoding the
IIb and 3 integrin subunits, was either
partially or completely inhibited by PD98059, the MAPKK1 specific
inhibitor. In addition, constitutive activation of the pathway via
expression of the constitutively active mutants of MAPKK1 or MAPKK2
induced the expression of the platelet/megakaryocytic-specific genes
IIb and 3. The regulatory elements
required for expression of megakaryocyte-specific proteins IIb3, platelet factor 4 (PF4),
glycoprotein Ib, and c-mpl have been mapped.23-28 Adjacent
GATA and ets consensus binding sites identified in the promoters of the
IIb, PF4, glycoprotein Ib, and c-mpl are essential for
transcriptional activation of these genes.27,28,46-48 GATA
and ets transcription factors are activated by the MAPK cascade.
The important role that adjacent GATA and ets binding sites play in
activation of many megakaryocyte-specific genes led us to evaluate the
role of the adjacent GATA and AP2 sites in enhancer activity of the
2 integrin gene. Surprisingly, the GATA and AP2 sites
did not serve enhancer function and served to slightly repress the
activity of the distal enhancer of the 2 integrin gene.
Although the architecture of the 2 promoter/enhancer
that consists of a promoter, a silencer, and a distal enhancer
resembles other megakaryocytic genes, the enhancer is distinctly
different and consists of two adjacent but inversely oriented AP1
binding sites.
Mutation of either or both AP1 sites markedly reduced enhancer activity
in the irrelevant promoter construct SV40pCAT, as well as in the intact
2 promoter/enhancer construct extending through the
distal 5' flank of the 2 integrin gene. Consensus AP1 binding sites have not been demonstrated to play an important role
in the enhancer activity of other megakaryocytic genes. However, the
inverse orientation of the two AP1 sites resembles the dyad symmetry
element (DSE) required for c-fos induction after 12-0-tetradecanoyl phorbol-13-acetate (TPA)-induced megakaryocytic differentiation of K562
cells.42 AP1, a sequence-specific transcription factor composed of c-fos and c-jun family members, was initially identified as
the factor that mediates TPA induction.43,49,50 Activation and expression of AP1 is induced by multiple stimuli, including growth
factors, cytokines, UV irradiation, and the different members of the
MAPK cascade.38,50
The NF-E2 binding site consists of either one or tandem, extended AP1
binding sites.51-54 A possible role for NF-E2 in
2 integrin gene regulation was
considered.51-55 The NF-E2 binding sites in the
hypersensitivity site 2 (HS2) enhancer of the globin gene resemble
the two tandem AP1 binding sites in the 2 integrin
enhancer.54,56 However, the additional -G- in the
GCTGAGTC sequence that is required for NF-E2 binding is
absent from both of the tandem AP1 binding sites in the
2 integrin enhancer. In addition, although nuclear proteins from induced K562 cells bind to an oligonucleotide containing the consensus NF-E2 binding site and the unlabeled oligonucleotide containing the NF-E2 consensus binding site successfully inhibits the
binding of nuclear proteins to NF-E2, the unlabeled NF-E2 oligonucleotide fails to compete for binding to the megakaryocyte enhancer region (data not shown). In addition, expression of the hematopoietic restricted component p45 failed to induce megakaryocytic differentiation or expression of the 21
integrin (data not shown).
Based on the studies presented here, the MAPK pathway is required for
2 integrin gene expression in cells induced to
differentiate along the megakaryocytic lineage. Two tandem and
inversely oriented AP1 sites in the 2 integrin gene
enhancer bind to members of the AP1 family of transcription factors. We
propose a model by which either PDB, under experimental conditions, or
growth factors such as thrombopoietin (TPO), under
physiologic conditions, activate the MAPK cascade and
ultimately lead to 2 integrin gene expression in
cells induced to be megakaryocytic. The mechanisms by which PDB
activates c-fos/c-jun family members is well delineated.38 TPO has been shown to directly activate the MAPK cascade after binding
to c-Mpl on megakaryocytic progenitors.57,58 This model provides a physiologically relevant series of events that lead to
expression of the 21 integrin.
| |
ACKNOWLEDGMENT |
The authors thank Dr Samuel A. Santoro for encouragement and
constructive criticism of the manuscript. We also thank Mary Beth Flynn
for excellent secretarial assistance.
| |
FOOTNOTES |
Submitted January 29, 1998; accepted October 19, 1998.
Supported by National Institutes of Health Grant No. R01 HL51450-04.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to Mary M. Zutter, MD, Washington University
School of Medicine, Department of Pathology, St Louis, MO 63110.
| |
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J. R. Sevinsky, A. M. Whalen, and N. G. Ahn
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Top
Abstract
Introduction