Tài liệu Báo cáo khoa học: Characterization of the promoter for the mouse a3 integrin gene Involvement of the Ets-family of transcription factors in the promoter activity doc

Characterization of the promoter for the mouse a3 integrin gene
Involvement of the Ets-family of transcription factors in the promoter activity
Takumi Kato
1
, Kouji Katabami
1
, Hironori Takatsuki
1
, Seon Ae Han
2
, Ken-ichi Takeuchi
2
, Tatsuro Irimura
2
and Tsutomu Tsuji
1,2
1
Department of Microbiology, Hoshi University School of Pharmacy and Pharmaceutical Sciences, Tokyo, Japan;
2
Laboratory of
Cancer Biology and Molecular Immunology, Graduate School of Pharmaceutical Sciences, University of Tokyo, Japan
The a3b1 integrin is an adhesion receptor for extracellular
matrix proteins including isoforms of laminin, and the
changes of its expression level in various cancer cells are
thought to cause their malignant phenotypes. We have
cloned an approximately 4 kb DNA fragment of the 5¢-
flanking region of the murine a3 integrin gene and analyzed
its promoter activity. Transfection of MKN1 gastric carci-
noma cells with serially truncated segments of the 5¢-flanking
region linked to a luciferase gene indicated that a 537-bp
SalI/SacI fragment upstream of exon 1 was sufficient to

promote high level gene expression. By 5¢-rapid amplifica-
tion of cDNA ends (5¢-RACE) using a cap site-labeled
cDNA library, we determined one major and one minor
transcription start sites in this region. The murine a3integrin
gene was found to contain a CCAAT box, but to lack a
TATA box. Luciferase assay following transfection with a
series of deletion constructs of the SalI/SacI fragment
revealed that the sequence between positions )260 and
)119 bp (relative to the major transcription start site) is
required for efficient transcription in gastric carcinoma cells.
The sequence analysis of this segment showed the presence
of several consensus sequences for transcription factors
including Ets, GATA and MyoD/E-box binding factors.
The introduction of mutation in one of the Ets-binding
sequences greatly decreased its promoter activity, suggesting
that the transcription of the a3 integrin gene in these cells is
regulated by the Ets-family of transcription factors.
Keywords: integrin; gene promoter; luciferase assay;
Ets-transcription factor; gastric carcinoma cell.
The a3b1integrin(VLA-3) is a transmembrane glycopro-
tein consisting of a noncovalently associated heterodimer
(a3andb1 subunits), and serves as an adhesion receptor
that mediates both cell-extracellular matrix and cell–cell
interactions. It has been suggested that this integrin is a
promiscuous receptor for a variety of extracellular matrix
proteins such as fibronectin, collagen, and laminin-1 (a
prototype of laminin), and for cell surface counter-ligands
[1–5]. Several recent studies have demonstrated that the
a3b1 integrin functions as a high-affinity receptor for
isoforms of laminin, i.e. laminin-5 and laminin-10/11 [6–

9]. More recently, thrombospondin-1 has been reported to
be a ligand for a3b1integrin[10].Thea3 integrin-deficient
mice die at birth, with lung, kidney, and skin defects,
suggesting that this integrin plays a crucial role in their
development and differentiation [11]. It has also been
reported that the a3b1 integrin forms complexes with
other cell-surface proteins, including transmembrane-4
superfamily (TM4SF, tetraspanin) proteins, and that these
complexes may play key roles in cell adhesion, motility,
signaling, transport, and other cell membrane functions
[1]. The cDNA for the hamster, human, and mouse
integrin a3 subunit has been cloned [12–15]. A variant of
the integrin a3 subunit with a different cytoplasmic
sequence has been detected [16], and its specific tissue
distribution has been reported [17]. We previously isolated
mouse genomic clones encoding the integrin a3 subunit
and found that the gene was encoded by 26 exons
spanning over 40 kb [18]. We have demonstrated that the
splicing variants of the a3 subunits (a3A and a3B) are
generated by an alternative exon usage.
Our previous reports showed that the expression of the
a3b1 integrin at both protein and mRNA levels is increased
after the oncogenic transformation of fibroblasts by SV40
or polyoma virus [12,13]. The enhanced expression of this
integrin receptor on transformed cells is likely to be related
to their oncogenic phenotypes. A number of studies have
demonstrated the aberrant expression of a3b1integrinin
various tumor cells in association with changes in their
invasive and metastatic potentials [19–27]. In gastric carci-
noma, melanoma, and glioma, the expression of the a3b1

integrin in these cells was positively correlated with their
malignancy [28–30]. It has also been reported that a3b1
integrin expression is closely related to the cell invasion and
metastatic potentials of gastric carcinoma cells [24]. Thus,
the regulatory mechanism for a3b1 integrin expression in
these cancer cells seems to be of considerable interest. In the
present study, we characterized the promoter region of the
mouse integrin a3 subunit gene, and present evidence
showing that its expression is regulated by the Ets-family of
transcription factors in carcinoma cells.
Correspondence to T. Tsuji, Department of Microbiology, Hoshi
University School of Pharmacy and Pharmaceutical Sciences,
2-4-41 Ebara, Shinagawa-ku, Tokyo 142–8501, Japan.
Fax: + 81 3 5498 5753, E-mail:
Abbreviations: SV40, simian virus 40; EMSA, electrophoretic mobility
shift assay.
Note: nucleotide sequence data are available in the DDBJ/EMBL/
GenBank databases under the accession number AB080229
(Received 1 May 2002, revised 19 July 2002, accepted 26 July 2002)
Eur. J. Biochem. 269, 4524–4532 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03146.x
MATERIALS AND METHODS
Reagents
Restriction endonucleases and modifying enzymes were
purchased from TaKaRa (Osaka, Japan), TOYOBO
(Osaka, Japan) and Gibco BRL (Rockville, MD, USA).
p-Nitrophenyl b-
D
-galactopyranoside was from Sigma (St.
Louis, MO, USA). Luciferase Assay System and Tfx-20
TM

were purchased from Promega Corp. (Madison, WI, USA).
Oligonucleotides were synthesized by Amersham-Pharma-
cia Biotech (Tokyo, Japan).
Cells
Human gastric carcinoma cell lines, MKN1, MKN28 and
MKN45, were supplied by RIKEN Cell Bank (Tsukuba,
Japan). A human gastric carcinoma cell line, KATO III,
was supplied by Health Science Research Resources Bank
(Osaka, Japan). These cells were cultured in RPMI 1640
medium (Gibco BRL) supplemented with 10% fetal bovine
serum (HyClone, Logan, UT, USA) at 37 °C under 5%
CO
2
.
Flow cytometric analysis
The expression of the a3 integrin was measured using a flow
cytometer (FACSCalibur, Becton-Dickinson, San Jose, CA,
USA) employing a monoclonal anti-human a3integrin
antibody (SM-T1) and FITC-labeled anti-mouse IgG (ICN
Pharmaceuticals Inc., Costa Mesa, CA, USA) as described
previously [4].
Cloning of the 5¢-flanking region of mouse integrin a3
subunit gene
A mouse (BALB/c) genomic library constructed in
kEMBL3 was screened with the cDNA for the mouse
integrin a3 subunit as described previously [18]. The
restriction fragments obtained by the digestions with
BamHI, EcoRI, and/or HindIII from positive clones were
subcloned into pBluescript SK(+) (Stratagene, La Jolla,
CA, USA), and analyzed by restriction enzyme mapping

and Southern hybridization using mouse integrin a3 subunit
cDNA. From the results of these analyses, an EcoRI/
HindIII fragment (3aEH70) was found to contain the 5¢-
flanking region and exon 1 of the mouse integrin a3 subunit
gene (Fig. 1).
Construction of reporter plasmids
The 4.0 kb EcoRI/SacI fragment of clone 3aEH70 was
inserted into the luciferase gene-containing plasmid pGL3-
basic (Promega), which lacks eukaryotic promoter and
enhancer sequences. A series of deletions was prepared by
use of restriction sites (PstI, XbaI, and SalI) (Fig. 1) and by
the method using exonuclease III [31] (Deletion Kit,
TaKaRa, Osaka, Japan). To obtain additional deletion
constructs, PCR was performed by using Taq DNA
polymerase (TaKaRa Ex Taq
TM
), 3aEH70 plasmid as a
template, and the sets of primers listed in Table 1. After the
PCR products were digested with KpnIandSacI, the
fragments were inserted into the KpnI/SacIsiteof
pGL3-basic vector. We confirmed by sequencing analysis
that no mutation due to PCR had occurred.
PCR-based site-directed mutagenesis was performed
according to the method described by Weiner et al. [32].
The PCR was performed using pfu DNA polymerase
(Stratagene) with K3S2 plasmid ()260/)119 in pGL-3
basic) as a template and a double-stranded oligonucleotide,
which has a mutation at the consensus binding sequence for
Ets ()248 or )133), MyoD/E-box binding factors ()241) or
GATA ()212) (Table 1). The conditions for the PCR were

as follows: 95 °C, 1 min; 56 °C, 1 min; 72 °C, 6 min; 20
cycles. The PCR products were sequentially treated with
DpnIandwithKpnI/SacI. The digested fragment after
electrophoretic separation on an agarose gel was subcloned
into the KpnI/SacI site of pGL-3-basic plasmid. The
introduction of the mutation was confirmed by the nucleo-
tide sequencing.
DNA sequencing
Nucleotide sequence was determined using a DNA
sequencer (Applied Biosystems model 377, Foster City,
CA, USA) by means of the BigDye
TM
terminator cycle
sequencing method. The primers used are as follows: M13
Fig. 1. Structures of the 5¢-flanking region of mouse a3 integrin gene.
The map (upper line) shows the organization of exons 1–3 and the
5¢-flanking region with the positions for HindIII (H) and EcoRI (E).
The restriction map for the 5¢-flanking region is also shown at a higher
magnification (lower line). The translation initiation site is indicated by
ATG.
Table 1. Oligodeoxynucleotide primers used in PCR experiments.
Mutated bases are underlined.
Primer Sequence
K4 5¢-
GTGGTACCAGTAGCAGCCGCCGCAAG-3¢
K3 5¢-
ATGGTACCGGGCTTTAAGGGTTCCCG-3¢
K2 5¢-
ATGGTACCGGAAGGAAAGCAGAGCCC-3¢
K1 5¢-

ATGGTACCTGGTGATCCAGGGCTTGC-3¢
Sac 5¢-
CCGTTCCGAGCTCCGAGCAC-3¢
S3 5¢-
ATGAGCTCGGGAACCCTTAAAGCCCG-3¢
S2 5¢-
ATGAGCTCTGCTTTCCTTCCGGGGA-3¢
S1 5¢-
ATTGAGCTCACCAGGAGGGCAGGAGG-3¢
mEts-R* 5¢-GACACCTGTCGGTAACCCTTAAAGCC-3¢
mGATA* 5¢-
CGGAGTCGCCTAAGGAGAGATGGAGA-3¢
mE-box* 5¢-
AGGGTTCCCGATCGGTGTCTGAGAGA-3¢
mEts-F* 5¢-
TTTTCTCTTTCCCCGTAAGGAAAGCA-3¢
Ó FEBS 2002 Integrin a3 gene promoter (Eur. J. Biochem. 269) 4525
()21) universal primer for pBluescript SK(+); 5¢-CTT
TATGTTTTTGGCGTCTTCC-3¢ (GL primer) and
5¢-CTAGCAAAATAGGCTGTCCC-3¢ (RV primer) for
plasmids constructed in pGL3-basic.
Transfection and luciferase assay
Luciferase assay was conducted using a Luciferase Assay
System (Promega) along with reporter plasmids constructed
in pGL3-basic plasmid. Carcinoma cells (5 · 10
5
cells) were
seeded in a 35-mm dish and cultured for 20 h. The cells were
then transfected with the mixture of the plasmid construct in
pGL3 vector (3.0 lg) and pRSV-b-Gal (1.0 lg) (used as an

internal control) using the lipofection method employing
Tfx-20
TM
(Promega) in serum-free media (ASF-104, Ajino-
moto, Tokyo, Japan) for 1 h, and subsequently cultured for
48 h in RPMI-1640/10% fetal bovine serum. After the cells
were harvested, the cell extracts were assayed for luciferase
activity with a luminometer. An aliquot of the cell extract
was assayed for b-galactosidase by using 2 m
M
p-nitrophe-
nyl b-
D
-galactoside as a substrate in 20 m
M
sodium
phosphate buffer (pH 7.5) in order to estimate the trans-
fection efficiency in each sample.
Determination of transcription start sites
A modified method of 5¢-rapid amplification cDNA ends
(5¢-RACE) with a cap site-labeled cDNA library was
employed for the determination of transcription start sites
[33]. The cap site-labeled cDNA library derived from
murine kidney was supplied by Nippon Gene Co., Ltd.
(Toyama, Japan). The library was prepared by the cleavage
of the cap structures of mRNA with Tobacco acid
pyrophosphatase followed by ligation with a synthetic
oligoribonucleotide (5¢-GUUGCGUUACAAGGUACGC
CACAGCGUAUGAUGCGUAA-3¢) and the reverse
transcription with a Moloney murine leukemia virus reverse

transcriptase. By using the cap site-labeled cDNA library as
a template, PCR was performed with a set of two primers;
5¢-CAAGGTACGCCACAGCGTATG-3¢ (1RC primer,
corresponding to a part of the sequence in the above
synthetic oligoribonucleotide) and 5¢- CGCTGCACCGGT
AGTCAGGCAAT-3¢ (antisense primer 1, complementary
to +217/+195 of the murine a3 integrin gene). Subse-
quently, nested PCR was carried out with 5¢-GTACGCCA
CAGCGTATGATGC-3¢ (2RC primer, corresponding to
an inner sequence of the synthetic oligoribonucleotide) and
5¢-CCGTTCCGAGCTCCGAGCAC-3¢ (antisense primer
2, complementary to +90/+71 of the murine a3integrin
gene). The condition for the PCR was as follows: 95 °C,
20 s; 60 °C, 20 s; 72 °C, 30 s; 30 cycles. The products were
separated by 2.5% agarose gel electrophoresis in 40 m
M
Tris/acetate buffer (pH 8.0) containing 1 m
M
EDTA
(1 · Tris/acetate/EDTA), and subcloned into a pGEM-T
easy vector (Promega).
Electrophoretic mobility shift assay (EMSA)
Preparation of nuclear extracts from MKN1 cells and
EMSA were performed essentially as described by Ko et al.
[34]. The DNA fragments containing the putative Ets-
binding sequence of the 5¢-flanking region of the mouse a3
integrin gene were synthesized; 5¢-TTTTCTCTTTCCCCG
GAAGGAAAGCAGAG-3¢ (wild-type) and 5¢-TTTTCTC
TTTCCCCG
TAAGGAAAGCAGAG-3¢ (mutant). The

double-stranded oligonucleotides were labeled with
[c-
32
P]ATP (Amersham Biosciences) and T4 polynucleotide
kinase (TaKaRa), and used as probes.
32
P-labeled probes
(15 000 d.p.m.) and nuclear extracts (5 lgprotein)were
mixedin0.02mLof25m
M
Tris/HCl (pH 7.9), 65 m
M
KCl, 6 m
M
MgCl
2
,0.25m
M
EDTA and 10% glycerol in
the presence of 400 ng of dI-dC, and incubated for 30 min
at room temperature. The mixture was then subjected to
polyacrylamide gel (6%) electrophoresis using 10 m
M
Tris/
acetate (pH 7.8) containing 0.25 m
M
EDTA (0.25 · Tris/
acetate/EDTA) as running buffer.
RESULTS
Structure and transient expression analysis of the

5¢-flanking region of mouse integrin a3 subunit gene
We previously cloned the 5¢-flanking region of the integrin
a3 subunit gene from a mouse genomic library [18]. The
restriction map for this region is shown in Fig. 1. The clone
contains exon 1 and approximately 4.0 kb of the 5¢-flanking
region of exon 1 of the mouse integrin a3 subunit gene. We
prepared a chimeric construct (pGL-ES), in which the
4.0 kb EcoRI/SacI fragment upstream of exon 1 was
inserted into the luciferase gene-containing plasmid pGL3-
basic in order to examine its promoter activity. Luciferase
expression was measured following the transfection of pGL-
ES into four human gastric carcinoma cell lines, which
differently express the a3 integrin subunit. When the
construct was introduced into these cell lines, it promoted
higher levels of luciferase activity than the background levels
in all cell lines tested (Table 2). The relative luciferase
activity induced by the transfection in each cell line roughly
parallels the level of a3 integrin expression as measured by
flow cytometry (Fig. 2), suggesting that this region includes
elements that up-regulate the expression of the integrin a3
subunit gene in gastric carcinoma cells.
To specify the region of the 5¢-flanking segment essential
for the promoter activity, we prepared serially deleted
constructs and analyzed the transient expression of luci-
ferase activity after transfection into MKN1 cells, which
were established from gastric cancer metastasis [35] (Fig. 3).
L2.5 and L2.3 induced similar levels of luciferase activity to
the original pGL-ES (L4.0) in these cells, and further deleted
Table 2. Transient expression analysis of integrin a3 subunit gene pro-
moter activity in gastric carcinoma cell lines.

Host cell line
a3 integrin
expression
a
Relative luciferase
activity
b
KATO III + 11.3 ± 0.5
MKN28 ++ 16.5 ± 2.4
MKN45 ++ 30.5 ± 3.1
MKN1 +++ 90.4 ± 8.2
a
The expression of the integrin a3 was measured by flow cyto-
metric analysis using a monoclonal anti-integrin a3 antibody
(Fig. 2).
b
Values (mean ± SD) are normalized to b-galactosidase
activity and expressed in relation to the activity of pGL3-basic
taken as 1.0. Triplicate transfections were performed in each
experiment.
4526 T. Kato et al.(Eur. J. Biochem. 269) Ó FEBS 2002
constructs (L1.8, L1.5 and L1.3) showed higher levels of
luciferase activity than did L4.0. Among the deletion
constructs tested, L1.2 had the highest relative luciferase
activity. L0.5 also showed a comparable high activity. These
results indicate that strong promoter activity is located
within the 0.5 kb stretch of the sequence between the SalI
and SacI sites upstream of exon 1, and that putative
suppressor elements are present between the PstI(approxi-
mately 2.5 kb upstream of the SacIsite)andXbaI

(approximately 1.2 kb upstream of the SacI) sites (Fig. 1).
Sequence analysis of the 5¢-flanking region and
determination of transcription start sites of mouse
integrin a3 subunit gene
The nucleotide sequence of the 0.5 kb SalI/SacIfragment
and a part of exon 1 is shown in Fig. 4. A TRANSFAC
TM
(GBF-AGBIN, Braunschweig, Germany) database search
of this sequence revealed the presence of a number of
potential regulatory elements, including consensus binding
sequences for GATA, NF-jB/Rel, Sp1, Ets, and MyoD/
E-box binding transcription factors. No canonical TATA
box but a CCAAT box was found in the mouse integrin a3
subunit flanking sequence. The integrin a subunit genes so
far characterized contain no CCAAT box except for human
integrin a4 subunit gene, which includes a GCAAT
sequence in its promoter region. The presence of a CCAAT
box seems to be a characteristic feature of the a3integrin
gene among integrin a subunit genes.
Fig. 2. Flow cytometric analysis of the expression of the integrin a3
subunit in gastric carcinoma cells. Profiles of control experiments
without anti-integrin a3 subunit antibody are also shown by thin lines.
(A) KATO III; (B) MKN28; (C) MKN45; (D) MKN1.
Fig. 3. Promoter activity of serial deletion constructs of the 5¢-flanking
region of the mouse integrin a3 subunit gene. Relative luciferase activity
was determined following the introduction of various deletion con-
structs derived from pGL-ES (L4.0, a construct with the 4.0 kb EcoRI/
SacI fragment) into MKN1 gastric carcinoma cells. The activity was
normalized to b-galactosidase activity induced by cotransfection with
pRSV-b-Gal plasmid. The assays were carried out in triplicate, and the

error bars indicate the standard deviation.
Fig. 4. Nucleotide sequence of the 5¢-flanking region of the mouse a3
integrin gene. Major and minor transcription start sites determined by
the cap site-labeled method are marked by closed and open triangles,
respectively. Bases are numbered with respect to the major starting site.
Potential binding sites for transcription factors are underlined and a
consensus sequence for C/EBP (CCAAT) is boxed. The translation
start site (ATG) and the cleavage site in the processing of the poly-
peptide (arrow) are also shown. The nucleotide sequence of the
5¢-flanking region and exon 1 has been deposited in DDBJ/EMBL/
GenBank (accession number AB080229).
Ó FEBS 2002 Integrin a3 gene promoter (Eur. J. Biochem. 269) 4527
To determine the transcription start sites for the integrin
a3 subunit gene, a modified method of 5¢-RACE using a cap
site-labeled cDNA library was employed, recently devel-
oped for rapid examination of 5¢-end of genes [33]. After the
amplification by PCR, the products were separated on 2.5%
agarose gel electrophoresis (Fig. 5). Three bands were
observed when the PCR reaction was performed in the
presence of the cap site-labeled cDNA library (Fig. 5, lane
1), whereas the PCR reaction mixture in the absence of the
cDNA library gave one band corresponding to that with
the highest mobility (Fig. 5, lane 2). Thus, we conclude that
the most prominent band with the highest mobility repre-
sents primers used for PCR. The other two bands, which
seem to be derived from the 5¢-cap site-labeled cDNA for
the integrin a3 subunit gene, were separately excised and
DNA fragments were extracted. Subcloning the fragments
into pGEM-T easy vector followed by sequence analysis
revealed that major and minor transcription start sites are

332 bp and 276 bp, respectively, upstream of the translation
initiation ATG. We hereafter refer to the major transcrip-
tion initiation C residue as +1 (indicated by the closed
triangle in Fig. 4).
The two transcription start sites were surrounded by GC-
rich sequences including the binding sites for transcription
factor Sp1, as frequently found in promoters without a
TATA box. Transcription from so-called TATA-less gene
promoters initiates at a consensus sequence designated as
the initiator sequence [36]. The sequences surrounding the
two transcription start sites of the mouse integrin a3 subunit
gene resembled the pyrimidine-rich initiator consensus
sequence, as found in most integrin a subunit genes lacking
a TATA box (Fig. 6). It should be noted that a consensus
CCT sequence was found at 3–8 bases downstream of the
transcription start sites for integrin a3, a5, a7, aL, aM, aX,
and aIIb subunit genes, all of which lack a TATA box in
their promoter regions [37–48]; i.e. the consensus sequence
can be represented by Py
2
A/CN
2)7
CCT.
Promoter activity of deletions and mutations derived
from the 5¢-flanking segment
We next prepared a series of deletion constructs and
analyzed their promoter activity in MKN1 cells. The L0.5
construct which includes the SalI/SacI fragment upstream
of the a3 integrin gene with high promoter activity (Fig. 3)
was deleted stepwisely from its 5¢-end. As shown in Fig. 7A,

L0.5, L0.4 and L0.3 were almost equally active as a
promoter in these cells. However, the promoter activity of
L0.2 was greatly diminished and that of L0.1 was almost
completely abolished when compared with L0.3. This result
indicates that segments essential for regulating the expres-
sion of the integrin a3 subunit gene are present between
)260 and )134. To confirm that this region is responsible
for the regulation of a3 integrin expression, we subsequently
prepared several constructs with or without this segment by
PCR and successive subcloning into pGL3-basic vector.
The transfection experiments using MKN1 cells demon-
strated that the constructs including the )260/)119 region
(L0.4, K4S1, K4S2, K3S1 and K3S2) showed high luci-
ferase activity, but those without this region (K4S3 and
K2S1) did not (Fig. 7B). These results indicate that the
elements located between )260 and )119 promote efficient
transcription.
As several consensus binding sequences for known
transcription factors such as GATA, Ets, and MyoD/
E-box binding factors were present within )260/)119, we
Fig. 5. Agarose gel electrophoresis of the PCR products of a cap site
region of the mouse a3integrinmRNA.PCR was carried out using a
cap site-labeled cDNA library as a template and primers as described
in Materials and methods. The products were separated in 2.5%
agarose gel in 40 m
M
Tris/acetate buffer containing 1 m
M
EDTA
(pH 8.0). Lane 1, PCR products in the presence of a cDNA library

derived from mouse kidney mRNA; lane 2, PCR products in the
absence of the cDNA library.
Fig. 6. Transcription start sites in the integrin a subunit genes. The
sequences flanking the transcription start sites in the integrin a subunit
genes are shown; chicken a1 [37], human a2[38],humana4[39],
human a5 [40], human a6 [41,42], murine a7 [43], human aL[44],
human aM [45,46], human aX [47], and human aIIb [48]. A consensus
CCT sequence present in most of the integrin a subunit genes that lack
a TATA box is underlined. The transcription start site (+1 position) is
indicated by a triangle. *GCAAT; **GATAAA.
4528 T. Kato et al.(Eur. J. Biochem. 269) Ó FEBS 2002
attempted to introduce mutations into these sequences. As
shown in Fig. 8, the introduction of mutation into one of
the Ets-binding sequences at )133 (GGAA to G
TAA)
greatly decreased the promoter activity, whereas mutations
in the other Ets-binding site at )248 (TTCC to TT
AC), the
E-box at )241 (CAGGTG to
TCGGTG), or the GATA-
binding site at )212 (GATA to
CTAA) showed no
substantial effect.
Electrophoretic mobility shift assay (EMSA) using
the Ets consensus site at )133
As the involvement of the Ets consensus binding site at )133
in the promoter activity of the mouse a3 integrin gene was
suggested by the luciferase assay, this region was further
studied using EMSA. An oligonucleotide corresponding to
the a3 integrin promoter region ()147 to )119) and

containing the wild-type or mutant Ets-binding site was
used as a probe to detect binding activity in MKN1 cells.
The mutant oligonucleotide differs from the wild-type by
single base substitution at the Ets consensus core sequence
as shown in Materials and methods. In the mobility shift
assay, we detected one band with the wild-type oligonucleo-
tide, but it was absent when the mutant oligonucleotide was
used as a probe (Fig. 9). The binding activity appeared to be
specific for the Ets consensus site as the binding competed
with the excess unlabeled wild-type, but not with the mutant
oligonucleotide.
DISCUSSION
The a3b1 integrin has been thought to play crucial roles in
various physiological and pathological processes including
cellular proliferation, differentiation, development, wound
healing, angiogenesis, transformation, and apoptosis [1]. A
vital role of the a3b1 integrin in organogenesis has been
suggested, as mice deficient in this integrin receptor die
during the neonatal period with kidney and lung defects and
skin blistering [11]. Additional abnormalities in the mor-
phogenesis of limbs were observed in integrin a3/a6-
deficient mice; e.g. the absence of digit separation and the
fusion of preskeletal elements [49]. These observations
suggest that the a3b1 integrin plays essential roles in
multiple processes during embryogenesis. The promoter
should thus contain elements directing the expression of this
integrin in the kidney, lung, and skin. A number of studies
Fig. 7. Promoter activity of serial deletion constructs of the 5¢-flanking
region of the mouse integrin a3 subunit gene. Relative luciferase activity
was determined following the introduction of various deletion con-

structs derived from L0.5 (a construct with the 0.5 kb SalI/SacI
fragment) into MKN1 cells. The activity was normalized to b-galac-
tosidase activity induced by co-transfection with pRSV-b-Gal plasmid.
The assays were carried out in triplicate, and the error bars indicate the
standard deviation.
Fig. 8. Effects of mutations in the Ets- and GATA-binding sites and the
E-box of the mouse a3 integrin gene on promoter activity. MKN1 cells
were transfected with wild type (K3S2) or mutated constructs. Relative
luciferase activity was determined in triplicate, and data were nor-
malized to b-galactosidase activity.
Fig. 9. Electrophoretic mobility shift assay using probes containing a
putative Ets binding site at –133. A
32
P-labeled oligonucleotide probe
(W, wild-type; M, mutant) was incubated with nuclear extracts from
MKN1 cells. For competition analysis, 20-fold molar excess of the
unlabeled oligonucleotide (W, wild-type; M, mutant) was added before
the incubation.
Ó FEBS 2002 Integrin a3 gene promoter (Eur. J. Biochem. 269) 4529
also demonstrated the relationship between the aberrant
expression of a3b1 integrin in tumor cells and their
malignant behavior. The increased expression of a3b1
integrin in gastric carcinoma cells is associated with their
increased invasion and metastatic potentials [24,28]. Thus,
the transcriptional regulation for the integrin a3 subunit is
one of crucial issues to be resolved in cancer biology. We
previously reported the structures of the mouse a3integrin
subunit gene including the exon/intron organization and the
alternative exon usage for the generation of variants of the
a3 subunits (a3A and a3B) [18]. In the present study, we

characterized the promoter region for this integrin receptor.
Most integrin a subunit genes lack both TATA and
CCAAT boxes, except for the integrin a4 subunit gene
which includes both TATA and CCAAT boxes and for the
integrin a6 subunit gene which contains a TATA-like box
but lacks a CCAAT box (Fig. 6). By contrast, the promoter
for the mouse integrin a3 gene was found to lack a TATA
box, but does contain a CCAAT box at 324 bp upstream of
the major transcription start site. The presence of a CCAAT
box and the absence of a TATA box seem to be one of the
characteristics of the mouse integrin a3 gene.
We identified two transcription start sites using a
modified method of 5¢-RACE employing a cap site-labeled
cDNA library. The sequences around these transcription
start sites of the mouse integrin a3 subunit gene showed
considerable homology to those of known integrin a
subunit genes (Fig. 6). Most integrin a subunit genes
without a TATA box (a3, a5, a7, aL, aM, aXandaIIb
subunit genes) contain a consensus Py
2
A/CN
2)7
CCT
sequence (where A/C is the transcription start site). The
role of the sequence containing CCT is unknown but it
might play a role in the initiation of transcription.
The active promoter region of the mouse integrin a3gene
in MKN1 cells was mapped in )260/)119. The sequence
analysis of this region revealed the presence of consensus
binding sequences for several transcription factors including

Ets, GATA, and MyoD/E-box binding factors. The
introduction of mutation into one of the putative Ets-
binding sequences suppressed the promoter activity. In
addition, the specific binding of a nuclear protein to the
oligonucleotide containing the Ets consensus sequence was
detected in EMSA. These results suggest that the transcrip-
tion of the mouse integrin a3 subunit gene is regulated by
the Ets-family transcription factors in these cells. A
homology search between human and mouse a3integrin
genes revealed that the Ets consensus core sequence and its
flanking sequences were well conserved and present at
approximately 460 bp upstream of the translation initiation
ATG in the human a3 integrin gene (DDBJ/EMBL/
GenBank database; accession number AC002401). How-
ever, it remains to be identified which transcription factor of
the Ets-family is involved in the regulation. The members of
the Ets-family of transcription factors bind to specific
purine-rich sequences with a core motif of GGAA/T and
control the expression of numerous genes that are critical
for various biological processes including cellular prolifer-
ation, differentiation, development, transformation, and
apoptosis [50].
It has been reported that Ets transcription factors were
involved in tumor metastasis through angiogenesis and the
expression of metalloproteinases or collagenases [51–53]. It
was recently reported that these transcription factors
regulated the expression of the aV integrin in mouse
melanoma cells [54] and the a5 integrin in human glioma
cells [55]. These factors have also been shown to regulate the
expression of N-acetylglucosaminyltransferase V [34,56] and

a(1,3) fucosyltransferase IV [57]. The former enzyme is
responsible for the synthesis of the b1–6 branch in
N-acetyllactosamine units in cell surface N-glycans, and
the latter enzyme is involved in the synthesis of cell surface
ligands for E-selectin; both carbohydrate structures have
been reported to be associated with cellular metastatic
potential. The invasion and metastasis of cancer cells are
thought to include complicated processes. Extracellular
matrix-degrading enzymes are crucial for cell invasion and
angiogenesis. Cell adhesion molecules and carbohydrate
chains present on cell membranes also define the cell–
substratum interaction in the initial attachment of cancer
cells to target tissues in the metastatic process. The
overexpression of a3b1 integrin as well as matrix metallo-
proteinases and collagenases may cooperatively potentiate
cellular metastatic activity.
ACKNOWLEDGEMENTS
We thank Dr Kensuke Suzuki (Pharmaceutical Frontier Research
Laboratories, Japan Tobacco Inc.) for his helpful discussion. We are
also grateful Ms Nami Kawai and Ms Yoko Kawame for their
technical assistance. This work was supported in part by a grant from
the Ministry of Education, Culture, Sports, Science and Technology of
Japan.
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