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

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

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

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¢-

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

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

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

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

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

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-

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

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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