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J Biol Chem, Vol. 273, Issue 50, 33848-33855, December 11, 1998
From the The cell-surface adhesion molecule
Integrins comprise a superfamily of cell-cell and cell-matrix
adhesion molecules, each consisting of a dimer of specific In contrast to other By contrast, Although several studies have shown the significance of altered
Cloning and Nucleotide Sequencing of the
Northern Blot Analysis--
Total RNA (15 µg/lane) was
fractionated by electrophoresis and transferred to Hybond N (Amersham
Corp., UK) by capillary blotting. Hybridization was performed by using
a radiolabeled human Primer Extension Analysis--
Primer extension was carried out
as described previously (31). The antisense oligonucleotide PE-0
(5'-GCGCGGGCGAGCAGGGACTGTCCGAGGGCGGGCGC-3') was hybridized with 3 µg
of mRNA from Plasmid Construction--
A 5.5-kb
HindIII-FspI fragment ( Cell Lines--
Five human urinary bladder carcinoma cell lines,
DAB1, UMUC6-dox, EJ, KU7, and UMUC2 (32-34), were used. The
Transfection and Dual Luciferase Assay--
Cells were seeded at
a density of 3-6 × 104 per 16-mm well and
transfected 24 h later with complexes containing 1.2-1.9 µl of
LipofectAMINE, 4 µl Plus reagent (Life Technologies, Inc.), 42 ng
pRL-TK vector (Promega, Madison, WI), which contained the Renilla luciferase gene as a transfection efficiency
control, and 420 ng of firefly luciferase reporter plasmid per well.
Lysates were prepared 24-48 h after transfection by adding 100 µl of
reporter lysis buffer (dual luciferase reporter system, Promega), and
their luciferase activity was determined with an analytical luminometer (model TD-20/20, Turner Designs, Sunnyvale, CA) and expressed as
relative luciferase units (RLU), calculated by determining the ratio of
the intensity of the light produced by the firefly luciferase reporter
plasmid to that produced by the Renilla luciferase pRL-TK
plasmid. Triplicate luciferase assays of all of the samples were performed.
Gel Retardation Assay--
Nuclear extracts were prepared as
described previously (36), and the gel retardation assays were carried
out using a gel-shift assay kit (Stratagene) according to the
manufacturer's instructions. The following gel retardation probes were
constructed by annealing complementary synthetic oligonucleotides and
end-labeling them with [32P]ATP using T4 polynucleotide
kinase: F0 ( DNase I Footprinting Assay--
DNase I footprinting was
performed with a Core Footprinting System (Promega) according to the
manufacturer's instructions. Briefly, 10-20 fmol of DNA probes (P1,
Structure of the 5'-Flanking Region of the Human
The transcription initiation site was determined by primer extension
analysis using a primer antisense to the known 5'-end of
The nucleotide sequence of the Transcriptional Activity of the Human
In an attempt to identify transcription factors with the potential to
interact with this region (
The above reporter and gel retardation analyses suggest that multiple
proteins bind to separate sites on the promoter and that some of these
complexes were needed simultaneously for transcriptional activation of
the Binding of AP1, Ets, and MyoD to the
One of several DNA-protein complexes that bound to the sequence D1 (
The region surrounding the transcription initiation site was also
examined by DNase I footprinting analysis. A footprint was clearly
visible with recombinant c-Jun protein from
In an attempt to identify the Jun and Fos family members that
interacted with the AP1 site of the
In order to further validate the involvement of AP1 and Ets in the
regulation of Enhancer of the Accumulated evidence has unveiled important roles of
We mapped the basic promoter region of the AP1 comprises either a homo- or heterodimer of two Jun proteins or a
heterodimer of one Jun and one Fos protein. Gel retardation assays
using specific antibodies revealed that c-Jun, JunB, JunD, and a Fos
family protein, Fra-2, bound to the By further delimitation of reporter constructs, we were able to narrow
the minimal promoter region down to nucleotides Complicated and interdependent interactions between multiple factors in
other genes have been reported. For example, AP1, Ets1, and NFkB
synergistically activate the transcription of granuclocyte-macrophage colony-stimulating factor (67). How this synergy is accomplished is not
understood, but altered binding affinity, formation of new binding
sites for additional factors, and a conformational change in DNA have
been suggested (67, 68). AP1 was demonstrated to mediate DNA bending
upon binding (69, 70), and the recruitment of a third molecule from
outside by binding transcription factors is a fascinating mechanism
that may account for the variety of target genes regulated by rather
common transcription factors, such as AP1, although a great deal of
further research will be needed to verify this hypothesis.
It is possible that The presence of separate enhancers is an interesting feature of
In addition to the mechanisms described above, the altered expression
of In conclusion, we have cloned and characterized the
*
This research was supported in part by a Grant-in-Aid for
the Second Term Comprehensive 10-Year Strategy for Cancer Control from
Ministry of Health and Welfare, Japan.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB012286.
¶
Awardee of a Research Resident Fellowship from the Foundation
for Promotion of Cancer Research.
The abbreviations used are:
bp, base pair(s); kb, kilobase pair(s); MMP, matrix metalloproteinase; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; RLU, relative luciferase units.
2
T. Harabayashi, Y. Kanai, T. Yamada, M. Sakamoto, A. Ochiai, T. Kakizoe, T. Koyanagi, and S. Hirohashi,
submitted for publication.
Cloning and Characterization of the Human
4-Integrin Gene Promoter and Enhancers*
§¶,,,,
Pathology Division,
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
6
4-integrin is a receptor for
laminins and a component of hemidesmosomes. 4-Integrin expression is restricted to proliferating basal keratinocytes in the
epidermis and is suppressed when differentiation commences. Altered
4-integrin expression levels correlate significantly with the aggressive behavior of cancers. In order to clarify the mechanisms that regulate transcription of the 4-integrin
gene, we cloned its 5'-flanking region. This 5'-flanking region was found to have a high G + C content and not to contain either TATA or
CAAT boxes. Nested delimitation and reporter analyses mapped a basal
promoter to nucleotides 
106 to +105, surrounding the most proximal
transcription initiation site. Gel retardation and mutational analyses
revealed that cooperation between AP1 and Ets, interacting with other
factors, mediated the promoter activity. In addition to the promoter
element, enhancer activity was found in the first intron (+1905/+3933)
and in a sequence upstream of the promoter region (414/107). These
findings should facilitate our understanding of the regulation of
4-integrin gene expression in processes such as cell
growth and differentiation, apoptosis, and cancer development and metastasis.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
- and
-subunits (1). The 4-subunit specifically associates with the
6-subunit and functions as a receptor for laminins 1, 2, 4, and 5 (2). 64-Integrin is expressed at the
basal surface of most epithelia and binds to the anchoring filaments as
a component of the hemidesmosome, a rigid adhesive microstructure (3). The functional importance of 4-integrin in cell adhesion
was demonstrated by a recent study on 4-knock-out mice, in which the
hemidesmosomes were disrupted, resulting in erosion and blistering of
the skin (4, 5). Patients with a hereditary skin blistering disorder
called junctional epidermolysis bullosa with pyloric atresia show
reduced numbers, or even absence, of hemidesmosomes in the skin due to
mutation of this gene (6). 4-Integrin expression has
been found to be restricted to proliferating basal keratinocytes and to
be suppressed when differentiation commences (7).
4-Integrin was initially identified as a
tumor-associated antigen, defined by the monoclonal antibody A9 raised
against human squamous cell carcinoma (8, 9), in which
4-integrin is no longer restricted to the epidermal
basal cell layer but extends to the suprabasal and upper layers (8, 10,
11). Altered 4-integrin expression during pulmonary,
pancreatic, and uterine cervical carcinogenesis has also been observed
(12-14). An extreme example of altered expression is seen in thyroid
carcinomas; normal thyroid follicular cells do not express
4-integrin at all, but thyroid cancer cells contain high
levels (15). An association between enhanced 4-integrin
expression and early recurrence of squamous cell carcinoma has been
demonstrated (8), and 4-integrin expression levels have
been found to correlate with the degree of invasiveness of colon
carcinoma cells (16) and with high metastatic potential of lung cancer
and melanoma cell lines (17). Introduction of 4-integrin
into colon carcinoma cells enhances their ability to invade laminin
matrices (18).
-subunits, 4-integrin has an
exceptionally large cytoplasmic domain, which associates with
cytoskeletal and signaling molecules (19), and it regulates cell
proliferation through Ras-mitogen-activated protein kinase signaling
pathways (20). Recently, 64-integrin was
found to activate phosphatidylinositol-3-OH kinase pathways upon
binding to laminin-1 (21). The enhanced cell migration resulting from
formation and stabilization of actin-containing structures may account
for the aggressive behavior of 4-integrin-expressing cancers (22).
4-integrin expression has been shown to be
reduced in basal cell carcinoma of the skin (23) and adenocarcinoma of
the breast (24) and prostate (25). Invasive types of urinary bladder
carcinoma have been found to have lost co-localized
4-integrin and collagen-VII (26), and transfection of
4-integrin into human urinary bladder carcinoma cells
has been found to inhibit cell growth and lead to apoptosis (27) by
inducing a cell cycle inhibitor, p21 (28). These findings indicate that
4-integrin has complicated dual functions.
4-integrin expression, the mechanism responsible for
regulating 4-integrin expression has not been
elucidated. 4-Integrin expression seems to be regulated,
at least partly, at the transcription level (7). In order to elucidate
the mechanism responsible for 4-integrin gene expression
and the transcriptional basis for cancer invasion and metastasis, we
cloned the 5'-flanking region of the 4-integrin gene and
identified several cis-acting elements. Below, we report that cooperative transactivation by AP1, Ets, MyoD, and other factors
is involved in the complicated transcriptional regulation of the
4-integrin gene.
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
4-Integrin Gene 5'-Flanking Region--
A human
placental genomic DNA library constructed in the phage vector 
-FIX
II (Stratagene, La Jolla, CA) was screened by plaque hybridization
using a 623-bp1 fragment
corresponding to nucleotides 287-909 of 4-integrin cDNA (29). One positive plaque was identified, and its DNA insert was subjected to characterization by restriction endonuclease mapping
and Southern blot analysis. Digestion with HindIII
restriction endonuclease and Southern blot hybridization with a
synthetic oligonucleotide specific to the 5'-end of
4-integrin cDNA (5'-ATCAGCGTCAGCCTCTCTGG-3') revealed that an 8.8-kb fragment contained the 5'-flanking region of
the 4-integrin gene. This 8.8-kb fragment was subcloned
into the HindIII site of pUC118 (the resulting plasmid was
designated p8.8Hind) and sequenced with an ABI 377 autosequencer
(Perkin-Elmer).
4-integrin cDNA fragment
(nucleotides 287-909) (29). The quality and quantity of the
electrophoresed RNA were determined by rehybridization of the same blot
with a 1.0-kb human glyceraldehyde-3-phosphate dehydrogenase (G3PDH)
cDNA (30). The signal intensity of both the
4-integrin and G3PDH autoradiograms was measured with an image densitometer (model GS-700; Bio-Rad). The relative expression level of 4-integrin was calculated as the ratio to
G3PDH, after subtracting the background signals.
4-integrin-expressing human epidermoid
carcinoma cell line A431 (29). The first strand cDNA was
synthesized with SuperScript II reverse transcriptase (Life Technologies, Inc.) at 50 °C for 30 min in the presence of 0.5 µCi/µl [-32P]dCTP, and the fragments produced were
analyzed by 6% denaturing sequencing gel (Long Ranger, FMC
BioProducts, Rockland, ME) electrophoresis.
5197/+333) was incised
from p8.8Hind, blunted with T4 DNA polymerase, and ligated with an EcoRI/NotI/BamHI adapter (TaKaRa
Shuzo, Japan). After BamHI digestion, the fragment was fused
upstream of the firefly luciferase gene in the pGL3-basic vector
(Promega, Madison, WI). The following 11 nested deletion mutants were
generated from this 5.5-kb fragment using various restriction
endonucleases as follows: L5.5K (HindIII, 5197), L2
(BlnI, 2688), L3 (SacI, 1805), L6
(BstXI, 1050), L7 (ApaI, 414), L8
(SmaI, 257), L9 (NheI, 138), L10
(SacII, 106), L11 (BssHII, +105), La (L5.5K
5197/+333 lacking 106 to +105), and Lc (L10 lacking 35 to +105)
(Fig. 5). In addition, a double-stranded oligonucleotide containing an
MluI site at the 5'-end and a BglII site at the
3'-end was constructed by annealing complementary synthetic sequences
and designated as F4 (96/+3). Constructs harboring mutations at the
Ets- or AP1-binding sites of F4 (96/+3), or both, were also
generated, and these fragments were inserted into the
MluI/BglII site of pGL3-basic. A 3.2-kb FspI-HindIII fragment (+333/+3566), which
contained intron 1 of the 4-integrin gene, was added to
the BamHI site downstream of the firefly luciferase gene in
L5.5K, L7, and L10 in forward (L5.5K-F, L7-F, and L10-F) and reverse
(L5.5K-R, L7-R, and L10-R) orientations (Fig. 11). The following intron
1 deletion constructs were made by digesting the above
intron-1-containing fragment with appropriate restriction endonucleases
and inserting the resulting fragments into the BamHI site of
L10 in both orientations as follows: I1-F and R
(FspI/SacI, +333/+1328), I2-F and R
(SacI/NdeI, +1328/+1905), I3-F and R
(NdeI/HindIII, +1905/+3566), I4-F and R
(FspI/NdeI, +333/+1905), I5-F and R
(NdeI/Tth111I, +1905/+2933), and I6-F and R
(Tth111I/HindIII, +2933/+3566). The composition
of all of the constructs was confirmed by restriction endonuclease
digestion and DNA sequencing.
4-integrin-expressing human epidermoid carcinoma cell
line A431 (29) and three human fibroblast cell lines, WI38, HFL-1, and
NB1RGD, which do not express 4-integrin, were obtained
from Riken Cell Bank (Tsukuba, Japan) (35). All of the cells were
maintained at 37 °C under a humidified atmosphere of 5%
CO2 and 95% air. The urinary bladder carcinoma cells and
A431 were maintained in Dulbecco's minimal essential medium
supplemented with 10% fetal calf serum, and the fibroblast cell lines
were maintained in minimal essential medium supplemented with 10%
fetal calf serum.
106/36), F1 (35/+4), F2 (+5/+44), F3 (+39/+108), D1
(95/77), D2 (56/30), and D3 (19/+4). The following
double-stranded oligonucleotides containing either canonical or mutated
consensus sequences were used for competition assays: AP1
(5'-CTAGTGATGAGTCAGCCGGATC-3') (37), Ets
(5'-GGGCTGCTTGAGGAAGTATAAGAAT-3') (38), MyoD
(5'-GATCCCCCCAACACCTGCTGCCTG-3') (39), SP1
(5'-GATCGATCGGGGCGGGGCGATC-3') (40), NFkB
(5'-GATCGAGGGGACTTTCCCTAGC-3') (41), AP2
(5'-GATCGAACTGACCGCCCGCGGCCCGT-3') (42), mutated AP1
(5'-CTAGTGATGAGTTGGCCGGATC-3'), mutated Ets
(5'-GGGCTGCTTGAGAGAGTATAAGAAT-3'), and mutated MyoD
(5'-GATCCCCCCAACACGGTAACCCTG-3'). For each competition assay, an approximately 100-fold molar excess of unlabeled competitor oligonucleotide was preincubated with the required nuclear extract for
5 min at room temperature before the labeled probe was added. An
anti-Jun antibody that reacts with the DNA binding domain common to
c-Jun, JunB, and JunD and specific anti-c-Jun, JunB, JunD, Fra-2, and
c-Fos antibodies were purchased from Santa Cruz Biotechnology (Santa
Cruz, CA).
137/+114 and P2, 137/+232) labeled at only one end were incubated
with 0-160 µg of nuclear extract or 0.55 µg of recombinant c-Jun
protein (Promega) for 10 min on ice. Freshly diluted DNase I was added
for 1-2 min at room temperature. The reaction was stopped,
phenol:chloroform-extracted, and ethanol-precipitated. The dried DNAs
were suspended in formamide loading dye and resolved on a 6%
denaturing sequencing gel.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
4-Integrin Gene--
An 8.8-kb HindIII
fragment of a 4-integrin genomic clone was isolated from
a human placental genomic library and found to contain an 8.5-kb
sequence upstream of the translation initiation site and a 5.2-kb
5'-flanking region preceding two exons that were separated by the first
intron, which was approximately 3.2 kb (Fig.
1). Exon 1 contained 159 untranslated
nucleotides, including the previously described 5'-end of
4-integrin cDNA (29), and exon 2 contained 89 nucleotides, including the translation initiation codon.
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Fig. 1.
Restriction map of the
4-integrin genomic clone p8.8Hind. The black
boxes represent exons 1 and 2. Exon 2 contains the translation
initiation codon ATG, and the arrow points to the most
proximal transcription initiation site (position +1). The restriction
endonucleases H, HindIII; S,
SacI; Sm, SmaI; N,
NheI; Sa, SacII; B,
BssHII, and F, FspI were used to make
reporter constructs.
4-integrin cDNA, PE0 (dotted arrow in
Fig. 2). Multiple elongation products
were identified from the mRNA of
4-integrin-expressing human epidermoid carcinoma cell
line A431 (Fig. 3, lane 1),
but they were not produced when the primer extension reaction was carried out without mRNA (lane 2). We also confirmed
these results with the mRNA of human lung carcinoma cell line PC10,
which expressed a high level of 4-integrin (data not
shown). Hereafter, we refer to the most proximal transcriptional
initiation site at the cytosine residue of the
4-integrin gene as +1 (arrow in Fig. 2).
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Fig. 2.
Partial nucleotide sequence of the 5'-region
of the human 4-integrin gene. The DNA sequence of
the genomic region surrounding the most proximal transcription
initiation site (arrow) is shown along with the
oligonucleotide (PE0) used for primer extension analysis (long
dotted arrow). Potential binding sites for transcription factors
and the restriction endonuclease recognition sites used to make
reporter constructs are indicated. The full nucleotide sequence
determined in this study has been deposited in GenBankTM
(accession number AB012286).
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Fig. 3.
Mapping of the human
4-integrin mRNA transcription initiation site by
primer extension analysis. mRNA of the human epidermoid
carcinoma cell A431 (lane 1) (29) was annealed to the
antisense oligonucleotide PE0 (dotted arrow in Fig. 2). The
extension reaction was also performed without mRNA, as a negative
control (lane 2). The extension products were analyzed by
electrophoresis on a 6% denaturing sequencing gel. The
arrows indicate anticipated transcription initiation sites,
and +1 corresponds to the most proximal site at the cytosine residue of
the 4-integrin gene (arrows in Figs. 1 and
2). The four lanes to the left contain the
M13mp18 sequencing product used as a size marker.
4-integrin gene
5'-flanking region did not show either TATA or CAAT boxes, and the
sequence between nucleotides 248 and +1 had a high G + C content
(80%). A data base search of the 5'-flanking region revealed the
presence of several putative binding sites for transcription factors,
including AP1, Ets, MyoD, NFkB, and SP1, as shown in Fig. 2.
4-Integrin mRNA
Expression--
4-Integrin mRNA expression was
analyzed by Northern blotting analysis (Fig.
4). The human urinary bladder carcinoma
cell lines DAB1 (lane 1) and UMUC6-dox (lane 2)
expressed 4-integrin mRNA, whereas three other
urinary bladder carcinoma cell lines, EJ (lane 3), KU7
(lane 4), and UMUC2 (lane 5), and the
fibroblast cell lines WI38 (lane 6), HFL-1 (lane
7), and NB1RGB (lane 8) did not express detectable
amounts. These findings agreed with the 4-integrin
protein expression results (data not shown), suggesting that
4-integrin expression in these cells is regulated at the transcription level. The results of the analyses of
4-integrin protein expression and the biological
behavior of these urinary bladder carcinoma cell lines will be
described elsewhere.2
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Fig. 4.
4-Integrin mRNA expression
by human cell lines. 4-Integrin mRNA expression
was examined by Northern blot analysis. The quality and quantity of the
template cDNA were determined by rehybridization of the same blot
with G3PDH cDNA (30). Lane 1, DAB1; lane 2,
UMUC6-dox; lane 3, EJ; lane 4, KU7; lane
5, UMUC2; lane 6, WI38; lane 7, HFL-1; and
lane 8, NB1RGD.
4-Integrin
Promoter--
In order to assess the promoter activity of the
4-integrin 5'-flanking region, nested deletion mutants
were cloned into the firefly luciferase reporter vector pGL3-basic.
Each resulting recombinant plasmid DNA was then transiently transfected
into 4-integrin-expressing DAB1 cells (Fig.
5), and the vector pGL3-basic, which has
no promoter, was used as a control. Among the constructs containing
5197- (L5.5K), 2688- (L2), 1805- (L3), 1050- (L6), and 414- (L7) bp
upstream sequences, L7 showed the highest relative luciferase activity.
Luciferase activity decreased progressively to 25% of L7 as the
sequence length increased, suggesting the presence of an upstream
suppressor activity in 5197/415. Removal of the upstream region
(414/106) from L10 led to a 36% decrease in luciferase activity,
suggesting the presence of a weak enhancer activity in this region.
When the sequence as far as +105 bp downstream was deleted (L11),
luciferase activity fell to that of pGL3-basic. Deletion of the regions
106/+105 and 35/+105 from L5.5K (La) and L10 (Lc), respectively,
virtually abolished their luciferase activity. These results indicate
the presence of a basal promoter at 106/+105. As expected, the
activity of this promoter (determined by L10/L11 ratios) was high in
the 4-integrin-expressing DAB1 and UMUC6-dox cells and
low in EJ, K7, UMUC2, WI38, and NB1RGB cells, which do not express
4-integrin (Table I). The
further delimited construct lacking the 3'-half (+4/+105) of L10,
designated as F4 (96/+3), retained significant promoter activity
compared with L11, but it had only about one-fifth the activity of L10 (Fig. 5), indicating that the downstream region from +4 to +105 is also
necessary for full promoter activity. On the basis of these reporter
assay results, we focused first on the promoter region between 106
and +105, which was critical for 4-integrin transcription.
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Fig. 5.
Promoter activity of serial deletion
constructs of the 4-integrin gene. DAB1 cells were
transfected with sequentially deleted reporter constructs. La is a
construct of L5.5K lacking the nucleotide sequence from 106 to +105,
and Lc is a construct of L10 lacking the nucleotide sequence from 35
to +105. F4 is a construct containing the nucleotide sequence from 96
to +3. The pGL3-basic (denoted by Basic) is a firefly luciferase
reporter vector lacking promoter activity. Transient transfection and
luciferase assays were performed in triplicate, and the data were
normalized to Renilla luciferase activity (RLU
indicates relative luciferase units, i.e. the ratio of
firefly luciferase activity to Renilla luciferase activity)
and are expressed as means ± S.D.
Activity of 4-integrin promoter in various human cell lines
106/+105), four synthetic double-stranded
DNA probes, designated F0 (106/36), F1 (35/+4), F2 (+5/+44), and
F3 (+39/+108), were subjected to gel retardation assays. Each probe
used contained one or two known consensus sequences, i.e.
Ets and MyoD in F0, AP1 in F1, SP1 and Cdx in F2, and NFkB and SP1 in
F3. Nuclear extracts of DAB1 and WI38, representative 4-integrin-positive and negative cell lines,
respectively, were analyzed. Several DNA-protein complexes (
in Fig.
6) were more abundant in the nuclear
extract from DAB1 than from WI38 cells, whereas several rapidly
migrating complexes (
in Fig. 6) with F0 and F1 were more abundant
in WI38 than in DAB1 cells.
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Fig. 6.
Binding of nuclear factors to the
4-integrin promoter. Gel retardation assays were
performed using F0 (106/36), F1 (35/+4), F2 (+5/+44), and F3
(+39/+108) probes and nuclear extracts (NE) from DAB1
(D) and WI38 (W) cells. Lanes 1, 6, 11, and 16 contained control samples incubated without
NE (denoted by ()). 100-Fold molar excesses of competing
oligonucleotides were added as indicated (F0 for lanes 3 and
5; F1 for lanes 8 and 10; F2 for
lanes 13 and 15; and F3 for lanes 18 and 20). Complexes that were more abundant in DAB1 than in
WI38 are indicated by , and those that were more abundant in WI38
are indicated by .
4-integrin gene.
4-Integrin
Promoter Region--
Further gel retardation assays were performed
using probes D1, D2, and D3, which contained the consensus Ets-, MyoD-,
and AP1-binding sites, respectively (Fig.
7). Competition assays using canonical
and mutated consensus oligonucleotides were also performed in order to
verify the identity of the factors in the DNA-protein complexes.
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Fig. 7.
Identification of nuclear factors that bind
to the 4-integrin promoter. A, probe D1
(95/77, containing an Ets-binding site), was incubated with
(lanes 2-5) or without (lane 1) nuclear extract
(NE) from DAB1 cells, and an unlabeled probe (D1)
or a canonical (E) or mutated (mE) Ets
oligonucleotide was added as a competitor (lanes 3-5). The
specific complex detected and subjected to competitive inhibition by
Ets is denoted by 
. B, probe D2 (56/30, containing
two MyoD-binding sites) was incubated with (lanes 7-10) or
without (lane 6) nuclear extract from DAB1 cells, and an
unlabeled probe (D2) or a canonical (M) or
mutated (mM) MyoD oligonucleotide was added as a competitor
(lanes 8-10). The specific complex detected and subjected
to competitive inhibition by MyoD is denoted by . C,
probe D3 (19/+4, containing an AP1-binding site) was incubated with
(lanes 12-15) or without (lane 11) nuclear
extract from DAB1 cells, and an unlabeled probe (D3) or a
canonical (A) or mutated AP1 (mA) oligonucleotide
was added as a competitor (lanes 13-15). The specific
complex detected and subjected to competitive inhibition by AP1 is
denoted by . The arrowheads indicate the undetermined
complexes that were not competitively inhibited by Ets, MyoD, or AP1.
The long arrows indicate the nonspecific bands that were not
competed for by the excess of unlabeled oligonucleotides the same as
the radiolabeled probes. No competitor, indicated by , was added to
the samples in lanes 2, 7, and 12.
in lanes 2-5 in Fig. 7A) was subjected to
competition by the unlabeled canonical Ets oligonucleotide, whereas
mutated Ets (GAGAGAGT instead of GAGGAAGT) did
not inhibit this binding reaction. When D2 was used as a probe, several
complexes were observed, and the binding of one of them ( in
lanes 7-10 in Fig. 7B) was inhibited by
canonical MyoD but not by mutated MyoD (CACGGTAAC instead
of CACCTGCTG). When D3 was used as a probe, a major complex ( in lanes 12-15 in Fig. 7C) was formed, and
its binding was inhibited competitively by canonical AP1 but not by
mutated AP1 (TGAGTTG instead of TGAGTCA). These
results indicate that the binding sites for AP1, Ets, and MyoD were
involved in the transcriptional activity of the
4-integrin promoter.
11 to +7 bp (Fig.
8, lane 5). Although less
apparent, a footprint extending from 10 to +2 bp was also present
with the crude nuclear extracts from DAB1 cells (Fig. 8, lanes
2-4). The protected region corresponds to the AP1 site.
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Fig. 8.
DNase I footprinting assay of the
4-integrin promoter. DNase I footprinting analysis
of the 137/+114 fragment. Shown are the digestion patterns in the
absence of nuclear extract (lane 1), in the presence of 40, 80, or 160 µg of DAB1 nuclear extract (lanes 2-4,
respectively), or in the presence of recombinant c-Jun protein
(lane 5). The sequence at the right corresponds
to the region protected from DNase I digestion by DAB1 nuclear extract
binding. The AP1 site within the protected region is
boxed.
4-integrin promoter,
the DAB1 nuclear extract was incubated with the D3 probe in the
presence and absence of antibodies against Jun and Fos family members
(Fig. 9). An antibody that reacts with
the DNA binding domain common to Jun family members specifically
inhibited the formation of the DNA-protein complex (long arrow,
lane J). Specific antibodies against c-Jun (lane cJ),
JunB (lane B), JunD (lane D), and Fra-2 (lane F) produced supershifted bands
(arrowheads), whereas no supershift was observed with a
specific anti-c-Fos antibody (lane cF) or normal rabbit IgG
(lane N).
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Fig. 9.
Identification of AP1 family members that
interacted with the 4-integrin promoter. Probe D3
(19/+4, containing an AP1-binding site) was incubated with nuclear
extract from DAB1 cells in the absence () or presence of antibodies
against c-Jun (cJ), the DNA binding domain common to the Jun
family (J), c-Fos (cF), JunB (B), JunD
(D), or Fra-2 (F). Normal rabbit IgG is
represented by N. The long arrow to the
left indicates the DNA-protein complex formed in the absence
of antibodies, and the arrowheads indicate supershifted
bands.
4-integrin transcription, we examined
whether mutations in either the Ets- or AP1-binding sites, or both,
affected the promoter activity of F4 (96/+3), because transcriptional regulation of a variety of genes by AP1 and Ets acting cooperatively has been reported (43-47). A mutation in Ets alone did not influence the transcriptional activity, whereas a mutation in the AP1 site reduced the activity by 35%, and mutations in both the Ets and AP1
sites reduced it by 56% (Fig. 10).
Although complete suppression did not occur, AP1, in cooperation with
Ets, is involved at least partly in 4-integrin gene
expression.
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Fig. 10.
Effects of mutations in the AP1- and
Ets-binding sites of the 4-integrin promoter.
Wild-type and mutant promoter constructs of F4 were transfected into
DAB1 cells (mE, mutant Ets-binding site; mA,
mutant AP1-binding site; mE+A, mutant Ets- and AP1-binding
sites). The wild-type and mutant sequences are indicated on the
left, the Ets- and AP1-binding sequences are
underlined (37, 38), and the mutated nucleotides are written
in italics. Triplicate luciferase assays were performed, and
the data were normalized to Renilla luciferase activity
(RLU) and expressed as means ± S.D.
4-Integrin Gene in the First
Intron--
As described above, intron 1 of the
4-integrin gene comprised 3.2 kb and was located
upstream of the translation start site in exon 2. Transcriptional
regulation by their first intron has been reported for several genes
(48-51), and so we examined the enhancer activity of intron 1. The
3.2-kb FspI/HindIII fragment was inserted
downstream of the luciferase gene in both forward and reverse
orientations (L5.5K-F, L5.5K-R, L7-F, L7-R, L10-F, and L10-R).
Transfection of the luciferase gene containing intron 1 in either
orientation into DAB1 cells significantly enhanced their luciferase
activity (Fig. 11). Six deletion
fragments were constructed (Fig. 12) to
better localize the region of the downstream enhancers, but only two of
them, I3 (+1905/+3566) and I5 (+1905/+2933), enhanced luciferase
activity to the same extent as the L10-F and L10-R constructs. Almost
identical results were obtained with UMUC6-dox cells
(Table II). We therefore concluded that
region I5 was responsible for the downstream enhancer activity of
intron 1. This region contained various putative consensus sequences, AP1, Ets, SP1, NFkB, and MyoD. Further delimitation of I5, however, abolished its enhancer activity (data not shown), suggesting that, as
with the promoter, the cooperation of multiple factors is necessary for
enhancer activity of 4-integrin.
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Fig. 11.
Enhancer activity within the first intron of
the 4-integrin gene. The 3.2-kb first intron of the
4-integrin gene was inserted into three representative
constructs containing the core promoter (L10), upstream
enhancer (L7), or full 5.5-kb upstream (L5.5K)
region. The arrows indicate the directions of the intron
relative to that of transcription of the 4-integrin
promoter (R and F, reverse and forward
directions, respectively). The transcription initiation site is
designated +1, and the shaded boxes represent
exons 1 and 2. Triplicate luciferase assays were performed, and the
data were normalized to Renilla luciferase activity
(RLU) and are expressed as means ± S.D.
View larger version (11K):
[in a new window]
Fig. 12.
Deletion analysis of the
4-integrin downstream enhancer. The following
restriction endonucleases were used to make these constructs:
F, FspI; S, SacI;
N, NdeI; T, Tth111I; and
H, HindIII, and the digested fragments are
designated I1, I2, I3, I4, I5, and I6,
respectively. The forward and reverse directions of the inserted
fragments relative to that of transcription of the
4-integrin promoter are indicated by F and
R, respectively. The transcription initiation site is
designated +1, and the shaded boxes represent
exons 1 and 2. Triplicate luciferase assays were performed, and the
data were normalized to Renilla luciferase activity
(RLU) and are expressed as means ± S.D.
Activity of 4-integrin upstream and downstream enhancers
in DAB1 and UMUC6-dox
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
4-integrin in various physiological and pathological
conditions, including cell differentiation, proliferation (11, 20),
adhesion and migration (3, 18, 21, 52), apoptosis, and signal
transduction (27, 28, 53). The exon-intron organization and boundary sequences flanking 41 exons of the human 4-integrin gene
have recently been elucidated (54, 55), but the promoter region and
mechanism that regulates transcription of this intriguing integrin were
unknown prior to our study. The 5'-region of the human
4-integrin gene possesses several features, such as the lack of TATA and CAAT boxes, a high G + C content, and multiple transcription initiation sites, that are commonly seen in a variety of
housekeeping genes (56). These features have also been found in several
non-housekeeping genes, such as epidermal growth factor receptor (57),
1-integrin (58), and DR-nm23, a member of the putative
metastatic suppressor nm23-H1 gene family (59).
4-integrin
gene to 106/+105 by nested deletion constructs. The basic promoter activities, determined by their L10/L11 ratios (Table I), consistently paralleled expression of the 4-integrin gene in urinary
bladder carcinoma cell lines (Fig. 4). The
4-integrin-expressing DAB1 and UMUC6-dox cells showed
high promoter activity, whereas the non-expressing EJ, K7, UMUC2, WI38,
and NB1RGB cells had low promoter activity (Table I). Competitive gel
retardation assays showed that AP1 and Ets bound to this basic promoter
region and that introduction of mutations into the AP1- and Ets-binding
sites partially reduced promoter activity, confirming that these two factors are involved in 4-integrin transactivation to
some extent. Cooperation between Ets and AP1 in transcriptional
regulation of a variety of genes, including matrix metalloproteinase-1
(MMP) (44, 45), MMP-3 (43, 44), urokinase (46), and the mammary tumor
suppressor gene Maspin (47), has been established. The simultaneous activation of these matrix-degrading proteases and a
cell-matrix adhesion molecule, such as 4-integrin, may
act synergistically to cause cancer invasion and metastasis (21, 22).
4-integrin promoter. Zonal transition of Jun and Fos family members was demonstrated to play
an important role in the regulation of epidermal differentiation genes
(60), and consistent with our results, basal keratinocytes have been
reported to express JunB, JunD, and Fra-2 (61). The Ets family of
oncoproteins has been implicated in the generation of several types
of cancer, and expression of the protooncogenes c-met (62)
and c-erb-B2 (63) has been found to be regulated by
transcription factors belonging to the Ets family. Overexpression of
Ets-1 in invasive gastric carcinomas has been observed (64), and Ets-1
has been detected in endothelial cells, particularly during tumor
vascularization (65). Another Ets family member, E1AF, regulates MMP-9
expression in human breast cancer cells and confers the invasive
phenotype on them (66). However, the member of the Ets family that
participates in 4-integrin promoter activity remains to
be identified.
96/+3 (F4
in Fig. 5). The fact that further division of this region failed to
reproduce transcriptional activity (data not shown) indicates that
multiple factors are required for full promoter activity. The construct
lacking the 3'-half (+4/+105) of L10, designated as F4 (96/+3),
retained significant promoter activity compared with L11, but its level
was only about one-fifth that of L10 (Fig. 5), indicating that the
downstream transcribable region from +4 to +105 is also necessary for
full promoter activity. Gel retardation assays detected several
DNA-protein complexes in this region (, lanes 12 and
17 in Fig. 6), suggesting that other, undetermined factors
are also present.
4-integrin-negative cells possess
molecules that suppress the promoter activity of
4-integrin. Our gel retardation assays revealed that
certain DNA-protein complexes were more abundant in nuclear extracts of
WI38 cells ( in Fig. 6) than of DAB1 cells, although it should be
noted that the different DNA-protein complexes in these cell lines may
reflect the protein contents of the nuclear extracts.
4-integrin gene regulation. We discovered that intron 1 possesses enhancer activity, and we tried to narrow down the enhancer
region by preparing delimited constructs. The results showed that
further delimitation to less than 1 kb abolished the enhancer activity, which, as observed with the promoter region, seems to be dependent on
multiple factors within this 1-kb region. In addition to the downstream
enhancer activity, there appears to be a modest upstream enhancer
located between nucleotides 414 and 107. There are several reports
about transcriptional regulation by multiple enhancers (49, 51, 71,
72), and cooperative regulation of certain genes through their
physically distant upstream and intronic enhancers has been reported
(49, 51). The upstream enhancer of the epidermal growth factor receptor
gene is necessary for the downstream enhancer to function, whereas the
two enhancers of the 4-integrin gene seem to function
independently. Furthermore, some inhibitors further upstream of the
promoter in the region from 5197 to 415 seem to be present in the
4-integrin gene. The mechanisms that regulate transcription of the extracellular matrix-associated glycoprotein osteonectin and 4-integrin are similar (51), as
osteonectin has an upstream suppressor, an upstream enhancer, and a
downstream enhancer in intron 1.
4-integrin observed in carcinoma cells may be due to
genetic and epigenetic changes. Sequence analyses of the promoter regions have revealed nucleotide alterations (G to C) at position 57
in DAB1, EJ, and UMUC2 (data not shown), but they showed no correlations with the 4-integrin expression level and
were suggested to be due to polymorphism. Hypermethylation of the
cytosine residues in the promoter was observed in
4-integrin non-expressing KU7 and UMUC2 cells (data not
shown), suggesting the involvement of epigenetic mechanisms in
silencing the 4-integrin gene. Further experiments are
necessary to resolve this issue.
4-integrin promoter and found that AP1 and Ets,
cooperating with other undetermined elements located in the separate
promoter regions, regulate 4-integrin expression. We
have also shown the presence of enhancer elements in intron 1 and
upstream of the promoter region, and a suppressor element upstream of
the upper enhancer region. These findings should facilitate our
understanding of the complicated mechanisms that regulate
4-integrin transcription. In view of the important roles
of 4-integrin, our findings have prompted us to carry
out further investigations on this adhesion molecule.
FOOTNOTES
To whom correspondence should be addressed: Pathology
Division, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan. Tel.: 81-3-3542-2511 (ext. 4102); Fax:
81-3-3248-2737; E-mail: shirohas{at}gan2.ncc.go.jp.
REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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