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: tsuji@hoshi.ac.jp 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. 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Characterization of the promoter for the mouse a3 integrin gene Involvement of the Ets-family of transcription factors in the promoter activity Takumi. 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;