©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Novel Promoter for Vascular Endothelial Growth Factor Receptor (flt-1) That Confers Endothelial-specific Gene Expression (*)

(Received for publication, May 18, 1995)

Kaoru Morishita (§) Daniel E. Johnson (¶) Lewis T. Williams (**)

From the Cardiovascular Research Institute, Box 0130, University of California San Francisco, San Francisco, California 94143

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The human transmembrane fms-like receptor tyrosine kinase Flt-1 is one of the receptors for vascular endothelial growth factor, a growth factor which induces endothelial proliferation and vascular permeability. Flt-1 is expressed specifically in endothelium and is likely to play a role in tumor angiogenesis and embryonic vascularization. To elucidate the molecular basis for the endothelial specific expression of Flt-1, the promoter region has been isolated and functionally characterized. The promoter region contains a TATA box, a GC-rich region, and putative transcription factor binding elements such as cAMP response element binding protein/activating transcription factor (CREB/ATF) and ets. Adenovirus-mediated transient expression of the flt-1 promoter/luciferase fusion gene in endothelial cells and other cell types demonstrated that a 1-kilobase fragment of the 5`-flanking region of flt-1 is involved in the endothelial-specific expression. A CREB/ATF element was found to be essential for basal transcription of the flt-1 expression. In addition, we also showed that the first intron negatively regulates flt-1 promoter activity. The flt-1 promoter will be useful in functional studies on the regulation of endothelial-specific gene expression and also as a tool in targeting the expression of exogenously introduced genes to the endothelium.


INTRODUCTION

Vascular endothelial growth factor is not only a specific mitogen for vascular endothelial cells but also a potent mediator of vascular permeability(1, 2) . We as well as other groups have shown that Flt-1 (^1)(fms-like tyrosine kinase) and Flk-1 (fetal liver kinase-1; mouse homologue of kinase insert domain-containing receptor (KDR)) are receptors for vascular endothelial growth factor(3, 4, 5, 6) . These receptors and Flt-4 (7, 8) are members of a family of tyrosine kinases, which is characterized by proteins containing seven immunoglobulin-like domains, a single transmembrane region, and a kinase insert sequence.

We have recently shown that flt-1 is expressed specifically in the endothelium in adult mouse tissues by in situ hybridization(9) . We have also shown that flt-1 is expressed in the endothelium during neovascularization of healing skin wounds and during early vascular development in mouse embryos. Therefore, expression of flt-1 is highly restricted to vascular endothelial cells. However, little is known about the molecular regulation of endothelial-specific gene expression as yet.

As a first step to address this issue, we identified the promoter region of flt-1 and characterized this promoter in a transient expression assay. Here we show in a series of transfection assays that a 1-kb DNA fragment of a 5`-flanking sequence of flt-1 demonstrates functional activity in vascular endothelial cells but not in epithelial cells, vascular smooth muscle cells, or fibroblasts. The study also demonstrates that the flt-1 CREB/ATF element is essential for basal transcription and the first intron of flt-1 contains negative regulatory elements.


EXPERIMENTAL PROCEDURES

Materials

The recombinant adenovirus Adex1CAlacZ, the adenovirus cosmid vector pAdex1W and EcoT22I-digested adenoviral DNA-terminal protein complex were obtained from Dr. Izumi Saito at the Institute of Medical Science, the University of Tokyo.

Cell Cultures

Bovine adrenal endothelial cells (BAEC) were obtained from Dr. Richard Weiner at the University of California, San Francisco, and maintained in Dulbecco's modified essential medium supplemented with 1 mg/ml glucose, 1 ng/ml basic fibroblast growth factor, and 10% fetal bovine serum (FBS). Human umbilical vein endothelial cells (HUVEC), human aortic endothelial cells, human pulmonary arterial endothelial cells, human aortic smooth muscle cells, and human mammary epithelial cells were obtained from Clonetics and maintained according to the manufacturer's recommendation. NIH-3T3 cells and human foreskin fibroblasts (HFF) were maintained in Dulbecco's modified essential medium supplemented with 10% FBS. NCI-H292 human pulmonary mucoepidermoid carcinoma cells were maintained in RPMI 1640 supplemented with 10% FBS. Rat aortic smooth muscle cells (Sprague-Dawley rats) were isolated from explants as described previously (31) and maintained in Dulbecco's modified essential medium supplemented with 10% FBS.

Cloning of the 5`-Flanking Region of the Human flt-1 Gene

A human placenta genomic library in EMBL-3 phage (Clontech) was screened with a 600-bp EcoRI/AccI fragment from the 5`-end of the flt-1 cDNA. After three rounds of screening, 13 positive clones were isolated. Two sets of overlapping synthetic oligonucleotides, 5`-GGACACTCCTCTCGGCTCCTCCCCGGCAGCGGCGGCGGCTCGG-3` (oligo-E) and 5`-CGCTGGCCGCTGCACCCGAGCCCCGGAGCCCGCTCCGAGCCGCCGC-3` (oligo-F), corresponding to the 5`-end of the flt-1 cDNA between positions +3 and +79 (designated as probe A) and 5`-GGTCTTTGCCTGAAATGGTGAGTAAGGAAAGCGAAAGGCTGAGCATAACT-3` (oligo-J) and 5`-CAGAATTGTTTGCCATTTCTTCCACAGGCAGATTTAGTTATGCTCAGCCT-3` (oligo-K) corresponding to the sequence of the flt-1 cDNA between positions +427 and +502 (designated as probe B) were annealed, followed by filling-in with Klenow fragment in the presence of [alpha-P]dCTP. Four of the 13 clones hybridized with probe A, but not with probe B. In contrast, the other clones hybridized with probe B, but not with probe A. Three different clones which hybridized with probe A were selected for restriction endonuclease and Southern blot analyses. The 3-kb EcoRI/XhoI fragments from all three clones and a 7-kb EcoRI fragment from clone 5-11 were subcloned into Bluescript KS+ (Stratagene) to generate pBKS3.0 and pBKS7.0. These plasmids were used for further restriction enzyme mapping, nucleotide sequencing analysis, subcloning, and expression studies as described below.

Construction of p(-2.5k/+284)-luc Containing the 5`-Flanking Region of flt-1 and Luciferase-fusion Gene

The plasmid pMC-luc was generated by cloning annealed complementary oligonucleotides including restriction sites for SwaI, I-PpoI, PmeI, SmaI, AscI, NotI, XhoI, SrfI, SfiI, and HindIII (5`-ATTTAAATCTCTCTTAAGGTAGCGTTTAAACCCGGGCGCGCCGCGGCCGCTCGAGCCCGGGCGGCCTCACTGGCCATTTAAATA-3` and 5`-AGCTTATTTAAATGGCCAGTGAGGCCGCCCGGGCTCGAGCGGCCGCGGCGCGCCCGGGTTTAAACGCTACCTTAAGAGAGATTTAAAT-3`) into the SmaI and HindIII sites of a luciferase expression vector (pGL2-basic, Promega). A 3-kb NotI/XhoI fragment from pBKS3.0 which contains the 5`-flanking region, the first exon, and part of the first intron of flt-1 was inserted into the NotI and XhoI sites of pMC-luc to generate p(-2.5k/+550)-luc. A 3-kb SacI/KpnI fragment from pBKS3.0 was inserted into the KpnI and SacI sites of pGL2-basic to generate p(+550/-2.5k)-luc which contains the 5`-flanking region oriented in the reverse direction. The plasmid p(-2.5k/+284)-luc, in which most of the first intron was deleted, the constructed by digesting p(-2.5k/+550)-luc with NcoI, XhoI, and mung bean nuclease followed by self-ligation.

Construction of 5`-Deletion Mutant Plasmids

The plasmid p(-1.9k/+284)-luc was constructed by digesting p(-2.5k/+284)-luc with PstI followed by self-ligation. The plasmid p(-1195/+284)-luc was constructed by digesting p(-2.5k/+284)-luc with PstI, BstXI, treating with T4 DNA polymerase, and self-ligating. To generate a series of deletion mutants, The plasmid p(-2.5k/+284)-luc was treated with BstXI, T4 DNA polymerase, and PstI, and digested with exonuclease III from the 5`-end followed by self-ligation using the Erase-a-Base kit (Promega). Several 5`-deletion mutants were produced: p(-962/+284)-luc, p(-748/+284)-luc, p(-583/+284)-luc, p(-386/+284)-luc, p(-356/+284)-luc, p(-333/+284)-luc, p(-239/+284)-luc, p(-219/+284)-luc, and p(+151/+286)-luc. To construct another deletion mutant, p(-2.5k/+284)-luc was digested with AatII and PstI, treated with T4 DNA polymerase, and then self-ligated to generate p(-75/+284)-luc.

Construction of Luciferase-fusion Plasmids Containing a 5`-Flanking Region, Exon 1, and a Hybrid Intron

The plasmid p(-2.5k/+550)sp-luc which contains a hybrid intron composed of the 5` portion of the first intron of flt-1 and the 3` portion of mouse immunoglobulin heavy chain gene (10) was constructed by cloning annealed complementary oligonucleotides corresponding to a mouse immunoglobulin heavy chain variable region (5`-TCGAGGCTTGAGGTCTGGACATATACATGGGTGACAATGACATCCACTTTGCCTTTCTCTCCACAGGTGTCCA C T C C CAGGTCCAACTGCAG-3` and 5`-CTGCAGTTGGACCTGGGAGTGGACACCTGTGGAGAGAAAGGCAAAGTGGATGTCATTGTCACCCATGTATATGTCCAGACCTCAAGCC-3`) into the XhoI and SrfI sites of p(-2.5k/+550)-luc. The resulting plasmid p(-2.5k/+550)sp-luc was treated with XhoI, Klenow fragment, and NcoI and ligated with a 4-kb NcoI/EcoRV fragment from pBKS7.0 which contains the 5` portion of the first intron of flt-1.

Construction of CREB/ATF Element-deleted Mutant Plasmids

The plasmid p(-962/+284)-luc was digested with AatII and treated with T4 DNA polymerase, resulting in pDeltaCRE(-962/+284)-luc. Four internal bases of CREB/ATF element (ACGT out of TGACGTCA) were deleted in pDeltaCRE(-962/+284)-luc.

Transient Transfection Experiments and Enzyme Assays

For transfection analyses, plasmids were purified with Wizard Megaprep (Promega) followed by cesium chloride gradient ultracentrifugation. BAEC and NIH-3T3 cells were seeded onto 6-well plates or, for HFF, onto 100-mm dishes at a density adjusted so that they reached 40-60% confluence prior to transfection. For BAEC and NIH-3T3 cells, 5 µg of DNA of test plasmid, 5 µg of pSV-CAT (pCAT-promoter, Promega), and 10 µl (5 µl for NIH-3T3 cells) of Lipofectin (Life Technologies, Inc.) were incubated in 0.1 ml of OptiMEM (Life Technologies, Inc.) for 20 min. Similarly, for HFF, 25 µg of plasmid construct DNA and 5 µg of DNA of pSV-CAT, and 50 µl of Lipofectin were incubated in 0.4 ml of OptiMEM. The resulting transfection mixture was added to the medium and incubated for 6-10 h at 37 °C. Then, the medium was replaced by complete medium for an additional 3 days. For the stimulation experiments, 10 µM forskolin and 0.5 mM 3-isobutyryl-1-methylxanthine (IBMX) or 0.5 mM dibutyryl-cAMP and 0.5 mM IBMX were added directly to media 16-17 h prior to harvest. Cells were washed with ice-cold phosphate-buffered saline twice, and lysed with 80 µl (6-well plate) or 400 µl (10-cm dish) of 100 mM potassium phosphate (pH 7.8), 0.5% Triton X-100. After removing the insoluble cell debris by centrifugation, each cell lysate was used to measure luciferase and CAT activities. The luciferase activity was measured with a Monolight 2010 luminometer in the presence of 1 mM dithiothreitol using Luciferase Assay Reagent (Promega). CAT activity was determined by the phase-extraction procedure using [^3H]chloramphenicol (DuPont NEN) and xylenes after endogenous deacetylating activity was destroyed by heating the lysates for 10 min at 65 °C(11) . The efficiency of transfections was normalized with activities of CAT assay.

Construction of a Recombinant Adenovirus Containing the flt-1 Promoter-Luciferase Fusion Gene and Enzyme Assays

The plasmid p(-748/+284)-luc was digested with SalI, treated with T4 DNA polymerase, and digested with BamHI to generate a 3.7-kb fragment. The fragment was cloned into the SwaI site of pAdex1W adenovirus cosmid vector to generate pAdexFLTP-luc. pAdexFLTP-luc and the EcoT22I-digested adenoviral DNA-terminal protein complex were co-transfected into 293 cells to prepare a replication-negative recombinant adenovirus AdexFLTP-luc(32) . A recombinant adenovirus Adex1CAlacZ which contains a CAG promoter (modified chicken beta-actin promoter with CMV-IE enhancer) (33) and a beta-galactosidase gene and AdexFLTP-luc were used to coinfect with various cell lines. Briefly, AdexFLTP-luc (1.1 times 10^5 plaque-forming units/ml) and Adex1CAlacZ (8.4 times 10^4 plaque-forming units/ml) were incubated with cells in 0.5 ml of Dulbecco's modified essential medium supplemented with 10% FBS in 24-well plates. Luciferase activity was measured as described above and beta-galactosidase activity was measured with chlorophenol red beta-D-galactopyranoside (Boehringer Mannheim Biochemica). The efficiencies of transfections were normalized with activities of beta-galactosidase assays.

Primer Extension and S1 Mapping

Primer extension analysis was carried out according to described methods(12) . Briefly, oligo-F was end-labeled with T4 polynucleotide kinase. Approximately 5 ng of labeled primer was hybridized to 50 µg of total RNA from HUVEC, human lung tissue (Clontech), and yeast tRNA in hybridization buffer (80% formamide, 40 mM PIPES (pH 6.4), 400 mM NaCl, 1 mM EDTA) at 30 °C overnight. The extension reaction was carried out with 50 units of avian myeloblastosis virus reverse transcriptase (Promega) in 50 mM Tris-HCl (pH 7.6), 60 mM KCl, 10 mM MgCl(2), 1 mM dNTPs, 1 mM dithiothreitol, 1 units/µl of RNase Block (Stratagene), 50 µg/ml actinomycin D for 2 h at 37 °C. The extended products were analyzed on denaturating 6% gel polyacrylamide gels. Sequence reactions on flt-1 with the same primer were run in parallel for accurate determination of the extension termination site.

S1 mapping analysis was carried out as described(13) . Briefly, end labeled oligo-F was hybridized with pBKS3.0 and incubated with 4 units of Klenow fragment in the presence of 4 mM dNTPs for 30 min at 37 °C. After heat inactivation, the extended product was digested with SmaI, separated on an alkaline agarose gel, and purified by phenol extraction and ethanol precipitation. The probe (5 times 10^4 Cerenkov counts) was then hybridized to 50 µg of total RNA from HUVEC, human lung tissue (Clontech), or yeast tRNA in hybridization buffer at 30 °C overnight. The reaction mixture was digested with 300 units of S1 nuclease in 280 mM NaCl, 50 mM sodium acetate (pH 4.5), 4.5 mM ZnSO(4) for 60 min at 30 °C. The protected products were analyzed on denaturating 6% polyacrylamide gels.


RESULTS

Restriction Map and Exon-Intron Organization of 5`-Specific Human flt-1 Genomic Clones

Genomic clones from a human placental genomic library were obtained by using the human flt-1 cDNA 5`-end 600-bp DNA fragment (described under ``Experimental Procedures''). Three overlapping but not identical genomic clones were selected for further analysis based on the result of Southern analyses using the human flt-1 cDNA 5`-end oligo DNA probe. The restriction maps of these clones were determined by the partial restriction method (Fig. 1). The 3-kb EcoRI/XhoI fragments from all three clones were subcloned into pBluescript-KS(+). Detailed restriction maps and partial sequences showed that these 3-kb fragments were identical. Although the restriction pattern of the 5` region of clone 4-18 was different from that of 5-21A and 5-11, the reason for this diversity remains unclear.


Figure 1: Structure and restriction map of the flt-1 genomic clones containing a 5`-flanking region, exon 1, and a 5` portion of intron 1. Three clones contained overlapping genomic segments. Solid boxes indicate the position of exon 1. The 3-kb EcoRI/XhoI fragments from all three clones were subcloned into pBluescript and restriction sites were determined. Restriction patterns of the three fragments were identical. The nucleotide sequence of the 5`-flanking region of flt-1 between positions -1195 (BstXI) and +550 (XhoI) indicated by the dotted line was determined by the Sanger method. Restriction sites for enzymes are indicated as follows: Ap, ApaI; At, AatII; B, BamHI; Bs, BstXI; E, EcoRI; N, NaeI; Nc, NcoI; P, PstI; S, SmaI; Sp, SpeI; X, XhoI.



Sequence Analysis of the Promoter Region of flt-1

The nucleotide sequence of a 1.8-kb BstXI/XhoI fragment from clone 4-18 (Fig. 1) was determined by the Sanger method. This fragment contains exon 1, a 5` portion of intron 1, and the 5`-flanking region of flt-1 containing putative transcription factor binding sites such as a TATA box, a CREB/ATF element, and an ets binding site (Fig. 2). The first intron contains a putative transcription arrest site as discussed below.


Figure 2: Nucleotide sequence of the 5`-flanking region, exon 1, and a 5` portion of intron 1 of the flt-1 gene. The transcription initiation site identified by primer extension and S1 mapping is indicated by an asterisk and designated as +1 (Fig. 3). The consensus sequence of a TATA box, the putative binding sites for CREB/ATF, and ets, and the putative transcription arrest site are underlined. A unique separated palindromic sequence is boxed. The nucleotide sequence of the synthetic oligonucleotide oligo-F used for primer extension is also underlined. The 5`-end of intron 1 is indicated by an arrow.




Figure 3: Identification of the transcription initiation site of flt-1 by primer extension analysis. Total RNAs from BAEC, yeast tRNA, or lung tissue RNA were analyzed using the synthetic oligonucleotide oligo-F as a primer. Sequence reactions of the flt-1 genomic DNA using the same primer were run in parallel. An asterisk indicates the transcription initiation site.



Transcription Initiation Site

To identify the transcription initiation site of flt-1, primer-extension analysis was performed with total RNA from HUVEC and human lung tissue (Fig. 3). The transcription initiation site was mapped to an adenosine residue 25-bp downstream from the TATA box. This result was confirmed by S1 mapping analysis (data not shown).

The flt-1 Promoter Activity Is Endothelial Cell Specific

To determine the sequences essential for efficient transcription of the flt-1 promoter, a DNA segment extending from +284 bp to -2.5 kb was fused to a luciferase gene in the pMC-luc vector. The construct designated as p(-2.5k/+284)-luc contains 2.5 kb of the promoter region, 230 bp of exon 1, and 54 bp of the 5`-end of the first intron. This construct was used to generate a series of 5`-end deletions ( Fig. 4and Fig. 6A). The resultant constructs are referred to as p(X/Y)-luc. For each, X and Y represent the 5`- and 3`-end positions in nucleotides. Each construct was transfected into BAEC.


Figure 4: Expression of luciferase fusion gene constructs containing 5`-deleted flt-1 promoter sequences. The constructs were transiently transfected into BAEC (solid), NIH-3T3 cells (open), and HFF (shaded). Luciferase activities were normalized to internal pSV-CAT control in each extract to adjust for differences in transfection efficiencies. The luciferase activity obtained with transfection of pSV-luc is arbitrarily set at 1 for each cell type. All other luciferase activities are given relative to this value. Each value is the mean of at least three independent experiments.




Figure 6: Effect on flt-1 promoter activity by: (A) further deletion of a 5`-flanking region and (B) internal deletion of CREB/ATF element. A, constructs containing shorter promoter regions than the constructs shown in Fig. 4and Fig. 5were analyzed. B, four internal bases of the CREB/ATF element (ACGT out of TGACGTCA) were deleted in DeltaCRE(-962/+284)-luc. These constructs were transiently transfected into BAEC. The data are presented in the same manner as in Fig. 4. Each value is the mean of two independent experiments. Filled triangles indicate the CREB/ATF element.




Figure 5: Expression of the flt-1 promoter (-748/+284)-luciferase fusion gene in various human primary cells and established cell lines. The replication-deficient recombinant adenovirus AdexFLTP-luc carrying the flt-1 promoter (-748/+284)-luciferase-fusion gene and Adex1CAlacZ were coinfected into various human primary endothelial cells (human aortic endothelial cells (HAEC), human pulmonary arterial endothelial cells (HPEC), HUVEC), BAEC, human primary mammary epithelial cells (HMEC), human primary aortic smooth muscle cells (AOSMC), NCI-H292 cells, HFF, rat primary aortic smooth muscle cells (rat SMC), and NIH-3T3 cells. Luciferase activities were normalized to beta-galactosidase activities in each extract. Each value is the mean of at least three independent experiments.



Deletion mutant p(-748/+248)-luc showed the highest activity. Transfection of the series of constructs deleted from -2.5 kb to +151 suggested the presence of at least two regions, 2500 to -1195 and -356 to -333, containing negative regulatory sequences, and two regions, -748 to -583 and -239 to -75, containing positively regulatory sequences. Deletion to +151 decreased luciferase activity to the level of the promoterless plasmid pMC-luc.

To determine if the 2.5-kb promoter region used in these experiments was sufficient to confer cell-type specificity, the deletion constructs were also transfected into NIH 3T3 cells and HFF. Relative luciferase activities in these cells were much weaker than those in BAEC (Fig. 4).

To clarify endothelial specificity of the flt-1 promoter further, we employed various human primary cells. However, because we could not efficiently transfect primary cells by conventional methods, we introduced the construct using a replication-deficient recombinant adenovirus. The recombinant adenovirus AdexFLTP-luc carrying the flt-1 promoter (-748/+284)-luciferase fusion gene was used to infect various human primary cells and established cell lines. Following infection, relative luciferase activities seen in human primary endothelial cells such as aortic endothelial cells, pulmonary arterial endothelial cells, umbilical vein endothelial cells, and bovine adrenal endothelial cells were much higher than activities seen in human primary mammary epithelial cells, human primary aortic smooth muscle cells, NCI-H292 cells, human foreskin fibroblasts, rat primary aortic smooth muscle cells, and NIH-3T3 cells (Fig. 5). These results showed that the flt-1 promoter region between positions -748 and +284 conferred endothelial-specific gene expression.

The CREB/ATF Element Is Essential for flt-1 Promoter Activity

In some genes, a CREB/ATF element has been shown to be involved in not only transcriptional activation by a cAMP-dependent protein kinase A but also basal transcription. To characterize the CREB/ATF element of the flt-1 promoter, we constructed a deletion mutant in the CREB/ATF element. Deletion of 4 internal bases in the CREB/ATF element of the flt-1 promoter (ACGT out of TGACGTCA) diminished relative luciferase activity in BAEC by 85% (Fig. 6B). However, we also failed to detect any stimulation of luciferase activity in response to forskolin/IBMX and dibutyryl-cAMP/IBMX in BAEC transfected with either p(-962/+284)-luc or pDeltaCRE(-962/+284)-luc (data not shown). Therefore, the CREB/ATF element of the flt-1 promoter is important for basal transcription of flt-1, but may not be important in the transcriptional activation in response to cAMP elevation.

The First Intron of flt-1 Negatively Regulated Transcription

Transfection of the p(-2.5k/+550)-luc construct, which contains 220 bp of the first intron of flt-1 (containing the 5` splice site but not the 3` splice site), resulted in no luciferase activity (Fig. 7). This may be due to the production of an undesirable protein instead of luciferase since the first intron contains an ATG at +286 which is upstream of the initiation codon of the luciferase gene and may not be spliced out because of the lack of a 3` splice site. When a 3` splice site from a mouse immunoglobulin gene (10) was introduced downstream of the first intron to generate a hybrid intron (p(-2.5k/550)sp-luc), luciferase activity was partially restored. The idea to make this construct was based on results obtained with a hybrid intron consisting of a 5` splice site from the first exon of the adenovirus tripartite leader and a 3` splice site from a mouse immunoglobulin gene on pMT2 expression vector(14) . These studies showed that the hybrid intron was completely spliced out when eukaryotic initiation factor 2 was expressed on pMT2 vector. Thus, it appears likely that the decrease of luciferase activity seen in p(-2.5k/550)sp-luc (Fig. 7) does not result from a deficiency in splicing. Therefore, we conclude that the first intron of flt-1 negatively regulated the transcription.


Figure 7: Negative effect of intron 1 on the flt-1 promoter activity. Both (-2.5k/+550)sp-luc and (-2.5k/+4.5k)sp-luc contain a hybrid intron consisting of 220 bp or 2.3 kb of the 5` portion of the first intron of flt-1 and a 3` splice site of a mouse immunoglobulin gene. By contrast, (-2.5k/+550)-luc contains only 220 bp of the 5` portion of the first intron of flt-1. (+550/-2.5k)-luc has the promoter oriented in the reverse direction. These constructs were transiently transfected into BAEC. The data are presented in the same manner as in Fig. 4. Each value is the mean of two independent experiments. The shaded box indicates a 3` splice site of a mouse immunoglobulin gene (10) .




DISCUSSION

In this report, we identified the flt-1 promoter region and showed that a 1-kb fragment of the 5`-flanking sequence of the flt-1 gene demonstrated functional activity in vascular endothelial cells but limited activity in epithelial cells, vascular smooth muscle cells, and fibroblasts. Deletion studies of the flt-1 sequence indicated that the regions from -2500 to -1195 and -356 to -333 contained negative regulatory sequences and regions from -748 to -583 and -239 to -75 contained positive regulatory sequences.

We determined that the transcription initiation site is located 25 bp downstream of a TATA box by primer extension and S1 mapping. The regions surrounding the transcription initiation site is similar to other initiator sequences. These results indicated that the initiation site of flt-1 is a typical one.

The 5`-flanking sequence of flt-1 exhibits common features of a promoter because it contains a TATA box, a GC-rich region, and potential binding sites for transcription factors. Internal deletion studies showed that the consensus CREB/ATF element (TGACGTCA) located at -74 was essential for basal transcription of flt-1. However, many attempts have failed to transactivate the flt-1 promoter in response to cAMP elevation by treatments with forskolin/IBMX and dibutyryl-cAMP/IBMX, suggesting that CREB is not involved in regulation of the flt-1 expression. As many transcription factors are known to bind the consensus CREB/ATF element, other members of the basic region-leucine zipper protein family may bind and activate transcription of flt-1(15) . The CREB/ATF-like element of the TGF-beta2 promoter has been shown to be essential for basal level promoter activity, but does not confer responsiveness from either protein kinase A or C(16) .

The promoters of genes whose expression is limited to endothelial cells and certain other cells, such as endothelin-1(17) , endothelial-leukocyte adhesion molecule 1(18) , P-selectin(19) , vascular cell adhesion molecule 1(20, 21, 22) , and thrombomodulin (23) contain ets-binding site(s)(24) . Similarly, flt-1 expression is limited to endothelial cells and its promoter contains at least four putative ets-binding sites at -36, -49, -125, and -141. Ets-1 expression is observed in endothelial cells during the early stages of blood vessel formation (25) and tumor angiogenesis(26) . flt-1 is expressed in endothelial cells from the early stages of mouse embryo development (9) and is up-regulated in tumor endothelial cells(27) . These results suggest that Ets-1 plays a role in regulation of the flt-1 expression during embryonic vascularization and tumor angiogenesis.

We have also shown that the first intron of flt-1 negatively regulated gene expression. There are several mechanisms by which introns have been shown to regulate gene expression: 1) transcriptional attenuation by a silencer, 2) formation of double-stranded RNA by antisense transcripts(28, 29) , and 3) transcription arrest(30) . In the case of flt-1, the first intron contains a sequence which is very similar to the transcription arrest site in the first intron of the adenosine deaminase gene (Fig. 8). Thus, we predict that the negative regulation conferred by intron 1 may be due to transcriptional arrest.


Figure 8: Comparison between the sequence surrounding the transcription arrest site in the first intron of the adenosine deaminase (30) and the homologous region in the first intron of flt-1. Asterisks indicate identical bases. The core sequence of the transcription arrest site is boxed. The underlined bases have been shown to be important for full transcription arrest activity by point mutational analysis (30) .



In conclusion, we showed in a series of transfection assays that a 1-kb DNA fragment of the 5`-flanking sequence of flt-1 has functional activity in vascular endothelial cells but limited activity in epithelial cells, vascular smooth muscle cells, and fibroblasts. We also showed that the flt-1 CREB/ATF element was essential for basal transcription and the first intron of flt-1 negatively regulated gene expression.


FOOTNOTES

*
This work was supported in part by a fund provided by Daiichi Pharmaceuticals and National Institutes of Health Grant HL43821 (to L. T. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by 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 GenBank(TM)/EMBL Data Bank with accession number(s) D64016[GenBank].

§
Present address: Daiichi Pharmaceutical Co., Ltd. 1-16-13, Kita-Kasai, Edogawa-ku, Tokyo, 134, Japan.

Present address: Dept. of Medicine, University of Pittsburgh, Pittsburgh Cancer Institute, 211 Lothrop St., Pittsburgh, PA 15261-2592.

**
To whom correspondence should be addressed: University of California San Francisco, Cardiovascular Research Institute, 505 Parnassus Ave., L-1332, Box 0130, San Francisco, CA 94143-0130. Tel.: 415-476-4402; Fax: 415-476-0429.

(^1)
The abbreviations used are: Flt-1, fms-like receptor tyrosine kinase; Flk, fetal liver kinase; HUVEC, human umbilical vein endothelial cells; BAEC, bovine adrenal endothelial cells; HFF, human foreskin fibroblast; CAT, chloramphenicol acetyltransferase; CREB, cAMP response element binding protein; ATF, activating transcription factor; IBMX, 3-isobutyryl-1-methylxanthine; FBS, fetal bovine serum; kb, kilobase pairs; bp, base pairs; PIPES, 1,4-piperazinediethanesulfonic acid.


ACKNOWLEDGEMENTS

We thank for Dr. Izumi Saito for the gift of pAdex1w, EcoT22I-digested adenoviral DNA-terminal protein complex, and the recombinant adenovirus Adex1CAlacZ and the members of this laboratory for many helpful discussions. We thank Betty Cheung for her assistance in preparing this manuscript.


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