(Received for publication, May 18, 1995)
From the
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.
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 ()(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.
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
10
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
for 60 min at 30 °C. The protected products were analyzed on
denaturating 6% polyacrylamide gels.
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.
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.
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 CRE(-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 -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.
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) .
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-2 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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) D64016[GenBank].