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Circulation Research. 1996;79:277-285

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(Circulation Research. 1996;79:277-285.)
© 1996 American Heart Association, Inc.

Articles

Characterization of the Endothelium-Specific Murine Vascular Endothelial Growth Factor Receptor-2 (Flk-1) Promoter

Volker Ronicke, Werner Risau, Georg Breier

the Max-Planck-Institut fur Physiologische und Klinische Forschung, Bad Nauheim, Germany.

Correspondence to Dr Georg Breier, Max-Planck-Institut fur Physiologische und Klinische Forschung, Parkstr. 1, 61231 Bad Nauheim, Germany. E-mail gbreier@alpha.kerckhoff.mpg.de.


*    Abstract
up arrowTop
 *Abstract
down arrowIntroduction
 down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Flk-1, a high-affinity signaling receptor for vascular endothelial growth factor (VEGF), is strongly and specifically expressed on endothelial cells during embryonic development of the vascular system and during tumor angiogenesis. Disruption of Flk-1 gene function has recently been shown to prevent completely endothelial cell differentiation during murine embryonic development. To gain insights into the mechanisms that regulate the endothelium-specific Flk-1 expression, we have isolated the 5'-flanking region of the murine Flk-1 gene. RNase protection and primer extension analyses revealed a single transcriptional start site located 299 bp upstream from the translational start site in an initiator-like pyrimidine-rich sequence. The 5'-flanking region is rich in GC residues and lacks a typical TATA or CAAT box. A luciferase reporter construct containing a fragment from nucleotides -1900 to +299 showed strong endothelium-specific activity in transfected bovine aortic endothelial cells. Deletion analyses revealed that endothelium-specific Flk-1 expression is stimulated by the 5'-untranslated region of the first exon, which contains an activating element between nucleotides +137 and +299. In addition, two endothelium-specific negative regulatory elements were identified between nucleotides -4100 and -623. Two strong general activating elements were present in the region between nucleotides -96 and -37, which contains one potential NF{kappa}B and three potential AP-2 binding sites. This study shows that Flk-1 expression in endothelial cells is mainly regulated by an endothelium-specific activating element in the long 5'-untranslated region of the first exon and by negative regulatory elements located further upstream.


Key Words: endothelium • Flk-1 • mouse • promoter • vascular endothelial growth factor receptor


*    Introduction
up arrowTop
 up arrowAbstract
 *Introduction
down arrowMaterials and Methods
 down arrowResults
 down arrowDiscussion
 down arrowReferences
 
The endothelium is a highly specialized tissue that constitutes the inner lining of all blood vessels. It fulfills multiple functions; eg, it supplies every organ with oxygen and nutrients, forms a highly selective permeability barrier for substances that travel between tissues and the bloodstream, and has a prominent role during clotting and wound healing. During embryonic development, the endothelium arises from the primitive mesoderm. The formation of blood vessels is mainly accomplished by two processes, vasculogenesis and angiogenesis. Vasculogenesis, the differentiation of new vessels from angioblasts in situ, takes place during development of major vascular structures of the yolk sac and of the embryo, eg, the dorsal aorta and the heart.1 Angiogenesis, the sprouting of new capillaries from preexisting vessels, is the mechanism by which the brain and the spinal cord are vascularized.2 3 Angiogenesis is also important for vascularization of tumors, which enables continuous growth of solid tumors. It was postulated that this process, which is based on migration and proliferation of endothelial cells, is regulated by a paracrine mechanism.4 5

Several lines of evidence suggest that VEGF and its receptors are important regulators of both vasculogenesis and angiogenesis (for reviews, see Ferrara6 and Klagsbrun and Soker7 ). VEGF is a specific mitogen for endothelial cells8 and acts also as a potent vascular permeability factor (VPF).9 In the mouse, three isoforms of 120, 164, and 188 amino acids have been described,10 which are derived from a single gene by alternative splicing.11 The two smaller peptides form secreted dimers. Two high-affinity tyrosine kinase receptors for VEGF, which are exclusively expressed on endothelial cells, are known. VEGF receptor-1 is encoded by the fms-like tyrosine kinase (Flt-1) gene,12 13 and VEGF receptor-2 is encoded by the murine fetal liver kinase (Flk-1) gene14 and its human homologue, the kinase insert domain–containing (KDR) receptor.15 The observation that both receptors were already expressed in the primitive mesoderm at the early stages of vascular development suggested that they play an important role in the differentiation of endothelial cells from their precursors.13 14 16 This hypothesis was supported by recent gene-targeting experiments, which showed that the disruption of the Flt-1 gene locus led to a disorganized assembly of endothelial cells,17 whereas the inactivation of the Flk-1 gene completely abolished endothelial cell differentiation.18 This phenotype is consistent with the observation that Flk-1 is the earliest known marker for endothelial cell precursors in the mouse.14 16 Therefore, Flk-1 seems to be particularly important for the early stages of vascular development. The observation that the Flk-1 receptor was upregulated in human gliomas suggested that it may play a crucial role also in glioma angiogenesis.19 This hypothesis was confirmed by the observation that the inhibition of signal transduction by the Flk-1 receptor led to the inhibition of tumor growth and angiogenesis in a nude mouse model.20

To obtain more insight into the mechanisms that lead to differentiation and proliferation of endothelial cells, it is important to understand how the endothelium-specific expression of genes is controlled at the transcriptional level. Although some endothelium-specific promoters have been characterized, eg, promoters of the genes for von Willebrand factor,21 endothelin-1,22 E-selectin,23 Tie-2,24 vascular cell adhesion molecule-1,25 and endothelial NO synthase,26 these genes are neither specific for proliferating endothelium nor necessary for endothelial cell determination. Since the Flk-1 receptor shows both these properties, we decided to isolate the murine Flk-1 promoter and to identify cis-acting elements that are involved in transcriptional regulation of the Flk-1 gene.

In the present study, we isolated the 5'-flanking region of the murine Flk-1 gene and identified the transcriptional start site. Cis-acting elements were characterized in transient transfection assays. Regions from bp -96 to -72 and from bp -71 to -37 upstream from the transcriptional start site contained strong general activating elements. In this area, consensus sequences for AP-2 and NF{kappa}B binding sites were detected. Endothelium-specific expression was found to be regulated by the 5'-untranslated region of the first exon, which contained an endothelium-specific activating element, and by endothelium-specific negative regulatory elements in the further upstream region.


*    Materials and Methods
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 *Materials and Methods
down arrowResults
 down arrowDiscussion
 down arrowReferences
 
Library Screening and DNA Cloning
Five phage clones were isolated from a genomic library prepared from mouse strain 129/SvJ in {lambda}-DashII vector. The library was a gift from Drs Tom Doetschman and Marcia Shull. The screening was performed under high-stringency conditions using a 32P-labeled EcoRI–Sal I restriction fragment of an Flk-1 cDNA clone14 spanning nucleotides 1 to 540.27 Phages were analyzed by restriction mapping, and lower fragments were subcloned into pBluescript KS II plasmid (Stratagene). Sequencing was performed using an automatic sequencer (373A, Applied Biosystems). Both strands of a region from -623 to +457 bp relative to the transcriptional start site were sequenced. Luciferase reporter constructs were generated by inserting Flk-1 genomic fragments into pGL2-basic plasmid (Promega). The fragment ranging from -1.9 kbp to +136 bp was an EcoRI–Sac II subclone of P6, which was blunt-ended and inserted into the Nhe I site of the pGL2-basic vector. The construct containing a fragment from -4100 to +136 was generated by insertion of a HindIII-EcoRI fragment (-4.1 to -1.9 kbp) of P6 into the former plasmid. Flk-1 genomic fragments for all other luciferase reporter constructs were generated by PCR amplification using oligonucleotide primers containing either a Kpn I site or a HindIII site. A plasmid containing a 7-kbp Xba I–Xba I fragment of P6 was used as a template. DNA was amplified by PCR (25 cycles: 94°C, 1 minute; 60°C, 1 minute; 72°C, 1 minute or 2 minutes for fragments >1 kbp). Reaction products were purified from agarose gels, digested with Kpn I and HindIII, and subcloned into pGL2-basic vector cut with the same enzymes. The primers used were as follows: -1900, 5'-GGG GTA CCG AAT TCT AAA TGG GGC GAT TAC C-3'; -623, 5'-TCG GTA CCG ACC CAG CCA GGA AGT TC-3'; -258, 5'-ATG GTA CCC AGG TTG CTG GGG GCA G-3'; -96, 5'-CCG GTA CCT GGT ATC CAG TGG GGG G-3'; -71, 5'-GCG GTA CCG GAC GCA GGG AGT CCC C-3'; -36, 5'-TAG GTA CCC CGC CCC CAT TCG CTA G-3'; +299, 5'-TTG CTA AGC TTC CTG CAC CTC GCG CTG GG-3'; and +8, 5'-GAG GAA AGC TTC AGA AAG AGA GCG CCG GCT A-3'. All clones were verified by restriction enzyme digestion and DNA sequence analysis.

RNA Isolation, RT-PCR, and RNase Protection and Primer Extension Analyses
RNA isolation was performed using the guanidinium thiocyanate method.28 RT-PCR analysis was carried out as previously described29 except that each cycle consisted of the following: 94°C, 1 minute; 55°C, 1 minute; and 72°C, 1 minute. Flk-1 fragments were amplified using primers 5'-CGC TCT GTG GTT CTG CGT G-3' (+326 bp to +344 bp) and 5'-CAT CCG GAA CAA ATC TCT TTT C-3' (+804 bp to +825 bp, reverse). These primers span a genomic region that contains introns, which would result in larger amplification products in the case of contamination of RNA samples with genomic DNA. ß-Actin primers were as previously described.29 Reaction products were separated by agarose gel electrophoresis and analyzed by Southern blot hybridization. The probe used for Flk-1 detection was an EcoRI–Sal I cDNA fragment14 containing nucleotides 1 to 540.27

RNase protection experiments were carried out using an RNase protection kit (Boehringer Mannheim). The 32P-labeled riboprobe was generated by in vitro transcription of a deletion subclone of P6 containing an EcoRI–Nhe I fragment (-1850 to +322 bp) ligated to an Nhe I–EcoRI fragment (+1300 to +1400 bp). The plasmid was linearized with HindIII, and single-stranded RNA was generated in a 20-µL reaction containing 1 µg template DNA, 7 µL [32P]UTP (Amersham, 800 Ci/mmol), 2 µL 5 mmol/L ATP/CTP/GTP, 1 µL of 200 mmol/L dithiothreitol, 0.8 µL RNAguard (Pharmacia), and 1 U T3 RNA polymerase (Stratagene) in 1x transcription buffer at 37°C for 1 hour. The probe was extracted with phenol/chloroform/isoamyl alcohol, precipitated with ethanol, and purified on a denaturating 6% polyacrylamide gel. Radioactive probe (3x105 cpm) was used for each RNase protection reaction with samples of total bEnd5 RNA (30 µg), total 14.5-day mouse embryo RNA (50 µg), or tRNA (50 µg). The following controls were performed: undigested probe, RNase digestion of the probe without cellular RNA, and digestion after incubation with a sense in vitro transcript of a Flk-1 cDNA subclone (nucleotides 1 to 540). In all cases, the expected results were obtained (data not shown). 32P-labeled Msp I restriction fragments of pBluescript were used as size marker.

Primer extension analysis was performed using 0.6 ng 32P-labeled oligonucleotide (5'-TCA GTC CTG CCG GGT AGC -3', nucleotides +65 to +82). Primer annealing was performed in a 10-µL reaction containing 50 µg of total RNA, 100 mmol/L NaCl, 20 mmol/L Tris-Cl (pH 8.3), and 0.1 mmol/L EDTA. After incubating at 90°C for 3 minutes, the reaction was transferred to a water bath at 60°C for 10 minutes and then slowly cooled to room temperature. Four microliters of 5x first-strand-buffer (GIBCO/BRL), 1 µL of 10 mmol/L dATP/dCTP/dGTP/dTTP, 2 µL of 0.1 mol/L dithiothreitol, 1 µL RNAguard (Pharmacia), and 1 µL Superscript reverse transcriptase (GIBCO/BRL) were added to a final volume of 20 µL. The reaction was incubated for 1 hour at 37°C and was stopped by adding 1 µL of 0.5 mol/L EDTA. Samples were digested with 1 U RNase A (Boehringer Mannheim) at 37°C for 30 minutes, extracted with phenol/chloroform/isoamyl alcohol, precipitated with ethanol, and separated on a denaturating 6% polyacrylamide gel. A dideoxy sequencing reaction30 of a 7-kbp Xba I–Xba I fragment (-6550 to +457 bp) of P6 performed using the same primer was included as a size marker.

Tissue Culture and Transient Transfections
All cells were cultured in DMEM+ supplemented with 10% FCS (Sigma Chemical Co). bEnd5 cells were generated by transformation with the polyoma middle-T oncogene as described earlier.31 BAECs were prepared as previously described.32 NIH 3T3, C2C12, and L cells were obtained from American Type Culture Collection. Transient transfections were performed using the CaPO4 precipitation method according to Chen and Okayama (1987).33 Each construct was transfected at least six times in three independent experiments. Cells were grown to 70% confluence in 6-cm dishes before transfection. Cells were washed 16 hours after addition of CaPO4 precipitate and incubated for a further 48 hours. In each experiment, 6 µg luciferase reporter construct and 1 µg pCMV5LacZ were used. The pCMV5LacZ contained the bacterial ß-galactosidase gene inserted into the pCMV5 plasmid.34 The plasmid was kindly provided by Dr U. Deutsch. Cells were washed two times with ice-cold PBS, collected in 1 mL PBS, and stored on ice. Cells were lysed in 1x reporter-lysis buffer (Promega) for 15 minutes on a test tube rotator. After centrifugation, the supernatant was transferred to a fresh tube and stored at -80°C or taken for luciferase and lacZ assay immediately.

Reporter gene assays for ß-galactosidase activity were performed according to Eustice et al (1991).35 Chlorophenol red/ß-D-galactopyranoside was used as a substrate, and the conversion was measured at 575 nm in an enzyme-linked immunosorbent assay reader (Biometra). Extracts were diluted to obtain values at an optical density of 575 nm between 0.2 and 0.8. These values were used to standardize for transfection efficiency after subtracting a background value, as determined from a cell extract of a transfection without lacZ reporter plasmid but with a luciferase reporter plasmid. Luciferase reporter gene assays were performed with the same extracts as described by the manufacturer (Promega). Luciferase activity was measured with a luminometer (LB96P, Berthold) and calculated as percentage of the activity of the pGL2 promoter plasmid (Promega).


*    Results
up arrowTop
 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 *Results
down arrowDiscussion
 down arrowReferences
 
Cloning of the 5' Region of the Murine Flk-1 Gene
To isolate the 5'-flanking region of the murine Flk-1 gene, a genomic DNA library prepared from 129/SvJ mice was screened using an Flk-1 cDNA-fragment,14 comprising nucleotides 1 to 540,27 as a probe. Five independent phage clones were isolated. Two phages, P6 and P16, contained overlapping inserts and were analyzed by restriction enzyme mapping. Subcloning and sequencing of restriction fragments revealed that the inserts spanned 32 kbp of the genomic Flk-1 locus (Fig 1Down). The DNA insert of P6 contained 15 kbp of upstream sequence, the first exon and a 2.5-kbp part of the 3.8-kbp first intron. P16 included 2 kbp of the first intron, the 94-bp second exon, and the 14-kbp downstream sequence. To exclude the possibility of recombination artifacts, genomic DNA isolated from the same mouse strain was analyzed by Southern blot hybridization and showed identical restriction fragment sizes (data not shown). A 7-kbp Xba I–Xba I fragment that contained the first exon and 6.5-kbp 5'-flanking sequence was subcloned and used for further analysis.



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  		Figure 1. Structure of the 5' region of the murine Flk-1 gene. Restriction map of a 21-kbp region of the Flk-1 gene is shown. The first and the second exons are represented as hatched boxes and are separated by the 3.8-kbp first intron. The genomic DNA inserts of two {lambda} phages, P6 and P16, are shown on the top. P16 extends 12 kbp further downstream, which is not shown. A 7-kbp Xba I–Xba I subclone of P6 containing the first exon was further analyzed and is depicted on the bottom. B indicates BamHI; E, EcoRI; H, HindIII; P, Pst I; and X, Xba I.

Sequence Analysis of the Murine Flk-1 Gene
The DNA sequence of a 1080-bp stretch of the Flk-1 upstream region was determined. A comparison with the cDNA sequence showed that this DNA contained the first exon, including the first ATG codon of the protein-coding region, part of the first intron, and {approx}600 bp of the 5'-flanking region (Fig 2Down). The coding sequences of the first and the second exons (Fig 2Down and data not shown) were identical to the corresponding part of the published cDNA sequence.14 27 The sequence of the 5'-untranslated region differed at positions 2, 18, 20, and 258 (base exchanges) and at positions 53, 83, 183, and 246 (insertion of one additional G) from the longest published cDNA.36 The 5'-flanking sequence did not contain a TATA box or a CAAT element. The upstream sequence was rich in GC residues and contained four potential Sp1-binding sites,37 four potential AP-2–binding sites,38 39 one potential NF{kappa}B-binding site,40 41 42 and five E-box motifs.43 Two additional AP-2 consensus sequences, a potential Krox-24–binding site,44 and an atypical GATA element (GGATAA instead of A/TGATAG/A)45 46 were present in the 5'-untranslated region of the first exon. Another consensus sequence for an Sp1-binding site was identified in the first intron.



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  		Figure 2. Nucleotide sequence of the 5' region of the murine Flk-1 gene. The nucleotide sequence of a 1080-bp region, containing the first exon with the 5'-untranslated region and part of the first intron, is indicated. The transcriptional start site is marked as +1. The translational start site is located at position +300. The amino acid sequence encoded by the first exon is shown in single-letter code under the corresponding nucleotide sequence. The asterisk marks the beginning of the first intron. Potential binding sites for transcription factors are underlined.

Determination of the Transcriptional Start Site of the Murine Flk-1 Gene
The transcriptional start site was determined by RNase protection experiments and primer extension analysis. First, it was investigated whether the endothelioma cell line, bEnd5, expressed sufficient amounts of Flk-1 mRNA for this analysis. Flk-1 mRNA expression was analyzed by a semiquantitative RT-PCR using Flk-1 oligonucleotide primers, which led to the amplification of a 500-bp fragment. Amplification of genomic DNA would lead to larger products, since the corresponding region of the Flk-1 gene includes introns. ß-Actin oligonucleotide primers were used in the same reaction to standardize for mRNA quantity. A Southern blot of the RT-PCR is shown in Fig 3Down. The reaction with the bEnd5 sample showed a strong signal. The lowest amount of Flk-1 mRNA was detected in 14.5-day total embryo RNA, which reflects the fact that Flk-1 expression is restricted to endothelial cells in the mouse embryo.14 The strongest Flk-1 signal was observed with a stable Flk-1–expressing 3T3 cell line,20 which served as a positive control. No Flk-1 transcript was detectable in the negative-control untransfected 3T3 cells.



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  		Figure 3. Expression of Flk-1 in endothelioma cells and in mouse embryos. A Southern blot of RT-PCR products was hybridized with probes specific for Flk-1 and ß-actin. Total RNA samples (1 µg) derived from bEnd5 cells, 14.5-day embryos, stably transfected Flk-1–expressing 3T3 cells, and normal 3T3 cells were used. H2O was used as a negative control instead of cDNA. The size of the expected amplification product for Flk-1 was 500 bp. PCR products were separated by gel electrophoresis, transferred to nylon membrane, and hybridized with a 32P-labeled cDNA probe for Flk-1 (bp 1 to 540) or ß-actin.

RNase protection analysis of RNA samples from the same sources was performed to identify the 5' end of the mRNA in the first exon. Total RNA samples from bEnd5 cells or from 14.5-day embryos were incubated with an antisense RNA probe generated from a 2.2-kbp EcoRI–Nhe I subclone, which contained part of the first exon and 5'-flanking sequence (-1850 to +322 bp). A 322-bp fragment was protected in bEnd5 cells, indicating that the transcriptional start site is located 299 nucleotides 5' of the ATG start codon (Fig 4Down, left). A protected fragment of the same size was also seen in the reaction with the embryo RNA but not in the negative control (tRNA). The weaker signal obtained with the 14.5-day embryo RNA reflected the lower amount of Flk-1 mRNA, which is consistent with the RT-PCR analysis (Fig 3Up). The result of the RNase protection analysis indicated a total length of 366 bp for the first exon. The 5' end of the first exon has the same position in cultured endothelioma cells and in endothelial cells in vivo and will be numbered as +1.




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  		Figure 4. Identification of the transcriptional start site of the murine Flk-1 gene. The transcriptional start site was mapped by RNase protection and primer extension experiments. Left, RNase protection analysis. Total RNA samples from bEnd5 cells (30 µg), 14.5-day embryos (50 µg), or tRNA (50 µg) were incubated with 32P-labeled antisense RNA probe generated from an Nhe I–EcoRI (from bp -1850 to +322) genomic subclone of P6. The protected fragments were separated on a denaturating 6% polyacrylamide gel. The arrow indicates the size of the protected fragment. The size marker was a 32P-labeled Msp I digest of pBluescript. The gel was exposed for 48 hours. The reaction of 14.5-day embryo RNA was exposed for 1 week. Right, Primer extension analysis. Total RNA (50 µg) from 14.5-day mouse embryos was incubated with a 32P-labeled antisense oligonucleotide primer spanning the region from nucleotides +82 to +65. tRNA or 3T3 RNA was used as a template in negative control reactions. RT reaction was performed, and the 32P-labeled products were separated on a denaturating 6% polyacrylamide gel. A DNA sequencing reaction of a 7-kbp Xba I–Xba I subclone of P6 (-6550 to +457 bp) generated using the same primer is shown in the left four lanes. The nucleotide sequence surrounding the start site is depicted at the left side. The arrow marks the transcriptional start site. The additional band seen immediately below could be due to premature termination of the RT reaction because of methylated nucleotides in the cap structure, or it could represent a second transcriptional start site.

To investigate whether an additional untranslated exon existed upstream from the ATG-containing exon, primer extension analyses were performed. A radiolabeled antisense oligonucleotide spanning nucleotides +82 to +65 was used as a primer. Total RNA samples isolated from 14.5-day mouse embryos were analyzed. RNA from 3T3 cells and tRNA served as negative controls. Two major extension products were obtained with 14.5-day embryo RNA (Fig 4Up, right). The longer extension product ended at a T residue included in the pyrimidine-rich sequence, CTCTCTTTC, which showed a high degree of homology with the weak consensus sequence for initiator elements (PyPyAT/APyPy).47 The additional signal immediately below the mentioned band might be due to a premature termination of the RT reaction, possibly because of methylation of nucleotides near the cap structure.48 Alternatively, it could represent a second transcriptional start site at the C residue directly downstream from the mentioned T. The weaker signals in this lane were unspecific, because they were also seen in the reaction with 3T3 RNA, which lacks any detectable Flk-1 mRNA. This result demonstrated that the ATG-containing exon is the first exon and confirmed the localization of the transcriptional start site, as determined by RNase protection.

Functional Analysis of the Murine Flk-1 Promoter In Vitro
To investigate whether the Flk-1 promoter was located in the characterized 5'-flanking region, transient transfection assays using luciferase reporter gene constructs were performed in BAECs, which are known to express the bovine homologue of Flk-1.49 3T3 cells were used as a control to determine whether the promoter activity was endothelium specific. The transfection efficiency was standardized by using an expression vector containing the ß-galactosidase gene under the control of the cytomegalovirus promoter/enhancer (pCMV5LacZ).

The activity in BAECs of an Flk-1 promoter fragment ranging from -1.9 kbp to the translational start site at nucleotide +300 (126±35%) was stronger than the activity of the SV40 promoter (100%) (Fig 5ADown). In contrast, the Flk-1 promoter activity in 3T3 cells (22±7%) was considerably lower compared with the SV40 promoter activity.




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  		Figure 5. Functional analysis of the murine Flk-1 promoter in vitro. A, Endothelium-specific promoter activity of the Flk-1 5'-region. The graph shows the results of transient transfection assays in BAECs or 3T3 cells of an Flk-1 promoter fragment from -1.9 kbp to +299 bp contained in pGL2-basic luciferase vector (Promega). Values represent percentage of the luciferase activity of the pGL2 promoter vector, which contains a luciferase reporter gene driven by the SV40 promoter. The transfection efficiency was determined by cotransfection of a pCMV5LacZ reporter construct. B, Potential transcription factor–binding sites in the murine Flk-1 promoter. The transcriptional start site is indicated by an arrow. Positions of various deletion constructs used for reporter gene analysis are indicated at the top. C and D, 5'-Deletion analysis (C) and 3'-deletion analysis (D) of the murine Flk-1 promoter. Constructs in pGL2-basic vector were generated as described in "Material and Methods." These constructs were transfected into BAECs and 3T3 cells. Transfection efficiency was calculated by cotransfection of pCMV5LacZ.

In order to identify and characterize functional sequence elements of the Flk-1 promoter, a deletion analysis of the 5'-flanking region was performed (Fig 5CUp). Promoter activity was standardized against SV40 promoter activity. All constructs investigated showed stronger promoter activity in endothelial cells than in 3T3 cells. Deletion of the DNA fragment from -1.9 kbp to -624 bp led to an increase of promoter activity by 60% in BAECs (from 126±35% to 203±69%, P=.017) and by 120% in 3T3 cells (from 22±7% to 50±10%, P=.0002). Further deletion of the upstream sequence to nucleotide -96 had only a minor effect on the promoter activity. Deletion of the fragment between bp -96 and -72 led to an almost 30% decrease of promoter activity in both cell types (BAECs, from 171±40% to 116±36%; 3T3 cells, from 42±7% to 30±4%). An additional deletion of the upstream region to bp -36 decreased the promoter activity to 10±4% of the SV40 promoter in BAECs and to 3±0.5% in 3T3 cells.

These results revealed the presence of a silencing element between bp -1900 and -623. This element also controlled endothelium specificity, because deletion of this region led to a decrease of the ratio of promoter activity in BAECs relative to 3T3 cells from 5.7 to 4.1 (TableDown); ie, this element repressed the Flk-1 promoter in nonendothelial cells stronger than in endothelial cells. The further deletion to bp -96, of a fragment including all potential E-box motifs and three potential Sp1-binding sites, decreased the promoter activity only weakly (from 203±69% to 171±40% in BAECs and from 50±10% to 42±7% in 3T3 cells). However, the deletion of an additional 25-bp fragment to nucleotide -71, eliminating one potential AP-2–binding site, decreased promoter activity in both cell types. A further deletion of a fragment to bp -36, which contains one potential NF{kappa}B and two potential AP-2 sites, reduced the promoter activity in both cell types to a very low level.


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  		Table 1. Ratio of Flk-1 Promoter Activity in BAECs to Activity in 3T3 Cells

To determine whether regulatory elements downstream from the transcriptional start site have a function in promoter activity, a deletion analysis of the 5'-untranslated region of the first exon was performed. Fig 5DUp shows the results of this experiment. The deletion of a fragment ranging from bp +137 to +299 led to a reduction of promoter activity by 63% in BAECs (from 126±35% to 46±16%) but only by 36% in 3T3 cells (from 22±7% to 14±4%). Further deletion at the 3' end to bp +8 showed an increase in promoter activity (from 46±16% to 78±22% in BAECs and from 14±4% to 18±5% in 3T3 cells). These results showed the presence of a positive regulatory element between bp +136 and +299 and of a negative regulatory element between bp +136 and +8. The deletion of the 5'-untranslated part of the first exon had stronger effects in BAECs, indicating a role of these elements in endothelium-specific gene expression. The GATA motif in the region between bp +8 and +136 (Fig 2Up) showed no positive regulatory function in this assay (Fig 5DUp). The fragment from bp +137 to +299 contained two possible AP-2–binding sites and one possible Krox-24–binding site, which could play a role in promoter activity.

The analysis of a construct ranging from -4.1 kbp to +136 bp showed a weaker promoter activity than the construct from -1.9 kbp to +136 bp. The reduction of the promoter activity by insertion of the 5' region from -1.9 to -4.1 kbp was stronger in 3T3 cells than in BAECs (from 14±4% to 4±1% in 3T3 cells and from 46±16% to 31±9% in BAECs). This increased the ratio of the promoter activity in BAECs to 3T3 cells from 3.3 to 7.8 (TableUp), revealing another endothelium-specific element located in a relative large distance upstream of the transcriptional start site.

Analysis of an Flk-1 promoter construct from -623 to +299 bp in different cell lines further confirmed endothelium specificity (Fig 6Down). This construct showed clearly stronger promoter activity in BAECs (203±69%) than in 3T3 cells (50±10%), L cells (55±9%), or C2C12 myoblastic cells (59±18%) relative to the SV40 promoter.



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  		Figure 6. Comparison of Flk-1 promoter activity in different cell lines. A pGL2-basic luciferase vector (Promega) containing an Flk-1 promoter fragment from -623 to +299 bp was transfected in different cell lines (BAECs, 3T3, L, and C2C12 cells). Transfection efficiency was standardized by cotransfection of a pCMV5LacZ reporter construct.


*    Discussion
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 up arrowAbstract
 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
The endothelial Flk-1 receptor is essential for the formation of the vascular system during embryonic development18 and for tumor angiogenesis.20 The cloning and characterization of the murine Flk-1 promoter provides the opportunity to study the mechanisms of endothelium-specific gene transcription.

We show in the present study that the 5'-flanking sequence of the Flk-1 gene is GC rich and lacks a typical CAAT box and a TATA box, which is responsible for accurate transcriptional initiation of many genes. Nevertheless, RNase protection and primer extension analysis revealed a single transcriptional start site, which lies in a pyrimidine-rich sequence, CTCTCTTTCT. This region has similarity with an initiator element (PyPyAT/APyPy).47 An initiator element, first described for the terminal desoxynucleotide transferase gene, is known to determine the transcriptional start site in promoters that lack a TATA box.50 Interestingly, there is a potential Sp1-binding site 30 bp upstream from the transcriptional start site of the Flk-1 gene. A similar combination of an initiator element and an Sp1-binding site was found in the cell type–specific and developmentally regulated promoter of the {alpha}2-integrin gene and seems to be generally required for the function of initiator elements.47 51 52 This structural similarity further supports the assumption that the pyrimidine-rich sequence surrounding the transcriptional start site represents a functional initiator element of the Flk-1 gene. Transient transfection assays of Flk-1 promoter fragments in BAECs showed that a fragment between nucleotides -36 and +8 contains a functional basal promoter.

The Flk-1 promoter shows a complex composition of positive and negative regulatory elements. The 5'-flanking sequence contains at least four cis-acting elements: two strong activating elements in the regions from nucleotides -96 to -72 and from nucleotides -71 and -37, and two negative regulatory elements in the regions between -4.1 and -1.9 kbp and between -1.9 kbp and -623 bp. A comparison of expression levels in BAECs and in NIH 3T3 cells showed that the two positive-acting elements do not mediate endothelium specificity. The sequence of the stronger activating element, located between bp -71 and -36, contains two potential AP-2–binding sites 38 and one NF{kappa}B-binding site.41 The NF{kappa}B family of transcription factors comprises ubiquitously expressed DNA-binding proteins, which are present in an inactive form in the cytoplasm and translocate to the nucleus after activation. Several genes expressed in endothelial cells, like E-selectin,53 54 vascular cell adhesion molecule-1,25 mucosal vascular addressin cell adhesion molecule-1,55 and tissue factor,56 also contain NF{kappa}B-binding sites in their promoters. These genes are inducible by the cytokine TNF{alpha}. The TNF{alpha}-dependent induction is thought to be mediated by NF{kappa}B. Whether Flk-1 expression is also induced by TNF{alpha} via NF{kappa}B binding to the Flk-1 promoter remains to be determined. In any case, this is unlikely to contribute to endothelium-specific expression.

Endothelium-specific Flk-1 expression is controlled by a positive regulatory element in the 5'-untranslated region of the first exon and two negative regulatory elements, located between nucleotides -4100 and -1900 and between nucleotides -1900 and -623, which repress transcription in nonendothelial cells stronger than in endothelial cells. The 299-bp 5'-untranslated region of the first exon contained an endothelium-specific activating element in the region from nucleotide +137 to +299 and a general negative regulatory element in the region from nucleotide +8 to +136. Similarly, the promoter of the human von Willebrand factor gene contains cis-acting regulatory elements in the 5'-untranslated region, including a GATA motif, which is necessary for endothelium-specific expression.21 The GATA motif was first identified in the erythroid-specific promoter of chicken ß-globin gene and is a binding site for a class of transcription factors.45 46 In contrast, the imperfect GATA motif (GGATAA instead of A/TGATAG/A) at position bp +112 in the Flk-1 promoter showed no detectable activating function in vitro. The organization of cis-acting elements in the 5'-untranslated region of the Flk-1 gene resembles the composition of the erythroid-specific human erythropoietin promoter.57 This promoter bears an erythroid-specific cis-acting element composed of a positive regulatory element in the region from nucleotide +1 to nucleotide +18 and a negative regulatory element between nucleotides +79 and +135. The positive endothelium-specific regulatory region of the Flk-1 promoter between nucleotides +136 and +299 shows consensus binding sites for AP-2 and Krox-24.44 Krox-24 is strongly expressed in brain and thymus but is also detectable in most other organs. It is upregulated in tissue culture after serum stimulation as an immediate-early gene.58 59 Whether these transcription factors represent functional regulators of Flk-1 gene expression remains to be determined, eg, by footprinting analyses.

After submission of the present article, the characterization of the human VEGF receptor-2 promoter was reported, which contains positive endothelium-specific cis-acting elements located in the near 5'-upstream region and a general activating element in the 5'-untranslated region.60 The murine Flk-1 promoter, in contrast, bears two negative regulatory endothelium-specific cis-acting elements in the far upstream region and an endothelium-specific positive cis-acting element in the 5'-untranslated region. Based on these observations, it is evident that at least the organization of endothelium-specific regulatory elements is dissimilar in mouse and human. Whether endothelium-specific expression in both species is mediated by similar regulatory elements remains to be investigated. Our preliminary transgenic mouse experiments showed that sequences between -623 bp and -4.1 kbp, which we have demonstrated to contribute to endothelial specificity in vitro, are necessary for directing transgene expression into the vasculature; however, additional sequences seem to be required in order to obtain the complete expression pattern of the endogenous Flk-1 gene. The availability of the murine Flk-1 promoter will be a valuable tool for studying the mechanisms of endothelial cell determination during embryonic development.


*    Selected Abbreviations and Acronyms
 
BAEC = bovine aortic endothelial cell
PCR = polymerase chain reaction
RT = reverse transcriptase
TNF = tumor necrosis factor
VEGF = vascular endothelial growth factor
VPF = vascular permeability factor


*    Acknowledgments
 
We thank Drs T. Doetschman and M. Shull for providing the genomic library of mouse strain 129/SvJ; Dr U. Deutsch for providing the pCMV5LacZ plasmid; Dr S. Wizigmann-Voos for characterization of the bEnd5 cells; M. Fahrig, S. Hennig, S. Pebler, and M. v. Reutern for excellent sequencing service; and U. Hofmann for technical assistance.

Received February 7, 1996; accepted May 15, 1996.


*    References
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