(Received for publication, April 19, 1995; and in revised form, July 21, 1995)
From the
KDR/flk-1 is one of two receptors for vascular endothelial
growth factor, a potent angiogenic peptide. KDR/flk-1 is an early
marker for endothelial cell progenitors, and its expression is
restricted to endothelial cells in vivo. To investigate the
molecular mechanisms regulating expression of KDR/flk-1, we cloned and
characterized the promoter of the human KDR/flk-1 gene. The
transcription start site was localized by primer extension and
ribonuclease protection to a nucleotide 303 base pairs (bp) 5` of the
initiation methionine codon. The 5`-flanking sequence is rich in G and
C residues and contains five Sp1 elements but no TATA consensus
sequence. By reporter gene transfection experiments, we found that
4 kilobases of KDR/flk-1 5`-flanking sequence directed high level
luciferase activity in bovine aortic endothelial cells; further
deletion analysis revealed positive regulatory elements between bp
-225 to -164, -95 to -77, -77 to
-60, and +105 to +127. Mutation of an atypical GATA
sequence between bp +105 and +127 did not affect promoter
activity, suggesting that GATA elements are not essential for the high
level promoter activity of this gene. Consistent with endothelial
cell-restricted expression of KDR/flk-1 mRNA, we found that the
4-kilobase flanking sequence directed high level promoter activity in
endothelial cells but not in other cell types. To our knowledge this is
the first report characterizing the KDR/flk-1 promoter. Understanding
the KDR/flk-1 promoter will allow us to investigate endothelial
cell-specific gene regulation and to uncover methods for targeting gene
delivery specifically to endothelial cells.
Vascular endothelial growth factor (VEGF) ()is a
potent and specific endothelial cell mitogen(1, 2) .
Through interactions with its receptors KDR/flk-1 and flt1, VEGF has
critical roles in the growth and maintenance of vascular endothelial
cells and in the development of new blood vessels in physiologic and
pathologic states(3, 4, 5) . The patterns of
embryonic expression of VEGF suggest that it is crucial for
differentiation of endothelial cells from hemangioblasts and for
development of blood vessels at all stages of
growth(6, 7) . Among many potentially angiogenic
factors, VEGF is the only one whose pattern of expression, secretion,
and activity suggests a specific angiogenic function in normal
development(8) .
High affinity receptors for VEGF are found only on endothelial cells, and VEGF binding has been demonstrated on macro- and microvascular endothelial cells and in quiescent and proliferating endothelial cells, suggesting that these receptors are important for both growth and maintenance of all endothelial cells(6, 9) . The tyrosine kinases KDR/flk-1 and flt1 have been identified as candidate VEGF receptors by affinity cross-linking and competition binding assays(10, 11, 12) . These two receptor tyrosine kinases contain seven similar extracellular immunoglobulin domains and a conserved intracellular tyrosine kinase domain interrupted by a kinase insert(10, 13, 14) ; they are expressed specifically by endothelial cells in vivo(11, 15, 16, 17) . In situ hybridization in the developing mouse has demonstrated that KDR/flk-1 is expressed in endothelial cells at all stages of development, as well as in the blood islands in which endothelial cell precursors first appear(11) , and that KDR/flk-1 specifies endothelial cell precursors at their earliest stages of development(17) .
The vascular endothelium is critical for physiologic responses including thrombosis and thrombolysis, lymphocyte and macrophage homing, modulation of the immune response, and regulation of vascular tone. The endothelium is also intimately involved in the pathogenesis of vascular diseases such as atherosclerosis(18) . Although a number of genes expressed in the endothelium have been characterized(19, 20, 21, 22) , expression of these genes is either not limited to vascular endothelium (e.g. the genes encoding von Willebrand factor, endothelin-1, vascular cell adhesion molecule-1, platelet/endothelial cell adhesion molecule-1) or is restricted to specific subpopulations of endothelial cells (e.g. the gene for endothelial-leukocyte adhesion molecule-1). (A fragment of the promoter for Tek/Tie2, another developmentally regulated endothelial cell receptor tyrosine kinase, has recently been shown to direct transgene expression in subpopulations of endothelial cells during mouse embryonic development but not in endothelial cells of adult mice(23) . This suggests that the Tek/Tie2 promoter fragment used in this study is sufficient to direct gene expression to subpopulations of endothelial cells during specific periods of development, although functional elements within this promoter have not yet been identified.) In contrast with cells derived from the skeletal muscle and hematopoietic lineages, little is known about the mechanisms of specification and differentiation of endothelial cells. To understand the molecular mechanisms regulating cell type specificity and activation of the differentiation pathway in endothelial cells, we studied the control of KDR/flk-1 transcription.
As a first step we cloned and characterized the promoter for the human KDR/flk-1 gene. We report here the sequence of the 5`-flanking region of the gene and identify a single transcription start site located 303 bp 5` of the initiation methionine codon. Four kilobases of KDR/flk-1 5`-flanking sequence were found to have promoter activity similar to that of the potent SV40 promoter/enhancer in reporter gene transfection experiments in endothelial cells. Deletion analysis in endothelial cells showed the presence of positive regulatory elements in regions from bp -225 to -164, -95 to -77, -77 to -60, and +105 to +127. We found that KDR/flk-1 mRNA was expressed specifically in endothelial cells in culture and that 4 kb of the KDR/flk-1 5`-flanking sequence had cell type-specific promoter activity in transient transfection assays.
Figure 1: KDR/flk-1 promoter sequences. Panel A, restriction map and nucleotide sequence of the human promoter. Nucleotide sequences are numbered on the left. The transcription start site is indicated by a curved arrow. Potential cis-acting elements are underlined and discussed under ``Results.'' PstI sites used to generate the riboprobe are double underlined, and the oligonucleotide used for primer extension is underlined with an arrow. Panel B, restriction map and nucleotide sequence of the mouse promoter. Nucleotide sequences and potential cis-acting elements are indicated as above. An asterisk marks the end of the cDNA.
Figure 2:
Identification of transcription start
site. Panel A, primer extension analysis of the KDR/flk-1
transcription start site. The oligonucleotide underlined with
an arrow in Fig. 1A was hybridized to 20
µg of total RNA from HUVEC and HeLa cells or 3 µg of poly(A)
HUVEC RNA and yeast tRNA. Extension products were analyzed on an 8%
polyacrylamide gel. A Sanger sequencing reaction primed on a plasmid
DNA template (with the same primer) was run next to it. Panel
B, strategy for mapping the transcription start site of the
KDR/flk-1 gene by ribonuclease protection. The predicted length of the
protected fragment based on the results of primer extension is 145 bp. Panel C, ribonuclease protection analysis of the KDR/flk-1
transcription start site. Total RNA from HUVEC and HeLa cells or
poly(A) HUVEC RNA and yeast tRNA was incubated with a 559-bp P-labeled riboprobe spanning the immediate 5` region of
the human KDR/flk-1 gene, and the annealing products were digested with
RNase. The size marker (bp) was prepared by radiolabeling
X174
replicative form DNA digested with HaeIII. Protected fragments
were analyzed on a 4% polyacrylamide gel. The arrow denotes a
single protected fragment of approximately 145 bp which is observed
only in endothelial cells, confirming the results of primer
extension.
Reporter constructs containing fragments of the human KDR/flk-1 5`-flanking region were inserted into pGL2 Basic and named according to the length of the fragment (from the transcription start site) in the 5` and 3` directions. (For example, plasmid pGL2-4kb+296 contained a human KDR/flk-1 promoter fragment extending from approximately -4 kb 5` of the transcription start site to position +296 inserted into pGL2 Basic.) Plasmids pGL2-4kb+296 and pGL2-900+296 were created by restriction digestion of purified phage DNA by using 5` BamHI and PvuII sites, respectively, and the 3` XhoI site at position +296. Plasmids pGL2-716+268, pGL2-570+268, pGL2-323+268, pGL2-225+268, pGL2-164+268, pGL2-37+268, pGL2-225+127, pGL2-225+105, pGL2-225+56, and pGL2-225+5 were created from promoter fragments generated by PCR of human KDR/flk-1 phage DNA. Plasmids pGL2-116+268, pGL2-95+268, pGL2-77+268, pGL2-60+268, and pGL2-12+268 were created by digesting the promoter fragment contained in plasmid pGL2-164+268 from the 5` end with exonuclease III (Pharmacia Biotech Inc.). Plasmid pGL2 GATA-MUT was identical to pGL2-225+268 except that bp +108 to +110 were mutated in the former (see below). All constructs were sequenced from the 5` and 3` ends to confirm orientation and sequence.
The ratio of luciferase activity to
-galactosidase activity in each sample served as a measure of the
normalized luciferase activity. The normalized luciferase activity was
divided by that of pGL2 Control and expressed as relative luciferase
activity. Each construct was transfected at least six times, and data
for each construct are presented as the mean ± S.E. Relative
luciferase activity among constructs was compared by a factorial
analysis of variance followed by Fisher's least significant
difference test. Statistical significance was accepted at p < 0.05.
To confirm the results of the primer extension studies, we performed ribonuclease protection analysis with an antisense riboprobe generated from a 559-bp genomic PstI-PstI fragment extending 5` from position +145 (Fig. 2B; the PstI sites are double underlined in Fig. 1A). Incubation of this probe with HUVEC poly(A) RNA and HUVEC total RNA, but not with total RNA from HeLa cells, resulted in protection of a single fragment corresponding in length to the distance between the 3` PstI site and the transcription start site identified by primer extension (Fig. 2C). Despite the absence of a TATA consensus sequence, transcription of the human KDR/flk-1 gene appears to begin from a single site located 303 bp 5` of the translation initiation codon (Fig. 1A, curved arrow).
We also compared the human and mouse KDR/flk-1 promoters
to identify conserved consensus sequences for nuclear proteins (Fig. 1B). Elements conserved between the two species
include two Sp1 sites located at positions -244 and -124
relative to the 5` end of the reported mouse cDNA
sequence(13) , two AP-2 sites at positions -168 and
-148, a noninverted NFB site at position -153, and the
keratinocyte element AAACCAAA at position -195. An atypical GATA
element (GGATAA) is found in the untranslated portion of the first exon
of the mouse promoter at position +18; an atypical GATA element
(GGATAT) is located similarly in the human promoter. Also, a CANNTG
sequence is present 12 bp 5` of the G- and C-rich sequences of the
promoter at mouse KDR/flk-1 position -257, a location analogous
to that of the CANNTG element at position -175 of the human
promoter. Conservation of these elements across species suggests that
some may have functional significance.
Figure 3:
5` deletion analysis of the KDR/flk-1
promoter in BAEC. Panel A, representation of deletion sites in
relation to consensus sequences for known nuclear proteins are
discussed under ``Results.'' Panel B, functional
analysis of the human KDR/flk-1 promoter by transfection into BAEC of
luciferase reporter constructs containing serial 5` deletions.
Constructs are described in the text. All constructs were cotransfected
with pSVgal to correct for transfection efficiency, and luciferase
activity was expressed as a percentage of pGL2 Control (mean ±
S.E.). Significant differences are noted under
``Results.''
To determine whether sequences in the first exon of human KDR/flk-1 are important for basal expression, we created a series of 3` deletion constructs from the vector pGL2-225+268, which is the smallest construct that possessed full promoter activity (Fig. 4). A fragment was identified between bp +105 and +127 which, when deleted, caused a 5-fold reduction in promoter activity (p < 0.05), indicating the presence of a positive regulatory element in this region.
Figure 4: 3` deletion analysis of the KDR/flk-1 promoter in BAEC. Panel A, representation of deletion sites in relation to consensus sequences for known nuclear proteins discussed in the text. Panel B, analysis effect of 3` deletions on KDR/flk-1 promoter activity in BAEC. Constructs are described in the text, and luciferase activity is presented as a percentage of pGL2 Control. Significant differences are noted under ``Results.''
Because GATA-2 is a key
regulatory factor in endothelial cell-specific gene
expression(21, 39) , we examined the functional
importance of the atypical GATA site located between bp +105 and
+127 of human KDR/flk-1. Three bp of the GATA motif in the
fragment -225 to +268 were mutated to GTCG by PCR (28) to create pGL2 GATA-MUT. Mutation of these bp in the GATA
motif has been observed to eliminate GATA-2 binding activity in the
endothelin-1 gene promoter. ()In comparison with the native
pGL2-225+268 promoter construct, the pGL2 GATA-MUT construct
containing the mutated atypical GATA sequence did not have
significantly decreased promoter activity in BAEC (p >
0.05; Fig. 5).
Figure 5: Mutation of the GATA site at +107 does not decrease the ability of the KDR/flk-1 promoter to direct transcription. When transfected into BAEC, the plasmid pGL2-225+268 directed luciferase expression comparable to that directed by pGL2 Control, which contains the SV40 promoter and enhancer. When 3 bp of the GATA motif at +107 were mutated to create pGL2 GATA-MUT there was no significant difference in promoter activity.
Figure 6: KDR/flk-1 RNA expression is restricted to endothelial cells in culture. RNA was extracted from cells in culture and analyzed by Northern blotting as described under ``Experimental Procedures'' with a human KDR/flk-1 cDNA probe. HASMC, human aortic smooth muscle cells; HISMC, human intestinal smooth muscle cells; Fibroblast, human cultured fibroblasts. A photograph of ethidium bromide-stained ribosomal RNA of the agarose gel is provided to assess loading.
To determine whether 5`-flanking sequences of the KDR/flk-1 gene confer endothelial cell-specific expression in cultured cells, we transfected pGL2-4kb+296, which contains more than 4 kb of the human KDR/flk-1 5`-flanking sequence and includes most of the untranslated portion of the first exon, into a variety of cell types in culture (Fig. 7). In accord with our previous experiments in BAEC, reporter gene expression driven by the pGL2-4kb+296 promoter fragment was similar to that driven by the potent SV40 promoter/enhancer. In JEG-3, Saos-2, A7r5, 3T3, and HeLa cells, however, expression driven by the pGL2-4kb+296 promoter was markedly lower, demonstrating that induction of high level expression by this promoter is specific to endothelial cells. We observed a similar expression pattern with a reporter plasmid containing 15.5 kb of KDR/flk-1 5`-flanking sequence (data not shown).
Figure 7:
High level activity of the KDR/flk-1
promoter is specific to endothelial cells. The luciferase reporter
construct pGL2-4kb+296 was transfected into cells in
culture, and transfection efficiency was corrected by cotransfection
with pSVgal. Results are expressed as a percentage of pGL2 Control
activity for each cell type.
Finally, in an attempt to establish the function of regulatory elements within the KDR/flk-1 5`-flanking sequence in other cell types, we transfected into JEG-3 and Saos-2 cells the promoter constructs that defined positive regulatory elements in endothelial cells. In JEG-3 cells, promoter activity was reduced significantly (p < 0.05) when elements from bp -77 to -60 and +127 to +105 were removed (Fig. 8). Because similar reductions were obtained in endothelial cells, these two positive regulatory elements do not appear to be endothelial cell specific. In contrast, no significant changes were noted after deletion of the elements from -225 to -164 and -95 to -77, suggesting that these fragments may define endothelial cell-specific regulatory elements. (Deletion of the region from -164 to -95 resulted in a reduction in promoter activity in JEG-3 cells but not BAEC, which may reflect differential usage of core promoter elements in nonendothelial cells.) Identical studies were done in Saos-2 cells, and the results were similar (data not shown). Because promoter activity in nonendothelial cells is so low, we are reluctant to overinterpret the cell type specificity of regulatory elements in the KDR/flk-1 promoter. However, these results exclude the possibility that the cell type specificity of this promoter is due to the presence of silencer elements in the 5`-flanking region of this gene.
Figure 8: Deletion analysis of the KDR/flk-1 promoter in JEG-3 cells. Panel A, effect of 5` deletions on KDR/flk-1 promoter activity in JEG-3 cells. Constructs are described in the text, and luciferase activity is presented as a percentage of pGL2 Control. Deletion of bp -164 to -95 and -77 to -60 significantly reduced luciferase activity in JEG-3 cells (p < 0.05). No other significant differences were noted. Panel B, effect of 3` deletions on KDR/flk-1 promoter activity in JEG-3 cells. Deletion of bp +127 to +105 significantly reduced luciferase activity in JEG-3 cells (p < 0.05).
As a receptor for VEGF, KDR/flk-1 plays an essential role in angiogenesis and endothelial cell growth, and it is among the earliest markers of endothelial cell differentiation during development. Moreover, in situ analysis and immunocytochemistry have shown that KDR/flk-1 expression is restricted to endothelial cells in vivo; presumably this restricted pattern of expression determines the pattern of VEGF activity. Despite the importance of the KDR/flk-1 gene in endothelial cell growth, the mechanisms that regulate and restrict its expression are not known. We report for the first time the cloning and characterization of the human and mouse KDR/flk-1 promoters, and we identify regions containing positive regulatory elements within the 5`-flanking region of the human gene.
Analysis
of the human KDR/flk-1 5`-flanking region reveals that the
transcription start site is located 303 bp 5` of the methionine
initiation codon. Like constitutive endothelial nitric oxide synthase (40) , another gene expressed in endothelial cells, KDR/flk-1
lacks a TATA box, is rich in G and C residues, and has numerous
putative binding sites for Sp1, a ubiquitous nuclear protein that can
initiate transcription of TATA-less genes(41) . We identified
by deletion analysis three sequences within the 5`-flanking region of
the KDR/flk-1 gene which appear to contain elements important for its
expression in endothelial cells. Deletion of sequences between bp
-225 and -164 reduced activity to 63% that of the
full-length promoter, deletion between -95 and -77 further
reduced promoter activity to 20%, and deletion from -77 to
-60 reduced promoter activity to less than 5%. Because potential
binding sites for Sp1, AP-2, NFB, and E-box proteins located
within these three positive regulatory elements in the human KDR/flk-1
gene are also present in the mouse 5`-flanking sequence, they may
represent functional binding domains. AP-2 is a developmentally
regulated trans-acting factor (42) without a
demonstrated role in endothelial cell gene regulation. NF
B,
however, trans-activates the inducible expression of vascular
cell adhesion molecule-1 and tissue factor in endothelial cells (20, 43) and is known to be a mediator of
tissue-specific gene regulation(33) . Nuclear proteins that
bind the E-box motif include the basic helix-loop-helix family of trans-acting factors. E-box-binding proteins have not been
clearly associated with endothelial cell gene expression, although
members of this family are critical for proper maturation of many cell
types, including skeletal muscle and B
lymphocytes(36, 44) . Further experiments will be
necessary to determine if these or other unidentified nuclear proteins
specifically trans-activate the KDR/flk-1 gene.
Four zinc
finger-containing transcription factors in the GATA protein family bind
to the consensus sequence (A/T)GATA(A/G) and regulate cell
type-specific gene expression in many cell lineages(45) ; among
these GATA-2 has been most closely linked to endothelial cell gene
expression. GATA-2 functions as an enhancer of endothelin-1 gene
expression (39) and acts to restrict expression of von
Willebrand factor to endothelial cells(21) . We observed that
the human KDR/flk-1 5`-flanking region has two potential GATA-binding
sequences, at positions -759 and +107, and that loss of the
element located at -759 had no effect on expression of KDR/flk-1
in endothelial cells. The potential GATA element at position +107
is in a region of the first exon which we have identified as a powerful
positive regulatory element. Although this GATA sequence (GGATAT)
differs from the GATA-binding sequences of endothelin-1 and von
Willebrand factor and from the consensus GATA sequence (A/T)GATA(A/G),
we speculated that it might be the functional motif in the region
between +105 and +127 because the functional GATA site in the
von Willebrand factor gene is located similarly in the first exon and
because a similar GATA element is found in the first exon of the mouse
KDR/flk-1 gene. To our surprise, mutation of 3 bp in this element (GATA
to GTCG), which had been observed to prevent trans-activation
of the GATA cis-acting element in the endothelin-1
promoter, had no significant effect on KDR/flk-1 promoter
activity (Fig. 5). Thus, our deletion analysis and mutagenesis
studies do not support a functional role for the two GATA sequences in
the human promoter in its high level activity in endothelial cells.
These observations are consistent with the finding that early stages of
endothelial cell development are normal in mice deficient in GATA-2 (46) and suggest that other transcription factors are necessary
for expression of the human KDR/flk-1 gene.
We demonstrate in this study that expression of KDR/flk-1 is restricted to endothelial cells in culture, as it is in vivo. Moreover, we show that the activity of the KDR/flk-1 promoter in endothelial cells is similar to that of the potent SV40 promoter/enhancer and that this high level activity is specific to endothelial cells: activity in other cell types is markedly diminished. We do not yet understand why we observed low but detectable promoter activity in transient transfection assays of cell types that do not express the KDR/flk-1 gene in vivo; however, this situation is not unique among cell type-specific genes(47) . It is possible that other silencer elements outside of the 15.5-kb 5`-flanking region are necessary to block promoter activity completely in nonendothelial cells. Alternatively, the context of the promoter in relation to normal chromatin structure may be essential for precise regulation of the gene. An example of this type of regulation can be found in the control of MyoD expression. MyoD, like KDR/flk-1, is developmentally regulated, and it marks skeletal muscle precursors at an early stage(48) . The MyoD 5`-flanking region contains an enhancer element that increases MyoD expression in many cell types in culture, even though MyoD expression is specific to skeletal muscle in vivo(47) . In contrast, transgenic constructs containing the MyoD enhancer are skeletal muscle-specific, implying that chromatin structure modifies the activity of this enhancer and regulates cell type specificity. Our results suggest that tissue-specific regulation of KDR/flk-1 involves a complex interaction between known, widely distributed nuclear factors and other, unknown elements. Therefore a complete explanation of the mechanisms of endothelial cell-specific expression of KDR/flk-1 may require integration of in vivo and in vitro observations.
Identification of the regulatory mechanisms responsible for KDR/flk-1 expression is likely to provide important information about the specification and differentiation of endothelial cells early in embryogenesis. Moreover, knowledge about DNA elements that restrict gene expression to endothelial cells may be useful for deciphering the function of proteins in this cell type and, potentially, for directing or preventing expression of genes specifically in endothelial cells.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) X89776 [GenBank]and X89777[GenBank].