From the ** Beetham Eye Institute and Research
Division, Joslin Diabetes Center, Boston, Massachusetts 02215, the
Department of Ophthalmology, Harvard
Medical School, Boston, Massachusetts 02215, and
Hybridon,
Inc., Cambridge, Massachusetts 02139
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ABSTRACT |
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Kinase domain receptor (KDR) is a
high affinity, endothelial cell-specific, autophosphorylating tyrosine
kinase receptor for vascular endothelial growth factor. This
transcriptionally regulated receptor is a critical mediator of
endothelial cell (EC) growth and vascular development. In this study,
we identify a DNA element modulating KDR promoter activity and evaluate
the nuclear binding proteins accounting for a portion of the cell-type
specificity of the region. KDR promoter luciferase activity was
retained within 85/+296 and was 10-30-fold higher in EC than non-EC.
Electrophoretic mobility shift assays demonstrated specific nuclear
protein binding to
85/
64, and single point mutations suggested
important binding nucleotides between
79/
68 with five critical
bases between
74/
70 (5'-CTCCT-3'). DNA-protein complexes were
displaced by Sp1 consensus sequence oligodeoxynucleotides and
supershifted by Sp1- and Sp3-specific antibodies. Sp1 and Sp3 protein
in EC nuclear extracts bound the
79/
68 region even when all
surrounding classic Sp1 recognition sites were removed. Sp1 protein in
nuclear extracts was 4-24-fold higher in EC than non-EC, whereas Sp3
was 3-7-fold higher. Sp1/Sp3 ratios in EC were 2-10-fold higher.
Overexpression of Sp1 protein increased KDR promoter activity 3-fold in
both EC and non-EC, whereas simultaneous co-expression of Sp3
attenuated this response. An Sp1 consensus sequence cis
element "decoy" reduced EC KDR promoter activity and mRNA
expression by 85 and 69%, respectively. An antisense phosphorothioate
oligodeoxynucleotide to Sp1 inhibited Sp1 and KDR protein expression by
66 and 68%, respectively, without changing Sp3 protein expression.
These data illustrate that Sp1 and Sp3 modulate KDR promoter activity through a novel recognition binding sequence. However, since Sp1-mediated promoter activation is attenuated by Sp3, endothelial selective KDR promoter activity may be partially regulated by variations in the Sp1/Sp3 ratio.
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INTRODUCTION |
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Vascular endothelial growth factor (VEGF)1 (1), also known as vascular permeability factor (2) or vasculotropin (3), is a hypoxia-inducible, endothelial cell-selective mitogen and potent vasopermeability factor. VEGF is thought to play a central role in mediating the formation of new blood vessels during fetal development (4) and during other physiologic and pathologic conditions associated with angiogenesis such as wound healing (5), collateral vascular formation (6), and tumorigenesis (7). The role of VEGF in mediating pathologic neovascularization (8, 9) and vascular permeability (10-13) in the eye has been well documented. Numerous retinal cell types produce VEGF, and expression can be increased up to 30-fold under hypoxic conditions (14, 15). Intraocular VEGF concentrations are highly correlated with neovascularization within the human eye resulting from common sight-threatening conditions (9, 16-22). Inhibition of VEGF activity can suppress retinal (23, 24) and iris (25) neovascularization as well as tumor growth in animals (26, 27).
VEGF exerts its action through two high affinity, tyrosine
phosphorylating, transmembrane receptors named KDR/flk-1 and
flt-1 (28, 29). KDR/flk-1 expression is selective
for endothelial cells in vivo (29, 30), whereas
flt-1 is expressed both in endothelial cells and in some
cells of non-endothelial origin such as retinal pericytes (31-32),
renal glomerular mesangial cells (33), and mononuclear phagocytes (34).
Both receptors possess seven extracellular immunoglobulin-like domains
and an intracellular tyrosine kinase region containing a kinase insert
(29, 35). However, KDR is much more efficiently phosphorylated in
vitro in response to VEGF stimulation than is flt-1 (36). VEGF
binding to either receptor can also activate numerous intracellular
signaling molecules including phosphatidylinositol 3-kinase,
phospholipase C, and protein kinase C
, -
, and -
isoforms
although the signal transduction pathways may differ between the
receptors (36-38).
Recent analysis of the human KDR promoter has demonstrated that maximal
activity resides in the 225 to +127 5'-flanking region of the KDR
gene relative to the transcriptional start site and that deletions of
95 to
37 result in complete loss of promoter activity (39, 40).
This region of the KDR promoter contains putative AP2-, Sp1-, and
NF
B-binding sites. DNase I footprinting experiments by Patterson
et al. (40) suggest that transcription factor Sp1 binds the
human KDR promoter in this region and that Sp1 binding is endothelial
cell-specific in vivo, potentially due to cell-specific
alterations in chromatin structure. Nevertheless, it has not been
documented that Sp1 binding actually alters either KDR promoter
activity or KDR expression, and the involvement of other nuclear
proteins modulating expression of this gene have not been fully
defined.
Due to the critical role of KDR in mediating important physiologic and pathologic processes and the incomplete understanding of the mechanisms of endothelial cell-specific KDR gene expression, we have performed a detailed functional characterization of the DNA-binding proteins regulating KDR promoter activity in both endothelial and non-endothelial cells. In this article we provide evidence that transcription factor Sp1 increases KDR promoter activity in both endothelial and non-endothelial cells through binding to a novel recognition sequence, whereas simultaneous co-expression of Sp3 attenuates this response. These data suggest that portions of KDR promoter activity and endothelial selective expression of KDR may in part be regulated by variations in the Sp1/Sp3 ratio. In addition, our data demonstrate that the majority of KDR promoter activity, mRNA expression, and protein synthesis require Sp1 activity in endothelial cells.
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MATERIALS AND METHODS |
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Cell Culture-- Primary cultures of bovine retinal endothelial cells (BREC), retinal pericytes (BRPC), retinal pigment epithelial cells (BRPE), smooth muscle cells (BSMC), fibroblasts (BFibro), and aortic endothelial cells (BAEC) were isolated from fresh slaughterhouse tissues by homogenization and a series of filtration steps as described previously (14). BREC were cultured in Endothelial Basal Medium (Clonetics, San Diego) with 10% plasma-derived horse serum (Wheaton Scientific), heparin (50 mg/liter), and 50 µg/ml endothelial cell growth factor (Boehringer Mannheim). BRPC were cultured in Dulbecco's modified Eagle's medium with 5.5 mM glucose and 20% fetal bovine serum (HyClone). BAEC, BRPE, BSMC, BFibro, COS, Chinese hamster ovary, and 3T3 cells were cultured in Dulbecco's modified Eagle's medium with 10% calf serum (Life Technologies, Inc.). HUVEC were cultured in M199 media with 10% fetal bovine serum. All cells were cultured at 37 °C in 5% CO2, 95% air, and media were changed every 2-3 days.
Plasmids--
The pGL2 Basic plasmid (Promega) contains the
firefly luciferase gene with no promoter or enhancer. Plasmid
pSV40gal (Promega) contains the
-galactosidase gene driven by the
SV40 promoter and enhancer. A 100-kb human genomic DNA fragment
encompassing the KDR gene was obtained by P1 phage cloning (42) and
confirmed by sequence comparison to previously published data (39). A luciferase reporter construct containing 4.3 kb of the KDR 5'-flanking region was created by restriction digestion of the purified P1 phage
DNA with BamHI and XhoI, corresponding to the
4-kb and +296 sites of the KDR gene, respectively. In this article,
all KDR gene nucleotides are expressed relative to the KDR
transcriptional start site which is defined as +1. The fragment was
inserted into the pGL2 Basic plasmid. Similar luciferase reporter
constructs were created from human KDR regions
101/+296 and
85/+296
(Fig. 1A). These were obtained
by polymerase chain reaction amplification of KDR P1 phage DNA and were
inserted into plasmid pGL2 Basic. All constructs were sequenced from
both the 5' and 3' ends to confirm orientation and sequence. The Sp1
and Sp3 expression vectors were generously provided by Dr. Jonathan M. Horowitz (Duke University Medical Center) and were constructed from Sp1
and Sp3 cDNA cloned into pCMV-4, an expression vector containing
the cytomegalovirus immediate early promoter, as described previously
(43-45).
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Mutagenesis--
Nineteen different double-stranded 22-mer
oligodeoxynucleotides representing the KDR promoter region 85/
64
were constructed with each incorporating 1-6 nucleotide substitutions
by synthesis of complementary single-stranded fragments (Oligos Etc.)
and subsequent annealing at room temperature after heating to 95 °C
(Fig. 1B). Site-directed mutagenesis of nucleotides within
the human KDR
85/+296 luciferase reporter construct was performed
using polymerase chain reaction and the human KDR P1 phage DNA as
template. The nucleotides mutated (
85 to
80,
76 to
74, or
73
to
71) were identical to those changed in oligodeoxynucleotide
mutations 1, 3, and 4, respectively, as shown in Fig. 1B.
Internal mismatches in the 5' primer were used in conjunction with a 3'
primer matched to the XhoI site of the coding area. The
amplified polymerase chain reaction fragment was gel-purified and
digested with XhoI at nucleotide +296 and cloned directly
into the pGL2 Basic plasmid. All mutation constructs were sequenced
from the 5' and 3' ends to confirm orientation and sequence.
Transfections--
Plasmid DNA was introduced into all cell
types with the LipofectAMINE reagent (Life Technologies, Inc.) as
instructed by the manufacturer. The appropriate luciferase reporter
construct (1 µg) was always co-transfected with 1 µg of pSV40gal
to normalize for transfection efficiency in the 1.5-3.0 × 105 cells used. Cells were harvested 48 h after
transfection, and luciferase activity was measured using the Luciferase
Assay System (Promega) and an ML3000 microtiter plate luminometer
(Dynatech Laboratories).
-Galactosidase activity was assayed as
described previously (46). For each transfection, luciferase activity was divided by
-galactosidase activity to obtain normalized
luciferase activity.
Co-transfections of Sp1 and Sp3 Expression Plasmids--
The Sp1
and Sp3 expression plasmids and transfection conditions are described
above. All cells were transfected with a total of 2 µg of expression
plasmids for 48 h in one of the following combinations: 2 µg of
pCMV4 (control), 1 µg of Sp1-pCMV4, and 1 µg of pCMV4, 1 µg of
Sp3-pCMV4 and 1 µg of pCMV4, or 1 µg of Sp1-pCMV4 and 1 µg of
Sp3-pCMV4. All cells were also co-transfected with 1 µg of
pSV40gal to normalize for transfection efficiency.
Electrophoretic Mobility Shift Assays--
Preparation of
nuclear extracts was performed as described previously (47). Protein
concentrations were determined by the method of Bradford (48), and the
same amount of nuclear protein was subjected to electrophoretic
mobility shift assays as described previously (49). Double-stranded
oligodeoxynucleotides were constructed by annealing commercially
synthesized complementary single-stranded oligodeoxynucleotides
corresponding to published human sequences (39, 41, 50-52). The KDR
sequences used are detailed in Fig. 1. Oligodeoxynucleotide sequences
corresponding to DNA binding consensus sites were as follows: Sp1,
5'-ATTCGATCGGGGCGGGGCGAGC-3'; NFB, 5'-AGTTGAGGGGACTTTCCCAGGC-3';
AP1, 5'-CGCTTGATGAGTCAGCCGGAA-3'; and AP2,
5'-GATCGAACTGACCGCCCGCGGCCCGT-3'.
Northern and Western Blot Analysis-- Northern and Western blot analyses were performed as described previously (53, 54). Quantitation of Northern blots was performed using a computing PhosphorImager with ImageQuant software analysis (Molecular Dynamics). Lane loading differences were normalized by hybridization with radiolabeled 36B4 cDNA (55). For Western blot analyses, rabbit anti-human polyclonal antibodies specific for Sp3 and flk-1 (Santa Cruz Biotechnology) and mouse anti-human monoclonal antibody specific for Sp1 (PharMingen) were used. Visualization was performed using Amersham Pharmacia Biotech ECL detection system per manufacturer's instructions.
Cis Element Phosphorothioate "Decoys"--
Double-stranded
phosphorothioate-modified oligodeoxynucleotides corresponding to the
consensus binding sequences of Sp1 (PS-Sp1) and NFB (PS- NF
B)
were made by annealing complementary single-stranded phosphorothioate
oligodeoxynucleotides (Oligos, Etc.). Decoys were co-transfected into
cells along with the
101/+296 KDR-luciferase pGL2 construct and the
pSV40
gal plasmid. Luciferase and
-galactosidase activity were
measured after 48 h as described above.
Sp1 Inhibition Using Antisense Oligodeoxynucleotides-- A phosphorothioate-modified antisense oligodeoxynucleotide (5'-ATATTAGGCATCACTCCAGG-3') directed against the Sp1 transcription start site and its sense control (5'-CCTGGAGTGATGCCTAATAT-3') were commercially synthesized (Oligos, Etc.). Cells were transfected with 1 µM oligodeoxynucleotide using LipofectAMINE reagent (Life Technologies, Inc.) as instructed by the manufacturer. Total protein was harvested for Western blot analysis of Sp1, Sp3, and KDR expression after 48 h as described above.
Statistical Analysis-- All experiments were repeated at least three times with similar findings, and results are expressed as mean ± S.E. unless otherwise indicated. The unpaired t test was used for comparisons of groups with equal variance and normal distribution. The Mann-Whitney Rank Sum test was used for comparison of groups with unequal variance or non-normal distributions. A p value of less than 0.05 was considered statistically significant.
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RESULTS |
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Endothelial Cell-specific Expression Is Retained within the 85 to
+296 Region of the Human KDR Promoter--
To confirm endothelial
specific expression of KDR in the primary cell cultures of interest in
this study, Northern blot analysis using a human KDR cDNA probe was
performed on total RNA (20 µg/lane) extracted from various cell types
of both endothelial and non-endothelial origin (Fig.
2A). KDR mRNA was detected
from all endothelial cell cultures (BREC, BAEC, and HUVEC), but no
signal was observed from non-endothelial cells (BSMC, BRPC, BRPE, or
BFibro). KDR expression was also undetectable using polyadenylated RNA
in non-endothelial cells (data not shown). The hybridization signal was
greatest for HUVEC probably since both cDNA probe and cell type
were of human origin. Between the two bovine endothelial cell types,
KDR expression was 2-fold greater in BREC than BAEC. This finding is
consistent with our previous report that KDR receptor number is 3-fold
higher in BREC than in BAEC (56).
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KDR Promoter Nucleotides 79 to
68 Permit Specific Nuclear
Protein Binding within the Endothelial Cell-selective
Region--
Previous reports have shown a near complete loss of KDR
promoter activity when the region 5' to nucleotide
60 is deleted (39). These data and those described above suggest that the region
regulating endothelial specific activity resides between
85 and
60.
To determine if nuclear proteins could specifically bind this region,
EMSA was performed using nuclear extract from BREC and radiolabeled KDR
promoter fragment
101/
56 (Fig.
3A). Specific DNA-protein
binding complexes were evident as a major slower migrating band
(immediately above a weak nonspecific band) and a faster migrating
doublet band. The slower migrating band was usually resolved as a
closely migrating doublet on careful examination. These bands were
competed by 100-fold molar excess of unlabeled
101/
56 promoter
fragment but not by the nonspecific
225/
164 fragment. Unlabeled
promoter fragments with 5' deletions within nucleotide
85 (
81/
64)
and 3' deletions beyond residue
64 (
85/
68) were unable to compete
with nuclear protein binding to region
101/
56, whereas fragments
containing the
85/
64 region
95/
56,
85/
56,
101/
64, and
85/
64 competed well.
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Nuclear Proteins Sp1 and Sp3 Bind to a Novel Recognition Sequence
in the KDR Promoter--
Since the 150 base region 5' to the
initiation start site of the KDR gene contains three potential AP2, two
potential NFB-, and five potential Sp1-binding sites, and since the
region conveying endothelial cell specificity both potential Sp1 and
NF
B sites, we evaluated whether DNA consensus sequences for various
transcription factors could compete for nuclear protein binding to the
KDR promoter. EMSA was performed using BREC nuclear extracts,
radiolabeled promoter fragment
101/
56, and competition with
100-fold molar excess of nonradioactive KDR promoter fragments
(
225/
164,
101/
56,
85/
64), or NF
B, Sp1, AP2, and AP1 DNA
consensus sequences (Fig. 4a).
It is known that Sp1 and Sp3 can bind the same Sp1 consensus sequence
(44, 45). The specific DNA-protein binding complexes were only competed
by unlabeled promoter fragments which included the intact binding
region (
101/
56,
85/
64) and by the Sp1 consensus sequence. These
data suggest that members of the Sp family of transcription factors
known to bind the Sp1 consensus sequence may be binding the
101/
56
region of the KDR promoter.
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Nuclear Protein Levels and Binding to the KDR Promoter Correlate
with KDR Expression--
If the nuclear protein-DNA interactions in
this region are important for KDR promoter activity, then the
DNA-protein binding might be expected to be higher in endothelial cell
nuclear extracts. EMSA was performed using the radiolabeled 101/
56
promoter fragment and nuclear extracts from a variety of endothelial
(BREC, BAEC, and HUVEC) and non-endothelial cells types (BSMC, BRPC,
BRPE, and BFibro) (Fig. 6A).
The specific DNA-protein complexes were evident in all endothelial cell
nuclear extracts tested but were >9-fold less prevalent in extracts
from non-endothelial cells.
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Nuclear Proteins Sp1 and Sp3 Partially Regulate Endothelial
Cell-specific Expression of KDR--
To determine if elevated Sp1
expression is sufficient to increase KDR promoter activity, BREC and
BSMC were transiently transfected with the 101/+296 human KDR
promoter luciferase reporter construct and expression plasmids for
either Sp1, Sp3, neither, or both (Fig.
7). Overexpression of Sp1 increased KDR
promoter activity nearly 3-fold in both BREC and the non-endothelial
BSMC. Expression of Sp3 alone reduced basal KDR promoter activity in
BREC; however, co-transfection of Sp3 along with Sp1 attenuated the Sp1
activation of the KDR promoter in both cell types. Similar results were
obtained in BRPC, BRPE, and BFibro (data not shown).
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DISCUSSION |
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In this study, we demonstrate that expression of KDR is induced by
transcription factor Sp1 and attenuated by Sp3 binding to a novel
recognition sequence located within the 79 to
68 region of the
human KDR promoter. To our knowledge, this is the first demonstration
of Sp3 binding to the human KDR promoter. Furthermore, we show that Sp1
is necessary for maximal expression of both KDR mRNA and protein in
endothelial cells and that overexpression of Sp1 in non-endothelial
cells activates the KDR promoter. These data suggest that a portion of
the endothelial cell-selective expression of KDR is mediated by the
relative binding of Sp1 and Sp3 to a novel recognition sequence.
Our findings demonstrate that both Sp1 and Sp3 bind to the 79/
64
promoter sequence 5'-AGCCC[CTCCT]CC(GCCC)-3' where the 5 residues in
the square brackets are the most critical for binding, the 7 unmarked
bases partially affect binding, and the 4 nucleotides in parentheses
are required for binding but do not need to form a classic Sp1
consensus site. The
79/
64 KDR promoter sequence has some similarity
to the positively acting homopyrimidine CT element (5'-CCCTCCCCA-3')
found upstream of the c-myc gene which is known to bind Sp1
and related factors (43, 63). However, the CT region usually consist of
4 imperfect direct repeats of the CT element and a fifth downstream
repeat separated by 9 base pairs. At least two nonadjacent units are
required for transcription factor binding. Such repeats are not present
in the KDR promoter (39).
Although Sp1 has recently been shown to bind the KDR promoter by EMSA
(40), it has not been previously demonstrated that Sp1 binding actually
alters either KDR promoter activity or KDR expression. The critical
role of Sp1 and Sp3 binding to the KDR promoter on KDR promoter
activity and KDR mRNA expression was demonstrated using
phosphorothioate-modified cis element Sp1 consensus sequence
decoys. Decoy cis elements block the binding of nuclear factors to promoter regions of genes by specific high affinity binding
to the targeted transcription factors resulting in inhibition of gene
transactivation (59-62). Phosphorothioate modification reduces
degradation of the molecules within the cell. The Sp1 consensus
sequence decoy markedly inhibited KDR promoter activity and mRNA
expression in endothelial cells, whereas a NFB consensus sequence
decoy did not (Fig. 8, A and B). These results
demonstrate that proteins which bind the Sp1 consensus sequence are
important for KDR promoter activity and mRNA expression in
endothelial cells. Since both Sp1 and Sp3 bind the same sequence, these
results do not help differentiate the relative contributions of Sp1 and
Sp3. However, an Sp1 antisense phosphorothioate oligodeoxynucleotide markedly reduced both Sp1 and KDR protein expression without affecting Sp3 protein levels (Fig. 9), thus demonstrating the critical role of
Sp1 in mediating KDR protein expression in endothelial cells.
Several observations in the current study suggest that Sp1 may modulate a portion of the endothelial selectivity of KDR expression. Overexpression of Sp1 protein by transient transfection in both endothelial and non-endothelial cells resulted in a 3-fold increase in KDR promoter activity. Although Sp1 expression in non-endothelial cells did not increase KDR promoter activity to the same level of BREC, it did increase it to roughly that of BAEC. BREC have nearly 3-fold higher KDR promoter activity than BAEC, which correlates with the 2-fold higher KDR mRNA (Fig. 2A) and 3-fold higher protein levels in BREC (56). It is not possible to determine from these experiments whether additional elevation of Sp1 protein over that obtained with the expression vector would further increase non-endothelial cell KDR promoter activity.
Co-expression of Sp1 and Sp3 protein resulted in attenuation of the Sp1-mediated increase in KDR promoter activity. Sp3 has previously been shown to produce repression of Sp1-mediated activation of numerous Sp1-responsive promoters by competition with Sp1 for their common binding sites (45, 57). This finding might also account for the high KDR promoter activity in BREC since protein levels of Sp1 were 35% greater in nuclear extracts from BREC than those from BAEC, whereas levels of Sp3 were 10% greater in BAEC (Fig. 6B). Thus, the Sp1/Sp3 stimulatory ratio was 48% greater in BREC than in BAEC, a result consistent with the increased KDR promoter activity observed in BREC.
The Sp1/Sp3 ratios for the various cells of endothelial origin were greater than for all the non-endothelial cell types studied. This observation combined with the markedly lower total Sp1 expression in non-endothelial ocular cells could partially account for the endothelial cell-specific expression of KDR. However, there could also be other factors contributing to the endothelial selectivity of KDR expression.
Although Sp1 is generally considered a ubiquitous transcription factor, there is considerable evidence that Sp1 participates in cell type-specific gene expression (16, 40, 58, 64, 65), is developmentally (66) and functionally regulated (67, 68), and is highly expressed during vasculogenesis (66). These attributes are similar to those observed for KDR (30, 31). The mechanism by which Sp1 preferentially binds to some promoters in specific cell types is not clear (40, 58, 65). Most studies have not observed differences in the ability of nuclear extracts from different cell types to protect Sp1-binding sites in DNase I footprinting experiments in vitro. Similarly, EMSA results and evaluation of nuclear protein levels of Sp1 in some cell types have not demonstrated significant differences in Sp1 levels. It has been proposed that another protein may be binding near the Sp1 site, thus preventing Sp1 binding in cells where the promoter is inactive. However, in vivo DNase I footprinting has confirmed that DNA-protein interactions occur within Sp1 elements in HUVEC but not in other non-endothelial cells, and changes in nucleosomal positioning are present (40). This has led to the hypothesis that distant elements of the KDR promoter alter the chromatin structure and thus permit specific Sp1-mediated expression of KDR in endothelial cells (40). Sp1 activity can be influenced by other transcription factors such as AP1, Egr-1, GATA, NF-E1, NS-1, and Pit-1 (58, 69, 70-73). Sp1-mediated transcription is also modulated by the tumor-suppressor gene product Rb (44, 74) which is thought to bind a 20-kDa Sp1-negative regulator, thus liberating active Sp1 (74). Indeed, we did not detect a supershifted complex when using a Rb-specific antibody (Fig. 4A) suggesting that Rb is neither directly bound to this region of the KDR promoter nor tightly bound to either Sp1 or Sp3 while they are bound to the promoter. Sp1 can also be glycosylated with multiple O-linked N-acetylglucosamine monosaccharide residues which may alter its transcriptional activation (72, 75).
Although these mechanisms for Sp1 cell-specific activity could occur in the cell types we evaluated in this study, our results differ somewhat from those presented above in that they suggest that either the absolute levels of nuclear Sp1 and Sp3 and/or the Sp1/Sp3 ratio might be partially responsible for differences in promoter binding and cellular KDR expression. This possibility is supported by the findings that in endothelial cells both Sp1 and Sp3 levels (as determined by Western blot analysis) were up to 24-fold higher, and the Sp1/Sp3 ratios were up to 10-fold higher as compared with those of non-endothelial cells. In addition, the specific KDR promoter DNA-protein interactions were 9-fold more prevalent using endothelial cell-derived nuclear extracts than when using those from non-endothelial cells. Finally, overexpression of Sp1 in non-endothelial cells resulted in increased KDR promoter activity.
Our results might reflect the use of ocular bovine cells which have not been evaluated in previous studies. These cell types are biologically relevant since VEGF plays a critical role in ocular development and in mediating a wide variety of intraocular neovascular disorders such as diabetic retinopathy, central retinal vein occlusion, retinopathy of prematurity, age-related macular degeneration, and numerous others. Biochemical results obtained from the ocular bovine cell types used in this study have been very similar to those observed in humans with regard to both VEGF action, glucose reactivity, and biochemical responses such as protein kinase C activation, endothelin production, etc. (8, 10, 14, 23, 25, 38, 55, 76). In addition, it has been previously suggested that Sp1 promoter specificity could be the result of both the abundance of Sp family members and the relative ratios of Sp1 and Sp3 (45). Finally, our results could be explained by the unlikely presence of novel Sp1 and Sp3-like molecules which have different binding specificity and cell type expression but share the specific Sp1 and Sp3 antigenic epitopes recognized by the antisera used in this study.
In summary, our studies are the first to demonstrate that expression of
KDR is partially mediated by Sp1 binding to a novel recognition
sequence within the 79 to
68 region of the KDR promoter. We show
that Sp3 binds to the region of the KDR promoter which retains
endothelial cell specificity and attenuates Sp1-mediated activation.
These data also demonstrate that Sp1 is critical for maximal expression
of both KDR mRNA and protein in endothelial cells and that
increased expression of Sp1 is sufficient to partially increase KDR
promoter activity in non-endothelial cells. The exact mechanism by
which Sp1 exerts its endothelial selectivity on the expression of KDR
remains unclear and is the focus of future studies.
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ACKNOWLEDGEMENTS |
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The very generous gift of Sp1 and Sp3 expression vectors by Jonathan M. Horowitz is gratefully acknowledged. We thank Dr. George L. King, Dr. Edward P. Feener, Dr. Jerry D. Cavallerano, and Ann Kopple for their insightful advice and technical assistance with manuscript preparation.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant EY-10827 (to L. P. A.) and a Juvenile Diabetes Foundation Fellowship (to E. D.). The Joslin Diabetes Center is the recipient of National Institutes of Health Diabetes and Endocrinology Research Center Grant 36836.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Both authors contributed equally to this work.
¶ Present address: Wilmer Ophthalmological Institute, Johns Hopkins Hospital, Baltimore, MD 21287.
§§ To whom correspondence should be addressed: Joslin Diabetes Center, One Joslin Place, Boston, MA 02215. Tel.: 617-732-2427; Fax: 617-735-1960; E-mail: aiellol{at}joslab.harvard.edu.
1 The abbreviations used are: VEGF, vascular endothelial growth factor; KDR, kinase domain receptor; BREC, bovine retinal endothelial cells; BRPC, bovine retinal retinal pericytes; BRPE, bovine retinal retinal pigment epithelial cells; BSMC, bovine aortic smooth muscle cells; BFibro, bovine fibroblasts; BAEC, bovine aortic endothelial cells; HUVEC, human umbilical vein endothelial cells; kb, kilobase pair(s); Rb, retinoblastoma protein; EMSA, electrophoretic mobility shift assays.
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REFERENCES |
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