Shear Stress Induces Expression of Vascular Endothelial Growth Factor Receptor Flk-1/KDR Through the CT-Rich Sp1 Binding Site
Fluid shear stress is 1 of the major factors that control gene expression in vascular endothelial cells. We investigated the role of shear stress in the regulation of the expression of fetal liver kinase-1/kinase domain region (Flk-1/KDR), a vascular endothelial growth factor receptor, by using human umbilical vein endothelial cells. Laminar shear stress (15 dyne/cm2) elevated Flk-1/KDR mRNA levels by ≈3-fold for 8 hours, and the expression was upregulated within the range of 5 to 40 dyne/cm2. Deletion analysis of the 5′-flanking region of the Flk-1/KDR gene promoter by use of a luciferase reporter vector revealed that a shear stress–responsive element resided in the sequence between −94 and −31 bp, which contained putative nuclear factor-κB, activator protein-2, and GC-rich Sp1 and CT-rich Sp1 binding sites. Electrophoretic mobility shift assay demonstrated that nuclear extract was bound to the GC-rich Sp1 sites and the CT-rich Sp1 site with a similar pattern. However, shear stress enhanced the DNA-protein interactions only on the CT-rich Sp1 site but not on the GC-rich Sp1 sites. A 3-bp mutation in the CT-rich Sp1 site eliminated the response to shear stress in electrophoretic mobility shift assay and luciferase reporter assay. These results suggest that shear stress induces Flk-1/KDR expression through the CT-rich Sp1 binding site.
Vascular endothelial growth factor (VEGF) is a potent angiogenic protein that enhances vascular permeability and promotes endothelial cell proliferation. VEGF stimulates 2 types of tyrosine kinase receptors, namely, the fms-like tyrosine kinase-1 (Flt-1) and the fetal liver kinase-1/kinase domain region (Flk-1/KDR). Although both are indispensable for the normal development of vascular structure,1,2⇓ Flk-1/KDR is more potent than Flt-1 in stimulating endothelial cell proliferation and migration.3 Therefore, to understand the physiological roles of VEGF, it is essential to know the mechanism by which Flk-1/KDR expression is regulated. Basic fibroblast growth factor (bFGF),4 tumor necrosis factor-α,5 and highly confluent cell culture6 induce Flk-1/KDR expression in endothelial cells, whereas transforming growth factor-β1 reduces it.7 Cloning of the 5′-flanking promoter region of the Flk-1/KDR gene revealed that this gene is TATA-less and contains repeated GC boxes. Deletion analysis of the 5′-flanking region has demonstrated that the −95/−37 region, containing putative binding sites for activator protein-2 (AP-2), nuclear factor-κB (NF-κB), and Sp1, is essential for its transcription.8 DNase I footprinting analysis and electrophoretic mobility shift assay (EMSA) have demonstrated that Sp1 is the major nuclear protein binding to the Flk-1/KDR core promoter region.9 EMSA has demonstrated that expression of human Flk-1/KDR is mediated by Sp1 binding to a CT-rich recognition sequence within the −79 to −68 region of the Flk-1/KDR promoter.10 The Sp1-mediated promoter activity is attenuated by Sp3, suggesting that variations in the Sp1/Sp3 ratio may be relevant in the transcriptional regulation.10
Shear stress has been implicated in the regulation of gene expression in endothelial cells. It has been demonstrated that several important molecules on vascular function and integrity, such as platelet-derived growth factor (PDGF)-A,11 PDGF-B,12,13⇓ monocyte chemoattractant protein-1,14 and tissue factor (TF),15,16⇓ are induced by shear stress through the activation of transcription factors: AP-1,14 NF-κB,13 Sp1,15 and/or Egr-1.11,16⇓ However, there may be more genes responsive to shear stress, inasmuch as a study using an mRNA differential display method has suggested that numerous genes, far more than reported to date, are shear stress responsive.17 Recently, it was shown that 65 genes were upregulated and 140 genes were downregulated by laminar shear stress during an exposure period of 24 hours among 11 397 unique human genes.18 In consideration of the fact that Sp1 and NF-κB binding sites reside in the Flk-1/KDR promoter region, it was important to clarify whether shear stress influences Flk-1/KDR gene expression. In the present study, we showed for the first time that shear stress induces Flk-1/KDR expression in endothelial cells and that the induction is mediated through the CT-rich Sp1 binding site.
Human umbilical vein endothelial cells (HUVECs) isolated from umbilical cords by collagenase digestion were cultured in DMEM containing 20% (vol/vol) FBS, 5 ng/mL bFGF (Amersham Pharmacia Biotech), 100 μg/mL streptomycin, 100 U/mL penicillin, and 1 μg/mL amphotericin B as described previously.19 HUVECs were used for experiments between passages 3 and 8.
Shear Stress Apparatus
Confluent HUVECs grown on a 0.1% gelatin–coated plastic sheet were exposed to steady laminar shear stress in a parallel-plate flow chamber as described previously.20 Because bFGF enhanced Flk-1/KDR expression, DMEM containing 10% (vol/vol) FBS without bFGF was used for shear stress experiments. HUVECs under static control were also cultured in the same component medium.
Northern Blot Analysis
Equal amounts (10 μg) of total cellular RNA isolated from HUVECs were subjected to Northern blot analysis as described previously.21 A 565-bp fragment of human Flk-1/KDR cDNA (GenBank accession No. X61656, 2261 to 2825 bp) generated by reverse transcription–polymerase chain reaction was used as the probe. The generated DNA fragment was verified by sequence analysis. Blotting images were analyzed with a bio-imaging analyzer BAS-2500 (Fuji Photo Film Co), and the levels of Flk-1/KDR mRNA were normalized with 28S rRNA or β-actin mRNA levels in the same blots.
Western Blot Analysis
Cell lysates (100 μg of protein) were immunoprecipitated with a polyclonal antibody to Flk-1 (Santa Cruz Biotechnology) as described previously.21 The precipitates were subjected to SDS-PAGE under reduced conditions, transferred to a filter, and immunoblotted with a monoclonal antibody to Flk-1 (Santa Cruz Biotechnology) as described previously.21 The blots were visualized by using an enhanced chemiluminescence detection system (Amersham Pharmacia Biotech).
Luciferase Reporter Assay
Human Flk-1/KDR 5′-flanking regions (−742/+265, −185/+265, −94/+265, and −31/+265 bp; GenBank accession No. X89776) were amplified by PCR from genomic DNA obtained from HUVECs and subcloned into the SacI-XhoI site of PGV-B2 (Toyo Ink Mfg Co), a firefly luciferase reporter vector. The DNA constructs were verified by sequence analysis. The plasmid DNAs (1 μg) were transfected into 1.0×106 HUVECs (50% to 70% confluence) in serum-free medium by using Trans IT polyamine transfection reagents (PanVera Corp) according to the manufacturer’s instructions. Simultaneously, 0.05 μg of pRL-SV40, a Renilla luciferase expression vector (Toyo Ink Mfg Co), was cotransfected as a control for transfection efficiency. After transfection for 4 to 8 hours, cells were trypsinized and transferred to a gelatin-coated plastic sheet to be exposed to shear stress. After loading shear stress, luciferase activities were measured by using a double luciferase assay system (Toyo Ink Mfg Co) with a CT-9000D microtiter plate luminometer (DIA-IATRON). Firefly luciferase activities were normalized to those of Renilla luciferase.
Electrophoretic Mobility Shift Assay
Nuclear extracts were prepared as described previously.22 The oligonucleotide probes from the Flk-1/KDR sequence were as follows: NF-κB (−89/−70), 5′-CGCACGGGAGAGCCCCTCAA-3′; Sp1(CT) (−85/−64), 5′-CGGGAGAGCCCCTCCTCCGCCC-3′; AP-2/Sp1(GC) (−74/−55), 5′-AACCTCCGCCCCGGCCCCGC-3′; and Sp1(GC)-Sp1(GC) (−63/−34), 5′-CGGCCCCGCCCCGCATGGCCCCGCCTCCGC-3′. Because the −89/−70 NF-κB probe and the −74/−55 AP-2/Sp1(GC) probe contained the neighboring Sp1(CT) binding site (−74/−70), 2-bp mutations in the Sp1(CT) binding site [CT changed into AA (−71/−70) in the −89/−70 NF-κB probe, and CT changed into AA (−74/−73) in the −74/−55 AP-2/Sp1(GC) probe] were constructed to eliminate the effect of the Sp1(CT) binding site. The oligonucleotide probe containing the Sp1(GC) consensus sequence, commercially available, was 5′-ATTCGATCGGGGCGGGGCGAGC-3′ (Santa Cruz Biotechnology). The oligonucleotide probe containing the Sp1(CT) 3-bp mutant (−73/−71; TCC changed into AAG) was 5′-GCACGGGAGAGCCCCAGGT-CCGCCCCGGCCCCG-3′. Ten micrograms of nuclear extract was incubated with 2×104 cpm of 32P-labeled oligonucleotide probes and 2.0 μg poly(dI-dC) for 30 minutes on ice and electrophoresed as described previously.22 For competition, a 100-fold molar excess of unlabeled oligonucleotide was added. To identify DNA binding proteins, nuclear extracts were incubated with 2 μg of anti-Sp1, anti-Sp3, anti–AP-2, anti–NF-κB p50, or anti–NF-κB p65 antibodies (Santa Cruz Biotechnology) for 1 hour on ice before the addition of the probes. Dried gels were analyzed for radioactivity with BAS-2500.
Site-directed mutagenesis was performed by using a QuickChange mutagenesis kit (Stratagene) as instructed by the manufacturer. The luciferase reporter construct containing the −94/+265 region of the Flk-1/KDR promoter was used for the DNA template. The 3-bp mutation (−73/−71; TCC changed to AAG) in the luciferase reporter vector was verified by sequence analysis.
Values are expressed as the mean±SD from at least 3 independent experiments. The paired or unpaired t test was used for comparisons, and a significant difference was defined as P<0.05.
Effect of Shear Stress on Flk-1/KDR Expression
A steady laminar shear stress of 15 dyne/cm2, a strength within the physiological range in arterial vasculature, was loaded on HUVECs to examine the effect of shear stress on Flk-1/KDR expression (Figure 1A). Although the effect of shear stress was not evident at 2 hours, the expression level of Flk-1/KDR mRNA was elevated at 8 hours by 3.2-fold compared with the static control. At 24 hours, the high expression level still remained 2.6-fold as high as that of the static control. The protein level of Flk-1/KDR was also increased by loading shear stress for 8 to 24 hours (Figure 1B). To examine shear strength dependence of Flk-1/KDR mRNA expression, various strengths of shear stress from 2 to 40 dyne/cm2 were loaded on HUVECs for 8 hours. Upregulation of Flk-1/KDR mRNA expression was evident in cells subjected to a shear stress of 5 dyne/cm2, and the mRNA level increased up to 40 dyne/cm2 (Figure 1C). To examine whether a de novo protein synthesis is required for shear stress–induced expression of Flk-1/KDR mRNA, cycloheximide, a protein synthesis inhibitor, was added to the medium in the shear stress experiments. Cycloheximide did not prevent the shear stress–induced upregulation of Flk-1/KDR mRNA (Figure 2). Therefore, shear stress appeared to be able to stimulate Flk-1/KDR expression without synthesizing new proteins.
Deletion Analysis of Flk-1/KDR Gene Promoter
Luciferase reporter constructs containing the 5′-flanking regions of the Flk-1/KDR gene (−742/+265, −185/+265, −94/+265, and −31/+265 bp) were transfected into HUVECs, and the promoter activities were measured after loading shear stress (15 dyne/cm2). The basal activity was sharply reduced by deleting the sequence from −94 to −31 bp (Figure 3). Shear stress induced a 1.7- to 2.0-fold increase in the luciferase activity of −742/+265–, −185/+265–, and −94/+265–bp constructs, but it did not increase the activity of the −31/+265–bp construct. Therefore, sequences required for the basal expression and the response to shear stress both appeared to reside between −94 and −31 bp in the 5′-flanking region.
EMSA for Flk-1/KDR Promoter Sequences
We carried out EMSA to determine what sequences and what transcription factors are involved in the shear stress–induced Flk-1/KDR expression. The probes were designed to examine DNA-protein interactions on the NF-κB, Sp1(CT), AP-2/Sp1(GC), and Sp1(GC)-Sp1(GC) sites in the Flk-1/KDR promoter. Because Flk-1/KDR mRNA was upregulated at 8 hours by shear stress, we examined DNA-protein interactions under 15 dyne/cm2 of shear stress at multiple time points within 8 hours, ie, at 0 (static control), 1, 3, and 6 hours. Consistent with the findings of a previous study,9 the nuclear extract from HUVECs was bound to the probes containing Sp1 sites, except for the −74/−55 AP-2/Sp1(GC) probe. The −85/−64 Sp1(CT) and −63/−34 Sp1(GC)-Sp1(GC) probes were shifted into 2 DNA-protein complexes (the slower migrating complex was more prominent than the faster one) similar to the Sp1 consensus probe (Figure 4A). When the −89/−70 NF-κB probe was used in EMSA, a very faint complex was formed in the static control and sheared cells (Figure 4A). Shear stress increased DNA-protein interactions on the −85/−64 Sp1(CT) probe ≈3-fold as high as the basal level at 3 to 6 hours, whereas no change in the DNA-protein interactions was observed on the −63/−34 Sp1(GC)-Sp1(GC) probe and the Sp1 consensus probe (Figure 4A and 4B).
To further characterize DNA-protein interactions on the −85/−64 Sp1(CT) probe under shear stress conditions, competition experiments and supershift experiments were carried out with nuclear extracts from HUVECs loaded with shear stress for 3 hours. In competition experiments, all of the bands disappeared after adding a 100-fold excess of the unlabeled probe (Figure 4C). Despite the failure to detect supershifted bands, the anti-Sp1 antibody and anti-Sp3 antibody reduced DNA-protein interactions (Figure 4C). In contrast to anti-Sp1 and -Sp3 antibodies, anti–AP-2, anti–NF-κB p50, and anti–NF-κB p65 antibodies showed no influence on the interactions (Figure 4C). These results suggested that Sp1 and Sp3, but not AP-2 or NF-κB, are the major transcription factors that bind to the −85/−64 Sp1(CT) probe in response to shear stress.
Effect of a Mutated CT Element on the Responses to Shear Stress
To determine whether the CT element in the Sp1 binding site is responsible for the shear stress–induced elevation of Flk-1/KDR expression, we generated a 3-bp mutation in the CT element (−73/−71; TCC changed into AAG). As shown in Figure 5A, when we used the mutant probe in EMSA, the DNA-protein binding activity was conspicuously diminished, and the shear stress–induced elevation was completely lost. The same mutation made in the luciferase reporter construct containing the −94/+265 region of the Flk-1/KDR promoter reduced the basal promoter activity to some extent and completely eliminated the increase in the activity induced by shear stress (Figure 5B). These results suggested that the CT element is essential for the shear stress–induced transcriptional activation of the Flk-1/KDR gene.
Shear stress upregulated Flk-1/KDR mRNA expression with a relatively slow and gradual time course. This time course made us consider the possibility that a certain intermediate molecule could be involved in the upregulation. For example, bFGF23 and tumor necrosis factor-α5 induce Flk-1/KDR mRNA expression in cultured endothelial cells, which is evident after 4 hours and more prominent after 24 hours. And indeed, shear stress (15 dyne/cm2) stimulates bFGF synthesis in endothelial cells, with a moderate mRNA increase at 0.5 to 6 hours.24 However, it is unlikely that de novo synthesized molecules, such as bFGF, are responsible for the Flk-1/KDR expression in the present study, inasmuch as suppression of protein synthesis by cycloheximide did not disturb the shear stress to induce Flk-1/KDR mRNA expression.
In addition to Flk-1/KDR, some other molecules such as endothelial NO synthase (eNOS),25,26⇓ C-type natriuretic peptide,27 and integrins α5 and β128 are also slowly and gradually upregulated in mRNA levels by shear stress. Among these molecules, the mechanism by which shear stress upregulates the mRNA has been investigated for eNOS. Shear stress–induced eNOS upregulation is not affected by cycloheximide, and shear stress increases eNOS promoter activity,26 which is similar to the findings on Flk-1/KDR obtained in the present study. Recently, it has been demonstrated that shear induction of eNOS mRNA is regulated by c-Src through divergent signal pathways involving transcriptional activation and mRNA stabilization.29 Although the possibility that mRNA stabilization is involved in the Flk-1/KDR upregulation has not been excluded, the shear induction of the Flk-1/KDR gene is at least in part transcriptional, because shear stress clearly increased Flk-1/KDR promoter activity and DNA-protein interaction on the CT-rich site.
Shear stress–responsive element was first identified in the PDGF-B gene promoter.12 The element containing a core sequence GAGACC is essential to shear stress–induced PDGF-B upregulation and interacts with NF-κB.13 The human Flk-1/KDR gene promoter also contains 2 putative NF-κB binding sites.8 However, the present study showed that the −89/−70 NF-κB probe did not form an apparent DNA-nuclear protein complex in either the static control or sheared cells (Figure 4A). A study of NF-κB activation in human aortic endothelial cells has demonstrated that steady high shear (16 dyne/cm2) does not induce a significant increase in NF-κB binding activity compared with steady low shear (2 dyne/cm2) and pulsatile low shear (2±2 dyne/cm2).30 Accordingly, at least under high shear stress conditions, NF-κB appears to be unrelated to shear stress–mediated activation of the Flk-1/KDR gene.
In addition to the original shear stress–responsive element (GAGACC), the Egr-1 site in the PDGF-A promoter11 and the Sp1 sites in the TF promoter15 were found to be responsive to shear stress. It has been demonstrated that hyperphosphorylation of Sp1, without an increase in DNA-protein interactions, may be relevant to the induction of TF expression.15 However, shear stress enhanced DNA-protein interactions on the CT-rich Sp1 site but not on the GC-rich Sp1 sites in the Flk-1/KDR gene promoter. Therefore, there appear to be differences in the mechanisms underlying the shear stress–induced increase of transcriptional activity between the GC-rich Sp1 sites in the TF promoter and the CT-rich Sp1 site in the Flk-1/KDR promoter. The differences may be related to the finding that the shear stress response of the Flk-1/KDR expression is relatively slow and gradual compared with that of the TF expression.
Hata et al10 have suggested that the CT element (−74/−70, CTCCT) is important for the basic expression of the Flk-1/KDR gene. In the present study, we showed that this element was also required for the response to shear stress. Therefore, the CT element is likely to be critical for the basic transcription and the shear stress–promoted transcription of the Flk-1/KDR gene. However, the CT element may not be the only sequence regulating Flk-1/KDR gene expression. There are 2 reasons for the suggestion that GC-rich Sp1 sites also are involved in the regulation of Flk-1/KDR expression. First, EMSA clearly showed that the GC-rich Sp1 sites were bound to nuclear extract. Second, the 3-bp mutation in the CT element was not able to completely eliminate the luciferase activity. The remaining activity may have been mediated by intact GC-rich Sp1 sites residing in the −94/+265 region. Thus, the basal transcription may be controlled by both of the GC-rich Sp1 sites and the CT-rich Sp1 site; however, the shear stress–induced upregulation of the transcription may be mediated by the CT-rich Sp1 site.
Sp3 has been suggested to antagonize Sp1 in Flk-1/KDR transcriptional regulation.10 Sp3 is bound to a GC box with an affinity and specificity comparable to those of Sp1 and, thereby, represses Sp1-mediated transcription.31 To clarify whether shear stress influences the expression levels of Sp1 and Sp3, we performed Western blotting by using nuclear extracts from HUVECs subjected to shear stress. However, there were no significant changes in the Sp1 and Sp3 protein levels between static and sheared cells (data not shown). Therefore, in addition to the expression levels of Sp1 and Sp3, the activities of these transcription factors may be controlled by acetylation, phosphorylation, nuclear translocation of coactivators, or some other posttranslational modification as recently suggested.32
The findings of the present study suggested that shear stress could augment the functions of VEGF by upregulating its receptor. For example, when blood flow increases in developing organs or in growing tumors, increasing shear stress may urge the endothelial cells to express Flk-1/KDR and thereby promote vasculogenesis or angiogenesis. Alternatively, an increase in the strength of shear stress by postischemic reperfusion may stimulate Flk-1/KDR expression, resulting in an increase in vascular permeability. However, further studies are needed to determine whether the shear stress–induced Flk-1/KDR expression is relevant to physiological or pathological processes in blood vessels.
This study was supported by grants from the Ministry of Health and Welfare, Japan (Research Grants for Cardiovascular Diseases 11C-1 and 12C-3), the Mochida Memorial Foundation for Medical and Pharmaceutical Research, and the Japan Heart Foundation/Pfizer Grant for Research on Hypertension and Vascular Metabolism.
Received October 29, 2001; revision accepted March 25, 2002.
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