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Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:377-384

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:377.)
© 2000 American Heart Association, Inc.

Vascular Biology

Zinc Finger Transcription Factor Egr-1 Activates Flt-1 Gene Expression in THP-1 Cells on Induction for Macrophage Differentiation

Nobuhiro Akuzawa; Masahiko Kurabayashi; Yoshio Ohyama; Masashi Arai; Ryozo Nagai

From the Second Department of Internal Medicine, Gunma University School of Medicine, Gunma, Japan.

Correspondence to Masahiko Kurabayashi, MD, Second Department of Internal Medicine, Gunma University School of Medicine, 3-39-15 Showa-machi, Maebashi 371-8511, Gunma, Japan. E-mail mkuraba{at}news.sb.gunma-u.ac.jp


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Abstract—Activation of macrophages is a hallmark of atherosclerosis. Stimulation of human monocytic leukemia THP-1 cells with phorbol 12-myristate 13-acetate (PMA) is known to induce a variety of genes whose function is relevant to activated macrophages. Flt-1, a receptor tyrosine kinase for vascular endothelial growth factor, is expressed in macrophages as well as in endothelial cells and mediates the biological response to vascular endothelial growth factor. In this study, we investigated the molecular mechanisms underlying the inducible expression of the flt-1 gene during the activation of THP-1 cells. Reverse transcription—polymerase chain reaction analysis showed that exposure of THP-1 cells to PMA increases flt-1 mRNA and protein levels. A transfected reporter gene, consisting of the human flt-1 promoter region coupled to the luciferase gene, indicated a direct effect of PMA on transcriptional activity. Transfection analysis of a series of 5'-deletion constructs and site-directed mutants localized the PMA-responsive region to a DNA stretch from -174 to -166, which represents overlapping Egr-1/Sp1 transcription factor–binding sites. Competitive gel mobility shift assays and supershift assays showed that PMA induces the binding of Egr-1 to this site. Consistent with these findings, the Egr-1 expression plasmid strongly induced flt-1 promoter activity in a sequence-specific manner. Taken together, our data demonstrate that PMA induces flt-1 gene transcription through an induction of Egr-1 in THP-1 cells, thus providing new evidence that the flt-1 gene is a direct target of Egr-1, the transcription factor primarily induced on macrophage differentiation.


Key Words: flt-1 • Egr-1 • macrophages • monocytes • vascular endothelial growth factor


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 *Introduction
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Activation of cellular components in the vessel walls is a hallmark of atherosclerosis.1 This concept is derived from studies showing that migration of vascular smooth muscle cells (SMCs), accompanied by phenotypic changes, plays a pivotal role in neointimal lesion formation. In addition, an inflammatory response of the vessel wall, which is characterized by the activation of macrophages, monocytes, and T lymphocytes, is a key feature of atherosclerotic plaques.2 Previous studies have identified many peptide growth factors, including platelet-derived growth factor (PDGF), basic fibroblast growth factor, angiotensin II, and transforming growth factor-ß, and inflammatory cytokines, such as tumor necrosis factor-{alpha} and interleukin-1ß, as being responsible for the activation of vascular cells.3 In addition, emerging evidence indicates that vascular endothelial growth factor (VEGF), a critical regulator of embryonic blood vessel growth and tubular formation,4 5 plays an important role in the development of atherosclerosis. For example, enhanced VEGF expression has been documented in atherosclerotic plaques of human coronary arteries,6 and it has been reported that VEGF exerts its effects on vascular SMCs and accelerates SMC migration through enhanced production of matrix metalloproteinases.7

The kinase insert domain–containing receptor/fetal liver kinase (KDR/flk-1) and fms-like tyrosine kinase-1 receptor (flt-1) constitute the 2 major tyrosine kinase receptors for the signaling of VEGF in endothelial cells.8 9 Gene-targeting studies unequivocally showed that these receptors are essential for the development of normal blood vessels but that their biological functions are considerably different. Targeted disruption of the KDR/flk-1 gene resulted in a severe defect involving endothelial cell growth, whereas flt-1 knockout mice displayed normal development of endothelial cells but no tubular formation.10 11

Expression profiles are also quite distinct between KDR/flk-1 and flt-1; whereas the KDR/flk-1 gene is specifically expressed in endothelial cells,12 13 the expression of the flt-1 gene is observed in a variety of cell types, including macrophages, proliferating vascular SMCs, and uterine SMCs, and in the pancreatic duct.7 14 15 Furthermore, although endothelial expression of the KDR/flk-1 gene is not responsive to hypoxic stimulation, flt-1 gene expression is significantly elevated by hypoxia.16 These results indicate that regulatory mechanisms underlying cell type–specific expression and inducible expression are largely distinct between these 2 types of high-affinity VEGF receptors.

Although the mitogenic activity of VEGF has been reported to be endothelial cell specific, recent reports have documented that VEGF is able to stimulate chemotaxis and thrombogenic protein tissue factor production in monocytes.14 17 These studies have indicated that VEGF-stimulated activity in monocytes is mediated by flt-1 expression.

In view of the ample evidence indicating the critical role of macrophages in the formation of atherosclerotic lesions,18 the accumulation of circulating monocytes and macrophages within the vascular wall is of particular importance for the initiation and progression of atherosclerosis.19 20 21 22 23 In addition, an increased production of tissue factor by macrophages contributes to the development of vaso-occlusive disease, which tends to culminate in acute coronary syndrome.2 24 In this regard, flt-1–mediated monocyte migration and tissue factor production are particularly relevant to the pathobiology of vascular disease. However, the molecular mechanisms by which flt-1 gene expression is regulated in monocytes and macrophages remain to be elucidated.

To determine the molecular mechanisms of gene expression during the process of macrophage differentiation, previous studies took advantage of the in vitro system in which human myeloblastic leukemia HL-60 cells can be induced for macrophage differentiation with phorbol 12-myristate 13-acetate (PMA). THP-1 human monocytic leukemia cells proliferate autonomously and are induced with PMA to undergo terminal differentiation along the macrophage lineage and to experience growth arrest.25 The zinc finger transcription factor Egr-1 was initially identified as an immediate-early growth response gene that is activated by serum stimulation.26 27 28 29 Subsequent studies have shown that Egr-1 is induced in response to B-cell maturation and during differentiation of nerve, bone, and myeloid cells.30 31 32 Libermann and colleagues33 34 35 have cloned Egr-1 cDNA among primary response genes that are activated in the absence of de novo protein synthesis after induction for macrophage differentiation. They have demonstrated that antisense oligomers for Egr-1 block the macrophage differentiation of HL-60 cells and that ectopic expression of Egr-1 restricts differentiation of HL-60 cells or M1 myeloid leukemia cells along the macrophage lineage, thus suggesting that Egr-1 is essential for macrophage differentiation. To date, however, few genes have been identified as Egr-1 target genes in monocytes and macrophages.

In the present study, we have used THP-1 cells to investigate the molecular mechanisms by which the flt-1 gene is activated during macrophage differentiation in the presence of PMA. We report that flt-1 mRNA expression is rapidly induced on PMA stimulation before an induction of macrophage-specific differentiation markers. Transient transfection analyses coupled with the gel mobility shift assays indicate that Egr-1 plays a major role in inducing flt-1 gene expression. The results of the present study provide evidence that the flt-1 gene is a direct target of Egr-1 and further substantiate the importance of Egr-1 in regulating the expression of genes relevant to vascular disease.


*    Methods
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Cell Cultures
THP-1 cells (obtained from American Type Culture Collection [ATCC]) were grown in RPMI 1640 medium supplemented with 10% (vol/vol) FBS and 25 mmol/L HEPES (pH 7.45). Suspension cell cultures of THP-1 cells were grown to a density of 1x106 cells per milliliter, and other cultured cells were grown to confluence. THP-1 cells were incubated in serum-free RPMI 1640 for at least 12 hours before PMA treatment. Human embryonic kidney cells (designated 293 cells) obtained from ATCC were grown in DMEM supplemented with 10% (vol/vol) FBS. Cells were incubated in a humidified incubator equilibrated with 5% CO2 at 37°C.

Reagents
PMA was purchased from Sigma Chemical Co. Affinity-purified rabbit polyclonal antibodies for flt-1 (C-17), Sp1 (PEP2), Sp3 (D-20), and Egr-1 (588) were purchased from Santa Cruz Biotechnology.

Northern Blot Analysis
Total RNA (15 µg per lane) was electrophoresed on 1.2% agarose gels containing 2.2 mol/L formaldehyde and then transferred onto Hybond-N+ membranes (Amersham). Northern blots were hybridized with Egr-1 cDNA probe labeled with [{alpha}-32P]dCTP (Amersham) by using random hexanucleotide primers (Boehringer-Mannheim), washed stepwise to a final stringency of 0.1x SSC and 0.1% SDS at 50°C, and exposed to Kodak XAR-5 film. Densitometric scanning was performed to quantify the amounts of mRNA with the use of Fuji BAS 2000 image analyzer (Fuji Photo Film). Mouse Egr-1 cDNA and human lipoprotein lipase (LPL) cDNA were obtained from ATCC and Riken Gene Bank, respectively.

Reverse Transcription–Polymerase Chain Reaction
Total RNA (2.5 µg per sample) was reverse-transcribed with oligo(dT) primer with the use of AMV reverse transcriptase. Reverse-transcribed materials were amplified with Taq DNA polymerase (Takara) by adding sense and antisense primers specific for human flt-1 (sense primer, 5'-CAGCGGCTTTTGTGGAAG-ACTCAC-3'; antisense primer, 5'-ACATCTCGGTGTCACTT-CTTGGAC-3') and human VEGF (sense primer, 5'-GAACTT-TCTGCTGTCTTGGG-3'; antisense primer, 5'-TCACCGCCTCG-GC-TTGTCAC-3'). GAPDH amplification was performed in the same way to allow relative quantification of polymerase chain reaction (PCR) products (sense primer, 5'-ACCACAGTCC-ATGCCATCAC-3'; antisense primer, 5'-TCCACCACCCTG-TTGCTGTA-3'). For PCR, 30 cycles were used at 94°C for 30 seconds, 60°C for 30 seconds, and 72°C for 90 seconds. The amplified fragments were separated in a 1% agarose gel and detected by Southern blotting.

Western Blot Analysis
Whole-cell protein extracts from THP-1 cells were loaded onto SDS–7.5% polyacrylamide gels and electroblotted on nitrocellulose membranes. The membrane was blocked with 5% nonfat dry milk in Tris-buffered saline (TBS)-T and then probed with anti–flt-1, anti–Egr-1, and anti-Sp1 antibodies for 30 minutes at room temperature. After incubation with primary antibody, the blot was washed 3 times in TBS-T, incubated for another 1 hour with 1:3000 of goat anti-rabbit horseradish peroxidase antibody, and developed with a chemiluminescence kit (Amersham) at room temperature, followed by exposure to Kodak XAR-5 film.

Preparation of Nuclear Extracts
Nuclear extracts were prepared from THP-1 cells according to the modified procedure described previously.36 Briefly, 1x106 cells were washed with 10 mL TBS and pelleted by centrifugation at 1500 rpm for 5 minutes. Then the pellet was resuspended in 1 mL TBS, transferred into an Eppendorf tube, and pelleted again by spinning for 15 seconds in a microfuge. TBS was removed, and the cell pellet was resuspended in 400 µL cold buffer A (mmol/L: HEPES 10 [pH 7.9], KCl 10, EDTA 0.1, EGTA 0.1, dithiothreitol 1, phenylmethylsulfonyl fluoride 0.5, leupeptin 1, and aprotinin 1) by gentle pipetting. The cells were allowed to swell on ice for 15 minutes, after which 25 µL of a 10% solution of Nonidet NP-40 (Calbiochem) was added, and the tube was vigorously vortexed for 10 seconds. The homogenate was centrifuged for 30 seconds in a microfuge. Then the supernatant was removed, and the nuclear pellet was resuspended in 50 µL of ice-cold buffer C (20 mmol/L HEPES [pH 7.9], 0.4 mol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1 mmol/L dithiothreitol, 1 mmol/L phenylmethylsulfonyl fluoride, 1 mmol/L leupeptin, and 1 mmol/L aprotinin), and the tube was vigorously rocked at 4°C for 15 minutes on a shaking platform. The nuclear extract was then centrifuged for 10 minutes in a microfuge at 4°C.

Electrophoretic Mobility Shift Assay
The sequences of the sense strand of double-stranded oligonucleotides used as probes or competitors in electrophoretic mobility shift assays (EMSAs) were as follows, with the consensus motif underlined and mutations of the wild-type sequence in boldface: -180/-160, 5'-CTCGTCGGCCCCCGCCCCTCT-3'; -180/-160M, 5'-CTCGTCGGCAAACGCCCCTCT-3'; Egr-1 consensus, 5'-CGC-CCTCGCCCCCGCGCCGGG-3'; Sp1 consensus, 5'-ATTCGAT-CGGGGCGGGGCGAGC-3'; AP-2 consensus, 5'-GATCGAA-CTGACCGCCCGCGGCCCGT-3'; and cAMP-responsive element (CRE) binding protein (CREB) consensus, 5'-AGAGATTGCCTG-ACGTCAGAGAGCTAG-3'.

All probes were 5'-end labeled with T4-polynucleotide kinase and [{gamma}-32P]ATP (Amersham). For competition experiments, unless otherwise indicated, 50 ng of unlabeled oligonucleotide was mixed with 0.5 ng of the labeled probe before addition of nuclear extracts. For supershift assay, nuclear extracts were preincubated with 1 to 2 µL of anti–Egr-1, anti-Sp1, anti-Sp3, or anti-CREB polyclonal antibodies (Santa Cruz Biotechnology) in the binding buffer for 1 hour at 4°C before initiation of the binding reaction. 32P-labeled probes were then added, the incubation was continued for an additional 20 minutes before electrophoretic separation on 4.5% to 5% polyacrylamide gel at 100 V, and gels were dried and exposed to Kodak XAR-5 film.

Expression Plasmids
For generation of the Egr-1 expression plasmid, the coding region of mouse Egr-1 cDNA (obtained from ATCC) was amplified by PCR by using the upstream primer with an EcoRI site (underlined), 5'-GGGGAATTCTCCAGCTCGCTG GTCCGGGAT-3', and the reverse primer with an XbaI site (underlined), 5'-GGGTCTAGA-CCTTTAGCAAATTTCAATTGT-3'. The PCR product was gel-purified, digested, and subcloned into the EcoRI/XbaI sites of pcDNA3 (Invitrogen).

Preparation of Plasmids for Luciferase Assay
The human flt-1 promoter coupled to the chloramphenicol acetyltransferase (CAT) reporter gene pPN00CAT (-1629 to +278) was kindly provided by Dr M. Shibuya (Tokyo University, Tokyo, Japan).37 For generation of luciferase reporter genes, the following forward primers with a KpnI site (underlined) preceded by several nucleotides were used in a PCR reaction with a plasmid pPN00CAT (-1629 to +278) as a template with the reverse primer (nucleotide +62) with a HindIII site (underlined), 5'-GGGAAGCTTGCCGGG-GAGGAGCCGAGAGGAGTGTCC-3'. Sequences for forward primers were as follows: -591Luc, 5'-GGGGGTACCTTGTGCC-GAGGGTCTCCGGTGCCTTCC-3'; -298Luc, 5'-TTTGGTACCA-GGAGGAGGGGCAAGGGCAAGAGG-3'; and -110Luc, 5'-CCCGGTACCGAGGCGGATGAGGGGTGG-3'.

PCR products were gel-purified, digested, and subcloned into the KpnI/HindIII sites of the promoterless luciferase reporter gene vector, pGL3 (Promega).

For generation of site-directed mutants of the Egr-1 binding sequence in the flt-1 promoter, recombinant PCR with 2 rounds of amplification was performed as described previously.38 The PCR primers (mutations of wild-type sequence appear in boldface) for mutation were 5'-CTCGTCGGCAAACGCCCCTCT-3' (sense) and 5'-AGAGGGGCGTTTGCCGACGAG-3'(antisense). In brief, sense and antisense primers with the corresponding mutations were synthesized and incubated in separate reaction tubes with -1629Luc as template and with an upstream primer (nucleotide -298) and reverse primer (nucleotide +62), thus yielding 2 subfragments that each contained the appropriate mutation. Subfragments were gel-purified and annealed, and a second round of PCR was performed with the upstream primer (nucleotide -298) and reverse primer (nucleotide +62). The PCR products were isolated and subcloned into the KpnI/HindIII sites of pGL3 as described above. The resultant plasmid was designated as flt-298(Egr-1m/Sp1m). For generation of the Egr-1 promoter luciferase construct, the mouse Egr-1 genomic sequence spanning from -363 to +80 relative to the transcription start site was amplified by using the following forward and reverse primers with the KpnI site (underlined) and HindIII site (underlined), respectively: forward primer, 5'-CCCGGTACCTTTCCC-CAGCGCCTTATATGG-3'; reverse primer, 5'-CCCAAGCTTCCG-ATCTTGCGGCGGCGGAAG-3'. The resultant plasmid was designated as Egr-1Luc. All constructs were verified by sequencing the inserts and flanking region in the plasmids.

DNA Transfection and Luciferase Assay
Transfection of the THP-1 cells has been performed by the electroporation method, as previously described.39 Briefly, suspension cultures of 5x106 cells were transfected with 5 µg of reporter gene along with 5 µg of expression plasmid or empty vector by setting the capacitance at 950 µF and voltage at 250 V. One hour after transfection, THP-1 cells were exposed to PMA (100 ng/mL) for 4 hours and were harvested for luciferase assays. Transfection of the 293 cells has been performed by the calcium phosphate precipitation method as previously described.40 Cells were plated at 1x106 cells per 6-cm plate, and 1 µg of the appropriate reporter construct was transfected along with 1 µg of Egr-1/pcDNA3 or pcDNA3 (Invitrogen). After 24 hours, transfected 293 cells were exposed to PMA (100 ng/mL) for 4 hours and were harvested for luciferase assays. Luciferase activity was measured in duplicate for all samples with a Lumat LB9507 luminometer and the Promega luciferase assay system.


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Expression of Flt-1 mRNA and Protein Is Induced by PMA in THP-1 Cells
To determine whether flt-1 mRNA levels are regulated during the differentiation of THP-1 cells to macrophages, we measured the flt-1 mRNA levels at the various time points after induction of differentiation with PMA (100 ng/mL). As shown in Figure 1Down, flt-1 mRNA expression (normalized to GAPDH), as measured by reverse transcription–PCR analysis, was relatively low in untreated THP-1 cells and was markedly increased a maximum of {approx}14.0-fold (P<0.05, n=5) compared with the unstimulated control in response to PMA within 1 hour. Flt-1 expression remained above baseline at all later time points tested (2 to 24 hours). In contrast, flt-1 ligand VEGF mRNA levels appeared to be unchanged by PMA stimulation in THP-1 cells. Western blot analysis showed a significant induction of flt-1 protein at 2 hours after PMA stimulation (bottom of Figure 1ADown).



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  		Figure 1. Time course of PMA-induced flt-1, VEGF, Egr-1, and LPL expression in THP-1 cells. A, THP-1 cells were cultured in 10-cm dishes for 2 days and then were made quiescent for 24 hours before stimulation with PMA (100 ng/mL). For reverse transcription (RT)-PCR, total RNA (2.5 µg) was extracted at indicated times after stimulation and reverse-transcribed by using the oligo(dT) as a primer. Resulting cDNAs were amplified by PCR with specific primers for flt-1, VEGF, and GAPDH. Data are representative of 3 independent experiments. For Northern blot analysis, total RNA (10 µg) was extracted from THP-1 cells at indicated times after stimulation with PMA and analyzed by Northern blotting for Egr-1 and LPL mRNAs. 18S ribosomal RNA stained by methylene blue indicates that comparable amounts of total RNA actually blotted onto a membrane. For Western blot analysis, flt-1, Egr-1, and Sp1 protein expression in whole cell-extracts from THP-1 cells were analyzed at indicated times after PMA stimulation. B, Each value represents the relative flt-1 (•, normalized to GAPDH) and Egr-1 ({circ}, normalized to 18S) mRNA levels expressed by mean±SE of 3 experiments performed. *P<0.05 vs untreated controls.

Induction of Egr-1 mRNA and Protein by PMA in THP-1 Cells
PMA rapidly induces Egr-1 mRNA expression during terminal monocytic differentiation of HL-60 and U937.33 To determine whether monocytic leukemia THP-1 cells are capable of expressing Egr-1 after exposure to PMA, we isolated total RNA from THP-1 cells treated with vehicle (dimethyl sulfoxide) or with PMA for various lengths of time. Northern blot analysis showed that PMA caused a large and rapid increase in Egr-1 mRNA expression in THP-1 cells (middle of Figure 1AUp). At 1 hour, Egr-1 expression (normalized to 18S) increased 11.5-fold (P<0.05, n=4) in THP-1 cells compared with untreated cells. Egr-1 expression levels returned to control levels at 4 hours. Consistent with these findings, Western blot analysis showed a robust increase in Egr-1 protein levels at 2 to 4 hours after stimulation with PMA. In contrast, Sp1 protein levels were not affected by PMA treatment.

PMA-Induced Flt-1 Expression Precedes Induction of Macrophage-Specific Gene Expression
THP-1 cells are capable of expressing macrophage-specific genes after induction of macrophage differentiation with PMA. We examined the time course of expression of the macrophage-specific genes, such as LPL and scavenger receptor-A, by Northern blot analysis. As shown in Figure 1Up, an induction of LPL mRNA levels was evident at 24 hours after stimulation. Expression of scavenger receptor-A exhibited the same time course (data not shown) as that of LPL. These results suggest that induced expression of the flt-1 gene, which occurs within 1 hour after PMA treatment, is regulated by mechanisms distinct from those of macrophage-specific genes.

PMA Induces Flt-1 Promoter Activity
To determine whether PMA induces flt-1 mRNA expression at the transcriptional level, we transfected THP-1 cells with the luciferase-reporter construct containing the flt-1 promoter region (from -591 to +62) coupled to the 5' end of the luciferase reporter gene, designated as flt-591Luc. As shown in Figure 2Down, measurement of the luciferase activity of the transfected cells revealed that PMA increases the luciferase activity of flt-591Luc by 2.2-fold (P<0.05, n=6) compared with the activity in cells treated with vehicle. Likewise, luciferase activity derived from the Egr-1 promoter (from -363 to +80) was increased by 2.6-fold after PMA stimulation (P<0.05, n=6). In contrast, luciferase activity driven by thymidine kinase basal promoter was only minimally affected by PMA, thus suggesting that PMA induces luciferase activity from flt-1 and Egr-1 promoters in a promoter-specific manner in THP-1 cells. To examine whether induction of the flt-1 promoter by PMA is THP-1 cell specific, 293 cells (an epithelial cell line derived from human embryonic kidney) were transfected with flt-591Luc and treated with PMA. The results showed that PMA increases flt-1 promoter activity in 293 cells by 3.3-fold (Figure 2Down, P<0.05, n=5), suggesting that an induction of flt-1 expression is not cell-type specific.



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  		Figure 2. Induction of flt-1 and Egr-1 promoter activity by PMA in THP-1 cells. THP-1 cells and 293 cells were transfected with the indicated reporter genes by electroporation and calcium phosphate methods, respectively. Transfected cells were incubated with vehicle or PMA (100 ng/mL) for 4 hours as described in Methods. The fold induction was calculated by dividing the luciferase activity values of samples treated with PMA by the activity value of untreated control samples. TK-Luc represents the thymidine kinase promoter coupled to luciferase vector. All results represent mean±SE of at least 3 experiments in duplicate.

Mapping of PMA Response Region Within Flt-1 Promoter
To delineate the promoter region that mediates PMA-induced THP-1 promoter activation, transient transfection of THP-1 cells with a series of 5'-deletion mutants was performed. As shown in Figure 3Down, the construct flt-298Luc, which contains the flt-1 promoter sequence from -298 to +62, displayed the modest but reproducible induction of luciferase activity in response to PMA. Further deletion to -110, however, resulted in a significant attenuation of PMA-mediated induction of flt-1 promoter activity. Similar but more remarkable results were obtained by transfection into 293 cells. These results suggest that the PMA-responsive region is localized to the sequence between -298 and -110 in THP-1 cells and in 293 cells.



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  		Figure 3. Mapping of cis-regulatory elements involved in an induction of flt-1 promoter by PMA. THP-1 cells (top) or 293 cells (bottom) were transfected with a series of 5'-deletion constructs of human flt-1 luciferase reporter plasmids. Transfected cells were incubated with vehicle or PMA (100 ng/mL) for 4 hours as described in Methods. The fold induction was calculated by dividing the luciferase activity values of samples treated with PMA by the activity value of untreated control samples. All results represent mean±SE of at least 3 experiments in duplicate.

Identification of PMA-Dependent Complex Containing Egr-1 by EMSAs
The observations described above prompted us to search for the sequence elements that can function as potential binding sites for PMA-inducible nuclear factors between -298 and -110. As shown in Figure 4ADown, the flt-1 promoter contains a G+C-rich sequence between -180 and -160 that includes the GGCCCCCGC motif (from -174 to -166), which is similar to the Egr-1 binding site, and the CCCCGCCCC motif, which is identical to the Sp1 binding site (from -171 to -163). To determine whether this G+C-rich sequence can serve as a binding site for Egr-1 and Sp1, EMSA was performed. After incubation of the radiolabeled oligonucleotide probe spanning this site (-180 and -160) with the nuclear extracts from vehicle-treated and PMA-treated THP-1 cells, reaction solutions were separated in 5.0% polyacrylamide gel. The results shown in Figure 4CDown demonstrate the presence of 5 major complexes in unstimulated control cells (Figure 4CDown, C1 to C5). These complexes, except for C4, were sequence specific, because a molar excess of unlabeled specific oligonucleotide but not of unrelated oligonucleotide CREB impaired the complex formation. The complex C4 was a nonspecific complex because it was not affected by a molar excess of unlabeled probe. Note that C1, C3, and C5 appear to contain members of the Sp family, because binding displayed efficient competition with an unlabeled Sp1 consensus motif. Complex C2 seems to contain Egr-1 because the Egr-1 consensus motif completely abolished the complex formation. It is of importance to note that PMA treatment induced a major complex with a mobility identical to that of C2. This complex is completely competed by the unlabeled Egr-1 consensus motif but not by the Sp1, AP-2, and CREB consensus motifs.



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  		Figure 4. EMSA of the potential binding site for Egr-1. A, Nucleotide sequence of the human flt-1 promoter region between -310 and -71. Sequence elements similar to Egr-1 and Sp1 are boxed and underlined, respectively. The Ets binding site and CRE are indicated by double underlining and boldface letters, respectively. B, Nucleotide sequences of double-stranded -180/-160 probe and its mutation oligonucleotide used as a competitor. The putative Egr-1 binding site (GGCCCCCGC), which closely matches consensus Egr-1 binding motif (CGCCCCCGC), and consensus Sp1-binding site (CCCGCC) were boxed and underlined, respectively. Three point-mutations introduced are indicated by boldface letters. C, Nuclear extracts (10 µg) prepared from either unstimulated or PMA-treated THP-1 cells and incubated with 32P-labeled -180/-160 probe, which contains a sequence similar to the consensus Egr-1 binding site, in the absence or presence of a 100-fold molar excess of indicated unlabeled competitors. Positions of the sequence-specific DNA-protein complexes (C1, C2, C3, and C5) and nonspecific bindings (NS) are indicated. Sp denotes the factors that belong to the Sp family of transcription factors.

To further demonstrate the effects of mutation, which disrupts Egr-1 and Sp1 consensus sequences on the nuclear factor bindings, an unlabeled oligonucleotide bearing 3 point-mutations within the overlapping Egr-1/Sp1 binding sites was used as a competitor (Figure 4BUp). Addition of a 100-fold molar excess of mutant competitor did not interfere with any of the specific complexes (C1, C2, C3, or C5), thus indicating that 3 bases (CCC, from -171 to -169) within the Egr-1/Sp1 site are involved in the binding of Egr-1 and Sp1.

Identification of Nuclear Protein Components in Shifted Complexes
To directly determine the nuclear proteins present in the shifted complexes that we have identified, gel supershift experiments were performed. Nuclear extracts from unstimulated THP-1 cells were incubated with antibodies against Egr-1, Sp1, Sp3, and unrelated CREB before binding to the -180/-160 probe. After adding the probe, DNA-protein complexes were separated on the 4.5% acrylamide gel instead of the 5% gel to better detect the supershifted complexes. As seen in Figure 5Down, complex C1 turned out to be composed of 2 distinct complexes, C1a and C1b. Supershift assays revealed that C1a and C1b contain Sp1 and Sp3, respectively, because anti-Sp1 antibody supershifted complex C1 and anti-Sp3 antibody supershifted complex C1b. It should be noted that the binding of the PMA-dependent complex (C2) is affected by anti–Egr-1 but not by anti-Sp1, anti-Sp3, or anti-CREB antibodies, thus indicating that Egr-1 is induced by PMA to bind to the Egr-1 site. Sp3 is a major participant in complex C3. Complexes C4 and C5, which were detected on 5% gel, did not form discrete bands on 4.5% gel (data not shown).



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  		Figure 5. Supershift assay for the potential binding site for Egr-1. Nuclear extracts (10 µg) from either unstimulated or PMA-stimulated THP-1 cells were incubated with 1 µL of specific antibodies against Sp1, Sp3, Egr-1, or CREB for 1 hour at 4°C before adding the 32P-labeled -180/-160 probe. The DNA binding and gel mobility shift assays were performed as described in Methods. Positions of the supershifted complexes and sequence-specific DNA-protein complexes (C1a, C1b, C2 and C3) are indicated.

Overexpression of Egr-1 Activates Flt-1 Promoter
To determine the functionality of the Egr-1 binding site located at -174, THP-1 cells were cotransfected with the flt-298Luc construct and Egr-1 expression vector. As can been seen from Figure 6Down, luciferase expression of flt-298Luc was substantially activated by the Egr-1 expression vector but not by an empty vector. The role of the Egr-1/Sp1 site was further evaluated by introducing a mutation that disrupts the binding of Egr-1. Mutation of the site completely eliminated the induction of Egr-1–induced promoter activity. This mutation also abrogated the effect of PMA on flt-1 promoter activity. These data demonstrate the importance of a sequence containing the Egr-1/Sp1 site for inducible expression of the flt-1 promoter in response to PMA and Egr-1 expression. We obtained the same results with 293 cells (bottom of Figure 6BDown).



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  		Figure 6. Effect of the mutation disrupting the consensus Egr-1–binding sequence on flt-1 promoter activity. A, Schematic representation shows the wild-type -298Luc and -298(Egr-1m/Sp1m)Luc, in which 3 point-mutations (indicated by boldface letters) are introduced to overlapping Egr-1/Sp1 site (boxed). B, THP-1 cells (top) or 293 cells (bottom) were transfected with the indicated reporter plasmids and, if indicated, along with the Egr-1 expression plasmid (Egr-1/pcDNA3) or the empty vector pcDNA3. Transfected cells were incubated with vehicle or PMA (100 ng/mL) for 4 hours as described in Methods. The fold induction was calculated by dividing the luciferase activity values of samples treated with PMA by the activity value of untreated control samples (solid bars). Effects of Egr-1 expression vector were expressed by fold induction calculated by dividing the luciferase activity values of samples cotransfected with Egr-1/pcDNA3 by the activity value of samples cotransfected with pcDNA3 (open bars). All results represent mean±SE of at least 3 experiments in duplicate.


*    Discussion
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In the present study, we demonstrate that expression of the flt-1 gene is transcriptionally activated by Egr-1 on stimulation of THP-1 human monocytic leukemia cells with PMA. Because THP-1 cells undergo differentiation exclusively along the macrophage lineage by PMA, these findings provide the first experimental evidence that flt-1 gene expression is regulated during macrophage differentiation. Previous studies have demonstrated that Egr-1 is essential for restricting differentiation to macrophage differentiation.33 34 35 They showed that forced expression of Egr-1 in HL-60 cells or in hematopoietic precursor cell line 32Dcl3, which can be induced to undergo either granulocytic or macrophage differentiation, restricts differentiation of the cells to the macrophage lineage and that blocking Egr-1 expression by antisense oligonucleotides results in repression of macrophage differentiation. However, Egr-1 target genes have not yet been documented. Because expression of the flt-1 gene is cell-type–restricted, the finding that the flt-1 gene is a direct target of Egr-1 will expand our knowledge of the role of Egr-1 in promoting macrophage differentiation.

At present, the functional consequence of an enhanced expression of the flt-1 gene during macrophage differentiation cannot be evaluated in depth. Recent studies,14 17 however, have shown that VEGF stimulates chemotaxis and production of thrombogenic protein tissue factor in monocytes. Interestingly, Hiratsuka et al41 have recently demonstrated that flt-1 tyrosine kinase–deficient homozygous mice, which express the extracellular domain but not the kinase domain of flt-1, survive normally without marked abnormality of blood vessels but exhibit impaired macrophage migration in response to VEGF. In this regard, an increase in expression of the flt-1 gene in response to PMA may be a relevant event to the increased monocytic recruitment and procoagulant activity seen in atherosclerotic lesions even though macrophage differentiation per se would not be a direct consequence of an induction of flt-1 gene expression.

Accumulating evidence suggests that Egr-1 plays a major part in the induction of many genes in endothelial cells in response to sheer stress and vascular injury.42 It has been demonstrated that vascular injury by balloon catheter rapidly and transiently induces Egr-1, which, in turn, trans-activates PDGF-B chain gene expression through binding to cognate binding sites located within the proximal promoter region.42 43 Besides the PDGF-B promoter, the Egr-1 binding sites are found in tissue factor, transforming growth factor-ß1, and tissue plasminogen activator genes, all of which are relevant to vascular disease.42 44 Because inflammation involving activation of macrophages is a critical event for plaque formation,2 the results of the present study will further support the notion that Egr-1 functions as a primary response gene in vascular disease, corroborating the findings with endothelial cells.

Recent studies have shown that the flt-1 gene transcript and its protein and the VEGF transcript are detected in proliferating vascular SMCs in the neointima.15 45 Furthermore, flt-1 has been postulated to mediate the VEGF-mediated upregulation of matrix metalloproteinase (MMP1 and MMP9) expression in vascular SMCs,7 although the molecular mechanisms underlying flt-1 gene expression have not been fully defined. It is likely that Egr-1 is responsible for an increase in flt-1 gene expression in vascular SMCs because our recent immunohistochemistry revealed that Egr-1 is strongly induced in neointimal SMCs after balloon injury of the rat aorta (data not shown). Furthermore, results of the transient transfection assays indicate that the Egr-1 binding site is functional in vascular SMCs (data not shown), 293 cells, and THP-1 cells (Figure 6Up). Thus, activation of the flt-1 promoter by Egr-1 does not require cell-type–specific transcription factors. In this regard, stimuli that induce Egr-1 expression are sufficient for upregulation of flt-1 gene expression.

We showed that the G+C-rich region identified as a PMA-responsive region can serve as a binding sequence for Egr-1, Sp1, and Sp3. Cis-regulatory elements, which are identical or similar to the Egr-1 consensus sequence, overlap the Sp1/Sp3 binding sites in many Egr-1 target genes. Khachigian et al42 have shown that Egr-1 binds to a cryptic element overlapping the Sp1 site in the PDGF-B promoter. They proposed a model in which Sp1 occupies the GC-box in the PDGF-B promoter and Egr-1 displaces prebound Sp1 on denudation of aortic endothelium. In these studies, Sp1 contributes to basal levels of expression, whereas Egr-1 functions as an inducible transcription in response to vascular injury. Likewise, an induction of tissue factor gene expression by 12-0-tetradecanoylphorbol-13-acetate in HeLa cells or sheer stress in endothelial cells is mediated through inducible binding to the Egr-1 site with the concomitant displacement of Sp1 from the overlapping Sp1 site.46 47 Unlike Sp1, Sp3 has been identified as a bifunctional transcription factor that can both activate and repress transcription.48 49 In the present study, the binding affinity of Sp1 and Sp3 to the probe is not reproducibly affected by PMA. Thus, Sp1/Sp3 may not play a major role in PMA-mediated flt-1 gene expression, although the regulation of the trans-activation function of Sp1/Sp3 by phosphorylation cannot be excluded.

Previous studies50 51 have reported that the flt-1 promoter contains a functional Ets-binding site as well as a CRE. Because the Ets-binding site has been reported to mediate the effects of PMA on the expression of the target genes,52 it is formally possible that the Ets-binding site plays a role in inducing flt-1 promoter activity in response to PMA. However, our data indicate that the PMA responsiveness relies solely on the Egr-1/Sp1 site at -174 because mutations of this site completely abrogated the response to PMA.

In summary, we demonstrate that Egr-1 plays an important role in inducing transcription of the flt-1 gene on induction of THP-1 for macrophage differentiation. Identification of the Egr-1 target gene whose expression is cell-type–restricted and is relevant to the function of macrophage will considerably increase our understanding of Egr-1 as a key regulator in the process of macrophage activation and differentiation. Given that activation of monocytes/macrophages is a critical process in inflammatory reaction and immune response, the results of the present study suggest that Egr-1 plays an important role in the development of atherosclerosis in which macrophages as well as T cells actively participate.


*    Acknowledgments
 
This study was supported in part by a grant-in-aid for scientific research from the Ministry of Education, Science, Sports, and Culture of Japan and a grant from the Japan Cardiovascular Foundation (to Drs Kurabayashi and Nagai). We are grateful to Dr M. Shibuya for a generous gift of the plasmid containing human flt-1 promoter.

Received July 6, 1999; accepted September 1, 1999.


*    References
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