The Egr-1 gene is induced by epidermal growth factor in ECV304 cells and primary endothelial cells

Jo C. Tsai, Lixin Liu, Jiazhen Guan, and William C. Aird

Department of Molecular Medicine, Beth Israel Deaconess Medical Center, Boston, Massachusetts 02215


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The early growth response (Egr)-1 transcription factor serves to couple changes in the extracellular environment to alterations in gene expression. An understanding of the mechanisms that underlie Egr-1 gene regulation should provide important insights into how environmental signals are transduced by endothelial cells. The aim of the present study was to determine whether epidermal growth factor (EGF) induces Egr-1 expression in endothelial cells. In ECV304 cells, Egr-1 mRNA and protein levels were increased in response to EGF. In stable transfection assays, the 1,200-bp promoter of the mouse Egr-1 gene contained information for EGF response via a protein kinase C-independent, mitogen-activated protein kinase-dependent pathway. The endogenous Egr-1 gene was similarly responsive to EGF in primary human umbilical vein endothelial cells, human coronary artery endothelial cells, and rat fat pad endothelial cells, but not in bovine aortic endothelial cells, calf pulmonary artery endothelial cells, or PY-4-1 endothelial cells. Together, these results suggest that the Egr-1 gene is responsive to EGF in a subset of endothelial cells.

gene regulation; growth factors; endothelium


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE ENDOTHELIAL LINING OF the vascular wall participates in a wide variety of homeostatic processes, including vasomotor control, hemostasis, and the transfer of cells and nutrients between blood and underlying tissue (7). Endothelial cell morphology and function vary among different segments of the vascular tree. Phenotypic heterogeneity is largely programmed by signals residing in the microenvironment (1, 7, 29). Indeed, endothelial cells have been shown to respond to a wide array of growth factors, cytokines, and hemodynamic forces. At the present time, the mechanisms by which extracellular signals are transduced by the endothelium are poorly defined. An understanding of the pathways that mediate phenotypic expression in endothelial cells would provide insight into the molecular basis of vascular diversity in both health and disease states.

One approach to this problem is to study the mechanisms of endothelial cell-specific gene expression. The immediate-early genes include members of the Fos, Jun, and early growth response (Egr) families. Egr-1 (also designated zif268, TIS 8, NFGI-A, Krox 24) encodes a zinc finger-containing DNA-binding protein in many cell types (5, 12, 21, 22, 27, 38, 42). Egr-1 mRNA and protein levels are upregulated in response to a wide variety of mitogenic and nonmitogenic stimuli, including peptide growth factors, shear stress, urea, and hypotonicity (2, 4, 6, 8, 15, 28, 30, 41). Egr-1 binds to 5'-GCG(G/T)GGGCG-3' consensus sequences within the promoter region of target genes, resulting in transcriptional activation or repression of the gene. Therefore, Egr-1 functions as an intermediary in signal transduction, coupling short-term changes in the environment to long-term changes in gene expression.

Egr-1 has been implicated in the transcriptional regulation of multiple genes within the endothelium, including platelet-derived growth factor (PDGF)-A, PDGF-B, tissue factor, transforming growth factor-beta (TGF-beta ), and urokinase-type plasminogen activator (18, 36). However, the mechanisms by which the Egr-1 gene is itself regulated in endothelial cells remain poorly understood. The Egr-1 gene has been shown to be induced in human umbilical arterial endothelial cells by basic fibroblast growth factor (bFGF) and in bovine aortic endothelial cells (BAECs) by acidic FGF (aFGF) (9, 20). In BAECs, the response of Egr-1 to injury has been shown to be mediated by a bFGF-dependent signaling pathway (32). Egr-1 expression in human aortic endothelial cells is increased in response to fluid shear stress (34). Finally, under in vivo conditions, levels of Egr-1 protein and mRNA rapidly increase at the wound edges of denuded rat aortic endothelium (19). Together, these findings suggest that the Egr-1 transcription factor may be involved in coordinating expression of environmentally dependent gene programs.

In the present study, we show that the Egr-1 gene is induced by epidermal growth factor (EGF) in both the ECV304 cell line and primary endothelial cells. Induction of Egr-1 by EGF is mediated by a protein kinase C (PKC)-independent, mitogen-activated protein kinase (MAPK)-dependent pathway that converges on the serum response elements (SREs) within the 5' flanking region of the Egr-1 promoter. Importantly, we demonstrate that the response of the Egr-1 gene to EGF and other growth factors varies among different types of cultured endothelial cells. These results suggest that the Egr-1 gene is regulated by cell type-specific signal transduction pathways.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Plasmid construction. A Sal I DNA fragment containing the 1,200-bp murine Egr-1 promoter was isolated from pEgr-1p1.2 [a generous gift from Vikas Sukhatme, Beth Israel Deaconess Medical Center (BIDMC), Boston, MA] and ligated into the Xho I site of the pGL2 basic vector (Promega, Madison, WI) to create Egr-1-Luc. The Egr-1 promoter constructs A-G and V were kindly provided by David Cohen (Oregon Health Sciences University). These latter constructs contain variable lengths of the Egr-1 promoter coupled to the luciferase reporter gene in the promoterless vector pXP2 (8). The full-length promoter construct (A) and Egr-1-Luc contain the same 1,200-bp region of the Egr-1 promoter. For stable transfection experiments, Egr-1-Luc was linearized with Sal I, whereas the Egr-1 promoter constructs A-G and V were linearized with Hind III.

The SRE heterologous promoter constructs were generated by PCR. The Egr-1-Luc plasmid served as a template, and the sense and antisense primers contained internal Kpn I and Hind III sites, respectively. The SRE-A fragment, corresponding to sequences between -425 and -250 of the mouse Egr-1 promoter, was synthesized with the forward primer (Kpn I site underlined) (5'-AATTGGTACCGCGCCGACCCGGAAACAC) and the reverse primer (Hind III site underlined) (5'-AATTAAGCTTGGGCTAGGCTCCTGGAGT). The SRE-B fragment, corresponding to sequences between -125 and -70, was synthesized with the primer sets (sense, 5'-AATTGGTACCGTCCTCCCGGTCGGTCCT; antisense, 5'-AATTAAGCTTCATGGCCATATATGGGAA), whereas the SRE-C fragment, corresponding to sequences between -425 and -70, was synthesized with the primer sets (sense, 5'-AATTGGTACCGCGCCGACCCGGAAACAC; antisense, 5'-AATTAAGCTTCATGGCCATATATGGGAA). The PCR products were digested with Kpn I and Hind III, and the resulting DNA fragments were ligated into the Kpn I and Hind III sites of a pGL2-based construct that contained promoter sequences of the human von Willebrand factor (vWF) gene between -90 and +10 coupled to the luciferase cDNA (vWF-90-Luc). All three constructs were linearized with Sal I for stable transfections.

Cell culture. ECV304 cells [American Type Culture Collection (ATCC) CRL-1998] were maintained in M199 medium supplemented with 10% fetal bovine serum (FBS; Life Technologies, Gaithersburg, MD) at 37°C and 5% CO2. The cells were grown to 50-60% confluence and then placed in serum-free M199 medium. Forty-eight hours later, the cells were overlaid with growth factor-supplemented or mock-treated serum-free M199 medium (1:10 volume), and the culture plates were gently swirled. NIH/3T3 cells (ATCC CRL-1658), BAECs (kindly provided by Robert D. Rosenberg, Massachusetts Institute of Technology, Cambridge, MA), calf pulmonary artery endothelial cells (CPAECs), Py-4-1 mouse endothelial cells (a generous gift of Victoria Bautch, University of North Carolina, Chapel Hill, NC), and rat fat pad endothelial cells (RFPECs; a generous gift of Michael Simons, BIDMC, Boston, MA) were grown in DMEM with 10% FBS. At 80-90% confluence, the cells were serum starved in DMEM containing 0.5% FBS for 24 h and subsequently treated with growth factor-supplemented medium for the times indicated. Human umbilical vein endothelial cells (HUVECs) and human coronary artery endothelial cells (HCAECs; Clonetics, San Diego, CA) were grown in endothelial growth medium-2-MV (EGM-2-MV) BulletKit (Clonetics) to confluence and were serum starved in M199 medium supplemented with 0.5% FBS overnight before various treatments with growth factors as described above for ECV304 cells. Human recombinant EGF, PDGF-BB, PDGF-AA, PDGF-AB, aFGF, tumor necrosis factor-alpha (TNF-alpha ), and TGF-beta were purchased from PeproTech (Rocky Hill, NJ). Human recombinant bFGF and vascular endothelial growth factor (VEGF) were generous gifts of Michael Simons (BIDMC, Boston, MA). Phorbol 12-myristate 13-acetate (PMA) was purchased from Calbiochem (La Jolla, CA). In inhibition studies, the serum-starved ECV304 cells were preincubated with 100 µM PD-98059 (New England Biolabs, Beverly, MA), a specific MAPK kinase (MEK) inhibitor, for 1 h and then incubated in the absence or presence of 10 ng/ml EGF for another 1 h. An equal amount of DMSO was added to the control plates.

Western blot analysis of Egr-1 expression. For Western blot analyses, whole cell lysates were prepared from serum-starved cells at various time points following addition of growth factor-supplemented or mock-treated medium. Cells were washed twice with cold PBS, harvested with a cell scraper, and lysed in ice-cold buffer containing 20 mM HEPES, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS, 1 mM EDTA, 1 mM dithiothreiotol, 0.5 mM phenylmethylsulfonyl fluoride, and a protease inhibitor cocktail (Boehringer Mannheim) for 1 h. The resulting lysates were centrifuged at 10,000 g for 10 min, and the supernatants were saved as whole cell lysates. Protein quantitation was carried out with the microprotein determination system (Sigma, St. Louis, MO). Forty micrograms of the whole cell lysates were separated by 10% SDS-PAGE and were electrotransferred to nitrocellulose membranes (Bio-Rad Laboratories, Hercules, CA). Membranes were blocked with 5% nonfat dry milk in Tris-buffered saline with 0.1% Tween 20 for 1 h at room temperature. The blot was incubated with primary rabbit polyclonal anti-Egr-1 IgG (1:1,000 dilution; Santa Cruz Biotechnology, CA) overnight at 4°C, followed by secondary antibody goat anti-rabbit horseradish peroxidase conjugate (1:1,000 dilution; Pierce, Rockford, IL). The blot was washed extensively between each incubation step. Peroxidase activity was visualized with an enhanced chemiluminescence (ECL) substrate system (Amersham, Arlington Heights, IL).

RT-PCR analysis of Egr-1 expression. Total RNA was prepared from ECV304 cells and HUVECs by guanidine thiocyanate-phenol-chloroform extraction (Stratagene, La Jolla, CA). Random-primed first-strand cDNA was synthesized from 2 µg of total RNA using the SuperScript preamplification system (Life Technologies) according to the manufacturer's instructions. One-tenth of the first-strand cDNA was then amplified by PCR with primer sets specific for human Egr-1 (sense, 5'-CAGCAGTCCCATTTACTCAG; antisense, 5'-GACTGGTAGCTGGTATTG) (20) and beta -actin (sense, 5'-GTGGGCCGCTCTAGGCACCAA; antisense, 5'-CTCTTTGATGTCACGCACGATTTC) (Clontech, Palo Alto, CA). PCR reactions were carried out with the following cycle conditions: 95°C for 4 min, 30 cycles of 95°C for 1 min, 60°C for 1 min, and 72°C for 1 min, followed by a final incubation at 72°C for 7 min. One-fifth of the PCR products were electrophoresed on a 1.2% agarose gel, and the PCR products were visualized by ethidium bromide staining. For actinomycin D experiments, ECV304 cells were pretreated with 10 µg/ml actinomycin D (Sigma) for 30 min before addition of EGF and were harvested for total RNA 1 h later.

Stable and transient transfections and luciferase assays. For stable transfections, 107 ECV304 or NIH/3T3 cells were electroporated with 20 µg of linearized Egr-1 luciferase constructs and a 1:10 molar ratio of Dra I-linearized pGK-neo (13) (Gene Pulser II; 300 V and 600 µF; Bio-Rad Laboratories). The cells were grown for 16 days in medium containing 1 mg/ml G418 (Life Technologies). Over 200 G418-resistant clones were trypsinized and pooled, and equal numbers of cells were seeded in 12-well tissue culture plates. The ECV304 transfectants were starved in serum-free M199 medium for 48 h, whereas the NIH/3T3 transfectants were serum starved in DMEM with 0.5% FBS for 24 h before various treatments. The cells were then incubated for 2-26 h with medium containing EGF, aFGF, bFGF, PDGF-AB, PDGF-AA, PDGF-BB, TNF-alpha , TGF-beta , or VEGF at the doses indicated. In inhibition studies, the serum-starved cells were pretreated with the MEK inhibitor PD-98059 at a final concentration of 50 µM or a specific PKC inhibitor bisindolylmaleimide I (BIM; Calbiochem) at a final concentration of 5 µM for 30 min before addition of growth factors. Cells were harvested for luciferase activity according to the manufacturer's instructions (Promega). Light activity was measured in a luminometer (Lumat LB 9507; EG&G Berthold, Germany) and was expressed as relative light units (RLU) ± SD. All experiments were carried out in triplicate and repeated at least four times.

ECV304 cells were transiently cotransfected with the Elk-Gal4 and Gal4-Luc constructs (Stratagene) using Lipofectamine reagents (Life Technologies). Elk-Gal4 consists of the cytomegalovirus (CMV) promoter coupled to a cDNA in which the DNA-binding domain of Gal4 has been fused upstream of the Elk1 activation domain. In the Gal4-Luc construct, the firefly luciferase cDNA is driven by a promoter in which five tandem repeats of the Gal4-binding element are located upstream of a TATA box. Cells were seeded in 12-well plates 24 h before transfection, and all transfections were carried out in triplicate. At 60-70% confluence, the cells were incubated with Lipofectamine:DNA complexes containing 3 µl Lipofectamine, 250 ng Elk-Gal4, 250 ng Gal4-Luc plasmids, and 50 ng of a control plasmid containing the Renilla luciferase reporter gene under the control of a CMV enhancer/promoter (Promega). Six hours later, the transfection mixture was replaced by regular growth medium for 18 h. The transfected cells were then serum deprived for 24 h and treated with growth factors for 4 h as described above. The cells were harvested using the dual-luciferase assay system according to the manufacturer's protocol (Promega). Standard firefly and Renilla luciferase activities were serially measured in a luminometer. Standard luciferase activity was normalized to the activity of the Renilla luciferase reporter gene to correct for transfection efficiency.


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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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The Egr-1 gene is induced in ECV304 cells by replacement of the medium. Simple replacement of serum-free medium after 48 h of serum starvation in ECV304 cells resulted in significant increases in Egr-1 mRNA and protein, with peak levels occurring between 30 and 60 min (Fig. 1, A and B). The Egr-1 gene was also induced by the addition of 10% volume of serum-free medium followed by gentle mixing (data not shown). Preincubation of serum-starved ECV304 cells with a MAPK-specific inhibitor (PD-98059) or a PKC-specific inhibitor (BIM) abolished Egr-1 induction by replacement of medium (Fig. 1C). The lower-than-baseline level of Egr-1 protein in the PD-98059-treated cells suggests that constitutive expression of Egr-1 is mediated by a MAPK signaling pathway. To establish a role of the Egr-1 promoter in transducing this response, we stably transfected ECV304 cells with the Egr-1-Luc construct. Over 200 G418-resistant clones were pooled and seeded at the same density in 12-well tissue culture plates. Luciferase activity was increased 1.8-fold by simply changing the serum-deprived medium (Fig. 1D, Delta  vs. C). The induction was abrogated by the addition of PD-98059 or BIM. These results suggest that the change-of-medium effect is transduced by a PKC-dependent MAPK signal transduction pathway operating through the 5' flanking region of the Egr-1 promoter. The sensitivity of the Egr-1 gene to these manipulations may be explained by transient alterations in hemodynamic forces or perhaps by subtle changes in the microenvironment of the freshly exchanged media. Regardless of the underlying mechanism, the results underscore the importance of including appropriate controls in studies of Egr-1 gene regulation.


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Fig. 1.   The effect of media change on Egr-1 gene expression in ECV304 cells. A: ECV304 cells were incubated in serum free-medium for 48 h, at which time the medium was replaced. Cells were harvested for total RNA and whole cell protein extracts at the times indicated. The control (C) cells were harvested without a change of medium. In RT-PCR assays, RNA-derived cDNA was amplified by PCR with primer pairs specific to human Egr-1 and beta -actin gene transcripts. The size of the transcripts are Egr-1, 345 bp; beta -actin, 540 bp. B: in Western blot analysis, 40 µg of cell lysates were separated by 10% SDS-PAGE, transferred to a nitrocellulose membrane, and incubated with a polyclonal anti-Egr-1 antibody as described in MATERIALS AND METHODS. The Egr-1 protein bands were visualized with the enhanced chemiluminescence system. C: serum-deprived ECV304 cells were preincubated with no inhibitor (Delta ), with 75 µM PD-98059 (Delta +PD), or with 5 µM bisindolylmaleimide (Delta +BIM) for 30 min before a change of medium. Cells were harvested 30 min later for total protein. Control cells (C) were harvested without a change of medium. Egr-1 protein levels were assayed by Western blot analysis. D: ECV304 cells were stably transfected with a construct that contains the 1,200-bp Egr-1 promoter fused to the luciferase reporter gene (Egr-1-Luc). Pools of Egr-1-Luc transfectants were seeded at the same cell density into 12-well tissue culture plates, serum deprived for 48 h, and then replaced with medium containing no inhibitor (Delta ), 50 µM PD-98059 (Delta +PD), or 5 µM BIM (Delta +BIM). Control cells (C) were unmanipulated. All cells were harvested and assayed for luciferase activity 4 h later. Luciferase activity is expressed as relative light units (RLU) ± SD.

The Egr-1 gene is induced in ECV304 cells by EGF but not by PDGF-BB or bFGF. Serum-deprived ECV304 cells were gently overlaid with 10% volume of serum-free M199 medium with or without 10 ng/ml EGF, 10 ng/ml PDGF-BB, or 40 ng/ml bFGF. Growth factor- and mock-treated cells were incubated for 60 or 120 min and then harvested for total RNA and whole cell protein extracts. Egr-1 mRNA and protein levels were significantly increased in ECV304 cells by treatment with EGF but not with PDGF-BB or bFGF, with peak levels occurring at 60 min (Fig. 2A shows Egr-1 mRNA at 60 min; Fig. 2B shows Egr-1 protein at 60 min and 120 min). To determine whether EGF-mediated induction of Egr-1 was dependent on new mRNA synthesis, ECV304 cells were pretreated with 10 µg/ml actinomycin D for 30 min before a 60-min incubation with EGF. Actinomycin D completely abolished EGF-mediated induction of Egr-1 mRNA (Fig. 2C), indicating that Egr-1 induction by EGF requires de novo mRNA synthesis. The addition of actinomycin also reduced basal levels of Egr-1 in untreated cells, suggesting a rapid turnover of Egr-1 mRNA.


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Fig. 2.   The effect of growth factors on Egr-1 expression in ECV304 cells. A: ECV304 cells were serum starved for 48 h and then overlaid with serum-free medium (10% vol) containing no growth factor (C), 10 ng/ml epidermal growth factor (EGF; E), 10 ng/ml platelet-derived growth factor BB (PDGF BB; P), or 40 ng/ml basic fibroblast growth factor (bFGF; F). Total RNA was isolated at 60 min, and RT-PCR reactions were carried out with primers specific to human Egr-1 and beta -actin genes. B: ECV304 cells were treated as described above, cell lysates were isolated at 60 or 120 min, and Egr-1 protein levels were assayed by Western blot analyses. C: ECV304 cells were serum starved for 48 h, overlaid with serum-free medium (10% vol) containing no growth factor (C) or 10 ng/ml EGF (E), and harvested 1 h later for total RNA. Alternatively, serum-starved cells were preincubated with 10 µg/ml actinomycin D (Act) for 30 min, overlaid with serum-free medium containing vehicle (Act) or 10 ng/ml EGF (Act + E), and harvested 1 h later for total RNA. Egr-1 gene transcripts were assayed by RT-PCR analysis as described above.

The 1,200-bp 5' flanking region of the Egr-1 promoter confers responsiveness to EGF in ECV304 cells. We next tested whether the 1,200-bp Egr-1 promoter contained information for EGF response. ECV304 cells stably transfected with Egr-1-Luc construct were serum deprived for 48 h, incubated in the presence or absence of growth factor-supplemented serum-free medium for 4 h, and harvested for luciferase activity. To control for the change-of-medium effect, the medium was replaced in both treated and untreated cells. The Egr-1-Luc reporter gene activity was induced by an average of 3.2-fold in the presence of 10 ng/ml EGF but was unchanged by the addition of 40 ng/ml aFGF, 40 ng/ml bFGF, 10 ng/ml PDGF-AB, 10 ng/ml PDGF-AA, 10 ng/ml PDGF-BB, 25 ng/ml TNF-alpha , 10 ng/ml TGF-beta , or 40 ng/ml VEGF (Fig. 3A). Incubation of transfected cells with up to 100 ng/ml of PDGF-BB or bFGF for 2, 4, 14, and 24 h had no effect on reporter gene activity (data not shown). As expected, the Egr-1 promoter was induced by the addition of either 10% FBS (2.6-fold) or 100 nM PMA (14-fold) (Fig. 3A shows the response to 10% FBS). The Egr-1 promoter induction by EGF was dose and time dependent (Fig. 3, B and C, respectively). Induction was maximal (3.2-fold) at 4 h of treatment with 10 ng/ml EGF. This effect was abolished by 50 µg/ml anti-EGF polyclonal antibody (Fig. 3B). In cells that were not exposed to EGF, basal levels of Egr-1 promoter activity were unaffected by the addition of anti-EGF antibody (data not shown). In summary, the 5' flanking region of the Egr-1 gene contains information for response to EGF, serum, and PMA but not to other growth factors.


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Fig. 3.   Growth factor response of the 1,200-bp mouse Egr-1 promoter in ECV304 cells. ECV304 cells stably transfected with Egr-1-Luc were serum deprived for 48 h and incubated with growth factors or 10% FBS as described in MATERIALS AND METHODS. Luciferase activity is expressed as RLU ± SD. Data presented are representatives of at least 4 independent experiments, each performed in triplicate. A: pools of serum-starved ECV304 stable transfectants were treated for 4 h by replacing the serum-free medium containing no growth factor (C), 10 ng/ml EGF, 40 ng/ml acidic FGF (aFGF), 40 ng/ml bFGF, 10 ng/ml PDGF-AB, 10 ng/ml PDGF-AA, 10 ng/ml PDGF-BB, 25 ng/ml tumor necrosis factor-alpha (TNF-alpha ), 10 ng/ml transforming growth factor-beta (TGF-beta ), 40 ng/ml vascular endothelial growth factor (VEGF), or 10% fetal bovine serum (FBS). B: pools of serum-starved ECV304 stable transfectants were treated for 4 h with serum-free medium containing no growth factor (C), 2 ng/ml EGF, 5 ng/ml EGF, 10 ng/ml EGF, 40 ng/ml EGF, or 40 ng/ml EGF in the presence of 50 µg/ml anti-EGF. C: pools of serum-starved ECV304 stable transfectants were treated with serum-free medium containing no growth factor (C) or 10 ng/ml EGF for 2, 4, 14, or 26 h.

Progressive deletion of SREs in the Egr-1 5' flanking sequence diminishes response to EGF in ECV304 cells. To delineate the promoter regions responsible for mediating Egr-1 response to EGF, a series of 5' and internal deletion mutants of the Egr-1 promoter (Fig. 4A) were stably transfected into ECV304 cells. A promoter construct that contains the 1,200-bp full-length Egr-1 5' flanking sequence (Egr-1-A) was induced 2.7-fold by EGF (Fig. 4B). Deletion of the upstream AP-1 binding sites (Egr-1-C) did not affect the level of EGF inducibility. On the other hand, deletion of all five SREs (Egr-1-B) from the full-length promoter resulted in a complete loss of response to EGF. A construct that lacked the 5' SRE cluster but contained the 3' SRE cluster (Egr-1-E) was induced a modest 1.8-fold, whereas a construct that contained an internal deletion of the 3' SRE cluster (Egr-1-D) was induced 2.4-fold in the presence of EGF. A construct that contained only the most proximal of the 3' SREs (Egr-1-F) failed to respond to EGF (Fig. 4B). The differential response of the SRE clusters to EGF was preserved in the context of a heterologous promoter. When coupled to the human vWF core promoter, the 5' SRE cluster was induced 2.2-fold, whereas the 3' SRE cluster was induced only 1.4-fold with EGF (Fig. 5; compare SRE-A-Luc with SRE-B-Luc). A construct that contained both the 5' and 3' clusters (SRE-C-Luc) was induced 2.5-fold by EGF (Fig. 5). The vWF-90-Luc construct by itself did not respond to EGF or to other growth factors in ECV304 stable transfectants (data not shown). These results suggest that while the 3' SRE cluster in the Egr-1 promoter contributes to EGF inducibility, the 5' SRE cluster is more efficient in transducing the EGF signal.


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Fig. 4.   Effect of progressive deletions of the mouse Egr-1 promoter on EGF response in ECV304 cells. A: a series of 5' or internal deletion mutants of the Egr-1 promoter were fused upstream of a luciferase reporter plasmid (pXP2), and the resulting constructs (A-G, and V) (8) were stably transfected into ECV304 cells. Sequences are numbered relative to the transcription start site. The serum response elements (SREs) 1-5 are shown in bars. B: pools of ECV304 stable transfectants containing the various deletion constructs were serum starved for 48 h and then treated with 10 ng/ml EGF for 4 h. The results are presented as mean fold induction ± SD, from 4 independent experiments, each performed in triplicate.



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Fig. 5.   Effect of growth factors on SRE heterologous promoter constructs. A: Egr-1 promoter SRE fragments were generated by PCR, fused upstream of the human von Willebrand Factor (vWF) core promoter-luciferase construct (vWF-90-Luc). SRE-A-Luc, SRE-B-Luc, and SRE-C-Luc constructs contain Egr-1 promoter fragments corresponding to sequences between -425 and -250, -125 and -70, and -425 and -70, respectively. The 3 constructs were stably transfected into ECV304 cells as described in MATERIALS AND METHODS. B: pools of ECV304 stable transfectants were serum deprived for 48 h and treated with 10 ng/ml EGF (E), 10 ng/ml PDGF-BB (P), or 40 ng/ml bFGF (F) for 4 h. Luciferase activity is expressed as fold induction ± SD, compared with control cells. Data presented are representative of 4 independent experiments.

Egr-1 induction by EGF in ECV304 cells is mediated by the MAPK pathway. To elucidate the signaling pathways that mediate EGF inducibility of Egr-1 in ECV304 cells, Egr-1-Luc stable transfectants were treated with protein kinase inhibitors. ECV304 Egr-1-Luc stable transfectants were pretreated with 50 µM PD-98059 or 5 µM BIM for 30 min and then incubated with serum-free medium in the absence or presence of 100 nM PMA or 10 ng/ml EGF for another 4 h. Basal reporter gene activity was downregulated by the addition of PD-98059. These results are consistent with those of the endogenous gene (see Figs. 1C and 6B) and suggest that constitutive Egr-1 expression requires MAPK-dependent signaling. EGF-mediated induction of the Egr-1 promoter was completely abolished by PD-98059, whereas the PKC inhibitor BIM had no effect (Fig. 6A). In contrast, PMA-mediated induction of the Egr-1 promoter was abrogated both by PD-98059 and BIM (Fig. 6A). EGF-mediated induction of the endogenous Egr-1 gene was also abolished by 100 µM PD-98059 in ECV304 cells (Fig. 6B). These results suggest that Egr-1 induction by EGF in ECV304 cells is mediated by a PKC-independent MAPK signaling pathway. To provide functional support for these observations, an Elk-1 expression vector was cotransfected with an Elk-1-responsive luciferase reporter gene construct in ECV304 cells. Treatment with EGF, but not with PDGF-BB or bFGF, resulted in increased Elk-1 transactivation activity (Fig. 6C). These results imply that EGF-mediated activation of MAPK is coupled to an increase in Elk-1 activity.


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Fig. 6.   Role of the MAPK pathway in transducing EGF-mediated induction of Egr-1. A: pools of serum-starved ECV304 stable transfectants containing the full-length Egr-1 promoter (Egr-1-Luc) were treated for 4 h with serum-free medium containing 0.1% DMSO (C), 10 ng/ml EGF, or 100 nM phorbol 12-myristate 13-acetate (PMA). Alternatively, the serum-starved cells were preincubated with 50 µM PD-98059 (PD) or 5 µM BIM for 30 min and then incubated for 4 h with serum-free medium containing no growth factor, 10 ng/ml EGF, or 100 nM PMA. B: serum-deprived ECV304 cells were pretreated with 100 µM PD-98059 for 45 min and then overlaid with serum-free medium containing no growth factor (PD) or 10 ng/ml EGF (E+PD). Alternatively, serum-deprived cells were preincubated with vehicle and subsequently overlaid with serum-free medium containing no growth factor (C) or 10 ng/ml EGF (E). Cells were processed for total protein 1 h later, and Egr-1 levels were measured by Western blot analysis. C: ECV304 cells were transiently transfected with Elk-Gal4 and Gal4-Luc constructs, serum starved, and treated with 10 ng/ml EGF, 40 ng/ml PDGF-BB, or 40 ng/ml bFGF for 4 h, as described in MATERIALS AND METHODS. Standard firefly and Renilla luciferase activity were assayed by the dual-luciferase assay system. The results are expressed as fold induction after correction for transfection efficiency.

Differential Egr-1 gene regulation in endothelial cells. In the next set of experiments, we determined whether EGF responsiveness of the Egr-1 gene is preserved in other endothelial cell types or whether the pattern of growth factor-mediated response varies among different endothelial cell types. Serum-starved HUVECs were growth factor or mock treated for 60 min and subjected to RT-PCR and Western blot analyses. Egr-1 protein and mRNA levels were induced by EGF and bFGF but not by PDGF-BB (Fig. 7, A and B, respectively). Egr-1 protein was rapidly induced by EGF, with peak levels occurring at 1 h and returning to baseline after 2 h (Fig. 7C). Similarly, in HCAECs, Egr-1 levels were upregulated by EGF and bFGF but not by PDGF-BB. In RFPECs, Egr-1 was induced by EGF and PDGF-BB but not by bFGF. In BAECs and Py-4-1 mouse endothelial cells, Egr-1 was induced by bFGF alone, whereas, in CPAECs, Egr-1 was only slightly induced by PDGF-BB alone (Fig. 7A). The response of Egr-1 to aFGF and VEGF also varied among different endothelial cells (data not shown). Together, these results show that the Egr-1 gene is regulated by endothelial cell subtype-specific mechanisms.


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Fig. 7.   Differential Egr-1 expression in cultured endothelial cells. A: serum-starved endothelial cells from different sources were overlaid with the starving medium (10% vol) containing no growth factor (C), 10 ng/ml EGF (E), 10 ng/ml PDGF-BB (P), or 40 ng/ml bFGF (F) for 1 h, and Egr-1 levels were assayed by Western blot analysis. The results are representative of at least 3 independent experiments. B: RT-PCR analysis of Egr-1 expression in human umbilical vein endothelial cells (HUVECs) after treatments with growth factors for 1 h. C: time course of Egr-1 induction by EGF in HUVECs. Serum-starved HUVECs were incubated with 10 ng/ml EGF-supplemented medium (10% vol) for the time indicated, while the control (C) was untouched serum-starved cells. HCAECs, human coronary artery endothelial cells; CPAECs, calf pulmonary artery endothelial cells; BAECs, bovine aortic endothelial cells; RFPECs, rat fat pad endothelial cells.

The Egr-1 gene is induced in NIH/3T3 cells by EGF, PDGF-BB, and bFGF. The Egr-1 gene is expressed in a variety of nonendothelial cell types. To compare the pattern of response in endothelial and nonendothelial cells, NIH/3T3 cells were serum starved in 0.5% FBS medium for 24 h and treated with EGF, PDGF-BB, and bFGF as described for ECV304 cells. In Western blot analyses, Egr-1 protein levels were induced in all treated cells, with highest levels occurring in the presence of PDGF-BB and bFGF (Fig. 8A). To determine whether the 1,200-bp Egr-1 promoter has the capacity to respond to other growth factors, NIH/3T3 cells were stably transfected with the Egr-1-Luc construct. Pooled transfectants were serum starved in 0.5% FBS for 24 h and incubated with various growth factors for 4 h, and the luciferase activity was assayed. Under these conditions, reporter gene activity was induced 6.6-fold by bFGF, 7.6-fold and 8.3-fold by PDGF-BB and PDGF-AB, respectively, and 3.4-fold by EGF (Fig. 8B). Together with the results of the ECV304 cells and other endothelial cells, these findings suggest that the Egr-1 gene is regulated in a cell type-specific fashion.


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Fig. 8.   Effect of growth factors on Egr-1 expression in NIH/3T3 fibroblast cells. A: serum-starved NIH/3T3 cells were mock treated (C) or treated with 10 ng/ml EGF (E), 10 ng/ml PDGF BB (P), or 40 ng/ml bFGF (F), for 60 or 120 min. Whole cell protein lysates were processed for Western blot analysis and probed with anti-Egr-1 antibody as described in MATERIALS AND METHODS. B: NIH/3T3 cells were stably transfected with the Egr-1-Luc reporter. Stable transfectants were serum starved for 24 h and incubated for 4 h with serum-starving medium containing no growth factor (C), 10 ng/ml EGF, 40 ng/ml aFGF, 40 ng/ml bFGF, 10 ng/ml PDGF-AB, 10 ng/ml PDGF-AA, 10 ng/ml PDGF-BB, 40 ng/ml VEGF, 100 nM PMA, or 10% FBS. Luciferase activity is expressed in RLU ± SD. Data presented are representative of 4 independent experiments, each performed in triplicate.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Far from being a passive barrier, the endothelium plays an active role in many homeostatic processes. Endothelial cells respond to signals residing in the local microenvironment and thereby adapt to the spatially and temporally diverse needs of the underlying tissue. Endothelial cell response involves the activation of intracellular signaling pathways, alterations in gene expression patterns, and changes in cellular phenotype. Because the Egr-1 gene lies at the convergence of a wide array of signals and in turn mediates the expression of multiple gene products, an understanding of its regulation may provide insight into how extracellular signals modulate gene expression and ultimately lead to changes in cell phenotype.

In the present study, the Egr-1 gene was shown to be induced by EGF in ECV304 cells and in a subset of primary endothelial cells. EGF-mediated induction of Egr-1 was conferred by DNA sequences within the 1,200-bp promoter region. The 5' flanking region of the Egr-1 gene contains five SREs that are organized into two discrete clusters, a 5' cluster that contains SREs 3, 4, and 5, and a 3' cluster that contains SREs 1 and 2. As originally defined in the context of the c-fos promoter, the SRE consensus sequence, CC(A/T)6GG, is recognized by the serum response factor (SRF), whereas neighboring Ets motifs form a complex with the ternary complex factor (TCF). In response to extracellular signals, the SRF and TCF bind to their cognate DNA elements and enhance transcription. Each of the SRE consensus sites within the Egr-1 promoter has been shown to be a functional SRE and to compete with the c-fos SRE sequences for binding by nuclear proteins (6). However, our findings suggest that EGF-mediated transactivation of the Egr-1 gene is mediated primarily by the 5' SRE cluster (SREs 3, 4, and 5). The same cluster of SREs has been shown to mediate response to shear stress and urea in HeLa and medullary collecting duct cells, respectively (8, 34). In contrast, the 3' SRE cluster has been implicated in granulocyte macrophage colony-stimulating factor-mediated induction of the Egr-1 gene (26, 31). Finally, all five SREs play a more equal role in transducing the PDGF-BB response in rat mesangial cells (30). Together, these observations suggest that the SREs in the upstream promoter region of the Egr-1 gene may cooperate in various combinations to transduce extracellular signals.

To our knowledge, the present study is the first to show that Egr-1 is responsive to EGF in endothelial cells. EGF, a 53-amino acid peptide, is a member of a large family of EGF receptor ligands that includes amphiregulin (35), heparin-binding EGF (14), and epiregulin (40). EGF upregulates the expression of genes involved in the adhesion, attachment, migration, and proliferation of many cell types. Transcriptional activation is mediated by a variety of EGF-responsive elements, including SREs (11), Sp1 (25), cAMP-response element (CRE) (24), and a 22-bp palindromic EGF response element (ERE) sequence (43). In endothelial cells, EGF signaling has been shown to play a role in angiogenesis. For example, under in vitro conditions, the proliferative and migratory responses of endothelial cells are stimulated by EGF (3, 44). These effects have been associated with increased levels of the Ets-1 transcription factor (17). In in vivo models, the EGF peptide has been shown to be integrally involved in the angiogenic process (33, 37). Together, these studies support an important role for EGF in the endothelial cell function. The results of the present investigation suggest that, in endothelial cells, the effect of EGF may be coupled to the activation of the immediate early gene Egr-1 and a downstream cascade of Egr-1 responsive genes.

ECV304 cells were reported to have originated from HUVECs by spontaneous transformation (39) and have been shown to retain many properties of endothelial cells, including the capacity to respond to VEGF (23) and to form capillary-like tubular structures in matrigel systems (16). However, in contrast to HUVECs, ECV304 cells do not express the vWF and CD31 antigens and display autonomous growth in the absence of growth factor-supplemented medium (16, 39). This latter property is presumably coupled to changes in growth factor receptor-mediated signaling pathways and may account for the loss of bFGF response of the Egr-1 gene in ECV304 cells. More recently, molecular genetic methods have uncovered evidence of cell line cross-contamination involving the ECV304 cell line (10). Until the precise nature of the ECV304 cell line is resolved, results derived from this cell type must be interpreted with caution and confirmed in primary endothelial cells.

The above limitations notwithstanding, our results clearly show that the Egr-1 gene is induced by EGF in some but not all cultured endothelial cells. Moreover, the response to PDGF-BB and bFGF also differed among endothelial cells. These differences are not fully explained by the species of origin. For example, bovine aortic and calf pulmonary artery endothelial cells displayed unique patterns of growth factor-mediated Egr-1 response. Our findings raise the question of whether the cell subtype-specific transcriptional networks reflect physiologically important differences or whether they are artifacts of the in vitro system. On the one hand, it is possible that diverse signal transduction networks within the endothelium are preserved when endothelial cells are uncoupled from their native tissue environment. On the other hand, cell type-specific differences in the mechanisms of Egr-1 gene regulation may arise from variable degrees of phenotypic drift. In other words, the expression of cell surface receptors and downstream signal transduction networks may be modified at different rates in different types of cultured endothelial cells.

Regardless of the underlying mechanism, our findings have wider implications for the interpretation of in vitro-based endothelial cell studies. In many cases, the response of an endothelial cell, whether to growth factors, to hemodynamic factors, or to hypoxia, is assumed to be a representative property of all endothelial cells. The observation that Egr-1 expression in endothelial cells from different sources responds to different growth factors is a clear example of how the results in one endothelial cell type do not necessarily apply to other cell subtypes. Data that are derived from in vitro studies of endothelial cells should be qualified according to the nature of the assays as well as the origin of the endothelial cell.

On a final note, we have recently shown that systemic administration of EGF to mice results in the upregulation of the Egr-1 gene in restricted populations of endothelial cells (23a). Together with the findings of the present investigation, these results suggest that the Egr-1 gene is indeed regulated by cell subtype-specific mechanisms in vivo. Rather than serving a common function throughout the endothelium, the Egr-1 gene may serve to regulate target gene expression according to the unique demands of the local tissue environment.


    ACKNOWLEDGEMENTS

We thank Vikas Sukhatme for helpful suggestions. We also thank David Cohen for providing the Egr-1 constructs.


    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grant HL-60585-01. J. C. Tsai is the recipient of a Judith Graham Pool Postgraduate Research Fellowship Award from the National Hemophilia Foundation. W. C. Aird is the recipient of an American Society of Hematology Scholar Award.

Address for reprint requests and other correspondence: W. C. Aird, Molecular Medicine, RW-663, Beth Israel Deaconess Medical Center, 330 Brookline Ave., Boston MA 02215 (E-mail: waird{at}caregroup.harvard.edu).

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.

Received 24 June 1999; accepted in final form 19 May 2000.


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