Epidermal growth factor induces Egr-1 promoter activity in hepatocytes in vitro and in vivo

Jo C. Tsai1, Lixin Liu1, Jie Zhang1, Katherine C. Spokes1, James N. Topper2, and William C. Aird1

1 Department of Molecular Medicine, Beth Israel Deaconess Medical Center, Boston, Massachusetts 02215; and 2 Department of Medicine, Stanford University School of Medicine, Stanford, California 94305


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Early growth response-1 (Egr-1) is a transcription factor that couples short-term changes in the extracellular milieu to long-term changes in gene expression. Under in vitro conditions, the Egr-1 gene has been shown to respond to many extracellular signals. In most cases, these findings have not been extended to the in vivo setting. The goal of the present study was to explore the role of epidermal growth factor (EGF) in mediating Egr-1 expression in hepatocytes under both in vitro and in vivo conditions. In HepG2 cells, Egr-1 protein and mRNA were upregulated in the presence of EGF. In stable transfections of HepG2 cells, a 1,200-bp Egr-1 promoter contained information for EGF response via a protein kinase C-independent, mitogen-activated protein kinase-dependent signaling pathway. A promoter region containing the two most proximal serum response elements was sufficient to transduce the EGF signal. In transgenic mice that carry the Egr-1 promoter coupled to the LacZ reporter gene, systemic delivery of EGF by intraperitoneal injection resulted in an induction of the endogenous Egr-1 gene and the Egr-1-lacZ transgene in hepatocytes. Together, these results suggest that the 1,200-bp promoter contains information for EGF response in hepatocytes both in vitro and in intact animals.

immediate-early genes; transgenic mice; HepG2 cells


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

EARLY GROWTH RESPONSE-1 (Egr-1) is a member of the immediate-early gene family that includes FOS, JUN, and early growth response genes (7, 12, 21, 23, 27, 39, 46). Egr-1 encodes a zinc finger-containing DNA-binding protein in many cell types and is upregulated in response to a wide variety of mitogenic and nonmitogenic stimuli, including peptide growth factors, shear stress, urea, and hypotonicity (2, 5, 6, 8, 14, 29, 33, 45). Once activated, Egr-1 binds to 5'-GCGGGGGCG-3' consensus sequences within the promoter region of target genes, resulting in transcriptional activation or repression.

Little is known about the mechanisms that underlie Egr-1 gene regulation. In transient transfection assays, the upstream promoter region has been shown to transduce both mitogenic and nonmitogenic signals. In cultured cells, the environmentally responsive DNA elements have typically been mapped to serum response elements (SREs) in the 5'-flanking region (8, 34, 36, 47). However, the role of these SREs or other transcriptional control elements in mediating constitutive and/or inducible expression of Egr-1 in vivo has yet to be defined.

To establish a role for the upstream promoter region in mediating physiological expression of the Egr-1 gene, we recently generated transgenic mice with a DNA construct containing 1,200 bp of the mouse promoter coupled to a nuclear localized LacZ reporter gene (43). Reporter gene activity was detected in subsets of endothelial cells, vascular smooth muscle cells, cardiomyocytes, neurons, and hepatocytes in a pattern that is similar to that of the endogenous Egr-1 gene. Moreover, expression of the Egr-1 transgene in hepatocytes was significantly upregulated in response to partial hepatectomy (43).

Epidermal growth factor (EGF) has been implicated in the proliferative response of hepatocytes under both in vitro and in vivo conditions (3, 37, 38, 40). In a recent report (24), we demonstrated that the systemic administration of EGF in mice results in increased phosphorylation of the EGF receptor and extracellular signal-related kinase (ERK)1/2 in the liver. These changes were associated with an upregulation of Egr-1 protein and mRNA levels and increased immunodetectable Egr-1 in hepatocytes.

In the present study, our goal was to determine whether the 1,200-bp Egr-1 promoter contains information for EGF-mediated response in the liver. Using a combination of in vitro and in vivo approaches, we show that the promoter does indeed transduce the EGF signal in hepatocytes. On the basis of these results, we believe that the transgenic mouse assay will serve as a valuable tool for dissecting growth factor-responsive DNA elements within the Egr-1 promoter.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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Transgenic mice. All mouse protocols were approved by the Institutional Animal Care and Use Committee of the Beth Israel Deaconess Medical Center. Two independent lines of Egr-1-lacZ transgenic mice (Egr-1-lacZ-#9 and -#20) were generated as previously described (43). Briefly, a 1,200-bp fragment of the mouse Egr-1 promoter was coupled to nuclear localized LacZ and the resulting DNA fragment was introduced into the germline of FVB mice by standard transgenic techniques. LacZ staining of whole mounts and cryosections was carried out as previously described (1).

Intraperitoneal injection of EGF. Egr-1-lacZ-#9 and -#20 transgenic mice between 6 and 8 wk of age were injected intraperitoneally with EGF (2.0 µg/g body wt) or 0.9% normal saline in a total volume of 300 µl. Animals were killed 1, 2, 4, 16, 24, and 48 h later, and the tissues were processed for LacZ staining or harvested for protein and RNA as described in Western blot analysis of Egr-1 expression and Northern blot analysis of Egr-1 expression, respectively.

Cell culture. Human hepatoma HepG2 cells (HB-8065, American Type Culture Collection, Manassas, VA) were maintained at 37°C and 5% CO2 in DMEM supplemented with 10% fetal bovine serum (FBS), 100 U/ml penicillin, and 100 µg/ml streptomycin (Life Technologies, Gaithersburg, MD). The cells were grown to 50-60% confluence and then placed in serum-free DMEM medium. Twenty-four hours later, the cells were overlaid with growth factor- or mock-treated DMEM media at the doses and times indicated. Human recombinant EGF, platelet-derived growth factor (PDGF)-BB, acidic fibroblast growth factor (aFGF), tumor necrosis factor-alpha (TNF-alpha ), and transforming growth factor-beta (TGF-beta ) were purchased from PeproTech (Rocky Hill, NJ). Phorbol 12-myristate 13-acetate (PMA) was purchased from Calbiochem (La Jolla, CA). Human recombinant basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) were generous gifts of Michael Simons (Beth Israel Deaconess Medical Center, Boston, MA). In inhibition studies, serum-starved HepG2 cells were pretreated with the mitogen-activated protein kinase (MAPK) kinase inhibitor PD-98059 (New England Biolabs) at a final concentration of 50 µM or a specific protein kinase C (PKC) inhibitor bisindolylmaleimide I (BIM; Calbiochem) at a final concentration of 5 µM for 30 min before addition of EGF. An equal amount of DMSO was added to the control plates.

Western blot analysis of Egr-1 expression. Whole cell protein extracts were prepared from serum-starved HepG2 cells after addition of growth factor-supplemented or mock-treated media. Cells were washed twice with cold phosphate-buffered saline, harvested with a cell scraper, and lysed in ice-cold lysis buffer containing 20 mM HEPES, pH 7.5, 150 mM NaCl, 1% NP-40, 0.1% SDS, 1 mM EDTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), and the protease inhibitor cocktail (Boehringer Mannheim, Mannheim, Germany) for 1 h. The resulting lysates were centrifuged at 10,000 g for 20 min, and the supernatants were saved as whole cell protein extracts. Forty micrograms of protein were separated by 10% SDS-PAGE and electrotransferred to nitrocellulose membranes. 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 donkey anti-rabbit horseradish peroxidase conjugate (1:1,000 dilution; Amersham, Arlington Heights, IL). The blot was washed extensively between each incubation step. Peroxidase activity was visualized with an enhanced chemiluminescence substrate system (Amersham). The membrane was subsequently stripped and reprobed with an anti-beta -actin antibody (Sigma, St. Louis, MO) to control for loading.

Northern blot analysis of Egr-1 expression. Total RNA was prepared from the HepG2 cells by guanidine thiocyanate-phenol-chloroform extraction (Stratagene, La Jolla, CA). Ten micrograms of total RNA was loaded on a 0.7% formaldehyde-containing agarose gel. The RNA was transferred to nylon membrane, covalently cross-linked with ultraviolet radiation, prehybridized for 6 h, and then hybridized for 18 h at 42°C with a [32P]dCTP-labeled cDNA probe containing mouse Egr-1 sequence (a generous gift from Vikas Sukhatme, Beth Israel Deaconess Medical Center, Boston, MA). The membranes were subsequently stripped and probed with a radiolabeled 18S ribosome probe.

Stable transfections and luciferase assays. The Egr-1-Luc construct was generated by ligating the SalI fragment of the 1,200-bp murine Egr-1 promoter to the XhoI site of the luciferase reporter pGL-2-basic vector (Promega, Madison, WI). The Egr-1 promoter constructs A, C-G, and V were kindly provided by David Cohen (Oregon Health Sciences University, Portland, OR). 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 SalI, whereas Egr-1 promoter constructs A, C-G, and V were linearized with HindIII. HepG2 cells were grown in 10-cm tissue culture dishes to 30% confluence and overlaid with a mixture containing 100 µl of lipofectamine (Life Technologies), 20 µg of linearized Egr-1 promoter constructs, and 2 µg of DraI-linearized pGK-neo plasmids (42). Forty-eight hours later, the transfected HepG2 cells were replated in 1:30 dilution and subsequently grown in selection medium containing 1 mg/ml G418 (Life Technologies). After 21 days of selection, >50 G418-resistant clones were trypsinized, pooled, and seeded at the same density in 12-well plates. The HepG2 stable transfectants were serum deprived for 24 h and then incubated for 4 h with medium containing EGF, PDGF-BB, bFGF, aFGF, TNF-alpha , TGF-beta , PMA, or VEGF at the doses indicated. Cells were harvested for luciferase activity according to the manufacturer's instructions (Promega), and light activity was measured in a luminometer (Lumat LB 9507; EG&G Berthold). All experiments were carried out in triplicate and repeated at least four times. A Student's t-test was carried out for statistical analyses.

RT-PCR analysis of Egr-1 expression. Total RNA was prepared from the liver as described for HepG2 cells. Random-primed first-strand cDNA synthesis was prepared 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 murine Egr-1 (sense, 5'-CAGCAGTCCCATCTACTCGG; antisense, 5'-GCTGGGATTGGTAGGTGGTA), beta -actin (Clontech, Palo Alto, CA) (sense, 5'-GTGGGCCGCTCTAGG CACCAA; antisense, 5'-CTCTTTGATGTCACGCACGATTTC), and LacZ (sense, 5'-ACGTAACCTATCCCATTACGGTCAATC; antisense, 5'-AATATTGGCTTCATCCACCACATACAG). PCR reactions were carried out with the following cycle conditions for Egr-1 and beta -actin: 95°C for 4 min, followed by 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. The PCR condition for LacZ was 95°C for 3 min, 30 cycles of 95°C for 1 min, 68°C for 3 min, followed by a final incubation of 7 min at 72°C. One-fifth of the PCR product was electrophoresed on a 1.2% agarose gel and visualized by ethidium bromide staining.

beta -Galactosidase assays. Liver pieces excised from mice were frozen in liquid nitrogen, homogenized in lysis buffer (100 mM potassium phosphate, pH 7.8, 0.2% Triton X-100; Tropix, Bedford, MA) plus 0.5 mM PMSF and a protease inhibitor cocktail (Boehringer Mannheim), and incubated on ice for 2 h. The lysates were clarified by centrifugation at 10,000 g for 30 min, and the cell debris was removed. Protein quantitation was carried out with the microprotein determination system (Sigma). beta -Galactosidase activity was assayed using the Galacto-Star chemiluminescent reporter assay system (Tropix) according to the manufacturer's instructions. Light units were normalized to protein concentration and to endogenous beta -galactosidase activity from wild-type FVB mouse livers.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
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Endogenous Egr-1 gene is induced by EGF in HepG2 cells. Previous studies demonstrated that TGF-beta (19), bFGF (25), aFGF (41), PDGF (18), and EGF (3, 37, 38, 40) are mitogenic for cultured hepatocytes. In addition, we showed (24) that the systemic delivery of VEGF to the liver of mice results in increased Egr-1 protein levels in hepatocytes, raising the possibility that VEGF may interact directly with hepatocytes. To study the effect of these various mediators on Egr-1 expression in hepatocytes, serum-starved HepG2 cells were incubated with mock-treated or cytokine/growth factor-supplemented media. The cells were harvested for total protein and RNA 1 h later, and Egr-1 levels were assayed by Western and Northern blot analyses, respectively. Egr-1 protein and mRNA were significantly induced by EGF and PMA and to a lesser extent by bFGF and aFGF. Egr-1 protein and/or RNA was unchanged in response to PDGF-BB, VEGF, TNF-alpha , or TGF-beta (Fig. 1). EGF-mediated induction of Egr-1 protein was time and dose dependent, with maximum levels occurring at 1-2 h of treatment with 10 ng/ml EGF (Fig. 2, A and B). Response to EGF was inhibited by PD-98059 but not by the PKC inhibitor BIM (Fig. 2C), suggesting that EGF induces Egr-1 through a MAPK-dependent, PKC-independent signaling pathway.


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Fig. 1.   Effect of epidermal growth factor (EGF) on endogenous early growth response-1 (Egr-1) expression in HepG2 cells. HepG2 cells were serum starved for 24 h and then overlaid with serum-free medium containing no growth factor (C), 10 ng/ml EGF (E), 10 ng/ml platelet-derived growth factor (PDGF)-BB (P), 40 ng/ml basic fibroblast growth factor (bFGF; bF), 40 ng/ml acidic fibroblast growth factor (aFGF; aF), 40 ng/ml vascular endothelial growth factor (VEGF; V), 25 ng/ml tumor necrosis factor-alpha (TNF-alpha ), 100 ng/ml phorbol 12-myristate 13-acetate (PMA), or 10 ng/ml transforming growth factor-beta (TGF-beta ). The cells were harvested for total protein and RNA 1 h later, and Egr-1 levels were assayed by Western blot (A) and Northern blot (B) analyses, respectively. The blots were subsequently stripped and reprobed with a beta -actin antibody (A) or radiolabeled 18S ribosome probe (B) to control for loading.



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Fig. 2.   Time-dose effect and signaling of EGF response in HepG2 cells. A: HepG2 cells were serum starved for 24 h and then overlaid with serum-free medium containing no growth factor (C) or 10 ng/ml EGF for 15, 30, 60, 120, or 240 min. B: HepG2 cells were serum-starved for 24 h and then overlaid with serum-free medium containing no growth factor (C) or 1, 2, 5, 10, or 20 ng/ml EGF for 1 h. C: serum-starved HepG2 cells were treated for 1 h with serum-free medium containing no growth factor (C) or 10 ng/ml EGF (-). Alternatively, the serum-starved cells were preincubated with 50 µM PD-98059 or 5 µM bisindolylmaleimide I (BIM) for 30 min and then incubated for 1 h with serum-free media containing no growth factor or 10 ng/ml EGF in the presence of PD or BIM. The blots were subsequently stripped and reprobed with a beta -actin antibody to control for loading.

Egr-1 promoter is induced by EGF in HepG2 cells. To determine whether the 5'-flanking region of the Egr-1 promoter transduces the EGF signal, HepG2 cells were stably transfected with the 1,200-bp mouse Egr-1 promoter fragment coupled to the luciferase reporter gene. Pools of >50 G418-resistant clones were serum starved and incubated in the presence or absence of growth factors. Cells were harvested for luciferase activity 4 h later. Consistent with the results of endogenous gene activity, reporter gene expression was significantly induced by EGF (9-fold), PMA (34-fold; not shown), bFGF (3-fold), and aFGF (4.4-fold) but not by VEGF, TNF-alpha , or TGF-beta (Fig. 3A). EGF-mediated induction of the Egr-1 promoter was abrogated by PD-98059 but not by BIM (Fig. 3B). In contrast, PMA-mediated induction of the Egr-1 promoter was inhibited by both PD-98059 and BIM (Fig. 3B). Together, these results suggest that the 1,200-bp Egr-1 promoter transduces the EGF signal in HepG2 cells via a MAPK-dependent, PKC-independent signaling pathway.


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Fig. 3.   The effect of EGF on Egr-1 promoter activity in HepG2 cells. HepG2 cells stably transfected with the Egr-1-Luc construct were serum deprived for 24 h and incubated with growth factors or 10% FBS as described in MATERIALS AND METHODS. Luciferase activity is expressed as relative light units (RLU) ± SD. The data presented are representative of at least 4 independent experiments, each performed in triplicate. A: pools of serum-starved HepG2 stable transfectants were treated for 4 h by replacing the serum-free medium with medium containing no growth factor (C), 10 ng/ml EGF, 10 ng/ml PDGF-BB, 40 ng/ml bFGF, 40 ng/ml aFGF, 40 ng/ml VEGF, 25 ng/ml TNF-alpha , 10 ng/ml TGF-beta , or 10% FBS. B: pools of serum-starved HepG2 stable transfectants containing the full length Egr-1 promoter (Egr-1-Luc) were treated for 4 h with serum-free medium containing 0.01% DMSO (C), 10 ng/ml EGF, or 100 nM PMA. Alternatively, the serum-starved cells were incubated for 4 h with serum-free media containing no growth factor (PD, BIM, respectively), 10 ng/ml EGF (EGF+PD, EGF+BIM, respectively) or 100 nM PMA (PMA+PD, PMA+BIM, respectively). *Significant induction compared with control untreated cells, P < 0.05. dagger Significant inhibition of induction by EGF or PMA, P < 0.001.

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 HepG2 cells. A promoter construct that contains the 1,200-bp full-length Egr-1 5'-flanking sequence (Egr-1-A) was induced 3.3-fold by EGF (Fig. 4B). Deletion of the upstream AP-1 sites (Egr-1-C) or the 5' SRE cluster (SRE 3, 4, and 5; Egr-1-E) had no affect on the level of EGF inducibility (3.6-fold and 3.7-fold, respectively), whereas an internal deletion of the 3' SRE cluster (SRE 1 and 2; Egr-1-D) had a modest effect on EGF response (2.3-fold) (Fig. 4B). A construct that contained the most proximal of the SREs (SRE 1; Egr-1-F) was induced only 1.4-fold by EGF (Fig. 4B). These results suggest that although the 5' SRE cluster contributes to the EGF response, the promoter region containing the 3' SRE cluster is more important 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 HepG2 cells. A: a series of 5' or internal deletion mutants of the Egr-1 promoter were coupled to the luciferase reporter gene and the resulting constructs (A, C-G) and the promoterless vector, V (not shown) (8) were stably transfected into HepG2 cells as described in MATERIALS AND METHODS. Sequences are numbered relative to the transcriptional start site. Serum response elements (SREs) 1-5 are shown as bars. B: pools of stably transfected HepG2 cells containing the various deletion constructs were serum starved for 24 h and then treated with 10 ng/ml of EGF for 4 h. The results are from a representative experiment performed in triplicate and are expressed as mean ± SD fold induction.

Egr-1 transgene is induced by EGF. In a recent study (24), we demonstrated that systemically administered EGF induced phosphorylation of the EGF receptor and ERK1/2 in the liver and upregulated Egr-1 levels in liver hepatocytes. To determine whether the Egr-1 promoter was capable of mediating EGF response in vivo, 50 µg of EGF was administered to Egr-1-lacZ transgenic mice by intraperitoneal injection. Both LacZ and Egr-1 mRNA levels were upregulated in response to EGF, with peak levels occurring at 1 h (Fig. 5A). Whole mount preparations of livers from EGF-treated mice revealed increased LacZ staining compared with normal saline-treated animals (Fig. 5B). LacZ staining of liver sections from EGF-treated mice revealed increased intensity of beta -galactosidase activity in hepatocytes and increased numbers of LacZ-positive hepatocytes (Fig. 5, C-F). The LacZ-positive hepatocytes appeared to be randomly distributed throughout the hepatic nodules. In quantitative beta -galactosidase assays, reporter gene activity was induced two- to fourfold in response to EGF (Fig. 5G). Together, these results suggest that the EGF signal is transduced by the 1,200-bp Egr-1 promoter in vivo.


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Fig. 5.   Effect of EGF on Egr-1 promoter in vivo. Fifty micrograms of EGF was administered to Egr-1-lacZ transgenic mice by intraperitoneal injection. Egr-1 mRNA, LacZ mRNA, and reporter gene activity were measured as described in MATERIALS AND METHODS. A: LacZ and Egr-1 mRNA levels as measured in RT-PCR assays 1, 2, or 4 h after EGF injections. B: LacZ staining is compared in a normal saline-treated Egr-1-lacZ-#20 mouse liver (left) and a 4-h EGF-treated Egr-1-lacZ-#20 mouse liver (right). LacZ staining of normal saline (C, low power; E, high power) and EGF (D, low power; F, high power)-treated liver sections. These specimens were processed and stained in parallel. G: beta -galactosidase activity of livers from EGF-treated Egr-1-lacZ transgenic mice was compared with that in normal saline-treated controls and expressed as fold induction. The control bar (C) represents mean ± SD of the 4-h beta -galactosidase activity in 12 normal saline-injected Egr-1-lacZ-#20 animals. Each of the other bars represents the average of duplicate samples from an individual animal. *Egr-1-lacZ-#9 mice; all other values were obtained from Egr-1-lacZ#20 mice.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The transcription factor Egr-1 is a nuclear signal transducer that couples short-term changes in the extracellular milieu to long-term changes in gene expression. Under in vitro conditions, the Egr-1 gene is responsive to a multitude of extracellular signals, including urea (8), heat shock (22), growth factors (10, 11, 20, 30, 32, 35), angiotensin II (32), thrombin (30), hypoxia (47, 48), and shear stress (36). In many cases, these extracellular signals have been shown to be transduced by SREs located in the upstream promoter region. The Egr-1 promoter contains five functional SRE sites that are organized into two clusters. The 5' SRE cluster has been shown to mediate Egr-1 response to shear stress (36), growth hormone, urea (8), hypotonicity, and hypoxia (47), whereas the 3' SRE cluster plays a predominant role in transducing response to granulocyte-macrophage colony-stimulating factor (34, 45). All five SREs play a more equal role in mediating response to PDGF-BB (33).

In contrast to our understanding of the transcriptional regulation of Egr-1 in vitro, there is little information about how the gene is regulated in the intact animal. To address this question, we recently generated transgenic mice that harbor an Egr-1-lacZ construct (43). In adult transgenic animals, the 1,200-bp promoter fragment was shown to direct authentic constitutive expression. Moreover, in response to partial hepatectomy, the transgene was significantly upregulated in hepatocytes (43). Together, these results suggest that the Egr-1 promoter contains information for appropriate spatial and temporal expression.

In another study aimed at understanding Egr-1 regulation in vivo, we recently examined (24) the effect of systemically administered growth factors in the intact animal. In these experiments, intraperitoneal injections of EGF or VEGF resulted in widespread delivery of the growth factor to the various tissues as well as widespread receptor phosphorylation and MAPK activation (24). Systemic delivery of growth factors led to organ-specific changes in Egr-1 expression. In the livers of EGF-treated mice, EGF receptor phosphorylation, ERK1/2 phosphorylation, and Egr-1 protein and mRNA levels were all increased. In immunofluorescence studies, the Egr-1 gene product was restricted to hepatocytes (24).

With the above foundation, we have studied EGF-mediated regulation of the Egr-1 promoter in hepatocytes under both in vitro and in vivo conditions. In HepG2 cells, we have shown that the endogenous Egr-1 gene and the 1,200-bp Egr-1 promoter are induced by EGF and, to a lesser extent, by bFGF or aFGF. Using a series of 5' and internal deletion mutants, we demonstrated that a promoter region containing the 3' SRE cluster was sufficient to transduce the EGF signal. EGF-mediated induction of Egr-1 was found to be governed by a MAPK-dependent, PKC-independent signaling pathway. Finally, in Egr-1-lacZ transgenic mice, the administration of EGF resulted in significant induction of both the endogenous Egr-1 gene and the transgene.

These findings are consistent with previous reports of EGF response in hepatocytes. For example, the addition of EGF to cultured rat hepatocytes results in a rapid burst in EGF receptor phosphorylation with subsequent activation of MAPK signaling pathways and accumulation of phosphorylated or activated target proteins (40, 44). Using in vivo models, we (24) and others (4, 31) showed that the intraperitoneal injection of EGF results in receptor phosphorylation and MAPK signaling in the adult liver. More importantly, the systemic administration of EGF resulted in Egr-1 induction in intact hepatocytes. In the present study, we have extended these observations by showing that EGF signaling in the liver is transduced by a defined region of the upstream Egr-1 promoter.

Although the above results support a link between EGF and Egr-1 expression in the liver, they do not prove a role for EGF in mediating either basal or inducible levels of Egr-1. That said, both EGF and Egr-1 have been implicated in the process of hepatic regeneration. For example, partial hepatectomy results in increased synthesis of EGF, increased phosphorylation of the EGF receptor (9), and upregulation of Egr-1 levels (13, 28). On the basis of the results of our studies, it is tempting to speculate that the proliferative effects of EGF are mediated at least in part by Egr-1 activity. More recent studies have pointed to a potential role of Egr-1 in the nonregenerating liver (17). Together with our results, these observations raise the possibility that EGF-mediated regulation of Egr-1 may contribute to programmed gene expression in mature hepatocytes.

Finally, it is interesting to note that the Egr-1 gene and the 1,200-bp promoter responded to aFGF and bFGF in HepG2 cells. aFGF is considered to be a physiological modulator of liver regeneration. Studies have shown that aFGF levels increase in the liver after partial hepatectomy (16). Moreover, aFGF and, to a lesser extent, bFGF are mitogenic for primary hepatocytes (15, 41). In in vitro wound models, bFGF has been shown to stimulate proliferation and migration of primary hepatocytes (26). Although our results were derived from a hepatoma cell line rather than primary hepatocytes or in vivo assays, they suggest that aFGF- and bFGF-mediated signaling in the liver may involve activation of the Egr-1 transcription factor.

In summary, we have shown that the Egr-1 gene is induced by EGF in hepatocytes and that the signal is transduced by the upstream promoter region under in vitro and in vivo conditions. On the basis of these results, it should now be possible to use the stable transfection and the transgenic mouse assays to map the growth factor-responsive DNA elements.


    ACKNOWLEDGEMENTS

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. This work was supported by National Heart, Lung, and Blood Institute Grant HL-60585-02.


    FOOTNOTES

Address for reprint requests and other correspondence: W. C. Aird, Beth Israel Deaconess Medical Center, Molecular Medicine, RW-663, 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 31 July 2000; accepted in final form 18 July 2001.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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