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
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ABSTRACT |
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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
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INTRODUCTION |
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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.
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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- (TNF-
), and transforming growth factor-
(TGF-
) 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--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-, TGF-
, 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), -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
-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.
-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).
-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
-galactosidase activity from
wild-type FVB mouse livers.
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RESULTS |
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Endogenous Egr-1 gene is induced by EGF in HepG2 cells.
Previous studies demonstrated that TGF- (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-
, or TGF-
(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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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-, or TGF-
(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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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 -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
-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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DISCUSSION |
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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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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