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INTRODUCTION |
The phosphatase of regenerating liver-1
(PRL-1)1 gene
encodes an evolutionarily conserved nuclear protein-tyrosine
phosphatase, and its expression is highly induced in liver regeneration
and mitogen-stimulated fibroblasts (1-5). Overexpression of PRL-1 in
transfected NIH 3T3 cells modifies cell growth (3). PRL-1 may play an
important role in cell growth via regulation of protein tyrosine
phosphorylation and dephosphorylation of specific substrates that
remain unknown (4). Expression of the PRL-1 gene is
positively associated not only with cellular growth during liver
regeneration, development, and oncogenesis but also with
differentiation in intestine and other tissues (5).
Following a partial hepatectomy or toxic liver injury, remnant liver
cells that are normally quiescent rapidly reenter the cell cycle within
minutes followed by DNA replication and restoration of liver mass
within a few days. The factors regulating this process are incompletely
understood, but a number of growth factors and cellular signals have
been implicated (1). We showed that interleukin-6 (IL-6) which is
increased in the liver posthepatectomy is required for normal liver
regeneration (2). We found that IL-6 specifically regulates the
expression of a subset of genes that are transcriptionally activated in
the regenerating liver, but other genes such as PRL-1 are
induced normally posthepatectomy even in the absence of IL-6. We
reasoned that examination of the regulation of PRL-1 gene
expression during liver regeneration would help us better understand
the IL-6 independent signals that are critical for liver regeneration.
Functional promoter analysis demonstrated the presence of two
promoters, P1 and P2, in the human PRL-1 gene, P1 directed
by a TATA box upstream of the non-coding first exon (6). A
non-canonical internal promoter, P2, was found in the first intron that
results in a PRL-1 transcript beginning 8 bp downstream of
the 5' end of exon 2 and causes no alteration in the encoded protein.
The first 200-bp region upstream of promoter P1 and P2 confers high basal transcriptional activity. An enhancer that binds a
developmentally regulated factor was localized to the first intron of
the human PRL-1 gene.
Promoter P1 of the PRL-1 gene contained two potential Egr-1
DNA-binding elements. Early growth response gene 1 (Egr-1,
also named NGFI-A, krox-24, zif268, Cef5, and
Tis8) is an immediate-early gene and encodes a transcription
factor with three zinc fingers recognizing a GC-rich sequence,
5'-CGCCCCCGC-3', which has been identified in the promoter regions of a
number of genes (7-12). Egr-1 expression is rapidly and transiently
induced during the transition of cells from the G0 to
G1 phase in response to various mitogens such as growth
factors, cytokines, injury, and partial hepatectomy.(7, 9, 13-19)
In this study, we demonstrated that the expression of PRL-1
gene was dramatically induced at a transcriptional level in the remnant
liver following partial hepatectomy. Egr-1 protein expression and Egr-1
binding activity increased in concert with PRL-1 gene transcription in regenerating liver. Egr-1 transactivated the PRL-1 promoter in the presence of an intact not mutant Egr-1
site, and the Egr-1 site at
99 was responsible for mitogen
stimulation of PRL-1 promoter activity.
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MATERIALS AND METHODS |
Rat Tissue Preparation--
For regenerating liver, female
Fisher rats (160-220 g, Charles River) were anesthetized with metofane
(Pitman-Moore, Inc.) and subjected to midventral laparotomy with
approximately 70% liver (left lateral and median lobes) (20). For
cycloheximide-treated samples, rats were pretreated 15 min prior to the
laparotomy with 50 mg of cycloheximide per kg of body weight (5%
solution in phosphate-buffered saline) intraperitoneally. Sham
surgeries were performed by subjecting rats to midventral laparotomy
and closure. Animals were allowed to recover for the times indicated in
the figure legends prior to the isolation of the remaining liver lobes
for purification of RNA, preparation of nuclei, and nuclear protein extracts.
RNA and RNA Analysis--
Regenerating liver RNA was prepared
exactly as reported (21). To make the RNA probe for the RNase
protection assay, EcoRI-linearized Bluescript KS plasmid
template containing EcoRI/PstI fragment of rat
PRL-1 cDNA was transcribed in vitro with T7
RNA polymerase in the presence of [
-32P]UTP using an
in vitro transcription kit (Stratagene, La Jolla, CA). The
probe used contains 32 bp of exon 1 and 180 bp of exon 2 (see Fig. 1).
50-70 µg of total regenerating liver RNA was used for the assay.
After overnight hybridization of 0.5-1 × 106 cpm of
urea-denatured 5% polyacrylamide gel-purified probe to the RNA, the
hybrids were digested according to the described protocol (22). The
protected fragments were then analyzed on 6% polyacrylamide gel
containing 8 M urea.
Nuclear Run-on Assays--
Nuclei preparation and nuclear run-on
assays were carried out by following the previously described procedure
(21, 23). Frozen nuclei were quickly thawed and pelleted, resuspended
in 100 µl of nuclear transcription buffer (50 mM
Tris·HCl, pH 7.9, 5 mM MnCl2, 50 mM NaCl, 0.35 M
(NH4)2SO4, 2 mM EDTA)
plus heparin (50 mg/ml), 100 µCi of [
-32P]UTP
(specific activity, 800 Ci/mmol, NEN Life Science Products), 1 mM of each ATP, GTP, and CTP. The reaction was allowed to
proceed at 30 °C for 30 min with shaking. The reaction was stopped
by the addition of 400 µl of a solution containing 10 mM
Tris·HCl, pH 7.4, 0.5 M NaCl, 50 mM
MgCl2, 2 mM CaCl2, and 50 µg/ml
RNase-free DNase I was incubated at 30 °C for 10 min with shaking.
Following proteinase K digestion and phenol/chloroform extraction, the
in vitro synthesized RNA was subsequently precipitated with
ethanol-sodium acetate and redissolved in H2O. The same
amount of the radiolabeled RNA (approximately 1-5 × 106 cpm) was hybridized to nitrocellulose membrane applied
with 20 µg of linearized insert-bearing plasmids using a slot blot
apparatus in the exactly same way as reported (22). We used ATP
synthase and
2-microglobulin as positive controls and
Bluescript SK(
) as negative control.
Cell Culture and DNA Transfections--
Human hepatoma cell line
HepG2 cells, mouse embryo testicular carcinoma cell line F9 cells, and
mouse fibroblast NIH 3T3 cells were grown in Dulbecco's modified
Eagle's medium (DMEM, Life Technologies, Inc.) supplemented with 10%
fetal bovine serum (Life Technologies, Inc.), 2 mM
L-glutamine, 100 units of penicillin, and 50 units of
streptomycin (Flow Laboratories) as described previously (6, 23). Rat
hepatoma cell line H35 cells were grown in low-glucose Dulbecco's
modified Eagle's medium (DMEM, Life Technologies, Inc.) supplemented
with 10% fetal bovine serum, 2 mM L-glutamine,
100 units of penicillin, and 50 units of streptomycin. To produce quiescent H35 and NIH 3T3 cells, the medium was changed to 0.5% serum/DMEM for 72 h. Following serum deprivation, NIH 3T3 and H35
cells were treated with 20% serum and insulin (10
8
M), respectively, for the indicated times and harvested for
preparation of nuclear extract. Cycloheximide treatment was performed
as described previously (21). For the transient transfection assays (6, 23), HepG2, NIH 3T3, or F9 cells were plated in 60-mm dishes at a
density of 1-5 × 105 cells/cm per dish for 16-24 h
prior to transfection. Cells were incubated with calcium phosphate
precipitates containing the indicated reporter plasmids,
pSV-
-galactosidase reference plasmid (Promega) as an internal
standard for transfection efficiency. After 16-18 h, the cells were
washed twice with serum-free culture medium, complete medium was added,
and then the cells were incubated for an additional 24 h before
harvesting for luciferase activity assays performed with luciferase
assay system (Promega) using the recommended procedure of the supplier.
For serum stimulation assay, NIH 3T3 cells were serum-deprived (0.5%
fetal calf serum) for 24 h after 16-18 h of CaPO4
transfection. The cells were harvested with or without 4 h of 20%
fetal calf serum induction.
Construction of Deletion Mutations and Site-directed
Mutagenesis--
PRL-1 promoter constructs were cloned into pGL2
luciferase (Promega). Bal-31 nuclease was used to make deletion mutant
constructs of PRL-1 promoter P1. The
SmaI-linearized plasmid pSma I (6) was incubated with Bal-31
exonuclease at 30 °C for various lengths of time, and the reaction
was terminated with addition of EGTA to 20 mM. After
phenol/chloroform extraction and ethanol precipitation, the resuspended
DNA was digested with XbaI and gel-purified on a 6% native
polyacrylamide gel. The resulting DNA fragments were cloned into the
luciferase report vector between SmaI and XbaI sites. The mutation of the consensus Egr-1-binding site was performed using QuickChangeTM Site-directed Mutagenesis Kit from
Stratagene following the standard procedure provided with the kit. The
overlapping primers used for the site mutation are
5'-GAGGGGGCGGGCCTCCGCCCtttCCTGTCGGCT-3' (forward) and 5'-AGCCGACAGGaaaGGGCGGAGGCCCGCCCCCTC-3' (backward). The correct orientation, mutation sites, and the 5' and 3'
boundaries of the insert of all plasmids were verified by DNA sequencing.
Preparation of Nuclear Protein Extracts--
All steps were
performed at 4 °C, and 2 µg/ml proteinase inhibitors antipain,
aprotinin, bestatin, and leupeptin were added into all buffers except
phosphate-buffered saline, pH 7.4. Nuclei from regenerating liver and
from cell lines HepG2, H35, and NIH 3T3 were prepared by using the
described procedure (6, 24, 25). Nuclei were lysed by the dropwise
addition of 1/9 volume of 4.2 M NaCl and shaken for 30 min
before being centrifuged at 50,000 × g for 50 min. The
supernatant was dialyzed against Buffer D (20 mM Hepes, pH
7.6, 0.2 mM EDTA, 0.1 M KCl, 1 mM
dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 20%
glycerol) for at least 6 h. Finally, the extract was centrifuged
at 12,000 × g for 10 min, and aliquots were
quick-frozen in dry ice, 95% ethanol mixture and stored at
70 °C.
Protein concentration were determined using Bio-Rad protein assay reagent.
Electrophoresis Mobility Shift Assays (EMSA)--
End labeling
of oligonucleotides was carried out with T4 polynucleotide kinase and
[
-32P]ATP and purified by Sephadex G-25 spin column.
Binding reactions were performed essentially as reported (6, 24).
Nuclear protein extracts (3-6 µg) were incubated with 2 µg of
poly(dI-dC) in 10 µl of binding buffer containing 20 mM
Hepes, pH 7.9, 1 mM EDTA, 60 mM KCl, 1 mM dithiothreitol, 5 mM MgCl2, 12%
glycerol for 15 min on ice. [
-32P]ATP-labeled probe
(0.5-1 × 104 cpm) was then added to the reaction,
and incubation was continued for another 20 min. DNA-protein binding
complexes were analyzed on 6% native polyacrylamide gel at 300 V using
0.5× TBE as electrophoresis buffer. Gels were dried and exposed to
Kodak X-Omat AR film overnight. Supershift experiments were performed
by incubating 1 µl of primary antibody with nuclear extracts in
binding buffer for 1 h at 4 °C prior to the addition of labeled
oligonucleotides. Potato acid phosphatase treatment experiments were
performed as described (26).
Antibodies against Egr-1 and Sp1 were purchased from Santa Cruz Biotechnology.
Oligonucleotides--
The oligonucleotides were synthesized by
Life Technologies, Inc. The synthesized complementary polynucleotides
were annealed in 10 mM Tris·HCl, pH 8.0, 1 mM
EDTA, 0.3 M NaCl at 90-95 °C for 10 min and then cooled
down to room temperature. After being kept at room temperature for
24 h, the annealed oligonucleotides were purified on 15% native
polyacrylamide gels. These oligonucleotides were PRL/B (
118,
5'-TCCGGAGGGGGCGGGCCTCCGCCCCCGCCTGT-3'
87); PRL/B-mA
(5'-TCCGGAGGGttaGGGCCTCCGCCCCCGCCTGT-3');
PRL/B-mB
(5'-TCCGGAGGGGGCGGGCCTCCGCCCtttCCTGT-3'); PRL/B-mAB
(5'-TCCGGAGGGttaGGGCCTCCGCCCtttCCTGT-3');
CRE(PRL/A) (
128, 5'-CGGAGTGACGTCCGGAGG-3'
111); E2, PRL/C
(
68, 5'-TCCAGACCGCGATTGGTGGCT-3'
48). These oligonucleotides
correspond to the specific elements of the human PRL-1 gene
and were used as competitor DNAs in gel mobility shift assays.
Consensus Sp1 (5'-ATTCGATCGGGGCGGGGCGAGC-3') and Egr-1 oligonucleotides
(5'-GGATCCAGCGGGGGCGAGCGGGGGCGA-3') were from Promega and Santa Cruz Biotechnology, respectively. In
addition, double-stranded oligonucleotides corresponding to Ap1, Ap2,
and CRE consensus binding sites were purchased from Promega.
Immunoblots--
50-70 µg of whole nuclei or nuclear extracts
were electrophoresed on a 10% SDS-polyacrylamide gel and transferred
to nitrocellulose. Primary antibody anti-Egr 1 antiserum (Santa Cruz
Biotechnology) was diluted 1:1000 and incubated with the membrane for
1 h at room temperature. Horseradish peroxidase-linked secondary
antibody was added at a dilution of 1:10000 for 30 min and then
detected by chemiluminescence (Amersham Pharmacia Biotech) according to the instructions of the manufacturer (27).
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RESULTS |
Transcriptional Regulation of the PRL-1 Gene during Liver
Regeneration--
PRL-1 is expressed as an immediate-early response
gene in regenerating liver and mitogen-stimulated 3T3 fibroblasts. To
assess the relative induction of the two rat PRL-1 gene
promoters in liver regeneration, an RNase protection assay was
performed. Like the human gene in which two promoters were identified
(6), two protected fragments of approximately 212 and 172 bp (Fig. 1A), which respectively
represented the products transcribed from PRL-1 promoter
(P1) and internal promoter (P2) located in the first intron, were
increased during liver regeneration. Both PRL-1 transcripts
were rapidly induced, reached a peak by 4 h, with 17.9- and
6.3-fold induction for P1 and P2, respectively, and remained elevated
through 8 h posthepatectomy.

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Fig. 1.
Induced expression of PRL-1
mRNA following partial hepatectomy. A,
top, schematic diagram of the relevant portion of the rat
PRL-1 gene along with the probe for RNase protection assay;
bottom, RNase protection assay of PRL-1 gene
expression at the indicated time points in regenerating liver (50 µg
of total RNA per lane). Lanes G and A,
bacteriophage m13mp18 sequencing marker. B, nuclear run-on
assay of PRL-1 gene transcription in nuclei at indicated
time points following partial hepatectomy. Bluescript KS as negative
control, 2-microglobulin and ATP synthase as positive
controls. Quantification of bands was by densitometry.
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Because the regulation of gene expression can be both transcriptional
and post-transcriptional, we tested whether the induction of
PRL-1 gene expression was transcriptionally regulated.
Nuclear run-on assays indicated that PRL-1 gene
transcription, which was virtually absent in normal liver, increased by
14.8-fold at 30 min posthepatectomy and remained elevated (10-fold) at
2 h posthepatectomy (Fig. 1B). As a control, there was
no induction of the constitutively expressed genes for ATP synthase and
2-microglobulin and no signal detected for vector
Bluescript SK. This indicated that PRL-1 was transcriptionally up-regulated during liver regeneration and that an
increase in transcription could largely explain the induced expression
of PRL-1 mRNA at early times posthepatectomy.
Nuclear Egr-1 Activity during Liver Regeneration--
Because of
its larger transcriptional induction, we focused on determining the
regions upstream of promoter P1 responsible for transcriptional
activation posthepatectomy. Transient transfection experiments had
revealed that virtually all of the basal trans-acting elements are
located within the first 200 bp upstream of the human P1 promoter in
both human hepatoma HepG2 and mouse NIH 3T3 cell lines (6). A number of
potential DNA-binding sites were identified within this region. Gel
mobility shift experiments were performed to characterize the various
DNA-binding proteins that might contribute to PRL-1 gene
transcription during liver regeneration. EMSA experiments with nuclear
extracts from normal and regenerating liver confirmed the binding of
transcription factors including CRE (PRL/A,
123 to
116), Sp1, Egr-1
(PRL/B,
118 to
87), and E2 (28) (PRL/C,
68 to
48) proteins to
the consensus sequences (not shown). Most of these DNA binding
activities were constitutive in normal and regenerating liver. The AP1
complex that bound the CRE is known to increase in liver regeneration
(14). However, transfection with c-Jun and c-Fos expression vectors did
not have a major effect on the PRL-1 promoter P1 activity in
NIH 3T3 cells or HepG2 cells (data not shown) nor did deletion of this
region reduce mitogenic stimulation of the promoter (see Fig. 7), so no
further analyses of PRL/A were performed.
PRL/B bound Egr-1 and SP1, and Egr-1 activity increased posthepatectomy
indicating a possible correlation with the transcriptional induction of
the PRL-1 gene posthepatectomy. Two potential overlapping Egr/Sp1 sites were found in region PRL/B. Gel mobility shift assays were employed to examine Egr-1 and Sp1 binding activities with nuclear
extract from regenerating rat liver at 30 min posthepatectomy. Egr-1
and Sp1 specifically bound to oligonucleotide PRL/B (
118 to
87)
containing the two potential overlapping Egr-1/Sp1 sites (Fig.
2, A and B). PRL/B
oligonucleotide specifically competed Sp1 and Egr-1 binding to the
consensus Sp1 and Egr-1 oligonucleotides (Fig. 2A, lanes
8 and 12).

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Fig. 2.
Characterization of proteins binding to
region 118 to 87 (PRL/B) in PRL-1 promoter
P1. A, EMSAs were performed using 5 µg of nuclear
extracts from rat regenerating liver at 30 min posthepatectomy. Probes
were [ -32P]dATP-labeled oligonucleotides corresponding
to potential Egr-1- and SP1-binding sites in the PRL-1
promoter P1 PRL/B ( 118 to 87) and the consensus Sp1 and Egr-1
binding sequence oligonucleotides. As indicated in the figure,
unlabeled oligonucleotides and antibodies against Sp1 and Egr-1 were
employed to characterize protein binding specificity. B,
EMSA assay of Egr-1 binding specificity with 200-fold molar excess of
the unlabeled Sp1 oligonucleotides in all of the reactions. Lane
1, probe alone; lane 2, no competition; lane
3, the unlabeled PRL/B; lane 4, preimmune serum
(negative control); lane 5, supershift, antibody specific
for Egr-1. The unlabeled competitor oligonucleotides were included as
indicated: lane 6, Ap1; lane 7, Sp1; lane
8, Ap2; lane 9, cAMP response element
(CRE).
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Sp1 is a ubiquitous protein that is constitutively expressed at very
high levels in normal and regenerating liver (see Fig. 4), and its DNA
binding profile is well characterized. Therefore, we focused on Egr-1
interactions with the PRL-1 promoter region PRL/B. 200-fold
molar excess of unlabeled Sp1 oligonucleotides was routinely included
in EMSA experiments to prevent Sp1 from binding to the overlapping Sp1
sites in PRL/B. As shown (Fig. 2B), Egr-1 binding to PRL/B
was specific because excess unlabeled PRL/B oligonucleotides
(lane 3) and antibody against Egr-1 (lane 5)
inhibited complex formation, whereas preimmune serum (lane 4) or a large excess of unrelated oligonucleotides Ap1 (lane
6), Sp1 (lane 7), Ap2 (lane 8), and CRE
(lane 9) did not.
To assess the role of two potential Egr-1 binding motifs in
PRL-1 promoter, we carried out in vitro binding
studies to compare Egr-1 binding affinity to the two Egr-1-binding
sites. Nuclear extracts from mitogen-stimulated NIH 3T3 cells were used
in gel mobility shift assays. As shown mB oligonucleotide bound Egr-1 much less efficiently than either wild type or mA (16- versus 72-h exposure) (Fig.
3A). The unlabeled wild type
oligonucleotide and mA oligonucleotide efficiently competed Egr-1
binding (Fig. 3A, lanes 2, 3, 7, and 8), whereas
mB competed Egr-1 binding weakly (Fig. 3A, lane 4).
Titration of increasing amounts of unlabeled competitor oligonucleotide
confirmed the relative affinities of mA and mB (Fig. 3B).
Addition of as little as a 50-fold molar excess of wild type or
mA-mutated oligonucleotides resulted in a complete inhibition (Fig.
3B, lanes 3 and 9). In contrast, over a 250-fold
molar excess of mB oligonucleotides was necessary to effectively
compete Egr-1 binding to the wild type oligonucleotides (Fig. 3B,
lane 8). The results demonstrated that Egr-1 has an estimated
binding affinity for the site B which is over 5-fold higher than for
the site A. On the other hand, Sp1 binding activity was well competed
in the presence of mB and wild type oligonucleotides but not mA or mAB,
indicating that Sp1 binds most strongly to site A (Fig. 3C).
Mutation of the
99 site does not have a major impact on total Sp1
binding to the region (Fig. 3C, and data not shown).

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Fig. 3.
Effects of selective mutagenesis of Egr-1
consensus motifs on Egr-1 binding abilities. A, left,
the effects of selective mutations to Egr-1 sites were analyzed by gel
mobility shift assays. Films were exposed at room temperature for 16 h
(left panel) and 72 h at 70 °C (right
panel); right, sequences of oligonucleotides used in
gel mobility shift assays. Sp1 and Egr-1 motifs are
underlined and the mutated nucleotides are represented with
lowercase letters. B, the competitive
(Comp.) gel mobility shift assays were performed to
determine Egr-1 binding affinity by using increasing molar excess of
the indicated competitors (25 times for lanes 2 and
4; 50 times for lanes 3, 5, and 9; 100 times for lane 6; 150 times for lane 7; and 250 times for lane 8). C, impact of oligonucleotide
mutagenesis on Sp1 binding to consensus Sp1 binding motif. The wild
type and mutated PRL-1 Egr-1 binding motifs were employed to
compete Sp1 binding to consensus Sp1 binding motif with increasing
molar excess (10 times for lanes 2, 5, 8, and 11;
20 times for lanes 3, 6, 9, and 12; and 50 times
for lanes 4, 7, 10, and 13).
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Rapid Activation of Egr-1 DNA Binding in Regenerating Liver and
Mitogen-stimulated H35 and NIH 3T3 Cells--
We then examined the
time course of induction of Egr-1 binding activity following
hepatectomy and mitogen-stimulated NIH 3T3 and H35 cells. As shown
(Fig. 4A), some Egr-1 binding
activity was present in normal rat liver, and Egr-1 binding activity
was rapidly induced 30 min posthepatectomy with a 5-7-fold induction at the 1-h peak. The induction of Egr-1 binding activity was specific to regenerating liver and not simply due to the stress of surgery or
induction of an acute-phase response, as sham operation did not
significantly increase Egr-1 binding during this time (Fig. 4A,
lane 6 and 7). Cycloheximide, a protein synthesis
inhibitor, did not block the increase in Egr-1 binding activity at 30 min posthepatectomy (Fig. 4A, lane 8) but inhibited Egr-1
retardation complex formation at 3 h posthepatectomy (Fig.
4A, lane 9).

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Fig. 4.
EMSA analysis of Egr-1 binding activity in
mitogen-activated cells. EMSAs were performed with the PRL/B
oligonucleotide using 5 µg of nuclear extracts from rat regenerating
liver at the indicated time points after hepatectomy (A).
The lower panel shows constitutive SP1 binding in the
absence of excess cold SP1 oligonucleotide. B, insulin
(10 8 M)-treated H35 cells; C, 20%
serum-treated NIH 3T3 cells in the presence or absence of cycloheximide
(CHX) at indicated times after insulin or serum
treatment. 200-fold molar excess of the unlabeled Sp1 oligonucleotides
was routinely included to keep Sp1 from binding to the overlapping Sp1
site. Quantification was by densitometry.
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In contrast, no Egr-1 binding activity was detected in quiescent NIH
3T3 cells and H35 cells, and there was little or no induction during
the first 30 min after serum or insulin (10
8
M) stimulation, respectively (Fig. 4, B and
C, lanes 1-3). Egr-1 retardation complexes were induced by
serum or insulin at 1 and 2 h (Fig. 4, B and
C, lanes 4 and 5) and were completely
abolished in the presence of cycloheximide (Fig. 4, B and
C, lanes 6-9). These results are in agreement
with those previously reported in which de novo synthesized
Egr-1 protein fully accounts for the induced Egr-1 binding activities
in mitogen-induced NIH 3T3 cells (29).
Immunoblot analysis was used to further examine Egr-1 protein
expression in the early phases of liver regeneration. As shown (Fig.
5A), Egr-1 protein was present
at a very low level in normal liver nuclear extracts and increased
dramatically over 30-fold at 30 min posthepatectomy (Fig. 5A,
lanes 1 and 2). However, gel mobility shift assays
showed only a 5-fold increase in Egr-1 DNA binding during this same
period (Fig. 4A). Cycloheximide treatment inhibited 80% of
the increase in Egr-1 protein at 30 min posthepatectomy (Fig. 5A,
lanes 2 and 4). On the other hand, a similar amount of
Egr-1 DNA binding activity was present at 30 min posthepatectomy with
or without cycloheximide treatment (Fig. 4A, compare
lanes 2 and 8). These results suggested that the
increase in Egr-1 DNA binding activity at 30 min posthepatectomy was
caused at least in part by post-translational activation of Egr-1 and
was not dependent on the absolute level of nuclear Egr-1 protein.

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Fig. 5.
Immunoblot analysis of nuclear extracts from
regenerating liver with polyclonal antibody specific for Egr-1.
A, nuclear extracts were prepared at indicated times
following partial hepatectomy in the presence or absence of
cycloheximide pretreatment. Lower panel, longer exposure.
B, the effect of phosphatase treatment on Egr-1 binding.
Nuclear extracts (5 µg) from 1 h posthepatectomy
(posthep) were incubated with or without 1 µg potato
phosphatase (PAP, Boehringer-Mannheim) for 20 min
at 37 °C, prior to EMSA assay as described under "Materials and
Methods." Where indicated the phosphatase inhibitor
Na2MoO4 was incubated at 20 mM with
the phosphatase for 10 min prior to adding nuclear extracts.
Quantification was by densitometry.
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Protein phosphorylation and dephosphorylation are the most common
post-translational modifications of protein function and play an
important role in regulation of gene expression, cell proliferation,
and differentiation (4). We therefore examined the effect of
phosphorylation on Egr-1 DNA binding by treating nuclear extracts with
potato acid phosphatase. We found that potato acid phosphatase
abolished most Egr-1 binding (Fig. 5B, lane 4). This effect
was largely blocked (Fig. 5B, lane 2) by preincubation with
phosphatase inhibitor Na2MoO4, indicating that
the effect of potato acid phosphatase was specific. These data
suggested that phosphorylation of Egr-1 protein was required for its
DNA binding activity.
Egr-1 Activation of PRL-1 Promoter P1--
Egr-1 DNA binding
activity increased in regenerating liver and mitogen-treated NIH 3T3
cells at the same time PRL-1 gene expression and/or
transcription increased. Because it is difficult to assess transcriptional activity of liver in vivo, we ascertained
whether Egr-1 could regulate the PRL-1 P1 promoter in cell
culture systems. First, we further delineated the sequences within the
first 200 bp 5' of the promoter essential for transcription of the
human PRL-1 gene in cell culture systems, and we then
assessed the relative amount of Egr-1 DNA binding in HepG2 and NIH 3T3
cells under normal growth conditions. Detailed deletions of the first
205 bp of the 5'-flanking region with exonuclease Bal-31 were performed
(Fig. 6A). Transfection and
luciferase assays demonstrated the presence of several potential
regulatory sequences in regions
198 to
110,
105 to
6, and
65
to
58 as the luciferase activity fell more than 20-fold following
serial deletions along this region. Loss of the strong Egr-1-binding
site at
99 did not have a major effect on the transcriptional
activity of the PRL-1 promoter in either HepG2 or NIH 3T3
cells. EMSA analyses confirmed that little Egr-1 binding activity is
present in these cells under conditions used to assess PRL-1
reporter activity (Fig. 6B), consistent with the lack of
impact of deletion of the
99 Egr-1 site. Therefore these cells
provided an appropriate background in which the role of Egr-1 in
PRL-1 promoter activity could be assessed.

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Fig. 6.
Promoter activity of the P1 region of the
PRL-1 gene and Egr-1 binding activity in growing HepG2
and 3T3 cells. A, left, the schematic
representation of the pGL2-Basic luciferase constructs used for
transfections. Right, graphical presentation of relative
luciferase activity after normalization for -galactosidase. HepG2
cells and NIH 3T3 cells were transfected using the calcium phosphate
method. 5 µg of luciferase reporter constructs were transiently
cotransfected with 1 µg of pSV- -galactosidase expression plasmid
as internal control to normalize transfection efficiency. Relative
luciferase activity was reported after normalization for transfection
efficiency. Six independent determinations were made for each construct
by performing duplicate analyses in three separate experiments.
Standard deviations were determined from the duplicate values in a
single experiment and were representative of the deviation in all six
determinations. B, EMSAs showing Egr-1 binding activity from
HepG2 and NIH 3T3 cells as compared with regenerating liver. EMSAs were
performed with the PRL/B oligonucleotide using 5 µg of nuclear
extracts from growing NIH 3T3 cells (lanes 1-3), HepG2
cells (lanes 4-6), and regenerating liver (RL)
30 min posthepatectomy (lanes 7-9). As indicated, 50-fold
excess of the unlabeled consensus Sp1 and Egr-1 oligonucleotides were
used as cold competitors.
|
|
As shown (Fig. 7A),
cotransfection of PRL-1 P1 promoter plasmid pSma I or p-D1
containing the
99 Egr-1 binding element with Egr-1 expression plasmid
pCMV-Egr-1 resulted in 5-7-fold induction in luciferase activity. No
obvious induction was observed when the Egr-1 site-deleted plasmid p-D3
was cotransfected with pCMV-Egr-1. Mutation of the Egr-1 site at
99
(mB in Fig. 3) abolished most Egr-1-mediated induction.

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Fig. 7.
Egr-1 activation of PRL-1
promoter P1. A, left, the schematic
representation of the pGL2-Basic luciferase constructs used for
transfections; right, graphical presentation of relative
luciferase activity after normalization for -galactosidase. 2 µg
of luciferase reporter constructs were transiently transfected into
growing NIH 3T3 cells with 7 µg of either parent expression plasmid
pCMV (vector) or Egr-1 expression plasmid pCMV-Egr-1 and 1 µg of
pSV- -galactosidase expression plasmid totaling 10-µg plasmids in
each transfection. B, serum induction of PRL-1
promoter. Left, the schematic representation of the
pGL2-Basic luciferase constructs used for transfections;
right, graphical presentation of relative luciferase
activity after normalization for -galactosidase. NIH 3T3 cells were
transfected with 2 µg of luciferase reporter constructs, 1 µg of
pSV- -galactosidase expression plasmid, and 7 µg of either
pCMV-Egr-1 or pCMV vector totaling 10-µg plasmids in each
transfection. At 16-18 h after CaPO4 transfection, cells
were serum-deprived (0.5% fetal calf serum) for 24 h. Serum
stimulation was executed by treating serum-deprived cells with 20%
serum in DMEM for 4 h. Cells were then harvested for luciferase
and -galactosidase assays. Relative luciferase activity was reported
after normalization for transfection efficiency. Six independent
determinations were made for each construct by performing duplicate
analyses in three separate experiments. Standard deviations were
determined from the duplicate values in a single experiment and were
representative of the deviation in all six determinations.
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The importance of the Egr-1 binding motifs in mitogen-induced
transcription of the PRL-1 gene was assessed following serum induction of serum-deprived NIH 3T3 cells when physiological levels of
Egr-1 protein are induced (see Fig. 4C). As shown (Fig.
7B), PRL-1 promoter activity was induced 3-fold
after stimulation with 20% serum, and this induction was entirely
dependent on the presence of an intact Egr-1 site. The high affinity
Egr-1 site B mutation or deletion of Egr-1 motifs in PRL-1
promoter resulted in loss of serum and Egr-1 inducibility of the
PRL-1 promoter under serum-starved conditions (Fig.
7B). These results suggest that Egr-1 plays an important
role in the mitogen-induced activation of the PRL-1 gene.
 |
DISCUSSION |
We demonstrated that PRL-1 mRNA expression is
up-regulated at the transcriptional level during the first few hours
posthepatectomy. PRL-1 run-on transcripts, which are
virtually absent in normal liver, were increased by 14.8-fold at 30 min
posthepatectomy and returned to base line by 6 h. However,
Northern blot and RNase protection assays indicate that
PRL-1 mRNA expression remains elevated through 24 h
posthepatectomy (3). This suggests that PRL-1 gene
expression may be regulated at the post-transcriptional level at later
times posthepatectomy. RNA stabilization could account for the
maintenance of PRL-1 mRNA at later times posthepatectomy as has been seen for other induced transcripts (30).
Egr-1 is transiently and rapidly expressed during the transition of
cell from the G0 to G1 phase in response to
various mitogens such as growth factors, cytokines, injury, and partial
hepatectomy (7, 9, 13, 14). Egr-1 bound to an oligonucleotide
containing the consensus site at
99 with 5-fold greater affinity than
for the
114 site of PRL-1 promoter P1. Egr-1 activated the
PRL-1 promoter primarily via the
99 site. The Egr-1
binding motif was necessary for transcriptional induction of the
PRL-1 gene following mitogen stimulation of NIH 3T3 cells
(5). This result supports the involvement of Egr-1 in PRL-1
gene transcription during liver regeneration as well when Egr-1 is even
more rapidly induced than in mitogen-treated 3T3 cells.
The region of the PRL-1 promoter with which Egr-1 interacts
is complicated by the presence of overlapping Sp1 binding motifs. Overlapping Egr-1/Sp1-binding sites have been found in many
Egr-1-regulated genes such as the genes for tumor necrosis factor a
,
basic fibroblast growth factor, the human IL-2 and human
platelet-derived growth factors A-chain and B-chain and others (9, 13,
31, 32). Competition between Egr-1 and Sp1 for the site plays a role in regulation of some of these genes (33). We have not formally addressed
competition between these factors for the motifs in PRL-1
promoter as Sp1 is constitutively expressed in normal liver, and its
binding activity does not change during liver regeneration. Although
these results do not support a role for Sp1 in the inducible expression
of PRL-1 gene in regenerating liver, it is possible that Sp1
is important for basal expression of the PRL-1 gene and Sp1,
resident on the promoter in normal liver, may be displaced by
increasing amounts of Egr-1, which confers higher transcriptional activation.
Egr-1 binding activity was rapidly induced during the early phase of
rat liver regeneration at 30 min with peak at 1 h, a good
correlation with the temporal increase in PRL-1 gene
transcription. The initial increase in Egr-1 DNA binding activity in
liver regeneration may be caused at least in part by post-translational
activation of Egr-1, because cycloheximide, a protein synthesis
inhibitor, blocked most of the increase in the absolute level of
nuclear Egr-1 protein but did not affect the increase in Egr-1 binding activity at 30 min posthepatectomy. Post-translational activation of
transcription factors existing in a latent state in the normal liver is
characteristic of signals that initiate liver regeneration. Some
examples of transcription factors that are activated by
post-translational mechanisms in the remnant liver immediately
posthepatectomy include NF-kB (24), STAT3 (33), and c-Jun (34) among
others. In addition to post-translational activation, many of these
transcription factors (NF-kB, STAT3, and c-Jun) are also induced at the
level of gene expression and de novo translation as is
Egr-1.
Potato acid phosphatase, a general phosphatase, abolished Egr-1
binding, and this effect was largely reversed by the phosphatase inhibitor Na2MoO4. Further studies with more
specific phosphatases have not allowed us to define further the
specific nature of the phosphorylation-dependent DNA
binding (not shown). Previous studies indicate that the regulation of
Egr-1 DNA binding activity is under the control of protein phosphatases
and kinases. Binding of cellular Egr-1 to its consensus sequence is
transiently stimulated by phorbol ester, which stimulates protein
kinase C, okadaic acid (35), and calyculin (36) which are inhibitors of
serine/threonine phosphoprotein phosphatases I and 2A. Egr-1 is
phosphorylated by the protein kinase casein kinase II, which has a
negative effect on its DNA binding and transcriptional activities (37).
Casein kinase II may be one of several kinases that regulate Egr-1
function, since the casein kinase II phosphorylation pattern of Egr-1
does not exactly coincide with that of serum-induced Egr-1. As another example, hyperphosphorylation of Egr-1 on tyrosine residues is seen in
v-Sis-induced transformation of NIH 3T3 cells (38). Thus multiple
activities of Egr-1 may be determined by site-specific phosphorylation.
Further research is necessary to define critical regulatory sites that
are phosphorylated in Egr-1, and their delineation may ultimately
provide insight into early signals in liver regeneration.
Although Egr-1 may play an important role in PRL-1 gene
transcription in regenerating liver and mitogen-treated fibroblasts, there is no obvious correlation between Egr-1 expression and/or activation and high expression of PRL-1 gene in normal
tissues such as brain and muscle or tumor cell lines like H35 cells,
where PRL-1 expression is constitutive, HepG2 cells, HeLa
cells and CV1 cells (3). For example, in intestinal epithelia that
contain both terminally differentiated and proliferating cells,
PRL-1 is expressed in the terminally differentiated villus
but not proliferating crypt enterocytes (5). As consensus CdxA sites
are present in PRL-1 promoter P1, Cdx homeodomain proteins,
which control intestinal differentiation, may contribute to this
differentiation related expression of the PRL-1 gene (6,
39). Likewise in other tissues, distinct transcriptional regulatory
factors are predicted to be major regulators of PRL-1 gene expression.