(Received for publication, December 21, 1994; and in revised form, May 12, 1995)
From the
We have isolated the promoter of the rat C/EBP During development, acquisition of the differentiated phenotype
involves expression of function-specific genes along with entry of the
cells into a quiescent phase of the cell cycle. Maintenance of this
state not only requires continual activation of the differentiation
program, but suppression of various growth related processes. In this
regard, investigators have proposed that the protein products of
certain function-specific genes also inhibit cell
proliferation(1, 2, 3) . To understand the
molecular mechanisms controlling this balance between growth and
differentiation, we are studying the proliferation of hepatocytes in
the regenerating liver and in culture. The liver is a highly
specialized organ composed primarily of hepatocytes which are normally
in a state of growth arrest (G While this growth process is
occurring, the regenerating liver is still capable of performing its
normal physiological functions by maintaining (7) and, in some
cases, inducing the expression of liver-specific genes (8, 9) . Consequently, we have focused on a family of
transcription factors, the C/EBP The four C/EBP proteins, C/EBP C/EBP Consistent with the notion
that C/EBP
Figure 3:
Ectopic expression of C/EBP
Figure 1:
Analysis of the
5`-upstream region of the rat C/EBP
To characterize the rat liver proteins that can
associate with elements within the C/EBP Fig. 2A shows that recombinant
C/EBP
Figure 2:
Identification and characterization of a
C/EBP binding site in the rat C/EBP
In an attempt to identify some of the polypeptides
within these complexes, we performed supershift analysis using
anti-C/EBP
Figure 4:
Analysis of C/EBP related proteins in
regenerating liver. A, changes in DNA binding activity. Equal
amounts (10 µg) of nuclear extracts from livers at the indicated
times following a partial hepatectomy were preincubated with either
anti-C/EBP
To demonstrate that C/EBP Fig. 3C also shows that deletion of either of two
regions from within construct 1 corresponding to -764 to
-330 (construct 8) or -764 to -232 (construct 9)
significantly reduces the activity (both basal and C/EBP
The DNA binding activity of the
C/EBP Expression of C/EBP
Polypeptides during Liver Regeneration-To determine whether
these changes in C/EBP binding activity can be accounted for by changes
in the abundance of the corresponding polypeptides, we analyzed nuclear
extracts from regenerating livers by Western blot using anti-C/EBP This decrease in the expression of these polypeptides
corresponds to a drop in DNA binding activity of the C/EBP In the case of C/EBP It appears that the decrease in the DNA binding activity of the
To more precisely define the factors responsible for this
pattern of transcription factor activity and to identify extracellular
effectors that may control the process, we have turned to freshly
isolated hepatocytes maintained in culture on a substratum of dried rat
tail collagen. It should be noted that events in the cell cycle
progress more slowly in hepatocytes in culture than in vivo.
Within the animal, DNA synthesis peaks steeply at 22-24 h
posthepatectomy, whereas in hepatocyte cultures G Fig. 5A shows an EMSA profile of nuclear proteins
isolated from hepatocytes, cultured for various times in the presence
or absence of EGF, that are capable of binding to the CB
oligonucleotide. In 4-h cultures there are abundant quantities of all
the C/EBP containing complexes identified in normal liver, but by 24 h
as the G
Figure 5:
Analysis of C/EBP related proteins in
cultured hepatocytes. A, changes in DNA binding activity.
Equal amounts (10 µg) of nuclear extracts obtained from hepatocytes
cultured for the indicated times in the presence or absence of EGF (10
ng/ml) were analyzed by EMSA as described in the legend to Fig. 4.
The
Western blot analysis of these nuclear extracts (Fig. 5B) shows that the drop in DNA binding activity
of the In the
case of C/EBP It is important to note,
however, that during the initial 24 h in culture, the changes in
expression of the C/EBP proteins do substantially reflect the changes
that occur in the regenerating liver: during early G
We first examined the effects of EGF, known to be a
potent hepatocyte mitogen both in vivo and in
vitro(33, 34) . Although EGF appears to have
little or no effect on the C/EBP-DNA binding activities at 4 h, by 24 h
and especially at 48 h (G The supershift analysis in Fig. 6A shows a decrease in the DNA binding activity of
C/EBP
Figure 6:
Effect of EGF and TGF
Addition of growth inhibiting doses of TGF In these 24-h
hepatocyte cultures, analysis by Western blots showed that EGF, in
agreement with the binding assays, also furthers reduction in
C/EBP Previous studies have shown that hepatocyte proliferation in
the regenerating liver (22, 30) and in culture (35) is accompanied by an extensive down-regulation of
C/EBP
Within the liver, the supershift analysis
with anti-C/EBP The role of these
different C/EBP
A second possibility is that C/EBP Recent studies have also suggested that C/EBP A similar change in C/EBP
binding activity is observed in nuclear extracts isolated from
hepatocytes proliferating in culture. As mentioned previously, the
growth related events progress much more slowly in culture than in
regenerating liver. Based on the time required for these cultured cells
to enter S phase and the pattern of gene expression, we estimate that
the initial 6 h of G Although these changes in C/EBP
It is important to note that
C/EBP Whereas the induction of the Addition of TGF The data in Fig. 6B suggest that the regulation of the C/EBP The parallel behavior of C/EBP With regard to the induction and progression of liver regeneration,
our data are consistent with significant roles for both C/EBP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
gene and
find a high degree of homology with the mouse gene, particularly in
putative regulatory domains. Transactivation of this promoter by
ectopic expression of rat C/EBP
occurs through a C/EBP regulatory
domain at position -170 to -195. An oligonucleotide
corresponding to this domain binds to complexes expressed in rat liver
that comprise C/EBP
-C/EBP
heterodimers (
) as well
as C/EBP
complexed with itself and/or other unidentified nuclear
factors (
1,
2, and
3). The DNA binding activity of these
complexes changes both qualitatively and quantitatively following
partial hepatectomy. Within 2-5 h postsurgery, the binding
activity of the
complexes drops severalfold, reaching a
nadir by 20 h. During the ensuing 3-8 days, as regeneration nears
completion, this activity slowly returns to normal quiescent liver
levels. Western blot analysis shows 3 major C/EBP
polypeptide
species (42, 40, and 30 kDa), whose abundance in general parallels the
decrease and recovery in DNA binding activity. In contrast to
C/EBP
behavior, the DNA binding activity of the
complexes is
transiently induced severalfold during the early G
period
between 2 and 6 h posthepatectomy. The major C/EBP
polypeptide is
the 32-kDa LAP protein, whereas the LIP protein (21 kDa) is weakly
expressed. Both remain essentially constant throughout the course of
regeneration, suggesting that changes in DNA binding activity may
reflect changes in the complexed proteins rather than the C/EBP
polypeptides themselves. In primary hepatocyte cultures, under growth
supporting conditions, in the absence of growth factors proliferation
is negligible; C/EBP
is abundantly expressed at the outset, but is
then extensively down-regulated. Epidermal growth factor causes further
decay of C/EBP
polypeptides and DNA binding activity, and
down-regulates C/EBP
DNA binding activity as well. Addition of
transforming growth factor
completely antagonizes the effects of
epidermal growth factor on C/EBP
activity, and partially overcomes
the effect on C/EBP
. These results demonstrate that the DNA
binding activity of C/EBP
and C/EBP
complexes is regulated in
the regenerating liver, and in hepatocyte cultures responding to growth
factors that regulate their proliferation.
), expressing an array of
differentiated functions that are crucial to the overall physiology of
the organism. Although growth and differentiation are considered
mutually exclusive, the hepatocytes can be induced to proliferate by a
metabolic overload imposed by the body, which, for experimental
purposes, is most readily initiated by a partial
hepatectomy(4, 5, 6) . This procedure
involves excision of the two large lobes of the liver in the rat (68%
of the whole organ), which induces the proliferation of the remaining
cells in the two small lobes. The excised lobes are not restored.
Within the remnant, the hepatocytes are activated first, moving rapidly
out of the G
into G
phase and progressing into
S phase at 14-16 h posthepatectomy. The peak of DNA synthesis
occurs approximately 8 h later and by 24-36 h most of the
hepatocyte population has undergone at least one cell division. It is
at this stage that the non-parenchymal cells start to proliferate while
some of the hepatocytes enter a second cell cycle. Growth of all the
cells continues throughout the liver remnant, preserving the
histological architecture, until the original mass of the liver is
restored within 9-12 days.
(
)proteins,
that are thought to support the differentiated state through regulating
many programs of gene expression including metabolism and growth.
,
,
, and
, are members
of a diverse group of nuclear factors that contain a leucine zipper
domain required for dimer formation and a basic DNA binding domain
which binds to the regulatory domains of promoters and/or enhancers of
target genes (10, 11) . Expression of the C/EBP genes
is regulated in such a way that three of the proteins (
,
,
and
) are restricted to a limited number of tissues (10) while C/EBP
(Ig/EBP) appears to be
ubiquitous(12) .
is produced in tissues capable
of gluconeogenesis and lipogenesis, especially liver and
fat(13) , and it is considered to play a direct role in
regulating transcription of some of the enzymes involved in controlling
these metabolic processes(14) . Recent data also suggest that
C/EBP
is a key player in the differentiation of preadipocytes into
fat cells(15, 16, 17) . This function
includes the activation of a program of fat-specific genes and
inhibition of cell growth(1) . In fact, Umek et al.(1) found that expression of a conditional form of
C/EBP
in preadipocytes arrested cell growth in a
differentiation-independent manner. Additionally, other investigators
have reported that forced expression of C/EBP
in other cell types
prevents the isolation of stable cell lines due to the growth
suppressing activity of this protein(18) .
is
also expressed in abundance in liver (19) but it is more widely
expressed among cell types than is C/EBP
(10) . In the case
of fat, C/EBP
appears to play a role at the early stages of
preadipocyte differentiation and it is then down-regulated as the
adipocytes adopt the complete fat-specific
phenotype(10, 20) . The role of C/EBP
in liver is
not known, but it is induced somewhat, along with C/EBP
, during
the acute phase response(21) .
is a growth suppressor(1) , we have shown that
C/EBP
gene transcription is inhibited during hepatocyte
proliferation in the regenerating liver and in culture(22) .
Additionally, studies by us and others have suggested that C/EBP
and immediate early genes are reciprocally expressed both in the liver (23) and in other cell types(18) . In fact, Freytag and
Geddes (18) have shown that overexpression of MYC can suppress
C/EBP
expression. To determine the mechanisms controlling this
apparent growth related expression of the C/EBP
gene, we have
isolated the rat gene and characterized the 5`-upstream region. We have
focused on a domain within the promoter that binds to a family of
hepatic nuclear factors which include C/EBP
and C/EBP
in
association with other proteins. Expression of these proteins changes
dramatically during liver regeneration and in response to growth factor
activation of hepatocyte proliferation in culture. This pattern of
C/EBP binding activity may contribute to the previously reported growth
related suppression of C/EBP
transcription, as well as to the
continuing expression of other liver-specific genes during hepatic
growth. As regards liver regeneration, our findings support the notion
that a role of C/EBP
may be to transactivate C/EBP
, and that
particular immediate early growth associated genes, as yet
unidentified, are likely partners in the C/EBP
heterodimers found
to undergo cell cycle associated changes in the regenerating
hepatocytes. The effects of two well known opposing growth factors (EGF
and TGF
) on particular aspects of C/EBP
and C/EBP
activities, illuminate specific ways in which growth factors may serve
to regulate hepatic regeneration at the molecular level.
Isolation of a Rat Genomic Clone Encoding C/EBP
A rat partial SauIIIA
genomic library obtained from Dr. Richard Hynes, MIT, was screened with
a rat C/EBP
mRNA and DNA Sequence Analysis
cDNA(24) . Twelve single plaques were positive
after the third screening and DNA isolated from each one was digested
to completion with EcoRI. Southern blot analysis using the
C/EBP
cDNA as a probe identified plaques that contained a positive
9.6-kilobase EcoRI fragment previously shown by Landschulz et al.(24) to correspond to the rat C/EBP
gene.
This fragment was gel purified, cloned into the Bluescript II
KS(+) vector to generate a subclone BK#9 which was used to produce
several additional subclones of the entire gene within the same
bluescript vector. One subclone, BK#1, corresponding to -1230 bp (BamHI) of the 5`-upstream region and 133 bp (NcoI)
of transcribed 5`-untranslated sequences was used to generate a series
of deletion constructs corresponding to various restriction enzyme
sites within the promoter (the NcoI site corresponds to the
initiation AUG for translation of the full-length C/EBP
polypeptide). Sequencing was performed on these double stranded DNA
subclones by the Sanger method(25) , using a Sequenase kit (U.
S. Biochemical Corp.) and a set of internal oligonucleotide primers
(Genosys Biotechnologies Inc., The Woodlands, TX).
Reporter Plasmids, Transfections, and CAT Assays
A
1.36-kilobase BamHI/NcoI fragment was isolated from
the Bluescript subclone BK#1 (see above) and cloned into the pCAT basic
reporter plasmid (Promega) to generate a C/EBP promoter/CAT
expression vector (construct 1, see Fig. 3C). A series
of deletion constructs (2-9, see Fig. 3C) were
generated from this parental vector 1 using a variety of restriction
enzymes identified from the sequence of the entire 1230 nucleotides of
BK#1 (-1230 = BamHI, -764 = AvrII, -720 = NotI, -449 = StuI, -330 = AvrII, -270 = PmlI, -232 = EspI, and
+133-NcoI). Construct 7 was generated unexpectedly during
the construction of the other constructs as a result of ★
restriction enzyme activity. DNA sequence analysis confirmed that the
promoter region of this reporter plasmid corresponded to -110 to
+133 of the 5`-upstream region of the C/EBP
gene. The other
constructs produced by deletion of different regions within construct 1
using the restriction enzymes listed above is shown in Fig. 3C. HepG2 cells maintained at 50-60%
confluency in Dulbecco's medium supplemented with 10% fetal calf
serum were transfected using the calcium-phosphate DNA coprecipitation
method(26) . The transfection mixture contained 10 µg of
the promoter plasmid DNA, 5 µg of MSV-C/EBP
expression vector,
and 2 µg of
-galactosidase plasmid per 100-mm dish. Cells were
harvested 40 h later and lysed by freeze-thawing. CAT assays were
performed as described in Promega technical bulletin TB 84:8/89. The
-galactosidase activity of the cell lysates was used to normalize
variabilities in the efficiency of transfection.
in HepG2
cells activates transcription of C/EBP
promoter/CAT reporter gene
constructs. A and B, ectopic expression of rat C/EBP
mRNA and DNA binding activity in HepG2 cells. The MSV-C/EBP
and
expression vectors were transiently transfected into HepG2 cells,
48 h later total RNA or nuclear extracts were isolated and analyzed by
Northern blot or EMSA, respectively. A, a Northern blot
containing equal amounts of total RNA (25 µg) corresponding to
normal rat liver (first lane), untransfected (second
lane), or transfected HepG2 cells (MSV-C/EBP
, third
lane, and MSV-C/EBP
, fourth lane) was hybridized
with a rat C/EBP
(upper panel) or C/EBP
(lower
panel) cDNA probe. B, nuclear extracts from untransfected (lane 1) or transfected (MSV-C/EBP
, lane 2, or
MSV-C/EBP
, lane 3) HepG2 cells were incubated with
anti-C/EBP
antibody for 2 h prior to addition of the radiolabeled
CB oligonucleotide. The C/EBP
containing complexes are
supershifted to the top of the gel (
) while the
complexes
migrate in the middle of the gel. These
complexes were shown to
contain predominantly C/EBP
proteins by an anti-C/EBP
supershift (data not shown). C, schematic maps and functional
analysis of the 5`-upstream region of the C/EBP
promoter. The
restriction fragment BamHI (at -1230) and NcoI
(at +133) corresponding to the promoter of the C/EBP
gene was
cloned into the promoterless reporter plasmid pCAT (Promega). Various
subclones of this expression plasmid were generated by creation of
deletions using a variety of restriction enzymes identified as a result
of sequencing the entire 1230 nucleotides of the 5`-flanking region of
the rat C/EBP
gene. These constructs were transiently transfected
into human hepatoma (HepG2) cells in the presence or absence of the
MSV-C/EBP
cDNA expression plasmid. CAT assays were performed as
described under ``Experimental Procedures.'' The data shown
represent numerical averages of duplicate
experiments.
Animals
Rats were maintained under veterinary
supervision, and experimental procedures were in accordance with BUSM
guidelines. Adult livers used for hepatectomy and cell culture studies
were obtained from male Sprague-Dawley rats (Taconic Farms, Germantown,
NY) weighing around 200 g, maintained under standardized light,
temperature, and feeding conditions. Partial hepatectomies, with
excision of 70% of the liver mass, were carried out under ether
anesthesia by the method of Higgins and Anderson (27) and the
residual lobes were allowed to regenerate over a period of 1 week. To
compensate for individual variation among rats, three animals were
sacrificed at each regenerating time point and their livers pooled for
isolation of nuclei.Hepatocyte Isolation and Culturing
Hepatocytes
were isolated by a modified collagenase perfusion method as described
previously(23, 28) . The final hepatocyte suspension
was either analyzed immediately or plated at 5.4 10
cells per 100-mm Lux culture dish precoated with dried rat tail
collagen. The culture medium was M-199 with Hank's salts and
added 26 mM sodium bicarbonate, 10 mM HEPES (pH 7.4),
10
M dexamethasone, 5 µg of sodium
linoleate (Aldrich), 0.1 mg/ml fatty acid free bovine serum albumin
(Pentax, Miles, Kankakee, IL), 20 mM sodium pyruvate, L-proline to a final concentration of 1 mM, 50
µg/ml sodium ascorbate, 20 milliunits/ml insulin, 1.5% gelatin, 100
units/ml penicillin, and 100 µg/ml streptomycin. Unless otherwise
stated, 10 ng/ml EGF (Peprotech, Rocky Hill, NJ) was always present,
and TGF
(R& Systems Inc., Minneapolis, MN) was added at the
doses indicated.
Isolation of Nuclear Proteins from Liver and Cultured
Hepatocytes
Nuclei were isolated from hepatocytes in the liver
or in culture as outlined by Mischoulon et al.(22) and Rana et al.(23) , respectively.
In each case, the nuclei were stored in nuclear suspension buffer (50
mM Tris-HCl, pH 7.5, 10 mM magnesium acetate, 40%
glycerol, 1 mM dithiothreitol) at -80 °C until ready
for use. To extract nuclear proteins, the stored nuclei were thawed on
ice, harvested by centrifugation at 1000 g for 10 min,
and protein extraction buffer (0.4 M NaCl, 5 mM EDTA,
10 mM sodium HEPES, pH 7.5, 0.2 mM phenylmethylsulfonyl fluoride, 1 mM dithiothreitol) was
added to the nuclear pellet, and the mixture was incubated on ice for
15 min, with occasional vortexing followed by centrifugation for 15
min. The nuclear protein supernatant was stored at -80 °C
after addition of glycerol to a final concentration of 15%. The protein
concentration was determined with the Bio-Rad protein assay kit
(Bio-Rad). All the steps of nuclei isolation and the subsequent protein
extraction were carried out in the cold room.
Electrophoretic Mobility Shift Assay (EMSA) and Western
Blot Analysis
These procedures were performed as described
previously (23) . The sequences of the double-stranded
oligonucleotides used in the EMSA studies are as follows: C/EBP,
5`-gatccGCGTTGCGCCACGATc-3`; and E Box, 5`-gatcCACGGACCACGTGTGTG-3`
(see Fig. 1for location of each sequence within C/EBP gene
promoter).
gene. DNA sequence of the
initial 400 bp of the promoter compared to the same region in the mouse
gene(31) . Underlined regions correspond to putative
protein recognition sites.
DNA Footprint Analysis
An oligonucleotide
corresponding to bases -316 to -301 of the rat C/EBP
gene (see Fig. 1) was 5`-end labeled with
[
-
P]ATP. Using this labeled oligonucleotide
and a second one corresponding to bases -3 to +18, a DNA
fragment (-316 to +18) was synthesized in a polymerase chain
reaction containing TaqI polymerase (Promega, Madison, WI). The
sequence of the resulting radiolabeled DNA probe was confirmed using
the Sanger dideoxynucleotide termination procedure(25) . DNase
I footprinting was performed on this probe as described by Graves et al.(29) .
Characterization of the 5`-Upstream Region of the
C/EBP
Studies by us (22) and others (30) have recently shown that the decrease in C/EBP Gene
gene
expression accompanying the proliferation of hepatocytes in the
regenerating rat liver is controlled at the level of transcription.
Seeking the mechanisms involved, we have isolated the C/EBP
gene
from a genomic library corresponding to rat liver DNA using as a probe
a cDNA complementary to the full-length (2.65 kilobases) rat C/EBP
mRNA. Southern blot analysis of several recombinant phage identified a
common 9.6-kilobase EcoRI fragment that contained the entire
C/EBP
cDNA as shown previously by Landschulz et
al.(24) . Subclones of this fragment were constructed and
the region corresponding to the promoter was identified by screening
with a probe corresponding to the 5`-end of the cDNA. The region
upstream from the start of transcription was sequenced bidirectionally
by the Sanger method using sequential oligonucleotides as primers.
Analysis of the initial 370 bp of this region revealed a high degree of
sequence conservation between this rat gene and a previously
characterized mouse gene (31) (see Fig. 1). Domains that
are completely homologous correspond to binding sites for particular
transcription factors including a region that can bind C/EBP proteins
in mouse cells, as well as an E box and a GC-rich region that has the
potential to associate with the egr-1 family of immediate early gene
products. The level of conservation within these domains suggests that
they are playing an important role in regulating C/EBP
gene
expression.
promoter, and to identify
any proteins that may be involved in regulating C/EBP
transcription during hepatocyte growth and differentiation, we have
performed a series of DNase I footprint and gel mobility shift assays
(EMSA) using a variety of polymerse chain reaction generated fragments
and oligonucleotides homologous to the putative regulatory domains. For
this study, we have focused on the region homologous to that in the
mouse gene that can bind to C/EBP related proteins. To map this region
within the rat gene, we performed a DNase I protection assay using a
polymerse chain reaction fragment extending from +15 to
-316.
protein footprints the region encompassing -170 to
-195 bp upstream of the start of transcription. In addition,
heat-treated nuclear extracts isolated from normal rat liver also
protects this region from DNase I digestion. Since C/EBP
is a
heat-stable protein, these data suggest that C/EBP
is at least one
of the proteins in liver nuclear extracts that can associate with this
site. To begin to identify these proteins, we performed EMSA using a
radiolabeled oligonucleotide (CB) corresponding to the footprinted
region shown in Fig. 2A. This CB oligonucleotide binds
avidly to recombinant C/EBP
to form a DNA-protein complex which
can be completely supershifted to the top of the gel by an
anti-C/EBP
antibody (Fig. 2B). The oligonucleotide
also associates with nuclear proteins isolated from rat liver which
migrate as a series of 5-6 DNA-protein complexes on a
nondenaturing polyacrylamide gel (Fig. 2C, first lane).
The specificity of this binding is demonstrated by the selective
inhibition of complex formation by a 100-fold molar excess of the
unlabeled CB oligonucleotide in the binding reaction (Fig. 2C, second lane). In contrast, a 100-fold molar
excess of an unrelated oligonucleotide corresponding to the E box and
its flanking region within the C/EBP
promoter (see Fig. 1)
has no effect on the association of the radiolabeled CB oligonucleotide
with the rat liver nuclear proteins (Fig. 2C, third
lane).
gene promoter. A,
DNase I footprint analysis. A polymerase chain reaction generated
fragment corresponding to the region between +18 and -316
was incubated with 5 µg of bacterially expressed C/EBP
(second lane) or with 10 µg of heat-treated (65 °C for
5 min) liver nuclear extracts (third lane) and then was
digested with DNase I. The protected region between -170 and
-195 is indicated by the box. B, association of
the putative C/EBP binding site with recombinant C/EBP
protein. A
double stranded oligonucleotide (CB) corresponding to the C/EBP
footprint was incubated with 5 µg of bacterially expressed
C/EBP
without(-) or with an anti-C/EBP
antibody (a/b
= C/E) and subjected to EMSA as described under
``Experimental Procedures.'' C, binding of the CB
oligonucleotide to nuclear proteins isolated from rat liver. Nuclear
extract isolated from rat liver at 5 h posthepatectomy was incubated
with radiolabeled CB oligonucleotide in the presence of a 100-fold
molar excess of unlabeled CB oligonucleotide or 100-fold molar excess
of an oligonucleotide encompassing the E Box at position -270 in
the rat C/EBP
promoter (see Fig. 1) and subjected to EMSA
as described under ``Experimental Procedures.'' D,
supershift analysis of C/EBP complexes. To identify specific proteins
within the complexes resolved in the EMSA shown in C, rat
liver nuclear extract was left untreated (first lane) or
preincubated for 2 h with anti-C/EBP
(second lane),
anti-C/EBP
(third lane), or c-Jun (fourth lane)
antibodies prior to addition of the radiolabeled CB oligonucleotide.
, correspond to complexes that are supershifted with both
anti-C/EBP antibodies, and
represent complexes that only interact
with anti-C/EBP
.
and anti-C/EBP
antibodies. This involved
preincubating liver nuclear extracts with the antibody for 2 h at room
temperature in order to produce a large C/EBP-antibody complex which is
still capable of associating with the CB oligonucleotide. Preincubation
with anti-C/EBP
antibody resulted in supershift of the slow
migrating species labeled
which now migrate as a discrete
band at the top of the gel (Fig. 2D, second lane). The
lower bands (labeled
) appear to be resistant to supershift with
the anti-C/EBP
antibody. All of the complexes react with the
anti-C/EBP
antibody to generate a supershifted complex which
migrates as a doublet at the top of the gel (Fig. 2D, third
lane). These data suggest that the larger complexes (
)
contain both C/EBP
and C/EBP
proteins, and possibly arise
from the association of three different C/EBP
polypeptides (42,
40, and 30 kDa) with the 32-kDa C/EBP
protein (shown below in Fig. 4B). The smaller complexes labeled
do not
contain C/EBP
polypeptides, and therefore, consist of either
homodimers of C/EBP
and/or heterodimers of C/EBP
with other
nuclear proteins. An antibody against c-Jun, which supershifts
complexes formed between an AP-1 oligonucleotide and c-Jun (data not
shown), has no effect on the migration of the C/EBP complexes
(
and
) within the gel (Fig. 2D, fourth
lane) demonstrating the specificity of the anti-C/EBP supershifts.
or anti-C/EBP
antibody for 2 h prior to addition
of a
P-labeled oligonucleotide corresponding to the C/EBP
site in the C/EBP
promoter as shown in Fig. 2. Binding of
the oligonucleotide to the different complexes was analyzed by EMSA as
described under ``Experimental Procedures.''
*,
C/EBP
supershifted band;
*, C/EBP
supershifted band;
1,
2, and
3 correspond to complexes resistant to
supershift by the anti-C/EBP
antibody but reactive with the
anti-C/EBP
antibody. B, Western blot analysis. Equal
amounts of nuclear extracts isolated from livers at the indicated times
following a partial hepatectomy were fractionated by SDS-12.5%
polyacrylamide gel electrophoresis, transferred to nitrocellulose
paper, and probed with either anti-C/EBP
or anti-C/EBP
antibodies as described under ``Experimental
Procedures.''
C/EBP
The EMSA data in Fig. 2D shows that
C/EBP Can Transactivate the Rat C/EBP
Promoter
when compared to C/EBP
plays a more prominent role in
directing the binding of rat liver proteins to the C/EBP site within
the C/EBP
promoter. It is likely, therefore, that C/EBP
plays
a role in controlling the transcription of the C/EBP
gene during
the growth and differentiation of hepatocytes in the liver. To explore
this possibility, we have determined whether C/EBP
protein can
transactivate the rat C/EBP
promoter in transiently transfected
human hepatoma cells (HepG2). To demonstrate that transient
co-transfection of this expression vector results in the ectopic
production of rat C/EBP
in human HepG2 cells, we performed the
experiment illustrated in Fig. 3, A and B. A
MSV-C/EBP
expression vector as well as a MSV-C/EBP
vector,
used as a control, were transiently transfected into separate cultures
of HepG2 cells at the same time that the transactivation-CAT assays (Fig. 3C, below) were being performed. Forty-eight
hours following the transfection, total RNA and nuclear proteins were
isolated from control non-transfected, MSV-C/EBP
, and
MSV-C/EBP
transfected cells and appropriate samples were subjected
to Northern blot analysis and EMSA. In Fig. 3A, the
same Northern blot corresponding to rat liver RNA (first lane)
as well as nontransfected (second lane) and transfected (right two lanes) HepG2 cell RNAs was probed with rat
C/EBP
(upper panel) and rat C/EBP
(lower
panel) cDNAs. The lower panel of Fig. 3A shows that HepG2 cells express appreciable amounts of C/EBP
mRNA which migrates slightly slower (larger mRNA) in the gel than the
corresponding rat liver mRNA (compare lanes 1 and 2).
Transfection of the rat C/EBP
expression vector results in the
production of abundant amounts of the corresponding mRNA to a level
that is even greater than that expressed in the same quantity of rat
liver RNA (compare first and fourth lanes). The upper panel shows barely detectable amounts of the human
C/EBP
mRNA in nontransfected HepG2 cells (second lane) as
expected for hepatoma cells which have down-regulated the C/EBP
gene(13) . Tranfection of the rat C/EBP
expression vector
results in the production of significant amounts of the corresponding
rat mRNA (third lane). Cells transfected with the rat
C/EBP
expression vector produce very low amounts of the human
C/EBP
mRNA (fourth lane) demonstrating that ectopic
expression of rat C/EBP
is not capable of activating the
endogenous human C/EBP
gene. The EMSA data in Fig. 3B shows that nuclear extracts isolated from nontransfected HepG2
cells (first lane) contain complexes that can bind to the
oligonucleotide that corresponds to the C/EBP binding site within the
C/EBP
gene promoter. Ectopic expression of the C/EBP mRNAs as
shown in Fig. 3A results in the synthesis of the
corresponding proteins that are capable of binding to the CB
oligonucleotide (second and third lanes). These data
indicate that the cotransfected C/EBP
cDNA is synthesizing large
amounts of functional rat C/EBP
protein within the human hepatoma
cells.
can transactivate the rat
C/EBP
gene promoter, we generated a variety of deletion constructs
corresponding to restriction fragments of the 5`-flanking region of the
start of transcription cloned upstream of a CAT reporter gene. These
constructs were transfected into HepG2 cells in the presence or absence
of the MSV-C/EBP
expression vector. Fig. 3C shows
that construct 1, corresponding to +133 to -1230 in the rat
C/EBP
promoter, supports a modest level of transcriptional
activity which was enhanced 3-4-fold when transfected into HepG2
cells along with the C/EBP
expression vector. Deletion of the
region -1230 to -720 results in an enhancement of the basal
activity (without C/EBP
), suggesting the removal of a negative
element. This resulting construct 2 (-720 to +133) is still
responsive to transactivation by C/EBP
protein. Deletion of a
region between -720 and -449 (construct 3) reduces the
activity slightly, without preventing the transactivation by
C/EBP
. Removal of nucleotides between -449 to -330
(construct 4) greatly elevates the basal (non-C/EBP
) activity to
such an extent that it is minimally responsive to C/EBP
.
Constructs 5 and 6 corresponding to -270 and -232
nucleotides upstream of the start of transcription, respectively, have
extremely low levels of basal activity suggesting that the small domain
between -330 and -232 is needed for promoter activity in
the absence of C/EBP
. More importantly, C/EBP
protein can
transactivate these minimal promoters, probably as a result of the
C/EBP binding site at position -190 to -170 (see Fig. 1and 2A) within each construct. To demonstrate the
importance of this C/EBP binding domain in regulating expression of
these promoters, we generated construct 7 which lacks the C/EBP site
and corresponds to -110 to +133 nucleotides of the
C/EBP
gene. Deletion of 122 nucleotides from the minimal promoter
6 (-232 to +133) to give rise to construct 7 (-110 to
+133) significantly reduced the transactivation of this promoter
by C/EBP
(compare the CAT activity of construct 6 with 7 in the
presence of C/EBP
). The only region within this 122-nucleotide
stretch that can bind to C/EBP proteins is the site at -170 to
-195 (see Fig. 2A). These data strongly suggest
that this C/EBP binding site is a regulatory domain responsible for
facilitating the transactivation of the C/EBP
gene by C/EBP
.
transactivated) of the entire 1230nucleotide upstream region (construct
1). These constructs (8 and 9) are still minimally responsive to
cotransfection with C/EBP
cDNA, probably due to the presence of
the C/EBP binding site at -170 to -195. These data suggest
that there is a prominent negative element between -720 and
-1230 which can inhibit the activity of the minimal promoter
-330 to +133. They also suggest that this negative element
can be neutralized by an internal (-764 to -330) positive
element. Taken together, these data strongly support the notion that
the C/EBP domain (-170/-195) contributes significantly to
the C/EBP
-dependent induction of C/EBP
promoter activity, and
this activity can be modulated by additional upstream elements.
Regenerative Changes in the Binding Activity of Rat Liver
Nuclear Proteins That Associate with the C/EBP Site
The data in Fig. 2D demonstrate that several protein complexes
containing both C/EBP and C/EBP
can associate with the domain
located at -170 to -195 within the 5`-upstream region of
the C/EBP
gene, and that this element appears to contribute to the
enhanced activity of the C/EBP
promoter when exposed to
ectopically expressed C/EBP
protein (Fig. 3C). To
determine whether there is a change in the binding activity of
complexes that bind to this site during liver regeneration, we
performed a series of EMSAs using nuclear extracts isolated from rat
livers at times following a partial hepatectomy. Activation of hepatic
growth appears to alter the overall binding pattern of the complexes
found in quiescent liver. To resolve these changes in overall binding
activity, we performed supershift analysis using anti-C/EBP
and
anti-C/EBP
antibodies throughout an entire regeneration time
course. As discussed above, the anti-C/EBP
antibody completely
supershifts the upper, slower migrating complexes (
) allowing
for the analysis of the lower complexes (labeled
) within the
center of the gel. This high resolution supershift facilitates,
therefore, an analysis of all C/EBP
containing complexes separate
from all the other C/EBP complexes during liver regeneration. Fig. 4A shows that hepatocyte proliferation in the
liver causes both a quantitative and qualitative change in the binding
properties of the nuclear proteins that associate with the C/EBP site.
It is clear from this supershift EMSA that the activity of C/EBP
complexes (Fig. 4A, left panel,
*) drops
significantly during the course of the regeneration. The drop is first
apparent at 2-6 h and reaches a nadir by 20 h as the hepatocytes
are progressing through S phase.
complexes also responds to hepatocyte proliferation.
Specifically, there is an extensive increase in activity during the
initial 6 h posthepatectomy, a time corresponding to entry of the
quiescent hepatocytes into G
phase of the cell cycle. At 2
h postsurgery there is an increase in the binding activity of a fast
migrating species labeled
3, which is virtually undetectable in
normal liver (see Fig. 4A, left panel, lane 2). The
most pronounced increase in C/EBP
binding activity is due to the
induction of a species labeled
1. The peak binding activity of
this species occurs at approximately 6 h, gradually returning to the
low levels detected in the resting liver by 72 h postsurgery. The
supershift analysis shown in the right panel of Fig. 4A confirms that these
species all contain
C/EBP
polypeptides. There are additional proteins that do
consistently associate with the CB oligonucleotide but they are
resistent to supershift with anti-C/EBP
,
, and
antibodies (the
data are not shown).
and anti-C/EBP
antibodies. Fig. 4B shows an
immunoblot corresponding to equal amounts of nuclear protein subjected
to SDS-polyacrylamide gel electrophoresis and probed with each
antibody. In the case of C/EBP
(Fig. 4B, upper
panel), the major immunoreactive species present in normal
quiescent liver migrate at 42, 40, and 30 kDa. The abundance of all
three polypeptides diminishes during the regeneration process, the
initial drop occurring between 2 and 5 h posthepatectomy, corresponding
to the early G
phase of the hepatocyte cell cycle. It is
important to note, however, that the 42- and 40-kDa species decrease to
a greater extent during the initial 5 h postsurgery than the 30-kDa
protein.
containing complexes. As mentioned above (see Fig. 2D),
it is quite likely that the complexes detected in the EMSA labeled
correspond to the three C/EBP
polypeptides, 42, 40, and
30 kDa, complexed with C/EBP
. Additionally, this drop in
C/EBP
protein levels seen in Fig. 4B occurs soon
after a corresponding decrease in the mRNA which we reported
previously(22) . As the accompanying burst of hepatocyte
proliferation subsides, there is a gradual return of the C/EBP
proteins and mRNA (data not shown) to near normal liver levels by 8
days (192 h).
(Fig. 4B, lower
panel), the major immunoreactive species is the 32-kDa polypeptide
corresponding to the LAP protein(19) ; there is a very small
amount of the 21-kDa species or LIP protein (32) expressed in
normal liver. Unlike the C/EBP
species, the abundance of both LAP
and LIP remains essentially constant throughout the entire regeneration
process (8 days). These data suggest that both the qualitative and
quantitative changes in the DNA binding activity of the C/EBP
complexes during the early phases of hepatocyte proliferation are
likely due to changes in the abundance of associated proteins rather
than changes in the abundance of C/EBP
polypeptides themselves.
heterodimers is due to a decrease in the C/EBP
rather
than the C/EBP
components. On the other hand, any
post-translational modifications of C/EBP
or C/EBP
polypeptides during liver regeneration could contribute to changes in
the binding activity of the
or other
dimers.
Changes in the Binding Activity of Nuclear Proteins That
Associate with the C/EBP Site during the Proliferation of Hepatocytes
in Culture
The changes in DNA binding activity as well as the
steady state levels of C/EBP polypeptides correlates closely with the
progression of hepatocytes through the cell cycle in the regenerating
liver. The extensive drop in C/EBP expression is consistent with
the hypothesis that C/EBP
is a growth suppressor and its activity
needs to be attenuated in order for the hepatocytes to transit the cell
cycle.
lasts
through the first and well into the second day postplating, followed by
S phase which becomes maximal in the earlier part of day 3.
phase progresses, there is a significant drop in
the activity of the upper
species and a slight increase in
the activity of the lower
species. During the following 24 h (48
h postplating), there is an additional decrease in
DNA
binding activity and a noticeable drop in the activity of the
complexes (Fig. 5A, lanes minus EGF).
correspond to complexes which are
supershifted by anti-C/EBP
antibody (see Fig. 2D);
correspond to complexes that are resistant to supershift by
anti-C/EBP
but contain C/EBP
. B, Western blot
analysis. Equal amounts of nuclear proteins isolated from hepatocytes
cultured for the indicated times in the presence of 10 ng/ml EGF were
analyzed on Western blots as described in the legend to Fig. 4.
complexes within the first 24 h can be accounted for
by an extensive decrease in the abundance of the three C/EBP
polypeptides. By 48-72 h of culture, the amounts of these
anti-C/EBP
immunoreactive polypeptides have dropped to
undetectable levels. This is similar to their behavior during G
progression into S phase in the regenerating liver.
, instead of a drop in abundance during the initial
24 h postplating, as observed for C/EBP
, there is an extensive
induction of the 32-kDa C/EBP
polypeptide (LAP) and a noticeable
enhancement in the 21-kDa LIP protein expression. During the following
24 h of culture (48 h time point), these elevated levels of C/EBP
polypeptides have rapidly dropped to amounts that are not detectable by
the Western blot procedure. It appears, therefore, that the change in
DNA binding activity of the complexes containing C/EBP
(labeled
) during the 24-72 h period are due to a corresponding
decrease in C/EBP
polypeptides. This pattern of C/EBP
expression during the late stages of hepatocyte proliferation in
culture deviates significantly from that observed during hepatocyte
cell cycle progression in the liver. This is probably due to the
extensive dedifferentiation of hepatocytes cultured on collagen that
does not occur in regenerating liver.
, both
in the liver (at 2-6 h) and in culture (at 24 h), there is an
extensive decline in C/EBP
polypeptides. Likewise, C/EBP
proteins continue to be expressed in liver, and in culture albeit at
somewhat enhanced levels (Figs. 4B and 5B).
Effects of Growth Factors (EGF and TGF
To determine whether the program
of C/EBP gene expression is responsive to growth factors that influence
hepatocyte proliferation, we analyzed the effect of both growth
promoting (EGF) and growth inhibiting (TGF) on C/EBP
Activity in Hepatocyte Cultures
) factors on the binding
activity of the C/EBP complexes in hepatocytes cultured for 24 h. We
chose this time because we believe that these isolated hepatocytes
cultured for 1 day represent an appropriate model to study the events
occurring during the G
phase of the hepatocyte cell cycle
in the regenerating liver. Short term cultures were utilized because at
longer times hepatocytes rapidly deviate from their in vivo counterparts due to the accompanying suppression of liver-specific
functions.
to early S phase) binding
activity of
and
complexes is well below the activities
of cells deprived of the growth factor (Fig. 5A). By 72
h (peak of S phase), overall binding activity has dropped to virtually
undetectable levels where EGF is present. In its absence significant
levels persist, but are still manyfold lower than in freshly isolated
cells. Although these changes in C/EBP
binding appear consonant
with those seen in regenerating liver, the discrepancies in C/EBP
binding and polypeptides, already noted, pointed to a 24-h limit for
reliable data from cultures.
containing complexes (
*) during the 24-h culture
period, even in the absence of EGF (compare lanes 1 and 2). Addition of a mitogenic dose (10 ng/ml) of EGF depresses
this activity even further (lane 3). On the other hand, there
is a large increase in the DNA binding activity of C/EBP
complexes
(
1,
2), which is reduced by EGF to the levels expressed in
normal liver.
on C/EBP
related proteins in primary cultures of hepatocytes. The hepatocytes
were cultured for 24 h in the absence or presence of the indicated
doses of EGF and TGF
. Nuclear proteins were extracted, and
analyzed by supershift EMSA (A) using anti-C/EBP
antibody or by Western blot (B) as described in the
legend to Fig. 4. NL, normal liver nuclear
extract.
(0.6
and 1.0 ng/ml) to the mitogenic dose of EGF antagonizes the EGF,
amplifying the C/EBP
activity severalfold and promoting its return
toward a normal level of expression. TGF
acts similarly in the
case of C/EBP
, overwhelming the inhibitory effect of EGF and
greatly augmenting its DNA binding activity.
protein (Fig. 6B, upper panel, compare lanes 1 and 2) and this effect is similarly reversed
by addition of TGF
. TGF
therefore seems to antagonize the EGF
at a level preceding translation. In contrast, these two growth factors
have little or no effect on the abundance of either of the C/EBP
proteins, LAP (32 kDa) or LIP (21 kDa) (Fig. 6B, lower
panel). Thus, it appears that the respective growth stimulatory
and growth inhibitory influences of EGF and TGF
on hepatocytes,
demonstrable both in the animal and in culture, are likewise evident in
their effects on C/EBP
and C/EBP
at the molecular level, both in vivo and in vitro. The evidence points to
regulation of C/EBP
at the pre-translational and C/EBP
at the
post-translational level.
gene transcription. In the present studies, we show that a
regulatory domain present in the promoter of the rat C/EBP
gene is
capable of binding to a family of hepatic nuclear protein complexes
that contain either C/EBP
or C/EBP
polypeptides.
Additionally, the DNA binding activity of these complexes is strikingly
altered in regenerating liver, and similarly in hepatocyte cultures
responding to growth factors that regulate their proliferation.
Complexity of the Proteins Binding to the C/EBP Site in the Rat
C/EBP
The C/EBP transcription factor was
first identified as a nuclear protein that could associate with CCAAT
motifs within two viral DNAs(29) . Comparison of DNA sequences
in additional genes led to early speculation that the optimal core
element for C/EBP-DNA interactions consists of directly abutted
half-sites of the sequence GCAAT. During the last few years, many
C/EBP-DNA binding sites have been discovered in the promoters and
enhancers of genes expressed in a variety of cell types. Some of these
genes code for proteins that have a defined function and are expressed
in a limited number of tissues (e.g. albumin in the liver (36) and insulin responsive glucose transporter,
GLUT4(37) , in fat and muscle), whereas other genes code for
ubiquitous proteins (e.g. Fos(38) ). Analysis of the
C/EBP sites within this diverse population of genes shows a significant
degree of variability among DNA sequences. The transcription factors
that bind to these sites are dimers, with a potentially variable
subunit. Regulation of the C/EBP-DNA binding activity can therefore be
affected by the ability of each C/EBP protein to dimerize with other
members of the C/EBP family and with other nuclear factors as well.
Consequently, different dimers may bind to specific sites in various
genes, depending on the sequence, thereby conferring specificity in
regulation of particular genes. It was therefore important in the
present study to analyze the protein complexes that associate with the
C/EBP site within the C/EBP Gene Promoter
promoter rather than with a consensus
DNA sequence, in view of the likely possibility that the expression of
the hepatic C/EBP
gene may be regulated in particular ways during
quiescence, growth, and differentiation due to association of different
C/EBP containing dimers with its C/EBP site. In this regard, it is
interesting that Diehl and Yang (39) using an oligonucleotide
corresponding to the C/EBP site in the c-Fos promoter concluded that
there was no significant decrease in C/EBP
DNA binding activity
during liver regeneration. It is possible that their probe associates
with a subset of
dimers that respond differently to
hepatocyte mitogens than the
dimers analyzed in Fig. 4A.
antibody (Fig. 2D) shows the
existence of at least 3 complexes, labeled
, which contain
both C/EBP
and C/EBP
polypeptides. As previously mentioned,
these heterodimers probably arise from the association of the different
C/EBP
proteins with the 32-kDa C/EBP
polypeptide (Fig. 4B). These C/EBP
polypeptides are
synthesized from the same full-length C/EBP
mRNA by initiation of
translation at internal methionine residues. Recent studies (40) have shown that the 42-kDa polypeptide corresponds to the
expected full-length C/EBP
protein encoded by the open reading
frame of the C/EBP
mRNA (+133 to +1207, i.e. 1074 nucleotides synthesizing 358 amino acids), whereas the 30-kDa
species is generated by the initiation of translation from an internal
alternative start site at methionine 118 terminating at the same
termination codon as the 42-kDa species. Analysis of the nucleotide
sequence of the C/EBP
mRNA reveals an additional internal
methionine at residue 15 that could explain the existence of the 40-kDa
polypeptide. The polypeptides produced all contain the leucine zipper
and basic DNA binding region that are located at amino acids
284-346, and are therefore all capable of forming heterodimers
with other leucine zippers and binding to DNA.
polypeptides is not known. It is possible that
elimination of particular amino terminus sequences may alter the
transactivation properties of the C/EBP
/C/EBP
heterodimers
present in liver nuclei. Lin et al.(40) have shown
that the NH
-terminal 12 kDa of the p42 C/EBP
is needed
for the anti-mitotic activity of the C/EBP
protein in
preadipocytes. Thus, p30 is no longer capable of suppressing growth but
it still retains its ability to transactivate reporter genes containing
the C/EBP site. The change in the ratio of p42 and p30 during the early
phase of liver regeneration (Fig. 4B, lane 5 h) may
therefore have some functional significance. For instance, it may be
important to lower the abundance of p42 before p30, which lacks the
anti-mitotic activity, to facilitate entry into G
. It is
also possible that the domains in p42 versus p30 may affect
other transcriptional events during liver regeneration.
Role of C/EBP Proteins in Regulating C/EBP
The changes in the binding of C/EBP containing
complexes to the C/EBP site within the promoter of the C/EBP Gene
Transcription
gene
that occur in proliferating cells suggests that this family of
transcription factors may play a role in regulating C/EBP
transcription during cell growth and differentiation. An important
question is to what extent does C/EBP
autoregulate its own
transcription? Our earlier studies (22) have shown that
C/EBP
gene transcription and steady state levels of C/EBP
mRNA begin to subside within the initial 2-5 h posthepatectomy
and we now find a corresponding drop in the abundance of the C/EBP
polypeptides and DNA binding activity (Fig. 4). There is no
indication that the C/EBP
protein decreases sooner than the drop
in transcription of the gene. It is reasonable to propose therefore
that the decrease in C/EBP
DNA binding activity is regulated
primarily by a decrease in the transcription of the C/EBP
gene.
This may involve an early, transient inhibitory event resulting in an
extensive decrease in C/EBP
protein that may then contribute to
the continued suppression of the C/EBP
gene in the proliferating
cells. Although, our previous data (22) show that restoration
of transcription, once the growth impetus subsides, occurs before the
protein begins to accumulate, further supporting the notion that
transcription of the C/EBP
gene is not autoregulated during
hepatic proliferation.
may
be a regulator of C/EBP
transcription. The induction of C/EBP
binding activity occurs within the initial 2 h posthepatectomy and
precedes the drop in C/EBP
expression (Fig. 4A, left
panel). This event coincides with the activation of the immediate
early gene program as indicated by the expression of c-Fos, c-Jun, and
JunB mRNAs(8) . This enhanced expression of C/EBP
consists
of at least three different complexes, referred to as
1,
2,
and
3, which are all induced transiently during the early G
period and then return to normal liver levels after S phase has
subsided (72 h post-surgery) (Fig. 4A). The nature of
these C/EBP
complexes is not known. They are either homodimers of
C/EBP
or heterodimers of C/EBP
with other hepatic nuclear
factors because monomers can not bind to DNA (41) . The fact
that these complexes are resistant to a supershift with anti-C/EBP
antibody suggests that they do not contain C/EBP
. LIP, a truncated
form of C/EBP
(LAP), can dimerize with C/EBP
and in so doing
can repress its ability to activate transcription of target
genes(32) ; in fact, only a moderate increase in LIP/LAP ratios
will have a significant inhibitory effect on C/EBP
(LAP) activity.
Although the level of LIP expression in the regenerating liver is very
low (Fig. 4B, lower panel), it is possible that one of
the
complexes, perhaps
3, corresponds to LAP/LIP
heterodimers.
can
associate with other transcription factors including Fos, Jun, and
NF-
B(42, 43) . In the case of Fos and Jun,
formation of heteromeric complexes between C/EBP
and one of these
other B-ZIP proteins represses transcriptional activation of reporter
genes by C/EBP
(42) . It is possible that the induction of
these immediate early gene products, Fos and Jun, as well as a possible
increase in LIP, during the initial 2 h of liver regeneration may
inhibit the transcriptional activation of C/EBP
by C/EBP
. The
data in Fig. 2D, however, suggests that c-Jun is not a
component of these C/EBP complexes in liver nuclei at 5 h
posthepatectomy since an anti-c-Jun antibody had no effect on the
migration of these complexes in the EMSA.
phase in the liver corresponds
approximately to the 18-24 h of culture of hepatocytes. In this
regard, it is worth noting that the pattern of C/EBP
binding
activity observed in the regenerating liver at 6 h (Fig. 4A,
lane 3) is very similar to the pattern in hepatocytes cultured for
24 h, with or without EGF (Fig. 6A, lanes 2 and 3).
DNA binding
activity during G
phase of the hepatocyte cell cycle may
contribute to the suppression of the C/EBP
gene transcription,
additional roles may come into play. The early increase in C/EBP
binding activity may contribute importantly to the regulation of other
liver-specific genes during this phase of regeneration, because several
genes which are known to contain C/EBP binding sites within their
promoters and enhancers continue to be transcribed at near normal
levels during regeneration. The enhanced activity of C/EBP
may
therefore function to compensate for the decrease in C/EBP
activity. It is also likely that C/EBP
can mediate the induction
of liver-specific genes. For instance, Taub and collaborators (8) have shown a large induction of phosphoenolpyruvate
carboxykinase that coincides with the G
/G
transition in regenerating liver. The promoter of the
phosphoenolpyruvate carboxykinase gene contains several C/EBP binding
sites which participate in the C/EBP dependent induction of the
gene(44) .
Growth Factor Regulation of C/EBP DNA Binding
Activity
We have recently shown that exposure of hepatocyte
cultures to mitogenic doses of EGF (10 ng/ml) depresses C/EBP mRNA
expression 3-4-fold during the initial 4 h(22) . We now
observe a similar depression of both C/EBP
protein steady state
levels and DNA binding activity during the initial 24 h of culture (Fig. 6). Taken together the data suggest that the change in
C/EBP
protein activity is primarily regulated at the level of
C/EBP
mRNA expression, and that this activity correlates with the
growth promoting activity of EGF.
mRNA, protein, and DNA binding activity all subside to
extremely low levels in hepatocytes cultured in the absence of growth
factors, while at least several of the immediate early growth
responsive genes remain activated, indicating that these cells are not
in a state of growth arrest in G
(23) . These
immediate early growth response genes may inhibit the transcriptional
activation of the C/EBP
gene either directly or indirectly through
association with C/EBP
.
1 and
2 C/EBP
complexes occurs during the initial 24 h of culture
in the absence of EGF (Fig. 6A, lane 2), the
3
species, which was prominent at 2 h posthepatectomy (Fig. 4A, lane 2), is absent. This observation suggests
that the 24-h cultures have progressed beyond this very early phase of
G
, and represent a phase approximating at the 6-h time
point in regenerating liver in which
3 is absent and
1 and
2 species are abundant. On the other hand, mitogenic doses of EGF
appear to reduce the activity of the
1 and
2 complexes in the
24-h hepatocyte cultures to a level approximating the regenerating
liver at 20 h posthepatectomy (compare Fig. 6A, lane 3, with Fig. 4A, left panel, lane 4). An
interpretation of these data is that hepatocytes cultured in the
absence of EGF are arrested in a stage of G
in which
abundant levels of
1 and
2 complexes are present and
equivalent to 6 h posthepatectomy. Exposure of cells to EGF, however,
stimulates progression through this control point during which time
1 and
2 activity is down-regulated as is apparent between 6
and 20 h posthepatectomy.
, which is known to
block cell cycle progression in mid to late G
phase (45) prevents this EGF dependent down-regulation of
1 and
2 activity and restores it to the abundant levels seen in early
G
cells (either at 6 h posthepatectomy or 24 h in culture
without EGF). The down-regulation of
1 and
2 activity may
therefore contribute to processes that control further progression of
hepatocytes through G
into S phase (Fig. 4A).
binding activity
occurs at a post-translational level, which raises the possibility that
the C/EBP proteins are targets of signal transduction pathways
activated by EGF or TGF
.
and C/EBP
in hepatocytes proliferating within the regenerating
liver and in growth factor stimulated cultures supports the
physiological relevance of the results, lending support to the data on
growth factor effects that can only be explored in vitro.
and
C/EBP
. C/EBP
appears to be an activator of C/EBP
. The
latter, as an important regulator of metabolism, may directly or
indirectly act as a growth suppressor and promoter of the
differentiated state. Our exploration of the protein products of these
two genes, and their DNA binding activities and associated quantitative
and qualitative changes in behavior of their protein heterodimers bring
to light a number of ways in which these two genes may operate. Growth
activation may involve participation of immediate early growth response
genes as partners in C/EBP heterodimers. Our results also show that two
oppositely acting growth factors, EGF and TGF
, exert specific
effects in cultures which parallel those occurring in the liver
following partial hepatectomy. Multiple growth factors and hormonal
modulators have been put forward as regulators of hepatic regeneration.
Mechanisms are now emerging at the molecular level that may eventually
clarify which of these agents are major players, at what stages, and in
what ways they function in this tightly controlled growth process.
We thank Dr. Richard Hynes for the Rat Genomic
library. We acknowledge Drs. Vassilis Zannis and Dimitris Kardassis for
recombinant C/EBP protein and for valuable advice. We also thank
Babette Radner, Kimberly Stielglitz, and Dezhung Zhao for excellent
technical assistance.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.