(Received for publication, July 31, 1995; and in revised form, October 30, 1995)
From the
CCAAT/enhancer-binding protein (C/EBP) isoforms are thought to
be important regulators of the hepatocyte phenotype. However, the
specific physiological roles of different isoforms are poorly
understood because hepatocytes express multiple C/EBPs, and various
isoforms have overlapping functions. To identify the functions of
C/EBP in mature hepatocytes, replication-defective adenovirus
vectors were used to efficiently and homogeneously overexpress the
mouse C/EBP
gene in a SV40 virus-conditionally transformed rat
hepatocyte line that can be induced to express C/EBP
and
C/EBP
but that has little endogenous C/EBP
expression.
Hepatocytes were infected with a recombinant adenovirus vector carrying
the cDNA for C/EBP
driven by Rous sarcoma virus promoter elements
(AdCEBP
) or a similar vector carrying the Escherichia coli
lacZ gene (Ad
gal). Staining for
-galactosidase
demonstrated an infection efficiency of 100% at a multiplicity of
infection of 25 plaque-forming units/cell and persistence of foreign
gene expression for at least 9 days. Cultures infected with AdCEBP
had 50-fold higher levels of C/EBP
mRNA and protein than those
infected with Ad
gal, but similar expression of C/EBP
.
Infection with AdCEBP
inhibited proliferation in cells expressing
little C/EBP
, even when proliferation was driven by the SV40
transforming antigen, and also blunted mitogenic induction of the
c-myc proto-oncogene in nontransformed cells with high levels
of C/EBP
. Although overexpression of C/EBP
consistently
increased C/EBP
DNA binding activity, it was not sufficient for
albumin expression. Infection with AdCEBP
only increased albumin
mRNA levels in nontransformed cells that also expressed relatively high
levels of C/EBP
. Thus, in hepatocytes, C/EBP
has a dominant
antiproliferative function, but must interact with other factors to
regulate hepatocyte-specific gene expression.
CCAAT/enhancer-binding proteins (C/EBPs) ()are
members of the basic leucine zipper superfamily of transcription
factors. To date, five distinct genes that encode different C/EBP
isoforms have been
identified(1, 2, 3, 4, 5, 6, 7, 8, 9) .
These genes vary in their pattern of tissue distribution. For example,
C/EBP
is expressed predominately in tissues that regulate
metabolic homeostasis (e.g. liver, fat, and intestine), while
C/EBP
and C/EBP
are more
ubiquitous(10, 11, 12, 13) . Work in
several cell lines indicates that, although they bind to similar
regulatory elements, unique C/EBP isoforms differ in their ability to
activate various tissue-specific promoters. In cells that express more
than one C/EBP isoform, different C/EBPs interact to regulate gene
expression by forming homo- and heterodimers(14, 15) .
Since the regulatory regions of many tissue-specific genes contain
C/EBP-binding sites, such cells are able to variably titrate the
transcriptional rates of a wide array of genes by modifying the
relative activities of different C/EBP
isoforms(5, 12, 16, 17) .
Mature
hepatocytes express at least three C/EBP isoforms (,
, and
) that interact to regulate their phenotype(12) . However,
since these different C/EBP isoforms have overlapping
functions(14) , the specific physiological role of each isoform
in these cells is not well understood. Attempts to modulate the
relative levels of these isoforms in vitro and to examine the
resulting phenotype have been thwarted, in part, because the
transfection efficiency of both primary adult and neoplastic
hepatocytes is relatively poor, in the range of
20-30%(18, 19, 20) . Furthermore, basal
expression of C/EBP
is low in these cell lines(21) ,
unlike in mature hepatocytes, where C/EBP
is strongly and
constitutively expressed(10, 11) . To overcome these
experimental difficulties and to clarify the role of C/EBP
in
mature hepatocytes, replication-defective adenovirus vectors were used
to efficiently overexpress the mouse C/EBP
gene in a conditionally
transformed rat hepatocyte line that can be induced to express
C/EBP
and C/EBP
but that has little endogenous C/EBP
expression. In vitro adenoviral gene transfer has a
significant advantage over traditional transient transfection
strategies in that up to 100% of the targeted cells can be
infected(22) . The subsequent analysis reflects changes in a
homogeneous population of cells and indicates that C/EBP
has a
dominant antiproliferative function in hepatocytes, but must interact
with other factors to regulate the expression of hepatocyte-specific
genes.
For large-scale preparations, 9-18 T-150 flasks of
HEK293 cells were infected with AdCEBP or Ad
gal at a
multiplicity of infection (m.o.i.) of 1-5 plaque-forming
units/cell. For detection of
-galactosidase activity, cells were
fixed with 0.05% glutaraldehyde for 5 min at room temperature and
stained with 5-bromo-4-chloro-3-indoyl
-D-galactoside
solution. The cells were lysed by freeze-thawing, and the supernatant
was purified on a CsCl step cushion and dialyzed against
phosphate-buffered saline as described previously(25) .
Figure 1: Temperature-dependent differences in mRNA levels of several hepatocyte genes in RALA255 cells. Shown are the results of Northern blot analysis of total RNA (20 µg/lane) harvested from cells grown to 70-80% confluence at 33 °C or similarly confluent cultures maintained for varying periods of time (1-4 days (D1-D4)) at 39 °C. A, the autoradiographs shown were obtained by repeated hybridizations of a single blot. Results are representative of triplicate experiments. B, the effect of temperature on albumin mRNA levels persists when mRNA levels are normalized for lane-lane differences in 18 S RNA levels on each of three different immunoblots (*, p < 0.001 for day 0 versus days 3 and 4). Similar analysis confirms that expression of the C/EBP mRNAs is temperature-insensitive.
In contrast to the relatively temperature-insensitive message
levels, immunoblot analysis reveals that levels of certain C/EBP
isoforms increase at 39 °C (Fig. 2). Although C/EBP
expression varies <2-fold, the nuclear concentrations of C/EBP
and C/EBP
increase 5-10-fold when cultures are shifted to
the higher temperature (p < 0.05 for expression at 33
°C versus expression during days 1-9 at 39 °C).
These increases in C/EBP
and C/EBP
expression are paralleled
by increases in DNA binding activity of these isoforms in gel mobility
shift experiments with an oligonucleotide probe that contains the
C/EBP-binding site in the albumin gene (Fig. 3)(36) .
Complex formation with this oligonucleotide is completely eliminated
when a mixture of antibodies to C/EBP
, C/EBP
, and C/EBP
is added to the reaction mixture, but is unaltered by preimmune sera
(see Fig. 7, lanes 9 and 10), confirming
previous reports (36) that this probe specifically identifies
C/EBP binding activity in liver nuclear extracts.
Figure 2:
Temperature-dependent differences in C/EBP
protein concentrations in RALA255 cells. Nuclear extracts were prepared
from cells grown to 70-80% confluence at 33 °C or from
similarly confluent cultures maintained for varying periods of time
(1-9 days) at 39 °C, separated by SDS-polyacrylamide gel
electrophoresis, and transferred to nylon membranes. A, C/EBP
expression was analyzed by immunoblotting (40 µg of nuclear
protein/lane). The last lane is a standard and contains
reticulocyte-translated C/EBP protein (40 µg/lane) that was
prepared by incubating rabbit reticulocytes with RNA prepared from the
respective cDNAs for C/EBP, C/EBP
, and
C/EBP
(44) . B, triplicate immunoblots were
performed and analyzed by scanning laser densitometry. Levels of each
C/EBP isoform at the various time points during culture at 39 °C
were normalized for the expression of that isoform in 33 °C nuclear
extracts on the same blot. Results were analyzed by analysis of
variance (*, p < 0.05 versus C/EBP
expression
at 33 °C; #, p < 0.05 versus C/EBP
expression at 33 °C).
Figure 3: Temperature-dependent differences in C/EBP binding activity for the albumin enhancer in RALA255 cells. Gel mobility shift assays were performed using 23-base pair oligonucleotides that included the C/EBP-binding site in the albumin enhancer plus nuclear extracts (8 µg of protein/lane) harvested from cells grown to 70-80% confluence at 33 °C or from similarly confluent cultures that had been maintained at 39 °C for 3 days. Antisera specific for different C/EBP isoforms were added to some reaction mixtures (lanes 2-4 and 6-8) to determine which isoforms were binding to C/EBP sites under the different culture conditions.
Figure 7:
Effect of adenovirus-mediated gene
transfer on C/EBP binding activity of RALA255 cells. Gel mobility and
supershift assays were performed using the 23-base pair oligonucleotide
described in the legend to Fig. 3plus nuclear extracts (8
µg of protein/lane) from cultures infected with either AdCEBP
or Ad
gal. Lanes 1-8 illustrate results in extracts
harvested 3 days after infection at 33 °C. Lanes 9 and 10 show results obtained when identical assays were performed
with extracts harvested from Ad
gal-infected cultures maintained at
39 °C for 3 days. These are included as a control to demonstrate
that addition of a mixture of C/EBP
, C/EBP
, and C/EBP
antisera (total volume of 3 µl) virtually eliminates complex
formation with the same probe (lane 10) but that addition of
an equal volume of preimmune serum (IgG) has little
effect.
Total C/EBP
binding activity is increased in nuclear extracts prepared from 39
°C cultures compared with extracts from 33 °C cultures. In
experiments with extracts obtained from 39 °C cultures, addition of
anti-C/EBP antibodies has little effect on complex formation;
anti-C/EBP
antibodies result in a small supershifted band, and
anti-C/EBP
antibodies disrupt most of the protein-probe complexes.
Thus, almost all of the increased binding activity at 39 °C is
secondary to C/EBP
. Because C/EBP
accounts for most of the
C/EBP binding activity in these cells at both 33 and 39 °C,
subsequent experiments focused on identifying effects of C/EBP
gene transfer on the expression and function of the C/EBP
isoform.
Figure 4:
-Galactosidase expression in RALA255
cultures 1 day after infection with Ad
gal. Infection with
Ad
gal (m.o.i. = 25) at 33 °C for 2 h resulted in
expression of the heterologous gene in 100% of the cells 1 day later.
Similar results were obtained when cultures were infected at 39 °C.
Heterologous gene expression persisted for at least 9
days.
Figure 5:
Effect
of adenovirus-mediated gene transfer on RALA255 cell expression of
several hepatocyte mRNAs. 30-40% confluent cultures of RALA255
were infected with either AdCEBP or Ad
gal (m.o.i. =
25) for 2 h at 33 °C and then maintained at this temperature for an
additional 1-3 days (D1-D3). In parallel
experiments, 70-80% confluent cultures of RALA255 cells were
infected with the same vectors for 2 h at 37 °C and then shifted to
39 °C and maintained at this temperature for 3 days. By day 3 at 39
°C, T antigen had been degraded, and albumin mRNA levels were
increased in uninfected cultures (see Fig. 1). A, total
RNA was harvested from cultures at various time points, and
treatment-related differences in mRNA levels of C/EBP
, C/EBP
,
and albumin were evaluated by Northern blot analysis (20 µg of
RNA/lane). The autoradiographs shown were obtained by repeated
hybridization of a single blot. The bottom panel illustrates
the same blot after staining with methylene blue. This, coupled with
normalization of results to 18 S RNA expression at the same time points
(data not shown), confirms that differences in C/EBP
and albumin
expression are not due to uneven RNA loading or transfer. The results
shown are representative of triplicate experiments. B,
densitometric analysis of these results and those from two other blots
confirms that infection with AdCEBP
increases C/EBP
mRNA
levels 50-80-fold and albumin mRNA levels 4.5-7-fold (p < 0.01 for AdCEBP
at days 1-3 at 33 °C and day 3
at 39 °C versus Ad
gal at the same time points).
, Ad
gal; &cjs2113;, AdCEBP
.
Figure 6:
Effect of adenovirus-mediated gene
transfer on RALA255 cell expression of C/EBP and C/EBP
proteins. Cultures were infected with either AdCEBP
(CEBP
) or Ad
gal (
gal) as described in
the legend to Fig. 5and harvested after either 2 days of
culture at 33 °C or 3 days of culture at 39 °C. Whole cell and
nuclear proteins were isolated as described under ``Experimental
Procedures.'' Immunoblot data from whole cell extracts (200 µg
of protein/lane) are shown. Similar results were obtained when nuclear
extracts were analyzed. Results shown are representative of triplicate
experiments in which protein expression was analyzed during 3 days of
culture at 33 °C and during days 3-6 of culture at 39 °C.
At every time point evaluated, C/EBP
protein concentrations were
greater in AdCEBP
-infected cultures than in parallel controls (p < 0.01).
Virus-mediated overexpression of C/EBP also has functional
relevance. Gel mobility and supershift experiments demonstrate
increased complex formation between C/EBP
and the C/EBP-binding
site in the albumin enhancer in extracts from AdCEBP
-treated
cultures compared with Ad
gal-infected cultures at both 33 and 39
°C. As shown in Fig. 7, complex formation is qualitatively
different in extracts obtained from 33 °C cultures that overexpress
C/EBP
(lane 5) compared with extracts obtained from
parallel Ad
gal-infected cultures (lane 1). Comparison of
the residual bands that are left in lanes 2 and 6 (after addition of anti-C/EBP
and anti-C/EBP
antibodies)
demonstrates more residual slow migrating bands that contain C/EBP
in lane 6 (from AdCEBP
cultures) than in lane 2 (from Ad
gal cultures). Comparison of lane 2 with lane 4 and of lane 6 with lane 8 confirms
that the slow migrating bands contain C/EBP
because they disappear
when antibodies to C/EBP
are added to the reaction mixture. Data
shown in lanes 3 and 7 (after addition of antibodies
to C/EBP
and C/EBP
) indicate that the fastest moving residual
bands contain C/EBP
. Presumably, the residual slow moving bands in lane 7 also contain C/EBP
since addition of antibodies to
all three C/EBP isoforms totally eliminates complex formation with this
probe (lane 10). Taken together, these experiments indicate
that, although infection of cultures with AdCEBP
results in
increased C/EBP
mRNA, increased C/EBP
protein, and increased
C/EBP
DNA binding activity, this is not sufficient to result in
increased expression of albumin, a C/EBP-regulated gene, at 33 °C.
Similarly, C/EBP
binding activity is not associated with albumin
gene expression at this temperature.
Since C/EBP has been
identified as a growth arrest gene in adipocytes, studies were carried
out to determine if overexpression of C/EBP
could influence the
proliferative activity of hepatocytes. As shown in Fig. 8A, infection with AdCEBP
inhibits
proliferation in cultures maintained at 33 °C. Differences in the
rate of [
H]thymidine incorporation are very
obvious after 1 day of culture, when the concentrations of DNA are not
different statistically in the two treatment groups, but become less
apparent over time, as DNA concentrations increase in the cultures
treated with Ad
gal (Fig. 8B). Infection with
AdCEBP
does not overtly influence the expression of T antigen (Fig. 9), which is thought to drive proliferation in cultures
maintained at 33 °C(28) .
Figure 8:
Effect of adenovirus-mediated gene
transfer on T antigen-driven proliferation of RALA255 cells at 33
°C. A, RALA255 cells were plated at low density (10%
confluence) and infected with either AdCEBP
(CEBP
)
or Ad
gal (
gal). Photomicrographs were taken at the
time of infection (day 0) and 1 or 2 days postinfection. These results
are representative of those obtained in three other experiments and
demonstrate the antiproliferative effects of AdCEBP
. B,
30-40% confluent cultures were infected with either AdCEBP
or Ad
gal as described above. One, two, or three days later,
triplicate cultures were incubated for 2 h in medium containing
[
H]thymidine and then immediately harvested. DNA
was isolated, and treatment-related effects on proliferation were
measured by comparing differences in the rate of incorporation of
[
H]thymidine. Results of four separate
experiments are shown.
Figure 9:
Effect of adenovirus-mediated gene
transfer on T antigen expression in RALA255 cultures at 33 °C.
Immunocytochemistry was used to evaluate the expression of T antigen in
AdCEBP- and Ad
gal-infected cultures. These experiments were
performed in parallel with those described in the legend to Fig. 8. Monoclonal antibodies to SV40 T antigen were generous
gifts of Thomas Kelly (Johns Hopkins University). Immunohistochemical
evaluation of T antigen expression was performed as described by
Hayward et al.(61) . T antigen expression in cultures
maintained at 33 °C for 1 day after infection is shown. Identical
results were obtained in cultures maintained for up to 3 days at 33
°C.
When nearly confluent RALA255
cells are infected with AdCEBP or Ad
gal and cultures are
shifted to 39 °C, T antigen expression disappears after 2 days
(data not shown)(28) , C/EBP
is induced (Fig. 6),
and proliferative activity declines in both groups (from 4413 ±
176 to 179 ± 2 and from 5198 ± 160 to 169 ± 11
cpm/µg DNA, respectively, by the third day of culture at 39
°C). As shown in Fig. 10, changing these cultures (which do
not express T antigen) to growth factor-enriched medium increases
steady-state levels of c-myc 3-5-fold in
Ad
gal-infected cultures (p < 0.01 versus Ad
gal cultures without mitogens). AdCEBP
-mediated
expression of C/EBP
blunts this mitogenic induction of c-myc by at least 50%, such that c-myc mRNA levels are
increased <1.5-fold (range of 0.9-1.5-fold greater than
without mitogens; p = not significant). Results of
triplicate experiments indicate that, after treatment with growth
factors, levels of c-myc mRNA are significantly lower in
AdCEBP
-infected cultures than in Ad
gal-infected controls (p < 0.05).
Figure 10:
Effect of adenovirus-mediated gene
transfer on mitogenic induction of c-myc in RALA255 cells at
39 °C. 70-80% confluent cultures were infected with either
AdCEBP or Ad
gal at 37 °C for 2 h and then shifted to 39
°C and maintained under conditions that supported differentiated
gene expression. By the third day of culture under these conditions, T
antigen had been degraded so that proliferative activity was very low
in both groups. Growth factor-enriched medium was added to cultures at
the beginning of day 3, and cultures were harvested at various times,
ranging from 1 h to 3 days thereafter. Total RNA was isolated, and
treatment-related differences in c-myc expression were
evaluated by Northern blot analysis (20 µg of RNA/lane) in
triplicate experiments. Upper panel, c-myc expression
in representative Ad
gal- or AdCEBP
-infected cells after 3
days of culture at 39 °C before (first and fifth
lanes) or 3 h after (second through fourth and sixth through eighth lanes) addition of mitogens,
when c-myc is maximally induced. Bottom panel, 18 S
RNA on the same blot. Densitometry of triplicate autoradiographs and
normalization of c-myc expression to 18 S RNA levels at the
same time point indicated that c-myc mRNA levels were similar
in AdCEBP
- and Ad
gal-infected cultures before addition of
growth factors. Mitogens increased c-myc mRNA levels 4.3
± 1-fold in Ad
gal-infected cultures (p < 0.05),
but did not alter c-myc mRNA levels in AdCEBP
-infected
cultures (c-myc mRNA levels after mitogens = 1.1
± 0.3-fold greater than before mitogens; p = not
significant). Thus, c-myc mRNA levels were at least 50% lower
in AdCEBP
-infected cultures than in Ad
gal-infected cultures
at any time point evaluated after addition of mitogens (p <
0.05 for AdCEBP
versus Ad
gal).
C/EBP has been incriminated as a component of a
``differentiation-proliferation switch'' based on work in
adipocyte cell lines(15, 38) . Proliferating
undifferentiated adipoblasts do not express C/EBP
, and increased
C/EBP
expression accompanies hormonal induction of differentiation
in these
cells(5, 10, 23, 39, 40) .
Transfection of adipoblasts with inducible expression vectors for
C/EBP
arrests their proliferation and promotes differentiation in
the presence of adipogenic hormones(15) . In addition, during
hormonally induced differentiation of preadipocytes, plasmid
vector-directed expression of C/EBP
antisense RNA blocks
expression of C/EBP
and inhibits the induction of
adipocyte-specific genes(41, 42) . However, whether
C/EBP
regulates proliferation and differentiated gene expression
in other highly differentiated cells is less clear. Indeed, in some
cells, including myeloid precursors, C/EBP
expression actually
declines during terminal differentiation (43) .
The adult
liver expresses more C/EBP than any other tissue(10) , yet
the physiological role of C/EBP
as a regulator of the hepatocyte
phenotype remains undefined. In rats, hepatic expression of C/EBP
increases during development and is greater postnatally than earlier
during embryogenesis(10, 11, 44) . However,
hepatocytes continue to proliferate actively for the first several
weeks after birth(44) . Furthermore, at birth, liver morphology
appears normal in C/EBP
knockout mice, and these animals express
many, albeit not all, hepatocyte-specific genes(45) . In the
adult, hepatic concentrations of C/EBP
mRNA and protein remain
relatively stable during liver regeneration after 70% partial
hepatectomy(21, 30, 46, 47) ,
although both the rate of hepatocyte proliferation and the
transcription of some hepatocyte-specific genes increase
significantly(48) . Finally, in hepatoma cell lines and
hepatocyte-derived hybridomas, loss of C/EBP
does not extinguish
the hepatocyte phenotype(49, 50) . Thus, although
C/EBP
has a clear antiproliferative effect in adipocytes, its role
in the liver appears more complex.
It is conceivable that, in
hepatocytes, identification of potentially important C/EBP actions
may be obscured by non-
C/EBP isoforms. Several metabolic and
inflammatory mediators induce expression of C/EBP
and C/EBP
in hepatocytes, and it has been postulated that the resulting changes
in C/EBP dimerization increase the transcription of some genes while
decreasing the trans-activation of
others(6, 8, 12, 16, 17, 44) .
However, since the differences among C/EBP isoform transcriptional
activities appear more qualitative than quantitative(14) , it
has been difficult to attribute unique functions to C/EBP
. Work in
neoplastic hepatocyte lines and primary hepatocytes has not resolved
this dilemma because these systems express relatively high levels of
C/EBP
, but very little C/EBP
endogenously(21, 46) . Furthermore, standard
transfection techniques transfer exogenous C/EBP
to <20% of the
cultured cells(18, 19, 20) , making it
difficult to identify treatment-related differences because of low
signal-to-noise ratios. While recently developed C/EBP
knockout
mice offer exciting opportunities to study C/EBP interactions, progress
in this model has been slow because the
-null phenotype is lethal
within a few days of birth(45) .
We have exploited the high
efficiency of adenovirus-mediated gene transfer (22, 25) to demonstrate that C/EBP inhibits
proliferation in hepatocytes, as it does in adipocytes. In hepatocytes,
following infection with AdCEBP
, the antiproliferative actions of
C/EBP
are particularly apparent when C/EBP
expression is low
(at 33 °C). Indeed, at this temperature, overexpression of
C/EBP
is sufficient to inhibit proliferation driven by T antigen.
Immunohistochemistry indicates that T antigen expression persists in
cells that overexpress C/EBP
. Although this technique is not
sensitive enough to detect small treatment-related differences in T
antigen levels, our results suggest that C/EBP
may interfere with
the function of this transforming protein. Given evidence that the
mechanism of action of T antigen involves alteration of events that
regulate G
to S phase transition(51, 52) ,
this implies that C/EBP
may retard cell cycle progression. Present
evidence that mitogenic induction of c-myc expression is
inhibited by overexpression of C/EBP
in hepatocytes lacking
functional T antigen further supports this theory and suggests that
C/EBP
may regulate entry into early G
phase. Since
c-myc is expressed in mid-G
(53) , this
could explain why C/EBP
and c-myc are considered opposing
elements in a differentiation-proliferation switch that regulates the
phenotype of transformed adipocytes(15, 54) . Although
additional experiments are necessary to confirm this theory, it is
tempting to speculate that, in mature hepatocytes, high constitutive
expression of C/EBP
(46, 47, 55) may
help to maintain these cells in their normal growth-arrested state.
Our results also provide direct evidence that interactions among
C/EBP isoforms modulate differentiated gene expression in hepatocytes
by demonstrating that albumin mRNA levels rise when C/EBP
concentrations are increased in cells expressing high levels of
C/EBP
at 39 °C. Since overexpression of C/EBP
does not
increase albumin mRNA levels at 33 °C, despite inhibiting
proliferative activity at this temperature, and some albumin mRNA is
expressed in uninfected RALA255 cells at 39 °C, it is unlikely the
effects of C/EBP
on albumin expression are a nonspecific
reflection of its ability to block hepatocyte proliferation.
Furthermore, extracts obtained from uninfected or Ad
gal-infected
RALA255 cells grown at 33 °C demonstrate some binding activity for
C/EBP sites in gel mobility shift assays, and this is mainly due to
C/EBP
. However, although complex formation between C/EBP
and
C/EBP sites is greater in 33 °C cultures infected with AdCEBP
,
albumin expression is not increased.
Liu et al.(37) reported that at least five distinct DNase-sensitive
sites in the albumin enhancer element are differentially protected in
RALA255-like hepatocytes grown at permissive and restrictive
temperatures. Our data suggest that some of the factors that bind to
these other sites may be differentially expressed in RALA255 cells
cultured at the two temperatures. Since multiple trans-acting
factors interact to promote transcription of the albumin
gene(56, 57) , this may explain why overexpression of
C/EBP increases albumin expression only at the restrictive
temperature. Adult hepatocytes in the healthy liver normally express
the full complement of trans-acting factors necessary to
activate albumin transcription(58) . In these cells, C/EBP
and C/EBP
can each trans-activate the albumin
gene(6, 59, 60) . However, our findings
indicate that albumin transcription is greater when cells express more
C/EBP
.
In summary, efficient adenovirus-mediated introduction
of the mouse C/EBP gene into cultures of a rat hepatocyte-derived
cell line under conditions that variably induce C/EBP
expression
has clarified the functions of these C/EBP isoforms. Proliferative
activity is inhibited by overexpression of C/EBP
. Hence,
C/EBP
functions as a growth arrest gene in hepatocytes. In
nonproliferating hepatocytes, C/EBP
also cooperates with other
factors to regulate the expression of certain hepatocyte-specific
genes, such as albumin. Of note, however, another C/EBP isoform
(C/EBP
) is capable of supporting hepatocyte-specific gene
transcription when C/EBP
expression declines. Thus, induction of
C/EBP
may be one mechanism whereby adult hepatocytes maintain
their differentiated phenotype while proliferating. Taken together, the
results of our gene transfer experiments help to explain the
significance of the reciprocal variations in C/EBP
and C/EBP
DNA binding activity that have been noted during liver regeneration (30, 46, 47) and suggest that C/EBP isoforms
interact to regulate the hepatocyte phenotype during a physiological
growth response.