©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The DNA Binding Activity of C/EBP Transcription Factors Is Regulated in the G Phase of the Hepatocyte Cell Cycle (*)

(Received for publication, December 21, 1994; and in revised form, May 12, 1995)

Basabi Rana (1) Yuhong Xie (1) (2) David Mischoulon (1) Nancy L. R. Bucher (2) Stephen R. Farmer (1)(§)

From the  (1)Departments of Biochemistry and (2)Pathology, Boston University School of Medicine, Boston, Massachusetts 02118

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have isolated the promoter of the rat C/EBP 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.


INTRODUCTION

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), 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.

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()proteins, that are thought to support the differentiated state through regulating many programs of gene expression including metabolism and growth.

The four C/EBP proteins, , , , 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) .

C/EBP 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) .

C/EBP 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) .

Consistent with the notion that C/EBP 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.


EXPERIMENTAL PROCEDURES

Isolation of a Rat Genomic Clone Encoding C/EBP mRNA and DNA Sequence Analysis

A rat partial SauIIIA genomic library obtained from Dr. Richard Hynes, MIT, was screened with a rat C/EBP 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.


Figure 3: Ectopic expression of C/EBP 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), 10M 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).


Figure 1: Analysis of the 5`-upstream region of the rat C/EBP 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) .


RESULTS

Characterization of the 5`-Upstream Region of the C/EBP Gene

Studies by us (22) and others (30) have recently shown that the decrease in C/EBP 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.

To characterize the rat liver proteins that can associate with elements within the C/EBP 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.

Fig. 2A shows that recombinant C/EBP 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).


Figure 2: Identification and characterization of a C/EBP binding site in the rat C/EBP 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.



In an attempt to identify some of the polypeptides within these complexes, we performed supershift analysis using 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.


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 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 Can Transactivate the Rat C/EBP Promoter

The EMSA data in Fig. 2D shows that C/EBP 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.

To demonstrate that C/EBP 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.

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 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.

The DNA binding activity of the C/EBP 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).

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 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.

This decrease in the expression of these polypeptides corresponds to a drop in DNA binding activity of the C/EBP 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).

In the case of C/EBP (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.

It appears that the decrease in the DNA binding activity of the 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.

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 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.

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 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).


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. 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.



The Western blot analysis of these nuclear extracts (Fig. 5B) shows that the drop in DNA binding activity of the 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.

In the case of C/EBP, 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.

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, 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) on C/EBP Activity in Hepatocyte Cultures

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) 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.

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 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.

The supershift analysis in Fig. 6A shows a decrease in the DNA binding activity of C/EBP 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.


Figure 6: Effect of EGF and TGF 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.



Addition of growth inhibiting doses of TGF (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.

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 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.


DISCUSSION

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 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 Gene Promoter

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 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.

Within the liver, the supershift analysis with anti-C/EBP 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.

The role of these different C/EBP 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 Gene Transcription

The changes in the binding of C/EBP containing complexes to the C/EBP site within the promoter of the C/EBP 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.

A second possibility is that C/EBP 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.

Recent studies have also suggested that C/EBP 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.

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 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).

Although these changes in C/EBP 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.

It is important to note that C/EBP 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.

Whereas the induction of the 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.

Addition of TGF, 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).

The data in Fig. 6B suggest that the regulation of the C/EBP 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.

The parallel behavior of C/EBP 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.

With regard to the induction and progression of liver regeneration, our data are consistent with significant roles for both C/EBP 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.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants DK45048 and CA39099. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Biochemistry, Boston University School of Medicine, 80 E. Concord St, Boston, MA 02118. Tel.: 617-638-4186; Fax: 617-638-5339.

The abbreviations used are: C/EBP, CCAAT/enhancer binding protein; EGF, epidermal growth factor; TGF, transforming growth factor; bp, base pair(s); MSV, moloney sarcoma virus; CAT, chloramphenicol acetyltransferase; EMSA, electrophoretic mobility shift assay; LAP, liver activatory protein; LIP, liver inhibitory protein.


ACKNOWLEDGEMENTS

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.


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