Role of the CCAAT/Enhancer Binding Protein-alpha Transcription Factor in the Glucocorticoid Stimulation of p21waf1/cip1 Gene Promoter Activity in Growth-arrested Rat Hepatoma Cells*

Erin J. CramDagger , Ross A. RamosDagger , Edward C. Wang, Helen H. Cha, Yukihiro Nishio, and Gary L. Firestone§

From the Department of Molecular and Cell Biology and The Cancer Research Laboratory, University of California, Berkeley, California 94720

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

The preceding paper (Cha, H. H., Cram, E. J., Wang, E. C., Huang, A. J., Kasler, H. G., and Firestone, G. L. (1998) J. Biol. Chem. 273, 0000-0000(478563) defined a glucocorticoid responsive region within the promoter of the p21 CDK inhibitor gene that contains a putative DNA-binding site for the transcription factor CCAAT/enhancer binding protein-alpha (C/EBPalpha ). Wild type rat BDS1 hepatoma cells as well as as4 hepatoma cells, which express antisense sequences to C/EBPalpha and ablate its protein production, were utilized to investigate the role of this transcription factor in the glucocorticoid regulation of p21 gene expression. The stimulation of p21 protein levels and promoter activity, as well as inhibition of CDK2-mediated retinoblastoma protein phosphorylation, by the synthetic glucocorticoid, dexamethasone, required the expression of C/EBPalpha . Overexpression of C/EBPalpha in as4 cells rescued the dexamethasone responsiveness of the p21 promoter. Site-directed mutagenesis of the p21 promoter revealed that dexamethasone stimulation of p21 promoter activity required the C/EBP consensus DNA-binding site. Furthermore, in glucocorticoid receptor-defective EDR1 hepatoma cells, dexamethasone failed to stimulate C/EBPalpha and p21 protein expression and promoter activities. Our results have established a functional link between the glucocorticoid receptor signaling pathway that mediates a G1 cell cycle arrest of rat hepatoma cells and the transcriptional control of p21 by a cascade that requires the steroid induction of C/EBPalpha gene expression.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Treatment with glucocorticoids, one class of steroid hormones, can inhibit both the in vivo and in vitro growth of many different types of normal and transformed cells. Normal hepatocytes and certain hepatoma cell lines are acutely sensitive to the anti-proliferative effects of glucocorticoids (1-9). We have previously established that in specific types of steroid responsive rat hepatoma cells (10, 11) and rat mammary tumor cells (12), glucocorticoids induce an early G1 block in cell cycle progression suggesting the existence of a unique hormone-regulated G1 restriction point in these transformed cells. Furthermore, the loss of G1 cell cycle control has been implicated in the uncontrolled proliferation of a variety of neoplastically transformed cells (13, 14). Given that steroid receptors are transcriptional regulators (15-20), this G1 cell cycle arrest is likely to be controlled in part by a glucocorticoid-mediated transcriptional cascade in which the glucocorticoid receptor directly alters the transcription of a small subset of genes which then regulate the subsequent expression and/or activity of specific sets of downstream proteins. Important final targets of this glucocorticoid growth suppression pathway are likely to be G1-acting components of the cell cycle which define a critical checkpoint in cell cycle progression. However, the molecular basis for the functional relationship between the glucocorticoid control of early events within this signaling cascade and the final cell cycle arrest of hepatoma cells is poorly understood.

To investigate the cellular signaling pathways mediating the glucocorticoid growth arrest of epithelial cells, we have isolated glucocorticoid growth suppressible and non-suppressible hepatoma cell proliferation variants derived from the rat Reuber hepatoma (2, 9). Characterization of these hepatoma cell variants revealed that the anti-proliferative effect of glucocorticoids is a receptor-dependent process that does not affect cell viability, decrease total cell number, or induce an apoptotic response (3, 4). Moreover, glucocorticoids induce an early G1 block in cell cycle progression within one cell doubling time in BDS1 hepatoma cells (4). By using this cell system, we have defined some of the earliest transcriptional events associated with the G1 arrest of the tumor cell line. Most significantly, the CCAAT/enhancer binding protein-alpha (C/EBPalpha )1 gene expression is specifically required for the glucocorticoid-mediated G1 cell cycle arrest of hepatoma cells. Ablation of C/EBPalpha protein by expression of antisense sequences precluded glucocorticoids from inducing the G1 cell cycle arrest and overexpression of C/EBPalpha suppressed hepatoma cell growth in the absence of glucocorticoids (11). In addition to the well established role of the C/EBP family of transcription factors in normal liver function (21, 22), the glucocorticoid stimulation of C/EBPalpha gene expression is a rapid response that represents an early and crucial intermediate in the glucocorticoid-stimulated anti-proliferative cascade that governs the cell cycle of liver-derived epithelial tumor cells (11).

G1-acting cell cycle components may be important downstream targets of the glucocorticoid growth suppression pathway in rat hepatoma cells. For example, C/EBPalpha , or other steroid responsive transcriptional regulators, may inhibit the transcription of components necessary for cell cycle progression, such as the cyclins or cyclin-dependent kinases (CDK), or stimulate expression of cell cycle inhibitors that inactivate specific CDKs (23). In the preceding accompanying paper (57), we established that glucocorticoids stimulate p21waf1/cip1 promoter activity through multiple elements within a glucocorticoid responsive region of promoter. One of the glucocorticoid responsive fragments contains a canonical C/EBP DNA-binding site. Because C/EBPalpha expression is required for glucocorticoids to induce a G1 cell cycle arrest of hepatoma cells (11), we examined if C/EBPalpha expression is linked to the glucocorticoid stimulation of p21 promoter activity. In this study, by using glucocorticoid responsive rat hepatoma cells in which we ablated C/EBPalpha expression with antisense sequences for this transcription factor, as4 cells (11), and a glucocorticoid-resistant hepatoma cell line, EDR1 (2), we demonstrate that the glucocorticoid stimulation of p21 promoter activity and induction of protein levels required the regulated expression of the C/EBPalpha transcription factor.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Materials-- Dulbecco's modified Eagle's/F12 (1:1) medium, fetal bovine serum, calcium- and magnesium-free phosphate-buffered saline, and trypsin-EDTA were supplied by BioWhittaker (Walkersville, MD). Dexamethasone was obtained from Sigma. [3H]Thymidine (84 Ci/mmol), [3H]acetyl coenzyme A (200 mCi/mmol), [alpha -32P]dCTP (3,000 Ci/mmol), and [alpha -32P]dATP (3,000 Ci/mmol) were obtained from NEN Life Science Products. Anti-p21, anti-C/EBPalpha , anti-CDK2, and horseradish peroxidase-conjugated donkey anti-goat antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Horseradish peroxidase-conjugated goat anti-rabbit antibodies were purchased from Bio-Rad. The enhanced chemiluminescence protein detection system and the Multiprime DNA labeling kit were purchased from Amersham Corp. The chimeric p21 promoter CAT reporter plasmid containing 2.326 kilobase pairs of p21 promoter sequences upstream of the RNA start site linked to the bacterial chloramphenicol acetyltransferase (CAT) gene or the firefly luciferase (luc) gene were generous gifts from Dr. Bert Vogelstein (Molecular Genetics Laboratory, Johns Hopkins Oncology Center, Baltimore) and has been described previously (24). The C/EBPalpha promoter luciferase reporter plasmid was generously provided by Dr. Kleanthis G. Xanthopoulos (Center for Biotechnology, Karolinska Institute, Sweden) and has been described previously (25). The C/EBPalpha eukaryotic expression vector (pCD-mC/EBP) and parental blank expression vector (pCD) were generously provided by Dr. Heinz Baumann (Department of Molecular and Cellular Biology, Roswell Park Cancer Institute, Buffalo, NY) and have been described previously (26).

Hepatoma Cell Lines and Methods of Culture-- Glucocorticoid-sensitive BDS1 cells are epithelial tumor cells derived from the rat Reuber hepatoma (2). The as4 and vector control (vc) cell lines were derived by single cell subcloning of BDS1 cells transfected with the pBCMG-AS antisense C/EBPalpha expression vector or the pBCMGneo parental vector, respectively. The EDR1 glucocorticoid-resistant cells are epithelial tumor cells derived from the rat Reuber hepatoma and were selected for their inability to be growth-arrested by glucocorticoids (2). All of the hepatoma cell lines were routinely grown in Dulbecco's modified Eagle's medium/F-12/10% fetal bovine serum at 37 °C in humidified air containing 5% CO2. The stably transfected cell lines were maintained in 400 µg/ml G418. Cell culture medium was routinely changed every 48 h. Dexamethasone was added to a final concentration of 1 µM as indicated.

Glucocorticoid Treatment of Rats and Analysis of Liver Gene Expression-- Two-month-old female virgin Lewis rats (Harlan Sprague-Dawley, Indianapolis, IN) were anesthetized with an intraperitoneal injection of ketamine, xylazine, and acepromazide (55 mg/kg body weight). A subscapular silastic capsule containing 20 mg of cortisol, or cholesterol as a control, was aseptically placed subcutaneously in an incision at the dorsal midline which was subsequently closed with wound clips. After 2 weeks, the rats were killed by anesthesia overdose, and the liver was isolated and quick-frozen in liquid nitrogen. Approximately 0.5 g of frozen tissue were minced with a sterile razor blade and lysed with 1 ml of RIPA buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS, 1% sodium deoxycholate) containing aprotinin, pepstatin, and leupeptin. Following centrifugation at 1.4 × 104 rpm for 20 min, the protein content for each lysate was determined by the Bradford assay (Bio-Rad). For each sample, 30 µg of protein were mixed with 15 µl of sample buffer (62.5 mM Tris-HCl, pH 6.8, 8% glycerol, 5% beta -mercaptoethanol, 3% SDS, 0.01% bromphenol blue) prior to resolution by electrophoresis and Western blotting (described below).

Assay of DNA Synthesis by [3H]Thymidine Incorporation-- Triplicate samples of asynchronously growing BDS1, vc, and as4 cells were treated with dexamethasone for the indicated times and pulse-radiolabeled as described in the accompanying paper (57).

Western Blot Analysis-- Western blots were prepared essentially as described in the accompanying paper (57) with the following procedural variations for blotting with the anti-C/EBPalpha antibodies. For each sample, 30 µg of protein were mixed with 15 µl of sample buffer and fractionated on 10% polyacrylamide, 0.1% SDS resolving gels by electrophoresis. Blots were subsequently incubated in TBST at room temperature with 1 mg/ml rabbit anti-C/EBPalpha for 1 h. Horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies were diluted in TBST, 1% non-fat dry milk 1:10000, and membranes were incubated with the diluted antibodies for 1 h at room temperature.

Transfection Procedures-- Logarithmically growing hepatoma cells were transfected by electroporation essentially as described in the preceding paper (57) with the following changes. In all transfection experiments, the cells were electroporated with 14 µg of pBLCAT2 empty vector or 16 µg of p21 promoter-CAT reporter construct alone or with 10 µg of DNA encoding a C/EBPalpha expression vector and plated into pre-warmed Dulbecco's modified Eagle's medium/F12, 10% fetal bovine serum. For the assays involving stably transfected cell lines (as4, vc), cells were cultured with medium supplemented with 400 µg/ml G418.

Reporter Gene Assays-- All CAT and luciferase assays were conducted exactly as described in the preceding paper (57).

PCR Mutagenesis of the C/EBP DNA-binding Site in the 199-bp -1.383/-1.184 Fragment of the p21waf1/cip1 Promoter-- The ATCCTCTGCAATTT wild type C/EBP DNA-binding site at -1.270 in the p21 promoter was mutated to ATCCTCCCATGGTT. Two separate PCR reactions were set up to amplify a 135-bp 5' fragment with mutations at the 3' end within the C/EBP binding site and a 90 bp-3' fragment with mutations at the 5' end within the C/EBP binding site using -1383/-1184 p21-tkCAT as a template. The C/EBP sites in the 5' and 3' fragments are overlapping by 26 nucleotides and can subsequently be annealed together to serve as templates for further amplification of a full-length 199-bp fragment containing selective point mutations in the C/EBP binding site. The 5' and 3' fragments were amplified in independent reactions containing 1 ng of -1333/-1184p21-tkCAT, 25 pmol of sense and antisense oligonucleotide primers, 250 µM nucleotide (62.5 µM dTTP, dATP, dCTP, and dGTP), 10 mM KCl, 10 mM (NH4)2SO4, 20 mM Tris-HCl, pH 8.75, 0.1% Triton X-100, 100 µg/ml bovine serum albumin (BSA), and 2.5 units of cloned Pfu DNA polymerase (Stratagene, La Jolla, CA) in a total reaction volume of 50 µl and overlaid with 50 µl of mineral oil. DNA was amplified for 30 cycles (denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, and extension at 75 °C for 30 s). The following specific primers were used for PCR and sequencing: 5' fragment, pC/EBPmutR-5'-CTTTTAAAAACCATGGGAGGATGGAT-3'; 3' fragment, pC/EBPmutF-5'-ATCCATCCTCCCATGGTTTTTAAAAG-3'.

DNA fragments of the predicted mobility were excised from 1.5% agarose gels and purified with the Qiaex II gel extraction kit (Qiagen, Chatsworth, CA) and eluted into 40 µl of 10 mM Tris-Cl, 0.1 mM EDTA, pH 8.0. To generate a full-length -1333/-1184 p21 promoter fragment with mutations within the C/EBP binding site, 1 µl of each of the purified 5' and 3' DNA fragments was annealed in a reaction mixture containing 250 µM nucleotides (62.5 µM dTTP, dATP, dCTP, and dGTP), 10 mM KCl, 10 mM (NH4)2SO4, 20 mM Tris-HCl, pH 8.75, 0.1% Triton X-100, and 100 µg/ml BSA by denaturing for 5 min at 95 °C and incubating at 50 °C for 5 min. 2.5 units of Pfu DNA polymerase were added to the reaction mixture, and DNA was amplified for 10 cycles (1 cycle = denaturation at 95 °C for 1 min, annealing at 50 °C for 1 min, extension at 75 °C for 1 min). Twenty-five pmol of primers 5'-ACTGGAAGCTTGCATGTCTGGGCAGAGATTT-3' and 5'-AATTTGGATCCATCTACCTCACACCCCTGAC-3' were subsequently added to the reaction mixture, and DNA was amplified for an additional 30 cycles (1 cycle = denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, extension at 75 °C for 30 s) with a final extension at 75 °C for 5 min. The PCR products were precipitated, washed twice in 70% ethanol, and digested with BamHI and HindIII. The digestion products were electrophoretically fractionated in 1.5% agarose, 1 × TAE, and a 199-bp DNA fragment was purified as described above and cloned into the plasmid, pTk-CAT, by ligation with the Takara DNA ligation kit (Panvera, Madison, WI). The resulting plasmid was designated C/EBP mut -1383/-1184 p21-tkCAT. The mutation was confirmed by DNA sequencing.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Dexamethasone Stimulation of p21 Protein Levels and Inhibition of CDK2 Activity Requires the Expression of the C/EBPalpha Transcription Factor-- We have previously demonstrated that the G1 block in cell cycle progression in rat hepatoma cells induced by the synthetic glucocorticoid, dexamethasone, requires the steroid-regulated expression of C/EBPalpha . This analysis was accomplished using hepatoma cells in which C/EBPalpha expression had been ablated by stable transfection of an antisense C/EBPalpha expression vector, generating the as4 cells, and with vc hepatoma cells transfected with an empty expression vector (11). As described in the accompanying paper (57), glucocorticoids stimulate the expression of the p21 CDK inhibitor gene which contains a canonical C/EBP DNA-binding site in its promoter. Therefore, as4 and vc hepatoma cells, as well as the BDS1 parental cell line, were utilized to functionally examine the mechanistic relationships between C/EBPalpha expression and the regulated expression of the p21 CDK inhibitor gene within the glucocorticoid growth suppression response.

The levels of p21 and C/EBPalpha protein were initially examined by Western blots of cell extracts isolated from dexamethasone-treated or untreated BDS1, as4, and vc hepatoma cells. As shown in Fig. 1, dexamethasone induced the level of p21 protein by approximately 5-fold in nontransfected BDS1 cells and in vc hepatoma cells, which also show a 5-10-fold induction of C/EBPalpha protein. In contrast, in as4 hepatoma cells, which fail to induce C/EBPalpha protein due to the expression of antisense C/EBPalpha sequences, dexamethasone treatment had no effect on p21 protein levels. In each of these three cell lines, CDK2 levels did not change and provided a loading control for the Western blot. The incorporation of [3H]thymidine was examined in parallel and revealed that dexamethasone strongly inhibited DNA synthesis (by approximately 90%) in BDS1 and vc hepatoma cells. However, dexamethasone had no effect on as4 cell DNA synthesis (Fig. 1, graph). Consistent with this effect on DNA synthesis, flow cytometry showed that dexamethasone induces a G1 cell cycle arrest of BDS1 and vc control cells but not the as4 hepatoma cells (11). Characterization of C/EBPalpha and p21 protein levels in liver isolated from cortisol-treated and untreated rats demonstrated that glucocorticoids can induce both proteins in adult normal hepatic tissue (Fig. 1, right panels). Thus, the correlation in glucocorticoid-regulated expression of C/EBPalpha and p21 may be a general biological response that is not limited to cultured hepatoma cells.


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Fig. 1.   Glucocorticoid stimulation of p21 protein levels requires the expression of C/EBPalpha in growth suppressible hepatoma cells. Upper panels, BDS1 hepatoma cells, vector control (vc) transfected cells, and antisense C/EBPalpha expressing as4 hepatoma cells were treated with or without dexamethasone (Dex) for 6 h, and cell extracts were fractionated by polyacrylamide gel electrophoresis. Western blots were probed with antibodies to p21, C/EBPalpha , or CDK2 and protein levels detected by autoradiography. Western blots were similarly used to examine the production of the same three proteins in extracts of liver tissue isolated from cortisol-treated and untreated rats. Lower panel, BDS1, vector control and as4 hepatoma cells were treated with dexamethasone, and at the indicated times, the cells were radiolabeled with [3H]thymidine for 2 h. The percent incorporation of [3H]thymidine was determined relative to cells not treated with dexamethasone. The reported values are an average of three independent experiments each done in triplicate, and the error bars represent the standard deviation.

To functionally test if the glucocorticoid-mediated increase in p21 protein in hepatoma cells had an effect on CDK activity, the in vitro activity of immunoprecipitated CDK2 was examined in dexamethasone-treated and untreated BDS1, as4, and vc cells. Consistent with the effects on p21 protein levels, dexamethasone inhibited the ability of CDK2 immunoprecipitated from extracts of BDS1 and vc cells, but not as4 hepatoma cells, to phosphorylate Rb (Fig. 2). In addition, significantly less p21 protein co-immunoprecipitated with CDK2 from dexamethasone-treated as4 cells compared with either vc or BDS1 cells which produce C/EBPalpha .2 Taken together, our results show a functional correlation between expression of the C/EBPalpha transcription factor in growth suppressible hepatoma cells and the glucocorticoid-regulated production and function of the p21 cell cycle inhibitor.


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Fig. 2.   Ablation of C/EBPalpha production prevents the dexamethasone inhibition of CDK2 kinase activity. BDS1, as4, and vc hepatoma cells were cultured with or without 1 µM dexamethasone (Dex) for 12 h, and at the indicated times, CDK2 was immunoprecipitated from cell lysates and assayed for in vitro kinase activity using the C terminus of the Rb protein as a substrate. One control kinase assay contained rabbit anti-IgG with no added anti-CDK2 antibodies (No IP). The kinase reaction mixtures were electrophoretically fractionated, and the level of [32P]Rb was analyzed by autoradiography.

Dexamethasone Activation of p21 Promoter Activity Can Be Functionally Complemented by Overexpression of C/EBPalpha in as4 Cells That Lack Induction of This Transcription Factor-- The preceding paper (57) demonstrated that the dexamethasone stimulation of p21 gene products in BDS1 hepatoma cells resulted from the transcriptional stimulation of the p21 gene. The requirement of C/EBPalpha transcription factor expression for the induction of p21 protein, the induction of C/EBPalpha by dexamethasone (11), and the presence of a canonical C/EBP DNA-binding site at -1270 bp in the p21 promoter implicated this transcription factor in the activation of p21 promoter activity. To test directly this possibility, as4, vc, and BDS1 hepatoma cells were transiently cotransfected with a C/EBPalpha expression vector (11, 26) and a chimeric reporter plasmid containing 2326 bp of the p21 promoter upstream of the transcription start site linked to the CAT reporter gene. Hepatoma cells transfected with an empty expression vector served as a negative control. Analysis of CAT activity in cells treated with or without dexamethasone for 48 h revealed that in vc and BDS1 hepatoma cells, this p21 promoter fragment conferred glucocorticoid responsiveness to the CAT reporter gene (Fig. 3, right and left panels). In contrast, in as4 cells, which lack C/EBPalpha , dexamethasone treatment failed to induce p21 promoter activity (Fig. 3, middle panel). Importantly, cotransfection of a C/EBPalpha expression vector into as4 cells rescued the defective glucocorticoid induction of p21 promoter activity (Fig. 3, middle panel) and caused a modest increase in the absolute level of both the basal and steroid-induced levels of p21 promoter activity in BDS1 or vc hepatoma cells (Fig. 3, left and right panels). This functional complementation of glucocorticoid-inducible p21 promoter activity demonstrates the requirement for expression of the C/EBPalpha transcription factor in this steroid response.


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Fig. 3.   Glucocorticoids stimulate p21 promoter activity in hepatoma cells in which C/EBPalpha is glucocorticoid responsive. BDS1 hepatoma cells, vector control (vc) transfected cells, and antisense C/EBPalpha expressing as4 hepatoma cells were transiently transfected with the -2.326 p21-CAT reporter plasmid and treated with or without 1 µM dexamethasone (Dex) for 48 h. CAT activity was assayed by a quantitative method that measures the conversion of [3H]acetyl coenzyme A and unlabeled chloramphenicol into [3H]acetyl chloramphenicol. CAT-specific activity is the CAT activity produced per µg of protein present in the corresponding cell lysates and is described under "Experimental Procedures." The reported values are an average of three independent experiments of triplicate samples, and the error bars represent the standard deviation.

Dexamethasone-stimulated Activity of the p21 Promoter Fragment Requires the C/EBPalpha DNA-binding Site and Expression of the C/EBPalpha Transcription Factor-- Sequence analysis of the promoter region of p21 revealed no canonical glucocorticoid response elements, but the promoter does contain a putative C/EBP DNA-binding site between nucleotides -1270 to -1256. As detailed in the preceding paper (57), this transcription factor site is located within one of the glucocorticoid responsive subfragments of the p21 promoter. To test directly if the C/EBP DNA-binding site in the p21 promoter contributes to the glucocorticoid-regulated transcriptional activity, the ATCCTCTGCAATTT wild type C/EBP DNA-binding site in the -1.380- to -1.184-bp fragment of p21 promoter was mutated to ATCCTCCCATGGTT eliminating the key nucleotides required for transcription factor binding (27). These p21 promoter fragments, containing either the wild type sequence or the C/EBP DNA-binding site mutation, were linked immediately upstream of the thymidine kinase (tk) minimal promoter sequences driving the bacterial CAT gene, forming -1.380/-1.184 p21-tkCAT and C/EBPmut-1.380/-1.184 p21-tkCAT, respectively (see diagrams in Fig. 4). The vc control cells and C/EBPalpha -deficient as4 hepatoma cells were cotransfected with one of the reporter plasmids in the presence or the absence of a C/EBPalpha expression vector. As shown in Fig. 4, dexamethasone failed to induce CAT activity from either of the p21 promoter fragments in transfected as4 cells. Transient ectopic expression of C/EBPalpha reinstated the dexamethasone activation of p21 promoter activity only in cells transfected with the wild type p21 promoter fragment and not in cells transfected with the reporter plasmid mutated in the C/EBP DNA site. In vc hepatoma cells, the wild type -1.380/-1.184 p21-tkCAT reporter plasmid was dexamethasone inducible in the presence or absence of cotransfected C/EBPalpha , whereas the reporter plasmid containing the mutated C/EBPalpha DNA-binding site was nonresponsive to glucocorticoids. In all experiments, reporter gene activity in cells transfected with the minimal promoter pTk-CAT alone was low and unaffected by dexamethasone treatment (data not shown). Thus, the presence of an intact C/EBP DNA-binding site and expression of a functional C/EBPalpha transcription factor is required for dexamethasone to confer glucocorticoid responsiveness of the -1.380 to -1.184 p21 promoter fragment to a heterologous promoter.


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Fig. 4.   Mutation of the C/EBP DNA-binding site abrogates glucocorticoid inducibility of a glucocorticoid responsive fragment of p21 promoter. BDS1 hepatoma cells, vector control (vc) transfected cells, and antisense C/EBPalpha expressing as4 hepatoma cells were transiently cotransfected with either the -1383/-1184 p21-tkCAT reporter plasmid (containing the wild type C/EBP DNA-binding site) or with the C/EBP mut-1383/-1184 p21-tkCAT (containing the indicated mutations in the C/EBP DNA-binding site) along with either a C/EBPalpha expression plasmid or an empty vector. Cells were treated with or without 1 µM dexamethasone (Dex) for 48 h, and CAT activity was determined as described in Fig. 3. The reported values are representative of three independent experiments of triplicate samples, and the error bars represent the standard deviation.

Dexamethasone Stimulation of C/EBPalpha and p21 Promoter Activities and Gene Expression Are Ablated in a Glucocorticoid-resistant Hepatoma Cell Variant-- A glucocorticoid-resistant hepatoma cell variant, EDR1, that was selected for its inability to be growth-suppressed (2), and which produces a glucocorticoid receptor with a point mutation in its zinc finger region,3 was utilized to test the functional relationship between the glucocorticoid stimulation of C/EBPalpha and p21 gene expression. Western blot analysis of dexamethasone-treated and untreated hepatoma cells revealed that under conditions in which both C/EBPalpha and p21 protein are stimulated by glucocorticoids in BDS1 hepatoma cells, neither protein was induced in the EDR1 hepatoma cell variant (Fig. 5, upper panel). BDS1 and EDR1 hepatoma cells were transiently transfected with luciferase reporter plasmids containing either fragments of the p21 promoter (-2.4 p21-Luc) or the C/EBPalpha promoter (-350 C/EBPalpha -Luc). Determination of relative luciferase-specific activity in both sets of transfections revealed that dexamethasone stimulated both the p21 promoter and the C/EBPalpha promoter activity to approximately the same extent in growth suppressible BDS1 hepatoma cells (Fig. 5, lower panel). In the EDR1 hepatoma cell line, which does not undergo a G1 cell cycle arrest (11), neither promoter was regulated by dexamethasone (Fig. 5, lower panel). These results suggest a direct functional connection between the glucocorticoid stimulation of C/EBPalpha gene expression and that the steroid responsiveness of p21 promoter activity is a key process involved in the cell cycle control of hepatoma cells.


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Fig. 5.   Glucocorticoid stimulation of p21 and C/EBPalpha promoter activity and protein production is defective in a glucocorticoid-resistant hepatoma cell variant. Upper panels, glucocorticoid responsive BDS1 hepatoma cells and glucocorticoid-resistant EDR1 cells were treated with or without 1 µM dexamethasone for 6 h, and cell extracts were fractionated by polyacrylamide gel electrophoresis. Western blots were probed with antibodies to p21, C/EBPalpha , or CDK2 and protein levels detected by autoradiography. Lower panels, BDS1 and EDR1 cells were transiently transfected with either the -2.4 p21-Luc or the -350 C/EBPalpha -Luc reporter plasmids, treated with or without 1 µM dexamethasone (DEX) for 48 h, and luciferase-specific activity measured as described under "Experimental Procedures." The relative light units per µg of protein were calculated as an average of three independent experiments of triplicate samples, and the error bars represent the standard deviation.

    DISCUSSION
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Abstract
Introduction
Procedures
Results
Discussion
References

An intricate network of growth inhibitory and stimulatory signals transduced from the extracellular environment converge on specific sets of cell cycle components which, through their concerted action, either drive cells through critical cell cycle transitions or inhibit cell cycle progression (28-32). Our results have established that the glucocorticoid receptor-mediated signaling pathway induces a G1 cell cycle arrest of rat hepatoma cells (4) with the coordinate transcriptional control of the p21 CDK inhibitor gene. We propose that glucocorticoid receptors activate two distinct types of transcriptional cascades that together target and activate the p21 gene promoter (Fig. 6). In one branch of this pathway, the glucocorticoid receptor is proposed to target the p21 promoter through receptor-transcription factor interactions involving pre-existing promoter-bound proteins. This feature of the pathway is based on the ability of dexamethasone to stimulate p21 transcript expression in the absence of ongoing de novo protein synthesis and on the existence of at least three distinct glucocorticoid responsive subregions of the p21 promoter that contain DNA recognition sequences for members of transcription factor families known to interact with the glucocorticoid receptor (see preceding accompanying paper (57)). The p21 promoter does not contain a canonical glucocorticoid response element, suggesting that the glucocorticoid receptor does not act through direct DNA binding (16, 18, 19, 33-35). Consistent with our results, it is well established that protein-protein interactions of steroid receptors with other transcription factors and accessory factors can effectively regulate gene transcription in the absence of DNA-binding sites for the receptor (15, 17, 19, 36, 37).


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Fig. 6.   Model for the transcriptional control of p21 gene expression and cell cycle control by glucocorticoid receptor signaling. We propose that glucocorticoids stimulate p21 gene expression through two distinct signaling pathways that target a glucocorticoid responsive region of the promoter. In a mechanism that does not require de novo protein synthesis, glucocorticoid receptors (GR) are proposed to interact with preexisting transcription factors (ovals) located within the glucocorticoid responsive region of the promoter and participate in the formation of an active transcription complex. A second cycloheximide-sensitive pathway (CHX) involves the glucocorticoid induction of the C/EBPalpha transcription factor that subsequently binds to its consensus DNA sequence within the p21 promoter. As a result of these interactions, p21 gene expression is stimulated by glucocorticoids which causes an inhibition of CDK activity and facilitates the G1 cell cycle arrest.

We hypothesize that there is a second branch of the glucocorticoid signaling pathway in which the promoter of the p21 CDK inhibitor gene is a direct downstream target of the glucocorticoid responsive C/EBPalpha transcription factor (Fig. 6). The proposed requirement for the C/EBPalpha transcription factor in this pathway is based on our observation that antisense ablation of C/EBPalpha production in as4 hepatoma cells abolished the glucocorticoid stimulation of p21 promoter activity. Moreover, mutation of the C/EBP DNA-binding site eliminated glucocorticoid responsiveness in wild type hepatoma cells, and ectopic expression of C/EBPalpha rescued the defective glucocorticoid stimulation of p21 promoter activity in as4 hepatoma cells. Although the antisense C/EBPalpha expressing as4 cells have functional glucocorticoid receptors (11), dexamethasone was unable to induce p21 promoter activity. This suggests that the glucocorticoid receptor alone is not sufficient to activate the p21 promoter and functionally requires C/EBPalpha or an additional factor which interacts with C/EBPalpha . The ability of dexamethasone to induce p21 transcripts in the presence of cycloheximide likely reflects biological redundancy between the two branches of the cellular cascade that target the glucocorticoid responsive region of the p21 gene promoter. The precise interactions between C/EBPalpha , p21 promoter elements, and promoter bound proteins may be complex because C/EBP family members contain a leucine zipper domain and can selectively interact with a variety of other transcription factors including the glucocorticoid receptor and members of the Ets transcription factor family (20, 38). Therefore, specific transcriptional regulators of the p21 promoter may be utilized by both branches of the glucocorticoid receptor transcriptional cascade, although the molecular details of this signaling mechanism have not been explored.

The transcriptional effects of steroid receptors on specific target genes can be enhanced, reduced, or inhibited depending on the availability of other transcription factors and accessory factors that target the promoter (15, 37, 39). We propose that C/EBPalpha represents a tissue-specific factor that is involved in the glucocorticoid regulation of p21 promoter activity and that its use as a transcriptional regulator of the cell cycle is restricted to a subset of cell types. Consistent with this possibility, our preliminary evidence has shown that different regions of the p21 promoter are glucocorticoid responsive in growth-arrested mammary tumor cells compared with the hepatoma cells.2 In liver-derived cells, C/EBPalpha plays a unique role in the glucocorticoid-induced cell cycle arrest, whereas in other growth suppressible cells (9, 12, 40, 41), such as mammary cells, osteosarcoma cells, fibroblasts, and lymphoid-derived cells, glucocorticoid receptors may induce the expression or interact with other tissue-specific transcription factors to affect cell cycle control.

The activities of the cyclin-dependent kinases (CDKs), which drive progression through the cell cycle, are regulated in part by the formation of protein complexes with appropriate cyclin and the cyclin-dependent kinase inhibitor binding partners (30). A key consequence of the glucocorticoid growth arrest pathway in hepatoma cells is an elevation in the level of p21, which inhibits the ability of the G1-acting CDKs to phosphorylate the Rb protein, and thereby helps induce the G1 cell cycle arrest. Of the two families of CDK inhibitors, the p21 family (p21, p27, and p57) forms complexes with a wider range of cyclins and CDKs (42, 43). The expression of p21 appears to be important in normal liver development because the targeted expression of this gene in hepatocytes of transgenic mice resulted in an aberrant organization of liver tissue, a decreased number of adult hepatocytes, reduced liver growth, and the failure of partial hepatectomy to stimulate liver cell proliferation (44). Mice deficient in C/EBPalpha also have defects in the control of hepatic growth, as well as lung development (45). We have observed that glucocorticoids induce both C/EBPalpha and p21 protein production in normal liver, suggesting a shared in vivo mechanism of regulation. Thus, consistent with our observations in rat hepatoma cells, which demonstrate a direct functional connection between C/EBPalpha and expression of p21, these in vivo studies suggest a potential mechanistic link between C/EBPalpha and p21 in the growth and development of normal liver tissue.

Only a few studies have directly assessed the regulation of p21 promoter activity and the function of the many potential transcription factor binding sites in the p21 promoter (46-51). For example, both vitamin D3 and retinoic acid, which act through members of the steroid/thyroid hormone receptor family, directly stimulate p21 transcription through their cognate DNA-binding sites in the p21 gene promoter (52-54). In addition, C/EBPalpha has been shown to stimulate p21 promoter activity in adipocytes (55). Our results have established that the p21 promoter is regulated by glucocorticoids through two distinct transcriptional mechanisms, one of which utilizes the regulated expression of C/EBPalpha to induce a G1 block in cell cycle progression of liver epithelial tumor cells (11). Because C/EBPalpha plays a key role in normal liver and adipocyte function (45, 56), it is tempting to speculate that the glucocorticoid-regulated production of p21 may play a key role in the differentiated functions of normal and transformed cells. We are currently attempting to identify other targets of C/EBPalpha that may complement the function of glucocorticoid responsive cell cycle components such as p21, thereby mediating or maintaining the growth-arrested and differentiated state of hepatic-derived tissue.

    ACKNOWLEDGEMENTS

We express our appreciation to Carolyn Cover, Anita Maiyar, and Paul Woo for critical reading of the manuscript. We also thank Khanh Tong, Vinh Trinh, Linda Yu, and Wei-Ming Kao for their technical assistance. We are grateful to Jerry Kapler for excellent photography.

    FOOTNOTES

* This work was supported by American Cancer Society Grant RPG-90-001-08-BE.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Contributed equally to this work.

§ To whom correspondence and reprint requests should be addressed: Dept. Molecular and Cell Biology, 591 LSA, University of California, Berkeley, Berkeley, CA 94720. Tel.: 510-642-8319; Fax: 510-643-6791; E-mail: glfire{at}uclink4.berkeley.edu.

1 The abbreviations used are: C/EBPalpha , CCAAT/enhancer binding protein-alpha ; CDK, cyclin-dependent kinase; Rb, retinoblastoma protein; tk, thymidine kinase; CAT, chloramphenicol acetyltransferase; PCR, polymerase chain reaction; bp, base pair(s); vc, vector control.

2 E. J. Cram and G. L. Firestone, unpublished results.

3 R. A. Ramos and G. L. Firestone, unpublished results.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

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