Repression of Glucocorticoid Receptor Transactivation and DNA Binding of a Glucocorticoid Response Element within the Serum/Glucocorticoid-Inducible Protein Kinase (sgk) Gene Promoter by the p53 Tumor Suppressor Protein

Anita C. Maiyar, Phan T. Phu, Arthur J. Huang1 and Gary L. Firestone

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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
sgk is a novel member of the serine/threonine protein kinase family that is transcriptionally regulated by serum and glucocorticoids in Rat2 fibroblasts and in mammary epithelial cells. 5'-Deletion analysis of the sgk promoter, using a series of sgk-CAT (chloramphenicol acetyltransferase) chimeric reporter gene plasmids, defined a glucocorticoid-responsive region that contains a glucocorticoid response element (sgkGRE) between -1000 and -975 bp. The sgkGRE is specifically bound by glucocorticoid receptors and is sufficient to confer glucocorticoid responsiveness to a heterologous promoter in several cell lines. Strikingly, cotransfection of either the murine or human wild type p53, but not a mutant p53, repressed the dexamethasone-stimulated transactivation of reporter plasmids containing either the sgkGRE or a consensus GRE. Gel shift analysis revealed that in vitro synthesized p53 prevented binding of the glucocorticoid receptor both to the sgkGRE as well as to a consensus GRE. The p53-mediated repression of dexamethasone-induced sgkGRE activity required both the DNA binding and transactivation functions of the p53 protein. Activation of endogenous p53, by exposure to UV light, repressed the glucocorticoid receptor transactivation of a consensus GRE-CAT reporter plasmid in transfected cells. Conversely, activated glucocorticoid receptors suppressed the transactivation function of p53, while transrepression by p53 was largely unaffected. The presented data demonstrate that sgk is a primary glucocorticoid-responsive protein kinase gene that implicates a new pathway of cross-talk between steroid receptor signaling and cellular phosphorylation cascades. In addition, our study provides the first evidence of mutual interference of transactivation functions of p53 and the glucocorticoid receptor, possibly through their direct interaction.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Glucocorticoid hormones govern a wide spectrum of cellular functions and physiological processes in animal tissues by interacting with their cognate intracellular receptors (1, 2, 3, 4, 5). These steroid receptor complexes act as ligand-activated transcriptional stimulators or repressors of primary response genes either by their specific binding to glucocorticoid-responsive elements (GRE), within the promoter of steroid-sensitive genes (3, 6, 7), and/or by direct protein-protein interactions with other transcription factors (6, 8, 9, 10, 11). It has been proposed that pleiotropic responses of steroids occur through cellular cascades in which changes in the expression and activity of a network of early-response regulatory molecules control a subsequent series of events. From this viewpoint, transcriptional modulations in the activities of primary steroid-responsive gene products provide mechanisms for potential cross-talk with signal transduction pathways that functionally complement or compete with steroid-dependent processes. For example, phosphorylation-dephosphorylation networks are commonly used by a variety of extracellular regulators, such as growth and differentiation factors, to rapidly and reversibly transduce signals from the extracellular environment to the cytoplasm and nucleus (12). There are many potential mechanisms by which the regulation of protein kinase and phosphatase signaling can be coordinated with steroid-responsive pathways at the cellular level. One direct, but not well characterized, mechanism of regulation is steroid-dependent changes in transcription of components of phosphorylation-dephosphorylation pathways. Consistent with this concept, we previously reported the cloning of a novel serine/threonine protein kinase gene, sgk, that is transcriptionally stimulated by glucocorticoid hormones and by serum in both Rat2 fibroblasts and in rat mammary tumor epithelial cells (13, 14), which implicates the existence of a direct interplay between glucocorticoid receptor (GR)-mediated and phosphorylation-dependent cell signaling.

The sgk gene encodes a 49-kDa putative protein kinase, which exhibits strong homology (45–55% amino acid identity) with the catalytic domains of the protein kinase C family, cAMP-dependent protein kinase, rac protein kinase, and ribosomal protein S6 kinase (13). Nuclear run-on and Northern blot analysis demonstrated that the induction of sgk transcripts by glucocorticoids and serum in both fibroblasts and epithelial cells are rapid transcriptional responses that do not require de novo protein synthesis (13, 14). These studies strongly indicate that sgk may be a primary glucocorticoid-responsive gene with its transcription regulated by a cis-acting GRE located within the promoter of sgk. Interestingly, in Rat2 fibroblasts, serum induces a rapid and transient increase in sgk transcription, reminiscent of the well characterized immediate early class of genes expressed during the transit of mitogen-stimulated cells through the G1 phase of cell cycle (14, 15). Apart from the serum and glucocorticoid up-regulation of sgk transcription, expression of sgk is strongly modulated by a combination of androgen, testosterone, and FSH in rat ovarian granulosa cells (16). Moreover, using differential display, sgk was isolated from a rat brain tissue after central nervous system injury, implicating a role for sgk in axonal regeneration upon central nervous system injury and development of specific groups of neurons in postnatal brain (17). Thus, diverse sets of cellular signals generated by steroid and peptide hormones, as well as serum-derived growth factors, appear to transcriptionally regulate the expression of sgk in a tissue-specific manner.

sgk is the second described member of a newly emerging subfamily of serine/threonine protein kinases that are predominantly regulated at the transcriptional level (18, 19, 20, 21), with sgk, snk, and fnk the only known mitogen-inducible immediate early response protein kinase genes (13, 19, 21). However, relatively little is known about the transcriptional control of this potentially important subfamily of protein kinases. To elucidate the molecular basis of the transcriptional regulation of sgk by trans-acting regulatory factors, 4.0 kb of the sgk promoter region upstream of the transcriptional start site was cloned from a rat genomic library. Consistent with the nuclear run-on results, showing that dexamethasone strongly stimulates sgk gene transcription, sequence analysis revealed the existence of a putative GRE approximately 1.0 kb upstream of sgk promoter (13). In addition, the sgk promoter contains multiple Sp-1 sites, TATA sequence at -35 bp relative to the transcriptional start site as well as several putative binding sites for transcription factors implicated in cell proliferation, development and differentiation such as the AP-1 transcriptional complex, GATA, hunchback, and Kruppel proteins, CCAAT enhancer binding protein (C/EBP), and Ets-2 factors (our unpublished data). A feature unique to this kinase promoter is the presence of putative p53 DNA recognition sequences dispersed throughout the 5'-flanking region of sgk promoter. We have recently shown that four of the p53 binding sites are specifically recognized by the p53 protein and that at least one of these sites within the sgk promoter (between -1380 and -1345 bp) can confer p53 transactivation to a heterologous promoter in mammary epithelial cells (22). Our observations are consistent with the p53 protein being a transcriptional regulator implicated in a wide range of cellular processes including cell proliferation, DNA replication, and apoptosis (23, 24, 25, 26, 27). The structure of this phospho-nucleoprotein contains features typical of transcription factors including DNA binding and transactivation domains (28, 29, 30), and several studies have documented the ability of p53 to either activate or repress a variety of cellular target genes (23, 24, 25, 26). Moreover, in addition to its sequence-specific DNA-binding properties, p53 also selectively interacts with several cellular proteins that include transcription factors, such as TATA-binding factor (TBF), CCAAT-binding factor (CBF), specificity protein 1 (Sp1), and the thyroid hormone receptor, as well as viral proteins via specific domains (31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41).

The availability of particular sets of transcription factors has been shown to dramatically modulate the transcriptional effects of steroid receptors on specific target genes in a tissue-specific manner (6, 9, 10, 11). The regulation of sgk gene expression by glucocorticoids and several other extracellular signals and the existence of many putative regulatory elements in the sgk promoter implicate important combinatorial actions of trans-acting transcription factors in mediating sgk gene expression. Given the existence of functional p53 DNA-binding sites and a near-consensus GRE in the sgk promoter and the known involvement of protein kinases and p53 in complex cellular functions, an important issue was to examine whether GRs and p53 coordinately regulate sgk promoter activity. In this study, we show that the sgk promoter is a direct target of GRs through a functional GRE sequence, and that in Rat2 fibroblasts, cotransfection of p53 inhibits the glucocorticoid-stimulated transactivation of the sgk promoter. The molecular basis for this inhibitory effect is a reciprocal repression of transactivation activities of p53 and the GR. Our results provide evidence for functional interactions between the GR and the p53 protein that implicates a potential coupling between these two classes of transcriptional regulators.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The sgk Promoter Contains a Functional GRE that Confers Dexamethasone Responsiveness to a Heterologous Promoter
In both Rat2 fibroblasts and Con8 mammary epithelial cells, dexamethasone stimulated transcription of the sgk serine/threonine protein kinase gene without an apparent time lag and in the absence of de novo protein synthesis, suggesting that sgk is a primary target gene of GRs (13, 14). To functionally determine which region within the sgk promoter is responsible for its glucocorticoid-mediated transcriptional activation, -4.0 kb of sgk 5'-flanking region was cloned from a rat genomic library (13), and a series of 5'-progressive deletions starting at -4000 bp and terminating at +51 were generated by controlled exonuclease III digestions. Rat2 cells were transiently transfected with sgk-CAT reporter constructs, which were generated by linking the various sgk promoter fragments to the CAT reporter gene, transfected cells treated with or without 1 µM dexamethasone for 24 h and cell lysates assayed for CAT activity. As shown in Fig. 1Go, dexamethasone stimulated transcriptional activity of the sgk-CAT constructs containing the four largest sgk promoter regions (deletions ending at -4000, -2321, -1428, and -1148 bp). Maximum stimulation (4- to 5-fold) of sgk-CAT activity by dexamethasone was observed with both the -1428sgk-CAT and -1148sgk-CAT constructs. Glucocorticoid inducibility of the sgk promoter was completely lost in the deletions at and beyond -901 bp, implicating sequences between -1148 and -901 bp of the sgk promoter as the key glucocorticoid-responsive region. The variation in basal level of CAT activity between the deletion constructs was likely due to the selective elimination of functional transcription factor-binding sites.



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Figure 1. Identification of a Dexamethasone-Responsive Region within the sgk Promoter

A series of sgk-CAT reporter plasmids containing the indicated 5'-deletions of sgk promoter fused to the bacterial CAT reporter gene were transfected by electroporation into Rat2 fibroblasts. Cells received 10 µg of reporter plasmid and 20 µg of the promoterless CAT vector, pBLCAT3, and then were incubated for 24 h with (+) or without (-) 1 µM dexamethasone. CAT activity was assayed by quantification of the conversion of [3H]acetylCoA into [3H]acetylchloramphenicol by the two phase fluor diffusion assay described in Materials and Methods and normalized to protein levels to determine the CAT-specific activity. Experiments were performed in triplicate, and the reported values represent the means and SD values derived from at least three separate transfections.

 
Sequence analysis of 4.0 kb of the sgk promoter revealed the presence of one putative GRE between -1000 and -975 within the -1148 to -901 glucocorticoid-responsive region functionally defined by the deletion studies. The sgkGRE bears strong homology with the consensus GRE sequence (Fig. 2Go, upper panel) and contains the base pairs critical for direct contact with the GR (6, 42). To functionally test whether the sgkGRE can confer dexamethasone responsiveness to a heterologous promoter, a 25-bp oligonucleotide representing the regions between -1000 to -975 bp of the sgk promoter was linked immediately upstream of the herpes simplex virus thymidine kinase minimal promoter sequences (-105 to +51) driving the bacterial CAT gene (sgkGREtk-CAT). Rat2 cells were transiently transfected with either sgkGREtk-CAT, tk-CAT containing only the -105 to +51 minimal promoter sequences, GRE-CAT containing six copies of a functional GRE encoded by the mouse mammary tumor virus (MMTV) gene, or the constitutively expressed Rous sarcoma virus (RSV)-CAT reporter gene. CAT reporter gene activity was monitored in cell extracts isolated from dexamethasone-treated and untreated cells. As shown in Fig. 2Go (lower panel), dexamethasone induced CAT expression driven by the sgkGREtk-CAT chimeric reporter plasmid by 6-fold compared with Rat2 fibroblasts not treated with dexamethasone. Reporter gene activity in cells transfected with the minimal promoter thymidine kinase (tk)-CAT alone was low and unaffected by dexamethasone treatment. As expected, the positive control GRE-CAT reporter plasmid was strongly induced (30-fold) by dexamethasone, whereas the constitutive RSV-CAT reporter construct displayed high basal activity that was unresponsive to dexamethasone. These results establish that the putative DNA element located between -1000 and -975 bp within the sgk promoter is a functional GRE capable of rendering hormone responsiveness to a heterologous promoter. The hormone sensitivity of the sgkGREtk-CAT reporter construct was also observed in several other glucocorticoid responsive epithelial cell lines (data not shown).



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Figure 2. The sgk GRE Confers Dexamethasone Inducibility to a Heterologous Gene Promoter

The upper panel shows the sequence of sgkGRE present between -1000 and -975 within sgk promoter in comparison to the consensus GRE. An oligonucleotide corresponding to the sgkGRE was fused upstream of the tk-CAT reporter gene driven by the tk minimal promoter to form sgkGREtk-CAT. Lower panel, Rat2 fibroblasts were transfected either with 10 µg each of sgkGREtk-CAT, tk-CAT containing only the tk minimal promoter, GRE-CAT containing six copies of the MMTV GRE, or a constitutively active RSV-CAT reporter plasmid. Transfected cells were treated with 1 µM dexamethasone for 24 h and assayed for CAT activity as described in Materials and Methods. CAT specific activity was determined as the amount of [3H]acetylchloramphenicol formed per µg of protein. The data represent the means and SD obtained from three separate transfections each carried out in triplicate.

 
Gel Shift Analysis of GR Binding to the sgkGRE
Gel shift analysis of 32P-labeled oligonucleotides corresponding to the sgkGRE and the consensus GRE demonstrated that GR can specifically recognize the sgkGRE. The radiolabeled oligonucleotides were incubated with cytoplasmic extracts prepared from Rat2 fibroblasts, which were transfected with a GR expression plasmid, and electrophoretically fractionated by native polyacrylamide gel electrophoresis. As shown in Fig. 3Go, a single protein/DNA complex was detected with the sgkGRE-labeled probe (No competitor). Addition of excess unlabeled oligomer corresponding to the sgkGRE abolished the complex formation, while the same amount of unlabeled consensus GRE (Con GRE) completely impaired formation of specific protein-DNA complexes. This competition was specific for GRE oligonucleotides because the addition of excess unlabeled DNA fragments containing an unrelated sequence was unable to compete for protein binding (Non-specific DNA). Importantly, the addition of BuGR-2 antibodies specific for the GR caused a supershift of the protein-DNA complex (BuGR-2) demonstrating the presence of GRs in this protein/DNA complex. A parallel set of gel shift reactions carried out with the [32P]oligonucleotide corresponding to the Con GRE revealed formation of specific protein/DNA complexes that was competed off with excess unlabeled Con GRE as well as with excess unlabeled sgkGRE but not with an unrelated sequence (Non-specific DNA). Binding reactions containing anti-GR antibodies caused supershift of this specifically retarded band (BuGR-2), establishing the specificity of binding. Our results show that the GR is able to specifically bind to the sgkGRE with nearly the same efficiency as the Con GRE.



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Figure 3. Gel Shift Analysis of GR Binding to the sgk GRE and a Consensus GRE

Cytoplasmic extracts (10 µg), prepared from Rat2 fibroblasts transfected with a GR expression plasmid, were incubated either with 32P-end-labeled double-stranded oligonucleotide DNA probes containing the GRE sequences between -1000 to -975 of the sgk promoter shown in Fig. 2Go (sgk GRE probe) or the consensus GRE sequence shown in Fig. 2Go (Consensus GRE probe). One set of reactions for each radiolabeled probe did not contain any cytoplasmic extracts (No extract lanes). The reaction mixtures contained no unlabeled competitor DNA (No competitor) or 100-fold molar excess of double-stranded oligonucleotides corresponding to GRE in the sgk promoter (sgk GRE), the consensus GRE (Con GRE), or an unrelated oligonucleotide (Non-specific DNA). One binding reaction was preincubated for 1 h with anti-GR antibodies (BuGR-2). The protein-DNA complexes formed were resolved by 4% native polyacrylamide gel electrophoresis and visualized by autoradiography. Arrows indicate the position of protein-DNA complexes and free probe.

 
Wild Type p53 Protein Represses the Dexamethasone-Induced sgk Promoter Activity in Rat2 Fibroblasts
The presence of specific transcription factors and the promoter context of a given GRE have been shown to profoundly influence the direction and magnitude of glucocorticoid regulation of target genes (6, 9, 11). The magnitude and kinetics of glucocorticoid-regulated sgk gene expression differ among several cell types (13), suggesting that nuclear factors along with GRs may have combinatorial or antagonistic effects on sgk transcription. Consistent with this possibility, four DNA recognition sites for the p53 protein, located at -1380/-1345, -1155/-1125, -285/-255, and -235/-205, are all contained within the glucocorticoid-responsive -1428sgk promoter fragment, and one of these sites, at -1380/-1345, can independently mediate p53 responsiveness in mammary epithelial cells (22). To functionally test whether p53 modulates glucocorticoid responsiveness of the sgk promoter, Rat2 fibroblasts were transfected with the -1428sgk-CAT reporter plasmid alone or along with expression plasmids encoding either the murine wild type p53 or a mutant p53 that is defective in DNA binding. Transfected cells were treated with or without 1 µM dexamethasone for 24 h,and cell extracts were assayed for CAT activity. As shown in Fig. 4Go (upper panel), cotransfection of an expression plasmid encoding wild type p53 abolished the 4-fold dexamethasone-induced promoter activity of the -1428sgk-CAT reporter plasmid. Transfection of wild type p53 had only a minor inhibitory effect on basal promoter activity. In contrast to the effects of wild type p53, dexamethasone-inducible activity of -1428sgk-CAT was only partially attenuated by cotransfection of the murine mutant p53 gene (Fig. 4Go, upper panel). The wild type p53-dependent inhibition of dexamethasone-induced -1428sgk-CAT activity occurred in the presence of a 3-fold molar excess of mutant p53 (data not shown).



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Figure 4. Repression of Dexamethasone-Induced sgk Promoter and AGP Promoter Activities by Wild Type p53

Rat2 fibroblasts were transfected with 10 µg of the -1428sgk-CAT reporter plasmid (upper panel) or AGP3xGRE-CAT (lower panel) either alone or along with 10 µg of expression plasmids encoding either wild type p53 (wt p53), or mutant p53 (mt p53). Transfected cells were treated with (+) or without (-) 1 µM dexamethasone for 24 h, and CAT activity was determined and normalized to protein levels as described in Fig. 1Go. The results represent the mean and SD of three independent transfections performed in triplicate.

 
To evaluate whether the repressive effects of p53 on GR transactivation are unique to the sgk promoter or are representative of a more general biological regulation of glucocorticoid responsiveness, the effects of wild type and mutant p53 were examined on the transcriptional activity of the glucocorticoid-responsive {alpha}-1-acid glycoprotein (AGP) gene promoter (43). Cells were cotransfected with the AGP-CAT reporter plasmid, which contains a small fragment of the AGP promoter and includes three GREs, but no p53-binding sites, along with the expression vectors for either the wild type or mutant p53 gene. As also shown in Fig. 4Go (lower panel), the 5-fold induction of the AGP promoter by dexamethasone was strongly inhibited in cells transfected with wild type p53, while the basal activity of the AGP-CAT reporter was unaffected. The dexamethasone-dependent activation of the AGP-CAT construct was mildly inhibited in the presence of mutant p53. Taken together, these results demonstrate that p53 is capable of inhibiting the GR transactivation of two distinct glucocorticoid-responsive genes, implicating potential functional interactions between p53 and GR activities.

The direction and magnitude of transcriptional activity observed with certain gene promoters can differ depending on the amount of transfected p53 (44). To test the dose-dependent effects of wild type p53 on glucocorticoid-responsive sgk promoter activity, Rat2 fibroblasts were cotransfected with -1428sgk-CAT along with various amounts of expression plasmids for either the wild type or mutant p53 protein, and CAT activity was monitored in cells treated with or without dexamethasone for 24 h. As shown in Fig. 5Go, cotransfection of increasing amounts of wild type p53-encoding plasmid evoked only a dose-dependent inhibition of the dexamethasone-induced -1428sgk-CAT activity without any activation effects in Rat2 fibroblasts. In contrast, coexpression of mutant p53 had little effect on the dexamethasone-induced sgk promoter activity except at the highest tested amounts of transfected plasmid.



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Figure 5. Dose-Dependent Inhibition of Dexamethasone-Induced -1428sgk-CAT Reporter Plasmid Activity by Wild Type p53

Rat2 fibroblasts were transfected with 10 µg of -1428sgk-CAT reporter plasmid and the indicated amounts of either wild type or mutant p53 expression plasmids. Cells were cultured in the presence or absence of 1 µM dexamethasone for 24 h, and CAT specific activity was determined as described in Fig. 1Go. Reporter gene activity is expressed as fold induction by dexamethasone. The data are expressed as the means and SD obtained from three separate transfections performed in triplicate.

 
Wild Type p53 Represses Dexamethasone-Stimulated Activity of the sgkGRE in the Absence of a p53 DNA-Binding Site
One explanation for the p53-mediated inhibition of dexamethasone-induced -1428sgk-CAT activity is that p53 may directly or indirectly interfere with receptor-stimulated transcriptional events converging on the GRE located at -1000 bp within the sgk promoter. To directly test whether p53 can disrupt the GR activation of the sgkGRE, in the absence of a p53-binding site, Rat2 fibroblasts were transfected with CAT reporter plasmids driven either by the minimal tk promoter containing the sgkGRE (sgkGREtk-CAT) or without this GRE sequence (tk-CAT). Cells were transfected with the reporter plasmids alone or along with expression plasmids for either the murine wild type p53 or mutant p53. Extracts isolated from dexamethasone-treated or untreated cells were assayed for CAT activity. As shown in Fig. 6Go, the 6-fold dexamethasone-stimulated activation of sgkGREtk-CAT was strongly repressed by cotransfection of wild type p53, whereas, this reporter plasmid was glucocorticoid-inducible in the presence of the mutant p53. Both the wild type and mutant p53 caused a minor reduction in the basal activity of sgkGREtk-CAT, which was also observed in cells transfected with the glucocorticoid nonresponsive tk-CAT reporter plasmid. These results show that the murine wild type p53 can functionally interfere with the ability of the GR to transactivate a simple GRE present within the sgk promoter.



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Figure 6. p53-Mediated Inhibition of Dexamethasone-Inducible sgkGRE Activity

Rat2 fibroblasts were transfected with 10 µg of the sgkGREtk-CAT or tk-CAT reporter plasmids, as described in Fig. 2Go, either alone or cotransfected with 10 µg of either the murine wild type p53 (wt p53) or the murine mutant p53 (mt p53). Transfected cells were treated with (+) or without (-) 1 µM dexamethasone for 24 h, and CAT activity was determined and normalized to protein levels as described in Fig. 1Go. The results represent the mean and SD of three independent transfections performed in triplicate.

 
Inhibition of Dexamethasone-Induced sgkGRE Activity Requires the DNA Binding and N-Terminal Domains of the p53 Protein
Cytomegalovirus (CMV)-driven expression plasmids for the human wild type p53 and two of its mutant counterparts were used to determine whether either the DNA binding and/or the N-terminal transactivation domains of the p53 protein play a role in the repression of dexamethasone-induced sgkGRE activity. Rat2 fibroblasts were transfected with sgkGREtk-CAT or with the minimal promoter tk-CAT lacking the sgkGRE sequence either alone or in combination with expression vectors encoding either the human wild type p53 (CMV-p53wt), a human mutant p53 (CMV-p53179) that is incapable of binding DNA (45), or a human mutant p53 (CMV-p53{Delta}43) that lacks the N-terminal 43 amino acids, rendering it incapable of its transactivation function (45). Transfected cells were treated with or without dexamethasone for 24 h and asssayed for CAT activity. Cotransfection of the human wild type p53 expression plasmid strongly repressed the dexamethasone-induced activity of sgkGREtk-CAT (Fig. 7Go) without any detectable effects on the basal reporter gene activity. In contrast, the dexamethasone-mediated induction of the sgkGREtk-CAT construct was completely retained in cells cotransfected with either the CMV-p53179 DNA- binding mutant or the transactivation deletion mutant, CMV-p53{Delta}43 (Fig. 7Go). Cotransfection of the wild type or both mutant p53 expression plasmids had a negligible effect on the tk-CAT reporter gene activity both in the presence or absence of dexamethasone (Fig. 7Go, right panel). These data demonstrate that the wild type p53-mediated repression of dexamethasone-induced sgkGRE activity requires both the DNA binding and transactivation functions of the p53 protein.



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Figure 7. Requirement of p53 DNA Binding and Transactivation Domains for the Inhibition of sgkGRE Activity

Rat2 fibroblasts were transiently transfected with 10 µg of the sgkGREtk-CAT reporter plasmid or tk-CAT minimal promoter construct either alone or cotransfected with 10 µg of expression plasmids for the human wild type p53 (p53wt), a mutant human p53 with a point mutation in the DNA-binding region (p53179), or a truncated p53 protein that lacks the transactivation region (p53{Delta}43). Cells were treated with (+) or without (-) 1 µM dexamethasone for 24 h and assayed for CAT activity as described in Fig. 1Go. The CAT activity is expressed relative to protein content and the values shown are the means and SD from three different transfections done in triplicate.

 
Repression of Dexamethasone-Induced Consensus GRE Activity by Wild Type p53
Although the sgkGRE sequence is highly homologous to the consensus GRE (Fig. 2Go, upper panel), the actual sequence differs by 2 bp. Therefore, we examined whether the transcriptional inhibition by p53 is unique to the GRE within the sgk promoter or is a more general phenomenon that modulates transcriptional activity of other functional GREs. Several glucocorticoid- responsive cell lines were cotransfected with the consensus GRE-CAT reporter plasmid alone or in combination with p53 expression plasmids encoding either the murine wild type or mutant p53 protein. CAT reporter activity was monitored in cells treated with or without dexamethasone for 24 h. As shown in Table 1Go, coexpression of wild type p53, but not mutant p53, strongly repressed the 80-fold stimulation of GRE-CAT activity by dexamethasone in nontumorigenic NMuMG mammary epithelial cells and the 30- to 35-fold stimulated CAT activities observed in transformed Con8Hd6 mammary epithelial cells, BDS1 hepatoma cells, and Rat2 fibroblasts, respectively. Neither the wild type nor the mutant p53 had any appreciable effect on basal activity of the GRE-CAT reporter plasmid in the different cell lines tested. As expected for a receptor-dependent response, the p53-mediated inhibition of GR transactivation was notably absent in hepatoma cells that lack the GR, and upon rescue of these cells with cotransfection of GR expression plasmid, transfected p53 was able to inhibit the GR-induced transactivation of a GRE-CAT reporter plasmid (data not shown). Thus, the functional consequences of coexpression of wild type p53 in different cell types is to blunt glucocorticoid responsiveness by preventing GR transactivation of it’s corresponding GRE.


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Table 1. Wild Type p53 Inhibits Dexamethasone-Inducible GRE-CAT Activity in GR-Positive Cell Lines

 
Wild Type p53 Inhibits DNA Binding of the GR either to the sgkGRE or to a Consensus GRE
One explanation for the p53-mediated repression of GR transactivation of a simple GRE is an inhibition of GR DNA-binding activity. To test this possibility, the effect of p53 on GR DNA binding in gel shift assays was examined using in vitro synthesized p53 protein from rabbit reticulocyte lysates in the presence of unlabeled amino acids. Cytoplasmic extracts (10 µg) prepared from GR-transfected cells were incubated with either the 32P-labeled sgkGRE or the consensus GRE oligomers in the absence or presence of in vitro translated p53, and the protein-DNA complexes were resolved by native polyacrylamide gel electrophoresis. Preincubation of extracts with either 5 µl or 15 µl of in vitro synthesized p53 (wt p53, 5 µl and 15 µl lanes) but not with unprogrammed lysate (Un. lysate) resulted in a profound inhibition of binding of GR either to the sgkGRE (Fig. 8Go, upper panel) or to the consensus GRE (middle panel)-labeled probes as compared with samples with extract alone (Extract). Moreover, the specific GR-retarded protein-DNA complexes formed with the sgkGRE and the consensus GRE probes were retained in p53-containing reaction mixtures preincubated with anti-p53 antibodies (wt p53+PAb421) or in which the in vitro translated p53 was heat inactivated for 15 min at 90 C before incubation with the extracts (wt p53, heat treated). The specificity of GR binding to both DNA probes was confirmed by preincubation of extracts with anti-GR antibodies (BuGR-2), which caused the disappearence of the protein-DNA complex in samples containing GR antibodies (BuGR-2) but not in extracts preincubated with preimmune sera (Preimmune). The observed protein-DNA complexes were shown to be specific by competition with 100-fold excess of unlabeled self-oligonucleotides but not with an unrelated DNA sequence (data not shown). As expected, no binding was observed when in vitro synthesized p53 alone (no extract) or unprogrammed lysate alone (no extract) was present in the binding reaction without any added extracts.



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Figure 8. Wild Type p53 Inhibits Binding of the GR to the sgkGRE and the Consensus GRE in Gel Shift Assays

Cytoplasmic extracts (10 µg) prepared from NMuMG mammary cells cotransfected with a GR expression plasmid were preincubated for 1 h on ice either with rabbit reticulocyte lysates containing in vitro synthesized unlabeled wild type p53 (wt p53) or unprogrammed lysate followed by the addition of radiolabeled sgkGRE (upper panel) or consensus GRE (middle panel) probes and incubated for 30 min on ice. The protein-DNA complexes were resolved on low ionic strength native 4% polyacrylamide gels. Extracts were incubated either with anti-GR antibodies (BuGR-2), preimmune sera (Preimmune), 15 µl unprogrammed lysate (Un. lysate), 5 µl and 15 µl of wt p53 (wt p53), with 5 µl wt p53 and 20 µl of anti-p53 antibody PAb421 (wt p53+PAb421), or with wt p53 inactivated by heating at 90 C for 10 min (wt p53, heat treated) before addition of the radiolabeled probes. As negative controls, unprogrammed lysate alone or wt p53 alone without added extracts were incubated with the sgkGRE or consensus GRE-labeled probes. Each binding reaction was adjusted to equal amounts of reticulocyte lysate by adding appropriate amounts of the unprogrammed lysate. All of the shifted protein-DNA complexes were shown to be specific by competition with unlabeled DNA (data not shown). In the lower panel, extracts were incubated with increasing amounts of reticulocyte lysate containing wt p53 (0.5 µl, 1.0 µl, 2.5 µl, 5.0 µl, 10.0 µl, and 15 µl) or 15 µl of unprogrammed lysate (Un. lysate), followed by addition of labeled consensus GRE probe. Arrows indicate position of specific protein-DNA complexes and free probe.

 
Preincubation of extracts with increasing amounts of in vitro synthesized p53 (0.5 µl to 15 µl) prevented GR binding to the consensus GRE probe in a dose-dependent fashion (Fig. 8Go, lower panel) whereas addition of unprogrammed lysate (15 µl) did not affect GR binding to the DNA probe (Un. lysate). Similar dose-dependent inhibition of GR DNA binding by p53 was observed with the sgkGRE probe (data not shown). Take together, our data show that p53 inhibits the specific binding of GR to the sgk-derived GRE or to a consensus GRE, probably by physically interacting with GR and thereby rendering it incapable of DNA binding.

Inhibition of GR-Dependent Transactivation by UV Activation of Endogenous p53
The repression of GR transactivation by ectopic expression of wild type p53 suggests that one of the functions of endogenous p53 is to inhibit or modulate GR activity. It is well established that the transient induction of p53 protein is one of the key cellular responses to DNA-damaging agents such as UV radiation (46, 47, 48). Therefore, to test whether the p53 repression of GR transactivation is a physiologically relevant response, the ability of activated endogenous p53 to alter GR transactivation was examined during a time course of UV treatment of mammary epithelial cells. The optimal time of induction of endogenous p53 in response to UV treatment was first ascertained in the untransformed mammary cells (NMuMG). At various time points after exposure to UV at a dose of 40 J/m2 the level of nuclear p53 was examined by Western analysis. The accumulation of p53 protein was first observed at 3 h after UV treatment, peaking at approximately 6–8 h, and then declining beyond 14 h after UV exposure (Fig. 9Go, upper panel). To directly test whether endogenously activated p53 can repress GR-dependent transactivation, NMuMG mammary cells were transfected with the consensus GRE-CAT reporter plasmid and 24 h later one set of cells was exposed to UV treatment and a second set maintained as a nonexposed control. Cells were treated with dexamethasone either during the transient peak in p53 (from 3 h to 10 h post-UV treatment) or after the level of p53 protein subsides to near basal levels (22 to 29 h post-UV treatment). Analysis of CAT activities revealed that the GR-mediated transactivation of the consensus GRE-CAT reporter plasmid was strongly inhibited when dexamethasone was added at a time coincident with the peak induction of p53 protein after UV treatment (Fig. 9Go, lower panel). In contrast, the fold activation of the GRE-CAT reporter plasmid was unaffected when dexamethasone was added after the p53 protein level had returned to near basal levels (Fig. 9Go, lower panel). Thus, endogenously activated p53 is capable of inhibiting GR-mediated transactivation of a consensus GRE-CAT reporter plasmid in a manner similar to that observed with ectopically expressed p53, suggesting that the inhibition of GR activity by p53 is biologically important.



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Figure 9. Time Course of Induction of p53 by UV Radiation and Concomitant Suppression of Dexamethasone-Dependent Transactivation of the GRE-CAT Reporter Plasmid in NMuMG Mammary Cells

NMuMG mammary epithelial cells were exposed to 40 J/m2 UV radiation and harvested at the indicated times (upper panel). Nuclear protein extracts (30 µg) were isolated, electrophoretically fractionated, blotted onto nitrocellulose membrane, and analyzed for p53 protein by Western blotting as described in Materials and Methods. The protein molecular mass standards shown on the left correspond to albumin (69 kDa), ovalbumin (46 kDa), and carbonic anhydrase (28 kDa), respectively. The arrow indicates the position of the p53-specific band. NMuMG cells were transfected with the GRE-CAT reporter plasmid (lower panel) and 24 h later exposed to UV (40 J/m2) (+) or remained untreated (-). Cells were then either treated with or without dexamethasone from 3 h to 10 h post UV (during the peak in p53 protein levels) or 22 to 29 h post UV (after p53 protein returns to basal levels) and harvested for monitoring CAT activity. Results are expressed as fold activation by dexamethasone, which is ratio of dexamethasone-induced CAT activity to the uninduced CAT activity. Data presented represent mean ± SD of at least three independent transfections done in triplicate.

 
GRs Antagonize p53 Transactivation without Affecting the p53 Transrepression Function
To determine whether activated GR, in a reciprocal manner, affects either the transactivation or transrepression functions of p53, NMuMG mammary cells were transfected either with a p53-responsive reporter plasmid (sgk p53tk-CAT) that is induced by wild type p53 (22) or with CMV-CAT reporter that is known to be transcriptionally repressed by p53 (49) alone or along with expression plasmids encoding wild type 53, mutant p53, or GR. The p53 expression plasmids were transfected individually or in combination with the GR-encoding expression vector. Reporter gene activity was monitored in cell lysates after treatment with or without dexamethasone for 24 h. As shown in Fig. 10Go (left panel), the p53 activation of the sgk p53tk-CAT reporter was significantly reduced by activated GR in cells expressing endogenous GR as well as in cells exogenously transfected with GR expression vector (p53wt and p53wt+GR). Negligible activation of this p53-responsive reporter plasmid was observed in cells transfected with the mutant p53 gene alone or along with GR vector (p53mt and p53mt+GR). Similarly, endogenously expressed or exogenously transfected GR had no effect on the p53-inducible reporter plasmid in the absence of wild type p53 (None and GR). The reporter plasmid lacking the p53- responsive element (tk-CAT) displayed low basal activity under all conditions tested and was not p53 or GR responsive (data not shown). In contrast to the GR inhibition of p53 transactivation, the p53-dependent repression of CMV-CAT activity was completely retained and unaffected by dexamethasone activation of either the endogenous or exogenous GR (Fig. 10Go, right panel). Mutant p53 exhibited a mild repression of CMV-CAT activity regardless of dexamethasone treatment or the presence of the transfected GR expression vector, whereas dexamethasone treatment in the absence of either wild type or mutant p53 had little effect on the CMV-CAT activity. These results demonstrate that activated GR is capable of attenuating the p53-dependent transactivation function but not its transrepression activity and that both GR and p53 display a mutual inhibition of their respective transactivation functions.



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Figure 10. Activated GR Inhibits p53-Dependent Transactivation Function but not the Transrepression Activity

NMuMG mammary epithelial cells were transiently transfected with sgkp53tk-CAT reporter (left panel), or with CMV-CAT reporter construct (right panel), either alone or cotransfected with expression vectors encoding GR, wild type p53 (p53wt), mutant p53 (p53mt) individually or as combinations (p53wt+GR), (p53mt+GR). Cells were either treated (+) or untreated (-) with dexamethasone for 24 h and harvested for assaying CAT activity. The mean levels of CAT activity with SD determined in at least three separate transfections are presented normalized to protein levels.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Our previous studies have demonstrated that the sgk serine/threonine protein kinase gene displays unique transcriptional stimulation by glucocorticoids (13) and is the second of four known protein kinases to be under transcriptional control by serum or mitogens (18, 19, 21). By functional dissection of cis-acting regulatory elements in the sgk promoter, we now demonstrate that GRs stimulate sgk transcription through a GRE located at -1.0 kb of the sgk promoter. The existence of this GRE provides a direct molecular basis for the transcriptional activation of sgk by glucocorticoid hormones (13, 14) and establishes a direct link between GR-dependent signaling and control of cellular phosphorylation/dephosphorylation cascades. We have also identified the p53 transcription factor as an important modulator of glucocorticoid-dependent transcriptional control of the sgk gene promoter. Transient transfection studies revealed a mutual antagonism of transactivation functions between p53 and GR. In addition, p53 specifically abrogates the ability of GR to bind to either the sgkGRE or to a consensus GRE. Taken together, our findings demonstrate functional interactions between the p53 protein and GRs and implicate an interplay of disparate transcription factor families in the control of cellular processes.

A functional GRE within the sgk promoter was identified within a 25-bp segment spanning nucleotides -1000 bp to -975 bp which, when linked to a reporter gene, confers glucocorticoid inducibility of sgk transcription, both in the context of the natural promoter and when placed upstream of a heterologous promoter in a variety of GR-positive cell lines. The sgkGRE sequence 5'-AGGACAgaaTGTTCT-3', although not a perfect palindrome, bears significant homology with the consensus GRE sequence 5'-GGTACAnnnTGTTCT-3', which is well established by previous studies to mediate glucocorticoid enhancement of gene transcription (6). Importantly, gel shift analysis demonstrated that the GR can specifically recognize the sgkGRE sequence in vitro. The receptor/DNA complexes can be supershifted by GR-specific antibodies and efficiently competed by unlabeled consensus GRE for binding the sgkGRE probe, establishing that the GR transactivation involves direct interaction with the sgkGRE. The precise functions and physiological substrates of this novel glucocorticoid-regulated kinase are not known. However, preliminary studies suggest the involvement of sgk in the control of mammary cell growth. Consistent with this concept, dexamethasone has recently been shown to inhibit the activity of the mitogen-activated pp70 S6 kinase, by mechanisms requiring ongoing transcription (50).

In addition to the regulation of the sgk promoter by glucocorticoids, our studies demonstrate a role for the p53 protein in the GR-dependent transcriptional activation of sgk promoter. The ectopic expression of wild type p53 strongly disrupted the GR-mediated transactivation of the sgk promoter, which contains several p53-binding sites. Strikingly, the suppression of glucocorticoid-responsive sgk promoter activity by wild type p53 was observed regardless of whether the p53 DNA binding sites were present or not in the reporter plasmid. In fact, this suppression was discernible using reporter plasmids containing only the sgkGRE, or a consensus GRE, transfected into glucocorticoid-responsive epithelial cell lines as well as Rat2 fibroblasts, which suggests a generality of the response and a minimal requirement for the presence of a GRE. The p53-dependent repression of another GRE-containing natural promoter, such as the AGP gene promoter, but lacks any p53-binding sites (43), further substantiates the view that the repression of GR transactivation by p53 does not entail binding to specific p53 recognition elements, but instead likely involves regulation at the protein-protein level. Our results also exclude the possibility of p53 evoking repression via affecting general transcription machinery, since basal promoter activity of the synthetic GRE-CAT construct was unaltered by coexpression of p53. Cotransfection of mutated human p53 genes revealed that the DNA-binding and transactivation domains of p53 are important for the inhibition of GRE activity. Receptor reconstitution experiments using a GR-negative cell line, demonstrated that the GR is required for the p53-dependent repression of transactivation (data not shown).

The repression of GR transactivation by p53 is not due to the unusually high levels of p53 protein that occurs after its ectopic expression since the same functional antagonism of GR function was observed in cells after the activation of endogenous p53 protein by exposure to UV stress. The transient induction of stable p53 protein is a well characterized protective cellular response to genotoxic agents including UV radiation (46, 47, 48). Inhibition of dexamethasone-induced GRE-CAT activity was observed at the time frame in which p53 protein is induced by UV, whereas, no effects on GR transactivation were observed when dexamethasone was added either preceding or subsequent to the expression of UV-induced p53. The ability of endogenously activated p53 to repress glucocorticoid-stimulated transactivation of the consensus GRE in transfected cells suggests that this transcriptional repression may be a widely used cellular pathway to modulate glucocorticoid responsiveness in a physiologically appropriate context.

Although the detailed molecular mechanism of p53-mediated repression of GR activity remains unknown, our data indicate that p53 impairs GR DNA binding capacity either to the sgkGRE or to a consensus GRE oligomer. One possible explanation for such inhibition of GR DNA binding by p53 may be competition for response element binding, which has been suggested as a likely mechanism for GR ß-mediated repression of GR activity (51). However, in our studies, data from both transfection experiments and DNA binding assays demonstrate that the inhibition of GR function by p53 was not due to a direct competition of p53 with GR for binding to the GRE because p53 neither bound to nor transactivated a GRE. In addition, the fact that the position of the GR- specific gel shifted band was unaltered in the presence of p53 suggest that any physical interaction of p53 with GR likely precludes GR from binding to DNA but does not alter the GR-DNA interaction once the receptor is bound to DNA. Although p53 could indirectly influence functionality of the receptor-DNA complex by altering the expression or activity of another protein, the strong inhibition of GR DNA binding in vitro by p53 argues against an indirect effect of p53. In this regard, p53 has been recently shown to physically interact with another member of the zinc finger family of hormone receptors, the human thyroid receptor ß-1, causing a repression of thyroid receptor-dependent transactivation (39). In another study, transcription of the orphan receptor TR2, which is also a member of the steroid receptor superfamily of transcription factors, was strongly suppressed by p53 (37), implying functional interactions between p53 and members of steroid receptor superfamily. It is interesting to note that in previous studies, functional interactions between p53 and other zinc finger proteins such as Sp1, WT1, and mdm2 have been reported, resulting in either transcriptional synergy or inhibition (35, 38, 41). Compatible with these findings, our results demonstrate another example of p53 modulating the transcriptional activity of the GR, which is a well characterized zinc finger transcription factor.

The strong inhibition of p53-dependent transactivation by overexpression of GR or endogenous activation of GR shows that p53 and GR mutually antagonize each other’s function, implicating a direct interaction between the two proteins. Conceivably, GR may be affecting DNA binding and/or transactivation function of p53. One of our future approaches will be to functionally define the specific structural domains within the GR protein that are required for its interaction with p53. In this context, GRs have been shown to interact with a select number of transcription factors resulting in either synergistic (such as C/EBP family members) or antagonistic (such as members of the AP-1 family or NF-KB) regulation of GR responsiveness (8, 10, 52, 53, 54, 55, 56). In addition, the tumor suppressor protein Rb has also been shown to potentiate transactivation by the GR (57). Interestingly, our results have further shown that activated GR had no significant influence on the transrepression function of p53. The reasons for this lack of effect of GR are not clear, especially since the mechanisms of p53-dependent transcriptional repression are poorly understood. Transcriptional repression by p53 requires the carboxy-terminal end of the protein and is believed to occur by interaction with components of the core transcriptional machinery (58, 59). On the other hand, p53-dependent transactivation involves specific DNA binding and the presence of an intact transactivation and DNA-binding domains, within the p53 molecule (29, 30). Therefore, it is likely that the selective inhibition of transactivation function of p53 by GR may be related to distinct differences in the mechanisms underlying p53-dependent transactivation vs. transrepression.

The repression of GR-dependent transactivation by p53 implicates, at a functional level, a new pathway by which this tumor suppressor can elicit its pleiotropic effects on mammalian cells. Both glucocorticoid hormones and p53 play important physiological roles in the control of cellular growth and differentiation in a variety of tissue and cell types (24, 25, 26, 27, 60, 61, 62, 63). The functional interactions between a growth regulator such as p53 and the GR, also implicated in growth control, may serve to provide a framework for understanding how transcription factor interactions affect diverse growth- regulatory networks. For instance, the degree of responsiveness to glucocorticoids varies widely not only between different tissues but also within specific cell types (5). Modulation of GR activity by p53 may partly account for such differences in tissue sensitivity to glucocorticoids, reflecting another level of receptor regulation. The inhibited expression of a glucocorticoid-responsive protein kinase gene, sgk, by p53 likely represents a possible target of cross-talk between two distinct signaling pathways that regulate cellular phosphorylation cascades as an appropriate response to specific extracellular signals. In addition to the multiple p53 DNA-binding sites and a GRE, the sgk promoter also contains potential binding sites for several other important regulatory factors implicated in growth and differentiation. These putative regulatory sites include the AP-1 complex recognition sequence, a consensus C/EBP site, which is recognized by a family of transcription factors involved in growth control, and a serum response element as well as DNA sites for factors involved in developmental programs, such as the kruppel and hunch-back proteins. Thus, important future approaches will attempt to determine the critical combinatorial effects, functional roles, and biological significance of the multiple cis-acting promoter elements that regulate transcription of the sgk gene in a cell type-specific manner.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cells and Materials
Rat2 cells are an untransformed rat fibroblast cell line generously provided by G. S. Martin (Department of Molecular and Cell Biology, University of California, Berkeley, CA). Con8Hd6 mammary epithelial tumor cells and Rat2 fibroblasts were maintained in DMEM/Ham’s F-12 medium (50:50) supplemented with 10% calf serum. The BDS1 cell line is an epithelial tumor cell line derived from the minimal deviation rat Reuber hepatoma (64) and cultured in DMEM/F-12 supplemented with 10% FBS. NMuMG cells are a nontransformed mouse mammary epithelial cell line originally derived from normal glandular tissue of an adult NAMRU mouse (65). NMuMG cells were cultured in DMEM supplemented with 10% FBS and insulin (10 µg/ml). All cell lines were routinely grown at 37°C in humidified air containing 5% CO2. Cell culture media were changed every 48 h, and dexamethasone was added to a final concentration of 1 µM as indicated. DMEM/F12 (1:1), the calf serum, and fetal bovine serum, the calcium- and magnesium-free PBS, and trypsin-EDTA were supplied by BioWhittaker (Walkersville, MD). Dexamethasone was obtained from Sigma Chemical Co. (St. Louis, MO). [3H]Acetyl coenzyme A (200 mCi/mmol) was purchased from DuPont/NEN (Wilmington, DE) and [{gamma}-32P]dATP was procured from ICN Biomedicals Inc. (Costa Mesa, CA). The BuGR-2 GR-specific antibody (66) was generously provided by Dr. John Forte (Department of Molecular and Cell Biology, University of California, Berkeley, CA). The anti-p53 monoclonal antibody PAb421 was purchased from Oncogene Science Inc. (Cambridge, MA). The GRE-CAT chimeric reporter plasmid containing six copies of GREs from the mouse mammary tumor virus (MMTV) promoter linked to the CAT reporter gene and the 6RGR expression plasmid containing the GR cDNA under the control of the RSV promoter were generous gifts from Dr. Keith R. Yamamoto (Department of Biochemistry & Biophysics, University of California, San Francisco, CA). The RSV-CAT reporter plasmid encodes the bacterial CAT gene constitutively driven by the long terminal repeat of the RSV promoter and was a gift from Dr. Marc Montimony (Salk Institute, La Jolla, CA). In the CMV-CAT plasmid construct, the CAT gene is driven by the CMV promoter. The sgk p53tk-CAT reporter plasmid contains the p53-responsive element from the sgk gene fused upstream of tk minimal promoter linked to the CAT gene (22). The AGP reporter plasmid designated as AGP3x(GRE)-CAT was kindly provided by Dr. Heinz Baumann (Department of Molecular and Cellular Biology, Roswell Park Cancer Institute, Buffalo, NY) and comprises 127 bp of the native AGP promoter sequences containing three copies of GRE linked to a CAT reporter (43). The murine wild type p53 expression plasmid driven by the CMV promoter, CMVp53wt, and the transforming mutant p53 expression plasmid, CMVp53mt, derived from Meth A fibrosarcoma cells bearing point mutations at positions corresponding to residues 168 and 234 of the protein (67) and incapable of DNA binding (68) were obtained from Dr. Moshe Oren (Weisman Institute for Science, Rehevot, Israel). The human p53 expression plasmids CMV-p53wt, CMV-p53179, and CMV-p53{Delta}43 were generous gifts of Dr. Peter Howley (National Cancer Institute, Rockville, MD). CMV-p53wt encodes the wild type p53 protein. CMV-p53179 was isolated from a human lung tumor and has a His-to Gln substitution at codon 179, and CMV-p53{Delta}43 has the first 43 amino acids from the N terminus of the protein deleted (45). All other reagents were of highest available purity.

Plasmid Constructions
Construction of the plasmid p-4.0sgk-CAT, which contains -4.0 kb of 5'-flanking sequences of rat sgk gene, ending at +51(-4000/+51), relative to transcriptional start site and fused to the coding region of bacterial CAT gene in the vector pBLCAT3, has been previously described (13). Unilateral 5'-deletions of p-4.0sgk-CAT were generated by ExonucleaseIII/S1 nuclease digestion of the HindIII and AflII-double-digested p-4.0sgk-CAT construct according to manufacturers’ instructions (Promega, Madison, WI). The 5'-protruding end of the HindIII site was protected from ExoIII by filling in the 3'-recessed end with {alpha}-phosphorothioate deoxynucleoside triphosphate. These deleted fragments were filled in using Klenow enzyme and religated and sequenced to determine endpoints of deletion. The sgkGREtk-CAT reporter plasmid was created by inserting double-stranded annealed oligonucleotide encompassing 5'-flanking sgk gene sequences from -1000 to -975 (5'- tgcggAGGACAgaaTGTTCTcggag-3') with the HindIII site at the 5'-end and the BamHI site at the 3'-end into HindIII/BamHI sites of the plasmid HSVtk-CAT, which contains the tk promoter (-105/+57) upstream of CAT gene (69). The plasmid was confirmed by DNA sequencing.

Transfection and UV Treatment of Cells
Logarithmically growing Rat2 fibroblasts and Con8Hd6 mammary epithelial tumor cells were incubated in DMEM/F12 (1:1)/10% calf serum and transfected by electroporation (13). Briefly, single-cell suspensions were generated by trypsinization, washed twice with sterile 1x PBS, and resuspended in an electroporation buffer containing 270 mM sucrose, 7 mM sodium phosphate buffer, pH 7.4, 1 mM MgCl2. The cells (1–2 x 107 cells per sample) contained in 250 µl of electroporation buffer were dispensed into sterile cuvettes. In most experiments, cells were transfected with 10 µg of reporter plasmid and 10 µg of appropriate expression plasmid. In all transfections, the total amount of DNA was adjusted to 30 µg with the promoterless pBLCAT3 vector DNA. All plasmids used for DNA transfections were purified twice by a CsCl banding method. The cells and DNA were gently mixed and electrically pulsed five times (700 V square wave pulse for 99 µsec for Rat2 cells and 400 V square wave pulse for 99 µsec for Con8Hd6 cells) using a BTX 800 Transfector apparatus (BTX Inc., San Diego, CA), and the cells were incubated on ice for 10 min. Transfected cells were plated into prewarmed DMEM-F12 (1:1)/10% calf serum in 100-mm Corning plastic tissue culture dishes and incubated at 37 C. Typically, 5 h after transfection, the medium was removed by aspiration, and the cells were washed in PBS and then incubated with fresh medium in the presence or absence of 1 µM dexamethasone. After 24 h, cells were harvested for CAT assays, and the protein contents of the resulting cell extracts were determined by the Bradford protein assay (70). NMuMG mammary epithelial cells and BDS1 hepatoma cells from a logarithmically growing culture were transfected by the calcium phosphate precipitation method (71). Briefly, cells were plated into 100-mm tissue culture plates after dispersion by treatment with trypsin-EDTA 16–20 h before transfections. Four hours before transfection, cells were replenished with fresh medium. Typically 10 µg of reporter plasmid, along with 10 µg of appropriate expression plasmid, were used to prepare the DNA-CaPO4 precipitates for each plate and added individually while rocking the plate gently. The total amount of DNA used in CaPO4 transfections for CAT assays was held constant at 20 µg and, in appropriate transfections, the total DNA adjusted to this amount using the empty CAT vector plasmid, pBLCAT3. After 4 h of incubation at 37 C, the cells were briefly shocked with 15% glycerol solution for 3 min at 37 C, followed by two PBS washes and subsequently incubated with fresh medium. UV treatment of cells was carried out using a UV Stratalinker (1800 series, Stratagene, La Jolla, CA) where the energy delivered was precisely controlled by the cross-linker. The medium was aspirated, and cells were rinsed in PBS twice and exposed to 40 J/m2 of UV energy. After UV treatment, cells were grown in fresh serum-replete medium for defined periods of time.

CAT Reporter Gene Assays
A quantitative nonchromatographic assay (72) was used to measure CAT activity in the cell extracts as detailed elsewhere (13). Briefly, cells were harvested by washing twice in PBS, resuspended in 0.1 M Tris-HCl, pH 7.8, and lysed by three cycles of freeze-thawing (alternating between an ethanol-dry ice bath and a 37°C water bath, 5 min per cycle). Cell lysates were heated at 68°C for 15 min and centrifuged at 12,000 x g for 10 min, and supernatants were recovered. CAT enzymatic assay was monitored in 50–75 µg of cell extracts and was carried out in 0.1 M Tris-HCl, pH 7.8, 1 mM aqueous chloramphenicol, and 1 µCi [3H]acetyl-coenzyme A (final reaction volume of 250 µl) at 37°C for 4 h. The reaction mixture was gently overlaid with 4 ml of Econofluor (DuPont/NEN), and the production of [3H]acetylated chloramphenicol was quantitated by liquid scintillation counting. The enzyme activity was expressed as [3H]acetylated chloramphenicol produced per µg protein present in corresponding cell lysates (counts per min/µg protein/4 h). Transfections were done in triplicate and repeated at least three times.

Gel Mobility Shift Assays
Cytoplasmic extracts from either NMuMG cells or Rat2 fibroblasts transiently transfected with GR-encoding plasmids were prepared essentially by the method of Dignam et al. (73). Protein contents were evaluated by the Bradford procedure (70). In assays utilizing in vitro synthesized protein, the TNT system (Promega) was used. The CMV-p53wt expression plasmid engineered for in vitro expression was used to synthesize wild type p53 either from labeled or unlabeled amino acids according to the manufacturers’ instructions. The purity of the synthesized wild type p53 from labeled amino acids was confirmed by resolving the product on 7.8% SDS polyacrylamide gels. Subsequently, unlabeled amino acids were used to generate in vitro synthesized protein for gel shift analysis. The sense strands of the oligonucleotides used to synthesize 32P-labeled DNA probes for gel shift assays included: 1) sgkGRE, 5'-tgcggAGGACAgaaTGTTCTcggag-3' derived from sequences within -1000 to -975 of sgk promoter. 2) Con GRE 5'-tcgacGGTACAggaTGTTCTagctact-3' has consensus GRE sequence and has been shown to specifically interact with the GR (74). In addition, unlabeled oligonucleotides corresponding to the sgkGRE, Con GRE, and a nonspecific sequence were utilized as competitor DNA in the gel shift assays. All oligonucleotides were synthesized by model 394 synthesizer in the University of California at Berkeley Cancer Research Laboratory Microchemical facility.

Radiolabeling of 5'-ends of the oligonucleotides involved incubation of equal amounts (10 pm), of sense and antisense strands with [{gamma}-32P]ATP, 7000 Ci/mmole (ICN), and T4 polynucleotide kinase (Boeringer Mannheim, Indianapolis, IN) for 30 min, at 37 C. Annealing of complementary strands either for labeled or unlabeled DNA probes was carried out by mixing equal amounts of sense and antisense oligonucleotides in 0.1 M NaCl, heated at 70 C for 10 min followed by gradual cooling to room temperature. Double-stranded labeled oligonucleotides were purified from single-stranded DNA, and unincorporated nucleotides were purified by electrophoretic fractionation on a 8% native polyacrylamide gel. The radioactive bands were excised, eluted in 400 µl TE buffer (10 mM Tris, 1 mM EDTA, pH 7.4) and 40 µl 3 M sodium acetate, pH 5.0, ethanol precipitated, washed in 70% ethanol, resuspended in TE buffer, and stored at -70 C.

The binding reactions of cell extracts with DNA were carried out in a 20 µl reaction volume, containing 10 µg of cytoplasmic extracts, 0.5 ng of [32P]labeled (5 x 104 cpm) DNA probe, 500 ng of poly(deoxyinosinic-deoxycytidylic)acid, 7 µl of 2x binding buffer (20% glycerol, 20 mM HEPES, pH 7.9, 50 mM KCl, 6.25 mM MgCl2, 0.5%NP-40, 0.2 mM EDTA, 4 mM spermidine) and incubated for 20 min on ice. In specific competition experiments, 100-fold molar excess of annealed double stranded unlabeled competing oligonucleotides were added before the addition of radiolabeled DNA probes. In some cases, the extracts were preincubated with either specific antibodies or in vitro translated proteins for 60 min before addition of radiolabeled probes. Antibodies used in the binding reactions included anti-GR antibody, BuGR-2, or the anti-p53 antibody, PAb421. The DNA- binding reactions were analyzed by electrophoretic fractionation on a 4% nondenaturing polyacrylamide gel (80:1 acrylamide/bis-acrylamide) in 0.25x TAE buffer (0.04 M Tris-acetate, 1 mM EDTA, pH 7.4) and 1 mM EDTA and 0.05% NP-40, at 180 V at 4 C. Gels were routinely prerun for 2 h, at 180 V at 4 C. After electrophoresis, gels were dried and protein-DNA complexes were visualized by autoradiography using Amersham Hyperfilm.

Western Blotting
Nuclear extracts were prepared according to previously described procedures (73). Thirty micrograms of nuclear protein extracts from UV-treated NMuMG cells were resolved on 7.8% SDS-polyacrylamide gel and transferred to nitrocellulose membrane (Micron Separations, Westborough, MA). The membrane was blocked overnight at 4 C with TBST blocking solution (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20) containing 5% nonfat dry milk, and then incubated for 2 h at room temperature with primary anti-p53 monoclonal antibody PAb421 at a 1:100 dilution in TBST blocking solution. The secondary antibody used was directed against mouse IgG conjugated to horse radish peroxidase (Zymed Laboratories, Inc., South San Francisco, CA) at a 1:10,000 dilution in TBST blocking solution containing 1% nonfat dry milk and incubated for 1 h. The signal was detected by enhanced chemiluminescence on Hyperfilm ECL (Amersham Corp.) in accordance with the manufacturer’s instructions. Equivalent protein loading was verified in parallel sets of samples by Coomassie Blue staining of the protein gel.


    ACKNOWLEDGMENTS
 
We express our appreciation to Paul Woo, Helen Cha, Yukihiro Nishio, and Ross Ramos, for critical reading of the manuscript. We also wish to thank Marina Chin, Althaea Yronwode, Khanh Tong, Thai Truong, Vinh Trinh, Charles C. Jackson, and William J. Meilandt for their technical assistance. We are grateful to Jerry Kapler for his excellent photography.


    FOOTNOTES
 
Address requests for reprints to: Gary L. Firestone, Department of Molecular and Cell Biology, Box 591 LSA, University of California at Berkeley, Berkeley, California 94720.

This work was supported by NIH Grant CA-71514 awarded by the National Cancer Institute. In the initial stages of this work, A.C.M. was a postdoctoral trainee supported by National Research Service Grant CA-09041 awarded by the NIH.

1 Supported by a summer research fellowship from the Biology Fellows Program at the University of California at Berkeley, sponsored by the Howard Hughes Medical Institute. Back

Received for publication January 31, 1996. Revision received October 23, 1996. Accepted for publication December 4, 1996.


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 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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