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J. Biol. Chem., Vol. 280, Issue 30, 27638-27644, July 29, 2005

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Regulation of the Expression of Caspase-9 by the Transcription Factor Activator Protein-4 in Glucocorticoid-induced Apoptosis^*

Kyoko Tsujimoto, Takeshi Ono, Masaki Sato, Takashi Nishida, Takemi Oguma, and Takushi Tadakuma

From the Department of Parasitology and Immunology, National Defense Medical College, 3-2 Namiki, Tokorozawa 359-8513, Japan

Received for publication, February 3, 2005 , and in revised form, May 24, 2005.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Caspase-9 (Casp-9) induces death signals by triggering other types of caspase activation, and its expression greatly influences the onset of apoptosis. During the isolation of apoptosis-related genes involved in glucocorticoid (GC)-induced cell death in murine thymic lymphomas, we found that the antisense gene of the transcription factor activator protein-4 (AP-4) inhibited dexamethasone-induced apoptosis. Western blot analysis revealed that the expression of Bcl-x_L, Bax, and Apaf-1 was not affected in cells transfected with sense or antisense AP-4 genes. In contrast, both the expression and activation of Casp-9 were inhibited in the antisense AP-4 transfectants. We isolated the 2.4-kb 5'-flanking region of Casp-9, and the promoter activity was investigated. We found the AP-4-binding sites at –1.55 and –1.38 kb to be responsible for the promoter activity. Furthermore, a negative cis-element was expected to exist between bases –1140 and –944. When the cells were treated with dexamethasone, a rapid down-regulation of AP-4 and Casp-9 was observed whether the cells were GC-sensitive lymphomas or GC-insensitive L929 fibroblast cells. In addition, L929 cells pretreated with dexamethasone were found to be resistant to subsequent treatment with etoposide, an apoptosis-inducing reagent. GC has a two-sided effect on apoptosis, i.e. a pro-apoptotic effect on certain cell types and a prosurvival effect on other cell types. Our findings will explain, at least in part, this effect.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Apoptosis is physiological cell death, and it is essential for successful embryonic development and the maintenance of normal tissue homeostasis (1). Many apoptosis-inducing stimuli induce the apoptotic mechanism by causing the release of cytochrome c from the mitochondria and by inducing the oligomerization of Apaf-1, followed by the recruitment of procaspase-9 into a large complex apoptosome (2, 3). After activation by Apaf-1, caspase (Casp)¹-9 initiates a proteolytic cascade by cleaving and activating both Casp-3 and the downstream caspases and thereby inducing cell death. As a result, both Apaf-1 and Casp-9 play a central role in initiating the events of apoptosis, and the level of expression of these two factors also greatly influences the onset of apoptosis.

Among the various apoptosis-inducing agents, glucocorticoid (GC) is one of the most well known and is the most widely prescribed compound in current medical practice (4). GC is used in high doses for the treatment of leukemia and lymphoma. GC also plays a major role in attenuating inflammatory responses. GC is expected to exert its apoptotic effect on the lymphocytes and macrophages that are responsible for inflammation. Furthermore, GC also physiologically helps to maintain homeostasis. Thymocytes are highly sensitive to GC-induced apoptosis, especially at the CD4⁺CD8⁺ stage (5). GC is thought to play an important role in eliminating any discarded cells during the selection of T cells in the thymus (6). In addition, some studies have reported the balance of the signals via the T cell and GC receptors to contribute to the positive selection in the thymus (7, 8).

We established a dexamethasone-sensitive CD4⁺CD8⁺ thymocyte cell line (2-257-20). With this cell line, we tried to isolate the apoptosis-related genes involved in GC-induced cell death (9). During this investigation, we identified the antisense (AS) activator protein-4 (AP-4) gene as one of the candidates that work to inhibit GC-induced apoptosis. A further analysis revealed that, in the AS AP-4 transfectants, both the expression and activation of Casp-9 were inhibited.

In this study, we isolated the 5'-flanking region of Casp-9 and demonstrated the promoter activity of Casp-9 to be dependent on the presence of AP-4. In addition, we demonstrated that dexamethasone treatment rapidly induced AP-4 down-regulation, followed by down-regulation of Casp-9 in both 2-257-20 cells and L929 fibroblast cells, which are resistant to dexamethasone-induced cell death. From the viewpoint of apoptosis, GC has an ambivalent effect depending on the target cells (10). GC induces apoptosis in cells of the hematopoietic system such as monocytes and lymphocytes, whereas there is increasing evidence that GC in cells such as hepatocytes and fibroblasts plays a protective role against the apoptotic signals evoked by various other apoptosis-inducing agents. Our findings are discussed with reference to this two-sided effect of GC.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Cell Lines—The CD3⁺CD4⁺CD8⁺ thymic cell line 2-257-20 was established as described previously (11). The cells are sensitive to apoptosis induced by dexamethasone, cAMP, and anti-CD3 antibody. They were maintained in RPMI 1640 medium containing 10% fetal calf serum. NIH/3T3 and L929 cells were obtained from American Type Culture Collection and were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum.

Cloning of the AS AP-4 Gene—2-257-20 cells were treated with ethyl methanesulfonate (Sigma), and the dexamethasone-resistant cell line 7-15 was selected. The cDNA library was established from this cell line and then inserted into the pPyori-SR-BX vector. These constructs were transfected into 2-257-20 cells, which express polyomavirus large T antigen upon transfection with the pPyLT-BSD vector, and selected with both blasticidin S hydrochloride (10 µg/ml; Funakoshi, Tokyo, Japan) and dexamethasone (1 µM). Transfection was performed by the DEAE-dextran method as modified by Takai and Ohmori (12). Three weeks after addition of 1 µM dexamethasone, the surviving cells were isolated, and episomal DNA was collected by the Hirt method. The rescued plasmids were pooled, re-transfected, and then further selected in the presence of blasticidin S hydrochloride and dexamethasone. After a third screening, AP-4 was isolated as an antisense gene. The sample was purified using a BigDye^TM DNA sequencing kit (Applied Biosystems), and AP-4 sequence was analyzed using the Applied Biosystems Model 310 DNA sequencing system.

Establishment of AP-4 Stable Transfectants—2-257-20 cells were harvested and washed with phosphate-buffered saline. 25 µg of pcDNA-BSD containing an antisense or sense cDNA fragment was added to the cell suspension. Thereafter, electroporation was carried out at 300 V and 960 microfarads with a Gene Pulsar (Bio-Rad). The cells were selected in the presence of blasticidin S hydrochloride (10 µg/ml). In this study, the mock transfectants V30 and V43; the sense AP-4 transfectants S5 and S6; and the AS AP-4 transfectants AS3, AS15, AS23, and AS40 were used.

Cell Viability and Determination of Apoptosis-associated Alterations—2-257-20 cells (2 x 10⁵ cells/ml) were incubated with 1 µM dexamethasone for 24 or 48 h to induce cell death. The cells were harvested, and viability was determined by trypan blue staining. The frequency of subdiploid cells was determined by staining with propidium iodide after permeabilization with Nonidet P-40 and analyzed by flow cytometry. The mitochondrial membrane potential was assessed by flow cytometry after staining with 20 nM 3,3'-dihexyloxacarbocyanine iodide (Molecular Probes, Inc.) for 15 min at 37 °C in 5% CO₂. The results of flow cytometry were analyzed using the CellQuest software package. To observe etoposide-induced cell death upon dexamethasone treatment, L929 cells were pretreated with 1 µM dexamethasone for 48 h. Thereafter, 10 or 30 µM etoposide (Sigma) was added, and cell viability was determined after additional incubation for 48 h. When a steroid receptor antagonist was included, 10 µM RU-486 (mifepristone, Sigma) was added to the culture medium 1 h before addition of 1 µM dexamethasone.

Antibodies—Antibodies for Western blotting were purchased as follows: anti-Apaf-1 polyclonal antibody (rabbit IgG) from Imgenex Corp., anti-Casp-9 polyclonal antibody (rabbit IgG) from Cell Signaling Technology, anti-Bcl-x_L monoclonal antibody (mouse IgG) from Pharmingen, and anti-Bax monoclonal antibody (mouse IgG) and anti--actin monoclonal antibody (mouse IgG) from Sigma. Anti-AP-4 antibody was obtained by injecting rabbits with glutathione S-transferase-AP-4 fusion protein produced using the GST gene fusion system (Amersham Biosciences).

Western Blot Analysis—2-257-20 cells and their AP-4 transfectants treated with or without 1 µM dexamethasone were harvested and lysed in lysis buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, and 5 mM EDTA). Total protein was electrophoresed on 10% SDS-polyacrylamide gels and transferred to polyvinylidene difluoride membranes. The membranes were blocked using Block Ace for 1 h at room temperature and then incubated with anti-AP-4, anti-Bcl-x_L, anti-Bax, anti-Apaf-1, or anti-Casp-9 antibody overnight at 4 °C or with anti--actin antibody for 30 min at room temperature. After washing, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody for 1 h at room temperature and then washed again. Each specific band was detected by ECL (Amersham Biosciences). L929 cells were seeded at 5 x 10⁵ cells/60-mm dish (Falcon 3004) the day before assay. Cells treated with or without dexamethasone were harvested after treatment with trypsin/EDTA and then subjected to immunoblotting.

Quantification of AP-4 and Caspase-9 mRNA Expression by Real-time PCR—Total RNA was extracted from 2-257-20 or L929 cells using RNeasy (Qiagen Inc.). mRNA was reverse-transcribed, and quantitative PCR was performed using a TaqMan Gold RT-PCR kit (Applied Biosystems) on an ABI Prism 7700 thermal cycler (Applied Biosystems). Both the primers and probes were designed using the Primer Express software package (Applied Biosystems). The primers and probes were as follows: AP-4, 5'-AGTCCCTCAAGACCCTCATTCC-3' (forward primer), 5'-TCTCCAGCAGACAGCAGAATACA-3' (reverse primer), and 5'-carboxyfluorescein-ACACAGATGGAGAGAAGCTCAGCAAGGCA-carboxytetramethylrhodamine-3' (TaqMan probe); Casp-9, 5'-ATGACCACCACAAAGCAGTCC-3' (forward primer), 5'-CGTGACCATTTTCTTGGCAG-3' (reverse primer), and 5'-carboxyfluorescein-TTCCGGTGTGCCATCTCCATCAAAG-carboxytetramethylrhodamine-3' (TaqMan probe); and glyceraldehyde-3-phosphate dehydrogenase, 5'-ATGTGTCCGTCGTGGATCTGAC-3' (forward primer), 5'-TCAAGAAGGTGGTGAAGCAGG-3' (reverse primer), and 5'-carboxyfluorescein-CGCCTGGAGAAACCTGCCAAGTATGATGAC-carboxytetramethylrhodamine-3' (TaqMan probe).

Reverse transcription and PCR were performed as a one-step reaction. The reverse transcription conditions were 48 °C for 30 min and AmpliTaq Gold activation at 95 °C for 10 min, and the PCR cycling conditions were 40 cycles at 95 °C for 15 s and 60 °C for 1 min. A relative standard curve was generated for glyceraldehyde-3-phosphate dehydrogenase in 2-257-20 or L929 cells. Relative quantification of their AP-4, Casp-9, and glyceraldehyde-3-phosphate dehydrogenase mRNAs was determined from the curve, and AP-4 and Casp-9 mRNAs were normalized to glyceraldehyde-3-phosphate dehydrogenase mRNA.

Isolation of the Mouse Casp-9 Promoter Region—Genomic DNA was purified from 2-257-20 cells using a DNeasy^TM tissue kit (Qiagen Inc.). Primary and nested PCRs were performed with ExTaq^TM (hot start version, TaKaRa, Tokyo). The forward primers were designed based on the rat Casp-9 promoter region sequence (GenBank^TM accession number AY027666 [GenBank] ): 5'-CTCATTACTGCTCTCCCCAGATTTCCTGT-3' for the primary reaction and 5'-ACCTCAGCTGCTCCTCCCGAGTGTACATCC-3' for the nested reaction. The reverse primers were designed based on the mouse Casp-9 mRNA: 5'-CGACATGATCGAGGATATTCAGGTGCG-3' for the primary reaction and 5'-CGAGAGCTCTTCACGCGCGACATGATCGA-3' for the nested reaction. 1.67 kb of DNA was isolated, and the sequence was clarified. In addition, 1 kb of the upstream region was isolated. The forward primers were designed based on the mouse Casp-9 promoter sequence draft data (UCSC Genome Bioinformatics Human Genome Project working draft, available at genome.ucsc.edu/): 5'-GACTGTACAGAGAAACCCTGTCTTG-3' for the primary reaction and 5'-AAGAACCGAGGGAAGAGGGCGTGGTGAATA-3' for the nested reaction. The reverse primers were the same as those of the 1.67-kb construct. Ultimately, 2.4 kb of DNA was isolated. The three deduced AP-4-binding sites were all submitted to MOTIF: Searching Protein and Nucleic Acid Sequence Motifs (available at www.motif.genome.jp).

Mouse Casp-9 Promoter Mutants and Promoter Activity—The 2.4-kb promoter region was inserted into the pCR2.1 vector (Invitrogen) by TA cloning and then digested with HindIII and NcoI restriction sites. The multiple cloning site of pCR2.1 has no NcoI site, but the mouse Casp-9 translational start codon was CCATGG. This fragment was cloned into pGL3-Basic at the cohesive ends and then used as a template to generate the AP-4-binding site point mutants and 5'-sequential deletion mutants. Seven AP-4-binding site point mutants were prepared: three of them had a one point mutation; three had two point mutations; and the last had three point mutations. The forward primers of the one-point mutants were 5'-ACCCTCCTCCCGAGTGTACATCCT-3', 5'-ACCTCATCAAGGAATAACTATATCCCTGTC-3', and 5'-ACCGTCCTCTTTACCCTGAACCATCCATCTC-3', and the reverse primers were 5'-CCAGATTTCCTGTTTGTAAATTCACCTTAT-3', 5'-CGCTGTGAGATCCCTGACACATGTCTATAT-3', and 5'-GACCTCTGGAAGAATAT-3', respectively. The underlined regions indicate the replaced bases that have been described in a previous study (13). First, one-point mutants were generated; then these constructs were used as templates for the two-point mutants; and one of the three two-point mutants were used as a template for the three-point mutant.

The 5'-sequential deletion mutants were 1.55, 1.37, 1.14, and 0.94 kb. The forward primers were 5'-ACCTCAGCTGCTCCTCCCGAGTGTACATCC-3', 5'-ATCAAGGAATAACTATATCCCTGTCAGGA-3', 5'-CCTCTTTACCCTGAACCATCCATCTC-3', and 5'-GTGTTCTTAACCGCTGAGCCATCTCTCCAG-3', respectively. The reverse primer was the same for the four constructs, 5'-CTGCGATCTAAGTAAGCTT-3'.

These constructs and pGL3-Basic were transfected with phRL (inner control DNA; Promega) into 2-257-20 cells by the DEAE-dextran method or into NIH/3T3 cells by jetPEI (Polyplus Transfection). The luciferase activities were measured after 48 h with the Dual-Luciferase reporter assay system (Promega).

Electrophoretic Mobility Shift Assay—2-257-20 cell nuclear extracts were purified with NE-PER^TM nuclear and cytoplasmic extraction reagent (Pierce). Each probe (double-strand DNA) was designed for each AP-4-binding site to be located in the center of the probe and labeled with ³²P. A negative control that could not bind to the AP-4-binding sites was also generated (13). Nuclear extract, poly(dI-dC), binding buffer (10 mM Tris-HCl (pH 7.5), 40 mM NaCl, 1 mM EDTA, 10% glycerol, 0.4% Nonidet P-40, and 4 mM dithiothreitol), and the ³²P-labeled probe were mixed on ice for 1 h to achieve the binding reaction. Both the competition and supershift tests were performed by incubating nuclear extract with a large number of unlabeled probes or anti-AP-4 antibody for 1 h before the binding reaction. Samples were electrophoresed on 4% polyacrylamide gel. The dried gels were exposed on an image plate and analyzed using a Fuji BAS2000 image analyzer.

Statistical Analysis—Differences were analyzed using the Mann-Whitney test. All data are expressed as the mean ± S.E., and a p value <0.05 was considered to be significant.

View larger version (36K): [in this window] [in a new window]   FIG. 1. Dexamethasone-induced cell death is suppressed in the AS AP-4 transfectants. A, AS AP-4 transfectants (AS3, AS15, AS23, and AS40) and vector-alone transfectants (V30 and V43) were treated with 1 µM dexamethasone, and viability was assessed by trypan blue staining after 24 and 48 h. B, mock and antisense AP-4 transfectants were incubated with 1 µM dexamethasone for 24 h, and the appearance of a sub-G1 fraction was analyzed after staining with propidium iodide. C, the mitochondrial membrane potential was determined after staining with 20 nM 3,3'-dihexyloxacarbocyanine iodide (DiOC6(3)).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Several vector-alone and AS AP-4 transfectants were established, and cell death was induced by treatment with 1 µM dexamethasone. At 0, 24, and 48 h, the cells were harvested, and cell viability was determined (Fig. 1A). All four AS AP-4 stable transfectants showed a reduced susceptibility to dexamethasone-induced apoptosis in comparison with the vector-alone transfectants, especially at the 24-h assessment. Sense AP-4 transfectants were also established and similarly investigated. An increase in cell death was observed at various doses of dexamethasone (data not shown).

The induction of apoptosis causes various alterations in cells. One of the typical events in apoptosis is the fragmentation of nuclear DNA, and this phenomenon can be detected as the appearance of subdiploid cells by flow cytometry. As shown in Fig. 1B, the frequency of subdiploid cells at 24 h was reduced in the AS AP-4 transfectants. Another typical event during apoptosis is the disruption of the mitochondrial transmembrane potential. This disruption was also reduced in the AS AP-4 transfectants (Fig. 1C).

Expression of the Factors Involved in the Apoptosis Pathway in the AP-4 Transfectants—Dexamethasone-induced apoptosis is mediated by the mitochondrial apoptosis pathway, and in various cells, the balance of repressive and promotive factors of the Bcl-2 family is known to influence the mitochondrial pathway (14). Bcl-2 expression is barely detectable in CD4⁺CD8⁺ thymocytes (15–17), and in this experiment, the expression of Bcl-x_L and Bax was examined (Fig. 2A). There were no big differences in their expression levels among the vector-alone and sense and AS AP-4 transfectants. Furthermore, we could not detect any differences in the mRNA expression of Bcl-x_L, Bak, Bax, and Bad in an RNase protection assay (data not shown).

Apaf-1 and Casp-9 are critical factors in the initiation of the cascade of cleavage and activation of other caspases, thus resulting in eventual cell death. The expression of Apaf-1 did not differ substantially among the AP-4 transfectants (Fig. 2B). In contrast, the expression of procaspase-9 was suppressed in the AS AP-4 transfectants (Fig. 2C). Furthermore, the kinetics of Casp-9 expression after treatment with 1 µM dexamethasone showed the appearance of the activated form of Casp-9 to be delayed (Fig. 2D). Subsequently, we evaluated the expression of Casp-9 mRNA by real-time PCR, and its expression was increased in sense AP-4 transfectants, but decreased in AS AP-4 transfectants (data not shown).

AP-4 and Promoter Activity of Casp-9—We isolated 2.4 kb of the upstream region of the mouse Casp-9 gene by the nested PCR method (Supplemental Fig. 1). Three AP-4-binding sites were presumed to be on the 5'-flanking region of Casp-9. To evaluate the relationship between these sites and the Casp-9 promoter activity, we prepared five deletion and seven point mutation constructs. They were inserted into the pGL3-Basic reporter gene vector containing firefly luciferase cDNA and then transfected into 2-257-20 cells. The control vector containing Renilla luciferase cDNA was cotransfected, and Dual-Luciferase reporter assay was performed.

A remarkable reduction in the promoter activity was observed when the two distal AP-4-binding sites at –1.55 and –1.38 kb were mutated (Fig. 3A). In addition, in the deletion constructs containing 1373- and 1140-bp promoter sequences, the promoter activities decreased remarkably (Fig. 3B). These results suggest that a negative cis-element exists between bases –1140 and –944. The experiments were repeated with NIH/3T3 cells (Fig. 3C). Again, the results clearly demonstrated the AP-4-binding sites at –1.55 and –1.38 kb to be important. Furthermore, the presence of a negative cis-element between bases –1140 and –944 was confirmed.

View larger version (28K): [in this window] [in a new window]   FIG. 2. Expression of Casp-9 is decreased in the AS AP-4 transfectants. The expression of AP-4, Bcl-xL, and Bax (A), Apaf-1 (B), and procaspase-9 (C) was assessed in mock (V30) and sense (S5 and S6) and AS (A15 and A23) AP-4 transfectants. The kinetics of Casp-9 expression after 1 µM dexamethasone treatment are shown (D).

Kinetics of AP-4 and Casp-9 Expression after Dexamethasone Treatment—To evaluate the physiological function of dexamethasone treatment and AP-4, the expression of AP-4 mRNA and protein was examined at various times after dexamethasone treatment. The expression of mRNA in 2-257-20 cells rapidly decreased, and a significant reduction was observed at 2 h (Fig. 5A). The down-regulation of AP-4 at the protein level was detected at 4 h (Fig. 5B). Following AP-4 down-regulation, the Casp-9 mRNA level also decreased after 2 h (Fig. 5C). To investigate whether the differences in the AP-4 levels in various transfectants correlated with both the level of Casp-9 and the induction of apoptosis, we treated three transfectants with 1 µM dexamethasone (Fig. 5D). The sense and AS AP-4 plasmids are driven by the cytomegalovirus promoter, and therefore, their expression would not be much influenced by dexamethasone treatment. In the sense AP-4 transfectant, down-regulation of Casp-9 was less because of the presence of AP-4, and the induction of apoptosis was accelerated. In contrast, in the antisense AP-4 transfectant, the low level of Casp-9 from the start delayed the onset of apoptosis. We next examined whether this series of events, dexamethasone/AP-4/Casp-9, is observed in fibroblast cells, which are resistant to dexamethasone-induced apoptosis. We examined the expression of AP-4 mRNA and protein in mouse L929 fibroblast cells (Fig. 6, A and B), and a significant down-regulation of mRNA was clearly observed after 6 h and also at the protein level. L929 cells themselves are not induced to undergo cell death by dexamethasone. However, if AP-4 expression is down-regulated by dexamethasone treatment, it is expected that L929 cells pretreated with dexamethasone would be resistant to subsequent treatment with apoptosis-inducing agents. This assumption was tested in Fig. 6 (C and Fig. D). As shown in Fig. 6C, L929 cells pretreated with dexamethasone for 48 h demonstrated a decrease in the expression of AP-4 and Casp-9, and this decrease was blocked in the presence of the steroid receptor antagonist RU-486. In this situation, the cells were further treated with the anticancer drug etoposide, which is reported to induce apoptosis via Casp-9 (18). Suppression of cell death was observed in the cells pretreated with dexamethasone (Fig. 6D).

View larger version (23K): [in this window] [in a new window]   FIG. 3. The promoter activity of Casp-9 involves the AP-4-binding sites. The promoter activities in point mutation constructs (A) and deletion constructs (B) of Casp-9 were assessed using the Dual-Luciferase reporter assay system. The constructs were inserted into the pGL3-Basic vector and then cotransfected into 2-257-20 cells with the phRL vector as an inner control. In A, the wild type (W.T.; no mutation) is taken as 100%. In B, the results are expressed as the -fold activity relative to that of the control pGL3-Basic vector (B). Point mutation and deletion constructs were transfected into NIH/3T3 cells, and their promoter activities are expressed as a percent of the wild type (no mutation) (C). k, kb; Luc, luciferase.

View larger version (55K): [in this window] [in a new window]   FIG. 4. The AP-4-binding site is directly involved in the Casp-9 promoter region. The 32P-labeled 31-bp oligonucleotide probes corresponding to three AP-4-binding sites were incubated with nuclear extracts from 2-257-20 cells. A, for the competition test, specific unlabeled probes were added up to 100-fold (x). In the fifth, tenth, and fifteenth lanes, a 100-fold excess of the negative probe was included (neg). B, the AP-4 specific antibody (Ab) was included in the supershift test. k, kb.

View larger version (30K): [in this window] [in a new window]   FIG. 5. Kinetics of the expression of AP-4 and Casp-9 after dexamethasone treatment in 2-257-20 cells. The expression of AP-4 at the protein (A) and mRNA (B) levels was assessed at the indicated hours after 1 µM dexamethasone treatment. The expression of Casp-9 was also assessed at the mRNA level after dexamethasone treatment (C). To investigate the correlation between the levels of AP-4 and Casp-9 and the induction of apoptosis, the AP-4 transfectants V43, S6, and AS15 were treated with 1 µM dexamethasone (Dex) (D). The expression of AP-4 and Casp-9 was assessed at 6 h, and the induction of apoptosis was evaluated as the appearance of the sub-G1 fraction at 17 h. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

View larger version (26K): [in this window] [in a new window]   FIG. 6. Dexamethasone treatment results in the down-regulation of AP-4 in L929 cells and causes the cells to prevent apoptosis induced by etoposide. L929 cells were treated with 1 µM dexamethasone, and the expression of AP-4 at the mRNA (A) and protein (B) levels was assessed at various hours. L929 cells were treated with 1 µM dexamethasone (Dex) and/or 10 µM RU-486 for 48 h (C). RU-486 was added to the cells 1 h before addition of dexamethasone. The expression of AP-4 and Casp-9 was assessed by Western blot analysis. L929 cells pretreated with () or without () dexamethasone for 48 h were further incubated in the presence of 10 or 30 µM etoposide (D). Cell viability was determined 48 h after addition of etoposide. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Until now, most experiments on the regulation of apoptosis have concentrated on the interaction of pro-apoptotic and contra-apoptotic factors. However, several studies have already reported on regulation via transcription factors such as the expression of Bax and Noxa by p53 (22, 23). The enhancer binding protein AP-4 is a transcription factor that activates both viral and cellular genes by binding to symmetrical DNA sequences (CAGCTG) (24). AP-4 has a HLH motif. Like other members of this family, the AP-4 HLH motif and the adjacent basic domain are necessary to confer site-specific DNA binding. However, unlike other HLH motif-containing proteins, AP-4 also contains two additional protein dimerization motifs consisting of leucine repeat elements LR1 and LR2. AP-4 binds the other leucine repeats to each other and thereby makes a dimer, but AP-4 is not considered to make a heterodimer. AP-4 has been reported to bind many promoters and enhancers of various genes (25–31). As an enhancer, AP-4 binds to the specific genes of pancreatic cells; for example, it is well known that the amylase-2A gene is regulated by AP-4 (26). In the field of immunology, AP-4 has been reported to be a ligand of immunoglobulin -chain E-box elements (30), whereas the AP-4-binding site of Fcgr2b, which is responsible for the negative regulation of the B cell antigen receptor, is deleted in autoimmunodeficient New Zealand Black mice (31). However, there has yet to be a report on AP-4 concerning apoptosis-related genes as far as we could determine.

In AS AP-4 transfectants, Casp-9 expression decreased at both the protein and mRNA levels. In contrast, it was a surprise that the increase in the expression of Casp-9 was small in the sense AP-4 transfectants. AP-4 is a ubiquitously expressed transcription factor and probably works to maintain the basic level of Casp-9. Therefore, AP-4 at more than a certain level would not much influence the expression of Casp-9, although the precise mechanism must await further investigations, including the possibility of the involvement of other transcription factors. We isolated the 5'-flanking region of Casp-9 and thus found three AP-4-binding sites to exist. Electrophoretic mobility shift assay showed these binding sequences to be specific for AP-4, and two distal binding sites (–1.55 and –1.38 kb) had a strong promoter activity. The sequence of the Casp-9 promoter region has already been described in the rat (32). We found four AP-4-binding sites in MOTIF; two of them were located at –1.52 and –1.35 kb, and these positions were very similar to those in mouse Casp-9. Although the expression of Casp-9 mRNA has been reported to double under hypoxic conditions, the transcriptional regulation of AP-4 has not yet been examined. It will be interesting to clarify whether Casp-9 is similarly regulated in the rat and human.

During an analysis of the promoter activity in deletion construct experiments, we found a negative cis-element to exist between bases –1140 and –944. Therefore, a decrease in the quantity of AP-4 causes a reduction in Casp-9 expression. Quite a similar situation has been demonstrated in the Fas antigen, in which a silencer exists between bases –1035 and –1008, and the presence of an enhancer just adjacent to the silencer between bases –1007 and –964 was reported (33–35).

In this study, we have focused our efforts on AP-4 and Casp-9. However, as determined by the kinetics of AP-4 and Casp-9 down-regulation (Fig. 5, A–C), there is a possibility that dexamethasone treatment induces other factors that are involved in Casp-9 down-regulation. This possibility will be investigated in the future experiments.

GC plays a major role in the attenuation of the inflammatory response. Steroid hormones can induce apoptosis in hematopoietic cells such as T lymphocytes, which are involved in the inflammation reaction (36). In contrast, GC has been reported recently to protect fibroblasts, hepatocytes, etc., which are vulnerable to the inflammation reaction, against apoptosis induced by other reagents (37–39). Furthermore, GC is frequently used as a co-treatment with chemotherapy or radiation therapy for cancer cells to reduce side effects (40). In contrast to the pro-apoptotic effect of GC in malignant lymphoid cells, GC also sometimes induces resistance to cancer therapy mediated-apoptosis in solid tumors. Recently, Herr et al. (41) reported that the expression of most pro-apoptotic factors, including CD95L, Trail, Casp-9, and Casp-8, is down-regulated in carcinomas pretreated with dexamethasone. They further demonstrated that the transfection of Casp-8 and Casp-9 cDNAs or proteins neutralizes resistance to dexamethasone-induced apoptosis. In this work, we have demonstrated that dexamethasone treatment induced the down-regulation of AP-4 in 2-257-20 thymic lymphoma cells and in L929 fibroblast cells, followed by the down-regulation of Casp-9. In addition, apoptosis was not induced in L929 cells by dexamethasone treatment itself, but dexamethasone-treated L929 cells were found to be resistant to apoptosis induced by subsequent treatment with etoposide. Based on our observations, we speculate that, in GC-sensitive cells, the expression level of AP-4 will influence the sensitivity to apoptosis. However, once the pro-apoptotic signals are triggered by GC, the subsequent down-regulation of AP-4 and Casp-9 will not be sufficient for the prosurvival response. In contrast, in GC-insensitive cells, the pretreatment with GC induces the down-regulation of Casp-9 and thus contributes to the resistance against subsequent treatment with apoptosis-inducing agents. We are currently investigating how the down-regulation of AP-4 is induced by GC and whether our findings are related to the attenuation of inflammation observed upon GC treatment.

	FOOTNOTES

 
^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains Supplemental Fig. 1.

To whom correspondence should be addressed. Tel.: 81-4-2995-1576; Fax: 81-4-2996-5197; E-mail: tadakuma{at}ndmc.ac.jp.

¹ The abbreviations used are: Casp, caspase; GC, glucocorticoid; AS, antisense; AP-4, activator protein-4; HLH, helix-loop-helix.

	ACKNOWLEDGMENTS

 
We thank Dr. N. Kondo for help in performing the electrophoretic mobility shift assay and N. Sakamoto for excellent secretarial work.

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TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 

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