The Promyelocytic Leukemia Protein Represses A20-mediated Transcription*

The promyelocytic leukemia (PML) protein is a tumor suppressor that is disrupted by the chromosomal translocation t(15;17), a consistent cytogenetic feature of acute promyelocytic leukemia. A role of PML in multiple pathways of apoptosis was conclusively demonstrated using PML−/− animal and cell culture models. In a previous study, we found that PML sensitizes tumor necrosis factor-induced apoptosis in tumor necrosis factor (TNF)-resistant U2OS cells. This finding helped to explain the mechanism of PML-induced apoptosis. The zinc finger protein A20 is a target gene of NFκB inducible by TNFα, and it is a potent inhibitor of TNF-induced apoptosis. In the this study, we demonstrated that PML is a transcriptional repressor of the A20 promoter and that PML represses A20 expression induced by TNFα. We showed that PML inhibits A20 transactivation through the NFκB site by interfering with its binding to the promoter. We also showed that stable overexpression of A20 inhibits apoptosis and caspase activation induced by PML/TNFα. The results of this study suggest that A20 is a downstream target of PML-induced apoptosis and supports a role of A20 in modulating cell death induced by PML/TNFα in TNF-resistant cells.

The promyelocytic leukemia (PML) protein is a tumor suppressor that is disrupted by the chromosomal translocation t(15;17), a consistent cytogenetic feature of acute promyelocytic leukemia. A role of PML in multiple pathways of apoptosis was conclusively demonstrated using PML ؊/؊ animal and cell culture models. In a previous study, we found that PML sensitizes tumor necrosis factor-induced apoptosis in tumor necrosis factor (TNF)-resistant U2OS cells. This finding helped to explain the mechanism of PML-induced apoptosis. The zinc finger protein A20 is a target gene of NFB inducible by TNF␣, and it is a potent inhibitor of TNF-induced apoptosis. In the this study, we demonstrated that PML is a transcriptional repressor of the A20 promoter and that PML represses A20 expression induced by TNF␣. We showed that PML inhibits A20 transactivation through the NFB site by interfering with its binding to the promoter. We also showed that stable overexpression of A20 inhibits apoptosis and caspase activation induced by PML/TNF␣. The results of this study suggest that A20 is a downstream target of PML-induced apoptosis and supports a role of A20 in modulating cell death induced by PML/TNF␣ in TNF-resistant cells.
The disruption of the promyelocytic leukemia (PML) 1 gene by the t(15;17) chromosomal translocation is believed to play an important role in the pathogenesis of acute promyelocytic leukemia (APL) (1). PML is a multifunctional Ring finger protein normally localized within the nucleus as a macromolecular structure in a nuclear speckled pattern designated PML nuclear body (NB) or PML oncogenic domain (2). In APL cells, the normal PML NBs are disrupted as a result of forming heterodimer with the fusion protein PML-retinoic acid receptor ␣ to form a nuclear microspeckled pattern. Interestingly, alltrans-retinoic acid treatment of APL patients induced dramatic differentiation of the leukemia blasts associated with rapid degradation of PML-retinoic acid receptor ␣ and reorganization of the normal PML NB that presumably restore normal PML function (2). PML has been shown to be a cellular growth and tumor suppressor (3)(4)(5) by acting as a regulator of apoptosis (6,7) and cell-cycle progression (4,5). The expression of PML suppresses transformation by cooperative oncogenes of mouse embryo fibroblasts and inhibits the neu-induced growth and tumorigenicity of NIH3T3 cells and many cancer cell lines (1,2,8). Wang et al. (6) recently showed that PML plays a critical role in multiple pathways of apoptosis. Studies using a PML knock-out animal model demonstrated that PML is essential for apoptosis induced by TNF␣, ceramide, ␥-irradiation, interferons, and Fas (6). However, the mechanism by which PML induces apoptosis remains obscure. The PML protein is normally modified by SUMO-1, a small ubiquitin-like protein, at three different sites (9). SUMO-1 modification plays an essential role in the formation of PML NB and in its function in transcriptional regulation and apoptosis (10).
PML also functions as a transcriptional regulator (for review see Refs. 11 and 12). The transcription coactivator CREBbinding protein, a histone acetyltransferase, interacts with PML and colocalizes in the PML NB, suggesting a role of PML in mediating transcription (13,14). The highly acetylated chromatin presumably associated with actively transcribing genes was also found to be associated with PML NB (14). Moreover, PML has been shown to upregulate the transcription of genes related to the major histocompatibility complex (15), GATA-2 (16), AP-1 (17), and p53-mediated transcriptions (18 -20). On the other hand, when PML was fused downstream of the GAL4 DNA binding domain, it repressed GAL4-mediated transcription through a mechanism that is sensitive to trichostatin A, a specific inhibitor of the transcription corepressor histone deacetylases (21,22). PML has been shown to interact with histone deacetylases (22,23) and repress transcription by deacetylation of the target promoter (22). Another mechanism of transcription repression by PML was also reported recently. It was found that PML interacts directly with transcription factor Sp1 and interferes with its ability to bind DNA, leading to repression of the Sp1-dependent transcription of epidermal growth factor receptor promoter (24).
A20 is a target gene of NFB and was originally identified as a zinc finger protein induced by treatment with TNF␣ (25). A20 was characterized as a potent inhibitor of TNF-induced apoptosis (26). The overexpression of A20 significantly downregulated NFB activation and inhibited the expression of NFB target genes, indicating that A20 is a potent inhibitor of NFB (27,28). Together, these findings suggest that A20 plays a role as a negative feedback regulator of NFB activation. The recent finding that A20-deficient mice failed to regulate TNFinduced NFB activation and apoptosis supports this notion (29). This finding implies that A20 plays a critical role in mediating apoptosis and inflammation by terminating the TNF-induced NFB responses.
To further understand the mechanism of how PML induces apoptosis, our present study shows that PML significantly inhibits the expression of A20, a potent repressor of TNFinduced apoptosis. PML represses the A20 promoter induced by TNF␣ and phorbol 12-myristate 13-acetate (PMA) through the NFB site. Our study further shows that A20 inhibits apoptosis induced by PML/TNF␣ and explains the mechanism by which PML induces apoptosis by sensitizing the TNF death receptor pathway.

EXPERIMENTAL PROCEDURES
Plasmids Construction-The inducible expression plasmid pMEP4/ PML was constructed by subcloning the full-length PML cDNA (PML3) into the NotI/XhoI sites of the pMEP4 vector (Invitrogen). pMEP4/HA-A20 was constructed by cloning the polymerase chain reaction-amplified full-length A20 cDNA into the EcoRI site of the pCruz/HA vector to generate pCruz/HA-A20. The HA-A20 fragment was then excised by XhoI/BglII and subcloned into the pMEP4 vector. The A20 promoterluciferase reporter construct A20PR-Luc was generated by cloning the PCR-amplified fragment of the A20 promoter using the upstream primer 5Ј-CCCAGCCCGACCCAGAGAGTCACGTGAC-3Ј and the downstream primer 5Ј-CCCAGACTGCGCAGTCTGCTTTGCCCCG-3Ј and inserting them into the pGL3 vector (Promega, Madison, WI). Correct DNA sequence of all plasmids was confirmed by direct DNA sequencing.
Transfection and Luciferase Reporter Assay-Cells were cultured to semiconfluence and transfected with the expression plasmids using FuGENE 6 transfection reagent (Roche Diagnostics). Luciferase activity was determined using the luciferase reporter assay according to the manufacturer's protocol (Promega).
Generation of Stable Cell Lines-U2OS cells were transfected with each of the following plasmids: pMEP4 (negative control), pMEP4/HA-A20, and pMEP4/PML with FuGENE 6 reagent. Cells were then selected with hygromycin (200 g/ml) for 10 days. Pools of hygromycinresistant stable clones were selected. Inducible expression of the respective proteins in these stable cell lines was determined by induction with CdSO 4 (5 M) for 20 h followed by immunofluorescent staining and Western blot analysis. Cell death was examined by trypan blue exclusion assay.
RNA Preparation and cDNA Synthesis-Total RNA was prepared from cells using RNeasy mini-kit (Qiagen). cDNA was synthesized from 4 g of total RNA using the Superscript preamplification system obtained from Invitrogen.
Electrophoretic Mobility Shift Assay-The in vitro-translated PML or p65 proteins were synthesized by the TnT-coupled wheat germ translation system (Promega). Nuclear extracts were prepared from U2OS stable cell lines or cells treated with TNF␣ (20 ng/ml) for 1 h. The A20 promoter NFB probe was prepared by annealing the oligonucleotides 5Ј-GACTTTGGAAAGTCCCGTGGAAATCCCCGGG-3Ј and 5Ј-GGCCC-GGGGATTTCCACGGGACTTTCCAAAGT-3Ј, and the 3Ј-recessive ends were labeled with Klenow fragment fill-in reaction. Binding reactions contained 5 g of nuclear extracts, 1 g of poly(dI-dC), and 1 ng of NFB probe (1 ϫ 10 5 cpm) in 20 l of KCl binding buffer (10% glycerol, 1 mM EDTA, 5 mM KCl, 5 mM dithiothreitol, and 20 mM Tris-HCl, pH 8.0). The reaction was incubated for 20 min at room temperature and then resolved in a 5% polyacrylamide gel in Tris-glycine buffer (50 mM Tris, 0.4 M glycine, and 2 mM EDTA, pH 8.5). For competition or supershift assays, 50 ng of unlabeled probe or 1 g of anti-p65 monoclonal antibody, respectively, was added to the binding reactions.

PML Inhibits Upregulation of A20 Induced by TNF␣-Our
preliminary study showed that PML significantly sensitized TNF-induced apoptosis in the TNF-resistant cell line U2O. To understand the effects of PML on the TNF-induced apoptotic pathway, we investigated the effect of PML on the expression of A20 in the osteosarcoma cell line U2OS. We found that TNF␣ induces a significant increase in A1 and A20 expression in these cells but not c-FLIP (Fig. 1a). To examine the effects of PML on the expression of A20, stable clones of inducible PML in U2OS cells (U2OS/PML) and the control U2OS cells (pMEP4/U2OS) were established. These cells were treated with 5 M cadmium sulfate to induce PML expression followed by the treatment of TNF␣. The results of this study showed that expression of PML significantly repressed the expression of A20 induced by TNF␣ (Fig. 1b). No such effect on A20 expres-FIG. 1. Repression of A20 expression by PML in U2OS cells. a, A1 and A20 are inducible by TNF␣ in U2OS cells. U2OS cells were starved for 2 h in Dulbecco's modified Eagle's medium with 05% serum and then treated (or untreated) with 20 ng/ml TNF␣ for 2 h. Total RNA in these cells was isolated, and PCR was performed for 22 cycles with specific primer pairs. b, PML inhibits upregulation of A20 induced by TNF␣. Stable cell lines pMEP4/U2OS (control) and PML/ U2OS were treated (or untreated) with 5 M Cd 2ϩ for 20 h to induce PML expression and then cultured in Dulbecco's modified Eagle's medium with 0.5% serum in the presence or absence of 20 ng/ml TNF␣ for another 6 h. Total protein was isolated and subjected to Western blotting using specific antibodies against A20, PML, and ␣-tubulin. sion was found in the control cell line pMEP4/U2OS or in the PML/U2OS cells if Cd 2ϩ was not included in the culture medium to induce PML expression. This study suggests that PML is a specific inhibitor of A20 expression.
PML Represses the Promoter Activity of A20 Induced by TNF␣-To continue to investigate the effects of PML on A20 expression, the A20 promoter-luciferase construct (pA20PR-Luc) was created and used in a series of cotransfection experiments to investigate whether PML affects the transactivation of the A20 promoter. We first investigated the effects of PML expression on TNF␣-induced activation of the A20 promoter. In the cotransfection experiments, U2OS cells were transiently transfected with pcDNA3/PML or pcDNA3 and pA20PR-Luc. After 24 h, cells were treated with TNF␣ for 2 and 4 h. The results of this study presented in Fig. 2a demonstrate that the presence of PML significantly represses A20 promoter activity. A similar experiment was performed using the stable PML/ U2OS cells and the control pMEP4/U2OS cells transfected with pA20PR-Luc followed by treatment with PMA. This experiment further showed that PML significantly represses transactivation of the A20 promoter in PML/U2OS cells treated with Cd 2ϩ (Fig. 2b). No inhibitory effects of the A20 promoter were found in pMEP4/U2OS control and PML/U2OS cells not treated with Cd 2ϩ . These results demonstrated that PML represses the promoter activity of A20 induced by treatment with TNF␣ and PMA.
PML Inhibits Transcriptional Activation of the A20 Promoter-A20 is a downstream target gene of NFB inducible by TNF treatment. Therefore, we speculate that PML represses transcription of A20 through the inhibition of the NFB function. To test this hypothesis, a series of cotransfection experiments was performed with pA20PR-Luc in the presence of RelA/p65 expression plasmid and increasing concentrations of pcDNA/PML. The results presented in Fig. 3 demonstrate that the presence of RelA/p65 dramatically activated the promoter activity of A20. Cotransfection with pcDNA3/PML at increasing concentrations significantly repressed A20 promoter activity in a dose-dependent manner. This result indicates that PML represses the RelA/p65-dependent transactivation of the A20 promoter.
PML Interferes with NFB Binding to the A20 Promoter-The study described above suggests that PML represses RelAdependent transactivation by interfering with NFB binding to the A20 promoter. To further understand how PML represses transactivation of the A20 promoter through NFB, electrophoretic mobility shift assay was performed using nuclear protein isolated from stable inducible cell lines pMEP4/U2OS and PML/U2OS treated or untreated with TNF␣. In this assay, electrophoretic mobility shift assay was performed using the DNA sequence derived from the NFB site of the A20 promoter. The results presented in Fig. 4 demonstrate that the expression of PML inhibited the binding of p65/RelA to the consensus sequences ( lanes 5 and 6). Together, these studies suggest that PML inhibits NFB binding to the promoter of A20 and consequently represses its transactivation.
Overexpression of A20 Inhibits Apoptosis Induced by PML/ TNF␣-It is clear that A20 is a target gene of NFB inducible by TNF␣ (25). There is also an abundance of evidence to suggest that A20 acts as a potent inhibitor of NFB (27)(28)(29). This raises the possibility that A20 plays an important role as a negative feedback regulator of NFB functions. To investigate the possible functional interaction between PML and A20, we investigated whether the expression of A20 has any significant effects on PML/TNF␣-induced apoptosis in U2OS cells. To address this question, a pool of inducible stable cell lines of A20 in U2OS cells driven by the metallothionine promoter was established. The expression of A20 can be induced by a low concentration of Cd 2ϩ (Fig. 5a). A control cell line (pMEP4/U2OS) transfected with the empty vector alone was also established. The results presented in Fig. 5b show that a significant degree of cell death was induced after a 24-h infection with recombinant PML adenovirus (Ad-PML) and 8-h treatment with TNF␣. However, PML/TNF␣-induced cell death was significantly reduced in cells overexpressing A20, indicating that A20 inhibited apoptosis induced by PML/TNF␣. The results shown in Fig. 5c demonstrate that control pMEP4/U2OS cells treated with PML/TNF␣ significantly activated procaspase-7; however, in cells overexpressing A20, procaspase-7 activation was significantly reduced. Thus, this study demonstrated that A20 inhibits cell death induced by PML/TNF␣ in TNF-resistant cells. DISCUSSION PML was originally cloned and characterized for its involvement in the nonrandom chromosomal translocation t (15;17), which occurs in ϳ99% of APL cases. The importance of PML in maintaining normal cellular function was unambiguously demonstrated by its essential role in multiple pathways of apoptosis (4,5) and cell cycle progression (3,6,7). PML and PML NBs are induced by interferons and inflammatory stimuli (31)(32)(33), indicating that they have a role in response to inflammation and viral infection. PML is a cell growth and tumor suppressor; however, the mechanisms by which PML exerts its tumor sup-FIG. 2. PML inhibits A20 promoter activity induced by TNF␣ and PMA. a, U2OS cells were cotransfected with A20PR-Luc reporter (0.2 g) and pCDNA3/PML (0.8 g) or pCDNA3 (0.58 g) in 12-well plates. After 24 h, cells were treated with 20 ng/ml TNF␣ for the indicated times. Total proteins were isolated, and luciferase activity was determined using the luciferase assay kit (Promega). b, PML/U2OS and pMEP4/U2OS stable cells induced or uninduced with 5 M Cd 2ϩ were transfected with A20PR-Luc reporter plasmid. After 24 h, cells were treated with 10 ng/ml PMA for the indicated times. Luciferase activity was determined as described above. The expression plasmid pCMV/␤-galactosidase was included in each transfection assay to normalize transfection efficiencies.
pressor function remains unclear. In MCF-7 cells, the overexpression of PML induced G 1 cell-cycle arrest associated with an upregulation of p21, p53, cyclin D, and hypophosphorylation of the Rb (6). Unphosphorylated Rb binds the E2F transcription factor and limits its ability to activate the transcription of genes essential for the G 1 to S transition (34).
PML does not bind DNA directly; however, it plays a role in transcriptional regulation through its association with the transcription coactivator CREB-binding protein and interacts with the transcription corepressor histone deacetylases (13,14,22). PML activates transcription by binding and sequestering the negative regulator, Daxx (35)(36)(37), interacting with p53 (18 -20) and recruiting p53 to the PML NB to enhance transactivation of p53 target genes. PML also represses transcription by interacting with transcription factor Sp1 and interfering with its binding to the epidermal growth factor receptor promoter (24). The results presented in Fig. 4 suggest a similar mechanism of transcriptional repression by PML. In this case, PML represses transcription of the A20 gene by interfering with NFB binding to the promoter. It is not clear why and how PML is involved in these two opposite functions in transcriptional regulation. Recent studies suggest that PML NB may serve as the storage site of important cellular regulatory proteins. Increased expression of PML may sequester these protein factors to the PML NBs and consequently limit their normal functional roles as a transcription activators or repressors.
Although a role of PML in multiple pathways of apoptosis has been clearly established, the molecular mechanisms remain unclear. PML is required for Daxx-induced apoptosis in mouse splenocytes. In the absence of PML, cellular localization of Daxx was altered, and its proapoptotic function was impaired (36). In these cells, the ability of Daxx to sensitize Fas-induced apoptosis was abrogated (36). Daxx was also shown to specifically enhance Fas-induced apoptosis (38) in human cells by association with PML NB through a mechanism that involved the activation of caspase 8 and caspase 3; however, it is not known what role PML plays in TNF-induced apoptosis.
The results presented in this report support the novel mechanism of PML-induced apoptosis in inhibiting the expression of A20, a potent inhibitor of TNF-induced apoptosis. This study showed that PML represses the promoter activity of A20 induced by treatment with TNF␣ and PMA. PML inhibits transactivation of the A20 promoter by interfering with NFB binding to its consensus cis-acting element. This finding was further supported by the study showing that stable overexpres- FIG. 3. PML inhibits A20 promoter activity through NFB sites. U2OS cells were cotransfected with A20PR-Luc reporter (0.2 g), pCDNA3/p65 (0.05 g), and the indicated amount of pCDNA3/PML in 12-well plates. An equal molar of final DNA concentration in each cotransfection assay was adjusted by the addition of pcDNA3 plasmid. After 24 h, total protein was isolated, and luciferase activity was determined. The expression plasmid pCMV/␤-galactosidase was included in each transfection to normalize transfection efficiencies.
FIG. 4. PML interferes with NFB binding to A20 promoter. Nuclear extracts were isolated from the inducible stable cell lines pMEP4/U2OS and PML/U2OS pretreated with 5 M Cd 2ϩ for 20 h and activated with TNF␣ (20 ng/ml) for 20 min. electrophoretic mobility shift assay was performed using NFB probe derived from the A20 promoter. SS, supershift. sion of A20 significantly inhibited apoptosis induced by the combined effects of PML and TNF␣ (Fig. 5). This study indicates that PML sensitizes TNF␣-induced cell death by repressing the expression of A20 and inhibits its anti-apoptotic function.
A20 is a novel zinc finger protein first described as an early responsive gene inducible by TNF treatment (25). It was originally characterized as an inhibitor of TNF-induced apoptosis (26). A20 is also a potent inhibitor of NFB activation induced by not only TNF but also by interleukin-1, lipopolysaccharide, PMA, and hydrogen peroxide (39). This inhibitory effect may be mediated through its interaction with tumor necrosis factor receptor-associated factors (40) or other A20 binding proteins (41). Interestingly, the expression of A20 is controlled by NFB, suggesting that it is involved in a negative feedback mechanism in regulating NFB. NFB is an important transcription factor that regulates the expression of many mediators of inflammation (42) and anti-apoptotic proteins involved in the survival pathway (43,44). The finding that PML represses the transcription of the A20 promoter by interfering with NFB binding is interesting. It will be important to further investigate the mechanism by which PML inhibits the binding of NFB to its target DNA sequence and to examine whether the two proteins are functionally associated in vivo.