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• W1999535314 abstract "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 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. promyelocytic leukemia acute promyelocytic leukemia nuclear body tumor necrosis factor α phorbol 12-myristate 13-acetate luciferase hemagglutinin 1,4-piperazinediethanesulfonic acid cAMP-response element-binding protein 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) (1Mu Z.M. Chin K.V. Liu J.H. Lozano G. Chang K.S. Mol. Cell. Biol. 1994; 14: 6858-6867Crossref PubMed Scopus (294) Google Scholar). 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 (2Melnick A. Licht J.D. Blood. 1999; 93: 3167-3215Crossref PubMed Google Scholar). In APL cells, the normal PML NBs are disrupted as a result of forming heterodimer with the fusion protein PML-retinoic acid receptor α to form a nuclear microspeckled pattern. Interestingly, all-trans-retinoic acid treatment of APL patients induced dramatic differentiation of the leukemia blasts associated with rapid degradation of PML-retinoic acid receptor α and reorganization of the normal PML NB that presumably restore normal PML function (2Melnick A. Licht J.D. Blood. 1999; 93: 3167-3215Crossref PubMed Google Scholar). PML has been shown to be a cellular growth and tumor suppressor (3Wang Z.G. Delva L. Gaboli M. Rivi R. Giorgio M. Gordon-Cardo C. Grosweld F. Pandolfi P.P. Science. 1998; 279: 1547-1551Crossref PubMed Scopus (454) Google Scholar, 4Le X.F. Vallian S., Mu, Z.M. Hung M.C. Chang K.S. Oncogene. 1998; 16: 1839-1849Crossref PubMed Scopus (94) Google Scholar, 5Mu Z.M., Le, X.F. Vallian S. Glassman A.B. Chang K.S. Carcinogenesis. 1997; 18: 2063-2069Crossref PubMed Scopus (73) Google Scholar) by acting as a regulator of apoptosis (6Wang Z.G. Ruggero D. Ronchetti S. Zhong S. Gaboli M. Rivi R. Pandolfi P.P. Nat. Genet. 1998; 20: 266-272Crossref PubMed Scopus (99) Google Scholar, 7Quignon F., De Bels F. Koken M. Feunteun J. Ameisen J.C. de The H. Nat. Genet. 1998; 20: 259-265Crossref PubMed Scopus (340) Google Scholar) and cell-cycle progression (4Le X.F. Vallian S., Mu, Z.M. Hung M.C. Chang K.S. Oncogene. 1998; 16: 1839-1849Crossref PubMed Scopus (94) Google Scholar, 5Mu Z.M., Le, X.F. Vallian S. Glassman A.B. Chang K.S. Carcinogenesis. 1997; 18: 2063-2069Crossref PubMed Scopus (73) Google Scholar). The expression of PML suppresses transformation by cooperative oncogenes of mouse embryo fibroblasts and inhibits theneu-induced growth and tumorigenicity of NIH3T3 cells and many cancer cell lines (1Mu Z.M. Chin K.V. Liu J.H. Lozano G. Chang K.S. Mol. Cell. Biol. 1994; 14: 6858-6867Crossref PubMed Scopus (294) Google Scholar, 2Melnick A. Licht J.D. Blood. 1999; 93: 3167-3215Crossref PubMed Google Scholar, 8Liu J.H., Mu, Z.M. Chang K.S. J. Exp. Med. 1995; 181: 1965-1973Crossref PubMed Scopus (99) Google Scholar). Wang et al. (6Wang Z.G. Ruggero D. Ronchetti S. Zhong S. Gaboli M. Rivi R. Pandolfi P.P. Nat. Genet. 1998; 20: 266-272Crossref PubMed Scopus (99) Google Scholar) 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 TNFα, ceramide, γ-irradiation, interferons, and Fas (6Wang Z.G. Ruggero D. Ronchetti S. Zhong S. Gaboli M. Rivi R. Pandolfi P.P. Nat. Genet. 1998; 20: 266-272Crossref PubMed Scopus (99) Google Scholar). 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 (9Kamitani T. Kito K. Nyuyen H.P. Wada H. Fukuda-Kamitani T. Yeh E.T. J. Biol. Chem. 1998; 273: 26675-26682Abstract Full Text Full Text PDF PubMed Scopus (274) Google Scholar). SUMO-1 modification plays an essential role in the formation of PML NB and in its function in transcriptional regulation and apoptosis (10Seeler J.-S. Dejean A. Oncogene. 2001; 20: 7243-7249Crossref PubMed Scopus (136) Google Scholar). PML also functions as a transcriptional regulator (for review see Refs.11Lin R.J. Sternsdorf T. Tini M. Evans R.M. Oncogene. 2001; 20: 7204-7215Crossref PubMed Scopus (151) Google Scholar and 12Zhong S. Salomoni P. Pandolfi P.P. Nat. Cell Biol. 2000; 2: E85-E90Crossref PubMed Scopus (490) Google Scholar). The transcription coactivator CREB-binding protein, a histone acetyltransferase, interacts with PML and colocalizes in the PML NB, suggesting a role of PML in mediating transcription (13LaMorte V.J. Dyck J.A. Ochs R.L. Evans R.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4991-4996Crossref PubMed Scopus (222) Google Scholar, 14Boisvert F.-M. Kruhlak M.J. Box A.K. Hendzel M.J. J. Cell Biol. 2001; 152: 1099-1106Crossref PubMed Scopus (129) Google Scholar). The highly acetylated chromatin presumably associated with actively transcribing genes was also found to be associated with PML NB (14Boisvert F.-M. Kruhlak M.J. Box A.K. Hendzel M.J. J. Cell Biol. 2001; 152: 1099-1106Crossref PubMed Scopus (129) Google Scholar). Moreover, PML has been shown to upregulate the transcription of genes related to the major histocompatibility complex (15Zheng P. Guo Y. Niu Q. Levy D.E. Dyck J.A., Lu, S. Sheiman L.A. Liu Y. Nature. 1998; 396: 373-376Crossref PubMed Scopus (143) Google Scholar), GATA-2 (16Tsuzuki S. Towatari M. Saito H. Enver T. Mol. Cell. Biol. 2000; 20: 6276-6286Crossref PubMed Scopus (50) Google Scholar), AP-1 (17Vallian S. Gaken A.J. Gingold E.B. Kouzarides T. Chang K.S. Farzaneh F. Oncogene. 1998; 90: 3265-3269Google Scholar), and p53-mediated transcriptions (18Fogal V. Gostissa M. Sandy P. Zacchi P. Sternsdorf T. Jensen K. Pandolfi P.P. Will H. Schneider C. DelSal G. EMBO J. 2000; 22: 6186-6195Google Scholar, 19Guo A. Salomoni P. Luo J. Shih A. Zhong S., Gu, W. Pandolfi P.P. Nat. Cell Biol. 2000; 2: 730-736Crossref PubMed Scopus (387) Google Scholar, 20Pearson, M., and Pelicci, P. G. Oncogene 20, 7250–7256Google Scholar). 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 (21Vallian S. Gaken J.A. Trayner I.D. Gingold E.B. Kourizarides T. Chang K.S. Farzaneh F. Exp. Cell Res. 1997; 237: 371-382Crossref PubMed Scopus (41) Google Scholar, 22Wu W.S. Vallian S. Seto E. Yang W.M. Edmondson D. Roth S. Chang K.S. Mol. Cell. Biol. 2001; 21: 2259-2268Crossref PubMed Scopus (129) Google Scholar). PML has been shown to interact with histone deacetylases (22Wu W.S. Vallian S. Seto E. Yang W.M. Edmondson D. Roth S. Chang K.S. Mol. Cell. Biol. 2001; 21: 2259-2268Crossref PubMed Scopus (129) Google Scholar, 23Khan M.M. Nomura T. Kim H. Kaul S.C. Wadhwa R. Shinagawa T. Ichikawa-Iwata E. Zhong S. Pandolfi P.P. Ishii S. Mol. Cell. 2001; 7: 1233-1243Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar) and repress transcription by deacetylation of the target promoter (22Wu W.S. Vallian S. Seto E. Yang W.M. Edmondson D. Roth S. Chang K.S. Mol. Cell. Biol. 2001; 21: 2259-2268Crossref PubMed Scopus (129) Google Scholar). 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 (24Vallian S. Chin K.V. Chang K.S. Mol. Cell. Biol. 1998; 18: 7147-7156Crossref PubMed Scopus (102) Google Scholar). A20 is a target gene of NFκB and was originally identified as a zinc finger protein induced by treatment with TNFα (25Opipari A.W. Boguski M.S. Dixit V.M. J. Biol. Chem. 1990; 265: 14705-14708Abstract Full Text PDF PubMed Google Scholar). A20 was characterized as a potent inhibitor of TNF-induced apoptosis (26Jaattela M. Mouritzen H. Elling F. Bastholm L. J. Immunol. 1996; 156: 1166-1173PubMed Google Scholar). The overexpression of A20 significantly downregulated NFκB activation and inhibited the expression of NFκB target genes, indicating that A20 is a potent inhibitor of NFκB (27Cooper J.T. Stroka D.M. Brostjan C. Palmetshofer A. Bach F.H. Ferran C. J. Biol. Chem. 1996; 271: 18068-18073Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 28Ferran C. Stroka D.M. Badrichani A.Z. Cooper J.T. Wrighton C.J. Soares M. Grey S.T. Bach F.H. Blood. 1998; 91: 2249-2258Crossref PubMed Google Scholar). Together, these findings suggest that A20 plays a role as a negative feedback regulator of NFκB activation. The recent finding that A20-deficient mice failed to regulate TNF-induced NFκB activation and apoptosis supports this notion (29Lee E.G. Boone D.L. Chai S. Libby S.L. Chien M. Lodolce J.P. Ma A. Science. 2000; 289: 2350-2354Crossref PubMed Scopus (1189) Google Scholar). This finding implies that A20 plays a critical role in mediating apoptosis and inflammation by terminating the TNF-induced NFκB 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 TNF-induced apoptosis. PML represses the A20 promoter induced by TNFα and phorbol 12-myristate 13-acetate (PMA) through the NFκB site. Our study further shows that A20 inhibits apoptosis induced by PML/TNFα and explains the mechanism by which PML induces apoptosis by sensitizing the TNF death receptor pathway. 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 theEcoRI site of the pCruz/HA vector to generate pCruz/HA-A20. The HA-A20 fragment was then excised byXhoI/BglII and subcloned into the pMEP4 vector. The A20 promoter-luciferase reporter construct A20PR-Luc was generated by cloning the PCR-amplified fragment of the A20 promoter using the upstream primer 5′-CCCAGCCCGACCCAGAGAGTCACGTGAC-3′ and the downstream primer 5′-CCCAGACTGCGCAGTCTGCTTTGCCCCG-3′ and inserting them into the pGL3 vector (Promega, Madison, WI). Correct DNA sequence of all plasmids was confirmed by direct DNA sequencing. The U2OS cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Anti-HA monoclonal antibody was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Anti-caspase-7 antibody (number 66871A) was purchased from PharMingen. Anti-A20 antibody (number 40901) was obtained from Active & Motif, Inc. (Carlsbad, CA). 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). 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 μg/ml) for 10 days. Pools of hygromycin-resistant stable clones were selected. Inducible expression of the respective proteins in these stable cell lines was determined by induction with CdSO4 (5 μm) for 20 h followed by immunofluorescent staining and Western blot analysis. Cell death was examined by trypan blue exclusion assay. Total RNA was prepared from cells using RNeasy mini-kit (Qiagen). cDNA was synthesized from 4 μg of total RNA using the Superscript preamplification system obtained from Invitrogen. Cultured cells were washed twice with cold phosphate-buffered saline and resuspended in ice-cold digitonin extraction buffer (10 mm PIPES, pH 6.8, 0.015% (w/v) digitonin, 300 mm NaCl, 3 mmMgCl2, 5 mm EDTA, and 1 mmphenylmethylsulfonyl fluoride). Cells were permeabilized for 10 min, assessed by trypan blue exclusion assay, and centrifuged for 5 min at 480 × g at 4 °C. The supernatant contains the cytoplasmic protein. The digitonin-insoluble pellet was resuspended in ice-cold Triton X-100 extraction buffer (10 mm PIPES, pH 7.4, 0.5% (v/v) Triton X-100, 300 mm sucrose, 100 mm NaCl, 3 mm MgCl2, 5 mm EDTA, and 1 mm phenylmethylsulfonyl fluoride) and incubated on ice for 30 min. The nuclei were pelleted by centrifugation for 10 min at 5000 × g and resuspended in nuclear extraction buffer (50 mm PIPES, pH 7.5, 400 mm NaCl, 1 mm EDTA, 1 mm EGTA, 1% (v/v) Triton X-100, 0.5% (v/v) Nonidet P-40, and 10% (v/v) glycerol). The nuclear mixture was incubated for 30 min on ice and then centrifuged for 5 min (6780 × g) at 4 °C. The supernatant contains the nuclear proteins. 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 TNFα (20 ng/ml) for 1 h. The A20 promoter NFκB probe was prepared by annealing the oligonucleotides 5′-GACTTTGGAAAGTCCCGTGGAAATCCCCGGG-3′ and 5′-GGCCCGGGGATTTCCACGGGACTTTCCAAAGT-3′, and the 3′-recessive ends were labeled with Klenow fragment fill-in reaction. Binding reactions contained 5 μg of nuclear extracts, 1 μg of poly(dI-dC), and 1 ng of NFκB probe (1 × 105 cpm) in 20 μl of KCl binding buffer (10% glycerol, 1 mm EDTA, 5 mmKCl, 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 mmEDTA, pH 8.5). For competition or supershift assays, 50 ng of unlabeled probe or 1 μg of anti-p65 monoclonal antibody, respectively, was added to the binding reactions. 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 TNFα 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 μm cadmium sulfate to induce PML expression followed by the treatment of TNFα. The results of this study showed that expression of PML significantly repressed the expression of A20 induced by TNFα (Fig. 1b). No such effect on A20 expression was found in the control cell line pMEP4/U2OS or in the PML/U2OS cells if Cd2+ was not included in the culture medium to induce PML expression. This study suggests that PML is a specific inhibitor of A20 expression. 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 TNFα-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 TNFα for 2 and 4 h. The results of this study presented in Fig. 2ademonstrate 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 Cd2+ (Fig.2b). No inhibitory effects of the A20 promoter were found in pMEP4/U2OS control and PML/U2OS cells not treated with Cd2+. These results demonstrated that PML represses the promoter activity of A20 induced by treatment with TNFα and PMA. A20 is a downstream target gene of NFκB inducible by TNF treatment. Therefore, we speculate that PML represses transcription of A20 through the inhibition of the NFκB 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. The study described above suggests that PML represses RelA-dependent transactivation by interfering with NFκB binding to the A20 promoter. To further understand how PML represses transactivation of the A20 promoter through NFκB, electrophoretic mobility shift assay was performed using nuclear protein isolated from stable inducible cell lines pMEP4/U2OS and PML/U2OS treated or untreated with TNFα. In this assay, electrophoretic mobility shift assay was performed using the DNA sequence derived from the NFκB 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 NFκB binding to the promoter of A20 and consequently represses its transactivation. It is clear that A20 is a target gene of NFκB inducible by TNFα (25Opipari A.W. Boguski M.S. Dixit V.M. J. Biol. Chem. 1990; 265: 14705-14708Abstract Full Text PDF PubMed Google Scholar). There is also an abundance of evidence to suggest that A20 acts as a potent inhibitor of NFκB (27Cooper J.T. Stroka D.M. Brostjan C. Palmetshofer A. Bach F.H. Ferran C. J. Biol. Chem. 1996; 271: 18068-18073Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 28Ferran C. Stroka D.M. Badrichani A.Z. Cooper J.T. Wrighton C.J. Soares M. Grey S.T. Bach F.H. Blood. 1998; 91: 2249-2258Crossref PubMed Google Scholar, 29Lee E.G. Boone D.L. Chai S. Libby S.L. Chien M. Lodolce J.P. Ma A. Science. 2000; 289: 2350-2354Crossref PubMed Scopus (1189) Google Scholar). This raises the possibility that A20 plays an important role as a negative feedback regulator of NFκB functions. To investigate the possible functional interaction between PML and A20, we investigated whether the expression of A20 has any significant effects on PML/TNFα-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 Cd2+ (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 TNFα. However, PML/TNFα-induced cell death was significantly reduced in cells overexpressing A20, indicating that A20 inhibited apoptosis induced by PML/TNFα. The results shown in Fig. 5cdemonstrate that control pMEP4/U2OS cells treated with PML/TNFα 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/TNFα in TNF-resistant cells. PML was originally cloned and characterized for its involvement in the nonrandom chromosomal translocationt(15;17), which occurs in ∼99% of APL cases. The importance of PML in maintaining normal cellular function was unambiguously demonstrated by its essential role in multiple pathways of apoptosis (4Le X.F. Vallian S., Mu, Z.M. Hung M.C. Chang K.S. Oncogene. 1998; 16: 1839-1849Crossref PubMed Scopus (94) Google Scholar, 5Mu Z.M., Le, X.F. Vallian S. Glassman A.B. Chang K.S. Carcinogenesis. 1997; 18: 2063-2069Crossref PubMed Scopus (73) Google Scholar) and cell cycle progression (3Wang Z.G. Delva L. Gaboli M. Rivi R. Giorgio M. Gordon-Cardo C. Grosweld F. Pandolfi P.P. Science. 1998; 279: 1547-1551Crossref PubMed Scopus (454) Google Scholar, 6Wang Z.G. Ruggero D. Ronchetti S. Zhong S. Gaboli M. Rivi R. Pandolfi P.P. Nat. Genet. 1998; 20: 266-272Crossref PubMed Scopus (99) Google Scholar, 7Quignon F., De Bels F. Koken M. Feunteun J. Ameisen J.C. de The H. Nat. Genet. 1998; 20: 259-265Crossref PubMed Scopus (340) Google Scholar). PML and PML NBs are induced by interferons and inflammatory stimuli (31Gongora C. David G. Pintard L. Tissot C. Hua T.D. Dejean A. Mechit N. J. Biol. Chem. 1997; 272: 19457-19463Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 32Stadler M. Chelbi-Alix M.K. Koken M.H.M. Venturini L. Lee C. Saib A. Quignon F. Pelicano L. Guillemin M.C. Schindler C. de The H. Oncogene. 1995; 11: 2565-2573PubMed Google Scholar, 33Terris B. Baldin V. Dubois S. Degott C. Flejou J.F. Henin D. Dejean A. Cancer Res. 1995; 55: 1590-1597PubMed Google Scholar), 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 suppressor function remains unclear. In MCF-7 cells, the overexpression of PML induced G1 cell-cycle arrest associated with an upregulation of p21, p53, cyclin D, and hypophosphorylation of the Rb (6Wang Z.G. Ruggero D. Ronchetti S. Zhong S. Gaboli M. Rivi R. Pandolfi P.P. Nat. Genet. 1998; 20: 266-272Crossref PubMed Scopus (99) Google Scholar). Unphosphorylated Rb binds the E2F transcription factor and limits its ability to activate the transcription of genes essential for the G1 to S transition (34Weinberg R.A. Cell. 1995; 81: 323-330Abstract Full Text PDF PubMed Scopus (4308) Google Scholar). 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 (13LaMorte V.J. Dyck J.A. Ochs R.L. Evans R.M. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4991-4996Crossref PubMed Scopus (222) Google Scholar, 14Boisvert F.-M. Kruhlak M.J. Box A.K. Hendzel M.J. J. Cell Biol. 2001; 152: 1099-1106Crossref PubMed Scopus (129) Google Scholar, 22Wu W.S. Vallian S. Seto E. Yang W.M. Edmondson D. Roth S. Chang K.S. Mol. Cell. Biol. 2001; 21: 2259-2268Crossref PubMed Scopus (129) Google Scholar). PML activates transcription by binding and sequestering the negative regulator, Daxx (35Li H. Leo C. Zhu J., Wu, X.Y. O'Neil J. Park E.J. Chen J.D. Mol. Cell. Biol. 2000; 20: 1784-1796Crossref PubMed Scopus (306) Google Scholar, 36Lehembre F. Muller S. Pandolfi P.P. Dejean A. Oncogene. 2001; 20: 1-9Crossref PubMed Scopus (93) Google Scholar, 37Zhong S. Salomoni P. Ronchetti S. Guo A. Ruggero D. Pandolfi P.P. J. Exp. Med. 2000; 191: 631-639Crossref PubMed Scopus (193) Google Scholar), interacting with p53 (18Fogal V. Gostissa M. Sandy P. Zacchi P. Sternsdorf T. Jensen K. Pandolfi P.P. Will H. Schneider C. DelSal G. EMBO J. 2000; 22: 6186-6195Google Scholar, 19Guo A. Salomoni P. Luo J. Shih A. Zhong S., Gu, W. Pandolfi P.P. Nat. Cell Biol. 2000; 2: 730-736Crossref PubMed Scopus (387) Google Scholar, 20Pearson, M., and Pelicci, P. G. Oncogene 20, 7250–7256Google Scholar) 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 (24Vallian S. Chin K.V. Chang K.S. Mol. Cell. Biol. 1998; 18: 7147-7156Crossref PubMed Scopus (102) Google Scholar). 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 NFκB 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 (36Lehembre F. Muller S. Pandolfi P.P. Dejean A. Oncogene. 2001; 20: 1-9Crossref PubMed Scopus (93) Google Scholar). In these cells, the ability of Daxx to sensitize Fas-induced apoptosis was abrogated (36Lehembre F. Muller S. Pandolfi P.P. Dejean A. Oncogene. 2001; 20: 1-9Crossref PubMed Scopus (93) Google Scholar). Daxx was also shown to specifically enhance Fas-induced apoptosis (38Torii S. Egan D.A. Evans R.A. Reed J.C. EMBO J. 1999; 18: 6037-6049Crossref PubMed Scopus (235) Google Scholar) 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 TNFα and PMA. PML inhibits transactivation of the A20 promoter by interfering with NFκB binding to its consensus cis-acting element. This finding was further supported by the study showing that stable overexpression of A20 significantly inhibited apoptosis induced by the combined effects of PML and TNFα (Fig. 5). This study indicates that PML sensitizes TNFα-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 (25Opipari A.W. Boguski M.S. Dixit V.M. J. Biol. Chem. 1990; 265: 14705-14708Abstract Full Text PDF PubMed Google Scholar). It was originally characterized as an inhibitor of TNF-induced apoptosis (26Jaattela M. Mouritzen H. Elling F. Bastholm L. J. Immunol. 1996; 156: 1166-1173PubMed Google Scholar). A20 is also a potent inhibitor of NFκB activation induced by not only TNF but also by interleukin-1, lipopolysaccharide, PMA, and hydrogen peroxide (39Beyaert R. Heyninck K. Huffel S.V. Biochem. Pharmacol. 2000; 60: 1143-1151Crossref PubMed Scopus (262) Google Scholar). This inhibitory effect may be mediated through its interaction with tumor necrosis factor receptor-associated factors (40Song H.Y. Rothe M. Goeddel D.V. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 6721-6725Crossref PubMed Scopus (369) Google Scholar) or other A20 binding proteins (41Huffel V. Delaei F. Heyninck K., De Valck D. Beyaert R. J. Biol. Chem. 2001; 276: 30216-30223Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Interestingly, the expression of A20 is controlled by NFκB, suggesting that it is involved in a negative feedback mechanism in regulating NFκB. NFκB is an important transcription factor that regulates the expression of many mediators of inflammation (42Perkins N.D. Trends Biochem. Sci. 2000; 25: 434-440Abstract Full Text Full Text PDF PubMed Scopus (331) Google Scholar) and anti-apoptotic proteins involved in the survival pathway (43Rahman A. Anwar K.N. True A.L. Malik A.B. J. Immunol. 1999; 162: 5466-5476PubMed Google Scholar, 44Barkett M. Gilmore T.D. Oncogene. 1999; 18: 6910-6924Crossref PubMed Scopus (1075) Google Scholar). The finding that PML represses the transcription of the A20 promoter by interfering with NFκB binding is interesting. It will be important to further investigate the mechanism by which PML inhibits the binding of NFκB to its target DNA sequence and to examine whether the two proteins are functionally associated in vivo. We thank Vickie Williams for critical reading of the paper." @default.
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• W1999535314 title "The Promyelocytic Leukemia Protein Represses A20-mediated Transcription" @default.
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• W1999535314 doi "https://doi.org/10.1074/jbc.m201648200" @default.
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