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. 2001 May;21(9):3012-24.
doi: 10.1128/MCB.21.9.3012-3024.2001.

AP-1 repressor protein JDP-2: inhibition of UV-mediated apoptosis through p53 down-regulation

F Piu  1 , A Aronheim, S Katz, M Karin
Affiliations

AP-1 repressor protein JDP-2: inhibition of UV-mediated apoptosis through p53 down-regulation

F Piu et al. Mol Cell Biol. 2001 May.

Abstract

Members of the AP-1 transcription factor family, especially c-Jun and c-Fos, have long been known to mediate critical steps in the cellular response to ultraviolet (UV) irradiation. We sought to examine whether two newly discovered members of the AP-1 family, JDP-1 and JDP-2, also participate in the mammalian UV response. Here we report that JDP-2, but not JDP-1, is transiently induced upon UV challenge and that elevated levels of JDP-2 increase cell survival following UV exposure. This protective function of JDP-2 appears to be mediated through repression of p53 expression at the transcriptional level, via a conserved atypical AP-1 site in the p53 promoter.

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Figures

FIG. 1
FIG. 1
Induction of JDP-2 following UV irradiation. (A) JDP-2, but not JDP-1, is induced upon UV irradiation. c-jun+/+ or c-jun−/− fibroblasts were exposed to UV-C (40 J/m2) or left unexposed and lysed after 2 h. After separation by SDS-PAGE, Western blots of lysate proteins were probed with specific antibodies against JDP-1 or JDP-2 and visualized by ECL. Responses were estimated by densitometry using a phosphorimager and plotted as a graph. (B) Time course of JDP-2 induction by UV. c-jun+/+ fibroblasts were exposed to UV-C (40 J/m2) or left unexposed and lysed at the indicated times. When indicated, cells were incubated for 2 h with MG132 (50 μM) prior to UV irradiation. After separation by SDS-PAGE, blots of lysate proteins were probed with specific antibodies against JDP-2 and visualized by ECL. Responses were estimated by densitometry using a phosphorimager and plotted as a graph.
FIG. 2
FIG. 2
Transient expression of JDP-2 inhibits UV-induced apoptosis. Cell cycle distribution was determined by flow cytometry. Spontaneously immortalized c-jun+/+ and c-jun−/− mouse fibroblasts were transiently transfected with either JDP-1 or JDP-2 mammalian expression vectors or a control vector. Sixteen hours posttransfection, cells were UV irradiated (40 J/m2) or left unirradiated and harvested 48 h later. Ethanol-fixed cells were incubated for 30 min in the presence of an antibody directed against either JDP-1 or JDP-2 or a control antibody, washed, and then incubated with an FITC-conjugated secondary antibody. Cells were stained with propidium iodide and examined by flow cytometry. In the case of JDP-1 and JDP-2-transfected cells, FITC-positive cells were sorted and further analyzed. The vertical and horizontal axes represent cell count and relative DNA content, respectively.
FIG. 3
FIG. 3
Stable expression of JDP-2 delays and reduces UV-mediated cell death. (A) Constitutive JDP-2 expression reduces apoptosis after UV irradiation. NIH 3T3 cells expressing elevated levels of either JDP-1 or JDP-2 were generated (2). Cells were UV-C irradiated (40 J/m2), and their cell cycle distribution was determined at 0, 1, 2, and 3 days postirradiation by flow cytometry. (B) Recapitulation of results from panel A. Values are indicative of apoptosis. (C) JDP-2 enhances clonogenic survival following UV irradiation. Clonogenic survival assays were performed on parental NIH-3T3 cells and cells that stably express JDP-1 and JDP-2, following exposure to the indicated doses of UV-C. After irradiation of a fixed number of cells, the number of surviving cells was determined by the number of colonies detected after 2 weeks.
FIG. 4
FIG. 4
p53 expression in JDP-2-overexpressing cells upon UV irradiation. (A) Induction of p53 is reduced and delayed in JDP-2-overexpressing cells. Parental NIH 3T3 cells and cells overexpressing JDP-1 or JDP-2 were exposed to UV-C (40 J/m2). Protein lysates were prepared at the indicated times postirradiation (in hours) and analyzed by Western blotting for expression of p53. To detect low levels of endogeneous p53 proteins, 200 μg of cell lysates was loaded per lane. The amounts of protein loaded on the gel were compared by probing with anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) antibodies. (B) Stability of p53 protein is not affected by JDP-2 upon UV. Parental NIH 3T3 cells and cells overexpressing JDP-1 or JDP-2 were subjected to UV-C irradiation (40 J/m2). Two hours later, cells were briefly labeled (45 min) with [35S]methionine and then chased with an excess of cold methionine for the indicated times. Protein lysates were immunoprecipitated with a specific antibody against p53 and then analyzed by SDS-PAGE. The relative amounts of p53-labeled proteins were quantified using a phosphorimager. Amounts were normalized for each cell line. (C) Accumulation of p53 transcripts is reduced and delayed in JDP-2-overexpressing cells. NIH 3T3 and JDP-2-overexpressing cells were exposed to 40 J of UV-C/m2. Total cellular RNA was harvested at the indicated times (in hours) and analyzed by Northern blotting for expression of p53 mRNA. Adequate loading was ensured by probing with a control GAPDH probe. rRNA are indicated.
FIG. 5
FIG. 5
Transcriptional regulation of p53 by UV and AP-1 proteins. (A) c-jun−/− and c-jun+/+ cells were transiently transfected with a wild-type or PF-1 deletion-containing p53 promoter fused to a luciferase reporter and JDP-1, JDP-2, and c-Jun mammalian expression vectors. A β-galactosidase reporter driven by a β-actin promoter was included to normalize for transfection efficiency. Sixteen hours after transfection, cells were exposed to UV-C (40 J/m2), and they were collected 9 h later to determine luciferase and β-galactosidase activities. The results are average relative luciferase induction levels, where the level expressed by untreated c-jun+/+ cells transfected with wild-type p53-luciferase was given an arbitrary value of 1.0. (B) Specific binding of c-Jun and JDP-2 to an intact PF-1 site. Gel retardation (EMSA) experiments were performed using in vitro-translated c-Jun and JDP-2 against 32P-radiolabeled PF-1 or ΔPF-1 primers. JDP-2 binding specificity to an intact PF-1 motif was confirmed through competition studies with a 100-fold excess of cold nonlabeled competitor (PF1 or ΔPF-1). Arrows indicate c-Jun–PF-1 and JDP-2–PF-1 DNA-protein complexes.
FIG. 6
FIG. 6
JDP-2 represses p53 induction following UV in vivo. Parental NIH 3T3 cells and JDP-1- or JDP-2-overexpressing cells were transiently transfected with either wild-type (A) or a PF-1 site deletion-containing (ΔPF-1) (B) p53 promoter-luciferase reporter. An actin–β-galactosidase reporter was included to normalize for transfection efficiency. Sixteen hours after transfection the cells were UV-C irradiated (40 J/m2), and luciferase and β-galactosidase activities were determined at the indicated times postirradiation. The normalized levels of luciferase expression in NIH 3T3 cells transfected with the wild-type p53-luciferase reporter were given an arbitrary value of 1.0. All other values are expressed relative to that value and are averages for three separate experiments.
FIG. 7
FIG. 7
Characterization of the AP-1 dimers timely involved in the UV response. (A) JDP-2, c-Jun, and c-Fos have different expression profiles upon UV irradiation. Exponentially growing c-jun+/+ cells were UV-C irradiated (40 J/m2), and protein extracts were prepared at the indicated times. Expression and phosphorylation of JDP-2, c-Jun, and c-Fos were analyzed by Western blotting using specific antibodies. (B) c-Jun–c-Fos and c-Jun–JDP-2 heterodimers accumulate with different kinetics upon UV irradiation. Exponentially growing c-jun+/+ cells were irradiated with UV-C (40 J/m2). Protein extracts were harvested at the indicated times and immunoprecipitated (IP) with specific c-Jun or JDP-2 antibodies. The various immune complexes were then analyzed by Western blotting with antibodies directed against c-Jun, c-Fos, or JDP-2 and against c-Jun or JDP-2, respectively. The amount of proteins in the extracts (quantified by the Bradford method) was further confirmed by Western blotting with a specific anti-GAPDH antibody.
FIG. 8
FIG. 8
A dominant positive JDP-2 mutant (JDP-2mut) lacks the ability to inhibit UV-induced p53 transcriptional activation and apoptosis. (A) Characterization of the dominant positive JDP-2 mutant transcriptional properties. NIH 3T3 cells were transiently transfected with luciferase reporter genes containing either 3×TRE, mouse wild-type p53 promoter, or p53 (ΔPF-1) promoter, with various combinations of expression vectors for c-Jun, c-Fos, JDP-2, and JDP-2mut. The JDP-2 mutant was constructed by fusing in frame with JDP-2 the transcriptional activation domain of c-Fos. An actin–β-galactosidase reporter was included to normalize for transfection efficiency. Cells transfected with the p53 promoter (wild-type and ΔPF-1) reporter constructs were UV irradiated (40 J of UV-C/m2) or left unirradiated, and extracts were analyzed 9 h postirradiation. Data are averages for two independent experiments done in triplicate. (B) JDP-2mut expression does not affect UV-induced apoptosis. Cell cycle distribution was determined by flow cytometry as described for Fig. 2. Spontaneously immortalized c-jun+/+ and c-jun−/− mouse fibroblasts were transiently transfected with JDP-2mut mammalian expression vectors. JDP-2 FITC-positive cells were sorted and further analyzed. The vertical and horizontal axes represent cell count and relative DNA content, respectively.
FIG. 9
FIG. 9
JDP-2 does not affect UV-induced apoptosis in cells lacking p53. (A) Cell cycle analysis of p53 null cells transiently expressing JDP-1 or JDP-2. p53 null cells were either mock or transiently transfected with JDP-1 or JDP-2 expression vectors. Sixteen hours after transfection, cells were UV irradiated (40 J of UV-C/m2) and harvested 48 h later. Ethanol-fixed cells were then stained with propidium iodide and analyzed by flow cytometry. Cells expressing transiently transfected JDP-1 or JDP-2 were sorted for FITC staining as described above (Fig. 2). (B) Expression levels of JDP-2 in p53 wild-type and null cells. Exponentially growing p53+/+ JDP-2+/+ and p53−/− JDP-2+/+ cells were harvested, and whole-cell protein extracts were made and analyzed by SDS-PAGE. Western blots were probed with specific antibodies against JDP-2 and GAPDH and revealed using ECL. (C) JDP-2 requires p53 to inhibit UV-induced apoptosis. p53+/+ JDP-2+/+ and p53−/− JDP-2+/+ cells were mock or UV treated (40 J of UV-C/m2), and apoptosis was evaluated 48 h postirradiation. Apoptosis was quantified by measuring levels of caspase 3 and caspase 8 present in extracts using a colorimetric assay.
FIG. 10
FIG. 10
JDP-2 ability to inhibit UV mediated apoptosis is a general phenomenon and is dependent upon p53. (A) Cell cycle distribution of UV irradiated human cell lines expressing JDP-2. Human HEK293 (p53+/+) and Saos-2 (p53−/−) cells were UV irradiated (40 J/m2), and their cell cycle distribution was examined 24 h postirradiation by fluorescence-activated cell sorting. Cells transiently expressing JDP-2 or JDP-2mut were sorted and treated as described above (Fig. 2). (B) Recapitulation of results from panel A. (C) Apoptosis of p53−/− and p53+/+ human cell lines expressing JDP-2 and JDP-2mut. HEK293 (p53+/+) and Saos-2 (p53−/−) cells were UV irradiated (40 J of UV-C/m2), and their respective degree of apoptosis were quantified after 24 h by measuring endogeneous levels of caspase 3 and caspase 8 using a fluorescent substrate.
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References

    1. Angel P, Karin M. The role of Jun, Fos and the AP-1 complex in cell-proliferation and transformation. Biochim Biophys Acta. 1991;1072:129–157. - PubMed
    1. Aronheim A, Zandi E, Hennemann H, Elledge S, Karin M. Isolation of an AP-1 repressor by a novel method for detecting protein-protein interactions. Mol Cell Biol. 1997;17:3094–3102. - PMC - PubMed
    1. Bates S, Vousden K H. p53 in signaling checkpoint arrest or apoptosis. Curr Opin Genet Dev. 1996;6:12–18. - PubMed
    1. Bennett M R. Mechanisms of p53-induced apoptosis. Biochem Pharmacol. 1999;58:1089–1095. - PubMed
    1. Blattner C, Kannouche P, Litfin M, Bender K, Rahmsdorf H J, Angulo J F, Herrlich P. UV-induced stabilization of c-fos and other short-lived mRNAs. Mol Cell Biol. 2000;20:3616–3625. - PMC - PubMed

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