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. 2009 Jun 26;284(26):17428-37.
doi: 10.1074/jbc.M109.007823. Epub 2009 Apr 24.

Loss of Wip1 sensitizes cells to stress- and DNA damage-induced apoptosis

Yun Xia  1 , Pat Ongusaha, Sam W Lee, Yih-Cherng Liou
Affiliations

Loss of Wip1 sensitizes cells to stress- and DNA damage-induced apoptosis

Yun Xia et al. J Biol Chem. .

Abstract

In response to various environmental stresses, the stress-responsive MAPKs p38 and JNK are activated and phosphorylate ATF2 and c-Jun transcription factors, thereby affecting cell-fate decision. Targeted gene disruption studies have established that JNK-c-Jun signaling plays a vital role in stress-induced apoptosis. The oncogenic phosphatase Wip1 acts as an important regulator in DNA damage pathway by dephosphorylating a spectrum of proteins including p53, p38, Chk1, Chk2, and ATM. In this study we show that Wip1 negatively regulates the activation of MKK4-JNK-c-Jun signaling during stress-induced apoptosis. The loss of Wip1 function sensitizes mouse embryonic fibroblasts to stress-induced apoptosis via the activation of both p38-ATF2 and JNK-c-Jun signaling. Here we reveal that Wip1 has dual roles in alternatively regulating stress- and DNA damage-induced apoptosis through p38/JNK MAPKs and p38/p53-dependent pathways, respectively. Our results point to Wip1 as a general regulator of apoptosis, which further supports its role in tumorigenesis.

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Figures

FIGURE 1.
FIGURE 1.
Antagonistic function of Wip1 during stress-induced cell death. Both wild-type and Wip1−/>− MEFs were mock treated or treated with 10 μm anisomycin, 10 μg/ml etoposide, 15 μm H2O2, 50 J/m2 UV, and 1 μm staurosporine, respectively. A, after 12 h of treatment, MTS was applied to the cells for cytotoxicity assay. Live cell ratios were then determined by normalization of the readings for treated cells to the readings for mock treated cells. The data are expressed as the means ± S.D. from three independent experiments. B, after 18 h of treatment, the cells were harvested live and stained with 0.1 μm calcein AM and 1 μm EthD-1. The labeled cells were then subjected to FACS analysis immediately. The results are representative of three independent experiments. WT, wild type; KO, knock-out.
FIGURE 2.
FIGURE 2.
Sensitization of Wip1−/− MEFs to apoptosis induced by anisomycin and etoposide. Wild-type and Wip1−/>− MEFs were mock treated or treated with 10 μm anisomycin and 10 μg/ml etoposide, respectively, A, after 18 h of treatment, the cells were fixed and stained with 10 μg/ml Hoechst 33342 to reveal nuclei. B, after 12 h of treatment, the cells were harvested, labeled with propidium iodide, and subjected to FACS analysis. The results are representative of three independent experiments. C and D, wild-type and Wip1−/>− MEFs were mock treated or treated with 10 μm anisomycin and 10 μg/ml etoposide, respectively. Extracts prepared at different time points after treatment were analyzed with anti-cleaved PARP and anti-cleaved caspase-3 antibodies. α-Tubulin was used as a protein loading control.
FIGURE 3.
FIGURE 3.
Enhanced activation of p38 and JNK MAPK pathways in Wip1−/− MEFs. Wild-type and Wip1−/>− MEFs were mock treated or treated with 10 μm anisomycin (A) or 10 μg/ml etoposide (B), respectively. Extracts were prepared at different time points after treatments. The proteins were separated on SDS-PAGE and probed with anti-p38, anti-p-p38 Thr180/Tyr182, anti-p-ATF2 Thr69/71, anti-p-JNK Thr183/Tyr185, anti-c-Jun, anti-p-c-Jun Ser63, and anti-p-c-Jun Ser73 antibodies. α-Tubulin was used as a protein loading control.
FIGURE 4.
FIGURE 4.
Sensitization of Wip1 siRNA expressing cells to anisomycin-induced apoptosis. U2OS and MCF-7 cells were transfected with scrambled negative control siRNA or Wip1 siRNAs for 72 h. A and B, after 72 h of transfection, extracts were separated on SDS-PAGE and probed with anti-Wip1 antibody. α-Tubulin was used as a protein loading control. C, MCF-7 cells were subjected to 10 μm anisomycin treatment. After 12 h of treatment, the cells were harvested, labeled with propidium iodide, and subjected to FACS analysis. D, U2OS cells were subjected to 5 μm anisomycin treatment. After 5 h of treatment, the cells were fixed and stained with 10 μg/ml Hoechst 33342 to reveal nuclei. E, U2OS cell were subjected to 5 μm anisomycin treatment, and extracts were prepared at different time points after treatment. The proteins were separated on SDS-PAGE and probed with anti-cleaved PARP, anti-cleaved caspase-3, anti-p-JNK Thr183/Tyr185, anti-p-c-Jun Ser63, and anti-p-c-Jun Ser73 antibodies. α-Tubulin was used as a protein loading control.
FIGURE 5.
FIGURE 5.
Higher FasL responsiveness of Wip1−/− MEFs upon anisomycin and etoposide treatment. Wild-type and Wip1−/>− MEFs were mock treated or treated with 10 μm anisomycin (A) and 10 μg/ml etoposide (B), respectively. RNAs were prepared at different time points and reverse transcribed. Expression levels of FasL, Tp53, Cyclin D1, Ppm1d, and GADPH were determined by semi-quantitative reverse transcription-PCR.
FIGURE 6.
FIGURE 6.
Inhibition of anisomycin-induced but not etoposide-induced apoptosis by suppression of both the p38 and JNK. A, C, and E, anisomycin; B, D, and F, etoposide. A and B, wild-type and Wip1−/>− MEFs were subjected to treatment as indicated in the figure. Extracts prepared after treatment were separated on SDS-PAGE and probed with anti-p-ATF2 Thr69/71, anti-p-c-Jun Ser63, and anti-p-c-Jun Ser73 antibodies. α-Tubulin was used as a protein loading control. C and D, equal numbers of wild-type and Wip1−/>− MEFs were seeded and cultured for 36 h prior to treatment. The cells were subjected to treatment as indicated in the figure. After 12 h of treatment, MTS was applied to the cells for cytotoxicity assay. Live cell ratios were then determined by normalization of the readings for treated cells to the control cell measurements. The data are expressed as the means ± S.D. from three independent experiments. **, p < 0.01; *, p < 0.05. E and F, wild-type and Wip1−/>− MEFs were mock treated or treated with 10 μm anisomycin and 10 μg/ml etoposide, respectively. Extracts were prepared at different time points after treatment. The proteins were separated on SDS-PAGE and probed with anti-p-MKK4 Thr261 and anti-p-MKK7 Ser271/Thr275 antibodies. α-Tubulin was used as a protein loading control.
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