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• W2003199401 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. 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. Wip1 (wild-type p53-induced-phosphatase 1) is a relatively new member of the PP2C family and possesses oncogenic properties (1Fiscella M. Zhang H. Fan S. Sakaguchi K. Shen S. Mercer W.E. Vande Woude G.F. O'Connor P.M. Appella E. Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 6048-6053Crossref PubMed Scopus (461) Google Scholar, 2Takekawa M. Adachi M. Nakahata A. Nakayama I. Itoh F. Tsukuda H. Taya Y. Imai K. EMBO J. 2000; 19: 6517-6526Crossref PubMed Scopus (360) Google Scholar). It was initially found that the expression of Wip1 phosphatase is induced upon ionizing radiation and UV exposure in a p53-dependent manner (1Fiscella M. Zhang H. Fan S. Sakaguchi K. Shen S. Mercer W.E. Vande Woude G.F. O'Connor P.M. Appella E. Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 6048-6053Crossref PubMed Scopus (461) Google Scholar, 2Takekawa M. Adachi M. Nakahata A. Nakayama I. Itoh F. Tsukuda H. Taya Y. Imai K. EMBO J. 2000; 19: 6517-6526Crossref PubMed Scopus (360) Google Scholar). Wip1 inhibits UV-induced p38 activation by dephosphorylating the conserved Thr(P)180 residue in p38, thereby suppressing the activation of its downstream target p53 (2Takekawa M. Adachi M. Nakahata A. Nakayama I. Itoh F. Tsukuda H. Taya Y. Imai K. EMBO J. 2000; 19: 6517-6526Crossref PubMed Scopus (360) Google Scholar). Moreover, Wip1 dephosphorylates p53 at the Ser15 residue, which is targeted by the ATM 2The abbreviations used are: ATMataxia-telangiectasia mutatedsiRNAsmall interfering RNAMAPKmitogen-activated protein kinaseJNKc-Jun N-terminal kinaseMEFmouse embryonic fibroblastMTS3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazoliumFACSfluorescence-activated cell sorterPBSphosphate-buffered salinePARPpoly(ADP-ribose) polymerase. /ATR (ATM and RAD3-related) pathway (3Lu X. Nannenga B. Donehower L.A. Genes Dev. 2005; 19: 1162-1174Crossref PubMed Scopus (316) Google Scholar). Wip1 also mediates the base excision pathway by negatively regulating the phosphorylation of UNG2 (4Lu X. Bocangel D. Nannenga B. Yamaguchi H. Appella E. Donehower L.A. Mol. Cell. 2004; 15: 621-634Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). In addition, Wip1 serves as a homoeostatic regulator of checkpoint kinases Chk1 and Chk2 (3Lu X. Nannenga B. Donehower L.A. Genes Dev. 2005; 19: 1162-1174Crossref PubMed Scopus (316) Google Scholar, 5Fujimoto H. Onishi N. Kato N. Takekawa M. Xu X.Z. Kosugi A. Kondo T. Imamura M. Oishi I. Yoda A. Minami Y. Cell Death Differ. 2006; 13: 1170-1180Crossref PubMed Scopus (163) Google Scholar). The phosphorylation of ATM at the Ser1981 residue is also negatively regulated by Wip1 (6Shreeram S. Demidov O.N. Hee W.K. Yamaguchi H. Onishi N. Kek C. Timofeev O.N. Dudgeon C. Fornace A.J. Anderson C.W. Minami Y. Appella E. Bulavin D.V. Mol. Cell. 2006; 23: 757-764Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar). Recently, Mdm2 was identified as a novel substrate of Wip1, which dephosphorylates Mdm2 at the Ser395 residue and increases its stability, thus further destabilizing p53 (7Lu X. Ma O. Nguyen T.A. Jones S.N. Oren M. Donehower L.A. Cancer Cell. 2007; 12: 342-354Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar). Accumulating evidence has implicated that Wip1 can be an oncogene and plays critical roles in regulating DNA damage pathways. ataxia-telangiectasia mutated small interfering RNA mitogen-activated protein kinase c-Jun N-terminal kinase mouse embryonic fibroblast 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium fluorescence-activated cell sorter phosphate-buffered saline poly(ADP-ribose) polymerase. Wip1 is encoded by the Ppm1d (protein phosphatase magnesium-dependent 1delta) gene, which maps to chromosome 17q22-q23 (8Li J. Yang Y. Peng Y. Austin R.J. van Eyndhoven W.G. Nguyen K.C. Gabriele T. McCurrach M.E. Marks J.R. Hoey T. Lowe S.W. Powers S. Nat. Genet. 2002; 31: 133-134Crossref PubMed Scopus (216) Google Scholar). This locus is a hot spot for gene amplification in multiple primary human cancers, including breast cancer, neuroblastomas, and ovarian clear cell adenocarcinomas (8Li J. Yang Y. Peng Y. Austin R.J. van Eyndhoven W.G. Nguyen K.C. Gabriele T. McCurrach M.E. Marks J.R. Hoey T. Lowe S.W. Powers S. Nat. Genet. 2002; 31: 133-134Crossref PubMed Scopus (216) Google Scholar, 9Hirasawa A. Saito-Ohara F. Inoue J. Aoki D. Susumu N. Yokoyama T. Nozawa S. Inazawa J. Imoto I. Clin. Cancer Res. 2003; 9: 1995-2004PubMed Google Scholar, 10Saito-Ohara F. Imoto I. Inoue J. Hosoi H. Nakagawara A. Sugimoto T. Inazawa J. Cancer Res. 2003; 63: 1876-1883PubMed Google Scholar). The overexpression of Wip1 acts in concert with other oncogenes such as HRas1, ErbB2, and Myc to transform mouse embryonic fibroblasts (11Harrison M. Li J. Degenhardt Y. Hoey T. Powers S. Trends Mol. Med. 2004; 10: 359-361Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). In contrast, disruption of Wip1 can suppress HRas1- or ErbB2-induced transformation by activating p38, which activates p53 and Ink4a/ARF pathways consequently (12Bulavin D.V. Phillips C. Nannenga B. Timofeev O. Donehower L.A. Anderson C.W. Appella E. Fornace Jr., A.J. Nat. Genet. 2004; 36: 343-350Crossref PubMed Scopus (357) Google Scholar). A mouse model study further demonstrated that activation of MKK6-p38 is critical in rendering Wip1−/− mice resistant to mammary gland tumors driven by the ErbB2 and HRas1 oncogenes (13Demidov O.N. Kek C. Shreeram S. Timofeev O. Fornace A.J. Appella E. Bulavin D.V. Oncogene. 2007; 26: 2502-2506Crossref PubMed Scopus (86) Google Scholar). As a negative regulator of ATM, Wip1 controls the magnitude and duration of ATM phosphorylation and activity. Wip1−/− mice display a dramatic delay in the onset of lymphomas induced by the Myc oncogene in an ATM- and p53-dependent but not a p38- or ARF-dependent manner (14Shreeram S. Hee W.K. Demidov O.N. Kek C. Yamaguchi H. Fornace Jr., A.J. Anderson C.W. Appella E. Bulavin D.V. J. Exp. Med. 2006; 203: 2793-2799Crossref PubMed Scopus (112) Google Scholar). These findings suggest that Wip1 is an indispensible regulator of tumorigenesis. The biological function of Wip1 confers its oncogenic properties. It has been well established that Wip1 plays a vital role in controlling cell proliferation by dephosphorylating Chk1 and Chk2, thereby negatively regulating DNA damage-induced cell cycle checkpoints (3Lu X. Nannenga B. Donehower L.A. Genes Dev. 2005; 19: 1162-1174Crossref PubMed Scopus (316) Google Scholar, 5Fujimoto H. Onishi N. Kato N. Takekawa M. Xu X.Z. Kosugi A. Kondo T. Imamura M. Oishi I. Yoda A. Minami Y. Cell Death Differ. 2006; 13: 1170-1180Crossref PubMed Scopus (163) Google Scholar). Several groups have suggested that Wip1 is a negative regulator of apoptosis in response to DNA damage. Bulavin et al. (12Bulavin D.V. Phillips C. Nannenga B. Timofeev O. Donehower L.A. Anderson C.W. Appella E. Fornace Jr., A.J. Nat. Genet. 2004; 36: 343-350Crossref PubMed Scopus (357) Google Scholar) have shown that tumors derived from Wip1−/>− MMTV-ErbB2 mice possess a reduced mitotic index and increased levels of apoptosis compared with tumors derived from wild-type mice. In addition, Takekawa et al. (2Takekawa M. Adachi M. Nakahata A. Nakayama I. Itoh F. Tsukuda H. Taya Y. Imai K. EMBO J. 2000; 19: 6517-6526Crossref PubMed Scopus (360) Google Scholar) have reported that Wip1 suppresses UV-induced apoptosis by negatively regulating p38/p53 signaling. Ectopic expression of Wip1 results in the inhibition of Chk2-mediated apoptosis following ionizing radiation (5Fujimoto H. Onishi N. Kato N. Takekawa M. Xu X.Z. Kosugi A. Kondo T. Imamura M. Oishi I. Yoda A. Minami Y. Cell Death Differ. 2006; 13: 1170-1180Crossref PubMed Scopus (163) Google Scholar). On the contrary, the depletion of Wip1 by siRNA prolongs the E2F1-induced activation of p38 signaling pathway, resulting in an enhancement of E2F1-induced apoptosis (15Hershko T. Korotayev K. Polager S. Ginsberg D. J. Biol. Chem. 2006; 281: 31309-31316Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). Recently, it was found that the loss of Wip1 suppresses APCMin-driven polyposis by lowering the threshold for p53-dependent apoptosis of stem cells, thus preventing their conversion into tumor-initiating stem cells (16Demidov O.N. Timofeev O. Lwin H.N. Kek C. Appella E. Bulavin D.V. Cell Stem Cell. 2007; 1: 180-190Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Taken together, these results suggest that Wip1 acts as an important regulator of apoptosis upon environmental stresses. However, it is still obscure whether the regulation of apoptosis by Wip1 is induced by stresses other than DNA damage stress. Our study shows that Wip1−/>− MEFs are more sensitive to apoptosis induced by various environmental stresses, including ribotoxic, oxidative, and DNA damage stresses. In Wip1−/>− MEFs, the external stresses elicit more significant activation of p38 and JNK compared with wild-type MEFs. Herein, we demonstrate that in addition to p38 and p53 signaling, Wip1 negatively regulates the MKK4-JNK-c-Jun pathway. Upon stress induction, the loss of Wip1 function in cells causes an accumulation of MKK4 phosphorylation at the Thr261 residue, resulting in the transcriptional activation of c-Jun and subsequently leading to the up-regulation of its downstream gene FasL (17Kasibhatla S. Brunner T. Genestier L. Echeverri F. Mahboubi A. Green D.R. Mol. Cell. 1998; 1: 543-551Abstract Full Text Full Text PDF PubMed Scopus (667) Google Scholar, 18Kolbus A. Herr I. Schreiber M. Debatin K.M. Wagner E.F. Angel P. Mol. Cell. Biol. 2000; 20: 575-582Crossref PubMed Scopus (145) Google Scholar). We therefore propose that Wip1 acts as a negative regulator of both stress-induced and DNA damage-induced apoptosis. HEK293T, U2OS, and MCF-7 cells were obtained from ATCC. Mouse embryonic fibroblasts derived from wild-type and Wip1−/>− mice were kindly provided by Bulavin and co-workers (19Choi J. Nannenga B. Demidov O.N. Bulavin D.V. Cooney A. Brayton C. Zhang Y. Mbawuike I.N. Bradley A. Appella E. Donehower L.A. Mol. Cell. Biol. 2002; 22: 1094-1105Crossref PubMed Scopus (152) Google Scholar). MEFs were immortalized with SV40. The cells were maintained in Dulbecco's modified Eagle's medium, supplemented with 10% (v/v) fetal bovine serum. The cells were seeded and cultured for 36 h prior to treatments. Anisomycin and etoposide were obtained from Sigma. H2O2 was obtained from AppliChem. Staurosporine was purchased from Alexis Biochemicals. UV irradiations were performed at a dose rate of 50 J/m2 using an XL-1500 UV cross-linker. Two siRNAs designed to knock down Wip1 and a scrambled negative control siRNA were purchased from Invitrogen. The sequences of Wip1 siRNAs are as follows: Wip1 siRNA 1, 5′-UUG UGA GUG AGU CGA GGU CGU UUC C-3′; and Wip1 siRNA 2, 5′-UAU CCU UAA AGU CAG GGC UUU AGC G-3′. The siRNA oligonucleotides for Wip1 were transfected into U2OS and MCF-7 cells using oligofectamine (Invitrogen). The cells were solubilized in lysis buffer (50 mm Tris-HCl, pH 7.4, 10% glycerol, 1% (v/v) Triton X-100, 100 mm NaCl, 0.5 mm MgCl2, 1 mm Na3VO4, 10 μg/ml aprotinin, 1 mm phenylmethylsulfonyl fluoride, 1 μm pepstatin, and leupeptin), and the resulting lysates were prepared by centrifugation at 16,000 × g for 30 min at 4 °C. Protein concentrations of whole cell lysates were detected using protein assay reagent (Bio-Rad) prior to immunoblotting. The samples were separated on SDS-PAGE, and transferred onto polyvinylidene difluoride membrane filters. The immunoblots were probed with the primary antibodies listed in the supplementary information. The secondary antibodies were either horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG (Santa Cruz Biotechnology). The p38 inhibitor SB203580 and JNK inhibitor SP600125 were purchased from Sigma. Equal numbers of wild-type and Wip1−/>− MEFs were seeded and cultured for 36 h before treatment with anisomycin, etoposide, H2O2, UV, and staurosporine, respectively. 5 μm SB203580 and/or SP600125 were added to the MEFs and incubated for 30 min prior to stress treatment. After treatment with different types of stress stimuli for 12 h, MTS (inner salt) (Promega) was applied to MEFs according to the manufacturer's instructions. Absorption at 495 nm was recorded at the 4-h time point. The data were expressed as the means ± S.D. from three independent experiments. The live/dead assay was used to simultaneously investigate both the live and death ratios of cells by measuring two parameters of cell viability, namely intracellular esterase activity and plasma membrane integrity. Higher esterase activity (labeled with fluorescein isothiocyanate) indicated live cells, represented by the dots located in region 4. Higher membrane permeability (labeled with Texas Red) indicated dead cells, represented by the dots located in region 1 (supplemental Fig. S1). The dots located in region 3 indicated unstained cells, and the dots located in region 2 indicated double-stained cells. For the live/dead assay, wild-type and Wip1−/>− MEFs were seeded and cultured for 36 h before treatment. At 18 h after treatment, both the floating and attached cells were harvested and subjected to labeling with a live/dead assay kit (l-3224; Molecular Probes) according to the manufacturer's instructions. After labeling, analyses were performed using Dako Cytomation, and the results were analyzed with Summit 4.3. In each experiment, 30,000 events were recorded. For propidium iodide labeling, wild-type and Wip1−/>− MEFs were seeded and cultured for 36 h before treatment. At 18 h after treatment, both floating and attached cells were harvested, washed twice with PBS, and fixed in 70% ethanol. DNA was stained with 50 μg/ml propidium iodide (Sigma) in PBS containing 100 μg/ml RNaseA (Qiagen). Analyses were then performed using Dako Cytomation, and the results were analyzed with Summit 4.3. In each experiment, 10,000 events were recorded, and the results were representative of three independent experiments. The cells grown on coverslips were fixed in 4% paraformaldehyde/PBS for 15 min and then permeabilized with PBS containing 0.2% Triton X-100 for 15 min at room temperature. Fixed cells were incubated with 10 μg/ml Hoechst 33342 (Molecular probe) for 40 min. The cells were washed twice in PBS and then mounted with FluorSaveTM reagent (Calbiochem) and analyzed under a fluorescence microscope. Total RNA was extracted from cultured cells using TRIzol reagent (Invitrogen), and was reverse-transcribed using Superscript II reverse transcriptase (Invitrogen) according to the manufacturer's recommendations. The sequences of primers are shown in the supplementary information. To elucidate whether Wip1 is involved in cell death, we treated wild-type and Wip1−/>− MEFs with different external stimuli such as anisomycin, etoposide, UV radiation, H2O2, and staurosporine. The cytotoxic responses of MEFs were assessed by measuring the metabolic activities of viable cells using MTS. As shown in Fig. 1A, in response to anisomycin treatment, Wip1−/>− MEFs displayed a significantly lower live cell ratio compared with wild-type MEFs (29.6% of Wip1−/>− MEFs compared with 56.1% of wild-type MEFs). Upon etoposide treatment, wild-type MEFs displayed a live cell ratio as high as 97.4%, whereas in Wip1−/>− MEFs this ratio was much lower at 28.4%. Similarly, Wip1−/>− MEFs showed a lower live cell ratio than wild-type MEFs in response to UV radiation, H2O2, and staurosporine treatments (Fig. 1A). To exclude factors other than cell death as a cause of lower metabolic activity in Wip1−/>− MEFs, we performed a live/dead assay. As shown in Fig. 1B, in response to anisomycin treatment, wild-type MEFs displayed a dead cell ratio of 3.57% and a live cell ratio of 87.49%. However, Wip1−/>− MEFs displayed a higher dead cell ratio of 37.15% and a lower live cell ratio of 34.56%. In agreement with the results of the cytotoxicity assay, Wip1−/>− MEFs displayed a markedly higher dead cell ratio than wild-type MEFs upon exposure to all five tested stimuli (Fig. 1B). Our results indicated that the loss of Wip1 sensitizes MEFs to cell death response resulting from different types of stress stimuli. To further elucidate the possible role of Wip1 in apoptosis, we examined the apoptotic responses of wild-type and Wip1−/>− MEFs to anisomycin and etoposide, respectively. As shown in Fig. 2A, after 12 h of treatment with these stimuli, Wip1−/>− MEFs displayed more malformed nuclei with condensed DNA (white arrows) compared with wild-type MEFs. Propidium iodide staining was also performed to reveal the ratio of apoptotic cells in wild-type and Wip1−/>− MEFs upon stresses. As shown in Fig. 2B, after 12 h of treatment with anisomycin, the ratio of sub-G1 apoptotic cells increased from 3.24 to 11.48% in Wip1−/>− MEFs. Similarly, in response to etoposide, Wip1−/>− MEFs displayed a substantial increase in the apoptotic cell ratio (from 3.24 to 16.51%; Fig. 2B). In contrast, no significant changes were observed in wild-type MEFs treated with anisomycin as compared with the mock treated control. However, wild-type MEFs exhibited a strong G2/M arrest response and less apoptotic cells upon etoposide treatment, in agreement with previous reports (20Cliby W.A. Lewis K.A. Lilly K.K. Kaufmann S.H. J. Biol. Chem. 2002; 277: 1599-1606Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 21Rossi R. Lidonnici M.R. Soza S. Biamonti G. Montecucco A. Cancer Res. 2006; 66: 1675-1683Crossref PubMed Scopus (38) Google Scholar). Two apoptotic markers, cleaved caspase-3 and cleaved poly(ADP-ribose) polymerase (PARP), were examined in wild-type and Wip1−/>− MEFs, upon anisomycin or etoposide treatment. As shown in Fig. 2 (C and D), after 6 h of treatment, both compounds evoked a more dramatic accumulation of cleaved caspase-3 and cleaved PARP in Wip1−/>− MEFs compared with wild type. UV radiation and H2O2 treatments also stimulated a more significant increase in the protein levels of cleaved caspase-3 and cleaved PARP in Wip1−/>− MEFs compared with wild type (supplemental Fig. S2). Taken together, our results thus indicated that Wip1-deficient MEFs are more sensitive to external stress-induced apoptosis, suggesting that Wip1 may act as a negative regulator of apoptosis and regulates apoptosis induced by other stresses in addition to DNA damage. To investigate the role of Wip1 in regulating stress apoptotic pathways, we utilized anisomycin because it is a protein synthesis inhibitor, and its involvement in the p38 and JNK MAPK pathways has been well characterized (22Morton S. Davis R.J. Cohen P. FEBS Lett. 2004; 572: 177-183Crossref PubMed Scopus (77) Google Scholar). Consistent with previous reports, anisomycin induced a more considerable increase of the phospho-p38 protein levels but not the total p38 protein levels in Wip1−/>− MEFs compared with wild-type cells (Fig. 3A). Accordingly, Wip1−/>− MEFs displayed higher levels of phospho-ATF2, an important downstream target of p38, upon anisomycin treatment (Fig. 3A). In addition, although the protein levels of phospho-JNK were induced in both wild-type and Wip1−/>− MEFs, the phosphorylation of JNK was found to be sustained for a longer time in the Wip1−/>− MEFs (Fig. 3A, lower panel). Similarly, although the total protein levels of c-Jun were slightly increased in both wild-type and Wip1−/>− MEFs, the latter showed a substantially faster increase in the phosphorylation of c-Jun at Ser63 and Ser73 residues compared with wild-type MEFs in response to anisomycin treatment (Fig. 3A). Interestingly, comparable results were found for etoposide (Fig. 3B), UV (supplemental Fig. S3), and H2O2 treatments (supplemental Fig. S3). The activation of p38 and JNK MAPKs might happen within a short time period in response to extracellular stress. We therefore proceeded to examine the earlier response of wild-type and Wip1−/>− MEFs to anisomycin and etoposide treatments. Within 1 h of treatment, anisomycin stimulated a more substantial activation of p38 and JNK signaling in Wip1−/>− MEFs than in wild-type MEFs (supplemental Fig. S4). In addition, the treatment of etoposide induced activated p38 and JNK at relatively later time points and sustained for a longer time in the Wip1−/>− MEFs (supplemental Fig. S4). Hence, various stresses can cause a more significant up-regulation of p38 and JNK in Wip1−/>− MEFs as compared with wild-type MEFs, suggesting that Wip1 acts as a negative regulator not only in the p38 pathway but also in the JNK pathway. A previous study has shown that tumors derived from Wip1−/>− MMTV-ErbB2 mice displayed an increased level of apoptosis compared with tumors derived from wild-type mice (12Bulavin D.V. Phillips C. Nannenga B. Timofeev O. Donehower L.A. Anderson C.W. Appella E. Fornace Jr., A.J. Nat. Genet. 2004; 36: 343-350Crossref PubMed Scopus (357) Google Scholar). To verify whether Wip1 negatively regulates apoptosis in cancer cells, we depleted Wip1 protein in U2OS and MCF-7 cells by two different siRNA oligonucleotides specific for Wip1 and monitored their effects on anisomycin-induced apoptosis (Fig. 4, A and B). Knockdown of Wip1 protein significantly sensitized MCF-7 cells to anisomycin-induced apoptosis (Fig. 4C). MCF-7 cells transfected with Wip1 siRNA 1 displayed 37.1% sub-G1 apoptotic cells in response to 12 h of 10 μm anisomycin treatment; whereas cells transfected with negative control siRNA only displayed 12.6% of apoptotic cells (Fig. 4C). Similar phenomena were also observed in U2OS cells. As shown in Fig. 4D, Hoechst staining revealed that Wip1 siRNA 1-transfected U2OS cells had normal nuclei, which were comparable with those transfected with negative control siRNA. However, after 5 h of 5 μm anisomycin treatment, U2OS cells transfected with Wip1 siRNA 1 exhibited more apoptotic cells than those transfected with negative control siRNA (Fig. 4D, white arrows). Accordingly, in Wip1 knocked down U2OS cells, the protein levels of both cleaved caspase-3 and cleaved PARP showed a significant increase in response to anisomycin treatment (Fig. 4E). More importantly, anisomycin induced a considerably stronger activation of JNK-c-Jun signaling in Wip1 siRNA 1-transfected U2OS cells than in negative control siRNA-transected cells (Fig. 4E). Consistently, in MCF-7 cells, knockdown of Wip1 using both siRNA oligonucleotides caused a moderately higher activation of JNK-c-Jun signaling upon anisomycin treatment (supplemental Fig. S5), as seen in Wip1−/>− MEFs. These results demonstrate that Wip1 inhibits stress-induced apoptosis not only in normal cells but also in cancer cells, suggesting that Wip1 has a role in preventing cells from apoptosis via the inhibition of JNK-c-Jun signaling. To elucidate whether Wip1 regulates the transcription of genes that function downstream of c-Jun, we stimulated wild-type and Wip1−/>− MEFs with anisomycin or etoposide and examined the transcription of several c-Jun downstream genes, such as Tp53, cyclin D1, and FasL (23Eferl R. Wagner E.F. Nat. Rev. Cancer. 2003; 3: 859-868Crossref PubMed Scopus (1664) Google Scholar). As shown in Fig. 5, the mRNA levels of FasL were significantly up-regulated in Wip1−/>− MEFs, but not in wild-type MEFs at 3 h post-anisomycin treatment or 1 h post-etoposide induction. However, the mRNA levels of Tp53 did not change in both wild-type and Wip1−/>− MEFs upon these two treatments (Fig. 5). Initially, we expected that the transcription of cyclin D1 would be up-regulated in parallel with the activation of c-Jun, because cyclin D1 is a well established downstream gene of c-Jun (24Bakiri L. Lallemand D. Bossy-Wetzel E. Yaniv M. EMBO J. 2000; 19: 2056-2068Crossref PubMed Scopus (337) Google Scholar). However, there were no significant change in the mRNA levels of cyclin D1 in both the wild-type and Wip1−/>− MEFs in response to stress, which suggests that other factors may counteract the transcription of cyclin D1 in this circumstance. Hence, our results demonstrated that the loss of Wip1 specifically stimulates FasL transcription upon external stresses. To address whether Wip1 functions through p38 and JNK in apoptosis, we inhibited p38 and JNK with specific kinase inhibitors. The p38 inhibitor SB203580 inhibited the phosphorylation of ATF-2 induced by anisomycin and etoposide (Fig. 6, A and B, upper panels). In addition, the JNK inhibitor SP600125 effectively inhibited the phosphorylation of c-Jun induced by anisomycin and etoposide (Fig. 6, A and B, lower panels), suggesting that SB203580 and SP600125 specifically inhibited the activity of p38 and JNK kinases, respectively. Importantly, as shown in Fig. 6C, the inhibition of either p38 or JNK MAPK partially rescued the enhanced apoptosis phenotype in Wip1−/>− MEFs upon anisomycin treatment. Simultaneous inhibition of both p38 and JNK MAPKs effectively rescued the susceptibility of Wip1−/>− MEFs to anisomycin-induced apoptosis, indicating that Wip1 regulates this response specifically via the p38 and JNK MAPK pathways (Fig. 6C). To further confirm the results derived from the cytotoxicity assay, we also examined the protein levels of both uncleaved and cleaved caspase-3 in Wip1−/>− MEFs; consistently, inhibition of p38 and/or JNK kinases significantly suppressed the induction of cleaved caspase-3 protein levels in Wip1−/>− cells upon anisomycin treatment (supplemental Fig. S6). On the other hand, the inhibition of both p38 and JNK MAPKs failed to rescue the higher susceptibility of Wip1−/>− MEFs to etoposide-induced apoptosis, suggesting that the p53-dependent, but not p38/JNK-dependent, apoptotic pathway may play a major role in etoposide-induced apoptosis (Fig. 6D). Classic MAPK signaling pathways are comprised of three components that are sequentially activated upon extracellular signals. We therefore could not exclude the possibility that the upstream kinases of p38 and JNK are responsible for the activation of downstream apoptotic signal in the absence of Wip1. To further address the molecular mechanism, we examined the activation of upstream kinases such as MKK4 and MKK7 upon anisomycin and etoposide treatments, respectively. As shown in supplemental Fig. S6, phospho-MKK4 increased significantly within 1 h in response to anisomycin treatment, whereas phospho-MKK7 increased at later time points. Compared with anisomycin, etoposide induced a modest accumulation of phosphorylation in both MKK4 and MKK7 at relatively late time points (supplemental Fig. S6). Markedly, both anisomycin and etoposide treatments stimulated a more substantial increase of phospho-MKK4 protein levels in Wip1−/>− MEFs than in wild type (Fig. 6, E and F). However, the induction of phospho-MKK7 protein levels had no significant difference between wild-type and Wip1−/>− MEFs. Our results demonstrated that both anisomycin and etoposide selectively stimulated substantial phosphorylation of MKK4 but not MKK7 in Wip1−/>− MEFs than in wild-type MEFs, indicating that MKK4 may be preferentially regulated by Wip1 upon anisomycin and etoposide stimulations. It was reported that the activation of MKK4 kinase is dependent on the phosphorylation at the Ser257 and Thr261 residues (25Kyriakis J.M. Avruch J. Physiol. Rev. 2001; 81: 807-869Crossref PubMed Scopus (2897) Google Scholar, 26Herskowitz I. Cell. 1995; 80: 187-197Abstract Full Text PDF PubMed Scopus (867) Google Scholar). To further investigate how Wip1 regulates the phosphorylation of MKK4, we performed an in vitro phosphatase assay using phospho-peptides derived from MKK4. Our results showed that Wip1 preferentially dephosphorylated the peptide containing the Thr(P)261 residue of MKK4 but had little effect on the peptide containing the Ser(P)257 residue (su" @default.
• W2003199401 created "2016-06-24" @default.
• W2003199401 creator A5044107486 @default.
• W2003199401 creator A5063840832 @default.
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