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• W2062489468 abstract "Nuclear factor κB (NF-κB) transcriptionally activates genes that promote immunity and cell survival. Activation of NF-κB is induced by an IκB kinase (IKK) complex that phosphorylates and promotes dissociation of IκB from NF-κB, which then translocates into the nucleus. Activation of phosphatidylinositol (PI) 3-kinase/Akt signaling by tumor necrosis factor (TNF) activates IKK and NF-κB. The present study shows that PTEN, a tumor suppressor that inhibits PI 3-kinase function, impairs TNF activation of Akt and the IKK complex in 293 cells. Transient expression of PTEN suppressed IKK activation and TNF-induced NF-κB DNA binding and transactivation. Studies were conducted with PC-3 prostate cancer cells that do not express PTEN and DU145 prostate cancer cells that express PTEN. TNF activated Akt in PC-3 cells, but not in DU145 cells, and the ability of TNF to activate NF-κB was blocked by pharmacological inhibition of PI 3-kinase activity in PC-3 cells, but not in DU145 cells. Expression of PTEN in PC-3 cells to a level comparable with that endogenously present in DU145 cells inhibited TNF activation of NF-κB. The cell type-specific ability of PTEN to negatively regulate the PI 3-kinase/AKT/NF-κB pathway may be important to its tumor suppressor activity. Nuclear factor κB (NF-κB) transcriptionally activates genes that promote immunity and cell survival. Activation of NF-κB is induced by an IκB kinase (IKK) complex that phosphorylates and promotes dissociation of IκB from NF-κB, which then translocates into the nucleus. Activation of phosphatidylinositol (PI) 3-kinase/Akt signaling by tumor necrosis factor (TNF) activates IKK and NF-κB. The present study shows that PTEN, a tumor suppressor that inhibits PI 3-kinase function, impairs TNF activation of Akt and the IKK complex in 293 cells. Transient expression of PTEN suppressed IKK activation and TNF-induced NF-κB DNA binding and transactivation. Studies were conducted with PC-3 prostate cancer cells that do not express PTEN and DU145 prostate cancer cells that express PTEN. TNF activated Akt in PC-3 cells, but not in DU145 cells, and the ability of TNF to activate NF-κB was blocked by pharmacological inhibition of PI 3-kinase activity in PC-3 cells, but not in DU145 cells. Expression of PTEN in PC-3 cells to a level comparable with that endogenously present in DU145 cells inhibited TNF activation of NF-κB. The cell type-specific ability of PTEN to negatively regulate the PI 3-kinase/AKT/NF-κB pathway may be important to its tumor suppressor activity. tumor necrosis factor phosphatidylinositol nuclear factor κB IκB kinase tumor necrosis factor receptor NF-κB-inducing kinase glutathione S-transferase electrophoretic mobility shift assay TNF-α1 is a pleiotropic cytokine that promotes immunity, fibroblast proliferation and wound repair, insulin resistance, and the syndrome of wasting and malnutrition known as cachexia in some chronic diseases (1Beutler B. Bazzoni F. Blood Cells Mol. Dis. 1998; 24: 216-230Crossref PubMed Scopus (79) Google Scholar, 2Aggarwal B.B. Natarajan K. Eur. Cytokine Netw. 1996; 7: 93-124PubMed Google Scholar, 3Peraldi P. Spiegelman B. Mol. Cell. Biochem. 1998; 182: 169-175Crossref PubMed Scopus (241) Google Scholar). TNF also induces programmed cell death of many transformed cells. Unfortunately, however, many malignancies are resistant to the cytotoxic activity of TNF. Such resistance may derive from the coincident activation of signaling pathways that lead to apoptosis or cell survival (4Wallach D. Varfolomeev E.E. Malinin N.L. Goltsev Y.V. Kovalenko A.V. Boldin M.P. Annu. Rev. Immunol. 1999; 17: 331-367Crossref PubMed Scopus (1125) Google Scholar). Whether or not a cancer cell succumbs to TNF is largely determined by whether the life or death signaling events induced by the cytokine predominate in a cell. TNF promotes its actions by binding to TNF receptor (TNFR) type 1 and TNFR2 (4Wallach D. Varfolomeev E.E. Malinin N.L. Goltsev Y.V. Kovalenko A.V. Boldin M.P. Annu. Rev. Immunol. 1999; 17: 331-367Crossref PubMed Scopus (1125) Google Scholar). Binding to TNFR1 activates the receptor such that it recruits proteins that contain a recognition motif, the death domain. The first of these proteins to engage the receptor is the TNFR-associated death domain protein, which subsequently interacts with Fas-associated death domain protein. Fas-associated death domain protein binds the death domain of procaspase 8 and promotes its activation, thereby inducing cell death. The signaling cascade downstream of TNFR1 bifurcates at TNFR-associated death domain protein, which also plays an obligate role in activation of NF-κB (5Hsu H. Xiong J. Goeddel D.V. Cell. 1995; 81: 495-504Abstract Full Text PDF PubMed Scopus (1747) Google Scholar). TNFR-associated death domain protein, through association with TNFR-associated factor 2 and in concert with receptor-interacting protein, activates a serine-threonine kinase cascade (4Wallach D. Varfolomeev E.E. Malinin N.L. Goltsev Y.V. Kovalenko A.V. Boldin M.P. Annu. Rev. Immunol. 1999; 17: 331-367Crossref PubMed Scopus (1125) Google Scholar, 5Hsu H. Xiong J. Goeddel D.V. Cell. 1995; 81: 495-504Abstract Full Text PDF PubMed Scopus (1747) Google Scholar). One component of this cascade is NF-κB-inducing kinase (NIK) (6Malinin N.L. Boldin M.P. Kovalenko A.V. Wallach D. Nature. 1997; 385: 540-544Crossref PubMed Scopus (1165) Google Scholar), which phosphorylates and plays a role in the activation of the IKK complex in cells (4Wallach D. Varfolomeev E.E. Malinin N.L. Goltsev Y.V. Kovalenko A.V. Boldin M.P. Annu. Rev. Immunol. 1999; 17: 331-367Crossref PubMed Scopus (1125) Google Scholar, 7Regnier C.H. Song H.Y. Gao X. Goeddel D.V. Cao Z. Rothe M. Cell. 1997; 90: 373-383Abstract Full Text Full Text PDF PubMed Scopus (1072) Google Scholar, 8DiDonato J.A. Hayakawa M. Rothwarf D.M. Zandi E. Karin M. Nature. 1997; 388: 548-554Crossref PubMed Scopus (1913) Google Scholar). A second serine-threonine kinase cascade can also play a role in NF-κB activation (9Ozes O.N. Mayo L.D. Gustin J.A. Pfeffer S.R. Pfeffer L.M. Donner D.B. Nature. 1999; 401: 82-86Crossref PubMed Scopus (1898) Google Scholar, 10Kane L.P. Shapiro V.S. Stokoe D. Weiss A. Curr. Biol. 1999; 9: 601-604Abstract Full Text Full Text PDF PubMed Scopus (740) Google Scholar, 11Romashkova J.A. Makarov S.S. Nature. 1999; 401: 86-90Crossref PubMed Scopus (1667) Google Scholar). Binding of TNF to TNFR1 activates phosphatidylinositol (PI) 3-kinase and its downstream target, the Akt/ protein kinase B serine-threonine kinase (9Ozes O.N. Mayo L.D. Gustin J.A. Pfeffer S.R. Pfeffer L.M. Donner D.B. Nature. 1999; 401: 82-86Crossref PubMed Scopus (1898) Google Scholar). Active Akt phosphorylates IKKα, which promotes activation of its heterodimeric partner, IKKβ, in IKKα/IKKβ complexes (12Tanaka M. Fuentes M.E Yamaguchi K. Durnin M.H. Dalrymple S.A. Hardy K.L. Goeddel D.V. Immunity. 1999; 10: 421-429Abstract Full Text Full Text PDF PubMed Scopus (495) Google Scholar, 13O'Mahoney A. Lin X. Geleziunas R. Green W.C. Mol. Cell. Biol. 2000; 20: 1170-1178Crossref PubMed Scopus (102) Google Scholar, 14Yamamoto Y. Yin M.J. Gaynor R.B. Mol. Cell. Biol. 2000; 20: 3655-3656Crossref PubMed Scopus (52) Google Scholar). The IKK complex phosphorylates IκB, thereby promoting its dissociation from NF-κB (15Bauerle P.A. Baltimore D. Cell. 1996; 87: 13-20Abstract Full Text Full Text PDF PubMed Scopus (2931) Google Scholar). Dissociation of IκB unmasks the nuclear localization sequence of NF-κB, permitting it to enter the nucleus, where it binds DNA and activates genes that promote immunity and cell survival (15Bauerle P.A. Baltimore D. Cell. 1996; 87: 13-20Abstract Full Text Full Text PDF PubMed Scopus (2931) Google Scholar, 16Wang C.-Y. Mayo M.W. Baldwin Jr., A.S. Science. 1996; 274: 784-787Crossref PubMed Scopus (2512) Google Scholar, 17Van Antwerp D.J. Martin S.J. Kafri T. Green D.R. Verma I.M. Science. 1996; 274: 787-789Crossref PubMed Scopus (2449) Google Scholar, 18Wang C.-Y. Mayo M.W. Korneluk R.G. Goeddel D.V. Baldwin Jr., A.S. Science. 1998; 281: 1680-1683Crossref PubMed Scopus (2580) Google Scholar). Thus, PI 3-kinase/Akt signaling may render cells resistant to TNF-mediated toxicity through activation of NF-κB. Activated PI 3-kinase promotes the formation of the lipid second messenger phosphatidylinositol 3-phosphate, which plays an essential role in activation of Akt (19Ruderman N.B. Kapeller R. White M.F. Cantley L.C. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 1411-1415Crossref PubMed Scopus (395) Google Scholar, 20Alessi D.R. Downes C.P. Biochim. Biophys. Acta. 1998; 1436: 151-164Crossref PubMed Scopus (191) Google Scholar, 21Stokoe D. Stephensm L.R. Copeland T. Gaffney P.R. Reese C.B. Painter G.F. Holmes A.B. McCormick F. Hawkins P.T. Science. 1997; 277: 567-570Crossref PubMed Scopus (1048) Google Scholar, 22Stephens L. Anderson K. Stokoe D. Erdjument-Bromage H. Painter G.F. Holmes A.B. Gaffney P.R. Reese C.B. McCormick F. Tempst P. Coadwell J. Hawkins P.T. Science. 1998; 279: 710-714Crossref PubMed Scopus (914) Google Scholar). The PTEN tumor suppressor is a dual specificity phosphatase that impairs survival signaling emanating from PI 3-kinase by dephosphorylating and inactivating phosphatidylinositol 3-phosphate (23Stambolic V. Suzuki A. de la Pompa J.L. Brother G.M. Mirtsos C. Sasaki T. Ruland J. Penninger J.M. Siderovski D.P. Mak T.W. Cell. 1998; 95: 29-39Abstract Full Text Full Text PDF PubMed Scopus (2107) Google Scholar, 24Myers M.P. Pass I. Batty I.H. Van der Kaay J. Stolarov J.P. Hemmings B.A. Wigler M.H. Downes C.P. Tonks N.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13513-13518Crossref PubMed Scopus (1007) Google Scholar, 25Maehama T. Dixon J.E. J. Biol. Chem. 1998; 273: 13375-13378Abstract Full Text Full Text PDF PubMed Scopus (2601) Google Scholar). These observations led us to consider the possibility that PTEN might inhibit activation of NF-κB by TNF. Demonstration of a PTEN/NF-κB connection would identify a mechanism through which PTEN could manifest its tumor suppressor activity. Recombinant human tumor necrosis factor was a gift from Genentech Inc. (South San Francisco, CA). Antibodies to IKKγ, IKKα, and PTEN were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-phospho-Akt and anti-Akt were from New England Biolabs (Beverly, MA), and anti-RelA (p65) was from Upstate Biotechnology (Lake Placid, NY). 293 cells were transfected using the calcium phosphate procedure. Routinely, transfection efficiencies of >90% were obtained based on control experiments with β-galactosidase. PC-3 cells were transfected using the GenePorter reagent according to the directions of the manufacturer (Gene Therapy Systems, San Diego, CA). Control experiments with β-galactosidase showed that the PC-3 cell line was transfected with an efficiency of 27%. After transfections, cells were harvested and lysed by incubation for 30 min in ice-cold lysis buffer (50 mm Hepes, pH 7.0, 150 mm NaCl, 10% glycerol, 1.2% Triton X-100, 1.5 mm MgCl2, 1 m EGTA, 10 mm sodium pyrophosphate, 100 mm NaF, 1 mm phenylmethylsulfonyl fluoride, 0.15 unit/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin A, 1 mm sodium orthovanadate, and 1 mm dithiothreitol). Lysates were fractionated by SDS-polyacrylamide gel electrophoresis and transferred to Immobilon-P to prepare Western blots, or IKKγ was immunoprecipitated from the lysates. For immunoprecipitations, 1 µg of anti-IKKγ was preadsorbed to protein A/G-Sepharose for 1 h and then added to 750 µg of cell lysate. After a 4-h shake at 4 °C, incubates were centrifuged, and the pellets were washed twice with lysis buffer and twice with kinase assay buffer (10 mmHepes, pH 7.4, 1 mm MnCl2, 5 mmMgCl2, 12.5 mm β-glycero-2-phosphate, 50 mm sodium orthovanadate, 2 mm NaF, and 50 mm dithiothreitol). Immunoprecipitates in 15 µl of kinase assay buffer were then incubated with 0.25 µCi/µl [γ-32P]ATP and 1 µg of recombinant GST-IκBα(1–51) at 30 °C for 30 min. Reactions were stopped by being heated at 105 °C for 5 min in SDS-polyacrylamide gel electrophoresis loading buffer and were fractionated on 10% polyacrylamide gels and transferred to Immobilon-P. EMSAs were conducted on 6 µg of protein from 293 cells transiently transfected with pCMV expression constructs. NF-κB DNA binding was assayed using a double-stranded32P-labeleled κB probe (5′-AGTTGAGGGACTTTCCCAGG-3′). For reporter gene assays, cells were transiently co-transfected with β-galactosidase and 3xNF-κB, a reporter plasmid containing three consensus NF-κB binding sites inserted upstream of the luciferase reporter plasmid pGL2. Forty-eight h after transfection, cells were incubated with vehicle or TNF for 6 h before assaying luciferase activity, which was normalized on the basis of β-galactosidase activity. Because our previous work showed that TNF activates a PI 3-kinase/Akt signaling pathway that plays a role in activation of NF-κB in 293 embryonic kidney cells, we determined whether PTEN would inhibit this process. 293 cells transfected with an empty vector or PTEN were stimulated with TNF. Akt activation was determined by probing a Western blot of transfected cell lysates with an antibody specifically directed against the active form of Akt, which is phosphorylated on serine 473. As illustrated in Fig.1 A, TNF increased Akt phosphorylation in cells transfected with empty vector. However, basal Akt activity was diminished in cells overexpressing PTEN, and the capacity of TNF to activate Akt above the basal level was abrogated by transient PTEN expression. The observations just described are reinforced by the demonstration that pharmacological inhibition of PI 3-kinase using LY294002 blocked TNF-promoted activation (phosphorylation) of Akt (Fig. 1 B). The results in Fig. 1show that activation of Akt by TNF is blocked by the PTEN phosphatase, which dephosphorylates phosphatidylinositol 3-phosphate generated by PI 3-kinase or by inhibition of the catalytic activity of PI 3-kinase with LY294002. Because our previous observations showed that Akt activates the IKK complex (9Ozes O.N. Mayo L.D. Gustin J.A. Pfeffer S.R. Pfeffer L.M. Donner D.B. Nature. 1999; 401: 82-86Crossref PubMed Scopus (1898) Google Scholar), the results shown above indicated that PTEN would inhibit the ability of TNF to activate this complex. To test this, IKK kinase activity was assayed, based on phosphorylation of the IKK substrate IκB, in vector-transfected 293 cells and in 293 cells overexpressing PTEN. Because the IKK complex is part of a high molecular weight signaling complex that also includes IKKγ (26Rothwarf D.M. Zandi E. Natoli G. Karin M. Nature. 1998; 395: 297-300Crossref PubMed Scopus (853) Google Scholar), we incubated cells in the absence or presence of TNF and then immunoprecipitated IKKγ from cell lysates. The immunoprecipitates were subjected to an in vitro kinase assay using GST-IκBα(1–51) as substrate. Western blot analysis of equal amounts of IKK showed that TNF induced substantial phosphorylation of GST-IκBα in vector-transfected cells, and this was blocked in cells transiently overexpressing PTEN (Fig. 2). The phosphorylation and dissociation of IκBα from NF-κB unmasks the nuclear localization sequence of the transcription factor, thereby allowing it to translocate from the cytoplasm into the nucleus, where it binds DNA (15Bauerle P.A. Baltimore D. Cell. 1996; 87: 13-20Abstract Full Text Full Text PDF PubMed Scopus (2931) Google Scholar). To determine whether PTEN would inhibit NF-κB DNA binding, 293 cells transfected with empty vector or transiently overexpressing PTEN were serum-starved and then treated with 1 nm TNF or vehicle for 30 min. Proteins from treated cells were used for EMSAs. As illustrated by the results in Fig.3, top panel, TNF stimulated NF-κB DNA binding in cells transfected with an empty vector. However, in cells overexpressing PTEN, TNF-induced DNA binding was abrogated. A Western blot prepared from the extracts used for EMSA was probed with an antibody to p65 Rel, one of the subunits of NF-κB. The data (Fig. 3, bottom panel) show that equivalent amounts of protein were used for the EMSA. Overall, the data in Fig. 3demonstrate that PTEN blocked TNF-induced NF-κB DNA binding in 293 cells. The observations described above indicate that TNF-induced PI 3-kinase/Akt signaling activates the IKK complex, thereby promoting NF-κB DNA binding, and that PTEN inhibits these events. This led us to test whether TNF-induced transactivation of NF-κB could be inhibited by PTEN. A reporter gene assay was conducted in which 293 cells transfected with empty vector or transiently overexpressing PTEN were stimulated with TNF before NF-κB transactivation was assayed. As illustrated by Fig. 4 A, TNF activated NF-κB, and this was suppressed in cells expressing PTEN. The ability of PTEN to inhibit TNF-induced activation of NF-κB was also demonstrable in MCF-7 breast cancer cells (Fig. 4 B), showing that this effect is not unique to the 293 cell line. Because PTEN is mutated or deleted in many types of malignancies, we extended our investigation to evaluate NF-κB activation in prostate cancer cells in which functional PTEN was present (DU145 cells) or absent (PC-3 cells) (27McMenamin M.E. Soung P. Perera S. Kaplan I. Loda M. Sellers W.R. Cancer Res. 1999; 59: 4291-4296PubMed Google Scholar). The PTEN status of the DU145 and PC-3 cells used in the present study was confirmed by Western blot analysis (Fig.5). Western blot analysis of proteins fractionated from lysates of control or TNF-treated cells also showed that TNF did not activate Akt in DU145 cells but induced such activity in PC-3 cells (Fig. 5). Phosphorylation and activation of IKKα by induction of PI 3-kinase/Akt signaling is one mechanism through which TNF can activate NF-κB (9Ozes O.N. Mayo L.D. Gustin J.A. Pfeffer S.R. Pfeffer L.M. Donner D.B. Nature. 1999; 401: 82-86Crossref PubMed Scopus (1898) Google Scholar). The ability of TNF to activate Akt in PC-3 cells, but not in DU145 cells, suggested that this mechanism of NF-κB activation might be of significance in the former cell type, but not in the latter. To test this hypothesis, PC-3 and DU145 cells were treated with LY294002 and then with vehicle or TNF. EMSAs revealed that TNF induction of NF-κB DNA binding in PC-3 cells was sensitive to inhibition by LY294002 (Fig. 6). Results averaged from three independent experiments showed that TNF increased NF-κB DNA binding 6.6 ± 0.7-fold relative to control and that this was not affected by LY294002 alone (1.0 ± 0.2-fold relative to control). However, treatment of PC-3 cells with LY294002 suppressed the ability of TNF to increase NF-κB DNA binding (3.0 ± 0.4-fold stimulation relative to control). In contrast, TNF induction of NF-κB DNA binding in DU145 cells was not affected by LY294002. In DU145 cells, TNF increased NF-κB DNA binding 4.8 ± 0.4-fold relative to control. LY294002 had no effect of NF-κB DNA binding (1.2 ± 0.4-fold relative to control) and did not suppress NF-κB DNA binding induced by TNF (4.7 ± 0.6-fold relative to control). These observations show that the ability of TNF to utilize PI 3-kinase/Akt signaling to activate NF-κB is cell-specific and that other mechanisms can be used by TNF to effect activation of NF-κB.Figure 6Effect of PI 3-kinase inhibition on NF-κB DNA binding in prostate cancer cells. DU145 and PC-3 prostate cancer cells were treated with vehicle or LY294002 and then incubated in the absence or presence of TNF. NF-κB DNA binding was then assayed by EMSAs using 6 µg of protein from whole cell lysates. Anti-p65 was used for supershifting to demonstrate that p65 Rel was in the DNA binding complex.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The observations illustrated in Figs. 5 and 6 led us to test whether expression of PTEN in PC-3 cells would inhibit activation of NF-κB by TNF. To accomplish this, we transiently expressed PTEN to varying levels in PC-3 cells. Next, we compared the level of expression to that in DU145 cells by Western blot analysis. Western blot analysis revealed that transfection of PC-3 cells with 0.5 or 1 µg of a PTEN expression vector led to a level of expression comparable to that observed in untransfected DU145 cells (Fig.7 A). A gene reporter assay using PC-3 cells transfected with an empty vector or PTEN showed that PTEN inhibited the capacity of TNF to induce NF-κB transactivation (Fig. 7 B). Activation of PI 3-kinase and Akt by cytokines or growth factors promotes cell survival (28Ahmed N.N. Grimes H.L. Bellacosa A. Chan T.O. Tsichlis P.N. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3627-3632Crossref PubMed Scopus (486) Google Scholar, 29Kennedy S.G. Wagner A.J. Conzen S.D. Jordan J. Bellacosa A. Tsischlis P.N. Hay N. Genes Dev. 1997; 11: 701-713Crossref PubMed Scopus (980) Google Scholar, 30Dudek H. Datta S.R. Franke T.F. Birnbaum M.J. Yao R. Cooper G.M. Segal R.A. Kaplan D.R. Greenberg M.E. Science. 1997; 275: 628-630Crossref PubMed Scopus (2218) Google Scholar, 31Kulik G. Klippel A. Weber M.J. Mol. 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Science. 1998; 282: 1318-1321Crossref PubMed Scopus (2735) Google Scholar), and FKHRL1, a member of the forkhead family of transcription factors, suppressing its ability to induce expression of genes that promote apoptosis (36Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5434) Google Scholar). Akt also has the capacity to delay the onset of p53-dependent apoptosis in cell cultures (37Sabbatini P. McCormick F. J. Biol. Chem. 1999; 274: 24263-24269Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). PI 3-kinase/Akt signaling can also play a role in NF-κB activation (9Ozes O.N. Mayo L.D. Gustin J.A. Pfeffer S.R. Pfeffer L.M. Donner D.B. Nature. 1999; 401: 82-86Crossref PubMed Scopus (1898) Google Scholar, 10Kane L.P. Shapiro V.S. Stokoe D. Weiss A. Curr. Biol. 1999; 9: 601-604Abstract Full Text Full Text PDF PubMed Scopus (740) Google Scholar, 11Romashkova J.A. Makarov S.S. Nature. 1999; 401: 86-90Crossref PubMed Scopus (1667) Google Scholar) and thereby promote the up-regulation of antiapoptotic genes and cell survival (18Wang C.-Y. Mayo M.W. Korneluk R.G. Goeddel D.V. Baldwin Jr., A.S. Science. 1998; 281: 1680-1683Crossref PubMed Scopus (2580) Google Scholar). Activation of NF-κB by phorbol esters and lipolysaccharide is mediated by PI 3-kinase and is thus inhibited by wortmannin (38Manna S.K. Aggarwal B.B. FEBS Lett. 2000; 473: 113-118Crossref PubMed Scopus (54) Google Scholar). Signaling through CD40 (39Andjelic S. Shia C. Suzuki H. Kadowaki T. Koyasu S. Liou H.C. J. Immunol. 2000; 165: 3660-3667Crossref Scopus (71) Google Scholar), interleukin-1 (40Sizemore N. Leung S. Stark G. Mol. Cell. Biol. 1999; 19: 4798-4805Crossref PubMed Google Scholar) and G-protein-coupled receptors (41Xie P. Browning D.D. Hay N. Mackman N. Ye R.D. J. Biol. Chem. 2000; 275: 24907-24914Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar) also induces PI 3-kinase/Akt signaling, leading to activation of NF-κB. Activation of NF-κB by PI 3-kinase/Akt can be effected not only through activation of the IKK complex (9Ozes O.N. Mayo L.D. Gustin J.A. Pfeffer S.R. Pfeffer L.M. Donner D.B. Nature. 1999; 401: 82-86Crossref PubMed Scopus (1898) Google Scholar) but also by stimulation of the transactivation potential of the RelA/p65 subunit of NF-κB (40Sizemore N. Leung S. Stark G. Mol. Cell. Biol. 1999; 19: 4798-4805Crossref PubMed Google Scholar, 42Madrid L.V. Wang C.-Y. Guttridge D.C. Schottelius A.J.G. Baldwin Jr., A.S. Mayo M.W. Mol. Cell. Biol. 2000; 20: 1626-1638Crossref PubMed Scopus (587) Google Scholar). Furthermore, the effect of PI 3-kinase Akt signaling on NF-κB activation is cell type-specific. In human umbilical vein endothelial cells, a PI 3-kinase/Akt pathway activated by TNF or interleukin-1 inhibits apoptosis but does not activate NF-κB (43Madge L.A. Pober J.S. J. Biol. Chem. 2000; 275: 15458-15465Abstract Full Text Full Text PDF PubMed Scopus (189) Google Scholar). NIK and PI 3-kinase/Akt also do not mediate NF-κB activation in embryonic fibroblasts from NIK-mutant mice (44Matsushima A. Kaisho T. Rennert P.D. Nakano H. Kurosawa K. Uchida D. Takeda K. Akira S. Matsumoto M. J. Exp. Med. 2001; 193: 631-636Crossref PubMed Scopus (179) Google Scholar). The cell type-specific activation of NF-κB is further emphasized by the demonstration that NF-κB activation in dermal fibroblasts is not mediated by NIK/TNFR-associated factor 2 signaling but rather by a surrogate pathway in which acidic sphingomyelinase and calpain activities are implicated (45Kouba D.J. Nakano H. Nishiyama T. Kang J. Uitto J. Mauviel A. J. Biol. Chem. 2001; 276: 6214-6224Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). However, epidermal keratinocytes utilize the previously defined NIK-dependent pathway to activate NF-κB (45Kouba D.J. Nakano H. Nishiyama T. Kang J. Uitto J. Mauviel A. J. Biol. Chem. 2001; 276: 6214-6224Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). A compelling indication of how cell type-specific mechanisms mediate NF-κB activation is shown in our observation that PI 3-kinase/Akt signaling plays a role in NF-κB activation in PC-3 but not DU145 prostate cancer cells. The demonstration that the mechanisms that activate NF-κB are diverse and cell type-specific indicates that NF-κB activation is unlikely to be inhibited by a single mechanism. PTEN is a dual specificity phosphatase that dephosphorylates inositol lipids at the D3 position of the inositol ring, suggesting that PTEN antagonizes PI 3-kinase and phosphatidylinositol 3-phosphate signaling (23Stambolic V. Suzuki A. de la Pompa J.L. Brother G.M. Mirtsos C. Sasaki T. Ruland J. Penninger J.M. Siderovski D.P. Mak T.W. Cell. 1998; 95: 29-39Abstract Full Text Full Text PDF PubMed Scopus (2107) Google Scholar, 24Myers M.P. Pass I. Batty I.H. Van der Kaay J. Stolarov J.P. Hemmings B.A. Wigler M.H. Downes C.P. Tonks N.P. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13513-13518Crossref PubMed Scopus (1007) Google Scholar, 25Maehama T. Dixon J.E. J. Biol. Chem. 1998; 273: 13375-13378Abstract Full Text Full Text PDF PubMed Scopus (2601) Google Scholar). The PTEN gene is mutated in 40–50% of high-grade gliomas and in prostate, endometrial, breast, and lung cancers, as well as in other tumor types (46Li J. Yen C. Liaw D. Podsypanina K. Bose S. Wang S.I. Puc J. Miliaresis C. Rodgers L. McCombie R. Bigner S.H. Giovanella B.C. Ittmann M. Tycko B. Hibshoosh H. Wigler M.H. Parsons R. Science. 1997; 275: 1943-1947Crossref PubMed Scopus (4285) Google Scholar, 47Steck P.A. Pershouse M.A. Jasser S.A. Yng W.K. 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Reed J.C. Science. 1998; 282: 1318-1321Crossref PubMed Scopus (2735) Google Scholar, 36Brunet A. Bonni A. Zigmond M.J. Lin M.Z. Juo P. Hu L.S. Anderson M.J. Arden K.C. Blenis J. Greenberg M.E. Cell. 1999; 96: 857-868Abstract Full Text Full Text PDF PubMed Scopus (5434) Google Scholar, 57Datta S.R. Brunet A. Greenberg M.E. Genes Dev. 1999; 13: 2905-2927Crossref PubMed Scopus (3721) Google Scholar). Our demonstration that PI 3-kinase/Akt signaling promotes activation of IKKα and NF-κB (9Ozes O.N. Mayo L.D. Gustin J.A. Pfeffer S.R. Pfeffer L.M. Donner D.B. Nature. 1999; 401: 82-86Crossref PubMed Scopus (1898) Google Scholar) and the ability of PTEN to oppositely affect the PI 3-kinase/Akt survival pathway suggested that one manifestation of the tumor suppressor activity of PTEN would be an ability to suppress NF-κB activation by TNF. The observations presented here confirm this hypothesis, although, as suggested above, such suppression is cell type-specific. One manifestation of such cell type specificity is the ability of TNF to activate Akt in PC-3 cells but not in DU145 cells. Consequently, PTEN inhibits activation of NF-κB by TNF in PC-3 cells, but not in DU145 cells. Further confirmation that the transduction pathways used by TNF to activate NF-κB are cell type-dependent is found in the observation that the protein kinase C inhibitor calphostin C blocked NF-κB activation by TNF or interleukin-1 in Jurkat or NIH3T3 cells but not in MCF-7 A/Z cells (58Bonizzi G. Piette J. Schoonbroodt S. Merville M.P. Bours V. Biochem. Pharmacol. 1999; 57: 713-720Crossref PubMed Scopus (36) Google Scholar). Atypical isoforms of protein kinase C can participate in activation of NF-κB through selective action on the IKKβ subunit of the IKK complex (59Lin X. O'Mahoney A. Mu Y. Geleziunas R. Green W.C. Mol. Cell. Biol. 2000; 20: 2933-2940Crossref PubMed Scopus (230) Google Scholar). These observations suggest that signaling pathways not involving PI 3-kinase/Akt signaling and therefore insensitive to suppression by PTEN are likely to play a role in NF-κB activation. While this work was under review, it was reported that PTEN could inhibit cytokine-induced NF-κB activation without interfering with the IκB degradation pathway (60Koul D. Yao Y. Abbruzze J.L. Yung W.K.A. Reddy S.A.G. J. Biol. Chem. 2001; 276: 11402-11408Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Thus, PI 3-kinase can use a mechanism that may be independent of the function of the IKK complex to affect the activity of NF-κB (40Sizemore N. Leung S. Stark G. Mol. Cell. Biol. 1999; 19: 4798-4805Crossref PubMed Google Scholar, 60Koul D. Yao Y. Abbruzze J.L. Yung W.K.A. Reddy S.A.G. J. Biol. Chem. 2001; 276: 11402-11408Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). These observations reinforce the view that much remains to be understood regarding the mechanisms that activate and regulate NF-κB. The ability of PTEN to suppress that component of NF-κB activation mediated by PI 3-kinase/Akt signaling is important. NF-κB activation renders cells resistant to the cytotoxic activity of TNF and chemotherapy by inducing the expression of survival genes (16Wang C.-Y. Mayo M.W. Baldwin Jr., A.S. Science. 1996; 274: 784-787Crossref PubMed Scopus (2512) Google Scholar, 17Van Antwerp D.J. Martin S.J. Kafri T. Green D.R. Verma I.M. Science. 1996; 274: 787-789Crossref PubMed Scopus (2449) Google Scholar, 18Wang C.-Y. Mayo M.W. Korneluk R.G. Goeddel D.V. Baldwin Jr., A.S. Science. 1998; 281: 1680-1683Crossref PubMed Scopus (2580) Google Scholar). NF-κB activity also suppresses MyoD expression (61Guttridge D.C. Mayo M.W. Madrid L.V. Wang C.-Y. Baldwin Jr., A.S. Science. 2000; 289: 2363-2366Crossref PubMed Scopus (763) Google Scholar) and may thereby promote the muscle wasting associated with cancer, AIDS, and other chronic diseases. The demonstration that PTEN can suppress NF-κB activation by TNF suggests that its tumor suppressor activity derives, in part, through its capacity to inhibit expression of genes that suppress the function of the cellular apoptotic machinery. Furthermore, PTEN may impair the ability of cytokines, such as TNF, to produce metabolic alterations, such as cachexia, that often lead to host mortality." @default.
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• W2062489468 date "2001-07-01" @default.
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• W2062489468 title "The PTEN Tumor Suppressor Protein Inhibits Tumor Necrosis Factor-induced Nuclear Factor κB Activity" @default.
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