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Cytokine-induced Apoptosis in Epithelial HT-29 Cells Is Independent of Nitric Oxide Formation

EVIDENCE FOR AN INTERLEUKIN-13-DRIVEN PHOSPHATIDYLINOSITOL 3-KINASE-DEPENDENT SURVIVAL MECHANISM*
  • Karen Wright
    Affiliations
    From the Department of Pharmacy and Pharmacology, Bath University, Claverton Down, Bath, BA2 7AY, United Kingdom
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  • George Kolios
    Affiliations
    From the Department of Pharmacy and Pharmacology, Bath University, Claverton Down, Bath, BA2 7AY, United Kingdom
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  • Author Footnotes
    ‡ Current address: Novartis Horsham Research Centre, Wimblehurst Rd., Horsham, West Sussex, RH125AB, UK.
    John Westwick
    Footnotes
    ‡ Current address: Novartis Horsham Research Centre, Wimblehurst Rd., Horsham, West Sussex, RH125AB, UK.
    Affiliations
    From the Department of Pharmacy and Pharmacology, Bath University, Claverton Down, Bath, BA2 7AY, United Kingdom
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  • Stephen G. Ward
    Correspondence
    To whom correspondence should be addressed. Tel.: 44-0-1225-323641; Fax: 44-0-1225-826114;
    Affiliations
    From the Department of Pharmacy and Pharmacology, Bath University, Claverton Down, Bath, BA2 7AY, United Kingdom
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  • Author Footnotes
    * The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
    ‡ Current address: Novartis Horsham Research Centre, Wimblehurst Rd., Horsham, West Sussex, RH125AB, UK.
Open AccessPublished:June 11, 1999DOI:https://doi.org/10.1074/jbc.274.24.17193
      A combination of the pro-inflammatory cytokines interleukin (IL)-1α, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α induces nitric oxide synthase mRNA expression and nitric oxide (NO) generation in the human colon carcinoma cell line HT-29. This can be inhibited by pretreatment with IL-13 via a phosphatidylinositol (PI) 3-kinase-dependent mechanism (Wright, K., Ward, S. G., Kolios, G., and Westwick, J. (1997)J. Biol. Chem. 272, 12626–12633). Since NO has been implicated in regulating mechanisms leading to cell death, while activation of PI 3-kinase-dependent signaling cascades are thought to be involved with promoting cell survival events, we have investigated the outcome of these cytokine treatments on apoptosis and cell survival of HT-29 cells. Initiation of apoptosis can be achieved by the combinations of IFN-γ/TNF-α, IFN-γ/CD95, IL-1α/IFN-γ, and IL-1α/IFN-γ/TNF-α to varying extents. Induction of apoptotic markers by HT-29 cells in response to cytokine treatment is not dependent on NO production. Pretreatment with IL-13 protects against IL-1α/IFN-γ/TNF-α- and IFN-γ/TNF-α- as well as IFN-γ/CD95-induced (but not IL-1α/IFN-γ-induced) cell death. In addition, IFN-γ/TNF-α and IL-1α/IFN-γ/TNF-α stimulate activation of caspase-8 and caspase-3, which IL-13 pretreatment was able to partially inhibit and delay. IL-13 also stimulates activation of the major PI 3-kinase effector, protein kinase B. The PI 3-kinase inhibitors wortmannin and LY294002 inhibit IL-13 stimulation of protein kinase B as well as the cell survival effects of IL-13. These data demonstrate that cytokine-induced apoptosis of HT-29 cells is NO-independent and that the activation of a PI 3-kinase-dependent signaling cascade by IL-13 is a key signal responsible for the inhibition of apoptosis.
      Interleukin-13 (IL-13)
      The abbreviations used are: IL, interleukin; IFN, interferon; IL-4/IL-13R, IL-4/IL-13 receptor; mAb, monoclonal antibody; iNOS, inducible nitric oxide synthase; NO, nitric oxide; TNF, tumor necrosis factor; PI, phosphatidylinositol; PKB, protein kinase B; pNA, p-nitroanilide; PAGE, polyacrylamide gel electrophoresis; IAP, inhibitor of apoptosis
      1The abbreviations used are: IL, interleukin; IFN, interferon; IL-4/IL-13R, IL-4/IL-13 receptor; mAb, monoclonal antibody; iNOS, inducible nitric oxide synthase; NO, nitric oxide; TNF, tumor necrosis factor; PI, phosphatidylinositol; PKB, protein kinase B; pNA, p-nitroanilide; PAGE, polyacrylamide gel electrophoresis; IAP, inhibitor of apoptosis
      is a pleiotropic cytokine secreted by activated Th-2 lymphocytes which regulates a variety of immune target cells (
      • Minty A.
      • Chalon P.
      • Derocq J.M.
      • Dumont X.
      • Guillemot J.C.
      • Kaghad M.
      • Labit C.
      • Leplatois P.
      • Liauzun P.
      • Miloux B.
      • Minty C.
      • Casellas P.
      • Loison G.
      • Lupker J.
      • Shire D.
      • Ferrara P.
      • Caput D.
      ,
      • McKenzie A.N.J.
      • Culpepper J.A.
      • de Waal Malefyt R.
      • Briere F.
      • Punnonen J.
      • Aversa G.
      • Sato A.
      • Dang W.
      • Cocks B.G.
      • Menon S.
      • Devries J.E.
      • Banchereau J.
      • Zurawski G.
      ). In B lymphocytes, IL-13 induces proliferation and differentiation, promotes CD23 expression and production of certain immunoglobulins such as IgG4 and IgE (
      • Cocks B.G.
      • Malefyt R.D.
      • Galizzi J.P.
      • Devries J.E.
      • Aversa G.
      ,
      • Defrance T.
      • Carayon P.
      • Billian G.
      • Guillemot J.C.
      • Minty A.
      • Caput D.
      • Ferrara P.
      ,
      • Punnonen J.
      • Aversa G.
      • Cocks B.G.
      • McKenzie A.N.J.
      • Menon S.
      • Zurawski G.
      • de Waal Malefyt R.
      • de Vries J.E.
      ). In monocytes, IL-13 induces morphological changes (
      • McKenzie A.N.J.
      • Culpepper J.A.
      • de Waal Malefyt R.
      • Briere F.
      • Punnonen J.
      • Aversa G.
      • Sato A.
      • Dang W.
      • Cocks B.G.
      • Menon S.
      • Devries J.E.
      • Banchereau J.
      • Zurawski G.
      ), up-regulates expression of members of the integrin superfamily and major histocompatability complex class II antigen expression, and down-regulates expression of CD14 and FcγR receptors (
      • de Waal Malefyt R.D.
      • Figdor C.G.
      • Huijbens R.
      • Mohan-Peterson S.
      • Bennet B.
      • Culpepper J.
      • Dang W.
      • Zurawski G.
      • de Vries J.E.
      ). In lipopolysaccharide-stimulated monocytes, IL-13 also acts as a suppresser of proinflammatory cytokines (e.g. TNF type α, IL-1, and IL-6), chemokines (e.g. IL-8 and macrophage inflammatory protein-1α), and hematopoietic growth factors (e.g. granulocyte/macrophage-colony stimulating factor and granulocyte-colony stimulating factor) expression by activated monocytes/macrophages or endothelial cells (
      • Minty A.
      • Chalon P.
      • Derocq J.M.
      • Dumont X.
      • Guillemot J.C.
      • Kaghad M.
      • Labit C.
      • Leplatois P.
      • Liauzun P.
      • Miloux B.
      • Minty C.
      • Casellas P.
      • Loison G.
      • Lupker J.
      • Shire D.
      • Ferrara P.
      • Caput D.
      ,
      • de Waal Malefyt R.D.
      • Figdor C.G.
      • Huijbens R.
      • Mohan-Peterson S.
      • Bennet B.
      • Culpepper J.
      • Dang W.
      • Zurawski G.
      • de Vries J.E.
      ). Another target of IL-13 is epithelial cells and we have recently demonstrated that IL-13 can modulate chemokine generation from the human colonic epithelial cell line HT-29 (
      • Kolios G.
      • Robertson D.A.F.
      • Jordan N.J.
      • Minty A.
      • Caput D.
      • Ferrara P.
      • Westwick J.
      ,
      • Kolios G.
      • Wright K.L.
      • Jordan N.J.
      • Leithead J.B.
      • Robertson D.A.F.
      • Westwick J.
      ) and inhibit iNOS expression and NO generation in this system (
      • Kolios G.
      • Rooney N.
      • Murphy C.T.
      • Robertson D.A.F.
      • Westwick J.
      ). Activation of the signaling enzyme phosphatidylinositol 3-kinase (PI 3-kinase) by IL-13 is important for mediating these effects (
      • Kolios G.
      • Wright K.L.
      • Jordan N.J.
      • Leithead J.B.
      • Robertson D.A.F.
      • Westwick J.
      ,
      • Wright K.
      • Ward S.G.
      • Kolios G.
      • Westwick J.
      ). PI 3-kinase and its major downstream effector, the serine/threonine kinase protein kinase B (PKB), have been shown to be key mediators of growth factor-induced cell survival and protection against c-Myc-induced cell death in fibroblasts (
      • Kauffman-Zeh A.
      • Rodriguez-Viciana P.
      • Ulrich E.
      • Gilbert C.
      • Coffer P.
      • Downward J.
      • Evan G.
      ,
      • Dudek H.
      • Datta S.R.
      • Franke T.F.
      • Birnbaum M.J.
      • Yao R.
      • Cooper G.M.
      • Segal R.A.
      • Kaplan D.R.
      • Greenberg M.E.
      ,
      • Kulik G.
      • Klippel A.
      • Weber M.J.
      ). In addition, IL-13 has been reported to up-regulate the cell survival factors Bcl-xL and Mcl-1 as well as protect B lymphocytes from apoptosis (
      • Lømo J.
      • Blomhoff H.K.
      • Jacobsen S.E.
      • Krajewski S.
      • Reed J.C.
      • Smeland E.B.
      ). Moreover, the related cytokine, IL-4, also enhances cell survival (
      • Zamorano J.
      • Wang H.Y.
      • Wang L.-M.
      • Pierce J.H.
      • Keegan A.D.
      ) and this correlates well with observations that both IL-13 and IL-4 stimulate PI 3-kinase (
      • Wright K.
      • Ward S.G.
      • Kolios G.
      • Westwick J.
      ,
      • Gold M.R.
      • Duronio V.
      • Saxena S.P.
      • Schrader J.W.
      • Aebersold R.
      ) and with the notion that the IL-13 receptor and IL-4 receptor share a common subunit in signal transduction (
      • Aversa G.
      • Punnonen J.
      • Cocks B.G.
      • Malefyt R.D.
      • Vega F.
      • Zurawski S.M.
      • Zurawski G.
      • Devries J.E.
      ,
      • Obiri N.I.
      • Leland P.
      • Murata T.
      • Debinski W.
      • Puri R.K.
      ,
      • Lefort S.
      • Vita N.
      • Reeb R.
      • Caput D.
      • Ferrara P.
      ).
      We have previously shown that a combination of pro-inflammatory cytokines (IL-1α/IFN-γ/TNF-α) up-regulates iNOS expression and generates NO in a human colonic epithelial cell line HT-29 (
      • Kolios G.
      • Brown Z.
      • Robson R.L.
      • Robertson D.A.F.
      • Westwick J.
      ). Also, it has recently been shown that IFN-γ, in combination with TNF-α or anti-CD95, induces apoptosis in HT-29 cells (
      • Abreu-Martin M.T.
      • Vidrich A.
      • Lynch D.H.
      • Targan S.R.
      ) and an increased frequency of epithelial apoptosis mediated by the CD95-CD95L system is seen in ulcerative colitis (
      • Sträter J.
      • Wellisch I.
      • Riedl S.
      • Walczak H.
      • Koretz K.
      • Tandara A.
      • Krammer P.H.
      • Möller P.
      ,
      • Iwamoto M.
      • Koji T.
      • Makiyama K.
      • Kobayashi N.
      • Nakane P.K.
      ), which is an inflammatory bowel disease of unknown etiology. However, colonic epithelial cell injury, resulting in impaired barrier function, could contribute to the pathogenesis of inflammatory bowel disease (
      • Gardiner K.R.
      • Anderson N.H.
      • Rowlands B.J.
      • Barbul A.
      ). It has been postulated that overproduction of nitric oxide (NO) by inflamed mucosa may play a role in the pathophysiology of inflammatory bowel disease due to the increased expression of the inducible form of nitric oxide synthase (iNOS) found in biopsies taken from patients with active ulcerative colitis as compared with normal colon (
      • Kolios G.
      • Rooney N.
      • Murphy C.T.
      • Robertson D.A.F.
      • Westwick J.
      ). The production of NO might play a critical role in the resolution of inflammation (
      • McCafferty D.M.
      • Mudgett J.S.
      • Swain M.G.
      • Kubes P.
      ), possibly by inducing apoptosis in the leukocytic population recruited to the area (e.g. neutrophils) (
      • McCafferty D.M.
      • Mudgett J.S.
      • Swain M.G.
      • Kubes P.
      ). While NO has also been reported to inhibit apoptosis in several settings (
      • Dimmeler S.
      • Haendeler J.
      • Nehls M.
      • Zeiher A.M.
      ,
      • Genaro A.M.
      • Hortelano S.
      • Alvarez A.
      • Martı́nez-A C.
      • Boscá L.
      ,
      • Mannick J.B.
      • Miao X.Q.
      • Stamler J.S.
      ,
      • Kim Y.M.
      • De Vera M.E.
      • Watkins S.C.
      • Billiar T.R.
      ), it has also been reported to mediate cell death through mechanisms consistent with apoptosis in various cells including peritoneal macrophages (
      • Messmer U.K.
      • Reed J.C.
      • Brune B.
      ,
      • Albina J.E.
      • Cui S.J.
      • Mateo R.B.
      • Reichner J.S.
      ,
      • Sarih M.
      • Souvannavong V.
      • Adam A.
      ), β-cells (
      • Kaneto H.
      • Fujii J.
      • Seo H.G.
      • Suzuki K.
      • Matsuoka T.
      • Nakamura M.
      • Tatsumi H.
      • Yamasaki Y.
      • Kamada T.
      • Taniguchi N.
      ,
      • Ankarcrona M.
      • Dypbukt J.M.
      • Brune B.
      • Nicotera P.
      ), and thymocytes (
      • Fehsel K.
      • Kroncke K.D.
      • Meyer K.L.
      • Huber H.
      • Wahn V.
      • Kolbbachofen V.
      ). Also, overproduction of NO may lead to oxidant-induced injury of the colon epithelial crypt (
      • McKenzie S.J.
      • Baker M.S.
      • Buffinton G.D.
      • Doe W.F.
      ), possibly by the reaction with superoxide to form peroxynitrite which in turn results in the nitration of proteins on tyrosine residues (
      • Singer I.I.
      • Kawka D.W.
      • Scott S.
      • Weidner J.R.
      • Mumford R.A.
      • Riehl T.E.
      • Stenson W.F.
      ).
      In this study, we sought to ascertain whether there is a relationship between NO production and apoptosis of HT-29 epithelial cells observed in response to a combination of cytokines and/or CD95 ligation. In addition, given that the ability of IL-13 to inhibit iNOS expression and NO generation in this system is driven by PI 3-kinase-dependent pathway, we investigated whether IL-13 could provide a cell survival signal through PI 3-kinase to protect against cytokine-driven apoptotic signals.

      DISCUSSION

      In this report we demonstrate that a combination of proinflammatory cytokines, namely IL-1α/IFN-γ/TNF-α, stimulates the expression of apoptotic markers in approximately 25–80% of cytokine-treated HT-29 cells (depending on time of analysis) as evidenced by assays that detect DNA fragmentation and externalization of phosphatidylserine. Apoptosis can also be stimulated to varying extents by the combination of IL-1α/IFN-γ or IFN-γ/TNF-α. Moreover, we present evidence that the induction of these apoptotic markers is not dependent on the expression of iNOS and NO production. Furthermore, pretreatment with the anti-inflammatory cytokine IL-13 which is known to prevent induction of NO production by HT-29 cells in response to IL-1α/IFN-γ/TNF-α (
      • Kolios G.
      • Rooney N.
      • Murphy C.T.
      • Robertson D.A.F.
      • Westwick J.
      ,
      • Wright K.
      • Ward S.G.
      • Kolios G.
      • Westwick J.
      ), also protects against IL-1α/IFN-γ/TNF-α-, IFN-γ/TNF-α-, and IFN-γ/CH11-induced (but not IL-1α/IFN-γ-induced) cell death in this system via a PI 3-kinase-dependent mechanism. This also correlates with the first demonstration that IL-13 stimulates activation of the major downstream PI 3-kinase effector PKB, which is thought to mediate the promotion of cell survival by this signaling pathway in a number of cell systems (
      • Kauffman-Zeh A.
      • Rodriguez-Viciana P.
      • Ulrich E.
      • Gilbert C.
      • Coffer P.
      • Downward J.
      • Evan G.
      ,
      • Dudek H.
      • Datta S.R.
      • Franke T.F.
      • Birnbaum M.J.
      • Yao R.
      • Cooper G.M.
      • Segal R.A.
      • Kaplan D.R.
      • Greenberg M.E.
      ,
      • Kulik G.
      • Klippel A.
      • Weber M.J.
      ). However, IL-13-induced phosphorylation of Bad (a known downstream target of PKB) was barely detectable, suggesting that this is an unlikely target for PKB activity in this system. In addition, IL-13 pretreatment partially inhibited, but did not prevent cytokine-stimulated activation of either caspase-8 or caspase-3.
      Individually, IL-1α, IFN-γ, and TNF-α do not induce apoptosis of HT-29 cells. However, our observation that combinations of IL-1α, IFN-γ, and TNF-α induce apoptosis of HT-29 cells, correlates well with recent reports that IFN-γ increases the sensitivity of HT-29 cells to pro-apoptotic agents such as TNF-α by directly and indirectly inducing select apoptosis-related genes (
      • Ossina N.K.
      • Cannas A.
      • Powers V.C.
      • Fitzpatrick P.A.
      • Knight J.D.
      • Gilbert J.R.
      • Shekhtman E.M.
      • Tomei L.D.
      • Umansky S.R.
      • Kiefer M.C.
      ). In addition, we have previously reported that this combination of cytokines stimulates NO production from HT-29 cells and there is considerable evidence that NO can promote apoptosis in other systems (
      • Brüne B.
      • Von Knethen A.
      • Sandau K.B.
      ). Indeed, iNOS transcripts can be detected 6 h after cytokine treatment (
      • Kolios G.
      • Brown Z.
      • Robson R.L.
      • Robertson D.A.F.
      • Westwick J.
      ) and this appears to precede cell death which is detectable at 8 h post-cytokine stimulation. However, there are several lines of evidence to indicate that cytokine-driven iNOS and apoptosis are independent functional events. First, the iNOS inhibitor aminoguanidine prevented IL-1α/IFN-γ/TNF-α and IL-1α/IFN-γ -induced NO production, but had no effect on the apoptosis stimulated by these combinations of cytokines. Second, apoptosis can also be stimulated by IFN-γ/TNF-α and IFN-γ/CH11 which are unable to stimulate NO production. Third, inhibition of IL-1α/IFN-γ/TNF-α-induced apoptosis by Z-VAD-FMK had no effect on NO production induced by these cytokines. Although markers of cytokine-stimulated apoptosis, such as DNA fragmentation and phosphatidylserine externalization, are detectable from 8 to 24 h, time course experiments have revealed that other functional responses continue unabated. For instance, identical cytokine treatment can also stimulate up-regulation of iNOS and chemokine mRNA up to 24 h post-stimulation (
      • Kolios G.
      • Robertson D.A.F.
      • Jordan N.J.
      • Minty A.
      • Caput D.
      • Ferrara P.
      • Westwick J.
      ,
      • Kolios G.
      • Brown Z.
      • Robson R.L.
      • Robertson D.A.F.
      • Westwick J.
      ) and these responses can be down-regulated by pretreatment with IL-13 (
      • Kolios G.
      • Robertson D.A.F.
      • Jordan N.J.
      • Minty A.
      • Caput D.
      • Ferrara P.
      • Westwick J.
      ,
      • Kolios G.
      • Wright K.L.
      • Jordan N.J.
      • Leithead J.B.
      • Robertson D.A.F.
      • Westwick J.
      ,
      • Wright K.
      • Ward S.G.
      • Kolios G.
      • Westwick J.
      ). So, cytokine-induced expression of apoptotic markers and events do not necessarily correlate with abrogated cell function, at least in the time frame studied here.
      Activation of the proteolytic cascade by caspases appears to be essential to cytokine-induced apoptosis of HT-29 cells, given our observation that pretreatment with the caspase inhibitor Z-VAD-FMK completely prevents apoptosis induced by IL-1α/IFN-γ/TNF-α, IL-1α/IFN-γ, IFN-γ/TNF-α, and IFN-γ/CH11. This is particularly interesting given that both IL-1α and TNF-α (
      • Schutze S.
      • Machleidt T.
      • Kronke M.
      ) and CD95 (
      • Cifone M.G.
      • DeMaria R.
      • Roncaioli P.
      • Rippo M.R.
      • Azuma M.
      • Lanier L.L.
      • Santoni A.
      • Testi R.
      ) ligation have been reported to activate the ceramide pathway, which has also been implicated as a signaling pathway involved in apoptosis (
      • Obeid L.M.
      • Linardic C.M.
      • Karolak L.A.
      • Hannun Y.A.
      ,
      • Pushkareva M.
      • Obeid L.M.
      • Hannun Y.A.
      ). However, since the apoptosis of HT-29 cells stimulated by these cytokine combinations is completely inhibited by the caspase inhibitor Z-VAD-FMK, this may indicate that ceramide production is not sufficient for cell death in this system. Indeed, it has recently been shown that IFN-γ was unable to induce changes in sphingolipid levels in HT-29 cells (
      • Veldman R.J.
      • Klappe K.
      • Hoekstra D.
      • Kok J.W.
      ), suggesting that ceramide-mediated signaling pathways may be cell-type specific. It is also interesting to note that while treatment of HT-29 cells with TNF-α or IFN-γ resulted in modest stimulation of caspases -8 and -3, this is insufficient to drive cell death, since neither TNF-α nor IFN-γ stimulated apoptosis in this system. This is in marked contrast to the TNF-α-induced apoptosis observed in neutrophils and T lymphocytes which correlates well with caspase activation (
      • Aggarwal S.
      • Goollapudi S.
      • Gupta S.
      ,
      • Yamashita K.
      • Takahashi A.
      • Kobayashi S.
      • Hirata H.
      • Mesner P.
      • Kaufmann S.H.
      • Yonehara K.
      • Uchiyama T.
      • Sasada M.
      ).
      Even though IL-13 exerts a protective effect against cell death induced by IL-1α/IFN-γ/TNF-α, IFN-γ/TNF-α, and IFN-γ/CH11, IL-13 pretreatment was unable to completely inhibit cytokine-activated caspase-8 and caspase-3. Rather, it appears that IL-13 pretreatment delays activation of these caspases by IL-1α/IFN-γ/TNF-α and in this respect it is interesting to note that IL-13 provides only partial protection against cell death induced by IL-1α/IFN-γ/TNF-α and IFN-γ/TNF-α. Caspase activation is required for the execution of cell death in an apoptotic manner (reviewed in 44), but the order of caspase activation cascades is not absolute and the commitment to live or die may originate from the mitochondria (reviewed in Ref.
      • Green D.
      • Kroemer G.
      ). Hence, while IL-13 partially inhibits and possibly delays activation of caspase-8 and caspase-3, there may well be additional targets of IL-13-activated biochemical signals that mediate cell survival at some point distal to the apical caspase-8 and the downstream caspase-3, possibly involving mitochondrial activity. It is certainly possible that other upstream and downstream caspases are activated by the cytokine combinations used in this study. Indeed, it has recently been shown that PKB can phosphorylate caspase-9 and inhibit its protease activity (
      • Cardone M.H.
      • Roy N.
      • Stennicke H.R.
      • Salvesen G.S.
      • Franke T.F.
      • Stanbridge E.
      • Frisch S.
      • Reed J.C.
      ). This would fit nicely with our observations that IL-13 can provide only partial protection against cell death induced by IL-1α/IFN-γ/TNF-α and IFN-γ/TNF-α, whereas apoptosis stimulated by IL-1α/IFN-γ was unaffected by IL-13. Hence, it appears that multiple death promoting pathways with different sensitivity to IL-13-activated cell survival mechanisms are activated by the cytokine combinations used in this study.
      The protective effects of IL-13 against IL-1α/IFN-γ/TNF-α-, IFN-γ/TNF-α-, and IFN-γ/CH11-stimulated apoptosis are dependent on the PI 3-kinase-dependent signaling pathway, since the PI 3-kinase inhibitors wortmannin and LY29002 abrogated the protective effects of IL-13. These observations are consistent with demonstrations that the PI 3-kinase-dependent signaling pathway and in particular its downstream effector PKB are involved in growth factor-dependent cell survival (
      • Kauffman-Zeh A.
      • Rodriguez-Viciana P.
      • Ulrich E.
      • Gilbert C.
      • Coffer P.
      • Downward J.
      • Evan G.
      ,
      • Dudek H.
      • Datta S.R.
      • Franke T.F.
      • Birnbaum M.J.
      • Yao R.
      • Cooper G.M.
      • Segal R.A.
      • Kaplan D.R.
      • Greenberg M.E.
      ,
      • Kulik G.
      • Klippel A.
      • Weber M.J.
      ,
      • Yao R.J.
      • Cooper G.M.
      ). Indeed, we have previously shown that IL-13 strongly activates PI 3-kinase as evidenced by PtdIns (3,4,5)-P3 accumulation (
      • Wright K.
      • Ward S.G.
      • Kolios G.
      • Westwick J.
      ). Moreover, data in this study demonstrates that IL-13 also activates PKB and this activation is abrogated by pretreatment with PI 3-kinase inhibitors. PKB is now known to promote cell survival by phosphorylating a critical serine residue (136Ser) on the death-promoting protein Bad, causing it to dissociate from and thus allow activation of the cell survival factor, Bcl-xL (
      • del Peso L.
      • GonzálezGarcı́a M.
      • Page C.
      • Herrera R.
      • Nuñez G.
      ,
      • Datta S.R.
      • Dudek H.
      • Tao X.
      • Masters S.
      • Fu H.
      • Gotoh Y.
      • Greenberg M.E.
      ). However, consistent with observations from other groups (
      • Ossina N.K.
      • Cannas A.
      • Powers V.C.
      • Fitzpatrick P.A.
      • Knight J.D.
      • Gilbert J.R.
      • Shekhtman E.M.
      • Tomei L.D.
      • Umansky S.R.
      • Kiefer M.C.
      ), Bad is expressed at very low levels in HT-29 cells, such that the band shift of Bad to the serine-phosphorylated form was barely detectable. It would seem unlikely, therefore, that the cell survival effects of IL-13 are solely mediated by PKB phosphorylation of Bad in the system described here. However, there are two alternative explanations to account for IL-13-stimulated PI 3-kinase/PKB-dependent cell survival mechanisms. First, other death promoting Bcl-2 family proteins may be regulated by PKB-dependent phosphorylation in a manner similar to that described for the regulation of Bad (
      • del Peso L.
      • GonzálezGarcı́a M.
      • Page C.
      • Herrera R.
      • Nuñez G.
      ,
      • Datta S.R.
      • Dudek H.
      • Tao X.
      • Masters S.
      • Fu H.
      • Gotoh Y.
      • Greenberg M.E.
      ). Indeed, expression of the related Bcl-2 family member Bak, which can also promote cell death, has been reported to be directly induced by IFN-γ (
      • Ossina N.K.
      • Cannas A.
      • Powers V.C.
      • Fitzpatrick P.A.
      • Knight J.D.
      • Gilbert J.R.
      • Shekhtman E.M.
      • Tomei L.D.
      • Umansky S.R.
      • Kiefer M.C.
      ). However, while Bak is expressed, we could not detect any IL-13-stimulated hyperphosphorylation of Bak by immunoblotting (data not shown). Nevertheless, it remains possible that other Bcl-2 family proteins may act as targets for IL-13-activated PKB. Second, an alternative target for the PI 3-kinase-dependent cell survival signals provided by IL-13 may be the transcription factors of the NF-κB family which have been reported to be important in cell survival by regulating unidentified, antiapoptotic genes (
      • Wu M.X.
      • Ao Z.
      • Prasad K.V.S.
      • Wu R.
      • Schlossman S.F.
      ). Recent evidence has identified the inhibitor of apoptosis (IAP) proteins c-IAP1 and c-IAP-2 as gene targets of NF-κB transcriptional activity (
      • Wang C.Y.
      • Mayo M.W.
      • Korneluk R.G.
      • Goeddel D.V.
      • Baldwin A.S.
      ). The c-IAP1 and c-IAP2 proteins specifically inhibit the active forms of caspase-3 and caspase-7 (
      • Wu M.X.
      • Ao Z.
      • Prasad K.V.S.
      • Wu R.
      • Schlossman S.F.
      ). In other systems such as T lymphocytes, activation of NF-κB has been reported to be dependent on p70 S6 kinase (
      • Lai J.H.
      • Tan T.H.
      ), which in turn has been reported to be a target for phosphorylation by either PKB (
      • Burgering B.M.T.
      • Coffer P.J.
      ) and/or its upstream kinase(s) PDK-1 and the putative PDK-2 (
      • Alessi D.R.
      • Kozlowski M.T.
      • Weng Q.P.
      • Morrice N.
      • Avruch J.
      ,
      • Downward J.
      ). Hence, one possibility is that the observed cell survival effects of IL-13 involves PI 3-kinase-dependent activation of NF-κB transcriptional activity, although this hypothesis does not fit easily with the recent report demonstrating that IL-13 down-regulates TNF-α-mediated activation of NF-κB (
      • Manna S.K.
      • Aggarwal B.B.
      ).
      In summary, apoptosis of HT-29 epithelial cells observed in response to a combination of cytokines and/or CD95 ligation is not dependent on NO production. In addition, IL-13 can provide a PI 3-kinase-dependent cell survival signal to HT-29 cells which protects against cytokine-driven apoptotic signals. The mechanism underlying this cell survival effect of IL-13 is unclear and apparently distinct from the known cell survival signals provided by PKB-dependent phosphorylation of Bad. Nevertheless, our observations indicate a potential role for IL-13 in regulating the controlled program of cell death and survival, a process which plays an important role during several stages of normal colonic epithelial cell development and maturation. Hence, dysregulation of cell survival and death may be important in the pathogenesis of inflammatory bowel disease and carcinogenesis in the large bowel.

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