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Phosphatidylinositol 3-Kinase-dependent Pathways Oppose Fas-induced Apoptosis and Limit Chloride Secretion in Human Intestinal Epithelial Cells

IMPLICATIONS FOR INFLAMMATORY DIARRHEAL STATES*
  • Maria T. Abreu
    Correspondence
    To whom correspondence should be addressed: Inflammatory Bowel Disease Center, Cedars-Sinai Medical Center, 8631 West 3rdSt., Suite 245E, Los Angeles, CA 90048. Tel.: 310-423-4100; Fax: 310-423-0147
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  • Elizabeth T. Arnold
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  • Jimmy Y.C. Chow
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  • Kim E. Barrett
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  • Author Footnotes
    * This work was supported by Grants K08 DK02635 and 1R03 DK59469 from the Natonal Institutes of Health (to M. T. A.), a Crohn's and Colitis Foundation of America First Award (to M. T. A.), and Grant DK 28305 from the Natonal Institutes of Health (to K. E. B.). Some of this work was performed using a laser scanning confocal microscope provided by Grant NCRR 1 S10 RR13717-01 from the Natonal Institutes of Health.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.
Open AccessPublished:December 14, 2001DOI:https://doi.org/10.1074/jbc.M106226200
      The epithelial lining of the intestine serves as a barrier to lumenal bacteria and can be compromised by pathologic Fas-mediated epithelial apoptosis. Phosphatidylinositol (PI)3-kinase signaling has been described to limit apoptosis in other systems. We hypothesized that PI3-kinase-dependent pathways regulate Fas-mediated apoptosis and barrier function in intestiynal epithelial cells (IEC). IEC lines (HT-29 and T84) were exposed to agonist anti-Fas antibody in the presence or absence of chemical inhibitors of PI3-kinase (LY294002 and wortmannin). Apoptosis, barrier function, changes in short circuit current (ΔI sc), and expression of adhesion molecules were assessed. Inhibition of PI3-kinase strongly sensitized IEC to Fas-mediated apoptosis. Expression of constitutively active Akt, a principal downstream effector of the PI3-kinase pathway, protected against Fas-mediated apoptosis to an extent that was comparable with expression of a genetic caspase inhibitor, p35. PI3-kinase inhibition sensitized to apoptosis by increasing and accelerating Fas-mediated caspase activation. Inhibition of PI3-kinase combined with cross-linking Fas was associated with increased permeability to molecules that were <400 Da but not those that were >3,000 Da. Inhibition of PI3-kinase resulted in chloride secretion that was augmented by cross-linking Fas. Confocal analyses revealed polymerization of actin and maintenance of epithelial cell adhesion molecule-mediated interactions in monolayers exposed to anti-Fas antibody in the context of PI3-kinase inhibition. PI3-kinase-dependent pathways, especially Akt, protect IEC against Fas-mediated apoptosis. Inhibition of PI3-kinase in the context of Fas signaling results in increased chloride secretion and barrier dysfunction. These findings suggest that agonists of PI3-kinase such as growth factors may have a dual effect on intestinal inflammation by protecting epithelial cells against immune-mediated apoptosis and limiting chloride secretory diarrhea.
      PI
      phosphatidylinositol
      TER
      transepithelial resistance
      EpCAM
      epithelial cell adhesion molecule
      Ab
      antibody
      PIP3
      phosphotidylinositol 3,4,5-triphosphate
      FITC
      fluorescein isothiocyanate
      GFP
      green fluorescent protein
      PBS
      phosphate-buffered saline
      ZO-1
      zona occludens-1
      Animal models of inflammatory bowel disease suggest that derangements in either T cell function or epithelial barrier function play a causal role in the pathophysiology of inflammatory bowel disease (
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      We have developed an in vitro model of immune-mediated epithelial cell apoptosis that permits us to study the effect of Fas-mediated apoptosis on epithelial barrier function (
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      ). As with colonic crypt epithelial cells, T84 cells express the Fas receptor and are sensitive to Fas-mediated apoptosis (
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      ). These cells have also been used to model crypt abscesses (
      • Nash S.
      • Parkos C.
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      ). Basolateral cross-linking of Fas on T84 monolayers induces apoptosis. Despite massive cell death, the barrier function to relatively small macromolecules remains intact. Our findings suggested that the intestinal epithelium is quite resilient in the face of apoptotic damage and is able to repair the wound created by apoptotic cell loss. Using this model, we wished to investigate the molecular mechanisms that protect against dysregulated apoptosis and perturbed barrier function in the intestine. The current study focuses on the inter-relationship between the Fas death receptor and phosphatidylinositol (PI)1 3-kinase signaling pathways in intestinal epithelial cells. We have chosen to study this interplay because Fas-mediated apoptosis of crypt intestinal epithelial cells is associated with human inflammatory bowel disease (
      • Strater J.
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      ), and PI3-kinase-dependent pathways protect against Fas-mediated apoptosis in the immune system (
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      Several lines of evidence support a role for PI3-kinase-dependent signaling in regulation of apoptosis in the intestine. In response to a variety of extracellular stimuli, PI3-kinase phosphorylates the 3 position of the inositol ring of membrane inositol phospholipids, resulting in the generation of 3-phosphorylated products including phosphotidylinositol 3,4,5-triphosphate (PIP3) (
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      ,
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      ). The generation of PIP3 activates signaling pathways downstream of PI3-kinase, including Akt (also known as protein kinase B), a kinase with anti-apoptotic properties (
      • Datta S.R.
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      ,
      • Franke T.F.
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      • Yao R.
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      ). A recently identified lipid phosphatase, PTEN, down-regulates PI3-kinase signaling by dephosphorylating PIP3, thereby inhibiting the recruitment and activation of Akt (
      • Whang Y.E.
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      ). Mutations in PTEN are responsible for the genetic syndrome of Cowden's disease, characterized by hamartomatous polyps of the gastrointestinal tract (
      • Chi S.G.
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      • Gorlin R.J.
      • Hodgson S.V.
      • Huson S.
      • Lacombe D.
      • Eng C.
      • et al.
      ). PTEN+/− mice develop intestinal polyps and dysplasia of the colonic epithelium (
      • Chi S.G.
      • Kim H.J.
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      • Lee J.I.
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      ,
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      ). These mice also develop lymphomas as a result of defective Fas-mediated apoptosis (
      • Di Cristofano A.
      • Kotsi P.
      • Peng Y.F.
      • Cordon-Cardo C.
      • Elkon K.B.
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      ). Mice deficient in the catalytic subunit of PI3-kinase γ develop invasive colorectal cancer (
      • Sasaki T.
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      ). Recent data demonstrate that PI3-kinase opposes intestinal epithelial differentiation in vitro (
      • Wang Q.
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      ). Taken together these data suggest that PI3-kinase-dependent pathways play a role in the regulation of apoptosis in the intestinal epithelium.
      PI3-kinase and Akt may also play important roles in epithelial adhesion and barrier function. Cellular adhesion to the extracellular matrix or neighboring cells results in the activation of PI3-kinase, recruitment of Akt, and protection against apoptosis (
      • Watton S.J.
      • Downward J.
      ,
      • Tamura M.
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      • Takino T.
      • Miyamoto S.
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      ,
      • Lee J.W.
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      ). Membrane-bound Akt and PIP3 accumulate at the leading edge of cells responding to chemotactic factors, suggesting that cell movement is regulated by Akt (
      • Hirsch E.
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      ). Finally, PI3-kinase activation is involved in actin cytoskeletal rearrangements leading to cell spreading (
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      ,
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      ).
      In this study, we tested the hypothesis that PI3-kinase-dependent mechanisms protect intestinal epithelial cells from Fas-mediated apoptosis and barrier dysfunction. We found that inhibition of PI3-kinase sensitizes intestinal epithelial cells to Fas-mediated apoptosis and exacerbates the barrier dysfunction associated with Fas-mediated apoptosis. Caspase inhibition protects against both apoptosis and increased monolayer permeability. Inhibition of PI3-kinase results in increased epithelial conductance and chloride secretion, which is increased further by activation of Fas. Despite marked apoptosis, the intestinal epithelial monolayer is able to maintain a relatively impermeable barrier by undergoing cytoskeletal changes and maintaining cell-cell adhesion. Our studies identify molecular mechanisms by which peptide growth factors may exert a beneficial clinical effect in patients with inflammatory diarrheal states.

      DISCUSSION

      In health, the intestinal epithelium is replaced every 3–5 days suggesting balanced processes of cell death and proliferation. Thus, the intestinal epithelium must integrate a variety of growth and death signals while maintaining an uninterrupted barrier. Inflammatory bowel diseases, nonsteroidal anti-inflammatory drug-induced injury, and celiac disease are associated with an increased rate of intestinal epithelial cell apoptosis. This study examines two inter-related intestinal epithelial responses: regulation of apoptosis and repair of the defect created by apoptotic cell loss. In human and murine studies, colonic epithelial cell apoptosis occurs at the base of the crypt near the dividing stem cell and at the lumenal surface (
      • Strater J.
      • Wellisch I.
      • Riedl S.
      • Walczak H.
      • Koretz K.
      • Tandara A.
      • Krammer P.H.
      • Moller P.
      ,
      • Potten C.S.
      ). We have previously described a model of intestinal epithelial apoptosis using T84 cells. In this model, basolateral cross-linking of the Fas receptor results in apoptotic cell loss (
      • Abreu M.T.
      • Palladino A.A.
      • Arnold E.T.
      • Kwon R.S.
      • McRoberts J.A.
      ). Although T84 cells reproduce the crypt cell phenotype in many respects, a homogenous cell line cannot replicate all stages of colonic epithelial cell differentiation. Because we use T84 cells that have been cultured on semi-permeable supports to confluence, little cell division is occurring during the time of our experiments. We speculate, therefore, that our model represents an intermediate stage between the dividing stem cell and the most differentiated lumenal colonocytes. Using this system, we found that loss of cells through apoptosis resulted in dramatic cell flattening and maintenance of E-cadherin-mediated cell-cell interactions of the remaining viable cells. The outcome is a remarkable preservation of barrier function despite apoptotic cell loss. Our studies suggested that this model epithelium undergoes a process similar to epithelial restitution to repair the wound created by apoptotic cell loss.
      In an effort to understand the signaling pathways by which intestinal epithelial cells limit apoptosis and promote epithelial restitution, we examined the role of PI3-kinase. Our study suggests that signaling via the PI3-kinase pathway tonically protects intestinal epithelial cells from apoptotic injury in a hostile environment. Exactly how inhibition of PI3-kinase sensitizes intestinal epithelial cells to Fas-mediated apoptosis remains to be elucidated fully. HT-29 colon cancer cells are susceptible to Fas-mediated apoptosis but are partially protected against apoptosis by interleukin-13 through its effect on PI3-kinase (
      • Wright K.
      • Kolios G.
      • Westwick J.
      • Ward S.G.
      ). Studies have shown that PI3-kinase, acting through downstream kinases, especially Akt, exerts an anti-apoptotic effect through inhibition of mitochondrial permeability, caspase 8, and FADD availability and pro-caspase 9 protease activity (
      • 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.
      ,
      • del Peso L.
      • Gonzalez-Garcia M.
      • Page C.
      • Herrera R.
      • Nunez G.
      ,
      • Cardone M.H.
      • Roy N.
      • Stennicke H.R.
      • Salvesen G.S.
      • Franke T.F.
      • Stanbridge E.
      • Frisch S.
      • Reed J.C.
      ,
      • Suzuki A.
      • Hayashida M.
      • Kawano H.
      • Sugimoto K.
      • Nakano T.
      • Shiraki K.
      ,
      • Li H.
      • Zhu H.
      • Xu C.J.
      • Yuan J.
      ). Our study demonstrates that inhibition of PI3-kinase sensitizes cells to Fas-mediated apoptosis in a caspase-dependent fashion. We have also shown that inhibition of PI3-kinase accelerates caspase activation in response to cross-linking Fas. Our data suggest a model in which PI3-kinase-dependent pathways, by limiting caspase availability, dampen an apoptotic stimulus, such as Fas receptor engagement in intestinal epithelial cells. Future studies will examine the caspase activation sequence in intestinal epithelial cells and the effect of PI3-kinase on its dynamics.
      In this study, we hypothesized that PI3-kinase-dependent pathways might play a role in intestinal epithelial barrier function, either as a result of modulating cellular sensitivity to an apoptotic signal or through an effect on epithelial cell-matrix and cell-cell signaling pathways (
      • Watton S.J.
      • Downward J.
      ,
      • Tamura M.
      • Gu J.
      • Danen E.H.
      • Takino T.
      • Miyamoto S.
      • Yamada K.M.
      ). We were surprised to find that when marked apoptosis was induced during inhibition of PI3-kinase, relatively small macromolecules (3,000 Da) were still unable to traverse the injured barrier. Thus, whole bacteria or lipopolysaccharide (10,000–20,000 Da) would be excluded, but deficient barrier function to smaller molecules could explain the increased permeability to disaccharides in patients with Crohn's disease (
      • Hilsden R.J.
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      • Hardin J.
      • Gall D.G.
      • Sutherland L.R.
      ). We have also previously shown that the barrier function of this model intestinal epithelium is quite resilient in the face of Fas-mediated apoptotic injury. The data presented in the current study demonstrate that repair of apoptotic injury even in the case of monolayers undergoing >60% apoptotic cell death. Furthermore, PI3-kinase-dependent pathways do not seem to be required for repair of apoptotic injury because actin polymerization and dramatic cell flattening are even more pronounced when Fas is cross-linked in combination with PI3-kinase inhibitors than when cells are treated with anti-Fas alone. Although we have not directly examined the role of actin polymerization in repair of the apoptotic monolayer, studies in which the actin cytoskeleton has been disrupted have demonstrated diminished epithelial barrier function, suggesting that actin plays a major role in maintenance of an impermeable epithelial barrier (
      • Madara J.L.
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      ,
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      • Smith J.A.
      • Awtrey C.S.
      ,
      • Youakim A.
      • Ahdieh M.
      ). Our model of T84 cell apoptosis parallels some of the findings seen during epithelial restitution with respect to actin polymerization and cell flattening (
      • Nusrat A.
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      • Carnes D.K.
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      ,
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      • Mercurio A.M.
      ,
      • Nusrat A.
      • Delp C.
      • Madara J.L.
      ). The signal(s) required for an intact epithelial cell to sense and respond to the death of its neighbor are not understood. However, our data suggest that intestinal epithelial cells do not require PI3-kinase for the cytoskeletal changes involved in repairing the defect created by apoptotic cell loss.
      Our study is the first to examine the expression of the adhesion molecule EpCAM in a model intestinal epithelium in the intact and apoptotic states. EpCAM, also known as the KSA antigen, was first described as a cell surface antigen present on the majority of epithelial-derived tumors. Characterization of EpCAM revealed that it is involved in homophilic cell-cell interactions and that its cytolasmic tail interacts with the actin cytoskeleton (
      • Balzar M.
      • Bakker H.A.
      • Briaire-de-Bruijn I.H.
      • Fleuren G.J.
      • Warnaar S.O.
      • Litvinov S.V.
      ,
      • Litvinov S.V.
      • Velders M.P.
      • Bakker H.A.
      • Fleuren G.J.
      • Warnaar S.O.
      ,
      • Litvinov S.V.
      • Balzar M.
      • Winter M.J.
      • Bakker H.A.
      • Briaire-de Bruijn I.H.
      • Prins F.
      • Fleuren G.J.
      • Warnaar S.O.
      ). A monoclonal Ab to EpCAM is currently being evaluated as treatment for patients with advanced colon cancer (
      • Haller D.G.
      ). We have examined the spatial expression pattern of EpCAM in T84 cells and found that in intact monolayers, expression of EpCAM is basal and lateral (Fig. 7 B), suggesting that EpCAM is involved in both cell-cell as well as cell-matrix interactions in the intestine. We have previously shown that apoptotic monolayers maintain E-cadherin-mediated junctions between the remaining intact cells (
      • Abreu M.T.
      • Palladino A.A.
      • Arnold E.T.
      • Kwon R.S.
      • McRoberts J.A.
      ). Our current studies with EpCAM demonstrate that apoptotic bodies continue to be lined by EpCAM and are in continuity with the intact cells. This finding suggests that apoptotic and intact cells continue to interact even as the apoptotic cell is fragmenting and lifting away from the remainder of the monolayer. The finding of EpCAM-lined apoptotic bodies in continuity with intact cells may help to explain why the apoptotic cell does not leave a hole as it separates from the monolayer and may provide a signal for the neighboring intact cells to fill an imminent void. The interaction of EpCAM with the actin cytoskeleton may integrate the signals required for an intact cell to recognize the retraction of its apoptotic neighbor, polymerize actin, and change its morphology. The function of EpCAM in intestinal epithelial barrier function deserves further investigation.
      In addition to exploring the role of PI3-kinase in barrier function, we have examined the role of PI3-kinase in chloride secretion. We have previously shown that inhibition of PI3-kinase in T84 cells blocks epidermal growth factor-mediated inhibition of chloride secretion, suggesting that PI3-kinase-dependent pathways play a role in the regulation of this transport process (
      • Chow J.Y.
      • Uribe J.M.
      • Barrett K.E.
      ). Our studies extend this observation by suggesting that PI3-kinase-dependent pathways may tonically inhibit chloride secretion in intestinal epithelial cells, at least under certain circumstances. Our data further suggest that in the response to infection, cross-linking Fas may play a coregulatory role in chloride secretion in the intestinal epithelium. Recent studies in an animal model of pseudomonal pneumonia demonstrate that Fas-mediated death of lung epithelial cells controls the primary infection, whereas Fas mutant mice succumb to disseminated bacterial infection (
      • Grassme H.
      • Kirschnek S.
      • Riethmueller J.
      • Riehle A.
      • von Kurthy G.
      • Lang F.
      • Weller M.
      • Gulbins E.
      ). Enteroinvasive bacteria have been shown to induce apoptosis in intestinal epithelial cells in vitro, supporting the notion that apoptosis may be a means to prevent the spread of infection (
      • Kim J.M.
      • Eckmann L.
      • Savidge T.C.
      • Lowe D.C.
      • Witthoft T.
      • Kagnoff M.F.
      ). Fas-mediated signaling in the intestinal epithelium may play a similar role, with infected intestinal epithelial cells undergoing apoptosis and the remaining cells secreting chloride in an effort to expel the remaining organisms. These hypotheses await testing in animal models.
      The combined results of our studies have several implications for the medical therapy of inflammatory bowel diseases. In the setting of inflammation, diminished growth factor signals may render intestinal epithelial cells susceptible to immune-mediated apoptosis. Studies performed in animal models of colitis demonstrate that growth factors, such as growth hormone, epidermal growth factor, and keratinocyte growth factor, ameliorate the ulceration of the mucosa in colitic animals (
      • Miceli R.
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      • Fuller C.R.
      • Dieleman L.A.
      • Da C.C.
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      ). A recent study also demonstrated clinical benefit from growth hormone therapy in the treatment of Crohn's disease (
      • Slonim A.E.
      • Bulone L.
      • Damore M.B.
      • Goldberg Y.
      • Wingertzahn M.A.
      • McKinley M.J.
      ). These trophic hormones are agonists of PI3-kinase (
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      • Birkenheager J.C.
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      ). Our studies suggest that one benefit of these growth factors is in protecting intestinal epithelial cells from immune-mediated apoptosis. Another potential benefit may be the inhibition of chloride secretion by intestinal epithelial cells. Indeed, Crohn's disease patients treated with growth hormone had a marked lessening of diarrhea (
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      • Bulone L.
      • Damore M.B.
      • Goldberg Y.
      • Wingertzahn M.A.
      • McKinley M.J.
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