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Interleukins 4 and 13 Increase Intestinal Epithelial Permeability by a Phosphatidylinositol 3-Kinase Pathway

LACK OF EVIDENCE FOR STAT 6 INVOLVEMENT*
Open AccessPublished:September 15, 2000DOI:https://doi.org/10.1074/jbc.M003516200
      Interleukins 4 and 13 can affect their target cells by activation of signal transducer and activator of transcription 6 (STAT 6) or phosphatidylinositol 3-kinase (PI3K). We examined the signal transduction events involved in IL-4 and IL-13 regulation of epithelial paracellular permeability using T84 cells, a model human colonic epithelium. T84 cells treated with IL-4 or IL-13 displayed virtually identical dose- and time-dependent STAT 6 activation as assessed by electrophoretic mobility shift assay (EMSA) and decreases in transepithelial resistance (TER). STAT 6 DNA binding activity was maximal in nuclear extracts 30 min after exposure to IL-4 or IL-13, and TER was maximally reduced by 24 h post-treatment. Pretreatment of epithelia with transcription factor decoys (phosphorothioated DNA oligonucleotides containing the STAT 6 binding site) dramatically reduced STAT 6 activation as detected by EMSA, but did not attenuate the TER reduction by IL-4 or IL-13. In contrast, although the PI3K inhibitors wortmannin and LY294002 did not affect IL-4 or IL-13 STAT 6 activation, they significantly inhibited the ability of either cytokine to lower TER. Thus, we provide evidence for PI3K as the major proximal signaling event in IL-4 and IL-13 regulation of TER and speculate that pharmacological targeting of enterocytic PI3K activity may represent a means to manipulate epithelial permeability.
      IFNγ
      interferon γ
      EMSA
      electrophoretic mobility shift assay
      IL
      interleukin
      PI3K
      phosphatidylinositol 3-kinase
      PKC
      protein kinase C
      STAT
      signal transducer and activator of transcription
      TER
      transepithelial resistance
      TFDs
      transcription factor decoys
      TEMED
      N,N,N′,N′-tetramethylethylenediamine
      ERK
      extracellular signal-regulated kinase
      The intestinal epithelium is not a passive entity but rather is a dynamic tissue which, along with other functions, serves as a regulated barrier between the external and internal environments. Loss of epithelial barrier function has been reported in many enteropathies, including the inflammatory bowel diseases: Crohn's disease and ulcerative colitis (
      • Soderholm J.D.
      • Holmgren-Peterson K.
      • Olaison G.
      • Franzen L.E.
      • Westrom B.
      • Magnusson K.-E.
      • Sjodahl R.
      ,
      • Schmitz H.
      • Barmeyer C.
      • Fromm M.
      • Runkel N.
      • Foss H.-D.
      • Bentzel C.J.
      • Riecken E.-O.
      • Schulzke J.-D.
      ). Whether this loss of barrier function is associated with the etiology of disease or acts as a potentiating factor is a point of debate. It has been shown that Crohn's disease is often accompanied by increased local, or circulating, levels of interferon-γ (IFNγ)1 and tumor necrosis factor-α and that IL-4 can be increased in cohorts of patients with ulcerative colitis or Crohn's disease (
      • Inoue S.
      • Matsumoto T.
      • Iida M.
      • Mizuno M.
      • Kuroki F.
      • Hosika K.
      • Shimizu M.
      ,
      • Desreumaux P.
      • Brandt E.
      • Gambiez L.
      • Emilie D.
      • Geboes K.
      • Klein O.
      • Ectors N.
      • Cortot A.
      • Capron M.
      • Colombel J.-F.
      ). Furthermore, several in vitro studies have demonstrated that cytokines, including IFNγ, IL-4, and IL-13, can increase the permeability characteristics of intestinal epithelial monolayers (
      • Madara J.L.
      • Stafford J.
      ,
      • Zund G.
      • Madara J.L.
      • Dzus A.
      • Awtrey C.
      • Colgan S.P.
      ,
      • Lu J.
      • Philpott D.J.
      • Saunders P.R.
      • Perdue M.H.
      • Yang P.-C.
      • McKay D.M.
      ).
      Several cytokines exert their biological effects via the signal transducer and activator of transcription (STAT) system (). Briefly, cytokine ligation of receptor causes receptor dimerization and phosphorylation by associated Janus kinase proteins. Subsequently, latent cytoplasmic STAT protein monomers bind to the receptor where they are phosphorylated, dimerize, and then translocate to the nucleus to modulate gene transcription. Studies with immune cells have illustrated that IL-4 and IL-13 specifically activate STAT 6 (). Additional investigations showed that IL-4 and IL-13 also signal along the insulin receptor substrate-1/2, phosphatidylinositol 3-kinase (PI3K) pathway; for example, to support proliferation of B cells (
      • Nelms K.
      • Keegan A.D.
      • Zamorano J.
      • Ryan J.J.
      • Paul W.E.
      ). Although IL-4 and IL-13 signal transduction events are being precisely characterized in immune cells considerably less information is available with specific reference to epithelial cells.
      Given the ability of IL-4 and IL-13 to directly affect epithelial permeability, the present study was designed to: (a) define STAT 6 kinetics in model intestinal epithelia, primarily the human transformed T84 cell line, and (b) to use a pharmacological approach to explore the intracellular signal transduction pathway events that govern the IL-4- and IL-13-induced increase in epithelial permeability. The data presented here support the conclusions that IL-4 and IL-13 mobilize STAT 6 in similar dose- and time-dependent manners and activate the PI3K signal transduction pathway in T84 cells. Also, our in vitroobservations suggest that the IL-4- and IL-13-induced increase in T84 epithelial permeability occur, at least in part, via a PI3K-dependent mechanism.

      DISCUSSION

      Cytokines directly affect a variety of epithelial functions, including electrolyte transport, barrier function, and mediator synthesis (
      • McKay D.M.
      • Baird A.W.
      ). Recent studies have begun to explore epithelial intracellular signaling cascades in response to cytokine exposure (
      • Guo F.H.
      • Uetani K.
      • Haque S.J.
      • Williams B.R.G.
      • Dweik R.A.
      • Thunnissen F.B.J.M.
      • Calhoun W.
      • Erzurum S.C.
      ,
      • Jobin C.
      • Panja A.
      • Hellerbrand C.
      • Iimuro Y.
      • Didonato J.
      • Brenner D.A.
      • Sartor R.B.
      ). Here we used a pharmacological approach to provide evidence for a PI3K-dependent, STAT 6-independent proximal signal transduction pathway in the IL-4 and IL-13 regulation of paracellular permeability in T84 cells, a model gut epithelium.
      Cell-mediated immunity is typified by IFNγ production (i.e. T helper 1 cell events), and humoral immunity is characterized by IL-4 and IL-10 synthesis (i.e. Th2 events). Investigations with patients with airways inflammation have shown elevated serum levels of Th2-type cytokines and, somewhat unexpectedly, concomitant increases in gut permeability (
      • Bernard A.
      • Desreumeaux P.
      • Huglo D.
      • Hoorelbeke A.
      • Tonnel A.
      • Wallaert B.
      ). Increases in IL-4 mRNA in ileal resections from some patients with Crohn's disease and colonic tissue from cohorts of patients with ulcerative colitis have been shown (
      • Inoue S.
      • Matsumoto T.
      • Iida M.
      • Mizuno M.
      • Kuroki F.
      • Hosika K.
      • Shimizu M.
      ,
      • Desreumaux P.
      • Brandt E.
      • Gambiez L.
      • Emilie D.
      • Geboes K.
      • Klein O.
      • Ectors N.
      • Cortot A.
      • Capron M.
      • Colombel J.-F.
      ). Also, the colitis that develops in mice lacking the α-chain of the T cell receptor is less severe in animals that also lack IL-4, although gut permeability has not been examined in this model (
      • Mizoguchi A.
      • Mizoguchi E.
      • Bhan A.K.
      ). Moreover, in vitro studies have unequivocally shown that cytokines can directly alter epithelial permeability (
      • McKay D.M.
      • Baird A.W.
      ,
      • Colgan S.P.
      • Resnick M.B.
      • Parkos C.A.
      • Delp-Archer C.
      • McGuirk D.
      • Bacarra A.E.
      • Weller P.F.
      • Madara J.L.
      ): IL-4 and IL-13 often exert similar biological activities and both decrease the barrier function of T84 monolayers (
      • Zund G.
      • Madara J.L.
      • Dzus A.
      • Awtrey C.
      • Colgan S.P.
      ,
      • Takeda K.
      • Tanaka T.
      • Shi W.
      • Matsumoto M.
      • Minami M.
      • Kashiwamura S.-I.
      • Nakanishi K.
      • Yoshida N.
      • Kisimoto T.
      • Akira S.
      ). Consequently, excess IL-4 (or IL-13) in vivo may be important in the regulation of gut permeability and the development and/or progression of inflammatory disease.
      The IL-4 and IL-13 receptors share a common chain, designated the IL-4Rα chain, and studies with immune cells have revealed that following receptor ligation both cytokines activate the STAT 6 and PI3K pathways (
      • Nelms K.
      • Keegan A.D.
      • Zamorano J.
      • Ryan J.J.
      • Paul W.E.
      ). Although cell-specific events have been described, the bulk of the available data indicate that IL-4/STAT 6 events control transcriptional regulation of genes for CD23, major histocompatibility complex II, and IgE as well as facilitating the generation of Th2 cells (
      • Takeda K.
      • Tanaka T.
      • Shi W.
      • Matsumoto M.
      • Minami M.
      • Kashiwamura S.-I.
      • Nakanishi K.
      • Yoshida N.
      • Kisimoto T.
      • Akira S.
      ,
      • Shimoda K.
      • van Deursen J.
      • Sangster M.Y.
      • Sarawar S.R.
      • Tripp R.A.
      • Chu C.
      • Quelle F.W.
      • Nosaka T.
      • Vignali D.A.A.
      • Doherty P.C.
      • Grosveld G.
      • Paul W.E.
      • Ihle J.N.
      ). The PI3K pathway has been shown to regulate vesicle trafficking and, by inference, cytoskeletal rearrangements (
      • Corvera S.
      • Czech M.P.
      ), proliferation, and rescue from apoptosis (
      • Chen R.-H.
      • Chang M.-C.
      • Su Y.-H.
      • Fsai Y.-T.
      • Kuo M.-L.
      ). The aims of our investigation were to assess if IL-4 and IL-13 activate STAT 6 to the same degree and to determine if IL-4 and IL-13 regulate epithelial permeability via a STAT 6 or a PI3K pathway.
      Initial studies revealed that IL-4 and IL-13 activation of STAT 6 (as defined by DNA binding activity on EMSA) was time- and dose-dependent and virtually identical for both cytokines. The identity of STAT 6 on EMSA was confirmed by anti-STAT 6 antibody supershifts and competitive binding by a non-radiolabeled STAT 6 DNA oligonucleotide. Although these data fit with the predicted effect of IL-4 and IL-13, they are nevertheless the first demonstration of STAT 6 activation in enteric epithelium. Indeed, it is only recently that STAT signaling has piqued the interest of the epithelial biologist (
      • Pfeffer L.M.
      • Yang C.H.
      • Pfeffer S.R.
      • Murti A.
      • McCormack S.A.
      • Johnston L.R.
      ,
      • Liang Q.
      • Mohan R.R.
      • Chen L.
      • Wilson S.E.
      ,
      • Walter M.J.
      • Look D.C.
      • Tidwell R.M.
      • Roswit W.T.
      • Holtzman M.J.
      ,
      • Reinecker H.-C.
      • MacDermott R.P.
      • Mirau S.
      • Dignass A.
      • Podolsky D.K.
      ). Additionally, a faster migrating constitutive STAT 6-like band was identified on EMSA. This may be a truncated STAT 6 isoform analogous to one of the STAT 6 isoforms (designated STAT 6, 6b, and 6c) that have been identified in a murine mast cell line (
      • Sherman M.A.
      • Secor V.H.
      • Brown M.A.
      ), a human lung fibroblast cell line (
      • Patel B.K.R.
      • Pierce J.H.
      • LaRochelle W.J.
      ), and primary isolates of human blood-derived natural killer and T cells (
      • McKay D.M.
      • Baird A.W.
      ).
      Currently there are no specific pharmacological inhibitors of STAT 6, with the possible exception of the immunosuppressive drug leflunomide, which was found to block STAT 6 activation in IL-4-treated B cells (
      • Siemasko K.
      • Chong A.S.-F.
      • Jack H.-M.
      • Gong H.
      • Williams J.W.
      • Finnegan A.
      ). Following the published leflunomide treatment protocol, we were unable to block either IL-4- or IL-13-induced STAT 6 activation in T84 cells or the drop in TER.
      P. J. M. Ceponis, F. Botelho, C. D. Richards, and D. M. McKay, personal observation.
      These preliminary data may reflect cell-specific effects of leflunomide. Rather, we employed transcription factor decoys (TFDs) to block STAT 6 activation. With this strategy, activated STAT dimers will bind to the TFDs in the cytoplasm, effectively blocking their binding to host cell nuclear DNA (
      • Bielinska A.
      • Shivdasani R.A.
      • Zhang L.
      • Nabel G.J.
      ,
      • Boccaccio C.
      • Ando M.
      • Tamagnone L.
      • Bardelli A.
      • Michielli P.
      • Battistini C.
      • Comoglio P.M.
      ,
      • Khaled A.R.
      • Butfiloski E.J.
      • Sobel E.S.
      • Schiffenbauer J.
      ). A recent report (
      • Park Y.G.
      • Nesterova M.
      • Agrawal S.
      • Cho-Chung Y.S.
      ) suggests that TFDs may remain stable in the cytoplasm for up to 48 h. Importantly, TFDs bearing the particular binding element specific for STAT 6 used in our study (i.e. TTC-N4-GAA) have been used to functionally disrupt the IL-4-induced, STAT 6-driven proliferation of CD4+ T cells (
      • Wang L.H.
      • Yang X.Y.
      • Kirken R.A.
      • Resau J.H.
      • Farrar W.L.
      ). Pretreatment of T84 cells with the appropriate TFD, but not a scrambled form of the DNA sequence, significantly blocked the activation of STAT 6 by 100 ng/ml IL-4 or IL-13 as determined by subsequent EMSA. Similarly, the constitutive STAT 6-like band was also reduced by TFD exposure. However, TFD pretreatment did not interfere to any significant extent with the ability of a 10-fold less dose of IL-4 or IL-13 to reduce the TER across T84 monolayers. Thus, we present no data in favor of the involvement of the inducible, or constitutive, form of STAT 6 in the regulation of epithelial paracellular permeability. The possibility remains that the EMSA was unable to detect low levels of STAT 6 that were capable of transducing a biological signal; that is, small amounts of STAT 6 below the EMSA detection limit, but enough to affect transcription may have escaped the TFDs. Unequivocal statements on the role of STAT 6 in regulating epithelial paracellular permeability await the advent of a stable and, preferably, inducible STAT 6 dominant-negative transfected enterocytic cell line suitable for electrophysiological studies.
      Ligation of the IL-4 and IL-13 receptors results in sequential binding of the docking protein insulin receptor substrate-1/2 and then PI3K, followed by the production of lipid inositols that can activate a variety of signaling molecules, including PKC (
      • Dutil E.M.
      • Toker A.
      • Newton D.
      ). Use of T84 cells and other epithelia have implicated PKC activity in the regulation of the epithelial tight junction function, the rate-limiting step governing paracellular permeability (
      • Turner J.R.
      • Angle J.M.
      • Black E.D.
      • Joyal J.L.
      • Sacks D.B.
      • Madara J.L.
      ,
      • Philpott D.J.
      • McKay D.M.
      • Mak W.
      • Perdue M.H.
      • Sherman P.M.
      ,
      • Anderson J.M.
      • van Itallie C.M.
      ). Recently, biphasic PI3K activity with characteristic early and late functions has been described, suggesting that PI3K is capable of supporting immediate and sustained cellular responses (
      • Hershberg R.M.
      • Mayer L.F.
      ). We found that inclusion of the inhibitors of PI3K activity, wortmannin, or the more specific compound LY294002, resulted in a significant ablation of the ability of IL-4 or IL-13 to decrease T84 monolayer TER. Indeed, these observations are in accordance with the recognized ability of PI3K to affect the cytoskeleton, which is a pivotal determinant in the control of epithelial tight junction activity (
      • Corvera S.
      • Czech M.P.
      ). Additionally, rescue of the HT-29 epithelial cell line from apoptosis by exposure to IL-13 was shown to occur via a PI3K pathway (
      • Wright K.
      • Kolios G.
      • Westwick J.
      • Ward S.G.
      ).
      A recent report (
      • Pan Z.K.
      • Chen L.-Y.
      • Cochrane C.G.
      • Zuraw B.L.
      ) indicates that wortmannin and LY294002 can inhibit NFκB activation in stimulated immune cells, raising the possibility that these drugs affect STAT 6 activation in response to IL-4 or IL-13. However, neither wortmannin nor LY294002, when used at the highest doses employed in the physiological studies, affected the STAT 6 activation by IL-4 or IL-13. Similarly, others have shown that wortmannin did not affect IL-4-evoked STAT 6 activation as assessed by Western blotting (
      • Ahmad F.
      • Gao G.
      • Wang L.M.
      • Landstrom T.R.
      • Degerman E.
      • Pierce J.H.
      • Manganiello V.C.
      ). Collectively, these data support the hypothesis that the proximal signal transduction events elicited by IL-4 and IL-13, namely STAT 6 and PI3K activation, are separate and distinct. The virtually indistinguishable kinetics of IL-4- and IL-13-induced STAT 6 mobilization, along with the pivotal role of PI3K in their modulation of epithelial paracellular permeability, adds support to the hypothesis that the common IL-4Rα chain in the IL-4 and IL-13 receptors is crucial for signal transduction in enteric epithelia.
      In conclusion, we have identified PI3K activity as a major proximal signaling event involved in the IL-4- and IL-13-induced increase in epithelial paracellular permeability. The data raise questions pertinent to downstream events from PI3K in the regulation of paracellular permeability such as the involvement of PKC isoforms in the modulation of epithelial tight junctions by cytokines. These issues are the focus of ongoing studies. Finally, our data lead to the tantalizing speculation that future therapies for enteropathies characterized by an epithelial permeability defect could specifically target PI3K while leaving STAT 6-driven events (e.g. major histocompatibility complex II expression) intact.

      Acknowledgments

      We thank Jun Lu for technical assistance at the outset of this study.

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