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• W2059796847 abstract "The signaling pathways mediating human intestinal epithelial cell differentiation remain largely undefined. Phosphatidylinositol 3-kinase (PI3K) is an important modulator of extracellular signals, including those elicited by E-cadherin-mediated cell-cell adhesion, which plays an important role in maintenance of the structural and functional integrity of epithelia. In this study, we analyzed the involvement of PI3K in the differentiation of human intestinal epithelial cells. We showed that inhibition of PI3K signaling in Caco-2/15 cells repressed sucrase-isomaltase and villin protein expression. Morphological differentiation of enterocyte-like features in Caco-2/15 cells such as epithelial cell polarity and brush-border formation were strongly attenuated by PI3K inhibition. Immunofluorescence and immunoprecipitation experiments revealed that PI3K was recruited to and activated by E-cadherin-mediated cell-cell contacts in confluent Caco-2/15 cells, and this activation appears to be essential for the integrity of adherens junctions and association with the cytoskeleton. We provide evidence that the assembly of calcium-dependent adherens junctions led to a rapid and remarkable increase in the state of activation of Akt and p38 MAPK pathways and that this increase was blocked in the presence of anti-E-cadherin antibodies and PI3K inhibitor. Therefore, our results indicate that PI3K promotes assembly of adherens junctions, which, in turn, control p38 MAPK activation and enterocyte differentiation. The signaling pathways mediating human intestinal epithelial cell differentiation remain largely undefined. Phosphatidylinositol 3-kinase (PI3K) is an important modulator of extracellular signals, including those elicited by E-cadherin-mediated cell-cell adhesion, which plays an important role in maintenance of the structural and functional integrity of epithelia. In this study, we analyzed the involvement of PI3K in the differentiation of human intestinal epithelial cells. We showed that inhibition of PI3K signaling in Caco-2/15 cells repressed sucrase-isomaltase and villin protein expression. Morphological differentiation of enterocyte-like features in Caco-2/15 cells such as epithelial cell polarity and brush-border formation were strongly attenuated by PI3K inhibition. Immunofluorescence and immunoprecipitation experiments revealed that PI3K was recruited to and activated by E-cadherin-mediated cell-cell contacts in confluent Caco-2/15 cells, and this activation appears to be essential for the integrity of adherens junctions and association with the cytoskeleton. We provide evidence that the assembly of calcium-dependent adherens junctions led to a rapid and remarkable increase in the state of activation of Akt and p38 MAPK pathways and that this increase was blocked in the presence of anti-E-cadherin antibodies and PI3K inhibitor. Therefore, our results indicate that PI3K promotes assembly of adherens junctions, which, in turn, control p38 MAPK activation and enterocyte differentiation. mitogen-activated protein kinase phosphatidylinositol 3-kinase zonula occludens-1 phosphate-buffered saline green fluorescent protein 1,4-piperazinediethanesulfonic acid adenomatous polyposis coli The epithelium of the small intestine is characterized by its rapid and constant renewal. This process involves cell generation and migration from the stem cell population located at the bottom of the crypt to the extrusion of terminally differentiated cells at the tip of the villus (1Babyatsky M.W. Podolsky D.K. Yamada T. Growth and Development of the Gastrointestinal Tract. 3rd Ed. J. B. Lippincott Co., Philadelphia1999: 547-584Google Scholar). Thus, the crypt is mainly composed of proliferative and poorly differentiated cells, whereas the villus is lined with functional absorptive, goblet, and endocrine cells (1Babyatsky M.W. Podolsky D.K. Yamada T. Growth and Development of the Gastrointestinal Tract. 3rd Ed. J. B. Lippincott Co., Philadelphia1999: 547-584Google Scholar). The molecular and cellular mechanisms responsible for the fine coordination between proliferation, migration, and differentiation along the crypt-villus axis are still largely unknown. E-cadherin-mediated cell-cell attachment plays an important role in the differentiation, polarization, and homeostasis of many epithelia (2Takeichi M. Curr. Opin. Cell Biol. 1995; 7: 619-627Crossref PubMed Scopus (1262) Google Scholar, 3Larue L. Antos C. Butz S. Huber O. Delmas V. Dominis M. Kemler R. Development. 1996; 122: 3185-3194Crossref PubMed Google Scholar, 4Vleminckz K. Kemler R. Bioessays. 1999; 21: 211-220Crossref PubMed Scopus (302) Google Scholar). Cadherins are responsible for cell-cell adhesion through a calcium-dependent interaction of their extracellular domains. Their cytoplasmic tails are linked to the cytoskeleton through a complex of proteins that includes α-, β-, and γ-catenins. This link is involved in the strengthening of cell-cell adhesion and in the cohesion of epithelial tissues (5Jou T.S. Stewart D.B. Stappert J. Nelson W.J. Mars J.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5067-5071Crossref PubMed Scopus (305) Google Scholar). The importance of cadherins in the renewal of the intestinal epithelium has been demonstrated in vivo in two mouse models. Overexpression of E-cadherin in the crypts of the small intestine reduces cell proliferation and migration (6Hermiston M.L. Gordon J.I. J. Cell Biol. 1995; 129: 489-506Crossref PubMed Scopus (385) Google Scholar). Conversely, expression of a dominant-negative N-cadherin leads to over-proliferation, uncoordinated differentiation, and a Crohn's disease phenotype (7Hermiston M.L. Gordon J.I. Science. 1995; 270: 1203-1207Crossref PubMed Scopus (593) Google Scholar). The intracellular signaling pathways that transmit extracellular cues for epithelial differentiation along the crypt-villus axis of the intestine remain poorly defined. We recently reported that p38 MAPK1 plays a crucial role in intestinal epithelial cell differentiation by enhancing the transactivation capacity of CDX2 (8Houde M. Laprise P. Jean D. Blais M. Asselin C. Rivard N. J. Biol. Chem. 2001; 276: 21885-21894Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar), an intestine-specific homeobox gene product well known for its broad effect on enterocyte differentiation (9Traber P.G. Silberg D.G. Annu. Rev. Physiol. 1996; 58: 275-297Crossref PubMed Scopus (135) Google Scholar). However, the upstream signaling pathways activating p38 MAPK in committed intestinal cells induced to differentiate remain to be defined. Interestingly, in vitroexperiments have shown that the establishment of cell-cell contacts in intestinal cell cultures could be a critical step in initiating p38 MAPK action (8Houde M. Laprise P. Jean D. Blais M. Asselin C. Rivard N. J. Biol. Chem. 2001; 276: 21885-21894Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar), cell cycle arrest (10Evers B.M. Ko T.C. Li J. Thompson E.A. Am. J. Physiol. 1996; 271: G722-G727PubMed Google Scholar, 11Deschênes C. Vézina A. Beaulieu J.-F. Rivard N. Gastroenterology. 2001; 120: 423-438Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar), and induction of the differentiation process (8Houde M. Laprise P. Jean D. Blais M. Asselin C. Rivard N. J. Biol. Chem. 2001; 276: 21885-21894Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 12Pinto M. Robine-Leon S. Appay M.D. Kedinger M. Triadou N. Bussaulx N. Lacroix B. Simon-Assman P. Haffen K. Fogh J. Zweibaum A. Biol. Cell. 1983; 47: 323-330Google Scholar, 13Vachon P.H. Beaulieu J.-F. Gastroenterology. 1992; 103: 414-423Abstract Full Text PDF PubMed Scopus (186) Google Scholar, 14Peterson M.D. Bement W.M. Mooseker M.S. J. Cell Sci. 1993; 105: 461-472Crossref PubMed Google Scholar, 15Aliaga J.C. Deschênes C. Beaulieu J.-F. Calvo E.L. Rivard N. Am. J. Physiol. 1999; 277: G631-G641PubMed Google Scholar). An important role for p38 MAPK in various mammalian cell differentiation processes has recently been proposed (16Nebrada A.R. Porras A. Trends Biochem. Sci. 2000; 26: 257-260Abstract Full Text Full Text PDF Scopus (500) Google Scholar). For instance, differentiation of C2C12 and L8 myoblasts into myotubes has been found to be mediated by p38 activation (17Cuenda A. Cohen P. J. Biol. Chem. 1999; 274: 4341-4346Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar, 18Zetser A. Gredinger E. Bengal E. J. Biol. Chem. 1999; 274: 5193-5200Abstract Full Text Full Text PDF PubMed Scopus (393) Google Scholar). Although this skeletal muscle differentiation requires phosphatidylinositol 3-kinase (PI3K), it is not yet clear whether PI3K and p38 MAPK act in a common pathway (19Chun Y.K. Kim J. Kwon S. Choi S.H. Hong F. Moon K. Kim J.M. Choi S.L. Kim B.M. Kim S.S. Biochem. Biophys. Res. Comm. 2000; 276: 502-507Crossref PubMed Scopus (25) Google Scholar, 20Li Y. Jiang B. Ensign W.Y. Vogt P.K. Han J. Cell. Signal. 2000; 11–12: 751-757Crossref Scopus (97) Google Scholar). The PI3K family members are lipid kinases that phosphorylate phosphoinositides at position 3 of the inositol ring (21Rameh L.E. Cantley L.C. J. Biol. Chem. 1999; 274: 8347-8350Abstract Full Text Full Text PDF PubMed Scopus (852) Google Scholar), acting as membrane anchors that locate and activate pleckstrin homology domain-containing effectors such as the well characterized serine/threonine kinase Akt (22Datta S.R. Brunet A. Greenberg M.E. Genes Dev. 1999; 13: 2905-2927Crossref PubMed Scopus (3729) Google Scholar). Class I PI3Ks are generally composed of a p85 regulatory subunit and a p110 catalytic subunit (21Rameh L.E. Cantley L.C. J. Biol. Chem. 1999; 274: 8347-8350Abstract Full Text Full Text PDF PubMed Scopus (852) Google Scholar). This class of PI3Ks can be activated by a wide variety of extracellular stimuli, including those elicited by E-cadherin-mediated cell-cell adhesion (23Pece S. Chiariello M. Murga C. Gutkind J.S. J. Biol. Chem. 1999; 274: 19347-19351Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar). In this study, the role and regulation of PI3K in intestinal epithelial cells were investigated. Using several approaches, we demonstrated that PI3K is necessary for the functional and morphological differentiation of intestinal epithelial cells. We also found that PI3K is recruited to and activated by E-cadherin-mediated cell-cell contacts in confluent Caco-2/15 cultures and that this activation appears to be essential for the integrity of the adherens junctions and the association of their components with the cytoskeleton. Finally, we have provided evidence that the assembly of the adherens junctions stimulates Akt and p38 MAPK in a PI3K-dependent manner. [γ-32P]ATP and the enhanced chemiluminescence immunodetection system (ECL) were obtained from Amersham Biosciences, Inc. (Baie d'Urfé, Québec, Canada). Antiserum that specifically recognizes p38α MAPK on Western blots (24Guay J. Lambert H. Gingras-Breton G. Lavoie J.N. Huot J. Landry J. J. Cell Sci. 1997; 110: 357-368Crossref PubMed Google Scholar) was a kind gift from Dr. J. Landry (Université Laval, Laval, Québec). Monoclonal antibody HSI-14 (25Beaulieu J.-F. Nichols B. Quaroni A. J. Biol. Chem. 1989; 264: 20000-20011Abstract Full Text PDF PubMed Google Scholar) against sucrase-isomaltase was kindly provided by Dr. A. Quaroni (Cornell University, Ithaca, NY). Monoclonal antibody CII10 recognizing the 89-kDa apoptotic fragment and the 113-kDa non-cleaved fragment of poly(ADP-ribose) polymerase was a kind gift from Dr. G. G. Poirier (Université Laval). Fluorescein isothiocyanate-labeled goat anti-rabbit IgG and rhodamine-labeled goat anti-mouse IgG were from Roche Molecular Biochemicals (Laval). Antibodies raised against villin, E-cadherin (used in Western blotting and immunofluorescence), and β-catenin were purchased from Transduction Laboratories (Mississauga, Ontario, Canada). Antibody recognizing the phosphorylated and active form of p38 MAPK was from Promega (Nepean, Ontario). Antibody directed against the PI3K p85 regulatory subunit was obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). Anti-Akt and anti-phospho-Akt(Ser473) antibodies were purchased from Cell Signaling (Mississauga). Antibody directed against total actin was from Roche Molecular Biochemicals. The anti-ZO-1 antibody and the anti-E-cadherin antibody used in antibody inhibition experiments were from Zymed Laboratories Inc. (South San Francisco, CA). The specific inhibitor of PI3K (LY294002) was purchased fromCalbiochem (Mississauga). All other materials were obtained from Sigma (Oakville, Ontario) unless otherwise stated. The Caco-2/15 cell line was obtained from Dr. A. Quaroni. This clone of the parent Caco-2 cell line (HTB37; American Type Culture Collection, Manassas, VA) has been extensively characterized elsewhere (13Vachon P.H. Beaulieu J.-F. Gastroenterology. 1992; 103: 414-423Abstract Full Text PDF PubMed Scopus (186) Google Scholar, 15Aliaga J.C. Deschênes C. Beaulieu J.-F. Calvo E.L. Rivard N. Am. J. Physiol. 1999; 277: G631-G641PubMed Google Scholar, 26Beaulieu J.-F. Quaroni A. Biochem. J. 1991; 280: 599-608Crossref PubMed Scopus (134) Google Scholar) and was originally selected for expressing the highest level of sucrase-isomaltase among 16 clones obtained by random cloning. This cell line was routinely cultured in plastic dishes in Dulbecco's modified Eagle's medium (Invitrogen, Burlington, Ontario) containing 10% fetal bovine serum, 4 mm glutamine, 20 mm HEPES, 50 units/ml penicillin, and 50 μg/ml streptomycin. Caco-2/15 cells were used between passages 53 and 78. Studies were performed on cultures at subconfluence (50%) and confluence and between days 2 and 15 post-confluence. Cells were lysed in SDS sample buffer (62.5 mm Tris-HCl (pH 6.8), 2.3% SDS, 10% glycerol, 5% β-mercaptoethanol, and 0.005% bromphenol blue). Proteins (10–50 μg) from whole cell lysates were separated by SDS-PAGE on 7.5 or 10% gels. Proteins were detected immunologically following electrotransfer onto nitrocellulose membranes (Amersham Biosciences, Inc.). Protein and molecular mass markers (Bio-Rad, Mississauga) were revealed by Ponceau red staining. Membranes were blocked in PBS containing 5% powdered milk and 0.05% Tween 20 for at least 1 h at 25 °C. Membranes were then incubated overnight at 4 °C with primary antibodies in blocking solution and then with horseradish peroxidase-conjugated goat anti-mouse or anti-rabbit IgG (1:1000) for 1 h. The blots were visualized using the Amersham Biosciences ECL system. Protein concentrations were measured using a modified Lowry procedure with bovine serum albumin as the standard (27Peterson G.L. Anal. Biochem. 1977; 83: 346-356Crossref PubMed Scopus (7141) Google Scholar). The sucrase-isomaltase reporter construct (provided by Dr. P. G. Traber, University of Pennsylvania, Philadelphia) used for luciferase assays contained the human sucrase-isomaltase promoter from residues −183 to +54 cloned upstream of the luciferase gene of the pGL2 reporter construct as described previously (9Traber P.G. Silberg D.G. Annu. Rev. Physiol. 1996; 58: 275-297Crossref PubMed Scopus (135) Google Scholar). The pRL-SV40 Renilla luciferase vector was from Promega. Expression vectors for a constitutively active form of PI3K (p110*) and a dominant-negative form of PI3K (Δp85) were obtained from Dr. Julian Downward (Imperial Cancer Research Fund, London, United Kingdom) and Dr. Masato Kasuga (Kobe University School of Medicine, Kobe, Japan). Green fluorescent protein (GFP) (CLONTECH, Palo Alto, CA) was subcloned into the expression vector pLHCX (CLONTECH). Newly confluent Caco-2/15 cells grown on glass coverslips in six-well plates were cotransfected by lipofection (LipofectAMINE 2000, Invitrogen) with 0.5 μg of pLHCX-GFP and 0.5 μg of pcDNA3 containing or not the dominant-negative form of PI3K (Δp85). Two days following transfection, cells were fixed for GFP fluorescence and immunofluorescence with either anti-E-cadherin antibody or anti-total actin antibody. Confluent Caco-2/15 cells grown in 24-well plates were transfected by lipofection (LipofectAMINE 2000) as described previously (15Aliaga J.C. Deschênes C. Beaulieu J.-F. Calvo E.L. Rivard N. Am. J. Physiol. 1999; 277: G631-G641PubMed Google Scholar) with 0.1 μg of sucrase-isomaltase/luciferase reporter. One day following transfection, cells were treated with 0, 1, 2, 5, 10, and 20 μm LY294002 for 24 h, and luciferase activity was measured. In other experiments, the sucrase-isomaltase reporter gene vector was cotransfected with 0.1 μg of pcDNA3 containing or not the dominant-negative (Δp85) or constitutively active (p110*) form of PI3K. Luciferase activity was measured 36 h after transfection. The pRL-SV40 Renilla luciferase vector was used as a control for transfection efficiency. Cell cultures were rinsed with PBS, prefixed for 15 min with a 1:1 mixture of culture medium (Dulbecco's modified Eagle's medium) and freshly prepared 2.8% glutaraldehyde in cacodylate buffer (0.1 m cacodylate and 7.5% sucrose), and then fixed for 30 min with 2.8% glutaraldehyde at room temperature. After two rinses, specimens were post-fixed for 60 min with 2% osmium tetroxide in cacodylate buffer. The cells were then dehydrated using increasing ethanol concentrations (40, 70, 90, 95, and 100%; three times each) and covered twice for 3 h with a thin layer of Araldite 502 resin (for ethanol substitution). Finally, the resin was allowed to polymerize at 60 °C for 48 h. The specimens were detached from the plastic vessels, inverted in embedding molds, covered with Araldite 502, and reincubated at 60 °C for 48 h. Thin sections were prepared using an ultramicrotome, contrasted with lead citrate and uranyl acetate, and observed in a blind fashion on a Jeol 100 CX transmission electron microscope. All reagents were purchased from Electron Microscopy Sciences (Cedarlane, Hornby, Ontario). First, the cells were washed twice with ice-cold PBS, and then soluble proteins were extracted on ice with cold lysis/cytoskeleton stabilization buffer (0.5% Triton X-100, 50 mm NaCl, 10 mm PIPES (pH 6.8), 300 mm sucrose, and 3 mmMgCl2). The cytoskeleton-associated proteins (insoluble fraction) were harvested by centrifugation (13,000 rpm at 4 °C for 20 min) and solubilized in SDS buffer (15 mm Tris (pH 7.5), 5 mm EDTA, 2.5 mm EGTA, and 1% SDS) (28Somasiri A. Colleen W. Ellchuk T. Turley S. Rosekelley C.D. Differentiation. 2000; 66: 116-1125Crossref PubMed Scopus (24) Google Scholar). Finally, E-cadherin and β-catenin levels were determined by immunoblotting of the cytoskeleton and total fractions (equal amounts of the soluble and cytoskeleton fractions). Cells were washed twice with ice-cold PBS and lysed in chilled lysis buffer (150 mmNaCl, 1 mm EDTA, 40 mm Tris (pH 7.6), 1% Triton X-100, 0.1 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 1 μg/ml pepstatin, 10 μg/ml aprotinin, 0.1 mm orthovanadate, and 40 mmβ-glycerophosphate). Cell lysates were then cleared of cellular debris by centrifugation. Primary antibodies were added to 600 μg of each cell lysate and incubated for 2 h at 4 °C under agitation. Forty μg of protein A-Sepharose (Amersham Biosciences, Inc.) were subsequently added for 1 h (4 °C under agitation). Immunocomplexes were then harvested by centrifugation and washed four times with ice-cold lysis buffer. Proteins were solubilized with Laemmli buffer and separated by SDS-PAGE. Caco-2/15 cells grown on sterile glass coverslips were washed twice with ice-cold PBS. Cultures were then fixed with 30% methanol and 70% acetone for 15 min at −20 °C, permeabilized with a solution of 0.1% of Triton X-100 in PBS for 10 min, and blocked with PBS and 2% bovine serum albumin for 20 min at room temperature. Cells were finally immunostained for 1 h with the primary antibody and for 30 min with the secondary antibody. For F-actin staining, fixed cells were incubated with 1 μg/ml fluorescein isothiocyanate-phalloidin for 30 min. For total actin staining, fixed cells were incubated with antibody raised against total actin and then with rhodamine-labeled goat anti-mouse IgG. Negative controls (no primary antibody) were included in all experiments. Cells were lysed for 10 min on ice with 1 ml/dish lysis buffer (150 mm NaCl, 1 mm EDTA, 40 mm Tris (pH 7.60), and 1% Triton X-100) supplemented with protease inhibitors (0.1 mm phenylmethylsulfonyl fluoride, 10 μg/ml leupeptin, 1 μg/ml pepstatin, and 10 μg/ml aprotinin) and phosphatase inhibitors (0.1 mm orthovanadate and 40 mm β-glycerophosphate). p38 MAPK was immunoprecipitated from 400 μg of cell lysate. Immunocomplexes were then washed four times with ice-cold lysis buffer and three times with ice-cold kinase buffer (20 mm p-nitrophenyl phosphate, 10 mm MgCl2, 1 mmdithiothreitol, and 30 mm HEPES (pH 7.4)) prior to the kinase assay. The kinase reaction was initiated by incubating the immunocomplexes at 30 °C in the presence of myelin basic protein and [γ-32P]ATP at 2 μg and 2 μCi/assay, respectively. After 30 min, the reaction was stopped by adding Laemmli buffer. Radiolabeled substrates were separated from immunocomplexes by SDS-12.5% PAGE and autoradiographed. Incorporation of 32P by myelin basic protein was linear over the course of the kinase assay. Day 2 post-confluent Caco-2/15 cells were serum-starved for 16 h in Dulbecco's modified Eagle's medium supplemented with 20 mm HEPES, 50 units/ml penicillin, 50 μg/ml streptomycin, and 4 mm glutamine. The adherens junctions were then disrupted by treatment with 4 mm EGTA for 30 min at 37 °C. Intercellular contacts were subsequently allowed to re-establish by restoration of the extracellular calcium concentration by replacing the EGTA-containing medium with fresh medium (1.8 mm CaCl2) (23Pece S. Chiariello M. Murga C. Gutkind J.S. J. Biol. Chem. 1999; 274: 19347-19351Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar, 29Volberg T. Geiger B. Kartenbeck J. Franke W.W. J. Cell Biol. 1986; 102: 1832-1842Crossref PubMed Scopus (180) Google Scholar). In some experiments, the fresh medium contained 100 μg/ml anti-E-cadherin antibody or mouse IgG purified from nonimmune serum. After selected time intervals of calcium restoration, cells were washed twice with ice-cold PBS and lysed to detect phospho-Akt and to assay p38 MAPK activity or were fixed for immunofluorescence. To investigate the role of PI3K in intestinal epithelial cell differentiation, we evaluated the impact of its inhibition on the spontaneous enterocytic differentiation of the human colon cancer cell line Caco-2/15. This cell line provides a unique and well characterized model for the study of intestinal epithelial differentiation because these cells undergo differentiation to a small bowel-like phenotype with microvilli, dome formation, and the expression of sucrase-isomaltase several days after reaching confluence (12Pinto M. Robine-Leon S. Appay M.D. Kedinger M. Triadou N. Bussaulx N. Lacroix B. Simon-Assman P. Haffen K. Fogh J. Zweibaum A. Biol. Cell. 1983; 47: 323-330Google Scholar, 13Vachon P.H. Beaulieu J.-F. Gastroenterology. 1992; 103: 414-423Abstract Full Text PDF PubMed Scopus (186) Google Scholar, 14Peterson M.D. Bement W.M. Mooseker M.S. J. Cell Sci. 1993; 105: 461-472Crossref PubMed Google Scholar, 15Aliaga J.C. Deschênes C. Beaulieu J.-F. Calvo E.L. Rivard N. Am. J. Physiol. 1999; 277: G631-G641PubMed Google Scholar). To block PI3K signaling, we used LY294002, a compound that acts as a competitive inhibitor of the adenosine triphosphate-binding site of PI3K and has been shown to cause specific inhibition with an IC50 of 1.4–5 μm in intact cells (30Vlahos C.J. Matter W.F. Hui K.Y. Brown R.F. J. Biol. Chem. 1994; 269: 5241-5248Abstract Full Text PDF PubMed Google Scholar). Daily addition of 10 μm LY294002 beginning at confluence strongly attenuated the expression levels of two enterocytic differentiation markers, viz. sucrase-isomaltase and villin, compared with untreated cells at days 3, 6, and 12 post-confluence (Fig. 1 A). Loss of PI3K activity did not interfere with overall protein expression, as shown by similar actin levels in LY294002-treated and untreated cells (Fig.1 A). To ascertain that the loss of differentiation marker expression was not a consequence of increased apoptosis, we evaluated the expression of poly(ADP-ribose) polymerase, a well known caspase-3 substrate (31Casciola-Rosen L. Nicholson D.W. Chong T. Rowan K.R. Thornberry N.A. Miller D.K. Rosen A. J. Exp. Med. 1996; 183: 1957-1964Crossref PubMed Scopus (578) Google Scholar), in cells treated with LY294002. As shown in Fig.1 A, chronic treatment of confluent Caco-2/15 cells with the PI3K inhibitor had no effect on poly(ADP-ribose) polymerase cleavage, suggesting that persistent inhibition of PI3K did not affect Caco-2/15 cell survival. The role of PI3K in sucrase-isomaltase expression was further investigated by transient transfection of newly confluent Caco-2/15 cells with a luciferase reporter gene under the control of the human sucrase-isomaltase promoter. As shown in Fig. 1 B, sucrase-isomaltase gene expression was inhibited in a dose-dependent manner by the PI3K inhibitor LY294002, with a maximal effect observed at 20 μm (91% inhibition). Furthermore, overexpression of a dominant-negative form of the regulatory p85 subunit (Δp85) reduced sucrase-isomaltase gene expression by 50%. Conversely, overexpression of a constitutively active p110 subunit (p110*) slightly enhanced sucrase-isomaltase gene expression. Caco-2/15 cell cultures were characterized by transmission electron microscopy day 14 post-confluence. As shown in Fig.2 (panels 1, 3, and5), post-confluent Caco-2/15 cells exhibited ultrastructural characteristics similar to those found in the intact villus epithelium, including well organized brush borders, terminal webs at the luminal aspect of absorptive cells, and typical junctional complexes. Interestingly, treatment of confluent Caco-2/15 cells with the PI3K inhibitor remarkably affected cell polarization and brush-border formation. Indeed, LY294002-treated cells exhibited a less polarized and flat phenotype compared with untreated cells (Fig. 2, panels 1 and 2). The morphology of the brush border was altered markedly, as visualized by a reduction in the number of microvilli (Fig. 2, panels 3 and 4). Interestingly, poorly defined apical junctional complexes were observed in LY294002-treated cells (Fig. 2, panels 5 and6). Taken together, these results indicate that PI3K activity is necessary for functional and morphological differentiation of intestinal epithelial cells. Cell-cell adhesion plays a crucial role in the polarization and differentiation of epithelial cells (2Takeichi M. Curr. Opin. Cell Biol. 1995; 7: 619-627Crossref PubMed Scopus (1262) Google Scholar, 3Larue L. Antos C. Butz S. Huber O. Delmas V. Dominis M. Kemler R. Development. 1996; 122: 3185-3194Crossref PubMed Google Scholar, 4Vleminckz K. Kemler R. Bioessays. 1999; 21: 211-220Crossref PubMed Scopus (302) Google Scholar). Our observation that LY294002-treated cells exhibited a less polarized and differentiated phenotype as well as poorly defined apical junctions prompted us to investigate whether PI3K might control the assembly of adherens and tight junctions in Caco-2/15 cells. We performed E-cadherin and ZO-1 staining in a two-step experiment in which tight and adherens junctions of day 2 post-confluent Caco-2/15 cells were disrupted by chelating extracellular calcium and subsequently allowed to re-establish by restoration of extracellular calcium (23Pece S. Chiariello M. Murga C. Gutkind J.S. J. Biol. Chem. 1999; 274: 19347-19351Abstract Full Text Full Text PDF PubMed Scopus (232) Google Scholar, 29Volberg T. Geiger B. Kartenbeck J. Franke W.W. J. Cell Biol. 1986; 102: 1832-1842Crossref PubMed Scopus (180) Google Scholar) in the presence or absence of LY294002. The untreated cells showed typical honeycomb E-cadherin and ZO-1 staining (Fig.3 A, panels 1 and5). After a 30-min EGTA treatment, cells became rounded, whereas E-cadherin and ZO-1 staining formed a diffuse ring at the cell periphery (Fig. 3 A, panels 2 and 6). Following calcium restoration, E-cadherin and ZO-1 redistributed at the sites of cell-cell contact, and the cells reacquired their epithelial shape (Fig. 3 A, panels 3 and 7), suggesting that tight and adherens junctions were almost completely reformed. However, in LY294002-treated cells, most of the immunoreactive E-cadherin remained diffusely distributed (Fig.3 A, panel 4). Interestingly, similar results were also noted in Caco-2/15 cells transiently transfected with the dominant-negative mutant of p85 (Fig. 3 B), indicating that PI3K inhibition interfered with the assembly of adherens junctions. This phenomenon appears to be specific for adherens junctions because ZO-1 redistributed at the sites of cell-cell contact even in the presence of LY294002 (Fig. 3 A, panel 8). Previous studies have shown that the E-cadherin and β-catenin associated with functional adherens junctions are indirectly linked to the cytoskeleton (32Hülsken J. Birchmeier W. Behrens J. J. Cell Biol. 1994; 127: 2061-2069Crossref PubMed Scopus (586) Google Scholar). Thus, they cannot be extracted with a solution of 0.5% of the nonionic detergent Triton X-100, but are found in the insoluble fraction. Inhibition of PI3K resulted in a significant reduction in the proportion of E-cadherin and β-catenin associated with the cytoskeleton after 16–72 h of treatment in newly confluent Caco-2/15 cells (Fig. 4 A,upper panels). Total amounts of E-cadherin or β-catenin proteins remained comparable. Interestingly, inhibition of PI3K activity by LY294002 or by ectopic expression of the dominant-negative mutant of p85 in newly confluent cells also led to a partial disruption of F-actin at the periphery of the cytoplasmic membrane (Fig.4 B, rig" @default.
• W2059796847 created "2016-06-24" @default.
• W2059796847 creator A5042038598 @default.
• W2059796847 creator A5051818638 @default.
• W2059796847 creator A5073838701 @default.
• W2059796847 creator A5074804060 @default.
• W2059796847 creator A5077433784 @default.
• W2059796847 creator A5083287608 @default.
• W2059796847 date "2002-03-01" @default.
• W2059796847 modified "2023-10-17" @default.
• W2059796847 title "Phosphatidylinositol 3-Kinase Controls Human Intestinal Epithelial Cell Differentiation by Promoting Adherens Junction Assembly and p38 MAPK Activation" @default.
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