Phosphatidylinositol 3-kinase-dependent pathways oppose Fas-induced apoptosis and limit chloride secretion in human intestinal epithelial cells. Implications for inflammatory diarrheal states.
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 (DeltaI(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.
Abreu
M T
MT
Inflammatory Bowel Disease Center and Burns and Allen Research Institute, Cedars-Sinai Medical Center, 8631 West 3rd Street, Suite 245E, Los Angeles, CA 90048, USA. Maria.Abreu@cshs.org
Arnold
E T
ET
Chow
J Y
JY
Barrett
K E
KE
D016475:3T3 Cells; D000199:Actins / Q000378:metabolism; D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D020169:Caspases / Q000378:metabolism; D002448:Cell Adhesion; D002460:Cell Line; D002478:Cells, Cultured; D002713:Chlorine / Q000378:metabolism*; D002867:Chromones / Q000494:pharmacology; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003598:Cytoskeletal Proteins / Q000096:biosynthesis; D051180:Desmoplakins; D003967:Diarrhea / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004847:Epithelial Cells / Q000378:metabolism*; D006801:Humans; D007249:Inflammation; D007413:Intestinal Mucosa / Q000378:metabolism*; D008565:Membrane Proteins / Q000096:biosynthesis; D051379:Mice; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D009025:Morpholines / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010750:Phosphoproteins / Q000096:biosynthesis; D015398:Signal Transduction; D013997:Time Factors; D000077191:Wortmannin; D062826:Zonula Occludens-1 Protein; D019014:fas Receptor / Q000378:metabolism*
D000199:Actins; D000730:Androstadienes; D002867:Chromones; D003432:Cross-Linking Reagents; D003598:Cytoskeletal Proteins; D051180:Desmoplakins; D004791:Enzyme Inhibitors; D008565:Membrane Proteins; D009025:Morpholines; D010750:Phosphoproteins; C569042:TJP1 protein, human; C569043:Tjp1 protein, mouse; D062826:Zonula Occludens-1 Protein; D019014:fas Receptor; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D002713:Chlorine; D019869:Phosphatidylinositol 3-Kinases; D020169:Caspases; D000077191:Wortmannin