title: Proteasome inhibition of pathologic shedding of enterocytes to defend barrier function requires X-linked inhibitor of apoptosis protein and nuclear factor κB.
abstract: OBJECTIVE
  Although we are beginning to understand where, when, and how intestinal epithelial cells are shed, physiologically, less is understood about alterations in cell fate during minimally invasive epithelial infections. We used a piglet model of Cryptosporidium parvum infection to determine how elimination of infected enterocytes is balanced with the need to maintain barrier function.
  
  
  METHODS
  We studied the effects of enterocyte shedding by C parvum-infected ileum on barrier function ex vivo with Ussing chambers. The locations and activities of caspase-3, nuclear factor κB (NF-κB), and inhibitor of apoptosis proteins (IAP) were assayed by enzyme-linked immunosorbent assay, immunoblot, and tissue immunoreactivity analyses and using specific pharmacologic inhibitors. The location, specificity, and magnitude of enterocyte shedding were quantified using special stains and light microscopy.
  
  
  RESULTS
  Infection with C parvum activated apoptotic signaling pathways in enterocytes that resulted in cleavage of caspase-3. Despite caspase-3 cleavage, enterocyte shedding was confined to villus tips, coincident with apoptosis, and observed more frequently in infected cells. Epithelial expression of X-linked inhibitor of apoptosis protein (XIAP), activation of NF-κB, and proteasome activity were required for control of cell shedding and barrier function. The proteasome blocked activity of caspase-3; this process was mediated by expression of XIAP, which bound to cleaved caspase-3.
  
  
  CONCLUSIONS
  We have identified a pathway by which villus epithelial cells are maintained during C parvum infection. Loss of barrier function is reduced by active retention of infected enterocytes until they reach the villus tip. These findings might be used to promote clearance of minimally invasive enteropathogens, such as by increasing the rate of migration of epithelial cells from the crypt to the villus tip.
authors.affiliation: Department of Population Health, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina 27607, USA.
authors.forename: Derek M
authors.initials: DM
authors.lastname: Foster
authors.forename: Stephen H
authors.initials: SH
authors.lastname: Stauffer
authors.forename: Maria R
authors.initials: MR
authors.lastname: Stone
authors.forename: Jody L
authors.initials: JL
authors.lastname: Gookin
mesh_terms: D000818:Animals; D017209:Apoptosis; D053148:Caspase 3 / Q000378:metabolism; D003457:Cryptosporidiosis / Q000378:metabolism; D016785:Cryptosporidium parvum; D004195:Disease Models, Animal; D020895:Enterocytes / Q000378:metabolism* / Q000382:microbiology; D007082:Ileum / Q000382:microbiology / Q000503:physiopathology*; D007413:Intestinal Mucosa / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D015398:Signal Transduction; D013552:Swine; D051636:X-Linked Inhibitor of Apoptosis Protein / Q000378:metabolism*
chemical_list: D016328:NF-kappa B; D051636:X-Linked Inhibitor of Apoptosis Protein; D053148:Caspase 3; D046988:Proteasome Endopeptidase Complex