Prevention of mucosal atrophy: role of glutamine and caspases in apoptosis in intestinal epithelial cells.
Glutamine starvation induces apoptosis in enterocytes; therefore glutamine is important in the maintenance of gut mucosal homeostasis. However, the molecular mechanisms are unknown. The caspase family of proteases constitutes the molecular machinery that drives apoptosis. Caspases are selectively activated in a stimulus-specific and tissue-specific fashion. The aims of this study were to (1) identify specific caspases activated by glutamine starvation and (2) determine whether a general caspase inhibitor blocks glutamine starvation-induced apoptosis in intestinal epithelial cells. Rat intestinal epithelial (RIE-1) cells were deprived of glutamine. Specific caspase activation was measured using fluorogenic substrate assay. Apoptosis was quantified by DNA fragmentation and Hoechst nuclear staining. Glutamine starvation of RIE-1 cells resulted in the time-dependent activation of caspases 3 (10 hours) and 2 (18 hours), and the induction of DNA fragmentation (12 hours). Caspases 1 and 8 remained inactive ZVAD-fluoromethyl ketone, a general caspase inhibitor, completely blocked glutamine starvation-induced caspase activation, DNA fragmentation, and nuclear condensation. These results indicate that glutamine starvation selectively activates specific caspases, which leads to the induction of apoptosis in RIE-1 cells. Furthermore, inhibition of caspase activity blocked the induction of apoptosis, suggesting that caspases are potential molecular targets to attenuate apoptotic responses in the gut.
Papaconstantinou
H T
HT
Department of Surgery, The University of Texas Medical Branch, Galveston, TX 77555-0542, USA.
Chung
D H
DH
Zhang
W
W
Ansari
N H
NH
Hellmich
M R
MR
Townsend
C M
CM
Ko
T C
TC
D000590:Amino Acid Chloromethyl Ketones / Q000494:pharmacology; D000704:Analysis of Variance; D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D001284:Atrophy; D020170:Caspase 1 / Q000187:drug effects; D053143:Caspase 2; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D061945:Caspase Inhibitors; D020169:Caspases / Q000187:drug effects* / Q000502:physiology; D002467:Cell Nucleus / Q000648:ultrastructure; D004396:Coloring Agents; D046508:Culture Techniques; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D053938:DNA Fragmentation; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000187:drug effects; D004847:Epithelial Cells / Q000187:drug effects / Q000201:enzymology / Q000473:pathology; D005973:Glutamine / Q000037:antagonists & inhibitors / Q000494:pharmacology* / Q000502:physiology; D006706:Homeostasis / Q000502:physiology; D007413:Intestinal Mucosa / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D051381:Rats; D013997:Time Factors
D000590:Amino Acid Chloromethyl Ketones; D061945:Caspase Inhibitors; D004396:Coloring Agents; D015853:Cysteine Proteinase Inhibitors; D004792:Enzyme Precursors; C096713:benzyloxycarbonylvalyl-alanyl-aspartyl fluoromethyl ketone; D005973:Glutamine; C505410:Casp3 protein, rat; C505528:Casp8 protein, rat; C506435:Casp9 protein, rat; D053143:Caspase 2; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases; D020170:Caspase 1