Clostridium difficile toxin A-induced colonocyte apoptosis involves p53-dependent p21(WAF1/CIP1) induction via p38 mitogen-activated protein kinase.
OBJECTIVE
Clostridium difficile toxin A causes marked apoptosis of colonocytes in vivo and in vitro, which contributes to the formation of ulcers and pseudomembranes. We investigated the role of p53-dependent pathways and p38 mitogen-activated protein kinase (p38) in toxin A-induced colonocyte apoptosis.


METHODS
The effects of the activation of p53 and p53-dependent pathways including p21(WAF1/CIP1) were assessed in nontransformed human colonic NCM460 epithelial cells exposed to toxin A. Phosphorylation of p53 protein by p38 was measured by in vitro kinase assay, whereas p21 induction by activated p53 was determined by gel shift assays and RNA silencing (small interfering RNA). The relationship between colonocyte apoptosis and p38/p53-dependent pathways was studied in intact mice.


RESULTS
Toxin A stimulated p38 and p53 activation and induced cell cycle arrest (G(2)-M) with persistent expression of p21(WAF1/CIP1). Blockage of p38 by SB203580 inhibited p53 phosphorylation and induction of p21(WAF1/CIP1). In intact mice, p38 blockade suppressed toxin A-mediated destruction of intestinal villi, p21(WAF1/CIP1) expression, and enterocyte apoptosis. In addition, toxin A-mediated p21(WAF1/CIP1) and Bak induction, cytochrome c release, and caspase-3 activation were markedly attenuated in p53-silenced colonocytes, despite active p38. Overexpression of p21(WAF1/CIP1) triggered apoptosis and increased toxin A-associated colonocyte apoptosis.


CONCLUSIONS
The signaling pathway for colonocyte apoptosis following toxin A exposure involves p38-dependent activation of p53 and subsequent induction of p21(WAF1/CIP1), resulting in cytochrome c release and caspase-3 activation through Bak induction.
Kim
Ho
H
Division of Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215, USA.
Kokkotou
Efi
E
Na
Xi
X
Rhee
Sang Hoon
SH
Moyer
Mary P
MP
Pothoulakis
Charalabos
C
Lamont
J Thomas
JT
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D001427:Bacterial Toxins / Q000633:toxicity*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D002453:Cell Cycle / Q000502:physiology; D002460:Cell Line; D003106:Colon / Q000166:cytology*; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics / Q000378:metabolism*; D045304:Cytochromes c / Q000378:metabolism; D004768:Enterotoxins / Q000633:toxicity*; D004791:Enzyme Inhibitors / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology* / Q000378:metabolism; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006801:Humans; D007413:Intestinal Mucosa / Q000166:cytology*; D020935:MAP Kinase Signaling System; D008297:Male; D051379:Mice; D012694:Serine / Q000378:metabolism; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism*; D050998:bcl-2 Homologous Antagonist-Killer Protein / Q000235:genetics / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism*
C494772:BAK1 protein, human; D001427:Bacterial Toxins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D004768:Enterotoxins; D004791:Enzyme Inhibitors; D016159:Tumor Suppressor Protein p53; D050998:bcl-2 Homologous Antagonist-Killer Protein; C041348:tcdA protein, Clostridium difficile; D012694:Serine; D045304:Cytochromes c; D048051:p38 Mitogen-Activated Protein Kinases; C505407:CASP3 protein, human; C505409:Casp3 protein, mouse; D053148:Caspase 3; D020169:Caspases