Role of the ATM-checkpoint kinase 2 pathway in CDT-mediated apoptosis of gingival epithelial cells.
The cytolethal distending toxin (CDT) of the oral pathogen Aggregatibacter actinomycetemcomitans induces cell cycle arrest and apoptosis in various cell types. Western analysis, pharmacological inhibition and siRNA silencing were performed in human immortalized gingival keratinocytes (HIGK) to dissect the functional role of the ataxia telangiectasia mutated (ATM) pathway in the signal transduction steps triggered by the CDT. Infection of HIGK was associated with a time-dependent induction of cytoplasmic histone-associated DNA fragmentation. However, in the absence of CDT, infected HIGK underwent reversible DNA strand breaks but not apoptosis, while caspase 3 activity, p21 levels, and HIGK viability were unaffected. Caspase 9 activity was attenuated in the CDT mutant-infected HIGK compared to wild-type infected cells. Pharmacological inhibition and siRNA-silencing of the ATM downstream effector, the protein kinase checkpoint kinase 2 (Chk2), significantly impacted CDT-mediated apoptosis. Together, these findings provide insight on the specificity of the ATM-Chk2 pathway in response to the CDT of A. actinomycetemcomitans in oral epithelial cells, which ultimately leads to apoptosis. We further propose the existence of an unidentified factor that is distinct from the CDT, and involved with a reversible DNA fragmentation that does not trigger terminal apoptosis in oral epithelial cells. This model potentially explains conflicting reports on the biological activity of the A. actinomycetemcomitans CDT.
Alaoui-El-Azher
Mounia
M
Department of Oral Biology and Center for Molecular Microbiology, College of Dentistry, University of Florida, Gainesville, Florida, United States of America.
Mans
Jeffrey J
JJ
Baker
Henry V
HV
Chen
Casey
C
Progulske-Fox
Ann
A
Lamont
Richard J
RJ
Handfield
Martin
M
D016976:Aggregatibacter actinomycetemcomitans / Q000737:chemistry; D017209:Apoptosis / Q000187:drug effects* / Q000235:genetics; D064007:Ataxia Telangiectasia Mutated Proteins; D001427:Bacterial Toxins / Q000494:pharmacology*; D015153:Blotting, Western; D053148:Caspase 3 / Q000235:genetics / Q000378:metabolism; D053453:Caspase 9 / Q000235:genetics / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D064447:Checkpoint Kinase 2; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000235:genetics / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded / Q000187:drug effects; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D004847:Epithelial Cells / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism; D005881:Gingiva / Q000166:cytology*; D006801:Humans; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D034741:RNA, Small Interfering; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism*
D001427:Bacterial Toxins; C493956:CDKN1A protein, human; D018797:Cell Cycle Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D004268:DNA-Binding Proteins; D034741:RNA, Small Interfering; D025521:Tumor Suppressor Proteins; C067044:cytolethal distending toxin; D064447:Checkpoint Kinase 2; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C578634:CHEK2 protein, human; D017346:Protein Serine-Threonine Kinases; D053148:Caspase 3; D053453:Caspase 9