Activation of NF-kappaB following detachment delays apoptosis in intestinal epithelial cells.
We reported earlier that IL-1beta, an NF-kappaB-regulated cytokine, was made by intestinal epithelial cells during detachment-induced apoptosis (anoikis) and that IL-1 was antiapoptotic for detached cells. Since surviving anoikis is a prerequisite for cancer progression and metastases, we are further exploring the link between anoikis and cytokines. Here we determined that multiple genes are expressed following detachment including a number of NF-kappaB-regulated products and therefore aimed to determine whether NF-kappaB signalling plays any role in regulating apoptosis. Using Western blotting, we detected that IkappaBalpha becomes phosphorylated immediately following detachment and that levels of phospho-IkappaBalpha peaked within 20 min. Phosphorylation of IkappaBalpha was followed by Rel A (p65) nuclear translocation. Increased NF-kappaB activity following detachment was confirmed using the detection of NF-kappaB-promoted luciferase gene expression delivered by adenovirus infection. Infection of cells with adenovirus expressing a super-repressor IkappaBalpha protein and pharmacological inhibitors of NF-kappaB resulted in the failure to phosphorylate IkappaBalpha, a more rapid activation of caspases and earlier apoptosis. We also detected that IkappaB kinase alpha (IKKalpha) and not IKKbeta became phosphorylated following detachment. Since IKKalpha is activated by NF-kappaB-inducing kinase (NIK), we overexpressed native NIK using an adenovirus vector that resulted in enhanced phospho-IkappaBalpha and nuclear p65 in detached cells compared to control detached cells but did not result in a significantly greater number of cells surviving to 24 h. We conclude that detachment directly activates NF-kappaB, which, in addition to launching an inflammatory cytokine wave, contributes to a delay in apoptosis in intestinal epithelial cells.
Yan
Sen Rong
SR
Department of Pediatrics, Dalhousie University, Halifax, NS, Canada B3H4R2.
Joseph
Robbie Randle
RR
Rosen
Kirill
K
Reginato
Mauricio J
MJ
Jackson
Amanda
A
Allaire
Norman
N
Brugge
Joan S
JS
Jobin
Christian
C
Stadnyk
Andrew W
AW
D021581:Active Transport, Cell Nucleus; D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D002232:Carbocyanines / Q000378:metabolism; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D018925:Chemokines / Q000235:genetics; D016207:Cytokines / Q000235:genetics; D004789:Enzyme Activation; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism; D020869:Gene Expression Profiling; D051550:I-kappa B Kinase; D020672:I-kappa B Proteins / Q000235:genetics / Q000378:metabolism; D007413:Intestinal Mucosa / Q000378:metabolism; D007422:Intestines / Q000166:cytology*; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000378:metabolism*; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D051381:Rats; D051996:Transcription Factor RelA; D000097782:NF-kappaB-Inducing Kinase
D002232:Carbocyanines; D020169:Caspases; D018925:Chemokines; D016207:Cytokines; D051550:I-kappa B Kinase; D020672:I-kappa B Proteins; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B; D017346:Protein Serine-Threonine Kinases; D051996:Transcription Factor RelA; D000097782:NF-kappaB-Inducing Kinase; C000606206:Nfkbia protein, rat; C007392:3,3'-dihexyl-2,2'-oxacarbocyanine