Activation of NF-kappaB following detachment delays apoptosis in intestinal epithelial cells

Oncogene. 2005 Sep 29;24(43):6482-91. doi: 10.1038/sj.onc.1208810.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Active Transport, Cell Nucleus
  • Animals
  • Apoptosis / physiology*
  • Carbocyanines / metabolism
  • Caspases / metabolism
  • Cell Line
  • Chemokines / genetics
  • Cytokines / genetics
  • Enzyme Activation
  • Epithelial Cells / cytology
  • Epithelial Cells / metabolism
  • Gene Expression Profiling
  • I-kappa B Kinase
  • I-kappa B Proteins / genetics
  • I-kappa B Proteins / metabolism
  • Intestinal Mucosa / metabolism
  • Intestines / cytology*
  • NF-KappaB Inhibitor alpha
  • NF-kappa B / metabolism*
  • Phosphorylation
  • Protein-Serine-Threonine Kinases / metabolism
  • Rats
  • Transcription Factor RelA

Substances

  • Carbocyanines
  • Chemokines
  • Cytokines
  • I-kappa B Proteins
  • NF-kappa B
  • Nfkbia protein, rat
  • Transcription Factor RelA
  • NF-KappaB Inhibitor alpha
  • 3,3'-dihexyl-2,2'-oxacarbocyanine
  • Protein-Serine-Threonine Kinases
  • I-kappa B Kinase
  • NF-kappa B kinase
  • Caspases