Lipopolysaccharide-induced activation of NF-κB non-canonical pathway requires BCL10 serine 138 and NIK phosphorylations.
OBJECTIVE
B-cell lymphoma/leukemia (BCL)-10 and reactive oxygen species mediate two pathways of NF-κB (RelA) activation by lipopolysaccharide (LPS) in human colonic epithelial cells. The pathway for LPS activation of RelB by the non-canonical pathway (RelB) in non-myeloid cells was not yet reported, but important for understanding the range of potential microbial LPS-induced effects in inflammatory bowel disease.


METHODS
Experiments were performed in human colonic epithelial cells and in mouse embryonic fibroblasts deficient in components of the IkappaB kinase (IKK) signalosome, in order to detect mediators of the non-canonical pathway of NF-κB activation, including nuclear RelB and p52 and phospho- and total NF-κB inducing kinase (NIK). BCL10 was silenced by siRNA and effects of mutations of specific phosphorylation sites of BCL10 (Ser138Gly and Ser218Gly) were determined.


RESULTS
By the non-canonical pathway, LPS exposure increased nuclear RelB and p52, and phospho-NIK, with no change in total NIK. Phosphorylation of BCL10 serine 138 was required for NIK phosphorylation, since mutation of this residue eliminated the increases in phospho-NIK and nuclear RelB and p52. Mutations of either serine 138 or serine 218 reduced RelA, p50, and phospho-IκBα of the canonical pathway. Effects of LPS stimulation and BCL10 silencing on NIK phosphorylation were demonstrated in confocal images.


CONCLUSIONS
LPS induces activation of both canonical and non-canonical pathways of NF-κB in human colonic epithelial cells, and the non-canonical pathway requires phosphorylations of BCL10 (serine 138) and NIK. These findings demonstrate the important role of BCL10 in mediating LPS-induced inflammation in human colonic epithelial cells and may open new avenues for therapeutic interventions.
Bhattacharyya
Sumit
S
Department of Medicine, University of Illinois at Chicago and Jesse Brown VA Medical Center, Chicago, IL 60612-7227, USA.
Borthakur
Alip
A
Dudeja
Pradeep K
PK
Tobacman
Joanne K
JK
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D000368:Aged; D000818:Animals; D000074018:B-Cell CLL-Lymphoma 10 Protein; D003106:Colon / Q000166:cytology; D004847:Epithelial Cells / Q000187:drug effects / Q000201:enzymology; D005347:Fibroblasts / Q000378:metabolism; D020868:Gene Silencing; D006801:Humans; D020672:I-kappa B Proteins / Q000172:deficiency / Q000378:metabolism; D016209:Interleukin-8 / Q000378:metabolism; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008297:Male; D051379:Mice; D009154:Mutation / Q000235:genetics; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D010768:Phosphoserine / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects*; D013329:Structure-Activity Relationship; D000097782:NF-kappaB-Inducing Kinase
D048868:Adaptor Proteins, Signal Transducing; D000074018:B-Cell CLL-Lymphoma 10 Protein; D020672:I-kappa B Proteins; D016209:Interleukin-8; D008070:Lipopolysaccharides; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B; D010768:Phosphoserine; D017346:Protein Serine-Threonine Kinases; D000097782:NF-kappaB-Inducing Kinase; C118591:BCL10 protein, human; C488088:Bcl10 protein, mouse; C083487:I kappa B beta protein; C000606204:NFKBIA protein, human; C000606205:Nfkbia protein, mouse
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