Platelet-activating factor-induced NF-kappaB activation and IL-8 production in intestinal epithelial cells are Bcl10-dependent.
BACKGROUND
Platelet-activating factor (PAF), a potent proinflammatory phospholipid mediator, has been implicated in inducing intestinal inflammation in diseases such as inflammatory bowel disease (IBD) and necrotizing enterocolitis (NEC). However, its mechanisms of inducing inflammatory responses are not fully understood. Therefore, studies were designed to explore the mechanisms of PAF-induced inflammatory cascade in intestinal epithelial cells.


METHODS
Nuclear factor kappa B (NF-kappaB) activation was measured by luciferase assay and enzyme-linked immunosorbent assay (ELISA), and interleukin 8 (IL-8) production was determined by ELISA. B-cell lymphoma 10 (Bcl10), caspase recruitment domain-containing membrane-associated guanylate kinase protein 3 (CARMA3), and mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) mRNA and protein levels were assessed by real-time reverse-transcription polymerase chain reaction (RT-PCR) and Western blot, respectively. siRNA silencing of Bcl10 was used to examine its role in PAF-induced NF-kappaB activation and IL-8 production. The promoter region of the Bcl10 gene was cloned with the PCR method and promoter activity measured by luciferase assay.


RESULTS
The adaptor protein Bcl10 appeared to play an important role in the PAF-induced inflammatory pathway in human intestinal epithelial cells. Bcl10 was required for PAF-induced I kappaB alpha phosphorylation, NF-kappaB activation, and IL-8 production in NCM460, a cell line derived from normal human colon, and Caco-2, a transformed human intestinal cell line. PAF also stimulated Bcl10 interactions with CARMA3 and MALT1, and upregulated Bcl10 expression in these cells via transcriptional regulation.


CONCLUSIONS
These findings highlight a novel PAF-induced inflammatory pathway in intestinal epithelial cells, requiring Bcl10 as a critical mediator and involving CARMA3/Bcl10/MALT1 interactions. The proinflammatory effects of PAF play prominent roles in the pathogenesis of IBD and this pathway may present important targets for intervention in chronic inflammatory diseases of the intestine.
Borthakur
Alip
A
Section of Digestive Diseases and Nutrition, Department of Medicine, University of Illinois at Chicago and Jesse Brown VA Medical Center, Chicago, Illinois 60612, USA. alipb@uic.edu
Bhattacharyya
Sumit
S
Alrefai
Waddah A
WA
Tobacman
Joanne K
JK
Ramaswamy
Krishnamurthy
K
Dudeja
Pradeep K
PK
D048868:Adaptor Proteins, Signal Transducing / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000074018:B-Cell CLL-Lymphoma 10 Protein; D015153:Blotting, Western; D053468:CARD Signaling Adaptor Proteins / Q000235:genetics / Q000378:metabolism; D020169:Caspases / Q000235:genetics / Q000378:metabolism; D002478:Cells, Cultured; D003106:Colon / Q000166:cytology / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D016209:Interleukin-8 / Q000378:metabolism*; D007413:Intestinal Mucosa / Q000187:drug effects* / Q000378:metabolism; D000074884:Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism*; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D010972:Platelet Activating Factor / Q000494:pharmacology*; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction
D048868:Adaptor Proteins, Signal Transducing; D000074018:B-Cell CLL-Lymphoma 10 Protein; C118591:BCL10 protein, human; D053468:CARD Signaling Adaptor Proteins; C430880:CARD10 protein, human; D016209:Interleukin-8; D016328:NF-kappa B; D009363:Neoplasm Proteins; D010972:Platelet Activating Factor; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D020169:Caspases; C400109:MALT1 protein, human; D000074884:Mucosa-Associated Lymphoid Tissue Lymphoma Translocation 1 Protein