Complement component C3 production in IL-1beta-stimulated human intestinal epithelial cells is blocked by NF-kappaB inhibitors and by transfection with ser 32/36 mutant IkappaBalpha.

Moon MR, Parikh AA, Pritts TA, Fischer JE, Cottongim S, Szabo C, Salzman AL, Hasselgren PO
J Surg Res. 1999 82 (1): 48-55

PMID: 10068525 · DOI:10.1006/jsre.1998.5503

BACKGROUND - Recent studies suggest that interleukin-1beta (IL-1beta) stimulates the production of the acute phase protein complement component C3 in human intestinal epithelial cells. The transcription factor NF-kappaB activates different genes involved in the response to cytokines. It is not known if IL-1beta-induced C3 production in the enterocyte is regulated by NF-kappaB.

MATERIALS AND METHODS - Cultured Caco-2 cells, a human intestinal epithelial cell line, were treated with one of the NF-kappaB inhibitors, tosyl-lys-chloromethylketone (TLCK), genistein, or pyrrolidine dithiocarbamate (PDTC), or with N-acetyl-leu-leu-norleucinal (LLnL), a proteasome inhibitor known to block the degradation of Ikappabeta, the cytosolic inhibitor of NF-kappaB. Following this treatment, the Caco-2 cells were stimulated with IL-1beta, and C3 levels in the culture medium were measured after 24 h by ELISA. C3 mRNA levels were determined after 4 h by Northern blot analysis. In other experiments, Caco-2 cells were transfected with a mutant IkappaBalpha in which serines 32 and 36 were substituted by alanine. This mutation prevents IkBalpha phosphorylation and subsequent NF-kappaB nuclear translocation. After transfection, the cells were stimulated with IL-1beta, and C3 levels in the culture medium were measured after 24 h. Cytosolic IkappaBalpha was determined by Western blot analysis.

RESULTS - TLCK, genistein, and LLnL each inhibited IL-1beta-induced C3 production in a dose-dependent fashion. These responses were associated with decreased C3 mRNA levels. In contrast, PDTC did not influence C3 production or C3 mRNA in the Caco-2 cells. Transfection of the Caco-2 cells with the Ser 32/36 mutant IkBalpha resulted in maintained IkappaBalpha levels and decreased IL-beta-induced C3 production.

CONCLUSIONS - IL-1beta-stimulated C3 production in the enterocyte may be regulated by NF-kappaB.

Copyright 1999 Academic Press.

MeSH Terms (19)

Base Sequence Caco-2 Cells Complement C3 DNA-Binding Proteins DNA Primers Genistein Humans I-kappa B Proteins Interleukin-1 Intestinal Mucosa Leupeptins Mutation NF-kappa B NF-KappaB Inhibitor alpha Pyrrolidines RNA, Messenger Thiocarbamates Tosyllysine Chloromethyl Ketone Transfection

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