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J Surg Res. 1999 Mar;82(1):48-55.

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.

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1
University of Cincinnati Medical Center, Children's Hospital Medical Center, Cincinnati, Ohio, 45267, USA.

Abstract

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.

PMID:
10068525
DOI:
10.1006/jsre.1998.5503
[Indexed for MEDLINE]
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