白细胞介素-1β刺激的人肠上皮细胞中补体成分C3的产生被NF-κB抑制剂以及通过转染丝氨酸32/36突变型IκBα所阻断。

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.

作者信息

Moon M R, Parikh A A, Pritts T A, Fischer J E, Cottongim S, Szabo C, Salzman A L, Hasselgren P O

机构信息

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

出版信息

J Surg Res. 1999 Mar;82(1):48-55. doi: 10.1006/jsre.1998.5503.

DOI:10.1006/jsre.1998.5503

PMID:10068525

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.

摘要

背景

近期研究表明，白细胞介素-1β（IL-1β）可刺激人肠上皮细胞中急性期蛋白补体成分C3的产生。转录因子核因子κB（NF-κB）可激活参与细胞因子应答的不同基因。目前尚不清楚肠上皮细胞中IL-1β诱导的C3产生是否受NF-κB调控。

材料与方法

用NF-κB抑制剂甲苯磺酰-L-赖氨酸氯甲基酮（TLCK）、染料木黄酮或吡咯烷二硫代氨基甲酸盐（PDTC）之一，或用N-乙酰-L-亮氨酰-L-亮氨酰-正亮氨酸（LLnL）（一种已知可阻断NF-κB胞质抑制剂IκBα降解的蛋白酶体抑制剂）处理人肠上皮细胞系Caco-2细胞。处理后，用IL-1β刺激Caco-2细胞，24小时后通过酶联免疫吸附测定法（ELISA）测量培养基中的C3水平。4小时后通过Northern印迹分析确定C3 mRNA水平。在其他实验中，用丝氨酸32和36被丙氨酸取代的突变型IκBα转染Caco-2细胞。这种突变可阻止IκBα磷酸化及随后的NF-κB核转位。转染后，用IL-1β刺激细胞，24小时后测量培养基中的C3水平。通过蛋白质印迹分析确定胞质IκBα。