Regulation of NF‐κB activity in murine macrophages: Effect of bacterial lipopolysaccharide and phorbol ester-猫眼课题宝

小鼠巨噬细胞中 NF-κB 活性的调节：细菌脂多糖和佛波酯的作用

DOI：

发表时间：

1992

期刊：

影响因子：

通讯作者：

中科院分区：

文献类型：

作者：

M. Vincenti;T. Burrell;S. Taffet

核因子κ B（NF-κB）已被证明在巨噬细胞中LPS介导的几种基因诱导中发挥重要作用。一些研究表明蛋白激酶C（PKC）或cAMP依赖性蛋白激酶参与NF-κB活性的调节。在这项研究中，我们研究了小鼠巨噬细胞中NF-κB诱导的机制。将含有多拷贝TNF-α NF-κB元件的氯霉素乙酰转移酶（CAT）表达载体转染至RAW 264巨噬细胞样细胞系中，并评估诱导型CAT活性。LPS处理的转染细胞导致CAT活性的显着诱导。用佛波醇肉豆蔻酸酯乙酸酯（PMA）或cAMP类似物8-溴cAMP处理不会诱导CAT活性。为了进一步研究NF-κB诱导，从RAW 264细胞制备核提取物。通过电泳迁移率变动分析（EMSA）测定，LPS处理30 min至2 h的RAW 264细胞提取物的NF-κB结合活性显著增加。用PMA处理这些细胞30分钟至2小时未产生这种结合活性。DNA结合活性的UV交联分析证实了这些结果，并表明LPS诱导了55 KD的DNA结合蛋白。用PKC抑制剂H-7预处理不抑制这种NF-κB结合活性的诱导。H-7确实抑制LPS或PMA对TPA反应元件结合的诱导。长期暴露于佛波酯（一种下调PKC的治疗）对这些细胞中LPS诱导NF-κB活性没有影响。这些结果表明，LPS诱导的巨噬细胞NF-κB的表达不依赖于PKC。

Nuclear factor kappa‐B (NF‐κB) has been shown to play an important role in LPS‐mediated induction of several genes in macrophages. Several studies have implicated protein kinase C (PKC) or cAMP‐dependent protein kinase in the regulation of NF‐κB activity. In this study we have investigated the mechanism of NF‐κB induction in murine macrophages. A chloramphenicol acetyl transferase (CAT) expression vector containing multiple copies of the TNF‐α NF‐κB element was transfected into the RAW264 macrophage‐like cell line and assessed for inducible CAT activity. LPS treatment of the transfected cells resulted in a significant induction of CAT activity. CAT activity was not induced by treatment with phorbol myristate acetate (PMA) or the cAMP analogue 8‐bromo cAMP. To further study NF‐κB induction, nuclear extracts were prepared from RAW264 cells. Extracts from RAW264 cells that were treated from 30 min to 2 hr with LPS had a significant increase in NF‐κB binding activity as determined by the electrophoresis mobility shift assay (EMSA). Treatment of these cells from 30 min to 2 hr with PMA did not result in such binding activity. U.V. crosslinking analysis of the DNA‐binding activity confirmed these results and indicated that LPS induced a 55 KD DNA‐binding protein. Induction of this NF‐κB binding activity was not inhibited by pretreatment with the PKC inhibitor H‐7. H‐7 did inhibit induction of TPA responsive element binding by either LPS or PMA. Prolonged exposure to phorbol ester, a treatment which down‐regulates PKC, had no effect on LPS induction of NF‐κB activity in these cells. These results suggest that the induction of NF‐κB in macrophages by LPS is independent of PKC.