NF-κB factors are essential, but not the switch, for pathogen-related induction of the bovine β-defensin 5-encoding gene in mammary epithelial cells
NF-κB factors are essential, but not the switch, for pathogen-related induction of the bovine β-defensin 5-encoding gene in mammary epithelial cells
复制标题
DOI：
10.1016/j.molimm.2005.02.003
复制
发表时间：
2006-02-01
影响因子：
3.6
通讯作者：
Seyfert, HM
中科院分区：
文献类型：
--
作者：
Yang, W;Molenaar, A;Seyfert, HM
杀菌肽β-防御素5（BNBD 5）的表达由乳房的细菌感染（乳腺炎）强烈诱导。原位杂交结果表明，细菌引起的乳腺上皮细胞（MEC）的BNBD 5强，局部限制性表达。我们通过引物延伸定义了BNBD 5启动子，并在小鼠HC-11和原代牛乳腺上皮细胞（pbMEC）培养物中用报告基因测定表明，热灭活细菌、LPS、IL-1 β和TNF α可诱导1 kb的启动子片段约3倍。启动子的一系列缺失和点突变表明，NF-IL 6增强了诱导，但NF-κ B B必须顺式结合，以刺激病原体相关的BNBD 5基因表达。EMSA分析显示，未刺激的MEC模型以及来自健康乳房的提取物已经显示出相当水平的结合活性NF-κ B。细菌刺激使该水平增加约3倍，如用pbMEC中NF-κ B驱动的报告基因所测量的，定量地匹配BNBD 5报告基因诱导的程度。相比之下，内源BNBD 5基因的表达在乳房和pbMEC中被刺激得更多（> 30倍），这表明除了有结合能力的NF-κ B因子水平升高之外的因素决定了天然基因的诱导。支持这一结论，我们发现，牛TLR2或TLR4在HEK 293细胞中的表达可以重建NF-κ B表达构建体的细菌激活，但不是BNBD 5报告基因。我们的数据表明，通过TLR病原体抑制机制介导的具有结合能力的NF-κ B因子的水平升高不是MEC中病原体相关诱导BNBD 5编码基因的关键开关。(c)2005年由Elsevier Ltd.出版
Expression of the bactericidal peptide beta-defensin 5 (BNBD5) is strongly induced by bacterial infections of the udder (mastitis). In situ hybridizations showed that bacteria elicit a strong, locally restricted expression of BNBD5 in mammary epithelial cells (MEC). We defined the BNBD5 promoter by primer extension and showed with reporter gene assays in murine HC-11 and primary bovine mammary epithelial cell (pbMEC) cultures that a 1 kb segment of the promoter is induced about 3-fold by heat-killed bacteria, LPS, IL-1 beta and TNF alpha. Deletion series and point mutations of the promoter showed that NF-IL6 augments the induction, but that NF-kappa B must be bound in cis for pathogen-related stimulation of BNBD5 gene expression. EMSA analyses revealed that both un-stimulated MEC models as well as extracts from healthy udders already display considerable levels of binding competent NF-kappa B. The bacterial stimulus increased this level about 3-fold, as measured with a NF-kappa B driven reporter gene in pbMEC, matching quantitatively the extent of the BNBD5-reporter gene induction. In contrast, expression of the endogenous BNBD5-gene is stimulated much more (> 30-fold) in udders and pbMEC indicating that factors other than elevated levels of binding-competent NF-kappa B factors determine the induction of the native gene. Supporting this conclusion, we found that expression of bovine TLR2 or TLR4 in HEK293 cells can reconstitute the bacterial activation of the NF-kappa B expression construct, but not that of the BNBD5-reporter gene. Our data suggest that elevated levels of binding competent NF-kappa B factors mediated via TLR pathogen recognitions mechanisms are not the key switch for pathogen related induction of the BNBD5-encoding gene in MEC. (c) 2005 Published by Elsevier Ltd.