Furlong E E, Keon N K, Thornton F D, Rein T, Martin F
Department of Pharmacology and Biotechnology Center, University College Dublin, Dublin 4, Ireland.
J Biol Chem. 1996 Nov 22;271(47):29688-97. doi: 10.1074/jbc.271.47.29688.
Testosterone repressed prostate message-2 (TRPM-2)/clusterin gene expression is rapidly induced in early involution of the mouse mammary gland, after weaning, and in the rat ventral prostate, after castration. A search for involution-enhanced DNaseI footprints in the proximal mouse TRPM-2/clusterin gene promoter led to the identification and characterization (by DNase I footprinting and EMSA) of a twin nuclear factor 1 (NF1) binding element at -356/-309, relative to the proposed transcription start site; nuclear extracts from 2-day involuting mouse mammary gland showed an enhanced footprint over the proximal NF1 element; extracts from involuting prostate showed enhanced occupancy of both NF1 binding elements. Subsequent EMSA and Western analysis led to the detection of a 74-kDa NF1 protein whose expression is triggered in early involution in the mouse mammary gland; such an induced protein is not found in the involuting rat ventral prostate. This protein was not found in lactation where three other NF1 proteins of 114, 68, and 46 kDa were detected. Reiteration of the epithelial cell apoptosis associated with early mammary gland involution, in vitro, in a primary cell culture system, triggered the appearance of the 74-kDa NF1. Overlaying the cells with laminin-rich extracellular matrix suppressed the apoptosis and the expression of the 74-kDa NF1 and, in the presence of lactogenic hormones, initiated milk protein gene expression and the expression of two of the lactation-associated NF1 proteins (68 and 46 kDa). This study, thus, identifies for the first time the occurrence of a switch in expression of different members of the family of NF1 transcription factors as mammary epithelial cells move from the differentiated to the involution/apoptotic state, and it is likely that the involution-specific 74-kDa NF1 accounts for the enhanced NF1 footprint detected on the TRPM-2/clusterin promoter with extracts of mouse mammary gland.
睾酮抑制前列腺信息-2（TRPM-2）/聚集素基因表达在小鼠乳腺断奶后的早期退化以及大鼠去势后的腹侧前列腺中迅速被诱导。在近端小鼠TRPM-2/聚集素基因启动子中寻找退化增强的DNaseI足迹，导致在相对于假定转录起始位点的-356/-309处鉴定并表征（通过DNase I足迹分析和电泳迁移率变动分析（EMSA））了一个双细胞核因子1（NF1）结合元件；来自2天退化期小鼠乳腺的核提取物在近端NF1元件上显示出增强的足迹；来自退化期前列腺的提取物显示两个NF1结合元件的占据增强。随后的EMSA和蛋白质免疫印迹分析导致检测到一种74 kDa的NF1蛋白，其表达在小鼠乳腺早期退化中被触发；在退化期大鼠腹侧前列腺中未发现这种诱导蛋白。在泌乳期未发现该蛋白，而检测到另外三种分别为114 kDa、68 kDa和46 kDa的NF1蛋白。在体外原代细胞培养系统中，与早期乳腺退化相关的上皮细胞凋亡的重复发生触发了74 kDa NF1蛋白的出现。用富含层粘连蛋白的细胞外基质覆盖细胞可抑制细胞凋亡和74 kDa NF1蛋白的表达，并且在存在催乳激素的情况下，启动乳蛋白基因表达以及两种与泌乳相关的NF1蛋白（68 kDa和46 kDa）的表达。因此，本研究首次确定了随着乳腺上皮细胞从分化状态转变为退化/凋亡状态，NF1转录因子家族不同成员表达发生切换；并且很可能退化特异性的74 kDa NF1蛋白解释了用小鼠乳腺提取物在TRPM-2/聚集素启动子上检测到的增强的NF1足迹。
