Steroid hormones modulate H19 gene expression in both mammary gland and uterus
Steroid hormones modulate H19 gene expression in both mammary gland and uterus
复制标题
DOI：
10.1038/sj.onc.1202819
复制
发表时间：
1999-08-05
期刊：
影响因子：
8
通讯作者：
Curgy, JJ
中科院分区：
文献类型：
--
作者：
Adriaenssens, E;Lottin, S;Curgy, JJ
H19是一种印迹和发育调控基因，其产物显然未被翻译。在之前的一项关于乳腺腺癌的研究中，我们报道了H19基因的过表达与类固醇受体的存在显著相关，这表明激素在H19转录中可能起作用。为了确定类固醇的作用方式，我们检测了H19 RNA的水平。通过原位杂交（ISH）在乳腺发育过程中合成：青春期和怀孕期间出现两个H19转录高峰。此外，我们通过ISH证明，在发情期和发情期子宫内H19 RNA合成较高。为了检测类固醇对H19转录的控制，切除卵巢和肾上腺的小鼠在术后1周补充17- β -雌二醇（E2, 20 μ g/kg/天）、黄体酮（P， 1 mg/kg/天）或皮质酮（B， 0.3 mg/kg/天）2周。根据ISH的数据，E2和B在较小程度上刺激了子宫内H19的转录，而P则抑制了它。为了证实体内结果，我们利用MCF-7细胞（一种激素敏感的乳腺细胞系）进行了体外实验。E2刺激了该细胞系的内源性H19基因，而他莫昔芬抑制了这一作用。此外，我们在存在或不存在类固醇激素的情况下，对MCF-7、HBL-100（另一种激素敏感的乳腺细胞系）和BT-20（一种激素不敏感的乳腺细胞系）进行了短暂共转染，转染了不同结构的ER α （WT或突变）和PR-A。我们证明了ER α上调MCF-7和HBL-100中的H19启动子，而PR-A没有任何影响。此外，在MCF-7中，PR-A明显拮抗ER α介导的启动子增强，但在HBL-100中未发现这种拮抗ER α上调的作用。有趣的是，在BT-20细胞系中进行的相同实验提供了与MCF-7细胞非常相似的结果，PR-A介导了H19启动子的明显下调。所有这些体外实验数据与体内实验结果一致。此外，从ERI突变体获得的数据表明，H19启动子的激活既依赖于配体，也不依赖于配体。因此，我们已经证明H19基因表达受类固醇激素控制；此外，当激素刺激伴随形态修复时，该基因在激素敏感器官中高度表达。
H19 is an imprinted and developmentally regulated gene whose product remains apparently untranslated. In a previous study on breast adenocarcinomas, we reported that overexpression of the H19 gene was significantly correlated with the presence of steroid receptors, suggesting the putative role of hormones in H19 transcription. To determine the mode of steroid action, we have detected levels of H19 RNA. synthesis during mammary gland development by in situ hybridization (ISH): two peaks of H19 transcription occur during puberty and pregnancy. Furthermore, we demonstrated by ISH that in the uterus H19 RNA synthesis is high during estrus and metestrus phases. To test steroid control of H19 transcription, ovariectomized and adrenalectomized mice were supplemented, 1 week after surgery, with 17-beta-estradiol (E2, 20 mu g/kg/day), progesterone (P, 1 mg/kg/day) or corticosterone (B, 0.3 mg/kg/day) for 2 weeks. According to ISH data, E2 and to a lesser extent B stimulated H19 transcription in the uterus, whereas P inhibited it. To confirm the in vivo results, in vitro experiments were performed using cultures of MCF-7 cells (a hormone-sensitive mammary cell line). E2 stimulated the endogenous H19 gene of this cell line and tamoxifen inhibited this effect. Furthermore, we performed transient cotransfections in MCF-7, in HBL-100 (another hormone-sensitive mammary cell line) and in BT-20 (a hormone-insensitive mammary cell line) with various constructs of ER alpha (WT or mutated) and PR-A, in presence or absence of steroid hormones. We demonstrated that ER alpha up-regulated the H19 promoter in MCF-7 and in HBL-100, whereas PR-A did not have any effect pel se. Moreover, in MCF-7, PR-A antagonized clearly the ER alpha-mediated promoter enhancement, but in HBL-100 this counteracting effect on the ER alpha up-regulation was not found. Interestingly, the same experiments performed in BT-20 cell line provided very similar results as those obtained in MCF-7 cells, with a clear down-regulation mediated by PR-A on the H19 promoter. All these in vitro data are in agreement with irt vivo results. In addition, data obtained with ERI mutants indicate that H19 promoter activation is both ligand-dependent and ligand-independent. We have thus demonstrated that H19 gene expression is controlled by steroid hormones; furthermore, this gene is highly expressed in hormone-sensitive organs when the hormonal stimulation is accompanied, with a morphological repair.