Steroid hormones modulate H19 gene expression in both mammary gland and uterus

H19 is an imprinted and developmentally regulated gene whose product remain s apparently untranslated. In a previous study on breast adenocarcinomas, w e reported that overexpression of the H19 gene was significantly correlated with the presence of steroid receptors, suggesting the putative role of ho rmones in H19 transcription. To determine the mode of steroid action, we ha ve detected levels of H19 RNA. synthesis during mammary gland development b y in situ hybridization (ISH): two peaks of H19 transcription occur during puberty and pregnancy. Furthermore, we demonstrated by ISH that in the uter us H19 RNA synthesis is high during estrus and metestrus phases. To test st eroid control of H19 transcription, ovariectomized and adrenalectomized mic e were supplemented, 1 week after surgery, with 17-beta-estradiol (E2, 20 m u 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 stimul ated H19 transcription in the uterus, whereas P inhibited it. To confirm th e in vivo results, in vitro experiments were performed using cultures of MC F-7 cells (a hormone-sensitive mammary cell line). E2 stimulated the endoge nous H19 gene of this cell line and tamoxifen inhibited this effect. Furthe rmore, we performed transient cotransfections in MCF-7, in HBL-100 (another hormone-sensitive mammary cell line) and in BT-20 (a hormone-insensitive m ammary cell line) with various constructs of ER alpha (WT or mutated) and P R-A, in presence or absence of steroid hormones. We demonstrated that ER al pha 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 t he ER alpha-mediated promoter enhancement, but in HBL-100 this counteractin g effect on the ER alpha up-regulation was not found. Interestingly, the sa me experiments performed in BT-20 cell line provided very similar results a s those obtained in MCF-7 cells, with a clear down-regulation mediated by P R-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 ha ve thus demonstrated that H19 gene expression is controlled by steroid horm ones; furthermore, this gene is highly expressed in hormone-sensitive organ s when the hormonal stimulation is accompanied, with a morphological repair .