Regulation of keratin 19 gene expression by estrogen in human breast cancer cells and identification of the estrogen responsive gene region.

Estrogens regulate the proliferation, cytoarchitectural, and invasive properties of estrogen receptor (ER)-containing breast cancer cells. To identify genes under direct regulation by estrogen in breast cancer cells, we have used representational difference analysis (RDA) of cDNAs. In this way, we have identified (cyto)keratin 19 (K19), a major component of cell intermediate filaments, as being under rapid and direct regulation by estrogen in MCF-7 cells. Stimulation by estradiol (E2) of K19 mRNA is rapid, with maximal increase at 3 h, and is not blocked by cycloheximide, suggesting that it is a primary response to the hormone. Increased accumulation of K19 protein is observable by 8 h after E2 and levels continue to increase at 24-48 h after E2 treatment. Suppression of E2-induced K19 gene expression by the antiestrogen ICI 182,780 suggests that ER mediates this regulation. Analysis of the human K19 chromosomal gene, by transient transfection assays employing reporter gene constructs with the 5' and 3' flanking regions and portions of the body of the K19 gene, has resulted in identification of a complex enhancer region in the first intron. This enhancer region consists of a near-consensus estrogen response element (K19 ERE, which differs by only 1 bp from the consensus ERE) and two ERE half sites, as well as two AP1-like sites. The results of transfections with either the K19 gene promoter or the heterologous thymidine kinase promoter and constructs containing mutated or deleted portions of the enhancer region show that the K19 ERE is responsible for the E2-dependent transactivation of the keratin 19 gene and for the synergism that is observed between E2 and TPA with both ER alpha and ER beta. These studies document ER regulation of the K19 gene, localize the estrogen responsive region, and suggest that up-regulation of keratin 19 gene expression by estrogen may contribute to the cytoskeletal and nuclear matrix reorganization, and increased metastatic potential of ER-containing breast cancer cells upon exposure to estrogens.