AN ESTROGEN-RECEPTOR BINDING-SITE WITHIN THE HUMAN GALANIN GENE

Regulation of galanin gene expression in the anterior pituitary (AP) i s positively influenced by estrogen in rodents and undetermined in hum ans. The objective of this study was to investigate the meChanism behi nd estrogen induction of galanin by identifying any putative estrogen receptor (ER) binding sequences within tile human galanin promoter tha t may function as estrogen response elements (ERE). Two regions, gERE1 and gERE2, were identified in the galanin 6'-flanking sequence with s imilarity to the full 13-base ERE consensus previously defined in the vitellogenin gene (vERE). Both sequences were tested in mobility shift assays for the ability to bind nuclear proteins isolated from rat AP tissue or MtTW-10 pituitary tumors. Only the distal sequence at -527 ( gERE1) yielded an ERE-specific DNA/protein complex distinguished by mo bility and cross-competition with vERE. The gel mobility pattern of th e DNA/protein complex was comparable between the pituitary tissue and tumor extracts. However, DNA/protein affinity estimations demonstrated a greater affinity of pituitary proteins for gERE1 over the vERE sequ ence. Evidence that the human ER (hER) does recognize the gERE1 sequen ce in the human galanin gene was provided by electrophoretic mobility shift. assays (EMSAs) with Sf9 extracts enriched in recombinant hER. I n addition, antibodies specific for the hER recognized the gERE1/prote in complex in supershift, experiments.Enhancer activity by gERE1 was d etected in transient transfections of the rat GH(3) pituitary cell lin e, resulting in a 4-fold induction of expression driven by the heterol ogous thymidine kinase promoter inthe presence of estrogen. Evidence f or ER regulation of the gERE1 enhancer was demonstrated by: 1) inhibit ion of enhancement using the specific ER antagonist ICI 164,384; and 2 ) enhancement in HeLa cells that was dependent upon coexpression with hER. Enhancement by gERE1 was half the magnitude as that from the vERE element and may reflect a difference in affinity or composition of th e ER complex between the two sequences. These data demonstrate the pre sence of a functional ERE sequence within the human galanin gene that could potentially function as a regulatory element for estrogen action in the AP.