Howard G, Peng L, Hyde J F
College of Pharmacy, University of Kentucky Medical Center, Lexington 40536, USA.
Endocrinology. 1997 Nov;138(11):4649-56. doi: 10.1210/endo.138.11.5507.
Regulation of galanin gene expression in the anterior pituitary (AP) is positively influenced by estrogen in rodents and undetermined in humans. The objective of this study was to investigate the mechanism behind estrogen induction of galanin by identifying any putative estrogen receptor (ER) binding sequences within the human galanin promoter that may function as estrogen response elements (ERE). Two regions, gERE1 and gERE2, were identified in the galanin 5'-flanking sequence with similarity 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 mobility and cross-competition with vERE. The gel mobility pattern of the 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 sequence. Evidence that the human ER (hER) does recognize the gERE1 sequence in the human galanin gene was provided by electrophoretic mobility shift assays (EMSAs) with Sf9 extracts enriched in recombinant hER. In addition, antibodies specific for the hER recognized the gERE1/protein complex in supershift experiments. Enhancer activity by gERE1 was detected in transient transfections of the rat GH3 pituitary cell line, resulting in a 4-fold induction of expression driven by the heterologous thymidine kinase promoter in the presence of estrogen. Evidence for ER regulation of the gERE1 enhancer was demonstrated by: 1) inhibition 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 the ER complex between the two sequences. These data demonstrate the presence of a functional ERE sequence within the human galanin gene that could potentially function as a regulatory element for estrogen action in the AP.
在啮齿动物中，雌激素对前脑垂体（AP）中甘丙肽基因表达具有正向调节作用，而在人类中这一作用尚未明确。本研究的目的是通过鉴定人类甘丙肽启动子内可能作为雌激素反应元件（ERE）发挥作用的任何假定雌激素受体（ER）结合序列，来探究雌激素诱导甘丙肽的机制。在甘丙肽5'-侧翼序列中鉴定出两个区域，gERE1和gERE2，它们与卵黄生成素基因（vERE）中先前定义的完整13碱基ERE共有序列相似。对这两个序列进行迁移率变动分析，以检测它们结合从大鼠AP组织或MtTW - 10垂体肿瘤中分离的核蛋白的能力。只有位于-527的远端序列（gERE1）产生了一种ERE特异性DNA /蛋白质复合物，其通过迁移率以及与vERE的交叉竞争得以区分。垂体组织和肿瘤提取物之间DNA /蛋白质复合物的凝胶迁移模式具有可比性。然而，DNA /蛋白质亲和力估计表明，垂体蛋白对gERE1的亲和力高于vERE序列。用富含重组人ER的Sf9提取物进行的电泳迁移率变动分析（EMSA）提供了证据，证明人ER（hER）确实能识别人类甘丙肽基因中的gERE1序列。此外，在超迁移实验中，针对hER的特异性抗体识别gERE1 /蛋白质复合物。在大鼠GH3垂体细胞系的瞬时转染中检测到gERE1的增强子活性，在雌激素存在的情况下，由异源胸苷激酶启动子驱动的表达诱导了4倍的增加。gERE1增强子受ER调节的证据表现为：1）使用特异性ER拮抗剂ICI 164,384抑制增强作用；2）在HeLa细胞中的增强作用依赖于与hER的共表达。gERE1的增强作用是vERE元件的一半，这可能反映了两个序列之间ER复合物在亲和力或组成上的差异。这些数据表明，人类甘丙肽基因中存在一个功能性ERE序列，它可能作为AP中雌激素作用的调节元件发挥作用。
