Musso R, Maggi A, Eva C
Section of Pharmacology, Department of Anatomy, Pharmacology and Forensic Medicine, Torino, Italy.
Neuroendocrinology. 2000 Dec;72(6):360-7. doi: 10.1159/000054605.
Several studies have shown that neuropeptide Y (NPY) is involved in the stimulation of gonadotropin hormone releasing hormone (GnRH) and luteinizing hormone (LH) secretion and that these effects are modulated by gonadal steroid feedback. The NPY regulation of GnRH release is probably mediated by the activation of the Y(1) receptor subtype. In this study we examined the regulation of the Y(1) receptor gene transcription by estrogens in transiently transfected NG108-15 neuroblastoma glioma cells. A chimeric plasmid containing the murine Y(1) receptor promoter fused to the firefly luciferase reporter gene was induced by approximately 2-fold in response to 17 beta-estradiol treatment. The estrogen-mediated enhancement of luciferase activity was dose-dependent, blocked by the estrogen receptor (ER) antagonist ICI 182,780, and was strictly dependent on the presence of ER alpha, since it occurred only in NG108-15 cells cotransfected with an expression vector for the human ER. Mutational analysis was performed to investigate whether the hemipalindromic estrogen-responsive elements (EREs) flanking the Y(1) receptor gene are responsible for conferring estradiol inducibility to the Y(1) receptor gene promoter. Mutation of the ERE1 half site at position -932, or mutation of the ERE2 half site at position -809, relative to the ATG, failed to affect the 17 beta-estradiol-mediated enhancement of luciferase activity. Conversely, mutation of both ERE1 and ERE2 half sites completely abolished activation of luciferase activity induced by estrogen. We also examined whether 17 beta-estradiol stimulates the transcriptional activity of the Y(1) receptor gene by binding to ER beta. Results demonstrated that luciferase activity was not modulated by estrogens when cells were transfected with the expression plasmid bearing the human ER beta. Moreover coexpression of both ER alpha and ER beta completely abolished the estrogen-induced activation of luciferase activity observed in the presence of ER alpha. Our data suggest that estrogens activate Y(1) receptor gene transcription possibly via a direct interaction of ER alpha with the hemipalindromic EREs flanking the Y(1) receptor gene.
多项研究表明，神经肽Y（NPY）参与促性腺激素释放激素（GnRH）和促黄体生成素（LH）分泌的刺激，且这些作用受性腺类固醇反馈调节。NPY对GnRH释放的调节可能是通过Y(1)受体亚型的激活介导的。在本研究中，我们检测了雌激素对瞬时转染的NG108 - 15神经母细胞瘤胶质瘤细胞中Y(1)受体基因转录的调节。一个含有与萤火虫荧光素酶报告基因融合的小鼠Y(1)受体启动子的嵌合质粒，在17β - 雌二醇处理下被诱导约2倍。雌激素介导的荧光素酶活性增强是剂量依赖性的，被雌激素受体（ER）拮抗剂ICI 182,780阻断，并且严格依赖于ERα的存在，因为它仅在与人类ER表达载体共转染的NG108 - 15细胞中发生。进行了突变分析以研究Y(1)受体基因侧翼的半回文雌激素反应元件（ERE）是否负责赋予Y(1)受体基因启动子雌二醇诱导性。相对于ATG，-932位的ERE1半位点突变或-809位的ERE2半位点突变均未影响17β - 雌二醇介导的荧光素酶活性增强。相反，ERE1和ERE2半位点的突变完全消除了雌激素诱导的荧光素酶活性激活。我们还检测了17β - 雌二醇是否通过与ERβ结合来刺激Y(1)受体基因的转录活性。结果表明，当用携带人类ERβ的表达质粒转染细胞时，荧光素酶活性不受雌激素调节。此外，ERα和ERβ的共表达完全消除了在ERα存在下观察到的雌激素诱导的荧光素酶活性激活。我们的数据表明，雌激素可能通过ERα与Y(1)受体基因侧翼的半回文ERE直接相互作用来激活Y(1)受体基因转录。
