EXPRESSION OF FUNCTIONAL ESTROGEN-RECEPTORS AND GALANIN MESSENGER-RIBONUCLEIC-ACID IN IMMORTALIZED LUTEINIZING-HORMONE-RELEASING HORMONE NEURONS - ESTROGENIC CONTROL OF GALANIN GENE-EXPRESSION

The activity of estradiol on the LHRH neuronal network is crucial in t he regulation of reproduction. In vivo, estradiol induces galanin (GAL ) gene expression in LHRH neurons and GAL/LHRH colocalization is sexua lly dimorphic and neonatally determined by steroid exposure. The effec ts of estradiol on LHRH neurons, however, are considered to be indirec t because estrogen receptors (ER) have not been detected in LHRH neuro ns in vivo. Using immortalized mouse LHRH neurons (GT1-7 cells), we de monstrated by RT-PCR and Southern blotting that GT1-7 cells express ER messenger RNA (mRNA). Sequencing of the amplification products indica ted that GT1-7 ER is of the alpha-subtype (ER alpha). Additionally, es trogen receptors in GT1-7 cells were characterized by competitive radi oligand receptor binding and IC50 values for 17 beta-estradiol and ICI -182,780 were found to be 0.24 and 4.1 nhl, respectively. The ability of endogenous GT1-7 cell ER to regulate transcription was determined i n transient transfection studies using a construct that consisted of a luciferase reporter gene that is driven by tandem estrogen response e lements (ERE) and a minimal herpes simplex virus thymidine kinase prom oter. 17 beta-Estradiol was found to enhance luciferase activity by 2. 5-fold at physiological concentrations with an ED50 value of 47 pM. Th is induction was completely inhibited by ICI-182,780 which had an IC50 value of 4.8 nM. Raloxifene, tamoxifen, 4-hydroxytamoxifen, and drolo xifene also fully blocked estrogen-mediated luciferase induction with IC50 values of 58.4, 89.2, 33.2, and 49.8 nM, respectively. In additio n, GAL mRNA was detected and identified by RT-PCR followed by Southern blotting using a rat GAL complementary DNA (cDNA) probe. The ability of 17 beta-estradiol to modulate expression of the endogenous GAL gene in immortalized LHRH neurons was also determined. Quantitative RT-PCR demonstrated that physiological concentrations of estrogen increase G AL gene expression by 2-fold with an ED50 value of 23 par. ICI-182,780 , raloxifene, and droloxifene completely blocked this induction. In su mmary, our data demonstrate the presence of ER alpha and GAL mRNA in G T1-7 cells. The ER in GT1-7 cells is biologically active because 17 be ta-estradiol enhances both endogenous GAL,gene expression and an ERE-d riven reporter gene. These results suggest that estrogenic control of GAL gene expression in immortalized LHRH neurons may be transduced by ER. Thus, hypothalamic-derived LHRH neurons appear to have the capacit y to be directly regulated by estrogen.