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Expression of Functional Estrogen Receptors and Galanin Messenger Ribonucleic Acid in Immortalized Luteinizing Hormone-Releasing Hormone Neurons: Estrogenic Control of Galanin Gene Expression

Peptide Pharmacology (E.S.S., E.H.M., D.C.D., F.J.L.) and Cell Biology (M.C.P.) Sections, Women’s Health Research Institute, Wyeth-Ayerst Research, Radnor, Pennsylvania 19087

Address all correspondence and requests for reprints to: Francisco J. López, M.D., Ph.D., Ligand Pharmaceuticals, Inc., 10255 Science Center Drive, San Diego, California 92121. E-mail: lopezfj@msn.com or flopez{at}ligand.com

The activity of estradiol on the LHRH neuronal network is crucial in the regulation of reproduction. In vivo, estradiol induces galanin (GAL) gene expression in LHRH neurons and GAL/LHRH colocalization is sexually dimorphic and neonatally determined by steroid exposure. The effects of estradiol on LHRH neurons, however, are considered to be indirect because estrogen receptors (ER) have not been detected in LHRH neurons in vivo. Using immortalized mouse LHRH neurons (GT1–7 cells), we demonstrated by RT-PCR and Southern blotting that GT1–7 cells express ER messenger RNA (mRNA). Sequencing of the amplification products indicated that GT1–7 ER is of the -subtype (ER). Additionally, estrogen receptors in GT1–7 cells were characterized by competitive radioligand receptor binding and IC₅₀ values for 17ß-estradiol and ICI-182,780 were found to be 0.24 and 4.1 nM, respectively. The ability of endogenous GT1–7 cell ER to regulate transcription was determined in transient transfection studies using a construct that consisted of a luciferase reporter gene that is driven by tandem estrogen response elements (ERE) and a minimal herpes simplex virus thymidine kinase promoter. 17ß-Estradiol was found to enhance luciferase activity by 2.5-fold at physiological concentrations with an ED₅₀ value of 47 pM. This induction was completely inhibited by ICI-182,780 which had an IC₅₀ value of 4.8 nM. Raloxifene, tamoxifen, 4-hydroxytamoxifen, and droloxifene also fully blocked estrogen-mediated luciferase induction with IC₅₀ values of 58.4, 89.2, 33.2, and 49.8 nM, respectively. In addition, GAL mRNA was detected and identified by RT-PCR followed by Southern blotting using a rat GAL complementary DNA (cDNA) probe. The ability of 17ß-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 GAL gene expression by 2-fold with an ED₅₀ value of 23 pM. ICI-182,780, raloxifene, and droloxifene completely blocked this induction.

In summary, our data demonstrate the presence of ER and GAL mRNA in GT1–7 cells. The ER in GT1–7 cells is biologically active because 17ß-estradiol enhances both endogenous GAL gene expression and an ERE-driven 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 capacity to be directly regulated by estrogen.

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