title: Glucocorticoid repression of gonadotropin-releasing hormone gene expression and secretion in morphologically distinct subpopulations of GT1-7 cells.
abstract: Two morphologically distinct subpopulations of GT1-7 cells have been characterized and examined for their responsiveness to glucocorticoids. Type I cells have a neuronal phenotype, extending many lengthy processes, and express neuronal, but not glial, markers. Type II cells show weaker or negative immunostaining for neuronal markers and exhibit fewer processes. The effect of glucocorticoids on gonadotropin-releasing hormone (GnRH) secretion and gene expression was compared in type I and type II GT1-7 cells. For secretion studies, cells were attached to Cytodex beads and perifused with control medium or medium containing dexamethasone (dex). The high level of GnRH secreted by type I cells was slightly enhanced in the presence of dex, whereas dex rapidly and profoundly decreased the already low level of GnRH secreted by type II cells. Immunocytochemistry for GnRH showed dark reaction product in the cell bodies and processes of type I cells and little or no immunoreactivity in type II cells. Both the endogenous mouse GnRH mRNA and the transcriptional activity of a mouse GnRH promoter luciferase reporter gene plasmid were suppressed to a greater extent in type II cells than in type I. In electrophoretic mobility shift assays, there was no difference between type I and type II nuclear extracts in the pattern of protein-DNA complexes formed on two previously identified negative glucocorticoid response elements located at -237 to -201 and -184 to -150 bp of the mouse promoter. Both cell types contained glucocorticoid receptors (GR) by Western blot analysis. Cytosols from type I or type II cells were incubated with [3H]dex to obtain GR binding parameters. Binding data were consistent with a one-site model for dex binding in each case. Small differences in Kd (1.7 nM, type I; 3.1 nM, type II) or Bmax (approximately 3600 sites/cell, type I; approximately 1800 sites/cell, type II) were not likely to account for the differential sensitivity to dex treatment. In conclusion, nuclear alterations in type II cells leading to greater transcriptional susceptibility to dex, coupled with low GnRH storage levels, may be reflected in exquisite sensitivity of GnRH secretion to glucocorticoid repression. This represents the first example of a steroid hormone acting directly on GnRH-producing cells to alter GnRH secretion.
authors.affiliation: Department of Medicine, University of Pittsburgh School of Medicine, PA 15213, USA.
authors.forename: B
authors.initials: B
authors.lastname: Attardi
authors.forename: T
authors.initials: T
authors.lastname: Tsujii
authors.forename: R
authors.initials: R
authors.lastname: Friedman
authors.forename: Z
authors.initials: Z
authors.lastname: Zeng
authors.forename: J L
authors.initials: JL
authors.lastname: Roberts
authors.forename: T
authors.initials: T
authors.lastname: Dellovade
authors.forename: D W
authors.initials: DW
authors.lastname: Pfaff
authors.forename: U R
authors.initials: UR
authors.lastname: Chandran
authors.forename: M W
authors.initials: MW
authors.lastname: Sullivan
authors.forename: D B
authors.initials: DB
authors.lastname: DeFranco
mesh_terms: D000818:Animals; D015153:Blotting, Western; D004247:DNA / Q000378:metabolism; D003907:Dexamethasone / Q000378:metabolism / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects*; D005938:Glucocorticoids / Q000494:pharmacology*; D007987:Gonadotropin-Releasing Hormone / Q000235:genetics* / Q000378:metabolism; D008156:Luciferases / Q000235:genetics; D051379:Mice; D008822:Mice, Transgenic; D009474:Neurons / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism; D010751:Phosphopyruvate Hydratase / Q000032:analysis; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins; D014162:Transfection; D014407:Tumor Cells, Cultured
chemical_list: D005938:Glucocorticoids; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D007987:Gonadotropin-Releasing Hormone; D003907:Dexamethasone; D004247:DNA; D008156:Luciferases; D010751:Phosphopyruvate Hydratase