Glucocorticoid repression of gonadotropin-releasing hormone gene expression and secretion in morphologically distinct subpopulations of GT1-7 cells

Barbara Attardi, Toshihiko Tsujii, Robert Friedman, Zhouwen Zeng, James Roberts, Tammy Dellovade, Donald W. Pfaff, Uma R. Chandran, Michael W. Sullivan, Donald B. DeFranco

Research output: Contribution to journalArticle

30 Citations (Scopus)

Abstract

Two morphologically distinct subpopulations of GT1-7 cells have been characterized and examined for their responsiveness to glucocorticoid. 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 promoterluciferase 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 K(d) (1.7 nM, type I; 3.1 nM, type II) or B(max) (~3600 sites/cell, type I; ~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.

Original languageEnglish (US)
Pages (from-to)241-255
Number of pages15
JournalMolecular and Cellular Endocrinology
Volume131
Issue number2
DOIs
StatePublished - Aug 8 1997
Externally publishedYes

Fingerprint

Gene expression
Gonadotropin-Releasing Hormone
Glucocorticoids
Gene Expression
Dexamethasone
Glucocorticoid Receptors
Steroid hormones
Electrophoretic mobility
Response Elements
Reaction products
Assays
Plasmids
Somatotypes
Genes
Cells
Electrophoretic Mobility Shift Assay
Messenger RNA
Reporter Genes
Neuroglia
Cytosol

Keywords

  • Glucocorticoid receptors
  • GnRH secretion
  • Gonadotropin-releasing hormone
  • GT1 cells

ASJC Scopus subject areas

  • Endocrinology
  • Endocrinology, Diabetes and Metabolism

Cite this

Glucocorticoid repression of gonadotropin-releasing hormone gene expression and secretion in morphologically distinct subpopulations of GT1-7 cells. / Attardi, Barbara; Tsujii, Toshihiko; Friedman, Robert; Zeng, Zhouwen; Roberts, James; Dellovade, Tammy; Pfaff, Donald W.; Chandran, Uma R.; Sullivan, Michael W.; DeFranco, Donald B.

In: Molecular and Cellular Endocrinology, Vol. 131, No. 2, 08.08.1997, p. 241-255.

Research output: Contribution to journalArticle

Attardi, B, Tsujii, T, Friedman, R, Zeng, Z, Roberts, J, Dellovade, T, Pfaff, DW, Chandran, UR, Sullivan, MW & DeFranco, DB 1997, 'Glucocorticoid repression of gonadotropin-releasing hormone gene expression and secretion in morphologically distinct subpopulations of GT1-7 cells', Molecular and Cellular Endocrinology, vol. 131, no. 2, pp. 241-255. https://doi.org/10.1016/S0303-7207(97)00102-0
Attardi, Barbara ; Tsujii, Toshihiko ; Friedman, Robert ; Zeng, Zhouwen ; Roberts, James ; Dellovade, Tammy ; Pfaff, Donald W. ; Chandran, Uma R. ; Sullivan, Michael W. ; DeFranco, Donald B. / Glucocorticoid repression of gonadotropin-releasing hormone gene expression and secretion in morphologically distinct subpopulations of GT1-7 cells. In: Molecular and Cellular Endocrinology. 1997 ; Vol. 131, No. 2. pp. 241-255.
@article{feccd787d2204ae397c590e86895f31b,
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 glucocorticoid. 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 promoterluciferase 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 K(d) (1.7 nM, type I; 3.1 nM, type II) or B(max) (~3600 sites/cell, type I; ~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.",
keywords = "Glucocorticoid receptors, GnRH secretion, Gonadotropin-releasing hormone, GT1 cells",
author = "Barbara Attardi and Toshihiko Tsujii and Robert Friedman and Zhouwen Zeng and James Roberts and Tammy Dellovade and Pfaff, {Donald W.} and Chandran, {Uma R.} and Sullivan, {Michael W.} and DeFranco, {Donald B.}",
year = "1997",
month = "8",
day = "8",
doi = "10.1016/S0303-7207(97)00102-0",
language = "English (US)",
volume = "131",
pages = "241--255",
journal = "Molecular and Cellular Endocrinology",
issn = "0303-7207",
publisher = "Elsevier Ireland Ltd",
number = "2",

}

TY - JOUR

T1 - Glucocorticoid repression of gonadotropin-releasing hormone gene expression and secretion in morphologically distinct subpopulations of GT1-7 cells

AU - Attardi, Barbara

AU - Tsujii, Toshihiko

AU - Friedman, Robert

AU - Zeng, Zhouwen

AU - Roberts, James

AU - Dellovade, Tammy

AU - Pfaff, Donald W.

AU - Chandran, Uma R.

AU - Sullivan, Michael W.

AU - DeFranco, Donald B.

PY - 1997/8/8

Y1 - 1997/8/8

N2 - Two morphologically distinct subpopulations of GT1-7 cells have been characterized and examined for their responsiveness to glucocorticoid. 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 promoterluciferase 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 K(d) (1.7 nM, type I; 3.1 nM, type II) or B(max) (~3600 sites/cell, type I; ~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.

AB - Two morphologically distinct subpopulations of GT1-7 cells have been characterized and examined for their responsiveness to glucocorticoid. 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 promoterluciferase 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 K(d) (1.7 nM, type I; 3.1 nM, type II) or B(max) (~3600 sites/cell, type I; ~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.

KW - Glucocorticoid receptors

KW - GnRH secretion

KW - Gonadotropin-releasing hormone

KW - GT1 cells

UR - http://www.scopus.com/inward/record.url?scp=0030884544&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0030884544&partnerID=8YFLogxK

U2 - 10.1016/S0303-7207(97)00102-0

DO - 10.1016/S0303-7207(97)00102-0

M3 - Article

C2 - 9296383

AN - SCOPUS:0030884544

VL - 131

SP - 241

EP - 255

JO - Molecular and Cellular Endocrinology

JF - Molecular and Cellular Endocrinology

SN - 0303-7207

IS - 2

ER -