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 language | English (US) |
|---|---|
| Pages (from-to) | 241-255 |
| Number of pages | 15 |
| Journal | Molecular and Cellular Endocrinology |
| Volume | 131 |
| Issue number | 2 |
| DOIs | |
| State | Published - Aug 8 1997 |
| Externally published | Yes |
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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 journal › Article
}
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 -