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Identification of glucocorticoid response element of the rat TRH gene.

Lee GC, Yang IM, Kim BJ, Woo JT, Kim SW, Kim JW, Kim YS, Choi YK - Korean J. Intern. Med. (1996)

Bottom Line: Although there is no typical palindromic glucocorticoid response element (GRE) on the rat TRH gene promoter, a perfect GRE half-site is found between -205 bp and -211 bp.On the contrary, deletion of the region between -242 to -200 bp reduced the basal transcriptional activity by 90% and also completely abolished the DEX-induced transcriptional activation of the luciferase gene.While the co-transfection of glucocorticoid receptor expression vector (pRSV-GR) did not increase the basal transcriptional activity of pMAMneo-LUC, it increased the basal transcription of pTRH(-242/84)-LUC by 1.8 fold.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, College of Medicine, KyungHee University, Seoul, Korea.

ABSTRACT

Objectives: It was suggested that glucocorticoid exerts cell-specific effects on thyrotropin-releasing hormone (TRH) gene expression at the transcriptional level. Although there is no typical palindromic glucocorticoid response element (GRE) on the rat TRH gene promoter, a perfect GRE half-site is found between -205 bp and -211 bp. We investigated whether the segment between -242 bp and -200 bp of the rat TRH gene promoter is responsible for glucocorticoid response.

Methods: For the 5' deletion study of the TRH gene, four different plasmid constructs, pTRH(- 554/84), pTRH(-242/84), pTRH(-200/6), pTRH(-113/84) were used for transient transfection study. The plasmid pMAMneo-LUC was used as a positive control and pRSV-GR expression vector, as a co-transfection study. Transfection was performed by the modified calcium precipitation method with 2 micrograms of each lasmid on the HeLa cells.

Results: Dexamethasone (DEX) stimulated the transcriptional activity of pTRH(-554/84)-LUC and pTRH(-242/84)-LUC by approximately 3.2 fold at 10(-8) M and 5.4 fold at 10(-8) M. On the contrary, deletion of the region between -242 to -200 bp reduced the basal transcriptional activity by 90% and also completely abolished the DEX-induced transcriptional activation of the luciferase gene. The DEX-induced transcriptional activation of pTRH(-242/84)-LUC was in dose-dependent manner. While the co-transfection of glucocorticoid receptor expression vector (pRSV-GR) did not increase the basal transcriptional activity of pMAMneo-LUC, it increased the basal transcription of pTRH(-242/84)-LUC by 1.8 fold. The pRSV-GR co-transfection and DEX treatment further increased the transcription of pTRH(-242/84)-LUC by 2-4 fold at the concentration of 10(-8) M.

Conclusion: These findings suggest that a cis-acting element(s) which is important for the basal transcriptional activation and glucocorticoid response of the rat TRH gene is located between -242 bp and -200 bp. The gene has a weak GRE glucocorticoid response and seems to be mediated by an interaction between glucocorticoid receptor and other transcriptional factor (s).

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Dexamethasone increased luciferase activity of the pTRH(−242/84)-LUC and pMAMneo-LUC in dose-dependant manner without pRSV-GR co-transfection in transient transfection assays. Relative luciferase activity shown represents the multiplies of the pMAMneo-IUC without dexamethasone as a control, and mean of the duplicate transfection obtained in 6 separate experiments.
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f2-kjim-11-2-138-7: Dexamethasone increased luciferase activity of the pTRH(−242/84)-LUC and pMAMneo-LUC in dose-dependant manner without pRSV-GR co-transfection in transient transfection assays. Relative luciferase activity shown represents the multiplies of the pMAMneo-IUC without dexamethasone as a control, and mean of the duplicate transfection obtained in 6 separate experiments.

Mentions: The basal transcription of pMAMneo-LUC, a positive control for GRE, was only about 3.2 fold stimulated by DEX at the physiologic concentration of 10−8 M and 5.4 fold at 10−6 M (Fig. 2). However, co-transfection of GR expression vector (pRSV-GR) enhanced the DEX-stimulated transcriptional activity of pMAMneo-LUC by approximately 83 fold (Fig. 4).


Identification of glucocorticoid response element of the rat TRH gene.

Lee GC, Yang IM, Kim BJ, Woo JT, Kim SW, Kim JW, Kim YS, Choi YK - Korean J. Intern. Med. (1996)

Dexamethasone increased luciferase activity of the pTRH(−242/84)-LUC and pMAMneo-LUC in dose-dependant manner without pRSV-GR co-transfection in transient transfection assays. Relative luciferase activity shown represents the multiplies of the pMAMneo-IUC without dexamethasone as a control, and mean of the duplicate transfection obtained in 6 separate experiments.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4532014&req=5

f2-kjim-11-2-138-7: Dexamethasone increased luciferase activity of the pTRH(−242/84)-LUC and pMAMneo-LUC in dose-dependant manner without pRSV-GR co-transfection in transient transfection assays. Relative luciferase activity shown represents the multiplies of the pMAMneo-IUC without dexamethasone as a control, and mean of the duplicate transfection obtained in 6 separate experiments.
Mentions: The basal transcription of pMAMneo-LUC, a positive control for GRE, was only about 3.2 fold stimulated by DEX at the physiologic concentration of 10−8 M and 5.4 fold at 10−6 M (Fig. 2). However, co-transfection of GR expression vector (pRSV-GR) enhanced the DEX-stimulated transcriptional activity of pMAMneo-LUC by approximately 83 fold (Fig. 4).

Bottom Line: Although there is no typical palindromic glucocorticoid response element (GRE) on the rat TRH gene promoter, a perfect GRE half-site is found between -205 bp and -211 bp.On the contrary, deletion of the region between -242 to -200 bp reduced the basal transcriptional activity by 90% and also completely abolished the DEX-induced transcriptional activation of the luciferase gene.While the co-transfection of glucocorticoid receptor expression vector (pRSV-GR) did not increase the basal transcriptional activity of pMAMneo-LUC, it increased the basal transcription of pTRH(-242/84)-LUC by 1.8 fold.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine, College of Medicine, KyungHee University, Seoul, Korea.

ABSTRACT

Objectives: It was suggested that glucocorticoid exerts cell-specific effects on thyrotropin-releasing hormone (TRH) gene expression at the transcriptional level. Although there is no typical palindromic glucocorticoid response element (GRE) on the rat TRH gene promoter, a perfect GRE half-site is found between -205 bp and -211 bp. We investigated whether the segment between -242 bp and -200 bp of the rat TRH gene promoter is responsible for glucocorticoid response.

Methods: For the 5' deletion study of the TRH gene, four different plasmid constructs, pTRH(- 554/84), pTRH(-242/84), pTRH(-200/6), pTRH(-113/84) were used for transient transfection study. The plasmid pMAMneo-LUC was used as a positive control and pRSV-GR expression vector, as a co-transfection study. Transfection was performed by the modified calcium precipitation method with 2 micrograms of each lasmid on the HeLa cells.

Results: Dexamethasone (DEX) stimulated the transcriptional activity of pTRH(-554/84)-LUC and pTRH(-242/84)-LUC by approximately 3.2 fold at 10(-8) M and 5.4 fold at 10(-8) M. On the contrary, deletion of the region between -242 to -200 bp reduced the basal transcriptional activity by 90% and also completely abolished the DEX-induced transcriptional activation of the luciferase gene. The DEX-induced transcriptional activation of pTRH(-242/84)-LUC was in dose-dependent manner. While the co-transfection of glucocorticoid receptor expression vector (pRSV-GR) did not increase the basal transcriptional activity of pMAMneo-LUC, it increased the basal transcription of pTRH(-242/84)-LUC by 1.8 fold. The pRSV-GR co-transfection and DEX treatment further increased the transcription of pTRH(-242/84)-LUC by 2-4 fold at the concentration of 10(-8) M.

Conclusion: These findings suggest that a cis-acting element(s) which is important for the basal transcriptional activation and glucocorticoid response of the rat TRH gene is located between -242 bp and -200 bp. The gene has a weak GRE glucocorticoid response and seems to be mediated by an interaction between glucocorticoid receptor and other transcriptional factor (s).

Show MeSH