J Korean Soc Endocrinol.  1999 Jun;14(2):265-277.

The Characterization of Glucocoritcoid Response Element(GRE) on the Promoter of Thyrotropin-Releasing Hormone(TRH) Gene

• Kim SW
• Kim JW
• Kim YS
• Choi YK
• Yang IM
• Woo JT
• Chung WW

Abstract

BACKGROUND: We previously demonstrated that the promoter of rat TRH gene has GRE half site (TGTTCT) between -210 bp and -205 bp flanking with similar sequences of TPA response element (TRE), TAGTCA, at a distance of several base pairs from the GRE half site. It promps us to hypothesize that this composite GRE/TRE sequence can provide a site for interaction between glucocorticoid receptor (GR) and c-Jun. Thus, we investigated whether the composite sequence mediates transcriptional regulation induced by dexamethasone (DEX) and 12-O-tetradecanoyl phobol-13-acetate (TPA), and whether it binds GR and c-Jun. METHODS: A luciferase expressing plasmids that contain a part of rat TRH promoter including the composite sequence or their mutants were transfected into HeLa cells by Fugene 6. After the cells were incubated overnight with DEX and TPA, the luciferase activity was measured in a chemiluminometer. A gel retardation assay was performed after binding of the labeled composite sequence or its mutants with GR and c-Jun. RESULTS: DEX increased the transcriptional activity of the plasmid containing the wild type GRE by 2.5 folds, and TPA increased the transcriptional activity by 4 folds. The simultaneous stimulation with DEX and TPA synergistically increased the transcriptional activity by 10 folds. Two mutants whose GRE half sits were altered showed no responses to DEX, and suppressed the TPA-induced or both agents-induced transcriptional activity by 50%. Two mutants whose TRE-like sites were altered suppressed the DEX-induced transcriptional activity by 20%, TPA-induced trarptional activity by 25%, and both agents-induced transcriptional activity by 50%. Gel retardation assay showed that the composite sequence fonned a complex with GR and its mutants bound to GR with remarkably less affinity. c-Jun also bound to the composite sequence to form two cornplexes with less affinity compared to the AP-1 consensus sequence. The mutants of the TRE-like sequence bound to c-Jun with a significantly lower affinity compared to that of the wild type. Simulateous binding of the composite sequence with GR and c-Jun did not form any larger complex. The complex of GR and the composite sequence was much smaller than that formed by c-Jun, suggesting that GR binds to the composite sequence as a monomer. CONCLUSION: These results suggest that the composite sequence of GRE half site and TRE-like site on the promoter of rat TRH gene provides binding sites for GR and c-Jun, which mediate the interaction between two signal transduction pathways. (J Kor Soc Endocrinol 14:265-277, 1999)

MeSH Terms

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid
Animals
Base Pairing
Binding Sites
Consensus Sequence
Dexamethasone
Electrophoretic Mobility Shift Assay
HeLa Cells
Humans
Luciferases
Plasmids
Rats
Receptors, Glucocorticoid
Response Elements
Signal Transduction
Transcription Factor AP-1
4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid
Dexamethasone
Luciferases
Receptors, Glucocorticoid
Transcription Factor AP-1