Stimulation of the Preprothyrotropin-Releasing Hormone Gene by Epidermal Growth Factor
Regulation of the expression of the prepro-TRH (ppTRH) gene by epidermal growth factor (EGF) was investigated. The ip injection of EGF significantly stimulated hypothalamic ppTRH messenger RNA levels in rats. To clarify whether this stimulatory effect of EGF could be exerted at the level of gene tra...
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Published in | Endocrinology (Philadelphia) Vol. 139; no. 1; pp. 195 - 203 |
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Main Authors | , , , , , , |
Format | Journal Article |
Language | English |
Published |
Endocrine Society
01.01.1998
|
Online Access | Get full text |
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Summary: | Regulation of the expression of the prepro-TRH (ppTRH) gene by
epidermal growth factor (EGF) was investigated. The ip injection of EGF
significantly stimulated hypothalamic ppTRH messenger RNA levels in
rats. To clarify whether this stimulatory effect of EGF could be
exerted at the level of gene transcription, the 5′-flanking region
(−1893/+127) of the mouse ppTRH gene fused to a luciferase reporter
gene was transiently transfected into pituitary
GH4C1 cells, and the effect of EGF on gene
transcription was measured by a luciferase assay. EGF stimulated ppTRH
gene promoter activity in a time- and dose-dependent manner. Deletion
analysis revealed that two different regions of the promoter, between−
254 and −218 [EGF response element-1 (EGFRE1)] and between −130
and −84 (EGFRE2) were required for full stimulation by EGF. The two
EGFREs possessed putative binding sequences for the transcription
factor Sp1, and they functioned cooperatively in heterologous
promoters. Nuclear extracts from GH4C1 cells
specifically bound those two EGFREs in gel retardation assays. Two
protein-DNA complexes were found on EGFRE1, whereas four complexes were
observed on EGFRE2. Although the binding of nuclear extracts to EGFRE1
was competed for by the consensus Sp1 binding sequence, the complexes
on EGFRE1 were not supershifted by an Sp1 antibody. Formation of the
slower migrating protein complex on EGFRE1 was prevented by EDTA,
suggesting that one of the EGFRE1-binding proteins might be an
Sp1-related zinc finger protein. Competition and supershift experiments
demonstrated that the EGFRE2-binding protein showing that the slowest
migration possessed a characteristic similar to that of Sp1. Selective
mutations of the Sp1-binding site in EGFRE2 markedly diminished the
EGF-induced stimulation. These results suggest that EGF may function as
a positive regulator of ppTRH gene expression, and that the stimulatory
effect may be mediated through a cooperative interaction between Sp1 or
Sp1-related proteins and additional factors that bind to two separate
DNA regions. |
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ISSN: | 0013-7227 1945-7170 |
DOI: | 10.1210/endo.139.1.5703 |