STIMULATION OF THE PREPROTHYROTROPIN-RELEASING HORMONE GENE BY EPIDERMAL GROWTH-FACTOR

Regulation of the expression of the prepro-TRH (ppTRH) gene by epiderm al growth factor (EGF) was investigated. The ip injection of EGF signi ficantly stimulated hypothalamic ppTRH messenger RNA levels in rats. T o clarify whether this stimulatory effect of EGF could be exerted at t he level of gene transcription, the 5'-flanking region (-1893/+127) of the mouse ppTRH gene fused to a luciferase reporter gene was transien tly transfected into pituitary GH(4)C(1) cells, and the effect of EGF on gene transcription was measured by a luciferase assay. EGF stimulat ed ppTRH gene promoter activity in a time-and dose-dependent manner. D eletion analysis revealed that two different regions of the promoter, between -254 and -218 [EGF response element-1 (EGFRE1)] and between -1 30 and -84 (EGFRE2) were required for full stimulation by EGF. The two EGFREs possessed putative binding sequences for the transcription fac tor Spl, and they functioned cooperatively in heterologous promoters. Nuclear extracts from GH(4)C(1) cells specifically bound those two EGF REs 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 far by the consensu s 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-bin ding proteins might be an Sp1-related zinc finger protein. Competition and supershift experiments demonstrated that the EGFRE2-binding prote in showing that the slowest migration possessed a characteristic simil ar to that of Sp1. Selective mutations of the Sp1-binding site in EGFR E2 markedly diminished the EGF-induced stimulation. These results sugg est that EGF may function as a positive regulator of ppTRH gene expres sion, and that the stimulatory effect may be mediated through a cooper ative interaction between Sp1 or Sp1-related proteins and additional f actors that bind to two separate DNA regions.