Stimulation of the Preprothyrotropin-Releasing Hormone Gene by Epidermal Growth Factor

Abstract

Regulation of the expression of the prepro-TRH (ppTRH) gene by epidermal growth factor (EGF) was investigated. The i.p. 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.

Activation of peroxisome proliferator-activated receptor-γ stimulates the growth arrest and DNA-damage inducible 153 gene in non-small cell lung carcinoma cells

“…Nuclear protein extracts were prepared from SQ-5 cells treated with troglitazone or DMSO as previously described (Ren et al, 1998). Protein concentrations were determined by Bradford's method.…”

“…The expression vector for human PPAR-g (pKCR2PPAR-g) was described previously (Monden et al, 1999). Transient transfection was performed into SQ-5 cells cultured in 6-well plates by the calcium phosphate precipitation method using 1.67 mg of the reporter construct and/or 83 ng of the expression vector for PPAR-g/well as described previously (Ren et al, 1998;Satoh et al, 1996;Monden et al, 1999). The total amounts of plasmids transfected into each plate were adjusted by adding an empty pKCR2 vector.…”

“…Sixteen hours after transfection, cells were washed with PBS and incubated with MEM containing 10% FBS stripped with AG1-X8 resin (Bio-Rad) and activated charcoal to eliminate bioactive substances such as retinoids. The luciferase assay was carried out after incubation in the presence or absence of 10 75 M troglitazone for 24 h as described previously (Ren et al, 1998;Satoh et al, 1996). The luciferase activities were normalized by protein concentrations measured by Bradford's method, and were expressed as light units per mg of protein as described previously (Ren et al, 1998;Satoh et al, 1996).…”

“…The nucleotide sequence of the upper strand of the consensus PPAR response element (PPRE) probe was 5'-AGCTTCAGGTCAGAGGTCAGA-GAGCT-3' (underlines indicate the core half-site sequence). The double-stranded oligonucleotide was radiolabeled using a Klenow fragment of the DNA polymerase I, and EMSA was performed in conditions described previously (Ren et al, 1998;Satoh et al, 1996).…”

Expression of the human melanocortin-4 receptor gene is controlled by several members of the Sp transcription factor family

“…Sp1 is abundantly expressed in most cell types including those of the hypothalamus (Ren et al 1998), and its level of expression changes during development and varies among different cell types (Lania et al 1997, Suske 1999. Expression of Sp1 protein and mRNA appears to be highest in fetal cells (Saffer et al 1991) and Sp1 / embryos display growth retardation and die early in gestation (Marin et al.…”