Abstract
Several treatments which regulate tyrosine hydroxylase (TH) transcription, such as stress in vivo, or 12-O-tetradecanoylphorbol-13-acetate (TPA) in cell culture, induce both Egr1 and AP1 factors. Previously, we identified that a functional Egr1 motif overlapped with Sp1 site in the rat TH promoter and determined that induction of TH promoter activity by Egr1 also required an AP1/Ebox motif in addition to the Egr1 motif. Here, we examined the mechanism of the cross-talk between these motifs. Increasing the distance between the AP1/Ebox and the Sp1/Egr1 motifs, as well as mutation of either or both motifs, reduced the ability of Egr1 to up-regulate the reporter activity controlled by proximal 272 nucleotides of the rat TH promoter. Electrophoretic mobility shift assays with nuclear extract from TPA treated PC12 cells were used to identify the composition of the factors which bound the AP1/Ebox motif. The complexes formed with labeled AP1/Ebox oligonucleotide were reduced or supershifted with antisera to Fos family, Fra-2, c-Fos and Jun D. Excess Sp1/Egr1 oligonucleotide or anti Egr1 antisera did not compete. Transfection of PC12 cells with Fra-2 induced reporter activity requiring the AP1, but not the Egr1 motif. However, when cotransfected with Fra-2, Egr1 expression vectors elicited lower induction of luciferase activity than observed with Egr1 alone. These results suggest that although Egr1 does not compete for binding of AP1 factors to the promoter, it can modulate the regulation of TH transcription by AP1 factors. [Jpn J Physiol 54 Suppl:S212 (2004)]
