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. 2012 Jul 9;7(7):e40437. doi: 10.1371/journal.pone.0040437

Figure 4. Stimulation of the promoter activity of the TSHβ gene by NR4A1 in vitro.

Figure 4

A) The human TSHβ promoter region between bp –1192 and +37 linked to a luciferase reporter gene (pA3TSHβ(−1192∼+37)-Luc) was transfected into CV-1 cells. Expression of Pit1 and GATA2 was necessary for the basal promoter activity of the TSHβ gene. When an expression vector for Pit1 was co-transfected, the promoter activity of the TSHβ was stimulated by 10-fold, but co-transfection with GATA2 did not significantly alter it. However, synergistic stimulation of the promoter activity was observed when Pit1 and GATA2 were simultaneously expressed. B) Under the above conditions expressing Pit1 and GATA2, the TSHβ promoter activity was stimulated by NR4A1 in a dose-dependent manner. When 1 μg of expression vector of NR4A1/3 wells was co-trasfected, the promoter activity was stimulated about 2-fold, and when 3 μg, it reached a plateau at about 6-fold. Relative luciferase activity against the value without expression of NR4A1 is shown. C) Stimulation of the promoter activity by NR4A1 was specific for the TSHβ gene (TSHB). The promoter activities of the thyrotropin-releasing hormone (TRH), TSHα, and prolactin (PRL) genes was not stimulated by overexpression of NR4A1. Relative luciferase activity against the value without expression of NR4A1 in each promoter is shown. Values are represented as the mean ± SEM from at least three experiments. **, p<0.01; N.S., not significant.