TRH/TRH-R1 stimulates GATA2/Pit1-dependent activity of the human TSHβ gene.

(A) Schematics of TSHβ(−1193/+37)-, TSHβ(−128/+37)- and TSHβ-DX1-CAT. Putative TRH-responsive elements (TSH-A, nt. −274/−258 and TSH-C, nt. −402/−384) and PKA-sensitive element (TSH-B, nt. −333/−325) reported in the rat TSHβ gene are indicated as boxes in TSHβ(−1193/+37)-CAT. Pit1-US, a functional Pit1 binding site upstream of GATA-REs. GATA-REs, GATA responsive elements. SR, suppressor region. (B) and (C) Two µg TSHβ(−1193/+37)-CAT (B) or TSHβ(−128/+37)-CAT (C) was cotransfected into CV1 cells together with the expression plasmids for Pit1 (0.1 µg), GATA2 (0.2 µg), TRH-R1(412) or TRH-R1(387) (0.2 µg) and β-galactosidase-based reporter gene (pCH111, 1.1 µg). After 24h of culture, the cells were treated with 1–1000 nM TRH. (D) TPA, but not forskolin, enhances the Pit1/GATA2-dependent activity of TSHβ(−128/+37)-CAT. CV1 cells were cotransfected with 2.0 µg of TSHβ(−128/+37)-CAT together with the expression plasmids for Pit1 (0.1 µg), GATA2 (0.2 µg), TRH-R1 (0.2 µg; 412 aa or 387 aa) and pCH111 (1.1 µg). After 24h of culture, the cells were treated with 1–100 nM TPA or 1–100 µM forskolin. (E) AP-1-like sequence overlapping with putative nTRE is dispensable for TRH-induced activation of the TSHβ gene. Reporter assay with TSHβ-DX1-CAT was performed as described in (C). CAT activity was normalized with β-galactosidase activity. CAT activity for pCMV-CAT (5 ng/well) was taken as 100%. Data are expressed as the mean ± S.D. of at least three independent experiments. *, P<0.05; **, P<0.01; ***, P<0.001.