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

By Kenji Ohba (255704), Shigekazu Sasaki (335241), Akio Matsushita (335242), Hiroyuki Iwaki (335243), Hideyuki Matsunaga (335244), Shingo Suzuki (67899), Keiko Ishizuka (335246), Hiroko Misawa (335240), Yutaka Oki (308231) and Hirotoshi Nakamura (335250)

Abstract

<p>(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.</p

Topics: Molecular Biology, Chemistry, stimulates
Year: 2013
DOI identifier: 10.1371/journal.pone.0018667.g001
OAI identifier: oai:figshare.com:article/453200
Provided by: FigShare
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