GATA2 alone mediates TRH signaling for transcription of the SR-less TSHβ promoter.

<p>(A) Schematics of TSHβ(−128/+37)-CAT, TSHβ-M3-CAT and TSHβ-M1-mXmYmZ-CAT. SR was deleted in both constructs and AP-1-like sequence/nTRE was mutated in TSHβ-M1-mXmYmZ-CAT. (B) and (C) Two µg TSHβ(−128/+37)-CAT, TSHβ-M3-CAT (B) and TSHβ-M1-mXmYmZ-CAT (C) were transfected into CV1 cells along with the expression plasmids for Pit1 (0.1 µg), GATA2 (0.2 µg), TRH-R1(412) (0.2 µg), and pCH111 (1.1 µg) in the presence or absence of 100 nM TRH. Basal and TRH-induced activities of TSHβ-M1-mXmYmZ-CAT were abolished with mutant GATA2, C349A (inset). (D) Expression level of wild-type GATA2 and C349A mutant. CV1 cells in 10 cm dish were transfected with an equal amount (10 µg/dish) of expression plasmids for wild-type or mutant GATA2s. Whole cell extracts were fractionated by SDS-PAGE and subjected to Western blot with anti-GATA2 antibody. (E) Schematic representation of hET1-CAT (upper panel). Two µg of hET1-CAT was transfected into CV1 cells along with expression plasmids for GATA2 (0.2 µg), TRH-R1(412) (0.2 µg), and pCH111 (1.1 µg) in the presence or absence of 100 nM TRH (lower panel). (F) Schematic representation of hαGSU-CAT (upper panel). Reporter assay was carried out as described in (E) in the presence or absence of 0–10 µM forskolin (lower panel). 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.