TR4 attenuates the Dex- or GR-mediated POMC suppression.

A, Murine pituitary corticotroph tumor AtT20 cells were cotransfected with POMC-promoter luciferase reporter and TR4-expressing or control plasmids for 18 hours, followed by treatment with Dex (10 or 100 nm) or vehicle for another 6 hours. The results are expressed as POMC promoter induction relative to vector control. B, shRNA TR4 stable transfectants or nonsense control-transfected AtT20 cells were cotransfected with POMC-promoter luciferase reporter. Twenty-four hours later, cells were treated with vehicle or Dex (10 or 100 nm) for another 4 hours, after which POMC luciferase activities were measured and expressed as POMC promoter induction relative to vector control. C, POMC luciferase activities were measured in AtT20 cells transfected with either TR4 or GR alone or with TR4 in combination with GR in the presence or absence of Dex treatment for 4 hours. D and E, POMC mRNA expression was measured by RT-PCR in AtT20 cells overexpressing TR4 (D) or knockdown TR4 (E) after treatment with vehicle or Dex for 6 hours. F, AtT20 cells were cotransfected with TR4 or GR plasmids alone or in combination. Twenty-four hours later, cells were treated with vehicle or Dex for 6 hours, and mRNA was harvested for real-time PCR analysis of POMC mRNA expression. G, Cell supernatants from latter cells were collected for measurement of ACTH secretion by ELISA assay. H–J, Primary cultures of freshly resected human corticotroph tumor cells (representative of five individual tumors studied) were transfected with TR4 or GR alone or with TR4 in combination with GR for 18 hours with or without Dex treatment. POMC mRNA expression was measured by RT-PCR (H and I). ACTH secretion was detected using an ACTH ELISA kit (J). Data shown were representative of at least three independently repeated experiments. Bars indicate the mean ± standard error of the mean of triplicate tests. *, P < .05; **, P < .01; ***, P < .005. ns, Not significant.