Role of a pituitary-specific transcription factor (Pit-1/GHF-1) or a closely related protein in cAMP regulation of human thyrotropin-β subunit gene expression

Hans J. Steinfelder, Sally Radovick, Melisa A. Mroczynski, Peter Hauser, John H. McClaskey, Bruce D. Weintraub, Fredric E. Wondisford

Research output: Contribution to journalArticlepeer-review


cAMP regulation of the human thyrotropin-β (TSHβ) gene cAMP was studied in two heterologous cell lines, a human embryonal kidney cell line (293) and a rat pituitary cell line (GH3). In 293 cells, human TSHβ gene expression was not stimulated by the adenylate cyclase activator forskolin or the cAMP analogue 8-bromo-cAMP (8-Br-cAMP). On the other hand, these agents induced human TSHβ gene expression 4-12-fold in GH3 cells. Deletion analysis demonstrated that the regions from +3 to 18 bp and from -128 to 61 bp were both necessary for cAMP stimulation. The latter region contains three DNA sequences homologous to a pituitary-specific transcription factor, Pit-1/GHF-1, DNA-binding site. Gel-mobility assays demonstrated that a radiolabeled human TSHβ probe (-128 to -61 bp) formed five specific DNA-protein complexes with mouse thyrotropic tumor (MTT) nuclear extract and two specific complexes with in vitro translated Pit-1/GHF-1. Four of the five MTT complexes and both in vitro Pit-1/GHF-1 complexes were reduced or eliminated by excess of an unlabeled Pit-1/GHF-1 DNA-binding site from the rat growth hormone gene, but not a mutated version of the same DNA fragment, suggesting that Pit-1/GHF-1 or a closely related thyrotroph protein binds to these DNA sequences. In 293 cells, co-transfection of an expression vector containing the Pit-1/GHF-1 cDNA restored cAMP-responsiveness to the human TSHβ promoter (5.2- and 6.6-fold maximal stimulation by 8-Br-cAMP and forskolin, respectively) but not the herpes virus thymidine kinase promoter (1.2-fold maximal stimulation by either agent). Thus we conclude that the human TSHβ gene is positively regulated by cAMP in GH3 but not 293 cells. Since the human TSHβ gene contains at least one high-affinity binding site for Pit-1/GHF-1 in a region necessary for cAMP stimulation and cAMP stimulation could be restored to the human TSHβ promoter in a previously nonresponsive cell line by the addition of Pit-1/GHF-1, this suggests that Pit-1/GHF-1, or a closely related protein in the thyrotroph, may be a trans-acting factor for cAMP stimulation of the TSHβ gene.

Original languageEnglish (US)
Pages (from-to)409-419
Number of pages11
JournalJournal of Clinical Investigation
Issue number2
StatePublished - 1992
Externally publishedYes


  • Phosphorylation
  • Pit-1
  • TSH-B
  • Transcription factor
  • cAMP

ASJC Scopus subject areas

  • Medicine(all)


Dive into the research topics of 'Role of a pituitary-specific transcription factor (Pit-1/GHF-1) or a closely related protein in cAMP regulation of human thyrotropin-β subunit gene expression'. Together they form a unique fingerprint.

Cite this