Phosphorylated Cyclic-AMP-Response Element-Binding Protein and Thyroid Hormone Receptor Have Independent Response Elements in the Rat Thyrotropin-Releasing Hormone Promoter: An Analysis in Hypothalamic Cells

Background: Thyrotropin-releasing hormone (TRH) from the hypothalamic paraventricular nucleus (PVN) controls the activity of the hypothalamus-pituitary-thyroid axis. TRH is expressed in other hypothalamic nuclei but is downregulated by 3,3',5-L-triiodothyronine (T-3) exclusively in the PVN. Thyroid hormone receptors (TRs) bind TRH promoter at Site-4 (-59/-52), also proposed to bind phosphorylated cAMP response element-binding protein (pCREB). However, nuclear extracts from 8Br-cAMP-stimulated hypothalamic cells showed no binding to Site-4 and instead to cAMP response element (CRE)-2 (-101/-94). Methods: We characterized, by DNA footprinting and chromatin immunoprecipitation, the sites in the rat (-242/+34) TRH promoter that bind to nuclear factors of hypothalamic primary cultures incubated with 8Br-cAMP and/or T-3. Results: In primary cultures of fetal hypothalamic cells, TRH mRNA levels rapidly diminished with 10 nM T-3 while they increased by 1 m M 8Br-cAMP (+/- T-3). Site-4 was protected from DNase I digestion with nuclear extracts from T-3-incubated cells but not from controls or from those incubated with 8Br-cAMP, which protected CRE-2; T-3 + 8Br-cAMP coincubation caused no interference. The region protected by nuclear extracts from cAMP-stimulated cells included sequences adjacent to CRE-2-containing response elements of the SP/Kruppel family. A TR beta 2 antibody immunoprecipitated chromatin containing Site-4 but not CRE-2, from cells incubated with T-3. A pCREB antibody immunoprecipitated CRE-2 containing chromatin in controls and more in 8Br-cAMP-stimulated cells but none when cells were incubated only with T-3. Recruitment of the 2 transcription factors was preserved in cells simultaneously exposed to 8Br-cAMP and T-3. Discussion: These results show that pCREB binds to a response element in the TRH promoter (CRE-2) that is independent of Site-4 where TR beta 2 is bound; pCREB and TR do not present mutual interference on their binding sites. Copyright (C) 2009 S. Karger AG, Basel