Endocrine Abstracts

The activity of hypothalamus–pituitary–thyroid (HPT) axis is essential for energy homeostasis. TRH expression in the PVN is rapidy stimulated by energy demanding situations as cold exposure or locomotion, but blunted by stress-induced increased corticosterone serum levels. In primary cultures of rat hypothalamic cells (RHC), PKA activators as forskolin (Fsk) increase TRH mRNA levels, effect reverted by coincubation with dexamethasone. Fsk-dex coincubation also impedes binding of GR and pCREB to GRE and CRE in TRH-promoter (Díaz Gallardo, J.Neuroendocrinol 2010). In an attempt to elucidate the mechanism involved, we determined whether 1 h coincubation with dex or Fsk-dex induced chromatin condensation. ChIP analyses of RHC-isolated chromatin from Fsk treated cells revealed increased PCR signal of TRH promoter (−246/+69) in immunoprecipitates of pCREB, RNA-Pol II, H3ACK10 or H3ACK14 antibodies (Ab), and of GR from dex-treatment; these signals were diminished in Fsk-dex coincubates. No recruitment was obtained with Ab against deacetylases HDAC3 H99 in Fsk-dex compared to that detected in cells treated T³. These results demonstrate that simultaneous activation of GR and PKA does not affect chromatin remodeling but impede GR and pCREB binding to TRH promoter. Antibodies against the catalytic subunit of PKA (PKAc), or against pCREB, incubated with a GR-immunoprecipitate from subcellular fractions of RHC treated with dex-Fsk allowed to demonstrate the interaction of GR:PKAc. In living Fsk treated RHC, cytoimmunochemical analysis demonstrated increased pCREB in nuclei that was diminished with Fsk-Dex treatment. Furthermore, co-transfecting GR and TRH-Luc on SH-SY5Y cells reproduced the interfering effect dex-Fsk on Fsk stimulation; dex interference on Fsk stimulation was lost transfecting GR mutated on DBD site (L501P), and with GR-T171A or GR-S224A mutants that affect nuclear translocation and interaction with coregulators, supporting GR intrinsic characteristics for protein-protein interactions. These results demonstrate that GR interaction with PKAc provides a fast mechanism for avoiding pCREB activation of TRH transcription.

Declaration of interest: The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project.

Funding: This work was supported, however funding details unavailable.