Abstract
Purpose
Corticosterone prevents cold-induced stimulation of thyrotropin-releasing hormone (Trh) expression in rats, and the stimulatory effect of dibutyryl cyclic-adenosine monophosphate (dB-cAMP) on Trh transcription in hypothalamic cultures. We searched for the mechanism of this interference.
Methods
Immunohistochemical analyses of phosphorylated cAMP-response element binding protein (pCREB) were performed in the paraventricular nucleus (PVN) of Wistar rats, and in cell cultures of 17-day old rat hypothalami, or neuroblastoma SH-SY5Y cells. Cultures were incubated 1h with dB-cAMP, dexamethasone and both drugs combined; their nuclear extracts were used for chromatin immunoprecipitation; cytosolic or nuclear extracts for coimmunoprecipitation analyses of catalytic subunit of protein kinase A (PKAc) and of glucocorticoid receptor (GR); their subcellular distribution was analyzed by immunocytochemistry.
Results
Cold exposure increased pCREB in TRH neurons of rats PVN, effect blunted by corticosterone previous injection. Dexamethasone interfered with forskolin increase in nuclear pCREB and its binding to Trh promoter; antibodies against histone deacetylase-3 precipitated chromatin from nuclear extracts of hypothalamic cells treated with tri-iodothyronine but not with dB-cAMP + dexamethasone, discarding chromatin compaction as responsible mechanism. Co-immunoprecipitation analyses of cytosolic or nuclear extracts showed protein:protein interactions between activated GR and PKAc. Immunocytochemical analyses of hypothalamic or SH-SY5Y cells revealed diminished nuclear translocation of PKAc and GR in cells incubated with forskolin + dexamethasone, compared to either forskolin or dexamethasone alone.
Conclusions
Glucocorticoids and cAMP exert mutual inhibition of Trh transcription through interaction of activated glucocorticoid receptor with protein kinase A catalytic subunit, reducing their nuclear translocation, limiting cAMP-response element binding protein phosphorylation and its binding to Trh promoter.
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Acknowledgements
Authors thank the helpful comments of Drs. M. Díaz-Gallardo, M. Zurita and technical assistance of A. Saralegui, A. Pimentel, F. Romero, M. Ramírez-Yarza, R. Rodríguez-Bahena and E. López-Bustos; and to the Instituto Nacional de Medicina Genómica (INMEGEN) for the genetic profile analysis of the neuroblastoma cells SH-SY5Y.
Funding
This work was partially supported by grants from CONACYT (CB180009) and DGAPA-UNAM (IN204913; 204316). ISR, PhD student of Biochemical Sciences (UNAM), received a CONACYT fellowship.
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Protocols followed the NIH ethics guidelines for care and use of laboratory animals, and were approved by the Institute’s Bioethics Committee.
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Sotelo-Rivera, I., Cote-Vélez, A., Uribe, RM. et al. Glucocorticoids curtail stimuli-induced CREB phosphorylation in TRH neurons through interaction of the glucocorticoid receptor with the catalytic subunit of protein kinase A. Endocrine 55, 861–871 (2017). https://doi.org/10.1007/s12020-016-1223-z
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DOI: https://doi.org/10.1007/s12020-016-1223-z