title: Glucocorticoids curtail stimuli-induced CREB phosphorylation in TRH neurons through interaction of the glucocorticoid receptor with the catalytic subunit of protein kinase A.
abstract: OBJECTIVE
  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.
authors.affiliation: Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México (UNAM), UNAM, A.P. 510-3, Cuernavaca, Morelos, 62271, Mexico.
authors.forename: Israim
authors.initials: I
authors.lastname: Sotelo-Rivera
authors.affiliation: Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México (UNAM), UNAM, A.P. 510-3, Cuernavaca, Morelos, 62271, Mexico.
authors.forename: Antonieta
authors.initials: A
authors.lastname: Cote-Vélez
authors.affiliation: Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México (UNAM), UNAM, A.P. 510-3, Cuernavaca, Morelos, 62271, Mexico.
authors.forename: Rosa-María
authors.initials: RM
authors.lastname: Uribe
authors.affiliation: Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México (UNAM), UNAM, A.P. 510-3, Cuernavaca, Morelos, 62271, Mexico.
authors.forename: Jean-Louis
authors.initials: JL
authors.lastname: Charli
authors.affiliation: Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México (UNAM), UNAM, A.P. 510-3, Cuernavaca, Morelos, 62271, Mexico. joseph@ibt.unam.mx.
authors.forename: Patricia
authors.identifier: http://orcid.org/0000-0003-4794-6816
authors.initials: P
authors.lastname: Joseph-Bravo
mesh_terms: D000818:Animals; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D003080:Cold Temperature; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D003907:Dexamethasone / Q000494:pharmacology*; D005938:Glucocorticoids / Q000494:pharmacology*; D007031:Hypothalamus / Q000187:drug effects / Q000378:metabolism; D009474:Neurons / Q000187:drug effects / Q000378:metabolism*; D010286:Paraventricular Hypothalamic Nucleus / Q000187:drug effects / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D051381:Rats; D017208:Rats, Wistar; D011965:Receptors, Glucocorticoid / Q000378:metabolism*; D013973:Thyrotropin-Releasing Hormone / Q000378:metabolism*
chemical_list: D017362:Cyclic AMP Response Element-Binding Protein; D005938:Glucocorticoids; D011965:Receptors, Glucocorticoid; D013973:Thyrotropin-Releasing Hormone; D003907:Dexamethasone; D017868:Cyclic AMP-Dependent Protein Kinases
keywords: Catalytic subunit of PKA; Cold stimulation; Glucocorticoid receptor; Protein-protein interaction; TRH; pCREB