Dexamethasone represses cAMP rapid upregulation of TRH gene transcription: identification of a composite glucocorticoid response element and a cAMP response element in TRH promoter.
Hypothalamic proTRH mRNA levels are rapidly increased (at 1 h) in vivo by cold exposure or suckling, and in vitro by 8Br-cAMP or glucocorticoids. The aim of this work was to study whether these effects occurred at the transcriptional level. Hypothalamic cells transfected with rat TRH promoter (-776/+85) linked to the luciferase reporter showed increased transcription by protein kinase (PK) A and PKC activators, or by dexamethasone (dex), but co-incubation with dex and 8Br-cAMP decreased their stimulatory effect (as observed for proTRH mRNA levels). These effects were also observed in NIH-3T3-transfected cells supporting a characteristic of TRH promoter and not of hypothalamic cells. Transcriptional regulation by 8Br-cAMP was mimicked by noradrenaline which increased proTRH mRNA levels, but not in the presence of dex. PKA inhibition by H89 avoided 8Br-cAMP or noradrenaline stimulation. TRH promoter sequences, cAMP response element (CRE)-like (-101/-94 and -59/-52) and glucocorticoid response element (GRE) half-site (-210/-205), were analyzed by electrophoretic mobility shift assays with nuclear extracts from hypothalamic or neuroblastoma cultures. PKA stimulation increased binding to CRE (-101/-94) but not to CRE (-59/-52); dex or 12-O-tetradecanoylphorbol-13-acetate (TPA) increased binding to GRE, a composite site flanked by a perfect and an imperfect activator protein (AP-1) site in the complementary strand. Interference was observed in the binding of CRE or GRE with nuclear extracts from cells co-incubated for 3 h with 8Br-cAMP and dex; from cells incubated for 1 h, only the binding to GRE showed interference. Rapid cross-talk of glucocorticoids with PKA signaling pathways regulating TRH transcription constitutes another example of neuroendocrine integration.
Cote-Vélez
A
A
Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México, AP 510-3, Cuernavaca, Mor. 62250, México.
Pérez-Martínez
L
L
Díaz-Gallardo
M Y
MY
Pérez-Monter
C
C
Carreón-Rodríguez
A
A
Charli
J-L
JL
Joseph-Bravo
P
P
D000818:Animals; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D003907:Dexamethasone / Q000494:pharmacology*; D005786:Gene Expression Regulation / Q000502:physiology*; D005938:Glucocorticoids / Q000378:metabolism; D007031:Hypothalamus / Q000378:metabolism; D051379:Mice; D041681:NIH 3T3 Cells; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D020218:Response Elements; D013973:Thyrotropin-Releasing Hormone / Q000096:biosynthesis / Q000235:genetics*; D015854:Up-Regulation
D005938:Glucocorticoids; D012333:RNA, Messenger; D013973:Thyrotropin-Releasing Hormone; D003907:Dexamethasone; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases