Creb and Sp/Krüppel response elements cooperate to control rat TRH gene transcription in response to cAMP.
Expression of hypophysiotropic TRH, that controls thyroid axis activity, is increased by cold exposure; this effect is mimicked in rat hypothalamic cells incubated with norepinephrine or cAMP analogs. TRH proximal promoter contains three putative CRE: Site-4 or CRE-1 that overlaps an element recognized by thyroid hormone receptors, CRE-2 with adjacent sequences GC box or CACCC recognized by Sp/Krüppel factors (extended CRE-2), and AP-1 sites flanking a GRE(1/2). To evaluate the role of each element in the cAMP response, these sites were mutated or deleted in rat TRH promoter linked to luciferase gene (TRH-luc) and co-transfected with β-gal expression vector in various cell lines; C6 cells gave the highest response to forskolin. Basal activity was most affected by mutations or deletion of CRE-2 site, or CACCC (50-75% of wild type-WT). Forskolin-induced 3× stimulation in WT which decreased 25% with CRE-1 or AP-1 deletions, but 50% when CRE-2 or its 5' adjacent GC box was altered. SH-SY5Y cells co-transfected with CREB-expression vector increased dB-cAMP response in the wild type but not in the CRE-2 mutated plasmid; cotransfecting CREB-A (a dominant negative expression vector) strongly diminished basal or cAMP response. Primary cultures of hypothalamic cells transfected with plasmids containing deletions of CRE-1, CRE-2, or extended CRE-2 failed to respond to forskolin when CRE-2 was modified. These results corroborate the CRE-2 site as the main cAMP-response element of rat TRH promoter, not exclusive of transcription factors of hypothalamic cells, and stress the relevance of adjacent Sp-1 sites, important mediators of some metabolic hormones.
Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México (UNAM), Cuernavaca, México.
Antonieta
A
Cote-Vélez
Adrián
A
Pérez-Maldonado
Joel
J
Osuna
Benito
B
Barrera
Jean-Louis
JL
Charli
Patricia
P
Joseph-Bravo
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D000242:Cyclic AMP / Q000494:pharmacology*; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics*; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D051741:Kruppel-Like Transcription Factors / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D017354:Point Mutation / Q000235:genetics; D051381:Rats; D020218:Response Elements / Q000235:genetics*; D051703:Sp Transcription Factors / Q000235:genetics*; D013973:Thyrotropin-Releasing Hormone / Q000235:genetics*; D014158:Transcription, Genetic / Q000187:drug effects*; D014162:Transfection
D017362:Cyclic AMP Response Element-Binding Protein; D051741:Kruppel-Like Transcription Factors; D051703:Sp Transcription Factors; D013973:Thyrotropin-Releasing Hormone; D000242:Cyclic AMP