Creb and Sp/Krüppel response elements cooperate to control rat TRH gene transcription in response to cAMP

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• 作者：Antonieta Cote-Vé ; lez^a ; Adriá ; n Pé ; rez-Maldonado^a ; Joel Osuna^b ; Benito Barrera^a ; Jean-Louis Charli^a ; Patricia Joseph-Bravo^a ; ^{joseph@ibt.unam.mx}
• 关键词：TRH ; CRE ; cAMP ; Sp/KFL ; Hypothalamus ; Transcription
• 刊名：Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms
• 出版年：2011
• 出版时间：March 2011
• 年：2011
• 卷：1809
• 期：3
• 页码：191-199
• 全文大小：790 K

文摘

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.