Involvement of transducer of regulated cAMP response element-binding protein activity on corticotropin releasing hormone transcription.
We have recently shown that phospho-cAMP response element-binding protein (CREB) is essential but not sufficient for activation of CRH transcription, suggesting the requirement of a coactivator. Here, we test the hypothesis that the CREB coactivator, transducer of regulated CREB activity (TORC), is required for activation of CRH transcription, using the cell line 4B and primary cultures of hypothalamic neurons. Immunohistochemistry and Western blot experiments in 4B cells revealed time-dependent nuclear translocation of TORC1,TORC 2, and TORC3 by forskolin [but not by the phorbol ester, phorbol 12-myristate 13-acetate (PMA)] in a concentration-dependent manner. In reporter gene assays, cotransfection of TORC1 or TORC2 potentiated the stimulatory effect of forskolin on CRH promoter activity but had no effect in cells treated with PMA. Knockout of endogenous TORC using silencing RNA markedly inhibited forskolin-activated CRH promoter activity in 4B cells, as well as the induction of endogenous CRH primary transcript by forskolin in primary neuronal cultures. Coimmunoprecipitation and chromatin immunoprecipitation experiments in 4B cells revealed association of CREB and TORC in the nucleus, and recruitment of TORC2 by the CRH promoter, after 20-min incubation with forskolin. These studies demonstrate a correlation between nuclear translocation of TORC with association to the CRH promoter and activation of CRH transcription. The data suggest that TORC is required for transcriptional activation of the CRH promoter by acting as a CREB coactivator. In addition, cytoplasmic retention of TORC during PMA treatment is likely to explain the failure of phorbolesters to activate CRH transcription in spite of efficiently phosphorylating CREB.
Liu
Ying
Y
Section on Endocrine Physiology, Developmental Endocrinology Branch, National Institute of Child Health and Human Development, National Institutes of Health, 10 Center Drive, Bethesda, Maryland 20892, USA.
Coello
Ana G
AG
Grinevich
Valery
V
Aguilera
Greti
G
D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D005576:Colforsin; D003346:Corticotropin-Releasing Hormone / Q000235:genetics / Q000378:metabolism*; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D017930:Genes, Reporter; D007031:Hypothalamus / Q000378:metabolism; D011401:Promoter Regions, Genetic; D034741:RNA, Small Interfering; D051381:Rats; D013755:Tetradecanoylphorbol Acetate; D015534:Trans-Activators; D014157:Transcription Factors / Q000378:metabolism*; D015533:Transcriptional Activation
C508137:Creb1 protein, rat; C545700:CRTC2 protein, rat; C519510:Crtc1 protein, rat; D017362:Cyclic AMP Response Element-Binding Protein; D034741:RNA, Small Interfering; D015534:Trans-Activators; D014157:Transcription Factors; D005576:Colforsin; D003346:Corticotropin-Releasing Hormone; D013755:Tetradecanoylphorbol Acetate