Cyclic adenosine-3',5'-monophosphate-mediated activation of a glutamine synthetase composite glucocorticoid response element.
The glutamate synthetase gene (GS) contains a composite glucocorticoid response element (cGRE) comprised of a GRE and an adjacent element with features of both a cAMP-response element (CRE) and a 12-O-tetradecanoylphorbol 13-acetate (TPA) response element (TRE). The CRE/TRE element of the cGRE contributed to two modes of transcriptional activation: 1) enhancement of the response to cortisol and 2) a synergistic response to cortisol and increased cAMP. COS-7 cells transfected with a cGRE-luciferase construct show minimal expression under basal conditions or forskolin treatment. After cortisol treatment, luciferase activity from the cGRE is enhanced 4- to 8-fold greater than the GRE portion of the cGRE or a GRE from the tyrosine aminotransferase gene. Treatment with both forskolin and cortisol produced a 2- to 4-fold synergistic response over cortisol alone. Synergy is also seen with 8-bromo-cAMP, is specific for the cGRE, and occurs in a number of established cell lines. Elimination of the GRE or CRE/TRE reduces the synergy by 70-100%. Altering the CRE/TRE to GRE spacing changed both enhancement and synergy. Moving the elements 3 bp closer or extending 15 bp reduced enhancement. Synergy was markedly reduced when elements were one half of a helical turn out of phase. Western blots verified that CREB (cAMP-responsive binding protein) and ATF-1 (activating transcription factor-1) binds to the cGRE sequence. A specific dominant negative inhibitor of the CREB family, A-CREB, reduced synergy by 50%. These results suggest that the GS cGRE can potentially integrate signaling from both the cAMP and glucocorticoid receptor transduction pathways and that CREB/ATF-1 may play an important role in this process.
Richardson
J
J
Department of Physiology, Dartmouth Medical School, Lebanon, New Hampshire 03756, USA.
Vinson
C
C
Bodwell
J
J
D051697:Activating Transcription Factor 1; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells / Q000187:drug effects / Q000378:metabolism; D019556:COS Cells / Q000187:drug effects / Q000378:metabolism; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D006224:Cricetinae; D000242:Cyclic AMP / Q000378:metabolism*; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D004268:DNA-Binding Proteins; D004357:Drug Synergism; D005938:Glucocorticoids / Q000378:metabolism*; D005974:Glutamate-Ammonia Ligase / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D006854:Hydrocortisone / Q000494:pharmacology; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D020218:Response Elements / Q000187:drug effects / Q000502:physiology*; D013755:Tetradecanoylphorbol Acetate / Q000378:metabolism / Q000494:pharmacology; D014157:Transcription Factors / Q000378:metabolism
D051697:Activating Transcription Factor 1; D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D005938:Glucocorticoids; D014157:Transcription Factors; D005576:Colforsin; D000242:Cyclic AMP; D005974:Glutamate-Ammonia Ligase; D013755:Tetradecanoylphorbol Acetate; D006854:Hydrocortisone