Upregulation of AKT1 protein expression in forskolin-stimulated macrophage: Evidence from ChIP analysis that CREB binds to and activates the AKT1 promoter

• Published in: Journal of cellular biochemistry 2007-03, Vol.100 (4), p.1022-1033
• Main Authors: Misra, Uma Kant, Pizzo, Salvatore Vincent
• Format: Article
• Language: English
• Subjects: Animals; Cells, Cultured; Chromatin Immunoprecipitation - methods; Colforsin - pharmacology; CREB and Akt promoter activation; Cyclic AMP Response Element-Binding Protein - metabolism; Cycloheximide - pharmacology; Dactinomycin - pharmacology; Gene Expression Regulation - drug effects; Isoquinolines - pharmacology; Macrophages, Peritoneal - cytology; Macrophages, Peritoneal - drug effects; Macrophages, Peritoneal - metabolism; Mice; Mice, Inbred C57BL; Models, Biological; Promoter Regions, Genetic - genetics; Protein Binding; Proto-Oncogene Proteins c-akt - genetics; Proto-Oncogene Proteins c-akt - metabolism; RNA Interference; RNA, Messenger - genetics; RNA, Messenger - metabolism; silencing of CREB gene expression by RNAi and Akt1 regulation; Sulfonamides - pharmacology; synthesis of radiolabeled Akt1; transcriptional upregulation of Akt synthesis; Up-Regulation - drug effects; Up-Regulation - genetics
• ISSN: 0730-2312; 1097-4644
• DOI: 10.1002/jcb.21086

Recently, we reported that silencing CREB gene expression by RNAi significantly attenuates forskolin‐induced activation of Akt1. We now provide evidence that forskolin‐treatment causes transcriptional and translational upregulation of Akt1 in macrophages. Akt synthesis was demonstrated by [14C]leucine or [35S] incorporation into newly synthesized Akt1 protein. Akt protein levels increased by ∼1.5‐fold after only a 5 min exposure of macrophages to forskolin. Akt1 levels thereafter rapidly returned to basal values (t1/2 ∼ 15 min). Maximal upregulation of Akt1 occurred in cells treated with 10 µM forskolin. Forskolin‐dependent Akt1 synthesis was abolished by pretreating the cells with CREB‐directed dsRNA as demonstrated at both the message and protein level, as well as by determining the synthesis of [35S]‐labeled Akt1 protein. The PKA inhibitor H‐89, greatly attenuated forskolin‐induced Akt1 synthesis. Transcriptional and translational inhibitors also greatly reduced Akt1 synthesis in forskolin‐stimulated [14C]leucine‐labeled macrophages. Using a chromatin immunoprecipitation assay, we demonstrate that CREB binds to a CRE binding domain of the Akt1 gene promoter. In conclusion, we show here for the first time transcriptional upregulation of Akt1 by CREB, based upon Akt1 protein synthesis and its modulation by transitional and translational inhibitors in forskolin‐stimulated cells, Akt1 protein. and mRNA levels upon silencing CREB gene expression, and binding of CREB to the Akt1 gene promoter. J. Cell. Biochem. 100: 1022–1033, 2007. © 2006 Wiley‐Liss, Inc.