Transcriptional activation of the human Galphai2 gene promoter through nuclear factor-kappaB and antioxidant response elements.
Very little is known regarding molecular mechanism(s) underlying transcriptional regulation of any G-protein gene despite the importance of G-protein expression in modulating cellular processes. Here we show that phorbol myristate acetate (PMA) and tert-butylhydroquinone (tBHQ), which induce oxidative stress in cells, up-regulate transcription of Galpha(i2) in K562 cells. Redox-sensing chemicals abrogated this transcriptional effect. A dominant negative I-kappaB double mutant (S32A/S36A) suppressed PMA-induced transcription by 54-62%, suggesting involvement of nuclear factor-kappaB (NF-kappaB). SN50, a cell-permeable peptide that inhibits nuclear import of stress-responsive transcription factors (such as NF-kappaB), inhibited PMA- and tBHQ-induced transcription. Deletion of an NF-kappaB-binding motif that maps at +10/+19 in the promoter resulted in 55-60% suppression of PMA-induced transcription, and 81% suppression of tBHQ-induced transcription. Mutation of an antioxidant response element (ARE) that maps at -84/-76 in the promoter resulted in 51 and 86% decrease in PMA- and tBHQ-induced transcription, respectively. In electrophoretic mobility shift assays, this element formed complexes with the transcription factors NF-E2p45 and Nrf2 that are prototypic for binding to the ARE, as well as with c-Fos, which can also interact with the ARE. Chromatin immunoprecipitation analysis demonstrated recruitment of these transcription factors to the promoter. Exogenously transfected Nrf2 transactivated the Galpha(i2) gene promoter; the cytoskeleton-associated protein, Keap1, abrogated this effect. Taken together, the present studies reveal that transcription factors that bind NF-kappaB and/or antioxidant response elements play an activating role in the transcription of the human Galpha(i2) gene.
Arinze
Ifeanyi J
IJ
Department of Biochemistry, Meharry Medical College, 1005 David B. Todd Jr., Blvd, Nashville, Tennessee 37208-3599, USA. iarinze@mmc.edu
Kawai
Yumiko
Y
D020816:Amino Acid Motifs; D000975:Antioxidants / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D015153:Blotting, Western; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D047369:Chromatin Immunoprecipitation; D004268:DNA-Binding Proteins / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D051960:GTP-Binding Protein alpha Subunit, Gi2; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000235:genetics / Q000378:metabolism*; D017353:Gene Deletion; D006801:Humans; D006873:Hydroquinones / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins; D020014:K562 Cells; D000072019:Kelch-Like ECH-Associated Protein 1; D020738:Leptin / Q000378:metabolism; D008957:Models, Genetic; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D051267:NF-E2-Related Factor 2; D016328:NF-kappa B / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D011506:Proteins / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D016760:Proto-Oncogene Proteins c-fos / Q000378:metabolism; D020218:Response Elements; D013755:Tetradecanoylphorbol Acetate; D013997:Time Factors; D015534:Trans-Activators / Q000378:metabolism; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection; D015854:Up-Regulation
D000975:Antioxidants; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D006873:Hydroquinones; D047908:Intracellular Signaling Peptides and Proteins; C431364:KEAP1 protein, human; D000072019:Kelch-Like ECH-Associated Protein 1; D020738:Leptin; D051267:NF-E2-Related Factor 2; D016328:NF-kappa B; C495635:NFE2L2 protein, human; D010455:Peptides; D011506:Proteins; D011518:Proto-Oncogene Proteins; D016760:Proto-Oncogene Proteins c-fos; D015534:Trans-Activators; C018855:2-tert-butylhydroquinone; C498015:GNAI2 protein, human; D051960:GTP-Binding Protein alpha Subunit, Gi2; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D013755:Tetradecanoylphorbol Acetate