Functional mapping of the promoter region of the GNB2L1 human gene coding for RACK1 scaffold protein.
RACK1 (Receptor for Activated C Kinase 1) is a scaffold protein for different kinases and membrane receptors. Previously, we characterized an age-dependent decline of RACK1 protein expression which could be counteracted with DHEA (dehydroepiandrosterone) [Corsini, E., et al. 2002. In vivo dehydroepiandrosterone restores age-associated defects in the protein kinase C signal transduction pathway and related functional responses. J. Immunol. 168, 1753-1758. and Corsini, E., et al. 2005. Age-related decline in RACK-1 expression in human leukocytes is correlated to plasma levels of dehydroepiandrosterone. J. Leukoc. Biol. 77, 247-256.]. Hypothesizing a direct control of RACK1 expression by DHEA we studied the not yet characterized human promoter region of its coding gene GNB2L1. The FLOE (Fluorescently Labeled Oligonucleotide Extension) was used to map the transcription start site and a novel Gateway luciferase vector (GW luc basic; Del Vecchio, I., Zuccotti, A., Canneva, F., Lenzken, S.C., Racchi, M., 2007. Development of the first Gateway firefly luciferase vector and use of reverse transcriptase in FLOE (Fluorescently Labeled Oligonucleotide Extension) reactions. Plasmid 58, 269-274.) to obtain promoter region mutants. Human SH-SY5Y, THP1 and lymphoblastoid cells were used for transient transfections and treatments with lipopolysaccharide (LPS), phorbol myristate acetate (PMA), DHEA and cortisol (the first two molecules to differently activate NF-kB, a transcription complex able to regulate the murine Gnb2l1 gene expression, whereas DHEA and cortisol since they are known to be imbalanced during the aging and possess counteracting actions on the immune function). The primer extension demonstrated the existence of two alternative start sites of transcription respectively located at about 230 and 300 nt 5' of the Genbank mRNA entry for GNB2L1. Moreover, as a result of the luciferase study we were able to demonstrate that a little region of approximately 300 nt conserved sufficient elements for reporter expression. We also reported that the DHEA modulation of GNB2L1 endogenous expression could not be recapitulated with the luciferase assays. Indeed, the promoter was significantly modulated by means of LPS and PMA treatments but not using DHEA. Differently the use of cortisol led us to demonstrate a biologically significant decrease of luciferase activity only in the presence of a binding site for nuclear receptors of glucocorticoids. Interestingly, other binding sites for transcriptional factors were identified in silico: different c-Rel (NF-kB) and some cardiomyocitic specific cis-acting elements. All this data suggest that the DHEA mediated GNB2L1 regulation is modulated by distant elements (enhancers/silencers), whereas LPS, PMA and cortisol effect can act directly on the mapped GNB2L1 promoter. In conclusion we hypothesize that the imbalance between DHEA and cortisol during aging could be important in the previously demonstrated recovery of the RACK1 expression.
Del Vecchio
Igor
I
University of Pavia, Department of Experimental and Applied Pharmacology, Centre of Excellence in Applied Biology, Viale Taramelli 14, 27100 Pavia, Italy. idv@fastwebnet.it
Zuccotti
Annalisa
A
Pisano
Federica
F
Canneva
Fabio
F
Lenzken
Silvia C
SC
Rousset
Francoise
F
Corsini
Emanuela
E
Govoni
Stefano
S
Racchi
Marco
M
D001483:Base Sequence; D002460:Cell Line; D002874:Chromosome Mapping; D019295:Computational Biology; D017931:DNA Primers / Q000378:metabolism; D003687:Dehydroepiandrosterone / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D006854:Hydrocortisone / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008156:Luciferases / Q000378:metabolism; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000235:genetics* / Q000378:metabolism; D016366:Open Reading Frames / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D055550:Protein Stability / Q000187:drug effects; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface / Q000235:genetics* / Q000378:metabolism; D017384:Sequence Deletion; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D024363:Transcription Initiation Site; D014158:Transcription, Genetic / Q000187:drug effects
D017931:DNA Primers; D005456:Fluorescent Dyes; D008070:Lipopolysaccharides; D009363:Neoplasm Proteins; C419368:RACK1 protein, human; D012333:RNA, Messenger; D000075370:Receptors for Activated C Kinase; D011956:Receptors, Cell Surface; D003687:Dehydroepiandrosterone; D008156:Luciferases; D019204:GTP-Binding Proteins; D013755:Tetradecanoylphorbol Acetate; D006854:Hydrocortisone