Synergistic action of upstream elements and a promoter-proximal CRE is required for neuroendocrine cell-specific expression and second-messenger regulation of the gene encoding the human secretory protein secretogranin II.
Secretogranin II (SgII) is a secretory polypeptide stored in large dense core vesicles of neuroendocrine and neuronal cells. In order to characterize the molecular mechanisms underlying the tissue-specific expression of the SgII gene and its regulation by second-messenger pathways in endocrine and neuronal cells, we have cloned and characterized the human SgII gene. Sequence analysis revealed the existence of numerous putative cis-regulatory elements in the SgII gene promoter, including a consensus cyclic AMP-responsive element (CRE). Constructs containing different portions of the human SgII promoter fused to the luciferase reporter were transfected in AtT-20, SH-SY5Y, LLC-PK1 or COS-7 cells. Northern blot analysis showed that the endogenous SgII gene is more highly expressed in AtT-20 cells than in SH-SY5Y cells, and not expressed at all in LLC-PK1 cells. Treatment by forskolin or 12-O-tetradecanoylphorbol-13-acetate (TPA) caused a 1.5- and 10-fold increase, respectively, in SgII mRNA levels in SH-SY5Y cells but not in AtT-20 cells. Transfection experiments revealed that 4 kb of the human SgII promoter is sufficient to impart cell-specific expression of the reporter gene in the four cell lines studied. Specifically, in AtT-20 cells, a positive element located between -1.38 and -4 kb, in addition to the CRE, is responsible for the high expression of the SgII gene. In SH-SY5Y cells, a negative element located between -0.66 and -1.4 kb represses the activating effect of the CRE leading to an overall lower activity of fusion genes in these cells compared to the activity in AtT-20 cells. Finally, the promoter activity was very low in LLC-PK1 and COS-7 cells. Forskolin and TPA stimulated the activity of a SgII-luciferase fusion gene in SH-SY5Y but not in AtT-20 cells. Disruption of the CRE abolished the stimulatory effect of forskolin and TPA. These data suggest that the basal activity of the human SgII gene relies on cell-specific trans-acting factors in addition to factors that bind to the CRE and show that the regulation of this gene by second messengers is cell-specific and requires an intact CRE.
Desmoucelles
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European Institute for Peptide Research (IFRMP 23), INSERM U413, UA CNRS, University of Rouen, Mont-Saint-Aignan, France.
Vaudry
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Eiden
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Anouar
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Y
D001483:Base Sequence; D002460:Cell Line; D002864:Chromogranins; D003001:Cloning, Molecular; D005576:Colforsin / Q000494:pharmacology; D000242:Cyclic AMP / Q000235:genetics*; D005786:Gene Expression Regulation; D005796:Genes / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009490:Neurosecretory Systems / Q000737:chemistry* / Q000166:cytology; D011401:Promoter Regions, Genetic / Q000187:drug effects; D011506:Proteins / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis / Q000096:biosynthesis / Q000187:drug effects; D020218:Response Elements; D015290:Second Messenger Systems / Q000235:genetics*; D017422:Sequence Analysis, DNA; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects
D002864:Chromogranins; D011506:Proteins; D012333:RNA, Messenger; D005576:Colforsin; D000242:Cyclic AMP; D013755:Tetradecanoylphorbol Acetate