Insulin-like growth factor I increases rat peptide YY promoter activity through Sp1 binding sites.
Studies in rodents demonstrate that the mitogen, IGF-I, stimulates intestinal peptide YY (PYY) expression. To investigate whether the stimulatory influence of IGF-I is exerted at the level of gene transcription, rat PYY 5'-upstream sequences (-2800/+37 bp, -770/+37 bp, -127/+37 bp) fused to the firefly luciferase (luc) reporter gene were transfected into rat pheochromocytoma cells (PC12) and luc activity measured after IGF-I treatment. IGF-I increased transcriptional activity of all constructs similarly; the PYY (-127/+37 bp)-luc construct was used in subsequent experiments. IGF-I increased PYY (-127/+37 bp)-luc activity in a time- and dose-dependent fashion. Sequence analysis detected five putative Sp1 binding sites in the -127/+37-bp sequence. EMSA and supershift experiments using two oligonucleotide fragments of the -127/+37 region showed that Sp1 and Sp3 proteins bound to putative Sp1 sites. Overexpression of Sp1 greatly increased PYY (-127/+37 bp)-luc activity and site-directed mutagenesis of putative Sp1 binding sites decreased basal and IGF-I-induced elevations in PYY (-127/+37 bp)-luc activity. IGF-I treatment also increased Sp1 protein levels and binding activity. Blockade of the IGF-I receptor (IGF-IR) with an IGF-IR antibody decreased the stimulatory influence of IGF-I on Sp1 protein levels and PYY (-127/+37 bp)-luc activity. Together, these findings indicate that IGF-I functions as a positive regulator of PYY gene expression and that the stimulatory effect may be mediated by Sp1 proteins that bind to the proximal PYY promoter region.
Wang
Guiyun
G
Department of Surgery, The University of Texas Medical Branch, 301 University Boulevard, Galveston, TX 77555-0725, USA.
Leiter
Andrew B
AB
Englander
Ella W
EW
Greeley
George H
GH
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000378:metabolism; D015870:Gene Expression; D005786:Gene Expression Regulation / Q000187:drug effects*; D017930:Genes, Reporter / Q000235:genetics; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology*; D007700:Kinetics; D008156:Luciferases / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016716:PC12 Cells; D019894:Peptide YY / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D051381:Rats; D011993:Recombinant Fusion Proteins; D016329:Sp1 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D014162:Transfection
D011993:Recombinant Fusion Proteins; D016329:Sp1 Transcription Factor; D019894:Peptide YY; D007334:Insulin-Like Growth Factor I; D004247:DNA; D008156:Luciferases