Insulin-like growth factor I increases rat peptide YY promoter activity through Sp1 binding sites

Endocrinology. 2004 Feb;145(2):659-66. doi: 10.1210/en.2003-0770. Epub 2003 Oct 30.

Abstract

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.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Base Sequence
  • Binding Sites
  • DNA / metabolism
  • Gene Expression
  • Gene Expression Regulation / drug effects*
  • Genes, Reporter / genetics
  • Insulin-Like Growth Factor I / pharmacology*
  • Kinetics
  • Luciferases / genetics
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • PC12 Cells
  • Peptide YY / genetics*
  • Promoter Regions, Genetic / genetics*
  • Rats
  • Recombinant Fusion Proteins
  • Sp1 Transcription Factor / genetics
  • Sp1 Transcription Factor / metabolism*
  • Transfection

Substances

  • Recombinant Fusion Proteins
  • Sp1 Transcription Factor
  • Peptide YY
  • Insulin-Like Growth Factor I
  • DNA
  • Luciferases