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 (lue) reporter gene were transfected into rat pheochromocytoma cells (PC12) and lue 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.
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