Identification of an Insulin-responsive Element in the Rat Insulin-like Growth Factor-binding Protein-3 Gene

Betty C Villafuerte,Weidong Zhao,Adrian C Herington,Richard Saffery,Lawrence S Phillips

doi:10.1074/jbc.272.8.5024

Abstract

The hepatic expression and serum levels of insulin-like growth factor-binding protein-3 (IGFBP-3) are decreased in insulin-dependent and insulin-resistant diabetes. Insulin increases hepatic IGFBP-3 expression by enhancing gene transcription. This report identifies sequences within the IGFBP-3 promoter that are necessary and sufficient for the response to insulin in hepatic nonparenchymal cells. By transient transfection, we mapped the insulin response element to the -1150 to -1124 base pair (bp) region of the rat IGFBP-3 promoter. Three tandem repeats of the -1150 to -1117 bp region conferred insulin responses in a heterologous promoter. Gel shift analyses revealed a 3-fold increase in DNA-protein complex formation with nuclear extracts obtained from insulin-stimulated nonparenchymal cells compared with cells incubated without insulin and revealed 3-4-fold decrease in complex formation with nuclear extracts obtained from the livers of streptozotocin-diabetic rats compared with control rats. Mutational analysis of this 34-bp region showed a core sequence of 10 bp (-1148 to -1139) that is critical for interaction with insulin-induced trans-acting factors. Southwestern blotting revealed a approximately 90-kDa protein that was increased 2-3-fold by the addition of insulin. Thus, we have identified cis-acting DNA sequences that are responsible for regulation of IGFBP-3 transcription by insulin and essential for binding of insulin-responsive nuclear factors.

Highlights

The hepatic expression and serum levels of insulinlike growth factor-binding protein-3 (IGFBP-3) are decreased in insulin-dependent and insulin-resistant diabetes
Effect of Insulin on Transcription of IGF-binding proteins (IGFBPs)-3 5Ј-Deletion Mutants—To investigate the sequences required for insulin to increase rat IGFBP-3 gene transcription, we developed a series of 5Ј-deletions of the promoter region between nt Ϫ1600 and ϩ34
Insulin increased luciferase activity 58 Ϯ 2% with plasmids containing the Ϫ99/ ϩ34 nt, presumably representing effects on the basal promoter. These results indicate that the deletion of bases Ϫ1201 to Ϫ1061 largely eliminated the ability of insulin to stimulate IGFBP-3 expression in liver cells, suggesting that an insulinresponsive element is located within this 140-bp region

Summary

EXPERIMENTAL PROCEDURES

Materials—The reagents listed were obtained from the following sources: collagenase type 1 from Worthington; Pronase-E from Calbiochem; type 1 rat tail collagen, human transferrin, fetal bovine serum, and Williams’ E medium from Sigma; Lipofectin reagent, human recombinant insulin, medium 199, and deoxyribonuclease 1 (DNase I) from Life Technologies, Inc.; [␥-32P]ATP from Amersham Corp. For Southwestern blotting, 20 ␮g/lane of nuclear extracts from cultured cells treated or untreated with 10Ϫ6 M insulin and from pooled liver extracts from normal and diabetic rats were subjected to SDSpolyacrylamide gel electrophoresis, blotted to nitrocellulose, and denatured by incubating in a solution containing 6 M guanidine HCl, 25 mM Hepes, pH 7.6, 12.5 mM MgCl2, 20% glycerol, 0.1% Nonidet P-40, 0.1 M KCl, and 1 mM dithiothreitol This was followed by gradual renaturation using decreasing concentrations of guanidine HCl and incubation of the filter with 32P-labeled oligonucleotide probe (Ϫ1150 to Ϫ1117 bp region of rat IGFBP-3), washing, and autoradiography.

RESULTS

TABLE II Sequences of the sense strand of oligonucleotides used

DISCUSSION