STRUCTURE AND FUNCTION OF THE MOUSE INSULIN-LIKE GROWTH-FACTOR BINDING-PROTEIN-5 GENE PROMOTER

The actions of insulin-like growth factors I and II (IGF-I and -II) ar e modulated by interactions with one or more of a family of secreted I GF binding proteins (IGFBPs). IGFBP-5, the most conserved of the six k nown IGFBPs, is a 252-amino-acid protein that has been shown both to p otentiate and inhibit IGF action. In previous studies, we have cloned and characterized the mouse IGFBP-5 gene and demonstrated that it is e xpressed in a hierarchical pattern in different adult mouse tissues an d during rodent embryonic development. In this report, we describe the initial analysis of the IGFBP-5 gene promoter. By transient gene tran sfer studies, we show the orientation-specific activity of DNA fragmen ts containing from 31 to 4,100 bp from the 5'-flanking region of the m onse IGFBP-5 gene in directing expression of the heterologous reporter gene luciferase in Hep G2 cells. DNA fragments with only 156 bp of 5' -flanking sequence mediated over 60% of maximal promoter activity, and a segment containing the TATA box and the first 120 bp of exon 1 stil l conferred some promoter function. Within the highly active 156-bp re gion, we identified a 37-bp segment from -70 to -34 that exhibited spe cific binding in DNase I footprinting and gel-mobility shift experimen ts with Hep G2 nuclear protein extracts. The footprinted region, which is almost completely conserved in the rat and human IGFBP-5 genes, wa s responsible for at least 70% of the activity of the intact promoter, as evidenced by the deleterious consequences of small internal deleti ons within this sequence on promoter function. Taken together, our res ults demonstrate that the mouse IGFBP-5 gene promotes has a simple str ucture, and defines a framework for examining the mechanisms responsib le for the multifactorial regulation of IGFBP-5 gene transcription dur ing development and in the adult.