Identification of growth hormone response sequence in rat IGFBP-1 promoter

Abstract

Hepatic transcription of insulin-like growth factor binding protein-1 (IGFBP-1) is enhanced in hypophysectomized (hypox) rat and can be rapidly down regulated by administration of GH. Here we have used DNase-1 protection and transient transfection studies to identify DNA sequences mediating the suppressive effect of GH on the IGFBP-1 promoter. Hepatic nuclear extracts prepared from pituitary intact, hypox and GH-treated hypox rats were analyzed. Three DNase-1 protected regions were identified where there were reproducible differences in the footprint pattern obtained with nuclear extracts from pituitary intact and hypox rats. These regions, F4, F3 and F2, correspond to the nucleotides $-$235 to $-$245, $-$447 to $-$500 and $-$748 to $-$788, respectively. F4 was not responsive to GH whereas the F3 and F2 footprints were acutely responsive to GH, in that a reversion to the normal pattern was apparent within 30 min after administration of GH, at 50 $\mu$g/100g body weight to hypox rats. In each case the response after a single injection of GH was transient with a reversion to the hypox pattern apparent after 1h. When F2 region was removed by deletion of fragment $-$824 to $-$557 bp, the suppressive effect of GH on the IGFBP-1 promoter CAT activity was lost. Since GH induces hepatic expression of c-fos and c-jun, the effect of AP-1 on the IGFBP-1 promoter was also investigated by co-transfection of the IGFBP-1 promoter CAT plasmid with c-fos or c-jun expression vectors. Co-expression of c-fos suppressed IGFBP-1 CAT activity 4 to 6 fold, while c-jun suppressed activity by less then 2 fold. Co-transfection of c-fos and c-jun together suppressed activity 5 to 7 fold. However, the IGFBP-1 promoter 5$\sp\prime$ and internal deletion constructs still retained c-fos suppressive effect. (Abstract shortened by UMI.)