Involvement of Sp1 in the transcriptional regulation of the rat insulin-like growth factor-1 gene

Most insulin-like growth factor-I (IGF-I) transcripts are initiated in exon 1, but mechanisms of regulation are not well understood. Since potential S pl sites are found in footprinted regions within similar to 360 bp upstream and downstream from the major initiation sites in exon 1, we explored the involvement of Spl and Sp3 in regulation of IGF-I expression. Gel shift ass ays showed strong Spl binding to the downstream site, but binding to the up stream site was weak; Spl bound to a CCTGCCCA sequence in downstream footpr int region V, and Sp3 binding was centered on the same sequence. IGF-I basa l promoter constructs containing a mutation in the downstream Spl site exhi bited a 32% decrease in expression in CHO cells and a 75% decrease in HepG2 cells, indicating the importance of Spl for expression in vivo. Spl and Sp 3 expression vectors provided three- to five-fold stimulation of wild-type IGF-I constructs, but had little effect on a construct containing a mutatio n in the downstream Spl site, and Spl had comparable effects in Drosophila SL2 cells. IGF-I heterologous promoter constructs exhibited similar respons es: in both SL2 cells and CHO cells, stimulation by Spl was enhanced with c onstructs containing downstream region V. Since Spl also stimulated express ion of concatamers of putative cis-acting sites fused to the SV40 promoter- enhancer in pGL3, the results in combination indicate that the presence of IGF-I region V is sufficient to permit stimulation by Spl. Conclusion: Spl and related factors may play an important role in the regulation of IGF-I g ene transcription, through interactions with region V downstream from the m ajor initiation sites in exon 1. (C) 2000 Elsevier Science Ireland Ltd. All rights reserved.