Isolation and characterization of the zebrafish Danio rerio insulin-like growth factor binding protein-3 promoter region

Abstract

Insulin-like growth factor binding protein-3 (IGFBP-3) is expressed in many different cell types and has a regulatory function in the insulin-like growth factor (IGF) system. To further understand the molecular mechanism, we cloned and sequenced the 5′-promoter region of zebrafish Danio rerio IGFBP-3 and characterized its activity using firefly luciferase transient transfection expression assays. Different fragments of the zebrafish IGFBP-3 5′-flanking region were transfected into HeLa and ZFL cells. In these cell lines, maximum promoter activity was located within 1218 bp of the zebrafish IGFBP-3 flanking region in the HeLa cell line and within 304 bp of the zebrafish IGFBP-3 flanking region in the ZFL cell line. Several putative transcription factors were revealed in the zebrafish IGFBP-3 promoter region, such as organic cation transporter-1, GATA-1 and yin and yang-1. Further study of the in vivo expression of the IGFBP-3 promoter during development was carried out in transgenic zebrafish expressing an IGFBP-3 promoter driven green fluorescent protein (GFP) encoding the GFP cDNA transgene. The GFP transcripts appeared for the first time in the 32-cell stage. These results indicate that the IGFBP-3 promoter is active in a development-specific manner and suggest that the IGFBP-3 promoter plays an important role in teleost embryo growth. Finally, IGFBP-3 has important IGF-independent effects on cell growth and may involve nuclear localization. By transiently transfecting HeLa cells with various zebrafish IGFBP-3 segments, we identified one nuclear localization signal (NLS): a basic amino acid rich sequence (PSKGRKR) between amino acids 256 and 262 was able to direct enhanced green fluorescence protein predominantly into the nucleus, whereas a deletion of this motif abrogated this nuclear localization property. These data suggest that zebrafish IGFBP-3 contains a NLS around the second NLS sequence, whereas the putative NLS at the first NLS is non-functional.