Molecular cloning and characterization of the human lactoferrin receptor gene promoter

Abstract

Lactoferrin (Lf) is an iron‐binding protein in human milk. Multiple functions of Lf are postulated to be mediated by a Lf receptor (LfR). We have demonstrated that the Lf receptor (LfR) plays an important role in Lf endocytosis by Caco‐2 cells via a clathrin‐mediated pathway, which focused our interest to investigate the transcriptional regulation of LfR. In the present study, we cloned and characterized the promoter from a 3100 bp 5′‐flanking region of the human LfR gene. The transcription start site was identified as 298 bp upstream of the translation start site (+1) by 5′ RLM‐RACE. The promoter region contains neither a TATA box nor a CCAAT box. A series of deletions of 5′‐flanking sequence were cloned into the promoter‐less luciferase reporter vector and transiently transfected into Caco‐2 cells. The transfection assays demonstrated a sequence of −419/+1 sufficient for eliciting maximal promoter activity and the region −507/−729 exhibited inhibitory effects on reporter gene expression in transiently transfected Caco‐2 cells. The −419/+1 fragment contains a Sp1 binding site, which was confirmed by electrophoretic mobility supershift assay. Mutations of this binding site decreased Sp1 binding affinity in gel shift assays. In conclusion, the isolated LfR gene promoter contains downstream core promoter elements and the Sp1 binding site plays a critical role in transcriptional regulation of the LfR gene.