Prostasin is a glycosylphosphatidylinositol (GPT)-anchored serine protease and a suppressor of tumor cell invasion. We recently reported that the human prostasin gene is up-regulated by the transcription factor sterol regulatory element-binding protein-2 (SREBP-2). In the present study, we identified multiple SREBP-2 binding sites, known as sterol regulatory elements (SREs), located at positions -897, -538, +8, +71, and +98 (named SRE-897, SRE-538, SRE+8, SRE+71, and SRE+98) in the human prostasin gene promoter. Prostasin promoter-reporter constructs, representing serial deletions of the 5'-flanking region of the human prostasin gene, were transiently transfected into HEK-293 cells for evaluation of promoter activities. The region defined by nucleotides -17 to +232 of the prostasin gene promoter was shown to be essential for the basal transcriptional activity of the human prostasin gene. Mutagenesis of the five SREs was carried out for evaluation of their roles in SREBP-2 up-regulation. SRE+98, a novel functional sterol regulatory element, was found to be the major site for the stimulatory response of prostasin gene expression to SREBP-2. (c) 2006 Elsevier Inc. All rights reserved.