Characterization of the human peptide transporter PEPT1 promoter: Sp1 functions as a basal transcriptional regulator of human PEPT1

Abstract

H+-coupled peptide transporter 1 (PEPT1, SLC15A1) localized at the brush-border membranes of intestinal epithelial cells plays an important role in the intestinal absorption of small peptides and a variety of peptidemimetic drugs. PEPT1 is regulated by various factors, including hormones, dietary conditions, some pharmaceutics, and diurnal rhythm. But there is little information about the transcriptional regulation of PEPT1. In the present study, therefore, we cloned the human (h)PEPT1 promoter region and examined its promoter activity using a human intestinal cell line, Caco-2. Deletion analysis of the hPEPT1 promoter suggested that the region spanning -172 to -35 bp was essential for basal transcriptional activity. This region lacked a TATA-box but contained some GC-rich sites that supposedly bind with the transcription factor Sp1. Mutational analysis revealed that three of these putative Sp1 sites contributed to the transcriptional activity. EMSA showed that Sp1 bound to two GC-rich sites. Furthermore, inhibition of Sp1 binding by mithramycin A treatment significantly reduced the transcriptional activity. Finally, overexpression of Sp1 increased the transcriptional activity in a dose-dependent manner. This study reports the first characterization of the hPEPT1 promoter and shows the significant role of Sp1 in the basal transcriptional regulation of hPEPT1.

Leptin Transcriptionally Enhances Peptide Transporter (hPepT1) Expression and Activity via the cAMP-response Element-binding Protein and Cdx2 Transcription Factors

“…For example, CREB-binding protein CBP/p300 acts as a cofactor for transcriptional activation by CREB (43). The Cdx proteins are involved in the regulation of intestine-specific genes, such as sucrase-isomaltase, intestinal phospholipase A/lysophospholipase, lactase-phlorizin hydrolase, p21, claudin-2, and the iron transport protein hephaestin (2,7,26,32,43). Cdx2 itself appears to play critical roles in gut differentiation, proliferation, and neoplasia (44), and it was recently demonstrated that the gastric mucosa of transgenic Cdx2-overexpressing mice are morphologically changed to intestinal metaplastic mucosa, including microvilli, and PepT1 expression (44).…”

“…Furthermore, we previously demonstrated that short term leptin treatment increased hPepT1 activity in vivo and in vitro (26). The 5Ј-flanking region of the hPepT1 gene has been cloned, and investigations into the regulation of its transcription have indicated that the Sp1 transcription factor functions in basal transcriptional regulation of hPepT1 (32). However, no previous study has investigated transcriptional regulation of hPepT1 in response to hormones, particularly during intestinal inflammation.…”

Long-Term Effect of Leptin on H+-Coupled Peptide Cotransporter 1 Activity and Expression in Vivo: Evidence in Leptin-Deficient Mice

“…These transcription factors then translocate into the nucleus and activate the transcription of a number of target genes (Morton et al, 1998), among which the PepT1 gene could be a candidate. The promoter regions of the rat, mouse and human PepT1 gene (Shiraga et al, 1999;Fei et al, 2000;Shimakura et al, 2005) have been characterized, and they contain a number of potential binding sites for regulatory factors of gene transcription that can be targeted by leptin signaling pathways. Likewise, Nduati et al (2007) showed that cAMP response element-binding protein (CREB) and caudal-related homeobox 2 (Cdx2) are involved in PepT1 regulation by leptin in vitro.…”

Human Organic Anion Transporter 3 Gene Is Regulated Constitutively and Inducibly via a cAMP-Response Element

“…Nuclear extract was prepared from OK cells according to the method of Shimakura et al (2005). The double-stranded oligonucleotides used in the EMSA are listed in Table 1.…”