Submitted on November 27, 2008
Revised on February 17, 2009
Accepted on February 18, 2009
Hepatocyte nuclear factor-4
and bile acids regulate human concentrative nucleoside transporter-1 gene expression
Kerstin Klein1, Gerd A. Kullak-Ublick1, Martin Wagner2, Michael Trauner2, and Jyrki J Eloranta1*
1 University Hospital Zurich
2 Medical University of Graz, Austria
* To whom correspondence should be addressed. E-mail: jyrki.eloranta{at}usz.ch.
The concentrative nucleoside transporter-1 (CNT1) is a member of the solute carrier 28 (SLC28) gene family, and is expressed in the liver, intestine, and kidneys. CNT1 mediates the uptake of naturally occurring pyrimidine nucleosides, but also nucleoside analogues used in anticancer and antiviral therapy. Thus, expression levels of CNT1 may affect the pharmacokinetics of these drugs and the outcome of drug therapy. Because little is known about the transcriptional regulation of human CNT1 gene expression, we have characterized the CNT1 promoter with respect to DNA response elements and their binding factors. The transcriptional start site of the CNT1 gene was determined by 5'-RACE. In silico analysis revealed the existence of three putative binding sites for the nuclear receptor hepatocyte nuclear factor-4
(HNF-4) within the CNT1 promoter. A luciferase reporter gene construct containing the CNT1 promoter region was transactivated by HNF-4 in human cell lines derived from the liver, intestine, and kidneys. Consistent with this, we showed in electromobility shift assays that HNF-4 specifically binds to two conserved direct repeat-1 (DR-1) motifs within the proximal CNT1 promoter. In cotransfection experiments, the transcriptional coactivator peroxisome proliferator-activated receptor-
coactivator-1 (PGC-1) further increased, whereas the bile acid-inducible corepressor small heterodimer partner (SHP) reduced, HNF-4-dependent CNT1 promoter activity. Consistent with the latter phenomenon, CNT1 mRNA expression levels were suppressed in primary human hepatocytes upon bile acid treatment. Supporting the physiological relevance and species-conservation of this effect, ileal Cnt1 mRNA expression was decreased upon bile acid feeding and increased upon bile duct ligation in mice.
