Characterization of the human OATP-C (SLC21A6) gene promoter and regulation of liver-specific OATP genes by hepatocyte nuclear factor 1 alpha.
OATP-C (SLC21A6) is the predominant Na(+)-independent uptake system for bile salts and bilirubin of human liver and is expressed exclusively at the basolateral (sinusoidal) hepatocyte membrane. To investigate the basis of liver-specific expression of OATP-C, we studied promoter function in the two hepatocyte-derived cell lines HepG2 and Huh7 and in nonhepatic HeLa cells. OATP-C promoter constructs containing from 66 to 950 nucleotides of 5'-regulatory sequence were active in HepG2 and Huh7 but not HeLa cells, indicating that determinants of hepatocyte-specific expression reside within the minimal promoter. Deoxyribonuclease I footprint analysis revealed a single region that was protected by HepG2 and Huh7 but not HeLa cell nuclear extracts. The liver-enriched transcription factor hepatocyte nuclear factor 1 alpha (HNF1 alpha) was shown by mobility shift assays to bind within this footprint. Coexpression of HNF1 alpha stimulated OATP-C promoter activity 30-fold in HepG2 and 49-fold in HeLa cells. Mutation of the HNF1 site abolished promoter function, indicating that HNF1 alpha is critical for hepatocyte-specific OATP-C gene expression. The human OATP8 (SLC21A8) and mouse Oatp4 (Slc21a6) promoters were also responsive to HNF1 alpha coexpression in HepG2 cells. These data support a role for HNF1 alpha as a global regulator of liver-specific bile salt and organic anion transporter genes.
Jung
D
D
Division of Clinical Pharmacology and Toxicology, University Hospital, CH-8091 Zurich, Switzerland .
Hagenbuch
B
B
Gresh
L
L
Pontoglio
M
M
Meier
P J
PJ
Kullak-Ublick
G A
GA
D000818:Animals; D027321:Anion Transport Proteins / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002478:Cells, Cultured; D004247:DNA / Q000032:analysis; D004268:DNA-Binding Proteins; D005786:Gene Expression Regulation; D051537:Hepatocyte Nuclear Factor 1; D051538:Hepatocyte Nuclear Factor 1-alpha; D051539:Hepatocyte Nuclear Factor 1-beta; D006801:Humans; D008099:Liver / Q000502:physiology*; D051379:Mice; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009687:Nuclear Proteins; D009928:Organ Specificity; D029362:Organic Anion Transporters, Sodium-Independent / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D000074542:Solute Carrier Organic Anion Transporter Family Member 1B3; D014157:Transcription Factors / Q000502:physiology*; D014158:Transcription, Genetic; D015533:Transcriptional Activation
D027321:Anion Transport Proteins; D004268:DNA-Binding Proteins; C496487:HNF1A protein, human; C496495:HNF1B protein, human; D051538:Hepatocyte Nuclear Factor 1-alpha; C496488:Hnf1a protein, mouse; C496496:Hnf1b protein, mouse; D009687:Nuclear Proteins; D029362:Organic Anion Transporters, Sodium-Independent; C421209:SLCO1B3 protein, human; D000074542:Solute Carrier Organic Anion Transporter Family Member 1B3; D014157:Transcription Factors; D051537:Hepatocyte Nuclear Factor 1; D051539:Hepatocyte Nuclear Factor 1-beta; D004247:DNA