Mechanisms of human hepatobiliary transporter gene regulation in the hepatocyte-like cell line HepaRG

Abstract

Background&Aim: Functional studies on human hepatic gene regulation are limited due to the lack of suitable models. Currently used human hepatoma cell lines such as HepG2 or Huh7 are deficient in several transporters such as NTCP or BSEP whereas primary human hepatocytes and freshly prepared liver slices are limited in availability. The highly differentiated hepatic cell line HepaRG, which exhibits a hepatocyte-like equipment with various CYP P450s, nuclear receptors and a broad range of hepatobiliary transporters, represents an ideal model system to study human transporter gene regulation (LeVee et al. 2006, Aninat et al. 2006). The aim of the present study was to functionally characterize inflammatory and bile salt-induced mechanisms of human transporter gene regulation in the unique HepaRG cell system (kindly provided by Biopredic). Methods: Highly differentiated HepaRG cells maintained in DMSO containing medium were treated with either 100ng/ml LPS or 10ng/ml IL–1s or 10µM taurocholate (exemplary for inflammatory conditions or bile salt overload) for 16 hours. RNA and protein were prepared and analyzed by real-time RT-PCR and Western blotting. Results: Upon LPS-treatment NTCP mRNA expression decreased to 13% whereas OATP-C and BSEP expression showed a moderate reduction of 56% and 39%, respectively, compared to untreated controls. Effects of IL–1s treatment were even more pronounced (NTCP 4%, OATP-C 10% and BSEP 18%). MRP2 is down-regulated by inflammatory signals to 40–60% at the protein level whereas mRNA was 65–77% for IL–1s and LPS stimulation, respectively. MRP4 mRNA is increased around 2-fold while MRP3 expression remained stable under these conditions. Taurocholate treatment increased BSEP mRNA to 258%, slightly enhanced NTCP as well as MRP4 expression while OATP-C, MRP3 and MRP2 were unaffected. Conclusions: Human bile salt transporters NTCP and BSEP are suppressed by similar inflammatory mechanisms as their rat counterparts. Inflammation-induced MRP2 down-regulation occurs by posttranscriptional mechanisms. During bile salt accumulation MRP4 appears to be the primary human rescue system whereas other transporters are rather moderately suppressed or unaffected. Our data allow a comprehensive insight into cholestatic human transporter gene regulation and enable further mechanistic studies in the future.