Regulation of Hepatic Uptake and Efflux Transporters in vitro

The nuclear receptors, pregnane X receptor, PXR (Nr1i2), and farnesoid X receptor, FXR (Nr1h4), have been implicated in the regulation of hepatic uptake transporters: the multispecific organic anion transporter, Oatp1a4 (Slco1a4), the sodium-dependent taurocholate co-transporting polypeptide, Ntcp (Slc10a1), and efflux transporters: multidrug resistance-associated protein 2, Mrp2 (Abcc2), the bile salt export pump, Bsep (Abcb11)¹. We have previously shown differential expression of Oatps in response to PXR ligands, with a correlation between Oatp1a4 mRNA, protein and enzyme activity ². The aim of this study was to establish the relative roles of PXR and FXR in the regulation of these transporters using ligands specific for these nuclear receptors. Hepatocytes isolated from male Sprague Dawley rats and cultured in a sandwich culture system for 48h were dosed with the PXR ligands: dexamethasone (DEX) (10µM), spironolactone (SPR) (10µM) and pregnenolone 16a-carboninitrile (PCN) (10µM) and the FXR ligands: chenodeoxycholic acid (CDCA) (10µM), GW4064 (GW) (10µM) and 6-ethyl chenodeoxycholic acid (6-ECDCA) (10µM). After 96h the cells were harvested, lysed and mRNA levels quantified using quantitative Taqman® real-time PCR and expressed as copy number/ng total RNA. The PXR ligands caused 4-6 fold induction of PXR (Control=162) without showing any effect on FXR (control=174) expression, whilst FXR ligands resulted in 5-7 fold induction of FXR along with up regulation of PXR expression, suggesting a potential role of FXR in the regulation of PXR. The PXR ligands induced the expression of Oatp1a4 (Control=210), Mrp2 (Control=175) and Bsep (Control=316) 5-8 fold and resulted in 2-3 fold up regulation of Ntcp (Control=118), whereas the FXR ligands induced Mrp2 and Bsep expression 7-9 fold and down-regulated Oatp1a4 and Ntcp expression. We concluded from this study that the expression of PXR and FXR is increased at the transcriptional level in vitro and FXR appears to regulate PXR expression. PXR up regulates both uptake and efflux transporters, whereas FXR up regulates efflux transporters and down regulates the uptake transporters. These results correlate with our previously reported in vivo findings and have implications for both drug-drug interactions (Oatp1a4 and Mrp2) and drug-induced cholestasis (Ntcp and Bsep).
References:
1 Cui, Y.J., Aleksunes, L., Tanaka, Y., Goedken, M.J., Klaassen, C.D. 2009 Toxicology 110(1), 47–60
2 Cowie, D.E., Weaver, R.J., Hawksworth, G.M. 2006 Toxicology 226(1), 42-43