Addition of the known LXR-activator TO-901317 (10 μM) resulted in LXR-dependent

enhanced luciferase activation of all constructs containing the first part of +53 to at least −128 ABC294640 clinical trial bp of the SLCO1B1 gene (Fig. 3C). However, we did not observe any enhancement of luciferase activity in the fragment containing the potential distal enhancer module (−5450 bp to −4620 bp). Similar results were obtained treating the HepG2 cells with the LXRα agonist GW3965 (Fig. 3C). Because all of the constructs include at least the +53 to −128 bp fragment of the SLCO1B1 gene, we focused on the two possible LXR response elements located in this region (Fig. 6A). Interestingly, mutation of the hexamere DR4 DNA motifs (localized at DR4-1 +22 to +37 and DR4-2 +32 to +47) resulted in the complete loss of agonist-stimulated, LXRα-dependent luciferase reporter activity in HepG2 cells (Fig. 6B) in all fragments. These findings suggest that this part of the SLCO1B1 promoter is transactivated by agonist-bound LXRα. To further confirm the role of the DR4 elements in the inductive regulation of OATP1B1 expression, we performed an LXR-specific chromatin immunoprecipitation assay (Fig. 6C-E). These results demonstrate that agonist-activated LXR

binds to the SLCO1B1 promoter. In accordance with the findings KU-57788 concentration concerning the RXRα–FXR interaction, experiments were conducted using LXRα. As shown in Supporting Fig. 2, there is no additional effect of RXRα on SLCO1B1 promoter response. In order to confirm that our cell line–based findings are reflective of an in vivo situation, we Astemizole performed ex vivo experiments using freshly isolated human hepatocytes. The inductive capacity of the human hepatocyte preparations for LXRα and FXR agonists were confirmed by assessing the effects of such agonists

to bona fide target genes such as BSEP16 (Fig. 7C) and OATP1B317, 18 (Fig. 7D) for FXR and ABCA1 for LXRα19 (Fig. 7B). As shown in Fig. 7A, treatment of freshly isolated human hepatocytes with CDCA and TO-901317 resulted in a significant induction of OATP1B1 expression. Addition of CDCA and TO-091317 to two additional human hepatocytes preparations revealed a moderate induction of OATP1B1 protein expression (Fig. 7E). Functional assessment of OATP1B1 in the same hepatocytes showed significantly greater cellular accumulation of [3H]taurocholate acid in the presence of CDCA or TO-091317, respectively (Fig. 7F). Uptake of CCK-8, a specific substrate of OATP1B3, was increased in hepatocytes treated with the FXR ligand CDCA (CCK-8 uptake percentage of dimethyl sulfoxide control 159.73 ± 19.07), but not TO-091317 (127.13 ± 24.12) (data not shown), suggesting the LXR effect is OATP1B1-specific. OATP1B1 is now emerging as a key transporter for the hepatic uptake of many compounds.1 Although much is currently known regarding the functional relevance of coding region SNPs in this drug transporter, remarkably little is known regarding the mechanisms that mediate its transcriptional regulation.