Fig. 11.

IR1b is completely conserved among species, whereas IR1a is primarily responsible for the species difference in transactivation of BSEP by FXRα1 and FXRα2. (A) Sequences of the IR1a and IR1b from seven species including mouse, rat, dog, cat, horse, primate, and human were aligned. The overall conservation percentages of IR1a and IR1b are indicated. (B) The hIR1a in human BSEP promoter reporter phBSEP(-2.6kb) was replaced with mouse/rat m/rIR1a, generating a chimeric reporter pBSEP(-2.6kb)-m/rIR1a. (C) The rIR1a in rat bsep promoter reporter prBSEP(-2kb) was replaced with human hIR1a, resulting a chimeric reporter prBSEP(-2kb)-hIR1a. Human and rat bsep promoter wt and chimeric reporters were cotransfected with FXRα1 or FXRα2 into Huh 7 cells, followed by treatment of transfected cells with DMSO (0.1%) or CDCA (10 μM) for 30 h. Reporter activities were detected by the dual-luciferase reporter assay system. The data are presented as mean ± SD of at least three repeated experiments. (D) Female CD-1 mice were randomly divided into four groups. Each group of mice was hydrodynamically injected with either phBSEP(-2.6kb), prBSEP(-2kb), phBSEP(-2.6kb)-m/rIR1a, or prBSEP(-2kb)-hIR1a promoter reporter. Luciferase activities as luminescent signals were detected by living imaging IVIS 48 h postinjection and captured by a CCD camera with an exposure of 45 s. (E) BSEP mRNA levels in human and mouse liver tissues were quantified by TaqMan real-time PCR. Using GAPDH mRNA levels as internal standards, the relative mRNA expression levels of BSEP in human and mouse liver tissues were compared. Median value of each group is indicated by a short line. Mann-Whitney nonparametric test was used for pair-wise comparison.