1). Induction of

Shp and Fgf15 by Fxr Lumacaftor in vivo activation is known to suppress Cyp7a1 and Cyp8b1 gene expression in the liver. However, it is unclear to what degree Shp and Fgf15 contributes to this suppression. Therefore, this study used Shp KO and Shp Tg mice to determine the contribution of Shp in suppressing Cyp7a1 and Cyp8b1 gene expression. In Shp KO mice, activation of Fxr still markedly suppressed Cyp7a1 to approximately 10% of the levels in vehicle-treated mice (Fig. 2A), though the degree was smaller than that in WT mice (99% suppression). In Shp Tg mice with Shp overexpressed in hepatocytes, basal Cyp7a1 mRNA levels did not change, nor did the degree of Cyp7a1 suppression after Fxr activation (Fig. 2B). Furthermore, in the FXR KO/Shp Tg mice that are deficient in Fxr but overexpressed Shp, levels of Cyp7a1 mRNA were not affected by Shp overexpression either (Fig. 2B). These results indicate that with Fxr activation, Shp may play only a minor role in suppressing Cyp7a1 gene expression. Interestingly, in Shp KO mice, basal Cyp8b1 mRNA levels were 2.5-fold higher than those in WT mice. Fxr activation suppressed Cyp8b1 mRNA levels in Shp KO mice, but the degree of suppression (an approximate 30% decrease) in Shp KO mice was smaller than that in WT mice (50% decrease) (Fig. 2A). Furthermore, in Shp Tg mice,

Cyp8b1 mRNA levels were only slightly reduced and were further decreased upon Fxr activation, but the degree of suppression was similar to that in WT mice (Fig. 2B). Fgf15 has been shown to be important in suppressing Cyp7a1 and Cyp8b1 gene PS-341 ic50 expression. Furthermore, it was reported that the Fgf15-mediated suppression

requires Shp.9-11 Therefore, in the current study, we determined to what degree Fgf15 mediates Terminal deoxynucleotidyl transferase the suppression of Cyp7a1 and Cyp8b1 gene expression after Fxr activation. In addition, by using Shp KO mice, we tested whether the Fgf15-mediated suppression of Cyp7a1 and Cyp8b1 requires Shp. The effect of purified recombinant Fgf15 protein on Cyp7a1 mRNA levels was determined in WT, Fxr WB KO, Shp KO, Shp Tg, and Fxr KO/Shp Tg mice. Compared with vehicle treatment, the Fgf15 protein resulted in a strong reduction of Cyp7a1 mRNA levels in all strains (Fig. 3), with 1%, 10%, 10%, 5%, and 10% of Cyp7a1 mRNA left, respectively, indicating that Fgf15 is downstream of Fxr and Shp. Fgf15 protein also suppressed Cyp8b1 gene expression in Fxr WB KO, Shp KO, and Fxr WB KO/Shp Tg mice; in addition, Fgf15 tended to suppress Cyp8b1 gene expression in WT mice, but not in Shp Tg mice (Fig. 3). Interestingly, Shp mRNA levels were also reduced markedly by Fgf15 treatment, indicating that Fgf15 regulates Shp expression in the liver. Next, by using Fgfr4 KO mice, we determined to what degree the induction of Fgf15 by Fxr activation contributes to the suppression of Cyp7a1 and Cyp8b1 gene expression.