Two farnesoid X receptor alpha isoforms in Japanese medaka (Oryzias latipes) are differentially activated in vitro

The nuclear receptor farnesoid X receptor alpha (FXR alpha, NR1H4) is activated by bile acids in multiple species including mouse, rat, and human and in this study we have identified two isoforms of Fxr alpha in Japanese medaka (Oryzias latipes), a small freshwater teleost. Both isoforms share a high amino acid sequence identity to mammalian FXR alpha (similar to 70% in the ligand-binding domain). Fxr alpha 1 and Fxr alpha 2 differ within the AF1 domain due to alternative splicing at the fourth intron-exon boundary. This process results in Fxr alpha 1 having an extended N-terminus compared to Fxr alpha 2. A Gal4DBD-Fxr alpha LBD fusion construct was activated by chenodeoxycholic, cholic, deoxycholic and lithocholic acids, and the synthetic agonist GW4064 in transient transactivation assays. Activation of the Gal4DBD-Fxr alpha aLBD fusion construct was enhanced by addition of PGC-1 alpha, as demonstrated through titration assays. Surprisingly, when the full-length versions of the two Fxr alpha isoforms were compared in transient transfection assays, Fxr alpha 2 was activated by C-24 bile acids and GW4064, while Fxr alpha 1 was not significantly activated by any of the compounds tested. Since the only significant difference between the full-length constructs was sequence in the AP1 domain, these experiments highlight a key functional region in the Fxr alpha AF1 domain. Furthermore, mammalian two-hybrid studies demonstrated the ability of Fxr alpha 2, but not Fxr alpha 1 to interact with PGC-1 alpha and SRC-1, and supported our results from the transient transfection reporter gene activation assays. These data demonstrate that both mammalian and teleost FXR (Fxr alpha 2 isoform) are activated by primary and secondary bile acids. (C) 2010 Elsevier B.V. All rights reserved.