FXR agonists up-regulate OSTα and OSTβ mRNA and protein in HepG2 cells. (A) mRNA was isolated from HepG2 cells treated with CDCA for the indicated time periods. Quantitative RT-PCR indicates that treatment with 50 μM CDCA up-regulates mRNA for OSTα (closed bars) and OSTβ (open bars) in a time dependent manner, peaking between 12 and 24 hrs. (B) Lysates from HepG2 cells treated with CDCA for the indicated time periods were combined with Laemmli sample buffer and subjected to SDS-PAGE. Protein expression for both subunits is detectable between 12 and 24 hrs and increases significantly at 48 hrs as demonstrated by Western blotting using the specific polyclonal antibodies described in Methods. (C) Immunofluorescence on cells treated for 48 hrs with vehicle alone, 50 μM CDCA or 2 μM 6-ethyl CDCA demonstrates that both the up-regulated OSTα and OSTβ subunits are correctly expressed on the plasma membrane. (D) Transport studies in HepG2 cells using 3H-taurocholate (1 μM) and 3H-estrone 3-sulfate (50 nM) indicate that treatment with 50 μM CDCA up-regulates functional activity 3–4 fold over non-treated cells.
Soroka et al. BMC Cell Biology 2008 9:57 doi:10.1186/1471-2121-9-57