Figure 3. FXR-dependent increase in fetuin-B expression is mediated through the P2 promoter.

(A) Three human fetuin-B transcripts, which originate from the P1 promoter, have previously been described by Hsu et al. [28]. Fetuin-B P1α contains exons 1–7. Fetuin-B P1β is missing exon 2. Fetuin-B P1γ is missing exons 2 and 3. Fetuin-B P2 α, β and γ isoforms arise from the alternative P2 promoter. Fetuin-B P2α contains the untranslated exon P2, the translated part (black box) of exon 1 and exons 2–7. Fetuin-B P2β is missing exon 3. Fetuin-B P2γ is missing exons 1–3. Solid boxes and empty boxes indicate translated and untranslated regions respectively. Arrows indicate P1 and P2 promoters. Arrowheads indicate PCR primers. (B) The differential splicing pattern of the fetuin-B gene was validated by PCR analysis with P1- or P2-specific primers in human primary hepatocytes. PCR products were analysed by gel electrophoresis on 1.5% agarose with TBE and verified by sequencing. Selective, promoter-dependent fetuin-B regulation by FXR in both HepG2 cells (C) and in human primary hepatocytes (D). Relative expression level of human fetuin-B transcripts, which originate either at the P1 or at the P2 promoter, was measured by real-time QPCR and normalized to 28S mRNA. Results are expressed as the fold change compared with DMSO in each cell type; results are means±S.E.M. (n=3).