Volume 124, Issue 7, Pages 1926-1940 (June 2003) FXR induces the UGT2B4 enzyme in hepatocytes: a potential mechanism of negative feedback control of FXR activity  Olivier Barbier, Ines Pineda Torra, Audrey Sirvent, Thierry Claudel, Christophe Blanquart, Daniel Duran-Sandoval, Folkert Kuipers, Vladimir Kosykh, Jean-Charles Fruchart, Bart Staels  Gastroenterology  Volume 124, Issue 7, Pages 1926-1940 (June 2003) DOI: 10.1016/S0016-5085(03)00388-3

Figure 1 Bile acids increase UGT2B4 mRNA levels in primary human hepatocytes. Primary human hepatocytes were treated for 24 hours with ethanol (vehicle) or chenodeoxycholic acid (CDCA; 30 μmol/L). (A ) Northern blot analysis of human UGT2B mRNA (36B4 RNA was measured as control). (B) Semiquantitative RT-PCR analyses of the human UGT2B isoforms (28S RNA was measured as control). C, RT-PCR negative control. (C ) Real-time RT-PCR analysis of UGT2B4 RNA. Values are expressed as means ± SD (n = 6) relative to the control set as 1. Statistically significant differences between vehicle- and CDCA-treated cells are indicated by asterisks (Mann-Whitney test: ∗∗∗P < 0.001). Gastroenterology 2003 124, 1926-1940DOI: (10.1016/S0016-5085(03)00388-3)

Figure 2 Bile acid and synthetic FXR agonists induce UGT2B4 gene expression in human hepatoblastoma HepG2 cells in a time- and dose-dependent manner via a direct transcriptional mechanism. (A ) HepG2 cells were incubated with increasing concentrations of CDCA (10, 25, and 50 μmol/L) or vehicle (ethanol) for 24 hours. (B) HepG2 cells were incubated with CDCA (50 μmol/L) or vehicle (ethanol) for 6, 12, 24, and 36 hours. (C ) HepG2 cells were incubated for 24 hours with ethanol (vehicle) or CDCA (50 μmol/L) in the absence and presence of cycloheximide (CHX; 20 μg/mL) or actinomycin D (Act. D; 1 μg/mL). (D) Cells were incubated for 24 hours with vehicle (ethanol), CDCA (50 μmol/L), or GW4064 (5 μmol/L). RNA levels were measured by real-time (bars) or semiquantitative (ethidium bromide-stained gels) RT-PCR. Values are means ± SD (n = 6). Statistically significant differences between vehicle- and FXR agonist-treated cells are indicated by asterisks (Mann-Whitney test: ∗P < 0.05; ∗∗∗P < 0.001; ns, not significant). Gastroenterology 2003 124, 1926-1940DOI: (10.1016/S0016-5085(03)00388-3)

Figure 3 Bile acids increase UGT2B protein levels and activity in HepG2 cells. (A ) HepG2 cells were incubated with vehicle (ethanol) or CDCA (50 μmol/L) for 36 hours. Microsomal proteins were subsequently purified and immunoblotted with an anti-UGT2B antibody (upper panel). Equal loading of protein in each lane was assessed by Ponceau S staining (lower panel). (B) HepG2 cells were incubated for 36 hours with CDCA (50 μmol/L) or vehicle (ethanol). Cell homogenates (100 μg) were incubated with [14C ]UDPGA (15 μmol/L), unlabeled UDPGA (485 μmol/L), and hyodeoxycholic acid (HDCA; 200 μmol/L) for 8 hours at 37°C. Radiolabeled HDCA/glucuronide was subsequently analyzed by thin-layer chromatography and quantified by PhosphorImager analysis. Values represent means ± SD (n = 3). Gastroenterology 2003 124, 1926-1940DOI: (10.1016/S0016-5085(03)00388-3)

Figure 4 Identification of a bile acid response element (B4-BARE) in the human UGT2B4 promoter. (A ) HepG2 cells were transfected with the indicated human UGT2B4 promoter-driven luciferase (Luc) reporter plasmids (100 ng) in the absence or presence of pcDNA3-hFXR (30 ng) and a cytomegalovirus-driven β-galactosidase expression plasmid (pCMV-β-gal; 50 ng). Cells were subsequently treated with CDCA (50 μmol/L) or vehicle for 24 hours. (B) HepG2 cells were transfected with the indicated plasmids (100 ng) containing 3 copies of the wild-type or mutated B4-BARE upstream of the thymidine kinase (TK) minimal promoter-driven luciferase reporter (TKpGL3) and pCMV-β-gal (50 ng) and in the absence or presence of hFXR (30 ng). Cells were subsequently treated with CDCA (50 μmol/L) or vehicle for 24 hours. Values are expressed as -fold induction of controls (pGL3 or TKpGL3) set at 1, normalized to internal β-galactosidase activity as described in Materials and Methods. Values represent the means ± SD. Gastroenterology 2003 124, 1926-1940DOI: (10.1016/S0016-5085(03)00388-3)

Figure 5 FXR binds the B4-BARE as a monomer. (A ) Electromobility shift assays (EMSA) were performed with end-labeled consensus IR-1 or B4-BAREwt oligonucleotides in the presence of RXR, FXR, both RXR and FXR, or unprogrammed reticulocyte lysate, as indicated. Supershift experiments were performed with anti-FXR antibody (0.2 μg). (B) EMSA assays were performed on radiolabeled B4-BAREwt and B4-BAREmt oligonucleotides by using in vitro transcribed/translated RXR (lanes 2 and 6), FXR (lanes 3 and 7 ), both RXR and FXR (lanes 4 and 8 ), or unprogrammed reticulocyte lysate (lanes 1 and 5 ). (C ) Competition EMSAs on B4-BAREwt as radiolabeled probe were performed by adding 1-, 10-, 50-, or 100-fold molar excess of the indicated cold IR-1, B4-BAREwt, or B4-BAREmt oligonucleotides in EMSA and FXR, RXR, FXR and RXR, or unprogrammed reticulocyte lysate. (D) Soluble chromatin was prepared from HepG2 cells treated with vehicle (ethanol) or CDCA (50 μmol/L) for 6 hours and immunoprecipitated with antibodies directed against FXR and RXR or with anti-UGT2B antibody as a negative control. The final DNA extractions were amplified by using pairs of primers covering the B4-BARE, a distal region of the UGT2B4 gene promoter, or the β-actin gene as negative control. As a positive control for FXR/RXR binding, a 235-bp DNA fragment encompassing the IR-1 of the SHP gene promoter was amplified. (E ) EMSAs were performed on end-labeled B4-BAREwt oligonucleotides by using in vitro transcribed/translated FXR (lane 1 ), both RXR and FXR (lanes 2 and 3 ), or 2.5 μg of nuclear extract from immortalized human hepatocytes (IHH-NE) (lanes 4 and 5 ). Supershift analyses were performed by adding the anti-FXR antibody (lanes 3 and 5 ). Gastroenterology 2003 124, 1926-1940DOI: (10.1016/S0016-5085(03)00388-3)

Figure 5 FXR binds the B4-BARE as a monomer. (A ) Electromobility shift assays (EMSA) were performed with end-labeled consensus IR-1 or B4-BAREwt oligonucleotides in the presence of RXR, FXR, both RXR and FXR, or unprogrammed reticulocyte lysate, as indicated. Supershift experiments were performed with anti-FXR antibody (0.2 μg). (B) EMSA assays were performed on radiolabeled B4-BAREwt and B4-BAREmt oligonucleotides by using in vitro transcribed/translated RXR (lanes 2 and 6), FXR (lanes 3 and 7 ), both RXR and FXR (lanes 4 and 8 ), or unprogrammed reticulocyte lysate (lanes 1 and 5 ). (C ) Competition EMSAs on B4-BAREwt as radiolabeled probe were performed by adding 1-, 10-, 50-, or 100-fold molar excess of the indicated cold IR-1, B4-BAREwt, or B4-BAREmt oligonucleotides in EMSA and FXR, RXR, FXR and RXR, or unprogrammed reticulocyte lysate. (D) Soluble chromatin was prepared from HepG2 cells treated with vehicle (ethanol) or CDCA (50 μmol/L) for 6 hours and immunoprecipitated with antibodies directed against FXR and RXR or with anti-UGT2B antibody as a negative control. The final DNA extractions were amplified by using pairs of primers covering the B4-BARE, a distal region of the UGT2B4 gene promoter, or the β-actin gene as negative control. As a positive control for FXR/RXR binding, a 235-bp DNA fragment encompassing the IR-1 of the SHP gene promoter was amplified. (E ) EMSAs were performed on end-labeled B4-BAREwt oligonucleotides by using in vitro transcribed/translated FXR (lane 1 ), both RXR and FXR (lanes 2 and 3 ), or 2.5 μg of nuclear extract from immortalized human hepatocytes (IHH-NE) (lanes 4 and 5 ). Supershift analyses were performed by adding the anti-FXR antibody (lanes 3 and 5 ). Gastroenterology 2003 124, 1926-1940DOI: (10.1016/S0016-5085(03)00388-3)

Figure 6 RXR activation reduces the bile acid-induced up-regulation of UGT2B4 in HepG2 cells. (A ) EMSAs were performed with the radiolabeled B4-BAREwt oligonucleotide in the presence of RXR (lane 2 ), FXR (lane 3 ), FXR and RXR (lane 4 ), FXRLeu433Arg (lane 5 ), FXRLeu433Arg and RXR (lane 6 ), or unprogrammed reticulocyte lysate (lane 1 ). (B) Chromatin immunoprecipitation experiments on DNA from HepG2 cells treated with CDCA (50 μmol/L), CD4064 (2.5 μmol/L), both CDCA and CD4064, or vehicle (ethanol) for 6 hours. Soluble chromatin was prepared as described in Materials and Methods and immunoprecipitated with anti-FXR, anti-RXR, or anti-UGT2B antibodies. The extracted DNA was amplified with pairs of primers that cover B4-BARE, a distal region of the UGT2B4 gene promoter, or the β-actin gene as a negative control. As a positive control, a 235-bp DNA fragment encompassing the IR-1 of the SHP gene promoter was amplified. (C ) HepG2 cells were transfected with the TK-pGL3 reporter plasmid or the TK-pGL3 plasmid containing 3 copies of the B4-BARE and pCMV-β-gal in the absence or presence of FXR, FXRLeu433Arg, and/or RXR. Cells were subsequently treated with CDCA (50 μmol/L) or vehicle (ethanol) for 24 hours. (D and E ) HepG2 cells were treated with vehicle (ethanol), CDCA (50 μmol/L), CD3640 (2.5 μmol/L), or both CDCA and CD3640 for 24 hours. Total RNA was extracted, and UGT2B4 (D) and SHP (E ) mRNA levels were measured by real-time PCR. Values are means ± SD (n = 4). Values followed by different letters are statistically significantly different (analysis of variance followed by the Mann-Whitney test; P < 0.05). Gastroenterology 2003 124, 1926-1940DOI: (10.1016/S0016-5085(03)00388-3)

Figure 7 UGT2B4 overexpression in HepG2 cells interferes negatively with the bile acid induction of SHP gene expression. Stable overexpressing UGT2B4- and UGT1A6-HepG2 cells were obtained as described in Materials and Methods. Cells were treated with vehicle (ethanol) or CDCA (50 μmol/L) for 24 hours. Levels of SHP, UGT2B4, and UGT1A6 mRNAs were analyzed by semiquantitative RT-PR (A ) and quantified with real-time RT-PCR (B). Statistically significant differences of SHP mRNA levels between vehicle- and FXR agonist-treated cells are indicated by asterisks (Mann-Whitney test; ∗∗∗P < 0.001; ns, not significant). Gastroenterology 2003 124, 1926-1940DOI: (10.1016/S0016-5085(03)00388-3)

Figure 8 CDCA-induced UGT2B4 expression is delayed compared with SHP in HepG2 cells. HepG2 cells were incubated with CDCA or ethanol for 6, 12, and 24 hours. RNA levels were measured by real-time RT-PCR. Values are means ± SD (n = 4). Statistically significant differences between UGT2B4 and SHP inductions are indicated by asterisks (Mann-Whitney test; ∗∗P < 0.005; ∗∗∗P < 0.001). Gastroenterology 2003 124, 1926-1940DOI: (10.1016/S0016-5085(03)00388-3)