Vitamin D Receptor As an Intestinal Bile Acid Sensor Makoto Makishima, Timothy T. Lu, Wen Xie, G. Kerr Whitfield, Hideharu Domoto, Ronald M. Evans, Mark R. Haussler, and David J. Mangelsdorf

Supplementary Material

Materials and Methods
Luciferase cotransfection assays. Cotransfection assays were performed in HEK293 cells as described (1). In Fig. 1, B and C, cells were cotransfected with a GAL4-responsive luciferase reporter plasmid and cytomegalovirus-based (CMX) expression plasmids for GAL4-SRC-1 and VP16 chimeras containing the full-length mammalian nuclear receptors. Receptors tested were: FXR (farnesoid X receptor, NR1H4), PXR (pregnane X receptor, NR1I2), VDR (vitamin D receptor, NR1I1), LXR (liver X receptor , NR1H3), LXR (liver X receptor , NR1H2), RXR (retinoid X receptor , NR2B1), PPAR (peroxisome proliferator activated receptor , NR1C1), PPAR (peroxisome proliferator activated receptor , NR1C2), PPAR (peroxisome proliferator activated receptor , NR1C3), RAR (retinoic acid receptor , NR1B1), TR (thyroid hormone receptor , NR1A2), ER (estrogen receptor , NR3A2), GR (glucocorticoid receptor, NR3C1), LRH-1 (liver receptor homolog-1, NR5A2) or SHP (short heterodimer partner, NR0B2) (2). Other nuclear receptor tested but not shown include CAR (constitutively active receptor, NR1I3), HNF4 (hepatocyte nuclear receptor 4, NR2A1), and COUP-TFI (chicken ovalbumin upstream promoter transcription factor 1, NR2F1). CMX-VP16 alone was used as control (no receptor). In Fig. 1, D and E, cells were cotransfected with the GAL4-responsive luciferase reporter and expression vectors containing the GAL4 DNA-binding domain fused to the ligand-binding domain of human VDR (residues 90-427) or human FXR (residues 193-472). Transfection data were normalized to an internal -galactosidase control and expressed as fold-induction or relative light units (RLU) from triplicate assays ±: SD. Bile acid (Sigma and Steraloids, Inc.) abbreviations: cholic acid, CA; taurocholic acid, TCA; glycocholic acid, GCA; chenodeoxycholic acid, CDCA; taurochenodeoxycholic acid, TCDCA; glycochenodeoxycholic acid, GCDCA; dehydrocholic acid, DHCA; deoxycholic acid, DCA; taurodeoxycholic acid, TDCA; lithocholic acid, LCA; taurolithocholic acid, TLCA; glycolithocholic acid, GLCA; 5-sulfate-glycolithocholic acid, SGLCA; tauroursodeoxycholic acid, TUDCA; ursodeoxycholic acid, UDCA; glycoursodeoxycholic acid, GUDCA; hyodeoxycholic acid, HDCA; taurohyodeoxycholic acid, THDCA; muricholic acid, MCA.

Competitive ligand binding assay. Ligand binding was performed as described (3) using lysates from COS-7 cells transfected with expression plasmids for VDR or RXR. Binding was performed overnight at 4°C in lysate buffer (3) with 0.71 nM (18 Ci/mmol) [³H]1,25(OH)₂D₃ (Amersham Pharmacia Biotech) and bile acid competitor. Unbound [³H]1,25(OH)₂D₃ was removed by adsorption to dextran-coated charcoal and the supernatant removed for scintillation counting. K_i values were calculated from a computer fit of competition curves from triplicate assays ±: SD.

---

Supplemental Figure 1. Fluorescence polarization assay of bile acids binding to VDR (5).

Medium version | Full size version

---

Supporting references and notes
1. M. Makishima et al., Science 284, 1362 (1999).

2. J. Auwerx et al., Cell 97, 161 (1999).

3. S. Nakajima et al., J. Biol. Chem. 271, 5143 (1996).

4. J. R. Schultz et al., Genes Dev. 14, 2831 (2000).

5. Fluorescence polarization assay. A fluorescein-labeled peptide (1 nM), which consists of LLRYLLDK (residues 748-755 of SRC-1), was incubated with purified glutathione-S-transferase-tagged human VDR (residues 90-427) and candidate ligands in 100 l of buffer (150 mM NaCl, 10 mM KPO₄, 2 mM CHAPS, 2 mM EDTA, 1 mM DTT at pH 7.3) in a 96-well plate on a shaker for 1 hr (4). Ligand-dependent recruitment of the coactivator peptide to VDR was then monitored as an increase in fluorescence polarization (mP) using an LJL analyst reader (LJL Biosystems). Means of triplicate data points ±: SD are plotted.