Unusual binding of ursodeoxycholic acid to ileal bile acid binding protein: role in activation of FXRα.

Ursodeoxycholic acid (UDCA, ursodiol) is used to prevent damage to the liver in patients with primary biliary cirrhosis. The drug also prevents the progression of colorectal cancer and the recurrence of high-grade colonic dysplasia. However, the molecular mechanism by which UDCA elicits its beneficial effects is not entirely understood. The aim of this study was to determine whether ileal bile acid binding protein (IBABP) has a role in mediating the effects of UDCA. We find that UDCA binds to a single site on IBABP and increases the affinity for major human bile acids at a second binding site. As UDCA occupies one of the bile acid binding sites on IBABP, it reduces the cooperative binding that is often observed for the major human bile acids. Furthermore, IBABP is necessary for the full activation of farnesoid X receptor α (FXRα) by bile acids, including UDCA. These observations suggest that IBABP may have a role in mediating some of the intestinal effects of UDCA.

UDCA to partially block the transmembrane apical Na + dependent bile acid transporter ( 7 ). Thus far, no intracellular receptor for UDCA has been reported.
Ileal bile acid binding protein (IBABP) is a small cytoplasmic protein mainly expressed in ileal epithelium. Although initially categorized as a member of the fatty acid binding protein family ( 19 ), IBABP binds exclusively to bile acids ( 20 ). The bile acid selectivity arises from the fact that IBABP has a larger binding pocket than other members of the fatty acid binding protein family ( 21,22 ). The precise biological function of IBABP is not clear, but it is presumed to coordinate with the apical bile acid transporter in the uptake of bile acids into ileocytes. IBABP also associates with FXR ␣ ( 23 ) and could potentially help mediate the transcriptional response to bile acids.
IBABP binds two bile acids in a cooperative manner ( 21,24 ). The basis of cooperative binding is the formation of a hydrogen bond between the 3-OH of the bile acid occupying site 1, and either the 7-OH or 12-OH of Abstract Ursodeoxycholic acid (UDCA, ursodiol) is used to prevent damage to the liver in patients with primary biliary cirrhosis. The drug also prevents the progression of colorectal cancer and the recurrence of high-grade colonic dysplasia. However, the molecular mechanism by which UDCA elicits its benefi cial effects is not entirely understood. The aim of this study was to determine whether ileal bile acid binding protein (IBABP) has a role in mediating the effects of UDCA. We fi nd that UDCA binds to a single site on IBABP and increases the affi nity for major human bile acids at a second binding site. As UDCA occupies one of the bile acid binding sites on IBABP, it reduces the cooperative binding that is often observed for the major human bile acids. Furthermore, IBABP is necessary for the full activation of farnesoid X receptor ␣ (FXR ␣ ) by bile acids, including UDCA. These observations suggest that IBABP may have a role in mediating some of the intestinal effects of UDCA. Supplementary key words farnesoid X receptor ␣ • colorectal cancer • bile salts Ursodeoxycholic acid (UDCA) is a bile acid that is abundant in the bile of black bears ( Ursudae ), and it is the active component of an ancient Chinese remedy for liver disorders ( 1 ). In Western society, UDCA is approved as a drug for treating primary biliary cirrhosis ( 1 ). It improves the symptoms and decreases the biochemical abnormalities in patients with primary sclerosing cholangitis (PSC) ( 2 ). Patients with PSC are at high risk for developing colorectal cancer, and it has been observed that UDCA also mitigates this risk ( 3,4 ). Recently, UDCA was shown to reduce the recurrence of high-grade dysplasia in patients with colorectal cancer ( 5 ). Much of the clinical benefi t of UDCA has been attributed to a reduction in the hydrophobicity of the systemic bile acid pool ( 6 ) and to the ability of in cold room. The columns were then washed with 180 ml PBS at 1 ml/min, injected with 12 ml thrombin protease solution at 20 U/ml (Amersham), and incubated at room temperature for 20 h. A PBS-equilibrated 1 ml HiTrap Benzamidine FF (high sub) column (Amersham) was connected after GSTrap column to remove thrombin protease, and the recombinant IBABP was eluted using PBS at 0.5 ml/min. The protein preparations were then delipidated by passing through hydroxyalkoxypropyl-dextran (type VI; Sigma) column preequilibrated with PBS at 37°C. 15 Nlabeled IBABP was expressed and purifi ed similarly, except that the M9 minimal medium supplemented with 15 NH 4 Cl was used. The purity of IBABP was estimated as >98% by SDS-PAGE gel and analytical gel-fi ltration chromatography. The protein was correctly folded as indicated by the sharp melting curve in differential scanning calorimetry (DSC) assay. Protein concentration was determined by BCA protein assay (Pierce).

Tryptophan fl uorescence spectroscopy
Tryptophan fl uorescence was measured in volts at 20°C with 450 volt input using MOS 250 fast UV/Vis spectrometer (Bio-Logic). IBABP (250-270 µl of 10-20 µM) in PBS was titrated stepwise at 1-2 µl increments with 2.5-5.0 mM of the different bile acids and UDCA in the same buffer. The detailed concentration of ligand and protein, and the titration volume and step are specifi ed in the legend of each fi gure. After each titration, the protein and ligand mixture was incubated for 5 min to allow the binding to reach equilibrium. Emission spectra were recorded in triplicate from 310 to 400 nm at a rate of 125 nm/s, with excitation at 280 nm. Both excitation and emission slits were 10 nm. Fluorescence gain ( ⌬ F) at 336 nm was calculated by subtracting the fl uorescence intensity of apo-protein from that of the holo-protein. The binding data were analyzed with two independent approaches. The Hill equation, H N ), was used to obtain binding affi nity, and the Hill coeffi cient from a plot of the normalized fl uorescence change ⌬ F/ ⌬ F max (specifi c binding) was plotted against bile acid concentration [BA]. In a second analysis, the Scatchard plot of ⌬ F/ ⌬ F max /[BA] versus ⌬ F/ ⌬ F max was used to identify binding cooperativity. In these plots, convex downward curvature indicates cooperativity. The value of the ordinate at the maximum abscissa value on these curves can also be used to calculate the Hill coeffi cient, H N (H N =1/(1-⌬ F/ ⌬ F max ).

NMR spectroscopy
Protein-observed NMR experiments were performed on 0.03 Ϫ 0.1 mM uniformly 15  the bile acid occupying site 2 (supplementary Fig. I). The binding of UDCA to these sites on IBABP has never been examined. Here we show that UDCA binds to IBABP, but unlike major human bile acids, UDCA binds only to a single site. Importantly though, binding of UDCA promotes binding of major human bile acids at the second binding site. This binding mechanism is evident in whole cells, where UDCA potentiates the activation of FXR ␣ by major human bile acids. These fi ndings explain how UDCA can promote the transcriptional response of FXR ␣ without binding to it.

Reagents
All free, glycine-, and taurine-conjugated cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), and ursodeoxycholic acid (UDCA) in sodium salt form were purchased from Sigma and Calbiochem. 15 N-labeled bile acids were synthesized in-house via coupling of 15 N-labeled glycine (Cambridge Isotope Laboratories) to unconjugated bile salts by the method of Momose et al. ( 25 ). Rabbit antiserum to human IBABP was raised by our laboratory using recombinant protein as antigen. Oligonucleotide primers were synthesized by Integrated DNA Technologies, Inc. siRNA SMARTpools were purchased from Dharmacon. Lipofectamine 2000 was ordered from Invitrogen. The molecular cloning reagents were purchased from Promega and New England Biolabs, unless otherwise indicated.

Plasmid constructs
The IBABP promoter containing a FXR ␣ binding element was amplifi ed from human genomic DNA as previously described ( 26 ) and inserted into pGL3-Basic vector (Promega) to produce the FXRE-Luc reporter gene for monitoring FXR ␣ activity. The pCDNA3.1-hFXR ␣ 2 ( 27 ) plasmid was a gift from Dr. Peter A. Edwards (University of California at Los Angeles). To construct the mammalian expression vector for human IBABP, the open reading frame (ORF) of IBABP was amplifi ed from IMAGE clone 1916019 (Research Genetics) using pfu DNA polymerase (Stratagene), and then inserted into pcDNA6/His C (Invitrogen) between BamH I and Xho I sites (pcDNA6/His-IBABP). To construct the recombinant IBABP expression vector, the ORF of IBABP was inserted into the prokaryotic expression vector pGEX-KG-1 between EcoR I and Xho I sites (pGEX-KG-1/IBABP). The ORF was fused in-frame in N terminus with glutathione S-transferase (GST) coding sequence separated by a thrombin site and a glycine linker.

Protein expression and purifi cation
pGEX-KG-1/IBABP was transformed into E. coli strain BL21(DE3) (Stratagene). The recombinant IBABP was expressed in LB medium with induction of 25 µM isopropyl-␤ -Dthiogalactopyranoside (IPTG) at 28°C for 4 h. Recombinant IBABP was purifi ed in mild buffer by affi nity chromatography on glutathione agarose, coupling with on-column cleavage by thrombin protease to remove the GST tag. Briefl y, 5 g of cells were suspended in 30 ml of ice-cold PBS (pH = 7.4) with 1.5 ml of bacterial protease inhibitor cocktail (Sigma) and lysed by the French Pressure Cell Press (Aminco). After brief sonication to break the host DNA, the crude bacterial extract was centrifuged at 13,000 rpm for 30 min at 4°C, and the supernatant was adjusted with 1 M DTT to a fi nal concentration of 5 mM, and loaded onto PBS-equilibrated 2 × 5 ml GSTrap HP columns (Amersham) at 0.5 ml/min overnight

Statistical analysis
All values are reported as mean ± SEM. Data from multiple treatment groups were compared using two-way ANOVA with Bonferroni posttest. Data from two treatment groups were compared using unpaired t -test. A probability value of P < 0.05 was considered statistically signifi cant.

UDCA binds to a single site on IBABP
The ability of IBABP to bind bile acids found in humans is well established, but its binding to UDCA has never been tested. Binding between IBABP and UDCA was measured with tryptophan fl uorescence spectroscopy. IBABP contains a single tryptophan, making interpretation of the binding data straightforward. Purifi ed IBABP was titrated with UDCA in a stepwise manner, yielding a typical hyperbolic binding curve with Hill coeffi cient of 1, indicative of a single class of binding site(s) ( Fig. 1A ). The affi nity of UDCA for IBABP is 63 µM. The glyco (G) and tauro (T) conjugates of UDCA, which are observed in humans treated with UDCA, have similar affi nity (81 µM for GUDCA, and 56 µM for TUDCA). We also used ligand-observed 1 H, 15 N HSQC NMR spectroscopy to probe the number of binding sites for UDCA on IBABP. Changes to the 2D 1 H, 15 N correlation spectra of 15 N-bile acids were monitored in response to addition of IBABP ( Fig. 1B, C ). Using the glyco conjugates of bile acids, two peaks of bound 15 N-GCA are observed in the presence of IBABP ( Fig. 1B ), whereas only a single bound peak of 15 N-GUDCA is detected ( Fig. 1C ). In conjunction with the experiments from tryptophan fl uorescence, this fi nding suggests that IBABP contains a single binding site for UDCA.
IBABP contains two binding sites for major human bile acids and binds them in a cooperative manner ( 21 ). Because UDCA appeared to bind only a single site on IBABP, we tested its effect on binding to major human bile acids. The binding experiments were conducted by saturating IBABP with GUDCA, and then titrating with other bile acids. GUDCA was used as the ligand because it is considered to be the active form of UDCA in humans ( 29 ). GUDCA shifted the binding curve between IBABP and other bile acids to the left ( Fig. 2A -C ), increasing the affi nity of IBABP for major human bile acids by a factor of 2-to 5-fold ( Table 1 ). In addition, when calculated with the Hill equation, we observe a 50-60% decrease in binding cooperativity of major human bile acids in the presence of UDCA ( Table 2 ). This is consistent with the fact that UDCA already occupies one binding site on IBABP. To confi rm the loss of cooperative binding, the data were also plotted according to the method of Scatchard ( Fig. 2D-F ). These plots show a convex downward curve in the absence of UDCA, with the ordinate of the apex ‫ف‬ 0.5, which equates to a Hill coeffi cient of 2, a value very similar to that obtained with the Hill equation ( Table 2 ). However, in the presence of UDCA, the Scatchard plots for other bile acids lack convex curvature, indicating that binding is far less cooperative. The affi nities and cooperativity of the major human bile acids for IBABP measured in these Cell culture Caco-2 cells were grown in complete Dulbecco's modifi ed Eagle's media (DMEM; Mediatech) supplemented with antibiotics (Omega Scientifi c) and 10% fetal bovine serum (Invitrogen). Cells were maintained in 100 mm standard cell culture dishes (BD Biosciences) and grown at 37°C under a humidifi ed 5% CO 2 atmosphere. Cells were split twice a week.

Overexpression of IBABP in cells and bile acid treatment
All transfections were performed with 12,000 cells per well in 24-well plate (for luciferase assay) or scaled up in 6-well plate (for Western blot) using Lipofectamine 2000 (Invitrogen). The day before transfection, proliferating Caco-2 cells were seeded in antibiotics-free DMEM with 10% FBS. pcDNA6/His-IBABP was introduced into cells with FXRE-Luc reporter and pRL-CMV using Lipofectamine 2000 according to manufacturer's protocol. The plasmid load was 0.9 µg per well in a 24-well plate and 4.0 µg per well in a 6-well plate. The cells were incubated in OptiMEM (Invitrogen) containing the DNA-lipid complexes for 24 h, and then treated with 125 µM UDCA or control medium for 5 h, followed by addition of 25 µM CDCA or DCA alone or plus 125 µM UDCA for an additional 24 h. In bile acid treatment experiments, cells were fed with phenol red-free DMEM (Mediatech) supplemented with 10% charcoal-stripped fetal bovine serum (Hyclone).

RNA interference and bile acid treatment
Two days before siRNA transfection, Caco-2 cells were seeded in antibiotics-free DMEM with 10% FBS. pCDNA3.1-hFXR ␣ 2 was introduced into Caco-2 cells. After 24 h incubation in OptiMEM, IBABP-specifi c siRNAs were transfected into Caco-2 cells using Lipofectamine 2000 according to manufacturer's protocol; the fi nal siRNA concentration was 40 nM. After 24 h incubation in OptiMEM, cells were transfected with FXRE-Luc reporter and pRL-CMV, and incubated for 24 h. The cells were then treated with 125 µM UDCA or control medium for 5 h, followed by addition of 25 µM CDCA alone or plus 125 µM UDCA for an additional 24 h in phenol red-free DMEM supplemented with 10% charcoal-stripped fetal bovine serum.

Determination of FXR ␣ activity with a luciferase reporter construct
The activation of FXR ␣ in cells was measured by luciferase reporter construct FXRE-Luc that contains coding sequence for fi refl y luciferase regulated by IBABP promoter. Renilla luciferase under CMV promoter control (pRL-CMV) was also included for normalization of transfection effi ciency. After 24 h bile acid treatment, cells were harvested in 1× Passive Lysis Buffer (Promega), and fi refl y and Renilla luciferase activities were measured using the Dual-Luciferase Assay System with Veritas Microplate Luminometer (Promega) according to the manufacturer's manual.

Western blotting and quantitative PCR
The cellular proteins were extracted with CelLytic buffer (Sigma), and 50 µg of total proteins were separated in 10-20% Criterion SDS-PAGE gel (Bio-Rad). The protein level of IBABP was determined by Western blot with standard procedures. The protein level of ␤ -actin was used a loading control. Cellular RNA was isolated using the RNeasy Mini Kit (Qiagen) according to manufacturer instruction. The mRNA expression of organic solute transporter ␣ (OST ␣ ) was measured by qPCR using Mx3000P qPCR system (Stratagene) and normalized to housekeeping gene ARPP0 as described previously ( 28 ). The qPCR primers for OST ␣ are 5 ′ -TTG CTT GTT CGC CTC CCT ATT CCT C-3 ′ and 5 ′ -GTC TTT CCT TCG GTA GTA CAT TCG TG-3 ′ . substantial conformational changes in IBABP ( Fig. 3A , B ) and that the conformation of IBABP is signifi cantly different when bound to TCDCA compared with TUDCA ( Fig. 3C ).
From the same HSQC spectra, we focused on resonances for an amino acid indicative of site 1 of IBABP (Gly66), which was previously reported to exhibit strong changes to the chemical shift upon binding of major human bile acids ( 30 ). A signifi cant shift is observed upon binding of TUDCA (compare Fig. 4A , B ), and TCDCA (compare Fig. 4A, C ). These fi ndings are consistent with the idea that this site (site 1) can bind either TUDCA or TCDCA. In contrast, we observed that an amino acid indicative of experiments are entirely consistent with values reported from published reports using different detection methods and kinetic models ( 20,22 ).

UDCA induces unique conformational changes in IBABP
We used protein-observed 1 H, 15 N HSQC NMR spectroscopy to monitor structural changes of IBABP induced by the stereoisomers TUDCA and TCDCA. The 7-hydroxyl group in TCDCA is in the ␣ -conformation, but in TUDCA, it is in the ␤ -conformation. The HSQC spectra of IBABP were obtained in its apo state when bound to TUDCA and when bound to TCDCA. The spectra show that both bile acids induce  colonocytes. CDCA was chosen as a representative bile acid for two reasons. One, it is the best bile acid agonist of FXR ␣ ( 8-10 ). Two, it is the most abundant bile acid in human colon tissue and colon polyps ( 31,32 ). In these experiments, we chose to preincubate the cells with UDCA because it is lipophobic and has a much slower diffusion rate than CDCA ( 33,34 ). In our view, this preincubation is more likely to recapitulate the human clinical condition in which UDCA is administered daily to maintain its concentration at a relatively steady-state. These experiments were performed on both Caco-2 cells where IBABP cannot be detected by Western blot and on Caco-2 cells transfected with an expression vector encoding IBABP. UDCA increased the effect of DCA and CDCA on activation of FXR ␣ in Caco-2 cells ( Fig. 5 , white bars). However, this effect was far more pronounced when IBABP was overexpressed ( Fig. 5 , black bars).
To confi rm that IBABP is necessary for the enhanced activation of FXR ␣ by UDCA, the expression of endogenous IBABP was suppressed with RNAi. Caco-2 cells have no detectable IBABP by Western blot ( Fig. 5 ). Because IB-ABP is a target gene of FXR ␣ , the cells were transfected with an FXR ␣ construct to increase the endogenous expression of IBABP ( Fig. 6 ). The cells were then treated with IBABP-specifi c siRNA to knock down the induced IB-ABP. This knockdown eliminated the ability of UDCA to potentiate the activation of FXR ␣ by CDCA. These effects are indicated by changes in the activity of the Luciferase reporter ( Fig. 6A ), and by changes to the expression of the OST ␣ , a target gene of FXR ␣ ( Fig. 6B ). The expression levels of FXR ␣ and RXR ␣ remained constant in these studies (not shown), proving that the primary effect on activation results from a reduction in IBABP.

DISCUSSION
The results of the study support the following conclusions: i ) UDCA binds to a single site on IBABP; ii ) occupation of this site by UDCA increases the affi nity of a second binding site for major human bile acids; iii ) UDCA augments the activation of FXR ␣ by major human bile acids; and iv ) this augmentation requires IBABP.
The primary fi nding of this study is that UDCA binds to a single site on IBABP. In a series of papers published by Cistola's group, IBABP has been shown to have two bile acid binding sites. The selectivity and cooperativity of these sites are governed by the hydroxylation pattern on steroid rings of major human bile acids ( 21,22,24,35 ). Here, we found that UDCA binds only one of the bile acid binding sites in IBABP. This conclusion is strongly supported by the binding isotherms evident in tryptophan fl uorescence studies, which show that UDCA binds to a single class of sites. The conclusion is further substantiated by the ligandobserved NMR spectrum from 15 N-GUDCA bound to IBABP, which shows a single peak corresponding to bound GUDCA. Additional support for the presence of only a single binding site for UDCA can be taken from the fact that TUDCA has little infl uence on protein resonance shift perturbation of an amino acid that is an indicator of site 2 (Val37) undergoes only a minor chemical shift perturbation in response to TUDCA (compare Fig. 4E, F); however, it is perturbed upon binding of TCDCA (compare Fig. 4E, G ). These fi ndings are consistent with the idea that major human bile acids, but not UDCA, bind to site 2 on IBABP.
We also performed an experiment with NMR to determine if TUDCA could be displaced from IBABP by TCDCA. Gly66, the indicator of occupancy at binding at site 1, exhibits two resonances when both of these bile acids are present (compare Fig. 4A, D ). These resonances are in equal portion, consistent with the conclusion that half the IBABP population has TUDCA at site 1 and the other half contains TCDCA. This displacement is independent of the order of the ligand addition because both experiments resulted in the same end state. In contrast, the resonance of Val37 is the same in the presence of TCDCA versus TCDCA and TUDCA (compare Fig. 4G, H ). Taken together, these results show that, at these concentrations of TUDCA and TCDCA, two populations of IBABP are possible. One population contains TUDCA at site 1 and TCDCA at site 2. The other population contains TCDCA at both sites.

Activation of FXR ␣ by major human bile acids is potentiated by UDCA
As we show that UDCA binds to IBABP and a recent study showed that IBABP interacts with FXR ␣ ( 23 ), we hypothesized that IBABP may have a role in mediating the activation of FXR ␣ by UDCA. This idea was tested in Caco-2 cells, which are commonly used as model The binding affi nity K D of CA, CDCA, DCA and their glycine (G) and taurine (T) conjugates with IBABP was calculated using the Hill equation. The binding cooperativity H N of CA, CDCA, DCA and their glycine (G) and taurine (T) conjugates with IBABP was calculated using the Hill equation. K D . In patients treated with UDCA, its levels in fecal water are 2-fold higher than DCA levels ( 37 ), which has a similar affi nity for the protein. Consequently, in patients treated with UDCA, it is likely that much of the IBABP has UDCA at site 1 and another bile acid at site 2.
We constructed a model of IBABP bound to CDCA ( Fig. 7A ) based on the structure of chicken liver bile acid binding protein (LBABP), bound to two cholic acids ( 38 ), to provide structural basis for the selectivity of UDCA for binding site 1. The model shows that the 7 ␣ -OH of the steroid ring of CDCA, occupying binding site 2, makes two important contacts. One contact is a hydrogen bond with the 3-OH of CDCA at site 1. The second contact includes Van der Waals interactions between the 7 ␤ -H and the two methyl groups of Ile69 in IBABP. If CDCA at site 2 is replaced with UDCA, the 7-OH group is in the ␤ rather than the ␣ conformation, so neither of these key contacts can occupancy at site 2. Because CDCA induced shifts in site 2, the lack of resonance shifts upon UDCA binding are consistent with the conclusion that it binds at only one site on IBABP. Finally, the fact that TUDCA fails to displace TCDCA bound to site 2 also supports the idea that UDCA cannot bind to site 2.
The second important fi nding of this study is that occupation of site 1 by UDCA increases the affi nity of IBABP at the second binding site for the major human bile acids by 2-to 5-fold. In patients treated with UDCA, the concentration of this bile acid in fecal water is ‫ف‬ 50 M ( 36 ), which is near the affi nity we report here. Therefore, in patients treated with UDCA, one would expect a substantial fraction of the IBABP to have UDCA at site 1. Although we observed displacement of TUDCA from site 1 by TCDCA, those experiments were conducted at equimolar concentrations of each bile acid and at concentrations above their  The fi ndings presented here are counter to the report of Campana et al. ( 43 ), who concluded that UDCA could compete for the binding of CDCA to FXR ␣ and thereby block the transcriptional response of FXR ␣ to CDCA. There are several potential explanations for the discrepancy in the two studies. One, the conclusion that FXR ␣ be made. In addition, Cistola's group showed that bile acids lacking a 12-OH preferentially bind to site 1 ( 35 ). As UDCA lacks a 12-OH, it falls into this category.
Importantly though, when UDCA is bound at site 1, the 3-OH group of its steroid ring is in the same conformation as major human bile acids. Therefore, UDCA can still engage in cooperative binding with major human bile acids at site 2. In this case, a hydrogen bond can form between the 3-OH of UDCA and the 7-OH or 12-OH group of the bile acid at site 2. These hydrogen bonds are illustrated in Fig. 7 and supplementary Fig. I. This conclusion is also consistent with the work of Cistola's group, which showed that binding cooperativity is governed by patterns of hydroxylation in the steroid B-and C-rings of bile acids ( 21 ).
Interestingly, binding cooperativity is also determined by two amino acid residues, 99 and 101, located in the inner cavity of IBABP ( 39 ). Chicken IBABP (H99/A101) has a rigid H-bond pattern and disfavors conformational fl exibility needed for coupling between the two sites, and indeed, chicken IBABP shows noncooperative binding of bile acids ( 39 ). On the other hand, human IBABP (A99/S101) has an extended H-bond network, which allows cooperative bile acid binding. Although the H-bond network does not appear to affect the bile acid selectivity, the presence of UDCA in site 1 is likely be communicated through this H-bond network.
The third important observation of this study is that UDCA augments the activation of FXR ␣ by major human bile acids. Our results show that IBABP mediates UDCA's enhancement of activation of FXR ␣ by other bile acids in cells. However, the mechanism by which IBABP bridges UDCA and FXR ␣ is not entirely clear. A prior report shows that IBABP directly interacts with FXR ␣ ( 23 ), so this binding could enable bile acid transfer from IBABP to FXR ␣ in the nucleus. Other proteins from the lipid binding protein family appear to act in this manner. For example, cellular retinoic acid binding protein II (CRABP II) transfers retinoic acid to the retinoic acid receptor and directly binds to this nuclear receptor ( 40 ). Similarly, liver fatty acid binding protein (LFABP) increases the intracellular levels of fatty acids and directly binds to peroxisome proliferator-activated receptor (PPAR) ( 41,42 ).  The discrepancy between our conclusions and those of Campana et al. ( 43 ) also underscore the confl icting views on the ability of UDCA to bind directly to FXR ␣ . Only one study has reported direct binding between UDCA and FXR ␣ ( 18 ). In that report, the affi nity was measured by an in vitro scintillation proximity assay. Weak binding between UDCA and FXR ␣ was detected (affi nity of ‫ف‬ 185 M ). Importantly though, in the same study, the affi nity of CDCA for FXR ␣ was reported to be 7-fold higher than that reported in other publications ( 44 ), so scintillation proximity appears to overestimate affi nity. Furthermore, UDCA also fails to recruit coactivators for FXR ␣ ( 10, 18 ), and it fails to activate FXR ␣ in cultured cells (8)(9)(10). Altogether, the overwhelming body of evidence indicates that UDCA is unlikely to directly bind to and activate FXR ␣ and supports our conclusion that IBABP is a necessary bridge between these two molecules.
To piece together a precise mechanism of action for IB-ABP, we will need to know the intracellular concentrations and movement of bile acids, especially in the presence of clinically relevant levels of UDCA. However, technical challenges currently preclude the measurement of intracellular bile acid concentrations and the tracking of bile acid movement in cells, especially combinations of bile acids. Although fl uorescently labeled bile acids have been synthesized, their value as intracellular probes is uncertain because they are transported differently than natural bile acids and because their choleretic properties differ (45)(46)(47). To our best knowledge, there is no report on the measurement of intracellular bile acid concentration in intestinal cells. Consequently, these measures remain at the forefront of key hurdles to overcome to enable a complete understanding of the cellular function of IBABP.
Given the fi ndings of this study, any interpretation of the therapeutic effects of UDCA should take IBABP into account. As outlined above, the concentration of UDCA in fecal water is near its K D for IBABP, so a signifi cant fraction of IBABP is occupied by this bile acid at its therapeutic concentration. On the basis of the observations presented here, UDCA bound to IBABP is likely to modulate the activity of FXR ␣ in ileocytes and thus enhance the excretion of major human bile acids ( 7,48 ). In addition, UDCA may increase the buffering capacity of IB-ABP in colonocytes. Due to the effi cient absorption at distal small intestine, colon bile acids are present at low concentration ( 49 ), and IBABP is not fully bound by major human bile acids due to their low affi nity. UDCA increases the binding affi nity of major human bile acids, thus reducing the levels of free bile acids in colonocytes. This would reduce cytotoxic stress placed on the gastrointestinal system. In colorectal cancer, this effect would protect against bile acid-induced mutations in the genome ( 50 ) and the acquisition of bile acid resistance, a property that is essential for disease progression ( 50 ). Although the clinical effects of UDCA have been attributed to its ability to reduce the hydrophobicity and, therefore, cytotoxicity of the systemic bile acid pool ( 6 ), the stimulation of FXR ␣ could be another reason for its clinical benefi t. binds to UDCA comes from binding studies performed on cell lysates rather than purifi ed protein, and it runs counter to three other reports (8)(9)(10). Two, we interpret the binding isotherms of Campana et al. ( 43 ) to indicate that CDCA actually promotes binding of UDCA to proteins in the lysate, rather than inhibiting binding. If this is the case and the binding in the lysate is attributed to IBABP, then this result would be consistent with our observations. Three, and most signifi cantly, Campana et al. used different concentrations of bile acids and different treatment times when measuring the effect of UDCA on the transcriptional response of FXR ␣ . This is an important point, and it may have clinical relevance. It is entirely conceivable that the effects of UDCA on cells are dose-dependent and that there may be multiple effects. Low concentrations of UDCA, which are not toxic and do not appreciably change the hydrophobicity of the extracellular milieu, are likely to potentiate the stimulation of FXR ␣ by other bile acids (this study). At higher concentrations, UDCA may have altogether different effects, like those observed by Campana et al. ( 43 ). Consequently, it may ultimately prove useful to monitor the FXR ␣ response in patients treated with UDCA to determine whether there is a more appropriate dosing regimen.