Chenodeoxycholic Acid-Mediated Activation of the Farnesoid X Receptor Negatively Regulates Hydroxysteroid Sulfotransferase

Drug Metab. Pharmacokinet. 21 (4): 315–323 (2006).

Regular Article Chenodeoxycholic Acid-mediated Activation of the Farnesoid X Receptor Negatively Regulates Hydroxysteroid Sulfotransferase

Masaaki MIYATA1,* Yoshiki MATSUDA1, Hiroyuki TSUCHIYA1,HirotakaKITADA1, Takanori AKASE1,MikiSHIMADA1, Kiyoshi NAGATA1, Frank J. GONZALEZ2 and Yasushi YAMAZOE1,3 1Division of Drug Metabolism and Molecular Toxicology, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan 2Laboratory of Metabolism, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA 3CRESCENDO, The Tohoku University 21st Century ``Center of Excellence'' Program, Sendai, Japan

Full text of this paper is available at http://www.jstage.jst.go.jp/browse/dmpk

Summary: Hydroxysteroid sulfotransferase catalyzing bile acid sulfation plays an essential role in protection against lithocholic acid (LCA)-induced liver toxicity. Hepatic levels of Sult2a is up to 8-fold higher in farnesoid X receptor-null mice than in the wild-type mice. Thus, the in‰uence of FXR ligand (chenodeoxycholic acid (CDCA) and LCA) feeding on hepatic Sult2a expression was examined in FXR- null and wild-type mice. Hepatic Sult2a protein content was elevated in FXR-null and wild-type mice fed aLCA(1z and 0.5z) diet. Treatment with 0.5z CDCA diet decreased hepatic Sult2a to 20z of the control in wild-type mice, but increased the content in FXR-null mice. Liver Sult2a1 (St2a4) mRNA levels were reduced to 26z in wild-type mice after feeding of a CDCA diet, while no decrease was observed on Sult2a1 mRNA levels in FXR-null mice after CDCA feeding. A signiˆcant inverse relationship (r2＝0.523) was found between hepatic Sult2a protein content and small heterodimer partner (SHP) mRNA level. PCN-mediated increase in Sult2a protein levels were attenuated by CDCA feeding in wild-type mice, but not in FXR-null mice. Human SULT2A1 protein and mRNA levels were decreased in HepG2 cells treated with the FXR agonists, CDCA or GW4064 in dose-dependent manners, although SHP mRNA levels were increased. These results suggest that SULT2A is negatively regulated through CDCA-mediated FXR activation in mice and humans.

Key words: hydroxysteroid sulfotransferase; FXR; bile acid; sulfation; negative regulation; Sult2a

tion is a main metabolic pathway for bile acid excretion, Introduction SULT2A is a critical enzyme for bile acid homeostasis. Sulfotransferases (SULT) are phase II metabolizing About 95z of bile acids excreted in bile are reabsorbed enzymes that catalyzes sulfation of various endogenous in the intestine and thus changes in the level of hepatic and exogenous chemicals.1,2) Hydroxysteroid sulfotran- SULT2A can disrupt bile acid homeostasis in entero- sferase (SULT2A) is a cytosolic enzyme expressed hepatic tissues. SULT2A is also considered to play a abundantly in enterohepatic tissues such as liver and major role in the protection against toxic bile acid- intestine, and also in steroidogenic tissue such as the induced liver injury in enterohepatic tissues. SULT2A adrenal cortex.3) SULT2A catalyzes the sulfation of preferably catalyzes the sulfation of hydrophobic and drugs, environmental chemicals and endogenous toxic bile acids such as LCA. A protective role of Sult2a steroids such as dehydroepiandrosterone (DHEA), against LCA-induced hepatotoxicity was demonstrated estrogen and bile acids.4,5) SULT2A-mediated sulfation by using farnesoid X receptor (FXR)-null female mice increases the hydrophilicites of bile acids and facilitates constitutively expressing higher levels of Sult2a pro- their clearance from the body. Because bile acid sulfa- tein.6) The increase in the production of hepatic Sult2a-

Received; February 13, 2006, Accepted; April 7, 2006 *To whom correspondence should be addressed: Masaaki MIYATA,Ph.D.,Division of Drug Metabolism and Molecular Toxicology, Graduate School of Pharmaceutical Sciences, Tohoku University, 6-3, Aoba, Aramaki, Aoba-ku, Sendai, 980-8578, Japan. Tel. ＋81-22-795-6829, Fax. ＋81-22-795-6826, E-mail: miyata＠mail.pharm.tohoku.ac.jp

315 316 Masaaki MIYATA et al. mediated 3a-sulfated TLCA and the e‹cient fecal matched groups of 8- to 12-week-old animals were used excretion contributes to the protection against LCA- for all experiments. The mice were fed the control diet induced toxicity (submitted for publication). (CE-2)mixedwith0.5z and 1.0z (wWw) LCA for 9 Two Sult2a mRNAs (Sult2a1 (St2a4) and Sult2a2 days or 0.5z CDCA for 5 days. The mice were fed the (St2a9)) that share high homology, were isolated from control diet or the diet mixed with 0.5z (wWw) CDCA mice7,8) and one mRNA (SULT2A1) has been isolated for 5 days and injected with PCN (100 mgWkg, ip) for from humans.5,9,10). Mouse Sult2a enzymes are the last 3 days. expressed to higher extents in the female. Because of the Hepatic bile acid concentration: Hepatic 3a-hydro- e‹cient bile acid sulfating activity in female mice as well xy bile acid concentrations were estimated by an as humans, female mice represent a feasible model for enzyme-colorimetric method using the Total bile acid- bile acid sulfation in humans. test kit from Wako (Wako Pure Chemicals, Osaka, FXR, a member of the nuclear receptor superfamily, Japan). Hepatic LCA and CDCA contents were is expressed abundantly in liver, intestine and adrenal.11) measured by HPLC as previously described.24) The involvement of FXR in bile acid and lipid Cell culture: HepG2 cells were plated in 10 cm homeostasis was demonstrated by using FXR-null culture dishes (Falcon Scientiˆc Co., Oxnard, CA) at mice.12) FXR is activated by several bile acids such as 3×106 cells and cultured in Dulbecco's modiˆed Eagle's CDCA.13–15) FXR regulates a variety of genes involved in medium (DMEM) containing 10z fetal calf serum. The the metabolism and transport of the lipids such as bile cells were cultured for 24-h and then treated with acid and cholesterol. FXR stimulates the expression CDCA, GW4064 or vehicle (DMSO) for 48-h. The cells of Bsep, small heterodimer partner (SHP) and Mrp2, were cultured in serum-free DMEM during chemical whereas FXR directly represses the expression of treatment. apolipoprotein A-I and apolipoprotein C-III, and Immunoblot analysis: Cytosolic proteins (30 mgW indirectly suppresses the expression of CYP7A1 and lane) were loaded onto a 9z polyacrylamide gel, isolat- Ntcp through the induction of SHP.16–18) ed and transferred to nitrocellulose ˆlters. The ˆlter was CDCA activation of FXR has been shown to stimu- immunostained with a polyclonal antibody prepared late rat SULT2A1 transcription via the FXR response against the puriˆed recombinant rat SULT2A1 protein element (IR0) in HepG2 cells.19).Furthermore,itwas (1:1000 dilution) that reacts with mouse Sult2a25) and a demonstrated that pregnane X receptor (PXR), polyclonal antibody against the puriˆed recombinant constitutive androstane receptor (CAR) and vitamin D human SULT2A1 protein26) (1:1000 dilution) for human receptor (VDR) also bind the rat and mouse IR0 SULT2A1. The stained ˆlters were scanned with an elements and enhance the reporter gene activity.20–23) Epson GT-8700 scanner, and the band intensities The Sult2a expression level, however, was increased in measured by use of the NIH image (version 1.59) FXR-null and PXR-null female mice as compared with software (Bethesda, MD). that of wild-type mice.6) Expression and puriˆcation of Sult2a proteins: In the present study, SULT2A expression levels were Mouse Sult2a cDNAs were obtained from wild-type determined using FXR-null mice and HepG2 cells to mouse liver by reverse transcription-polymerase chain clarify the involvement of FXR signaling in the suppres- reaction (RT-PCR). Sult2a cDNA fragments, contain- sion of SULT2A gene expression. These results suggest ing a sequence of the recognition site of enterokinese that SULT2A is negatively regulated by activation of next to the N-terminal methionine of the native form, FXR with CDCA. were obtained by RT-PCR and ligated into the prokaryotic expression vector, pQE30 (QIAGEN). The Materials and Methods plasmid DNA was transformed into Escherichia coli, Materials: CDCA, LCA, tauroLCA (TLCA), M15 (pREP4). Recombinant Sult2a proteins were glycoLCA (GLCA), cholic acid (CA), deoxycholic acid expressed and puriˆed from bacterial cytosols by nickel- (DCA), ursodeoxycholic acid (UDCA), pregnenolone nitrilotriacetic acid a‹nity chromatography. The fused 16a-carbonitrile (PCN) and dehydroepiandrosterone portion of recombinant Sult2a proteins was removed (DHEA) were purchased from Sigma-Aldrich Co. (St. to yield native proteins for standard of immunoblot Louis, MO). GW4064 was kindly provided by Dr. analyses by enterokinase. Timothy M. Willson (GlaxoSmithKline, Research Analysis of mRNA levels: Total RNAs were Triangle Park, NC). prepared from livers and HepG2 cells using the RNA- Animal treatment and sample collection: FXR-null gents Total RNA Isolation System (Promega, Madison, female mice12) were housed under standard 12-h lightW WI), and RNA concentrations were determined by 12-h dark cycle. Prior to the administration of special measuring the absorbance at 260 nm using a spec- diets, the mice were fed standard rodent chow (CE-2, trophotometer (Beckman DU 640). Messenger RNA Clea Japan) and water ad libitum for acclimation. Age- levels of diŠerentially expressed genes were analyzed FXR Signaling Represses SULT2A Expression 317 using RT-PCR as previously described.24) Single strand dNTPs and Taq polymerase. The PCR cycle numbers cDNAs were constructed using an oligo(dT) primer with were titrated for each primer pair to assure ampliˆca- the Ready-to-Go You-Prime First-strand Beads kit tion in linear range. The reaction was completed at 7 (Amersham Pharmacia Biotech AB, Uppsala, Sweden). min incubation at 729C and PCR products were These cDNAs provided templates for PCRs using analyzed in a 2z agarose gel (wWv) containing ethidium speciˆc primers at a denaturation temperature of 949C bromide for visualization. Their intensities were for 30 sec, an annealing temperature of 579C–619C measured by use of the NIH image (version 1.59) (619C for Sult2a1) for 30 sec, and an elongation software (Bethesda, MD). The speciˆc forward and temperature of 729C for 30 sec in the presence of reversal primers for the genes examined by PCR were

Fig. 1. In‰uence of bile acid feeding on hepatic Sult2a protein expression levels. A. Immunoblot analyses of hepatic Sult2a proteins. Hepatic cytosol was prepared from FXR-null and wild-type mice fed a LCA diet for 9 days or fed a CDCA diet for 5 days. Hepatic cytosols (30 mg) were analyzed by immunoblotting with antibody to rat SULT2A1. Intensiˆed staining was shown on the top. B. Quantiˆcation of hepatic Sult2a protein levels of mice fed a LCA diet, or a CDCA diet. Data are shown as the mean±S.D. (n＝4). *, signiˆcant diŠerence from control group (pº0.05). **,(pº0.01). UT, untreated.

Table 1. Primers for RT-PCR

Gene Forward Primer Reverse Primer

mGAPDH 5?-TGCATCCTGCACCACCAACTG-3? 5?-GTCCACCACCCTGTTGCTGTAG-3? mSult2a1 5?-CGATCTATCTCGTGAGAAATCCC-3? 5?-TCTCTTCATAGTACAGTACCAAA-3? mSHP 5?-CTAGCCAAGACACTAGCCTTCC-3? 5?-TTCAGTGATGTCAACGTCTCC-3? hGAPDH 5?-TTCAACGGCACAGTCAAGG-3? 5?-CACACCCATCAAACATG-3? hSHP 5?-GCTGTCTGGAGTCCTTCTGG-3? 5?-GAGCCTCCTGCTGCAGGTGC-3? hSULT2A1 5?-TGGACAAAGCACAACTTCTG-3? 5?-TTATTCCCCATGGGAACAGCTC-3? m; mouse, h; human. 318 Masaaki MIYATA et al.

Fig. 2. Hepatic LCA and CDCA concentrations in bile acid-fed mice. Liver homogenates were prepared from FXR-null and wild-type mice fed a LCA diet for 9 days or fed a CDCA diet for 5 days. Hepatic LCA and CDCA concentrations were determined by HPLC. Combined amounts of tauro and free forms are shown. Data are shown as the mean±S.D. (n＝4). ND, not detected. UT, untreated. shown in Table 1. 9days(Fig. 2). In addition, CDCA was detected as the Statistical analysis: All values are expressed as the major bile acid in livers of FXR-null and wild-type mice means±SD. All data were analyzed by unpaired fed LCA (0.5z and 1z) diets. In the FXR-null mice fed Student's t test or Dunnett's multiple comparisons test a1z LCA diet, hepatic CDCA concentrations were for signiˆcant diŠerences between the mean values of higher than LCA. Furthermore, hepatic LCA concen- each group. trations were 3.4-fold higher in wild-type mice fed a 0.5 z LCA diet than in FXR-null mice fed a 1z LCA diet. Results In CDCA fed mice, CDCA was detected as the main bile In‰uence of bile acid feeding on hepatic content of acid component in liver together with trace amounts of Sult2a protein: Hepatic Sult2a expression levels were LCA. Hepatic CDCA concentrations were 1.8-fold increased in FXR-null and PXR-null female mice as higher in FXR-null mice fed a 0.5z CDCA diet than compared to their wild-type counterparts.6) Hepatic those in wild-type mice. Hepatic levels of LCA and contents of Sult2a protein in bile acid-treated mice were CDCA in FXR-null and wild-type mice fed a control determined by immunoblotting using rat SULT2A1 diet were below the detection limits under our condi- antibody to assess the in‰uence of the bile acid-related tions. nuclear receptors in hepatic Sult2a expression. Consis- Expression and characterization of mouse Sult2a1 tent with our previous data, Sult2a protein contents and Sult2a2: Two distinct mRNAs of mouse Sult2a were markedly higher in livers of FXR-null female mice were isolated from C57BLW6 wild-type mice by than in wild-type mice (Fig. 1A, B). Hepatic Sult2a RT-PCR. One matched with Sult2a1 (Accession protein contents were increased 2.4-fold in wild-type Number:L02335), and the other showed two nucleotide mice fed a 1z LCA diet. The Sult2a contents were also diŠerences from the reported Sult2a2 cDNA (Accession increased 1.8-fold in wild-type mice fed a 0.5z LCA Number:L27121) isolated from BALBWc mice. This diet. Signiˆcant increases (2.4-fold) in Sult2a protein cDNAwasjudgedtocorrespondtoSult2a2inthe contents were also observed in FXR-null nice fed a 1z C57BLW6 strain. Although a clear correlation between LCA diet. Hepatic Sult2a protein contents were individual cytosolic LCA sulfation activity and Sult2a markedly decreased to 20z of control in wild-type mice protein content was demonstrated,6) the correspondence fed a 0.5z CDCA diet (Fig. 1B). In contrast to between Sult2a mRNA and protein remained unclear. wild-type mice, Sult2a was signiˆcantly increased 2.1- Thus, recombinant Sult2a (Sult2a1 and Sult2a2) fold in FXR-null mice after feeding CDCA. proteins were expressed in Escherichia coli.The Hepatic LCA and CDCA concentrations: To recombinant Sult2a1 protein showed the same elec- understand the mechanism of the change in hepatic trophoretic mobility on SDS-PAGE as the major cyto- Sult2a expression in bile acid-fed mice, hepatic LCA solic Sult2a protein detected by antibody generated and CDCA concentrations were measured in mice fed a against rat SULT2A1, whereas the recombinant Sult2a2 LCA or CDCA diet. FXR-null female mice constitutive- protein exhibited a mobility diŠerent from the cytosolic ly expressing high levels of Sult2a protein did not Sult2a protein (data not shown). These results indicate accumulate LCA in livers.6) As expected, hepatic LCA that Sult2a protein quantiˆed by Western blotting concentrations were 6.9-fold higher in wild-type mice corresponds to Sult2a1. than in FXR-null mice after feeding a 1z LCA diet for Hepatic Sult2a1 mRNA levels in CDCA fed mice: FXR Signaling Represses SULT2A Expression 319

Fig. 3. In‰uence of CDCA feeding on expression levels of Sult2a1 and SHP mRNAs. A. Changes in mRNA levels of hepatic mouse Sult2a1 and mouse SHP were analyzed by RT-PCR. Hepatic mRNAs were prepared from FXR- nullandwild-typemicefeda0.5z CDCA diet for 5 days. Speciˆc primers described in Table 1 were used. B. Quantiˆcation of Sult2a1 and SHP mRNA levels. Data are shown as the mean±S.D. (n＝4). *, signiˆcant diŠerence from control group (pº0.05). UT, untreated.

Fig. 5. In‰uence of CDCA feeding on Sult2a expression in PCN- Fig. 4. Relationship between hepatic Sult2a protein and SHP treated mice. mRNA levels. Wild-type mice and FXR-null mice were fed a control diet or the Data on FXR-null and wild-type mice fed a 0.5z CDCA diet or a diet containing 0.5z CDCA for 5 days and injected with PCN (100 control diet are indicated. mgWkg, ip) the last 3 days. Hepatic cytosols were analyzed by immunoblotting with antibody to rat SULT2A1. The expression level of Sult2a protein was normalized to that of control wild-type mice. Hepatic contents of Sult2a protein were higher in female Data are shown as the mean±S.D. (n＝4). **, signiˆcant diŠerence FXR-null mice than in the corresponding wild-type from corresponding PCN-treated groups (pº0.01). mice. These results suggest the possibility that FXR mediated suppression of Sult2a expression. Clear consistent with the change in Sult2a protein levels. decreases in hepatic Sult2a content in CDCA-fed mice However, no signiˆcant decrease in Sult2a1 mRNA further support this ˆnding. Liver Sult2a1 mRNA level levels were observed in the FXR-null mice fed a CDCA was detected by RT-PCR. Under the conditions diet (Fig. 3A, B). As expected, the prototypical FXR employed, Sult2a2 mRNA was undetectable. Hepatic target gene, SHP mRNA levels were increased 1.9-fold levels of Sult2a1 mRNA were reduced to 26z of the in wild-type mice fed a CDCA diet. The same treatment wild-type control in mice fed a CDCA diet, which was decreased SHP mRNA levels in FXR-null mice, suggest- 320 Masaaki MIYATA et al.

Sult2a contents were observed between both the groups in FXR-null mice. Human SULT2A1 expression in HepG2 cells: The in‰uence of FXR agonists on human SULT2A1 levels in HepG2 cells was assessed to determine whether human SULT2A1 was also negatively regulated by FXR signal- ing. Human SULT2A1 mRNA levels were signiˆcantly decreasedinHepG2cellsafteradditionofCDCAina dose-dependent manner (Fig. 6A, B). In contrast, SHP mRNA levels were increased in these cells. These results are consistent with the in vivo data in the mice fed a 0.5z CDCA diet. Human SULT2A1 mRNA levels were also decreased in HepG2 cells treated with GW4064, a selective nonsteroidal FXR agonist. Human SULT2A1 protein levels were also decreased in HepG2 cells after addition of 100 mMofCDCA(Fig. 6C). Discussion The present study demonstrates that SULT2A is negatively regulated by CDCA signaling in vivo.The negative in‰uence of CDCA feeding on Sult2a expression is likely to be FXR-dependent as suggested by the inverse relationship (r2＝0.523) between levels of SHP mRNA and Sult2a protein. Human SULT2A1 mRNA levels were also reduced in HepG2 cells after treatment with CDCA or GW4064, similar to the results Fig. 6. In‰uence of FXR agonist on expression levels of human obtained with mouse Sult2a1 expression in vivo. SULT2A1 mRNA and protein in HepG2 cells. Consistent with these results, human SULT2A1 mRNA Total mRNAs and cytosols were prepared from HepG2 cells treated levels are decreased in cultured human primary hepato- with CDCA, GW4064 or vehicle (DMSO) for 48 hrs. A. Human cytes treated with CDCA.28) Although some diŠerences SULT2A1 and SHP mRNA levels. Human SULT2A1 and SHP mRNA levels were analysed by RT-PCR using speciˆc primers in transcriptional regulation has been demonstrated in described in Table 1. B. Relative SULT2A mRNA levels. The levels rodent and human SULT2A genes, human SULT2A1 were analysed by conventional PCR method. Data are shown as the expression is also likely to be suppressed by CDCA mean±S.D. (n＝4). *, signiˆcant diŠerence from control group activation of FXR. However, it cannot be excluded that (pº0.05). **,(pº0.01). C. Human SULT2A1 protein contents. a CDCA-mediated FXR-independent mechanism such Human SULT2A1 protein was detected by immunoblots with 29,30) antibody to human recombinant SULT2A1. as the c-Jun N-terminal kinase (JNK) signaling plays a role in suppression of SULT2A expression. It should also be noted that Sult2a protein contents ing a CDCA-mediated suppression of SHP expression. were increased in mice fed a LCA diet, thus supporting A signiˆcant inverse relationship (r2＝0.523) was found the view that Sult2a is involved in protection against between Sult2a protein levels and SHP mRNA levels LCA-induced toxicity through sulfation of LCA. LCA (Fig. 4). Furthermore, r2 value is increased to 0.672 if serves as an agonist for PXR and VDR31–33) and it was two points that show more than 1.5 of SHP mRNA level shown that transcription of rodent SULT2A is positive- are omitted. ly regulated by several nuclear receptors such as PXR, In‰uence of CDCA feeding on Sult2a expression in CAR, VDR and PPARa.20–23,28,34,35) Thus, hepatic Sult2a PCN-treated wild-type mice: Rodent SULT2A in liver protein is likely at least in part induced by PXR and is increased after treatment with PXR ligands such as CAR signaling through LCA. dexamethasone and PCN.27) Thus, the in‰uence of Although 3- to 4-fold higher hepatic LCA concentra- CDCA feeding on Sult2a induction by PCN was tions were observed in wild-type mice fed a 0.5z LCA examined. Sult2a protein contents were increased 9.0- diet, as compared to LCA in FXR-null mice fed a 1z fold in PCN-treated wild-type mice, compared to those LCA diet, the extents of increase in Sult2a protein was in control mice. (Fig. 5). The contents were reduced in lower in the former mice than that in the latter. These PCN-treated wild-type mice fed a 0.5z CDCA diet to results also support the idea that CDCA-mediated FXR 35z level of PCN-treated wild-type mice fed a control activation negatively regulates hepatic Sult2a expression diet. On the other hand, no signiˆcant diŠerences in because CDCA as well as LCA were detected as the FXR Signaling Represses SULT2A Expression 321 main bile acid component in these mice. LCA serves as a positively and negatively regulated by several bile acids weak agonist for FXR, while it strongly antagonizes through a complex nuclear receptor network to main- CDCA-mediated FXR activation.36) Thus, Sult2a1 tain bile acid homeostasis. expression in LCA-fed mice might be regulated by The hepatic bile acid components and concentration complex networks with negative FXR signaling and were analyzed and correlated with the hepatic Sult2a positive PXRWCAR signaling through CDCA and LCA, expression proˆle. Hepatic bile acid concentrations were respectively. Furthermore, PCN-mediated increases in signiˆcantly diŠerent between wild-type mice and FXR- Sult2a protein levels were attenuated by CDCA feeding null mice after feeding bile acids. Furthermore, unlike in wild-type mice. This result also suggests the possibili- in vitro LCA treatment, CDCA was detected as the ty of CDCA-mediated negative FXR signaling and the main bile acid component in livers of LCA-fed mice, potential for cross-talk between FXR and PXR in the suggesting hepatic accumulation of the LCA metabo- regulation of Sult2a expression. lite, CDCA in vivo. Hepatic bile acids are critical It was reported that CDCA-mediated FXR activation determinants for hepatic gene expression regulated by stimulates reporter CAT expression directed via the rat nuclear receptors such as PXR, CAR and FXR. The SULT2A1 promoter from －366 to ＋38 in HepG2 analyses of hepatic bile acids are thus important in order cells.19) An FXR binding site, designated IR0, in this to compare wild-type mice and null mice treated with promoter region was also demonstrated in this study. bile acids. Furthermore, PXR, CAR and VDR were found to bind In the present study, we have demonstrated that to this element and enhance reporter activity. The IR0 SULT2A-mediated LCA sulfation is negatively element has also been detected in the mouse Sult2a1 regulated by CDCA-mediated FXR activation. Further promoter, but not in the human SULT2A1 promoter. studies are needed to elucidate the precise mechanism Our results using FXR-null mice and HepG2 cells for this suppression and to determine the physiological suggest that the mouse and human SULT2As are signiˆcance of this response. negatively regulated by FXR signaling. Two possible mechanisms for negative transcriptional regulation of Acknowledgements: We thank Dr. Timothy M. Willson target genes by FXR activation have been proposed. (GlaxoSmithKline, Research Triangle Park, NC) for One is an indirect mechanism via FXR-mediated providing GW4064. induction of negative transcription factor such as SHP. This study was supported by a Grant-in Aid from the The other is a direct mechanism via FXR binding to the Ministry of Education, Science and Culture, Japan and negative response element of the target gene. Further by a Grant-in Aid from the Ministry of Health, Labor studies are needed to understand whether FXR regulates and Welfare, Japan. SULT2A expression directly or indirectly. References SULT2A seems to facilitate the excretion of toxic bile acids such as LCA from body because human and 1) Falany, C. N.: Sulfation and sulfotransferases. mouse SULT2A predominantly catalyze LCA sulfation, Introduction: changing view of sulfation and the cytosol- but not primary bile acid, CA and CDCA sulfation.6) ic sulfotransferases. Faseb J., 11: 1–2 (1997). Under physiological conditions, the main components 2) Nagata, K. and Yamazoe, Y.: Pharmacogenetics of sulfotransferase. Annu. Rev. Pharmacol. 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