Note Rapid and Drastic Induction of CYP3A4 Mrna Expression Via Vitamin D Receptor in Human Intestinal LS180 Cells

Drug Metab. Pharmacokinet. 22 (5): 377–381 (2007).

Note Rapid and Drastic Induction of CYP3A4 mRNA Expression via Vitamin D Receptor in Human Intestinal LS180 Cells

Shiro FUKUMORI, Toshiya MURATA, Masato TAGUCHI and Yukiya HASHIMOTO* Graduate School of Pharmaceutical Sciences, University of Toyama, Toyama, Japan

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

Summary: The aim of this study was to evaluate the usefulness of human intestinal LS180 cells for studying the induction of CYP3A4 mRNA expression via vitamin D receptor (VDR). CYP3A4 mRNA ex-

pression in LS180 cells treated with 100 nM 1a,25-dihydroxyvitamin D3 (1a,25(OH)2D3)for6and24h was approximately 80- and 500-fold higher than the control, respectively. A protein kinase (PK) inhibitor (staurosporine), c-jun N-terminal kinase (JNK) pathway inhibitor (curcumin), and JNK inhibitor

(SP600125) attenuated 1a,25(OH)2D3-induced CYP3A4 mRNA expression, suggesting that the PK-JNK pathway contributed to the rapid and drastic induction of CYP3A4 expression via VDR in LS180 cells. The ability of CYP3A4 mRNA induction in LS180 cells was highly dependent on the site and number of

vitamin D3 and D2 hydroxylation. In addition, short-time (6 h) treatment of LS180 cells with cytotoxic secondary bile acids, lithocholic acid (LCA) and 3-keto-LCA also signiˆcantly induced the mRNA expression of CYP3A4. LS180 cells may be useful to quickly investigate the CYP3A4-inducing eŠect of drugs, xenobiotics, and/or endogenous substrates in the intestinal epithelia.

Key words: 1a,25-dihydroxyvitamin D3; CYP3A4; VDR; LS180 cells

centration.4) Introduction The expression of CYP3A4 protein in the liver is in- Humans have 17 known cytochrome P450 (CYP) duced by the nuclear pregnane X receptor (PXR), which gene families, among which only the ˆrst three, CYP1, is activated by a wide variety of structurally diverse ex- CYP2, and CYP3, are involved in the metabolism of ogenous and endogenous chemicals, including drugs drugs and xenobiotics.1) The major adult CYP3A en- such as rifampicin, phenobarbital, and hyperforin.1,5) zyme, CYP3A4, is predominately expressed in human On the other hand, the expression of CYP3A4 in the in- liver and is the most abundant hepatic CYP protein.2) testine is regulated by the nuclear vitamin D receptor CYP3A4 is involved in the systemic and presystemic (VDR) in addition to PXR.5,6) VDR is expressed abun- (ˆrst-pass) metabolism of many drugs and xenobiotics, dantly in the human intestine, and may act as a physio- converting them to more hydrophilic compounds that logical sensor of certain molecules which are metabo- are more easily eliminated from the body. Recently, it lized by CYP3A4.2) Recently, Makishima et al. have was shown that CYP3A4 is expressed not only in the reported that VDR functions as a receptor for cytotoxic liver but also in the intestinal epithelia, and that the in- secondary bile acid, lithocholic acid (LCA) and its hibition of intestinal CYP3A4 function causes a sig- major metabolite 3-keto-LCA, and that these com- niˆcant increase in the blood concentration of orally ad- pounds induce the expression of CYP3A in vivo.7) The ministered therapeutic compounds.3) These ˆndings in- ˆnding suggests that CYP3A4 in conjunction with VDR dicate that hepatic and also intestinal ˆrst-pass serves as a potential detoxiˆcation system in the enteric metabolism mediated by CYP3A4 is a primary factor tract.7) which determines drug bioavailability and blood con- In the previous report, we examined the induction

eŠect of 1a,25-dihydroxyvitamin D3 (1a,25(OH)2D3)on This work was supported in part by a Grant-in-Aid for Scientiˆc CYP3A4 mRNA expression under various culture con- Research from the Japan Society for the Promotion of Sciences ditions in two human intestinal cell lines, Caco-2 and (JSPS). LS180.8) Caco-2 cells formed a monolayer expressing

Received; June 4, 2007, Accepted; July 9, 2007 *To whom correspondence should be addressed: Yukiya HASHIMOTO,Ph.D.,Graduate School of Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan. Tel. ＋81-76-434-7585, Fax. ＋81-76-434-7587, E-mail: yukiya＠pha.u-toyama.ac.jp

377 378 Shiro FUKUMORI, et al.

CYP3A4 by treatment with 100–250 nM 1a,25(OH)2D3 curcumin), or a JNK inhibitor (15 mM SP600125) at 30 8,9) 10,11) for 2–3 weeks. In contrast, LS180 cells responded min before the addition of 30 nM 1a,25(OH)2D3. quickly to 1a,25(OH)2D3 treatment by increasing their Real-time PCR assay of CYP3A4 mRNA: Total mRNA expression of CYP3A4. That is, it takes only a RNA was isolated from the cells using an RNA extrac- few days to obtain more than 100-fold induction of tion kit (RNeasyMini Kit, QIAGEN, Valencia, CA, CYP3A4 mRNA by treatment of LS180 cells with USA) according to the manufacturer's instructions.8) 8) 100–250 nM 1a,25(OH)2D3. In the present study, we Reverse transcription of extracted total RNA was per- further evaluated the usefulness of LS180 cells for formed as previously described.8) Real-time PCR was studying the induction of CYP3A4 in the intestine. carried out on an Mx3000P (Stratagene, La Jolla, CA, USA) using SYBRPremix Ex Taq (TaKaRa, Tokyo, Methods Japan) according to the manufacturer's instructions.

Materials: 1a,25(OH)2D3, rifampicin, staurospo- Primer sequences for CYP3A4 and b-actin have been rine, and curcumin were purchased from Wako Pure reported previously.8) Cycling conditions were 1 cycle

Chemicals (Osaka, Japan), and vitamin D3 was ob- for30-sat959C, followed by 45 cycles of 5-s denatura- tained from Nacalai Tesque (Kyoto, Japan). SP600125, tion at 959C, 20-s annealing at 609C, and 15-s extension

25-hydroxyvitamin D3 (25(OH)D3), and 1a-hydroxyvit- at 609C. The mRNA level of CYP3A4 was normalized amin D2 (1a(OH)D2) were purchased from Calbiochem according to the b-actin mRNA level, and the ratio is 8) (La Jolla, CA, USA). 1a-hydroxyvitamin D3 presented using a common logarithm. (1a(OH)D3) was purchased from Alexis Biochemicals Data analysis: Values are expressed as the mean±

(San Diego, CA, USA), and vitamin D2 was purchased S.E. Multiple comparisons were performed using from Tokyo Kasei Kogyo (Tokyo, Japan). LCA and 3- ScheŠáe's test following one-way ANOVA provided that keto LCA were purchased from Sigma (St. Louis, MO, the variances of groups were similar. If this was not the USA). Dulbecco's modiˆed Eagle's medium (DMEM) case, ScheŠáe-type test was applied following Kruskal- was acquired from Nihon Pharmaceutical (Tokyo, Wallis analysis. pº0.05 was considered statistically sig- Japan). Fetal bovine serum (FBS) was acquired from niˆcant. Valley Biochemical (Winchester, VA, USA). All other Results and Discussion chemicals were of the ˆnest grade available. Cell culture: LS180 cells at passage number 38 were Thummel et al. have reported that 48-h treatment of purchased from the American Type Culture Collection LS180 cells with 1 to 100 nM 1a,25(OH)2D3 increased (Manassas, VA, USA), and all experiments were con- the CYP3A4 protein and CYP3A4 mRNA expression in ducted with LS180 cells between passages 43 to 55. The a concentration dependent manner.6) In the present cells were maintained with DMEM supplemented with study, we ˆrst investigated CYP3A4 mRNA expression 10z heat-inactivated FBS, 100 mM nonessential amino following short-time (6–24 h) treatment of LS180 cells acids, 100 units/mL penicillin G, and 100 mg/mL strep- with 1a,25(OH)2D3. Table 1 shows the log(CYP3A4/ tomycininanatmosphereof5z CO2-95z air at 379C. b-actin) value of cells treated with 10, 30, and 100 nM

When the cells reached 80-90z con‰uence, they were 1a,25(OH)2D3. Treatment of LS180 cells with subcultured using a 0.05z trypsin/0.02z EDTA solu- 1a,25(OH)2D3 did not aŠect the expression of b-actin tion.8) LS180 cells were seeded at a density 5×105 mRNA. CYP3A4 mRNA expression after only 6-h 2 2 cells/cm on a 3.8 cm plastic dish using a Falconmul- treatment with 1a,25(OH)2D3 increased approximately tiwellTM plate (BD Bioscience, Bedford, U.S.A.), and 60-, 70-, and 80-fold at concentrations of 10, 30, and were maintained for 2 days.8) 100 nM, respectively. Prolonged treatment of the cells Treatment of cells: We evaluated the CYP3A4 mRNA-inducing eŠect of 1a,25(OH)2D3, rifampicin, 1a(OH)D3,25(OH)D3, vitamin D3, vitamin D2, Table 1. Log(CYP3A4/b-actin) values in LS180 cells treated with

1a(OH)D2, LCA, and 3-keto-LCA. The drugs were 1a,25(OH)2D3 and rifampicin added to culture medium supplemented with 45 nM (±) Treatment period -a-tocopherol, 0.1 mM sodium selenite, 3 mMzincsul- Treatment (mM) fate, and 5 mM ferrous sulfate. The cells were main- 6h 15h 24h tained with drug-containing culture medium for 6–24 h Control －4.77±0.04 －4.15±0.10 －4.59±0.04 in order to evaluate the induction of CYP3A4 mRNA 1a,25(OH)2D3 0.01 －2.99±0.03* －2.20±0.03* －2.23±0.06* expression.8) To evaluate the eŠects of the cellular phos- 0.03 －2.93±0.03* －2.03±0.04* －2.05±0.04* 0.1 －2.87±0.04* －1.98±0.03* －1.87±0.03* phorylation state on 1a,25(OH)2D3-induced CYP3A4 Rifampicin 10 — — －4.08±0.03* mRNA expression, LS180 cells were treated with a protein kinase (PK) inhibitor (500 nM staurosporine), a c- Values are expressed as the mean±S.E. for 3 experiments. jun N-terminal kinase (JNK) pathway inhibitor (15 mM *pº0.05: signiˆcantly diŠerent from each control. Dramatic CYP3A4 Induction by VDR Ligands in LS180 379

with 1a,25(OH)2D3 resulted in further increases of VDR ligands, we further examined the CYP3A4-induc- CYP3A4 mRNA expression. In particular, CYP3A4 ing eŠect of commercially available vitamin D analogs mRNA expression increased by approximately 500-fold in LS180 cells (Table 2). The rank order of CYP3A4-in- following 24-h treatment with 100 nM 1a,25(OH)2D3 ducing activity of vitamin D analogs was 1a,25(OH)2D3 (Table 1). On the other hand, CYP3A4 mRNA expression in cells treated with a PXR ligand, rifampicin, increased marginally as compared with the control (Table Table 2. The induction eŠect of vitamin D analogs on CYP3A4 1). These results indicated that 1a,25(OH)2D3 induced mRNA expression in LS180 cells CYP3A4 expression rapidly and drastically in LS180 Vitamin D Concentration cells, and that VDR played an important role in the analogs (mM) log(CYP3A4/b-actin) regulation of CYP3A4 expression in LS180 cells. Control －3.82±0.02 (45) Recent studies revealed that the cellular phosphoryla- 1a,25(OH)2D3 0.01 －1.88±0.05 (6) tion state was a key event in the xenobiotics-induced ex- 0.03 －1.65±0.05 (6) pression of CYP1A, 2B, and 3A subfamilies.12,13) Spe- 0.1 －1.65±0.05 (6) ciˆcally, Hara et al. have reported that alteration of the 1a(OH)D3 0.01 －3.59±0.09 (6) cellular phosphorylation state mediated by PK and JNK 0.03 －2.64±0.07 (6) 0.1 －1.98±0.02 (6) aŠected 1a,25(OH) D -induced CYP3A4 mRNA ex- 2 3 25(OH)D 0.1 －3.41±0.04 (6) 10,11) 3 pression in Caco-2 cells. In the present study, to 0.3 －2.15±0.03 (6) clarify whether PK and JNK modulate CYP3A4 mRNA 1 －1.75±0.04 (6) a expression via VDR in LS180 cells, we next evaluated Vitamin D3 10 －3.28±0.02 (6) the eŠect of staurosporine, curcumin, and SP600125 on 30 －3.37±0.02 (6) 100 －2.50±0.03 (6) 30 nM 1a,25(OH)2D3-induced CYP3A4 mRNA expres- 1a(OH)D2 0.01 －2.55±0.05 (6) sion. Treatment of LS180 cells with 500 nM staurospo- 0.03 －1.83±0.09 (6) rine caused a 46z decrease in the 1a,25(OH)2D3-in- 0.1 －1.50±0.10 (6) b duced CYP3A4 mRNA expression (Fig. 1A). Further- Vitamin D2 3 －3.23±0.02 (6) more, treatment with 15 mM curcumin and SP600125 10 －2.87±0.04 (6) 30 －2.57±0.03 (6) caused the 55z and 79z decreases, respectively, in the

1a,25(OH)2D3-induced CYP3A4 mRNA expression Number of experiments is shown in parentheses. (Fig. 1B, 1C). These results suggested that the PK-JNK pathway contributed signiˆcantly to the rapid and drastic induction of CYP3A4 mRNA expression in LS180 cells treated with 1a,25(OH)2D3. To evaluate the structure-activity relationship of

Fig. 1. EŠect of staurosporine (St), curcumin (Cur), and SP600125 (SP) on 1a,25(OH)2D3-induced CYP3A4 mRNA expression. LS180 cells were pretreated with 500 nM St, 15 mM Cur, and 15 mM SP for 30 min (A, B, C), followed by incubation of the cells with 30 nM

1a,25(OH)2D3 for 6 h. Each column represents the mean±S.E. for 4-6 experiments. *pº0.05: signiˆcantly diŠerent from 1a,25(OH)2D3-treated cells. 380 Shiro FUKUMORI, et al.

CA at concentrations of 3, 10, and 30 mM, respectively (Fig. 2). In conclusion, the present study indicated that the PK-JNK pathway was involved in the rapid and drastic induction of CYP3A4 expression via VDR in LS180 cells. LS180 cells may be useful to quickly investigate the CYP3A4-inducing eŠect of drugs, xenobiotics, and/or endogenous substances in the intestinal epithelia. References 1) Pascussi, J. M., Gerbal-Chaloin, S., Drocourt, L., Maurel, P. and Vilarem, M. J.: The expression of CYP2B6, CYP2C9 and CYP3A4 genes: a tangle of net- works of nuclear and steroid receptors. Biochim. Biophys. Acta., 1619: 243–253 (2003). 2) Bodin, K., Lindbom, U. and Diczfalusy, U.: Novel path- ways of bile acid metabolism involving CYP3A4. Biochim. Biophys. Acta., 1687: 84–93 (2005). Fig. 2. Dose-dependent eŠect of lithocholic acid (LCA) and 3-keto- 3) Paine, M. F., Khalighi, M., Fisher, J. M., Shen, D. D., LCA on CYP3A4 mRNA levels in LS180 cells. Kunze, K. L., Marsh, C. L., Perkins, J. D. and Thum- Cells were treated with LCA (open column) and 3-keto-LCA (hatched mel, K. E.: Characterization of interintestinal and in- column) at the indicated concentrations for 6 h. Each column traintestinal variations in human CYP3A-dependent represents the means±S.E. for 6 experiments. *pº0.05: signiˆcantly metabolism. J. Pharmacol. Exp. Ther., 283: 1552–1562 diŠerent from the control value. (1997). 4) Andersen, V., Pedersen, N., Larsen, N. E., Sonne, J. and Larsen, S.: Intestinal ˆrst pass metabolism of midazolam in liver cirrhosis-eŠect of grapefruit juice. Æ1a(OH)D2À1a(OH)D3Æ25(OH)D3Àvitamin D2Ævi- Br.J.Clin.Pharmacol., 54: 120–124 (2002). tamin D (Table 2). The ˆnding indicated that the abil- 3 5) Lehmann, J. M., McKee, D. D., Watson, M. A., ity of CYP3A4 induction was highly dependent on the Willson,T.M.,Moore,J.T.andKliewer,S.A.:The site and number of vitamin D2 and D3 hydroxylation. In human orphan nuclear receptor PXR is activated by addition, it should be noted that the CYP3A4-inducing compounds that regulate CYP3A4 gene expression and eŠect of vitamin D2 was higher than that of vitamin D3, cause drug interactions. J. Clin. Invest., 102: 1016–1023 and that the eŠect of 1a(OH)D2 washigherthanthatof (1998). 1a(OH)D3. Vitamin D2 and vitamin D3 share a common 6) Thummel, K. E., Brimer, C., Yasuda, K., Thottassery, steroid-like nucleus (carbon atoms 1–19) but have diŠer- J.,Senn,T.,Lin,Y.,Ishizuka,H.,Kharasch,E., ent side chains (carbon atoms 20–25).14) That is, vitamin Schuetz, J. and Schuetz, E.: Transcriptional control of intestinal cytochrome P-4503A by 1alpha,25-dihydroxy D2 possesses a methyl group in the 24 position and a double bond between carbon atoms 22 and 23.14) The vitamin D3. Mol. Pharmacol., 60: 1399–1406 (2001). structural diŠerence in the side chains may also be partly 7) Makishima,M.,Lu,T.T.,Xie,W.,Whitˆeld,G.K., Domoto,H.,Evans,R.M.,Haussler,M.R.andMan- responsible for the diŠerence in the ability of CYP3A4 gelsdorf, D. J.: Vitamin D receptor as an intestinal bile induction among vitamin D analogs. acid sensor. Science, 296: 1313–1316 (2002). The induction of CYP3A4 mRNA expression via 8) Aiba,T.,Susa,M.,Fukumori,S.andHashimoto,Y.: 8) VDR in Caco-2 cells requires at least several days. We The eŠects of culture conditions on CYP3A4 and MDR1 tried to evaluate the eŠect of cytotoxic secondary bile mRNA induction by 1a,25-dihydroxyvitamin D3 in hu- acids on CYP3A4 mRNA expression in Caco-2 cells; man intestinal cell lines, Caco-2 and LS180. Drug however, treatment of the cells with 30 mM LCA result- Metab. Pharmacokinet., 20: 268–274 (2005). ed in cell death within a few days (unpublished observa- 9) Schmiedlin-Ren, P., Thummel, K. E., Fisher, J. M., tion). In the present study, therefore, we utilized LS180 Paine, M. F., Lown, K. S. and Watkins, P. B.: Expres- cells to evaluate the CYP3A4-inducing eŠect of cytotox- sion of enzymatically active CYP3A4 by Caco-2 cells grown on extracellular matrix–coated permeable sup- ic secondary bile acids. We treated LS180 cells with ports in the presence of 1a,25-dihydroxyvitamin D . 3–100 mM LCA and 3-keto-LCA for 6 h. CYP3A4 3 Mol. Pharmacol., 51: 741–754 (1997). mRNA expression increased 4-, 16-, and 20-fold by 10) Hara,H.,Yasunami,Y.andAdachi,T.:Alterationof treatmentwith10,30,and100mM LCA, respectively cellular phosphorylation state aŠects vitamin D receptor- (Fig. 2). On the other hand, mRNA expression in- mediated CYP3A4 mRNA induction in Caco-2 cells. creased 5-, 23-, and 56-fold by treatment with 3-keto-L- Dramatic CYP3A4 Induction by VDR Ligands in LS180 381

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