Post-transcriptional Regulation of Human Pregnane X Receptor by Micro-RNA Affects the Expression of Cytochrome P450 3A4

Pregnane X receptor (PXR) is a major transcription factor regulating the inducible expression of a variety of transporters and drug-metabolizing enzymes, including CYP3A4 (cytochrome P450 3A4). We first found that the PXR mRNA level was not correlated with the PXR protein level in a panel of 25 human livers, indicating the involvement of post-transcriptional regulation. Notably, a potential miR-148a recognition element was identified in the 3′-untranslated region of human PXR mRNA. We investigated whether PXR might be regulated by miR-148a. A reporter assay revealed that miR-148a could recognize the miR-148a recognition element of PXR mRNA. The PXR protein level was decreased by the overexpression of miR-148a, whereas it was increased by inhibition of miR-148a. The miR-148a-dependent decrease of PXR protein attenuated the induction CYP3A4 mRNA. Furthermore, the translational efficiency of PXR (PXR protein/PXR mRNA ratio) was inversely correlated with the expression levels of miR-148a in a panel of 25 human livers, supporting the miR-148a-dependent regulation of PXR in human livers. Eventually, the PXR protein level was significantly correlated with the CYP3A4 mRNA and protein levels. In conclusion, we found that miR-148a post-transcriptionally regulated human PXR, resulting in the modulation of the inducible and/or constitutive levels of CYP3A4 in human liver. This study will provide new insight into the unsolved mechanism of the large interindividual variability of CYP3A4 expression. Pregnane X receptor (PXR) is a major transcription factor regulating the inducible expression of a variety of transporters and drug-metabolizing enzymes, including CYP3A4 (cytochrome P450 3A4). We first found that the PXR mRNA level was not correlated with the PXR protein level in a panel of 25 human livers, indicating the involvement of post-transcriptional regulation. Notably, a potential miR-148a recognition element was identified in the 3′-untranslated region of human PXR mRNA. We investigated whether PXR might be regulated by miR-148a. A reporter assay revealed that miR-148a could recognize the miR-148a recognition element of PXR mRNA. The PXR protein level was decreased by the overexpression of miR-148a, whereas it was increased by inhibition of miR-148a. The miR-148a-dependent decrease of PXR protein attenuated the induction CYP3A4 mRNA. Furthermore, the translational efficiency of PXR (PXR protein/PXR mRNA ratio) was inversely correlated with the expression levels of miR-148a in a panel of 25 human livers, supporting the miR-148a-dependent regulation of PXR in human livers. Eventually, the PXR protein level was significantly correlated with the CYP3A4 mRNA and protein levels. In conclusion, we found that miR-148a post-transcriptionally regulated human PXR, resulting in the modulation of the inducible and/or constitutive levels of CYP3A4 in human liver. This study will provide new insight into the unsolved mechanism of the large interindividual variability of CYP3A4 expression. A key function of the liver is the metabolism and elimination of xenobiotics or endobiotics. The expression of genes involved in these processes is largely regulated by transcription factors belonging to the nuclear receptor family. Pregnane X receptor (PXR 2The abbreviations used are:PXRpregnane X receptorAsOantisense oligonucleotideCARconstitutive androstane receptormiRNAmicro-RNARXRαretinoid X receptor αUTRuntranslated regionRTreverse transcriptionGAPDHglyceraldehyde-3-phosphate dehydrogenasesnRNAsmall nuclear RNA.; alternate names SXR, PAR, and NR1I2), a member of the nuclear receptor family, is a crucial regulator of drug metabolism and elimination. It is predominantly expressed in liver and small intestine. PXR is activated by a broad spectrum of xenobiotics, including antibiotics, antimycotics, and herbal components (1Kliewer S.A. Goodwin B. Willson T.M. Endocr. Rev. 2002; 23: 687-702Crossref PubMed Scopus (731) Google Scholar); dimerizes with retinoid X receptor α (RXRα); and binds to response elements of target genes, including cytochrome P450s, UDP-glucuronosyltransferases, glutathione S-transferases, sulfotransferases, and various transporters, such as MDR1 (multidrug resistance 1) and MRP-2 (multidrug resistance-associated protein 2) to induce them (2Meijerman I. Beijnen J.H. Schellens J.H. Oncologist. 2006; 11: 742-752Crossref PubMed Scopus (183) Google Scholar). Thus, PXR is recognized as a xenosensor for the detoxification of foreign compounds. However, it also plays a role as a physiological sensor of bile acids to protect the body from toxicity by regulating the expression of target genes that decreases the synthesis and increases the elimination of bile acids (3Staudinger J.L. Goodwin B. Jones S.A. Hawkins-Brown D. MacKenzie K.I. LaTour A. Liu Y. Klaassen C.D. Brown K.K. Reinhard J. Willson T.M. Koller B.H. Kliewer S.A. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3369-3374Crossref PubMed Scopus (1152) Google Scholar, 4Xie W. Radominska-Pandya A. Shi Y. Simon C.M. Nelson M.C. Ong E.S. Waxman D.J. Evans R.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 3375-3380Crossref PubMed Scopus (685) Google Scholar). pregnane X receptor antisense oligonucleotide constitutive androstane receptor micro-RNA retinoid X receptor α untranslated region reverse transcription glyceraldehyde-3-phosphate dehydrogenase small nuclear RNA. One of the best known genes regulated by PXR is CYP3A4, the most abundant P450 in human liver that catalyzes the metabolism of over 50% of current prescription drugs (5Blumberg B. Sabbagh Jr., W. Juguilon H. Bolado Jr., J. van Meter C.M. Ong E.S. Evans R.M. Genes Dev. 1998; 12: 3195-3205Crossref PubMed Scopus (820) Google Scholar, 6Lehmann J.M. McKee D.D. Watson M.A. Willson T.M. Moore J.T. Kliewer S.A. J. Clin. Invest. 1998; 102: 1016-1023Crossref PubMed Scopus (1389) Google Scholar, 7Goodwin B. Hodgson E. Liddle C. Mol. Pharmacol. 1999; 56: 1329-1339Crossref PubMed Scopus (592) Google Scholar). A large interindividual difference (∼50-fold) has been reported for the CYP3A4 level in the general population (8Ozdemir V. Kalowa W. Tang B.K. Paterson A.D. Walker S.E. Endrenyi L. Kashuba A.D. Pharmacogenetics. 2000; 10: 373-388Crossref PubMed Scopus (364) Google Scholar), which cannot be explained by genetic polymorphisms (9Lamba J.K. Lin Y.S. Thummel K. Daly A. Watkins P.B. Strom S. Zhang J. Schuetz E.G. Pharmacogenetics. 2002; 12: 121-132Crossref PubMed Scopus (296) Google Scholar, 10Floyd M.D. Gervasini G. Masica A.L. Mayo G. George Jr., A.L. Bhat K. Kim R.B. Wilkinson G.R. Pharmacogenetics. 2003; 13: 595-606Crossref PubMed Scopus (239) Google Scholar). The CYP3A4 expression is largely regulated at the transcriptional level by transcriptional factors, such as CCAAT/enhancer-binding proteins, C/EBPα and C/EBPβ, and hepatocyte nuclear factors, HNF4α and HNF3γ, as well as constitutive androstane receptor (CAR) and PXR (11Martínez-Jiménez C.P. Jover R. Donato M.T. Castell J.V. Gómez-Lechón M.J. Curr. Drug Metab. 2007; 8: 185-194Crossref PubMed Scopus (113) Google Scholar). However, the cause of the large interindividual variability in CYP3A4 level is poorly understood and is an urgent issue to be solved. The regulation by PXR may, in part, be responsible for such variability, since PXR is activated by endogenous compounds, such as steroid hormones and bile acids (1Kliewer S.A. Goodwin B. Willson T.M. Endocr. Rev. 2002; 23: 687-702Crossref PubMed Scopus (731) Google Scholar, 12Kretschmer X.C. Baldwin W.S. Chem. Biol. Interact. 2005; 155: 111-128Crossref PubMed Scopus (244) Google Scholar). PXR regulates many targets controlling pharmacokinetics, but its own regulation is not fully understood, with reports showing only that human PXR is induced by dexamethasone through glucocorticoid receptor (13Pascussi J.M. Drocourt L. Fabre J.M. Maurel P. Vilarem M.J. Mol. Pharmacol. 2000; 58: 361-372Crossref PubMed Scopus (330) Google Scholar) or by clofibrate through peroxisome proliferator-activated receptor α (14Aouabdi S. Gibson G. Plant N. Drug Metab. Dispos. 2006; 34: 138-144Crossref PubMed Scopus (61) Google Scholar). Employing an on-line search using the miRBase Target data base (15Griffiths-Jones S. Nucleic Acids Res. 2004; 32: D109-D111Crossref PubMed Google Scholar) (available on the World Wide Web), we found some potential recognition sites for micro-RNAs (miRNAs) in the 3′-untranslated region (UTR) of the human PXR. miRNAs are a recently discovered family of short noncoding RNA whose final product is an ∼22-nucleotide functional RNA molecule (16Bartel D.P. Cell. 2004; 116: 281-297Abstract Full Text Full Text PDF PubMed Scopus (29863) Google Scholar). They play important roles in the regulation of target genes by binding to complementary regions of transcripts to repress their translation or regulate degradation. At present, more than 400 miRNAs have been identified in humans, and miRNAs are predicted to control about 30% of the genes within the human genome (17Lewis B.P. Burge C.B. Bartel D.P. Cell. 2005; 120: 15-20Abstract Full Text Full Text PDF PubMed Scopus (9936) Google Scholar, 18Xie X. Lu J. Kulbokas E.J. Golub T.R. Mootha V. Lindblad-Toh K. Lander E.S. Kellis M. Nature. 2005; 434: 338-345Crossref PubMed Scopus (1540) Google Scholar). The roles of miRNAs have received attention especially in the cancer field but hardly yet in the field of pharmacokinetics. In the present study, we investigated whether human PXR might be post-transcriptionally regulated by miRNA and its impact on CYP3A4 expression. Chemicals and Reagents—Rifampicin was obtained from Wako Pure Chemicals (Osaka, Japan). The pGL3-promoter vector, pGL4.74-TK plasmid, Tfx-20 reagent, and dual luciferase reporter assay system were purchased from Promega (Madison, WI). Lipofectamine 2000 and Lipofectamine RNAiMAX were from Invitrogen. Pre-miR miRNA precursors for miR-148a and for the negative control were from Ambion (Austin, TX). Locked nucleic acid-modified antisense oligonucleotides (AsOs) for miR-148a (5′-ACAAAGTTCTGTAGTGCACTGA-3′; locked nucleic acid is indicated by the underline) and for the negative control (5′-AGACUAGCGGUAUCUUAAACC-3′) were commercially synthesized at Greiner Bio-One (Tokyo, Japan). All primers and oligonucleotides were commercially synthesized at Hokkaido System Sciences (Sapporo, Japan). Goat anti-human PXR polyclonal antibodies (N-16), rabbit anti-human RXRα polyclonal antibodies (D-20), and goat anti-human HNF4α polyclonal antibodies (S-20) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Rabbit anti-CYP3A4 polyclonal antibodies were from BD Gentest (Worburn, MA), and rabbit anti-human CAR polyclonal antibodies were from CHEMICON (Temecula, CA). Rabbit anti-human GAPDH polyclonal antibodies were from IMGENEX (San Diego, CA). Alexa Fluor 680 donkey anti-goat IgG was from Invitrogen. IRDye 680 goat anti-rabbit IgG was from LI-COR Biosciences (Lincoln, NE). All other chemicals and solvents were of the highest grade commercially available. Human Livers and Cell Culture Conditions—Human liver samples from 25 donors were obtained from the Human and Animal Bridging Research Organization (Chiba, Japan). The human hepatocellular carcinoma cell lines HepG2 and HuH7 were obtained from Riken Gene Bank (Tsukuba, Japan), and HLE was from the Japanese Collection of Research Bioresources (Tokyo, Japan). The human colon carcinoma cell lines LS180 and Caco-2, the human embryonic kidney cell line HEK293, and the human breast adenocarcinoma cell line MCF-7 were obtained from American Type Culture Collection (Manassas, VA). HepG2, HuH7, and HLE cells were cultured in Dulbecco's modified Eagle's medium (Nissui Pharmaceutical, Tokyo, Japan) supplemented with 10% fetal bovine serum (Invitrogen). LS180, Caco-2, and MCF-7 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 0.1 mm nonessential amino acid (Invitrogen) and 10% fetal bovine serum. Differentiated Caco-2 (Caco-2/D) cells were obtained by culture for 3 weeks postconfluence. HEK293 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 4.5 g/liter glucose, 10 mm HEPES, and 10% fetal bovine serum. These cells were maintained at 37 °C an of for PXR and RNA was from 25 human liver samples using Tokyo, Japan) to the The were synthesized from RNA using Japan). The and reverse primers for CYP3A4 were and The and reverse primers for human PXR were and The was using the with as an at °C for the was by at °C for and and at °C for for The mRNA levels were with GAPDH mRNA by as Y. M. S. M. Res. 2004; PubMed Scopus Google Scholar). and of PXR and cell were from 25 human liver samples by with mm mm mm mm The protein were using protein assay with as a The cell were with and to The were with goat anti-human PXR, rabbit anti-human CYP3A4, goat anti-human rabbit anti-human or rabbit anti-human GAPDH antibodies and the and the were with an system from the HepG2 and cells were also used to the PXR protein for the of miR-148a, and reverse transcription were using the miRNA first synthesis (Invitrogen) to the The for miR-148a was and the reverse was the supplemented The were as an at °C for the was by at °C for 10 and and at °C for 10 for The miR-148a level was with by as Y. M. S. Res. 2006; PubMed Scopus Google Scholar). of luciferase reporter various target were into the of the luciferase in the pGL3-promoter The from to in the human PXR mRNA was the miR-148a recognition element The of the is was into the pGL3-promoter The complementary of of the was also into the pGL3-promoter A the with the miR-148a, of miR-148a is was into the pGL3-promoter The of the were by luciferase reporter were with pGL4.74-TK into HEK293 or HepG2 the the cells were into of plasmid, of pGL4.74-TK plasmid, and of the precursors for miR-148a or control were into HEK293 cells using Lipofectamine HepG2 of plasmid, of pGL4.74-TK plasmid, and 10 of the for miR-148a or control were using Tfx-20 for the cells were in and the luciferase was with a using the dual luciferase reporter assay of or for miR-148a into HepG2 and and of and the of miR-148a on the expression of PXR or for miR-148a or control was into HepG2 cells using Lipofectamine RNA was using and the miR-148a levels were by as was using the nuclear and to the LS180 cells were with for miR-148a or control using Lipofectamine the cells were with or for RNA and nuclear were of the of PXR in HepG2 reporter the region to including the by and the region to including the by of the CYP3A4 to which PXR binds M. M. M. K. Y. Drug Metab. 2004; PubMed Scopus Google Scholar). The HepG2 cells were into of of pGL4.74-TK plasmid, and various of the precursors and for miR-148a or control were using Tfx-20 for the cells were with 10 or for and the luciferase was was by of by or of was with an were by A of was PXR with PXR mRNA in Human first the PXR mRNA level in a panel of 25 human by assay and investigated the with the PXR protein in was the PXR mRNA and protein levels indicating the involvement of post-transcriptional regulation of human PXR. the mechanism of the post-transcriptional we to the involvement of regulation. Employing an on-line search using the miRBase Target data base (15Griffiths-Jones S. Nucleic Acids Res. 2004; 32: D109-D111Crossref PubMed Google Scholar) (available on the World Wide Web), potential recognition elements for of such as miR-148a, and were found in the in human PXR. we on miR-148a it is and expressed in liver E. A. R. A. I. U. S. P. Y. E. M. P. Res. 2004; PubMed Scopus Google Scholar) and has in the at including the The potential miR-148a target is of the of the human PXR mRNA. The of with the of human PXR mRNA was using M. P. M. R. RNA. 2004; 10: PubMed Scopus Google Scholar) (available on the World Wide We investigated whether the miR-148a recognition element might be involved in the regulation of PXR by regulation of human PXR by miR-148a. of miR-148a to the predicted target of human PXR. The potential miR-148a recognition element is on to in the of human PXR the to the of mRNA as the miR-148a levels in HepG2, HuH7, Caco-2, LS180, HEK293, and MCF-7 cells were by using an miRNA first synthesis The were the miR-148a levels with the levels to that in HLE and luciferase were to whether is functional in the regulation by miR-148a. The reporter were with of the precursors for miR-148a or control into HEK293 cells or 10 of the for miR-148a or control into HepG2 cells The data were the luciferase with the luciferase to that of the of with with for of miR-148a in Human Cell using the miRNA first synthesis was to the expression levels of miR-148a in of human cancer cell lines The miR-148a was in cell lines in study, with large variability cell lines The which were used for in the than the miR-148a levels. The miR-148a was in HepG2 and Caco-2 cells than the other cell It was also in Caco-2 the expression level of miR-148a was increased with Thus, the expression levels of miR-148a were the human cancer cell of PXR by miR-148a in Human Cell whether is functional in the regulation by miR-148a, luciferase were with HEK293 cells We first that the luciferase of the plasmid, in which the miR-148a complementary was of the luciferase was significantly decreased by the with the for miR-148a. The luciferase of plasmid, in which of the potential miR-148a recognition were of the luciferase was also significantly decreased by with the for miR-148a of whereas that of with the recognition was not In HepG2 which the highest expression of miR-148a the luciferase of and were significantly than of the control These were significantly by the of for miR-148a. These that miR-148a to decrease the expression. of or of miR-148a on the PXR in a Human Cell the in endogenous PXR protein expression by the overexpression or inhibition of miR-148a. the of the for miR-148a into HepG2 cells that the increased level of miR-148a, the PXR protein level was significantly decreased with the control by the of the for miR-148a into HepG2 the expression of miR-148a was the PXR protein level was significantly increased with the control the expression level of RXRα a of PXR, was not by the overexpression or inhibition of miR-148a. It is well known that PXR the transcription of targets by binding to the the the element as a reporter the in the PXR protein levels were with the reporter and The luciferase of was increased by the with in HepG2 cells of the for miR-148a significantly decreased the and transcriptional resulting in a decrease of the In the of antisense for miR-148a significantly increased the and transcriptional resulting in a of the induction These that miR-148a regulates the expression of PXR protein the induction of its of miR-148a-dependent PXR in the of CYP3A4 mRNA in a Human Cell to whether the miR-148a-dependent of the PXR protein level the CYP3A4 induction in human cells were cell line expressed CYP3A4 mRNA than the other cell lines not the of the for miR-148a into the the PXR protein level was significantly decreased with a of the miR-148a whereas the PXR mRNA level was not decreased at the The RXRα protein level was not by the overexpression of miR-148a in the CYP3A4 mRNA level was significantly increased by the with However, induction was by the overexpression of miR-148a, the CYP3A4 mRNA level was not These that the miR-148a-dependent regulation of PXR the induction of The miR-148a-dependent PXR CYP3A4 in Human the of the miR-148a-dependent regulation of PXR in human liver the the expression levels of miR-148a, PXR, and CYP3A4 were investigated using a panel of 25 human The expression levels of miR-148a were in the panel of human livers. The miR-148a level in liver was with that in HepG2 The PXR mRNA and CYP3A4 mRNA were also in the PXR mRNA level was not correlated with the PXR protein In the CYP3A4 mRNA level was significantly correlated with the CYP3A4 protein level the PXR protein/PXR mRNA were as an of the translational efficiency of PXR, were inversely correlated with the miR-148a level that PXR is regulated by miR-148a in human liver. The PXR protein level was significantly correlated with the CYP3A4 mRNA level and the CYP3A4 protein level in the post-transcriptional regulation of PXR by miR-148a to have impact on the CYP3A4 level in human livers. PXR regulates at genes responsible for the metabolism and elimination of drugs (14Aouabdi S. Gibson G. Plant N. Drug Metab. Dispos. 2006; 34: 138-144Crossref PubMed Scopus (61) Google Scholar). The study of PXR regulation in the of the and variability in the of many have found variability in the PXR mRNA levels in human liver the with its protein level has not fully been In study, we first that was them in human livers. human PXR, including or the of the have been reported G. J. K. M. L. M. R. H. P. A. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar, H. E. Watson P. Clin. Res. 1999; Google Scholar), which cannot be with However, since the expression levels of these were in not with a by V. K. J.K. M. J. Strom S. Schuetz E.G. Pharmacol. 2004; PubMed Scopus Google Scholar), the of the PXR protein level with its mRNA level is not an of the miRNA recognition element in the human PXR the involvement of miRNA in the regulation of PXR. The luciferase that the endogenous and miR-148a regulated the through In the endogenous PXR protein level was by the overexpression of miR-148a and by its These indicated that human PXR is post-transcriptionally regulated by miR-148a. The miR-148a-dependent of PXR protein the induction of CYP3A4 in LS180 whether the miR-148a the induction of other targets of PXR, we the expression levels of MDR1 and in LS180 cells not induced the MDR1 and known targets of PXR I. 2001; PubMed Scopus Google Scholar, B. Moore C.M. McKee D.D. Kliewer S.A. Mol. Pharmacol. 2001; Google Scholar), and the induction was attenuated by the overexpression of miR-148a. Thus, the miR-148a-dependent regulation of PXR to its target genes in the miR-148a recognition element is also present in the of CYP3A4 mRNA. The of CYP3A4 with was than that of human PXR whether CYP3A4 is regulated by miR-148a, luciferase were using a of the miR-148a recognition element in the CYP3A4 However, the the element in CYP3A4 not to miR-148a indicating that CYP3A4 is not regulated by miR-148a. In the panel of human livers, the expression level of miR-148a was inversely correlated with the translational efficiency of PXR, supporting the role of miR-148a in the regulation of PXR in liver. The the CYP3A4 mRNA and the CYP3A4 protein level in human in study, in with A. S. A. C. I. M. Drug Metab. Dispos. 2003; PubMed Scopus Google Scholar, M. N. M. S. S. J. Pharmacol. Sci. 2004; PubMed Scopus Google Scholar), the that miRNA not regulate the CYP3A4 expression. The PXR protein level was correlated with the CYP3A4 mRNA level in human indicating that miR-148a the CYP3A4 expression through PXR expression. The PXR protein level was not correlated with the or MDR1 mRNA levels in the panel of human not in to Thus, we that the PXR not largely the constitutive expression of and MDR1 in the liver. In panel of human livers, the CYP3A4 mRNA level was not correlated with the HNF4α protein level or CAR protein level indicating a of PXR to the constitutive CYP3A4 of the genes in the nuclear receptor are The of PXR amino acid of human and which the key for PXR the of PXR more than amino acid (1Kliewer S.A. Goodwin B. Willson T.M. Endocr. Rev. 2002; 23: 687-702Crossref PubMed Scopus (731) Google Scholar), a of miRNAs are which that the regulation of genes be The is also identified in the in PXR and PXR and are not at but it has only a difference from the at of the the of PXR is poorly and It is that PXR also be regulated by miR-148a, that might be to the role of miR-148a in drug metabolism and elimination in In conclusion, we found that human PXR is post-transcriptionally regulated by miR-148a the CYP3A4 level in human liver. This study provide new insight into the unsolved mechanism of the large interindividual variability of CYP3A4 expression. We are to and of Drug and of Japan) for We for the with