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• W2020872262 abstract "It is a long-standing observation that inflammatory responses and infections decrease drug metabolism capacity in human and experimental animals. Cytochrome P-450 3A4 cyp304 is responsible for the metabolism of over 50% of current prescription drugs, and cyp3a4 expression is transcriptionally regulated by pregnane X receptor (PXR), which is a ligand-dependent transcription factor. In this study, we report that NF-κB activation by lipopolysaccharide and tumor necrosis factor-α plays a pivotal role in the suppression of cyp3a4 through interactions of NF-κB with the PXR·retinoid X receptor (RXR) complex. Inhibition of NF-κB by NF-κB-specific suppressor SRIκBα reversed the suppressive effects of lipopolysaccharide and tumor necrosis factor-α. Furthermore, we showed that NF-κB p65 disrupted the association of the PXR·RXRα complex with DNA sequences as determined by electrophoretic mobility shift assay and chromatin immunoprecipitation assays. NF-κB p65 directly interacted with the DNA-binding domain of RXRα and may prevent its binding to the consensus DNA sequences, thus inhibiting the transactivation by the PXR·RXRα complex. This mechanism of suppression by NF-κB activation may be extended to other nuclear receptor-regulated systems where RXRα is a dimerization partner. It is a long-standing observation that inflammatory responses and infections decrease drug metabolism capacity in human and experimental animals. Cytochrome P-450 3A4 cyp304 is responsible for the metabolism of over 50% of current prescription drugs, and cyp3a4 expression is transcriptionally regulated by pregnane X receptor (PXR), which is a ligand-dependent transcription factor. In this study, we report that NF-κB activation by lipopolysaccharide and tumor necrosis factor-α plays a pivotal role in the suppression of cyp3a4 through interactions of NF-κB with the PXR·retinoid X receptor (RXR) complex. Inhibition of NF-κB by NF-κB-specific suppressor SRIκBα reversed the suppressive effects of lipopolysaccharide and tumor necrosis factor-α. Furthermore, we showed that NF-κB p65 disrupted the association of the PXR·RXRα complex with DNA sequences as determined by electrophoretic mobility shift assay and chromatin immunoprecipitation assays. NF-κB p65 directly interacted with the DNA-binding domain of RXRα and may prevent its binding to the consensus DNA sequences, thus inhibiting the transactivation by the PXR·RXRα complex. This mechanism of suppression by NF-κB activation may be extended to other nuclear receptor-regulated systems where RXRα is a dimerization partner. Inflammatory responses and infections suppress the biotransformation of drugs and decrease the hepatointestinal capacity of drug clearance. This results in alterations of therapeutic indices and increases the toxicity of certain administered drugs. Inflammatory responses also play important roles in liver pathological conditions such as drug-induced hepatitis and cholestatic diseases (1Lehmann V. Freudenberg M.A. Galanos C. J. Exp. Med. 1987; 165: 657-663Crossref PubMed Scopus (490) Google Scholar, 2Pirovino M. Meister F. Rubli E. Karlaganis G. Gastroenterology. 1989; 96: 1589-1595Abstract Full Text PDF PubMed Scopus (39) Google Scholar). The mechanisms of these clinically important effects have not been well understood. In human liver, the first pass of biotransformation is mainly carried out by cytochrome P-450 (CYP) 2The abbreviations used are: CYP3A4, cytochrome P-450 3A4; hPXR, human pregnane X receptor; hPXR, human PXR; RXR, retinoid X receptor; RIF, rifampicin; SRIκBα, super repressor IκBα; LPS, lipopolysachride; ChIP, chromatin immunoprecipitation; EMSA, electrophoretic mobility shift assay; TNF, tumor necrosis factor; PBS, phosphate-buffered saline; GST, glutathione S-transferase; PIPES, 1,4-piperazinediethanesulfonic acid. 2The abbreviations used are: CYP3A4, cytochrome P-450 3A4; hPXR, human pregnane X receptor; hPXR, human PXR; RXR, retinoid X receptor; RIF, rifampicin; SRIκBα, super repressor IκBα; LPS, lipopolysachride; ChIP, chromatin immunoprecipitation; EMSA, electrophoretic mobility shift assay; TNF, tumor necrosis factor; PBS, phosphate-buffered saline; GST, glutathione S-transferase; PIPES, 1,4-piperazinediethanesulfonic acid. 3A4, which is the predominant isoform of monooxygenases that are expressed in the adult hepatointestinal system. It is estimated that CYP3A4 is responsible for the metabolism of over 50% of drugs in use today, many of which are either metabolically activated and/or metabolically broken down (detoxified) through this enzyme. Therefore, transcriptional and post-transcriptional alterations of CYP3A4 activity have direct effects on the efficacy of drugs and detoxification of xenobiotics (reviewed in Refs. 3Guengerich F.P. Annu. Rev. Pharmacol. Toxicol. 1999; 39: 1-17Crossref PubMed Scopus (1045) Google Scholar and 4Quattrochi L.C. Guzelian P.S. Drug Metab. Dispos. 2001; 29: 615-622PubMed Google Scholar). Recent molecular and pharmacological studies have demonstrated that transcriptional activation of cyp3a4 is mediated by the nuclear receptor PXR (pregnane X receptor). The rodent PXR (5Kliewer S.A. Moore J.T. Wade L. Staudinger J.L. Watson M.A. Jones S.A. McKee D.D. Oliver B.B. Willson T.M. Zetterstrom R.H. Perlmann T. Lehmann J.M. Cell. 1998; 92: 73-82Abstract Full Text Full Text PDF PubMed Scopus (1317) Google Scholar) and its human homolog hPXR (6Lehmann J.M. McKee D.D. Watson M.A. Willson T.M. Moore J.T. Kliewer S.A. J. Clin. Investig. 1998; 102: 1016-1023Crossref PubMed Scopus (1365) Google Scholar), also known as steroid and xenobiotic receptor (7Blumberg 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 (812) Google Scholar) or hPAR (8Bertilsson G. Heidrich J. Svensson K. Asman M. Jendeberg L. Sydow-Backman M. Ohlsson R. Postlind H. Blomquist P. Berkenstam A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12208-12213Crossref PubMed Scopus (785) Google Scholar), were identified as xenobiotic receptors that can be activated by certain xenobiotics and endobiotics. PXR regulates the expression of cyp3a4 by associating with its obligate partner RXR, and the heterodimer binds to the nuclear receptor response elements found in the regulatory regions of these genes. Genes that are regulated by PXR include multiple drug-resistant genes such as MDR1 (9Synold T.W. Dussault I. Forman B.M. Nat. Med. 2001; 7: 584-590Crossref PubMed Scopus (755) Google Scholar) and MRP2 (10Kast H.R. Goodwin B. Tarr P.T. Jones S.A. Anisfeld A.M. Stoltz C.M. Tontonoz P. Kliewer S. Willson T.M. Edwards P.A. J. Biol. Chem. 2002; 277: 2908-2915Abstract Full Text Full Text PDF PubMed Scopus (766) Google Scholar) as well as genes involved in metabolism and transport of endogenous molecules, including bilirubin, bile acids, thyroid hormone, fatty acids, and steroids (11Goodwin B. Moore J.T. Trends Pharmacol. Sci. 2004; 25: 437-441Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar, 12Guo G.L. Staudinger J. Ogura K. Klaassen C.D. Mol. Pharmacol. 2002; 61: 832-839Crossref PubMed Scopus (122) Google Scholar, 13Kullak-Ublick G.A. Stieger B. Meier P.J. Gastroenterology. 2004; 126: 322-342Abstract Full Text Full Text PDF PubMed Scopus (543) Google Scholar). PXR·RXR can also interact with pathways regulated by other nuclear receptors such as the constitutive androstane receptor·RXR by mutual binding to the consensus regulatory DNA sequences, thus forming a redundant, compensatory network for the metabolism and disposition of xenobiotic and endobiotics (14Xie W. Barwick J.L. Simon C.M. Pierce A.M. Safe S. Blumberg B. Guzelian P.S. Evans R.M. Genes Dev. 2000; 14: 3014-3023Crossref PubMed Scopus (460) Google Scholar). The mechanisms of cyp3a4 suppression caused by inflammatory responses and infections have been investigated (15Morgan E.T. Drug Metab. Rev. 1997; 29: 1129-1188Crossref PubMed Scopus (422) Google Scholar, 16Renton K.W. Curr. Drug Metab. 2004; 5: 235-243Crossref PubMed Scopus (153) Google Scholar). Several aspects of the transcriptional regulation may be involved including decreases of PXR and RXR mRNA levels or induction of the liver inhibitory protein, which suppresses cyp3a4 through a distal flanking region (17Martinez-Jimenez C.P. Gomez-Lechon M.J. Castell J.V. Jover R. Mol. Pharmacol. 2005; 67: 2088-2101Crossref PubMed Scopus (60) Google Scholar). It is likely that modulation of transcriptional activation by several pathways leads to down-regulation of PXR-regulated gene expression. It has been shown that most inflammatory cytokines induced during sepsis and aseptic responses lead to suppression of CYP3A4 gene expression. We hypothesize that there may be immediate, early events at transcriptional level where the effects of the proinflammatory responses converge. One of the critical responses to acute infections and inflammations is the activation of NF-κB (18Aggarwal B.B. Cancer Cell. 2004; 6: 203-208Abstract Full Text Full Text PDF PubMed Scopus (1363) Google Scholar, 19Karin M. Greten F.R. Nat. Rev. Immunol. 2005; 5: 749-759Crossref PubMed Scopus (2495) Google Scholar, 20Xiao C. Ghosh S. Adv. Exp. Med. Biol. 2005; 560: 41-45Crossref PubMed Scopus (134) Google Scholar), which has pleiotropic functions and has been shown to down-regulate the transcriptional activity of multiple steroid/nuclear receptors (21McKay L.I. Cidlowski J.A. Endocr. Rev. 1999; 20: 435-459Crossref PubMed Google Scholar). NF-κB regulates innate as well as adaptive immune systems. One of the pivotal functions of NF-κB is its swift activation in response to LPS or proinflammatory cytokines, which is an evolutionally conserved defensive mechanism against infections. The classic NF-κB consists of p65 (RelA) and p50 heterodimer, and it is activated in response to various stimuli including LPS, TNF-α, double-stranded RNA, and UV radiation. In this study, we investigate the role of NF-κB in regulation of the transcriptional activity of the PXR·RXRα complex in an attempt to address the mechanism of suppression of cyp3a4 by LPS and proinflammatory cytokine TNF-α. The results reveal that NF-κB plays an important role in suppression of PXR·RXRα-regulated gene expression by interfering with the binding of PXR·RXRα to the regulatory DNA sequences. The mechanism may have general implications in gene expressions regulated by nuclear receptors where RXRα is a common dimerization partner. Oligonucleotides as the PCR primers and ER6 EMSA probe, the DNA modifying enzymes, and Lipofectamine were from Invitrogen. Dulbecco's modified Eagle's medium was from Invitrogen or HyClone (Logan, UT), fetal bovine serum was from Atlanta Biologicals (Lawrenceville, GA). Plasmid DNA purification kits, rifampicin, lipopolysachride, and monoclonal antibody against the FLAG tag were from Sigma. Recombinant human TNF-α was purchased from Roche Applied Science (Indianapolis, IN). The polyclonal antibodies against RXRα and p65 were from Santa Cruz (Santa Cruz, CA). The human HepG2 cell line was purchased from the American Type Culture Collection (Manassas, VA). Plasmid Constructs—The reporter plasmid pGL3–3A4-Luc was constructed via the following steps. First, the promoter module (–362/+53)-containing DNA fragment was generated by PCR amplification using human genomic DNA as the template with the primers (5′-CATTGCTGGCTGAGGTGGTT-3′ and 5′-CATAAGCTTTGTTGCTCTTTGCTGGGCTATGTGC-3′). The 1.13-kb PCR product was restricted with BglII and Hind III, and the resultant 415-bp fragment was cloned into pGL3-basic vector (Promega) to yield pGL3-3A4 (–362/+53). The DNA fragment corresponding to the XREM region (–7836 to –7208) (23Goodwin B. Hodgson E. Liddle C. Mol. Pharmacol. 1999; 56: 1329-1339Crossref PubMed Scopus (585) Google Scholar) were generated by PCR with the primer oligonucleotides CYP3A4-3 (5′-GGGGTACCATTCTAGAGAGATGGTTCATTCC-3′) and CYP3A4-4 (5′-CCGCTCGAGATCTTCGTCAACAGGTTAAAGGAG-3′), 5′ KpnI site and 3′ BglII site were created by restriction digestion. The KpnI and BglII fragment was then inserted into the KpnI- and BglII-restricted pGL3-3A4 plasmid to yield the pGL3-3A4-Luc reporter gene. The expression vector for hPXR, pCI-hPXR, and FLAG-PXR was generated as follows, DNA fragment corresponding to the coding region of hPXR (amino acids 1–434) was generated by reverse transcription-PCR using total RNA from HepG2 cells. For pCI-PXR, the PCR primers were 5′-GGGAATTCCCACCAGGAGGTGAGACCCAAAGAAAGCTGG-3′ and 5′-GGGGTCGACGCGGCCGCTCAGCTACCTGTGATGCCGAACA-3′; for FLAG-PXR, the PCR primers were 5′-ATAAGAATGCGGCCGCCTGGAGGTGAGACCCAAAGA-3′ and 5′-CGGGATCCTCAGCTACCTGTGATGCCG-3′, designed based on published hPXR sequence (6Lehmann J.M. McKee D.D. Watson M.A. Willson T.M. Moore J.T. Kliewer S.A. J. Clin. Investig. 1998; 102: 1016-1023Crossref PubMed Scopus (1365) Google Scholar). The PCR product was modified with EcoRI and NotI or with NotI and BamHI and cloned into the pCI-neo vector (Promega) or p3XFLAG-myc-CMV-26 vector (Sigma). Cell Culture and Transient Transfection—For primary human hepatocyte culture, cell suspension was purchased from Cambrex BioScience (Walkersville, MD). The donor of the human hepatocytes was a 26-year-old male without heart disease or hypertension. Serological tests showed negative for human immunodeficiency virus, types 1 and 2, hepatitis B surface antigen (HBsAg), hepatitis C virus, human T-cell lymphotropic I/II virus, and syphilis. Upon arrival the cells were resuspended in Dulbecco's modified Eagle's medium containing 5% fetal bovine serum, antibiotics, 4 μg/ml insulin, and 1 μmol/liter dexamethasone, plated in collagen-coated plate for attachment, and then maintained in Williams' E medium containing ITS+ (insulin, transferrin, selenium, borine serum albumin, and linoleic acid), 0.1 μmol/liter dexamethasone and antibiotics overnight for recovery, and then the cells were treated with Me2SO, RIF, RIF+LPS, and RIF+TNF-α. 24 h after the treatment, the cells were harvested for isolation of total RNA for real time reverse transcription-PCR analysis. HepG2 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 1% antibiotic-antimycotic (100 units/ml penicillin G sodium, 100 μg/ml streptomycin sulfate, and 0.25 μg/ml amphotericin B) in 5% CO2 at 37 °C. For transient transfection, HepG2 cells were seeded in 12-well plates at 30% confluence. On the next day transfection was performed using Lipofectamine (Invitrogen). 6 h after transfection, cells were treated with RIF and other reagents. 48 h later, cells were harvested to determine the luciferase activity using the luciferase assay system (Promega). Conclusions were made based on three or more independent transfection experiments. In Vitro Transcription Coupled to Translation and EMSA—Human PXR and human RXRα polypeptides were generated by in vitro transcription coupled to translation using TnT-coupled reticulocyte lysate system (Promega, Madison, WI). Oligonucleotides used for EMSA were the ER6 consensus sequences in cyp3a4 promoter region as described (14Xie W. Barwick J.L. Simon C.M. Pierce A.M. Safe S. Blumberg B. Guzelian P.S. Evans R.M. Genes Dev. 2000; 14: 3014-3023Crossref PubMed Scopus (460) Google Scholar). The double-stranded oligonucleotide was labeled with [α-32P]dCTP using Klenow enzyme (USB Corp., Cleveland, OH). For EMSA assay, PXR and RXRα and recombinant p50 (Promega) and p65 (produced by baculoviral expressions) in various combinations were incubated for 30 min in a reaction mixture containing 40 mm KCl, 1 mm MgCl2, 0.1 mm EGTA, 0.5 mm dithiothreitol, 20 mm Hepes, pH 7.9, 4% Ficoll (400 K), and ∼30000 cpm of radiolabeled double-stranded oligonucleotide probe. After incubation for 30 min at room temperature, the reaction mixtures were separated by electrophoresis in 4.5% nondenaturing polyacrylamide gel. The results were recorded by autoradiography. Real Time Quantitative PCR—For real time quantitative PCR, total RNA samples were reverse-transcribed by using Moloney murine leukemia virus reverse transcriptase (Invitrogen), and the cDNA samples were used for quantification by PCR. Amplifications were performed in the ABI Prism 7900HT (Applied Biosystems) by using SYBR Green Master Mix (Applied Biosystems). The PCR primers used were: PXR, 5′-GGCCACTGGCTATCACTTCAA-3′ and 5′-TTCATGGCCCTCCTGAAAA-3′; RXRα, 5′-TCAATGGCGTCCTCAAGGTC-3′ and 5′-TTGCCTGAGGAGCGGTCC-3′; CYP3A4, 5′-CCACAAAGCTCTGTCCGATCT-3′ and 5′-GAACACTGCTCGTGGTTTCACA-3′; and β-actin, 5′-CCATCGAGCACGGCATC-3′ and 5′-ATTGTAGAAGGTGTGGTGCCAGA-3′. The β-actin was used as a housekeeping gene for normalization with the rest of the samples. Immunocytochemistry—Primary human hepatocytes growing in 24-well plates were treated with LPS or TNF-α for 1 h. The cells were washed three times with cold PBS and then fixed with fresh 4% formaldehyde in PBS for 10 min at room temperature. After washing three times with PBS, the cells were permeablized with 0.2% Triton X-100 for 10 min at room temperature. After washing with PBS (three times for 5 min each), the cells were blocked with 5% bovine serum albumin in PBS/Tween 20 for 1 h at room temperature. Then primary antibody against p65 (Santa Cruz, sc-109X) diluted (1:500) in PBS/Tween 20 was added, and the reaction was incubated at room temperature for 1 h. After three washes with PBS/Tween 20, 10 min each, secondary antibody conjugated with Alexa Fluo-568 (Molecular Probe, A11011) diluted in PBS/Tween 20 (1:1000) was added and incubated for 1 h at room temperature. The cells were washed with PBS/Tween 20 three times for 10 min each. 4′,6′-Diamino-2-phenylindole was added to stain the cells. The images were visualized, and representative views of the cells were recorded by fluorescence microscopy with an Olympus IX71 microscope. GST Pull-down Analysis—The GST pull-down assay was essentially as described (22Tian Y. Ke S. Chen M. Sheng T. J. Biol. Chem. 2003; 278: 44041-44048Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). [35S]Methionine-labeled full-length p65 protein was generated with a TnT-coupled reticulocyte lysate system (Promega) using the T7 promoter-driven cDNA plasmid as the template. PCR-generated cDNA fragments of RXRα corresponding to the domains of RXRα (see Fig. 5A for details and the sequences of PCR primer used are available upon request) were inserted into pGEX-5X-3 (Amersham Biosciences), yielding the expression plasmids for GST-RXRα fusion peptides. The plasmids were expressed in Escherichia coli (BL21), and fusion polypeptides were purified with the glutathione-Sepharose 4B affinity matrix (Amersham Biosciences) according to the manufacturer's instructions. Ten micrograms of each fusion polypeptides (estimated by comparison with bovine serum albumin in an SDS-PAGE gel with Coomassie staining) was incubated with 20 μl of radiolabeled p65 in a total of 250 μl of binding reaction buffer (20 mm Hepes, pH 7.9, 1% Triton X-100, 20 mm dithiothreitol, 0.5% bovine serum albumin, and 100 mm KCl) for 2 h at 4°C. After incubation, the beads were washed with the same buffer without bovine serum albumin five times. The bound proteins were eluted by boiling in the SDS-PAGE sample buffer and resolved by 8% SDS-PAGE gel electrophoresis. The signals were detected by autoradiography. Chromatin Immunoprecipitation (ChIP) Assay—The ChIP assay was based on published procedure with modification (11Goodwin B. Moore J.T. Trends Pharmacol. Sci. 2004; 25: 437-441Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). HepG2 cells were transfected with FLAG-tagged PXR and pGL3-3A4-Luc and were maintained in 10-cm plates under standard cell culture conditions. At 95% confluence formaldehyde was added directly to tissue culture medium to a final concentration of 1% for cross-linking, and the plates were incubated for 15 min at room temperature on a rocker. The cross-linking reaction was stopped by adding glycine to a final concentration of 0.125 m. The plates were incubated at room temperature for 5 min. The plates were then rinsed twice with ice-cold phosphate-buffered saline. The cells were scraped off the plates and collected into 50-ml conical tubes by centrifugation (600 × g for 5 min at 4 °C), and the pellet was washed once with phosphate-buffered saline containing 1 mm phenylmethylsulfonyl fluoride and resuspended in 2 ml of cell lysis buffer (5 mm PIPES, pH 8, 1 mm EDTA, 0.5 mm EGTA, 85 mm KCl, 0.5% Nonidet P-40, 1 mm phenylmethylsulfonyl fluoride, 1 mm dithiothreitol, and 5 μg/ml each of leupeptin and aprotinin) and incubated for on ice for 10 min. The cells were homogenized on ice using an B type pestle by processing in a Dounce homogenizer 200 times to aid the release of nuclei. The nuclei were collected by centrifugation (5000 × g for 10 min at 4 °C) and then resuspended them in nuclei lysis buffer (50 mm Tris-HCl, pH 8.1, 10 mm EDTA, 0.5 mm EGTA, 1% SDS, 1 mm phenylmethylsulfonyl fluoride, 1 mm dithiothreitol, 5 μg/ml each of leupeptin and aprotinin) and incubated again on ice for 10 min. The samples were sonicated into DNA fragments of 0.5–1.5 kilobase pairs (checked by agarose gel electrophoresis/ethidium bromide staining) and microcentrifuged at 14,000 rpm for 10 min at 4 °C. The supernatant was cleared by incubation with Staph A cells (2.5 μg/sample; Roche Applied Science) for 15 min and AG beads for 30–60 min sequentially at 4 °C on a rotating platform. The supernatant was aliquoted after centrifugation at 12,000 × g for 5 min to the clean tubes. Appropriate antibodies (1 μg each) were added to the aliquots and then 25 μl of precleared 50% protein A/G beads (Amersham Biosciences) was added. The final volume of each sample was adjusted to no more than 500 μl with the same amount of immunoprecipitation dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mm EDTA, 16.7 mm Tris-Cl, pH 8.1, 167 mm NaCl, 100 μg/ml sonicated salmon sperm DNA) as the nuclei lysis buffer. The mixtures were incubated on the rotating platform at 4 °C, overnight. After incubation, the beads were collected by centrifugation at 5000 rpm for 1 min in a microcentrifuge, and pellets were washed once with 1 ml of 1× dialysis buffer (2 mm EDTA; 50 mm Tris-Cl, pH 8.0) with 100 μg/ml sonicated salmon sperm DNA, twice with 1× dialysis buffer and three times with 1 ml of immunoprecipitation wash buffer (100 mm Tris-Cl, pH 9.0, 500 mm LiCl, 1% Nonidet P-40, 1% deoxycholic acid) for 10 min with rotation. After the wash, 200 μl of protein kinase digestion buffer (50 mm Tris, pH 8.0, 1 mm EDTA, 100 mm NaCl, 0.5% SDS, 100 mg/ml proteinase K) was added to each sample, and the reaction was incubated at 55 °C for 3 h and then at 65 °C for 6 h to reverse the cross-linking. The sample was extracted once with phenol-chloroform-isoamyl alcohol and precipitated with ethanol in the presence of 20 μg of glycogen overnight. The precipitated pellets were collected by centrifugation at 14,000 × g in microcentrifuge, and the pellets were resuspended in 20 μl of TE buffer. Aliquots from each tube were amplified by PCR, and PCR products were separated by 1.2% agarose gel electrophoresis and visualized by ethidium bromide staining. The PCR primer pairs were 5′-TTGGACTCCCCAGTAACATTG-3′ and 5′-TGCATGGAGCTTTCCTGC-3′ for amplifying the cyp3a4 promoter region and 5′-ACTCATGTCCCAATTAAAGGTC-3′ and 5′-TGTTCTTGTCAGAAGTTCAGC-3′ for amplifying the enhancer module. Suppression of PXR-mediated Gene Activation by LPS and TNF-α in Human Liver Cells—The effects of LPS and TNF-α on the expression of PXR, RXRα, and CYP3A4 were investigated in a primary human hepatocyte cell culture model by quantitative real time PCR. Treatment of the hepatocytes with the prototypical human PXR agonist RIF induced a 34-fold increase in CYP3A4 mRNA; the RIF-induced CYP3A4 mRNA levels were suppressed by more than 50 and 90% after cotreatment with either TNF-α (2 ng/ml, 24 h) and LPS (5 μg/ml, 24 h), respectively (Fig. 1A). In contrast, PXR mRNA levels were unchanged by TNF-α treatment, and there was an approximately 30% decrease in hPXR mRNA in LPS-treated samples (Fig. 1B). RXRα mRNA levels were not significantly changed after treatments with either LPS or TNF-α (Fig. 1C). Activation of NF-κB by LPS or TNF-α was confirmed by immunocytochemistry for p65 nuclear translocation (Fig. 1D). The RNA samples were also analyzed by microarray profiling, and the results were consistent with those obtained by the quantitative PCR, with respect to the changes of PXR, RXRα levels, and the suppression of cyp3a4 by LPS or TNF-α (data not shown). To further investigate the effects of proinflammatory agents on the transcriptional activity of PXR and to avoid donor variability in PXR-regulated genes in the human primary hepatocytes, we constructed the luciferase reporter gene driven by PXR-responsive enhancer modules for analysis of the PXR-regulated gene expression in a human hepatoma cell line (HepG2) based on the published information (Fig. 2A) (23Goodwin B. Hodgson E. Liddle C. Mol. Pharmacol. 1999; 56: 1329-1339Crossref PubMed Scopus (585) Google Scholar). HepG2 cells were transiently cotransfected with pGL3-3A4-Luc and hPXR expression plasmids pCI-hPXR. The transfected cells were then treated with RIF alone or cotreated with RIF and LPS or RIF and TNF-α. TNF-α and LPS caused significant suppressions of the luciferase gene expression (Fig. 2B) that are consistent with the results from the primary human hepatocyte culture model (Fig. 1). These results using the HepG2 cell line also confirmed the utility of the HepG2 cell culture model in analysis of PXR-regulated transcription. NF-κB Plays a Critical Role in Down-regulation of cyp3a4 Expression by Inflammatory Mediators—NF-κB is an immediate early gene, which is activated in response to various stress stimuli including infections and inflammatory responses. NF-κB plays a pivotal role in mediating the pathological effects of TNF-α and LPS. It has been demonstrated that NF-κB regulates several nuclear/steroid receptors through physical and function interactions, resulting in transrepression of the gene expressions regulated by these receptors (24Tian Y. Ke S. Denison M.S. Rabson A.B. Gallo M.A. J. Biol. Chem. 1999; 274: 510-515Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar) (reviewed in Ref. 21McKay L.I. Cidlowski J.A. Endocr. Rev. 1999; 20: 435-459Crossref PubMed Google Scholar). To test the role of NF-κB in mediating the suppression of PXR transcriptional activity, we first transiently cotransfected NF-κB p65 with PXR-driven luciferase reporter gene in HepG2 cells. Coexpression of NF-κB p65 potently suppressed PXR-driven luciferase reporter gene activity, suggesting a role for NF-κB in mediating suppression (Fig. 3). To further demonstrate that NF-κB is specifically involved in the suppression of cyp3a4 expression, we coexpressed the NF-κB super repressor, SRIκBα, in transient transfection assays and analyzed the effects of NF-κB inhibition on TNF-α- and LPS-treated cells. SRIκBα is a mutant of IκBα with a serine to alanine mutation at residues 32 and 36. These mutations render the IκBα unable to be phosphorylated at serines 32 and 36 and therefore resistant to degradation by the proteosome pathway, thus causing constitutive inhibition of NF-κB. In transient transfection assays, HepG2 cells were cotransfected with plasmids pCI-PXR, pGL3-3A4-Luc reporter gene and increasing amounts of SRIκBα expression plasmid. As expected, activation of NF-κB by either TNF-α or LPS caused suppression of the reporter gene activity. However, the LPS or TNFα-induced suppression of reporter gene was reversed by coexpression of SRIκBα (Fig. 3), indicating that NF-κB activation was directly responsible for the suppression of the PXR-regulated gene expression. NF-κB Regulates PXR Transcriptional Activity by Disrupting the Association between PXR·RXRα Complex and DNA Sequences—It has been shown that NF-κB regulates the transcriptional activity of steroid/nuclear receptors through direct protein-protein interaction. Na et al. (25Na S.Y. Kang B.Y. Chung S.W. Han S.J. Ma X. Trinchieri G. Im S.Y. Lee J.W. Kim T.S. J. Biol. Chem. 1999; 274: 7674-7680Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar) reported that NF-κB directly interacts with RXR. The association of NF-κB with nuclear receptors may potentially have a functional impact on the transcriptional activity of the PXR·RXR complex. One possible effect is that the binding of p65 with RXRα may interfere with the formation of the enhancersome consisting of the PXR·RXR complex and consensus DNA sequences. To test this hypothesis, we performed EMSA. PXR and RXRα proteins were generated through in vitro transcription coupled to translation. PXR and RXRα bound to the ER6 probe as dimer (Fig. 4, lanes 5 and 6). Addition of the recombinant p65 protein disrupted the binding of PXR·RXRα to the consensus ER6 sequence (Fig. 4, compare lane 5 with lanes 10 and 11). Interestingly, disruption by p65 could be reversed upon the addition of p50 protein, which is a cognate p65 partner known to negatively regulate p65 activity (Fig. 4, compare lane 10 with lane 12). As expected, the addition of bovine serum albumin had no effects (compare lane 5 with 7) on retarded band formation, suggesting that the p65 disrupted the binding of the PXR·RXRα complex to DNA in this assay. The results of EMSA are consistent with the hypothesis that association between RXRα and p65 prevents RXRα binding to the DNA sequences. To further analyze the interaction between RXRα and p65, we mapped the domains of RXRα responsible for association with p65 using GST pull-down assay. The known functional modular domains were fused with GST in various combinations and expressed as fusion peptides in E. coli (Fig. 5, A and C). The p65 was radiolabeled by in vitro transcription-coupled translation in the presence of [35S]methionine. Interestingly, the DNA-bindin" @default.
• W2020872262 created "2016-06-24" @default.
• W2020872262 creator A5012408586 @default.
• W2020872262 creator A5014524647 @default.
• W2020872262 creator A5022920979 @default.
• W2020872262 creator A5038698115 @default.
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• W2020872262 date "2006-06-01" @default.
• W2020872262 modified "2023-10-11" @default.
• W2020872262 title "Role of NF-κB in Regulation of PXR-mediated Gene Expression" @default.
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