Role of NF-κB in Regulation of PXR-mediated Gene Expression

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 DNA was by PCR human DNA as the with the primers and The PCR was with and and the was to The DNA to the region to B. E. C. Mol. Pharmacol. 1999; PubMed Scopus Google Scholar) were by PCR with the and and were by The and was the and plasmid to the reporter The expression for hPXR, and was as DNA to the region of hPXR was by from HepG2 the PCR primers were and for the PCR primers were and on 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). The PCR was modified with and or with and and the or Culture and human cell was purchased from The of the human was a or showed for human and hepatitis hepatitis human and the were in Dulbecco's modified Eagle's medium fetal bovine and in for and in medium serum and and for and the were with RIF, and the the were for of for HepG2 were in Dulbecco's modified Eagle's medium with fetal bovine serum and and in at HepG2 were in at the was Lipofectamine were with and other were to the activity the assay were on or In to and PXR and human RXRα were by in transcription to Oligonucleotides used for EMSA were the ER6 consensus sequences in cyp3a4 region as (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 was with EMSA PXR and RXRα and p50 and p65 by in various were for in a and of double-stranded for at the were by in The results were by were by and the were used for by were in the by The PCR primers used and and CYP3A4, and and and The was used as a gene for with the of the human in were with LPS or TNF-α for The were with and with in for at with PBS, the were with for at with for the were with bovine serum in for at antibody against p65 (Santa Cruz, in was and the was at for with antibody with in was and for at The were with for was to the The were and of the were by with an assay was as S. M. T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). p65 was with a the plasmid as the of RXRα to the of RXRα for and the sequences of PCR used are were the expression for The were expressed in and were with the to the of by with bovine serum in an with was with of p65 in a of of binding bovine serum and for at the were with the bovine serum The were by in the and by The were by assay was on with (11Goodwin B. Moore J.T. Trends Pharmacol. Sci. 2004; 25: 437-441Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). HepG2 were with PXR and and were in cell was directly to medium to a of for and the were for at on a The was by to a of The were at for The were with phosphate-buffered The were the and by for at and the was with phosphate-buffered and in of cell PIPES, and of and and for on for The were on an by in a to the of The were by for at and in of and and on for The were DNA of by and at for at The was by with Roche Applied for and for at on a The was at for to the antibodies were to the and of 50% was The of was to with the of immunoprecipitation as the The were on the at the were by at for in a and were with of with with and with of immunoprecipitation for with the of was to and the was at for and at for to the The was with and with in the of of The were by at in and the were in of from were by and PCR were by and by The PCR were and for the cyp3a4 region and and for the of by LPS and TNF-α in effects of LPS and TNF-α on the expression of and CYP3A4 were investigated in a human cell by of the with the human PXR induced a in CYP3A4 the CYP3A4 mRNA levels were by and with either TNF-α and LPS In PXR mRNA levels were by TNF-α and there was an decrease in hPXR mRNA in RXRα mRNA levels were not with either LPS or TNF-α of NF-κB by LPS or TNF-α was by for p65 nuclear The were also by and the results were with by the with to the of RXRα and the suppression of cyp3a4 by LPS or TNF-α not investigate the effects of proinflammatory on the transcriptional activity of PXR and to in PXR-regulated genes in the human we constructed the reporter gene by for of the PXR-regulated gene expression in a human cell line on the B. E. C. Mol. Pharmacol. 1999; PubMed Scopus Google Scholar). HepG2 were with and hPXR expression The were with or with and LPS or and TNF-α. TNF-α and LPS caused of the gene expression that are with the results from the human results the HepG2 cell line also the of the HepG2 cell in of PXR-regulated NF-κB a in of cyp3a4 by Inflammatory is an early which is activated in response to various stimuli including infections and inflammatory NF-κB plays a pivotal role in the pathological effects of TNF-α and It has been demonstrated that NF-κB regulates several receptors through and in of the gene expressions regulated by these receptors S. M.A. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar) (reviewed in L.I. Cidlowski J.A. Endocr. Rev. 1999; 20: 435-459Crossref PubMed Google Scholar). the role of NF-κB in the suppression of PXR transcriptional we first NF-κB p65 with reporter gene in HepG2 of NF-κB p65 reporter gene a role for NF-κB in suppression that NF-κB is involved in the suppression of cyp3a4 we the NF-κB super SRIκBα, in and the effects of NF-κB on and SRIκBα is a of with a to at and the to be at and and to by the thus constitutive of In HepG2 were with reporter gene and of SRIκBα expression activation of NF-κB by either TNF-α or LPS caused suppression of the reporter gene the LPS or suppression of reporter gene was reversed by of SRIκBα that NF-κB activation was directly responsible for the suppression of the PXR-regulated gene expression. NF-κB PXR by the PXR·RXRα and DNA has been shown that NF-κB regulates the transcriptional activity of steroid/nuclear receptors through direct G. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar) that NF-κB directly with The association of NF-κB with nuclear receptors may have a on the transcriptional activity of the PXR·RXR complex. One is that the binding of p65 with RXRα may with the of the of the PXR·RXR complex and consensus DNA sequences. this we PXR and RXRα were through in transcription to PXR and RXRα to the ER6 as and of the p65 disrupted the binding of PXR·RXRα to the consensus ER6 with and by p65 be reversed the of p50 protein, which is a p65 partner known to p65 activity with the of bovine serum effects with on that the p65 disrupted the binding of the PXR·RXRα complex to DNA in this The results of EMSA are with the that association RXRα and p65 RXRα binding to the DNA sequences. the RXRα and we the of RXRα responsible for association with p65 The known were with in various and expressed as in E. and The p65 was by in in the of the DNA-binding domain is critical in the domain and that domain interacted with the the that not domain not with The association of p65 with RXRα DNA-binding domain may the binding of RXRα to thus of the gene expression. this in we a assay on HepG2 with LPS and TNF-α as well as of p65 the association of RXRα with the regulatory regions of the cyp3a4 the that NF-κB with the binding of the PXR·RXRα complex to regulatory sequences of inhibiting the PXR-regulated gene expression. CYP3A4 is a predominant human liver of the drugs in use and post-transcriptional of the expression of this are of in therapeutic as well as the of Recent studies have demonstrated that the ligand-dependent transcription hPXR plays a pivotal role in regulation of enzymes, and at the transcriptional level (reviewed in Refs. C. Pharmacol. Rev. PubMed Scopus Google Scholar and P. T. M. Med. PubMed Scopus (153) Google it is important to and pathological conditions that may the PXR and inflammatory responses have been to suppress P-450 as well as enzymes, in capacity of drug in and experimental (reviewed in Refs. 16Renton K.W. Curr. Drug Metab. 2004; 5: 235-243Crossref PubMed Scopus (153) Google Scholar and E.T. T. 2002; PubMed Scopus Google Scholar). clinically important have been investigated Several mechanisms have been to the and suppression of cyp3a4 expression. it has been that LPS the PXR mRNA levels in and C. 2002; PubMed Scopus Google Scholar), and this may in suppression of cyp3a4 expression. the levels of the nuclear receptors may not be an in transcriptional PCR and with human we found that a decrease of PXR mRNA and RXRα mRNA level was The decrease in PXR mRNA may not for the suppression of the CYP3A4 mRNA by LPS and TNF-α and and E.T. J. Pharmacol. Exp. 2005; PubMed Scopus Google Scholar) have shown that caused the levels of suppression of P-450 in as in the that nuclear receptors PXR and are not for the suppression of the P-450 including at in the have not been induced by there have been nuclear the compensatory roles of other nuclear receptors in the suppression to be This is in of the current that the suppression of the nuclear receptors may be through a general mechanism where the functions of a common partner (RXR) for nuclear receptors is NF-κB Recent studies have shown that the DNA sequences at of cyp3a4 regulatory region sequences, which can be regulated by liver inhibitory (17Martinez-Jimenez C.P. Gomez-Lechon M.J. Castell J.V. Jover R. Mol. Pharmacol. 2005; 67: 2088-2101Crossref PubMed Scopus (60) Google Scholar), transcription has also been shown to the LPS suppressive effects of the Klaassen C.D. Mol. Pharmacol. 2004; Google Scholar). In current we found that reporter gene the sequences was also by NF-κB activation and of NF-κB the suppression that of the binding of PXR·RXRα complex to the consensus sequences is an important mechanism in to the regulation by liver inhibitory It is likely that mechanism may be responsible for the suppression of cyp3a4 gene expression. common transcriptional response to the of and is the induction of early genes. One of these genes is the pleiotropic transcription which is activated in response to various proinflammatory NF-κB has been shown to interact with the receptor (21McKay L.I. Cidlowski J.A. Endocr. Rev. 1999; 20: 435-459Crossref PubMed Google Scholar, S. M.A. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, S. M.A. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar) and the transcriptional activity of these receptors (reviewed in Refs. L.I. Cidlowski J.A. Endocr. Rev. 1999; 20: 435-459Crossref PubMed Google Scholar and M.A. Chem. Biol. 2002; PubMed Scopus Google Scholar). In the it has been shown that receptor regulates the suppression of the P-450 by LPS E. K.W. Drug Dispos. 2005; PubMed Scopus Google Scholar, A. K.W. J. 2005; PubMed Scopus Google Scholar). studies that NF-κB is involved in regulation of the it is NF-κB activation plays a role in the transcriptional activity of it was found that the common dimerization partner RXR for the nuclear receptors interacted with NF-κB G. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). We that NF-κB may play a direct role in suppression of cyp3a4 expression and a assay HepG2 cell for the of transcriptional regulation of PXR by proinflammatory In with human the of PXR activation by or of PXR by NF-κB activation were which may be to the of the cell It is well known that certain aspects of xenobiotic responses in the has to the transcriptional regulation by PXR and certain with human such as the and In this study, the important role of NF-κB in the suppression of cyp3a4 is demonstrated on the following TNF-α and LPS of human in activation of the NF-κB and with the down-regulation of and in reporter gene activation of NF-κB the reporter gene and of cyp3a4 activity was reversed by the NF-κB super repressor thus the of the mechanism the suppression of cyp3a4 by we EMSA, and to the NF-κB and PXR·RXRα EMSA we found that binding of PXR·RXRα heterodimer to the ER6 consensus sequences was by The inhibitory effects of p65 on the binding of PXR·RXRα to ER6 were by which is the partner for that the inhibitory of p65 be by which is with the that p65 with the association of PXR·RXRα with DNA sequences This was by the observation that the association RXR and NF-κB p65 was mediated through the RXR DNA-binding domain as determined by assay Furthermore, the we found that the association of RXRα with the regulatory regions of cyp3a4 was disrupted activation of NF-κB by either by LPS or expression of that the association PXR·RXRα complex with DNA sequences was disrupted by NF-κB in activation of gene expression consists of multiple a of transcriptional at steps. in regulation of gene the regulatory that have been investigated include and S. M.A. J. Biol. Chem. 2001; Full Text Full Text PDF PubMed Scopus Google Scholar, S. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar), of S. J. S. K. Mol. Cell. Biol. 2002; PubMed Scopus Google Scholar) and S. K. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar), and of the transcriptional which leads to of the domain of the of S. M. T. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). It is likely that transcriptional regulation of cyp3a4 is also to regulation at these critical by various mechanisms including NF-κB these in and in results that activation of NF-κB results in of the of the PXR·RXRα complex with the consensus DNA sequences in the regulatory regions of thus a for the suppression of cyp3a4 by LPS, proinflammatory cytokines, and other that are known to The mechanism is that NF-κB activation by and pathological stimuli leads to its the where it the binding of PXR·RXRα complex to the consensus DNA sequences, transcriptional suppression RXRα binding is with by this mechanism of suppression by NF-κB may be extended to other nuclear receptor-regulated systems where RXRα is a dimerization partner. We S. H. Safe for critical of the