During various inflammatory processes circulating cytokines including IL-6, IL-1β, and TNFα elicit a broad and clinically relevant impairment of hepatic detoxification that is based on the simultaneous downregulation of many drug metabolizing enzymes and transporter genes. To address the question whether a common mechanism is involved we treated human primary hepatocytes with IL-6, the major mediator of the acute phase response in liver, and characterized acute phase and detoxification responses in quantitative gene expression and (phospho-)proteomics data sets. Selective inhibitors were used to disentangle the roles of JAK/STAT, MAPK, and PI3K signaling pathways. A prior knowledge-based fuzzy logic model comprising signal transduction and gene regulation was established and trained with perturbation-derived gene expression data from five hepatocyte donors. Our model suggests a greater role of MAPK/PI3K compared to JAK/STAT with the orphan nuclear receptor RXRα playing a central role in mediating transcriptional downregulation. Validation experiments revealed a striking similarity of RXRα gene silencing versus IL-6 induced negative gene regulation (rs = 0.79; P<0.0001). These results concur with RXRα functioning as obligatory heterodimerization partner for several nuclear receptors that regulate drug and lipid metabolism.
References
[1]
Cray C, Zaias J, Altman NH. Acute Phase Response in Animals: A Review. Comp Med. 2009;59: 517–526. pmid:20034426
[2]
Aitken AE, Richardson TA, Morgan ET. Regulation of drug-metabolizing enzymes and transporters in inflammation. Annu Rev Pharmacol Toxicol. 2006;46: 123–149. doi: 10.1146/annurev.pharmtox.46.120604.141059. pmid:16402901
[3]
Morgan ET, Goralski KB, Piquette-Miller M, Renton KW, Robertson GR, Chaluvadi MR, et al. Regulation of drug-metabolizing enzymes and transporters in infection, inflammation, and cancer. Drug Metab Dispos 2008;36: 205–16. doi:36/2/205 doi: 10.1124/dmd.107.018747. pmid:18218849
[4]
Klein M, Thomas M, Hofmann U, Seehofer D, Damm G, Zanger UM. A Systematic Comparison of the Impact of Inflammatory Signaling on ADME Gene Expression and Activity in Primary Human Hepatocytes and HepaRG Cells. Drug Metab Dispos 2014;43: 273–283. doi: 10.1124/dmd.114.060962. pmid:25480923
[5]
Zanger UM, Turpeinen M, Klein K, Schwab M. Functional pharmacogenetics/genomics of human cytochromes P450 involved in drug biotransformation. Anal Bioanal Chem. 2008;392: 1093–1108. doi: 10.1007/s00216-008-2291-6. pmid:18695978
[6]
Evers R, Dallas S, Dickmann LJ, Fahmi OA, Kenny JR, Kraynov E, et al. Critical Review of Preclinical Approaches to Investigate Cytochrome P450–Mediated Therapeutic Protein Drug-Drug Interactions and Recommendations for Best Practices: A White Paper. Drug Metab Dispos. 2013;41: 1598–1609. doi: 10.1124/dmd.113.052225. pmid:23792813
[7]
Harvey RD, Morgan ET. Cancer, Inflammation, and Therapy: Effects on Cytochrome P450–Mediated Drug Metabolism and Implications for Novel Immunotherapeutic Agents. Clin Pharmacol Ther. 2014;96: 449–457. doi: 10.1038/clpt.2014.143. pmid:24987833
[8]
Morgan ET. Impact of infectious and inflammatory disease on cytochrome P450-mediated drug metabolism and pharmacokinetics. Clin Pharmacol Ther. 2009;85: 434–438. doi: 10.1038/clpt.2008.302. pmid:19212314
[9]
Slaviero KA, Clarke SJ, Rivory LP. Inflammatory response: an unrecognised source of variability in the pharmacokinetics and pharmacodynamics of cancer chemotherapy. Lancet Oncol. 2003;4: 224–232. pmid:12681266 doi: 10.1016/s1470-2045(03)01034-9
[10]
Jover R, Moya M, Gómez-Lechón MJ. Transcriptional regulation of cytochrome p450 genes by the nuclear receptor hepatocyte nuclear factor 4-alpha. Curr Drug Metab. 2009;10: 508–519. pmid:19689247 doi: 10.2174/138920009788898000
[11]
Zanger UM, Schwab M. Cytochrome P450 enzymes in drug metabolism: Regulation of gene expression, enzyme activities, and impact of genetic variation. Pharmacol Ther. 2013;138: 103–141. doi: 10.1016/j.pharmthera.2012.12.007. pmid:23333322
[12]
Pascussi J-M, Gerbal-Chaloin S, Duret C, Daujat-Chavanieu M, Vilarem M-J, Maurel P. The tangle of nuclear receptors that controls xenobiotic metabolism and transport: crosstalk and consequences. Annu Rev Pharmacol Toxicol. 2008;48: 1–32. doi: 10.1146/annurev.pharmtox.47.120505.105349. pmid:17608617
[13]
Xie W, editor. Nuclear receptors in drug metabolism. Hoboken, NJ: John Wiley & Sons; 2009.
[14]
Jover R, Bort, Lechon, Castell. Down-regulation of human CYP3A4 by the inflammatory signal interleukin-6: molecular mechanism and transcription factors involved. 2002;
[15]
Gu X, Ke S, Liu D, Sheng T, Thomas PE, Rabson AB, et al. Role of NF-kappaB in regulation of PXR-mediated gene expression: a mechanism for the suppression of cytochrome P-450 3A4 by proinflammatory agents. J Biol Chem. 2006;281: 17882–17889. doi: 10.1074/jbc.M601302200. pmid:16608838
[16]
Sun Kim M, Sweeney TR, Shigenaga JK, Chui LG, Moser A, Grunfeld C, et al. TNF and IL-1 Decrease RXRα, PPARα, PPARγ, LXRα, and the Coactivators SRC-1, PGC-1α, and PGC-1β in Liver Cells. Metabolism. 2007;56: 267–279. doi: 10.1016/j.metabol.2006.10.007. pmid:17224343
[17]
Congiu M, Mashford ML, Slavin JL, Desmond PV. Coordinate regulation of metabolic enzymes and transporters by nuclear transcription factors in human liver disease. J Gastroenterol Hepatol. 2009;24: 1038–1044. doi: 10.1111/j.1440-1746.2009.05800.x. pmid:19638083
[18]
Wang Z, Salih E, Burke PA. Quantitative Analysis of Cytokine-Induced Hepatocyte Nuclear Factor-4α Phosphorylation by Mass Spectrometry. Biochemistry 2011;50: 5292–5300. doi: 10.1021/bi200540w. pmid:21598922
[19]
Ghose R, Zimmerman TL, Thevananther S, Karpen SJ. Endotoxin leads to rapid subcellular re-localization of hepatic RXRα: A novel mechanism for reduced hepatic gene expression in inflammation. Nucl Recept. 2004;2: 4. doi: 10.1186/1478-1336-2-4. pmid:15312234
[20]
Lefebvre P, Benomar Y, Staels B. Retinoid X receptors: common heterodimerization partners with distinct functions. Trends Endocrinol Metab. 2010;21: 676–683. doi: 10.1016/j.tem.2010.06.009. pmid:20674387
[21]
Eulenfeld R, Dittrich A, Khouri C, Müller PJ, Mütze B, Wolf A, et al. Interleukin-6 signalling: more than Jaks and STATs. Eur J Cell Biol. 2012;91: 486–495. doi: 10.1016/j.ejcb.2011.09.010. pmid:22138086
[22]
Ryll A, Samaga R, Schaper F, Alexopoulos LG, Klamt S. Large-scale network models of IL-1 and IL-6 signalling and their hepatocellular specification. Mol Biosyst. 2011;7: 3253–3270. doi: 10.1039/c1mb05261f. pmid:21968890
[23]
Burgermeister E, Lanzendoerfer M, Scheuer W. Comparative analysis of docking motifs in MAP-kinases and nuclear receptors. J Biomol Struct Dyn. 2003;20: 623–634. doi: 10.1080/07391102.2003.10506879. pmid:12643765
[24]
Zordoky BNM, El-Kadi AOS. Role of NF-kappaB in the regulation of cytochrome P450 enzymes. Curr Drug Metab. 2009;10: 164–178. pmid:19275551 doi: 10.2174/138920009787522151
[25]
Machado D, Costa RS, Rocha M, Ferreira EC, Tidor B, Rocha I. Modeling formalisms in Systems Biology. AMB Express. 2011;1: 45. doi: 10.1186/2191-0855-1-45. pmid:22141422
[26]
Schr？der A, Wollnik J, Wrzodek C, Dr？ger A, Bonin M, Burk O, et al. Inferring statin-induced gene regulatory relationships in primary human hepatocytes. Bioinformatics 2011;27: 2473–2477. doi: 10.1093/bioinformatics/btr416. pmid:21757465
[27]
Samaga R, Klamt S. Modeling approaches for qualitative and semi-quantitative analysis of cellular signaling networks. Cell Commun Signal CCS. 2013;11: 43. doi: 10.1186/1478-811X-11-43. pmid:23803171
[28]
Aldridge BB, Saez-Rodriguez J, Muhlich JL, Sorger PK, Lauffenburger DA. Fuzzy logic analysis of kinase pathway crosstalk in TNF/EGF/insulin-induced signaling. PLoS Comput Biol. 2009;5: e1000340. doi: 10.1371/journal.pcbi.1000340. pmid:19343194
[29]
Morris MK, Saez-Rodriguez J, Clarke DC, Sorger PK, Lauffenburger DA. Training signaling pathway maps to biochemical data with constrained fuzzy logic: quantitative analysis of liver cell responses to inflammatory stimuli. PLoS Comput Biol. 2011;7: e1001099. doi: 10.1371/journal.pcbi.1001099. pmid:21408212
[30]
Bernardo-Faura M, Massen S, Falk CS, Brady NR, Eils R. Data-derived modeling characterizes plasticity of MAPK signaling in melanoma. PLoS Comput Biol. 2014;10: e1003795. doi: 10.1371/journal.pcbi.1003795. pmid:25188314
[31]
Lecluyse EL, Alexandre E. Isolation and culture of primary hepatocytes from resected human liver tissue. Methods Mol Biol. 2010;640: 57–82. doi: 10.1007/978-1-60761-688-7_3. pmid:20645046
[32]
Godoy P, Hewitt NJ, Albrecht U, Andersen ME, Ansari N, Bhattacharya S, et al. Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME. Arch Toxicol. 2013;87: 1315–1530. doi: 10.1007/s00204-013-1078-5. pmid:23974980
Terfve C, Cokelaer T, Henriques D, MacNamara A, Goncalves E, Morris MK, et al. CellNOptR: a flexible toolkit to train protein signaling networks to data using multiple logic formalisms. BMC Syst Biol. 2012;6: 133. doi: 10.1186/1752-0509-6-133. pmid:23079107
[35]
Gerhartz C, Heesel B, Sasse J, Hemmann U, Landgraf C, Schneider-Mergener J, et al. Differential activation of acute phase response factor/STAT3 and STAT1 via the cytoplasmic domain of the interleukin 6 signal transducer gp130. I. Definition of a novel phosphotyrosine motif mediating STAT1 activation. J Biol Chem. 1996;271: 12991–12998. pmid:8662591 doi: 10.1074/jbc.271.22.12991
[36]
Takahashi-Tezuka M, Yoshida Y, Fukada T, Ohtani T, Yamanaka Y, Nishida K, et al. Gab1 acts as an adapter molecule linking the cytokine receptor gp130 to ERK mitogen-activated protein kinase. Mol Cell Biol. 1998;18: 4109–4117. pmid:9632795 doi: 10.1128/mcb.18.7.4109
[37]
Chang F, Lee JT, Navolanic PM, Steelman LS, Shelton JG, Blalock WL, et al. Involvement of PI3K//Akt pathway in cell cycle progression, apoptosis, and neoplastic transformation: a target for cancer chemotherapy. Leukemia. 2003;17: 590–603. pmid:12646949 doi: 10.1038/sj.leu.2402824
[38]
Karaman MW, Herrgard S, Treiber DK, Gallant P, Atteridge CE, Campbell BT, et al. A quantitative analysis of kinase inhibitor selectivity. Nat Biotechnol. 2008;26: 127–132. doi: 10.1038/nbt1358. pmid:18183025
[39]
Harmsen S, Meijerman I, Maas-Bakker RF, Beijnen JH, Schellens JHM. PXR-mediated P-glycoprotein induction by small molecule tyrosine kinase inhibitors. Eur J Pharm Sci. 2013;48: 644–649. doi: 10.1016/j.ejps.2012.12.019. pmid:23277288
[40]
Lee SML, Schelcher C, Demmel M, Hauner M, Thasler WE. Isolation of human hepatocytes by a two-step collagenase perfusion procedure. J Vis Exp. 2013; 79:e50615. doi: 10.3791/50615.
[41]
Campbell JS, Prichard L, Schaper F, Schmitz J, Stephenson-Famy A, Rosenfeld ME, et al. Expression of suppressors of cytokine signaling during liver regeneration. J Clin Invest. 2001;107: 1285–1292. doi: 10.1172/JCI11867. pmid:11375418
[42]
Vee ML, Lecureur V, Stieger B, Fardel O. Regulation of Drug Transporter Expression in Human Hepatocytes Exposed to the Proinflammatory Cytokines Tumor Necrosis Factor-α or Interleukin-6. Drug Metab Dispos. 2009;37: 685–693. doi: 10.1124/dmd.108.023630. pmid:19074973
[43]
Blommaart EF, Krause U, Schellens JP, Vreeling-Sindelárová H, Meijer AJ. The phosphatidylinositol 3-kinase inhibitors wortmannin and LY294002 inhibit autophagy in isolated rat hepatocytes. Eur J Biochem FEBS. 1997;243: 240–246. doi: 10.1111/j.1432-1033.1997.0240a.x
[44]
Favata MF, Horiuchi KY, Manos EJ, Daulerio AJ, Stradley DA, Feeser WS, et al. Identification of a novel inhibitor of mitogen-activated protein kinase kinase. J Biol Chem. 1998;273: 18623–18632. pmid:9660836 doi: 10.1074/jbc.273.29.18623
[45]
Goueli SA, Hsiao K, Lu T, Simposn D. U0126: A Novel, Selective and Potent Inhibitor of MAP Kinase Kinase (MEK). Promega Notes. 1998; 6.
[46]
Schust J, Sperl B, Hollis A, Mayer TU, Berg T. Stattic: a small-molecule inhibitor of STAT3 activation and dimerization. Chem Biol. 2006;13: 1235–1242. doi: 10.1016/j.chembiol.2006.09.018. pmid:17114005
[47]
Spurgeon SL, Jones RC, Ramakrishnan R. High throughput gene expression measurement with real time PCR in a microfluidic dynamic array. PloS One. 2008;3: e1662. doi: 10.1371/journal.pone.0001662. pmid:18301740
[48]
Andersen CL, Jensen JL, ？rntoft TF. Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res. 2004;64: 5245–5250. doi: 10.1158/0008-5472.CAN-04-0496. pmid:15289330
[49]
Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001;25: 402–408. doi: 10.1006/meth.2001.1262. pmid:11846609
[50]
Poetz O, Ostendorp R, Brocks B, Schwenk JM, Stoll D, Joos TO, et al. Protein microarrays for antibody profiling: specificity and affinity determination on a chip. Proteomics. 2005;5: 2402–2411. doi: 10.1002/pmic.200401299. pmid:15887189
[51]
Braeuning A, Heubach Y, Knorpp T, Kowalik MA, Templin M, Columbano A, et al. Gender-Specific Interplay of Signaling through β-Catenin and CAR in the Regulation of Xenobiotic-Induced Hepatocyte Proliferation. Toxicol Sci. 2011;123: 113–122. doi: 10.1093/toxsci/kfr166. pmid:21705713
[52]
Warnes GR, Bolker B, Bonebakker L, Gentleman R, Liaw WHA, Lumley T, et al. gplots: Various R programming tools for plotting data [Internet]. 2013.
[53]
Hartigan JA. Clustering Algorithms. New York: John Wiley & Sons Inc.; 1975.
[54]
Saez-Rodriguez J, Alexopoulos LG, Epperlein J, Samaga R, Lauffenburger DA, Klamt S, et al. Discrete logic modelling as a means to link protein signalling networks with functional analysis of mammalian signal transduction. Mol Syst Biol. 2009;5: 331. doi: 10.1038/msb.2009.87. pmid:19953085
[55]
Castellano E, Downward J. RAS Interaction with PI3K. Genes Cancer. 2011;2: 261–274. doi: 10.1177/1947601911408079. pmid:21779497
[56]
Shannon P, Markiel A, Ozier O, Baliga NS, Wang JT, Ramage D, et al. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 2003;13: 2498–2504. doi: 10.1101/gr.1239303. pmid:14597658
