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Upregulation of CYP1B1 Expression by Inflammatory Cytokines Is Mediated by the P38 MAP Kinase Signal Transduction Pathway

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Upregulation of CYP1B1 Expression by Inflammatory Cytokines Is Mediated by the P38 MAP Kinase Signal Transduction Pathway Carcinogenesis vol.35 no.11 pp.2534–2543, 2014 doi:10.1093/carcin/bgu190 Advance Access publication September 18, 2014 Upregulation of CYP1B1 expression by inflammatory cytokines is mediated by the p38 MAP kinase signal transduction pathway Lenka Šmerdová1, Jana Svobodová1,2, Markéta Kabátková1,2, which, despite sharing ~40% homology with both CYP1A1 and Jiří Kohoutek3, Dalibor Blažek4, Miroslav Machala3 and CYP1A2, has several distinct properties, including its tissue-specific Jan Vondráček1,* pattern of expression. CYP1B1 metabolizes a number of procarcino- gens, including polycyclic aromatic hydrocarbons, N-heterocyclic 1Department of Cytokinetics, Institute of Biophysics, Academy of Sciences of amines, arylamines, amino azo dyes and several other compounds (2). the Czech Republic, Brno 62165, Czech Republic, 2Institute of Experimental CYP1B1 messenger RNA (mRNA) has been found at significant lev- 3 Biology, Faculty of Science, Brno 61137, Czech Republic, Department of els in a number of extrahepatic human tissues, including kidney and Chemistry and Toxicology, Veterinary Research Institute, Brno 62100, Czech colon, and also in hormonal tissues (including prostate, ovary, uterus Republic and 4Central European Institute of Technology (CEITEC), Masaryk University, Brno 62500, Czech Republic and mammary gland) (2). The expression of CYP1B1 in hormonal tissues could be related to the important role of CYP1B1 in the catab- *To whom correspondence should be addressed. Tel: +420 541517168; olism of 17β-estradiol, whose product, 4-hydroxyestradiol, may con- Downloaded from https://academic.oup.com/carcin/article/35/11/2534/418774 by guest on 29 September 2021 Fax: +420 41211293; tribute to estrogen-mediated carcinogenesis (3,4). CYP1B1 has been Email: [email protected] also proposed to have an impact on course of the tamoxifen therapy Cytochrome P450 1B1 (CYP1B1) is an enzyme that has a unique of hormone-dependent cancers since it catalyzes the conversion of tumor-specific pattern of expression and is capable of bioactivat- trans-4-hydroxytamoxifen to weakly estrogenic cis-4-hydroxytamox- ing a wide range of carcinogenic compounds. We have reported ifen (5). The presence of CYP1B1 in various fetal tissues points to its previously that coordinated upregulation of CYP1B1 by inflam- role in normal fetal development and it has been demonstrated that a matory cytokines, such as tumor necrosis factor-α (TNF-α) and mutation in the CYP1B1 is linked with the development of primary the aryl hydrocarbon receptor ligands, may increase bioactiva- congenital glaucoma (6). tion of promutagens, such as benzo[a]pyrene (BaP) in epithelial CYP1B1 has been found to be overexpressed in a wide range of cells. Here, we extend those studies by describing a novel mech- malignant tumors of different tissue origin, including breast, colon, anism participating in the regulation of CYP1B1 expression, lung, brain, skin, prostate, ovary and liver cancers (7–9). Based on which involves activation of the p38 mitogen-activated protein this phenomenon, it has been suggested that CYP1B1 may have an kinase (p38) and mitogen- and stress-activated protein kinase endogenous function in tumor cells and contribute to drug resistance 1 (MSK1). Using inhibitors of p38 and MSKs, as well as mouse (10). Upregulation of CYP1B1 often correlates with upregulation of embryonic cells derived from p38α-deficient and MSK1/2 double the aryl hydrocarbon receptor (AhR), its major transcriptional regula- knockout mice, we show here that TNF-α potentiates CYP1B1 tor, suggesting that AhR may participate in the maintenance of high upregulation via the p38/MSK1 kinase cascade. Effects of this baseline CYP1B1 levels in tumors (11,12). The AhR ligands, such inflammatory cytokine on CYP1B1 expression further involve the as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) or benzo[a]pyrene positive transcription elongation factor b (P-TEFb). The inhibi- (BaP), are potent CYP1B1 inducers and CYP1B1 plays an impor- tion of the P-TEFb subunit, cyclin-dependent kinase 9 (CDK9), tant role in bioactivation of BaP and formation of the reactive anti- which phosphorylates RNA polymerase II (RNAPII), prevented 7α,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydro-BaP (BPDE), the enhanced CYP1B1 induction by a combination of BaP and which binds covalently to cellular macromolecules, including DNA, inflammatory cytokine. Furthermore, using chromatin immuno- and causes mutations (13). Although activation of the AhR is a major precipitation assays, we found that cotreatment of epithelial cells mechanism responsible for CYP1B1 induction, additional signal- with TNF-α and BaP resulted in enhanced recruitment of both ing pathways have been implicated in either inducing CYP1B1 or in CDK9 and RNAPII to the Cyp1b1 gene promoter. Overall, these maintaining its constitutive level of expression (reviewed in ref. 14). results have implications concerning the contribution of inflam- However, the mechanisms of transcriptional or post-transcriptional matory factors to carcinogenesis, since enhanced CYP1B1 induc- regulation of CYP1B1 are still not fully clear. tion during inflammation may alter metabolism of exogenous Recently, we have observed that CYP1B1 is significantly upregu- carcinogens, as well as endogenous CYP1B1 substrates playing lated in epithelial cells cotreated by AhR ligands and inflammatory role in tumor development. cytokine tumor necrosis factor-α (TNF-α) (15–17). We have also found that via CYP1B1 upregulation, inflammatory mediators may accelerate BaP metabolism and production of its genotoxic metabo- lites (18). This type of transcriptional regulation is in sharp contrast Introduction with a majority of other CYPs involved in the metabolism of carcino- gens or in drug clearance, including CYP1A1 and CYP1A2 enzymes, Cytochrome P450 family 1 (CYP1) heme-thiolate monooxygenases whose activity/expression is generally downregulated by infection participate in the bioactivation of a variety of potent hydrophobic pro- or inflammation (19). It is presently unclear what mechanisms are carcinogens in the liver, lungs and other organs with a high metabolic responsible for the opposite CYP1B1 regulation under inflammatory capacity (1). CYP1B1 is a unique member of this enzyme family, conditions. Chronic inflammation, accompanied with deregulated and sustained Abbreviations: AhR, aryl hydrocarbon receptor; BaP, benzo[a]pyrene; CDK9, production of TNF-α, has been proposed to contribute to carcinogen- cyclin-dependent kinase 9; ChIP, chromatin immunoprecipitation; CREB, esis via mechanisms participating in tumor promotion, progression cyclic adenosine 3′,5′-monophosphate response element-binding protein; and, in some cases, also tumor initiation (20). Deregulated expres- CYP1, cytochrome P450 family 1; CYP1B1, cytochrome P450 1B1; DRB, sion/activity of enzymes, which bioactivate procarcinogens, may 5,6-dichlorobenzimidazole-1-β-d-ribofuranoside; EDTA, ethylenediaminetet- provide one of the mechanisms potentially contributing to tumor ini- raacetic acid; EGF, epidermal growth factor; MAPKAPK-2, MAP kinase-acti- tiation/development. In the present study, we identified the signaling vated protein kinase 2; MEF, mouse embryonic fibroblast; mRNA, messenger pathway responsible for the upregulation of CYP1B1 expression by RNA; MSK, mitogen- and stress-activated protein kinase; NF-κB, nuclear fac- tor κ-light-chain-enhancer of activated B cells; PBS, phosphate-buffered saline; inflammatory cytokine(s). Our results suggest that activation of the P-TEFb, positive transcription elongation factor b; RNAPII, RNA polymer- p38 mitogen-activated protein (MAP) kinase p38/mitogen- and stress- ase II; SDS, sodium dodecyl sulfate; siRNA, short interfering RNA; TCDD, activated protein kinase (MSK) kinase cascade is responsible for 2,3,7,8-tetrachlorodibenzo-p-dioxin; TNF-α, tumor necrosis factor-α. the observed CYP1B1 upregulation by inflammatory cytokines. We © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected] 2534 P38 MAP kinase regulates CYP1B1 expression also demonstrate that TNF-α contributes to the increased CYP1B1 was confirmed by performing the qPCR experiments under same conditions expression via enhanced recruitment of the positive elongation fac- with the respective pCR4-ZeroBlunt-TOPO vectors with inserted PCR prod- tor b (P-TEFb) and RNA polymerase II (RNAPII) to the CYP1B1 ucts. The conditions of assay were based strictly on the protocol provided by promoter. This mechanism may help to explain a unique pattern of the supplier of the one-step qRT-PCR kit. The amplifications were run on the Rotor-Gene 6000 (Corbett Research, Sydney, Australia) using the following CYP1B1 regulation during inflammation that alters the metabolic program: reverse transcription at 50°C for 30 min and initial activation step at activation of procarcinogens, such as BaP. 95°C for 15 min, followed by 35 cycles of 95°C for 10 s and 60°C for 40 s. The absolute quantification was performed using pCR4-ZeroBlunt-TOPO vector Materials and methods with inserted mouse Cyp1a1 and Cyp1b1 PCR products (Generi-Biotech, Hradec Králové, Czech Republic) as standards. The total RNA content was Chemicals and reagents determined using NanoDrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA). BaP (purity 99.9%) was from Ehrenstorfer (Augsburg, Germany). TCDD (purity 99%) was purchased from Cambridge Isotope Laboratories (Andover, Short interfering RNA transfections MA). Stock solutions
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