Expression of Serpin Peptidase Inhibitor B2 (SERPINB2) is regulated by Aryl hydrocarbon receptor (AhR)

Chemico-Biological Interactions 309 (2019) 108700

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Expression of Serpin Peptidase Inhibitor B2 (SERPINB2) is regulated by Aryl hydrocarbon receptor (AhR)

T

Damian Brauzea,*, Katarzyna Kiwerskaa, Kinga Bednareka, Reidar Grenmanc, Joanna Janiszewskaa, Maciej Giefinga, Malgorzata Jarmuz-Szymczaka,b a

Institute of Human Genetics, Polish Academy of Sciences, Strzeszynska 32, 60-479, Poznań, Poland Department of Hematology, Poznan University of Medical Sciences, Szamarzewskiego 82/84, 60-569, Poznań, Poland c Department of Otorhinolaryngology - Head and Neck Surgery and Department of Medical Biochemistry, Turku University Central Hospital and Turku University, P.O. Box 52, FIN, 20521, Turku, Finland b

A R T I C LE I N FO

A B S T R A C T

Keywords: Aryl hydrocarbon receptor SERPINB2 CYP1A1 CYP1B1 Enhancer RNA

Aryl hydrocarbon receptor (AhR) is a highly conserved ligand-activated transcription factor with high affinity to aromatic planar compounds, such as β-naphthoflavone (BNF), benzo[a]pyrene (BaP) or dioxin (TCDD). After binding the ligand, AhR triggers induction of the expression of phase I and phase II drug-metabolizing genes, together with numerous other genes that are not directly involved in the metabolism of xenobiotics. Several studies have shown that AhR plays a role in tumor initiation, promotion and progression, but the molecular mechanisms involved in these processes are not fully understood. A previous study from our laboratory indicated that the SERPINB2 gene is presumably regulated by AhR. To prove that such induction is really AhR-dependent, in the present study we knocked down the expression of AhR by stable transfection of a laryngeal squamous cell carcinoma cell line (UT-SCC-34) with shRNA, resulting in 92% reduction of BNF-induced expression of SERPINB2. However, in silico analysis did not reveal AhR-dependent responsive elements in the promoter of the SERPINB2 gene. Therefore, to address this problem, we have used cycloheximide, an inhibitor of translation, and our results clearly indicate that an additional, newly synthesized protein is involved in AhR-dependent induction of SERPINB2 expression by BNF. So, to exclude that AhR binds to the putative xenobiotic-responsive elements (XREs) localized upstream of the SERPINB2 gene, we performed chromatin immunoprecipitation assays. As expected, we found no direct binding of AhR to its responsive elements in the vicinity of the SERPINB2 gene, further demonstrating the indirect SERPINB2 induction by AhR. However, the further analysis demonstrated that the expression of the enhancer RNA encoded by the region of DNA 20 kbp upstream from the SERPINB2 gene was AhR-dependent. Although AhR-mediated SERPINB2 induction clearly requires the synthesis of an additional protein, the kinetics of SERPINB2 induction is as fast as the kinetics of CYP1A1 and CYP1B1 induction (both genes directly regulated by AhR). Therefore, given previous studies regarding the induction of SERPINB2 expression by bacterial lipopolysaccharides (LPS), we think that, similarly, the interaction with pause-release proteins may be responsible for AhR-dependent regulation of SERPINB2 expression.

1. Introduction The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that mediates a variety of biological responses to ubiquitous environmental pollutants, such as polycyclic aromatic hydrocarbons (PAH) and chlorinated dibenzo-p-dioxins. After ligand binding, the receptor translocates from the cytosol to the nucleus and forms a heterodimer with the aryl hydrocarbon receptor nuclear translocator

(ARNT) protein. Then, the liganded AhR/ARNT heterodimer binds to xenobiotic-responsive element (XRE) sequences, which constitute enhancer DNA elements present in the 5′-flanking region of target genes. Elevated expression of target genes leads to altered metabolism, which often results in enhanced carcinogenesis and toxicity [reviewed in Refs. [1–4]]. Activation of procarcinogenic PAHs to ultimate carcinogens by AhR-regulated enzymes is traditionally considered as the first step in tumor initiation. Despite the variability observed between experiments

*

Corresponding author. E-mail addresses: [email protected] (D. Brauze), [email protected] (K. Kiwerska), [email protected] (K. Bednarek), reigre@utu.fi (R. Grenman), [email protected] (J. Janiszewska), maciej.giefi[email protected] (M. Giefing), [email protected] (M. Jarmuz-Szymczak). https://doi.org/10.1016/j.cbi.2019.06.013 Received 14 May 2018; Received in revised form 30 April 2019; Accepted 6 June 2019 Available online 07 June 2019 0009-2797/ © 2019 Published by Elsevier B.V.

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Abbreviations

CYP1A1 CYP1B1 XRE TSS ncRNA eRNA CHX LPS

SERPINB2 serpin peptidase inhibitor B2 AhR aryl hydrocarbon receptor BNF β-naphthoflavone B(a)P benzo[α]pyrene TCDD 2,3,7,8-tetrachlorodibenzodioxin SCC laryngeal squamous cell carcinoma

cytochrome P450 1A1 cytochrome P450 1B1 xenobiotic- (dioxin) responsive element transcription start site noncoding RNA enhancer RNA cycloheximide bacterial lipopolysaccharide

Browser [33]. The present study was therefore designed to investigate the role of AhR in the regulation of SERPINB2 expression. The human laryngeal squamous cell carcinoma UT-SCC-34 cell line was selected for the subsequent experiments because, among several SCC cell lines tested by us recently, the UT-SCC-34 line appeared to be the best responder to BNF stimuli of AhR-dependent genes [34]. The majority of abovementioned studies applied TCDD as an AhR ligand. However, substantial differences between diverse AhR ligands, including BNF and TCDD, were observed [17]. Consequently, we decided to use AhR ligands different from TCDD, such as the noncarcinogenic synthetic flavonoid BNF and the carcinogenic polycyclic aromatic hydrocarbon B(a) P. BNF, a well-known AhR agonist [35], is a widely used inducer of phase I and phase II enzymes in xenobiotic metabolism [36,37]. BNF has been also shown to suppress chemical carcinogenesis at numerous sites in mice [38]. In contrast, B(a)P is a prevalent carcinogen identified e.g. in cigarette smoke [39], the major etiological risk factor for laryngeal squamous cell carcinoma [40]. To verify if the BNF-induced changes in SERPINB2 expression were indeed AhR-dependent, we knocked down the expression of AhR by stable transfection of UT-SCC34 cells with a vector encoding shRNA directed against AhR. To explore if the observed effects depend on direct AhR binding to the SERPINB2 promoter or if some additional factors are required, we investigated the effects of cycloheximide (CHX) administration on AhR-dependent induction of SERPINB2 expression, and compared the kinetics of SERPINB2 induction after B(a)P treatment with the induction of expression of CYP1A1 and CYP1B1 genes, both well known as directly regulated by AhR. Additionally, because no XREs were identified near the gene promoter, we investigated AhR binding to its potential binding sites (located in enhancers upstream of the SERPINB2 gene) by chromatin immunoprecipitation assays. Furthermore, we studied induction of 774 enhancer RNA (eRNA) expression by AhR ligands.

aiming to discover AhR-dependent genes, a small subset of AhR target genes encoding phase I xenobiotic-metabolizing enzymes (e.g. cytochromes P450: CYP1A1, CYP1A2, CYP1B1) and several phase II conjugating enzymes (e.g. NQO1, ALDH3A1, UGT1A, and GSTA1), are commonly reported as upregulated following AhR activation. Those enzymes metabolize many of their substrates to more soluble and excretable products, but also – as the classic example of benzo[a]pyrene (B(a)P) shows – are responsible for their activation to ultimate carcinogenic metabolites [reviewed in Ref. [5]]. Experiments with knockout animals revealed that PAH-induced carcinogenicity is lost in AhR-deficient mice [6]. Moreover, functional analysis of AhR knockout mice revealed that AhR is involved in lethality, teratogenesis, immunotoxicity, hepatotoxicity and tumor promotion caused by 2,3,7,8tetrachlorodibenzo-p-dioxin (TCDD) [7–11]. Additionally, numerous studies have shown that AhR plays a role not only in tumor initiation but also in its promotion and progression [12,13], but the molecular mechanisms involved in those processes are not fully understood. Some pleiotropic effects of AhR activation could be partly explained by crosstalk with other signal transduction pathways. The ability of AhR to interfere with multiple signal transduction pathways, including those regulated by nuclear receptors, has been reported by many laboratories and involves multiple mechanisms [reviewed in Refs. [1–4]]. Although the best-studied AhR-responsive genes encode enzymes involved in xenobiotic metabolism, microarray-based gene expression profiling studies have identified a large number of other genes, supposedly not directly connected to the metabolism of xenobiotics, which are induced or repressed in an AhR-dependent and its ligand-dependent manner [14–19]. Exploration of the functions of those genes is especially interesting in light of recent reports implying AhR in the control of cell differentiation and its involvement in cell pluripotency [3,20,21]. SERPINB2 seems to be one of such genes, presumably under AhR control. SERPINB2 was reported in various human cell lines as inducible by TCDD treatment [22–25]. Recently, in order to investigate the AhRdependent effects of β-naphthoflavone (BNF), we have used expression microarrays to assess the global effects of this compound on RNA expression in the HepaRG cell line. Our study indicated that SERPINB2 is one of the genes most strongly induced by BNF treatment in the HepaRG cell line [26]. SERPINB2 (PAI-2) was originally described as an inhibitor of urokinase plasminogen activator, but its predominant cytoplasmic localization suggests some other intracellular function, given that both urokinase-type and tissue-type plasminogen activators (its only known protease targets) are exclusively extracellular [27,28]. Recent studies have shown that down-regulation of SERPINB2 is associated with acquired resistance to cisplatin in head and neck squamous cell carcinoma [29] and with gefitinib resistance in non-small cell lung cancer [30]. Furthermore, it has been suggested that the main function of SERPINB2 is to modulate proteotoxic stress [31], and the expression of the gene is strictly regulated by cooperation of an upstream silencer and distal transactivator region between −5100 and −3300 bp from the transcription start site (TSS) [32]. Additionally, it has been reported recently that expression of SERPINB2 is highly induced by bacterial lipopolysaccharide in human monocytes and noncoding RNAs, localized approximately 20 kbp upstream of the SERPINB2, and responsible for induction of the gene expression. The RNAs (772, 774, 775) formed a cluster near an enhancer, defined by H3K27ac in UCSC Genome

2. Materials and methods 2.1. Chemicals BNF, B(a)P, SYBR® Green I (10000x concentration), JumpStart Taq DNA Polymerase, Enhanced Avian RT First Strand Synthesis Kit (STR1), GenElute™ PCR Clean-Up Kit, GenElute™ HP Endotoxin-Free Plasmid Maxiprep Kit, PCR Low Ladder Marker Set, guanidine thiocyanate, ammonium thiocyanate, CHX, LB broth, and LB agar were supplied by Sigma-Aldrich Co (St. Louis, MO, USA). Maxima First Strand cDNA Synthesis Kit for RT-qPCR with dsDNase #K1671 was supplied by Thermo Scientific. GeneClip™ U1 Hairpin Cloning System Neomycin Vector and antibiotic G418 (Geneticin) were provided by Promega (Madison, WI, USA). ChromataChIP Kit and AhR monoclonal antibody (RPT9) were provided by Novus Biologicals (Littleton, CO, USA). Lipofectamine 2000 and Opti-MEM® I Reduced Serum Medium were provided by Invitrogen (Carlsbad, CA, USA). Fluorescein was obtained from Bio-Rad Laboratories (Hercules, CA, USA). Restriction endonucleases were purchased from Fermentas International Inc (Burlington, Canada). Deoxyribonucleotide triphosphates (dATP, dGTP, dCTP, dTTP) were provided by Roche Diagnostics (Mannheim, Germany). PCR primers were provided by the Institute of Biochemistry 2

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SCC-34 cells were treated for 8 h (Fig. 2) or 24 h (Table 1) with BNF dissolved in DMSO to a final concentration of 100 μM in the medium (8 μl of 50 mM BNF per 4 ml of the medium in 25-cm2 flasks; 0.2% of DMSO). The same amount of the solvent (DMSO) was added to control, untreated cells.

and Biophysics, Polish Academy of Sciences, Poland (oligo.pl) and Genomed, Poland. All the other compounds were readily available commercial products. 2.2. Cell line culture and treatment The UT-SCC-34 cell line, derived from a patient diagnosed with squamous laryngeal cancer at the University of Turku, was used in both time-course and AhR knockout experiments. The characteristics of the original material taken to establish the cell line are described elsewhere [41]. The cells were grown either in 75-cm2 (time-course) or in 25-cm2 (AhR knockout) flasks in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) at 37 °C under CO2 (5%). For the time-course experiment, the cells were treated for 1–72 h with B(a)P dissolved in DMSO to a final concentration of 1 μM in the medium (10 μl of 1 mM B(a)P per 10 ml of the medium in 75-cm2 flasks; 0.1% of DMSO). For the RNA interference experiment, transfected UT-

2.3. GeneClip Hairpin oligonucleotide design and transformation of E. coli The small hairpin (shRNA) sequence targeting AhR mRNA (NM_001621) was designed using the siDESIGN Center (http:// dharmacon.gelifesciences.com/design-center/?redirect=true), obeying the rules outlined in the technical manual from GeneClip™ U1 Hairpin Cloning System (Promega). Selection of oligonucleotides, ligation to the pGeneClip neomycin vector construct, and transformation of One Shot® TOP10 competent E. coli cells (Invitrogen) were described in detail earlier [26]. Sequences of AhR knockdown oligonucleotides and nonspecific control sequences (not complementary to any known Fig. 1. (a) Positioning of the xenobioticresponsive element sequences in promoters of the investigated genes. Location of the core consensus sequence “GCGTG” in both orientations: forward (Δ) and reverse (□), up to −4000 bp upstream and 500 bp downstream of the transcription start site (TSS) in CYP1A1, CYP1B1, and SERPINB2. Genome Reference Consortium Human Build 38 patch release 7 assembly from March 2016 was used for this analysis. (b) Positioning of the xenobiotic-responsive element sequences in a putative enhancer (defined by H3K27ac in UCSC Genome Browser) −20000 bp till −15500 bp upstream of the SERPINB2 TSS. Dashed line -– position of the 774 eRNA (from −18948bp to −15975bp). c) Positioning of the SERPINB2, H3K27Ac, and H3K4Me3 marks (UCSC Genome Browser) and positioning of primers used in chromatin immunoprecipitation assays (arrows).

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with BNF dissolved in DMSO to a final concentration of 100 μM or DMSO alone (0.2% of DMSO), following a 15-min pretreatment with CHX dissolved in DMSO to a final concentration of 100 μg/ml medium (4 μl of 100 mg/ml (355.4 mM) CHX per 4 ml of the medium, 0.1% of DMSO) or DMSO. Final concentration of DMSO in the medium was 0.3%.

2.6. Chromatin immunoprecipitation (ChIP) assay The ChIP assay was performed using the ChromataChIP Kit (Novus Biologicals), following the manufacturer's protocol. Briefly, UT-SCC34 cells from two 75-cm2 flasks (80% confluent) were treated with or without 100 μM BNF or DMSO for 4 h and afterwards protein/DNA complexes were cross-linked by 10-min incubation in 1% formaldehyde at room temperature with gentle agitation. Cells from two dishes were pelleted together and lysed by 15-min incubation on ice in RIPA lysis buffer containing protease inhibitors (400 μl). DNA in the cell lysate was then sheared into 200- to 900-bp fragments by sonication (6 × 10s impulses/1.5 min rest on ice between impulses, Misonix 3000 sonicator – power level 1.5). AhR/chromatin complexes were precipitated by overnight incubation with AhR monoclonal antibody (RPT9) and caught by protein A/G magnetic beads. H3K4Me3 rabbit polyclonal antibody (NB21-1023) was used as a positive control. DNA was purified by silica columns (ChromataChIP kit) and used for quantitative realtime polymerase chain reactions (PCRs) performed as described below.

Fig. 2. The effect of AhR knockdown on the relative expression of 774 eRNA in UT-SCC-34 cells, measured 8 h after BNF or DMSO treatment. The negative control (NC) shRNA is a scrambled artificial sequence, which does not match any human gene. The AhR(−) shRNA is a sequence that decreases the expression of AhR mRNA by RNA interference. Each value represents the mean ± standard error for mRNA from four individual 25-cm2 flasks; each measurement was performed in triplicate. Statistical significance was assessed by one-way ANOVA followed by Newman-Keuls multiple comparisons test, p < 0.05. * – significantly different from solvent-treated cells.

2.7. RNA isolation and cDNA synthesis Total RNA was isolated directly from monolayer cells in a culture dish, as described before [42]. Estimation of RNA quantity, purity, and integrity as well as cDNA synthesis was performed as described earlier [26], except that for experiments measuring eRNAs, the Thermo Scientific cDNA synthesis kit with DNase treatment was used.

human, rat, and mouse sequence) have been described by us recently [26]. 2.4. Transfection of the pGeneClip vector to UT-SCC-34 cells Stable transfection of the pGeneClip neomycin vector construct to UT-SCC-34 cells was carried out according to suggestions from the GeneClip™ U1 Hairpin Cloning System technical manual (Promega). We applied lipofection with Lipofectamine 2000 (Invitrogen) as a transfection method. Cell line cultures were grown as a monolayer in 500 μl of DMEM supplemented with 10% FBS (without standard antibiotics) to reach about 90% confluence of 2 cm2 flasks. Liposomes were prepared by mixing 0.8 μg plasmid DNA diluted in 50 μl Opti-MEM medium with 2 μl of Lipofectamine 2000 in 50 μl of Opti-MEM. After 20 min of incubation, DNA-lipid complexes (100 μl) were added to UT-SCC-34 cells. Cells were passaged at 1:10 dilution into a fresh growth medium with standard antibiotics 24 h after transfection. Selective antibiotic G418 was added to the medium 3–4 days later, when cells reached about 50% confluence.

2.8. Primer design for real-time PCR Sequences of SERPINB2 qPCR primers, published recently, were as follows: forward 5′-GAATGCTGTCTACTTCAA-3′ and reverse 5′-TCTTC TATGTATCCAATGTT-3’ [26]. Primer sequences for eRNAs 774 and 571 were published earlier [33]. Primer sequences for reference genes and other genes used herein were published in our previous work [34]. ChIP DNA was used as a template for qPCR reactions with primers 5′-TGA TCCAGTCTAGGCTTCT-3′ and 5′- CTGTCCAGTAAGGCTTAGTAAT-3′ for the SERPINB2 promoter region (H3K4Me3 mark, 1392 bp from TSS, prSERPINB2); 5′-GAATCCTGTGACCTCAAG-3′ and 5′-CGCTTAGCATTT TATGTCTT-3′ for DNA localized between enhancer 1 and enhancer 2, -11984 bp from the SERPINB2 TSS (enh1-enh2); and 5′-TATGACTGG AGCCGACTT-3′ and 5′-GGTTGAAGTTTCTGCTGT-3′ for the CYP1B1 promoter region (−790 bp from TSS, prCYP1B1). Positioning of primers used in CHIP assays is indicated in Fig. 1c. Sequences of RPL30 primers included in the ChromataChip kit as a positive control were Novus Biologicals proprietary.

2.5. Cycloheximide treatment The cells were grown in 25-cm2 flasks to reach about 80% of confluence, as described in section 2.2. UT-SCC-34 cells were treated for 8 h

Table 1 The effect of AhR knockdown on the relative expression of SERPINB2, CYP1A1, and CYP1B1 genes in UT-SCC-34 cells, measured 24 h after β-naphthoflavone (BNF) treatment. The negative control shRNA is a scrambled artificial sequence, which does not match any human gene. The AhR(−) shRNA is a sequence that decreases the expression of AhR mRNA by RNA interference. The mean values of relative mRNA expression for the negative control were adjusted as 100. Each value represents the mean ± standard error for mRNA from three individual 25-cm2 flasks; each measurement was performed in triplicate. Statistical significance was assessed by ttest, p < 0.05 was considered as significant. a - significantly different from cells transfected with negative shRNA. Transfected construct

SERPINB2

CYP1A1

CYP1B1

AhR

AhR(−) Negative control

8.25 ± 1.53a 100.0 ± 19.99

16.39 ± 1.64a 100.0 ± 25.82

24.41 ± 2.40a 100.0 ± 23.63

27.64 ± 4.38 100.0 ± 29.13

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sequences in promoters of both CYP1A1 and CYP1B1 genes, only one such a sequence was found in the promoter of the SERPINB2 gene. The sequence was located at 2848–2852 bp upstream of the SERPINB2 TSS. Additionally, as the sequence of the second AhR-dependent enhancer element named XRE-II was reported [47,48], we included this element in our screening. Similarly, there was no such a sequence in the screened part of the SERPINB2 promoter. Furthermore, we did not find any sequence similar to nonconsensus XRE, described for the promoter of the mouse PAI-1 gene [49], in the SERPINB2 promoter. TSSs for CYP1A1, CYP1B1 and SERPINB2 genes were localized using reference sequences NM_000499, NM_000104, and NM_001143818, respectively. Genome Reference Consortium Human Build 38 patch release 7 assembly from March 2016 was used for the analysis. We therefore searched for XRE sequences more distantly, in the putative enhancer −20 kbp till −15.5 kbp upstream of the SERPINB2 TSS (Fig. 1b). The putative enhancer was defined as a chromatin site with acetylation of 27 lysine from the N-terminal tail of histone H3 (H3K27ac) in UCSC Genome Browser. The enhancer and the 774 eRNA (dashed line, from −18948bp to −15975bp; Fig. 1b) were reported as important for the regulation of SERPINB2 expression [33]. A cluster of core consensus sequences (2 oriented forward and 6 reverse) exists in the putative enhancer, especially in the region spanned by 774 eRNA. This is the only large cluster identified up to −60 kbp upstream of the SERPINB2 TSS. However, additional 4 XRE sequences (2 oriented forward and 2 reverse) are present in the DNA region from −11444 to −11066 bp upstream of the SERPINB2 TSS, but this region is located in transcriptionally inactive chromatin, as can be deduced from H3K27ac or H3K4Me1 marks (UCSC Genome Browser).

2.9. Real-time PCR ARNT, ß-actin, CYP1A1, CYP1B1, SERPINB2, and 774 cDNA were amplified by real-time PCR in the iCycler iQ5 real-time PCR detection system with iQ5 Optical System Software 2.0 (Bio-Rad Laboratories; Hercules, CA, USA), using SYBR® Green I as the detection dye. Amplification was carried out in a total volume of 20 μl containing 0.2x SYBR® Green I, PCR buffer (50 mM KCl, 10 mM Tris-HCl, pH 8.3), 3.5 mM MgCl2, 10 nM fluorescein, 0.2 μM each primer, 0.2 mM each dNTPs, 0.5 U of JumpStart Taq DNA Polymerase, and 0.5 μl of cDNA (undiluted reverse-transcription product derived from 8 μg of RNA in 40-μl reaction). The reaction parameters for SERPINB2 amplification [26] and conditions of amplification of other sequences used herein were published earlier [34]. The reaction parameters for 774 eRNA amplification were as follows: annealing at 59 °C for 10s, elongation at 72 °C for 20 s, and 1 μl of cDNA (undiluted) per 20-μl reaction. At the end of the PCR, a melting curve was generated by heating the samples from 50 °C to 95 °C in 0.5 °C increments with a dwell time at each temperature of 10s, to verify the specificity of the product. Non-template controls were run with every assay and no indication of PCR contamination was observed. The lack of PCR products from the nonreverse transcribed RNA control indicated that the possible contamination of the genomic DNA has not served as an amplification template. ChIP DNA was amplified by qPCR using exactly the same master mix, conditions, and equipment as described above, except that 2 μl of DNA (undiluted, ChromataChip purified) were used. The reaction parameters for DNA encoding 571 ncRNA, enh1-enh2, and prSERPINB2 amplification were as follows: annealing at 56 °C 10 s, elongation at 72 °C for 20 s, whereas annealing temperature for prCYP1B1 was established as 59.5 °C. The reaction parameters for RPL30 primers were as follows: annealing at 52 °C for 10 s and elongation at 72 °C for 20 s. Primers used in ChIP PCR are detailed above.

3.2. Effects of AhR silencing on BNF-induced mRNA expression of SERPINB2, CYP1A1, CYP1B1, and 774 eRNA Given that a previous study from our laboratory documented a huge induction of SERPINB2 expression by BNF in HepaRG cells [26], to

2.10. Quantitative PCR data analysis and statistics Expression levels of the target genes were normalized with respect to two reference genes, ß-actin and ARNT, using a relative quantification method. In rats, ß-actin expression was not affected by treatment with TCDD [43]. The ARNT mRNA levels of rats were not altered as a result of in vivo treatment with TCDD, 3-MC, and BNF [44,45]. All calculations were performed using Gene Expression MacroTM 1.10 software (Bio-Rad Laboratories, CA, USA). The relative gene expression was calculated for the triplicate samples derived from each RT reaction by Gene Expression MacroTM 1.10 (Bio-Rad) software. The average of the three values was used for the calculation of the mean ± standard error for each treatment group. Statistical significance of differences was assessed by t-test (Table 1) or by one-way ANOVA (Figs. 1, 3 and 4) followed by Newman-Keuls multiple comparisons test, using GraphPad Prism for Windows version 6.01 (GraphPad Software, San Diego, CA); p < 0.05 was considered statistically significant. Analysis of chromatin immunoprecipitation data was performed according to Novus Biologicals suggestions, and the results were expressed as percentage of total DNA input into the experiment.

Fig. 3. Time-course of the induction of SERPINB2, CYP1A1, and CYP1B1 mRNA expression in UT-SCC-34 cell line cells treated with benzo[α]pyrene. Expression levels of SERPINB2 (Δ), CYP1A1 (○) or CYP1B1 (□) relative to β-actin and ARNT mRNA concentrations are presented as % of the highest value. The highest mean values of relative mRNA expression of particular mRNAs were adjusted as 100. Each value represents the mean ± standard error for mRNA from three individual 75-cm2 flasks; each measurement was performed in triplicate. If the error bar cannot be seen, then standard error is within the size of the symbol. Reverse transcription was performed with Sigma STR-1 kit without DNase treatment. * Significantly different from the “0” time point, as determined by one-way ANOVA followed by Newman-Keuls multiple comparisons test, p < 0.05.

3. Results 3.1. In silico search for XRE-like sequences in SERPINB2, CYP1A1, and CYP1B1 promoters The location of the core consensus XRE-like sequence in both directions (forward “GCGTG” and reverse “CACGC”), up to −4000 bp upstream and 500 bp downstream of the TSS in CYP1A1, CYP1B1, and SERPINB2, is presented in Fig. 1a. Several variants of XREs have been published, so for our analysis we have used a constant core consensus sequence, but not extended ones [46]. In contrast to multiple XRE-like 5

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reduced by 92% after AhR knockdown (Table 1). The high concentration of BNF (100 μM) in the medium used in this experiment resulted in maximum induction of expression of AhR-dependent genes after 24 h [34]. Moreover, the effect of AhR knockdown on the relative expression of 774 eRNA after the BNF treatment of UT-SCC-34 cells was analyzed. A significant induction of expression of 774 eRNA was observed in the negative control (NC) transfected cells, whereas the induction was almost completely abolished after AhR knockdown (Fig. 2), suggesting that eRNA 774 induction after BNF treatment is AhR-dependent.

3.3. Time course of mRNA expression after treatment with benzo(a)pyrene Changes in relative mRNA levels of SERPINB2, CYP1A1, and CYP1B1 were analyzed throughout the 72-h time course following B(a) P administration to UT-SCC-34 cells. We found a 13.7-fold significant (p < 0.05) increase in SERPINB2 mRNA after 4 h of incubation with B (a)P (Fig. 3). After this time point, the expression of SERPINB2 rapidly decreased. Time-course curves of CYP1A1 and CYP1B1 expression after B(a)P treatment were very similar to that obtained for induction of SERPINB2 expression. The maximal induction of CYP1A1 and CYP1B1 expression was observed 4 h after B(a)P treatment (6.4 and 4.0-fold increase, respectively). After this time point, the expression of CYP1A1 and CYP1B1 mRNAs rapidly decreased. Time point “0”, presented in Fig. 3, depicts the expression of mRNA from control, untreated cells. During the experiment, no differences in expression of all examined genes were observed between untreated cells at time point “0” and cells treated only with the vehicle. To prove that 774 eRNA is induced by B(a)P, we compared the induction of this RNA with the induction of SERPINB2 mRNA. Fig. 4 presents the 774 eRNA and SERPINB2 mRNA levels throughout the 24-h time course following B(a)P administration to UT-SCC-34 cells. The expression of both 774 eRNA and SERPINB2 mRNA was significantly induced after B(a)P treatment. However, the expression of 774 eRNA precedes that of SERPINB2 mRNA (Fig. 4). Despite our attempts, we found only trace levels of 772 and 775 eRNA expression, below the limit of reliable detection in our experimental models (data not shown). In conclusion, our results demonstrate that AhR-dependent induction of 774 eRNA precedes the induction of SERPINB2 mRNA.

Fig. 4. Time-course of the induction of 774 eRNA and SERPINB2 mRNA expression in the UT-SCC-34 cell line cells treated with B(a)P. Expression levels of 774 (□) and SERPINB2 (○) relative to β-actin and ARNT mRNA concentrations are presented as % of the highest value. Each value represents the mean ± standard error for mRNA from three individual 75-cm2 flasks; each measurement was performed in triplicate. If the error bar cannot be seen, then standard error is within the size of the symbol. Reverse transcription was performed with Thermo Scientific Maxima K1671 kit with DNase treatment. * Significantly different from “0” and “24” time points. ** Significantly different from the “0”, “1”, and “24” time points, as determined by one-way ANOVA followed by Newman-Keuls multiple comparisons test, p < 0.05.

prove that BNF-induced changes of the investigated genes were indeed AhR-dependent, we knocked down the expression of AhR by stable transfection of UT-SCC-34 cells with shRNA. Quantitative PCR analysis revealed that our AhR-silencing construct was effective, as AhR mRNA expression was silenced by about 72% in UT-SCC-34 cells (Table 1). Reduction of AhR mRNA resulted in a substantial reduction of AhR function, since expression of CYP1A1 and CYP1B1 genes, both of them known to be regulated by AhR, was reduced by 84% and 76%, respectively. Much the same, BNF-induced expression of SERPINB2 was

Fig. 5. The effect of cycloheximide (CHX) pretreatment on the relative expression of SERPINB2 (panel a) and CYP1B1 (panel b) in UT-SCC-34 cells, measured 8 h after β-naphthoflavone (BNF) or DMSO treatment. Each value represents the mean ± standard error for mRNA from three individual 25cm2 flasks; each measurement was performed in triplicate. Statistical significance was assessed by oneway ANOVA followed by Newman-Keuls multiple comparisons test, p < 0.05: a -– significantly different from DMSO (solvent), cycloheximide (CHX) DMSO, and CHX BNF-treated cells; b -– not different from DMSO and CHX DMSO- treated cells; c -– significantly lower than any other treatment group; d -– significantly higher than any other treatment group.

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completely lost in AhR-deficient mice [6]. So, AhR signaling is an important factor in the development of cigarette smoke-induced cancers. Involvement of AhR in PAH-induced carcinogenicity was traditionally attributed to its ability to regulate the expression of genes encoding phase I and phase II xenobiotic-metabolizing enzymes. These enzymes are responsible for the activation of xenobiotics to ultimate carcinogenic metabolites and initiation of carcinogenesis [4,5]. However, AhR has recently been linked to several “non-traditional” roles in tumor promotion and progression. Thus, AhR has been found to impact differentially a number of endpoints, such as apoptosis, proliferation, cell growth, and differentiation [1–3]. Therefore, search for AhRregulated genes not directly connected to the metabolism of xenobiotics and their potentially new mechanisms of action seems to be very important for better understanding of the involvement of AhR in carcinogenesis. Consequently, microarray-based gene expression profiling studies have identified a large number of other genes, not directly connected to the metabolism of xenobiotics, that are induced or repressed in an AhR- and ligand-dependent manner [14–19,26]. One of such genes seems to be SERPINB2. Recently, we have found that SERPINB2 belongs to the genes most inducible by BNF, AhR-dependent genes, in both undifferentiated and differentiated HepaRG cells [26]. More precisely, SERPINB2 was the most inducible gene in differentiated HepaRG cells and ranked second (after CYP1A1) in undifferentiated cells. A search of the literature revealed that SERPINB2 was previously reported in different human cell lines as inducible by TCDD treatment [22–25]. Additionally, TCDD-induced expression of SERPINB2 was inhibited by the AhR antagonist CH223191 [13]. However, the involvement of AhR and its diverse ligands in induction of SERPINB2 expression was not further explored. Therefore, in order to investigate more extensively the AhR-dependent effects of BNF treatment on SERPINB2 expression in the cell culture model, we studied the effect of AhR knockdown on the

3.4. Effects of cycloheximide (CHX) pretreatment on BNF-dependent induction of SERPINB2 expression In order to examine whether AhR-dependent induction of SERPINB2 expression relies on direct AhR binding to SERPINB2 promoter or synthesis of some intermediate protein is involved, we used CHX, a potent protein synthesis inhibitor. Fig. 5a presents the mRNA levels of SERPINB2 after treatment with BNF for 8 h, and/or after 15-min pretreatment with CHX. We found that BNF treatment caused a significant increase in SERPINB2 mRNA level and the effect was completely abolished by coincubation with CHX. In contrast to induction of SERPINB2 mRNA expression, CHX did not reduce BNF-induced expression of CYP1B1 (Fig. 5b). In fact, as shown in Fig. 5b, CHX administration caused an increase in CYP1B1 mRNA expression to a level comparable with administration of BNF. Coadministration of CHX and BNF to UTSCC-34 cells resulted in an additive effect, where the level of CYP1B1 mRNA expression was about 19-fold and 3-fold as high as in the vehicle control (DMSO) and in cells treated with BNF or CHX, respectively. Therefore, our results suggest indirect regulation of SERPINB2 expression by AhR, with involvement of translation of an additional protein. 3.5. Chromatin immunoprecipitation analysis for ligand–AhR binding to putative XREs To determine whether AhR binds directly to the putative SERPINB2 gene enhancers and promoter, we performed chromatin immunoprecipitation assays (Fig. 6). UT-SCC-34 cells were grown with or without BNF for 4 h and analyzed by ChIP assays, using AhR monoclonal antibody for immunoprecipitation. The immunoprecipitated DNA was amplified by qPCR, using primers to amplify the XRE-rich DNA regions preceding the SERPINB2 gene. The 774 eRNA primers are localized about −18000 bp upstream of the TSS, in close vicinity to 7 XREs (Fig. 1b), within a DNA area marked by H3k27Ac modification of histone 3 as enhancer 1 (UCSC Genome Browser, GRCh37/hg19). The enh1-enh2 primers are localized −11984 bp upstream of the SERPINB2 TSS, in close vicinity to 4 XREs, within transcriptionally inactive chromatin. The 571 ncRNA primers are localized about −4600 bp upstream of the SERPINB2 TSS, within enhancer2 (H3k27Ac), very close to DNA sequence annotated as an AhR/ARNT-binding site in the UCSC Genome Browser. The prSERPINB2 primers are localized +1392 bp from the TSS within the active promoter (H4K4Me3 mark, UCSC Genome Browser, Fig. 1c). As a positive control, primers localized within the promoter of the CYP1B1 gene (prCYP1B1, -790 bp from the TSS) were used. Immunoprecipitation with rabbit polyclonal antibody NB21-1023 directed against modification of histone 3, indicating an active promoter (H3K4Me3), was used as an additional positive control of the method. As expected, AhR was recruited to the XRE region of the CYP1B1 promoter. In addition, the anti-AhR antibody efficiently immunoprecipitated this region in a ligand-dependent manner (Fig. 6). Conversely, sequences of DNA containing putative XRSs and preceding SERPINB2 gene were not efficiently immunoprecipitated by the AhR antibody (Fig. 6). These results strongly suggest that AhR controls SERPINB2 expression by a yet unknown indirect mechanism, different from that utilized to control CYP1B1 expression. Our positive control anti-H3K4Me3 antibody efficiently immunoprecipitated promoter regions of SERPINB2, CYP1B1, and RPL30 genes (4%, 7%, and 40% enrichment, respectively), but not in a liganddependent manner (data not presented graphically).

Fig. 6. UT-SCC-34 cells were grown with or without β-naphthoflavone (BNF) for 4 h and analyzed by chromatin immunoprecipitation assays, using AhR monoclonal antibody for immunoprecipitation. The immunoprecipitated DNA was amplified by qPCR using primers to amplify the DNA regions rich in xenobiotic-responsive elements, preceding the SERPINB2 gene (774 eRNA, enh1enh2, 571 ncRNA, prSERPINB2) and, as a positive control, the promoter of the CYP1B1 gene (prCYP1B1). Positions of primers in relation to the transcription start site (TSS) of each gene are detailed in section 2.8. Open bars represent solvent (DMSO) treatment, whereas filled bars stand for BNF treatment of cells. Quantitative data shown as percentage of input, represent means ± standard error of three independent experiments. NC – negative control. * Significantly different from corresponding solvent-treated cells.

4. Discussion Tobacco smoke contains numerous carcinogenic pyrolytic products (which bind to DNA and cause mutations), including polycyclic aromatic hydrocarbons (PAH), such as the potent B(a)P [39]. Experiments with knockout animals revealed that PAH-induced carcinogenicity is 7

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eRNAs encoded therein, is responsible for induction of SERPINB2 expression by different stimuli and different transcription factors. Maybe such a phenomenon could, at least partly, explain multiple observations of crosstalk between the AhR pathway and different signal transduction pathways (reviewed in Refs. [1–4]). Second, it is possible that some component of the LPS complex could itself serve as an AhR ligand. The second possibility could shed light on the intriguing issue of how the receptor system utilizing mostly exogenous ligands has evolved. Interestingly, 774 eRNA was identified as a novel LPS-inducible, noncoding RNA locus with elevated expression in monocytes from systemic lupus erythematosus (SLE) patients, as compared to a healthy control group [53]. Additionally, the authors of the above-mentioned publication indicate that CYP1A1 is significantly upregulated in SLE monocytes [53]. Since CYP1A1 is a model gene directly regulated by AhR, this finding supports the second possibility: AhR may be responsible for the elevated expression of both 774 eRNA and SERPINB2, in monocytes from SLE patients. This suggestion seems to be plausible, as numerous interactions of the AhR-signaling pathway with the immunological system were reported earlier [54]. However, additional research is required to prove this hypothesis. Furthermore, we tested the possibility that AhR-dependent induction of SERPINB2 expression by BNF is indirect, and relies on prior synthesis of a yet unknown AhR-dependent protein. Therefore, to address this problem, we used CHX (an inhibitor of translation) to incubate the cells before BNF administration. We found that BNF treatment caused a significant increase in SERPINB2 mRNA level, whereas coincubation with CHX completely abolished the observed induction (Fig. 5a). Our results clearly indicate the involvement of a newly synthesized protein in AhR-dependent induction of SERPINB2 expression by BNF. Conversely, CHX did not inhibit BNF-dependent induction of CYP1B1 expression (Fig. 5b). What is more, CHX, on its own, caused a large increase in CYP1B1 mRNA levels (Fig. 5b) and the effect was additive with BNF treatment of the cells. Superinduction of CYP1A1 mRNA by the combination of CHX and an AhR agonist was reported earlier [55–57] and has been attributed to the fact that CHX treatment increases the level of AhR protein in cells by blocking AhR proteolysis [57]. However, other reports speak against such an explanation [56]. Thus, considering that the effect of CHX on CYP1B1 expression was not the priority of our research, we did not seek further for an explanation of the observed phenomenon. Trying to explore more deeply the mechanisms of SERPINB2 regulation, we compared the expression of SERPINB2 with the expression of CYP1A1 and CYP1B1 genes, regulated directly by AhR. Interestingly, we found that the time course of induction of mRNA by B(a)P treatment in the UT-SCC-34 cell line followed the same pattern for all 3 genes under examination, with a maximum of mRNA induction after 4 h (Fig. 3). We reported earlier in rats that the maximum induction of Cyp1a1 mRNA expression was observed after 8 h, whereas the maximum induction of Cyp1a1 enzymatic activity was 24 h after BNF treatments [44,58]. Therefore, assuming an indirect induction of SERPINB2 expression, we expected a similar delay. However, no delay was observed. We can only speculate that the newly translated protein is characterized by a very fast synthesis ratio. To determine whether AhR binds directly to the putative XREs localized upstream of the SERPINB2 gene, we performed chromatin immunoprecipitation assays (Fig. 6). We found no direct binding of AhR to its responsive elements in the vicinity of the SERPINB2 gene. These results support the findings of our CHX experiments and strongly suggest that AhR controls SERPINB2 expression by an indirect mechanism, different from that utilized to control CYP1B1 expression. These findings are in concordance with previous results that direct AhR binding is not required in about one third of TCDD inducible genes [59]. On the other hand, the localization of the cluster of XREs within the 774 eRNA sequences upstream of the SERPINB2 TSS suggests the opposite. It was earlier demonstrated that most inducible genes are regulated

expression of SERPINB2, in comparison to the expression of CYP1A1 and CYP1B1 (Table 1). Our results clearly indicated that AhR is the transcription factor responsible for the induction of SERPINB2 expression by BNF in UT-SCC-34 cells. In fact, reduction of gene expression after AhR knockdown was more pronounced in the case of SERPINB2 than in the case of reference AhR-dependent genes, such as CYP1A1 or CYP1B1 (Table 1). Furthermore, despite substantial differences between diverse AhR ligands, including BNF and TCDD reported elsewhere [17], our results are consistent with reports describing induction of SERPINB2 expression by TCDD treatment of different human cell lines [22–25]. Thus, involvement of AhR in induction of SERPINB2 expression seems to be well documented. However, in silico analysis of SERPINB2 promoter indicates substantial differences, compared to promoters of CYP1A1 or CYP1B1 genes (Fig. 1a). DNA sequence analyses of the human SERPINB2 promoter revealed only one XRE-like element located at 2848–2852 bp upstream of the SERPINB2 TSS. The putative XRE-like element ( 5′-TNGCGTGATC-3′) resembles an extended XRE but is not identical in sequence to a classical XRE, which contains the consensus sequence 5′T/GNGCGTGA/CG/CA-3’ [50,51]. Although the putative SERPINB2 XRE-like element contains the core nucleotides GCGTG, there is a significant variation in the flanking 3′ sequences. Since the flanking nucleotides also contribute to XRE activity [51], the putative SERPINB2 XRE-like element may not be functional. In contrast to SERPINB2, 14 and 10 XRE-like elements can be found in CYP1A1 and CYP1B1 promoters, respectively (Fig. 1a). Furthermore, we did not find any sequences similar to XRE-II [47] or to nonconsensus XRE [49] in the SERPINB2 gene promoter, which were reported as alternative responsive elements recognized by AhR. A single XRE-like element seems unlikely to be responsible for such an intense induction of SERPINB2 expression; however, we cannot completely exclude this possibility. On the basis of SERPINB2 induction by phorbol 12-myristate 13-acetate (PMA), it has been reported that the human SERPINB2 gene promoter is controlled by three major transcription regulatory domains: an inducible proximal promoter, an upstream silencer, and a distal transactivator region between −5100 bp and −3300 bp, which appears to overcome inhibition mediated by the silencer [32]. PMA-induced changes in gene transcription are primarily mediated through the specific binding of AP-1 complexes to the DNA sequence known as PMAresponsive element, and such elements were found in a distal transactivator region of the SERPINB2 gene. Conversely, we could not find any known AhR-dependent responsive elements in the transactivator region (−5100 bp to −3300 bp) of the SERPINB2 gene. Therefore, trying to elucidate the mechanism of AhR-dependent induction of the SERPINB2 gene, we had a closer look on more distant parts of DNA. We found a cluster of the classical XRE about −20000 bp upstream of the SERPINB2 TSS (Fig. 1b). Further in silico analysis determined that the XRE cluster is located within the H3K27ac mark of active chromatin (UCSC Genome Browser). H3K27ac is a modification to histone H3, which involves acetylation at the 27th lysine from the Nend of the protein and is defined as an active enhancer mark [52]. A search of the literature revealed that the −20 Kb enhancer is responsible for induction of SERPINB2 expression by bacterial LPS in human monocytes, and enhancer RNAs, namely 772, 774 and 775 eRNAs, are involved in this process [33]. This has led us to the hypothesis that maybe the same enhancer region of DNA and the same eRNA are responsible for the induction of SERPINB2 expression by AhR ligands as well. Indeed, we determined that AhR ligands were capable of induction of 774 eRNA expression (Figs. 2 and 4). Furthermore, the induction of 774 eRNA expression by AhR ligands precedes the induction of SERPINB2 expression, and this is consistent with results obtained for LPS [33]. Additionally, we demonstrated that AhR itself is responsible for the induction of 774 eRNA expression, as AhR knockdown diminished the effect (Fig. 2). A comparison of our results with results obtained by others [33] leads to interesting conclusions. First, it is possible that the same enhancer region of DNA, together with the 8

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at the level of pause-release and elongation [60–62]. According to this concept, paused RNA polymerase II (RNAPII) is held in place by the negative elongation factor (NELF) and can be released from pausing by recruitment of positive transcription elongation factor-b (P-TEFb) [63]. The eRNAs may induce the release of NELF from RNAPII [64]. In view of the above, studies of the SERPINB2 promoter demonstrated the presence of a NELF protein. Additionally, induction with LPS led to recruitment of the pause-releasing kinase P-TEFb and departure of the pause-inducing protein NELF, and the process is stimulated by 774 eRNA [33]. Therefore, given our findings of AhR-dependent induction of 774 eRNA expression by AhR ligands, it seems plausible that endpoint molecular mechanisms of induction of SERPINB2 expression by AhR ligands and LPS are very similar. In spite of the above hypothesis, we still do not know how AhR interacts with the pause-release machinery and, in particular, which newly-translated protein is involved in the process. In conclusion, our results indicate that AhR is involved in the regulation of SERPINB2 expression. We have demonstrated that the expression of SERPINB2 can be induced by diverse AhR ligands, the induction appears to rely on synthesis of a yet unknown protein, and no direct binding of AhR to sequences preceding SERPINB2 is necessary for the process. However, additional work is needed to unravel the complex interplay between AhR and diverse signal transduction pathways in the regulation of SERPINB2 expression. This is especially interesting as, on the one hand, the exact physiological function of SERPINB2 still remains mysterious and, on the other hand, numerous studies indicate its involvement in diverse pathological conditions.

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