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The Flavonoid Quercetin Induces AP-1 Activation in FRTL-5 Thyroid Cells

The Flavonoid Quercetin Induces AP-1 Activation in FRTL-5 Thyroid Cells antioxidants Brief Report The Flavonoid Quercetin Induces AP-1 Activation in FRTL-5 Thyroid Cells Cesidio Giuliani Unit of Endocrinology, Department of Medicine and Sciences of Aging, and Ce.S.I.-Me.T., University of Chieti-Pescara, 66100 Chieti, Italy; cesidio.giuliani@unich.it Received: 20 March 2019; Accepted: 23 April 2019; Published: 29 April 2019 Abstract: Previous studies have shown that quercetin inhibits thyroid function both in vitro and in vivo. An attempt to evaluate the e ect of quercetin at the promoter level of the thyroid-specific genes led to the observation that this compound induces the basal activity of the reporter vector. Therefore, the action of quercetin has been evaluated on the basal activity of several reporter vectors: The PGL3 basic, promoter and control vectors from Promega, and a pSV-based chloramphenicol acetyltransferase (CAT) reporter vector. In the Fisher Rat Thyroid cell Line FRTL-5 thyroid cells transiently transfected, quercetin 10 M increased the basal activity of all the reporter vectors evaluated, although the degree of the e ect was significantly di erent among them. The analysis of the di erence among the regulatory regions of these vectors identified the activator protein 1 (AP-1) binding site as one of the potential sites involved in the quercetin e ect. Electromobility shift assay experiments showed that the treatment with quercetin induced the binding of a protein complex to an oligonucleotide containing the AP-1 consensus binding site. This is the first study showing an e ect of quercetin on AP-1 activity in thyroid cells. Further studies are in progress to understand the role of AP-1 activation in the e ects of quercetin on thyroid function. Keywords: quercetin; thyroid; FRTL-5; AP-1; gene expressions; PGL3 vectors; pSV0-CAT vector 1. Introduction 0 0 Quercetin (3,3 ,4 ,5,7-pentahydroxyflavone) is one of the most widely distributed and abundant flavonoids present in fruits and vegetables. Several studies have shown that quercetin possesses many therapeutically relevant properties, and therefore its e ects have been evaluated at cellular and molecular level [1–7]. Several reports have shown that quercetin possesses anti-thyroid and goitrogenic e ects [8–11]. Indeed, quercetin inhibits iodide organification through a thiourea-like action inhibiting thyroid peroxidase (TPO) enzyme activity and interferes with thyroid hormone metabolism, particularly through the inhibition of type I 5 -deiodinase activity [8]. Besides this, quercetin down-regulates the expression of the thyroid-specific genes sodium/iodide symporter (NIS), thyrotropin receptor (TSHR), TPO, and thyroglobulin (TG) in the FRTL-5 rat thyroid cells in continuous culture [10,11]. An attempt to evaluate the e ect of quercetin at the promoter level of the aforementioned thyroid-specific genes showed a strong induction of the basal activity of the reporter gene (PGL3 Luciferase reporter basic vector) by quercetin, which interfered with the analysis of the promoter. This observation led to inquire the action of quercetin on the expression of several Luciferase reporter vectors of the PGL3 series: the basic, the promoter, and the control vectors. Furthermore, a di erent type of vector was also evaluated, the pSV0-CAT. For this study, the FRTL-5 thyroid cells were used. These cells are a nontransformed rat thyroid cell line in continuous culture that represents a well-defined and reproducible in vitro model of the thyroid gland [12–16]. Antioxidants 2019, 8, 112; doi:10.3390/antiox8050112 www.mdpi.com/journal/antioxidants Antioxidants 2019, 8, 112 2 of 10 The treatment with quercetin of the FRTL-5 cells transiently transfected with the PGL3 basic, promoter and control vectors, and with the pSV0-CAT vector, increased the activity of all the reporter vectors evaluated, although the degree of the e ect was significantly di erent among them. The analysis of the di erence among the regulatory regions of the aforementioned vectors has identified the activator protein 1 (AP-1) binding site as one of the potential transcription factors involved in the quercetin e ect. Indeed, quercetin treatment results in AP-1 activation in FRTL-5 cells. 2. Materials and Methods 2.1. Materials Quercetin was from Sigma-Aldrich Co (St. Louis, MO, USA). Heat-treated, mycoplasma-free calf serum was from Life Technologies Europe (Monza, Italy). [ - P]-ATP was from Perkin Elmer Italia (Monza, Italy). Antibodies against c-fos (sc-413) and c-jun (sc-44) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The source of all of the other materials was Sigma-Aldrich, unless otherwise specified. 2.2. Cell Culture The F1 subclone of FRTL-5 rat thyroid cells (American Type Culture Collection, CRL-8305) was a gift from the Interthyr Research Foundation (Marietta, OH, USA). These FRTL-5 cells were grown in the six-hormone (6H) medium of Coon’s modified Ham’s F-12 supplemented with 5% calf serum, 2 mM glutamine, 1 mM nonessential amino acids, and the 6H mixture (6H5% medium): 1 mU/mL bovine TSH (Thyroid-stimulating hormone), 10 g/mL insulin, 0.4 ng/mL cortisol, 5 g/mL transferrin, 10 ng/mL glycyl-L-histidyl-L-lysine acetate, and 10 ng/mL somatostatin. These cells were diploid, between the 5th and 25th passage, and had all of the functional properties described previously [12–16]. Fresh 6H5% medium was added to the cells every 2 to 3 days, and they were passaged every 7 days. In individual experiments, the cells were transferred to a five-hormone (5H) medium (i.e., without TSH), again with 5% calf serum (5H5% medium). The treatments were performed with 10 M quercetin. In all of the experiments with quercetin, the medium was changed every 24 h, with addition of fresh medium with quercetin. Quercetin was used from a stock solution in absolute ethanol, with control cells treated with the same amount of vehicle. The final ethanol concentration was thus identical in the control and treated samples and did not exceed 0.1% (v/v). 2.3. Plasmids and Transfection The Luciferase reporter vectors PGL3 basic, PGL3 control, and PGL3 promoter were from Promega Corp. (Madison, WI, USA). The pSV0-CAT vector was obtained from the pSV2-CAT vector by removing the SV40 (Simian virus 40) early promoter sequences [17]. The reporter vectors were transiently transfected into the FRTL-5 cells using the diethylaminoethyl (DEAE)-dextran procedure [18,19]. The cells were grown to 60% confluency, maintained in 5H5% medium for 6 days, and then returned to 6H5% medium 12 h before transfection. Twenty-four hours after transfection, the cells were treated with 10 M quercetin or the control vehicle for 48 h. 2.4. Luciferase and CAT Assay Luciferase assays were performed by using the luciferase assay system (Promega) following manufacturer instructions and a LUMAT LB 9507 Luminometer (EG & G Berthold, Bundoora, Australia). All values were normalized for total cell proteins. Chloramphenicol acetyltransferase (CAT) assays were performed using the CAT ELISA kit (Roche Diagnostics GmbH, Mannheim, Germany) following manufacturer instructions. All values were normalized for total cell proteins. Antioxidants 2019, 8, 112 3 of 10 2.5. Nuclear Extracts and Electrophoretic Mobility Shift Assay FRTL-5 cells were grown to 60% confluency and maintained in 5H5% medium for 6 days to become quiescent. They were cultured again in 6H5% medium for 24 h and then treated with 10 M quercetin or the control vehicle for 48 h. Nuclear extracts were prepared as previously described [20,21]. DNA probe was made labeling the AP-1 consensus oligonucleotide (Santa Cruz Biotechnology) with [ - P]-ATP using T4 polynucleotide kinase (New England Biolabs Inc., Ipswich, MA, USA) and purified by passing through a G-50 MicroSpin Sephadex column. The electrophoretic mobility shift assays (EMSAs) were performed as described previously [14,21]. The binding reactions were performed in high salts plus detergent and included 1.5 fmol of [ P]DNA, 4 g of nuclear extracts and 1 g poly(dI-dC) in 10 mM Tris-Cl at pH 7.9, 5mM MgCl , 50 mM KCl, 1 mM DTT, 1 mM EDTA, 0.1% Triton X-100, and 12.5% glycerol in a total volume of 20 L, with incubations at room temperature for 30 min. Where indicated, antibodies were added to the binding reactions, followed by incubation with the nuclear extracts for 20 min before the addition of the labeled DNA. Following the incubations, the reaction mixtures were electrophoresed on 5% native polyacrylamide gels at 160 V in 0.5% Tris-borate-EDTA bu er at room temperature. The gels were dried and autoradiographed. 2.6. Other Assays Protein concentrations were determined using bicinchoninic acid (BCA) protein assay kits (Pierce Biotechnology Inc., Rockford, IL, USA), with crystalline bovine serum albumin (BSA) as standard. 2.7. Statistical Analysis All experiments were repeated at least three times with independent batches of cells. The data are given as means  S.D. The significance between experimental values was determined by unpaired two-tailed t-test, with p < 0.05 or better when the data from all of the experiments were considered. 3. Results 3.1. Quercetin Up-Regulates the Activity of the Following Reporter Vectors: PGL3 Basic, PGL3 Control, PGL3 Promoter, and PSV0-CAT The FRTL-5 cells were grown in 6H5% medium until 60% confluent, and then switched to 5H5% medium for 6 days (i.e., without TSH) to become quiescent. They were cultured again in 6H5% medium for 12 h and then transiently transfected with the following Luciferase reporter vectors: PGL3 basic, PGL3 control, and PGL3 promoter. After 24 h from transfection, cells were treated with 10 M quercetin for 48 h. The dose and time of the treatment were chosen based on previous studies that had shown that these treatment conditions were characterized by maximal e ects of quercetin on thyroid genes expression, without toxic e ects on the FRTL-5 cells [10,11]. As shown in Figure 1, quercetin treatment significantly increased the activity of the aforesaid reporter vectors. First of all, it has to be noted that there is a significant di erence in the basal luciferase activities of the three vectors in the control cells (Figure 1a). This is due to the presence in the PGL3 promoter vector of the SV40 promoter region upstream of the luciferase gene (between 48 and 250 bp of the vector sequence) and to the presence in the PGL3 control vector of the SV40 enhancer region (between 2205 and 2441 bp of the vector sequence) in addition to the SV40 promoter region [22]. Therefore, the e ect of quercetin was evaluated as percentage of control for each vector. This analysis revealed that quercetin treatment increased the activity of the PGL3 basic vector about 2-fold (248.69% 15.68% of control value), whereas it increased the activities of the PGL3 promoter and PGL3 control vectors of more than 6-fold (703.06 22.3% and 697.39% 36.48% of control values, respectively) (Figure 1b). Antioxidants 2019, 8, 112 4 of 10 Antioxidants 2019, 8, x FOR PEER REVIEW 4 of 10 (a) (b) Figure 1. E ects of 10 M quercetin on Luciferase activity in Fischer Rat Thyroid cell Line FRTL-5 cells Figure 1. Effects of 10 M quercetin on Luciferase activity in Fischer Rat Thyroid cell Line FRTL-5 transiently transfected with PGL3 basic, PGL3 promoter and PGL3 control. (a) Data are expressed as cells transiently transfected with PGL3 basic, PGL3 promoter and PGL3 control. (a) Data are Relative Lights Units (RLU) to protein concentrations (proteins conc.) ratio and represent the means expressed as Relative Lights Units (RLU) to protein concentrations (proteins conc.) ratio and represent S.D. of three separate experiments; (b) Same data expressed relative to the control of each vector (set to the means ± S.D. of three separate experiments; (b) Same data expressed relative to the control of each 100%). Control, cells treated with the control vehicle (0.1% ethanol); Querc, cells treated with 10 M vector (set to 100%). Control, cells treated with the control vehicle (0.1% ethanol); Querc, cells treated quercetin. * p < 0.05 versus relevant control. with 10 M quercetin. * p < 0.05 versus relevant control. These data suggested that quercetin treatment induced the activation of one or more transcription These data suggested that quercetin treatment induced the activation of one or more factors able to increase the activity of the PGL3 vectors. The attention was focused on the potential transcription factors able to increase the activity of the PGL3 vectors. The attention was focused on factors binding the SV40 promoter region, since its presence was responsible for a stronger e ect of the potential factors binding the SV40 promoter region, since its presence was responsible for a quercetin on luciferase activity (compare PGL3 promoter with PGL3 basic in Figure 1b). Nevertheless, stronger effect of quercetin on luciferase activity (compare PGL3 promoter with PGL3 basic in Figure no further significant increase was brought by the presence of the SV40 enhancer region in the PGL3 1b). Nevertheless, no further significant increase was brought by the presence of the SV40 enhancer control vector compared to the PGL3 promoter vector (Figure 1b). Therefore, the putative transcription region in the PGL3 control vector compared to the PGL3 promoter vector (Figure 1b). Therefore, the factors binding the sequence of the SV40 promoter in the PGL3 promoter vector were analyzed. putative transcription factors binding the sequence of the SV40 promoter in the PGL3 promoter This sequence, spanning from 48 to 250 bp of the vector sequence [23], was analyzed with the web tools vector were analyzed. This sequence, spanning from 48 to 250 bp of the vector sequence [23], was LASAGNA-search 2.0 and AliBaba2.1 [24–26]. The analysis revealed several potential transcription analyzed with the web tools LASAGNA-search 2.0 and AliBaba2.1 [24–26]. The analysis revealed factors binding sites. Among them, the transcription factors characterized by the best matching were several potential transcription factors binding sites. Among them, the transcription factors AP-1, Oct-1 and Sp1, with AP-1 having the highest score, Table 1. Indeed, the SV40 promoter of the characterized by the best matching were AP-1, Oct-1 and Sp1, with AP-1 having the highest score, PGL vectors contains two characterized AP-1 binding sites [26], one with reverse orientation from 58 Table 1. Indeed, the SV40 promoter of the PGL vectors contains two characterized AP-1 binding sites to 64 bp and the other with forward orientation from 144 to 150 bp. [26], one with reverse orientation from 58 to 64 bp and the other with forward orientation from 144 to 150 bp. Table 1. Transcription factors with the best binding match with the SV40 promoter sequence. Table 1. Transcription fa Tc ranscription tors with the Factor best bindinScore g match witp h V th alue e SV40 promoter sequence. AP-1 13.33 0 Transcription Factor Score p Value Oct-1 13.00 0 AP-1 13.33 0 Sp1 12.32 0.00025 Oct-1 13.00 0 Data obtained with LASAGNA-search 2.0 [24]. Sp1 12.32 0.00025 Data obtained with LASAGNA-search 2.0 [24]. 0 0 The two AP-1 binding sites have an identical sequence: 5 -TGACTAA-3 , although in inverted The two AP-1 binding sites have an identical sequence: 5’-TGACTAA-3’, although in inverted orientation. This sequence has been first described in the human papilloma virus (HPV)-18 regulatory orientation. This sequence has been first described in the human papilloma virus (HPV)-18 regulatory region, where there are also two binding sites in inverted orientation [27,28]. region, where there are also two binding sites in inverted orientation [27,28]. Potential binding sites for the transcription factors AP-1, Oct-1, and Sp1 were also identified in Potential binding sites for the transcription factors AP-1, Oct-1, and Sp1 were also identified in the backbone of the PGL3 basic vector. These data could explain the e ect of quercetin in enhancing the backbone of the PGL3 basic vector. These data could explain the effect of quercetin in enhancing the luciferase activity even in the PGL3 basic vector. the luciferase activity even in the PGL3 basic vector. Quercetin action was also evaluated in a di erent reporter vector, the pSV0-CAT, a pSV2-derived Quercetin action was also evaluated in a different reporter vector, the pSV0-CAT, a pSV2- vector that does not contain promoter or enhancer elements and is characterized by a very low derived vector that does not contain promoter or enhancer elements and is characterized by a very background activity [17,29]. FRTL-5 cells were transfected with the pSV0-CAT vector, as described low background activity [17,29]. FRTL-5 cells were transfected with the pSV0-CAT vector, as above for the PGL3 vectors, and treated with quercetin 10 M for 48 h. As shown in Figure 2, quercetin described above for the PGL3 vectors, and treated with quercetin 10 μM for 48 h. As shown in Figure treatment increased 10-fold the CAT expression compared to control activity, 1135.5%  24.5% of 2, quercetin treatment increased 10-fold the CAT expression compared to control activity, 1135.5% ± control. Surprisingly, this e ect was even higher than that observed with the PGL3 control and 24.5% of control. Surprisingly, this effect was even higher than that observed with the PGL3 control promoter vectors. and promoter vectors. Antioxidants 2019, 8, 112 5 of 10 Antioxidants 2019, 8, x FOR PEER REVIEW 5 of 10 (a) (b) Figure 2. E ects of 10 M quercetin on CAT (Chloramphenicol acetyltransferase) expression in FRTL-5 Figure 2. Effects of 10 M quercetin on CAT (Chloramphenicol acetyltransferase) expression in FRTL- cells transiently transfected with pSV0-CAT vector. (a) CAT protein concentrations are normalized 5 cells transiently transfected with pSV0-CAT vector. (a) CAT protein concentrations are normalized with respect to total cell protein concentrations and represent the means  S.D. of three separate with respect to total cell protein concentrations and represent the means ± S.D. of three separate experiments; (b) Same data expressed relative to the control vector (set to 100%). Control, cells treated experiments; (b) Same data expressed relative to the control vector (set to 100%). Control, cells treated with the control vehicle (0.1% ethanol); Querc, cells treated with 10 M quercetin. * p < 0.05 versus with the control vehicle (0.1% ethanol); Querc, cells treated with 10 M quercetin. * p < 0.05 versus relevant control. relevant control. A possible explanation of the quercetin e ect on pSV0-CAT activity is the presence of cryptic A possible explanation of the quercetin effect on pSV0-CAT activity is the presence of cryptic promoters in the pBR322 backbone of the pSV0 vector [30,31]. Indeed, the analysis of the pSV0-CAT promoters in the pBR322 backbone of the pSV0 vector [30,31]. Indeed, the analysis of the pSV0-CAT vector revealed the presence of the potential binding sites for the aforementioned transcription factors vector revealed the presence of the potential binding sites for the aforementioned transcription AP-1, Oct-1, and Sp1. Of particular interest is the presence of these binding sites in a region of about factors AP-1, Oct-1, and Sp1. Of particular interest is the presence of these binding sites in a region of 1100 bp upstream of the CAT gene, since this region is important for the regulation of the CAT gene about 1100 bp upstream of the CAT gene, since this region is important for the regulation of the CAT transcription [30,31]. Notably, a non-canonical AP-1 binding site [32,33] was present from 1025 to 1031 gene transcription [30,31]. Notably, a non-canonical AP-1 binding site [32,33] was present from 1025 0 0 bp, whose sequence 5 -TGAGTAA-3 is similar to that contained in the SV40 promoter described above to 1031 bp, whose sequence 5’-TGAGTAA-3’ is similar to that contained in the SV40 promoter 0 0 (5 -TGACTAA-3 ). described above (5’-TGACTAA-3’). 3.2. Quercetin Induces AP-1 Binding to DNA 3.2. Quercetin Induces AP-1 Binding to DNA The transcription factors members of the AP-1 family are involved in the regulation of thyroid The transcription factors members of the AP-1 family are involved in the regulation of thyroid growth and thyroid genes expression [34–37]. Further, quercetin has an opposite role in AP-1 activation, growth and thyroid genes expression [34–37]. Further, quercetin has an opposite role in AP-1 since it increases AP-1 activity in some in vitro models [38,39], whereas it decreases it in other activation, since it increases AP-1 activity in some in vitro models [38,39], whereas it decreases it in models [40,41]. For these reasons, EMSA experiments were performed to evaluate the e ect of other models [40,41]. For these reasons, EMSA experiments were performed to evaluate the effect of quercetin on AP-1 activity in the FRTL-5 cells. Nuclear extracts from cells treated with 10 M quercetin on AP-1 activity in the FRTL-5 cells. Nuclear extracts from cells treated with 10 M quercetin for 48 h were incubated with a radiolabeled AP-1 consensus oligonucleotide. As shown in quercetin for 48 h were incubated with a radiolabeled AP-1 consensus oligonucleotide. As shown in Figure 3 (lane 3 versus lane 2), quercetin treatment induced the formation of a protein/DNA complex. Figure 3 (lane 3 versus lane 2), quercetin treatment induced the formation of a protein/DNA complex. To identify the transcription factors involved in the protein complex, nuclear extracts were preincubated To identify the transcription factors involved in the protein complex, nuclear extracts were with antisera to c-fos and c-jun. Preincubation of the extracts with c-jun antiserum decreased the preincubated with antisera to c-fos and c-jun. Preincubation of the extracts with c-jun antiserum protein/DNA complex to 52 4% of control (Figure 3, lane 5 versus lane 4), whereas a slight but not decreased the protein/DNA complex to 52 ± 4% of control (Figure 3, lane 5 versus lane 4), whereas a significant e ect was seen with the c-fos antiserum (90 8,5% of control), (Figure 3, lane 6 versus lane slight but not significant effect was seen with the c-fos antiserum (90 ± 8,5% of control), (Figure 3, 4). Although it was not possible to see any super shift, the decrease of the complex by the antibodies lane 6 versus lane 4). Although it was not possible to see any super shift, the decrease of the complex against c-jun suggests that this protein may either bind directly to the cis-acting element or interact by the antibodies against c-jun suggests that this protein may either bind directly to the cis-acting with other transcription factors involved in the protein/DNA complex formation. element or interact with other transcription factors involved in the protein/DNA complex formation. Antioxidants 2019, 8, 112 6 of 10 Antioxidants 2019, 8, x FOR PEER REVIEW 6 of 10 Figure 3. E ects of quercetin on the formation of protein/DNA complexes between FRTL-5 cell nuclear Figure 3. Effects of quercetin on the formation of protein/DNA complexes between FRTL-5 cell extracts and an AP-1 (Activator protein-1) consensus oligonucleotide. Radiolabeled oligonucleotide nuclear extracts and an AP-1 (Activator protein-1) consensus oligonucleotide. Radiolabeled was incubated with nuclear extracts from cells cultured in 6H5% medium (control), cells treated oligonucleotide was incubated with nuclear extracts from cells cultured in 6H5% medium (control), with 10 M quercetin (Querc) for 48 h; cells treated with quercetin plus normal rabbit polyclonal cells treated with 10 M quercetin (Querc) for 48 h; cells treated with quercetin plus normal rabbit antiserum (Querc-Cont) or antibodies against c-jun (+anti-c-jun) or c-fos (+anti-c-fos). Lane 1 contains polyclonal antiserum (Querc-Cont) or antibodies against c-jun (+anti-c-jun) or c-fos (+anti-c-fos). Lane the probe alone. 1 contains the probe alone. 4. Discussion 4. Discussion The present study is the consequence of an attempt to investigate the e ect of quercetin on the expression The prof esethe nt st thyr udy oid-specific is the conseq genes uence at otranscriptional f an attempt to level. investiga Indeed, te the the effec analysis t of querof ceti the n oe n th ect e of expr quer essi cetin on of on the the thyro NIS id pr -spec omoter ific genes gene inserted at transcri in pti the onPGL3 al level basic . Indee vector d, the was anaspoiled lysis of th by e a efstr fecong t of incr quer ease cetin of on the the basal NIS pr activity omoterof gene theiempty nsertedvector in the P induced GL3 basi by c vthe ectoquer r was cetin spoil tr ed eatment. by a stroThese ng incrdata ease wer of th e e surprising, basal activ as ity the of PGL3 the empty basic vector vector is inbelieved duced by toth be e transcriptionally quercetin treatmen neutral, t. These since datit a lacks were any surpr viral ising pr , as omoter the PG orLenhancer 3 basic vec region. tor is bHowever elieved to , even be trapr nscri omoterless ptionally vectors, neutralsuch , since as it the lacks PGL3 any basic, viral can prom be otransactivated ter or enhancer by reseveral gion. Ho stimuli weverin , etransiently ven promot transfected erless vecto cells, rs, suc due h ato s tthe he PG presence L3 basi of c, cryptic can be r tra egulatory nsactivasequences ted by sev contained eral stimin ulithe in vector transientl backbone y tran[sfe 42,cte 43]. d Notably cells, d,ue quer toceti the n had prese an str ceonger of cre ypti ect c on regul the ato transactivati ry sequence on s of con the taiPGL3 ned in pr th omoter e vecto vector r back . b This one e[ 4ect 2,43 could ]. Nota be bl explained y, querceti by n the hadp a r esence stronger in this effec vector t on th of e the tran SV40 sactiva promoter tion of r th egion e PGupstr L3 pr eam omo of ter the veluciferase ctor. Thisgene. effect These coulddata be ex led plto ain investigate ed by the the prese potential nce in th transcription is vector of th factors e SV40binding promoter sites regi contained on upstrea in mthe of th SV40 e luci pr fomoter erase gene and . These shared da by ta the led PGL3 to investi basic gavector te the .po Among tential them, transcri AP-1, ption Oct-1, factors and bin Sp1 ding wer sites e the con transcription tained in the factors SV40 pr characterized omoter and by shathe red best by th matching. e PGL3 basi These c vecto binding r. Amosites ng thar em, e also AP-1 pr , Oct esent -1, in anthe d Sp1 pSV0-CA were th Te vector transcri and ptiopr n ecisely factors in cha arr aegion cterized particularly by the besimportant t matching.for The the se CA binT digene ng sites a transcription, re also prese located nt in th ab e out pSV1100 0-CAbp T vupstr ector eam and fr pr om ecisel it [y 30i,n 31 a]. re Of gio particular n particul inter arly est impo is the rtan pr t f esence or the in CA the T gene SV40tra prn omoter scriptio of n,two loca AP-1 ted abinding bout 110sites, 0 bp 0 0 with upstre the am sequence from it [5 30-TGACT ,31]. Of AA-3 particul in ar inverted interest orientation, is the presen which ce in ar the e known SV40 pr to om play oter aof cr tw ucial o AP role -1 in bin the dincontr g siteol s, wi ofth viral the transcription sequence 5’-TGAC and rTA eplication A-3’ in i[n 44 ver ]. ted Significantly orientatio,na , wh similar ich are AP-1 known binding to pla site y a 0 0 sequence crucial rol (5 e -TGAGT in the co AA-3 ntrol )ois f v pr ira esent l train nscri thepti CA on T upstr and re eam plica region tion [of 44the ]. SipSV0-CA gnificantl Ty, vector a sim . iIt lashould r AP-1 be binunderlined ding site seq that uence the ( AP-1 5’-TGAGTA binding Asites -3’) iidentifie s presend t iin n th the e SV40 CAT upstre promoter am and regio in n pSV0-CA of the pST V0 vector -CAT 0 0 with vectothe r. It motif shoul 5d -TGA(C be und /G)T erlin A ed A-3 tha ar t eth identical e AP-1 b to inthat ding observed sites identi in the fied r egulatory in the SV4 region 0 prom ofothe ter genital and in HPVs-11, pSV0-CAT -16, vecto -18, r wi and th th -59 e m [27 oti ,33 f 5]. ’-TGA This(C/ motif G)TA pr A efer -3’ a entially re identi binds cal to the that AP-1 obserfamily ved in th subgr e regul oup ato jun ry pr reoteins gion of[ 33 the ,45 geni ]. tal HPVs-11, -16, -18, and -59 [27,33]. This motif preferentially binds the AP-1 family subgro The up pr jun esent pro study teins lacks [33,45 dir ]. ect data confirming an AP-1 involvement in the vectors’ transactivation. However The , pr this ese hypothesis nt study l is acks supported direct by data the co observation nfirming athat n A quer P-1 cetin involcan vement induce in AP-1 the binding vectors’ activity transaction vati an on enhancer . However element , this hypo of the thesi cytochr s is suppo omerte P4501A2 d by the gene obser containing vation that the quer AP-1 cetin binding can indsite uce 0 0 5 AP -TGACT -1 bindiAA-3 ng acti[v 46 ity ], o similar n an enh toan that cer el pr emen esentt on of th the e cy SV40 tochro prm omoter e P4501 and A2 gene in the con pSV0-CA taining T the vector AP-1 , as bin indicated ding site above. 5’-TGAC However TAA-3’, [further 46], sim studies, ilar to th such at pras esen mutations t on the S or V4 deletions 0 promoter of the and AP-1 in thbinding e pSV0-CA sites T vector, as indicated above. However, further studies, such as mutations or deletions of the AP-1 Antioxidants 2019, 8, 112 7 of 10 contained in the vectors, are needed to confirm the role of AP-1 in the vector transactivation and to evaluate the role of other potential transcription factors in the quercetin e ect. The present study particularly focused on AP-1 activation for two reasons: First, the AP-1 family of transcription factors is involved in the regulation of thyroid growth and thyroid genes expression [21,34–37], second, quercetin can increase AP-1 activity in several cell types [38,39,46,47], although no data were available about thyroid cells. This is the first study showing an e ect of quercetin on AP-1 activation in thyroid cells. Although still preliminary, the experiments performed show an involvement of c-jun in the protein/DNA complex induced by quercetin. This is in agreement with previous data executed in other cell types, showing the ability of quercetin to induce the binding of members of the jun subgroup to the AP-1 binding sites [46,47]. The data reported herein are of particular relevance to understand the mechanism of action of quercetin in thyroid cells. Indeed, previous studies using the FRTL-5 cells have demonstrated that quercetin has anti-thyroid e ects inhibiting the expression of the thyroid-specific genes NIS, TSHR, TPO and TG [10,11]. At least some of these e ects may be mediated by AP-1 activation, as suggested by the observation that jun activation is associated with a decrease of NIS gene expression [48]. Of course, more detailed studies are needed to know which factors of the AP-1 family are activated by quercetin in thyroid cells and how they interact with the regulatory regions of the genes involved. Indeed, the AP-1 family includes several members, such as c-fos, fosB, fra-1, fra-2, c-jun, junB, junD, which can have a di erent e ect on a single gene transcription, based on the di erent combinations of dimers that they can form and on the di erent sequences they can preferentially bind [14,21,37,49]. A final consideration that comes from the present study concerns the e ect of quercetin on the basal expression of the PGL3 and the pSV0-CAT reporter vectors in the FRTL-5 cells. This observation underlines the need to always use the empty vector as control in the transfection experiments. Indeed, it is not correct to assume that a vector reported as transcriptionally neutral would not respond to cellular treatments, as also demonstrated in previous experiments performed in other cellular models [42,43]. 5. Conclusions This study, performed in the thyroid cell line FRTL-5, shows an interference of quercetin on the activity of the plasmid reporter vectors. These data suggest caution in the use of plasmid reporter vectors when evaluating the transcriptional e ect of quercetin, due to the presence of numerous cryptic sequences for transcription factors. Moreover, the treatment with quercetin induces AP-1 activity in the FRTL-5 cells. 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The Flavonoid Quercetin Induces AP-1 Activation in FRTL-5 Thyroid Cells

Giuliani, Cesidio
Antioxidants , Volume 8 (5) – Apr 29, 2019
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Abstract

antioxidants Brief Report The Flavonoid Quercetin Induces AP-1 Activation in FRTL-5 Thyroid Cells Cesidio Giuliani Unit of Endocrinology, Department of Medicine and Sciences of Aging, and Ce.S.I.-Me.T., University of Chieti-Pescara, 66100 Chieti, Italy; cesidio.giuliani@unich.it Received: 20 March 2019; Accepted: 23 April 2019; Published: 29 April 2019 Abstract: Previous studies have shown that quercetin inhibits thyroid function both in vitro and in vivo. An attempt to evaluate the e ect of quercetin at the promoter level of the thyroid-specific genes led to the observation that this compound induces the basal activity of the reporter vector. Therefore, the action of quercetin has been evaluated on the basal activity of several reporter vectors: The PGL3 basic, promoter and control vectors from Promega, and a pSV-based chloramphenicol acetyltransferase (CAT) reporter vector. In the Fisher Rat Thyroid cell Line FRTL-5 thyroid cells transiently transfected, quercetin 10 M increased the basal activity of all the reporter vectors evaluated, although the degree of the e ect was significantly di erent among them. The analysis of the di erence among the regulatory regions of these vectors identified the activator protein 1 (AP-1) binding site as one of the potential sites involved in the quercetin e ect. Electromobility shift assay experiments showed that the treatment with quercetin induced the binding of a protein complex to an oligonucleotide containing the AP-1 consensus binding site. This is the first study showing an e ect of quercetin on AP-1 activity in thyroid cells. Further studies are in progress to understand the role of AP-1 activation in the e ects of quercetin on thyroid function. Keywords: quercetin; thyroid; FRTL-5; AP-1; gene expressions; PGL3 vectors; pSV0-CAT vector 1. Introduction 0 0 Quercetin (3,3 ,4 ,5,7-pentahydroxyflavone) is one of the most widely distributed and abundant flavonoids present in fruits and vegetables. Several studies have shown that quercetin possesses many therapeutically relevant properties, and therefore its e ects have been evaluated at cellular and molecular level [1–7]. Several reports have shown that quercetin possesses anti-thyroid and goitrogenic e ects [8–11]. Indeed, quercetin inhibits iodide organification through a thiourea-like action inhibiting thyroid peroxidase (TPO) enzyme activity and interferes with thyroid hormone metabolism, particularly through the inhibition of type I 5 -deiodinase activity [8]. Besides this, quercetin down-regulates the expression of the thyroid-specific genes sodium/iodide symporter (NIS), thyrotropin receptor (TSHR), TPO, and thyroglobulin (TG) in the FRTL-5 rat thyroid cells in continuous culture [10,11]. An attempt to evaluate the e ect of quercetin at the promoter level of the aforementioned thyroid-specific genes showed a strong induction of the basal activity of the reporter gene (PGL3 Luciferase reporter basic vector) by quercetin, which interfered with the analysis of the promoter. This observation led to inquire the action of quercetin on the expression of several Luciferase reporter vectors of the PGL3 series: the basic, the promoter, and the control vectors. Furthermore, a di erent type of vector was also evaluated, the pSV0-CAT. For this study, the FRTL-5 thyroid cells were used. These cells are a nontransformed rat thyroid cell line in continuous culture that represents a well-defined and reproducible in vitro model of the thyroid gland [12–16]. Antioxidants 2019, 8, 112; doi:10.3390/antiox8050112 www.mdpi.com/journal/antioxidants Antioxidants 2019, 8, 112 2 of 10 The treatment with quercetin of the FRTL-5 cells transiently transfected with the PGL3 basic, promoter and control vectors, and with the pSV0-CAT vector, increased the activity of all the reporter vectors evaluated, although the degree of the e ect was significantly di erent among them. The analysis of the di erence among the regulatory regions of the aforementioned vectors has identified the activator protein 1 (AP-1) binding site as one of the potential transcription factors involved in the quercetin e ect. Indeed, quercetin treatment results in AP-1 activation in FRTL-5 cells. 2. Materials and Methods 2.1. Materials Quercetin was from Sigma-Aldrich Co (St. Louis, MO, USA). Heat-treated, mycoplasma-free calf serum was from Life Technologies Europe (Monza, Italy). [ - P]-ATP was from Perkin Elmer Italia (Monza, Italy). Antibodies against c-fos (sc-413) and c-jun (sc-44) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The source of all of the other materials was Sigma-Aldrich, unless otherwise specified. 2.2. Cell Culture The F1 subclone of FRTL-5 rat thyroid cells (American Type Culture Collection, CRL-8305) was a gift from the Interthyr Research Foundation (Marietta, OH, USA). These FRTL-5 cells were grown in the six-hormone (6H) medium of Coon’s modified Ham’s F-12 supplemented with 5% calf serum, 2 mM glutamine, 1 mM nonessential amino acids, and the 6H mixture (6H5% medium): 1 mU/mL bovine TSH (Thyroid-stimulating hormone), 10 g/mL insulin, 0.4 ng/mL cortisol, 5 g/mL transferrin, 10 ng/mL glycyl-L-histidyl-L-lysine acetate, and 10 ng/mL somatostatin. These cells were diploid, between the 5th and 25th passage, and had all of the functional properties described previously [12–16]. Fresh 6H5% medium was added to the cells every 2 to 3 days, and they were passaged every 7 days. In individual experiments, the cells were transferred to a five-hormone (5H) medium (i.e., without TSH), again with 5% calf serum (5H5% medium). The treatments were performed with 10 M quercetin. In all of the experiments with quercetin, the medium was changed every 24 h, with addition of fresh medium with quercetin. Quercetin was used from a stock solution in absolute ethanol, with control cells treated with the same amount of vehicle. The final ethanol concentration was thus identical in the control and treated samples and did not exceed 0.1% (v/v). 2.3. Plasmids and Transfection The Luciferase reporter vectors PGL3 basic, PGL3 control, and PGL3 promoter were from Promega Corp. (Madison, WI, USA). The pSV0-CAT vector was obtained from the pSV2-CAT vector by removing the SV40 (Simian virus 40) early promoter sequences [17]. The reporter vectors were transiently transfected into the FRTL-5 cells using the diethylaminoethyl (DEAE)-dextran procedure [18,19]. The cells were grown to 60% confluency, maintained in 5H5% medium for 6 days, and then returned to 6H5% medium 12 h before transfection. Twenty-four hours after transfection, the cells were treated with 10 M quercetin or the control vehicle for 48 h. 2.4. Luciferase and CAT Assay Luciferase assays were performed by using the luciferase assay system (Promega) following manufacturer instructions and a LUMAT LB 9507 Luminometer (EG & G Berthold, Bundoora, Australia). All values were normalized for total cell proteins. Chloramphenicol acetyltransferase (CAT) assays were performed using the CAT ELISA kit (Roche Diagnostics GmbH, Mannheim, Germany) following manufacturer instructions. All values were normalized for total cell proteins. Antioxidants 2019, 8, 112 3 of 10 2.5. Nuclear Extracts and Electrophoretic Mobility Shift Assay FRTL-5 cells were grown to 60% confluency and maintained in 5H5% medium for 6 days to become quiescent. They were cultured again in 6H5% medium for 24 h and then treated with 10 M quercetin or the control vehicle for 48 h. Nuclear extracts were prepared as previously described [20,21]. DNA probe was made labeling the AP-1 consensus oligonucleotide (Santa Cruz Biotechnology) with [ - P]-ATP using T4 polynucleotide kinase (New England Biolabs Inc., Ipswich, MA, USA) and purified by passing through a G-50 MicroSpin Sephadex column. The electrophoretic mobility shift assays (EMSAs) were performed as described previously [14,21]. The binding reactions were performed in high salts plus detergent and included 1.5 fmol of [ P]DNA, 4 g of nuclear extracts and 1 g poly(dI-dC) in 10 mM Tris-Cl at pH 7.9, 5mM MgCl , 50 mM KCl, 1 mM DTT, 1 mM EDTA, 0.1% Triton X-100, and 12.5% glycerol in a total volume of 20 L, with incubations at room temperature for 30 min. Where indicated, antibodies were added to the binding reactions, followed by incubation with the nuclear extracts for 20 min before the addition of the labeled DNA. Following the incubations, the reaction mixtures were electrophoresed on 5% native polyacrylamide gels at 160 V in 0.5% Tris-borate-EDTA bu er at room temperature. The gels were dried and autoradiographed. 2.6. Other Assays Protein concentrations were determined using bicinchoninic acid (BCA) protein assay kits (Pierce Biotechnology Inc., Rockford, IL, USA), with crystalline bovine serum albumin (BSA) as standard. 2.7. Statistical Analysis All experiments were repeated at least three times with independent batches of cells. The data are given as means  S.D. The significance between experimental values was determined by unpaired two-tailed t-test, with p < 0.05 or better when the data from all of the experiments were considered. 3. Results 3.1. Quercetin Up-Regulates the Activity of the Following Reporter Vectors: PGL3 Basic, PGL3 Control, PGL3 Promoter, and PSV0-CAT The FRTL-5 cells were grown in 6H5% medium until 60% confluent, and then switched to 5H5% medium for 6 days (i.e., without TSH) to become quiescent. They were cultured again in 6H5% medium for 12 h and then transiently transfected with the following Luciferase reporter vectors: PGL3 basic, PGL3 control, and PGL3 promoter. After 24 h from transfection, cells were treated with 10 M quercetin for 48 h. The dose and time of the treatment were chosen based on previous studies that had shown that these treatment conditions were characterized by maximal e ects of quercetin on thyroid genes expression, without toxic e ects on the FRTL-5 cells [10,11]. As shown in Figure 1, quercetin treatment significantly increased the activity of the aforesaid reporter vectors. First of all, it has to be noted that there is a significant di erence in the basal luciferase activities of the three vectors in the control cells (Figure 1a). This is due to the presence in the PGL3 promoter vector of the SV40 promoter region upstream of the luciferase gene (between 48 and 250 bp of the vector sequence) and to the presence in the PGL3 control vector of the SV40 enhancer region (between 2205 and 2441 bp of the vector sequence) in addition to the SV40 promoter region [22]. Therefore, the e ect of quercetin was evaluated as percentage of control for each vector. This analysis revealed that quercetin treatment increased the activity of the PGL3 basic vector about 2-fold (248.69% 15.68% of control value), whereas it increased the activities of the PGL3 promoter and PGL3 control vectors of more than 6-fold (703.06 22.3% and 697.39% 36.48% of control values, respectively) (Figure 1b). Antioxidants 2019, 8, 112 4 of 10 Antioxidants 2019, 8, x FOR PEER REVIEW 4 of 10 (a) (b) Figure 1. E ects of 10 M quercetin on Luciferase activity in Fischer Rat Thyroid cell Line FRTL-5 cells Figure 1. Effects of 10 M quercetin on Luciferase activity in Fischer Rat Thyroid cell Line FRTL-5 transiently transfected with PGL3 basic, PGL3 promoter and PGL3 control. (a) Data are expressed as cells transiently transfected with PGL3 basic, PGL3 promoter and PGL3 control. (a) Data are Relative Lights Units (RLU) to protein concentrations (proteins conc.) ratio and represent the means expressed as Relative Lights Units (RLU) to protein concentrations (proteins conc.) ratio and represent S.D. of three separate experiments; (b) Same data expressed relative to the control of each vector (set to the means ± S.D. of three separate experiments; (b) Same data expressed relative to the control of each 100%). Control, cells treated with the control vehicle (0.1% ethanol); Querc, cells treated with 10 M vector (set to 100%). Control, cells treated with the control vehicle (0.1% ethanol); Querc, cells treated quercetin. * p < 0.05 versus relevant control. with 10 M quercetin. * p < 0.05 versus relevant control. These data suggested that quercetin treatment induced the activation of one or more transcription These data suggested that quercetin treatment induced the activation of one or more factors able to increase the activity of the PGL3 vectors. The attention was focused on the potential transcription factors able to increase the activity of the PGL3 vectors. The attention was focused on factors binding the SV40 promoter region, since its presence was responsible for a stronger e ect of the potential factors binding the SV40 promoter region, since its presence was responsible for a quercetin on luciferase activity (compare PGL3 promoter with PGL3 basic in Figure 1b). Nevertheless, stronger effect of quercetin on luciferase activity (compare PGL3 promoter with PGL3 basic in Figure no further significant increase was brought by the presence of the SV40 enhancer region in the PGL3 1b). Nevertheless, no further significant increase was brought by the presence of the SV40 enhancer control vector compared to the PGL3 promoter vector (Figure 1b). Therefore, the putative transcription region in the PGL3 control vector compared to the PGL3 promoter vector (Figure 1b). Therefore, the factors binding the sequence of the SV40 promoter in the PGL3 promoter vector were analyzed. putative transcription factors binding the sequence of the SV40 promoter in the PGL3 promoter This sequence, spanning from 48 to 250 bp of the vector sequence [23], was analyzed with the web tools vector were analyzed. This sequence, spanning from 48 to 250 bp of the vector sequence [23], was LASAGNA-search 2.0 and AliBaba2.1 [24–26]. The analysis revealed several potential transcription analyzed with the web tools LASAGNA-search 2.0 and AliBaba2.1 [24–26]. The analysis revealed factors binding sites. Among them, the transcription factors characterized by the best matching were several potential transcription factors binding sites. Among them, the transcription factors AP-1, Oct-1 and Sp1, with AP-1 having the highest score, Table 1. Indeed, the SV40 promoter of the characterized by the best matching were AP-1, Oct-1 and Sp1, with AP-1 having the highest score, PGL vectors contains two characterized AP-1 binding sites [26], one with reverse orientation from 58 Table 1. Indeed, the SV40 promoter of the PGL vectors contains two characterized AP-1 binding sites to 64 bp and the other with forward orientation from 144 to 150 bp. [26], one with reverse orientation from 58 to 64 bp and the other with forward orientation from 144 to 150 bp. Table 1. Transcription factors with the best binding match with the SV40 promoter sequence. Table 1. Transcription fa Tc ranscription tors with the Factor best bindinScore g match witp h V th alue e SV40 promoter sequence. AP-1 13.33 0 Transcription Factor Score p Value Oct-1 13.00 0 AP-1 13.33 0 Sp1 12.32 0.00025 Oct-1 13.00 0 Data obtained with LASAGNA-search 2.0 [24]. Sp1 12.32 0.00025 Data obtained with LASAGNA-search 2.0 [24]. 0 0 The two AP-1 binding sites have an identical sequence: 5 -TGACTAA-3 , although in inverted The two AP-1 binding sites have an identical sequence: 5’-TGACTAA-3’, although in inverted orientation. This sequence has been first described in the human papilloma virus (HPV)-18 regulatory orientation. This sequence has been first described in the human papilloma virus (HPV)-18 regulatory region, where there are also two binding sites in inverted orientation [27,28]. region, where there are also two binding sites in inverted orientation [27,28]. Potential binding sites for the transcription factors AP-1, Oct-1, and Sp1 were also identified in Potential binding sites for the transcription factors AP-1, Oct-1, and Sp1 were also identified in the backbone of the PGL3 basic vector. These data could explain the e ect of quercetin in enhancing the backbone of the PGL3 basic vector. These data could explain the effect of quercetin in enhancing the luciferase activity even in the PGL3 basic vector. the luciferase activity even in the PGL3 basic vector. Quercetin action was also evaluated in a di erent reporter vector, the pSV0-CAT, a pSV2-derived Quercetin action was also evaluated in a different reporter vector, the pSV0-CAT, a pSV2- vector that does not contain promoter or enhancer elements and is characterized by a very low derived vector that does not contain promoter or enhancer elements and is characterized by a very background activity [17,29]. FRTL-5 cells were transfected with the pSV0-CAT vector, as described low background activity [17,29]. FRTL-5 cells were transfected with the pSV0-CAT vector, as above for the PGL3 vectors, and treated with quercetin 10 M for 48 h. As shown in Figure 2, quercetin described above for the PGL3 vectors, and treated with quercetin 10 μM for 48 h. As shown in Figure treatment increased 10-fold the CAT expression compared to control activity, 1135.5%  24.5% of 2, quercetin treatment increased 10-fold the CAT expression compared to control activity, 1135.5% ± control. Surprisingly, this e ect was even higher than that observed with the PGL3 control and 24.5% of control. Surprisingly, this effect was even higher than that observed with the PGL3 control promoter vectors. and promoter vectors. Antioxidants 2019, 8, 112 5 of 10 Antioxidants 2019, 8, x FOR PEER REVIEW 5 of 10 (a) (b) Figure 2. E ects of 10 M quercetin on CAT (Chloramphenicol acetyltransferase) expression in FRTL-5 Figure 2. Effects of 10 M quercetin on CAT (Chloramphenicol acetyltransferase) expression in FRTL- cells transiently transfected with pSV0-CAT vector. (a) CAT protein concentrations are normalized 5 cells transiently transfected with pSV0-CAT vector. (a) CAT protein concentrations are normalized with respect to total cell protein concentrations and represent the means  S.D. of three separate with respect to total cell protein concentrations and represent the means ± S.D. of three separate experiments; (b) Same data expressed relative to the control vector (set to 100%). Control, cells treated experiments; (b) Same data expressed relative to the control vector (set to 100%). Control, cells treated with the control vehicle (0.1% ethanol); Querc, cells treated with 10 M quercetin. * p < 0.05 versus with the control vehicle (0.1% ethanol); Querc, cells treated with 10 M quercetin. * p < 0.05 versus relevant control. relevant control. A possible explanation of the quercetin e ect on pSV0-CAT activity is the presence of cryptic A possible explanation of the quercetin effect on pSV0-CAT activity is the presence of cryptic promoters in the pBR322 backbone of the pSV0 vector [30,31]. Indeed, the analysis of the pSV0-CAT promoters in the pBR322 backbone of the pSV0 vector [30,31]. Indeed, the analysis of the pSV0-CAT vector revealed the presence of the potential binding sites for the aforementioned transcription factors vector revealed the presence of the potential binding sites for the aforementioned transcription AP-1, Oct-1, and Sp1. Of particular interest is the presence of these binding sites in a region of about factors AP-1, Oct-1, and Sp1. Of particular interest is the presence of these binding sites in a region of 1100 bp upstream of the CAT gene, since this region is important for the regulation of the CAT gene about 1100 bp upstream of the CAT gene, since this region is important for the regulation of the CAT transcription [30,31]. Notably, a non-canonical AP-1 binding site [32,33] was present from 1025 to 1031 gene transcription [30,31]. Notably, a non-canonical AP-1 binding site [32,33] was present from 1025 0 0 bp, whose sequence 5 -TGAGTAA-3 is similar to that contained in the SV40 promoter described above to 1031 bp, whose sequence 5’-TGAGTAA-3’ is similar to that contained in the SV40 promoter 0 0 (5 -TGACTAA-3 ). described above (5’-TGACTAA-3’). 3.2. Quercetin Induces AP-1 Binding to DNA 3.2. Quercetin Induces AP-1 Binding to DNA The transcription factors members of the AP-1 family are involved in the regulation of thyroid The transcription factors members of the AP-1 family are involved in the regulation of thyroid growth and thyroid genes expression [34–37]. Further, quercetin has an opposite role in AP-1 activation, growth and thyroid genes expression [34–37]. Further, quercetin has an opposite role in AP-1 since it increases AP-1 activity in some in vitro models [38,39], whereas it decreases it in other activation, since it increases AP-1 activity in some in vitro models [38,39], whereas it decreases it in models [40,41]. For these reasons, EMSA experiments were performed to evaluate the e ect of other models [40,41]. For these reasons, EMSA experiments were performed to evaluate the effect of quercetin on AP-1 activity in the FRTL-5 cells. Nuclear extracts from cells treated with 10 M quercetin on AP-1 activity in the FRTL-5 cells. Nuclear extracts from cells treated with 10 M quercetin for 48 h were incubated with a radiolabeled AP-1 consensus oligonucleotide. As shown in quercetin for 48 h were incubated with a radiolabeled AP-1 consensus oligonucleotide. As shown in Figure 3 (lane 3 versus lane 2), quercetin treatment induced the formation of a protein/DNA complex. Figure 3 (lane 3 versus lane 2), quercetin treatment induced the formation of a protein/DNA complex. To identify the transcription factors involved in the protein complex, nuclear extracts were preincubated To identify the transcription factors involved in the protein complex, nuclear extracts were with antisera to c-fos and c-jun. Preincubation of the extracts with c-jun antiserum decreased the preincubated with antisera to c-fos and c-jun. Preincubation of the extracts with c-jun antiserum protein/DNA complex to 52 4% of control (Figure 3, lane 5 versus lane 4), whereas a slight but not decreased the protein/DNA complex to 52 ± 4% of control (Figure 3, lane 5 versus lane 4), whereas a significant e ect was seen with the c-fos antiserum (90 8,5% of control), (Figure 3, lane 6 versus lane slight but not significant effect was seen with the c-fos antiserum (90 ± 8,5% of control), (Figure 3, 4). Although it was not possible to see any super shift, the decrease of the complex by the antibodies lane 6 versus lane 4). Although it was not possible to see any super shift, the decrease of the complex against c-jun suggests that this protein may either bind directly to the cis-acting element or interact by the antibodies against c-jun suggests that this protein may either bind directly to the cis-acting with other transcription factors involved in the protein/DNA complex formation. element or interact with other transcription factors involved in the protein/DNA complex formation. Antioxidants 2019, 8, 112 6 of 10 Antioxidants 2019, 8, x FOR PEER REVIEW 6 of 10 Figure 3. E ects of quercetin on the formation of protein/DNA complexes between FRTL-5 cell nuclear Figure 3. Effects of quercetin on the formation of protein/DNA complexes between FRTL-5 cell extracts and an AP-1 (Activator protein-1) consensus oligonucleotide. Radiolabeled oligonucleotide nuclear extracts and an AP-1 (Activator protein-1) consensus oligonucleotide. Radiolabeled was incubated with nuclear extracts from cells cultured in 6H5% medium (control), cells treated oligonucleotide was incubated with nuclear extracts from cells cultured in 6H5% medium (control), with 10 M quercetin (Querc) for 48 h; cells treated with quercetin plus normal rabbit polyclonal cells treated with 10 M quercetin (Querc) for 48 h; cells treated with quercetin plus normal rabbit antiserum (Querc-Cont) or antibodies against c-jun (+anti-c-jun) or c-fos (+anti-c-fos). Lane 1 contains polyclonal antiserum (Querc-Cont) or antibodies against c-jun (+anti-c-jun) or c-fos (+anti-c-fos). Lane the probe alone. 1 contains the probe alone. 4. Discussion 4. Discussion The present study is the consequence of an attempt to investigate the e ect of quercetin on the expression The prof esethe nt st thyr udy oid-specific is the conseq genes uence at otranscriptional f an attempt to level. investiga Indeed, te the the effec analysis t of querof ceti the n oe n th ect e of expr quer essi cetin on of on the the thyro NIS id pr -spec omoter ific genes gene inserted at transcri in pti the onPGL3 al level basic . Indee vector d, the was anaspoiled lysis of th by e a efstr fecong t of incr quer ease cetin of on the the basal NIS pr activity omoterof gene theiempty nsertedvector in the P induced GL3 basi by c vthe ectoquer r was cetin spoil tr ed eatment. by a stroThese ng incrdata ease wer of th e e surprising, basal activ as ity the of PGL3 the empty basic vector vector is inbelieved duced by toth be e transcriptionally quercetin treatmen neutral, t. These since datit a lacks were any surpr viral ising pr , as omoter the PG orLenhancer 3 basic vec region. tor is bHowever elieved to , even be trapr nscri omoterless ptionally vectors, neutralsuch , since as it the lacks PGL3 any basic, viral can prom be otransactivated ter or enhancer by reseveral gion. Ho stimuli weverin , etransiently ven promot transfected erless vecto cells, rs, suc due h ato s tthe he PG presence L3 basi of c, cryptic can be r tra egulatory nsactivasequences ted by sev contained eral stimin ulithe in vector transientl backbone y tran[sfe 42,cte 43]. d Notably cells, d,ue quer toceti the n had prese an str ceonger of cre ypti ect c on regul the ato transactivati ry sequence on s of con the taiPGL3 ned in pr th omoter e vecto vector r back . b This one e[ 4ect 2,43 could ]. Nota be bl explained y, querceti by n the hadp a r esence stronger in this effec vector t on th of e the tran SV40 sactiva promoter tion of r th egion e PGupstr L3 pr eam omo of ter the veluciferase ctor. Thisgene. effect These coulddata be ex led plto ain investigate ed by the the prese potential nce in th transcription is vector of th factors e SV40binding promoter sites regi contained on upstrea in mthe of th SV40 e luci pr fomoter erase gene and . These shared da by ta the led PGL3 to investi basic gavector te the .po Among tential them, transcri AP-1, ption Oct-1, factors and bin Sp1 ding wer sites e the con transcription tained in the factors SV40 pr characterized omoter and by shathe red best by th matching. e PGL3 basi These c vecto binding r. Amosites ng thar em, e also AP-1 pr , Oct esent -1, in anthe d Sp1 pSV0-CA were th Te vector transcri and ptiopr n ecisely factors in cha arr aegion cterized particularly by the besimportant t matching.for The the se CA binT digene ng sites a transcription, re also prese located nt in th ab e out pSV1100 0-CAbp T vupstr ector eam and fr pr om ecisel it [y 30i,n 31 a]. re Of gio particular n particul inter arly est impo is the rtan pr t f esence or the in CA the T gene SV40tra prn omoter scriptio of n,two loca AP-1 ted abinding bout 110sites, 0 bp 0 0 with upstre the am sequence from it [5 30-TGACT ,31]. Of AA-3 particul in ar inverted interest orientation, is the presen which ce in ar the e known SV40 pr to om play oter aof cr tw ucial o AP role -1 in bin the dincontr g siteol s, wi ofth viral the transcription sequence 5’-TGAC and rTA eplication A-3’ in i[n 44 ver ]. ted Significantly orientatio,na , wh similar ich are AP-1 known binding to pla site y a 0 0 sequence crucial rol (5 e -TGAGT in the co AA-3 ntrol )ois f v pr ira esent l train nscri thepti CA on T upstr and re eam plica region tion [of 44the ]. SipSV0-CA gnificantl Ty, vector a sim . iIt lashould r AP-1 be binunderlined ding site seq that uence the ( AP-1 5’-TGAGTA binding Asites -3’) iidentifie s presend t iin n th the e SV40 CAT upstre promoter am and regio in n pSV0-CA of the pST V0 vector -CAT 0 0 with vectothe r. It motif shoul 5d -TGA(C be und /G)T erlin A ed A-3 tha ar t eth identical e AP-1 b to inthat ding observed sites identi in the fied r egulatory in the SV4 region 0 prom ofothe ter genital and in HPVs-11, pSV0-CAT -16, vecto -18, r wi and th th -59 e m [27 oti ,33 f 5]. ’-TGA This(C/ motif G)TA pr A efer -3’ a entially re identi binds cal to the that AP-1 obserfamily ved in th subgr e regul oup ato jun ry pr reoteins gion of[ 33 the ,45 geni ]. tal HPVs-11, -16, -18, and -59 [27,33]. This motif preferentially binds the AP-1 family subgro The up pr jun esent pro study teins lacks [33,45 dir ]. ect data confirming an AP-1 involvement in the vectors’ transactivation. However The , pr this ese hypothesis nt study l is acks supported direct by data the co observation nfirming athat n A quer P-1 cetin involcan vement induce in AP-1 the binding vectors’ activity transaction vati an on enhancer . However element , this hypo of the thesi cytochr s is suppo omerte P4501A2 d by the gene obser containing vation that the quer AP-1 cetin binding can indsite uce 0 0 5 AP -TGACT -1 bindiAA-3 ng acti[v 46 ity ], o similar n an enh toan that cer el pr emen esentt on of th the e cy SV40 tochro prm omoter e P4501 and A2 gene in the con pSV0-CA taining T the vector AP-1 , as bin indicated ding site above. 5’-TGAC However TAA-3’, [further 46], sim studies, ilar to th such at pras esen mutations t on the S or V4 deletions 0 promoter of the and AP-1 in thbinding e pSV0-CA sites T vector, as indicated above. However, further studies, such as mutations or deletions of the AP-1 Antioxidants 2019, 8, 112 7 of 10 contained in the vectors, are needed to confirm the role of AP-1 in the vector transactivation and to evaluate the role of other potential transcription factors in the quercetin e ect. The present study particularly focused on AP-1 activation for two reasons: First, the AP-1 family of transcription factors is involved in the regulation of thyroid growth and thyroid genes expression [21,34–37], second, quercetin can increase AP-1 activity in several cell types [38,39,46,47], although no data were available about thyroid cells. This is the first study showing an e ect of quercetin on AP-1 activation in thyroid cells. Although still preliminary, the experiments performed show an involvement of c-jun in the protein/DNA complex induced by quercetin. This is in agreement with previous data executed in other cell types, showing the ability of quercetin to induce the binding of members of the jun subgroup to the AP-1 binding sites [46,47]. The data reported herein are of particular relevance to understand the mechanism of action of quercetin in thyroid cells. Indeed, previous studies using the FRTL-5 cells have demonstrated that quercetin has anti-thyroid e ects inhibiting the expression of the thyroid-specific genes NIS, TSHR, TPO and TG [10,11]. At least some of these e ects may be mediated by AP-1 activation, as suggested by the observation that jun activation is associated with a decrease of NIS gene expression [48]. Of course, more detailed studies are needed to know which factors of the AP-1 family are activated by quercetin in thyroid cells and how they interact with the regulatory regions of the genes involved. Indeed, the AP-1 family includes several members, such as c-fos, fosB, fra-1, fra-2, c-jun, junB, junD, which can have a di erent e ect on a single gene transcription, based on the di erent combinations of dimers that they can form and on the di erent sequences they can preferentially bind [14,21,37,49]. A final consideration that comes from the present study concerns the e ect of quercetin on the basal expression of the PGL3 and the pSV0-CAT reporter vectors in the FRTL-5 cells. This observation underlines the need to always use the empty vector as control in the transfection experiments. Indeed, it is not correct to assume that a vector reported as transcriptionally neutral would not respond to cellular treatments, as also demonstrated in previous experiments performed in other cellular models [42,43]. 5. Conclusions This study, performed in the thyroid cell line FRTL-5, shows an interference of quercetin on the activity of the plasmid reporter vectors. These data suggest caution in the use of plasmid reporter vectors when evaluating the transcriptional e ect of quercetin, due to the presence of numerous cryptic sequences for transcription factors. Moreover, the treatment with quercetin induces AP-1 activity in the FRTL-5 cells. 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Journal

Antioxidants – Multidisciplinary Digital Publishing Institute

Published: Apr 29, 2019

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