author's personal copynature.cc.hirosaki-u.ac.jp/.../gazou2/ebina.2011.pdfauthor's...

This article appeared in a journal published by Elsevier. The attachedcopy is furnished to the author for internal non-commercial researchand education use, including for instruction at the authors institution

and sharing with colleagues.

Other uses, including reproduction and distribution, or selling orlicensing copies, or posting to personal, institutional or third party

websites are prohibited.

In most cases authors are permitted to post their version of thearticle (e.g. in Word or Tex form) to their personal website orinstitutional repository. Authors requiring further information

regarding Elsevier’s archiving and manuscript policies areencouraged to visit:

http://www.elsevier.com/copyright

http://www.elsevier.com/copyright

Author's personal copy

Correlation of dysfunction of nonmuscle myosin IIA with increased induction ofCyp1a1 in Hepa-1 cells

Masayuki Ebina a,b, Masahiko Shibazaki c, Kyoko Kudo a,b, Shuya Kasai a,b, Hideaki Kikuchi a,⁎a Science of Bioresources, United Graduate School of Agricultural Science, Iwate University, Morioka 020-8551, Japanb Department of Biochemistry and Biotechnology, Faculty of Agriculture and Life Science, Hirosaki University, Hirosaki 036-8561, Japanc Department of Molecular Genetics, Institute of Development, Aging and Cancer, Tohoku University, Sendai 980-8575, Japan

a b s t r a c ta r t i c l e i n f o

Article history:Received 1 August 2010Received in revised form 20 December 2010Accepted 3 January 2011Available online 7 January 2011

Keyword:TCDDAhRTranscriptionNMIIA

The aryl hydrocarbon receptor (AhR) is one of the best known ligand-activated transcription factors and itinduces Cyp1a1 transcription by binding with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Recent focus hasbeen on the relationship of AhR with signaling pathways that modulate cell shape and migration. Innonmuscle cells, nonmuscle myosin II is one of the key determinants of cell morphology, but it has not beeninvestigated whether its function is related to Cyp1a1 induction. In this study, we observed that (−)-blebbistatin, which is a specific inhibitor of nonmuscle myosin II, increased the level of CYP1A1-mRNA inHepa-1 cells. Comparison of (−)-blebbistatin with (+)-blebbistatin, which is an inactive enantiomer,indicated that the increase of CYP1A1-mRNA was due to nonmuscle myosin II inhibition. Subsequentknockdown experiments observed that reduction of nonmuscle myosin IIA, which is only an isoform ofnonmuscle myosin II expressed in Hepa-1 cells, was related to the enhancement of TCDD-dependent Cyp1a1induction. Moreover, chromatin immunoprecipitation assay indicated that the increase of Cyp1a1 inductionwas the result of transcriptional activation due to increased binding of AhR and RNA polymerase II to theenhancer and proximal promoter regions of Cyp1a1, respectively. These findings provide a new insight intothe correlation between the function of nonmuscle myosin II and gene induction.

© 2011 Elsevier B.V. All rights reserved.

1. Introduction

Halogenated or polycyclic aromatic hydrocarbons, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and benzo[a]pyrene (B[a]P), arewidespread environmental pollutants. The toxic effects of thesecompounds in experimental animals include carcinogenicity, immu-notoxicity and the development of malformations such as cleft palate[1–3].

The aryl hydrocarbon receptor (AhR) belongs to the basic helix–loop–helix/Per-Arnt-Sim (bHLH/PAS) superfamily, and functions as atranscription factor after bindingwith ligands such as TCDD and B[a]P.Unliganded AhR forms a cytosolic complex with heat shock protein 90(Hsp90), hepatitis B virus X-associated protein 2 (XAP2) [4], and a co-chaperone protein called p23 [5]. Upon ligand binding, AhR undergoesa conformational change, dissociates from the cytosolic complex, and

then translocates to the nucleus. Inside the nucleus, AhR dimerizeswith AhR nuclear translocator (ARNT), and the AhR/ARNT hetero-dimer recognizes its target sequence, the xenobiotic response element(XRE), in target genes such as Cyp1a1, Cyp1a2, and Nqo1, and thenrecruits several transcriptional cofactors for induction of their targetgenes [6–8].

AhR null mice display non-inducible carcinogenicity, a small liver,and eye and kidney anomalies [9,10]. Therefore, it is thought that, inaddition to being the central component in TCDD toxicity, AhR playsan important role in development. In relation to the developmentalfunction of AhR, a ligand-independent signaling pathway and anendogenous ligand for AhR activation have been proposed [11–13].

Recently, several reports have indicated the involvement of AhR incell physiology [14]. Diry et al. have reported that TCDD-activated AhRmodulates the morphology and plasticity of MCF-7 cells through amechanism that depends on c-Jun N-terminal kinase (JNK) [15], andmore recently, Bui et al. have reported that the upregulation of Hef-1by TCDD-activated AhR is involved in cell migration and plasticity[16]. Furthermore, Carvajal-Gonzalez et al. have reported that AhRregulates cell shape and adhesion by modulating Vav3-dependentsignaling [17].

Myosin II is a bipolar, hexameric protein complex that is composedof two heavy chains, two essential light chains, and two regulatorylight chains. Hydrolysis of ATP by the myosin II motor domain alters

Biochimica et Biophysica Acta 1809 (2011) 176–183

Abbreviations: TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; AhR, aryl hydrocarbonreceptor; XRE, xenobiotic responsive element; NMIIA, nonmuscle myosin heavy chainIIA or MYH9; qPCR, Quantitative real-time PCR; GAPDH, glyceraldehyde-3-phosphatedehydrogenase; LDH, lactase dehydrogenase; RNAPII, RNA polymerase II; ChIP,chromatin immunoprecipitation⁎ Corresponding author. Department of Biochemistry and Biotechnology, Faculty of

Agriculture and Life Science, Hirosaki University, 3 Bunkyo-cho, Hirosaki, Aomori,Japan. Tel.: +81 172 39 3586; fax: +81 172 39 3587.

E-mail address: [email protected] (H. Kikuchi).

1874-9399/$ – see front matter © 2011 Elsevier B.V. All rights reserved.doi:10.1016/j.bbagrm.2011.01.002

Contents lists available at ScienceDirect

Biochimica et Biophysica Acta

j ourna l homepage: www.e lsev ie r.com/ locate /bbagrm


the position of the domain relative to bound actin filaments (F-actin),which generates a contractile force that is thought to be important forcell motility, adhesion, and shape [18–21]. In vertebrates, the myosinII family consists of at least 15 different isoforms, and includesskeletal, cardiac and smooth muscle, and nonmuscle myosin heavychains. Moreover, nonmuscle myosin II comprises three isoforms:nonmuscle myosin IIA, IIB, and IIC (NMIIA, IIB, and IIC). The aminoacid sequence of these three isoforms is well conserved throughoutthe entire protein and they share certain cellular functions, butdifferences in expression pattern in embryonic and adult tissues[22,23], intracellular localization [24], and function [25] suggest thateach isoform has a distinct role. The discovery of (−)-blebbistatin,which is a specific inhibitor of nonmuscle myosin II, has allowed thefunction of nonmusclemyosin II to be investigated in more detail [26].Zhang and Rao have indicated that nonmusclemyosin II plays a role inthe maintenance of cell morphology [27]. In another study, Ivanov etal. have shown by siRNA-mediated downregulation that NMIIA has aunique role in maintaining cell morphology and intercellular junc-tions [28]. The functions of nonmuscle myosin II have been elucidatedgradually over time, but their correlation with gene regulationremains unclear.

In this study, we used (−)-blebbistatin and siRNA-mediateddownregulation to examine the role of nonmuscle myosin II in theCyp1a1 induction in Hepa-1 cells.

2. Materials and methods

2.1. Cell culture

The NIH 3T3 cell line was kindly provided by Prof. Ken Itoh(Hirosaki University, Japan). Hepa-1c1c7 (Hepa-1) and NIH 3T3 cellswere maintained in Dulbecco's Modified Eagle's Medium (Nissui,Tokyo, Japan) that contained 5% fetal bovine serum (BiosourceInternational, Inc., Camarillo, CA, USA), 100,000 U/l penicillin,100 mg/l streptomycin, and 3.7 g/l NaHCO3 in humidified 95% airand 5% CO2 at 37 °C.

2.2. Chemicals

TCDD was obtained from Cambridge Isotope Laboratories (And-over, MA, USA). (−)- and (+)-blebbistatin (≥98% purity) was fromCayman Chemical (Ann Arbor, MI, USA). These were diluted in DMSO.The toxicities of these chemicals were tested by examining their effecton the growth rate of Hepa-1 cells using 0.3% trypan blue in calcium–

magnesium free-PBS, and the chemicals were used within the non-toxic dose range (in terms of cell viability).

2.3. RNA extraction, cDNA synthesis, RT-PCR and quantitative real-timePCR (qPCR)

RNA extraction was performed as described previously [29]. cDNAwas synthesized by using M-MuLV reverse transcriptase (Fermentas,Hanover,MD, USA). To confirm the expression ofmRNA formembers ofthe myosin II family, PCR reactions were performed on cDNA derivedfrom Hepa-1 and NIH 3T3 cells and mouse lung tissue, using Phusion™RNApolymerase (Finnzymes,Keilaranta, Espoo, Finland) and theprimersets published previously [22]. The following primers for smoothmusclemyosinwere designedwith Primer Blast (http://www.ncbi.nlm.nih.gov/tools/primer-blast/): 5′-GGAAACGAGGCTTCATTTGTTCC-3′ and5′-ACCCTTTGTGCAGGGCTGTGG-3′. PCR amplification was as follows:an initial step at 98 °C for 30 s, then 35 cycles at 98 °C for 10 s, 60 °C for20 s, and 72 °C for 15 s, with a final step for 7 min at 72 °C. The resultingPCR products were separated by electrophoresis on 2% agarose gels thatincluded ethidium bromide. qPCR was performed in a reaction mixturethat contained Thunderbird™ SYBR qPCR Mix (Toyobo, Osaka, Japan),and 6 μM for each primer set. Amplification was performed in a

LightCycler (Roche Diagnostics GmbH, Mannheim, Germany), wherethe reaction mixture was heated to 95 °C for 30 s, and then underwent40 cycles of denaturing at 95 °C for 5 s, annealing at 55 °C for 10 s, andelongation at 72 °C for 15 s. Primer sets for qPCR were as describedpreviously: Cyp1a1 and Nqo1 [30], Cyp1a2 and Gapdh [31]. Gapdh wasalso amplified in each sample as an internal control for all the qPCRreactions.

2.4. Preparation of total cell extract, cytosolic fraction and nuclearfraction

Cells were suspended in a cell lysis buffer [15 mM HEPES pH 7.9,0.35 M KCl, 1 mM MgCl2, 0.15 mM EDTA, 10% glycerol, 0.35% NP-40,3 mM 2-mercaptoethanol, and 1× Complete™ Protease InhibitorCocktail (Roche Diagnostics GmbH, Mannheim, Germany)], kept onice for 10 min, and centrifuged at 20,000×g for 10 min at 4 °C. Thesupernatant was used as the total cell extract. Cytosolic fraction andnuclear fraction were prepared by our previously described method[32]. The protein concentrations of the total cell extract, cytosolicfraction and nuclear fraction were measured by the Bio-Rad proteinassay (Bio-Rad Laboratories, Inc., Hercules, CA, USA), and prepared forwestern blotting in a 5× SDS sample buffer (312.5 mM Tris–HCl pH6.8, 10% SDS, 25% 2-mercaptoethanol, 0.05% bromophenol blue). Toimmunoprecipitate NMIIA and actin interacting with NMIIA, 1 μg ofNMIIA antibody and 5 μl of Dynabeads Protein G (Invitrogen, Carlsbad,CA, USA) were added to the total cell extract (1.5 mg) that had beenprepared with a cell-lysis buffer of high ionic strength (0.5 M KCl and0.5% NP-40). The mixture was incubated for 2 h at 4 °C with gentleagitation. The bound complexes were washed three times with theabove-mentioned cell-lysis buffer, and then resuspended in a 1× SDSsample buffer.

2.5. SDS-PAGE and western blotting

The protein samples were resolved by 6 or 9% SDS-PAGE, andproteins were transferred to PVDF membranes (GE Healthcare, LittleChalfont, Bucks, UK). After Ponceau S staining to confirm an eventransfer, themembranes were blockedwith a blocking buffer [0.1% (v/v) Tween20-TBS (T-TBS) that contained 5% skimmed milk]. Themembranes were incubated with primary antibody, washed twice inT-TBS, and then incubated with secondary antibody followed by twowashes in T-TBS. Finally, immunoreactive bandswere visualized usingan enhanced chemiluminescence kit (GE Healthcare) and an X-rayfilm (Fujifilm Co., Tokyo, Japan). The following primary antibodieswere used: anti-AhR (M20), anti-GAPDH (FL-335) and anti-Lamin B(M-20; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA); anti-LDH(Chemicon Inc., USA); a Myosin II Isoform Antibody Sampler Kit (CellSignaling Technology, Inc., Danvers, MA, USA); anti-smooth musclemyosin heavy chain (G4; Santa Cruz Biotechnology); anti-actin (ACTN05; Thermo Scientific, San Jose, CA, USA); anti-vinculin (h-VIN1;Sigma–Aldrich Japan, Tokyo, Japan); and anti-RNAPII (05-623;Upstate Biotechnology, Charlottesville, VA, USA). Secondary antibo-dies were: horseradish-peroxidase-conjugated goat anti-rabbit, rabbitanti-mouse, and rabbit anti-goat IgG (DAKO, Cambridge, UK).

2.6. Preparation and transfection of siRNA

For the RNA interference (RNAi) experiments, siRNA duplexes weresynthesized by using a Silencer siRNA Construction Kit (Ambion Inc.,Austin, TX, USA) in accordance with the manufacturer's protocol.Oligonucleotides for mouse control siRNA (siCont) and siRNA againstNMIIAwere as described previously byVascotto et al. [33].We chose si1and si2 from that study, and named these as siNMIIA-1 and siNMIIA-2.Hepa-1 cells (3×105)were plated in culture dishes (6 cmdiameter) andtransfected by using Lipofectamine™ RNAiMAX (Invitrogen Corpora-tion, Carlsbad, CA, USA), to give a final concentration of 5 nM. After

177M. Ebina et al. / Biochimica et Biophysica Acta 1809 (2011) 176–183


transfection, the cells were incubated for 2–4 days, and harvested forwestern blotting or other experiments.

2.7. Immunofluorescence labeling and image analysis

Phase-contrast images were obtained with an Olympus IMT-2fluorescencemicroscope (Olympus Corporation, Tokyo, Japan). Chem-ical-treated or siRNA-transfected cells on coverslips coated with type Icollagen (Iwaki Co., Tokyo, Japan) were fixed with 4% paraformalde-hyde and permeabilized with 0.1% Triton X-100. To monitor focaladhesion and F-actin, the cells were first immunostained with anti-vinculin and Alexa 488-conjugated secondary antibody (MolecularProbes) and then labeled with rhodamine–phalloidin (CytoskeletonInc., Denver, CO, USA). Nuclei were stained with Hoechst 33258(Sigma). Fluorescence microscopy images were obtained using a laserscanning confocal microscope (FV-1000; Olympus Co., Tokyo, Japan).

2.8. Chromatin immunoprecipitation (ChIP) assay and qPCR analysis

The ChIP assay was performed as described by Schnekenburger etal. [30], with some modifications. After chemical treatment or siRNAtransfection, Hepa-1 cells were incubated for 10 min at roomtemperature with 1% formaldehyde, and then incubated for a further10 min with 0.125 M glycine. After harvesting by centrifugation, cellswere resuspended in a cell burst buffer (5 mM PIPES pH 8.0, 85 mMKCl, 0.5% NP-40, and 1× Protease Inhibitor Cocktail), and incubated onice for 10 min. The nuclei were pelleted by centrifugation, resus-pended in a nuclei lysis buffer (50 mM Tris–HCl pH 8.0, 10 mM EDTA,1% SDS, 0.1% Tween-20, and 1× Protease Inhibitor Cocktail), andincubated on ice for 10 min. Chromatin was sheared to a size range of0.3–0.6 kb by sonication. After centrifugation to remove cell debris, the

chromatin was diluted 1:10 in a ChIP dilution buffer (10 mM Tris–HClpH 8.0, 150 mM NaCl, 1% Triton X-100, 0.1% Tween-20, and 1×Protease Inhibitor Cocktail), and aliquots were removed as total input.The diluted chromatin was incubated overnight on a rotator at 4 °C,with 1 μg of antibodies specific for the protein of interest, or 1 μg ofnormal goat or rabbit IgG (Santa Cruz Biotechnology) and DynabeadsProtein G (Invitrogen). Procedures for ChIP assay after antibodyreactionswere as described by Shibazaki et al. [34]. DNAwas dissolvedin TE buffer (10 mM Tris–HCl pH 7.5, 1 mM EDTA) and subjected toqPCR with ChIP primer sets for the Cyp1a1 enhancer and proximalpromoter regions, which corresponded to the−0.8 kbp and−0.1 kbpprimer sets, respectively, that were described previously [30].

2.9. Statistics

Numerical values from individual experiments were pooled andexpressed throughout as the mean±standard deviation of the mean.The numbers obtained were compared by two-tailed Student's t test,with pb0.05 considered to be statistically significant.

3. Results

3.1. Effect of (−)-blebbistatin on Cyp1a1 induction in Hepa-1 cells

We used (−)-blebbistatin to examine whether inhibition ofnonmuscle myosin II functions affects the Cyp1a1 induction inHepa-1 cells. Hepa-1 cells have been used widely by both us andothers to examine in detail the mechanism of Cyp1a1 transcription byTCDD [7,8,30,34]. (−)-Blebbistatin increased Cyp1a1 induction inboth the presence and absence of TCDD (Fig. 1A). In the presence of40 μM (−)-blebbistatin, (−)-blebbistatin itself increased the basal

Fig. 1. Effects of nonmuscle myosin II inhibitor on CYP1A1 mRNA induction. (A and B) Hepa-1 cells were pretreated for 24 h with (A) DMSO (0) or various concentrations of(−)-blebbistatin [(−)-Bleb], and (B) DMSO (DM) or 40 μM (−)-blebbistatin [(−)-Bleb] or (+)-blebbistatin [(+)-Bleb], and then incubated for a further 9 h with DMSO or4 nM TCDD. After harvesting of the cells, total RNA was isolated and reverse transcribed to cDNA. Levels of CYP1A1 and GAPDH mRNA were determined by qPCR using specificprimer sets. The plotted values from three independent experiments (means±SD) represent the relative Cyp1a1 induction and correspond to the ratio of expression ofCYP1A1/GAPDH mRNA given relative to the mean value obtained after TCDD treatment in blebbistatin-untreated cells, which was arbitrarily set at 100. * and # indicate asignificant difference (pb0.05) from the values for TCDD and DMSO treatments in blebbistatin-untreated cells, respectively. (C) After treatment with DMSO (DM) or 40 μM(−)-blebbistatin [(−)-Bleb] for 1.5–24 h, Hepa-1 cells were harvested and total cell extracts were prepared. Equal amounts of total cell extract (5 μg) were subjected towestern blotting to examine the level of AhR expression. GAPDH was detected as a loading control. Upper panel shows AhR levels in cells treated with the inhibitor for 24 h.Lower panel shows AhR levels in cells treated with 40 μM (−)-blebbistatin [(−)-Bleb] or (+)-blebbistatin [(+)-Bleb] for 1.5–24 h, with a sample treated with DMSO for 24 hloaded as a control. (D) After treatment with DMSO or 40 μM (−)-blebbistatin [(−)-Bleb] for 24 h, Hepa-1 cells were treated for a further 1.5 h with DMSO (DM) or 4 nM TCDD(TC), and cytosolic and nuclear fractions were prepared. Aliquots of each fraction (10 μg) were used to determine the extent of nuclear localization of AhR by western blotting.LDH and Lamin B were detected as loading controls for the cytosolic and nuclear fractions, respectively. The AhR signal was detected by short and long exposures (Short-expo.and Long-expo. AhR, respectively) to X-ray film. (E) qPCR was performed to quantify the relative induction of Cyp1a2 and Nqo1 using the reverse-transcribed cDNA samplesdescribed in (B). □ and ■ indicate DMSO and TCDD treatments, respectively. The values plotted correspond to those plotted in (B).

178 M. Ebina et al. / Biochimica et Biophysica Acta 1809 (2011) 176–183


level of CYP1A1 mRNA, to a level that was approximately 40% of thatdetected in the presence of TCDD in (−)-blebbistatin-untreated cells.In addition, the level of CYP1A1 mRNA by TCDD was also increasedadditively. To clarify whether the increase of CYP1A1-mRNA was dueto the inhibition of nonmuscle myosin II, we compared the effect of(−)-blebbistatin and its enantiomer, (+)-blebbistatin, on theinduction of Cyp1a1. Although a faint increase of CYP1A1-mRNAwas observed by (+)-blebbistatin treatment, clear increase of Cyp1a1induction was observed both in the presence and absence of TCDD by(−)-blebbistatin treatment. Previously, we have reported thattyrosine kinase inhibitors suppress TCDD-triggered Cyp1a1 inductionthrough promoting the degradation of total AhR or inhibitingAhR nuclear localization [32]. Therefore, there is a possibility that(−)-blebbistatin treatment increases the amount of total AhR orits nuclear localization, which we examined by western blotting.Fig. 1C (upper panel) and D indicates that the increased inductionof Cyp1a1 by TCDD in (−)-blebbistatin-treated cells was not due to anincreased level of either total AhR or nuclear-localized AhR. Moreover,in the presence of (−)-blebbistatin the total amount of AhR decreasedgradually from 4 h, although after 24 h the level had stabilized (lowerpanel in Fig. 1C).

It is well known that several genes are induced by TCDD-activatedAhR, and these genes include phase I and phase II genes of xe-nobiotic metabolizing enzymes. To examine the effect of (−)- and(+)-blebbistatin on the induction of other AhR target genes, we choseCyp1a2 and Nqo1 as representative phase I and II genes, respectively,and compared them with Cyp1a1. As shown in Fig. 1E, whereasincreased induction of Cyp1a2 by (−)-blebbistatin was similar to thatof Cyp1a1, Nqo1 induction increased less in both the presence andabsence of TCDD. These results indicate that the inhibition ofnonmuscle myosin II functions is related to the increased inductionof AhR target genes.

3.2. NMIIA is the only isoform expressed in Hepa-1 cells

In addition to being a highly selective inhibitor of nonmusclemyosin II, (−)-blebbistatin also inhibits smooth muscle myosin athigh dose (IC50=80 μM) [35]. To determine the target for (−)-blebbistatin, we investigated which isoform of nonmuscle myosin IIwas expressed in Hepa-1 cells. Fig. 2A shows that mainly NMIIAmRNA was detected, and smooth muscle and NMIIC mRNA weredetected to a lesser extent, by RT-PCR. However, Fig. 2B shows that,at the protein level, only NMIIA was detected in Hepa-1 cells.Moreover, immunoprecipitation experiments showed that the inter-action of NMIIA and actin was disrupted by treatment with 40 μM(−)-blebbistatin, but not (+)-blebbistatin (Fig. 2C). These resultssuggest that NMIIA is the only target of (−)-blebbistatin in Hepa-1cells.

3.3. Knockdown of NMIIA enhances CYP1A1-mRNA induction by TCDD

We used RNAi (siRNA) to reduce the level of endogenous NMIIAand to examinewhether knockdown of NMIIA altered the induction ofCYP1A1 mRNA in a similar manner to (−)-blebbistatin treatment. Asshown in Fig. 3A, the level of NMIIA was reduced substantially at4 days after transfection with siNMIIA-1, whereas siNMIIA-2 had asmaller effect. In accordance with this, the induction of Cyp1a1 byTCDD was enhanced more by siNMIIA-1 than by siNMIIA-2 (Fig. 3B).Although (−)- or (+)-blebbistatin enhanced the basal level of Cyp1a1induction, knockdown by siNMIIA did not enhance it. In a similar way,the enhancement of TCDD-triggered induction was observed forCyp1a2 and Nqo1 (Fig. 3D). Fig. 3C shows that the enhancement ofAhR target gene induction by siNMIIA was not due to an increasedlocalization of AhR to the nucleus. These results indicate that thereduction of NMIIA by knockdown of the protein enhances theinduction of AhR target genes by TCDD.

3.4. (−)-Blebbistatin and knockdown of NMIIA elicit the samemorphological change in Hepa-1 cells

It is known that nonmuscle myosin II forms actomyosin throughits interaction with F-actin, and plays a fundamental role in deter-mining cell morphology. It has been reported that the inhibition ofnonmuscle myosin II by (−)-blebbistatin treatment or the knock-down by siRNA results in changes in cell morphology, with thereorganization of vinculin-containing focal adhesions and the F-actincytoskeleton [27,28]. However, the effects of the inhibition andknockdown of NMIIA have not been compared in the same cell line.Hence, we performed immunostaining of Hepa-1 cells in order tocompare the effects of (−)-blebbistatin and siNMIIA in terms of cellmorphology. Phase contrast images showed that (−)-blebbistatininduced the formation of fibroblast-like or dendritic extensions inHepa-1 cells (Fig. 4A, upper panel). To analyze cytoskeletal remodel-ing, we performed immunocytochemistry to monitor the status ofvinculin-containing focal adhesions and the F-actin cytoskeleton.The (−)-blebbistatin-treated cells contained fewer focal adhesionsand F-actin than the DMSO- or (+)-blebbistatin-treated cells,although the levels of vinculin and actin in the (−)-blebbistatin-treated cells did not differ from those in other cells (middle and lowerpanel in Fig. 4A and B). These changes were not affected by TCDDtreatment (Supplemental Fig. 3). Fig. 4C and D shows that themorphological changes and cytoskeletal reorganization that wereinduced by the knockdown of NMIIA were similar to those induced by(−)-blebbistatin treatment, and occurred in the absence of changes inthe levels of vinculin and actin. Furthermore, they were not affectedby TCDD treatment (data not shown). These results suggest that theinhibition and knockdown of NMIIA have similar effects on cellmorphology and cytoskeletal organization in Hepa-1 cells.

Fig. 2. Expression of nonmuscle myosin II isoforms and smoothmuscle myosin in Hepa-1 cells. mRNA (A) and protein (B) expression of nonmuscle myosin II (NMII) isoformsand smooth muscle (SM) myosin in Hepa-1 cells were analyzed by PCR using isoform-specific primer sets and western blotting with antibodies specific to each protein. NIH3T3 cells (3T3) and mouse lung tissue (lung) were used as positive controls for theexpression of NMIIA and NMIIB, and SM and NMIIC, respectively. Distilled water (DW)was used as a negative control for RT-PCR. (C) After cells had been treated with DMSO(DM) or 40 μM (−)-blebbistatin [(−)-Bleb] or (+)-blebbistatin [(+)-Bleb] for 24 h,total cell extracts were prepared in cell-lysis buffer of high ionic strength forimmunoprecipitation. Details are described in the Materials and methods section.Total cell extracts were immunoprecipitated using an antibody against NMIIA, and thensubjected to western blotting to examine the interaction between NMIIA and actin.



3.5. Binding of AhR and RNA polymerase II to each target region ofCyp1a1 is increased by NMIIA dysfunction

It is well known that the induction of transcription of Cyp1a1 byligand-activated AhR is accompanied by dynamic events on thegenomic promoter, which includes the binding of AhR to the enhancerregion, which contains an XRE, and RNA polymerase II (RNAPII) to theproximal region, which contains a TATA box [30]. The results of the

ChIP assay showed that TCDD-triggered binding of AhR and RNAPIIwas increased by (−)-blebbistatin treatment (Fig. 5A and B, TC and(+)-Bleb+TC vs. (−)-Bleb+TC). The ChIP assay also showed asimilar increase in siNMIIA-1-transfected cells (Fig. 5C and D, siCont+TC vs. siNMIIA-1+TC). Basal binding of AhR and RNAPII wasincreased by blebbistatin treatments (Fig. 5A and B, DM vs. (−)-Bleb+DM and (+)-Bleb+DM), but not by siNMIIA-1 transfection (Fig. 5Cand D, siCont+DM vs. siNMIIA-1+DM). These results indicate thatthe increase of Cyp1a1 induction by the inhibition or knockdown ofNMIIA is due to the increased binding of AhR and RNAPII to theenhancer and proximal regions of Cyp1a1, respectively.

4. Discussion

It is well known that nonmuscle myosin II plays an important rolein cell motility, migration, and shape [20], although it has not beeninvestigated thoroughly whether the function of the protein is relatedto gene induction. The inhibitor (−)-blebbistatin can be used toinvestigate the function of nonmuscle myosin II due to its cellpermeability and selectivity for the protein [26]. In this study, weexamined the correlation between the function of nonmuscle myosinII and the induction of AhR target genes. We observed that (−)-blebbistatin treatment increased the CYP1A1-mRNA expression bothin the presence and absence of TCDD, and then confirmed that thisoccurred through the inhibition of NMIIA, which was only an isoformthat was expressed in Hepa-1 cells.

Phase contrast imaging and immunofluorescence showed thattreatment of Hepa-1 cells with (−)-blebbistatin and transfection withsiNMIIA resulted in the same morphological changes and cytoskeletalreorganization, which included the formation of dendritic extensionsand loss of vinculin-containing focal adhesions and F-actin. Morerecently, Liu et al. have reported that treatment with (−)-blebbistatinsuppresses the contraction and accelerates the migration of mousehepatic stellate cells (HSCs), and this is accompanied by fewervinculin-containing focal adhesions and F-actin [36]. Although we didnot examine cell contraction and migration in the present study, weexpect that Hepa-1 cells in which NMIIA function is inhibited orknocked down would have similar characteristics to HSCs treatedwith (−)-blebbistatin.

We noticed that the amount of AhR in Hepa-1 cells was decreasedslightly by (−)-blebbistatin treatment or siNMIIA transfection(Figs. 1C and 3A). It has been suggested that unliganded AhR interactswith F-actin via binding to XAP2 [37]; therefore, AhR might becomesensitive to degradation after the disruption of F-actin as a result ofthe inhibition or knockdown of NMIIA.

Furthermore, in spite of the reduction in the amount of total AhRafter the inhibition or knockdown of NMIIA, it was clear that theamount of nuclear-localized AhR by TCDD in these cells was almostthe same as that in the control cells (Figs. 1D and 3C). This mightindicate that the reduced amount of AhR in cells that are dysfunctionalfor NMIIA is sufficient to translocate into the nucleus and induce thetarget genes of AhR. Therefore, the reduction of AhR might not affectthe amount of nuclear AhR.

Increase of Cyp1a1 induction by TCDD was not due to an increasedlevel of either total AhR or nuclear-localized AhR.Moreover, the kineticsof AhR nuclear localization by TCDD was similar between DMSO- and(−)-blebbistatin-treated cells, and between siCont and siNMIIA-1-transfected cells (Supplemental Fig. 1, middle and lower panel). As aconsequence, we focused on the binding of AhR and RNAPII to theenhancer and proximal promoter regions of the Cyp1a1 gene at 1.5 hafter TCDD treatment, which corresponds to the peak binding of AhRand RNAPII to each target region [30,32,34,38]. The ChIP assay indicatedthat the increase of Cyp1a1 induction by TCDD occurred via an increasein the binding of AhR and RNAPII to their target regions (Fig. 5). It hasbeen proposed that the induction of Cyp1a1 by ligand-bound AhRinvolves the formation of a chromatin loop due to interaction between

Fig. 3. Effect of reduction of NMIIA on the induction of Cyp1a1 transcription by TCDD inHepa-1 cells. (A) Hepa-1 cells were transfected with control siRNA (siCont) orsiRNA against NMIIA (siNMIIA-1 and -2) at a final concentration of 5 nM. After culturefor 2–4 days, cells were harvested and total cell extracts were prepared. Equal amountsof total cell extract (5 μg) were subjected to western blotting with each antibody.(B) Nine days after siRNA transfection, Hepa-1 cells were treated for 9 h with DMSO or4 nM TCDD. Cells were harvested and total RNA was isolated followed by reversetranscription. The amount of CYP1A1 and GAPDH mRNA was determined by qPCR. Theplotted values from three independent experiments (means±SD) represent therelative Cyp1a1 induction and correspond to the ratio of expression of CYP1A1/GAPDHmRNA given relative to the mean value obtained after TCDD treatment of siCont-transfected cells, which was set arbitrarily at 100. * indicates significant difference(pb0.05) from the value obtained after TCDD treatment of siCont-transfected cells.(C) After transfection with siCont or siNMIIA1 for 4 days, Hepa-1 cells were treated for afurther 1.5 h with DMSO (DM) or 4 nM TCDD (TC) and cytosolic and nuclear fractionswere prepared. Aliquots of each fraction (10 μg) were used to examine the extent ofnuclear localization of AhR by western blotting, as described in the legend for Fig. 1D.(D) qPCR was performed to quantify the relative induction of Cyp1a2 and Nqo1, usingreverse-transcribed cDNA samples derived from siCont- and siNMIIA-1-transfectedcells. □ and ■ indicate DMSO and TCDD treatments, respectively. The values plottedcorrespond to those plotted in (A).



the enhancer and proximal promoter region [39]. The results of our ChIPassay imply that dysfunction of NMIIA results in the formation of atighter chromatin loop, which increases the accessibility of transcrip-tional cofactors.Notably, treatmentwith (−)- or (+)-blebbistatin aloneincreased the binding of AhR and RNAPII to their target regions (Fig. 5Aand B), which could explain the increase of basal Cyp1a1 induction in(−)- and (+)-blebbistatin-treated cells (Fig. 1B). The low level of AhRthat was detected in the nuclear fraction of (−)- or (+)-blebbistatin-treated cells in the absence of TCDD (Fig. 1D and Supplemental Fig. 1,upper panel), and the results of the reporter gene assay thatused mutant F318A, which has no ligand binding ability [40], inthe Gal-4 AhR fusion system [41] (Supplemental Fig. 2) implied that(−)- and (+)-blebbistatin bind to AhR as a weak ligand. The findingsalso suggest that (−)-blebbistatin has twomechanisms of action: firstlyit acts as a weak ligand of AhR, and secondly it enhances gene inductionby inhibiting NMIIA.

Several members of the myosin superfamily have been identifiedin the nucleus [42]. Among these, nuclear myosin I interacts withRNAPII [43], and forms interchromatin networks [44]. Li and Sarnahave reported that the assembly of the RNAPII pre-initiation complexon the promoter of the intercellular adhesion molecule-1 (ICAM-1)gene is regulated by nuclear myosin II (smooth muscle andnonmuscle) [45]. In our study, we did not clarify the mechanism forthe increase of AhR and RNAPII binding to their target regions. IfNMIIA functions in the nucleus in a similar manner to the nuclearmyosins, we can speculate that it suppresses Cyp1a1 induction byrecruiting a co-repressor, such as histone deacetylase 1 (HDAC1), tothe proximal promoter region.

The cytoskeleton containsmicrotubules, aswell as actomyosin, and acrosstalk between actomyosin and microtubules is involved in main-taining cell motility and shape [46,47]. Dvorak et al. have implied thatTCDD-triggered Cyp1a1 induction is suppressed by colchicines, whichare known disruptors of microtubules [48]. From our results, wespeculate that the enhancement/suppression of Cyp1a1 induction byTCDD, which occurs through the disruption of actomyosin/microtu-bules, involves the crosstalk between the two cytoskeletal components.

Fig. 4. Effect of (−)-blebbistatin and siNMIIA on the morphology of Hepa-1 cells. Hepa-1 cells were treated with DMSO (DM) or 40 μM (−)-blebbistatin [(−)-Bleb] or (+)-blebbistatin[(+)-Bleb] for 1 day (A) orwere subjected to siRNA transfection (siCont or siNMIIA-1) for 4 days (C). Phase-contrast imagesof cellswere takenbeforefixation (upperpanel). Afterfixation,cells on the collagen type I- coated coverslip were fixed and examined for focal adhesions (middle panel) and actin stress fibers (lower panel) by stainingwith anti-vinculin antibody andrhodamine-conjugatedphalloidin, respectively. The scale bar represents10 μm. (BandD)Total cell extracts (5 μg)derived fromchemical-treatedor siRNA-transfectedcellswere subjectedto western blotting to evaluate changes in cytoskeletal components.

Fig. 5. (−)-Blebbistatin and siNMIIA increase AhR and RNA polymerase II binding toCyp1a1 promoter regions. After pretreatment with DMSO or 40 μM (−)-blebbistatin[(−)-Bleb] or (+)-blebbistatin [(+)-Bleb] for 1 day (A and B) or siRNA transfection(siCont or siNMIIA-1) for 4 days (C and D), Hepa-1 cells were treated with DMSO (DM)or 4 nM TCDD (TC) for another 1.5 h, and subjected to ChIP assay using normal IgG orantibodies against AhR (A and C) and RNA polymerase II (RNAPII; B and D). PrecipitatedDNA was quantified by qPCR using primer sets for the enhancer (−0.8 kbp) andproximal promoter (−0.1 kbp) regions. Relative binding to each region is expressed asthe percentage of total input DNA in each sample. Data are shown as means±SD fromthree independent experiments. * and # indicate significant differences (pb0.05) fromthe values for TCDD and DMSO treatment in blebbistatin-untreated cells (A and B), andsiCont-transfected cells (C and D), respectively.



NMIIA has been investigated as a potential clinical cause of MYH9-related diseases such as May–Hegglin anomaly, and Sebastian,Fechtner, and Epstein syndromes [49]. In addition to its cellularfunction and role in clinical conditions, NMIIA might also have adevelopmental role, which has been suggested by experiments inknockout mice and expression profiling of the protein [22,50].Recently, Martinelli and co-workers have observed a decrease inNMIIA in medial epithelial cells during palatal genesis and identifiedlinkage disequilibrium between specific polymorphic alleles and cleftpalate [51], which is one of the most common malformations that arecaused by TCDD. It is thought that gestational exposure to TCDDduring palatal genesis interferes with the firm adhesion and/ordegeneration of palatal clefts by preventing the programmed death ofmedial epithelial cells [52,53]. In the present study, the enhancementof induction of AhR target genes by TCDD was observed in cellstransfected with siNMIIA; therefore this type of abnormal geneinduction in medial epithelial cells might contribute to TCDD-inducedcleft palate.

In conclusion, we demonstrated that the function of NMIIA isrelated to the induction of AhR target genes in Hepa-1 cells. Althoughfurther investigation is needed to establish how dysfunction of NMIIAenhances the binding of AhR and RNAPII to their target regions, ourstudy sheds new light on the relationship between the function ofNMIIA and gene induction.

Supplementarymaterials related to this article can be found onlineat doi:10.1016/j.bbagrm.2011.01.002.

Acknowledgments

The authors express their appreciation to Prof. Sei-ichi Ishiguroand Dr. Taku Ozaki for helpful advice.

We thank the staff of the Gene Research Center and Center forInstrumental Analysis in Hirosaki University.

This workwas supported by Grants-in-aid for Science Research (B)(No.15310032) and (C) (No.21510065) from the Ministry of Educa-tion, Culture, Sports, Science and Technology (Monbu Kagakusho),and the Global Environment Research Fund from the Ministry of theEnvironment, Japan (C-0803).

References

[1] L.A. Couture, B.D. Abbott, L.S. Birnbaum, A critical review of the developmentaltoxicity and teratogenicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin: recentadvances toward understanding the mechanism, Teratology 42 (1990) 619–627.

[2] J. Huff, G. Lucier, A. Tritscher, Carcinogenicity of TCDD: experimental, mechanistic,and epidemiologic evidence, Annu. Rev. Pharmacol. Toxicol. 34 (1994) 343–372.

[3] J.G. Vos, C. De Heer, H. Van Loveren, Immunotoxic effects of TCDD and toxicequivalency factors, Teratog. Carcinog. Mutagen. 17 (1997) 275–284.

[4] B.K. Meyer, M.G. Pray Grant, J.P. Vanden Heuvel, G.H. Perdew, Hepatitis B virus X-associated protein 2 is a subunit of the unliganded aryl hydrocarbon receptor corecomplex and exhibits transcriptional enhancer activity, Mol. Cell. Biol. 18 (1998)978–988.

[5] A. Kazlauskas, L. Poellinger, I. Pongratz, Evidence that the co-chaperone p23regulates ligand responsiveness of the dioxin (aryl hydrocarbon) receptor, J. Biol.Chem. 274 (1999) 13519–13524.

[6] D.W. Nebert, A.L. Roe, M.Z. Dieter, W.A. Solis, Y. Yang, T.P. Dalton, Role of thearomatic hydrocarbon receptor and [Ah] gene battery in the oxidative stressresponse, cell cycle control, and apoptosis, Biochem. Pharmacol. 59 (2000) 65–85.

[7] J. Mimura, Y. Fujii Kuriyama, Functional role of AhR in the expression of toxiceffects by TCDD, Biochim. Biophys. Acta 1619 (2003) 263–268.

[8] O. Hankinson, Role of coactivators in transcriptional activation by the arylhydrocarbon receptor, Arch. Biochem. Biophys. 433 (2005) 379–386.

[9] Y. Shimizu, Y. Nakatsuru, M. Ichinose, Y. Takahashi, H. Kume, J. Mimura, Y. FujiiKuriyama, T. Ishikawa, Benzo[a]pyrene carcinogenicity is lost in mice lacking thearyl hydrocarbon receptor, Proc. Natl Acad. Sci. USA 97 (2000) 779–782.

[10] G.P. Lahvis, S.L. Lindell, R.S. Thomas, R.S. McCuskey, C. Murphy, E. Glover, M. Bentz,J. Southard, C.A. Bradfield, Portosystemic shunting and persistent fetal vascularstructures in aryl hydrocarbon receptor-deficient mice, Proc. Natl Acad. Sci. USA97 (2000) 10442–10447.

[11] H. Kikuchi, A. Hossain, H. Yoshida, S. Kobayashi, Induction of cytochrome P-4501A1 by omeprazole in human HepG2 cells is protein tyrosine kinase-dependentand is not inhibited by alpha-naphthoflavone, Arch. Biochem. Biophys. 358 (1998)351–358.

[12] M. Backlund, M. Ingelman Sundberg, Different structural requirements of theligand binding domain of the aryl hydrocarbon receptor for high- and low-affinityligand binding and receptor activation, Mol. Pharmacol. 65 (2004) 416–425.

[13] L.P. Nguyen, C.A. Bradfield, The search for endogenous activators of the arylhydrocarbon receptor, Chem. Res. Toxicol. 21 (2008) 102–116.

[14] R. Barouki, X. Coumoul, P.M. Fernandez Salguero, The aryl hydrocarbon receptor,more than a xenobiotic-interacting protein, FEBS Lett. 581 (2007) 3608–3615.

[15] M. Diry, C. Tomkiewicz, C. Koehle, X. Coumoul, K.W. Bock, R. Barouki, C. Transy,Activation of the dioxin/aryl hydrocarbon receptor (AhR)modulates cell plasticitythrough a JNK-dependent mechanism, Oncogene 25 (2006) 5570–5574.

[16] L.C. Bui, C. Tomkiewicz, A. Chevallier, S. Pierre, A.S. Bats, S. Mota, J. Raingeaud, J.Pierre, M. Diry, C. Transy, M. Garlatti, R. Barouki, X. Coumoul, Nedd9/Hef1/Cas-Lmediates the effects of environmental pollutants on cell migration and plasticity,Oncogene 28 (2009) 3642–3651.

[17] J.M. Carvajal Gonzalez, S.Mulero Navarro, A.C. Roman, V. Sauzeau, J.M.Merino, X.R.Bustelo, P.M. Fernandez Salguero, The dioxin receptor regulates the constitutiveexpression of the vav3 proto-oncogene and modulates cell shape and adhesion,Mol. Biol. Cell 20 (2009) 1715–1727.

[18] S.K.Maciver, Myosin II function in non-muscle cells, Bioessays 18 (1996) 179–182.[19] J.S. Berg, B.C. Powell, R.E. Cheney, A millennial myosin census, Mol. Biol. Cell 12

(2001) 780–794.[20] K. Clark, M. Langeslag, C.G. Figdor, F.N. van Leeuwen, Myosin II and mechan-

otransduction: a balancing act, Trends Cell Biol. 17 (2007) 178–186.[21] N. Prasain, T. Stevens, The actin cytoskeleton in endothelial cell phenotypes,

Microvasc. Res. 77 (2009) 53–63.[22] V. Marigo, A. Nigro, A. Pecci, D. Montanaro, M. Di Stazio, C.L. Balduini, A. Savoia,

Correlation between the clinical phenotype of MYH9-related disease and tissuedistribution of class II nonmuscle myosin heavy chains, Genomics 83 (2004)1125–1133.

[23] E. Golomb, X. Ma, S.S. Jana, Y.A. Preston, S. Kawamoto, N.G. Shoham, E. Goldin, M.A. Conti, J.R. Sellers, R.S. Adelstein, Identification and characterization ofnonmuscle myosin II-C, a new member of the myosin II family, J. Biol. Chem.279 (2004) 2800–2808.

[24] T. Saitoh, S. Takemura, K. Ueda, H. Hosoya, M. Nagayama, H. Haga, K. Kawabata, A.Yamagishi, M. Takahashi, Differential localization of non-muscle myosin IIisoforms and phosphorylated regulatory light chains in human MRC-5 fibroblasts,FEBS Lett. 509 (2001) 365–369.

[25] N.T. Swailes, M. Colegrave, P.J. Knight, M. Peckham, Non-muscle myosins 2A and2B drive changes in cell morphology that occur as myoblasts align and fuse, J. CellSci. 119 (2006) 3561–3570.

[26] A.F. Straight, A. Cheung, J. Limouze, I. Chen, N.J. Westwood, J.R. Sellers, T.J.Mitchison, Dissecting temporal and spatial control of cytokinesis with a myosin IIinhibitor, Science 299 (2003) 1743–1747.

[27] M. Zhang, P.V. Rao, Blebbistatin, a novel inhibitor of myosin II ATPase activity,increases aqueous humor outflow facility in perfused enucleated porcine eyes,Investig. Ophthalmol. Vis. Sci. 46 (2005) 4130–4138.

[28] A.I. Ivanov, M. Bachar, B.A. Babbin, R.S. Adelstein, A. Nusrat, C.A. Parkos, A uniquerole for nonmuscle myosin heavy chain IIA in regulation of epithelial apicaljunctions, PLoS ONE 2 (2007) e658.

[29] E. Sasamori, S. Shimoyama, S. Fukushige, H. Kikuchi, Involvement of CREM inCYP1A1 induction through ligand-independent activation pathway of arylhydrocarbon receptor in HepG2 cells, Arch. Biochem. Biophys. 478 (2008) 26–35.

[30] M. Schnekenburger, G. Talaska, A. Puga, Chromium cross-links histone deacety-lase 1-DNA methyltransferase 1 complexes to chromatin, inhibiting histone-remodeling marks critical for transcriptional activation, Mol. Cell. Biol. 27 (2007)7089–7101.

[31] Y. Yanagiba, Y. Ito, O. Yamanoshita, S.Y. Zhang, G. Watanabe, K. Taya, C.M. Li, Y.Inotsume, M. Kamijima, F.J. Gonzalez, T. Nakajima, Styrene trimer may increasethyroid hormone levels via down-regulation of the aryl hydrocarbon receptor(AhR) target gene UDP-glucuronosyltransferase, Environ. Health Perspect. 116(2008) 740–745.

[32] S. Kasai, H. Kikuchi, The inhibitory mechanisms of the tyrosine kinase inhibitorsherbimycin a, genistein, and tyrphostin B48 with regard to the function of the arylhydrocarbon receptor in Caco-2 cells, Biosci. Biotechnol. Biochem. 74 (2010)36–43.

[33] F. Vascotto, D. Lankar, G. Faure Andre, P. Vargas, J. Diaz, D. Le Roux, M.I. Yuseff, J.B.Sibarita, M. Boes, G. Raposo, E. Mougneau, N. Glaichenhaus, C. Bonnerot, B.Manoury, A.M. Lennon Dumenil, The actin-based motor protein myosin IIregulates MHC class II trafficking and BCR-driven antigen presentation, J. CellBiol. 176 (2007) 1007–1019.

[34] M. Shibazaki, T. Takeuchi, S. Ahmed, H. Kikuchi, Suppression by p38 MAP kinaseinhibitors (pyridinyl imidazole compounds) of Ah receptor target gene activationby 2,3,7,8-tetrachlorodibenzo-p-dioxin and the possible mechanism, J. Biol. Chem.279 (2004) 3869–3876.

[35] J. Limouze, A.F. Straight, T. Mitchison, J.R. Sellers, Specificity of blebbistatin, aninhibitor of myosin II, J. Muscle Res. Cell Motil. 25 (2004) 337–341.

[36] Z. Liu, L.A. van Grunsven, E. Van Rossen, B. Schroyen, J.P. Timmermans, A. Geerts,H. Reynaert, Blebbistatin inhibits contraction and accelerates migration in mousehepatic stellate cells, Br. J. Pharmacol. 159 (2010) 304–315.

[37] P. Berg, I. Pongratz, Two parallel pathways mediate cytoplasmic localization of thedioxin (aryl hydrocarbon) receptor, J. Biol. Chem. 277 (2002) 32310–32319.

[38] T. Suzuki, K. Nohara, Regulatory factors involved in species-specific modulation ofarylhydrocarbon receptor (AhR)-dependent gene expression in humans andmice, J. Biochem. 142 (2007) 443–452.

[39] Y. Tian, S. Ke, M. Chen, T. Sheng, Interactions between the aryl hydrocarbonreceptor and P-TEFb. Sequential recruitment of transcription factors and



differential phosphorylation of C-terminal domain of RNA polymerase II at cyp1a1promoter, J. Biol. Chem. 278 (2003) 44041–44048.

[40] K. Goryo, A. Suzuki, C.A. Del Carpio, K. Siizaki, E. Kuriyama, Y. Mikami, K. Kinoshita,K. Yasumoto, A. Rannug, A. Miyamoto, Y. Fujii Kuriyama, K. Sogawa, Identificationof amino acid residues in the Ah receptor involved in ligand binding, Biochem.Biophys. Res. Commun. 354 (2007) 396–402.

[41] K. Kudo, T. Takeuchi, Y. Murakami, M. Ebina, H. Kikuchi, Characterization of theregion of the aryl hydrocarbon receptor required for ligand dependency oftransactivation using chimeric receptor between Drosophila and Mus musculus,Biochim. Biophys. Acta 1789 (2009) 477–486.

[42] E. Louvet, P. Percipalle, Transcriptional control of gene expression by actin andmyosin, Int. Rev. Cell Mol. Biol. 272 (2009) 107–147.

[43] L. Pestic Dragovich, L. Stojiljkovic, A.A. Philimonenko, G. Nowak, Y. Ke, R.E.Settlage, J. Shabanowitz, D.F. Hunt, P. Hozak, P. de Lanerolle, A myosin I isoform inthe nucleus, Science 290 (2000) 337–341.

[44] Q. Hu, Y.S. Kwon, E. Nunez, M.D. Cardamone, K.R. Hutt, K.A. Ohgi, I. Garcia Bassets, D.W. Rose, C.K. Glass, M.G. Rosenfeld, X.D. Fu, Enhancing nuclear receptor-inducedtranscription requires nuclear motor and LSD1-dependent gene networking ininterchromatin granules, Proc. Natl Acad. Sci. USA 105 (2008) 19199–19204.

[45] Q. Li, S.K. Sarna,Nuclearmyosin II regulates the assembly of preinitiation complex forICAM-1 gene transcription, Gastroenterology 137 (2009) 1051–10608 1060.e1-.

[46] S. Etienne Manneville, Actin and microtubules in cell motility: which one is incontrol? Traffic 5 (2004) 470–477.

[47] S. Even Ram, A.D. Doyle, M.A. Conti, K. Matsumoto, R.S. Adelstein, K.M. Yamada,Myosin IIA regulates cell motility and actomyosin-microtubule crosstalk, Nat. CellBiol. 9 (2007) 299–309.

[48] Z. Dvorak, R. Vrzal, J. Ulrichova, J.M. Pascussi, P. Maurel, M. Modriansky,Involvement of cytoskeleton in AhR-dependent CYP1A1 expression, Curr. DrugMetab. 7 (2006) 301–313.

[49] S. Kunishima, H. Saito, Congenital macrothrombocytopenias, Blood Rev. 20 (2006)111–121.

[50] M.A. Conti, S. Even Ram, C. Liu, K.M. Yamada, R.S. Adelstein, Defects in celladhesion and the visceral endoderm following ablation of nonmuscle myosinheavy chain II-A in mice, J. Biol. Chem. 279 (2004) 41263–41266.

[51] M. Martinelli, M. Di Stazio, L. Scapoli, J. Marchesini, F. Di Bari, F. Pezzetti, F. Carinci,A. Palmieri, P. Carinci, A. Savoia, Cleft lip with or without cleft palate: implicationof the heavy chain of non-muscle myosin IIA, J. Med. Genet. 44 (2007) 387–392.

[52] R.M. Pratt, L. Dencker, V.M. Diewert, 2,3,7,8-Tetrachlorodibenzo-p-dioxin-induced cleft palate in the mouse: evidence for alterations in palatal shelf fusion,Teratog. Carcinog. Mutagen. 4 (1984) 427–436.

[53] B.D. Abbott, L.S. Birnbaum, TCDD alters medial epithelial cell differentiationduring palatogenesis, Toxicol. Appl. Pharmacol. 99 (1989) 276–286.


author's personal copynature.cc.hirosaki-u.ac.jp/.../gazou2/ebina.2011.pdfauthor's...

Documents