Journal of Dermatological Science 58 (2010) 211–216

B lymphocyte-induced maturation protein 1 is a novel target gene of arylhydrocarbon receptor

Togo Ikuta a,*, Motoi Ohba b, Christos C. Zouboulis c, Yoshiaki Fujii-Kuriyama d,e, Kaname Kawajiri a

a Research Institute for Clinical Oncology, Saitama Cancer Center, 818 Komuro, Ina-machi, Kitaadachi-gun, Saitama 362-0806, Japanb Institute of Molecular Oncology, Showa University, Hatanodai, Shinagawa-ku, Tokyo 142-8555, Japanc Departments of Dermatology, Venereology, Allergology and Immunology, Dessau Medical Center, Dessau, Germanyd Medical Research Institute Tokyo Medical and Dental University, Bunkyo-ku, Tokyo 113-8510, Japane Institute of Molecular and Cellular Biosciences, The University of Tokyo, Bunkyo-ku, Tokyo 113-0032, Japan

A R T I C L E I N F O

Article history:

Received 20 November 2009

Received in revised form 15 March 2010

Accepted 2 April 2010

Keywords:

Blimp1

Ah receptor

Sebocyte

Keratinocyte

A B S T R A C T

Background: The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor. When

environmental pollutants, including chemical carcinogens, bind to AhR, the receptor translocates to

nucleus and transcriptionally activates target genes including drug metabolizing enzymes such as P450s.

Recent studies have shown that AhR mediates various responses, including cellular growth,

differentiation, immune system and development.

Objective: In this study, we investigated the physiological function of AhR in skin.

Methods: Distribution of AhR in murine skin was examined by immunohistochemistry. Expression of a

target gene which is transcriptionally activated by AhR is analysed by RT-PCR.

Results: We found that AhR co-localizes with the transcriptional repressor B lymphocyte maturation

protein 1 (Blimp1) in sebaceous gland. In this report, we show that expression of Blimp1 is induced by

treatment with AhR ligands, such as methylcolanthrene (MC) in sebocyte and keratinocyte cell lines.

Exposure to ultraviolet B, which has been reported to generate AhR ligand intracellularly, also increased

Blimp1 mRNA. This ligand-dependent induction of Blimp1 requires the expression of both AhR and

ARNT, since transfection of siRNA specific to either AhR or ARNT significantly reduced Blimp1 mRNA in

response to MC. Analysis using kinase inhibitors revealed that ligand-dependent induction of Blimp1,

but not that of CYP1A1, is inhibited by staurosporine. TPA, a potent activator of protein kinase C,

increased Blimp1 mRNA but not CYP1A1.

Conclusion: These data indicate that Blimp1 is a novel AhR-target gene in epidermal keratinocyte and

sebocyte.

� 2010 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights

reserved.

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Journal of Dermatological Science

journa l homepage: www.e lsev ier .com/ jds

1. Introduction

The skin protects the internal body from various environmentalstimuli including chemicals, microbial infection, and ultravioletlight as a primary defense structure. Aryl hydrocarbon receptor(AhR) is a ligand-dependent transcription factor which has beendetected in various tissues containing skin [1], and upon binding toits ligand, AhR translocates to nucleus to form a heterodimer withAhR nuclear translocator (ARNT) [2,3]. Thus, the formed hetero-dimer AhR/ARNT binds to the xenobiotic responsive element (XRE)[4] in the promoter of the target genes to enhance the expression ofthe target genes. CYP1A1 is highly induced in the skin of animalstreated with topical application of carcinogens such as benzo[a]-

* Corresponding author. Tel.: +81 48 722 1111; fax: +81 48 722 1739.

E-mail address: togo@cancer-c.pref.saitama.jp (T. Ikuta).

0923-1811/$36.00 � 2010 Japanese Society for Investigative Dermatology. Published b

doi:10.1016/j.jdermsci.2010.04.003

pyrene (B[a]P), methylcolanthrene (MC), and 7,12-dimethylben-z[a]anthracene (DMBA). In most cases these chemicals aremetabolically activated to become mutagenic metabolites throughformation of epoxides by the activation of CYP1A1 [5]. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD), among the most toxicpollutants known to date, is a ligand of AhR with high affinity.Exposure of human skin to TCDD results in chloracne characterizedby an acne-like eruption. Histological changes are associated withincreased cellular proliferation in the infundibular portion of thehair follicles, and TCDD applied topically to mouse skin inducescutaneous changes that involve involution and rapid disappear-ance of sebaceous gland [6,7].

These investigations, including carcinogenesis and pathologicalstudies of skin disease, suggest that AhR plays an important role inskin homeostasis. It is reported that AhR function is associated withtoxicological effects of ozone [8], and is blocked by EGF signaling [9]in normal human epidermal kerationcytes. In addition, recent

y Elsevier Ireland Ltd. All rights reserved.

T. Ikuta et al. / Journal of Dermatological Science 58 (2010) 211–216212

studies have revealed the physiological roles of AhR in development[10], cellular growth [11] and differentiation [12,13], as well asimmune system [14] in the absence of exogenous ligands. In thisreport, we studied the biological functions of AhR in the skin andfound a novel target gene, B lymphocyte maturation protein 1(Blimp1), which is a transcriptional repressor that is a criticalregulator of maturation of epidermis [15] as well as determination ofcell fate in sebocytes [16].

2. Materials and methods

2.1. Cell culture

The human sebocyte cell line SZ95 [17] was maintained inSebomed basal medium (Biochrom AG, Berlin, Germany) supple-mented with 5 ng/ml EGF and 10% fetal calf serum. The humankeratinocyte cell line HaCaT [18], provided by Dr. N. Fusenig, wasgrown in DMEM supplemented with 10% fetal calf serum at 37 8Cunder 5% CO2 atmosphere.

2.2. Transfection of siRNA

The siRNA for AhR was purchased from Dharmacon (Chicago, IL)and that for ARNT was from Ambion (Austin, TX). The sequence ofthe oligonucleotides (sense strand) was as follows: AhR, GAAA-GUGGCAUGAUAGUUUUU, ARNT, GGAAUGUCCAUUUGCUGAGTT.Silencer Negative control #1 siRNA (Ambion) was used for negativecontrol. A total of 2 � 105 SZ95 cells were seeded in each well of a24-well multiplate on the day before transfection. The siRNA wasintroduced into the cells using Lipofectamine 2000 (Invitrogen,Carlsbad, CA) at a concentration of 50 nM in culture medium. Thecells were collected 48 h after transfection.

2.3. Reverse transcription and real-time quantitative PCR

Total RNA extracted from cultured cells with Isogen (NipponGene, Tokyo, Japan) was used for generation of the first-strand cDNAwith a Takara RNA PCR kit (Takara, Tokyo Japan). PCR analysis wasperformed using gene-specific primers and the first strand cDNA asthe template. The sequence of primers was as follows: GAPDH,50-ACATCGCTCAGACACCATGG-30 and 50-GTAGTTGAGGTCAAT-GAAGGG-30; CYP1A1, 50-TCCCTATTCTTCTTCGCTACCTA-30 and 50-TCTCTGTACCCTGGGGTT-30; Blimp1, 50-AAGTGTAACTCCAGCACTG-TG-30 and 50-CCAAAACGTGTGCCCTTTGGTATG-30; ARNT, 50-CGGAA-CAAGATGACAGCCTAC-30 and 50-ACAGAAAGCCATCTGCTGCC-30;AhR, 50-ATACTGAAGCAGAGCTGTGC-30 and 50-AAAGCAGGCGTG-CATTAGAC-30. The PCR products were fractionated on a 2% agarosegel and visualized by ethidium bromide staining. Quantitative real-time PCR was performed on a Light-Cycler FastStart DNA Master PLUS

SYBER Green I (Roche, Mannheim, Germany). The PCR was evaluatedby a melting curve analysis following the manufacture’s instructions.

2.4. Immunohistochemistry

Back skin of the AhR�/� mice [19] in which AhR gene wasreplaced with b-galactosidase gene was immediately frozen inO.C.T Compound (Sakura Finetek, Tokyo, Japan), and the frozensections were prepared. Staining of b-galactosidase (b-gal) wascarried out as follows. The sections were fixed with 0.2%glutaraldehyde, followed by staining with 0.2% 5-bromo-4-chloro-3-indolyl b-D-galactopyranoside in 10 mM sodium phos-phate (pH 7.0) containing 1 mM MgCl2, 150 mM NaCl, and 3.3 mMeach of potassium ferrocyanide and ferricyanide at 37 8C overnight.For immunostaining, sections were fixed with 4% formaldehydeand were blocked with Protein Block (Dako, Carpinteria, CA),followed by incubation with the first antibodies including anti-b-

galactosidase antibodies (MP Biomedicals, Aurora, OH), and anti-Blimp1 antibodies (Santa Cruz Biotechnology, Inc., Santa Cruz, CA).Immunofluorescent staining was performed using appropriatesecondary antibodies conjugated with fluorescein isothiocyanateor rhodamine. Hoechst 33258 was used to visualize nuclei. Thetissue sections were observed with fluorescent microscope.

3. Results and discussion

3.1. AhR co-localizes with Blimp1 in sebaceous gland

To investigate AhR expression in the skin, b-gal staining wasperformed with the AhR homozygous mutant (AhR�/�) [19]. In thismouse, a part of AhR genomic fragment was replaced by NLS-LacZso that b-gal expression could mimic the mode of AhR expression.As shown in Fig. 1A, the signals by b-gal staining were detected inepidermis, upper part of hair follicles and sebaceous gland.Apparent staining was especially noticeable in sebaceous gland,which was visualized by oil red O (Fig. 1B). This staining patternwas also observed in AhR+/�mice, suggesting that it was not due toAhR-deficiency. We noticed this staining was similar to the patternof expression of Blimp1 which had been reported by Horsely et al.[16]. So we performed double-staining using anti-b-galactosidaseand anti-Blimp1 antibodies to determine whether AhR co-localizeswith Blimp1. Immunofluorescence with anti-b-gal revealed theexpression of AhR in sebaceous gland (Fig. 1C). Interestingly, thenuclei stained by anti-b-gal antibodies were apparently alsorecognized by anti-Blimp1 antibodies (Fig. 1D and E). These datasuggest that AhR co-localizes with Blimp1 in sebaceous gland.

3.2. Blimp1 is transcriptionally activated by AhR ligands

The mouse we used here for immunostaining is AhR-deficient,and Blimp1 is expressed in the absence of AhR. However, it would beinteresting to consider that AhR transcriptionally activates Blimp1as a target gene with some lipids synthesized in sebaceous glands asphysiological ligands of AhR. So to study the functional relevancebetween AhR and Blimp1, we were interested to determine whetherAhR regulates expression of Blimp1, and we used human sebocytecell line, SZ95 [17]. RT-PCR analysis indicated that treatment withMC, a ligand for AhR, increased Blimp1 mRNA as well as CYP1A1mRNA, which is a typical target gene of AhR/ARNT heterodimer(Fig. 2A). Real-time PCR analysis revealed about 4-fold inductionafter the treatment for 90 min. Addition of b-naphthoflavone,another ligand for AhR, to culture medium also induced Blimp1mRNA (Fig. 2B). Pretreatment with actinomycin D before addition ofMC suppressed the induction of Blimp1 (Fig. 2C). In addition,induction of Blimp1 was also observed in human keratinocyte cellline HaCaT by treatment with MC (Fig. 2D). As Fritsche et al. hadreported, AhR ligands such as 6-formylindolo[3,2-b]carbazolederived from tryptophan are intracellularly generated by UVBirradiation [20]. In this context, we investigated the effect of UVBirradiation on Blimp1 expression. As shown in Fig. 2E, exposure ofHaCaT cells to 10 mJ/cm2 UVB activated the expression of Blimp1 aswell as CYP1A1. These data suggest that the expression of Blimp1 istranscriptionally regulated by AhR ligands.

3.3. Induction of Blimp1 mRNA depends on AhR and ARNT

We next wanted to know whether induction of Blimp1 dependson AhR and ARNT. SZ95 cells were transiently transfected witheither control siRNA or siRNA specific for either AhR or ARNT, andRT-PCR analysis was conducted to evaluate the amount of Blimp1mRNA after treatment with MC. As shown in Fig. 3A, transfection ofsiRNA for AhR significantly reduced AhR mRNA. In the presence ofcontrol siRNA, treatment with MC for 90 min increased Blimp1

Fig. 1. Localization of AhR in murine skin. (A and B) b-Galactosidase staining. Nuclei stained with high intensity in sebaceous gland are shown by arrowheads. Oil red O

staining was performed after b-galactosidase staining and a hair follicle is surrounded by dotted lines in (B). Sebaceous gland is inserted. (C–E) AhR co-localizes Blimp1 in

sebaceous gland. Immunostaining with anti-b-galactosidase (C), anti-Blimp1 (D), and merge (E). b-Galactosidase is detected in nucleus by nuclear localization signal which is

fused to b-galactosidase [19].

T. Ikuta et al. / Journal of Dermatological Science 58 (2010) 211–216 213

mRNA about 5-fold. In contrast, the presence of siRNA specific forAhR inhibited the induction of Blimp1 mRNA in response to MC(Fig. 3C). Induction of CYP1A1 mRNA was similarly inhibited bytransfection of siRNA for AhR (Fig. 3B). We next tested the effect ofthe siRNA specific for ARNT. Transfection of siRNA for ARNT

Fig. 2. Blimp1 mRNA is induced by AhR ligands. Reverse transcription-PCR (RT-PCR) anal

were separated on a 2% agarose gel and visualized by ethidium bromide staining. Real-

normalized to expression of GAPDH and are mean of triplicate samples�SD. (A) Expressio

methylcolanthrene (MC) for 0–120 min. One microgram RNA was used to synthesize cDNA,

AhR ligands on Blimp1 expression; SZ95 cells were incubated with MC, 1 mM a-naphthoflav

more Blimp1 mRNA (*P < 0.001; **P < 0.05 by Student’s t-test). (C) Effect of actinomycin D

60 min before addition of MC to the culture medium. After 90 min incubation, RNA was ext

mRNA (*P < 0.001). (D) Induction of Blimp1 mRNA in HaCaT cells which was incubated w

exposed with UVB (10 mJ/cm2) and incubated for 0–180 min before extraction of RNA.

significantly reduced ARNT mRNA by 60% of control (Fig. 3D), andin this status, induction of Blimp1 mRNA by MC was significantlyinhibited by 70% of control (Fig. 3F). Expression of CYP1A1 was alsoinhibited by transfection of siRNA for ARNT (Fig. 3E). These datasuggest that treatment with AhR ligands transcriptionally activates

ysis was used to determine the expression of each indicated gene. The PCR products

time PCR was used to determine the relative expression of Blimp1. The values are

n of Blimp1 and CYP1A1. Total RNA was prepared from SZ95 cells incubated with 1 mM

followed by amplification with PCR using an appropriate pair of primers. (B) Effects of

one (aNF), 1 mM b-naphthoflavone (bNF). Treatment of ligands produced significantly

on induction of Blimp1; SZ95 cells were pretreated with 1 mg/ml actinomycin D for

racted from the cells. Pretreatment of actinomycin D significantly reduced the Blimp1

ith MC for 0–120 min. (E) UVB treatment increased Blimp1 mRNA. HaCaT cells were

Fig. 3. Induction of Blimp1 mRNA depends on AhR and ARNT. SZ95 cells were transfected with siRNA for either AhR (A–C) or ARNT (D–F). Real-time PCR was used to determine

the relative expression of each indicated gene. The values are normalized to expression of GAPDH and are mean of triplicate samples�SD. Cells were incubated with 1 mM MC,

or 100 nM TPA (T) for 90 min before extraction of total RNA.

T. Ikuta et al. / Journal of Dermatological Science 58 (2010) 211–216214

Blimp1 expression in the heterodimer of AhR/ARNT-dependentmanner.

3.4. Staurosporine inhibits the induction of Blimp1

We were next interested to investigate the activation mechanismof Blimp1 by AhR/ARNT. There are several xenobiotic response

Fig. 4. Staurosporine inhibits Blimp1 induction. (A) SZ95 cells were pretreated with stauro

for 90 min. RT-PCR analysis was performed to detect Blimp1 and CYP1A1 expression. Tre

with ST for 60 min before exposure to UVB. After exposure, the cells were incubated for e

expression: SZ95 cells were pretreated with 0.2 mM ST for 60 min, before addition of eithe

of RNA. The values are normalized to expression of GAPDH and are mean of triplicate

elements (XRE), which is a short sequence containing an AhR/ARNTbinding motif, on the downstream of the human BLIMP1 transcrip-tion start site. In order to find out whether the AhR/ARNT complexbinds to these XRE motifs to induce Blimp1 expression, variousluciferase plasmids connected to BLIMP1 containing the XRE wereconstructed. However, we were unable to detect XRE-dependentexpression of the luciferase reporter gene.

sporine (ST) for 60 min, and then MC was added to the culture and further incubated

atment with 2 mM ST(*) showed a cytotoxic effect. (B) HaCaT cells were pretreated

ither 1.5 or 3 h before extraction of total RNA. (C) Real-time PCR analysis for Blimp1

r 1 mM MC or 100 nM TPA. Cells were further incubated for 90 min before extraction

samples �SD.

T. Ikuta et al. / Journal of Dermatological Science 58 (2010) 211–216 215

We next investigated the signaling pathway leading toexpression of Blimp1 by adding several kinase inhibitors to culturemedium in the presence of MC. We found that pretreatment ofSZ95 with staurosporine 1 h before addition of MC inhibitedBlimp1 expression in a dose-dependent manner, but expression ofCYP1A1 was not inhibited (Fig. 4A). Pretreatment of HaCaT cellswith staurosporine also caused inhibition of UVB-induced Blimp1mRNA expression but not CYP1A1 (Fig. 4B). These data imply thatinduction of Blimp1 by AhR ligands requires kinase(s) activity.Since staurosporine is a potent inhibitor of protein kinase C (PKC),we next examined the effect of TPA, an activator of PKC, on Blimp1expression. TPA treatment of SZ95 cells slightly increased Blimp1mRNA (Fig. 3C and F) but not CYP1A1 (Fig. 3B and E); this effect ofTPA is abolished by pretreatment with staurosporine (Fig. 4C).Induction of Blimp1 mRNA by TPA treatment was found to beindependent of AhR and ARNT because transfection of siRNAspecific for either AhR or ARNT did not decrease Blimp1 mRNA(Fig. 3C and F). These data suggest that induction of Blimp1 mRNAby AhR ligands, regardless of whether they are exogenous orintracellularly generated, requires staurosporine-sensitive kinasethat can also be activated by TPA treatment.

It has been reported that various protein kinase activities areinvolved in the transcriptional regulation by AhR. Carrier et al.showed that CYP1A1 mRNA induction by TCDD requires PKC-dependent phosphorylation using Hepa-1 mouse hepatoma cellline [21]. In our studies, staurosporine, a PKC inhibitor did notaffect CYP1A1 induction, suggesting cell-type specificity. WhileChen et al. found AhR-dependent activation of MAPK p38 [22], thekinase does not appear to be associated with induction of Blimp1mRNA since SB203580, a specific inhibitor of MAPK p38, did notaffect the expression of Blimp1. Although several XRE motifs arelocated downstream of the human BLIMP1 transcription start site,these XRE were not active when connected to luciferase reportergene, implying that induction of Blimp1 mRNA by AhR isindependent of XRE. The possible signaling cascade is summarizedin Fig. 5.

As shown above, when human skin is exposed by AhR ligandsuch as B[a]P, DMBA, or TCDD as well as UVB, it is possible thatBlimp1 mRNA is induced in keratinocytes and sebocytes. AhR hasbeen shown to be necessary for B[a]P-induced murine skincarcinogenesis [23], and Yan et al. reported repression of p53transcription by Blimp1 [24]. It is thus plausible that induction ofBlimp1 by ligand-activated AhR suppresses p53 expression,leading to a hyperproliferative state.

Transcription of c-myc, which is a target gene of Blimp1transcriptional repressor, is an important regulator of sebocytedifferentiation [25]. Overexpression of c-myc in the epidermisleads to sebaceous hyperplasia [26] while inactivation of c-mycresults in hypoplasia of the sebaceous glands [27]. Furthermore,loss of Blimp1 results in the elevation of c-myc expression andhyperplasia of the sebaceous gland [16]. It is also reported that

Fig. 5. The possible signal cascade leading to Blimp1 induction by AhR and ARNT:

When epidermal keratinocytes or sebocytes are exposed to AhR ligands such as

chemical carcinogen as well as UVB, AhR/ARNT heterodimer binds to XRE, which is

an enhancer DNA element located in the 50-flanking region of the target genes such

as CYP1A1. On the other hand, AhR/ARNT heterodimer co-operates with

staurosporine-sensitive and TPA-activated kinase to induce Blimp1 mRNA.

topical application of TCDD or polycyclic aromatic hydrocarbonsinduce degeneration of the sebaceous gland [7]. So it is quiteinteresting to consider that the topical application of AhR ligandscauses hypoplasia of the sebaceous gland by induction of Blimp1that represses c-myc required for cell-fate determination ofsebocytes. Further studies are required for the characterizationof the specific nature of the Blimp1 stimulated by AhR in the skin.

Acknowledgement

This study was supported by the Grants-in-Aid for ScientificResearch from the Japanese Ministry of Education, Culture, Sports,Science and Technology.

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