Differential Regulation of Karyopherin Α 2 Expression by TGF-Β1 and IFN-Γ in Normal Human Epidermal Keratinocytes

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Differential Regulation of Karyopherin a 2 Expression by TGF-b1 and IFN-c in Normal Human Epidermal Keratinocytes: Evident Contribution of KPNA2 for Nuclear Translocation of IRF-1 Noriko Umegaki1,3, Katsuto Tamai2, Hajime Nakano1, Ryuta Moritsugu1, Takehiko Yamazaki2, Katsumi Hanada1, Ichiro Katayama3 and Yasufumi Kaneda2

Despite a number of studies on signal transduction in epidermal keratinocytes, very little is known about how signals move from the cytosole to the nucleus during the course of keratinocyte proliferation and differentiation. In this study, we first compared the expression patterns of the karyopherin a (KPNA) subtypes, and found that KPNA2, KPNA3, and KPNA4 were the major subtypes in both normal human epidermal keratinocytes (NHEKs) and normal human dermal fibroblasts (NHDFs). Stimulation with either transforming growth factor (TGF)-b1 or IFN-g for 24 hours resulted in the downregulation of KPNA2 expression specifically in NHEK at both the mRNA and protein levels. Interestingly, IFN-g, but not TGF-b1, specifically downregulated KPNA2 expression at the promoter level, suggesting differential regulation of KPNA2 expression by IFN-g and TGF-b1. We then demonstrated that KPNA2 physically bound to IFN regulatory factor-1 (IRF-1), a transcription factor induced by IFN-g, and induced nuclear translocation of IRF-1 in NHEKs. We finally performed microarray and quantitative real-time PCR analysis for the mRNA expression pattern of NHEK with either overexpression or knockdown of KPNA2, and indicated KPNA2 involvement for various epidermal gene regulations such as involucrin. Our data suggest that KPNA2 may play an important role in the signal-transduction pathways that regulate epidermal proliferation and differentiation. Journal of Investigative Dermatology (2007) 127, 1456–1464. doi:10.1038/sj.jid.5700716; published online 25 January 2007

INTRODUCTION karyopherin a (KPNA) family interact specifically with Recent reports have indicated that there are multiple pathways karyopherin b1 to form karyopherin a–b heterodimers, which for nucleocytoplasmic protein transport, which involve affect the receptors’ affinity for NLSs and docking at the specific transporters recognizing specific sequences in target nuclear pore complex. At least six KPNA isoforms have been proteins, such as nuclear localization signals (NLSs). The NLS reported to be ubiquitously expressed in human cells (Quensel protein–receptor complex is then transported through the et al., 2004): KPNA1/importin a5(Corteset al., 1994; O’Neil nuclear pore in an energy-dependent manner (Goldfarb et al., and Palese, 1995), KPNA2/importin a1 (Cuomo et al., 1994; 2004; Pemberton and Paschel, 2005). The importin/karyo- Weis et al., 1995; Guillemain et al., 2002), KPNA3/importin pherin superfamily consists of cytosolic receptors that recog- a4(Ko¨hler et al., 1997; Takeda et al., 1997; Nachury et al., nize NLSs and dock the NLS-containing proteins at the nuclear 1998), KPNA4/importin a3(Ko¨hler et al., 1997; Seki et al., pore complex on the nuclear membrane. Members of the 1997), KPNA5/importin a6(Ko¨hler et al., 1997), and KPNA6/ importin a7(Ko¨hler et al., 1997; Tsuji et al., 1997). The 1Department of Dermatology, Hirosaki University School of Medicine, different KPNA proteins show distinct NLS-binding specificities Hirosaki, Japan; 2Division of Gene Therapy Science, Osaka University (Nadler et al., 1997; Ko¨hler et al., 1999), and members of this 3 Graduate School of Medicine, Suita, Japan and Department of Dermatology, gene family exhibit selective expression in various tissues at Osaka University Graduate School of Medicine, Suita, Japan both the mRNA and protein levels (Takeda et al., 1997; Correspondence: Dr Katsuto Tamai, Division of Gene Therapy Science, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita-city, Nachury et al., 1998; Ko¨hler et al., 2002). There are, however, Osaka 565-0871, Japan. E-mail: [email protected] few reports concerning the expression and function of Abbreviations: ANOVA, analysis of variance; CHX, cycloheximide; particular KPNA isoforms in the skin. Moreover, whether IRF-1, IFN regulatory factor-1; KPNA, karyopherin a; NHDF, normal human there are specific roles for each KPNA isoform during signal dermal fibroblast; NHEK, normal human epidermal keratinocyte; transduction in various tissues, including the skin, has not yet NLS, nuclear localization signal; PBS, phosphate-buffered saline; RT, reverse transcription; siRNA, small interfering RNA been determined. Received 15 May 2006; revised 6 November 2006; accepted 15 November Epidermal keratinocytes regulate their proliferation and 2006; published online 25 January 2007 differentiation by transducing signals from outside the cell

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membrane to the nucleus through nuclear pores, thereby regulating the expression of epidermal proliferation- and NHEKHaCaTNHDF differentiation-specific genes. To maintain epidermal homeo- KPNA1 stasis and to respond to pathogenic processes, such as wounds or inflammatory conditions, entry of the signal mediators into the nucleus seems to be tightly regulated by KPNA2 KPNAs. This suggests that each KPNA may have a unique set of target proteins, although the precise mechanism has not KPNA3 been identified. Here we report the first examination of the expression patterns of KPNAs in normal human epidermal keratinocytes KPNA4 (NHEKs) and normal human dermal fibroblasts (NHDFs). In this study, we determined that KPNA2 expression in NHEKs, KPNA5 but not in NHDFs, is differentially regulated by trans- GAPDH forming growth factor (TGF)-b1 and IFN-g, both of which are established modulators of epidermal proliferation and differentiation. We also provide evidence that KPNA2 Figure 1. Expression patterns of KPNA mRNAs in NHEK, HaCaT, and NHDF may mediate the translocation of epidermal differentia- cells. mRNA extracted from sub-confluent NHEK, HaCaT, and NHDF cells tion-inducing signals into the nucleus, at least in part, by was subjected to Northern blot analysis for the human KPNA1, KPNA2, KPNA3, KPNA4, KPNA5, and GAPDH genes. Exposure time: KPNA2, recruiting transcription factors such as IFN regulatory KPNA3, and KPNA4 for 24 hours; KPNA1 and KPNA5 for 7 days. Data are factor-1 (IRF-1), a mediator of IFN-g-induced epidermal representative of two independent experiments. differentiation (Odanagi et al., 2004). The data shown here provide novel insights into the signal transduction in epidermal keratinocytes mediated by the nuclear trans- a NHEK 0 13612 24 48 (hours) NHEK porter KPNA2. KPNA2 120 NHDF 100 GAPDH 80 RESULTS NHDF 60 Expression patterns of KPNA mRNAs in NHEK, HaCaT, KPNA2 40 ** ** and NHDF cells 20

GAPDH KPNA2-mRNA (%) 0 The expressions of KPNA1, KPNA2, KPNA3, and KPNA4 0 1 3 6 122448 mRNAs were detected in NHEK, HaCaT, and NHDF cells by (hours) Northern blot analysis. KPNA5 mRNA, however, did not b NHEK 01510(ng/ml) produce sufficiently strong signals using this method, and NHEK KPNA2 120 NHDF was detected only by reverse transcription (RT)-PCR. In 100 * GAPDH 80 ** all three cell types, KPNA2, KPNA3, and KPNA4 seemed ** to be strongly expressed, whereas KPNA1 and KPNA5 60 NHDF 40 were expressed with relatively low signals (Figure 1). After KPNA2 20 correction with the glyceraldehyde-3-phosphate dehydrogen- KPNA2-mRNA (%) 0 GAPDH 0 1510 ase (GAPDH) expression level, relative mRNA expression (ng/ml) levels of KPNA1 and KPNA2 in NHEK cells were stronger c NHEK 150 than in NHDF cells. TGF- 1 – – ++ CHK – + –+ 100 Downregulation of KPNA2 mRNA expression by TGF-b1 ** KPNA2 50 in NHEK cells 0 TGF-b1 treatment (5 ng/ml) of NHEK cells resulted in the KPNA2-mRNA (%) GAPDH + selective and time-dependent downregulation of KPNA2 TGF- 1 ––+ CHX – + – + mRNA expression; a nearly 70% inhibition was observed Figure 2. Specific regulation of KPNA2 mRNA expression by TGF-b in after 24 hours (Po0.001) (Figure 2a). Moreover, this TGF-b1- 1 mediated inhibition was found to be dose dependent; an NHEK cells. (a) Time-course analysis of KPNA2 mRNA expression regulated b approximately 50% inhibition was observed after 24 hours by TGF- 1 in NHEK and NHDF cells. (b) Dose analysis of KPNA2 mRNA expression was obtained after 24-hour incubations with TGF-b1 in NHEK and with a dose of 5 ng/ml TGF-b1 (Po0.001). At a dose of 10 ng/ NHDF cells. (c) Downregulation of KPNA2 mRNA expression by TGF-b1 ml TGF-b1, however, the KPNA2 mRNA level was not (5 ng/ml) was antagonized by CHX treatment (10 mg/ml), which started 1 hour 7 significantly different from that observed at a dose of 5 ng/ml. before TGF-b1 administration. Data are expressed as means SD from three On the contrary, there were no remarkable changes in the independent experiments (*Po0.05 and **Po0.01 compared with the initial KPNA2 mRNA levels in NHDF cells (Figure 2a and b). The expression level by one-way ANOVA). addition of cycloheximide (CHX) to the culture medium antagonized the effects of TGF-b1 on KPNA2 mRNA expression in NHEK cells (Figure 2c). For the other members

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of the KPNA family, no significant changes in mRNA and b). CHX treatment antagonized both IFN-g-dependent expression levels were observed following TGF-b1 stimula- upregulation and downregulation of KPNA2 mRNA expres- tion (data not shown). sion in NHEKs (Figure 3c). No significant change in the expression levels of other KPNA family members was Dual modulation of KPNA2 mRNA expression by observed after IFN-g stimulation (data not shown). IFN-c in NHEKs Stimulation of NHEKs with 100 U/ml IFN-g specifically Time-dependent regulation of KPNA2 protein expression by upregulated KPNA2 mRNA expression within 12 hours TGF-b1 and IFN-c (Figure 3a). The maximal expression level, which was TGF-b1 stimulation (5 ng/ml) of NHEKs resulted in the down- approximately twice the initial expression level, was ob- regulation of KPNA2 protein expression after 24 hours served after 6-hour incubation (Po0.001). The expression (Po0.001). Stimulation of NHEKs with 100 U/ml IFN-g initially level then gradually declined to about 40% of the initial upregulated KPNA2 protein expression after 6 hours (Po0.01), expression level (Po0.05) after 48 hours, suggesting before the expression level gradually declined and was dual regulation of KPNA2 by IFN-g. Stimulation of cells completely suppressed after 48 hours (Po0.001), suggesting with various concentrations of IFN-g for 48 hours resulted in dual regulation of KPNA2 protein expression by IFN-g (Figure 4). the selective downregulation of KPNA2 mRNA expression; This KPNA2 protein regulation by IFN-g and TGF-b corresponds the smallest effective dose of IFN-g was 10 U/ml (Po0.001). to the results obtained in the Northern blot analysis. There were no remarkable changes of KPNA2 mRNA expression in NHDF cells following IFN-g treatment (Figure 3a Suppression of KPNA2 promoter activity by IFN-c, not by TGF-b1 To confirm the direct effects of TGF-b1 and IFN-g for KPNA2 NHEK 0136122448(hours) a NHEK *** *** transcription, NHEKs were transiently transfected with 200 ** ** NHDF KPNA2 KPNA2 promoter-luc plasmid pKPNA2-luc followed by 150 GAPDH TGF-b and IFN-g inoculation in the culture medium, and 100 NHDF luciferase assays were performed. KPNA2 promoter activity * KPNA2 50 was significantly suppressed by IFN-g (100 U/ml) after 12 and KPNA2-MRNA (%) 0 24 hours (P 0.001), but no significant promoter regulation GAPDH 0 1 3 6 12 24 48 o (hours) was observed in the early time point of IFN-g treatment or by TGF-b stimulations (Figure 5). b NHEK 0 10 100 1,000 (U/ml) KPNA2 NHEK 120 NHDF 100 Nuclear transport of IRF-1 with KPNA2 GAPDH 80 The induction of KPNA2 expression by IFN-g raised the *** 60 *** NHDF *** possibility that KPNA2 functions to transport IFN-g-inducing 40 KPNA2 20 transcription factors such as IRF-1, which is known to KPNA2-MRNA (%) 0 be upregulated by IFN-g in NHEK cells, into the nucleus. GAPDH 100 100 1,000 (U/ml) To test for an interaction between IRF-1 and KPNA2, we co-transfected COS-7 cells with expression vectors coding for c NHEK IFN- (6 hours) ––+ + myc-tagged IRF-1 and FLAG-tagged KPNA2, and performed 200 CHX – + – + *** immunoprecipitation assays using anti-FLAG M2 agarose. 150 Immunoblotting analysis with both anti-myc antibodies 100 50 1.5 TGF- 1 0 12 24 48 (hours) KPNA2-mRNA (%) 0 IFN- (6 hours) – – + + 1.0 CHX – + – + KPNA2 ** IFN- (24 hours) – + + 0.5 – CHX – + 120 -Actin ** 100 0 80 TGF- 0122448(hours) 60 1 40 ** 20 ** IFN- 0 6 12 24 48 (hours) 2.5 ** 0 KPNA2-mRNA (%) 2.0 IFN- (24 hours) – ++ KPNA2 * 1.5 CHX –– + 1.0 -Actin Figure 3. Dual regulation of KPNA2 mRNA expression by IFN-c in NHEKs. 0.5 ** (a) Time-course analysis of KPNA2 mRNA expression regulated by IFN-g in 0 IFN- 0 6 12 24 48 (hours) NHEK and NHDF cells. (b) Dose-dependent downregulation of KPNA2

mRNA expression in NHEK cells was evident after 24-hour incubations with Figure 4. Regulation of KPNA2 protein expression by TGF-b1 and IFN-c.

IFN-g.(c) CHX antagonized the effects of IFN-g on the regulation of KPNA2 NHEK cells were treated with either 5 ng/ml TGF-b1 or 100 U/ml IFN-g, and mRNA expression after both 6 and 24 hours. Data are expressed as subjected to Western blot analysis for KPNA2 expression. Data are expressed means7SD from three independent experiments (**Po0.01 and ***Po0.001 as means7SD from three independent experiments (*Po0.05 and **Po0.01 compared with the initial expression level by one-way ANOVA). compared with the initial expression level by one-way ANOVA).

1458 Journal of Investigative Dermatology (2007), Volume 127 N Umegaki et al. KPNA2 for Nuclear Translocation of IRF-1

** a Immunoprecipitation Input 100 150 yc G G yc G G F1-m LA IR A2-FLAA2-FLA F1-m2-F 2-FLA PN N IR NA NA 100 K KP NC P KP NC 50 K 100 50 75 IRF-1-myc 0 0 a:Anti-myc IFN- ––++TGF- ––++ 50 12 hours 24 hours 12 hours 24 hours 100 Figure 5. Regulation of KPNA2 promoter activity by IFN-c. NHEK cells were transfected with KPNA2 promoter expression vector and treated with 5 ng/ml 75 KPNA2-FLAG b:Anti-FLAG TGF-b1 or 100 U/ml IFN-g for 12 and 24 hours, and subjected to luciferase 50 assay. Data are expressed as means7SD (*Po0.001 compared with the IFN-g or TGF-b1 absent expression level by one-way ANOVA). b Immunoprecipitation Input yc yc 2-m G 2-m G NA A KP KPN F1-FLA F1-FLAG F1-FLA F1-FLAG indicated that IRF-1 precipitated with anti-FLAG M2 agarose, IR IR NC IR IR NC demonstrating an association between IRF-1 and KPNA2 100

(Figure 6a). Moreover, COS-7 cells co-transfected with the 75 KPNA2-myc myc-tagged KPNA2 and FLAG-tagged IRF-1 expression a:Anti-myc vectors also revealed co-precipitation of IRF-1 with KPNA2 50 (Figure 6b). Interaction of endogenous KPNA2 and IRF-1 in 100

NHEK was then detected by immunoprecipitation using 75 anti-IRF-1 antibody and Western blotting using anti- b:Anti-FLAG IRF1-myc KPNA2 antibody (Figure 6c). These data suggest that KPNA2 50 might act to import IRF-1 into the nucleus in response to IFN-g stimulation. c F-1 To investigate the roles of KPNA2 in IRF-1 localization, we Anti-IR NC stimulated NHEK cells with 100 U/ml IFN-g with or without 75

KPNA2 knockdown with KPNA2-directed small interfering KPNA2 RNA (siRNA). IRF-1, which was primarily observed in the 50 cytoplasm of NHEK cells without stimulation, translocated to the nucleus after IFN-g stimulation (Figure 7). After KPNA2 Figure 6. Evidence for KPNA2 association with IRF-1. (a) FLAG-tagged knockdown, however, IRF-1 was retained in the cytoplasm KPNA2 (KPNA-FLAG) and myc-tagged IRF-1 (IRF-1-myc) expressed in even after IFN-g stimulation, and some seemed to be COS-7 cells were co-precipitated with anti-FLAG M2-agarose. IRF-1-myc colocalized with KPNA2 (Figure 7), suggesting that IRF-1 was detected with both anti-myc and anti-IRF antibodies in the precipitants. localization into the nucleus following IFN-g treatment may (b) The association of IRF-1-FLAG with KPNA2-myc was also demonstrated be mediated by KPNA2. by immunoprecipitation with anti-FLAG M2-agarose, followed by immunoblotting with anti-myc antibodies. (c) Endogenous association of IRF-1 and KPNA2 in NHEK. NC: negative control. Possible roles of KPNA2 in keratinocyte differentiation To investigate the effects of KPNA2 on NHEK gene expression, we performed a single experiment of microarray of GAPDH gene expression in the same mRNA samples used analysis in conditions with either a high or a low KPNA2 in this study (data not shown). These data indicated that the expression level. One hundred and fifty-one genes were function of KPNA2 is, at least in part, to recruit transcriptional upregulated when KPNA2 was overexpressed in NHEK cells, regulators for keratinocyte differentiation to the nucleus. whereas 608 genes were downregulated as a result of KPNA2 knockdown. Fifty-four genes were modulated under both DISCUSSION conditions and therefore seemed to be putative genes In this paper, we demonstrated that among the KPNA specifically regulated by KPNA2 expression (Table 1). Data subunits in NHEK cells, the expression of KPNA2 is mining with the 54 genes was performed using keratinocyte specifically regulated by TGF-b1 and IFN-g. TGF-b1, which differentiation, a structural component of the epidermis, and is naturally expressed in the suprabasal cells of the epidermis, epidermal development as key terms. The database mining induces reversible growth arrest at the G1 phase of the extracted 10 genes, including involucrin, keratin 1, keratin cell cycle (Shipley et al., 1986; Alexandrow et al., 1995), 10, proline-rich protein 1A (SPRR1A), and small proline-rich but does not contribute to terminal differentiation (George protein 1B (SPRR1B) (Figure 8a). To confirm the data of et al., 1990; Arany et al., 1996). In contrast, IFN-g, which the microarray analysis, real-time RT-PCR was performed for is primarily produced and secreted by Langerhans cells involucrin mRNA expression in NHEKs. Involucrin mRNA in normal epidermis, induces irreversible growth arrest in was significantly upregulated in the condition of high KPNA2 cultured keratinocytes (Saunders and Jetten, 1994) and expression level and downregulated in that of KPNA2 promotes aberrant terminal differentiation (Brysk et al., knockdown (Figure 8b). We also confirmed no modulation 1991; Tamai et al., 1995).

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IRF-1 KPNA2 DAPI Marge KPNA2 promoter assay. These results may be explained by the secondary effect caused by induced changes in growth IFN (–) arrest and keratinocyte differentiation, or this 1 kb promoter scramble region may not contain a cis-element responsible for the modulation. CHX antagonized the effects of both TGF-b1 and IFN-g on KPNA2 mRNA expression, indicating that protein synthesis IFN (–) was required for KPNA2 regulation by these cytokines. There si KPNA2 is evidence that, following IFN-g activation, STAT-1 enters the nucleus via a mechanism that is KPNA1/NPI-1 dependent and KPNA2/Rch1 independent (Sekimoto et al., 1997). This IFN (+) STAT1/KPNA1 complex may regulate the protein synthesis scramble required for the modulation of KPNA2 by IFN-g. In this study, we also demonstrated for the first time that KPNA2 physically interacts with IRF-1, which is specifically induced in NHEK cells by IFN-g stimulation, IFN (+) and regulates the nuclear entry of IRF-1. Whereas IRF-1 si KPNA2 mRNA expression remains high after treatment of keratinocytes with IFN-g after 48 hours (Odanagi et al., 2004), KPNA2 expression was suppressed at this time point, suggesting Figure 7. KPNA2-mediated nuclear import of IRF-1 after IFN-c stimulation. To detect the localization of IRF-1 in NHEK cells, immunohistochemistry was that KPNA2 may play a pivotal role, if not exclusive, for the performed. Without IFN-g stimulation, IRF-1 was mainly located in the fine-tuning of IFN-g/IRF-1-dependent signal transduc- cytoplasm (IFN-g () scramble and IFN-g () si KPNA2), whereas IRF-1 tion during epidermal differentiation. A single microarray accumulated in the nucleus after IFN-g stimulation (100 U/ml) for 12 hours experiment with mRNA from KPNA2-overexpressed/ (IFN ( þ ) scramble). IRF-1 in NHEK cells pretreated with siRNA, however, downregulated NHEKs extracted a putative list of 10 genes remained in the cytoplasm. Bar ¼ 50 mm. relating to keratinocyte differentiation, suggesting again that KPNA2 may function for the nuclear entry of signals for keratinocyte differentiation. Repeated experiments, however, Previous studies have revealed that transcription factors must be performed to provide a final list of the genes such as Myc, NF-kB (Nadler et al., 1997), and Epstein-Barr regulated by KPNA2. virus nuclear antigen (EBNA) (Fischer et al., 1997) are target Although KPNA2 overexpression resulted in induction of proteins of KPNA2. The expression and nuclear localization several keratinocyte differentiation-marker genes, we could of c-myc, an essential transcription factor required to not observe apparent morphological changes as seen in maintain cell growth in the basal layer of the epidermis differentiated keratinocytes. Taken together, KPNA2 seems to (Pietenpol et al., 1990; Alexandrow et al., 1995), are rapidly be involved in keratinocyte differentiation, but may not be downregulated when keratinocytes are treated with TGF-b1 sufficient for the full-differentiation induction by itself. (Coffey et al., 1988), resulting in growth arrest in the G1 Aberrant expression of KPNA2 induced by IFN-g in the phase of the cell cycle (Pietenpol et al., 1990). Therefore, we epidermis may be involved in disorders such as psoriasis, in speculate that the inhibition of proliferation observed in which both proliferation and differentiation of epidermal NHEK cells following TGF-b1 and IFN-g treatment may be a keratinocytes are disorganized. Further elucidation of the result of the downregulation of KPNA2 and impaired nuclear functions of KPNAs in epidermal keratinocytes during import of c-myc. physiological and pathological conditions may provide Whereas TGF-b1 caused only the downregulation of clues for developing novel therapeutic approaches to skin KPNA2 expression, IFN-g treatment induced KPNA2 expres- diseases. sion within an initial 12 hours followed by downregulation after 24 hours at both mRNA and protein levels. A previous MATERIALS AND METHODS study showed that transglutaminase and cornifin, both of Reagents which are marker genes of keratinocyte differentiation, were Recombinant human TGF-b1 and recombinant human IFN-g were upregulated by IFN-g in 12–36hours, indicating the promo- purchased from Roche (Mannheim, Germany). Anti-human KPNA2 tion of keratinocyte terminal differentiation (Saunders and goat polyclonal antiserum, anti-human IRF-1 rabbit polyclonal Jetten, 1994). The parallel induction of KPNA2 and kerati- antiserum, and horseradish peroxidase-conjugated donkey anti-goat nocyte differentiation marker genes by IFN-g suggest that IgG were purchased from Santa Cruz Biotechnology (Santa Cruz, increased nuclear entry of KPNA2-mediated signals may be CA). Anti-myc-tag monoclonal mouse antibodies and anti-FLAG involved in the keratinocyte differentiation. mouse antibodies were obtained from MBL (Nagoya, Japan) and IFN-g directly inhibited KPNA2 promoter activity followed Sigma-Aldrich (St Louis, MO), respectively. Finally, horseradish by suppression of KPNA2 expression at both mRNA and peroxidase-conjugated donkey anti-rabbit IgG and horseradish protein levels. However, early upregulation by IFN-g and peroxidase-conjugated sheep anti-mouse IgG were purchased from downregulation by TGF-b1 were not observed by the Amersham (Arlington Heights, IL).

1460 Journal of Investigative Dermatology (2007), Volume 127 N Umegaki et al. KPNA2 for Nuclear Translocation of IRF-1

Table 1. Lists of genes upregulated in response to Table 1. continued KPNA2 overexpression and downregulated by siRNA- Gene name Systematic name mediated KPNA2 knockdown MGC13102 NM_032323 Gene name Systematic name IVL NM_005547 CDSN L20815 LOC344332 XM_293018 KLK7 NM_005046 FLJ21511 NM_025087 DEFB1 NM_005218 A_24_P821693 A_24_P821693 GJB6 NM_006783 UNQ467 NM_207392 BF337308 BF337308 SPRR2C M21539 ZD52F10 NM_033317 LOC144501 NM_182507 KPNA2 NM_002266 KLK5 NM_012427 KRT23 NM_173213 IL1F5 NM_012275 TUBB2 NM_001069 PGLYRP4 NM_020393 FABP5 NM_001444 KPNA2, karyopherin a2; siRNA, small interfering RNA. SERPINB3 NM_006919 Fifty-four genes were identified by microarray analysis. SPINK5 NM_006846 HOP NM_139211 AF113013 AF113013 a FABP5 NM_001444 KRT1 NM_006121 Downregulated genes SLPI NM_003064 Common genes by siKPNA2 regulated by KPNA2 and A_23_P247 A_23_P247 Upregulated genes 608 genes siKPNA2 by KPNA2 SPRR1A NM_005987 54 genes (Table 1) 151 genes KLK11 NM_144947 SERPINB4 NM_002974 GLTP NM_016433 TMEM45A NM_018004 Keratinocyte differentiation, SERPINB7 NM_003784 structural constitute of epidermis and epidermis development RDH12 NM_152443 10 genes KLK7 NM_005046 Kallikrein 7 THC2172764 THC2172764 FABP5 NM_001444 Fatty acid binding protein in 5 (psoriasis-associated) (FABP5) KRTI NM_006121 Keratin 1 ANKRD35 NM_144698 SPRR1A NM_005987 Proline-rich protein 1A (SPRR1A) SPRR1B NM_003125 Small proline-rich protein 1B (cornifin) (SPRR1B) ANKRD22 NM_144590 KRT16 NM_005557 Keratin 16 KRT10 NM_000421 Keratin 10 KLK5 NM_012427 Kallikerin 5 C1orf42 NM_019060 PKP1 NM_000299 Plakophilin 1 IVL NM_005547 Involucrin LOC346910 XM_294456 DSC1 NM_004948 2 10 b * * SPRR1B NM_003125 1 5 CENTB5 NM_030649 0 0 Control KPNA2 up Control KPNA2 up RHCG NM_016321 3 1 2 * * 0.5 KLK10 NM_002776 1 0 0 ENST00000238875 ENST00000238875 Control siKPNA2 Control siKPNA2 A_24_P290087 A_24_P290087 Involucrin KPNA2 KRT16 NM_005557 Figure 8. Keratinocyte differentiation is modulated by KPNA2 expression. (a) To evaluate the effects of the KPNA2 expression level on NHEK gene SLPI NM_003064 expression, microarray analysis was performed. Ten of these genes, which are KRT10 NM_000421 listed in the figure, were extracted by data mining in the database of identified genes using keratinocyte differentiation, structural component of the SLC6A14 NM_007231 epidermis, and epidermis development as key terms. (b) Expression analysis of SPRR2B NM_006945 involucrin and keratin 5 by quantitative real-time PCR in the condition of high 7 S100A9 NM_002965 or low KPNA2 expression levels. Data are expressed as means SD from three independent experiments (*Po0.05).

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Cell culture primary antibodies for at least 1 hour at room temperature, washed NHEKs obtained from neonatal foreskin were purchased from five times (5 minutes each) with Tris-buffered saline containing Clonetics (San Diego, CA) and Cascade Biologics (Portland, OR). 0.05% Tween 20, and incubated with horseradish peroxidase- The cells were propagated in keratinocyte growth medium conjugated secondary antibodies for 1 hour at room temperature. (Clonetics) containing recombinant human EGF (0.1 mg/ml), insulin After washing, the membranes were developed with an enhanced (0.5 mg/ml), hydrocortisone (0.5 mg/ml), gentamicin (50 mg/ml), am- chemiluminescence system (ECL-plus; Amersham). Normalized photericin B (50 ng/ml), and bovine pituitary extract (0.1 mg/ml), or in densitometric values for the individual lanes in three independent standard medium containing 0.06 mmol/ml Ca2 þ (Epilife-KGM; experiments were averaged; data are represented as the means7SD. Cascade Biologics) supplemented with human EGF (0.1 mg/ml), insulin (10 mg/ml), hydrocortisone (0.5 mg/ml), gentamicin (50 mg/ml), Construction of KPNA2 promoter-luciferase plasmid amphotericin B (50 ng/ml), and 4% bovine pituitary extract at 371C PCR primers for amplification of KPNA2 promoter regions were 0 under 5% CO2–95% air. Cells were passed by trypsinization and designed to obtain 5 flanking DNA at positions from 1to1,000. 50–60% confluents of the keratinocytes were used for each PCR-amplified fragments were then inserted into the KpnI-HindIII experiment at the third or fourth passage. site of the pGL3-basic vector (Promega, San Luis, CA) to generate HaCaT, an immortalized, non-tumorigenic keratinocyte cell line pKPN2-luc plasmid. The sequences inserted into pGL3-basic vector (kindly provided by Dr Husenig), NHDF, and COS-7 cells were were confirmed by direct sequencing. cultured in DMEM (Nakarai, Tokyo, Japan) containing 10% fetal

bovine serum at 371C under 5% CO2–95% air. NHDFs at the fifth Transient transfection and luciferase assay passage were used for the experiments. Before cytokine stimulation, NHEK cells placed into 12-well plates were transfected with 0.43 mg sub-confluent NHDF cells were incubated for 24 hours in serum-free of pKPN2-luc plasmid and 0.17 mg of pRL-TK vector using TransIT-

medium. Various doses of TGF-b1 or IFN-g were then added to the LT1 (Mirus, Bienne, Bern, Switzerland). At the time of the

medium and the cells were incubated as indicated. The study was transfection, 5 ng/ml of TGF-b1 or 100 U/ml of IFN-g were added conducted according to the Declaration of Helsinki Principles. into the medium and harvested by 100 ml of passive cell lysis buffer (Promega) after each time course. The dual luciferase reporter assay RNA isolation and Northern blot analysis was performed by a Dual-Glo luciferase Assay kit (Promega). pRL-TK Total RNA was extracted from cells using an RNA isolation kit vector was used as internal control, and data were normalized by (Qiagen, Hilden, Germany) according to the manufacturer’s Renilla luciferase luminescence intensity. The experiments were instructions. mRNA (15–20 mg) from each sample were electrophore- performed in triplicate, and the differences were statistically sed on 1% formaldehyde/agarose gels, transferred to nylon examined using one-way ANOVA. membranes (Zeta-Probe blotting membrane, Bio-Rad, Hercules, CA), and immobilized by shortwave UV irradiation (UV-Stratalinker Construction of KPNA2 and IRF-1 expression vectors, and 1800, Stratagene, La Jolla, CA). All KPNA probes were generated by siRNA-mediated knockdown of KPNA2 RT-PCR, sequenced, and labeled with 32P using a random prime To create the pCY4B-KPNA2, pCAGIp-FLAG-KPNA2, pCAGIp-myc- labeling kit (Stratagene). Blots were hybridized with 32P-labeled KPNA2, pCAGIp-FLAG-IRF-1, and pCAGIp-myc-IRF-1 expression cDNA probes for KPNA1, KPNA2, KPNA3, KPNA4, and KPNA5 vectors, full-length human KPNA2 and IRF-1 cDNAs were generated overnight at 431C in 50% deionized formamide and re-hybridized by RT-PCR with PfuUltra High-Fidelity DNA polymerase with GAPDH. The filters were washed in 2 Â standard sodium (Stratagene). The PCR products were sequenced and their citrate and 0.1% SDS at room temperature and exposed to film at sizes were verified by agarose gel electrophoresis. The products 801C for approximately 24 hours for KPNA2, KPNA3, and KPNA4 were then cut from the gel, purified using a QIAquick gel extrac- or for 1 week for KPNA1 and KPNA5. Hybridization signals were tion kit (Qiagen), and cloned into the pCR-Blunt-TOPO vector quantified with a densitometer. The differences were statistically (Invitrogen, Carlsbad, CA). To complete the recombination sites, an examined using one-way analysis of variance (ANOVA). adapter PCR was performed as described in the Gateway manual (Invitrogen). The purified PCR products were also cloned Western blot analysis into the pENTER/SD/D-TOPO vector (Invitrogen), and cDNA Cells were washed twice with cold phosphate-buffered saline (PBS) plasmids were converted into other expression vectors using the and then lysed in radioimmunoprecipitation assay buffer (20 mM Gateway LR reaction (Invitrogen). The medical ethical committee of Tris-HCl, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, and 0.5 M Osaka University Graduate School of Medicine approved all EDTA at pH 7.4), supplemented with protease inhibitor cocktail described studies. (complete mini, Roche). After ultrasonication and removal of siRNAs specific for KPNA2 and its control stealth siRNA were insoluble cell debris, the protein concentration in the supernatant designed and obtained from Invitrogen. We performed KPNA2 was determined with Bio-Rad Protein Assay Dye Reagent Concen- knockdown studies with siRNA, which ensured more than 50% trate (Bio-Rad). For Western analysis, 100 mg of the proteins were suppression of KPNA2 mRNA and protein. electrophoresed on a 10% SDS polyacrylamide gel. After electrophoresis, the proteins were transferred to nitrocellulose membranes Immunoprecipitation analysis (Hybond-P, Amersham) in transfer buffer containing 100 mM Tris- COS-7 cells transiently transfected using FuGene 6 (Roche) with HCl, 192 mM glycine, and 20% methanol. Residual binding sites on pCAGIp-FLAG-KPNA2 and pCAGIp-myc-IRF-1, or pCAGIp-FLAG- the membranes were blocked with 2% skim milk in Tris-buffered IRF-1 and pCAGIp-myc-KPNA2 were lysed in ice-cold EBL buffer 0 saline overnight at room temperature. The blots were incubated with (150 mM NaCl, 50 mM N-2-hydroxyethylpiperazine-N -2-ethanesul-

1462 Journal of Investigative Dermatology (2007), Volume 127 N Umegaki et al. KPNA2 for Nuclear Translocation of IRF-1

fonic acid, 5 mM EDTA, 0.1% Nonidet P-40 at pH 7.5 supplemented Quantitative real-time PCR with protease inhibitors) for 15 minutes. After precleaning The NHEK cells that were used for microarray analysis were also with mouse IgG agarose (Santa Cruz Biotechnology), the examined by quantitative real-time PCR. After RNA isolation, RT lysates were immunoprecipitated with anti-FLAG M2-agarose reactions were performed using SuperScript III (Invitrogen). All (Sigma) and extensively washed. Immunoprecipitates were analyzed TaqMan probes (KPNA2: Hs 00818252g1; GAPDH: by Western blotting with anti-myc, anti-IRF-1, and anti-KPNA2 Hs99999905m1; involucrin: Hs00846307s1) were ordered from antibodies. ‘‘Gene Expression Assays’’ (Applied Biosystems Inc., Foster City, NHEK cells cultured on 100 mm dishes were lysed in PBS CA). GAPDH was used as an internal control for all PCR containing 0.5% Triton X and 5 mM EDTA with protease inhibitor amplification reactions. Relative mRNA expression was quantified DDCt cocktail and phosphatase inhibitor (Sigma). Cell extracts were using the comparative Ct (DCt) method and expressed as 2 . incubated overnight with 2 mg of anti-IRF-1 antibodies or in the Each assay was carried out in triplicate and expressed as mean7SD. absence of antibodies at 41C. The immunoprecipitates were collected on protein A–Sepharose beads (Amersham) for 4 hours at CONFLICT OF INTEREST 41C. After washing five times with lysis buffer, proteins bound to The authors state no conflict of interest. Sepharose beads were eluted by boiling in sample buffer. Western blotting was performed with anti-KPNA2 antibodies. ACKNOWLEDGMENTS This work was supported by a Grant-in-Aid of Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and Immunohistochemistry by Health and Labor Sciences research grants from the Ministry of Health and NHEK cells were grown on tissue-culture-treated slides (Falcon, Welfare of Japan. Becton Dickinson, Franklin Lakes, NJ) and transfected with or without KPNA2 siRNAs 24 hours before stimulation with 100 U/ml IFN-g. After about 12 hours, cells were fixed with 4% paraformalde- REFERENCES hyde at room temperature for 1 hour. Cells were permeabilized using Alexandrow MG, Kawabata M, Aakre M, Moses HL (1995) Overexpression of 0.1% Triton X-100 in PBS for 3 minutes. Slides were washed in PBS, the c-Myc oncoprotein blocks the growth-inhibitory response but is required for the mitogenic effects of transforming growth factor b1. 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