Toxicology and Applied Pharmacology 253 (2011) 235–243

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Toxicology and Applied Pharmacology

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Nickel-induced down-regulation of ΔNp63 and its role in the proliferationof keratinocytes

Zhuo Zhang b,⁎, Wenqi Li b, Senping Cheng a, Hua Yao c, Fan Zhang a, Qingshan Chang a, Zunji Ke d,Xin Wang a, Young-Ok Son a, Jia Luo d, Xianglin Shi a

a Graduate Center for Toxicology, University of Kentucky, 1095 VA Drive, Lexington, KY 40536, USAb Department of Preventive Medicine and Environmental Health, University of Kentucky, 121 Washington Avenue, Lexington, KY 40536, USAc Department of Stomatology, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, Zhejiang 310003, Chinad Department of Internal Medicine, University of Kentucky, 800 Rose Street, Lexington, KY 40536, USA

⁎ Corresponding author. Fax: +1 859 323 1059.E-mail address: zhuo.zhang@uky.edu (Z. Zhang).

0041-008X/$ – see front matter © 2011 Elsevier Inc. Aldoi:10.1016/j.taap.2011.03.024

a b s t r a c t

a r t i c l e i n f o

Article history:Received 16 February 2011Revised 23 March 2011Accepted 25 March 2011Available online 3 April 2011

Keywords:Nickel chlorideΔNp63NF-κBInterferon regulatory factor (IRF)p21Cell proliferationKeratinocytes

Epidemiological, animal, and cell studies have demonstrated that nickel compounds are human carcinogens.The mechanisms of their carcinogenic actions remain to be investigated. p63, a close homologue of the p53tumor suppressor protein, has been linked to cell fate determination and/or maintenance of self-renewingpopulations in several epithelial tissues, including skin, mammary gland, and prostate. ΔNp63, a dominantnegative isoform of p63, is amplified in a variety of epithelial tumors including squamous cell carcinomas andcarcinomas of the prostate and mammary glands. The present study shows that nickel suppressed ΔNp63expression in a short-time treatment (up to 48 h). Nickel treatment caused activation of NF-κB. Blockage ofNF-κB partially reversed nickel-induced ΔNp63 suppression. Nickel decreased interferon regulatory factor(IRF) 3 and IRF7, IKKε, and Sp100. Over-expression of IRF3 increased ΔNp63 expression suppressed by nickel.Nickel was able to activate p21, and its activationwas offset by the over-expression ofΔNp63. In turn, elevatedp63 expression counteracted the ability of nickel to restrict cell growth. The present study demonstrated thatnickel decreased interferon regulatory proteins IRF3 and IRF7, and activated NF-κB, resulting in ΔNp63suppression and then p21 up-regulation. ΔNp63 plays an important role in nickel-induced cell proliferation.

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Introduction

Human exposure to nickel occurs predominantly in mining,refining, alloy production, electroplating, and welding. Exposure ofworkers to nickel compounds can produce a variety of adverse effectson human health, such as nickel allergy in the form of contactdermatitis, lung fibrosis, cardiovascular and kidney diseases, andcancer of the respiratory tract (Uddin et al., 2007). Epidemiologicalstudies demonstrate increased mortality from cancers of the lung andnasal cavities in nickel-containing dusts and fumes (Roberts et al.,1989). Nickel-induced human carcinogenesis may be due to itsmultiple mechanisms, including both genetic and epigenetic routes(Salnikow and Costa, 2000; Clevers, 2004; Zhao et al., 2004).

For many years, p53 has been considered the prototypical tumorsuppressor and remains the subject of intensive research. p53responds to cellular stress such as DNA damage and hypoxia andplays important roles in regulating cell cycle, cell differentiation,genomic stability, and apoptosis (Levine, 1997; Oren, 1999). p63, amember of p53 family, shares extensive homology to p53 and

produces multiple transcripts with varying functions (Osada et al.,1998; Schmale and Bamberger, 1997; Trink et al., 1998; Yang et al.,1998). The p63 family includes two major classes of proteins, TA63,which contains the N-terminal transactivation (TA) domain, andN-terminal truncated (ΔN) p63, which lacks the transactivationdomain (Yang et al., 2002). Alternative splicing at the 3′ end of thetranscripts generates three different C-termini: α, β and γ (Candiet al., 2007). p63 exhibits a rather tissue-specific distribution in thatits expression is higher in the basal layer of stratified epithelia,including the epidermis (Yang et al., 1998). The most highlyexpressed p63 isoform in the epidermis is ΔNp63α (Yang et al.,1998). This isoform lacks 5′ region that exhibits extensive homologyto the transactivation domain of p53. ΔNp63α inhibits p53transcriptional activity. Deficiency of ΔNp63α resulted in severelimb, craniofacial, and epithelial defects, leading to death shortlyafter birth (Mills et al., 1999; Yang et al., 1999). Newborns lackingfunctional p63 display abnormal epidermis and hair follicles with athin single cell layer covering the body.

p63 is essential to normal epidermal development and ishomologue of the p53 tumor suppressor. It has been linked to cellfate determination and/or maintenance of self-renewing populationsin several epithelial tissues, including skin, mammary gland, andprostate (Yang et al., 2002). ΔNp63 is over-expressed in a variety of

236 Z. Zhang et al. / Toxicology and Applied Pharmacology 253 (2011) 235–243

epithelial tumors including oral and skin squamous cell carcinomas(Westfall and Pietenpol, 2004). While elevated p63 expression canpromote cell proliferation (King et al., 2006; Koster et al., 2004), theunderlying mechanisms are still unknown. In this study weinvestigated the role of p63 in nickel-reduced cell proliferationusing epidermal cells.

Materials and methods

Cells and chemicals. Spontaneously immortalized human kerati-nocytes (HaCats) were cultured in monolayers at 37 °C, 5% CO2 usingDulbecco's modified Eagle's medium (DMEM) containing 10% fetalbovine serum (FBS), 2 mML-glutamine, and 25 μg of gentamicin/mL.Human primary keratinocytes (HPKs) were purchased from Lonza(Walksville, MD). The cells were cultured in the growth mediumcontaining BPE (Bovine Pitutary Extract), hEGF, Insulin, Hydrocortisone,and GA-1000 (Gentamicin, Amphotericin B) according to themanufacture's manual. Nickel chloride (Ni2+) was purchased fromSigma (St. Louis, MO).

Viruses. Human cDNA for p63α was obtained by RT-PCR cloning inpCDNA3.1 (Invitrogen) according to the manufactory manual. Properexpression was confirmed by transient transfection in 293 cells andimmunoblotting with p63 pan-antibodies (Santa Cruz). ΔNp63 cDNAinsert was then sub-cloned into the BamHI site of the PINCO retroviralvector (Nocentini et al., 1997) and was used to make retrovirus in 293GPG cells. Expression of the retrovirally transduced p63 cDNA wasverified by immunoblotting of infected human keratinocytes withanti-p63 antibodies.

ΔNp63 siRNA cloning. To knock down the specific genes of interest,artificial miRNA sequences were designed using BLOCK-iT RNAiDesigner (Invitrogen). The oligonucleotides were synthesized,annealed, and inserted into the pcDNA6.2-GW/miR vector (BLOCK-iTPol II miR RNAi Expression Vector, Invitrogen) according to theprotocol provided by the manufacturer. The artificial miRNA sequencesinserted into the BLOCK-iT Pol II miR RNAi Expression Vector areas follow: 5′-TGCTGTTTGATGCGCTGTTGCTTATTGTTTTGGCCA-CTGACTGACAATAAGCAAGCGCATCAAA-3′. The expression of the vec-tors was confirmed by Western blot.

Plasmids. The human ΔNp63 promoter region (1.477 kb) wascloned by PCR using human genomic DNA as template. The followingprimers were used to amplify nucleotides −1477 to −4 from theinitiation codon, plus an additional BglII or HindIII restriction site:BglII-5′-AGCCAATGACCACGTCATCC-3′ , reverse HindIII 5′-AGCTGTAAGATTGATCAA TGC-3′. The PCR product was then clonedinto the pGL3 basic vector (Promega) digested by BglII–HindIII. Theconstruct was verified by sequencing. pCR-FLAG-IKKβ-KM (K44A)was from Dr. Hiroyasu Nakano (Juntendo University, Japan). NF-κBluciferase reporter was purchased from Clontech (Mountain View,CA). p21 luciferase reporter and pCMV4-IRF3 over-expression vectorwere from Addgene Inc (Cambridge, MA).

Promoter activity assays. Transient transfection promoter activityassays were performed as previously described (Rangarajan et al.,2001; Talora et al., 2002), using co-transfection with a TK-renillareporter for internal normalization. Total quantities of plasmid DNAwere kept constant by adding appropriate amounts of empty vectorswithout inserts. Transfected cells were harvested at 24 h aftertransfection and relative luciferase activities were normalized forRenilla luciferase activity. All conditions were tested in triplicate wells,and experiments were repeated for a minimum of three times.

Analysis of gene expression. Total RNA preparations (1–2 μg) wereused in a reverse transcriptase reaction with random primers, followed

by real-time PCRwith gene-specific primers, using an Icycler IQTM Real-Time detection System (Bio-Rad) according to the manufacturer'srecommendations, with SYBR Green (Bio-Rad) for detection. ΔNp63primers, forward 5′-CGTGTCCTTCCAGCAGTC-3′, reverse 5′-GCAATTT-GGCAGTAGAGTT-3′. IRF3 primers, forward 5′-GTGGCCTGGGTG-AACAAGAG, reverse 5′-CTGGAAGATTCCG AAATCCTCC. IRF7 primers,forward 5′-GAGCCGTACCTGTCACCCT, reverse 5′-GGGCCGTAT-AGGAACGTGC. IKKε primers, forward 5′-ACAAGGCCCGCAACAAGAAA,reverse 5′-TTGACGATGTTCTGGTGGTTC. Sp100 primers, forward 5′-TGAGGTGTGCAA CAAATGGG-3′ , reverse 5′-AAGATGCAAC-TCCACGGGTTC-3′. p21 primers, forward 5′-CCTGTCACTGTCT-TGTACCCT, reverse 5′-GCGTTTGGAGTGGTAGAAATCT. GAPDH primers,forward 5′-ATCATCAGCAATGCCTCC, reverse 5′-AGTCCTTCCA-CGATACCAA. Each sample was tested in triplicate and results werenormalized using amplification of the same cDNAs with human GAPDHprimer.

Western blotting. After designed treatments, both HaCat and HKCcells were collected and washed with PBS twice. The cells were lysedin RIPA buffer (150 mM NaCl, 100 mM Tris at pH8.0, 1% Triton X-100,1% deoxycholic acid, 0.1% SDS, 5 mM EDTA and 10 mM NaF, 2 mMDTT, 0.5 mM PMSF, 1 mM sodium vanadate, 1 μg/ml leupeptin). 30 μgof protein was loaded into 4–12% NuPAGE Bis–Tris gel (Invitrogene).After blocking with 5% milk, the membrane was probed with variousantibodies and developed with ECL (Pierce).

Cell growth analysis. HaCat cells were infected with recombinantretroviruses expressing either a full-length ΔNp63α cDNA togetherwith GFP (Pinco-ΔNp63α) or GFP alone (Pinco). Two days afterinfection, GFP-positive cells were purified by cell sorting using flowcytometry and then replated to normal cell culture. After 3 days offurther cultivation, cells were trypsinized, counted, and split intotriplicate dishes. The cells were treated with 100 μM nickel orremained untreated as control. The fresh medium with 100 μM nickelwas added every 3 days. After 3 weeks of further cultivation,clonogenic growth was evaluated by staining of dishes and countingof macroscopically visible colonies (containing N50 cells).

Statistical analysis. Statistical analyses were performed using theone-way ANOVA. Results were expressed as average values±SD. Thedifference was considered significant if pb0.05.

Results

ΔNp63 expression is down-modulated by short term nickel treatment

The molecular mechanism involved in the control of p63expression in growing versus differentiating keratinocytes is notknown. Real-time PCR study showed that ΔNp63 mRNA expressionwas sharply down-modulated by treatment with nickel in bothimmortalized human keratinocytes and primary human keratinocytes(Figs. 1A and B). A similar down-modulation was found byimmunoblotting at the protein level (Fig. 1C). It displayed a dose-and time-dependant manner. Phosphorylations of both p53ser15 andp73were activated after nickel treatment, while the total protein levelof actin remained essentially the same (Fig. 1D). The analysis of 10-kbnucleotide sequence of human ΔNp63 promoter found that ΔNp63promoter contains multiple binding sites of NF-κB-binding andinterferon-responsive factors (IRF) (Nguyen et al., 2006). In thepresent study, the 10-kb ΔNp63 promoter assay was conducted tomeasure transcriptional activity of ΔNp63. The results showed thatnickel treatment caused a dose-dependent decrease in ΔNp63 activity(Fig. 1E). While the major isoform expressed in keratinocytes afterbirth is ΔNp63α (Yang et al., 1998), the TAp63, another isoform ofp63, was also expressed in these cells at very low levels that were notquantifiable by real-time PCR (data not shown).

Fig. 1. Negative control of p63 expression in keratinocytes by nickel treatment. (A) and (B), Down-modulation of p63 mRNA expression by nickel. Both HaCat and HPK cells weretreated with different doses of nickel for 24 hr (A), and treated with 1.0 mM for different time (B). ΔNp63 mRNA levels were quantified by real-time PCR. Values are expressed asrelative arbitrary units, after internal normalization for GAPDHmRNA expression. (C) Down-modulation of p63 protein expression by nickel. Both HaCat and HPK cells were treatedwith nickel for different times and doses. The cells then were analyzed for ΔNp63 protein level by immunoblotting with the corresponding antibodies. Immunoblotting for β-actinwas used for equal loading control. (D) Up-regulation of both p53 and p73 by nickel treatment. HaCat cells were treated with nickel for 24 h. The cells were harvested, p53ser15,phospho-p73 and β-actin expression were analyzed by immunoblotting. (E) Down-modulation of p63 activity in keratinocytes in response to nickel treatment. Both the HaCat andHPK cells were transfected with 0.5 μg of ΔNp63 luciferase reporter DNA followed by nickel treatment. The cells were harvested for the measurement of luciferase activity after 24 h.Values are expressed as relative units after internal normalization for protein concentration. *, pb0.05 compared to control without nickel treatment (one-way ANOVA test).

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Nickel suppresses ΔNp63 expression through negative regulation of theinterferon signaling pathway

To gain further insights into regulation of p63 expression, weanalyzed a 10 kb nucleotide sequence of the human ΔNp63αpromoters for common transcription factor-binding motifs. The

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Fig. 2. Role of NF-κB and interferon signaling pathways in nickel-induced down-regulation owere transfected with 0.5 μg of NF-κB-responsive reporter (pNF-κB-luc) with increasing amactivity was measured as described in Materials and methods. (B), (C), (D), and (E) Down-nickel treatment. HaCat cells were treated with different doses of nickel for 24 h, followed(E) by real-time PCR analysis. (F) Induction of p63 expression by inhibition of NF-κB. HaCat cas indicated. ΔNp63 mRNA levels were determined by real-time PCR. (G) Partial restoration20 μg of IRF3 expression vector or empty vector for 4 h. Then 1 mM nickel was added as indcontrol without nickel treatment (one-way ANOVA test). #, pb0.05 compared to nickel tre

presence of multiple NF-κB-binding sites, interferon-stimulatedresponsive elements (ISRE) (Levy et al., 1988) and binding sites forinterferon-responsive factors (IRF) (Taniguchi et al., 2001) was foundto be a characteristic of both promoters (Nguyen et al., 2006). Nickelexposure caused activation of NF-κB (Cruz et al., 2004). The activationof NF-κB by nickel resulted in significant modulation of cellular and

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f ΔNp63 expression. (A) Induction of NF-κB transcriptional activity by nickel. HaCat cellsounts of nickel as indicated. Cells were collected after 24 h treatment. The promoter

modulation of endogenous interferon-responsive genes IRF3, IRF7, IKKε, and Sp100 byby determination of mRNA expression levels of IRF3 (B), IRF7 (C), IKKε (D), and Sp100ells were transfected with IKKβ-KM, either alone or in combination with 1 mM of nickelof IRF3 on suppression of p63 expression by nickel. HaCat cells were transfected withicated. ΔNp63 mRNA levels were determined by real-time PCR. *, pb0.05 compared toatment (one-way ANOVA test).

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tissue responses (Denkhaus and Salnikow, 2002). Moreover, variousnickel-induced allergic effects and contact skin hypersensitivity inindustrialized countries are able to be interpreted as the activation ofNF-κB (Goebeler et al., 1995). However, its possible impact on theinterferon signaling pathway in this cell type has not been reported.Among NF-κB suppressing genes in human cells were the ones forIRF7, a key regulator of the interferon-dependent transcriptioncascade with oncogenic potential (Zhang and Pagano, 2002; Hondaet al., 2005), and Sp100, an essential component of nuclear bodies(NBs) (Moller et al., 2003). IRF7 physically interacts and functionallyoverlaps with IRF3 (Servant et al., 2002). IKKε is a key kinase thatpositively regulates the interferon response (Fitzgerald et al., 2003).Fig. 2A showed that nickel treatment caused an increase of NF-κBactivities. RT-PCR analysis showed that IRF3, IRF7, IKKε and Sp100expression levels were significantly decreased in nickel treated HaCatcells (Figs. 2B–E).

To assess whether the observed changes in both NF-κB andinterferon signaling pathways contribute to down-modulated p63expression by nickel, HaCat cells were transfected with expressingvector IKKβ kinase mutant (IKKβ-KM), which functions as inhibitor ofNF-κB, or the full-length IRF3 protein. IRF3 is a key downstreammediator of the interferon response (Honda and Taniguchi, 2006;Zhang et al., 2004). Expression of IKKβ-KM resulted in a partialrestoration of nickel-reduced p63 expression (Fig. 2F), indicating thatthe NF-κB pathway functions as a negative regulator of p63 inkeratinocytes under nickel stimulation. Transfection with IRF3 causeda partial restoration of p63 expression reduced by nickel treatment(Fig. 2G).

Fig. 3. Over-expression of ΔNp63 expression restores nickel-induced cell growth inhibitiongrowth was evaluated by staining of dishes. (B) Quantification of colony formation. *, pb0compared to nickel treatment with Pinco vector (one-way ANOVA test).

Down-modulation of ΔNp63 expression by nickel cell proliferation

It has been reported that the down-modulation of ΔNp63expressionmay be essential for its ability to decrease cell proliferation(Pellegrini et al., 2001). The clonogenic behavior of keratinocytesprovides a widely used assay for their growth potential (Rochat et al.,1994). We examined whether nickel treatment was able to suppresscolonogenicity and if it did, whether this suppression depended onp63 down-modulation. HaCat cells were infected with a recombinantretrovirus expressing the ΔNp63 gene together with GFP (Pinco-ΔNp63) or a retrovirus expressing GFP alone (Pinco, empty vector). Ineach case, GFP-positive keratinocytes were purified by sorting andsubsequently by treatment with 100 μM nickel. HaCat cells infectedwith the p63 retrovirus formed essentially the same colonies as cellsinfected with the retrovirus control, indicating that p63 over-expression per se is not sufficient to increase the number ofclonogenic keratinocytes (Fig. 3). However, while treatment withnickel caused a drastic drop in number of clonogenic cells, a muchlesser reduction was observed with cells that had been previouslytransduced with the ΔNp63 retrovirus (Fig. 3), indicating the role ofp63 in the maintenance of the keratinocyte growth potential undernickel stimulation.

Nickel treatment causes growth arrest through induction of AP-1and NFAT by direct p38-dependent mechanisms (Ding et al., 2009;Huang et al., 2001). Figs. 4A and B showed that nickel treatmentincreased p21 expression at both mRNA and protein levels. Over-expression of ΔNp63 decreased p21 protein level in both untreated andnickel treated cells (Fig. 4B). Similarly, in transient transfection assays,

. The detailed procedure is described in “Materials and methods”. (A) The clonogenic

.05 compared to control without nickel treatment (one-way ANOVA test). #, pb0.05

240 Z. Zhang et al. / Toxicology and Applied Pharmacology 253 (2011) 235–243

the ability of nickel to induce the 2.4-kb promoter of the p21 gene wassuppressed by over-expression of ΔNp63 in a dose-dependent fashion(Fig. 4C). This suppression may result from the demonstrated ability ofΔNp63α to bind directly to p53/p63-binding sites in the p21 promoter(Westfall et al., 2003), thereby interfering with nickel-induced p21 up-regulation. However, induction of a minimal p21 promoter region notcontaining the binding site for p53/p63 was also suppressed by ΔNp63,to a similar extent as the full-length promoter (Fig. 4D). These resultsshow that nickel-inducedup-regulation of p21, leading todecreased cellproliferation.ΔNp63 is a negative regulator of p21. The inhibitory effectof ΔNp63 on p21 promoter may be through either p53 binding site orother site beyond p53.

Control p21 by endogenous ΔNp63

To assess whether p21 is under negative control of endogenousp63, HaCat cells were transfected with miRNA for the coding region ofhuman p63 versus a scrambled miRNA control. This approach caused80%–90% reduction in p63 mRNA and p63 protein levels 2–3 daysafter transfection. In cells treated with nickel, the p63 miRNAs causedan additional p63 reduction (Figs. 5A and B). p21 expression was up-regulated in cells with p63 knockdown (Fig. 5C). This knockdownresulted in a substantial increase in p21 expression under basalconditions, with a much higher induction in response to nickeltreatment (Fig. 5C).

Discussion

Although the molecular mechanisms by which nickel compoundscause cancer are still under investigation, recent studies show that thecarcinogenic actions of nickel compound include oxidative stress,genomic DNA damage, epigenetic effects, and the regulation of gene

Fig. 4. Counteracting effects of ΔNp63 on the nickel-induced p21WAF1/Cip1 activation. (A) IndThe cells were harvested for analysis of p21 mRNA level by real-time PCR. (B–D) CounterinΔNp63 followed by nickel treatment. Immunobloting was used to measure p21 expression (Bpromoter region of the p21 gene (C), and a minimal region of the p21 promoter devoid of2001) (D). The cells were treated with 1 mM nickel, plus/minus an expression vector for Δ*, pb0.05 compared to control (one-way ANOVA test). #, pb0.05 compared to nickel treatm

expression by activation of certain transcription factors related tocorresponding signal transduction pathways (Lu et al., 2005). It hasbeen reported that nickel caused reactive oxygen species (ROS)generation, leading to cell transformation (Costa et al., 1994; Huanget al., 1994). Nickel was able to activate NF-κB (Goebeler et al., 1995)and NFAT (Huang et al., 2001), induce inflammation mediators suchas COX-2 and TNF-α (Ding et al., 2009), and increase HIF-α, VEGF, andangiogenesis (Ouyang et al., 2009).

The present study investigated the role of p63 in the epidermalnickel response. First, we found that ΔNp63 isoform was predomi-nantly expressed in the epidermis. ΔNp63 expression was dramati-cally reduced at both transcription and translation levels in responseto nickel treatment. p53, a tumor suppress gene, was activated uponnickel treatment. Similarly, another member of the p53 family, p73was also up-regulated by nickel. Nickel was able to activate NF-κB.Inhibition of NF-κB partially restored ΔNp63 mRNA level reduced bynickel treatment. Furthermore, nickel treatments caused decreases inIRF3, IR7, IKKε, and Sp100 expressions in a dose-dependent manner.Over-expression of IRF3 reversed nickel-induced suppression ofΔNp63 mRNA level. Cell proliferation assay showed that nickeltreatment reduced the cell growth. Over-expression of ΔNp63counteracted the effect of nickel, causing an increase of cellproliferation compared to nickel treatment only. The present studyfound that p21, a downstream regulatory protein of p53, played a rolein ΔNp63 signaling. Nickel increased p21 activity, and ΔNp63eliminated the p21 activity induced by nickel. Blockade of ΔNp63dramatically increased p21 expression.

It has been shown that in p63 family, ΔNp63α is the mostabundantly expressed isotype in tumor cells. This protein has beenimplicated in cell proliferation and oncogenic growth (Crook et al.,2000; Hibi et al., 2000; Park et al., 2000). The studies of p63 null micehave demonstrated that p63 plays a critical role in regulating cell

uction of p21 by nickel. HaCat cells were treated with different doses of nickel for 24 h.g effects of ΔNp63 on nickel-induced p21activation. HaCat cells were transfected with). HaCat cells were transiently transfected with reporter plasmids containing the 2.4-kbp53-binding sites but containing a fully conserved RBP-binding site (Rangarajan et al.,Np63 in increasing amounts as indicated. After 24 h promoter activity was measured.ent (one-way ANOVA test).

Fig. 5. Control of p21 by endogenous ΔNp63. (A–B) Knockdown of endogenous ΔNp63 expression. HaCat cells were transfected with miRNA human ΔNp63 or miRNA control.Parallel cultures were treated with1 mM nickel for 24 h after miRNA transfection. Cells were analyzed for measurement of p63 expression by real-time PCR (A) or immunoblottingwith the corresponding antibodies (B). (C) Up-regulation of p21 expression as a consequence of ΔNp63 knockdown. The procedure is the same as (A). HaCat cells were harvested foranalysis of p21 mRNA level by real-time PCR. *, pb0.05 compared to control (one-way ANOVA test). #, pb0.05 compared to nickel treatment (one-way ANOVA test).

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proliferation and in mediating epidermal stem cell renewal duringearly development (Yang et al., 1999). These findings raise theintriguing possibility that p63 functions as a regulator of cell growthin normal tissues and in cancer (Patturajan et al., 2002). The results inthe present study showed that short-time (up to 48 h) nickeltreatment caused a decrease of ΔNp63 at both transcription andtranslation levels, and promoter activity. However, our preliminaryresults showed that 8 weeks and 16 weeks exposure to low dosenickel (100 μM) increased ΔNp63 expression in HaCat cells (data notshown). Over-expression of ΔNp63 synergistically promoted nickel-induced cell transformation and tumorigenesis. This is consistent withthe study that low dose arsenic treatment in normal RWPE cellscaused a decrease of ΔNp63 in early stage and dramatic increase ofΔNp63 in late stage due to the cells being transformed (Tokar et al.,2010). It has been observed that normal cell self-renewal was initiallylost during early transformation, potentially due to aberrant differ-entiation, and then regained with malignant transformation, consis-tent with distorted self-renewal common to cancer cells (Pardal et al.,2003). Another study has also shown that ΔNp63α is reduced inresponse to UV-B treatment (Liefer et al., 2000). Nickel is able to causeDNA damage.When a cell incurs DNA damage, p63 is down-regulated,allowing p53 to step into action, protecting the cells from damagedDNA. Deregulated p63 expressionwould interfere with this protectiverole of p53, possibly by competingwith p53 for binding to DNA targetsand/or exclusion from the nucleus and degradation via the actions ofnewly synthesized p63.

ROS is one of the important determinants in the regulation of cellsignaling pathways involved in proliferation, apoptosis, transforma-tion, and senescence (Tokar et al., 2010; Westfall et al., 2005; Daltonet al., 1999). Many of these effects contribute to nickel-inducedcarcinogenesis (Chen and Shi, 2002). It was reported that p63−/−MEFs (mouse embryo fibroblasts) had significantly lower levels of

ROS compared with cells from wild-type or heterozygous littermates(Ellisen et al., 2002). Over-expression of ΔNp63α in the p63−/−MEFs led to a marked enhancement of H2O2-induced ROS (Ellisenet al., 2002). 100 J/m2 of UV exposure to primary human keratinocytesdecreased ΔNp63α expression level, concordant with increased ROSgeneration (Westfall et al., 2005). Our preliminary data showed thatnickel treatmentwas able to cause ROS generation in HaCat cells (datanot shown). Pretreatment with ROS scavengers, SOD and catalaseincreased ΔNp63 expression reduced by nickel. In addition, we alsofound that nickel treatment increased SOD2 expression. Inhibition ofSOD2 expression partially restored ΔNp63 expression reduced bynickel. Those indicate thatΔNp63may act as one of the target genes ofROS, promoting cell proliferation.

NF-κB is sequestered in the cytoplasm in an inactive form bindingto the I-κBα inhibitor. NF-κB activation requires the I-κB kinase (IKK)to mediate I-κBα phosphorylation, an event leading to I-κBαdegradation and consequently freeing NF-κB to translocate to thenucleus for regulating the transcription of its target genes (Haydenand Ghosh, 2004; Karin and Greten, 2005). The mechanism by whichp63 initiates or directs the differentiation program and the keydownstream targets involved are still poorly understood. p63 hasbeen linked with many genes that have been shown to be importantfor promoting differentiation, a key protein being IKKα (Truong andKhavari, 2007). IKKα was identified as a direct downstream target ofp63 (Candi et al., 2006). IKKα is a component of the multi-unit IKKcomplex that phosphorylates the inhibitor molecule IκB, therebytargeting it for ubiquitination and degradation. This relieves theinhibitory unit of NF-κB, resulting in its activation (Zandi et al., 1997).There are several NF-κB binding sites in both mouse and humanpromoters of ΔNp63 (Nguyen et al., 2006). Since NF-κB is activated inkeratinocytes differentiation and is involved in carcinogenesis, it islikely that NF-κB is involved in inhibition of ΔNp63 by nickel.

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Blockade of NF-κB by expressing of IKKβ kinase mutant reversednickel-induced suppression of ΔNp63, indicating that ΔNp63 is adownstream protein which is negatively regulated by NF-κB inresponse to nickel treatment.

In addition to NF-κB binding sides on ΔNp63 promoter, there arebinding sites for interferon-responsive elements, where a synergisticmulti-protein complex is formed by NF-κB subunits and Interferonregulatory factors 3 and 7 (IRF3/IRF7), two key mediators of theinterferon response (Nguyen et al., 2006). IRF3 and IRF7 participate inthe formation of a large protein complex called an IFN-β enhance-some/DRAF1 that are also NF-κB, AP-1, and CREB binding protein(CBP)/p300 to activate transcription of IFN-β gene (Honda andTaniguchi, 2006; Wathelet et al., 1998). IRF3 is believed to play a rolein DNA damage-induced apoptosis as IRF3 protein is phosphorylatedand translocates from the cytoplasm to the nucleus in response toDNA damage (Kim et al., 1999). In addition, IRF3 may function as atumor suppressor gene (Savitsky et al., 2010). An induction of IFN-γwas observed in nickel contacting patients (Bordignon et al., 2008). Invitro study also showed that treatment with nickel sulfate inducedsecretion of IFN-gamma by splenic natural killer cells (Kim et al.,2009). It has been reported that nickel compounds are able to up-regulate both TNFα and NF-κB (Huang et al., 1994). However, theregulation of interferon pathway remains unclear. It has beenreported that up-regulation of IKKε depends on NF-κB activity(Hemmi et al., 2004; Kravchenko et al., 2003; Shimada et al., 1999).The mechanistic role of IKKε in NF-κB activation is still not fullyunderstood. It is possible that IKKε-dependent activation of NF-κBtargets genes in certain cells is mediated through IRFs. IRF3 is requiredfor the activation of certain NF-κB target genes (Covert et al., 2005).Our data suggested that IKKε, IRF3 and IRF7 mRNA levels decreasedafter nickel treatment. Over-expression of IRF3 itself indeed reducedΔNp63 mRNA level. Treatment of the cells by nickel together withIRF3 over-expression restored ΔNp63 expression reduced by nickel.These results demonstrate that IRF3 is involved in nickel-inducedsuppression of ΔNp63 expression.

Abundant genetic evidence indicates that one of p63 functions is tomaintain regenerative capacity of base epithelia at several sitesthroughout the body. Down-modulation of ΔNp63 expression bynickel treatment may be important for the cell growth inhibition.The clonogenic assay provides a widely used method to determinethe cell growth potential (Rochat et al., 1994). Using this method, wehave shown that treatment with the low dose of 100 μM nickelchloride (NiCl2) for 2 weeks caused a dramatic growth inhibitioncompared to control without nickel treatment. Moreover, over-expression of ΔNp63 restored cell growth inhibited by nickel,indicating that ΔNp63 counteracts the ability of nickel to restrictcell growth.

Induction of p21 protein by nickel is responsible for the cell cyclearrest, whereas p63may function as an inhibitor of p21 (Nguyen et al.,2006). The results from the present study indicate that nickeltreatment caused an increase of p21 promoter activity. Nickel wasstill able to activate truncated p21 without p53 response elements.ΔNp63 counteracted the effect of nickel, causing the reduction of p21activity. The reduction effect of ΔNp63 was p53-independent. It hasbeen reported that ΔNp63 binds to the p21 other than p53 bindingsites. Loss of ΔNp63α-dependent transcriptional repression ofp21WAF1 correlates with ΔNp63α down-regulation during keratino-cyte differentiation (Craig et al., 2010). Consistently, the present studyshows that inhibition of ΔNp63 increased nickel-induced p21expression. Inhibition of ΔNp63 alone caused a decrease of p21mRNA expression. There are two modes of transcriptional suppres-sion by ΔNp63. First, ΔNp63 can compete with p53 activity (ortransactivating p63 isoforms) for DNA target sites (Yang et al., 1998;Davison et al., 1999). A second mode of inhibition might involve theformation of transactivation-incompetent heterocomplexes betweenDNA binding domains of p53 and the ΔNp63 (Ratovitski et al., 2001).

Our results suggested that reduction ofΔNp63 by nickel may act on itsdownstream genes other than p53 binding sites.

In summary, our results showed that in keratinocytes nickel is ableto decrease IRF3 and IRF7, and then activate NF-κB. The activation ofNF-κB suppressed ΔNp63, resulting in an increase of p21 expressionand decrease in cell growth.

Conflict of Interest

This manuscript will not be submitted to any other scientificjournal prior to the decision by Toxicology and Applied Pharmacology.Each listed author on the manuscript is aware of and agrees to thecontents of the manuscript, including the authorship. None of thelisted authors has any financial or other interests that could be ofconflict.

Acknowledgment

This research is supported by NIH grant R01ES015518.

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