clic4 mediates and is required for ca2+-induced keratinocyte … › content › joces › 120 ›...

2631Research Article

IntroductionThe CLIC family of chloride intracellular channels iscomposed of seven differentially expressed proteins thatlocalize to the cytoplasm and intracellular organelles in manycell types (Suh and Yuspa, 2005). Five members of the CLICfamily are highly homologous in size and sequence (CLIC1-5), whereas p64 (CLIC5B) and parchorin (CLIC6) haveextended amino-terminal domains and are considerably larger.Crystallographic analysis of CLIC1 and CLIC4 indicates thatthe protein exists in both soluble and membrane bound formsand is structurally related to the omega class of glutathione S-transferases (Harrop et al., 2001; Littler et al., 2005). SeveralCLIC proteins have Cl– selective channel activity (Ashley,2003), but it is not yet clear if they form pores by themselvesor participate in ion flux indirectly (Jentsch et al., 2002).

Recent data from several laboratories have suggested thatCLIC family members also participate in cell signaling. CLIC3interacts with ERK7 in the nucleus of mammalian cells andstimulates chloride conductance (Qian et al., 1999). p64 is asubstrate for Fyn kinase, and the combination enhanceschloride channel activity when co-expressed in HeLa cells(Edwards and Kapadia, 2000). CLIC1 is associated with TNF�release in amyloid-�-treated rat microglial cells (Novarino etal., 2004). CLIC4 is induced by TGF� in mammary fibroblastsas they transdifferentiate into myofibroblasts (Ronnov-Jessenet al., 2002), and CLIC4 binds to dynamin 1 in a complexinvolving actin, tubulin and 14-3-3� in rat brain extracts(Suginta et al., 2001). CLIC4 also interacts with nuclear

transport factor 2 (NTF2), Ran and importin-�, activecomponents of the nuclear import machinery, suggesting thatthe nuclear localization signal (NLS)-recognition proteincomplex may be involved in CLIC4 nuclear trafficking (Suh etal., 2004). The physiological consequences of theseinteractions is under study, but CLIC1 and CLIC4 has beenimplicated in regulation of the G2-M phase of the cell cycle inCHO cells (Berryman and Goldenring, 2003), and CLIC4 isassociated with adipocyte differentiation in 3T3-L1 cells(Kitamura et al., 2001).

In keratinocytes, an increase in CLIC4 expression isassociated with Ca2+-induced differentiation, DNA damage,p53 upregulation and exposure to TNF� (Fernandez-Salas etal., 1999; Fernandez-Salas et al., 2002; Suh et al., 2004). Inaddition to cytoplasmic residence, CLIC4 is a component ofthe inner mitochondrial membrane, and maintenance of CLIC4levels is essential for viability of keratinocytes (Fernandez-Salas et al., 2002). Thus, those conditions that elevate CLIC4are associated with cell cycle arrest or cell death. CLIC4elevation in differentiating keratinocyte could participate in theterminal phase of the differentiation process whenkeratinocytes undergo cell cycle arrest, karyorrhexis and die.Cytoplasmic CLIC4 translocates to the nucleus in stressed cells(Suh et al., 2004), and nuclear CLIC4 could also participate inother aspects of the differentiation response such as the geneexpression changes that are associated with differentiation.Previous studies have suggested that changes in intracellularchloride and pH are associated with keratinocyte

Keratinocyte differentiation requires integrating signalingamong intracellular ionic changes, kinase cascades,sequential gene expression, cell cycle arrest, andprogrammed cell death. We now show that Cl– intracellularchannel 4 (CLIC4) expression is increased in both mouseand human keratinocytes undergoing differentiationinduced by Ca2+, serum and the protein kinase C (PKC)-activator, 12-O-tetradecanoyl-phorbol-13-acetate (TPA).Elevation of CLIC4 is associated with signaling by PKC�,and knockdown of CLIC4 protein by antisense or shRNAprevents Ca2+-induced keratin 1, keratin 10 and filaggrinexpression and cell cycle arrest in differentiatingkeratinocytes. CLIC4 is cytoplasmic in activelyproliferating keratinocytes in vitro, but the cytoplasmicCLIC4 translocates to the nucleus in keratinocytesundergoing growth arrest by differentiation, senescence or

transforming growth factor � (TGF�) treatment. TargetingCLIC4 to the nucleus of keratinocytes via adenoviraltransduction increases nuclear Cl– content and enhancesexpression of differentiation markers in the absence ofelevated Ca2+. In vivo, CLIC4 is localized to the epidermisin mouse and human skin, where it is predominantlynuclear in quiescent cells. These results suggest that CLIC4participates in epidermal homeostasis through bothalterations in the level of expression and subcellularlocalization. Nuclear CLIC4, possibly by altering the Cl–

and pH of the nucleus, contributes to cell cycle arrest andthe specific gene expression program associated withkeratinocyte terminal differentiation.

Key words: CLIC4, Chloride channel, PKC, Calcium,Differentiation, Keratinocytes, AP1

Summary

CLIC4 mediates and is required for Ca2+-inducedkeratinocyte differentiationKwang S. Suh, Michihiro Mutoh, Tomoko Mutoh, Luowei Li, Andrew Ryscavage, John M. Crutchley,Rebecca A. Dumont, Christina Cheng and Stuart H. Yuspa*Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute, Bethesda, MD20892, USA*Author for correspondence (e-mail: [email protected])

Accepted 29 May 2007Journal of Cell Science 120, 2631-2640 Published by The Company of Biologists 2007doi:10.1242/jcs.002741

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differentiation (Mauro et al., 1990; Mauro et al., 1993). Thisstudy was undertaken to document the changes in CLIC4protein that may be associated with keratinocyte differentiationand to explore the potential function of these changes in thedifferentiation response of keratinocytes to increasingextracellular Ca2+.

ResultsCLIC4 is Ca2+ responsive and PKC dependent in mouseand human keratinocytesPrevious results had indicated that CLIC4 transcripts andprotein increased in mouse keratinocytes induced todifferentiate by Ca2+ (Fernandez-Salas et al., 1999). In mousekeratinocytes, CLIC4 protein and transcript levels increaseafter 24 hours relative to the level of extracellular Ca2+ (Fig.1A,B). Both Ca2+ and serum also elevate CLIC4 in humanforeskin keratinocytes (Fig. 1A,C). Ca2+ is known to activateseveral protein kinase C (PKC) isoforms in keratinocytes

(Denning et al., 1995). To determine if PKC signaling isinvolved in CLIC4 expression, mouse and human keratinocyteswere treated with the PKC activator TPA. As shown in Fig. 1D,this treatment increased CLIC4 levels within 24 hours also,suggesting a PKC-regulated process. To explore the connectionamong Ca2+ signaling, PKC activation, keratinocytedifferentiation and CLIC4 induction, mouse keratinocytesinduced to differentiate by 0.5 mM Ca2+ were treated withselective PKC inhibitors and probed for expression of CLIC4,keratin 1 (K1) and keratin 10 (K10; Fig. 2A). In control cells,both CLIC4 and the differentiation markers increase by 24hours after Ca2+ induction. The PKC� selective inhibitorGO6976 did not inhibit either CLIC4 or the differentiationmarker response to Ca2+. In fact, both responses increased withGO6976, consistent with the negative effect of PKC�activation on markers of spinous differentiation reported earlier(Lee et al., 1997). By contrast, the general PKC inhibitorbisindoylmaleimide-I at 10 �M and the PKC� selectiveinhibitor rottlerin reduced expression of both CLIC4 and K1,and K10, implying that PKC� regulates CLIC4 expression.Fig. 2B supports this conclusion since both differentiationmarkers and CLIC4 expression in Ca2+-induced keratinocyteswere depressed by infection with an adenovirus encoding aPKC� dominant negative construct previously shown toeffectively and selectively inhibit PKC� activity inkeratinocytes (Li et al., 1999). In the presence of the dominantnegative construct or the pharmacological inhibitor rottlerin,CLIC4 expression in high Ca2+ culture conditions was reducedtoward or below the level in low Ca2+ culture. To test if CLIC4is a specific and direct substrate for PKC, we incubatedrecombinant CLIC4 and CLIC1 with recombinant PKC�, �,�, � and � in an in vitro kinase reaction (Fig. 2C). In all cases,phosphorylation was detected on both substrates, indicating alack of specificity. Furthermore, we were unable to detectphosphorylation of CLIC4 after treatment of keratinocytes withTPA (data not shown). Taken together, the results suggest thatCLIC4 is regulated by activation of PKC� but not by a specificdirect phosphorylation of CLIC4 by PKC�. Instead, PKC� mayregulate the expression or stability of CLIC4 indirectly and theupregulation of CLIC serves as a downstream mediator ofkeratinocyte differentiation.

Upregulation of CLIC4 is required for Ca2+-inducedkeratinocyte differentiationTo determine if upregulation of CLIC4 is essential for Ca2+-induced differentiation in keratinocytes, we reduced CLIC4level by expressing CLIC4 antisense in an adenoviral vector(AS-CLIC4 Ad) in keratinocytes exposed to 0.5 mM (Hi) Ca2+

medium. We have previously shown that this vector reducesCLIC4 expression in a dose-dependent manner (Suh et al.,2005). Under conditions in which AS-CLIC4 adenovirus (Ad)reduced CLIC4 and prevented the Ca2+-induced increase inCLIC4 expression, K1 and K10 proteins were not elevated at24 hours after Ca2+ addition, and the expression of the latedifferentiation marker filaggrin was inhibited after 48 hours(Fig. 3A). Expression of AS-CLIC4 did not alter theendogenous level of PKC� (Fig. 3A), suggesting that CLIC4upregulation is an important component of the differentiationresponse to elevated Ca2+. To verify this observation by anindependent differentiation marker, cell cycle parameters weremonitored to determine if the rapid G1 cell cycle arrest

Journal of Cell Science 120 (15)

Fig. 1. CLIC4 is upregulated in differentiating keratinocytes.(A) Newborn mouse and human foreskin keratinocytes were culturedin 0.05 mM Ca2+ medium for 4 days, then treated for 24 hours withthe indicated concentrations of Ca2+ in the culture medium andlysates were examined by immunoblotting. (B) RNA isolated frommouse keratinocytes from A was amplified by RT-PCR, and CLIC4and actin transcripts were detected by ethidium bromide staining ofagarose gels. (C) Human foreskin keratinocytes were cultured inserum-free medium for 4 days and exposed to 5% FBS deleted ofCa2+ serum for the indicated times. (D) Mouse and humankeratinocyte were treated with indicated doses of TPA for 24 hours,and lysates were analyzed by immunoblotting. The fold change foreach band was determined by densitometry as described in Materialsand Methods.

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associated with Ca2+-induced differentiation, was altered(Dotto, 1999). Within 24 hours of 0.5 mM Ca2+ exposure,BrdU pulse-labeled cells were absent when assayed by flowcytometry whereas the number in G0-G1 increasedsubstantially (Fig. 3B,C). However, these changes wereprevented by expression of CLIC4 antisense, and theproliferating population was maintained up to 48 hours as inthe 0.05 mM Ca2+ medium (Fig. 3C). To verify CLIC4 is aspecific CLIC family member that mediates the keratinocytedifferentiation response, non-specific (NS) and specific shRNAconstructs were developed for CLIC1 (sR-1), CLIC4 (sR-4)and CLIC5 (sR-5) and used for a transient knockdown of CLICmembers (Fig. 3D,E). When transfected cells are induced todifferentiate by 0.5 mM Ca2+, CLIC4 shRNA preventedupregulation of K10 but the other shRNAs did not. This resultalso distinguishes CLIC4 from other CLIC proteins that might

have been influenced by AS-CLIC4 expression (Suh et al.,2005).

Fig. 2. CLIC4 is a downstream target of PKC� in keratinocytesinduced to differentiate by Ca2+. (A) Mouse keratinocytes were pre-treated with the indicated doses of PKC inhibitors for 1 hour, andthen 0.5 mM Ca2+ was added in the medium for 24 hours prior tolysis and immunoblotting. (B) Mouse keratinocytes were infectedwith PKC� DN Ad for 17 hours before the treatment with 0.5 mMCa2+ for 24 hours. Actin was used as a loading control. Bis,bisindolylmaleimide I; Ro, rottlerin; Go, Go 6976. Similar data wereobtained from two independent experiments. The numbers over theCLIC4 bands in A and B represent fold change determined bydensitometry. (C) Purified recombinant CLIC1 or CLIC4 proteinswere incubated with purified, kinase active recombinant PKCisoforms in appropriate reaction buffers with [�-32P]ATP, the reactionmixture was resolved by SDS-PAGE and then the phosphorylatedproteins were visualized by phospho-imaging methods. Un, norecombinant PKC.

Fig. 3. CLIC4 upregulation is required for Ca2+-induceddifferentiation in keratinocytes. K1, K10, Filaggrin, CLIC1, CLIC4,PKC� and actin protein levels in mouse keratinocytes weredetermined by immunoblot analysis. (A) Mouse keratinocytes wereco-infected with 5 MOI of Tet-Off Ad (to induce expression of theconditional AS) and increasing MOI (5 and 10, shaded triangle) ofAS-CLIC4 Ad or Null Ad (N) for 17 hours before treatment with 0.5mM Ca2+ for 24 hours. (B) Duplicate sets of mouse keratinocyteswere treated with BrdU, and anti-BrdU-FITC stained cells of eachsample were analyzed with flow cytometry. Cell populations: nullAd-infected without BrdU (upper left, N/(–)/ Lo Ca2+), with BrdU(upper right, N/(+)/Lo Ca2+) both in 0.05 mM Ca2+; with 0.5 mMCa2+ and BrdU (lower left, N/(+)/Hi Ca2+); co-infected with AS-CLIC4 Ad and Tet-Off Ad followed by Ca2+ and BrdU treatment(lower right, AS/(+)/ Hi Ca2+). (C) The key indicates the gatedpopulation in each cell cycle phase. The percentage of cells in eachcell cycle phase from the total cell population in N/(+)/Lo Cal2+,N/(+)/Hi Ca2+ and AS/(+)/Hi Ca2+ at 24 hours and 48 hours afterCa2+ induction is represented in a bar graph format. (D) Human 293cells were transfected with constructs expressing nonspecific (NS) orCLIC4 (sR-CLIC4) shRNA for 48 hours, and specificity of CLIC4knockdown was analyzed by immunoblots of cell lysates. (E) Mousekeratinocytes were transfected with constructs expressing shRNA fornonspecific (NS), CLIC1 (sR-1), CLIC4 (sR-4) or CLIC5 (sR-5) for24 hours, and then treated with 0.05 mM (Lo) or 0.5 mM (Hi) Ca2+.Actin was used as a loading control. Similar data were obtained fromat least two independent experiments. The numbers over the CLIC4bands in A and B represent fold changes determined bydensitometry.

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CLIC4 translocates to the nucleus in differentiatingkeratinocytes in vitro and is localized to the nucleus indifferentiating keratinocytes in vivoPrevious data have shown that cytoplasmic CLIC4 translocatesto the nucleus of cells undergoing a variety of stress responses(Suh et al., 2004). Similarly, CLIC4 translocates to the nucleusin both mouse and human keratinocytes that are induced todifferentiate by Ca2+ (Fig. 4A). Interestingly, CLIC4 alsotranslocates to the nucleus in keratinocytes undergoing G1arrest because of senescence (Fig. 4B) or serum starvation (Fig.4C) or when arrested by TGF� (Fig. 4D) or induced todifferentiate by TPA (Fig. 4E) treatment. This intracellulartrafficking of CLIC4 is dynamic since the nuclear CLIC4 inthe serum-starved cells translocates back to the cytoplasmwithin 30 minutes upon addition of serum to the medium,suggesting that CLIC4 has a physiological function during cellcycle arrest and release (Fig. 4C). Translocation of CLIC4 tothe nucleus in keratinocytes undergoing differentiation andsenescence in vitro prompted us to investigate the localizationof CLIC4 in vivo. In contrast to the cytoplasmic pattern ofCLIC4 seen in the actively proliferating keratinocytes inculture, immunohistochemical studies demonstrate that CLIC4is predominantly nuclear in the epidermis of adult human andmouse skin (Fig. 4F). CLIC4 is often nuclear in basal cells, butis excluded from the nucleus in a subpopulation of basal cellsand some spinous cells. Overall CLIC4 cellular staininggenerally increases in the suprabasal region wheredifferentiation is occurring. Almost all granular layerkeratinocytes express CLIC4 in the nucleus. This stainingpattern suggests that cycling keratinocytes are regulating thesubcellular localization of CLIC4. It should be noted thatnuclear CLIC4 can be detected in certain regions of the stromalcompartment, most likely in endothelial cells where CLIC4 isknown to be important for blood vessel tubulogenesis (Bohmanet al., 2005).

Nuclear CLIC4 enhances the differentiation responseTo address whether upregulation and nuclear translocation ofCLIC4 is sufficient to induce expression of differentiationmarkers, keratinocytes in 0.05 mM Ca2+ medium were infectedwith adenoviruses encoding intact CLIC4 (Cyt-CLIC4) orCLIC4 with a nuclear targeting signal (Nuc-CLIC4). Theseadenoviral vectors infect keratinocytes with high efficiency andtarget appropriate organelles as described previously (Suh etal., 2004). At relatively low levels of expression overendogenous CLIC4 levels, both constructs upregulated CLIC4,K1 and K10 in the absence of a Ca2+ switch, but expression ofthe markers was greater when CLIC4 was targeted to thenucleus (Fig. 5A). Thus, a small increase in the exogenousNuc-CLIC4 expression, above the endogenous level of CLIC4,caused a significant increase in the keratinocyte differentiationresponse. Primary keratinocytes cultured in basal medium(0.05 mM Ca2+) actively proliferate but individual cellsspontaneously differentiate and detach from the culture dish.Confocal studies (Fig. 5B) show that the increased and nuclearexpression of CLIC4 coincides (merged image) with theupregulation of K1 in keratinocytes that are spontaneouslyundergoing differentiation in the absence of extracellular Ca2+.In this setting, the nuclear CLIC4 is detected only in thesubpopulation of differentiating keratinocytes while thecytoplasmic CLIC4 is detected in the neighboring cells. Taken

together, the results shown in Fig. 5A and B suggest thatnuclear translocation of CLIC4 coincides with the expressionof early differentiation markers during keratinocytedifferentiation.


Fig. 4. CLIC4 translocates to the nucleus in differentiating andsenescing keratinocytes in vitro and is primarily nuclear in epidermisin vivo. (A) Mouse primary keratinocytes and human foreskinkeratinocytes were treated with indicated Ca2+ for 24 hours andimmunostained with CLIC4 antibody. (B) Mouse keratinocytes weremaintained in 0.05 mM Ca2+ medium and either fixed on the first dayof culture (1 Day) or after 7 days of culture (7 Day) to allow forsenescence. Fixed cultures were immunostained with CLIC4antibodies. Duplicate sets of cells were subjected to assay by �-galSA, and the number of senescing cells (7 Day) is presented as a foldincrease over the control (1 Day; lower panel). (C) Mousekeratinocytes were incubated in the serum-depleted medium for 16hours (0 min), rinsed and treated with the serum-containing medium(30 min). (D,E) Mouse keratinocytes were treated with TGF�(1 ng/ml; D) or TPA (25 ng/ml; E) for the times indicated andimmunostained for CLIC4. Insets in Fig. 4C-E represent DAPInuclear staining. (F) Formalin-fixed human breast skin and acetone-fixed mouse skin frozen sections were stained with CLIC4 antibodiesby immunohistochemical methods as described in the Materials andMethods. The mouse skin section was from a hyperplastic area toenhance the resolution of strata. Red and green arrows indicatekeratinocytes in the basal layers (blue nucleus) and the differentiatinglayers (brown nucleus, CLIC4 stain) respectively. In allimmunofluorescent and immunohistochemical staining experiments,secondary antibody alone was used as controls.

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Nuclear-targeted CLIC4 causes Cl– flux into the nucleusin keratinocytes, and Cl– flux is required for expressionof Ca2+-induced K10CLIC proteins are involved in flux of chloride ions in various

intracellular compartments (Tulk et al., 2002). To investigate ifnuclear-specific CLIC4 alters chloride ion flux, the chlorideion-sensitive fluorescent dye MQAE was used. With this dye,the fluorescent light emission is inversely related to theCl– concentration of the MQAE-containing intracellularcompartment, and the fluorescent output can be measuredsemi-quantitatively and visualized by fluorometer and confocalmicroscopy. Fig. 5C indicates that targeting CLIC4 to thenucleus of keratinocytes via an adenovirus reducesfluorescence of nuclear MQAE detected by confocalmicroscopy, indicating influx of chloride ion into the nucleus.By contrast, expression of �-galactosidase, cytoplasmic CLIC4or targeting GFP to the nucleus does not cause significantMQAE quenching, indicating that the CLIC4 result is not aconsequence of overexpression of any exogenous protein in thecytoplasm or nucleus. This dye quenching effect can beblocked by treating the cells with a chloride channel inhibitorNPPB [5-nitro-2-(3-phenylpropylamino) benzoic acid] prior toAd infection (Fig. 5C, first, second and third panel insets). Inthis experimental setting, overloading the culture medium withNaF (Cl– depletory agent) was used as a control to maximizethe fluorescence. When NaF-treated cells were washed andreloaded with excess NaCl, the fluorescence was quenchedagain in the nucleus of nuclear-targeted CLIC4 cells but not inthe cytoplasmic CLIC4 control, suggesting that thefluorescence is quenched by the nuclear CLIC4 (data notshown). When whole cell MQAE fluorescence is measured byfluorometry, the cells expressing Nuc-CLIC exhibit significantquenching of MQAE compared with the cells expressing wild-type (Cyt) CLIC4 (Fig. 5D). To investigate whether chlorideion flux is required for Ca2+-induced keratinocytedifferentiation, cells were treated with a chloride channelinhibitor prior to Ca2+ induction. Treatment of keratinocyteswith NPPB significantly reduced the expression level of K10upon Ca2+ induction (Fig. 5E). At the highest concentration ofNPPB (2 mM), both K10 and CLIC4 are reduced, a changethat was associated with morphological evidence of toxicity.

CLIC4 is a down-stream target of AP1AP1 signaling is involved in Ca2+- and TPA-inducedkeratinocyte differentiation through the regulation oftranscription of multiple maturation markers (Eckert andWelter, 1996; Angel et al., 2001). In previous work we haveshown that the expression of a highly specific AP1 dominantnegative recombinant protein A-Fos blocks expression ofseveral keratinocyte differentiation markers (Rutberg et al.,1997). A-Fos binds to Jun family members with high affinitybut does not transactivate, thus serving as a pan AP1 inhibitorfor keratinocytes (Gerdes et al., 2006). Inhibition of AP1activity by A-Fos expression also blocks the upregulation ofCLIC4 protein in keratinocytes induced to differentiate by Ca2+

(Fig. 6A,B). Inhibition of CLIC4 transcripts coincides withinhibition of Ca2+-induced transcripts for involucrin and K1 asdemonstrated by quantitative PCR (Fig. 6B). CLIC4expression is regulated by elements 5 from the transcriptionstart site of the first exon (Fernandez-Salas et al., 2002).Contained within this region are functional binding sites forp53 and Myc (Fernandez-Salas et al., 2002; Shiio et al., 2006).Further analysis reveals two consensus AP1 binding sites at–1507 to –1499 (site A) and –840 to –832 (site B; Fig. 6D). Aluciferase reporter driven by the active human CLIC4 promoter

Fig. 5. Nuclear CLIC4 induces the expression of keratinocytedifferentiation markers, and intracellular chloride influenceskeratinocyte differentiation. (A) Mouse keratinocytes in 0.05 mMCa2+ medium were pre-treated with null, wild-type CLIC4 (Cyt) ornuclear-targeted CLIC4 (Nuc) adenovirus for 12 hours, and celllysates were analyzed by immunoblots using K1, K10, CLIC4 andactin antibodies. Gray arrow indicates the endogenous CLIC4 (Endo-CLIC4) and the black arrows indicate the two exogenous CLIC4bands. (B) Mouse keratinocytes were cultured in 0.05 mM Ca2+

medium and double-immunostained with CLIC4 and K1 antibodies.Examples of spontaneously differentiating keratinocytes are indicatedby white arrows. All immunostained cells were analyzed by confocalmicroscopy as described in the Materials and Methods. Insets showDAPI staining. (C) Mouse keratinocytes in 0.05 mM Ca2+ mediumwere infected with adenoviruses encoding �-gal, wild-type CLIC4(Cyt-CLIC4), CLIC4 (Nuc-CLIC4) or nuclear-targeted GFP (Nuc-GFP) for 16 hours, treated with MQAE fluorescent dyes and assayedby confocal microscopy. (Insets) The chloride ion content of the �-galactosidase, Cyt-CLIC4 or Nuc-CLIC4 Ad infected cells treatedwith NPPB was visualized by confocal microscopy. (D) Thefluorescence intensity was also measured in similarly treated cells.Fluorescence on the y axis is shown in arbitrary units and decreasingvalues (quenching) indicates greater Cl– content. (E) Mousekeratinocytes were treated with increasing amount of NPPB (gradienttriangle, 200 �M for 1 and 2 mM for 10) for 2 hours prior tocalcium (0.5 mM) induction. After 24 hours, cell lysates wereanalyzed by immunoblots using K10, CLIC4 and actin antibodies.

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(Fig. 6C) incorporating these sites is induced by high Ca2+

medium when transfected into mouse keratinocytes, and this isprevented by A-Fos. Analogous sites for AP1 regulation aredetected in the mouse CLIC4 promoter (Fig. 6D). When eithersite A or B, or both, in the human CLIC4 promoter is mutated,luciferase reporter activity in response to 0.5 mM Ca2+ isabrogated (Fig. 6E). In addition, nuclear extracts fromkeratinocytes bind to each of these sites, and binding isenhanced in extracts derived from keratinocytes induced todifferentiate by Ca2+ (Fig. 6F). Mutation of either site A or siteB abrogates binding to that site, and A-Fos expression inkeratinocytes prevents binding to either site. Together, thesedata reveal the functional importance of AP1 in the regulationof CLIC4 and indicate that AP1 activity is sufficient to increaseCLIC4 expression in keratinocytes induced to differentiate byCa2+.

DiscussionFlux of different ions that are specifically modulated bymembrane transport proteins including channels, pumps andcarriers sets an environment that favors the initiation ofdifferentiation programs in keratinocytes and other cell types(Hennings et al., 1983a; Mauro et al., 1993; Debska et al.,2001; Ronnov-Jessen et al., 2002). The murine intracellularchloride channel protein CLIC4 was identified as adifferentially expressed gene in p53-wild-type versus p53-nullkeratinocytes that were induced to differentiate by Ca2+.Subsequently, CLIC4 was proved to be a direct downstreamtarget of p53 in keratinocytes that was also regulated

independently of p53 (Fernandez-Salas et al., 1999;Fernandez-Salas et al., 2002). We now report that CLIC4 isdirectly involved in mouse and human keratinocytedifferentiation, and nuclear translocation of CLIC4 is anintegral part of the Ca2+-induced differentiation response.Thus, expression of keratinocyte differentiation markers (K1,K10 and filaggrin) and a rapid G1 cell cycle arrest are stronglyassociated with CLIC4 upregulation and nuclear translocation.Reduction of CLIC4 by antisense or shRNA approachesprevents expression of these differentiation markers andprevents cell growth arrest associated with differentiation.CLIC4 is also frequently found in the nucleus of differentiatingcells from mouse and human epidermis in vivo. The findingsreported here also suggests that nuclear translocation of CLIC4is associated with increased Cl– ion content in the nucleus, andthis ionic flux could be related to the nuclear events that areassociated with activation of intrinsic differentiation programs.The signals downstream from Ca2+ that regulate expression andtranslocation of CLIC4 in differentiating keratinocytes remainto be delineated. However, our data suggest that PKC� is animportant intermediary in the regulation of CLIC4, and CLIC4expression is more directly regulated by AP1 factors, joininga host of other differentiation-induced AP1-regulated markers


Fig. 6. CLIC4 is regulated by AP1 in keratinocytes undergoingCa2+-induced differentiation. (A) Mouse keratinocytes in 0.05 mMCa2+ medium were infected with �-gal or A-Fos adenovirus for 12hours prior to induction with 0.5 mM Ca2+ medium for 24 hours.Duplicate sets of cells were analyzed by real-time quantitative RT-PCR using involucrin, K1 and CLIC4 gene-specific primer sets.Bars represent standard error. (B) After 24 hours, the treated cellsfrom A were analyzed by immunoblotting using K10, CLIC4 andactin antibodies. Asterisks (*) in A-Fos lanes of both CLIC4 andK10 blots indicate ‘undetectable’ CLIC4 and K10 bands that arewithin the linear exposure range of chemiluminescence. With longerexposures CLIC4 and K1 are detected in the A-Fos group (data notshown). (C) Triplicate sets of mouse keratinocytes were transfectedwith a reporter plasmid containing a 2.5 kb human CLIC4 promoterreporter construct prior to the treatment as described in A. Thepromoter responses were analyzed by measuring luciferase activity16 hours after Ca2+ induction. (D) Schematic diagram of mouse andhuman CLIC4 promoters; solid black arrow indicates transcriptionstart site, solid gray circles indicate AP1 binding sites and locations5 upstream from the transcription start site. Two AP1 consensussites are shown in the human promoter (A: –1507 and B: –840).(E) Luciferase reporter constructs containing the wild-type (WT),AP1-A site mutant (Mt A), AP1-B site mutant (Mt B), both AP1-Aand AP1-B sites mutant (Mt A+B) of human CLIC4 promoter andan empty reporter construct (pGL3; Mock) were used to transfectmouse primary keratinocytes. Triplicate sets of transfected cellswere induced with 0.5 mM calcium, and the promoter activitieswere measured by luciferase assay and presented as a fold increasein the promoter activities in high calcium versus low calcium-treatedcells. (F) Biotinylated DNA oligos containing the wild-type (WT Aand WT B) or the mutant (Mt A and Mt B) AP1 consensus bindingsites derived from human CLIC4 promoter sequence were used todetect AP1 binding activities from the nuclear extracts generatedfrom low or high calcium-treated cells in the absence or presence ofA-Fos expression. AP1 proteins and the DNA oligo complex formedin the nuclear extracts were detected by ELISA-based assays asdescribed in the Materials and Methods section, and the signalswere normalized by the protein concentration of the nuclearextracts.

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in keratinocytes (Eckert and Welter, 1996; Angel et al., 2001).Further studies are required to determine if PKC� is workingthrough AP1 to regulate CLIC4 expression. The human CLIC4promoter encompasses two AP1 consensus binding sites(–1507 to –1499; gTGAaTCAg and –840 to –832;gTGAgTCAa). These sites are conserved in a similar regionupstream from the first exon in the mouse CLIC4 promoter.Mutation of these sites abrogates the activation of the CLIC4promoter in response to Ca2+, indicating they are functionalregulatory sites, and ELISA-based EMSA assays confirmbinding activity. AP1 factors now join with p53 and Myc askey regulators of this multifunctional protein (Fernandez-Salaset al., 2002; Shiio et al., 2006).

CLIC family members have been associated with celldifferentiation and development in other models. CLIC4 ishighly expressed when mouse 3T3-L1 cells differentiate intoadipocytes (Kitamura et al., 2001). CLIC4 is also upregulatedduring the TGF�1-mediated differentiation of fibroblasts intomyofibroblasts in breast cancer cells (Ronnov-Jessen et al.,2002). Similarly, chloride channel activity is required inTGF�-induced differentiation of human lung fibroblast tomyofibroblast (Yin and Watsky, 2005). EXC-4, a homologueof CLIC proteins, plays a critical role in digestive/excretorytubulogenesis in C. elegans (Berry et al., 2003), and CLIC4 isa key factor in VEGF-induced tubular morphogenesis ofendothelial cells (Bohman et al., 2005). Tubulogenesis involvesmany cellular processes, including differentiation, apoptosis,cytoskeletal organization and ion fluxes (Zegers et al., 2003).CLIC1 is also involved in differentiation induced byinsulin/IGFI-mediated signaling in human hematopoietic cells(Saeki et al., 2005). These reports and our studies support thatimportance of CLICs in several cell differentiation programs.Whether this is related to their chloride channel functionsremains to be determined, but the multiple levels of regulationof CLIC4 and its subcellular trafficking suggest this protein isimportant in maintaining homeostasis.

Ion flux that is specifically modulated by membranetransport proteins including channels, pumps and carriers hasbeen associated with differentiation in other cell types. hERGK+ channel activation is mediated by integrin signals and is adetermining factor for osteoclastic differentiation (Arcangeli etal., 2004). Several anion channels are also reported to beinvolved in differentiation in multiple cell types; these channelsinclude swelling-activated Cl– channels, volume-activated Cl–

channels, voltage-dependent intracellular chloride channelClCs and P-glycoprotein mdr-1 (Nilius et al., 1996; Nilius andDroogmans, 2003; d’Anglemont de Tassigny et al., 2003;Remillard and Yuan, 2005). Chloride ion flux is also relevantto cancer since re-expression of detachment-inducible chloridechannel (mCLCA5) suppresses growth of metastatic breastcancer cells (Beckley et al., 2004), and swelling-activated Cl–

current is associated with neuroendocrine differentiation ofprostate cancer cells (Lemonnier et al., 2005).

The work of Mauro et al. and Bikle et al. have implicatedseveral channels and ion transporters in the regulation ofepidermal differentiation (Mauro et al., 1993). Ca2+ is a wellestablished regulator of keratinocyte maturation, and a Ca2+

gradient across the living epidermis in vivo supports thesignificance of Ca2+ in epidermal homeostasis. The presenceof a Ca2+ sensing signaling receptor on keratinocytes hasprovided significant insight into the mechanisms involved in

Ca2+ signaling (Komuves et al., 2002). Ablation of this receptorappears to work in concert with channels for other ions thatmay be the proximal regulators of the differentiation signal.For example, Ca2+-induced differentiation activates K+

conductance, and K+ channel inhibitors block keratinocytematuration (Hennings et al., 1983b; Mauro et al., 1997). Ofparticular interest to our studies are reports that Ca2+ activatesvoltage gated Cl– channels in keratinocytes (Mauro et al.,1990), and the activated Cl– current is associated withdifferentiation. Understanding the relationship of ion transportto Ca2+ signaling in keratinocytes remains an important tasksince reports of Ca2+ signaling abnormalities in psoriasis(McKenzie et al., 2003), Hailey-Hailey (Hu et al., 2000) andDarier’s disease (Sakuntabhai et al., 1999) suggest thatmodulation of downstream signaling could be an approach totherapy.

Nuclear translocation of CLIC4 as a coincident event inkeratinocyte differentiation was particularly striking in ourstudy. The presence of CLIC4 in the nucleus amplifies theexpression of differentiation markers. We previously reportedthat CLIC4 translocates to the nucleus by the interaction of anuclear localization signal with nuclear transport proteins(Ran, importin and NTF2) (Suh et al., 2004). Thus, nucleartranslocation is a physiological response. The function ofCLIC4 in the nucleus has not been clarified at this time. CLIC4binds with multiple partners, including actin, tubulin, dynamin,AKAPs and 14-3-3 isoforms (Suginta et al., 2001; Berrymanand Goldenring, 2003). These multiple protein-proteininteractions imply that CLIC4 has a broader molecular functionbeyond a chloride channel/regulator, and these interactingproteins, and others to be identified, may contribute to thephysiological functions of CLIC4. Immunoelectronmicroscopy indicates that nuclear CLIC4 resides in the nuclearmembrane and the nucleoplasm so that several functions maybe served by translocation (Suh et al., 2004).

In the nuclear membrane, CLIC4 could be involved inchloride channel activity. In support of this possibility,qualitative analyses of Cl– flux with MQAE dye indicatenuclear CLIC4 is associated with accumulation of Cl– thatwould subsequently acidify the microenvironment. Thenucleus may be particularly sensitive to pH changes asacidification could alter chromatin structure and the bindingefficiency of transcription factors (Eastman, 1995; Li andWeinman, 2002). For example, Runx transcription factors aresensitive to Cl– content in the nucleus for binding to targetsequences (Backstrom et al., 2002), and Runx activation isassociated with multiple cellular responses, including TFG�signals, cell cycle regulation and terminal differentiation inseveral cell types (Ito and Miyazono, 2003; Blyth et al., 2005).In fact, Runx3 is expressed in epidermis and is required fornormal hair follicle development (Raveh et al., 2005).

Additional studies will be required to define the functions ofCLIC4 in the nucleus and the signals that send it there. It isclear that CLIC4 contributes an essential regulatory role inepidermal homeostasis. Both in vivo and in vitro, by virtue ofthe level of expression and subcellular localization, CLIC4 isimportant for keratinocyte proliferation and terminaldifferentiation. Nuclear CLIC4 in particular is associated withkeratinocyte growth arrest under conditions of differentiation,senescence and inhibitory growth factors suggesting a broadrole of nuclear CLIC4 in growth control of skin keratinocytes.

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Materials and MethodsChemicalsBisindolylmaleimide I, Go 6976 and rottlerin were obtained from Calbiochem (SanDiego, CA). 12-O-tetradecanoyl-phorbol-13-acetate (TPA) was obtained fromSigma (St Louis, MO).

Cell cultureKeratinocytes were isolated and cultured as described previously (Dlugosz et al.,1995). Human keratinocytes from foreskin were isolated and cultured in EpiLifekeratinocyte medium as described by the manufacturer (Cascade Biologics,Portland, OR). For Ca2+ and TGF� induction, keratinocytes were treated withmedium containing Ca2+ levels higher than 0.1 mM (typically 0.5 or 1 mM) orTGF� (1 ng/ml). To induce serum-mediated differentiation in human keratinocytes,Ca2+-chelexed 5% FBS was added to EpiLife medium. To promote and assaysenescence of primary mouse keratinocytes, the �-galactosidase senescence assay(�-gal SA) was used as described previously (Vijayachandra et al., 2003).

Construction of shRNA for CLIC family membersHuman cDNA sequences of CLIC members were used as a query on public web-based programs (The RNAi Web) to determine preliminary sites and then pick onesequence (18-22 bp) from each group that does not overlap with other CLICmembers by manual cDNA sequence alignment and use of other publicly availablebioinformatic programs (e.g. siDirect). RNA interference compatibility for mousesequences was also considered, and the choice of sequences was manuallydetermined based on cDNA sequence alignments of mouse and human CLICmembers. Sequences are as follows: nonspecific shRNA (sR-NS) 5-gttctccgaacgtgtcacg-3, CLIC1 shRNA (sR-1) 5-gtctgattgagcttgtgttg-3, CLIC4 shRNA (sR-4) 5-gctgaaggaggaggacaaaga-3, CLIC5 shRNA (sR-5) 5-gacactgatctctcagac-3.All shRNA sequences have 5-uucaagaga-3 sequence as a loop prior to the directinverse repeat sequences to make the short-hairpin loop. The siRNA version ofCLIC4 shRNA was used and its efficiency in knockdown of CLIC4 was reportedin previous works (Bohman et al., 2005; Shiio et al., 2006). These shRNA sequenceswere inserted into pENTR-H1/TO vectors (BLOCK-iT system; Invitrogen) andverified as described by the manufacturer. Lipofectamine and Plus reagents wereused for transfection as described by the manufacturer (Invitrogen).

Production and purification of adenovirusesConstruction of antisense, sense or organelle-specific CLIC4 adenoviral vectors wasdescribed elsewhere (Suh et al., 2004). The use of dominant negative PKC� and A-Fos adenovirus (Ad) were described previously (Li et al., 1999; Bonovich et al.,2002). The adenoviral vector without a recombinant insert (null virus) or with �-gal was used as a control in experiments where adenoviruses were used. All theadenovirus was plaque purified, amplified as described by Clontech and purified bytwo rounds of CsCl centrifugation by Gene Expression Laboratory/NCI Frederick,Adenovirus Core Facility. To be consistent throughout the experiments, 10multiplicity of infection (MOI) was used in all adenoviral infections, except for MOIdose studies.

Immunoblot analysisProtein expression was analyzed by immunoblot as described previously (Suh et al.,2004). Monospecific rabbit antibodies (Abs) against mouse K1 (1:2000), K10(1:2000) and filaggrin (1:2000) were used to detect specific bands, as describedpreviously (Dlugosz and Yuspa, 1993). The following antibodies were also used:Anti-PKC� Ab (1:500) was from Sigma. Anti-�-actin mouse monoclonal antibodywas from Chemicon International Inc. (Temecula, CA). For densitometry analyses,ImageMaster TotalLab-v1.11 (Amersham-Pharmacia/GE Healthcare Bioscience,NJ) was used to quantify the fold changes of CLIC4 expression by normalizingagainst the actin signals. All immunoblots analyzed were within the linear range ofchemiluminescence exposure.

Immunofluorescent stainingKeratinocytes were stained as described in the previous report (Suh et al., 2004).Keratinocytes were exposed to 0.05 mM or higher Ca2+ medium for 24 hours afterinfection with null, wild-type CLIC4 (Cyt-CLIC4) and nucleus-targeted CLIC4(Nuc-CLIC4) Ad for 17 hours. Cells were subjected to immunofluorescenceanalysis with anti-K1or anti-CLIC4 antibodies at 1:200 dilutions followed byappropriate secondary antibodies conjugated to FITC or Texas Red (VectorLaboratories). The specificity of the CLIC4 and K1 primary antibodies has beendescribed in previous publications (Dlugosz and Yuspa, 1993; Fernandez-Salas etal., 1999). Cells stained with secondary antibody alone or purified control rabbitIgG were used as controls, and no specific immunofluorescent staining wasdetected. For detection of chloride flux, MQAE (Molecular Probes/Invitrogen) wasused as described by the manufacturer.

Cell cycle analysisMouse primary keratinocytes (5105) were infected with Null or AS-CLIC4 for 17hours and further incubated with or without additional Ca2+ for 24 hours or 48 hours.2 hours before harvesting the cells, 10 �M 5-bromo-2-deoxyuridine (BrdU) was

added to the medium and the trypsinized cells were analyzed by flow cytometry asdescribed by the manufacturer (BD Bioscience, FACScan). Data acquisition andanalysis were performed using Cell Quest software.

Immunodetection of AP1-oligo-transcription factor complexAn ELISA-based detection method that is equivalent of EMSA/SuperShift assay wasused to quantify AP1 binding to the biotinylated oligos that encode the sequencesderived from the human CLIC4 promoter with putative AP1 binding consensussequences. The oligo pairs generated from the AP1-A site (capitalized, –1507)sequences were 5-biotin-ggcttagatctgctttgaaacTGATTCAccatttggttcctttttg-3 for thewild type (WT A) and 5-biotin-ggcttagatctgctttgaaac GACTCAGccatttggttcctttttg-3for the mutant (Mt A). The oligo pairs generated from the AP1-B site (capitalized,–840) sequences were 5-biotin-gaagttgggaggagtagctgTGAGTCAagtattatg gaaag -tcag-3 for the wild type (WT B) and 5-biotin-gaagttgggaggagtagctg GACGC AG -agtattatggaaagtcag-3 for the mutant (Mt B). PAGE-purified sense and antisenseoligos were hybridized to form double strand DNA fragments and were incubated ata final concentration of 1.0 nM with the nuclear extracts at room temperature for 2hours. As positive control for the experiments, the procedure was repeated withrecombinant Fos-Jun mixture (data not shown, Active Motif, Calsbad, CA) andnuclear extracts of Jurkat (data not shown, untreated vs TPA treated) or U937 (datanot shown, untreated vs TPA treated). These nuclear extracts and the nuclearextraction kit were purchased from Active Motif. The mixture was then added tostreptavidin-coated plates (Pierce) that were pretreated with blocking buffer [1%BSA, 100 �g/ml sperm DNA, 1 APK (Ventana, AZ), 0.5% PMSF and 0.5 mMDTT], incubated at 4°C overnight and washed several times with wash buffer [1%BSA, 10 �g/ml Sperm DNA, 1 APK, 0.5% PMSF, Complete Protease Cocktail(Roche), and 50nM DTT]. A mixture of Fos and Jun polyclonal antibodies (bothfrom Santa Cruz Biotechnology)were added at 50 ng/ml to the wells, after whichthey were incubated for 2 hours at room temperature, extensively washed with thewash buffer and further incubated with donkey anti-rabbit HRP (GE HealthcareBioscience, Piscataway, NJ) at 1:50,000 dilution for 2 hours. The wells wereextensively washed with the wash buffer, chemiluminescent reagent (Dura, Pierce,IL) was added and the signal was measured using an Infinite M200 plate reader(Tecan, CA). Triplicate samples were processed for all experiments.

Detection of chloride ion flux and semi-quantitativemeasurementsNuclear chloride content was measured semi-quantitatively and qualitativelydetected by confocal microscopy using the chloride-ion-sensitive fluorescent dyeN-(6-methoxyquinolyle) acetoethyl ester (MQAE; Molecular Probes/Invitrogen)based on the methods described by the manufacturer and Maglova et al. (Maglovaet al., 1998a; Maglova et al., 1998b). The chloride channel inhibitor NPPB waspurchased from BioMol (Plymouth Meeting, PA). Briefly, cells were incubated withserum-free culture medium containing 10 mM MQAE for 2 hours and gently butquickly washed several times with Rinse-Buffer (20 mM HEPES, 100 mM NaCl,5 mM KCl, 1 mM MgCl2, 0.05 mM CaCl2, 10 mM glucose, pH 7.4). Backgroundfluorescence was determined by confocal microscopy after incubating the cells withthe Quenching-Buffer [Rinse-Buffer without NaCl, 100 mM NaNO3, 10 �Mnigericin (Biomol), 10 �M valinomycin (Biomol)] and used for normalization. Tovisualize chloride ion flux qualitatively, keratinocytes grown on 60 mm platesinfected with adenovirus expressing �-gal, wild-type CLIC4 (Cyt-CLIC4) andnuclear-targeted CLIC4 (Nuc-CLIC4) for 16 hours were treated as stated above, andfluorescent images were captured by using the 40 Achroplan objective on theZeiss-510 confocal microscope.

Fluorescence from the washed cells was measured using a Molecular DevicesfMax fluorometric plate reader with parameters set at 350 nm for excitation and 460nm for emission. 355 nm excitation filter and 460 nm emission filters (MolecularDevices) were used. Raw data were collected from the plate reader in triplicate andSoftMax Pro and Excel programs were used to calculate the average value that isrepresented in two digit numbers as arbitrary units for simplicity.

Immunostaining of skin sectionsAdult skin samples from humans or mice were formalin fixed, or snap-frozen andthen acetone fixed, respectively. For immunohistochemical staining of fixedsections, slides were deparafinized with HistoChoice (Amresco, Solon, OH), andthe antigen was retrieved with Target Retrieval Solution-High pH (Dako,Carpinteria, CA) as described by the manufacturer. Envision-secondary antibodyconjugated to horseradish peroxidase (Dako) and DAB (Pierce) were used to detectthe antigen as described by the manufacturer. Slides were analyzed with bright-fieldmicroscopy using Leitz-DMRB (Leica, Bannockburn, IL) and OpenLab(Improvision, Lexington, MA) software. Tissue sections stained with secondaryantibody alone or purified control rabbit IgG were used as controls, and no specificstaining was detected.

Production of recombinant CLIC1 and CLIC4 proteins and invitro kinase assayPlasmid pGEX-6P or pGEX-4T (Amersham Bioscience, NY) containing CLIC4 orCLIC1 cDNA, respectively, was used to produce the recombinant GST-CLIC4 or


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GST-CLIC1 protein, and the proteins were purified as described by themanufacturer. The purified proteins were phosphorylated in vitro with severalrecombinant and kinase-active human PKC isoforms (Invitrogen) as described bythe manufacturer. The kinase reaction mixture was resolved by SDS-PAGE, stainedand visualized with Coomassie Blue dye, and then the gel was exposed to PhosphorScreen (GE Healthcare Life Bio-Science, Piscataway, NJ). The phosphorylatedCLIC proteins were detected using Storm imager (Molecular Dynamics).

Real-time PCR analysisBio-Rad iQ iCycler and Gene Expression Macro (version 1.1) from Bio-Rad wereused to measure transcript expression levels. cDNA (diluted 1:50 in the final volumereaction) was measured using iQ SYBR Green Supermix (Bio-Rad) and includedexperimental duplicate reactions. The primer sequences were as follows: L32(forward) 5-tggttttcttgttg-3, (reverse) 5-gggtgcggagaagg-3, mouse CLIC4(forward) 5-tccttgagttatactcccagaacc-3 (reverse) 5-gtgagtttcactaacaaacgctc-3.Mouse keratin 1 and mouse involucrin primers were obtained from Superarray(Frederick, MD). Relative standard curves were generated from log input (serialdilutions of pooled cDNA) versus the threshold cycle (Ct). The linear correlationcoefficient (r2) was 0.81, 0.99 and 0.99 for CLIC4, K1 and involucrin respectively.The slope of the standard curve was used to determine the efficiency of targetamplification (E) using the equation E=(10–1/slope–1)100. Similar high efficiencywas obtained for all primers allowing for the comparative Ct method to be used.Relative quantitation was used to calculate the 2–(��Ct) formula where ��Ctrepresents the difference corrected for L32 used as internal control (Livak andSchmittgen, 2001). Electrophoresis analysis of amplified product from real-timePCR showed a single band.

CLIC4 promoter construction and reporter assay3.5 kb of human CLIC4 promoter (accession number: NT_004610, on humanchromosome 1p36.11 region, with VEGA coordinates contig 24,944,435-25,043,402, start of transcript location is AL445648.18.1.133587) was cloned bygenomic PCR and used as an insert to ligate with pGEM-Teasy (Promega). Theprimer pairs used for the genomic PCR were 5-ttaacatagactaatttatcaatgatccta-3 forforward and 5-ccgtgctgctcgctggactgtccggcgtcg-3 for reverse, and Expand HighFidelity PCR kit (Roche) was used. Separate primer sets 5-gagaGCTAGC -gtgtaccatgagctgtcctctgagccaggaatatagccataaacaaaaacc-3 (NheI-engineered sitecapitalized) for forward and 5-cttggtgtgtttcaggctctgagctagcccttgg-3 for reversewas used to subclone the PCR fragment into pGlow-TOPO (Invitrogen). A 2.5 kb(–2720 bp to –369 bp) fragment was further isolated by NheI digestion, subclonedinto pGL3 (Promega) and used for reporter assays. Mouse primary keratinocytes(2106/plate) were transfected with the reporter plasmid in triplicate usingLipofectamine (Invitrogen), treated with or without A-Fos Ad or 0.5 mM Ca2+

overnight, and the luciferase activities were measured using the Enhance LuciferaseAssay Kit as described by the manufacturer (BD Pharmingen, San Jose, CA). Thenormalization of the promoter assays was done by cell number and proteinconcentration. Putative AP1 sites were determined by Genomatrix software withinthe promoter fragment and designated AP1-A (–1507) and AP1-B (–840) upstreamfrom the putative transcription start site. To determine whether these two sites areresponsible for CLIC4 promoter activity during Ca2+ shift, the two sites weremutated separately (Mt A or Mt B) or together (Mt A+B) by deleting five bases(capitalized) out of seven AP1 consensus sequence (Mt A: TGATTca and Mt A:TGAGTca).

This research was supported by the Intramural Research Programof the NIH, National Cancer Institute, Center for Cancer Research.We thank Joanna Anders for assistance with references, Ulrike Lichtifor differential fractionations of mouse skin, Narayan Bhat of theScience Applications International Corporation, Inc. (SAIC),Frederick, NCI, Molecular Biology, Gene Expression Laboratory foradenoviral amplification, and Barbara Taylor of the CCR FACS CoreFacility. M. Mutoh was the recipient of JSPS Research Fellow inBiomedical and Behavioral Research at NIH (2003-2005).

ReferencesAngel, P., Szabowski, A. and Schorpp-Kistner, M. (2001). Function and regulation of

AP-1 subunits in skin physiology and pathology. Oncogene 20, 2413-2423.Arcangeli, A., Becchetti, A., Cherubini, A., Crociani, O., Defilippi, P., Guasti, L.,

Hofmann, G., Pillozzi, S., Olivotto, M. and Wanke, E. (2004). Physical andfunctional interaction between integrins and hERG potassium channels. Biochem. Soc.Trans. 32, 826-827.

Ashley, R. H. (2003). Challenging accepted ion channel biology: p64 and the CLIC familyof putative intracellular anion channel proteins (review). Mol. Membr. Biol. 20, 1-11.

Backstrom, S., Wolf-Watz, M., Grundstrom, C., Hard, T., Grundstrom, T. and Sauer,U. H. (2002). The RUNX1 Runt domain at 1.25A resolution: a structural switch andspecifically bound chloride ions modulate DNA binding. J. Mol. Biol. 322, 259-272.

Beckley, J. R., Pauli, B. U. and Elble, R. C. (2004). Re-expression of detachment-

inducible chloride channel mCLCA5 suppresses growth of metastatic breast cancercells. J. Biol. Chem. 279, 41634-41641.

Berry, K. L., Bulow, H. E., Hall, D. H. and Hobert, O. (2003). A C. elegans CLIC-likeprotein required for intracellular tube formation and maintenance. Science 302, 2134-2137.

Berryman, M. A. and Goldenring, J. R. (2003). CLIC4 is enriched at cell-cell junctionsand colocalizes with AKAP350 at the centrosome and midbody of cultured mammaliancells. Cell Motil. Cytoskeleton 56, 159-172.

Blyth, K., Cameron, E. R. and Neil, J. C. (2005). The RUNX genes: gain or loss offunction in cancer. Nat. Rev. Cancer 5, 376-387.

Bohman, S., Matsumoto, T., Suh, K., Dimberg, A., Jakobsson, L., Yuspa, S. andClaesson-Welsh, L. (2005). Proteomic analysis of vascular endothelial growth factor-induced endothelial cell differentiation reveals a role for chloride intracellular channel4 (CLIC4) in tubular morphogenesis. J. Biol. Chem. 280, 42397-42404.

Bonovich, M., Olive, M., Reed, E., O’Connell, B. and Vinson, C. (2002). Adenoviraldelivery of A-FOS, an AP-1 dominant negative, selectively inhibits drug resistance intwo human cancer cell lines. Cancer Gene Ther. 9, 62-70.

d’Anglemont de Tassigny, A., Souktani, R., Ghaleh, B., Henry, P. and Berdeaux, A.(2003). Structure and pharmacology of swelling-sensitive chloride channels,I(Cl,swell). Fundam. Clin. Pharmacol. 17, 539-553.

Debska, G., Kicinska, A., Skalska, J. and Szewczyk, A. (2001). Intracellular potassiumand chloride channels: an update. Acta Biochim. Pol. 48, 137-144.

Denning, M. F., Dlugosz, A. A., Williams, E. K., Szallasi, Z., Blumberg, P. M. andYuspa, S. H. (1995). Specific protein kinase C isozymes mediate the induction ofkeratinocyte differentiation markers by calcium. Cell Growth Differ. 6, 149-157.

Dlugosz, A. A. and Yuspa, S. H. (1993). Coordinate changes in gene expression whichmark the spinous to granular cell transition in epidermis are regulated by protein kinaseC. J. Cell Biol. 120, 217-225.

Dlugosz, A. A., Glick, A. B., Tennenbaum, T., Weinberg, W. C. and Yuspa, S. H.(1995). Isolation and utilization of epidermal keratinocytyes for oncogene research. InMethods in Enzymology (ed. P. K. Vogt and I. M. Verma), pp. 3-20. New York:Academic Press.

Dotto, G. P. (1999). Signal transduction pathways controlling the switch betweenkeratinocyte growth and differentiation. Crit. Rev. Oral Biol. Med. 10, 442-457.

Eastman, A. (1995). Assays for DNA fragmentation, endonucleases, and intracellular pHand Ca2+ associated with apoptosis. Methods Cell Biol. 46, 41-55.

Eckert, R. L. and Welter, J. F. (1996). Transcription factor regulation of epidermalkeratinocyte gene expression. Mol. Biol. Rep. 23, 59-70.

Edwards, J. C. and Kapadia, S. (2000). Regulation of the bovine kidney microsomalchloride channel p64 by p59fyn, a Src family tyrosine kinase. J. Biol. Chem. 275,31826-31832.

Fernandez-Salas, E., Sagar, M., Cheng, C., Yuspa, S. H. and Weinberg, W. C. (1999).p53 and tumor necrosis factor � regulate the expression of a mitochondrial chloridechannel protein. J. Biol. Chem. 274, 36488-36497.

Fernandez-Salas, E., Suh, K. S., Speransky, V. V., Bowers, W. L., Levy, J. M., Adams,T., Pathak, K. R., Edwards, L. E., Hayes, D. D., Cheng, C. et al. (2002).mtCLIC/CLIC4, an organellular chloride channel protein, is increased by DNA damageand participates in the apoptotic response to p53. Mol. Cell. Biol. 22, 3610-3620.

Gerdes, M. J., Myakishev, M., Frost, N. A., Rishi, V., Moitra, J., Acharya, A., Levy,M. R., Park, S. W., Glick, A., Yuspa, S. H. and Vinson, C. (2006). Activator protein-1 activity regulates epithelial tumor cell identity. Cancer Res. 66, 7578-7588.

Harrop, S. J., DeMaere, M. Z., Fairlie, W. D., Reztsova, T., Valenzuela, S. M.,Mazzanti, M., Tonini, R., Qiu, M. R., Jankova, L., Warton, K. et al. (2001). Crystalstructure of a soluble form of the intracellular chloride ion channel CLIC1 (NCC27)at 1.4-A resolution. J. Biol. Chem. 276, 44993-45000.

Hennings, H., Holbrook, K. A. and Yuspa, S. H. (1983a). Factors influencing calcium-induced terminal differentiation in cultured mouse epidermal cells. J. Cell. Physiol.116, 265-281.

Hennings, H., Holbrook, K. A. and Yuspa, S. H. (1983b). Potassium mediation ofcalcium induced terminal differentiation of epidermal cells in culture. J. Invest.Dermatol. 81, 50s-55s.

Hu, Z., Bonifas, J. M., Beech, J., Bench, G., Shigihara, T., Ogawa, H., Ikeda, S.,Mauro, T. and Epstein, E. H., Jr (2000). Mutations in ATP2C1, encoding a calciumpump, cause Hailey-Hailey disease. Nat. Genet. 24, 61-65.

Ito, Y. and Miyazono, K. (2003). RUNX transcription factors as key targets of TGF-betasuperfamily signaling. Curr. Opin. Genet. Dev. 13, 43-47.

Jentsch, T. J., Stein, V., Weinreich, F. and Zdebik, A. A. (2002). Molecular structureand physiological function of chloride channels. Physiol. Rev. 82, 503-568.

Kitamura, A., Nishizuka, M., Tominaga, K., Tsuchiya, T., Nishihara, T. andImagawa, M. (2001). Expression of p68 RNA helicase is closely related to the earlystage of adipocyte differentiation of mouse 3T3-L1 cells. Biochem. Biophys. Res.Commun. 287, 435-439.

Komuves, L., Oda, Y., Tu, C. L., Chang, W. H., Ho-Pao, C. L., Mauro, T. and Bikle,D. D. (2002). Epidermal expression of the full-length extracellular calcium-sensingreceptor is required for normal keratinocyte differentiation. J. Cell. Physiol. 192, 45-54.

Lee, Y. S., Dlugosz, A. A., McKay, R., Dean, N. M. and Yuspa, S. H. (1997). Definitionby specific antisense oligonucleotides of a role for protein kinase C alpha in expressionof differentiation markers in normal and neoplastic mouse epidermal keratinocytes.Mol. Carcinog. 18, 44-53.

Lemonnier, L., Lazarenko, R., Shuba, Y., Thebault, S., Roudbaraki, M., Lepage, G.,Prevarskaya, N. and Skryma, R. (2005). Alterations in the regulatory volumedecrease (RVD) and swelling-activated Cl- current associated with neuroendocrinedifferentiation of prostate cancer epithelial cells. Endocr. Relat. Cancer 12, 335-349.

Jour

nal o

f Cel

l Sci

ence

2640

Li, L., Lorenzo, P. S., Bogi, K., Blumberg, P. M. and Yuspa, S. H. (1999). Proteinkinase C� targets mitochondria, alters mitochondrial membrane potential, and inducesapoptosis in normal and neoplastic keratinocytes when overexpressed by an adenoviralvector. Mol. Cell. Biol. 19, 8547-8558.

Li, X. and Weinman, S. A. (2002). Chloride channels and hepatocellular function:prospects for molecular identification. Annu. Rev. Physiol. 64, 609-633.

Littler, D. R., Assaad, N. N., Harrop, S. J., Brown, L. J., Pankhurst, G. J., Luciani,P., Aguilar, M. I., Mazzanti, M., Berryman, M. A., Breit, S. N. et al. (2005). Crystalstructure of the soluble form of the redox-regulated chloride ion channel proteinCLIC4. FEBS J. 272, 4996-5007.

Livak, K. J. and Schmittgen, T. D. (2001). Analysis of relative gene expression datausing real-time quantitative PCR and the 2(–Delta Delta C(T)) Method. Methods 25,402-408.

Maglova, L. M., Crowe, W. E., Altamirano, A. A. and Russell, J. M. (1998a). Humancytomegalovirus infection stimulates Cl-/HCO-3 exchanger activity in humanfibroblasts. Am. J. Physiol. 275, C515-C526.

Maglova, L. M., Crowe, W. E., Smith, P. R., Altamirano, A. A. and Russell, J. M.(1998b). Na+-K+-Cl– cotransport in human fibroblasts is inhibited by cytomegalovirusinfection. Am. J. Physiol. 275, C1330-C1341.

Mauro, T. M., Pappone, P. A. and Isseroff, R. R. (1990). Extracellular calcium affectsthe membrane currents of cultured human keratinocytes. J. Cell. Physiol. 143, 13-20.

Mauro, T. M., Isseroff, R. R., Lasarow, R. and Pappone, P. A. (1993). Ion channelsare linked to differentiation in keratinocytes. J. Membr. Biol. 132, 201-209.

Mauro, T., Dixon, D. B., Komuves, L., Hanley, K. and Pappone, P. A. (1997).Keratinocyte K+ channels mediate Ca2+-induced differentiation. J. Invest. Dermatol.108, 864-870.

McKenzie, R. C., Oda, Y., Szepietowski, J. C., Behne, M. J. and Mauro, T. (2003).Defective cyclic guanosine monophosphate-gated calcium channels and thepathogenesis of psoriasis. Acta Derm. Venereol. 83, 414-418.

Nilius, B. and Droogmans, G. (2003). Amazing chloride channels: an overview. ActaPhysiol. Scand. 177, 119-147.

Nilius, B., Eggermont, J., Voets, T. and Droogmans, G. (1996). Volume-activated Cl-channels. Gen. Pharmacol. 27, 1131-1140.

Novarino, G., Fabrizi, C., Tonini, R., Denti, M. A., Malchiodi-Albedi, F., Lauro, G.M., Sacchetti, B., Paradisi, S., Ferroni, A., Curmi, P. M. et al. (2004). Involvementof the intracellular ion channel CLIC1 in microglia-mediated beta-amyloid-inducedneurotoxicity. J. Neurosci. 24, 5322-5330.

Qian, Z., Okuhara, D., Abe, M. K. and Rosner, M. R. (1999). Molecular cloning andcharacterization of a mitogen-activated protein kinase-associated intracellular chloridechannel. J. Biol. Chem. 274, 1621-1627.

Raveh, E., Cohen, S., Levanon, D., Groner, Y. and Gat, U. (2005). Runx3 is involvedin hair shape determination. Dev. Dyn. 233, 1478-1487.

Remillard, C. V. and Yuan, J. X. (2005). ClC-3: more than just a volume-sensitive Cl–

channel. Br. J. Pharmacol. 145, 1-2.

Ronnov-Jessen, L., Villadsen, R., Edwards, J. C. and Petersen, O. W. (2002).Differential expression of a chloride intracellular channel gene, CLIC4, in transforminggrowth factor-beta1-mediated conversion of fibroblasts to myofibroblasts. Am. J.Pathol. 161, 471-480.

Rutberg, S. E., Saez, E., Lo, S., Jang, S. I., Markova, N., Spiegelman, B. M. andYuspa, S. H. (1997). Opposing activities of c-Fos and Fra-2 on AP-1 regulatedtranscriptional activity in mouse keratinocytes induced to differentiate by calcium andphorbol esters. Oncogene 15, 1337-1346.

Saeki, K., Yasugi, E., Okuma, E., Breit, S. N., Nakamura, M., Toda, T., Kaburagi,Y. and Yuo, A. (2005). Proteomic analysis on insulin signaling in human hematopoieticcells: identification of CLIC1 and SRp20 as novel downstream effectors of insulin. Am.J. Physiol. Endocrinol. Metab. 289, E419-E428.

Sakuntabhai, A., Ruiz-Perez, V., Carter, S., Jacobsen, N., Burge, S., Monk, S., Smith,M., Munro, C. S., O’Donovan, M., Craddock, N. et al. (1999). Mutations inATP2A2, encoding a Ca2+ pump, cause Darier disease. Nat. Genet. 21, 271-277.

Shiio, Y., Suh, K. S., Lee, H., Yuspa, S. H., Eisenman, R. N. and Aebersold, R. (2006).Quantitative proteomic analysis of myc-induced apoptosis: a direct role for Mycinduction of the mitochondrial chloride ion channel, mtCLIC/CLIC4. J. Biol. Chem.281, 2750-2756.

Suginta, W., Karoulias, N., Aitken, A. and Ashley, R. H. (2001). Chloride intracellularchannel protein CLIC4 (p64H1) binds directly to brain dynamin I in a complexcontaining actin, tubulin and 14-3-3 isoforms. Biochem. J. 359, 55-64.

Suh, K. S. and Yuspa, S. H. (2005). Intracellular chloride channels: critical mediatorsof cell viability and potential targets for cancer therapy. Curr. Pharm. Des. 11, 2753-2764

Suh, K. S., Mutoh, M., Nagashima, K., Fernandez-Salas, E., Edwards, L. E., Hayes,D. D., Crutchley, J. M., Marin, K. G., Dumont, R. A., Levy, J. M. et al. (2004).The organellular chloride channel protein CLIC4/mtCLIC translocates to the nucleusin response to cellular stress and accelerates apoptosis. J. Biol. Chem. 279, 4632-4641.

Suh, K. S., Mutoh, M., Gerdes, M., Crutchley, J. M., Mutoh, T., Edwards, L. E.,Dumont, R. A., Sodha, P., Cheng, C., Glick, A. et al. (2005). Antisense suppressionof the chloride intracellular channel family induces apoptosis, enhances tumor necrosisfactor {alpha}-induced apoptosis, and inhibits tumor growth. Cancer Res. 65, 562-571.

Tulk, B. M., Kapadia, S. and Edwards, J. C. (2002). CLIC1 inserts from the aqueousphase into phospholipid membranes, where it functions as an anion channel. Am. J.Physiol. Cell Physiol. 282, C1103-C1112.

Vijayachandra, K., Lee, J. and Glick, A. B. (2003). Smad3 regulates senescence andmalignant conversion in a mouse multistage skin carcinogenesis model. Cancer Res.63, 3447-3452.

Yin, Z. and Watsky, M. A. (2005). Chloride channel activity in human lung fibroblastsand myofibroblasts. Am. J. Physiol. Lung Cell. Mol. Physiol. 288, L1110-L1116.

Zegers, M. M., O’Brien, L. E., Yu, W., Datta, A. and Mostov, K. E. (2003). Epithelialpolarity and tubulogenesis in vitro. Trends Cell Biol. 13, 169-176.


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