Research Article 2631 CLIC4 mediates and is required for Ca2+-induced keratinocyte differentiation

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

Summary Keratinocyte differentiation requires integrating signaling transforming growth factor ␤ (TGF␤) treatment. Targeting among intracellular ionic changes, kinase cascades, CLIC4 to the nucleus of keratinocytes via adenoviral sequential expression, cell cycle arrest, and transduction increases nuclear Cl– content and enhances programmed cell death. We now show that Cl– intracellular expression of differentiation markers in the absence of channel 4 (CLIC4) expression is increased in both mouse elevated Ca2+. In vivo, CLIC4 is localized to the epidermis and human keratinocytes undergoing differentiation in mouse and human skin, where it is predominantly induced by Ca2+, serum and the protein kinase C (PKC)- nuclear in quiescent cells. These results suggest that CLIC4 activator, 12-O-tetradecanoyl-phorbol-13-acetate (TPA). participates in epidermal homeostasis through both Elevation of CLIC4 is associated with signaling by PKC␦, alterations in the level of expression and subcellular and knockdown of CLIC4 protein by antisense or shRNA localization. Nuclear CLIC4, possibly by altering the Cl– prevents Ca2+-induced keratin 1, keratin 10 and filaggrin and pH of the nucleus, contributes to cell cycle arrest and expression and cell cycle arrest in differentiating the specific program associated with keratinocytes. CLIC4 is cytoplasmic in actively keratinocyte terminal differentiation. proliferating keratinocytes in vitro, but the cytoplasmic CLIC4 translocates to the nucleus in keratinocytes Key words: CLIC4, , PKC, Calcium, undergoing growth arrest by differentiation, senescence or Differentiation, Keratinocytes, AP1

Introduction transport factor 2 (NTF2), Ran and importin-␣, active

Journal of Cell Science The CLIC family of chloride intracellular channels is components of the nuclear import machinery, suggesting that composed of seven differentially expressed proteins that the nuclear localization signal (NLS)-recognition protein localize to the cytoplasm and intracellular organelles in many complex may be involved in CLIC4 nuclear trafficking (Suh et cell types (Suh and Yuspa, 2005). Five members of the CLIC al., 2004). The physiological consequences of these family are highly homologous in size and sequence (CLIC1- interactions is under study, but CLIC1 and CLIC4 has been 5), whereas p64 (CLIC5B) and parchorin (CLIC6) have implicated in regulation of the G2-M phase of the cell cycle in extended amino-terminal domains and are considerably larger. CHO cells (Berryman and Goldenring, 2003), and CLIC4 is Crystallographic analysis of CLIC1 and CLIC4 indicates that associated with adipocyte differentiation in 3T3-L1 cells the protein exists in both soluble and membrane bound forms (Kitamura et al., 2001). and is structurally related to the omega class of glutathione S- In keratinocytes, an increase in CLIC4 expression is transferases (Harrop et al., 2001; Littler et al., 2005). Several associated with Ca2+-induced differentiation, DNA damage, CLIC proteins have Cl– selective channel activity (Ashley, p53 upregulation and exposure to TNF␣ (Fernandez-Salas et 2003), but it is not yet clear if they form pores by themselves al., 1999; Fernandez-Salas et al., 2002; Suh et al., 2004). In or participate in ion flux indirectly (Jentsch et al., 2002). addition to cytoplasmic residence, CLIC4 is a component of Recent data from several laboratories have suggested that the inner mitochondrial membrane, and maintenance of CLIC4 CLIC family members also participate in cell signaling. CLIC3 levels is essential for viability of keratinocytes (Fernandez- interacts with ERK7 in the nucleus of mammalian cells and Salas et al., 2002). Thus, those conditions that elevate CLIC4 stimulates chloride conductance (Qian et al., 1999). p64 is a are associated with cell cycle arrest or cell death. CLIC4 substrate for Fyn kinase, and the combination enhances elevation in differentiating keratinocyte could participate in the chloride channel activity when co-expressed in HeLa cells terminal phase of the differentiation process when (Edwards and Kapadia, 2000). CLIC1 is associated with TNF␣ keratinocytes undergo cell cycle arrest, karyorrhexis and die. release in amyloid-␤-treated rat microglial cells (Novarino et Cytoplasmic CLIC4 translocates to the nucleus in stressed cells al., 2004). CLIC4 is induced by TGF␤ in mammary fibroblasts (Suh et al., 2004), and nuclear CLIC4 could also participate in as they transdifferentiate into myofibroblasts (Ronnov-Jessen other aspects of the differentiation response such as the gene et al., 2002), and CLIC4 binds to 1 in a complex expression changes that are associated with differentiation. involving , and 14-3-3␨ in rat brain extracts Previous studies have suggested that changes in intracellular (Suginta et al., 2001). CLIC4 also interacts with nuclear chloride and pH are associated with keratinocyte 2632 Journal of Cell Science 120 (15)

differentiation (Mauro et al., 1990; Mauro et al., 1993). This (Denning et al., 1995). To determine if PKC signaling is study was undertaken to document the changes in CLIC4 involved in CLIC4 expression, mouse and human keratinocytes protein that may be associated with keratinocyte differentiation were treated with the PKC activator TPA. As shown in Fig. 1D, and to explore the potential function of these changes in the this treatment increased CLIC4 levels within 24 hours also, differentiation response of keratinocytes to increasing suggesting a PKC-regulated process. To explore the connection extracellular Ca2+. among Ca2+ signaling, PKC activation, keratinocyte differentiation and CLIC4 induction, mouse keratinocytes Results induced to differentiate by 0.5 mM Ca2+ were treated with CLIC4 is Ca2+ responsive and PKC dependent in mouse selective PKC inhibitors and probed for expression of CLIC4, and human keratinocytes keratin 1 (K1) and keratin 10 (K10; Fig. 2A). In control cells, Previous results had indicated that CLIC4 transcripts and both CLIC4 and the differentiation markers increase by 24 protein increased in mouse keratinocytes induced to hours after Ca2+ induction. The PKC␣ selective inhibitor differentiate by Ca2+ (Fernandez-Salas et al., 1999). In mouse GO6976 did not inhibit either CLIC4 or the differentiation keratinocytes, CLIC4 protein and transcript levels increase marker response to Ca2+. In fact, both responses increased with after 24 hours relative to the level of extracellular Ca2+ (Fig. GO6976, consistent with the negative effect of PKC␣ 1A,B). Both Ca2+ and serum also elevate CLIC4 in human activation on markers of spinous differentiation reported earlier foreskin keratinocytes (Fig. 1A,C). Ca2+ is known to activate (Lee et al., 1997). By contrast, the general PKC inhibitor several protein kinase C (PKC) isoforms in keratinocytes bisindoylmaleimide-I at 10 ␮M and the PKC␦ selective inhibitor rottlerin reduced expression of both CLIC4 and K1, and K10, implying that PKC␦ regulates CLIC4 expression. Fig. 2B supports this conclusion since both differentiation markers and CLIC4 expression in Ca2+-induced keratinocytes were depressed by infection with an adenovirus encoding a PKC␦ dominant negative construct previously shown to effectively and selectively inhibit PKC␦ activity in keratinocytes (Li et al., 1999). In the presence of the dominant negative construct or the pharmacological inhibitor rottlerin, CLIC4 expression in high Ca2+ culture conditions was reduced toward or below the level in low Ca2+ culture. To test if CLIC4 is a specific and direct substrate for PKC, we incubated recombinant 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 a lack of specificity. Furthermore, we were unable to detect

Journal of Cell Science phosphorylation of CLIC4 after treatment of keratinocytes with TPA (data not shown). Taken together, the results suggest that CLIC4 is regulated by activation of PKC␦ but not by a specific direct phosphorylation of CLIC4 by PKC␦. Instead, PKC␦ may regulate the expression or stability of CLIC4 indirectly and the upregulation of CLIC serves as a downstream mediator of keratinocyte differentiation.

Upregulation of CLIC4 is required for Ca2+-induced keratinocyte differentiation To determine if upregulation of CLIC4 is essential for Ca2+- induced differentiation in keratinocytes, we reduced CLIC4 level by expressing CLIC4 antisense in an adenoviral vector (AS-CLIC4 Ad) in keratinocytes exposed to 0.5 mM (Hi) Ca2+ Fig. 1. CLIC4 is upregulated in differentiating keratinocytes. medium. We have previously shown that this vector reduces (A) Newborn mouse and human foreskin keratinocytes were cultured CLIC4 expression in a dose-dependent manner (Suh et al., in 0.05 mM Ca2+ medium for 4 days, then treated for 24 hours with 2005). Under conditions in which AS-CLIC4 adenovirus (Ad) the indicated concentrations of Ca2+ in the culture medium and reduced CLIC4 and prevented the Ca2+-induced increase in lysates were examined by immunoblotting. (B) RNA isolated from CLIC4 expression, K1 and K10 proteins were not elevated at mouse keratinocytes from A was amplified by RT-PCR, and CLIC4 24 hours after Ca2+ addition, and the expression of the late and actin transcripts were detected by ethidium bromide staining of differentiation marker filaggrin was inhibited after 48 hours agarose gels. (C) Human foreskin keratinocytes were cultured in (Fig. 3A). Expression of AS-CLIC4 did not alter the serum-free medium for 4 days and exposed to 5% FBS deleted of ␦ Ca2+ serum for the indicated times. (D) Mouse and human endogenous level of PKC (Fig. 3A), suggesting that CLIC4 keratinocyte were treated with indicated doses of TPA for 24 hours, upregulation is an important component of the differentiation 2+ and lysates were analyzed by immunoblotting. The fold change for response to elevated Ca . To verify this observation by an each band was determined by densitometry as described in Materials independent differentiation marker, cell cycle parameters were and Methods. monitored to determine if the rapid G1 cell cycle arrest CLIC4 modulation of Ca2+-induced differentiation 2633

Fig. 2. CLIC4 is a downstream target of PKC␦ in keratinocytes induced to differentiate by Ca2+. (A) Mouse keratinocytes were pre- treated with the indicated doses of PKC inhibitors for 1 hour, and 2+ then 0.5 mM Ca was added in the medium for 24 hours prior to Fig. 3. CLIC4 upregulation is required for Ca2+-induced lysis and immunoblotting. (B) Mouse keratinocytes were infected differentiation in keratinocytes. K1, K10, Filaggrin, CLIC1, CLIC4, with PKC␦ DN Ad for 17 hours before the treatment with 0.5 mM ␦ 2+ PKC and actin protein levels in mouse keratinocytes were Ca for 24 hours. Actin was used as a loading control. Bis, determined by immunoblot analysis. (A) Mouse keratinocytes were Journal of Cell Science bisindolylmaleimide I; Ro, rottlerin; Go, Go 6976. Similar data were co-infected with 5 MOI of Tet-Off Ad (to induce expression of the obtained from two independent experiments. The numbers over the conditional AS) and increasing MOI (5 and 10, shaded triangle) of CLIC4 bands in A and B represent fold change determined by AS-CLIC4 Ad or Null Ad (N) for 17 hours before treatment with 0.5 densitometry. (C) Purified recombinant CLIC1 or CLIC4 proteins mM Ca2+ for 24 hours. (B) Duplicate sets of mouse keratinocytes were incubated with purified, kinase active recombinant PKC ␥ 32 were treated with BrdU, and anti-BrdU-FITC stained cells of each isoforms in appropriate reaction buffers with [ - P]ATP, the reaction sample were analyzed with flow cytometry. Cell populations: null mixture was resolved by SDS-PAGE and then the phosphorylated Ad-infected without BrdU (upper left, N/(–)/ Lo Ca2+), with BrdU proteins were visualized by phospho-imaging methods. Un, no (upper right, N/(+)/Lo Ca2+) both in 0.05 mM Ca2+; with 0.5 mM recombinant PKC. Ca2+ 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 gated 2+ population in each cell cycle phase. The percentage of cells in each associated with Ca -induced differentiation, was altered 2+ (Dotto, 1999). Within 24 hours of 0.5 mM Ca2+ exposure, cell cycle phase from the total cell population in N/(+)/Lo Cal , N/(+)/Hi Ca2+ and AS/(+)/Hi Ca2+ at 24 hours and 48 hours after BrdU pulse-labeled cells were absent when assayed by flow Ca2+ induction is represented in a bar graph format. (D) Human 293 cytometry whereas the number in G0-G1 increased cells were transfected with constructs expressing nonspecific (NS) or substantially (Fig. 3B,C). However, these changes were CLIC4 (sR-CLIC4) shRNA for 48 hours, and specificity of CLIC4 prevented by expression of CLIC4 antisense, and the knockdown was analyzed by immunoblots of cell lysates. (E) Mouse proliferating population was maintained up to 48 hours as in keratinocytes were transfected with constructs expressing shRNA for the 0.05 mM Ca2+ medium (Fig. 3C). To verify CLIC4 is a nonspecific (NS), CLIC1 (sR-1), CLIC4 (sR-4) or CLIC5 (sR-5) for specific CLIC family member that mediates the keratinocyte 24 hours, and then treated with 0.05 mM (Lo) or 0.5 mM (Hi) Ca2+. differentiation response, non-specific (NS) and specific shRNA Actin was used as a loading control. Similar data were obtained from constructs were developed for CLIC1 (sR-1), CLIC4 (sR-4) at least two independent experiments. The numbers over the CLIC4 and CLIC5 (sR-5) and used for a transient knockdown of CLIC bands in A and B represent fold changes determined by densitometry. members (Fig. 3D,E). When transfected cells are induced to differentiate by 0.5 mM Ca2+, CLIC4 shRNA prevented upregulation of K10 but the other shRNAs did not. This result have been influenced by AS-CLIC4 expression (Suh et al., also distinguishes CLIC4 from other CLIC proteins that might 2005). 2634 Journal of Cell Science 120 (15)

CLIC4 translocates to the nucleus in differentiating keratinocytes in vitro and is localized to the nucleus in differentiating keratinocytes in vivo Previous data have shown that cytoplasmic CLIC4 translocates to the nucleus of cells undergoing a variety of stress responses (Suh et al., 2004). Similarly, CLIC4 translocates to the nucleus in both mouse and human keratinocytes that are induced to differentiate by Ca2+ (Fig. 4A). Interestingly, CLIC4 also translocates to the nucleus in keratinocytes undergoing G1 arrest because of senescence (Fig. 4B) or serum starvation (Fig. 4C) or when arrested by TGF␤ (Fig. 4D) or induced to differentiate by TPA (Fig. 4E) treatment. This intracellular trafficking of CLIC4 is dynamic since the nuclear CLIC4 in the serum-starved cells translocates back to the cytoplasm within 30 minutes upon addition of serum to the medium, suggesting that CLIC4 has a physiological function during cell cycle arrest and release (Fig. 4C). Translocation of CLIC4 to the nucleus in keratinocytes undergoing differentiation and senescence in vitro prompted us to investigate the localization of CLIC4 in vivo. In contrast to the cytoplasmic pattern of CLIC4 seen in the actively proliferating keratinocytes in culture, immunohistochemical studies demonstrate that CLIC4 is predominantly nuclear in the epidermis of adult human and mouse skin (Fig. 4F). CLIC4 is often nuclear in basal cells, but is excluded from the nucleus in a subpopulation of basal cells and some spinous cells. Overall CLIC4 cellular staining generally increases in the suprabasal region where differentiation is occurring. Almost all granular layer keratinocytes express CLIC4 in the nucleus. This staining pattern suggests that cycling keratinocytes are regulating the subcellular localization of CLIC4. It should be noted that nuclear CLIC4 can be detected in certain regions of the stromal compartment, most likely in endothelial cells where CLIC4 is Fig. 4. CLIC4 translocates to the nucleus in differentiating and senescing keratinocytes in vitro and is primarily nuclear in epidermis known to be important for blood vessel tubulogenesis (Bohman in vivo. (A) Mouse primary keratinocytes and human foreskin Journal of Cell Science et al., 2005). keratinocytes were treated with indicated Ca2+ for 24 hours and immunostained with CLIC4 antibody. (B) Mouse keratinocytes were Nuclear CLIC4 enhances the differentiation response maintained in 0.05 mM Ca2+ medium and either fixed on the first day To address whether upregulation and nuclear translocation of of culture (1 Day) or after 7 days of culture (7 Day) to allow for CLIC4 is sufficient to induce expression of differentiation senescence. Fixed cultures were immunostained with CLIC4 markers, keratinocytes in 0.05 mM Ca2+ medium were infected antibodies. Duplicate sets of cells were subjected to assay by ␤-gal with adenoviruses encoding intact CLIC4 (Cyt-CLIC4) or SA, and the number of senescing cells (7 Day) is presented as a fold CLIC4 with a nuclear targeting signal (Nuc-CLIC4). These increase over the control (1 Day; lower panel). (C) Mouse adenoviral vectors infect keratinocytes with high efficiency and keratinocytes were incubated in the serum-depleted medium for 16 hours (0 min), rinsed and treated with the serum-containing medium target appropriate organelles as described previously (Suh et (30 min). (D,E) Mouse keratinocytes were treated with TGF␤ al., 2004). At relatively low levels of expression over (1 ng/ml; D) or TPA (25 ng/ml; E) for the times indicated and endogenous CLIC4 levels, both constructs upregulated CLIC4, immunostained for CLIC4. Insets in Fig. 4C-E represent DAPI 2+ K1 and K10 in the absence of a Ca switch, but expression of nuclear staining. (F) Formalin-fixed human breast skin and acetone- the markers was greater when CLIC4 was targeted to the fixed mouse skin frozen sections were stained with CLIC4 antibodies nucleus (Fig. 5A). Thus, a small increase in the exogenous by immunohistochemical methods as described in the Materials and Nuc-CLIC4 expression, above the endogenous level of CLIC4, Methods. The mouse skin section was from a hyperplastic area to caused a significant increase in the keratinocyte differentiation enhance the resolution of strata. Red and green arrows indicate response. Primary keratinocytes cultured in basal medium keratinocytes in the basal layers (blue nucleus) and the differentiating (0.05 mM Ca2+) actively proliferate but individual cells layers (brown nucleus, CLIC4 stain) respectively. In all immunofluorescent and immunohistochemical staining experiments, spontaneously differentiate and detach from the culture dish. secondary antibody alone was used as controls. Confocal studies (Fig. 5B) show that the increased and nuclear expression of CLIC4 coincides (merged image) with the upregulation of K1 in keratinocytes that are spontaneously undergoing differentiation in the absence of extracellular Ca2+. together, the results shown in Fig. 5A and B suggest that In this setting, the nuclear CLIC4 is detected only in the nuclear translocation of CLIC4 coincides with the expression subpopulation of differentiating keratinocytes while the of early differentiation markers during keratinocyte cytoplasmic CLIC4 is detected in the neighboring cells. Taken differentiation. CLIC4 modulation of Ca2+-induced differentiation 2635

intracellular compartments (Tulk et al., 2002). To investigate if nuclear-specific CLIC4 alters chloride ion flux, the chloride ion-sensitive fluorescent dye MQAE was used. With this dye, the fluorescent light emission is inversely related to the Cl– concentration of the MQAE-containing intracellular compartment, and the fluorescent output can be measured semi-quantitatively and visualized by fluorometer and confocal microscopy. Fig. 5C indicates that targeting CLIC4 to the nucleus of keratinocytes via an adenovirus reduces fluorescence of nuclear MQAE detected by confocal microscopy, indicating influx of chloride ion into the nucleus. By contrast, expression of ␤-galactosidase, cytoplasmic CLIC4 or targeting GFP to the nucleus does not cause significant MQAE quenching, indicating that the CLIC4 result is not a consequence of overexpression of any exogenous protein in the cytoplasm or nucleus. This dye quenching effect can be blocked by treating the cells with a chloride channel inhibitor NPPB [5-nitro-2-(3-phenylpropylamino) benzoic acid] prior to Ad infection (Fig. 5C, first, second and third panel insets). In this experimental setting, overloading the culture medium with NaF (Cl– depletory agent) was used as a control to maximize the fluorescence. When NaF-treated cells were washed and reloaded with excess NaCl, the fluorescence was quenched again in the nucleus of nuclear-targeted CLIC4 cells but not in the cytoplasmic CLIC4 control, suggesting that the fluorescence is quenched by the nuclear CLIC4 (data not shown). When whole cell MQAE fluorescence is measured by fluorometry, the cells expressing Nuc-CLIC exhibit significant quenching of MQAE compared with the cells expressing wild- type (Cyt) CLIC4 (Fig. 5D). To investigate whether chloride 2+ Fig. 5. Nuclear CLIC4 induces the expression of keratinocyte ion flux is required for Ca -induced keratinocyte differentiation, cells were treated with a chloride channel differentiation markers, and intracellular chloride influences 2+ keratinocyte differentiation. (A) Mouse keratinocytes in 0.05 mM inhibitor prior to Ca induction. Treatment of keratinocytes Ca2+ medium were pre-treated with null, wild-type CLIC4 (Cyt) or with NPPB significantly reduced the expression level of K10 2+ Journal of Cell Science nuclear-targeted CLIC4 (Nuc) adenovirus for 12 hours, and cell upon Ca induction (Fig. 5E). At the highest concentration of lysates were analyzed by immunoblots using K1, K10, CLIC4 and NPPB (2 mM), both K10 and CLIC4 are reduced, a change actin antibodies. Gray arrow indicates the endogenous CLIC4 (Endo- that was associated with morphological evidence of toxicity. CLIC4) and the black arrows indicate the two exogenous CLIC4 2+ bands. (B) Mouse keratinocytes were cultured in 0.05 mM Ca CLIC4 is a down-stream target of AP1 medium and double-immunostained with CLIC4 and K1 antibodies. AP1 signaling is involved in Ca2+- and TPA-induced Examples of spontaneously differentiating keratinocytes are indicated by white arrows. All immunostained cells were analyzed by confocal keratinocyte differentiation through the regulation of microscopy as described in the Materials and Methods. Insets show transcription of multiple maturation markers (Eckert and DAPI staining. (C) Mouse keratinocytes in 0.05 mM Ca2+ medium Welter, 1996; Angel et al., 2001). In previous work we have were infected with adenoviruses encoding ␤-gal, wild-type CLIC4 shown that the expression of a highly specific AP1 dominant (Cyt-CLIC4), CLIC4 (Nuc-CLIC4) or nuclear-targeted GFP (Nuc- negative recombinant protein A-Fos blocks expression of GFP) for 16 hours, treated with MQAE fluorescent dyes and assayed several keratinocyte differentiation markers (Rutberg et al., by confocal microscopy. (Insets) The chloride ion content of the ␤- 1997). A-Fos binds to Jun family members with high affinity galactosidase, Cyt-CLIC4 or Nuc-CLIC4 Ad infected cells treated but does not transactivate, thus serving as a pan AP1 inhibitor with NPPB was visualized by confocal microscopy. (D) The for keratinocytes (Gerdes et al., 2006). Inhibition of AP1 fluorescence intensity was also measured in similarly treated cells. activity by A-Fos expression also blocks the upregulation of Fluorescence on the y axis is shown in arbitrary units and decreasing 2+ values (quenching) indicates greater Cl– content. (E) Mouse CLIC4 protein in keratinocytes induced to differentiate by Ca (Fig. 6A,B). Inhibition of CLIC4 transcripts coincides with keratinocytes were treated with increasing amount of NPPB (gradient 2+ triangle, 200 ␮M for 1ϫ and 2 mM for 10ϫ) for 2 hours prior to inhibition of Ca -induced transcripts for involucrin and K1 as calcium (0.5 mM) induction. After 24 hours, cell lysates were demonstrated by quantitative PCR (Fig. 6B). CLIC4 analyzed by immunoblots using K10, CLIC4 and actin antibodies. expression is regulated by elements 5Ј from the transcription start site of the first exon (Fernandez-Salas et al., 2002). Contained within this region are functional binding sites for Nuclear-targeted CLIC4 causes Cl– flux into the nucleus p53 and Myc (Fernandez-Salas et al., 2002; Shiio et al., 2006). in keratinocytes, and Cl– flux is required for expression Further analysis reveals two consensus AP1 binding sites at of Ca2+-induced K10 –1507 to –1499 (site A) and –840 to –832 (site B; Fig. 6D). A CLIC proteins are involved in flux of chloride ions in various luciferase reporter driven by the active human CLIC4 promoter 2636 Journal of Cell Science 120 (15)

Fig. 6. CLIC4 is regulated by AP1 in keratinocytes undergoing Ca2+-induced differentiation. (A) Mouse keratinocytes in 0.05 mM Ca2+ medium were infected with ␤-gal or A-Fos adenovirus for 12 hours 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 cells from A were analyzed by immunoblotting using K10, CLIC4 and actin antibodies. Asterisks (*) in A-Fos lanes of both CLIC4 and K10 blots indicate ‘undetectable’ CLIC4 and K10 bands that are within the linear exposure range of chemiluminescence. With longer exposures CLIC4 and K1 are detected in the A-Fos group (data not shown). (C) Triplicate sets of mouse keratinocytes were transfected with a reporter plasmid containing a 2.5 kb human CLIC4 promoter reporter construct prior to the treatment as described in A. The promoter responses were analyzed by measuring luciferase activity 16 hours after Ca2+ induction. (D) Schematic diagram of mouse and human CLIC4 promoters; solid black arrow indicates transcription start site, solid gray circles indicate AP1 binding sites and locations 5Ј upstream from the transcription start site. Two AP1 consensus sites 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-A and AP1-B sites mutant (Mt A+B) of human CLIC4 promoter and an empty reporter construct (pGL3; Mock) were used to transfect mouse primary keratinocytes. Triplicate sets of transfected cells were induced with 0.5 mM calcium, and the promoter activities were measured by luciferase assay and presented as a fold increase in the promoter activities in high calcium versus low calcium-treated cells. (F) Biotinylated DNA oligos containing the wild-type (WT A and WT B) or the mutant (Mt A and Mt B) AP1 consensus binding sites derived from human CLIC4 promoter sequence were used to detect AP1 binding activities from the nuclear extracts generated from low or high calcium-treated cells in the absence or presence of A-Fos expression. AP1 proteins and the DNA oligo complex formed (Fig. 6C) incorporating these sites is induced by high Ca2+ in the nuclear extracts were detected by ELISA-based assays as medium when transfected into mouse keratinocytes, and this is described in the Materials and Methods section, and the signals were normalized by the protein concentration of the nuclear

Journal of Cell Science prevented by A-Fos. Analogous sites for AP1 regulation are extracts. detected in the mouse CLIC4 promoter (Fig. 6D). When either site A or B, or both, in the human CLIC4 promoter is mutated, luciferase reporter activity in response to 0.5 mM Ca2+ is abrogated (Fig. 6E). In addition, nuclear extracts from independently of p53 (Fernandez-Salas et al., 1999; keratinocytes bind to each of these sites, and binding is Fernandez-Salas et al., 2002). We now report that CLIC4 is enhanced in extracts derived from keratinocytes induced to directly involved in mouse and human keratinocyte differentiate by Ca2+ (Fig. 6F). Mutation of either site A or site differentiation, and nuclear translocation of CLIC4 is an B abrogates binding to that site, and A-Fos expression in integral part of the Ca2+-induced differentiation response. keratinocytes prevents binding to either site. Together, these Thus, expression of keratinocyte differentiation markers (K1, data reveal the functional importance of AP1 in the regulation K10 and filaggrin) and a rapid G1 cell cycle arrest are strongly of CLIC4 and indicate that AP1 activity is sufficient to increase associated with CLIC4 upregulation and nuclear translocation. CLIC4 expression in keratinocytes induced to differentiate by Reduction of CLIC4 by antisense or shRNA approaches Ca2+. prevents expression of these differentiation markers and prevents cell growth arrest associated with differentiation. Discussion CLIC4 is also frequently found in the nucleus of differentiating Flux of different ions that are specifically modulated by cells from mouse and human epidermis in vivo. The findings membrane transport proteins including channels, pumps and reported here also suggests that nuclear translocation of CLIC4 carriers sets an environment that favors the initiation of is associated with increased Cl– ion content in the nucleus, and differentiation programs in keratinocytes and other cell types this ionic flux could be related to the nuclear events that are (Hennings et al., 1983a; Mauro et al., 1993; Debska et al., associated with activation of intrinsic differentiation programs. 2001; Ronnov-Jessen et al., 2002). The murine intracellular The signals downstream from Ca2+ that regulate expression and chloride channel protein CLIC4 was identified as a translocation of CLIC4 in differentiating keratinocytes remain differentially expressed gene in p53-wild-type versus p53-null to be delineated. However, our data suggest that PKC␦ is an keratinocytes that were induced to differentiate by Ca2+. important intermediary in the regulation of CLIC4, and CLIC4 Subsequently, CLIC4 was proved to be a direct downstream expression is more directly regulated by AP1 factors, joining target of p53 in keratinocytes that was also regulated a host of other differentiation-induced AP1-regulated markers CLIC4 modulation of Ca2+-induced differentiation 2637

in keratinocytes (Eckert and Welter, 1996; Angel et al., 2001). Ca2+ signaling (Komuves et al., 2002). Ablation of this receptor Further studies are required to determine if PKC␦ is working appears to work in concert with channels for other ions that through AP1 to regulate CLIC4 expression. The human CLIC4 may be the proximal regulators of the differentiation signal. promoter encompasses two AP1 consensus binding sites For example, Ca2+-induced differentiation activates K+ (–1507 to –1499; gTGAaTCAg and –840 to –832; conductance, and K+ channel inhibitors block keratinocyte gTGAgTCAa). These sites are conserved in a similar region maturation (Hennings et al., 1983b; Mauro et al., 1997). Of upstream from the first exon in the mouse CLIC4 promoter. particular interest to our studies are reports that Ca2+ activates Mutation of these sites abrogates the activation of the CLIC4 voltage gated Cl– channels in keratinocytes (Mauro et al., promoter in response to Ca2+, indicating they are functional 1990), and the activated Cl– current is associated with regulatory sites, and ELISA-based EMSA assays confirm differentiation. Understanding the relationship of ion transport binding activity. AP1 factors now join with p53 and Myc as to Ca2+ signaling in keratinocytes remains an important task key regulators of this multifunctional protein (Fernandez-Salas since reports of Ca2+ signaling abnormalities in psoriasis et al., 2002; Shiio et al., 2006). (McKenzie et al., 2003), Hailey-Hailey (Hu et al., 2000) and CLIC family members have been associated with cell Darier’s disease (Sakuntabhai et al., 1999) suggest that differentiation and development in other models. CLIC4 is modulation of downstream signaling could be an approach to highly expressed when mouse 3T3-L1 cells differentiate into therapy. adipocytes (Kitamura et al., 2001). CLIC4 is also upregulated Nuclear translocation of CLIC4 as a coincident event in during the TGF␤1-mediated differentiation of fibroblasts into keratinocyte differentiation was particularly striking in our myofibroblasts in breast cancer cells (Ronnov-Jessen et al., study. The presence of CLIC4 in the nucleus amplifies the 2002). Similarly, chloride channel activity is required in expression of differentiation markers. We previously reported TGF␤-induced differentiation of human lung fibroblast to that CLIC4 translocates to the nucleus by the interaction of a myofibroblast (Yin and Watsky, 2005). EXC-4, a homologue nuclear localization signal with nuclear transport proteins of CLIC proteins, plays a critical role in digestive/excretory (Ran, importin and NTF2) (Suh et al., 2004). Thus, nuclear tubulogenesis in C. elegans (Berry et al., 2003), and CLIC4 is translocation is a physiological response. The function of a key factor in VEGF-induced tubular morphogenesis of CLIC4 in the nucleus has not been clarified at this time. CLIC4 endothelial cells (Bohman et al., 2005). Tubulogenesis involves binds with multiple partners, including actin, tubulin, dynamin, many cellular processes, including differentiation, apoptosis, AKAPs and 14-3-3 isoforms (Suginta et al., 2001; Berryman cytoskeletal organization and ion fluxes (Zegers et al., 2003). and Goldenring, 2003). These multiple protein-protein CLIC1 is also involved in differentiation induced by interactions imply that CLIC4 has a broader molecular function insulin/IGFI-mediated signaling in human hematopoietic cells beyond a chloride channel/regulator, and these interacting (Saeki et al., 2005). These reports and our studies support that proteins, and others to be identified, may contribute to the importance of CLICs in several cell differentiation programs. physiological functions of CLIC4. Immunoelectron Whether this is related to their chloride channel functions microscopy indicates that nuclear CLIC4 resides in the nuclear remains to be determined, but the multiple levels of regulation membrane and the nucleoplasm so that several functions may

Journal of Cell Science of CLIC4 and its subcellular trafficking suggest this protein is be served by translocation (Suh et al., 2004). important in maintaining homeostasis. In the nuclear membrane, CLIC4 could be involved in Ion flux that is specifically modulated by membrane chloride channel activity. In support of this possibility, transport proteins including channels, pumps and carriers has qualitative analyses of Cl– flux with MQAE dye indicate been associated with differentiation in other cell types. hERG nuclear CLIC4 is associated with accumulation of Cl– that K+ channel activation is mediated by integrin signals and is a would subsequently acidify the microenvironment. The determining factor for osteoclastic differentiation (Arcangeli et nucleus may be particularly sensitive to pH changes as al., 2004). Several anion channels are also reported to be acidification could alter chromatin structure and the binding involved in differentiation in multiple cell types; these channels efficiency of transcription factors (Eastman, 1995; Li and include swelling-activated Cl– channels, volume-activated Cl– Weinman, 2002). For example, Runx transcription factors are channels, voltage-dependent intracellular chloride channel sensitive to Cl– content in the nucleus for binding to target ClCs and P-glycoprotein mdr-1 (Nilius et al., 1996; Nilius and sequences (Backstrom et al., 2002), and Runx activation is Droogmans, 2003; d’Anglemont de Tassigny et al., 2003; associated with multiple cellular responses, including TFG␤ Remillard and Yuan, 2005). Chloride ion flux is also relevant signals, cell cycle regulation and terminal differentiation in to cancer since re-expression of detachment-inducible chloride several cell types (Ito and Miyazono, 2003; Blyth et al., 2005). channel (mCLCA5) suppresses growth of metastatic breast In fact, Runx3 is expressed in epidermis and is required for cancer cells (Beckley et al., 2004), and swelling-activated Cl– normal hair follicle development (Raveh et al., 2005). current is associated with neuroendocrine differentiation of Additional studies will be required to define the functions of prostate cancer cells (Lemonnier et al., 2005). CLIC4 in the nucleus and the signals that send it there. It is The work of Mauro et al. and Bikle et al. have implicated clear that CLIC4 contributes an essential regulatory role in several channels and ion transporters in the regulation of epidermal homeostasis. Both in vivo and in vitro, by virtue of epidermal differentiation (Mauro et al., 1993). Ca2+ is a well the level of expression and subcellular localization, CLIC4 is established regulator of keratinocyte maturation, and a Ca2+ important for keratinocyte proliferation and terminal gradient across the living epidermis in vivo supports the differentiation. Nuclear CLIC4 in particular is associated with significance of Ca2+ in epidermal homeostasis. The presence keratinocyte growth arrest under conditions of differentiation, of a Ca2+ sensing signaling receptor on keratinocytes has senescence and inhibitory growth factors suggesting a broad provided significant insight into the mechanisms involved in role of nuclear CLIC4 in growth control of skin keratinocytes. 2638 Journal of Cell Science 120 (15)

Materials and Methods added to the medium and the trypsinized cells were analyzed by flow cytometry as Chemicals described by the manufacturer (BD Bioscience, FACScan). Data acquisition and Bisindolylmaleimide I, Go 6976 and rottlerin were obtained from Calbiochem (San analysis were performed using Cell Quest software. Diego, CA). 12-O-tetradecanoyl-phorbol-13-acetate (TPA) was obtained from Sigma (St Louis, MO). Immunodetection of AP1-oligo-transcription factor complex An ELISA-based detection method that is equivalent of EMSA/SuperShift assay was Cell culture used to quantify AP1 binding to the biotinylated oligos that encode the sequences Keratinocytes were isolated and cultured as described previously (Dlugosz et al., derived from the human CLIC4 promoter with putative AP1 binding consensus 1995). Human keratinocytes from foreskin were isolated and cultured in EpiLife sequences. The oligo pairs generated from the AP1-A site (capitalized, –1507) sequences were 5Ј-biotin-ggcttagatctgctttgaaacTGATTCAccatttggttcctttttg-3Ј for the keratinocyte medium as described by the manufacturer (Cascade Biologics, Ј Ј Portland, OR). For Ca2+ and TGF␤ induction, keratinocytes were treated with wild type (WT A) and 5 -biotin-ggcttagatctgctttgaaac GACTCAGccatttggttcctttttg-3 medium containing Ca2+ levels higher than 0.1 mM (typically 0.5 or 1 mM) or for the mutant (Mt A). The oligo pairs generated from the AP1-B site (capitalized, Ј TGF␤ (1 ng/ml). To induce serum-mediated differentiation in human keratinocytes, –840) sequences were 5 -biotin-gaagttgggaggagtagctgTGAGTCAagtattatg gaaag - Ј Ј Ca2+-chelexed 5% FBS was added to EpiLife medium. To promote and assay tcag-3 for the wild type (WT B) and 5 -biotin-gaagttgggaggagtagctg GACGC AG - Ј senescence of primary mouse keratinocytes, the ␤-galactosidase senescence assay agtattatggaaagtcag-3 for the mutant (Mt B). PAGE-purified sense and antisense (␤-gal SA) was used as described previously (Vijayachandra et al., 2003). oligos were hybridized to form double strand DNA fragments and were incubated at a final concentration of 1.0 nM with the nuclear extracts at room temperature for 2 Construction of shRNA for CLIC family members hours. As positive control for the experiments, the procedure was repeated with recombinant Fos-Jun mixture (data not shown, Active Motif, Calsbad, CA) and Human cDNA sequences of CLIC members were used as a query on public web- nuclear extracts of Jurkat (data not shown, untreated vs TPA treated) or U937 (data based programs (The RNAi Web) to determine preliminary sites and then pick one not shown, untreated vs TPA treated). These nuclear extracts and the nuclear sequence (18-22 bp) from each group that does not overlap with other CLIC extraction kit were purchased from Active Motif. The mixture was then added to members by manual cDNA sequence alignment and use of other publicly available streptavidin-coated plates (Pierce) that were pretreated with blocking buffer [1% bioinformatic programs (e.g. siDirect). RNA interference compatibility for mouse ␮ ϫ sequences was also considered, and the choice of sequences was manually BSA, 100 g/ml sperm DNA, 1 APK (Ventana, AZ), 0.5% PMSF and 0.5 mM DTT], incubated at 4°C overnight and washed several times with wash buffer [1% determined based on cDNA sequence alignments of mouse and human CLIC ␮ ϫ members. Sequences are as follows: nonspecific shRNA (sR-NS) 5Ј-gttctccga BSA, 10 g/ml Sperm DNA, 1 APK, 0.5% PMSF, Complete Protease Cocktail acgtgtcacg-3Ј, CLIC1 shRNA (sR-1) 5Ј-gtctgattgagcttgtgttg-3Ј, CLIC4 shRNA (sR- (Roche), and 50nM DTT]. A mixture of Fos and Jun polyclonal antibodies (both 4) 5Ј-gctgaaggaggaggacaaaga-3Ј, CLIC5 shRNA (sR-5) 5Ј-gacactgatctctcagac-3Ј. from Santa Cruz Biotechnology)were added at 50 ng/ml to the wells, after which All shRNA sequences have 5Ј-uucaagaga-3Ј sequence as a loop prior to the direct they were incubated for 2 hours at room temperature, extensively washed with the inverse repeat sequences to make the short-hairpin loop. The siRNA version of wash buffer and further incubated with donkey anti-rabbit HRP (GE Healthcare CLIC4 shRNA was used and its efficiency in knockdown of CLIC4 was reported Bioscience, Piscataway, NJ) at 1:50,000 dilution for 2 hours. The wells were in previous works (Bohman et al., 2005; Shiio et al., 2006). These shRNA sequences extensively washed with the wash buffer, chemiluminescent reagent (Dura, Pierce, were inserted into pENTR-H1/TO vectors (BLOCK-iT system; Invitrogen) and IL) was added and the signal was measured using an Infinite M200 plate reader verified as described by the manufacturer. Lipofectamine and Plus reagents were (Tecan, CA). Triplicate samples were processed for all experiments. used for transfection as described by the manufacturer (Invitrogen). Detection of chloride ion flux and semi-quantitative Production and purification of adenoviruses measurements Construction of antisense, sense or organelle-specific CLIC4 adenoviral vectors was Nuclear chloride content was measured semi-quantitatively and qualitatively described elsewhere (Suh et al., 2004). The use of dominant negative PKC␦ and A- detected by confocal microscopy using the chloride-ion-sensitive fluorescent dye Fos adenovirus (Ad) were described previously (Li et al., 1999; Bonovich et al., N-(6-methoxyquinolyle) acetoethyl ester (MQAE; Molecular Probes/Invitrogen) 2002). The adenoviral vector without a recombinant insert (null virus) or with ␤- based on the methods described by the manufacturer and Maglova et al. (Maglova gal was used as a control in experiments where adenoviruses were used. All the et al., 1998a; Maglova et al., 1998b). The chloride channel inhibitor NPPB was adenovirus was plaque purified, amplified as described by Clontech and purified by purchased from BioMol (Plymouth Meeting, PA). Briefly, cells were incubated with two rounds of CsCl centrifugation by Gene Expression Laboratory/NCI Frederick, serum-free culture medium containing 10 mM MQAE for 2 hours and gently but Journal of Cell Science Adenovirus Core Facility. To be consistent throughout the experiments, 10 quickly washed several times with Rinse-Buffer (20 mM HEPES, 100 mM NaCl, multiplicity of infection (MOI) was used in all adenoviral infections, except for MOI 5 mM KCl, 1 mM MgCl2, 0.05 mM CaCl2, 10 mM glucose, pH 7.4). Background dose studies. fluorescence was determined by confocal microscopy after incubating the cells with the Quenching-Buffer [Rinse-Buffer without NaCl, 100 mM NaNO3, 10 ␮M Immunoblot analysis nigericin (Biomol), 10 ␮M valinomycin (Biomol)] and used for normalization. To Protein expression was analyzed by immunoblot as described previously (Suh et al., visualize chloride ion flux qualitatively, keratinocytes grown on 60 mm plates ␤ 2004). Monospecific rabbit antibodies (Abs) against mouse K1 (1:2000), K10 infected with adenovirus expressing -gal, wild-type CLIC4 (Cyt-CLIC4) and (1:2000) and filaggrin (1:2000) were used to detect specific bands, as described nuclear-targeted CLIC4 (Nuc-CLIC4) for 16 hours were treated as stated above, and ϫ previously (Dlugosz and Yuspa, 1993). The following antibodies were also used: fluorescent images were captured by using the 40 Achroplan objective on the Anti-PKC␦ Ab (1:500) was from Sigma. Anti-␤-actin mouse monoclonal antibody Zeiss-510 confocal microscope. was from Chemicon International Inc. (Temecula, CA). For densitometry analyses, Fluorescence from the washed cells was measured using a Molecular Devices ImageMaster TotalLab-v1.11 (Amersham-Pharmacia/GE Healthcare Bioscience, fMax fluorometric plate reader with parameters set at 350 nm for excitation and 460 NJ) was used to quantify the fold changes of CLIC4 expression by normalizing nm for emission. 355 nm excitation filter and 460 nm emission filters (Molecular against the actin signals. All immunoblots analyzed were within the linear range of Devices) were used. Raw data were collected from the plate reader in triplicate and chemiluminescence exposure. SoftMax Pro and Excel programs were used to calculate the average value that is represented in two digit numbers as arbitrary units for simplicity. Immunofluorescent staining Keratinocytes were stained as described in the previous report (Suh et al., 2004). Immunostaining of skin sections Keratinocytes were exposed to 0.05 mM or higher Ca2+ medium for 24 hours after Adult skin samples from humans or mice were formalin fixed, or snap-frozen and infection with null, wild-type CLIC4 (Cyt-CLIC4) and nucleus-targeted CLIC4 then acetone fixed, respectively. For immunohistochemical staining of fixed (Nuc-CLIC4) Ad for 17 hours. Cells were subjected to immunofluorescence sections, slides were deparafinized with HistoChoice (Amresco, Solon, OH), and analysis with anti-K1or anti-CLIC4 antibodies at 1:200 dilutions followed by the antigen was retrieved with Target Retrieval Solution-High pH (Dako, appropriate secondary antibodies conjugated to FITC or Texas Red (Vector Carpinteria, CA) as described by the manufacturer. Envision-secondary antibody Laboratories). The specificity of the CLIC4 and K1 primary antibodies has been conjugated to horseradish peroxidase (Dako) and DAB (Pierce) were used to detect described in previous publications (Dlugosz and Yuspa, 1993; Fernandez-Salas et the antigen as described by the manufacturer. Slides were analyzed with bright-field al., 1999). Cells stained with secondary antibody alone or purified control rabbit microscopy using Leitz-DMRB (Leica, Bannockburn, IL) and OpenLab IgG were used as controls, and no specific immunofluorescent staining was (Improvision, Lexington, MA) software. Tissue sections stained with secondary detected. For detection of chloride flux, MQAE (Molecular Probes/Invitrogen) was antibody alone or purified control rabbit IgG were used as controls, and no specific used as described by the manufacturer. staining was detected.

Cell cycle analysis Production of recombinant CLIC1 and CLIC4 proteins and in Mouse primary keratinocytes (5ϫ105) were infected with Null or AS-CLIC4 for 17 vitro kinase assay hours and further incubated with or without additional Ca2+ for 24 hours or 48 hours. Plasmid pGEX-6P or pGEX-4T (Amersham Bioscience, NY) containing CLIC4 or 2 hours before harvesting the cells, 10 ␮M 5-bromo-2Ј-deoxyuridine (BrdU) was CLIC1 cDNA, respectively, was used to produce the recombinant GST-CLIC4 or CLIC4 modulation of Ca2+-induced differentiation 2639

GST-CLIC1 protein, and the proteins were purified as described by the inducible chloride channel mCLCA5 suppresses growth of metastatic breast cancer manufacturer. The purified proteins were phosphorylated in vitro with several cells. J. Biol. Chem. 279, 41634-41641. recombinant and kinase-active human PKC isoforms (Invitrogen) as described by Berry, K. L., Bulow, H. E., Hall, D. H. and Hobert, O. (2003). A C. elegans CLIC-like the manufacturer. The kinase reaction mixture was resolved by SDS-PAGE, stained protein required for intracellular tube formation and maintenance. Science 302, 2134- and visualized with Coomassie Blue dye, and then the gel was exposed to Phosphor 2137. Screen (GE Healthcare Life Bio-Science, Piscataway, NJ). The phosphorylated Berryman, M. A. and Goldenring, J. R. (2003). CLIC4 is enriched at cell-cell junctions CLIC proteins were detected using Storm imager (Molecular Dynamics). and colocalizes with AKAP350 at the centrosome and midbody of cultured mammalian cells. Cell Motil. Cytoskeleton 56, 159-172. Blyth, K., Cameron, E. R. and Neil, J. C. (2005). The RUNX : gain or loss of Real-time PCR analysis function in cancer. Nat. Rev. Cancer 5, 376-387. Bio-Rad iQ iCycler and Gene Expression Macro (version 1.1) from Bio-Rad were Bohman, S., Matsumoto, T., Suh, K., Dimberg, A., Jakobsson, L., Yuspa, S. and used to measure transcript expression levels. cDNA (diluted 1:50 in the final volume Claesson-Welsh, L. (2005). Proteomic analysis of vascular endothelial growth factor- reaction) was measured using iQ SYBR Green Supermix (Bio-Rad) and included induced endothelial cell differentiation reveals a role for chloride intracellular channel experimental duplicate reactions. The primer sequences were as follows: L32 4 (CLIC4) in tubular morphogenesis. J. Biol. Chem. 280, 42397-42404. (forward) 5Ј-tggttttcttgttg-3Ј, (reverse) 5Ј-gggtgcggagaagg-3Ј, mouse CLIC4 Bonovich, M., Olive, M., Reed, E., O’Connell, B. and Vinson, C. (2002). Adenoviral (forward) 5Ј-tccttgagttatactcccagaacc-3Ј (reverse) 5Ј-gtgagtttcactaacaaacgctc-3Ј. delivery of A-FOS, an AP-1 dominant negative, selectively inhibits drug resistance in Mouse keratin 1 and mouse involucrin primers were obtained from Superarray two human cancer cell lines. Cancer Gene Ther. 9, 62-70. (Frederick, MD). Relative standard curves were generated from log input (serial d’Anglemont de Tassigny, A., Souktani, R., Ghaleh, B., Henry, P. and Berdeaux, A. dilutions of pooled cDNA) versus the threshold cycle (Ct). The linear correlation (2003). Structure and pharmacology of swelling-sensitive chloride channels, coefficient (r2) was 0.81, 0.99 and 0.99 for CLIC4, K1 and involucrin respectively. I(Cl,swell). Fundam. Clin. Pharmacol. 17, 539-553. The slope of the standard curve was used to determine the efficiency of target Debska, G., Kicinska, A., Skalska, J. and Szewczyk, A. (2001). Intracellular potassium and chloride channels: an update. Acta Biochim. Pol. 48, 137-144. amplification (E) using the equation E=(10–1/slope–1)ϫ100. Similar high efficiency Denning, M. F., Dlugosz, A. A., Williams, E. K., Szallasi, Z., Blumberg, P. M. and was obtained for all primers allowing for the comparative Ct method to be used. Yuspa, S. H. (1995). Specific protein kinase C isozymes mediate the induction of –(⌬⌬Ct) ⌬⌬ Relative quantitation was used to calculate the 2 formula where Ct keratinocyte differentiation markers by calcium. Cell Growth Differ. 6, 149-157. represents the difference corrected for L32 used as internal control (Livak and Dlugosz, A. A. and Yuspa, S. H. (1993). Coordinate changes in gene expression which Schmittgen, 2001). Electrophoresis analysis of amplified product from real-time mark the spinous to granular cell transition in epidermis are regulated by protein kinase PCR showed a single band. C. J. Cell Biol. 120, 217-225. Dlugosz, A. A., Glick, A. B., Tennenbaum, T., Weinberg, W. C. and Yuspa, S. H. CLIC4 promoter construction and reporter assay (1995). Isolation and utilization of epidermal keratinocytyes for oncogene research. In 3.5 kb of human CLIC4 promoter (accession number: NT_004610, on human Methods in Enzymology (ed. P. K. Vogt and I. M. Verma), pp. 3-20. New York: 1p36.11 region, with VEGA coordinates contig 24,944,435- Academic Press. 25,043,402, start of transcript location is AL445648.18.1.133587) was cloned by Dotto, G. P. (1999). Signal transduction pathways controlling the switch between genomic PCR and used as an insert to ligate with pGEM-Teasy (Promega). The keratinocyte growth and differentiation. Crit. Rev. Oral Biol. Med. 10, 442-457. Eastman, A. (1995). Assays for DNA fragmentation, endonucleases, and intracellular pH primer pairs used for the genomic PCR were 5Ј-ttaacatagactaatttatcaatgatccta-3Ј for and Ca2+ associated with apoptosis. Methods Cell Biol. 46, 41-55. forward and 5Ј-ccgtgctgctcgctggactgtccggcgtcg-3Ј for reverse, and Expand High Ј Eckert, R. L. and Welter, J. F. (1996). Transcription factor regulation of epidermal Fidelity PCR kit (Roche) was used. Separate primer sets 5 -gagaGCTAGC - keratinocyte gene expression. Mol. Biol. Rep. 23, 59-70. Ј gtgtaccatgagctgtcctctgagccaggaatatagccataaacaaaaacc-3 (NheI-engineered site Edwards, J. C. and Kapadia, S. (2000). Regulation of the bovine kidney microsomal capitalized) for forward and 5Ј-cttggtgtgtttcaggctctgagctagcccttgg-3Ј for reverse chloride channel p64 by p59fyn, a Src family tyrosine kinase. J. Biol. Chem. 275, was used to subclone the PCR fragment into pGlow-TOPO (Invitrogen). A 2.5 kb 31826-31832. (–2720 bp to –369 bp) fragment was further isolated by NheI digestion, subcloned Fernandez-Salas, E., Sagar, M., Cheng, C., Yuspa, S. H. and Weinberg, W. C. (1999). into pGL3 (Promega) and used for reporter assays. Mouse primary keratinocytes p53 and tumor necrosis factor ␣ regulate the expression of a mitochondrial chloride (2ϫ106/plate) were transfected with the reporter plasmid in triplicate using channel protein. J. Biol. Chem. 274, 36488-36497. Lipofectamine (Invitrogen), treated with or without A-Fos Ad or 0.5 mM Ca2+ Fernandez-Salas, E., Suh, K. S., Speransky, V. V., Bowers, W. L., Levy, J. M., Adams, overnight, and the luciferase activities were measured using the Enhance Luciferase T., Pathak, K. R., Edwards, L. E., Hayes, D. D., Cheng, C. et al. (2002). Assay Kit as described by the manufacturer (BD Pharmingen, San Jose, CA). The mtCLIC/CLIC4, an organellular chloride channel protein, is increased by DNA damage Journal of Cell Science normalization of the promoter assays was done by cell number and protein and participates in the apoptotic response to p53. Mol. Cell. Biol. 22, 3610-3620. concentration. Putative AP1 sites were determined by Genomatrix software within 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- the promoter fragment and designated AP1-A (–1507) and AP1-B (–840) upstream 1 activity regulates epithelial tumor cell identity. Cancer Res. 66, 7578-7588. from the putative transcription start site. To determine whether these two sites are 2+ Harrop, S. J., DeMaere, M. Z., Fairlie, W. D., Reztsova, T., Valenzuela, S. M., responsible for CLIC4 promoter activity during Ca shift, the two sites were Mazzanti, M., Tonini, R., Qiu, M. R., Jankova, L., Warton, K. et al. (2001). Crystal mutated separately (Mt A or Mt B) or together (Mt A+B) by deleting five bases structure of a soluble form of the intracellular chloride CLIC1 (NCC27) (capitalized) out of seven AP1 consensus sequence (Mt A: TGATTca and Mt A: at 1.4-A resolution. J. Biol. Chem. 276, 44993-45000. TGAGTca). Hennings, H., Holbrook, K. A. and Yuspa, S. H. (1983a). Factors influencing calcium- induced terminal differentiation in cultured mouse epidermal cells. J. Cell. Physiol. This research was supported by the Intramural Research Program 116, 265-281. Hennings, H., Holbrook, K. A. and Yuspa, S. H. (1983b). Potassium mediation of of the NIH, National Cancer Institute, Center for Cancer Research. calcium induced terminal differentiation of epidermal cells in culture. J. Invest. We thank Joanna Anders for assistance with references, Ulrike Lichti Dermatol. 81, 50s-55s. for differential fractionations of mouse skin, Narayan Bhat of the Hu, Z., Bonifas, J. M., Beech, J., Bench, G., Shigihara, T., Ogawa, H., Ikeda, S., Science Applications International Corporation, Inc. (SAIC), Mauro, T. and Epstein, E. H., Jr (2000). Mutations in ATP2C1, encoding a calcium pump, cause Hailey-Hailey disease. Nat. Genet. 24, 61-65. Frederick, NCI, Molecular Biology, Gene Expression Laboratory for Ito, Y. and Miyazono, K. (2003). RUNX transcription factors as key targets of TGF-beta adenoviral amplification, and Barbara Taylor of the CCR FACS Core superfamily signaling. Curr. Opin. Genet. Dev. 13, 43-47. Facility. M. Mutoh was the recipient of JSPS Research Fellow in Jentsch, T. J., Stein, V., Weinreich, F. and Zdebik, A. A. (2002). Molecular structure Biomedical and Behavioral Research at NIH (2003-2005). and physiological function of chloride channels. Physiol. Rev. 82, 503-568. Kitamura, A., Nishizuka, M., Tominaga, K., Tsuchiya, T., Nishihara, T. and Imagawa, M. (2001). Expression of p68 RNA helicase is closely related to the early stage of adipocyte differentiation of mouse 3T3-L1 cells. Biochem. Biophys. Res. References Commun. 287, 435-439. Angel, P., Szabowski, A. and Schorpp-Kistner, M. (2001). Function and regulation of Komuves, L., Oda, Y., Tu, C. L., Chang, W. H., Ho-Pao, C. L., Mauro, T. and Bikle, AP-1 subunits in skin physiology and pathology. Oncogene 20, 2413-2423. D. D. (2002). Epidermal expression of the full-length extracellular calcium-sensing Arcangeli, A., Becchetti, A., Cherubini, A., Crociani, O., Defilippi, P., Guasti, L., receptor is required for normal keratinocyte differentiation. J. Cell. Physiol. 192, 45- Hofmann, G., Pillozzi, S., Olivotto, M. and Wanke, E. (2004). Physical and 54. functional interaction between integrins and hERG potassium channels. Biochem. Soc. Lee, Y. S., Dlugosz, A. A., McKay, R., Dean, N. M. and Yuspa, S. H. (1997). 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