QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 17 July 2007
doi: 10.1242/jcs.002741

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Suh, K. S. Articles by Yuspa, S. H. Search for Related Content PubMed PubMed Citation Articles by Suh, K. S. Articles by Yuspa, S. H. Social Bookmarking                         What's this?

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

Laboratory of Cancer Biology and Genetics, Center for Cancer Research, National Cancer Institute, Bethesda, MD20892, USA

View larger version (28K): [in this window] [in a new window]   Fig. 1. CLIC4 is upregulated in differentiating keratinocytes. (A) Newborn mouse and human foreskin keratinocytes were cultured in 0.05 mM Ca2+ medium for 4 days, then treated for 24 hours with the indicated concentrations of Ca2+ in the culture medium and lysates were examined by immunoblotting. (B) RNA isolated from mouse keratinocytes from A was amplified by RT-PCR, and CLIC4 and actin transcripts were detected by ethidium bromide staining of agarose gels. (C) Human foreskin keratinocytes were cultured in serum-free medium for 4 days and exposed to 5% FBS deleted of Ca2+ serum for the indicated times. (D) Mouse and human keratinocyte were treated with indicated doses of TPA for 24 hours, and lysates were analyzed by immunoblotting. The fold change for each band was determined by densitometry as described in Materials and Methods.

View larger version (46K): [in this window] [in a new window]   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 then 0.5 mM Ca2+ was added in the medium for 24 hours prior to lysis and immunoblotting. (B) Mouse keratinocytes were infected with PKC DN Ad for 17 hours before the treatment with 0.5 mM Ca2+ for 24 hours. Actin was used as a loading control. Bis, bisindolylmaleimide I; Ro, rottlerin; Go, Go 6976. Similar data were obtained from two independent experiments. The numbers over the CLIC4 bands in A and B represent fold change determined by densitometry. (C) Purified recombinant CLIC1 or CLIC4 proteins were incubated with purified, kinase active recombinant PKC isoforms in appropriate reaction buffers with [-32P]ATP, the reaction mixture was resolved by SDS-PAGE and then the phosphorylated proteins were visualized by phospho-imaging methods. Un, no recombinant PKC.

View larger version (38K): [in this window] [in a new window]   Fig. 3. CLIC4 upregulation is required for Ca2+-induced differentiation in keratinocytes. K1, K10, Filaggrin, CLIC1, CLIC4, PKC and actin protein levels in mouse keratinocytes were determined by immunoblot analysis. (A) Mouse keratinocytes were co-infected with 5 MOI of Tet-Off Ad (to induce expression of the conditional AS) and increasing MOI (5 and 10, shaded triangle) of AS-CLIC4 Ad or Null Ad (N) for 17 hours before treatment with 0.5 mM Ca2+ for 24 hours. (B) Duplicate sets of mouse keratinocytes were treated with BrdU, and anti-BrdU-FITC stained cells of each sample were analyzed with flow cytometry. Cell populations: null Ad-infected without BrdU (upper left, N/(–)/Lo Ca2+), with BrdU (upper right, N/(+)/Lo Ca2+) both in 0.05 mM Ca2+; with 0.5 mM 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 population in each cell cycle phase. The percentage of cells in each cell cycle phase from the total cell population in N/(+)/Lo Cal2+, N/(+)/Hi Ca2+ and AS/(+)/Hi Ca2+ at 24 hours and 48 hours after Ca2+ induction is represented in a bar graph format. (D) Human 293 cells were transfected with constructs expressing nonspecific (NS) or CLIC4 (sR-CLIC4) shRNA for 48 hours, and specificity of CLIC4 knockdown was analyzed by immunoblots of cell lysates. (E) Mouse keratinocytes were transfected with constructs expressing shRNA for nonspecific (NS), CLIC1 (sR-1), CLIC4 (sR-4) or CLIC5 (sR-5) for 24 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 from at least two independent experiments. The numbers over the CLIC4 bands in A and B represent fold changes determined by densitometry.

View larger version (66K): [in this window] [in a new window]   Fig. 4. CLIC4 translocates to the nucleus in differentiating and senescing keratinocytes in vitro and is primarily nuclear in epidermis in vivo. (A) Mouse primary keratinocytes and human foreskin keratinocytes were treated with indicated Ca2+ for 24 hours and immunostained with CLIC4 antibody. (B) Mouse keratinocytes were maintained in 0.05 mM Ca2+ medium and either fixed on the first day of culture (1 Day) or after 7 days of culture (7 Day) to allow for senescence. Fixed cultures were immunostained with CLIC4 antibodies. Duplicate sets of cells were subjected to assay by -gal SA, and the number of senescing cells (7 Day) is presented as a fold increase over the control (1 Day; lower panel). (C) Mouse keratinocytes were incubated in the serum-depleted medium for 16 hours (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 and immunostained for CLIC4. Insets in Fig. 4C-E represent DAPI nuclear staining. (F) Formalin-fixed human breast skin and acetone-fixed mouse skin frozen sections were stained with CLIC4 antibodies by immunohistochemical methods as described in the Materials and Methods. The mouse skin section was from a hyperplastic area to enhance the resolution of strata. Red and green arrows indicate keratinocytes in the basal layers (blue nucleus) and the differentiating layers (brown nucleus, CLIC4 stain) respectively. In all immunofluorescent and immunohistochemical staining experiments, secondary antibody alone was used as controls.

View larger version (51K): [in this window] [in a new window]   Fig. 5. Nuclear CLIC4 induces the expression of keratinocyte differentiation markers, and intracellular chloride influences keratinocyte differentiation. (A) Mouse keratinocytes in 0.05 mM Ca2+ medium were pre-treated with null, wild-type CLIC4 (Cyt) or nuclear-targeted CLIC4 (Nuc) adenovirus for 12 hours, and cell lysates were analyzed by immunoblots using K1, K10, CLIC4 and actin antibodies. Gray arrow indicates the endogenous CLIC4 (Endo-CLIC4) and the black arrows indicate the two exogenous CLIC4 bands. (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 indicated by white arrows. All immunostained cells were analyzed by confocal microscopy as described in the Materials and Methods. Insets show DAPI staining. (C) Mouse keratinocytes in 0.05 mM Ca2+ medium were 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 assayed by confocal microscopy. (Insets) The chloride ion content of the -galactosidase, Cyt-CLIC4 or Nuc-CLIC4 Ad infected cells treated with NPPB was visualized by confocal microscopy. (D) The fluorescence intensity was also measured in similarly treated cells. Fluorescence on the y axis is shown in arbitrary units and decreasing values (quenching) indicates greater Cl– content. (E) Mouse keratinocytes were treated with increasing amount of NPPB (gradient triangle, 200 µM for 1x and 2 mM for 10x) for 2 hours prior to calcium (0.5 mM) induction. After 24 hours, cell lysates were analyzed by immunoblots using K10, CLIC4 and actin antibodies.

View larger version (34K): [in this window] [in a new window]   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 5' 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 in the nuclear extracts were detected by ELISA-based assays as described in the Materials and Methods section, and the signals were normalized by the protein concentration of the nuclear extracts.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter