The Organellular Chloride Channel Protein CLIC4/mtCLIC Translocates to the Nucleus in Response to Cellular Stress and Accelerates Apoptosis
2004; Elsevier BV; Volume: 279; Issue: 6 Linguagem: Inglês
10.1074/jbc.m311632200
ISSN1083-351X
AutoresKwang S. Suh, Michihiro Mutoh, Kunio Nagashima, Ester Fernández‐Salas, Lindsay E. Edwards, Daniel Hayes, John Crutchley, Keith G. Marin, Rebecca A. Dumont, Joshua M. Levy, Christina Cheng, Susan H. Garfield, Stuart H. Yuspa,
Tópico(s)Ion channel regulation and function
ResumoCLIC4/mtCLIC, a chloride intracellular channel protein, localizes to the mitochondria and cytoplasm of keratinocytes and participates in the apoptotic response to stress. We now show that multiple stress inducers cause the translocation of cytoplasmic CLIC4 to the nucleus. Immunogold electron microscopy and confocal analyses indicate that nuclear CLIC4 is detected prior to the apoptotic phenotype. CLIC4 associates with the Ran, NTF2, and Importin-α nuclear import complexes in immunoprecipitates of lysates from cells treated with apoptotic/stress-inducing agents. Deletion or mutation of the nuclear localization signal in the C terminus of CLIC4 eliminates nuclear translocation, whereas N terminus deletion enhances nuclear localization. Targeting CLIC4 to the nucleus via adenoviral transduction accelerates apoptosis when compared with cytoplasmic CLIC4, and only nuclear-targeted CLIC4 causes apoptosis in Apaf null mouse fibroblasts or in Bcl-2-overexpressing keratinocytes. These results indicate that CLIC4 nuclear translocation is an integral part of the cellular response to stress and may contribute to the initiation of nuclear alterations that are associated with apoptosis. CLIC4/mtCLIC, a chloride intracellular channel protein, localizes to the mitochondria and cytoplasm of keratinocytes and participates in the apoptotic response to stress. We now show that multiple stress inducers cause the translocation of cytoplasmic CLIC4 to the nucleus. Immunogold electron microscopy and confocal analyses indicate that nuclear CLIC4 is detected prior to the apoptotic phenotype. CLIC4 associates with the Ran, NTF2, and Importin-α nuclear import complexes in immunoprecipitates of lysates from cells treated with apoptotic/stress-inducing agents. Deletion or mutation of the nuclear localization signal in the C terminus of CLIC4 eliminates nuclear translocation, whereas N terminus deletion enhances nuclear localization. Targeting CLIC4 to the nucleus via adenoviral transduction accelerates apoptosis when compared with cytoplasmic CLIC4, and only nuclear-targeted CLIC4 causes apoptosis in Apaf null mouse fibroblasts or in Bcl-2-overexpressing keratinocytes. These results indicate that CLIC4 nuclear translocation is an integral part of the cellular response to stress and may contribute to the initiation of nuclear alterations that are associated with apoptosis. Chloride intracellular channel (CLIC) 1The abbreviations used are: CLICchloride intracellular channelERendoplasmic reticulumTNFtumor necrosis factorNLSnuclear localization signalPBSphosphate-buffered salineORFopen reading frameCMVcytomegalovirusHAhemagglutininFACSfluorescence-activated cell sortingPIpropidium iodideTUNELterminal deoxynucleotidyl transferase-mediated dUTP nick end labelingGFPgreen fluorescent proteinNTFnuclear transport factorTMtransmembraneAdadenovirusApafApoptotic protease activating factorEMelectron microscopy.1The abbreviations used are: CLICchloride intracellular channelERendoplasmic reticulumTNFtumor necrosis factorNLSnuclear localization signalPBSphosphate-buffered salineORFopen reading frameCMVcytomegalovirusHAhemagglutininFACSfluorescence-activated cell sortingPIpropidium iodideTUNELterminal deoxynucleotidyl transferase-mediated dUTP nick end labelingGFPgreen fluorescent proteinNTFnuclear transport factorTMtransmembraneAdadenovirusApafApoptotic protease activating factorEMelectron microscopy. is a recently identified family of proteins with seven members (p64, CLIC1–5, and parchorin) that are localized in various cellular compartments and expressed in multiple tissue types. The cellular functions of CLIC family members may involve organellar volume regulation and ionic homeostasis and regulation of electro-neutrality (1Jentsch T.J. Nature. 2002; 415: 276-277Crossref PubMed Scopus (43) Google Scholar). Five of the CLIC proteins are similar in size and highly homologous to the C terminus (CLIC module) of p64 and parchorin, which in addition consist of distinct N-terminal domains (2Ashley R.H. Mol. Membr. Biol. 2003; 20: 1-11Crossref PubMed Scopus (104) Google Scholar). All members have one major hydrophobic stretch in the CLIC module that may be used to span the membrane, but some members are also found in a soluble form in the cytoplasm (3Qian Z. Okuhara D. Abe M.K. Rosner M.R. J. Biol. Chem. 1999; 274: 1621-1627Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 4Edwards J.C. Am. J. Physiol. 1999; 276: F398-F408PubMed Google Scholar, 5Mizukawa Y. Nishizawa T. Nagao T. Kitamura K. Urushidani T. Am. J. Physiol. 2002; 282: C786-C795Crossref PubMed Scopus (24) Google Scholar, 6Tulk B.M. Kapadia S. Edwards J.C. Am. J. Physiol. 2002; 282: C1103-C1112Crossref PubMed Scopus (93) Google Scholar). Soluble CLIC proteins are structural homologues of soluble omega-class glutathione S-transferase family proteins, and they may behave as an “active” anion channel and/or channel regulator when “autoinserted” to form integral membrane proteins (7Harrop S.J. DeMaere M.Z. Fairlie W.D. Reztsova T. Valenzuela S.M. Mazzanti M. Tonini R. Qiu M.R. Jankova L. Warton K. Bauskin A.R. Wu W.M. Pankhurst S. Campbell T.J. Breit S.N. Curmi P.M. J. Biol. Chem. 2001; 276: 44993-45000Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). chloride intracellular channel endoplasmic reticulum tumor necrosis factor nuclear localization signal phosphate-buffered saline open reading frame cytomegalovirus hemagglutinin fluorescence-activated cell sorting propidium iodide terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling green fluorescent protein nuclear transport factor transmembrane adenovirus Apoptotic protease activating factor electron microscopy. chloride intracellular channel endoplasmic reticulum tumor necrosis factor nuclear localization signal phosphate-buffered saline open reading frame cytomegalovirus hemagglutinin fluorescence-activated cell sorting propidium iodide terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling green fluorescent protein nuclear transport factor transmembrane adenovirus Apoptotic protease activating factor electron microscopy. Mitochondrial CLIC (mtCLIC/CLIC4) was the first homologue of p64 identified, and its mRNA is ubiquitously expressed with the highest expression in lung, brain, liver, kidney, and skin (8Fernandez-Salas E. Sagar M. Cheng C. Yuspa S.H. Weinberg W.C. J. Biol. Chem. 1999; 274: 36488-36497Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). CLIC4 was described as a mitochondrial and cytoplasmic protein in mouse keratinocytes and localized to the inner mitochondrial membrane in human keratinocytes (9Fernandez-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. Steven A.C. Weinberg W.C. Yuspa S.H. Mol. Cell. Biol. 2002; 22: 3610-3620Crossref PubMed Scopus (141) Google Scholar). Other reports indicate CLIC4 is also localized in the trans-Golgi network in pancreatic cells, endoplasmic reticulum (ER) in rat hippocampal HT-4 cells, and large dense core vesicles in neurosecretory cells (10Chuang J.Z. Milner T.A. Zhu M. Sung C.H. J. Neurosci. 1999; 19: 2919-2928Crossref PubMed Google Scholar, 11Duncan R.R. Westwood P.K. Boyd A. Ashley R.H. J. Biol. Chem. 1997; 272: 23880-23886Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 12Edwards J.C. Tulk B. Schlesinger P.H. J. Membr. Biol. 1998; 163: 119-127Crossref PubMed Scopus (54) Google Scholar). Similar to other family members (parchorin, p64, CLIC1, CLIC3), CLIC4 has a putative Cl--selective channel activity exhibiting a single channel conductance (2Ashley R.H. Mol. Membr. Biol. 2003; 20: 1-11Crossref PubMed Scopus (104) Google Scholar). CLIC4 homologues are expressed in many species with 85–95% identity at the amino acid level suggesting physiological functions are evolutionarily conserved (13Shorning B.Y. Wilson D.B. Meehan R.R. Ashley R.H. Dev. Genes Evol. 2003; 213: 514-518Crossref PubMed Scopus (13) Google Scholar). CLIC4 has also been associated with the actin cytoskeleton in membrane ruffles and lamellipodia and interacts dynamically with signaling proteins involved in cell membrane remodeling, including dynaminI, actin, tubulin, and 14-3-3ϵ. O. isoforms in neuronal cells (14Suginta W. Karoulias N. Aitken A. Ashley R.H. Biochem. J. 2001; 359: 55-64Crossref PubMed Scopus (90) Google Scholar). Recently, substantial up-regulation of CLIC4 was reported in transforming growth factor-β and serum-activated human breast fibroblasts, where up-regulation of CLIC4 was associated with transdifferentiation to myofibroblasts (15Ronnov-Jessen L. Villadsen R. Edwards J.C. Petersen O.W. Am. J. Pathol. 2002; 161: 471-480Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Our previous studies have shown that CLIC4 is a direct response gene for p53 transactivation and involved in TNF-α- and p53-mediated signaling pathways. CLIC4 overexpression induces apoptosis associated with loss of mitochondrial membrane potential, cytochrome c release, and caspase activation suggesting that apoptosis is mediated by mitochondrial dysfunction (9Fernandez-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. Steven A.C. Weinberg W.C. Yuspa S.H. Mol. Cell. Biol. 2002; 22: 3610-3620Crossref PubMed Scopus (141) Google Scholar). During the course of our studies on CLIC4 and apoptosis, we have noted that cytoplasmic CLIC4 translocates to the nucleus in cells undergoing p53-mediated apoptosis. Further studies have indicated that nuclear translocation of CLIC4 was detected in cells responding to various stress and apoptotic signals. This dynamic trafficking occurs early after stress signals and is mediated by the nuclear localization signal (NLS) and the nuclear import machineries. When CLIC4 is targeted directly to the nucleus, apoptosis is accelerated and proceeds in the absence of mitochondrial-dependent caspase activation. These results suggest that CLIC4 participates in the stress-induced apoptotic response in several cellular compartments. Keratinocytes were isolated from newborn Balb/C mice and cultured as basal cells in medium with 0.05 mm calcium as described previously (16Dlugosz A.A. Glick A.B. Tennenbaum T. Weinberg W.C. Yuspa S.H. Methods Enzymol. 1995; 254: 3-20Crossref PubMed Scopus (139) Google Scholar). The non-tumorigenic keratinocyte S1 cell line was derived from normal mouse keratinocytes and maintained in the same medium as primary keratinocytes. The human osteosarcoma-derived p53 Tet-On Saos-2 cell line (17Phillips A.C. Bates S. Ryan K.M. Helin K. Vousden K.H. Genes Dev. 1997; 11: 1853-1863Crossref PubMed Scopus (242) Google Scholar) was maintained in Dulbecco's modified Eagle's medium (BioWhittaker) containing 8% fetal bovine serum (Gemini), 20 units/ml penicillin-streptomycin (Invitrogen), and addition of 800 ng/ml doxycycline induced p53 transgene expression. Cells were treated with apoptotic/stress inducers adriamycin (17 μm, Sigma), mitomycin (30 μm, Sigma), etoposide (100 μm, Sigma), camptothecin (2 μm, MBL International Corp.), actinomycin D (10 μm, MBL), cycloheximide (100 μm, Sigma), and TNF-α (50 ng/ml, Calbiochem) in culture medium, and the treated cells were harvested at various time points for analysis. Bcl-2 adenovirus was purchased from InVivogen. Apaf-null mouse embryo fibroblasts were a generous gift of Dr. Tak Mak and Dr. Scott Lowe and were cultured as previously described (18Soengas M.S. Alarcon R.M. Yoshida H. Giaccia A.J. Hakem R. Mak T.W. Lowe S.W. Science. 1999; 284: 156-159Crossref PubMed Scopus (592) Google Scholar). All experiments shown were repeated at least twice. Anti-C terminus CLIC4 antibodies were generated, and antisera were affinity-purified by chromatography using the immunogenic peptides as described previously (8Fernandez-Salas E. Sagar M. Cheng C. Yuspa S.H. Weinberg W.C. J. Biol. Chem. 1999; 274: 36488-36497Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). The affinity-purified anti-C terminus CLIC4 antibodies are mono-specific for CLIC4 and were used in all experiments, including immunoprecipitation and Western analysis. PBS-washed cells were harvested by adding cell lysis buffer (1% SDS, M-Per (Pierce), Protease inhibitor mixture (Roche Applied Science), and 200 μm NaVO3), sheared with 25-gauge needle several times, and centrifuged at 14,000 × g for 1 h. Cleared lysates were resolved by gradient SDS-PAGE (8–16%), blotted onto a polyvinylidene difluoride membrane, and the protein expression was analyzed by Western blot. In some experiments, subcellular fractions were isolated as described previously (8Fernandez-Salas E. Sagar M. Cheng C. Yuspa S.H. Weinberg W.C. J. Biol. Chem. 1999; 274: 36488-36497Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). Primary antibodies against cytochrome c and LaminA/B (BD Biosciences), actin (Calbiochem), p53 (Oncogene), MARCKS (Santa Cruz Biotechnology), Ran (BD Biosciences), NTF2 (BD Transduction Laboratories), Importin-α (Amersham Biosciences), and cytochrome c subunit IV (Molecular Probes) were used at dilutions suggested by the manufacturer. Anti-mouse, -rabbit, and -goat secondary antibodies conjugated to horseradish peroxidase (Amersham Biosciences) and Super-Signal chemiluminescent substrate (Pierce) were used for the antigen detection. For immunoprecipitation, cells were washed with cold PBS, lysed in IP buffer (M-Per (Pierce), Protease inhibitor mixture (Roche Applied Science), 1 mm phenylmethylsulfonyl fluoride, 200 μm NaVO3, and 1mm NaF), sheared with a 25-gauge needle several times, and then centrifuged at 14,000 × g for 1 h, and the cleared supernatant was collected as the starting material. The cell lysate (1 mg of total protein) was pre-cleared at 4 °C for 2 h with protein A/G Plus-agarose beads and incubated with the protein A/G beads conjugated with CLIC4 antibody overnight at 4 °C. These beads were washed in IP buffer, centrifuged, resuspended, and boiled prior to electrophoresis. Organelle-targeted CLIC4 Plasmids and Adenoviral Vectors—Full-length CLIC4 open reading frame (ORF) was PCR-amplified with 5′-forward and 3′-reverse primer sets containing SalI and NotI restriction sites, respectively. The amplified DNA fragment was gel-purified, ligated to pGEM-Teasy vector (CLIC4-Teasy, Promega), and then resub-cloned into the cytoplasm (Cyt)-, mitochondria (Mit)-, and nucleus (Nuc)-targeted vectors (pShooter vectors, Invitrogen) in-frame with the C terminus “myc-tag” using methods suggested by the manufacturer. Insert in-frame fusion integrity, orientation, and organelle-targeting capability were confirmed by DNA sequencing, restriction analysis, and confocal microscopy, respectively. Organelle-specific CLIC4 constructs were subsequently used as templates to generate organelle-targeted CLIC4 adenovirus. To construct these adenoviral vectors, pCMV Forward and pcDNA3.1/BGH Reverse primers (Invitrogen) were used to amplify the organelle-specific CLIC4 DNA fragment and ligated to pGEM-Teasy (T-Shooter-CLIC4). NotI-digested and gel-purified inserts from T-Shooter-CLIC4 were subcloned into NotI-digested pTRE-Shuttle vector (pTRE-Shooter, BD Biosciences). Insert integrity, orientation, and tetracycline inducibility were confirmed by DNA sequencing, restriction digestion, and Western blot analysis, respectively. Shooter-CLIC4 inserts were isolated from pTRE-Shooter vectors by I-CeuI/PISceI digestion and ligated to the pAdeno-X vector (BD Biosciences) using methods suggested by the manufacturer. Adenoviruses were amplified in the Science Applications International Corp., Inc., Frederick, NCI, National Institutes of Health, Molecular Biology, Gene Expression Laboratory. Deletion Mutant Constructs—Three of the 5′ forward primers and four of the 3′ reverse primers that are specific to various regions of mouse CLIC4 were synthesized to generate deletion mutant constructs. Forward primers included 5′-GGCCATG-3′ in the upstream of the CLIC4 gene-specific sequence to provide sufficient Kozak motif and the starting methionine codon. PCR-amplified fragments were agarose gel-purified and ligated to pcDNA5/FRT/V5-His-TOPO vector (Invitrogen), and the positive clones were screened and verified by PCR, DNA sequencing, and restriction digestion analysis. Inducible deletion mutants were constructed by using a T7-promoter forward and BGH reverse primer set (Invitrogen) to PCR-amplify CLIC4-V5-His inserts. Amplified DNA fragments were agarose gel-purified and ligated to pcDNA5/FRT/TO-TOPO vector (Invitrogen), and the positive clones were screened and verified with the methods mentioned above, including Western blots and confocal microscopy. Deletion mutant CLIC4 proteins were induced in transfected cells with Tet-On (+Dox) or Tet-Off (-Dox) adenovirus (Clontech) infection, and the mutant proteins were expressed at desired levels by controlling the viral multiplicity of infection. NLS Mutagenesis—Full-length CLIC4 ORF from CLIC4-Teasy plasmid was digested with NotI, agarose-gel purified, and subcloned into NotI site of pAlterMax (CLIC4-AlterMax, Promega) in-frame with the N terminus HA tag (wild-type CLIC4). Insert orientation and integrity was determined by DNA sequencing, restriction analysis, and Western blotting. NLS mutagenesis primer (5′-GCTCCACATTGTCACGGTGGTGGCCATAACATACGGCAACTTTGATATTCC-3′) was designed to substitute four amino acids (lysine and arginine) in the NLS motif (KVVAKKYR to TVVAITYG) to obliterate the motif consensus sequence. The primer was synthesized and PAGE-purified (Invitrogen). The mutagenesis was performed as described by the manufacturer, and the mutated CLIC4 (-NLS CLIC4) was verified by DNA sequencing and Western blotting. SigmaStat (SSPS Science) was used as a statistical analysis program for all data represented by multiple numerical measurements. FACS Analysis—LipofectAMINE Plus reagent (Invitrogen) alone or with null (empty)-Adenovirus as a carrier was used to transfect plasmids into cell lines as described previously (9Fernandez-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. Steven A.C. Weinberg W.C. Yuspa S.H. Mol. Cell. Biol. 2002; 22: 3610-3620Crossref PubMed Scopus (141) Google Scholar). Primary keratinocytes and S1 cells transfected or infected with organelle-targeted CLIC4 vectors were analyzed by flow cytometry on a FACSCalibur instrument (BD Biosciences). At various times after transfection or infection, cells were trypsinized and collected in 1 ml of medium and assayed in the presence or absence of propidium iodide (0.5 μg/ml, PI). AnnexinV—Transfected cultures were analyzed for the presence of cell surface annexinV as a measure of apoptotic death. Briefly, cells were trypsinized from dishes at specific times after transfection, centrifuged and incubated with an allophycocyanin-conjugated annexinV (BD Biosciences) as described by the manufacturer. Cells were then washed, fixed in 10% neutral buffered formalin for 10 min, diluted in buffer, and analyzed by flow cytometry. To analyze annexinV staining by confocal microscopy, cells were cultured in 60-mm plates, initially stained with propidium iodide/PBS (2 μg/ml) for 2 min, washed with PBS once, then stained with annexinV reagents (BD Biosciences) as described by the manufacturer. Stained cells were fixed using 2% paraformaldehyde dissolved in annexinV-binding buffer (BD Biosciences), washed with PBS ten times, mounted with fluorescent protective medium (Dako), and analyzed using the Zeiss-510 confocal microscope (Zeiss). The annexinV-positive cells were manually counted from the confocal micrograph and divided by the number of Hoechst-stained cells to generate the percentage of annexinV-positive cells. Eight randomly picked fields were used to generate the average percentage of the annexinV-positive cells for each treatment. TUNEL—Cells were fixed in 2% paraformaldehyde for 30 min and then washed with PBS several times. After dehydrating the surface of the plate with methanol, quadrants were drawn on plates using a PAP pen, washed with PBS ten times, and then the fixed cells were rehydrated with PBS. The cells were permeabilized using PBS/0.1% Triton X-100 solution for 10 min, then washed twice with PBS, and a TUNEL assay (Roche Applied Science) was performed as described by the manufacturer. Stained cells were mounted with Vectashield (Vector Laboratory) and analyzed by confocal microscopy. The average percentage of TUNEL-positive cells was quantified using the random field method as described for the annexinV assay. Active Caspase3 Assay—Primary keratinocytes were infected with organelle-targeted CLIC4 adenovirus, harvested at 16 h post-infection, and subjected to caspase3 assay (BioVision) as described by the manufacturer. Fluorescence was measured by using a fluorometric plate reader (fMax, Molecular Devices). Cells were fixed on culture dishes in 2% paraformaldehyde/PBS for 30 min and extensively washed with PBS. Fixed cells were permeabilized with cold methanol for 6 min, and dried briefly, and desired quadrants were drawn using a PAP pen. Cells were re-hydrated and extensively washed with PBS, treated in 100 mm glycine for 30 min, equilibrated with 0.2% Triton X-100 for 10 min, washed with PBS, and then blocked for 1 h or overnight in 0.5% bovine serum albumin/PBS. The cells were incubated with primary antibody of interest for 2 h, washed three times with PBS, and incubated in the dark with secondary antibody for 1 h. The plates were washed three times in PBS, treated with Hoechst nuclear stain/PBS (1 μg/ml) for 3 min, washed three times with PBS, and finally washed twice with ddH2O for 3 min. Plates were mounted with fluorescent protective mounting solution (Dako), and cells were analyzed with the Zeiss-510 confocal microscope. Post embedding immunoelectron microscopy (19Tobin G.J. Li G.H. Fong S.E. Nagashima K. Gonda M.A. Virology. 1997; 236: 307-315Crossref PubMed Scopus (19) Google Scholar) was performed using LR White resin (Polysciences, Inc.) slightly modified from the manufacture's procedures. Briefly, cell pellets were fixed in 4% paraformaldehyde (Tousimis Research) in PBS for 2 h, rinsed in cold PBS, dehydrated in a series of ice-cold graded ethanol, infiltrated in 1:1 mixture of 100% ice-cold ethanol and LR White resin, then in pure LR White resin overnight in 4 °C, and cured at 55 °C for 24 h. Thin sections were mounted on 200-mesh nickel grids and incubated in a serial dilution of primary antibody overnight at 4 °C. The grids were washed with PBS containing 0.1% (w/v) bovine serum albumin and 0.05% Tween 20 for 2 h, then incubated in 1:100 dilution of immunogoldconjugated secondary antibody (Amersham Pharmacia Biotech) for 1 h at room temperature. The grids were washed as above and stained in uranyl acetate and lead citrate. The digital images were obtained with an electron microscope (Hitachi H7000) equipped with a digital camera system (Gatan). A Variety of Stress-inducing Agents Cause Endogenous CLIC4 Nuclear Translocation in Keratinocytes—In resting keratinocytes, ∼60% of CLIC4 is localized in the perinuclear area in mitochondria and 40% is cytosolic (8Fernandez-Salas E. Sagar M. Cheng C. Yuspa S.H. Weinberg W.C. J. Biol. Chem. 1999; 274: 36488-36497Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar). To determine if CLIC4 intracellular localization was altered in stressed cells, agents that are DNA-damaging, metabolic inhibitors or cytotoxic were tested on primary mouse keratinocytes. Translocation of endogenous CLIC4 from the cytoplasmic compartment into the nucleus was detected in virtually all of the cells (see inset for cell density) after DNA-damaging agents (etoposide, adriamycin, and mitomycin), metabolic inhibitors (cycloheximide and actinomycin D), camptothecin, and TNF-α (Fig. 1A). Time required for the nuclear translocation was different for each chemical treatment (Fig. 1, legend). Nevertheless, the translocation occurred prior to appearance of apoptotic bodies indicating that endogenous CLIC4 nuclear translocation may be a common event preceding the cellular response to stress. When apoptosis-inducing chemicals were used at higher concentrations, the nuclear translocation occurred more rapidly (data not shown). Within the limits of confocal analysis, the level of endogenous mitochondrial CLIC4 did not appear to be altered. CLIC4 antibody is a monospecific anti-peptide and affinity-purified antibody, and the immunostained signal can be eliminated with the addition of blocking peptide (data not shown), indicating the confocal image of immunostained CLIC4 is not an artifact. Exogenous HA-CLIC4 (see Fig. 4C) and GFPCLIC4 (Fig. 1B) fusion proteins also translocated to the nucleus upon TNF-α or etoposide treatment, further supporting physical translocation of CLIC4 from the cytosol to the nucleus after stimuli. In contrast, cytoplasmic or nuclear-targeted GFP were localized in the appropriate intracellular compartment and did not migrate to the nucleus in response to TNF-α or etoposide treatment (Fig. 1B). Typically, greater than a 7-fold increase of endogenous CLIC4 nuclear content was detected by subcellular fractionation early after treatment of primary keratinocytes with TNF-α or etoposide (Fig. 1C), supporting the confocal microscopy data. However, we do not see a corresponding decrease of cytosolic CLIC4 in the fractionation experiments as suggested in the confocal analysis. This could be due to residual mitochondrial contamination of the cytosolic fraction or reflect a concentration difference within the nucleoplasm and cytoplasm resulting in more intense nuclear staining by microscopy. Alternatively, nuclei from a stressed cell might be leaky during the subcellular fractionation procedure.Fig. 4CLIC4 nuclear translocation is regulated by N terminus transmembrane domain (TM) and C terminus nuclear localization signal (NLS).A, series of CLIC4 deletion mutant proteins with “His-V5 C terminus tag” (designated with the shaded circle) were constructed. The constructs shown here include wild-type (Full), lacking NLS domain (-NLS), missing the C terminus half (-C), lacking N terminus TM (-TM), and missing the N terminus half (-N). B, S1 keratinocytes were transfected with the deletion constructs and 36 h later they received no other treatment (-TNF) or were exposed to TNF-α (+TNF) for 2 h, and CLIC4 subcellular localization was detected by immunostaining with anti-V5 antibody, followed by confocal microscopy. C, the CLIC4-NLS mutant construct (HA-CLIC4ΔNLS) with “HA N terminus tag” containing four amino acid substitutions at the NLS motif (KVVAKKYR to TVVAITYG) was used to transfect S1 keratinocytes, followed by TNF-α (4 h) or etoposide (6 h) exposure at 24 h post-transfection. Subcellular location was detected by immunostaining using HA tag antibody and confocal microscopy. Hoechst nuclear staining is represented in the inset. D, S1 keratinocytes transfected with deletion mutant plasmid constructs were assayed by AnnexinV or TUNEL at 24 h post-transfection, and the apoptotic cells were detected by confocal microscopy. The average percentage of apoptotic cells was calculated by manually counting AnnexinV- or TUNEL-positive cells from eight random fields of confocal images.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Endogenous CLIC4 Translocates to the Nucleus in Human Osteosarcoma Cells Undergoing p53-mediated Apoptosis—The human CLIC4 promoter contains functional p53-binding elements, and CLIC4 is up-regulated during doxycycline-induced p53-mediated apoptosis in p53 Tet-On Saos-2 cells (9Fernandez-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. Steven A.C. Weinberg W.C. Yuspa S.H. Mol. Cell. Biol. 2002; 22: 3610-3620Crossref PubMed Scopus (141) Google Scholar). In response to doxycycline treatment, endogenous CLIC4 translocates from the cytoplasm to the nucleus within 24 h, a time when these cells enter the apoptotic program (Fig. 1D). Thus CLIC4 nuclear translocation is associated with apoptosis induced through an endogenous pathway as well as from treatment with exogenous cytotoxic agents. The response is also detected in human cells as well as murine cells. Because endogenous CLIC4 nuclear translocation also occurs in p53 null cell lines (Saos-2 and Ak1B) following TNF-α or etoposide, that translocation itself is a p53 independent event (Ref. 8Fernandez-Salas E. Sagar M. Cheng C. Yuspa S.H. Weinberg W.C. J. Biol. Chem. 1999; 274: 36488-36497Abstract Full Text Full Text PDF PubMed Scopus (122) Google Scholar and data not shown). Endogenous CLIC4 Physically Interacts with the Nuclear Transport Complexes during the Nuclear Translocation Process—Treatment of mouse keratinocytes with TNF-α or etoposide induced an interaction of endogenous CLIC4 with Ran, Importin-α, and NTF-2 (Fig. 2A). Nuclear transport factor (NTF)-2 is an active component of the nuclear import machinery, suggesting that CLIC4 is transported into the nucleus by an NTF-2 nuclear transport complex. Reverse immunoprecipitation verified the physical interaction of CLIC4 with Ran, Importin-α, and NFT-2 (data not shown). In Saos-2 cells (data not shown) and mouse keratinocytes, a low level of interaction with NTF-2 and Ran can also be detected in untreated cells, and the interaction with Importin-α may be constitutive (Fig. 2A), suggesting that nuclear trafficking may be occurring to some extent in resting cells. Confocal images and cell fractionation both suggest a low level of nuclear CLIC4 is constitutive, particularly in p53 Tet-On Saos cells (Fig. 1D). Confocal analyses sh
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