Kwang S. Suh

mtCLIC/CLIC4, an Organellular Chloride Channel Protein, Is Increased by DNA Damage and Participates in the Apoptotic Response to p53

ABSTRACT mtCLIC/CLIC4 (referred to here as mtCLIC) is a p53- and tumor necrosis factor alpha-regulated cytoplasmic and mitochondrial protein that belongs to the CLIC family of intracellular chloride channels. mtCLIC associates with the inner mitochondrial membrane. Dual regulation of mtCLIC by two stress response pathways suggested that this chloride channel protein might contribute to the cellular response to cytotoxic stimuli. DNA damage or overexpression of p53 upregulates mtCLIC and induces apoptosis. Overexpression of mtCLIC by transient transfection reduces mitochondrial membrane potential, releases cytochrome c into the cytoplasm, activates caspases, and induces apoptosis. mtCLIC is additive with Bax in inducing apoptosis without a physical association of the two proteins. Antisense mtCLIC prevents the increase in mtCLIC levels and reduces apoptosis induced by p53 but not apoptosis induced by Bax, suggesting that the two proapoptotic proteins function through independent pathways. Our studies indicate that mtCLIC, like Bax, Noxa, p53AIP1, and PUMA, participates in a stress-induced death pathway converging on mitochondria and should be considered a target for cancer therapy through genetic or pharmacologic approaches.

Proteomic Analysis of Vascular Endothelial Growth Factor-induced Endothelial Cell Differentiation Reveals a Role for Chloride Intracellular Channel 4 (CLIC4) in Tubular Morphogenesis

Formation of new vessels from pre-existing capillaries demands extensive reprogramming of endothelial cells through transcriptional and post-transcriptional events. We show that 120 protein spots in a two-dimensional isoelectric focusing/electrophoretic analysis were affected during vascular endothelial growth factor-A-induced endothelial cell tubular morphogenesis in vitro, as a result of changes in charge or expression level of the corresponding proteins. For about 22% of the spots, the protein products could be identified, of which several previously have been implicated in cytoskeletal reorganization and angiogenesis. One such protein was heat shock protein 27, a chaperone involved in β-actin rearrangement that was identified as regulated in degree of serine phosphorylation. We also identified regulation of chloride intracellular channel 4 (CLIC4), the expression of which decreased during tubular morphogenesis. CLIC4 was expressed at high levels in resting vessels, whereas expression was modulated during pathological angiogenesis such as in tumor vessels. The subcellular localization of CLIC4 in endothelial cells was dependent on whether cells were engaged in proliferation or tube formation. Antisense- and small interfering RNA-mediated suppression of CLIC4 expression led to arrest in tubular morphogenesis. Our data implicate CLIC4 in formation of a vessel lumen.

p21WAF1/CIP1 Is a Common Transcriptional Target of Retinoid Receptors PLEIOTROPIC REGULATORY MECHANISM THROUGH RETINOIC ACID RECEPTOR (RAR)/RETINOID X RECEPTOR (RXR) HETERODIMER AND RXR/RXR HOMODIMER

The divergent response and the molecular mechanisms underlying the anti-cancer effects of retinoid X receptor (RXR) ligand (rexinoid) therapy are poorly understood. This study demonstrates that ligand-activated RXR homodimer facilitated G1 arrest by up-regulation of p21 in vitro and in vivo but failed to induce G1 arrest when p21 expression was blocked by p21 small interfering RNA. RXR ligand-dependent p21 up-regulation was transcriptionally controlled through the direct binding of RXR homodimers to two consecutive retinoid X response elements in the p21 promoter. Structural overlap of a retinoic acid response element with these retinoid X response elements led to a high affinity binding of retinoic acid receptor/RXR heterodimer to the retinoic acid response element, resulting in the prevention of RXR ligand-mediated p21 transactivation. These data show that p21 is a potential and novel molecular target for RXR ligand-mediated anti-cancer therapy and that the expression level of retinoic acid receptor and RXR in tumors may be crucial to induce p21-mediated cell growth arrest in RXR ligand therapy.

Reciprocal Modifications of CLIC4 in Tumor Epithelium and Stroma Mark Malignant Progression of Multiple Human Cancers

Purpose: CLIC4, a member of a family of intracellular chloride channels, is regulated by p53, c-Myc, and tumor necrosis factor-α. Regulation by factors involved in cancer pathogenesis, together with the previously shown proapoptotic activity of CLIC4, suggests that the protein may have a tumor suppressor function. To address this possibility, we characterized the expression profile, subcellular localization, and gene integrity of CLIC4 in human cancers and determined the functional consequences of CLIC4 expression in tumor epithelium and stromal cells. Experimental Design: CLIC4 expression profiles were analyzed by genomics, proteomics, bioinformatics, and tissue microarrays. CLIC4 expression, as a consequence of crosstalk between stroma and epithelium, was tested in vitro by coculture of breast epithelial tumor cells and normal fibroblasts, and the functional consequences of CLIC4 expression was tested in vivo in xenografts of human breast tumor cell lines reconstituted with CLIC4 or mixed with fibroblasts that overexpress CLIC4 transgenically. Results: In cDNA arrays of matched human normal and tumor tissues, CLIC4 expression was reduced in renal, ovarian, and breast cancers. However, CLIC4 protein levels were variable in tumor lysate arrays. Transcript sequences of CLIC4 from the human expressed sequence tag database and manual sequencing of cDNA from 60 human cancer cell lines (NCI60) failed to reveal deletion or mutations in the CLIC4 gene. On matched tissue arrays, CLIC4 was predominantly nuclear in normal human epithelial tissues but not cancers. With advancing malignant progression, CLIC4 staining became undetectable in tumor cells, but expression increased in stromal cells coincident with up-regulation of α-smooth muscle actin, suggesting that CLIC4 is up-regulated in myofibroblasts. Coculture of cancer cells and fibroblasts induced the expression of both CLIC4 and α-smooth muscle actin in fibroblasts adjacent to tumor nests. Introduction of CLIC4 or nuclear targeted CLIC4 via adenovirus into human breast cancer xenografts inhibited tumor growth, whereas overexpression of CLIC4 in stromal cells of xenografts enhanced tumor growth. Conclusion: Loss of CLIC4 in tumor cells and gain in tumor stroma is common to many human cancers and marks malignant progression. Up-regulation of CLIC4 in tumor stroma is coincident with myofibroblast conversion, generally a poor prognostic indicator. Reactivation and restoration of CLIC4 in tumor cells or the converse in tumor stromal cells could provide a novel approach to inhibit tumor growth.

Quantitative proteomic analysis of Myc-induced apoptosis: A direct role for Myc induction of the mitochondrial chloride ion channel, mtCLIC/CLIC4

Myc is a key regulatory protein in higher eukaryotes controlling important cellular functions such as proliferation, differentiation, and apoptosis. Myc is profoundly involved in the genesis of many human and animal cancers, and the abrogation of Myc-induced apoptosis is a critical event in cancer progression. Because the mechanisms that mediate Myc-induced apoptosis are largely unknown, we analyzed protein expression during Myc-induced apoptosis using an isotope-coded affinity tag quantitative proteomics approach and identified that a proapoptotic mitochondrial chloride ion channel, mtCLIC/CLIC4, is induced by Myc. Myc binds to the mtCLIC gene promoter and activates its transcription. Suppression of mtCLIC expression by RNA interference inhibited Myc-induced apoptosis in response to different stress conditions and abolished the cooperative induction of apoptosis by Myc and Bax. We also found that Myc reduces the expression of Bcl-2 and Bcl-xL and that the apoptosis-inducing stimuli up-regulate Bax expression. These results suggest that up-regulation of mtCLIC, together with a reduction in Bcl-2 and Bcl-xL, sensitizes Myc-expressing cells to the proapoptotic action of Bax.

Intracellular Chloride Channels: Critical Mediators of Cell Viability and Potential Targets for Cancer Therapy

The passage of ions to form and maintain electrochemical gradients is a key element for regulating cellular activities and is dependent on specific channel proteins or complexes. Certain ion channels have been the targets of pharmaceuticals that have had impact on a variety of cardiovascular and neurological diseases. Chloride channels regulate the movement of a major cellular anion, and in so doing they in part determine cell membrane potential, modify transepithelial transport, and maintain intracellular pH and cell volume. There are multiple families of chloride channel proteins, and respiratory, neuromuscular, and renal dysfunction may result from mutations in specific family members. Interest in chloride channels related to cancer first arose when the multidrug resistance protein (MDR/P-glycoprotein) was linked to volume-activated chloride channel activity in cancer cells from patients undergoing chemotherapy. More recently, CLC, CLIC, and CLCA intracellular chloride channels have been recognized for their contributions in modifying cell cycle, apoptosis, cell adhesion, and cell motility. Moreover, advances in structural biology and high-throughput screening provide a platform to identify chemical compounds that modulate the activities of intracellular chloride channels thereby influencing chloride ion transport and altering cell behavior. This review will focus on several chloride channel families that may contribute to the cancer phenotype and suggest how they may serve as novel targets for primary cancer therapy.

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

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

Genomic structure and promoter analysis of PKC-δ

Protein kinase C-δ (PKC-δ) is a ubiquitously expressed kinase involved in a variety of cellular signaling pathways including cell growth, differentiation, apoptosis, tumor promotion, and carcinogenesis. While signaling pathways downstream of PKC-δ are well studied, the regulation of the gene has not been extensively analyzed. A mouse genomic DNA fragment containing the PKC-δ gene was sequenced by the primer-walking method, and the subsequent DNA sequence data were used as a query to clone Caenorhabditis elegans and human genomic homologs from the publicly available genomic databases. The genomic structures of C. elegans, mouse, rat, and human PKC-δ were analyzed, and the result revealed that PKC-δ genes comprise 12, 18, 19, and 18 exons for C. elegans, mouse, rat, and human, respectively. The translation start methionine resides in the second exon in mouse and human and in the third exon in rat. The first intron between the first exon and the exon with the translation start methionine in mammalian genes represents a very large gap, as long as 17 kb in human, indicating a complexity involved in gene splicing. Overall exon–intron genomic structure is highly conserved among mammals, while significantly diverged in C. elegans. Putative transcription factor binding sites on the 1.7-kb promoter region of the mouse gene suggest that PKC-δ might be involved in spermatogenesis, embryogenesis, development, brain generation, immune response, oxidative environment, and oncogenesis. Studies on the promoter and subsequent biological testing on mouse keratinocytes indicate that tumor necrosis factor (TNF)-α increases the expression of PKC-δ, and this correlates with the time of NFκB nuclear translocation and activation. This TNF-α-mediated upregulation of PKC-δ is repressed in keratinocytes that are preinfected with IκB superrepressor adenovirus, suggesting that NFκB is involved directly in PKC-δ expression.

S-Nitrosylation Regulates Nuclear Translocation of Chloride Intracellular Channel Protein CLIC4

Nuclear translocation of chloride intracellular channel protein CLIC4 is essential for its role in Ca2+-induced differentiation, stress-induced apoptosis, and modulating TGF-β signaling in mouse epidermal keratinocytes. However, post-translational modifications on CLIC4 that govern nuclear translocation and thus these activities remain to be elucidated. The structure of CLIC4 is dependent on the redox environment, in vitro, and translocation may depend on reactive oxygen and nitrogen species in the cell. Here we show that NO directly induces nuclear translocation of CLIC4 that is independent of the NO-cGMP pathway. Indeed, CLIC4 is directly modified by NO through S-nitrosylation of a cysteine residue, as measured by the biotin switch assay. NO enhances association of CLIC4 with the nuclear import proteins importin α and Ran. This is likely a result of the conformational change induced by S-nitrosylated CLIC4 that leads to unfolding of the protein, as exhibited by CD spectra analysis and trypsinolysis of the modified protein. Cysteine mutants of CLIC4 exhibit altered nitrosylation, nuclear residence, and stability, compared with the wild type protein likely as a consequence of altered tertiary structure. Moreover, tumor necrosis factor α-induced nuclear translocation of CLIC4 is dependent on nitric-oxide synthase activity. Inhibition of nitric-oxide synthase activity inhibits tumor necrosis factor α-induced nitrosylation and association with importin α and Ran and ablates CLIC4 nuclear translocation. These results suggest that S-nitrosylation governs CLIC4 structure, its association with protein partners, and thus its intracellular distribution.

CLIC4, skin homeostasis and cutaneous cancer: Surprising connections

Chloride intracellular channel 4 (CLIC4) is a putative chloride channel for intracellular organelles. CLIC4 has biological activities in addition to or because of its channel activity. In keratinocytes, CLIC4 resides in the mitochondria and cytoplasm, and CLIC4 gene expression is regulated by p53, TNF‐α, and c‐Myc. Cytoplasmic CLIC4 translocates to the nucleus in response to cellular stress conditions including DNA damage, metabolic inhibition, senescence, and exposure to certain trophic factors such as TNF‐α and LPS. Nuclear translocation is associated with growth arrest or apoptosis, depending on the level of expression. In the nucleus CLIC4 interacts with several nuclear proteins as demonstrated by yeast two‐hybrid screening and co‐immunoprecipitation. Nuclear CLIC4 appears to act on the TGF‐β pathway, and TGF‐β also causes CLIC4 nuclear translocation. In human and mouse cancer cell lines, CLIC4 levels are reduced, and CLIC4 is excluded from the nucleus. CLIC4 soluble or membrane‐inserted status is dependent on redox state, and redox alterations in cancer cells could underly the defect in nuclear translocation. CLIC4 is reduced and excluded from the nucleus of many human epithelial neoplasms. Paradoxically, CLIC4 is reciprocally upregulated in tumor stroma in conjunction with the expression of α‐smooth muscle actin in the fibroblast to myofibroblast transition. Overexpression of CLIC4 in cancer cells inhibits tumor growth in vivo. Conversely, overexpression of CLIC4 in tumor stromal cells stimulates tumor growth in vivo. Thus, CLIC4 participates in normal and pathological processes and may serve as a useful target for therapies in disturbances of homeostasis and neoplastic transformation.