Chloride intracellular channel 4 participates in the regulation of lipopolysaccharide-induced inflammatory responses in human bronchial epithelial cells.

The airway epithelium is located at the interactional boundary between the external and internal environments of the organism and is often exposed to harmful environmental stimuli. Inflammatory response that occurs after airway epithelial stress is the basis of many lung and systemic diseases. Chloride intracellular channel 4 (CLIC4) is abundantly expressed in epithelial cells. The purpose of this study was to investigate whether CLIC4 is involved in the regulation of lipopolysaccharide (LPS)-induced inflammatory response in airway epithelial cells and to clarify its potential mechanism. Our results showed that LPS induced inflammatory response and decreased CLIC4 levels in vivo and in vitro. CLIC4 silencing aggravated the inflammatory response in epithelial cells, while overexpression of CLIC4 combined with LPS exposure significantly decreased the inflammatory response compared with cells exposed to LPS without CLIC4 overexpression. By labeling intracellular chloride ions with chloride fluorescent probe MQAE, we showed that CLIC4 mediated intracellular chloride ion-regulated LPS-induced cellular inflammatory response.

Retrograde AAV-mediated gene modulation reveals chloride intracellular channel proteins as potent regulators of retinal ganglion cell death.

Most projection neurons, including retinal ganglion cells (RGCs), undergo cell death after axotomy proximal to the cell body. Specific RGC subtypes, such as ON-OFF direction selective RGCs (ooDSGCs) are particularly vulnerable, whereas intrinsically photosensitive RGCs (ipRGCs) exhibit resilience to axonal injury. Through the application of RNA sequencing and fluorescent in situ hybridization, we show that the expression of chloride intracellular channel protein 1 and 4 (Clic1 and Clic4) are highly increased in the ooDSGCs after axonal injury. Toward determining a gene's role in RGCs, we optimized the utility and efficacy of adenovirus associated virus (AAV)-retro expressing short hairpin RNA (shRNA). Injection of AAV2-retro into the superior colliculus results in efficient shRNA expression in RGCs. Incorporating histone H2B gene fused with mGreenLantern results in bright nuclear reporter expression, thereby enhancing single RGC identification and cell quantitation in live retinas. Lastly, we demonstrate that AAV2-retro mediated knockdown of both Clic1 and Clic4 promotes RGC survival after injury. Our findings establish an integrated use of AAV2-retro-shRNA and real-time fundus imaging and reveal CLICs' contribution to RGC death.

CLIC4 Function in the Epithelial-Mesenchymal Transition of Epithelial Odontogenic Lesions.

BACKGROUND: Odontogenic lesions constitute a heterogeneous group of lesions. CLIC4 protein regulates different cellular processes, including epithelial-mesenchymal transition and fibroblast-myofibroblast transdifferentiation. This study analyzed CLIC4, E-cadherin, Vimentin, and α-SMA immunoexpression in epithelial odontogenic lesions that exhibit different biological behavior. METHODS: It analyzed the immunoexpression of CLIC4, E-cadherin, and Vimentin in the epithelial cells, as well as CLIC4 and α-SMA in the mesenchymal cells, of ameloblastoma (AM) (n = 16), odontogenic keratocyst (OKC) (n = 20), and adenomatoid odontogenic tumor (AOT) (n = 8). Immunoexpressions were categorized as score 0 (0% positive cells), 1 (< 25%), 2 (≥ 25% - < 50%), 3 (≥ 50% - < 75%), or 4 (≥ 75%). RESULTS: Cytoplasmic CLIC4 immunoexpression was higher in AM and AOT (p < 0.001) epithelial cells. Nuclear-cytoplasmic CLIC4 was higher in OKC's epithelial lining (p < 0.001). Membrane (p = 0.012) and membrane-cytoplasmic (p < 0.001) E-cadherin immunoexpression were higher in OKC, while cytoplasmic E-cadherin expression was higher in AM and AOT (p < 0.001). Vimentin immunoexpression was higher in AM and AOT (p < 0.001). Stromal CLIC4 was higher in AM and OKC (p = 0.008). Similarly, α-SMA immunoexpression was higher in AM and OKC (p = 0.037). Correlations in these proteins' immunoexpression were observed in AM and OKC (p < 0.05). CONCLUSIONS: CLIC4 seems to regulate the epithelial-mesenchymal transition, modifying E-cadherin and Vimentin expression. In mesenchymal cells, CLIC4 may play a role in fibroblast-myofibroblast transdifferentiation. CLIC4 may be associated with epithelial odontogenic lesions with aggressive biological behavior.

The stromal immunoexpression of CLIC4 may be related to the difference in the biological behavior between oral squamous cell carcinoma and oral verrucous carcinoma

Background: Oral squamous cell carcinoma (OSCC) has high morbidity and mortality rates while oral verrucous carcinoma (OVC), an uncommon variant of OSCC, exhibits a distinct biological behavior. CLIC4 protein plays a role in the cell cycle and apoptosis regulation and participates in the myofibroblasts transdifferentiation process, which are the main cells of the tumor stroma. This study analyzed the immunoexpression of CLIC4 and α-SMA in 20 OSCC cases and 15 OVC cases. Material and methods: A semiquantitative analysis of CLIC4 and α-SMA immunoexpression was performed in the parenchyma and stroma. Nuclear and cytoplasmic reactivity was analyzed separately for the CLIC4 immunostaining. The data were submitted to Pearson's chi-square and Spearman's correlation tests (p ≤ 0.05). Results: In the CLIC4 analysis, there was a significant difference in the immunoexpression of this protein between OSCC and OVC stroma (p < 0.001). It was observed a higher expression of α-SMA in the OSCC stroma. There was a positive and significant correlation between CLIC4 and α-SMA immunoexpression in the OVC stroma (r = 0,612; p = 0,015). Conclusions: The decrease or absence of nuclear CLIC4 immunoexpression in the neoplastic epithelial cells and the increase of its expression in the stroma may influence the difference in biological behavior between OSCC and OVC. (AU)

Imunoexpressão de clic4, α-sma, e-caderina e vimentina em lesões odontogênicas epitelias benígnas / Immunoexpression of clic4, α-sma, e-cadherin and vimentin in benign epithelial odontogenic lesions

As lesões odontogênicas epiteliais benignas constituem um grupo heterogêneo de lesões. A proteína CLIC4 atua na regulação dos processos de parada de crescimento e apoptose, participando também do processo de transdiferenciação dos fibroblastos em miofibroblastos que passam a expressar α-SMA. Além disso, a expressão de CLIC4 pode interferir no processo de transição epitélio-mesenquima (TEM) em neoplasias. Este trabalho avaliou a imunoexpressão de CLIC4, α-SMA, E-caderina e Vimentina em ameloblastomas (AM) (n = 16), ceratocistos odontogênicos (n = 20) e tumores odontogênicos adenomatóides (TOA) (n = 8). A análise da expressão imunoistoquímica das proteínas CLIC4, E-caderina e vimentina no componente epitelial das lesões e de CLIC4 e α-SMA no tecido conjuntivo foi realizada de forma semi-quantitativa por um avaliador previamente calibrado. A expressão no componente epitelial de CLIC4 foi analisada separadamente no núcleo e no citoplasma, bem como a marcação de E-caderina que foi avaliada na membrana e no citoplasma. As comparações dos percentuais de imunorreatividade em relação aos grupos estudados foram realizadas por meio dos testes não paramétricos de Kruskal-Wallis e Mann-Whitney. Possíveis correlações entre a expressão de CLIC4, α-SMA, E-caderina e Vimentina foram avaliadas por meio do teste de correlação de Spearman. O nível de significância foi estabelecido em 5% (p < 0,05). Foram observados diferentes padrões de marcação entre os grupos analisados, observando-se que a imunoexpressão exclusivamente citoplasmática da CLIC4 no componente epitelial dos AM (p < 0,001) e TOA (p < 0,001) foi significativamente superior a dos CO, não demonstrarando significância estatística entre os AM e TOA. A imunoexpressão (nuclear e citoplasmática) da CLIC4 no revestimento epitelial CO foi significativamente superior à encontrada no componente epitelial dos AM (p < 0,001) e dos TOA (p < 0,001). A imunoexpressão estromal de CLIC4 foi significativamente superior nos AM (p = 0,009) e CO (p = 0,004) quando comparados aos TOA. A imunoexpressao de α-SMA significativamente maior em AM (p = 0,016) e CO (p = 0,034) quando comparados aos TOA. Para a imunoexpressão membranar da E-caderina em CO foi significativamente superior em comparação à encontrada nos AM (p = 0,009) e nos TOA (p = 0,024). Foi observada maior imunoexpressão de E-caderina (membranar e citoplasmática) nos COs, quando comparados aos AM (p < 0,001) e aos TOAs (p < 0,001). A expressão de Ecaderina citoplasmática foi significativamente maior nos AM e TOA (p < 0,001) quando comparados aos CO. Observou-se diferença estatisticamente significativa na imunoexpressão de vimentina entre os casos de AM e os casos de TOA (p = 0,038) e CO (p < 0,001), bem como entre o TOA e CO (p < 0,001). As correlações testadas entre os escores das proteínas estudadas evidenciou que no grupo dos AM foi possível evidenciar moderada correlação positiva e estatisticamente significativa (r = 0,527; p = 0,036) entre a expressão citoplasmática da CLIC4 e a expressão citoplasmática da E-caderina. Também foi verificada fraca correlação negativa e estatisticamente significativa (r = -0,499; p = 0,049) entre a expressão núcleo-citoplasmática da CLIC4 e a expressão citoplasmática da E-caderina nos AM. Além disso, uma moderada correlação positiva e estatisticamente significativa entre a expressão estromal da CLIC4 e a expressão da α-SMA nos AM (r = 0,648; p = 0,007) e nos CO (r = 0,541; p = 0,014). Foi observada forte correlação negativa e estatisticamente significativa (r = -0,813; p < 0,001) entre a expressão da E-caderina e a expressão da vimentina nos AM. Os resultados deste estudo sugerem um potencial envolvimento de CLIC4 no processo de transdiferenciação de miofibroblastos, e que a presença destas células é mais frequentemente associada a lesões de comportamento biológico mais agressivo como os AM e CO, além de uma possível atuação desta proteína na regulação do ciclo celular e na TEM nas lesões estudadas (AU).

Benign epithelial odontogenic lesions constitute a heterogeneous group of lesions. the CLIC4 protein acts in the regulation of growth arrest and apoptosis processes, also participating in the process of transdifferentiation of fibroblasts Into myofibroblasts that begin to express α-SMA. Furthermore, CLIC4 expression can interfere with the epithelialmesenchymal transition (EMT) process in neoplasms. This work evaluated the immunoexpression of CLIC4, α-SMA, e-cadherin and vimentin in ameloblastomas (AM) (n = 16), odontogenic keratocysts (OK) (n = 20) and adenomatoid odontogenic tumors (AOT) (n = 8). The analysis of the immunohistochemical expression of the proteins CLIC4, ecadherin and vimentin in the epithelial component of the lesions and of CLIC4 and α-SMA in the connective tissue was carried out in a semi-quantitative way by a previously calibrated evaluator. Expression in the epithelial component of CLIC4 was analyzed separately in the nucleus and cytoplasm, as well as e-cadherin labeling, which was evaluated in the membrane and cytoplasm. Comparisons of the percentages of immunoreactivity in relation to the studied groups were carried out using the nonparametric kruskal-wallis and mann-whitney tests. Possible correlations between the expression of CLIC4, α-SMA, e-cadherin and vimentin were evaluated using the spearman correlation test. The significance level was set at 5% (p < 0.05). Different staining patterns were observed between the groups analyzed, observing that the exclusively cytoplasmic immunoexpression of CLIC4 in the epithelial component of AM (p < 0.001) and AOT (p < 0.001) was significantly higher than that of OK, not demonstrating statistical significance between the AM and AOT. The immunoexpression (nuclear and cytoplasmic) of CLIC4 in the co epithelial lining was significantly higher than that found in the epithelial component of AM (p < 0.001) and AOT (p < 0.001). Stromal CLIC4 immunoexpression was significantly higher in AM (p = 0.009) and OK (p = 0.004) when compared to AOT. The immunoexpression of α-SMA is significantly higher in AM (p = 0.016) and OK (p = 0.034) when compared to AOT. For e-cadherin membrane immunoexpression in co was significantly higher compared to that found in AM (p = 0.009) and AOT (p = 0.024). Greater immunoexpression of e-cadherin (membrane and cytoplasmic) was observed in OK, when compared to AM (p < 0.001) and AOT (p < 0.001). Cytoplasmic ecadherin expression was significantly higher in AM and AOT (p < 0.001) when compared to OK. A statistically significant difference in vimentin immunoexpression was observed between cases of AM and cases of AOT (p = 0.038) and OK (p < 0.001), as well as between AOT and OK (p < 0.001). The correlations tested between the scores of the proteins studied showed that in the am group it was possible to demonstrate a moderate positive and statistically significant correlation (r = 0.527; p = 0.036) between the cytoplasmic expression of clic4 and the cytoplasmic expression of e-cadherin. A weak and statistically significant negative correlation (r = -0.499; p = 0.049) was also found between the nucleus-cytoplasmic expression of clic4 and the cytoplasmic expression of e- cadherin in AM. Furthermore, a moderate positive and statistically significant correlation between the stromal expression of CLIC4 and the expression of α-SMA in AM (r = 0.648; p = 0.007) and OK (r = 0.541; p = 0.014). Additionally, a strong negative and statistically significant correlation (r = -0.813; p < 0.001) was observed between the expression of ecadherin and the expression of vimentin in AM. The results of this study suggest a potential involvement of CLIC4 in the myofibroblast transdifferentiation process, and that the presence of these cells is more frequently associated with lesions with more aggressive biological behavior such as AM and OK, in addition to a possible role of this protein in the regulation of cell cycle and EMT in the lesions studied (AU).

Nuclear localisation sequences of chloride intracellular channels 1 and 4 facilitate nuclear import via interactions with import mediator importin-α: An empirical and theoretical perspective.

Chloride intracellular channel proteins (CLICs) display ubiquitous expression, with each member exhibiting specific subcellular localisation. While all CLICs, except CLIC3, exhibit a highly conserved putative nuclear localisation sequence (NLS), only CLIC1, CLIC3 and CLIC4 exist within the nucleus. The CLIC4 NLS, 199-KVVAKKYR-206, appears crucial for nuclear entry and interacts with mouse nuclear import mediator Impα isoform 1, omitting the IBB domain (mImpα1ΔIBB). The essential nature of the basic residues in the CLIC4 NLS has been established by the fact that mutating out these residues inhibits nuclear import, which in turn is linked to cutaneous squamous cell cancer. Given the conservation of the CLIC NLS, CLIC1 likely follows a similar import pathway to CLIC4. Peptides of the CLIC1 (Pep1; Pep1_S C/S mutant) and CLIC4 (Pep4) NLSs were designed to examine binding to human Impα isoform 1, omitting the IBB domain (hImpα1ΔIBB). Molecular docking indicated that the core CLIC NLS region (KKYR) forms a similar binding pattern to both mImpα1ΔIBB and hImpα1ΔIBB. Fluorescence quenching demonstrated that Pep1_S (Kd ≈ 237 µM) and Pep4 (Kd ≈ 317 µM) bind hImpα1ΔIBB weakly. Isothermal titration calorimetry confirmed the weak binding interaction between Pep4 and hImpα1ΔIBB (Kd ≈ 130 µM) and the presence of a proton-linked effect. This weak interaction may be due to regions distal from the CLIC NLS needed to stabilise and strengthen hImpα1ΔIBB binding. Additionally, this NLS may preferentially bind another hImpα isoform with different flexibility properties.

Crosstalk between tumor and stroma modifies CLIC4 cargo in extracellular vesicles.

Mouse models of breast cancer have revealed that tumor-bearing hosts must express the oxidoreductase CLIC4 to develop lung metastases. In the absence of host CLIC4, primary tumors grow but the lung premetastatic niche is defective for metastatic seeding. Primary breast cancer cells release EVs that incorporate CLIC4 as cargo and circulate in plasma of wildtype tumor-bearing hosts. CLIC4-deficient breast cancer cells also form tumors in wildtype hosts and release EVs in plasma, but these EVs lack CLIC4, suggesting that the tumor is the source of the plasma-derived EVs that carry CLIC4 as cargo. Paradoxically, circulating EVs are also devoid of CLIC4 when CLIC4-expressing primary tumors are grown in CLIC4 knockout hosts. Thus, the incorporation of CLIC4 (and perhaps other factors) as EV cargo released from tumors involves specific signals from the surrounding stroma determined by its genetic composition. Since CLIC4 is also detected in circulating EVs from human breast cancer patients, future studies will address its association with disease.

Chloride Intracellular Channel Proteins (CLICs) and Malignant Tumor Progression: A Focus on the Preventive Role of CLIC2 in Invasion and Metastasis.

CLICs are the dimorphic protein present in both soluble and membrane fractions. As an integral membrane protein, CLICs potentially possess ion channel activity. However, it is not fully clarified what kinds of roles CLICs play in physiological and pathological conditions. In vertebrates, CLICs are classified into six classes: CLIC1, 2, 3, 4, 5, and 6. Recently, in silico analyses have revealed that the expression level of CLICs may have prognostic significance in cancer. In this review, we focus on CLIC2, which has received less attention than other CLICs, and discuss its role in the metastasis and invasion of malignant tumor cells. CLIC2 is expressed at higher levels in benign tumors than in malignant ones, most likely preventing tumor cell invasion into surrounding tissues. CLIC2 is also expressed in the vascular endothelial cells of normal tissues and maintains their intercellular adhesive junctions, presumably suppressing the hematogenous metastasis of malignant tumor cells. Surprisingly, CLIC2 is localized in secretory granules and secreted into the extracellular milieu. Secreted CLIC2 binds to MMP14 and inhibits its activity, leading to suppressed MMP2 activity. CLIC4, on the other hand, promotes MMP14 activity. These findings challenge the assumption that CLICs are ion channels, implying that they could be potential new targets for the treatment of malignant tumors.

The oxidoreductase CLIC4 is required to maintain mitochondrial function and resistance to exogenous oxidants in breast cancer cells.

The chloride intracellular channel-4 (CLIC4) is one of the six highly conserved proteins in the CLIC family that share high structural homology with GST-omega in the GST superfamily. While CLIC4 is a multifunctional protein that resides in multiple cellular compartments, the discovery of its enzymatic glutaredoxin-like activity in vitro suggested that it could function as an antioxidant. Here, we found that deleting CLIC4 from murine 6DT1 breast tumor cells using CRISPR enhanced the accumulation of reactive oxygen species (ROS) and sensitized cells to apoptosis in response to H2O2 as a ROS-inducing agent. In intact cells, H2O2 increased the expression of both CLIC4 mRNA and protein. In addition, increased superoxide production in 6DT1 cells lacking CLIC4 was associated with mitochondrial hyperactivity including increased mitochondrial membrane potential and mitochondrial organelle enlargement. In the absence of CLIC4, however, H2O2-induced apoptosis was associated with low expression and degradation of the antiapoptotic mitochondrial protein Bcl2 and the negative regulator of mitochondrial ROS, UCP2. Furthermore, transcriptomic profiling of H2O2-treated control and CLIC4-null cells revealed upregulation of genes associated with ROS-induced apoptosis and downregulation of genes that sustain mitochondrial functions. Accordingly, tumors that formed from transplantation of CLIC4-deficient 6DT1 cells were highly necrotic. These results highlight a critical role for CLIC4 in maintaining redox-homeostasis and mitochondrial functions in 6DT1 cells. Our findings also raise the possibility of targeting CLIC4 to increase cancer cell sensitivity to chemotherapeutic drugs that are based on elevating ROS in cancer cells.

Induction of Pro-Fibrotic CLIC4 in Dermal Fibroblasts by TGF-ß/Wnt3a Is Mediated by GLI2 Upregulation.

Chloride intracellular channel 4 (CLIC4) is a recently discovered driver of fibroblast activation in Scleroderma (SSc) and cancer-associated fibroblasts (CAF). CLIC4 expression and activity are regulated by TGF-ß signalling through the SMAD3 transcription factor. In view of the aberrant activation of canonical Wnt-3a and Hedgehog (Hh) signalling in fibrosis, we investigated their role in CLIC4 upregulation. Here, we show that TGF-ß/SMAD3 co-operates with Wnt3a/ß-catenin and Smoothened/GLI signalling to drive CLIC4 expression in normal dermal fibroblasts, and that the inhibition of ß-catenin and GLI expression or activity abolishes TGF-ß/SMAD3-dependent CLIC4 induction. We further show that the expression of the pro-fibrotic marker α-smooth muscle actin strongly correlates with CLIC4 expression in dermal fibroblasts. Further investigations revealed that the inhibition of CLIC4 reverses morphogen-dependent fibroblast activation. Our data highlights that CLIC4 is a common downstream target of TGF-ß, Hh, and Wnt-3a through signalling crosstalk and we propose a potential therapeutic avenue using CLIC4 inhibitors.

Comparative kinetic analysis of ascorbate (Vitamin-C) recycling dehydroascorbate reductases from plants and humans.

Ascorbate is an important cellular antioxidant that gets readily oxidized to dehydroascorbate (DHA). Recycling of DHA is therefore paramount in the maintenance of cellular homeostasis and preventing oxidative stress. Dehydroascorbate reductases (DHARs), in conjunction with glutathione (GSH), carry out this vital process in eukaryotes, among which plant DHARs have garnered considerable attention. A detailed kinetic analysis of plant DHARs relative to their human counterparts is, however, lacking. Chloride intracellular channels (HsCLICs) are close homologs of plant DHARs, recently demonstrated to share their enzymatic activity. This study reports the highest turnover rate for a plant DHAR from stress adapted Pennisetum glaucum (PgDHAR). In comparison, HsCLICs 1, 3, and 4 reduced DHA at a significantly lower rate. We further show that the catalytic cysteine from both homologs was susceptible to varying degrees of oxidation, validated by crystal structures and mass-spectrometry. Our findings may have broader implications on crop improvement using pearl millet DHAR vis-à-vis discovery of cancer therapeutics targeting Vitamin-C recycling capability of human CLICs.

Circular RNA_0033596 aggravates endothelial cell injury induced by oxidized low-density lipoprotein via microRNA-217-5p /chloride intracellular channel 4 axis.

In recent years, the modulatory functions of some circular RNAs (circRNAs) in the pathogenesis of atherosclerosis (AS) have been reported. Nonetheless, the role of circular RNA_0033596 (circ_0033596) in AS and its mechanism remains unclarified. In this study, oxidized low-density lipoprotein (ox-LDL) was applied to treat human umbilical vein endothelial cells (HUVECs) to establish a cell model of endothelial cell injury. Western blot and quantitative real-time polymerase chain reaction (qRT-PCR) were employed to detect the expression of circ_0033596, microRNA-217-5p (miR-217-5p), and chloride intracellular channel 4 (CLIC4) in HUVECs. The binding sites between circ_0033596 and miR-217-5p, as well as between miR-217-5p and CLIC4 mRNA 3'UTR were determined through a dual-luciferase reporter gene assay. It was found that circ_0033596 expression was increased in ox-LDL-induced HUVECs. After ox-LDL stimulation, HUVEC viability and cell cycle progression were inhibited, and the apoptosis was promoted, while circ_0033596 overexpression aggravated these effects. MiR-217-5p was identified as a downstream target of circ_0033596, and circ_0033596 negatively regulated miR-217-5p expression. CLIC4 was identified as miR-217-5p's downstream target gene and could be positively modulated by circ_0033596. All in all circ_0033596 aggravates ox-LDL-induced HUVEC apoptosis by regulating the miR-217-5p/CLIC4 axis, by which circ_0033596 participates in the pathogenesis of AS.

Targeted proteomic analysis dataset of archival core human kidney biopsies to investigate the biology of hypertensive nephropathy.

Hypertensive nephropathy is the second most frequent cause of end-stage renal disease in western societies. In previous experiments in our laboratory with proteomic analysis of renal parenchyma of SHR hypertensive animals, we identified two molecules, namely SGLT2 and CLIC4, associated with the development of hypertension. Here, we apply the methodology of targeted proteomic analysis in kidney biopsies from patients with hypertensive nephropathy to study the role of SGLT2 and CLIC4 in the pathogenesis of the disease. Relative quantification data of SGLT2 and CLIC4 via means of targeted proteomic analysis in human kidney biopsies from hypertensive patients and normotensive controls are reported. In addition, validation data of the proteomic results via immunofluorescence are presented. Renal tissue biopsies (N = 17) from archival material of patients with hypertensive nephropathy and normotensive controls were used. Targeted proteomic analysis was performed using the method: ``Parallel Reaction Monitoring'' (PRM) in renal parenchyma of hypertensive and normotensive patients for the selective identification of SGLT2 and CLIC4 and the relative quantification of their expression using proteotypic peptides for each protein. The expression of SGLT2 molecule was also confirmed by immunofluorescence followed by quantification of fluorescence intensity. According to PRM, the SGLT2 protein was found with reduced and the CLIC4 protein with increased expression levels in hypertensive patients compared to normotensive controls. Comparison of representative immunofluorescence images confirmed a decrease in the expression of SGLT2 in the brush border of proximal tubular epithelial cells in hypertensive patients. Our data show changes in the tubular compartment of the kidney and especially in the proximal tubules associated with the hypertensive nephropathy. The clinical significance of these findings should be further explored for the discovery and/or confirmation of novel therapeutic approaches and biomarkers in the development of hypertensive kidney disease.

Characterization of chloride intracellular channel 4 in the regulation of human trophoblast function.

INTRODUCTION: Proper placentation requires well controlled extravillous trophoblast cell (EVT) migration and invasion. Transforming growth factor ß (TGFß) signaling has been well characterized as negatively regulating EVT migration and invasion. CLIC4 is an enhancer of TGFß signaling, however CLIC4's function in placentation and its association to placental TGFß signaling is unknown. Here we aimed to investigate the role of CLIC4 on trophoblast cell function and its relationship to TGFß signaling. METHODS: CLIC4 was immunolocalized in human placenta throughout gestation and the first trimester decidua. siRNA was used to knockdown CLIC4 in a human trophoblast cell line (HTR8/SVneo) to reveal functional consequences of CLIC4 loss on cell adhesion, proliferation, migration and invasion via xCELLigence. qPCR was used to identify downstream targets of CLIC4 in HTR8/SVNeo cells. RESULTS: CLIC4 was widely expressed in the syncytiotrophoblast, cytotrophoblast and decidual cells across all trimesters of pregnancy with no significant difference in staining intensity in the different cellular compartments both across gestation and between compartments. Using immunofluorescent co-localization of CLIC4 and EVT marker HLA-G, we confirmed that CLIC4 localized to the cytoplasm of cell column EVTs in the first trimester decidua and nuclei of some EVTs that invaded in the decidua. Knockdown of CLIC4 in HTR8/SVneo cells significantly elevated cell adhesion, migration and invasion. Analysis of TGFß signaling downstream targets identified that CDH2 and BAMBI expression were significantly increased after CLIC4 knockdown in HTR8/SVneo cells. DISCUSSION: Our data support an inhibitory role for CLIC4 in regulating trophoblast migration and invasion, likely acting in part via BAMBI and CDH2.

Chloride intracellular channel 4 (CLIC4) expression profile in the mouse medial prefrontal cortex and its regulation by ethanol.

BACKGROUND: Chloride intracellular channel 4 (CLIC4) is a multifunctional metamorphic protein for which a growing body of evidence supports a major role in the brain's molecular and behavioral responses to ethanol (EtOH). Although key to understanding the functional biology underlying this role, little is known about the cellular and subcellular expression patterns of CLIC4 in brain and how they are affected by EtOH. METHODS: We used qRT-PCR to assess Clic4 mRNA expression in the medial prefrontal cortex (mPFC) of C57BL/6J mice in the absence and presence of acute EtOH exposure. Two complementary immunohistochemical techniques were employed to assess the subcellular localization of the CLIC4 protein and its pattern of expression across brain cell types in the mPFC in the absence and presence of acute EtOH. RESULTS: Through immunohistochemical and stereological techniques, we show that CLIC4 protein is robustly expressed by oligodendrocytes (most abundant), microglia, and astrocytes, with minimal expression in neurons. Following acute EtOH exposure, we observed a rapid increase in Clic4 mRNA expression in female but not male mice and an overall increase in the number of oligodendrocytes and astrocytes expressing the CLIC4 protein. CONCLUSIONS: These findings suggest that Clic4 functions as an early response gene for acute EtOH in brain, which likely underlies its ability to modulate EtOH behavior. Our results also suggest that the role of CLIC4 in the brain's response to EtOH is mediated through oligodendrocytes.

Dexmedetomidine Alleviates Lipopolysaccharide-Induced Hippocampal Neuronal Apoptosis via Inhibiting the p38 MAPK/c-Myc/CLIC4 Signaling Pathway in Rats.

Dexmedetomidine (DEX) has multiple biological effects. Here, we investigated the neuroprotective role and molecular mechanism of DEX against lipopolysaccharide (LPS)-induced hippocampal neuronal apoptosis. Sprague Dawley rats were intraperitoneally injected with LPS (10 mg/kg) and/or DEX (30 µg/kg). We found that DEX improved LPS-induced alterations of hippocampal microstructure (necrosis and neuronal loss in the CA1 and CA3 regions) and ultrastructure (mitochondrial damage). DEX also attenuated LPS-induced inflammation and hippocampal apoptosis by inhibiting the increase of interleukin-1ß, interleukin-6, interleukin-18, and tumor necrosis factor-α levels and downregulating the expression of mitochondrial apoptosis pathway-related proteins. Moreover, DEX prevented the LPS-induced activation of the c-Myc/chloride intracellular channel 4 (CLIC4) pathway. DEX inhibited the p38 MAPK pathway, but not JNK and ERK. To further clarify whether DEX alleviated LPS-induced neuronal apoptosis through the p38 MAPK/c-Myc/CLIC4 pathway, we treated PC12 cells with p38 MAPK inhibitor SB203582 (10 µM). DEX had the same effect as SB203582 in reducing the protein and mRNA expression of c-Myc and CLIC4. Furthermore, DEX and SB203582 diminished LPS-induced apoptosis, indicated by decreased Bax and Tom20 fluorescent double-stained cells, reduced annexin V-FITC/PI apoptosis rate, and reduced protein expression levels of Bax, cytochrome C, cleaved caspase-9, and cleaved caspase-3. Taken together, the findings indicate that DEX attenuates LPS-induced hippocampal neuronal apoptosis by regulating the p38 MAPK/c-Myc/CLIC4 signaling pathway. These findings provide new insights into the mechanism of Alzheimer's disease and depression and may help aid in drug development for these diseases.

DNA Hypermethylation Involves in the Down-Regulation of Chloride Intracellular Channel 4 (CLIC4) Induced by Photodynamic Therapy.

The altered expression of chloride intracellular channel 4 (CLIC4) was reported to correlate with tumor progression. Previously, we have shown that the reduced cellular invasion induced by photodynamic therapy (PDT) is associated with suppression of CLIC4 expression in PDT-treated cells. Herein, we attempted to decipher the regulatory mechanisms involved in PDT-mediated CLIC4 suppression in A375 and MDA-MB-231 cells in vitro. We found that PDT can increase the expression and enzymatic activity of DNA methyltransferase 1 (DNMT1). Bisulfite sequencing PCR further revealed that PDT can induce hypermethylation in the CLIC4 promoter region. Silencing DNMT1 rescues the PDT-induced CLIC4 suppression and inhibits hypermethylation in its promoter. Furthermore, we found tumor suppressor p53 involves in the increased DNMT1 expression of PDT-treated cells. Finally, by comparing CLIC4 expression in lung malignant cells and normal lung fibroblasts, the extent of methylation in CLIC4 promoter was found to be inversely proportional to its expression. Taken together, our results indicate that CLIC4 suppression induced by PDT is modulated by DNMT1-mediated hypermethylation and depends on the status of p53, which provides a possible mechanistic basis for regulating CLIC4 expression in tumorigenesis.

Long non-coding RNA TDRG1 facilitates cell proliferation, migration and invasion in breast cancer via targeting miR-214-5p/CLIC4 axis.

Accumulated studies have revealed the critical role of long non-coding RNAs (lncRNAs) in the carcinogenesis and progression of various cancers. LncRNA TDRG1 has been reported to exhibit oncogenic potential in some cancers. However, its underlying mechanism regulating breast cancer (BC) remains obscure. QRT-PCR was used to measure the relative expression of mRNAs, and western blot was used to detect protein expression levels. CCK8 and CFSE assays were utilized to testify cell proliferation ability. Flow cytometry assay was used for cell apoptosis ability investigation. Transwell and tube formation assays were implemented to test cell migrating and invasive abilities. Relevant mechanism experiments were implemented to determine the molecular mechanism. TDRG1 was remarkably overexpressed in BC cell lines. TDRG1 knockdown suppressed cell proliferation, migration and invasion, but enhanced BC cell apoptosis. Mechanistically, TDRG1 acted as a miR-214-5p sponge to up-regulate CLIC4 expression. MiR-214-5p inhibition or CLIC4 overexpression could revive the tumor-suppressing effects induced by TDRG1 knockdown. TDRG1 promoted cell proliferation, migration, and invasion in BC, suggesting that TDRG1 could promisingly be a therapeutic target for BC.

Astragaloside IV alleviates atherosclerosis through targeting circ_0000231/miR-135a-5p/CLIC4 axis in AS cell model in vitro.

Non-coding RNAs (ncRNAs) have shown to act as crucial mediators in atherosclerosis (AS) development. The purpose of our study was to explore the role of Astragaloside IV (ASV) and circular RNA_0000231 (circ_0000231) in AS using AS cell model. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry were used to analyze cell viability and apoptosis. Migration ability was assessed by transwell migration assay and wound healing assay. The inflammatory response was evaluated via enzyme-linked immunosorbent assay (ELISA). Oxidative status was assessed via matching commercial kits. Western blot assay was conducted to detect the expression of monocyte chemoattractant protein 1 (MCP1), intercellular adhesion molecule 1 (ICAM1), and chloride intracellular channel 4 (CLIC4). The levels of circ_0000231, its linear form Rho GTPase activating protein 12 (ARHGAP12), microRNA-135a-5p (miR-135a-5p), and CLIC4 messenger RNA (mRNA) were detected by quantitative real-time polymerase chain reaction (qRT-PCR). Circ_0000231-miRNA interactions were established using Starbase and Circbank softwares, while the targets of miR-135a-5p were explored by Starbase software. Dual-luciferase reporter assay and RNA-pull down assay were used to verify these target interactions. ASV suppressed the apoptosis, inflammation, and oxidative stress while recovered the viability and migration ability of HUVECs which were mediated by oxidized low-density lipoprotein (ox-LDL). Circ_0000231 overexpression antagonized the protective role of ASV in ox-LDL-induced HUVECs. MiR-135a-5p was verified as a direct target of circ_0000231, and circ_0000231 contributed to ox-LDL-induced cell injury of HUVECs through down-regulating miR-135a-5p. MiR-135a-5p directly interacted with the 3' untranslated region (3'-UTR) of CLIC4 mRNA in HUVECs, and miR-135a-5p protected HUVECs against ox-LDL-induced injury through down-regulating CLIC4. ASV protected HUVECs against ox-LDL-induced injury through targeting circ_0000231/miR-135a-5p/CLIC4 axis. Targeting circ_0000231/miR-135a-5p/CLIC4 axis might provide a novel insight to develop effective strategy for AS treatment.

Aberrant Chloride Intracellular Channel 4 Expression Is Associated With Adverse Outcome in Cytogenetically Normal Acute Myeloid Leukemia.

Background and Methods: Acute myeloid leukemia (AML), which starts in the bone marrow, is a group of hematopoietic stem cell disorders. Chloride intracellular channel 4 (CLIC4) is regulated by p53, c-Myc, and TGF-ß. It induces the NF-κB-dependent activation of HIF (hypoxia-inducible factor) and participates in tumor growth through its microenvironmental function. However, its prognostic value in AML remains unclear, as well as its co-expression biomarkers. In this study, we evaluated the prognostic significance of CLIC4 expression using two independent large cohorts of cytogenetically normal AML (CN-AML) patients. Multivariable analysis and multi-omics analysis with weighted correlation network analysis (WGCNA) in the CN-AML group were also presented. Based on CLIC4 and its related genes, microRNA-target gene interaction network analysis and downstream gene ontology analysis were performed to unveil the complex functions behind CLIC4. Results: We demonstrated that the overexpression of CLIC4 was notably associated with unfavorable outcome in the two independent cohorts of CN-AML patients [overall survival (OS) and event-free survival (EFS): P < 0.0001, n = 185; OS: P = 0.016, n = 232], as well as in the European LeukemiaNet (ELN) Intermediate-I group (OS: P = 0.015, EFS: P = 0.012, n = 115), the National Comprehensive Cancer Network Intermediate Risk AML group (OS and EFS: P < 0.0001, n = 225), and the non-M3 AML group (OS and EFS: P < 0.0001, n = 435). Multivariable analysis further validated CLIC4 as a high-risk factor in the CN-AML group. Multi-omics analysis presented the overexpression of CLIC4 as associated with the co-expression of the different gene sets in leukemia, up/downregulation of the immune-related pathways, dysregulation of microRNAs, and hypermethylation around the CpG islands, in open sea regions, and in different gene structural fragments including TSS1500, gene body, 5'UTR region, 3'UTR region, and the first exon. By further performing WGCNA on multi-omics data, certain biomarkers that are co-expressed with CLIC4 were also unveiled. Conclusion: We demonstrated that CLIC4 is a novel, potential unfavorable prognosticator and therapeutic target for CN-AML. As having a key role in CN-AML, the interactions between CLIC4 and other genomics and transcriptomics data were confirmed by performing microRNA-target gene interaction network analysis and gene ontology enrichment analysis. The experimental result provides evidence for the clinical strategy selection of CN-AML patients.