Article

Chloride Intracellular Channel Protein-4 Functions in Angiogenesis by Supporting Acidification of Vacuoles Along the Intracellular Tubulogenic Pathway

Department of Medicine, University of North Carolina, Chapel Hill, NC 27514-7155, USA.
American Journal Of Pathology (Impact Factor: 4.59). 03/2009; 174(3):1084-96. DOI: 10.2353/ajpath.2009.080625
Source: PubMed

ABSTRACT

Endothelial cells form capillary tubes through the process of intracellular tubulogenesis. Chloride intracellular channel (CLIC) family proteins have been previously implicated in intracellular tubulogenesis, but their specific role has not been defined. In this study, we show that disruption of the Clic4 gene in mice results in defective angiogenesis in vivo as reflected in a Matrigel plug angiogenesis assay. An angiogenesis defect is also apparent in the retina, both in the decreased spontaneous development of retinal vasculature of unstressed mice and in the dramatically decreased angiogenic response of retinal vessels to an oxygen toxicity challenge. We found that endothelial cells derived from Clic4(-/-) mice demonstrated impaired tubulogenesis in three-dimensional fibrin gels compared with cells derived from wild-type mice. Furthermore, we found that tubulogenesis of wild-type cells in culture was inhibited by both an inhibitor of CLICs and an inhibitor of the vacuolar proton ATPase. Finally, we showed that vacuoles along the endothelial tubulogenesis pathway are acidic in wild-type cells, and that vacuolar acidification is impaired in Clic4(-/-) cells while lysosomal acidification is intact. We conclude that CLIC4 plays a critical role in angiogenesis by supporting acidification of vacuoles along the cell-hollowing tubulogenic pathway.

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Available from: Mary Elizabeth Hartnett, Jan 15, 2014
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    • "CLICs are multifunctional proteins playing a role in membrane trafficking , cytoskeletal function (Berryman et al., 2004), apoptosis (Fernandez-Salas et al., 2002; Suh et al., 2004), cell cycle control (Valenzuela et al., 2000), tubulogenesis (Berry et al., 2003), VEGFmediated angiogenesis of endothelial cells (Tung et al., 2009), modulation of ryanodine receptors (Board et al., 2004; Takano et al., 2012) and cell differentiation (Suh et al., 2007). CLICs are also implicated in modulating cardiovascular physiology, specifically, CLIC4 regulates vascular endothelial growth factor (VEGF)-mediated tubulogenesis in mammalian endothelial cells (Ulmasov et al., 2009). Recently, it was reported that inhibition of CLIC4 attenuates the development of pulmonary hypertension in chronically hypoxic mice (Wojciak-Stothard et al., 2014). "
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    ABSTRACT: Emerging evidences demonstrate significance of chloride channels in cardiac function and cardioprotection from ischemia–reperfusion (IR) injury. Unlike mitochondrial potassium channels sensitive to calcium (BKCa) and ATP (KATP), molecular identity of majority cardiac mitochondrial chloride channels located at the inner membrane is not known. In this study, we report the presence of unique dimorphic Chloride Intracellular Channel proteins (CLICs) namely CLIC1, CLIC4 and CLIC5 as abundant CLICs in the rodent heart. Further, CLIC4, CLIC5, and an ortholog present in Drosophila (DmCLIC) localizes to adult cardiac mitochondria. We found that CLIC4 is enriched in the outer mitochondrial membrane, whereas CLIC5 is present in the inner mitochondrial membrane. Also, CLIC5 plays a direct role in regulating mitochondrial reactive oxygen species (ROS) generation. Our study highlights that CLIC5 is localized to the cardiac mitochondria and directly modulate mitochondrial function.
    Full-text · Article · Jan 2016 · Mitochondrion
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    • "The most experimentally supported direct result of a CLIC protein action is in the formation of the gut lumen in C. elegans (the CLIC homologue exc-4 gene product) and tube formation in mouse blood vessels (CLIC4); while the loss of exc-4 in C. elegans causes expansion of the gut lumen into a large cyst, deficiency of CLIC4 in mice and vascular endothelial cells prevents normal vascular tubulogenesis [17,18,30,31] Mice devoid of CLIC4 also have skin and cornea wound healing defects. It has been suggested that the vascular abnormalities result from a defect in acidification of vacuoles possibly dependent on ion transport functions of CLIC4 while the wound healing defects likely result from the involvement of soluble CLIC4 in TGF-β signaling [18,26]. Although prior tissue lysate analyses have revealed presence of CLIC4 protein or transcripts in skin, lung, liver, kidney, heart, brain, spleen, testis, an extensive immunohistochemical analysis for cellular or developmental expression has been lacking [26,27]. "
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    ABSTRACT: Background Chloride Intracellular Channel 4 (CLIC4) is one of seven members in the closely related CLIC protein family. CLIC4 is involved in multiple cellular processes including apoptosis, cellular differentiation, inflammation and endothelial tubulogenesis. Despite over a decade of research, no comprehensive in situ expression analysis of CLIC4 in a living organism has been reported. In order to fulfill this goal, we generated a knock-in mouse to express Green Fluorescent Protein (GFP) from the CLIC4 locus, thus substituting the GFP coding region for CLIC4. We used GFP protein expression to eliminate cross reaction with other CLIC family members. Results We analyzed CLIC4 expression during embryonic development and adult organs. During mid and late gestation, CLIC4 expression is modulated particularly in fetal brain, heart, thymus, liver and kidney as well as in developing brown adipose tissue and stratifying epidermis. In the adult mouse, CLIC4 is highly expressed globally in vascular endothelial cells as well as in liver, lung alveolar septae, pancreatic acini, spermatogonia, renal proximal tubules, cardiomyocytes and thymic epithelial cells. Neural expression included axonal tracks, olfactory bulb, Purkinje cell layer and dentate gyrus. Renal CLIC4 expression was most pronounced in proximal tubules, although altered renal function was not detected in the absence of CLIC4. Myeloid cells and B cells of the spleen are rich in CLIC4 expression as are CD4 and CD8 positive T cells. Conclusions In a comprehensive study detailing CLIC4 expression in situ in a mouse model that excludes cross reaction with other family members, we were able to document previously unreported expression for CLIC4 in developing fetus, particularly the brain. In addition, compartmentalized expression of CLIC4 in specific adult tissues and cells provides a focus to explore potential functions of this protein not addressed previously.
    Full-text · Article · May 2014 · BMC Developmental Biology
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    • "In humans, CLIC family proteins consist of six members, designated as CLIC 1e6, which have a conserved C-terminal 240 residue module and one major transmembrane domain [20]. CLIC proteins are a subgroup of the glutathione-S-transferase (GSTs) superfamily. "
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    ABSTRACT: Chloride intracellular channel (CLIC) proteins constitute a subgroup of the glutathione-S-transferase (GSTs) superfamily. In humans, the CLIC family of proteins consists of six members, designated CLIC 1-6, which have a conserved C-terminal 240 residue module and one major transmembrane domain. CLIC proteins regulate fundamental cellular processes including regulation of chloride ion concentration, stabilization of cell membrane potential, trans-epithelial transport, regulation of cell volume and stimulation of apoptotic processes in response to cellular stress. Previously, we described the expression profile of a member of the CLIC family of proteins, CLIC3, in human placentae and fetal membranes. In the current study, we determined CLIC3 expression in placentae from pregnancies complicated with either fetal growth restriction (FGR, n=19), pre-eclampsia (PE, n=16) or both FGR and PE combined (n=12) compared to gestation-matched controls (n=13) using real-time PCR and a CLIC3 specific immunoassay. Significantly increased CLIC3 mRNA and protein were detected in placental extracts from pregnancies with FGR, PE and PE with FGR compared to controls. Our results suggest that increased expression of CLIC3 may play a role in abnormal placental function associated with the human pregnancy disorders FGR and PE.
    Full-text · Article · Jul 2012 · Placenta
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