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


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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    • "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.
    BMC Developmental Biology 05/2014; 14(1):24. DOI:10.1186/1471-213X-14-24 · 2.67 Impact Factor
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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.
    Placenta 07/2012; 33(9):741-4. DOI:10.1016/j.placenta.2012.06.011 · 2.71 Impact Factor
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    • "The role of CLIC4 in autophagy may be associated with autophagic gene regulation by nuclear translocation or the interruption of protein-protein interactions. Furthermore, the presence of CLIC4 by nuclear translocation may participate in altering pH and chloride ion content that could be involved in pH relative events in the organelles, cytoplasm and nucleus [38]. It is worth mentioning that CLIC4 can also translocate to the plasma membrane upon receptor stimulation [39]. "
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    ABSTRACT: CLIC4/mtCLIC, a chloride intracellular channel protein, localizes to mitochondria, endoplasmic reticulum (ER), nucleus and cytoplasm, and participates in the apoptotic response to stress. Apoptosis and autophagy, the main types of the programmed cell death, seem interconnected under certain stress conditions. However, the role of CLIC4 in autophagy regulation has yet to be determined. In this study, we demonstrate upregulation and nuclear translocation of the CLIC4 protein following starvation in U251 cells. CLIC4 siRNA transfection enhanced autophagy with increased LC3-II protein and puncta accumulation in U251 cells under starvation conditions. In that condition, the interaction of the 14-3-3 epsilon isoform with CLIC4 was abolished and resulted in Beclin 1 overactivation, which further activated autophagy. Moreover, inhibiting the expression of CLIC4 triggered both mitochondrial apoptosis involved in Bax/Bcl-2 and cytochrome c release under starvation and endoplasmic reticulum stress-induced apoptosis with CHOP and caspase-4 upregulation. These results demonstrate that CLIC4 nuclear translocation is an integral part of the cellular response to starvation. Inhibiting the expression of CLIC4 enhances autophagy and contributes to mitochondrial and ER stress-induced apoptosis under starvation.
    PLoS ONE 06/2012; 7(6):e39378. DOI:10.1371/journal.pone.0039378 · 3.23 Impact Factor
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