CLIC4/Arf6 Pathway

Abstract

Rationale: Increased expression of CLIC4 (chloride intracellular channel 4) is a feature of endothelial dysfunction in pulmonary arterial hypertension, but its role in disease pathology is not fully understood.

Objective: To identify CLIC4 effectors and evaluate strategies targeting CLIC4 signaling in pulmonary hypertension.

Methods and results: Proteomic analysis of CLIC4-interacting proteins in human pulmonary artery endothelial cells identified regulators of endosomal trafficking, including Arf6 (ADP ribosylation factor 6) GTPase activating proteins and clathrin, while CLIC4 overexpression affected protein regulators of vesicular trafficking, lysosomal function, and inflammation. CLIC4 reduced BMPRII (bone morphogenetic protein receptor II) expression and signaling as a result of Arf6-mediated reduction in gyrating clathrin and increased lysosomal targeting of the receptor. BMPRII expression was restored by Arf6 siRNA, Arf inhibitor Sec7 inhibitor H3 (SecinH3), and inhibitors of clathrin-mediated endocytosis but was unaffected by chloride channel inhibitor, indanyloxyacetic acid 94 or Arf1 siRNA. The effects of CLIC4 on NF-κB (nuclear factor-kappa B), HIF (hypoxia-inducible factor), and angiogenic response were prevented by Arf6 siRNA and SecinH3. Sugen/hypoxia mice and monocrotaline rats showed elevated expression of CLIC4, activation of Arf6 and NF-κB, and reduced expression of BMPRII in the lung. These changes were established early during disease development. Lung endothelium-targeted delivery of CLIC4 siRNA or treatment with SecinH3 attenuated the disease, reduced CLIC4/Arf activation, and restored BMPRII expression in the lung. Endothelial colony-forming cells from idiopathic pulmonary hypertensive patients showed upregulation of CLIC4 expression and Arf6 activity, suggesting potential importance of this pathway in the human condition.

Conclusions: Arf6 is a novel effector of CLIC4 and a new therapeutic target in pulmonary hypertension.

Keywords: chloride channels; endocytosis; endothelial cells; endothelial progenitor cells; hypertension, pulmonary.

Figure 1.

CLIC4 increases NF-κB activity and reduces BMPRII signaling in HPAECS. A, NF-κB activity in HPAECs overexpressing CLIC4 or CLIC4shRNA in normoxia, hypoxia, and in cells treated with TNF-α (10 μg/L, 24 h) or NF-κB inhibitor, BAY 117085 (10 μmol/L, 24 h); luciferase reporter assay. (B) BMPRII protein levels, (C) Smad1/5 phosphorylation, and (D) Smad3 phosphorylation in HPAECs infected with Adcontrol or AdCLIC4. In (C), the cells were treated with BMP9 (10 μg/L, 1 h) and in (D) with TGF-β (10 μg/L, 1 h). Representative Western blots are shown underneath the graphs. E, The effect of BMPRII and PPM1A overexpression on CLIC4-induced activation of NF-κB in cells treated, as indicated. *P<0.05; **P<0.01, ***P<0.001, ****P<0.0001, comparisons with Adcontrol; #P<0.05; ##P<0.01; ###P<0.001, comparisons with CLIC4+TNF-α or as indicated. Data are presented as mean±SEM; n=4–8. Student t test or 1-way ANOVA with Tukey’s post-test, as appropriate. BMP9 indicates bone morphogenetic protein 9; BMPRII, bone morphogenetic protein receptor II; CLIC4, chloride intracellular channel 4; HPAECs, human pulmonary artery endothelial cells; PPM1A, protein phosphatase 1A; NF-κB, nuclear factor kappa B; and TNF-α, tumor necrosis factor α.

Figure 2.

The effect of CLIC4 on intracellular localization of BMPRII, lysosomal function, and clathrin-mediated vesicular trafficking. Confocal images and graph in (A) show increased colocalization of BMPRII and CLIC4 in CLIC4-overexpressing cells; proximity ligation assay (PLA). B, Colocalization of CLIC4 and BMPRII in LAMP1-positive vesicles, PLA. Boxed area in the left image is magnified in the middle and right images, which show colocalization of BMPRII with CLIC4 (white pixels, middle image) or colocalization of CLIC4 and BMPRII (red) with LAMP1 (green) in the right image. Arrowheads indicate colocalization points with 1 enlarged colocalization point in the inset in the right image. C, Endosomal/lysosomal acidification; D, clathrin-mediated uptake of Alexa488-transferrin. E, Single confocal images of YFP-GGA1 (yellow fluorescent protein–labelled golgi-associated, gamma adaptin ear-containing, ARF-binding protein 1) vesicles (left), sum projection images from image stacks (middle), and trajectories of YFP-GGA1 vesicles (right) in cells treated, as indicated; F, levels of gyrating clathrin (G-clathrin) in control and CLIC4-overexpressing HPAECs, with or without Sec7 inhibitor H3 (SecinH3; 10 mg/L), as indicated. In all images Bar=10 μm. **P<0.01, ***P<0.001, Student t test, or 1-way ANOVA with Tukey’s post-test, as appropriate; n=4–6. Data are presented as mean±SEM. BMPRII indicates bone morphogenetic protein receptor II; CLIC4, chloride intracellular channel 4; HPAECs, human pulmonary artery endothelial cells; and LAMP1, lysosomal-associated membrane protein 1.

Figure 3.

Arf6 mediates the effects of CLIC4. A, Arf6 activity; B, BMPRII expression in control HPAECs (Adcontrol), HPAECs overexpressing CLIC4 (AdCLIC4), with or without Sec7 inhibitor H3 (SecinH3; 10 mg/L, 24 h), as indicated. Representative Western blots are shown on the right. Graph in (C) and corresponding representative Western blots show the effect of control siRNA (ctrl siRNA), Arf6 siRNA, and Arf1 siRNA on BMPRII levels in Adcontrol- and AdCLIC4-overexpressing cells. D, The effect of SecinH3 on TNF-α-induced activation of NF-κB in control and CLIC4-overexpressing HPAECs; E, The effect of Arf6 siRNA and Arf1 siRNA on TNF-α-induced activation of NF-κB in cells treated, as indicated. F, Arf6 activity and CLIC4 expression in ECFCs from IPAH patients, n=6. *P<0.05, **P<0.01, ***P<0.001, comparisons with Adcontrol; #P<0.05; ##P<0.01; ###P<0.001, comparisons with AdCLIC4+TNF-α or as indicated. Data are presented as mean±SEM; n=4–6. Student t test or 1-way ANOVA with Tukey post hoc test, except for (F), where data were analyzed with Mann-Whitney U test. Arf6 indicates ADP ribosylation factor 6; BMPRII, bone morphogenetic protein receptor II; CLIC4, chloride intracellular channel 4; HPAECs, human pulmonary artery endothelial cells; NF-κB, nuclear factor kappa B; and TNF-α, tumor necrosis factor α.

Figure 4.

Effects of CLIC4siRNA on development of pulmonary hypertension, Arf activation, BMPRII expression, and NF-κB activity in Sugen/hypoxia mice. (A) RVSP; (B) RV/LV+S; and (C) percentage of muscularized vessels in lungs of control mice and Sugen/hypoxia mice treated with nontargeting siRNA (control siRNA) or CLIC4 siRNA/DACC lipoplex, as indicated. D, Confocal images showing endothelial localization of nontargeting fluorescent siRNA delivered by DACC delivery vehicle (siRNACy3/DACC); Bar=10 μm. E, αSMA staining in mouse lung sections. Arrowheads point to small intrapulmonary vessels. Bar=25 μm. F–H, Graphs and corresponding representative Western blots show Arf6 and Arf1 activity, CLIC4, and BMPRII expression in lungs of the untreated and CLIC4siRNA-treated mice, as indicated. I, NF-κB activity in mice treated, as indicated. *P<0.05, **P<0.01, ***P<0.001, comparisons with controls; #P<0.05, ###P<0.001 comparisons with control siRNA Sugen/hypoxia group or as indicated. Data are presented as mean±SEM; n=7–8. One-way ANOVA with Tukey post hoc test. Arf6 indicates ADP ribosylation factor 6; BMPRII, bone morphogenetic protein receptor II; CLIC4, chloride intracellular channel 4; NF-κB, nuclear factor kappa B; LV, left ventricle; RV, right ventricular; RVSP, right ventricular systolic pressure; and VWF, von Willebrand Factor.

Figure 5.

Effects of Sec7 inhibitor H3 (SecinH3) on development of pulmonary hypertension, Arf activation, BMPRII expression, and NF-κB activity in Sugen/hypoxia mice. A, RVSP; (B) RV/LV+S, and (C) percentage of muscularized vessels in lungs of control mice and Sugen/hypoxia mice treated with SecinH3, as indicated. D, αSMA staining in mouse lung sections. Arrowheads point to small intrapulmonary vessels. Bar=25 μm. (E) Arf6 activity, (F) Arf1 activity, and (G) BMPRII expression in the lungs of untreated and SecinH3-treated mice, as indicated. Representative Western blots are shown below the graphs. H, NF-κB activity in mice. *P<0.05, **P<0.01, ***P<0.001, comparisons with controls; #P<0.05, ##P<0.01, ###P<0.001 comparisons, as indicated. Data are presented as mean±SEM; n=8. One-way ANOVA with Tukey post hoc test. Arf6 indicates ADP ribosylation factor 6; BMPRII, bone morphogenetic protein receptor II; NF-κB, nuclear factor kappa B; LV,left ventricle; RV, right ventricle; and RVSP, right ventricular systolic pressure.

Figure 6.

Effects of Sec7 inhibitor H3 (SecinH3) on development of pulmonary hypertension, Arf activation, BMPRII, and CLIC4 expression in lungs of MCT rats. (A) mPAP; (B) RV/LV+S; and (C) percentage of remodeled vessels in lungs of control and MCT rats treated with SecinH3, as indicated. D, Elastic van Gieson (EVG) staining showing fully muscularized peripheral arteries with double elastic laminae in MCT rat lung but single elastic laminae in control and SecinH3-treated rat lung (arrowheads). Bar=25 μm. E, Arf6 activity, (F) Arf1 activity, (G) BMPRII expression, and (H) NF-κB activity. Representative Western blots are shown below the graphs. *P<0.05, **P<0.01, ***P<0.001, comparisons with controls; #P<0.05, ##P<0.01, ###P<0.001 comparisons, as indicated. Data are presented as mean±SEM; n=6. One-way ANOVA with Tukey post hoc test. Arf6 indicates ADP ribosylation factor 6; BMPRII, bone morphogenetic protein receptor II; CLIC4, chloride intracellular channel 4; MCT, monocrotaline; and NF-κB, nuclear factor kappa B.

Figure 7.

Proposed CLIC4/Arf6 signaling pathway. Arf6 indicates ADP ribosylation factor 6; BMPR, bone morphogenetic protein receptor; CLIC4, chloride intracellular channel 4; GAPs, GTPase-activating proteins; GEFs, guanine exchange factors; PAH, pulmonary arterial hypertension; PPM1A, protein phosphatase 1A; NF-κB, nuclear factor kappa B; and TGF-β, transforming growth factor.