CLIC4/Arf6 Pathway - A New Lead in BMPRII Inhibition in Pulmonary Hypertension.

RATIONALE: Increased expression of chloride intracellular channel 4 (CLIC4) 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 signalling in pulmonary hypertension.

METHODS AND RESULTS: Proteomic analysis of CLIC4-interacting proteins in human pulmonary artery endothelial cells (HPAECs) identified regulators of endosomal trafficking, including ADP Ribosylation Factor 6 (Arf6) GTPase activating proteins and clathrin, while CLIC4 overexpression affected protein regulators of vesicular trafficking, lysosomal function and inflammation. CLIC4 reduced BMPRII expression and signalling 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 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 NFkB, HIF 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 NFκB and reduced expression of BMPRII in the lung. These changes were established early during disease development. Lung endothelium-targeted delivery of CLIC4siRNA 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 importane of this pathway in the human condition.

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