ORP2, a cholesterol transporter, regulates angiogenic signaling in endothelial cells

Abstract

Oxysterol-binding protein-related protein 2 (ORP2), a cholesterol-PI(4,5)P 2 countercurrent transporter, was recently identified as a novel regulator of plasma membrane (PM) cholesterol and PI(4,5)P 2 content in HeLa cells. Here, we investigate the role of ORP2 in endothelial cell (EC) cholesterol and PI(4,5)P 2 distribution, angiogenic signaling, and angiogenesis. We show that ORP2 knock-down modifies the distribution of cholesterol accessible to a D4H probe, between late endosomes and the PM. Depletion of ORP2 from ECs inhibits their angiogenic tube formation capacity, alters the gene expression of angiogenic signaling pathways such as VEGFR2, Akt, mTOR, eNOS, and Notch, and reduces EC migration, proliferation, and cell viability. We show that ORP2 regulates the integrity of VEGFR2 at the PM in a cholesterol-dependent manner, the depletion of ORP2 resulting in proteolytic cleavage by matrix metalloproteinases, and reduced activity of VEGFR2 and its downstream signaling. We demonstrate that ORP2 depletion increases the PM PI(4,5)P 2 coincident with altered F-actin morphology, and reduces both VEGFR2 and cholesterol in buoyant raft membranes. Moreover, ORP2 knock-down suppresses the expression of the lipid raft-associated proteins VE-cadherin and caveolin-1. Analysis of the retinal microvasculature in ORP2 knockout mice generated during this study demonstrates the subtle alterations of morphology characterized by reduced vessel length and increased density of tip cells and perpendicular sprouts. Gene expression changes in the retina suggest disturbance of sterol homeostasis, downregulation of VE-cadherin, 14672 | KOPONEN Et al.

ORP2 couples LDL‐cholesterol transport to FAK activation by endosomal cholesterol/PI(4,5)P2 exchange

“…Considering that ORP2 exchanges PI(4,5)P 2 , we also investigated whether acute ORP2 degradation alters PM PI(4,5)P 2 . Based on PLCdelta PH‐domain distribution, no obvious changes in PM PI(4,5)P 2 were observed during the first 4 h upon ORP2 removal; however, after 3 days of ORP2 ablation, more PM PI(4,5)P 2 was detected (Fig EV4E–G), in consistence with cells silenced for ORP2 for 3 days (Koponen et␣al , 2020). In addition, we tested whether a well‐known PM PI(4,5)P 2 ‐dependent membrane transport process, clathrin‐mediated endocytosis of transferrin was affected, and found it not to be altered in 4 h of ORP2 removal (Fig EV4H).…”

Protrudin regulates FAK activation, endothelial cell migration and angiogenesis

“…Human Aortic Endothelial cells (HAEC) were procured from Lonza (Basel, CH). Cells were cultured in ECGM2 as described earlier [ 27 ]. Briefly, cells were seeded onto flasks/plates pre-coated with fibronectin 10 µg/ml–gelatin 0.05% (Sigma-Aldrich, Saint Louis, MO) and maintained in ECGM2 media at 37 °C in a humidified atmosphere with 5% CO 2 until confluent.…”

“…Migration of HUVECs transduced with shNT or shProtrudin lentiviruses was measured using an xCELLigence RTCA DP instrument (ACEA Biosciences) equipped with CIM-plate 16 [ 27 ]. Prior to the xCELLigence measurements, cells were starved for 2 h in ECM2 media containing 0.1% BSA.…”

“…Migration of HUVECs was assessed using IncuCyte™ ZOOM Scratch Wound assay (Essen BioScience, Hertfordshire, UK) as described earlier [ 27 ]. 3 × 10 4 HUVECs were seeded in a 96-well Image Lock plate along with shNT or shProtrudin lentivirus in ECGM2 media.…”

Mutations in OSBPL2 cause hearing loss associated with primary cilia defects via sonic hedgehog signaling

“…OSBPL2 is one of the short ORPs comprising only 2 domains: 2 phenylalanines in an acidic tract motif (FFAT) and the OSBP-related domain (ORD). The FFAT motif is an ER-targeting residue and the ORD domain refers to a highly conserved region shared by OSBP/ORPs family members, which was the most functional part of OSBPL2 for binding and transferring sterols, oxysterols, and PIs ( 23 , 25 , 33 ). When Flag-OSBPL2 and Flag-ΔFFAT were reexpressed in Osbpl2 –/– HEI-OC1 cells, the length of primary cilia was restored in Flag-OSBPL2–expressing cells, but not significantly changed in Flag-ΔORD–expressing cells ( Figure 4, A and B ).…”