Highlights d ORP4L is highly expressed in LSCs and essential for LSC survival d ORP4L extracts and presents PIP 2 from the plasma membrane for PLCb3 catalysis in LSCs d LYZ-81 is identified as a specific inhibitor of ORP4L

The enhanced expression of miR-31 has been observed in many human malignancies including lung cancer, and this microRNA regulates several aspects of oncogenesis. However, the role of miR-31-5p in energy metabolism remains elusive. Here, we confirm that H1299 and A549 cells, 2 lung cancer cell lines, relay on aerobic glycolysis as main source of ATP. Inhibition of miR-31-5p leads to decreased glycolysis and ATP production, while miR-31-5p overexpression increases them. Hypoxia inducible factor 1 (HIF-1) up-regulates the expression of glycolytic enzymes, and the HIF-1α inhibitor (FIH) inhibits HIF-1 activity. Because FIH is a direct target of miR-31-5p, inhibition of miR-31-5p results in enhanced FIH expression and suppression of HIF-1 signaling, while overexpression of miR-31-5p has the opposite effects. Via this mechanism, miR-31-5p up-regulates aerobic glycolytic genes and maintains energy homeostasis. To further validate the mechanism of miR-31-5p in glycolysis regulation, we show that overexpression or knockdown of FIH rescued the effects of miR-31-5p or miR-31-5p inhibitor on HIF activation and its target gene expression, respectively. Finally, by means of an A549 cell xenograft mouse model, we demonstrate that the miR-31-5p promotes cell proliferation via enhancing glycolysis. In summary, this study reveals that miR-31-5p promotes the Warburg effect via direct targeting of FIH.-Zhu, B., Cao, X., Zhang, W., Pan, G., Yi, Q., Zhong, W., Yan, D. MicroRNA-31-5p enhances the Warburg effect via targeting FIH.

Oxysterol-binding protein related-protein 5 and 8 (ORP5/8) localize to the membrane contact sites (MCS) of the endoplasmic reticulum (ER) and the mitochondria, as well as to the ER-plasma membrane (PM) MCS. The MCS are emerging as important regulators of cell signaling events, including calcium (Ca) signaling. ORP5/8 have been shown to interact with phosphatidylinositol-4,5-bisphosphate (PIP) in the PM, and to modulate mitochondrial respiration and morphology. PIP is the direct precursor of inositol trisphosphate (IP), a key second messenger responsible for Ca-release from the intracellular Ca stores. Further, mitochondrial respiration is linked to Ca transfer from the ER to the mitochondria. Hence, we asked whether ORP5/8 would affect Ca signaling in these cell compartments, and employed genetically engineered aequorin Ca probes to investigate the effect of ORP5/8 in the regulation of mitochondrial and caveolar Ca. Our results show that ORP5/8 overexpression leads to increased mitochondrial matrix Ca as well as to increased Ca concentration at the caveolar subdomains of the PM during histamine stimulation, while having no effect on the cytoplasmic Ca. Also, we found that ORP5/8 overexpression increases cell proliferation. Our results show that ORP5/8 regulate Ca signaling at specific MCS foci. These local ORP5/8-mediated Ca signaling events are likely to play roles in processes such as mitochondrial respiration and cell proliferation.

Rationale: Macrophage survival within the arterial wall is a central factor contributing to atherogenesis. Oxysterols, major components of oxidized low-density lipoprotein, exert cytotoxic effects on macrophages. Objective: To determine whether oxysterol-binding protein–related protein 4 L (ORP4L), an oxysterol-binding protein, affects macrophage survival and the pathogenesis of atherosclerosis. Methods and Results: By hiring cell biological approaches and ORP4L − /− mice, we show that ORP4L coexpresses with and forms a complex with Gα q/11 and phospholipase C (PLC)-β3 in macrophages. ORP4L facilitates G-protein–coupled ligand-induced PLCβ3 activation, IP 3 production, and Ca 2+ release from the endoplasmic reticulum. Through this mechanism, ORP4L sustains antiapoptotic Bcl-XL expression through Ca 2+ -mediated c-AMP responsive element binding protein transcriptional regulation and thus protects macrophages from apoptosis. Excessive stimulation with the oxysterol 25-hydroxycholesterol disassembles the ORP4L/Gα q/11 /PLCβ3 complexes, resulting in reduced PLCβ3 activity, IP 3 production, and Ca 2+ release, as well as decreased Bcl-XL expression and increased apoptosis. Overexpression of ORP4L counteracts these oxysterol-induced defects. Mice lacking ORP4L exhibit increased apoptosis of macrophages in atherosclerotic lesions and a reduced lesion size. Conclusions: ORP4L is crucial for macrophage survival. It counteracts the cytotoxicity of oxysterols/oxidized low-density lipoprotein to protect macrophage from apoptosis, thus playing an important role in the development of atherosclerosis.

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.

Oxysterol-binding protein-related protein (ORP) 4L acts as a scaffold protein assembling CD3-«, G-a q/11 , and PLC-b3 into a complex at the plasma membrane that mediates inositol (1,4,5)-trisphosphate (IP 3 )-induced endoplasmic reticulum (ER) Ca 2+ release and oxidative phosphorylation in T-cell acute lymphoblastic leukemia cells. Here, we offer new evidence that ORP4L interacts with the carboxyl terminus of the IP 3 receptor type 1 (ITPR1) in Jurkat T cells. ORP4L enables IP 3 binding to ITPR1; a truncated construct that lacks the ITPR1-binding region retains the ability to increase IP 3 production but fails to mediate IP 3 and ITPR1 binding. In association with this ability of ORP4L, it enhances Ca 2+ release from the ER and subsequent cytosolic and mitochondrial parallel Ca 2+ spike oscillations that stimulate mitochondrial energetics and thus maintains cell survival. These data support a novel model in which ORP4L is a cofactor of ITPR1, which increases ITPR1 sensitivity to IP 3 and enables ER Ca 2+ release.

Phosphoinositide phospholipases C (PLCs) are a family of eukaryotic intracellular enzymes with important roles in signal transduction. In addition to their location at the plasma membrane, PLCs also exist within the cell nucleus where they are stored. We previously demonstrated that OSBP-related protein 4L (ORP4L) anchors cluster of differentiation 3⑀ (CD3⑀) to the heterotrimeric G protein subunit (G␣ q/11 ) to control PLC␤3 relocation and activation. However, the underlying mechanism by which ORP4L facilitates PLC␤3 translocation remains unknown. Here, using confocal immunofluorescence microscopy and coimmunoprecipitation assays, we report that ORP4L stimulates PLC␤3 translocation from the nucleus to the plasma membrane in Jurkat T-cells in two steps. First, we found that ORP4L is required for the activation of Ras-related nuclear protein (RAN), a GTP-binding nuclear protein that binds to exportin 1 and eventually promotes the nuclear export of PLC␤3. Second, we also observed that ORP4L interacts with vesicleassociated membrane protein-associated protein A (VAPA) through its two phenylalanines in an acidic tract (FFAT) motif. This complex enabled PLC␤3 movement to the plasma membrane, indicating that PLC␤3 translocation occurs in a VAPAdependent manner. This study reveals detailed mechanistic insight into the role of ORP4L in PLC␤3 redistribution from storage within the nucleus to the plasma membrane via RAN activation and interaction with VAPA in Jurkat T-cells.

Human T-cell leukemia virus 1 (HTLV-1) causes adult T-cell leukemia (ATL), but the mechanism underlying its initiation remains elusive. Here we report that ORP4L is expressed in ATL cells but not normal T-cells. ORP4L ablation completely blocks T-cell leukemogenesis induced by the HTLV-1 oncoprotein Tax in mice while engineering ORP4L expression in T-cells results in T-cell leukemia in mice, suggesting the oncogenic properties and prerequisite of ORP4L for the initiation of T-cell leukemogenesis. For molecular insight, loss of miR-31 caused by HTLV-1 induces ORP4L expression in T-cells. ORP4L interacts with PI3Kδ to promote PI(3,4,5)P3 generation, contributing to AKT hyperactivation, NF-κB-dependent p53 inactivation induced pro-oncogenes expression and T-cell leukemogenesis. Consistently, ORP4L ablation eliminates human ATL cells in patient-derived xenograft ATL models. These results reveal a plausible mechanism of T-cells deterioration by HTLV-1 that can be therapeutically targeted.