Orp8 deficiency in bone marrow-derived cells reduces atherosclerotic lesion progression in LDL receptor knockout mice.

INTRODUCTION: Oxysterol binding protein Related Proteins (ORPs) mediate intracellular lipid transport and homeostatic regulation. ORP8 downregulates ABCA1 expression in macrophages and cellular cholesterol efflux to apolipoprotein A-I. In line, ORP8 knockout mice display increased amounts of HDL cholesterol in blood. However, the role of macrophage ORP8 in atherosclerotic lesion development is unknown. METHODS AND RESULTS: LDL receptor knockout (KO) mice were transplanted with bone marrow (BM) from ORP8 KO mice and C57Bl/6 wild type mice. Subsequently, the animals were challenged with a high fat/high cholesterol Western-type diet to induce atherosclerosis. After 9 weeks of Western-Type diet feeding, serum levels of VLDL cholesterol were increased by 50% in ORP8 KO BM recipients compared to the wild-type recipients. However, no differences were observed in HDL cholesterol. Despite the increase in VLDL cholesterol, lesions in mice transplanted with ORP8 KO bone marrow were 20% smaller compared to WT transplanted controls. In addition, ORP8 KO transplanted mice displayed a modest increase in the percentage of macrophages in the lesion as compared to the wild-type transplanted group. ORP8 deficient macrophages displayed decreased production of pro-inflammatory factors IL-6 and TNFα, decreased expression of differentiation markers and showed a reduced capacity to form foam cells in the peritoneal cavity. CONCLUSIONS: Deletion of ORP8 in bone marrow-derived cells, including macrophages, reduces lesion progression after 9 weeks of WTD challenge, despite increased amounts of circulating pro-atherogenic VLDL. Reduced macrophage foam cell formation and lower macrophage inflammatory potential are plausible mechanisms contributing to the observed reduction in atherosclerosis.

Silencing of OSBP-related protein 8 (ORP8) modifies the macrophage transcriptome, nucleoporin p62 distribution, and migration capacity.

ORP8 is an oxysterol/cholesterol binding protein anchored to the endoplasmic reticulum and the nuclear envelope, and is abundantly expressed in the macrophage. We created and characterized mouse RAW264.7 macrophages with ORP8 stably silenced using shRNA lentiviruses. A microarray transcriptome and gene ontology pathway analysis revealed significant alterations in several nuclear pathways and ones associated with centrosome and microtubule organization. ORP8 knockdown resulted in increased expression and altered subcellular distribution of an interaction partner of ORP8, nucleoporin NUP62, with an intranuclear localization aspect and association with cytoplasmic vesicular structures and lamellipodial edges of the cells. Moreover, ORP8 silenced cells displayed enhanced migration, and a more pronounced microtubule cytoskeleton than controls expressing a non-targeting shRNA. ORP8 was shown to compete with Exo70 for interaction with NUP62, and NUP62 knockdown abolished the migration enhancement of ORP8-silenced cells, suggesting that the endogenous ORP8 suppresses migration via binding to NUP62. As a conclusion, the present study reveals new, unexpected aspects of ORP8 function in macrophages not directly involving lipid metabolism, but rather associated with nuclear functions, microtubule organization, and migration capacity.

OSBP-related protein 8 (ORP8) regulates plasma and liver tissue lipid levels and interacts with the nucleoporin Nup62.

We earlier identified OSBP-related protein 8 (ORP8) as an endoplasmic reticulum oxysterol-binding protein implicated in cellular lipid homeostasis. We now investigated its action in hepatic cells in vivo and in vitro. Adenoviral overexpression of ORP8 in mouse liver induced a decrease of cholesterol, phospholipids, and triglycerides in serum (-34%, -26%, -37%, respectively) and liver tissue (-40%, -12%, -24%), coinciding with reduction of nuclear (n)SREBP-1 and -2 and mRNA levels of their target genes. Consistently, excess ORP8 reduced nSREBPs in HuH7 cells, and ORP8 overexpression or silencing by RNA interference moderately suppressed or induced the expression of SREBP-1 and SREBP-2 target genes, respectively. In accordance, cholesterol biosynthesis was reduced by ORP8 overexpression and enhanced by ORP8 silencing in [(3)H]acetate pulse-labeling experiments. ORP8, previously shown to bind 25-hydroxycholesterol, was now shown to bind also cholesterol in vitro. Yeast two-hybrid, bimolecular fluorescence complementation (BiFC), and co-immunoprecipitation analyses revealed the nuclear pore component Nup62 as an interaction partner of ORP8. Co-localization of ORP8 and Nup62 at the nuclear envelope was demonstrated by BiFC and confocal immunofluorescence microscopy. Furthermore, the impact of overexpressed ORP8 on nSREBPs and their target mRNAs was inhibited in cells depleted of Nup62. Our results reveal that ORP8 has the capacity to modulate lipid homeostasis and SREBP activity, probably through an indirect mechanism, and provide clues of an entirely new mode of ORP action.