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.
Minerva Foundation Institute for Medical Research, FI-00290 Helsinki, Finland.
Olivier
O
Béaslas
Terhi
T
Vihervaara
Jiwei
J
Li
Pirkka-Pekka
PP
Laurila
Daoguang
D
Yan
Vesa M
VM
Olkkonen
D000818:Animals; D001483:Base Sequence; D001667:Binding, Competitive; D002453:Cell Cycle; D002460:Cell Line; D002465:Cell Movement; D002467:Cell Nucleus / Q000378:metabolism; D049109:Cell Proliferation; D017931:DNA Primers / Q000235:genetics; D055785:Gene Knockdown Techniques; D008264:Macrophages / Q000166:cytology / Q000502:physiology*; D008562:Membrane Glycoproteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008870:Microtubules / Q000378:metabolism; D028861:Nuclear Pore Complex Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D020411:Oligonucleotide Array Sequence Analysis; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D011987:Receptors, Steroid / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000502:physiology*; D059467:Transcriptome; D033921:Vesicular Transport Proteins / Q000378:metabolism; D000098825:Oxysterol Binding Proteins
D017931:DNA Primers; C522783:Exo70 protein, mouse; D008562:Membrane Glycoproteins; D028861:Nuclear Pore Complex Proteins; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D011987:Receptors, Steroid; D033921:Vesicular Transport Proteins; C049312:nuclear pore protein p62; D000098825:Oxysterol Binding Proteins