Internalized group V secretory phospholipase A2 acts on the perinuclear membranes

Abstract

Mammalian secretory phospholipases A2 (sPLA2) have been implicated in cellular eicosanoid biosynthesis but the mechanism of their cellular action remains unknown. To elucidate the spatiotemporal dynamics of sPLA2 mobilization and determine the site of its lipolytic action, we performed time-lapse confocal microscopic imaging of fluorescently labeled sPLA2 acting on human embryonic kidney (HEK) 293 cells the membranes of which are labeled with a fluorogenic phospholipid, N-((6.(2,4-dinitrophenyl)amino)hexanoyl)-1-hexadecanoyl-2-(4,4-difluoro-5, 7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoyl)-sn-glycero-3- phosphoethanolamine. The Western blotting analysis of HEK293 cells treated with exogenous sPLA2s showed that not only the affinity for heparan sulfate proteoglycan but also other factors, such as sPLA2 hydrolysis products or cytokines, are necessary for the internalization of sPLA2 into HEK293 cells. Live cell imaging showed that the hydrolysis of fluorogenic phospholipids incorporated into HEK293 cell membranes was synchronized with the spatiotemporal dynamics of sPLA2 internalization, detectable initially at the plasma membrane and then at the perinuclear region. Also, immunocytostaining showed that human group V sPLA2 induced the translocation of 5-lipoxygenase to the nuclear envelope at which they were co-localized. Together, these studies provide the first experimental evidence that the internalized sPLA2 acts on the nuclear envelope to provide arachidonate for other enzymes involved in the eicosanoid biosynthesis.