Group V Secretory Phospholipase A(2) Regulates Endocytosis of Acetylated LDL by Transcriptional Activation of PGK1 in RAW264.7 Macrophage Cell Line

Aims: It was suggested that group V secretory phospholipase A(2) (sPLA(2)-V) existed in the nucleus. This study examined whether nuclear sPLA(2)-V plays a role in endocytosis of acetylated low-density lipoprotein (AcLDL) in monocyte/macrophage-like cell line RAW264.7 cells. Methods: RAW264.7 cells were transfected with shRNA vector targeting sPLA(2)-V (sPLA(2)-V-knockdown [KD] cells) or empty vector (sPLA(2)-V-wild-type [WT] cells). AcLDL endocytosis was assessed by incubation with(125)I-AcLDL or AcLDL conjugated with pHrodo. Actin polymerization was assessed by flow cytometry using Alexa Fluor 546-phalloidin. Results: In immunofluorescence microscopic studies, sPLA(2)-V was detected in the nucleus. ChIP-Seq and ChIP-qPCR analyses showed binding of sPLA(2)-V to the promoter region of thephosphoglycerate kinase 1 (Pgk1) gene. In the promoter assay, sPLA(2)-V-KD cells had lower promoter activity of thePgk1 gene than sPLA(2)-V-WT cells, and this decrease could be reversed by transfection with a vector encoding sPLA(2)-V-H48Q that lacks enzymatic activity. Compared with sPLA(2)-V-WT cells, sPLA(2)-V-KD cells had decreased PGK1 protein expression, beclin 1 (Beclin1) phosphorylation at S30, and class III PI3-kinase activity that could also be restored by transfection with sPLA(2)-V-H48Q. sPLA(2)-V-KD cells had impaired actin polymerization and endocytosis, which was reversed by introduction of sPLA(2)-V-H48Q or PGK1 overexpression. In sPLA(2)-V-WT cells, siRNA-mediated depletion of PGK1 suppressed Beclin1 phosphorylation and impaired actin polymerization and intracellular trafficking of pHrodo-conjugated AcLDL. Conclusions: Nuclear sPLA(2)-V binds to thePgk1 gene promoter region and increases its transcriptional activity. sPLA(2)-V regulates AcLDL endocytosis through PGK1-Beclin1 in a manner that is independent of its enzymatic activity in RAW264.7 cells.