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

• Published in: Journal of atherosclerosis and thrombosis 2022-05, Vol.29 (5), p.692
• Main Authors: Fujioka, Daisuke, Watanabe, Yosuke, Nakamura, Takamitsu, Yokoyama, Takashi, Miyazawa, Keiji, Murakami, Makoto, Kugiyama, Kiyotaka
• Format: Article
• Language: English
• Subjects: Actins - genetics; Beclin-1 - metabolism; Cell Line; Endocytosis; Humans; Lipoproteins, LDL - metabolism; Macrophages - metabolism; Phosphoglycerate Kinase - metabolism; Phospholipases A2, Secretory - metabolism; Transcriptional Activation
• ISSN: 1880-3873
• DOI: 10.5551/jat.62216

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