Group V Secretory Phospholipase A 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 (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.

METHODS

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.

RESULTS

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.

CONCLUSIONS

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.