Group V Secretory Phospholipase A<sub>2</sub> Regulates Endocytosis of Acetylated LDL by Transcriptional Activation of PGK1 in RAW264.7 Macrophage Cell Line
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- Fujioka Daisuke
- Department of Internal Medicine II, University of Yamanashi, Faculty of Medicine
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- Watanabe Yosuke
- Department of Internal Medicine II, University of Yamanashi, Faculty of Medicine
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- Nakamura Takamitsu
- Department of Internal Medicine II, University of Yamanashi, Faculty of Medicine
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- Yokoyama Takashi
- Department of Biochemistry, University of Yamanashi, Faculty of Medicine
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- Miyazawa Keiji
- Department of Biochemistry, University of Yamanashi, Faculty of Medicine
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- Murakami Makoto
- Laboratory of Microenvironmental and Metabolic Health Science, Center for Disease Biology and Integrative Medicine, Graduate School of Medicine, University of Tokyo AMED-CREST, Japan Agency for Medical Research and Development
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- Kugiyama Kiyotaka
- Department of Internal Medicine II, University of Yamanashi, Faculty of Medicine AMED-CREST, Japan Agency for Medical Research and Development
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<p> Aims: It was suggested that group V secretory phospholipase A2 (sPLA2-V) existed in the nucleus. This study examined whether nuclear sPLA2-V plays a role in endocytosis of acetylated low-density lipoprotein (AcLDL) in monocyte/macrophage-like cell line RAW264.7 cells.</p><p>Methods: RAW264.7 cells were transfected with shRNA vector targeting sPLA2-V (sPLA2-V-knockdown [KD] cells) or empty vector (sPLA2-V-wild-type [WT] cells). AcLDL endocytosis was assessed by incubation with 125I-AcLDL or AcLDL conjugated with pHrodo. Actin polymerization was assessed by flow cytometry using Alexa Fluor 546-phalloidin.</p><p>Results: In immunofluorescence microscopic studies, sPLA2-V was detected in the nucleus. ChIP-Seq and ChIP-qPCR analyses showed binding of sPLA2-V to the promoter region of the phosphoglycerate kinase 1 (Pgk1) gene. In the promoter assay, sPLA2-V-KD cells had lower promoter activity of the Pgk1 gene than sPLA2-V-WT cells, and this decrease could be reversed by transfection with a vector encoding sPLA2-V-H48Q that lacks enzymatic activity. Compared with sPLA2-V-WT cells, sPLA2-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 sPLA2-V-H48Q. sPLA2-V-KD cells had impaired actin polymerization and endocytosis, which was reversed by introduction of sPLA2-V-H48Q or PGK1 overexpression. In sPLA2-V-WT cells, siRNA-mediated depletion of PGK1 suppressed Beclin1 phosphorylation and impaired actin polymerization and intracellular trafficking of pHrodo-conjugated AcLDL.</p><p>Conclusions: Nuclear sPLA2-V binds to the Pgk1 gene promoter region and increases its transcriptional activity. sPLA2-V regulates AcLDL endocytosis through PGK1-Beclin1 in a manner that is independent of its enzymatic activity in RAW264.7 cells.</p>
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- Ｊｏｕｒｎａｌ ｏｆ Ａｔｈｅｒｏｓｃｌｅｒｏｓｉｓ ａｎｄ Ｔｈｒｏｍｂｏｓｉｓ
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Ｊｏｕｒｎａｌ ｏｆ Ａｔｈｅｒｏｓｃｌｅｒｏｓｉｓ ａｎｄ Ｔｈｒｏｍｂｏｓｉｓ 29 (5), 692-718, 2022-05-01
一般社団法人 日本動脈硬化学会