- 著者
- Daisuke Fujioka Yosuke Watanabe Takamitsu Nakamura Takashi Yokoyama Keiji Miyazawa Makoto Murakami Kiyotaka Kugiyama
- 出版者
- Japan Atherosclerosis Society
- 雑誌
- Journal of Atherosclerosis and Thrombosis (ISSN:13403478)
- 巻号頁・発行日
- vol.29, no.5, pp.692-718, 2022-05-01 (Released:2022-05-01)
- 参考文献数
- 44
- 被引用文献数
- 1 2
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.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.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.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.
- 著者
- Yosuke Watanabe Richard A. Cohen Reiko Matsui
- 出版者
- 日本循環器学会
- 雑誌
- Circulation Journal (ISSN:13469843)
- 巻号頁・発行日
- pp.CJ-16-0317, (Released:2016-05-06)
- 参考文献数
- 64
- 被引用文献数
- 23
Antioxidants are expected to improve cardiovascular disease (CVD) by eliminating oxidative stress, but clinical trials have not shown promising results in chronic CVD. Animal studies have revealed that reactive oxygen species (ROS) exacerbate acute CVDs in which high levels of ROS are observed. However, ROS are also necessary for angiogenesis after ischemia, because ROS not only damage cells but also stimulate the cell signaling required for angiogenesis. ROS affect signaling by protein modifications, especially of cysteine amino acid thiols. Although there are several cysteine modifications, S-glutathionylation (GSH adducts; -SSG), a reversible cysteine modification by glutathione (GSH), plays an important role in angiogenic signal transduction by ROS. Glutaredoxin-1 (Glrx) is an enzyme that specifically removes GSH adducts in vivo. Overexpression of Glrx inhibits, whereas deletion of Glrx improves revascularization after mouse hindlimb ischemia. These studies indicate that increased levels of GSH adducts in ischemic muscle are beneficial in promoting angiogenesis. The underlying mechanism can be explained by multiple targets of S-gluathionylation, which mediate the angiogenic effects in ischemia. Increments in the master angiogenic transcriptional factor, HIF-1α, reduction of the anti-angiogenic factor sFlt1, activation of the endoplasmic reticulum Ca2+pump, SERCA, and inhibition of phosphatases may occur as a consequence of enhanced S-glutathionylation in ischemic tissue. In summary, inducing S-glutathionylation by inhibiting Glrx may be a therapeutic strategy to improve ischemic angiogenesis in CVD.