Dataset Information

Group V Secretory Phospholipase A₂ Regulates Endocytosis of Acetylated LDL by Transcriptional Activation of PGK1 in RAW264.7 Macrophage Cell Line.

ABSTRACT:

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.

SUBMITTER: Fujioka D

PROVIDER: S-EPMC9135649 | biostudies-literature | 2022 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

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

Fujioka Daisuke D Watanabe Yosuke Y Nakamura Takamitsu T Yokoyama Takashi T Miyazawa Keiji K Murakami Makoto M Kugiyama Kiyotaka K

Journal of atherosclerosis and thrombosis 20210327 5

<h4>Aims</h4>It was suggested that group V secretory phospholipase A<sub>2</sub> (sPLA<sub>2</sub>-V) existed in the nucleus. This study examined whether nuclear sPLA<sub>2</sub>-V plays a role in endocytosis of acetylated low-density lipoprotein (AcLDL) in monocyte/macrophage-like cell line RAW264.7 cells.<h4>Methods</h4>RAW264.7 cells were transfected with shRNA vector targeting sPLA<sub>2</sub>-V (sPLA<sub>2</sub>-V-knockdown [KD] cells) or empty vector (sPLA<sub>2</sub>-V-wild-type [WT] cell ...[more]

PMID: 33775979

Similar Datasets

Project description:ObjectiveHighly elevated plasma levels of interleukin-10 (IL-10) are causally associated with "Disappearing HDL Syndrome" and low plasma LDL-cholesterol, but the underlying mechanism is poorly understood. Fluid-phase endocytosis, a process highly dependent on actin dynamics, enables cells to internalize relatively high amounts of extracellular fluids and solutes. We sought to investigate whether IL-10 induces lipoprotein uptake by fluid-phase endocytosis in macrophages.Methods and resultsMacrophages (RAW264.7, Kupffer and human) were incubated with vehicle (PBS) or IL-10 (20 ng/ml) for 7 days. Uptake of HDL, LDL, and/or fluid-phase endocytosis probes (albumin-Alexa680®, 70 kDa FITC-Dextran and Lucifer Yellow, LY) was evaluated by FACS. Intracellular cofilin and phosphorylated cofilin (p-cofilin) levels were determined by immunoblotting. Macrophage uptake of lipoproteins and probes was non-saturable and increased after IL-10 incubation (p < 0.0001). Furthermore, pre-incubation with fluid-phase endocytosis inhibitors (LY294002, Latrunculin A, and Amiloride) significantly reduced uptake (p < 0.05). IL-10 increased the cofilin/p-cofilin ratio (p = 0.021), signifying increased cofilin activation and hence filamentous actin. Consistently, phalloidin staining revealed increased filamentous actin in macrophages after IL-10 treatment (p = 0.0018). Finally, RNA-seq analysis demonstrated enrichment of gene sets related to actin filament dynamics, membrane ruffle formation and endocytosis in IL-10-treated macrophages (p < 0.05). IL-10 did not alter mRNA levels of Ldlr, Vldlr, Scarb1, Cd36 or Lrp1. In primary human monocyte-derived macrophages and murine Kupffer cells, IL-10 incubation also increased uptake of lipoproteins, albumin and LY (p < 0.01).ConclusionsInterleukin-10 induces the uptake of HDL and LDL by fluid-phase endocytosis by increasing actin-filament rearrangement in macrophages, thus providing a plausible mechanism contributing to "Disappearing HDL Syndrome".

Project description:Objective- Inflammation is a causal risk factor for cardiovascular disease (CVD). sPLA2-IIA (group IIA secretory phospholipase A2) plays an integral role in regulating vascular inflammation. Although studies investigated sPLA2-IIA in secondary prevention, we prospectively evaluated sPLA2-IIA mass and genetic variants with CVD events in a primary prevention population with chronic inflammation. Approach and Results- The JUPITER trial (Justification for the Use of Statins in Prevention: An Intervention Trial Evaluating Rosuvastatin) randomized participants with LDL (low-density lipoprotein) <130 mg/dL and hsCRP (high-sensitivity C-reactive protein) ≥2 mg/L to high-intensity rosuvastatin versus placebo. Baseline and 1-year plasma sPLA2-IIA mass was measured (N=11 269 baseline; N=9620 1 year). We also identified genetic variants influencing sPLA2-IIA using genome-wide association and examined them with CVD. Three hundred thirteen incident CVD events occurred during follow-up. Baseline sPLA2-IIA mass (median, 25th-75th percentile: 3.81, 2.49-6.03 ng/mL) was associated with increased risk of CVD: risk factor-adjusted hazard ratio (95% CI; P) per SD increment: 1.22 (1.08-1.38; P=0.002). This remained significant (1.18; 1.04-1.35; P=0.01) after incrementally adjusting for hsCRP. Similar estimates were observed in rosuvastatin and placebo groups ( P treatment interaction>0.05). The rs11573156C variant in PLA2G2A (encoding sPLA2-IIA) had the strongest effect on sPLA2-II: median (25th-75th percentile, ng/mL) for CC and GG genotypes: 2.79 (1.97-4.01) and 7.38 (5.38-10.19), respectively; and had nonsignificant trend for higher CVD risk (hazard ratio, 1.11; 95% CI, 0.89-1.38; P=0.34). Conclusions- In the JUPITER population recruited on chronic inflammation, sPLA2-IIA mass was associated with CVD risk relating to vascular inflammation not fully reflected by hsCRP. Additional studies, including larger functional genetic and clinical studies, are needed to determine whether sPLA2-IIA may be a potential pharmacological target for primary prevention of CVD. Clinical Trial Registration- URL: http://www.clinicaltrials.gov . Unique identifier: NCT00239681.

Dataset Information

Group V Secretory Phospholipase A₂ Regulates Endocytosis of Acetylated LDL by Transcriptional Activation of PGK1 in RAW264.7 Macrophage Cell Line.

Aims

Methods

Results

Conclusions

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Dataset Information

Group V Secretory Phospholipase A2 Regulates Endocytosis of Acetylated LDL by Transcriptional Activation of PGK1 in RAW264.7 Macrophage Cell Line.

Aims

Methods

Results

Conclusions

Publications

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets

Group V Secretory Phospholipase A₂ Regulates Endocytosis of Acetylated LDL by Transcriptional Activation of PGK1 in RAW264.7 Macrophage Cell Line.