Dataset Information

Lysosomal Cholesterol Hydrolysis Couples Efferocytosis to Anti-Inflammatory Oxysterol Production.

ABSTRACT: RATIONALE:Macrophages face a substantial amount of cholesterol after the ingestion of apoptotic cells, and the LIPA (lysosomal acid lipase) has a major role in hydrolyzing cholesteryl esters in the endocytic compartment. OBJECTIVE:Here, we directly investigated the role of LIPA-mediated clearance of apoptotic cells both in vitro and in vivo. METHODS AND RESULTS:We show that LIPA inhibition causes a defective efferocytic response because of impaired generation of 25-hydroxycholesterol and 27-hydroxycholesterol. Reduced synthesis of 25-hydroxycholesterol after LIPA inhibition contributed to defective mitochondria-associated membrane leading to mitochondrial oxidative stress-induced NLRP3 (NOD-like receptor family, pyrin domain containing) inflammasome activation and caspase-1-dependent Rac1 (Ras-related C3 botulinum toxin substrate 1) degradation. A secondary event consisting of failure to appropriately activate liver X receptor-mediated pathways led to mitigation of cholesterol efflux and apoptotic cell clearance. In mice, LIPA inhibition caused defective clearance of apoptotic lymphocytes and stressed erythrocytes by hepatic and splenic macrophages, culminating in splenomegaly and splenic iron accumulation under hypercholesterolemia. CONCLUSIONS:Our findings position lysosomal cholesterol hydrolysis as a critical process that prevents metabolic inflammation by enabling efficient macrophage apoptotic cell clearance.

SUBMITTER: Viaud M

PROVIDER: S-EPMC6034181 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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Lysosomal Cholesterol Hydrolysis Couples Efferocytosis to Anti-Inflammatory Oxysterol Production.

Viaud Manon M Ivanov Stoyan S Vujic Nemanja N Duta-Mare Madalina M Aira Lazaro-Emilio LE Barouillet Thibault T Garcia Elsa E Orange Francois F Dugail Isabelle I Hainault Isabelle I Stehlik Christian C Marchetti Sandrine S Boyer Laurent L Guinamard Rodolphe R Foufelle Fabienne F Bochem Andrea A Hovingh Kees G KG Thorp Edward B EB Gautier Emmanuel L EL Kratky Dagmar D Dasilva-Jardine Paul P Yvan-Charvet Laurent L

Circulation research 20180309 10

<h4>Rationale</h4>Macrophages face a substantial amount of cholesterol after the ingestion of apoptotic cells, and the LIPA (lysosomal acid lipase) has a major role in hydrolyzing cholesteryl esters in the endocytic compartment.<h4>Objective</h4>Here, we directly investigated the role of LIPA-mediated clearance of apoptotic cells both in vitro and in vivo.<h4>Methods and results</h4>We show that LIPA inhibition causes a defective efferocytic response because of impaired generation of 25-hydroxyc ...[more]

PMID: 29523554

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Project description:AimThe effects of 24(S),25-epoxycholesterol (24,25EC) on aspects of cholesterol homeostasis is well-documented. When added to cells, 24,25EC decreases cholesterol synthesis and up-regulates cholesterol efflux genes, including ABCA1. Synthesis of 24,25EC occurs in a shunt of the mevalonate pathway which also produces cholesterol. Therefore, 24,25EC synthesis should be subject to the same negative feedback regulation as cholesterol synthesis. To date, no role has been ascribed to 24,25EC in light of the fact that increased accumulation of cholesterol should decrease formation of this oxysterol through feedback inhibition. This leads to the intriguing paradox: why inhibit production of an apparently important regulator of cholesterol homeostasis when it is needed most?MethodsWe used a combination of pharmacological and genetic approaches in Chinese Hamster Ovary cell-lines to investigate this paradox. Endogenous synthesis of 24,25EC was manipulated using partial inhibition of the enzyme, Oxidosqualene Cyclase. Changes in cholesterol and 24,25EC synthesis were determined using metabolic labelling with [1-14C]-acetate, thin-layer chromatography and phosphorimaging. Transcriptional effects mediated via SREBP and LXR were analysed by luciferase reporter assays.ResultsWe showed that cholesterol addition to cells lead to a rapid and preferential inhibition of 24,25EC synthesis. Addition of 24,25EC resulted in parallel inhibition of 24,25EC and cholesterol synthesis. Furthermore, we used a variety of approaches to examine the relationship between cholesterol and 24,25EC synthesis, including cell-lines with different rates of cholesterol synthesis, varying cholesterol synthetic rates by pre-treatment with a statin, or lipoprotein cholesterol loading of macrophages. In all cases, we showed that 24,25EC synthesis faithfully tracked cholesterol synthesis. Moreover, changes in 24,25EC synthesis exerted downstream effects, reducing SREBP transcriptional activity whilst increasing ABCA1 and LXR transcriptional activity.ConclusionOur results show that 24,25EC synthesis parallels cholesterol synthesis, consistent with this oxysterol functioning as a safety valve to protect against the accumulation of newly-synthesised cholesterol (as opposed to exogenously-derived cholesterol). Considering that 24,25EC is capable of being produced in all cholesterogenic cells, we propose that production of 24,25EC may represent a ubiquitous defence mechanism.

Project description:Mitochondria are key organelles for cellular metabolism, and regulate several processes including cell death and macroautophagy/autophagy. Here, we show that mitochondrial respiratory chain (RC) deficiency deactivates AMP-activated protein kinase (AMPK, a key regulator of energy homeostasis) signaling in tissue and in cultured cells. The deactivation of AMPK in RC-deficiency is due to increased expression of the AMPK-inhibiting protein FLCN (folliculin). AMPK is found to be necessary for basal lysosomal function, and AMPK deactivation in RC-deficiency inhibits lysosomal function by decreasing the activity of the lysosomal Ca2+ channel MCOLN1 (mucolipin 1). MCOLN1 is regulated by phosphoinositide kinase PIKFYVE and its product PtdIns(3,5)P2, which is also decreased in RC-deficiency. Notably, reactivation of AMPK, in a PIKFYVE-dependent manner, or of MCOLN1 in RC-deficient cells, restores lysosomal hydrolytic capacity. Building on these data and the literature, we propose that downregulation of the AMPK-PIKFYVE-PtdIns(3,5)P2-MCOLN1 pathway causes lysosomal Ca2+ accumulation and impaired lysosomal catabolism. Besides unveiling a novel role of AMPK in lysosomal function, this study points to the mechanism that links mitochondrial malfunction to impaired lysosomal catabolism, underscoring the importance of AMPK and the complexity of organelle cross-talk in the regulation of cellular homeostasis. Abbreviation: ΔΨm: mitochondrial transmembrane potential; AMP: adenosine monophosphate; AMPK: AMP-activated protein kinase; ATG5: autophagy related 5; ATP: adenosine triphosphate; ATP6V0A1: ATPase, H+ transporting, lysosomal, V0 subbunit A1; ATP6V1A: ATPase, H+ transporting, lysosomal, V0 subbunit A; BSA: bovine serum albumin; CCCP: carbonyl cyanide-m-chlorophenylhydrazone; CREB1: cAMP response element binding protein 1; CTSD: cathepsin D; CTSF: cathepsin F; DMEM: Dulbecco's modified Eagle's medium; DMSO: dimethyl sulfoxide; EBSS: Earl's balanced salt solution; ER: endoplasmic reticulum; FBS: fetal bovine serum; FCCP: carbonyl cyanide-p-trifluoromethoxyphenolhydrazone; GFP: green fluorescent protein; GPN: glycyl-L-phenylalanine 2-naphthylamide; LAMP1: lysosomal associated membrane protein 1; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MCOLN1/TRPML1: mucolipin 1; MEF: mouse embryonic fibroblast; MITF: melanocyte inducing transcription factor; ML1N*2-GFP: probe used to detect PtdIns(3,5)P2 based on the transmembrane domain of MCOLN1; MTORC1: mechanistic target of rapamycin kinase complex 1; NDUFS4: NADH:ubiquinone oxidoreductase subunit S4; OCR: oxygen consumption rate; PBS: phosphate-buffered saline; pcDNA: plasmid cytomegalovirus promoter DNA; PCR: polymerase chain reaction; PtdIns3P: phosphatidylinositol-3-phosphate; PtdIns(3,5)P2: phosphatidylinositol-3,5-bisphosphate; PIKFYVE: phosphoinositide kinase, FYVE-type zinc finger containing; P/S: penicillin-streptomycin; PVDF: polyvinylidene fluoride; qPCR: quantitative real time polymerase chain reaction; RFP: red fluorescent protein; RNA: ribonucleic acid; SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis; shRNA: short hairpin RNA; siRNA: small interfering RNA; TFEB: transcription factor EB; TFE3: transcription factor binding to IGHM enhancer 3; TMRM: tetramethylrhodamine, methyl ester, perchlorate; ULK1: unc-51 like autophagy activating kinase 1; ULK2: unc-51 like autophagy activating kinase 2; UQCRC1: ubiquinol-cytochrome c reductase core protein 1; v-ATPase: vacuolar-type H+-translocating ATPase; WT: wild-type.

Dataset Information

Lysosomal Cholesterol Hydrolysis Couples Efferocytosis to Anti-Inflammatory Oxysterol Production.

Publications

Lysosomal Cholesterol Hydrolysis Couples Efferocytosis to Anti-Inflammatory Oxysterol Production.

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