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Etomoxir Actions on Regulatory and Memory T Cells Are Independent of Cpt1a-Mediated Fatty Acid Oxidation.

ABSTRACT: T cell subsets including effector (Teff), regulatory (Treg), and memory (Tmem) cells are characterized by distinct metabolic profiles that influence their differentiation and function. Previous research suggests that engagement of long-chain fatty acid oxidation (LC-FAO) supports Foxp3+ Treg cell and Tmem cell survival. However, evidence for this is mostly based on inhibition of Cpt1a, the rate-limiting enzyme for LC-FAO, with the drug etomoxir. Using genetic models to target Cpt1a specifically in T cells, we dissected the role of LC-FAO in primary, memory, and regulatory T cell responses. Here we show that the ACC2/Cpt1a axis is largely dispensable for Teff, Tmem, or Treg cell formation, and that the effects of etomoxir on T cell differentiation and function are independent of Cpt1a expression. Together our data argue that metabolic pathways other than LC-FAO fuel Tmem or Treg differentiation and suggest alternative mechanisms for the effects of etomoxir that involve mitochondrial respiration.

SUBMITTER: Raud B

PROVIDER: S-EPMC6747686 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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Etomoxir Actions on Regulatory and Memory T Cells Are Independent of Cpt1a-Mediated Fatty Acid Oxidation.

Raud Brenda B Roy Dominic G DG Divakaruni Ajit S AS Tarasenko Tatyana N TN Franke Raimo R Ma Eric H EH Samborska Bozena B Hsieh Wei Yuan WY Wong Alison H AH Stüve Philipp P Arnold-Schrauf Catharina C Guderian Melanie M Lochner Matthias M Rampertaap Shakuntala S Romito Kimberly K Monsale Joseph J Brönstrup Mark M Bensinger Steven J SJ Murphy Anne N AN McGuire Peter J PJ Jones Russell G RG Sparwasser Tim T Berod Luciana L

Cell metabolism 20180628 3

T cell subsets including effector (Teff), regulatory (Treg), and memory (Tmem) cells are characterized by distinct metabolic profiles that influence their differentiation and function. Previous research suggests that engagement of long-chain fatty acid oxidation (LC-FAO) supports Foxp3+ Treg cell and Tmem cell survival. However, evidence for this is mostly based on inhibition of Cpt1a, the rate-limiting enzyme for LC-FAO, with the ...[more]

PMID: 30043753

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Project description:Nasopharyngeal carcinoma (NPC) has a particularly high prevalence in southern China, southeastern Asia and northern Africa. Radiation resistance remains a serious obstacle to successful treatment in NPC. This study aimed to explore the metabolic feature of radiation-resistant NPC cells and identify new molecular-targeted agents to improve the therapeutic effects of radiotherapy in NPC. Methods: Radiation-responsive and radiation-resistant NPC cells were used as the model system in vitro and in vivo. Metabolomics approach was used to illustrate the global metabolic changes. 13C isotopomer tracing experiment and Seahorse XF analysis were undertaken to determine the activity of fatty acid oxidation (FAO). qRT-PCR was performed to evaluate the expression of essential FAO genes including CPT1A. NPC tumor tissue microarray was used to investigate the prognostic role of CPT1A. Either RNA interference or pharmacological blockade by Etomoxir were used to inhibit CPT1A. Radiation resistance was evaluated by colony formation assay. Mitochondrial membrane potential, apoptosis and neutral lipid content were measured by flow cytometry analysis using JC-1, Annexin V and LipidTOX Red probe respectively. Molecular markers of mitochondrial apoptosis were detected by western blot. Xenografts were treated with Etomoxir, radiation, or a combination of Etomoxir and radiation. Mitochondrial apoptosis and lipid droplets content of tumor tissues were detected by cleaved caspase 9 and Oil Red O staining respectively. Liquid chromatography coupled with tandem mass spectrometry approach was used to identify CPT1A-binding proteins. The interaction of CPT1A and Rab14 were detected by immunoprecipitation, immunofluorescence and in situ proximity ligation analysis. Fragment docking and direct coupling combined computational protein-protein interaction prediction method were used to predict the binding interface. Fatty acid trafficking was measured by pulse-chase assay using BODIPY C16 and MitoTracker Red probe. Results: FAO was active in radiation-resistant NPC cells, and the rate-limiting enzyme of FAO, carnitine palmitoyl transferase 1 A (CPT1A), was consistently up-regulated in these cells. The protein level of CPT1A was significantly associated with poor overall survival of NPC patients following radiotherapy. Inhibition of CPT1A re-sensitized NPC cells to radiation therapy by activating mitochondrial apoptosis both in vitro and in vivo. In addition, we identified Rab14 as a novel CPT1A binding protein. The CPT1A-Rab14 interaction facilitated fatty acid trafficking from lipid droplets to mitochondria, which decreased radiation-induced lipid accumulation and maximized ATP production. Knockdown of Rab14 attenuated CPT1A-mediated fatty acid trafficking and radiation resistance. Conclusion: An active FAO is a vital signature of NPC radiation resistance. Targeting CPT1A could be a beneficial regimen to improve the therapeutic effects of radiotherapy in NPC patients. Importantly, the CPT1A-Rab14 interaction plays roles in CPT1A-mediated radiation resistance by facilitating fatty acid trafficking. This interaction could be an attractive interface for the discovery of novel CPT1A inhibitors.

Dataset Information

Etomoxir Actions on Regulatory and Memory T Cells Are Independent of Cpt1a-Mediated Fatty Acid Oxidation.

Publications

Etomoxir Actions on Regulatory and Memory T Cells Are Independent of Cpt1a-Mediated Fatty Acid Oxidation.

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