Title

 

Authors

C. Bolego¹, T. Nishizawa^2,3, K. Bornfeldt².

¹Dept. of Pharmacology and Anesthesiology, School of Pharmacy, University of Padova, Italy; ²Dept. of Pathology, Diabetes and Obesity Center of Excellence, School of Medicine, University of Washington, Seattle, USA; ³Biological Research Laboratories, R&D Division, Daiichi-Sankyo, Tokyo, Japan. 

 

Abstract

Acyl CoA thioesterases (ACOTs) catalyze the hydrolysis of fatty acyl-CoA to free fatty acid and CoA, and in concert with acyl CoA synthases (ACS), which produce acyl-CoA from free fatty acid and CoA, regulate lipid metabolism and cellular signaling in different tissues.A number of different ACOTs (1-7) have been described until now, which display different tissue distribution, intracellular localization and/or fatty acid preferences. ACOT7 is an arachidonic acid preferring ACOT isoform. We therefore hypothesized that ACOT7 may contribute to phospholipid remodeling and arachidonic acid (AA) release, thus affecting the inflammatory capacity of macrophages. We used bone marrow (BM) cells that were differentiated into macrophages with MCSF for 7 days and polarized into M1 and M2 macrophages using LPS+IFNγ and IL-4, respectively. Gene expression data obtained with quantitative PCR showed higher expression of proinflammatory markers (iNOS, COX-2, TNFα, IL-1β) in M1 macrophages already after 4 hours polarization. Relative gene expression of the different ACOTs (1-7) indicated that ACOT7, differently from other isoforms, was expressed to a greater extent in M1- with respect to M2-polarized cells. Treatment of BM cells differentiated into macrophages with M1 or M2 stimuli for different times (0-48 hours) led to ACOT7 protein accumulation over time peaking in M1 cells after 24- and 48-hour stimulation. In order to evaluate specific activity, ACOT7 was immunoprecipitated with a specific antibody and enzyme activity was measured on immunoprecipitates from M1 or M2 polarized cells. ACOT7 thioesterase activity was significantly higher in M1 with respect to M2 stimulated macrophages after 24 hours stimulation. To further evaluate the functional role of ACOT7, we obtained a stable ACOT7-overexpressing line transfecting J774 macrophages by retroviral infection. For this purpose, Phoenix cells were transfected with ACOT7 cDNA and selected with puromycin. Therefore, ACOT7 is likely to play an important regulatory role in M1-polarized rodent macrophages. Ongoing experiments for the analysis of prostanoid production (e.g. PGs, resolvins) in ACOT7-overexpressing J774 as well as in BM macrophages will help clarify the role of this protein as a potential target for regulating inflammatory phenotype in macrophages.