Cloning, Identification and Functional Characterization of Bovine Free Fatty Acid Receptor-1 (FFAR1/GPR40) in Neutrophils

March Cloning, Identification and Functional Characterization of Bovine Free Fatty Acid Receptor-1 (FFAR1/GPR40) in Neutrophils Carolina Manosalva 0 1 2 Jaqueline Mena 0 1 2 Zahady Velasquez 0 1 2 Charlotte K. Colenso 0 1 2 Sebastian Brauchi 0 1 2 Rafael A. Burgos 0 1 2 Maria A. Hidalgo 0 1 2 0 Current address: Institute of Histology, Pathology and Anatomy, Faculty of Medicine, Universidad Austral de Chile , Valdivia , Chile 1 1 Laboratory of Molecular Pharmacology, Institute of Pharmacology, Faculty of Veterinary Science, Universidad Austral de Chile , Valdivia , Chile , 2 Department of Biology, Universidad de Narino, Pasto, Colombia, 3 Institute of Physiology, Faculty of Medicine, Universidad Austral de Chile , Valdivia , Chile 2 Academic Editor: Thierry Alquier, CRCHUM- Montreal Diabetes Research Center , CANADA Long chain fatty acids (LCFAs), which are ligands for the G-protein coupled receptor FFAR1 (GPR40), are increased in cow plasma after parturition, a period in which they are highly susceptible to infectious diseases. This study identified and analyzed the functional role of the FFAR1 receptor in bovine neutrophils, the first line of host defense against infectious agents. We cloned the putative FFAR1 receptor from bovine neutrophils and analyzed the sequence to construct a homology model. Our results revealed that the sequence of bovine FFAR1 shares 84% identity with human FFAR1 and 31% with human FFAR3/GPR41. Therefore, we constructed a homology model of bovine FFAR1 using human as the template. Expression of the bovine FFAR1 receptor in Chinese hamster ovary (CHO)-K1 cells increased the levels of intracellular calcium induced by the LCFAs, oleic acid (OA) and linoleic acid (LA); no increase in calcium mobilization was observed in the presence of the short chain fatty acid propionic acid. Additionally, the synthetic agonist GW9508 increased intracellular calcium in CHO-K1/bFFAR1 cells. OA and LA increased intracellular calcium in bovine neutrophils. Furthermore, GW1100 (antagonist of FFAR1) and U73122 (phospholipase C (PLC) inhibitor) reduced FFAR1 ligand-induced intracellular calcium in CHO-K1/ bFFAR1 cells and neutrophils. Additionally, inhibition of FFAR1, PLC and PKC reduced the FFAR1 ligand-induced release of matrix metalloproteinase (MMP)-9 granules and reactive oxygen species (ROS) production. Thus, we identified the bovine FFAR1 receptor and demonstrate a functional role for this receptor in neutrophils activated with oleic or linoleic acid. - Funding: This study was supported by the following grants: Grant Fondo Nacional de Desarrollo Cientfico y Tecnolgico (FONDECYT) No. 11100413 and Direccin de Investigacin y Desarrollo (DID)-UACH S-2014-23. CM is a Ph.D. student supported by scholarship Comisin Nacional de Investigacin Cientfica y Tecnolgica (CONICYT) No. 21100559 and 24121495, and Mecesup Program AUS1203. JM is a Ph.D. student supported by scholarship Mecesup No. AUS0704 and DID-UACH D-201302. CKC is supported by Grant FONDECYT No. 3140233. MAH and SB Laboratories are part of CISNe-UACh, and SB Laboratory is part of the UACh Program in Cellular Dynamics and Microscopy. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. oxidative and non-oxidative mechanisms; oxidative mechanisms are associated with ROS production, such as superoxide, via activating the NADPH oxidase complex [1], whereas the nonoxidative mechanisms include the release of granules that contain proteolytic proteins, such as MMP-9 [2]. Granule release is triggered by several stimuli that, upon coupling to exposed extracellular receptors, produce signals across the plasma membrane, which increase the intracellular generation of second messengers. The process involved in the secretion of MMP-9 granules is only partially understood, but several authors have demonstrated that the increase in intracellular calcium is an important signal [3,4]. Fatty acids regulate immune and inflammatory responses in humans [5,6]. In the bovine, free fatty acids are significantly increased in the plasma between 1 to 2 weeks postpartum [7], the same period during which cows are more susceptible to acquire infectious diseases that the innate immune system actively opposes. Linoleic acid (C18:2), a polyunsaturated long chain fatty acid (LCFA), is the main fatty acid in plasma and its percentage significantly increases two weeks postpartum compared with values before parturition. Similarly, the unsaturated LCFA oleic acid (C18:1), also shows an increase until two weeks postpartum [7]. In vitro, fatty acids affect ROS production in neutrophils. Oleic acid, linoleic acid and -linolenic acid, markedly increase intracellular and extracellular ROS levels in rat and human neutrophils [8]. In bovine neutrophils, oleic acid increases intracellular superoxide levels in an intracellular calcium-dependent manner [4]. Oleic and linoleic acids also rapidly increase MMP-9 activity in bovine neutrophils in an extracellular calcium-dependent manner [4,9]. Similarly, oleic acid promotes MMP-9 secretion in breast cancer cells through PKC, Src and EGFR-dependent pathways [10]. Despite the fact that oleic and linoleic acids induce MMP-9 release in breast cancer cells and bovine neutrophils, the mechanisms underlying this response are not well understood. Recent studies demonstrated that free fatty acids are ligands for seven-transmembrane domain receptors. Free Fatty Acid Receptor 1 (FFAR1), also known as G Protein-coupled Receptor 40 (GPR40), is a receptor for medium and long chain fatty acids, such as docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), oleic acid and linoleic acid [9,11,12,13]. FFAR1 mediates insulin secretion from pancreatic -cells; however, more recent evidence suggests that FFAR1 plays a role in cellular proliferation and innate immunity because it is present in human and bovine mammary epithelial cells [14,15] and bovine neutrophils [4]. In pancreatic -cells and breast cancer cells, FFAR1 is coupled to an intracellular Gq protein that activates the PLC and phosphatidylinositol-4,5-bisphosphate signaling pathways [16,17]. FFAR1 is also coupled to Gi protein in bovine neutrophils because pertussis toxin only partially reduces oleic acid-induced intracellular calcium mobilization [4]. Several effects of oleic and linoleic acid have been described in bovine neutrophils [4,9], and treatment with the synthetic FFAR1 antagonist, GW1100, suggested that FFAR1 plays a role in neutrophil activation [9]. Recently, several discrepancies regarding the identity of the FFAR1 cDNA sequence in bovine have arose. The first antecedents of FFAR1 showed a cDNA sequence obtained from bovine mammary epithelial cells with 84% identity to human FFAR1 [15]. In bovine neutrophils, we observed a product of FFAR1 amplification that was similar to that observed in (...truncated)