12/15-Lipoxygenase Translocation Enhances Site-specific Actin Polymerization in Macrophages Phagocytosing Apoptotic Cells

Abstract

The enzyme 12/15-lipoxygenase (12/15-LO) introduces peroxyl groups in a position-specific manner into unsaturated fatty acids in certain cells, but the role of such enzymatic lipid peroxidation remains poorly defined. Here we report a novel function for 12/15-LO in mouse peritoneal macrophages. When macrophages were coincubated with apoptotic cells, the enzyme translocated from cytosol to the plasma membrane and was more extensively concentrated at sites where macrophages bound apoptotic cells, colocalizing with polymerized actin of emerging filopodia. Disruption of F-actin did not prevent the 12/15-LO translocation. In contrast, inhibition of the 12/15-LO activity, or utilization of genetically engineered macrophages in which the 12/15-LO gene has been disrupted, greatly reduced actin polymerization in phagocytosing macrophages. Lysates of 12/15-LO-deficient macrophages had significantly lower ability to promote in vitro actin polymerization than the lysates of wild type macrophages. These studies suggest that the 12/15-LO enzyme plays a major role in local control of actin polymerization in macrophages in response to interaction with apoptotic cells. 12/15-Lipoxygenase (LO)1 is a member of the LO family of enzymes that insert peroxyl groups into double bonds of free and phospholipid-bound polyunsaturated fatty acids. The exact role of these enzymes in biological processes has remained elusive, but increasingly evidence has accumulated that they play important roles in specific cellular functions. For example, 15-LO activity in reticulocytes at the stage of organelle degradation may contribute to membrane destabilization and contribute to pore formation in intracellular membranes (1, 2). Fatty acid products of 12/15-LO are powerful agonists for the nuclear receptor PPAR-␥, which helps regulate glucose metabolism and adipocyte and macrophage differentiation and function (3). A remarkable feature of 12/15-LO is that its expression is not constant during the cell life span but rather turns on at certain points during cell development. While circulating human monocytes do not express 15-LO, monocyte-derived macrophages exposed to interleukin-4 or interleukin-13 express 15-LO (4, 5). In addition, mouse macrophages residing for a long time in the peritoneum (resident macrophages) also highly express the mouse homologue, 12/15-LO, although the pathway leading to 12/15-LO expression may differ somewhat from that which occurs with human monocytes (6).Macrophages of atherosclerotic lesions express high levels of 15-LO (7), and recent evidence utilizing apoE Ϫ/Ϫ mice in which the 12/15-LO gene was disrupted demonstrated its importance in the pathogenesis of atherosclerosis (8). Another characteristic of atherosclerotic tissue but not of normal vascular wall is the high abundance of apoptotic cells (9). This fact might reflect either an increased rate of formation of such cells, a decreased rate of clearance, for example by arterial macrophages, or both. In either case, phagocytosis and the degradation and metabolism of th...

Eicosanoids participate in the regulation of cardiac glucose transport by contribution to a rearrangement of actin cytoskeletal elements

“…It may be speculated that, under these conditions, the enhanced production of 12(S)-HETE may operate to retain proper actin network organization in the cardiac cell, providing the basis for ongoing GLUT4 trafficking. This view is supported by recent data from Funk, Witztum and co-workers [35], who demonstrated that specific inhibition of the 12\15-LO isoform, or disruption of the corresponding gene, led to a strong reduction in actin polymerization. Thus it is suggested that this enzyme plays an important role in the local control of actin polymerization.…”

Wnt5a Signaling Promotes Host Defense againstLeishmania donovaniInfection

“…1). Cleavage and destruction of host proteins by GP63, a parasite-specific protease, have been reported (28). In light of the role of Wnt5a signaling in the modulation of cytoskeletal dynamics and immune homeostasis (15,16) (Fig.…”

“…F-actin of macrophages was stained with Alexa Fluor 555-labeled phalloidin (28). Briefly, isolated PMFs or RAW 264.7 cells were plated on a six-well plate or confocal dish and incubated overnight under standard tissue culture conditions.…”

Low Density Lipoprotein Receptor-related Protein-mediated Membrane Translocation of 12/15-Lipoxygenase Is Required for Oxidation of Low Density Lipoprotein by Macrophages

“…However, the 12/ 15-lipoxygenase is predominantly present in cytoplasm and not in the membranes in various cells (15). Recent study has shown that the translocation of 12/15-lipoxygenase from the cytosol to the plasma membrane was observed in macrophages when incubated with apoptotic cells (16). Here we demonstrate that the binding of LDL to LRP expressed in normal macrophages and 12/15-lipoxygenase-expressing macrophage-like J774A.1 cells translocates the enzyme from cytoplasm to the plasma membrane.…”