The Mechanism of Docosahexaenoic Acid-induced Phospholipase D Activation in Human Lymphocytes Involves Exclusion of the Enzyme from Lipid Rafts

Abstract

Docosahexaenoic acid (DHA), an n-3 polyunsaturated fatty acid that inhibits T lymphocyte activation, has been shown to stimulate phospholipase D (PLD) activity in stimulated human peripheral blood mononuclear cells (PBMC). To elucidate the mechanisms underlying the DHA-induced PLD activation, we first characterized the PLD expression pattern of PBMC. We show that these cells express PLD1 and PLD2 at the protein and mRNA level and are devoid of oleate-dependent PLD activity. DHA enrichment of PBMC increased the DHA content of cell phospholipids, which was directly correlated with the extent of PLD activation. The DHA-induced PLD activation was independent of conventional protein kinase C but inhibited by brefeldin A, which suggests ADP-ribosylation factor (ARF)-dependent mechanism. Furthermore, DHA enrichment dosedependently stimulated ARF translocation to cell membranes. Whereas 50% of the guanosine 5-3-O-(thio)triphosphate plus ARF-dependent PLD activity and a substantial part of PLD1 protein were located to the detergent-insoluble membranes, so-called rafts, of nonenriched PBMC, DHA treatment strongly displaced them toward detergent-soluble membranes where ARF is present. Collectively, these results suggest that the exclusion of PLD1 from lipid rafts, due to their partial disorganization by DHA, and its relocalization in the vicinity of ARF, is responsible for its activation. This PLD activation might be responsible for the immunosuppressive effect of DHA because it is known to transmit antiproliferative signals in lymphoid cells.

Disruption of Lipid Rafts Stimulates Phospholipase D Activity in Human Lymphocytes: Implication in the Regulation of Immune Function

“…These lipid domains characterized by their insolubility in cold nonionic detergents are also present in cells devoid of caveolae such as lymphoid cells (4). We shown have recently that a substantial part of PLD1 activity and protein was located to the detergent-insoluble membranes of human PBMCs (5). Furthermore, we have also shown that the partial disorganization of PBMC rafts by enrichment of the cells with the polyunsaturated fatty acid docosahexaenoic acid (DHA) induces a shift of PLD1 out of the rafts and a concomitant activation of the enzyme.…”

“…However, this early peak could be attributed to PA derived from DAG through the sequential activation of phospholipase C-␥ and DAG kinase because it was totally suppressed by DAG kinase inhibitors (10,11). Only when phospholipids of lymphocyte membrane were modified by enrichment with DHA (5,12) or the monohydroxylated derivative of arachidonic acid, 12-HETE (13), were we able to observe PLD activation following mitogenic stimulation. In both cases, this was accompanied by an inhibition of the proliferative response (13,14).…”

“…Lipid rafts were isolated as described previously (5). Briefly, control or SMase-treated PBMCs (5 ϫ 10 8 cells) were homogenized in 1 ml of icecold lysis buffer (25 mM Tris (pH 7.5), 150 mM NaCl, and 5 mM EDTA) supplemented with a mixture of protease inhibitors (protease inhibitor mixture; Sigma-Aldrich) and 1 mM orthovanadate.…”

“…Primers for the amplification of PLD1a, PLD1b, and PLD2 were designed on the basis of published human sequences as described in Ref. 5 (expected sizes of amplified fragments: 446, 332 and 329 bp, respectively). For IL-2, the sense and antisense primers were 5Ј-CACTAAGTCTTGCACTTGTCAC-3Ј and 5Ј-CCTTCTTGGGCATGTA AAACT-3Ј, respectively (expected size of amplified fragment: 186 bp).…”

Mechanism of Angiotensin II-induced Superoxide Production in Cells Reconstituted with Angiotensin Type 1 Receptor and the Components of NADPH Oxidase

“…RNA Extraction and RT-PCR-Total RNA was isolated from PLD1 or PLD2 siRNA-transfected HEK293 cells using Trizol Reagent (Invitrogen) and reverse-transcribed and amplified with SuperScript TM III One-Step RT-PCR System with Platinum TaqDNA Polymerase(Invitrogen). Specific primers and conditions used for the amplification of PLD1 or PLD2 were described previously (55). RT-PCR primer and control were purchased for setSpecific primers for actin (Invitrogen).…”