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

Abstract

Recent evidence suggests that phospholipase D (PLD) can be regulated through its association/dissociation to lipid rafts. We show here that modifying lipid rafts either by cholesterol depletion using methyl-β-cyclodextrin and filipin or by conversion of sphingomyelin to ceramide with exogenous bacterial sphingomyelinase (bSMase) markedly activated the PLD of human PBMC. bSMase was the most potent PLD activator, giving maximal 6- to 7-fold increase in PLD activity. Triton X-100-treated lysates prepared from control PBMC and from bSMase-treated cells were fractionated by centrifugation on sucrose density gradient. We observed that bSMase treatment of the cells induced a larger ceramide increase in raft than in nonraft membranes and displaced both the Src kinase Lck and PLD1 out of the raft fractions. In addition, the three raft-modifying agents markedly inhibited the lymphoproliferative response to mitogenic lectin. To examine further the potential role of PLD activation in the control of lymphocyte responses, we transiently overexpressed either of the PLD1 and PLD2 isoforms in Jurkat cells and analyzed the phorbol ester plus ionomycin-induced expression of IL-2 mRNA, which is one of the early responses of lymphocyte to activation. We observed a 43% decrease of IL-2 mRNA level in Jurkat cells overexpressing PLD1 as compared with mock- or PLD2-transfected cells, which indicates that elevated PLD1, but not PLD2, activity impairs lymphocyte activation. Altogether, the present results support the hypothesis that PLD1 is activated by exclusion from lipid rafts and that this activation conveys antiproliferative signals in lymphoid cells.

Phospholipase D as a potential target for the antiproliferative effects of polyunsaturated fatty acids in rat thymocytes

“…4). These results support further our current hypothesis that PLD activity conveys antiproliferative signals in lymphoid cells [8,15] and suggest that 20:5n-3 inhibits thymocyte proliferation by a particular mechanism unrelated to that of the other FAs.…”

“…In contrast, when lymphocyte membranes were enriched with docosahexaenoate or 12-HETE prior to mitogenic stimulation, PLD was activated, leading to an inhibition of the proliferation response. The hypothesis that PLD is antiproliferative in lymphoid cells has been strongly reinforced by our recent work showing that overexpression of the PLD1 isoform in Jurkat cells inhibits IL-2 mRNA expression in response to phorbol 12-myristate 13-acetate (PMA) and ionomycin activation [15].…”

Effects of Active and Inactive Phospholipase D2 on Signal Transduction, Adhesion, Migration, Invasion, and Metastasis in EL4 Lymphoma Cells

“…It is noteworthy that fibroblasts express endogenous PLD1 and PLD2. A study of overexpression of PLD1 and PLD2 in Jurkat T-cells, using transient transfection, concluded that PLD1 (but not PLD2) impairs mitogenic signaling (Diaz et al, 2005). The Jurkat cell line used expresses endogenous PLD2 but low levels of PLD1.…”

Therapeutic Levels of the Hydroxmethylglutaryl-Coenzyme A Reductase Inhibitor Lovastatin Activate Ras Signaling via Phospholipase D2

“…on May 12, 2018 by guest http://mcb.asm.org/ Previous work has shown that PLD2 may be associated with cholesterol-dependent plasma membrane nanodomains, commonly called lipid rafts (10), and that cholesterol depletion leads to PLD2 activation (11). Collecting these data together with the new results presented in this paper, we propose that PLD2 is a cholesterol-sensitive regulator of the Ras/MAPK signaling pathway.…”