Galectin-8 induces apoptosis in Jurkat T cells by phosphatidic acid-mediated ERK1/2 activation supported by protein kinase A down-regulation

Abstract

Galectins have been implicated in T cell homeostasis playing complementary pro-apoptotic roles. Here we show that galectin-8 (Gal-8) is a potent pro-apoptotic agent in Jurkat T cells inducing a complex phospholipase D/phosphatidic acid signaling pathway that has not been reported for any galectin before. Gal-8 increases phosphatidic signaling, which enhances the activity of both ERK1/2 and type 4 phosphodiesterases (PDE4), with a subsequent decrease in basal protein kinase A activity. Strikingly, rolipram inhibition of PDE4 decreases ERK1/2 activity. Thus Gal-8-induced PDE4 activation releases a negative influence of cAMP/protein kinase A on ERK1/2. The resulting strong ERK1/2 activation leads to expression of the death factor Fas ligand and caspase-mediated apoptosis. Several conditions that decrease ERK1/2 activity also decrease apoptosis, such as anti-Fas ligand blocking antibodies. In addition, experiments with freshly isolated human peripheral blood mononuclear cells, previously stimulated with anti-CD3 and anti-CD28, show that Gal-8 is pro-apoptotic on activated T cells, most likely on a subpopulation of them. Anti-Gal-8 autoantibodies from patients with systemic lupus erythematosus block the apoptotic effect of Gal-8. These results implicate Gal-8 as a novel T cell suppressive factor, which can be counterbalanced by function-blocking autoantibodies in autoimmunity.

FIGURE 1.

Gal-8 induces apoptosis in Jurkat T cells. Jurkat T cells maintained for 2 h in serum-free medium were seeded on GST-Gal-8 (referred to as Gal-8)-coated coverslips or treated in suspension with 2 μg/ml of soluble Gal-8 (0.03 μm) for 6 h at 37 °C in the absence or presence of TDG and then seeded on lysine-coated coverslips. A, adhered cells assessed for apoptotic signs. Hoechst staining of condensed nuclei and indirect immunofluorescence of activated caspase 3. B, percentage of apoptotic cells (means ± S.E. of triplicates). Gal-8 induces apoptosis both as matrix (60% of the cells) and in suspension (30% of the cells). TDG inhibits the Gal-8 effect indicating dependence on cell surface interaction with glycoconjugates. C, GST-Gal-8 and isolated Gal-8 have similar pro-apoptotic effects, whereas GST has no effect. Therefore, in all other experiments we used GST-Gal-8 and referred to as Gal-8. D, DNA fragmentation in Jurkat T cells incubated in suspension in the absence or presence of 0.03 μm Gal-8 for 12 h. Ethidium bromide staining shows that Gal-8 increases DNA fragmentation. E, anti-Gal-8 autoantibodies abrogate Gal-8-induced apoptosis. The cells were treated with either Gal-8 (0.03 μm) or Gal-8 preincubated with 20 μg/ml of affinity-purified anti-Gal-8 autoantibodies from SLE patients. Apoptosis is expressed as a percentage of the maximal levels achieved under Gal-8 treatment.

FIGURE 2.

Gal-8 activates ERK1/2 in Jurkat T cells in suspension. A, Jurkat T cells maintained for 2 h in serum free medium and then incubated in suspension in the absence (control, C) or presence of 0.5 μg/ml (0.008 μm), 2 μg/ml (0.03 μm), or 5 μg/ml (0.08 μm) Gal-8 for 30 min at 37 °C were analyzed for ERK1/2 activation by immunoblot against phosphorylated-ERK1/2 (p-ERK) or for total ERK1 (ERK). B, TDG abrogates Gal-8-induced ERK1/2 activation. C, time course of Gal-8 effect shows persistent ERK1/2 activation for up to 4 h. The numbers below the blots represent the ratio phosphorylated ERK1/2/ERK1 estimated from the densitometric analysis of the bands. p-ERK1/2, phosphorylated ERK1/2.

FIGURE 3.

Gal-8 activates ERK1/2 via PLD-generated PA pathway. A, TLC analysis of PA levels. Jurkat T cells were first incubated with [³H]myristic acid for 16 h at 37 °C and then treated with 0.03 μm Gal-8 for 5–15 min, as indicated, in the absence or presence of 1-butanol (1-but). Increased levels of PA are detected after 15 min of Gal-8 stimulation, sensitive to inhibition by 1-butanol. B, ERK1/2 activation in Jurkat T cells treated with 2 μg/ml of Gal-8 (0.03 μm) for 30 min decreased in the presence of 1-butanol (1%) but not 2-butanol (2-but), indicating its dependence on PLD-generated PA. C, Jurkat T cells directly incubated with PA micelles (10–200 μg/ml) for 30 min show ERK1/2 activation. D, transfection with dominant negative (DN) PLD2 but not with wild type (WT) PLD2 decreases ERK1/2 response to Gal-8. E, propranolol (150 μg/ml), used to inhibit PAP, decreases ERK1/2 activation. p-ERK1/2, phosphorylated ERK1/2.

FIGURE 4.

Gal-8 increases PDE4 activity leading to PKA inhibition and maximal ERK1/2 activation. Jurkat T cells were treated with 0.03 μm Gal-8 at 37 °C for the indicated time periods. A, PDE activity increases reaching a maximal at 15 min and being sensitive to inhibition by 50 μm rolipram (black circle), a type 4 PDE specific inhibitor. B, progressive decrease in PKA activity. C, ERK1/2 activation is sensitive to inhibition by rolipram. pERK1/2, phosphorylated ERK1/2.

FIGURE 5.

Apoptosis induced by Gal-8 in Jurkat T cells involves ERK1/2 and the PLD/PA/DAG pathway. Jurkat T cells incubated in the absence or presence of 0.03 μm Gal-8 for 6 h. A, PD98059 (PD; 25 μm) inhibits by 60% Gal-8-induced ERK1/2 activation. B, PD98059 decreases by 50% the apoptotic effect of Gal-8. C, preincubation (5 min) with either 1-but or (D) propranolol decreases Gal-8-induced apoptosis. Cells directly incubated with PA micelles (PA) in conditions that activate ERK1/2 (see Fig. 3C) show increased apoptosis (average percentages of three independent experiments ± S.E.). p-ERK1/2, phosphorylated ERK1/2; C, control.

FIGURE 6.

Gal-8-induced ERK1/2 activity leads to enhanced expression of IL-2 and FasL, which mediates apoptosis. Jurkat T cells incubated in the absence or presence of 0.03 μm Gal-8 for 6 h were analyzed for IL-2 and FasL mRNA expression by RT-PCR (A and B) and for apoptosis sensitivity to a blocking anti-FasL antibody (C). A, the increase of IL-2 and FasL mRNA mediated by Gal-8 is inhibited by PD98059 (PD) and TDG. B, quantitative assessment of RT-PCR bands (averages of three independent experiments ± S.E.). C, co-incubation of Gal-8 with 10 or 40 μg/ml of anti-FasL (α FasL) decreased the apoptotic effect of Gal-8.

FIGURE 7.

Gal-8 induces PA-mediated ERK1/2 and apoptosis in human PBMC and the apoptotic effect increases after T cell activation. PBMC freshly isolated from healthy human donors were incubated with 0.03 μg/ml Gal-8 for 30 min at 37 °C. A, ERK1/2 activation sensitive to inhibition by 1-butanol involves PA produced by PLD. B, apoptosis in activated T cells. Gal-8 induces apoptosis in 5% of freshly isolated PBMC and in 13% of the cells after 48 h of T cell activation with anti-CD3 and anti-CD28 antibodies. p-ERK1/2, phosphorylated ERK1/2.