Glatiramer acetate triggers PI3Kδ/Akt and MEK/ERK pathways to induce IL-1 receptor antagonist in human monocytes

Abstract

Glatiramer acetate (GA), an immunomodulator used in multiple sclerosis (MS) therapy, induces the production of secreted IL-1 receptor antagonist (sIL-1Ra), a natural inhibitor of IL-1β, in human monocytes, and in turn enhances sIL-1Ra circulating levels in MS patients. GA is a mixture of peptides with random Glu, Lys, Ala, and Tyr sequences of high polarity and hydrophilic nature that is unlikely to cross the blood-brain barrier. In contrast, sIL-1Ra crosses the blood-brain barrier and, in turn, may mediate GA anti-inflammatory activities within the CNS by counteracting IL-1β activities. Here we identify intracellular signaling pathways induced by GA that control sIL-1Ra expression in human monocytes. By using kinase knockdown and specific inhibitors, we demonstrate that GA induces sIL-1Ra production via the activation of PI3Kδ, Akt, MEK1/2, and ERK1/2, demonstrating that both PI3Kδ/Akt and MEK/ERK pathways rule sIL-1Ra expression in human monocytes. The pathways act in parallel upstream glycogen synthase kinase-3α/β (GSK3α/β), the knockdown of which enhances sIL-1Ra production. Together, our findings demonstrate the existence of signal transduction triggered by GA, further highlighting the mechanisms of action of this drug in MS.

Fig. 1.

GA triggers both PI3K and MAPK pathways in human monocytes. (A) Monocytes were stimulated by GA. After the indicated time, cell lysates were subjected to Western blot analysis with the indicated antibody. (B) Monocytes were preincubated for 30 min in the presence or absence of 10 μM cycloheximide (CHX) before activation by GA for 2 h. Cell lysates were subjected to Western blot analysis.

Fig. 2.

PI3Kδ controls sIL-1Ra induction by GA in human monocytes. (A) Monocytes were stimulated by GA. Cell membrane and cytoplasm fractions were analyzed by Western blot. (B) Monocytes were preincubated or not preincubated with 10 μM Ly294002 (Ly), 5 μM IC87114 (IC), 50 nM PI3Kα inhibitor, 50 nM PI3Kβ inhibitor, or 500 nM PI3Kγ inhibitor as indicated. Cells were activated with GA (gray columns) or left unactivated (white column). Production of sIL-1Ra was measured in supernatants and presented as percentage of production observed in the absence of inhibitor (no inh. = 100% = 1989 ± 867 pg/mL sIL-1Ra). (C) Monocytes were nucleofected with stealth siRNA for PI3Kδ or negative control (mock). PI3Kδ silencing was assessed by Western blot (Lower). PI3Kδ knockdown or mock transfected monocytes were activated (gray columns; 100% = 1806 ± 634 pg/mL sIL-1Ra) or not activated (white column; sIL-1Ra concentration = 1173 ± 696 pg/mL) with GA and sIL-1Ra measured in culture supernatants. (D) Monocytes were treated with 10 μM Ly294002 (Ly) or 5 μM IC87114 (IC) before activation (gray columns) by GA or left unactivated (white column). sIL-1Ra mRNA was analyzed by qPCR and presented as percentage of transcript level induced by GA in the absence of inhibitor. Results are either representative of three experiments (Western blots) or mean ± SD of three independent experiments carried out with monocytes isolated from three different individuals. **P < 0.01, *P < 0.05 as determined by Student t test.

Fig. 3.

GA triggers the production of sIL-1Ra through a PI3Kδ/Akt pathway. (A) Monocytes were preincubated with or without 10 μM Ly294002 (Ly) or 5 μM IC 87114 (IC) before stimulation by GA. Cell lysates were subjected to Western blot analysis. (B) Monocytes were nucleofected with stealth siRNA for Akt1/2 or negative control (mock). The efficiency of Akt1/2 silencing was assessed by Western blot (Lower); a representative experiment out of 3 is presented. Akt1/2 knockdown or mock transfected monocytes were GA activated (gray columns; 100% = 2174 ± 467 pg/mL sIL-1Ra) or left unactivated (white column; sIL-1Ra concentration = 1,346 ± 467 pg/mL). sIL-1Ra was measured in culture supernatants and presented as described in legend of Fig. 2B. Data are mean ± SD of three independent experiments. *P < 0.05 as determined by Student t test.

Fig. 4.

MEK1/2 controls GA-induced sIL-1Ra production. (A) Monocytes were preincubated in the absence or presence of increasing concentration of U0126 (○) or PD98059 (●) before the addition of GA. sIL-1Ra production was measured in supernatants. Results are presented as described in Fig. 2B (no inh. = 100% = 2177 ± 837 pg/mL sIL-1Ra). (B) Monocytes were treated with 5 μM U0126 (U0) or 5 μM PD98059 (PD) before addition (gray columns) or no addition (white column) of GA. mRNA was isolated and analyzed by real-time qPCR. Results are presented as in Fig. 2D. (C) Monocytes were nucleofected with stealth siRNA for MEK1 and MEK2 or negative control (mock). Efficiency of MEK1/2 silencing was assessed by Western blot (Lower), and sIL-1Ra production by MEK1/2 knocked-down or mock transfected monocytes activated (gray columns; 100% = 1933 ± 656 pg/mL sIL-1Ra) or not activated (white column; sIL-1Ra concentration = 1211 ± 547 pg/mL) by GA was measured in culture supernatants. Results are presented as mean ± SD of three independent experiments. **P < 0.01, *P < 0.05 as determined by Student t test.

Fig. 5.

ERK1/2 controls GA-induced sIL-1Ra production. Monocytes were nucleofected with stealth siRNA for ERK1/2 or negative control (mock). Efficiency of ERK1/2 silencing was assessed by Western blot (Lower); one representative experiment among three is presented. ERK1/2 knocked-down or mock-transfected monocytes were activated (gray columns; 100% = 2,334 ± 556 pg/mL sIL-1Ra) or not activated (white column; sIL-1Ra concentration = 1,555 ± 767 pg/mL) with GA and sIL-1Ra measured in culture supernatants. Results are presented as mean ± SD of three independent experiments; *P < 0.05 as determined by Student t test.

Fig. 6.

PI3Kδ and MEK1/2 are part of two different pathways controlling sIL-1Ra production. (A) Monocytes were preincubated in the absence or presence of 0.5 μM U0126 (U0, MEK1/2 inhibitor), 0.5 μM IC87114 (IC, PI3Kδ inhibitor) or a mixture of both inhibitors (U0 + IC). Cells were then activated (gray columns) or not activated (white column) by GA. Production of sIL-1Ra was measured in harvested supernatants. Results are presented as described in Fig. 2B (no inh. = 100% = 2720 ± 1726 pg/mL sIL-1Ra). Data are mean ± SD of three experiments carried out with monocytes prepared from blood of three different donors. **P < 0.01 as determined by Student t test.

Fig. 7.

GSK3α/β controls GA-induced sIL-1Ra production. (A) Monocytes were stimulated by GA for indicated time, and cell lysates were subjected to Western blot analysis. (B and C) Monocytes were preincubated for 45 min in the presence or absence of 10 μM SB216763 (SB) and then cultured in the absence (white columns) or presence (gray columns) of GA. (B) Production of sIL-1Ra was measured in harvested supernatants (no inh. = 100% = 2020 ± 636 pg/mL sIL-1Ra) and (C) mRNA analyzed by real-time quantitative PCR; results are presented as described in Fig. 2 B and D, respectively. (D) Monocytes were nucleofected with stealth siRNA for GSK3α and GSK3β (siGSK3) or negative control (mock). Efficiency of GSK3α/β silencing was assessed by Western blot (Lower). sIL-1Ra was measured in culture supernatants of GSK3α/β knocked-down or mock-transfected monocytes activated (gray columns; 100% = 2174 ± 467 pg/mL sIL-1Ra) or not activated (white column; sIL-1Ra concentration = 1,344 ± 676 pg/mL) by GA. (E) Monocytes were preincubated in the absence or presence of 10 μM Ly294002 (Ly); 5 μM IC87114 (IC); 500 nM PI3Kγ inhibitor, 5 μM U0126 (U0), or 5 μM PD98059 (PD) before activation by GA. Cell lysates were subjected to Western blot analysis. **P < 0.01 as determined by Student's t test.