The Selective Agonist for Sphingosine-1-Phosphate Receptors Siponimod Increases the Expression Level of NR4A Genes in Microglia Cell Line

Abstract

Fingolimod (FTY720) and siponimod (BAF312) are selective agonists for sphingosine-1-phosphate (S1P) receptors approved for the treatment of relapsing-remitting (RR) and secondary progressive (SP) multiple sclerosis (MS), respectively. BAF312 exerts pro-myelination and neuro-protective functions on CNS resident cells, although the underlying molecular mechanism is not yet fully understood. NR4A2 is an anti-inflammatory gene, belonging to the NR4A family, whose expression is reduced in blood from treatment-naïve patients with RRMS, but is restored in patients treated with FTY720 for more than two years. We performed an in vitro study to investigate the potential involvement of the NR4A genes in the protective and restorative effects of BAF312. We showed that BAF312 enhances the expression of NR4A1 and NR4A2 in the N9 microglial cell line, but has no effect in the peripheral blood mononuclear cells and oligodendrocytes. This study suggests a novel molecular mechanism of action for the selective agonists for S1P receptors within the CNS.

Keywords: CNS resident cells; NR4A; NURR1; fingolimod; multiple sclerosis; siponimod; sphingosine-1-phosphate (S1P) receptors.

Figure 1

BAF312 does not influence the expression of the NR4A genes in primary cultures of PBMCs. Gene expression levels of NR4A1 (A), NR4A2 (B), and NR4A3 (C) in human primary cultures of PBMCs. Comparison of gene expression levels between not-treated (n = 4) and BAF312-treated (n = 4) cells. Two-tailed t-test for (A,C), and Mann–Whitney U test for (B).

Figure 2

BAF312 up-regulates NR4A1, NR4A2, and TREM-2 expression in the N9 microglial cell line. Gene expression levels of NR4A1 (A), NR4A2 (B), NR4A3 (C), IL 1-beta (D), iNOS (E), TNF-alfa (F), TREM-2 (G), and IGF-1 (H) in N9 murine microglial cell line. Comparison of gene expression levels between not-treated (n = 4), BAF312 (n = 4), LPS (n = 4), and BAF312/LPS (n = 4) -stimulated cells. One-way ANOVA followed by Tukey’s HSD post hoc test for (A,B,D–H), and Kruskal-Wallis followed by Dunn’s post hoc test for (C). * 0.05 > p value ≥ 0.01; ** 0.01 > p value ≥ 0.001; *** p value < 0.001.

Figure 3

BAF312 does not influence the expression of the NR4A genes in the oligodendrocytic cell line. (A) Representative Western blots of CNPase, NG2, and S100 proteins extracted from the MO3.13 oligodendrocytic cell line under control conditions (T0), after differentiation with PMA for 3, 5, and 7 days in vitro (T3, T5, T7 PMA), and with FBS for 7 days in vitro (FBS T7). Tubulin served as the loading control. (B) Representative images of the MO3.13 oligodendrocytic cell line under control conditions (T0), after differentiation with PMA (T7 PMA) and FBS (FBS T7) for 7 days in vitro, and stained with tubulin antibody (red). DAPI (blue) counterstained the cell nuclei. Magnification 20×. Gene expression levels of NR4A1 (C), NR4A2 (D), and NR4A3 (E) in the MO3.13 oligodendrocytic cell under the PMA T7 condition. Comparison of the gene expression levels between the not-treated (n = 4) and BAF312-treated (n = 4) cells. Two-tailed t-test.