Neuroprotection of interleukin-6 against NMDA-induced neurotoxicity is mediated by JAK/STAT3, MAPK/ERK, and PI3K/AKT signaling pathways

Abstract

We have previously shown that interleukin-6 (IL-6) has neuroprotective effect against N-methyl-D-aspartate (NMDA)-induced excitotoxicity. The current study aimed to reveal signal transduction pathways involved in the IL-6 neuroprotection. Cerebellar granule neurons (CGNs) from postnatal 8-day infant rats were exposed to IL-6 (120 ng/ml) for 8 days and stimulated with NMDA (100 μM) for 15 or 30 min. Dynamic intracellular Ca(2+) fluorescence intensity, cytosolic Ca(2+)-dependent phospholipase A2 (cPLA2) expression, and apoptosis and necrosis in cultured CGNs were measured by laser scanning confocal microscope, real-time PCR and Western blot, and annexin V-FITC/propidium iodide staining, respectively. NMDA stimulation of neurons evoked an intracellular Ca(2+) overload, an upregulated expression of cPLA2, and an increase in cell death. Chronic IL-6 exposure prevented the NMDA-evoked neuronal Ca(2+) overload, cPLA2 expression upregulation, and apoptosis and necrosis. Anti-gp130 monoclonal antibody (mAb), a blocker of gp130 that is a 130-kDa signal-transducing β-subunit of IL-6 receptor complex, blocked these effects of IL-6 preventing NMDA neurotoxicity. AG490, PD98059, or LY294002, inhibitors specific for the intracellular signals, JAK, MAPK, and PI3K, respectively, partially blocked these IL-6 neuroprotective effects. Phosphorylation levels of STAT3, ERK1/2, and AKT, the downstream proteins for these enzymes of JAK, MAPK, and PI3K, respectively, were elevated by IL-6 pretreatment. The enhanced activation of STAT3, ERK1/2, and AKT by IL-6 was abolished by AG490, PD98059, and LY294002, respectively. Anti-gp130 mAb attenuated the activation of all the three detected signaling molecules. The present findings suggest that IL-6 neuroprotection is jointly mediated by the cellular signal transduction pathways, gp130-JAK-STAT3, gp130-MAPK-ERK, and gp130-PI3K-AKT.

Fig. 1

Effects of anti-gp130 mAb, AG490, PD98059, and LY294002 on IL-6 preventing NMDA-evoked neuronal Ca²⁺ overload. Cultured CGNs were exposed to IL-6 alone or co-exposed to IL-6 and either of anti-gp130 mAb, AG490, PD98059, and LY294002 for 8 days and then stimulated with NMDA for 15 min. Dynamic changes in intracellular Ca²⁺ levels were recorded by LSCM before and after NMDA stimulation. In each treatment, thirty neurons were randomly selected to obtain their fluorescence intensity reflecting intracellular Ca²⁺ levels. The intracellular fluorescence intensity before NMDA stimulation was used as a basal Ca²⁺ level for the respective treatment. The images of a1, b1, c1, and d1 are representative for dynamic changes in intracellular Ca²⁺ fluorescence intensity of randomly selected thirty neurons with different treatments. The a2, b2, c2, and d2 are statistical charts showing mean and SD of three repeated experiments as exhibited in a1, b1, c1, and d1, respectively. The data in a3, b3, c3, and d3 are from the maximal values in a2, b2, c2, and d2, respectively, and they were compared for the statistical significance. The arrows denote NMDA-added time. Anti-gp anti-gp130 mAb, AG AG490, PD PD98059, LY LY294002. **P < 0.01 versus baseline; ⁺⁺ P < 0.01 versus control; ^& P < 0.05, ^&& P < 0.01 versus IL-6 treatment

Fig. 2

Effects of anti-gp130 mAb, AG490, PD98059, and LY294002 on IL-6 preventing NMDA-induced upregulation of cPLA2 expression. Cultured CGNs were exposed to IL-6 alone or co-exposed to IL-6 and either of anti-gp130 mAb, AG490, PD98059, and LY294002 for 8 days and then stimulated with NMDA for 30 min. a Statistical chart for mean and standard deviation of four separate experiments showing cPLA2 gene expression measured by real-time PCR. b, c Representative immunoblots of cPLA2 and compilation of data for mean and standard deviation of five respective experiments. Anti-gp anti-gp130 mAb, AG AG490, PD PD98059, LY LY294002. **P < 0.01 versus control; ⁺⁺ P < 0.01 versus NMDA; ^& P < 0.05, ^&& P < 0.01 versus IL-6+ NMDA group

Fig. 3

Protective effect of IL-6 against NMDA-induced increase in neuronal death is blocked by anti-gp130 mAb, AG490, PD98059, or LY294002. CGNs were double-stained with the fluorescent dyes, annexin V-FITC and PI, for 15 min after NMDA stimulation. In the representative photomicrograph, the green annexin V-FITC-stained cells are apoptotic neurons, and the red PI-stained cells show necrotic neurons. The yellow annexin V-FITC/PI double-labeled cells reflect a late apoptosis. In the statistical diagram, the percentage of dead neurons including apoptotic and necrotic ones was counted in total cells. For each sample, five visual fields were randomly selected and 100 cells were minimally counted. The data were from three independent experiments. Anti-gp anti-gp130 mAb, AG AG490, PD PD98059, LY LY294002. **P < 0.01 versus control; ⁺⁺ P < 0.01 versus NMDA; ^&& P < 0.01 versus IL-6+ NMDA group

Fig. 4

Effects of anti-gp130 mAb, AG490, PD98059, and LY294002 on IL-6-induced enhancement of STAT3, ERK1/2, and AKT activation. Cultured CGNs were exposed to IL-6 alone or co-exposed to IL-6 and either of anti-gp130 mAb, AG490, PD98059, and LY294002 for 8 days and then stimulated with NMDA for 30 min. Left panels exhibit examples of Western blots for phosphorylation levels of a variety of cellular signal transduction proteins. Right panels show statistical charts relative to left panels, respectively. The data are mean and standard deviation of five separate experiments. Anti-gp anti-gp130 mAb, AG AG490, PD PD98059, LY LY294002. **P < 0.01 versus control; ⁺⁺ P < 0.01 versus NMDA; ^&& P < 0.01 versus IL-6+ NMDA group