Calcium Ion Channel Mechanisms Underlying IL-6Exerting Neuroprotection And Its Signaling Pathway

Interleukin-6(IL-6), one of the dozens of cytokines, has been known to be involved inthe induction, growth, and differentiation of cells in the immune and hematopoietic system.But now IL-6is also found and produced within the central nervous system (CNS),especially by cells of the brain. It has been recognized that in the normal brain, IL-6levelsremain low, but are elevated rapidly and markedly in many neurological disorders, e.g.,brain injury, neurodegeneration, infection, ischemia and multiple sclerosis. This suggeststhat IL-6is closely related to neurological diseases. It has been reported that IL-6has aneuroprotective role in the CNS. We have previously shown that IL-6has a neuroprotectiveeffect against glutamate or NMDA induced excitotoxicity and intracellular Ca2+overload.But the mechanisms through which IL-6inhibits intracellular Ca2+overload are primarilyunknown. To this end, this study aimed to investigate the possible mechanism underlyingIL-6inhibition of intracellular Ca2+overload induced by NMDA by means of patch clamp,calcium imaging, morphology and molecular biology technique. Calcium influx fromvoltage-gated calcium channel (VGCC) and NMDAR ion channel was selected to explorethe mechanism underlying IL-6inhibition of Ca2+overload and the links between IL-6signaling pathway and NMDAR ion channel function was to be further explored. Thepresent findings suggested that IL-6inhibited intracellular Ca2+overload by suppressingL-type VGCC and NMDAR subunits NR2B and NR2C activities via JAK-STAT3-CaNsignaling pathway. This study may offer more and new evidence of IL-6effects in the CNSdisorders.Part One: IL-6Decreases Intracellular Ca2+Overload via Inhibiting L-typeVoltage-gated Calcium Channel ActivityObjective:To investigate the type of VGCC and mechanisms underlying IL-6inhibition ofneuronal Ca2+overload.Methods: 1. The cultured cerebellar granule neurons(CGNs) were pretreated with IL-6(120ng/ml) for24h. Whole-cell patch clamp technique was used to observe the effects of IL-6on whole cell currents of VGCC and LCC in cultured CGNs.2. Cultured CGNs were incubated with Ca2+fluorescence probe Fluo-3/AM and werescanned by laser scanning confocal microscope to observe the effect of IL-6onintracellular Ca2+level changes evoked by high K+solution.3. Western blotting analysis was used to detect the effect of IL-6on LCC subunitCav1.2level in cultured CGNs.Results:1. The whole-cell VGCC current density was significantly smaller in IL-6exposedneurons than in control neurons by depolarization from-80mV to-20mV,-10mV,0mVand+10mV at a holding potential of-80mV, suggesting IL-6pretreatment inhibits VGCCactivity.2. The whole-cell LCC current density was significantly decreased in IL-6pretreatedneurons compared to control neurons by depolarization from-80mV to-10mV at aholding potential of-80mV. While non-LCC current density had no significant differencesbetween IL-6pretreated neurons and control neurons at the same depolarization pulse.Meanwhile, voltage-dependent activation curves obtained by Boltzmann equation andfitting parameters showed no significant differences between control and IL-6-treatedneurons. This suggested that IL-6inhibited LCC activity and did not modify thevoltage-dependent activation feature of LCC.3. High K+stimulation evoked a significant elevation of the intracellular Ca2+fluorescence intensity in CGNs. After exposure to nifedipine, an LCC antagonist, high K+stimulation resulted in a reduction of intracellular Ca2+fluorescence intensity compared tocontrol neurons. This suggested that high K+prompted LCC open and increased Ca2+influx.4. High K+stimulation evoked a significant less elevation of the intracellular Ca2+fluorescence intensity in IL-6pretreated neurons than in control neurons. Furthermore,IL-6suppressed nifedipine-dependent LCC Ca2+influx.5. The LCC subunit Cav1.2expression was remarkably downregulated by IL-6pretreatment. Conclusion:1. IL-6inhibits LCC activity through downregulation of LCC subunit Cav1.2proteinexpression, followed by decreasing Ca2+influx via LCC, which suggests that theneuroprotective role of IL-6is implemented, at least partially, by suppression of Ca2+overload via LCC.Part Two: Receptor Mechanism and Signaling Pathway of Interleukin-6Inhibiting Intracellular Ca2+Overload and Apoptosis Induced by NMDAObjective:To illustrate the role of IL-6inhibiting neuronal Ca2+overload and apoptosis inducedby NMDA through suppressing NMDAR subunits NR2B and NR2C activity viaJAK-STAT3-CaN signaling pathway.Methods:1. Cultured CGNs were pretreated with IL-6(120ng/ml) for24h, followed bytreatment with Ca2+store depleter thapsigargin(TG), or NMDAR subunit antagonistsNVP-AAM077(NVP), ifenprodil and PPDA, or inhibitors of JAK-STAT3-CaN signalingpathway AG490and FK506.2. Cultured CGNs were loaded with Fluo-3/AM and then scanned by laser scanningconfocal microscope to observe the effects of IL-6on intracellular Ca2+fluorescenceintensity changes evoked by NMDA(100μM) combined application of other drugsabove-mentioned, respectively.3. Whole-cell patch clamp technique was applied to observe the effect of IL-6andother drugs above-mentioned on NMDA-induced current in cultured CGNs.4. Mitochondrial membrane potential was measured by JC-1assay kit. Andmitochondrial membrane potential was assessed when cells were stimulated with NMDAin the presence or absence of IL-6.5. TUNEL assay was applied to detect the effects of IL-6and other signalingmolecules against NMDA-induced apoptosis in cultured CGNs.6. Western blotting analysis was used to detect the NMDAR subunits NR1, NR2A,NR2B, NR2C and phosphorylation level with NMDA stimulation in the presence of IL-6.7. Western blotting analysis and colorimetric assay were used to detect the CaNprotein expression and activity. And the CaN activity and protein level were detected when cells were pretreated with AG490in the presence or absence of IL-6under NMDAstimulation.Results:1. NMDA stimulation of cultured CGNs evoked an acute increase of intracellular Ca2+level by1.84fold of basal level. After exposure to TG, an inhibitot of Ca2+pump, NMDAstimulation also triggered an elevation of intracellular Ca2+level, which suggested NMDAstimulation induced Ca2+influx via NMDAR ion channel. However, IL-6pretreatmentfurther decreased the elevation of intracellular Ca2+level evoked by NMDA when neuronswere exposed to TG before. These results indicated that IL-6inhibited extracellular Ca2+influx via NMDAR ion channel.2. NMDAR antagonist MK-801signifcantly blocked the NMDA-induced currents,indicating NMDA-induced currents were mediated by NMDAR. IL-6pretreatment resultedin a smaller current density compared to control neurons without IL-6pretreatment,indicating IL-6inhibiting the NMDAR-mediated inward current.3. Ifenprodil and PPDA, the antagonists for NR2B and NR2C, significantly abolishedNMDA-induced intracellular Ca2+overload, while NR2A antagonist NVP could notsignificantly reduced NMDA-induced intracellular Ca2+overload, which suggested thatNMDAR subunits NR2B and NR2C play a major role in mediating Ca2+influx induced byNMDA. Meanwhile, application of ifenprodil or PPDA resulted in a lesser inhibition ofNMDA-induced intracellular Ca2+increase in neurons pretreated with IL-6than in controlneurons without IL-6pretreatment. But NVP showed the same inhibition rate ofNMDA-induced intracellular Ca2+increase between neurons pretreated with IL-6andcontrol neurons. These results suggested IL-6suppressed the NR2B and NR2C activity.4. Application of ifenprodil or PPDA resulted in a lesser inhibition of NMDA-evokedwhole-cell currents in neurons pretreated with IL-6than in control neurons. While NVPshowed the same inhibition rate of NMDA-evoked currents between neurons pretreatedwith IL-6and control neurons. These results further suggested IL-6suppressed the NR2Band NR2C activity.5. IL-6prevented the NMDA-induced neuronal apoptosis and application ofifenprodil or PPDA similarly resulted in a lesser inhibition of apoptosis rate in neuronspretreated with IL-6than in control neurons. These results suggested that IL-6exerted ananti-apoptosis effect by inhibiting the NR2B and NR2C subunits function. 6. IL-6pretreatment significantly reduced the level of NR2B, NR2C andphospho-NR2C in cultured CGNs, but had no significant effect on NR2A andphospho-NR2A.7. JAK-STAT3inhibitor AG490and CaN inhibitor FK506both blocked the effect ofIL-6on prevention of NMDA-induced currents and neuronal Ca2+overload.8. IL-6significantly prevented the NMDA-induced a loss of mitochondrial membranepotential, while AG490and FK506blocked the effect of IL-6.9. IL-6significantly prevented the NMDA-induced neuronal apoptosis. AG490andFK506signifcantly blocked the effect of IL-6suppressing NMDA-triggered neuronalapoptosis.10. IL-6pretreatment upregulated expression of CaNβprotein level in cultured CGNs.AG490blocked the effect of IL-6on CaNβexpression. Meanwhile, IL-6pretreatmentprevented the NMDA-induced a decrease of CaN activity and AG490also blocked theeffect of IL-6on CaN activity.Conclusions:1. IL-6reduces extracellular Ca2+influx induced by NMDA via inhibiting NMDARchannel activity2. IL-6inhibits NMDA-induced neuronal Ca2+overload and apoptosis by suppressingNMDAR subunits NR2B and NR2C activity and expression.3. JAK-STAT3-CaN signaling pathway may be involved in IL-6preventingNMDA-triggered neuronal Ca2+overload and apoptosis.