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Glucosamine suppresses platelet-activating factor-induced activation of microglia through inhibition of store-operated calcium influx
Environmental Toxicology and Pharmacology 42 (2016) 1–8
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Glucosamine suppresses platelet-activating factor-induced activation of microglia through inhibition of store-operated calcium influx Jae-Hyung Park a,1 , Jeong-Nam Kim a,1 , Byeong-Churl Jang b , Seung-Soon Im a , Dae-Kyu Song a , Jae-Hoon Bae a,∗ a b
Department of Physiology, Keimyung University School of Medicine, Daegu, South Korea Department of Molecular Medicine, Keimyung University School of Medicine, Daegu, South Korea
a r t i c l e
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Article history: Received 31 August 2015 Received in revised form 17 December 2015 Accepted 19 December 2015 Available online 23 December 2015 Keywords: Glucosamine Platelet-activating factor Microglia Intracellular calcium concentration Store-operated calcium channel
a b s t r a c t Microglia activation and subsequent release of inflammatory mediators are implicated in the pathophysiology of neurodegenerative diseases. Platelet-activating factor (PAF), a potent lipid mediator synthesized by microglia, is known to stimulate microglia functional responses. In this study, we determined that endogenous PAF exert autocrine effects on microglia activation, as well as the underlying mechanism involved. We also investigated the effect of d-glucosamine (GlcN) on PAF-induced cellular activation in human HMO6 microglial cells. PAF induced sustained intracellular Ca2+ ([Ca2+ ]i ) increase through store-operated Ca2+ channels (SOC) and reactive oxygen species (ROS) generation. PAF also induced proinflammatory markers through NFB/COX-2 signaling. GlcN significantly inhibited PAF-induced Ca2+ influx and ROS generation without significant cytotoxicity. GlcN downregulated excessive expression of pro-inflammatory markers and promoted filopodia formation through NFB/COX-2 inhibition in PAFstimulated HMO6 cells. Taken together, these data suggest that GlcN may offer substantial therapeutic potential for treating inflammatory and neurodegenerative diseases accompanied by microglial activation. © 2015 Elsevier B.V. All rights reserved.
1. Introduction In the central nervous system (CNS), microglial cells are the resident macrophages. That have a key part in the regulation of innate immune responses in the healthy or degenerating CNS (Khoo et al., 2001). While actively scanning the microenvironment with their long protrusions (Nimmerjahn et al., 2005), loss of inhibitory signals and the recognition of damage-associated molecular patterns from degenerating neurons lead to the activation of microglia (Cardona et al., 2006; Heneka et al., 2014). When microglial cells are activated, the cells exhibit proliferation and phagocytosis. Moreover, the activated cells can secrete pro- and anti-inflammatory
Abbreviations: CNS, central nervous system; [Ca2+ ]i , intracellular Ca2+ ; COX-2, cyglooxygenase-2; GlcN, d-glucosamine; IB-␣, inhibitor of B-␣; iNOS, inducible nitric oxide synthase; IL-1, interleukin-1; PAF, platelet-activating factor; PGE2 , prostaglandin E2 ; ROS, reactive oxygen species; SOC, store-operated Ca2+ channels; TNF-␣, tumor necrosis factor-␣. ∗ Corresponding author at: Department of Physiology, Keimyung University School of Medicine, 1095 Dalgubeol-Daero, Dalseo-Gu, Daegu 704-701, South Korea. E-mail address: [email protected] (J.-H. Bae). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.etap.2015.12.014 1382-6689/© 2015 Elsevier B.V. All rights reserved.
cytokines, chemokines, and neurotrophic factors (Heneka et al., 2014). Therefore, reactive microgliosis is a common hallmark of various neurodegenerative diseases including Alzheimer’s disease (Perry et al., 2010), Parkinson’s disease (Orr et al., 2002), and multiple sclerosis (Raivich and Banati, 2004). Platelet-activating factor (PAF) is a potent proinflammatory phospholipid with diverse pathological and physiological effects (Yue and Feuerstein, 1994). PAF is produced by a variety of cells, but especially those involved in host defense, such as platelets, endothelial cells, neutrophils, monocytes, and macrophages (Zimmerman et al., 2002). In the brain, this endogenous phospholipid is produced by microglia in response to ischemic injury or various neurodegenerative diseases (Bate et al., 2006; Prescott et al., 2000; Tuttolomondo et al., 2008). Moreover, PAF stimulates microglia itself (Mori et al., 1996) and induces arachidonic acid metabolism, resulting in the production of neurotoxic factors. It also mediates pathological inflammation in the brain (Mori et al., 1996). It has been reported that PAF immediately induced an increase in intracellular Ca2+ ([Ca2+ ]i ) levels in human microglial cells (Khoo et al., 2001; Sattayaprasert et al., 2005; Wang et al., 1999). These prolonged increase of [Ca2+ ]i by PAF treatment can increase the expression of inflammatory factors, including
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cyglooxygenase-2 (COX-2), tumor necrosis factor-␣ (TNF-␣), interleukin-1 (IL-1), inducible nitric oxide synthase (iNOS) and prostaglandin E2 (PGE2 ) (Choi et al., 2002; Hoffmann et al., 2003). d-Glucosamine (GlcN) is a dietary supplement widely used for the prevention and/or treatment of rheumatoid arthritis and osteoarthritis (de los Reyes et al., 2000). GlcN can be distributed to the brain at relevant quantities 8 h after oral administration (Setnikar et al., 1984). Our previous study demonstrated that GlcN inhibits exogenous lipopolysaccharide-induced microglia activation (Yi et al., 2005), thus implicating this agent as part of a new inhibitory strategy in targeting activated microglial cells in the CNS. However, the effect of GlcN on endogenous PAF-induced activation of microglia remains largely unknown. Therefore, the aim of the present study was to investigate the effects of GlcN on endogenous PAF-induced increase in [Ca2+ ]i and expression of inflammatory factors in HMO6 microglial cells and to evaluate the inhibitory mechanisms involved. 2. Materials and methods 2.1. Materials FBS and DMEM medium were purchased from Life Technologies (Carlsbad, CA). GlcN was from Sigma–Aldrich (St. Louis, MO). PAF, PK11195 and SKF96365 were from Tocris Bioscience (Bristol, UK). Fura-2 acetoxymethyl (fura-2/AM) ester was obtained from Molecular Probes (Eugene, OR). All other chemicals were obtained from Sigma–Aldrich. 2.2. Cell culture Human microglial cells (HMO6) were a gift from S.U. Kim (University of British Columbia, Vancouver, Canada). HMO6 cells were established as immortalized clonal cells of human microglia from human embryonic telencephalon tissue by using a retroviral vector encoding v-myc and were investigated by immunochemistry and fluorescence-activated cell sorting analyses (Nagai et al., 2005). HMO6 cells were cultured at 37 ◦ C in a humidified incubator with 5% CO2 in DMEM medium containing 5% heat-inactivated FBS, 5% heat-inactivated horse serum and 20 g/mL gentamycin. 2.3. Measurement of intracellular calcium concentration ([Ca2+ ]i ) Microfluorescent imaging of [Ca2+ ]i was performed on HMO6 cells loaded with the calcium indicator dye fura-2/AM. Some of the procedures used in calcium imaging in this experiment have been described (Yi et al., 2005). Fura-2/AM (3 M) was added to HMO6 cells bathed in 1.8 mM Ca2+ containing physiological saline solution (126 mM NaCl, 5 mM KCl, 1.8 mM CaCl2 , 1.2 mM MgCl2 , 10 mM HEPES [pH 7.4], 0.2% BSA and 10 mM glucose) at room temperature for 30 min followed by a 30-min wash in dye-free saline solution to allow esterase conversion to the free form of fura-2. Cover slips were placed on the stage of an inverted microscope and imaging was performed with a dual-wavelength system (Intracellular Imaging, Cincinnati, OH). [Ca2+ ]i was calculated as the relationship between the ratio of emissions at 510 nm from excitation at 340 and 380 nm, respectively. Ratio images were processed every 5 s and converted to [Ca2+ ]i as compared to a range of such ratios obtained by measurement of fura-2 in the presence of known concentrations of calcium (Calcium Calibration Buffer Kit, Molecular Probe, Eugene, OR). When Ca2+ -free solution was used, Ca2+ was omitted and 2 mM EGTA was added. After the establishment of a stable baseline [Ca2+ ]i level, the cells were pre-treated with or without 1 mM GlcN for 2 min prior to stimulation with 300 nM PAF for 1 min. Each experimental data point represents the mean [Ca2+ ]i calculated from at least 12 individually measured cells from three separate
cultures. All imaging experiments were done at room temperature (20–22 ◦ C). 2.4. Western blot analysis Cells were treated with 300 nM PAF in the presence or absence of 1 mM GlcN for 4 h. Crude cell extracts were subjected to SDSPAGE and immunoblotted with anti-COX-2 (Abcam, Cambridge, UK), anti-Ib-␣ (Cell Signaling, Danvers, MA) and anti- actin (Sigma–Aldrich) antibodies. The immunoreactive bands were visualized using a horseradish peroxidase-conjugated secondary antibody (Abcam) and enhanced chemiluminescence (Amersham Biosciences, Little Chalfont, UK). The experiments were repeated at least three times. 2.5. Quantitative real-time PCR analysis After exposure of cells to 300 nM PAF in the presence or absence of 1 mM GlcN for 4 h, total cellular RNA was extracted from HMO6 cells using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. The RNA was eluted with Rnase-free water. Reverse transcription was performed using the first strand cDNA synthesis kit (Fermentas, Glen Burnie, MD) according to the manufacturer’s protocols. Real-time PCR amplification was performed using the SYBR Green master mix (Applied Biosystems, Foster City, CA) and the Prism 7500 real-time PCR detection system. Relative amounts of mRNA were normalized by use of the gene encoding glyceraldehyde 3-phosphate dehydrogenase and calculated using the CT (cycle threshold) method. The specific primers for iNOS were 50-GTT CTC AAG GCA CAG GTC TC-30 (forward) and 50-GCA GGT CAC TTA TGT CAC TTA TC-30 (reverse). The primers for IL-1 were 50-TTA CAG TGG CAA TGA GGA TGA-30 (forward) and 50-TGT AGT GGT GGT CGG AGA TT-30 (reverse). The primers for TNF-␣ were 50-GGA GAA GGG TGA CCG ACT CA-30 (forward) and 50-CTG CCC AGA CTC GGC AA-30 (reverse). 2.6. Measurement of reactive oxygen species (ROS) by confocal microscopy Intracellular ROS were measured using the cell-permeable fluorescent dye H2 DCFDA. First, HMO6 cells were treated with 300 nM PAF in the presence or absence of 1 mM GlcN for 1 h. After incubation, wells were loaded with 5 M H2 DCFDA and incubated for 30 min at 37 ◦ C. The cells were then washed twice with Krebs Ringer Bicarbonate buffer to ensure the removal of unbound dye. After washing, the cells were incubated for an additional 10 min. Images were obtained by subjecting the cells to confocal laser microscopy (LSM 5 EXCITER; Carl Zeiss, Jena, Germany) using excitation and emission wavelengths of 488 and 525 nm, respectively. 2.7. Measurement of PGE2 The levels of PGE2 were measured using ELISA kits (Cayman Chemical Co., Ann Arbor, MI) according to the manufacturer’s instructions. Briefly, HMO6 cells were loaded in 24-well plates and treated with 300 nM PAF in the presence or absence of 1 mM GlcN for 1 h. A total of 100 L of culture medium supernatant was collected to determine PGE2 concentration by ELISA. 2.8. Statistical analyses Results were expressed as the mean ± SEM. SPSS version 20.0 (SPSS, Chicago, IL) was used for statistical analyses. The AUC was calculated using Microcal Origin software version 9.1 (Northampton, MA). Comparisons between the two groups were performed using a Student’s two-tailed t test. For comparisons involving more
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than two groups, the significance was tested using ANOVA with Bonferroni correction. Statistical significance was set at p < 0.05. 3. Results 3.1. PAF induces activation of human microglia through [Ca2+ ]i increase To evaluate the effect of PAF on [Ca2+ ]i , HMO6 microglial cells were treated with PAF for 1 min in Ca2+ -containing physiological saline or Ca2+ -free solution. As shown in Fig. 1A, PAF immediately induced transient increases of [Ca2+ ]i and the increase of [Ca2+ ]i was sustained even after removal of PAF from the Ca2+ -containing solution. In the Ca2+ -free solution, PAF immediately induced transient increases of [Ca2+ ]i but sustained increases of [Ca2+ ]i were not observed. After pre-treatment with two different types of ER blockers, U73122 (an inhibitor of phospholipase C-coupled inositol triphosphate formation) and dantrolene-Na+ (an inhibitor of ryanodine receptor), transient increases of [Ca2+ ]i by PAF were largely blocked in the Ca2+ -free solution (Fig. 1B). After pre-treatment with one of the non-specific store-operated Ca2+ channels (SOC) inhibitors, SKF96365 or PK11195, sustained increases of [Ca2+ ]i by PAF were blocked in the Ca2+ -containing solution (Fig. 1C). These results suggest that treatment with PAF causes a biphasic increase of [Ca2+ ]i and that the initial rapid increase by PAF is due to Ca2+ release from ER stores followed by a sustained increase due to Ca2+ entry mediated by SOC. Because an up-regulation of COX-2 expression is considered as a marker for microglial activation (Hong et al., 2006; McLarnon et al., 2005; Sattayaprasert et al., 2005), we next investigated whether PAF induces an up-regulation of COX-2 expression. Treatment with 300 nM PAF effectively increased the expression level of COX-2 (Fig. 1D and E). To confirm the effect of PAF on the activation of HMO6 microglia, we next measured the expression levels of inflammatory cytokines after PAF treatment
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for 4 h. As shown in Fig. 1F, mRNA levels of iNOS, IL-1 and TNF-␣ were increased after PAF treatment. Furthermore, secreted levels of PGE2 were significantly increased after treatment with PAF while PAF-induced PGE2 secretion was significantly inhibited by SOC inhibitors (Fig. 1G). 3.2. GlcN suppresses PAF-induced [Ca2+ ]i increase and activation of microglia We next determined the effect of GlcN on PAF-induced [Ca2+ ]i increase in HMO6 cells. GlcN were treated for 5 min prior to the addition of PAF in Ca2+ -containing solution. As shown in Fig. 2A and B, GlcN suppressed PAF-induced Ca2+ influx from the extracellular space in a dose-dependent manner, whereas GlcN had no effect on transient Ca2+ release by PAF. Because Ca2+ overload can stimulate ROS generation in mitochondria (Adam-Vizi and Starkov, 2010), we measured intracellular ROS levels by using the fluorescent probe H2 DCFDA. We found that PAF effectively stimulated ROS generation, and that GlcN markedly decreased cellular ROS levels in PAF-treated HMO6 cells (Fig. 2D). These data suggest that PAFinduced ROS generation contributed to PAF-induced activation of microglia and that GlcN may inhibit the PAF-induced activation of microglia by inhibiting Ca2+ influx and ROS generation. 3.3. GlcN reduces the PAF-induced pro-inflammatory microglia response To confirm the inhibitory effect of GlcN on the PAF-induced activation of microglia, we investigated the effects of GlcN on PAFinduced mRNA expression of the proinflammatory mediators, iNOS, IL-1 and TNF-␣, using quantitative real-time RT-PCR. GlcN significantly suppressed mRNA levels of iNOS, IL-1 and TNF-␣ (Fig. 3A) in PAF-treated HMO6 cells. Stimulating the cells with PAF also resulted in a significant increase in PGE2 production; however,
Fig. 1. Effect of platelet-activating factor (PAF) on the activation of HMO6 microglia. (A) Changes in [Ca2+ ]i were measured after treatment with 300 nM PAF for 1 min in Ca2+ -containing or Ca2+ -free solution. (B) Changes in [Ca2+ ]i were measured after treatment with PAF in Ca2+ -free solution containing 10 M dantrolene-Na+ and 2 M U73122. (C) Changes in [Ca2+ ]i were measured after treatment with PAF in Ca2+ -containing solution containing 20 M PK11195 or 50 M SKF96365. The values represent the mean ± SEM of three independent experiments (n = 25–30 cells). (D) HMO6 cells were treated with different concentrations of PAF for 4 h. (E) HMO6 cells were treated with 300 nM PAF for the indicated times. The expression level of COX-2 was detected by Western blot analysis of whole cell lysates. (F) The expression levels of iNOS, IL-1 and TNF-␣ were determined using quantitative real-time PCR analysis in HMO6 cells treated with PAF for 4 h. (G) The amount of PGE2 production was determined in HMO6 cells treated with PAF for 4 h. The values represent the mean ± SEM (n = 6). *p < 0.05 vs cells treated with vehicle; # p < 0.05 vs cells treated with PAF alone; NS, not significant.
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Fig. 2. Effects of glucosamine (GlcN) on PAF-induced Ca2+ influx and reactive oxygen species (ROS) generation in HMO6 microglia. (A) Cells were treated with different concentrations of GlcN for 2 min prior to treatment with 300 nM PAF in Ca2+ -containing solution. The values represent the mean ± SEM of three independent experiments (n = 25–30 cells). (B) Area under the curve (AUC) is depicted as the percentage of the values in the presence of PAF alone. The values represent the mean ± SEM. *p < 0.05 and **p < 0.01 vs cells treated with PAF alone. (C) Confocal laser microscopy was used to analyze cellular ROS levels in HMO6 cells. Cells were treated with 300 nM PAF in the presence or absence of 1 mM GlcN for 1 h. Scale bars, 50 m.
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Fig. 3. Effects of GlcN on PAF-induced pro-inflammatory HMO6 microglia response. The cells were treated with 300 nM PAF in the presence or absence of 1 mM GlcN for 4 h. (A) The expression levels of iNOS, IL-1 and TNF-␣ were determined using quantitative real-time PCR analysis. (B) The amount of PGE2 production was determined. The values represent the mean ± SEM (n = 6). *p < 0.05 vs cells treated with vehicle; # p < 0.05 vs cells treated with PAF alone. (C) Confocal laser microscopy was used to analyze morphologic changes in HMO6 cells.
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treatment with GlcN decreased PGE2 production (Fig. 3B). After pre-treatment with one of SOC inhibitors, SKF96365 or PK11195, PAF-induced increase of PGE2 secretion was inhibited (Fig. 3B). As filopodia formation is a hallmark of homeostatic microglia, we next investigated the effect of GlcN on microglia morphology. PAFactivated or control HMO6 cells were cultured in the absence or presence of GlcN, and their morphology was observed. GlcN treatment induced filopodia formation in control microglia and even reversed the amoeboid phenotype observed after PAF activation (Fig. 3C).
3.4. GlcN inhibits PAF-induced degradation of IB-˛ and up-regulation of COX-2 To investigate the effects of GlcN on microglial NFB/COX-2 signaling, we analyzed the protein levels of inhibitor of B-␣ (IB␣) and COX-2. HMO6 microglia cells were treated with 300 nM PAF in the presence or absence of 1 mM GlcN for 4 h. Activation of NFB is closely linked to the proteolytic degradation of the cytosolic protein IB-␣. Western blot analysis showed that treatment with PAF for 30 min led to strong IB-␣ degradation in HMO6 cells, indicating NF-B activation by PAF (Fig. 4A). Treatment with GlcN effectively prevented PAF-induced degradation of IB-␣ (Fig. 4A) and up-regulation of COX-2 (Fig. 4B). To investigate the effects of the SOC inhibitors on the PAF-induced degradation of IB-␣ and upregulation of COX-2, the SOC inhibitor was pretreated for 1 h before PAF application. Each SOC inhibitor also prevented PAF-induced degradation of IB-␣ (Fig. 4A) and up-regulation of COX-2 (Fig. 4B).
4. Discussion PAF is known to induce the activation of microglia through the increase of [Ca2+ ]i (Sattayaprasert et al., 2005; Wang et al., 1999). However, little is known about the mechanism underlying PAF activation of microglia. In the present study, we demonstrated that PAF induced the increase of [Ca2+ ]i through the activation of SOCs, which in turn potentiated intracellular ROS generation in human HMO6 microglial cells. The increased ROS subsequently stimulated NFB/COX-2 signaling, which increased the expression levels of iNOS, IL-1 and TNF-␣, as well as the secreted levels of PGE2 (Fig. 4C). Previous studies have demonstrated that PAF elicits a biphasic response in [Ca2+ ]i consisting of an initial rapid increase of [Ca2+ ]i due to release from ER, followed by a sustained increase of [Ca2+ ]i due to influx from SOCs (Sattayaprasert et al., 2005; Wang et al., 1999). Here, we show that PAF induced a biphasic increase of [Ca2+ ]i in HMO6 cells. In addition, PAF-induced PGE2 secretion and COX-2 up-regulation were significantly inhibited by SOC inhibitors. These results suggest that PAF-induced increase of [Ca2+ ]i due to influx from SOCs was more predominant than PAF-induced increase of [Ca2+ ]i due to release from ER in the stimulatory effect of PAF on the activation of microglia. In the microglial cells, SOC-mediated Ca2+ entry could serve as an important mechanism in mediating cellular inflammatory responses. It is known that excessive [Ca2+ ]i stimulates ROS generation in mitochondria which provides the main source of physiological ROS production (Dichmann et al., 2000; Yan et al., 2006). Here, we demonstrate that ROS production in PAF-treated microglia appear to require Ca2+ influx from extracellular sources. In addition, ROS have been reported to activate NF-B signaling through the modulation of IB kinase or IB-␣ activity (Kabe et al., 2005; Morgan and Liu, 2011). Consistent with these reports, the current study demonstrated that PAF induced IB-␣ degradation in microglia, which could be dependent on the [Ca2+ ]i –ROS signaling pathway.
Fig. 4. Effects of GlcN on IB-␣ and COX-2 expressions in PAF-treated HMO6 microglia cells. The cells were treated with 300 nM PAF in the presence or absence of 1 mM GlcN for 4 h. The expression levels of IB-␣ (A) and COX-2 (B) were detected by Western blot analysis of whole cell lysates. (C) Proposed mechanism to explain the effects of exogenous GlcN in endogenous PAF-induced pro-inflammatory microglia response. ER, endoplasmic reticulum; Mito, mitochondria; SOC, store operated Ca2+ channels; IP3 R, IP3 receptors; RYR, ryanodine receptors.
In inflammation, activated microglia have a change in morphology and release various cytotoxic mediators, such as NO, TNF-␣, IL-1, and PGE2 . Overproduction of these mediators results in neuronal death. NFB signaling is a major pathway in the activated microglia that may result in chronic neuroinflammation (Mattson and Camandola, 2001). Activation of NF-B is induced by the phosphorylation and subsequent degradation of IB. And then activated NF-B subsequently moves in the nucleus where it promotes the expression of proinflammatory cytokines, COX-2 and iNOS (Lee, 2013). Our results show that PAF significantly induces the production of proinflammatory cytokines, COX-2 and iNOS through the [Ca2+ ]i –ROS signaling pathway in microglial cells.
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GlcN is known to exert a neuroprotective effect by inducing microglial inactivation (Hwang et al., 2010, 2013; Yi et al., 2005). However, little is known about the effect of GlcN on endogenous PAF-induced activation in microglial cells. In the present study, GlcN inhibited the PAF-induced Ca2+ influx from the extracellular space in a dose-dependent manner, but did not elicit any change in the initial [Ca2+ ]i release from ER. GlcN-induced inhibition of the SOC-mediated Ca2+ entry led to microglial inactivation in PAF-treated microglial cells (Fig. 4C). GlcN was reported to inhibit SOC activity in rat cardiomyocytes (Hunton et al., 2004) and macrophages (Vemuri and Marchase, 1999). Consistent with these reports, the current study demonstrated that GlcN has a strong inhibitory action on the SOC-mediated Ca2+ entry in microglial cells. GlcN might blunt SOC-mediated Ca2+ entry directly through protein modifications. Treatment with GlcN increases hexosamine biosynthesis pathway and leads to increased O-GlcNAcylation levels. And O-GlcNAc is an intracellular carbohydrate that dynamically modifies proteins in the nucleus and cytoplasm on the serine and threonine residues (Hart et al., 2011). Stromal interaction molecule-1 (STIM1) and Orai1 calcium channel are the primary mediators of store-operated Ca2+ entry. And STIM1 as an ER Ca2+ sensor is a key player in regulating the SOC activity (Prakriya and Lewis, 2015). Furthermore, STIM1 is a target for O-GlcNAc modification and that this is associated with impaired STIM1 function and blunted the SOC-mediated Ca2+ entry (Zhu-Mauldin et al., 2012). Activation of O-GlcNAcylation by the treatment with GlcN might attenuate the SOC-mediated Ca2+ entry via STIM1 O-GlcNAcylation in microglial cells. However, it remains unclear whether GlcN interferes directly with PAF-induced IB-␣ degradation (Shin et al., 2013) and COX-2 N-glycosylation (Jang et al., 2007) in HMO6 cells. 5. Conclusion In this study, we have shown that PAF is an endogenous pro-inflammatory phospholipid of microglial cells that acts in a paracrine fashion. PAF increased SOC-mediated Ca2+ influx in HMO6 microglial cells. The excess [Ca2+ ]i induced the generation of intracellular ROS and the activation of NFB/COX-2 signaling, which increased the expression levels of iNOS, IL-1 and TNF-␣, and the secreted levels of PGE2 . We also have provided evidence to show that GlcN produces anti-inflammatory effects on PAF-activated microglia by inhibiting SOC-mediated Ca2+ influx. Furthermore, GlcN inhibits pro-inflammatory gene expression, reduces microglial neurotoxicity, and promotes filopodia formation. In conclusion, our data suggest that GlcN has a neuroprotective effect in microglia and that GlcN may be a potential therapeutic to inhibit detrimental microglial phenotypes in neurodegenerative diseases of the brain. Conflict of interest The authors declare that there are no conflicts of interest associated with this manuscript. Transparency document The Transparency document associated with this article can be found in the online version. Acknowledgements This study was financially supported by a National Research Foundation (NRF) Grant funded by the Korean Government (MSIP) (2014R1A5A2010008).
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Environmental Toxicology and Pharmacology journal homepage: www.elsevier.com/locate/etap
Glucosamine suppresses platelet-activating factor-induced activation of microglia through inhibition of store-operated calcium influx Jae-Hyung Park a,1 , Jeong-Nam Kim a,1 , Byeong-Churl Jang b , Seung-Soon Im a , Dae-Kyu Song a , Jae-Hoon Bae a,∗ a b
Department of Physiology, Keimyung University School of Medicine, Daegu, South Korea Department of Molecular Medicine, Keimyung University School of Medicine, Daegu, South Korea
a r t i c l e
i n f o
Article history: Received 31 August 2015 Received in revised form 17 December 2015 Accepted 19 December 2015 Available online 23 December 2015 Keywords: Glucosamine Platelet-activating factor Microglia Intracellular calcium concentration Store-operated calcium channel
a b s t r a c t Microglia activation and subsequent release of inflammatory mediators are implicated in the pathophysiology of neurodegenerative diseases. Platelet-activating factor (PAF), a potent lipid mediator synthesized by microglia, is known to stimulate microglia functional responses. In this study, we determined that endogenous PAF exert autocrine effects on microglia activation, as well as the underlying mechanism involved. We also investigated the effect of d-glucosamine (GlcN) on PAF-induced cellular activation in human HMO6 microglial cells. PAF induced sustained intracellular Ca2+ ([Ca2+ ]i ) increase through store-operated Ca2+ channels (SOC) and reactive oxygen species (ROS) generation. PAF also induced proinflammatory markers through NFB/COX-2 signaling. GlcN significantly inhibited PAF-induced Ca2+ influx and ROS generation without significant cytotoxicity. GlcN downregulated excessive expression of pro-inflammatory markers and promoted filopodia formation through NFB/COX-2 inhibition in PAFstimulated HMO6 cells. Taken together, these data suggest that GlcN may offer substantial therapeutic potential for treating inflammatory and neurodegenerative diseases accompanied by microglial activation. © 2015 Elsevier B.V. All rights reserved.
1. Introduction In the central nervous system (CNS), microglial cells are the resident macrophages. That have a key part in the regulation of innate immune responses in the healthy or degenerating CNS (Khoo et al., 2001). While actively scanning the microenvironment with their long protrusions (Nimmerjahn et al., 2005), loss of inhibitory signals and the recognition of damage-associated molecular patterns from degenerating neurons lead to the activation of microglia (Cardona et al., 2006; Heneka et al., 2014). When microglial cells are activated, the cells exhibit proliferation and phagocytosis. Moreover, the activated cells can secrete pro- and anti-inflammatory
Abbreviations: CNS, central nervous system; [Ca2+ ]i , intracellular Ca2+ ; COX-2, cyglooxygenase-2; GlcN, d-glucosamine; IB-␣, inhibitor of B-␣; iNOS, inducible nitric oxide synthase; IL-1, interleukin-1; PAF, platelet-activating factor; PGE2 , prostaglandin E2 ; ROS, reactive oxygen species; SOC, store-operated Ca2+ channels; TNF-␣, tumor necrosis factor-␣. ∗ Corresponding author at: Department of Physiology, Keimyung University School of Medicine, 1095 Dalgubeol-Daero, Dalseo-Gu, Daegu 704-701, South Korea. E-mail address: [email protected] (J.-H. Bae). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.etap.2015.12.014 1382-6689/© 2015 Elsevier B.V. All rights reserved.
cytokines, chemokines, and neurotrophic factors (Heneka et al., 2014). Therefore, reactive microgliosis is a common hallmark of various neurodegenerative diseases including Alzheimer’s disease (Perry et al., 2010), Parkinson’s disease (Orr et al., 2002), and multiple sclerosis (Raivich and Banati, 2004). Platelet-activating factor (PAF) is a potent proinflammatory phospholipid with diverse pathological and physiological effects (Yue and Feuerstein, 1994). PAF is produced by a variety of cells, but especially those involved in host defense, such as platelets, endothelial cells, neutrophils, monocytes, and macrophages (Zimmerman et al., 2002). In the brain, this endogenous phospholipid is produced by microglia in response to ischemic injury or various neurodegenerative diseases (Bate et al., 2006; Prescott et al., 2000; Tuttolomondo et al., 2008). Moreover, PAF stimulates microglia itself (Mori et al., 1996) and induces arachidonic acid metabolism, resulting in the production of neurotoxic factors. It also mediates pathological inflammation in the brain (Mori et al., 1996). It has been reported that PAF immediately induced an increase in intracellular Ca2+ ([Ca2+ ]i ) levels in human microglial cells (Khoo et al., 2001; Sattayaprasert et al., 2005; Wang et al., 1999). These prolonged increase of [Ca2+ ]i by PAF treatment can increase the expression of inflammatory factors, including
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cyglooxygenase-2 (COX-2), tumor necrosis factor-␣ (TNF-␣), interleukin-1 (IL-1), inducible nitric oxide synthase (iNOS) and prostaglandin E2 (PGE2 ) (Choi et al., 2002; Hoffmann et al., 2003). d-Glucosamine (GlcN) is a dietary supplement widely used for the prevention and/or treatment of rheumatoid arthritis and osteoarthritis (de los Reyes et al., 2000). GlcN can be distributed to the brain at relevant quantities 8 h after oral administration (Setnikar et al., 1984). Our previous study demonstrated that GlcN inhibits exogenous lipopolysaccharide-induced microglia activation (Yi et al., 2005), thus implicating this agent as part of a new inhibitory strategy in targeting activated microglial cells in the CNS. However, the effect of GlcN on endogenous PAF-induced activation of microglia remains largely unknown. Therefore, the aim of the present study was to investigate the effects of GlcN on endogenous PAF-induced increase in [Ca2+ ]i and expression of inflammatory factors in HMO6 microglial cells and to evaluate the inhibitory mechanisms involved. 2. Materials and methods 2.1. Materials FBS and DMEM medium were purchased from Life Technologies (Carlsbad, CA). GlcN was from Sigma–Aldrich (St. Louis, MO). PAF, PK11195 and SKF96365 were from Tocris Bioscience (Bristol, UK). Fura-2 acetoxymethyl (fura-2/AM) ester was obtained from Molecular Probes (Eugene, OR). All other chemicals were obtained from Sigma–Aldrich. 2.2. Cell culture Human microglial cells (HMO6) were a gift from S.U. Kim (University of British Columbia, Vancouver, Canada). HMO6 cells were established as immortalized clonal cells of human microglia from human embryonic telencephalon tissue by using a retroviral vector encoding v-myc and were investigated by immunochemistry and fluorescence-activated cell sorting analyses (Nagai et al., 2005). HMO6 cells were cultured at 37 ◦ C in a humidified incubator with 5% CO2 in DMEM medium containing 5% heat-inactivated FBS, 5% heat-inactivated horse serum and 20 g/mL gentamycin. 2.3. Measurement of intracellular calcium concentration ([Ca2+ ]i ) Microfluorescent imaging of [Ca2+ ]i was performed on HMO6 cells loaded with the calcium indicator dye fura-2/AM. Some of the procedures used in calcium imaging in this experiment have been described (Yi et al., 2005). Fura-2/AM (3 M) was added to HMO6 cells bathed in 1.8 mM Ca2+ containing physiological saline solution (126 mM NaCl, 5 mM KCl, 1.8 mM CaCl2 , 1.2 mM MgCl2 , 10 mM HEPES [pH 7.4], 0.2% BSA and 10 mM glucose) at room temperature for 30 min followed by a 30-min wash in dye-free saline solution to allow esterase conversion to the free form of fura-2. Cover slips were placed on the stage of an inverted microscope and imaging was performed with a dual-wavelength system (Intracellular Imaging, Cincinnati, OH). [Ca2+ ]i was calculated as the relationship between the ratio of emissions at 510 nm from excitation at 340 and 380 nm, respectively. Ratio images were processed every 5 s and converted to [Ca2+ ]i as compared to a range of such ratios obtained by measurement of fura-2 in the presence of known concentrations of calcium (Calcium Calibration Buffer Kit, Molecular Probe, Eugene, OR). When Ca2+ -free solution was used, Ca2+ was omitted and 2 mM EGTA was added. After the establishment of a stable baseline [Ca2+ ]i level, the cells were pre-treated with or without 1 mM GlcN for 2 min prior to stimulation with 300 nM PAF for 1 min. Each experimental data point represents the mean [Ca2+ ]i calculated from at least 12 individually measured cells from three separate
cultures. All imaging experiments were done at room temperature (20–22 ◦ C). 2.4. Western blot analysis Cells were treated with 300 nM PAF in the presence or absence of 1 mM GlcN for 4 h. Crude cell extracts were subjected to SDSPAGE and immunoblotted with anti-COX-2 (Abcam, Cambridge, UK), anti-Ib-␣ (Cell Signaling, Danvers, MA) and anti- actin (Sigma–Aldrich) antibodies. The immunoreactive bands were visualized using a horseradish peroxidase-conjugated secondary antibody (Abcam) and enhanced chemiluminescence (Amersham Biosciences, Little Chalfont, UK). The experiments were repeated at least three times. 2.5. Quantitative real-time PCR analysis After exposure of cells to 300 nM PAF in the presence or absence of 1 mM GlcN for 4 h, total cellular RNA was extracted from HMO6 cells using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. The RNA was eluted with Rnase-free water. Reverse transcription was performed using the first strand cDNA synthesis kit (Fermentas, Glen Burnie, MD) according to the manufacturer’s protocols. Real-time PCR amplification was performed using the SYBR Green master mix (Applied Biosystems, Foster City, CA) and the Prism 7500 real-time PCR detection system. Relative amounts of mRNA were normalized by use of the gene encoding glyceraldehyde 3-phosphate dehydrogenase and calculated using the CT (cycle threshold) method. The specific primers for iNOS were 50-GTT CTC AAG GCA CAG GTC TC-30 (forward) and 50-GCA GGT CAC TTA TGT CAC TTA TC-30 (reverse). The primers for IL-1 were 50-TTA CAG TGG CAA TGA GGA TGA-30 (forward) and 50-TGT AGT GGT GGT CGG AGA TT-30 (reverse). The primers for TNF-␣ were 50-GGA GAA GGG TGA CCG ACT CA-30 (forward) and 50-CTG CCC AGA CTC GGC AA-30 (reverse). 2.6. Measurement of reactive oxygen species (ROS) by confocal microscopy Intracellular ROS were measured using the cell-permeable fluorescent dye H2 DCFDA. First, HMO6 cells were treated with 300 nM PAF in the presence or absence of 1 mM GlcN for 1 h. After incubation, wells were loaded with 5 M H2 DCFDA and incubated for 30 min at 37 ◦ C. The cells were then washed twice with Krebs Ringer Bicarbonate buffer to ensure the removal of unbound dye. After washing, the cells were incubated for an additional 10 min. Images were obtained by subjecting the cells to confocal laser microscopy (LSM 5 EXCITER; Carl Zeiss, Jena, Germany) using excitation and emission wavelengths of 488 and 525 nm, respectively. 2.7. Measurement of PGE2 The levels of PGE2 were measured using ELISA kits (Cayman Chemical Co., Ann Arbor, MI) according to the manufacturer’s instructions. Briefly, HMO6 cells were loaded in 24-well plates and treated with 300 nM PAF in the presence or absence of 1 mM GlcN for 1 h. A total of 100 L of culture medium supernatant was collected to determine PGE2 concentration by ELISA. 2.8. Statistical analyses Results were expressed as the mean ± SEM. SPSS version 20.0 (SPSS, Chicago, IL) was used for statistical analyses. The AUC was calculated using Microcal Origin software version 9.1 (Northampton, MA). Comparisons between the two groups were performed using a Student’s two-tailed t test. For comparisons involving more
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than two groups, the significance was tested using ANOVA with Bonferroni correction. Statistical significance was set at p < 0.05. 3. Results 3.1. PAF induces activation of human microglia through [Ca2+ ]i increase To evaluate the effect of PAF on [Ca2+ ]i , HMO6 microglial cells were treated with PAF for 1 min in Ca2+ -containing physiological saline or Ca2+ -free solution. As shown in Fig. 1A, PAF immediately induced transient increases of [Ca2+ ]i and the increase of [Ca2+ ]i was sustained even after removal of PAF from the Ca2+ -containing solution. In the Ca2+ -free solution, PAF immediately induced transient increases of [Ca2+ ]i but sustained increases of [Ca2+ ]i were not observed. After pre-treatment with two different types of ER blockers, U73122 (an inhibitor of phospholipase C-coupled inositol triphosphate formation) and dantrolene-Na+ (an inhibitor of ryanodine receptor), transient increases of [Ca2+ ]i by PAF were largely blocked in the Ca2+ -free solution (Fig. 1B). After pre-treatment with one of the non-specific store-operated Ca2+ channels (SOC) inhibitors, SKF96365 or PK11195, sustained increases of [Ca2+ ]i by PAF were blocked in the Ca2+ -containing solution (Fig. 1C). These results suggest that treatment with PAF causes a biphasic increase of [Ca2+ ]i and that the initial rapid increase by PAF is due to Ca2+ release from ER stores followed by a sustained increase due to Ca2+ entry mediated by SOC. Because an up-regulation of COX-2 expression is considered as a marker for microglial activation (Hong et al., 2006; McLarnon et al., 2005; Sattayaprasert et al., 2005), we next investigated whether PAF induces an up-regulation of COX-2 expression. Treatment with 300 nM PAF effectively increased the expression level of COX-2 (Fig. 1D and E). To confirm the effect of PAF on the activation of HMO6 microglia, we next measured the expression levels of inflammatory cytokines after PAF treatment
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for 4 h. As shown in Fig. 1F, mRNA levels of iNOS, IL-1 and TNF-␣ were increased after PAF treatment. Furthermore, secreted levels of PGE2 were significantly increased after treatment with PAF while PAF-induced PGE2 secretion was significantly inhibited by SOC inhibitors (Fig. 1G). 3.2. GlcN suppresses PAF-induced [Ca2+ ]i increase and activation of microglia We next determined the effect of GlcN on PAF-induced [Ca2+ ]i increase in HMO6 cells. GlcN were treated for 5 min prior to the addition of PAF in Ca2+ -containing solution. As shown in Fig. 2A and B, GlcN suppressed PAF-induced Ca2+ influx from the extracellular space in a dose-dependent manner, whereas GlcN had no effect on transient Ca2+ release by PAF. Because Ca2+ overload can stimulate ROS generation in mitochondria (Adam-Vizi and Starkov, 2010), we measured intracellular ROS levels by using the fluorescent probe H2 DCFDA. We found that PAF effectively stimulated ROS generation, and that GlcN markedly decreased cellular ROS levels in PAF-treated HMO6 cells (Fig. 2D). These data suggest that PAFinduced ROS generation contributed to PAF-induced activation of microglia and that GlcN may inhibit the PAF-induced activation of microglia by inhibiting Ca2+ influx and ROS generation. 3.3. GlcN reduces the PAF-induced pro-inflammatory microglia response To confirm the inhibitory effect of GlcN on the PAF-induced activation of microglia, we investigated the effects of GlcN on PAFinduced mRNA expression of the proinflammatory mediators, iNOS, IL-1 and TNF-␣, using quantitative real-time RT-PCR. GlcN significantly suppressed mRNA levels of iNOS, IL-1 and TNF-␣ (Fig. 3A) in PAF-treated HMO6 cells. Stimulating the cells with PAF also resulted in a significant increase in PGE2 production; however,
Fig. 1. Effect of platelet-activating factor (PAF) on the activation of HMO6 microglia. (A) Changes in [Ca2+ ]i were measured after treatment with 300 nM PAF for 1 min in Ca2+ -containing or Ca2+ -free solution. (B) Changes in [Ca2+ ]i were measured after treatment with PAF in Ca2+ -free solution containing 10 M dantrolene-Na+ and 2 M U73122. (C) Changes in [Ca2+ ]i were measured after treatment with PAF in Ca2+ -containing solution containing 20 M PK11195 or 50 M SKF96365. The values represent the mean ± SEM of three independent experiments (n = 25–30 cells). (D) HMO6 cells were treated with different concentrations of PAF for 4 h. (E) HMO6 cells were treated with 300 nM PAF for the indicated times. The expression level of COX-2 was detected by Western blot analysis of whole cell lysates. (F) The expression levels of iNOS, IL-1 and TNF-␣ were determined using quantitative real-time PCR analysis in HMO6 cells treated with PAF for 4 h. (G) The amount of PGE2 production was determined in HMO6 cells treated with PAF for 4 h. The values represent the mean ± SEM (n = 6). *p < 0.05 vs cells treated with vehicle; # p < 0.05 vs cells treated with PAF alone; NS, not significant.
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Fig. 2. Effects of glucosamine (GlcN) on PAF-induced Ca2+ influx and reactive oxygen species (ROS) generation in HMO6 microglia. (A) Cells were treated with different concentrations of GlcN for 2 min prior to treatment with 300 nM PAF in Ca2+ -containing solution. The values represent the mean ± SEM of three independent experiments (n = 25–30 cells). (B) Area under the curve (AUC) is depicted as the percentage of the values in the presence of PAF alone. The values represent the mean ± SEM. *p < 0.05 and **p < 0.01 vs cells treated with PAF alone. (C) Confocal laser microscopy was used to analyze cellular ROS levels in HMO6 cells. Cells were treated with 300 nM PAF in the presence or absence of 1 mM GlcN for 1 h. Scale bars, 50 m.
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Fig. 3. Effects of GlcN on PAF-induced pro-inflammatory HMO6 microglia response. The cells were treated with 300 nM PAF in the presence or absence of 1 mM GlcN for 4 h. (A) The expression levels of iNOS, IL-1 and TNF-␣ were determined using quantitative real-time PCR analysis. (B) The amount of PGE2 production was determined. The values represent the mean ± SEM (n = 6). *p < 0.05 vs cells treated with vehicle; # p < 0.05 vs cells treated with PAF alone. (C) Confocal laser microscopy was used to analyze morphologic changes in HMO6 cells.
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treatment with GlcN decreased PGE2 production (Fig. 3B). After pre-treatment with one of SOC inhibitors, SKF96365 or PK11195, PAF-induced increase of PGE2 secretion was inhibited (Fig. 3B). As filopodia formation is a hallmark of homeostatic microglia, we next investigated the effect of GlcN on microglia morphology. PAFactivated or control HMO6 cells were cultured in the absence or presence of GlcN, and their morphology was observed. GlcN treatment induced filopodia formation in control microglia and even reversed the amoeboid phenotype observed after PAF activation (Fig. 3C).
3.4. GlcN inhibits PAF-induced degradation of IB-˛ and up-regulation of COX-2 To investigate the effects of GlcN on microglial NFB/COX-2 signaling, we analyzed the protein levels of inhibitor of B-␣ (IB␣) and COX-2. HMO6 microglia cells were treated with 300 nM PAF in the presence or absence of 1 mM GlcN for 4 h. Activation of NFB is closely linked to the proteolytic degradation of the cytosolic protein IB-␣. Western blot analysis showed that treatment with PAF for 30 min led to strong IB-␣ degradation in HMO6 cells, indicating NF-B activation by PAF (Fig. 4A). Treatment with GlcN effectively prevented PAF-induced degradation of IB-␣ (Fig. 4A) and up-regulation of COX-2 (Fig. 4B). To investigate the effects of the SOC inhibitors on the PAF-induced degradation of IB-␣ and upregulation of COX-2, the SOC inhibitor was pretreated for 1 h before PAF application. Each SOC inhibitor also prevented PAF-induced degradation of IB-␣ (Fig. 4A) and up-regulation of COX-2 (Fig. 4B).
4. Discussion PAF is known to induce the activation of microglia through the increase of [Ca2+ ]i (Sattayaprasert et al., 2005; Wang et al., 1999). However, little is known about the mechanism underlying PAF activation of microglia. In the present study, we demonstrated that PAF induced the increase of [Ca2+ ]i through the activation of SOCs, which in turn potentiated intracellular ROS generation in human HMO6 microglial cells. The increased ROS subsequently stimulated NFB/COX-2 signaling, which increased the expression levels of iNOS, IL-1 and TNF-␣, as well as the secreted levels of PGE2 (Fig. 4C). Previous studies have demonstrated that PAF elicits a biphasic response in [Ca2+ ]i consisting of an initial rapid increase of [Ca2+ ]i due to release from ER, followed by a sustained increase of [Ca2+ ]i due to influx from SOCs (Sattayaprasert et al., 2005; Wang et al., 1999). Here, we show that PAF induced a biphasic increase of [Ca2+ ]i in HMO6 cells. In addition, PAF-induced PGE2 secretion and COX-2 up-regulation were significantly inhibited by SOC inhibitors. These results suggest that PAF-induced increase of [Ca2+ ]i due to influx from SOCs was more predominant than PAF-induced increase of [Ca2+ ]i due to release from ER in the stimulatory effect of PAF on the activation of microglia. In the microglial cells, SOC-mediated Ca2+ entry could serve as an important mechanism in mediating cellular inflammatory responses. It is known that excessive [Ca2+ ]i stimulates ROS generation in mitochondria which provides the main source of physiological ROS production (Dichmann et al., 2000; Yan et al., 2006). Here, we demonstrate that ROS production in PAF-treated microglia appear to require Ca2+ influx from extracellular sources. In addition, ROS have been reported to activate NF-B signaling through the modulation of IB kinase or IB-␣ activity (Kabe et al., 2005; Morgan and Liu, 2011). Consistent with these reports, the current study demonstrated that PAF induced IB-␣ degradation in microglia, which could be dependent on the [Ca2+ ]i –ROS signaling pathway.
Fig. 4. Effects of GlcN on IB-␣ and COX-2 expressions in PAF-treated HMO6 microglia cells. The cells were treated with 300 nM PAF in the presence or absence of 1 mM GlcN for 4 h. The expression levels of IB-␣ (A) and COX-2 (B) were detected by Western blot analysis of whole cell lysates. (C) Proposed mechanism to explain the effects of exogenous GlcN in endogenous PAF-induced pro-inflammatory microglia response. ER, endoplasmic reticulum; Mito, mitochondria; SOC, store operated Ca2+ channels; IP3 R, IP3 receptors; RYR, ryanodine receptors.
In inflammation, activated microglia have a change in morphology and release various cytotoxic mediators, such as NO, TNF-␣, IL-1, and PGE2 . Overproduction of these mediators results in neuronal death. NFB signaling is a major pathway in the activated microglia that may result in chronic neuroinflammation (Mattson and Camandola, 2001). Activation of NF-B is induced by the phosphorylation and subsequent degradation of IB. And then activated NF-B subsequently moves in the nucleus where it promotes the expression of proinflammatory cytokines, COX-2 and iNOS (Lee, 2013). Our results show that PAF significantly induces the production of proinflammatory cytokines, COX-2 and iNOS through the [Ca2+ ]i –ROS signaling pathway in microglial cells.
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GlcN is known to exert a neuroprotective effect by inducing microglial inactivation (Hwang et al., 2010, 2013; Yi et al., 2005). However, little is known about the effect of GlcN on endogenous PAF-induced activation in microglial cells. In the present study, GlcN inhibited the PAF-induced Ca2+ influx from the extracellular space in a dose-dependent manner, but did not elicit any change in the initial [Ca2+ ]i release from ER. GlcN-induced inhibition of the SOC-mediated Ca2+ entry led to microglial inactivation in PAF-treated microglial cells (Fig. 4C). GlcN was reported to inhibit SOC activity in rat cardiomyocytes (Hunton et al., 2004) and macrophages (Vemuri and Marchase, 1999). Consistent with these reports, the current study demonstrated that GlcN has a strong inhibitory action on the SOC-mediated Ca2+ entry in microglial cells. GlcN might blunt SOC-mediated Ca2+ entry directly through protein modifications. Treatment with GlcN increases hexosamine biosynthesis pathway and leads to increased O-GlcNAcylation levels. And O-GlcNAc is an intracellular carbohydrate that dynamically modifies proteins in the nucleus and cytoplasm on the serine and threonine residues (Hart et al., 2011). Stromal interaction molecule-1 (STIM1) and Orai1 calcium channel are the primary mediators of store-operated Ca2+ entry. And STIM1 as an ER Ca2+ sensor is a key player in regulating the SOC activity (Prakriya and Lewis, 2015). Furthermore, STIM1 is a target for O-GlcNAc modification and that this is associated with impaired STIM1 function and blunted the SOC-mediated Ca2+ entry (Zhu-Mauldin et al., 2012). Activation of O-GlcNAcylation by the treatment with GlcN might attenuate the SOC-mediated Ca2+ entry via STIM1 O-GlcNAcylation in microglial cells. However, it remains unclear whether GlcN interferes directly with PAF-induced IB-␣ degradation (Shin et al., 2013) and COX-2 N-glycosylation (Jang et al., 2007) in HMO6 cells. 5. Conclusion In this study, we have shown that PAF is an endogenous pro-inflammatory phospholipid of microglial cells that acts in a paracrine fashion. PAF increased SOC-mediated Ca2+ influx in HMO6 microglial cells. The excess [Ca2+ ]i induced the generation of intracellular ROS and the activation of NFB/COX-2 signaling, which increased the expression levels of iNOS, IL-1 and TNF-␣, and the secreted levels of PGE2 . We also have provided evidence to show that GlcN produces anti-inflammatory effects on PAF-activated microglia by inhibiting SOC-mediated Ca2+ influx. Furthermore, GlcN inhibits pro-inflammatory gene expression, reduces microglial neurotoxicity, and promotes filopodia formation. In conclusion, our data suggest that GlcN has a neuroprotective effect in microglia and that GlcN may be a potential therapeutic to inhibit detrimental microglial phenotypes in neurodegenerative diseases of the brain. Conflict of interest The authors declare that there are no conflicts of interest associated with this manuscript. Transparency document The Transparency document associated with this article can be found in the online version. Acknowledgements This study was financially supported by a National Research Foundation (NRF) Grant funded by the Korean Government (MSIP) (2014R1A5A2010008).
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