Spleen tyrosine kinase-dependent Nrf2 activation regulates oxidative stress-induced cell death in WiL2-NS human B lymphoblasts

Sojin Park, Ju-Won Jang & Eun-Yi Moon

To cite this article: Sojin Park, Ju-Won Jang & Eun-Yi Moon (2018): Spleen tyrosine kinase- dependent Nrf2 activation regulates oxidative stress-induced cell death in WiL2-NS human B lymphoblasts, Free Radical Research, DOI: 10.1080/10715762.2018.1505044
To link to this article: https://doi.org/10.1080/10715762.2018.1505044

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https://doi.org/10.1080/10715762.2018.1505044

ORIGINAL ARTICLE Image
Spleen tyrosine kinase-dependent Nrf2 activation regulates oxidative stress-induced cell death in WiL2-NS human B lymphoblasts
Sojin Park, Ju-Won Jang and Eun-Yi Moon
Department of Bioscience and Biotechnology, Sejong University, Seoul, Republic of Korea

ABSTRACT
Autoimmune rheumatic lesions are often characterised by the immune cell recruitment including B lymphocytes and the presence of reactive oxygen species (ROS), which increase antioxidant gene transcription via nuclear factor (erythroid-derived 2)-like 2 (Nrf2). Spleen tyrosine kinase (Syk) has a major role in the signal transmission of all haematopoietic lineage cells including B/T cells, mast cells, and macrophages. In this study, we investigated whether B cell survival is regu- lated by Nrf2 via ROS-mediated Syk activation in WiL2-NS human B lymphoblast cells. When WiL2-NS cells were incubated with 1% foetal bovine serum (FBS), the survival rate and mitochon- drial membrane potential (MMP) were reduced. In addition, 1% FBS increased caspase 3 activity, cytochrome C release, nuclear localisation of Nrf2, and ROS production. N-acetylcysteine attenu- ated ROS production and nuclear translocation of Nrf2. It also inhibited cell death, caspase 3 acti- vation, MMP collapse, and cytochrome C release. Results from the 1% FBS treatment were consistent with those of H2O2 treatment. Syk phosphorylation at tyrosine 525/526 was increased by incubation with 1% FBS or treatment with 100 mM H2O2. Nuclear translocation of Nrf2 by H2O2 was inhibited by treatment with BAY61-3606, a Syk inhibitor. BAY61-3606 also promoted MMP collapse, cytochrome C release, caspase 3 activation, and cell death. Taken together, these results implicate that Syk controls oxidative stress-induced human B cell death via nuclear trans- location of Nrf2 and MMP collapse. These results suggest that Syk is a novel regulator of Nrf2 activation.
ARTICLE HISTORY
Received 3 March 2018
Revised 11 July 2018
Accepted 24 July 2018

KEYWORDS
B cell; MMP; nuclear factor (erythroid-derived 2)-like 2 (Nrf2); ROS; spleen tyrosine kinase (Syk)

Introduction
At most disease sites, the inflammatory response pro- motes increased levels of reactive oxygen species (ROS) [1–3]. Higher ROS levels cause tissue damage and col- lapse of mitochondrial membrane potential (MMP), which is crucial for ATP production [4] and cell survival [5]. Together, these processes can ultimately lead to cel- lular apoptosis or necrosis [5,6].
Nuclear factor erythroid 2-like 2 (Nrf2) is a master transcription factor involved in antioxidant and detoxifi- cation responses [7,8]. Nrf2 contributes to cytoprotec- tion against exogenous and endogenous insults including xenobiotics, oxidative stress, hyperoxia, nitro- sative stress, and endoplasmic reticulum (ER) stress [9–12]. Under homeostatic conditions, Kelch-like ECH- associated protein 1 (Keap1) binds to the Nrf2 protein and induces proteasome-mediated degradation of Nrf2. When cells undergo stress, Nrf2 dissociates from Keap1 and translocate into the nucleus [13,14]. Nrf2 then binds to the antioxidant responsive element (ARE), acis-acting regulatory element in genes that express cytoprotective or antioxidant enzymes such as heme- oxygenase-1, NAD(P)H quinone oxidoreductase, peroxir- edoxin 1, superoxide dismutase-2, and glutamate cyst- eine ligases [12,13].
It has been reported that Nrf2 inhibits apoptosis in osteoarthritic chondrocytes [15], Fas-induced apoptosis [16], nitric oxide-induced apoptosis [17], and cell death in an in vitro model of ischemia/reperfusion injury [18]. Nrf2 protects mitochondria from oxidant injury via dir- ect interactions with mitochondria. Overexpression of Nrf2 prevented a decrease in mitochondrial metabolism after hydrogen peroxide (H2O2) exposure [19]. The Nrf2/ ARE pathway is of great interest as an attractive drug target for the pharmacological control of the oxidative and apoptotic response in different cell types and organ-specific diseases [20,21]. The oncogenic functions of Nrf2 make it a potential powerful therapeutic target in cancer treatment [9]. Nrf2 modulates immune responses in numerous rodent models of inflammation, but its effects on human immune cells are not well

CONTACT Eun-Yi Moon Image [email protected] ImageDepartment of Bioscience and Biotechnology, Sejong University, 98 Kunja-Dong Kwangjin-Gu, Seoul 143-747, Republic of Korea
© 2018 Informa UK Limited, trading as Taylor & Francis Group
characterised. Activation of Nrf2 by tert-butylhydroqui- none inhibits murine CD4(þ) T-cell differentiation and activation of primary human CD4 T cells [22,23]. In add-ition, Nrf2 regulated the activation and early differenti- ation of B cells in mice, and Nrf2 deficiency impaired IgM secretion by plasma cells [21,24]. With respect to the regulation of B cell survival, however, little is known about the role of ROS-induced Nrf2.
Spleen tyrosine kinase (Syk) is a major signal trans- mitter contributing to B cell survival and differentiation [25,26]. Oxidative stress-induced Syk activation initiates several different pathways, including proapoptotic and survival pathways. The balance between proapoptotic and survival pathways is important for determining the fate of cells exposed to oxidative stress [3]. Syk was demonstrated to protect MCF7/MDA-MB-231 breast cancer and DG75 B-lymphoma cells from oxidative stress- and genotoxic stress-induced apoptosis, respect- ively [27]. Syk-deficient B cells can survive in the periph- eral circulation for an extended period [28]. The survival pathways of Syk-deficient primary mouse B cells are severely defective [29], and this is mediated by Syk- dependent phospholipase C gamma 2, the activation of which is required for inducing apoptosis following oxi- dative stress [3]. However, little is known about the role of ROS-induced Syk on Nrf2 activation in the regulation of human B cell survival.
In this study, we investigated whether ROS-mediated Syk activation regulates the nuclear localisation of Nrf2 and, consequently, B cell survival. Using WiL2-NS human B lymphoblast cells, we demonstrated that Syk inhibitors ameliorate B cell death via enhancement of MMP collapse and inhibition of Nrf2. These results sug- gest that Syk is a novel protein regulating Nrf2 activa- tion in human B cells.

Materials and methods
Reagents
BAY61-3606, Syk inhibitor, was purchased from Adooq bioscience (Irvine, CA, USA). N-acetyl-L-cysteine (NAC), hydrogen peroxide (H2O2) and 3,30-dihexyloxacarbocya-
nine iodide (DiOC6) were purchased from Sigma-Aldrich
(St. Louis, MO). 5,50,6,60-Tetrachloro-1,10,3,30-tetraethyl- benzimidazolocarbocyanine iodide (JC-1) was obtained from Invitrogen (Eugene, OR). 20,70-dichlorofluorescin
diacetate (DCF-DA) was purchased from Molecular Probe (Eugene, Oregon, USA). Ac-DEVD-pNA, caspase 3 substrate, and antibodies to Nrf2 and cytochrome C were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Antibodies to Syk and phospho-Syk (Y525/ 526) were purchased from Cell Signaling Technology
(Berverly, MA). Except indicated, all chemicals were obtained from Sigma-Aldrich.

Cell cultures
WiL2-NS, a human B lymphoblast cells (ATCC, CRL- 8155) were provided from the Korea Research Institute of Bioscience and Biotechnology (KRIBB) cell bank (Daejeon, Korea). Cells were incubated in RPMI 1640 medium (Gibco, Grand Island, NY) with 10% heat-inacti- vated foetal bovine serum (FBS) (Gibco), 2 mM L-glu-
tamine and 100 units/ml of penicillin/streptomycin (Gibco) at 37 ◦C humidified incubator under the 5% CO2 condition. For the induction of cell death, cells were
incubated in the medium with 1% FBS or treated with 100 mM H2O2 for 12 or 24 h.

Trypan blue staining
Cell suspension was mixed with equal volume of 0.4% trypan blue in PBS. Dying or dead cells were stained with blue colour and viable cells were unstained. Each cell was counted by using haemocytometer under light microscope (Olympus, Korea Co, Ltd., Seoul, Republic of Korea).

Measurement of caspase 3 activity
1 × 107 cells were harvested and lysed in 100-ll lysis buffer containing 50 mM 2-[4-(2-hydroxyethyl)pipera- zine-1-yl]ethanesulfonic acid (HEPES, pH 7.4), 5 mM (3-
[(3-cholamidopropyl)dimethylammonio]-1-propanesul- fonate (CHAPS), and 5 mM dithiothreitol (DTT), for 20 min on ice. Then, cell lysates were centrifuged by 16,000 × g for 10 min at 4 ◦C. After centrifugation,
supernatants were transferred into new tube. Each 5-ll
supernatant sample was incubated with 200 mM of cas- pase 3 substrate (Ac-DEVD-pNA) in assay buffer contain- ing 20 mM HEPES (pH 7.4), 0.1% CHAPS, 5 mM DTT, and
2 mM ethylenediaminetetraacetic acid (EDTA). Total incubation volume was 100 ml per each well in 96-well plate, After incubation for 2 h, optical density was meas- ured by ELISA reader (Molecular Devices, Sunnyvale, CA, USA) at 405 nm. Each sample was normalised by protein concentration.

Measurement of mitochondria membrane potential
To measure MMP, cells were stained with 2.5 mg/ml JC- 1 or DiOC6 for 10 min at 37 ◦C. Cells stained with JC-1 were observed with 485-nm excitation filter of fluores-
cence microscope and the emission was collected for J-monomer (kem ¼ 527 nm) and the J-aggregates
(kem ¼ 590 nm). Then, five areas were pictured to count number of high red fluorescent (J-aggregate) live cells. Dead or dying cells exhibit yellow or green fluorescence (J-monomer) with collapsed mitochondrial potential, respectively. Data were represented by the percentage of high red-fluorescence cells. In addition, living cells were labelled with DiOC6 that is a fluorescent dye used for the staining of intracellular membrane structures including mitochondria. The fluorescence of DiOC6 was greatly enhanced when incorporated into membranes or bound to lipophilic biomolecules such as proteins[30,31]. Cells stained with DiOC6 were analysed by FACSCaliburTM (Becton Dickinson, San Joes, CA, USA). Cells with MMP collapse showed a decrease in DiOC6 fluorescence intensity.

Purification of nuclear fraction
Cells were harvested and suspended with buffer A [25 mM Tris-Cl (pH 8.0), 10 mM KCl, 1 mM DTT, 0.5 mM
PMSF] followed by the incubation on ice for 15 min. Cell suspension was treated with 0.75% Nonidet P-40, mixed with vortexing for 30 s and centrifuged at 1400 × g for 1 min. Then, pellet was separated from
supernatant. Nuclear fraction was obtained by the lysis
of pellet in buffer C [50 mM Tris-Cl (pH 8.0), 400 mM NaCl, 1 mM DTT, 1 mM PMSF], the centrifugation at 15,000 × g for 30 min and the collection of supernatant.

Cytoplasmic fraction was obtained by the centrifuga-
tion of supernatant at 1500 × g for 15 min and the col- lection of its supernatant. Nuclear and cytosolic fractions were validated by Western blot analysis for lamin B and tubulin, respectively.

Separation of cytosolic and heavy- membrane fractions
Cells were suspended in hypotonic buffer [20 mM HEPES (N-2-hydroxyethylpiperazine-N0-2-ethanesulfonic acid), pH 7.5, 10 mM MgCl2, 2 mM EDTA, 1 mM DTT,
1 mM Na4VO3, 250 mM sucrose, and proteinase inhibi- tors]. Then, cells were sonicated and supernatant con- taining cytosol fraction was obtained by centrifugation at 10,000 × g for 20 min at 4 ◦C. The pellet fraction con-
taining mitochondria was dissolved in lysis buffer
[10 mM Tris-acetate (pH 8.0), 0.5% Nonidet P-40, 5 mM CaCl2, 1 mM Na4VO3] and incubated for 30 min on ice before centrifugation at 14,000 × g for 5 min at 4 ◦C.

Measurement of reactive oxygen species
Intracellular ROS level was measured by incubating cells with or without 10 mM DCF-DA at 37 ◦C for 30 min.
Fluorescence intensity of 10,000 cells was analysed by FACSCaliburTM (Becton Dickinson, San Joes, CA).

Western blot analysis
Cellular proteins were extracted by 0.5% Nonidet P-40 lysis buffer containing 20 mM Tris-HCl (pH 8.2), 150 mM NaCl, protease inhibitor (2 mg/ml aprotinin, 2 mg/ml pepstatin, 1 mg/ml leupeptin, 1 mM phenylmethylsul- fonyl fluoride) and phosphatase inhibitor (1 mM sodium
vanadate and 5 mM sodium fluoride). Cells were lysed for 30 min on ice and centrifuged at 13,000 × g for 20 min at 4 ◦C. Protein concentrations of lysates were determined by using SMARTTM BCA protein assay kit
(iNtRON, Gyeonggido, Korea).
Equal amounts of cellular proteins in sodium dodecyl sulphate (SDS) sample buf- fer were denatured by boiling at 100 ◦C for 5 min. Samples were separated according to protein size by
SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis). Separated samples were trans- ferred to nitrocellulose membrane. Membranes were blocked with 2% skim milk in Tris buffered saline con- taining 0.5% Tween 20. After blocking, protein expres- sion of each sample was probed by immune-reaction using enhanced chemiluminescence.

Statistical analyses
Experimental differences were examined separately for statistical significance using ANOVA and students’ t-dis- tribution. The p value of <.05 or <.01 was considered to be significant.

Results
Serum deprivation (SD)-induced oxidative stress affects cell death, MMP collapse, and nuclear localisation of Nrf2. The incidence of autoimmune disease is associ- ated with an increase in B cell survival [32–34] and SD, which lead to apoptotic cell death [35,36]. Since Nrf2 regulates intracellular oxidative stress [7,8], we exam- ined the effect of SD-induced oxidative stress on B cells using WiL2-NS human B lymphoblast cells. When the cells were incubated with medium containing 1% FBS, no tubulin bands appear or at least, minimal expression of tubulin was detected in the nuclear fraction (data not shown), which implicates that the fractionation is efficient. Nuclear localisation of Nrf2 was increased as compared with 10% FBS-containing medium (Figure 1(A)). This treatment also affected cell death and MMP collapse. As shown in Figure 1(B), we observed a 9.5% increase in cell death at the subG0/G1

Serum deprivation induced cell death, MMP collapse and nuclear localisation of Nrf2. (A–E) WiL2-NS cells were incu- bated in the RPMI 1640 medium with 10 or 1% FBS for 3 (A) or 12 (B–F) h. Nuclear fraction was prepared and western blotting was used to detect Nrf2 (A). Cells were fixed, stained with propidium iodide, and analysed with flow cytometry (B). Dead cells were estimated by trypan blue staining (C). Cell lysates were prepared and caspase 3 activity was measured by using Ac-DEVD- pNA, caspase 3 substrate. Caspase 3 activity was normalised with protein concentration (D). Cells were stained with MitoProbeTM JC-1 reagent for the detection of MMP. Cells were photographed with 400 magnification under fluorescence microscope (E, left) and number of high-red fluorescent cells was counted (E, right). Cytosolic and heavy membrane fractions were separated at 12 h after the incubation with 10 or 1% FBS. Western blotting was used to detect cytochrome C (F). Western blots for each pro- tein band (A,F) and pictures for MMP (E) were the representative of four experiments. Data in bar graphs were represented as means ± SEM of four experiments. Experimental differences were examined for statistical significance using ANOVA one-way test.

ωp < .05; ωωp < .01; significant difference as compared to control with 10% FBS (B, right, C, D, and E, right).

phase following incubation with 1% FBS compared with 3% cell death in the control group treated with 10% FBS. The percentage of cells in the G0/G1 phase increased to ~41% after incubation with 1% FBS, which was a 5% greater increase than that in the 10% FBS
control group. The percentages of cells in the S and G2/ M phases were decreased to ~24 and 23%, respectively, following incubation with 1% FBS, which were lowerthan those in the 10% FBS control group (29 and 31% in the S and G2/M phases, respectively). When the cells were incubated with 10 or 1% FBS-containing medium and subjected to trypan blue staining to assess viability, we found that the percentage of cell survival was reduced to ~80%, compared with ~97% after treat- ment with 10% FBS (Figure 1(C)). Cell death was con- firmed by a caspase 3 assay, which showed that caspase 3 activity was 1.8-fold greater in cells incubated with 1% than 10% FBS (Figure 1(D)). These results sug- gest that oxidative stress induced by 1% FBS-containing medium activates Nrf2 to regulate B cell survival and death.

It has been reported that SD induces apoptosis of rat retinal ganglion cells via mitochondrial changes [35,37]. We determined changes in MMP using MitoProbeTM JC-1 reagent in WiL2-NS cells incubated with 1% FBS. After incubating the cells with 1% FBS and MitoProbeTM JC-1 reagent, MMP was assessed by counting the num- ber of live cells with high red fluorescence. MMP was
lowered to ~40% in cells treated with 1% FBS, com-
pared with 90% in cells treated with 10% FBS (Figure 1(E)). MMP collapse by 1% FBS was confirmed by the release of cytochrome C into the cytosol (Figure 1(F)). These observations suggest that oxidative stress-associ- ated B cell survival and death might be regulated by nuclear localisation of Nrf2 via changes in MMP.

NAC attenuated Nrf2 activation, MMP collapse, and cell death
It has been reported previously that ROS generation is the main cause of MMP collapse [35,37] and that the presence of Nrf2 can protect mitochondria from MMP

N-acetylcysteine inhibited nuclear localisation of Nrf2. (A D) WiL2-NS cells were incubated in the RPMI 1640 medium with 10 or 1% FBS for 12 h (A C) or 3 h (D) in the presence or absence of 10 mM N-acetylcysteine (NAC). Then, cells were incu- bated with DCF-DA and analysed by flow cytometry (A–B). MFI was analysed by WinMDI 2.8 for ROS level of each sample (C). Nuclear fraction was prepared and western blotting was used to detect Nrf2. Western blots for each protein band were the repre- sentative of four experiments (D, top). Each band was quantified by using ImageJ 1.34 and the results were represented as fold changes to control (D, bottom). Data in bar graphs were represented as means ± SEM of four experiments. Experimental differen-
ces were examined for statistical significance using ANOVA one-way test. ωp < .05; significant difference as compared to control
with 10% FBS. #p < .05; significant difference as compared to NAC-untreated control with 1% FBS (C and D, bottom).

collapse after H2O2 exposure [19]. We examine the changes in intracellular ROS levels after incubation with 1% FBS using DCF-DA. ROS levels were significantly increased by 1% FBS compared with 10% FBS (Figure 2(A)). When cells were incubated with 1% FBS in the presence of the ROS scavenger NAC, ROS levels were significantly decreased (Figure 2(B)). The mean fluorescence intensity (MFI) was measured in each sam- ple as an indicator of ROS levels and is presented in a bar graph in Figure 2(C). The MFI was 1.8 fold higher in
samples incubated with 1% FBS compared with 10% FBS. This value was reduced to ~65% by treatment with NAC compared with 1% FBS treatment. In add-
ition, nuclear localisation of Nrf2 by 1% FBS was also reduced by NAC treatment (Figure 2(D)). However, no tubulin bands appear or at least, minimal expression of tubulin was detected in the nuclear fraction (data not shown), which implicates that the fractionation is effi- cient. When cells were incubated with 1% FBS-contain- ing medium in the presence or absence of NAC, the
percentage of cell survival was recovered to ~94% fol-lowing NAC treatment (Figure 3(A)). Cell death induced
reagent treatment. MMP was lowered to ~44% by 1% FBS, which was attenuated to ~84% by NAC treatment, compared with ~95% by 10% FBS treatment (Figure 3(C)). The inhibition of MMP collapse by NAC treatment
was confirmed using DiOC6, a fluorescent dye used to stain intracellular membrane structures including those of mitochondria [31]. As expected, the fluorescence intensity of DiOC6 was decreased by incubation with
1% FBS. The percentage of the cell population with low DiOC6 fluorescence intensity was ~23% and signifi- cantly larger than that in the 10% FBS control (2%).

When cells were incubated with 1% FBS in the presence of NAC, the cell population with low DiOC6 fluores- cence intensity was reduced to ~9.4% (Figure 3(D)).
The MFI of cells incubated with 1% FBS was 80%, which
was enhanced to ~88% by NAC treatment, compared with 10% FBS treatment (Figure 3(E)). Together, these results suggest that ROS might be associated with theregulation of MMP changes and nuclear localisation of Nrf2, leading to regulation of B cell survival.
H O -induced cell death is associated with MMP
by 1% FBS was confirmed by caspase 3 activation. 2 2
Caspase 3 activity following treatment with NAC was attenuated to a level similar to that in the 10% FBS con- trol (Figure 3(B)). MMP collapse was assessed by count- ing the number of live cells with high red fluorescence intensity after staining the cells with MitoProbeTM JC-1
collapse and nuclear localisation of Nrf2
To confirm the role of ROS in SD-induced oxidative stress in B cells, WiL2-NS cells were incubated with 100 mM H2O2. Trypan blue staining was used to assess cell death. Incubation with H2O2 resulted in a reduction

N-acetylcysteine reduced MMP collapse and cell death. (A E) WiL2-NS cells were incubated in the RPMI 1640 medium with 10 or 1% FBS in the presence or absence of 10 mM N-acetylcysteine (NAC) for 12 h. Dead cells were estimated by trypan blue staining (A). Cell lysates were prepared and caspase 3 activity was measured by using Ac-DEVD-pNA, caspase 3 substrate. Caspase 3 activity was normalised with protein concentration (B). Cells were stained with MitoProbeTM JC-1 reagent (C) or DiOC6 (D and E) for the detection of MMP. Cells were photographed with 400 magnification under fluorescence microscope (C, top) and number of high-red fluorescent cells was counted (C, bottom). Cells were analysed by flow cytometry analysis (D and E). Pictures (C) and histograms (D) were the representative of four experiments. MFI was analysed by WinMDI 2.8 for MMP of each sample (E). Data in bar graphs were represented as means ± SEM of four experiments. Experimental differences were examinedfor statistical significance using ANOVA one-way test. ωp < .05; ωωp < .01; significant difference as compared to control with 10%FBS. #p < .05; ##p < .01; significant difference as compared to NAC-untreated control with 1% FBS (A, B, C, bottom, and E)in cell survival to ~80 and 10% by 12 and 24 h, respect- ively (Figure 4(A)). Cell death by H2O2 was also con- firmed by assay of caspase 3 activity, which was enhanced ~1.2- and 1.7-fold by 50 and 100 mM H2O2 treatment, respectively, for 24 h (Figure 4(B)). MMP col- lapse was once again assessed by counting the numberof live cells with high red fluorescence intensity after staining the cells with MitoProbeTM JC-1 reagent. MMP was lowered to ~72, 48, and 13% by H2O2treatment for 6, 12, and 24 h, respectively, compared with 82% in the control group (Figure 4(C)). MMP collapse by H2O2 treatment for 12 h was also confirmed by the release of cytochrome C into the cytosol (Figure 4(D)). The nuclear localisation of Nrf2 was also increased by H2O2 treatment for 3 h compared with the control (Figure 4(E)). However, no tubulin bands appear or at least, minimal expressionof tubulin was detected in the nuclear fraction (data not shown), which implicates that the fractionation is effi- cient. These observations suggest that H2O2 regulates B cell survival by controlling MMP collapse via nuclear localisation of Nrf2. They also suggest that B cell survivalis regulated by MMP collapse via Nrf2 activation, and that ROS potentially regulate Nrf2 activation in B cells.

Nrf2 activation was reduced by inhibition of Syk
It has been reported previously that Syk plays an important role in B cell survival [29]. Tyrosine (Y) 525/ 526 in the Syk domain is an essential residue for kinase activity [38,39]. Thus, we examined whether Nrf2 activa- tion is regulated by phosphorylation of Syk at Y525/
526. We observed an increase in Syk phosphorylation at Y525/526 in cells incubated with 1% FBS-containing medium (Figure 5(A)). When cells were incubated with 1% FBS in the presence of the ROS scavenger NAC, the increase in Syk phosphorylation at Y525/526 in cells incubated with 1% FBS alone was inhibited by NAC treatment (Figure 5(B)). Syk phosphorylation at Y525/
526 was also enhanced by incubation with 100 mM H2O2 (Figure 5(C)). In addition, when cells were incu- bated with 1% FBS-containing medium in the presence of the Syk inhibitor BAY61-3606, nuclear localisation of

H2O2 induced cell death, MMP collapse, and nuclear localisation of Nrf2. (A D) WiL2-NS cells were incubated in the RPMI 1640 medium with 100 mM H2O2 for 6, 12, and 24 h. Then, dead cells were estimated by trypan blue staining (A). Cells were incubated for 24 h, cell lysates were prepared and caspase 3 activity was measured by using Ac-DEVD-pNA, caspase 3 sub- strate. Caspase 3 activity was normalised with protein concentration (B). Cells were stained with MitoProbeTM JC-1 reagent for the detection of MMP. Cells were observed and photographed with 400 magnification under fluorescence microscope (C, left). Number of high-red fluorescent cells was counted (C, right). WiL2-NS cells were treated with 100 mM H2O2 for 12 h. Cytosolic and heavy membrane fractions were separated and western blotting was used to detect cytochrome C (D). Cells were incubated for 1 h, nuclear fraction was prepared and western blotting was used to detect Nrf2 (E). Pictures for MMP (C) and western blots for each protein band (D, E) were the representative of four experiments. Each band was quantified by using ImageJ 1.34 and the results were represented as fold changes to control (D, E). Data in bar graphs were represented as means ± SEM of fourexperiments. Experimental differences were examined for statistical significance using ANOVA one-way test. ωp < .05; ωωp < .01;significant difference as compared to H2O2-untreated control (A, B, and C–E, right).

Nrf2 was prevented (Figure 5(D)), implying that Syk phosphorylation activates Nrf2 by inducing its nuclear localisation. In addition, no tubulin bands appear or at least, minimal expression of tubulin was detected in the nuclear fraction (data not shown), which implicates that the fractionation is efficient. The data suggest that B cell survival is regulated by oxidative stress via Syk- mediated Nrf2 activation.

A Syk inhibitor enhanced MMP collapse and cell death under SD conditions
To confirm the effect of Syk on oxidative stress-induced B cell death, WiL2-NS cells were incubated with 1% FBS-containing medium in the presence or absence of BAY61-3606. MMP was assessed by flow cytometry analysis after staining cells with DiOC6. As shown in Figure 6(A), the fluorescence intensity of DiOC6 was decreased after incubation with 1% FBS. The percent- age of the cell population with low DiOC6 fluorescence
intensity was increased to ~10% by 1% FBS, comparedwith 3% by 10% FBS treatment. While the values do not match the 23% measured in Figure 3(D), there was a consistent decrease in DiOC6 fluorescence intensity when the cells were incubated with 1% FBS. The MFI was decreased to ~90% following incubation with 1% FBS and further decreased to ~66% by treatment with BAY61-3606, compared with 10% FBS treatment (Figure 6(B)). When cells were incubated with 1% FBS in the presence of BAY61-3606, the cell population with low DiOC6 fluorescence intensity was increased to
~39%. A 6% increase was detected by treatment with BAY61-3606 in 10% FBS-containing medium (Figure
6(A)). These changes suggest that MMP collapse is regu- lated by Syk activation. Caspase 3 activity was enhanced ~1.5 fold by 1% FBS compared with 10% FBS. The caspase 3 activity induced by 1% FBS treat- ment was further enhanced ~2.3-fold by treatment with BAY61-3606 (Figure 6(C)). Caspase 3 activity was also enhanced ~1.5 fold by 100 mM H2O2 and further enhanced ~2.2 fold by BAY61-3606 (Figure 6(D)). According to trypan blue staining, the percentage of

Nuclear localisation of Nrf2 was inhibited by the treatment with BAY61-3606, an inhibitor of spleen tyrosine kinase (Syk). WiL2-NS cells were incubated in the RPMI 1640 medium with 10 or 1% FBS in the absence (A) or presence (B) of 10 mM N-acetylcysteine (NAC) for 3 h. WiL2-NS cells were incubated in the RPMI 1640 medium with 100 mM H2O2 in the absence (C) or presence (D) of 10 mM BAY61-3606, Syk inhibitor for 1 h. Cell lysates were prepared and western blotting was used to detect phosphorylated Syk at tyrosine (Y) 525/526 (A, B and C). Nuclear fraction was prepared and western blotting was used to detect Nrf2 (D). Data were the representative of four experiments. Each band was quantified by using ImageJ 1.34 and the results wererepresented as fold changes to control. Data in bar graphs were represented as means ± SEM of four experiments. Experimental differences were examined for statistical significance using ANOVA one-way test. ωp < .05; ωωp < .01; significant difference as compared to control group with 10% FBS (A, B) or H2O2-untreated control (C, D). #p < .05; ##p < .01; significant difference as com- pared to NAC-untreated control with 1% FBS (C) or BAY61-3606-untreated control with 100 mM H2O2 (D).

Syk inhibitor enhanced MMP collapse and cell death in serum deprived condition. (A C) WiL2-NS cells were incubated in the RPMI 1640 medium with 10 or 1% FBS in the presence or absence of 10 mM BAY61-3606, Syk inhibitor. (D–E) WiL2-NS cells were incubated in the RPMI 1640 medium with 100 mM H2O2 in the absence or presence of 10 mM BAY61-3606, Syk inhibi- tor. Then, cells were stained with DiOC6 for the detection of MMP at 12 h after the incubation with an appropriate condition. Percentage of cells with low fluorescence of DiOC6 was analysed by flow cytometry. Data were the representative of four experi- ments (A). MFI was analysed by WinMDI 2.8 for MMP of each sample (B). Cell lysates were prepared at 24 h after the incubation with an appropriate condition. Caspase 3 activity was measured by using Ac-DEVD-pNA, caspase 3 substrate and was normalised with protein concentration (C, D). Dead cells were estimated by trypan blue staining (E). Data in bar graphs were represented as means ± SEM of four experiments. Experimental differences were examined for statistical significance using ANOVA one-way test.
ωp < .05; significant difference as compared to control with 10% FBS. #p < .05; significant difference as compared to BAY61-3606-
untreated control with 1% FBS (B, C) or 100 mM H2O2 (D, E).

Scheme for the regulation of SD-associated B cell survival via ROS-associated Syk activation and nuclear localisa- tion of Nrf2. Our findings are indicated by grey-dotted lines. Black lines are from the results reported already in the literatures.cell survival was reduced to ~70% by 100 mM H2O2 and further reduced to 59% after treatment with BAY61- 3606 (Figure 6(E)). Minimal cell death (2%) was detected
by treatment with BAY61-3606 alone in 10% FBS-con- taining medium. Together, these results suggest that SD-induced Syk activation regulates MMP and Nrf2 acti- vation to regulate B cell survival and death (Figure 7).

Discussion
In the progression of autoimmune diseases, B cell sur- vival is an important contributor to the production of inflammatory cytokines and antibodies, presentation of autoantigens, and interactions with T cells [40]. Increased ROS levels at most inflammatory disease sites can affect cell fate [3,5,6]. Upon cell exposure to oxida- tive stress, Nrf2 translocate to the nucleus [14], which leads to regulation of cellular resistance against exogenous and endogenous insults, including xenobi- otics, oxidative stress, hyperoxia, nitrosative stress, and ER stress [8–12]. In addition, ROS induce B cell activa- tion via changes in Syk activity [41,42]. However, little is known about the regulation of B cell survival via Syk- associated Nrf2 activation. Here, we investigated whether B cell survival is regulated by oxidative stress- mediated nuclear localisation of Nrf2 and MMP collapse via Syk activation using WiL2-NS human B lymphoblast cells. As intracellular functional events are time depend- ent under physiological conditions, we evaluated Syk phosphorylation, Nrf2 nuclear translocation, MMP col- lapse, and oxidative stress-mediated cell death at differ- ent time points. These experiments were repeated at least four times to ensure reproducibility, and the pooled results are depicted in Figure 7. Our data show that Syk controls oxidative stress-induced human B cell death via nuclear translocation of Nrf2 and MMP
collapse. These results suggest that Syk is a novel regu- lator of Nrf2 activation.
Nrf2 plays a dual role in health and disease [9]. The Nrf2-mediated antioxidant response allows cell survival in the face of toxic insults. Nrf2 protects mitochondria from oxidant injury, likely through direct interaction with mitochondria [19]. Nrf2 signalling is turned off by the absence of insults in the cellular microenvironment, which can result in the vulnerability of the cell to vari- ous insults under pathological conditions [14]. Overactive Nrf2 induces metabolic reprogramming towards cell proliferation [43] and confers protection of tumour cells to apoptosis [44,45]. It is possible that Syk tightly regulates Nrf2-mediated antioxidant responses such as the expression of antioxidant enzymes; how- ever, further studies are needed to confirm this hypothesis.

It is also possible that Syk activation by SD-induced oxidative stress affects Nrf2/Keap1 interactions. Under normal conditions, Nrf2/Keap1 interaction induces pro- teasomal degradation of Nrf2. When cells are exposed to oxidative stress, Nrf2 dissociates from Keap1, leading to nuclear translocation of Nrf2 [13,14]. Our results showed that nuclear translocation of Nrf2 was enhanced by oxidative stress-induced phosphorylation of Syk at Y525/526. It cannot be ruled out that the aber- rant accumulation of proteins may disrupt Nrf2/Keap1 interactions via Syk activation. Thus, it is necessary to determine whether Nrf2/Keap1 interaction is depend- ent on Syk activation.
H2O2 phosphorylates Lyn and Syk proteins residing in the mitochondrial intermembrane space [46]. Following stimulation of FceRI or other immune recep- tors, the activation of Syk is also associated with its phosphorylation on several Y residues: Y317, Y342, Y346, Y519, and Y520 [47,48]. Residues Y323, Y352, andY525/526 are the most important in human Syk [38,39,49,50]. It has been also reported that proteins other than Syk regulate Nrf2 signalling. Evidence indi- cates that Nrf2 is regulated by multiple mechanisms, such as phosphorylation by several kinases, including protein kinase C (PKC), MAPK/ERK/JNK, JUN/MYC, and PI3K [51]. PKCd positively modulates nitric oxide (NO)- induced Nrf2/ARE-dependent signalling in neurons [17]. However, further studies are required to investigate Syk phosphorylation at sites other than Y525/526.

Syk activation is associated with many signalling molecules other than Nrf2. Oxidative stress- induced Caþþ responses and JNK activity are partly dependenton Syk [41]. Both Lyn and Syk may regulate Caþþmobilisation via the phosphatidylinositol pathway in B cells [42]. Akt is activated in a Syk-dependent pathwayand also plays a role in B cell survival [52]. Previous studies have demonstrated that H2O2 exposure rapidly leads to necrosis via tyrosine phosphorylation of FAK downstream of Lyn and Syk signalling [3]. Therefore, it is necessary to determine whether Y525/526 phosphor- ylation of Syk regulates Nrf2 directly or indirectly.

In conclusion, our data demonstrate that SD-induced oxidative stress regulates MMP collapse and nuclear localisation of Nrf2 via Syk activation. The data suggest that Syk regulates oxidative stress-induced human B cell death via nuclear translocation of Nrf2 and MMP collapse, and that Syk is a novel kinase regulating Nrf2 activation in human B cells. Syk may prove to be a valu- able target in the development of effective therapies against B cell-mediated autoimmune disease.

Disclosure statement
No potential conflict of interest was reported by the authors.

Funding
This work was supported by Grant from Mid-Career Researcher Program (#2016-R1A2B400746) through the National Research Foundation (NRF) funded by the Ministry of Education, Science and Technology (MEST), Republic of Korea.

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