Mol Neurobiol DOI 10.1007/s12035-015-9314-z

Epigenetic Regulation of Cytosolic Phospholipase A2 in SH-SY5Y Human Neuroblastoma Cells Charlene Siew-Hon Tan 1 & Yee-Kong Ng 1 & Wei-Yi Ong 1,2

Received: 29 April 2015 / Accepted: 23 June 2015 # Springer Science+Business Media New York 2015

Abstract Group IVA cytosolic phospholipase A2 (cPLA2 or PLA2G4A) is a key enzyme that contributes to inflammation via the generation of arachidonic acid and eicosanoids. While much is known about regulation of cPLA2 by posttranslational modification such as phosphorylation, little is known about its epigenetic regulation. In this study, treatment with histone deacetylase (HDAC) inhibitors, trichostatin A (TSA), valproic acid, tubacin and the class I HDAC inhibitor, MS-275, were found to increase cPLA2α messenger RNA (mRNA) expression in SH-SY5Y human neuroblastoma cells. Co-treatment of the histone acetyltransferase (HAT) inhibitor, anacardic acid, modulated upregulation of cPLA2α induced by TSA. Specific involvement of class I HDACs and HAT in cPLA2α regulation was further shown, and a Tip60-specific HAT inhibitor, NU9056, modulated the upregulation of cPLA2α induced by MS-275. In addition, co-treatment of with histone methyltransferase (HMT) inhibitor, 5′-deoxy-5′-methylthioadenosine (MTA) suppressed TSA-induced cPLA2α upregulation. The above changes in cPLA2 mRNA expression were reflected at the protein level by Western blots and immunocytochemistry. Chromatin immunoprecipitation (ChIP) showed TSA increased binding of trimethylated H3K4 to the proximal promoter region of the cPLA2α gene. Cell injury after TSA treatment as indicated by lactate dehydrogenase (LDH) release

* Wei-Yi Ong [email protected] 1

Department of Anatomy, National University of Singapore, Singapore 119260, Singapore

2

Neurobiology and Ageing Research Programme, National University of Singapore, Singapore 119260, Singapore

was modulated by anacardic acid, and a role of cPLA2 in mediating TSA-induced injury shown, after co-incubation with the cPLA 2 selective inhibitor, arachidonoyl trifluoromethyl ketone (AACOCF3). Together, results indicate epigenetic regulation of cPLA2 and the potential of such regulation for treatment of chronic inflammation. Keywords Cytosolic phospholipase A2 . Histone deacetylase . Histone acetyltransferase . Anacardic acid . Epigenetic regulation . Arachidonic acid . Excitotoxicity . Neurons . Brain Neuroinflammation

Introduction The phospholipase A2 (PLA2) superfamily of enzymes play an important role in lipid metabolism in the brain, catalyzing the hydrolysis of glycerophospholipids at the sn-2 position to generate free fatty acids and lysophospholipids [1, 2]. PLA2 activation and in particular, cPLA2 upregulation, is one of the earliest events in brain damage pathways leading up to various forms of acute and chronic brain injury, including head injury, cerebral ischemia, epilepsy, and Alzheimer’s disease (AD) [3, 4]. The cPLA2 protein has a Ca2+-dependent phospholipidbinding domain at the N-terminal region to allow for membrane binding [5, 6]. It generates arachidonic acid from glycerophospholipids and plays a key role in production of pro-inflammatory eicosanoids such as leukotrienes, prostaglandins, thromboxanes, and lipoxins [7]. cPLA2 is expressed at relatively low levels in the normal forebrain [8], but is upregulated in neurons and astrocytes after kainate induced excitotoxic injury [9]. Elevated cPLA2 expression and enzyme activity is found in the cerebral cortex of subjects with AD, which could lead to increased release of arachidonic acid, generation of eicosanoids, and neuronal injury [4, 10–12].

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Transcriptional regulation plays an important role in cPLA2 expression [13]. The cPLA2 gene is located on chromosome 1q25, adjacent to the COX-2 locus [14]. The primary promoter region of the gene occurs between 31 and 73 positions upstream of the transcriptional start site [13]. Several binding sites at this transcriptional start site have been identified and proposed to be important for gene regulation [13]. Cytokines, thrombin and growth factors interact at these sites to influence transcription of cPLA2 [13, 15]. Glucocorticoid growth factors stimulate cPLA2α expression in human amnion fibroblasts [16], and IL-1β induces its upregulation in human rheumatoid arthritis synovial fibroblasts [17]. Posttranscriptionally, the cPLA2 messenger RNA (mRNA) can be alternately spliced to produce cPLA2α, cPLA2β, cPLA2γ, cPLA2δ, cPLA2ε, and cPLA2ζ isoforms [18]. Posttranslational modifications also play a role in regulation of cPLA2 [14]. Phosphorylation of the cPLA2 protein is involved in translocation from the cytosol to intracellular membranes where it carries out its function [19–21]. Treatment of primary mouse astrocyte cultures with cytokines and ATP promotes the PKC and ERKmediated phosphorylation of cPLA2, followed by arachidonic acid release [22]. Tumor necrosis factor-α induces cPLA2 phosphorylation and arachidonic acid release via activation of the MAP kinase cascade and NF-κB [23]. Specific phosphorylation events at the serine-515 and serene-505 residues are required for arachidonic acid release in vascular smooth muscle cells [20]. Thus far, however, little is known about epigenetic regulation of cPLA2. Epigenetic modifications are typically characterized by changes in gene expression following environmental stimuli [24, 25] or dietary changes [26]. They include DNA methylation, histone modifications such as acetylation and methylation, nucleosome remodeling, and RNA-mediated pathways [27, 28]. Enzymes such as histone acetyltransferases (HATs), histone deacetylases (HDACs), histone methyltransferases (HMTs), and histone demethylases mediate epigenetic histone modifications and control the state of lysine residues (K) on histone tails [28]. Acetylation of lysine 9 residue of histone H3 (H3K9ac) is typically associated with activated genes [28]. In addition, trimethylation of the lysine 4 residue of histone 3 (H3K4me3) is associated with open chromatin conformation and increased gene expression [28–30]. HDAC inhibitors such as TSA significantly alters the expression of approximately 2 % of genes [31] and recent studies highlighted it as a potential drug candidate, especially in the field of cancer therapy [32]. The present study was carried out to investigate possible epigenetic regulation of cPLA2 in SH-SY5Y neuroblastoma cells. The effects of histone acetylation and methylation on cPLA2 expression were explored, and possible changes in cell viability as a result of these changes, elucidated.

Materials and Methods Materials SH-SY5Y cells were treated with dimethyl sulfoxide (DMSO), TSA, valproic acid, MS-275, tubacin, anacardic acid, curcumin (CCM), NU9056, C646, butyrolactone-3 (MB3), 5′-deoxy-5′-methylthioadenosine (MTA) and arachidonyl trifluoromethyl ketone (AACOCF3). DMSO, TSA, valproic acid, tubacin, CCM, C646 and MTA were purchased from Sigma (St. Louis, USA). MS-275 and MB-3 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anacardic acid was purchased from Calbiochem (San Diego, CA). NU9056 and the cPLA2 selective inhibitor AACOCF3 were purchased from Tocris Bioscience (Bristol, UK). Stock solutions were prepared in DMSO and diluted in cell culture medium for use, except valproic acid, whereby distilled H2O was used due to better solubility. Cell Culture and Treatments Human SH-SY5Y neuroblastoma cells were obtained from American Type Culture Collection (ATCC, Manassas, VA) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10 % heat-inactivated fetal bovine serum (FBS) and 1 % penicillin/streptomycin (GibcoInvitrogen, Carlsbad, CA). SH-SY5Y cells are generally locked in an early neuronal differentiation phase [33, 34]. Retinoic-acid-differentiated SH-SY5Y cells displayed increased tau phosphorylation besides displaying neuron-like characteristics [35]. In addition, retinoic acid reduced binding of histone deacetylase 1 (HDAC1) to neuronal genes to bring about changes to the epigenome [36]. Thus, SH-SY5Y cells were not further differentiated with retinoic acid, so that responses to treatments affecting histones can be evaluated. The cells used were of a constant N-type origin, as interconversion to S-type was shown in previous literature not to occur due to their copy number variants and genetic variation [37, 38]. This excludes the possibility of changes in gene expression due to spontaneous interconversions between cell types. Cells were grown in 100-mm2 cell culture dishes and incubated at 37 °C, 100 % humidity with 95 % air and 5 % CO2. Dose-Dependent Treatments with HDAC Inhibitors To study the effects of different HDAC inhibitor treatments on cPLA2 expression, dose-dependent treatments were administered to four groups of SH-SY5Y cells, with the first group treated with vehicle controls, and the next three groups with increasing doses of the HDAC inhibitors. Each group consisted of four replicates. Increasing doses of TSA were administered up to the IC50 value of 0.5 μM [39]. Valproic acid was administered up to 1000 μM as considerable

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epigenetic effects were observed in SH-SY5Y cells up to that dose [40]. Tubacin is known to inhibit HDAC6 at micromolar doses [41], while MS-275 is known to display epigenetic effects in SH-SY5Y cells at 1–10 μM [42]. Cells were incubated with respective HDAC inhibitors or vehicle for 24 h before harvesting. Dose-Dependent Treatment with General HAT Inhibitor, Anacardic Acid To examine the effect of the general HAT inhibitor, anacardic acid, on cPLA2 expression, dose-dependent treatments of 10, 20, and 30 μM were administered to four groups of SH-SY5Y cells, according to known doses where it exerts significant epigenetic effects [43]. DMSO was used as vehicle control, and each group consists of four replicates. Anacardic acid is a well-known HAT inhibitor that is a flavonoid extract derived from cashew nuts and has been reported to possess antiinflammatory properties [44]. Cells were incubated with anacardic acid or vehicle for 24 h before harvesting. Treatment with General HAT Inhibitor, Anacardic Acid, and TSA To investigate the effect of the general HAT inhibitor, anacardic acid, and TSA on cPLA2 expression, SH-SY5Y cells were divided into four groups and treated as follows: (1) DMSO as vehicle control, (2) 0.5 μM TSA, (3) 20 μM anacardic acid, and (4) 20 μM anacardic acid and 0.5 μM TSA. Each group consists of four replicates. Cells were coincubated with anacardic acid and TSA or vehicle for 24 h before harvesting. Treatment with Natural P300-Specific HAT Inhibitor, CCM, and TSA

consists of four replicates. Cells were co-incubated with C646 and TSA or vehicle for 24 h before harvesting. Treatment with GCN5-Specific HAT Inhibitor, MB-3, and TSA To investigate the effect of the GCN5-specific HAT inhibitor, MB-3, and TSA on cPLA2 expression, SH-SY5Y cells were divided into four groups and treated as follows: (1) DMSO as vehicle control, (2) 0.5 μM TSA, (3) 20 μM MB-3, and (4) 20 μM MB-3 and 0.5 μM TSA. Each group consists of four replicates. Cells were co-incubated with MB-3 and TSA or vehicle for 24 h before harvesting. Treatment with Tip60-Specific HAT Inhibitor, NU9056, and TSA To study the effect of the Tip60-specific HAT inhibitor, NU9056, and TSA on cPLA2 expression, SH-SY5Y cells were divided into four groups and treated as follows: (1) DMSO as vehicle control, (2) 0.5 μM TSA, (3) 20 μM NU9056, and (4) 20 μM NU9056 and 0.5 μM TSA. Each group consists of four replicates. Cells were co-incubated with NU9056 and TSA or vehicle for 24 h before harvesting. Treatment with Tip60-Specific HAT Inhibitor, NU9056, and MS-275 To examine the effect of the Tip60-specific HAT inhibitor, NU9056, and MS-275 on cPLA2 expression, SH-SY5Y cells were divided into four groups and treated as follows: (1) DMSO as vehicle control, (2) 5 μM MS-275, (3) 20 μM NU9056, (4) 20 μM NU9056 and 5 μM MS-275. Each group consists of four replicates. Cells were co-incubated with NU9056 and MS-275 or vehicle for 24 h before harvesting. Treatment with HMT Inhibitor, MTA, and TSA

To study the effect of the natural p300-specific HAT inhibitor, CCM, and TSA on cPLA2 expression, SH-SY5Y cells were divided into four groups and treated as follows: (1) DMSO as vehicle control, (2) 0.5 μM TSA, (3) 20 μM CCM, and (4) 20 μM CCM and 0.5 μM TSA. Each group consists of four replicates. Cells were co-incubated with CCM and TSA or vehicle for 24 h before harvesting. Treatment with Synthetic P300-Specific HAT Inhibitor, C646, and TSA To examine the effect of the synthetic p300-specific HAT inhibitor, C646, and TSA on cPLA2 expression, SH-SY5Y cells were divided into four groups and treated as follows: (1) DMSO as vehicle control, (2) 0.5 μM TSA, (3) 20 μM C646, and (4) 20 μM C646 and 0.5 μM TSA. Each group

To investigate the effect of the HMT inhibitor, MTA, and TSA on cPLA2 expression, SH-SY5Y cells were divided into four groups and treated as follows: (1) DMSO as vehicle control, (2) 0.5 μM TSA, (3) 200 μM MTA, and (4) 200 μM MTA and 0.5 μM TSA. MTA reduces trimethylation of H3K4 at micromolar doses via the inhibition of Set1 methyltransferases [45, 46]. Each group consists of four replicates. Cells were co-incubated with MTA and TSA or vehicle for 24 h before harvesting. Real-Time RT-PCR Total RNA from SH-SY5Y cells was extracted with the RNeasy Mini kit (Qiagen, Hamburg, Germany). Reverse transcription of

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RNA to complementary DNA (cDNA) was performed using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA) with the thermal cycler of reaction conditions set at 25 °C for 10 min, 37 °C for 120 min, and 85 °C for 5 min. The cDNA obtained was quantified by real-time RTPCR using the TaqMan® Universal PCR Master Mix (Applied Biosystems, Foster City, CA), with TaqMan® Gene Expression Assay Probes for cPLA2α (Hs00233352_m1), iPLA2β (Hs00185926_m1), and β-actin (#4326315E) (Applied Biosystems, Foster City, CA). Real-time PCR was performed using a MicroAmp® 96-Well Optical Reaction Plate (Applied Biosystems, Foster City, CA) and run on the Applied Biosystem 7500 Real-Time PCR system. Reaction conditions were as follows: an initial incubation of 50 °C for 2 min and 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. The relative amount of gene transcript was estimated after normalization to the endogenous control gene, β-actin. This is a suitable control, as it was shown to be unaffected by TSA treatment in a previous study [47].Using the 2−ΔΔCT method [48], relative fold changes were quantified by first obtaining the threshold cycle, CT, that inversely correlates with levels of mRNA present in the sample. All reactions were performed in triplicates and the mean and standard error calculated. Statistical differences were analyzed using one-way ANOVA with Bonferroni’s multiple comparison post hoc test, where P

Epigenetic Regulation of Cytosolic Phospholipase A2 in SH-SY5Y Human Neuroblastoma Cells.

Group IVA cytosolic phospholipase A2 (cPLA2 or PLA2G4A) is a key enzyme that contributes to inflammation via the generation of arachidonic acid and ei...
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