NMDA receptor signaling mediates the expression of protein inhibitor of activated STAT1 (PIAS1) in rat hippocampus

Neuropharmacology 65 (2013) 101e113

Contents lists available at SciVerse ScienceDirect

Neuropharmacology journal homepage: www.elsevier.com/locate/neuropharm

NMDA receptor signaling mediates the expression of protein inhibitor of activated STAT1 (PIAS1) in rat hippocampus S.Y. Liu a, b, Y.L. Ma b, E.H.Y. Lee b, * a b

Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan Institute of Biomedical Sciences, Academia Sinica, Taipei 115, Taiwan

a r t i c l e i n f o

a b s t r a c t

Article history: Received 12 March 2012 Received in revised form 14 August 2012 Accepted 26 August 2012

Protein inhibitor of activated STAT1 (PIAS1) was shown to play an important role in inflammation and innate immune response, but how PIAS1 is regulated is not known. We have recently demonstrated that PIAS1 enhances spatial learning and memory performance in rats. In this study, we examined the signaling pathway and neural mechanism that regulate PIAS1 expression in the brain by using pharmacological and molecular approaches. Our results revealed that pias1 gene expression is rapidly induced upon NMDA receptor activation in rat hippocampus, but this effect is blocked by transfection of sub-threshold concentrations of ERK1 siRNA/ERK2 siRNA or CREB siRNA. Pias1 gene expression is similarly induced by overexpression of the ERK1/ERK2 plasmids in rat hippocampus, and this effect is also blocked by sub-threshold concentration of CREB siRNA transfection. On the other hand, transfection of ERK1 siRNA/ERK2 siRNA or CREB siRNA at a higher concentration is sufficient to downregulate PIAS1 expression. Inhibition of PI-3 kinase signaling and CaMKII signaling, which both result in CREB inactivation, similarly decreases PIAS1 expression. But NMDA and MK-801 do not affect the expression of IL-6 and TNFa. NMDA also did not affect the expression of PIAS2, PIAS3 and PIAS4. Further, pias1 mRNA has a similar degradation rate to that of the zif268 gene. These results together suggest that pias1 may function as an immediate early gene in an activity-dependent manner and PIAS1 expression is regulated by the NMDA-MAPK/ERK-CREB signaling pathway implicated in neuronal plasticity. Ó 2012 Elsevier Ltd. All rights reserved.

Keywords: NMDA MAPK/ERK CREB PIAS1 expression Hippocampus

1. Introduction Protein inhibitor of activated STAT1 (PIAS1) was initially identified as an inhibitor of STAT1 that blocks the DNA-binding activity of STAT1 and inhibits STAT1-mediated gene transcription (Liu et al., 1998; Liao et al., 2000). PIAS1 was well known to play a role in inflammation and innate immune response through negative regulation of STAT1 (Liu et al., 2004a). In addition to STAT1, PIAS1 was found to also regulate the activity of other transcription factors to regulate immunity. For example, proinflammatory stimuli were found to activate PIAS1 through IKKa. Phosphorylated IKKa further inhibits the DNA binding activity of STAT1 and NF-kB to reduce inflammation (Liu et al., 2007). But other than its role involved in the immune response, other functions that PIAS1 also participates in is less known. In a recent study, we have found that PIAS1 also plays an important role in learning and memory function. Spatial training * Corresponding author. Tel: þ886 2 2789 9125; fax: þ886 2 2782 9224. E-mail address: [email protected] (E.H.Y. Lee). 0028-3908/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuropharm.2012.08.024

was found to increase the expression of PIAS1 in rat hippocampus. Overexpression of PIAS1 enhances spatial learning and memory whereas knockdown of PIAS1 expression by PIAS1 siRNA transfection impairs spatial learning and memory performance in rats (Tai et al., 2011). Further, PIAS1 was found to enhance spatial learning and memory through decreased phosphorylation of STAT1 and increased sumoylation of STAT1, and this latter result is consistent with the report that PIAS1 possesses small ubiquitin-like modifier (SUMO) E3 ligase activity and functions as a SUMO E3 ligase (Kahyo et al., 2001). This result is also consistent with the notion that sumoylation may involve in the regulation of certain neuronal functions, such as synaptic transmission (Scheschonka et al., 2006). Despite the fact that PIAS1 plays an important role in neural plasticity, how the pias1 gene is regulated is not known. Because the glutamate N-methyl-D-aspartate (NMDA) receptor plays a critical role in mammalian learning and memory as well as synaptic plasticity (Collingridge, 1987; Izquierdo, 1991), we suspected that the NMDA receptor signaling may regulate the expression of PIAS1 in rat hippocampus. The present study was aimed to examine this hypothesis.

102

S.Y. Liu et al. / Neuropharmacology 65 (2013) 101e113

2. Methods 2.1. Animals Adult male Sprague-Dawley rats (250e350 g) bred at the Animal Facility of the Institute of Biomedical Sciences, Academia Sinica in Taiwan were used. Animals were housed in a room maintained on a 12 h/12 h light/dark cycle (light on at 6:30 am) with food and water continuously available. Experimental procedures follow the Guidelines of Animal Use and Care of the National Institute of Health. Efforts were made to minimize animal suffering. Alternative approach was also adopted to reduce the number of animals used, such as the mRNA stability assay and the chromatin immunoprecipitation (ChIP) PCR assay carried out in HEK293T cells and the pias1 promoter activity assay carried out in Neuro2A cells. 2.2. Drugs The NMDA receptor agonist NMDA and the NMDA receptor antagonist MK-801 were purchased from Tocris Bioscience (St. Louis, MO). The PI-3 kinase inhibitor LY294002 and wortmannin as well as the CaMKII inhibitor KN-62 and KN-93 were purchased from SigmaeAldrich (St. Louis, MO). Actinomycin-D was also purchased from SigmaeAldrich. NMDA and MK-801 were dissolved in PBS, and LY294002 and KN-62 were dissolved in 0.1% DMSO immediately before use. 2.3. Plasmid DNA construction and DNA/polyethyleneimine (PEI) complex preparation

water to a stock concentration of 20 pmol/ml. Branched PEI of 25 kDa (Sigmae Aldrich) was diluted to 0.1 M concentration in 5% glucose. Immediately before injection, 0.1 M PEI was added to the designated siRNA (0.5 ml each). The final concentration of the designated siRNA is 10 pmol/ml. CREB siRNA (7 pmol in 0.7 ml), ERK1 siRNA and ERK2 siRNA (3.5 pmol in 0.35 ml each) or control siRNA (0.7 ml) was transfected to each side of the CA1 area. For the Myc-ERK1, MycERK2 and CREB siRNA co-transfection experiment, 0.25 ml each was injected and the concentration for CREB siRNA was 5 pmol/ml. The reason why we used a lower concentration of CREB siRNA for the co-transfection experiment is that we aimed to examine whether CREB siRNA at a sub-threshold concentration blocks the effect of ERK1 and ERK2 overexpression on PIAS1 expression. For CREB siRNA and EGFP-PIAS1WT co-transfection experiment, equal volume (0.35 ml) of CREB siRNA (3.5 pmol) and EGFP-PIAS1WT plasmid (0.55 mg) was cotranfected to rat CA1 area. Animals were subjected to perfusion 48 h later and processed for immunohistochemistry. The inner diameter of the injection needle is 0.31 mm and the wall thickness of the injection needle is 0.12 mm each side. The injection needle was left in place for 5 min to limit the diffusion of injected DNA and siRNA. CREB siRNA was transfected 24 h before ERK1/ERK2 plasmid transfection. Animals were sacrificed 48 h after ERK1/ERK2 plasmid transfection. This procedure was adopted because we have previously demonstrated that siRNA transfection lasts longer (>72 h) than plasmid DNA transfection does (>48 h) (Tai et al., 2011). Their brains were removed and the hippocampal tissue slices (2 mm thickness each slice, two slices in all) were dissected out by using a brain slicer. The CA1 tissue was further punched out by using a punch with 1.4 mm in diameter. 2.6. Western blot

For construction of the Myc-tagged erk1 and erk2 plasmids, full-length erk1 and erk2 was cloned by amplifying the rat hippocampal erk1 cDNA and erk2 cDNA with primers 50 -GCTCGGATCCACGCCACCATGGCGGCGGCGGCGGCG-30 and 50 -CTCGAGCG GCCGCCAGGGGGCCTCTGGTGCCCCTGG-30 (for erk1) and primers 50 -GCTCGGATCCACGCCACCATGGCGGCGGCGGCGGCG-30 and 50 -CTCGAGCGGCCGCCAAGATCTGTAT CCTGGCTGGAA-30 (for erk2), respectively. The PCR products were sub-cloned between the BamHI and Not1 sites of the mammalian expression vector pcDNA3.1-Myc-His (Invitrogen, Carlsbad, CA). For construction of the EGFP-tagged erk1 and EGFP-tagged erk2 plasmids, full-length erk1 and erk2 were sub-cloned into the pEGFP-C1 expression vector between the XhoI and HindIII sites. The method used for plasmid DNA transfection to brain tissues was adopted from that of a previous study (Abdallah et al.,1996) with some modification. The non-viral vector transfection reagent polyethyleneimine (PEI) was used because we have previously demonstrated that PEI does not produce toxicity to hippocampal neurons (Chao et al., 2011). Before injection, plasmid DNA was diluted in 5% glucose to a stock concentration of 2.77 mg/ml. Branched PEI of 25 kDa (SigmaeAldrich) was diluted to 0.1 M concentration in 5% glucose. Immediately before injection, 0.1 M PEI was added to the DNA solution (0.45 ml PEI and 0.55 ml plasmid DNA) to reach a ratio of PEI nitrogen per DNA phosphate equals to 10. The final concentration of the plasmid DNA is 1.5 mg/ml. The injection volume is 0.7 ml each side of the hippocampus. The mixture was subjected to vortex for 30 s and allowed to equilibrate for 15 min before transfection. 2.4. RNA interference The rat ERK1 siRNA and ERK2 siRNA were used to knockdown ERK1 and ERK2 expression in CA1 area, respectively. The CREB siRNA was used to knockdown CREB expression in the same area. For ERK1, the sequence for the sense strand is: 50 -GGACCUAAAUUGUAUCAUUtt-30 and that for the antisense strand is: 50 -AAUGAUACAAUUUAGGUCCtc-30 . For ERK2, the sequence for the sense strand is: 50 GGGUAUUCUUGGAUCUCCAtt-30 and that for the antisense strand is: 50 -UGGAGAUCCAAGAAUACCCag-30 . For CREB, the sense and antisense sequences used were adopted from a previous study (Peng et al., 2009). The sense sequence is: 50 -GCACUUAAGGACCUUUACUtt-30 and the antisense sequence is: 50 -AGUAAAGGUCCUUAAGUGCtt-30 . Further, the sense sequence for NR2A is: 50 -GGACUGUAGUGAGGUCGAGtt30 and the antisense sequence for NR2A is: 50 -CUCGACCUCACUACAGUCCtt-30 , which is adopted from the study of Watanabe et al. (2008). The sense sequence for NR2B is: 50 GGAUGAGUCCUCCAUGUUCtt-30 and the antisense sequence for NR2B is: 50 -GAACAUGGAGGACUCAUCCtt-30 , which is adopted from the study of Zhao et al. (2005). The Silencer Negative Control number 1 siRNA (control siRNA) was used as a control. These are the siRNAs with sequences that do not target any gene product (Ambion, Austin, TX). All the siRNAs were synthesized from Ambion. 2.5. Intra-hippocampal drug injection, gene transfection and siRNA transfection Rats were anesthetized with pentobarbital (40 mg/kg, i.p.) and subjected to stereotaxic surgery. Two 23-gauge, stainless-steel, thin-wall cannulae were implanted bilaterally to the CA1 area of the dorsal hippocampus at the following coordinates: 3.5 mm posterior to the bregma, 2.5 mm lateral to the midline, and 3.4 mm ventral to the skull surface. After recovery from the surgery, various drugs (0.7 ml each side), erk1 and erk2 plasmid DNA complex (0.35 ml each, both at 1.5 mg/ml) was injected to the CA1 area at a rate of 0.1 ml/min (total of 0.7 ml each side). For siRNA transfection, the designated siRNA was diluted in distilled

The CA1 tissue lysate was lysed in RIPA buffer (50 mM TriseHCl (pH 7.4), 150 mM NaCl, 2 mM EDTA, 1% IGEPAL CA-630, 1 mM phenymethylsulfonyl fluoride (PMSF), 20 mg/ml pepstatin A, 20 mg/ml leupeptin, 20 mg/ml aprotinin, 50 mM NaF and 1 mM Na3VO4). The lysate was resolved by 8% SDS-PAGE. The proteins resolved by SDS-PAGE were transferred to the polyvinylidene fluoride (PVDF) membrane (Millipore, Bedford, MA) and western blot was conducted by using the following antibodies: rabbit anti-PIAS1 (Epitomics, Burlingame, CA), anti-PIAS2, anti-IL6, anti-TNFa (Abcam, Cambridge, UK), anti-ERK1/2, anti-phospho-ERK1/2, anti-CREB, anti-phospho-CREB, anti-PIAS3, anti-PIAS4, anti-NR2A, anti-NR2B, anti-Akt, antiphospho-Akt (all from Cell Signaling, Danvers, MA), anti-CaMKII, anti-phosphoCaMKII (Millipore), anti-Myc (Signalway Antibody, Pearland, TX) and anti-Actin (Chemicon, Temecula, CA). The secondary antibody used was HRP-conjugated goatanti-rabbit and goatanti-mouse IgG antibodies (Chemicon). Membrane was developed by reacting with chemiluminescence HRP substrate and exposed to the LAS-3000 image system (Fujifilm, Tokyo, Japan) for visualization of protein bands. The protein bands were quantified by using the NIH Image J Software. 2.7. Quantitative real-time PCR Total RNA from CA1 tissue was isolated by using the RNAspin mini kit (GE Healthcare, Barrington, IL). The cDNA was generated from total RNA with Superscript III reverse transcriptase (Invitrogen). Real-time PCR analysis was performed by using the ABI PRISM 7500 real-time PCR system with Power SYBR Green PCR Master Mix according to the instruction manual (Applied Biosystems (ABI), Foster City, CA). The primers for rat pias1 were designed according to a previous report (Kawai-Kowase et al., 2005). The forward sequence is 50 -TCCTGCTGTAGATACAAGCTAC-30 and the reverse sequence is 50 -TGCCAAAGATGGACGCTGTGTC-30 . The primers for rat HPRT were adopted from a previous study (Tai et al., 2011). The forward sequence is 50 GCCGACCGGTTCTGTCAT-30 and the reverse sequence is 50 -TCATAACCTGGTT CATCATCACTAATC-30 . The cycle threshold (Ct) value and data were analyzed by using the 7500 system Sequence Detection Software (ABI). The amount of pias1 gene expression was normalized to that of HPRT gene expression. The relative expression level (in fold) was determined by using the 2(DDCt) method (Livak and Schmittgen, 2001). Q-PCR was also adopted for the mRNA stability assay. The primers for human pias1 were adopted from the study of Munarriz et al. (2004). The forward sequence is 50 -CCAAGCCTTCCTGCTGTAGA-30 and the reverse sequence is 50 -TATCACACAGGCAGTCTTAGAT-30 . The primers for human zif268 were adopted from a previous study (Covington et al., 2010). The forward primer sequence is 50 GCGAGCAGCCCTACGAGCAC-30 and the reverse primer sequence is 50 -TGAGGACGAGGAGGCCGGTG-30 . The primers for human b-actin were adopted from the study of Barber et al. (2005). The forward primer sequence is 50 -TCACCCACACTGT GCCCATCTACGA-30 and the reverse primer sequence is 50 -CAGCGGAACCGCTCATTGCCAATGG-30 . HPRT was used as an internal control. The forward primer sequence for HPRT is 50 -CTTCCTCCTCCTGAGGAGTC-30 and the reverse primer sequence for HPRT is 50 -CCTGACCAAGGAAAGCAAAG-30 . 2.8. Immunohistochemistry Rats were anesthetized with pentobarbital (100 mg/kg, i.p.) and perfused with ice-cold phosphate-buffered saline followed by 4% paraformaldehyde. Brains were removed and post-fixed in 20% sucrose/4% paraformaldehyde solution for 20e

S.Y. Liu et al. / Neuropharmacology 65 (2013) 101e113 48 h. Brains were then frozen, cut into 30-mm sections on a cryostat and mounted on gelatin-coated slides. Brain sections were rinsed with 1 PBS for 10 min and permeabilized with pre-cold EtOH/CH3COOH (95%:5%) for 10 min followed by 1 PBS for 10 min for three times. The sections were preincubated in a blocking solution containing 3% normal goat serum, 3% BSA, and 0.2% Triton X-100 in 1 PBS for 2 h followed by 1 PBS for 10 min for three times. For visualization of endogenous PIAS1 distribution in hippocampal CA1 cell layer, brain sections were incubated with the PIAS1 antibody (1:250, Epitomics) at 4  C overnight. Brain sections were then washed with 1 PBS for 10 min for three times and then incubated with the goat-anti-rabbit secondary antibody conjugated with FITC (Genetex, San Antonio, TX) for 1 h. Immunohistochemistry for MAP2 was used as a neuronal process marker in CA1 cell layer. Similarly, brain sections were incubated with the MAP2 antibody (1:1000, Millipore) at 4  C overnight. Brain sections were then washed with 1 PBS for 10 min for three times and then incubated with the goat-anti-mouse antibody conjugated with Cy3 (Genetex) for 1 h. No control tissue sections were prepared because no fluorescence is present without incubation with the primary and secondary antibody. For immunofluorescence detection of the nucleus, tissue sections were added with 20 ml of the VECTASHIELD mounting medium with DAPI (1.5 mg/ml) (Vector Laboratories, Burlingame, CA). To confirm CREB siRNA transfection, the Cy3 labeled CREB siRNA (synthesized and conjugated by Ambion) was used for intra-hippocampal injection and brain sections were prepared 72 h after siRNA injection for visualization of Cy3 fluorescence under a confocal microscope. To verify ERK plasmid transfection, the EGFP-ERK2 plasmid was used for intra-hippocampal injection and brain sections were prepared 48 h later for visualization of EGFP fluorescence. Digital photomicrographs were taken with an Olympus digital C-3030 camera mounted on a Zeiss confocal microscope. The method used for quantification of PIAS1 fluorescence intensity was adopted from that of Hiscock et al. (2004) with modifications, and the PIAS1 and DAPI fluorescence intensity was quantified using the Metamorph software (version 7.7, Universal Imaging Corporation, Downington, PA). Briefly, two consecutive hippocampal tissue sections containing the CA1 area were used for quantification for each rat. Within each tissue section, three squares covering the CA1 cell layer were chosen for fluorescence quantification (Fig. 1I). The mean value from six squares was adopted. For fluorescence quantification using the Metamorph software, an absolute threshold value was set. Only cells that show fluorescence intensity greater than this value were included in the quantification. The value of PIAS1 fluorescence intensity was normalized with that of DAPI fluorescence intensity and is expressed as the ratio of PIAS1 over DAPI fluorescence intensity. 2.9. Chromatin immunoprecipitation assay (ChIP assay) ChIP assay was performed according to the protocol of Millipore ChIP assay kit (Catalog no. 17-10085). Briefly, HEK293 cells were transfected with the pGL4.10-rat pias1 promoter (2 kb in length) plasmid (0.4 mg) by LipofectamineÔ 2000 (Invitrogen) (4 ml) for 48 h. Cells were then fixed with 1% formaldehyde by adding formaldehyde to the culture medium at room temperature for 10 min. After adding glycine to quench the un-reacted formaldehyde, cells were collected and resuspended in cell lysis buffer plus protease inhibitor cocktail II, and then changed to nuclear lysis buffer plus protease inhibitor cocktail II for sonication. The chromatin was immunoprecipitated using anti-rabbit CREB antibody (Cell Signaling) and rabbit IgG antibody (GeneTex). DNA purified from the immunoprecipitated samples was subjected to PCR reaction using the following primers for the pias1 promoter. The forward primer sequence is: 50 -cagtgacagggatatggaggaaagttc-30 (nucleotide 1446 to 1420) and the reverse primer sequence is: 50 -acctccttccttaacagaatcattttc-30 (nucleotide 1053 to 1079). The PCR product was 393 bp in length and was separated by agarose gel electrophoresis. 2.10. Promoter activity assay Neuro2A cells were maintained in Dulbecco’s modified Eagle’s medium containing 10% fetal calf serum and incubated at 37  C in a humidified atmosphere with 5% CO2. For pias1 reporter assay, the 1.4 kb length of the rat pGL4.10 pias1-luciferase reporter plasmid (0.4 mg) was co-transfected with the CREB siRNA (or control siRNA) (20 pmol) to Neuro2A cells using LipofectamineÔ 2000 (4 ml). The primer set used to obtain the 1.4 kb length rat pias1 promoter is as follows. The forward primer sequence is: 50 -aattaggtaccataggtggaaaggtcagtgc-30 (nucleotide 1248 to 1228) and the reverse primer sequence is: 50 -aattagatatcattcgagctgcgctttac-30 (nucleotide 66 to 43). Luciferase activity assay was performed 48 h later using the Dual-Glo luciferase assay system (Promega) and the TD-20/20 Luminometer (Turner Designs Hydrocarbon Instruments). 2.11. Statistics Biochemical data were analyzed with Student’s t-test or one-way analysis of variance followed by post-hoc NewmaneKeuls comparisons between any two given groups (represented by the q value).

103

3. Results 3.1. NMDA receptor activation increases PIAS1 expression In this study, we examined the role of NMDA receptor involved in PIAS1 expression. In the first experiment, we examined whether blockade of NMDA receptors decreases PIAS1 expression. Separate groups of animals were randomly divided into two groups (n ¼ 6 each group) to receive PBS or a high concentration of MK-801 (20 mM) injection to CA1 area. They were sacrificed at different time points and their hippocampal CA1 tissue was subjected to real-time PCR determination of pias1 mRNA level (1 h) and western blot determination of various protein expressions (30 min for pERK1/2 and pCREB and 2 h for PIAS1). Results revealed that MK801 treatment markedly decreased pias1 mRNA level 1 h later (t1,10 ¼ 5.57, p < 0.001) (Fig. 1A). A representative gel pattern for various western blots is shown in Fig. 1B. Quantitative analyses revealed that MK-801 decreased the level of MAPK/ERK phosphorylation, as indicated by pERK1 and pERK2, 30 min later (t1,10 ¼ 6.06 and t1,10 ¼ 17.14 for pERK1 and pERK2, respectively, both p < 0.001) (Fig. 1C). Meanwhile, it also decreased the level of CREB phosphorylation, as indicated by pCREB at Ser133, 30 min later and decreased the level of PIAS1 2 h later (t1,10 ¼ 10.14 and t1,10 ¼ 20.67 for pCREB and PIAS1, respectively, both p < 0.001) (Fig. 1C). On the other hand, administration of an NMDA receptor agonist produced an opposite effect on these measures in separate groups of animals (n ¼ 6 each group). Results revealed that NMDA treatment (12.5 mM) significantly increased pias1 mRNA level 30 min later (t1,10 ¼ 6.2, p < 0.001) (Fig. 1D, left). Because pias1 mRNA expression is rapidly induced by NMDA treatment, we suspected that the pias1 gene may function as an immediate early gene, which is rapidly induced upon proper stimulation and it regulates neuronal functions through further post-translational modification mechanisms, such as sumoylation of substrate proteins. To address this issue, we have measured the stability of the pias1 mRNA in HEK293T cells in comparison with that of another immediate early gene zif268. The mRNA degradation of a cytoskeleton protein bactin was also measured for comparison. Our result revealed that the pias1 mRNA has a similar degradation rate to that of the zif268 gene after actinomycin D inhibition of transcription over 4 h. But the b-actin mRNA remains more stable (Fig. 1D, right). In different groups of animals, various western blots were conducted at different time points after NMDA treatment and a representative gel pattern is shown in Fig. 1E. Quantitative analyses revealed that NMDA treatment markedly increased the level of pERK1 and pERK2 as early as 15 min after injection (t1,10 ¼ 7.55 and t1,10 ¼ 8.48 for pERK1 and pERK2, respectively, both p < 0.001) (Fig. 1F). Meanwhile, it also increased the level of pCREB at Ser133 15 min later (t1,10 ¼ 9.39, p < 0.001) (Fig. 1F). The PIAS1 protein level was similarly increased by NMDA and it reached a significant level 1 h later (t1,10 ¼ 3.95, p < 0.01) (Fig. 1F). In addition, the effect of NMDA on ERK activation was still observed 30 min later (t1,10 ¼ 4.28, p < 0.01 for pERK1 and t1,10 ¼ 3.21, p < 0.01 for pERK2) and it gradually declined 60 min later (t1,10 ¼ 0.46, p > 0.05 for pERK1 and t1,10 ¼ 0.22, p > 0.05 for pERK2). Similar situation happened to CREB activation. The effect of NMDA on CREB activation was still observed 30 min later (t1,10 ¼ 3.35, p < 0.01), but it is diminished 60 min later (t1,10 ¼ 0.54, p > 0.05). Moreover, the effect of NMDA on PIAS1 expression was not observed 15 min and 30 min later (t1,10 ¼ 0.67, p > 0.05 for 15 min and t1,10 ¼ 0.25, p > 0.05 for 30 min), and it is apparent 60 min later (Fig. 1F). Because both ERK1/2 and CREB are downstream molecules of NMDA receptormediated signaling (Thomas and Huganir, 2004), these results suggest that PIAS1 expression may be up-regulated by the NMDA-

104

S.Y. Liu et al. / Neuropharmacology 65 (2013) 101e113

Fig. 1. NMDA receptor activation up-regulates PIAS1 expression. The NMDA receptor antagonist MK-801 (20 mM) was injected to rat hippocampal CA1 area directly and (A) pias1 mRNA level in CA1 area was measured by quantitative real-time PCR (Q-PCR) 1 h later. (B) A representative gel pattern for various western blots after MK-801 injection is shown. The effect of MK-801 injection on (C) pERK1 and pERK2 levels, pS133CREB and PIAS1 levels at various time points in CA1 area is shown. NMDA (12.5 mM) was directly injected to CA1 area and (D) pias1 mRNA level in CA1 area was measured by Q-PCR 30 min later (left). mRNA degradation of pias1, zif268 and b-actin at various time points after actinomycin D treatment is shown (right). Results are from five independent experiments. (E) A representative gel pattern for various western blots after NMDA injection is shown. The effect of NMDA injection on (F) pERK1 and pERK2 levels, pS133CREB and PIAS1 levels at various time points in CA1 area is shown. N ¼ 6 each group. (G) A representative illustration showing the location of needle placement and dye distribution (3 mg/ml methylene blue) in CA1 area. Arrows indicate the area of dye distribution. Scale bar equals 500 mm. (H) Immunohistochemistry showing the distribution of PIAS1, MAP2 and their merged image (right) in hippocampal CA1 cell layer. Scale bar equals 50 mm (I) A representative illustration showing the square areas selected for PIAS1 and DAPI fluorescence intensity measure in control and NMDA-treated (12.5 mM) animals. Scale bar equals 100 mm. The quantified result is shown on the right. N ¼ 2 each group. Data are expressed as mean  SEM. **p < 0.01 and ***p < 0.001.

S.Y. Liu et al. / Neuropharmacology 65 (2013) 101e113

MAPK/ERK-CREB signaling pathway. Further, because the CA1 tissues from the above experiments were used for western blot analysis, to confirm that the injected drugs were target to the CA1 area, in separate animals we have injected methylene blue dye (3 mg/ml) to the same coordinates. Fig. 1G is a representative illustration showing the location of needle placement and dye distribution in CA1 area. Fig. 1H shows the endogenous distribution of PIAS1 in hippocampal CA1 cell layer. The merged image of PIAS1 and MAP2 showing their co-localization revealed that PIAS1 is mainly distributed in neurons in the CA1 cell layer. Fig. 1I is a representative illustration showing the square areas in CA1 selected for PIAS1 and DAPI fluorescence intensity measure from control and NMDA-treated animals (three squares for each tissue section). The quantified result showed that NMDA treatment significantly increased the fluorescence intensity measure of PIAS1 (relative to DAPI) compared with the PBS control in CA1 pyramidal cell layer (t1,2 ¼ 10.29, p < 0.01) (Fig. 1I, right). 3.2. Role of NMDA receptor subunit NR2A and NR2B in regulation of PIAS1 expression The NMDA receptor is composed of different subunits, including NR2A and NR2B, and they play different roles in the polarity of synaptic plasticity of CA1 neurons (Liu et al., 2004b). Thus, here we examined whether NR2A and NR2B may also play different roles in regulation of PIAS1 expression. Two separate experiments were carried out to examine this issue. In the first experiment, animals were randomly divided into two groups (n ¼ 5 each group) to receive control siRNA or NR2A siRNA transfection. They were sacrificed 72 h later and the CA1 tissue was subjected to western blot analysis of PIAS1 expression. A representative gel pattern is shown in Fig. 2A (left). Statistical analysis revealed that NR2A siRNA transfection markedly decreased the level of NR2A expression (t1,8 ¼ 18.55, p < 0.001). It also decreased the level of PIAS1 expression (approximately 40%, t1,8 ¼ 5.16, p < 0.001) (Fig. 2A, right). Effect of NR2B siRNA transfection on NR2B expression and PIAS1 expression is shown in Fig. 2B (left). Similarly, NR2B transfection markedly decreased the expression level of NR2B (t1,8 ¼ 8.97, p < 0.001). It also decreased the level of PIAS1 expression (approximately 60%, t1,8 ¼ 11.16, p < 0.001) (Fig. 2B, right). 3.3. Effect of NMDA receptor activation on the expression of PIAS1, PIAS2, PIAS3, PIAS4 and IL-6 as well as TNFa The PIAS1 family consists of different members of the PIAS protein, including PIAS1, PIAS2, PIAS3 and PIAS4 (Rytinki et al., 2009). Here, we examined whether NMDA receptor activation may also regulate the expression of the other three PIAS family proteins. Animals were randomly divided into two groups (n ¼ 4 each group) to receive PBS or NMDA injection (12.5 mM). They were sacrificed 1 h later and their CA1 tissues were subjected to western blot analysis of PIAS1, PIAS2, PIAS3 and PIAS4 expression. A representative gel pattern is shown in Fig. 2C (left). Statistical analysis revealed that NMDA treatment consistently increased the expression of PIAS1 (t1,6 ¼ 6.72, p < 0.001), but it did not alter the expression of PIAS2, PIAS3 and PIAS4 from the same rats in CA1 area (t1,6 ¼ 1.54, t1,6 ¼ 1.15 and t1,6 ¼ 0.57 for PIAS2, PIAS3 and PIAS4, respectively; all p > 0.05) (Fig. 2C, right). Because PIAS1 is involved in inflammation, here we also examined whether NMDA receptor-mediated signaling may possibly affect the immune system to alter PIAS1 expression in the hippocampus. Western blot for IL-6 and TNFa, two neuro-inflammatory cytokines in the central nervous system (Arvin et al., 1996), was determined after NMDA and MK-801 treatments. In the first experiment, animals were randomly divided into two groups (n ¼ 6

105

each group) to receive PBS or NMDA (12.5 mM) injection. They were sacrificed 1 h later and the CA1 tissue was subjected to western blot analysis of IL-6 and TNFa expression. A representative gel pattern is shown in Fig. 2D (left). Results revealed that NMDA did not apparently affect the expression of both IL-6 and TNFa (t1,10 ¼ 0.33 for IL-6 and t1,10 ¼ 1.43 for TNFa, both p > 0.05). The effectiveness of NMDA injection is confirmed by an apparent increase in the phosphorylation level of ERK1 and ERK2 15 min later in separate animals (t1,10 ¼ 13.77 for pERK1 and t1,10 ¼ 5.26 for pERK2, both p < 0.001) (Fig. 2D, right). In the second experiment, animals were randomly divided into two groups (n ¼ 6 each group) to receive PBS or MK-801 (20 mM) injection. They were sacrificed 2 h later and the CA1 tissue was subjected to western blot analysis of IL-6 and TNFa expression. A representative gel pattern is shown in Fig. 2E (left). Results revealed that MK-801 did not affect the expression of either IL-6 or TNFa (t1,10 ¼ 0.69 for IL-6 and t1,10 ¼ 0.76 for TNFa, both p > 0.05). The effectiveness of MK-801 injection is confirmed by an apparent decrease in the phosphorylation level of ERK1 and ERK2 30 min later in separate animals (t1,10 ¼ 7.1 for pERK1 and t1,10 ¼ 9.86 for pERK2, both p < 0.001) (Fig. 2E, right). 3.4. ERK1/2 and CREB mediate the expression of PIAS1 In this experiment, we examine whether ERK1/2 activation regulates the expression of PIAS1. Animals were randomly divided into two groups to receive control siRNA or ERK1/2 siRNA (3.5 pmol each) transfection (n ¼ 6 each group). Animals were sacrificed 72 h later and their hippocampal CA1 tissue was subjected to western blot analysis of various protein expressions and real-time PCR determination of pias1 mRNA level. Results revealed that ERK1/2 siRNA transfection markedly decreased pias1 mRNA level in CA1 area (t1,10 ¼ 6.13, p < 0.001) (Fig. 3A). A representative gel pattern of western blot is shown in Fig. 3B. Quantitative analyses revealed that ERK1/2 siRNA decreased the level of ERK1 and ERK2 (t1,10 ¼ 7.41 and t1,10 ¼ 12.26 for ERK1 and ERK2, respectively, both p < 0.001) (Fig. 3C), confirming the knockdown effect of ERK1/2 siRNA transfection. It also decreased the level of pCREB at Ser133 and the level of PIAS1 (t1,10 ¼ 17.33 and t1,10 ¼ 9.69 for pCREB and PIAS1, respectively, both p < 0.001) (Fig. 3C). We next examined whether PIAS1 is regulated by CREB. A separate group of animals were randomly divided into two groups to receive control siRNA or CREB siRNA (7 pmol) transfection (n ¼ 6 each group). Animals were sacrificed 72 h later and their hippocampal CA1 tissue was subjected to western blot analysis of CREB and PIAS1 protein expression and real-time PCR analysis of pias1 mRNA expression. Results revealed that CREB siRNA transfection markedly decreased pias1 mRNA level (t1,10 ¼ 6.99, p < 0.001) (Fig. 3D). A representative gel pattern for western blot is shown in Fig. 3E (left). Quantitative analyses revealed that CREB siRNA markedly decreased the level of CREB (t1,10 ¼ 9.93, p < 0.001) (Fig. 3E), confirming the knockdown effect of CREB siRNA transfection. It also decreased the level of PIAS1 (t1,10 ¼ 9.48, p < 0.001) (Fig. 3E). Immunohistochemistry against Cy3 indicated that CREB siRNA was indeed transfected to the CA1 area (Fig. 3F). The average area of CREB siRNA transfection is approximately 33% of total CA1 area viewed from a single plane as indicated by arrows (Fig. 3F, upper-left panel). Tissue sessions were also added with DAPI for visualization of transfection to individual cells at higher magnifications. Cells that showed double staining of red fluorescence (Cy3) and blue fluorescence (DAPI) are cells successfully transfected with the Cy3-CREB siRNA (Fig. 3F, upper-right panel). The entire transfection area is also shown in consecutive tissue sections (Fig. 3F, lower panel). It is approximately 830 mm in length. To confirm that PIAS1 expression is significantly reduced in CREB siRNAtransfected cells, we have co-transfected Cy3-CREB siRNA

106

S.Y. Liu et al. / Neuropharmacology 65 (2013) 101e113

Fig. 2. NR2A and NR2B both regulate the expression of PIAS1, and NMDA receptor activation preferentially up-regulates PIAS1 expression without affecting the expression of IL-6 and TNFa. (A) NR2A siRNA was transfected to rat hippocampal CA1 area and the expression of NR2A and PIAS1 was measured by western blot 72 h later. (B) NR2B siRNA was transfected to rat CA1 area and the expression of NR2B and PIAS1 was measured by western blot 72 h later. N ¼ 5 each group. (C) The effect of NMDA injection (12.5 mM) on the expression of PIAS1, PIAS2, PIAS3 and PIAS4 was measured by western blot 1 h later. A representative gel pattern is shown on the left and the quantified result is shown on the right. N ¼ 4 each group. (D) The effect of NMDA injection (12.5 mM) on the expression of IL-6, TNFa, pERK1 and pERK2 was measured by western blot at various time points. A representative gel pattern is shown on the left and the quantified result is shown on the right. N ¼ 6 each group. (E) The effect of MK-801 injection (20 mM) on the expression of IL6, TNFa, pERK1 and pERK2 was measured by western blot at various time points. A representative gel pattern is shown on the left and the quantified result is shown on the right. N ¼ 6 each group. Data are expressed as mean  SEM. ***p < 0.001.

(3.5 pmol, 0.35 ml) with EGFP-PIAS1WT plasmid (0.55 mg, 0.35 ml) to rat CA1 area. Control animals received control siRNA (0.35 ml)þ EGFP-PIAS1WT plasmid (0.55 mg, 0.35 ml) transfection (n ¼ 2 each group). We then compared the EGFP fluorescence intensity in these two groups of rats (two consecutive tissue sections from each rat were taken for fluorescence intensity quantification). A representative immunohistochemistry illustration for each treatment is shown in Fig. 3G. Results revealed that for the Cy3-CREB siRNA and EGFP-PIAS1WT co-transfected animals, the EGFP fluorescence

intensity is reduced for approximately 58% compared with the control siRNA and EGFP-PIAS1WT co-transfected animals (t1,2 ¼ 10.95, p < 0.01) (Fig. 3G, right). Next, we examined whether pias1 is a CREB target gene. Rat pias1 promoter (2 kb) was transfected to HEK293T cells and ChIP PCR assay was performed 48 h later using rabbit anti-CREB antibody (or rabbit IgG antibody) and the pias1 promoter (2 kb in length), and the result revealed that CREB indeed binds to the pias1 promoter (Fig. 3H, upper panel). We next performed the pias1

S.Y. Liu et al. / Neuropharmacology 65 (2013) 101e113

107

Fig. 3. ERK1/2 siRNA interference and CREB siRNA interference both down-regulate PIAS1 expression. ERK1 siRNA and ERK2 siRNA were transfected to rat CA1 area and (A) pias1 mRNA level in CA1 area was measured by Q-PCR. (B) A representative gel pattern for various western blots after ERK1/ERK2 siRNA transfection is shown. (C) The effect of ERK1/ERK2 siRNA transfection on ERK1 and ERK2 levels, pS133CREB and PIAS1 levels in CA1 area is shown. (D) CREB siRNA was transfected to CA1 area and pias1 mRNA level was measured by Q-PCR. N ¼ 6 each group. (E) CREB siRNA was transfected to CA1 area and the expression of CREB and PIAS1 was shown. (F) Immunohistochemistry staining against Cy3-CREB siRNA and DAPI showing the area of CREB siRNA transfection in CA1 layer (as indicated by arrows) and in individual neurons in CA1 area at different magnifications. Scale bar equals 500 mm and 20 mm for the upper-left and upper-right panels, respectively and scale bar equals 25 mm for the lower panel. (G) EGFP-PIAS1WT plasmid was co-transfected with CREB siRNA (or control siRNA) to rat CA1 area and the EGFP (PIAS1) fluorescence intensity was measured. A representative illustration showing the square areas selected for EGFP (PIAS1) and DAPI fluorescence intensity measure is shown on the left. Scale bar equals 25 mm. The quantified result is shown on the right. N ¼ 2 each group. (H) Chromatin immunoprecipitation (ChIP) PCR assay conducted in HEK293T cells shows that endogenous CREB binds to the pias1 promoter. Experiments are in triplicates (upper). Bioinformatic analysis of the pias1 promoter shows that there are two CRE elements (CREB binding sites) within 1.4 kb length of the pias1 promoter. The locations of these CRE elements relative to the ATG start codon are also shown (lower). This pias1-luciferase promoter construct was used for the promoter activity assay. (I) CREB siRNA (20 pmol, 1 ml) (or control siRNA) was cotransfected with the pias1 promoter construct (0.4 mg) to Neuro2A cells and pias1 promoter activity was measured by luciferase activity assay (left). Effect of CREB siRNA transfection on the expression level of CREB is shown. Results are from five independent experiments. Data are expressed as mean  SEM. **p < 0.01 and ***p < 0.001.

108

S.Y. Liu et al. / Neuropharmacology 65 (2013) 101e113

promoter activity assay to examine whether CREB regulates the pias1 promoter activity. Bioinformatic analysis revealed that the pias1 promoter (1.4 kb in length) contains two CRE elements (Fig. 3H, lower panel). This length of the pias1 promoter was cloned to the pGL4.10 vector and co-transfected with the CREB siRNA (or control siRNA) to Neuro2A cells. Results revealed that transfection of CREB siRNA significantly decreased pias1 promoter activity (t1,8 ¼ 16.91, p < 0.001) (Fig. 3I, left). The effectiveness of CREB siRNA transfection was confirmed by a marked reduction of the CREB expression level (t1.8 ¼ 14.73, p < 0.001) (Fig. 3I, right). 3.5. NMDA receptor activation enhances the expression of PIAS1 through ERK1/2 The above results demonstrated that both NMDA receptor activation and ERK1/2 mediate the expression of PIAS1, but it is

not known whether NMDA receptor activation enhances PIAS1 expression through the mediation of ERK1/2. This hypothesis was tested here. Animals were randomly divided into three groups to receive PBS þ control siRNA, NMDA (12.5 mM)þcontrol siRNA or NMDA (12.5 mM)þERK1/2 siRNA (1.75 pmol each) treatments (n ¼ 6 each group). A total of 3.5 pmol ERK1/2 siRNA (equally mixed) was adopted here because we aimed to use a subthreshold concentration of ERK1/2 siRNA to examine whether it interferes with the activation effect of NMDA. ERK1/2 siRNA was transfected 72 h before NMDA injection and animals were sacrificed 15 min after NMDA treatment. The expression level of pERK1, pERK2 and pCREB was determined by western blot. In a separate group of animals, rats were divided into the same groups and were sacrificed 60 min after NMDA injection for determination of ERK1, ERK2, CREB and PIAS1 protein levels. A representative gel pattern of various western blots is shown in

Fig. 4. ERK1/2 siRNA blocks the enhancing effect of NMDA on PIAS1 expression. NMDA (12.5 mM) was injected to rat CA1 area along with ERK1/2 siRNA transfection (or control siRNA transfection). (A) A representative gel pattern for various western blots after these treatments is shown. Effects of these treatments on protein expression of (B) pERK1 and pERK2 (C) ERK1 and ERK2 (D) pS133CREB and PIAS1 are shown. N ¼ 6 each group. (E) ERK1/2 siRNA (or control siRNA) was transfected to rat CA1 area and PIAS1 fluorescence intensity was measured. A representative illustration showing the square areas selected for PIAS1 and DAPI fluorescence intensity measure is illustrated on the left. Scale bar equals 50 mm. The quantified result is shown on the right. Data are expressed as mean  SEM. ***p < 0.001.

S.Y. Liu et al. / Neuropharmacology 65 (2013) 101e113

Fig. 4A. Further analyses indicated that NMDA treatment significantly increased the level of pERK1 and pERK2 15 min later (F2,15 ¼ 43.36, p < 0.001; q ¼ 10.53, p < 0.001 for pERK1 and F2,15 ¼ 48.91, p < 0.001; q ¼ 12.46, p < 0.001 for pERK2), but these effects were blocked by prior ERK1/2 siRNA transfection (q ¼ 12.12, p < 0.001 for pERK1 and q ¼ 11.73, p < 0.001 for pERK2 comparing the NMDA þ ERK1/2 siRNA group with NMDA group) (Fig. 4B). NMDA treatment also increased the level of pCREB (F2,15 ¼ 41.33, p < 0.001; q ¼ 11.31, p < 0.001) and PIAS1 (F2,15 ¼ 771.96, p < 0.001; q ¼ 26.67, p < 0.001), and these effects were similarly blocked by prior ERK1/2 siRNA transfection (q ¼ 10.96, p < 0.001 and q ¼ 55.55, p < 0.001 for pCREB and PIAS1 comparing the NMDA þ ERK1/2 siRNA group with NMDA group) (Fig. 4D). NMDA alone did not apparently affect the level of ERK1 and ERK2, and the effectiveness of ERK1/2 siRNA transfection is verified by a reduced level of ERK1 and ERK2 expression (F2,15 ¼ 37.1, p < 0.001; q ¼ 11.09, p < 0.001 for ERK1 and F2,15 ¼ 50.3, p < 0.001; q ¼ 12.64, p < 0.001 for ERK2 comparing the NMDA þ ERK1/2 siRNA group with NMDA group) (Fig. 4C). Because ERK1/2 siRNA completely blocked the effect of NMDA on CREB activation and PIAS1 expression, we have further analyzed the effect of ERK1/2 siRNA on PIAS1 expression using immunohistochemsitry fluorescence quantification. Fig. 4E is a representative illustration showing the square areas in CA1 selected for PIAS1 and DAPI fluorescence intensity measure from control siRNA and ERK1/2 siRNA-treated animals (three squares for each tissue section). The quantified result showed that ERK1/2 siRNA transfection significantly decreased the fluorescence intensity measure of PIAS1 (relative to DAPI) in CA1 cell layer for approximately 60% (t1,2 ¼ 23.96, p < 0.001) (Fig. 4E, right). These results together suggest that ERK1/2 strongly regulates the expression of PIAS1 and that knockdown of ERK1/2 expression for approximately 50% completely blocked the effect of NMDA receptormediated signaling. 3.6. NMDA receptor activation enhances the expression of PIAS1 through CREB The above results indicated that NMDA receptor-mediated signaling to MAPK/ERK regulates the expression of PIAS1 and CREB also mediates PIAS1 expression, but it is not known whether NMDA receptor activation regulates the expression of PIAS1 through the activation of CREB. This issue was examined here. Animals were randomly divided into three groups to receive PBS þ control siRNA, NMDA (12.5 mM) þ control siRNA or NMDA (12.5 mM) þ CREB siRNA (3.5 pmol) treatments (n ¼ 6 each group). For the same reason as described above, a sub-threshold concentration of CREB siRNA was used here and it was transfected 72 h before NMDA injection. Animals were sacrificed 15 min after NMDA treatment for pCREB determination by western blot. In a separate group of animals, rats were divided into the same groups and were sacrificed 60 min after NMDA injection for determination of CREB and PIAS1 protein levels. A representative gel pattern of western blot is shown in Fig. 5A. Further analyses indicated that NMDA treatment significantly increased the level of pCREB (F2,15 ¼ 63.78, p < 0.001; q ¼ 14.62, p < 0.001), but this effect was blocked by prior CREB siRNA transfection (q ¼ 12.88, p < 0.001 comparing the NMDA þ CREB siRNA group with NMDA group) (Fig. 5B). NMDA also increased PIAS1 protein level (F2,15 ¼ 52.61, p < 0.001; q ¼ 8.17, p < 0.001) and this effect is similarly blocked by prior CREB siRBA transfection (q ¼ 14.47, p < 0.001 comparing the NMDA þ CREB siRNA group with NMDA group) (Fig. 5C). NMDA alone did not affect the CREB expression level and the effectiveness of CREB siRNA transfection is confirmed by a decreased level of CREB expression (F2,15 ¼ 29.17, p < 0.001;

109

Fig. 5. CREB siRNA blocks the enhancing effect of NMDA on PIAS1 expression. NMDA (12.5 mM) was injected to rat CA1 area together with CREB siRNA transfection (or control siRNA transfection). (A) A representative gel pattern for various western blots after these treatments is shown. Effects of these treatments on protein expression of (B) pS133CREB and CREB (C) PIAS1 are shown. N ¼ 6 each group. Data are expressed as mean  SEM. ***p < 0.001.

q ¼ 8.98, p < 0.001 comparing the NMDA þ CREB siRNA group with NMDA group) (Fig. 5B). 3.7. MAPK/ERK signaling to CREB mediates the expression of PIAS1 The above results demonstrated that PIAS1 expression is regulated by MAPK/ERK-mediated signaling and by CREB, but it is not known whether MAPK/ERK signaling to CREB mediates the expression of PIAS1. This hypothesis was examined here. Animals were randomly divided into three groups to receive Mycvector þ control siRNA, Myc-ERK1 and Myc-ERK2 (0.55 mg each)þ control siRNA or Myc-ERK1 and Myc-ERK2 (0.55 mg each)þCREB siRNA (3.5 pmol) treatment (n ¼ 6 each group). Similarly, a subthreshold concentration of CREB siRNA was used here. CREB siRNA was transfected 24 h before the Myc-ERK1/2 plasmids were transfected, and animals were sacrificed 48 h after Myc-ERK1/2 plasmids transfection. A representative gel patter for western blot is shown in Fig. 6A. Results revealed that Myc-ERK1 and Myc-ERK2 plasmid transfection markedly increased the level pERK1 (F2,15 ¼ 8.82, p < 0.01; q ¼ 5.28, p < 0.01) and pERK2 (F2,15 ¼ 34.66, p < 0.001; q ¼ 10.39, p < 0.001), but CREB siRNA transfection did not affect these measures (q ¼ 0.29, p > 0.05 for pERK1 and q ¼ 0.39, p > 0.05 for pERK2 comparing the Myc-ERK þ CREB siRNA group with the Myc-ERK group) (Fig. 6B). Myc-ERK plasmid transfection also increased the level of pCREB (F2,15 ¼ 43.75, p < 0.001; q ¼ 12.19, p < 0.001), but this effect was blocked by CREB siRNA co-transfection (q ¼ 10.55, p < 0.001 comparing the MycERK þ CREB siRNA group with the Myc-ERK group) (Fig. 6C). Meanwhile, Myc-ERK plasmid transfection also increased the level of PIAS1 (F2,15 ¼ 80.67, p < 0.001; q ¼ 17.76, p < 0.001), and this effect was similarly blocked by CREB siRNA co-transfection (q ¼ 11.22, p < 0.001 comparing the Myc-ERK þ CREB siRNA group with the Myc-ERK group) (Fig. 6C). Myc-ERK1/2 plasmid transfection and expression was confirmed by anti-Myc western blot (Fig. 6A). In addition, immunofluorescence staining of EGFP-

110

S.Y. Liu et al. / Neuropharmacology 65 (2013) 101e113

3.8. PI-3 kinase signaling and CaMK signaling also regulate the expression of PIAS1 In addition to NMDA receptor signaling, other pathways are also implicated in neuronal plasticity and memory function, such as the PI-3 kinase signaling (Lin et al., 2001; Horwood et al., 2006) and the CaMKII signaling pathways (Cammarota et al., 2002; Colbran and Brown, 2004). Therefore, we also examined whether PIAS1 expression could be regulated by the PI-3 kinase signaling and CaMKII signaling. Results revealed that at a concentration of LY294002 (1.5 mg/ml) that apparently decreased Akt phosphorylation (t1,10 ¼ 9.28, p < 0.001), it also significantly decreased the level of CREB phosphorylation (t1,10 ¼ 3.69, p < 0.01) and PIAS1 expression (t1,10 ¼ 5.80, p < 0.001) (n ¼ 6 each group) (Fig. 7A). Since LY294002 may have off target effects, here we also examined the effect of another PI-3K inhibitor wortmannin on PIAS1 expression. Results revealed that wortmannin (1 mg/ml) similarly decreased Akt phosphorylation (t1,8 ¼ 5.42, p < 0.001), it also decreased the level of CREB phosphorylation (t1,8 ¼ 4.33, p < 0.01) and PIAS1 expression (t1,8 ¼ 8.55, p < 0.001) (n ¼ 5 each group) (Fig. 7B). Moreover, at a concentration of KN-62 (7.2 ng/ml) that decreased CaMKII phosphorylation (t1,10 ¼ 21.85, p < 0.001), it also markedly decreased CREB phosphorylation (t1,10 ¼ 12.71, p < 0.001) and PIAS1 expression (t1,10 ¼ 12.29, p < 0.001) (n ¼ 6 each group) (Fig. 7C). In a separate group of animals, we have also examined the effect of another CaMKII inhibitor KN-93 on the level of CREB phosphorylation and PIAS1 expression. Results revealed that KN-93 injection (5 mg/ml) decreased CaMKII phosphorylation (t1,8 ¼ 4.35, p < 0.01). Meanwhile, it also markedly decreased CREB phosphorylation (t1,8 ¼ 3.54, p < 0.01) and PIAS1 expression (t1,8 ¼ 7.89, p < 0.001) (n ¼ 5 each group) (Fig. 7D). 4. Discussion

Fig. 6. CREB siRNA blocks the enhancing effect of ERK1/2 overexpression on PIAS1 expression. Myc-ERK1 and Myc-ERK2 plasmids were transfected to rat CA1 area together with CREB siRNA (or control siRNA). (A) A representative gel pattern for various western blots after these treatments is shown. Effects of these treatments on protein expression of (B) pERK1 and pERK2 (C) pS133CREB and PIAS1 are shown. N ¼ 6 each group. Data are expressed as mean  SEM. (D) Immunohistochemistry staining against EGFP and DAPI shows the area of EGFP-ERK1 and EGFP-ERK2 transfection and expression in CA1 cell layer (as indicated by arrows) and in individual neurons in CA1 area at different magnifications. Scale bar equals 400 mm and 50 mm for the upper-left and upper-right panels, respectively and scale bar equals 25 mm for the lower panel. **p < 0.01 and ***p < 0.001.

ERK1 and EGFP-ERK2 (mixed plasmid) showed that the ERK plasmid is indeed transfected and expressed in CA1 neurons, as indicated by the arrows (Fig. 6D, upper-left panel). The entire transfection area is approximately 500 mm in length that is about 22% of total CA1 area viewed from a single plane (Fig. 6D, lower panel).

The present results demonstrated that NMDA receptor activation up-regulates PIAS1 expression via the activation of MAPK/ERK and CREB. These results are consistent with the literature that NMDA receptors play an important role in synaptic plasticity as well as learning and memory function. They are also congruent with the notion that MAPK/ERK and CREB play key roles in mediating long-term memory formation (Sweatt, 2004; Silva et al., 1998) and that MAPK/ERK signaling targets to CREB to control long-term potentiation-dependent gene expression (Davis et al., 2000). These results also support our previous finding that PIAS1 facilitates spatial learning and memory in rats (Tai et al., 2011). On the other hand, there is usually little NMDA receptor activation under basal conditions, but we have observed a significant reduction of PIAS1 expression under MK-801 treatment. This is probably because that we have used a high concentration of MK-801 for this experiment (20 mM) which completely suppresses the basal activity of NMDA receptor. In addition, certain degree of NMDA receptor activation may still be present because MK-801 was injected to awake and slightly moving animals under handling. When we have examined the effect of lower concentrations of MK-801 (5 mM and 10 mM), they did not apparently alter PIAS1 expression (Supplemental Fig. S1). Moreover, other than the pivotal role that NMDA receptors play in synaptic plasticity and learning and memory function (Collingridge et al., 2013), NMDA (10 nmol) is also known to produce moderate excito-toxicity to hippocampal neurons both in vitro and in vivo (Kimonides et al., 1998). Thus, we have also examined the possible excito-toxicity of NMDA on CA1 neurons in vivo by TUNEL assay. Immunohistochemical results revealed that under the concentration (12.5 mM, equivalent to 10 nmol when 0.8 ml is injected) and time interval (1 h) of NMDA treatment adopted in the present study, it did not produce toxicity to CA1

S.Y. Liu et al. / Neuropharmacology 65 (2013) 101e113

111

Fig. 7. PI-3 kinase signaling and CaMKII signaling regulate PIAS1 expression. (A) The PI-3 kinase inhibitor LY294002 (1.5 mg/ml) was directly injected to rat CA1 area, and a representative gel pattern for various western blots as well as the quantitative results for these measures are shown (N ¼ 6 each group). (B) Another PI-3 kinase inhibitor wortmannin (1 mg/ml) was similarly injected to rat CA1 area. A representative gel pattern for various western blots is shown on the left. The quantified results of these measures are shown on the right (N ¼ 5 each group). (C) A CaMKII inhibitor KN-62 (7.2 ng/ml) was directly injected to rat CA1 area. A representative gel pattern for various western blots is shown on the left. The quantified results of these measures are shown on the right (N ¼ 6 each group). (D) Another CaMKII inhibitor KN-93 (5 mg/ml) was similarly injected to rat CA1 area. A representative gel pattern for various western blots is shown on the left. The quantified results of these measures are shown on the right (N ¼ 5 each group). Data are expressed as mean  SEM. **p < 0.01 and ***p < 0.001.

neurons. Kainic acid treatment (0.4 mg) was used as a positive control (Supplemental Fig. S2). This discrepancy is probably because that in the study of Kimonides et al. (1998), a 3-day postinjection interval was used; while in the present study, various biochemical assays were examined 15 min to 1 h after NMDA injection. If there is any possible excito-toxicity of NMDA, it may not have developed yet. Furthermore, since NMDA up-regulated the expression of PIAS1, we also examined whether overexpression of PIAS1 may produce any toxicity to CA1 neurons. Results revealed that transfection of PIASWT plasmid to CA1 neurons did not

produce toxicity to these neurons as determined by TUNEL assay (Supplemental Fig. S2, the same kainic acid treatment was used as a positive control). Because both NMDA and PIAS1 did not produce excito-toxicity to hippocampal neurons, we have excluded the possibility that PIAS1 may be involved in NMDA-driven cell death. The present results together suggest that the pias1 gene is involved in neuronal plasticity. This suggestion is supported by the findings that PIAS1 enhances the sumoylation of focal adhesion kinase (FAK) to activate its autophosphorylation (Kadare et al., 2003) and FAK activation plays an important role in synaptic

112

S.Y. Liu et al. / Neuropharmacology 65 (2013) 101e113

plasticity (Yang et al., 2003). It is also supported by the findings that PIAS1 promotes the sumoylation of metabotropic glutamate receptor 8 (Tang et al., 2005), whereas metabotropic glutamate receptors are involved in regulating synaptic activity (Luscher and Huber, 2010). Furthermore, PIAS1 was found to interact with the CREB-binding protein (CBP) (Yin et al., 2005), whereas CBP is a transcriptional coactivator for CREB and the CREB/CBP complex is suggested to mediate the memory-enhancing effect of histone deacetylase (HDAC) inhibitors through regulation of target gene expression (Vecsey et al., 2007). Moreover, our results revealed that PIAS1 expression is also regulated by PI-3 kinase signaling through the mediation of CREB because both LY294002 and wortmannin treatments down-regulated the expression of PIAS1 accompanied by a decreased phosphorylation level of CREB. In addition, PIAS1 expression is perhaps regulated by the CaMK family signaling through the regulation of CREB also. This is supported by our results that both KN-62 and KN-93 treatments inhibited the expression of PIAS1 and the phosphorylation level of CREB. Further, CREB was found to be a downstream effector of CaMKI that regulates the expression of Wnt-2 (Wayman et al., 2006). These results are congruent with the report that CREB is a regulatory target for Akt (Du and Montminy, 1998) and they together suggest the important role of CREB in converging a diverse upstream signaling to regulate PIAS1 expression. Further, our results from ChIP assay and promoter activity assay revealed that there is a direct effect of CREB in regulation of pias1 gene expression. In addition, previous studies have shown that PIAS1 blocks the DNA binding activity of STAT1 and inhibits STAT1-mediated gene transcription (Liu et al., 1998; Liao et al., 2000). We have presently found that NMDA receptor activation up-regulates PIAS1 expression; thus, we have further examined the effect of NMDA receptor activation on STAT1 function. Consistent with our previous report that overexpression of PIAS1 decreases STAT1 DNA binding activity (Tai et al., 2011), the present result revealed that NMDA administration decreased STAT1 DNA binding activity at the same time it increased PIAS1 expression without markedly alter the level of STAT1 expression (Supplemental Fig. S3). In the present study, we found that pias1 mRNA expression is rapidly induced upon NMDA administration. But NMDA administration no longer increased pias1 mRNA level at 60 min later (Supplemental Fig. S4A). Further, NMDA no longer enhanced PIAS1 protein expression at 3 h later (Supplemental Fig. S4B). These results are congruent with our present result that PIAS1 expression is rapidly induced by NMDA stimulation and are consistent with our previous report that PIAS1 expression is rapidly induced after spatial training in rats, but this effect is significantly diminished at later time points (Tai et al., 2011). Moreover, because the PIAS family contains other PIAS proteins in addition to PIAS1, we also examined whether NMDA receptor signaling may regulate the expression of the other three PIAS family proteins. Our results revealed that PIAS1 is preferentially regulated by stimuli that activate the NMDA receptor-mediated signaling, for example, glutamate release. But this result does not exclude the possibility that PIAS2, PIAS3 and PIAS4 may be regulated by other non-NMDAmediated signaling, such as brain-derived neurotrophic factor. In this study, CREB siRNA was transfected to approximately onethird of the CA1 cell layer (approximately 830 mm, Fig. 3F) and ERK plasmid was transfected to only about 22% of the CA1 cell layer (approximately 500 mm, Fig. 6D) viewed from a single plane of 200 mm-thickness tissue sessions, but with this limited area of transfection, significant biochemical effects from western blot were observed. One explanation is that the inner diameter of the tissue punch is 1.4 mm, thus, the transfected area counts for approximately 35e60% (in diameter) of the punched area for biochemical assay. The detailed illustration of the transfection area and the

punched tissue is shown elsewhere (Chao et al., 2011, Supplemental Fig. S2). On the other hand, within the punched CA1 area, there are presumably more neuronal processes and fibers than cells (pyramidal cells), but the protein extraction method we have adopted is suitable for extraction of proteins in the cell. When western blot was performed, total amount of proteins, instead of total tissue volume, was adopted for the assay. Therefore, the percentage of transfected cells should be much higher than we estimated. When we have used immunohistochemistry fluorescence quantification to assess the effect of CREB siRNA on PIAS1 expression, we have observed about 58% decrease in PIAS1 fluorescence intensity (Fig. 3G), which is comparable to the effect of CREB siRNA on PIAS1 expression analyzed by western blot (about 62%) (Fig. 3E). In another study, the CREB plasmid was similarly transfected to approximately 20% of amygdala cells, but it successfully rescues fear memory deficits in CREB-deficient mice (Han et al., 2007). These results together suggest that activation of a specific sub-population of neurons is sufficient to mediate the signaling cascade they transduce. But the molecular mechanisms and communications within this sub-group of neurons require further investigation. In summary, PIAS1 is well documented to play a role in inflammation and the innate immune response, but PIAS1 is rarely studied in the brain. How is PIAS1 regulated is also not known. Here we first demonstrated that the NMDA receptor-mediated signaling pathway regulates pias1 gene expression in the brain. Because CREB integrates diverse upstream signals for transcriptional activation (Shaywitz and Greenberg,1999), it is possible that PIAS1 expression is also regulated by other stimuli in addition to NMDA receptor activation. Besides, it is as important to identify other transcription factors that may also regulate PIAS1 expression and identify other physiological functions that PIAS1 may also participate in future studies. Acknowledgments This work was supported by a Grant funded by the National Science Council of Taiwan (NSC100-2321-B-001-014) and research fund from the Institute of Biomedical Sciences, Academia Sinica, Taiwan. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.neuropharm.2012.08.024. References Abdallah, B., Hassas, A., Benoist, C., Goula, D., Behr, J.P., et al., 1996. A powerful nonviral vector for in vivo gene transfer into the adult mammalian brain: polyethylenimine. Hum. Gene Ther. 7, 1947e1954. Arvin, B., Neville, L.F., Barone, F.C., Feuerstein, G.Z., 1996. The role of inflammation and cytokines in brain injury. Neurosci. Biobehav. Rev. 20, 445e452. Barber, R.D., Harmer, D.W., Coleman, R.A., Clark, B.J., 2005. GAPDH as a housekeeping gene: analysis of GAPDH mRNA expression in a panel of 72 human tissues. Physiol. Genomics 21, 389e395. Cammarota, M., Bevilaqua, L.R.M., Viola, H., Kerr, D.S., Reichmann, B., et al., 2002. Participation of CaMKII in neuronal plasticity and memory formation. Cell. Mol. Neurobiol. 22, 259e267. Chao, C.C., Ma, Y.L., Lee, E.H.Y., 2011. BDNF enhances Bcl-xL expression through protein kinase CK2-activated and NF-kB-mediated pathway in rat hippocampus. Brain Pathol. 21, 150e162. Colbran, R., Brown, A.M., 2004. Calcium/calmodulin-dependent protein kinase II and synaptic plasticity. Curr. Opin. Neurobiol. 14, 318e327. Collingridge, G.L., 1987. Synaptic plasticity: the role of NMDA receptors in learning and memory. Nature 330, 604e605. Collingridge, G.L., Volianskis, A., Bannister, N., France, G., Hanna, L., et al., 2013. The NMDA receptor as a target for cognitive enhancement. Neuropharmacology 64, 13e26. Covington 3rd, H.E., Lobo, M.K., Maze, I., Vialou, V., Hyman, J.M., et al., 2010. Antidepressant effect of optogenetic stimulation of the medial prefrontal cortex. J. Neurosci. 30, 16082e16090.

S.Y. Liu et al. / Neuropharmacology 65 (2013) 101e113 Davis, S., Vanhoutte, P., Pages, C., Caboche, J., Laroche, S., 2000. The MAPK/ERK cascade targets both Elk-1 and cAMP response element-binding protein to control long-term potentiation-dependent gene expression in the dentate gyrus in vivo. J. Neurosci. 20, 4563e4572. Du, K., Montminy, M., 1998. CREB is a regulatory target for the protein kinase Akt/ PKB. J. Biol. Chem. 273, 32377e32379. Han, J.H., Kushner, S.A., Yiu, A.P., Cole, C.J., Matynia, A., et al., 2007. Neuronal competition and selection during memory formation. Science 316, 457e460. Hiscock, N., Chan, M.H.S., Bisucci, T., Darby, I.A., Febbraio, M.A., 2004. Skeletal myocytes are a source of interleukin-6 mRNA expression and protein release during contraction: evidence of fiber type specificity. FASEB J. 18, 992e994. Horwood, J.M., Dufour, F., Laroche, S., Davis, S., 2006. Signalling mechanisms mediated by the phosphoinositide 3-kinase/Akt cascade in synaptic plasticity and memory in rats. Eur. J. Neurosci. 23, 3375e3384. Izquierdo, I., 1991. Role of NMDA receptors in memory. Trends Pharmacol. Sci. 12, 128e129. Kadare, G., Toutant, M., Formstecher, E., Corvol, J.C., Carnaud, M., et al., 2003. PIAS1mediated sumoylation of focal adhesion kinase activates its autophosphorylation. J. Biol. Chem. 278, 47434e47440. Kahyo, T., Nishida, T., Yasuda, H., 2001. Involvement of PIAS1 in the sumoylation of tumor suppressor p53. Mol. Cell. 8, 713e718. Kawai-Kowase, K., Kumar, M.S., Hoofnagle, M.H., Yoshida, T., Owens, G.K., 2005. PIAS1 activates the expression of smooth muscle cell differentiation marker genes by interacting with serum response factor and class I basic helix-loophelix proteins. Mol. Cell. Biol. 25, 8009e8023. Kimonides, V.G., Khatibi, N.H., Svendsen, C.N., Sofroniew, M.V., Herbert, J., 1998. Dehydroepiandrosterone (DHEA) and DHEA-sulfate (DHEAS) protect hippocampal neurons against excitatory amino acid-induced neurotoxicity. Proc. Natl. Acad. Sci. U.S.A 95, 1852e1857. Liao, J., Fu, Y., Shuai, K., 2000. Distinct roles of the NH2- and COOH-terminal domains of the protein inhibitor of activated signal transducer and activator of transcription (STAT1) (PIAS1) in cytokine-induced PIAS1-Stat1 interaction. Proc. Natl. Acad. Sci. U.S.A 97, 5267e5572. Lin, C.H., Yeh, S.H., Lin, C.H., Lu, K.T., Leu, T.H., et al., 2001. A role for the PI-3 kinase signaling pathway in fear conditioning and synaptic plasticity in the amygdale. Neuron 31, 841e851. Liu, B., Liao, J., Rao, X., Kushner, S.A., Chung, C.D., et al., 1998. Inhibition of stat1mediated gene activation by PIAS1. Proc. Natl. Acad. Sci. U.S.A 95, 10626e10631. Liu, B., Mink, S., Wong, K.A., Stein, N., Getman, C., et al., 2004a. PIAS1 selectively inhibits interferon-inducible genes and is important in innate immunity. Nat. Immunol. 5, 891e898. Liu, L., Wong, T.P., Pozza, M.F., Lingenhoehl, K., Wang, Y., et al., 2004b. Role of NMDA receptor subtypes in governing the direction of hippocampal synaptic plasticity. Science 304, 1021e1024. Liu, B., Yang, Y., Chernishof, V., Ogorzalek Loo, R.R., Jang, H., et al., 2007. Proinflammatory stimuli induce IKKa-mediated phosphorylation of PIAS1 to restrict inflammation and immunity. Cell 129, 903e914.

113

Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2DDCT method. Methods 25, 402e408. Luscher, C., Huber, K.M., 2010. Group 1 mGluR-dependent synaptic long-term depression: mechanisms and implications for circuitry and disease. Neuron 65, 445e459. Munarriz, E., Barcaroli, D., Stephanou, A., Townsend, P.A., Maisse, C., et al., 2004. PIAS-1 is a checkpoint regulator which affects exit from G1 and G2 by sumoylation of p73. Mol. Cell. Biol. 24, 10593e10610. Peng, G., Han, M., Du, Y., Lin, A., Yu, L., Zhang, Y., Jing, N., 2009. SIP30 is regulated by ERK in peripheral nerve injury-induced neuropathic pain. J. Biol. Chem. 284, 30138e30147. Rytinki, M.M., Kaikkonen, S., Pehkonen, P., Jaaskelainen, T., Palvimo, J.J., 2009. PIAS proteins: pleiotropic interactors associated with SUMO. Cell. Mol. Life Sci. 66, 3029e3041. Scheschonka, A., Tang, Z., Betz, H., 2006. Sumoylation in neurons: nuclear and synaptic roles? Trends Neurosci. 30, 85e91. Shaywitz, A.J., Greenberg, M.E., 1999. CREB: a stimulus-induced transcription factor activated by a diverse array of extracellular signals. Annu. Rev. Biochem. 68, 821e861. Silva, A.J., Kogan, J.H., Frankland, P.W., Kida, S., 1998. CREB and memory. Annu. Rev. Neurosci. 21, 127e148. Sweatt, J.D., 2004. Mitogen-activated protein kinase in synaptic plasticity and memory. Curr. Opin. Neurobiol. 14, 311e317. Tai, D.J.C., Hsu, W.L., Liu, Y.C., Ma, Y.L., Lee, E.H.Y., 2011. Novel role and mechanism of protein inhibitor of activated STAT1 in spatial learning. EMBO J. 30, 205e220. Tang, Z., Far, O.E., Betz, H., Scheschonka, A., 2005. Pias1 interaction and sumoylation of metabotropic glutamate receptor 8. J. Biol. Chem. 280, 38153e38159. Thomas, G., Huganir, R.L., 2004. MAPK cascade signaling and synaptic plasticity. Nat. Rev. Neurosci. 5, 173e183. Vecsey, C.G., Hawk, J.D., Lattal, K.M., Stein, J.M., Fabian, S.A., et al., 2007. Histone deacetylase inhibitors enhance memory and synaptic plasticity via CREB: CBPdependent transcriptional activation. J. Neurosci. 27, 6128e6140. Watanabe, K., Kanno, T., Oshima, T., Miwa, H., Tashiro, C., et al., 2008. The NMDA receptor NR2A subunit regulates proliferation of MKN45 human gastric cancer cells. Biochem. Biophysical. Res. Commun. 367, 487e490. Wayman, G.A., Impey, S., Marks, D., Saneyoshi, T., Grant, W.F., et al., 2006. Activitydependent dendritic arborization mediated by CaM-kinase I activation and enhanced CREB-dependent transcription of Wnt-2. Neuron 50, 897e909. Yang, Y.C., Ma, Y.L., Chen, S.K., Wang, C.W., Lee, E.H.Y., 2003. Focal adhesion kinase is required, but not sufficient, for the induction of long-term potentiation in dentate gyrus neurons in vivo. J. Neurosci. 23, 4072e4080. Yin, X., Warner, D.R., Roberts, E.A., Pisano, M.M., et al., 2005. Novel interaction between nuclear coactivator CBP and the protein inhibitor of activated stat1 (PIAS1). J. Interferon. Cytokine Res. 25, 321e327. Zhao, M.G., Toyoda, H., Lee, Y.S., Wu, L.J., Ko, S.W., et al., 2005. Roles of NMDA NR2B subtype receptor in prefrontal long-term potentiation and contextual fear memory. Neuron 47, 859e872.