J Mol Neurosci (2012) 48:209–218 DOI 10.1007/s12031-012-9809-2 Glucocorticoid Receptor and Myocyte Enhancer Factor 2 Cooperate to Regulate the Expression of c-JUN in a Neuronal Context Niels Speksnijder & Kenneth V. Christensen & Michael Didriksen & E. Ronald De Kloet & Nicole A. Datson Received: 17 April 2012 /Accepted: 7 May 2012 /Published online: 24 May 2012 # The Author(s) 2012. This article is published with open access at Springerlink.com Abstract The glucocorticoid receptor (GR) and myocyte c-JUN. In conclusion, for the first time, we show that enhancer factor 2 (MEF2) are transcription factors involved activated GR requires MEF2 to regulate c-JUN. At the in neuronal plasticity. c-JUN, a target gene of GR and same time, GR influences MEF2 activity and DNA binding. MEF2, plays a role in regulating both synaptic strength These results give novel insight into the molecular interplay of and synapse number. The aim of this study was to investi- GR and MEF2 in the control of genes important for neuronal gate the nature of this dual regulation of c-JUN by GR and plasticity. MEF2 in a neuronal context. First, we showed that GR mediates the dexamethasone-induced suppression of c- Keywords Glucocorticoid receptor . Myocyte enhancer JUN mRNA expression. Next, we observed that GR activa- factor 2 . c-JUN . Dexamethasone . PC-12 cells tion resulted in an increase in phosphorylation of MEF2, a post-translational modification known to change MEF2 Abbreviations from a transcriptional enhancer to a repressor. In addition, CDK5 Cyclin-dependent kinase 5 we observed an enhanced binding of MEF2 to genomic sites ChIP Chromatin immuno-precipitation directly upstream of the c-JUN gene upon GR activation. DEX Dexamethasone Finally, in primary hippocampal neuronal cultures, knock- GBS GR-binding site down of MEF2 not only reduced c-JUN expression levels GR Glucocorticoid receptor but abolished GR regulation of c-JUN expression. This GRE Glucocorticoid response element suggests that MEF2 is necessary for GR regulation of HPA-axis Hypothalamic–pituitary–adrenal axis IEG Immediate-early gene MBS MEF2 binding site Electronic supplementary material The online version of this article (doi:10.1007/s12031-012-9809-2) contains supplementary material, MEF2 Myocyte enhancer factor 2 which is available to authorized users. PC-12 Pheochromocytoma cells N. Speksnijder : E. R. De Kloet : N. A. Datson (*) Division of Medical Pharmacology, Leiden/Amsterdam Center for Drug Research (LACDR), Leiden University Medical Center, P.O. Box 9502, 2300 RA Leiden, The Netherlands Introduction e-mail: [email protected] : Neuronal plasticity, a change in the structure, function, and K. V. Christensen M. Didriksen organization of neurons in response to environmental stim- H. Lundbeck A/S R&D, 9 Ottiliavej, uli, underlies many key processes such as learning and DK-2500 Copenhagen-Valby, Denmark memory, adaptation, and behavioral sensitization. Changes in gene expression, governed by key transcription factors, N. A. Datson such as the glucocorticoid receptor (GR) and myocyte en- Department of Human Genetics, Leiden University Medical Center, hancer factor 2 (MEF2), underlie neuroplasticity. GR is Leiden, The Netherlands activated by glucocorticoid stress hormones, released by 210 J Mol Neurosci (2012) 48:209–218 the HPA-axis in response to stress. Upon activation, GR acts For GR blockade, cells were pretreated with VEH (0.1% as a ligand-activated transcription factor to influence expres- ethanol) or 1 mM RU486 (M8046, Sigma-Aldrich) for sion of a wide variety of genes, including genes involved in 60 min before addition of DEX or VEH. neuronal plasticity (Datson et al. 2008). MEF2 comprises a family of four members, MEF2a–d, showing distinct but Hippocampal Cultures partly overlapping expression patterns and is activated by neuronal activity. Upon activation, MEF2 regulates the ex- Newborn pups from NMRI mice were killed at postnatal pression of genes that control dendritic remodeling, result- day 1. Brains were isolated and kept in Hank’s balanced salt ing in the inhibition of synapse formation. Conversely, a solution on ice until dissection. Hippocampi were dissected decrease in MEF2 activity increases spine density (Flavell et in ice-cold dissection solution consisting of Krebs buffer al. 2006; Shalizi et al. 2006). supplemented with 3 mg/ml BSA, 1.2 mM MgSO4,and We previously showed that GR and MEF2 have several 2 mM HEPES. Hippocampi (n012) were transferred to a target genes in common, including the c-JUN gene (Datson conical tube containing 1.5 ml of dissection solution sup- et al. 2011a). c-JUN is a subunit of the transcription factor plemented with 184 μg/ml trypsin. The tissue was incubated AP-1 and is a ubiquitously expressed IEG with important at 37°C for 6 min. Subsequently, 3.5 ml of dissection solu- functions in cell death, differentiation, and inflammation tion supplemented with 0.65 mg/ml soyabean trypsin inhib- (Beck et al. 2009; Sun et al. 2005). The AP-1 family of itor; 10 μg/ml DNAse and 0.19 mM MgSO4 were added. transcription factors is recruited in the activation of neuronal The trypsinated and DNase-treated hippocampi were centri- circuits leading to long-term changes, such as long-term fuged at 100×g for 3 min. The supernatant was discarded, memory formation (Alberini 2009). MEF2 is known to and the conical part of the tube was filled with 1.5 ml of induce transcription of c-JUN (Kato et al. 1997; Aude- dissection solution supplemented with 5.2 mg/ml soyabean Garcia et al. 2010; Han and Prywes 1995), while GR trypsin inhibitor, 80 μg/ml DNAse, and 1.5 mM MgSO4. on the other hand is known to repress the expression of c-JUN The cells were dissociated by pipetting and left for 5 min at in vitro in AtT-20 cells and mouse fibroblast cells (Autelitano room temperature (RT), allowing remaining tissue to settle. 1994; Wei et al. 1998). The aim of this study was to The supernatant was transferred to a new tube containing investigate the molecular interplay of GR and MEF2 in a 3.5 ml of dissection solution supplemented with 132 μM neuronal context, using the shared target gene c-JUN as a CaCl2 and 120 μMMgSO4 and centrifuged for 10 min at proof-of-principle. 100×g. The cell pellet was resuspended in 3.5 ml of MEM II+ B27 (MEM buffer supplemented with 0.5% D-glucose, 0.22% bicarbonate, penicillin–streptomycin, 2 mM L-glutamate, Materials and Methods 10% NU-serum, and 2% B27). After resuspension, the concentrated cell solution was diluted to 7.5 ml MEM II+ Cell Culture and Treatment B27. Cells were plated at a density of 50,000 live cells/well in poly-D-lysine coated 96-well dishes. The yield from one pup Rat pheochromocytoma (PC-12) cells (passage # 15–29) (two hippocampi) was approximately 400,000 living cells. were cultured as described earlier (Morsink et al. 2006b). The day after plating, media was changed to MEM II+B27 In short, cells were grown in DMEM medium, supple- buffer supplemented with 1 μM AraC (cytosine arabinoside). mented with 0–10% fetal bovine serum and 0–10% horse The cells were left for 14 days in vitro before assaying. serum, dependent on the stage of neuronal differentiation. For mRNA and protein analysis cells were seeded at a Lentiviral shRNA Transduction, Stimulation, and RNA confluency of 30–50% in pre-coated six-well plates Purification (356400, BD Biosciences, San Jose, CA, USA). For ChIP experiments, the cells were seeded at 50% confluency in High titer batches (>5×10−8 TU/ml) of lentiviral particles pre-coated 175 cm2 plates (356478, BD Biosciences). Neu- harboring gene-specific short hairpin RNA (shRNA) target- ronal differentiation was achieved by giving 50 ng/ml ing MEF2A (Sigma, TRCN0000095959) as well lentiviral Nerve Growth Factor Beta (NGF-ß) (N2513, Sigma- particles harboring control shRNA (Sigma, SHC002V) were Aldrich, St. Louis, MO, USA) every other day for 10 days. purchased from Sigma. The day after plating, hippocampal Medium at day 9 of the differentiation was supplemented with cultures were transduced with lentiviral particles at the charcoal-stripped serum to deprive the medium of endoge- following concentrations: 150,000 lentiviral particles/well, nous steroids (Sarabdjitsingh et al. 2010b). At day 10, the cells 75,000 lentiviral particles/well, and 37,500 lentiviral particles/ were treated for 30, 60, 90, or 180 min, dependent on the well. At day 14, the hippocampal cultures were stimulated for experiment, with either vehicle (VEH) (0.1% ethanol) or 90 min with 100 nM dexamethasone diluted in astrocyte- 100 nM dexamethasone (DEX) (D1756, Sigma-Aldrich). conditioned media. The latter was to avoid glutamate-induced J Mol Neurosci (2012) 48:209–218 211 excitotoxicity by the media change. Subsequently, cells 1% NaDOC), and 2× with TE buffer (1 mM EDTA pH 8.0; were processed for RNA isolation using the Aurum Total 10 mM Tris–HCl pH 8.0). Subsequently, the DNA com- RNA 96 Kit (BioRad). plexes were eluted from the beads with 0.1 M NaHCO3 and 1% SDS, and the DNA was reverse-crosslinked o/n at 4°C in Real-Time Quantitative PCR 0.2 M NaCl. The samples were then treated for 1 h with RNAse at 37°C and the DNA purified using Nucleospin Total RNA was isolated using Trizol (15596, Invitrogen) columns. The DNA was eluted in TE buffer for RT-qPCR according to the manufacturer’s instructions. RNAwas diluted analysis. RT-qPCR on chromatin immunoprecipitation to 50 ng/μl, and cDNA was synthesized using the iScript (ChIP) material was performed directly on purified DNA. cDNA synthesis kit (170–8897, Bio-Rad, Hercules, CA, ChIP results were obtained by performing three individual USA) according to the manufacturer’s protocol.