[21]. Upon activation of a cell surface receptor, such as the IL-1R, a variety of signaling pathways are activated, including the mitogen-activated protein kinases (MAPKs) and the IkB kinases (IKKs). Activation of the IKKs induces phosphorylation of the IkBs, which leads to their subsequent degradation through ubiquitin-mediated pathways. The degradation of IkBs reveals the nuclear localization signals in NF-kB; dimerization and nuclear accumulation of combinations of NF-kB subunit proteins ensues, thus facilitating signal-mediated regulation of gene transcription within a given cell type, including those that contribute to inflammatory responses [20,22]. The CCL2 gene contains NF-kB response elements in its proximal gene promoter and is responsive to IL-1b and other stimuli [23,24]. However, the transcription factors and associated signaling pathways responsible for controlling expression of CCL2 in pancreatic b-cells have not been established. Signaling through the MAPKs often links extracellular signals to specific gene promoters [25]. For example, the p38 MAPK is linked to inflammation in multiple tissues, including the pancreatic b-cell [26,27] and systemic inhibition of p38 delays diabetes progression in the non-obese diabetic (NOD) mouse [28]. Thus, strategies to downregulate p38 MAPK may be a therapeutic approach to prevent chemokine release and subsequent immune cell recruitment. The MAPK phosphatases, a subset of the family of dual specificity phosphatases (DUSPs), could be one such targetable approach. The genes encoding several of these phosphatases are regulated by glucocorticoids (GCs) in a variety of tissues [29,30,31]. GCs are often used in a variety of clinical situations to decrease inflammation. These steroids activate the intracellular glucocor- ticoid receptor (GR), leading to suppression of many outcomes controlled by the NF-kB pathway [32]. GR activation coordi- nately alters transcriptional patterns within many different cell types, leading to both activation and repression of a multitude of genes, and shifts cellular phenotype towards an anti-inflammatory state [32,33]. MKP-1 (also known as DUSP1) gene transcription is increased by glucocorticoids [29,34]. The increase in MKP-1 protein promotes p38 MAPK dephosphorylation, thus diminishing stimulus-specific kinase activity. Since p38 MAPK integrates cell surface receptor-mediated signaling pathways to inflammatory responses [27], upregulation of MKP-1 in pancreatic b-cells may represent a viable strategy to decrease inflammation-associated pathologies, such as cytokine-mediated increases in chemokine production. We undertook this study to investigate the regulation of the CCL2 gene by IL-1b and GCs in rat islets and b-cell lines. We hypothesized that expression of the MKP-1 gene, which we show is induced by multiple GCs in rat islets and b-cell lines, would provide anti-inflammatory actions that decrease expression of the CCL2 gene. We discovered that augmenting MKP-1 levels partially mimicked the inhibition of the IL-1b-mediated increase in CCL2 gene expression and secretion seen with GCs. Importantly, the anti-inflammatory actions associated with MKP-1 overexpression were obtained without suppressing glu- cose-stimulated insulin secretion, demonstrating that this gene is a possible target for therapeutic intervention with preservation of b- cell function. Experimental Procedures Cell Culture, Islet Isolation, Glucose-stimulated Insulin Secretion, and Reagents The establishment of the 832/13 and INS-1E rat insulinoma cells has been described [35,36]. These cell lines were maintained Regulation of CCL2 Gene Expression in RPMI-1640 (Mediatech; Manassas, VA) with 10% fetal bovine serum (FBS; Life Technologies Co., Carlsbad, CA). Islets were isolated from Wistar rats as outlined in prior studies [37]. All animal experiments were conducted in accordance with Duke University IACUC guidelines using approved procedures in appropriately accredited facilities (protocol #: A309-10-12). Buffer components and procedures for measuring insulin release into the media following secretagogue exposure were performed as previously published [35]. IL-1b was from Thermo Fisher Scientific (Waltham, MA) and c-IFN was purchased from Shenandoah Biotechnology Inc. (Warwick, PA). Dexamethasone, hydrocortisone, budesonide, fluticasone propionate, TPCA, and all MAPK inhibitors used herein were from Tocris Bioscience (Ellisville, MO). Recombinant adenoviruses expressing 5X NF-kB- luciferase [38], b-Galactosidase [39], p65 wild-type and S276A [40], IkBa super-repressor [41] and MKP-1 [42] have all been described. The CCL2-luciferase reporter [24], the 3X GAS- luciferase reporter [43], and the MKP-1-luciferase reporter constructs [34] have also been documented. The adenovirus expressing hIKKb S177E/S181E was a kind gift from Dr. Haiyan Xu (Brown University). Isolation of RNA, cDNA Synthesis and Real-time RT-PCR Total RNA was isolated using Isol-RNA Lysis Reagent (5 Prime Inc, Gaithersburg, MD), cDNA synthesized and real-time RT- PCR performed using SYBR Green (Applied Biosystems, Carlsbad, CA) as previously described [38]. Primers for COX2, CCL2, MKP-1, and RS9 used to detect transcript levels via RT- PCR reactions are available upon request. Isolation of Protein and Immunoblot Analysis Whole cell lysates were prepared using M-PER (Thermo Fisher Scientific) supplemented with protease and phosphatase inhibitor cocktails (Thermo Fisher Scientific). Proteins were quantified using the BCA method (Thermo Fisher Scientific). SDS-PAGE, transfer to PVDF, and blocking of membranes prior to antibody incubation as well as subsequent downstream detection has been described [44]. Antibodies used in this study were from the following sources: MKP-1 was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), while PO42 and total p38, PO42 and total JNK, PO42 and total ERK, as well as p65 were all from Cell Signaling Technology, Inc. (Danvers, MA). Anti-b Actin and anti- FLAG were from Sigma Aldrich. Plasmid and siRNA Transfection and Luciferase Assays Transient transfections of plasmids and siRNA duplexes into 832/13 cells and cell lysis for luciferase assays were as described [45]. Reporter gene activity was analyzed as previously described [46]. The Silencer Select siRNA duplexes were obtained from Life Technologies Co.(siRNA ID for p65: s159517; siRNA ID for MKP-1: s137873) and negative control siRNA sequence (catalog no. M4611). Delivery was via Dharmafect reagent 1 (Dharmacon, Lafayette, CO) according to the manufacturer’s protocol for duplexes and FuGene6 for plasmids. Dharmafect Duo (Dharma- con) was used in all experiments requiring simultaneous delivery of plasmids and siRNA duplexes. ELISA Detection of CCL2 secreted into the media was performed using the Quantikine kit from R & D Systems, Inc. (Minneapolis, MN) according to their suggested protocol. Chemokine release into the media was normalized to total protein to account for any potential differences in cell number. PLOS ONE | www.plosone.org 2 October 2012 | Volume 7 | Issue 10 | e46986