overexpression of MKP-1 did not fully mimic the potency of GCs. These data are consistent with another recent report using a different cellular population [72]. Moreover, it is likely that glucocorticoids employ multiple mechanisms in order to suppress inflammatory responses and induction of phosphatase proteins, such as MKP-1, represent only one important component of GR action. It is possible, even likely, that other phosphatases of the dual specificity family may be coordinately regulated by glucocorticoids to tailor inflammatory responses to the individual needs of a particular cell. Furthermore, ligand-bound GR may directly interfere with transcriptional machinery at various promoters, as has been described in other systems [73]. Thus, MKP-1 overexpression would perhaps be predicted to only partially recapitulate the actions associated with liganded GR, a postulate consistent with what we observed in the present study. It thus remains to be determined how many of the DUSP family members are responsive to GCs in the pancreatic b- cell and if any of these other phosphatases represent anti- inflammatory actions that could be exploited to protect b-cells from pro-inflammatory signals. Moreover, a direct repressive effect of liganded GR on specific genes activated by IL-1b cannot be ruled out at the present time. It is entirely possible, even likely, that GCs use multiple mechanisms to suppress inflammation in pancreatic b-cells, while MKP-1 simply targets a highly specific pathway (e.g., MAPKs). Finally, the preservation of b-cell secretory capacity is a significant consideration for any anti-inflammatory strategy aimed at maintaining or restoring functional b-cell mass. Notably, neither glucose-stimulated insulin secretion nor the potentiating effects of cAMP on glucose-stimulated insulin secretion were impaired with MKP-1 overexpression. Elevated cAMP levels are essential for the actions of incretin hormones on pancreatic b-cell function. The insulin secretion results obtained after delivering the MKP-1 gene to rat islets and b-cell lines are fundamentally important considering that decreases in b-cell function are a known side effect of chronic glucocorticoid exposure [74,75]. We have shown here that MKP-1 gene delivery modestly enhances islet b-cell function, which is consistent with studies that have measured insulin secretion in isolated islets in the presence of p38 MAPK inhibitors [26,76]. Finally, we note that annexin A1, itself a glucocorticoid-responsive anti-inflammatory gene in some tissues [77], is not responsive to dexamethasone in the pancreatic b-cell (JJC, unpublished data). Therefore, individual tissues clearly use distinct strategies to combat inflammation; accordingly, glucocor- ticoids induce and/or repress discrete and overlapping genes in different tissues towards achieving this goal. In summary, the separation of the anti-inflammatory properties of glucocorticoids from their side effects is an ongoing pursuit of many laboratories; consequently, glucocorticoid responsive genes might represent a novel approach for therapeutic intervention in many tissues. While we have described the role of MKP-1 in the current study, future studies will include additional dissection of glucocorticoid receptor-mediated actions, including analysis of other downstream target genes, in an attempt to identify additional strategies which can protect pancreatic b-cells from pro-inflam- matory signals. Thus, we conclude that increased expression of the MKP-1 gene, either via GC exposure or via viral overexpression, is a negative regulator of IL-1b-mediated p38 activation. Interfering with p38 activity by pharmacologic or gene delivery approaches reduces the expression of the CCL2 gene in response References Regulation of CCL2 Gene Expression to IL-1b, which results in diminished levels of secreted CCL2 protein. Importantly, these actions were achieved with full retention of b-cell function.. Supporting Information Figure S1 Cycle threshold (Ct) values reflecting relative expression patterns of the CCL2, Pdx1, Nkx6.1 and GADD45 genes in 832/13 rat insulinoma cells either at basal levels or stimulated with 1 ng/ml IL-1b for 6 hrs. Numerical values are average Ct values, representing three independent RT-PCR runs. (TIF) Figure S2 832/13 cells were transfected with either an siRNA duplex targeting p65 or non-targeting siScramble control. Following 48 h incubation with duplexes cells were harvested and separated into cytoplasmic and nuclear fractions. Shown is a representative immunoblot of p65 protein abundance, with b-Actin serving as a loading control. (TIF) Figure S3 832/13 cells were pre-treated for 1 hr with 1 mM SP600125 (JNKi) followed by a 6 h stimulation with 1 ng/ml IL-1b alone or in combination with 1 U/ml c- IFN for 6 hrs. GADD45 mRNA abundance was measured and normalized to RS9. Data are means 6 SEM from 3 individual experiments. *p,0.05. (TIF) Figure S4 832/13 cells were transfected with the 3X GAS- luciferase construct; 24 h post-transfection cells were treated for 1 h with 20 mM ERK inhibitor prior to 4 h stimulation with 100 U/mL c-IFN. Luciferase reporter activity was normalized to protein content. Data are represented as means 6 SEM from 3 individual experiments. *p,0.05. (TIF) Figure S5 832/13 cells were treated with 10 nM Dex for 1 h then stimulated with 1 ng/ml IL-1b for 15 mins. Immunofluorescence assay was used to track nuclear localization of p65 (Scale bars represent 50 mm on the main image and 5 mm scale bars within the magnified inlays). Immunofluorescence experiments were conducted on three individual occasions and representative images are shown. (TIF) Acknowledgments We thank Drs. Guoxun Chen, Christian Jobin, Mary Law, Naima Moustaid-Moussa, Christopher Newgard, Yoshihiko Nishio, Thomas Spencer, Wayne Vedeckis, Wally Wang, Jay Whelan, and Haiyan Xu for reagents. We also express thanks to Dr. Robert Noland and members of the Collier lab for critical reading of the manuscript. We are grateful to Dr. Bob Rider in the College of Education, Health, and Human Sciences and Dr. Greg Reed in the Office of Research for their support of the Train-to- Retain program. Author Contributions Conceived and designed the experiments: SJB JJC. Performed the experiments: SJB MRG BLU DL PLB SCM LZ JJC. Analyzed the data: SJB MRG SCM DL MDK JJC. Contributed reagents/materials/analysis tools: SJB JPB DL LZ MDK JJC. Wrote the paper: SJB JJC. 1. Tisch R, McDevitt H (1996) Insulin-dependent diabetes mellitus. Cell 85: 291– 297. 2. Gepts W (1965) Pathologic anatomy of the pancreas in juvenile diabetes mellitus. Diabetes 14: 619–633. PLOS ONE | www.plosone.org 17 October 2012 | Volume 7 | Issue 10 | e46986