Regulation of CCL2 Gene Expression Figure 5. The expression of the CCL2 gene is sensitive to p38 MAPK inhibition. A. 832/13 cells were treated for 15, 30 or 60 min with 1 ng/ mL IL-1b. Immunoblot analysis was performed on three separate occasions with the indicated antibodies and a representative image is shown. B. Rat islets were pre-treated for 1 h with 10 mM of SB202190 (p38i = p38i inhibitor) followed by a 6 h incubation with 10 ng/ml IL-1b. *p,0.05. C, D. 832/13 cells were pre-treated for 1 h with 10 mM of SB202190, SB203580, SB239063, 1 mM SP600125 (JNK inhibitor; JNKi), or 20 mM PD98059 (ERK inhibitor; ERKi) followed by a 6 h incubation with 1 ng/mL IL-1b. Steady-state mRNA abundance for CCL2 and COX2 was quantified and normalized to RS9. #p,0.001 vs. DMSO E. 832/13 cells were transfected with 3.6-Luc; 24 h post-transfection cells were pre-treated for 1 h with the respective MAPK inhibitors (10 mM p38 inhibitors, 20 mM ERKi or 1 mM JNKi), followed by a 4 h incubation with 1 ng/mL IL-1b. Luciferase reporter activity was normalized to protein content. #p,0.001 vs. DMSO. F. 832/13 cells were pre-treated for 1 h with 10 mM SB202190, followed by 12 h stimulation with 1 ng/mL IL-1b. An ELISA was performed to determine CCL2 release into the media, and results were normalized to total protein content. #p,0.001. RNA abundance, luciferase promoter activity and ELISA data are expressed as means 6 SEM from 3–4 individual experiments. doi:10.1371/journal.pone.0046986.g005 1b-mediated increase in CCL2 secretion was reduced by 85% in the presence of p38 inhibitor SB202190 (Figure 5F). We interpret these data collectively to indicate that while the CCL2 and COX2 genes are both NF-kB responsive, there are specific and distinct signaling inputs required for their expression in the pancreatic b- cell in response to IL-1b. Activation of the Glucocorticoid Receptor Suppresses IL- 1b-mediated Induction of the CCL2 Gene After discovering that the p38 MAPK was required for activation of the CCL2 gene by IL-1b, we next tested whether anti-inflammatory mechanisms targeting this pathway could block this induction. Because glucocorticoids are often used clinically for their potent anti-inflammatory properties, we treated 832/13 cells with a panel of known glucocorticoid receptor ligands: dexameth- asone (Dex), hydrocortisone (HC), budesonide (BD), and flutica- sone propionate (FP). As shown in Figure 6A, each GR agonist strongly inhibited the ability of IL-1b to induce the transcription of a multimerized NF-kB luciferase reporter gene. The effect was even more striking on the IL-1b-mediated activation of the 3.6 kb proximal CCL2 luciferase reporter gene (Figure 6B). We next observed that the IL-1b-mediated increase in CCL2 mRNA levels was diminished by 74%, 61%, and 73% by Dex, BD, and FP, respectively (Figure 6C). To begin to address the mechanism of GC-mediated repression, we examined the sensi- tivity of the CCL2 gene to Dex. We observed decreases of 5%, 46%, and 63% in CCL2 mRNA levels in response to increasing increments in dexamethasone concentration (Figure 6D). Dex exposure also diminished the IL-1b-mediated phosphorylation of the p38 MAPK (Figure 6E). In turn, this resulted in a 60% decrease in secreted CCL2 (Figure 6F), which is congruent with the results in Figure 5. Furthermore, while CCL2 secretion was driven by the constitutive-active IKKb, release of peptide was markedly repressed by both Dex and BD (Figure 6G). Thus, these results are consistent with a role for IKKb and p38 MAPK to induce expression and secretion of CCL2 and demonstrate further that synthetic glucocorticoids interrupt this process, likely by interfering with the signaling mechanisms used by IL-1b. Because Dex blocks p65 nuclear translocation in mast cells [53], we next tested the hypothesis that glucocorticoids have a similar function in b-cells, which could explain the repressive effects on CCL2 gene expression. Using 832/13 cells, IL-1b stimulated the nuclear translocation of p65 to a similar level in both the presence and absence of Dex (Figure S5). Thus, the immunofluorescence assays reveal that in contrast to the mast cell, where Dex prevents nuclear translocation of p65 [53], no such blockade in trafficking exists in glucocorticoid-treated 832/13 rat insulinoma cells that are also exposed to IL-1b. We therefore conclude that the repressive actions of GCs in pancreatic b-cells, such as seen in Figure 6 A–G must exist through mechanisms other than cytoplasmic retention of p65. Induction of the MKP-1 Gene by Glucocorticoids Promotes Dephosphorylation of the p38 MAPK GCs promote expression of the MKP-1 gene [29,31] and MKP- 1 dephosphorylates the p38 MAPK [54]. Because GCs do not impair IL-1b-mediated translocation of p65 from cytoplasm to nucleus (Figure S5), and because expression of the CCL2 gene is p38 MAPK dependent (Figure 5), we next investigated whether the MKP-1 gene was responsive to GCs in 832/13 cells and isolated rat islets. First, we examined the effects of each glucocorticoid on a synthetic glucocorticoid response element (GRE) promoter luciferase construct. As shown in Figure 7A, Dex, BD, and FP induced GRE-luciferase promoter activity by 19.4, 17.9 and 16.6-fold respectively. Next, we used approximately 700 bases of the human MKP-1 proximal gene promoter, which contains multiple glucocorticoid responsive elements [34], and discovered that these sites supported a 1.8, 2.5 and 2.9-fold increase in transcriptional activity by the respective glucocorticoids (Figure 7B). We then examined expression of the endogenous MKP-1 gene and discovered that Dex, BD and FP increased MKP-1 mRNA 11.2-fold, 13.5-fold, and 12-fold, respectively in 832/13 cells (Figure 7C). The MKP-1 gene was also induced by the panel of glucocorticoids in isolated rat islets; although to a lesser extent than 832/13 cells (Figure 7D). Further examination of the MKP-1 gene in response to Dex revealed dose-dependent increases in MKP-1 mRNA accumulation (Figures 7E). We also observed a rapid increase in MKP-1 mRNA (not shown) and protein abundance in response to 10 nM dex (Figure 7F). The increase in MKP-1 protein does not directly correlate with the magnitude of induction seen for the mRNA encoding this protein. We interpret these data to indicate that signals downstream of transcription may also be involved in controlling accumulation of MKP-1 protein. Transcriptional and post-transcriptional regula- tion are not uncommon for genes involved in regulating sensitive cellular functions [55].Taken together, these observations indicate that GCs induce expression of the MKP-1 gene in pancreatic b- cells. Overexpression of MKP-1/DUSP1 Diminishes the IL-1b- mediated Increase in CCL2 Gene Expression and Secretion with Full Retention of b-cell Function Using siRNA duplexes targeting the MKP-1 gene, we observed a 51% decrease in MKP-1 mRNA levels (Figure 8A); this reduction in MKP-1 coincided with an enhancement in IL-1b- stimulated p38 MAPK phosphorylation signal (not shown). Further, the siRNA-directed reduction in MKP-1 protein (Figure 8B -inset) eliminated 56% of the glucocorticoid-mediated suppression of the CCL2 gene (Figure 8B - graph). Moreover, this interference with Dex-mediated suppression resulted in 43% more CCL2 secretion (Figure 8C), demonstrating that the effectiveness of Dex to prevent CCL2 expression and release from the cell is diminished when MKP-1 abundance decreases. PLOS ONE | www.plosone.org 10 October 2012 | Volume 7 | Issue 10 | e46986