Regulation of CCL2 Gene Expression Figure 6. Activation of the glucocorticoid receptor suppresses IL-1b-mediated induction of the CCL2 gene. A,B. 832/13 cells were transfected with 5X NF-kB-Luc (A) or 3.6-Luc (B) followed by co-treatment with either 10 nM Dexamethasone (Dex), 100 nM Hydrocortisone (HC), 10 nM Budesonide (BD) or 10 nM Fluticasone Propionate (FP) and 1 ng/mL IL-1b for 4 h. Luciferase promoter activity was quantified and normalized to total cellular protein. #p,0.001 vs. DMSO (black bar). C. 832/13 cells were co-stimulated for 6 h with 10 nM Dex, BD or FP in the presence of 1 ng/ mL IL-1b. D. 832/13 cells were treated with increasing concentrations of Dex (0.1, 1, 10 nM) for 6 h in the presence of 1 ng/mL IL-1b. C, D. CCL2 mRNA accumulation was assessed via RT-PCR and normalized to RS9 mRNA abundance. #p,0.001 vs. DMSO., @ p,0.1 vs. DMSO, *p,0.05 vs DMSO. E. 832/ 13 cells were pre-treated with 0.1, 1 or 10 nM Dex for 6 h followed by 15 min stimulation with IL-1b. Immunoblot analysis was performed with antibodies against p38, PO4-p38, JNK, PO4-JNK and b Actin. F. ELISA was used to quantify CCL2 release into the media following a12 h incubation with media alone, media containing 1 ng/mL IL-1b or 1 ng/mL IL-1b plus 10 nM Dex. #p,0.01. G. 832/13 cells were transduced with recombinant adenoviruses expressing either bGAL or increasing concentrations of CA IKKb for 12 h in the presence of either 10 nM Dex or BD. CCL2 release into the media was quantified using an ELISA. @ p,0.1, *p,0.05. The bar graphs represent means 6 SEM from 3–4 individual experiments. doi:10.1371/journal.pone.0046986.g006 To examine whether MKP-1 induction per se is sufficient to mimic the effects of Dex, we overexpressed MKP-1 in 832/13 cells using recombinant adenovirus (Figure 8D). Treatment with either Dex or overexpression of MKP-1 prevented the IL-1b-stimulated increase in p38 MAPK phosphorylation (Figure 8E). The dephosphorylation of p38 correlated with a reduction in IL-1b- mediated accumulation of CCL2 mRNA (Figure 8F). There was not a dose-dependent decrease in CCL2 expression in response to increasing concentrations of the MKP-1 virus (the range of suppression was 25–35%). Even more striking was the decrease in CCL2 gene transcription (Figure 8G), which was also not dose- dependent and included a range of reduction of 48–61%. Importantly, the secretion of CCL2 in response to IL-1b was also decreased by 30% via overexpression of MKP-1 (Figure 8H). These results are consistent with the model that inhibiting signaling through the p38 MAPK decreases the ability of IL-1b to induce expression of the gene and subsequent secretion of the CCL2 protein. Of significant note, MKP-1 overexpression does not impair glucose-stimulated insulin secretion in 832/13 cells (Figure 9A). Moreover, the ability of forskolin (FSN), an adenylyl cyclase activator, to potentiate the stimulus-secretion coupling response by an additional 2.6-fold was also retained (Figure 9A). In addition, overexpression of MKP-1 in isolated rat islets also did not impair insulin secretion (Figure 9B). We observed that MKP-1 overex- pression actually enhanced glucose-stimulated insulin secretion by 16.8% relative to the control virus expressing b-Galactosidase. Thus, an important finding here is that the anti-inflammatory actions associated with MKP-1 overexpression occur with full retention of b-cell function. Discussion Chemokines play a crucial role in recruiting leukocytes to areas of inflammation [56,57]. The chemokine CCL2 likely influences the islet immune cell recruitment related to T1DM, as well as with islet graft rejection [4,8,10,18,19,58]. Thus, the present study was designed to examine the signals required to activate and repress expression of the CCL2 gene in pancreatic b-cells. Several novel observations emerged: 1) expression of the CCL2 gene in response to IL-1b requires the p65 subunit of NF-kB and signaling through the kinases IKKb and p38 MAPK; 2) overexpression of p65 is sufficient to drive transcription of the CCL2 gene, leading to increased synthesis and secretion of CCL2; 3) multiple glucocor- ticoid receptor ligands diminish the IL-1b-mediated induction of the CCL2 gene; 4) overexpression of the glucocorticoid responsive gene MKP-1 partially impairs the ability of IL-1b to induce expression of the CCL2 gene and to promote CCL2 release; 5) overexpression of MKP-1 does not interfere with glucose- stimulated insulin secretion, demonstrating that the anti-inflam- matory activity of this phosphatase occurs with preservation of b- cell secretory function. The results reported herein are consistent with MKP-1 knockout animals which display exaggerated responses to innate immune receptor stimuli, including prolonged p38 phosphoryla- tion [59]; this inability to dephosphorylate p38 in a normal manner is associated with an increase in circulating pro- inflammatory factors, including CCL2 [60]. In pancreatic b-cells, the exquisite sensitivity of the CCL2, but not the COX2 gene, to p38 inhibition is indicative of distinct signaling inputs required to facilitate efficient communication of the same signal (e.g., IL-1b). We note that while JNK is activated in pancreatic b-cells in response to IL-1b, this kinase does not appear to be modulated by MKP-1. However, the JNK pathway is required for repair of DNA damage after pro-inflammatory signals by upregulation of GADD45 expression in pancreatic b-cells [61] and thus represents the diverse actions associated with IL-1b signaling. Alternatively, the expression of the COX2 gene, which also requires p65 for induction by IL-1b [38], does not require p38 to communicate the IL-1b response (Figure 5C). We interpret these findings to indicate a tissue specific and/or signal specific phenomenon, as p38 is required to induce the expression of the COX2 gene in macrophages exposed to encephalomyocarditis virus [62]. We suspect that signal integration at various promoters is controlled by discrete mechanisms in order to finely tune homeostasis versus inflammation in a tissue specific manner. The discovery that p65 overexpression is sufficient to drive synthesis and secretion of CCL2 from b-cells in the absence of a pro-inflammatory stimulus (Figure 3) has important implications for both major forms of diabetes. In our view, this observation indicates that any number of stimuli promoting degradation of the regulatory IkB proteins (e.g., cytokines, TLR-2 and -4 ligands, etc.) or otherwise facilitating p65 entry into the nucleus could potentially induce the expression of the CCL2 gene. In support of this view, fatty acids promote the synthesis and secretion of IL-1b through TLR-2 and -4 activation [63] leading to auto-inflamma- tory feed foward mechanisms that promote islet leukocyte accumulation [64]. CCL2 is one such gene that may be a contributor to this phenotype. Conversely, other IL-1b responsive genes, such as COX2, do not respond to simple increases in p65 abundance (not shown), indicating that the b-cell uses distinct and selective mechanisms to control the expression of genes associated with inflammation. The coupling of CCL2 gene transcription with CCL2 release allows the b-cell to rapidly respond to fluctuations in NF-kB activity with corresponding increases or decreases in chemoattractant potential. Our observations are also congruent with several in vivo mouse studies in addition to findings related to islet transplantation. For example, transgenic overexpression of CCL2 in islet b-cells promotes massive immune cell infiltration into the islets [65] and the quantity of CCL2 synthesized and secreted from b-cells into the serum correlates with diabetes development [8]. Intriguingly, however, transgenic expression of CCL2 on the non-obese diabetic mouse background, a model of autoimmune- mediated islet destruction, delays diabetes development despite increased immune cell accumulation in islets [19]. Thus, genetic background potentially dictates the role of CCL2 involvement in diabetes onset and progression by potentially controlling the type PLOS ONE | www.plosone.org 12 October 2012 | Volume 7 | Issue 10 | e46986