Regulation of CCL2 Gene Expression Figure 2. The p65 subunit of NF-kB is required for IL-1b- mediated expression of the CCL2 gene. A. 832/13 cells were transduced with adenoviruses expressing either b-Galactosidase (bGAL) or IkBa S32A/S36A for 24 h followed by stimulation with 1 ng/mL IL-1b for 6 h. *p,0.05. B. Cells were transduced with the indicated adenoviruses for 24 h, transfected with 3.6-Luc promoter construct for 4 h, followed by a 4 h stimulation with IL-1b. *p,0.05. C. 832/13 cells were transduced with the indicated adenoviruses; 24 h post-transduction cells were treated with IL-1b for 12 h. CCL2 release into the media was measured by ELISA and was normalized to protein content via BCA assay. *p,0.05. D. 832/13 cells were transfected with either 3.6-Luc (3.6 -WT) or 3.6–kBm; 24 h post-transfection cells were treated with IL-1b for 4 h. **p,0.01. E. 832/13 cells were co-transfected with either two different siRNA duplexes targeting p65 and 3.6-Luc promoter construct. 24 h post-transfection cells were treated with 1 ng/mL IL-1b for 4 h. #p,0.001. F. 832/13 cells were transfected with two duplexes against p65 using a scrambled siRNA sequence duplex as a control. 24 h post- transfection cells were treated for 6 h with IL-1b. #p,0.001. A, F. Total RNA was isolated, and RT-PCR was performed using CCL2 primers; expression of CCL2 was normalized to RS9. B, D, E. Promoter activity was analyzed via luciferase assay and values normalized to protein content using a BCA assay. G. DNA binding using nuclear extracts from cells treated as indicated on the top of the image. The specific antisera or cold-competitor oligos are indicated on the bottom of the image. The arrow indicates the specific complex bound to the NF-kB DNA sequence from the CCL2 gene promoter. All data in A–F represent means 6 SEM from 3–4 individual experiments, while the image in G. is representative of 2–3 experiments. 50M X, 50-fold molar excess of unlabeled (cold) oligonucleotide. doi:10.1371/journal.pone.0046986.g002 Collectively, we interpret these results to indicate that the IL-1b- mediated activation of the CCL2 gene in pancreatic b-cells requires the p65 subunit of NF-kB. Because p65 is necessary for the IL-1b-mediated induction of the CCL2 gene (Figure 2E and F), we next examined whether p65 overexpression was sufficient to drive the expression of the CCL2 gene. Figure 3A shows the increase in p65 abundance produced by the viral transgene. This increase in p65 protein driven by the viral transgene enhanced the expression of a synthetic multimerized NF-kB luciferase gene in a dose-dependent fashion (Figure 3B; dashed line indicates promoter activity induced by 1 ng/mL IL- 1b). Next, we examined the expression of the CCL2 gene in response to overexpression of p65 and observed an 18.5, 53.6 and 215.2-fold enhancement in mRNA levels (Figure 3C), demon- strating that simply enhancing p65 abundance is also sufficient to increase the expression of the gene in a dose-dependent manner. By contrast, removal of a phosphoacceptor site at Ser276 within p65, which is known to control recruitment of the CBP/p300 co- activators, diminished the ability of p65 to drive expression of the CCL2 gene (Figure 3C) and 3.6 kb of the proximal CCL2 gene promoter (Figure 3D). The expression levels of CCL2 driven by p65 overexpression are similar to the effect seen with IL-1b exposure (note that the dashed line in Figure 3B and C represents the induction by IL-1b). We observed that while the 3.6 kb promoter construct containing mutations within the NF-kB elements is unresponsive to p65 overexpression (data not shown), transcription of the wild-type CCL2 gene promoter is markedly enhanced by this maneuver (Figure 3D). By contrast, the COX2 gene, which requires p65 for induction by IL-1b [38], is not activated simply by p65 overexpression (data not shown). Similar results were obtained in isolated rat islets (Figure 3E). Importantly, expression of the manganese superoxide dismutase (MnSOD) gene was not different between the wild-type and S276A forms of p65 (Figure 3F), demonstrating that both wild-type p65 and the S276A mutant proteins are transcriptionally competent in the b-cell. Finally, we detected a dose-dependent increase in secretion of CCL2 protein with overexpression of p65 (Figure 3G). Therefore, we conclude that augmenting p65 abundance was sufficient to drive the expression of the CCL2 gene, which resulted in secretion of CCL2 protein. IKKb Drives the Expression of the CCL2 Gene We next examined specific signaling pathways responsible for activation of the CCL2 gene. Using 2-[(Aminocarbonyl)amino]-5- (4-fluorophenyl)-3-thiophenecarboxamide (TPCA), a pharmaco- logical inhibitor of IKKb, we found that the ability of IL-1b to induce expression of the CCL2 gene was diminished by 57, 73 and 77% (Figure 4A). To further demonstrate the involvement of IKKb, we overexpressed a constitutively-active (S177E/S181E) form of the kinase (CA-IKKb) and examined the degradation of the IkBa protein, a known target of IKKb. We detected a decrease in IkBa protein abundance in the present of the CA-IKKb (Figure 4B; left side of dashed line), which was similar to the degradation induced by IL-1b (Figure 4B; right side of dashed line). Accordingly, we discovered that expression of the CA-IKKb increased the transcriptional activity of a synthetic NF-kB reporter gene by 15.7 and 29.9-fold (Figure 4C). Examination of CCL2 mRNA levels revealed a 15-fold increase in the presence of the CA-IKKb (Figure 4D). To ensure specificity for NF-kB-mediated induction of the CCL2 gene by IKKb, we combined overexpres- sion of the IkBa super-repressor with CA-IKKb expression; this experiment revealed a 73% and 84% reduction in the ability of CA-IKKb to induce the expression of CCL2 (Figure 4D). Thus, we demonstrate for the first time in pancreatic b-cells that IKKb is a key participant in the regulation of the CCL2 gene by NF-kB. The Expression of the CCL2 Gene is Sensitive to Inhibition of the p38 MAPK IL-1b activation of the IL-1 receptor is linked to a number of different signaling pathways, including the IKKs (see [20] and Figure 4 above) and the MAPKs [51]. To further examine the specific signaling pathways involved with IL-1b-mediated increas- es in the expression of the CCL2 gene in pancreatic b-cells, we examined phosphorylation of p38, JNK, and ERK in 832/13 cells. Both JNK and p38 were rapidly phosphorylated (within 15 min) upon IL-1b exposure (Figure 5A). By contrast, we found that ERK is not activated in response to IL-1b out to 60 mins (data not shown). The lack of ERK phosphorylation by IL-1b in pancreatic b-cells observed in our study is consistent with a prior report [52]. We next used pharmacological inhibitors that interfere with p38 kinase activity to determine if this maneuver hindered IL-1b- mediated increases in CCL2 gene expression and promoter activity. We discovered that pharmacological inhibition of p38 by SB202190 diminished the IL-1b-induced expression of the CCL2 gene in rat islets by 65% (Figure 5B). Moreover, in 832/13 cells, each of the pyridinyl imidazole based p38 inhibitors used - SB202190, SB203580, and SB239063 - markedly inhibited the IL- 1b-stimulated expression of the CCL2 gene (Figure 5C). Although JNK is phosphorylated in response to IL-1b, blocking JNK activity using SP600125 (JNKi) had no impact on IL-1b- mediated induction of the CCL2 gene (Figure 5C); however, this inhibitor decreased expression of the GADD45 gene (Figure S3). Similarly, inhibition of ERK activity using 20 mM PD98059 did not impair IL-1b- mediated expression of the CCL2 gene (Figure 5C); these findings are congruent with our lack of observed ERK phosphor- ylation in response to IL-1b in pancreatic b-cells. The findings described herein are also consistent with the lack of ERK phosphorylation by IL-1b in a previous study [52]. Furthermore, PLOS ONE | www.plosone.org 6 October 2012 | Volume 7 | Issue 10 | e46986