Regulation of CCL2 Gene Expression Figure 10. Pharmacological or phosphatase-mediated inhibition of IL-1b-stimulated p38 MAPK phosphorylation decreases synthesis and secretion of CCL2. A. IL-1b induces release of NF-kB subunits from regulatory proteins by activating the IKKs. In addition, IL-1b promotes phosphorylation of p38 MAPK, increases CCL2 transcription using the NF-kB pathway, culminating with secretion of CCL2 protein. B. The activation of p38 by IL-1b is critical for induction of CCL2 gene expression and secretion of CCL2 protein. Inhibiting p38 MAPK activity, either through the use of pyridinyl-imidazole based inhibitors or via MKP-1 mediated dephosphorylation, impairs synthesis and secretion of CCL2. doi:10.1371/journal.pone.0046986.g010 plausible that manipulation of GC responsive genes may be one such strategy. Towards this end, we examined the GR controlled gene MKP-1 (aka DUSP-1) as a potential regulatory factor controlling IL-1b- mediated signaling in the pancreatic b-cell. We selected MKP-1 because it has been well studied as both a gene induced by GCs [29,34] and because the knockout mice displayed enhanced sensitivity to inflammatory stimuli [59,60]. Since both glucocor- ticoids and p38 inhibitors were effective strategies to oppose the effects of IL-1b, it was reasonable to postulate that MKP-1 might be an effective and targeted anti-inflammatory strategy. We demonstrate here for the first time that multiple GR ligands induced the expression of the MKP-1 gene in isolated rat islets and b-cell lines, leading to a decrease in IL-1b-stimulated p38 MAPK phosphorylation (Figure 10). Interfering with GR-mediated induction of MKP-1 by siRNA duplexes decreased the effective- ness of GCs to suppress CCL2 gene expression (Figure 8). We interpret this finding to indicate that upregulation of this phosphatase by GCs is a key component to controlling signal intensity to the CCL2 gene, and perhaps other genes. Moreover, the effects of MKP-1 overexpression to oppose p38 signaling also suppressed CCL2 release (Figure 8). We note that MKP-1 overexpression had a more modest impact on the CCL2 gene when compared to pharmacological inhibitors of p38 MAPK or to the effects of glucocorticoids. We attribute this finding to the pharmacological inhibitors being able to suppress both stimulated and tonic activity of the p38 MAPK (by virtue of competing with ATP for active site binding), while the phosphatase can only diminish stimulus-enhanced activity by removing phosphate groups from specific amino acids. On the other hand, GCs can potentially employ a variety of mechanisms to suppress inflam- mation, of which inducing MKP-1 is only one such possibility. Indeed, silencing MKP-1 only partially inhibited the effectiveness of GCs to suppress CCL2 expression and secretion, while PLOS ONE | www.plosone.org 16 October 2012 | Volume 7 | Issue 10 | e46986