Regulation of the CCL2 Gene in Pancreatic b-Cells by IL- 1b and Glucocorticoids: Role of MKP-1 Susan J. Burke1, Matthew R. Goff1, Barrett L. Updegraff1, Danhong Lu 2 , Patricia L. Brown 3 , Steven C. Minkin Jr3, John P. Biggerstaff3, Ling Zhao1,5, Michael D. Karlstad1,4,5, J. Jason Collier1,5* 1 Department of Nutrition, University of Tennessee, Knoxville, Tennessee, United States of America, 2 Sarah W. Stedman Nutrition and Metabolism Center, Duke University Medical Center, Durham, North Carolina, United States of America, 3 Advanced Microscopy and Imaging Center, University of Tennessee, Knoxville, Tennessee, United States of America, 4 Department of Surgery, Graduate School of Medicine, University of Tennessee Medical Center, Knoxville, Tennessee, United States of America, 5 University of Tennessee Obesity Research Center, Knoxville, Tennessee, United States of America Abstract Release of pro-inflammatory cytokines from both resident and invading leukocytes within the pancreatic islets impacts the development of Type 1 diabetes mellitus. Synthesis and secretion of the chemokine CCL2 from pancreatic b-cells in response to pro-inflammatory signaling pathways influences immune cell recruitment into the pancreatic islets. Therefore, we investigated the positive and negative regulatory components controlling expression of the CCL2 gene using isolated rat islets and INS-1-derived b-cell lines. We discovered that activation of the CCL2 gene by IL-1b required the p65 subunit of NF-kB and was dependent on genomic response elements located in the 23.6 kb region of the proximal gene promoter. CCL2 gene transcription in response to IL-1b was blocked by pharmacological inhibition of the IKKb and p38 MAPK pathways. The IL-1b-mediated increase in CCL2 secretion was also impaired by p38 MAPK inhibition and by glucocorticoids. Moreover, multiple synthetic glucocorticoids inhibited the IL-1b-stimulated induction of the CCL2 gene. Induction of the MAP Kinase Phosphatase-1 (MKP-1) gene by glucocorticoids or by adenoviral-mediated overexpression decreased p38 MAPK phosphorylation, which diminished CCL2 gene expression, promoter activity, and release of CCL2 protein. We conclude that glucocorticoid-mediated repression of IL-1b-induced CCL2 gene transcription and protein secretion occurs in part through the upregulation of the MKP-1 gene and subsequent deactivation of the p38 MAPK. Furthermore, the anti- inflammatory actions observed with MKP-1 overexpression were obtained without suppressing glucose-stimulated insulin secretion. Thus, MKP-1 is a possible target for anti-inflammatory therapeutic intervention with preservation of b-cell function. Citation: Burke SJ, Goff MR, Updegraff BL, Lu D, Brown PL, et al. (2012) Regulation of the CCL2 Gene in Pancreatic b-Cells by IL-1b and Glucocorticoids: Role of MKP-1. PLoS ONE 7(10): e46986. doi:10.1371/journal.pone.0046986 Editor: Vassiliki A. Boussiotis, Beth Israel Deaconess Medical Center, Harvard Medical School, United States of America Received March 28, 2012; Accepted September 7, 2012; Published October 9, 2012 Copyright: 2012 Burke et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by start-up funds provided by the University of Tennessee, Knoxville (to JJC). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Jason.Collier@utk.edu Introduction Type 1 diabetes mellitus (T1DM) results from selective elimination of the insulin-producing b-cells within the pancreatic islets via an autoimmune mediated process that requires infiltra- tion of T-lymphocytes and activation of resident macrophages [1,2,3]. Accumulation of immune cells within pancreatic islets is also a major contributor to tissue rejection after islet transplan- tation [4,5]. One of the primary signals leading to immune cell infiltration into tissues, including the pancreatic islets, is the release of chemotactic cytokines, usually referred to as chemokines [6,7]. Synthesis and secretion of chemokines from the b-cell population is a major signal for islet immune cell invasion [8,9,10] and chemokines are critical factors associated with the development of autoimmune diabetes [11,12,13,14]. One chemokine that participates in islet immune cell recruit- ment is CCL2, also known as monocyte chemoattractant protein-1 [15]. CCL2 is a member of the CC chemokine family and recruits specific leukocytes, such as dendritic cells, monocytes, macro- phages, and T-cells to tissues from which it was initially released [13,16]. Each of the immune cell types recruited by CCL2 influences the islet destruction that precedes onset of T1DM [17]. Polymorphisms that increase the expression of the CCL2 gene negatively correlate with pancreatic islet function [18] and transgenic overexpression of CCL2 specifically in islet b-cells promotes insulitis and progression to diabetes in the B6D2 genetic background [8]. Alternatively, transgenic expression of CCL2 in the NOD mouse decreases autoimmune-mediated b-cell destruc- tion [19]. Thus, recruitment of leukocytes into the islet can lead to either immune cell-mediated destruction of the pancreatic b-cell or sparing of b-cell mass through non-destructive insulitis, depending on the genetic environment. Hence, understanding the molecular determinants controlling expression of the CCL2gene may offer insights into the factors regulating islet immune cell invasion. One of the major signals controlling expression of the CCL2 gene is the cytokine IL-1b.The pro-inflammatory outcomes associated with IL-1b are often signaled through the NF-kB pathway [20]. NF-kB is composed of dimers of the transcriptional regulatory subunits RelA/p65, RelB, c-Rel, p50, and p52. The inhibitor of kB proteins (IkBs) bind to NF-kB proteins and mask their nuclear localization signal which promotes cytosolic retention PLOS ONE | www.plosone.org 1 October 2012 | Volume 7 | Issue 10 | e46986