## 3 Transcriptional Regulation of Individual Chemokine Genes in Pancreatic \(\beta\)-Cells
### Chemokine C-C Motif Ligand 2 (CCL2 Aka Monocyte Chemoattractant Protein-1/MCP-1)  
CCL2 is a powerful signal promoting recruitment of various immune cells (e.g., monocytes and macrophages) into secreting tissues, including pancreatic islets [52,53]. The CCL2 gene is regulated positively by IL-1\(\beta\) and negatively by glucocorticoids [5]. Importantly, CCL2 co-localizes with insulin in pancreatic islets, demonstrating \(\beta\)-cells are capable of direct chemokine production [8]. Increasing the secretion of CCL2 from islet \(\beta\)-cells influences monocyte and macrophage recruitment and activity via CCR2 [52]. CCL2 is induced by IL-1\(\beta\) in the clonal 832/13 and INS-1E \(\beta\)-cell lines, two distinct rat insulinoma cell culture models, based on our unpublished microarray experiments [54]. In addition, CCL2 expression is increased in rat and human islets exposed to IL-1\(\beta\)[5] and in islets isolated from a human subject with T1 DM [9]. The elevated expression of CCL2 in response to \(\beta\)-cell exposure to IL-1\(\beta\) requires the p65 subunit of NF-kB [5]. CCL2 has a baseline expression in \(\beta\)-cells, indicating it may have both homeostatic and inflammatory actions. By contrast, expression of the CCL20 gene, encoding a distinct chemokine, is largely undetectable in healthy islets and clonal \(\beta\)-cell lines that have not received an inflammatory stimulus. However, CCL20 is highly inducible by pro-inflammatory signals, such as IL-1\(\beta\) or palmitate, in rodent and human islets, and in rodent \(\beta\)-cell lines [7,32], indicating responsiveness in specific settings. For example, both CCL20 and CCL2 are elevated in circulation during obesity [32,55], a condition associated with chronic, low-grade inflammation.  
IL-1\(\beta\)-dependent increases in CCL2 gene expression, promoter activity and secretion are decreased by low molecular weight inhibitors of the p38 MAPK but not by inhibitors of ERK and JNK kinases [5]. Furthermore, the inhibitor of nuclear factor kappa-B kinase subunit beta (IKK\(\beta\)), which is located upstream of p65 in the NF-kB signaling pathway, is also involved in regulating expression of the CCL2 gene. Inhibitionof IKK\(\beta\) with the small molecule 2-[(Aminocarbonyl)amino]-5-(4-fluorophenyl)-3-thiophenecarboxamide (TPCA) prevents IL-1\(\beta\)-mediated expression in \(\beta\)-cell lines while overexpression of a constitutively-active IKK\(\beta\) drives CCL2 mRNA synthesis and protein secretion [5]. These _in vitro_ data are consistent with transgenic overexpression of constitutively-active IKK\(\beta\), which also leads to elevated CCL2 expression in islets _in vivo_[56].  
The CCL2 gene contains bona fide NF-\(\kappa\)B responsive elements that control gene expression in response to inflammatory signals. Preventing nuclear translocation of the primary DNA binding NF-\(\kappa\)B transcription factor subunit p65 blocks the IL-1\(\beta\)-mediated increases in CCL2 gene transcription [5]. Specific and targeted deletion of p65 protein by siRNA delivery also prevents CCL2 induction by IL-1\(\beta\), while siRNA-directed decreases in the p50 subunit do not have this effect [5]. Moreover, Ser276, within the Rel homology domain of p65, is critical for IL-1\(\beta\) to increase production of CCL2. Thus, the p65 subunit of NF-\(\kappa\)B is the major transcription factor used to induce the expression of the CCL2 gene in pancreatic \(\beta\)-cells, leading to mRNA accumulation and secretion of CCL2 protein [5]. As proof of principle for inflammatory disease, elevated expression of CCL2 and other chemokines is observed in islets of NOD mice [10], hIAPP transgenic mice [57,58], obese mice [11,32], humans with T1 DM [9], and humans with T2 DM [12]. Notably, these are all situations where IL-1\(\beta\) levels are also enhanced.