Molecular Regulation of Monocyte Chemoattractant Protein-1 Expression in Pancreatic β-Cells
- 1Laboratory of Experimental Medicine, Université Libre de Bruxelles, Brussels, Belgium
- 2Gene Expression Unit, Diabetes Research Center, Vrije Universiteit Brussel, Brussels, Belgium
Pancreatic β-cells are selectively destroyed during the course of type 1 diabetes. In the early stages of the disease, inflammatory infiltrates of mononuclear cells, containing predominantly monocytes and T-cells, are present in the islets (insulitis). Chemokines, such as monocyte chemoattractant protein-1 (MCP-1), play a key role in the recruitment and activation of these immunocytes. We have previously described cytokine-induced MCP-1 gene expression in human and rat pancreatic islets. In the present study, the transcriptional regulation by cytokines of the rat MCP-1 gene in fluorescence-activated cell sorting-purified rat β-cells, insulin-producing INS-1E cells, and RINm5F cells was investigated. Transient transfections with luciferase-reporter constructs identified an interleukin (IL)-1β-responsive enhancer region between -2,180 bp and −2,478 bp. Mutation of either of the two nuclear factor (NF)-κB sites present in this region abrogated IL-1β-induced MCP-1 promoter activity. Binding of NF-κB to the two sites was shown in vitro by gel shift assays, while supershift assays revealed the presence of p65/p50 heterodimers and p65 homodimers. In vivo binding of NF-κB was confirmed by chromatin immunoprecipitation assay. Blocking of NF-κB activation in cytokine-exposed primary β-cells by an adenovirus overexpressing a nondegradable form of IκBα or by pyrrolidine dithiocarbamate decreased IL-1β-induced MCP-1 mRNA expression. We conclude that NF-κB plays an important role for MCP-1 expression in β-cells. This transcription factor may be an interesting target for ex vivo gene therapy before islet transplantation.
Address correspondence and reprint requests to Décio L. Eizirik, Laboratory of Experimental Medicine, Université Libre de Bruxelles, Route de Lennik, 808, B-1070, Brussels, Belgium CP 618. E-mail:.
Received for publication 25 July 2002 and accepted in revised form 13 November 2002.
ChIP, chromatin immunoprecipitation; EMSA, electrophoretic mobility shift assay; FACS, fluorescence-activated cell sorting; GAS, γ-activated sequence; IFN, interferon; IL, interleukin; IRIS, IFN response-inhibitory sequence; MCP-1, monocyte chemoattractant protein-1; NF, nuclear factor; OD, optical density; PDTC, pyrrolidine dithiocarbamate; TNF, tumor necrosis factor.