Molecular regulation of monocyte chemoattractant protein-1 expression in pancreatic beta-cells.
Pancreatic beta-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 beta-cells, insulin-producing INS-1E cells, and RINm5F cells was investigated. Transient transfections with luciferase-reporter constructs identified an interleukin (IL)-1beta-responsive enhancer region between -2,180 bp and -2,478 bp. Mutation of either of the two nuclear factor (NF)-kappaB sites present in this region abrogated IL-1beta-induced MCP-1 promoter activity. Binding of NF-kappaB 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-kappaB was confirmed by chromatin immunoprecipitation assay. Blocking of NF-kappaB activation in cytokine-exposed primary beta-cells by an adenovirus overexpressing a nondegradable form of IkappaBalpha or by pyrrolidine dithiocarbamate decreased IL-1beta-induced MCP-1 mRNA expression. We conclude that NF-kappaB plays an important role for MCP-1 expression in beta-cells. This transcription factor may be an interesting target for ex vivo gene therapy before islet transplantation.
Kutlu
Burak
B
Laboratory of Experimental Medicine, Université Libre de Bruxelles, Belgium.
Darville
Martine I
MI
Cardozo
Alessandra K
AK
Eizirik
Décio L
DL
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D018932:Chemokine CCL2 / Q000235:genetics*; D018925:Chemokines / Q000235:genetics; D017931:DNA Primers; D005786:Gene Expression Regulation / Q000502:physiology*; D007515:Islets of Langerhans / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D016328:NF-kappa B / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism
D018932:Chemokine CCL2; D018925:Chemokines; D017931:DNA Primers; D016328:NF-kappa B; D011994:Recombinant Proteins; D012097:Repressor Proteins