Molecular regulation of monocyte chemoattractant protein-1 expression in pancreatic β-cells
Molecular regulation of monocyte chemoattractant protein-1 expression in pancreatic β-cells
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DOI：
10.2337/diabetes.52.2.348
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发表时间：
2003-02-01
期刊：
影响因子：
7.7
通讯作者：
Eizirik, DL
中科院分区：
文献类型：
--
作者：
Kutlu, B;Darville, MI;Eizirik, DL
在1型糖尿病的病程中，胰岛β细胞被选择性地破坏。在疾病的早期阶段，单核细胞的炎性渗入，主要包含单核细胞和T细胞，出现在胰岛(岛炎)。趋化因子，如单核细胞趋化蛋白-1(MCP-1)，在这些免疫细胞的募集和激活中起着关键作用。我们之前已经描述了细胞因子诱导的人和大鼠胰岛MCP-1基因的表达。在本研究中，研究了细胞因子对大鼠MCP-1基因在荧光激活细胞分选纯化的大鼠胰岛β细胞、胰岛素分泌的INS-1E细胞和RINm5F细胞中的转录调控。用荧光素酶报告基因的瞬时转染法在-2,180~-2,478个碱基之间发现了一个白细胞介素1β反应增强子区。该区域存在的两个核因子-kappaB位点中的任何一个的突变都会取消IL-1β诱导的单核细胞趋化蛋白-1启动子活性。凝胶移位实验显示核因子-kappaB与这两个位点结合，超移位实验显示存在p65/p50异源二聚体和p65同源二聚体。染色质免疫沉淀实验证实了核因子-kappaB的体内结合。通过过度表达不可降解形式的IkappaBalpha的腺病毒或吡咯烷二硫代氨基甲酸酯阻断细胞因子暴露的原代β细胞中核因子-kappaB的激活，可降低IL-1β诱导的MCP-1mRNA的表达。我们认为，核因子-kappaB在β细胞MCP-1的表达中起重要作用。这种转录因子可能是胰岛移植前体外基因治疗的一个有趣的靶点。
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.