核因子-κB 和 Nurr-1 在细胞因子诱导的 AtT20 嗜铬细胞瘤细胞前阿黑皮素原基因转录中的作用。

Involvement of nuclear factor-kB and Nurr-1 in cytokine-induced transcription of proopiomelanocortin gene in AtT20 corticotroph cells.

机构信息

Department of Endocrinology and Metabolism, Hirosaki University School of Medicine, Hirosaki, Japan.

出版信息

Neuroimmunomodulation. 2010;17(2):88-96. doi: 10.1159/000258691. Epub 2009 Nov 17.

DOI:10.1159/000258691

PMID:19923853

Abstract

OBJECTIVE

The precise mechanism whereby proinflammatory cytokines activate the hypothalamo-pituitary-adrenal axis is still unclear. We examined whether transcription factors nuclear factor (NF)-kappaB and Nurr-1 are involved in the cytokine-induced proopiomelanocortin (POMC) gene expression.

METHODS

The mouse corticotropinoma cell line AtT20 was treated with tumor necrosis factor-alpha (TNF-alpha) or interleukin-1beta (IL-1beta). Real-time PCR, luciferase assay and Western blotting were conducted to assess the gene expression, promoter activity and protein expression in various conditions.

RESULTS

Intrinsic expression of NF-kappaB was confirmed by RT-PCR. An active component of NF-kappaB (p65) was upregulated in the nuclear fraction by both TNF-alpha and IL-1beta treatment in a dose- and time-related manner. These cytokines potently stimulated the promoter activity of NF-kappaB and Nurr-1. We also found rapid upregulation of the Nurr-1 gene and protein after treatment with these cytokines. Cotreatment of the cells with either of the cytokines and corticotropin-releasing hormone resulted in additive effects. Cytokine-induced Nurr-1 transcription and Nurr-1 transcription induced by overexpression of NF-kappaB were both blunted by mutagenesis within the NF-kappaB responsive element, which implies that Nurr-1 upregulation specifically requires NF-kappaB binding to its own DNA-binding site. Proinflammatory cytokines exert positive effects on POMC gene expression, which were inhibited by pretreatment with a specific NF-kappaB inhibitor.

CONCLUSION

These results together imply that Nurr-1 expression is a connecting point between neuroendocrine and immune systems in mediating cytokine-induced POMC gene expression.

摘要

目的

炎症细胞因子激活下丘脑-垂体-肾上腺轴的确切机制仍不清楚。我们研究了转录因子核因子（NF）-κB 和 Nurr-1 是否参与细胞因子诱导的前阿黑皮素原（POMC）基因表达。

方法

用肿瘤坏死因子-α（TNF-α）或白细胞介素-1β（IL-1β）处理小鼠嗜铬细胞瘤细胞系 AtT20。进行实时 PCR、荧光素酶测定和 Western blot，以评估各种条件下的基因表达、启动子活性和蛋白表达。

结果

通过 RT-PCR 证实了 NF-κB 的内在表达。TNF-α 和 IL-1β 处理以剂量和时间相关的方式上调了核部分中的 NF-κB （p65）的活性成分。这些细胞因子强烈刺激 NF-κB 和 Nurr-1 的启动子活性。我们还发现，这些细胞因子处理后，Nurr-1 基因和蛋白迅速上调。用这些细胞因子和促肾上腺皮质激素释放激素共同处理细胞会产生相加作用。细胞因子诱导的 Nurr-1 转录和 NF-κB 过表达诱导的 Nurr-1 转录都被 NF-κB 反应元件内的突变所阻断，这表明 Nurr-1 的上调特别需要 NF-κB 与其自身 DNA 结合位点结合。促炎细胞因子对 POMC 基因表达有积极影响，用特定的 NF-κB 抑制剂预处理可抑制其作用。