Department of Systems Biology, University of Texas - MD Anderson Cancer Center, Houston, 77030, USA.
Breast Cancer Res. 2011 Jun 20;13(3):R65. doi: 10.1186/bcr2902.
The c-Jun coactivator, Jun activation-domain binding protein 1 (Jab1) also known as the fifth component of the COP9 signalosome complex (CSN5), is a novel candidate oncogene whose aberrant expression contributes to the progression of breast carcinoma and other human cancers. The mechanism of Jab1 gene expression and its deregulation in cancer cells remains to be identified. We therefore investigated the transcriptional regulatory mechanisms of Jab1 expression in human breast carcinoma cells.
To identify potential regulators of Jab1 transcription, we cloned the 5' upstream region of the human Jab1 gene and mapped its transcriptional start site. We identified binding sequences for the CCAAT/enhancer binding protein (C/EBP) and GATA, as well as a signal transducer and activator of transcription-3 (Stat3) consensus sequence overlapping the C/EBP site, using 5'- deletion analysis and a gene reporter assay. Mutational analysis of these binding sites was performed to confirm their roles in promoting Jab1 transcription in breast cancer cells. We further confirmed these binding sites using electrophoretic mobility shift assays (EMSAs) and chromatin immunoprecipitation (ChIP) assays. We also analyzed whether the siRNA-mediated inactivation of Stat3 and Src could reduce Jab1-promoter activity and whether interleukine-6 (IL-6) could mediate increased Jab1 expression through Stat3 signaling.
We identified binding sequences for C/EBP, GATA, as well as a Stat3 consensus sequence overlapping the C/EBP site in the promoter region of Jab1. C/EBP-beta2 is a potential transcriptional activator of Jab1 and mutation of the C/EBP/Stat3 binding site significantly reduced Jab1-promoter activity. In addition, inhibiting Stat3 significantly reduced Jab1-promoter activation. EMSA and ChIP assays confirmed that C/EBP, GATA1 and Stat3 bind to Jab1 promoter in breast carcinoma cells. We also found that Src, an activator of Stat3, is involved in Jab1-promoter activation. siRNA knockdown of Src reduced the Jab1-promoter activity, similar to the results seen when Stat3 was inhibited in breast carcinoma cells. Interestingly, reactivation of Stat3 in normal mammary epithelial cells (MCF-10A, MCF-10F) is sufficient to reactivate Jab1 expression. Treatment with the cytokine IL-6 resulted in increased Jab1 expression that was blocked by inhibition of Stat3.
These findings reveal a novel mechanism of Jab1 gene regulation and provide functional and mechanistic links between the Src/Stat3 and IL-6/Stat3 signaling axes that are involved in the activation of Jab1 transcription and regulation of this novel oncogenic protein.
c-Jun 共激活因子，Jun 激活结构域结合蛋白 1（Jab1）也称为 COP9 信号体复合物（CSN5）的第五个组成部分，是一种新的候选癌基因，其异常表达有助于乳腺癌和其他人类癌症的进展。Jab1 基因表达及其在癌细胞中的失调的机制仍有待确定。因此，我们研究了人乳腺癌细胞中 Jab1 表达的转录调控机制。
为了鉴定 Jab1 转录的潜在调节剂，我们克隆了人 Jab1 基因的 5'上游区并绘制了其转录起始位点。我们使用 5'-缺失分析和基因报告基因测定鉴定了 CCAAT/增强子结合蛋白（C/EBP）和 GATA 的结合序列，以及重叠 C/EBP 位点的信号转导和转录激活因子 3（Stat3）的共有序列。对这些结合位点进行突变分析以确认它们在促进乳腺癌细胞中 Jab1 转录中的作用。我们进一步使用电泳迁移率变动分析（EMSA）和染色质免疫沉淀（ChIP）分析证实了这些结合位点的作用。我们还分析了 siRNA 介导的 Stat3 和 Src 失活是否可以降低 Jab1 启动子活性，以及白细胞介素 6（IL-6）是否可以通过 Stat3 信号转导介导 Jab1 表达的增加。
我们在 Jab1 启动子区域中鉴定了 C/EBP、GATA 以及重叠 C/EBP 位点的 Stat3 共有序列的结合序列。C/EBP-beta2 是 Jab1 的潜在转录激活因子，C/EBP/Stat3 结合位点的突变显著降低了 Jab1 启动子活性。此外，抑制 Stat3 显著降低了 Jab1 启动子的激活。EMSA 和 ChIP 分析证实 C/EBP、GATA1 和 Stat3 在乳腺癌细胞中结合 Jab1 启动子。我们还发现，Stat3 的激活剂 Src 参与 Jab1 启动子的激活。Src 的 siRNA 敲低降低了 Jab1 启动子活性，这与在乳腺癌细胞中抑制 Stat3 时的结果相似。有趣的是，在正常乳腺上皮细胞（MCF-10A、MCF-10F）中重新激活 Stat3 足以重新激活 Jab1 表达。用细胞因子 IL-6 处理导致 Jab1 表达增加，该增加被 Stat3 抑制所阻断。
这些发现揭示了 Jab1 基因调控的新机制，并提供了 Src/Stat3 和 IL-6/Stat3 信号轴之间的功能和机制联系，这些联系涉及 Jab1 转录的激活和这种新的致癌蛋白的调节。
