Overexpression of Kpnβ1 and Kpnα2 importin proteins in cancer derives from deregulated E2F activity.
Overexpression of Kpnβ1 and Kpnα2 importin proteins in cancer derives from deregulated E2F activity.
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DOI：
10.1371/journal.pone.0027723
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发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Leaner VD
中科院分区：
文献类型：
--
作者：
van der Watt PJ;Ngarande E;Leaner VD
核粘蛋白超家族包括核转运蛋白，参与运送某些货物蛋白进出细胞核。核粘蛋白β1 （Kpnβ1）和核粘蛋白α2 （Kpnα2）是两种进口蛋白，它们协同工作将它们的货物运输到细胞核中。我们之前发现，与正常上皮和转化细胞相比，Kpnβ1和Kpnα2在宫颈肿瘤中的表达增加。因此，本研究旨在确定高水平的Kpnβ1和Kpnα2在癌细胞中的转录调控机制。将Kpnβ1（−2013至+100）和Kpnα2（−1900至+69）启动子片段分别克隆到报告载体pGL3-basic中，荧光素酶测定显示，与正常细胞相比，这两种启动子片段在癌细胞和转化细胞中的活性显著提高。一系列缺失构建确定了−637至−271 Kpnβ1和−180至−24 Kpnα2启动子区域负责差异启动子活性，并且在这些区域内鉴定了许多高度保守的E2F结合位点。突变分析证实了启动子活性需要E2F位点，ChIP分析证实了体内E2F2/Dp1与Kpnβ1和Kpnα2启动子结合。Dp1抑制导致各自蛋白水平下降，证实了E2F在癌症中Kpnβ1和Kpnα2蛋白过表达中的作用。由于E2F的抑制因子Rb被HPV E7抑制，已知E2F活性在宫颈癌细胞中被解除调控。使用siRNA抑制E7导致Kpnβ1和Kpnα2启动子活性降低，Rb过表达也是如此。总之，本研究首次表明，在癌细胞中升高的Kpnβ1和Kpnα2表达与E2F/Rb活性解除相关的转录调控改变有关
The Karyopherin superfamily comprises nuclear transport proteins, involved in the shuttling of certain cargo proteins into and out of the nucleus. Karyopherin β1 (Kpnβ1) and Karyopherin α2 (Kpnα2) are importin proteins, which work in concert to transport their cargo into the nucleus. We previously identified increased expression of Kpnβ1 and Kpnα2 in cervical tumours compared to normal epithelium and in transformed cells compared to their normal counterparts. This study therefore aimed to identify the transcription regulatory mechanisms associated with high Kpnβ1 and Kpnα2 levels in cancer cells. Kpnβ1 (−2013 to +100) and Kpnα2 (−1900 to +69) promoter fragments were separately cloned into the reporter vector, pGL3-basic, and luciferase assays revealed both as significantly more active in cancer and transformed cells compared to normal. A series of deletion constructs identified the −637 to −271 Kpnβ1 and −180 to −24 Kpnα2 promoter regions as responsible for the differential promoter activity, and a number of highly conserved E2F binding sites were identified within these regions. Mutation analysis confirmed the requirement of E2F sites for promoter activity, and ChIP analysis confirmed E2F2/Dp1 binding to the Kpnβ1 and Kpnα2 promoters in vivo. Dp1 inhibition resulted in decreased levels of the respective proteins, confirming the role of E2F in the overexpression of Kpnβ1 and Kpnα2 proteins in cancer. E2F activity is known to be deregulated in cervical cancer cells due to the inhibition of its repressor, Rb, by HPV E7. The inhibition of E7 using siRNA resulted in decreased Kpnβ1 and Kpnα2 promoter activities, as did the overexpression of Rb. In conclusion, this study is a first to show that elevated Kpnβ1 and Kpnα2 expression in cancer cells correlates with altered transcriptional regulation associated with deregulated E2F/Rb activities
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