Department of Molecular Biology, Institute of Basic Medicine, Chinese PLA General Hospital, Beijing, China.
PLoS One. 2011 Mar 31;6(3):e18157. doi: 10.1371/journal.pone.0018157.
Nuclear factor κB (NF-κB)-mediated pathways have been widely implicated in cell survival, development and tumor progression. Although the molecular events of determining NF-κB translocation from cytoplasm to nucleus have been extensively documented, the regulatory mechanisms of NF-κB activity inside the nucleus are still poorly understood. Being a special member of macro domain proteins, LRP16 was previously identified as a coactivator of both estrogen receptor and androgen receptor, and as an interactor of NF-κB coactivator UXT. Here, we investigated the regulatory role of LRP16 on NF-κB activation.
GST pull-down and coimmunoprecipitation (CoIP) assays assessed protein-protein interactions. The functional activity of NF-κB was assessed by luciferase assays, changes in expression of its target genes, and its DNA binding ability. Annexin V staining and flow cytometry analysis were used to evaluate cell apoptosis. Immunohistochemical staining of LRP16 and enzyme-linked immunosorbent assay-based evaluation of active NF-κB were performed on primary human gastric carcinoma samples.
We demonstrate that LRP16 integrates into NF-κB transcriptional complex through associating with its p65 component. RNA interference knockdown of the endogenous LRP16 in cells leads to impaired NF-κB activity and significantly attenuated NF-κB-dependent gene expression. Mechanistic analysis revealed that knockdown of LRP16 did not affect tumor necrosis factor α (TNF-α)-induced nuclear translocation of NF-κB, but blunted the formation or stabilization of functional NF-κB/p300/CREB-binding protein transcription complex in the nucleus. In addition, knockdown of LRP16 also sensitizes cells to apoptosis induced by TNF-α. Finally, a positive link between LRP16 expression intensity in nuclei of tumor cells and NF-κB activity was preliminarily established in human gastric carcinoma specimens.
Our findings not only indicate that LRP16 is a crucial regulator for NF-κB activation inside the nucleus, but also suggest that LRP16 may be an important contributor to the aberrant activation of NF-κB in tumors.
核因子 κB（NF-κB）介导的途径已广泛涉及细胞存活、发育和肿瘤进展。尽管已经广泛记录了确定 NF-κB 从细胞质向核内易位的分子事件，但 NF-κB 活性在核内的调节机制仍知之甚少。LRP16 作为宏结构域蛋白的特殊成员，先前被鉴定为雌激素受体和雄激素受体的共激活子，也是 NF-κB 共激活子 UXT 的相互作用蛋白。在这里，我们研究了 LRP16 对 NF-κB 激活的调节作用。
GST 下拉和免疫共沉淀（CoIP）测定评估蛋白质-蛋白质相互作用。通过荧光素酶测定、其靶基因表达的变化以及其 DNA 结合能力评估 NF-κB 的功能活性。使用 Annexin V 染色和流式细胞术分析评估细胞凋亡。对原发性人胃癌样本进行 LRP16 的免疫组织化学染色和基于酶联免疫吸附测定的活性 NF-κB 评估。
我们证明 LRP16 通过与 p65 成分结合而整合到 NF-κB 转录复合物中。细胞内内源性 LRP16 的 RNA 干扰敲低导致 NF-κB 活性受损，并显著减弱 NF-κB 依赖性基因表达。机制分析表明，LRP16 敲低不影响 TNF-α 诱导的 NF-κB 核内易位，但削弱了功能性 NF-κB/p300/CREB 结合蛋白转录复合物在核内的形成或稳定。此外，LRP16 的敲低也使细胞对 TNF-α 诱导的凋亡敏感。最后，在人胃癌标本中初步建立了肿瘤细胞核内 LRP16 表达强度与 NF-κB 活性之间的正相关关系。
我们的发现不仅表明 LRP16 是核内 NF-κB 激活的关键调节剂，还表明 LRP16 可能是肿瘤中 NF-κB 异常激活的重要贡献者。
