Laboratory of DNA Damage Signaling, Chiba Cancer Center Research Institute, Chiba, Japan.
Cell Death Dis. 2013 Apr 25;4(4):e610. doi: 10.1038/cddis.2013.127.
Runt-related transcription factor 2 (RUNX2) is the best known as an essential protein for osteoblast differentiation. In this study, we have found for the first time that RUNX2 acts as a negative regulator for p53 in response to DNA damage. On DNA damage mediated by adriamycin (ADR) exposure, p53 as well as RUNX2 was induced at protein and mRNA level in human osteosarcoma-derived U2OS cells in association with a significant upregulation of various p53-target genes. Indirect immunostaining and co-immunoprecipitation experiments demonstrated that RUNX2 colocalizes with p53 in cell nucleus and forms a complex with p53 following ADR treatment. Chromatin immunoprecipitation assays revealed that RUNX2/p53 complex is efficiently recruited onto p53-target promoters in response to ADR, suggesting that RUNX2 might be involved in the regulation of transcriptional activation mediated by p53. Indeed, forced expression of RUNX2 resulted in a remarkable downregulation of p53-target genes. Consistent with these observations, knockdown of RUNX2 enhanced ADR-mediated apoptosis and also elevated p53-target gene expression in response to ADR. On the other hand, depletion of RUNX2 in p53-deficient human lung carcinoma-derived H1299 cells had an undetectable effect on p53-target gene expression regardless of ADR treatment, indicating that RUNX2-mediated downregulation of p53-target genes is dependent on p53. Furthermore, RUNX2/p53 complex included histone deacetylase 6 (HDAC6) and HDAC6 was also recruited onto p53-target promoters following ADR exposure. Of note, HDAC6-specific chemical inhibitor tubacin treatment enhanced ADR-mediated upregulation of p53-target gene expression, indicating that deacetylase activity of HDAC6 is required for RUNX2-mediated downregulation of p53-target gene. Taken together, our present findings strongly suggest that RUNX2 inhibits DNA damage-induced transcriptional as well as pro-apoptotic activity of p53 through the functional collaboration with HDAC6 and therefore might be an attractive therapeutic target for cancer treatment.
runt 相关转录因子 2（RUNX2）是成骨细胞分化所必需的蛋白，这是最为人所知的。在这项研究中，我们首次发现 RUNX2 在 DNA 损伤时作为 p53 的负调控因子发挥作用。在阿霉素（ADR）暴露介导的 DNA 损伤中，人骨肉瘤衍生的 U2OS 细胞中 p53 和 RUNX2 的蛋白和 mRNA 水平均被诱导，同时各种 p53 靶基因的显著上调。间接免疫染色和共免疫沉淀实验表明，RUNX2 与 p53 在细胞核中共定位，并在 ADR 处理后与 p53 形成复合物。染色质免疫沉淀分析显示，RUNX2/p53 复合物可有效地募集到 ADR 响应的 p53 靶启动子上，提示 RUNX2 可能参与 p53 介导的转录激活调节。事实上，强制表达 RUNX2 可显著下调 p53 靶基因。与这些观察结果一致，RUNX2 的敲低增强了 ADR 介导的细胞凋亡，并提高了 ADR 响应时的 p53 靶基因表达。另一方面，在 p53 缺失的人肺癌衍生的 H1299 细胞中耗尽 RUNX2 对 p53 靶基因表达没有检测到影响，无论 ADR 处理如何，这表明 RUNX2 介导的 p53 靶基因下调依赖于 p53。此外，RUNX2/p53 复合物包含组蛋白去乙酰化酶 6（HDAC6），并且在 ADR 暴露后，HDAC6 也被募集到 p53 靶启动子上。值得注意的是，HDAC6 特异性化学抑制剂 tubacin 处理增强了 ADR 介导的 p53 靶基因表达上调，表明 HDAC6 的去乙酰化酶活性对于 RUNX2 介导的 p53 靶基因下调是必需的。总之，我们的研究结果强烈表明，RUNX2 通过与 HDAC6 的功能协作抑制 DNA 损伤诱导的 p53 的转录和促凋亡活性，因此可能是癌症治疗的有吸引力的治疗靶标。
