Cell and Molecular Biology, Karolinska Institutet, Stockholm, Sweden; Department of Transfusion Medicine, Saga University Hospital, Saga, Japan.
Department of Gene Expression Regulation, Institute of Development, Aging and Cancer, Tohoku University, Sendai, Japan.
J Biol Chem. 2022 Jul;298(7):102137. doi: 10.1016/j.jbc.2022.102137. Epub 2022 Jun 14.
Upregulation of Notch3 expression has been reported in many cancers and is considered a marker for poor prognosis. Hypoxia is a driving factor of the Notch3 signaling pathway; however, the induction mechanism and role of hypoxia-inducible factor-1α (HIF-1α) in the Notch3 response are still unclear. In this study, we found that HIF-1α and poly [ADP-ribose] polymerase 1 (PARP-1) regulate Notch3 induction under hypoxia via a noncanonical mechanism. In the analyzed cancer cell lines, Notch3 expression was increased during hypoxia at both the mRNA and protein levels. HIF-1α knockdown and Notch3 promoter reporter analyses indicated that the induction of Notch3 by hypoxia requires HIF-1α and also another molecule that binds the Notch3 promoter's guanine-rich region, which lacks the canonical hypoxia response element. Therefore, using mass spectrometry analysis to identify the binding proteins of the Notch3 promoter, we found that PARP-1 specifically binds to the Notch3 promoter. Interestingly, analyses of the Notch3 promoter reporter and knockdown of PARP-1 revealed that PARP-1 plays an important role in Notch3 regulation. Furthermore, we demonstrate that PARP inhibitors, including an inhibitor specific for PARP-1, attenuated the induction of Notch3 by hypoxia. These results uncover a novel mechanism in which HIF-1α associates with PARP-1 on the Notch3 promoter in a hypoxia response element-independent manner, thereby inducing Notch3 expression during hypoxia. Further studies on this mechanism could facilitate a better understanding of the broader functions of HIF-1α, the roles of Notch3 in cancer formation, and the insights into novel therapeutic strategies.
Notch3 表达的上调已在许多癌症中得到报道，被认为是预后不良的标志物。缺氧是 Notch3 信号通路的驱动因素；然而，缺氧诱导因子-1α（HIF-1α）在 Notch3 反应中的诱导机制和作用尚不清楚。在本研究中，我们发现 HIF-1α 和聚[ADP-核糖]聚合酶 1（PARP-1）通过非经典机制调节缺氧下的 Notch3 诱导。在分析的癌细胞系中，Notch3 在缺氧时在 mRNA 和蛋白质水平上均增加。HIF-1α 敲低和 Notch3 启动子报告分析表明，缺氧诱导 Notch3 需要 HIF-1α，还需要另一种结合 Notch3 启动子富含鸟嘌呤区域的分子，该区域缺乏经典的缺氧反应元件。因此，使用质谱分析来鉴定 Notch3 启动子的结合蛋白，我们发现 PARP-1 特异性结合 Notch3 启动子。有趣的是，Notch3 启动子报告分析和 PARP-1 的敲低表明 PARP-1 在 Notch3 调节中起重要作用。此外，我们证明 PARP 抑制剂，包括 PARP-1 的抑制剂，可减轻缺氧诱导的 Notch3 诱导。这些结果揭示了一种新的机制，其中 HIF-1α 以缺氧反应元件独立的方式与 Notch3 启动子上的 PARP-1 结合，从而在缺氧期间诱导 Notch3 表达。对这种机制的进一步研究可以促进更好地理解 HIF-1α 的更广泛功能、 Notch3 在癌症形成中的作用以及对新治疗策略的深入了解。
