Miri Seyed Mohammad, Ata Büşra N, Çimen Şeyma, Barakat Sarah, Ghaffari Zaki Asal, Armouch Joudi, Vatandaşlar Emre, Vilain Sven, Öztürk Gürkan, Eroğlu Emrah
Regenerative and Restorative Medicine Research Center (REMER), Research Institute for Health Sciences and Technologies (SABITA), Istanbul Medipol University, Istanbul 34810, Turkey.
Molecular Biology, Genetics and Bioengineering Program, Faculty of Engineering and Natural Sciences, Sabanci University, Istanbul 34956, Turkey.
ACS Sens. 2025 May 23;10(5):3402-3411. doi: 10.1021/acssensors.4c03167. Epub 2025 Feb 28.
Reactive oxygen species, particularly hydrogen peroxide (HO), play crucial roles in cellular signaling, with Nrf2 serving as a key transcription factor in maintaining redox homeostasis. However, the precise influence of HO on Nrf2 activity under physiological normoxia remains unclear due to the limitations of oxygen-sensitive imaging methods. To address this, we developed and validated an oxygen-insensitive Nrf2 reporter named pericellular oxygen-insensitive Nrf2 transcriptional performance reporter (POINTER). We employed this reporter in human cerebral microvascular endothelial cells (hCMEC/D3). Using POINTER, we investigated how varying intracellular HO concentrations affect Nrf2 regulation under normoxia (5 kPa O) compared to hyperoxia (ambient air, 21 kPa O). We manipulated intracellular HO levels through exogenous application, chemogenetic production using a modified amino acid oxidase, and pharmacological induction with Auranofin. Our findings reveal that Nrf2 transcriptional activity is significantly lower under normoxia than under hyperoxia, supporting previous literature and expectations. Using POINTER, we found that both antioxidant pathway inhibition and sustained HO elevation are essential for modulating Nrf2 activity. These findings provide new insights into the regulation of Nrf2 by HO.
活性氧物种，尤其是过氧化氢（H₂O₂），在细胞信号传导中发挥着关键作用，其中核因子E2相关因子2（Nrf2）是维持氧化还原稳态的关键转录因子。然而，由于氧敏感成像方法的局限性，在生理常氧条件下H₂O₂对Nrf2活性的确切影响仍不清楚。为了解决这一问题，我们开发并验证了一种对氧不敏感的Nrf2报告基因，称为细胞周围氧不敏感Nrf2转录性能报告基因（POINTER）。我们在人脑血管内皮细胞（hCMEC/D3）中使用了该报告基因。使用POINTER，我们研究了与高氧（环境空气，21 kPa O₂）相比，在常氧（5 kPa O₂）条件下不同的细胞内H₂O₂浓度如何影响Nrf2的调控。我们通过外源应用、使用改良氨基酸氧化酶的化学遗传产生以及用金诺芬进行药理学诱导来操纵细胞内H₂O₂水平。我们的研究结果表明，在常氧条件下Nrf2的转录活性显著低于高氧条件下，这支持了先前的文献和预期。使用POINTER，我们发现抗氧化途径抑制和持续的H₂O₂升高对于调节Nrf2活性都是必不可少的。这些发现为H₂O₂对Nrf2的调控提供了新的见解。
