Herbal Research Laboratory, Food, Drug & Chemical Toxicology Group, CSIR-Indian Institute of Toxicology Research (CSIR-IITR), Vishvigyan Bhawan 31, Mahatma Gandhi Marg, Lucknow 226001, Uttar Pradesh, India; Babu Banarasi Das University, Lucknow, Uttar Pradesh, India.
Herbal Research Laboratory, Food, Drug & Chemical Toxicology Group, CSIR-Indian Institute of Toxicology Research (CSIR-IITR), Vishvigyan Bhawan 31, Mahatma Gandhi Marg, Lucknow 226001, Uttar Pradesh, India; Academy of Scientific and Innovative Research, CSIR-IITR Campus, Lucknow, Uttar Pradesh, India.
Free Radic Biol Med. 2018 May 20;120:414-424. doi: 10.1016/j.freeradbiomed.2018.04.550. Epub 2018 Apr 12.
NF-E2 p45-related factor 2 (Nrf2), is a major redox sensitive transcription factor that plays an essential role in regulating glucose metabolism. Inactivation of Nrf2 has been associated with diabetic complications however, mechanisms warranting Nrf2 suppression are incompletely understood. We hypothesized that PHLPP1 activates GSK3β to induce β-TrCP mediated Nrf2 phosphorylation and degradation. In vivo study was carried out in STZ-NA induced type 2 diabetic male Wistar rats. GSK3β mediated Nrf2 ubiquitination was confirmed by administration of GSK3β inhibitor (LiCl; 60 mg/kg bwt.) which rapidly enhanced Nrf2 protein levels in STZ-NA treated diabetic rats. In addition, high glucose (30 mM; 48 h) treated renal proximal tubular cells NRK52E showed decreased Nrf2 nuclear localization, enhanced oxidative stress and caspase3 activation. While specific inhibition with GSK3β inhibitor SB216763 in vitro restored cellular homeostasis, glucose uptake and decreased apoptotic cell death. Immunoblotting and immunocytochemistry data demonstrated that aberrant renal glucose fluxes are associated with p53 mediated modulation in glucose transporter levels where expression of p53 is indirectly targeted through Nrf2 responsive MDM2 protein. Gene knockdown of PHLPP1 in NRK52E cells enhanced Nrf2-responsive antioxidant enzymes HO-1 and NQO-1 which suggested that PHLPP1 up-regulation during hyperglycemia lowers Nrf2 stability via GSK3β activation. More significantly, GSK3β inhibition enhanced Nrf2-ARE binding compared to diabetic rats, providing further confirmation for GSK3β/β-TrCP pathway in suppressing Nrf2 activation during diabetic renal injury. Taken together, our results indicate that PHLPP1 up-surged Nrf2 nuclear instability by promoting Nrf2/β-TrCP association and its inhibition may be critical in the management of diabetic nephropathy.
NF-E2 p45 相关因子 2 (Nrf2) 是一种主要的氧化还原敏感转录因子，在调节葡萄糖代谢中发挥着重要作用。Nrf2 的失活与糖尿病并发症有关，然而，导致 Nrf2 抑制的机制尚未完全阐明。我们假设 PHLPP1 通过激活 GSK3β 诱导 β-TrCP 介导的 Nrf2 磷酸化和降解。在 STZ-NA 诱导的 2 型糖尿病雄性 Wistar 大鼠中进行了体内研究。通过给予 GSK3β 抑制剂 (LiCl; 60mg/kg bwt.) 证实了 GSK3β 介导的 Nrf2 泛素化，该抑制剂可迅速增强 STZ-NA 处理的糖尿病大鼠中 Nrf2 蛋白水平。此外，高葡萄糖 (30mM; 48 小时) 处理的肾近端小管细胞 NRK52E 显示 Nrf2 核定位减少、氧化应激增强和 caspase3 激活增强。而在体外用 GSK3β 抑制剂 SB216763 特异性抑制时，可恢复细胞内稳态、葡萄糖摄取和减少细胞凋亡死亡。免疫印迹和免疫细胞化学数据表明，异常的肾葡萄糖通量与 p53 介导的葡萄糖转运体水平调节有关，其中 p53 的表达通过 Nrf2 反应性 MDM2 蛋白间接靶向。NRK52E 细胞中 PHLPP1 的基因敲低增强了 Nrf2 反应性抗氧化酶 HO-1 和 NQO-1 的表达，这表明高血糖时 PHLPP1 的上调通过激活 GSK3β 降低了 Nrf2 的稳定性。更重要的是，与糖尿病大鼠相比，GSK3β 抑制增强了 Nrf2-ARE 结合，这进一步证实了 GSK3β/β-TrCP 通路在抑制糖尿病肾损伤过程中 Nrf2 激活的作用。综上所述，我们的研究结果表明，PHLPP1 通过促进 Nrf2/β-TrCP 结合而上调 Nrf2 核不稳定性，其抑制可能在糖尿病肾病的治疗中具有重要意义。
