Nuclear factor erythroid 2 related factor-1 is stimulated by the nutraceutical celastrol at proteasome inhibiting doses

The mechanism by which many nutraceuticals promote health is incompletely understood. Some act by inhibiting the proteasome. Nuclear factor erythroid 2 related factor 1 (NRF1) is a stress-protective transcription factor that is stimulated by suppressed proteasome activity, such as upon exposure to the proteasome inhibitor bortezomib. Here, we investigated whether nutraceutical compounds with proteasome inhibitory properties can stimulate NRF1 to regulate stress-protective genes. Using a luciferase-based promoter reporting the activity of NRF1 and its homolog NRF2 in Hepa 1-6 cells, we performed a small nutraceutical screen and identified that 2 and 4 μM treatment with celastrol exhibited robust stimulatory effect. Using the same reporter in wild type and gene deleted murine embryonic fibroblasts, we found that Nrf1 deficiency reduced celastrol-stimulated luciferase activity. In Hep3B cells, we found 2 μM celastrol, but not lower concentrations, increased NRF1 processing and nuclear accumulation while also suppressing the trypsin-like, chymotrypsin-like, and caspase-like activities of the proteasome, similar to proteasome inhibiting drugs. However, using control and NRF1 deficient Hep3B cells treated with 2 μM celastrol, MG132, bortezomib, or vehicle, we found distinct effects by celastrol versus MG132 and bortezomib on NRF1-dependent regulation of stress-protective genes. Hence, our results show that celastrol can stimulate NRF1 at concentrations that suppress proteasome activity, but its effect on gene regulation appear distinct compared to proteasome inhibitor. Overall, our findings reveal that that NRF1 may play a role in the health promoting effects of celastrol and possibly other nutraceuticals with proteasome inhibitory properties.许多营养保健品（nutraceuticals）促进健康的具体机制尚未完全阐明。部分营养保健品可通过抑制蛋白酶体发挥作用。核因子红细胞系2相关因子1（Nuclear factor erythroid 2 related factor 1, NRF1）是一种应激保护性转录因子，当蛋白酶体活性受到抑制时（例如暴露于蛋白酶体抑制剂硼替佐米（bortezomib）时），该因子会被激活。本研究旨在探究具有蛋白酶体抑制特性的营养保健品化合物是否能够激活NRF1，从而调控应激保护性基因的表达。我们采用基于荧光素酶的报告基因启动子体系，在Hepa 1-6细胞中检测NRF1及其同源蛋白NRF2的活性，通过小型营养保健品筛选实验，发现雷公藤红素（celastrol）以2 μM和4 μM浓度处理时，可产生显著的激活效应。我们在野生型及基因敲除小鼠胚胎成纤维细胞中使用相同的报告基因体系，发现Nrf1基因缺失会削弱雷公藤红素诱导的荧光素酶活性。在Hep3B细胞中，2 μM浓度的雷公藤红素（而非更低浓度）可促进NRF1的剪切与核积累，同时抑制蛋白酶体的胰蛋白酶样、糜蛋白酶样及半胱天冬酶样活性，这与蛋白酶体抑制剂的作用效果类似。不过，我们分别用2 μM雷公藤红素、MG132、硼替佐米或溶剂处理对照组与NRF1缺陷型Hep3B细胞后发现，雷公藤红素与MG132、硼替佐米在NRF1依赖的应激保护性基因调控方面存在显著差异。综上，本研究结果表明，雷公藤红素可在抑制蛋白酶体活性的浓度范围内激活NRF1，但其对基因表达的调控作用与经典蛋白酶体抑制剂存在明显区别。整体而言，我们的研究揭示，NRF1可能在雷公藤红素以及其他具有蛋白酶体抑制特性的营养保健品的健康促进效应中发挥作用。