Department of Molecular and Developmental Biology, Institute of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, 305-8575, Japan.
Department of Molecular and Developmental Biology, Institute of Medicine, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, 305-8575, Japan; Division of Aquaculture Biotechnology, Biotechnology Center of Ho Chi Minh City, Ho Chi Minh City, Viet Nam.
Redox Biol. 2023 Jun;62:102673. doi: 10.1016/j.redox.2023.102673. Epub 2023 Mar 14.
The Keap1-Nrf2 pathway is an evolutionarily conserved mechanism that protects cells from oxidative stress and electrophiles. Keap1 is a repressor of Nrf2 in normal cellular conditions but also a stress sensor for Nrf2 activation. Interestingly, fish and amphibians have two Keap1s (Keap1a and Keap1b), of which Keap1b is the ortholog of mammalian Keap1. Keap1a, on the other hand, is a gene found only in fish and amphibians, having been lost during the evolution to amniotes. We have previously shown that keap1b-knockout zebrafish have increased Nrf2 activity and reduced response to certain Nrf2-activating compounds but that they grow normally to adulthood. This may be because the remaining keap1a suppresses the hyperactivation of Nrf2, which is responsible for the post-natal lethality of Keap1-knockout mice. In this study, we analyzed keap1a;keap1b-double-knockout zebrafish to test this hypothesis. We found that keap1a;keap1b-double-knockout zebrafish, like Keap1-knockout mice, showed eating defects and were lethal within a week of hatching. Genetic introduction of the Nrf2 mutation rescued both the eating defects and the larval lethality, indicating that Nrf2 hyperactivation is the cause. However, unlike Keap1-knockout mice, keap1a;keap1b-double-knockout zebrafish showed no physical blockage of the food pathway; moreover, the cause of death was not directly related to eating defects. RNA-sequencing analysis revealed that keap1a;keap1b-double-knockout larvae showed extraordinarily high expression of known Nrf2-target genes as well as decreased expression of visual cycle genes. Finally, trigonelline or brusatol partially rescued the lethality of keap1a;keap1b-double-knockout larvae, suggesting that they can serve as an in vivo evaluation system for Nrf2-inhibiting compounds.
Keap1-Nrf2 通路是一种进化上保守的机制，可保护细胞免受氧化应激和亲电物质的侵害。在正常细胞条件下，Keap1 是 Nrf2 的抑制剂，但也是 Nrf2 激活的应激传感器。有趣的是，鱼类和两栖动物有两种 Keap1（Keap1a 和 Keap1b），其中 Keap1b 是哺乳动物 Keap1 的同源物。另一方面，Keap1a 是仅在鱼类和两栖动物中发现的基因，在进化为羊膜动物的过程中丢失了。我们之前已经表明，keap1b 敲除斑马鱼具有增加的 Nrf2 活性和对某些 Nrf2 激活化合物的反应降低，但它们正常生长到成年。这可能是因为剩余的 keap1a 抑制了 Nrf2 的过度激活，这是 Keap1 敲除小鼠出生后致死的原因。在这项研究中，我们分析了 keap1a;keap1b 双敲除斑马鱼，以验证这一假设。我们发现，keap1a;keap1b 双敲除斑马鱼与 Keap1 敲除小鼠一样，表现出进食缺陷，并在孵化后一周内死亡。Nrf2 突变的遗传引入挽救了进食缺陷和幼虫致死，表明 Nrf2 过度激活是原因。然而，与 Keap1 敲除小鼠不同的是，keap1a;keap1b 双敲除斑马鱼没有食物途径的物理阻塞；此外，死亡的原因与进食缺陷直接无关。RNA 测序分析显示，keap1a;keap1b 双敲除幼虫表现出已知 Nrf2 靶基因的异常高表达以及视觉循环基因的表达降低。最后，瓜氨酸或布瑞沙托醇部分挽救了 keap1a;keap1b 双敲除幼虫的致死率，表明它们可以作为 Nrf2 抑制化合物的体内评估系统。
