Nrf2 activation attenuates genetic endoplasmic reticulum stress induced by a mutation in the phosphomannomutase 2 gene in zebrafish Katsuki Mukaigasaa,1,2, Tadayuki Tsujitaa,b,1,3, Vu Thanh Nguyena,1,LiLia,1,4, Hirokazu Yagic, Yuji Fusea, Yaeko Nakajima-Takagia,5, Koichi Katoc,d,e, Masayuki Yamamotob,f, and Makoto Kobayashia,b,6 aDepartment of Molecular and Developmental Biology, Faculty of Medicine, University of Tsukuba, 305-8575 Tsukuba, Japan; bExploratory Research for Advanced Technology Environmental Response Project, Japan Science and Technology Agency, University of Tsukuba, 305-8575 Tsukuba, Japan; cGraduate School of Pharmaceutical Sciences, Nagoya City University, Mizuho-ku, 467-8603 Nagoya, Japan; dOkazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, Okazaki, 444-8787 Aichi, Japan; eInstitute for Molecular Science, National Institutes of Natural Sciences, Okazaki, 444-8787 Aichi, Japan; and fDepartment of Medical Biochemistry, Tohoku University Graduate School of Medicine, Aoba-ku, 980-8575 Sendai, Japan Edited by Igor B. Dawid, The Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, and approved January 31, 2018 (received for review August 9, 2017) Nrf2 plays critical roles in animals’ defense against electrophiles the novel Nrf2-inducing cellular stresses and elucidate the mo- and oxidative stress by orchestrating the induction of cytoprotec- lecular basis of their regulation. tive genes. We previously isolated the zebrafish mutant it768,which In this study, we identified the gene responsible for it768, one displays up-regulated expression of Nrf2 target genes in an uninduced of the six mutant lines, and elucidated the molecular mechanism state. In this paper, we determine that the gene responsible for it768 for how the mutation activates the Keap1–Nrf2 system. The was the zebrafish homolog of phosphomannomutase 2 (Pmm2), gene responsible for it768 was phosphomannomutase 2 (pmm2), which is a key enzyme in the initial steps of N-glycosylation, and its which encodes the enzyme required for the biosynthesis of mutation in humans leads to PMM2-CDG (congenital disorders of gly- mannose-1-phosphate, an essential metabolite for an early step cosylation), the most frequent type of CDG. The pmm2it768 larvae in the N-glycosylation process. pmm2it768 homozygous larvae exhibited mild defects in N-glycosylation, indicating that the showed impaired and insufficient N-glycosylation, followed by an pmm2it768 mutation is a hypomorph, as in human PMM2-CDG pa- increase in the endoplasmic reticulum (ER) stress. We found tients. A gene expression analysis showed that pmm2it768 larvae that the ER stress induces the up-regulation of the Keap1– display up-regulation of endoplasmic reticulum (ER) stress, sug- Nrf2 system through the double-stranded RNA-activated protein gesting that the activation of Nrf2 was induced by the ER stress. Indeed, the treatment with the ER stress-inducing compounds up- Significance regulated the gstp1 expression in an Nrf2-dependent manner. Fur- thermore, the up-regulation of gstp1 by the pmm2 inactivation Nrf2 is a master regulator of the antioxidant response and xeno- was diminished by knocking down or out double-stranded RNA- biotic metabolism. In this paper, we demonstrate that Nrf2 also activated protein kinase (PKR)-like ER kinase (PERK), one of the main plays a critical role in the endoplasmic reticulum (ER) stress response ER stress sensors, suggesting that Nrf2 was activated in response to usingazebrafishmutantinwhichNrf2isspontaneouslyactivated. the ER stress via the PERK pathway. ER stress-induced activation of The gene responsible for this mutant was phosphomannomutase 2 Nrf2 was reported previously, but the results have been controver- (pmm2), the enzyme required for the N-glycosylation. Human sial. Our present study clearly demonstrated that ER stress can indeed PMM2 is known to be the gene responsible for PMM2-CDG activate Nrf2 and this regulation is evolutionarily conserved among (congenital disorders of glycosylation), which currently has no vertebrates. Moreover, ER stress induced in pmm2it768 mutants was therapeutic options. pmm2 mutant larvae showed up-regulated ameliorated by the treatment of the Nrf2-activator sulforaphane, in- ER stress and ER stress-dependent Nrf2 activation. Of note, the dicating that Nrf2 plays significant roles in the reduction of ER stress. ER stress in mutant larvae was attenuated following treatment with the Nrf2 activator sulforaphane, suggesting that the ER stress | Nrf2 | PMM2-CDG | sulforaphane | zebrafish mutant pathological conditions of ER stress-associated diseases may be improved by taking Nrf2-activating foods. he Keap1–Nrf2 system senses various environmental stresses Tand induces cytoprotective genes to protect cells from these Author contributions: K.M., T.T., V.T.N., L.L., M.Y., and M.K. designed research; K.M., T.T., stresses (1–3). The main components of the system are Nrf2 and V.T.N., L.L., H.Y., Y.F., Y.N.-T., K.K., and M.K. performed research; K.M., T.T., V.T.N., and Keap1; Nrf2 heterodimerizes with small Maf proteins and func- L.L. analyzed data; and M.K. wrote the paper. tions as a transcription activator while Keap1 represses Nrf2 The authors declare no conflict of interest. functions as an adaptor for Cul3-based E3 ligase to regulate This article is a PNAS Direct Submission. the proteasomal degradation of Nrf2. In addition to the Nrf2- Published under the PNAS license. inhibiting effects, Keap1 also functions as a sensor molecule for a 1K.M., T.T., V.T.N., and L.L. contributed equally to this work. variety of Nrf2-activating compounds. One of the most interesting 2Present address: Department of Neuroanatomy and Embryology, School of Medicine, points in the Keap1–Nrf2 system is that it can respond to cellular Fukushima Medical University, 960-1295 Fukushima, Japan. stresses, such as autophagy impairment and mitochondria stress 3Present address: Department of Applied Biochemistry and Food Science, Saga University, (4, 5), in addition to well-studied oxidative stress and electrophiles. 840-8502 Saga, Japan. The Keap1–Nrf2 system is well conserved among vertebrates, 4Present address: School of Life Science and Technology, Harbin Institute of Technology, including zebrafish (6–8), in light of its molecular basis (9–11), 150080 Harbin, China. activating chemicals and stress (12–15), target genes (16–18), 5Present address: Department of Cellular and Molecular Medicine, Graduate School of and physiological functions (15, 19). We previously isolated six Medicine, Chiba University, 260-8670 Chiba, Japan. mutant zebrafish lines in which Nrf2 is activated without treat- 6To whom correspondence should be addressed. Email: [email protected]. ment with Nrf2 activators (12). We concluded that the mutations This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. in these lines generate some endogenous cellular stresses that 1073/pnas.1714056115/-/DCSupplemental. can activate Nrf2. The analysis of these mutants will help clarify Published online February 22, 2018. 2758–2763 | PNAS | March 13, 2018 | vol. 115 | no. 11 www.pnas.org/cgi/doi/10.1073/pnas.1714056115 Downloaded by guest on September 29, 2021 kinase (PKR)-like ER kinase (PERK) pathway. The Nrf2 acti- type (5mis-pmm2MO). Conversely, the overexpression of pmm2 vation by ER stress has been demonstrated previously using cultured reduced the up-regulated expression of gstp1 in pmm2it768 homo- cells (20), but whether or not this is part of general regulation is zygous larvae (Fig. S2D). These results indicated that the mutation controversial, since negative data were also reported by other groups of pmm2 is indeed the reason why the gstp1 expression was up- (21–23). regulated in it768 mutants. Genomic sequencing of pmm2 demonstrated that there were Results no marked differences in the coding region between WT and pmm2 Is the Gene Responsible for it768. it768 mutant embryos and it768 mutants, but the G in the beginning of intron 5 was A in the larvae showed no obvious defects during embryogenesis and mutant (Fig. 1B). Since it is the first G in the GU–AG rule (25), hatching process in bright-field images while the gstp1 expression a splicing defect was suggested. We therefore next analyzed the in the gills was up-regulated in 5-d-postfertilization (dpf) larvae pmm2 mRNA in WT and mutant larvae by reverse transcription- (Fig. 1A). The up-regulation of other Nrf2 targets, such as PCR (RT-PCR) and found that the WT mRNA was a bit longer hmox1a, was also observed in the liver of 7-dpf larvae so Nrf2 may than that from the it768 larvae, which lost the corresponding have been activated constitutively in the mutants. The expression sequences to exon 5, including the EcoRI site (Fig. S2E). To of gstp1 and hmox1a in the mutant larvae was reduced when nrf2a investigate this splicing defect in detail, we subcloned cDNA was knocked down by morpholino oligonucleotide injection from 2-dpf embryos and 5-dpf larvae and determined their nu- (nrf2aMO) (Fig. 1A), suggesting that up-regulation of these genes cleotide sequences (Fig. S2F). Normal type was not observed in is indeed mediated by the Keap1–Nrf2 system. To confirm the the homozygous mutants; instead, four types of abnormal pmm2 Nrf2 dependence of the up-regulation, we crossed the it768 mu- cDNAs with different splicing types were identified, including fh318 “+3,” which is probably translated into enzymatic-active