Nrf2 activation attenuates genetic endoplasmic reticulum stress induced by a mutation in the phosphomannomutase 2 gene in zebrafish

Abstract

Nrf2 plays critical roles in animals' defense against electrophiles and oxidative stress by orchestrating the induction of cytoprotective genes. We previously isolated the zebrafish mutant it768, which displays up-regulated expression of Nrf2 target genes in an uninduced state. In this paper, we determine that the gene responsible for it768 was the zebrafish homolog of phosphomannomutase 2 (Pmm2), which is a key enzyme in the initial steps of N-glycosylation, and its mutation in humans leads to PMM2-CDG (congenital disorders of glycosylation), the most frequent type of CDG. The pmm2^it768 larvae exhibited mild defects in N-glycosylation, indicating that the pmm2^it768 mutation is a hypomorph, as in human PMM2-CDG patients. A gene expression analysis showed that pmm2^it768 larvae display up-regulation of endoplasmic reticulum (ER) stress, suggesting that the activation of Nrf2 was induced by the ER stress. Indeed, the treatment with the ER stress-inducing compounds up-regulated the gstp1 expression in an Nrf2-dependent manner. Furthermore, the up-regulation of gstp1 by the pmm2 inactivation was diminished by knocking down or out double-stranded RNA-activated protein kinase (PKR)-like ER kinase (PERK), one of the main ER stress sensors, suggesting that Nrf2 was activated in response to the ER stress via the PERK pathway. ER stress-induced activation of Nrf2 was reported previously, but the results have been controversial. Our present study clearly demonstrated that ER stress can indeed activate Nrf2 and this regulation is evolutionarily conserved among vertebrates. Moreover, ER stress induced in pmm2^it768 mutants was ameliorated by the treatment of the Nrf2-activator sulforaphane, indicating that Nrf2 plays significant roles in the reduction of ER stress.

Keywords: ER stress; Nrf2; PMM2-CDG; sulforaphane; zebrafish mutant.

Fig. 1.

Up-regulated expression of Nrf2 target genes in it768 larvae that have splicing defects in the pmm2 gene. (A) Expression of gstp1 and hmox1a in WT or pmm2^it768 larvae injected or not with 1 pmol nrf2aMO. The closed and open arrowheads indicate the gills and liver, respectively. The numbers in each picture indicate the larvae exhibiting strong expression of gstp1 or hmox1a/tested larvae. (B) Nucleotide and deduced amino acid sequences of the major cDNA isolated from WT and pmm2^it768 larvae. The red “a” indicates the G-to-A mutation site of the it768 mutant in the zebrafish pmm2 genome. The green “R” indicates the amino acid residue corresponding to Arg141 in human PMM2. EcoRI indicates the EcoRI site only present in the WT cDNA.

Fig. 2.

Lethality and glycosylation defects in pmm2^it768 larvae. (A) The survival of pmm2^it768 mutants calculated using the Kaplan–Meier method (78). (B) Mannose-related metabolic pathways in N-glycan synthesis. On the ER membrane, 14 sugars [2 N-acetylglucosamine (GlcNAc), 9 mannose (Man), and 3 glucose (Glc)] from nucleotide-activated sugars (UDP-GlcNAc, GDP-Man, Dol-P-Man, and Dol-P-Glc) are sequentially transferred to the lipid-like precursor dolichol phosphate (Dol-P) to synthesize the lipid-linked oligosaccharides (LLO) required for the N-glycosylation of proteins. PMM2 is an essential enzyme for generating mannose-1-phosphate (Man-1-P), which is required for the formation of both GDP-Man and Dol-P-Man. Tunicamycin (TM) blocks the transfer of GlcNAc to Dol-P. (C) Pyridylamino-glycans derived from the zebrafish larvae. The glycan content in WT and pmm2^it768 was calculated based on the peak areas of the chromatograms on the amide column. (D) The HPLC profiles on the amide column of N-linked glycans from whole bodies of WT (black) or pmm2^it768/it768 larvae (pink) at 5 dpf. MX (X = numbers) indicates Man_xGlcNAc₂-asn, and a, b, and c indicate pauci-mannose–type glycans. (E) N-glycosylation profiles on the octadecylsilyl column of fraction c separated on the amide column. The N-glycan structures were identified using the HPLC mapping method, as described in Materials and Methods. circles, mannose; squares, N-acetylglucosamine. Of note, the peak c1 was not detected in WT larvae.

Fig. 3.

PERK-dependent activation of Nrf2 in 5-dpf larvae by genetic- and chemical-induced ER stress. The numbers in each picture indicate the larvae exhibiting strong bip expression in the liver (open arrowheads)/tested larvae (A) or strong gstp1 expression in the gills (closed arrowheads)/tested larvae (D–G). (A) bip expression in WT sibling and pmm2^it768 larvae. (B) The expression of bip and chop was analyzed by RT-PCR in WT, pmm2^it768/+ (m/+), or pmm2^it768/it768 (m/m) larvae. The amount of cDNA used for RT-PCR was standardized by the ef1α expression. (C) The relative expression levels of bip and chop to ef1α was evaluated by qPCR in WT, pmm2^it768/+ (m/+), or pmm2^it768/it768 (m/m) larvae. a and b indicate statistically significant differences (Tukey’s test, P < 0.001; n = 3 for each genotype). (D) Induced expression of gstp1 after 12-h treatment of 5 μg/mL tunicamycin (TM) or 2 μM thapsigargin (TG) in WT AB larvae injected or not with 1 pmol nrf2aMO. (E) Induced expression of gstp1 after 12-h treatment of 1 μM thapsigargin (TG) in WT sibling and nrf2a^fh318 larvae. (F) gstp1 expression in WT sibling and pmm2^it768 larvae injected or not with 1 pmol perkMO. (G) gstp1 expression in WT sibling and perk^it312 larvae injected or not with 1 pmol pmm2MO. The graph shows the percentages of gstp1-positive larvae of the indicated genotypes. The numbers of larvae tested are indicated above the bars. Asterisks denote statistical significance (Fisher’s exact test; *P < 0.001). The data were obtained based on the results of the WISH analysis shown in the Upper panels.

Fig. 4.

The Nrf2-dependent attenuation of the up-regulated ER stress by sulforaphane treatment. The graphs show the percentages of bip-positive 5-dpf larvae of the indicated genotypes and their expression after 12-h treatment of 40 μM sulforaphane (SF). The numbers of larvae tested are indicated above the bars. An asterisk denotes statistical significance (Fisher’s exact test; *P < 0.001). The data were obtained based on the results of the WISH analysis shown in the Right panels. The strength of the bip staining was determined by double-blind scoring. The arrowheads indicate the liver.