Physiological IRE-1-XBP-1 and PEK-1 signaling in Caenorhabditis elegans larval development and immunity

Abstract

Endoplasmic reticulum (ER) stress activates the Unfolded Protein Response, a compensatory signaling response that is mediated by the IRE-1, PERK/PEK-1, and ATF-6 pathways in metazoans. Genetic studies have implicated roles for UPR signaling in animal development and disease, but the function of the UPR under physiological conditions, in the absence of chemical agents administered to induce ER stress, is not well understood. Here, we show that in Caenorhabditis elegans XBP-1 deficiency results in constitutive ER stress, reflected by increased basal levels of IRE-1 and PEK-1 activity under physiological conditions. We define a dynamic, temperature-dependent requirement for XBP-1 and PEK-1 activities that increases with immune activation and at elevated physiological temperatures in C. elegans. Our data suggest that the negative feedback loops involving the activation of IRE-1-XBP-1 and PEK-1 pathways serve essential roles, not only at the extremes of ER stress, but also in the maintenance of ER homeostasis under physiological conditions.

Figure 1. XBP-1 deficiency results in a dramatic increase in IRE-1 activity.

(A) Detection of xbp-1 mRNA splicing by IRE-1 in the C. elegans xbp-1(zc12) mutant. Schematic of the unspliced and spliced xbp-1 mRNA, noting the position of the premature termination codon present in the xbp-1(zc12) allele. The smg-2(qd101) mutation results in inactivation of the NMD pathway, stabilizing the xbp-1(zc12) mRNA for detection. (B) Quantitative real-time PCR measurements of mRNA levels in the xbp-1(zc12) and smg-2(qd101);xbp-1(zc12) mutants relative to the smg-2(qd101) mutant synchronized in the L3 stage. (C) Quantitative real-time PCR measurements of levels of total and spliced xbp-1 mRNA in the smg-2(qd101) strain relative to WT grown to the L3 stage and then shifted to plates with or without tunicamycin (5 µg/mL) for 4 h. (D) Larval development and survival assay showing the proportion of animals of each of the indicated strains that reach the indicated stage after 4 d of development from eggs laid on plates containing tunicamycin (2.5 µg/mL) at 16°C. (E) Quantitative real-time PCR measurements of levels of total and spliced xbp-1 mRNA in the smg-2(qd101); xbp-1(zc12) strain relative to the smg-2(qd101) strain treated as in C. (In B, C, and E, values represent fold change ± s.e.m., n = 3 independent experiments, *P<0.05, ***P<0.001, two-way ANOVA with Bonferroni post test).

Figure 2. XBP-1 deficiency increases PEK-1 dependent phosphorylation of eIF2α.

(A) PEK-1 dependent phosphorylation of eIF2α is induced by high dose (50 ug/ml) tunicamycin. Western blot of P-eIF2α, total eIF2α and tubulin in WT and pek-1(ok275) strains grown to the L4 stage and then shifted to plates with or without tunicamycin (50 µg/mL) for 4 hours. (B) PEK-1 dependent phosphorylation of eIF2α is induced by XBP-1 deficiency. Western blot of P-eIF2α, total eIF2α and tubulin in WT, xbp-1(tm2482), xbp-1(tm2482); pek-1(ok275) and pek-1(ok275) strains grown to the L4 stage.

Figure 3. Pathogen-induced immune activation exacerbates ER stress levels in XBP-1 deficiency in C. elegans.

Quantitative real-time PCR measurements of levels of total and spliced xbp-1 mRNA in the indicated strains grown from synchronized L1s for 23 h at 20°C and then shifted to plates with or without P. aeruginosa PA14 at 25°C for (A) 4 h or (B) 11 h. Values represent fold change ± s.e.m. (n = 2 independent experiments, ***P<0.001, two-way ANOVA with Bonferroni post test). The WT strain exposed to either treatment was normalized to WT without P. aeruginosa; all other strains were normalized to smg-2(qd101) without P. aeruginosa.

Figure 4. Temperature-sensitive lethality of the xbp-1;pek-1 double mutant.

(A) Development of the xbp-1(tm2482); pek-1(ok275) mutant across physiological temperatures. Values represent average fraction of eggs developed to the indicated stage ± s.e.m. (n = 3 independent experiments at 20°C, 2 independent experiments at all other temperatures; ***P<0.001, two-way ANOVA with Bonferroni post test). (B) Lifespan of indicated strains grown at 16°C to the L4 stage, then shifted to either 16°C to 25°C. Results are representative of two independent experiments.

Figure 5. XBP-1 and PEK-1 each protect against elevated physiological temperature and immune activity.

(A) Development of indicated mutants from eggs to the L4 larval stage or older after 4 d at 16°C on P. aeruginosa PA14. Values represent mean ± s.d. from 1 of 2 representative experiments (n = 4 plates with 20–50 eggs each, ***P<0.001, one-way ANOVA with Bonferroni post test). (B) Development of xbp-1(tm2482); pek-1(ok275) and xbp-1(tm2482); pmk-1(km25); pek-1(ok275) mutants from eggs on E. coli OP50. Values represent average fraction of eggs developed to the indicated stage ± s.e.m. (n = 2 independent experiments, **P<0.01, ***P<0.001, two-way ANOVA with Bonferroni post test). (C) Development of indicated mutants from eggs to the L4 larval stage or older after 2 d at 27°C on E. coli OP50. Values represent mean ± s.e.m. (n = 3 independent experiments, ***P<0.001, one-way ANOVA with Bonferroni post test). (D) Development of indicated mutants from eggs to the L4 larval stage or older after 2 d at 27°C on P. aeruginosa PA14. Values represent mean ± s.d. from 1 of 2 representative experiments (n = 3–4 plates with 20–60 eggs each, ***P<0.001, one-way ANOVA with Bonferroni post test).

Figure 6. PMK-1 protects against exogenously induced ER stress.

Larval development and survival assay showing the proportion of animals of each of the indicated strains that reach the indicated stage after 4 d of development from eggs laid on plates containing tunicamycin at 16°C. Values are from either one experiment (0.5 µg/ml and 2.5 µg/ml tunicamycin) or combined from two experiments with similar results (1.5 µg/ml tunicamycin).

Figure 7. Maintenance of ER homeostasis through activation of the IRE-1 and PEK-1 pathways under basal physiological conditions during development.

(A) The increase in both IRE-1 and PEK-1 activities in XBP-1 deficiency in the absence of exogenous compounds to impose ER stress, combined with the temperature-sensitive lethality of the UPR mutants, implies that UPR signaling maintains ER homeostasis not only in response to the extremes of ER stress, but also under basal physiological conditions. (B) Infection, basal growth and development, and elevated physiological temperature all contribute to ER stress, leading to lethality of UPR mutants as indicated by dashed lines.