Integration of Stress Signaling in Caenorhabditis elegans Through Cell-Nonautonomous Contributions of the JNK Homolog KGB-1

Abstract

Dealing with physiological stress is a necessity for all organisms, and the pathways charged with this task are highly conserved in Metazoa . Accumulating evidence highlights cell-nonautonomous activation as an important mode of integrating stress responses at the organism level. Work in Caenorhabditis elegans highlighted the importance of such regulation for the unfolded protein response (UPR) and for gene expression downstream of the longevity-associated transcription factor DAF-16 Here we describe a role for the JNK homolog KGB-1 in cell-nonautonomous regulation of these two response modules. KGB-1 protects developing larvae from heavy metals and from protein folding stress (which we found to be independent of canonical UPR pathways), but sensitizes adults to the same stress, further shortening life span under normal conditions. This switch is associated with age-dependent antagonistic regulation of DAF-16 Using transgenic tissue-specific KGB-1 expression or tissue-specific KGB-1 activation we examined the contributions of KGB-1 to gene regulation, stress resistance, and life span. While cell-autonomous contributions were observed, particularly in the epidermis, cell-nonautonomous contributions of neuronal KGB-1 (and also in muscle) were effective in driving intestinal gene induction, age-dependent regulation of intestinal DAF-16, and stress resistance, and did not require KGB-1 expression in the target tissue. Additional genetic analyses revealed requirement for UNC-13 in mediating neuronal contributions, indicating involvement of neurotransmission. Our results expand the role of KGB-1 in stress responses from providing local cellular protection to integrating stress responses at the level of the whole organism.

Keywords: Caenorhabditis elegans; DAF-16; KGB-1; autonomous; nonautonomous; stress.

Figure 1

KGB-1 is broadly expressed. Images (not to scale) of kgb-1p::kgb-1::gfp worms from the larval L3 stage to day 4 of adulthood (D4). (A) Pharyngeal (ph) and intestinal (int) expression, with prominent localization to intestinal nuclei and to intestinal cell apical membranes (ap, arrowheads). (B) Epidermal (epi) expression, with arrowheads marking nuclei. (C) Muscle (m) expression; fluorescence intensity is blown up to show association with muscle fibers (arrowheads). (D) Expression in head neurons (n). (E) Expression in neuronal commissures (nc). (F) Vulval (v) expression.

Figure 2

Tissue-specific expression and activation of KGB-1. (A) kgb-1 tissue-specific expression constructs, and the resulting expression in distinct lines of integrated transgenic (Tg) animals with the kgb-1(km21) background; RNA levels were measured in L4 larvae. Averages ± SDs from two to three qRT-PCR experiments, each performed in duplicate. (B) Immunoblotting of protein extracts from L3/L4 larvae, using designated antibodies, demonstrates activation of KGB-1 in either one of the tested expressing tissues following vhp-1 knockdown. (C and D) Quantification of signal in immunoblots: (C) KGB-1 activation, shown as normalized values in worms treated with vhp-1 RNAi compared to controls; (D) levels of activated KGB-1 in vhp-1 RNAi-treated animals. Values from an additional immunoblot are factored in when available, for the wild-type, kgb-1 mutants, and the neuronal KGB-1 strains; shown are averages and SDs.

Figure 3

Tissue-specific expression of KGB-1 partially rescues stress resistance in kgb-1 mutants. Development of worms (3 days at 20°) of designated strains grown in the presence of (A) 1 μg/ml tunicamycin, or (B) 50 μM cadmium. Shown are averages ± SDs for two independent experiments, each performed in duplicates with 100–400 worms per strain per duplicate. * denotes significant differences in the fraction of worms of a developmental stage compared to the respective value in kgb-1 mutants (P < 0.05, t-test).

Figure 4

Tissue-specific KGB-1 contributions to detrimental effects in adults. (A) Infection resistance or (B) life span for adults (time 0) of the designated strains, assessed following exposure to control RNAi (solid lines), or vhp-1 RNAi (dashed) for 3 days (in infection experiments with tissue-specific KGB-1 expression shown in A), or for 2 days (in all other panels in A and B). Shown are averages ± SDs of fraction survival measured in triplicates with a total of 38–233 worms per group per experiment (Tables S1 and S2). Shown in each panel is a representative of two to three independent experiments, or a single experiment for tissue-specific RNAi experiments.

Figure 5

Cell-autonomous and -nonautonomous effects of KGB-1 activation on gene expression. (A) Representative images of L4 cpr-3p::gfp transgenics, with wild-type genetic background, kgb-1(km21), or tissue-specific KGB-1, fed throughout development with control (EV), or vhp-1 RNAi. Images were acquired using identical magnification and exposure settings. (B) Quantification of GFP signal. Averages ± SDs for two independent experiments (n = 9–11 worms per group per experiment). (C) Quantification of similar experiments as in B, with egg to young adult exposure to RNAi mixtures, as designated; cdc-25.1 RNAi was additionally included in all RNAi mixes, to abolish differences associated with selective sterility, previously shown for fos-1 knockdown; previous work showed that mixtures of the RNAi clones in use gave rise to efficient knockdown (Zhang et al. 2017). Shown are averages ± SDs (wild-type, n = 24–31 worms per group; neuronal, n = 16–19 worms per group; intestinal, n = 7–15 worms per group; * P < 0.05, ** P < 0.01, *** P < 0.001, t-test). (D–I) qRT-PCR measurements of gene induction in L4 larvae following KGB-1 activation by (D–H) vhp-1 knockdown (D–H), or exposure to cadmium (1 mM, 1 hr); tissue-specificity was achieved through restricted expression of KGB-1 or RNAi machinery in the designated mutants. Averages ± SDs for measurements from two to three independent experiments, each measured in duplicates. Asterisks mark significant induction following vhp-1 knockdown or cadmium treatment (* P < 0.05, ** P < 0.01, *** P < 0.001, paired t-test).

Figure 6

KGB-1 regulates DAF-16 cell-nonautonomously. (A and D) Representative images showing DAF-16::GFP nuclear localization in (A) wild-type L4 or (D) day 2 adults following 2 days of exposure to RNAi as designated, and further including cdc-25.1 RNAi in the experiment shown in D, to drive DAF-16 nuclear localization. Arrowheads mark worms with nuclear localization. (B) Quantification of DAF-16::GFP nuclear localization in L4 worms of the designated genetic backgrounds. Averages ± SDs of three independent experiments, each with 20–380 worms per group (N shown on columns). (C) qRT-PCR measurements of mtl-1 gene induction following vhp-1 knockdown in larvae expressing tissue-specific KGB-1. Averages ± SDs for two to nine independent experiments, each measured in duplicates. (E) DAF-16 nuclear localization quantified as in B, in day 2 adult worms treated during development with cdc-25.1 RNAi, and then shifted to cdc-25.1+EV or vhp-1 RNAi; averages of two experiments, each with N = 21–92 per group. (F) qRT-PCR measurements in day 2 adults; shown is fold repression relative to values in the respective EV-treated worms. Averages ± SDs for two to five independent experiments for each strain, each measured in duplicates. Asterisks mark significant induction following vhp-1 knockdown. * P < 0.05, ** P < 0.01, *** P < 0.001; t-test for nuclear localization, paired t-test for qRT-PCR measurements.

Figure 7

Disruption of neuronal secretion impairs cell-nonautonomous contributions of neuronal KGB-1 (A). Intestinal gene induction in L4 larvae following vhp-1 RNAi-mediated activation of neuronal KGB-1, in animals with the designated genetic backgrounds. (B) Quantification of GFP signal in animals with only neuronal KGB-1. Averages ± SDs of values in three independent experiments with N = 9–30 worms per group per experiment; N shown on columns (P = 1.7 × 10⁻²⁴, t-test). (C) qRT-PCR measurements of endogenous cpr-3 induction following KGB-1 activation in L4 larvae of the designated strains. Averages ± SDs for measurements from two independent experiments, each measured in duplicates; * P < 0.01, t-test.