G protein-coupled receptor kinase 2 modifies the ability of Caenorhabditis elegans to survive oxidative stress

Abstract

Survival and adaptation to oxidative stress is important for many organisms, and these occur through the activation of many different signaling pathways. In this report, we showed that Caenorhabditis (C.) elegans G protein-coupled receptor kinases modified the ability of the organism to resist oxidative stress. In acute oxidative stress studies using juglone, loss-of-function grk-2 mutants were more resistant to oxidative stress compared with loss-of-function grk-1 mutants and the wild-type N2 animals. This effect was Ce-AKT-1 dependent, suggesting that Ce-GRK2 adjusted C. elegans oxidative stress resistance through the IGF/insulin-like signaling (IIS) pathway. Treating C. elegans with a GRK2 inhibitor, the selective serotonin reuptake inhibitor paroxetine, resulted in increased acute oxidative stress resistance compared with another selective serotonin reuptake inhibitor, fluoxetine. In chronic oxidative stress studies with paraquat, both grk-1 and grk-2 mutants had longer lifespan compared with the wild-type N2 animals in stress. In summary, this research showed the importance of both GRKs, especially GRK2, in modifying oxidative stress resistance.

Keywords: Aging; Caenorhabditis elegans (C. elegans); G protein coupled receptor kinase (GRK); Oxidative stress; Resistance; Stress response.

Fig. 1

Loss-of-function grk-2 animals show increased resistance to increasing oxidative stress exposure. a Protein expression of Ce-GRK1 and Ce-GRK2 in N2, loss-of-function grk-1 or grk-2 animals. b–d Responsiveness of N2, grk-1 or grk-2 animals after 1 (b, n = 6), 2 (c, n = 5) or 4-h exposure (d, n = 5) to 0–300 μM juglone

Fig. 2

Loss of function grk-2 animals show increased resistance to oxidative stress exposure over time. a, b Responsiveness of N2, grk-1 or grk-2 animals after either 120 μM (a, n = 5) or 240 μM juglone (b, n = 5) exposure from 0 to 4 h

Fig. 3

Partial grk-2 knockout RT97 animals also show increased resistance to oxidative stress. a Protein expression of Ce-GRK2 in N2 and grk-2 RT97 animals. b Responsiveness of N2, grk-2 and grk-2 RT97 animals after 1-h exposure to 120 μM juglone (n = 5). ***p < 0.01 in using one-way ANOVA. c Responsiveness of grk-2 and grk-2 RT97 animals after 2-h exposure to 0–300 μM juglone (n = 5). d Responsiveness of grk-2 and grk-2 RT97 animals after 0–4 h treatment with 120 μM juglone (n = 4)

Fig. 4

Dual grk-1 and grk-2 loss-of-function animals show increased resistance to oxidative stress. a Expression of Ce-GRK1 and Ce-GRK2 in loss-of-function animals. b Responsiveness of N2, grk-1, grk-2, and grk-1+ grk-2 animals after 1-h exposure to 120 μM juglone (n = 4). ***p < 0.01 in using one-way ANOVA. c Responsiveness of grk-1 and grk-1 + grk-2 animals after 2-h exposure to 0–300 μM juglone (n = 5). d Responsiveness of grk-1 and grk-1 + grk-2 animals after 0–4 h treatment with 120 μM juglone (n = 5)

Fig. 5

Loss-of-function grk-2 animals show increased resistance to chronic oxidative stress exposure. Longevity of wild type N2 or loss of function grk-1 or grk-2 animals in 0.5 mM paraquat (a, n = 5) or 4 mM paraquat (b, n = 5)

Fig. 6

Increased DAF-16 translocation from cytoplasm to nuclei in Ce-GRK2 RNAi fed GFP-DAF16 animals upon oxidative stress exposure. a Distribution of GFP-DAF-16, either in cytosol (left) or partially between cytosol and nuclei (right). Arrows indicate nucleus-localized GFP-DAF-16. b % animals showing partial cytosol/nuclei GFP-DAF-16 distribution after 0–8 h 100 mM paraquat exposure, n = 6. *p < 0.05 significant difference by Student t test between untreated and Ce-GRK2 RNAi treated GFP-DAF-16 animals at each time point

Fig. 7

Ce-AKT-1 is a key mediator of the ability of Ce-GRK2 to modify oxidative stress resistance. a, b Oxidative stress responsiveness of loss-of-function akt-1, akt-2, or pdk-1 loss-of-function animals fed with Ce-GRK2 RNAi to 120 μM juglone at 2 h (a, n = 4) or 240 μM juglone for 1 h (b, n = 4). *p < 0.05 significant difference by Student t test between untreated and Ce-GRK2 RNAi treated transgenic (pdk-1, akt-1, or akt-2) animals

Fig. 8

The GRK2 inhibitor paroxetine increased the oxidative stress resistance of C. elegans. L2/3-staged wild-type N2 C. elegans were pre-treated with either 100 nM fluoxetine or paroxetine for 24 h and then exposed to either increasing concentrations of juglone, with their responsiveness queried after 1- (a, n = 7) or 2-h 0–240 μM juglone (b, n = 7) treatment or either at a single concentration, either 120 μM (c, n = 7) or 240 μM (d, n = 7) of juglone, from 0 to 3 h