Feeding a ROS-generator to Caenorhabditis elegans leads to increased expression of small heat shock protein HSP-16.2 and hormesis

Abstract

Reactive oxygen species (ROS) are thought to be a driving force in the aging process. In transgenic Caenorhabditis elegans expressing green fluorescent protein (GFP) under control of the hsp-16.2 promoter (CL2070) 100 muM of the ROS-generator juglone induced GFP-expression. This was associated with translocation of DAF-16 to the nucleus as visualized in a transgenic strain expressing a DAF-16::GFP fusion protein (TJ356) and with increased cellular levels of reduced glutathione. RNA-interference for DAF-16 in CL2070 blocked the juglone-induced HSP-16.2 expression and the increase in glutathione levels. Higher concentrations of juglone did not further increase the adaptive responses but caused premature death, indicating hormetic adaptations unless the stressor exceeds the intrinsic protective capacity. The addition of the ROS-scavenger ascorbic acid finally blocked lifespan reductions and all of the adaptations to juglone stressing that ROS are indeed the molecular species that require protective response.

Fig. 1

HSP-16.2 expression as visualized by GFP-fluorescence in a hsp-16.2p::GFP transgenic strain (CL2070). Green fluorescence of GFP in CL2070 exposed for 4 h to 100 μM juglone either on agar plates with E. coli as food source (left panel) or in axenic liquid culture medium (right panel) can be visualized from the fluorescence images as given in (a), left panels, and the overlays of fluorescence and light microscopy images (a, right panels). Time-dependent GFP-fluorescence is shown in (b) when CL2070 nematodes were exposed to 100 μM juglone in liquid axenic medium. Intensities of GFP expression in CL2070 cultured in liquid axenic medium for 4 h at various juglone concentrations is shown in (c). *P < 0.05, **P < 0.01, ***P < 0.001 versus the control

Fig. 2

Lifespan curves of N2 wildtype animals grown on agar plates with E. coli as a food source in comparison to those measured in liquid axenic medium is shown in (a). Lifespans of CL2070 in axenic liquid medium either in the absence (control) or presence of 100 or 250 μM juglone are given in (b). P-values for significances of differences of survival curves were P = 0.7907 for 100 μM juglone and P < 0.0001 for 250 μM juglone versus the untreated CL2070 nematodes

Fig. 3

HSP-16.2 expression is dependent on DAF-16. In TJ356 DAF-16 localization in the nucleus is increased by 100 and 250 μM juglone (a). Knock-down of DAF-16 by RNA-interference abolished juglone-induced activation of the hsp-16-2 promoter in CL2070 as is evidenced by the lack of GFP-fluorescence in (b). Left panels show the fluorescence images and right panels an overlay of fluorescence and light microscopic images

Fig. 4

GSH-levels but not SOD- or catalase activities are increased by juglone in CL2070. Levels of GSH were increased by the ROS-generator (a) with *P < 0.05, and **P < 0.01 versus the control, but DAF-16 RNAi inhibited the juglone-induced increase in GSH-levels (b). GSSG-levels remained unaffected by juglone (c). SOD-activities (d) and catalase-activities (e) did not differ significantly between untreated CL2070 or those treated with 100 or 250 μM juglone

Fig. 5

Ascorbic acid prevents juglone-induced effects in CL2070. Ascorbic acid at 250 μM blocks nuclear translocation of DAF-16 in TJ356 (a), and in CL2070 hsp-16.2p::GFP expression (b), increase in GSH-levels (c), and reductions in survival under heat-stress (d), that were all caused by 250 μM juglone. ***P < 0.001 versus juglone-treated nematodes. P-value for significance of difference of survival curve of CL2070 treated with 250 μM juglone plus 250 μM ascorbic acid and those treated with 250 μM juglone alone was P < 0.0001