Lifespan extension by calorie restriction relies on the Sty1 MAP kinase stress pathway

Abstract

Either calorie restriction, loss-of-function of the nutrient-dependent PKA or TOR/SCH9 pathways, or activation of stress defences improves longevity in different eukaryotes. However, the molecular links between glucose depletion, nutrient-dependent pathways and stress responses are unknown. Here, we show that either calorie restriction or inactivation of nutrient-dependent pathways induces lifespan extension in fission yeast, and that such effect is dependent on the activation of the stress-dependent Sty1 mitogen-activated protein (MAP) kinase. During transition to stationary phase in glucose-limiting conditions, Sty1 becomes activated and triggers a transcriptional stress programme, whereas such activation does not occur under glucose-rich conditions. Deletion of the genes coding for the SCH9-homologue, Sck2 or the Pka1 kinases, or mutations leading to constitutive activation of the Sty1 stress pathway increase lifespan under glucose-rich conditions, and importantly such beneficial effects depend ultimately on Sty1. Furthermore, cells lacking Pka1 display enhanced oxygen consumption and Sty1 activation under glucose-rich conditions. We conclude that calorie restriction favours oxidative metabolism, reactive oxygen species production and Sty1 MAP kinase activation, and this stress pathway favours lifespan extension.

Figure 1

Sty1-dependent lifespan promotion only occurs on calorie restriction. (A) Growth in minimal media (MM; 2% glucose), but not in complex media (YE; 3% glucose), induces life extension in a Sty1-dependent manner. Strains 972 (WT) and AV18 (Δsty1) were grown in standard MM or YE-3% glucose media. Serial dilutions of logarithmic (Log) or stationary phase (Day 6) cultures were spotted onto YE plates. (B) Concentrations of glucose below 1% in YE media induce chronological lifespan extension. Strain 972 (WT) was grown in MM, or in YE with the indicated concentrations of glucose (Glu). Serial dilutions of logarithmic (Log) and stationary phase (Days 4 and 5) cultures were spotted onto YE plates. (C) Life extension by low glucose concentrations is dependent on Sty1. Survival percentages of wild-type strain 972 (WT) and AV18 (Δsty1) are indicated in the graphs.

Figure 2

Sty1 activation at stationary phase only occurs on calorie restriction. (A) Growth curves and glucose concentrations of YE-1% (calorie restriction) and YE-4% glucose (glucose-rich) wild-type cultures. Wild-type strain (972) was grown in both media; OD₆₀₀ (triangles) and glucose concentration (circles) were recorded at the times indicated in hours. (B) Sty1 is barely activated at stationary phase in cells grown in YE-4% glucose media. At the time points indicated in (A) (A–E for growth in YE-1% and A′–E′ for YE-4% cultures), cells were collected, TCA protein extracts were prepared and proteins were analysed by western blot using anti-p38-P (Sty1-P, top panels) or anti-Sty1 antibodies as a loading control (Sty1, bottom panels). As a control of activated Sty1, logarithmic cultures of wild-type cells were treated with 1 mM H₂O₂ for 10 min (H). (C) Growth curves and glucose concentrations of MM (calorie restriction) and YE-3% glucose (glucose-rich) wild-type cultures. OD₆₀₀ and glucose concentrations were recorded as indicated in (A). (D) Sty1 phosphorylation is barely observed in YE-3% glucose culture. Levels of activated Sty1 (Sty1-P) were determined as indicated in (B). (E) Sty1-dependent gene transcription is not activated at stationary phase in cells grown in YE-3% glucose media. RNA from the same time points as in (D) were obtained and hybridized with probes against atf1, gpx1, cta1 and fbp1.

Figure 3

Heat shock resistance of stationary phase cells is calorie restriction dependent and Sty1 dependent. Strains 972 (WT) and AV18 (Δsty1) were grown in YE-1% or YE-4% glucose media. At logarithmic phase (Log), 48 h (Day 2) and 72 h (Day 3) after reaching stationary phase, liquid cultures were heat shocked at 48°C during 2 h or left untreated (Unt.). Serial dilutions of the cultures were spotted onto YE plates.

Figure 4

Activation of the transcription factor Atf1 by Sty1 is required for life extension by calorie restriction. (A) Scheme of the stress-, Sty1-, Atf1-dependent activation of gene expression. (B) Lifespan of cells lacking Atf1. Strains 972 (WT), AV18 (Δsty1) and AV15 (Δatf1) were grown in YE-1% glucose media. At the logarithmic phase (Log) or several days after reaching stationary phase (Day 3 and Day 5), serial dilutions of the cultures were plated onto YE plates. (C) Life extension by low glucose concentrations is dependent on Atf1. Survival percentages of strains as in (B) are indicated in the graphs. (D) Heat shock resistance of stationary phase cells is calorie restriction dependent and Atf1 dependent. Strains as in (B) were heat shocked and analysed as described in Figure 3.

Figure 5

Strain AZ107, expressing an analogue-sensitive mutant Sty1, shows shorter viability at stationary phase on addition of the inhibitory ATP analogue 1NM-PP1. (A) Addition of 1NM-PP1 inhibits phosphorylation of the Sty1-dependent transcription factor Atf1 on H₂O₂ treatment. Strain AZ107, harbouring the sty1.T97A mutation at its genomic locus, was grown in YE-1% glucose. At an OD₆₀₀ of 0.2, 10 μM 1NM-PP1 was added (1NM-PP1) or not to the cultures for 2 h. Then, when indicated, a 15 min treatment with 1 mM H₂O₂ was performed (H₂O₂) or not (Unt.). The phosphorylation of Sty1 (Sty1-P) and the accumulation/phosphorylation of Atf1 (Atf1 and Atf1-P) were determined from TCA extracts using anti-p38-P and anti-Atf1 antibodies. Anti-Sty1 antibody was used as a loading control (Sty1). (B) Addition of 1 NM-PP1 to strain AZ107 inhibits calorie restriction-dependent phosphorylation of Atf1 at stationary phase. Cultures of strain AZ107 (sty1.T97A), grown in YE-1% glucose media to an OD₆₀₀ of 2, were left untreated or were treated with 10 μM of 1NM-PP1. Cells were collected at the indicated OD₆₀₀. The accumulation and phosphorylation of Atf1 were determined as in (A). (C) Inactivation of Sty1 by 1NM-PP1 impairs heat shock resistance acquisition. Two days (Day 2) after reaching stationary phase, cultures from (B) were heat shocked (48°C, 2 h) or not (Unt.), and viability was determined by sequential spotting. (D) Inactivation of Sty1 by 1NM-PP1 decreases cell viability during stationary phase. At the logarithmic phase (Log) or several days after reaching stationary phase (Day 3 and Day 5), viability of cultures from (B) was assayed by sequential spotting.

Figure 6

Oxygen consumption and H₂O₂ levels of S. pombe cells are higher in cells grown in YE-1% than in YE-4% glucose media. (A) Oxygen consumption along the growth curve is higher in YE-1% than in YE-4% glucose cultures. Wild-type strain (972) was grown in YE-1% or YE-4% glucose media; OD₆₀₀ (squares) and oxygen consumption (circles) were recorded at different times during the growth curve. (B) Relative intracellular H₂O₂ levels of cells grown in YE-1% and YE-4% glucose media. Wild-type strain (972) was grown in YE-1% and YE-4% glucose media to reach the relative OD₆₀₀ indicated on the right y axis (% OD₆₀₀ max). At the indicated time points from the starting OD₆₀₀ (around 5% of the maximum OD₆₀₀ of each culture), cells were incubated with the redox-sensitive dye dihydrorhodamine 123 (DHR123) and with the permeability-dependent dye propidium iodide (PI), and the fluorescence of live cells was analysed by flow cytometry, as described in ‘Materials and methods'. The DHR123 green fluorescence was normalized to the PI red fluorescence and to the cell size (left y axis: Relative DHR123/IP/cell size), and all the values are referred to that of YE-4% glucose cultures at 2 h, with an assigned value of 1; s.e.m. of three replicates are indicated.

Figure 7

Loss-of-function of the glucose-dependent Pka1 kinase triggers enhanced respiratory rates, activation of the Sty1 pathway and life extension at high glucose concentrations. (A) Lack of Sck2 and Pka1 kinases promotes stationary phase cell survival under glucose-rich conditions in a Sty1-dependent manner. Strains 972 (WT), MC22 (Δpka1), MC24 (Δpka1 Δsty1), MC25 (Δsck2) and MC27 (Δsck2 Δsty1) were grown in YE-4% media. Serial dilutions of the logarithmic (Log) and stationary phase (Day 5) cultures were spotted onto YE plates. (B) Survival percentages of strains as in (A) grown in YE-3% glucose media are indicated in the graph. Similar results were obtained with three different biological replicates. (C) Oxygen consumption of wild-type and pka1 cells. Oxygen consumption of strains 972 (WT), grown in YE-1% and YE-4% glucose media, and MC22 (Δpka1), grown in YE-4%, was determined from logarithmic cultures at low OD₆₀₀ (10–30% of the maximum OD₆₀₀); s.e.m. of seven replicates are indicated. Significant difference between wild-type and pka1 cells respiration in YE-4% glucose was determined by the Student's t-test. ^*P<0.05. (D) pka1 cells show enhanced levels of phosphorylated Sty1 and increased protein levels of Atf1. Strains as in (C) were grown in YE-4% media. As a control of activated Sty1, wild-type cells at the logarithmic phase were treated with 1 mM H₂O₂ for 10 min (H₂O₂), or were left untreated (Unt.). Western blot analysis of TCA extracts is shown, using anti-p38-P (Sty1-P, top panel), anti-Atf1 (Atf1, middle panel) or anti-Sty1 antibodies (Sty1, bottom panel). (E) Sty1-dependent gene transcription is constitutively activated in cells lacking Pka1. RNA from strains and conditions as in (D) was obtained, and probed with gpx1 (activated by Sty1 and Atf1) or fbp1 (repressed by Pka1). rRNA are shown as a loading control. As a control of activated Sty1, wild-type cells were treated (WT H₂O₂) or not (Unt.) with 1 mM H₂O₂ for 30 min.

Figure 8

Constitutively active Sty1 is sufficient to promote life extension of cells grown under glucose-rich conditions. (A) Scheme of the regulation of Sty1 phosphorylation. (B) Strains 972 (WT), AZ103 (Δpyp1), AZ104 (Δpyp1 Δsty1), AZ98 (wis1DD) and AZ102 (wis1DD Δsty1) were grown in YE-3% glucose media. Serial dilutions of logarithmic (Log) and stationary phase (Day 6) cultures were spotted onto YE plates. (C) Survival percentages of strains as in (B) are indicated in the graph. Similar results were obtained with three different biological replicates.