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. 2023 Dec 7;24(24):17209.
doi: 10.3390/ijms242417209.

Mitochondrial p38 Mitogen-Activated Protein Kinase: Insights into Its Regulation of and Role in LONP1-Deficient Nematodes

Eirini Taouktsi  1   2 , Eleni Kyriakou  1 , Evangelia Voulgaraki  1 , Dimitris Verganelakis  1   3 , Stefania Krokou  1 , Stamatis Rigas  2 , Gerassimos E Voutsinas  4 , Popi Syntichaki  1
Affiliations

Mitochondrial p38 Mitogen-Activated Protein Kinase: Insights into Its Regulation of and Role in LONP1-Deficient Nematodes

Eirini Taouktsi et al. Int J Mol Sci. .

Abstract

p38 Mitogen-Activated Protein Kinase (MAPK) cascades are central regulators of numerous physiological cellular processes, including stress response signaling. In C. elegans, mitochondrial dysfunction activates a PMK-3/p38 MAPK signaling pathway (MAPKmt), but its functional role still remains elusive. Here, we demonstrate the induction of MAPKmt in worms deficient in the lonp-1 gene, which encodes the worm ortholog of mammalian mitochondrial LonP1. This induction is subjected to negative regulation by the ATFS-1 transcription factor through the CREB-binding protein (CBP) ortholog CBP-3, indicating an interplay between both activated MAPKmt and mitochondrial Unfolded Protein Response (UPRmt) surveillance pathways. Our results also reveal a genetic interaction in lonp-1 mutants between PMK-3 kinase and the ZIP-2 transcription factor. ZIP-2 has an established role in innate immunity but can also modulate the lifespan by maintaining mitochondrial homeostasis during ageing. We show that in lonp-1 animals, ZIP-2 is activated in a PMK-3-dependent manner but does not confer increased survival to pathogenic bacteria. However, deletion of zip-2 or pmk-3 shortens the lifespan of lonp-1 mutants, suggesting a possible crosstalk under conditions of mitochondrial perturbation that influences the ageing process. Furthermore, loss of pmk-3 specifically diminished the extreme heat tolerance of lonp-1 worms, highlighting the crucial role of PMK-3 in the heat shock response upon mitochondrial LONP-1 inactivation.

Keywords: C. elegans; LONP-1; PMK-3; ZIP-2; ageing; heat stress response; mitochondria.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Activation of the mitochondrial MAPK (MAPKmt) signaling pathway in lonp-1-deficient worms. (A) Schematic representation of the MAPKmt pathway consisting of the DLK-1/SEK-3/PMK-3 kinase cascade and the downstream _target gene, tbb-6. (B) Relative mRNA quantification of endogenous tbb-6 gene expression levels in wt and lonp-1-knockout (ko) mutants. (C) Representative microscopy images and GFP fluorescence quantification of the tbb-6p::gfp reporter in wt and lonp-1(ko) worms. (D,E) Quantification of endogenous tbb-6 gene expression or the tbb-6p::gfp reporter in wt and lonp-1(ko) mutants upon disruption of the MAPKmt signaling components. Experiments were performed on 1-day-old adult worms grown on NGM plates, seeded with E. coli OP50 or with E. coli HT115 bacteria transformed with the indicated RNAi construct (and empty vector as a control) at 20 °C. Scale bar indicates 100 µm. Values are presented as means ± SEM, and asterisks denote statistical significance assessed with an unpaired Student’s t-test or with a two-way ANOVA followed by a post hoc Tukey’s test, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.
Figure 2
Figure 2
ATFS-1 inhibits activation of the MAPKmt pathway upstream of PMK-3. (A) Relative mRNA quantification of endogenous tbb-6 gene expression levels in wt and atfs-1(gk3094) loss-of-function mutants, or in wt and lonp-1(ko) animals subjected to RNAi against atfs-1. (B) Representative microscopy images and GFP fluorescence quantification of the tbb-6p::gfp reporter in wt, lonp-1(ko), pmk-3(ok169) and lonp-1(ko);pmk-3(ok169) worms subjected to atfs-1(RNAi). Experiments were performed on 1-day-old adult worms grown on NGM plates seeded with E. coli OP50 or with E. coli HT115 bacteria transformed with the indicated RNAi construct (and empty vector as a control) at 20 °C. Scale bar indicates 100 µm. Values are presented as means ± SEM, and asterisks denote statistical significance assessed with an unpaired Student’s t-test or with a two-way ANOVA followed by a post hoc Tukey’s test, ** p ≤ 0.01, **** p ≤ 0.0001.
Figure 3
Figure 3
CBP-3 positively regulates MAPKmt signaling and ATFS-1 suppresses cbp-3 induction in lonp-1 mutants. (A) Representative microscopy images and GFP fluorescence quantification of the tbb-6p::gfp reporter in wt and lonp-1(ko) worms subjected to RNAi against cbp-3. (B) Relative mRNA quantification of endogenous tbb-6 gene expression levels in wt and lonp-1(ko) worms subjected to RNAi against cbp-3. (C) Relative mRNA quantification of cbp-3 and cbp-1 gene expression levels in wt, lonp-1(ko), pmk-3(ok169) and lonp-1(ko);pmk-3(ok169) worms. (D) Relative mRNA quantification of cbp-3 gene expression levels in wt and lonp-1(ko) animals subjected to RNAi against atfs-1 or skn-1. Experiments were performed on 1-day-old adult worms grown on NGM plates seeded with E. coli OP50 or with E. coli HT115 bacteria transformed with the indicated RNAi construct (and empty vector as control) at 20 °C. Scale bar indicates 100 µm. Values are presented as means ± SEM, and asterisks denote statistical significance assessed with a two-way ANOVA followed by post hoc Tukey’s test, * p ≤ 0.05, ** p ≤ 0.01, **** p ≤ 0.0001.
Figure 4
Figure 4
The MAD pathway does not mediate activation of the MAPKmt pathway in lonp-1 mutants. (A) Representative microscopy images and GFP fluorescence quantification of the tbb-6p::gfp reporter in 1-day adults of wt, cdc-48.2(tm659), lonp-1(ko) and lonp-1(ko);cdc-48.2(tm659), at 20 °C. (B) Relative mRNA quantification of endogenous tbb-6 gene expression levels in 1-day adults of the aforementioned strains, grown on NGM plates seeded with E. coli OP50 or with E. coli HT115 bacteria transformed with the cdc-48.1(RNAi) construct (and empty vector as control), at 20 °C. Scale bar indicate 100 µm. Values were presented as mean ± SEM, and numeric data were analyzed with a two-way ANOVA followed by post hoc Tukey’s test, showing no significant (ns) differences.
Figure 5
Figure 5
PMK-3-dependent activation of the bZIP transcription factor ZIP-2 does not confer resistance to pathogen infection. (A) Relative mRNA quantification of zip-2, irg-1 and irg-2 gene expression levels in 1-day-old adults of wt, lonp-1(ko), zip-2(ok3730), lonp-1(ko);zip-2(ok3730), pmk-3(ok169) and lonp-1(ko);pmk-3(ok169) at 20 °C. Values are presented as means ± SEM and statistical significance was assessed with an unpaired Student’s t-test, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001 and **** p ≤ 0.0001. (B) Survival of slow killing Pseudomonas aeruginosa PA14 pathogen infection assay in 1-day-old adults of wt, lonp-1(ko), pmk-3(ok160) and lonp-1(ko);pmk-3(ok169) on NGM plates seeded with E. coli OP50 in the presence of FUdR at 20 °C. Statistical significance was assessed via a Kaplan–Meier survival curve, **** p ≤ 0.0001.
Figure 6
Figure 6
Loss of ZIP-2 or PMK-3 shortened the lifespan of lonp-1 mutants and PMK-3 promotes organismal survival under stress. (A) Lifespan assays of wt, lonp-1(ko), zip-2(ok3730), lonp-1(ko);zip-2(ok3730), pmk-3(ok169) and lonp-1(ko);pmk-3(ok169) worms on NGM plates seeded with E. coli OP50 at 20 °C. Replicates and statistical analysis of lifespan assays are shown in Table S3, ** p ≤ 0.01, *** p ≤ 0.001. (B) Survival of 1-day-old adults of wt, lonp-1(ko), pmk-3 ok160) and lonp-1(ko);pmk-3(ok160) strains treated with tert-butyl hydroperoxide (tBHP, 10 mM), Antimycin A (40 μm for 24 h), or subjected to heat shock (at 35 °C for 5.5, 7.5 or 8 h). The percentage survival for all biological replicates was plotted, and an unpaired Student’s t-test was used to assess significance, * p ≤ 0.05, ** p ≤ 0.01 and **** p ≤ 0.0001.
Figure 7
Figure 7
A schematic representation of the genetic interactions and molecular functions of MAPKmt in worms deficient in the lonp-1 gene.
See this image and copyright information in PMC

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