The C. elegans TGF-beta Dauer pathway regulates longevity via insulin signaling

Abstract

Background: Previous genetic evidence suggested that the C. elegans TGF-beta Dauer pathway is responsible solely for the regulation of dauer formation, with no role in longevity regulation, whereas the insulin/IGF-1 signaling (IIS) pathway regulates both dauer formation and longevity.

Results: We have uncovered a significant longevity-regulating activity by the TGF-beta Dauer pathway that is masked by an egg-laying (Egl) phenotype; mutants in the pathway display up to 2-fold increases in life span. The expression profiles of adult TGF-beta mutants overlap significantly with IIS pathway profiles: Adult TGF-beta mutants regulate the transcription of many DAF-16-regulated genes, including genes that regulate life span, the two pathways share enriched Gene Ontology categories, and a motif previously associated with DAF-16-regulated transcription (the DAE, or DAF-16-associated element) is overrepresented in the promoters of TGF-beta regulated genes. The TGF-beta Dauer pathway's regulation of longevity appears to be mediated at least in part through insulin interactions with the IIS pathway and the regulation of DAF-16 localization.

Conclusions: Together, our results suggest there are TGF-beta-specific downstream targets and functions, but that the TGF-beta and IIS pathways might be more tightly linked in the regulation of longevity than has been previously appreciated.

Fig. 1. Hierarchical clustering and Correlations between the TGF-β dauer and Insulin/IGF-1 Signaling pathways

(A) TGF-β dauer stage (Liu, et al.) and TGF-β adult profiles (Pearson correlation = 0.004); (B) TGF-β adults and IIS adults (8 IIS arrays), Pearson corr. = 0.35. Note: TGF-β adults and IIS adults Pearson correlations remained high with six IIS arrays (0.42; as in Supp. Fig. 1), when only the adult profiles for the two pathways were compared (0.348), and when all of the TGF-β arrays are forced into a single clade and compared with the IIS clade (0.32).

Fig. 2. Gene Ontology analysis of TGF-β targets in dauer and adults

SAM-determined significantly-changed genes in TGF-β dauers (Liu, et al.), TGF-β dauer pathway adults (Supp. Table. 2), and IIS mutants were submitted for GO analysis. (A) GO categories of genes upregulated by TGF-β dauers (1381), TGF-β adults (2181 genes), and IIS mutants (1390); (B) GO categories of genes downregulated by TGF-β dauers (2725), TGF-β adults (top 3000 genes), and IIS mutants (1054). Asterisks indicate GO categories that are known to function in longevity regulation.

Fig. 3. Transcriptional targets of TGF-β dauer stage, TGF-β adults, and IIS adults with WebLogos of associated motifs

1) TGF-β adult-specific up/dauer downregulated targets; 2) TGF-β/IIS shared downregulated targets; 3) TGF-β adult/IIS shared upregulated; 4) TGF-β adult-specific down/IIS upregulated; 5) TGF-β/IIS shared upregulated; and 6) TGF-β dauer stage upregulated targets. Arrays deposited into Princeton University MicroArray database (PUMA, puma.princeton.edu). Arrays are log2 expression ratios as indicated on scale bar. 1.5 kb of promoter sequences from genes in each cluster were submitted to BioProspector [12] and Weeder [12] to identify overrepresented motifs; high-scoring motifs are depicted by WebLogo [31].

Fig. 4. Longevity and thermotolerance are regulated by TGF-β Dauer pathway signaling in a DAF-3- and DAF-16-dependent manner (see Supp. Data for expanded legend including lifespan data)

(A) daf-7(e1372) matricide rates (20°C, no FUdR). (B–G) Lifespans of hermaphrodites treated with 50 µM FUdR during early adulthood to prevent progeny development. (B, C, D) Daf-c mutants (daf-7, daf-4, daf-8, daf-1, and daf-14) are long-lived, while Daf-d mutants (daf-3 and daf-5) are short-lived. (E, F) daf-7(e1372) mutant lifespan extension is dependent on both daf-3 (E, 8 replicates, Supp. Table 4) and daf-16 activity (F, 6 replicates, Supp. Table 5). (G) daf-2 longevity is independent of daf-3 (7 replicates, Supp Table 6). (H) TGF-β mutants are thermotolerant (35°C) in a daf-3-, daf-16-dependent manner. (daf-2(e1370) was very thermotolerant in this assay, with a mean of 21.2 hours.)

Fig. 5. Temporal Analysis of daf-7 Longevity Regulation

(A) Lifespan of wild type (14.2 ± 0.4), daf-7(e1372) (18.9 ± 0.7, p<0.0001), and daf-7(m62) (18 ± 0.5, p<0.0001) worms at 20°C their entire life. (B) Lifespan of wild type (20.1 ± 0.5), daf-7(e1372) (20.3 ± 0.6, p=0.9), and daf-7(m62) (20.8±0.5, p=0.9) worms raised at 20°C until L4/young adulthood then shifted to 15°C. (C) Lifespan of wild type (15.9 ± 0.5), daf-7(e1372) (21.6 ± 0.9, p<0.0001), and daf-7(m62) (20.8 ± 0.5, p<0.0001) worms raised at 15°C until L4/young adulthood, then shifted to 20°C. (D) Lifespan of wild type worms treated with vector control (16.7 ± 0.4), or daf-7 RNAi during adulthood only (21.1 ± 1.1, p<0.0001).

Fig. 6. The TGF-β Dauer pathway regulates DAF-16 localization and sod-3 transcription

(A) 100× and (B) 400× images of DAF-16::GFP animals. DAF-16::GFP remains diffuse in a wild-type background, but is excluded from nuclei in many daf-3 animals, and is partially nuclearly localized in many daf-7 mutants. (C) Ratios of animals with diffuse (black), cytoplasmic (red), and nuclearly-localized (blue) DAF-16::GFP in wild type and daf-7(e1372) and daf-3(mgDf90) mutant backgrounds. (D) Psod-3::gfp, Psod-3::gfp;daf-7(e1372), Psod-3::gfp;daf-3(mgDf90), and daf-2(e1370);Psod-3::gfp animal treated with daf-3 RNAi. (E) Model of TGF-β Dauer and IIS pathway interactions regulating longevity.