PDP-1 links the TGF-β and IIS pathways to regulate longevity, development, and metabolism

Abstract

The insulin/IGF-1 signaling (IIS) pathway is a conserved regulator of longevity, development, and metabolism. In Caenorhabditis elegans IIS involves activation of DAF-2 (insulin/IGF-1 receptor tyrosine kinase), AGE-1 (PI 3-kinase), and additional downstream serine/threonine kinases that ultimately phosphorylate and negatively regulate the single FOXO transcription factor homolog DAF-16. Phosphatases help to maintain cellular signaling homeostasis by counterbalancing kinase activity. However, few phosphatases have been identified that negatively regulate the IIS pathway. Here we identify and characterize pdp-1 as a novel negative modulator of the IIS pathway. We show that PDP-1 regulates multiple outputs of IIS such as longevity, fat storage, and dauer diapause. In addition, PDP-1 promotes DAF-16 nuclear localization and transcriptional activity. Interestingly, genetic epistasis analyses place PDP-1 in the DAF-7/TGF-β signaling pathway, at the level of the R-SMAD proteins DAF-14 and DAF-8. Further investigation into how a component of TGF-β signaling affects multiple outputs of IIS/DAF-16, revealed extensive crosstalk between these two well-conserved signaling pathways. We find that PDP-1 modulates the expression of several insulin genes that are likely to feed into the IIS pathway to regulate DAF-16 activity. Importantly, dysregulation of IIS and TGF-β signaling has been implicated in diseases such as Type 2 Diabetes, obesity, and cancer. Our results may provide a new perspective in understanding of the regulation of these pathways under normal conditions and in the context of disease.

Figure 1. PDP-1 regulates daf-2 dauer formation independent of the PDHc.

Error bars indicate the standard deviation among the different RNAi plates within one experiment. Data shown are from one representative experiment. (A) pdp-1 RNAi suppresses daf-2(e1370) dauer formation similar to daf-18 RNAi. Dauer formation of daf-2(e1370) is 56.5±8.0% (n = 278) on vector RNAi, 18.9±0.8% (n = 79) on daf-18 RNAi (p<0.05) and 10.5±5.3% (n = 293) on pdp-1 RNAi (p<0.05). (B) pdp-1 RNAi suppresses dauer formation of daf-2(e1368) worms similar to daf-18 RNAi. Dauer formation of daf-2(e1368) is 77.1±13.2% dauers (n = 297) on vector RNAi, daf-2(e1368) worms form only 9.4±6.4% (n = 258) dauers on daf-18 RNAi (p<0.06) and 25.9±3.9% (n = 636) dauers on pdp-1 RNAi (p<0.05). (C) RNAi of other components of the PDHc including the E1α subunit does not affect daf-2(e1370) dauer formation. Dauer formation of daf-2(e1370) on PDHc RNAi is 23.3±4.1% (n = 282) on vector RNAi, 1.3±0.2% (n = 219) on daf-18 RNAi (p<00.04), 1.6±0.6% (n = 185) on pdp-1 RNAi (p<0.03), 13.1±1.0% (n = 233) on pdhk-2 on RNAi (p<0.05), 18.2±2.0% (n = 193) on E1α RNAi, 23.5±0.5% (172) on a combination of E1α and E1β RNAi and 33.3±7.1% (n = 25) on E2 RNAi.

Figure 2. PDP-1 regulates multiple outputs of the IIS pathway.

Data shown are from one representative experiment. (A) pdp-1 RNAi does not significantly reduce the lifespan of wild-type worms. Mean lifespan of wild-type worms is 23.8±0.5 days (n = 93) on vector RNAi, 14.5±0.9 days (n = 34) on daf-18 RNAi (p<0.0001) and 22.6±0.6 days (n = 68) on pdp-1 RNAi (p<0.08). (B) The increased lifespan of daf-2(e1370) worms is reduced by pdp-1 RNAi. Mean lifespan of daf-2(e1370) worms is 38.9±0.9 days (n = 75) on vector RNAi, 24.5±0.5 days (n = 59) on daf-18 RNAi (p<0.0001) and 31.7±0.8 days (n = 66) on pdp-1 RNAi (p<0.0001). (C) pdp-1 RNAi reduces the increased lifespan of age-1(hx546) mutants. Mean lifespan of daf-2(e1370) worms is 42.8±0.8 days (n = 84) on vector RNAi, 28.0±0.9 days (n = 81) on daf-18 RNAi (p<0.0001) and 36.5±1.0 days (n = 67) on pdp-1 RNAi (p<0.0001). (D) pdp-1 overexpression increases the lifespan of wild-type and daf-2(e1370) worms while pdp-1 mutants live slightly shorter than wild-type animals. Mean lifespan of wild-type worms is 29.4±0.5 days (n = 104), pdp-1(tm3734) mutants was 27.1±0.7 days (n = 98), p<0.05, pdp-1::gfp mutants is 34.5±0.8 days (n = 92) p<0.0001, daf-2(e1370) is 38.7±0.7 days (n = 108) and daf-2(e1370); pdp-1::gfp is 42.8±0.7 days (n = 105) days p<0.0001. (E) PDP-1 regulates thermotolerance. Mean survival of wild-type worms is 18.3±0.7 hours (n = 37), pdp-1(tm3734) mutants is 17.1±0.8 hours (n = 27) p<0.2, pdp-1::gfp worms is 19.7±0.9 days (n = 25) p<0.09, daf-2(e1370) worms is 21.6±0.6 hours (n = 30) and pdp-1(tm3734); daf-2(e1370) worms is 18.6±0.9 hours (n = 19), p<0.0007). (F) Oil Red O staining reveals that pdp-1(tm3734); daf-2(e1370) worms store less fat than daf-2 worms across different stages in the worm life cycle: dauers (left), L3 worms (middle) and adults (right). Arrows indicate the lower bulb of the pharynx. (G) Quantification of Oil Red O staining in L3 and young adults of daf-2(e1370), pdp-1(tm3734); daf-2(e1370) and daf-2(e1370); pdp-1::gfp worms. A mutation in pdp-1 significantly reduces daf-2(e1370) fat storage in both, L3s (p<0.0001) and young adults (p<0.01). In adult worms, daf-2(e1370); pdp-1::gfp worms store slightly more fat than daf-2(e1370) not in younger L3 animals (p<0.02).

Figure 3. PDP-1 regulates DAF-16 nuclear localization and transcriptional activity.

(A) DAF-16::GFP localization visualized in daf-2(e1370); daf-16::gfp worms on vector, daf-18 and pdp-1 RNAi (top panel, 100× magnification) and quantification of DAF-16::GFP nuclear-cytosolic localization (lower panel). Data shown are from one representative experiment. (n = 68 on vector RNAi, n = 88 on daf-18 RNAi and n = 79 on pdp-1 RNAi). (B) Representative images of high, medium and low GFP expression in daf-2(e1370); Psod-3::gfp worms (top panel, 100× magnification). Quantification of GFP expression in daf-2(e1370);Psod-3::gfp worms on vector, daf-18, pdp-1 and daf-16 RNAi (Lower panel). Data shown are from one representative experiment (n = 31 on vector RNAi, n = 35 on pdp-1 RNAi, n = 27 on daf-18 RNAi and n = 28 on daf-16 RNAi). (C) Levels of known DAF-16 targets are reduced in pdp-1(tm3734); daf-2(e1370) worms when compared to daf-2(e1370) worms. Data shown is an average of three independent repeats. * p<0.05, **p<0.01.

Figure 4. Crosstalk between IIS and TGF-β signaling pathways.

Data shown are from one representative experiment. For the dauer assays, error bars indicate the standard deviation among the different plates within one experiment. (A) Lifespan graph showing the opposite effects of daf-3 and daf-5 mutations in a daf-2(e1370) background. Mean survival of wild-type worms is 20.1±0.2 days (n = 108), daf-2(e1370) worms is 35.6±0.5 days (n = 111), daf-16(e1370); daf-2(e1370) worms is 14.9±0.6 days (n = 74) p<0.0001, daf-2(e1370); daf-3(e1376) worms is 37.2±0.8 days (n = 76) p<0.02 , daf-2(e1370); daf-3(mgDf90) is 40.5±0.7 days (n = 57) p<0.0001 and daf-5(e1386); daf-2(e1370) worms is 13.0±0.2 days (n = 104), p<0.0001). (B) Top panel: Oil Red O staining showing the modulation of fat stores in daf-2(e1370) by mutations in daf-16, daf-3 and daf-5. Arrows indicate the lower bulb of the pharynx. Lower panel: Quantification of Oil Red O staining shows a significant reduction in daf-2(e1370) fat storage by a mutation in daf-16 (p<0.0001) and daf-5 (p<0.0001). (C) daf-2(e1370) dauer formation is enhanced or decreased by mutations in daf-3 and daf-5. i) Dauer formation of daf-2(e1370) is 19.2±0.7% (n = 1062) and daf-2(e1370); daf-3(mgDf90) is 95.3±0.3% (n = 393), p<0.0001. (ii) Dauer formation of daf-2(e1370) is 57.1±9.8% (n = 176) and daf-5(e1386); daf-2(e1370) is 43.8±5.4% (n = 396), p<0.02.

Figure 5. PDP-1 modulates the expression of insulin genes that possibly feed into the IIS pathway.

Data are representative of one experiment. Error bars represent standard error of the mean within triplicates. All experiments were performed at least twice. (A) The expression of several insulins is elevated in both pdp-1(tm3734) and daf-3(mgDf90) mutants. * p<0.05, ** p<0.007, ^# a significant reduction in ins-30 levels is observed in pdp-1(tm3734) worms this set but not in others, p<0.03. (B) The same insulins show decreased expression on daf-14(m77) mutants. *p<0.008, **p<0.005, ***p<0.0001. (C) In contrast to the mutants, daf-3::gfp and pdp-1::gfp worms show reduced levels of the insulins tested. *p<0.05, **p<0.005. (D) The trend in Insulin levels are similar between pdp-1(tm3734); daf-2(e1370) and daf-16(mgDf50); daf-2(e1370) double mutants compared to the daf-2(e1370) parental strain. *p<0.03, **p<0.01, ^# a significant reduction in ins-18 levels is observed in daf-16(mgDf50; daf-2(e1370) mutants this set but not in others, p<0.001. (E) ins-7 levels are drastically elevated in daf-16(mgDf50); daf-2(e1370) and pdp-1(tm3734); daf-2(e1370) worms as compared daf-2(e1370) worms. *p<0.01, **p<0.004.

Figure 6. PDP-1 links TGF-β signaling to the IIS pathway and DAF-16.

Top panel: Under favorable environmental conditions, signaling through the TGF-β pathway activates the R-SMAD proteins DAF-8 and DAF-14, which regulate insulin gene expression while antagonizing DAF-3 and DAF-5 function. These insulins may act as agonists and activate IIS, thereby promoting phosphorylation and suppression of DAF-16 activity. In this feed-forward model, the worm undergoes reproductive growth and has a normal life span. Middle panel: PDP-1 negatively regulates TGF-β signaling through dephosphorylation of DAF-8 and DAF-14. Under these conditions, DAF-3 and DAF-5 repress the transcription of agonistic insulins as well as expression of the daf-7 TGF-β ligand and daf-8, leading to further downregulation of the TGF-β pathway. Alternatively, DAF-3 and DAF-5 may promote transcription of potential antagonistic insulins. This results in reduced signaling through the IIS pathway, enhancing DAF-16 nuclear localization. Lower panel: Under low IIS conditions, DAF-16 localization is predominantly nuclear, where it regulates the transcription of hundreds of target genes that act in combination to regulate longevity, stress resistance, dauer formation and the response to stress. Paradoxically, under low IIS conditions, DAF-3 and DAF-5 play opposite roles. DAF-5 is likely to synergize with DAF-16 and modulate the activity of its target genes. DAF-3 acts to antagonize DAF-16, either directly or through suppression of DAF-16 target genes. Therefore, the role of DAF-3 in modulating IIS depends upon the level of signaling through the pathway.