An overview of stress response and hypometabolic strategies in Caenorhabditis elegans: conserved and contrasting signals with the mammalian system

Abstract

Studies of the molecular mechanisms that are involved in stress responses (environmental or physiological) have long been used to make links to disease states in humans. The nematode model organism, Caenorhabditis elegans, undergoes a state of hypometabolism called the 'dauer' stage. This period of developmental arrest is characterized by a significant reduction in metabolic rate, triggered by ambient temperature increase and restricted oxygen/ nutrients. C. elegans employs a number of signal transduction cascades in order to adapt to these unfavourable conditions and survive for long times with severely reduced energy production. The suppression of cellular metabolism, providing energetic homeostasis, is critical to the survival of nematodes through the dauer period. This transition displays molecular mechanisms that are fundamental to control of hypometabolism across the animal kingdom. In general, mammalian systems are highly inelastic to environmental stresses (such as extreme temperatures and low oxygen), however, there is a great deal of conservation between the signal transduction pathways of nematodes and mammals. Along with conserving many of the protein targets in the stress response, many of the critical regulatory mechanisms are maintained, and often differ only in their level of expression. Hence, the C. elegans model outlines a framework of critical molecular mechanisms that may be employed in the future as therapeutic targets for addressing disease states.

Keywords: Apoptosis; Diapause; Hypometabolism; Lifespan extension; Post-translational modification; Transcriptional regulation.

Figure 1

Summary of the insulin/IGF signaling pathway, using the terminology for mammalian systems. Binding of the ligand to the insulin like growth factor-I Receptor initiates the signaling cascade, which is propagated by PI3K. Akt acts as the signaling hub for the majority of the downstream effects such as cell cycle control, glycogen synthesis and apoptotic suppression. The phosphorylation state of Akt is critically regulated by protein phosphatases (PTEN/PP2) and protein kinases (PDK-1).

Figure 2

Regulation and downstream targets of the forkhead box (subtype O) transcription factors. Protein kinases (Akt and SGK) phosphorylate FoxO on up to three sites, including one proximal to the nuclear localization sequence (NLS). This prevents nuclear translocation or, in the event that the FoxO is phosphorylated in the nucleus, nuclear expulsion. Phosphorylation may stimulate binding of the 14-3-3 protein to the transcription factor, covering its NLS and DNA binding domains and blocking transcriptional regulatory capacities.

Figure 3

Insulin/IGF signaling in C. elegans controls entry into the dauer larval stage. Dauer entry is prevented through AKT mediated phosphorylation of the DAF-16 (FoxO homolog) transcription factor. Like the mammalian model, AKT phosphorylation is the key to canonical DAF-2 signaling, and is regulated by protein kinases (PDK-1) and phosphatases (DAF-18 and PPTR-1).

Figure 4

TGF-β signaling in mammalian models. Signaling begins with ligand dimerization and interaction with the type II receptor. This recruits and activates (via phosphorylation) the type I receptor. The active type I receptor phosphorylates downstream Smad transcription factors, which form complexes and enter the nucleus. There are numerous inhibitory pathways that act on both the type I receptor, and the Smad transcription factors, typically resulting in loss of phosphorylation state or ubiquitin ligase mediated degradation.

Figure 5

TGF-β signaling in C. elegans. The TGF-β ligand (DAF-7) stimulates the type II receptor (DAF-4), which in turn, recruits and phosphorylates the type I receptor (DAF-1). DAF-1 phosphorylates (activates) downstream Smads (DAF-14), which complex with DAF-8 and enter the nucleus. In the nucleus, the DAF-8/-14 complex inhibits pro-dauer Smads (DAF-3/-5) and promotes development, rather than dauer formation. An alternate Smad based pathway involves hetero-trimer formation of a combination of SMA-2/-3 and 4. These trimers enter the nucleus, associate with transcriptional co-factors and regulate body size development of C. elegans.

Figure 6

Mammalian target of rapamycin (mTOR) activation pathway. Formation of the mTOR complexes (mTORC1 and C2) is dependent on regulation of the tuberin/hamartin (TSC1/2) complex. This is regulated by numerous kinases, which can inhibit (AKT) or activate the complex (GSK-3β and AMPK). Active TSC1/2, interacting with Rheb-GTP, inhibits mTOR complex formation, and subsequent downstream activities. mTOR activation is often based on nutrient availability.

Figure 7

mTOR complex formation combines either Raptor (C1) or Rictor (C2). Inhibition of mTORC1 by FKBP38 can be reversed by direct interaction with Rheb-GTP. Both complexes have downstream targets to promote cell cycle progression and protein translation, but mTORC2 also has feedback activation of mTOR activation through AKT phosphorylation.

Figure 8

The C. elegans pathway of mTOR production is dependent on insulin/IGF response. TOR homologs DAF-15 (Raptor) and LET-363 (TOR) are regulated by DAF-16 (FoxO) transcription factors which are also responsible for entry into dauer. A. Shows high nutrient conditions, with insulin signaling, through active AGE-1(PI3K) and AKT phosphorylating DAF-16, impeding its entry into the nucleus and allowing transcription of the TOR genes. B. When phosphorylation of AKT is blocked by DAF-18 (PTEN), DAF-16 is able to enter the nucleus, and can inhibit the transcription of TOR genes.

Figure 9

TOR interactions with SGK1 in both mammalian and C. elegans models. A. Phosphorylation of SGK1 by mTORC1 and then PDK activates the kinase, before it acts upon p27. Phosphorylated p27 is unable to bind E-type Cyclin - CDK2 complexes, which in turn allow progression into the S phase of the cell cycle. B. The C. elegans TORC2 complex phosphorylates SGK-1 which modifies DAF-16 to prevent entry into dauer, and increases lipid stores, possibly as a preparation for dauer.

Figure 10

Mammalian STAT signaling controls multiple developmental processes. Key members of the STAT transcription factors, STAT-1 and 3, also regulate apoptosis. STAT-1 transcriptionally up-regulates apoptosis signaling genes, as well as interacting with the P53 tumour suppressor. This interaction increases P53 activity. Active P53 may also inhibit STAT-3 activation, which would prevent the downstream up-regulation of anti-apoptotic/anti-STAT-1 proteins.

Figure 11

STA-1 Signaling in C. elegans. Standard STA-1 activity mirrors the TGF-β pathway, in terms of contribution to development, but there is an alternate mechanism in which STA-1 directly interacts with members of this path. In the absence of DAF-7, 4 or 14, STA-1 is able to directly contribute to TGF-β signaling through interactions with the DAF-1 receptor or the downstream Smad, DAF-8. There is also a proposed feedback inhibition of dauer initiation through (pro-dauer) DAF-3 regulated transcription of sta-1.

Figure 12

Apoptotic signaling in C. elegans (A) and mammalian (B) models. The anti-apoptotic Bcl-2 family members (CED-9, Bcl-2 and Bcl-xl) bind pro-apoptotic proteins attempting to locate to the outer mitochondrial membrane (OMM), neutralizing their activity. At the OMM, pro-apoptotic BH3 containing proteins will form a pore (OMMP) which allows mitochondrial proteins and second messengers to enter the cytoplasm, signaling downstream caspase activity (though it is unknown if cytochrome C release is a factor in C. elegans apoptosis). In both models, apoptosis and autophagy (via Bax/DRP-1 and Beclin-1) may be inhibited by Bcl-2/CED-9 binding. In the mammalian model, this can be reversed through the phosphorylation of Bcl-2 by JNK.

Figure 13

Formation of the autophagosome, and subsequently, the autophagolysosome. Formation begins with the binding of isolated membrane by the Beclin-1 complex. This recruits further Atg proteins to the membrane, via nucleation, and initiates the elongation of the membrane by the Atg 12/5/16L complex. The cleaved Atg8 (LC3)/Phosphatidyl ethanolamine (PE) complex finalizes the formation of the autophagosome. Binding to and incorporation of the lysosome creates the autophagolysosome which is then able to degrade/recycle its contents through hydrolase activities.

Figure 14

Regulation of autophagy by mTOR and Beclin-1. Both compete for Vsp34, with Beclin-1 requiring it for its complex formation, and mTOR requiring it for its Rheb-GTP based activation. While Beclin-1 promotes autophagy, mTOR blocks autophagosome formation via Atg-1 kinase inhibition, by phosphorylating Atg13.

Figure 15

Activation of the p53 transcription factor begins with stress signaling (primarily DNA Damage). This triggers post-translational modification of the (inactive) p53-MDM2 complex, releasing and activating p53. P53 triggers death by halting the cell cycle, through p21, and triggering the caspase cascade, through Caspase 8.

Figure 16

The protein-protein interaction between P53 and STAT1-p induces apoptosis and suppresses STAT3 protective mechanisms. STAT1-p inhibits transcription of anti-apoptosis BCL genes and enhances apoptosis signaling by p53 through binding.

Figure 17

P53, via recruitment of AMPK, regulates mTOR which in turn, is unable to inhibit the p53 family member, p73. P73 is known to up-regulate transcription of autophagy genes.

Figure 18

The C. elegans p53 ortholog, CEP-1, is stimulated by DNA damage, but can be inhibited by both SIR- 2.1 (deacetylase) and SCF^FSN-1 (through an unknown). Its transcription factor activity is controlled by post-translational modification. While the sirtuin like protein, SIR-2.1, can deacetylate and destabilize Cep-1, under certain conditions, it is also seen to trigger germline apoptosis; either upstream of or parallel to CEP-1.

Figure 19

Basic regulation of the mammalian cell cycle. The cyclin/CDK complexes which act to push the cell cycle across the G₁/S barrier act primarily on the Rb protein. The complexes themselves are subject to inhibition from CKIs. Rb phosphorylation prevents its binding (and inhibition) of E2Fa (activating) transcription factors. It similarly inhibits its activities with E2Fr (repressing) transcription factors, which would compete with the E2Fa factors for promoter binding sites on critical S phase genes.

Figure 20

Reversible phosphorylation regulation of the cell cycle. Cyclin/CDK complexes may be inactivated through phosphorylation, mediated by the Wee kinase family. Phosphorylation may be reversed by the Cdc-25 phosphatase. This phosphatase may also be inactivated through Chk-1 mediated phosphorylation. DNA checkpoint kinases are regulated by the ATM kinase, which in turn may receive signals from DNA damage and the insulin response pathway. The Akt kinase of the insulin signaling pathway, phosphorylates numerous cell cycle targets, including Cip/Kips and GSK-3 (which targets cyclin D).

Figure 21

Regulation in the C. elegans cell cycle. Like the mammalian system, the primary role of the cyclin/CDK complexes is to phosphorylate the retinoblastoma protein (LIN-35), which negatively regulates S-phase progression genes. The cyclin/CDK complexes are susceptible to phosphorylation (which can be reversed by CDC-25.1) and inhibition by CKIs, which act to attenuate the cell cycle.