Date Published: September 15, 2011
Publisher: Impact Journals LLC
Author(s): Christopher Rongo.
Epidermal Growth Factor (EGF) is known for its role in promoting cell division and cellular differentiation in developing animals, but we know surprising little about what EGF does in vivo in mature adult animals. Here I review EGF signaling, emphasizing several recent studies that uncovered an unexpected role for EGF in promoting longevity and healthspan in mature adult C. elegans. EGF, acting through phospholipase Cγ and the IP3 receptor signaling, maintains pharyngeal and body wall muscle function in aging adults, and delays the accumulation of lipofuscin-enriched aging pigments within intestinal cells. EGF also acts through the Ras/ERK pathway to regulate protein homeostasis by promoting the expression of antioxidant genes, stimulating the activity of the Ubiquitin Proteasome System (UPS), and repressing the expression of small heat shock protein chaperones. The effects of EGF signaling on lifespan are largely independent of Insulin/IGF-like Signaling (IIS), as the effects of EGF signaling mutants on lifespan and heathspan are not affected by mutations in the DAF-2 insulin receptor or the DAF-16 FOXO transcription factor. Nevertheless, these two signal pathways have multiple points of overlap, coordination, and cross regulation. I propose that the IIS and EGF signaling pathways respond to environment and to developmental timing, respectively, so as to coordinate the appropriate physiological strategy that cells use to maintain protein homeostasis.
Many diverse functional roles for Epidermal Growth Factor (EGF) have been uncovered since its discovery by Stanley Cohen more than half a century ago . Cohen had noted that the injection of crude submaxillary gland preparations into newborn mice resulted in increased epidermal growth and keratinization, triggering the premature opening of the eyelid [1, 2]. Using precocious eyelid opening as a bioassay, Cohen isolated and identified EGF as a small secreted protein rich in disulphide bonds [3-6]. Latter experiments showed that EGF could induce mitosis of cultured epidermal cells, promote DNA synthesis, stimulate translation, and increase protein phosphorylation [7-9]. A rush to identify the receptor for EGF and its downstream signaling components ensued.
A combination of many years of biochemical and genetic studies has elucidated several parallel signal transduction pathways used by EGF and its receptor (Figure 1). Upon EGF binding, the EGFR becomes autophosphorylated, resulting in the recruitment of adaptors like Grb2/SEM-5 and activation of the Ras/ERK signal transduction cascade . In C. elegans, LIN-3/EGF activates LET-60/Ras and MPK-1/ERK to promote vulval differentiation through the modulation of multiple transcription factors . EGFR activation also stimulates phospholipase C gamma (PLCγ) [24, 25], resulting in the production of inositol 1,4,5-trisphosphase (IP3) and the release of calcium from intracellular stores via the IP3 receptor. The C. elegans PLCγ and IP3 receptor are encoded by PLC-3 and ITR-1, respectively, and C. elegans uses this pathway to promote ovulatory contractions in response to LIN-3/EGF [21, 26]. Finally, EGFR activation can in turn promote phosphoinositide 3-kinase (PI3K) activity either directly, with the p85 regulatory subunit of PI3K recognizing the phosphorylated receptor, or indirectly, with p85 interacting through a Grb2/GAB complex. Activated Ras can also activate the PI3K p110 catalytic subunit . Once activated, PI3K converts phosphatidylinositol [4,5]-bisphosphate (PIP2) into phosphatidylinositol [3,4,5]-triphosphate (PIP3). PIP3 in turn binds the pleckstrin homology (PH) domain of Akt, stimulating its kinase activity and promoting the phosphorylation of proteins that regulate cell growth, cell cycle entry, and cellular survival . This includes mammalian target of rapamycin (mTOR), a positive regulator of translation . It is unknown whether C. elegans LIN-3/EGF signaling activates PI3K (AAP-1, PDK-1, and AGE-1 in C. elegans) or Akt (AKT-1 and AKT-2 in C. elegans); however, Insulin/IGF Signaling (IIS) has been implicated in regulating longevity through PI3K and Akt .
Several recent studies have implicated a novel function for EGF signaling in promoting C. elegans longevity. One of these studies was originally prompted from analysis of the IIS pathway, another signaling pathway long implicated in aging. Central to the IIS pathway in C. elegans is the DAF-2 insulin receptor, which signals through the AGE-1 PI3K to inhibit longevity . In well-fed animals, DAF-2 becomes activated, triggering the phosphorylation of the DAF-16 FOXO transcription factor by AKT-1, AKT-2, and SGK-1, which prevents it from accumulating in the nucleus [32,36]. By contrast, stress or acute nutrient deprivation depresses IIS activity, releasing DAF-16/FOXO to enter into the nucleus and regulate the transcription of genes involved in dauer formation (a diapause state), metabolism, lipid storage, stress response, and lifespan extension [37,42]. While there is a single DAF-2 insulin receptor, there are forty putative insulin-like ligands in the C. elegans genome, as well as multiple insulin receptor-related proteins that are predicted to be secreted molecules [43,45]. Iwasa et al. recently screened these proteins by RNAi-mediated knockdown to identify candidates that might have a role in promoting healthy longevity . Either RNAi-mediated knockdown or deletion mutations for two of these genes, named HPA-1 and HPA-2 for high performance in old age, resulted in increased healthy longevity based on locomotory activity, pharyngeal activity, and age-pigment accumulation in older animals compared to wild-type controls. While HPA-1 and HPA-2 both contain sequences similar to the ligand binding region of the insulin receptor, genetic analysis between hpa-1, hpa-2,daf-2, and daf-16 mutants indicated that HPA-1 and HPA-2, at least in part, normally function to shorten healthy lifespan by a mechanism that is independent of IIS. Interestingly, HPA-1 and HPA-2 contain leucine-rich domains similar to those of mammalian EGFR-related protein (ERRP), a secreted negative regulator of the EGFR, raising the possibility that HPA-1/2 might inhibit lifespan by antagonizing the LET-23/EGFR .
Activation of SKN-1 is not the only way by which EGF signaling through Ras/ERK affects lifespan. Indeed, a role for EGF in longevity was also recently demonstrated by Liu et al., using a completely different approach . These researchers were examining protein homeostasis regulation by the Ubiquitin Proteasome System (UPS) in C. elegans using an UbG76V-GFP chimeric reporter for UPS activity. Chimeric UbG76V-GFP protein contains an amino-terminal ubiquitin fused in frame with GFP, but with a mutation at glycine 76 that prevents the cotranslational cleavage that would otherwise release ubiquitin from GFP after synthesis [56-58]. The resulting uncleaved UbG76V-GFP protein mimics a monoubiquitinated GFP and is an efficient, non-specific substrate for additional polyubiquitination and proteolysis by the 26S proteasome. UbG76V-GFP essentially acts as an inverse reporter for UPS activity, yielding high GFP fluorescence when UPS activity is low and vice versa. Liu et al. used different cell-type specific promoters to express UbG76V-GFP in different C. elegans tissues. They noticed that UbG76V-GFP levels in larvae remained relatively high in epithelia, but that epithelial UbG76V-GFP was rapidly degraded as animals matured into fertile adults, suggesting that the steady state levels of UPS activity are low during early development, but become enhanced at a specific point in adult maturation. To identify the biological signal that triggers this augmentation in UPS activity in adulthood, they undertook a candidate gene approach looking at known genes that had been previously implicated in regulating protein turnover. A role for fibroblast growth factor (FGF) and Ras/ERK signaling in protein turnover had previously been demonstrated in C. elegans body wall muscle . Liu et al. found that whereas Ras/ERK signaling was required for UbG76V-GFP turnover in adult epithelia, FGF was not required. Reasoning that the FGF ligand might be specific for muscle, and that different tissues might use different signaling ligands to regulate UPS activity, they examined EGF signaling mutants, including lin-3 and let-23. Loss of either LIN-3/EGF or LET-23/EGFR prevented the rapid degradation of UbG76V-GFP as animals entered adulthood. By contrast, the let-23(sa62) gain of function mutation in the EGFR resulted in precocious turnover of UbG76V-GFP during larval development. Thus, EGF signaling through the Ras/ERK pathway, but not through the PLCγ/IP3 pathway, was directing protein turnover in adult epithelia.
Taken together, it would appear that EGF is used as a signal not only for tissue morphogenesis during development, but for regulating tissue physiology as well. Initially, nematodes use LIN-3/EGF to induce epithelia morphogenesis during larval development. As nematodes enter adulthood, their epithelia resynthesize LIN-3/EGF, perhaps as an autocrine signal, to trigger the activation of multiple signal transduction pathways, altering calcium homeostasis, translation, protein folding and anti-aggregation, and UPS activity and protein turnover (Figure 2). This represents a strategic, genetically programmed switch in cellular physiology as animals mature into adulthood, with timing that closely parallels the final maturation of the germline, suggesting that these two events might be coupled. Given the close timing to germline maturity, its reasonable to speculate that this EGF-triggered switch in physiology might have evolved to maximize organismal health at the period of peak fecundity. If this is the case, then the impact of EGF signaling on aging might simply be a lucky consequence of optimized fitness during young adulthood. By maximizing organismal health during the period of fecundity, the animal is able to ward off physiological decline associated with aging even long after it has depleted its germ cells and fertility no longer matters, with the added benefit of extended longevity, at least under laboratory conditions.