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The hypoxic response is a well-studied and highly conserved biological response to low oxygen availability. First described more than 20 y ago, the traditional model for this response is that declining oxygen levels lead to stabilization of hypoxia-inducible transcription factors (HIFs), which then bind to hypoxia responsive elements (HREs) in target genes to mediate the transcriptional changes collectively known as the hypoxic response.1,2 Recent work in C. elegans has forced a re-evaluation of this model by indicating that the worm HIF (HIF-1) can mediate effects in a cell non-autonomous fashion and, in at least one case, increase expression of an intestinal hypoxic response target gene in cells lacking HIF-1.
In most animals, HIFs function as a heterodimer consisting of a regulated HIF-α subunit and a constitutive HIF-β subunit.2 Under normoxic conditions, the HIF-α subunit is targeted for degradation by the proteasome via post-translational hydroxylation and ubiquitination reactions.3 In worms, this regulation is mediated by the EGL-9 prolyl hydroxylase and the VHL-1 ubiquitin ligase. Loss of function mutations in either of these proteins causes constitutive stabilization of HIF-1 and expression of hypoxia-inducible HIF-1 target genes under normoxic conditions.4,5 Conversely, loss of function mutations in hif-1 result in the inability to induce hypoxic response genes under low oxygen conditions.6 Phenotypically, this manifests itself as an inability to develop into reproductive adults at oxygen levels below about 0.5%.6,7
Multiple studies over the past several years have implicated the worm HIF-1 in a variety of processes not directly related to sensing and responding to changes in environmental oxygen. The first of these studies found that HIF-1 is necessary for worms to acclimate to high temperatures.8 The authors showed that not only does the loss of HIF-1 prevent worms at higher temperatures from improving their heat tolerance (adapting), but that constitutively stabilized HIF-1 alone is sufficient to improve heat tolerance. A later paper described how the nematode hypoxic response protects against the pathogenesis of bacteria that use pore-forming toxins, implicating a role for HIF-1 in immunity that was later confirmed by other groups.9,10 A report in Nature in 2010 associated HIF-1 with the well-studied oncogene, p53, and showed that HIF-1 signaling through an intermediate tyrosinase can prevent germline apoptosis.11 An additional study from the Lee and Kenyon labs showed that inhibiting respiration by mutating mitochondrial proteins increases HIF-1 activity, and that this increase is necessary for these mutations to increase longevity in some cases.12 Most recently, worm HIF-1 has been shown to be a key regulator of iron homeostasis.13,14 Together, there is accumulated research that the hypoxic response is important for many processes beyond its canonical role in cellular adaptation to low oxygen.
Our interest in the worm hypoxic response stemmed initially from an RNAi screen aimed at identifying components of the ubiquitin proteasomal system that modulate aging and protein homeostasis. We had previously observed that dietary restriction by bacterial deprivation was able to both increase lifespan and enhance resistance to polyglutamine and amyloid β toxicity in worms,15-17 and we postulated that alterations in proteasomal function might underlie these phenotypes. From this screen, we found that RNAi knockdown of vhl-1 led to enhanced resistance to both polyglutamine and amyloid β toxicity, as well as increased lifespan by about 30%.18 In contrast to our original hypothesis, however, these effects were genetically distinct from the dietary restriction pathway and were instead mediated by activation of HIF-1.18 Soon after our initial publication, two other labs reported similar findings by showing that either knockdown of vhl-1 or constitutive stabilization of HIF-1 could increase lifespan in C. elegans.19,20 These studies and others that have followed have established HIF-1 and the hypoxic response as a pro-longevity pathway that is genetically distinct from the other major longevity pathways such as insulin-like signaling and dietary restriction.21,22
Intriguingly, although constitutive activation of HIF-1 is sufficient to increase lifespan, loss of function mutations in hif-1 can also increase lifespan under certain conditions.19,23 The effect of hif-1 deletion on lifespan appears to be largely dependent on temperature; hif-1(ia4) animals are long-lived at 25C but not at 15C.24 The effect of hif-1 deletion on lifespan at 20C appears somewhat variable and may depend on additional experimental conditions that have yet to be defined.25 Two distinct mechanisms have been proposed by which loss of hif-1 extends lifespan at higher temperature. Chen et al. reported that deletion of hif-1 results in enhanced ER stress resistance and promotes longevity by a mechanism akin to dietary restriction,23 while our group and the Powell-Coffman lab have published studies indicating that loss of hif-1 extends lifespan by activating the FOXO transcription factor DAF-16 and independently of dietary restriction.18,19,24,26
In order to better understand the mechanisms by which HIF-1 promotes longevity in C. elegans, we set out to establish which tissues and which HIF-1 target genes were directly involved in promoting longevity when HIF-1 is stabilized. Perhaps not surprisingly given the emerging importance of neuronal expression of HIF-1 for other physiological responses in worms,27-29 we found that stabilization of HIF-1 in neurons was sufficient to increase lifespan.30 In fact, expression of stabilized HIF-1 only in serotonergic neurons under control of the tryptophan hydroxylase (tph-1) promoter was sufficient to enhance longevity. Interestingly, this effect is maintained even when HIF-1 is deleted in non-neuronal cells.
In parallel studies, we also found that transgenic overexpression of a single HIF-1 target gene, fmo-2, was sufficient to increase lifespan.30 The fmo-2 gene encodes one of a family of flavin-containing monooxygenases in C. elegans that are conserved from yeast to humans.31 This gene represents one of the most highly induced mRNAs in vhl-1 mutant animals or in N2 animals subjected to hypoxia.4,32 In contrast to hif-1, however, overexpression of fmo-2 in neurons had no effect on lifespan while overexpression of fmo-2 in intestine extended lifespan.
Given these two sets of results, we wondered whether stabilization of HIF-1 in neurons could be inducing FMO-2 in the intestine to increase lifespan. Utilizing a GFP reporter of fmo-2 expression, we were able to confirm this hypothesis by observing increased GFP in intestinal cells when stabilized HIF-1 is expressed pan-neuronally.30 Strikingly, this induction occurred even when hif-1 is deleted in intestinal cells. Based on this latter observation, we proposed that fmo-2, and perhaps other “HIF-1 target genes” can be regulated by HIF-1 cell non-autonomously, independently of HIF-1 activity in the target cells (Fig. 1).
The paradigm for the hypoxic response in both worms and other animals has been that HIFs become stabilized by hypoxia or other stimuli (such as reactive oxygen species) and bind directly to the promoter of hypoxic response genes in order to regulate their transcription. The fact that at least one canonical hypoxic response gene in the worm is induced by HIF-1 via a cell non-autonomous mechanism that does not require HIF-1 in the target tissue raises several important questions about this model. For example, to what extent is this mechanism in play for other hypoxic response genes? It seems unlikely that fmo-2 is the only hypoxic response gene regulated in this manner in the worm; however, future studies will be required to determine this for certain. Assuming that fmo-2 is not unique, this will require a significant revision of the model for hypoxia signaling in C. elegans. It will also set the stage for related studies in other metazoans, to determine whether hypoxic response genes can be similarly regulated cell non-autonomously in those organisms.
Although it seems clear that fmo-2 does not require direct binding by HIF-1 for induction, at least in the intestine, it remains possible that in wild-type C. elegans HIF-1 is still a direct regulator of intestinal fmo-2 in response to hypoxia and other stimuli. In other words, it may be that our results reflect the ability of some other transcription factor to activate fmo-2 transcription when HIF-1 is absent, but this mechanism does not normally occur in wild type animals. One approach to assess this is to determine which transcription factors induce fmo-2 in animals where HIF-1 is restricted to neurons and determine whether these factors modulate fmo-2 expression in wild type animals subjected to hypoxia or genetic stabilization of HIF-1. Our study identified HLH-30 as one likely candidate for such a role,30 and we are currently searching for others.
There are also numerous open questions surrounding how HIF-1 signals with specificity across tissues. Our data support a model whereby the monoamine neurotransmitter serotonin plays a necessary role in signaling between the neurons and the intestine through the ser-7 receptor.30 It is unclear, however, how many (if any) steps lie between neuronal serotonin production and release and intestinal activation of fmo-2 and potentially other genes. Since serotonin is commonly used for signaling outside of hypoxic signaling, it would seem likely that mechanisms have evolved to increase specificity for this response. Future work will answer many of these questions, including 1) Are other neurotransmitters and/or neuropeptides involved?33; 2) in what tissue(s) is SER-7 important?; 3) How is serotonin released from the relevant neurons?; and 4) Which neurons are essential for the signal?
The data supporting roles for the hypoxic response in regulating multiple processes, including responding to varying oxygen levels, are abundant. Studies in worms suggest that HIF proteins utilize multiple mechanisms to regulate the hypoxic response in various tissues. These mechanisms include cell autonomous responses to modify metabolism and escape hypoxia, and cell non-autonomous signals that can modify stress resistance and longevity. Since the hypoxic response is a well-conserved pathway, the conservation of hypoxic cell autonomous and cell non-autonomous signaling can be postulated but is not yet proven. We look forward to future studies delineating HIF signaling in worms that will lead to further insights about the evolutionary conserved roles of this important pathway.
No potential conflicts of interest were disclosed.
This work was supported by NIH grant R01AG038518 to MK and NIH grant K99AG045200 to SFL.