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Keywords:

  • Birt–Hogg–Dubé;
  • Celegans;
  • folliculin;
  • hif;
  • longevity;
  • Vhl

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. Author contributions
  9. References
  10. Supporting Information

Signaling through the hypoxia-inducible factor hif-1 controls longevity, metabolism, and stress resistance in Caenorhabditis elegans. Hypoxia-inducible factor (HIF) protein levels are regulated through an evolutionarily conserved ubiquitin ligase complex. Mutations in the VHL gene, encoding a core component of this complex, cause a multitumor syndrome and renal cell carcinoma in humans. In the nematode, deficiency in vhl-1 promotes longevity mediated through HIF-1 stabilization. However, this longevity assurance pathway is not yet understood. Here, we identify folliculin (FLCN) as a novel interactor of the hif-1/vhl-1 longevity pathway. FLCN mutations cause Birt–Hogg–Dubé syndrome in humans, another tumor syndrome with renal tumorigenesis reminiscent of the VHL disease. Loss of the C. elegans ortholog of FLCN F22D3.2 significantly increased lifespan and enhanced stress resistance in a hif-1-dependent manner. F22D3.2, vhl-1, and hif-1 control longevity by a mechanism distinct from insulin-like signaling. Daf-16 deficiency did not abrogate the increase in lifespan mediated by flcn-1. These findings define FLCN as a player in HIF-dependent longevity signaling and connect organismal aging, stress resistance, and regulation of longevity with the formation of renal cell carcinoma.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. Author contributions
  9. References
  10. Supporting Information

Hypoxia signaling has recently been identified as an important modifier of longevity and stress resistance in Caenorhabditis elegans. It has been demonstrated that upregulation of hypoxia-inducible factor (HIF) decreases susceptibility to the deleterious effects of various insults, increases stress resistance, and enhances lifespan (Mehta et al., 2009; Müller et al., 2009; Zhang et al., 2009). HIF exists as a heterodimer of alpha and beta subunits (Jiang et al., 1996). Under normoxic conditions, the alpha subunit is ubiquitylated by a cullin–ubiquitin ligase complex containing the von Hippel Lindau protein (VHL) substrate recognition subunit and targeted for proteosomal degradation. VHL-mediated ubiquitylation is inhibited in hypoxic conditions because prolyl hydroxylases required for HIF modification and degradation depend on oxygen for their enzymatic activity. Thus, hypoxia induces stabilization of HIF allowing HIF-dependent signaling (Jaakkola et al., 2001). This signaling mechanism is highly conserved in evolution from nematodes to humans. Interestingly, pVHL is a known oncoprotein. Mutations in the VHL gene account for the majority of hereditary and sporadic renal cell carcinomas (Gnarra et al., 1994; Kaelin, 2002). Whereas the loss of VHL in humans causes a tumor syndrome, genetic deletion of vhl-1 in the nematode significantly increases the lifespan, in a predominantly hif-1-dependent manner (Mehta et al., 2009; Müller et al., 2009; Zhang et al., 2009).

Another multitumor syndrome that goes along with the formation of renal tumors is the Birt–Hogg–Dubé (BHD) syndrome. Just like von Hippel Lindau syndrome, BHD is an autosomal dominantly inherited monogenic condition in which, according to the Knudson hypothesis, sporadic mutation of the second allele induces the formation of various cancerous lesions. BHD was first described in 1977 as the inherited clinical triad of skin lesions (fibrofolliculomas), pulmonary cysts leading to recurrent pneumothorax, and a predisposition to kidney tumors (Birt et al., 1977). In 2002, Nickerson et al. identified mutations in the BHD gene product folliculin (FLCN) to be the genetic cause of this disorder (Nickerson et al., 2002). To date, 140 unique sequence variations have been described in FLCN, around 100 of which are actually pathogenic affecting approximately 200 families (www.lovd.nl/flcn; BHD Foundation, 2012; (Lim et al., 2010)). The so-called folliculin domain is highly conserved across species but shows no significant similarity to known other protein domains, thus providing no insight into the protein's molecular function. Just recently, the structure of the FLCN C-terminal domain has been published, indicating that it may serve as a guanine nucleotide-exchange factor (Nookala et al., 2012). Even though the molecular function of FLCN is poorly understood, it has been closely linked to the mammalian target of rapamycin (mTOR) pathway and to HIF activity (Baba et al., 2006; Hasumi et al., 2009). Both pathways are also important regulators of lifespan in the worm (Leiser & Kaeberlein, 2010; Bjedov & Partridge, 2011). The fact that BHD syndrome is dominantly inherited and predisposes to cancer as well as benign tumors suggests that the entity is a hamartoma syndrome like VHL syndrome, Peutz–Jeghers syndrome, or tuberous sclerosis that are all caused by a dysregulation of HIF or mTOR signaling (Kaelin, 2002; Inoki et al., 2005). F22D3.2 is a homolog of FLCN in C. elegans, yet its function in the nematode has not been studied. Thus, we decided to examine the role of F22D3.2 in nematode lifespan regulation and to establish C. elegans as a model organism for deciphering the signaling network associated with FLCN.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. Author contributions
  9. References
  10. Supporting Information

To test whether the tumor suppressor protein FLCN may act in a similar way as pVHL, we searched for a C. elegans ortholog of FLCN and identified F22D3.2. This nematode protein shows a significant homology to human FLCN, which is even more striking when focusing on the so-called folliculin domain (Fig. 1A). This domain does not show any significant homology to any other proteins but is unique to FLCN and highly conserved among species. Its molecular function is unclear. We obtained a strain harboring a deletion in F22D3.2 (from here on referred to as flcn-1) that had been created by the C. elegans Gene Knockout Consortium. We were able to confirm the mutant allele (ok975) by PCR showing that FLCN-1 mRNA is still transcribed and does not get entirely degraded (Fig. 1B). The mutation deletes 817 nucleotides in the genomic DNA and inserts a short stretch of 16 nucleotides. When sequencing flcn-1-mutant cDNA, we found exons 5 and 6 to be entirely deleted; on the level of mRNA, none of the inserted nucleotides remained, but had been removed by splicing (data not shown). The resulting protein – if translated – starts with 121 amino acids that do not differ from wild-type protein. This stretch is followed by 18 amino acids not present in wild-type and a premature stop codon at position 140 of the mutant protein (Figs 1C and S1).

image

Figure 1. Characterization of C. elegans F22D3.2. (A) Alignment of human (NP_659534) and C. elegans (F22D3.2b) folliculin protein shows a significant conservation especially when regarding the folliculin domain as defined by Pfam. (B) PCR gel pictures from worm cDNA data showing expression of folliculin mRNA in the two strains. When running PCRs using primers for full-length folliculin CDS, a shorter band results in the mutant strain, which corresponds to the loss of exons 5 and 6 as confirmed by sequencing (see Fig. S1). Consequently when using one primer lying in the assumed deletion, a PCR product is detected only when using WT cDNA [the low molecular band seen in the mutant lane is a result of primer dimerization and can be detected in no template controls as well (data not shown)]. (C) Graphical illustration of the mutated protein in the folliculin-mutant strain RB1035. The protein is truncated which leads to an entire loss of the folliculin domain (see Fig. S1). (D) Representative images of adult TMB100 carrying a Pflcn-1::GFP transgene (bar represents 25 μm). 1st panel: head region showing expression in head neurons; 2nd panel: midbody showing expression in the vulva; 3rd panel: midbody showing expression in the spermatheca and the intestine; 4th panel: tail region showing expression in tail neurons.

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Due to this early frameshift, the mutation leads to the expression of a very short truncated protein that lacks the folliculin domain (Figs 1C and S1). Thus, the mutant allele is likely a severe loss-of-function mutation. Furthermore, sequencing of flcn-1 cDNA from WTN2 revealed slight differences compared to the published sequence on Wormbase due to usage of alternative splice sites (Fig. S1). In order to gain an insight into the tissue-specific activity of the flcn-1 promoter, we generated a strain expressing GFP under the control of a promoter consisting of 2 kb upstream of the flcn-1-coding region. This strain exhibited GFP expression in the majority of tissues throughout development (L1, L2/3, L4) with the highest levels observed in the adult nervous system, the excretory cell, spermatheca, vulva, intestine, and body wall muscles (Fig. 1D and Movies S1–S4). No expression was observed in the germline, eggs, and seam cells.

When studying the flcn-1 loss-of-function allele in lifespan experiments, we found the flcn-1-deficient worms to live significantly longer than the wild-type strain (Fig. 2A). In order to exclude the influence of possible background mutations, all worms had been backcrossed to wild-type at least eight times. Using RNA interference (RNAi), we were able to confirm this effect because wild-type worms grown on RNAi against flcn-1 lived significantly longer than wild-type worms grown on an empty vector control (Fig. 2B). This shows the longevity phenotype to be specific for a loss of function of flcn-1.

image

Figure 2. Loss of folliculin induces longevity. (A) Lifespan analysis comparing RB1035 to WTN2 shows a significant increase in mean and maximum lifespan (for data and significance testing, see Table S2A). (B) Downregulation of flcn-1 using RNAi confers a similar effect on nematode lifespan, confirming that the effect observed in flcn-1-mutant worms is specific (for data and significance testing, see Table S2B).

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As mentioned before, increased activity of hif-1 is a potent inducer of longevity in C. elegans. To test whether the increased lifespan observed in flcn-1-mutant worms may be mediated by altered HIF-1 signaling, we examined a possible genetic interaction between these two genes. Depletion of hif-1 with RNAi abrogated the effect of loss of flcn-1 on lifespan, suggesting that longevity in the flcn-1-deficient worms depends on and may be mediated by hif-1 (Fig. 3A). Moreover, this finding could further be strengthened by demonstrating that wild-type and flcn-1-deficient worms grown on RNAi against vhl-1 display a nearly identical lifespan (Fig. 3B). In order to confirm dependency of lifespan extension in flcn-1-mutant worms on hif-1, this strain was crossed with a strain harboring the hif-1 loss-of-function allele ia4. Loss of hif-1 not only entirely abrogated longevity of the flcn-1-mutant strain, but shortened the lifespan of these worms compared to WTN2, underlining hif-1 dependency of lifespan assurance upon loss of flcn-1 (Fig. 3C). Flcn-1-deficient worms exhibit a significant developmental delay. This phenotype is – just as lifespan extension – abrogated upon concomitant loss of hif-1 (Fig. 3D). Furthermore, loss of flcn-1 did not alter the brood size and the pharyngeal pumping rate of the worms (Fig. S4A,B). Surprisingly, flcn-1-mutant worms did not show increased levels of the canonical HIF-1 target genes nhr-57 and F22B5.4, pointing out that flcn-1 may not be a classical activator of HIF-1, but rather act as a modulator of its function (Fig. S2).

image

Figure 3. Genetic interaction of folliculin with hypoxia-inducible factor signaling. (A) When growing flcn-1-mutant worms on RNAi against hif-1, the increase in lifespan compared to WTN2 is abrogated, showing the effect depends on the presence of HIF-1 (for data and significance testing, see Table S3A). (B) Performing a lifespan assay on RNAi against vhl-1 shows that the lifespan increase reached through the depletion of VHL-1 does not increase significantly upon the loss of flcn-1, confirming the longevity phenotype of these worms to be mediated by HIF-1 (for data and significance testing, see Table S3B). (C) As expected, lifespan extension of flcn-1-mutant worms is lost entirely when combined with a hif-1 loss-of-function allele (for data and significance testing, see Table S3C). (D) Flcn-1-deficient worms show a significant developmental delay as compared to WTN2. Concomitant loss of hif-1 entirely abrogates this phenotype.

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We next went on to test whether additional pathways would mediate the longevity effect of folliculin. The insulin/IGF-1-like signaling pathway is one of the most extensively studied pathways in lifespan determination. To test whether the longevity phenotype of flcn-1-defective C. elegans could also be explained by modulation of the insulin/IGF-1-like signaling pathway, we used RNAi to downregulate daf-2 (insulin receptor) and daf-16 (FOXO) expression in wild-type and flcn-1-defective worms. As expected, knockdown of daf-2 resulted in an increased lifespan in the wild-type worms. Interestingly, lifespan of flcn-1-deficient worms was increased in a similar manner, demonstrating that flcn-1 acts in an insulin-independent pathway (Fig. 4B). Growing the two strains on daf-16 RNAi shortens lifespan in a nearly identical way, which further supports this conclusion (Fig. 4A). In accordance with these findings, independence from DAF-16 signaling is underlined by the fact that daf-16; flcn-1 double-mutant worms live significantly longer than the daf-16 single-mutant strain (Fig. 4C). Moreover – in contrast to RNAi against daf-2 – depletion of flcn-1 did not induce nuclear translocation of a DAF-16::GFP fusion protein (Fig. S3). As expected, the expression of the daf-16 target sod-3 was not increased in the flcn-1-mutant strain (Fig. S2).

image

Figure 4. Genetic interaction of Folliculin with insulin signaling. (A) Lifespan analysis of WTN2 and flcn-1-mutant worms using RNAi against daf-16. As expected, loss of DAF-16 leads to a decrease in lifespan of both strains, yet the difference in lifespan between the two strains remains (for data and significance testing, see Table S4A). (B) Growing these two strains on RNAi against the insulin receptor daf-2 increases lifespan in a similar manner maintaining the difference between the two strains. This finding confirms – just as Fig. 4A – the lifespan extension in RB1035 to be mediated by a pathway independent from insulin signaling (for data and significance testing, see Table S4B). (C) Analysis of daf-16; flcn-1 double-mutant worms confirms the RNAi experiment in A. (for data and significance testing, see Table S4C.)

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We and others have previously shown that enhanced stress resistance may contribute to the life extension observed in vhl-1-deficient or hif-1-overactive worms (Chen et al., 2009; Mehta et al., 2009; Müller et al., 2009; Zhang et al., 2009). To test cellular stress resistance in our flcn-1-mutant strain, we exposed these worms to heat stress (35 °C). Upon growth at elevated temperatures, flcn-1-mutant worms showed a significantly better outcome than wild-type worms, nearly all of which had died after 8 h at 35 °C (Fig. 5A). In the flcn-1-mutant strain, up to 50% of the worms survived under these conditions, clearly showing an increased heat resistance of this strain. Just as lifespan extension, increased thermotolerance did not depend on daf-16 as shown by using daf-16; flcn-1 double mutants (Fig. 5B). Overexpression of flcn-1 using a transgene did not affect heat resistance when compared to wild-type worms (Fig. S5). We then went on to address the question whether heat resistance of flcn-1-deficient worms depended on intact hif-1. Loss of flcn-1 did not increase heat resistance of hif-1 mutants, pointing toward the genes acting in the same pathway (Figs 5C and S4C). Hif-1 has been described as a dual modulator of longevity with both gain- and loss-of-function mutations increasing lifespan and stress resistance through different pathways (Leiser & Kaeberlein, 2010). In hif-1-mutant worms, this phenotype is mediated by daf-16 [3, 19, (Leiser et al., 2011). In order to strengthen both this finding and hif-1 dependency of heat resistance in flcn-1-mutant worms, we performed genetic interaction experiments depleting daf-16 through RNAi. Loss of DAF-16 strongly attenuated heat resistance in both hif-1 mutants and flcn-1; hif-1 double-mutant worms, but did not show any effect in the flcn-1 loss-of-function strain (Fig. 5D–F). Thus, the combined loss of daf-16 and hif-1 is epistatic to heat resistance in this strain. Due to the complex role of hif-1 regarding this phenotype, entire independence of increased stress resistance in flcn-1-mutant worms from daf-16 cannot be proven using thermotolerance assays. However, taking into account the lifespan data (Figs 3 and 4), these results strengthen the notion that flcn-1 genetically interacts with the hypoxia-inducible factor signaling pathway, while it is independent from insulin-like signaling.

image

Figure 5. Increased stress resistance of flcn-1-mutant worms. (A) When growing the WTN2 strain at an elevated temperature of 35 °C, nearly all of these worms are dead after 8 h. However, the flcn-1-mutant worms showed significant resistance to heat, establishing a link between longevity and stress resistance upon the loss of flcn-1. (B) Enhanced thermotolerance of the flcn-1-mutant strain does not depend on daf-16 as shown by testing the daf-16; flcn-1 double-mutant strain (all P-values were calculated in comparison with the wild-type control). (C) Both loss of flcn-1 and loss of hif-1 increase heat stress resistance to a similar extent. Double mutants do not show any additive effect (n = 3; the number of asterisks represents the maximum P-value of either of the genotypes under the bar compared to the wild-type control). (D) As shown in B for the double-mutant strain, depletion of DAF-16 through RNAi does not affect stress resistance in the flcn-1-mutant strain. (E) However as expected, increased survival in the hif-1-mutant strain is strongly dependent on DAF-16. (F) Similarly, flcn-1; hif-1 double-mutant worms lose their increased heat resistance upon depletion of DAF-16, underlining the effect observed in flcn-1-mutant worms to be dependent on intact hif-1.

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vhl-1 and egl-9 regulate hif-1 post-translationally through ubiquitylation-dependent proteolysis. Western blotting shows a clear increase in HIF-1 in both the vhl-1- and egl-9-mutant strains as expected. Loss of flcn-1 does not change total HIF-1 protein levels (Fig. 6A,B), which points toward an interaction between hif-1 and flcn-1 that differs from vhl-1. In order to find mediators of longevity in flcn-1-mutant worms, we performed whole-genome microarrays establishing a list of differentially regulated genes compared to WTN2 (Table S7). This list shows no significant overlap with published transcriptional targets of HIF-1 and confirms our qPCR data regarding nhr-57, F22B5.4, and sod-3 (Fig. S7). We then performed a functional clustering analysis of the data, which yielded a cluster of four genes that had previously been implicated in lifespan control (Fig. S6). When performing qPCR to validate the microarray data and address the question whether regulation of these genes was dependent on hif-1, we found only lys-7 to be significantly increased in a hif-1-dependent fashion (Figs 6C and S6). Thus, we asked whether loss of this gene would affect the phenotype of flcn-1-mutant worms. Interestingly, when grown on lys-7 RNAi, flcn-1-deficient worms do not show any increase in thermotolerance compared to WTN2, indicating lys-7 to be involved in the increased stress resistance of our mutant strain (Fig. 6D).

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Figure 6. Loss of flcn-1 does not alter overall HIF-1 protein levels and lys-7 is a putative downstream regulator of stress resistance in flcn-1-mutant worms. (A) Western blot analysis shows no increase in HIF-1 protein in the flcn-1-deficient strain in contrast to egl-9- and vhl-1-knockout worms. (B) Densitometry of Western blots of three independent experiments containing the datapoints shown in A. (error bars represent SEM; significance was calculated in comparison with WTN2; * P-value < 0.05). (C) lys-7 is upregulated in flcn-1-mutant worms. This upregulation is lost in flcn-1; hif-1 double mutants. (n = 3 biological replicates; error bars represent SEM; **P-value <0.01, *P-value < 0.05). (D) Enhanced heat resistance of flcn-1-mutant worms relies on lys-7. When grown at 35 °C, the flcn-1-mutant strain shows an about twofold increased survival after 8 h compared to WTN2. This effect is lost when the worms are grown on RNAi targeting lys-7 (error bars represent SEM; **P-value < 0.01; n = 3).

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. Author contributions
  9. References
  10. Supporting Information

Here, we describe FLCN – the gene mutated in BHD syndrome – as a novel modulator of C. elegans lifespan. Longevity of flcn-1 mutants requires the presence of hif-1 and is independent of insulin/IGF-1-like receptor signaling. Besides the fact that folliculin adds to the growing list of C. elegans longevity genes, our findings have several important implications. Although recent evidence suggested that the vhl-1/hif-1 pathway is a novel and independent pathway regulating lifespan in worms, not much was known about its regulators and the responsible signaling network. Our data now show that folliculin may be an upstream regulator of the hif-1 effects on longevity. Moreover, the vhl-1/hif-1 pathway is a crucial pathway in the formation of renal cell carcinoma in humans (Gnarra et al., 1994). The mechanisms that determine longevity and link regulation of lifespan with tumorigenesis in general are poorly understood. The identification of FLCN as another gene, which causes renal tumors when mutated but is also involved in life extension in C. elegans, may provoke the fascinating hypothesis that stable tumor suppression may come at the expense of losing tissue-regenerative capacity (and shorter life) (Campisi & Yaswen, 2009). C. elegans – due to its short lifespan and missing cell division of adult somatic cells – does not face the problem of tumorigenesis. Consequently, the predominant read-out of loss of vhl-1 and flcn-1 is extended lifespan and enhanced stress resistance. Nonetheless, loss of these genes is expected to come at a price for the nematode as well. For vhl-1 mutants, a decreased brood size has been reported (Mehta et al., 2009). Even though further studies will be required in order to decipher the advantageous functions of flcn-1 for C. elegans, we already observed a marked developmental delay in flcn-1-mutant worms.

We now show that – in contrast to vhl-1-knockout worms – flcn-1 mutants do not exhibit increased HIF-1 protein levels, explaining why canonical hif-1 targets are not upregulated in our mutant. On the one hand, this finding could point toward a merely cell-specific stabilization of HIF-1 that cannot be detected in whole worm lysates. On the other hand, flcn-1 may modulate the transcriptional activity of HIF-1, a phenomenon that has been shown for egl-9 before. Intriguingly, the significantly upregulated genes in our microarray analysis of flcn-1-mutant worms contain a cluster of genes that had previously been shown to be mediators of longevity downstream of daf-16. In the light of our genetic interaction data, this finding may appear peculiar at first. However, one of these genes – dod-3 – had been identified as a target of hif-1 by a previous study (Shen et al., 2005). With another daf-16 target gene – dod-21 – being among the genes upregulated in a vhl-1-mutant strain, these findings point toward a more complex interplay between hypoxia and insulin-like signaling (Bishop et al., 2004). Furthermore, upregulation of the genes in this cluster depended on the presence of hif-1 in our analysis. hif-1-dependent upregulation of lys-7 is essential for increased thermotolerance of the flcn-1-mutant strain over WTN2, providing a possible regulator downstream of hif-1. Moreover –because lys-7 encodes an antimicrobial lysozyme – this finding provides another putative link between hif-1 and the defense against bacterial pathogens (Shao et al., 2010; Luhachack et al., 2012). Yet, the question whether hif-1 is an essential mediator of longevity in flcn-1-mutant worms or whether the mere presence of hif-1 is required for this phenotype cannot be finally answered. flcn-1; hif-1 double-mutant worms do not only lose longevity but live even shorter than WTN2, a finding that may point toward synthetic lethality induced by the loss of hif-1 in a flcn-1-deficient background. Because synthetic lethality is a novel and promising concept in the hunt for targeted cancer therapy, this is an intriguing hypothesis (Reinhardt et al., 2009). However, this hypothesis will need extensive further investigation, including studies in cell culture and mouse models of the BHD syndrome.

Downstream effectors and upstream regulators of the vhl-1/hif-1 pathway are not entirely clear as yet. Kenyon and colleagues showed that inhibition of mitochondrial respiration increased hif-1 activity and prolonged life in C. elegans (Lee et al., 2010). hif-1 was required for both the transcriptional changes and the increase in lifespan mediated through mitochondrial deficiency. Interestingly, when comparing the gene expression patterns from renal tumors in patients with BHD to sporadic renal tumors of the same histological subtype, Klomp et al. found mitochondrial genes to be highly dysregulated in the FLCN-deficient samples with a marked deregulation of the PGC-1α–TFAM axis and OXPHOS-related gene expression (Klomp et al., 2010). The enhanced level of mitochondrial gene expression may be a sign of increased mTOR signaling that has been closely linked to BHD syndrome on the one hand (Cunningham et al., 2007). On the other hand, it may reflect a response to mitochondrial dysfunction, which indicates FLCN to be a putative regulator of mitochondrial function.

Very recently, it was shown that activation of hif-dependent transcription occurred in a FLCN-deficient human cell line derived from a renal tumor of a patient with BHD (Preston et al., 2011). These data suggest that the genetic interaction in C. elegans may well be correlated with human disease. However, FLCN has been linked to a number of additional signal transduction pathways (BHD Foundation, 2012). Most studies available focused on a modulation of mTOR signaling through FLCN (Sarbassov et al., 2005; Landau et al., 2009). FLCN has two major interaction partners – FNIP1 and FNIP2 – through which it appears to interact with AMP-activated protein kinase (AMPK) (Baba et al., 2006; Hasumi et al., 2008). AMPK is a key player in dietary restriction-mediated longevity as it inhibits the function of mTORC1 upon an increased AMP/ATP ratio. Lifespan extension through dietary restriction has been shown to function through modulation of HIF-1 signaling providing yet another link to the folliculin signaling network (Chen et al., 2009). On a functional level, the role of the interaction between the FLCN complex and the two mTOR complexes is unclear because contradictory results depending on the experimental model used have been reported (Hartman et al., 2009; Hasumi et al., 2009; Hudon et al., 2010). As both mTOR signaling and the FLCN interaction partners – with the closely related proteins FNIP1 and FNIP2 being represented by T04C4.1 – are conserved in C. elegans, the nematode provides an ideal model to elucidate the function of folliculin. Because mTORC1 is an activator of the transcriptional activity of hypoxia-inducible factors and mTOR signaling plays a major role in longevity regulation, it will be very interesting to study a possible involvement of mTOR signaling in our longevity model (Linehan et al., 2010).

Mitogen-activated protein kinase (MAPK) signaling is yet another key signaling pathway in cellular stress resistance and has been implicated in nematode lifespan regulation (Neumann-Haefelin et al., 2008; Müller et al., 2009; Okuyama et al., 2010). Interestingly, an activation of p44/42 signaling has been reported in folliculin-knockout kidneys (Baba et al., 2008). Activation of MAPK signaling just as hypoxia-inducible factor and mTOR signaling is a common feature of a number of different tumors and thus goes along well with the formation of multiple tumors in patients harboring mutations in FLCN. We have previously shown that activation of p38 signaling may be involved in mediating HIF-1-dependent longevity in a vhl-1-mutant strain of C. elegans (Müller et al., 2009). p44/42-dependent signaling events have been reported in long-lived nematodes to mediate longevity through the transcription factor skn-1 and insulin-like signaling, yet a link to hypoxia-inducible factor signaling of p44/42 in longevity has so far not been established. Thus, it will be important to study a possible involvement of MAPK signaling in long-lived flcn-1 mutants.

Resistance to apoptotic cell death is another hallmark of tumorigenesis. It has been reported that neuronal expression of hif-1 inhibits germline apoptosis in C. elegans through a paracrine mechanism (Sendoel et al., 2010). FLCN itself has been linked to modulation of apoptosis as well. FLCN-deficient cells showed defects in cell-intrinsic apoptosis, which was the result of highly decreased expression of the pro-apoptotic protein Bim (Cash et al., 2011). Loss of Bim expression was not a consequence of increased mTOR or MAPK signaling but resulted from a highly decreased activity of TGFβ signaling. Folliculin had already before been associated with regulating TGFβ signaling in a study by Hong et al. (2010). Whether an altered activity of TGFβ signaling and an increased resistance to apoptosis play a role in FLCN-deficient C. elegans strains is unknown but should clearly be subject to further studies because it can be easily addressed analyzing apoptosis in germline cells.

Because only few families suffering from BHD syndrome have been described and studies using human material have several important limitations, the use of model organisms is crucial on the way to a better understanding of FLCN tumor suppressor protein function. Various animal models have been developed to study BHD gene function from mammals including mouse, rat, and dog models to Drosophila and yeast (BHD Foundation, 2012). Here, we describe the use of C. elegans as a model organism in BHD research. This animal model will be an amazingly versatile addition to the choice of model organisms in BHD research. Future research will clarify the link between tumor suppression and lifespan regulation. Both apoptosis and cellular senescence are crucial for tumor suppression and regression, yet both processes probably severely impair the regenerative capacity of tissues and whole organisms (Campisi & Sedivy, 2009). Thus, it is intriguing to speculate that increased tumor suppressor activity, which is vital in the aging individual who accumulates DNA damage over time, induces an aging phenotype. Our work further supports the hypothesis that a stable protection from tumorigenesis may come at the expense of altered regenerative capacity and aging. It appears crucial to study these phenomena in order to unravel their potential that holds the promise to turn an extended nematode lifespan into increased human healthspan.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. Author contributions
  9. References
  10. Supporting Information

Strains and growth conditions

Strains were maintained according to standard methods (Brenner, 1974) and grown on OP50 feeding bacteria except for RNAi experiments. A list of strains used in this study can be found in the Data S1 (Supporting information).

Feeding RNAi

RNAi by feeding was performed as described by Fire et al. RNAi clones for daf-16 and daf-2 knockdown were used as contained in the library established by Marc Vidal (Rual et al., 2004). RNAi clones for flcn-1, vhl-1, and hif-1 were used as contained in the library established by Julie Ahringer (Kamath et al., 2003). All clones used were confirmed by sequencing. Co RNAi refers to the empty vector control.

Lifespan assays

Lifespan assays were performed as described previously (Müller et al., 2009). All lifespan assays were carried out at 20 °C.

Lifespan graphs were produced using Prism 5.0 (GraphPad, La Jolla, CA, USA). Statistics and significance calculations were determined using the OASIS online tool (Yang et al., 2011).

Heat shock experiments

D1 adult hermaphrodites were selected by picking and placed at 35 °C for the time indicated. Worms that did not respond to repeated gentle prodding with a platinum wire after 30 min of recovery at 20°C were scored as dead. Every datapoint shown consists of at least three independent experiments including a minimum of 20 worms each. Significance testing was performed using a two-tailed Student's t-test.

Imaging

Worms were immobilized using sodium azide and placed on an agar pad for imaging. Imaging was performed using a Zeiss Axiovert 200 microscope (Zeiss, Jena, Germany) and the Plan Apochromat 20x objective for TJ356. The Pflcn-1::GFP transcriptional fusion-harboring strain was imaged using a confocal microscope (Zeiss LSM2000). To generate movies, Z-stacks with a constant distance of 1 μm were converted into the QuickTime format using AxioVision (Zeiss) at a speed of five frames per second. The same Z-stacks were processed using the SUM algorithm in ImageJ (http://rsbweb.nih.gov/ij/) to create representative images of different regions in adult worms.

Quantification of progeny

For the quantification of progeny, L4 hermaphrodites were singularized on NGM plates seeded with OP50. After entering adult stage, the worms were placed onto a new plate every day and the number of offspring per worm was counted. This procedure was continued until the hermaphrodites stopped laying eggs.

Quantification was performed in five independent experiments counting the offspring of at least five worms each time. Significance was calculated using a two-tailed Student's t-test.

Pharyngeal pumping

For the quantification of pharyngeal pumping, day 1 adult hermaphrodites were singularized on NGM plates seeded with OP50 and filmed using a Leica M80 stereo microscope. Movies of at least 2-min duration were recorded, and pumping movements were counted in slow motion. Quantification of pharyngeal pumping was performed in three independent experiments counting at least five worms each time. Significance was calculated using a two-tailed Student's t-test.

Developmental timing

Gravid hermaphrodites were lysed and single eggs were placed onto small NGM dishes seeded with OP50 and cultured at 20 °C. After 10 h (when no eggs had hatched yet), eggs were checked for hatching every hour. Another 34 h later, worms were checked hourly for reaching the L4 stage. Another 10 h later, worms were checked every hour for reaching adulthood. All counts were performed in at least three independent experiments using a minimum of 10 viable eggs per experiment. Significance was calculated using a two-tailed Student's t-test.

Statistical analysis

If not indicated otherwise, the following symbols represent the respective P-values: *< 0.05; **P < 0.01; ***P < 0.001; ****P > 0.0001. n.s. represents a P-value > 0.05. Unless indicated otherwise, error bars represent the SEM. The statistical test used for significance testing is indicated in the methods section of the according experiment.

Bombardment

Microparticle bombardment of C. elegans unc-119(ed3) (strain HT1593 from CGC) hermaphrodites was carried out using a BioRad Biolistic PDS-1000/HE gene gun, according to the protocol from Praitis et al. (2001). Briefly, 10–15 μg of circular DNA was coupled to 4.2 mg of 0.3- to 3-μm microcarrier gold beads (chemPUR) and bombarded onto more than 10 000 L4 and young adult hermaphrodites.

Worms were allowed to recover for 1 h at 20 °C after bombardment and were then transferred onto six 10-cm OP50 seeded dishes and incubated at 25 °C for at least 10 days. From each plate, rescued worms for the unc-119 mutation were cloned and their F1 progeny scored for the presence of unc-119 mutants. Rescued worms were screened for GFP expression using a stereo microscope (Lumar.V12, Zeiss).

Germline injection

Microinjection and antibiotic selection was performed as described by Giordano-Santini et al. (2010). Briefly, 25 ng μL−1 Pflcn-1::flcn-1::flcn-1(3′UTR) in pPB1 and 50 ng μL−1 coinjection marker pmyo-2::mCherry were injected into EN5271 strain. Positive transgenic F1 worms were moved onto selective plates at the critical concentration of G-418 (0.4 mg mL−1) and allowed to proliferate. Resistant lines were maintained on 10-cm selective plates by chunking. For the experiments performed in this publication, we did not screen integrants but used a line still containing extrachromosomal arrays.

A detailed description of the molecular biology techniques involved can be found in Data S1 (Supporting information).

Acknowledgments

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. Author contributions
  9. References
  10. Supporting Information

Thomas Benzing was supported by DFG BE2212 and SFB 832. Bernhard Schermer was supported by DFG SCHE1562 and SFB832. We thank members of the Benzing and Antebi laboratory for helpful discussions and critically reading the manuscript. Special thanks to the C. elegans Gene Knockout Consortium (Dr. Barstead, Oklahoma Medical Research Foundation) for the creation of the flcn-1-mutant allele and to the Caenorhabditis Genetics Center for providing the strains. Jasmin Manz, Kathrin Riehl, and Ruth Herzog provided excellent technical assistance that helped performing the experiments described. Regarding the RNAi libraries, we thank the Dana-Farber Cancer Institute and Source BioScience LifeSciences. We are grateful to Ataman Sendoel, Michael Hengartner, Peter Ratcliffe, Mark Roth, and Denis Dupuy for providing antibodies and reagents and to Martin Höhne for help with image processing.

Author contributions

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. Author contributions
  9. References
  10. Supporting Information

HG performed the majority of the experiments; FF conducted some and supervised all experiments. PB, SB, and MR performed the experiments. PF analyzed the microarray data. VB, BS, and TB contributed to study design, data interpretation, and revised the manuscript. RUM designed the study, analyzed the data, and wrote the manuscript.

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  7. Acknowledgments
  8. Author contributions
  9. References
  10. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgments
  8. Author contributions
  9. References
  10. Supporting Information
FilenameFormatSizeDescription
acel12081-sup-0001-FigS1.epsimage/eps2274KFig. S1 Conservation of FLCN and C. elegans sequence determination by sequencing.
acel12081-sup-0002-FigS2.epsimage/eps113KFig. S2 qPCR analysis reveals expression levels of HIF-1 and DAF-16 target genes not to be affected in the flcn-1-mutant strain.
acel12081-sup-0003-FigS3.tifimage/tif9049KFig. S3 Loss of flcn-1 does not induce nuclear translocation of DAF-16.
acel12081-sup-0004-FigS4.epsimage/eps202KFig. S4 Phenotyping flcn-1-mutant worms.
acel12081-sup-0005-FigS5.epsimage/eps128KFig. S5 Overexpression of flcn-1 does not alter thermotolerance.
acel12081-sup-0006-FigS6.epsimage/eps1941KFig. S6 Functional annotation clustering.
acel12081-sup-0007-FigS7.epsimage/eps945KFig. S7 Comparison to published microarrays.
acel12081-sup-0008-TableS1.epsimage/eps1453KTable S1 Statistics of lifespan experiments examining the role of flcn-1.
acel12081-sup-0009-TableS2.epsimage/eps1614KTable S2 Statistics of lifespan experiments examining the genetic interaction with hif-1.
acel12081-sup-0010-TableS3.epsimage/eps1421KTable S3 Statistics of lifespan experiments examining the genetic interaction with daf-16.
acel12081-sup-0011-TableS4.xlsapplication/msexcel24KTables S4 Pooled lifespan data underlying the lifespan curves in Figure 2 specifying the number of worms that entered, died, or were censored during the interval.
acel12081-sup-0012-TableS5.xlsapplication/msexcel39KTables S5 Pooled lifespan data underlying the lifespan curves in Figure 3 specifying the number of worms that entered, died, or were censored during the interval.
acel12081-sup-0013-TableS6.xlsapplication/msexcel30KTables S6 Pooled lifespan data underlying the lifespan curves in Figure 4 specifying the number of worms that entered, died, or were censored during the interval.
acel12081-sup-0014-TableS7.xlsxapplication/msexcel67KTable S7 Differentially expressed genes in flcn-1-mutant worms compared to WTN2 as determined by Agilent whole-genome microarrays.
acel12081-sup-0015-MovieS1.MOVvideo/mov1438KMovie S1 Tissue-specific activity of the flcn-1 promoter. Movies show Z-stacks with a fixed distance of 1 μm of TMB100 (unc-119 (ed3); klnIs2 [Pflcn-1::GFP + unc-119 (+)]). 1. Adult hermaphrodite head.
acel12081-sup-0016-MovieS2.MOVvideo/mov1761KMovie S2 Tissue-specific activity of the flcn-1 promoter. Movies show Z-stacks with a fixed distance of 1 μm of TMB100 (unc-119 (ed3); klnIs2 [Pflcn-1::GFP + unc-119 (+)]). 2. Adult hermaphrodite midbody.
acel12081-sup-0017-MovieS3.MOVvideo/mov4842KMovie S3 Tissue-specific activity of the flcn-1 promoter. Movies show Z-stacks with a fixed distance of 1 μm of TMB100 (unc-119 (ed3); klnIs2 [Pflcn-1::GFP + unc-119 (+)]). 3. Adult hermaphrodite midbody.
acel12081-sup-0018-MovieS4.MOVvideo/mov1347KMovie S4 Tissue-specific activity of the flcn-1 promoter. Movies show Z-stacks with a fixed distance of 1 μm of TMB100 (unc-119 (ed3); klnIs2 [Pflcn-1::GFP + unc-119 (+)]). 4. Adult hermaphrodite tail.
acel12081-sup-0019-Methods.docWord document35KData S1 Suppl. methods.

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