While increasing evidence suggests that mammalian FLCN acts in a number of signalling pathways such as mTOR signalling, Rho signalling, membrane trafficking and stem cell exit from pluripotency, FLCN’s precise function, and how it causes the symptoms seen in BHD is not known.
The FLCN gene is highly conserved between species, with homologues in the fruit fly, nematode worm and fission yeast. Studies in these organisms have suggested roles for FLCN in germ-cell stem cell maintenance, lifespan regulation and Tor signalling (Gharbi et al., 2013; Singh et al., 2006; van Slegtenhorst et al., 2007). A paper published in PLoS ONE last week by Liu et al. describes the first constitutive knock out of the Drosophila homologue of FLCN, DBHD.
RNAi has been previously used to knock down DBHD expression in Drosophila (Singh et al., 2006). However, this method of knock down resulted in residual DBHD expression; thus Liu et al. decided to generate a knock out of DBHD. Only when both alleles were deleted did any mutant phenotype become evident in this study, suggesting that DBHD acts recessively in Drosophila. Although BHD syndrome is inherited in an autosomal dominant manner, it has been suggested that somatic loss of FLCN or PTEN, or dominant negative effects, may be required for oncogenic transformation in BHD syndrome (Pradella et al., 2013; Vocke et al., 2005), suggesting that FLCN may also act recessively in BHD patients, with respect to a subset of disease symptoms.
DBHD-null flies did not develop into adulthood, but remained in a small, early larval form for an extended period of up to three weeks, as opposed to three days in wild type and heterozygous flies. Similarly, a recent study showed that C.elegans nematode worms lacking FLCN display mild developmental delay and have an extended lifespan, although they did develop into adulthood (Gharbi et al., 2013). Increased lifespan in C.elegans has been previously associated with increased autophagy (Schiavi et al., 2013), which was observed in the larvae in the Liu et al. study, suggesting that there is significant overlapping function between the FLCN homologues in the two organisms.
Yeast rich food, specifically increased dietary leucine, partially reversed this growth phenotype, allowing flies to reach the pupal stage, although they still died during metamorphosis. The mTOR pathway maintains cellular homeostasis in response to environmental stimuli and nutrients, such as leucine and other amino acids. Indeed, Rapamycin treatment abrogated the positive effect of dietary leucine, suggesting that reduced mTOR signalling is responsible for the starvation phenotype observed in the DBHD-null larvae. Introduction of the human FLCN gene also partially rescued the phenotype, suggesting that FLCN and DBHD do share some functionality.
mTOR activation occurs at the surface of lysosomes (Sancak et al., 2010). Thus, Liu et al. suggest a model whereby DBHD acts to sequester Leucine in lysosomes, where it can be used over time to activate mTOR signalling. The authors suggest that in the DBHD-null larvae, a maternal contribution of Leucine in the oocyte allows embryos to develop into larvae. At this point, excess dietary Leucine was sufficient to allow DBHD-null larvae to pupate, while DBHD-null larvae not given Leucine stopped developing and died. DBHD-null larvae that pupated had insufficient stores of Leucine, and died during metamorphosis as they were no longer feeding.
This is a compelling theory of how DBHD might act as FLCN has recently been shown to be a GEF protein, implicating it in membrane trafficking (Nookala et al., 2012). Additionally, FLCN has been shown to both activate and inhibit mTOR signalling in different contexts (Hudon et al., 2010). If FLCN functions to sequester Leucine, it is possible that under certain conditions this allows mTOR activation at lysosomes, but perhaps under non-permissive conditions sequestering Leucine in this way could preclude mTOR access, thus inhibiting its activation.
However, FLCN has recently been shown to regulate stem exit from pluripotency (Betschinger et al., 2013). During metamorphosis, cells in larval imaginal discs proliferate and differentiate to form adult fly tissues. Therefore, dysregulation of stem cell differentiation could be a factor in the lethality seen during metamorphosis in these flies. Indeed, DBHD-null larvae had very small imaginal discs, if any at all, which was found to be due to reduced mitosis. Singh et al. have previously shown that DBHD is required to maintain populations of germ cell stem cells in Drosophila. It is therefore possible that DBHD is also required to maintain pools of imaginal disc stem cells to ensure correct metamorphosis into adulthood.
This study describes the first Drosophila knock out model of BHD syndrome and shows how powerful animal models can be in furthering medical research. Determining the exact mechanism that links DBHD, Leucine and mTOR signalling will shed light on how FLCN interacts with the mTOR pathway and how this becomes dysregulated in BHD Syndrome, and may identify new druggable targets.
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Betschinger J, Nichols J, Dietmann S, Corrin PD, Paddison PJ, Smith A (2013). Exit from pluripotency is gated by intracellular redistribution of the bHLH transcription factor Tfe3. Cell, 153 (2), 335-47 PMID: 23582324
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Gharbi H, Fabretti F, Bharill P, Rinschen MM, Brinkkötter S, Frommolt P, Burst V, Schermer B, Benzing T, Müller RU (2013). Loss of the Birt-Hogg-Dubé gene product folliculin induces longevity in a hypoxia-inducible factor-dependent manner. Aging Cell. PMID: 23566034
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Hudon V, Sabourin S, Dydensborg AB, Kottis V, Ghazi A, Paquet M, Crosby K, Pomerleau V, Uetani N, Pause A (2010). Renal tumour suppressor function of the Birt-Hogg-Dubé syndrome gene product folliculin. Journal of Medical Genetics, 47 (3), 182-9 PMID: 19843504
- Liu W, Chen Z, Ma Y, Wu X, Jin Y, & Hou S (2013). Genetic Characterization of the Drosophila Birt-Hogg-Dubé Syndrome Gene. PloS one, 8 (6) PMID: 23799055
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Nookala RK, Langemeyer L, Pacitto A, Ochoa-Montaño B, Donaldson JC, Blaszczyk BK, Chirgadze DY, Barr FA, Bazan JF, Blundell TL (2012). Crystal structure of folliculin reveals a hidDENN function in genetically inherited renal cancer. Open Biology, 2 (8), 120071 PMID: 22977732
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Pradella LM, Lang M, Kurelac I, Mariani E, Guerra F, Zuntini R, Tallini G, Mackay A, Reis-Filho JS, Seri M, Turchetti D, Gasparre G (2013). Where Birt-Hogg-Dubé meets Cowden Syndrome: mirrored genetic defects in two cases of syndromic oncocytic tumours. European journal of human genetics. PMID: 23386036
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Sancak Y, Bar-Peled L, Zoncu R, Markhard AL, Nada S, Sabatini DM (2010). Ragulator-Rag complex targets mTORC1 to the lysosomal surface and is necessary for its activation by amino acids. Cell, 141 (2), 290-303 PMID: 20381137
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Schiavi A, Torgovnick A, Kell A, Megalou E, Castelein N, Guccini I, Marzocchella L, Gelino S, Hansen M, Malisan F, Condò I, Bei R, Rea SL, Braeckman BP, Tavernarakis N, Testi R, Ventura N (2013). Autophagy induction extends lifespan and reduces lipid content in response to frataxin silencing in C. elegans. Experimental gerontology, 48 (2), 191-201 PMID: 23247094
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Singh SR, Zhen W, Zheng Z, Wang H, Oh SW, Liu W, Zbar B, Schmidt LS, Hou SX (2006). The Drosophila homolog of the human tumor suppressor gene BHD interacts with the JAK-STAT and Dpp signaling pathways in regulating male germline stem cell maintenance. Oncogene, 25 (44), 5933-41 PMID: 16636660
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van Slegtenhorst M, Khabibullin D, Hartman TR, Nicolas E, Kruger WD, Henske EP (2007). The Birt-Hogg-Dube and tuberous sclerosis complex homologs have opposing roles in amino acid homeostasis in Schizosaccharomyces pombe. The journal of biological chemistry, 282 (34), 24583-90 PMID: 17556368
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Vocke CD, Yang Y, Pavlovich CP, Schmidt LS, Nickerson ML, Torres-Cabala CA, Merino MJ, Walther MM, Zbar B, Linehan WM (2005). High frequency of somatic frameshift BHD gene mutations in Birt-Hogg-Dubé-associated renal tumors. Journal of the National Cancer Institute, 97 (12), 931-5 PMID: 15956655
www.bhdsyndrome.org – the primary online resource for anyone interested in BHD Syndrome.