Loss of the Birt–Hogg–Dub  e gene product folliculin induces longevity in a hypoxia-inducible factor–dependent manner Hakam Gharbi, 1,2 Francesca Fabretti, 1 Puneet Bharill, 1 Markus M. Rinschen, 1 Sibylle Brinkk€ otter, 1 Peter Frommolt, 2,4 Volker Burst, 1 Bernhard Schermer, 1,2,3 Thomas Benzing 1,2,3 and Roman-Ulrich M€ uller 1,2,3 1 Department 2 of Internal Medicine and Center for Molecular Medicine Cologne, 2 Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases, 3 Systems Biology of Ageing Cologne, University of Cologne, Cologne, Germany 4 Cologne Center for Genomics, University of Cologne, Cologne, Germany Summary Signaling through the hypoxia-inducible factor hif-1 controls longevity, metabolism, and stress resistance in Caenorhabd- itis 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. How- ever, 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– Dub e 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 mech- anism 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. Key words: Birt–Hogg–Dub e; C. elegans; folliculin; hif; lon- gevity; Vhl. Introduction 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; M€ uller 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 nema- todes 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; M€ uller et al., 2009; Zhang et al., 2009). Another multitumor syndrome that goes along with the forma- tion of renal tumors is the Birt–Hogg–Dube (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 predis- position 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 approxi- mately 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 dysregu- lation of HIF or mTOR signaling (Kaelin, 2002; Inoki et al., 2005). Correspondence Dr. Roman-Ulrich M€ uller, Department 2 of Internal Medicine, University of Cologne, Kerpener Str. 62, 50937 Cologne, Germany. Tel.: 0049 221 478 4480; fax: 0049 221 478 89041; e-mail: roman-ulrich.mueller@uk-koeln.de Hakam Gharbi and Francesca Fabretti contributed equally to this work. Accepted for publication 28 March 2013 ª 2013 John Wiley & Sons Ltd and the Anatomical Society 593 Aging Cell (2013) 12, pp593–603 Doi: 10.1111/acel.12081 Aging Cell