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• W3131707355 abstract "Birt–Hogg–Dubé (BHD) syndrome is a multiorgan disorder caused by inactivation of the folliculin (FLCN) protein. Previously, we identified FLCN as a binding protein of Rab11A, a key regulator of the endocytic recycling pathway. This finding implies that the abnormal localization of specific proteins whose transport requires the FLCN-Rab11A complex may contribute to BHD. Here, we used human kidney-derived HEK293 cells as a model, and we report that FLCN promotes the binding of Rab11A with transferrin receptor 1 (TfR1), which is required for iron uptake through continuous trafficking between the cell surface and the cytoplasm. Loss of FLCN attenuated the Rab11A–TfR1 interaction, resulting in delayed recycling transport of TfR1. This delay caused an iron deficiency condition that induced hypoxia-inducible factor (HIF) activity, which was reversed by iron supplementation. In a Drosophila model of BHD syndrome, we further demonstrated that the phenotype of BHD mutant larvae was substantially rescued by an iron-rich diet. These findings reveal a conserved function of FLCN in iron metabolism and may help to elucidate the mechanisms driving BHD syndrome. Birt–Hogg–Dubé (BHD) syndrome is a multiorgan disorder caused by inactivation of the folliculin (FLCN) protein. Previously, we identified FLCN as a binding protein of Rab11A, a key regulator of the endocytic recycling pathway. This finding implies that the abnormal localization of specific proteins whose transport requires the FLCN-Rab11A complex may contribute to BHD. Here, we used human kidney-derived HEK293 cells as a model, and we report that FLCN promotes the binding of Rab11A with transferrin receptor 1 (TfR1), which is required for iron uptake through continuous trafficking between the cell surface and the cytoplasm. Loss of FLCN attenuated the Rab11A–TfR1 interaction, resulting in delayed recycling transport of TfR1. This delay caused an iron deficiency condition that induced hypoxia-inducible factor (HIF) activity, which was reversed by iron supplementation. In a Drosophila model of BHD syndrome, we further demonstrated that the phenotype of BHD mutant larvae was substantially rescued by an iron-rich diet. These findings reveal a conserved function of FLCN in iron metabolism and may help to elucidate the mechanisms driving BHD syndrome. Loss of folliculin (FLCN) has been associated with Birt–Hogg–Dubé (BHD) syndrome, which is characterized by frequent development of skin tumors, lung cysts, and a high risk of kidney cancer. FLCN regulates a wide range of cellular processes, such as amino acid homeostasis, energy metabolism, biogenesis of lysosomes and mitochondria, membrane transport, cytoskeletal remodeling, and primary cilia formation (1Nickerson M.L. Warren M.B. Toro J.R. Matrosova V. Glenn G. Turner M.L. Duray P. Merino M. Choyke P. Pavlovich C.P. Sharma N. Walther M. Munroe D. Hill R. Maher E. et al.Mutations in a novel gene lead to kidney tumors, lung wall defects, and benign tumors of the hair follicle in patients with the Birt-Hogg-Dubé syndrome.Cancer Cell. 2002; 2: 157-164Abstract Full Text Full Text PDF PubMed Scopus (674) Google Scholar). However, the primary cause of BHD is still unknown. This uncertainty is partially due to the observation that both the growth-promoting protein mTOR (2Baba M. Furihata M. Hong S.B. Tessarollo L. Haines D.C. Southon E. Patel V. Igarashi P. Alvord W.G. Leighty R. Yao M. Bernardo M. Ileva L. Choyke P. Warren M.B. et al.Kidney-targeted Birt-Hogg-Dubé, gene inactivation in a mouse model: Erk1/2 and Akt-mTOR activation, cell hyperproliferation, and polycystic kidneys.J. Natl. Cancer Inst. 2008; 100: 140-154Crossref PubMed Scopus (191) Google Scholar, 3Chen J. Futami K. Petillo D. Peng J. Wang P. Knol J. Li Y. Khoo S.K. Huang D. Qian C.N. Zhao P. Dykema K. Zhang R. Cao B. Yang X.J. et al.Deficiency of FLCN in mouse kidney led to development of polycystic kidneys and renal neoplasia.PLoS One. 2008; 3e3581Crossref PubMed Scopus (113) Google Scholar, 4Wu M. Si S. Li Y. Schoen S. Xiao G.Q. Li X. Teh B.T. Wu G. Chen J. Flcn-deficient renal cells are tumorigenic and sensitive to mTOR suppression.Oncotarget. 2015; 6: 32761-32773Crossref PubMed Scopus (9) Google Scholar, 5Gijezen L.M. Vernooij M. Martens H. Oduber C.E. Henquet C.J. Starink T.M. Prins M.H. Menko F.H. Nelemans P.J. van Steensel M.A. Topical rapamycin as a treatment for fibrofolliculomas in Birt-Hogg-Dubé syndrome: A double-blind placebo-controlled randomized split-face trial.PLoS One. 2014; 9e99071Crossref PubMed Scopus (24) Google Scholar, 6Hartman T.R. Nicolas E. Klein-Szanto A. Al-Saleem T. Cash T.P. Simon M.C. Henske E.P. The role of the Birt-Hogg-Dubé protein in mTOR activation and renal tumorigenesis.Oncogene. 2009; 28: 1594-1604Crossref PubMed Scopus (166) Google Scholar, 7Petit C.S. Roczniak-Ferguson A. Ferguson S.M. Recruitment of folliculin to lysosomes supports the amino acid-dependent activation of Rag GTPases.J. Cell Biol. 2013; 202: 1107-1122Crossref PubMed Scopus (210) Google Scholar, 8Tsun Z.Y. Bar-Peled L. Chantranupong L. Zoncu R. Wang T. Kim C. Spooner E. Sabatini D.M. The folliculin tumor suppressor is a GAP for the RagC/D GTPases that signal amino acid levels to mTORC1.Mol. Cell. 2013; 52: 495-505Abstract Full Text Full Text PDF PubMed Scopus (334) Google Scholar, 9Wu X. Zhao L. Chen Z. Ji X. Qiao X. Jin Y. Liu W. FLCN maintains the leucine level in lysosome to stimulate mTORC1.PLoS One. 2016; 11e0157100Crossref PubMed Scopus (18) Google Scholar) and the energy sensor AMP-activated protein kinase (10Yan M. Gingras M.C. Dunlop E.A. Nouët Y. Dupuy F. Jalali Z. Possik E. Coull B.J. Kharitidi D. Dydensborg A.B. Faubert B. Kamps M. Sabourin S. Preston R.S. Davies D.M. et al.The tumor suppressor folliculin regulates AMPK-dependent metabolic transformation.J. Clin. Invest. 2014; 124: 2640-2650Crossref PubMed Scopus (96) Google Scholar, 11Possik E. Jalali Z. Nouët Y. Yan M. Gingras M.C. Schmeisser K. Panaite L. Dupuy F. Kharitidi D. Chotard L. Jones R.G. Hall D.H. Pause A. Folliculin regulates ampk-dependent autophagy and metabolic stress survival.PLoS Genet. 2014; 10e1004273Crossref PubMed Scopus (78) Google Scholar, 12Yan M. Audet-Walsh É. Manteghi S. Dufour C.R. Walker B. Baba M. St-Pierre J. Giguère V. Pause A. Chronic AMPK activation via loss of FLCN induces functional beige adipose tissue through PGC-1alpha/ERRalpha.Genes Dev. 2016; 30: 1034-1046Crossref PubMed Scopus (65) Google Scholar, 13Hasumi Y. Baba M. Hasumi H. Huang Y. Lang M. Reindorf R. Oh H.B. Sciarretta S. Nagashima K. Haines D.C. Schneider M.D. Adelstein R.S. Schmidt L.S. Sadoshima J. Linehan M.W. Folliculin (Flcn) inactivation leads to murine cardiac hypertrophy through mTORC1 deregulation.Hum. Mol. Genet. 2014; 23: 5706-5719Crossref PubMed Scopus (41) Google Scholar) can be either activated or suppressed upon FLCN loss. It has thus been speculated that FLCN regulates these two pathways through varied mechanisms, depending on the specific cellular and tissue contexts. FLCN is conserved from yeast to humans, implying that it necessarily regulates certain fundamental cellular processes. Consistent with this hypothesis, emerging evidence has revealed important roles of FLCN during vesicular trafficking, which is a highly conserved process in eukaryotic cells. Indeed, before FLCN was initially identified in humans and subsequently linked to BHD, the Kaiser group had identified a group of yeast genes governing the movement of the amino acid permease Gap1p from the Golgi apparatus to the plasma membrane (14Roberg K.J. Bickel S. Rowley N. Kaiser C.A. Control of amino-acid permease sorting in the late secretory pathway of Saccharomyces cerevisiae by SEC13, LST4, LST7, and LST8.Genetics. 1997; 147: 1569-1584Crossref PubMed Google Scholar). Among these genes are LST7, an ortholog of the mammalian FLCN (15van Slegtenhorst M. Khabibullin D. Hartman T.R. Nicolas E. Kruger W.D. Henske E.P. The Birt-Hogg-Dube and tuberous sclerosis complex homologs have opposing roles in amino acid homeostasis in Schizosaccharomyces pombe.J. Biol. Chem. 2007; 282: 24583-24590Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar), and LST4, an ortholog of FNIP1/2 (16Péli-Gulli M.-P. Sardu A. Panchaud N. Raucci S. De Virgilio C. Amino acids stimulate TORC1 through Lst4-Lst7, a GTPase-activating protein complex for the Rag family GTPase Gtr2.Cell Rep. 2015; 13: 1-7Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar, 17Pacitto A. Ascher D.B. Wong L.H. Blaszczyk B.K. Nookala R.K. Zhang N. Dokudovskaya S. Levine T.P. Blundell T.L. Lst4, the yeast Fnip1/2 orthologue, is a DENN-family protein.Open Biol. 2015; 5: 150174Crossref PubMed Scopus (23) Google Scholar); the latter has been found to form a complex with FLCN in most (if not all) functions of FLCN (18Baba M. Hong S.B. Sharma N. Warren M.B. Nickerson M.L. Iwamatsu A. Esposito D. Gillette W.K. Hopkins 3rd R.F. Hartley J.L. Furihata M. Oishi S. Zhen W. Burke Jr T.R. Linehan W.M. et al.Folliculin encoded by the BHD gene interacts with a binding protein, FNIP1, and AMPK, and is involved in AMPK and mTOR signaling.Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 15552-15557Crossref PubMed Scopus (358) Google Scholar, 19Hasumi H. Baba M. Hong S.B. Hasumi Y. Huang Y. Yao M. Valera V.A. Linehan W.M. Schmidt L.S. Identification and characterization of a novel folliculin-interacting protein FNIP2.Gene. 2008; 415: 60-67Crossref PubMed Scopus (136) Google Scholar, 20Takagi Y. Kobayashi T. Shiono M. Wang L. Piao X. Sun G. Zhang D. Abe M. Hagiwara Y. Takahashi K. Hino O. Interaction of folliculin (Birt-Hogg-Dubé gene product) with a novel Fnip1-like (FnipL/Fnip2) protein.Oncogene. 2008; 27: 5339-5347Crossref PubMed Scopus (103) Google Scholar, 21Hasumi H. Baba M. Hasumi Y. Lang M. Huang Y. Oh H.F. Matsuo M. Merino M.J. Yao M. Ito Y. Furuya M. Iribe Y. Kodama T. Southon E. Tessarollo L. et al.Folliculin-interacting proteins Fnip1 and Fnip2 play critical roles in kidney tumor suppression in cooperation with Flcn.Proc. Natl. Acad. Sci. U. S. A. 2015; 112: E1624-E1631Crossref PubMed Scopus (52) Google Scholar). Both LST7 and LST4 had been believed to be components of specific transport machinery (14Roberg K.J. Bickel S. Rowley N. Kaiser C.A. Control of amino-acid permease sorting in the late secretory pathway of Saccharomyces cerevisiae by SEC13, LST4, LST7, and LST8.Genetics. 1997; 147: 1569-1584Crossref PubMed Google Scholar), but the precise mechanisms remained unknown. In 2012, a structural study uncovered a differentially expressed in normal cells and neoplasia (DENN) domain in the FLCN C terminus (22Nookala R.K. Langemeyer L. Pacitto A. Ochoa-Montaño B. Donaldson J.C. Blaszczyk B.K. Chirgadze D.Y. Barr F.A. Bazan J.F. Blundell T.L. Crystal structure of folliculin reveals a hidDENN function in genetically inherited renal cancer.Open Biol. 2012; 2: 120071Crossref PubMed Scopus (85) Google Scholar). Because some DENN-containing proteins are activators of Rab proteins (Rabs), which are key regulators of vesicular trafficking, FLCN has been suspected to regulate protein transport via Rabs (22Nookala R.K. Langemeyer L. Pacitto A. Ochoa-Montaño B. Donaldson J.C. Blaszczyk B.K. Chirgadze D.Y. Barr F.A. Bazan J.F. Blundell T.L. Crystal structure of folliculin reveals a hidDENN function in genetically inherited renal cancer.Open Biol. 2012; 2: 120071Crossref PubMed Scopus (85) Google Scholar). Shortly after this discovery, two studies demonstrated that FLCN regulates the intracellular transport of EGFR. In one study (23Zheng J. Duan B. Sun S. Cui J. Du J. Zhang Y. Folliculin interacts with Rab35 to regulate EGF-induced EGFR degradation.Front. Pharmacol. 2017; 8: 688Crossref PubMed Scopus (10) Google Scholar), FLCN was found to promote the movement of EGFR from early endosomes to the cell surface by acting as a guanine nucleotide exchange factor (GEF) of Rab35; in the other study, FLCN was found to be a GTPase-activating protein (GAP) of Rab7 and to prevent the transport of EGFR from early to late endosomes (24Laviolette L.A. Mermoud J. Calvo I.A. Olson N. Boukhali M. Steinlein O.K. Roider E. Sattler E.C. Huang D. Teh B.T. Motamedi M. Haas W. Iliopoulos O. Negative regulation of EGFR signalling by the human folliculin tumour suppressor protein.Nat. Commun. 2017; 8: 15866Crossref PubMed Scopus (25) Google Scholar). At almost the same time, our group found that the amino acid permease PAT1 (also called slc36A1) is a cargo protein of FLCN (9Wu X. Zhao L. Chen Z. Ji X. Qiao X. Jin Y. Liu W. FLCN maintains the leucine level in lysosome to stimulate mTORC1.PLoS One. 2016; 11e0157100Crossref PubMed Scopus (18) Google Scholar). We further demonstrated that FLCN promotes the localization of PAT1 on the plasma membrane and inhibits its localization on lysosomes through the Rab11A-mediated pathway (25Zhao L. Ji X. Zhang X. Li L. Jin Y. Liu W. FLCN is a novel Rab11A-interacting protein that is involved in the Rab11A-mediated recycling transport.J. Cell Sci. 2018; 131jcs218792Crossref PubMed Scopus (5) Google Scholar). This relocalization of PAT1 helps cells to acquire amino acids from the environment while maintaining a robust lysosomal amino acid pool that stimulates mTOR. This function of FLCN seems to be conserved in yeast (14Roberg K.J. Bickel S. Rowley N. Kaiser C.A. Control of amino-acid permease sorting in the late secretory pathway of Saccharomyces cerevisiae by SEC13, LST4, LST7, and LST8.Genetics. 1997; 147: 1569-1584Crossref PubMed Google Scholar), fly (26Liu W. Chen Z. Ma Y. Wu X. Jin Y. Hou S. Genetic characterization of the Drosophila Birt-Hogg-Dube syndrome gene.PLoS One. 2013; 8e65869Crossref PubMed Scopus (16) Google Scholar) and human cells (25Zhao L. Ji X. Zhang X. Li L. Jin Y. Liu W. FLCN is a novel Rab11A-interacting protein that is involved in the Rab11A-mediated recycling transport.J. Cell Sci. 2018; 131jcs218792Crossref PubMed Scopus (5) Google Scholar). Regarding the underlying mechanism, we did not find clear GEF or GAP activity of FLCN toward Rab11A. Instead, we found that FLCN promotes the binding of PAT1 to Rab11A in a dose-dependent manner (25Zhao L. Ji X. Zhang X. Li L. Jin Y. Liu W. FLCN is a novel Rab11A-interacting protein that is involved in the Rab11A-mediated recycling transport.J. Cell Sci. 2018; 131jcs218792Crossref PubMed Scopus (5) Google Scholar). Through a similar mechanism, FLCN has been found to promote the interaction between Rab34 and its effector, RILP, during the positioning of lysosomes (27Starling G.P. Yip Y.Y. Sanger A. Morton P.E. Eden E.R. Dodding M.P. Folliculin directs the formation of a Rab34–RILP complex to control the nutrient-dependent dynamic distribution of lysosomes.EMBO Rep. 2016; 17: e201541382-e201541841Crossref Scopus (54) Google Scholar). Based on these findings, one appealing hypothesis is that the diversified BHD phenotypes might be due to the abnormal localization of specific FLCN substrates. In the current study, we identified transferrin receptor 1 (TfR1, or CD71) as a new substrate of FLCN and thus linked FLCN to iron metabolism. Iron is an essential nutrient for many biological processes, such as oxygen delivery and storage, DNA metabolism, and energy production. Both iron deficiency and iron overload can cause pathological changes (28Muckenthaler M.U. Rivella S. Hentze M.W. Galy B. A red carpet for iron metabolism.Cell. 2017; 168: 344-361Abstract Full Text Full Text PDF PubMed Scopus (476) Google Scholar). TfR1 is a ubiquitously expressed membrane protein. Most mammalian cells obtain iron from plasma via TfR1. Most Fe3+ ions in the plasma are loaded on transferrin (Tf), a ferric iron carrier produced mainly in the liver. The Tf-Fe3+ complex (called holo-Tf) binds to TfR1 and then enters the cell through membrane invagination. Once Fe3+ is released inside the cell, free Tf-TfR1 is sent back to the cell surface for another round of iron uptake. Blocking the cellular trafficking of TfR1 inhibits iron uptake and causes iron deficiency disorders (29Lim J.E. Jin O. Bennett C. Morgan K. Wang F. Trenor 3rd C.C. Fleming M.D. Andrews N.C. A mutation in Sec15l1 causes anemia in hemoglobin deficit (hbd) mice.Nat. Genet. 2005; 37: 1270-1273Crossref PubMed Scopus (77) Google Scholar, 30Chen C. Garcia-Santos D. Ishikawa Y. Seguin A. Li L. Fegan K.H. Hildick-Smith G.J. Shah D.I. Cooney J.D. Chen W. King M.J. Yien Y.Y. Schultz I.J. Anderson H. Dalton A.J. et al.Snx3 regulates recycling of the transferrin receptor and iron assimilation.Cell Metab. 2013; 17: 343-352Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). On the other hand, high iron can be toxic by generating deleterious reactive oxygen species (ROS). Therefore, cells have evolved sophisticated mechanisms to control the iron pool (31Kawabata H. Transferrin and transferrin receptors update.Free Radic. Biol. Med. 2019; 133: 46-54Crossref PubMed Scopus (142) Google Scholar). The promoter region of the TfR1 gene contains a hypoxia response element (HRE). Hypoxia-inducible factor (HIF) family transcription factors bind this HRE and directly activate TfR1 transcription (32Bianchi L. Tacchini L. Cairo G. HIF-1-mediated activation of transferrin receptor gene transcription by iron chelation.Nucleic Acids Res. 1999; 27: 4223-4227Crossref PubMed Scopus (145) Google Scholar). Via a feedback mechanism, iron deficiency can stabilize the HIF protein by inactivating prolyl hydroxylases (PHDs), which utilize iron as a cofactor to target HIF for degradation (33Wang G.L. Semenza G.L. Desferrioxamine induces erythropoietin gene expression and hypoxia-inducible factor 1 DNA-binding activity: Implications for models of hypoxia signal transduction.Blood. 1993; 82: 3610-3615Crossref PubMed Google Scholar). The 3’ noncoding region of TfR1 mRNA forms stem-loop structures called iron-responsive elements (IREs). Iron regulatory proteins (IRP1 and IRP2) bind these IREs and prevent degradation of the mRNA (34Owen D. Kühn L.C. Noncoding 3’ sequences of the transferrin receptor gene are required for mRNA regulation by iron.EMBO J. 1987; 6: 1287-1293Crossref PubMed Scopus (167) Google Scholar). A robust iron pool increases iron–sulfur cluster production. The binding of IPRs with iron–sulfur clusters either converts IRP1 into a functional aconitase, which translocates to mitochondria to catalyze respiratory chain reactions, or directs IRP2 for degradation (35Anderson C.P. Shen M. Eisenstein R.S. Leibold EA Mammalian iron metabolism and its control by iron regulatory proteins.Biochim. Biophys. Acta. 2012; 1823: 1468-1483Crossref PubMed Scopus (316) Google Scholar, 36Kühn L.C. Iron regulatory proteins and their role in controlling iron metabolism.Metallomics. 2015; 7: 232-243Crossref PubMed Google Scholar). TfR1 can be regulated at the protein level. The TfR1 protein is constantly degraded in lysosomes (37Matsui T. Itoh T. Fukuda M. Small GTPase Rab12 regulates constitutive degradation of transferrin receptor.Traffic. 2011; 12: 1432-1443Crossref PubMed Scopus (62) Google Scholar, 38Raiborg C. Bache K.G. Gillooly D.J. Madshus I.H. Stang E. Stenmark H. Hrs sorts ubiquitinated proteins into clathrin-coated microdomains of early endosomes.Nat. Cell Biol. 2002; 4: 394-398Crossref PubMed Scopus (559) Google Scholar, 39Fujita H. Iwabu Y. Tokunaga K. Tanaka Y. Membrane-associated RING-CH (MARCH) 8 mediates the ubiquitination and lysosomal degradation of the transferrin receptor.J. Cell Sci. 2013; 126: 2798-2809Crossref PubMed Scopus (46) Google Scholar). Depletion of iron by iron chelators such as desferrioxamine (DFO) increases the TfR1 protein level, but the mechanism is not fully understood (40Tong X. Kawabata H. Koeffler H.P. Iron deficiency can upregulate expression of transferrin receptor at both the mRNA and protein level.Br. J. Haematol. 2002; 116: 458-464Crossref PubMed Scopus (35) Google Scholar). Here, we report that FLCN regulates TfR1 transport through the Rab11A-mediated pathway. Loss of FLCN delays the recycling of TfR1 and decreases the iron pool. These findings provide new insights for deciphering the mechanisms underlying BHD syndrome. In a previous study, we carried out coimmunoprecipitation (co-IP) assays and discovered that FLCN promotes the binding of PAT1 to Rab11A (25Zhao L. Ji X. Zhang X. Li L. Jin Y. Liu W. FLCN is a novel Rab11A-interacting protein that is involved in the Rab11A-mediated recycling transport.J. Cell Sci. 2018; 131jcs218792Crossref PubMed Scopus (5) Google Scholar). To determine whether FLCN specifically regulates PAT1, we also examined TfR1, which is continuously transported by Rab11A. Surprisingly, we found that FLCN also bound to TfR1, and loss of FLCN decreased the TfR1-Rab11A interaction (Fig. 6D in (25Zhao L. Ji X. Zhang X. Li L. Jin Y. Liu W. FLCN is a novel Rab11A-interacting protein that is involved in the Rab11A-mediated recycling transport.J. Cell Sci. 2018; 131jcs218792Crossref PubMed Scopus (5) Google Scholar)). However, these data were obtained in cells with GFP-Rab11A overexpression. To eliminate the potential artifacts caused by GFP-Rab11A, we repeated these assays in normal HEK293 cells using a monoclonal anti-TfR1 antibody for the precipitation experiment. Under steady-state conditions, TfR1 showed little interaction with endogenous Rab11A or FLCN (Fig. 1A), probably because under physiological conditions, only a small amount of TfR1 is transported by Rab11A and FLCN. Then, we decided to induce iron deficiency before the assay, based on the following two considerations. First, low iron levels can increase TfR1 expression (31Kawabata H. Transferrin and transferrin receptors update.Free Radic. Biol. Med. 2019; 133: 46-54Crossref PubMed Scopus (142) Google Scholar); thus, co-IP would be easier to perform under these conditions. Second, we speculated that low iron levels may stimulate iron uptake by enhancing the TfR1-Rab11A interaction. To decrease the iron pool, we incubated cells with DFO, which is a clinically used iron chelator (41Chaston T.B. Richardson D.R. Iron chelators for the treatment of iron overload disease: Relationship between structure, redox activity, and toxicity.Am. J. Hematol. 2003; 73: 200-210Crossref PubMed Scopus (142) Google Scholar). We detected both Rab11A and FLCN in the TfR1 precipitates from cells treated with 100 μM DFO for 12 h. Moreover, the amount of TfR1-bound Rab11A was decreased in FLCN−/− cells (Fig. 1B). These results demonstrate that FLCN promotes the TfR1-Rab11A interaction, particularly under the iron-deficient conditions. FLCN has been found to bind to different Rabs via its C-terminal DENN domain (23Zheng J. Duan B. Sun S. Cui J. Du J. Zhang Y. Folliculin interacts with Rab35 to regulate EGF-induced EGFR degradation.Front. Pharmacol. 2017; 8: 688Crossref PubMed Scopus (10) Google Scholar, 24Laviolette L.A. Mermoud J. Calvo I.A. Olson N. Boukhali M. Steinlein O.K. Roider E. Sattler E.C. Huang D. Teh B.T. Motamedi M. Haas W. Iliopoulos O. Negative regulation of EGFR signalling by the human folliculin tumour suppressor protein.Nat. Commun. 2017; 8: 15866Crossref PubMed Scopus (25) Google Scholar, 25Zhao L. Ji X. Zhang X. Li L. Jin Y. Liu W. FLCN is a novel Rab11A-interacting protein that is involved in the Rab11A-mediated recycling transport.J. Cell Sci. 2018; 131jcs218792Crossref PubMed Scopus (5) Google Scholar, 27Starling G.P. Yip Y.Y. Sanger A. Morton P.E. Eden E.R. Dodding M.P. Folliculin directs the formation of a Rab34–RILP complex to control the nutrient-dependent dynamic distribution of lysosomes.EMBO Rep. 2016; 17: e201541382-e201541841Crossref Scopus (54) Google Scholar). It is thus conceivable that FLCN might bind to TfR1 via a region other than its DENN domain. To test this hypothesis, we constructed plasmids expressing different forms of FLCN proteins, including wild-type FLCN (1–579), the N-terminal region (1–340), and the DENN domain (341–579), fused to a C-terminal HA tag. To exclude the influence of endogenous FLCN, we transfected these plasmids into FLCN−/− cells. Twenty-four hours after plasmid transfection, the cells were deprived of iron by incubation with 100 μM DFO for 12 h. The cell lysates were precipitated with an anti-HA antibody, followed by western blot (WB) analysis. As anticipated, both FLCN and its N-terminal region bound to TfR1; however, there was only a weak interaction between TfR1 and the FLCN DENN domain (Fig. 1C). We used a fluorescence conjugated transferrin (FITC-Tf) to monitor the trafficking of TfR1. Cells were incubated with 20 μg/ml FITC-Tf on ice for 20 min. At low temperatures, Tf still bound to TfR1 on the cell surface, but endocytosis was terminated. After the unbound FITC-Tf was washed away, the cells were transferred to 37 °C to reinitiate endocytosis. When the cells were transferred to 37 °C for 5 min, we observed similar levels of FITC-Tf in both wild-type and FLCN−/− cells. In addition, FITC-Tf translocated to an intracellular location very similar to the perinuclear recycling center (PRC, arrows in Fig. 2A), where Rab11A normally accumulates (42Grant B.D. Donaldson J.G. Pathways and mechanisms of endocytic recycling.Nat. Rev. Mol. Cell Biol. 2009; 10: 597-608Crossref PubMed Scopus (946) Google Scholar, 43Stenmark H. Rab GTPases as coordinators of vesicle traffic.Nat. Rev. Mol. Cell Biol. 2009; 10: 513-525Crossref PubMed Scopus (2137) Google Scholar). This finding implies that FLCN has little influence on the translocation of TfR1 from the cell surface into the cell. After membrane trafficking was reinitiated by incubation at 37 °C for 50 min, the level of FITC-Tf was markedly decreased in wild-type cells (Fig. 2A), but a substantial amount of FITC-Tf was retained in FLCN−/− cells (Fig. 2, A and B), suggesting that loss of FLCN delayed the recycling of TfR1. To examine the uptake rate of Tf-bound iron, we employed a recently developed method using calcein-AM, a membrane-permeable, fluorescent iron probe whose fluorescence is quenched by binding with iron. Cells were first stained with 0.4 μM calcein-AM for 10 min and then incubated with 10 μg/ml holo-Tf for 3 h. After holo-Tf enters the cells and releases the Tf-bound iron, the intracellular calcein-AM signal decreases. The calcein signal can be measured by flow cytometric analysis, and the reduction in the signal after incubation with holo-Tf (representing the quenchable iron pool, QIP) correlates with the amount of holo-Tf taken up by the cells (44Riedelberger M. Kuchler K. Analyzing the quenchable iron pool in murine macrophages by flow cytometry.Bio-protocol. 2020; 10e3552Crossref PubMed Google Scholar). The results revealed that the QIP was decreased in FLCN−/− cells, indicating reduced uptake of holo-Tf (Fig. 2C). We carried out a colorimetric ferrozine-based assay to measure the total cellular iron concentration directly. Compared with wild-type cells, FLCN−/− cells exhibited a trend of reduced total iron levels (Fig. 3A), although the difference was not statistically significant (p = 0.136, based on three repeated experiments). We used calcein-AM staining to assess the labile iron pool. FLCN−/− cells had stronger calcein signals than wild-type cells, suggesting that the labile iron pool was decreased by FLCN loss (Fig. 3B). When the iron pool is depleted, the cellular ferritin is degraded to release stored iron. Indeed, the level of FTH, a component of the ferritin protein complex, was decreased in FLCN−/− cells (Fig. 3C). In response to iron deficiency, the expression of iron assimilation genes, including TfR1 and the ferrous iron (Fe2+) transporter, also called divalent metal transporter 1 (DMT1, or SLC11A2), increases. These increases can be achieved through multiple mechanisms, including transcriptional activation by HIF (32Bianchi L. Tacchini L. Cairo G. HIF-1-mediated activation of transferrin receptor gene transcription by iron chelation.Nucleic Acids Res. 1999; 27: 4223-4227Crossref PubMed Scopus (145) Google Scholar, 45Wang D. Wang L.H. Zhao Y. Lu Y.P. Zhu L. Hypoxia regulates the ferrous iron uptake and reactive oxygen species level via divalent metal transporter 1 (DMT1) Exon1B by hypoxia-inducible factor-1.IUBMB Life. 2010; 62: 629-636Crossref PubMed Scopus (29) Google Scholar) and inhibition of mRNA degradation by the IRP-IRE system (34Owen D. Kühn L.C. Noncoding 3’ sequences of the transferrin receptor gene are required for mRNA regulation by iron.EMBO J. 1987; 6: 1287-1293Crossref PubMed Scopus (167) Google Scholar). The mRNA levels of these genes can be used as indicators of the iron status. We performed real-time PCR (RT-PCR) to measure mRNA levels. A control assay showed that deprivation of iron by DFO chelation increased the expression of both TfR1 and DMT1 (Fig. 3D). Importantly, loss of FLCN produced a similar result but to a lesser extent than DFO treatment, suggesting that FLCN deficiency is less potent than DFO in decreasing the iron pool. Consistent with previous reports that FLCN inhibits the transcriptional coactivator PGC-1α (12Yan M. Audet-Walsh É. Manteghi S. Dufour C.R. Walker B. Baba M. St-Pierre J. Giguère V. Pause A. Chronic AMPK activation via loss of FLCN induces functional beige adipose tissue through PGC-1alpha/ERRalpha.Genes Dev. 2016; 30: 1034-1046Crossref PubMed Scopus (65) Google Scholar, 13Hasumi Y. Baba M. Hasumi H. Huang Y. Lang M. Reindorf R. Oh H.B. Sciarretta S. Nagashima K. Haines D.C. Schneider M.D. Adelstein R.S. Schmidt L.S. Sadoshima J. Linehan M.W. Folliculin (Flcn) inactivation leads to murine cardiac hypertrophy through mTORC1 deregulation.Hum. Mol. Genet. 2014; 23: 5706-5719Crossref PubMed Scopus (41) Google Scholar, 46Klomp J.A. Petillo D. Niemi N.M. Dykema K.J. Chen J. Yang X.J. Sääf A. Zickert P. Aly M. Bergerheim U. Nordenskjöld M. Gad S. Giraud S. Denoux Y. Yonneau L. et al.Birt-Hogg-Dubé renal tumors are genetically distinct from other renal neoplasias and are associated with up-regulation of mitochondrial gene expression.BM" @default.
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• W3131707355 title "FLCN regulates transferrin receptor 1 transport and iron homeostasis" @default.
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