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Haploinsufficiency of the Folliculin Gene Leads to Impaired Functions

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Haploinsufficiency of the Folliculin Gene Leads to Impaired Functions Physiological Reports ISSN 2051-817X ORIGINAL RESEARCH Haploinsufficiency of the folliculin gene leads to impaired functions of lung fibroblasts in patients with Birt–Hogg–Dube syndrome Yoshito Hoshika1,2, Fumiyuki Takahashi1, Shinsaku Togo1, Muneaki Hashimoto3, Takeshi Nara3, Toshiyuki Kobayashi4, Fariz Nurwidya1, Hideyuki Kataoka2,5, Masatoshi Kurihara2,5, Etsuko Kobayashi1,2, Hiroki Ebana1,2, Mika Kikkawa6, Katsutoshi Ando1,2, Koichi Nishino1, Okio Hino4, Kazuhisa Takahashi1 & Kuniaki Seyama1,2 1 Divisions of Respiratory Medicine, Juntendo University Faculty of Medicine and Graduate School of Medicine, Tokyo, Japan 2 The Study Group of Pneumothorax and Cystic Lung Diseases, Tokyo, Japan 3 Department of Molecular and Cellular Parasitology, Juntendo University School of Medicine, Tokyo, Japan 4 Department of Pathology and Oncology, Juntendo University School of Medicine, Tokyo, Japan 5 Pneumothorax Research Center and Division of Thoracic Surgery, Nissan Tamagawa Hospital, Tokyo, Japan 6 Biomedical Research Center, Laboratory of Proteomics and Biomolecular Science, Juntendo University Faculty of Medicine and Graduate School of Medicine, Tokyo, Japan Keywords Abstract Chemotaxis, fibroblast, folliculin, – – haploinsufficiency, TGF-b1. Birt Hogg Dube syndrome (BHDS) is an autosomal dominant inherited disorder caused by germline mutations in the FLCN gene, and characterized Correspondence by skin fibrofolliculomas, multiple lung cysts, spontaneous pneumothorax, Kuniaki Seyama, Division of Respiratory and renal neoplasms. Pulmonary manifestations frequently develop earlier Medicine, Juntendo University Faculty of than other organ involvements, prompting a diagnosis of BHDS. However, Medicine & Graduate School of Medicine, the mechanism of lung cyst formation and pathogenesis of pneumothorax 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 Japan. have not yet been clarified. Fibroblasts were isolated from lung tissues n = n = Tel: (+81) 3-5802-1063 obtained from patients with BHDS ( 12) and lung cancer ( 10) as con- Fax: (+81) 3-5802-1617 trols. The functional abilities of these lung fibroblasts were evaluated by the E-mail: [email protected] tests for chemotaxis to fibronectin and three-dimensional (3-D) gel contrac- tion. Fibroblasts from BHDS patients showed diminished chemotaxis as com- Funding Information pared with fibroblasts from controls. Expression of fibronectin and TGF-b1 This study was supported in part by: Grant- was significantly reduced in BHDS fibroblasts when assessed by qPCR. Addi- in-Aid for Scientific Research No. 22790767 b and No. 24591177 (Mika Kikkawa) from the tion of TGF- 1 in culture medium of BHDS lung fibroblasts significantly Ministry of Education, Culture, Sports, restored these cells’ abilities of chemotaxis and gel contraction. Human fetal Science, and Technology; the High lung fibroblasts (HFL-1) exhibited reduced chemotaxis and 3-D gel contrac- Technology Research Center Grant from the tion when FLCN expression was knocked down. To the contrary, a significant Ministry of Education, Culture, Sports, increase in chemotactic activity toward to fibronectin was demonstrated when Science and Technology; and the Institute for wild-type FLCN was overexpressed, whereas transduction of mutant FLCN Environmental and Gender-Specific Medicine, showed no effect on chemotaxis. Our results suggest that FLCN is associated Juntendo University Graduate School of with chemotaxis in lung fibroblasts. Together with reduced TGF-b1 expression Medicine. by BHDS lung fibroblasts, a state of FLCN haploinsufficiency may cause lung Received: 27 September 2016; Revised: 7 fibroblast dysfunction, thereby impairing tissue repair. These may reveal one October 2016; Accepted: 10 October 2016 mechanism of lung cyst formation and pneumothorax in BHDS patients. doi: 10.14814/phy2.13025 Physiol Rep, 4 (21), 2016, e13025, doi: 10.14814/phy2.13025 ª 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of 2016 | Vol. 4 | Iss. 21 | e13025 The Physiological Society and the American Physiological Society. Page 1 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Lung Fibroblast Functions in BHD Syndrome Y. Hoshika et al. Introduction gels. Our aims were to evaluate the functions of primary cultured lung fibroblasts in BHDS and then to clarify Birt–Hogg–Dube syndrome (BHDS) is an autosomal how a state of haploinsufficiency of the FLCN gene affects dominant inherited disorder caused by germline muta- lung fibroblasts. tions in the FLCN (folliculin) gene. The distinguishing features of BHDS are skin fibrofolliculomas, multiple lung Materials and Methods cysts, spontaneous pneumothorax, and renal neoplasms (Birt et al. 1977). The FLCN gene is located on chromo- Study population some 17p11.2 and encodes a 64 kDa protein called folli- culin with no characteristic functional domains Twelve patients with BHDS and 10 patients with lung (Nickerson et al. 2002). Folliculin forms a complex with cancer as a control for BHDS were included in this study novel folliculin-interacting proteins 1 and 2 (FNIP1 and (Table 1). All BHDS subjects had undergone surgery for FNIP2), and 50-AMP-activated protein kinase (AMPK), pneumothorax, and all subjects with lung cancer were an important energy sensor in cells that negatively regu- operated for tumor resection. Portion of resected lung tis- lates mechanistic target of rapamycin (mTOR) (Baba sues were used for a primary culture of lung fibroblasts as et al. 2006; Takagi et al. 2008). The FLCN gene is thought described below. Each subject provided written, informed to be a tumor suppressor because a second somatic muta- consent for the acquisition of material for research. This tion inactivating wild-type FLCN is demonstrated in study was approved by institutional ethics committee at BHDS-associated renal tumors (Vocke et al. 2005). How- Juntendo University and Tamawaga hospital. ever, fibrofolliculomas from BHDS patients do not neces- sarily have FLCN loss of heterozygosity (LOH), meaning that tumor-like lesions of the hair follicle are evoked by a Isolation and primary culture of lung haploinsufficiency of FLCN (van Steensel et al. 2007). fibroblasts Similarly, lung cysts are likely to develop because of hap- Human fetal lung fibroblasts (HFL-1) were purchased loinsufficiency status of the FLCN gene since no neoplas- from the American Type Culture Collection (CCL-153, tic cells have been identified in cyst walls or throughout Manassas, VA). The human lung parenchymal fibroblasts lungs. Accordingly, we theorized that investigating the were cultured from the resected lung tissues of patients effect exerted by haploinsufficiency of the FLCN gene on with BHDS (BHDS lung fibroblasts) and patients with the cellular function of lungs would disclose pathogenesis lung cancer (control lung fibroblasts), using the method of lung cyst and pneumothorax that underlie BHDS. by Holz et al. (2004). In patients with lung cancer, only Lung fibroblasts are considered crucial for maintaining lungs without visible or palpable lung metastases were the integrity of alveolar structures by producing extracel- used to avoid isolating cancer-associated fibroblasts. lular matrix proteins required for attachment, structure, Briefly, the portion of lung parenchymal tissue that was and function of alveolar epithelial cells (Dunsmore and free of the pleural surface was minced and placed in cul- Rannels 1996). Warren et al. demonstrated that FLCN ture with Dulbecco’s modified eagle’s medium (DMEM) mRNA was strongly expressed in stromal cells within the supplemented with 10% FCS, 100 lg/mL penicillin, and connective tissue (Warren et al. 2004). After histopatho- 250 lg/mL streptomycin (complete medium) in a humid- logical and morphometric analysis of the BHDS cysts, we ified atmosphere of 5% CO2 and passaged every 4–5 days recently reported that pulmonary cysts are likely to at 1:4 ratios. They were used for chemotaxis and three- develop in the periacinar region, an anatomically weak dimensional (3-D) collagen gel contraction assays at the site in a primary lobule, where alveoli attach to connec- fourth to fifth passages after isolation to exclude the effect tive tissue septa (Kumasaka et al. 2014). Additionally, of differences in passage number. characteristic distribution of the BHDS cysts in the basilar medial and lateral regions on CT of the chest (Tobino FLCN et al. 2011) may imply that shear stress imposed by respi- Mutation analysis of the gene and LOH FLCN ratory movement of the lungs and heartbeats participates analysis of the gene-associated region in cyst formation. Accordingly, we hypothesized that Genomic DNA isolated from peripheral blood leukocytes impaired tissue repair responses or fragility of the lungs was utilized to identify a germline FLCN mutation due to FLCN haploinsufficiency are involved in BHD cyst according to the method described previously (Gunji formation. To assess these possibilities, we performed two et al. 2007). Briefly, each exon of the FLCN gene was sep- functional-phenotype assays: fibroblast chemotaxis and arately amplified and then screened by denaturing high- fibroblast contraction of three-dimensional (3-D) collagen performance liquid chromatography. When a mobility 2016 | Vol. 4 | Iss. 21 | e13025 ª 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of Page 2 The Physiological Society and the American Physiological Society. Y. Hoshika et al. Lung Fibroblast Functions in BHD Syndrome Table 1. Patient characteristics of study population with BHDS. Patient no. Age Gender Exon Nucleotide change No. and location of PTX Lung cysts Skin lesions Renal tumors 1 38 M 4 c.119delG R(5) + ÀÀ 2 39 F 7 c.769_771delTCC R(5)L(1) + ÀÀ 3 48 F 9 c.932_933delCT R(2)L(1) + + À 4 38 M 11 c.1285dupC R(10)L(5) + ÀÀ 5 43 F 11 c.1285dupC R(1)L(1) + ÀÀ 6 50 M 11 c.1285dupC R(2)L(1) + ÀÀ 7 39 M 11 c.1285dupC R(2) + ÀÀ 8 62 F 11 c.1285dupC R(3) + ÀÀ 9 61 M 11 c.1285dupC L(6) + ÀÀ 10 57 F 12 c.1347_1353dupCCACCCT R(3)L(2) + ÀÀ 11 34 M 13 c.1533_1536delGATA R(3)L(1) + ÀÀ 12 38 M 13 c.1533_1536delGATA R(3) + ÀÀ No, number; PTX, pneumothorax.
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