DOI: 10.5061/dryad.6djh9w19q
title: Cigarette smoking is a secondary cause of folliculin loss

description: Background: Birt-Hogg-Dubé syndrome (BHD) is a clinical syndrome
 manifesting with cystic lung disease and pneumothorax. Features of BHD
 result from the loss-of-function mutations of the folliculin (FLCN) gene.
 Chronic obstructive pulmonary disease (COPD), characterised by an
 irreversible airflow limitation, is primarily caused by cigarette smoking.
 Objective: Given that COPD often shares structural features with
 BHD, we investigated the link between COPD, cigarette smoke (CS) exposure
 and FLCN expression.MethodsWe measured the expression of FLCN in human
 COPD lungs and CS-exposed mouse lungs, as well as in CS extract
 (CSE)-exposed immortalised human airway epithelial cells by
 immunoblotting. Results: We found that the lung FLCN protein
 levels in smokers with COPD and CS exposure mice exhibit a marked decrease
 compared with smokers without COPD and room air exposure mice,
 respectively. We confirmed CS induced degradation of FLCN in immortalised
 human bronchial epithelial Beas-2B cells via ubiquitin proteasome system.
 Further, siRNA targeting FLCN enhanced CSE-induced cytotoxicity. By
 contrast, FLCN overexpression protected cells from CSE-induced
 cytotoxicity. We found that FBXO23, the ubiquitin E3 ligase subunit,
 specifically binds to and targets FLCN for degradation. Inhibition of ATM
 (ataxia‐telangiectasia mutated) attenuated CSE induced FLCN degradation,
 suggesting a role of ATM in FLCN proteolysis. We further confirmed that
 the mutant of major FLCN phosphorylation site serine 62A is resistant to
 CSE-induced degradation and cytotoxicity. Conclusions: Our study
 demonstrates that CS exposure is a secondary cause of FLCN deficiency due
 to the enhanced proteolysis, which promoted airway epithelial cell death.
description: # Cigarette smoking is a secondary cause of folliculin loss
 [https://doi.org/10.5061/dryad.6djh9w19q](https://doi.org/10.5061/dryad.6djh9w19q) ## Description of the data and file structure 1.     The project’s title: Smoking is a secondary cause of folliculin loss 2.     Grant information: American Heart Association (19TPA34830061); NHLBI R01 HL149719 3.     Links/information to other repository data: NA 4.      Dataset contents a.    All stat analysis data (FLCN-Figures_Stat_Analysis.pzfx):  For statistical analysis, we used GraphPad Prism 9 software. Viewing and using the PRISM files requires access to this software, which must be installed on your device. For comparisons between two groups, we conducted an unpaired t-test. For comparisons among multiple groups, we applied one-way ANOVA with Bonferroni correction. In this prism stat file there are a total of 12 tables as below.  1. Fig 1B human-protein: Relative Fold Difference of FLCN/GAPDH mRNA levels in the lungs of control smokers (n=5) vs. COPD subjects (n=6). A student t test was used for the comparison. 2. Fig 1C human-mRNA: Relative Densitometry of Folliculin (FLCN)/β-actin protein expression in the lungs of mice exposed to cigarette smoke (CS) or room air (RA) for 6 months (n=4 per group). An unpaired t test was used for the comparison. 3. Fig 2B mice-protein: Relative Densitometry of Folliculin (FLCN)/β-actin protein expression in the lungs of mice exposed to cigarette smoke (CS) or room air (RA) for 6 months. A student t test was used for the comparison. 4. Fig 2C mice-mRNA:  Relative Fold Difference of FLCN/GAPDH mRNA levels in the lungs of control mice exposed to cigarette smoke (CS) or room air (RA) for 6 months (n=4 per group). An unpaired t test was used for the comparison. 5. Fig 3D: FLCN protein CSE time course: BEAS-2B cells were treated with 2% CSE for up to 8 hours and analyzed for FLCN or β-actin by immunoblotting. The densitometry data (FLCN/β-Actin) obtained from (C) are expressed as mean±SEM. 6. Fig 3E: FLCN mRNA CSE time course:  Total RNA was prepared from BEAS-2B cells treated with 2% CSE for up to 8 hours. Levels of FLCN mRNA were measured by qPCR. The relative fold difference compared with GAPDH (control) was expressed. Data are expressed as mean±SEM. 7. Fig 3G: Cell viability was measured by MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay in BEAS-2B cells were transfected with FLCN siRNA (siFLCN) or scrambled siRNA (siCtrl) for 48 hours and further cultured with or without 2% of CSE for 24 hours. Data are expressed as mean±SEM for three independent experiments with triplicate samples. A one-way analysis of variance with Bonferroni’s correction was used for the comparison between siFLCN with CSE and siCtrl with CSE. 8. Fig 3J: Cell viability was measured by MTT (3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide) assay in BEAS-2B cells were transfected with HA-tagged FLCN (FLCN) or empty vector (vector) for 24 hours and further cultured with or without 2% of CSE for 24 hours. Data are expressed as mean±SEM for three independent experiments with triplicate samples. A one-way analysis of variance with Bonferroni’s correction was used for the comparison between vector with CSE and FLCN with CSE. 9. Fig 4B Half life: BEAS-2B cells were treated with CHX (20 µg/mL) or leupeptin (20 µM) or MG132 (40 µM) for indicated times. Cell lysates were subjected to FLCN and β-actin immunoblotting. The densitometry data (FLCN/β-Actin) are expressed as mean±SEM.  10. Fig 6E WT-S62A-CSE: HA-tagged FLCN (WT) or S62A mutant plasmid was transfected into BEAS-2B cells for 24 hours. The cells were treated with 2% CSE for up to 8 hours. Cell lysates were immunoblotted with HA or β-actin antibodies. The densitometry data (HA/β-Actin) obtained from are expressed as mean±SEM. 11. Fig 6G: WT-S62A-CSE: Cell viability was measured by MTT assay in BEAS-2B cells transfected with FLCN WT or S62A and treated with 2% CSE overnight. Data are expressed as a mean±SEM for three independent experiments with triplicate samples. A one-way analysis of variance with Bonferroni’s correction was used for the comparison between WT with CSE and S62A with CSE. 12. A library of V5-tagged F-box O family plasmids were transfected in BEAS-2B cells and cell lysates were immunoblotted for FLCN, V5 or β-actin antibodies.  The densitometry data (FLCN/β-Actin) obtained from a are expressed as mean±SEM. b.    Human subject demographic data (FLCN_Human_Demographic_Data.pdf) Table A:  COPD stage 4 subjects (n=6) – age, sex, smoking history (pack-years), ratio (forced expiratory volume in 1 second [FEV1] divided by forced vital capacity [FVC]), diffusing capacity of the lungs for carbon monoxide (DLCO), post FEV1 (% predicted values), and post FVC (% predicted values) Table B: Control smoking subjects (n=5) – age, sex and smoking history c.     All original immunoblotting data  Fig 1A (FLCN_original_Fig1_WB_data.pdf): The original immunoblot analysis of FLCN and HPRT1 for control smokers (n=5) vs. COPD subjects (n=6) Fig 2A (FLCN_original_Fig2_WB_data.pdf): The original immunoblot analysis of FLCN and β-actin protein expression in the lungs of mice exposed to cigarette smoke (CS) or room air (RA) for 6 months (n=4 per group). Fig 3B (FLCN_original_Fig3_WB_data.pdf): The original immunoblot analysis of FLCN and β-actin in Beas-2B cells exposed to different concentrations of fresh CSE (0, 1, 2, 4, 6%) for 8 hours. Fig 3C (FLCN_original_Fig3_WB_data.pdf):  The original immunoblot analysis of FLCN and β-actin in Beas-2B cells exposed to 2% CSE for up to 8 hours. Fig 4A (FLCN_original_Fig4_WB_data.pdf): The original immunoblot analysis of FLCN and β-actin in BEAS-2B cells treated with CHX (20 µg/mL) or leupeptin (20 µM) or MG132 (40 µM) for 0, 2, 4, 6, and 8 hours. Fig 4C (FLCN_original_Fig4_WB_data.pdf) : The original immunoblot analysis of FLCN, HA, and β-actin in BEAS-2B cells transfected with HA-tagged ubiquitin plasmid (0, 1, 3, and 5 µg). Fig 5A (FLCN_original_Fig5_WB_data.pdf) :  The original immunoblot analysis of FLCN, V5, and β-actin in BEAS-2B cells transfected with a library of V5-tagged F-box O family plasmids (O23, O25, O27, O28, O38, O41,O42, and O44). Fig 5C (FLCN_original_Fig5_WB_data.pdf) : The original immunoblot analysis of FLCN, V5, and β-actin in BEAS-2B cells transfected with V5-tagged FBXO23 or FBXO41 plasmid (0, 1, 2, and 3 µg). Fig 5E (FLCN_original_Fig5_WB_data.pdf): The original immunoblot analysis of normal mouse IgG or V5 immunoprecipitation in BEAS-2B cells transfected with V5-tagged FBXO23 or FBXO41 plasmid. Fig 6A (FLCN_original_Fig6_WB_data.pdf): The original immunoblot analysis of FLCN, phosphorylated ATM (pATM) or total ATM in BEAS-2B cells treated with 2% CSE together with Nu7441(10 µM), VE-821(10 µM) or KU-55933 (10 µM) for 24 hours. Fig 6B (FLCN_original_Fig6_WB_data.pdf): The original immunoblot analysis of ATM, FLCN, or β-actin in BEAS-2B cells transfected with ATM siRNA (siATM) or scrambled siRNA (siCtrl) for 48 hours and further cultured with or without 2% of CSE for 8 hours.