Cigarette smoking is a secondary cause of folliculin loss

Abstract

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.

Methods: We 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.

Keywords: airway epithelium; emphysema; rare lung diseases; tobacco and the lung.

Figure 1

Smokers with COPD exhibit decreased pulmonary FLCN (A): The stage of COPD was determined by the Global Initiative for Obstructive Lung Disease (GOLD) criteria. Stage 3, severe and stage 4, very severe. Smokers with normal lung function serve as control. Whole lung parenchyma lysates were prepared from a total of 11 smokers with various gold stages of COPD and analysed for FLCN by immunoblotting. (B): The densitometry data (FLCN/HPRT1) obtained from (A) are expressed as a median (lines) with IQR (IQR, whiskers) and analysed using Mann-Whitney test. (C): Total RNA was prepared from whole lung parenchymal tissues obtained from the same donors (control and stage 3/4) as in (A). Levels of FLCN mRNA were measured by qPCR. The relative fold difference compared with GAPDH (control) was expressed. Data are expressed as a median (lines) and IQR (whiskers). COPD, chronic obstructive pulmonary disease; FLCN, folliculin; mRNA, messenger RNA; qPCR, quantitative PCR.

Figure 2

Pulmonary FLCN reduced in CS treated mice (A): Whole lung tissue were prepared from mice exposure to cigarette smoke (CS) or room air (RA) for 6 months. The samples were analysed by immunoblotting for FLCN. (B): The densitometry data (FLCN/β-Actin) obtained from (A) are expressed as a median (lines) with IQR (whiskers) and analysed using Mann-Whitney test. (C): Total RNA was prepared from whole lung tissues obtained from the same mice (RA and CS) as in (A). Levels of FLCN mRNA were measured by qPCR. The relative fold difference compared with GAPDH (control) was expressed. Data are expressed as a median (lines) and IQR (whiskers). FLCN, folliculin; mRNA, messenger RNA; qPCR, quantitative PCR.

Figure 3

FLCN is required to protect against cigarette smoke-induced cytotoxicity in BEAS-2B cells (A): Primary human alveolar epithelial cells (pHAECs) and primary human bronchial epithelial cells (pHBECs) were cultured with or without 2% CSE for 24 hours, and analysed by immunoblotting for FLCN or β-actin. (B): Beas-2B cells were exposed to different concentrations of fresh CSE (0, 1, 2, 4, 6%) for 8 hours, and analysed by immunoblotting for FLCN or β-actin. (C): BEAS-2B cells were treated with 2% CSE for up to 8 hours and analysed for FLCN or β-actin by immunoblotting. (D): The densitometry data (FLCN/β-Actin) obtained from (C) are expressed as mean±SEM. (E): Total RNA was prepared from cells as in (C). Levels of FLCN mRNA were measured by qPCR. The relative fold difference compared with GAPDH (control) was expressed. Data are expressed as mean±SEM. (F): BEAS-2B cells were transfected with siCrtl or siFLCN for 72 hours and cell lysates were immunoblotted for FLCN, or β-actin antibodies. (G): 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. MTT assay was performed for analysis of cell viability. (H): BEAS-2B cells were transfected with vector or FLCN-HA plasmid for 48 hours, and cell lysates were immunoblotted with FLCN or β-actin antibodies. Immunoblotting data are representative of three independent experiments. (I): BEAS-2B were transfected with FLCN-HA plasmid for 24 hours, and cells were fixed and stained with HA and Alexa Fluor 488 antibodies. (J): 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. Cell viability was measured by MTT assay. Data are expressed as mean±SEM for three independent experiments with triplicate samples. CSE, cigarette smoke extract; FLCN, folliculin; HA, haemagglutinin; mRNA, messenger RNA; qPCR, quantitative PCR.

Figure 4

FLCN undergoes proteasomal degradation (A): 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. (B): The densitometry data (FLCN/β-Actin) obtained from (A) are expressed as mean±SEM. (C): BEAS-2B cells were transfected with indicated amounts of HA-tagged ubiquitin plasmid and the cell lysates were immunoblotted for FLCN, HA, or β-actin antibodies. Immunoblotting data are representative of three independent experiments. CHX, cycloheximide; FLCN, folliculin; HA, haemagglutinin.

Figure 5

FBXO23 targets FLCN for proteasomal degradation (A): 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. (B): The densitometry data (FLCN/β-Actin) obtained from a are expressed as mean±SEM. (C): BEAS-2B cells were transfected with indicated amounts of V5-tagged FBXO23 or FBXO41 plasmid and cell lysates were immunoblotted for FLCN, V5, or β-actin antibodies. (D): The densitometry data (FLCN/β-Actin) obtained from (C) are expressed as mean±SEM. (E): V5-tagged FBXO23 or FBXO41 plasmid was transfected into BEAS-2B cells, and cell lysates were subjected to normal mouse IgG or V5 immunoprecipitation. The immunoprecipitates were immunoblotted for FLCN, V5 or GAPDH antibodies. Immunoblotting data are representative of three independent experiments. FLCN, folliculin.

Figure 6

Phosphorylation regulates FLCN degradation induced by the cigarette smoke (A): BEAS-2B cells were treated with 2% CSE together with Nu7441(10 µM), VE-821(10 µM) or KU-55933 (10 µM) for 24 hours. Cell lysates were immunoblotted with FLCN, phosphorylated ATM (pATM) or total ATM antibodies. (B): BEAS-2B cells were transfected with ATM siRNA (siATM) or scrambled siRNA (siCtrl) for 48 hours and further cultured with or without 2% of CSE for 8 hours. The cell lysates were immunoblotted for ATM, FLCN, or β-actin antibodies. (C): BEAS-2B cells were transfected with siATM or siCtrl for 24 hours, and cells were further transfected with FBXO23 plasmid and cultured another 48 hours. These cells were treated with 2% of CSE for 4 hours, and cell lysates were subjected to V5 immunoprecipitation. The immunoprecipitates were immunoblotted for V5, FLCN, GAPDH, or ATM, antibodies. Immunoblotting data are representative of three independent experiments. (D): 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. (E): The densitometry data (HA/β-Actin) obtained from (D) are expressed as mean±SEM. (F): FLCN WT or FLCN S62A plasmid was transfected into BEAS-2B cells, and cell lysates were subjected to HA immunoprecipitation. The immunoprecipitates were immunoblotted for phosphoserine, HA, or GAPDH antibodies. Immunoblotting data are representative of three independent experiments. (G): BEAS-2B cells were transfected with FLCN WT or S62A and treated with 2% CSE overnight. Cell viability was measured by MTT assay. Data are expressed as a mean±SEM for three independent experiments with triplicate samples. ATM, ataxia‐telangiectasia mutated; CSE, cigarette smoke extract; FLCN, folliculin; HA, haemagglutinin; WT wild type.