Haploinsufficiency of the folliculin gene leads to impaired functions of lung fibroblasts in patients with Birt–Hogg–Dubé syndrome

Abstract

Birt–Hogg–Dubé syndrome (BHDS) is an autosomal dominant inherited disorder caused by germline mutations in the FLCN gene, and characterized by skin fibrofolliculomas, multiple lung cysts, spontaneous pneumothorax, and renal neoplasms. Pulmonary manifestations frequently develop earlier than other organ involvements, prompting a diagnosis of BHDS. However, the mechanism of lung cyst formation and pathogenesis of pneumothorax have not yet been clarified. Fibroblasts were isolated from lung tissues obtained from patients with BHDS (n = 12) and lung cancer (n = 10) as controls. The functional abilities of these lung fibroblasts were evaluated by the tests for chemotaxis to fibronectin and three‐dimensional (3‐D) gel contraction. Fibroblasts from BHDS patients showed diminished chemotaxis as compared with fibroblasts from controls. Expression of fibronectin and TGF‐β1 was significantly reduced in BHDS fibroblasts when assessed by qPCR. Addition of TGF‐β1 in culture medium of BHDS lung fibroblasts significantly restored these cells' abilities of chemotaxis and gel contraction. Human fetal lung fibroblasts (HFL‐1) exhibited reduced chemotaxis and 3‐D gel contraction when FLCN expression was knocked down. To the contrary, a significant increase in chemotactic activity toward to fibronectin was demonstrated when wild‐type FLCN was overexpressed, whereas transduction of mutant FLCN showed no effect on chemotaxis. Our results suggest that FLCN is associated with chemotaxis in lung fibroblasts. Together with reduced TGF‐β1 expression by BHDS lung fibroblasts, a state of FLCN haploinsufficiency may cause lung fibroblast dysfunction, thereby impairing tissue repair. These may reveal one mechanism of lung cyst formation and pneumothorax in BHDS patients.

Epithelial folliculin is involved in airway inflammation in workers exposed to toluene diisocyanate

“…Knockout of FLCN induced apoptosis, downregulated the expression of E-cadherin in mouse lung alveolar cells and damaged several signaling pathways in human airway epithelial cells (HAECs). 7 , 8 These findings suggest that FLCN may involve in chronic airway diseases that involve airway damage. However, to date, the involvement of FLCN in OA and NOA is unknown.…”

Folliculin Haploinsufficiency Causes Cellular Dysfunction of Pleural Mesothelial Cells

“…Regarding pulmonary manifestations, no neoplastic cells have been detected either on cyst walls or in any other parts of the lungs of BHDS patients. Instead, BHDS lung broblasts showed diminished chemotaxis with reduced TGF-β expression in a state of FLCN haploinsu ciency 11 . According to these reports 10,11 , other constituent cells of the lungs are also likely to be affected by the FLCN haploinsu ciency and consequently, lung cysts may develop and result in the development of pneumothorax.…”

“…Immuno uorescence phalloidin staining was performed as previously described. 11 In our study, PMCs were seeded in triplicate at a density of 3×10 5 cells on a 35 mm glass-bottom dish and cultured in the complete medium until the cells became con uent. Immuno uorescence images were obtained using a Leica TCS SP5 II confocal microscope (Leica Biosystems, Wetzlar, Germany).…”

“…The levels of active GTP-bound RhoA (RhoA-GTP) and total RhoA were determined using the G-LISA™ RhoA Activation Assay Biochem Kit (Cytoskeleton Inc., Denver, CO, USA) and the Total RhoA ELISA Assay (Cytoskeleton Inc.), respectively, as done in previous research 11 . In our study, PMCs were seeded at a density of 3×10 5 cells/well onto a collagen-coated 6-well plate (Corning Incorporated) and cultured until approximately 70% con uence was reached.…”

“…Conversely, RhoA-GTP was decreased by 60% in a FLCN-null kidney cancer cell line (UOK257) compared with a FLCN-expressing isogenic kidney cancer cell line (UOK257-2) 8 . Furthermore, FLCN haploinsu ciency has been found to cause the decrease of the organization of actin stress bers as well as RhoA-GTP in BHDS lung broblasts 11 . Accordingly, we evaluated the organization of actin laments and RhoA activity in BHDS-PMCs.…”