Birt–Hogg–Dubé syndrome (BHD) is caused by germline mutations in the FLCN gene and characterized by fibrofolliculomas, lung cysts, spontaneous pneumothorax and renal tumours. The stretch hypothesis for pulmonary cyst formation proposes that cysts in BHD arise from defects in cell–cell adhesion, leading to repeated respiration-induced physical stretch-induced stress and, over time, expansion of alveolar spaces particularly in vulnerable regions of lung (Kennedy et al., 2016). A new study by Hoshika et al. (2016) has shed some light on this mechanism. The authors isolated lung fibroblasts from BHD patients and evaluated them by testing chemotaxis to fibronectin and three-dimensional (3-D) gel contraction. They showed that FLCN is associated with chemotaxis in lung fibroblasts and that, together with reduced TGF-β1 expression by BHD lung fibroblasts, FLCN haploinsufficiency seems to cause lung fibroblast dysfunction, impairing tissue repair.
FLCN is often described as a tumour suppressor in the kidney, because renal tumours are associated with a somatic second hit mutation in BHD (Vocke et al., 2005). Here, the authors hypothesize that lung cysts develop due to haploinsufficiency since no neoplastic cells have been identified.
The authors isolate lung fibroblasts from BHD patients and confirmed that they were FLCN haploinsufficient. FLCN expression was confirmed by RT-PCR and protein expression levels of folliculin measured by western blot was lower in BHD lung fibroblasts. Both chemotaxis and contraction of 3-D collagen gel bioassays evaluate fibroblast function by modelling the cellular response to injury and subsequent tissue repair. BHD lung fibroblasts were less active in migration toward fibronectin than controls. The extent of 3-D collagen gel contraction was also assessed and results showed that BHD lung fibroblasts contracted the gels to ~64% of the original size compared to ~50% contraction in controls.
To evaluate the tissue-repair ability of lung fibroblasts, the authors assessed mRNA expression of several genes including TGFB1 (TGF-β1) and FN1 (fibronectin). The expression of TGFB1 and FN1 was significantly lower in BHD lung fibroblasts and therefore the release of TGF-β1 and fibronectin by cultured lung fibroblasts was measured. BHD fibroblasts released a significantly smaller amount of TGF-β1 than controls and fibronectin release also tended to be lower.
Because less folliculin was expressed by BHD lung fibroblasts than by controls, the authors investigated if knockdown of FLCN expression in normal fibroblasts results in the phenotype seen in BHD samples. FLCN expression was knocked down in normal fetal lung fibroblasts (HFL-1 cells). Knocked down cells exhibited reduction of both chemotaxis and 3-D collagen gels contraction. In addition, the release of TGF-β1 and fibronectin decreased. Chemotactic activity of HFL-1 cells toward fibronectin significantly increased by overexpression of FLAG-tagged wild-type FLCN, but no effect followed the induced expression of mutated FLCN, suggesting that the specific mutant used did not exert a dominant negative effect. These results support the idea that the amount of wildtype folliculin protein rather than mutant protein is important.
The authors then examined if FLCN-knockdown HFL-1 cells could increase repair responses when exposed to TGF-β1. No significant increase in chemotaxis was found in FLCN-knockdown HFL-1 or control when exposed to exogenous TGF-β1. In contrast, both FLCN-knockdown HFL-1 and control showed increased 3-D collagen gel contraction when exposed to exogenous TGF-β1. Exogenously added TGF-β1 effectively enhanced both chemotaxis and 3-D collagen gel contraction on BHD lung fibroblasts. The authors suggest that chemotaxis and gel contraction are impaired in FLCN-knockdown HFL-1, but their chemotactic responsiveness to TGF-β1 might need a longer period of folliculin-deficiency. Dysregulation of TGF-β signalling by FLCN inactivation has been shown to be important for tumorigenesis in BHD (Hong et al., 2010). Here, the authors consider such dysregulation also to be implicated in pulmonary manifestations. The organization of RhoA activity was also assessed in BHD lung fibroblasts. The levels of GTP-bound RhoA was lower in BHD lung fibroblasts than in controls. These results suggest that the haploinsufficiency state of the FLCN gene in patients with BHD causes lung fibroblast dysfunction via impaired TGF-β1/RhoA signalling pathway.
In summary, BHD lung fibroblasts presenting FLCN haploinsufficiency, showed lower ability to migrate, contract, and produce extracellular matrix proteins. These phenotypes did not result from a dominant negative effect of mutated folliculin, but instead, from the reduced amount of wild-type folliculin protein itself. The present study adds new insight to understanding the mechanisms of lung cyst formation in BHD.
- Hoshika, Y., Takahashi, F., Togo, S., Hashimoto, M., Nara, T., Kobayashi, T., Nurwidya, F., Kataoka, H., Kurihara, M., Kobayashi, E., Ebana, H., Kikkawa, M., Ando, K., Nishino, K., Hino, O., Takahashi, K., & Seyama, K. (2016). Haploinsufficiency of the gene leads to impaired functions of lung fibroblasts in patients with Birt–Hogg–Dubé syndrome. Physiological Reports, 4 (21) DOI: 10.14814/phy2.13025