Roughly 90% of BHD patients develop lung cysts, which are thought to also cause spontaneous pneumothorax in 1 in 4 BHD patients (Houweling et al., 2011, Predina et al., 2011). However, although lung cysts are a highly penetrant symptom of BHD, how losing one copy of the FLCN gene causes cysts to develop is currently unknown. A recent study from Professor Vera Krymskaya’s lab at the University of Pennsylvania may have shed light this question.
By comparing resected BHD lung tissue with healthy lung tissue, Goncharova et al. found that in healthy tissue FLCN expression co-localises with surfactant protein C (SP-C) expression, which is only expressed in Type II alveolar epithelial cells (ATII cells), the cells that line and maintain the structure and function of alveoli (Fujino et al., 2011). BHD lungs lacked FLCN expression in ATII cells, and showed disrupted lung parenchyma, suggesting that FLCN function in ATII cells is required for correct lung morphology. In order to further investigate the role of FLCN in ATII cells, the authors generated a mouse model where, using a floxed FLCN allele and a tet-O Cre allele under the control of the SP-C promoter, FLCN is conditionally deleted in these cells following exposure to doxycycline.
Mice with FLCN-null ATII cells showed alveolar enlargement and reduced lung function compared with control littermates, while overall lung structure and organisation was unaffected. Loss of FLCN in these cells also led to increased apoptosis and cell permeability, reduced transepithelial resistance and visible disruption of the alveolar monolayer, indicating that FLCN is necessary for ATII cell survival and function. ATII cells secrete phospholipids into alveoli to reduce surface tension, thus increased apoptosis of these cells led to increased alveolar surface tension in these mice. The authors also observed increased inflammation and MMP3 and MMP9 expression, suggesting that FLCN loss caused an inflammatory response in the lungs of these mice. Mice with FLCN-null Type 1 alveolar epithelial cells did not show any of these phenotypic changes, indicating that this phenotype is specifically caused by the loss of FLCN in ATII cells.
Molecular analysis of three different isogenic cell lines showed that loss of FLCN led to a reduction of E-cadherin at cell membranes, which led to reduced LKB1 phosphorylation and a consequent reduction in AMPK phosphorylation. Treating cells with the AMPK activator AICAR, or transfecting them with a constitutively active form of AMPK reversed all of the cellular phenotypes caused by FLCN deletion, meaning that reduced AMPK function, via LKB1 and E-cadherin, is the likely cause of the lung pathology seen in the FLCN-null ATII mice in this study. Indeed, treating these mice with AICAR suppressed ATII cell apoptosis, lung inflammation and MMP expression.
This study draws together a number of earlier observations: a number of studies have suggested a role for FLCN in apoptosis; increased MMP9 expression has been reported in the lung cysts of a BHD patient; and collagen – an important structural and signalling protein known to be active in the lungs – has numerous overlaps with FLCN signalling pathways. Furthermore, it has been hypothesised that loss of FLCN causes alveolar walls to become weak and vulnerable to mechanical stress, ultimately causing lung cysts to form. The increased alveolar surface tension observed by Goncharova et al. may add strength to this hypothesis.
Finally, the loss of FLCN leads to disrupted cell junction formation and E-cadherin mislocalisation and FLCN may play a role in the localisation of proteins via its DENN domain. Indeed, Rab11a is known to control the localisation of E-cadherin and interacts with PKP4, which is itself a known FLCN-interactor (Medvetz et al., 2012, Nahorski et al., 2012). Thus it is tempting to speculate that FLCN may control E-cadherin localisation – and subsequent ATII cell survival and function – by interacting with PKP4 and Rab11a.
- Fujino N, Kubo H, Suzuki T, Ota C, Hegab AE, He M, Suzuki S, Suzuki T, Yamada M, Kondo T, Kato H, & Yamaya M (2011). Isolation of alveolar epithelial type II progenitor cells from adult human lungs. Laboratory investigation; a journal of technical methods and pathology, 91 (3), 363-78 PMID: 21079581
- Goncharova EA, Goncharov DA, James ML, Atochina-Vasserman EN, Stepanova V, Hong SB, Li H, Gonzales L, Baba M, Linehan WM, Gow AJ, Margulies S, Guttentag S, Schmidt LS, & Krymskaya VP (2014). Folliculin Controls Lung Alveolar Enlargement and Epithelial Cell Survival through E-Cadherin, LKB1, and AMPK. Cell reports, 7 (2), 412-23 PMID: 24726356
- Houweling AC, Gijezen LM, Jonker MA, van Doorn MB, Oldenburg RA, van Spaendonck-Zwarts KY, Leter EM, van Os TA, van Grieken NC, Jaspars EH, de Jong MM, Bongers EM, Johannesma PC, Postmus PE, van Moorselaar RJ, van Waesberghe JH, Starink TM, van Steensel MA, Gille JJ, & Menko FH (2011). Renal cancer and pneumothorax risk in Birt-Hogg-Dubé syndrome; an analysis of 115 FLCN mutation carriers from 35 BHD families. British journal of cancer, 105 (12), 1912-9 PMID: 22146830
- Medvetz DA, Khabibullin D, Hariharan V, Ongusaha PP, Goncharova EA, Schlechter T, Darling TN, Hofmann I, Krymskaya VP, Liao JK, Huang H, & Henske EP (2012). Folliculin, the product of the Birt-Hogg-Dube tumor suppressor gene, interacts with the adherens junction protein p0071 to regulate cell-cell adhesion. PloS one, 7 (11) PMID: 23139756
- Nahorski MS, Seabra L, Straatman-Iwanowska A, Wingenfeld A, Reiman A, Lu X, Klomp JA, Teh BT, Hatzfeld M, Gissen P, & Maher ER (2012). Folliculin interacts with p0071 (plakophilin-4) and deficiency is associated with disordered RhoA signalling, epithelial polarization and cytokinesis. Human molecular genetics, 21 (24), 5268-79 PMID: 22965878
- Predina JD, Kotloff RM, Miller WT, & Singhal S (2011). Recurrent spontaneous pneumothorax in a patient with Birt-Hogg-Dubé syndrome. European journal of cardio-thoracic surgery : official journal of the European Association for Cardio-thoracic Surgery, 39 (3), 404-6 PMID: 20692178
www.bhdsyndrome.org – the primary online resource for anyone interested in BHD Syndrome.