Birt-Hogg-Dubé syndrome is caused by a mutation in the gene folliculin (FLCN). Unravelling the functions of FLCN has been a focus of research into BHD, because if we can determine how FLCN mutations cause the characteristics of BHD we can potentially reverse them or even prevent them from happening. Lung cysts are a common feature of BHD with cysts being present in up to 80% of patients. An exciting study by Ikue Tai-Nagara and colleagues discovered a new function of folliculin and hypothesised that when lost it may result in lung cyst formation (1).
The body contains different systems, working in harmony to keep us functioning including the cardiovascular and lymphatic systems. The cardiovascular system transports blood throughout the body whereas the lymphatic system collects excess fluid, clears waste, and transports immune cells. The vessels of these two systems are very similar and both are lined with a layer of cells known as endothelial cells. However, throughout the body, the lymphatic and blood vessels are mostly separated. Imagine they are two different train tracks, structurally similar but carrying different cargo.
The research team investigated what would happen if FLCN was knocked out (removed) in endothelial cells. They studied mouse models at different developmental stages and found that knocking out FLCN in mice that were still developing resulted in enlarged and blood-filled lymphatic vessels. Examining both the lymphatic vessels and blood vessels under a microscope they observed that the lymphatic vessels were developing offshoots towards veins (the blood vessels that transport blood from the organs to the heart) and the veins were developing offshoots towards the lymphatic vessels. It appeared that there was an attraction between the two systems that had not previously been present. Additionally, the lymphatic endothelial cells were dividing rapidly which was the likely cause for the enlargement of the lymphatic vessels.
Next, the researchers investigated why knocking out FLCN caused a change in the relationship between the lymphatic vessels and veins and one key gene stood out. PROX1 controls the development of lymphatic endothelial cells. In mice where FLCN was knocked out not only was PROX1 expressed in the lymphatic endothelial cells (where it should be) but it was also inappropriately expressed in the endothelial cells of the veins. The significance of this expression was highlighted when PROX1 was deleted from the endothelial cells and the characteristics of the vessels normalised.
Next, they needed to connect how knocking out FLCN caused PROX1 expression to increase. They focused on TFE3, which is well documented as being regulated by FLCN (find out more in our recent blog post). Firstly they found that TFE3 was highly expressed in the endothelial cells of the FLCN knockout mice. They then looked at human endothelial cells and found that knocking down FLCN increased PROX1 and that this could be reversed by also knocking down TFE3. They also showed that TFE3 bound and regulated the PROX1 gene. Therefore, when FLCN was knocked down and could no longer regulate TFE3, PROX1 increased resulting in changes to the lymphatic vessels and veins.
So, what has all this got to do with the lungs? The final experiment involved looking at lung samples from three BHD patients. The samples were compared with normal lung specimens and non-BHD cyst-filled lungs. Significantly, only the BHD lung specimens showed evidence of lymphatic vessels filled with blood, inappropriate PROX1 expression and increased TFE3 expression. This suggests that the structural changes in the lungs such as cysts could be linked with the disruption of the lymphatic and cardiovascular systems.
Altogether this research demonstrates a new role of folliculin as a gatekeeper of the lymphatic and cardiovascular system. New research is on the horizon looking further into how lung cysts form and how these pathways can be targeted therapeutically to improve the lung symptoms associated with BHD.
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