BHD is an autosomal dominant disease associated with lung cysts, skin tumors, and kidney cancer, primarily of the chromophobe subtype 
. Germline BHD
mutations also occur in families with hereditary lung collapse (pneumothorax) without renal or skin disease 
. Somatic BHD
mutations occur in ~10% of sporadic chromophobe RCC 
. We report here that FLCN interacts with the adherens junction protein p0071 to regulate cell-cell adhesion, cell migration, cell polarity, and RhoA signaling. Furthermore, inactivation of FLCN in the basal layer of the epidermis, which is classically used to study the in vivo impact of cell adhesion proteins on the integrity of epithelial architecture, results in striking phenotypes including wavy fur, epidermal hyperplasia and inflammation, and defective eyelid opening.
These data provide a critical new component to our emerging understanding of the pathogenesis of BHD. BHD is one of the least studied monogenic tumor suppressor gene disorders, with fewer than 12 publications on the functions of FLCN. FLCN has been linked to AMPK signaling via a direct interaction with FNIP1/2, to HIF signaling and mitochondrial dysfunction, to aberrant TGF-beta signaling, and to mTOR complex 1 signaling 
, but the fundamental mechanisms through which loss of FLCN leads to abnormalities in three epithelial organs, the lung, the kidney, and the skin, remain incompletely understood. Our data point toward a completely new model in which regulation of the integrity of the epithelium via cell-cell junctions is a critical function of FLCN.
Our finding that FLCN-deficient and p0071-deficient cells have enhanced cell-cell adhesion is unexpected, given that cell-cell junctions are critical for epithelial tissue homeostasis and loss of the integrity of these junctions is associated with cancer progression 
. p120-catenin, the closest homolog of p0071, localizes to adherens junctions and positively regulates cell adhesion in prostate cancer-derived 
and lung cancer-derived cells 
. p0071 (also called PKP4) localizes to adherens junctions, but its function in cell-cell adhesion has not been previously studied. Additionally, there is controversy regarding the desmosomal localization of p0071. We have observed desmosomal defects in FLCN-deficient cells (), but at this stage we cannot ascribe the FLCN effects exclusively to one type of junction. Furthermore, the effects of loss of FLCN or p0071 may result in part from the impact on p0071’s other interacting partners, which include E-cadherin 
, a critical component of adherens junctions. Germline loss-of-function mutations in the CDH1
gene, which encodes E-cadherin, cause human gastric cancer 
In addition to their localization to adherens junctions, both p120-catenin and p0071 localize to the cytoplasm where they regulate RhoA activity 
. Our data that FLCN-deficient cells have low levels of RhoA and ROCK activity are consistent with the known role of p0071 as a positive regulator of RhoA, but surprising given that RhoA is often activated in human cancer 
. The TGF-beta signaling network, which is often activated in human cancer 
, is also downregulated in FLCN-deficient cells, and mTORC1 signaling can be either up or downregulated in FLCN-deficient cells depending on the cell type and context 
. Therefore the signaling consequences of FLCN deficient cells do not appear to align precisely with those of other tumors, or of other genetic diseases associated with kidney cancer and skin tumors, such as tuberous sclerosis complex 
. Some of the signaling functions of FLCN for which a molecular mechanism is not yet clearly identified, including mTORC1 and TGF-beta, could be downstream of the FLCN-p0071 interaction, either through a mechanism arising at the cell-cell junction or via RhoA.
Patients with mutations in the BHD
gene develop renal cell carcinomas (RCC), the majority of which are chromophobe and chromophobe/oncocytic hybrid tumors, and somatic BHD
mutations have been found in sporadic chromophobe RCC, which is a rare disease affecting ~1500 individuals in the United States each year. There appear to be at least two potential links between defective cell junctions and sporadic RCC. First, we found that FLCN-deficient cells have more desmosomes than FLCN-expressing cells, and Kim et al. found that desmosomes are increased in RCC compared to normal kidney tissue and identified an association between desmosome number and tumor grade 
. Second, Walter et al. found that p0071, which is expressed in human and mouse kidney epithelium 
, appears to be lost in 41% of chromophobe RCC 
; we found that p0071 interacts with FLCN and that loss of p0071 phenocopies loss of FLCN in cell-based assays. These fundamental mechanistic connections between cell-cell junctions and the pathogenesis of sporadic chromophobe RCC could eventually lead to an improved understanding of disease pathogenesis and the development of targeted strategies for metastatic chromophobe RCC, for which there are currently no known effective therapies.
The cellular and physiologic mechanisms that lead to cystic and emphysematous airspace enlargement in BHD are not known. Respiration exposes the lung to unique mechanical forces. Cell-cell junctions allow the lung to expand in response to these forces and then “snap back” to their original shape and organization. Mechanical-induced stretch of pulmonary alveolar type II cells leads to widened cell-cell gaps and is speculated to be a protective mechanism in response to lung injury, likely due to a flexibility effect 
, and defects in tight junctions lead to lung injury and fibrosis in mice 
. We hypothesize that the defects in cell-cell adhesion in FLCN-deficient cells underlie the pathogenesis of airspace enlargement in BHD. If correct, this could lead to an improved understanding of airspace enlargement in other diseases including emphysema and chronic obstructive pulmonary disease (COPD), the third leading cause of death in the world.
Finally, we have developed a novel mouse model in which Bhd
is inactivated in the epidermis via the Keratin-14 (K14) promoter. K14-cre driven inactivation of adhesion proteins has been used to study other junctional proteins in vivo
. Most notably, K14-Cre driven inactivation of p120-catenin, the closest homolog of p0071, leads to epidermal hyperplasia and hair loss 
that resemble those of FLCN inactivation. Rho signaling is known to regulate keratinocyte differentiation and proliferation 
, and may contribute to the epidermal consequences of p120-catenin and FLCN inactivation.
In conclusion, we have uncovered a physical interaction between FLCN and p0071 that is critical for the proper regulation of cell-cell adhesion, RhoA activity, and epithelial cell polarity. A mouse model of FLCN inactivation in the epidermis leads to wavy hair and whiskers, delays in eyelid opening, hair loss, and increased epidermal thickness, phenotypes which resemble the inactivation of p120-catenin, the closest homolog of p0071. Taken together, these findings reveal a novel paradigm in which loss of the FLCN-p0071 interaction and consequent defects in cell-cell adhesion, cell polarity and RhoA activity participates in the pathogenesis of renal cell carcinoma, skin tumors, and cystic airspace enlargement ().