Folliculin, the Product of the Birt-Hogg-Dube Tumor Suppressor Gene, Interacts with the Adherens Junction Protein p0071 to Regulate Cell-Cell Adhesion

Birt-Hogg-Dube (BHD) is a tumor suppressor gene syndrome associated with fibrofolliculomas, cystic lung disease, and chromophobe renal cell carcinoma. In seeking to elucidate the pathogenesis of BHD, we discovered a physical interaction between folliculin (FLCN), the protein product of the BHD gene, and p0071, an armadillo repeat containing protein that localizes to the cytoplasm and to adherens junctions. Adherens junctions are one of the three cell-cell junctions that are essential to the establishment and maintenance of the cellular architecture of all epithelial tissues. Surprisingly, we found that downregulation of FLCN leads to increased cell-cell adhesion in functional cell-based assays and disruption of cell polarity in a three-dimensional lumen-forming assay, both of which are phenocopied by downregulation of p0071. These data indicate that the FLCN-p0071 protein complex is a negative regulator of cell-cell adhesion. We also found that FLCN positively regulates RhoA activity and Rho-associated kinase activity, consistent with the only known function of p0071. Finally, to examine the role of Flcn loss on cell-cell adhesion in vivo, we utilized keratin-14 cre-recombinase (K14-cre) to inactivate Flcn in the mouse epidermis. The K14-Cre-Bhdflox/flox mice have striking delays in eyelid opening, wavy fur, hair loss, and epidermal hyperplasia with increased levels of mammalian target of rapamycin complex 1 (mTORC1) activity. These data support a model in which dysregulation of the FLCN-p0071 interaction leads to alterations in cell adhesion, cell polarity, and RhoA signaling, with broad implications for the role of cell-cell adhesion molecules in the pathogenesis of human disease, including emphysema and renal cell carcinoma

Identification of the Junctional Plaque Protein Plakophilin 3 in Cytoplasmic Particles Containing RNA-binding Proteins and the Recruitment of Plakophilins 1 and 3 to Stress Granules

Recent studies on the subcellular distribution of cytoplasmic plaque proteins of intercellular junctions have revealed that a number of such proteins can also occur in the cyto- and the nucleoplasm. This occurrence in different, and distant locations suggest that some plaque proteins play roles in cytoplasmic and nuclear processes in addition to their involvement in cell–cell adhesive interactions. Plakophilin (PKP) 3, a member of the arm-repeat family of proteins, occurs, in a diversity of cell types, both as an architectural component in plaques of desmosomes and dispersed in cytoplasmic particles. In immuno-selection experiments using PKP3-specific antibodies, we have identified by mass spectrometric analysis the following RNA-binding proteins: Poly (A) binding protein (PABPC1), fragile-X-related protein (FXR1), and ras-GAP-SH3-binding protein (G3BP). Moreover, the RNA-binding proteins codistributed after sucrose gradient centrifugation in PKP3-containing fractions corresponding to 25–35 S and 45–55 S. When cells are exposed to environmental stress (e.g., heat shock or oxidative stress) proteins FXR1, G3BP, and PABPC1 are found, together with PKP3 or PKP1, in “stress granules” known to accumulate stalled translation initiation complexes. Moreover, the protein eIF-4E and the ribosomal protein S6 are also detected in PKP3 particles. Our results show that cytoplasmic PKP3 is constitutively associated with RNA-binding proteins and indicate an involvement in processes of translation and RNA metabolism

Bhd^f/f K14-Cre mice have wavy hair, erythematous skin, and delayed eyelid opening.

<p><b>A)</b> Bhd^f/f K14-cre mice at three weeks of age display coarse, wavy fur and wavy whiskers compared to Bhd^f/f wild-type mice. <b>B)</b> A representative Bhd^f/f K14-cre mouse at three weeks of age with erythematous skin and hair loss (arrow). <b>C)</b> 100% of Bhd^f/f K14-cre mice have closed eyelids at three weeks of age (arrow) compared to 0% of wild-type mice.</p

FLCN-deficiency leads to decreased Rho activity in sub-confluent cells and delays in wound closure.

<p><b>A)</b> Rho-associated kinase (ROCK) activity was assayed by measuring the phosphorylation of myosin-binding subunit (MBS), a downstream substrate of ROCK. Cells were grown in DMEM supplemented with 10% FBS (C), stimulated with Lysophosphatidic acid (LPA, 30 uM, 30 min), or treated with the ROCK inhibitor Hydroxyfasudil (HF, 20 uM, 30 min). The phosphorylation of MBS was decreased in the FLCN-null UOK257 cells compared to the FLCN-expressing UOK257-2 cells. FLCN levels were analyzed by western blot. <b>B)</b> Rho activity was measured using a Rhotekin binding assay at 70% confluence. The FLCN-null UOK257 cells had a 2.5-fold decrease in active Rho levels (Rho GTP) compared to the FLCN re-expressing UOK257-2 cells (n = 3, p<0.05). The data are expressed as mean ± standard error by ANOVA. <b>C)</b> A wound assay was used to measure migration in UOK257 cells compared to UOK257-2 cells. A scratch wound was administered (0 hr), and the wound size was measured at 8, 24, 48, 80, and 92 hr post wound induction. At 24 and 48 hours UOK257 cells had a larger wound area than UOK257-2 cells (n = 3, p<0.05) indicating that the FLCN-null UOK257 cells migrate more slowly than the FLCN re-expressing UOK257-2 cells. Data are expressed as mean ± standard deviation. <b>D)</b> A scratch wound assay was performed as described in (C) in A549 cells stably expressing FLCN shRNA or control shRNA. Images are shown at wound induction (0 hr), 48 hours post wound induction (48 hr), and 90 hours post wound induction (90 hr). FLCN downregulation was monitored by western blot (bottom).</p

Working Model.

<p>In wild-type cells (left) the FLCN-p0071 interaction is required for maintenance of normal epithelial cell-cell adhesion and proper cell polarity. Red arrows indicate cell-cell adhesion forces. In FLCN-deficient cells (right), loss of the FLCN-p0071 interaction leads to enhanced cell-cell adhesion, though we do not currently understand which cellular junctions are critical for this defect, and is accompanied by defects in RhoA signaling and cell polarity.</p

p0071 loss phenocopies FLCN-deficiency.

<p><b>A)</b> HEK293 cells expressing control non-targeting shRNA, FLCN shRNA, or p0071 shRNA were cultured in anchorage-independent growth conditions using a rotary shaker and sheared (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047842#s4" target="_blank">Methods</a> for details). A representative phase contrast image is shown pre- and post-shear. <b>B)</b> Quantification of cell cluster size from (A) using pixel number. After shearing, 65% of the control cells sheared into clusters of less than 10,000 pixels. In contrast, only 8% (n = 3, p<0.01) of the FLCN shRNA cells and 12% (n = 2, p<0.01) of the p0071 shRNA cells sheared into clusters of less than 10,000 pixels. Data in (A) and (C) are expressed as mean ± standard deviation. <b>C)</b> Western blot analysis of HEK293 cells showing decreased levels of p0071 using two different shRNAs (79, 80). Clone 80 was used in (A). <b>D)</b> UOK257 cells and UOK257-2 cells were analyzed by immunofluorescence using pan-keratin antibodies (20×magnification). Pan-keratin immunofluorescence was stronger in FLCN-null UOK257 cells. ZO-1 was used to identify cell borders. <b>E</b>) UOK257 cells and UOK257-2 cells were analyzed by immunofluorescence using keratin-18 antibodies (60×magnification). Keratin 18 immunofluorescence was also stronger in FLCN-null UOK257 cells. <b>F)</b> pan-Keratin levels were compared by immunoblot in UOK257 and UOK257-2 cells. Keratin levels were increased in the FLCN-null UOK257 cells. <b>G</b>) Keratin-18 levels were compared by immunoblot in UOK257 and UOK257-2 cells. Keratin-18 levels were also increased in the FLCN-null UOK257 cells. <b>H)</b> Keratin levels were analyzed by immunoblot in T84 cells expressing p0071 shRNA (+) or non-targeting control (-) shRNA. Keratin levels were elevated in p0071-deficient T84 cells. <b>I)</b> FLCN and p0071 regulate lumen formation in a 3-dimensional matrigel assay. T84 cells stably expressing either non-targeting (control), FLCN, or p0071 shRNA were stained for F-actin (red) and counter stained with DAPI (blue) to visualize nuclei. Downregulation of FLCN or p0071 leads to disorganized, irregularly shaped cell clusters with smaller lumens. The bars represent 20 um.</p

Bhd^f/f K14-Cre mice exhibit increased epidermal thickness.

<p>A) Bhd^f/f K14-cre mice exhibit acanthosis and hyperkeratosis (arrows) compared to littermate control Bhd^f/f wild-type mice at three weeks of age. H&E stain, 20×magnification. <b>B)</b> Skin region from (A) at 40×magnification with the epidermal layer outlined and vertical bars showing the thickness from the epidermal basement membrane to the granular layer. Scale bars are 100 µm for both (A) and (B). <b>C)</b> Quantification of the thickness of the epidermis. The Bhd^f/f K14-cre mice have a 3-fold increase in thickness of the epidermis (n = 4, p<0.001). Data are expressed as mean ± standard deviation. <b>D)</b> Bhd^f/f K14-cre mice exhibit elevated levels of phosphorylated ribosomal protein S6 (S235/236, red staining), a readout or mTORC1 activity, compared to littermate control Bhd^f/f wild-type mice at three weeks of age. 20×magnification. E) A representative western blot of the skin of a 7 month old Bhd^f/f K14-cre mouse compared to the skin of a littermate control at 7 months of age. beta-catenin, E-cadherin, p120 catenin, and phospho-NF-kB levels are increased in the mutant skin.</p

FLCN interacts with p0071.

<p><b>A)</b> Schematic of the localization and known functions of p0071 and p120-catenin. <b>B)</b> The homology of p0071 compared to the armadillo repeat proteins p120-catenin, the closest homolog of p0071, and beta-catenin. <b>C)</b> A representative structure of the armadillo repeats of p0071 (AA 415–993). FLCN interacts with armadillo repeats 2–6 based on the yeast-two-hybrid data. <b>D)</b> p0071 interacts with GST-FLCN. MDCK cell lysates were incubated with immobilized GST or GST-FLCN overnight. WCL – whole cell lysate input. <b>E)</b> myc-FLCN and FLAG-p0071 or myc-FLCN and FLAG-vector were expressed in HEK293 cells. Myc-FLCN was immunoprecipitated with anti-myc and detected with anti-FLCN. p0071 was detected using anti-p0071 antibodies. IgG antibodies were used as a control (IgG). <b>F)</b> Endogenous p0071 immunoprecipitation (left) and FLCN immunoprecipitation (right) in HEK293 cells showing co-immunoprecipitation of FLCN and p0071. IgG antibodies were used as a control (IgG).</p

FLCN influences p0071 protein expression, negatively regulates desmosome formation, and negatively regulates cell-cell adhesion.

<p><b>A)</b> The hanging drop assay (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047842#s4" target="_blank">Methods</a> for details) was performed in UOK257 and UOK257-2 cells. After shearing, 60% of the FLCN-null UOK257 cells were present in clusters of 6 or more cells. In contrast, none of the FLCN-expressing UOK257-2 cells remained in clusters of 6 or more cells (n = 3, p<0.001) and more than 80% sheared into single cells. (<b>B–C</b>) FLCN-null UOK257 cells and FLCN re-expressing UOK257-2 cells (<b>D–E</b>) were grown to confluence and analyzed by electron microscopy (50,000×magnification). Desmosomes had normal (B) and abnormal (C) morphology. No desmosomes were seen in the UOK257-2 cells; examples of cell-cell borders (marked by arrows) are shown (D–E). <b>F)</b> T84 (colon carcinoma-derived) cells stably expressing either non-targeting (control) or FLCN shRNA were grown in adherence-free conditions (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047842#s4" target="_blank">Methods</a>). FLCN shRNA cells form large, spherical clusters suggesting increased cell-cell adhesion compared to non-targeting control shRNA cells. G–I) p0071, plakoglobin (JUP), ZO-1, and p120 catenin levels were analyzed by western blot in UOK257 cells compared to UOK257-2 cells.</p