7 research outputs found
Bhd<sup>f/f</sup> K14-Cre mice exhibit increased epidermal thickness.
<p>A) Bhd<sup>f/f</sup> K14-cre mice exhibit acanthosis and hyperkeratosis (arrows) compared to littermate control Bhd<sup>f/f</sup> 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<sup>f/f</sup> 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<sup>f/f</sup> 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<sup>f/f</sup> wild-type mice at three weeks of age. 20×magnification. E) A representative western blot of the skin of a 7 month old Bhd<sup>f/f</sup> 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
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
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
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<sup>f/f</sup> K14-Cre mice have wavy hair, erythematous skin, and delayed eyelid opening.
<p><b>A)</b> Bhd<sup>f/f</sup> K14-cre mice at three weeks of age display coarse, wavy fur and wavy whiskers compared to Bhd<sup>f/f</sup> wild-type mice. <b>B)</b> A representative Bhd<sup>f/f</sup> K14-cre mouse at three weeks of age with erythematous skin and hair loss (arrow). <b>C)</b> 100% of Bhd<sup>f/f</sup> K14-cre mice have closed eyelids at three weeks of age (arrow) compared to 0% of wild-type mice.</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