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

p53 induction and activation of DDR1 kinase counteract p53-mediated apoptosis and influence p53 regulation through a positive feedback loop

DDR1, discoidin domain receptor 1, belongs to a subfamily of tyrosine kinase receptors with an extracellular domain homologous to Dictyostellium discoideum protein discoidin 1. We showed that DDR1 is a direct p53 transcriptional target, and that DNA damage induced a p53-dependent DDR1 response associated with activation of its tyrosine kinase. We further demonstrated that DDR1 activated the MAPK cascade in a Ras-dependent manner. Whereas levels of p53, phosphoserine-15 p53, p21, ARF and Bcl-X(L) were increased in response to exogenous overexpression of activated DDR1, dominant-negative DDR1 inhibited irradiation-induced MAPK activation and p53, phosphoserine-15 p53, as well as induced p21 and DDR1 levels, suggesting that DDR1 functions in a feedforward loop to increase p53 levels and at least some of its effectors. Nonetheless, inhibition of DDR1 function resulted in strikingly increased apoptosis of wild-type p53-containing cells in response to genotoxic stress through a caspase-dependent pathway. These results strongly imply that this p53 response gene must predominately act to alleviate the adverse effects of stress induced by p53 on its target cell

p53-mediated induction of Cox-2 counteracts p53- or genotoxic stress-induced apoptosis

The identification of transcriptional targets of the tumor suppressor p53 is crucial in understanding mechanisms by which it affects cellular outcomes. Through expression array analysis, we identified cyclooxygenase 2 (Cox-2), whose expression was inducible by wild-type p53 and DNA damage. We also found that p53-induced Cox-2 expression results from p53-mediated activation of the Ras/Raf/MAPK cascade, as demonstrated by suppression of Cox-2 induction in response to p53 by dominant-negative Ras or Raf1 mutants. Furthermore, heparin-binding epidermal growth factor-like growth factor (HB- EGF), a p53 downstream target gene, induced Cox-2 expression, implying that Cox-2 is an ultimate effector in the p53→HB-EGF→Ras/Raf/MAPK→Cox-2 pathway. p53-induced apoptosis was enhanced greatly in Cox-2 knock-out cells as compared with wild-type cells, suggesting that Cox-2 has an abrogating effect on p53-induced apoptosis. Also, a selective Cox-2 inhibitor, NS-398, significantly enhanced genotoxic stress-induced apoptosis in several types of p53+/+ normal human cells, through a caspase-dependent pathway. Together, these results demonstrate that Cox-2 is induced by p53-mediated activation of the Ras/Raf/ERK cascade, counteracting p53-mediated apoptosis. This anti-apoptosis effect may be a mechanism to abate cellular stresses associated with p53 induction

Identification of ROCK1 as an upstream activator of the JIP-3 to JNK signaling axis in response to UVB damage

Although apoptosis triggered by ultraviolet B (UVB)-mediated activation of the c-Jun N-terminal kinase (JNK) pathway is mediated by both intrinsic and extrinsic pathways, the mechanism of initiation of JNK activation remains obscure. Here, we report the characterization of the JNK-interacting protein 3 (JIP-3) scaffolding protein as an interacting partner of Rho-associated kinase 1 (ROCK1), as determined by tandem affinity protein purification. Upon UVB-induced stress in keratinocytes, ROCK1 was activated, bound to JIP-3, and activated the JNK pathway. Moreover, phosphorylation of JIP-3 by ROCK1 was crucial for the recruitment of JNK. Inhibition of the activity of ROCK1 in keratinocytes resulted in decreased activation of the JNK pathway and thus a reduction in apoptosis. ROCK1(+/-) mice exhibited decreased UVB-mediated activation of JNK and apoptosis relative to wild-type mice. Our findings present a new molecular mechanism by which ROCK1 functions as a UVB sensor that regulates apoptosis, an important event in the prevention of skin cancer

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

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