Plakophilin3 Loss Leads to an Increase in PRL3 Levels Promoting K8 Dephosphorylation, Which Is Required for Transformation and Metastasis

The desmosome anchors keratin filaments in epithelial cells leading to the formation of a tissue wide IF network. Loss of the desmosomal plaque protein plakophilin3 (PKP3) in HCT116 cells, leads to an increase in neoplastic progression and metastasis, which was accompanied by an increase in K8 levels. The increase in levels was due to an increase in the protein levels of the Phosphatase of Regenerating Liver 3 (PRL3), which results in a decrease in phosphorylation on K8. The increase in PRL3 and K8 protein levels could be reversed by introduction of an shRNA resistant PKP3 cDNA. Inhibition of K8 expression in the PKP3 knockdown clone S10, led to a decrease in cell migration and lamellipodia formation. Further, the K8 PKP3 double knockdown clones showed a decrease in colony formation in soft agar and decreased tumorigenesis and metastasis in nude mice. These results suggest that a stabilisation of K8 filaments leading to an increase in migration and transformation may be one mechanism by which PKP3 loss leads to tumor progression and metastasis

Apert′s Syndrome: A Rare Case Report

Apert′s syndrome /Acrocephalosyndactyly is a rare, congenital disorder characterized by craniosynostosis, midfacial malformations and symmetrical syndactyly. It is caused by a genetic mutation in the FGFR2 gene on chromosome 10. Although the syndrome has typical clinical features, the relative rarity of the condition still poses a diagnostic dilemma. Considering the general paucity of cases in the Indian literature, we present a case report of a 14-year-old female having all the features of classical Apert′s syndrome

High K8 levels in the PKP3 knockdown clones are due to a decrease in phosphorylation.

<p><b>A.</b> Protein extracts from the vector control (pTU6) and PKP3 knockdown clones (S9 and S10) were resolved on SDSPAGE gels and Western blots performed with the indicated antibodies. <b>B.</b> S9 cells were transfected with either dsRed or the shRNA resistant dsRed PKP3 3.7R cDNA. 48 hours post transfection cells were stained with antibodies to K8 (green) and visualized by confocal microscopy. Note that dsRed PKP3 3.7R localizes to the border as previously described (indicated by arrow) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038561#pone.0038561-Gosavi1" target="_blank">[28]</a>. Original magnification is 630X with a 2X optical zoom. Scale bar 5 µm. <b>C.</b> A real time PCR analysis to determine the mRNA levels of K8 and K18 was performed on RNA isolated from the vector control and PKP3 knockdown clones. GAPDH was used as an internal control for normalization. The Ct values for all samples are shown on the Y-axis. <b>D.</b> Protein extracts from the PKP3 knockdown clones or the vector control were subjected to 2-dimensional gel electrophoresis and Western blots performed with antibodies to K8. <b>E.</b> Protein extracts from the vector control cells or the PKP3 knockdown clones were resolved on SDS-PAGE gels followed by Western blotting with antibodies to K8 or phosphospecific antibodies against S73 (α-S73), S431 (α-S431) and actin. <b>F.</b> HCT116 cells transfected with GFPK8 or GFPS73A or GFP S431A were resolved on two dimensional gels followed by Western blots with antibodies to K8. MW markers are indicated. Protein extracts from the transfected cells were resolved on SDS-PAGE gels followed by Western blotting with antibodies to actin to serve as loading controls.</p

Plakophilin3 loss leads to an increase in PRL3 levels.

<p><b>A.</b> RNA prepared from the vector control or PKP3 knockdown clones was used as a template in reverse transcriptase coupled PCR reactions to determine the mRNA levels of PRL3 and PKP3. A PCR for GAPDH served as a loading control. <b>B.</b> S9 cells were transfected with either dsRed or the shRNA resistant dsRed PKP3 3.7R cDNA. 48 hours post transfection cells were stained with antibodies to PRL3 (green) and visualized by confocal microscopy. Note that dsRed PKP3 3.7R localizes to the border as previously described (indicated by arrow) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038561#pone.0038561-Gosavi1" target="_blank">[28]</a>. Original magnification is 630X with a 2X optical zoom. Scale bar 5 µm. <b>C.</b> The vector control or PKP3 knockdown clones were treated with either the vehicle control (DMSO) or the indicated concentrations of the PRL3 inhibitor. Protein extracts were resolved on gels followed by Western blotting with antibodies to K8 and β-actin. <b>D.</b> GFP PRL3 was transfected into either vector control (pTU6) or PKP3 knockdown clones (S9 and S10). 48 hours post transfection, the cells were stained with antibodies to K8 (red) and visualized by confocal microscopy. Note that GFP PRL3 shows a marginally enhanced localization to the border in S9 and S10 cells in contrast to pTU6 and doesn’t show increased localization on K8 filaments. Original magnification is 630X with a 2X optical zoom. Scale bar 5 µm. <b>E.</b> Total cell extracts (T), Soluble fractions (S) and the filament fractions (F) were prepared as described from either the vector control or PKP3 knockdown clones. Equal cell equivalents of these extracts were loaded on 10% SDS-PAGE gels followed by Western blotting with antibodies to K8 (top four panels). A Western blot for β-actin was performed in the total cell extracts as a loading control (fifth panel). 100 µg of soluble fractions were resolved on SDS-PAGE gels followed by Western blotting for K8 or β-actin (bottom two panels).</p

K8 knockdown leads to a decrease in migration.

<p><b>A.</b> Protein extracts from the S10 derived K8 (8.21, 8.24 and 8.28) knockdown clones or the vector alone (S10P3) were resolved on SDSPAGE gels followed by Western blotting with antibodies to PKP3, K8, K18, rac, rhoA and β-actin. <b>B and C.</b> Scratch wound healing assays were performed on the S10 derived K8 knockdown clones or the vector control and the distance migrated measured. The data shown is the average from three independent experiments with the mean and standard error plotted as shown (* p<0.03 by students t-test). <b>D.</b> Scratch wound healing assays were performed on the double knockdown clone 8.21, transfected with either GFP alone or GFP K8 res and the distance migrated measured. The data shown is the average of three independent experiments (p<0.01 by students t-test). <b>E.</b> Scratch wound healing assays were performed on the vector control (pTU6) or PKP3 knockdown clones (S9 and S10) or the S10 derived K8 (8.21, and 8.28) knockdown clones or the vector control (S10P3). The cells were fixed and stained with FITC labeled phalloidin to visualize actin filaments, followed by confocal microscopy. Arrows indicate cells showing lamellipodia formation (Original magnification is 630X with a 2X optical zoom. Scale bar 10 µm).</p

K8 downregulation leads to an inhibition of transformation in vitro and in vivo.

<p><b>A.</b> The S10 derived K8 (8.21, 8.24 and 8.28) knockdown clones or the vector alone (S10P3) were plated in soft agar and colony formation determined after 2–3 weeks. The number of colonies formed by the clones per 20 low power fields (10X) was counted in triplicate in each experiment and the mean and standard deviation of three independent experiments is plotted as shown. <b>B.</b> 10⁶ cells from the S10 derived K8 (8.21 and 8.28) knockdown clones or the vector alone (S10P3) were injected subcutaneously into 5 different nude mice and tumor size determined every week as described. Tumor volume is plotted on the Y-axis and the time in weeks on the X-axis. <b>C.</b> Protein extracts from primary tumors from mice injected with the S10 derived K8 (8.21 and 8.28) knockdown clones or the vector alone (S10P3) were resolved on SDS-PAGE gels followed by Western blotting with antibodies to K8 and β-actin. The numbers indicate different mice injected with the single or double knockdown clones. All the samples were run on the same gel and the Western blots performed at the same time. <b>D.</b> Haematoxylin and eosin staining of paraffin embedded sections of lung tissue from nude mice injected with 10⁶ cells of the vector alone (S10P3) or the double knockdown clones (8.21 and 8.28). Lung section from mice injected with S10P3 cells show extensive metastasis with thickening of alveolar walls from deposition and aggregation of metastasized tumor cells, whereas lungs from mice injected with the double knockdown cells show normal lungs with thin walled alveoli, with a few metastatic tumor cells. The images in the top row are at magnification x100 and images in the bottom row are at magnification x 400. <b>E.</b> PCR reactions were performed on DNA isolated from paraffin sections for the presence of Alu repeats in genomic DNA. Genomic DNA was purified from normal lung tissue, lung tissue from mice injected with cells with PKP3 knockdown alone (S10P3) and lung tissue from mice injected with the double knockdown clones (8.21 and 8.28). Lung tissues from uninjected mice (N) were used as a negative control for the Alu PCR. A PCR for the mouse patch gene was performed as a loading control. <b>F.</b> Immunohistochemical staining was performed with antibody against K8 on sections of paraffin embedded tissue of tumor and lungs of mice injected with S10P3 vector control cells or the double knockdown clones, 8.21 and 8.28. Images a, b, c, g, h and i are taken at a magnification x 200 and the respective magnified images at magnification x 400, of the indicated areas in the white rectangles, are represented by images d, e, f, j, k and l. Tumor tissue from mice injected with S10P3 (a and d) show K8 staining at levels higher compared to tumors from mice injected with the double knockdown clones, 8.21 and 8.28 (b, c, e and f). In contrast lung tissue, from mice injected with the vector control S10P3 cells (g and j) and the double knockdown clones (8.21 and 8.28) (h, i, k and l) show elevated K8 staining in metastatic areas of the lungs with infiltrated tumor cells.</p

PKP3 CK8 double knockdown clones show a decrease in lung metastasis.

<p>Hematoxylin-eosin stained sections of lungs from mice injected with the indicated clones were examined for the presence of infiltrating tumor cells and the percentage of area occupied by the tumor cells determined microscopically. Five mice were tested for each clone.</p