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Plakophilin3 loss leads to an increase in PRL3 levels promoting K8 dephosphorylation, which is required for transformation and metastasis.

Khapare N, Kundu ST, Sehgal L, Sawant M, Priya R, Gosavi P, Gupta N, Alam H, Karkhanis M, Naik N, Vaidya MM, Dalal SN - PLoS ONE (2012)

Bottom Line: 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.Inhibition of K8 expression in the PKP3 knockdown clone S10, led to a decrease in cell migration and lamellipodia formation.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.

View Article: PubMed Central - PubMed

Affiliation: Tata Memorial Centre, Kharghar Node, Navi Mumbai, Maharashtra, India.

ABSTRACT
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.

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K8 knockdown leads to a decrease in migration.A. 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 and C. 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). D. 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). E. 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).
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pone-0038561-g003: K8 knockdown leads to a decrease in migration.A. 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 and C. 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). D. 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). E. 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).

Mentions: To determine whether the elevated K8 levels were required for transformation upon PKP3 loss, we inhibited K8 expression using RNA interference using previously described shRNA constructs [19]. These experiments were performed in the S10 clone as previous results from the laboratory have demonstrated that the S9 and S10 clones are indistinguishable in terms of their ability to initiate desmosome formation and the transformed phenotype [28], [33]. The PKP3 knockdown clone (S10) was transfected with either the vector control or the K8 knockdown construct and single cell clones with a decrease in PKP3 and K8 expression (8.21, 8.24, 8.28) as compared to a clone transfected with the vector alone (S10P3) were identified (Figure S5A and figure 3A). As reported previously [42], [43], a decrease in the levels of K8 lead to a decrease in the levels of K18 (figure 3A), despite the fact that these constructs do not inhibit K18 [19]. Further, the level of the other type I keratin was also altered in the double knockdown clone 8.21 suggesting that it forms a complex with K8 (Figure S1B). An immunofluorescence analysis demonstrated that filament formation was not affected in the knockdown clones (Figure S5B), though the knockdown cells showed a decrease in the immunofluorescence signal as compared to the vector controls. These results show that the alteration in K8 levels does not lead to alterations in the ability of K8 to form filaments. Similarly, knockdown experiments were performed for K18 in the PKP3 knockdown clone. The knockdown of K18 did not lead to a huge decrease in the levels of K8 (Figure S5C), presumably because of the presence of another type I keratin in the cells (Figure S1B).


Plakophilin3 loss leads to an increase in PRL3 levels promoting K8 dephosphorylation, which is required for transformation and metastasis.

Khapare N, Kundu ST, Sehgal L, Sawant M, Priya R, Gosavi P, Gupta N, Alam H, Karkhanis M, Naik N, Vaidya MM, Dalal SN - PLoS ONE (2012)

K8 knockdown leads to a decrease in migration.A. 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 and C. 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). D. 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). E. 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).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3368841&req=5

pone-0038561-g003: K8 knockdown leads to a decrease in migration.A. 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 and C. 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). D. 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). E. 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).
Mentions: To determine whether the elevated K8 levels were required for transformation upon PKP3 loss, we inhibited K8 expression using RNA interference using previously described shRNA constructs [19]. These experiments were performed in the S10 clone as previous results from the laboratory have demonstrated that the S9 and S10 clones are indistinguishable in terms of their ability to initiate desmosome formation and the transformed phenotype [28], [33]. The PKP3 knockdown clone (S10) was transfected with either the vector control or the K8 knockdown construct and single cell clones with a decrease in PKP3 and K8 expression (8.21, 8.24, 8.28) as compared to a clone transfected with the vector alone (S10P3) were identified (Figure S5A and figure 3A). As reported previously [42], [43], a decrease in the levels of K8 lead to a decrease in the levels of K18 (figure 3A), despite the fact that these constructs do not inhibit K18 [19]. Further, the level of the other type I keratin was also altered in the double knockdown clone 8.21 suggesting that it forms a complex with K8 (Figure S1B). An immunofluorescence analysis demonstrated that filament formation was not affected in the knockdown clones (Figure S5B), though the knockdown cells showed a decrease in the immunofluorescence signal as compared to the vector controls. These results show that the alteration in K8 levels does not lead to alterations in the ability of K8 to form filaments. Similarly, knockdown experiments were performed for K18 in the PKP3 knockdown clone. The knockdown of K18 did not lead to a huge decrease in the levels of K8 (Figure S5C), presumably because of the presence of another type I keratin in the cells (Figure S1B).

Bottom Line: 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.Inhibition of K8 expression in the PKP3 knockdown clone S10, led to a decrease in cell migration and lamellipodia formation.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.

View Article: PubMed Central - PubMed

Affiliation: Tata Memorial Centre, Kharghar Node, Navi Mumbai, Maharashtra, India.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus